A eucalyptane-type sesquiterpene dimer compound, its preparation method and application

Abstract

This invention discloses a eucalyptane-type sesquiterpene dimer compound, its preparation method, and its applications, belonging to the field of pharmaceutical preparation technology. The six eucalyptane-type sesquiterpene dimers (compounds 1-6) disclosed in this invention are novel compounds with defined stereostructures and optically pure composition, exhibiting varying degrees of cytotoxic activity against HepG2, Hep3B, and Huh7 cells. Using sorafenib as a positive control, compounds 1, 5, and 6 showed stronger cytotoxic activity against HepG2 cells than the positive control sorafenib, compound 5 exhibited the strongest cytotoxic activity against Hep3B cells, and compounds 1, 4, 5, and 6 showed stronger cytotoxic activity against Huh7 cells than the positive control sorafenib. Therefore, compounds 1-6 disclosed in this invention have the potential to be developed into anti-hepatocellular carcinoma drugs, showing promising application prospects, and also provide important theoretical support for the further development of Atractylodes macrocephala medicinal plant resources.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a eucalyptane-type sesquiterpene dimer compound, its preparation method, and its application. Background Technology

[0002] Sesquiterpene dimers in Asteraceae plants have attracted widespread attention due to their novel structures and pharmacological activities. Atractylodes macrocephala is rich in sesquiterpene dimers such as atractylodes bis(atractylodes) and atractylodes bis(epiota)-atractylodes. Furthermore, the structures of sesquiterpenes in Atractylodes macrocephala are diverse, mainly eucalyptane-type sesquiterpenes, which are considered the main active ingredient group. In addition, atractylodes bis(atractylodes)-III can inhibit the proliferation of HepG2 and SMMC7721 hepatocellular carcinoma cells by inhibiting FGFR1 and inducing apoptosis. However, research on the pharmacodynamic material basis and mechanism of action of Atractylodes macrocephala against hepatocellular carcinoma is still relatively rare. Therefore, research on the active sesquiterpene dimers in Atractylodes macrocephala against hepatocellular carcinoma and the development of anti-hepatocellular carcinoma drugs is promising, with high scientific value and practical significance, and also provides important theoretical support for the further development of Atractylodes macrocephala medicinal plant resources. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a eucalyptane-type sesquiterpene dimer compound, its preparation method and application, so as to solve the technical problem that the existing anti-hepatocellular carcinoma drugs do not have strong enough cytotoxic activity.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a eucalyptane-type sesquiterpene dimer compound, which is any one of the following compounds 1 to 6, and the chemical structural formulas of compounds 1 to 6 are shown below:

[0005]

[0006] This invention also discloses a method for preparing eucalyptane-type sesquiterpene dimers, comprising the following steps:

[0007] S1. The rhizome of Atractylodes macrocephala was dried, pulverized, and then extracted with ethanol. The extracts were combined to obtain an ethanol extract. Then, it was extracted with ethyl acetate. The extract was separated by normal-phase silica gel column chromatography. Finally, gradient elution was performed with a volume ratio of petroleum ether:ethyl acetate = 100:0 to 0:100 to obtain 7 fractions from Fr.A to Fr.G.

[0008] S2. The Fr.C fraction was separated by normal phase silica gel column chromatography, followed by gradient elution with petroleum ether:ethyl acetate = 50:1 to 0:100 (volume ratio) to obtain Fr.C1 to Fr.C22 fractions;

[0009] S3. The Fr.C14 fraction was separated by medium-pressure column chromatography, followed by gradient elution with methanol to obtain Fr.C14-1 to Fr.C14-22 fractions; the Fr.C14-12 fraction was separated by normal-phase silica gel column chromatography, followed by semi-preparative high-performance liquid chromatography to obtain compound 3; the Fr.C14-13 fraction was separated by medium-pressure column chromatography and semi-preparative high-performance liquid chromatography to obtain compound 2;

[0010] S4. The Fr.C19 fraction was first separated by normal-phase silica gel column chromatography, followed by gradient elution with petroleum ether:ethyl acetate at a volume ratio of 50:1 to 0:1 to obtain Fr.C19-1 to Fr.C19-11; Fr.C19-4 was separated by medium-pressure column chromatography and semi-preparative high-performance liquid chromatography to obtain compound 5; Fr.C19-5 was separated by medium-pressure column chromatography and semi-preparative high-performance liquid chromatography to obtain compounds 1, 4 and 6.

[0011] Based on the above technical solution, the present invention can be further improved as follows:

[0012] Furthermore, the steps for separation by normal-phase silica gel column chromatography are as follows: gradient elution is performed using a volume ratio of petroleum ether:ethyl acetate = 100:0 to 0:100, with each gradient elution consisting of 3 to 6 column volumes. Elution fractions are collected at 1 / 3 to 1 / 4 of the column volume. Fractions with the same characteristics are combined according to the TLC detection results to obtain subfractions.

[0013] Furthermore, the steps of medium-pressure column chromatography separation are as follows: the chromatographic packing material is ODS, and gradient elution is performed using a volume ratio of methanol:water = 50:50 to 100:0. Each gradient elution is performed for 3 to 6 column volumes, and the eluted fractions are collected at 1 / 5 to 1 / 10 of the column volume. Based on the TLC detection results, the same fractions are combined to obtain the subcomponents.

[0014] Furthermore, the semi-preparative high-performance liquid chromatography separation steps are as follows: using a semi-preparative C18 column or cholesterol column, isocratic elution is performed with 60%–90% methanol or acetonitrile to obtain the subfraction.

[0015] This invention also discloses the application of eucalyptane-type sesquiterpene dimers in the preparation of drugs for treating hepatocellular carcinoma.

[0016] The beneficial effects of this invention are as follows:

[0017] The six eucalyptane-type sesquiterpene dimers (compounds 1-6) disclosed in this invention are novel compounds with defined stereostructures and optically pure composition, exhibiting varying degrees of cytotoxic activity against HepG2, Hep3B, and Huh7 cells. Using sorafenib as a positive control, compounds 1, 5, and 6 showed stronger cytotoxic activity against HepG2 cells than the positive control sorafenib, with compound 6 exhibiting the strongest activity (IC50).50 Compound 5 (IC50 = 5.78 μM) showed the strongest cytotoxic activity against Hep3B cells (IC50 = 5.78 μM). 50 =7.33 μM), compounds 1, 4, 5 and 6 showed stronger cytotoxic activity against Huh7 cells than the positive control drug Sorafenib, with compound 6 exhibiting the strongest cytotoxic activity against Huh7 cells (IC50 = 7.33 μM). 50 =3.71 μM). Therefore, compounds 1-6 disclosed in this invention have the potential to be developed into anti-hepatocellular carcinoma drugs and have good application prospects. At the same time, they also provide important theoretical support for the further development of Atractylodes macrocephala medicinal plant resources. Attached Figure Description

[0018] Figure 1 For compound 1 1 H NMR spectrum;

[0019] Figure 2 For compound 1 13 C NMR spectrum;

[0020] Figure 3 For compound 2 1 H NMR spectrum;

[0021] Figure 4 For compound 2 13 C NMR spectrum;

[0022] Figure 5 For compound 3 1 H NMR spectrum;

[0023] Figure 6 For compound 3 13 C NMR spectrum;

[0024] Figure 7 For compound 4 1 H NMR spectrum;

[0025] Figure 8 For compound 4 13 C NMR spectrum;

[0026] Figure 9 For compound 5 1 H NMR spectrum;

[0027] Figure 10 For compound 5 13 C NMR spectrum;

[0028] Figure 11 For compound 6 1 H NMR spectrum;

[0029] Figure 12 For compound 6 13C NMR spectrum;

[0030] Figure 13 The X-ray single-crystal diffraction ORTEP pattern of compound 1;

[0031] Figure 14 The X-ray single-crystal diffraction ORTEP pattern of compound 2;

[0032] Figure 15 The X-ray single-crystal ORTEP pattern of compound 3;

[0033] Figure 16 The X-ray single-crystal diffraction ORTEP pattern of compound 4 is shown. Detailed Implementation

[0034] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0035] Example

[0036] A method for preparing a eucalyptane-type sesquiterpene dimer compound includes the following steps:

[0037] S1. 100 kg of Atractylodes macrocephala rhizomes were dried, pulverized, and then extracted three times with 10 times the amount of 95% ethanol. The extracts were combined and the solvent was recovered to obtain an ethanol extract. The ethanol extract was then suspended in an appropriate amount of water and extracted with ethyl acetate. The extract (1.8 kg) was passed through a normal-phase silica gel column and eluted with a gradient of petroleum ether:ethyl acetate at volume ratios of 100:0, 50:1, 15:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, and 0:100. Each gradient eluted 3 to 6 column volumes. The eluted fractions were collected at 1 / 3 to 1 / 4 of the column volume. According to the TLC identification results, the same fractions were combined to obtain 7 components from Fr.A to Fr.G.

[0038] S2. The Fr.C fraction (337g) was separated by normal-phase silica gel column chromatography, followed by gradient elution with petroleum ether:ethyl acetate at volume ratios of 50:1, 30:1, 15:1, 10:1, 8:1, 6:1, 4:1, 1:1 and 0:100 to obtain the Fr.C1 to Fr.C22 fractions.

[0039] S3. The Fr.C14 fraction (25.2 g) was separated by medium-pressure column chromatography (1300 mL ODS, flow rate 30 mL / min, mobile phase using methanol:water = 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0 volume ratios for gradient elution). Each gradient elution was performed for 3 to 6 column volumes. The eluted fractions were collected at 1 / 5 to 1 / 10 of the column volume. Fractions with the same characteristics were combined according to the TLC identification results to obtain the Fr.C14-1 to Fr.C14-22 fractions.

[0040] The Fr.C14-12 fraction was separated by normal-phase silica gel column chromatography (gradient elution with petroleum ether:ethyl acetate = 10:1, 8:1, 6:1, 4:1 and 1:1 volume ratios), and then separated by semi-preparative high-performance liquid chromatography (semi-preparative C18 column, flow rate of 3 mL / min, mobile phase isocratic elution with 88% methanol) to obtain compound 3;

[0041] Fr.C14-13 fraction was separated by medium-pressure column chromatography (200 mL ODS, flow rate 20 mL / min, 0-180 min, mobile phase 80%-100% methanol) and semi-preparative high-performance liquid chromatography (semi-preparative C18 column, flow rate 3 mL / min, mobile phase 88% methanol isocratic elution) to obtain compound 2;

[0042] S4. The Fr.C19 fraction was first separated by normal-phase silica gel column chromatography, followed by gradient elution with petroleum ether:ethyl acetate at volume ratios of 50:1, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 to obtain Fr.C19-1 to Fr.C19-11; Fr.C19-4 was separated by medium-pressure column chromatography (400 mL ODS, flow rate 15 mL / min, 0-360 min, mobile phase 60%-100% methanol) and semi-preparative high-performance liquid chromatography (cholesterol column, flow rate 3 mL / min, isocratic elution with 68% acetonitrile) to obtain compound 5;

[0043] Fr.C19-5 was separated by medium-pressure column chromatography (400 mL ODS, flow rate 15 mL / min, 0–360 min, mobile phase 60%–100% methanol) and semi-preparative high-performance liquid chromatography (using a semi-preparative C18 column, flow rate 3 mL / min, mobile phase 80% methanol isocratic elution and a cholesterol column, flow rate 3 mL / min, mobile phase 70% acetonitrile isocratic elution) to obtain compounds 1, 4, and 6.

[0044] Compound 1: Atramacronin A, colorless bulk crystals. UV(CH3OH)λ max(logε)218(4.13),IR(KBr)cm -1 :3445,2974,2945,2841,1749,1697,1647,1443,1389,1323,989,943,881; HR ESIMS m / z 519.2711(calcd.for C 30 H 40 O6Na + ,519.2718). X-ray crystal data: C 30 H 40 O6,M=496.62g / mol,orthorhombic,space group P212121, α=90, β=90, γ=90, Z = 4, μ(Cu Kα) = 0.672 mm -1 ,ρ calc =1.219g / cm 3 ,F(000)=1072.0;crystal size:0.24×0.22×0.2mm 3 ;12727reflections measured(4.191°≤2Θ≤65.080°),4454unique(R int =0.0748), finalR1=0.0622(I>2σ(I)), wR2=0.1646 (all data), flack parameter = 0.0 (3).

[0045] Compound 2: Atramacronin B, colorless bulk crystals. UV(CH3OH)λ max (logε)219(4.20),IR(KBr)cm -1 :3446,2976,2934,2870,1774,1689,1649,1453,1445,1323,1151,1119,991,941,889; HR ESIMS m / z 519.2712(calcd.for C 30 H 40 O6Na + ,519.2718). X-ray crystal data: C 30 H 40O6,M=496.62g / mol,orthorhombic,space group P21212, α=90, β=90, γ=90, Z=4, T=169.99(10)K, μ(Cu Kα)=0.553mm -1 ,ρ calc =1.002g / cm 3 ,F(000)=1072.0;crystal size:0.16×0.12×0.09mm 3 ;22485reflections measured(4.852°≤2Θ≤133.17°),5831unique(R int =0.0525,R sigma = 0.0391), final R1 = 0.0412 (I > 2σ(I)), wR2 = 0.1045 (all data), flack parameter = 0.08 (11).

[0046] Compound 3: Atramacronin C, colorless bulk crystals. UV(CH3OH)λ max (logε)219(4.11),IR(KBr)cm -1 :3487,3452,2987,2928,2849,1769,1691,1649,1441,1385,1319,1109,997,931; HR ESIMS m / z 519.2711(calcd.for C 30 H 40 O6Na + ,519.2718). X-ray crystal data: C 30 H 40 O6,M=496.62g / mol,orthorhombic,space group P212121, α=90, β=90, γ=90, Z=4, T=150.00(10)K, μ(Cu Kα)=0.663mm -1 ,ρ calc =1.203g / cm 3,F(000)=1072.0;crystal size:0.16×0.14×0.12mm 3 ;10825reflections measured(8.614°≤2Θ≤147.524°),5319unique(R int =0.0291,R sigma = 0.0339), final R1 = 0.0382 (I > 2σ(I)), wR2 = 0.1031 (all data), flack parameter = -0.03 (8).

[0047] Compound 4: Atramacronin D, colorless bulk crystals. UV(CH3OH)λ max (logε)219(4.00),IR(KBr)cm -1 :3449,2976,2932,2864,1778,1689,1649,1450,1381,1319,1284,1149,1089,898,941,885; HR ESIMS m / z 519.2708 (calcd.for C 30 H 40 O6Na + ,519.2718). X-ray crystal data: C 30 H 40 O6,M=496.62g / mol,monoclinic,space group P21, α=90, β=91.617(2), γ=90, Z=4, T=169.99(10)K, μ(Cu Kα)=0.669mm -1 ,ρ calc =1.214g / cm 3 ,F(000)=1072.0;crystal size:0.15×0.12×0.11mm 3 ;27704reflections measured(7.682°≤2Θ≤147.836°),10678unique(R int =0.0416,R sigma=0.0436), final R1=0.0507(I>2σ(I)), wR2=0.1424(all data), flack parameter=-0.09(10).

[0048] Compound 5: Atramacronin E, amorphous powder. UV(CH3OH)λ max (logε)219(4.00),IR(KBr)cm -1 :3524,2972,2932,2856,1776,1689,1647,1443,1381,1319,1092,991,956,931,887; HR ESIMS m / z 519.2712(calcd.for C 30 H 40 O6Na + ,519.2718).

[0049] Compound 6: Atramacronin F, amorphous powder. UV(CH3OH)λ max (logε)217(4.06),IR(KBr)cm -1 :3505,3445,3383,2930,2862,1776,1695,1651,1443,1387,1327,1234,1147,1094,960,893; HR ESIMS m / z 519.2712 (calcd.for C 30 H 40 O6Na + ,519.2718).

[0050] Compounds 1-6 1 H NMR data and 13 C NMR data are shown in Table 1, Table 2 and Figures 1-12 As shown, the X-ray single-crystal ORTEP diffraction patterns of compounds 1–4 are as follows: Figures 13-16 As shown.

[0051] Table 1. Compounds Atramacronins AF(1-6) 1 H NMR data (CDCl3, 400 MHz, J in Hz)

[0052]

[0053]

[0054] Table 2. Compounds Atramacronins AF(1-6) 13 C10 NMR data (CDCl3, 100MHz)

[0055]

[0056]

[0057] Experimental Example: MTT assay was used to determine the cytotoxic activity of compounds 1–6 against HepG2, Hep3B, and Huh7 liver cancer cells.

[0058] Using sorafenib as a positive control, the anti-hepatocellular carcinoma activity of compounds 1-6 prepared in this invention was investigated. HepG2, Hep3B, and Huh7 cell lines were inoculated into MEM-α medium supplemented with 10% fetal bovine serum and 1% penicillin antibiotics, and then cultured in a 5% CO2, 37°C cell culture incubator. HepG2, Hep3B, and Huh7 cells in the logarithmic growth phase were collected for cell counting, and the cell density was adjusted based on the counting results. Cells were then seeded into 96-well plates at 1.0 × 10⁶ cells per well. 4 Cells were cultured in a 5% CO2, 37°C incubator for 24 h. After cell attachment, different concentrations of the compound (final concentrations of 20, 15, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0 μM) were added to each well, and incubation continued for 48 h. Then, 20 μL of MTT (5 mg / mL) was added to each well, and the cells were cultured for another 4 h. The supernatant was discarded, and 100 μL of DMSO was added to each well to dissolve formazan crystals. The absorbance (A) at 490 nm was measured using a microplate reader, and the inhibition rate and IC50 of each group were calculated. 50 The values ​​are shown in Table 3.

[0059] Table 3 shows the cytotoxic activities (IC50) of compounds 1–6 against HepG2, Hep3B, and Huh7. 50 μM)

[0060] compound HepG2 Hep3B Huh7 1 8.47±0.66 9.18±0.11 4.30±0.19 2 15.98±0.44 9.34±0.60 17.41±0.64 3 >40 >40 22.85±1.89 4 11.30±0.12 8.81±0.29 8.12±0.21 5 6.34±0.62 7.33±0.67 8.99±0.04 6 5.78±0.42 8.89±0.44 3.71±0.02 Sorafenib 9.48±0.50 3.19±0.39 9.53±0.23

[0061] MTT assay results showed that compounds 1–6 exhibited varying degrees of cytotoxicity against HepG2, Hep3B, and Huh7 cells. Compounds 1, 5, and 6 showed stronger cytotoxic activity against HepG2 cells than the positive control drug Sorafenib, with compound 6 exhibiting the strongest activity (IC50). 50 Compound 5 (IC50 = 5.78 μM) showed the strongest cytotoxic activity against Hep3B cells (IC50 = 5.78 μM). 50 =7.33 μM), compounds 1, 4, 5 and 6 showed stronger cytotoxic activity against Huh7 cells than the positive control drug Sorafenib, with compound 6 exhibiting the strongest cytotoxic activity against Huh7 cells (IC50 = 7.33 μM).50 =3.71 μM). Therefore, compounds 1 to 6 disclosed in this invention have the potential to be developed into anti-hepatocellular carcinoma drugs.

Claims

1. An eudesmane sesquiterpene dimer compound characterized by, The eudesmane type sesquiterpene dimer compound is any one of compounds 1-6, and the chemical structural formulas of the compounds 1-6 are shown as follows:

2. The method of producing the eudesmane-type sesquiterpene dimer compound according to claim 1, characterized by, The method comprises the following steps: S1, drying the atractylodes rhizome, crushing, then extracting with ethanol, and combining the extract to obtain an ethanol extract; Then extracting with ethyl acetate, separating the extract by normal phase silica gel column chromatography, and finally performing gradient elution with petroleum ether: ethyl acetate = 100:0-0:100 by volume to obtain 7 components Fr.A-Fr.G; S2, separating the Fr.C component by normal phase silica gel column chromatography, then performing gradient elution with petroleum ether: ethyl acetate = 50:1-0:100 by volume to obtain Fr.C1-Fr.C22 components; S3, separating the Fr.C14 component by medium pressure column chromatography, then performing gradient elution with methanol to obtain Fr.C14-1-Fr.C14-22 components; separating the Fr.C14-12 component by normal phase silica gel column chromatography, then by semi-preparative high performance liquid chromatography to obtain compound 3; separating the Fr.C14-13 component by medium pressure column chromatography, semi-preparative high performance liquid chromatography to obtain compound 2; S4, separating the Fr.C19 component by normal phase silica gel column chromatography, then performing gradient elution with petroleum ether: ethyl acetate = 50:1-0:1 by volume to obtain Fr.C19-1-Fr.C19-11; separating the Fr.C19-4 by medium pressure column chromatography, semi-preparative high performance liquid chromatography to obtain compound 5; separating the Fr.C19-5 by medium pressure column chromatography, semi-preparative high performance liquid chromatography to obtain compounds 1, 4 and 6.

3. The method of preparing eudesmane-type sesquiterpene dimer compounds according to claim 2, characterized by, The step of normal phase silica gel column chromatography separation is: gradient elution with petroleum ether: ethyl acetate = 100:0-0:100 by volume, 3-6 column volumes for each gradient elution, collecting elution fractions at 1 / 3-1 / 4 of the column volume, and combining the same fractions according to TLC identification results to obtain sub-components.

4. The method for preparing the eucalyptane-type sesquiterpene dimer compound according to claim 2, characterized in that, The step of medium pressure column chromatography separation is: the chromatographic filler is ODS, gradient elution with methanol: water = 50:50-100:0 by volume, 3-6 column volumes for each gradient elution, collecting elution fractions at 1 / 5-1 / 10 of the column volume, and combining the same fractions according to TLC identification results to obtain sub-components.

5. The method for preparing the eucalyptane-type sesquiterpene dimer compound according to claim 2, characterized in that, The step of semi-preparative high performance liquid chromatography separation is: using a semi-preparative C18 column or a cholesterin column, isocratic elution with 60%-90% methanol or acetonitrile to obtain sub-components.

6. Use of the eudesmane type sesquiterpene dimer compound in claim 1 in the preparation of a drug for resisting hepatocellular carcinoma.

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