A cucurbitacin compound and its preparation method and application

Abstract

The present invention provides a cucurbitacin compound, its preparation method, and application, belonging to the field of pharmaceutical chemistry. The cucurbitacin compound represented by Formula I is extracted and isolated from Scrophularia purpurogenum. This furanocoumarin compound 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 in 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. #imgabs0# Formula I.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and in particular 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 uncontrollably and form tumors. It is one of the most common cancers among women worldwide. It usually originates 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 specific type of breast cancer characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This type of breast cancer accounts for approximately 15% of all breast cancers and is generally more aggressive than other types of breast cancer, with a relatively poor prognosis. Developing drugs to treat triple-negative breast cancer is of great significance.

[0004] Scrophulariaceae, also known as ground gall grass, fish gall grass, bitter grass, ground small gold coins, and tetracycline grass, is the dried whole herb of Picria felterrae Lour., a plant of the Scrophulariaceae family. As the only plant in the genus Picriae, Picriae is listed in the Chinese Pharmacopoeia and has a history of medicinal use spanning over 200 years. In my country, Picriae is primarily found in Guangdong, Guangxi, Guizhou, and Yunnan provinces. It has the properties of clearing heat and detoxifying, reducing swelling and relieving pain. It is used to treat wind-heat colds, sore throats, laryngeal paralysis, mumps, abdominal pain, dysentery, traumatic injuries, carbuncle, and snake bites. Various preparations using the whole herb have been developed, including Wantong Yankang Tablets, Yinju Liyan Lozenges, Fuyanjing Capsules, and Liyan Qinghuo Lozenges. Literature reports suggest that extracts of Picriae felterrae exhibit antitumor activity, but the underlying mechanism remains 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] .

[0008] The present invention also provides a method for preparing the cucurbitacin compound, which comprises the following steps:

[0009] (1) crushing the bitter figwort, extracting with an organic solvent, and concentrating to obtain an extract;

[0010] (2) Extracting the extract with petroleum ether and ethyl acetate in sequence to obtain the ethyl acetate extract;

[0011] (3) The ethyl acetate extract was subjected to silica gel column chromatography to obtain Fr.1-Fr.17, and Fr.13 was selected;

[0012] (4) Fr.13 was subjected to reverse silica gel preparative column chromatography, and fractions at different retention times were combined to obtain Fr.13-1 to Fr.13-8, and Fr.13-5 was selected;

[0013] (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;

[0014] (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;

[0015] (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.

[0016] "Fractions" refer to the effluent.

[0017] Furthermore, in step (1), the organic solvent is ethanol; the mass ratio of bitter ginseng to the organic solvent is 1:5-15;

[0018] 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;

[0019] 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.

[0020] Furthermore, in step (1), the organic solvent is 90% ethanol; the mass ratio of bitter ginseng to the organic solvent is 1:10;

[0021] 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;

[0022] 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 minutes.

[0023] 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;

[0024] 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;

[0025] 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.

[0026] Furthermore, in step (5), the reverse silica gel preparative column chromatography is performed by isocratically eluting methanol, acetonitrile and water in 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 minutes;

[0027] 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;

[0028] 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.

[0029] The present invention also provides use of the cucurbitacin compound in preparing a drug for preventing and / or treating cancer.

[0030] Furthermore, the cancer is triple-negative breast cancer.

[0031] Furthermore, the drug is a drug that reduces the phosphorylation level of STAT3 and increases the expression of Caspase-3 and PARP.

[0032] The present invention also provides a pharmaceutical composition, which is a preparation prepared by taking the cucurbitacin compound as an active ingredient and adding commonly used pharmaceutical auxiliary materials.

[0033] The present invention has achieved the following beneficial effects:

[0034] 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 46O6. 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.

[0035] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0036] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the ESI-MS spectrum of compound 1.

[0038] Figure 2 is the UV spectrum of compound 1.

[0039] Figure 3 is the ECD spectrum of compound 1.

[0040] Figure 4 is the IR spectrum of compound 1.

[0041] Figure 5 For compound 1 1 H NMR spectrum.

[0042] Figure 6 For compound 1 1 C NMR spectrum.

[0043] Figure 7 For compound 1 1 H- 1 H COSY spectrum.

[0044] Figure 8 is the HMBC spectrum of compound 1.

[0045] Figure 9 is the HSQC spectrum of compound 1.

[0046] Figure 10 This is the NOSEY spectrum of compound 1.

[0047] Figure 11 This is the DP4+ prediction result of the isomer of compound 1.

[0048] Figure 12 This is the inhibitory effect of compound 1 on BT549 cells.

[0049] Figure 13 Compound 1 inhibits STAT3 phosphorylation and promotes Caspase-3 expression and PARP cleavage: (A) Protein immunoblot analysis of the pY705-STAT3 site in BT549 cells inhibited by FN13422 for 4 hours; (B) Quantitative statistical bar graph of protein immunoblot analysis of the control group and each drug-treated group in (A); (C) Protein immunoblot analysis of PARP, Cleaved-PARP, Caspase-3, and Cleaved Caspase-3 in BT549 cells regulated by FN13422 for 24 hours; (D) Quantitative statistical bar graph of protein immunoblot analysis of Caspase-3 in the control group and each drug-treated group; (E) Quantitative statistical bar graph of protein immunoblot analysis of Cleaved Caspase-3 in the control group and each drug-treated group; (F) Quantitative statistical bar graph of protein immunoblot analysis of PARP in the control group and each drug-treated group; (G) Quantitative statistical bar graph of protein immunoblot analysis of Cleaved-PARP in the control group and each drug-treated group. DETAILED DESCRIPTION

[0050] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0051] The experimental instruments used in the present invention are shown in Table 1:

[0052] Table 1 Experimental instruments list

[0053]

[0054] The spectral data and physicochemical parameters used in the present invention are shown in Table 2:

[0055] Table 2 Spectral data and physicochemical parameters

[0056]

[0057] Example 1. Preparation of cucurbitacin compounds of the present invention

[0058] 5 kg of Scrophularia purpurogena was crushed and extracted with 10 times the volume of 90% ethanol under reflux. After concentration, 440.0 g of extract was obtained. This extract was dispersed in distilled water and extracted sequentially with petroleum ether and ethyl acetate. The specific procedure was as follows: After extraction with petroleum ether and water, the aqueous layer was extracted with ethyl acetate, and the ethyl acetate layer was collected. The solvent was recovered to obtain 130.5 g of ethyl acetate extract. The ethyl acetate fraction was purified by silica gel column chromatography using a gradient elution of petroleum ether and ethyl acetate (100:0 → 10:90, v / v). After TLC analysis, the combined fractions yielded Fr.1-Fr.17, with Fr.13 being the fraction collected in the petroleum ether and ethyl acetate (25:75, v / v) elution. Fr.13 was repeatedly prepared by HPLC (i.e., reverse-phase silica gel preparative column chromatography) and isocratically eluted with methanol-acetonitrile-water (45.5:19.5:35, v / v, flow rate 4 mL / min). Fractions at different retention times were combined to obtain Fr.13-1 to Fr.13-8. Fr.13-5 was the fraction with a retention time of 12-13 minutes. Fr.13-5 was repeatedly prepared by HPLC using methanol-acetonitrile-water (18:42:40, v / v, flow rate 4 mL / min). Fractions at different retention times were combined to obtain Fr.13-5-1 to Fr.13-5-9. Fr.13-5-4 was the fraction with a retention time of 11-12 minutes. Fr.13-5-4 was repeatedly prepared by HPLC, and isocratic elution was performed with acetonitrile-water (41:59, v / v, flow rate 4 mL / min). Fractions at different retention times were combined to obtain Fr.13-5-4-1 to Fr.13-5-4-3, of which Fr.13-5-4-2 was the fraction with a retention time of 11.5 min to 12.5 min. Fr.13-5-4-2 was repeatedly prepared by HPLC, and isocratic elution was performed with methanol-water (65:35, v / v, flow rate 4 mL / min). Fractions with a retention time of 18.5 min to 20 min were combined to obtain compound 1.

[0059]

[0060] Structural characterization data of compound 1:

[0061] 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).

[0062] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0063] Experimental Example 1: Characterization and Biological Activity Test of Cucurbitacin Compounds of the Present Invention

[0064] 1. Experimental methods

[0065] 1.1 Structural characterization: ESI-MS, UV, ECD, IR, 1 H NMR, 1 C NMR, 1 H- 1 The structural characterization of compound 1 was carried out by H COSY, HMBC, HSQC, NOSEY and DP4+ prediction.

[0066] 1.2 Biological activity test:

[0067] (1) CCK8 test

[0068] To assess 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. Compound 1 was then introduced into each well at varying doses and incubated for an additional 48 hours. Subsequently, 10 μl of CCK-8 reagent was added to each well and incubated for a further 2–4 hours, until the untreated control wells turned orange. Absorbance at 450 nm was measured using a Varioskan® Flash (Thermo, USA). Cell viability was calculated as follows: Cell viability (%) = (OD value of the treatment group - OD value of the blank well) / (OD value of the blank control group - OD value of the blank well) × 100%.

[0069] (2) Western Blot test

[0070] Cells were lysed at 4°C for 30 minutes 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 minutes 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 sequentially with a primary antibody and an HRP-conjugated secondary antibody. The target proteins were visualized using Immobilon Western chemiluminescent HRP substrate (Millipore, USA), and the western blots were captured and analyzed using a chemiluminescent imaging system (SAGECREATION, Beijing, China).

[0071] 2. Experimental results

[0072] 2.1 Structural characterization

[0073] Figure 1-11 The results of the structural test of compound 1 indicate that the present invention has successfully prepared compound 1. 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).

[0074] 2.2 Biological activity results

[0075] (1) CCK8 results

[0076] Figure 12 The results showed that compound 1 had a strong inhibitory effect on the activity of BT549 triple-negative breast cancer cell line, IC 50 =0.05559μM.

[0077] (2) Western Blot results

[0078] like Figure 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.

[0079] In summary, the present invention provides a cucurbitacin compound and its preparation method and application. 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 sequence to obtain the ethyl acetate extract; (3) The ethyl acetate extract was subjected to silica gel column chromatography to obtain Fr.1-Fr.17, and Fr.13 was selected; (4) Fr.13 was subjected to reverse silica gel preparative column chromatography, and 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 minutes.

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 in 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 minutes; 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 triple-negative breast cancer.

8. A pharmaceutical composition, characterized in that The preparation is prepared by taking the cucurbitacin compound described in claim 1 as an active ingredient and adding common pharmaceutical auxiliary materials.

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