A novel C20 type diterpenoid alkaloid compound, its preparation method and application

Abstract

This invention discloses a novel C20-type diterpenoid alkaloid compound, its preparation method, and its applications. The molecular formula of the C20-type diterpenoid alkaloid compound is C... 29 H 36 N2O2. This invention, through systematic and in-depth research on the chemical composition of Delphinium spp., uses the whole herb of Delphinium spp. as raw material. Through alcohol extraction, silica gel column chromatography, and preparative liquid chromatography purification, multi-spectral data analysis shows that a new C20 diterpenoid alkaloid compound was isolated from Delphinium spp. This compound is a novel compound reported for the first time. Antitumor activity studies have shown that this C20 diterpenoid alkaloid compound has good inhibitory activity against colon adenocarcinoma cells and can be used to develop plant-derived antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of diterpenoid alkaloid technology, and in particular relates to a C20 type diterpenoid alkaloid compound, its preparation method and application. Background Technology

[0002] *Delphinium brunonianum* Royle is the whole herb of *Delphinium brunonianum*, a plant belonging to the genus *Delphinium* in the family Ranunculaceae. It grows in grasslands or rocky areas at altitudes of 4500-6000m. The Tibetan medicine "Qia Gao Bei" refers to its dried aerial parts, which are cold in nature and bitter and astringent in taste. *Commonly Used Tibetan Herbal Medicines* records that it "cools the blood and detoxifies, dispels wind and relieves itching. It is mainly used to treat influenza, itchy skin rashes, snake bites, etc." Research shows that the chemical components of *Delphinium brunonianum* are mainly C19 and C20 diterpenoid alkaloids. C19 diterpenoid alkaloids are the most abundant type, structurally belonging to the pentacyclic diterpenoid class, mainly of the aconitine and taurine types. Currently, research on *Delphinium brunonianum* is limited both domestically and internationally.

[0003] Natural products have always been an important source of new drugs, and previous phytochemical studies of the genus *Delphinium* have shown it to be a rich natural resource of structurally diverse diterpenoid alkaloids (DAs). Diterpenoid alkaloids (DAs) are a class of structurally diverse natural products derived from the amination of natural tetracyclic diterpenoids, characterized by multiple stereogenic centers, and mainly distributed in the genera *Aconitum* and *Delphinium*. However, most reported compounds belong to the C19 type, such as aconitine and luteolin. On the other hand, C20 type diterpenoid alkaloids remain extremely rare among DAs. DAs possess a wide range of pharmacological activities, including antitumor, anti-inflammatory, analgesic, neuroprotective, and antiarrhythmic effects. Notably, C19 type diterpenoid alkaloids are often highly toxic, hindering drug development. For example, a dose of 0.2 mg of pure aconitine can cause poisoning, and an oral dose of 3-5 mg of aconitine can be fatal. In contrast, C20 diterpenoid alkaloids exhibit lower cytotoxicity and are considered among the most structurally complex natural products due to their cage-like and polycyclic ring systems. Therefore, this invention provides a novel C20 diterpenoid alkaloid compound, its preparation method, and its applications. This C20 diterpenoid alkaloid compound exhibits three unique features within its skeletal framework: a cyano group, tetrahydropyran, and phenethyl, which are extremely rare and not documented in existing literature. Summary of the Invention

[0004] The purpose of this invention is to provide a novel C20 type diterpenoid alkaloid compound, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a novel C20-type diterpenoid alkaloid compound, wherein the molecular formula of the C20-type diterpenoid alkaloid compound is C 29 H 36 N2O2 has the following chemical structure:

[0007]

[0008] This invention also provides a novel method for preparing C20 type diterpenoid alkaloids, the specific preparation steps of which are as follows:

[0009] (1) The dried whole plant of Delphinium serratum was extracted with 95 wt% ethanol solution 4-6 times at room temperature, each time for 6-8 days. Finally, the extract was concentrated under reduced pressure. The extract was then dissolved in water and the pH of the aqueous phase was adjusted to 2-3 with 36 wt% hydrochloric acid solution. Petroleum ether and ethyl acetate were added for extraction, and the petroleum ether phase, ethyl acetate phase and acidic aqueous phase were separated.

[0010] (2) The acidic aqueous phase was alkalized with 25wt% ammonia to make its pH value 9-10, and then extracted with dichloromethane to obtain an aqueous phase and a dichloromethane phase. The dichloromethane phase was concentrated under reduced pressure to remove the solvent and obtain total alkaloids.

[0011] (3) The total alkaloids were eluted by chloroform-methanol gradient elution, and the eluent was then eluted by a forward silica gel column using a petroleum ether-acetone system to obtain ten fractions from Fr2-1 to Fr2-10. Fr2-3 was eluted by dichloromethane-acetone gradient elution to obtain five fractions from Fr2-3-1 to Fr2-3-5.

[0012] (4) Fr2-3-4 was prepared by separation and high-performance liquid chromatography (HPLC) using a C10 column. 18 The column was prepared using acetonitrile-water-triethylamine as the mobile phase, with a flow rate of 2-4 mL / min. -1 Isocratic elution was performed at detection wavelengths of 254 nm and 210 nm and a column temperature of 35-40 °C to obtain the desired C20 type diterpenoid alkaloid compound.

[0013] Furthermore, in step (1), the solid-liquid ratio of the dried whole plant of Delphinium septemlobum to the ethanol solution is 1 kg: (20-30) L, and the solid-liquid ratio of the extract to water is 1 kg: (15-25) L.

[0014] Further, in step (3), the volume ratio of chloroform to methanol in chloroform-methanol is (1-100):1, the volume ratio of petroleum ether to acetone in petroleum ether-acetone is (0.5-5):1, and the volume ratio of dichloromethane to acetone in dichloromethane-acetone is (1-300):1.

[0015] Furthermore, in step (3), diethylamine is also added to the petroleum ether-acetone, and the amount of diethylamine added is 0.5-1.5% of the sum of the volumes of petroleum ether and acetone.

[0016] Furthermore, in step (3), gradient elution is performed using a 200-300 mesh silica gel column.

[0017] Furthermore, in step (4), C 18 The column specifications are: column length 240-260mm, inner diameter 8-12mm, and packing diameter 4-6μm.

[0018] Further, in step (4), the volume ratio of acetonitrile, water and triethylamine in acetonitrile-water-triethylamine is (70-80):(20-30):(0.05-0.15).

[0019] This invention also provides the application of a novel C20 type diterpenoid alkaloid compound in the preparation of drugs for treating colon adenocarcinoma.

[0020] Furthermore, the C20 type diterpenoid alkaloid compound, combined with pharmaceutical excipients and a pharmaceutically or physiologically acceptable carrier, can be prepared into any pharmaceutical dosage form using conventional preparation processes.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention, through systematic and in-depth research on the chemical composition of Delphinium spp., uses the whole herb of Delphinium spp. as raw material. Through alcohol extraction, silica gel column chromatography, and preparative liquid chromatography purification, comprehensive multispectral data analysis shows that a new C20 diterpenoid alkaloid compound was isolated from Delphinium spp. This compound is a novel compound reported for the first time. Antitumor activity studies have shown that this C20 diterpenoid alkaloid compound has good inhibitory activity against colon adenocarcinoma cells and can be used to develop plant-derived antitumor drugs. Attached Figure Description

[0023] Figure 1 The 1H-NMR spectrum of the C20 type diterpenoid alkaloid compound prepared in this invention;

[0024] Figure 2 The 13C-NMR spectrum of the C20 type diterpenoid alkaloid compound prepared in this invention;

[0025] Figure 3 HSQC spectra of C20 type diterpenoid alkaloids prepared in this invention;

[0026] Figure 4 The HMBC spectrum of the C20 type diterpenoid alkaloids prepared in this invention;

[0027] Figure 5The 1H-1HCOSY spectrum of the C20 type diterpenoid alkaloids prepared in this invention;

[0028] Figure 6 The NOESY spectrum of the C20 type diterpenoid alkaloids prepared in this invention;

[0029] Figure 7 The IR spectrum of the C20 type diterpenoid alkaloid compound prepared in this invention;

[0030] Figure 8 The UV spectrum of the C20 type diterpenoid alkaloid compound prepared in this invention;

[0031] Figure 9 HR-ESI-MS spectra of C20 type diterpenoid alkaloids prepared in this invention;

[0032] Figure 10 ECD spectra of C20 type diterpenoid alkaloids prepared in this invention;

[0033] Figure 11 The ORTEP spectrum of the C20 type diterpenoid alkaloid compound prepared in this invention. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] Preparation of C20 type diterpenoid alkaloids:

[0037] (1) 10 kg of dried whole plant of Delphinium serratum was extracted 5 times at room temperature with 250 L of 95 wt% ethanol solution for 7 days each time. Finally, 3 kg of extract was obtained by vacuum concentration. Then, 3 kg of extract was dissolved in 60 L of water and the pH of the aqueous phase was adjusted to 2.5 with 36 wt% hydrochloric acid solution. Petroleum ether and ethyl acetate were added for extraction respectively to separate the petroleum ether phase, ethyl acetate phase and acidic aqueous phase.

[0038] (2) The acidic aqueous phase was alkalized with 25wt% ammonia to make its pH value 9.5, and then extracted with dichloromethane to obtain an aqueous phase and a dichloromethane phase. The dichloromethane phase was concentrated under reduced pressure to remove the solvent, and 208.4g of total alkaloids were obtained.

[0039] (3) The total alkaloids were eluted by chloroform-methanol gradient elution with a volume ratio of chloroform to methanol of 50:1. The gradient elution was carried out using a 250-mesh silica gel column. The eluent was then eluted by a forward silica gel column of a petroleum ether-acetone system with a volume ratio of petroleum ether to acetone of 3:1. Diethylamine was also added to the petroleum ether-acetone system at a volume ratio of 1% of the sum of the volumes of petroleum ether and acetone. The gradient elution was carried out using a 250-mesh silica gel column to obtain ten fractions from Fr2-1 to Fr2-10. Fr2-3 was eluted by dichloromethane-acetone gradient elution with a volume ratio of dichloromethane to acetone of 150:1. The gradient elution was carried out using a 250-mesh silica gel column to obtain five fractions from Fr2-3-1 to Fr2-3-5.

[0040] (4) Fr2-3-4 was prepared by separation and high-performance liquid chromatography (HPLC) using a C10 column. 18 The column was prepared using acetonitrile-water-triethylamine (volume ratio: 75:25:0.1) as the mobile phase, with a flow rate of 3.0 mL / min. -1 Isocratic elution was performed at detection wavelengths of 254 nm and 210 nm and a column temperature of 40 °C. 18 The column specifications are as follows: column length 250mm, inner diameter 10mm, packing diameter 5μm, and volume ratio of acetonitrile, water, and triethylamine 75:25:0.1, thus obtaining the desired C20 type diterpenoid alkaloid compound.

[0041] Example 2

[0042] Structural analysis of C20 type diterpenoid alkaloids:

[0043] The compound is a colorless crystal (MeOH). The ion peak measured by HR-ESI-MS is 445.2837 (calculated value [M+H]). + 445.2850) determined its molecular formula to be C 29 H 36 N₂O₂ indicates the presence of 13 degrees of unsaturation. According to... 1 H and 13 CNMR data (see Table 1) show that the five unsaturations originate from one double bond and one benzene ring group. Infrared spectroscopy reveals secondary alcohols (1090 cm⁻¹). -1 ) and cyano (2235cm) -1 The unique absorption band of the compound is evident. Therefore, the remaining six unsaturated groups confirm that the compound is a six-membered ring. In addition to the resonances of the benzene ring group and the cyano group, there are 20 carbon signals, including one methyl group, nine methylene groups, seven methyl groups, three quaternary carbons, and a characteristic outer ring double bond unit [δ]. H 5.11(1H,d,J=1.2,H-17),5.02(1H,d,J=2.5,H-17); δ C[157.1, 108.2], which means the compound is likely an atrazine alkaloid. Typically, atrazine diterpenoid alkaloids contain one monomolecular methyl group and one trimolecular methyl group, but in δ... H Only one single methyl group was found at 1.13 (3H, s, Me-18) (see Table 1). This compound contains the characteristic hydrogen and carbon atoms of the benzene ring, therefore, according to HMBC, the methyl group can be found at H2-21 (δ). H 3.25,m) to C-1'(δ C 139.8), and H2-22 (δ H 2.88, 2.78, m) to C-2', C-6' (δ C The correlation of 128.6) indicates that a benzene ring functional group is attached to the ethylamine group. The position of the hydroxyl group is determined by the H-14 (δ) on the HMBC. H 4.20, dt, J = 6.9, 2.3 Hz) to quaternary carbon C-13 (δ C 157.1), C-12(δ) C 36.9) and from H2-17 (δ H The correlation between 5.02) and C-14 was determined. Furthermore, the cyano group (δ) C 118.8) showed an HMBC association with H-19 (δ H 3.52, s), H-19 / C-3 / C-5 are also correlated, indicating that the C-19 position is substituted with a cyano group. This is also the first report of cyano substitution at the C-19 position in atrazine diterpenoid alkaloids. The relative configuration of the compound was determined by NOESY spectral data. The NOESY correlation of H-14 / H-16 / H-15 indicates that they are all on the same face, therefore the hydroxyl group is β-oriented. Further, it can be found that H-19 (δ... H 3.52, s) and H-21 (δ) H 3.25, m), H-22 (δ) H 2.78, m, 2.88, m) and H-2α (δ H The relative configuration of the C-19 position is determined to be R configuration, based on the correlation between 1.88 and m.

[0044] The absolute configuration of a compound can be preliminarily determined through ECD calculations. Figure 10 Observations showed that the measured CD value exhibited a positive Cotton effect at 201 nm and a negative Cotton effect at 216 nm (see...). Figure 10 This is consistent with the calculated values ​​for the 14R and 19S conformations. Fortunately, we obtained a single crystal of the compound, and XRD [Flack parameter = 0.03(19), Cu-Kα] analysis (see...). Figure 10The absolute configurations of the compounds (4R, 5R, 7R, 8S, 9R, 10S, 12S, 14R, 19R) were confirmed to be consistent with the ECD calculations.

[0045] Based on the above analysis, the compound was identified as a C20 type diterpenoid alkaloid. A SCIFinder search revealed no literature reports, leading to the preliminary conclusion that it is a new compound.

[0046] Table 1 1 H(600MHz)and 13 CNMR(150MHz)data of 1in CDCl3(J in Hz,δin ppm)

[0047]

[0048]

[0049]

[0050] Example 3

[0051] Experimental analysis of the antitumor activity of C20 type diterpenoid alkaloids:

[0052] The cell lines used in the experiment were internationally recognized tumor cell lines: A549: human non-small cell lung cancer cells, Caco-2: human colon adenocarcinoma cells, H460: human large cell lung cancer cells, and Skov-3: human ovarian cancer cells.

[0053] The experimental method adopted the internationally accepted CCK-8 assay method:

[0054] (1) Cell passage: When the cell density reaches approximately 80-90%, the cells are passaged. The culture dish is transferred to a clean bench, the original culture medium is discarded, and the cells are washed once with 3 mL of PBS. The PBS is discarded, and 2 mL of 0.25% trypsin containing EDTA is added for digestion. Since the digestion time varies for different cell types, cell morphology should be observed under a microscope. When the cells become round and bright, the dish is quickly transferred to a clean bench, the trypsin is discarded, and 3 mL of the corresponding culture medium is added to stop the digestion. The bottom of the dish is repeatedly pipetted to form a single-cell suspension (HepG-2 cells tend to clump together, so the pipetting time should be longer). 1 mL of the cell suspension is transferred to a new 10 cm culture dish, 7-8 mL of the corresponding culture medium is added, and the dish is gently shaken back and forth and side to side to mix. The dish is then placed in a 37°C, 5% CO2 incubator.

[0055] (2) Half-inhibition concentration IC 50Experiment: ① Cells in the logarithmic growth phase were digested with trypsin, and culture medium was added to form a single-cell suspension. Cells were counted using a counting chamber. Both the control and experimental groups were seeded at 5000 cells / well in 96-well plates. 100 μL of culture medium was added to each well, and the 96-well plates were incubated at 37℃ in a 5% CO2 incubator for 24 h. ② Culture media containing different concentrations of the compound were prepared: the compound was weighed and dissolved completely in DMSO to obtain a stock solution with an initial concentration of 2.0 mM. A certain volume of the stock solution was taken and diluted with the corresponding culture medium at concentration gradients of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. ③ After the cells had completely adhered to the plates, the 96-well plates were removed and transferred to a clean bench. The original culture medium in the experimental group wells was discarded, and 100 μL of culture medium containing different concentrations of the compound was added to each well. The culture medium containing gradient compounds was prepared in 6 concentration gradients, with 3 replicates. The 96-well plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours, the original culture medium for both the experimental and control groups was discarded. 100 μL of culture medium containing 10% CCK-8 solution was added to each well. The blank group received 100 μL of culture medium containing 10% CCK-8 solution without cells, while the control group received 100 μL of culture medium containing 10% CCK-8 solution with normal cancer cells. The plates were incubated at 37°C in a 5% CO2 incubator for 2 hours. The OD value of each well was measured at 450 nm using a microplate reader. The cell growth inhibition rate was calculated using the following formula:

[0056] (3) Inhibition rate (%) = (average OD value of blank wells - average OD value of control wells) / (average OD value of blank wells - average OD value of experimental wells) × 100%.

[0057] Table 2 shows the cytotoxic IC50 of the compounds. 50 Value (μM)

[0058]

[0059] Experimental results: The inhibitory activities of the C20 type diterpenoid alkaloids prepared according to Example 1 against four common tumor cell types are shown in Table 2. As can be seen from Table 2, the C20 type diterpenoid alkaloids showed poor inhibitory activity against A549 (human non-small cell lung cancer cells) and Skov-3 (human ovarian cancer cells), but showed good inhibitory activity against Caco-2 (human colon adenocarcinoma cells) and H460 (human large cell lung cancer cells), especially against Caco-2.

[0060] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A novel C20 type diterpenoid alkaloid compound, characterized in that, The molecular formula of the C20 type diterpene alkaloid compound is C 29 H 36 N2O2, the chemical structure of which is as follows:

2. The method for preparing a novel C20 type diterpenoid alkaloid compound according to claim 1, characterized in that, The specific preparation steps are as follows: (1) The dried whole plant of Delphinium serratum was extracted with 95 wt% ethanol solution 4-6 times at room temperature, each time for 6-8 days. Finally, the extract was concentrated under reduced pressure. The extract was then dissolved in water and the pH of the aqueous phase was adjusted to 2-3 with 36 wt% hydrochloric acid solution. Petroleum ether and ethyl acetate were added for extraction, and the petroleum ether phase, ethyl acetate phase and acidic aqueous phase were separated. (2) The acidic aqueous phase was alkalized with 25wt% ammonia to make its pH value 9-10, and then extracted with dichloromethane to obtain an aqueous phase and a dichloromethane phase. The dichloromethane phase was concentrated under reduced pressure to remove the solvent and obtain total alkaloids. (3) The total alkaloids were eluted by chloroform-methanol gradient elution, and the eluent was then eluted by a forward silica gel column using a petroleum ether-acetone system to obtain ten fractions from Fr2-1 to Fr2-10. Fr2-3 was eluted by dichloromethane-acetone gradient elution to obtain five fractions from Fr2-3-1 to Fr2-3-5. (4) Fr2-3-4 was prepared by separating it by high performance liquid chromatography under the conditions that the column was C 18 18, acetonitrile-water-triethylamine was the mobile phase, the volume flow rate was 2-4 mL·min -1 -1, the detection wavelength was 254 nm and 210 nm, and the column temperature was 35-40℃.

3. The method for preparing a novel C20 type diterpenoid alkaloid compound according to claim 2, characterized in that, In step (1), the solid-liquid ratio of the dried whole plant of Delphinium septemlobum to the ethanol solution is 1 kg: (20-30) L, and the solid-liquid ratio of the extract to water is 1 kg: (15-25) L.

4. The method for preparing a novel C20 type diterpenoid alkaloid compound according to claim 2, characterized in that, In step (3), the volume ratio of chloroform to methanol in chloroform-methanol is (1-100):1, the volume ratio of petroleum ether to acetone in petroleum ether-acetone is (0.5-5):1, and the volume ratio of dichloromethane to acetone in dichloromethane-acetone is (1-300):

1.

5. The method for preparing a novel C20 type diterpenoid alkaloid compound according to claim 2, characterized in that, In step (3), diethylamine is also added to the petroleum ether-acetone, and the amount of diethylamine added is 0.5-1.5% of the sum of the volumes of petroleum ether and acetone.

6. The method for preparing a novel C20 type diterpenoid alkaloid compound according to claim 2, characterized in that, In step (3), gradient elution is performed using a 200-300 mesh silica gel column.

7. The method for preparing a novel C20 type diterpenoid alkaloid compound according to claim 2, characterized in that, In step (4), C 18 The column specifications are: column length 240-260mm, inner diameter 8-12mm, and packing diameter 4-6μm.

8. The method for preparing a novel C20 type diterpenoid alkaloid compound according to claim 2, characterized in that, In step (4), the volume ratio of acetonitrile, water and triethylamine in acetonitrile-water-triethylamine is (70-80):(20-30):(0.05-0.15).

9. The use of a novel C20 diterpenoid alkaloid compound as described in any one of claims 1-8 in the preparation of an anti-colon adenocarcinoma drug.

10. The application according to claim 9, characterized in that, The C20 type diterpenoid alkaloid compound, combined with pharmaceutical additives and pharmaceutically or physiologically acceptable carriers, can be prepared into any pharmaceutical dosage form using conventional preparation processes.

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