Cyclic peptide compounds extracted from sponges and methods of making and using the same

Abstract

The application discloses a cyclic peptide compound extracted from a sponge, and the structure is selected from one of the following compounds 1-4: the application also provides application of the cyclic peptide compound in preparation of an antitumor drug. The cyclic peptide compound shows strong growth inhibition activity on human lung cancer cells NCI-H460. The application provides a new lead compound for research and development of a new antitumor drug, and provides a scientific basis for development and utilization of marine medicinal resources in China.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a cyclic peptide compound extracted from sponges, its preparation method, and its application. Background Technology

[0002] Cyclic peptides possess a wide range of diverse biochemical and therapeutic properties and have achieved significant success in drug therapy. Of the more than 60 peptide drugs approved by the FDA and EMA, two-thirds are cyclic peptides. Notably, the marine environment fosters a diverse array of structurally diverse and biologically active cyclic peptides. Plitidepsin, one of the most representative marine-derived molecules, was approved by the EMA in 2018 for the treatment of relapsed / refractory multiple myeloma and has recently been identified as a potential therapeutic candidate for COVID-19.

[0003] Sponges have long been an important source of bioactive cyclic peptides in the ocean. Literature reports that sponges rich in cyclic peptides mainly include genera such as *Phakellia*, *Hymeniacidon*, *Stylissa*, and *Callyspongia*. Studies of their chemical composition have ultimately yielded cyclic peptides of types such as phakellistatins, hymenamides, stylissamides, and callyaerins. Interestingly, these cyclic peptides share common structural features: they are rich in proline, with leucine and / or isoleucine making up a large proportion of their structure. Further structure-activity analysis revealed that leucine and / or isoleucine often link with proline to form Pro-Leu / Ile fragments, and cyclic peptides with more Pro-Leu / Ile fragments tend to exhibit better cytotoxic activity. Based on the characteristic of cyclic peptides losing fixed neutral fragment ions, the powerful precursor ion scanning function of triple quadrupole mass spectrometry (QQQ) can facilitate targeted research and directed acquisition of cyclic peptides containing Pro-Leu / Ile fragments, thereby efficiently discovering potential new antitumor lead compounds and accelerating the development of marine drugs. Summary of the Invention

[0004] The first objective of this invention is to provide a cyclic peptide compound extracted from a sponge.

[0005] A second objective of this invention is to provide a method for preparing the cyclic peptide compound extracted from the sponge.

[0006] A third objective of this invention is to provide the application of the cyclic peptide compound extracted from the sponge in the preparation of an antitumor drug.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a novel cyclic peptide compound extracted from a sponge, the structure of which is selected from one of the following compounds 1-4:

[0009]

[0010] The sponge in question refers to the sponge sample Reniocharina sp.

[0011] A second aspect of the present invention provides a method for extracting the cyclic peptide compounds extracted from the sponge, comprising the following steps:

[0012] The first step, sample screening: Addressing the characteristic of cyclic peptide compounds losing fixed neutral fragments, a rapid localization method for selectively identifying characteristic structures of cyclic peptides in crude extracts was established by combining the PI scanning function of LC-QTRAP-MS / MS with IDA-triggered EPI scanning technology. This method allowed for the screening of sponge samples rich in cyclic peptide compounds, specifically Reniocharinasp., from the sponge sample library.

[0013] The second step is solvent extraction: After cutting the sponge sample Reniochalina sp. selected in the first step into pieces, add equal volumes of MeOH and CH2Cl2 / MeOH with a volume ratio of 1:1 respectively for ultrasonic extraction at least three times. Combine the extracts and concentrate under reduced pressure to obtain crude extract.

[0014] The third step is stepwise extraction: First, the crude extract prepared in the second step is suspended in 90% methanol-water and extracted 3 to 5 times with an equal volume of petroleum ether. Then, the 90% methanol-water is diluted to 60% methanol-water and extracted 3 to 5 times with an equal volume of dichloromethane. The extracts are combined and concentrated under reduced pressure to obtain the dichloromethane extract.

[0015] Step 4, Separation and Enrichment: The dichloromethane extract prepared in Step 3 was subjected to Sephadex LH-20 gel column chromatography. The first eluent was CH2Cl2 / MeOH at a volume ratio of 1:1, and the second eluent was n-hexane / CH2Cl2 / MeOH at a volume ratio of 4:5:1. High molecular weight compounds were enriched by mass spectrometry localization tracking. ODS medium-pressure column chromatography was used for separation, with a gradient elution of 10%-100% MeOH / H2O. Mass spectrometry localization analysis was used to obtain a series of fine fractions containing high molecular weight cyclic peptides.

[0016] Step 5: Screening target compounds: using m / z 211 (C 11 H 19N2O2 was used as a characteristic fragment ion of the Pro-Leu / Ile fragment. The fine fraction containing high molecular weight cyclic peptides prepared in the fourth step was analyzed by the parent ion scanning mass spectrometry method. Target compounds containing the Pro-Leu / Ile fragment were screened out. Combining molecular weight information and chromatographic retention behavior, the fine fraction containing the target compound was located by mass spectrometry analysis.

[0017] Step 6, mass spectrometry-guided separation: The fine fraction prepared in step 5 is separated by mass spectrometry-guided semi-preparative high-performance liquid chromatography to obtain the target compound.

[0018] In the sixth step, the separation conditions for mass spectrometry-guided semi-preparative high-performance liquid chromatography are as follows: Compound 1, 50-60% acetonitrile-water, flow rate 6.0 mL / min, molecular ion m / z 941.59 detected in positive ion mode; Compounds 2 and 3, 20-23% acetonitrile-water, flow rate 6.0 mL / min, molecular ion m / z 724.37 and 770.41 detected in positive ion mode, respectively; Compound 4, 28-35% acetonitrile-water, flow rate 6.0 mL / min, molecular ion m / z 794.48 detected in positive ion mode.

[0019] A third aspect of the invention provides the use of the cyclic peptide compound extracted from the sponge in the preparation of an antitumor drug.

[0020] The tumors mentioned are lung cancer, ovarian cancer, colon cancer, and liver cancer.

[0021] The tumor cells are human ovarian cancer cells A2780, human colon cancer cells HCT-8, human lung cancer cells NCI-H460, human colon cancer cells SW480, human lung cancer cells PC-9, and human liver cancer cells HepG2.

[0022] The cyclic peptide compounds exhibit strong inhibitory activity against human lung cancer cells NCI-H460, and therefore can be used to prepare antitumor drugs.

[0023] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0024] The medicinal material of this invention is the sponge *Reniochalina* sp., belonging to the family Axinellidae of the order Halichondrid in the class Demospongiae. Its unique living environment enables it to produce novel and highly active secondary metabolites. The cyclic peptide compounds provided by this invention are derived from *Reniochalina* sp. and are separated using a pre-ion scanning mass spectrometry and LC-MS-guided programmed separation method, which is highly efficient and simple. The cyclic peptide compounds of this invention exhibit significant inhibitory activity against human lung cancer cells NCI-H460. In summary, this invention provides new lead compounds for the research and development of novel antitumor drugs, offers a new strategy for the rapid identification and targeted tracking of trace cyclic peptide active ingredients in sponges, and provides a scientific basis for the development and utilization of my country's marine medicinal resources. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the discovery process of cyclic peptide compounds 1-4.

[0026] Figure 2 This is a schematic diagram illustrating the structure of cyclic peptide compound 1.

[0027] Figure 3 This is a schematic diagram illustrating the structure of cyclic peptide compound 2.

[0028] Figure 4 This is a schematic diagram illustrating the structure of cyclic peptide compound 3.

[0029] Figure 5 This is a schematic diagram illustrating the structure of cyclic peptide compound 4.

[0030] Figure 6 This is a schematic diagram illustrating the in vitro proliferation inhibitory effects of the cyclic peptide compounds 1-4 of the present invention on six human tumor cell lines. Detailed Implementation

[0031] 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.

[0032] Example 1

[0033] Discovery and extraction of sponge cyclic peptide compounds 1-4 from sponges

[0034] A sample of *Reniochalina* sp., rich in cyclic peptides, was obtained from a sponge library using precursor ion scanning. The sponge (54.8 g dry weight) was cut into pieces and extracted multiple times with an organic solvent using ultrasonication. The extracts were combined and concentrated under reduced pressure to obtain a crude extract. The crude extract was then subjected to stepwise extraction, gel column chromatography, and ODS medium-pressure column chromatography to obtain several fine fractions. Precursor ion scanning analysis was performed on the dichloromethane fraction rich in cyclic peptides to obtain a series of target cyclic peptides containing the Pro-Leu / Ile fragment. Combining molecular weight information and chromatographic retention behavior, mass spectrometry was used to locate the fine fraction containing the target compounds, and mass spectrometry-guided semi-preparative high-performance liquid chromatography (HPLC) was used to finally separate the sponge cyclic peptides.

[0035] The specific steps for extracting sponge cyclic peptide compounds 1-4 from sponges are as follows:

[0036] The first step, sample screening: Addressing the characteristic of cyclic peptide compounds losing fixed neutral fragments, a rapid localization method was established by combining the PI scanning function of LC-QTRAP-MS / MS with IDA-triggered EPI scanning technology to selectively identify characteristic structures of cyclic peptides in crude extracts. This method allowed for the screening of sponge samples rich in cyclic peptide compounds from a sponge sample library. This particular sample, collected in April 2021 from the waters near the Yongle Islands in the South China Sea (depth 25-30m), was orange-yellow in color and relatively hard. Dr. Qi Yang from the Marine Biological Products Development Center at Flinders University identified it as *Reniochalina* sp.

[0037] The second step, solvent extraction: The sponge sample Reniochalina sp. (dry weight 54.8g) selected in the first step was cut into pieces and then subjected to ultrasonic extraction three times with equal volumes of MeOH and CH2Cl2 / MeOH (v / v = 1:1). The extracts were combined and concentrated under reduced pressure to obtain crude extract (23.7g).

[0038] The third step is stepwise extraction: First, the crude extract prepared in the second step is suspended in 250 mL of 90% methanol-water and extracted 3 to 5 times with an equal volume of petroleum ether. Then, the 90% methanol-water is diluted to 60% methanol-water and extracted 3 to 5 times with an equal volume of dichloromethane. The extracts are combined and concentrated under reduced pressure to obtain the dichloromethane extract (1.2 g).

[0039] Step 4, Separation and Enrichment: The dichloromethane extract prepared in Step 3 was subjected to Sephadex LH-20 gel column chromatography. The first eluent was CH2Cl2 / MeOH (v / v = 1:1), and the second eluent was n-hexane / CH2Cl2 / MeOH (v / v / v = 4:5:1). High molecular weight compounds were enriched by mass spectrometry localization and tracking, yielding 600 mg. ODS medium-pressure column chromatography was used for separation, with a 10%-100% MeOH / H2O gradient elution. Mass spectrometry localization and tracking analysis was used to obtain a series of fine fractions containing high molecular weight cyclic peptide compounds.

[0040] Step 5: Screening target compounds: using m / z 211 (C 11 H 19 N2O2 was used as a characteristic fragment ion of the Pro-Leu / Ile fragment. The fine fraction containing high molecular weight cyclic peptides obtained in the fourth step was analyzed by parent ion scanning mass spectrometry. Target compounds containing the Pro-Leu / Ile fragment were screened out. Combining molecular weight information and chromatographic retention behavior, the fine fraction containing the target compound was located by mass spectrometry analysis.

[0041] Step 6, Mass-Spectrometry-Guided Separation: The fine fraction prepared in Step 5 was separated using mass-spectrum-guided semi-preparative high-performance liquid chromatography (HPLC) to obtain target compounds 1-4. The separation conditions for each compound are as follows:

[0042] Compound 1, 50-60% acetonitrile-water, flow rate 6.0 mL / min, molecular ion m / z 941.59 was detected in positive ion mode; Compounds 2 and 3, 20-23% acetonitrile-water, flow rate 6.0 mL / min, molecular ion m / z 724.37 and 770.41 were detected in positive ion mode, respectively; Compound 4, 28-35% acetonitrile-water, flow rate 6.0 mL / min, molecular ion m / z 794.48 was detected in positive ion mode.

[0043] Figure 1 This is a schematic diagram illustrating the discovery process of cyclic peptide compounds 1-4. As can be seen from the diagram, the precursor ion scanning mass spectrometry method established in this invention is accurate and effective, successfully and accurately identifying target cyclic peptides containing the Pro-Leu / Ile fragment from crude sponge extracts. Combined with mass spectrometry-guided separation techniques, it can efficiently and directionally track target cyclic peptides.

[0044] The structures of compounds 1-4 are shown below:

[0045]

[0046] The physicochemical properties and NMR data of the sponge cyclic peptide compounds 1-4 obtained through the above steps are as follows:

[0047] Compound 1: White amorphous powder; [α] 25 D -75.5(c 0.22, MeOH); IR(ATR)ν max 3297, 2953, 2869, 1620, 1514, 1435, 1384, 1239, 698cm -1 ; 1 H and 13 The C NMR data are shown in Table 1; the ESIMS / MS data are shown in Table 2. Figure 2 As shown in C; HRESIMS m / z 941.5872 [M+H] + (calcd for C 52 H 76 N8O8, 941.5864).

[0048] Compound 2: Pale yellow amorphous powder; [α] 25 D -62.4(c 0.25, MeOH); IR(ATR)ν max 3275, 2953, 1625, 1515, 1446, 1385, 1237, 512cm -1 ; 1 H and 13 The C NMR data are shown in Table 2; the ESIMS / MS data are shown in Table 2. Figure 3 As shown in C; HRESIMS m / z 724.3676 [M+H] + (calcd for C 36 H 49 N7O9, 724.3670).

[0049] Compound 3: Pale yellow amorphous powder; [α] 25 D -70.0(c 0.22, MeOH); UV(MeOH)λ max 279nm; IR(ATR)ν max 3396, 2956, 2873, 1625, 1520, 1448, 1244, 1201, 1161, 1026, 702, 538cm -1 ; 1 H and 13 The C NMR data are shown in Table 3; the ESIMS / MS data are shown in Table 4. Figure 4 As shown in C; HRESIMS m / z 770.4084 [M+H] + (calcdfor C38 H 55 N7O 10 ,770.4089).

[0050] Compound 4: Pale yellow amorphous powder; [α] 25 D -83.0(c 0.30, MeOH); UV(MeOH)λ max 279nm; IR(ATR)ν max 3239, 2956, 2873, 1636, 1515, 1446, 1345, 1241, 1026cm -1 ; 1 H and 13 CNMR data are shown in Table 4; ESIMS / MS data are shown in Table 4. Figure 5 As shown in C; HRESIMS m / z 794.4824 [M+H] + (calcd for C 42 H 63 N7O8, 794.4816).

[0051] The NMR spectral data of sponge cyclic peptide compounds 1-4 are shown in Tables 1, 2, 3 and 4, respectively.

[0052] Table 1: NMR spectral data of cyclic peptide compound 1 (DMSO-d6)

[0053]

[0054]

[0055]

[0056] Detailed analysis of 2D NMR (COSY, TOCSY, and HMBC) spectra revealed that the eight amino acid residues constituting compound 1 include proline (2 residues), phenylalanine (2 residues), isoleucine (2 residues), and leucine (2 residues). The sequence of these amino acid residues was determined through careful analysis of HMBC, ROESY, and ESI-MS / MS mass spectrometry data. Specifically, the HMBC-related signal, such as Leucine, was used to determine the amino acid residue sequence. 1 -NH / Pro 2 -CO、Ile 1 -NH / Pro 1 -CO、Phe 1 -NH / Phe 2 -CO、Leu 2 -NH / Phe 1 -CO、Ile 2-NH / Leu 2 -CO and Phe 2 -NH / Leu 1 -CO can identify two structural segments: Ile 1 -Pro 1 and Ile 2 -Leu 2 -Phe 1 -Phe 2 -Leu 1 -Pro 2 According to ROESY related signals such as Ile 1 -Hα / Pro 2 -Hδ and Ile 2 -Hα / Pro 1 -Hδ, thus determining the structure of compound 1 as cyclo-(Pro 1 -Ile 1 -Pro 2 -Leu 1 -Phe 2 -Phe 1 -Leu 2 -Ile 2 This result was validated by ESI-MS / MS mass spectrometry data. The Δδ values ​​of the two proline residues... Cβ-Cγ Both have small Δδ Cβ-Cγ Value, where Pro 1 It's 4.8, Pro 2 The value of 3.9 indicates that they are all in the trans conformation. The molecular weight and retention time (t) of the amino acid derivative of compound 1 of this invention were compared using the advanced Marfey method with those of the standard derivative and the compound. R To determine the chirality of amino acid residues, the results showed that all amino acid residues in compound 1 were chiral. L - Configuration. A schematic diagram illustrating the structure of compound 1, a cyclic peptide of the present invention, is shown below. Figure 2 As shown, Figure 2 This is a schematic diagram illustrating the structure of cyclic peptide compound 1. In the diagram, A represents the key 2D NMR signal, B represents the MS / MS fragmentation mode, C represents the HRESI-MS / MS spectrum, and D represents the advanced Marfey method based on LC-MS.

[0057] Table 2: NMR spectral data of cyclic peptide compound 2 (DMSO-d6)

[0058]

[0059]

[0060]

[0061] Detailed analysis of 2D NMR (COSY and HMBC) spectra revealed that the seven amino acid residues constituting compound 2 include leucine (1), aspartic acid (1), proline (3), glycine (1), and phenylalanine (1). The sequence of these amino acid residues was determined through careful analysis of HMBC, COSY correlation signals, and ESI-MS / MS mass spectrometry data. Specifically, the HMBC correlation signal, such as Gly-NH / Pro... 2 -CO, Asp-NH / Gly-CO, Leu-NH / Pro 1 -CO and Phe-NH / Leu-CO can identify two structural segments: Asp-Gly-Pro 2 and Phe-Leu-Pro 1 Based on ROESY related signals such as Asp-Hα / Pro 3 -Hδ、Pro 1 -Hδ / Pro 3 -Hα and Phe-Hα / Pro 2 -Hα, thus determining the structure of compound 2 as cyclo-(Pro 1 -Leu-Phe-Pro 2 -Gly-Asp-Pro 3 This result was validated by ESI-MS / MS mass spectrometry data. The Δδ of proline was used to determine the Δδ Cβ-Cγ The value determines its cis-trans conformation, where Pro 1 For 5.0, Pro 3 The value of 3.3 indicates that they are in the trans conformation, while Pro... 2 A value of 10.0 indicates that it is in the cis conformation. The molecular weight and retention time (t) of the standard derivative and the amino acid derivative of the compound of this invention were compared using the advanced Marfey method. R To determine the chirality of amino acid residues, the results showed that all amino acid residues in compound 2 were chiral. L - Configuration. A schematic diagram illustrating the structure of the cyclic peptide compound 2 of this invention is shown below. Figure 3 As shown, Figure 3 This is a schematic diagram illustrating the structure of cyclic peptide compound 2. In the diagram, A represents the key 2D NMR signal, B represents the MS / MS fragmentation mode, C represents the HRESI-MS / MS spectrum, and D represents the advanced Marfey method based on LC-MS.

[0062] Table 3: NMR spectral data of cyclic peptide compound 3 (DMSO-d6)

[0063]

[0064]

[0065]

[0066] Detailed analysis of 2D NMR (COSY, TOCSY, and HMBC) spectra revealed that the seven amino acid residues constituting compound 3 include leucine (2 residues), proline (2 residues), tyrosine (1 residue), alanine (1 residue), and aspartic acid (1 residue). The sequence of these amino acid residues was determined through careful analysis of HMBC, ROESY correlation signals, and ESI-MS / MS mass spectrometry data. Specifically, the HMBC correlation signal, such as Tyr-NH / Pro... 2 -CO, Asp-NH / Tyr-CO, Leu 2 -NH / Ala-CO and Ala-NH / Leu 1 -CO can identify two structural segments: Leu 2 -Ala-Leu 1 and Asp-Tyr-Pro 2 Based on ROESY related signals such as Asp-Hα / Pro 1 -Hδ、Leu 1 -Hα / Pro 1 -Hα and Leu 2 -Hβ / Pro 2 -Hα, thus determining the structure of compound 3 as cyclo-(Pro 1 -Leu 1 -Ala-Leu 2 -Pro 2 -Tyr–Asp). This result was validated by ESI-MS / MS mass spectrometry data. The two proline residues have different Δδ values. Cβ-Cγ Value, of which, Pro 1 It is 4.6, corresponding to the trans conformation, Pro 2 The value is 10.3, corresponding to the cis conformation. The molecular weight and retention time (t) of the 3-amino acid derivative of the compound of this invention were compared using the advanced Marfey method with those of the standard derivative and the compound of this invention. R To determine the chirality of amino acid residues, the results showed that all amino acid residues in compound 3 were chiral. L - Configuration. A schematic diagram illustrating the structure of the cyclic peptide compound 3 of this invention is shown below. Figure 4 As shown, Figure 4 This is a schematic diagram illustrating the structure of cyclic peptide compound 3. In the diagram, A represents the key 2D NMR signal, B represents the MS / MS fragmentation mode, C represents the HRESI-MS / MS spectrum, and D represents the advanced Marfey method based on LC-MS.

[0067] Table 4: NMR spectral data of cyclic peptide compound 4 (DMSO-d6)

[0068]

[0069]

[0070]

[0071] Detailed analysis of 2D NMR (COSY, TOCSY, and HMBC) spectra revealed that the seven amino acid residues constituting compound 4 include leucine (1), proline (3), isoleucine (2), and tyrosine (1). The linkage order between residues was determined through careful analysis of HMBC, ROESY correlation signals, and ESI-MS / MS mass spectrometry data. Specifically, the linkage order was determined based on the HMBC correlation signal. 2 -NH,Hα / Leu-CO identifies a key structural segment Ile 2 -Leu. The key signal missing in the HMBC spectrum was supplemented by the ROESY correlation signal, according to Ile 2 -Hα / Pro 1 -Hα,Leu-NH / Tyr-Hα,β,Pro 3 -Hα / Ile 1 -Hα,Tyr-NH / Pro 3 -Hα,γ,Pro 3 -Hα / Ile 1 -Hα and Pro 1 -Hα / Pro 2 -Hα determined the structure of compound 4 to be cycloPro 1 -Pro 2 -Ile 1 -Pro 3 -Tyr-Leu-Ile 2 This result was validated by ESI-MS / MS mass spectrometry data. All three proline residues exhibited large Δδ values. Cβ-Cγ Value, Pro 1 Pro 2 and Pro 3 The values ​​were 9.6, 8.4, and 9.8, respectively, indicating that they are all in the cis conformation. The molecular weight information and retention times (t) of the standard derivative and the 4-amino acid derivative of the present invention were compared using the advanced Marfey method. R The chirality of the amino acid residues was determined, and the results showed that all amino acid residues in compound 4 were in the L-configuration. A schematic diagram illustrating the structure of compound 4, a cyclic peptide of this invention, is shown below. Figure 5 As shown, Figure 5This is a schematic diagram illustrating the structure of cyclic peptide compound 4. In the diagram, A represents the key 2D NMR signal, B represents the MS / MS fragmentation mode, C represents the HRESI-MS / MS spectrum, and D represents the advanced Marfey method based on LC-MS.

[0072] Example 2

[0073] In vitro antitumor activity experiment

[0074] Initial screening method: The in vitro cytotoxic activity of the cyclic peptide compounds 1-4 of this invention was evaluated using the CCK-8 assay. The tumor cell lines used were A2780 (human ovarian cancer cells), HCT-8 (human colon cancer cells), NCI-H460 (human lung cancer cells), SW480 (human colon cancer cells), PC-9 (human lung cancer cells), and HepG2 (human liver cancer cells). A2780, HCT-8, and HepG2 cells were cultured in DMEM medium, while NCI-H460, SW480, and PC-9 cells were cultured in RPMI 1640 medium. Samples were dissolved in DMSO and stored at low temperature. The concentration of DMSO in the final system was controlled within a range that did not affect the detection activity, and the samples were serially diluted to a working concentration of 1-100 μg / mL. When the cell lines reached the logarithmic growth phase, the culture medium was used to prepare a single-cell suspension of 1×10⁻⁶ cells / mL. 6 The suspension was prepared at a concentration of 100 μL / mL and added to each well of a 96-well plate. After incubation for 24 h in a 5% CO2, 37°C incubator, the test drug (cyclic peptide compounds 1-4 of this invention) was added, with three replicates per sample. An equal volume of culture medium was used as a negative control, DMSO of the corresponding concentration as a solvent control, and cisplatin as a positive control. After incubation for 48 h in a 5% CO2, 37°C incubator, 10 μL of CCK-8 solution was added to each well. After another 4 h of incubation, the absorbance (OD value) of each well was measured at 450 nm, with each cell line measured at least three times. The corresponding cell viability (%) was calculated according to the cell viability formula. For compounds with an inhibition rate greater than 50%, their IC50 was calculated using GraphPad Prism 6.0 software. 50 The values ​​are expressed as mean ± standard deviation (SD).

[0075] Preliminary screening results: Preliminary screening of six human tumor cell lines at a concentration of 20 μM showed that among the four novel cyclic peptide compounds of this invention, only compound 1 exhibited significant cytotoxic activity, with growth inhibition rates against four tumor cell lines—NCI-H460, SW480, PC-9, and HepG2—far exceeding 50%. Figure 6 As shown, Figure 6 This is a schematic diagram illustrating the in vitro proliferation inhibitory effects of the cyclic peptide compounds 1-4 of the present invention on six human tumor cell lines.

[0076] Given that compound 1 exhibited good inhibitory activity against tumor cell proliferation in vitro during the initial screening, concentration gradients of 0.01, 0.03, 0.10, 0.30, 1.00, 3.00, 10.00, 20, and 40.00 μM were set to further investigate its activity dose-dependent relationship. The results are shown in Table 5, where the half-maximal inhibitory concentration (IC50) of compound 1 against NCI-H460 cells is shown. 50 The concentration is only 4.7 ± 0.35 μM. Therefore, it can be used to prepare antitumor drugs. This invention provides a new lead compound for the development of novel antitumor drugs.

[0077] Table 5 Cytotoxic activity of compound 1

[0078]

[0079] 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 cyclic peptide compound extracted from sponges, characterized in that, The structure is as follows:

2. The use of the cyclic peptide compound extracted from sponges according to claim 1 in the preparation of antitumor drugs, characterized in that, The tumors mentioned are lung cancer, ovarian cancer, colon cancer, and liver cancer.

3. The application of the cyclic peptide compound extracted from sponge according to claim 2 in the preparation of antitumor drugs, characterized in that, The tumor cells described are human ovarian cancer cells A2780, human colon cancer cells HCT-8, human lung cancer cells NCI-H460, human colon cancer cells SW480, human lung cancer cells PC-9, and human liver cancer cells HepG2.

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