Isoprenylated flavonoids, methods of making and uses thereof

Abstract

This invention relates to isopentenyl flavonoids, their preparation methods, and applications, belonging to the field of natural product chemistry. Specifically, it involves the separation of five isopentenyl flavonoids—daphengiratriprenylone D, (3"'R)-daphnegiravone F, (3"'S)-daphnegiravone F, daphnegiravone E, and daphnegiralin H—from *Daphne giraldii* Nitsche, a plant in the Thymelaeaceae family, using silica gel column chromatography, macroporous resin chromatography, ODS column chromatography, and HPLC. Their inhibitory activity against HepG2 and Hep3B cells was tested. Ultimately, it was found that these compounds exert their antitumor effect by inhibiting the growth activity of tumor cells. The preparation method of this invention is simple, reproducible, and yields high-purity compounds with significant antitumor activity.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to five new isopentenyl flavonoids isolated from the root bark and stem bark of Daphne odora, their preparation methods, and their uses in antitumor activity. Background Technology

[0002] Daphne giraldii Nitsche, a plant belonging to the genus Daphne in the family Thymelaeaceae, uses its root bark and stem bark in traditional Chinese medicine. It is one of the important source plants for this herb, primarily used to treat injuries from falls and blows, rheumatism, and rheumatoid arthritis, with significant therapeutic effects. It is a commonly used traditional Chinese medicine. The root bark and stem bark of Daphne giraldii are rich in chemical components, including coumarins, flavonoids, and diterpenoids, thus possessing diverse pharmacological activities. Summary of the Invention

[0003] 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 use in the preparation of antitumor drugs.

[0004] In the first aspect, the isopentenyl flavonoid compound of the present invention has the following structure:

[0005]

[0006] In a second aspect, the method for preparing the isopentenyl flavonoid compound of the present invention includes the following steps:

[0007] The dried root bark and stem bark of Daphne odora were extracted with ethanol, the extracts were concentrated under reduced pressure, 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 extract was separated using various chromatographic methods, including silica gel column chromatography, HP-20 macroporous resin, ODS, and HPLC, to finally obtain the above 5 new compounds.

[0008] The specific steps are as follows:

[0009] (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.

[0010] (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;

[0011] (3) The fraction Fr.A6 obtained after elution was subjected to semi-preparative HPLC and further purified by an acetonitrile-water system to obtain the above 5 isopentenyl flavonoids.

[0012] in:

[0013] In step (1), the gradient of dichloromethane-methanol used in silica gel column chromatography is 100:1-1:1; the ethanol is 70% industrial ethanol, and the extraction is carried out by reflux three times for two hours each time.

[0014] In step (2), the ethanol-water gradient is 20%-90%.

[0015] Structural analysis of the novel isopentenyl flavonoids (daphengiratriprenylone D, (3"'R)-daphnegiravone F, (3"'S)-daphnegiravone F, daphnegiravone E, daphnegiralin H).

[0016] Compound 1: Yellow amorphous powder (methanol). HRESIMS yields a quasi-molecular ion peak [M+H]. + m / z521.2567(calcd for C 31 H 37 O7,521.2573), combined 1 H-NMR, 13 The C10-NMR spectrum confirms that the molecular formula of this compound is C10. 31 H 36 O7, calculated unsaturation degree is 14, 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 previously unreported novel compound, which was named daphengiratriprenylone D. 1 HNMR, 13 The assignments of the 1C NMR spectral signals are shown in Tables 1 and 2, respectively, and the relevant spectra are shown in Tables 1 and 2. Figures 1-6 .

[0017] Compounds 2a / 2b: yellow amorphous powder (methanol); HRESIMS yields a quasi-molecular ion peak [M+H]. + m / z439.1762(calcd for C 25 H 27 O7,439.1751), combined 1 H-NMR, 13 The C10-NMR spectrum confirms that the molecular formula of this compound is C10. 25 H 26O7, calculated unsaturation degree is 13, 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 (3"'R)-daphnegiravone F and (3"'S)-daphnegiravone F, respectively. 1 H NMR, 13 The assignments of the 1C NMR spectral signals are shown in Tables 1 and 2, respectively, and the relevant spectra are shown in Tables 1 and 2. Figures 7-12 .

[0018] Compound 3: Yellow amorphous powder (methanol); HRESIMS yields a quasi-molecular ion peak [M+H]. + m / z435.1817(calcd for C 26 H 27 O6,435.1802), combined with 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 unsaturation degree is 14, 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 previously unreported novel compound, which was named daphnegiravone E. 1 HNMR, 13 The assignments of the 1C NMR spectral signals are shown in Tables 1 and 2, respectively, and the relevant spectra are shown in Tables 1 and 2. Figures 13-18 .

[0019] Compound 4: Brown amorphous powder (methanol). HRESIMS yields a quasi-molecular ion peak [M–H₂O+Na]. + m / z447.1814(calcd for C 25 H 28 O6Na, 447.1814), combined 1 H-NMR, 13 The C10-NMR spectrum confirms that the molecular formula of this compound is C10. 25 H 30 O7, calculated unsaturation degree is 11, and... 1 H-NMR, 13The 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 daphnegiralin H. 1 HNMR, 13 The assignments of the 1C NMR spectral signals are shown in Tables 1 and 2, respectively, and the relevant spectra are shown in Tables 1 and 2. Figures 19-24 .

[0020] Table 11, 2a / 2b, 3, 4 1 1H NMR (600MHz) spectral data (δinppm)

[0021]

[0022] Table 21, 2a / 2b, 3, 4 13 C10 NMR (150MHz) spectral data (δin ppm)

[0023]

[0024]

[0025] In a third aspect, the present invention provides a pharmaceutical composition comprising the isopentenyl flavonoid compound described in the first aspect above and a pharmaceutically acceptable carrier or excipient.

[0026] In a fourth aspect, the present invention provides the use of the isopentenyl flavonoid compound described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of an antitumor drug.

[0027] Preferably, in the intended use, the tumor is liver cancer.

[0028] Preferably, the isopentenyl flavonoid compound or the pharmaceutical composition achieves its anti-tumor effect by inhibiting the growth of tumor cells.

[0029] In a fifth aspect, the present invention provides an extract of the root bark and stem bark of Daphne odora, comprising the isopentenyl flavonoid compound, for use in the preparation of antitumor drugs.

[0030] The beneficial effects of this invention are:

[0031] The compound involved in this application exhibits better inhibitory activity against tumor cells than the positive control drug sorafenib, and can be prepared from the traditional Chinese medicine *Zushima*. This application 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

[0032] Figure 1 UV spectrum of compound 1;

[0033] Figure 2 HRESIMS spectrum of compound 1;

[0034] Figure 3 Compound 1 1 H NMR spectrum (DMSO-d6, 600MHz);

[0035] Figure 4 Compound 1 13 C10 NMR spectrum (DMSO-d6, 150MHz);

[0036] Figure 5 HSQC spectrum of compound 1 (DMSO-d6, 600MHz);

[0037] Figure 6 HMBC spectrum of compound 1 (DMSO-d6, 600MHz);

[0038] Figure 7 UV spectra of compounds 2a / 2b;

[0039] Figure 8 HRESIMS spectra of compounds 2a / 2b;

[0040] Figure 9 Compounds 2a / 2b 1 HNMR spectrum (DMSO-d6, 600MHz);

[0041] Figure 10 Compounds 2a / 2b 13 C10 NMR spectrum (DMSO-d6, 150MHz);

[0042] Figure 11 HSQC spectra of compounds 2a / 2b (DMSO-d6, 600MHz);

[0043] Figure 12 HMBC spectra of compounds 2a / 2b (DMSO-d6, 600MHz);

[0044] Figure 13 UV spectrum of compound 3;

[0045] Figure 14 HRESIMS spectrum of compound 3;

[0046] Figure 15 Compound 3 1H NMR spectrum (DMSO-d6, 600MHz);

[0047] Figure 16 Compound 3 13 C10 NMR spectrum (DMSO-d6, 150MHz);

[0048] Figure 17 HSQC spectrum of compound 3 (DMSO-d6, 600MHz);

[0049] Figure 18 HMBC spectrum of compound 3 (DMSO-d6, 600MHz);

[0050] Figure 19 UV spectrum of compound 4;

[0051] Figure 20 HRESIMS spectrum of compound 4;

[0052] Figure 21 Compound 4 1 H NMR spectrum (DMSO-d6, 600MHz);

[0053] Figure 22 Compound 4 13 C10 NMR spectrum (DMSO-d6, 150MHz);

[0054] Figure 23 HSQC spectrum of compound 4 (DMSO-d6, 600MHz);

[0055] Figure 24 HMBC spectrum of compound 4 (DMSO-d6, 600MHz). Detailed Implementation

[0056] Example 1

[0057] Preparation of isopentenyl flavonoids 1,2a / 2b,3,4

[0058] (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.

[0059] (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.

[0060] (4) The eluted fraction Fr.A6 was further purified by semi-preparative HPLC through an acetonitrile-water system to obtain isopentenyl flavonoids daphengiratriprenylone D (4.2 mg), (3"'R)-daphnegiravone F (1.3 mg), (3"'S)-daphnegiravone F (0.8 mg), daphnegiravone E (14.2 mg), and daphnegiralin H (5.9 mg).

[0061] Experiment Example 2

[0062] The inhibitory effects of compounds daphengiratriprenylone D, daphnegiravone F, daphnegiravone E, and daphnegiralin H on HepG2 and Hep3B cells were determined by in vitro experiments, thereby exploring their antitumor activity.

[0063] (1) Cell Culture

[0064] Human hepatocellular carcinoma cells HepG2 and Hep3B 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.

[0065] (2) Cell grouping

[0066] Blank group: No drugs were contained, and cultured only in DMEM complete culture medium.

[0067] Compound Group 1: After HepG2 and Hep3B cells were cultured in DMEM complete medium, different concentrations (5 μM, 12.5 μM, 25 μM, 50 μM) of compound daphengiratriprenylone D were added and cultured for 48 hours. Then, 20 μL MTT was added to each well and cultured for another 4 hours.

[0068] Compound Group 2: HepG2 and Hep3B cells were cultured in DMEM complete medium and then cultured for 48 hours with different concentrations (5 μM, 12.5 μM, 25 μM, 50 μM) of compound daphnegiravone F, respectively. Then, 20 μM LTT was added and the cells were cultured for another 4 hours.

[0069] Compound Group 3: HepG2 and Hep3B cells were cultured in DMEM complete medium and then cultured for 48 hours with different concentrations (5 μM, 12.5 μM, 25 μM, 50 μM) of the compound daphnegiravone E, respectively. Then, 20 μM LTT was added and the cells were cultured for another 4 hours.

[0070] Compound Group 4: After HepG2 and Hep3B cells were cultured in DMEM complete medium, different concentrations (5μM, 12.5μM, 25μM, 50μM) of the compound daphnegiralin H were added and cultured for 48 hours, followed by the addition of 20μM LTT and culture for another 4 hours.

[0071] Positive drug group: HepG2 and Hep3B cells were cultured in DMEM complete medium and then cultured for 48 hours with different concentrations (5μM, 12.5μM, 25μM, 50μM) of the positive drug sorafenib. Then, 20μM LTT was added and the cells were cultured for another 4 hours.

[0072] Add 150 μL of DMSO to each well, shake on a constant temperature shaker for 10 min, and measure the absorbance of each well at 490 nm using a microplate reader (Thermo Scientific Multiskan MK3, Shanghai, China).

[0073] Cell viability (%) = [A (给药组) -A (空白对照) ] / [A (阴性对照) -A (空白对照) ]×100%

[0074] (3) Experimental Results

[0075] Data on the tumor cell inhibitory effects of compounds 1, 2, 3, and 4 (IC50) 50 As shown in Table 3 below, the unit is μM.

[0076] Table 3

[0077]

[0078] The result is IC 50 The values ​​are expressed as mean ± standard deviation, in μM. The experiment was repeated three times.

[0079] Experimental results showed that compound 2 exhibited the best inhibitory activity against HepG2 and Hep3B cells, with an IC50 value of [missing value]. 50 The values ​​are IC 50 =11.63±0.22μM and IC 50 =0.10±0.02μM.

[0080] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. An isoprenylated flavonoid compound, characterized by, It is a compound as shown below; 。 2. The method of producing a prenylflavonoid compound according to claim 1, characterized by, The preparation method 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 four fractions were collected: Fr. A – Fr. D. (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. A6 obtained after elution was subjected to semi-preparative HPLC and further purified by an acetonitrile-water system to obtain the compound described in claim 1.

3. The method of preparing a prenylated flavonoid according to claim 2, wherein In step (1), the gradient of dichloromethane-methanol used in silica gel column chromatography is 100:1 - 1:1; the ethanol is 70% industrial ethanol, and the extraction is carried out by reflux three times, each time for 2 hours.

4. The method for preparing a prenylflavonoid according to claim 2, characterized by, In step (2), the ethanol-water gradient is 20% - 90%.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the isopentenyl flavonoid compound of claim 1 and a pharmaceutically acceptable carrier.

6. The use of the pentenyl flavonoid compound of claim 1 or the pharmaceutical composition of claim 5 in the preparation of an antitumor drug, characterized in that, The tumor is liver cancer.

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