CN116333054A - Cyclic peptide compound extracted from sponge and preparation method and application thereof - Google Patents
Cyclic peptide compound extracted from sponge and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a cyclic peptide compound extracted from sponge, the structure of which is selected from one of the following compounds 1-4:
the invention also provides application of the cyclopeptide compound in preparing antitumor drugs. The cyclic peptide compound has strong growth inhibition activity on human lung cancer cells NCI-H460. The inventionProvides a new lead compound for researching and developing new anti-tumor drugs and provides scientific basis for developing and utilizing ocean medicinal resources in China.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a cyclic peptide compound extracted from sponge, and a preparation method and application thereof.
Background
Cyclic peptides have many different biochemical and therapeutic properties and have achieved great success in drug therapy. Of the 60 peptide drugs approved for sale by the FDA and EMA, two thirds are cyclic peptides. Notably, the marine environment fosters a variety of structurally diverse and biologically active cyclic peptides. Plitidpsin is one of the most representative marine-derived molecules, approved by EMA in 2018 for the treatment of relapsed/refractory multiple myeloma, and has recently proven to be a potential therapeutic candidate for covd-19.
Sponges have been an important prolific source of marine active cyclic peptides. The sponges reported in the literature to be rich in cyclic peptides mainly comprise the genera Phakellia, hymenoccidon, stylissa and Callyspongia, and research on the chemical compositions of the sponges finally results in cyclic peptides of the types Phakellistatins, hymenomidades, stylissamidades and Callyerins. Interestingly, these cyclic peptides share common structural features: proline-rich, leucine and/or isoleucine account for a greater proportion of the structure. Further structure-activity analysis shows that leucine and/or isoleucine are/is frequently linked to proline to form Pro-Leu/Ile fragments, whereas cyclic peptides with more Pro-Leu/Ile fragments tend to have better cytotoxic activity. Based on the characteristic that the cyclopeptide compound loses fixed neutral fragment ions, the cyclopeptide compound is beneficial to target research and directional acquisition of the cyclopeptide containing Pro-Leu/Ile fragments by means of a strong parent ion scanning function of a composite liquid phase tandem triple quadrupole mass spectrum (Q-Q-Q), so that a potential new anti-tumor lead compound is efficiently discovered, and the development speed of ocean medicines is accelerated.
Disclosure of Invention
It is a first object of the present invention to provide a cyclic peptide compound extracted from a sponge.
The second object of the invention is to provide a method for preparing the cyclic peptide compound extracted from the sponge.
The third object of the invention is to provide an application of the cyclopeptide compound extracted from sponge in preparing antitumor drugs.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a novel cyclic peptide extracted from a sponge, the structure of which is selected from one of the following compounds 1 to 4:
the sponge refers to a sponge sample Reniochalina sp.
The second aspect of the present invention provides a method for extracting a cyclic peptide compound from a sponge, comprising the steps of:
first, sample screening: aiming at the characteristic that cyclic peptide compounds lose fixed neutral fragments, combining a PI scanning function of LC-QTRAP-MS/MS with an IDA triggered EPI scanning technology, establishing a rapid positioning method for selectively identifying cyclic peptide characteristic structures in crude extracts, and screening a sponge sample Reniochalina sp rich in cyclic peptide compounds from a sponge sample library;
second, solvent extraction: cutting the sponge sample Reniochalina sp obtained by the first step into pieces, and adding equal volume of MeOH and volume ratio of 1:1 CH respectively 2 Cl 2 Ultrasonic extracting with MeOH for at least three times, mixing extractive solutions, and concentrating under reduced pressure to obtain crude extract;
step three, step extraction: suspending the crude extract prepared in the second step in 90% methanol water, extracting for 3-5 times by using petroleum ether with the same volume, diluting the 90% methanol water into 60% methanol water, extracting for 3-5 times by using dichloromethane with the same volume, merging the extracting solutions, and concentrating under reduced pressure to obtain a dichloromethane extraction part;
fourth, separating and enriching: subjecting the dichloromethane extraction part prepared in the third step to Sephadex LH-20 gel column chromatography, wherein the volume ratio of CH to CH is 1:1 for the first time 2 Cl 2 MeOH as eluent, and n-hexane/CH with the volume ratio of 4:5:1 for the second time 2 Cl 2 MeOH as eluent, and enriching the compound with large molecular weight by mass spectrum positioning tracking; separating by ODS medium pressure column chromatography with 10% -100% MeOH/H 2 O gradient elution, adopting mass spectrum tracking positioning analysis to obtain a series of fine fractions containing the large molecular weight cyclopeptide compounds;
fifth, screening target compounds: at m/z 211 (C) 11 H 19 N 2 O 2 ) As characteristic fragment ions of the Pro-Leu/Ile fragment, adopting a parent ion scanning mass spectrometry to analyze the fine fraction containing the large molecular weight cyclopeptide compound prepared in the fourth step, screening out a target compound containing the Pro-Leu/Ile fragment, and tracking and positioning the fine fraction containing the target compound by combining molecular weight information and chromatographic retention behavior;
sixth, mass spectrum guided separation: and (3) separating the fine fraction prepared in the fifth step by adopting a mass spectrum guided semi-preparative high-liquid chromatography method, thereby obtaining the target compound.
In the sixth step, separation conditions of mass spectrometry-oriented semi-preparative high-performance liquid chromatography: compound 1, 50-60% acetonitrile-water, flow rate 6.0mL/min, molecular ion m/z 941.59 detected in positive ion mode; compound 2 and compound 3, 20-23% acetonitrile-water, flow rate 6.0mL/min, molecular ions m/z724.37 and 770.41 were detected in positive ion mode; compound 4, 28-35% acetonitrile-water, flow rate 6.0mL/min, molecular ion m/z 794.48 was detected in positive ion mode.
The third aspect of the invention provides an application of the cyclopeptide compound extracted from sponge in preparing antitumor drugs.
The tumor is lung cancer, ovarian cancer, colon cancer and liver cancer.
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.
The cyclic peptide compound shows strong inhibition activity on human lung cancer cells NCI-H460, so that the cyclic peptide compound can be used for preparing antitumor drugs.
By adopting the technical scheme, the invention has the following advantages and beneficial effects:
the drug source material is sponge Reniochalina sp of the Phakellidae (Halichondrid) of the Demospongiae (Armillarid) of the Phakellidae (Axisellidae), and the unique living environment enables the drug source material to generate secondary metabolites with novel structures and obvious activities. The cyclic peptide compound is from sponge Reniochalina sp, and is separated by a parent ion scanning mass spectrometry and LC-MS guided procedural separation means, and the preparation method is efficient and simple. The cyclic peptide compound has remarkable inhibitory activity on human lung cancer cells NCI-H460. In summary, the invention provides a new lead compound for researching and developing new antitumor drugs, a new strategy for rapidly identifying and directionally tracking trace cyclic peptides active ingredients in sponge, and a scientific basis for developing and utilizing marine medicinal resources in China.
Drawings
FIG. 1 is a schematic diagram of the discovery scheme for cyclic peptides 1-4.
FIG. 2 is a schematic diagram illustrating the structure of cyclic peptide compound 1.
FIG. 3 is a schematic diagram illustrating the structure of cyclic peptide compound 2.
FIG. 4 is a schematic diagram illustrating the structure of cyclic peptide compound 3.
FIG. 5 is a schematic diagram illustrating the structure of cyclic peptide compound 4.
FIG. 6 is a schematic diagram showing the in vitro proliferation inhibition effect of cyclopeptides 1-4 of the present invention on six human tumor cell lines.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
Example 1
Discovery and extraction of sponge cyclic peptides 1-4 from sponge
And adopting parent ion scanning to obtain a renal finger sponge sample Reniochalina sp. Cutting sponge (dry weight 54.8 g) into pieces, ultrasonic extracting with organic solvent for several times, mixing extractive solutions, and concentrating under reduced pressure to obtain crude extract. The crude extract is subjected to fractional extraction, gel column chromatography and ODS medium pressure column chromatography to obtain a plurality of fine fractions. And (3) performing parent ion scanning analysis on a dichloromethane part rich in the cyclopeptide compound to obtain a series of target cyclopeptides containing Pro-Leu/Ile fragments, tracking and positioning fine fractions of the target compounds by mass spectrometry in combination with molecular weight information and chromatographic retention behaviors, and separating by adopting mass spectrometry-guided semi-preparative high-performance liquid chromatography to finally obtain the sponge cyclopeptides.
The specific steps for extracting the spongy cyclic peptide compounds 1-4 from the sponge are as follows:
first, sample screening: aiming at the characteristic that cyclic peptide compounds lose fixed neutral fragments, the PI scanning function of LC-QTRAP-MS/MS is combined with IDA triggered EPI scanning technology, a rapid positioning method for selectively identifying cyclic peptide characteristic structures in crude extracts is established, and sponge samples rich in cyclic peptide compounds are screened from a sponge sample library. The sample was collected from the sea area (25-30 m depth) near the island of forever island in south China 4 months 2021, and was orange-yellow in color and harder in texture. The product was identified as Reniochalina sp.
Second, solvent extraction: after shearing the sponge sample obtained in the first step (Reniochalina sp. (dry weight: 54.8 g)) into pieces, equal volumes of MeOH and CH were added, respectively 2 Cl 2 MeOH (v/v=1:1) in turnUltrasonic extracting for three times, mixing the extractive solutions, and concentrating under reduced pressure to obtain crude extract (23.7 g);
step three, step extraction: suspending the crude extract prepared in the second step in 250mL of 90% methanol water, extracting for 3-5 times by using petroleum ether with equal volume, diluting the 90% methanol water into 60% methanol water, extracting for 3-5 times by using dichloromethane with equal volume, merging the extracting solutions, and concentrating under reduced pressure to obtain a dichloromethane extraction part (1.2 g);
fourth, separating and enriching: subjecting the dichloromethane extract prepared in the third step to Sephadex LH-20 gel column chromatography, wherein CH is used for the first time 2 Cl 2 MeOH (v/v=1:1) as eluent, second time n-hexane/CH 2 Cl 2 MeOH (v/v/v=4:5:1) as eluent and enrichment of the high molecular weight compounds by means of mass spectrometry localization tracking gave 600mg; separating by ODS medium pressure column chromatography with 10% -100% MeOH/H 2 O gradient elution, adopting mass spectrum tracking positioning analysis to obtain a series of fine fractions containing the large molecular weight cyclopeptide compounds;
fifth, screening target compounds: at m/z 211 (C) 11 H 19 N 2 O 2 ) As characteristic fragment ions of the Pro-Leu/Ile fragment, adopting a parent ion scanning mass spectrometry to analyze the fine fraction containing the large molecular weight cyclopeptide compound prepared in the fourth step, screening out a target compound containing the Pro-Leu/Ile fragment, and tracking and positioning the fine fraction containing the target compound by combining molecular weight information and chromatographic retention behavior;
sixth, mass spectrum guided separation: and separating the fine fraction prepared in the fifth step by adopting a mass spectrum guided semi-preparative high-liquid chromatography method, thereby obtaining target compounds 1-4. The isolation conditions for each compound were as follows:
FIG. 1 is a schematic diagram of the discovery scheme for cyclic peptides 1-4. The method for establishing the parent ion scanning mass spectrum is accurate and effective, can successfully and accurately identify the target cyclopeptide containing Pro-Leu/Ile fragments from the sponge crude extract, and can efficiently and directionally track the target cyclopeptide by combining a mass spectrum guided separation means.
The structures of compounds 1-4 are shown below:
the physicochemical properties and nuclear magnetic resonance data of the spongy cyclic peptide compounds 1-4 prepared by the steps are as follows:
compound 1: white amorphous powder; [ alpha ]] 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; ESIMS/MS data are shown in FIG. 2C; HRESIMS m/z 941.5872[ M+H ]] + (calcd for C 52 H 76 N 8 O 8 ，941.5864)。
Compound 2: a pale yellow amorphous powder; [ alpha ]] 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; ESIMS/MS data are shown in FIG. 3C; HRESIMS m/z 724.3676[ M+H ]] + (calcd for C 36 H 49 N 7 O 9 ，724.3670)。
Compound 3: a pale yellow amorphous powder; [ alpha ]] 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; ESIMS/MS data are shown in FIG. 4C; HRESIMS M/z770.4084[ M+H ]] + (calcd for C 38 H 55 N 7 O 10 ，770.4089)。
Compound 4: a pale yellow amorphous powder; [ alpha ]] 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 FIG. 5C; HRESIMS m/z 794.4824[ M+H ]] + (calcd for C 42 H 63 N 7 O 8 ，794.4816)。
Nuclear magnetic resonance spectrum data of the spongy cyclic peptides 1-4 are shown in tables 1, 2, 3 and 4, respectively.
Table 1: nuclear magnetic resonance spectrum data (DMSO-d) of cyclopeptide 1 6 )
By analyzing the 2D NMR (COSY, TOCSY and HMBC) spectra in detail, it was determined that the 8 amino acid residues constituting compound 1 include proline (2), phenylalanine (2), isoleucine (2) and leucine (2). The order of amino acid residues ligation was determined by careful analysis of HMBC, ROESY-related signals and ESI-MS/MS mass spectrometry data. Wherein, based on HMBC related signals such as Leu 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, two structural fragments can be determined: ile 1 -Pro 1 And Ile 2 -Leu 2 -Phe 1 -Phe 2 -Leu 1 -Pro 2 . Based on ROESY-related signals, e.g. Ile 1 -Hα/Pro 2 -Hdelta and Ile 2 -Hα/Pro 1 -hδ, thereby 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 verified by ESI-MS/MS mass spectrometry data. Delta of two prolines Cβ-Cγ All have smaller delta Cβ-Cγ Value of Pro 1 4.8, pro 2 3.9, indicating that they are all in the trans-conformation. Molecular weight information and retention time (t) of reference derivatives and amino acid derivatives of compound 1 of the present invention were compared using the advanced Marfey method R ) To determine the chirality of the amino acid residues, which shows that all amino acid residues of compound 1 are L -configuration. The structural elucidation schematic diagram of the cyclopeptide compound 1 is shown in fig. 2, and fig. 2 is the structural elucidation schematic diagram of the cyclopeptide compound 1. Wherein A is a key 2D NMR related signal, B is an MS/MS fragmentation pattern, C is an HRESI-MS/MS spectrogram, and D is an advanced Marfey method based on LC-MS.
Table 2: nuclear magnetic resonance spectrum data (DMSO-d) of cyclopeptide compound 2 6 )
By analyzing the 2D NMR (COSY and HMBC) spectra in detail, it was determined that 7 amino acid residues constituting compound 2 include leucine (1), aspartic acid (1), proline (3), glycine (1) and phenylalanine (1). Determination of amino acid residues by careful analysis of HMBC, ROESY-related signals and ESI-MS/MS mass spectrometry dataConnection sequence. Wherein, according to HMBC related signals such as Gly-NH/Pro 2 -CO、Asp-NH/Gly-CO、Leu-NH/Pro 1 -CO and Phe-NH/Leu-CO, two structural fragments can be determined: asp-Gly-Pro 2 And Phe-Leu-Pro 1 . According to ROESY-related signals, e.g. Asp-H.alpha./Pro 3 -Hδ、Pro 1 -Hδ/Pro 3 -Hα and Phe-Hα/Pro 2 -hα, thereby determining the structure of compound 2 as cyclo- (Pro) 1 -Leu-Phe-Pro 2 -Gly-Asp-Pro 3 ). This result was verified by ESI-MS/MS mass spectrometry data. Delta through proline Cβ-Cγ Values determine its cis-trans conformation, where Pro 1 5.0, pro 3 3.3, indicating that they are in trans-conformation, whereas Pro 2 10.0, indicating that it is in cis conformation. Molecular weight information and retention time (t) of reference derivatives and amino acid derivatives of compounds 2 of the invention were compared using the advanced Marfey method R ) To determine the chirality of the amino acid residues, which shows that all amino acid residues of compound 2 are L -configuration. The structural elucidation schematic diagram of the cyclopeptide compound 2 is shown in fig. 3, and fig. 3 is the structural elucidation schematic diagram of the cyclopeptide compound 2. Wherein A is a key 2D NMR related signal, B is an MS/MS fragmentation pattern, C is an HRESI-MS/MS spectrogram, and D is an advanced Marfey method based on LC-MS.
Table 3: nuclear magnetic resonance spectrum data (DMSO-d) of cyclopeptide 3 6 )
By analyzing the 2D NMR (COSY, TOCSY and HMBC) spectra in detail, it was confirmed that 7 amino acid residues constituting Compound 3 include leucine (2), prolineAcid (2), tyrosine (1), alanine (1) and aspartic acid (1). The order of amino acid residues ligation was determined by careful analysis of HMBC, ROESY-related signals and ESI-MS/MS mass spectrometry data. Wherein, according to HMBC related signals such as Tyr-NH/Pro 2 -CO、Asp-NH/Tyr-CO、Leu 2 -NH/Ala-CO and Ala-NH/Leu 1 -CO, two structural fragments can be determined: leu (Leu) 2 -Ala-Leu 1 And Asp-Tyr-Pro 2 . According to ROESY-related signals, e.g. Asp-H.alpha./Pro 1 -Hδ、Leu 1 -Hα/Pro 1 -hα and Leu 2 -Hβ/Pro 2 -hα, thereby determining the structure of compound 3 as cyclo- (Pro) 1 -Leu 1 -Ala-Leu 2 -Pro 2 -Tyr-Asp). This result was verified by ESI-MS/MS mass spectrometry data. Two prolines have different Δδ Cβ-Cγ Value of, wherein Pro 1 4.6, corresponding to trans conformation, pro 2 10.3, corresponding to the cis conformation. Molecular weight information and retention time (t) of reference derivatives and of amino acid derivatives of compounds 3 according to the invention were compared using the advanced Marfey method R ) To determine the chirality of the amino acid residues, which shows that all amino acid residues of compound 3 are L -configuration. The structural elucidation schematic diagram of the cyclopeptide compound 3 is shown in fig. 4, and fig. 4 is the structural elucidation schematic diagram of the cyclopeptide compound 3. Wherein A is a key 2D NMR related signal, B is an MS/MS fragmentation pattern, C is an HRESI-MS/MS spectrogram, and D is an advanced Marfey method based on LC-MS.
Table 4: nuclear magnetic resonance spectrum data (DMSO-d) of cyclopeptide 4 6 )
By analyzing the 2D NMR (COSY, TOCSY and HMBC) spectra in detail, it was determined that 7 amino acid residues constituting compound 4 include leucine (1), proline (3), isoleucine (2) and tyrosine (1). The order of attachment between residues was determined by careful analysis of HMBC, ROESY-related signals and ESI-MS/MS mass spectrometry data. Wherein, according to the HMBC related signal Ile 2 -NH, Hα/Leu-CO to define a critical structural fragment Ile 2 Leu. The missing key signals in the HMBC spectra are complemented by the ROESY-related signals, 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 Determination of the structure of Compound 4 as CycloPro by Hα 1 -Pro 2 -Ile 1 -Pro 3 -Tyr-Leu-Ile 2 ). This result was verified by ESI-MS/MS mass spectrometry data. All three prolines have a larger Δδ Cβ-Cγ Value, pro 1 、Pro 2 And Pro 3 9.6, 8.4 and 9.8, respectively, which indicate that they are all in cis-conformation. Molecular weight information and retention time (t) of reference derivatives and of amino acid derivatives of compounds 4 of the invention were compared using the advanced Marfey method R ) To determine the chirality of the amino acid residues, and the results show that all amino acid residues of compound 4 are in the L-configuration. The structural elucidation schematic diagram of the cyclopeptide compound 4 is shown in fig. 5, and fig. 5 is the structural elucidation schematic diagram of the cyclopeptide compound 4. Wherein A is a key 2D NMR related signal, B is an MS/MS fragmentation pattern, C is an HRESI-MS/MS spectrogram, and D is an advanced Marfey method based on LC-MS.
Example 2
In vitro anti-tumor Activity assay
The primary screening experiment method comprises the following steps: the CCK-8 method was used to evaluate the in vitro cytotoxic activity of the cyclopeptides 1-4 of this invention. The tumor cell lines used were A2780 (human ovarian cancer cell), HCT-8 (human colon cancer cell), NCI-H460 (human lung cancer cell), SW480 (human colon cancer cell), PC-9 (human lung cancer cell) and HepG2 (human liver cancer cell). Wherein, A2780, HCT-8 and HepG2 cells use DMEM medium, NCI-H460, SW480 and PC-9 were fineCells used RPMI 1640 medium. The sample is preserved at low temperature after being dissolved by DMSO, the concentration of the DMSO in a final system is controlled within a range which does not influence the detection activity, and the double ratio is diluted to a working concentration of 1-100 mug/mL. When the cell strain is cultured to logarithmic phase, the culture solution is taken to prepare single cell suspension 1 multiplied by 10 6 Each mL, and the suspension was added to a 96-well plate at 100. Mu.L per well. At 5% CO 2 After 24h incubation in 37℃incubator, 3 multiplex wells were added per sample, and the equivalent volume of medium was used as negative control, corresponding concentration of DMSO as vehicle control, and cisplatin as positive control. At 5% CO 2 After 48h of incubation in an incubator at 37℃10. Mu.L of CCK-8 solution was added to each well. After further culturing for 4 hours, absorbance (OD) was measured at 450nm for each well, and 3 times or more per cell line. The corresponding cell viability (%) was calculated according to the cell viability formula. For compounds with inhibition greater than 50%, IC was calculated using GraphPad prism6.0 software 50 Values, and the final results are expressed as mean.+ -. Standard Deviation (SD).
Preliminary screening test results: the results of preliminary screening of six human tumor cell lines at a concentration of 20 μm show that only compound 1 of the four novel cyclic peptide compounds of the present invention exhibits significant cytotoxic activity, and the growth inhibition rates of the four tumor cell lines NCI-H460, SW480, PC-9 and HepG2 are all much greater than 50%, as shown in fig. 6, and fig. 6 is a schematic diagram of the in vitro proliferation inhibition effect of the cyclic peptide compounds 1-4 of the present invention on the six human tumor cell lines.
In view of the fact that compound 1 shows a better inhibition effect on the proliferation of tumor cells in vitro in the primary screening step, the concentration gradient ranges of 0.01, 0.03, 0.10, 0.30, 1.00, 3.00, 10.00, 20 and 40.00 mu M are set, and the activity dose dependence relationship is further examined. The results are shown in Table 5, where compound 1 has a half-effective inhibitory concentration IC for NCI-H460 cells 50 The value was only 4.7.+ -. 0.35. Mu.M. Therefore, can be used for preparing antitumor drugs. The invention provides a new lead compound for developing new antitumor drugs.
TABLE 5 cytotoxic Activity of Compound 1
The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.
Claims (6)
1. A cyclic peptide compound extracted from a sponge, characterized by a structure selected from one of the following compounds 1-4:
2. a method for extracting a cyclic peptide compound from a sponge according to claim 1, comprising the steps of:
first, sample screening: aiming at the characteristic that cyclic peptide compounds lose fixed neutral fragments, combining a PI scanning function of LC-QTRAP-MS/MS with an IDA triggered EPI scanning technology, establishing a rapid positioning method for selectively identifying cyclic peptide characteristic structures in crude extracts, and screening a sponge sample Reniochalina sp rich in cyclic peptide compounds from a sponge sample library;
second, solvent extraction: cutting the sponge sample Reniochalina sp obtained by the first step into pieces, and adding equal volume of MeOH and volume ratio of 1:1 CH respectively 2 Cl 2 Ultrasonic extracting with MeOH for at least three times, mixing extractive solutions, and concentrating under reduced pressure to obtain crude extract;
step three, step extraction: suspending the crude extract prepared in the second step in 90% methanol water, extracting for 3-5 times by using petroleum ether with the same volume, diluting the 90% methanol water into 60% methanol water, extracting for 3-5 times by using dichloromethane with the same volume, merging the extracting solutions, and concentrating under reduced pressure to obtain a dichloromethane extraction part;
fourth, separating and enriching: subjecting the dichloromethane extraction part prepared in the third step to Sephadex LH-20 gel column chromatography, wherein the volume ratio of CH to CH is 1:1 for the first time 2 Cl 2 MeOH as eluent, and n-hexane/CH with the volume ratio of 4:5:1 for the second time 2 Cl 2 MeOH as eluent, and enriching the compound with large molecular weight by mass spectrum positioning tracking; separating by ODS medium pressure column chromatography with 10% -100% MeOH/H 2 O gradient elution, adopting mass spectrum tracking positioning analysis to obtain a series of fine fractions containing the large molecular weight cyclopeptide compounds;
fifth, screening target compounds: taking m/z 211 as characteristic fragment ions of Pro-Leu/Ile fragments, analyzing the fine fraction containing the large molecular weight cyclopeptide compound prepared in the fourth step by adopting a parent ion scanning mass spectrometry, screening out a target compound containing the Pro-Leu/Ile fragments, and tracking and positioning the fine fraction containing the target compound by adopting mass spectrometry in combination with molecular weight information and chromatographic retention behaviors;
sixth, mass spectrum guided separation: and separating the fine fraction prepared in the fifth step by adopting a mass spectrum guided semi-preparative high-liquid chromatography method, thereby obtaining the compounds 1-4.
3. The method for extracting cyclic peptide compounds from sponge as claimed in claim 2, wherein in the sixth step, separation conditions of mass spectrometry-guided semi-preparative high-liquid chromatography are as follows: compound 1, 50-60% acetonitrile-water, flow rate 6.0mL/min, molecular ion m/z 941.59 detected in positive ion mode; compound 2 and compound 3, 20-23% acetonitrile-water, flow rate 6.0mL/min, molecular ions m/z724.37 and 770.41 were detected in positive ion mode; compound 4, 28-35% acetonitrile-water, flow rate 6.0mL/min, molecular ion m/z 794.48 was detected in positive ion mode.
4. Use of a cyclic peptide extracted from a sponge according to claim 1 for the preparation of an antitumor agent.
5. The use of the cyclic peptide extracted from sponge according to claim 4 for preparing an antitumor drug, wherein the tumor is lung cancer, ovarian cancer, colon cancer or liver cancer.
6. The use of cyclic peptide extracted from sponge according to claim 5 for preparing antitumor drugs, wherein 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.
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