Anti-tumor cytisine alkaloids in caesalpinia sappan and application thereof

Abstract

The application provides an antitumor delphinine alkaloid in Caesalpinia minax, a core skeleton of angustifoline type, and provides an extraction method and application, and compound 3 is used for preparation of an antitumor drug. The natural delphinine with a new angustifoline type structural skeleton is obtained by separation and extraction, wherein compound 3 has a significant inhibitory effect on multiple tumor cell strains, and can be used for preparation of an antitumor drug.

Description

Technical Field

[0001] This invention belongs to the field of cycad alkaloid technology, specifically relating to an antitumor cycad alkaloid from the red bean tree and its application. Background Technology

[0002] Natural cytisine alkaloids can be classified into eight structural types based on their composition: cytisine, sparteine, albine, angustifoline, camoensidine, cytisine-like, tsukushinamine, and lupanacosmine. These natural cytisine alkaloids are mainly found in legumes and possess pharmacological activities such as anti-inflammatory, antitumor, insecticidal and antiviral effects, osteoclast inhibition, and central nervous system stimulation.

[0003] The red bean tree (Ormosia hosiei Hemsl. et Wils) is a plant belonging to the genus Ormosia in the legume family. Also known as the E'xi red bean or He's red bean, it is mainly distributed in Zhejiang, Fujian, Anhui, and Jiangxi provinces. The seeds are used medicinally; they are bitter, neutral in nature, and slightly toxic, primarily used to treat hernia, abdominal pain, blood stasis, and amenorrhea. The red bean tree exhibits various biological activities, including anti-inflammatory, anti-tumor, and central nervous system-influencing effects, making it a potentially valuable economic forest tree. Studies have shown that the main chemical components of the red bean tree include flavonoids, volatile oils, and alkaloids, with alkaloids being the primary components, mainly cytisine-like alkaloids. Currently, only 11 monomeric alkaloids have been reported in the red bean tree, including cytisine-like and angustifoline types. Nine cytisine-like alkaloids were reported, with the following structures and activities: hosieines AD showed significant affinity for the nicotinic acetylcholinesterase receptor (nAChR); hositisine B and hositisine E exhibited anti-inflammatory effects; and hosimonoal, hositisine C, and hositisine D showed moderate inhibitory activity against the human hepatocellular carcinoma cell line (HepG2 cells). Only two angustifoline alkaloids were reported, with the following structures or activities: hositisine A showed significant anti-inflammatory activity; and tinctorine showed moderate inhibitory activity against the HepG2 tumor cell line. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an angustifoline-type cytisine alkaloid from red bean tree and its application, which is a new compound obtained by separation and extraction. Compound 3 has a significant inhibitory effect on a variety of tumor cell lines and can be used for the preparation of antitumor drugs.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: an antitumor cytisine alkaloid from the red bean tree, wherein the core skeleton of the antitumor cytisine alkaloid is angustifoline type.

[0006] The structural framework of the angusifoline type is as follows:

[0007]

[0008] The R group is hydrogen, hydroxyl, or other substituted group; the stereoconfiguration at the C-6 position is R-type or S-type; there is a single or double bond between C-5 and C-6; the stereoconfiguration at the C-7 and C-9 positions is R-type or S-type; and the stereoconfiguration at the C-11 position is R-type or S-type.

[0009] Preferably, the antitumor cytisine alkaloids include compounds 1 to 4, which are new compounds with the following structural formulas in sequence:

[0010]

[0011] This invention also provides a method for extracting antitumor cytisine alkaloids from the red bean tree, the method being as follows:

[0012] Step 1: Crush the dried red bean tree seeds, then extract them three times with a 95% ethanol solution (volume ratio 1:3). After concentration, obtain the seed extract.

[0013] Step 2: Dissolve the seed extract obtained in Step 1 in water to obtain an aqueous solution of the seed extract. Adjust the pH to 2 with a 2% sulfuric acid solution, mix well, let stand, and then extract with petroleum ether to obtain an acidic aqueous solution after extraction.

[0014] Step 3: Adjust the pH of the acidic aqueous solution obtained in Step 2 to 10 with a 2 mol / L NaOH solution, mix well and let stand, then extract with chloroform to obtain a chloroform extract, which is then concentrated to obtain a chloroform layer extract.

[0015] Step 4: The chloroform extract from Step 3 was sequentially separated using normal-phase silica gel column chromatography, reversed-phase ODS column chromatography, and Sephadex LH-20 column chromatography, supplemented by HPLC purification to obtain compounds 1 to 4. Their structural formulas were identified as follows:

[0016]

[0017] The present invention also provides the application of the antitumor cytisine alkaloids extracted by the above extraction method, wherein compound 3 is used in the preparation of antitumor drugs.

[0018] Preferably, the antitumor drug includes drugs that inhibit human liver cancer cell line HepG2, human ovarian cancer cell line A2780, or human breast cancer cell line MCF-7.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention extracts four new natural angustifoline-type antitumor cycad alkaloids from the red bean tree. Among them, compound 3 has inhibitory effects on human liver cancer cell line HepG2, human ovarian cancer cell line A2780 and human breast cancer cell line MCF-7, and can be used for the preparation of antitumor drugs.

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] Figure 1 It is compound 1 of the present invention 1 H- 1 Correlation diagram of H COSY and HMBC.

[0023] Figure 2 These are the experimental and calculated ECD curves of compound 1 of the present invention.

[0024] Figure 3 These are the experimental and calculated ECD curves for compound 2 of the present invention.

[0025] Figure 4 These are the relevant signals of compounds 3 and 4HMBC and ROESY of the present invention.

[0026] Figure 5 It is compound 1 of the present invention 1 H NMR spectrum.

[0027] Figure 6 It is compound 1 of the present invention 13 C NMR spectrum.

[0028] Figure 7 This is the HSQC spectrum of compound 1 of the present invention.

[0029] Figure 8 It is compound 1 of the present invention 1 H- 1 H COSY spectrum.

[0030] Figure 9 This is the HMBC spectrum of compound 1 of the present invention.

[0031] Figure 10 This is the ROESY spectrum of compound 1 of the present invention.

[0032] Figure 11 It is compound 2 of the present invention 1 H NMR spectrum.

[0033] Figure 12 It is compound 2 of the present invention 13 C NMR spectrum.

[0034] Figure 13 This is the ROESY spectrum of compound 2 of the present invention.

[0035] Figure 14 It is compound 3 of the present invention 1 H NMR spectrum.

[0036] Figure 15 It is compound 3 of the present invention 13 C NMR spectrum.

[0037] Figure 16 It is compound 4 of the present invention. 1 H NMR spectrum.

[0038] Figure 17 It is compound 4 of the present invention. 13 C NMR spectrum.

[0039] Figure 18 These are the experimental and calculated ECD curves for compound 3 of the present invention.

[0040] Figure 19 These are the experimental and calculated ECD curves for compound 4 of the present invention. Detailed Implementation

[0041] Example 1

[0042] The preparation method of novel alkaloids from red bean trees, and the specific preparation steps are as follows:

[0043] Step 1: Crush the dried red bean seeds, then extract them three times with 95% ethanol at a volume ratio of 1:3, and concentrate the extract to obtain the seed extract.

[0044] Step 2: Dissolve the seed extract obtained in Step 1 in warm water to obtain an aqueous solution of the seed extract. Adjust the pH to 2 with a 2% sulfuric acid solution, mix well, let stand, and then extract with petroleum ether to obtain an acidic aqueous solution after extraction.

[0045] Step 3: Adjust the pH of the acidic aqueous solution obtained in Step 2 to 10 with a 2 mol / L NaOH solution, mix well and let stand, then extract with chloroform to obtain a chloroform extract, which is then concentrated to obtain a chloroform layer extract.

[0046] Step 4: The chloroform extract from Step 3 was separated by normal-phase silica gel column chromatography (dichloromethane-methanol-diethylamine) to obtain 3 fractions (Aa-Ac). Fraction Aa was then separated by reversed-phase ODS column chromatography (methanol-water) to obtain 5 subfractions (Aa1-Aa5). Subfraction Aa2 was further separated by normal-phase silica gel and Sephadex LH-20 gel electrophoresis (ethanol) to obtain 5 subfractions (Aa2a-Aa2e). Subfraction Aa2c was purified by HPLC (MeOH:H2O = 70:30) to obtain monomeric compounds 1 and 2. Using the same separation process, compounds 3 and 4 were obtained from subfractions Aa3a and Aa2e, respectively. Their structures were identified by NMR, MS, UV, and IR spectroscopy.

[0047] Compounds 1 to 4 are analyzed in Examples 2-5 below.

[0048] Example 2

[0049] Structural identification of compound 1:

[0050] Compound 1, isolated in Example 1, was a white powder. The quasi-molecular ion, 265.1911 [M+H], was determined by HR-ESI-MS. + (Calculated value 265.1917), the molecular formula is given as C 15 H 24 N₂O₂, with an unsaturation degree of 5. Compound 1 1 H-NMR spectrum (CD3OD, 600MHz, Table 1) Figure 5 The signal showed three olefin protons (δ). H 5.05, 1H, s), (δ H 5.73, 1H, m), (δ H 5.07, 1H, m) and 1 methyl signal (δ) H 2.34,3H,s). 13C-NMR spectrum (CD3OD, 150MHz, Table 1) Figure 6 ) and HSQC spectrum ( Figure 7 The results showed 15 carbon signals, including one methyl group (δ). C 41.3), 8 methylene groups (δ) C 115.7, 67.0, 50.5, 32.1, 30.5, 27.5, 19.1 and 15.6), 4 methines (δ C 136.3, 63.4, 37.1 and 30.6), 2 quaternary carbons (one carbonyl carbon δ) C 174.3 and an oxygen-bound quaternary carbon δ C 84.5). The aforementioned functional group occupies 2 unsaturation degrees, leaving 3 unsaturation degrees, indicating the presence of a tricyclic system in the structure.

[0051] 1 H- 1 H COSY spectrum ( Figure 1 , Figure 8 Compound 1 was analyzed to contain two structural fragments, a and b. The connection mode of the two fragments was determined by HMBC spectroscopy. Figure 9 The determination was made. Compound 1 possesses HMBC-related signals for H-3, H-4, and C-2. Figure 1 The presence of carbonyl groups at the C-2 position indicates that fragment a is connected to fragment a. The HMBC correlation signals at H-4 / C-6, H-5 / C-6 and C-7, and H-10 / C-6 and C-8 confirm that fragment a is connected to fragment b via C-6 and N-1. The HMBC correlation signals at H-11 / C-13 and H-13 / C-11 and C-17 indicate that C-11, C-13, and C-17 are connected to N-12. Therefore, the planar structure of compound 1 is N-methyl-11-allyl-6-hydroxy-tetrahydrocytisine.

[0052] The relative configuration of compound 1 was analyzed using a ROESY spectrum. Compound 1 contains four chiral centers: C-6, C-7, C-9, and C-11. The hydrogens on the bridgehead carbons C-7 and C-9 are in the cis configuration. If we consider H-7 and H-9 on the bridgehead carbons as α-positions, then C-11 and C-13 are in the β-positions. In the ROESY spectrum (… Figure 10 The ROESY correlation signals for H-5β / H-13β, H-13α / H-8β, H-8α / H-10α, and H-10β / H-11 indicate that the hydrogen atoms are spatially close, with H-11 located at the β-position. The relative configuration of the quaternary carbon C-6 connecting the hydroxyl group cannot be determined by ROESY mapping. To clarify the relative configuration of the C-6 hydroxyl group in the compound, two possible isomers, 1a (6α-OH) and 1b (6β-OH), were analyzed.1 H and 13 C NMR chemical shift calculations. Analysis using the DP4+ method showed that the DP4+ probability prediction value for structure 1a was 100%, indicating that structure 1a is correct.

[0053] After confirming the relative configuration as 1a, the ECD curve of compound 1 was calculated using TD-DFT at the B3LYP / 6-31+G(d,p) level. The calculated ECD curve of (6R,7R,9R,11R)-1 was basically consistent with the experimental ECD curve of compound 1. Figure 2 Based on this, the absolute configuration of compound 1 is determined to be 6R,7R,9R,11R. Therefore, the structure of compound 1 is (6R,7R,9R,11R)-N-methyl-11-allyl-6α-hydroxy-tetrahydrocytisine.

[0054] Table 1. Compounds 1 and 2 1 H-NMR (600MHz) and 13 C-NMR (150MHz) data, solvent CD3OD:

[0055]

[0056]

[0057] Example 3

[0058] Structural identification of compound 2

[0059] Compound 2 is a pale yellow oil. Its molecular formula was determined to be C2 based on the quasi-molecular ion 265.1911[M+H]+ (calculated value 265.1917) obtained by HR-ESI-MS. 15 H 24 N₂O₂ has an unsaturation degree of 5. Compound 2... 1 1H NMR spectra (Table 1, Figure 11 It shows 3 olefin proton signals and 1 methyl signal. 13 C NMR spectra (Table 1, Figure 12 The results showed the presence of 15 carbon signals. Comparison of compound 2 and compound 1... 13 The C10 NMR spectra revealed no significant difference in chemical shifts between the two compounds. Detailed analysis of their two-dimensional spectra showed that compound 2 and compound 1 have the same planar structure.

[0060] The relative configuration of compound 2 was analyzed using ROESY spectra. By setting H-7 and H-9 on the bridgehead carbons to the α-position, a key ROESY correlation signal was observed for H-10β / H-11. Figure 13From this, we can infer that H-11 is at the β position. Therefore, the configurations of compounds 2 and 1 at the C-7, C-9, and C-11 positions are identical, with the only difference being the configuration of the hydroxyl group at the C-6 position. Thus, the relative configuration of the 6-OH group in compound 2 is β-type. To further infer its absolute configuration, ECD calculations were performed using TD-DFT at the B3LYP / 6-31+G(d,p) level. The calculated ECD curve of (6S,7R,9R,11R)-2 is basically consistent with the experimental ECD curve of compound 2. Figure 3 Based on this, the absolute configuration of compound 2 is determined to be 6S,7R,9R,11R. Therefore, compound 2 is presumed to be (6S,7R,9R,11R)-N-methyl-11-allyl-6β-hydroxy-tetrahydrocytisine.

[0061] Example 4

[0062] Structural identification of compound 3

[0063] Compound 3 is a pale yellow oil. Its molecular formula was determined to be C1 by HR-ESI-MS using the quasi-molecular ion 247.1802[M+H]+ (calculated value 247.1811). 15 H 22 N₂O, with an unsaturation degree of 6. Compound 3. 1 1H NMR spectra (Table 2, Figure 14 The results show four olefin proton signals and one methyl signal. 13 C NMR spectra (Table 2, Figure 15 The spectrum showed 15 carbon signals. Combined with DEPT 135 and HSQC spectra, it revealed 1 methyl group, 7 methylene groups, 5 methine groups, and 2 quaternary carbons (one of which is a carbonyl carbon). These functional groups occupy 3 unsaturated regions, leaving 3 unsaturated regions, indicating that the compound also has a tricyclic skeleton.

[0064] Combination 1 H and 13 The 1 / 2C NMR data revealed that the most significant difference between compound 3 and compound 1 was the addition of an olefin signal [δ]. H 5.05 (1H, s), δ C 102.3; δ C [142.1], while reducing one oxygen-linked quaternary carbon (δ) C-6 84.5) and a secondary carbon (δ) C-5 32.1) Signals. HMBC related signals of H-3 / C-5, H-4 / C-5 and C-6, H-5 / C-6 and C-7 ( Figure 4From A), we know that the double bond is located at the C-5 and C-6 positions. In summary, compound 3 has a structure in which the C-6 hydroxyl group of compound 1 forms a 5,6 double bond.

[0065] Using ROESY maps ( Figure 4 B) Analysis of the relative configuration of compound 3. Compound 3 contains three chiral centers C-7, C-9, and C-11, with the hydrogens of the bridgehead carbons C-7 and C-9 in the cis configuration. Assuming H-7 and H-9 on the bridgehead carbons are at the α-position, then C-11 and C-13 are at the β-position. In the ROESY spectra, the ROESY correlation signals of H-13α / H-8β, H-8α / H-10α, and H-10β / H-11 confirm that the hydrogens are spatially close, with H-11 at the β-position. ECD calculations were performed using TD-DFT at the B3LYP / 6-31+G(d,p) level. The calculated ECD curve of (7S,9S,11S)-3 is basically consistent with the experimental ECD curve of compound 3. Figure 18 From this, we can determine that the absolute configuration of compound 3 is 7S,9S,11S. Therefore, the compound is presumed to be (7S,9S,11S)-N-methyl-11-allyl-5,6-dehydro-tetrahydrocytisine.

[0066] Compounds 3 and 4 in Table 2 1 H-NMR (600MHz) and 13 C-NMR (150MHz) data, solvent CD3OD:

[0067]

[0068] Example 5

[0069] Structural identification of compound 4

[0070] Compound 4 is a pale yellow oil. HR-ESI-MS yielded a quasi-molecular ion of 247.1803 [M+H]+ (calculated value 247.1811), confirming its molecular formula as C. 15 H 22 N₂O, with an unsaturation degree of 6. Compound 4. 1 1H NMR spectra (Table 2, Figure 16 The results show four olefin proton signals and one methyl signal. 13 C NMR spectra (Table 2, Figure 17 The spectrum showed 15 carbon signals. Combined with DEPT135 and HSQC spectra, it revealed one methyl group, seven methylene groups, five methine groups, and two quaternary carbons (one of which is a carbonyl carbon). Further comparison of the HMBC of compound 4 and compound 3... 1 H- 1HCOSY two-dimensional spectral correlation signals revealed that compound 4 and compound 3 have the same planar structure.

[0071] Using ROESY maps ( Figure 4 B) Analysis of the relative configuration of compound 4. Compound 4 has three chiral centers C-7, C-9, and C-11, with the hydrogens of the bridgehead carbons C-7 and C-9 in the cis configuration. Assuming H-7 and H-9 on the bridgehead carbons are at the α-position, then C-11 and C-13 are at the β-position. In the ROESY spectrum, the correlation signals of H-8β / H-13α, H-8β / H-11, and H-8α / H-10 confirm that the hydrogens are spatially close, with H-11 at the α-position. Therefore, compounds 4 and 3 are diastereomers of C-11. ECD calculations were performed using TD-DFT at the B3LYP / 6-31+G(d,p) level. The calculated ECD curve of (7S,9S,11R)-4 is basically consistent with the experimental ECD curve of compound 4. Figure 19 From this, we can determine that the absolute configuration of compound 4 is 7S,9S,11R. Therefore, the compound is presumed to be (7S,9S,11R)-N-methyl-11-allyl-5,6-dehydro-tetrahydrocytisine.

[0072] In summary, the cycad alkaloids extracted from the red bean tree in this invention have an angustifoline-type structural framework, and their molecular structures are as follows:

[0073]

[0074] In cytisine alkaloids with an angustifoline-type structural skeleton, the R group is hydrogen, hydroxyl, or other substituted groups; the stereoconfiguration at the C-6 position is R-type or S-type; there is a single or double bond between C-5 and C-6; the stereoconfiguration at the C-7 and C-9 positions is R-type or S-type; and the stereoconfiguration at the C-11 position is R-type or S-type.

[0075] The R group of compound 1 is a hydroxyl group, the stereoconfiguration at the C-6 position is R-type, there is a single bond between C-5 and C-6, the stereoconfiguration at the C-7 and C-9 positions is R-type, and the stereoconfiguration at the C-11 position is R-type.

[0076] The R group of compound 2 is a hydroxyl group, the stereoconfiguration at C-6 is S-type, there is a single bond between C-5 and C-6, the stereoconfiguration at C-7 and C-9 is R-type, and the stereoconfiguration at C-11 is R-type.

[0077] Compound 3 has a double bond between C-5 and C-6, and its stereoconfiguration at C-7 and C-9 is S-type, as is the stereoconfiguration at C-11.

[0078] Compound 4 has a double bond between C-5 and C-6, an S-type stereoconfiguration at C-7 and C-9, and an R-type stereoconfiguration at C-11.

[0079] The alkaloids 1-4 extracted from the red bean tree described in this invention are new compounds.

[0080] Example 6

[0081] Screening of antitumor activity of compounds in this invention

[0082] The antitumor activity of the compounds of this invention was screened using the MTT assay. Logarithmic growth phase cells were seeded into 96-well culture plates at a cell density of 1 × 10⁻⁶ cells / well. 4 Cells were cultured at 100 μL / mL with sterile PBS in each well, and incubated at 37°C with 5% CO2 until adherence. In the experimental group, 10 μM of the novel compound of this invention was added, and the 96-well plate was gently shaken to mix the medium and drug evenly. Incubation continued for 48 hours in a 37°C incubator with 5% CO2. A zero-incubation group (culture medium containing DMSO) was also set up to investigate the effect of drug addition on cells. After incubation, 20 μL of 5 mg / mL MTT solution was added to each well, and incubation continued for 4 hours. The culture was then terminated, the liquid in the wells was aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plate was shaken for 20 min to fully dissolve intracellular crystals, and the absorbance of each well was measured at 560 nm using a microplate reader to calculate the inhibition rate.

[0083] Compound 3, at a concentration of 10 μM, showed an inhibition rate of 11% against the human hepatocellular carcinoma cell line HepG2, 45.1% against the human ovarian cancer cell line A2780, and 20.6% against the human breast cancer cell line MCF-7.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for extracting antitumor cytisine alkaloids from the red bean tree, characterized in that, The method is as follows: Step 1: Crush the dried red bean tree seeds, then extract them three times with a 95% ethanol solution (volume ratio 1:3). After concentration, obtain the seed extract. Step 2: Dissolve the seed extract obtained in Step 1 in water to obtain an aqueous solution of the seed extract. Adjust the pH to 2 with a 2% sulfuric acid solution, mix well, let stand, and then extract with petroleum ether to obtain an acidic aqueous solution after extraction. Step 3: Adjust the pH of the acidic aqueous solution obtained in Step 2 to 10 with a 2 mol / L NaOH solution, mix well and let stand, then extract with chloroform to obtain chloroform extract, and concentrate to obtain chloroform extract. Step 4: The chloroform extract from Step 3 was sequentially separated using normal-phase silica gel column chromatography, reversed-phase ODS column chromatography, and Sephadex LH-20 column chromatography, supplemented by HPLC purification to obtain compounds 1 to 4. Their structural formulas were identified as follows: 。

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