Application of compounds and ACLY inhibitors or anticancer drugs

Abstract

The present invention provides natural compounds 2 and 4 and their use in preparing ATP citrate lyase (ACLY) inhibitors and anticancer drugs. Compounds 2 and 4 have the following structures: #imgabs0##imgabs1# The compounds 2 and 4 of the present invention have significant inhibitory effects on ATP citrate lyase (ACLY), IC 50 The results show that compound 4 can significantly inhibit the proliferation of lung cancer cells A549 and prostate cancer cells PC3, showing a good anticancer effect. Therefore, compound 4 can be used for the preparation of anticancer drugs, especially for the preparation of lung cancer and prostate cancer drugs. The present invention provides a new inhibitor for ATP citrate lyase and a new lead compound for tumor treatment, which has great application value.

Description

Technical Field

[0001] The invention belongs to the field of drug development, and specifically relates to two newly discovered ACLY inhibitors and their application in preparing anti-tumor drugs. Background Art

[0002] ATP citrate lyase (ACLY) is an enzyme localized in the cytosol. 2+ ACLY catalyzes the cleavage of citrate into acetyl-CoA and oxaloacetate in the presence of ATP. The substrate citrate in this cleavage reaction is an important participant in the tricarboxylic acid cycle, and the product acetyl-CoA is an important precursor molecule for the synthesis of fatty acids and cholesterol. Therefore, ACLY links carbohydrate metabolism and lipid metabolism and is a key enzyme in glucose and lipid metabolism (Chypre, et al. 2012).

[0003] ACLY is abnormally highly expressed in some metabolic diseases, cardiovascular diseases and various cancers. When ACLY activity is inhibited, the related symptoms are alleviated. Therefore, ACLY has become a potential drug target for hyperlipidemia, cardiovascular diseases and tumors (Khwairakpam, et al. 2015). The study of ACLY inhibitors is of great significance for the development of drugs targeting ACLY.

[0004] Natural products are a huge number with diverse structures and are an important source of drug molecules. Screening from natural product libraries is a way to discover new ACLY inhibitors. Through screening of natural products in the group, we found two new ACLY inhibitors, which have different structural types from the reported inhibitors. Therefore, they can be used in the preparation of ACLY inhibitors and the preparation of drugs for ACLY-related diseases. In the in vitro pharmacodynamic anticancer activity evaluation, compound 4 can significantly inhibit the proliferation of lung cancer and prostate cancer cells. Therefore, compound 4 can be used in the preparation of anticancer drugs and has great application value. Summary of the invention

[0005] The present invention provides the use of a compound in the preparation of an ATP citrate lyase inhibitor and / or an anti-tumor drug, wherein the compound is a compound having the following structural formula 2 or structural formula 4:

[0006]

[0007] The application of the present invention is characterized by: one or two of the compounds 2 and 4 are used in the preparation of drugs for one or more diseases of ACLY enzyme-related hyperlipidemia, atherosclerosis and tumors.

[0008] The application described in the present invention is characterized in that the tumor is one or both of lung cancer and prostate cancer.

[0009] The application described in the present invention is characterized in that the tumor drug is a pharmaceutical composition prepared by using one or two of compounds 2 and 4 (preferably compound 4) as active ingredients and a pharmaceutically acceptable carrier, including tablets, capsules, pills, granules or injections.

[0010] The ATP citrate lyase inhibitor or anti-tumor drug of the present invention is characterized in that it contains one or more of the compounds represented by the following structural formula 2 or structural formula 4 as active ingredients:

[0011]

[0012] The inhibitor or anti-tumor drug of the present invention is characterized in that: the inhibitor or anti-tumor drug is a pharmaceutical composition prepared by using one or two of compounds 2 and 4 (preferably compound 4) as active ingredients and a pharmaceutically acceptable carrier, including tablets, capsules, pills, granules or injections.

[0013] Compounds 2 and 4 of the present invention have significant inhibitory effects on ATP citrate lyase (ACLY), IC 50 The results show that compound 4 can significantly inhibit the proliferation of lung cancer cells A549 and prostate cancer cells PC3, showing a good anticancer effect. Therefore, compound 4 can be used for the preparation of anticancer drugs, especially for the preparation of lung cancer and prostate cancer drugs. The present invention provides a new inhibitor for ATP citrate lyase and a new lead compound for tumor treatment, which has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Inhibitory activity of compound 2 on ACLY.

[0015] Figure 2 : Inhibitory activity of compound 4 on ACLY.

[0016] Figure 3 : Inhibitory activity of compound 4 on lung cancer cell proliferation.

[0017] Figure 4 : Inhibitory activity of compound 4 on prostate cancer cell proliferation. DETAILED DESCRIPTION

[0018] The present invention is further described with reference to examples, which are intended to illustrate the present invention but not to limit the present invention.

[0019] The instruments and reagents used in the experiment mainly include: microplate reader (PerkinElmer), ADP-Glo TM KinaseAssay kit (Promega), CulturPlate 384-well plate (Perkinelmer), CCK kit (Beyotime), compound 2 (Kunming Institute of Botany, Chinese Academy of Sciences), compound 4 (Kunming Institute of Botany, Chinese Academy of Sciences), etc.

[0020] The preparation process of 2 and 4 is as follows:

[0021] 36.0 kg of dried maca root tubers were crushed and extracted with acetone at room temperature for 3 times, each time with 1 L, for 3 days each time, and the extract was concentrated to obtain 1.24 kg of extract, which was dispersed with water and extracted with ethyl acetate (EA) for many times. After the solvent was recovered, 484.0 g was obtained. The EA part was separated using a normal silica gel column (volume ratio, petroleum ether / ethyl acetate, 100:1→0:1) to obtain 9 components (Fr.AI). The Fr.F component (11.9 g) was separated using a reverse phase C18 silica gel column (volume ratio, methanol-water, 50%-100%) to obtain 8 parts (Fr.F1-Fr.F8). The Fr.F5 fraction (2.3 g) was treated with a normal phase silica gel column (volume ratio, petroleum ether / ethyl acetate = 8:1-0:1) and semi-preparative high performance liquid chromatography (volume ratio, 93% CH3CN / H2O, 2.5 mL / min) to obtain compound 2 (10.4 mg, tR = 17.3 min), which was named (10E, 12E)-9-keto-10,12-octadecadienoic acid. The Fr.G fraction (8.4 g) was treated with reverse phase RP-C18 silica gel and gradient eluted with methanol-water (50%-100%) to be divided into 12 fractions (Fr.G1-Fr.G12). Fr.G8 (672 mg) was treated with a normal phase silica gel column (PE / EA=6:1-0:1) and semi-preparative HPLC (93% CH3CN / H2O, 2.5 mL / min) to obtain compound 4 (10.3 mg, tR=22.9 min), which was named (9Z, 12Z, 15Z)-N-(3-methoxybenzyl) octadecanol-9,12,15-trienamide.

[0022] The characterization of 2 and 4 is as follows:

[0023] (10E,12E)-9-oxo-octadecadienoic acid(2)

[0024] Pale yellow oil; C18H30O3: 1H NMR (600MHz, CDCl3) δH: 2.32 (2H, t, J = 7.3Hz, H-2), 1.60 (4H, overlapped, H-3 / 7), 1.30 (10H, overlapped, H-4 / 5 / 6 / 16 / 17), 2.52 (2H, t, J = 7.3Hz, H-8), 7.12 (1H, dd, J = 15.4, 9.0Hz, H-11), 6.06 (1H, d, J = 15.4Hz, H-10), 6.16 (2H, m, H-12 / 13), 2.16 (2H, m, H-14), 1.42 (2H, m, H-15), 0.87 (3H, t, J = 6.9Hz, H-18); 13C NMR-DEPT(150MHz,CDCl3)δC:179.8(s,C-1),34.0(t,C-2),24.5(t,C-3),29.0(t,C-4),29 .0(t,C-5),31.4(t,C-6),24.3(t,C-7),40.3(t,C-8),201.3(s,C-9),127.7(d,C-10),143. 2(d,C-11),128.8(d,C-12),145.8(d,C-13),33.1(t,C-14),28.8(t,C-15),28.3(t,C-16),22.4(t,C-17),13.9(q,C-18). The above data are consistent with the 9-oxo-10E,12E-octadecadienoic acid data reported in the literature [1].

[0025] N-(3-Methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide(4):

[0026] Pale yellow oil; C26H39NO2; 1H NMR (600MHz, CDCl3) δH: 6.82 (1H, s, H-3′), 6.85 (1H, d, J=7.6Hz, H-5′), 7.24 (1H, m, H-6′), 6.82 (1H, overlapped, H-7′), 4.41 (2H, d, J=5.7Hz, H-1′), 5.73 (1H, br, s, NH), 2.20 (2H, t, J=7.5Hz, H-2), 1.65 (2 H,m,H-3),1.31(8H,overlapped,H-4 / 5 / 6 / 7),2.6(4H,overlapped,H-8 / 17),5.36(6H,overlapped,H- 9 / 10 / 12 / 13 / 15 / 16),2.80(4H,overlapped,H-11 / 14),0.97(3H,t,J=7.5Hz,H-18),3.80(3H,s,4′-OCH 3);13C NMR-DEPT(150MHz, CDCl3)δC:43.5(t,C-1′),139.9(s,C-2′),113.3(d,C-3′),159.8(s,C-4′),112.9(d,C-5′),129.7(d,C-6′),1 20.0(d,C-7′),172.9(s,C-1),36.7(t,C-2),25.5(t,C-3),29.1(t,C-4),29.2(t,C-5),29.3(t,C-6),29.5(t,C-7),27.2(t,C-8), 130.2(d,C-9),127.1(d,C-10),25.6(t,C-11),128.2(d,C-12),128.2(d,C-13),25.7(t,C-14),127.7(d,C-15),131.9(d,C-16),20.5(t,C-17),14.3(q,C-18),55.2(q,4′-OCH3). The above data are consistent with the data of N-(3-Methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide reported in literature [2].

[0027] [1]Xia C, Chen J, Deng JL, et al. Novel macamides from maca(Lepidiummeyenii Walpers) root and their cytotoxicity[J]. Phytochemistry Letters, 2018, 25: 65-69.

[0028] [2]Mccollom MM, Villinski JR, Mcphail KL, et al. Analysis of macamidesin samples of Maca(Lepidium meyenii) by HPLC-UV-MS / MS[J]. PhytochemicalAnalysis, 2005, 16(6): 463-469.

[0029] Example 1: Determination of ACLY inhibitory activity of compound 2

[0030] The stock solution concentration of 2 is 50mM, and the solvent is DMSO. Take 1μL of the stock solution and add it to 62.5μL of aqueous buffer, the buffer composition is 40mM tris (Tris), 4mM dithiothreitol (DTT), 0.01% lauryl alcohol polyoxyethylene ether (Brij35), 0.001% bovine serum albumin (BSA), 10mM MgCl2, 1% dimethyl sulfoxide (DMSO), pH = 8.0. The initial concentration of 2 obtained by the above operation was 200 μM, and then a shot gun was used to dilute the aqueous buffer in a 384-well plate to prepare 14 concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.156, 0.781, 0.391, 0.195, 0.098, 0.049 and 0.024 μM, and 2.5 μL was taken from each. The ACLY stock solution was diluted to 30 nM with aqueous buffer, and 2.5 μL was taken from each of the above compounds. The buffer was prepared as above, and the solvent composition of the enzyme (ACLY) stock solution was 50 mM Tris, 400 mM NaCl, and 10% glycerol by volume. The well plate was flicked to mix the system liquid evenly, and incubated at room temperature for 15 minutes. Then, 5 μL of substrate mixture solution was added, and the substrate mixture solution was 300 μM citric acid, 150 μM adenosine triphosphate (ATP), and 150 μM coenzyme A (CoA). Low-speed centrifugation (1200r, 1 min, 20°C) was performed to mix evenly, and the mixture was incubated in a 37°C incubator for 55 min. The mixture was taken out and allowed to stand at room temperature for 5 min. After returning to room temperature, 10 μL of ADP-Glo ​​reagent (Promega) was added, and the reaction was allowed to proceed at room temperature for 40 min to terminate the reaction. 20 μL of kinase detection reagent (Promega) was added to convert ADP into ATP, which emitted fluorescence under the catalysis of luciferase. The process lasted for 40 min. The Luminescence384 method provided by the instrument was selected on the PE Ensight microplate reader to detect the fluorescence intensity. GraphPad Prism 6.0 was used for data processing to obtain a dose-effect curve of 2 ( Figure 1), the horizontal axis is the logarithm of molar concentration, and the vertical axis is the fluorescence intensity value measured by the instrument. The inhibitory activity of 2 targeting ACLY is 3.86±0.62μM, indicating that 2 is an inhibitor of ACLY.

[0031] Example 2: ACLY inhibitory activity assay of compound 4

[0032] The stock solution concentration of 4 is 50 mM, and the solvent is DMSO. Take 1 μL of the stock solution and add it to 62.5 μL of aqueous buffer, the buffer composition is 40 mM tris (Tris), 4 mM dithiothreitol (DTT), 0.01% polyoxyethylene lauryl alcohol ether (Brij35), 0.001% bovine serum albumin (BSA), 10 mM MgCl2, 1% dimethyl sulfoxide (DMSO), pH = 8.0. The initial concentration of 4 obtained by the above operation was 200 μM, and then a 384-well plate was gradiently diluted with aqueous buffer using a shotgun to prepare 14 concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.156, 0.781, 0.391, 0.195, 0.098, 0.049 and 0.024 μM, and 2.5 μL was taken from each. The ACLY stock solution was diluted to 30 nM with aqueous buffer, and 2.5 μL was taken from each of the above compounds. The buffer was prepared as above, and the solvent composition of the enzyme (ACLY) stock solution was 50 mM Tris, 400 mM NaCl, and 10% glycerol by volume. The well plate was flicked to mix the system liquid evenly, and incubated at room temperature for 15 minutes. Then, 5 μL of substrate mixture solution was added, and the substrate mixture solution was 300 μM citric acid, 150 μM adenosine triphosphate (ATP), and 150 μM coenzyme A (CoA). Low-speed centrifugation (1200r, 1 min, 20°C) was performed to mix evenly, and the mixture was incubated in a 37°C incubator for 55 min. The mixture was taken out and allowed to stand at room temperature for 5 min. After returning to room temperature, 10 μL of ADP-Glo ​​reagent (Promega) was added, and the reaction was allowed to proceed at room temperature for 40 min to terminate the reaction. 20 μL of kinase detection reagent (Promega) was added to convert ADP into ATP, which emitted fluorescence under the catalysis of luciferase. The process lasted for 40 min. The Luminescence384 method provided by the instrument was selected on the PE Ensight microplate reader to detect the fluorescence intensity. GraphPad Prism 6.0 was used for data processing to obtain a dose-effect curve of 4 ( Figure 1 ), the horizontal axis is the logarithm of molar concentration, and the vertical axis is the fluorescence intensity value measured by the instrument. The inhibitory activity of 4 targeting ACLY is 3.86±0.62μM, indicating that 4 is an inhibitor of ACLY.

[0033] Example 3: Evaluation of the anti-lung cancer activity of compound 4

[0034] Lung cancer A549 cells were plated in 96-well plates with 100 μL F12 complete medium + 10% (volume fraction) fetal bovine serum, and the plate density was 5000 cells / well. After culturing in a 37°C incubator for 24 hours, drug treatment was added. Compound 4 was diluted with the culture medium to obtain the following 8 concentration gradients: 200, 100, 50, 25, 12.5, 6.250, 3.125 and 1.563 μM. It was added to the 96-well plate in the form of a replacement medium and incubated in a 37°C incubator for 72 hours. CCK reagent was diluted with F12 basal culture medium at a volume ratio of 9:1 and added to the 96-well plate in the form of a replacement medium. Incubate in a 37°C incubator for 2 hours, take out, and measure the absorbance at 450 nm with an enzyme reader. GraphPad Prism 6.0 was used for data processing ( Figure 3 ), get IC 50 =45.28±4.23μM, indicating that 4 has a good inhibitory effect on the proliferation of A549 cells.

[0035] Example 4: Evaluation of the anti-prostate cancer activity of compound 4

[0036] Prostate cancer cells PC3 were plated in 96-well plates with 100 μL F12 complete medium + 10% (volume fraction) fetal bovine serum, and the plate density was 5000 cells / well. After culturing in a 37°C incubator for 24 hours, drug treatment was added. Compound 4 was diluted with culture medium to obtain the following 8 concentration gradients: 200, 100, 50, 25, 12.5, 6.250, 3.125 and 1.563 μM. It was added to the 96-well plate in the form of liquid replacement and incubated in a 37°C incubator for 72 hours. CCK reagent was diluted with F12 basal culture medium at a volume ratio of 9:1 and added to the 96-well plate in the form of liquid replacement. Incubate in a 37°C incubator for 2 hours, take out, and measure the absorbance at 450 nm with an enzyme reader. GraphPad Prism 6.0 was used for data processing ( Figure 3 ), get IC 50 =45.28±4.23μM, indicating that 4 has a good inhibitory effect on the proliferation of A549 cells.

Claims

1. Use of a compound in the preparation of an anti-prostate cancer drug, characterized in that: The compound is a compound having the following structural formula 4: 。 2. The use according to claim 1, characterized in that: The anti-prostate cancer drug is a pharmaceutical composition prepared by using compound 4 as an active ingredient and a pharmaceutically acceptable carrier, including tablets, capsules, pills, granules or injections.

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