WO2007062568A1

WO2007062568A1 - Compounds used as mammalian cell amp-activated protein kinase(ampk) activators and their preparation methods and usages

Info

Publication number: WO2007062568A1
Application number: PCT/CN2006/002685
Authority: WO
Inventors: Fajun Nan; Jia Li; Bing Liu; Tao Pang; Lifang Yu; Jingya Li
Original assignee: Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences
Priority date: 2005-12-02
Filing date: 2006-10-12
Publication date: 2007-06-07
Also published as: CN1978445B; CN1978445A

Abstract

The present application has disclosed small molecule organic compounds which are used as mammalian cell AMP-activated protein kinase(AMPK) activators. The compounds can be used to prevent, delay or treat disorder caused by AMPK, especially type II diabetes, adiposity and tumour. The present application has also disclosed their preparation methods. The present application has also disclosed the usages of these compounds in activating mammalian cell AMP-activated protein kinase(AMPK) and promoting absorption of glucose in cell. The present application has also disclosed the usages of these compounds in preparation of medicament for metabolic disease.

Description

Compound useful as human adenosine mononucleotide activator protein kinase activator and preparation method and application thereof

FIELD OF THE INVENTION The present invention relates to a small molecule organic compound useful as a human adenosine mononucleotide activating protein kinase (AMPK) activator. Such compounds are useful for preventing, delaying, or treating AMPK-mediated disorders, particularly type II diabetes, obesity, and tumors. The invention also relates to a process for the preparation of this compound. Further, the present invention relates to the use of the compound for activating adenosine mononucleotide-activated protein kinase and for promoting intracellular glucose uptake and for the preparation of a medicament for treating a metabolic disease. BACKGROUND OF THE INVENTION Diabetes has become one of the three major diseases that endanger human health. According to the World Health Organization, in 1997, the number of diabetic patients worldwide reached 124 million, and it is expected to rise to 300 million by 2025, 97% of which are type II diabetes. patient. Diabetes is a general term for a group of heterogeneous endocrine and metabolic diseases characterized by different causes and pathogenesis of insulin deficiency or insulin in the body, and clinically characterized by abnormal glucose metabolism. (Ferrarmini, E. Insulin resistance versus insulin deficiency in non-insulin-dependent diabetes mellitus: problems and prospects. Endocr. Rev, 1998, 19: 477-490. Stefan. The cost of diabetes and diabetes care. Diabetes Research and Clinical Practice 54 Sullppl.l, 2001 : S13-S18.) If not treated promptly and effectively, it can lead to many complications such as blindness, cardiovascular and cerebrovascular diseases, renal failure, and serious harm to human health. Diabetes is highly prevalent in China and around the world. Therefore, it is important to study effective prevention and treatment of diabetes. Although a large amount of experimental and research data has been available to clarify its etiology, the underlying defects in most diabetic patients remain unclear. Due to the extremely complex etiology of the disease, The syndrome of life is also diverse, so that there is currently no drug on the market that can completely cure diabetes. Therefore, research in recent years has focused on finding new drug action points from the perspective of molecular biology to promote the development of new drugs. Among them, adenosine mononucleotide-activated protein kinase (AMPK) is a hot spot in this field in recent years, and it is a new target for the treatment of type 2 diabetes. (Winder, WW AMP-activated protein kinase: possible target for treatment of type 2 diabetes. Diabetes Technol Ther, 2000, 2(3): 441-448. Nicolas Musi, Nobuharu Fujii, Michael F. Hirshman, et al. AMP- Activated protein kinase (AMPK) is activated in muscle of subjects with Type 2 diabetes during exercise. Diabetes, 2001, 50: 921-927.

AMPK is a heterotrimer comprising an alpha catalytic subunit, two regulatory subunits, beta and gamma. For AMPK of mammalian cells, each subunit is encoded by 2-3 genes (α1, α2; β1, β2; γ1, γ2, γ3), respectively. The α-subunit contains a kinase catalytically active region at the terminus of the α-subunit, the C-terminal is an active regulatory region, and contains a self-inhibiting region and a binding region of a regulatory subunit; the β subunit acts as a scaffold for linking the α and γ subunits. (Hardie, D. G, and D. Carling. The AMP-activated protein kinase. Fuel gauge of the mammalian cell? Eur. J. Biochem, 1997, 246: 259-273. Kahn, BB, Alquier, T" Carling, D., and Hardie, DG AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism. Cell Metabolism. 2005, 1: 15-25) In type 2 diabetes, glucose metabolism and lipid metabolism are dysregulated, AMPK has an important regulatory effect on these generations. (Cmte, BE, Seefeld, K., Gamle, J., Kemp, BE, and Witters, LA Functional domain of the al catalytic subunit of the AMP-activated protein (1998) J. Biol. Chem. 273, 35347-35354) Studies have confirmed that activation of AMPK activates and enhances fat in muscle cells. Acid oxidation and glucose absorption, inhibit cholesterol and triglyceride synthesis, lipid production in hepatocytes, regulate insulin secretion of β cells, enhance insulin sensitivity of insulin target tissues, and activate glucose absorption in muscle cells. (Winder, WW, and Hardie, DG AMP-activated protein kinase, a metabolic master switch: possible roles in Type 2 diabetes. Am. J. Physiol, 1999, 277: E1-E10) AMPK in the body muscle tissue and other major Tissue enhances glucose uptake, enhances insulin sensitivity and plays a key role in glucose metabolism and lipid metabolism, making it an effective way to treat type 2 diabetes and obesity. Therefore, the use of human AMPK-al (aa 1-394) inactive fragments to screen AMPK's highly active, highly selective activators may provide an important basis for the study of AMPK molecular mechanisms and the development of new drugs for type II diabetes. . At present, there is no invention of AMPK activator, and currently used AICAR is only a non-specific AMPK activator. Therefore, research and development of a novel AMPK-specific activator for preventing type II diabetes and obesity has broad application prospects and Significant business sense. SUMMARY OF THE INVENTION In view of the above problems, the present invention contemplates and synthesizes a novel compound which can be used as a human AMPK activator and can effectively activate the activity of human AMPK-al (aa 1-394) to promote intracellular glucose uptake. Therefore, it can be used to prevent, delay or treat AMPK-mediated disorders, particularly type II diabetes, obesity and tumors. The compounds of the present invention are relatively simple in structure and easy to prepare. The novel compound of the present invention has a structure represented by the following formula 1: [Formula 1]

Where X is NH, O or S; is O or S;

Y ₂ is NR ₅ , O or S, wherein R ₅ is H;

Ri is H, phenyl or

a benzyl group, or a substituted or unsubstituted C1-C10 alkyl group or a cycloalkyl group; wherein R ₉ and Ru) are each independently H; F; C1; trifluoromethyl; ethynyl, propynyl; a carboxyl group, a C1-C4 alkyl group of a carboxylate group, and the like.

R ₃ , each independently H, F, Cl, pyrrolyl, pyridyl, thiosalt,

Thienyl, benzyl, phenyl, or

And wherein R ₇ > R ₈ are each independently H, F, Cl, trifluoromethyl, trifluoromethoxy, nitro, amine, benzoylamide, C2-C6 alkanoamide, and C3 a cycloalkyl-substituted amide group of C6, a C1-C4 alkyl group or the like. Wherein the unsubstituted C1-C10 alkyl or cycloalkyl group is cyclopropyl, cyclobutane, decyl, ethyl, n-propyl, n-butyl, n-pentyl, cyclopentanyl a substituent of a substituted C1-C10 alkyl or cycloalkyl group, including one or more alkoxy groups selected from the group consisting of F, Cl, C1-C10, a nitrile group, a carboxyl group, and a formic acid. a methyl ester group, a decanoic acid group or a substituent group of a hydroxyl group, and the like. The novel human AMPK activator of the present invention is prepared by the following three steps: Step a: The carboxyl group in Compound 1 is reduced to an alcohol group according to the following chemical reaction formula 1.

[Chemical reaction formula 1]

1 2 at 0 ~ 5 °C, using tetrahydrofuran as a solvent, compound 1 is reacted with lithium aluminum hydride to reduce the carboxyl group to an alcoholic hydroxyl group, and after quenching with water, extraction, drying, concentration, etc., to obtain compound 2;

[Chemical Reaction Formula 2]

twenty three The alcoholic hydroxyl group of the compound ² is oxidized to the aldehyde group with active manganese dioxide at room temperature by using chloroform as a solvent, and after treatment with concentration and concentration, the compound ^{3 is} obtained;

[Chemical Reaction Formula 3]

The compound 3, 4 is coupled with water acetic acid as a solvent and sodium acetate or with anhydrous ethanol as a solvent under the action of piperidine, and subjected to extraction, drying, concentration, silica gel column chromatography and the like, and obtained. The final compound ⁵ . In the above three steps, where X is ΝΉ, 0 or S; Ο or S;

Y ₂ is NR ₅ , O or S; wherein R ₅ is phenyl,

Wait

! ^ is phenyl or

a benzyl group, or an alkyl or cycloalkyl group of a substituted or unsubstituted Cl-ClO; Wherein R ₉ and R ₁₀ are each independently H; F; CI; trifluoromethyl; ethynyl, propynyl; CI-C4 alkyl having a carboxyl group or a carboxylate group.

R ₂ , R ₃ and R 4 are each independently H, F, Cl, pyrrolyl, pyridyl, thioenyl,

Thiol, benzyl, phenyl, or

Wherein R ₇ and R ₈ are each independently H, F, Cl, trifluoromethyl, trifluoromethoxy, nitro, amine, benzamide, C2-C6 alkanoamide, and C3- a cycloalkyl-substituted amide group of C6, a C1-C4 alkyl group or the like. Wherein the unsubstituted C1-C10 alkyl or cycloalkyl group is cyclopropyl, cyclobutane, decyl, ethyl, n-propyl, n-butyl, n-pentyl, cyclopentane a substituent, a n-hexane group, a cyclohexyl group or the like; a substituted C1-C10 alkyl or cycloalkyl substituent including one or more alkoxy groups selected from the group consisting of F, Cl, C1-C10, a nitrile group, a carboxyl group, and a formic acid a methyl ester group, a decanoic acid group or a substituent group of a hydroxyl group, and the like. Thin layer chromatography (TLC) is commonly used to track the completion of a reaction. The post-treatment methods generally employed after the completion of the reaction include cooling, concentration of the reaction solution to remove the solvent, extraction, column chromatography separation, and the like. The final product was detected by NMR. The novel compound of the present invention can be used as a human AMPK activator and can effectively activate the activity of human AMPK-al (aa 1-394), thereby preventing, delaying or treating AMPK-mediated disorders, especially II. Type 2 diabetes, obesity and cancer. Hereinafter, the present invention will be more specifically described by way of examples and comparative examples. However, the following examples are provided for the purpose of illustration only, and thus the invention is not limited to the embodiments. DRAWINGS Fig. 1 shows the measurement of AMPK activity without adding the compound of the present invention and adding the compounds 5f, 5r, 5s, 5t, and 5u of the present invention, respectively.

In Fig. 2, (-) indicates that the compound was not treated with the compound, (+) indicates that the compound was treated with the compound; (pAMPK) indicates the phosphorylation level of the AMPK catalytic subunit α-threonine (Thr) 172; (AMPK) ) indicates the expression level of intracellular AMPK catalytic subunit α; (pACC) indicates the phosphorylation level of intracellular AMPK substrate ACC; (ACC) indicates the expression level of intracellular ACC; (-Actin) indicates intracellular actin To show the total amount of intracellular protein. DETAILED DESCRIPTION OF THE INVENTION In the following examples, NMR was measured by a Mercury-Vx 300M instrument manufactured by Varian, NMR calibration: δ H/C 7.26/77.0 ppm (CDC1 ₃ ); reagents were mainly supplied by Shanghai Chemical Reagent Co., Ltd. Column chromatography, silica gel (200-300 mesh), the silica gel model used for column chromatography is coarse air (ZLX-II), produced by Qingdao Ocean Chemical Plant. Example 1: Preparation of Compound 5a

[Chemical Reaction Formula 4]

According to the above chemical reaction formula First step: Add 28 mg of AlLi to a 25 ml round bottom flask, disperse it in 10 ml of freshly steamed THF, and add 101 mg of compound 3a (0.37 mmol) in 3 ml of freshly steamed THF solution at room temperature. (24-26 V) The reaction was stirred for 30 minutes, and the completion degree of the reaction was measured by TLC. After the reaction was completed, 0.5 ml of water was carefully added, stirring was continued for 10 minutes, diluted with water, extracted with ethyl acetate. The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated to remove solvent to afford 2b.

Step 2: Add 68 mg of compound 2a, 20 ml of chloroform to a 50 ml round bottom flask, add active Mn0 ₂ 122 mg with stirring, stir the reaction at room temperature (24-26 ° C) for 5 hours, and use TLC to track the completion of the reaction. . After completion of the reaction, the insoluble matter was removed by filtration, and the solvent was concentrated to give 3a 38 mg. Step 3: In a 5 ml round bottom flask, add 38 mg of compound 3a (0.16 mmol), 60 mg of compound 4a (0.16 mmol), 27 mg of piperidine, 3 ml of absolute ethanol, reflux with nitrogen (80"C to 120 °C) After 3 hours, the completion degree of the reaction was measured by TLC. After the completion of the reaction, the reaction mixture was cooled, and the reaction mixture was concentrated to remove the solvent and purified by column chromatography to give the product 5a 30 mg. Compound 5a: ^! H NMR (CDC13, 300 MHz) δ 6.78 (dd , 2H), 7.11 (m, 4H), 7.43 (m, 3H), 7.69 (t, lH). Example 2: Preparation of compound 5b:

[Chemical Reaction Formula 5]

4b 5b According to the above chemical reaction formula: First step: Compound 2b was prepared in the same manner as in the first step of Example 1, except that the starting material lb was used instead of the starting material in the first step of Example 1. Second step: Compound 3b was prepared in the same manner as in the second step of Example 1 except that the starting material 2b was used instead of the starting material 2a in the second step of Example 1. Step 3: In a 5 ml round bottom flask, add 34 mg of compound 3b (0.28 mmol), 60 mg of compound 4b (0.28 mmol), 100 mg of AcONa, 2 ml of glacial acetic acid, and reflux with nitrogen (100 ° C to 120 ° C) 2 Hours, TLC tracks the response. After completion of the reaction, the mixture was cooled, filtered, washed and dried in vacuo to give the compound (yield: Compound 5b: 1H MR (CDC13, 300MHz) δ 1.27-1.43 (m, 4H), 1.69 (d, 4H), 1.87 (d, 2H), 2.10 (s, 3H), 2.27 (s, 3H), 5.10 ( Br,lH), 5.99(s,lH), 6.76(s,lH). Example 3: Preparation of Compound 5c: Compound 5c was prepared in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to Compound 5c and a carbonyl compound were used as a starting material. The structural formula of compound 5c:

Compound 5c: NMR (DMSO-d ₆ , 300 MHz) δ 2.32 (s, 6H), 3.66 (s, 3H), 3.75 (s, 3H), 3.93 (s, 2H), 6.78 (d, lH), 6.93 (d, lH), 7.01 (s, lH), 7.09 (s, lH), 7, 63 (s, lH), 7.73 (s, lH), 7.83 (d, lH). Example 4: Preparation of Compound 5d: Compound 5d was prepared in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to Compound 5d and a carbonyl compound were used. The structural formula of compound 5d:

Compound 5d: ^! H NMR (CDC13, 300MHz) δ 3.11(s,lH), 6.83(d ₅ lH), 7.02(d,lH), 7.16(s,lH), 7.37(m,6H), 7.51(s , lH), 7.80 (s, lH). Example 5: Preparation of Compound 5e: Compound 5e was prepared in the same manner as in Example 2 except that a carboxylic acid or a carboxylic acid ester corresponding to Compound 5e and a mercapto compound were used. The structural formula of compound 5e:

Compound 5e : ^ι NMR (DMSO-d ₆ , 30 MHz) δ 6.78 (d, lH), 7.05 (d, 1H), 7.21. (m, 2H), 7.40 (m, lH), 7.60 (m, 2H) , 7.79(d,lH), 7.99(d,lH). Example 6: Preparation of Compound 5f: Compound 5f was obtained in the same manner as in Example 2 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5f and a carbonyl compound were used as a starting material. The structural formula of compound 5f:

Compound 5f: NMR (DMSO-d ₆ , 300 MHz) δ 1.78, 1.94, 2.02, 2.49, 6.85 7.19, 7.28, 7.45, 7.72, 8.03, 8.39, 9.45. Example 7: Preparation of Compound 5g: Compound 5g was prepared in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5g and a carbonyl compound were used. Structural formula of compound 5g:

Compound _{5g: Ή NMR (CDCl 3,} 300MHz) δ 6.74 (d), 7.05-7.1 l (m), 7.16 (d), 7.31 (d), 7.36 (s), 7.58 (d). Example 8: Preparation of Compound 5h: Compound 5h was obtained in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5h and a carbonyl compound were used as a starting material. The structural formula of compound 5h:

Compound _{5h: Ή NMR (CDCl 3,} 300MHz) δ 6.85 (d), 7.02 (d), 7.20 (d): 7.33 (d), 7.54 (s), 7.75 (d). Example 9: Preparation of Compound 5i: Compound 5i was prepared in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5i and a carbonyl compound were used. The structural formula of compound 5i:

Compound 5i: 'Η NMR (DMSO-d ₆ , 300 MHz) δ 7.06, 7.18, 7.43, 7.56, 7.74, 8.01. Example 10: Preparation of Compound 5j: Compound 5j was prepared in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to Compound 5j and a thiol compound were used as a starting material. The structural formula of compound 5j:

Compound 5j: ¹ R NMR (CDCl ₃ , 300 MHz) δ 6.77, 6.94, 7.06, 7.27: 7.37, 7.68. Example 11: Preparation of Compound 5k: Compound 5k was prepared in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5k and a carbonyl compound were used. The structural formula of compound 5k:

Compound _{5k: ¾ NMR (CD 3 OD} -d 4, 300MHz) δ 6.70, 6.82, 7.06, 7.23, 7.43, 7.52,7.60. Example 12: Preparation of Compound 51: Compound 51 was obtained in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to Compound 51 and a carbonyl compound were used. The structural formula of compound 51:

Compound 51: _3⁄4 NMR (CD ₃ OD-d _4; 300 MHz) δ 7.13, 7.19, 7.32, 7.36 7.46, 7.60, 7.12, 7.81, 8.05. Example 13: Preparation of Compound 5m: Compound 5m was prepared in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5m and a base compound were used as a starting material. Structural formula of compound 5m:

Compound 5m: NMR (CD ₃ OD-d ₄ , 30 MHz) δ 2.18, 2.26, 2.30, 6.67, 6.72, 7.03, 7.05, 7.34, 7.47. Example 14: Preparation of Compound 5η: Compound 5n was produced in the same manner as in Example 1 except that a carboxylic acid or a carboxylic acid ester corresponding to Compound 5n and a base compound were used as a starting material. Structural formula of compound 5η:

Compound 5 η: NMR (DMSO-d ₆ , 300 MHz) δ 2.26, 2.31, 2.36, 7.01 7.04, 7.10, 7.29, 7.43, 7.56, 7.69, 7.85. Example 15: Preparation of Compound 5ο: Compound 5o was prepared in the same manner as in Example 2 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5o and a base compound were used as a starting material. The structural formula of compound 5o:

Compound 5o: NMR (CD ₃ OD-d ₄ , 300 MHz) δ 2.14, 2.28, 2.36, 6.87, 6.97, 7.10, 7.18, 7.50, 7.60, 7.95. Example 16: Preparation of Compound 5ΐ3: Compound 5p was prepared in the same manner as in Example 2 except that a carboxylic acid or a carboxylic acid ester corresponding to Compound 5p and a carbonyl compound were used as a starting material. The structural formula of compound 5ρ:

Compound 5ρ: Ή NMR (DMSO-d 6, 300MHz) δ 7.19, 7.39, 7.44, 7.54 7.65, 7.81, 7.96, 8.17. Example 17: Preparation of Compound 5q: Compound 5q was prepared in the same manner as in Example 2 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5q and a carbonyl compound were used. The structural formula of compound 5q:

Compound 5q: NMR (DMSO-d ₆ , 300 MHz) δ 6.67, 6.71, 6.97, 7.08, 7.24, 7.46, 7.49, 7.77, 8.00. Example 18: Preparation of Compound 5r: Compound 5r was prepared in the same manner as in Example 2 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5r and a base compound were used as a starting material. The structural formula of compound 5r:

Compound 5r: ^] H NMR (CD ₃ OD-d ₄ , 300 MHz) δ 2.14, 2.28, 2.36, 6.87, 6.97 7.10, 7.18, 7.35, 7.47, 7.52. Example 19: Preparation of Compound 5s: Compound 5s was prepared in the same manner as in Example 2 except that the carboxylic acid or the carboxylic acid ester corresponding to the compound 5s and the base compound were used as a raw material. The structural formula of compound 5s:

Compound 5s: ^] H NMR (CDCl ₃ , 300 MHz) δ 2.18, 3.91, 6.68, 7.16, 7.19, 7.37, 7.68, 7.84, 7.92, 8.01. Example 20: Preparation of Compound 5t: Compound 5t was prepared in the same manner as in Example 2 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5t and a carbonyl compound were used as a starting material. The structural formula of compound 5t:

Compound 5t: 3⁄4 NMR (DMSO-d ₆ , 300 MHz) δ 1.06, 2.19, 2.36, 6.99 7.12, 7.28, 7.44, 7.58, 8.04, 8.39. Example 21: Preparation of Compound 5u: Compound 5u was produced in the same manner as in Example 2 except that a carboxylic acid or a carboxylic acid ester corresponding to compound 5u and a mercapto compound were used as a starting material. The structural formula of compound 5u:

Compound 5u: ^! H NMR (DMSO-d ₆ , 300 MHz) δ 2.36, 6.71, 6.83, 7.22, 7.44, 7.58, 7.98. Example 22: Preparation of Compound 5ν: Compound 5ν was obtained in the same manner as in Example 2 except that the carboxylic acid or the carboxylic acid ester corresponding to the compound 5? The structural formula of the compound 5ν:

Compound 5ν: ^! H NMR (DMSO-d ₆ , 300MHz) δ 7.09, 7.20, 7.40, 7.58 7.98, 8.00, 8.31 » Test Example 1: Test for 激活 activity activation test

Preparation of ruthenium catalytic subunit αΐ inactive fragment cdd-394 amino acid): A gene sequence encoding the human AMPK catalytic subunit αΐ inactive fragment al (l-394 amino acid) was constructed in the pGEMEX-Ι vector. The correctly identified pGEMEX-AMPK-1 (1 -394) recombinant plasmid was transformed into the expression type Escherichia coli BL21-Codon-Plus (DE3)-RIL strain, and the monoclonal colony was picked at 1 L containing 50 g/mL ampicillin and LB rich medium of 25 g/mL chloramphenicol, 180 to 37 per minute. C was cultured overnight until the OD600 value of the bacterial solution was 0.4-0.6, and 0.1 mM IPTG was added at 22. C induced expression overnight. 4. C. The expression bacteria (about 2.0 g/L expressing bacteria) were harvested by centrifugation at 6000 xg for 3 minutes, and the bacteria were resuspended and washed three times with 20 mL Buffer A (50 mM Tris-HCl, pH 7.5, 1 mM DTT), and then lysate was used. Buffer A, 1% Triton X-100 resuspend the bacteria. In; 4. The bacterial solution was treated with ultrasonic waves for three minutes under C to fully lyse the cells. The above lysate containing the protein of interest was centrifuged at 12000 x g for 15 minutes at 4 ° C, centrifuged twice, and the precipitate was discarded, and the supernatant protein solution was passed through a Q column. It was carried out using a HiTrap-5ml QHP anion exchange column and an FPLC system. The solution passing through the Q column was Buffer A (50 mM Tris-HCl, pH 7.5, 1 mM DTT) and Buffer B (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 M NaCl). After equilibrating the HiTrap-5ml QHP column with Buffer A, the supernatant protein sample was injected, first equilibrated with Buffer A, and then eluted with a linear NaCl gradient consisting of Buffer A and 100% Buffei' B. The protein composition and purity of the collected fractions were verified by SDS-PAGE - and the purer fractions were combined according to the results and protein concentrations. The eluted target protein AMPK-ocl (l-394) was identified by 12% SDS-PAGE: the purity of the purified protein was about 95%. The recombinant mouse AMPK-al (aa 1-392), which is identified by its size and enzymatic properties, corresponds to the amino acid sequence reported in the literature. (Hamilton et al., AMP-activated protein kinase kinase: detection with recombinant AMPK al subunit (2002) Biochem. Biophys. Res. Commun. 293, 892-898; Crute et al, Functional domains of the al catalytic subunit of the AMP-activated protein kinase, (199S) J. Biol. Chem. 273, 35347-35354 ). ΑΜΡΚ-α 1 (1 - 394) activation test principle:

Using the above E. coli expression system, the inactive fragment al(l-394) of the catalytic subunit αΐ of human ΑΜΡΚ was purified, and the fragment contained its own self-inhibitory sequence for screening experiments. Based on the radioisotope technique, the activated hydrazine can phosphorylate the substrate SAMS (MW: 1779), and the γ-33 Ρ on the [γ- 33P]-ATP in the solution is covalently bonded to the substrate SAMS to make its Ser residue. Acidification, production of the isotope-labeled substrate 33P-SAMS, and finally the scintillation fluid was counted by liquid scintillation counter to quantify the enzyme activity. The activity of the compound was initially evaluated by observing the activation of its activity by different compounds.

Compare the activity of AMPK-al (aa 1-394) in the presence and absence of compound samples in the living system, and determine whether the sample has activation activity; if so, determine AMPK-al (aa 1_ ³ 9 ⁴ ) at different sample concentrations. Activity, calculate the EC50 of the sample, ie the half maximum concentration (50%).

ΑΜΡΚ-α 1 (1 -394) activation test method:

HsAMPK-al (aa 1-394) was prephosphorylated by the upstream kinase CaM Kp for 4 hours, and then added to the substrate-containing assay reaction solution (20 mM Tris-HCl, pH 7.5, 1 mM) in a volume ratio of 1:4. In DTT, 5 mM MgC12, 50 μΜ [γ-33Ρ]-ΑΤΡ (0.4 μα/assay), 50 μΜ SAMS), the total activity was 50 μΐ at 30. C was incubated for 10 min. After the reaction was terminated by 1% H3P04, the protein reaction solution was adsorbed on P30 filter paper by Harvester, washed three times with 0.1% H3P04, dried, added with scintillation liquid, and sealed, and counted by a 96-well microplate scintillation counter.

Add 35.5 reaction mixture (containing 20 mM Tris-HCl, pH 7.5, 1 mM DTT, 5 mM MgC12, 50 μΜ [γ-33Ρ]-ΑΤΡ (0.4 μα/assay), 50 μΜ SAMS) to each containing 2 xL In Example 6, 18 to 21, the compound 5f, 5r ~ 5u in the sample plate to be tested, and finally by 12. ⁵ μί, fully phosphorylated HsAMPK-al (aa 1-394) (100 The reaction was initiated by the addition of nM) and the activity temperature was 30. C. The reaction was terminated after 10 min, aspirate, washed, dried, and counted after sealing. The activation rate of the sample was determined by the following formula: % activation rate of the sample = [(compound CPM value - blank control) - (negative control - blank control)] / (negative control - blank control) XI 00.

The reaction rate obtained by the above measurement system is the reaction rate of the enzyme activity; DMSO (2 μl) in which the above compound is dissolved is substituted for DMSO (2 μ1) in the above system, and the reaction rate is determined as the reaction rate under the action of the compound.

At a preliminary screening concentration of 30 μΜ, the inventors found that the 22 compounds in the above examples showed an activation rate ranging from 3% to 30%, and selected the most representative of the five compounds 5f, 5r, 5s, 5t, 5u, the human AMPK-al (aa 1-394) activity was determined at different sample concentrations, and the EC50 of the sample, ie the half maximum concentration (50%), was calculated. This demonstrates that such compounds are capable of activating the activity of human AMPK-al (aa 1-394). Fig. 1 shows the measurement of AMPK activity without adding the compound of the present invention and adding the compounds 5f, 5r, 5s, 5t, and 5u of the present invention, respectively. Figure 2 is a graph showing the catalytic activity of the compound 5r prepared in the examples of the present invention at various concentrations. In Fig. 2, (-) indicates that the compound was not treated with the compound, (+) indicates that the compound was treated with the compound; (pAMPK) indicates the phosphorylation level of the AMPK catalytic subunit α-threonine (Thr) 172; (AMPK) ) indicates the expression level of intracellular AMPK catalytic subunit α; (pACC) indicates the phosphorylation level of intracellular AMPK substrate ACC; (ACC) indicates the expression level of intracellular ACC; (-Actin) indicates intracellular actin To show the total amount of intracellular protein. The compound 5r prepared in Example 18 has a significant effect on enhancing the phosphorylation level of intracellular AMPK phosphorylation and its downstream target protein ACC at the L6 cell level and exhibits a concentration-dependent manner, indicating that the compound can be at the cellular level. Activate AMPK.

Sample test results:

Claims

Claim

1. A compound having the structure of the following formula 1: [Formula 1]

Wherein X is NH, O or S;

^ is O or S;

Y ₂ is N, O or S, of which is 11, phenyl,

For 11, phenyl or

, benzyl, or substituted or unsubstituted C1-C10 alkyl or cycloalkyl; wherein R ₉ , R _{1 ()} are each independently H; F; C1; trifluoromethyl; ethynyl, propynyl a CI-C4 alkyl group having a carboxyl group, a decyl decanoate group, or an ethyl decanoate group;

R ₂ , R ₃ and R 4 are each independently H, F, Cl, pyrrolyl, pyridyl, thiazolyl,

Thienyl, benzyl, phenyl, or

Wherein R ₇ and R ₈ are each independently H, F, Cl, trifluoromethyl, trifluoromethoxy, nitro, amine, benzamide, C2-C6 alkanoamide, and C3-C6 Cycloalkyl substituted amide group, C1-C4 alkyl group.

The compound according to claim 1, characterized in that said unsubstituted C1-C10 alkyl or cycloalkyl group is cyclopropyl, cyclobutyl, decyl, ethyl, n-propyl, n-butyl a substituent selected from the group consisting of F, Cl, C1-C10 a substituent group of an oxy group, a nitrile group, a carboxyl group, a decyl decanoate group, an ethyl decanoate group or a hydroxyl group.

3. A process for the preparation of a compound according to claim 1, which comprises the following three steps:

Wherein, the definitions of the various groups are the same as those in claim 1; Step a. Reducing the carboxyl group to the alcoholic hydroxyl group by reacting the compound 1 with lithium aluminum hydride at a temperature of 0 to 5 ° C in tetrahydrofuran as a solvent, after conventional The treatment step, the compound 2 is obtained; Step b. The hydroxy group of the compound 2 is oxidized to the aldehyde group with active manganese dioxide at room temperature, and the compound 3 is obtained through a conventional post-treatment step; Step C. using water acetic acid or absolute ethanol as a solvent, and under the action of sodium acetate or piperidine,

The compound 3, 4 was coupled by refluxing at 80 ° C to 120 ° C, and a conventional post-treatment step was carried out to obtain the final compound 5.

4. Use of a compound according to claim 1 for activating a human adenosine mononucleotide activating protein kinase.

5. Use of a compound according to claim 1 in the manufacture of a medicament for the treatment of a metabolic disease.

6. Use according to claim 5 wherein said metabolic disease is type II diabetes, obesity and tumor.