3 alpha-O-acylated glaucocalyxin A derivative as well as preparation method and application thereof

Abstract

The embodiment of the invention discloses a 3 alpha-O-acylated glaucocalyxin A derivative as well as a preparation method and application thereof. The 3 alpha-O-acylated glaucocalyxin A derivative is characterized in that the 3 alpha-O-acylated glaucocalyxin A derivative has a structure as shown in a formula (I). The novel glaucocalyxin A derivative provided by the invention shows remarkable anti-tumor activity and has the potential of being developed into an anti-tumor drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to a 3alpha-O-acylated oridonin derivative, a preparation method and application thereof. BACKGROUND

[0002] Oridonin is a lead compound with significant anti-tumor activity, and its anti-tumor mechanism involves inducing cell apoptosis and inhibiting proliferation. However, the compound faces many challenges in clinical application, such as low water solubility, fast metabolism and low bioavailability. Current optimization researches on oridonin mainly focus on two directions: preparation technology and structural modification. The preparation technology mainly improves the solubility and bioavailability through strategies such as nano-carrier loading and inclusion compound formation, so as to systematically improve the pharmacokinetic characteristics. The structural modification researches mainly focus on 7 / 14-OH site modification, A ring introduction of heterocycle and D ring Michael addition. Researchers modify the structure of oridonin through various strategies: Yang introduces amine group into the D ring through Mannich reaction to prepare Mannich base derivative, and the activity of the prepared derivative is equivalent to or higher than that of oridonin; Zhang introduces N-alkyl amino thiazole group into the A ring of oridonin to modify, and the anti-tumor activity is significantly enhanced; Xiang performs acid alcohol esterification substitution on 7-OH or 14-OH of oridonin to synthesize six acid alcohol esterification derivatives, but the activity is not significantly improved; Xiang prepares 7, 14-di-glucoside derivative of oridonin through glycosylation modification. The results show that glycosylation can improve the cytotoxic activity while retaining the alpha-methylene cyclopentanone structure unit. Huang finds through biotin labeling that 7 / 14-OH biotinylation hinders the binding of the pharmacophore group to the corresponding protein receptor, resulting in reduced activity; Zhang introduces triazole fragments into 7, 14-OH, and the results show that under the premise of retaining the alpha, beta-unsaturated ketone pharmacophore group on the D ring, the introduction of 1, 2, 3-triazole aldehyde structure fragment substituted by meta-hydroxy or para-hydroxy phenyl can improve the anti-tumor activity of the lead compound oridonin.

[0003] Therefore, it is necessary to develop a new strategy for structural improvement of oridonin to break through the existing research framework and provide a new way for the optimization of drug property and clinical application of oridonin. SUMMARY

[0004] At present, the chemical conversion potential of the C-3 carbonyl group of oridonin has not been developed by the existing strategies. Therefore, the present application reduces the 3-position carbonyl group to hydroxyl group by taking oridonin as raw material, and then introduces ester bond through acylation with acyl chloride, which can obviously improve the solubility and other physical and chemical properties of the compound. This strategy is expected to break through the existing research framework and provide a new way for the optimization of drug property and clinical application of oridonin.

[0005] To achieve the above object, embodiments of the present application provide the following technical solutions:

[0006] According to a first aspect of embodiments of the present application, a 3α-O-acylated pluviatilols derivative has the structure shown in formula (I):

[0007]

[0008] wherein R is 2-chloroacetyl, hexanoyl, 3-chlorobenzoyl, 3,5-dichlorobenzoyl, propionyl, nonanoyl, 2,4-dichlorobenzoyl, 4-chlorobenzoyl, cinnamoyl, 3,4-dichlorobenzoyl, p-fluorobenzoyl, 3-bromobenzoyl, 2-bromobenzoyl, 4-bromobenzoyl, o-fluorobenzoyl, o-chlorobenzoyl, 3,5-difluorobenzoyl, 2,6-difluorobenzoyl, acryloyl, 2-thiophenecarbonyl, 3-methylcrotonoyl, 2-trifluorobenzoyl, butyryl or heptanoyl.

[0009] Further, R is 3,5-dichlorobenzoyl, 3,4-dichlorobenzoyl or o-fluorobenzoyl.

[0010] According to a second aspect of embodiments of the present application, a preparation method of the 3α-O-acylated pluviatilols derivative is provided, and a synthetic route is as follows:

[0011] The preparation method comprises the following steps:

[0012] (1) condensation reaction of pluviatilols and 2,2-dimethoxypropane in the presence of p-toluenesulfonic acid and acetone to obtain compound A-1;

[0013] (2) reduction reaction of compound A-1 and CeCl3·7H2O in the presence of NaBH4 and methanol to obtain compound A-26;

[0014] (3) acylation reaction of compound A-26 and R-Cl in the presence of triethylamine, DMAP and dichloromethane under nitrogen protection to obtain 3α-O-acyl-7,14-O-isopropylidenepluviatilols derivative, which is directly subjected to the next step without purification; hydrolysis reaction of 3α-O-acyl-7,14-O-isopropylidenepluviatilols derivative and hydrochloric acid in the presence of a solvent to obtain target compound D, i.e. the 3α-O-acylated pluviatilols derivative; wherein R in R-Cl is defined in claim 1.

[0015] Further, in step (1), the molar ratio of pluviatilols, 2,2-dimethoxypropane and p-toluenesulfonic acid is 1:(2-4):(0.03-0.1), and the condensation reaction temperature is 50-55°C.

[0016] Further, in step (2), the molar ratio of compound A-1, CeCl3·7H2O and NaBH4 is 1:3:(1.5-2), and the temperature of the reduction reaction is 0-25℃.

[0017] Further, in step (3), the molar ratio of compound A-26, R-Cl, triethylamine and DMAP is 1:(2-4):(8-10):(0.03-0.1), the temperature of the acylation reaction is 0-40℃, the concentration of the hydrochloric acid is 1M, the solvent is methanol and dichloromethane in a volume ratio of 1:1, and the temperature of the hydrolysis reaction is 15-25℃.

[0018] According to a third aspect of the embodiments of the present application, the present application provides a pharmaceutical composition comprising the 3α-O-acylated pluviatilols derivative as described above.

[0019] According to a fourth aspect of the embodiments of the present application, a pharmaceutical preparation comprises the 3α-O-acylated pluviatilols derivative as described above, and at least one pharmaceutically acceptable excipient or carrier.

[0020] According to a fifth aspect of the embodiments of the present application, the present application provides use of the 3α-O-acylated pluviatilols derivative as described above in the preparation of an antitumor drug.

[0021] Further, the tumor includes leukemia, cervical cancer, lung cancer and prostate cancer.

[0022] The embodiments of the present application have the following advantages:

[0023] (1) The present application realizes functional transformation of the C-3 carbonyl group of pluviatilol for the first time, reduces the C-3 carbonyl group to a hydroxyl group through stereoselective reduction, breaks through the limitation of 7 / 14-OH site modification in the prior research, constructs a new derivative library by coupling an ester bond with an aryl halogenated side chain or a fatty chain, and retains the active pharmacophore of the α-methylene cyclopentanone while improving the solubility and bioavailability by introducing an ester bond to improve the drug property.

[0024] (2) The 3α-O-acylated pluviatilols derivative provided by the present application has significantly improved activity, and the proliferation inhibition effect of most of the derivatives on tumor cells (Hela, K562, A549, HL-60 and LNCaP) is significantly better than that of the lead compound pluviatilol, and the half-inhibitory concentration (IC 50 value) of most of the derivatives is significantly lower than that of the positive control drug paclitaxel, wherein the derivatives D-4, D-10 and D-15 have the best activity on A549 cells, and the IC 50The values are (0.273 ± 0.018) μM, (0.299 ± 0.020) μM and (0.437 ± 0.041) μM, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only exemplary, and other drawings can be obtained by the provided drawings without creative labor for those skilled in the art.

[0026] Figure 1 A column chart of the inhibition rate of 3α-O-acylated glaucocalyxin B derivatives provided by the present application on cancer cells; DETAILED DESCRIPTION

[0027] The embodiments of the present application are described below by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the specification. Obviously, the described examples are part of the examples of the present application, not all. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0028] The reagents used in the present application are commercially available, generally purchased from reagent companies such as Adamas, Aladdin, Sigma-Aldrich, Acros, Greagent, Merck and TCI. All solvents used in synthesis are analytical grade, generally used directly without treatment, and anhydrous solvents are refluxed and evaporated after adding drying agent for standby. The raw material glaucocalyxin B (GLA) is extracted and separated from traditional Chinese medicinal material Wangzaozi. The traditional Chinese medicinal material Wangzaozi is purchased from Huizhou Pharmaceutical Material Market in Anhui Province, and is identified by Han Zhengbin of Suzhou Lv Yuan Chinese Medicine Technology Co., Ltd. as Isodonsuzhouensis of Isodon of Labiatae, and the specimen (Assrmyy 202108) is preserved in the pharmacy laboratory of Anshun People's Hospital.

[0029] Example 1: Preparation of 3α-O-acylated glaucocalyxin B derivatives (D-1~D-24)

[0030] Preparation of compound A-1

[0031] Compound GLA (7.33 g, 22.76 mmol, 1.0 eq) and p-toluenesulfonic acid (100 mg, 0.59 mmol, 0.03 eq) were weighed into a 500 mL dry round-bottom flask. Acetone (300 mL) was added and stirred to dissolve. 2,2-Dimethoxypropane (7.40 mL, 45.52 mmol, 2.0 eq) was then added. The mixture was refluxed at 55°C under nitrogen and progress was monitored by TLC. The reaction was complete after 24 hours. The solvent was partially removed by concentration under reduced pressure and then purified by recrystallization to obtain Compound A-1 (white powder, 7.1705 g) in a yield of 87.3%. 1 H NMR (500MHz, CDCl3) δ: 6.17 (1H, s), 5.40 (1H, s), 4.59 (1H, d, J = 1.4Hz), 4.21 (1H, dd, J = 12.7, 5.4 Hz),3.07(1H,s),2.55(1H,ddd,J=15.9,12.0,6.7Hz),2.39(1H,ddd,J=15.9,6.1,3.8Hz),2.09( 1H,q,J=12.7Hz),2.01–1.93(3H,m),1.88(1H,ddd,J=10.8,5.7,2.6Hz),1.62–1.54(4H,m),1.37 (1H,dd,J=12.4,2.2Hz),1.33–1.26(3H,m),1.24(3H,s),1.20(3H,s),1.11(3H,s),1.07(3H,s). 13 C NMR(125MHz, CDCl3)δ:215.7,205.6,146.5,117.4,97.5,71.1,70.7,54.7,51.5,51.4,4 7.2,43.2,38.4,38.0,34.1,31.2,30.5,28.4,26.1,25.6,21.6,18.2,17.0.ESI-MS:m / z 373[M+H] + (C 23 H 32 O).

[0032] Preparation of compound A-26

[0033] Compound A-1 (1.00 g, 2.69 mmol, 1.0 eq) was dissolved in methanol (150 mL) and then cerium chloride heptahydrate (3.00 g, 8.06 mmol, 3.0 eq) was added. Sodium borohydride (406.50 mg, 5.38 mmol, 2.0 eq) was added under nitrogen protection and ice bath, and the reaction was stirred at room temperature. TLC was used to monitor the progress of the reaction. After 3 hours, the starting material disappeared, and the reaction was quenched by adding acetone (20 mL). The solvent was concentrated under reduced pressure to obtain the crude product. The crude product was diluted with water (150 mL) and extracted with ethyl acetate (150 mL x 3). The combined organic phase was washed with saturated NaCl solution, dried over anhydrous Na2SO4, and concentrated. The product was purified by silica gel column chromatography (eluent: petroleum ether-ethyl acetate, V / V = 15:1-12:1) to obtain intermediate A-26 (white powder, 877.50 mg) in a yield of 87.3%. 1 H NMR (600 MHz, CDCl3) δ: 6.16 (1H, s), 5.38 (1H, s), 4.56 (1H, s), 4.18 (1H, dd, J = 12.7, 5.5 Hz), 3.20 (1H, dd, J = 11.6, 4.4 Hz), 3.04 (1H, s), 2.05 (1H, ddd, J = 12.7, 5.4, 1.7 Hz), 2.01–1.91 (2H, m), 1.86–1.81 (1H, m), 1.76 (1H, dt, J = 13.2, 3.4 Hz), 1.65 (1H, ddd, J = 11.8, 7.8, 3.8 Hz), 1.60 (3H, s), 1.58 (1H, s), 1.57–1.55 (1H, m), 1.52–1.44 (1H, m), 1.41 (1H, s), 1.24 (3H, s), 1.20 (1H, d, J = 8.9 Hz), 1.05 (3H, s), 1.01 (3H, s), 0.91–0.84 (2H, m), 0.82 (3H, s). 13 C NMR (150 MHz, CDCl3) δ: 206.2, 146.8, 117.2, 97.4, 78.6, 71.2, 54.9, 52.5, 51.2, 43.3, 38.9, 38.8, 37.7, 31.2, 30.8, 29.8, 28.1, 27.4, 25.5, 17.8, 17.5, 15.5. ESI-MS: m / z 375 [M+H] + (C 23 H 34 O4)。

[0034] Preparation of compounds D-1 to D-24

[0035] Reaction was carried out in anhydrous dichloromethane as solvent, with a small amount of DMAP as catalyst. Compound A-26 (30.0 mg, 0.08 mmol, 1.0 eq) and DMAP (5 mg) were weighed into a 50 mL round bottom flask, dissolved in dichloromethane under nitrogen protection, 100 μL of triethylamine was added under ice bath, and then the corresponding acyl chloride (2.0-4.0 eq) was added dropwise slowly. The reaction was carried out at room temperature to 40 °C for 2-72 h, and the reaction progress was monitored by TLC. After the disappearance of the starting material, the reaction was quenched by adding 30 mL of water, and then extracted with dichloromethane (3 x 30 mL). The combined dichloromethane layer was washed with 1 M hydrochloric acid solution once and then with saturated sodium chloride solution once, dried over anhydrous sodium sulfate, and concentrated directly for the next step. Methanol and dichloromethane (2 mL:2 mL) were used as solvent, 2 mL of 1 M HCl was added to deprotect the 7- and 14-hydroxyl groups, and the reaction was stirred at room temperature for 8-48 h. The reaction progress was monitored by TLC. After complete reaction, the reaction was concentrated in vacuum, 30 mL of saturated sodium bicarbonate solution was added, and then extracted with ethyl acetate (3 x 30 mL). The ethyl acetate layer was washed with saturated sodium chloride solution once, dried over anhydrous sodium sulfate, and concentrated to give the crude product. The crude product was purified by silica gel column chromatography to give the corresponding 3-OH acylated derivatives 24 (D-1-D-24) with different yields depending on the nature of the acyl chloride, but the overall yield was high, ranging from 30.5% to 98.9%. The acyl chlorides were 2-chloroacetyl chloride, hexanoyl chloride, 3-chlorobenzoyl chloride, 3,5-dichlorobenzoyl chloride, propionyl chloride, nonanoyl chloride, 2,4-dichlorobenzoyl chloride, 4-chlorobenzoyl chloride, cinnamoyl chloride, 3,4-dichlorobenzoyl chloride, p-fluorobenzoyl chloride, 3-bromobenzoyl chloride, 2-bromobenzoyl chloride, 4-bromobenzoyl chloride, o-fluorobenzoyl chloride, o-chlorobenzoyl chloride, 3,5-difluorobenzoyl chloride, 2,6-difluorobenzoyl chloride, acryloyl chloride, 2-thiophenecarbonyl chloride, 3-methylcrotonoyl chloride, 2-trifluorobenzoyl chloride, butyryl chloride, and heptanoyl chloride.

[0036] The spectral data of compounds D-1-D-24 are as follows:

[0037] Compound D-1: Petroleum ether-acetic ether (V / V) = (8:1-2:1) was eluted to give the product as a white powder 23.2 mg with a yield of 70.5%. 1H NMR (600 MHz, CDC13) δ: 6.17 (1H, s), 5.42 (1H, s), 4.87 (1H, s), 4.53 (1H, dd, J = 11.8, 4.6 Hz), 4.35 (1H, dd, J = 12.2, 4.1 Hz), 4.09 - 4.02 (2H, m), 3.08 (1H, s), 2.03 - 1.92 (2H, m), 1.83 - 1.73 (4H, m), 1.72 - 1.63 (1H, m), 1.58 (1H, dd, J = 15.7, 5.7 Hz), 1.46 - 1.36 (1H, m), 1.22 (1H, d, J = 8.6 Hz), 1.10 (3H, s), 1.05 (1H, s), 1.01 (1H, dd, J = 12.2, 1.4 Hz), 0.98 - 0.95 (1H, m), 0.94 (3H, s), 0.92 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 167.2, 147.5, 118.5, 82.5, 75.0, 61.7, 53.9, 52.68, 46.0, 41.3, 39.6, 38.0, 37.5, 31.1, 28.5, 27.6, 23.5, 18.2, 17.8, 16.8. ESI-MS: m / z 411 [M+H] + (C 22 H 31 ClO5).

[0038] Compound D-2: Petroleum ether-acetic ether (V / V) = (8:1 - 6:1) elution, yellow oily solid 19.6 mg, yield 55.1%. 1 H NMR (600 MHz, CDC13) δ: 6.17 (1H, s), 5.42 (1H, s), 4.87 (1H, s), 4.53 (1H, dd, J = 11.8, 4.6 Hz), 4.35 (1H, dd, J = 12.2, 4.1 Hz), 4.09 - 4.02 (2H, m), 3.08 (1H, s), 2.03 - 1.92 (2H, m), 1.83 - 1.73 (4H, m), 1.72 - 1.63 (1H, m), 1.58 (1H, dd, J = 15.7, 5.7 Hz), 1.46 - 1.36 (1H, m), 1.22 (1H, d, J = 8.6 Hz), 1.10 (3H, s), 1.05 (1H, s), 1.01 (1H, dd, J = 12.2, 1.4 Hz), 0.98 - 0.95 (1H, m), 0.94 (3H, s), 0.92 (3H, s). 13C NMR (150 MHz, CDC13) δ: 208.0, 173.8, 147.5, 118.4, 79.9, 75.0, 74.9, 61.7, 54.0, 52.6, 46.0, 39.6, 37.8, 37.6, 34.8, 31.4, 31.1, 28.5, 27.7, 24.9, 23.6, 22.4, 18.2, 17.7, 17.0, 14.1. ESI-MS: m / z 433 [M+H] + (C 26 H 40 O5)。

[0039] Compound D-3: Petroleum ether-acetic ether (V / V) = (6:1) eluted to white powder 20.0 mg, yield 54.5%; 1 H NMR (600 MHz, CDC13) δ: 7.98 (1H, s), 7.91 (1H, d, J = 7.8 Hz), 7.53 (1H, d, J = 8.0 Hz), 7.39 (1H, t, J = 7.9 Hz), 6.19 (1H, s), 5.43 (1H, s), 4.90 (1H, s), 4.70 (1H, dd, J = 11.7, 4.7 Hz), 4.39 (1H, dd, J = 12.2, 4.0 Hz), 3.09 (1H, s), 2.05 - 1.98 (2H, m,), 1.89 - 1.72 (6H, m), 1.61 (1H, dd, J = 15.7, 5.8 Hz), 1.44 (1H, tt, J = 15.4, 7.7 Hz), 1.27 (1H, s), 1.25 (1H, d, J = 4.0 Hz), 1.28 - 1.24 (2H, m), 1.15 (3H, s), 1.08 (1H, d, J = 12.2 Hz), 1.06 (3H, s), 1.02 (1H, dd, J = 13.3, 3.2 Hz), 0.99 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 165.1, 147.5, 134.7, 133.1, 132.5, 129.9, 129.7, 127.8, 118.4, 81.4, 75.1, 75.0, 61.7, 53.9, 52.7, 46.1, 39.6, 38.2, 37.6, 31.1, 28.7, 27.723.7, 18.3, 17.8, 17.2. ESI-MS: m / z 473 [M+H] + (C 27 H 33 ClO5)。

[0040] Compound D-4: Petroleum ether-ethyl acetate (V / V) = (6:1) eluted to get white powder 33.2 mg, yield 78.9%; 1 H NMR (600 MHz, CDC13) δ: 7.87 (2H, s), 7.54 (1H, s), 6.19 (1H, s), 5.43 (1H, s), 4.89 (1H, s), 4.71 (1H, dd, J = 11.7, 4.6 Hz), 4.39 (1H, dd, J = 12.2, 3.8 Hz), 3.09 (1H, s), 2.17 (1H, s), 2.05 - 1.97 (2H, m), 1.87 - 1.72 (6H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.2, 7.6 Hz), 1.26 (1H, d, J = 8.6 Hz), 1.15 (3H, s), 1.08 (1H, d, J = 12.4 Hz), 1.05 (3H, s), 1.05 - 1.00 (1H, m), 0.98 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 164.0, 147.5, 135.4, 133.5, 132.9, 128.1, 118.5, 81.9, 75.0, 61.7, 53.9, 52.7, 46.0, 39.6, 38.2, 37.6, 31.1, 28.7, 27.7, 23.6, 18.2, 17.8, 17.2. ESI-MS: m / z 507 [M+H] + (C 27 H 32 Cl2O5).

[0041] Compound D-5: Petroleum ether-ethyl acetate (V / V) = (6:1) eluted, yellow oil 13.7 mg, yield: 41.7%; 1H NMR (600 MHz, CDC13) δ: 6.18 (1H, s), 5.42 (1H, s), 4.87 (1H, s), 4.45 (1H, dd, J = 11.7, 4.5 Hz), 4.36 (1H, dd, J = 12.1, 3.7 Hz), 3.08 (1H, s), 2.33 (2H, ddd, J = 9.9, 7.6, 2.7 Hz), 1.99 (2H, d, J = 8.2 Hz), 1.82 - 1.71 (4H, m), 1.66 - 1.55 (3H, m), 1.45 - 1.37 (1H, m), 1.25 (1H, s), 1.22 (1H, d, J = 8.5 Hz), 1.14 (3H, t, J = 7.5 Hz), 1.10 (3H, s), 1.00 (1H, d, J = 12.1 Hz), 0.96 - 0.93 (1H, m), 0.91 (3H, s), 0.90 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 174.3, 147.5, 118.4, 79.9, 75.1, 75.0, 61.7, 54.0, 52.7, 46.1, 39.6, 37.9, 37.6, 31.1, 28.5, 28.1, 27.7, 23.6, 18.2, 17.8, 17.0, 9.4. ESI-MS: m / z 391 [M+H] + (C 23 H 34 O5)。

[0042] Compound D-6: Petroleum ether-ethyl acetate (V / V) = (8:1 - 6:1) yellow solid 32.3 mg, yield 82.7%; 1 H NMR (600 MHz, CDC13) δ: 6.18 (1H, s), 5.42 (1H, s), 4.87 (1H, s), 4.45 (1H, dd, J = 11.7, 4.5 Hz), 4.36 (1H, dd, J = 12.1, 3.7 Hz), 3.08 (1H, s), 2.33 (2H, ddd, J = 9.9, 7.6, 2.7 Hz), 1.99 (2H, d, J = 8.2 Hz), 1.82 - 1.71 (4H, m), 1.66 - 1.55 (3H, m), 1.45 - 1.37 (1H, m), 1.25 (1H, s), 1.22 (1H, d, J = 8.5 Hz), 1.14 (3H, t, J = 7.5 Hz), 1.10 (3H, s), 1.00 (1H, d, J = 12.1 Hz), 0.96 - 0.93 (1H, m), 0.91 (3H, s), 0.90 (3H, s). 13C NMR (150 MHz, CDC13) δ: 207.9, 173.8, 147.5, 118.4, 79.9, 75.1, 75.0, 61.7, 54.0, 52.7, 46.1, 39.6, 37.8, 37.6, 34.9, 31.9, 31.1, 29.3, 28.5, 27.7, 25.3, 23.7, 22.8, 18.2, 17.8, 17.0, 14.2. ESI-MS: m / z 475 [M+H] + (C 29 H 46 O5)。

[0043] Compound D-7: Petroleum ether-acetic ether (V / V) = (8:1) white powder 40.13 mg, yield 98.9%; 1 H NMR (600 MHz, CDC13) δ: 7.78 (1H, d, J = 8.4 Hz), 7.47 (1H, d, J = 2.0 Hz), 7.30 (1H, dd, J = 8.4, 2.0 Hz), 6.19 (1H, s), 5.43 (1H, s), 4.89 (1H, s), 4.72 (1H, dd, J = 11.7, 4.7 Hz), 4.39 (1H, dd, J = 12.2, 4.1 Hz), 3.09 (1H, s), 2.17 (1H, s), 2.05 - 1.97 (2H, m), 1.93 - 1.88 (1H, m), 1.86 - 1.72 (5H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.5, 7.7 Hz), 1.26 (1H, d, J = 8.7 Hz), 1.13 (3H, s), 1.07 (1H, dd, J = 12.1, 1.3 Hz), 1.02 (3H, s), 1.00 (3H, s), 0.99 - 0.97 (1H, m). 13 C NMR (150 MHz, CDC13) δ: 207.9, 164.8, 147.5, 138.4, 134.9, 132.6, 131.2, 128.9, 127.2, 118.5, 82.2, 75.1, 75.0, 61.7, 53.9, 52.7, 46.1, 39.6, 38.0, 37.7, 31.1, 28.7, 27.7, 23.6, 18.2, 17.8, 17.3. ESI-MS: m / z 507 [M+H] + (C 27 H 32 Cl2O5)。

[0044] Compound D-8: Eluted with petroleum ether-ethyl acetate (V / V) = (5:1), white powder 19.0 mg, yield 50.2%. 1 H NMR (600 MHz, CDC13) δ: 7.96 (2H, d, J = 8.5 Hz), 7.41 (2H, d, J = 8.5 Hz), 6.19 (1H, s), 5.42 (1H, d, J = 10.4 Hz), 4.89 (1H, s), 4.69 (1H, dd, J = 11.8, 4.7 Hz), 4.39 (1H, dd, J = 12.0, 2.8 Hz), 3.12 (1H, s), 3.09 (1H, s), 2.05 - 1.97 (2H, m), 1.89 - 1.71 (5H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.4, 7.6 Hz), 1.26 (2H, d, J = 8.8 Hz), 1.15 (3H, s), 1.08 (1H, d, J = 12.0 Hz), 1.05 (3H, s), 1.02 (1H, dd, J = 13.5, 3.5 Hz), 0.98 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 165.5, 147.5, 139.5, 131.1, 129.2, 128.9, 118.4, 81.2, 75.1, 75.0, 61.7, 53.9, 52.7, 46.1, 39.6, 38.2, 37.6, 31.1, 28.7, 27.7, 23.7, 18.2, 17.8, 17.2. ESI-MS: m / z 473 [M+H] + (C 27 H 33 ClO5)。

[0045] Compound D-9: Eluted with petroleum ether-ethyl acetate (V / V) = (15:1 - 8:1), white powder 17.4 mg, yield 46.8%. 1H NMR (600 MHz, CDC13) δ: 7.66 (1H, d, J = 16.0 Hz), 7.53 (2H, dd, J = 6.5, 2.8 Hz), 7.40-7.37 (3H, m), 6.43 (1H, d, J = 16.0 Hz), 6.17 (1H, s), 5.39 (1H, s), 4.59 (1H, d, J = 4.1 Hz), 4.58 (1H, s), 4.21 (1H, dd, J = 12.5, 5.6 Hz), 3.05 (1H, s), 2.09-2.04 (1H, m), 2.04-1.98 (1H, m), 1.98-1.92 (1H, m), 1.89-1.83 (1H, m), 1.83-1.77 (2H, m), 1.66 (2H, m), 1.56-1.47 (1H, m), 1.26 (1H, s), 1.24 (3H, s), 1.11 (3H, s), 0.98 (3H, s), 0.96 (1H, d, J = 1.5 Hz), 0.95 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 206.0, 166.7, 146.6, 144.7, 134.6, 130.4, 129.0, 128.2, 118.6, 117.3, 97.4, 80.4, 71.2, 71.0, 54.9, 52.3, 51.3, 43.3, 38.8, 37.9, 37.4, 31.2, 28.1, 27.3, 25.5, 23.6, 17.8, 17.5. ESI-MS: m / z 465 [M+H] + (C 29 H 36 O5)。

[0046] Compound D-10: Petroleum ether-ethyl acetate (V / V) = (6:1-5:1) elution, white powder 29.3 mg, yield 71.2%. 1H NMR (600 MHz, CDC13) δ: 8.07 (1H, d, J = 1.8 Hz), 7.84 (1H, dd, J = 8.3, 1.9 Hz), 7.52 (1H, d, J = 8.3 Hz), 6.18 (1H, s), 5.43 (2H, s), 4.89 (1H, s), 4.70 (1H, dd, J = 11.6, 4.6 Hz), 4.38 (1H, dd, J = 12.1, 3.7 Hz), 3.09 (1H, s), 2.05 - 1.97 (2H, m), 1.88 - 1.72 (6H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.3, 7.6 Hz), 1.25 (1H, d, J = 8.7 Hz), 1.15 (3H, s), 1.07 (1H, d, J = 12.2 Hz), 1.05 (3H, s), 1.03 - 0.99 (1H, m), 0.98 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 208.0, 164.5, 147.5, 137.7, 133.1, 131.6, 130.7, 130.6, 128.8, 118.5, 81.7, 75.0, 75.0, 61.7, 53.9, 52.7, 46.0, 39.6, 38.2, 37.6, 31.1, 28.7, 27.7, 23.7, 18.2, 17.8, 17.2. ESI-MS: m / z 507 [M+H] + (C 27 H 32 Cl2O5).

[0047] Compound D-11: Petroleum ether-acetic ether (V / V) = (5:1) elution, white powder 32.6 mg, yield 87.7%. 1H NMR (600 MHz, CDC13) δ: 8.05 - 8.02 (2H, m), 7.11 (2H, t, J = 8.6 Hz), 6.18 (1H, s), 5.43 (1H, s), 4.89 (1H, s), 4.68 (1H, dd, J = 11.8, 4.7 Hz), 4.38 (1H, dd, J = 12.1, 3.7 Hz), 3.09 (1H, s), 2.05 - 1.97 (2H, m), 1.89 - 1.85 (1H, m), 1.84 - 1.71 (6H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.3, 7.6 Hz) 1.26 (1H, d, J = 8.7 Hz), 1.14 (3H, s), 1.08 (1H, d, J = 11.1 Hz), 1.05 (3H, s), 1.02 (1H, dd, J = 13.6, 3.7 Hz), 0.99 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 208.0, 166.7, 165.4, 147.5, 132.2, 127.0, 118.4, 115.7, 115.6, 81.0, 75.0, 61.7, 53.9, 52.7, 46.1, 39.6, 38.2, 37.6, 31.1, 28.7, 27.7, 23.7, 18.2, 17.8, 17.2. ESI-MS: m / z 457 [M+H] + (C 27 H 33 FO5).

[0048] Compound D-12: Petroleum ether-ethyl acetate (V / V) = (5:1) elution, white powder 35.1 mg, yield 84.0%. 1H NMR (600 MHz, CDC13) δ: 8.14 (1H, s), 7.95 (1H, d, J = 7.8 Hz), 7.69 (1H, d, J = 8.0 Hz), 7.33 (1H, t, J = 7.9 Hz), 6.18 (1H, s), 5.43 (1H, s), 4.89 (1H, s), 4.70 (1H, dd, J = 11.7, 4.7 Hz), 4.39 (1H, dd, J = 12.2, 4.0 Hz), 3.09 (1H, s), 2.06 - 1.97 (2H, m), 1.89 - 1.72 (6H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.4, 7.7 Hz), 1.26 (1H, d, J = 8.7 Hz), 1.15 (3H, s), 1.08 (1H, d, J = 12.4 Hz), 1.06 (3H, s), 1.02 (1H, dd, J = 13.1, 3.3 Hz), 0.99 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 165.0, 147.5, 136.0, 132.7, 132.6, 130.1, 128.3, 122.61, 118.4, 81.4, 75.0, 61.7, 53.9, 52.7, 46.0, 39.6, 38.2, 37.6, 31.1, 28.7, 27.7, 23.7, 18.2, 17.8, 17.2. ESI-MS: m / z 517 [M+H] + (C 27 H 33 BrO5)。

[0049] Compound D-13: Petroleum ether-acetone (V / V) = (10:1) elution, 30.1 mg of white powder was obtained, the yield was 71.5%. 1H NMR (600 MHz, CDC13) δ: 7.75 (1H, dd, J = 7.6, 1.7 Hz), 7.65 (1H, dd, J = 7.8, 0.8 Hz), 7.39 - 7.31 (2H, m), 6.18 (1H, s), 5.43 (1H, s), 4.88 (1H, s), 4.73 (1H, dd, J = 11.8, 4.6 Hz), 4.38 (1H, dd, J = 12.0, 2.9 Hz), 3.08 (1H, s), 2.05 - 1.96 (2H, m), 1.96 - 1.90 (1H, m), 1.85 - 1.73 (7H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.4, 7.7 Hz), 1.25 (1H, d, J = 8.5 Hz), 1.13 (3H, s), 1.07 (1H, d, J = 11.4 Hz), 1.03 (3H, s), 1.01 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 208.0, 166.1, 147.5, 134.5, 132.8, 132.6, 131.3, 127.3, 121.6, 118.4, 82.0, 75.0, 61.7, 53.9, 52.8, 46.0, 39.6, 38.0, 37.7, 31.1, 28.7, 27.7, 23.6, 18.2, 17.8, 17.2. ESI-MS: m / z 517 [M+H] + (C 27 H 33 BrO5)。

[0050] Compound D-14: Petroleum ether-acetic ether (V / V) = (5:1) elution, white powder 23.0 mg, yield 54.8%. 1H NMR (600 MHz, CDCI3) δ: 7.90-7.86 (2H, m), 7.62-7.56 (2H, m), 6.19 (1H, s), 5.43 (1H, s), 4.89 (1H, s), 4.69 (1H, dd, J = 11.8, 4.7 Hz), 4.39 (1H, dd, J = 12.2, 4.1 Hz), 3.09 (1H, s), 2.05-1.98 (2H, m), 1.90-1.69 (7H, m), 1.61 (1H, dd, J = 15.7, 5.8 Hz), 1.48-1.39 (1H, m), 1.26 (1H, d, J = 8.6 Hz), 1.15 (3H, s), 1.09-1.06 (1H, m), 1.05 (3H, s), 1.02 (1H, dd, J = 13.8, 4.1 Hz), 0.98 (3H, s). 13 C NMR (150 MHz, CDCI3) δ: 207.9, 165.6, 147.5, 131.9, 131.2, 129.6, 128.2, 118.4, 81.2, 75.1, 75.0, 61.7, 53.9, 52.7, 46.1, 39.6, 38.2, 37.6, 31.1, 28.7, 27.7, 23.7, 18.2, 17.8, 17.2. ESI-MS: m / z 517 [M+H] + (C 27 H 33 BrO5)。

[0051] Compound D-15: Petroleum ether-acetic ether (V / V) = (6:1) elution, white powder 31.5 mg, yield 84.7%. 1H NMR (600 MHz, CDC13) δ: 7.93 (1H, td, J = 7.6, 1.7 Hz), 7.54 - 7.49 (1H, m), 7.24 - 7.19 (1H, m), 7.13 (1H, dd, J = 10.3, 8.7 Hz), 6.18 (1H, s), 5.43 (1H, s), 4.89 (1H, s), 4.71 (1H, dd, J = 11.8, 4.7 Hz), 4.39 (1H, dd, J = 12.2, 4.1 Hz), 3.09 (1H, s), 2.06 - 1.97 (2H, m), 1.94 - 1.89 (1H, m), 1.87 - 1.71 (5H, m), 1.61 (1H, dd, J = 15.7, 5.8 Hz), 1.48 - 1.39 (1H, m), 1.26 (2H, dd, J = 13.7, 4.9 Hz), 1.14 (3H, s), 1.08 (1H, d, J = 12.2 Hz), 1.03 (3H, s), 1.02 (3H, s), 0.98 (1H, d, J = 3.1 Hz). 13 C NMR (150 MHz, CDC13) δ: 207.9, 164.4, 161.2, 147.5, 134.6, 132.3, 124.1, 118.4, 117.2, 117.1, 81.6, 75.1, 75.0, 61.7, 54.0, 52.7, 46.1, 39.6, 38.0, 37.6, 31.1, 28.6, 27.7, 23.6, 18.3, 17.8, 17.1. ESI-MS: m / z 457 [M+H] + (C 27 H 33 FO5).

[0052] Compound D-16: Petroleum ether-acetone (V / V) = (10:1 - 8:1) elution, white powder 34.1 mg, yield 85.8%. 1H NMR (600 MHz, CDC13) δ: 7.80 (1H, dd, J = 7.7, 1.4 Hz), 7.42 (2H, ddd, J = 12.7, 9.3, 4.7 Hz), 7.36 - 7.29 (1H, m), 6.19 (1H, s), 5.43 (1H, s), 4.89 (1H, s), 4.74 (1H, dd, J = 11.6, 4.7 Hz), 4.39 (1H, dd, J = 12.2, 4.0 Hz), 3.09 (1H, s), 2.17 (1H, s), 2.06 - 1.97 (2H, m), 1.95 - 1.89 (1H, m), 1.86 - 1.73 (5H, m), 1.70 (1H, s), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.48 - 1.39 (1H, m), 1.26 (1H, d, J = 8.6 Hz), 1.13 (3H, d, J = 8.2 Hz), 1.08 (1H, d, J = 11.2 Hz), 1.04 (3H, s), 1.01 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 165.7, 147.5, 133.7, 132.6, 131.4, 131.2, 130.7, 126.7, 118.4, 81.9, 75.1, 75.0, 61.7, 53.9, 52.8, 46.1, 39.6, 38.0, 37.7, 31.1, 28.7, 27.7, 23.6, 18.3, 17.8, 17.2. ESI-MS: m / z 473 [M+H] + (C 27 H 33 ClO5)。

[0053] Compound D-17: Petroleum ether-acetic ether (V / V) = (5:1) elution, white powder 33.3 mg, yield 85.9%. 1H NMR (600 MHz, CDC13) δ: 7.52 (2H, dd, J = 7.1, 2.0 Hz), 7.01 (1H, tt, J = 8.4, 2.3 Hz), 6.19 (1H, s), 5.43 (2H, s), 4.89 (1H, s), 4.69 (1H, dd, J = 11.8, 4.6 Hz), 4.38 (1H, dd, J = 12.2, 3.8 Hz), 3.09 (1H, s), 2.05 - 1.97 (2H, m), 1.89 - 1.74 (5H, m), 1.73 - 1.68 (2H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.4, 7.7 Hz), 1.26 (1H, d, J = 8.7 Hz), 1.15 (3H, s), 1.08 (1H, d, J = 12.2 Hz), 1.05 (3H, s), 1.02 (1H, dd, J = 13.0, 3.0 Hz), 0.99 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 164.1, 163.7, 162.1, 147.5, 134.0, 118.5, 112.8, 112.6, 108.5, 81.9, 75.0, 61.7, 53.9, 52.7, 46.0, 39.6, 38.1, 37.6, 31.1, 28.7, 27.7, 23.6, 18.2, 17.8, 17.2. ESI-MS: m / z 475 [M+H] + (C 27 H 32 F2O5).

[0054] Compound D-18: Petroleum ether-acetic ether (V / V) = (5:1) elution, white powder 29.8 mg, yield 78.4%. 1H NMR (600 MHz, CDC13) δ: 7.40 (1H, tt, J = 8.4, 6.2 Hz), 6.95 (2H, t, J = 8.2 Hz), 6.18 (1H, s), 5.42 (1H, s), 4.88 (1H, s), 4.76 (1H, dd, J = 11.7, 4.7 Hz), 4.38 (1H, dd, J = 12.2, 4.1 Hz), 3.08 (1H, s), 2.17 (1H, s), 2.05 - 1.96 (2H, m), 1.94 - 1.90 (1H, m), 1.84 - 1.73 (5H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.47 - 1.39 (1H, m), 1.25 (1H, d, J = 8.6 Hz), 1.12 (3H, s), 1.08 - 1.05 (1H, m), 1.04 (3H, s), 1.03 - 0.97 (1H, m), 0.94 (3H, d, J = 6.7 Hz). 13 C NMR (150 MHz, CDC13) δ: 207.9, 161.6, 161.5, 159.9, 147.5, 132.7, 118.4, 112.1, 82.4, 75.0, 61.7, 53.9, 52.7, 46.0, 39.6, 37.9, 37.6, 31.1, 28.4, 27.7, 23.6, 18.2, 17.8, 16.9. ESI-MS: m / z 475 [M+H] + (C 27 H 32 F2O5).

[0055] Compound D-19: Petroleum ether-ethyl acetate (V / V) = (6:1) elution, white powder 26.0 mg, yield 81.6%. 1H NMR (600 MHz, CDC13) δ: 6.38 (1H, dd, J = 17.3, 1.1 Hz), 6.17 (1H, s), 6.11 (1H, dd, J = 17.3, 10.4 Hz), 5.82 (1H, dd, J = 10.4, 1.2 Hz), 5.44 (1H, s), 5.41 (1H, s), 4.87 (1H, s), 4.53 (1H, dd, J = 11.9, 4.3 Hz), 4.36 (1H, dd, J = 12.1, 3.5 Hz), 3.19 (1H, s), 3.07 (1H, s), 2.03 - 1.95 (2H, m), 1.83 - 1.74 (4H, m), 1.66 (1H, m), 1.60 (1H, d, J = 5.9 Hz), 1.45 - 1.37 (1H, m), 1.23 (1H, t, J = 7.1 Hz), 1.11 (3H, s), 1.02 (1H, d, J = 11.2 Hz), 0.99 - 0.95 (1H, m), 0.93 (6H, d, J = 1.8 Hz). 13 C NMR (150 MHz, CDC13) δ: 207.9, 166.1, 147.5, 130.7, 129.0, 118.4, 80.3, 75.0, 61.7, 53.9, 52.6, 46.0, 39.6, 38.0, 37.6, 31.1, 28.5, 27.7, 23.6, 18.2, 17.8, 17.0. ESI-MS: m / z 389 [M+H] + (C 23 H 32 O5)。

[0056] Compound D-20: Petroleum ether-acetone (V / V) = (12:1) elution, white powder 15.3 mg, yield 41.2%. 1H NMR (600 MHz, CDC13) δ: 7.79 (1H, d, J = 3.5 Hz), 7.55 (1H, d, J = 4.9 Hz), 7.15 - 7.07 (1H, m), 6.18 (1H, s), 5.43 (2H, s), 4.89 (1H, s), 4.64 (1H, dd, J = 11.7, 4.8 Hz), 4.38 (1H, dd, J = 12.1, 3.7 Hz), 3.09 (1H, s), 2.05 - 1.96 (2H, m), 1.91 - 1.84 (1H, m), 1.84 - 1.73 (4H, m), 1.73 - 1.66 (2H, m), 1.60 (1H, dd, J = 15.7, 5.8 Hz), 1.43 (1H, tt, J = 15.4, 7.7 Hz), 1.25 (1H, d, J = 8.5 Hz), 1.14 (3H, s), 1.07 (1H, d, J = 12.0 Hz), 1.02 (3H, s), 0.99 (3H, s). 13 C NMR (150 MHz, CDC13) δ: 207.9, 162.1, 147.5, 134.3, 133.4, 132.4, 127.9, 118.4, 81.1, 75.1, 75.0, 61.7, 53.9, 52.7, 46.1, 39.6, 38.2, 37.6, 31.1, 28.6, 27.7, 23.7, 18.2, 17.8, 17.1. ESI-MS: m / z 445 [M+H] + (C 25 H 32 O5S)。

[0057] Compound D-21: Petroleum ether-acetone (V / V) = (12:1) elution, white powder 32.7 mg, yield 98.0%. 1H NMR (600 MHz, CDC13) δ: 6.16 (1H, s), 6.10-6.09 (1H, m), 5.41 (1H, s), 4.85 (1H, s), 4.48 (1H, dd, J = 11.8, 4.5 Hz), 4.33 (1H, dd, J = 12.2, 4.0 Hz), 3.06 (1H, s), 2.13 (3H, s), 1.99-1.94 (2H, m), 1.87 (3H, s), 1.81-1.71 (7H, m), 1.62-1.54 (2H, m), 1.40 (1H, tt, J = 15.4, 7.7 Hz), 1.20 (1H, d, J = 8.6 Hz), 1.06 (3H, s), 0.98 (1H, d, J = 11.1 Hz), 0.89 (3H, d, J = 10.2 Hz), 0.77 (3H, d, J = 6.6 Hz). 13 C NMR (150 MHz, CDC13) δ: 208.0, 165.2, 156.4, 147.5, 125.4, 118.4, 80.7, 75.0, 61.7, 53.9, 52.7, 46.0, 39.6, 37.8, 37.6, 31.1, 28.4, 28.0, 27.7, 23.6, 22.6, 18.2, 17.7, 16.7. ESI-MS: m / z 417 [M+H] + (C 25 H 36 O5)。

[0058] Compound D-22: Petroleum ether-acetic ether (V / V) = (5:1 - 4:1) elution, white powder 39.8 mg, yield 97.7%. 1 H NMR (600 MHz, CDC13) δ: 6.16 (1H, s), 6.10-6.09 (1H, m), 5.41 (1H, s), 4.85 (1H, s), 4.48 (1H, dd, J = 11.8, 4.5 Hz), 4.33 (1H, dd, J = 12.2, 4.0 Hz), 3.06 (1H, s), 2.13 (3H, s), 1.99-1.94 (2H, m), 1.87 (3H, s), 1.81-1.71 (7H, m), 1.62-1.54 (2H, m), 1.40 (1H, tt, J = 15.4, 7.7 Hz), 1.20 (1H, d, J = 8.6 Hz), 1.06 (3H, s), 0.98 (1H, d, J = 11.1 Hz), 0.89 (3H, d, J = 10.2 Hz), 0.77 (3H, d, J = 6.6 Hz). 13C NMR(150MHz, CDCl3)δ:208.0,166.9,147.5,131.9,131.1,130.0,129.6,126.8,122.6,118.5,82.4, 75.0,61.7,53.9,52.8,46.0,39.6,37.9,37.6,31.1,28.5,27.7,23.2,18.2,17.8,16.9.ESI-MS:m / z 507[M+H] + (C 28 H 33 F3O5).

[0059] Compound D-23: eluted with petroleum ether-acetone (V / V) = (12:1-10:1) to obtain 16.5 mg of white powder with a yield of 30.5%. 1 H NMR (600MHz, CDCl3) δ6.17(1H,s),5.41(1H,s),4.87(1H,s),4.46(1H,dd,J=11.6,4.5Hz),4.36(1H,dd,J= 12.2,3.7Hz),3.08(1H,s),2.28(2H,t,J=7.5Hz),2.03–1.95(2H,m),1.82–1.70(4H,mH),1.65(2H,dt,J=1 4.2,7.1Hz),1.58(2H,dd,J=15.7,6.1Hz),1.55–1.47(1H,m),1.46–1.37(1H,m),1.22(1H,d,J=8.6Hz),1. 10(3H,s),1.00(1H,d,J=12.5Hz),0.95(3H,d,J=7.3Hz),0.93(2H,d,J=3.0Hz),0.91(3H,s),0.90(3H,s). 13 C NMR (150MHz, CDCl3) δ207.9,173.6,147.5,118.4,79.9,75.1,75.0,61.7,54.0,52.7,46.1 ,39.6,37.8,37.6,36.8,31.1,28.5,27.7,23.7,18.7,18.2,17.8,17.0,13.9.ESI-MS:m / z 405[M+H] + (C 24 H 36 O5).

[0060] Compound D-24: Petroleum ether-acetone (V / V) = (12:1 - 10:1) eluted to give white powder 19.7 mg, yield 55.1%. 1 H NMR (600 MHz, CDC13) δ 6.17 (1H, s), 5.41 (1H, s), 4.87 (1H, s), 4.45 (1H, dd, J = 11.8, 4.5 Hz), 4.36 (1H, dd, J = 12.1, 3.7 Hz), 3.07 (1H, s), 2.29 (2H, t, J = 7.5 Hz), 2.02 - 1.95 (1H, m), 1.81 - 1.70 (4H, m), 1.64 - 1.56 (4H, m), 1.41 (1H, td, J = 15.2, 8.0 Hz), 1.33 - 1.24 (8H, m), 1.22 (1H, d, J = 8.6 Hz), 1.10 (3H, s), 1.00 (1H, d, J = 12.1 Hz), 0.91 (3H, s), 0.90 (3H, s), 0.88 (5H, t, J = 6.0 Hz). 13 C NMR (150 MHz, CDC13) δ: 207.9, 173.7, 147.5, 118.3, 79.9, 75.1, 75.0, 61.7, 54.0, 52.7, 46.1, 39.6, 37.8, 37.6, 34.9, 31.6, 31.1, 29.0, 28.5, 27.7, 25.2, 23.7, 22.6, 18.2, 17.8, 17.0, 14.2. ESI-MS: m / z 447 [M+H] + (C 27 H 42 O5)。

[0061] Test Example 1: Anti-tumor activity test

[0062] 1.1 Drugs and reagents: test samples, culture medium (IMDM + 20% FBS, RPMI-1640, DMEM), fetal bovine serum (FBS), PBS buffer, dimethyl sulfoxide (DMSO), triple liquid (10% SDS + 5% isopropanol + 12mM HCl), tetramethyl azo blue (MTT), Taxol (positive control drug).

[0063] 1.2 Instruments: clean bench, CO2 incubator, multifunctional inverted microscope, centrifuge, automatic enzyme marker 96-hole culture plate.

[0064] 1.3 Cell lines: Cervical cancer cells (Hela), human chronic myeloid leukemia cells (K562), human non-small cell lung cancer cells (A549), human myeloblast leukemia cells (HL-60) and LNCaP (human prostate cancer cells) five tumor cell lines from Guizhou Province, China Academy of Sciences Key Laboratory of Natural Products (Guiyang, Guizhou).

[0065] 1.4 Cell culture: HL-60 cells were cultured in IMDM medium containing 20% FBS, K562 and LNCaP cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, A549 and Hela cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin, and cultured at 37℃, 5% CO2. Cells were passaged every 2-3 days, and cells in logarithmic growth phase and good condition were collected for subsequent detection.

[0066] 1.5 Sample preparation: accurately weigh the glaucocalyxin B derivatives, add 500 μL DMSO solvent, shake to dissolve completely, prepare the stock solution. According to the molecular weight of the compound, calculate the concentration of the stock solution. The required amount of stock solution is taken for subsequent experiments, and the target concentration is diluted with buffer or medium. The resulting drug solution is stored at -20℃ for standby.

[0067] 1.6 Experimental method

[0068] Step (1): Select Hela, K562, LNCaP, A549 and HL-60 five cell lines in logarithmic growth phase, take paclitaxel (Taxol) as positive control group, glaucocalyxin B (GLA) and glaucocalyxin B derivatives D-1~D-24 as experimental group. HL-60, LNCaP, A549, K562 and Hela cells were inoculated in 96-well plates at a density of 8×10 3 / well, and then gradient concentrations of test compounds were added after stable adhesion.

[0069] Step (2): Place at 37℃, 5% CO2 for 48 hours and observe the morphological changes of the cells.

[0070] Step (3): Add 20 μL of MTT solution to each well in the dark and continue to incubate in the cell incubator for 4 hours in the dark.

[0071] Step (4): After 4 hours, terminate the culture and use two treatment methods respectively: (1) add triplex liquid for 4 hours or overnight, and measure the OD value at a wavelength of 570 nm with a microplate reader. (2) 3000 rpm, centrifugation for 30 min, discard the supernatant, add 150 μL DMSO, fully dissolve for 15 min at 37℃ constant temperature shaking bed, and measure the OD value at a wavelength of 490 nm with a microplate reader.

[0072] Step (5): Calculate the proliferation inhibition rate of the derivative on tumor cells by formula: inhibition rate (%) = [1- (experimental group OD value-blank control group OD value) / (control group OD value-blank control group OD value)] x 100%. According to the tumor cell growth inhibition activity, compounds with significant inhibitory effect on tumor cells HL-60, K562, LNCaP, A549 and HeLa are screened, and IC 50 values are calculated by concentration-effect curve analysis. All experiments are set up with three biological repeats, and data processing is performed using GraphPad Prism 9.5.1 software, and the results are expressed as the mean ± standard deviation of three independent experiments .

[0073] 1.7 3α-O-acylated pluviatilols derivatives on tumor cell proliferation inhibition activity results

[0074] The results of the proliferation activity of 3α-O-acylated pluviatilols derivatives (D-1~D-24) provided by the present application on HL-60, K562, LNCaP, A549 and HeLa five kinds of tumor cells at a concentration of 5 μM are shown (see Table 1). Most of the 3α-O-acylated pluviatilols derivatives have an inhibition rate greater than that of the positive drug paclitaxel Figure 1 ) at a treatment concentration of 5 μM. The correlation between the compound concentration and the growth inhibition of cancer cells is evaluated for the derivatives with better inhibition rates, and the IC 50 values are calculated. The IC 50 values are shown in Table 2. The results show that the IC 50 values of some derivatives in the series are close to or lower than that of the positive drug paclitaxel. Derivatives D-4, D-10 and D-15 have better inhibitory activity on tumor cells, especially on A549 and HeLa cancer cells. Among them, D-4, D-10 and D-15 have the best A549 cell proliferation inhibition activity, and the IC 50 values are (0.273±0.018) μM, (0.299±0.020) μM and (0.437±0.041) μM, respectively.

[0075] Table 1 Inhibition rate of 3α-O-acylated pluviatilols derivatives on tumor cells at a concentration of 5 μM

[0076]

[0077]

[0078] Note: The inhibition rate is the average value ± SD of three independent experiments (x±s, n=3)

[0079] Table 2 Anti-tumor IC50 Values (48h)

[0080]

[0081]

[0082] Note: IC 50 Values are the average of three independent experiments ± SD (x ± s, n = 3)

[0083] Conclusion: The 3-OH acylation structural modification of GLA is carried out in the application, the activity of the obtained 3α-O-acylated glaucarubin derivatives is significantly improved, most of the derivatives show significant anti-tumor effect, among them, the derivatives D-4, D-10 and D-15 have the best activity on A549 cells, the IC 50 values are (0.273 ± 0.018) μM, (0.299 ± 0.020) μM and (0.437 ± 0.041) μM, respectively. In summary, the novel glaucarubin derivatives prepared in the application exhibit significant anti-tumor activity, and have the potential to be developed as anti-tumor drugs.

[0084] Although the application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the application, all belong to the scope of the application claimed.

Claims

1. A 3α-O-acylated cyanocalyxin derivative, characterized in that: It has the structure shown in formula (I): wherein R is 2-chloroacetyl, hexanoyl, 3-chlorobenzoyl, 3,5-dichlorobenzoyl, propionyl, nonanoyl, 2,4-dichlorobenzoyl, 4-chlorobenzoyl, cinnamoyl, 3,4-dichlorobenzoyl, p-fluorobenzoyl, 3-bromobenzoyl, 2-bromobenzoyl, 4-bromobenzoyl, o-fluorobenzoyl, o-chlorobenzoyl, 3,5-difluorobenzoyl, 2,6-difluorobenzoyl, acryloyl, 2-thenoyl, 3-methylcrotonyl, 2-trifluorobenzoyl, butyryl, or heptanoyl.

2. The 3α-O-acylated cyanocalyxin derivative according to claim 1, characterized in that R is 3,5-dichlorobenzoyl, 3,4-dichlorobenzoyl or o-fluorobenzoyl.

3. The method for preparing the 3α-O-acylated cyanocalyxin derivative according to claim 1, characterized in that: The synthetic route is as follows: The preparation method comprises the following steps: (1) condensation reaction of cyanocalyxin and 2,2-dimethoxypropane in the presence of p-toluenesulfonic acid and acetone to obtain compound A-1; (2) Compound A-1 is reduced with CeCl3·7H2O in the presence of NaBH4 and methanol to obtain compound A-26; (3) Under nitrogen protection, compound A-26 is subjected to an acylation reaction with R-Cl in the presence of triethylamine, DMAP, and dichloromethane to obtain a 3α-O-acyl-7,14-O-isopropylidene cyanocalyxin derivative, which is directly subjected to the next step without purification; the obtained 3α-O-acyl-7,14-O-isopropylidene cyanocalyxin derivative is subjected to a hydrolysis reaction with hydrochloric acid in the presence of a solvent to obtain the target compound D, i.e., the 3α-O-acylated cyanocalyxin derivative; Wherein, the definition of R in R-Cl is as described in claim 1.

4. The method for preparing the 3α-O-acylated cyanocalyxin derivative according to claim 3, characterized in that: In step (1), the molar ratio of cyanocalyxin, 2,2-dimethoxypropane and p-toluenesulfonic acid is 1:(2-4):(0.03-0.1), and the temperature of the condensation reaction is 50-55°C.

5. The method for preparing the 3α-O-acylated cyanocalyxin derivative according to claim 3, characterized in that: In step (2), the molar ratio of compound A-1, CeCl3·7H2O, and NaBH4 is 1:3:(1.5-2), and the temperature of the reduction reaction is 0-25°C.

6. The method for preparing the 3α-O-acylated cyanocalyxin derivative according to claim 3, characterized in that: In step (3), the molar ratio of compound A-26, R-Cl, triethylamine, and DMAP is 1:(2-4):(8-10):(0.03-0.1), and the temperature of the acylation reaction is 0-40°C; the concentration of the hydrochloric acid is 1M, the solvent is methanol and dichloromethane in a volume ratio of 1:1, and the temperature of the hydrolysis reaction is 15-25°C.

7. A pharmaceutical composition, characterized in that The invention comprises the 3α-O-acylated cyanocalyxin derivative as claimed in claim 1.

8. A pharmaceutical preparation, characterized in that The invention comprises the 3α-O-acylated cyanocalyxin derivative according to claim 1, and at least one pharmaceutically acceptable excipient or carrier.

9. Use of the 3α-O-acylated cyanocalyxin derivative according to claim 1, the pharmaceutical composition according to claim 7, or the pharmaceutical preparation according to claim 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that The tumors include leukemia, cervical cancer, lung cancer and prostate cancer.

Images