Scalarane type sesterterpenoids and anti-tumor application thereof

Abstract

The invention belongs to the technical field of marine organisms and medicines, and particularly relates to a scalarane type sesterterpenoids compound and anti-tumor application of the scalarane type sesterterpenoids compound. The pentacyclic scalarane type sesterterpenoid C32H46O6 provided by the invention has antitumor activity, provides a new lead compound for antitumor drugs, can be used for developing products for preventing and / or treating tumors, and has predictable market application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of marine biology and pharmaceutical technology, and specifically relates to a scalarane-type disesquiterpenoid compound and its anti-tumor application. The scalarane-type disesquiterpenoid compound discovered in the present invention is extracted and isolated from the marine animal Phyllospongia foliascens, which was collected from the waters of the Xisha Yongle Islands in the South China Sea. Background Art

[0002] Sponges are low-level multicellular animals, numbering approximately 10,000 species. Due to their unique habitat and lack of physical defenses, sponges have developed diverse enzyme reaction systems and biosynthetic pathways distinct from those of terrestrial organisms to protect against predation and transmit information between species. These interactions with symbiotic and environmental microorganisms produce numerous secondary metabolites with novel structures and unique activities. Consequently, sponges have long been a hot topic in marine pharmaceutical research.

[0003] The medicinal source material of the present invention is the cup-leaved sponge Phyllospongiafoliascens collected from the waters of the Yongle Islands in the Xisha Islands in the South China Sea. It is classified into the class Demospongiae, the order Dictyoceratida, and the family Spongiidae. The structures of the compounds isolated and identified from the sponges of the genus Phyllospongia mainly include terpenes, steroids, brominated diphenyl ethers, and sulfonic acids, among which the most representative are scalarane-type diterpenes. Scalarane-type diterpenes are a relatively rare class of natural products, and sponges are their main source. This type of compound exhibits a wide range of and significant biological activities, such as anti-tumor activity, anti-inflammatory, anti-tuberculosis, enzyme inhibitory activity, etc. The tetracyclic scalarane-type diterpenes carteriofenone D isolated from this species of sponge has obvious cytotoxic activity against mouse leukemia cells P388, human colon cancer cells HT-29, and human lung cancer cells A549, IC 50 The values ​​were 0.96, 1.43 and 3.72 μmoL, respectively (Cao F, Wu ZH, Shao CL, et al. Cytotoxic scalarane sesterterpenoids from the South China Seasponge Carteriospongia foliascens[J]. Org Biomol Chem. 2015; 13(13): 4016-24.).

[0004] However, no 6 / 6 / 6 / 6 / 5 pentacyclic scalarane-type diterpenoid compound C has been isolated from this sponge. 32 H 46 O6 was reported and found to have antitumor activity. Summary of the Invention

[0005] A first aspect of the present invention provides a scalarane-type sesquiterpenoid compound.

[0006] A second aspect of the present invention provides a method for preparing a scalarane-type sesquiterpenoid compound.

[0007] A third aspect of the present invention provides an application of a scalarane-type sesquiterpenoid compound.

[0008] In the first aspect, the technical solution of the present invention is:

[0009] A scalarane-type diterpenoid compound containing at least one of the following:

[0010]

[0011] a. a compound represented by formula Ⅰ;

[0012] b. derivatives of the compound of formula Ⅰ;

[0013] a biologically acceptable solvate of ca or b, preferably a pharmaceutically acceptable solvate of a or b;

[0014] A biologically acceptable salt of da or b, preferably a pharmaceutically acceptable salt of a or b.

[0015] The biological activity mentioned in the present invention refers to the ability of a substance or material to interact with an organism, or biological tissue, or substances or components within an organism and induce specific physiological, chemical or functional effects. It is widely used in the fields of medicine, biomaterials, environmental science, agricultural production, nutrition and health, to develop drugs with specific therapeutic effects, materials with the function of repairing and replacing biological tissues, substances or materials that participate in processes such as biodegradation, bioaccumulation, pollution assessment, and ecological restoration, and nutritional and health components that produce nutritional, metabolic or therapeutic effects in the human body, providing strong support for human health and ecological environmental protection.

[0016] Derivatives of the compound of Formula I refer to derivatives obtained by replacing one or more atoms or groups in the compound of Formula I with other atoms or groups, or derivatives with similar properties obtained by adjusting the structure of the compound of Formula I without changing the position and type of the original substituents. Derivatives of the compound of Formula I have at least the same or similar biological activity as the compound of Formula I, such as a hydroxyl group attached to the C-11 position.

[0017] The solvate of the compound represented by Formula I or the solvate of the derivative of the compound represented by Formula I refers to a solvate formed by the compound represented by Formula I or the derivative of the compound represented by Formula I binding to solvent molecules through hydrogen bonds, van der Waals forces, etc. The solvent is divided into inorganic solvents and organic solvents, including but not limited to water, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, acetone, dimethyl sulfoxide, propylene carbonate, etc. The solvate of the compound represented by Formula I or the solvate of the derivative of the compound represented by Formula I has at least the same or similar biological activity as the compound represented by Formula I.

[0018] The salt of the compound represented by Formula I or the salt of the derivative of the compound represented by Formula I refers to a salt compound formed by the combination of the compound represented by Formula I or the derivative of the compound represented by Formula I with inorganic ions or organic ions, including but not limited to inorganic acid salts, organic acid salts, alkali salts or neutral salts. Inorganic acids include but are not limited to hydrochloric acid, sulfuric acid, nitric acid, nitrous acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid, etc., organic acids include but are not limited to acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid or oxalic acid, etc., bases include but are not limited to lithium hydroxide, sodium hydroxide, calcium hydroxide, potassium hydroxide, ammonia water, sodium carbonate, sodium bicarbonate, etc., and neutral compounds in neutral salts include but are not limited to dimethyl sulfate, diethyl succinate, etc.

[0019] In a second aspect, the technical solution of the present invention is: a method for preparing a scalarane-type diterpenoid compound, comprising the following steps:

[0020] (1) Extract the sponge C. serrata with dichloromethane and methanol to obtain the total extract;

[0021] (2) extracting the total extract with methanol and petroleum ether to obtain a petroleum ether extract;

[0022] (3) The petroleum ether extract was separated and purified by normal phase silica gel chromatography using petroleum ether and ethyl acetate as the mobile phase for gradient elution. The eluent was selected from the range of 1:40 to 1:80 of petroleum ether to ethyl acetate, and was recorded as component Fr.8;

[0023] (4) Component Fr.8 was isolated and purified using a normal phase silica gel column chromatography column, using a gradient elution method with a mobile phase of dichloromethane and ethyl acetate, and the eluent was selected from a dichloromethane:ethyl acetate volume ratio within the range of 50:1-10:1, and was recorded as component Fr.8.2;

[0024] (5) Component Fr.8.2 was separated and purified by C8 reverse-phase high-performance liquid chromatography, eluted isocratically with acetonitrile:water = 86:14, and detected at a wavelength of 210 nm, and / or 254 nm, and / or 288 nm to obtain scalarane-type diterpenoid compounds.

[0025] In step (1), the volume ratio of dichloromethane to methanol is 1:0.6-2, preferably 1:1.

[0026] In step (1), ultrasonic extraction of the sponge is performed using a mixed solvent of dichloromethane and methanol, and the ultrasonic extraction is performed 4-6 times, preferably 6 times; the time for each ultrasonic extraction is 1.5-3 hours, preferably 2 hours.

[0027] In step (2), the volume ratio of methanol to petroleum ether is 1:0.6-2, preferably 1:1; the methanol is 75%-99% methanol, preferably 90% methanol.

[0028] In step (2), ultrasonic extraction is performed using a mixed solvent of methanol and petroleum ether, and the ultrasonic extraction is performed 2-4 times, preferably 3 times; the time for each ultrasonic extraction is 0.5-1.5h, preferably 0.5h.

[0029] In step (3), during the gradient elution process, the volume ratio of petroleum ether to ethyl acetate is 100:0, 100:1, 80:1, 60:1, 40:1, 20:1, 1:1, 1:20, 1:40, 1:60, 1:80, 1:100, and 0:100.

[0030] In step (4), during the gradient elution process, the volume ratio of dichloromethane to ethyl acetate is 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:20, 1:50, 1:100, and 0:100.

[0031] In step (5), the retention time of the scalarane-type diperquiterpenoid compound is 52 min, with an error within ±5%, further within ±2%, and further within ±1%.

[0032] The present invention has found that the scalarane-type diperpenoid compound provided by the present invention has inhibitory activity against human chronic myeloid leukemia cells K562, human Burkitt lymphoma cells Raji and Daudi, IC 50The values ​​were 4.70, 5.19, and 5.28 μmoL, respectively.

[0033] In the third aspect, the technical solution of the present invention is:

[0034] The present invention provides a scalarane-type diperquiterpenoid compound for use in preparing products for tumor prevention and / or treatment, including but not limited to medicines, and / or biomaterials, and / or medical devices, and / or health products, and / or foods.

[0035] The present invention provides a product containing a scalarane-type dipterpenoid compound provided by the present invention, wherein the product includes but is not limited to drugs for tumor prevention and / or treatment, and / or biomaterials for tumor prevention and / or treatment, and / or medical devices for tumor prevention and / or treatment, and / or health products for tumor prevention and / or treatment, and / or foods for tumor prevention and / or treatment.

[0036] The medicines described in the present invention refer to substances used for preventing, treating, and diagnosing diseases, purposefully regulating physiological functions, and having prescribed indications or functional indications, usage, and dosage, including traditional Chinese medicines, chemical drugs, and biological products.

[0037] The biomaterials described in the present invention refer to a class of natural or artificially synthesized special functional materials that are used to contact and interact with living systems and can diagnose, treat, replace, repair or induce regeneration of their cells, tissues and organs.

[0038] The medical devices referred to in the present invention refer to instruments, equipment, instruments, in vitro diagnostic reagents and calibrators, materials and other similar or related items that are used directly or indirectly on the human body, including but not limited to invasive, implantable, and human body contacting types, to play a preventive, and / or therapeutic, and / or restorative role.

[0039] The health care products described in the present invention contain a certain amount of effective ingredients that can regulate human functions and have specific effects. They are suitable for specific groups of people and are used as human function regulators, nutritional supplements, etc. They can be prepared in the form of tea, wine, bee products, beverages, soups, fresh juices, medicinal foods, etc.

[0040] The scalarane-type sesquiterpenoid compound provided by the present invention has a simple preparation method and significant anti-tumor activity. The present invention provides a new lead compound for the research and development of new anti-tumor drugs and provides a scientific basis for the development and utilization of my country's marine medicinal resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The scalarane type sesquiterpenoid compound C of the present invention 32 H 46Structural formula of O6.

[0042] Figure 2 The scalarane type sesquiterpenoid compound C of the present invention 32 H 46 O6 1 H-NMR spectrum (DMSO).

[0043] Figure 3 The scalarane type sesquiterpenoid compound C of the present invention 32 H 46 O6 13 C-NMR spectrum (DMSO). DETAILED DESCRIPTION

[0044] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0045] The Phyllospongia foliascens used in this invention was collected from the waters of the Yongle Islands in the Xisha Islands in the South China Sea in April 2019 and identified and confirmed by Mr. Yan Yizhen in the laboratory. Sample certificate (No. DS-PF01) is stored at the Marine Drug Research Center, Department of Pharmacy, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. The scalarane-type diterpenoid compounds prepared in this invention can also be prepared using Phyllospongia foliascens from other sources or by synthetic methods.

[0046] Example 1. Preparation of scalarane-type sesquiterpenoid compounds

[0047] 1. Preparation of crude petroleum ether extract

[0048] At room temperature, about 500 g of Phyllospongia foliascens was chopped and ultrasonically extracted with dichloromethane:methanol (1:1) for 2 h for a total of 6 times. The extracts were combined and concentrated under reduced pressure to obtain a total extract of about 40 g.

[0049] The total extract was ultrasonically extracted three times with 90% methanol: petroleum ether (1:1), and the extract was concentrated to obtain 5 g of petroleum ether extract.

[0050] 2. Separation and purification

[0051] 1) Isolation and purification: The petroleum ether extract was purified by normal phase silica gel chromatography using a mobile phase of petroleum ether:ethyl acetate (100:0, 100:1, 80:1, 60:1, 40:1, 20:1, 1:1, 1:20, 1:40, 1:60, 1:80, 1:100, 0:100) for gradient elution. The eluted portion was monitored by thin layer chromatography (TLC). The eluate in the range of petroleum ether:ethyl acetate 1:40-1:80 was recorded as component Fr.8.

[0052] 2) Component Fr.8 was subjected to normal phase silica gel chromatography with a mobile phase of dichloromethane:ethyl acetate (100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:20, 1:50, 1:100, 0:100) for gradient elution. The eluted portion was monitored by TLC, and the eluent from the dichloromethane:ethyl acetate range of 50:1-10:1 was recorded as component Fr.8.2.

[0053] 3) Component Fr.8.2 was subjected to reverse phase high performance liquid chromatography using acetonitrile:water=86:14, a semi-preparative C8 column, a flow rate of 2 mL / min, detection wavelengths of 210 nm, 254 nm and 288 nm, and a retention time of 52 min to obtain compound C of the present invention. 32 H 46 O6.

[0054] 3. Structural identification

[0055] Compound C of the present invention 32 H 46 O6 was confirmed to be compound C by NMR, HR-ESI-MS, UV and other modern spectral techniques. 32 H 46 The chemical structure of O6 is as follows Figure 1 shown.

[0056] Compound C of the present invention 32 H 46 O6: white powdery solid; UV(MeOH):λ max (logε)240(2.00),277(1.73)nm; ECD(MeOH):λ max (Δε)205(41.518),247(-30.916),282(15.424)nm; HR-ESI-MS m / z[MH] - 525.3223 (calculated value is C 32 H 45 O6,525.3216). 1 H-NMR and 13C-NMR data are shown in Table 1. Figure 2 and Figure 3 .

[0057] Table 1 Compound C 32 H 46 NMR spectrum data of O6

[0058]

[0059]

[0060] a Measured at 700MHz in DMSO-d6; b Measured at 175MHz in DMSO-d6

[0061] Example 2. In vitro antitumor activity test of the compounds of the present invention:

[0062] 1. Experimental cell lines:

[0063] Human Burkitt lymphoma cells Raji and human chronic myeloid leukemia cells K562 were purchased from the Cell Bank of the Chinese Academy of Sciences. Human Burkitt lymphoma cells Daudi were provided by Assistant Professor Yin Qianqian of ShanghaiTech University. The cells grew in suspension.

[0064] 2. Experimental steps:

[0065] Take out the cells from the incubator and observe under a microscope whether the cell morphology is normal, whether the cell culture medium is clean, and whether the cell number is sufficient for the number of plates. Take out the cells in the logarithmic growth phase from the culture flask and dilute 2 mL of cell suspension to 10 5 10 cells were seeded per well 4 Cells were plated in a 100 μL inoculum, with three replicates per well for each concentration. Dissolve the sample in DMSO and add 10 μL of the sample solution at different concentrations (20, 10, 5, 2.5, 1.25, 0.65, and 0.325 μmoL / L) to each well. A cisplatin-positive drug group was also set up, with three replicates per well for each concentration. A blank group (blank culture medium) and a control group (blank culture medium plus cells) were also set up. The culture plate was placed in a 37°C, 5% CO2 incubator and cultured for 72 hours. 10 μL of CCK-8 reagent was added to each well and detected at a wavelength of 450 nm using a microplate reader.

[0066] 3. Experimental data processing:

[0067] For the samples that showed activity in the initial screening under single concentration conditions, the activity dose-dependence relationship was further tested. The activity versus concentration was nonlinearly fitted using Graphpad to calculate the IC value of the sample. 50 value.

[0068] 4. Experimental results:

[0069] The cytotoxicity test was performed using CCK-8 assay, and the results showed that compound C 32 H 46 O6 has inhibitory activity against human chronic myeloid leukemia cells K562 and human Burkitt lymphoma cells Raji and Daudi. The specific activity data are shown in Table 2.

[0070] Table 2 Compound C 32 H 46 The half effective inhibitory concentration of O6 on tumor cells (μmoL)

[0071]

[0072] As shown in Table 2, compound C 32 H 46 O6 showed a certain inhibitory effect on a variety of different tumor cell lines. IC values ​​of human chronic myeloid leukemia K562 cells, human Burkitt lymphoma Raji and Daudi cells were 50 The values ​​were 4.70, 5.19 and 5.28 μmol L, respectively. Therefore, the compound of the present invention can be used to prepare anti-tumor drugs.

[0073] The present invention provides a new lead compound for developing new anti-tumor drugs.

[0074] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A scalarane-type sesquiterpenoid compound, characterized in that: Contains at least one of the following: a. a compound represented by formula Ⅰ; b. derivatives of the compound of formula Ⅰ; a biologically acceptable solvate of ca or b; A biologically acceptable salt of da or b.

2. The method for preparing a scalarane-type sesquiterpenoid compound according to claim 1, characterized in that the steps include: (1) Extract the sponge C. serrata with dichloromethane and methanol to obtain the total extract; (2) extracting the total extract with methanol and petroleum ether to obtain a petroleum ether extract; (3) The petroleum ether extract was separated and purified by normal phase silica gel chromatography using petroleum ether and ethyl acetate as the mobile phase for gradient elution. The eluent was selected from the range of 1:40 to 1:80 of petroleum ether to ethyl acetate, and was recorded as component Fr.8; (4) Component Fr.8 was isolated and purified using a normal phase silica gel column chromatography column, using a gradient elution method with a mobile phase of dichloromethane and ethyl acetate, and the eluent was selected from a dichloromethane:ethyl acetate volume ratio within the range of 50:1-10:1, and was recorded as component Fr.8.2; (5) Component Fr.8.2 was separated and purified by C8 reverse-phase high-performance liquid chromatography, eluted isocratically with acetonitrile:water = 86:14, and detected at a wavelength of 210 nm, and / or 254 nm, and / or 288 nm to obtain scalarane-type diterpenoid compounds.

3. The preparation method according to claim 2, characterized in that In step (1), the volume ratio of dichloromethane to methanol is 1:0.6-2.

4. The preparation method according to claim 2, characterized in that In step (2), the volume ratio of methanol to petroleum ether is 1:0.6-2, and the methanol is 75%-99% methanol.

5. The preparation method according to claim 2, characterized in that In step (3), during the gradient elution process, the volume ratio of petroleum ether to ethyl acetate is 100:0, 100:1, 80:1, 60:1, 40:1, 20:1, 1:1, 1:20, 1:40, 1:60, 1:80, 1:100, and 0:

100.

6. The preparation method according to claim 2, characterized in that In step (4), during the gradient elution process, the volume ratio of dichloromethane to ethyl acetate is 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:20, 1:50, 1:100, and 0:

100.

7. The preparation method according to claim 2, characterized in that In step (5), the retention time of the scalarane-type sesquiterpenoid compound was 52 min, with an error within ±5%.

8. The scalarane-type disesquiterpenoid compound according to claim 1 or the scalarane-type disesquiterpenoid compound prepared by the preparation method according to any one of claims 2 to 7 is used for preparing products for tumor prevention and / or treatment.

9. The use according to claim 8, characterized in that The products include medicines, and / or biological materials, and / or medical devices, and / or health products, and / or foods.

10. At least one of the following products, characterized in that: A scalarane-type disesquiterpenoid compound according to claim 1 or a scalarane-type disesquiterpenoid compound prepared by the preparation method according to any one of claims 2 to 7: a. Drugs for the prevention and / or treatment of tumors; b. Biomaterials for tumor prevention and / or treatment; c. Medical devices for the prevention and / or treatment of tumors; d. Health products for the prevention and / or treatment of tumors; e. Foods for the prevention and / or treatment of tumors.

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