Benzocarbaspirane mixene compounds, methods of making and using the same

Abstract

The present application relates to the technical field of mixed terpenoids, and is a kind of benzotetraquinabicyclic mixed terpenoid compound and its preparation method and application, wherein the compound is obtained by the following steps: crushing mushroom fruiting body into powder; then, soaking the mushroom fruiting body with ethyl acetate to obtain an extract, concentrating the extract to obtain an extract; then, using normal phase silica gel filler, eluting the extract with petroleum ether-dichloromethane to obtain an elution fraction; finally, separating and purifying the obtained elution fraction by reverse phase high performance liquid chromatography.The present application has significant anti-gastric cancer activity, and the preparation method is simple, environmentally friendly and low in cost.

Description

Benzo[a]-decacyclic mixed-source terpenoids, their preparation methods and applications Technical Field

[0001] This invention relates to the field of mixed-source terpenoids, specifically a benzo[a]-decacyclic carbon ring mixed-source terpenoid compound, its preparation method, and its applications. Background Technology

[0002] Benzo[a]-decacyclic mixed-source terpenes are a class of compounds with a benzo[a]-decacyclic structural unit, formed by two carbon-carbon bonds connecting a geranyl segment and an aromatic ring segment in the structural skeleton. Their unique characteristic lies in the presence of this benzo[a]-decacyclic structural unit. These compounds exhibit novel and unique structures, are rare in nature, but possess significant biological activity, making them invaluable for research and application in the field of innovative drug development. To date, only 24 related mixed-source terpenes have been discovered from plants and higher fungi, most of which are lead compounds with significant biological activity and potential development value. Since 2006, five teams—Barrett, Takao, Yoshimitsu, Li, and Lindel—have reported the total synthesis of these compounds in authoritative journals such as *J. Am. Chem. Soc.*, *Angew. Chem. Int. Ed.*, *Org. Lett.*, and *J. Org. Chem.*. Summary of the Invention

[0003] This invention discloses for the first time a benzo[a]-decacyclic carbon ring mixed-source terpene compound, its preparation method and application. The compound has significant anti-gastric cancer activity, and the preparation method is simple, environmentally friendly and low in cost.

[0004] One of the technical solutions of this invention is achieved through the following measures: a benzo[a]decane-membered carbon ring mixed-source terpene compound, the chemical structural formula of which is:

[0005] .

[0006] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions:

[0007] The above compounds are nitrogen-substituted benzene ring fused ten-membered carbon ring mixed-source terpenes.

[0008] The above-mentioned benzo[a]decacyclic mixed-source terpene compound was obtained by the following method:

[0009] First, crush the fruiting bodies of the mushroom Clitocybeclavipes into powder;

[0010] Then, the mushroom fruiting bodies were soaked in ethyl acetate to obtain an extract, which was then concentrated to obtain a paste.

[0011] Next, using normal-phase silica gel packing, the extract was eluted with petroleum ether-dichloromethane to obtain the eluted fraction;

[0012] Finally, the obtained elution fraction was separated and purified by reversed-phase high-performance liquid chromatography.

[0013] The above-mentioned normal-phase silica gel filler has a mesh size of 100 to 200 mesh, and the volume ratio of petroleum ether to dichloromethane is 4 to 6:1.

[0014] The second technical solution of the present invention is achieved through the following measures: a method for preparing a benzo[a]-decacyclic carbon ring mixed-source terpene compound, which is carried out according to the following method:

[0015] First, crush the fruiting bodies of the mushroom Clitocybeclavipes into powder;

[0016] Then, the mushroom fruiting bodies were soaked in ethyl acetate to obtain an extract, which was then concentrated to obtain a paste.

[0017] Next, using normal-phase silica gel packing, the extract was eluted with petroleum ether-dichloromethane to obtain the eluted fraction;

[0018] Finally, the obtained elution fraction was separated and purified by reversed-phase high-performance liquid chromatography.

[0019] The third technical solution of the present invention is achieved through the following measures: the application of a benzo[a]decacyclic carbon ring mixed-source terpene compound in the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0020] This invention discloses a benzo[a]-decacyclic mixed-source terpene compound isolated from the fruiting body of the mushroom *Clitocybeclavipes* using a natural product chemical separation method. This compound exhibits significant anti-gastric cancer activity, and the preparation method is simple, environmentally friendly, and low-cost. Experiments revealed that this compound could only be isolated by eluting the concentrated extract of *Clitocybeclavipes* fruiting bodies using a petroleum ether-dichloromethane solution at a volume ratio of 5:1. This may be related to the presence of heteroatom-substituted benzene rings in the monomer structure of this compound. Attached Figure Description

[0021] Figure 1 shows the 1H-NMR spectrum of the benzo[a]-decacyclic carbon ring mixed-source terpene compound prepared in Example 7 of the present invention;

[0022] Figure 2 shows the 13C-NMR spectrum of the benzo[a]-deca-carbon ring mixed-source terpene compound prepared in Example 7 of the present invention;

[0023] Figure 3 shows the 1H-1H COSY spectrum of the benzo[a]-decacyclic carbon ring mixed-source terpene compound prepared in Example 7 of the present invention;

[0024] Figure 4 shows the HSQC spectrum of the benzo[a]-decacyclic carbon ring mixed-source terpene compound prepared in Example 7 of the present invention;

[0025] Figure 5 shows the HMBC spectrum of the benzo[a]-decacyclic carbon-ring mixed-source terpene compound prepared in Example 7 of this invention. Detailed Implementation

[0026] This invention is not limited to the following embodiments; specific implementation methods can be determined based on the technical solution of this invention and actual circumstances. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are well-known and commonly used chemical reagents and chemical products in the prior art.

[0027] The present invention will be further described below with reference to embodiments:

[0028] Example 1: The chemical structural formula of this benzo[a]decacyclic mixed-source terpene compound is as follows:

[0029] .

[0030] Example 2: As an optimization of the above examples, the compound is a nitrogen-substituted benzene ring fused ten-membered carbon ring mixed-source terpene.

[0031] Example 3: As an optimization of the above examples, the benzo[a]-decacyclic mixed-source terpene compound was obtained by the following method:

[0032] First, crush the fruiting bodies of the mushroom Clitocybeclavipes into powder;

[0033] Then, the mushroom fruiting bodies were soaked in ethyl acetate to obtain an extract, which was then concentrated to obtain a paste.

[0034] Next, using normal-phase silica gel packing, the extract was eluted with petroleum ether-dichloromethane to obtain the eluted fraction;

[0035] Finally, the obtained elution fraction was separated and purified by reversed-phase high-performance liquid chromatography.

[0036] Example 4: As an optimization of the above example, the normal phase silica gel filler has a mesh size of 100 to 200 mesh, and the volume ratio of petroleum ether to dichloromethane is 4 to 6:1.

[0037] Example 5: This benzo[a]decacyclic mixed-source terpene compound was obtained by the following method:

[0038] First, crush the fruiting bodies of the mushroom Clitocybeclavipes into powder;

[0039] Then, the mushroom fruiting bodies were soaked in ethyl acetate to obtain an extract, which was then concentrated to obtain a paste.

[0040] Next, the extract was eluted with petroleum ether-dichloromethane at a volume ratio of 4:1 using normal-phase silica gel 100 mesh packing to obtain the eluted fraction;

[0041] Finally, the obtained elution fraction was separated and purified by reversed-phase high-performance liquid chromatography.

[0042] Example 6: This benzo[a]decacyclic mixed-source terpene compound was obtained by the following method:

[0043] First, crush the fruiting bodies of the mushroom Clitocybeclavipes into powder;

[0044] Then, the mushroom fruiting bodies were soaked in ethyl acetate to obtain an extract, which was then concentrated to obtain a paste.

[0045] Next, the extract was eluted with petroleum ether-dichloromethane at a volume ratio of 6:1 using normal-phase silica gel 200 mesh packing to obtain the eluted fraction;

[0046] Finally, the obtained elution fraction was separated and purified by reversed-phase high-performance liquid chromatography.

[0047] Example 7: This benzo[a]decacyclic mixed-source terpene compound was obtained by the following method:

[0048] First, crush the fruiting bodies of the mushroom Clitocybeclavipes into powder;

[0049] Then, the mushroom fruiting bodies were soaked in ethyl acetate to obtain an extract, which was then concentrated to obtain a paste.

[0050] Next, using normal-phase silica gel 200 mesh packing, the extract was eluted with petroleum ether-dichloromethane at a volume ratio of 5:1 to obtain the eluted fraction;

[0051] Finally, the obtained elution fraction was separated and purified by reversed-phase high-performance liquid chromatography.

[0052] Example 8: Structural confirmation of the benzo[a]-decacyclic mixed-source terpene compound prepared in Example 7 of the present invention: ESI-MS m / z: 326.31 [M+Na]+. 1H NMR (600 MHz, Methanol-d4) δ: 7.17 (1H, s, H-2), 6.15 (1H, s, H-6), 5.28 (1H, t, J=7.8Hz, H-11), 4.08 (1H, s, H-7), 3.76 (1H, d, J=15.6Hz, H-13a), 3.1 6 (1H, d, J=9.6Hz, H-13b), 2.76 (1H, dd, 13.2, 2.4Hz, H-9a), 2.51 (1H, m, H-10a), 2.23 (1H, m, H-10b), 1.60 (3H, s, H-15), 1.30 (1H, m, H-9b). 13C NMR (150 MHz, Methanol-d4) δ: 152.4 (C-1), 107.1 (C-2), 139.8 (C-3), 145.9 (C-4), 124.1 (C-5), 75.5 (C-6), 63.0 (C-7), 61.4 (C-8), 25.1 (C-9), 22.8 (C-10), 122.4 (C-11), 138.0 (C-12), 28.0 (C-13), 116.4 (C-14), 20.5 (C-15), 171.8 (C-16). Correlation data between hydrogen and carbon atoms were determined using two-dimensional HSQC spectroscopy. In the 1H-1H COSY spectrum, H-10 and H-9 and H-11 were correlated, H-11 and H-13 were correlated, and H-9 and H-7 were correlated, suggesting a ten-membered ring fragment. In the HMBC spectrum, H-2 was correlated with C-1, C-3, C-4, and C-5, respectively. The confirmed spectra are shown in Figures 1 to 5. Combining the hydrogen spectrum, carbon spectrum, 1H-1H COSY spectrum, HSQC spectrum, and HMBC spectrum, it was determined that the structure of this compound is consistent with the benzo[a] ten-membered carbon ring mixed-source terpene compound of this invention.

[0053] Comparative Example: Same as Example 7, except that petroleum ether-dichloromethane at volume ratios of 40:1, 2:1, and 10:1, and dichloromethane-ethyl acetate at volume ratios of 10:1, 5:1, and 4:1, were used instead of petroleum ether-ethyl acetate at a volume ratio of 5:1 to obtain 6 control fractions. These fractions were then further separated and purified by reversed-phase high-performance liquid chromatography. The obtained fractions were verified to be non-benzo[a]-decacyclic carbon ring mixed-source terpenoids of the present invention.

[0054] Example 9: Cytotoxicity Experiment

[0055] The in vitro anti-gastric cancer activity of the benzo[a]-decacyclic mixed-source terpene compound prepared in Example 7 of this invention was screened using the MTT assay. Human gastric cancer cells MGC-803 were selected, diluted in culture medium, and seeded at a density of 6 × 10⁴ / ml in 96-well plates, 100 μl per well. After normal incubation for 24 hours, the drug was added. The final drug concentrations were 2.5 μM (Group 1), 5 μM (Group 2), 10 μM (Group 3), 20 μM (Group 4), and 40 μM (Group 5), with adriamycin used as a positive control. A total of 5 concentrations were set, with 3 replicates for each concentration, and a blank control group was also included. After 48 hours of incubation, 10 μL of MTT was added to each well for staining. After culturing for another four hours, the original culture medium was discarded, and 100 μL of DMSO was added to each well. The well was then shaken at low speed for 10 min on a shaker to fully dissolve the crystals. The optical density was detected at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) instrument. The results are shown in Table 1. As can be seen from Table 1, the benzo[a]decacyclic carbon ring mixed-source terpene compound of the present invention has anti-gastric cancer activity.

[0056] In summary, the benzo[a]-decacyclic mixed-source terpene compound isolated from the fruiting body of mushroom Clitocybeclavipes by the present invention through natural product chemical separation has significant anti-gastric cancer activity. The preparation method is simple, environmentally friendly and low in cost.

[0057] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

[0058]

Claims

1. A benzo[a]decacyclic mixed-source terpene compound, characterized in that... The chemical structural formula is: 。 2. A method for preparing the benzo[a]decacyclic mixed-source terpene compound according to claim 1, characterized in that... The procedure is as follows: First, the fruiting bodies of the mushroom *Clitocybeclavipes* are pulverized into powder; then, the fruiting bodies are soaked in ethyl acetate to obtain an extract, which is then concentrated to obtain a paste; next, the paste is eluted with petroleum ether-dichloromethane at a volume ratio of 5:1 using normal-phase silica gel packing material with a mesh size of 100 to 200 to obtain an eluted fraction; finally, the obtained eluted fraction is separated and purified by reversed-phase high-performance liquid chromatography to obtain a benzo[a]-decacyclic carbon ring mixed-source terpene compound, which is a nitrogen-substituted benzene ring fused ten-membered carbon ring mixed-source terpene.

3. The use of the benzo[a]decacyclic mixed-source terpene compound according to claim 1 in the preparation of a drug for treating gastric cancer.

Images