Ailanthus altissima bark extract, quassin compound, and preparation method, application and pharmaceutical composition of ailanthus altissima bark extract and quassin compound

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a cortex ailanthi extract, a quassin compound, and a preparation method, application and a pharmaceutical composition thereof. The quassin compound shown in the formula I is extracted from cortex ailanthi, can be used as a novel JAK2 inhibitor, can be combined with JAK2 protein in a high affinity manner, and blocks JAK2 kinase activity and a downstream STAT3 signal channel, so that triple negative breast cancer cell proliferation and clone formation are inhibited, and cancer cell apoptosis is induced. Therefore, the quassin compound disclosed by the invention is expected to provide a new strategy for targeted therapy of the triple negative breast cancer, and has a good application prospect. Formula I

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an extract of Toona sinensis bark, a quassinolide compound, its preparation method, uses, and pharmaceutical composition. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer characterized by cancer cells that do not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Due to the lack of these receptors, TNBC is unresponsive to conventional endocrine therapy and HER2-targeted therapy. TNBC is typically highly aggressive, has a poor prognosis, and is prone to early distant metastasis; therefore, finding safe and effective new strategies and drugs for treating TNBC is of great significance.

[0003] Toona sinensis bark, or dried root bark, is the dried root bark or bark of *Ailanthus altissima* (Mill.) Swingle, a plant belonging to the Simaroubaceae family. It can be harvested year-round, but is best collected in spring when moisture is plentiful and the bark is easy to peel. It is sun-dried or obtained by scraping off the outer bark and then sun-drying. This product was first recorded in the *Xinxiu Bencao* (also known as *Tang Bencao*). In ancient times, Toona sinensis bark referred to the root bark and dried bark of both *Toona sinensis* and *Ailanthus altissima*, with *Ailanthus altissima* bark being considered superior. Currently, in the Chinese market, except for a few regions that use *Toona sinensis* bark, most use *Ailanthus altissima* bark as Toona sinensis bark. Toona sinensis bark has the effects of clearing heat and drying dampness, astringing and stopping leukorrhea, stopping diarrhea, and stopping bleeding. It is used for leukorrhea, damp-heat diarrhea, chronic diarrhea, hematochezia, and metrorrhagia.

[0004] To date, scholars both domestically and internationally have conducted extensive research on the chemical constituents, pharmacological effects, and biological activities of Ailanthus altissima. Approximately 221 compounds have been isolated from Ailanthus altissima, including alkaloids, quassinolides, phenylpropanes, triterpenoids, and other compounds. Quassinolides are its characteristic components and the most widely studied components in pharmacology, exhibiting good antitumor activity.

[0005] As is well known, the therapeutic effects of medicinal plants are closely related to their pharmacological effects, and the main effects of plant-based drugs are influenced by the active compounds contained in the plants. Therefore, it is crucial to isolate and identify more bioactive chemical components from the bark of *Toona sinensis*, select natural candidate drugs with therapeutic potential, and promote their development and application. Summary of the Invention

[0006] To address the problems of the prior art, the present invention provides an extract of Toona sinensis bark, a quassinolide compound, its preparation method, uses, and a pharmaceutical composition thereof.

[0007] This invention provides compounds of Formula I, or pharmaceutically acceptable salts thereof, or solvates thereof, or stereoisomers thereof, or geometric isomers thereof, or isotopic labels thereof, or crystal forms thereof, or prodrugs thereof: Formula I Among them, R1, R2, and R3 are independently selected from hydrogen and C1~C6 alkyl groups, respectively; R4, R5, and R6 are independently selected from hydrogen, halogen, C1-C6 alkoxy, and C1-C6 acyl groups, respectively.

[0008] Preferably, the compound has the structural formula shown in Formula II: Formula II.

[0009] The present invention also provides a method for preparing the compound of Formula II, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopic label thereof, or a crystal form thereof, or a prodrug thereof, comprising the following steps: Step 1: After crushing the bark of Toona sinensis, add an organic solvent for reflux extraction, concentrate under reduced pressure to obtain the primary extract of Toona sinensis bark; wherein, the material-to-liquid ratio is 1:5-1:20. Step 2: Take the initial extract of Toona sinensis bark and extract it with petroleum ether, ethyl acetate, n-butanol and water respectively to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction; Step 3: The ethyl acetate fraction was subjected to silica gel column chromatography, and after thin-layer chromatography analysis and merging, 17 fractions of extract were obtained. The 5th fraction of extract was taken to obtain the CpE.5 fraction extract. Step 4: The CpE.5 extract was eluted with a methanol-water gradient, and the fraction with a retention time of 16-18 min was collected to obtain the compound shown in Formula II.

[0010] Preferably, in step 1, the organic solvent is at least one of ethanol and methanol, with a volume fraction of 50-70%. And / or, in step 1, the extraction is performed 2-3 times, each time for 0.5-2 hours; the temperature for vacuum concentration is 45-50℃; And / or, in step 2, the volume ratio of the primary extract of Toona sinensis bark to petroleum ether is 1:0.85-1:1.15; the volume ratio of the primary extract of Toona sinensis bark to ethyl acetate is 1:0.85-1:2; the volume ratio of the primary extract of Toona sinensis bark to n-butanol is 1:0.85-1:1.15; and the number of extractions is 3-5. And / or, in step 3, the mobile phase of the silica gel column chromatography is dichloromethane and methanol, and the elution gradient is 100% CH2Cl2, 99.5% CH2Cl2, 99% CH2Cl2, 98.5% CH2Cl2, 98% CH2Cl2, 97.5% CH2Cl2, 97% CH2Cl2, 96.5% CH2Cl2, 96% CH2Cl2, 95.5% CH2Cl2, 94% CH2Cl2, 92% CH2Cl2, 80% CH2Cl2, and 40% CH2Cl2 (volume fractions). And / or, in step 4, the gradient elution conditions are as follows: 0-7 min using 50% methanol (volume fraction), 7-13 min using 50-70% methanol (volume fraction), 13-26 min using 70-90% methanol (volume fraction), 26-35 min using 50% methanol (volume fraction), and the flow rate is 1.5-2.5 ml / min.

[0011] This invention also provides an extract of *Toona sinensis* bark, containing the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotope label thereof, or a crystal form thereof, or a prodrug thereof, the preparation method of which includes the following steps: Step a: After crushing the bark of Toona sinensis, add an organic solvent for reflux extraction, concentrate under reduced pressure to obtain the primary extract of Toona sinensis bark; wherein the material-to-liquid ratio is 1:5-1:20. Step b: Take the initial extract of Toona sinensis bark and extract it with petroleum ether, ethyl acetate, n-butanol and water respectively to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction; Step c: The ethyl acetate fraction was subjected to silica gel column chromatography, and after thin-layer chromatography analysis and merging, 17 fractions of extract were obtained. The 5th fraction of extract was taken to obtain the extract.

[0012] Preferably, in step a, the organic solvent is at least one of ethanol and methanol, with a volume fraction of 50-70%; And / or, in step a, the extraction is performed 2-3 times, each time for 0.5-2 hours; the temperature for vacuum concentration is 45-50℃; And / or, in step b, the volume ratio of the primary extract of Toona sinensis bark to petroleum ether is 1:0.58-1.15; the volume ratio of the primary extract of Toona sinensis bark to ethyl acetate is 1:0.58-2; the volume ratio of the primary extract of Toona sinensis bark to n-butanol is 1:0.58-1.15; and the number of extractions is 3-5. And / or, in step c, the mobile phase of the silica gel column chromatography is dichloromethane-methanol, and the elution gradient is: 100% CH2Cl2, 99.5% CH2Cl2, 99% CH2Cl2, 98.5% CH2Cl2, 98% CH2Cl2, 97.5% CH2Cl2, 97% CH2Cl2, 96.5% CH2Cl2, 96% CH2Cl2, 95.5% CH2Cl2, 94% CH2Cl2, 92% CH2Cl2, 80% CH2Cl2, 40% CH2Cl2.

[0013] The present invention also provides a method for preparing the above-mentioned Toona sinensis bark extract, comprising the following steps: Step a: After crushing the bark of Toona sinensis, add an organic solvent for reflux extraction, concentrate under reduced pressure to obtain the primary extract of Toona sinensis bark; wherein the material-to-liquid ratio is 1:5-1:20. Step b: Take the initial extract of Toona sinensis bark and extract it with petroleum ether, ethyl acetate, n-butanol and water respectively to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction; Step c: The ethyl acetate fraction was subjected to silica gel column chromatography, and after thin-layer chromatography analysis and merging, 17 fractions of extract were obtained. The 5th fraction of extract was taken to obtain the extract.

[0014] The present invention also provides the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopic label thereof, or a crystal form thereof, or a prodrug thereof in the preparation of JAK2 inhibitors. And / or, the use of the above-mentioned ailanthus bark extract in the preparation of JAK2 inhibitors.

[0015] Preferably, the JAK2 inhibitor is a drug for treating tumors; the tumor is selected from at least one of breast cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, pancreatic cancer, head and neck cancer, and glioblastoma.

[0016] Preferably, the tumor is selected from breast cancer.

[0017] Preferably, the breast cancer is triple-negative breast cancer.

[0018] The present invention also provides a pharmaceutical composition, which is a formulation made by adding pharmaceutically acceptable excipients to the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotope label thereof, or a crystal form thereof, or a prodrug thereof as an active ingredient. Alternatively, it is a preparation made by adding pharmaceutically acceptable excipients to the above-mentioned Toona sinensis bark extract as the active ingredient.

[0019] This invention extracts quassin-like compound L1 from the bark of Toona sinensis, which can serve as a novel JAK2 inhibitor. It can bind to JAK2 protein with high affinity, blocking JAK2 kinase activity and its downstream STAT3 signaling pathway, thereby inhibiting the proliferation and colony formation of triple-negative breast cancer cells and inducing apoptosis of cancer cells, thus playing a therapeutic role in triple-negative breast cancer. This provides a new strategy for targeted therapy of triple-negative breast cancer in clinical practice and has good application prospects.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. 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-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 ESI-MS spectrum of compound L1.

[0023] Figure 2 UV spectrum of compound L1 in methanol.

[0024] Figure 3 ECD spectrum of compound L1 in methanol.

[0025] Figure 4 Infrared spectrum of compound L1 in potassium bromide.

[0026] Figure 5 Compound L1 in deuterated chloroform 1 H NMR spectrum.

[0027] Figure 6 Compound L1 in deuterated chloroform 13 C NMR spectrum.

[0028] Figure 7 Compound L1 in deuterated chloroform 1 H- 1 H COSY spectrum.

[0029] Figure 8 HMBC spectrum of compound L1 in deuterated chloroform.

[0030] Figure 9 HSQC spectrum of compound L1 in deuterated chloroform.

[0031] Figure 10Noise spectrum of compound L1 in deuterated chloroform.

[0032] Figure 11 The CCK-8 assay was used to detect the inhibitory effect of compound L1 on the cell viability of triple-negative breast cancer cell lines.

[0033] Figure 12 Compound L1 perturbs the genes of MDA-MB-231.

[0034] Figure 13 Compound L1 regulates the signaling pathway of MDA-MB-231.

[0035] Figure 14 Compound L1 inhibits the clonogenic ability of triple-negative breast cancer cell lines.

[0036] Figure 15 Compound L1 inhibits the proliferation of triple-negative breast cancer cell lines.

[0037] Figure 16 Compound L1 induces apoptosis in triple-negative breast cancer cell lines.

[0038] Figure 17 Compound L1 reduces protein levels in triple-negative breast cancer cell lines.

[0039] Figure 18 Compound L1 increases the thermal stability of JAK2 protein.

[0040] Figure 19 SPR detection results of compound L1 and protein JAK2. Among them, (A) shows the experimental results of response value change with the concentration of compound L1; (B) shows the trend of response value change with time under each compound L1 concentration condition.

[0041] Figure 20 The results of enrichment analysis of MDA-MB-231 treated with components CpE.1-CpE.17 and the gene set enrichment analysis of the "JAK2 inhibitor characteristic gene expression profile" are presented. Detailed Implementation

[0042] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0043] Example 1: Quassin-like compound L1 and its preparation method The structure of quassinolide compound L1 (formula II) is as follows: Preparation method: 1. Separation of ethyl acetate with silica gel column 15 kg of Toona sinensis root bark and stem bark were pulverized and placed in an extraction bottle. 70% ethanol was added at a material-to-liquid ratio of 1:10, and the mixture was refluxed twice, 1 hour each time, for a total extraction of approximately 280 L. The extracts were combined and concentrated under reduced pressure at 50°C to 20 L to obtain the primary extract of Toona sinensis bark. The primary extract was extracted four times with an aqueous phase and petroleum ether ratio of 1:1, collecting a total of 76 L of the petroleum ether fraction. This fraction was then concentrated under reduced pressure at 40°C and freeze-dried to obtain the petroleum ether fraction. The primary extract was also extracted four times with an aqueous phase and ethyl acetate ratio of 1:2, collecting a total of 150 L of the ethyl acetate fraction. This fraction was then concentrated under reduced pressure at 40°C and freeze-dried to obtain the ethyl acetate fraction. Finally, the primary extract was extracted four times with an aqueous phase and n-butanol ratio of 1:1, collecting a total of 70 L of the n-butanol fraction. This fraction was concentrated under reduced pressure at 60°C and freeze-dried to obtain the n-butanol fraction. A total of 22 L of the aqueous fraction was collected, concentrated under reduced pressure at 55°C, and freeze-dried to obtain the aqueous fraction. The total amount of the ethyl acetate fraction from the root bark and stem bark was approximately 394 g. The ethyl acetate fraction was obtained by silica gel column chromatography with gradient elution of dichloromethane-methanol (100% CH2Cl2, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 94%, 92%, 80%, 40%, v / v). The combined fractions were analyzed by TLC to obtain fractions CpE.1-CpE.17.

[0044] 2. Library construction and sequencing using the CpE.1-CpE.17 HiMAP technology platform Components CpE.1-CpE.17 were dissolved in DMSO (cell culture medium), and after being administered to MDA-MB-231 cells, libraries were constructed and sequenced on the HiMAP platform to obtain characteristic gene expression profiles of components CpE.1-CpE.17. A "JAK2 inhibitor characteristic gene expression profile" was pre-constructed: MDA-MB-231 cells were treated with JAK2 inhibitors (Gandotinib, Ilginatinib hydrochloride, WP1066, Fedratinib hydrochloride hydrate, TG101209, AZ960) and the control DMSO to obtain transcriptome sequencing data. Differentially expressed genes between JAK2 inhibitors and the control group (pvalue < 0.05, |logFoldChange| > 0) were defined as "JAK2 inhibitor characteristic gene expression profiles". Individual inhibitors with pvalue < 0.05 and logFoldChange > 0 were considered as an inhibitor upregulated gene set, and those with pvalue < 0.05 and logFoldChange < 0 were considered as an inhibitor downregulated gene set. GSEA enrichment was performed on the expression profiles of CpE.1-CpE.17 characteristic genes and the expression profiles of JAK2 inhibitor characteristic genes. The presence of potential JAK2 inhibitors was evaluated by NES values ​​and pvalues. Figure 20 Based on the numerical results of pvalue < 0.05 and NES value, it was found that the CpE.5 extract could well mimic the gene expression of several known JAK2 inhibitors, and therefore it was isolated.

[0045] 3. HPLC preparation of CpE.5 The CpE.5 fraction extract was eluted with methanol-water (0-7 min: 50% methanol; 7-13 min: 50-70% methanol; 13-26 min: 70-90% methanol; 26-35 min: 50% methanol, flow rate: 2 ml / min), and the fraction with a retention time of 17 min was collected to obtain compound L1.

[0046] Example 2: Extract of CpE.5 segment from Toona sinensis bark and its preparation method 1. Separation of ethyl acetate with silica gel column 15 kg of Toona sinensis root bark and stem bark were pulverized and placed in an extraction bottle. 70% ethanol was added at a material-to-liquid ratio of 1:10, and the mixture was refluxed twice, 1 hour each time, for a total extraction of approximately 280 L. The extracts were combined and concentrated under reduced pressure at 50°C to 20 L to obtain the primary extract of Toona sinensis bark. The primary extract was extracted four times with an aqueous phase and petroleum ether ratio of 1:1, collecting a total of 76 L of the petroleum ether fraction. This fraction was then concentrated under reduced pressure at 40°C and freeze-dried to obtain the petroleum ether fraction. The primary extract was also extracted four times with an aqueous phase and ethyl acetate ratio of 1:2, collecting a total of 150 L of the ethyl acetate fraction. This fraction was then concentrated under reduced pressure at 40°C and freeze-dried to obtain the ethyl acetate fraction. Finally, the primary extract was extracted four times with an aqueous phase and n-butanol ratio of 1:1, collecting a total of 70 L of the n-butanol fraction. This fraction was concentrated under reduced pressure at 60°C and freeze-dried to obtain the n-butanol fraction. A total of 22 L of the aqueous fraction was collected, concentrated under reduced pressure at 55°C, and freeze-dried to obtain the aqueous fraction. The total amount of the ethyl acetate fraction from the root bark and stem bark was approximately 394 g. The ethyl acetate fraction was obtained by silica gel column chromatography with gradient elution of dichloromethane-methanol (100% CH2Cl2, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 94%, 92%, 80%, 40%, v / v). The combined fractions were analyzed by TLC to obtain fractions CpE.1-CpE.17.

[0047] 2. Library construction and sequencing using the CpE.1-CpE.17 HiMAP technology platform Fragments CpE.1-CpE.17 were dissolved in DMSO (cell culture medium), and after being administered to MDA-MB-231 cells, libraries were constructed and sequenced on the HiMAP platform to obtain the characteristic gene expression profiles of fragments CpE.1-CpE.17. Subsequently, the CpE.5 fragment extract was obtained according to the method in Example 1.

[0048] The technical solution of the present invention will be further explained through experiments below.

[0049] Experimental Example 1: Characterization of the quassinolide compound L1 and its structure I. Experimental Methods 1. Extraction and separation 1.1 Separation of Ethyl Acetate with Silica Gel 15 kg of Toona sinensis root bark and stem bark were pulverized and placed in an extraction bottle. 70% ethanol was added at a material-to-liquid ratio of 1:10, and the mixture was refluxed twice, 1 hour each time, for a total extraction of approximately 280 L. The extracts were combined and concentrated under reduced pressure at 50°C to 20 L to obtain the primary Toona sinensis bark extract. The primary extract was extracted four times with an aqueous phase and petroleum ether ratio of 1:1, collecting a total of 76 L of the petroleum ether fraction. This fraction was then concentrated under reduced pressure at 40°C and freeze-dried to obtain the petroleum ether fraction. Four extractions were also performed with an aqueous phase and ethyl acetate ratio of 1:2, collecting a total of 150 L of the ethyl acetate fraction. This fraction was then concentrated under reduced pressure at 40°C and freeze-dried to obtain the ethyl acetate fraction. Four extractions were performed with an aqueous phase and n-butanol ratio of 1:1, collecting a total of 70 L of the n-butanol fraction. This fraction was then concentrated under reduced pressure at 60°C and freeze-dried to obtain the n-butanol fraction. Finally, 22 L of the aqueous fraction was collected and concentrated under reduced pressure at 55°C and freeze-dried to obtain the aqueous fraction. Approximately 394g of the ethyl acetate fractions of the root bark and stem bark of *Toona sinensis* were collected. The ethyl acetate fractions were analyzed by silica gel column chromatography using a gradient elution of dichloromethane-methanol (100% CH2Cl2, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 94%, 92%, 80%, 40%, v / v). The combined fractions were obtained by TLC analysis, yielding fractions CpE.1-CpE.17.

[0050] 1.2 Library preparation and sequencing using the HiMAP technology platform for CpE.1-CpE.17 Fragments CpE.1-CpE.17 were dissolved in DMSO (cell culture medium), and after being administered to MDA-MB-231 cells, libraries were constructed and sequenced on the HiMAP platform to obtain the expression profile data of characteristic genes of fragments CpE.1-CpE.17. Based on the expression profile data, the CpE.5 fragment was selected.

[0051] HPLC preparation of 1.3CpE.5 The CpE.5 fraction was eluted with methanol-water (0-7 min: 50% methanol; 7-13 min: 50-70% methanol; 13-26 min: 70-90% methanol; 26-35 min: 50% methanol, flow rate: 2 ml / min), and the fraction with a retention time of 17 min was collected to obtain compound L1.

[0052] 2. Experimental materials (Table 1) Table 1 List of Experimental Materials II. Experimental Results The quassin-like compound L1 was prepared, with the following structure: Its spectral data and physicochemical parameters: Pale yellow powder: C25 H 32 O 9， ESI HRMS m / z: 475.20 IR (KBr)ν max 3229 ,2924 ,2852 ,1747 ,1667 ,1631 ,1458 ,1377 ,1051 cm -1， 1 H NMR (600 MHz, CDCl3) δ0.96 (t, 3H,H-25), 1.18 (t, 3H,H-23),1.19 (s,3H,H-18), 1.25(s,1H,H-10), 1.53(m,1H,H-24a), 1.77(m,1H,H-24b), 2.01(m, 1H,H-6a), 2.03(s,3H.H-17), 2.37( dt, J = 14.7, 3.0 Hz, H-6b), 2.49(m,1H,H-22),2.87(d,J = 10.3 Hz,1H,H-14), 2.97(d, J = 12.8 Hz, 1H,H-5), 3.04(s,1H,H-9),3.56(d,J = 8.7 Hz,1H,H-19a), 3.92(d,J = 8.8 Hz,1H,H-19b), 4.06(s,1H,H-11),4.16(s,1H,H-1), 4.60(s,1H,H-7), 5.20(s,1H,H-20a), 5.39(s,1H,H-20b), 5.69(brs,1H,H-15), 6.17(s,1H,H-3). 13C NMR (151 MHz, CDCl3) δ9.82(CH3, C-18), 11.71(CH3, C-25), 16.64(CH3,C-23), 23.23(CH3,C-17), 25.51(CH2, C-6), 26.75(CH2,C-24), 41.08(CH, C-22),41.97(CH, C-5), 45.11(C-9), 45.36(C-10), 47.02(C-8), 51.99(CH, C-14), 67.83(CH, C-15),71.78(CH2, C-19) ,78.27(CH, C-7) 79.46(CH, C-11), 83.01(CH, C-1),108.73(C-13), 123.84(CH2, C-20), 124.61(CH,C-3), 139.63(C-12),164.11(C-4),166.67(C-16), 175.90(C-21), 195.96(C-2).

[0053] Figure 1-10 The structural characterization results of compound L1 are presented. Nuclear magnetic resonance (NMR) spectroscopy analysis revealed multiple methyl and methylene signals at δ 0.8–1.5 ppm, olefinic hydrogen signals at δ 5.0–6.0 ppm, and hydrogen signals bonded to oxygen atoms at δ 3.0–4.0 ppm by ¹³C NMR. olefinic carbon signals were observed at δ 120–140 ppm, and carbonyl carbon signals at δ 170–200 ppm. Mass spectrometry analysis showed a molecular ion peak of m / z 475.62, consistent with the molecular formula C1. 25 H 32 O9 is consistent. The infrared spectrum shows a carbonyl characteristic absorption peak at 1747.36 cm⁻¹ and an olefin characteristic absorption peak at 1600-1670 cm⁻¹. Based on these data, it is a quassinolide compound with a lower triterpenoid content.

[0054] Experiment Example 2: Effect of compound L1 on the viability of triple-negative breast cancer cell lines I. Experimental Methods Log-growth phase MDA-MB-231, BT549, HCC38, and 4T1 triple-negative breast cancer cell lines were selected and treated with 1×10⁻⁶ cells per cell line. 3The cells were seeded at a density of 100 μL / well in 96-well cell culture plates and cultured for 24 h in a cell culture incubator. Different concentrations of complete culture medium were prepared using DMEM complete medium according to the characteristics of different cell lines. For MDA-MB-231 and 4T1 cells, the concentrations of compound L1 were 0.01 μM, 0.1 μM, 0.4 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM, respectively. For HCC38 and BT549 cells, the concentrations of compound L1 were 0.01 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM, respectively. DMSO was used as a blank control. The original culture medium was replaced with 100 μL / well, and the cells were cultured for 48 h in a cell culture incubator. Under light-protected conditions, 100 μL of DMEM basal medium containing 10 μL of CCK-8 was added to each well, and the cells were incubated in a cell culture incubator for 1 hour in the dark. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. Result analysis: Cell viability % = (OD value of drug-treated group - OD value of blank well) / (OD value of blank control group - OD value of blank well) × 100%.

[0055] II. Experimental Results like Figure 11 As shown, compound L1 exhibited strong inhibitory effects on the viability of various triple-negative breast cancer cell lines (MDA-MB-231, BT549, HCC38, 4T1). This indicates that compound L1 has the potential to treat triple-negative breast cancer.

[0056] Experiment 3: Analysis of the effects of compound L1 on gene expression and enrichment of apoptosis signaling pathways in triple-negative breast cancer cells using the HiMAP platform. I. Experimental Methods Log-phase MDA-MB-231 triple-negative breast cancer cells were seeded at a density of 3000 cells per well in 384 culture plates and cultured for 24 h. Compound L1 (100 mM in DMSO) was diluted to 1 μM using DMEM complete medium, with DMSO as a blank control. Cells were then treated with the drug and cultured for another 24 h. Cells were lysed, and libraries were constructed using the HiMAP platform. Sequencing was performed to construct drug-perturbed gene expression profiles, followed by bioinformatics analysis.

[0057] II. Experimental Results like Figure 12 , 13As shown, compound L1 upregulated the expression of gene 339 and downregulated gene 306 in MDA-MB-231 cells, and enriched apoptosis-related signaling pathways, with the most significant upregulation of gene expression in the PI3K-Akt signaling pathway. This suggests that compound L1 may exert its anti-tumor effect by activating apoptosis signaling pathways to inhibit the viability of triple-negative breast cancer cells.

[0058] Experiment Example 4: Effect of compound L1 on triple-negative breast cancer cell clones I. Experimental Methods Log-phase MDA-MB-231, BT549, and HCC38 triple-negative breast cancer cell lines were seeded at a density of 1000 cells per well in 6-well cell culture plates and cultured for 24 hours. Compound L1 (100 mM) prepared in DMSO was diluted to the appropriate concentrations (0.05 μM, 0.25 μM, and 0.5 μM) using DMEM complete medium, with DMSO as a blank control. The original medium was replaced with 2 ml / well, and the cells were cultured for 7-14 days. Clonal clusters were observed; if they formed and reached a suitable size, culture was stopped. The supernatant was aspirated, washed twice with PBS, fixed with 4% paraformaldehyde for 10 minutes, discarded, and washed twice with pure water for 1 minute each time. Crystal violet was added for staining in the dark for 20 minutes, washed thoroughly with pure water, and dried for photographing.

[0059] II. Experimental Results like Figure 14 As shown, a colony formation assay was used to investigate the effects of compound L1 on triple-negative breast cancer (TNBC) cell lines MDA-MB-231, HCC38, and BT549. Compared with the control group, the addition of compound L1 reduced the number of colonies in MDA-MB-231, HCC38, and BT549 in a dose-dependent manner. This indicates that compound L1 can significantly inhibit colony formation in triple-negative breast cancer cell lines at low concentrations.

[0060] Experimental Example 5: Effects of Compound L1 on the Proliferation and Apoptosis of Triple-Negative Breast Cancer Cells I. Experimental Methods Log-phase MDA-MB-231, BT549, and HCC38 triple-negative breast cancer cell lines were seeded at a density of 500,000 cells per well in 6 cm cell culture plates and cultured for 24 h. Compound L1 (100 mM) prepared in DMSO was diluted to the corresponding concentrations (0.5 μM, 1 μM, and 3 μM) using DMEM complete medium, with DMSO as a blank control. The original medium was replaced with 2 ml / well, and the cells were cultured for another 24 h. EdU incubation, fixation, washing, and permeabilization were performed, followed by filtration and data processing using FlowJ software.

[0061] Log-phase MDA-MB-231 and BT549 triple-negative breast cancer cell lines were seeded at a density of 200,000 cells per well in 6-well cell culture plates and cultured for 24 hours. Compound L1 (100 mM) prepared in DMSO was diluted to the appropriate concentrations (0.5 μM, 1 μM, and 3 μM) using DMEM complete medium, with DMSO as a blank control. The original medium was replaced with 2 ml / well, and the cells were cultured for another 24 hours. Cell supernatant and cells were collected, stained with Annexin V-FITC and propidium iodide, filtered, and immediately analyzed. Data were processed using FlowJ software.

[0062] II. Experimental Results like Figure 15 As shown, compared with the control group, the cell proliferation rate of the compound L1 treatment group decreased in a dose-dependent manner; at the same time, the compound L1 treatment group significantly increased the apoptosis rate of triple-negative breast cancer cell lines. Figure 16 The results showed that compound L1 could significantly inhibit the proliferation of triple-negative breast cancer cell lines and induce apoptosis at low concentrations.

[0063] Experiment Example 6: Effect of compound L1 on protein expression in triple-negative breast cancer cells I. Experimental Methods Log-phase MDA-MB-231, BT549, and 4T1 triple-negative breast cancer cell lines were seeded at a density of 500,000 cells per well in 6cm cell culture plates and cultured for 24 hours. A 100mM solution of compound L1 prepared in DMSO was diluted to the appropriate concentration using DMEM complete medium, with DMSO serving as a blank control. The original medium was replaced with 2ml / well, and the cells were cultured for another 24 hours. Proteins were lysed, and BCA quantification was performed. Subsequent procedures included gel preparation, electrophoresis, transfer to a membrane, blocking, incubation with primary and secondary antibodies, and imaging.

[0064] II. Experimental Results like Figure 17As shown in the results, Western blot analysis revealed that with increasing concentrations of compound L1, the total protein levels and phosphorylation levels of JAK2 and STAT3 significantly decreased. This suggests that compound L1 may exert its antitumor effect by inhibiting the JAK2 / STAT3 signaling pathway.

[0065] Experimental Example 7: Binding of Compound L1 to Target JAK2 I. Experimental Methods Log-phase MDA-MB-231 triple-negative breast cancer cells were seeded at a density of 1 million cells per well in 10 cm cell culture plates and cultured for 24 h. A 100 mM solution of compound L1 prepared in DMSO was diluted to a concentration of 3 μM using DMEM complete medium, with DMSO as a blank control. The original medium was replaced with 5 ml / well, and the cells were cultured for 1 h. Cells were digested and aliquoted into 8-tube strips. After heating, the cells were lysed three times in liquid nitrogen, centrifuged, and the supernatant was collected for Western blot analysis.

[0066] II. Experimental Results like Figure 18 As shown, without the addition of compound L1, JAK2 protein began to denature significantly at 42.2–44 °C; after the addition of compound L1, compound L1 could increase the thermal stability of JAK2 protein levels, suggesting that it may bind to JAK2 protein and exert anti-triple-negative breast cancer activity.

[0067] Experiment Example 8: Detection of the binding ability of compound L1 to JAK2 protein using surface plasmon resonance (SPR) technique. I. Experimental Methods First, prepare a stock solution (4 mM) of JAK2 protein and compound L1. Use phosphate-buffered saline (PBS) or HEPES buffered saline (HBS) as the buffer system, optimizing the pH to 4.5. CM5 chips are used for the experiment. The chips are first rinsed with a low-concentration NaOH solution, then with the buffer solution to ensure uniform charge on the chip surface. Next, an NHS / EDC mixed solution is used to activate the carboxyl groups on the chip surface, enabling them to covalently couple with the amino groups of the JAK2 protein, thus immobilizing the ligand. To reduce non-specific binding, ethanolamine reagent is used to reactivate unbound activating groups. After pretreatment, small molecule analytes were injected into the flow cell at a constant flow rate of 5 μL / min and concentrations (200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM and 0.78125 μM). The binding and dissociation processes of the analytes with the JAK2 protein immobilized on the chip surface were monitored in real time, and SPR signal curves were generated to reveal the interaction behavior between the two.

[0068] II. Experimental Results like Figure 19 As shown, compound L1 binds with high affinity and specificity to JAK2 protein (Janus kinase 2 / JAK2 protein, Human(His), protein number O60674-1 (833A-1132G)), with a dissociation equilibrium constant (KD) of 1.076E-4 M. This indicates that compound L1 exerts its therapeutic effect on triple-negative breast cancer by directly targeting JAK2 protein, blocking its kinase activity, and thereby inhibiting the downstream STAT3 pathway.

[0069] In summary, this invention extracts quassin-like compound L1 from the bark of Toona sinensis, which can serve as a novel JAK2 inhibitor. It binds to the JAK2 protein with high affinity, blocking JAK2 kinase activity and its downstream STAT3 signaling pathway, thereby inhibiting the proliferation and colony formation of triple-negative breast cancer cells and inducing apoptosis of cancer cells. It can be used to treat triple-negative breast cancer and has broad application prospects.

Claims

1. A compound represented by Formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopic label thereof, or a crystal form thereof, or a prodrug thereof: Formula I in, R1, R2, and R3 are each independently selected from hydrogen and C1-C6 alkyl groups; R4, R5, and R6 are independently selected from hydrogen, halogen, C1-C6 alkoxy, and C1-C6 acyl groups, respectively.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopic label thereof, or a crystal form thereof, or a prodrug thereof, characterized in that, The structural formula of the compound is shown in Formula II: Formula II.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopic label thereof, or a crystal form thereof, or a method for preparing a prodrug thereof, characterized in that, Includes the following steps: Step 1: After crushing the bark of Toona sinensis, add an organic solvent for reflux extraction, concentrate under reduced pressure to obtain the primary extract of Toona sinensis bark; wherein, the material-to-liquid ratio is 1:5-1:

20. Step 2: Take the initial extract of Toona sinensis bark and extract it with petroleum ether, ethyl acetate, n-butanol and water respectively to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction; Step 3: The ethyl acetate fraction was subjected to silica gel column chromatography, and after thin-layer chromatography analysis and merging, 17 fractions of extract were obtained. The 5th fraction of extract was taken to obtain the CpE.5 fraction extract. Step 4: The CpE.5 extract was eluted with a methanol-water gradient, and the fraction with a retention time of 16-18 min was collected to obtain the compound shown in Formula II.

4. The preparation method according to claim 3, characterized in that, In step 1, the organic solvent is at least one of ethanol and methanol, with a volume fraction of 50-70%. And / or, in step 1, the extraction is performed 2-3 times, each time for 0.5-2 hours; the temperature for vacuum concentration is 45-50℃; And / or, in step 2, the volume ratio of the primary extract of Toona sinensis bark to petroleum ether is 1:0.85-1:1.15; the volume ratio of the primary extract of Toona sinensis bark to ethyl acetate is 1:0.85-1:2; the volume ratio of the primary extract of Toona sinensis bark to n-butanol is 1:0.85-1:1.15; and the number of extractions is 3-5. And / or, in step 3, the mobile phase of the silica gel column chromatography is dichloromethane and methanol, and the elution gradient is 100% CH2Cl2, 99.5% CH2Cl2, 99% CH2Cl2, 98.5% CH2Cl2, 98% CH2Cl2, 97.5% CH2Cl2, 97% CH2Cl2, 96.5% CH2Cl2, 96% CH2Cl2, 95.5% CH2Cl2, 94% CH2Cl2, 92% CH2Cl2, 80% CH2Cl2, and 40% CH2Cl2 (volume fractions). And / or, in step 4, the gradient elution conditions are as follows: 0-7 min using 50% methanol (volume fraction), 7-13 min using 50-70% methanol (volume fraction), 13-26 min using 70-90% methanol (volume fraction), 26-35 min using 50% methanol (volume fraction), and the flow rate is 1.5-2.5 ml / min.

5. An extract from Toona sinensis bark, characterized in that: It contains the compound of claim 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopic label thereof, or a crystal form thereof, or a prodrug thereof, and its preparation method includes the following steps: Step a: After crushing the bark of Toona sinensis, add an organic solvent for reflux extraction, concentrate under reduced pressure to obtain the primary extract of Toona sinensis bark; wherein the material-to-liquid ratio is 1:5-1:

20. Step b: Take the initial extract of Toona sinensis bark and extract it with petroleum ether, ethyl acetate, n-butanol and water respectively to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction; Step c: The ethyl acetate fraction was subjected to silica gel column chromatography, and after thin-layer chromatography analysis and merging, 17 fractions of extract were obtained. The 5th fraction of extract was taken to obtain the extract.

6. The toon bark extract according to claim 5, characterized in that, In step a, the organic solvent is at least one of ethanol and methanol, with a volume fraction of 50-70%. And / or, in step a, the extraction is performed 2-3 times, each time for 0.5-2 hours; the temperature for vacuum concentration is 45-50℃; And / or, in step b, the volume ratio of the primary extract of Toona sinensis bark to petroleum ether is 1:0.58-1.15; the volume ratio of the primary extract of Toona sinensis bark to ethyl acetate is 1:0.58-2; the volume ratio of the primary extract of Toona sinensis bark to n-butanol is 1:0.58-1.15; and the number of extractions is 3-5. And / or, in step c, the mobile phase of the silica gel column chromatography is dichloromethane-methanol, and the elution gradient is: 100% CH2Cl2, 99.5% CH2Cl2, 99% CH2Cl2, 98.5% CH2Cl2, 98% CH2Cl2, 97.5% CH2Cl2, 97% CH2Cl2, 96.5% CH2Cl2, 96% CH2Cl2, 95.5% CH2Cl2, 94% CH2Cl2, 92% CH2Cl2, 80% CH2Cl2, 40% CH2Cl2.

7. The method for preparing the Toona sinensis bark extract according to claim 5 or 6, characterized in that, Includes the following steps: Step a: After crushing the bark of Toona sinensis, add an organic solvent for reflux extraction, concentrate under reduced pressure to obtain the primary extract of Toona sinensis bark; wherein the material-to-liquid ratio is 1:5-1:

20. Step b: Take the initial extract of Toona sinensis bark and extract it with petroleum ether, ethyl acetate, n-butanol and water respectively to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction; Step c: The ethyl acetate fraction was subjected to silica gel column chromatography, and after thin-layer chromatography analysis and merging, 17 fractions of extract were obtained. The 5th fraction of extract was taken to obtain the extract.

8. Use of the compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopic label thereof, or a crystal form thereof, or a prodrug thereof, in the preparation of a JAK2 inhibitor; And / or, the use of the ailanthus bark extract as described in claim 5 or 6 in the preparation of JAK2 inhibitors.

9. The use according to claim 8, characterized in that: The JAK2 inhibitor is a drug used to treat tumors; the tumor is selected from at least one of breast cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, pancreatic cancer, head and neck cancer, and glioblastoma.

10. A pharmaceutical composition, characterized in that: It is a formulation made by adding pharmaceutically acceptable excipients to the compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a geometric isomer thereof, or an isotopic label thereof, or a crystal form thereof, or a prodrug thereof as the active ingredient; Alternatively, it is a formulation made with the toon bark extract as described in claim 5 or 6 as the active ingredient, and with the addition of pharmaceutically acceptable excipients.

Images