Compound for treating diseases caused by abnormal fat metabolism

Abstract

The invention discloses a compound for treating diseases caused by abnormal fat metabolism, such as a compound shown in a formula I, and application of the compound in preparation of drugs for treating diseases caused by abnormal fat metabolism. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry technology, but is not limited thereto, and in particular to a compound for treating diseases caused by abnormal fat metabolism. Technical Background

[0002] Abnormal lipid metabolism is an abnormality in the substance and amount of lipids and their metabolites in the blood and other tissues and organs caused by congenital or acquired factors. Lipid metabolism includes digestion and absorption of lipids in the small intestine, entry into the blood circulation from the lymphatic system (transported by lipoproteins), transformation by the liver, storage in adipose tissue, and utilization by tissues when needed. The main function of lipids in the body is oxidation for energy, and adipose tissue is the body's energy warehouse. Phospholipids are important structural components of all cell membranes, and cholesterol is a precursor of bile acid and steroid hormones (adrenal cortex hormones and gonadal hormones). Lipid metabolism is regulated by genetics, neurohumoral factors, hormones, enzymes, and tissues and organs such as the liver. When these factors are abnormal, lipid metabolism disorders and pathological and physiological changes in related organs can occur.

[0003] The liver is an important organ for the human body to metabolize fat. When fat metabolism is abnormal, fat can accumulate in liver cells, forming fatty liver, which mainly includes alcoholic fatty liver disease (ALD) and non-alcoholic fatty liver disease (collectively referred to as metabolic-associated fatty liver disease MAFLD from 2022).

[0004] There are more than 1.3 billion patients with liver disease in the world, of which more than 400 million are in China. Among these more than 400 million Chinese patients, nearly half (>200 million) are fatty liver patients. Fatty liver disease is seriously threatening the health of Chinese people and has become the second largest liver disease after viral hepatitis. It has been recognized as a common cause of latent cirrhosis. Therefore, finding drugs that can effectively regulate fat metabolism has important social significance and economic value. Summary of the invention

[0005] The inventors have developed a compound that can be decomposed into two different action targets and has the characteristics of synergistic effects on diseases caused by abnormal fat metabolism, providing a feasible solution to the current urgent clinical needs in the field of non-alcoholic fatty liver disease.

[0006] In one aspect, the present invention provides a compound of formula I:

[0007]

[0008] including isomers, solvates, isotopic derivatives, oxides, and salts thereof;

[0009] in:

[0010] M is

[0011]

[0012] L is

[0013] R x , R y are independently hydrogen, C1-C6 alkyl, or R x , R y Connected into a ring in any reasonable way;

[0014] G is

[0015]

[0016]

[0017] When M is

[0018]

[0019] When G is not

[0020]

[0021]

[0022] When L is When G is

[0023]

[0024] When L is hour,

[0025] G is

[0026]

[0027] In some embodiments, M is

[0028] L is in,

[0029] R x , R y are independently hydrogen, C1-C6 alkyl, or R x , R y are connected in any reasonable manner to form a ring; preferably, R x , R y is hydrogen;

[0030] G is

[0031] In some embodiments, M is

[0032] L is

[0033] G is

[0034] In some embodiments, the isomers, solvates, isotope derivatives, oxides, and salts thereof provided by the present invention are selected from the following compounds:

[0035]

[0036]

[0037]

[0038]

[0039] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned isomers, solvates, isotopic derivatives, oxides, and salts thereof.

[0040] In another aspect, the present invention provides the use of the above-mentioned isomers, solvates, isotope derivatives, oxides, and salts thereof, or the above-mentioned pharmaceutical compositions for preparing drugs for treating diseases caused by abnormal fat metabolism.

[0041] In some embodiments, the novel compounds of the present invention can be formulated into pharmaceutical compositions and administered to patients according to a variety of suitable administration routes, including systemic administration such as oral or parenteral, intravenous, intramuscular, transdermal or subcutaneous administration, etc.

[0042] The present invention provides the above-mentioned pharmaceutical composition which can be used to treat related diseases caused by abnormal fat metabolism, including obesity, hypothyroidism, thyroid cancer, diabetes, cardiovascular disease, hyperlipidemia, hypercholesterolemia, atherosclerosis, non-alcoholic steatohepatitis (NASH), hypertriglyceridemia, and fatty liver lesions.

[0043] The compounds disclosed in the present invention are metabolized in the body to release molecular fragments with different action mechanisms, which act synergistically to more quickly and efficiently treat diseases caused by fat metabolism. DETAILED DESCRIPTION

[0044] Many exemplary preparation methods of the compounds of the present invention are provided in the following examples. The present invention is described in detail below by way of example, but is not intended to be any adverse limitation to the present invention. The present invention has been described in detail herein, and specific embodiments thereof are disclosed therein, and it will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Certain compounds of the present invention can be used as intermediates for preparing other compounds of the present invention, and the structures of all compounds are determined by liquid chromatography.

[0045] Unless otherwise specified, the materials in the examples of this application were purchased through commercial channels.

[0046] Example 1: Synthesis of compound RES-01

[0047] Reaction:

[0048]

[0049] Preparation method:

[0050] Step 1: Preparation of compound RES-02:

[0051] Under nitrogen protection, 3,6-dichloro-4-isopropylpyridazine (5.00 g, 26.2 mmol) was dissolved in DMF (50 mL) at room temperature, and then 2,6-dichloro-4-aminophenol (4.66 g, 26.2 mmol) and potassium tert-butoxide (5.90 g, 52.6 mmol) were added to the above system. The reaction mixture was reacted at 90 ° C for 24 hours, then cooled to room temperature and poured into water (500 mL). The pH was adjusted to 7-8 with 1N dilute hydrochloric acid, and then ethyl acetate was added and stirred thoroughly. The system was filtered through diatomite and the filter cake was washed with ethyl acetate. The filtrate was refined and separated, and the organic phase was collected. The aqueous phase was extracted with ethyl acetate twice again, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound RES-02: 6.05 g, with a yield of 69.54%.

[0052] ESI-MS (+): m / z = 333.32;

[0053] 1 HNMR (400MHz) δ: 7.63 (s, 1H), 6.67 (s, 2H), 5.65 (brs, 2H), 3.11 (m, 1H), 1.24 (d, J = 6.8Hz, 6H).

[0054] Step 2: Preparation of compound RES-03:

[0055] Add glacial acetic acid (100 mL) to the reaction bottle, then add compound RES-02 (6.00 g, 18.04 mmol) and sodium acetate (4.91 g, 36.08 mmol) in sequence under stirring at room temperature. After the addition is complete, the system is heated to 100 ° C for 24 hours, and then cooled to room temperature for 2 days. The system is concentrated, the residue is diluted with water, and then the pH of the system is adjusted to 9 with 1N sodium hydroxide. The system is extracted twice with ethyl acetate, the aqueous phase is adjusted to pH 5 with concentrated hydrochloric acid, and then extracted twice with ethyl acetate again, all organic layers are combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was diluted with methanol, 1N sodium hydroxide (100 mL, 100 mmol) was added, heated to 120 ° C for 24 hours, then cooled and concentrated, an appropriate amount of purified water was added to the residue, extracted twice with ethyl acetate, the organic phases were combined, washed twice with dilute hydrochloric acid aqueous solution and saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column to obtain compound RES-03: 4.12 g, with a yield of 72.66%.

[0056] ESI-MS (+): m / z = 315.18;

[0057] 1 HNMR (400MHz) δ: 12.12 (s, 1H), 7.27 (s, 1H), 6.67 (s, 2H), 5.62 (brs, 2H), 3.01 (m, 1H), 1.18 (d, J = 6.8Hz, 6H).

[0058] Step 3: Preparation of compound RES-04:

[0059] Compound RES-03 (3.0 g, 9.55 mmol) was added to water (30 mL), and then concentrated hydrochloric acid (10 mL) was added. The reaction mixture was cooled to 0°C, and an aqueous solution (5 mL) containing sodium nitrite (0.79 g, 11.46 mmol) was slowly added, and then the above system was stirred at 0°C for 30 minutes. Then the above reaction solution was quickly filtered and the filtrate was quickly added to a mixed solution of N-cyanoacetylurea (1.97 g, 12.61 mmol), water (200 mL) and pyridine (50 mL) precooled to 0°C. After the addition was completed, it was stirred at 0°C for 30 minutes, filtered, and the filter cake was rinsed with water and petroleum ether in turn, and purified by column to obtain compound RES-04: 2.87 g, with a yield of 62.39%.

[0060] ESI-MS (+): m / z = 482.07;

[0061] 1HNMR(400MHz)δ:12.25(s,1H),12.22(s,1H),10.89(s,1H),7.99(s,2H),7.36( s, 1H), 4.21 (q, J = 7.1Hz, 2H), 3.06 (m, 1H), 1.27 (t, J = 7.1Hz, 3H), 1.20 (m, 6H).

[0062] Step 4: Preparation of compound RES-01:

[0063] At room temperature, compound RES-04 (2.0 g, 4.16 mmol) was added to glacial acetic acid (40 mL), and then sodium acetate (1.71 g, 20.8 mmol) was added. The reaction mixture was heated to 120°C and maintained for 1.5 hours, and then cooled to 0°C. The system was diluted with water and stirred for 30 minutes. Filtered, the filter cake was dissolved in hot acetonitrile and decolorized with activated carbon, filtered with diatomaceous earth, rinsed, and the filtrate was concentrated under reduced pressure. The residue was slurried with a mixed solution of water and acetonitrile, filtered, and dried to obtain compound RES-01: 1.03 g, with a yield of 56.91%.

[0064] ESI-MS (+): m / z = 436.22;

[0065] 1 HNMR (400MHz) δ: 13.20 (brs, 1H), 12.25 (s, 1H), 7.78 (s, 2H), 7.46 (s, 1H), 3.06 (m, 1H), 1.18 (d, J = 6.91Hz, 6H).

[0066] Example 2: Synthesis of compound RES-05

[0067]

[0068] Referring to the operation procedure of Example 1, RES-05 was obtained through four steps of synthesis reaction.

[0069] ESI-MS (+): m / z = 443.20;

[0070] 1HNMR (400MHz) δ: 12.00 (brs, 1H), 8.31 (d, J = 8.0Hz, 1H), 8.26 (d, J = 8.0Hz, 1H), 8.09 (t, J = 12.0Hz, 1H), 8.03 (t, J = 16.0Hz, 1H), 7.81 (s, 2H).

[0071] Example 3: Synthesis of compound RES-M01

[0072] Reaction:

[0073]

[0074] Preparation method:

[0075] Under nitrogen protection, compound RES-01 (0.50 g, 1.15 mmol) was dissolved in methanol (15 mL), and then formaldehyde aqueous solution (35-40%, 4 mL) was slowly added, the temperature was raised to 100 ° C for 8 h, TLC detected that the reaction was complete, the system was concentrated, water and dichloromethane were added, shaken, separated, the aqueous phase was extracted with dichloromethane again, the dichloromethane phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the concentrated residue was purified and separated by column to obtain compound RES-M01: 0.30 g, with a yield of 56.6%.

[0076] ESI-MS (+): m / z = 466.20;

[0077] 1 HNMR (400MHz) δ: 12.25 (s, 1H), 7.78 (s, 2H), 7.46 (s, 1H), 5.17 (s, 2H), 4.01 (brs, 1H), 3.06 (m, 1H), 1.18 (d, J = 6.91Hz, 6H).

[0078] Example 4: Synthesis of compound RES-M02

[0079] Reaction:

[0080]

[0081] Preparation method:

[0082] Step 1: Preparation of compound RES-MM

[0083] In a reaction flask, RES-01 (0.50 g, 1.15 mmol), pyridinium p-toluenesulfonate (28.9 mg, 0.12 mmol) and 3,4-dihydro-2H-pyran were added to 1,4-dioxane (10 mL) in sequence. After the addition was completed, the temperature was raised to 65 ° C for reaction for 16 hours, and then concentrated under reduced pressure. The concentrated residue was prepared for liquid separation to obtain compound RES-MM: 0.25 g, with a yield of 42.02%.

[0084] ESI-MS (+): m / z = 520.07;

[0085] 1HNMR(400MHz)δ:7.52(s,1H),6.87(s,2H),5.73(m,1H),5.37(s,2H),4.01(brs, 1H), 3.68-3.54 (m, 2H), 3.06 (m, 1H), 2.01-1.48 (m, 6H), 1.18 (d, J = 6.91Hz, 6H).

[0086] Step 2: Preparation of compound RES-M02

[0087] Referring to the operation procedure of Example 2, compound RES-M02 was obtained: 0.10 g, yield 38.46%. ESI-MS (+): m / z = 550.26.

[0088] Example 5: Synthesis of compound RES-M03

[0089] Reaction:

[0090]

[0091] Preparation method:

[0092] In a reaction flask, RES-01 (0.50 g, 1.15 mmol) was dissolved in acetonitrile (10 mL), potassium carbonate (0.32 g, 2.30 mmol) was added, and then the temperature was raised to 60°C. After reaching the reaction temperature, chloroiodomethane (0.41 g, 2.30 mmol) was slowly added dropwise. After the addition was completed, the temperature was maintained and the reaction was continued for 4 hours. The system was brought to room temperature, filtered through diatomaceous earth, and the filtrate was concentrated to remove the solvent. The concentrated residue was subjected to column chromatography to obtain RES-M03: 0.18 g, yield: 32%.

[0093] ESI-MS (+): m / z = 483.18;

[0094] 1 HNMR(400MHz)δ:10.89(s,1H),7.99(s,2H),7.36(s,1H),6.08(s,2H),3.06(m,1H),1.20(m,6H).

[0095] Example 6: Synthesis of compound RES-M04

[0096]

[0097] Referring to the operating procedures of Example 3, compound RES-M04 was prepared.

[0098] ESI-MS (+): m / z = 473.23;

[0099] 1HNMR (400MHz) δ: 8.31 (d, J = 8.0Hz, 1H), 8.26 (d, J = 8.0Hz, 1H), 8.09 (t, J = 12.0H z, 1H), 8.03 (t, J = 16.0Hz, 1H), 7.81 (s, 2H), 5.40 (s, 2H).

[0100] Example 7: Synthesis of compound RES-M05

[0101]

[0102] Referring to the operating procedures of Example 4, compound RES-M05 was prepared.

[0103] ESI-MS (+): m / z = 557.34;

[0104] 1 HNMR(400MHz)δ:8.31(d,J=8.0Hz,1H),8.26(d,J=8.0Hz,1H),8.09(t,J=12.0H z,1H),8.03(t,J=16.0Hz,1H),7.81(s,2H),5.70(t,J=12Hz,1H),5.40(s,2H),3.68-3.56(m,2H),1.98-1.45(m,6H).

[0105] Example 8: Synthesis of compound RES-M06

[0106]

[0107] Referring to the operation procedure in Example 5, compound RES-M06 was prepared.

[0108] ESI-MS (+): m / z = 491.27;

[0109] 1HNMR (400MHz) δ: 8.31 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 8.0 Hz, 2H), 7.81 (s, 2H), 6.05 (s, 2H).

[0110] Example 9: Synthesis of Compound DSC180-01

[0111] Reaction:

[0112]

[0113] Preparation method:

[0114] Step 1: Preparation of compound RES-M04:

[0115] In a reaction flask, P-01 (1.0 g, 2.38 mmol) was added to dichloromethane (10 mL), 1 drop of DMF was added to the system, and then a solution of thionyl chloride (0.34 g, 2.85 mmol) in dichloromethane (5 mL) was added at 0°C. The temperature was maintained and stirred for 5 hours. The system was concentrated under reduced pressure to obtain a white solid of RES-M04: 1.05 g, which was used directly in the next step without purification.

[0116] Step 2: Compound DSC180-01

[0117] In a reaction flask, RES-M01 (0.50 g, 1.07 mmol) was dissolved in anhydrous acetonitrile (10 mL), potassium carbonate (0.22 g, 1.61 mmol) was added, and the system was cooled to 0°C, and a solution of RES-M03 (0.52 g, 1.18 mmol) in anhydrous acetonitrile (5 mL) was added dropwise. After the addition was completed, the temperature was naturally raised to room temperature and reacted for 16 hours. The system was poured into water, and the aqueous phase was extracted three times with DCM. The organic phases were combined and dried and concentrated. After the liquid phase was prepared, the white solid was lyophilized to obtain DSC180-01: 0.37 g, yield: 39.78%.

[0118] ESI-MS (+): m / z = 867.27;

[0119] 1 HNMR(400MHz)δ:12.25(s,1H),7.78(s,2H),7.46(s,1H),5.17(s,2H),4.50(s,1 H),4.45(s,1H),3.57(m,1H)3.30(m,1H),3.06-2.85(m,3H),1.89-0.72(m,43H).

[0120] Example 10: Synthesis of Compound DSC180-02

[0121] Reaction:

[0122]

[0123] Preparation method:

[0124] Step 1: Preparation of compound DSC180-021:

[0125] Referring to the operating procedures of Example 4, compound DSC180-021 was prepared: 0.45 g, with a yield of 51.7%.

[0126] ESI-MS (+): m / z = 953.0.

[0127] Step 2: Preparation of compound DSC180-02:

[0128] In the reaction flask, DSC180-021 (0.30 g, 0.31 mmol) was dissolved in ethanol (10 mL), and 732 type strong acid cation exchange resin (1.5 g) was added. After stirring at room temperature for 18 hours, the mixture was filtered. After the filtrate was concentrated, the concentrated residue was prepared by preparative liquid phase to obtain compound DSC180-02: 0.12 g, yield: 43.96%.

[0129] ESI-MS (+): m / z = 867.16;

[0130] 1 HNMR(400MHz)δ:13.20(brs,1H),7.78(s,2H),7.46(s,1H),5.17(s,2H),4.50(s, 1H),4.45(s,1H),3.57(m,1H)3.30(m,1H),3.06-2.85(m,3H),1.89-0.72(m,43H).

[0131] Example 11: Synthesis of Compound DSC180-03

[0132] Reaction:

[0133]

[0134] Preparation method:

[0135] In the reaction bottle, RES-M03 (0.5 g, 1.04 mmol), P-01 (0.65 g, 1.55 mmol) and triethylamine (0.32 g, 3.12 mmol) were mixed and dissolved in acetonitrile (20 mL), heated to 70 ° C for 18 hours, and the system was cooled and filtered through diatomaceous earth. The filtrate was directly concentrated to remove the solvent, and the concentrated residue was subjected to column chromatography to obtain DSC180-03: 0.80 g, yield: 89%.

[0136] ESI-MS (+): m / z = 867.31;

[0137] 1 HNMR(400MHz)δ:12.25(s,1H),7.78(s,2H),7.46(s,1H),6.37(s,2H),4.50(s,1 H),4.45(s,1H),3.57(m,1H)3.30(m,1H),3.06-2.85(m,3H),1.89-0.72(m,43H).

[0138] Example 12: Synthesis of Compound DSC180-14

[0139] Reaction:

[0140]

[0141] Preparation method:

[0142] Step 1: Preparation of compound RES-M05

[0143] In the reaction flask, P-02 (0.50 g, 1.89 mmol) was dissolved in DCM (10 mL), pyridine (0.18 g, 2.27 mmol) was added dropwise, the system was cooled to 0°C, triphosgene (0.62 g, 2.08 mmol) was added in batches, and the temperature was maintained for 6 hours after the addition was completed. The system was directly concentrated, and the concentrated residue was compound RES-M05, which was used directly in the next step without treatment.

[0144] Step 2: Preparation of compound DSC180-14

[0145] In a reaction flask, RES-M01 (0.50 g, 1.07 mmol) and triethylamine (0.15 g, 1.5 mmol) were added to dichloromethane (10 mL) in sequence, and then a solution of RES-M05 (1.89 mmol, the yield in the previous step was 100%) in dichloromethane (5 mL) was added dropwise at 0°C. The temperature was maintained and stirred for 5 hours. The system was concentrated under reduced pressure, and the concentrated residue was prepared by preparative liquid phase to obtain compound DSC180-14: 0.27 g, with a yield of 33.3%.

[0146] ESI-MS (+): m / z = 755.21;

[0147] 1 HNMR(400MHz)δ:12.25(s,1H),7.78(s,2H),7.46(s,1H),5.17(s,2H),4.85(d,1H),4.65( s,1H),4.57(d,2H),4.32-3.97(m,6H),3.81-3.65(m,3H),3.38-2.57(m,3H),1.20(m,6H).

[0148] Example 13: Synthesis of Compound DSC180-15

[0149] Reaction:

[0150]

[0151] Preparation method:

[0152] Step 1: Preparation of compound RES-M05

[0153] RES-M05 was prepared by referring to the operation process of step 1 in Example 8;

[0154] Step 2: Preparation of compound DSC180-15

[0155] RES-M05 (0.5 g, 1.03 mmol), RES-M03 (0.53 g, 1.24 mmol) and triethylamine (0.52 g, 5.2 mmol) were mixed and dissolved in 1,4-dioxane (20 mL), heated to 80 ° C for 4 hours, and the system was cooled and poured into ice water. The aqueous phase was extracted with DCM. After separation, the organic phases were combined and concentrated. The concentrated residue was subjected to column chromatography to obtain DSC180-15: 0.61 g, yield: 78%.

[0156] ESI-MS (+): m / z = 755.28;

[0157] 1 HNMR(400MHz)δ:12.25(s,1H),7.78(s,2H),7.46(s,1H),6.07(s,2H),4.85(d,1H),4.65( s,1H),4.57(d,2H),4.32-3.97(m,6H),3.81-3.65(m,3H),3.38-2.57(m,3H),1.20(m,6H).

[0158] Example 14: Synthesis of Compound DSC180-20

[0159] Reaction:

[0160]

[0161] Preparation method:

[0162] RES-M01 (0.5 g, 1.07 mmol), P-04 (0.40 g, 1.61 mmol) and pyridine (1 mL) were mixed and dissolved in acetonitrile (10 mL). DCC (0.66 g, 3.21 mmol) was added and the temperature was raised to 50 °C for reaction for 24 hours. After diatomaceous earth filtration, the filtrate was concentrated to remove the solvent, and the concentrated residue was used to prepare a liquid phase to obtain DSC180-20: 0.13 g, yield: 17%.

[0163] ESI-MS (+): m / z = 692.13;

[0164] 1HNMR(400MHz)δ:12.25(s,1H),9.04(s,1H),8.37(s,2H),8.12(s,1H),7.78(s,2H),7.46(s,1H),5.17(s,2H ),4.85(t,1H),4.18(s,2H),4.01(s,2H),2.18(m,1H),1.87(m,2H),1.44(m,1H),1.20(d,6H),1.09(d,6H).

[0165] The following example compounds were synthesized by the same method as in the above examples using commercially available compounds or intermediate compounds appropriately synthesized from commercially available compounds.

[0166]

[0167]

[0168]

[0169]

[0170] Example 15: Testing of THRα or THRβ agonist activity based on reporter gene activity detection method

[0171] Construction of pGAL4-THR-LBD plasmid and preparation of pG5-Luc and pRL-Tk plasmids:

[0172] The pGAL4-THRα-LBD and pGAL4-THRβ-LBD reporter gene detection system plasmids used were constructed according to conventional molecular cloning methods. The main steps are as follows: using PCR technology, the cDNA sequences of THRα (NM-003250) and THRβ (NM-000461) corresponding to the amino acid sequences of THRα (163-407AA) and THRβ (217-461AA) were inserted into the BamHI and NotI restriction sites of the pGAL4 vector to obtain plasmids pGAL4-THRα-LBD and pGAL4-THRβ-LBD; plasmids pG5-Luc (#E249A) and pRL-Tk (#E2241) were purchased from Promega; Escherichia coli DH5α competent cells were prepared using the CaCl2 method, and the plasmids were transformed into DH5α competent cells. After further culture and amplification, the corresponding plasmid DNA was purified using a plasmid extraction kit (TIANGEN, #D107).

[0173] Plasmid co-transfection into HEK293T cells and compound treatment:

[0174] One day before plasmid transfection, HEK293T cells were cultured at 1×104 / well density inoculated in a 96-well plate, cultured at 37 ° C, 5% CO2 incubator, when the cells enter the exponential growth phase, according to the transfection reagent Cell transfection was performed according to the instructions of HD (Promega, #E2311).

[0175] The main steps are as follows: taking a single culture well as an example, add 20 ng of plasmid pGAL4-THRα-LBD or pGAL4-THRβ-LBD, 50 ng of pG5-Luc, and 5 ng of pRL-TK to 10 μL of Opti-MEMTMⅠ culture medium (Gibco, #11058021), mix thoroughly, and then add DNA transfection reagent. HD (Promega, #E2311) 0.25μL, after thorough mixing, stand at room temperature for 5min to obtain a mixture; take 10μL of this mixture and add it to the cell culture well containing 100μL culture medium for cell co-transfection. 6h after cell co-transfection, add a series of concentrations of compounds to the cell culture medium for 24h treatment. The compounds are divided into 10 concentrations, with the highest concentration being 1μM. They are diluted 3 times with DMSO and divided into 3 replicates. Triiodothyronine (T3) is used as a positive control, and a negative control group (containing cells, without drugs and plasmids) is set up.

[0176] Detection:

[0177] After the cells were treated with the compounds for 24 h, Detection was performed according to the instructions of Luciferase Assay System (Promega, #E2940).

[0178] The main steps are: aspirate 50 μL of culture medium from each culture well, discard it, and then add 50 μL Luciferase reagent, gently shake at room temperature for 10 minutes; take 80 μL of the cleavage reaction solution and add it to the white opaque optiPlate-96 well plate, and use the MD i3x multifunctional microplate reader to detect the luminescent signal value (Firefly-Luc) of firefly luciferase; then add Stop& 40 μL of the reagent was gently shaken at room temperature for 10 min; then the luminescent signal of Renilla luciferase (Renilla-Luc) was detected using an MD i3x multifunctional microplate reader.

[0179] The data is processed according to formula (1).

[0180] Formula (1): Calculation formula: F / R ratio = Firefly fluorescence signal / Renilla fluorescence signal

[0181] The agonist activity was obtained by processing according to formula (2).

[0182] Formula (2): Agonist activity (%) = (F / R test compound - F / R negative control) / (F / R positive compound - F / R negative control) × 100%, and then use GraphPadPrism software to draw a curve and calculate the THRβ-EC of each test compound 50 (nM).

[0183] The THRα-EC of the test compound was measured and calculated using the similar method of the above formula (1) and formula (2): 50 (nM).

[0184] The THRβ agonist selectivity (SI) was calculated according to formula (3).

[0185] Formula (3): THRβ agonist selectivity (SI) = THRα-EC 50 / THRβ-EC 50

[0186] The experimental results are shown in Table 1.

[0187] Table 1 Agonistic activity and agonistic selectivity of the compounds of the present invention on THR-β and THR-α

[0188]

[0189]

[0190]

[0191] Note: *The structural formula of positive compound 1 is:

[0192] The structural formula of positive compound 2 is:

[0193] The structural formula of reference compound 1 is:

[0194] The structural formula of reference compound 2 is:

[0195] The structural formula of Aramchol is:

[0196] Analysis of the experimental data in the above table shows that the test compounds all showed THRβ agonist activity superior to that of positive compound 1, positive compound 2, control compound 1 and control compound 2. Among them, DSC180-1, DSC180-7, DSC180-10, DSC180-13, DSC180-16, DSC180-20, DSC180-28, DSC180-31, DSC180-34, DSC180-44, and DSC180-47 had a selectivity for THRβ agonist activity that was more than twice that of the positive compounds and control compounds. The selectivity of DSC180-44 and DSC180-47 for THRβ agonist activity was more than three times that of the positive compound 2 and control compound 2, and both showed excellent agonist activity and agonist selectivity.

[0197] Example 16: Pharmacokinetic Study

[0198] 96 male SD rats weighing 200±20g were selected. The rats were allowed to eat and drink freely and were adaptively raised for 3 days at room temperature of 20-26℃, humidity of 40-70%, and light illumination: dark = 12h:12h. The experimental animals began to fast at 8 pm the day before administration, fasted for 12-13h before administration, and continued to fast for 4h after administration, for a total of 16-17h. No water was allowed during the entire experiment.

[0199] Preparation of oral administration samples: Weigh appropriate amounts of the test samples, place them in reagent bottles, add appropriate amounts of mixed solution (2% Klucel LF: 0.1% Tween 80 aqueous solution / 1:1 / v:v), and prepare the administration solution. Prepare and use immediately.

[0200] Preparation of intravenous injection samples: Weigh appropriate amounts of the test samples, place them in reagent bottles, and add appropriate amounts of mixed solvent (10% DMSO: 30% PEG400: 60% saline for injection / 1:1:1 / v:v:v). Prepare dosing solutions of corresponding concentrations, filter them with a microporous membrane on a sterile bench before use (if there is a transfer bottle, make sure it is sterile), and use them immediately after preparation.

[0201] Experimental grouping and administration method: 96 male SD rats were randomly divided into 32 groups, 3 rats in each group, with a total of 16 test articles. Each test article was administered by oral gavage and intravenous injection, respectively, for a single dose, as shown in Table 2.

[0202] Table 2 Dosage regimen for pharmacokinetic study

[0203]

[0204]

[0205] Note: The test compound and positive compound 1 or positive compound 2 were administered in equimolar amounts.

[0206] Blood collection after intragastric administration: About 200 μl of blood was collected from the orbital venous plexus of rats within 0.5 h before intragastric administration (0 h), and 0.5 h, 1.5 h, 3.0 h, 4.5 h, 6.0 h, 8.0 h, 10.0 h, and 24.0 h after administration. The animals were fed 4 h after administration, and water was not allowed during the whole process.

[0207] Blood collection for intravenous injection: About 200 μl of blood was collected from the orbital venous plexus of rats within 0.5 h (0 h) before single intravenous injection, and 0.15 h, 0.5 h, 1.0 h, 3.0 h, 5.0 h, 8.0 h, 10.0 h, and 24.0 h after administration. Animals were fed 4 h after administration, and water was not allowed during the whole process.

[0208] Plasma sample processing and testing:

[0209] Blood sample collection and processing: After each blood collection time point, immediately add blood to a centrifuge tube with sodium heparin precooled in ice water, then centrifuge at 4°C (4000rpm, 10min), take plasma and divide it into 50μL units, place it in a sterile EP tube, and store it at -80°C for later use. Determine the concentration of the target analyte in the plasma as soon as possible.

[0210] Test object information: control compound 1, DSC180-1, DSC180-7, DSC180-10, DSC180-13, DSC180-16, DSC180-20, DSC180-34, and DSC180-44 tested positive compound 1;

[0211] Positive compound 2 was tested against control compound 2, DSC180-28, DSC180-31, DSC180-36, and DSC180-47.

[0212] The bioavailability (%) of oral administration was calculated according to formula (4):

[0213] Formula (4): Oral administration bioavailability (%) = (AUC inf-ig / administered dose) / (AUC inf-iv / administered dose)×100%.

[0214] The pharmacokinetic parameters of injection administration are shown in Table 3, and the pharmacokinetic parameters of oral administration are shown in Table 4.

[0215] Table 4 Pharmacokinetic parameters of injection

[0216]

[0217]

[0218] Table 5 Pharmacokinetic parameters of oral administration

[0219]

[0220] The test compound was compared with positive compound 1 and positive compound 2, control compound 1 and control compound 2 in terms of exposure (AUC Inf ), peak blood concentration (C max ), half-life (T 1 / 2 ), mean residence time (MRT) and bioavailability were improved, showing better pharmacokinetic properties. Among them, the bioavailability of DSC180-07, DSC180-20, DSC180-34, DSC180-44, and DSC180-47 was increased by more than 20% compared with the positive compound 2, and the exposure (AUC Inf ) increased by more than 30%, and the peak blood concentration (C max ) increased by more than 35%, and the half-life (T 1 / 2 ) was extended by more than 14%, and the mean residence time (MRT) was extended by more than 25%.

[0221] Example 17: Efficacy test on HFD-induced NASH model in mice

[0222] Test compounds: Aramchol group, positive compound 1, positive compound 2, DSC180-44, DSC180-47.

[0223] Preparation of test compounds: Weigh appropriate amounts of test compounds respectively, place in reagent bottles, add appropriate amount of mixed solution (2% Klucel LF: 0.1% Tween80 aqueous solution / 1:1 / v:v) to prepare dosing solution, prepare before daily administration and vortex evenly before administration.

[0224] Experimental method: 80 C57BL / 6J mice, male, SPF grade, 6 weeks old, weighing 18g-20g, were randomly divided into a normal group (n=10) and a NASH modeling group (n=70) after 7 days of adaptive feeding. The normal group was fed with a normal feed (crude protein %≥20.0% and crude fat %≥4.0% in the main nutrients), and the NASH modeling group was fed with a high-fat feed (crude fat %≥60% in the main nutrients, crude protein %≥10%, and calories (carbohydrate or sugar) %≥25%). From the end of adaptive feeding, after 40 weeks of feeding, the body weight was weighed and blood was collected to test four blood lipids and liver blood biochemistry (TC, TG, HDL, LDL, ALT, AST). Mice with obvious abnormal indicators were eliminated, and mice with uniform indicators were selected for the experiment. Eight mice fed with normal diet were randomly selected from the normal group; 48 mice fed with high-fat diet were randomly selected from the NASH modeling group and randomly divided into 6 groups, 8 mice in each group, namely model group, positive compound group 1, positive compound group 2, Aramchol group, DSC180-44 group, and DSC180-47 group. The drug was administered by gavage once a day for 6 consecutive weeks. The administration schedule is shown in Table 5.

[0225] Table 6 Dosage regimen for efficacy test

[0226]

[0227]

[0228] After the last administration, the mice were fasted but not watered for 12 h, anesthetized and killed, and the intact liver tissue was isolated.

[0229] Observation, sample processing, and testing indicators:

[0230] 1) General clinical symptom observation and body weight measurement

[0231] The general clinical symptoms of the experimental animals were observed before administration every day (10 a.m.), and the body weight was measured once every Monday and Thursday, each measurement time was 8 a.m.

[0232] Weigh the liver wet weight and calculate the liver index according to formula (5):

[0233] Formula (5): Liver index = liver weight (g) / body weight (g) × 100%.

[0234] 2) Determination of triglyceride (TG) and total cholesterol (TC) content in the liver:

[0235] TG content measurement: According to the instructions of the triglyceride detection kit (enzymatic method) (Zhejiang Dongou Diagnostic Products Co., Ltd.), the test was performed using an enzyme reader;

[0236] TC content determination: According to the instructions of the total cholesterol content detection kit (cholesterol oxidase method) (Nanjing Jiancheng Bioengineering Institute), the detection was performed using an enzyme reader.

[0237] 3) Histopathological analysis

[0238] All liver samples were dehydrated by a dehydration instrument (Leica, Germany, ASP300), then embedded by a paraffin embedding machine (Leica, Germany, EG1160), and then sliced ​​by a data scanning slicer (Leica, Germany, SCN400). Liver tissues were observed and analyzed under a microscope, and the severity of liver inflammation, necrosis, and fibrosis in each group of mice was evaluated using the NAS scoring method (see Table 6).

[0239] Table 7 NASH scoring evaluation method

[0240]

[0241]

[0242] Lesion assessment criteria:

[0243] (1) Hepatocyte ballooning: Pathological changes similar to vacuoles are observed in hepatocytes. Due to vacuolar changes, the size of hepatocytes increases and the hepatocyte nuclei become centralized or deviated.

[0244] (2) Inflammatory cell infiltration: A large number of aggregated inflammatory cells, mainly neutrophils and macrophages, are found in the portal vein area, ventral venous area, or around the liver lobules.

[0245] (3) Fatty changes in hepatocytes: Regular round vacuoles were observed in hepatocytes of different sizes, with the hepatocyte nuclei located at the edges.

[0246] All picrosirius red-stained sections were scanned with a Leica Aperio AT2 Brightfield scanner, and the percentage of picrosirius red-positive staining area was calculated using the HALO AI system to assess the percentage area of ​​picrosirius red over the total scanned liver area.

[0247] Statistical analysis:

[0248] The experimental data were processed using SPSS 23.0 software, and the mean and standard deviation were The independent sample T test was used for comparison between the groups, and P < 0.05 or P < 0.01 indicated that the difference was statistically significant.

[0249] Test results:

[0250] The changes in animal body weight, liver wet weight and liver index after administration are shown in Table 7.

[0251] Table 8 Animal body weight, liver wet weight and liver index after administration

[0252]

[0253] Note: **P<0.01 compared with the normal group; #P<0.05, ##P<0.01 compared with the model group

[0254] Compared with the normal group, the body weight, wet liver weight and liver index of the model group mice increased significantly after 40 weeks of HFD diet induction. Compared with the model group, the body weight, wet liver weight and liver index of the positive compound 1 group, the positive compound 2 group, the Aramchol group, the DSC180-44 group, and the DSC180-47 group decreased; compared with the positive compound 1 group, the positive compound 2 group and the Aramchol group, the body weight, wet liver weight and liver index of the DSC180-44 group and the DSC180-47 group decreased, among which the DSC180-47 group decreased most significantly. In addition, compared with the use of positive compound 1, positive compound 2, and Aramchol alone, the body weight, wet liver weight and liver index of DSC180-47 alone decreased more significantly.

[0255] The data of the detection of triglyceride (TG) and total cholesterol (TC) contents in the liver of animals after administration are shown in Table 8.

[0256] Table 9 Triglyceride (TG) and total cholesterol (TC) content in animal liver after administration

[0257]

[0258]

[0259] Note: **P<0.01 compared with the normal group; # P<0.05, ## P<0.01

[0260] Compared with the normal group, the triglyceride (TG) and total cholesterol (TC) contents of the model group mice increased significantly after 40 weeks of HFD diet induction. Compared with the model group, the positive compound 1 group, the positive compound 2 group, the Aramchol group, the DSC180-44 group, and the DSC180-47 group all decreased significantly; compared with the positive compound 1 group, the positive compound 2 group, and the Aramchol group, the TG and TC contents of the DSC180-44 group and the DSC180-47 group decreased. Compared with the positive compound 1, the positive compound 2, and the Aramchol group, the use of DSC180-47 alone can significantly reduce the TC and TG contents.

Claims

1. A compound of formula I: or its isomers, solvates, isotopic derivatives, oxides, or salts thereof; in: M is L is R x , R y are independently hydrogen, C1-C6 alkyl, or R x , R y Connected into a ring in any reasonable way; G is When M is When G is not When L is hour, G is When L is When G is 2. The compound according to claim 1 or its isomer, solvate, isotope derivative, oxide, or salt thereof, wherein: L is R x and R y For hydrogen.

3. The compound according to claim 1 or its isomer, solvate, isotope derivative, oxide, or salt thereof, which is one of the compounds having the following structure:

4. A pharmaceutical composition comprising the compound of claims 1 to 3 or its isomer, solvate, isotope derivative, oxide, or salt thereof.

5. Use of the compound according to any one of claims 1 to 3 or its isomer, solvate, isotope derivative, oxide, or salt thereof, or the pharmaceutical composition according to claim 4 for preparing drugs for related diseases caused by abnormal fat metabolism.

6. The use according to claim 5, wherein the related diseases caused by abnormal fat metabolism are one or more of obesity, hypothyroidism, thyroid cancer, diabetes, cardiovascular disease, hyperlipidemia, hypercholesterolemia, atherosclerosis, non-alcoholic steatohepatitis (NASH), hypertriglyceridemia and liver fatty lesions.

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