Isoprenylated flavone compounds, methods of making and uses thereof

Abstract

The present application relates to isoprenyl flavonoids and its preparation method and application, belong to the medical technology field, specifically related to from the thymelaeaceae daphne giraldii nitsche, through silica gel column chromatography, macroporous resin, ODS column chromatography, HPLC and other chromatographic methods such as three isoprenyl flavonoids broussoflavonol C, daphnegiranol G, daphnegiranol E are obtained.The present application also tests the inhibition activity of the above three compounds on Hep3B cells.Finally, it is found that the above compounds play an anti-tumor activity by inhibiting the growth activity of tumor cells.The preparation method of the present application is simple, reproducible, and the obtained compounds have high purity, and some of the compounds have obvious anti-tumor activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to three novel isopentenyl flavonoids isolated from the root bark and stem bark of Daphne odora, their preparation methods, and their applications in anti-tumor treatment. Background Technology

[0002] *Daphne giraldii* Nitsche is a plant belonging to the genus *Daphne* in the family Thymelaeaceae. Its root bark and stem bark are used medicinally, and together with the root bark and stem bark of *Daphne tangutica* and *Daphne retusa*, they are collectively known as "Zushi Ma". It is mainly distributed in Shaanxi, Gansu, Qinghai, Sichuan, and Xinjiang provinces. It is warm in nature, pungent and bitter in taste, and slightly toxic. It has the effects of dispelling wind and dampness, relieving pain, and dispersing blood stasis. Modern pharmacological studies have shown that *Daphne giraldii* possesses various pharmacological activities, including anti-inflammatory and analgesic, antitumor, immunomodulatory, antioxidant, antibacterial, and neuroprotective effects. The root bark and stem bark of *Daphne giraldii* are rich in chemical components, including coumarins, flavonoids, and diterpenoids, among which isopentenyl flavonoids have good antitumor activity.

[0003] Prenylated flavonoids are alkylated products of flavonoids, mainly distributed in legumes and mulberry plants, and isolated from *Daphne odora* in the Thymelaeaceae family. They are characterized by one or more isopentenyl side chains linked to the flavonoid core via C-C or CO bonds, significantly increasing the lipophilicity of the flavonoids and thus improving their bioactivity and bioavailability. Due to the high reactivity of the ortho-hydroxyl groups on the benzene ring, the isopentenyl groups are mostly substituted at the C-6 and C-8 positions of ring A and the C-3' and C-5' positions of ring B. During the biosynthesis of natural plants, they often undergo cyclization, oxidation, and dehydration reactions with the ortho-phenolic hydroxyl groups to form six-membered pyran-cyclic or five-membered furan-cyclic isopentenyl flavonoids. Summary of the Invention

[0004] The purpose of this invention is to provide a series of isopentenyl flavonoids isolated from the root bark and stem bark of Daphne odora, their preparation methods, and their applications in antitumor drugs.

[0005] The isopentenyl flavonoid compounds of this invention have the following structures:

[0006]

[0007] The method for preparing the isopentenyl flavonoid compound of the present invention includes the following steps:

[0008] The dried root bark and stem bark of Daphne odora were extracted with ethanol, and the extracts were concentrated under reduced pressure. The extracts were combined and concentrated to obtain an extract. The extract was extracted with ethyl acetate and n-butanol in sequence. The ethyl acetate extract was then subjected to various chromatographic methods such as silica gel column chromatography, HP-20 macroporous resin, ODS, and HPLC to finally separate the above three new compounds.

[0009] The specific steps are as follows:

[0010] (1) The dried root bark and stem bark of Daphne odora were extracted with ethanol, and the extracts were combined and concentrated to obtain an extract. The extract was then extracted with ethyl acetate and n-butanol. The ethyl acetate fraction was subjected to silica gel column chromatography, and four fractions Fr.A–Fr.D were collected.

[0011] (2) Fraction Fr.A was subjected to HP-20 and ODS column chromatography with ethanol-water gradient elution, and a total of 7 fractions Fr.A1–Fr.A7 were collected;

[0012] (3) The fraction Fr.A5 obtained after elution was subjected to semi-preparative HPLC and further purified by an acetonitrile-water system to obtain the isopentenyl flavonoid compound.

[0013] As an optional approach, where:

[0014] In step (1), the gradient of dichloromethane-methanol is 100:1-1:1; the ethanol is industrial ethanol with a concentration of 70%, and the extraction is carried out by reflux three times, each time for 2 hours.

[0015] In step (2), the ethanol-water gradient is 20%-90%.

[0016] In step (3), the acetonitrile-water ratios used for the three compounds are: broussoflavonol C (38:62, v / v), daphnegiranol G (25:75, v / v), and daphnegiranol E (27:78, v / v).

[0017] The structural analysis of the novel isopentenyl flavonol compounds (broussoflavonol C, daphnegiranol G, daphnegiranol E) prepared in this invention is shown below.

[0018] Compound 1: Yellow amorphous powder (methanol). HRESIMS yields a quasi-molecular ion peak [M+H]. + m / z437.1972(calcd for C 26 H 29 O6,437.1959), combined 1 H-NMR, 13The C-NMR spectrum confirms that the molecular formula of this compound is C. 26 H 29 O6, calculated unsaturation degree is 13, and... 1 H-NMR, 13 The C-NMR signal was assigned, and the planar structure was determined using HMBC spectroscopy, ultimately identifying the compound as an isopentenyl flavonoid. A SciFinder search revealed this compound to be a previously unreported novel compound, which was named broussoflavonol C. 1 H NMR spectrum, 13 The C NMR spectral signal assignments are shown in Table 1, and the relevant spectra are shown in [Table 1]. Figures 1-6 .

[0019] Table 1. Broussoflavonol C 1 H NMR spectrum and 13 C NMR spectral data

[0020]

[0021]

[0022] Compound 2: Red amorphous powder (methanol); HRESIMS yields a quasi-molecular ion peak [M+Na]. + m / z415.1880 (calcd for C 25 H 28 O4Na, 415.1878), combined 1 H-NMR, 13 The C-NMR spectrum confirms that the molecular formula of this compound is C. 25 H 28 O4, calculated unsaturation degree is 12, and... 1 H-NMR, 13 The C-NMR signal was assigned, and the planar structure was determined by HMBC spectroscopy, ultimately identifying the compound as an isopentenyl flavonoid. A SciFinder search revealed this compound to be a novel, previously unreported compound, which was named daphnegiranol G. 1 H NMR spectrum, 13 The C NMR spectral signal assignments are shown in Table 2, and the relevant spectra are shown in [Table 2]. Figures 7-12 .

[0023] Table 2 daphnegiranol G 1 H NMR spectrum and 13 C NMR spectral data

[0024]

[0025] Compound 3: Red amorphous powder (methanol); HRESIMS yields a quasi-molecular ion peak [M+Na]. + m / z399.1917(calcd for C 25 H 28 O3Na,399.1931), combined 1 H-NMR, 13 C10-NMR and HSQC NMR can determine the molecular formula of this compound as C10-NMR. 25 H 28 O3, calculated to have an unsaturation degree of 12, and then... 1 H-NMR, 13 The C-NMR signal was assigned, and the planar structure was determined by HMBC spectroscopy, ultimately identifying the compound as an isopentenyl flavonoid. A SciFinder search revealed this compound to be a previously unreported novel compound, which was named daphnegiranol E. 1 H NMR spectrum, 13 The C NMR spectral signal assignments are shown in Table 3, and the relevant spectra are shown in [Table 3]. Figures 13-18 .

[0026] Table 3 daphnegiranol E 1 H NMR spectrum and 13 C NMR spectral data

[0027]

[0028] A pharmaceutical composition comprising the isopentenyl flavonoid compound and a pharmaceutically acceptable carrier or excipient.

[0029] The use of the isopentenyl flavonoids or the pharmaceutical composition described in this invention in the preparation of antitumor drugs.

[0030] Preferably, the tumor is liver cancer.

[0031] The isopentenyl flavonoids described in this invention achieve anti-tumor effects by inhibiting the growth of tumor cells.

[0032] The beneficial effects of this invention are:

[0033] The compounds involved in this invention exhibit better inhibitory effects on tumor cells than the positive control drug sorafenib, and can be prepared from the traditional Chinese medicine *Zushima*. This invention enriches the chemical research on *Rhizoma Cylindricae*, improves the pharmacological research on *Rhizoma Cylindricae*, and provides a research foundation for subsequent structural modification work targeting its activity. Attached Figure Description

[0034] Figure 1 UV spectrum of compound broussoflavonol C;

[0035] Figure 2 HRESIMS spectrum of compound broussoflavonol C;

[0036] Figure 3 compound broussoflavonol C 1 H NMR spectrum (DMSO-d6, 600MHz);

[0037] Figure 4 compound broussoflavonol C 13 C10 NMR spectrum (DMSO-d6, 150MHz);

[0038] Figure 5 HSQC spectrum of compound broussoflavonol C (DMSO-d6, 600MHz);

[0039] Figure 6 HMBC spectrum of compound broussoflavonol C (DMSO-d6, 600MHz);

[0040] Figure 7 UV spectrum of compound daphnegiranol G;

[0041] Figure 8 HRESIMS spectrum of compound daphnegiranol G;

[0042] Figure 9 compound daphnegiranol G 1 H NMR spectrum (DMSO-d6, 600MHz);

[0043] Figure 10 compound daphnegiranol G 13 C10 NMR spectrum (DMSO-d6, 150MHz);

[0044] Figure 11 HSQC spectrum of compound daphnegiranol G (DMSO-d6, 600MHz);

[0045] Figure 12 HMBC spectrum of compound daphnegiranol G (DMSO-d6, 600MHz);

[0046] Figure 13 UV spectrum of compound daphnegiranol E;

[0047] Figure 14 HRESIMS spectrum of compound daphnegiranol E;

[0048] Figure 15 compound daphnegiranol E 1 H NMR spectrum (DMSO-d6, 600MHz);

[0049] Figure 16 compound daphnegiranol E 13 C10 NMR spectrum (DMSO-d6, 150MHz);

[0050] Figure 17 HSQC spectrum of compound daphnegiranol E (DMSO-d6, 600MHz);

[0051] Figure 18 HMBC spectrum of compound daphnegiranol E (DMSO-d6, 600MHz). Detailed Implementation

[0052] Example 1

[0053] Preparation methods of isopentenyl flavonoids 1-3:

[0054] (1) Take 100 kg of dried root bark and stem bark of Daphne odora and grind them into fragments. Extract them three times with 70% industrial ethanol under reflux for 3 hours each time. Filter the crude extract and concentrate it under reduced pressure to obtain an extract. Add water to the extract and extract it successively with ethyl acetate and n-butanol. Separate the ethyl acetate layer (1200 g) by silica gel column chromatography (200-300 mesh) and elute with a gradient of dichloromethane-methanol (100:1-1:1) to obtain fractions Fr.A-Fr.D.

[0055] (2) The fraction Fr.A was eluted with ethanol-water (20%-90%) on HP-20 macroporous resin, and then subjected to ODS column chromatography with the same ethanol-water gradient elution. After analysis, seven fractions Fr.A1-Fr.A7 were obtained.

[0056] (4) The eluted fraction Fr.A5 was further purified by semi-preparative HPLC using an acetonitrile-water system to obtain isopentenyl flavonol compounds broussoflavonol C (48.6 mg), daphnegiranol G (33.9 mg), and daphnegiranol E (30.4 mg). The acetonitrile-water ratios used for the three compounds were broussoflavonol C (38:62, v / v), daphnegiranol G (25:75, v / v), and daphnegiranol E (27:78, v / v).

[0057] Experiment Example 2

[0058] The inhibitory effects of compounds broussoflavonol C, daphnegiranol G, and daphnegiranol E on Hep3B cells were determined in vitro to explore their antitumor activity.

[0059] (1) Cell Culture

[0060] Human hepatocellular carcinoma cells (Hep3B, purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody at 37°C in a 5% CO2 incubator. Cells that were stably passaged to logarithmic growth were used for experiments.

[0061] (2) Cell grouping

[0062] Control group: Hep3B cells in logarithmic growth phase were seeded at a density of 4000 cells / well in 96-well plates after trypsin digestion. After 12 hours of culture, 100 μL of blank culture medium was added to each well and cultured for 48 hours. Then, 50 μL of 30% TCA fixative was added to each well and fixed at 4°C for 1 hour.

[0063] Group 1: Hep3B cells in logarithmic growth phase were seeded at a density of 4000 cells / well in 96-well plates after trypsin digestion. After 12 h of culture, 100 μL of broussoflavonol C was added to each well and cultured for 48 h. Then, 50 μL of 30% TCA fixative was added to each well and the cells were fixed at 4 °C for 1 h.

[0064] Compound group 2: Hep3B cells in logarithmic growth phase were seeded at a density of 4000 cells / well in 96-well plates after trypsin digestion. After 12 h of culture, 100 μL of compound daphnegiranol G was added to each well and cultured for 48 h. Then, 50 μL of 30% TCA fixative was added to each well and the cells were fixed at 4 °C for 1 h.

[0065] Compound group 3: Hep3B cells in logarithmic growth phase were seeded at a density of 4000 cells / well in 96-well plates after trypsin digestion. After 12 h of culture, 100 μL of compound daphnegiranol E was added to each well and cultured for 48 h. Then, 50 μL of 30% TCA fixative was added to each well and fixed at 4°C for 1 h.

[0066] Positive drug group: Hep3B cells in the logarithmic growth phase were seeded at a density of 4000 cells / well in 96-well plates after trypsin digestion. After 12 hours of culture, 100 μL of the positive drug sorafenib (Sora) was added to each well and cultured for 48 hours. Then, 50 μL of 30% TCA fixative was added to each well and the cells were fixed at 4°C for 1 hour.

[0067] Discard the liquid from the wells and rinse with tap water. After drying, add 0.4% SRB (50-70 μL) and stain at room temperature in the dark for 30 minutes. After 30 minutes, dry the wells and wash the 96-well plate with 1% acetic acid until the liquid is colorless. After air drying, add 100 μL of Tris, vortex for 20 minutes, and detect the microplate at 540 nm using a microplate reader.

[0068] Growth inhibition rate (%) = [A 540 (Negative control)-A 540 (Drug-dosing group) / A 540 (Negative control) ×100%, and then the half-maximal inhibitory concentration (IC50) of the compound was determined using GraphPad Prism 8 data analysis software. 50 ).

[0069] (3) Experimental Results

[0070] Data on the tumor cell inhibitory effects of compounds 1-3 (IC50) 50 As shown in Table 4 below, the unit is μM:

[0071] Table 4: Results of the inhibitory effect of the compounds of the present invention on tumor cells

[0072]

[0073] Experimental results showed that daphnegiranol E exhibited the best inhibitory activity against Hep3B cells, with an IC50 value of [missing information]. 50 The value is 2.53±0.49μM.

[0074] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An isopentenyl flavonoid compound, characterized in that, It is a compound as shown below; 。 2. A method for preparing the isopentenyl flavonoid compound according to claim 1, characterized in that, Includes the following steps: (1) The dried root bark and stem bark of Daphne odora were extracted with ethanol, and the extracts were combined and concentrated to obtain an extract. The extract was then extracted with ethyl acetate and n-butanol. The ethyl acetate fraction was subjected to silica gel column chromatography and eluted with dichloromethane-methanol gradient. Four fractions, Fr. A – Fr. D, were collected. (2) Fraction Fr. A was subjected to HP-20 column chromatography and then ODS column chromatography with ethanol-water gradient elution, and a total of 7 fractions Fr. A1 – Fr. A7 were collected; (3) The fraction Fr. A5 obtained after elution was subjected to semi-preparative HPLC and further purified by an acetonitrile-water system to obtain the isopentenyl flavonoid compound.

3. The method for preparing isopentenyl flavonoids according to claim 2, characterized in that, In step (1), the gradient of dichloromethane-methanol is 100:1 - 1:1; the ethanol is industrial ethanol with a concentration of 70%, and it is refluxed and extracted 3 times, each time for 2 hours.

4. The method for preparing isopentenyl flavonoids according to claim 2, characterized in that, In step (2), the ethanol-water gradient is 20% - 90%.

5. The method for preparing isopentenyl flavonoids according to claim 2, characterized in that, In step (3), the acetonitrile-water ratio used for compound 3 is 27:

78. v / v .

6. A pharmaceutical composition, characterized in that, It comprises the isopentenyl flavonoid compound of claim 1 and a pharmaceutically acceptable carrier or excipient.

7. The use of the isopentenyl flavonoid compound according to claim 1 in the preparation of an anti-liver cancer drug.

8. Use of the pharmaceutical composition according to claim 6 in the preparation of an anti-liver cancer drug.

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