Taxoid compounds, methods of making, and use against cervical cancer

Abstract

The application discloses taxane-like compounds, a preparation method and application of the taxane-like compounds in treating cervical cancer, and particularly relates to three taxane-like polycyclic polyisopentenyl substituted phloroglucinol compounds (PPAPs) compounds, a preparation method and application of the taxane-like polycyclic polyisopentenyl substituted phloroglucinol compounds in treating cervical cancer. The compounds are separated from Hypericum elatoides R. Keller, wherein compounds 1 and 2 are new compounds. Subsequently, according to the structural characteristics of the compounds, a plurality of cancer cell strains are selected to evaluate the anti-tumor activity of the three separated compounds. The compound 2 has an obvious inhibitory effect on C-33A cells, and activates Caspase-3 through a mitochondrial pathway to induce cancer cell apoptosis. Meanwhile, in a zebrafish cervical cancer xenograft model, the compound 2 inhibits the proliferation, metastasis and tumor-induced angiogenesis of C-33A cells. The above research results show that the newly found compound 2 is a potential lead compound for treating cervical cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to taxane-like compounds, their preparation methods, and their application in treating cervical cancer. Background Technology

[0002] Complex diseases like cancer require a series of treatment methods to inhibit and stabilize their development. One approach involves directly targeting the tumor with chemotherapy drugs and microtubule inhibitors to kill actively differentiating tumor cells. This method is exemplified by drugs like paclitaxel and vincristine, which inhibit mitosis and induce apoptosis by acting on microtubules to disrupt the formation of spindle cells during mitosis. Another approach is to induce tumor cell apoptosis by using anti-angiogenic drugs to disrupt the nutrient delivery required for tumor growth and metastasis. Therefore, in clinical treatment, the discovery of microtubule-targeting and anti-angiogenic drugs has become crucial for cancer therapy.

[0003] Microtubules (MTs) are cellular components composed of α- and β-tubulin dimer subunits. MTs form hollow, cylindrical, filamentous structures with highly dynamic properties. MTs play a crucial role in cellular mechanisms, managing cell growth and division, cell motility, maintaining cell shape, and regulating intracellular transport (Downing et al. 1998). MT homeostasis is characterized by the non-covalent binding of tubulin dimers to growing MTs, followed by depolymerization to return to the dimer state. MTs produce the mitotic spindle, a vital structure required for the proper segregation of chromosomes during cell division in eukaryotic cells. Inhibition of tubulin polymerization and stabilization of MT degradation both disrupt homeostasis, leading to potentially fatal cell damage. In drug-treated cell cultures, the cellular marker of tubulin binding is a large number of cells appearing arrested in mitosis. These cells exhibit chromosome condensation, lack of a nuclear membrane, and deformed or missing mitotic spindles. Drugs that disrupt MT homeostasis, also known as antimitotics, have become a fruitful approach in the development of clinically applicable anticancer drugs.

[0004] Compounds that interfere with cell division by binding to α, β-dimers, oligomers, or polymers have been reported with increasing frequency over the past few decades. [8] Antimitotic agents include a variety of natural, synthetic, and semi-synthetic products with different chemical structures; even compounds that interact with tubulin in the same binding region exhibit structural diversity. Antimitotic agents mainly include: (1) microtubule assembly inhibitors. These agents bind at the CLC site, or to the Vinca domain, or alkylate the thiol group of tubulin; (2) microtubule stabilizers; and (3) microtubule degraders. These compounds bind with high affinity to polymerized tubulin compared to α,β-tubulin heterodimers.

[0005] *Hypericum qishanense* is a distinctive species belonging to the genus *Hypericum* within the family Hypericaceae, distributed in the Qinling Mountains and adjacent areas. It typically inhabits mountainous regions at altitudes of 1700-1900 m. Due to its relatively narrow geographical distribution and morphological tendency to be confused with other species in the same genus, research on the chemical constituents and bioactivities of *Hypericum qishanense* is currently lacking both domestically and internationally, except for previous findings by our research group (Yan et al. 2018; Yan et al. 2019). However, numerous studies have been reported on the chemical constituents and bioactivities of other species in the *Hypericum* genus, particularly the characteristic polycyclic polyisoprenyl substituted phloroglucinols (PPAPs). These studies indicate that *Hypericum* plants exhibit significant therapeutic effects on central nervous system diseases and anti-tumor activity, with their unique pharmacological efficacy closely related to their complex and diverse chemical composition. Summary of the Invention

[0006] This invention isolated three taxane-like compounds from the methanol extract of Hypericum qishanense. Based on the structural characteristics of these compounds, their antitumor activities were screened in various tumor cell lines. Compound 2 was found to exhibit the best inhibitory activity against cervical cancer cells C-33A, and it induced apoptosis in cancer cells by activating Caspase-3 via the mitochondrial pathway. Simultaneously, in a zebrafish cervical cancer xenograft model, compound 2 inhibited the proliferation, metastasis, and tumor-induced angiogenesis of C-33A cells.

[0007] The taxane-like compounds of the present invention include:

[0008] Compound 1:

[0009] Compound 2:

[0010] and / or compound 3:

[0011] The method for preparing taxane-like compounds according to the present invention is described in which the taxane-like compounds are isolated from St. John's wort.

[0012] Optionally, the taxane-like compounds are all isolated from the methanol extract of the aerial parts of Hypericum qishanense.

[0013] Optional, specifically including:

[0014] The aerial parts of St. John's wort from Qishan were extracted with methanol to obtain a methanol extract. The methanol extract was dissolved in water and extracted with petroleum ether to obtain a petroleum ether extract. The petroleum ether extract was continuously eluted by silica gel column chromatography under the following elution conditions: 100-200 mesh, petroleum ether-dichloromethane, 10:1→5:1; petroleum ether-acetone, 80:1→2:1; chloroform-methanol, 10:1→3:1. The products were combined into component AH based on TLC detection.

[0015] Fr.D was eluted by silica gel column chromatography with gradient elution under the following conditions: petroleum ether-ethyl acetate, 200:1 → 3:1; yielding 11 fractions Fr.D.01 to Fr.D.11.

[0016] Fr.D.11 via RP C 18 Column chromatography gradient elution was performed under acetone-water ratio of 5:1 to 20:1, yielding 13 fractions from Fr.D.11.01 to Fr.D.11.13.

[0017] Fr.D.11.06 was purified by dextran gel Sepedax LH-20 column chromatography and preparative thin-layer chromatography to obtain compound 1. The chromatographic solvent for dextran gel Sepedax LH-20 column chromatography was acetone, and the conditions for preparative thin-layer chromatography were dichloromethane-methanol, 100:1.

[0018] Fr.D.11.11 was purified by Sepedax LH-20 column chromatography and preparative thin-layer chromatography to obtain compound 2. The chromatographic solvent for Sepedax LH-20 column chromatography was acetone, and the conditions for preparative thin-layer chromatography were: petroleum ether-ethyl acetate, 10:1.

[0019] Fr.E was eluted by silica gel column chromatography gradient to obtain 7 fractions Fr.E.01 to Fr.E.07. The conditions for silica gel column chromatography gradient elution were: dichloromethane-methanol, 50:1 → 2:1.

[0020] Fr.E.03RP C 18 Six fractions, Fr.D.03.01 to Fr.D.03.06, were obtained by gradient elution using column chromatography; the elution conditions were: methanol-water, 5:1 → 10:1. Fr.D.03.05 was purified by gradient elution using silica gel column chromatography and preparative thin-layer chromatography to obtain compound 3. The gradient elution conditions for silica gel column chromatography were: petroleum ether-ethyl acetate, 15:1 → 4:1; dichloromethane-methanol, 50:1 → 15:1. The elution conditions for preparative thin-layer chromatography were: dichloromethane-methanol, 250:1.

[0021] The taxane-like compounds described in this invention are used in the preparation of anti-cervical cancer drugs.

[0022] The taxane-like compounds described in this invention are used in the preparation of drugs for treating cervical scleroderma.

[0023] A drug for treating cervical cancer, wherein the drug contains the taxane-like compounds described in this invention.

[0024] The taxane-like compounds of this invention have the potential for development and application as small molecule drugs for the prevention and treatment of cervical cancer. Attached Figure Description

[0025] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0026] Figure 1 The effects of compound 2 on the proliferation of different cell lines; (A) the effect of compound 2 at concentrations of 10 μM and 20 μM on the survival rate of five types of uterine cancer cells (HeLa, HCC94, SiHa, MF751, and C-33A); (B) the effect of compound 2 on the IC50 of three types of uterine cancer cells (HCC94, MF751, and C-33A). 50 Value; (C) Compound 2 induces cell death; (red arrows represent dead cells) (D) IC50 of compound 2 after 24h, 48h and 72h of treatment on C-33A cells 50 Value; (E) Effect of compound 2 on C-33A cell colony formation; (F) Quantitative analysis of colony formation; *P<0.05, **P<0.01.

[0027] Figure 2 The effects of compound 2 on apoptosis in C-33A cells; (A) Effect of compound 2 on apoptosis in C-33A cells; (B) Quantitative analysis of the effects of compound 2 on apoptosis in C-33A cells; (C) Effect of compound 2 on the expression of apoptosis-related proteins; (D) Quantitative analysis of the expression of apoptosis-related proteins; *P<0.05, **P<0.01.

[0028] Figure 3 The effect of compound 2 on the migration rate of C-33A cells; (A) Effect of compound 2 on the migration rate of C-33A cells after treatment for 24, 48 and 72 h; (B) Quantitative analysis of migration rate; (C) Effect of compound 2 on the expression of migration-related proteins; (D) Quantitative analysis of related proteins; *P<0.05, **P<0.01.

[0029] Figure 4Survival / teratogenicity of embryos treated with compound 2 and paclitaxel; (A) Typical malformation phenotypes of embryos treated with compound 2 are pericardial edema, yolk sac edema, and axial curvature; (B) Survival and teratogenicity of embryos treated with compound 2; (C) Survival and teratogenicity of embryos treated with paclitaxel; N = 30, scale bar: 200 μm.

[0030] Figure 5 To evaluate the antitumor activity of compound 2 and paclitaxel; C-33A cells were xenografted into the yolk sac of Tg(flk:FGFP) zebrafish embryos; C-33A cells were labeled with CM-DiI (red); (A) Representative photographs of zebrafish embryos treated with 0.1% dimethyl sulfoxide (control), 5 μM paclitaxel, 2.5 μM, 1.25 μM and 0.625 μM compound 2 for 3 days; (B) Quantitative results of fluorescence intensity of C-33A cells in zebrafish embryos after treatment with control or drug; N≥40, scale bar: 100 μm, *P<0.05.

[0031] Figure 6 The image shows zebrafish HE sections at 4 dpi (3 dpt) after xenografting of C-33A cells; the top image shows longitudinal sections of HE from the control group, the 5 μM paclitaxel group, and the 0.625 / 1.25 / 2.5 μM groups; the yellow dashed lines represent C-33A cells; the bottom image is a magnified view of the circle within the yellow dashed lines in the top image; the arrows point to necrotic or apoptotic C-33A cells; the scale bar is 50 μm.

[0032] Figure 7 Evaluation of the effects of compound 2 and paclitaxel on brain metastasis of C-33A cells in zebrafish embryos; (A) Schematic diagram of xenograft injection sites, with the black arrow indicating the injection site—periovolumic space; scale bar: 200 μm; (B) Representative images of brain metastasis in each group after treatment of zebrafish embryos with 0.1% dimethyl sulfoxide (control), 5 μM paclitaxel, 2.5 μM, 1.25 μM and 0.625 μM compound 2; the white dashed circle indicates the eye position of the zebrafish embryo, and the white arrow indicates the brain metastasis of C-33A cells; (C) Quantitative results of anti-brain metastasis activity in zebrafish embryos after treatment with control or drug; N≥34, scale bar: 50 μm, ***P<0.001, *P<0.05.

[0033] Figure 8Compound 2 and paclitaxel inhibited the activity of tumor-induced angiogenesis; angiogenesis was induced by xenografting of C-33A cells into the yolk sac of Tg(flk:FGFP) zebrafish embryos; C-33A cells were labeled with CM-DiI (red); (A) Representative images of SIVs after 24 hours of treatment with 0.1% dimethyl sulfoxide (control), 5 μM paclitaxel, 2.5 μM, 1.25 μM and 0.625 μM compound 2; (B) Quantitative results of anti-zebrafish SIV angiogenesis activity after treatment with control or drug; SIV region (white dashed line), N≥29, scale bar: 100 μm, ***P<0.001.

[0034] Figure 9 The hydrogen spectrum of compound 1;

[0035] Figure 10 The carbon spectrum of compound 1;

[0036] Figure 11 The DEPT 135 spectrum of compound 1;

[0037] Figure 12 The DEPT 90 spectrum of compound 1;

[0038] Figure 13 For compound 1 1 H- 1 H COSY spectrum;

[0039] Figure 14 The HSQC spectrum of compound 1;

[0040] Figure 15 The HMBC spectrum of compound 1;

[0041] Figure 16 The NOESY spectrum of compound 1;

[0042] Figure 17 The image shows the HRESIMS spectrum of compound 1.

[0043] Figure 18 The ECD spectrum of compound 1 is shown below.

[0044] Figure 19 The UV spectrum of compound 1;

[0045] Figure 20 The hydrogen spectrum of compound 2;

[0046] Figure 21 The carbon spectrum of compound 2;

[0047] Figure 22 The DEPT 135 spectrum of compound 2;

[0048] Figure 23The DEPT 90 spectrum of compound 2;

[0049] Figure 24 For compound 2 1 H- 1 H COSY spectrum;

[0050] Figure 25 The HSQC spectrum of compound 2;

[0051] Figure 26 The HMBC spectrum of compound 2;

[0052] Figure 27 The ROESY spectrum of compound 2;

[0053] Figure 28 The HRESIMS spectrum of compound 2;

[0054] Figure 29 The ECD spectrum of compound 2;

[0055] Figure 30 This is the UV spectrum of compound 2. Detailed Implementation

[0056] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only a part of the embodiments of the present invention, not all of them, and do not impose any limitations on the present invention. Any use of the technical solutions of this embodiment, including simple modifications to this embodiment, falls within the scope of protection of the present invention.

[0057] The compound structure of this invention is as follows:

[0058] Compound 1:

[0059] Compound 2:

[0060] and / or compound 3:

[0061] Physicochemical properties of compound 1 of the present invention: C 34 H 42 O6, colorless crystals; mp 144-147℃; (c0.05,MeOH); UV(MeOH)λ max (logε)200(4.53),251(4.02)nm; ECD(MeOH)λ max(Δε)202(-6.67),215(+2.62),233(-7.44),258(+3.75),282(-0.26),311(+4.87)nm; HRESIMS(positive)m / z 547.3063[M+H] + (Calculated value: C) 34 H 43 O6,547.3060). 1 H NMR (CDCl3, 500MHz) and 13 CNMR (CDCl3, 125MHz) data are shown in Table 1, and the spectrum is attached. Figure 9-19 .

[0062] Physicochemical properties of compound 2 of the present invention: C 34 H 42 O5, white powder; (c 0.05, MeOH); UV(MeOH)λ max (logε)200(4.69),241(4.08),292(4.03)nm; ECD(MeOH)λ max (Δε)212(+0.70),234(-4.51),275(+0.64),294(-0.29),322(+1.66)nm; HRESIMS(positive)m / z 553.2919[M+Na] + (Calculated value: C) 34 H 42 NaO (5,553.2930). 1 H NMR (CDCl3, 500MHz) and 13 CNMR (CDCl3, 125MHz) data are shown in Table 2, and the spectrum is attached. Figures 20-30 .

[0063] Physicochemical properties of compound 3 of the present invention: C 34 H 42 O5, colorless oil; ESI-MS m / z: 530.30M + .

[0064] All compounds were isolated from the petroleum ether layer of the methanol extract of St. John's wort from Qishan.

[0065] St. John's wort from Qishan was collected in Qishan County, Baoji City, Shaanxi Province in 2019 and dried in the shade at room temperature before use. The aerial parts of St. John's wort were extracted with methanol under reflux, followed by extraction with an equal volume of petroleum ether to obtain a petroleum ether extract.

[0066] The petroleum ether extract was continuously eluted by silica gel column chromatography (100-200 mesh, petroleum ether-dichloromethane, 10:1→5:1; petroleum ether-acetone, 80:1→2:1; chloroform-methanol, 10:1→3:1), and the products were combined into component AH according to TLC detection.

[0067] Fr.D was eluted by silica gel column chromatography with a gradient elution (petroleum ether-ethyl acetate, 200:1→3:1) to obtain 11 fractions Fr.D.01~Fr.D.11. Fr.D.11 was subjected to RP-C... 18 Column chromatography gradient elution (acetone-water, 5:1→20:1) yielded 13 fractions Fr.D.11.01~Fr.D.11.13. Fr.D.11.06 was purified by dextran gel Sephexax LH-20 column chromatography (acetone) and preparative thin-layer chromatography (dichloromethane-methanol, 100:1) to obtain compound 1 (2.6 mg).

[0068] Fr.D.11.11 was purified by Sephex LH-20 column chromatography (acetone) and thin-layer preparative chromatography (petroleum ether-ethyl acetate, 10:1) to give compound 2 (22.0 mg).

[0069] Fr.E was eluted by gradient elution using silica gel column chromatography (dichloromethane-methanol, 50:1→2:1) to obtain seven fractions Fr.E.01~Fr.E.07. Fr.E.03RP C 18 Column chromatography gradient elution (methanol-water, 5:1→10:1) yielded six fractions Fr.D.03.01 to Fr.D.03.06. Fr.D.03.05 was purified by silica gel column chromatography gradient elution (petroleum ether-ethyl acetate, 15:1→4:1; dichloromethane-methanol, 50:1→15:1) and preparative thin-layer chromatography (dichloromethane-methanol, 250:1) to obtain compound 3 (19.2 mg).

[0070] The taxane-like compounds of this invention are used in the preparation of antitumor drugs. In this invention, six tumor cell types were selected as research models based on the structural characteristics of the compounds; the taxane-like compounds exhibit pharmacological activity against cervical cancer, wherein compound 2, after acting on C-33A cervical cancer cells for 72 hours, showed an IC50 value... 50 With a value of 7.4 μM, the compounds of the present invention are particularly suitable for use in the preparation of drugs for treating cervical squamous cell carcinoma.

[0071] A drug for treating cervical cancer, comprising the taxane-like compounds of this invention.

[0072] Compounds 1, 2, and 3 of this invention were isolated from methanol extracts of the aerial parts of *Hypericum qishanense*. *Hypericum qishanense* was collected from Qishan County, Baoji City, Shaanxi Province. The sample was identified as *Hypericum qishanense* by Professor Jiang Zaimin of the College of Life Sciences, Northwest A&F University, and the specimen is preserved in the Northwest A&F University Herbarium (Specimen No.: Jiang1043).

[0073] Compounds 1, 2, and 3 are taxane-like compounds. Based on their structural characteristics, we found that compound 2 can promote apoptosis in cervical cancer cells C-33A. The taxane-like compounds of this invention have the potential for development and application as anti-tumor drugs in the field of oncology.

[0074] I. Extraction method, identification, determination of antitumor activity, and application of the compounds of this invention:

[0075] 1. Experimental Materials

[0076] Qishan St. John's wort: The above-ground parts of fresh Qishan St. John's wort are dried in the shade at room temperature.

[0077] Extraction of extract: extract St. John's wort from Qishan by heating and reflux with methanol, suspend the extract in water, extract five times with petroleum ether, concentrate the extract to obtain 1.37 kg of extract.

[0078] Reagents and instruments:

[0079] Analytical grade reagents: petroleum ether, dichloromethane, acetone, ethyl acetate, methanol.

[0080] Chromatographic grade reagents: methanol, acetonitrile.

[0081] Deuterated reagent: chloroform.

[0082] Colorimetric reagent: 10% sulfuric acid-ethanol and anisaldehyde solution.

[0083] Commonly used instruments: Nuclear magnetic resonance spectrometer (AVANCEⅢ 500MHz); Circular dichroism spectrometer (Chirascan spectrometer); High-resolution mass spectrometer (LC-30A+TripleTOF5600+); Infrared spectrometer (Tensor 27FT-IR); Polarimeter (Rudolph AutopolⅢ). Electronic balance (BSA124S); Low-temperature coolant circulation pump (DLSB); Ultrasonic cleaner (KQ5200); Rotary evaporator (N-1300DH-WB); High-performance liquid chromatograph (Agilent 1100); Dark box ultraviolet analyzer (ZF-20D); Cell incubator (Forma™ 310); Clean bench (SW-CJ-2F); Upright microscope (CKX41); Benchtop high-speed centrifuge (H1650-W); Multifunctional microplate reader (Synergy HTX); Laser scanning confocal microscope (LEICATCS SP8); Chemiluminescence imaging system (Alliance Q9 Advanced); High-speed sorting flow cytometer (BDFACSAria) TM III); High-speed rotary laser confocal (Revolution WD).

[0084] 2. Specific extraction and separation methods for taxane-like compounds (unless otherwise specified, all reagent ratios are volume ratios, and the volume unit is generally ml).

[0085] The aerial parts of dried St. John's wort from Qishan were extracted by reflux with methanol, followed by extraction with an equal volume of petroleum ether to obtain a petroleum ether extract. The petroleum ether extract was continuously eluted by silica gel column chromatography (100-200 mesh, petroleum ether-dichloromethane, 10:1→5:1; petroleum ether-acetone, 80:1→2:1; chloroform-methanol, 10:1→3:1). The products were combined into fraction AH based on TLC analysis. Fr.D was further eluted by gradient silica gel column chromatography (petroleum ether-ethyl acetate, 200:1→3:1) to obtain 11 fractions Fr.D.01~Fr.D.11. Fr.D.11 was analyzed by RP-C... 18Column chromatography gradient elution (acetone-water, 5:1→20:1) yielded 13 fractions Fr.D.11.01~Fr.D.11.13. Fr.D.11.06 was purified by Sephadax LH-20 dextran gel chromatography (acetone) and preparative thin-layer chromatography (dichloromethane-methanol, 100:1) to obtain compound 1 (2.6 mg). Fr.D.11.11 was purified by Sephadax LH-20 dextran gel chromatography (acetone) and preparative thin-layer chromatography (petroleum ether-ethyl acetate, 10:1) to obtain compound 2 (22.0 mg). Fr.E was purified by silica gel column chromatography gradient elution (dichloromethane-methanol, 50:1→2:1) to obtain 7 fractions Fr.E.01~Fr.E.07. Fr.E.03RP C 18 Column chromatography gradient elution (methanol-water, 5:1→10:1) yielded six fractions Fr.D.03.01 to Fr.D.03.06. Fr.D.03.05 was purified by silica gel column chromatography gradient elution (petroleum ether-ethyl acetate, 15:1→4:1; dichloromethane-methanol, 50:1→15:1) and preparative thin-layer chromatography (dichloromethane-methanol, 250:1) to obtain compound 3 (19.2 mg).

[0086] 3. Physicochemical properties of compounds 1 and 2

[0087] The physicochemical properties of compound 1 of the present invention are as follows:

[0088] C 34 H 42 O6, colorless crystals; mp 144-147℃; (c 0.05, MeOH); UV(MeOH)λ max (logε)200(4.53),251(4.02)nm; ECD(MeOH)λ max (Δε)202(-6.67),215(+2.62),233(-7.44),258(+3.75),282(-0.26),311(+4.87)nm; HRESIMS(positive)m / z 547.3063[M+H] + (Calculated value: C) 34 H 43 O6,547.3060). 1 H NMR (CDCl3, 500MHz) and 13 The C NMR (CDCl3, 125MHz) data are shown in Table 1, and the spectra are shown in the appendix. Figure 9-19 .

[0089] Table 1 Compound 1 1 H and 13C-NMR data (δin ppm, J in Hz)

[0090]

[0091]

[0092] The physicochemical properties of compound 2 of the present invention are as follows:

[0093] C 34 H 42 O5, white powder; (c 0.05, MeOH); UV(MeOH)λ max (logε)200(4.69),241(4.08),292(4.03)nm; ECD(MeOH)λ max (Δε)212(+0.70),234(-4.51),275(+0.64),294(-0.29),322(+1.66)nm; HRESIMS(positive)m / z553.2919[M+Na] + (Calculated value: C) 34 H 42 NaO (5,553.2930). 1 H NMR (CDCl3, 500MHz) and 13 The C NMR (CDCl3, 125MHz) data are shown in Table 2, and the spectra are attached. Figures 20-30 .

[0094] Table 2 Compound 2 1 H and 13 C-NMR data (δin ppm, J in Hz)

[0095]

[0096]

[0097] 4. Testing of antitumor activity

[0098] 4.1 Screening for the antitumor activity of compounds 1-3

[0099] By reviewing the literature, we first screened the activity of taxane-like compounds on six tumor cell lines (C-33A, MKN-45, LNCaP, A2780, MDA-MB-468, and LECPα-1). As shown in Table 3, compound 1 showed weak inhibitory activity against the six tumor cell lines; compound 3 showed no significant inhibitory activity against the six tumor cell lines at a concentration of 100 μM; while compound 2 showed significant inhibitory activity against C-33A cervical cancer cells. Subsequently, we selected various human uterine cancer cells (HeLa, HCC94, SiHa, MF751) and normal cervical cells (HcerEpic) to evaluate the cytotoxic activity of compound 2. Figure 1 Results A and 1B showed that compound 2 also inhibited the growth of HCC94 and MF751 cells, but exhibited the best inhibitory effect on C-33A cells. No inhibitory effect was observed on normal cervical cells (HcerEpic) (Table 3). The inhibitory effect of compound 2 on the proliferation of C-33A cells after treatment for 24 h, 48 h, and 72 h was tested. Figure 1 Results showed that the inhibitory effect increased with increasing treatment time, with the most significant inhibitory effect observed after 72 hours of treatment, and its IC50 value was [value missing]. 50 The value was 7.4 μM. These results indicate that compound 2 inhibits the proliferation of cervical cancer cells C-33A.

[0100] Colony formation assays can reflect the ability of tumor cells to form clonal colonies in vitro. Therefore, we further investigated the effect of compound 2 on the formation of clonal colonies in C-33A cells. Figure 1 As shown in E and 1F, compound 2 significantly inhibited the number of colonies formed by C-33A cell clones in a dose-dependent manner. The colony formation assay results further demonstrate that compound 2 can significantly inhibit the proliferation and growth of cervical cancer cells C-33A in vitro, indicating its potential ability to inhibit cervical cancer growth in vivo.

[0101] Table 3. Effects of compounds 1-3 on the proliferation of C-33A, MKN-45, LNCaP, A2780, MDA-MB-468, LECPα-1, and HcerEpic cells.

[0102]

[0103]

[0104] a Data are presented as means±SD (n=3).

[0105] b Positive control.

[0106] 4.2 Compound 2 induces apoptosis in C-33A cells via the mitochondrial pathway.

[0107] The effect of compound 2 on apoptosis in C-33A cells was analyzed using flow cytometry. (See attached image.) Figure 2 As shown in A and 2B, the apoptosis experiment demonstrated that, compared to the control group, the number of apoptotic cells in the 2-treatment group increased, with the most significant increase observed at a concentration of 10 μM. Subsequently, we investigated the expression of apoptosis-related proteins. The upstream mitochondrial proteins Bcl-2 and Bax can alter mitochondrial membrane permeability, and their overexpression can control the activation of downstream Caspase-3 protease and the release of Cyt-c, mediating cell survival or death. When stimulated by death signals, the mitochondrial membrane potential changes, and this potential is regulated by the balance between the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax. Dynamic changes in Bcl-2 and Bax can lead to mitochondrial depolarization. During mitochondrial depolarization, Caspase-3 is cleaved into its activated form, c-caspase 3, which further cleaves PARP to form c-PARP, while simultaneously losing its DNA repair function, leading to cellular DNA damage or apoptosis. Therefore, we further investigated the effect of compound 2 on apoptosis-related proteins. (See attached image) Figure 2 As shown in C and 2D, compound 2 reduced the Bcl-2 / Bax ratio and significantly increased the expression levels of c-Caspase-3 and c-PARP proteins, indicating that compound 2 can induce apoptosis by regulating these cell death-related proteins. These results suggest that compound 2 induces apoptosis by activating Caspase-3 via the mitochondrial membrane potential pathway.

[0108] 4.3 Compound 2 inhibits the migration of C-33A cells.

[0109] Cancer metastasis is the pathological process by which malignant tumor cells travel from their primary site to other sites via blood vessels, body cavities, or lymphatic systems, where they continue to proliferate. This characteristic of cancer is commonly referred to as cancer spread. Metastasis of malignant tumors is the primary cause of treatment failure in the vast majority of cancers. We investigated the effect of compound 2 on the migration of C-33A cells using a cell scratch assay, as shown in the attached figure. Figure 3 The results showed that C-33A cells were inhibited from migration by treatment with different concentrations and for different durations (24h, 48h, 72h), and the inhibitory effect was more pronounced with increasing concentration and treatment time. (See appendix) Figure 3 (A and B). Simultaneously, we examined the effect of compound 2 on the expression of cell migration-related proteins. The results showed that, after treatment of C-33A cells with compound 2, compared with the control group, the expression of MMP2, MMP9, and β-certain all showed a decreasing trend, and the decrease was more pronounced with increasing concentration (see appendix). Figure 3 (C and D). The above results indicate that compound 2 can significantly inhibit the migration of C-33A cells. Cell migration and movement are closely related to tubulin, which makes up the cytoskeleton. The above experimental results suggest that compound 2 may inhibit the migration of C-33A cells by acting on tubulin.

[0110] 4.4 Maximum tolerated concentration (MTD) of compound 2 in a zebrafish cervical cancer xenograft model

[0111] Before establishing a zebrafish xenotransplantation model, we first determined the maximum tolerated dose (MTD) of compound 2 and paclitaxel in zebrafish embryos. Compound 2 exhibited significant teratogenic toxicity (teratogenicity rate >10%) in zebrafish embryos at concentrations greater than 2.5 μM, and significant lethal toxicity at concentrations greater than 10 μM. The teratogenic phenotypes induced by compound 2 included pericardial edema, yolk sac edema, and body axis curvature, etc. (see appendix). Figure 4 A). As attached Figure 4 As shown in Figure B, the MTD of compound 2 in zebrafish embryos was 2.5 μM. The maximum concentration of paclitaxel selected (80 μM) did not cause significant teratogenic or lethal toxicity in zebrafish embryos (see attached figure). Figure 4 C). In our experiments, we observed significant drug precipitation after administration of paclitaxel (>5 μM). Considering the drug's solubility (<6.15 μM), we selected 5 μM as the MTD of paclitaxel in zebrafish embryos. In subsequent experiments, the positive control paclitaxel was used at a dose of 5 μM (MTD) in zebrafish embryos, and compound 2 was used at doses of 2.5 μM (MTD), 1.25 μM (1 / 2 MTD), and 0.625 μM (1 / 4 MTD).

[0112] 4.5 Compound 2 inhibits tumor proliferation in a zebrafish cervical cancer xenograft model.

[0113] Endometrial cancer cells C-33A were xenografted into the yolk sac of a 2-day-fart (dpf) zebrafish embryo. (Attached) Figure 5 A shows representative images of the control group and each treatment group 4 days after transplantation (3 days of drug administration). (See attached image.) Figure 5 As shown in Figure B, 4 days after embryo transfer (4 dpi, i.e., 3 dpt), the fluorescence intensity of C-33A cells in the control group embryos was 298.98 × 10⁻⁶. 5 The fluorescence intensity of C-33A cells in the paclitaxel group embryos was 231.76 × 10⁻⁶. 5 The fluorescence intensity of cells in the two treatment groups (0.625 μM, 1.25 μM, and 2.5 μM) was statistically different from that in the control group (P < 0.033). The fluorescence intensities of C-33A cells in the embryos of the two treatment groups were 238.92 × 10⁻⁶ and 2.5 μM, respectively. 5230.35×10 5 and 227.70×10 5 The fluorescence intensity of the cells in the control group was statistically different (P < 0.05).

[0114] The results showed that compound 2 exhibited inhibitory activity against tumor proliferation in a zebrafish cervical cancer xenograft model, and this activity was dose-dependent. The treatment effect in the high-dose group was comparable to that in the positive control group (paclitaxel). Compound 2 significantly inhibited the growth of cervical cancer in zebrafish.

[0115] 4.6 Effects of Compound 2 on Embryonic Tissue Lesions in a Zebrafish Cancer Xenograft Model

[0116] After determining the effects of compound 2 on the anti-proliferation, anti-angiogenesis, and anti-metastasis of C-33A cells in a zebrafish xenograft model, we selected zebrafish embryos four days after transplantation for histological sections (longitudinal sections). Hematoxylin-Eosin (HE) staining further confirmed the presence of cancer cells in the zebrafish embryos and the effect of compound 2 on C-33A cancer cells. HE staining primarily distinguishes normal and abnormal cells based on cell and nuclear morphology. (See attached image) Figure 6 As shown, HE staining of longitudinal sections of zebrafish embryos revealed that C-33A cells could be detected at almost single-cell resolution in zebrafish embryos at 4 dpi (3 dpt) (cells within the yellow dashed line in the image above). These cancer cells varied in size and morphology, with nuclei that were larger and irregularly shaped than normal cells (compared to cells in normal tissues and organs in the "Color Atlas of Zebrafish Histology and Cytology"), consistent with the atypia of cancer cells and the irregular enlargement of nuclei. The enlarged image of C-33A cells below shows that no significant apoptosis was observed in the control group, while some C-33A cells in the positive control group (paclitaxel 5 μM) and the group treated with compound 2 showed significant necrosis or apoptosis, such as cell shrinkage, cytoplasmic condensation, and round or oval nuclei. The arrows indicate necrotic or apoptotic cells. These data further indicate that compound 2 can promote cancer cell apoptosis in vivo, thereby inhibiting tumor cell growth.

[0117] 4.7 Compound 2 inhibits brain metastasis of tumors in a zebrafish cervical cancer xenograft model.

[0118] We investigated the effects of compound 2 on brain metastasis of cancer cells in zebrafish embryos. (See attached image) Figure 7 A shows a schematic diagram of the injection site. C-33A cells were xenografted into the periovarian space of 2-dpf zebrafish embryos, and changes in the transferred brain cells were observed. (Attached) Figure 7 B shows representative images of brain cancer cells in each group of embryos after drug administration. (For example...) Figure 7As shown in Figure C, four days after transplantation (three days after drug administration), the fold increase in brain metastatic cells in the control group embryos was 1.36, while the fold increase in brain metastatic cells in the paclitaxel group was 0.90, showing a statistically significant difference compared to the control group (P < 0.001). The fold increases in brain metastatic cells in the embryos treated with 0.625 μM, 1.25 μM, and 2.5 μM compounds were 1.10, 0.98, and 0.92, respectively, showing statistically significant differences in cell metastasis rates compared to the control group embryos (P < 0.05, P < 0.001, and P < 0.001). These results indicate that compound 2 significantly inhibited brain metastasis of cancer cells in a zebrafish cervical cancer xenograft model, with effects comparable to the positive control group.

[0119] Compound 2 inhibits tumor angiogenesis in a zebrafish cervical cancer xenograft model.

[0120] It is well known that xenografting of tumor cells into animals can induce the secretion of angiogenic factors, thereby mimicking the initial stages of tumor angiogenesis and metastasis. Therefore, a zebrafish xenograft model of the cervical cancer cell line C-33A was established. This model was used to evaluate the inhibitory activity of compound 2 on tumor-induced angiogenesis. Cervical cancer cell line C-33A was xenografted into the yolk sac of 2-dpf zebrafish embryos to induce angiogenesis. (Appendix) Figure 8 A shows representative images of the control group and each treatment group 24 hours after drug administration. (See attached image.) Figure 8 As shown in Figure B, 24 hours after drug administration, the area of ​​sub-intestinal vessels (SIV) in embryos of the control group was 186,922 pixels, while that of embryos of the paclitaxel group was 127,919 pixels, showing a statistically significant difference compared to the control group (P < 0.001). The SIV areas in embryos treated with 0.625 μM, 1.25 μM, and 2.5 μM were 122,665, 120,028, and 88,708 pixels, respectively, also showing statistically significant differences compared to the control group (P < 0.001). The results indicate that compound 2 significantly inhibits tumor-induced angiogenesis, and at the high-dose group (2.5 μM), it is significantly superior to the positive control group.

[0121] In conclusion, taxane-like compounds can be developed as promising lead compounds for the prevention and treatment of tumors.

[0122] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0123] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0124] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A taxane-like compound, characterized in that, The compound is selected from: Compound 1: ; and / or compound 2: .

2. A method for preparing a taxane-like compound, characterized in that, The aforementioned taxane-like compounds were all isolated from the methanol extract of the aerial parts of Hypericum qishanense, specifically including: The aerial parts of St. John's wort from Qishan were extracted with methanol to obtain a methanol extract. The methanol extract was dissolved in water and extracted with petroleum ether to obtain a petroleum ether extract. The petroleum ether extract was continuously eluted by silica gel column chromatography under the following elution conditions: 100-200 mesh, petroleum ether-dichloromethane, 10:1→5:1; petroleum ether-acetone, 80:1→2:1; chloroform-methanol, 10:1→3:

1. The products were combined into component AH based on TLC detection. Fr.D was eluted by silica gel column chromatography with gradient elution under the following conditions: petroleum ether-ethyl acetate, 200:1 → 3:1; yielding 11 fractions Fr.D.01 to Fr.D.

11. Fr.D.11 via RP C 18 Column chromatography gradient elution was performed under acetone-water ratio of 5:1 to 20:1, yielding 13 fractions from Fr.D.11.01 to Fr.D.11.

13. Fr.D.11.06 was purified by dextran gel Sepedax LH-20 column chromatography and preparative thin-layer chromatography to obtain compound 1. The chromatographic solvent for dextran gel Sepedax LH-20 column chromatography was acetone, and the conditions for preparative thin-layer chromatography were dichloromethane-methanol, 100:

1. The structural formula of compound 1 is ; Compound 2 was obtained from Fr.D.11.11 by purification using Sephex LH-20 dextran gel chromatography and preparative thin-layer chromatography. The chromatographic buffer for Sephex LH-20 column chromatography was acetone, and the preparative thin-layer chromatography conditions were petroleum ether-ethyl acetate, 10:

1. The structural formula of compound 2 is [insert structural formula here]. ; Fr.E was eluted by silica gel column chromatography gradient to obtain 7 fractions Fr.E.01~Fr.E.

07. The conditions for silica gel column chromatography gradient elution were: dichloromethane-methanol, 50:1→2:1; Fr.E.03 via RP C 18 Six fractions, Fr. E.03.01 to Fr. E.03.06, were obtained by gradient elution using column chromatography; the elution conditions were: methanol-water, 5:1 → 10:

1. Fr. E.03.05 was purified by gradient elution using silica gel column chromatography and preparative thin-layer chromatography to obtain compound 3. The gradient elution conditions for silica gel column chromatography were: petroleum ether-ethyl acetate, 15:1 → 4:1; dichloromethane-methanol, 50:1 → 15:1; the elution conditions for preparative thin-layer chromatography were: dichloromethane-methanol, 250:

1. The structural formula of compound 3 is .

3. The application of taxane-like compounds in the preparation of anti-cervical cancer drugs, wherein the taxane-like compound is compound 2. .

4. The application of taxane-like compounds in the preparation of drugs for treating cervical scleroderma, wherein the taxane-like compound is compound 2. .

5. A drug for treating cervical cancer, characterized in that, The drug contains a taxane-like compound, wherein the taxane-like compound is compound 2. .

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