A tanshinone- imidazole derivative, and a preparation method and use thereof

Abstract

The application discloses a tanshinone derivative, a preparation method and application thereof, wherein the tanshinone derivative has the following structural formula: wherein R1 is selected from allyl; R2 is selected from ethyl, propyl, n-butyl, sec-butyl, isobutyl, 2-methylallyl, 2-methoxyethyl, 2-ethoxyethyl, 3-methoxypropyl, 3-ethoxypropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, benzyl, p-fluorobenzyl, p-chlorobenzyl, p-bromobenzyl, p-methylbenzyl, p-methoxybenzyl, 2,3-dimethylbenzyl and 3,4-dichlorobenzyl. The application introduces a five-membered heterocyclic structure on a tanshinone molecular skeleton of tanshinone to obtain a tanshinone-derivative, and the bioactivity test result shows that the tanshinone-derivative has good lipid-lowering activity on human hepatoma cell HepG2.

Description

Technical Field

[0001] This invention relates to a pterostilbene-imidazolium derivative, specifically to a pterostilbene-imidazolium derivative, its preparation method, and its uses. Background Technology

[0002] Pterostilbene PTS, also known as 4'-hydroxy-3,5-stilbene, is a natural product with a stilbene backbone and two methylene groups in its structure. It is mainly found in citrus fruits, grapes, and blueberries, and possesses broad-spectrum biological activities including anti-inflammatory, anticancer, and antioxidant effects. Unlike another natural product, resveratrol, the two methoxy groups in its backbone exhibit high hydrophobicity, playing an important role in promoting small intestinal permeability and stabilizing liver function. Its lipophilicity enhances cellular absorption and limits metabolic clearance by intestinal epithelial cells and hepatocytes. Due to the presence of only one free hydroxyl group, pterostilbene exhibits good metabolic stability. Therefore, its bioavailability is higher than that of other natural products with similar structures. Furthermore, because pterostilbene has low toxicity and is less likely to cause side effects, it can be used as a core structure in the design and development of new drugs, as well as in research on the prevention and treatment of related diseases.

[0003] Aromatic heterocycles are a preferred structural feature in drug design. Imidazole, a five-membered aromatic heterocyclic compound containing two nitrogen atoms, is a common structural unit in the pharmaceutical field. Compounds based on imidazole exhibit a wide range of biological activities, including antibacterial, anti-inflammatory, antidiabetic, antiparasitic, antituberculosis, antifungal, antioxidant, antitumor, antimalarial, anticancer, and antidepressant effects. Imidazole has proven to be a very versatile medicinal chemical structure, playing a crucial role as a pharmacophore.

[0004] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a serine protease primarily produced in the liver and plays a crucial role in regulating cholesterol homeostasis. As a member of the proprotein convertase family, PCSK9 binds to the low-density lipoprotein receptor (LDLR), allowing it to enter lysosomes for degradation. This inhibits the recirculation of LDLR to the cell surface, thus preventing it from clearing low-density lipoprotein cholesterol (LDL-C) from the plasma, ultimately leading to hypercholesterolemia and a range of related diseases.

[0005] This invention employs a "combined pharmacophore" design strategy, introducing the active pharmacophore—the imidazole ring—onto the pterostilbene skeleton, and screening and synthesizing novel, highly efficient, and low-toxic structures as candidate compounds for lipid-lowering drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a pterostilbene-imidazolium derivative, its preparation method, and its uses. The pterostilbene-imidazolium compounds of this invention exhibit good lipid-lowering activity against HepG2 human liver cancer cells, and have potential application prospects in the treatment of non-alcoholic fatty liver disease.

[0007] The pterostilbene-imidazolium derivative of this invention has the following structure:

[0008] ;

[0009] Wherein, R1 is selected from 4-enbutyl; R2 is selected from ethyl, propyl, n-butyl, sec-butyl, isobutyl, 2-methylallyl, 2-methoxyethyl, 2-ethoxyethyl, 3-methoxypropyl, 3-ethoxypropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, benzyl, p-fluorobenzyl, p-chlorobenzyl, p-bromobenzyl, p-methylbenzyl, p-methoxybenzyl, 2,3-dimethylbenzyl, and 3,4-dichlorobenzyl.

[0010] Furthermore, the pterostilbene-imidazolium derivative preferably has the following structure:

[0011]

[0012]

[0013] The method for preparing the pterostilbene-imidazolium derivative of the present invention includes the following steps:

[0014] Step 1: Add 10 mmol of pterostilbene to a 100 mL round-bottom flask, add 20 mL of acetonitrile, add a magnetic stir bar, and stir to dissolve. Weigh anhydrous K₂CO₃ (10 mmol) and 4-bromobutene (15 mmol) into the above system, set the reaction temperature to 80 °C, reflux the resulting reaction mixture overnight, and monitor the reaction by TLC. After the reaction is complete, cool the reaction mixture to room temperature, pour it into a separatory funnel, add water and ethyl acetate, shake, and let stand until the liquid layer separates. Collect the upper organic phase, add anhydrous Na₂SO₄ to dry and remove water, and finally concentrate using a rotary evaporator. Separate and purify by silica gel column chromatography to obtain intermediate pterostilbene derivative A.

[0015] The structural formula of the pterostilbene derivative A is:

[0016] .

[0017] Step 2: Add pterostilbene derivative A to a round-bottom flask, add N,N-dimethylformamide and stir to dissolve. Under ice bath conditions, add POCl3 dropwise and stir for 0.5 h. Then, stir the reaction mixture at room temperature for 1.5 h. After the reaction is completed by TLC monitoring, pour the reaction mixture into ice water and add saturated sodium hydroxide while stirring until the pH reaches alkalinity. After stirring overnight, a pale yellow solid precipitates. Filter, collect the filter cake, dissolve in dichloromethane, then add Na2SO4 to dry to remove moisture. Dehydrate, concentrate by vacuum distillation to obtain the crude product, and purify by silica gel column chromatography to obtain the pale yellow solid pterostilbene benzaldehyde derivative B.

[0018] The structural formula of the pterostilbenealdehyde derivative B is:

[0019] .

[0020] Step 3: Weigh pterostilbenealdehyde derivative B and substituted amine into a round-bottom flask, add ethanol and stir at room temperature for 2 hours, then add anhydrous K2CO3 and p-toluenesulfonylmethylisocyanate sequentially, and stir overnight at 50°C; after the reaction is completed by TLC monitoring, cool the reaction mixture to room temperature, pour it into a separatory funnel, extract the aqueous phase with dichloromethane, wash the organic phase with brine, let stand until the liquid surface separates, collect the upper organic phase, then add anhydrous Na2SO4 to dry to remove water, obtain the crude product by vacuum distillation, separate by silica gel column chromatography, and recrystallize with petroleum ether to obtain the target compound.

[0021] The substituted amine has the structural formula: H2N-R2.

[0022] R2 is selected from ethyl, propyl, n-butyl, sec-butyl, isobutyl, 2-methylallyl, 2-methoxyethyl, 2-ethoxyethyl, 3-methoxypropyl, 3-ethoxypropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, benzyl, p-fluorobenzyl, p-chlorobenzyl, p-bromobenzyl, p-methylbenzyl, p-methoxybenzyl, 2,3-dimethylbenzyl, and 3,4-dichlorobenzyl.

[0023] In step 1, the molar ratio of pterostilbene to anhydrous K2CO3 and 4-bromobutene is 1:1:1.5.

[0024] In step 1, the eluent for column chromatography is ethyl acetate: petroleum ether = 1:3, v / v.

[0025] In step 2, 2 millimoles of POCl3 are added for every 1 millimole of pterostilbene derivative A.

[0026] In step 2, the eluent for column chromatography is dichloromethane:petroleum ether = 3:1, v / v.

[0027] In step 3, the molar ratio of pterostilbenealdehyde derivative B to substituted amine is 1:1.5.

[0028] In step 3, the molar ratio of pterostilbene derivatives, anhydrous K2CO3, and p-toluenesulfonylmethylisocyanate is 1:1.5:1.2.

[0029] In step 3, the eluent for column chromatography is dichloromethane:methanol = 30:1, v / v.

[0030] This invention relates to the application of pterostilbene-imidazol derivatives in the preparation of lipid-lowering drugs.

[0031] The lipid-lowering drug described above exhibits good lipid-lowering activity against human hepatocellular carcinoma cells (HepG2). HepG2 cells are frequently used in in vitro studies of liver metabolic diseases, drug metabolism, and in vitro hepatotoxicity to confirm the inhibitory activity of pterostilbene-imidazolium compounds on hepatic lipid accumulation.

[0032] Furthermore, the lipid-lowering drug can reduce FBS-induced lipid accumulation in liver cells.

[0033] Based on the principles of drug combination and structure-based drug molecule design, this invention introduces a five-membered heterocyclic structure onto the pterostilbene molecular skeleton, obtaining pterostilbene-imidazolium derivatives. Bioactivity tests show that the pterostilbene-imidazolium compounds of this invention exhibit good lipid-lowering activity against HepG2 human liver cancer cells. Attached Figure Description

[0034] Figure 1 The study investigated the cytotoxicity of the pterostilbene-imidazolium compound PIA1-21 (10 μM) against HepG2 human liver cancer cells.

[0035] Figure 2 The expression levels of PCSK9 and LDLR in cells were detected by Western blotting of the pterostilbene-imidazolium compound PIA1-21 (10 μM).

[0036] Figure 3 Oil Red O staining was used to observe the formation of intracellular lipid droplets using compounds PIA4, PIA5, PIA19, and PIA20, which have the most significant effects at the molecular level. Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail through the following specific embodiments, but it should be noted that the scope of the present invention is not limited by these embodiments.

[0038] Example 1: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-ethyl-1H-imidazolium (compound PIA1)

[0039]

[0040] 1. Weigh 10 mmol of pterostilbene into a 100 mL round-bottom flask, add 20 mL of acetonitrile, and stir with a magnetic stir bar to dissolve. Weigh 10 mmol of anhydrous K₂CO₃ and 15 mmol of 4-bromobutene into the above system, set the reaction temperature to 80 °C, and reflux the resulting reaction mixture overnight. Monitor the reaction by TLC. After the reaction is complete, cool the reaction mixture to room temperature, pour it into a separatory funnel, add water and ethyl acetate, shake, and let stand until the liquid layer separates. Collect the upper organic phase, add anhydrous Na₂SO₄ to dry and remove water, and finally concentrate using a rotary evaporator. Finally, concentrate and purify by silica gel column chromatography to obtain intermediate pterostilbene derivative A.

[0041] 2. In a 100 mL round-bottom flask, weighed intermediate A (5 mmol, 1.0 eq) was added, along with 20 mL of N,N-dimethylformamide. A magnetic stir bar was added, and the mixture was stirred until dissolved. Under ice bath conditions, POCl3 (10 mmol, 2.0 eq) was added dropwise and stirred for 0.5 h. Subsequently, the reaction mixture was stirred at room temperature for 1.5 h. After the reaction was completed by TLC monitoring, the solution was poured into ice water, and saturated sodium hydroxide was added with stirring until the pH reached alkalinity. After stirring overnight, a pale yellow solid precipitated. The solid was filtered, and the filter cake was collected, dissolved in 100 mL of dichloromethane, and then dried with Na2SO4 to remove moisture. The product was dehydrated and concentrated by vacuum distillation to obtain the crude product. The pale yellow pterostilbenealdehyde derivative B was purified by silica gel column chromatography.

[0042] 3. Weigh intermediate B (1.0 eq) and ethylamine (1.5 eq) into a 100 mL round-bottom flask, add 20 mL of ethanol, add a magnetic stir bar, and stir at room temperature for 2 h. Then add anhydrous K₂CO₃ (1.5 eq) and p-toluenesulfonylmethylisocyanate (1.2 eq) sequentially, and stir overnight at 50 °C. After the reaction is completed by TLC monitoring, cool the reaction mixture to room temperature, pour it into a separatory funnel, extract the aqueous phase with dichloromethane, wash the organic phase with brine, let stand until the liquid surface separates, collect the upper organic phase, and then add anhydrous Na₂SO₄ to dry to remove water. Obtain the crude product by vacuum distillation. Separate by silica gel column chromatography (dichloromethane / methanol = 30:1, v / v gradient elution), and recrystallize with petroleum ether to obtain target compound 1. Product 1 is a white solid with a yield of 71.3% and a melting point of 98.4-100 °C.1 H NMR (600 MHz, cdcl3) δ 7.64 (s, 1H), 7.27 (d, J = 7.5 Hz, 2H), 6.95 (s, 1H), 6.93 (s, 1H), 6.88 (s, 1H), 6.83 (d, J =8.7 Hz, 2H), 6.75 (s, 1H), 6.44 (s, 1H), 6.03 (s, 1H), 5.41 (s, 1H), 5.28 (s,1H), 4.52 (d, J = 5.3 Hz, 2H), 3.91 (s, 3H), 3.72 (s, 3H), 3.68 (d, J = 2.3Hz, 2H), 1.19 (s, 3H). HR-MS(ESI): calcd for C 25 H 28 N₂O₃, [M+H] + ,404.2100;found404.2102.

[0043] Example 2: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-propyl-1H-imidazolium (compound PIA2)

[0044]

[0045] The preparation method is the same as in Example 1. Propylamine was used instead of ethylamine to obtain a pale yellow solid with a yield of 69.6% and a melting point of 110-112.8℃. 1 H NMR (600 MHz, cdcl3) δ 7.63 (s, 1H), 7.25 (s, 1H), 6.95 (d, J =22.2 Hz, 2H), 6.88 (s, 1H), 6.84 (d, J = 8.6 Hz, 2H), 6.75 (s, 1H), 6.44 (s,1H), 6.04 (s, 1H), 5.41 (s, 1H), 5.29 (s, 1H), 4.52 (d, J = 5.3 Hz, 2H), 3.91(s, 3H), 3.72 (s, 3H), 3.61 (d, J = 7.1 Hz, 2H), 1.53 (d, J = 7.3 Hz, 2H), 0.75 (t, J = 7.4 Hz, 3H). HR-MS(ESI):calcd for C 26 H 30 N₂O₃, [M+H]+ ,418.2256;found418.2248.

[0046] Example 3: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-n-butyl-1H-imidazolium (compound PIA3)

[0047]

[0048] The preparation method is the same as in Example 1. Using n-butylamine instead of ethylamine, a white solid was obtained with a yield of 72.5% and a melting point of 93-96.1℃. 1 H NMR (600 MHz, cdcl3) δ 7.62 (s, 1H), 7.25 (s, 2H), 6.94 (d, J = 8.4Hz, 2H), 6.88 (s, 1H), 6.84 (d, J = 8.6 Hz, 2H), 6.75 (s, 1H), 6.44 (s, 1H), 6.04 (s, 1H), 5.42 (s, 1H), 5.29 (s, 1H), 4.52 (d, J = 5.2 Hz, 2H), 3.92 (s,3H), 3.72 (s, 3H), 3.64 (d, J = 12.4 Hz, 2H), 1.48 (s, 2H), 1.14 (d, J = 5.8Hz, 2H), 0.74 (d, J = 7.3 Hz, 3H). HR-MS(ESI):calcd for C 27 H 32 N₂O₃, [M+H] + , 432.2413; found 432.2424.

[0049] Example 4: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-sec-butyl-1H-imidazolium (compound PIA4)

[0050]

[0051] The preparation method is the same as in Example 1. Using sec-butylamine instead of ethylamine, a yellow solid was obtained with a yield of 74.1% and a melting point of 126.1-128.3℃. 1H NMR (600 MHz, cdcl3) δ 7.69 (s, 1H), 7.24 (s, 1H), 6.92 (dd,J = 36.0, 12.7 Hz, 4H), 6.82 (d, J = 5.0 Hz, 2H), 6.70 (s, 1H), 6.43 (s, 1H), 6.02 (s, 1H), 5.41 (s, 1H), 5.28 (s, 1H), 4.51 (d, J = 3.4 Hz, 2H), 3.91 (s,3H), 3.70 (d, J = 16.1 Hz, 3H), 3.61 (s, 1H), 1.63 (d, J = 13.2 Hz, 2H), 1.39(d, J = 6.3 Hz, 2H), 1.24 (s, 1H), 1.19 (s, 1H), 0.79 (s, 1H), 0.60 (s, 1H).HR-MS(ESI):calcd for C 27 H 32 N₂O₃, [M+H] + , 432.2413; found 432.2419.

[0052] Example 5: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-isobutyl-1H-imidazolium (compound PIA5)

[0053]

[0054] The preparation method is the same as in Example 1. Isobutylamine was used instead of ethylamine to obtain a yellow solid with a yield of 65.3% and a melting point of 150.1-152.6℃. 1H NMR (600 MHz, cdcl3) δ 7.60 (s, 1H), 7.24 (s, 2H), 6.94 (d, J= 15.8 Hz, 2H), 6.85 (s, 1H), 6.83 (d, J = 7.6 Hz, 2H), 6.77 (s, 1H), 6.41(s, 1H), 6.03 (s, 1H), 5.39 (d, J = 17.1 Hz, 1H), 5.26 (s, 1H), 4.75 (s, 1H), 4.59 (s, 1H), 4.51 (s, 2H), 4.17 (s, 1H), 4.13 (s, 1H), 3.89 (s, 3H), 3.70(s, 3H), 1.50 (s, 3H). HR-MS(ESI):calcd for C 27 H 32 N₂O₃, [M+H] + ,432.2413; found432.2430.

[0055] Example 6: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(2-methylallyl)-1H-imidazolium (compound PIA6)

[0056]

[0057] The preparation method is the same as in Example 1. Using 2-methylallylamine instead of ethylamine, a pale yellow solid was obtained with a yield of 76.5% and a melting point of 97.5-99.4℃. 1 H NMR (600 MHz, cdcl3) δ 7.60 (s, 1H), 7.24 (s, 2H), 6.94 (d,J = 15.8 Hz, 2H), 6.85 (s, 1H), 6.83 (d, J = 7.6 Hz, 2H), 6.77 (s, 1H), 6.41(s, 1H), 6.03 (s, 1H), 5.39 (d, J = 17.1 Hz, 1H), 5.26 (s, 1H), 4.75 (s, 1H), 4.59 (s, 1H), 4.51 (s, 2H), 4.17 (s, 1H), 4.13 (s, 1H), 3.89 (s, 3H), 3.70(s, 3H), 1.50 (s, 3H). HR-MS(ESI):calcd for C 27 H 30N₂O₃, [M+H] + ,430.2256;found430.2251.

[0058] Example 7: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(2-methoxyethyl)-1H-imidazolium (compound PIA7)

[0059]

[0060] The preparation method is the same as in Example 1. 2-Aminoethyl methyl etheramine was used instead of ethylamine to obtain a white solid with a yield of 77.3% and a melting point of 78.4-80.2℃. 1 H NMR (600 MHz, cdcl3) δ 7.77 (s, 1H), 7.27 (s, 2H), 6.96 (d,J = 16.3 Hz, 2H), 6.88 (s, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.75 (s, 1H), 6.44(s, 1H), 6.03 (s, 1H), 5.42 (s, 1H), 5.29 (s, 1H), 4.52 (d, J = 5.3 Hz, 2H), 3.91 (s, 3H), 3.81 (d, J = 24.9 Hz, 2H), 3.73 (s, 3H), 3.39 (s, 1H), 3.32 (s,1H), 3.17 (s, 3H). HR-MS(ESI):calcd for C 26 H 30 N₂O₄, [M+H] + ,434.2206; found434.2209.

[0061] Example 8: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(2-ethoxyethyl)-1H-imidazolium (compound PIA8)

[0062]

[0063] The preparation method is the same as in Example 1. Using 2-ethoxyethylamine instead of ethylamine, a yellow solid was obtained with a yield of 59.6% and a melting point of 64.7-67.6℃. 1H NMR (600 MHz, cdcl3) δ 7.75 (s, 1H), 7.27 (s, 1H), 7.26 (s,1H), 6.95 (d, J = 22.6 Hz, 2H), 6.88 (s, 1H), 6.83 (d, J = 8.6 Hz, 2H), 6.76(s, 1H), 6.43 (s, 1H), 6.04 (s, 1H), 5.41 (s, 1H), 5.29 (s, 1H), 4.52 (d, J =5.2 Hz, 2H), 3.91 (s, 3H), 3.80 (d, J = 18.4 Hz, 2H), 3.73 (s, 3H), 3.43 (s,1H), 3.35 (s, 1H), 3.29 (d, J = 2.6 Hz, 2H), 1.06 (t, J = 7.0 Hz, 3H). HR-MS(ESI):calcd for C 27 H 32 N₂O₄, [M+H] + , 448.2362; found 448.2367.

[0064] Example 9: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(3-methoxypropyl)-1H-imidazolium (compound PIA9)

[0065]

[0066] The preparation method is the same as in Example 1. Replacing ethylamine with 3-methoxypropylamine yielded a yellow solid with a yield of 58.4% and a melting point of 87.7-90.7℃. 1H NMR (600 MHz, cdcl3) δ 7.62 (s, 1H), 7.25 (s, 2H), 6.96 (d, J= 16.4 Hz, 2H), 6.87 (s, 1H), 6.84 (d, J = 8.6 Hz, 2H), 6.75 (s, 1H), 6.44(s, 1H), 6.04 (s, 1H), 5.41 (s, 1H), 5.29 (s, 1H), 4.52 (d, J = 5.3 Hz, 2H), 3.91 (s, 3H), 3.77 (d, J = 11.8 Hz, 2H), 3.72 (s, 3H), 3.19 (s, 3H), 3.16 (s,2H), 1.68 (d, J = 7.3 Hz, 2H). HR-MS(ESI):calcd for C 27 H 32 N₂O₄, [M+H] + , 448.2362; found 448.2369.

[0067] Example 10: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(3-ethoxypropyl)-1H-imidazolium (compound PIA10)

[0068]

[0069] The preparation method is the same as in Example 1. Using 3-ethoxypropylamine instead of ethylamine, a pale yellow oily droplet was obtained with a yield of 78.6% and a melting point of 98.2-100.7℃. 1H NMR (600 MHz, cdcl3) δ 7.62 (s, 1H), 7.25 (s, 2H), 6.96 (d, J = 15.5 Hz, 2H), 6.87 (s, 1H), 6.84 (d, J = 8.5 Hz, 2H), 6.75 (s,1H), 6.44 (s, 1H), 6.04 (s, 1H), 5.42 (s, 1H), 5.29 (s, 1H), 4.52 (d, J = 5.1Hz, 2H), 3.91 (s, 3H), 3.79 (d, J = 11.7 Hz, 2H), 3.72 (s, 3H), 3.30 (d, J =6.9 Hz, 2H), 3.18 (d, J = 3.5 Hz, 2H), 1.69 (d, J = 6.5 Hz, 2H), 1.09 (t, J =7.0 Hz, 3H). HR-MS(ESI):calcd for C 28 H 34 N₂O₄, [M+H] + , 462.2519; found 462.2512.

[0070] Example 11: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(cyclopropylmethyl)-1H-imidazolium (compound PIA11)

[0071]

[0072] The preparation method is the same as in Example 1. Cyclopropylmethylamine was used instead of ethylamine to obtain a white solid with a yield of 59.1% and a melting point of 97.2-100.8℃. 1H NMR (600 MHz, cdcl3) δ 7.77 (s, 1H), 7.27 (s, 2H), 6.96 (d, J= 12.0 Hz, 2H), 6.88 (s, 1H), 6.84 (d, J = 8.4 Hz, 2H), 6.77 (s, 1H), 0.98 (s, 1H), 0.48 (d, J = 6.8 Hz, 2H), 0.15 (s, 1H), 0.08 (s, 1H). HR-MS(ESI):calcd for C 27 H 31 N₂O₃, [M+H] + , 431.2326; found 431.2325.

[0073] Example 12: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-cyclobutyl-1H-imidazolium (compound PIA12)

[0074]

[0075] The preparation method is the same as in Example 1. Cyclobutylamine was used instead of ethylamine to obtain a white solid with a yield of 60.5% and a melting point of 128.9-130℃. 1 H NMR (600 MHz, cdcl3) δ 7.81 (s, 1H), 7.27 (s, 2H), 6.92 (s,2H), 6.84 (d, J = 8.3 Hz, 3H), 6.70 (d, J = 16.2 Hz, 1H), 6.43 (s, 1H), 6.03(s, 1H), 5.41 (s, 1H), 5.29 (s, 1H), 4.52 (d, J = 4.9 Hz, 2H), 4.19 (s, 1H), 3.91 (s, 3H), 3.71 (s, 3H), 2.32 (s, 2H), 2.17 (s, 2H), 1.69 (d, J = 49.0 Hz, 2H). HR-MS(ESI): calcd for C 27 H 30 N₂O₃, [M+H] +, 430.2256; found 430.2250.

[0076] Example 13: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-cyclopentyl-1H-imidazolium (compound PIA13)

[0077]

[0078] The preparation method is the same as in Example 1. Cyclopentylamine was used instead of ethylamine to obtain a pale yellow solid with a yield of 51.4% and a melting point of 168.4-170.6℃. 1 H NMR (600 MHz, cdcl3) δ 7.71 (s, 1H), 7.25 (s, 2H), 6.94 (s,1H), 6.91 (s, 1H), 6.88 (s, 1H), 6.83 (d, J = 8.6 Hz, 2H), 6.73 (s, 1H), 6.44(s, 1H), 6.04 (s, 1H), 5.41 (s, 1H), 5.28 (d, J = 10.5 Hz, 1H), 4.51 (s, 2H), 4.02 (s, 1H), 3.91 (s, 3H), 3.72 (s, 3H), 2.00 (s, 1H), 1.83 (d, J = 12.8 Hz,4H), 1.57 (d, J = 52.8 Hz, 3H). HR-MS(ESI):calcd for C 28 H 32 N₂O₃, [M+H] + , 444.2413; found 444.2420.

[0079] Example 14: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-benzyl-1H-imidazolium (compound PIA14)

[0080]

[0081] The preparation method is the same as in Example 1. Aniline was used instead of ethylamine to obtain a yellow solid with a yield of 76.2% and a melting point of 97.3-100.9℃. 1H NMR (600 MHz, cdcl3) δ 7.62 (s, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.12(s, 2H), 6.98 (s, 1H), 6.93 (d, J = 5.4 Hz, 2H), 6.85 (dd, J = 20.7, 12.3 Hz, 4H), 6.80 (s, 1H), 6.64 (s, 1H), 6.37 (s, 1H), 6.03 (s, 1H), 5.41 (s, 1H), 5.28 (s, 1H), 4.79 (d, J = 13.4 Hz, 2H), 4.50 (d, J = 4.7 Hz, 2H), 3.87 (s,3H), 3.59 (s,3H). HR-MS(ESI):calcd for C 30 H 30 N₂O₃, [M+H] + ,466.2256; found466.2259.

[0082] Example 15: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(4-fluorobenzyl)-1H-imidazolium (compound PIA15)

[0083]

[0084] The preparation method is the same as in Example 1. Using p-fluorobenzylamine instead of ethylamine, a pale yellow solid was obtained with a yield of 71.5% and a melting point of 138.8-140.4℃. 1 H NMR (600 MHz, cdcl3) δ 7.63 (s, 1H), 7.21 (d, J = 8.4 Hz,2H), 6.97 (s, 1H), 6.88 (s, 1H), 6.84 (dd, J = 13.2, 6.8 Hz, 4H), 6.80 (d, J= 8.9 Hz, 3H), 6.59 (s, 1H), 6.37 (s, 1H), 6.03 (s, 1H), 5.42 (s, 1H), 5.29(s, 1H), 4.77 (s, 1H), 4.74 (s, 1H), 4.52 (d, J = 5.1 Hz, 2H), 3.88 (s, 3H), 3.61 (s, 3H). HR-MS(ESI): calcd for C 30 H 29 FN₂O₃,[M+H]+ , 484.2162; found 484.2167.

[0085] Example 16: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(4-chlorobenzyl)-1H-imidazolium (compound PIA16)

[0086]

[0087] The preparation method is the same as in Example 1. Using p-chlorobenzylamine instead of ethylamine, a white solid was obtained with a yield of 49.3% and a melting point of 116-118.3℃. 1 H NMR (600 MHz, cdcl3) δ 7.73 (s, 1H), 7.21 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 8.3 Hz, 2H), 7.00 (s, 1H), 6.85 (d, J = 8.4 Hz, 3H), 6.82 (d, J= 6.2 Hz, 2H), 6.79 (s, 1H), 6.57 (s, 1H), 6.37 (s, 1H), 6.05 (s, 1H), 5.43(s, 1H), 5.28 (s, 1H), 4.78 (d, J = 15.2 Hz, 2H), 4.54 (d, J = 5.1 Hz, 2H), 3.90 (s, 3H), 3.61 (s, 3H). HR-MS(ESI):calcd for C 30 H 29 ClN2O3,[M+H] + , 500.1867; found 500.1861.

[0088] Example 17: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(4-bromobenzyl)-1H-imidazolium (compound PIA17)

[0089]

[0090] The preparation method is the same as in Example 1. Using p-bromobenzylamine instead of ethylamine yielded a yellow solid with a yield of 79.3% and a melting point of 112-114.3℃. 1H NMR (600 MHz, cdcl3) δ 7.86 (s, 1H), 7.24 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 8.7 Hz, 2H), 7.02 (s, 1H), 6.84 (t, J = 12.1 Hz, 3H), 6.78 (t, J= 5.3 Hz, 3H), 6.53 (d, J = 16.2 Hz, 1H), 6.37 (s, 1H), 6.04 (s, 1H), 5.43(s, 1H), 5.30 (s, 1H), 4.78 (d, J = 20.2 Hz, 2H), 4.54 (d, J = 5.3 Hz, 2H),3.90 (s, 3H), 3.61 (s, 3H). HR-MS(ESI):calcd for C 30 H 29 BrN2O3,[M+H] + , 544.1362; found 544.1365.

[0091] Example 18: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(4-methylbenzyl)-1H-imidazolium (compound PIA18)

[0092]

[0093] The preparation method is the same as in Example 1. Using p-methylbenzylamine instead of ethylamine, a pale yellow solid was obtained with a yield of 80.1% and a melting point of 81.4-84.2℃. 1 H NMR (600 MHz, cdcl3) δ 7.58 (s, 1H), 7.19 (d, J = 8.5 Hz,2H), 6.94 (s, 1H), 6.86 – 6.80 (m, 6H), 6.64 (d, J = 8.5 Hz, 2H), 6.60 (s,1H), 6.39 (s, 1H), 6.03 (s, 1H), 5.40 (s, 1H), 5.27 (s, 1H), 4.74 (s, 1H), 4.68 (s, 1H), 4.51 (d, J = 4.9 Hz, 2H), 3.88 (s, 3H), 3.63 (d, J = 7.7 Hz,6H). HR-MS(ESI):calcd for C 31 H 32 N₂O₃, [M+H] +, 480.2413; found 480.2418.

[0094] Example 19: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(4-methoxybenzyl)-1H-imidazolium (compound PIA19)

[0095]

[0096] The preparation method is the same as in Example 1. Using p-methoxybenzylamine instead of ethylamine, a white solid was obtained with a yield of 71.6% and a melting point of 106.9-110.7℃. 1 H NMR (600 MHz, cdcl3) δ 7.63 (s, 1H), 7.20 (d, J = 8.6 Hz,2H), 6.96 (s, 1H), 6.87 – 6.80 (m, 6H), 6.65 (d, J = 8.5 Hz, 2H), 6.58 (s,1H), 6.40 (s, 1H), 6.04 (s, 1H), 5.42 (s, 1H), 5.29 (s, 1H), 4.75 (s, 1H), 4.69 (s, 1H), 4.53 (s, 2H), 3.90 (s, 3H), 3.64 (d, J = 6.9 Hz, 6H). HR-MS(ESI):calcd for C 31 H 32 N₂O₃, [M+H] + , 496.2362; found 496.2368.

[0097] Example 20: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(2,3-dimethylbenzyl)-1H-imidazolium (compound PIA20)

[0098]

[0099] The preparation method is the same as in Example 1. 2,3-Dimethylbenzylamine was used instead of ethylamine to obtain a yellow solid with a yield of 64.4% and a melting point of 125.6-130.5℃. 1H NMR (600 MHz, cdcl3) δ 7.43 (s, 1H), 7.25 (s, 2H), 7.03(d, J = 15.5 Hz, 2H), 6.93 (d, J = 26.1 Hz, 2H), 6.84 (dd, J = 16.1, 11.3 Hz, 4H), 6.75 (s, 1H), 6.42 (s, 1H), 6.05 (s, 1H), 5.42 (s, 1H), 5.29 (s, 1H), 4.78 (d, J = 24.1 Hz, 2H), 4.54 (d, J = 5.2 Hz, 2H), 3.91 (s, 3H), 3.70 (s,3H), 2.18 (s, 3H), 1.94 (s, 3H). HR-MS(ESI):calcd for C 32 H 34 N₂O₃, [M+H] + , 494.2569; found 494.2562.

[0100] Example 21: Preparation of (E)-5-(2-(4-(enylbutoxy)styryl-4,6-dimethoxyphenyl)-1-(3,4-dichlorobenzyl)-1H-imidazolium (compound PIA21)

[0101]

[0102] The preparation method is the same as in Example 1. 3,4-Dichlorobenzylamine was used instead of ethylamine to obtain a yellow solid with a yield of 72.1% and a melting point of 100.1-102.3℃. 1 H NMR (600 MHz, cdcl3) δ 7.89 (s, 1H), 7.20 (d, J = 8.5 Hz,2H), 7.15 (s, 1H), 7.02 (s, 1H), 6.89 (s, 1H), 6.84 (d, J = 8.2 Hz, 3H), 6.78(d, J = 15.3 Hz, 2H), 6.52 (s, 1H), 6.38 (s, 1H), 6.04 (s, 1H), 5.43 (s, 1H), 5.30 (s, 1H), 4.78 (d, J = 24.2 Hz, 2H), 4.54 (d, J = 5.1 Hz, 2H), 3.90 (s, 3H), 3.63 (s, 3H). HR-MS(ESI):calcd for C 30 H 28Cl2N2O3,[M+H] + ,534.1477; found534.1472.

[0103] Example 22: HepG2 cell culture

[0104] We selected HepG2 human liver cancer cells for culture. HepG2 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 100 U / mL penicillin and streptomycin. The incubator conditions were set to 5% CO2, 37℃, with daily medium changes and cell growth observed. When HepG2 cells reached 70-80% confluence, the old cell culture medium was discarded, and the cells were washed twice with PBS. 0.25% trypsin was added, and cell morphology changes were observed under an inverted microscope. When cytoplasmic retraction, cell rounding, and increased intercellular spaces were observed, the digestion solution was discarded, PBS was added, and the adherent cells were gently pipetted repeatedly to detach and suspend them. After adjusting the cell density to a suitable level, the cells were seeded into new culture dishes and incubated in a 5% CO2, 37℃ incubator.

[0105] Example 23: Evaluation of lipid-lowering activity against HepG2 human liver cancer cells

[0106] We used the MTT assay to determine the cytotoxicity of compound PIA1-21 against human hepatocellular carcinoma HepG2 cells. HepG2 cells in logarithmic growth phase were seeded into 96-well plates, approximately 10,000 cells per well. After culturing for 16 h, 10 μM of compound PIA1-21 was added, with six replicates per compound. After 24 h of culture, the culture medium was aspirated, and MTT solution was added, followed by 3 h of further culture. Then, dimethyl sulfoxide was added, and the plates were shaken for 10 min. Finally, the absorbance (OD value) of the solution was measured at 550 nm using a microplate reader, and the effect of each drug on cell viability was calculated. MTT results ( Figure 1 The study showed that a 10 μM concentration of compound PIA1-21 had no significant toxicity to cells.

[0107] HepG2 cells in logarithmic growth phase were seeded into 12-well plates. After culturing for 16 h, all cells except the control group were replaced with DMEM medium containing 20% ​​FBS and co-treated with 10 μM compound PIA1-21. After culturing for 24 h, total cell protein was extracted, and the expression levels of PCSK9 and LDLR in the cells were detected by Western blotting. Figure 2 It can be seen that under 20% FBS induction, 10 μm compound PIA1-21 significantly inhibited the expression of PCSK9 in cells, while the expression of LDLR in cells increased.

[0108] Compounds PIA4, PIA5, PIA19, and PIA20, which showed the most significant effects, were selected for subsequent experiments. HepG2 cells in the logarithmic growth phase were seeded into 48-well plates pre-coated with glass slides. After culturing in an incubator for 16 h, except for the control group, the medium was replaced with DMEM containing 20% ​​FBS, and simultaneously treated with 10 μM compound 1-21. After culturing for 24 h, Oil Red O staining was performed to observe the formation of intracellular lipid droplets. Figure 3 It can be seen that under 20% FBS induction, intracellular lipid droplets increased significantly, resulting in lipid accumulation. Compounds PIA4, PIA5, PIA19, and PIA20 at a concentration of 10 μm can significantly reduce the formation of intracellular lipid droplets.

[0109] In summary, our study fully demonstrates that these compounds can reduce lipid accumulation in liver cells induced by 20% FBS, indicating that pterostilbene-imidazol compounds have potential application prospects in the treatment of non-alcoholic fatty liver disease.

[0110] Experimental Materials and Methods

[0111] MTT method:

[0112] The MTT assay was used to detect cell viability and growth levels. After specific treatment, cells were co-treated with MTT (0.5 mg / mL) in a cell culture incubator for 4 hours, then washed three times with sterile PBS, and infiltrated with DMSO (200 μL / well) at room temperature for 15 minutes. The absorbance (A) of the dissolved formazan in the cells was obtained using a microplate reader, and cell viability was characterized by the fold increase compared to the control group.

[0113] Western Blot:

[0114] Western blotting separates target proteins of different molecular weights from tissues or cells using gel electrophoresis. The target proteins are then transferred to a solid support (NC membrane) using blotting technology. Finally, the sample is stained by the specific binding of antigen and antibody, and after staining, the expression of the target protein can be detected and analyzed. Its basic operation is as follows:

[0115] Tissue protein extraction—protein concentration determination—gel preparation—sample preparation—electrophoresis—transfer membrane—incubation in 5% milk for blocking—antibody incubation and binding—exposure.

[0116] Oil Red O staining:

[0117] Oil Red O is a lipid-soluble dye with a strong affinity for neutral lipid components. It can specifically bind to triglycerides (TG) in tissues and cells to form lipid droplets-like red circles. When Oil Red O is used to stain cells or tissue sections, it binds to TG in the cells or tissues to form red lipid droplets.

[0118] The steps for cell oil red O staining are as follows:

[0119] 1) Slide preparation: Lay a sterile glass slide of appropriate size flat in the 48-well plate;

[0120] 2) Cell culture: Resuspend the cells in DMEM complete medium, add them to the prepared 48-well plates, and culture them in a 37°C, 5% CO2 incubator until the cells are completely adhered.

[0121] 3) Cell fixation: Aspirate the culture medium from the cells in the 48-well plate, wash the cells twice with PBS solution, 500 μL per well, 5 min each time. Add 400 μL of 4% paraformaldehyde and fix the cells at room temperature for 30 min;

[0122] 4) Oil Red O staining: After washing the cells twice with PBS, add 500 μl of Oil Red O working solution to each well and stain for 40-60 min;

[0123] 5) Destaining: Rinse the cells repeatedly with deionized water to remove excess staining solution;

[0124] 6) Counterstaining: Add 500 μL of hematoxylin staining solution, stain for 30 s, then remove the hematoxylin staining solution, rinse the staining solution with tap water, and place the cells in tap water for 5 min to turn the purple color of hematoxylin blue.

[0125] 7) Mounting: Add an appropriate amount of mounting solution to the slide, then remove the slide from the well plate, blot off excess water with filter paper, and place the side containing cells face down on the mounting solution, taking care to avoid air bubbles;

[0126] 8) Photography: After the film has dried, observe and photograph it under a Leica upright microscope.

Claims

1. A pterostilbene-imidazolium derivative, characterized in that... Its structure is as follows: ； Wherein, R1 is selected from 4-enbutyl; R2 is selected from ethyl, propyl, n-butyl, sec-butyl, isobutyl, 2-methylallyl, 2-methoxyethyl, 2-ethoxyethyl, 3-methoxypropyl, 3-ethoxypropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, benzyl, p-fluorobenzyl, p-chlorobenzyl, p-bromobenzyl, p-methylbenzyl, p-methoxybenzyl, 2,3-dimethylbenzyl, and 3,4-dichlorobenzyl.

2. The method for preparing the pterostilbene-imidazolium derivative according to claim 1, characterized in that... Includes the following steps: Step 1: Add pterostilbene to the reactor, add acetonitrile dropwise, and stir to dissolve; weigh anhydrous K2CO3 and 4-bromobutene and add them to the system, reflux at 80℃, and monitor the reaction by TLC; after the reaction is completed, cool the reaction mixture to room temperature, pour it into a separatory funnel, add water and ethyl acetate, collect the upper organic phase, add anhydrous Na2SO4 to dry to remove water, concentrate by rotary evaporation, and separate and purify by silica gel column chromatography to obtain intermediate pterostilbene derivative A; The structural formula of the pterostilbene derivative A is: ； Step 2: Add pterostilbene derivative A to the reactor, add N,N-dimethylformamide and stir to dissolve. Under ice bath conditions, add POCl3 dropwise and stir for 0.5 h. Then, stir the reaction mixture at room temperature for 1.5 h. After the reaction is completed by TLC monitoring, pour the reaction mixture into ice water and add saturated sodium hydroxide while stirring until the pH reaches alkalinity. After stirring for 8-12 h, a pale yellow solid precipitates. Filter, collect the filter cake, dissolve it in dichloromethane, then add Na2SO4 to dry to remove water, dehydrate, concentrate by vacuum distillation to obtain crude product, and purify by silica gel column chromatography to obtain pale yellow solid pterostilbene benzaldehyde derivative B. The structural formula of the pterostilbenealdehyde derivative B is: ； Step 3: Weigh pterostilbene derivative B and substituted amine into a reactor, add ethanol and stir at room temperature for 2 h, then add anhydrous K2CO3 and p-toluenesulfonylmethylisocyanate sequentially, and stir at 50 °C for 8-12 h; after the reaction is completed by TLC monitoring, cool the reaction mixture to room temperature, pour it into a separatory funnel, extract the aqueous phase with dichloromethane, wash the organic phase with brine, let stand until the liquid surface separates, collect the upper organic phase, then add anhydrous Na2SO4 to dry to remove water, obtain the crude product by vacuum distillation, separate by silica gel column chromatography, and recrystallize with petroleum ether to obtain the target compound; The structural formula of the substituted amine is: H2N-R2. R2 is selected from ethyl, propyl, n-butyl, sec-butyl, isobutyl, 2-methylallyl, 2-methoxyethyl, 2-ethoxyethyl, 3-methoxypropyl, 3-ethoxypropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, benzyl, p-fluorobenzyl, p-chlorobenzyl, p-bromobenzyl, p-methylbenzyl, p-methoxybenzyl, 2,3-dimethylbenzyl, and 3,4-dichlorobenzyl.

3. The preparation method according to claim 2, characterized in that: In step 1, the molar ratio of pterostilbene to anhydrous K2CO3 and 4-bromobutene is 1:1:1.

5.

4. The preparation method according to claim 2, characterized in that: In step 1, the eluent for column chromatography is ethyl acetate: petroleum ether = 1:3, v / v.

5. The preparation method according to claim 2, characterized in that: In step 2, 2 millimoles of POCl3 are added for every 1 millimole of pterostilbene derivative A.

6. The preparation method according to claim 2, characterized in that: In step 2, the eluent for column chromatography is dichloromethane:petroleum ether = 3:1, v / v.

7. The preparation method according to claim 2, characterized in that: In step 3, the molar ratio of the pterostilbene derivative B to the substituted amine is 1:1.5; the molar ratio of the pterostilbene derivative B, anhydrous K2CO3, and p-toluenesulfonylmethylisocyanate is 1:1.5:1.

2.

8. The preparation method according to claim 2, characterized in that: In step 3, the eluent for column chromatography is dichloromethane:methanol = 30:1, v / v.

9. The use of the pterostilbene-imidazol derivative of claim 1 in the preparation of lipid-lowering drugs.

10. The application according to claim 9, characterized in that: The lipid-lowering drug can reduce FBS-induced lipid accumulation in liver cells.

Images