Cucurbitacine compound as well as preparation method and application thereof

Abstract

The invention provides a cucurbitacine compound as well as a preparation method and application thereof, and belongs to the field of medicinal chemistry. The cucurbitacine compound shown in the formula I is extracted and separated from picria fel-terrae and is a furocoumarin compound, phosphorylation of STAT3 in triple negative breast cancer cells can be inhibited, expression of downstream apoptosis-related proteins PARP and Caspase3 is further affected, apoptosis of the triple negative breast cancer cells is promoted, and the cucurbitacine compound can be used for treating the triple negative breast cancer cells. Therefore, the effect of inhibiting the growth of the triple-negative breast cancer is achieved, and the compound has a wide application prospect in preparation of drugs for preventing and / or treating the triple-negative breast cancer. And the formula I of the # imgabs0 # is shown in the specification.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to a cucurbitacin compound and a preparation method and application thereof. Background Art

[0002] Breast cancer is a disease in which abnormal breast cells grow out of control and form tumors. It is one of the most common cancers in women worldwide. It usually starts in the milk ducts or lobules and can spread to the lymph nodes and other parts of the body.

[0003] Triple-negative breast cancer (TNBC) is a special type of breast cancer characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This type of breast cancer accounts for about 15% of all breast cancers and is usually more aggressive than other types of breast cancer, with a relatively poor prognosis. The development of drugs to treat triple-negative breast cancer is of great significance.

[0004] Scrophulariaceae, also known as ground gall grass, fish gall grass, bitter grass, small gold coins, tetracycline grass, is the dried whole herb of Picria felterrae Lour., a plant of the Scrophulariaceae family. Scrophulariaceae is the only plant of the genus Scrophulariaceae, a species included in the Chinese Pharmacopoeia, and has a history of more than 200 years of medicinal use. In my country, Scrophulariaceae is mainly distributed in Guangdong, Guangxi, Guizhou, Yunnan and other places. It has the effects of clearing heat and detoxifying, reducing swelling and relieving pain. It is used for wind-heat cold, sore throat, laryngeal paralysis, mumps, abdominal pain, dysentery, traumatic injury, furuncle, snake bites and other symptoms. Scrophulariaceae has developed a variety of preparations using the whole herb as raw materials, including Wantong Yankang Tablets, Yinju Liyan Lozenges, Fuyanjing Capsules, Liyan Qinghuo Lozenges and other varieties. There are literature reports that Scrophulariaceae extracts have anti-tumor activity, but its material basis is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide a cucurbitacin compound and a preparation method and application thereof.

[0006] The present invention provides a cucurbitacin compound, the structural formula of the cucurbitacin compound is: .

[0007] The present invention also provides a method for preparing the cucurbitacin compound, the method comprising the following steps: (1) crushing the bitter figwort, extracting with an organic solvent, and concentrating to obtain an extract; (2) extracting the extract with petroleum ether and ethyl acetate in turn to obtain an ethyl acetate extract; (3) subjecting the ethyl acetate extract to silica gel column chromatography to obtain Fr.1-Fr.17, and selecting Fr.13; (4) Fr.13 was subjected to reverse silica gel preparative column chromatography, and the fractions at different retention times were combined to obtain Fr.13-1 to Fr.13-8, and Fr.13-5 was selected; (5) Fr.13-5 was subjected to reverse silica gel preparative column chromatography, and the fractions at different retention times were combined to obtain Fr.13-5-1 to Fr.13-5-9, and Fr.13-5-4 was selected; (6) Fr.13-5-4 was subjected to reverse silica gel preparative column chromatography, and the fractions at different retention times were combined to obtain Fr.13-5-4-1 to Fr.13-5-4-3, and Fr.13-5-4-2 was selected; (7) Fr.13-5-4-2 was subjected to reverse silica gel preparative column chromatography, and the fractions at different retention times were combined to obtain the cucurbitacin compound.

[0008] "Fractions" refer to the effluent.

[0009] Furthermore, in step (1), the organic solvent is ethanol; the mass ratio of bitter ginseng to the organic solvent is 1:5-15; In step (3), the silica gel column chromatography is performed by gradient elution using petroleum ether and ethyl acetate in a volume ratio of 100:0 to 10:90; In step (4), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water in a volume ratio of 40-50:15-25:30-40 at a flow rate of 2-6 mL / min.

[0010] Furthermore, in step (1), the organic solvent is 90% ethanol; the mass ratio of bitter ginseng to the organic solvent is 1:10; In step (3), the Fr.13 is a fraction collected by eluting with petroleum ether and ethyl acetate in a volume ratio of 25:75; In step (4), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water in a volume ratio of 45.5:19.5:35 at a flow rate of 4 mL / min, and the Fr.13-5 is a fraction with a retention time of 12 to 13 min.

[0011] Furthermore, in step (5), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water at a volume ratio of 20-25:40-45:30-35 at a flow rate of 2-6 mL / min; In step (6), the reverse silica gel preparative column chromatography is performed by isocratically eluting acetonitrile and water at a volume ratio of 40-45:55-65 at a flow rate of 2-6 mL / min; In step (7), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol and water at a volume ratio of 60-70:30-40 at a flow rate of 2-6 mL / min.

[0012] Furthermore, in step (5), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water at a volume ratio of 18:42:40 at a flow rate of 4 mL / min, and the Fr.13-5-4 is a fraction with a retention time of 11 to 12 min; In step (6), the reverse silica gel preparative column chromatography is performed by isocratically eluting acetonitrile and water at a volume ratio of 41:59 at a flow rate of 4 mL / min, and the Fr.13-5-4-2 is a fraction with a retention time of 11.5 min to 12.5 min; In step (7), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol and water at a volume ratio of 65:35 at a flow rate of 4 mL / min, and the cucurbitacin compound is a fraction with a retention time of 18.5 min to 20 min.

[0013] The present invention also provides use of the cucurbitacin compound in preparing a drug for preventing and / or treating cancer.

[0014] Furthermore, the cancer is triple-negative breast cancer.

[0015] Furthermore, the drug is a drug that reduces the phosphorylation level of STAT3 and increases the expression of Caspase-3 and PARP.

[0016] The present invention also provides a pharmaceutical composition, which is a preparation prepared by taking the cucurbitacin compound as an active ingredient and adding auxiliary materials commonly used in pharmacy.

[0017] The present invention has achieved the following beneficial effects: The present invention extracts and separates a new cucurbitacin compound from Scrophularia odorata, and the molecular formula of the cucurbitacin compound is C 30 H 46 O6. This compound is a furanocoumarin compound that can inhibit the phosphorylation of STAT3 in triple-negative breast cancer cells, and further affect the expression of downstream apoptosis-related proteins PARP and Caspase3, promoting apoptosis of triple-negative breast cancer cells, thereby inhibiting the growth of triple-negative breast cancer. It has broad application prospects in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer.

[0018] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0019] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the ESI-MS spectrum of compound 1.

[0021] Figure 2 is the UV spectrum of compound 1.

[0022] Figure 3 is the ECD spectrum of compound 1.

[0023] Figure 4 is the IR spectrum of compound 1.

[0024] Figure 5 For compound 1 1 H NMR spectrum.

[0025] Figure 6 For compound 1 1 C NMR spectrum.

[0026] Figure 7 For compound 1 1 H- 1 H COSY spectrum.

[0027] Figure 8 is the HMBC spectrum of compound 1.

[0028] Fig. 9 is the HSQC spectrum of compound 1.

[0029] Fig.10 This is the NOSEY spectrum of compound 1.

[0030] Fig.11 This is the DP4+ prediction result of the isomer of compound 1.

[0031] Fig.12 This is the inhibitory effect of compound 1 on BT549 cells.

[0032] Fig.13Compound 1 inhibits STAT3 phosphorylation and promotes Caspase-3 expression and PARP cleavage: (A) Protein immunoblot of FN13422 inhibiting the pY705-STAT3 site in BT549 for 4 hours; (B) Quantitative statistical bar graph of protein immunoblot of the control group and each drug-treated group in (A); (C) Protein immunoblot of FN13422 regulating PARP, Cleaved-PARP, Caspase-3, and Cleaved Caspase-3 in BT549 cells for 24 hours; (D) Quantitative statistical bar graph of protein immunoblot of Caspase-3 in the control group and each drug-treated group; (E) Quantitative statistical bar graph of protein immunoblot of Cleaved Caspase-3 in the control group and each drug-treated group; (F) Quantitative statistical bar graph of protein immunoblot of PARP in the control group and each drug-treated group; (G) Quantitative statistical bar graph of protein immunoblot of Cleaved-PARP in the control group and each drug-treated group. DETAILED DESCRIPTION

[0033] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0034] The experimental instruments used in the present invention are shown in Table 1: Table 1 Experimental instruments list The spectral data and physicochemical parameters used in the present invention are shown in Table 2: Table 2 Spectral data and physicochemical parameters Example 1. Preparation of cucurbitacin compounds of the present invention 5kg of Scrophularia scabra was crushed and extracted with 10 times the amount of 90% ethanol reflux. After concentration, 440.0g of extract was obtained. After dispersion with distilled water, petroleum ether and ethyl acetate were used for extraction in sequence. The specific operation was as follows: After extraction with petroleum ether and water, the water layer was extracted with ethyl acetate, and the ethyl acetate layer was taken. The solvent was recovered to obtain 130.5g of ethyl acetate extract. The ethyl acetate part was chromatographed on a silica gel column with a gradient elution of petroleum ether-ethyl acetate (100:0→10:90, v / v). After TLC analysis and merging, Fr.1-Fr.17 were obtained, of which Fr13 was the fraction collected under the elution of petroleum ether-ethyl acetate (25:75, v / v). Fr.13 was repeatedly prepared by HPLC (i.e., reverse silica gel preparative column chromatography), and isocratically eluted with methanol-acetonitrile-water (45.5:19.5:35, v / v, flow rate 4mL / min), and the fractions at different retention times were combined to obtain Fr.13-1~Fr.13-8. Among them, Fr.13-5 was the fraction at the retention time of 12~13min. Fr.13-5 was selected for repeated HPLC preparation, and isocratically eluted with methanol-acetonitrile-water (18:42:40, v / v, flow rate 4mL / min), and the fractions at different retention times were combined to obtain Fr.13-5-1~Fr.13-5-9. Among them, Fr.13-5-4 was the fraction at the retention time of 11~12min. Fr.13-5-4 was selected for repeated HPLC preparation, and acetonitrile-water (41:59, v / v, flow rate 4 mL / min) was used for isocratic elution, and the fractions at different retention times were combined to obtain Fr.13-5-4-1~Fr.13-5-4-3, among which Fr.13-5-4-2 was the fraction at the retention time of 11.5min~12.5min. Fr.13-5-4-2 was selected for repeated HPLC preparation, and methanol-water (65:35, v / v, flow rate 4 mL / min) was used for isocratic elution, and the fractions at the retention time of 18.5min~20min were combined to obtain compound 1.

[0035] Structural characterization data of compound 1: White powder, C 30 H 46 O6; [α] D 20 +95.6 (c 0.1,ACN); IR (KBr) ν max 3445, 2972,1747, 1698, 1541, 1521, 1456, 1011, 668 cm -1 ; UV(ACN)λ max (log ε): 226(3.28).

[0036] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0037] Experimental Example 1: Characterization and biological activity test of cucurbitacin compounds of the present invention 1. Experimental methods 1.1 Structural characterization: ESI-MS, UV, ECD, IR, 1 H NMR, 1 C NMR, 1 H- 1 H COSY, HMBC, HSQC, NOSEY and DP4+ prediction were used to test the structural characterization of compound 1.

[0038] 1.2 Biological activity test: (1) CCK8 test To evaluate cell viability, the present invention used a cell counting kit 8 (CCK-8, MCE, HY-K0301, USA). BT549 cells (5×10³ cells / 100 μl / well) were seeded into 96-well plates (Costar, USA) and cultured for 24 hours. Thereafter, compound 1 was introduced into each well at different doses and incubated for another 48 hours. Subsequently, 10 μl of CCK-8 reagent was added to each well and further incubated for 2-4 hours until the untreated control wells turned orange. The absorbance at 450 nm was measured using Varioskan® Flash (Thermo, USA). The calculation formula for cell viability is: Cell viability % = (OD value of the drug group-OD value of the blank well) / (OD value of the blank control group-OD value of the blank well) × 100%.

[0039] (2) Western Blot test The cells were lysed at 4°C for 30 min using RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors (Beyotime, Shanghai, China) and phosphatase inhibitors (BOSTER, Wuhan, China). The lysate was then centrifuged to obtain the supernatant. The supernatant was heated at 95°C for 10 min to denature the proteins and then loaded onto a 10% SDS-polyacrylamide gel for electrophoresis. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked and then incubated with the primary antibody and HRP-conjugated secondary antibody in sequence. The target proteins were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA), and the protein blots were captured and analyzed using a chemiluminescent imaging system (SAGECREATION, Beijing, China).

[0040] 2. Experimental results 2.1 Structural characterization Figure 1-11 The results of the structural test of compound 1 indicate that the present invention successfully prepared compound 1, and the structural characterization data are: [α] D 20 +95.6 (c 0.1,ACN); IR (KBr) ν max 3445, 2972, 1747, 1698, 1541,1521, 1456, 1011, 668 cm -1 ; UV(ACN)λ max (log ε): 226(3.28).

[0041] 2.2 Biological activity results (1) CCK8 results Fig.12 The results showed that compound 1 had a strong inhibitory effect on the viability of BT549 triple-negative breast cancer cell line, IC 50 =0.05559μM.

[0042] (2) Western Blot results like Fig.13 As shown, compound 1 can reduce the phosphorylation level of STAT3 in a dose-dependent manner, increase the expression of activated Caspase-3 protein, and promote the cleavage of PARP, thereby promoting the apoptosis process of BT549 triple-negative breast cancer cells.

[0043] In summary, the present invention provides a cucurbitacin compound and a preparation method and application thereof. The molecular formula of the cucurbitacin compound is C 30 H 46 O6. This compound is a furanocoumarin compound that can inhibit the phosphorylation of STAT3 in triple-negative breast cancer cells, and further affect the expression of downstream apoptosis-related proteins PARP and Caspase3, promoting apoptosis of triple-negative breast cancer cells, thereby inhibiting the growth of triple-negative breast cancer. It has broad application prospects in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer.

Claims

1. A cucurbitacin compound, characterized in that: The structural formula of the cucurbitacin compound is: .

2. A method for preparing the cucurbitacin compound according to claim 1, characterized in that: The method comprises the following steps: (1) crushing the bitter figwort, extracting with an organic solvent, and concentrating to obtain an extract; (2) extracting the extract with petroleum ether and ethyl acetate in turn to obtain an ethyl acetate extract; (3) subjecting the ethyl acetate extract to silica gel column chromatography to obtain Fr.1-Fr.17, and selecting Fr.13; (4) Fr.13 was subjected to reverse silica gel preparative column chromatography, and the fractions at different retention times were combined to obtain Fr.13-1 to Fr.13-8, and Fr.13-5 was selected; (5) Fr.13-5 was subjected to reverse silica gel preparative column chromatography, and the fractions at different retention times were combined to obtain Fr.13-5-1 to Fr.13-5-9, and Fr.13-5-4 was selected; (6) Fr.13-5-4 was subjected to reverse silica gel preparative column chromatography, and the fractions at different retention times were combined to obtain Fr.13-5-4-1 to Fr.13-5-4-3, and Fr.13-5-4-2 was selected; (7) Fr.13-5-4-2 was subjected to reverse silica gel preparative column chromatography, and the fractions at different retention times were combined to obtain the cucurbitacin compound.

3. The method according to claim 2, characterized in that In step (1), the organic solvent is ethanol; the mass ratio of bitter ginseng to the organic solvent is 1:5-15; In step (3), the silica gel column chromatography is performed by gradient elution using petroleum ether and ethyl acetate in a volume ratio of 100:0 to 10:90; In step (4), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water in a volume ratio of 40-50:15-25:30-40 at a flow rate of 2-6 mL / min.

4. The method according to claim 3, characterized in that In step (1), the organic solvent is 90% ethanol; the mass ratio of bitter ginseng to the organic solvent is 1:10; In step (3), the Fr.13 is a fraction collected by eluting with petroleum ether and ethyl acetate in a volume ratio of 25:75; In step (4), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water in a volume ratio of 45.5:19.5:35 at a flow rate of 4 mL / min, and the Fr.13-5 is a fraction with a retention time of 12 to 13 min.

5. The method according to claim 2, characterized in that: In step (5), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water at a volume ratio of 10-25:40-45:35-45 at a flow rate of 2-6 mL / min; In step (6), the reverse silica gel preparative column chromatography is performed by isocratically eluting acetonitrile and water at a volume ratio of 40-45:55-65 at a flow rate of 2-6 mL / min; In step (7), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol and water at a volume ratio of 60-70:30-40 at a flow rate of 2-6 mL / min.

6. The method according to claim 5, characterized in that In step (5), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water at a volume ratio of 18:42:40 at a flow rate of 4 mL / min, and the Fr.13-5-4 is a fraction with a retention time of 11 to 12 min; In step (6), the reverse silica gel preparative column chromatography is performed by isocratically eluting acetonitrile and water at a volume ratio of 41:59 at a flow rate of 4 mL / min, and the Fr.13-5-4-2 is a fraction with a retention time of 11.5 min to 12.5 min; In step (7), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol and water at a volume ratio of 65:35 at a flow rate of 4 mL / min, and the cucurbitacin compound is a fraction with a retention time of 18.5 min to 20 min.

7. Use of the cucurbitacin compound according to claim 1 in the preparation of a medicament for preventing and / or treating cancer.

8. The use according to claim 7, characterized in that The cancer is triple-negative breast cancer.

9. The use according to claim 7, characterized in that: The drug is a drug that reduces the phosphorylation level of STAT3 and increases the expression of Caspase-3 and PARP.

10. A pharmaceutical composition, characterized in that The invention relates to a preparation prepared by taking the cucurbitacin compound described in claim 1 as an active ingredient and adding auxiliary materials commonly used in pharmacy.

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