A polypentylene-based adamantane-type benzophenone compound and its preparation method and application

Abstract

The invention discloses a polypentylene-based adamantane-type benzophenone compound and a preparation method and application thereof, and belongs to the field of medical technology. The polypentylene-based adamantane-type benzophenone compound of the invention is extracted with golden silk plum stems by acetone, concentrated to obtain a crude extract, suspended in water, extracted with petroleum ether and dichloromethane in sequence, the dichloromethane extract is loaded onto a silica gel chromatographic column, and after two silica gel chromatographic column purifications, the obtained eluent is recrystallized from petroleum ether to obtain. Experimental studies have shown that the compound has a very strong growth inhibitory effect on human acute promyelocytic leukemia cells HL-60, human prostate cancer cells PC-3, and human colon adenocarcinoma cells Caco-2, and has the prospect of preparing clinical tumor prevention and treatment drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to polypentylene adamantane-type benzophenone compounds extracted and separated from the medicinal plant Garcinia paucinervis and use thereof in preparing drugs for treating human acute promyelocytic leukemia, human prostate cancer and human colon adenocarcinoma. Background Art

[0002] In recent years, the plants of the Garcinia family have been favored by scholars and have become a research hotspot. The plants of this family are rich in benzophenone and polyisoprene. Ketones and other compounds. These two types of compounds have complex and novel structures and have extensive and significant pharmacological activities, such as anti-cancer, hypoglycemic, anti-parasitic, anti-AIDS, antioxidant, anti-inflammatory, anti-ulcer, antibacterial, antifungal, neuroprotective, anthelmintic and other effects, and have great application value.

[0003] Garcinia paucinervis is a tree of the genus Garcinia in the family Garciniae. It is a traditional Chinese medicine. It grows naturally only in limestone mountain forests and is mainly distributed in western and southwestern Guangxi and eastern Yunnan. It is a national second-level key protected plant and an endangered plant. At present, multiple benzophenones, Ketones, polycyclic isopentenyl phloroglucinol compounds, and have obvious inhibitory activity on the growth of tumor cells. The present invention further searches for novel anti-tumor active compounds in the plant, which is of great significance to the research and development of anti-tumor drugs. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a polypentylene adamantane type benzophenone compound and its preparation method and application. The present invention is the first to separate and obtain a new polypentylene adamantane type benzophenone derivative from the stem of golden silk plum, and study its anti-tumor activity. So far, there has been no patent or literature report.

[0005] The object of the present invention is to achieve the following:

[0006] The present invention provides a novel polypentylene-based adamantane-type benzophenone compound extracted and separated from the medicinal plant Garcinia paucinervis as a raw material, the structure of which is shown below:

[0007]

[0008] Wherein, R1, R2, R3, R4, R5, R6, R7 and R8 are independently one of H, OH, CH3, CH3 CH2.

[0009] Based on the above technical solution, further, the structure of the polypentylene-based adamantane-type benzophenone compound is as shown in Formula I:

[0010]

[0011] Another aspect of the present invention provides a method for preparing the above-mentioned polypentylene-based adamantane-type benzophenone compounds, comprising the following steps:

[0012] (1) extracting golden silk plum stems with 50-90% acetone aqueous solution, recovering the extraction solvent, and concentrating to obtain a crude extract;

[0013] (2) suspending the crude extract obtained in step (1) in water, and extracting with petroleum ether and dichloromethane in sequence;

[0014] (3) loading the dichloromethane extract obtained in step (2) onto a silica gel chromatography column and gradient eluting the eluent with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio gradually decreasing from 100:0 to 0:100, and collecting the eluent in a volume ratio of 100:5 to 100:10;

[0015] (4) The eluate obtained in step (3) was concentrated and loaded onto a silica gel chromatography column, and gradient eluted. The eluent was a mixed solvent of petroleum ether and ethyl acetate in a volume ratio gradually reduced from 100:0 to 0:100, and the eluate with a volume ratio of 100:10 to 100:30 was collected;

[0016] (5) The eluate obtained in step (4) is concentrated and then recrystallized from petroleum ether to obtain a polypentylene-based adamantane-type benzophenone compound.

[0017] Based on the above technical solution, further, before soaking in step (1), the golden silk plum stems are cut into thin slices, and the weight ratio of the golden silk plum stems to the volume ratio of the acetone aqueous solution is 1:1 to 1:5 g / mL.

[0018] Based on the above technical solution, further, the number of extractions in step (1) is 1-5 times, and each extraction lasts 48 to 96 hours.

[0019] Based on the above technical solution, further, in step (2), petroleum ether and dichloromethane are used for extraction three times in sequence; the volume ratio of the aqueous phase to the organic phase of each extraction is 1:1 to 1:5.

[0020] Based on the above technical solution, further, the mesh size of the silica gel in the silica gel chromatographic column in step (3) and step (4) is 200-300 mesh or 100-200 mesh.

[0021] Based on the above technical solution, further, in step (3), an eluate with a volume ratio of 100:7 is collected.

[0022] Based on the above technical solution, further, in step (4), an eluate with a volume ratio of 100:20 is collected.

[0023] The present invention also provides the use of the above-mentioned polypentylene adamantane type benzophenone compounds in the preparation of anti-tumor drugs.

[0024] Based on the above technical solution, further, the tumor includes leukemia, prostate cancer and colon adenocarcinoma.

[0025] The beneficial effects of the present invention compared with the prior art are as follows:

[0026] The polypentylene-based adamantane-type benzophenone compounds obtained by the present invention have a stable skeleton and a novel structure and have the activity of inhibiting the proliferation of tumor cells. The in vitro experimental results show that the polypentylene-based adamantane-type benzophenone compounds of the present invention have a strong inhibitory effect on the proliferation of human acute promyelocytic leukemia cells HL-60, human prostate cancer cells PC-3, and human colon adenocarcinoma cells Caco-2 in vitro. The IC 50 The values ​​were 2.47, 9.57 and 9.28 μM, respectively. Therefore, the polypentylene adamantane-type benzophenone compounds of the present invention have a prospect for preparing clinical tumor prevention and treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0028] Figure 1 : Preparation flow chart of compound of formula I;

[0029] Figure 2 : UV spectrum of the compound of formula I;

[0030] Figure 3 : High resolution ESIMS spectrum of the compound of formula I;

[0031] Figure 4 : Hydrogen spectrum of the compound of formula I;

[0032] Figure 5 : Carbon spectrum of the compound of formula I;

[0033] Figure 6 : HMQC spectrum of the compound of formula I;

[0034] Figure 7 : HMBC spectrum of the compound of formula I;

[0035] Figure 8 : The main HMBC-related signal of the compound of formula I;

[0036] Fig. 9: NOESY spectrum of the compound of formula I;

[0037] Fig.10 : Experimental and calculated ECD spectra of compounds of formula I. DETAILED DESCRIPTION

[0038] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0039] Example 1: Preparation of compounds of formula I:

[0040] 12.7 kg of dried medicinal material of Garcinia paucinervis stem is used as raw material, cut into small pieces, and cold-extracted with 80% acetone aqueous solution for 3 times, each time for 72 hours, the extract is concentrated, suspended in water, extracted with petroleum ether and dichloromethane for 3 times, and concentrated; the obtained dichloromethane soluble part (490 g) is separated by silica gel (200-300 mesh) column chromatography, and gradient eluted with petroleum ether-ethyl acetate (100:0-0:100), wherein the petroleum ether-ethyl acetate (100:7) elution part (5 g) is further separated by silica gel (200-300 mesh) column chromatography, and gradient eluted with petroleum ether-ethyl acetate (100:0-0:100), and the petroleum ether-ethyl acetate (100:20) elution part is treated with petroleum ether recrystallization to obtain the compound of formula I (15.15 mg).

[0041] The obtained compound of formula I was subjected to systematic spectral analysis, mainly using ultraviolet spectroscopy, high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR, 2D-NMR), etc. The structural identification results are as follows:

[0042] Colorless needle-shaped crystals (methanol), ferric chloride-potassium ferrocyanide solution appears blue, indicating the presence of phenolic hydroxyl groups in the structure. UV spectrum ( Figure 2 ) gives the maximum absorption peak as λ max :313(3.81), 276(3.95) and 233(4.22)nm;

[0043] HR-ESI-MS( Figure 3 ) gives the quasi-molecular ion peak m / z: 601.3520 [M+H] + (C 38 H 49 O6, calculated value is 601.3524), the molecular formula of the compound is determined to be C 38 H48 O6, unsaturation degree is 15; combined with NMR, it is inferred that the parent nucleus of the compound is a polyisoprenylidene adamantane type benzophenone derivative.

[0044] 1 H NMR (600 MHz, DMSO-d6) Figure 4 ) spectrum, two phenolic hydroxyl proton signals δ9.84, 9.26 (1H, s, OH-13, 14 respectively); one group of ABX coupled aromatic proton signals δ6.69 (1H, d, J = 1.9 Hz, H-12), 6.63 (1H, d, J = 8.4 Hz, H-15) and 6.44 (1H, dd, J = 8.4, 1.9 Hz, H-16); two groups of isopentyl proton signals Signal δ4.77 (1H, t, J = 6.0 Hz, H-28), 2.38, 2.30 (1H, m, H-27 each), 1.55 (3H, s, CH3-30) and 1.59 (3H, s, CH3-31); δ4.83 (1H, d, J = 7.7 Hz, H-35), 3.96 (1H, d, J = 7.7 Hz, H-34), 1.65 (3H, s , CH3-37) and 1.68 (3H, s, CH3-38); 1 methine proton signal δ1.82 (1H, d, J=2.1 Hz, H-7); 5 groups of methylene proton signals δ2.83, 2.64 (each 1H, d, J=14.9 Hz, H-17), 2.28, 2.19 (each 1H, m, H-6), 1.76 (2H, t-like, J=5.6H z, H-26), 1.23 (2H, m, H-25), 1.70 (2H, s, H-21); in addition, there are 5 methyl proton signals δ1.58 (3H, s, CH3-20), 1.11 (3H, s, CH3-32), 1.07 (3H, s, CH3-33), 0.83 (3H, s, CH3-23) and 0.80 (3H, s, CH3-24). 13 C NMR (150 MHz, DMSO-d6) Figure 5 ) spectrum gives 38 carbon signals, including 22 sp 3 Hybridized carbon signals, including 9 methyl carbon signals δ28.0 (C-23, 24), 25.7 (C-37), 25.6 (C-30), 22.4 (C-33), 21.7 (C-32), 20.0 (C-20), 18.1 (C-38), 17.9 (C-31), and 16 sp 2 Hybridized carbon signals, including four carbonyl carbon signals δ202.6 (C-9), 201.6 (C-4), 201.4 (C-2), 191.2 (C-10). 1 H NMR and13 C NMR data are similar to those of the known compound garcinialiptone A (Journal of Natural Products, 2010, 73: 557-562), indicating that the two compounds have the same parent nucleus, and the difference is the signal of the geranyl group in garcinialiptone A. Combined with HSQC ( Figure 6 ) and HMBC( Figure 7 ) data analysis, it can be determined that the side chain composed of two isopentenyl groups is cyclized to form 2,4,4-trimethylcyclohex-1-enylmethyl, which can be confirmed by the following HMBC correlation. In the HMBC spectrum, δ1.58 (3H, s, CH3-20) is remotely correlated with δ128.1 (C-18), 123.9 (C-19), 45.8 (C-21); δ1.70 (2H, s, H-21) is remotely correlated with δ128.1 (C-18), 123.9 (C-19), 35.6 (C-25); δ0.83 (3H, s, CH3-23) and 0.80 (3H, s, CH3-24) has a telecorrelation with δ45.8 (C-21), 35.6 (C-25), and 28.6 (C-22); δ1.23 (2H, m, H-25) has a telecorrelation with 28.8 (C-26); δ1.76 (2H, t-like, J=5.6Hz, H-26) has a telecorrelation with δ128.1 (C-18), 123.9 (C-19), and 35.6 (C-25). Through the long-range correlation of δ2.83 and 2.64 (each 1H, d, J = 14.9 Hz, H-17) with δ202.6 (C-9), 201.6 (C-4), 128.1 (C-18), 123.9 (C-19), 68.1 (C-5), 41.1 (C-6), 28.8 (C-26), it was determined that the 2,4,4-trimethylcyclohex-1-enyl methyl group was located at the C-5 position of the parent core. The HMBC correlation also further confirmed the parent core structure and substitution pattern ( Figure 8). Through the long-range correlation between δ2.38, 2.30 (each 1H, m, H-27) and δ202.6 (C-9), 201.4 (C-2), 132.4 (C-29), 120.4 (C-28), 76.3 (C-1), and the long-range correlation between δ4.77 (1H, t, J = 6.0 Hz, H-28) and δ76.3 (C-1), 22.8 (C-27), 25.6 (C-30), 17.9 (C-31), it was determined that an isopentenyl group was connected to the C-1 position; through δ4.83 (1H, d, J = 7.7 Hz, H-35) and δ78.6 (C-3), 50.8 (C-34), 25.7 (C-37) , 18.1 (C-38) has a long-range correlation, δ1.68 (3H, s, CH3-38) has a long-range correlation with δ133.0 (C-36), 121.1 (C-35), 25.7 (C-37), δ1.65 (3H, s, CH3-37) has a long-range correlation with δ133.0 (C-36), 121.1 (C-35), and δ3.96 (1H, d, J=7.7 Hz, H-34) has a long-range correlation with δ78.6 (C-3), 201.6 (C-4), 41.4 (C-6), 46.3 (C-7), 121.1 (C-35), 133.0 (C-36), confirming that the isobutylenyl group is at the C-34 position. In addition, δ6.69 (1H, d, J = 1.9 Hz, H-12) is correlated with δ150.1 (C-14), 144.7 (C-13), 122.1 (C-16), 6.63 (1H, d, J = 8.4 Hz, H-15) is correlated with δ150.1 (C-14), 144.7 (C-13), 126.2 (C-11), and δ6.64 (1H, dd, J = 8.4, 1.9 Hz, H-16) is correlated with δ191.2 (C-10), 150.1 (C-14), 116.4 (C-12), which further illustrates the existence of the ABX coupling system. According to HSQC and HMBC, the hydrogen and carbon spectrum signals were assigned (Table 1).

[0045] Table 1 Compounds of Formula I 1 H NMR and 13 C NMR chemical shift values ​​(solvent: DMSO-d6)

[0046]

[0047]

[0048]

[0049] The skeleton of this compound has been confirmed by NMR data to be the same as the known compound garcinialiptone A. The skeleton is very rigid and the relative configuration is fixed. It is only necessary to determine the relative configuration of the isobutylene group. Fig. 9 ), δ3.96 (1H, d, J = 7.7 Hz, H-34) and 1.82 (1H, d, J = 2.1 Hz, H-7); 4.83 (1H, d, J = 7.7 Hz, H-35) and 2.28 (1H, m, H-6) are correlated, and δ3.96 (1H, d, J = 7.7 Hz, H-34) and δ2.28, 2.19 (1H, m, H-6) are not correlated, indicating that H-34 and H-7 are in the same plane. Its absolute configuration is calculated by comparing the ECD with the measured ECD ( Fig.10 ), the absolute configuration was determined to be (1S, 3S, 5S, 7R, 34S). The optical rotation value of this compound is -49.86 (c 0.91MeOH).

[0050] Through the above analysis, it is finally determined that the structure of the adamantane-type benzophenone compound of the present invention is as shown in Formula I.

[0051] Example 2: In vitro growth inhibition experiment of the compound of formula (I) on human acute promyelocytic leukemia cells HL-60, human prostate cancer cells PC-3, and human colon adenocarcinoma cells Caco-2

[0052] HL-60 cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, 100 IU / mL penicillin, 100 μg / mL streptomycin and 1 mmol / L L-glutamine in a 37° C., 5% CO 2 saturated humidity incubator.

[0053] Weigh trypan blue, add a small amount of distilled water to grind, add ultrapure water to dilute to 4%, filter with filter paper, and store at 4° C. When used, dilute the mother solution to 0.4% with PBS.

[0054] In this example, 5-fluorouracil was used as a control drug, and each test monomer compound was prepared into a storage solution with a concentration of 100 mM using DMSO and stored at 4°C.

[0055] Take the test cells in the logarithmic growth phase and use (5-5.5)×10 4The cells were inoculated at a density of 100 cells / mL in a 24-well plate, with 2 mL in each well. After inoculation, different concentrations of the drug to be tested were added. After 72 hours of drug treatment (the group without drug addition was the control group), 50 μL of cell suspension was drawn from each well, added to 50 μL of 0.4% trypan blue solution and mixed. Within 3 minutes, the cells were observed under an optical microscope and the total number of cells in each well was counted. The percentage of the total number of cells in each well to the total number of cells in the control well is the growth inhibition rate of the drug at that concentration: 1-(total number of cells in the drug-added well / total number of cells in the control well) × 100%.

[0056] PC-3 and Caco-2 cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, 100 IU / mL penicillin, 100 μg / mL streptomycin and 1 mmol / L L-glutamine, and incubated in a 37°C, 5% CO2 saturated humidity incubator.

[0057] Accurately weigh MTT, add an accurate volume of PBS to dissolve it, and prepare a 5 mg / mL MTT solution. After stirring for 30 minutes in the dark, filter and sterilize it with a microporous filter membrane with a diameter of 0.22 μm, then divide it into portions and store it in a -20°C refrigerator in the dark.

[0058] Take the cells in the logarithmic growth phase, count them accurately with a cell / hemocytometer, and dilute them to 3×10 with RPMI-1640 culture medium. 4 The cells / mL density was inoculated in a 96-well culture plate (sterilized), 100 μL / well, and the 96-well plate was placed in an incubator for 24 hours. According to the pre-designed concentration gradient, it was added to the 96-well plate that had been incubated for 24 hours, 100 μL / well, and at least 3 wells were made in parallel for each concentration, and a control well was set up (only 100 μL blank RPMI-1640 culture medium was added after 24 hours of incubation). After continuing to culture for 96 hours, 50 μL of MTT solution was added to each well. After continuing to culture for 3-4 hours, the solution in the well was discarded, 200 μL of DMSO was added to each well, and it was placed on a timed micro-oscillator for 10 minutes. The absorbance value at a wavelength of 490 nm was measured with an enzyme marker. Among them, only 200 μL of DMSO was added to the A1 well during the entire experiment as a blank control of the 96-well plate, and the IC was calculated using SPSS software. 50 value, its IC 50 The results are shown in Table 2.

[0059] Table 2 In vitro growth inhibition of compounds of formula I on three human cancer cell lines

[0060]

[0061]

[0062] The experimental results in Table 2 show that the compound of formula I has a strong growth inhibitory effect on human acute promyelocytic leukemia cells HL-60, human prostate cancer cells PC-3, and human colon adenocarcinoma cells Caco-2 in vitro, and is expected to be used in the preparation of anti-tumor drugs.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a polypentylene-based adamantane-type benzophenone compound, characterized in that: The steps include: (1) extracting golden silk plum stems with 50-90% acetone aqueous solution, recovering the extraction solvent, and concentrating to obtain a crude extract; (2) suspending the crude extract obtained in step (1) in water, and extracting with petroleum ether and dichloromethane in sequence; (3) loading the dichloromethane extract obtained in step (2) onto a silica gel chromatography column and gradient eluting the eluent with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio gradually decreasing from 100:0 to 0:100, and collecting the eluent in a volume ratio of 100:5 to 100:10; (4) The eluate obtained in step (3) was concentrated and loaded onto a silica gel chromatography column, and gradient eluted. The eluent was a mixed solvent of petroleum ether and ethyl acetate in a volume ratio gradually reduced from 100:0 to 0:100, and the eluate with a volume ratio of 100:10 to 100:30 was collected; (5) concentrating the eluate obtained in step (4) and then recrystallizing it from petroleum ether to obtain a polypentylene-based adamantane-type benzophenone compound; The structure of the polypentylene-based adamantane-type benzophenone compound is shown in Formula I:

2. The preparation method according to claim 1, characterized in that: Before the immersion in step (1), the stems of golden silk plum are cut into thin slices, and the volume ratio of the weight of the golden silk plum stems to the acetone aqueous solution is 1:1-1:5 g / mL; the number of immersions is 1-5 times, and each immersion takes 48-96 hours.

3. The preparation method according to claim 1, characterized in that: In step (2), petroleum ether and dichloromethane are used for extraction three times in sequence; the volume ratio of the aqueous phase to the organic phase in each extraction is 1:1 to 1:

5.

4. The preparation method according to claim 1, characterized in that: The mesh size of the silica gel in the silica gel chromatographic column in step (3) and step (4) is 200-300 mesh or 100-200 mesh.

5. The preparation method according to claim 1, characterized in that: In step (3), the eluate having a volume ratio of 100:7 was collected.

6. The preparation method according to claim 1, characterized in that: In step (4), the eluate was collected at a volume ratio of 100:20.

Images