A abietane-type diterpenoid compound, preparation method, application and anti-tumor drug

Abstract

The present invention relates to the technical field of anti-tumor compounds, and discloses a abietane-type diterpenoid compound, a preparation method, an application and an anti-tumor drug. The compound has the chemical structure of formula I, or a pharmaceutical composition of a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug of the compound of formula I. The present invention can extract and prepare the abietane-type diterpenoid compound from Leucosceptrum canum, and the compound has an inhibitory effect on human liver cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor compounds, and particularly to a abietane-type diterpene compound, a preparation method, an application and an anti-tumor drug. Background Art

[0002] Leucosceptrum canum Smith, also known as Zitanghua and Mifengshu flower, is a shrub or small tree. Its young branches are densely covered with villi. The leaves are elliptic-lanceolate, 10-23 cm long and 5-9 cm wide, with a gradually pointed apex, a cuneate base, serrulate or serrate margins, and the abaxial surface is densely covered with grayish-white or pale yellow stellate villi and tufted hairs. The verticillaster is many-flowered and arranged into a terminal long cylindrical spike-like inflorescence; the bracts are reniform, entire or with irregular teeth, and are covered with stellate villi outside; the bracteoles are linear, about 1 mm long, and are densely covered with stellate villi; the calyx is campanulate, covered with pale yellow stellate villi and powdery cyst-like protuberances outside, glabrous inside, and the calyx teeth are triangular, about 1.5 mm long or slightly shorter; the corolla is white or pink to purplish-red, the corolla tube is included or slightly exserted, without a hairy annulus inside, the labial part is bilabiate, the upper lip is slightly emarginate at the apex, and the lower lip is 3-lobed, with the middle lobe being larger. The nutlets are oblong-trigonal, truncate at the apex, and the hilum is small and located at the base. The flowering period is from November to March of the following year, and the fruiting period is from March to May. Its nature and flavor are bitter and cool. This plant has the effects of clearing heat and detoxifying, promoting diuresis and detumescence, and stopping bleeding.

[0003] Abietane-type diterpene compounds have been used in pharmacy. For example, the patent with the application number CN101633660A discloses a new abietane-type diterpene compound, its preparation method and uses. It discloses the physicochemical properties and optical activity of oridonin, and uses the MTT method for in vitro activity screening. The results show that it has obvious inhibitory effects on mouse sarcoma cells and mouse liver cancer cells, and can be used as a lead compound for developing new anti-tumor drugs, and can also be used as a drug for developing drugs for treating various common and frequently-occurring cancers in clinical practice.

[0004] The above technical solution extracts abietane-type diterpene compounds from Rabdosia rubescens as anti-tumor drugs. However, more than 60 chemical components have been isolated from plants of the genus Leucosceptrum so far, including sesquiterpenoids, flavonoids, phenyl ethanol glycosides, acyl glycosides and phenols, etc., among which sesquiterpenoids and flavonoids are the main structural types. However, there is no report on the research of applying the chemical components of Leucosceptrum canum Smith branches to anti-tumor activity. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an abietane-type diterpene compound, a preparation method, an application and an anti-tumor drug, which can extract and prepare abietane-type diterpene compounds from Leucosceptrum canum Smith, and this compound has an inhibitory effect on human liver cancer cells.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] In a first aspect, the present invention provides a abietane-type diterpenoid compound Leucosceptrum B, with the chemical name of 3,11,12-trihydroxy-8,11,13,15-abietatetraene-7-one, and its structure is shown in the following formula I:

[0008] The present invention also provides a pharmaceutical composition of the compound of formula I in the form of a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug.

[0009] In a second aspect, the present invention also provides a method for preparing the above-mentioned abietane-type diterpenoid compound, adopting the following technical scheme:

[0010] Step (1): Using the air-dried branches of Leucosceptrum canum as raw materials, after pulverization, extract with an organic solvent, and concentrate under reduced pressure at low temperature to obtain an extract.

[0011] Step (2): Mix the extract with water to obtain a suspension, add ethyl acetate for extraction, and concentrate under reduced pressure at low temperature to obtain an ethyl acetate extraction phase.

[0012] Step (3): Separate the ethyl acetate extraction phase by silica gel column chromatography, and perform gradient elution using petroleum ether - ethyl acetate as the eluent. The volume ratios of petroleum ether to ethyl acetate are 9.8 - 10.2:1, 4.8 - 5.2:1, 2.8 - 3.2:1, and 0.8 - 1.2:1 in sequence. Collect the eluate corresponding to the volume ratio of petroleum ether to ethyl acetate of 0.8 - 1.2:1, and then analyze by thin-layer chromatography, and combine similar components to obtain different initial components F41 - F44.

[0013] Step (4): Separate the initial component F44 by preparative liquid chromatography, and perform isocratic elution using acetonitrile and water as the eluent to obtain the abietane-type diterpenoid compound Leucosceptrum B shown in formula I.

[0014] Further, in step (1), the organic solvent is ethanol with a volume fraction ≥ 90%, and the volume-mass ratio with the Leucosceptrum canum branches is 10 - 20 mL:1 g; the extraction temperature is 65 - 75 °C, and the extraction is carried out 2 - 3 times, 4 - 6 h each time.

[0015] Further, in steps (1) and (2), the temperature for concentration under reduced pressure at low temperature is 40 - 50 °C, and the vacuum degree is 0.07 - 0.09 MPa.

[0016] Further, in step (2), the mass ratio of the extract to water is 1:1 - 3, the volume ratio of ethyl acetate to the suspension is 0.8 - 1:1, and the extraction is carried out 2 - 4 times.

[0017] Further, in step (4), the volume fraction of acetonitrile in the eluent is 30 - 40%, and the rest is water.

[0018] The abietane - type diterpenoid compound Leucosceptrum B of the present invention is obtained from the branches of Leucosceptrum canum by crushing, extraction, vacuum concentration, extraction and multiple separation and purification. When preparing, dry branches of Leucosceptrum canum or dried branches of Leucosceptrum canum are selected for use, which is convenient for branch crushing. Using an eluent with equal volumes of petroleum ether and ethyl acetate as the target separation system, and at the same time, due to the large number of impurity components in Leucosceptrum canum, the abietane - type diterpenoid compound Leucosceptrum B cannot be obtained by elution with a single concentration; and if the concentration gradient is set unreasonably, too large or too small may result in the abietane - type diterpenoid compound Leucosceptrum B not being fully eluted. In the absence of reference materials providing indications for extracting the abietane - type diterpenoid compound Leucosceptrum B, there is great uncertainty in processing the branches of Leucosceptrum canum and obtaining Leucosceptrum B from them.

[0019] Thirdly, the present invention also provides the application of a pharmaceutical composition containing the above - mentioned abietane - type diterpenoid compound, or its pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug in the preparation of an anti - tumor drug.

[0020] Furthermore, the tumor cells corresponding to the tumor are human liver cancer SMMC7721 cells.

[0021] Fourthly, the present invention also provides an anti - tumor drug, which includes a pharmaceutically acceptable carrier and the above - mentioned compound, or a pharmaceutical composition of the compound's pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug.

[0022] The beneficial effects of the present invention are as follows:

[0023] The abietane - type diterpenoid compound Leucosceptrum B of the present invention is a natural anti - tumor compound extracted from the branches of Leucosceptrum canum, and has the biological activity of inhibiting tumor cells. For example, it has an obvious inhibitory effect on human liver cancer SMMC7721 cells, and the half - inhibitory concentration IC50 value is smaller than that of the broad - spectrum anti - tumor drug cisplatin on the market, indicating that it has strong anti - tumor activity and has great application prospects in the preparation of anti - tumor drugs, which plays an important role in enhancing the medical and economic value of Leucosceptrum canum. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the preparation flow chart of the abietane - type diterpenoid compound Leucosceptrum B. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The present invention will be described in detail below in combination with examples and drawings:

[0026] Unless otherwise specified, the materials used in the present invention can be obtained from the market or are commonly used in the art. Unless otherwise specified, the methods in the following examples are conventional methods in the art.

[0027] Example 1: Physicochemical Property Tests of Abietane-Type Diterpenoid Leucosceptrum B

[0028] 1.1 Determination of Molecular Structural Formula

[0029] The nuclear magnetic resonance instrument used was a Bruker AVANCE III 500-type nuclear magnetic resonance instrument manufactured by Bruker Corporation. Deuterated CDCl3 (deuterated chloroform) reagent was used for nuclear magnetic resonance. The hydrogen spectrum and carbon spectrum data of the abietane-type diterpenoid Leucosceptrum B are shown in Table 1 below.

[0030] Table 1 Hydrogen Spectrum 1H-NMR (500 MHz) and Carbon Spectrum 13C-NMR (125 MHz) Data of Leucosceptrum B

[0031]

[0032]

[0033] Note: Chemical shift is represented by δ; s represents singlet; br s represents broad singlet; m represents multiplet; dd represents double doublet.

[0034] 1.2 Molecular Weight Test

[0035] The accurate molecular weight of the compound was determined by high-resolution mass spectrometry HR-ESI-MS. The HR-ESI-MS [M-H]-m / z was 329.1753. According to the NMR data and mass spectrometry data in Table 1, the molecular formula of the abietane-type diterpenoid Leucosceptrum B was determined to be C20H26O4, with a molecular weight of 330, an unsaturation degree of 8, and the chemical name was 3,11,12-trihydroxy-8,11,13,15-abietatetraene-7-one, and the structure was as shown in Formula I.

[0036] 1.3 Melting Point Test

[0037] The melting point instrument used was a WRS-1C type melting point instrument manufactured by Shanghai Physical Optics; after testing, the melting point of the abietane-type diterpenoid Leucosceptrum B was m.p. 252 - 254 °C.

[0038] Example 2:

[0039] This example is for the preparation of abietane-type diterpenoid Leucosceptrum B

[0040] Using the method of the present invention to prepare compound Leucosceptrum B, as Figure 1 shown, includes the following steps:

[0041] (1) Air-dry and crush 5 kg of the branches of Leucosceptrum canum, add them to ethanol with a volume fraction of 95%, add ethanol according to the ratio of the mass of Leucosceptrum canum to the volume of ethanol of 1 g:10 mL, the extraction temperature is 70 °C, extract 3 times, each time for 5 hours; filter the insoluble matter and combine the filtrates, and then evaporate to dryness under reduced pressure at a low temperature under a vacuum of 0.08 MPa and a temperature of 45 °C to obtain an extract;

[0042] (2) Mix the obtained extract with water in a mass ratio of 1:2 to make a suspension, extract with an equal volume of ethyl acetate 3 times, combine the extracts, and evaporate to dryness under reduced pressure at a low temperature under a vacuum of 0.08 MPa and a temperature of 45 °C to obtain an ethyl acetate extraction phase;

[0043] (3) Mix the obtained ethyl acetate extraction phase with silica gel of equal mass, then place it in the middle layer of a silica gel column for chromatography, use petroleum ether - ethyl acetate as the eluent for gradient elution, and the volume ratios of petroleum ether to ethyl acetate in each gradient elution are 10:1, 5:1, 3:1, 1:1. Collect the eluent when the volume ratio of petroleum ether to ethyl acetate is 1:1, analyze by thin-layer chromatography and combine similar components to obtain different primary components F41 - F44;

[0044] (4) Separate the collected primary component F44 by preparative liquid chromatography, use acetonitrile: water with a volume ratio of 35:65 as the eluent for isocratic elution to obtain the diterpenoid compound A (10.2 mg) with antitumor activity shown in formula I.

[0045] Example 3:

[0046] This example is for the preparation of abietane-type diterpenoid compound Leucosceptrum B

[0047] Using the method of the present invention to prepare compound Leucosceptrum B, includes the following steps:

[0048] (1) Air-dry and crush 5 kg of the branches of Leucosceptrum canum, add them to ethanol with a volume fraction of 90%, the addition amount is 20 mL / g of Leucosceptrum canum, the extraction temperature is 65 °C, extract 3 times, each time for 4 hours; filter the insoluble matter and combine the filtrates, and then evaporate to dryness under reduced pressure at a low temperature under a vacuum of 0.07 MPa and a temperature of 50 °C to obtain an extract;

[0049] (2) Mix the obtained extract with water at a mass ratio of 1:1 to make a uniform suspension, extract with ethyl acetate of equal volume 4 times, combine the extraction solutions, and evaporate to dryness under reduced pressure at a vacuum degree of 0.07 MPa and a temperature of 50 °C to obtain the ethyl acetate extraction phase;

[0050] (3) Mix the obtained ethyl acetate extraction phase with silica gel of equal mass evenly, then place it in the middle layer of a silica gel column for chromatography, use petroleum ether - ethyl acetate as the eluent for gradient elution, and the volume ratios of petroleum ether to ethyl acetate in each gradient elution are 9.8:1, 4.8:1, 2.8:1, 0.8:1. Collect the eluent when the volume ratio of petroleum ether to ethyl acetate is 0.8:1, analyze by thin layer chromatography and combine similar components to obtain different preliminary components F41 - F44;

[0051] (4) Subject the collected preliminary component F44 separated by chromatography to preparative liquid chromatography separation, and perform isocratic elution with acetonitrile: water (volume ratio 30:70) as the eluent to obtain the diterpenoid compound A (7.0 mg) with antitumor activity shown in Formula I.

[0052] Example 4:

[0053] This example is for the preparation of abietane - type diterpenoid compound Leucosceptrum B

[0054] Use the method described in the present invention to prepare compound Leucosceptrum B, including the following steps:

[0055] (1) Air - dry and crush 5 kg of the branches of Leucosceptrum canum, add them to ethanol with a volume fraction of 95%, the addition amount is 15 mL of ethanol per gram of Leucosceptrum canum, the extraction temperature is 75 °C, extract 2 times, 4 hours each time; filter the insoluble substances and combine the filtrates, then evaporate to dryness under reduced pressure at a vacuum degree of 0.09 MPa and a temperature of 40 °C to obtain an extract;

[0056] (2) Mix the obtained extract with water at a mass ratio of 1:3 to make a uniform suspension, extract with ethyl acetate of equal volume 2 times, combine the extraction solutions, and evaporate to dryness under reduced pressure at a vacuum degree of 0.09 MPa and a temperature of 40 °C to obtain the ethyl acetate extraction phase;

[0057] (3) Mix the obtained ethyl acetate extraction phase with silica gel of equal mass evenly, then place it in the middle layer of a silica gel column for chromatography, use petroleum ether - ethyl acetate as the eluent for gradient elution, and the volume ratios of petroleum ether to ethyl acetate in each gradient elution are 10.2:1, 5.2:1, 3.2:1, 1.2:1. Collect the eluent when the volume ratio of petroleum ether to ethyl acetate is 1.2:1, analyze by thin layer chromatography and combine similar components to obtain different preliminary components F41 - F44;

[0058] (4) The initially collected chromatographically separated fraction F44 was separated by preparative liquid chromatography, and isocratically eluted with acetonitrile: water (40:60) as the eluent to obtain 7.5 mg of the diterpenoid compound A with antitumor activity shown in Formula I.

[0059] Example 5: Antitumor performance test of abietane-type diterpenoid Leucosceptrum B

[0060] The abietane-type diterpenoid Leucosceptrum B prepared in Example 2 was used for the antitumor ability test, and the process was as follows:

[0061] In vitro tumor cell proliferation inhibition test: Tumor cells in the logarithmic growth phase were taken, the cell suspension concentration was adjusted (50,000 - 100,000 cells / ml), 100 μl of the cell suspension was inoculated into a 96-well cell culture plate per well. After 24 h of inoculation, drugs were administered (100 μl / well). A blank control group, a cell control group, and six concentration groups (3.12, 6.25, 12.5, 25, 50, 100 μmol / L) of the test drug group were set up respectively. The drug in the cell control group was cisplatin. After continuous culture for 72 h, 100 μl of MTT (1 mg / ml, dissolved in DMEM culture medium) was added to each well, incubated at 37 °C for 4 h. After discarding the liquid in each well, 150 μl of acidified isopropanol (containing 0.04 mol / L HCl) was added, and it was placed in the dark for 30 min. The absorbance at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the proliferation inhibition rate of the test drug on tumor cells was calculated. The half-maximal inhibitory concentration (IC50) of the test substance on tumor cell proliferation (72 h) was calculated. To reduce the influence of experimental errors, three parallels were performed for each experiment, and the results were as follows.

[0062] 5.1 Human hepatocellular carcinoma SMMC7721 cells

[0063] (1) The inhibitory effect of cisplatin in the control group on SMMC7721 cells is shown in Tables 2, 3, and 4.

[0064] Table 2 Results of the first parallel test of the inhibitory effect of cisplatin on SMMC7721 cells

[0065]

[0066] The IC50 = 21.97 was obtained by Graphpad Prism 6.0.

[0067] Table 3 Results of the second parallel test of the inhibitory effect of cisplatin on SMMC7721 cells

[0068]

[0069]

[0070] The IC50 value obtained by Graphpad Prism 6.0 is 22.68.

[0071] Table 4 Results of the third parallel test on the inhibitory effect of cisplatin on SMMC7721 cells

[0072]

[0073] The IC50 value obtained by Graphpad Prism 6.0 is 18.72.

[0074] From the above data, the IC50 of cisplatin can be calculated as 21.12 ± 2.11.

[0075] (2) The inhibitory effect of compound Leucosceptrum B on SMMC7721 cells is shown in Tables 5, 6, and 7.

[0076] Table 5 Results of the first parallel test on the inhibitory effect of compound Leucosceptrum B on SMMC7721 cells

[0077]

[0078] The IC50 value obtained by Graphpad Prism 6.0 is 19.93.

[0079] Table 6 Results of the second parallel test on the inhibitory effect of compound Leucosceptrum B on SMMC7721 cells

[0080]

[0081]

[0082] The IC50 value obtained by Graphpad Prism 6.0 is 15.95.

[0083] Table 7 Results of the third parallel test on the inhibitory effect of compound Leucosceptrum B on SMMC7721 cells

[0084]

[0085] The IC50 value obtained by Graphpad Prism 6.0 is 16.04.

[0086] From the above data, the IC50 of Leucosceptrum B can be calculated as 17.31 ± 2.27.

[0087] 5.2 The half-maximal inhibitory concentration IC50 (μmol / L) of Leucosceptrum B and cisplatin on SMMC7721 cells for 72 hours is shown in Table 8.

[0088] Table 8 The 72-hour half-maximal inhibitory concentration IC50 (μmol / L) of Leucosceptrum B and cisplatin on SMMC7721 cells

[0089]

[0090] As can be seen from the above Tables 5-8, compounds of different concentrations of Leucosceptrum B have inhibitory effects on human liver cancer SMMC7721. The IC50 value of Leucosceptrum B after 72 h reaches 17.31 ± 2.27 μmol / L, and its IC50 value is lower than that of the cisplatin control group (21.12 ± 2.11 μmol / L), indicating that Leucosceptrum B has a very significant inhibitory effect on SMMC7721 cells.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

Claims

1. A method for preparing a abietane-type diterpenoid compound, characterized in that: The compound is a compound of formula I, Comprising the following steps: Step (1): Using the air-dried branches of Leucosceptrum canum as raw materials, pulverizing them, extracting with an organic solvent, and concentrating under reduced pressure at low temperature to obtain an extract. Step (2): Mixing the extract with water to obtain a suspension, adding ethyl acetate for extraction, and concentrating under reduced pressure at low temperature to obtain an ethyl acetate extraction phase. Step (3): Separating the ethyl acetate extraction phase by silica gel column chromatography, performing gradient elution with petroleum ether - ethyl acetate as the eluent, and the volume ratios of petroleum ether to ethyl acetate are successively (9.8 - 10.2):1, (4.8 - 5.2):1, (2.8 - 3.2):1, (0.8 - 1.2):1; collecting the eluate corresponding to the volume ratio of petroleum ether to ethyl acetate of (0.8 - 1.2):1, and then analyzing by thin-layer chromatography, and combining similar components to obtain different preliminary components F41 - F44. Step (4): Separating the preliminary component F44 by preparative liquid chromatography, performing isocratic elution with acetonitrile and water as the eluent to obtain the abietane-type diterpenoid compound. The organic solvent in step (1) is ethanol with a volume fraction ≥ 90%, the temperature for concentration under reduced pressure at low temperature in steps (1) and (2) is 40 - 50 °C, the volume fraction of acetonitrile in the eluent in step (4) is 30 - 40%, and the rest is water.

2. The preparation method of a abietane-type diterpenoid compound according to claim 1, characterized in that: In step (1), the volume-mass ratio of the organic solvent to the air-dried branches of Leucosceptrum canum is 10 - 20 mL:1 g; the extraction temperature is 65 - 75 °C, extracting 2 - 3 times, and each extraction is for 4 - 6 h.

3. The preparation method of a abietane - type diterpenoid compound according to claim 2, characterized in that: The vacuum degree for concentration under reduced pressure at low temperature in steps (1) and (2) is 0.07 - 0.09 MPa.

4. The preparation method of a abietane - type diterpenoid compound according to claim 3, characterized in that: In step (2), the mass ratio of the extract to water is 1:1 - 3, the volume ratio of ethyl acetate to the suspension is 0.8 - 1:1, and extracting 2 - 4 times.

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