Bis-benzoquinone compound as well as preparation method and application thereof

Abstract

The invention relates to a bisbenzoquinone compound and a preparation method and application thereof.The bisbenzoquinone compound is separated from a fermentation product of chaetomium trilateral, has a brand-new chemical structure, has remarkable inhibitory activity on proliferation of multiple tumor cells, and reaches the nM-level inhibitory activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a bisbenzoquinone compound, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease that seriously threatens human health. Despite the availability of various treatments such as surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, problems like tumor recurrence, metastasis, drug resistance, and toxic side effects during treatment make the development of novel, highly effective, and low-toxicity anti-tumor drugs an urgent priority. Discovering new compounds from natural products and exploring their biological activities and applications holds promise for providing a new material basis for the development of anti-tumor drugs. Natural products are secondary metabolites with specific activities extracted and isolated from plants, animals, microorganisms, and marine organisms. For a long time, researchers have been searching for compounds that can treat diseases from numerous natural products. With advancements in analytical and identification technologies, approximately 300,000 natural products have been identified by their structures. Studies show that the COCONUT database contains over 400,000 natural compounds, approximately 65% ​​of which are derived from plants and approximately 0.5% from animals or marine organisms. In conclusion, natural products are an important source for drug development.

[0003] Quinones are a class of natural products with important biological activities, showing potential application value in multiple fields such as antioxidation, antibacterial, anti-inflammatory, and antitumor activity, and have always been a research hotspot in the field of drug development. Among them, quinones with antitumor effects include: daunorubicin, doxorubicin, juglone, and shikonin.

[0004] Bisbenzoquinone compounds, due to their unique chemical structure and diverse biological activities, have shown great potential in the field of drug development. For example, oosporein can act on tumor cells through various mechanisms, such as interfering with intracellular signaling pathways, inducing tumor cell apoptosis, and inhibiting tumor angiogenesis. Previous studies have reported that the bisbenzoquinone compound oosporein has certain cytotoxicity against human non-small cell lung cancer A549 cells, with an IC50 value of [missing information]. 50 The value is 21 μM.

[0005] However, the limited availability of natural products and the high difficulty, cost, and limited antitumor effects of bibenzoquinone compounds in synthesis have greatly restricted the in-depth research and development of their antitumor activity. Summary of the Invention

[0006] The purpose of this invention is to provide a bisbenzoquinone compound, its preparation method, and its application.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] One of the technical solutions of the present invention is to provide a bisbenzoquinone compound, the structural formula of which is shown in formula (I) below:

[0009]

[0010] The second technical solution of the present invention is to provide a method for preparing a bisbenzoquinone compound as described in one of the above technical solutions, comprising the following steps:

[0011] S1. The fungus Trichoderma trilateralis was inoculated into PDA medium to obtain seed liquid, and the seed liquid was inoculated into fermentation medium for fermentation to obtain fermentation product.

[0012] S2. Extract the fermentation product obtained in step S1 with ethyl acetate to obtain the extract;

[0013] S3. The extract obtained in step S2 was separated and purified by column chromatography to obtain a bisbenzoquinone compound with the structure shown in formula (I).

[0014] In some specific embodiments, in step S1, the seed culture conditions are 200 rpm and 28°C for 3 to 5 days.

[0015] In some specific embodiments, in step S1, the fermentation medium consists of rice and water, with the ratio of rice to water being (70-90) g: (110-130) mL.

[0016] In some specific embodiments, in step S1, the inoculation amount of seed liquid in the fermentation medium is 5% to 10%.

[0017] In some specific implementations, in step S1, the fermentation culture is carried out at room temperature for 25 to 35 days.

[0018] In some specific embodiments, in step S3, the separation and purification process conditions are as follows: silica gel column chromatography is used, with a mixture of petroleum ether and ethyl acetate as the eluent, the volume ratio of petroleum ether to ethyl acetate in the mixture is gradually changed from 10:1 to 1:1, the component with a volume ratio of petroleum ether to ethyl acetate of 3:1 is collected and the solvent is removed to obtain the bisbenzoquinone compound with the structure shown in formula (I).

[0019] The third technical solution of the present invention is to provide the application of the bisbenzoquinone compound as described in one of the above technical solutions in the preparation of antitumor drugs.

[0020] In some specific embodiments, the tumor includes prostate cancer and lung cancer.

[0021] In some specific embodiments, the prostate cancer includes human prostate cancer 22RV1 cells and human prostate cancer LNCaP cells;

[0022] The lung cancers mentioned include human non-small cell lung cancer cells resistant to osimertinib H1975 cells and non-small cell lung cancer cells H1975 cells.

[0023] In some specific embodiments, the antitumor drug also includes a pharmaceutically acceptable carrier.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The bisbenzoquinone compound of the present invention has a novel chemical structure, which expands the structural types of benzoquinone compounds and provides a new structural template for the research and development of related compounds.

[0026] (2) The preparation method of the bibenzoquinone compound of the present invention is simple to operate and is obtained by fermentation of the fungus Trichoderma trilateralis. It is suitable for large-scale production and lays the foundation for the industrial production of the bibenzoquinone.

[0027] (3) The bisbenzoquinone compound of the present invention has a significant inhibitory effect on a variety of tumor cells, especially the inhibitory activity against lung cancer cells reaches the nM level, which is far higher than the μM level inhibitory activity of oosporin against lung cancer, and has certain application prospects in the field of anti-tumor drug development. Attached Figure Description

[0028] Figure 1 The novel dibenzoquinone compound of this invention 1 H NMR spectrum.

[0029] Figure 2 For dibenzoquinone compounds 13 C10 NMR spectrum.

[0030] Figure 3 This is the DEPT 135 spectrum of the bisbenzoquinone compound.

[0031] Figure 4 This is the HSQC spectrum of the bisbenzoquinone compound.

[0032] Figure 5 This is the HMBC spectrum of the bisbenzoquinone compound.

[0033] Figure 6 This is the 1H-1H COSY spectrum of the bisbenzoquinone compound.

[0034] Figure 7 This is the single-crystal X-ray diffraction pattern of the bisbenzoquinone compound. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0036] In the following embodiments, unless otherwise specified, the raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0037] Example 1

[0038] This embodiment provides a novel bisbenzoquinone compound and its preparation method, including the following steps:

[0039] (1) Commercially available Chaetomium trilaterale strain (CGMCC No.: 3.3781) was inoculated into potato dextrose agar (PDA) medium and activated by culturing at 30°C for 3 days.

[0040] (2) 200 mL of PDA medium was added to a 500 mL Erlenmeyer flask, and the final pH of the medium was adjusted to 6.5. The medium was then sterilized in an autoclave to obtain the inoculation medium. The activated Chaetomium trilaterale strain was inoculated into 10 inoculation medium flasks and cultured at 200 rpm and 28 °C for 4 days to prepare seed culture.

[0041] (3) Fermentation was carried out in 10 500 mL Ferment flasks, each containing 80 g of rice and 120 mL of sterile H2O. After cooling to room temperature, 10 mL of seed inoculum was added to each Ferment flask and cultured at room temperature for 30 days.

[0042] (4) After the culture was completed, ethyl acetate was added to the culture and extracted 5 times. The organic phases were combined and concentrated by rotary evaporation under reduced pressure. Impurities were removed by filtration to obtain 1.2g of extract.

[0043] (5) The extract was purified by silica gel column chromatography using petroleum ether-ethyl acetate (the volume ratio of petroleum ether to ethyl acetate gradually changed from 10:1 to 1:1) as the eluent. The eluent containing the target product was collected (the volume ratio of petroleum ether to ethyl acetate was 3:1). The collected eluent was distilled under reduced pressure to remove the solvent, yielding 25 mg of a red solid. The compound was analyzed by Agilent 1260Ⅱ high-performance liquid chromatography (column: Diamonsil Plus 5 μm C18 250 × 4.6 mm; method: 0–20 min 10%–100% methanol, 20–30 min 100% methanol, flow rate 1 mL / min). The purity of the compound was greater than 98% at detection wavelengths of 254, 280, and 365 nm.

[0044] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 H NMR, carbon spectrum 13 The structure of the obtained red solid was identified by C NMR and single-crystal X-ray diffraction (SC-XRD), such as Figures 1-7 As shown.

[0045] 1 H NMR (400MHz, CDCl3) δ2.46 (t, J = 7.7Hz, 2H), 1.50 (m, 2H), 1.31 (m, 8H), 0.89 (t, J = 6.7Hz, 3H).

[0046] 13 C NMR (100MHz, CDCl3) δ117.4,104.4,31.8,29.6,29.1,28.0,22.7,22.6,14.1.

[0047] C 26 H 34 O8 crystal data (molecular weight M = 474.53 g / mol): triclinic system, space group P-1 (No. 2), cell parameters α = 94.12, β = 95.55, γ = 92.79, cell volume With a cell number of molecules Z = 2, a temperature T = 273.15 K, and a linear absorption coefficient μ = 0.718 mm for Kα radiation from a copper target,... -1 The calculated density Dcalc = 1.180 g / cm³ 3 A total of 4670 diffraction points were collected, of which 4670 were independent diffraction points (R0). int =0,R sigma =0.0639), all of which were used for structural calculations. Finally, the correction factor R1 for strong diffraction points (I>2σ(I)) was 0.1338, and the weighted correction factor wR2 for all data was 0.2755.

[0048] The structure of the resulting red solid is shown in formula (I), representing a novel bisbenzoquinone compound:

[0049]

[0050] The prepared bisbenzoquinone compound was tested for antitumor activity.

[0051] Test Example 1: Antitumor activity of the bisbenzoquinone compound against 22RV1 cells

[0052] Adherent human prostate cancer 22RV1 cells in logarithmic growth phase were harvested, digested with trypsin, and transferred to 15 mL centrifuge tubes. The cell pellet was collected by centrifugation at 1000 rpm for 5 min. Cells were resuspended in RPMI-1640 medium and counted using a cell counting chamber under an inverted microscope. The cell density was adjusted to 1 × 10⁶ cells / mL. 4 Cells / 100μL. Seed the cell suspension into 96-well plates, 100μL per well. After the cells adhered, incubate at 37°C and 5% CO2 for 48 hours.

[0053] Discard the original culture medium in each well and add solutions of bisbenzoquinone compounds at concentrations of 25, 50, 75, 100, 125, 150, 175, and 200 μmol / L. A control group was also included. Each group had six replicates. Incubation was continued for 48 hours under the same conditions. After incubation, the culture medium in each well was discarded, and 100 μL of 10% CCK-8 serum-free culture medium was added. The 96-well plate was then returned to the incubator and incubated for another 4 hours.

[0054] The absorbance of each well was measured at 450 nm using a microplate reader. After subtracting background and blank control values, the cell viability percentage was calculated. The results showed that this bisbenzoquinone compound had an IC50 effect on 22RV1 cells. 50 The value was 93.14 ± 8.38 μM.

[0055] Test Example 2: Antitumor activity of the bisbenzoquinone compound against LNCaP cells

[0056] Logarithmic growth phase adherent human prostate cancer LNCaP cells were harvested, digested with trypsin, and transferred to 15 mL centrifuge tubes. The cell pellet was collected by centrifugation at 1000 rpm for 5 min. Cells were resuspended in RPMI-1640 medium, counted using a cell counting chamber under an inverted microscope, and the cell density was adjusted to 8 × 10⁶ cells / mL. 3 Cells / 100μL. Seed the cell suspension into 96-well plates, 100μL per well. After the cells adhered, incubate at 37°C and 5% CO2 for 48 hours.

[0057] Discard the original culture medium in each well, and prepare solutions of bisbenzoquinone compounds at concentrations of 25, 50, 75, 100, 125, 150, 175, and 200 μmol / L. Add these solutions and set up a culture medium control group. Each group has 6 replicates. Incubate under the same conditions for 48 hours. After incubation, discard the culture medium in each well, add 100 μL of 10% CCK-8 serum-free culture medium solution, and return the 96-well plate to the incubator for another 4 hours.

[0058] The absorbance of each well was measured at 450 nm using a microplate reader. After subtracting background and blank control values, the percentage of cell viability was calculated. The results showed that the compound had an IC50 effect on LNCaP cells. 50 The value was 30.08 ± 3.52 μM.

[0059] Test Example 3: Antitumor activity of the bisbenzoquinone compound against drug-resistant H1975 cells

[0060] Adherent human non-small cell lung cancer (NSCLC) cells in logarithmic growth phase, resistant to osimertinib (H1975), were digested with trypsin and transferred to 15 mL centrifuge tubes. The cell pellet was collected by centrifugation at 1000 rpm for 5 min. The cells were resuspended in RPMI-1640 medium, counted using a cell counting chamber under an inverted microscope, and the cell density was adjusted to 8 × 10⁶ cells / mL. 3 Cells / 100μL. Seed the cell suspension into 96-well plates, 100μL per well. After the cells adhered, incubate at 37°C and 5% CO2 for 24 hours.

[0061] Discard the original culture medium in each well, and prepare solutions of bisbenzoquinone compounds at concentrations of 25, 50, 75, 100, 125, 150, 175, and 200 nmol / L. Add these solutions and set up a culture medium control group. Each group has 6 replicates. Incubate under the same conditions for 24 hours. After incubation, discard the culture medium in each well, add 100 μL of 10% CCK-8 serum-free culture medium solution, and return the 96-well plate to the incubator for another 1 hour.

[0062] The absorbance of each well was measured at 450 nm using a microplate reader. After subtracting background and blank control values, the percentage of cell viability was calculated. The results showed that the compound had an IC50 effect on drug-resistant H1975 cells. 50 The value is 84.80±6.65 nM.

[0063] Test Example 4: Antitumor Activity of the Bibenzoquinone Compound on H1975 Cells

[0064] Adherent human non-small cell lung cancer (NSCLC) H1975 cells in logarithmic growth phase were harvested, digested with trypsin, and transferred to 15 mL centrifuge tubes. The cell pellet was collected by centrifugation at 1000 rpm for 5 min. Cells were resuspended in RPMI-1640 medium and counted using a cell counting chamber under an inverted microscope. The cell density was adjusted to 8 × 10⁶ cells / mL. 3 Cells / 100μL. Seed the cell suspension into 96-well plates, 100μL per well. After the cells adhered, incubate at 37°C and 5% CO2 for 24 hours.

[0065] Discard the original culture medium in each well, and prepare solutions of bisbenzoquinone compounds at concentrations of 25, 50, 75, 100, 125, 150, 175, and 200 nmol / L. Add these solutions and set up a culture medium control group. Each group has 6 replicates. Incubate under the same conditions for 24 hours. After incubation, discard the culture medium in each well, add 100 μL of 10% CCK-8 serum-free culture medium solution, and return the 96-well plate to the incubator for another 4 hours.

[0066] The absorbance of each well was measured at 450 nm using a microplate reader. After subtracting background and blank control values, the percentage of cell viability was calculated. The results showed that the compound had an IC50 effect on drug-resistant H1975 cells. 50 The value is 166.30±7.15nM.

[0067] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A bisbenzoquinone compound, characterized in that, Its structural formula is shown in equation (I) below:

2. A method for preparing the bisbenzoquinone compound as described in claim 1, characterized in that, Includes the following steps: S1. The fungus Trichoderma trilateralis was inoculated into PDA medium to obtain seed liquid, and the seed liquid was inoculated into fermentation medium for fermentation to obtain fermentation product. S2. Extract the fermentation product obtained in step S1 with ethyl acetate to obtain the extract; S3. The extract obtained in step S2 was separated and purified by column chromatography to obtain a bisbenzoquinone compound with the structure shown in formula (I).

3. The preparation method according to claim 2, characterized in that, In step S1, the seed culture conditions are 200 rpm and 28℃ for 3 to 5 days.

4. The preparation method according to claim 2, characterized in that, In step S1, the fermentation medium consists of rice and water, with a ratio of (70-90) g to (110-130) mL.

5. The preparation method according to claim 2, characterized in that, In step S1, the inoculation amount of seed culture in the fermentation medium is 5% to 10%.

6. The preparation method according to claim 2, characterized in that, In step S1, the fermentation culture is carried out at room temperature for 25 to 35 days.

7. The preparation method according to claim 2, characterized in that, In step S3, the separation and purification process conditions are as follows: silica gel column chromatography is used, with a mixture of petroleum ether and ethyl acetate as the eluent. The volume ratio of petroleum ether to ethyl acetate in the mixture is gradually changed from 10:1 to 1:

1. The fraction with a volume ratio of petroleum ether to ethyl acetate of 3:1 is collected and the solvent is removed to obtain the bisbenzoquinone compound with the structure shown in formula (I).

8. The use of the bisbenzoquinone compound as described in claim 1 in the preparation of an antitumor drug.

9. The application according to claim 8, characterized in that, The tumors include prostate cancer and lung cancer.

10. The application according to claim 8, characterized in that, The antitumor drugs also include pharmaceutically acceptable carriers.

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