A polycyclic polyisoprenyl phloroglucinol compound, a preparation method and application thereof

Abstract

The application relates to the technical field of natural medicines, and discloses a polycyclic multi-isopentenyl phloroglucinol compound as well as a preparation method and application thereof. The polycyclic multi-isopentenyl phloroglucinol compound is a compound shown in formula I. The polycyclic multi-isopentenyl phloroglucinol compound has significant reverse tumor drug resistance activity, has great potential application value, and provides a new thought and way for development of a new type of preparation for preventing or treating tumors.

Description

A polycyclic polyisoprene-based phloroglucinol compound, its preparation method and application Technical Field

[0001] This invention relates to the field of natural medicine technology, and in particular to a polycyclic polyisoprene-based phloroglucinol compound, its preparation method, and its application. Background Technology

[0002] Tumor drug resistance is a core mechanism of treatment failure in malignant tumors. Essentially, it involves tumor cells evading the killing effects of chemotherapy, targeted therapy, or immunotherapy through gene mutations, epigenetic regulation, activation of drug efflux pumps, and microenvironment remodeling. Taking non-small cell lung cancer as an example, approximately 50% of patients develop resistance after EGFR-TKI treatment due to T790M mutations, MET amplification, or epithelial-mesenchymal transition (EMT), leading to tumor progression. Ovarian cancer resistance to platinum-based drugs is closely related to the activation of DNA repair pathways and the enrichment of tumor stem cells. Studies have shown that active ingredients in traditional Chinese medicine, such as ginsenosides, curcumin, and resveratrol, can synergistically reverse drug resistance through multiple targets: inhibiting the expression of efflux pumps such as P-gp / MRP1 to enhance drug influx, blocking signaling pathways such as EGFR / ALK / MAPK to inhibit tumor proliferation, and simultaneously regulating the immune microenvironment (e.g., activating T cells and inhibiting MDSCs) to restore anti-tumor immune responses. Compared to single-target chemical drugs, traditional Chinese medicine, with its "multi-component-multi-target-multi-pathway" regulatory network, can delay the onset of drug resistance and reduce toxic side effects, while also having dual anti-tumor and immunomodulatory effects, providing a new strategy for developing tumor drug resistance reversal drugs that are both highly effective and safe.

[0003] Hypericum, a Chinese medicinal herb, refers to the whole plant of *Hypericum hypericum*, a member of the Clusiaceae family. Many species in the *Hypericum* genus are medicinal plants. Traditional Chinese medicine believes that plants in this genus have the effects of clearing heat and detoxifying, astringing and stopping bleeding, and promoting diuresis. They are used to treat hemoptysis, hemorrhage, intestinal bleeding, traumatic bleeding, and rheumatic bone pain. Recent studies have found that plants in this genus possess pharmacological activities such as antidepressant, antitumor, antiviral, analgesic, antibacterial, and anti-inflammatory effects. Research indicates that the main chemical components of *Hypericum hypericum* include two major categories: phloroglucinol and thiazolinone. Polycyclic isopentenyl phloroglucinol is an important chemical component of *Hypericum hypericum* and is considered the main material basis for its numerous pharmacological activities. Regarding the polycyclic isopentenyl phloroglucinol compound involved in this invention and its activity in reversing tumor drug resistance, no patents or literature reports have been found to date. Summary of the Invention

[0004] Based on the above, the present invention aims to provide a polycyclic isopentenyl phloroglucinol compound, its preparation method, and its applications. This polycyclic isopentenyl phloroglucinol compound exhibits good activity in reversing tumor drug resistance and can be used to prepare antitumor drugs.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] One of the technical solutions of this invention is a polycyclic polyisoprene-based phloroglucinol compound, with the structural formula shown in Formula I:

[0007] Formula I.

[0008] Based on this, the present invention also provides a pharmaceutically acceptable salt, solvate, polymorph or prodrug of a polycyclic polyisoprene phloroglucinol compound.

[0009] The second technical solution of the present invention is a method for preparing the above-mentioned polycyclic isopentenyl phloroglucinol compound, comprising the following steps:

[0010] (1) Using the dried aerial parts of Hypericum as raw material, an ethanol extract was obtained by ethanol percolation extraction;

[0011] (2) Extract the ethanol extract with petroleum ether to obtain a petroleum ether extract;

[0012] (3) The petroleum ether extract was eluted sequentially by silica gel column chromatography, MCI resin column chromatography, Sephadex LH-20 gel column chromatography and ODS medium-pressure column chromatography. The collected eluent was then purified by preparative ODS liquid chromatography and semi-preparative ODS liquid chromatography to obtain the polycyclic polyisoprenyl phloroglucinol compound.

[0013] In this invention, the dried aerial parts of St. John's wort are used as raw materials, and an ethanol extract is obtained by ethanol percolation extraction. The volume concentration of the ethanol solution is 70%-95%, the percolation extraction time is 48-168 hours, and the flow rate is 0.3-1.0 L / h.

[0014] In the preferred embodiment, the dried roots of Hypericum are extracted by percolation with 95% ethanol for 120 h at a flow rate of 0.5 L / h, and the extract is concentrated by rotary evaporation to obtain a paste-like ethanol extract.

[0015] In this invention, the ethanol extract is extracted with petroleum ether to obtain a petroleum ether extract. Specifically, the paste-like ethanol extract is suspended in water to obtain a suspension, and then an equal volume of petroleum ether is added for extraction. The extract solution is then concentrated to obtain a paste, which is the petroleum ether extract.

[0016] In step (3), the silica gel column chromatography is performed by gradient elution with a mixed solvent of petroleum ether and ethyl acetate, and the elution fractions with a volume ratio of (3:1) to (10:1) are collected.

[0017] The MCI resin column chromatography uses a methanol-water mixed solvent for gradient elution, and collects the elution fraction with a methanol volume fraction of 80%-95%.

[0018] The Sephadex LH-20 gel column chromatography was performed with isocratic elution using methanol, and the elution fraction was collected.

[0019] The ODS reversed-phase column chromatography uses a gradient elution with a methanol-water or acetonitrile-water mixed solvent. After collecting the target fraction, it is further purified by preparative or semi-preparative high-performance liquid chromatography, with the mobile phase being an acetonitrile-water or methanol-water system.

[0020] In a preferred embodiment, the elution method using silica gel column chromatography in this invention is gradient elution; the eluents used during elution are mixed solutions of petroleum ether and ethyl acetate with volume ratios of 10:1, 5:1, 3:1, 2:1, 1:1, and 1:2; and the eluent collected by silica gel column chromatography is the eluent obtained by elution with a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1.

[0021] In a preferred embodiment, the elution method using MCI resin column chromatography in this invention is gradient elution; the eluents used during elution are mixed solutions of methanol and water with volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10; and the eluent collected by MCI resin column chromatography is the eluent obtained by elution with a mixed solution of methanol and water with a volume ratio of 90:10.

[0022] In the preferred embodiment, the elution method of Sephadex LH-20 gel column chromatography in this invention is isocratic elution; the eluent is a pure methanol solution; the elution rate is 1 mL / min, the elution time is 10 h, and the eluent is collected every 2 h, and the eluent eluted by the eluent in the second time period is collected.

[0023] In the preferred embodiment, the elution method using ODS medium-pressure column chromatography in this invention is gradient elution; the eluent is a mixture of methanol and water with a volume ratio of 60:40 to 95:5; during elution, the volume ratio of methanol to water increases in a gradient from 60:40 to 95:5 at a rate of 5 increments per hour, the flow rate is 25 mL / min, and the eluent is collected every 1 hour, with a total elution time of 7 hours, yielding 7 eluents from Fr.1 to Fr.7; eluent Fr.6 is purified by preparative ODS liquid chromatography and semi-preparative ODS liquid chromatography to obtain the polycyclic polyisoprenyl phloroglucinol compound.

[0024] In this invention, when preparing ODS by liquid chromatography, the mobile phase is a mixed solution of acetonitrile and water; the volume ratio of acetonitrile to water is 82:18.

[0025] In this invention, during the preparation of semi-preparative ODS by liquid chromatography, the mobile phase is a mixed solution of methanol and water; the volume ratio of methanol to water is 96:4.

[0026] The third technical solution of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned polycyclic polyisoprene phloroglucinol compound, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0027] Its dosage form is an oral preparation or an injectable preparation, preferably a tablet, capsule, granule, oral liquid, injection or lyophilized powder for injection.

[0028] The fourth technical solution of the present invention provides the use of the above-mentioned polycyclic isopentenyl phloroglucinol compound or the pharmaceutical composition thereon in the preparation of a medicament for the prevention and / or treatment of tumors.

[0029] The tumor is a malignant tumor that has developed resistance to chemotherapy drugs, and the chemotherapy drugs are selected from at least one of taxanes, platinum drugs, antimetabolites, topoisomerase inhibitors, or anthracyclines.

[0030] The drug is used in combination with one or more other antitumor drugs to reverse multidrug resistance in tumors.

[0031] The present invention discloses the following technical effects:

[0032] This invention marks the first successful extraction of polycyclic isopentenyl phloroglucinol compounds from St. John's wort, which possess the ability to reverse tumor drug resistance. These compounds have significant development value as novel drugs for the prevention or treatment of drug-resistant tumors. The design concept of these compounds also provides new ideas and approaches for the development of novel drugs for the prevention or treatment of drug-resistant tumors. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 shows the polycyclic isopentenyl phloroglucinol compounds of the present invention. 1 H-NMR spectrum.

[0035] Figure 2 shows the polycyclic isopentenyl phloroglucinol compounds of the present invention.13 C-NMR and DEPT spectra.

[0036] Figure 3 illustrates the effects of the polycyclic isopentenyl phloroglucinol compounds of this invention on the proliferation and apoptosis of multidrug-resistant cells. (A) The combined action of paclitaxel (PTX) and the compounds resulted in a reduction in colony formation of SW620 / AD300 cells. (B) The combined use of the compounds and paclitaxel led to a reduction in the volume of three-dimensional tumor spheroids formed by drug-resistant tumor cells. (C) The combination of the compounds and paclitaxel promoted apoptosis in drug-resistant tumor cells SW620 / AD300.

[0037] Figure 4. Effects of the combined treatment of the polycyclic isopentenyl phloroglucinol compound and paclitaxel of the present invention on tumor growth. (A) Tumor after treatment. (B) Tumor weight after treatment. (C) Changes in tumor volume in mice during treatment. (D) Changes in body weight in mice during treatment. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0044] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0045] Example 1

[0046]

[0047] Formula I.

[0048] The preparation method of polycyclic isopentenyl phloroglucinol compounds of formula I includes the following steps:

[0049] (1) Using the dried aerial parts of St. John's wort (1 kg) as raw material, the extract was extracted with 95% ethanol for 120 h by percolation at a flow rate of 0.5 L / h. The extract was concentrated by rotary evaporation to obtain a paste-like ethanol extract. The ethanol extract was suspended in water to obtain a suspension. Petroleum ether of equal volume to the suspension was added for extraction. The extract was concentrated to obtain the petroleum ether extract.

[0050] (2) The petroleum ether extract was subjected to gradient elution by silica gel column chromatography. Specifically, the eluent was a petroleum ether-ethyl acetate volume ratio of 10:1, 5:1, 3:1, 2:1, 1:1 and 1:2. Six eluents were collected in fractions after gradient elution with the eluent.

[0051] (3) The eluent obtained by elution with petroleum ether-ethyl acetate volume ratio of 5:1 in step (2) was subjected to MCI resin column chromatography and gradient elution with methanol-water solution. Specifically, the eluent was a methanol-water volume ratio of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10. Seven eluents were collected in fractions after gradient elution with the eluent.

[0052] (4) Take the eluent from step (3) with a methanol-water volume ratio of 90:10 and elute it by Sephadex LH-20 gel column chromatography. Elute isocratically with pure methanol solvent. Specifically, the elution rate of the eluent is 1 mL / min and the elution time is 10 h. Collect the eluent every 2 h. Collect 5 eluents in fractions after elution with the eluent.

[0053] (5) Take the eluent from the second time period in step (4) and elute it by ODS medium pressure column chromatography. Use methanol-water gradient elution. Specifically, the eluent is methanol and water in a volume ratio of 60:40 to 95:5, with an increase rate of 5 ratios per hour and a flow rate of 25 mL / min. Collect once every 1 h. The total elution time is 7 h. Collect 7 eluents Fr.1 to Fr.7 by gradient elution.

[0054] (6) The Fr.6 fraction in step (5) was prepared by preparative ODS liquid chromatography using acetonitrile-water solution with a volume ratio of 82:18 as the mobile phase, and purified by semi-preparative ODS liquid chromatography using methanol-water solution with a volume ratio of 96:4 as the mobile phase to obtain compound I.

[0055] Example 2

[0056] The structure of the compound of formula I prepared in Example 1 was identified.

[0057] The results of the identification are as follows:

[0058] Compound I was detected by nuclear magnetic resonance (NMR), and the NMR data of compound I was compared and analyzed with the NMR data of the known compound Hyperion W, thus confirming that compound I is Hyperion W.

[0059] Compound I 13 C NMR and 1 The H NMR data are shown in Table 1.

[0060] Table 1. Compounds of Formula I 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150MHz, CDCl3) data

[0061]

[0062] Example 3 Pharmacological activity

[0063] Experimental methods and results

[0064] 1. The effect of compound Hypericin W (HPW) on reversing resistance to multiple antitumor drugs.

[0065] The effect of hypericin W on reversing resistance to the antitumor drugs paclitaxel (PTX), adriamycin (ADR), gemcitabine (GEM), capecitabine (CAP), and 5-fluorouracil (5-FU) in drug-resistant tumor cells SW620 / AD300 was determined using the CCK-8 assay.

[0066] CCK8 method: SW620 / AD300 cells were seeded in 96-well cell culture plates (5000 cells / well) and cultured overnight. After incubation with different concentrations of the drug for 72 h, the culture medium was discarded, and 100 μL of fresh culture medium mixed with CCK-8 (100 μL / mL) was added. The 96-well microplates were then incubated in the dark for 2 h. The absorbance at A450 nm was measured using a Varioskan LUX multi-functional microplate reader (ThermoFisher, Waltham, MA). The IC50 was calculated using GraphPad software. 50 The value is used to calculate the resistance reversal coefficient (RF = resistance IC) according to the formula. 50 / Combined use with anti-drug resistance IC 50 It is used to evaluate the actual level of drug resistance reversal of a compound.

[0067] As shown in Table 2, hypericin W increased the sensitivity of drug-resistant cells SW620 / AD300 to paclitaxel by 137.3 times, significantly reversing the drug resistance of these cells to paclitaxel. Simultaneously, it also exhibited some resistance reversal activity against antitumor drugs such as doxorubicin.

[0068] Table 2. Drug resistance reversal effect of hypericin W against multiple antitumor drugs

[0069]

[0070] 2. Hypericin W enhanced the inhibitory effect of paclitaxel on the proliferation of drug-resistant cell lines.

[0071] Hypericone W was evaluated for its resistance reversal activity against the antitumor drug paclitaxel in drug-resistant tumor cell lines using cell plate cloning, three-dimensional tumor sphere culture, and apoptosis analysis.

[0072] Cell plate cloning method: SW620 / AD300 cells were seeded in 6-well plates (2000 cells / well) and cultured overnight. Cells were treated with different concentrations of compounds, paclitaxel (1 μmol / L), or a combination thereof for 7 days. The culture medium was aspirated and the cells were rinsed with PBS. The cells were then fixed with 4% paraformaldehyde (Biossci, Wuhan, China) for 30 minutes and stained with 0.1% (w / v) crystal violet staining solution (Biosharp, Anhui, China) for 30 minutes. The cells were washed with ultrapure water, air-dried, and images were acquired.

[0073] Three-dimensional tumor spheroid culture method: SW620 / AD300 cells were diluted (200 cells / well) and seeded into 12-well plates treated with matrix gel and cultured overnight. Images were acquired after 14 days of treatment with different concentrations of compounds, paclitaxel (1 μmol / L), or a combination thereof.

[0074] Apoptosis assay: SW620 / AD300 cells were seeded at a concentration of 300,000 cells / well in 6-well plates and cultured overnight. Cells were then treated with different concentrations of compounds, paclitaxel (1 μmol / L), or a combination thereof. After 24 h, cells were collected and washed twice with PBS. Cells were stained using an apoptosis assay kit (Beyotime, Shanghai, China). Apoptosis levels were analyzed by flow cytometry.

[0075] As shown in Figure 3A, the combination of hypericin W and paclitaxel significantly reduced the number and size of cell clones formed by drug-resistant tumor cells SW620 / AD300, and the effect was superior to that of verapamil (VPM) as a positive control. In Figure 3B, compared with the blank group and the paclitaxel-only group, the hypericin W combined with paclitaxel group significantly reduced the average diameter of drug-resistant tumor spheroids and caused them to break down. In the apoptosis analysis shown in Figure 3C, the combined administration of hypericin W and paclitaxel significantly increased the total apoptosis level of cells, and this level was positively correlated with the concentration of hypericin W. Therefore, hypericin W largely reversed the drug resistance of tumor cells to paclitaxel, restoring the sensitivity of drug-resistant cells to anti-tumor drugs.

[0076] 3. Hypericin W showed the effect of reversing tumor drug resistance in xenograft tumor models.

[0077] A xenograft tumor model was established by subcutaneous injection of multidrug-resistant tumor cells SW620 / AD300 into the axilla of BALB / c nude mice. Thirty 6-week-old Balb / c nude mice were randomly assigned to groups of five and fed an acclimatization diet for one week. SW620 / AD300 cells in the logarithmic growth phase were collected and washed three times with PBS. The cells were then resuspended in serum-free medium and the cell density was adjusted to 10-1. 7 Cells were stored on ice at a concentration of 0.2 mL / mL. The cells were seeded subcutaneously into the axillae of mice. Tumor growth was observed and recorded regularly. Administration: 1) Blank group: 90% corn oil, 10% DMSO, 10 ml / kg / 3 days, intraperitoneal injection; 2) Paclitaxel control group: Paclitaxel 5 mg / kg / 3 days, dissolved in DMSO and suspended in corn oil; 3) Compound control group: Hypericin W 100 mg / kg / 3 days, dissolved in DMSO and suspended in corn oil; 4) Positive control group: Verapamil 5 mg / kg / 3 days and paclitaxel 5 mg / kg / 3 days, dissolved in DMSO and suspended in corn oil; 5) Drug-treated group: Hypericin W 100 mg / kg / 3 days and paclitaxel 5 mg / kg / 3 days, dissolved in DMSO and suspended in corn oil. Mouse body weight and tumor volume were measured and recorded daily. Fourteen days after administration, blood samples were collected using the enucleation method, and the mice were euthanized by cervical dislocation to remove the tumor.

[0078] As shown in Figures 4A and 4B, the combined use of hypericin W and paclitaxel significantly reduced the size and weight of tumors in the xenograft tumor model, and the effect was significantly better than that of paclitaxel alone, and also better than the positive control group. Figure 4C shows the inhibitory effect of combined administration of hypericin W and paclitaxel on tumor volume growth during the administration process. Figure 4D presents the changes in body weight of mice during various administration regimens, revealing the safety of hypericin HPW and paclitaxel.

[0079] In summary, the polycyclic isopentenyl phloroglucinol compounds prepared in this invention exhibit excellent activity in reversing tumor drug resistance and can be used to prepare drugs for drug-resistant tumors. These compounds have significant development value as novel drugs for the prevention or treatment of drug-resistant tumors, and the design concept of these compounds also provides new ideas and approaches for the development of novel drugs for the prevention or treatment of drug-resistant tumors.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a polycyclic isopentenyl phloroglucinol compound, characterized in that, Includes the following steps: (1) Using the dried aerial parts of Hypericum plants as raw materials, an ethanol extract was obtained by percolation extraction with an ethanol solution; (2) The ethanol extract was suspended in water and then extracted with petroleum ether to obtain a petroleum ether extract; (3) The petroleum ether extract was subjected to chromatography including silica gel column chromatography, MCI resin column chromatography, and Sephadex chromatography. The polycyclic isopentenyl phloroglucinol compounds were separated and purified by LH-20 gel column chromatography and ODS reversed-phase column chromatography. Gradient elution was performed by silica gel column chromatography with eluents of mixed solutions of petroleum ether and ethyl acetate at volume ratios of 10:1, 5:1, 3:1, 2:1, 1:1, and 1:

2. The eluent collected by silica gel column chromatography was a mixed solution of petroleum ether and ethyl acetate at a volume ratio of 5:

1. Gradient elution was then performed by MCI resin column chromatography with eluents of mixed solutions of methanol and water at volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:

10. The eluent collected by MCI resin column chromatography was a mixed solution of methanol and water at a volume ratio of 90:

10. The final eluent was obtained by Sephadex chromatography. Isocratic elution was performed using LH-20 gel column chromatography with a methanol solution of ≥98% purity as the eluent. The elution rate was 1 mL / min, and the elution time was 10 h. The eluent was collected every 2 h, and the eluent from the second time interval was also collected. Elution was then performed using ODS reversed-phase column chromatography with a methanol-water mixture in a volume ratio of 60:40 to 95:

5. The volume ratio of methanol to water was increased in a gradient from 60:40 to 95:5 at a rate of 5 increments per hour, with a flow rate of 25 mL / min. The eluent was collected every 1 h, for a total elution time of 7 h, yielding 7 eluents from Fr.1 to Fr.

7. Eluent Fr.6 was purified by preparative ODS liquid chromatography and semi-preparative ODS liquid chromatography to obtain the polycyclic polyisoprenyl phloroglucinol compound. The structural formula of the polycyclic polyisoprenyl phloroglucinol compound is shown in Formula I. Formula I.

2. The preparation method according to claim 1, characterized in that, The volume concentration of the ethanol solution in step (1) is 70%-95%, the percolation extraction time is 48-168 hours, and the flow rate is 0.3-1.0 L / h.

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