An antitumor polyketide-nonribosomal peptide hybrid, method of preparation and use

Abstract

The application belongs to the technical field of medical compounds, and particularly relates to an anti-tumor polyketide-nonribosomal peptide hybrid compound, a preparation method and application. The polyketide-nonribosomal peptide hybrid compound is compound 1 or compound 2, and structural formulas of the compound 1 and the compound 2 are as follows: The compound has anti-tumor activity, particularly anti-human breast cancer effect, and has relatively high anti-tumor activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical compound technology, specifically relating to an antitumor polyketide-nonribosomal peptide hybrid, its preparation method, and its application. Background Technology

[0002] Natural products are compounds isolated from nature (fungi, bacteria, plants, and animals, etc.) and play an important role in drug development. Fungal natural products are one of the main sources of bioactive drugs, such as the natural antibiotic penicillin, the antifungal drug griseofulvin, and the lipid-lowering drug lovastatin. Purifying and screening antitumor active ingredients from fungi, bacteria, and plants is an important research topic. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an antitumor polyketide-nonribosomal peptide hybrid, its preparation method and application. The compound of the present invention has antitumor activity, especially anti-human breast cancer effect, and has high antitumor activity.

[0004] This invention provides an antitumor polyketide-nonribosomal peptide hybrid, wherein the polyketide-nonribosomal peptide hybrid is compound 1 or compound 2, and the structural formulas of compound 1 and compound 2 are as follows:

[0005]

[0006] This invention provides a method for preparing the antitumor polyketide-nonribosomal peptide hybrid, wherein *Penicillium* is used. The culture medium after expansum fermentation was extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract. The crude extract was subjected to macroporous resin column chromatography, eluted with water and 80% methanol aqueous solution (preferably followed by pure methanol), to obtain an 80% methanol eluent. The 80% methanol eluent was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate gradient elution at mobile phase volume ratios of 5:0, 1:1, and 0:1. TLC analysis and combination yielded three fractions Fr.A-Fr.C (Fr.A-Fr.C arranged in the order of elution). Fr.C was further subjected to silica gel column chromatography with a dichloromethane-methanol gradient elution at mobile phase volume ratios of 50:1, 30:1, 20:1, 15:1, 8:1, 4:1, and 2:1 to obtain four fractions Fr.C1-Fr.C4.

[0007] Fr.C3 was subjected to gel column chromatography, eluted with pure methanol, and the fractions were combined to obtain 8 sub-fractions Fr.C3.1-Fr.C3.8 (Fr.C3.1-Fr.C3.8 are arranged in the order of elution). Fr.C3.5 was purified (preferred purification method: 72% methanol buffer solution, acetic acid:triethylamine = 1:1, 1000 mL water, 3 mL / min, 254 nm) to obtain compound 1.

[0008] Fr.C2 was subjected to gel column chromatography, eluted with pure methanol, and the fractions were combined to obtain 10 sub-fractions Fr.C2.1-Fr.C2.10. Fr.C2.4 was purified (preferred purification method: 72% methanol buffer solution, acetic acid:triethylamine = 1:1, 1000 mL water, 3 mL / min, 254 nm) to obtain compound 2.

[0009] In one embodiment, *Penicillium expansum* is *Penicillium expansu*, sourced from the China General Microbiological Culture Collection Center, with accession number CGMCC 3.15687.

[0010] In one embodiment, the culture medium after fermentation of Penicillium expansum is prepared by inoculating Penicillium expansum seed liquid into rice culture medium and then performing static fermentation at room temperature.

[0011] In one embodiment, the rice culture medium is prepared by mixing rice and water, then sterilizing the mixture.

[0012] In one embodiment, the method for preparing Penicillium expansum seed culture is as follows: Penicillium expansum mycelia are inoculated on SDA solid medium, cultured, and then inoculated on SDA liquid medium. After cultivation, Penicillium expansum seed culture is obtained.

[0013] This invention provides an application of the aforementioned antitumor polyketide-nonribosomal peptide hybrid for the preparation of a drug for treating cancer, preferably human breast cancer.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0015] 1. The polyketide-nonribosomal peptide hybrid prepared in this invention is a novel compound obtained for the first time. It is a hybrid of polyketide and nonribosomal peptide, and its chemical structure is completely different from any single-component drug already on the market. Specific cancer treatment drugs further developed based on this compound can circumvent existing marketed drug patents, providing the market with more options.

[0016] 2. This invention is the first to reveal that the secondary metabolite prepared by fermentation of Penicillium expansum—a polyketide-nonribosomal peptide hybrid—has cancer therapeutic activity, which helps the drug development field to focus on the antitumor protective activity of Penicillium expansum secondary metabolites and to find compounds with antitumor protective activity.

[0017] 3. The polyketide-nonribosomal peptide hybrid to be protected by this invention is abundant and can be obtained through microbial fermentation. The entire production process is free of chemical pollution and is green and environmentally friendly. Attached Figure Description

[0018] Figure 1 Compound 1 of the present invention 1 H NMR spectrum.

[0019] Figure 2 Compound 1 of the present invention 13 C NMR and DEPT spectra.

[0020] Figure 3 This is the HSQC spectrum of compound 1 of the present invention.

[0021] Figure 4 This is the HMBC spectrum of compound 1 of the present invention.

[0022] Figure 5 Compound 1 of the present invention 1 H- 1 H COSY spectrum.

[0023] Figure 6 This is the ROESY spectrum of compound 1 of the present invention.

[0024] Figure 7 This is the HRESIMS diagram of compound 1 of the present invention.

[0025] Figure 8 This is the IR spectrum of compound 1 of the present invention.

[0026] Figure 9 The main components of compounds 1 and 2 of this invention 1 H- 1 H COSY and HMBC are related.

[0027] Figure 10 Compound 2 of the present invention 1 H NMR spectrum.

[0028] Figure 11 Compound 2 of the present invention 13 C NMR and DEPT spectra.

[0029] Figure 12 Compound 2 of the present invention 1 H- 1 H COSY spectrum.

[0030] Figure 13 This is the HSQC spectrum of compound 2 of the present invention.

[0031] Figure 14 This is the HMBC spectrum of compound 2 of the present invention.

[0032] Figure 15 This is the ROESY spectrum of compound 2 of the present invention.

[0033] Figure 16 This is the IR spectrum of compound 2 of the present invention.

[0034] Figure 17 This is the HRESIMS diagram of compound 2 of the present invention.

[0035] Figure 18 A diagram showing the expression of ABCA1 protein in MDA-MB-231 cells induced by compound 1;

[0036] in, Figure 18 A is a volcano plot of differentially expressed mRNAs detected by RNA-Seq after treating MDA-MB-231 cells with compound 1. Figure 18 B is a heatmap of the top 10 differentially expressed mRNAs in MDA-MB-231 cells after treatment with compound 1, as determined by RNA-Seq. Figure 18 C represents the expression level of ABCA1 protein in MDA-MB-231 cells after treatment with compound 1 by Western blotting.

[0037] Figure 19 The total cholesterol content and cholesterol efflux in cells after compound treatment;

[0038] in, Figure 19 A represents the total cholesterol content in cells after treatment with the compound. Figure 19 B represents the cholesterol efflux from cells after compound treatment. Detailed Implementation

[0039] To make the purpose and content of this invention clearer, the applicant will now provide a clear and complete description of the technical solution of this invention in conjunction with specific embodiments.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] Experimental instruments, reagents and consumables

[0043]

[0044] Experimental materials

[0045] Rice culture medium preparation: Take a 500mL Erlenmeyer flask, put 80g of rice into each flask and add 100mL of tap water, mix well, cover with a breathable membrane and tie with a rubber band, put it into an autoclave and sterilize at 121℃ for 30min. After sterilization, cool to room temperature for later use (a total of 9.0kg of rice was used for this strain, which was evenly divided into 90 500mL Erlenmeyer flasks).

[0046] SDA solid medium consists of: glucose (20 g / L), peptone (10 g / L), yeast extract (1 g / L), and agar (15 g / L).

[0047] SDA liquid culture medium consists of glucose (20 g / L), peptone (10 g / L), and yeast extract (1 g / L).

[0048] Example 1

[0049] Mycelia of *Penicillium expansum* (purchased from the China General Microbiological Culture Collection Center, accession number CGMCC 3.15687) were inoculated onto SDA solid medium and cultured at 25°C for 5 days. After successful growth, under aseptic conditions, an appropriate amount was inoculated into SDA liquid medium (no more than 300 mL of SDA liquid medium in a 500 mL Erlenmeyer flask). The inoculated liquid culture was then incubated at room temperature and 120 rpm for 5 days to obtain a seed culture. This seed culture was then aseptically inoculated onto sterilized rice culture medium, approximately 5 mL per flask, and cultured statically at room temperature for 30 days. Observations were performed every 2 days to prevent contamination.

[0050] After fermentation of *Penicillium expansum*, the fermented culture medium was collected and extracted five times with 10 L of ethyl acetate. The extract was then concentrated under reduced pressure to obtain 98.0 g of crude extract. The total extract was subjected to macroporous resin D101 column chromatography, eluted with purified water (5 L), 80% methanol-water (12 L), and pure methanol (3 L), respectively. The eluent from the water wash was discarded, yielding two crude extracts: an 80% methanol eluent (62.0 g) and a 100% methanol eluent (13.5 g). The 80% methanol eluent was subjected to silica gel column chromatography. The sample was dry-mixed with 200-300 mesh silica gel and eluted using a petroleum ether-ethyl acetate gradient (mobile phase volume ratio v / v: 5:0 (4.5 L), 1:1 (6 L), 0:1 (2 L)). After TLC analysis and merging according to the elution order, three fractions were obtained, numbered Fr.A-Fr.C. Component C (i.e., Fr.C, 2.5 g) was further subjected to silica gel column chromatography. The sample was dry-mixed with 200-300 mesh silica gel and eluted with a dichloromethane-methanol gradient (mobile phase volume ratio v / v, 50:1 (1.5 L), 30:1 (2 L), 20:1 (2.2 L), 15:1 (2.5 L), 8:1 (2 L), 4:1 (1.5 L), 2:1 (2 L)) to obtain 4 components, which were numbered C1-C4 (i.e., Fr.C1-Fr.C4) in sequence. Component C3 (i.e., Fr.C3, 0.5 g) was subjected to gel column chromatography (LH-20) and eluted with pure methanol. The fractions were combined according to the order of elution to obtain 8 sub-fractions: Fr.C3.1-Fr.C3.8. Fr.C3.5 was purified by semi-preparative high performance liquid chromatography (72% methanol buffer solution, acetic acid:triethylamine = 1:1, 1000 mL water, 3 mL / min, 254 nm) to obtain target compound 1 (100.0 mg, t). R =27.0min).

[0051] Fraction C2 (i.e., Fr.C2, 0.8 g) was subjected to gel column chromatography (LH-20) with pure methanol elution. The fractions were combined in the order of elution to obtain 10 sub-fractions: Fr.C2.1-Fr.C2.10. Fr.C2.4 was purified by semi-preparative high-performance liquid chromatography (72% methanol buffer solution, acetic acid:triethylamine = 1:1, 1000 mL water, 3 mL / min, 254 nm) to obtain the target compound (6.0 mg, t). R =20.5min).

[0052] Structural identification of compound 1, such as Figure 1-8 As shown.

[0053] White amorphous powder, readily soluble in methanol, chloroform, acetonitrile, ethanol, acetone, and dimethyl sulfoxide, etc. CH3OH), UV(CH3CN)λ max =205,244,318nm, IR(KBr)υ max :3432,2927,1682,1407,1239,1153,749cm -1 HRESIMS indicates that its molecular formula is C 38 H 41 N5O7 (m / z: 680.3079 [M+H]) + ,calcd.for 680.3079), with an unsaturation degree of 21.

[0054] compound 1 The 1H NMR spectrum (500MHz, acetone-d6) showed 39 proton signals. Figure 1 (and Table 1), which includes: 5 olefin protons: δ H 6.15(1H,dd,J=17.7,10.5Hz,H-15),δ H 5.12(2H, overlapped, H-16), δ H 6.50 (1H, s, H-19), δ H 8.40 (1H, s, H-22); 7 aromatic protons: δ H 7.25 (1H, d, J = 7.5Hz, H⁻⁶), δ H 6.70 (1H,t,J=7.5Hz,H-7),δ H 7.05 (1H,t,J=7.5Hz,H⁻⁸),δ H 6.64 (1H, d, J = 7.5Hz, H-9), δ H 6.36 (1H, s, H-31), δ H 6.32 (1H, overlapped, H-33), δ H 6.30 (1H, overlapped, H-35); 5 methine protons: δ H 4.63(1H,m,H-23),δ H 4.02(1H,dd,J=11.1,5.9Hz,H-2),δ H 3.68 (1H, m, H-27), δ H 5.69 (1H, s, H-11), δ H 7.71 (1H, s, H-21); 5 methylene protons: δ H 1.35(2H,m,H-25),δ H 1.43(2H,m,H-26),δ H1.99(2H,m,H-24),δ H 2.47(2H,m,H-3),δ H 3.11 (2H, overlapped, H-37); 3 methyl protons: δ H 1.05(3H,s,H-17),δ H 1.17(3H,s,H-18),δ H 1.08 (3H, d, J = 6.2 Hz, H-38); 3 active protons: δ H 5.78 (1H, s, 10-NH), δ H 9.36 (1H, s, 13-NH), δ H 11.06 (1H, s, 30-OH).

[0055] compound 13 The C NMR spectrum (125 MHz, CDCl3) shows 37 carbon signals ( Figure 2 (As shown in Table 1), another carbon signal δ C 137.1 was read from the HMBC spectrum. Combined with the DEPT spectrum ( Figure 2 ), HSQC spectrum ( Figure 3 ) and HMBC spectrum ( Figure 4 This suggests that the compound contains two amide carbonyl carbons (δ-carbons). C 166.8, 158.9), 1 ester carbonyl carbon (δ C 166.7), 7 olefinic carbons (δ C 145.2, 114.4, 125.2, 117.3, 137.1, 122.0, 107.0), two groups of benzene ring aromatic carbons (δ C 130.1, 125.9, 118.9, 129.5, 109.7, 152.2, 99.6, 164.5, 102.7, 166.7, 103.8, 140.3), 4 methylene carbons (δ C 57.0, 59.5, 67.3, 78.6), 3 methyl carbons (δ C 23.4, 23.0, 24.1), 5 methylene carbons (δ C 38.3, 36.1, 23.3, 39.5, 40.7) and 4 quaternary carbons (δ C 62.1, 41.8, 136.6, 154.3).

[0056] compound 1 H- 1 H COSY spectrum ( Figure 6The following spin-coupled systems are shown: H3-38 / H-27 / H2-26 / H2-25 / H2-24 / H-23 / H2-37, H-15 / H2-16, and H-6 / H-8 / H-9. In the HMBC spectrum, H2-16 is correlated with C-14 and C-4; H3-18 is correlated with C-14 and C-4; H2-3, H-6, and H-11 are correlated with C-4 respectively; H-11 is correlated with C-12; H-19 is correlated with C-13, C-22, C-12, and C-20; H-22 is correlated with C-20; it can be inferred that the isoniazid fragment is present. By combining the correlations of H2-37 with C-35 and C-36 in the HMBC spectrum; the correlation of H-33 with C-34, C-35, and C-36; and the correlation of 30-OH with C-29, C-30, and C-31, along with the degree of unsaturation of the compound, the polyketide fragment can be deduced. The connection between the isoniazid patulin fragment and the polyketide fragment can be determined by the long-range correlations of H-23 with C-21, C-22, C-24, C-37, and C-36 in the HMBC spectrum. Figure 9 ).

[0057] The compound has five chiral centers (C-2, C-4, C-11, C-23, C-27). (ROESY spectrum) Figure 7 The ROESY spectrum shows correlations between H-11 and H3-18, H3-17, H-15, and H2-16, suggesting that the substituent at the C-4 position and H-11 are both at the β-position. Furthermore, the ROESY spectrum shows a correlation between H-19 and 13-NH, indicating that the double bond between C-13 and C-19 is in the E configuration, consistent with the literature on roquefortine C compound.

[35] The configurations of C-23 and C-27 on the polyketide fragment were further determined to be 23S,27R by chemical communication and biosynthetic pathways.

[0058] Structural identification of compound 2, such as Figure 10-17 As shown.

[0059] White amorphous powder, readily soluble in methanol, chloroform, acetonitrile and ethanol. UV(CH3CN)λ max =205,244nm, IR(KBr)υ max :3421,2928,1682,1459,1239,1172,1060,746cm -1 HRESIMS indicates the molecular formula is C 38 H 43 N5O7 (m / z: 682.3235 [M+H]) + ,calcd.for 682.3235), with an unsaturation degree of 20.

[0060] Compound 21 The 1H NMR spectrum (500MHz, CD3OD) showed 38 proton signals, with 5 active protons not detected. Figure 10 (and Table 1), which includes: 4 olefin protons: δ H 6.02(1H,dd,J=17.3,10.9Hz,H-15),δ H 5.10(2H,dd,J=14.4,11.0Hz,H-16),δ H 7.06 (1H, overlapped, H-22); 7 aromatic protons: δ H 7.18 (1H, d, J = 7.5Hz, H⁻⁶), δ H 6.71 (1H,t,J=7.5Hz,H-7),δ H 7.06 (1H, overlapped, H-8), δ H 6.59 (1H, d, J = 7.5Hz, H-9), δ H 6.21(1H,s,H-31),δ H 6.26(1H,s,H-33),δ H 6.12 (1H, s, H-35); 6 methine protons: δ H 4.48 (1H, m, H-23), δ H 3.94(1H,dd,J=11.0,6.0Hz,H-2),δ H 3.67(1H,td,J=12.7,6.2Hz,H-27),δ H 5.57 (1H, s, H-11), δ H 7.58 (1H, s, H-21), δ H 4.17 (1H, brs, H-13); 6 methylene protons: δ H 2.90(1H, overlapped, H) a -19),δ H 3.19(1H,dd,J=15.2,4.4Hz,H b -19),δ H 1.30(2H,m,H-25),δ H 1.42(2H,m,H-26),δ H 1.92(2H,m,H-24),δ H 2.30(1H,t,J=11.9Hz,H a -3),δ H 2.46 (1H,q,J=6.2Hz,H) b -3),δ H2.88(1H, overlapped, H) a -37),δ H 3.01(1H,dd,J=14.7,4.8Hz,H b -37); 3 methyl protons: δ H 0.98(3H,s,H-17),δ H 1.10(3H,s,H-18),δ H 1.09 (3H, overlapped, H-38).

[0061] Compound 2 13 The C NMR spectrum (125 MHz, CD3OD) shows 38 carbon signals ( Figure 12 (and Table 1). Combined with DEPT, HSQC, and HMBC spectra, it is suggested that compound 2 contains two amide carbonyl carbons (δ...). C 171.3, 167.0), 1 ester carbonyl carbon (δ C 167.4), 6 olefinic carbons (δ C 145.3, 114.7, 138.2, 116.6, 107.3, 154.4), two groups of benzene ring aromatic carbons (δ C 130.4, 126.1, 119.5, 130.0, 110.2, 152.3, 99.6, 164.9, 104.1, 167.3, 103.0, 140.6), 5 methylene carbons (δ C 56.5, 58.0, 59.9, 68.2, 78.7), 3 methyl carbons (δ C 23.5, 23.0, 23.6), 6 methylene carbons (δ C 38.0, 29.9, 36.1, 23.5, 39.5, 41.0) and 4 quaternary carbons (δ C 62.8, 78.7, 42.1, 138.5).

[0062] Comparing the NMR data of compounds 2 and 1, the difference lies in the fact that compound 2 has an additional methine (δ-methyl group). C 56.5) and a methylene (δ) C 29.9), missing a double bond signal. We speculate that compound 2 may be derived from the reduction of the double bond (C-13 / C-19) of compound 1. HSQC, HMBC and 1 H- 1 Two-dimensional correlation signals such as H COSY confirmed its planar structure. Figure 9 ). The Rosey spectrum of compound 2 ( Figure 15The correlations between H-11 and H-15, H2-16, H3-17, and H3-18 indicate that C-14 and H-11 are at the β-position. The ROSEY correlations between H-2 and H-13 indicate that H-2 and H-13 are at the α-position, thus allowing determination of the relative configurations of C-2, C-4, C-11, and C-13 in compound 2. Furthermore, the absolute configurations of C-23 and C-27, derived from the biosynthetic pathway, are identical to those of compound 1.

[0063] Table 1. Compounds 1 and 2 1 H NMR and 13 C NMR data

[0064]

[0065]

[0066] Notes a1 H NMR (500MHz) and 13 C NMR (125MHz) was tested in acetone-d6; b1 H NMR (600MHz) and 13 C NMR (150MHz) data were tested in CD3OD.

[0067] Example 2 Evaluation of antitumor activity

[0068] Cell culture

[0069] The breast cancer cell line MDA-MB-231 was purchased from Wuhan Pronosei Biotechnology Co., Ltd.; the culture medium was GIBCO DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-dextrose antibody. Cell culture was conducted in a cell culture incubator at 37℃ and 5% CO2 saturated humidity.

[0070] Solution preparation

[0071] Preparation of working fluid

[0072] Working stock solution: Weigh a certain amount of the compound, dissolve it in a certain amount of DMSO, and prepare a 5mM working stock solution for later use.

[0073] Cytotoxicity assay

[0074] Cytotoxicity assays were performed using the CCK8 kit. 10 4 Cells were seeded into 96-well plates and cultured overnight in a cell culture incubator for 12 hours. After cell attachment, different concentrations of compounds were added to the cells, and the cells were cultured for another 48 hours. Then, the cells were cultured with CCK8 for 1 hour. Finally, the absorbance at 450 nm was measured using a microplate reader.

[0075] RNA sequencing and analysis

[0076] Total RNA was extracted from MDA-MB-231 cells treated with TRLzol. RNA concentration and purity were determined using NanoDrop 2000, and RNA integrity was detected using the RNANano 6000 assay kit. Following the instructions of the Hieff NGSUltima Dual-mode mRNALibrary Prep Kit for Illumina, 1 μg of total RNA was used to generate a sequencing library. The library was sequenced using the Illumina NovaSeq platform, generating 150 bp paired-end sequences. Valid data were obtained through data filtering, and the data were aligned and annotated against a reference genome. Gene expression levels were estimated by the number of sequences mapped per million kilobase transcripts. Differential expression analysis was performed on the two groups using DESeq2, with thresholds for differentially expressed genes set at FDR < 0.01 and fold change ≥ 2. Finally, differentially expressed genes were subjected to functional enrichment analysis.

[0077] Protein immunoblotting analysis

[0078] MDA-MB-231 cells (0, 1, and 3 μM) treated with the compound were washed three times with PBS, then lysed with RIPA lysis buffer containing PMSF. The total protein obtained after centrifugation was added to protein loading buffer and boiled for 10 min. The denatured total protein was added to an SDS-PAGE gel for electrophoresis, then transferred to a PVDF membrane. After blocking, the membrane was incubated with primary antibody at 4°C overnight, followed by incubation with secondary antibody. Finally, the membrane was imaged using an ECL chemiluminescence analyzer.

[0079] Total cholesterol measurement

[0080] MDA-MB-231 cells treated with the compound (0, 1 and 3 μM) were washed three times with PBS, and then total cholesterol was measured using an automated biochemical analyzer according to the instructions of the total cholesterol assay kit.

[0081] BODIPY - Cholesterol Measurement

[0082] MDA-MB-231 cells were cultured in serum-free medium containing 5 μM BODIPY-cholesterol for 1 h, then washed twice with serum-free medium, and cultured for 48 h in serum-free medium containing different concentrations of compounds (0, 1, 3 μM). The cell culture supernatant was transferred to 96-well plates to measure fluorescence values; adherent cells were lysed with 1% SDS solution and fluorescence values ​​were measured.

[0083] % Cholesterol Efflux = ((Supernatant Fluorescence Value) / (Cell Fluorescence Value + Supernatant Fluorescence Value)) * 100.

[0084] Statistical processing

[0085] Data are expressed as mean and standard deviation. Comparisons between the two groups were performed using an independent samples t-test in SPSS 18.0 software. *p < 0.05 indicates a statistically significant difference.

[0086] Experimental results

[0087] 1. Cytotoxicity results of compounds 1 and 2 against MDA-MB-231 cells, IC50 of compound 1 50 The value was 3.23 ± 0.89 μM, and the IC50 of compound 2 was... 50 The value is 58±0.63μM.

[0088] 2. Compound 1 induces the expression of ABCA1 protein in MDA-MB-231 cells.

[0089] Figure 18 The image shows a volcano plot obtained from transcriptome sequencing of MDA-MB-231 cells after compound treatment. The plot shows 419 genes with significantly upregulated expression and 67 genes with significantly downregulated expression. Furthermore, the differentially expressed genes were sorted according to their fold change, and the top 10 genes with the highest upregulated and downregulated expression were selected. Figure 18 The heatmap shown in B illustrates that ABCA1 was significantly upregulated under the influence of the compound. Finally, Western blotting was used to detect the expression level of ABCA1 protein in MDA-MB-231 cells after treatment with the compound, and the results are as follows: Figure 18 As shown in C, with the increase of compound 1 concentration, the expression level of ABCA1 protein in MDA-MB-231 cells increased, consistent with the transcriptome sequencing results.

[0090] 3. Compound 1 reduces the total cholesterol content in MAD-MB-231 cells and increases their cholesterol efflux.

[0091] Studies have shown that ABCA1-mediated cholesterol efflux has anti-tumor activity, and ABCA1 may become a new target for cancer therapy. Experimental results showed that compound 1 induced ABCA1 expression in MDA-MB-231 cells. Furthermore, the total cholesterol content and cholesterol efflux in cells treated with the compound were measured, and the results are shown in the figure below. Figure 19 A showed that as the concentration of compound 1 increased, the total cholesterol concentration in MDA-MB-231 cells decreased, and the difference was statistically significant. Figure 19 B showed that as the concentration of compound 1 increased, the proportion of cholesterol efflux from MDA-MB-231 cells increased, and the difference was statistically significant.

[0092] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0093] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. An antitumor polyketide-nonribosomal peptide hybrid, characterized in that, The polyketide-nonribosomal peptide hybrid is compound 1 or compound 2, and the structural formulas of compound 1 and compound 2 are as follows: 。 2. A method for preparing the antitumor polyketide-nonribosomal peptide hybrid as described in claim 1, characterized in that, Will expand Penicillium Penicillium expansum The fermented culture medium was extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract. The crude extract was subjected to macroporous resin column chromatography, eluted with water and 80% methanol aqueous solution, respectively, to obtain an 80% methanol eluent. The 80% methanol eluent was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate gradient elution at mobile phase ratios of 5:0, 1:1, and 0:

1. TLC analysis and combination yielded three fractions Fr.A-Fr.C. Fr.C was further subjected to silica gel column chromatography with a dichloromethane-methanol gradient elution at mobile phase ratios of 50:1, 30:1, 20:1, 15:1, 8:1, 4:1, and 2:1 to obtain four fractions Fr.C1-Fr.C4. Fr.C3 was subjected to gel column chromatography, eluted with pure methanol, and the fractions were combined to obtain 8 sub-fractions Fr.C3.1-Fr.C3.

8. Fr.C3.5 was purified to obtain compound 1. Fr.C2 was subjected to gel column chromatography and eluted with pure methanol. After merging, 10 fractions Fr.C2.1-Fr.C2.10 were obtained. Fr.C2.4 was purified to obtain compound 2. Penicillium Penicillium expansum The source is the China General Microbiological Culture Collection Center, with accession number CGMCC 3.15687.

3. The preparation method according to claim 2, characterized in that, the preparation is extended. Penicillium Penicillium expansum The method for preparing the culture medium after fermentation is based on Penicillium expansum. Penicillium expansum The seed culture was inoculated into a rice culture medium and then statically fermented at room temperature.

4. The preparation method according to claim 3, characterized in that, The rice culture medium is prepared by mixing rice and water, then sterilizing the mixture.

5. The preparation method according to claim 3, characterized in that, the preparation is extended. Penicillium Penicillium expansum The method for preparing the seed solution is as follows: Penicillium extended Penicillium expansum Mycelia were inoculated on SDA solid medium and cultured. After inoculation, they were then inoculated on SDA liquid medium and cultured to obtain Penicillium expansum. Penicillium expansum Seed liquid.

6. The application of the antitumor polyketide-nonribosomal peptide hybrid as described in claim 1, characterized in that, Used to prepare drugs for treating human breast cancer.

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