Extraction method and application of centerline lipopeptide compounds in actinomycetes

Abstract

The invention discloses a linear lipopeptide compound, the structure of which is selected from one of the following structures: the invention not only provides the linear lipopeptide compound, but also establishes a complete technical system of strain fermentation, directional separation, structural analysis, activity verification and mechanism research; the industrial application of a marine actinomycete natural product in the anti-tumor field is promoted.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically, it relates to a method for extracting and applying lipopeptide compounds from actinomycetes. Background Technology

[0002] Acute promyelocytic leukemia (APL), a clinical subtype of acute myeloid leukemia (AML), is characterized by the PML-RARA fusion oncogene. Currently, the combination therapy of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) can significantly improve the prognosis of patients (Clin. Exp. Med. 25 (2025) 217. Leukemia 39 (2025) 1865-1870.).

[0003] The PI3K / Akt / mTOR signaling pathway has been identified as a key target for APL treatment. It plays a central role in leukemia development, cell survival, and chemotherapy resistance, and more than 50% of AML patients have abnormal activation of this pathway. Preclinical studies have confirmed that inhibiting this pathway can effectively induce apoptosis in leukemia cells (Cancer Gene Ther. 30 (2023) 1691-1701.).

[0004] Methylated peptide natural products, especially peptides containing β-methylleucine (β-Me-Leu), have attracted much attention among natural products targeting the PI3K / Akt / mTOR pathway due to their high membrane permeability and metabolic stability (Angew. Chem. Int. Ed. 52 (2013) 12256–12267.).

[0005] Currently, only eight peptides containing β-Me-Leu have been identified, including etamycin A, cyanogripeptides A-C, fijimycins AC, and aeronamide A.

[0006] For extracts from natural sources such as actinomycetes, traditional separation relies on a non-targeted approach of "solvent extraction-column chromatography-UV detection," using stepwise fractional enrichment combined with spectral analysis to screen for target compounds (Anal. Chem. 69 (1997) 5146−5151.). LC-MS / MS techniques based on feature recognition (such as predictor ion scanning and neutral loss scanning) have been applied to targeted screening of natural products. These techniques can identify target compounds from complex extracts through specific structural fragments (such as characteristic cleavage fragments), improving discovery efficiency.

[0007] Although the combination therapy of ATRA and ATO is a first-line treatment for APL, drug resistance (such as resistance caused by PML-RARA gene mutation) and serious adverse reactions (such as ATRA-induced differentiation syndrome and ATO cardiotoxicity) remain unresolved clinical challenges, and there is an urgent need for targeted therapies with novel mechanisms of action.

[0008] Currently, APL drug candidates targeting the PI3K / Akt / mTOR pathway are mainly synthetic small molecules, which have problems such as poor membrane permeability and low metabolic stability; the development of targeted peptide drugs from natural sources is lagging behind, and there is a lack of candidate molecules with novel structures and high activity.

[0009] Methylated peptides exhibit low structural diversity and abundance in natural products. Peptides containing β-Me-Leu have extremely low natural abundance, with only eight species reported so far, limiting their structural diversity and making it difficult to meet the demands of drug development for compound structure optimization and activity screening. Furthermore, the structure-activity relationship (SAR) is unclear: existing studies have not systematically validated β-Me-Leu, making it impossible to clarify the SAR and thus restricting directions for compound structure optimization.

[0010] Traditional methods for separating natural products are inefficient: Traditional "non-targeted separation" methods cannot target the structural features of β-Me-Leu (such as 127 Da characteristic cleavage fragments) for targeted screening. When separating trace amounts of β-Me-Leu peptides from complex actinomycete extracts, there are problems such as long separation cycles, low yields of target compounds, and easy omission of trace components. Limitations in the separation, detection, and identification of natural products: Some peptides containing β-Me-Leu have complex structures (such as the amino acid sequence and absolute configuration of linear lipopeptides). Traditional NMR and mass spectrometry techniques lack efficient and accurate methods for resolving their complete structures (especially the absolute configuration of β-Me-Leu). They rely on cumbersome derivatization and standard comparison, which is time-consuming and complicated.

[0011] While existing studies have confirmed that some methylated peptides have anti-tumor activity, there are no clear reports on whether peptides containing β-Me-Leu induce apoptosis in APL cells (such as NB4 cells) by inhibiting the PI3K / Akt / mTOR pathway or whether they affect cell cycle progression. The ambiguity of the mechanism of action makes it impossible to establish a "structure-activity-mechanism" association, which restricts the transformation of these compounds into clinical drugs. Summary of the Invention

[0012] The purpose of this invention is to provide a method for extracting lipopeptide compounds from actinomycetes.

[0013] Another object of the present invention is to provide the use of the aforementioned lipopeptide compound in the preparation of a medicament for treating tumors.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] In a first aspect, the present invention provides a linear lipopeptide compound having a structure selected from one of the following structures:

[0016]

[0017] A second aspect of the present invention provides a method for extracting the aforementioned lipopeptide compounds, comprising the following steps:

[0018] (1) Fermentation culture

[0019] Blue-gray heterotomycete LHW52806, which is epiphytic on brown flat sponges, was cultured in A1 liquid medium and statically cultured at room temperature (25℃) for 20-30 days (preferably 21 days) to obtain fermentation broth; it was extracted 3-6 times (preferably 5 times) with an equal volume of ethyl acetate, and the extract was concentrated under reduced pressure to obtain ethyl acetate layer.

[0020] (2) Extraction and separation: The ethyl acetate layer was suspended in 90% methanol-water (v / v) and extracted 3-6 times (preferably 4 times) with an equal volume of petroleum ether. The petroleum ether phase was discarded. The remaining phase was diluted to 60% (v / v) with 90% methanol-water and extracted 3-6 times (preferably 4 times) with an equal volume of dichloromethane. The dichloromethane phases were combined and concentrated to obtain a dichloromethane extract. The target compound was confirmed to be concentrated in the dichloromethane extract by LC-MS (neutral loss scanning).

[0021] (3) Column chromatography enrichment: Dichloromethane extract was loaded onto a Sephadex LH-20 column (2.6×100 cm) and eluted with methanol / dichloromethane (1:1, v / v) (flow rate 1.0 mL / min) to obtain 5 fractions Fr.1-5; Fr.3 was loaded onto a medium-pressure liquid chromatograph (ODS column, 20×250 mm) and eluted with a gradient of 5%-100% methanol / water (v / v) (600 min, flow rate 20 mL / min) and detected by 210 nm UV to obtain 10 subfractions Fr.3.AJ;

[0022] (4) LC-MS guided purification: The target subfraction was purified by semi-preparative RP-HPLC (Waters BEH C18 column, 5 μm, 10×250 mm): Fr.3.C: the mobile phase was 30%-40% acetonitrile-water containing 0.1% formic acid (v / v), the flow rate was 6 mL / min, and t was collected. R The fraction obtained after 20.5 min was freeze-dried to give compound 1; t was collected. R The fraction obtained after 21.3 min yielded compound 2; Fr.3.E: the mobile phase was 45%-50% acetonitrile-water containing 0.1% formic acid (v / v), the flow rate was 6 mL / min, and the fraction collected was...R The fraction obtained after 17.8 min yielded compound 3; t was collected. R The fraction obtained after 24.7 min yielded compound 4; t was collected. R The fraction obtained after 20.2 min yielded compound 5.

[0023] The composition of the A1 liquid culture medium is as follows: yeast extract, 4 g; soluble starch, 10 g; peptone, 2 g; sea salt, 33 g; deionized water, 1 L, pH: 7.0.

[0024] The neutral loss scan: Using neutral loss scan 127 Da mass spectrometry, five ion peaks were detected in the NL127 ion channel at m / z 832.5, 826.6, 818.5, 812.6, and 798.5 [M + H]. + This allows us to obtain information about its fragment ions and chromatographic retention behavior.

[0025] or,

[0026] (1) Resin loading: 2-CTC resin was swollen with anhydrous DC for 10~30 min (preferably 20 min), and a DCM solution of Fmoc-Leu-OH and DIEA was added. The molar ratio of 2-CTC resin, Fmoc-Leu-OH and DIEA was 1:2~4:2~8 (preferably 1:3:6). The mixture was vortexed at room temperature (25℃) for 1~5 h (preferably 3 h), quenched with MeOH, and washed with DCM, DMF and MeOH in sequence.

[0027] (2) Fmoc deprotection and peptide chain coupling: Fmoc protection was removed with 20% piperidine / DMF (preferably 30 min), followed by washing with DMF; Fmoc-protected amino acids were activated in DMF with HATU, HOAt, and DIEA, and then coupled with resin at room temperature (25°C) under nitrogen flow for 0.5~2 h (preferably 1 h), followed by washing with DMF, and the process was repeated until the peptide chain was complete;

[0028] (3) Cutting and purification: The resin-bound peptide was cut with 20% HFIP / DCM (twice, 10 min each time), concentrated and purified by LC-MS to obtain the side-chain protected peptide; TFA / H2O / TIS (95:2.5:2.5, v / v / v) was added for deprotection at room temperature (preferably 2 h), precipitated with ice-cold ether, and purified by LC-MS to obtain compound 8;

[0029] (4) Esterification and purification: Compound 8 was esterified with ethanol and methanol under EDCI and DMAP respectively, and the esterification was analyzed by preparative LC-MS (C 18The column was 5 μm, 10 × 250 mm; the mobile phase was H2O and ACN; the elution gradient was ACN from 30% to 80% for 30 min; the flow rate was 6 mL / min. Compounds 6 and 7 were purified.

[0030]

[0031] A third aspect of the present invention provides the use of the aforementioned lipopeptide compound in the preparation of a medicament for treating tumors.

[0032] The tumors are selected from colon cancer, ovarian cancer, liver cancer, desmoplastic small round cell tumor, leukemia, non-small cell lung cancer, prostate cancer, lymphoma, and rhabdomyosarcoma.

[0033] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0034] The present invention has high separation efficiency. The NL scan-guided technology specifically monitors novel peptide compounds containing β-Me-Leu 127 Da characteristic fragments. Compared with the traditional "blind separation", the separation cycle of target compounds is shortened and the yield is improved. It can be extended to the screening of other natural products containing special amino acids.

[0035] The compounds of this invention have clearly defined activity and mechanism: Compound 1 exhibits potent and selective toxicity to NB4 cells, with a clear mechanism of action (inhibition of the PI3K / Akt / mTOR pathway), avoiding the shortcomings of traditional natural products that have "clear activity but unclear mechanism," and providing high-quality lead compounds for the development of drugs against acute promyelocytic leukemia.

[0036] The present invention has broad application prospects: It not only provides linear lipopeptide compounds, but also establishes a complete technical system from "strain fermentation-directional isolation-structural analysis-activity verification-mechanism research", which promotes the industrial application of marine actinomycete natural products in the field of anti-tumor. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the extraction process for compounds 1-5 and compounds 6-8.

[0038] Figure 2 This is a schematic diagram illustrating the structure of compound 1.

[0039] Figure 3 This is a schematic diagram illustrating the structure of compound 2.

[0040] Figure 4 This is a schematic diagram illustrating the structure of compound 3.

[0041] Figure 5 This is a schematic diagram illustrating the structure of compound 4.

[0042] Figure 6 This is a schematic diagram illustrating the structure of compound 5.

[0043] Figure 7 This is a schematic diagram of the synthesis process of compounds 6-8.

[0044] Figure 8 This is a schematic diagram showing the inhibitory effects of compounds 1-8 on ten tumor cell lines.

[0045] Figure 9 This is a schematic diagram illustrating the effect of compound 1 on the NB4 cell cycle.

[0046] Figure 10 This is a schematic diagram showing the results of compound 1 inducing apoptosis in NB4 cells.

[0047] Figure 11 This is a schematic diagram illustrating the effect of compound 1 on the PI3K / Akt / mTOR pathway.

[0048] Figure 12 It is compound 6. 1 Schematic diagram of H NMR (DMSO-d6, 600 MHz) spectrum.

[0049] Figure 13 It is compound 6. 13 Schematic diagram of C NMR (DMSO-d6, 150 MHz) spectrum.

[0050] Figure 14 It is compound 7. 1 Schematic diagram of H NMR (DMSO-d6, 600 MHz) spectrum.

[0051] Figure 15 It is compound 7. 13 Schematic diagram of C NMR (DMSO-d6, 150 MHz) spectrum.

[0052] Figure 16 It is compound 8. 1 Schematic diagram of H NMR (DMSO-d6, 600 MHz) spectrum.

[0053] Figure 17 It is compound 8. 13 Schematic diagram of C NMR (DMSO-d6, 150 MHz) spectrum. Detailed Implementation

[0054] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0055] To address the shortcomings of existing technologies, this invention provides a β-methylleucine linear lipopeptide compound derived from actinomycetes, along with its extraction method and applications. The aim is to efficiently separate and purify novel β-Me-Leu linear lipopeptides using neutral loss (NL) scanning-guided technology; to clarify their structural characteristics and structure-activity relationship; and to verify their cytotoxic activity and mechanism of action against acute promyelocytic leukemia (APML) cells, providing new lead compounds and technical support for the development of anti-APML drugs.

[0056] This invention employs a targeted screening strategy combining neutral loss (NL) scanning (monitoring 127 Da characteristic fragments) with LC-MS to address the difficulty in identifying trace amounts of β-Me-Leu lipopeptides in complex extracts, thereby improving separation efficiency. Using HRESIMS, 1D / 2D NMR, and the advanced Marfey method, the planar structure and absolute configuration of the compounds were clarified, and for the first time, the configuration of β-Me-Leu in cyanogriamides AE (compounds 1-5) was determined to be (2S, 3R). Using the obtained compounds 1-5, analogs replacing β-Me-Leu with Leu (compounds 6-8) were synthesized, confirming that β-Me-Leu and 4-methylvaleric acid are key groups for maintaining cytotoxic activity, elucidating the structure-activity relationship. This invention clarifies that compound 1 induces S-phase arrest and apoptosis in NB4 cells by inhibiting the PI3K / Akt / mTOR pathway, providing a mechanistic basis for its potential as a candidate drug against acute promyelocytic leukemia.

[0057] Preservation instructions:

[0058] Strain name: LHW52806;

[0059] Accession number: CCTCC NO M 20241051;

[0060] Category naming;

[0061] Deposit date: May 23, 2024;

[0062] Depository: China Center for Type Culture Collection;

[0063] Address of the depositary institution: Wuhan University, Wuhan, China.

[0064] Example 1

[0065] To identify potential target metabolites in the crude extract of *Heterothecia bluegrass* LHW52806, five characteristic ion peaks (m / z 832.5, 826.6, 818.5, 812.6, and 798.5) were screened using neutral loss (NL) 127 scanning mode mass spectrometry. Subsequently, target molecules were precisely located in the dichloromethane extract using liquid chromatography-mass spectrometry (LC-MS). After chromatographic separation and mass spectrometry-guided fractionation of the dichloromethane extract, five novel linear lipopeptides containing β-methylleucine were successfully isolated and named cyanogriamides AE (compounds 1-5). To further verify whether β-Me-Leu is a pharmacophore, compounds 6-8 were synthesized using a solid-phase / solution-phase synthesis method, in which β-Me-Leu in cyanogriamides AC (compounds 1-3) was replaced by Leu. Figure 1 ).

[0066] The specific steps for extracting actinomycete lipopeptides 1-5 from actinomycetes are as follows:

[0067] (1) Fermentation culture

[0068] The blue-gray heterotheca actinomycete LHW52806, an epiphytic species of brown flat sponge, was cultured in A1 liquid medium (yeast extract, 4 g; soluble starch, 10 g; peptone, 2 g; sea salt, 33 g; deionized water, 1 L, pH: 7.0) and statically cultured at 25°C for 21 days, yielding a total of 60 L of fermentation broth. The broth was extracted five times with an equal volume of ethyl acetate, and the extracts were concentrated under reduced pressure to obtain an ethyl acetate layer (40.0 g).

[0069] (2) Extraction and separation: The ethyl acetate layer was suspended in 90% methanol-water (v / v), and extracted 4 times with an equal volume of petroleum ether. The petroleum ether phase (15.3 g, containing a large amount of lipid impurities) was discarded. The remainder was diluted to 60% (v / v) with 90% methanol-water, and extracted 4 times with an equal volume of dichloromethane. The dichloromethane phases were combined and concentrated to obtain 3.0 g of dichloromethane extract. The target compound was confirmed to be concentrated in the dichloromethane extract by LC-MS (NL 127 Da scan).

[0070] Neutral loss scanning: Using neutral loss scanning 127 Da mass spectrometry, five ion peaks were detected in the NL127 ion channel at m / z 832.5, 826.6, 818.5, 812.6, and 798.5 [M + H]. + This allows us to obtain information about its fragment ions and chromatographic retention behavior.

[0071] (3) Column chromatography enrichment: Dichloromethane extract was loaded onto a Sephadex LH-20 column (2.6×100 cm), with methanol / dichloromethane (1:1, v / v) as the eluent, at a flow rate of 1.0 mL / min, and one fraction was collected for every 10 mL, for a total of 5 fractions (Fr.1-5); Fr.3 (containing the target compound) was loaded onto a medium-pressure liquid chromatograph (ODS column, 20×250 mm), and eluted with a gradient of 5%-100% methanol / water (v / v) (600 min, flow rate 20 mL / min), and detected by 210 nm UV, and 10 subfractions (Fr.3.AJ) were obtained.

[0072] (4) LC-MS-guided purification: Based on the obtained molecular weight information and chromatographic retention behavior, the fine fractions of the linear lipopeptide compounds (m / z 832.5, 826.6, 818.5, 812.6, 798.5) detected in the above positive ion mode were separated and purified using an LC-MS-guided liquid chromatography-mass spectrometry system. Semi-preparative RP-HPLC (Waters BEH C) was used. 18 Purification of the target subfraction using a column (5 μm, 10 × 250 mm): Fr. 3. C: Mobile phase was 30%-40% acetonitrile-water (containing 0.1% formic acid, v / v), flow rate 6 mL / min, t R The fraction obtained after 20.5 min was freeze-dried to give compound 1 (18.1 mg); t R The fraction obtained after 21.3 min yielded compound 2 (15.2 mg); Fr.3.E: the mobile phase was 45%-50% acetonitrile-water (containing 0.1% formic acid, v / v), the flow rate was 6 mL / min, and the fraction collected was... R The fraction obtained after 17.8 min yielded compound 3 (5.2 mg); t was collected. R The fraction obtained after 24.7 min yielded compound 4 (1.5 mg); t was collected. R The fraction after 20.2 min yielded compound 5 (3.1 mg).

[0073] The specific steps for synthesizing compounds 6-8 are as follows ( Figure 7 This is a schematic diagram of the synthesis process for compounds 6-8.

[0074] (1) Resin loading: 2-CTC resin (400 mg, 1.0 mmol / g) was swollen with anhydrous DC for 20 min, and a DCM solution of Fmoc-Leu-OH (3 equiv) and DIEA (6 equiv) was added. The mixture was vortexed at 25 °C for 3 h, quenched with MeOH for 15 min, and washed with DCM, DMF and MeOH in sequence.

[0075] (2) Fmoc deprotection and peptide chain coupling: Fmoc protection was removed by 20% piperidine / DMF (30 mL) for 30 min, followed by washing with DMF; Fmoc-protected amino acids (3 equiv) were activated in DMF with HATU (3 equiv), HOAt (6 equiv) and DIEA (6 equiv), added to resin and coupled under nitrogen flow at 25℃ for 1 h, followed by washing with DMF, and repeated until the peptide chain was complete;

[0076] (3) Cutting and purification: The resin-bound peptide was cut with 20% HFIP / DCM (30 mL) (twice, 10 min each time), concentrated and purified by LC-MS to obtain the side-chain protected peptide; TFA / H2O / TIS (95:2.5:2.5, v / v / v) was added and deprotected at room temperature for 2 h, and precipitated with ice-cold diethyl ether to obtain compound 8 (200 mg, yield 23%).

[0077] (4) Esterification and purification: Compound 8 was esterified with ethanol and methanol in EDCI (1.0 equiv) and DMAP (0.1 equiv), respectively, and the esterification was analyzed by preparative LC-MS (C 18 The column was 5 μm, 10 × 250 mm; the mobile phase was H2O and ACN; the elution gradient was ACN from 30% to 80% for 30 min; the flow rate was 6 mL / min. Compound 6 (33 mg, yield 85%) and compound 7 (34 mg, yield 87%) were purified.

[0078] The structures of linear lipopeptides are shown below:

[0079]

[0080] Figure 1 This is a schematic diagram of the extraction process for compounds 1-5 and compounds 6-8. Figure 2 This is a schematic diagram illustrating the structure of compound 1. Figure 3 This is a schematic diagram illustrating the structure of compound 2. Figure 4 This is a schematic diagram illustrating the structure of compound 3. Figure 5 This is a schematic diagram illustrating the structure of compound 4. Figure 6 This is a schematic diagram illustrating the structure of compound 5.

[0081] The physicochemical properties and NMR data of linear lipopeptides 1-8 are as follows:

[0082] Compound 1: a colorless gel-like substance; [α] 20 D -14.7 (c 0.05, MeOH); IR (micro-IR) νmax were 3284, 2958, 1732, 1635, 1538, 1466, 1351, 1209, 1156 and 768 cm⁻¹.-1 ; 1 H NMR (DMSO-d6, 600 MHz) and 13 CNMR (DMSO-d6, 150 MHz) data are shown in Table 1; High-resolution electrospray ionization mass spectrometry (HRESIMS) shows a quasi-molecular ion peak at m / z 826.5783 [M+H]. + (Corresponding molecular formula C) 40 H 76 N9O9 + The theoretical calculated value is 826.5766.

[0083] Compound 2: a colorless gel-like substance; [α] 20 D -24.7 (c 0.05, MeOH); IR (micro-IR) νmax were 3285, 2958, 1734, 1633, 1537, 1466, 1382, 1346 and 1210 cm⁻¹. -1 ; 1 H NMR (DMSO-d6, 600 MHz) and 13 CNMR (DMSO-d6, 150 MHz) data are shown in Table 2; High-resolution electrospray ionization mass spectrometry (HRESIMS) shows a quasi-molecular ion peak at m / z 812.5661 [M+H]. + (Corresponding molecular formula C) 39 H 74 N9O9 + The theoretical calculated value is 812.5650.

[0084] Compound 3: a colorless gel-like substance; [α] 20 D -10.7 (c 0.05, MeOH); IR (micro-IR) νmax were 3287, 2959, 1647, 1544, 1468, 1388 and 1161 cm⁻¹. -1 ; 1 H NMR (DMSO-d6, 600 MHz) and 13 The C10 NMR (DMSO-d6, 150 MHz) data are shown in Table 3; high-resolution electrospray ionization mass spectrometry (HRESIMS) shows a quasi-molecular ion peak at m / z 798.5460 [M+H]. + (Corresponding molecular formula C) 38 H 72 N9O9 + The theoretical calculated value is 798.5453.

[0085] Compound 4: a colorless gel-like substance; [α] 20D-24.0 (c 0.05, MeOH); IR (micro-IR) νmax were 3394, 3187, 2918, 2849, 1731, 1643, 1468, 1418, 1245 and 720 cm⁻¹. -1 ; 1 H NMR (DMSO-d6, 600 MHz) and 13 The C10 NMR (DMSO-d6, 150 MHz) data are shown in Table 4; high-resolution electrospray ionization mass spectrometry (HRESIMS) shows a quasi-molecular ion peak at m / z 832.5316 [M+H]. + (Corresponding molecular formula C) 41 H 70 N9O9 + The theoretical calculated value is 832.5296.

[0086] Compound 5: a colorless gel-like substance; [α] 20 D -6.0 (c 0.05, MeOH); IR (micro-IR) νmax were 3394, 3186, 2919, 2849, 1642, 1467, 1418, 1118 and 719 cm⁻¹. -1 ; 1 H NMR (DMSO-d6, 600 MHz) and 13 The C10 NMR (DMSO-d6, 150 MHz) data are shown in Table 5; high-resolution electrospray ionization mass spectrometry (HRESIMS) shows a quasi-molecular ion peak at m / z 818.5143 [M+H]. + (Corresponding molecular formula C) 40 H 68 N9O9 + The theoretical calculated value is 818.5140.

[0087] Compound 6: Colorless gelatinous substance; hydrogen nuclear magnetic resonance (NMR) 1 ¹H NMR, solvent DMSO-d6, 600 MHz) and carbon NMR ( 13 C NMR (solvent DMSO-d6, 150 MHz) data are as follows Figure 12 and Figure 13 As shown; High-resolution electrospray ionization mass spectrometry (HRESIMS) reveals a quasi-molecular ion peak at m / z 798.5479 [M + H]. + (Corresponding molecular formula C) 38 H 72 N9O9 + The theoretical calculated value is 798.5453. Figure 12 It is compound 6. 1Schematic diagram of H NMR (DMSO-d6, 600 MHz) spectrum. Figure 13 It is compound 6. 13 Schematic diagram of C NMR (DMSO-d6, 150 MHz) spectrum.

[0088] Compound 7: Colorless gelatinous substance; hydrogen nuclear magnetic resonance (NMR) 1 ¹H NMR, solvent DMSO-d6, 600 MHz) and carbon NMR ( 13 C NMR (solvent DMSO-d6, 150 MHz) data are as follows Figure 14 and Figure 15 As shown; High-resolution electrospray ionization mass spectrometry (HRESIMS) reveals a quasi-molecular ion peak at m / z 784.5314 [M + H]. + (Corresponding molecular formula C) 37 H 70 N9O9 + The theoretical calculated value is 784.5296. Figure 14 It is compound 7. 1 Schematic diagram of H NMR (DMSO-d6, 600 MHz) spectrum. Figure 15 It is compound 7. 13 Schematic diagram of C NMR (DMSO-d6, 150 MHz) spectrum.

[0089] Compound 8: Colorless gelatinous substance; hydrogen nuclear magnetic resonance (NMR) 1 ¹H NMR, solvent DMSO-d6, 600 MHz) and carbon NMR ( 13 C NMR (solvent DMSO-d6, 150 MHz) data are as follows Figure 16 and Figure 17 As shown; High-resolution electrospray ionization mass spectrometry (HRESIMS) reveals a quasi-molecular ion peak at m / z 770.5125 [M + H]. + (Corresponding molecular formula C) 36 H 68 N9O9 + The theoretical calculated value is 770.5140. Figure 16 It is compound 8. 1 Schematic diagram of H NMR (DMSO-d6, 600 MHz) spectrum. Figure 17 It is compound 8. 13 Schematic diagram of C NMR (DMSO-d6, 150 MHz) spectrum.

[0090] The planar structure of compound 1 was analyzed by 2D NMR (TOCSY, COSY, and HMBC) spectra (Table 1), which identified seven spin-coupled amino acid structural units: one ethoxy-β-methylleucine (OEt-β-Me-Leu), one glycine (Gly), one threonine (Thr), one arginine (Arg), one leucine (Leu), one β-methylleucine (β-Me-Leu), and one 4-methylvaleric acid (4-MPA) residue.

[0091] The sequence of the seven amino acid residues was determined by analyzing HMBC and ROESY related signals and ESI-MS / MS. Key HMBC related signals: CH3CH2O / CH3CH2O, CH3CH2O / β-Me-Leu1-CO, β-Me-Leu1-NH / Gly-CO, Gly-NH / β-Me-Leu2-CO, β-Me-Leu2-NH / Thr-CO, Thr-NH / Arg-CO, Arg-NH / Leu-CO, Leu-NH / 4-MPA-CO. ROESY related signals: β-Me-Leu1-NH / Gly-NH, Gly-NH / β-Me-Leu2-NH, β-Me-Leu2-NH / Thr-NH, Thr-NH / Arg-NH, Arg-NH / Leu-NH, Leu-NH / 4-MPA-2. The connection sequence was further verified by tandem mass spectrometry (MS / MS) experiments: a series of "b"-type fragment ions (m / z 653.4348, 596.4133, 469.3141, 368.2646, 212.2632) and "y"-type fragment ions (m / z 728.4987, 615.4202, 459.3183, 358.2690, 231.1700) appeared in the MS / MS spectrum, which correspond to OEt-β-Me-Leu, respectively. 1 Between 4-MPA, between Gly and Leu, and between β-Me-Leu 2 Between Arg, between Thr and Thr, and between Arg and β-Me-Leu 2 The amide bond between the two bonds was broken, and the structure of compound 1 was finally determined to be OEt-β-Me-Leu. 1 -Gly-β-Me-Leu 2 -Thr-Arg-Leu-4-MPA ( Figure 2 ).

[0092] Table 1: Nuclear magnetic resonance spectral data of compound 1 (DMSO-d6)

[0093]

[0094]

[0095] a Overlapping signals.

[0096] Compound 2 is an analogue of compound 1. 2D NMR (TOCSY, COSY and HMBC) spectra were analyzed (Table 2), and seven spin-coupled amino acid structural units were identified, namely one methoxy-β-methylleucine (OMe-β-Me-Leu), one glycine (Gly), one threonine (Thr), one arginine (Arg), one leucine (Leu), one β-methylleucine (β-Me-Leu), and one 4-methylvaleric acid (4-MPA) residue.

[0097] The sequence of the seven amino acid residues was determined by analyzing HMBC and ROESY related signals and ESI-MS / MS. The spectrum of compound 2 lacked the methylene signal (δC 60.5, δH 4.09×2) and the methyl signal (δC 14.0, δH 1.18×3), while a new methyl signal (δC 51.8, δH 3.63×3) was added, suggesting that the methoxy group (OMe) in compound 2 replaces the ethoxy group (OEt) in compound 1. The amino acid sequence of compound 2 was confirmed to be OMe-β-Me-Leu. 1 -Gly-β-Me-Leu 2 -Thr-Arg-Leu-4-MPA ( Figure 3 ).

[0098] Table 2: Nuclear magnetic resonance spectral data of compound 2 (DMSO-d6)

[0099]

[0100]

[0101] a Overlapping signals.

[0102] Compound 3 is a homologue of compound 2 (with one less CH2 unit in the molecule). Seven spin-coupled amino acid structural units were identified, namely two β-methylleucine residues (β-Me-Leu), one glycine residue (Gly), one threonine residue (Thr), one arginine residue (Arg), one leucine residue (Leu), and one 4-methylvaleric acid residue (4-MPA).

[0103] The 1D NMR data of compound 3 measured in DMSO-d6 (Table 3) were compared with those of compound 2, revealing the absence of methoxy groups (δC 51.8, δH 3.63×3) in the spectrum of compound 3. Characteristic 2D NMR correlation signals and HR-MS / MS fragment ion analysis confirmed that the amino acid linkage sequence of compound 3 is β-Me-Leu. 1 -Gly-β-Me-Leu 2 -Thr-Arg-Leu-4-MPA ( Figure 4 ).

[0104] Table 3: Nuclear magnetic resonance spectral data of compound 3 (DMSO-d6)

[0105]

[0106]

[0107] an overlapping signal.

[0108] Compound 4 was analyzed by 2D NMR (TOCSY, COSY, and HMBC) spectra, which identified seven spin-coupled amino acid structural units: methoxy-β-methylleucine (OMe-β-Me-Leu), one glycine (Gly), one threonine (Thr), one arginine (Arg), one leucine (Leu), one β-methylleucine (β-Me-Leu), and one 2-phenylacetic acid (2-PAA) residue.

[0109] NMR data of compound 4 obtained in DMSO-d6 (Table 4) were compared with those of compound 2, revealing that the 4-methylpentanoic acid (4-MPA) residue in compound 4 was substituted with a 2-phenylacetic acid (2-PAA) residue. The amino acid linkage sequence of compound 4 was determined to be OMe-β-Me-Leu using key 2D NMR correlation signals. 1 -Gly-β-Me-Leu 2 -Thr-Arg-Leu-2-PAA, this sequence was further verified by HR-MS / MS fragment ions ( Figure 5 ).

[0110] Table 4: Nuclear magnetic resonance spectral data of compound 4 (DMSO-d6)

[0111]

[0112]

[0113] a Overlapping signals.

[0114] The 1D NMR data of compound 5 measured in DMSO-d6 (Table 5) were compared with those of compound 4, revealing the absence of a methoxy group signal in the spectrum of compound 5 (δC 51.9, δH 3.63×3). 2D NMR correlation signal and HR-MS / MS fragment ion analysis confirmed that the amino acid linkage sequence of compound 5 is β-Me-Leu1-Gly-β-Me-Leu2-Thr-Arg-Leu-2-PAA (…). Figure 6 ).

[0115] Table 5: Nuclear magnetic resonance spectral data of compound 5 (DMSO-d6)

[0116]

[0117]

[0118] an overlapping signal.

[0119] The absolute configurations of compounds 1–5 were determined using the advanced Marfey method. Figure 2 This is a schematic diagram illustrating the structure of compound 1, in which the stereoconfigurations of all its amino acids β-Me-Leu, Leu, Thr and Arg are (2S,3R)-β-Me-Leu, L-Leu, L-Thr and D-Arg, respectively. Figure 3 This is a schematic diagram illustrating the structure of compound 2, in which the stereoconfigurations of all its β-Me-Leu, Leu, Thr and Arg are (2S,3R)-β-Me-Leu, L-Leu, L-Thr and D-Arg, respectively. Figure 4 This is a schematic diagram illustrating the structure of compound 3, in which the stereoconfigurations of all its β-Me-Leu, Leu, Thr and Arg are (2S,3R)-β-Me-Leu, L-Leu, L-Thr and D-Arg, respectively. Figure 5 The structural diagram of compound 4 shows that the stereoconfigurations of all its β-Me-Leu, Leu, Thr and Arg are (2S,3R)-β-Me-Leu, L-Leu, L-Thr and D-Arg, respectively. Figure 6 This is a schematic diagram illustrating the structure of compound 5, in which the stereoconfigurations of all its β-Me-Leu, Leu, Thr, and Arg are (2S,3R)-β-Me-Leu, L-Leu, L-Thr, and D-Arg, respectively.

[0120] Compound 6: Compared with compound 1, the structural difference of compound 6 is that the two methyl signals (δC 11.5×2, δH 0.80×6) are replaced by methylene signals (δC / δH 40.5 / 1.49, 40.2 / 1.54), and its amino acid linkage sequence is determined to be OEt-L-Leu1-Gly-L-Leu2-L-Thr-D-Arg-L-Leu3-4-MPA.

[0121] Compound 7: NMR analysis showed that the key difference between compound 7 and compound 2 was the absence of two methyl signals (δC 11.6, δH 0.78×3; δC 11.1, δH 0.80×3), while two methylene signals were present (δC / δH 40.5 / 1.48, 40.1 / 1.55). Its amino acid linkage sequence was determined to be OMe-L-Leu1-Gly-L-Leu2-L-Thr-D-Arg-L-Leu3-4-MPA.

[0122] Compound 8: 1D NMR data analysis showed that, compared with compound 3, the structural difference of compound 8 is that the two methyl signals (δC / δH 12.1 / 0.68×3, 12.0 / 0.78×3) in compound 3 are replaced by two methylene signals (δC / δH 40.5 / 1.48, 40.2 / 1.61) in compound 8, and its amino acid linkage sequence is determined to be L-Leu1-Gly-L-Leu2-L-Thr-D-Arg-L-Leu3-4-MPA.

[0123] Example 2

[0124] Cytotoxicity experiments of compounds 1-8 prepared in this invention

[0125] The Cell Titer-Glo (CTG) assay was used to evaluate the in vitro cell viability effects of compounds 1–8 on 10 human cancer cell lines (NB4, PC9, PC3, Daudi, HT-29, A2780, U-937, HepG2, Dsrct, RH30) and 1 human keratinocyte cell line (HaCaT, normal control cell).

[0126] Culture medium selection: HT-29 (human colon cancer cells), A2780 (human ovarian cancer cells), HepG2 (human liver cancer cells), Dsrct (de novo cytotoxic small round cell tumor cells), and HaCaT (human normal keratinocytes) were cultured in DMEM medium; NB4 (human acute promyelocytic leukemia cells), PC9 (human non-small cell lung cancer cells), PC3 (human prostate cancer cells), Daudi (human Burkitt lymphoma cells), U-937 (human histiocytic lymphoma cells), and RH30 (human rhabdomyosarcoma cells) were cultured in RPMI-1640 medium.

[0127] Culture medium supplements: All culture media were supplemented with 10% fetal bovine serum (FBS), 1% antibiotics and 1% sodium pyruvate.

[0128] Culture environment: Cells were cultured in a constant temperature incubator at 37°C with 5% CO2.

[0129] Sample preparation and dosing: Test compounds 1-8 were dissolved in dimethyl sulfoxide (DMSO) and stored at low temperature. During the experiment, working solutions of various concentrations were prepared by serial dilution to ensure that the highest concentration of DMSO in the system did not affect cell viability.

[0130] Cell seeding and drug administration: Cells in the logarithmic growth phase are seeded at approximately 1 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of cells / well into 96-well plates. After adding cell suspension to each well, the plates were pre-cultured in an incubator. Then, different concentrations of the test compound were added, and the drug treatment time was 48 h.

[0131] Activity assay: After 48 h of drug treatment, 10 μL of CTG detection solution was added to each well, and the mixture was incubated at 37℃ and 5% CO2 for another 1 h. The absorbance (OD value) of each well was measured at 450 nm using a SpectraMax 190 microplate reader.

[0132] Data calculation: GraphPad Prism 9.0 software was used to calculate the half-maximal inhibitory concentration (IC50) of cell growth through linear regression analysis. 50 Value), IC 50 The value represents the drug concentration required to inhibit 50% of cell growth.

[0133] Figure 8 This is a schematic diagram illustrating the inhibitory effects of compounds 1-8 on ten tumor cell lines. The results show that compound 1 exhibited significant growth-inhibiting activity against all tested cancer cells, with potency comparable to the positive control cisplatin. Compound 1 showed an IC50 inhibitory effect on NB4 cells. 50 =0.5±0.06 μM (superior to cisplatin, IC50) 50Compound 1 (2.2 ± 0.16 μM) showed a selectivity index (SI) of 6.3 between HaCaT and NB4 cell lines, indicating good selectivity for NB4 cells. Compound 2 showed moderate cytotoxic activity against NB4, PC9, A2780, U-937, HepG2, and RH30 cells, with an IC50 concentration of 2.2 ± 0.16 μM. 50 The values ​​ranged from 2.53 ± 0.28 to 19.33 ± 0.69 μM. Compound 3 exhibited relatively selective growth-inhibiting activity against NB4 and PC9, with IC50 values ​​of 2.53 ± 0.28 to 19.33 ± 0.69 μM. 50 The values ​​were 12.88 ± 0.92 and 14.71 ± 0.83 μM. Compounds 4–8, at the highest tested concentration of 20 μM, showed IC50 values ​​against all 10 tumor cell lines. 50 All values ​​were greater than 20 μM, indicating that the cell inhibition rate at this concentration did not reach 50%.

[0134] Mechanism study of compound 1 of this invention:

[0135] Cell cycle analysis (PI staining method)

[0136] Cell treatment: NB4 cells (2×10⁻⁶) 5 Inoculate 6-well plates with 0, 0.5, 1.0, and 2.0 μM compound 1 for 48 h (3 replicates / group) and incubate at 37°C and 5% CO2.

[0137] Cell collection and fixation: Collect cells by centrifugation at 300×g for 5 min, wash twice with PBS, and fix overnight at 4℃ with 70% ethanol.

[0138] Staining and detection: RNA was degraded by incubation of RNase A (100 μg / mL) at 37℃ for 30 min, and PI (50 μg / mL) was stained at 25℃ in the dark for 30 min. Thermo Fisher flow cytometry was used for detection. CDK2 and Cyclin A2 proteins were detected by Western blot (primary antibody 1:1000, CST; secondary antibody 1:3000).

[0139] result: Figure 9 This is a schematic diagram illustrating the effect of compound 1 on the cell cycle of NB4 cells. Cell cycle: The proportion of cells in S phase increased from 50.65% in the control group to 68.80% in the 2 μM group, and the dose-dependent effect on G0 / G1 phase decreased.

[0140] Protein expression: CDK2 and Cyclin A2 were downregulated in a dose-dependent manner, confirming that compound 1 induced S-phase arrest.

[0141] Apoptosis detection (Annexin V-FITC / PI double staining method)

[0142] Cell treatment: NB4 cells were treated with 0, 0.5, 1.0, and 2.0 μM compound 1 for 48 h (3 replicates / group).

[0143] Staining detection: Collect cells by centrifugation at 300×g, wash twice with PBS, resuspend in 300 μL binding buffer, add 5 μL Annexin V-FITC and PI, incubate at 25℃ in the dark for 15 min, and detect by flow cytometry.

[0144] Protein validation: Western blot detection of Bax, Bcl-2, cleaved caspase-9 / 3, and PARP (primary antibody 1:1000, CST).

[0145] result: Figure 10 This is a schematic diagram showing the results of compound 1 inducing apoptosis in NB4 cells. Apoptosis rate: The total apoptosis rate in the 10 μM group reached 51.04% (compared to only 7.85% in the control group), showing a dose-dependent increase. Protein changes: Bax was upregulated, Bcl-2 was downregulated, and cleaved caspase-9 / 3 and PARP were significantly activated (p<0.001), confirming the induction of mitochondrial-dependent apoptosis.

[0146] PI3K / Akt / mTOR pathway detection (Western blot method)

[0147] Cell treatment: NB4 cells were treated with 0, 0.5, 1.0, and 2.0 μM compound 1 for 24 h (3 replicates / group).

[0148] Protein detection: Total protein was extracted with RIPA lysis buffer, quantified by BCA, separated by SDS-PAGE, and transferred to NC membrane; blocked with 5% skim milk for 1 h, added primary antibody (total PI3K, p-PI3K, etc., 1:1000, CST) and incubated overnight at 4℃, then incubated with secondary antibody (1:3000) at room temperature for 2 h, and developed by ECL.

[0149] result: Figure 11 This is a schematic diagram illustrating the effect of compound 1 on the PI3K / Akt / mTOR pathway. Phosphorylated proteins: p-PI3K, p-Akt, and p-mTOR protein levels were dose-dependently downregulated (2 μM group decreased to 28%, 21%, and 23% of the control, respectively, p<0.001). Total protein: There was no difference in total PI3K, Akt, and mTOR expression (p>0.05), confirming that compound 1 inhibits the PI3K / Akt / mTOR pathway.

[0150] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A linear lipopeptide compound, characterized in that, The structure is selected from one of the following: 。 2. A method for extracting the linear lipopeptide compound according to claim 1, characterized in that, Includes the following steps: (1) Fermentation culture The blue-gray heterotheca actinomycete LHW52806, which is epiphytic on brown flat sponges, was cultured in A1 liquid medium and statically cultured at room temperature for 20-30 days to obtain the fermentation broth; it was extracted 3-6 times with an equal volume of ethyl acetate, and the extract was concentrated under reduced pressure to obtain the ethyl acetate layer. (2) Extraction and separation: The ethyl acetate layer was suspended in 90% methanol and water, and extracted 3 to 6 times with an equal volume of petroleum ether. The petroleum ether phase was discarded. The remaining phase was diluted to 60% with 90% methanol and water, and extracted 3 to 6 times with an equal volume of dichloromethane. The dichloromethane phases were combined and concentrated to obtain a dichloromethane extract. The target compound was found to be concentrated in the dichloromethane extract by LC-MS. (3) Column chromatography enrichment: Dichloromethane extract was loaded onto a Sephadex LH-20 column and eluted with methanol / dichloromethane to obtain 5 fractions Fr.1-5; Fr.3 was loaded onto a medium-pressure liquid chromatograph and eluted with a gradient of 5%-100% methanol / water and detected by UV at 210 nm to obtain 10 subfractions Fr.3.AJ; (4) LC-MS-guided purification: The target subfraction was purified by semi-preparative RP-HPLC: Fr.3.C: the mobile phase was 30%-40% acetonitrile-water containing 0.1% formic acid, the flow rate was 6 mL / min, and t was collected. R The fraction obtained after 20.5 min was freeze-dried to give compound 1; t was collected. R The fraction obtained after 21.3 min yielded compound 2; Fr.3.E: the mobile phase was 45%-50% acetonitrile-water containing 0.1% formic acid, the flow rate was 6 mL / min, and the fraction collected was t. R The fraction obtained after 17.8 min yielded compound 3; t was collected. R The fraction obtained after 24.7 min yielded compound 4; t was collected. R The fraction obtained after 20.2 min yielded compound 5.

3. The method for extracting lipopeptide compounds according to claim 2, characterized in that, The composition of the A1 liquid culture medium is as follows: yeast extract, 4 g; soluble starch, 10 g; peptone, 2 g; sea salt, 33 g; deionized water, 1 L, pH: 7.

0.

4. The method for extracting lipopeptide compounds according to claim 2, characterized in that, The neutral loss scan: Using neutral loss scan 127 Da mass spectrometry, five ion peaks were detected in the NL127 ion channel at m / z 832.5, 826.6, 818.5, 812.6, and 798.5 [M + H]. + This allows us to obtain information about its fragment ions and chromatographic retention behavior.

5. A method for preparing the linear lipopeptide compound according to claim 1, characterized in that, Includes the following steps: (1) Resin loading: 2-CTC resin was swollen with anhydrous DC for 10~30 min, and then a DCM solution of Fmoc-Leu-OH and DIEA was added. The molar ratio of 2-CTC resin, Fmoc-Leu-OH and DIEA was 1:2~4:2~8. The mixture was vortexed at room temperature for 1~5 h, quenched with MeOH, and washed with DCM, DMF and MeOH in sequence. (2) Fmoc deprotection and peptide chain coupling: Fmoc protection was removed with 20% piperidine / DMF and washed with DMF; Fmoc-protected amino acids were activated in DMF with HATU, HOAt, and DIEA, added to resin and coupled under nitrogen flow at room temperature for 0.5-2 h, washed with DMF, and repeated until the peptide chain was complete; (3) Cutting and purification: The resin-bound peptide was cut with 20% HFIP / DCM, concentrated and purified by LC-MS to obtain the side-chain protected peptide; TFA / H2O / TIS was added to deprotect at room temperature, precipitated with ice-cold ether, and purified by LC-MS to obtain compound 8; (4) Esterification and purification: Compound 8 was esterified with ethanol and methanol under the action of EDCI and DMAP, respectively, and purified by preparative LC-MS to obtain compounds 6 and 7; 。 6. The use of the lipopeptide compound of claim 1 in the preparation of a medicament for treating tumors.

7. The application according to claim 6, characterized in that, The tumors are selected from colon cancer, ovarian cancer, liver cancer, desmoplastic small round cell tumor, leukemia, non-small cell lung cancer, prostate cancer, lymphoma, and rhabdomyosarcoma.

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