A pterostilbene paeonol acetic acid compound, its preparation method and medical use

Abstract

The present invention discloses a pterostilbene-paeonol acetic acid compound represented by general formula (I). The compound can regulate the apoptosis of tumor cells and the expression of related proteins to exert excellent anti-tumor effects. It can be used to treat various tumor diseases including osteosarcoma, lung cancer, and liver cancer. It has potential anti-tumor research value and application prospects. In addition, the method used to prepare these compounds has the characteristics of readily available raw materials, simple operation, and high yield.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a pterostilbene paeonol acetic acid compound, a preparation method and medical uses thereof. The compounds have an inhibitory effect on tumor cells, and some of the compounds have a more prominent inhibitory effect on HDAC1 and HDAC2 within a certain concentration range, and can be used to prepare anti-tumor drugs. Background Art

[0002] Pterostilbene is a natural active ingredient found primarily in red sandalwood, grapes, and blueberries. It exhibits anti-tumor, anti-inflammatory, and antioxidant activities. Compared to resveratrol, pterostilbene exhibits enhanced anti-tumor activity due to its two additional methoxy groups, which make it more lipid-soluble and have stronger cell-penetrating properties. However, pterostilbene's low structural stability and short biological half-life have hindered its further development and application.

[0003] Furthermore, the anti-tumor effects of paeonol, the main active ingredient in the traditional Chinese medicine (Moong Cortex Moutan), have also garnered significant attention. Studies have shown that paeonol exhibits anti-cancer effects against a variety of tumors, including liver cancer, gastric cancer, breast cancer, and ovarian cancer. Paeonol may exert its anti-tumor effects by inhibiting tumor cell proliferation, affecting the tumor cell cycle, and regulating apoptosis-related factors and pathways, thereby inhibiting tumor cell metastasis. However, paeonol's clinical applications are limited by its readily sublimable nature, poor water solubility, and rapid metabolic rate.

[0004] Therefore, both pterostilbene and paeonol, active ingredients with proven anti-tumor effects, have limitations in practical application, making it difficult to truly realize their anti-tumor potential. Combining the two through pharmaceutical chemical structure modification to design and synthesize novel pterostilbene compounds with enhanced anti-tumor activity, improved physicochemical properties, and enhanced applicability is of great significance in providing material support for the development of anti-tumor drugs. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a pterostilbene-paeonol acetic acid compound, its preparation method, and medical use. Utilizing the principles of bioisostere and bridging in medicinal chemistry, using pterostilbene as a lead compound and combining it with a structurally modified paeonol, a series of novel pterostilbene-paeonol acetic acid compounds with outstanding anti-tumor effects, good compound structural stability, and strong applicability were designed and synthesized. These compounds have very important clinical application prospects and practical value.

[0006] In the first aspect of the present invention, a pterostilbene paeonol acetic acid compound is provided, represented by the general formula (I);

[0007]

[0008] Wherein, R is a nitrogen-containing group.

[0009] According to a specific embodiment of the present invention, the nitrogen-containing group is selected from any one of morpholinyl, pyrrolidinyl, piperidinyl, N-(2-hydroxyalkyl)piperazinyl, N-alkylpiperazinyl, 1,2,3,4-tetrahydroisoquinolinyl, N-alkyl-1-phenylalkylamino, 4-hydroxypiperidinyl, and piperazine-1-carboxylic acid alkyl ester; preferably, the alkyl group in N-(2-hydroxyalkyl)piperazinyl, N-alkylpiperazinyl, N-alkyl-1-phenylalkylamino, and piperazine-1-carboxylic acid alkyl ester is a C1 to C4 alkyl, preferably a C1 to C2 alkyl.

[0010] The second aspect of the present invention provides a method for preparing the aforementioned pterostilbene-paeonol acetic acid compound, comprising the following steps:

[0011] S1. Under alkaline conditions, pterostilbene, a methyl etherification agent and a solvent are contacted to perform a methyl etherification reaction to obtain a compound represented by formula (1). The reaction formula is as follows:

[0012]

[0013] S2. In an ice bath, a formylating agent and an acid chloride are contacted with the compound represented by formula (1) to carry out a Vilsmeier-Haack reaction to obtain a compound represented by formula (2). The reaction formula is as follows:

[0014]

[0015] S3. Under alkaline conditions, the compound represented by formula (3) and the compound represented by formula (4) are contacted with a solvent to carry out a nucleophilic substitution reaction to obtain a compound represented by formula (5). The reaction formula is as follows:

[0016]

[0017] Wherein, X is a halogen;

[0018] S4. Under alkaline conditions, the compound represented by formula (2) and the compound represented by formula (5) are contacted with a solvent to carry out an aldol condensation reaction to obtain a compound represented by formula (6). The reaction formula is as follows:

[0019]

[0020] S5. Under alkaline conditions, the compound represented by formula (6), the compound represented by formula (7), a condensing agent and a solvent are contacted to carry out an acid-ammonium condensation reaction to obtain a compound represented by formula (I), and the reaction formula is as follows:

[0021]

[0022] Wherein, the compound represented by formula (7) is a nitrogen-containing compound, preferably any one of morpholine, pyrrolidine, piperidine, N-(2-hydroxyalkyl)piperazine, N-alkylpiperazine, 1,2,3,4-tetrahydroisoquinoline, N-alkyl-1-phenylalkylamine, 4-hydroxypiperidine, and piperazine-1-carboxylic acid alkyl ester; preferably, the alkyl group in N-(2-hydroxyalkyl)piperazine, N-alkylpiperazine, N-alkyl-1-phenylalkylamine, and piperazine-1-carboxylic acid alkyl ester is a C1-C4 alkyl group, preferably a C1-C2 alkyl group; and the definition of R is the same as that of claim 1.

[0023] According to a specific embodiment of the present invention, in step S1, the molar ratio of pterostilbene to the methyl etherification reagent is 1:2-4; preferably, the base includes sodium hydroxide, and the solvent includes N,N-dimethylformamide; preferably, the reaction temperature is 40-80° C., and the reaction time is 4-8 h.

[0024] According to a specific embodiment of the present invention, in step S2, the molar ratio of the compound represented by formula (1) to the acidic chloride is 1:5-10; preferably, the formylating agent includes N,N-dimethylformamide; preferably, the reaction temperature is 30-60° C., and the reaction time is 1-2 h.

[0025] According to a specific embodiment of the present invention, in step S3, the molar ratio of the compound represented by formula (4) to the compound represented by formula (3) is 1:2 to 4; preferably, the base includes sodium hydroxide, and the solvent includes N,N-dimethylformamide; preferably, the reaction temperature is 40 to 80° C., and the reaction time is 2 to 4 hours.

[0026] According to a specific embodiment of the present invention, in step S4, the molar ratio of the compound represented by formula (2) to the compound represented by formula (5) is 1:2-4; preferably, the base includes pyrrolidine, and the solvent includes anhydrous ethanol; preferably, the reaction temperature is 30-60°C, and the reaction time is 24-36h.

[0027] According to a specific embodiment of the present invention, in step S5, the molar ratio of the compound represented by formula (6) to the compound represented by formula (7) is 1:2.5-5; preferably, the base includes N,N-diisopropylethylamine, the condensing agent includes 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the solvent includes N,N-dimethylformamide; preferably, the reaction temperature is 30-60°C, and the reaction time is 4-8h.

[0028] The third aspect of the present invention provides a pharmaceutical preparation, which is any pharmaceutically acceptable dosage form prepared with the aforementioned pterostilbene paeonol acetic acid compound as the sole active ingredient or the main active ingredient and a pharmaceutically acceptable carrier; preferably, the dosage form of the pharmaceutical preparation is any one of tablets, pills, capsules, powders, syrups, liquids, suspensions, freeze-dried powder injections or injections, and nano preparations.

[0029] The fourth aspect of the present invention provides use of the aforementioned pterostilbene-paeonol acetic acid compound or pharmaceutical preparation in the preparation of an anti-tumor drug; preferably, the aforementioned tumor is any one of osteosarcoma, lung cancer, and liver cancer.

[0030] The beneficial effects of the present invention are:

[0031] The present invention designs and synthesizes a series of pterostilbene paeonol acetic acid compounds with novel structures. By methoxylating the phenolic hydroxyl group in the pterostilbene structure, the antioxidant stability is improved. By introducing an aldehyde group and combining it with paeonol acetic acid to form chalcone derivatives, the anti-tumor activity is enhanced. The compounds then form amide structures with different organic amines. The compounds of the present invention can regulate the apoptosis of tumor cells and the expression of related proteins, thereby exerting excellent anti-tumor effects. Among them, compound I1 has an IC of 1.5 for U-2OS cells. 50 The value reached 6.10±1.11 μmol / L. At 25 μmol / L, the compound achieved an inhibition rate of approximately 40% against HDAC1 and HDAC2, significantly superior to the positive control, demonstrating significant anti-tumor activity. The compounds of this invention can be used to treat a variety of tumor diseases, including osteosarcoma, lung cancer, and liver cancer, and have potential anti-tumor research value and application prospects. Furthermore, the methods used to prepare these compounds feature readily available raw materials, simple operation, and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic structural diagram of the pterostilbene paeonol acetic acid compound of the present invention;

[0033] Figure 2 Inhibition rates of compounds I1, I7, and chidamide on HDAC1;

[0034] Figure 3 is the inhibition rate of compounds I1, I7 and chidamide on HDAC2. DETAILED DESCRIPTION

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0036] Example 1: Synthesis of Compound I1

[0037] Synthesis of (E)-1,3-dimethoxy-5-(4-methoxyphenyl)benzene (Compound 1):

[0038]

[0039] Pterostilbene (5.00 g, 19.53 mmol) was added to a clean 100 mL round-bottom flask, followed by the addition of DMF (30 mL) and sodium hydroxide (1.56 g, 39.06 mmol) and stirring for 15 min. Dimethyl sulfate (3.70 mL, 39.06 mmol) was then added thereto. After reacting at 60°C for 4 h, the reaction was essentially complete as monitored by TLC. The reaction solution was poured into 300 mL of water and extracted with ethyl acetate, washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product, which was purified by column chromatography (DCM:PE = 1:3) to obtain 4.68 g of a white product, namely, compound 1, with a yield of 88.75%.

[0040] Characterization data of compound 1: HRMS (m / z): 271.1283 [M+H] + (Theoretical value: 271.1256); 1 H NMR (600MHz, Chloroform-d) δ7.45 (d, J = 8.2 Hz, 2H), 7.04 (d, J = 16.3 Hz, 1H), 6.94–6.88 (m, 3H), 6.65 (d, J = 2.2 Hz, 2H), 6.38 (t, J = 2.1 Hz, 1H), 3.83 (s, 9H).

[0041] Synthesis of (E)-2,4-dimethoxy-6-(4-methoxyphenyl)benzaldehyde (Compound 2):

[0042]

[0043] Compound 1 (4 g, 14.81 mmol) was added to a clean 100 mL round-bottom flask, followed by DMF (20 mL). The mixture was cooled to below 0°C in an ice-salt bath and phosphorus oxychloride (5.20 mL, 74.07 mmol) was slowly added dropwise. After the addition was complete, the temperature was raised to 40°C to initiate the reaction. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was slowly added dropwise to ice water, and the pH of the reaction solution was adjusted to neutral with NaHCO3. The crude product was filtered and recrystallized from ethyl acetate to obtain 2.17 g of a yellow solid, i.e., compound 2, with a yield of 49.17%.

[0044] Characterization data of compound b: HRMS (m / z): 299.1237 [M+H] + (Theoretical value: 299.1205); 1 H NMR(600MHz,Chloroform-d)δ10.53(d,J=5.5Hz,1H),7.53–7.47(m,2H),7.25(d,J=3.6Hz,1H),6.96(d,J=8.0 Hz,1H),6.92–6.86(m,2H),6.75(d,J=7.7Hz,1H),6.38(d,J=7.1Hz,1H),3.93–3.87(m,6H),3.85–3.81(m,3H).

[0045] Synthesis of 3-methoxy-5-acetyl-phenoxyacetic acid (Compound 5):

[0046]

[0047] Paeonol (5 g, 30.12 mmol) was added to a clean 100 mL round-bottom flask, and then DMF (20 mL) and NaOH (7.23 g, 180.72 mmol) were added to the reaction flask and stirred for 15 min. Bromoacetic acid (8.37 g, 60.24 mmol) was then added thereto. After reacting at 60°C for 2 h, the reaction was basically completed as monitored by TLC. The reaction solution was poured into ice water, and the pH was adjusted to 1-2 with dilute hydrochloric acid. The solution was filtered and recrystallized from anhydrous ethanol to obtain 3.92 g of a white solid, i.e., compound (5), with a yield of 58.10%.

[0048] Characterization data of compound (5): 1 H NMR(600MHz,Chloroform-d)δ7.80(d,J=8.8Hz,1H),6.64(d,J=8.1Hz,1H),6.46(d ,J=2.6Hz,1H),4.73(d,2H),3.88(s,3H),2.61(d,3H).HRMS(m / z): 225.0692[M+H] + (Theoretical value: 225.0685).

[0049] Synthesis of 2-(2-((E)-3-(2,4-dimethoxy-6-((E)-4-methoxyphenyl)phenyl)acryloyl)-5-methoxyphenoxy)acetic acid (Compound 6):

[0050]

[0051] Compound (2) (2 g, 6.71 mmol) was added to a clean 50 mL round-bottom flask, and then anhydrous ethanol (10 mL), compound (5) (2.25 g, 10.07 mmol), and pyrrolidine (1.11 mL, 13.42 mmol) were added to the reaction flask in sequence. After reacting at 40°C for 24 h, the reaction was basically completed as monitored by TLC. The reaction solution was poured into ice water, and the pH was adjusted to 1-2 with dilute hydrochloric acid. The solution was filtered and recrystallized from ethyl acetate to obtain 3.14 g of a yellow solid, i.e., compound (6), with a yield of 92.85%.

[0052] Characterization data of compound (6): 1 H NMR(600MHz,Chloroform-d)δ8.19(d,J=15.7Hz,1H),7.70–7.64(m,1H),7.48–7 .43(m,2H),7.37(d,J=15.7Hz,1H),7.29(d,J=16.0Hz,1H),6.96–6.91(m,2H),6. 91(d,J=2.2Hz,1H),6.72(d,J=2.4Hz,1H),6.56–6.52(m,2H),6.44(d,J=2.4Hz, 1H),4.79(s,2H),3.91(s,6H),3.85(d,J=2.2Hz,6H).HRMS(m / z):505.1798[M+H] + (Theoretical value: 505.1784).

[0053] Synthesis of compound I1:

[0054]

[0055] Morpholine (0.10 mL, 0.10 mmol) was added to a clean 25 mL round-bottom flask, followed by DMF (3 mL), and then compound (d) (0.20 g, 0.40 mmol), HATU (33.32 g, 0.87 mmol), and DIPEA (11.33 mg, 0.87 mmol) were added in that order. The reaction was stirred at 40°C and monitored by TLC until completion. The reaction solution was poured into water, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. The filtrate was filtered and recovered to obtain a yellow oil. Column chromatography (DCM:MeOH=60:1) gave 0.12 g of a yellow solid, i.e., compound I1, with a yield of 52.36% and mp 116.8-117.4°C.

[0056] Characterization data of compound I1: 1 H NMR(400MHz,Chloroform-d)δ7.99(d,J=15.8Hz,1H),7.69(d,J=8.5Hz,1H),7.42(d, J=8.1Hz,2H),7.33(d,J=30.3Hz,1H),7.26(d,J=2.4Hz,1H),6.91(d,J=11.7Hz,2H),6 .88(s,1H),6.71(d,J=2.4Hz,1H),6.60–6.49(m,2H),6.42(d,J=2.4Hz,1H),4.56(s,2 H),3.89(s,3H),3.86(s,3H),3.83(s,3H),3.82(s,3H),3.48(dt,J=15.4,5.4Hz,8H). 13 C NMR (125MHz, CDCl3) δ191.27,166.58,166.47,165.73,161.51,159.92,15 6.73,137.84,135.46,130.59,129.71,129.26,125.76,124.49,122.78,1 20.35,118.93,113.28,109.68,109.30,102.11,99.09,66.73,64.35,64. 33,55.21,55.10,54.64,54.32,43.29,43.27.HRMS(m / z):574.2375[M+H] + (Theoretical value: 574.2363).

[0057] Example 2: Synthesis of Compound I2

[0058]

[0059] The preparation method is the same as that of Example 1, except that morpholine in Example 1 is replaced by pyrrolidine to obtain compound I2.

[0060] Characterization data of compound Ⅰ2: 1 H NMR(400MHz,Chloroform-d)δ8.02(d,J=15.8Hz,1H),7.72(d,J=8.7Hz,1H),7.50–7.43(m,2H),7.42 (d,J=2.1Hz,1H),7.30(d,J=16.0Hz,1H),6.95–6.88(m,2H),6.87(s,1H),6.71(d,J=2.4Hz,1H),6.5 5(dd,J=8.7,2.2Hz,1H),6.48(d,J=2.2Hz,1H),6.41(d,J=2.3Hz,1H),4.45(s,2H),3.89(s,3H),3.8 6(s,3H),3.83(s,3H),3.81(s,3H),3.37(t,J=6.9Hz,2H),3.31(t,J=6.7Hz,2H),1.80–1.67(m,4H). 13 C NMR (125MHz, CDCl3) δ191.29,166.89,166.77,165.73,160.51,159.38,15 6.83,138.84,134.46,129.59,128.71,128.46,125.76,124.39,123.78,1 20.75,118.44,113.36,110.38,110.80,103.11,99.05,66.25,54.91,54. 73,54.64,54.32,45.43,45.58,24.86,22.47.HRMS(m / z):558.2436[M+H] + (Theoretical value: 558.2414).

[0061] Example 3: Synthesis of Compound I3

[0062]

[0063] The preparation method is the same as that of Example 1, except that the morpholine in Example 1 is replaced by piperidine to obtain compound I3.

[0064] Characterization data of compound Ⅰ3: 1H NMR(400MHz,Chloroform-d)δ8.02(d,J=15.8Hz,1H),7.73(d,J=8.5Hz,1H),7.50–7.43(m,2H),7.42( d,J=2.1Hz,1H),7.30(d,J=16.0Hz,1H),6.92(d,J=16.1Hz,2H),6.88(d,J=2.0Hz,1H),6.71(d,J=2.3H z,1H),6.57–6.51(m,2H),6.42(d,J=2.3Hz,1H),4.52(s,2H),3.89(s,3H),3.86(s,3H),3.83(s,3H),3 .81(s,3H),3.40(t,J=5.5Hz,2H),3.30(t,J=5.5Hz,2H),1.48(d,J=5.1Hz,2H),1.38(d,J=7.0Hz,4H). 13 C NMR (125MHz, CDCl3) δ191.27,166.57,166.17,165.82,160.21,159.64,156 .25,138.69,134.51,129.73,128.62,128.45,125.66,124.83,123.71,120. 69,118.49,113.29,110.25,110.05,103.61,99.41,66.35,54.74,54.58,5 4.37,54.28,44.59,44.53,24.63,24.61,22.25.HRMS(m / z):572.2593[M+H] + (Theoretical value: 572.2570).

[0065] Example 4: Synthesis of Compound I4

[0066]

[0067] The preparation method is the same as that of Example 1, except that the morpholine in Example 1 is replaced by N-(2-hydroxyethyl)piperazine to obtain Compound I4.

[0068] Characterization data of compound Ⅰ4: 1H NMR(400MHz,Chloroform-d)δ8.00(d,J=15.8Hz,1H),7.70(d,J=8.6Hz,1H),7.47(s,1H),7.43(d,J=1.8Hz,1H),7.41 (d,J=2.1Hz,1H),7.33–7.27(m,1H),6.94–6.88(m,2H),6.88(s,1H),6.71(d,J=2.4Hz,1H),6.56(dd,J=8.6,2.2Hz,1H ),6.51(d,J=2.2Hz,1H),6.42(d,J=2.3Hz,1H),4.55(s,2H),3.89(s,3H),3.86(s,3H),3.83(s,3H),3.82(s,3H),3.59 (q,J=5.8,5.3Hz,1H),3.51(q,J=4.5,3.8Hz,4H),3.44(t,J=4.9Hz,2H),2.39(t,J=5.3Hz,2H),2.31(p,J=4.8Hz,4H). 13 C NMR (125MHz, CDCl3) δ191.85,166.95,166.87,165.69,161.13,159.47,156.6 2,138.75,134.52,129.77,128.63,128.36,125.87,124.71,123.64,120.81, 118.55,113.47,110.64,110.35,103.29,99.15,66.37,58.71,57.61,54.85, 54.73,54.64,54.19,50.56,50.53,43.42,43.38.HRMS(m / z):617.2792[M+H] + (Theoretical value: 617.2785).

[0069] Example 5: Synthesis of Compound I5

[0070]

[0071] The preparation method is the same as that of Example 1, except that the morpholine in Example 1 is replaced by N-methylpiperazine to obtain Compound I5.

[0072] Characterization data of compound Ⅰ5: 1H NMR(400MHz,Chloroform-d)δ8.02(d,J=15.8Hz,1H),7.71(d,J=8.5Hz,1H),7.47(d,J=15.9Hz,1H ),7.43(d,J=8.2Hz,2H),7.30(d,J=15.8Hz,1H),6.95–6.86(m,3H),6.71(d,J=2.4Hz,1H),6.56(d d,1H),6.51(d,J=2.3Hz,1H),6.41(d,J=2.3Hz,1H),4.54(s,2H),3.89(s,3H),3.86(s,3H),3.83( s,3H),3.81(s,3H),3.52(t,J=5.0Hz,2H),3.43(t,J=5.0Hz,2H),2.26–2.17(m,4H),2.15(s,3H). 13 C NMR (125MHz, CDCl3) δ191.35,166.72,166.84,165.65,160.82,159.27,156 .73,138.93,134.58,129.58,128.65,128.55,125.73,124.47,123.86,120. 72,118.38,113.40,110.42,110.74,103.29,99.15,66.06,54.91,54.70,5 4.64,54.32,54.15,54.12,45.59,45.45,44.17.HRMS(m / z):587.2693[M+H] + (Theoretical value: 587.2679).

[0073] Example 6: Synthesis of Compound I6

[0074]

[0075] The preparation method is the same as that of Example 1, except that the morpholine in Example 1 is replaced by N-ethylpiperazine to obtain Compound I6.

[0076] Characterization data of compound Ⅰ6: 1H NMR(400MHz,Chloroform-d)δ8.02(d,J=15.9Hz,1H),7.71(d,J=8.6Hz,1H),7.48(d,J=15.9Hz,1H),7 .45–7.40(m,2H),7.33–7.27(m,1H),6.94–6.86(m,4H),6.71(d,J=2.4Hz,1H),6.56(dd,J=8.6,2.2Hz ,1H),6.51(d,J=2.2Hz,1H),6.41(d,J=2.3Hz,1H),4.54(s,2H),3.88(s,3H),3.86(s,3H),3.83(s,3H ),3.81(s,4H),3.52(t,J=5.2Hz,2H),3.43(t,J=5.0Hz,2H),2.30–2.19(m,4H),0.98(t,J=7.2Hz,3H). 13 C NMR (125MHz, CDCl3) δ191.85,166.38,166.77,165.68,160.35,159.28,15 6.73,138.76,134.39,129.68,128.73,128.66,125.71,124.27,123.53,1 20.75,118.64,113.56,110.33,110.65,100.26,99.15,66.36,54.81,54. 72,54.54,54.32,51.42,51.07,45.42,12.15.HRMS(m / z):601.2851[M+H] + (Theoretical value: 601.2836).

[0077] Example 7: Synthesis of Compound I7

[0078]

[0079] The preparation method is the same as that of Example 1, except that the morpholine in Example 1 is replaced by 1,2,3,4-tetrahydroisoquinoline to obtain compound I7.

[0080] Characterization data of compound Ⅰ7: 1H NMR(400MHz,Chloroform-d)δ8.03(dd,J=15.8,8.0Hz,1H),7.78–7.70(m,1H),7.54–7.27(m,4H),7.18–7.03(m,3H),7.02–6.77(m,4H),6.69(dd,J=1 1.2,2.3Hz,1H),6.61–6.49(m,2H),6.43–6.31(m,1H),4.69–4.40(m,4H),3 .96–3.86(m,3H),3.86–3.69(m,9H),3.69–3.59(m,2H),2.82–2.61(m,2H). 13 C NMR (125MHz, CDCl3) δ192.15,166.73,166.68,165.79,162.17,159.69,156.73,138. 94,134.59,132.87,130.42,129.37,128.86,128.58,125.71,124.51,124.42,124.3 2,124.07,123.43,122.71,120.88,118.52,113.46,110.32,110.73,103.14,99.06, 66.18,54.87,54.65,54.62,54.28,50.23,45.72,29.89.HRMS(m / z): 620.2692[M+H] + (Theoretical value: 620.2570).

[0081] Example 8: Synthesis of Compound I8

[0082]

[0083] The preparation method is the same as that of Example 1, except that the morpholine in Example 1 is replaced by N-methyl-1-phenylmethanamine to obtain compound I8.

[0084] Characterization data of compound Ⅰ8: 1H NMR(400MHz,Chloroform-d)δ8.01(dd,J=22.3,15.8Hz,1H),7.73(dd,J=8.6,1.2Hz,1H),7.54–7.27(m,4H) ,7.25–7.19(m,3H),7.10(dd,J=6.7,2.9Hz,1H),6.99–6.92(m,1H),6.92–6.78(m,3H),6.73–6.65(m,1H),6 .60–6.52(m,1H),6.50(d,J=2.3Hz,1H),6.41(t,J=2.6Hz,1H),4.55(d,J=13.9Hz,2H),4.44(d,J=6.6Hz,2H ),3.89(d,J=1.3Hz,3H),3.87–3.81(m,6H),3.80(d,J=2.8Hz,3H),2.80(t,J=6.4Hz,3H),0.92–0.80(m,1H). 13 C NMR (125MHz, CDCl3) δ191.30,166.65,166.53,165.72,161.52,159.32,156.81 ,139.77,138.81,134.52,129.54,128.71,128.54,125.68,125.14,124.35,12 4.17,124.06,123.78,120.71,118.36,113.73,110.46,110.92,103.12,99.27 ,66.57,54.81,54.70,54.64,54.32,52.18,32.49.HRMS(m / z): 608.2593[M+H] + (Theoretical value: 608.2570).

[0085] Example 9: Synthesis of Compound I9

[0086]

[0087] The preparation method is the same as that of Example 1, except that the morpholine in Example 1 is replaced by 4-hydroxypiperidine to obtain compound I9.

[0088] Characterization data of compound Ⅰ9: 1H NMR(400MHz,Chloroform-d)δ8.01(d,J=15.8Hz,1H),7.72(d,J=8.6Hz,1H),7.50–7.42(m,2H),7.42(t,J=2.1Hz,1 H),7.29(d,J=16.0Hz,1H),6.95–6.88(m,2H),6.88(t,J=2.1Hz,1H),6.71(d,J=2.4Hz,1H),6.56(dd,J=8.6,2.2Hz ,1H),6.51(d,J=2.2Hz,1H),6.42(d,J=2.4Hz,1H),4.53(s,2H),3.92(d,J=5.1Hz,1H),3.89(s,3H),3.86(s,3H),3 .83(s,3H),3.81(s,3H),3.78–3.71(m,1H),3.17–3.05(m,2H),1.75–1.66(m,2H),1.59(s,2H),1.37–1.26(m,2H). 13 C NMR (125MHz, CDCl3) δ191.76,166.25,166.13,165.69,160.51,159.42,156.73,138.92,134.55,129.68,128.82,128.52,125.81,124.25,123.8 3,120.57,118.51,113.39,110.45,110.71,103.16,99.07,66.05,64.2 3,54.71,54.60,54.44,54.32,41.02,32.03.HRMS(m / z):588.2537[M+H] + (Theoretical value: 588.2519).

[0089] Example 10: Compound I 10 Synthesis

[0090]

[0091] The preparation method is the same as that in Example 1, except that the morpholine in Example 1 is replaced by tert-butyl piperazine-1-carboxylate to obtain compound I 10 .

[0092] Compound Ⅰ 10 Characterization data: 1H NMR(400MHz,Chloroform-d)δ8.01(d,J=15.8Hz,1H),7.71(d,J=8.6Hz,1H),7.46–7.43(m,1H), 7.43–7.36(m,2H),7.29(d,J=12.0Hz,1H),6.95–6.89(m,2H),6.89(d,J=1.9Hz,1H),6.72(d,J=2 .4Hz,1H),6.57(dd,J=8.6,2.0Hz,1H),6.51(s,1H),6.42(d,J=2.4Hz,1H),4.56(s,2H),3.90(s ,3H),3.87(s,3H),3.84(s,3H),3.82(s,3H),3.48–3.35(m,4H),3.33–3.22(m,4H),1.44(s,9H). 13 C NMR (125MHz, CDCl3) δ191.36,166.50,166.41,165.72,160.51,159.29,156 .73,152.82,138.76,134.42,129.65,128.61,128.36,125.76,124.25,123. 66,120.74,118.41,113.28,110.34,110.82,103.11,99.05,76.41,66.06,5 4.51,54.46,54.24,54.12,44.54,43.97,26.39.HRMS(m / z):673.3071[M+H] + (Theoretical value: 673.3047).

[0093] Experimental example: Anti-tumor activity study

[0094] 1. In vitro anti-tumor cell activity experiment

[0095] The MTT assay was used to evaluate the in vitro antitumor activity of the target compounds against human hepatocellular carcinoma cells HepG2, human non-small cell lung cancer cells A549, and human osteosarcoma cells U-2OS, with the lead compounds pterostilbene, 5-fluorouracil, and cisplatin as positive controls. All three cell types in the logarithmic growth phase were seeded into 96-well plates at a cell density of approximately 3,000 cells per well. Drug concentrations were set at 6.25, 12.5, 25, 50, and 100 μmol / L. After 24 hours of exposure, 10 mL of MTT solution was added to each well and the cells were incubated under the same conditions for approximately 2 hours. Finally, the absorbance (OD value) at 450 nm was measured using a microplate reader. Each experiment was repeated three times, and each experiment was independent of each other.

[0096]

[0097] After calculating the inhibition rate corresponding to different concentrations of compounds using the formula, the inhibition curve was fitted using Origin software to obtain IC 50 IC 50 The values ​​are presented in Table 2 as mean ± standard deviation.

[0098] Table 2 Target compounds Ⅰ1~I 10 IC for three cell types 50 value

[0099]

[0100]

[0101] The experimental results showed that most of the target compounds showed good inhibitory activity against the selected tumor cells, and human osteosarcoma cells U-2OS cells were more sensitive to most of the target compounds, indicating that the target compounds had better inhibitory effects on U-2OS cells. The anti-tumor activity of compound I1 was significantly better than that of other compounds and pterostilbene, with an IC of 0.05 for HepG2, U-2OS, and A549 cells. 50 The values ​​were 17.02, 6.10, and 7.31 μM, respectively. Compared with compound I3, compound I7 had better inhibitory activity against the three cell lines than the lead compound pterostilbene, with the best inhibitory activity against human osteosarcoma cell U-2OS, IC 50 The value is 7.08 μM, indicating that the introduction of benzene ring can enhance the activity of the compound.

[0102] Therefore, compounds I1 and I7 were selected for in-depth in vitro antitumor activity studies.

[0103] 2. Histone deacetylase (HDAC) inhibition experiment

[0104] Adherent U-2OS cells in the logarithmic growth phase were plated at a cell suspension density of 1 × 10 / mL in six-well plates. Treatment groups with different concentrations and a negative control group were set up. After 24 hours of inoculation and complete cell attachment, the medium was aspirated and the drug was administered. After 24 hours of incubation, the medium was aspirated and washed with PBS. The adherent cells were then digested to a single-cell suspension using trypsin, and digestion was terminated by adding culture medium. The cell solution was then added to an EP tube, centrifuged, and the supernatant discarded. The cells were then washed once with pre-chilled PBS. Finally, 100 μL of lysis buffer was added, and the cells were sonicated for 30 seconds. The supernatant was then centrifuged and collected. Intracellular protein samples were obtained and stored at -20°C.

[0105] A human histone deacetylase enzyme-linked immunosorbent assay (ELISA) kit was used, with blank wells, negative control wells, standard wells, and dosing wells set up. After incubation, the OD value at a wavelength of 450 nm was measured using a microplate reader. A standard curve was generated using the standard sample, and the HDAC concentrations of the different dosing groups were calculated. Each experiment was repeated three times, and each experiment was independent of each other. The deacetylase activity inhibition rate was calculated using the following formula.

[0106]

[0107] HDACs are a class of proteases that play a crucial role in chromatin structural modification and gene expression regulation. In tumor cells, overexpression of HDACs leads to enhanced deacetylation, which is detrimental to the expression of specific genes, including some tumor suppressor genes. By inhibiting HDAC activity, apoptosis and the expression of related proteins are regulated, achieving anti-tumor effects. Using chidamide as a positive control, an enzyme-linked immunosorbent assay (ELISA) was used to calculate the concentrations of HDAC1 and HDAC2 in the groups treated with different concentrations of compounds I1 and I7. The inhibition rates of the groups treated with different concentrations of compounds I1 and I7 were also calculated.

[0108] The results are as follows Figure 2 and Figure 3 As shown in the figure, when the concentration reaches 25 μmol / L, the inhibition rate of compound I1 on HDAC1 and HDAC2 can reach about 40%, the inhibitory activity is equivalent to that of the positive control drug chidamide, and there is a dose-effect relationship between the inhibition rate and the concentration range.

[0109] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. Pterostilbene paeonol acetic acid compound represented by general formula (I); Formula (I); in, R is selected from morpholinyl, pyrrolidinyl, N -(2-hydroxyalkyl)piperazinyl, N -methylpiperazinyl, 1,2,3,4-tetrahydroisoquinolinyl, N -Any one of alkyl-1-phenylalkylamino and 4-hydroxypiperidinyl; described N -(2-hydroxyalkyl)piperazinyl, N The alkyl group in the -alkyl-1-phenylalkylamino group is a C1~C4 alkyl group.

2. The pterostilbene paeonol acetic acid compound according to claim 1, characterized in that described N -(2-hydroxyalkyl)piperazinyl, N The alkyl group in the -alkyl-1-phenylalkylamino group is a C1~C2 alkyl group.

3. A method for preparing the pterostilbene paeonol acetic acid compound according to claim 1, characterized in that: The following steps are involved: S1. Under alkaline conditions, pterostilbene, a methyl etherification agent and a solvent are contacted to perform a methyl etherification reaction to obtain a compound represented by formula (1). The reaction formula is as follows: ； S2. In an ice bath, a formylating agent and an acid chloride are contacted with the compound represented by formula (1) to carry out a Vilsmeier-Haack reaction to obtain a compound represented by formula (2). The reaction formula is as follows: ； S3. Under alkaline conditions, the compound represented by formula (3) and the compound represented by formula (4) are contacted with a solvent to carry out a nucleophilic substitution reaction to obtain a compound represented by formula (5). The reaction formula is as follows: Wherein, X is a halogen; S4. Under alkaline conditions, the compound represented by formula (2) and the compound represented by formula (5) are contacted with a solvent to carry out an aldol condensation reaction to obtain a compound represented by formula (6). The reaction formula is as follows: ； S5. Under alkaline conditions, the compound represented by formula (6), compound (7), a condensing agent and a solvent are contacted to carry out an acid-amine condensation reaction to obtain a compound represented by formula (I), and the reaction formula is as follows: ； Among them, compound (7) is morpholine, pyrrolidine, N -(2-hydroxyalkyl)piperazine, N -Methylpiperazine, 1,2,3,4-tetrahydroisoquinoline, N -alkyl-1-phenylalkylamine, 4-hydroxypiperidine, any one of the N -(2-hydroxyalkyl)piperazine, N The alkyl group in the -alkyl-1-phenylalkylamine is a C1~C4 alkyl group; the definition of R is the same as that in claim 1.

4. The preparation method according to claim 3, characterized in that described N -(2-hydroxyalkyl)piperazine, N The alkyl group in the -alkyl-1-phenylalkylamine is a C1~C2 alkyl group.

5. The preparation method according to claim 3, characterized in that In step S1, the molar ratio of pterostilbene to the methyl etherification reagent is 1:2-4.

6. The preparation method according to claim 3, characterized in that In step S1, the base is sodium hydroxide and the solvent is N,N -dimethylformamide.

7. The preparation method according to claim 3, characterized in that In step S1, the reaction temperature is 40-80°C, and the reaction time is 4-8 hours.

8. The preparation method according to claim 3, characterized in that In step S2, the molar ratio of the compound represented by formula (1) to the acidic chloride is 1:5-10.

9. The preparation method according to claim 3, characterized in that In step S2, the formylating agent is N , N -dimethylformamide.

10. The preparation method according to claim 3, characterized in that In step S2, the reaction temperature is 30-60° C., and the reaction time is 1-2 h.

11. The preparation method according to claim 3, characterized in that In step S3, the molar ratio of the compound represented by formula (4) to the compound represented by formula (3) is 1:2-4.

12. The preparation method according to claim 3, characterized in that In step S3, the base is sodium hydroxide and the solvent is N , N -dimethylformamide.

13. The preparation method according to claim 3, characterized in that In step S3, the reaction temperature is 40-80° C., and the reaction time is 2-4 h.

14. The preparation method according to claim 3, characterized in that In step S4, the molar ratio of the compound represented by formula (2) to the compound represented by formula (5) is 1:2-4.

15. The preparation method according to claim 3, characterized in that In step S4, the base is pyrrolidine, and the solvent is anhydrous ethanol.

16. The preparation method according to claim 3, characterized in that In step S4, the reaction temperature is 30-60° C., and the reaction time is 24-36 h.

17. The preparation method according to claim 3, characterized in that In step S5, the molar ratio of the compound represented by formula (6) to the compound (7) is 1:2.5-5.

18. The preparation method according to claim 3, characterized in that In step S5, the base is N , N -diisopropylethylamine, the condensing agent is 2-(7-azabenzotriazole)- N , N , N' , N' -tetramethyluronium hexafluorophosphate, the solvent is N , N -dimethylformamide.

19. The preparation method according to claim 3, characterized in that: In step S5, the reaction temperature is 30-60° C., and the reaction time is 4-8 h.

20. A pharmaceutical preparation, characterized in that: The invention relates to a pharmaceutically acceptable dosage form prepared with the pterostilbene paeonol acetic acid compound described in claim 1 or 2 as the main active ingredient and a pharmaceutically acceptable carrier. The dosage form of the pharmaceutical preparation is any one of tablets, pills, capsules, powders, syrups, suspensions and injections.

21. Use of the pterostilbene paeonol acetic acid compound according to claim 1 or 2 or the pharmaceutical preparation according to claim 20 in the preparation of an anti-tumor drug, characterized in that: The R of the compound is a morpholinyl group, N -(2-hydroxyalkyl)piperazinyl, 1,2,3,4- or tetrahydroisoquinolinyl, the tumor is any one of osteosarcoma, lung cancer, and liver cancer; When R of the compound is pyrrolidino, the tumor is any one of osteosarcoma and lung cancer; The R of the compound is N -methylpiperazinyl or 4-hydroxypiperidine, the tumor is any one of lung cancer and liver cancer; The R of the compound is N -alkyl-1-phenylalkylamino, the tumor is lung cancer.

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