A polyketide-xanthone hybrid compound, and a preparation method and application thereof

Abstract

The application discloses a polyketone-zoysenol ketone hybrid compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The polyketone-zoysenol ketone hybrid compound provided by the application has a structural formula as shown in formula (I). The half inhibitory concentration (IC 50 ) of the compound to human chronic myeloid leukemia cells (K562) and human colorectal adenocarcinoma cells (HT-29) reaches the nanomolar level in in-vitro anti-tumor activity evaluation. The compound has excellent tumor cell selectivity while maintaining high anti-tumor activity, and provides a valuable candidate molecule for the research and development of new anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polyketide-phenoxyketone hybrid compound, its preparation method, and its application. Background Technology

[0002] Tropolones are a class of natural products with a cycloheptatrienone core structure, widely distributed in plants, bacteria, and fungi. Studies have shown that these compounds exhibit a variety of biological activities, including antibacterial, antitumor, and antiviral activity, thus attracting significant attention in the field of drug lead compound discovery. Currently, the heterocyclic tropolone derivatives isolated and identified from nature are mainly concentrated in the sesquiterpene-tropolone hybrid form.

[0003] Microorganisms, especially fungi living in unique ecological environments (such as marine environments), are an important resource for discovering natural products with novel structures and unique biological activities, and their metabolites often exhibit rich chemical diversity. However, the structural types and chemical spaces of quinone compounds discovered from microbial resources still need further exploration. Therefore, discovering novel quinone derivatives from microbial resources is of great significance for enriching the structural types of this class of active compounds and further exploring their medicinal potential. Summary of the Invention

[0004] In view of this, the present invention provides a polyketide-phenylene ketone hybrid compound, its preparation method and application, which is derived from marine Fusarium oxysporum (… Fusarium A novel polyketone-glucanone hybrid compound was isolated from the rice fermentation products of sp. WA57. It was determined to have nanomolar inhibitory activity against human chronic myeloid leukemia cells (K562) and human colorectal adenocarcinoma cells (HT-29).

[0005] In a first aspect, the present invention provides a polyketone-phenanthone hybrid compound, the structural formula of which is shown in formula (I):

[0006] Formula (I).

[0007] Secondly, the present invention provides a method for preparing the above-mentioned polyketone-phenoxyketone hybrid compounds, comprising the following steps:

[0008] From marine Fusarium oxysporum ( Fusarium The polyketone-phenylene ketone hybrid compound was isolated from the fermentation culture of sp. WA57;

[0009] The marine Fusarium oxysporum ( Fusariumsp.) WA57 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on March 4, 2026, with accession number CGMCC NO. 42520.

[0010] Preferably, the preparation method specifically includes the following steps:

[0011] Marine Fusarium oxysporum ( Fusarium The WA57 sp. was used for fermentation culture to obtain a fermentation culture;

[0012] The fermentation culture was extracted to obtain a crude extract;

[0013] The crude extract was subjected to chromatographic separation and purification to obtain the polyketone-phenanthone hybrid compound.

[0014] Preferably, the fermentation culture is carried out using a solid culture medium for static culture; the solid culture medium contains rice, peptone, monosodium glutamate and water; the static culture time is 28 to 32 days and the culture temperature is 25 to 30°C.

[0015] Preferably, the solvent used for extraction is ethyl acetate, and after extraction, the extract is concentrated under reduced pressure to obtain a crude extract.

[0016] Preferably, the chromatographic separation and purification specifically includes: sequentially separating and purifying the crude extract using normal-phase silica gel column chromatography, medium-pressure preparative liquid chromatography, and preparative high-performance liquid chromatography.

[0017] Furthermore, the normal-phase silica gel column chromatography separation uses a methanol-dichloromethane system for gradient elution; the medium-pressure preparative liquid chromatography separation uses a methanol-water system for gradient elution; and the preparative high-performance liquid chromatography separation uses a methanol-trifluoroacetic acid aqueous solution system as the mobile phase.

[0018] Thirdly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described polyketide-phenoxyphenone hybrid compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0019] Fourthly, the present invention provides the use of the above-mentioned polyketone-phenoxyphenone hybrid compounds or the above-mentioned pharmaceutical compositions in the preparation of antitumor drugs.

[0020] Preferably, the tumor is selected from chronic myeloid leukemia and / or colorectal adenocarcinoma.

[0021] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0022] (1) This invention provides a polyketide-phenoxyphenone hybrid compound with the structure shown in formula (I). This compound is derived from marine Fusarium oxysporum (… Fusarium The natural product isolated from the fermentation culture of sp. WA57 contains two tetrahydropyran rings of quercetin linked by conjugated triene carbon chains, exhibiting a partially symmetrical structural feature. Compared with quercetin derivatives that are mainly concentrated in the sesquiterpene-quercetin hybrid form in the prior art, it has significant structural novelty and enriches the structural types and chemical space of quercetin natural products.

[0023] (2) The polyketone-phenanthone hybrid compounds of the present invention exhibit excellent inhibitory effects in in vitro antitumor activity evaluation. Specifically, the compounds showed a half-maximal inhibitory concentration (IC50) against human chronic myeloid leukemia cells (K562) and human colorectal adenocarcinoma cells (HT-29). 50 This compound reaches nanomolar levels, exhibiting activity comparable to paclitaxel, a first-line clinical antitumor drug. Notably, its cytotoxicity against normal human hepatocytes (L-02) and human embryonic kidney cells (HEK293T) is significantly lower than its cytotoxicity against the same tumor cells. Furthermore, its calculated selectivity (SI) is significantly higher than that of paclitaxel, indicating that it maintains highly potent antitumor activity while possessing excellent tumor cell selectivity. This demonstrates its ideal characteristics as an antitumor drug candidate or lead compound, providing a valuable candidate molecule for the development of new antitumor drugs. Attached Figure Description

[0024] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0025] Figure 1 This is the 1H NMR spectrum of fusariolone A, the compound of Example 1 of this invention;

[0026] Figure 2 This is the carbon NMR spectrum of fusariolone A, the compound of Example 1 of this invention;

[0027] Figure 3 This is the electronic circular dichroism spectroscopy of fusariolone A, the compound in Example 1 of this invention. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] This invention provides a polyketone-phenanthone hybrid compound, the structural formula of which is shown in formula (I):

[0030] Formula (I).

[0031] The above-mentioned compounds of the present invention are derived from marine Fusarium oxysporum (… Fusarium A novel polyketone-cyclophenolone hybrid was isolated from the secondary metabolites of sp. WA57. This invention utilizes modern spectroscopic techniques such as high-resolution mass spectrometry (HR-MS), nuclear magnetic resonance spectroscopy, and electron circular dichroism (ECD) calculations to comprehensively analyze its planar structure and stereoconfiguration, ultimately determining its unique chemical structure. The compound contains two cyclophenolone pyran rings linked by conjugated triene carbon chains, exhibiting a partially symmetrical structural feature.

[0032] The present invention also provides a method for preparing the above-mentioned polyketone-phenoxyketone hybrid compounds, comprising the following steps:

[0033] From marine Fusarium oxysporum ( Fusarium The polyketone-phenylene ketone hybrid compound was isolated from the fermentation culture of sp. WA57;

[0034] The marine Fusarium oxysporum ( Fusarium sp.) WA57 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on March 4, 2026, with accession number CGMCC NO. 42520.

[0035] As a preferred embodiment of the preparation method of the present invention, the preparation method specifically includes the following steps:

[0036] (1) Marine Fusarium oxysporum ( Fusarium The WA57 sp. was used for fermentation culture to obtain a fermentation culture;

[0037] (2) Extract the fermentation culture to obtain a crude extract;

[0038] (3) The crude extract was subjected to chromatographic separation and purification to obtain the polyketone-phenylene ketone hybrid compound.

[0039] In step (1), the preserved bacterial strain first needs to be activated. Typically, the strain from the cryopreservation tube is streaked onto potato dextrose agar (PDA) plates or slant culture media and incubated at a suitable temperature (e.g., 25-30°C, preferably 28°C) for 3-7 days, preferably 4-5 days, to obtain vigorous colonies rich in spores. Then, the activated strain is inoculated into a seed culture medium for amplification. The seed culture medium can be a common fungal culture medium in the art, such as potato dextrose liquid (PDB). The preferred culture conditions are 25-32°C, a shaking speed of 150-250 rpm, and a culture time of 2-6 days, for example, 4 days at 200 rpm, to obtain a homogeneous seed solution.

[0040] After obtaining the seed culture, it is inoculated into a fermentation medium for scale-up fermentation. Preferably, the fermentation culture is carried out using a solid culture medium for static incubation. The solid culture medium can simulate the natural growth environment of fungi, which is beneficial for inducing the synthesis of their secondary metabolites. More preferably, the solid culture medium contains rice, peptone, monosodium glutamate (MSG), and water. Among them, rice serves as the main carbon source and carrier, peptone provides nitrogen source and growth factors, and MSG can serve as a supplementary nitrogen source or metabolic precursor. The amount of each component can be adjusted according to the fermentation scale. For example, the ratio of rice to water can preferably be 1 g : (0.8~1.5) mL, such as 70 g of rice to 100 mL of water; the amount of peptone and MSG added can be 0.1%~1% and 0.05%~0.5% of the mass of rice, respectively. After the culture medium is prepared, it needs to be sterilized, usually by autoclaving at 115~121℃ for 20~40 minutes.

[0041] The inoculation volume of the seed culture can be optimized to 2-8 mL / bottle, for example, 4 mL / bottle. After inoculation, the fermentation flask is placed in a constant temperature incubator for static incubation. The static incubation period is 28-32 days, for example, 28, 30, or 32 days; the incubation temperature is 25-30℃, for example, 26℃, 28℃, or 30℃. During the incubation process, mycelium gradually grows and covers the surface of the culture medium, synthesizing and accumulating the target product at specific time points.

[0042] In step (2), after fermentation, the metabolites in the fermentation culture need to be extracted. Preferably, ethyl acetate is used as the solvent for extraction. The extraction operation may include: crushing or homogenizing the fermentation product, soaking it in ethyl acetate, and performing ultrasonic-assisted extraction to improve the extraction efficiency. The ultrasonic time can be 5-20 minutes, such as 10 minutes; then separating the organic phase by filtration or centrifugation. To improve the extraction rate, the extraction can be repeated 2-4 times, such as 3 times, and all extracts are combined. After extraction, the combined extracts are concentrated under reduced pressure to remove the solvent and obtain a crude extract. The temperature of the reduced pressure concentration can be controlled at 40-50°C, such as 45°C, to avoid damage to heat-sensitive components. The obtained crude extract is a complex mixture containing the target compound and other metabolites.

[0043] In step (3), the crude extract needs to be systematically separated and purified by chromatography to obtain the target compound with high purity. Preferably, the chromatographic separation and purification specifically includes: sequentially separating and purifying the crude extract by normal-phase silica gel column chromatography, medium-pressure preparative liquid chromatography, and preparative high-performance liquid chromatography.

[0044] Furthermore, the normal-phase silica gel column chromatography separation employs a methanol-dichloromethane gradient elution system. Normal-phase silica gel chromatography performs initial separation based on the differences in compound polarity. The crude extract is dissolved in a small amount of a suitable solvent (such as dichloromethane or methanol), mixed with silica gel (e.g., 100-200 mesh), evaporated, and then loaded onto a silica gel column. During elution, a gradient elution is performed using a methanol-dichloromethane mixed solvent with gradually increasing polarity, for example, starting with pure dichloromethane and gradually increasing the proportion of methanol. Elution ratios (volume ratio of methanol to dichloromethane) are used, such as 0:100, 1:99, 2:98, 3:97, 5:95, 1:10, 1:8, 1:4, 1:2, and 1:1. The eluents are collected in fractions. Thin-layer chromatography (TLC) is used to analyze each collected fraction, and similar fractions containing the target compound are combined to obtain a primary separator rich in the target component.

[0045] Next, the fraction rich in the target component is separated by medium-pressure preparative liquid chromatography (MLC). The MLC separation employs a methanol-water gradient elution system. MLC uses packing material with smaller particle size and higher separation efficiency (such as reversed-phase C18 silica gel), which can achieve better separation results under moderate pressure. The sample components separated by normal-phase silica gel are dissolved in solvents such as methanol-water and then injected into the MLC system. During elution, a methanol-water gradient elution system is used, for example, starting with a low proportion of methanol (e.g., 50%–65%) and gradually increasing the methanol proportion to achieve separation of the target peak and impurity peaks. The target fraction is collected based on the ultraviolet absorption signal of the chromatographic peak (e.g., monitored around 280 nm), concentrated under reduced pressure to remove the organic solvent, and then further purified to obtain the sample.

[0046] Finally, the sample purified by medium-pressure separation was further purified by preparative high-performance liquid chromatography (HPLC). The preparative HPLC separation used a methanol-trifluoroacetic acid aqueous solution system as the mobile phase. HPLC offers higher column efficiency and resolution, making it a crucial step in obtaining high-purity compounds. To improve separation efficiency and peak shape, a small amount of modifier, such as trifluoroacetic acid (TFA), can be added to the mobile phase, typically at a concentration of 0.01%–0.1% (volume ratio). For example, a methanol-0.1% trifluoroacetic acid aqueous solution can be used as the mobile phase for isocratic or gradient elution. Based on the retention time and UV absorption characteristics of the target compound, the corresponding chromatographic peak eluent was accurately collected. The collected solution was concentrated and dried under reduced pressure to obtain the pure product, namely the polyketide-fusariolone hybrid compound, denoted as fusariolone A. The entire separation and purification process can be monitored using a combination of TLC and HPLC to guide fraction merging and product purity assessment.

[0047] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described polyketide-phenoxyphenone hybrid compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0048] The compounds described in this invention can be used as active ingredients in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients to prepare pharmaceutical compositions. The term "pharmaceutically acceptable salt" refers to a conventional salt formed by the compounds of this invention with inorganic or organic acids, such as hydrochlorides, sulfates, phosphates, methanesulfonates, benzenesulfonates, acetates, citrates, succinates, tartrates, etc.; or a salt formed with a base, such as sodium salts, potassium salts, calcium salts, aluminum salts, ammonium salts, etc. The term "stereoisomer" includes enantiomers and diastereomers resulting from the presence of a chiral center in the molecule, as well as geometric isomers (such as Z / E isomers) resulting from differences in the configuration of the carbon-carbon double bond. The compounds of this invention can exist in free form, or in the form of their isomers or salts.

[0049] "Pharmaceuticalally acceptable excipients" refer to excipients and additives that are routinely used in pharmaceutical formulations, are compatible with the active ingredient, and are harmless to organisms. These include, but are not limited to: fillers such as lactose, sucrose, mannitol, starch, and microcrystalline cellulose; binders such as hydroxypropyl methylcellulose, polyvinylpyrrolidone, and pregelatinized starch; disintegrants such as sodium carboxymethyl starch, croscarmellose sodium, and croscarmellose; lubricants such as magnesium stearate, talc, and microcrystalline silica; wetting agents; or solubilizers such as polysorbate and poloxamer; pH adjusters; preservatives; and antioxidants. This pharmaceutical composition can be formulated into various dosage forms, such as tablets, capsules, granules, powders, oral liquids, injections, powders for injection, ointments, and suppositories, according to clinical needs and using methods known in the art, for systemic or local administration.

[0050] The present invention also provides the use of the above-mentioned polyketone-phenoxyphenone hybrid compounds or the above-mentioned pharmaceutical compositions in the preparation of antitumor drugs.

[0051] This invention demonstrates, through in vitro cytotoxicity experiments, that the compound exhibits significant inhibitory activity against various human tumor cell lines. This compound and its pharmaceutical compositions possess clear anti-tumor therapeutic potential and can be used to prepare drugs for treating various malignant tumors. This application encompasses situations where the compound of this invention is used as the sole active ingredient or in combination with other anti-tumor drugs.

[0052] As a preferred embodiment, the tumor is selected from chronic myeloid leukemia and / or colorectal adenocarcinoma. Based on specific experimental data, the compounds of this invention exhibit nanomolar levels of half-maximal inhibitory concentration (IC50) against human chronic myeloid leukemia cells (K562) and human colorectal adenocarcinoma cells (HT-29). 50 This compound exhibits activity comparable to paclitaxel, a first-line clinical antitumor drug, and shows relatively low toxicity to normal human cells (such as L-02 hepatocytes and HEK293T kidney cells), demonstrating excellent therapeutic selectivity. Therefore, this compound is particularly suitable for the preparation of drugs for the treatment of chronic myeloid leukemia and / or colorectal adenocarcinoma. Of course, its application is not limited to this; based on its broad-spectrum antitumor activity, the tumors may further include, but are not limited to, non-small cell lung cancer, cervical cancer, acute T-lymphoblastic leukemia, or pancreatic cancer.

[0053] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0054] Unless otherwise specified, "%" in the following examples refers to volume percentage, such as "65% methanol" which means that the volume fraction of methanol is 65%.

[0055] Example 1

[0056] This embodiment provides a method for preparing a polyketide-phenoxyketone hybrid compound.

[0057] 1. Marine Fusarium oxysporum ( Fusarium Fermentation culture of sp.) WA57

[0058] (1) Activation of microbial strains

[0059] Marine Fusarium oxysporum was extracted from a cryovial at -80°C. Fusarium The preserved strain of *Fusarium oxysporum* sp. WA57 was aseptically streaked onto potato dextrose agar (PDA) plates. The plates were incubated at 28°C for 4 days to obtain activated colonies. *Fusarium oxysporum* sp. Fusarium sp.) WA57 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on March 4, 2026, with accession number CGMCC NO. 42520.

[0060] Preparation of PDA medium: Take 200 g of fresh, peeled potatoes, cut them into small pieces, add 1 L of distilled water and boil until the potatoes are cooked but not mushy (about 25 minutes). Filter through double-layered gauze, collect the filtrate, and add distilled water to 1 L. Add 20 g of glucose and 20 g of agar, stir to dissolve, dispense into Erlenmeyer flasks, and autoclave at 115℃ for 30 minutes.

[0061] (2) Seed liquid preparation

[0062] Spores were scraped from the activated plate using a sterile cotton swab and transferred to a 1 L Erlenmeyer flask containing 300 mL of potato dextrose (PDB) medium. The Erlenmeyer flask was placed in a constant temperature shaker and cultured at 28°C and 200 rpm for 4 days to obtain the seed culture.

[0063] Preparation of PDB medium: Except for the absence of agar, the other components are the same as those of PDA medium in step (1). After dispensing, autoclave at 115℃ for 30 minutes and set aside.

[0064] (3) Scale up fermentation

[0065] Preparation of rice solid culture medium: Prepare the solid culture medium according to the following component ratio: 70 g rice, 0.3 g peptone, 0.1 g monosodium glutamate, and 100 mL distilled water. Add the above components to a 1 L Erlenmeyer flask, stir well, and autoclave at 115℃ for 30 minutes. Cool before use.

[0066] Inoculation and fermentation: In a sterile operating table, the cultured seed culture was inoculated into rice solid culture medium at an inoculation rate of 4 mL / bottle. The fermentation bottles were placed in a 28℃ constant temperature incubator and incubated statically for 30 days to obtain the fermentation culture.

[0067] 2. Extraction of fermentation products

[0068] The rice fermentation culture was crushed with a glass rod, and ethyl acetate (200 mL / bottle) was added. Extraction was performed with ultrasonic assistance for 10 minutes. The extract was then collected by vacuum filtration using a Buchner funnel. This extraction process was repeated three times, and all ethyl acetate extracts were combined.

[0069] The combined ethyl acetate extract was concentrated under reduced pressure in a water bath at 45°C to remove the solvent, yielding 14.0 g of a brown crude extract.

[0070] 3. Isolation and purification of compounds

[0071] (1) Preliminary separation by normal phase silica gel column chromatography

[0072] Dissolve the crude extract in an appropriate amount of methanol, add an equal mass of 100-200 mesh silica gel, stir well, evaporate the solvent in a fume hood, and prepare a dry sample for loading.

[0073] The sample was packed at the top of a normal-phase silica gel column, and gradient elution was performed using a methanol-dichloromethane system. The elution ratios (volume ratio of methanol to dichloromethane, v / v) were 0:100, 1:99, 2:98, 3:97, 4:96, 5:95, 1:10, 1:8, 1:4, 1:2, and 1:1, respectively. Each gradient elution consisted of 2-3 column volumes, and the eluent was collected in fractions.

[0074] Each collected fraction was detected by thin-layer chromatography (TLC), fluorescence was observed under ultraviolet light (254 nm and 365 nm), and color development was performed using 10% sulfuric acid ethanol solution. Fractions containing the same or similar components were combined and concentrated under reduced pressure to obtain 6 components (Fr.A ~ Fr.F).

[0075] (2) Medium-pressure preparative liquid chromatography separation

[0076] The component Fr.D, which was found to contain the target compound by TLC and high performance liquid chromatography (HPLC), was separated by medium-pressure preparative liquid chromatography.

[0077] Instrument: CombiFlash NextGen 300+ rapid preparative liquid chromatography system.

[0078] Chromatographic column: Pre-loaded C18 reversed-phase column.

[0079] Mobile phase: methanol-water system.

[0080] Gradient elution program: 65% methanol for 15 min, 80% methanol for 15 min, 100% methanol for 30 min.

[0081] Flow rate: 20 mL / min.

[0082] Detection wavelengths: 210 nm and 280 nm.

[0083] Each elution peak was collected based on the chromatographic peaks, and after detection and analysis by high performance liquid chromatography (HPLC), the same components were combined to obtain 6 subfractions (Fr.D1 ~ Fr.D6).

[0084] (3) Preparative high performance liquid chromatography fine purification

[0085] The subfraction Fr.D5 was purified by preparative high performance liquid chromatography.

[0086] Instrumentation: Preparative high performance liquid chromatography system (Agilent 1260 preparative binary pump, Agilent 1260 variable wavelength detector).

[0087] Column: HPLCONE ® 5C18F (20 mm × 250 mm, 5 µm).

[0088] Mobile phase: methanol-0.1% trifluoroacetic acid (TFA) aqueous solution.

[0089] Elution program: The methanol ratio was increased linearly from 60% to 100% within 0 to 25 min; then maintained at 100% methanol for 15 min.

[0090] Flow rate: 10 mL / min.

[0091] Detection wavelengths: 210 nm and 280 nm.

[0092] The chromatographic peak eluent at a retention time of 26.5 min was collected, concentrated under reduced pressure at 45 °C to remove the organic solvent, and the remaining aqueous phase was freeze-dried to obtain the pure compound, designated fusariolone A. The calculated yield was approximately 1.11 mg / g (based on rice fermentation), with a purity of 99%.

[0093] 4. Structural identification of compound fusariolone A

[0094] (1) Physicochemical properties

[0095] The compound fusariolone A of this invention is a brown solid. Its specific rotation ([ α ] 25D The concentration was -9.0 in methanol solution (sample concentration 0.02 g / 100 mL). Its ultraviolet (UV) spectrum was measured in methanol solution, with the maximum absorption wavelength being (…). λ max The corresponding molar absorptivity (log) is 275 nm and 363 nm, respectively. ε The figures are 2.30 and 1.58 respectively.

[0096] (2) High-resolution mass spectrometry

[0097] The molecular weight of the compound was determined using high-resolution electrospray ionization mass spectrometry (HRESIMS), and the results showed that its quasi-molecular ion peak was [missing information]. m / z 475.2115 [M + H] + (i.e., the protonated molecular ion peak), thus determining the molecular formula of the compound to be C. 29 H 30 O6 was identified, and its degree of unsaturation was calculated to be 15 based on its molecular formula, suggesting that there may be multiple unsaturated bonds and ring systems in the molecular structure.

[0098] (3) Nuclear magnetic resonance spectroscopy

[0099] The planar structure of the compound was analyzed in detail using nuclear magnetic resonance spectroscopy (NMR). 1 1H NMR (proton nuclear magnetic resonance) was measured at 600 MHz, using deuterated dimethyl sulfoxide (DMSO-). d 6) is a solvent; 13 3C NMR (carbon nuclear magnetic resonance) was measured at 150 MHz, also using DMSO- d 6 is the solvent. Detailed NMR spectral data are listed in Table 1. The corresponding original spectra are shown below. Figure 1 ( 1 H NMR spectrum) and Figure 2 ( 13 (C NMR spectrum).

[0100] Table 1 Nuclear Magnetic Resonance Spectroscopy Data

[0101]

[0102] Note: Nuclear magnetic resonance spectroscopy at 600 MHz ( 1 H) and 150 MHz ( 13 Determined under conditions C). Chemical shift. δ In ppm δ c This refers to the chemical shift of carbon. δ H This refers to the chemical shift of hydrogen; coupling constant. JMeasured in Hz. Quaternary carbon (C) is carbon not directly bonded to a hydrogen atom; tertiary carbon (CH) is carbon bonded to one hydrogen atom; methylene (CH2) is carbon bonded to two hydrogen atom groups; methyl (CH3) is carbon bonded to three hydrogen atom groups. Abbreviations for multiplicity in 1H NMR spectroscopy: s for singlet, d for doublet, t for triplet, dd for doublet of doublets, m for multiplet. Superscript α , β , γ This indicates overlapping or incompletely distinguishable signals.

[0103] (4) Electronic circular dichroism

[0104] The stereostructure of the compound was analyzed in detail using electronic circular dichroism (ECD) chromatography, such as... Figure 3 As shown. By comparing the consistency of the ECD curves obtained from calculation and experiment, the stereoconfiguration of compound fusariolone A was determined.

[0105] Based on the above structural data, the structural formula of the compound fusariolone A was finally determined as shown in formula (I):

[0106] Formula (I).

[0107] Example 2

[0108] This embodiment provides an evaluation of the in vitro antitumor cytotoxic activity of the compound fusariolone A obtained in Example 1.

[0109] 1. Experimental materials

[0110] (1) Test compound:

[0111] The compound fusariolone A prepared in Example 1 was accurately weighed in appropriate amounts, dissolved in biological-grade dimethyl sulfoxide (DMSO), and prepared into a 1 mM stock solution, which was stored at -20°C for later use. It was then diluted to the required concentration with the appropriate culture medium during experiments.

[0112] (2) Positive control drug: paclitaxel.

[0113] (3) Cell lines:

[0114] Tumor cell lines: human non-small cell lung cancer cells (A549), human chronic myeloid leukemia cells (K562), human colorectal adenocarcinoma cells (HT-29), human cervical adenocarcinoma cells (HeLa), human acute T-lymphoblastic leukemia cells (MOLT-4), and human pancreatic ductal adenocarcinoma cells (PANC-1).

[0115] Normal cell lines: human normal hepatocytes (L-02) and human embryonic kidney cells (HEK293T).

[0116] 2. Experimental Methods

[0117] (1) Cell culture

[0118] Based on the growth characteristics of each cell line, the appropriate culture medium was selected for culture:

[0119] A549, K562 and MOLT-4 cells: RPMI-1640 medium, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.;

[0120] HT-29 cells: McCoy's 5A medium, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.;

[0121] HeLa, PANC-1, L-02 and HEK293T cells: DMEM medium, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0122] All culture media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). Cells were cultured in a 37°C, 5% CO2 incubator. Cells were passaged or used in experiments when they reached the logarithmic growth phase.

[0123] (2) Cell inoculation

[0124] Logarithmic growth phase cells were harvested, and the cell suspension concentration was adjusted. Cells were seeded in 384-well plates at 50 μL / well. Seeding densities were as follows: HeLa cells 0.8 × 10⁸. 4 cells / mL; PANC-1 and MOLT-4 cells were 2 × 10⁶. 4 Cells / mL; A549, K562, HT-29, L-02 and HEK293T cells were 1.2 × 10⁻⁶. 4 per mL.

[0125] (3) Dosing treatment

[0126] The Echo 550 non-contact liquid handling system was used to transfer serially diluted test compounds and positive controls from the source plate to the cell plate. The final concentration of DMSO in each well was ensured to be less than 0.5% (v / v).

[0127] Drug concentration settings: The initial test concentration is 100 μM, and it is serially diluted by 2-fold to set a total of 8-10 concentration points. Each concentration point is set with 3 replicates.

[0128] The following control group was also set up:

[0129] Cell control group (negative control): culture medium containing an equal volume of DMSO was added, but no drugs were added;

[0130] Blank control group: culture medium containing no cells and drugs was added.

[0131] After drug administration, the cell plates were placed in a 37°C, 5% CO2 incubator for 72 hours for further incubation.

[0132] (4) CCK-8 detection

[0133] After incubation, add 3 μL of CCK-8 reagent to each well (avoid light). Gently vortex to mix, then return the cell plate to the incubator for another 3 hours of incubation.

[0134] After incubation, the absorbance (OD value) at a wavelength of 450 nm was measured using a microplate reader.

[0135] (5) Data processing and calculation

[0136] Calculate cell viability using the following formula:

[0137] Cell viability (%) = (OD) 药物处理组 - OD 空白对照组 ) / (OD 细胞对照组 - OD 空白对照组 ) × 100%;

[0138] Among them, OD 药物处理组 It is the absorbance of the drug-treated group at a wavelength of 450 nm; OD 空白对照组 This is the absorbance value of the blank control group at a wavelength of 450 nm; OD 细胞对照组 This is the absorbance value of the cell control group at a wavelength of 450 nm.

[0139] Plotting the logarithm of drug concentration on the x-axis and cell viability on the y-axis, a nonlinear regression model was used to fit the dose-response curve, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The experiment was independently repeated three times, and the results are expressed as mean ± standard deviation (Mean ± SD).

[0140] (6) Calculation of selection coefficient

[0141] The selectivity index (SI) is calculated using the following formula:

[0142] SI = IC 50 (Normal cells) / IC 50 (Tumor cells);

[0143] Among them, IC 50(Normal cells) refers to the IC50 of normal cells. 50 Value; IC 50 (Tumor cells) refers to the IC50 of tumor cells. 50 value.

[0144] Using L-02 and HEK293T normal cells as references, the selectivity coefficients (SI) of the compounds for different tumor cells were calculated. A higher SI value indicates higher selectivity of the compound for tumor cells and relatively lower toxicity to normal cells.

[0145] 3. Experimental Results

[0146] (1) Inhibitory activity against tumor cells and normal cells

[0147] Results of the in vitro inhibitory activity (IC50) of compound fusariolone A against 6 human tumor cell lines and 2 human normal cell lines. 50 The values ​​are shown in Table 2. The activity data of the positive control drug paclitaxel are also listed in the table.

[0148] Table 2. In vitro inhibitory activity (IC50) of compound fusariolone A against 6 tumor cell lines and 2 human normal cell lines. 50 (nM)

[0149]

[0150] Note: All data in Table 2 are from three independent repeated experiments and are expressed as mean ± standard deviation.

[0151] The results showed that compound fusariolone A exhibited varying degrees of inhibitory activity against all six tested tumor cell types. Among them, the inhibitory activity against human chronic myeloid leukemia cells K562 and human colorectal adenocarcinoma cells HT-29 was the most prominent, with an IC50 score of [missing value]. 50 The values ​​were 3.8 nM and 3.1 nM, respectively, reaching the nanomolar level, comparable to the inhibitory activity of the positive control drug paclitaxel on the same cells (3.4 nM and 1.6 nM). This compound also showed strong inhibitory activity (IC50) against A549 and HeLa cells. 50 The concentrations were 18.0 nM and 10.1 nM, respectively, and their inhibitory activity against MOLT-4 cells was moderate (IC50). 50 The concentration was 58.8 nM, indicating relatively weak inhibitory activity against PANC-1 cells (IC50). 50 (537.0 nM).

[0152] Regarding toxicity to normal cells, fusariolone A showed IC50 in L-02 hepatocytes and HEK293T kidney cells. 50The IC50 values ​​were 187.1 nM and 124.8 nM, respectively, significantly higher than their IC50 values ​​against sensitive tumor cells such as K562 and HT-29. 50 value.

[0153] (2) Selective evaluation

[0154] Using two types of normal cells as references, the selectivity coefficients of compound fusariolone A for different tumor cells were calculated, and the results are shown in Table 3.

[0155] Table 3. Selection coefficients (SI) of compounds fusariolone A and paclitaxel for six tumor cell lines.

[0156]

[0157] The results showed that fusariolone A exhibited extremely high selectivity for K562 and HT-29 cells. Using L-02 normal hepatocytes as a reference, the selection coefficients for K562 and HT-29 cells reached 49.2 and 60.4, respectively; using HEK293T normal kidney cells as a reference, the selection coefficients also reached 32.8 and 40.3, respectively. This selectivity was significantly superior to the positive control drug paclitaxel.

[0158] 4. Experimental Conclusions

[0159] Compound fusariolone A exhibits significant inhibitory activity against the proliferation of various human tumor cells in vitro, particularly showing potent nanomolar-level inhibition against human chronic myeloid leukemia cells K562 and human colorectal adenocarcinoma cells HT-29, with activity comparable to that of paclitaxel, a first-line clinical antitumor drug. More importantly, this compound demonstrates extremely high selectivity for these two tumor cell types, with a selectivity coefficient (SI>30) far superior to paclitaxel (SI<5), indicating that it effectively kills tumor cells while exhibiting low toxicity to normal cells, thus providing a wider therapeutic safety window. Therefore, compound fusariolone A has the potential for further development as an antitumor candidate drug or lead compound, especially in the preparation of drugs for the treatment of chronic myeloid leukemia and colorectal adenocarcinoma.

[0160] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polyketone-phenanthone hybrid compound, characterized in that, Its structural formula is shown in equation (I): Equation (I).

2. The method for preparing the polyketone-phenanthone hybrid compound as described in claim 1, characterized in that, Includes the following steps: From marine Fusarium oxysporum ( Fusarium The polyketone-phenylene ketone hybrid compound was isolated from the fermentation culture of sp. WA57; The marine Fusarium oxysporum ( Fusarium sp.) WA57 is deposited at the China General Microbiological Culture Collection Center on March 4, 2026, with accession number CGMCC NO. 42520.

3. The preparation method according to claim 2, characterized in that, The preparation method specifically includes the following steps: Marine Fusarium oxysporum ( Fusarium The WA57 sp. was used for fermentation culture to obtain a fermentation culture; The fermentation culture was extracted to obtain a crude extract; The crude extract was subjected to chromatographic separation and purification to obtain the polyketone-phenanthone hybrid compound.

4. The preparation method according to claim 2, characterized in that, The fermentation culture is carried out using a solid culture medium for static culture; the solid culture medium contains rice, peptone, monosodium glutamate and water; the static culture time is 28 to 32 days and the culture temperature is 25 to 30°C.

5. The preparation method according to claim 2, characterized in that, The solvent used for extraction was ethyl acetate. After extraction, the extract was concentrated under reduced pressure to obtain a crude extract.

6. The preparation method according to claim 2, characterized in that, The chromatographic separation and purification specifically includes: sequentially separating and purifying the crude extract using normal-phase silica gel column chromatography, medium-pressure preparative liquid chromatography, and preparative high-performance liquid chromatography.

7. The preparation method according to claim 6, characterized in that, The normal-phase silica gel column chromatography separation uses a methanol-dichloromethane system for gradient elution; the medium-pressure preparative liquid chromatography separation uses a methanol-water system for gradient elution; and the preparative high-performance liquid chromatography separation uses a methanol-trifluoroacetic acid aqueous solution system as the mobile phase.

8. A pharmaceutical composition, characterized in that, The compound comprises a therapeutically effective amount of the polyketide-phenoxyphenone hybrid compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

9. The use of the polyketide-phenoxyphenone hybrid compound of claim 1 or the pharmaceutical composition of claim 8 in the preparation of an antitumor drug.

10. The application as described in claim 9, characterized in that, The tumor was selected from chronic myeloid leukemia and / or colorectal adenocarcinoma.

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