CN102863506A - Four cassane type diterpenoid compounds with antineoplastic activities - Google Patents

Abstract

The present invention relates to the separation and separation of 4 new Kazanane-type diterpenoids and such compounds from the seed of Caesalpina minax Hance by means of phytochemical extraction and separation. Cytotoxic effect on cervical cancer and colon cancer, and its application in clinical prevention and treatment of tumors.

Description

Four carzane-type diterpenoids with antitumor activity

Technical field:

The present invention relates to extraction and separation of 4 new Kazanane-type diterpenoids and the like from the seed of Caesalpinia minax Hance through phytochemical extraction and separation means. The cytotoxic effect of the compound on cervical cancer and colon cancer, and its application in the clinical prevention and treatment of tumors. the

Background technique:

The bitter stone lotus is the seed of Caesalpinia minax Hance in the family Fabaceae. Commonly known as Nanshele and Dragon's Claw, it is a low shrub that grows in mountains, valleys, river valleys or roadside bushes at an altitude of 150-680m. In China, it is mainly distributed in Guangxi and Yunnan in Southwest and South China. , Guizhou, Guangxi, Guangdong, Hainan and other places. Shilian bitterness is cool in nature, bitter in taste, has the effects of clearing away heat and dampness, promoting blood circulation and dispelling blood stasis, and relieving pain. It is a commonly used herbal medicine in southern China. , influenza, hepatitis, dysentery, stranguria, carbuncle, itchy skin, skin ulcers, bruises, snake bites, etc., are included in the classics of Chinese medicine "Zhonghua Materia Medica", and are also included in the standards of Guangxi Chinese medicinal materials and Guizhou Chinese medicinal materials. , Sichuan Traditional Chinese Medicinal Materials Standards, etc., it is a valuable traditional strong medicine. Studies have shown that the characteristic and active ingredients in Echeveria chinensis are mainly cassane diterpenes and their lactones. According to pharmacological activity studies, this compound has good antibacterial, antiviral, antimalarial, antitumor and other activities. the

The compounds Minaxin A (I), Neocaesalpin AA (II), Neocaesalpin AB (III) and Neocaesalpin AD (IV) involved in the present invention are all new compounds, and their antitumor activity is discovered for the first time. So far, the above research results have not yet been published. Patents or literature reports. the

The purpose of the present invention is to make full use of our country's unique plant resources containing carzane-type diterpenes, conduct in-depth research and development, and find such compounds with unique chemical structures, strong anti-tumor activity, and small toxic and side effects, with a view to clinical research. Provide new anticancer drugs. the

Invention content:

The invention provides the application of the carzane-type diterpenoids in pharmacy. the

In our laboratory, we used modern separation techniques and structure determination methods to study the chemical constituents of the seeds of the genus Cercis rosacea, and obtained 4 new kashanane-type diterpenes from it, which are respectively Minaxin A (I), Neocaesalpin AA (II), Neocaesalpin AB (III), Neocaesalpin AD (IV). Through in vitro antitumor pharmacodynamic studies on these compounds, it was found that they have certain inhibitory effects on human cervical cancer Hela and human colon cancer HCT-8. The IC ₅₀ values of compounds Ⅰ-Ⅳ against human cervical cancer Hela and human colon cancer HCT-8 were 40.0, 39.8, 31.6, 32.8 μg/ml and 10.5, 33.0, 29.5, 35.5 μg/ml, respectively.

Technical scheme of the present invention comprises the following steps accordingly:

Nanning, Guangxi collected 8.0 kg of the seeds of A. chinensis, which was identified as [Caesalpiniaminax Hance] by Yuan Jingquan, a researcher at the Guangxi Institute of Medicinal Botany. After crushing 8.0 kg of Echeveria chinensis, heat and reflux extraction with methanol twice, each time for 2 hours, combine the extracts, recover the solvent under reduced pressure, and concentrate to obtain 2020 g of the total extract. After dissolving the total extract of Echeveria chinensis in chloroform-methanol, mix the sample with 100-200 mesh silica gel, elute with petroleum ether, chloroform, ethyl acetate, acetone, methanol in turn, and concentrate the eluent under reduced pressure to finally obtain petroleum ether Fraction 130g, fraction chloroform 325g, fraction ethyl acetate 160g, fraction acetone 305g, fraction methanol 1100g. (The extraction process is shown in Figure 5). The chloroform part was subjected to silica gel column chromatography, and eluted with petroleum ether-ethyl acetate gradient 1:0-1:1 to obtain 12 fractions (Fr.A-L), from which fraction C (15.4g) was selected, fraction D (8.9g), fraction E (6.8g) was subjected to LH20 gel column chromatography, and the pigment was eluted with chloroform-methanol 40:60. Fraction C was subjected to reverse-phase MCI column chromatography and eluted with methanol-water (30:70-100:0) gradient to obtain 8 fractions (C1-C8). C7-C8 were combined after thin-layer detection, and the combined components were separated by reverse-phase high-pressure preparative chromatography with methanol-water (60:40) to obtain compound Ⅰ (10.0 mg). Fraction D was subjected to reverse-phase MCI column chromatography and eluted with methanol-water (30:70-100:0) gradient to obtain 6 fractions (D1-D6). D2-D3 were combined after thin-layer detection, and the combined components were chromatographed on a 300-400 mesh silica gel column with chloroform-petroleum ether (1:1-1:0) gradient elution to obtain compound Ⅱ (8.0mg), compound III (5.3 mg). Fraction E was subjected to medium-pressure preparative liquid phase and eluted with methanol-water (40:70-100:0) gradient to obtain 4 fractions (E1-E4). Component E2 was separated by reverse-phase high-pressure preparative chromatography with methanol-water (68:32) to obtain compound IV (8.2 mg). the

HRESI-MS显示准分子离子峰为m/z 565.2255[M+Na]⁺(计算值565.2261)，提示其分子式为C₂₆H₃₈O₁₂。  Compound I, white powder,

HRESI-MS showed that the quasi-molecular ion peak was m/z 565.2255[M+Na] ⁺ (calculated value 565.2261), suggesting that its molecular formula was C ₂₆ H ₃₈ O ₁₂ .

The maximum UV absorption wavelength is 207nm, which is terminal absorption, showing that it has no strong conjugated system. 1R spectrum has strong absorption at 3350, 1733, 1704cm ^-1 which shows that it has hydroxyl and ketone carbonyl.

¹ H-NMR (DMSO-d ₆ , 600MHz, Table 1): δ1.03(3H, s), 1.04(3H, s), 1.12(3H, s), 1.13(3H, s) are the compounds on the skeleton The angle methyl signal of δ1.91(3H, s), 1.95(3H, s), 2.02(3H, s) is the methyl signal on the acetyl group, and it can be seen from ¹ H-NMR that there are 9 hydrogens above 3.5 Signal; ¹³ C-NMR spectrum (DMSO-d ₆ , 150MHz, Table 1): shows 26 carbon signals, including 4 angle methyl carbon signals (δ14.4, 16.4, 24.1, 30.1), 3 acetyl group Methyl signal (δ20.8, 21.2, 21.3), there are 5 oxymethines and 3 quaternary carbons between δ70-105, and 3 carbonyl carbon signals above 169.0 (δ169.6, 169.7, 169.8 ), the signal at δ209.2 should be the signal of ketone carbonyl carbon.

Comparing the ¹³ C-NMR data of this compound with the compound Magnicaesalpin ^[1] , it is found that the two are very similar, only the chemical shifts at C-15 and C-16 are quite different, indicating that the compound may be at C-15, The C-16 position is different from Magnicaesalpin (the chemical shifts of Magnicaesalpin at the C-15 and C-16 positions are δ42.7 and δ171.6, and the chemical shifts of the two positions of compound I are δ79.0 and δ97.3, respectively ); Therefore, it is speculated that compound I has hydroxyl substitution at C-15 and C-16.

HSQC found four -OH hydrogen signals when determining the connection relationship between carbon and hydrogen atoms: d, J=5.4Hz), through splitting, it is speculated that δ3.70 (1H, s) and δ5.38 (1H, s) are the hydroxyl hydrogen signals attached to the quaternary carbon, and δ5.85 (1H, d, J =9.6Hz), 5.90 (1H, d, J=5.4Hz) is the signal of the hydroxyl hydrogen attached to the methine. the

HMBC spectrum shows: δ5.38 (1H, s, -OH) hydroxyl hydrogen and C-12 (δ209.2), C-13 (δ86.4), C-14 (δ88.9), C-15 (δ79 .0), H-15 (δ3.68, t, 1H) is correlated with C-13 (δ86.4), C-14 (δ88.9), C-16 (δ97.2), H-16 ( δ5.22, dd, 1H) are related to C-13 (δ86.4), C-14 (δ88.9), C-15 (δ79.0); confirm that C-15 and C-16 have hydroxyl substitution infer. the

NOE spectrum shows: H-1, H-6 and 10-CH3 (β position) have NOE gain; H-7, H-15, H-16, 13-OH and 14-CH ₃ (α position) have NOE gain , indicating that 1-OAc, 6-OAc, 13-OH are at the α position; 7-OAc, 15-OH, 16-OH are at the β position.

In summary: Compound I was determined to be 1α, 6α, 7β-triacetoxy-5α, 13α, 15β, 16β-tetrahydroxy-12-oxocassane-14, 16-epoxy, named Minaxin C. the

Table 1. Hydrogen spectrum and carbon spectrum data of compound Ⅰ

Solvent: DMSO-d ₆ , the hydrogen spectrum data was measured at 600MHz, and the carbon spectrum data was measured at 150MHz.

HRESI-MS显示准分子离子峰为m/z 503.2237[M+Na]⁺(计算值503.2257)，提示其分子式为C₂₅H₃₆O₉。  Compound Ⅱ, white powder,

HRESI-MS showed that the quasi-molecular ion peak was m/z 503.2237[M+Na] ⁺ (calculated value 503.2257), suggesting that its molecular formula was C ₂₅ H ₃₆ O ₉ .

¹ H-NMR (CDCl ₃ , 600MHz, Table 2): δ1.09(3H, s), 1.10(3H, s), 1.15(3H, s), 1.42(3H, s) are the angles on the compound skeleton Methyl signal, δ1.97 (3H, s), 2.16 (3H, s) is the methyl signal on the ^acetyl group, and a methoxy signal δ3.14 (3H, s), two Hydrogen signal δ3.03, 3.49 for each hydroxyl group, hydrogen signal δ5.20, 5.27 for two oxygenated carbons, δ6.04 for proton signal on a double bond; ¹³ C-NMR spectrum (CDCl ₃ , 150MHz, Table 2) : Shows 25 carbon signals, including 4 angular methyl carbon signals (δ17.0, 20.3, 25.8, 28.3), 2 acetyl methyl signals (δ21.0, 21.1), and 1 methoxy methyl Signal (δ51.0), there are 2 oxymethines and 3 quaternary carbons between 60-110. In addition, there are 2 double bond carbon signals (δ115.5, 173.0), 3 carbonyl carbons signal (δ169.0, 169.1, 170.5) and the other 8 carbon signals.

Compared the carbon spectrum data of this compound with the carbon spectrum data of Neocaesalpin A ^[2] , it was found that the two are very similar, only the C-12 position has changed, and compound II has one more methoxyl methyl signal than Neocaesalpin A (δ51.0), it can be deduced that the hydroxyl substitution at C-12 in compound Neocaesalpin A becomes methoxyl substitution in compound II. This conclusion was proved by HSQC and HMBC tests.

In summary, the compound Ⅱ was determined to be 1α, 2α-diacetoxy-5α, 14β-dihydroxy-12α-methoxycass-13(15)-en-16, 12-olide, named Neocaesalpin AA. the

HRESI-MS显示准分子离子峰为m/z 445.2192[M+Na]⁺(计算值445.2202)，提示其分子式为C₂₃H₃₄O₇。  Compound Ⅲ, white powder,

HRESI-MS showed that the quasi-molecular ion peak was m/z 445.2192[M+Na] ⁺ (calculated value 445.2202), suggesting that its molecular formula was C ₂₃ H ₃₄ O ₇ .

The ¹ H-NMR (CDCl ₃ , 600MHz, Table 2) and ¹³ C-NMR spectrum (CDCl ₃ , 150MHz, Table 2) data of this compound are very similar to Compound II, only C-2, C-7, and C-14 Significant changes in chemical shifts occurred. Compared with compound II, the C-2 position of compound III changes from the original low-field δ67.2 to high-field δ22.9, and C-7 changes from the original high-field δ19.2 to low-field δ66.9, The C-14 position changed from the original low-field δ75.0 to high-field δ33.0. It can be inferred that there is no substitution at the C-2 position of compound III, a hydroxyl substitution at the C-7 position, and no hydroxyl substitution at the C-14 position. This conclusion was proved by HSQC and HMBC tests.

Combined with the NOE spectrum, the compound Ⅲ was determined to be 1α-acetoxy-5α, 7β-dihydroxy-12α-methoxycass-13(15)-en-16, 12-olide, named Neocaesalpin AB. the

HRESI-MS显示准分子离子峰为m/z 489.2070[M+Na]⁺(计算值489.2191)，提示其分子式为C₂₄H₃₄O₉。  Compound IV, colorless solid,

HRESI-MS showed that the quasi-molecular ion peak was m/z 489.2070[M+Na] ⁺ (calculated value 489.2191), suggesting that its molecular formula was C ₂₄ H ₃₄ O ₉ .

^{The 1} H-NMR (CDCl ₃ , 600MHz, Table 2) and ¹³ C-NMR spectrum (CDCl ₃ , 150MHz, Table 2) data of this compound are very similar to those of compound Ⅲ, only the chemical shift of C-14 has changed significantly . Compared with compound III, the C-14 position of compound IV changed from the original high-field δ33.0 to low-field δ49.2, and compound IV had one more methoxy methyl carbon signal than compound III (δ52. 6) and a carbonyl signal (δ 172.3). Therefore, it is inferred that the compound IVC-14 has a carboxymethyl substitution. This conclusion was proved by HSQC and HMBC tests.

Combined with the NOE spectrum, the compound IV was determined to be 1α-acetoxy-5α, 7β-dihydroxy-12α-methoxycass-13(15)-en-16, 12-olide-17β-Carboxylate, named Neocaesalpin AD. the

Table 2. Hydrogen spectrum and carbon spectrum data of compounds Ⅱ, Ⅲ, Ⅳ

Solvent: CDCl ₃ , the hydrogen spectrum data was measured at 600MHz, and the carbon spectrum data was measured at 150MHz.

Invention effect:

The present invention relates to the application of four carzane-type diterpenoids described in Formula 1 in the preparation of drugs for treating and/or preventing tumors. Experiments have proved that the compound of the present invention has the effect of treating and/or preventing tumors. The present invention has carried out an in vitro anti-tumor pharmacodynamics research test, and the test is as follows:

Detailed ways:

The following examples help those skilled in the art to better understand the present invention, but do not limit the present invention in any way. the

Example 1

Cytotoxicity test:

The activity of each compound was screened by MTT colorimetry. Human cervical cancer cell Hela and human colon cancer cell HCT-8 were diluted in culture medium and inoculated in a 96-well plate at a density of 6×10 ⁴ /ml, 100 μl per well. After normal culture in an incubator for 24 hours, drugs were added. The final concentrations of the drugs were 2.5 μg/ml (group 1), 5 μg/ml (group 2), 10 μg/ml (group 3), 20 μg/ml (group 4), and 40 μg/ml (group 5). A total of 5 concentrations were set up, with 3 replicate wells for each concentration, and a blank group was set up. After culturing for 48 hours, add 10 μl of MTT to each well for staining. After continuing to cultivate for four hours, discard the original culture solution, add 100 μl of DMSO to each well, shake it on a shaker at a low speed for 10 minutes to fully dissolve the crystals, and detect the optical density value at a wavelength of 570nm by an enzyme-linked immunosorbent detector. The results show that: Compound The IC _{50 values} of Ⅰ-Ⅳ against human cervical cancer Hela and human colon cancer HCT-8 were 40.0, 39.8, 31.6, 32.8 μg/ml and 10.5, 33.0, 29.5, 35.5 μg/ml, respectively.

references

[1] Yin Y, Ma L, Hu L.-H. Helvetica Chimica Acta.91, 2008, 972-977.

[2] Kinoshita T, Kaneko M, Noguchi H, Kitagawa I. Heterocycles 1996, 43, 409-414.

Description of drawings:

Figure 1: Structure of compound Ⅰ

Figure 2: Structure of compound Ⅱ

Figure 3: Structure of compound Ⅲ

Figure 4: Structure of Compound IV

Figure 5: The preparation process of compound Ⅰ-Ⅳ

Figure 6: ¹ H-NMR spectrum of compound Ⅰ

Figure 7: ¹³ C-NMR spectrum of compound Ⅰ

Figure 8: HSQC Spectrum of Compound Ⅰ

Figure 9: HMBC spectrum of compound Ⅰ

Figure 10: HRESI-MS spectrum of compound Ⅰ

Figure 11: ¹ H-NMR spectrum of compound Ⅱ

Figure 12: ¹³ C-NMR spectrum of compound II

Figure 13: HSQC spectrum of compound Ⅱ

Figure 14: HMBC spectrum of compound Ⅱ

Figure 15: HRESI-MS spectrum of compound Ⅱ

Figure 16: ¹ H-NMR spectrum of compound III

Figure 17: ¹³ C-NMR spectrum of compound III

Figure 18: HSQC spectrum of compound Ⅲ

Figure 19: HMBC spectrum of compound Ⅲ

Figure 20: HRESI-MS spectrum of compound Ⅲ

Figure 21: ¹ H-NMR spectrum of compound IV

Figure 22: ¹³ C-NMR spectrum of compound IV

Figure 23: HSQC spectrum of compound Ⅳ

Figure 24: HMBC spectrum of compound Ⅳ

Figure 25: HRESI-MS spectrum of compound Ⅳ

Claims (3)

1. There are four carzane-type diterpenoids with anti-tumor activity, which are characterized in that they are obtained by extracting and separating from the seed Lily chinensis of the leguminous plant Cercis cerevisiae, and the chemical structural formula is:

According to 4 new carzane type diterpenoids described in claim 1, it is characterized in that: Compound Ⅰ is Minaxin C[1α, 6α, 7β-triacetoxy-5α, 13α, 15β, 16β-tetrahydroxy-12-oxocassane-14, 16-epoxy], the molecular formula is C ₂₆ H ₃₈ O ₁₂ , it is a white powder, compound Ⅰ for new compounds. Compound Ⅱ is Neocaesalpin AA[1α, 2α-diacetoxy-5α, 14α-dihydroxy-12α-methoxycass-13(15)-en-16, 12-olide], the molecular formula is C ₂₅ H ₃₆ O ₉ , it is a white powder, compound II is a new compound. Compound Ⅲ is Neocaesalpin AB[1α-acetoxy-5α, 7β-dihydroxy-12α-methoxycass-13(15)-en-16, 12-olide], the molecular formula is C ₂₃ H ₃₄ O ₇ , it is white powder, and compound Ⅲ is new compounds. Compound IV is Neocaesalpin AD [1α-acetoxy-5α, 7β-dihydroxy-12α-methoxycass-13(15)-en-16, 12-olide-17β-carboxylate], the molecular formula is C ₂₄ H ₃₄ O ₉ , it is colorless Solid, compound IV is a new compound. 2. The four compounds I-IV according to claim 1 have been proved by tumor pharmacodynamic experiments to have anti-tumor activity. The IC ₅₀ of Minaxin A (compound Ⅰ), Neocaesalpin AA (compound Ⅱ), Neocaesalpin AB (compound Ⅲ), Neocaesalpin AD (compound Ⅳ) for human cervical cancer cell Hela and human colon cancer cell HCT-8 were 40.0, 39.8, 31.6, 32.8 μg/ml and 10.5, 33.0, 29.5, 35.5 μg/ml. 3. the preparation method of 4 carzane-type diterpenes (compound I-IV) described in claim 1, comprises the following steps: Nanning, Guangxi collected 8.0 kg of the seeds of A. chinensis, which was identified as [Caesalpiniaminax Hance] by Yuan Jingquan, a researcher at the Guangxi Institute of Medicinal Botany. After crushing 8.0 kg of Echeveria chinensis, heat and reflux extraction with methanol twice, each time for 2 hours, combine the extracts, recover the solvent under reduced pressure, and concentrate to obtain 2020 g of the total extract. After dissolving the total extract of Echeveria chinensis in chloroform-methanol, mix the sample with 100-200 mesh silica gel, elute with petroleum ether, chloroform, ethyl acetate, acetone, methanol in turn, and concentrate the eluent under reduced pressure to finally obtain petroleum ether Fraction 130g, fraction chloroform 325g, fraction ethyl acetate 160g, fraction acetone 305g, fraction methanol 1100g. (The extraction process is shown in Figure 5). The chloroform part was subjected to silica gel column chromatography, and eluted with petroleum ether-ethyl acetate gradient 1:0-1:1 to obtain 12 fractions (Fr.A-L), from which fraction C (15.4g) was selected, fraction D (8.9g), fraction E (6.8g) were subjected to LH20 gel column chromatography, and eluted with chloroform-methanol 40:60 to remove the pigment. Fraction C was subjected to reverse-phase MCI column chromatography and eluted with methanol-water (30:70-100:0) gradient to obtain 8 fractions (C1-C8). C7-C8 were combined after thin-layer detection, and the combined components were separated by reverse-phase high-pressure preparative chromatography with methanol-water (60:40) to obtain compound Ⅰ (10.0 mg). Fraction D was subjected to reverse-phase MCI column chromatography and eluted with methanol-water (30:70-100:0) gradient to obtain 6 fractions (D1-D6). D2-D3 were combined after thin-layer detection, and the combined components were chromatographed on a 300-400 mesh silica gel column, chloroform-petroleum ether (1:1-1:0) gradient elution, to obtain compound Ⅱ (8.0mg), compound III (5.3 mg). Fraction E was subjected to medium-pressure preparative liquid phase and eluted with methanol-water (40:70-100:0) gradient to obtain 4 fractions (E1-E4). Component E2 was separated by reverse-phase high-pressure preparative chromatography with methanol-water (68:32) to obtain compound IV (8.2 mg).

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