CN111704654B

CN111704654B - Peptide compound and preparation method and application thereof

Info

Publication number: CN111704654B
Application number: CN202010557972.6A
Authority: CN
Inventors: 林厚文; 王淑萍; 吴莹; 孙凡; 刘丽云
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2020-06-18
Filing date: 2020-06-18
Publication date: 2021-12-03
Anticipated expiration: 2040-06-18
Also published as: CN111704654A

Abstract

The invention belongs to the technical field of pharmaceutical chemistry. The invention discloses a linear peptide or cyclic peptide with tumor cell inhibiting activity, which contains a condensation segment of kynurenine and arginine. The invention also provides a mass spectrum rapid positioning method for selectively identifying the cyclic peptide compound containing kynurenine from the complex extract, and a novel cyclic peptide compound with anti-tumor activity is separated from the Phakellia fusca. And the total synthesis of the compound of the invention is realized by applying solid-phase synthesis, liquid-phase cyclization and final-stage ozonization strategies.

Description

Peptide compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a peptide compound and a preparation method and application thereof.

Background

Sponges are the oldest and simplest multicellular animals, have an evolutionary history of over 5 hundred million years, are fed by means of filtered seawater, lack real tissue, are immobile and lack physical defenses. In the harsh competitive environment of the ocean, sponges protect against foreign biological attack by producing active secondary metabolites. Sponges are one of the most productive and important sources of marine new compounds, and since the research on marine natural products started in the 60's of the 20 th century, active substances with novel structures were discovered from sponges every year, accounting for about one third of the marine natural products. At present, 49 active substances from the sea at home and abroad are approved to be on the market or enter the clinic, and 14 active substances are from sponges (3 on the market), so that the sponges become important sources of marine medicines and are always the research hotspots of marine natural product chemists.

Sponges are an important source of marine active peptides, mainly including linear peptides, depsipeptides, and cyclic peptides. The cyclic peptide compound is a cyclic compound formed by amido bonds, and is a complex molecule mainly formed by amino acid, glycopeptide, lipopeptide and nucleoside peptide after a series of modifications. The cyclic peptide compound is used as a medicine molecule and has wide biological activities of resisting cancer, resisting virus, resisting bacteria, inhibiting immunity and the like. Therefore, researches on cyclic peptide drugs are receiving more and more attention. At present, more than 40 cyclic peptide drugs are clinically used, and in recent decades, about one cyclic peptide drug is approved to be on the market every year, which accounts for 3 percent of the total number of new drugs on the market at the same time. Cyclic peptide compounds are widely developed in the field of drug development and are associated with several properties. First, due to the conformational change limitations imposed by the cyclic structure, cyclic peptides generally have a large surface area, which provides high affinity and recognition specificity for the target protein. The restriction of the conformational flexibility of the macrocyclic structure also reduces the entropy value of the combination of the drug and the target and improves the stability of the combination. Secondly, the amino acid composition characteristics determine that cyclic peptide compounds tend to have extremely low or even no cytotoxicity. Thirdly, the cyclic peptide compound is easy to realize production through an automatic chemical synthesis process, and is convenient for various modifications, treatments and monitoring, and the characteristics are very beneficial to the drug development process.

Although cyclic peptide compounds have wide market application prospect, most of the cyclic peptide compounds derived from sponges are low in content in organisms, and the targeted acquisition of the cyclic peptide compounds is extremely challenging. If the tracking separation under the guidance of activity is only relied on, the sensitivity of specific components is not enough, the misdirection is easy to generate, the steps are complex, time and labor are wasted, the flux is low, the omission of trace active components is easy to cause, and the discovery process of the cyclopeptide natural product with low content, novel structure and remarkable activity in the sponge is greatly limited. In recent years, due to the rapid development of mass spectrometry technology, the discovery speed of marine natural products is greatly promoted. Particularly, the sensitivity and selectivity of finding trace active ingredients from complex natural extracts are greatly improved through the functions of parent ion scanning, daughter ion scanning, neutral loss scanning, multi-ion reaction monitoring and the like of the composite liquid phase tandem triple quadrupole mass spectrometry (Q-Q-Q). The neutral loss scan refers to that the Q1 and the Q3 perform full scan at the same time, but the two always keep a certain fixed mass difference, and only the ions with the neutral parts lost in the collision cell Q2 meeting the fixed mass difference can be detected. Based on the characteristic that the cyclic peptide compounds lose fixed neutral fragments, a mass spectrum neutral loss scanning function is combined, a mass spectrum rapid positioning method of the cyclic peptide characteristic structure is established, and novel cyclic peptide compounds containing the skeleton fragments in the complex extract are rapidly screened out, so that a new research idea is provided for relevant research of marine complex compounds, and the speed of marine drug development is accelerated.

Disclosure of Invention

The first purpose of the invention is to disclose a peptide compound, which is a linear peptide or a cyclic peptide with tumor cell inhibiting activity, and contains the following condensed fragments of kynurenine and arginine:

preferably, it is composed of 3 to 20, preferably 4 to 12 amino acids.

Further, the compound is one of the following compounds 1-6:

the second purpose of the invention is to provide two methods for preparing the peptide compound.

One preparation method is to extract and separate the sponge, and the extraction and separation method comprises the steps of extraction, separation and enrichment, chromatographic purification and the like.

In some specific embodiments, the method comprises the following steps:

(a) extraction: crushing sponge, percolating and extracting with an organic solvent, mixing the extracting solutions, and concentrating under reduced pressure to obtain an extracting solution; the organic solvent is preferably alcohol solvent such as ethanol, methanol, propanol, etc., or combination of two or more alcohol solvents;

(b) extraction and separation: extracting the extracting solution by using ethyl acetate, suspending the extract obtained by concentrating the ethyl acetate layer in 80-90%, preferably 85-90% methanol-water, and extracting by using petroleum ether; diluting 80-90%, preferably 85-90% of methanol-water layer to 50-60%, preferably 55-60% of methanol-water, extracting with dichloromethane, combining extract liquor and concentrating under reduced pressure to obtain dichloromethane extraction part;

(c) separation and enrichment: adopting mass spectrum tracking and positioning analysis, and separating and enriching fractions containing high molecular weight compounds by one or more modes of gel column chromatography, reversed phase medium pressure column chromatography and full preparative high performance liquid chromatography;

(d) screening for the target compound: screening a target compound containing kynurenine by adopting a neutral loss scanning mass spectrometry, firstly detecting parent ions with neutral loss of 190Da in fractions, and then carrying out daughter ion scanning verification on the parent ions, wherein the parent ions with poor mass of 190Da in secondary fragment ions are the target compound parent ions, so as to obtain fragment ion information and chromatographic retention behavior;

(e) mass spectrum guided separation: on the basis of obtaining fragment ion information and chromatographic retention behavior, separating fractions by adopting mass spectrum guided semi-preparative high performance liquid chromatography to obtain the target compound.

More specifically, for compound 1, it can be extracted from the sponge by the following steps:

(a) extraction: crushing Phakellia fusca, percolating with ethanol, mixing extractive solutions, and concentrating under reduced pressure to obtain extractive solution;

(b) extraction and separation: extracting the extracting solution for 3-5 times and preferably 3 times by using equal volume of ethyl acetate, combining ethyl acetate layers, concentrating to obtain an extract, suspending the extract in 80-90% methanol-water, preferably 85-90%, and extracting for 3-5 times by using equal volume of petroleum ether; diluting 80-90%, preferably 85-90% of methanol-water layer to 50-60% of methanol-water, extracting for 3-5 times, preferably 3 times, with the same volume of dichloromethane, combining the extracts, and concentrating under reduced pressure to obtain dichloromethane extraction part;

(c) separation and enrichment: subjecting the dichloromethane extract to Sephadex LH-20 gel column chromatography with 50% CH₂Cl₂Eluting with MeOH eluent, and enriching the compound with large molecular weight by adopting a mass spectrum localization tracking mode; separating by ODS medium pressure column chromatography with MeOH/H₂Eluting with O gradient (10% -100%, 450min), and performing mass spectrum tracking and positioning analysis to obtain fine fraction containing cyclic peptide compounds with large molecular weight;

(d) screening for the target compound: screening kynurenine-containing target compounds by neutral loss scanning mass spectrometry, and detecting parent ion M/z884.48[ M + H ] with neutral loss of 190Da in the fraction]⁺Performing ion scanning verification on the fragment ions, and finding out the mass difference of 190Da in the secondary fragment ions so as to obtain the fragment ion information and the chromatographic retention behavior;

Another preparation method is a total synthesis method, comprising the following steps:

(A) loading the first amino acid into the 2-CTC resin according to the amino acid sequence on the peptide chain: swelling the 2-CTC resin, adding amino acid protected by Fmoc (9-fluorenylmethoxycarbonyl protecting group) and DIEA (N, N-diisopropylethylamine), and washing to remove unreacted raw materials after complete reaction;

(B) fmoc deprotection: performing Fmoc deprotection by using a DMF solution containing 15-30% and preferably 20% of piperidine at room temperature, and washing the resin by using DMF;

(C) peptide coupling: according to the amino acid sequence on the peptide chain, the rest amino acids are coupled through the following steps in sequence: the Fmoc protected amino acid, HATU (polypeptide condensation reagent, systematic name 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) and DIEA were gently vortexed with the resin in DMF at room temperature, then the resin was washed with DMF; the side chains of arginine and tryptophan in the rest of the amino acids are respectively protected by Pbf group and Boc group;

(D) fmoc deprotection: performing Fmoc deprotection by using a DMF solution containing 15-30% and preferably 20% of piperidine at room temperature, and washing the resin by using DMF;

(E) cracking: filtering the resin, cracking the resin by using an HFIP cracking solution, and purifying the cracked resin to obtain a linear peptide with a protected side chain;

(G) side chain deprotection: adding a cleavage mixture into linear peptide containing a protecting group for deprotection, and purifying after complete reaction to obtain precursor peptide; the cracking mixture contains 90-95% TFA (trifluoroacetic acid), 2.5-5% TIS (triisopropylsilane) and 2.5-5% H by volume percentage₂O, preferably 93-95% TFA, 2.5-3.5% TIS and 2.5-3.5% H₂O。

(H) Ozone oxidation: dissolving the precursor peptide in MeOH, cooling to-78 deg.C, and passing O through an ozone generator₃Blowing and reacting for 4-8 minutes; dimethyl sulfide was added to the reaction at-78 deg.CHeating the reaction mixture to room temperature within 0.8-1.2 hours; the reaction mixture was concentrated in vacuo and then dissolved in TFA/H₂Stirring the mixture of O for 0.8 to 1.2 hours at room temperature to obtain a target compound;

alternatively, prior to step (G), it further comprises the steps of:

(F) macrocyclization: a solution of linear peptide in DCM (dichloromethane) was added EDCI ((1-ethyl-3 (3-dimethylpropylamine) carbodiimide), HOAt (1-hydroxy-7-azabenzotriazole) and DIEA, the reaction was stirred at room temperature to effect ring closure, and then the solvent was removed in vacuo.

Taking compound 1 as an example, the following synthetic route can be used for preparation:

the method specifically comprises the following steps:

(A) loading a first amino acid into a 2-CTC resin: swelling the 2-CTC resin in a disposable container filled with anhydrous DCM for 15-30 minutes; adding a DCM solution of Fmoc-protected proline (1.5-2.5 equivalents) and DIEA (3-5 equivalents), and shaking the reaction vessel at room temperature in a vortex for 0.8-1.5 hours; adding MeOH into the reaction mixture, and rotating the resin for 10-20 minutes; the resin was filtered and washed with DCM (3mL × 5 times, 1 min/time), DCM/MeOH (v/v ═ 1: 1) (3mL × 5 times, 1 min/time) and MeOH (3mL × 2 times, 1 min/time);

(B) fmoc deprotection: fmoc deprotection is carried out for 15-30 minutes at room temperature using 15-30% piperidine in DMF, and then the resin is washed with DMF (3mL × 2 times, 1 min/time);

(C) peptide coupling: according to the amino acid sequence on the peptide chain, the rest amino acids are coupled through the following steps in sequence: gently swirling Fmoc-protected amino acid (2-4 equivalents), HATU (2-4 equivalents) and DIEA (5-7 equivalents) with the resin in DMF at room temperature for 0.8-1.2 hours, and then washing the resin with DMF (3mL × 5 times, 1 min/time); the side chains of arginine and tryptophan in the rest of the amino acids are respectively protected by Pbf group and Boc group;

(D) fmoc deprotection: fmoc deprotection is carried out for 15-30 minutes at room temperature using 15-30% piperidine in DMF, and then the resin is washed with DMF (3mL × 2 times, 1 min/time);

(E) cracking: filtering the resin, treating the resin with 3mL of 20% (v/v) HFIP cracking solution in anhydrous DCM for 0.8-1.5 h, and then repeating the step for 30 min; after filtration, the resulting lysates were combined, concentrated in vacuo, and purified by HPLC to give side chain-protected linear peptide 1 a;

(F) macrocyclization: a solution of linear peptide 1a (1 eq) in DCM was added to EDCI (3 eq), HOAt (3 eq) and DIEA (3 eq) and the solution was stirred at rt for 16h, then the solvent was removed in vacuo;

(G) side chain deprotection: addition of cleavage cocktail (TFA: TIS: H) to the protected cyclic peptide₂O95/2.5/2.5, v/v/v), stirred for 3h, and the reaction monitored by LC-MS; the solution was concentrated in vacuo and then purified by HPLC to give the precursor peptide 1 b;

(H) ozone oxidation: precursor peptide 1b was dissolved in MeOH, cooled to-78 deg.C, and O was added using an ozone generator₃The reaction was bubbled for 5 minutes; dimethyl sulfide was added to the reaction at-78 ℃ and the reaction mixture was allowed to warm to room temperature over 1 hour; the reaction mixture was concentrated in vacuo and then dissolved in TFA/H₂O (1/1, v/v), and stirring at room temperature for 1h to obtain the target compound 1.

The third purpose of the invention is to disclose the application of the peptide compound in preparing anti-tumor drugs.

The peptide compound of the invention shows strong inhibitory activity to four tumor cells of MCF-7, HeLa, NCI-H460 and PC9, wherein the IC50 values of the compound 1 on the four tumor cells are respectively 3.4, 6.2, 7.1 and 12 mu M, the activity of the compound is equivalent to that of a positive drug cis-platinum, the toxicity to a normal cell H9C2 is very low, IC50 is more than 100 mu M, and the research on the action mechanism shows that the compound 1 is a potential antagonist of RXR alpha, and can inhibit the proliferation of cancer cells and promote the apoptosis through a PI3K/AKT signal pathway. Analysis of structure-activity relationship, Kyn and guanidyl are pharmacophores of the compound, and the action targets and the pharmacophores are verified through a molecular docking experiment. Therefore, the compound can be used for preparing antitumor drugs.

The positive progress effects of the invention are as follows:

the invention provides a mass spectrum rapid positioning method for selectively identifying kynurenine-containing peptide compounds from complex extracts. From the mass spectrum cleavage mechanism of peptide compounds, it is found that secondary fragment ions formed after the dissociation of cyclic peptides induced by collision often result from the loss of certain neutral molecular fragments, most of which are the basic units-amino acid residues-constituting the cyclic peptides. According to this feature, secondary MS analysis revealed that kynurenine-containing cyclic peptides could be rapidly searched by parent ions with poor mass of 190Da between fragment ions. Scanning crude fraction of Phakellia fusca collected from the territorial waters near Hsisha Islands of China by using neutral loss mass spectrometry, and finding out that the parent ion is M/z884.48[ M + H ]]⁺On the basis of obtaining fragment ion information and chromatographic retention behavior, the peak is combined with mass spectrum to guide a preparation liquid phase to separate the fragment ion information and the chromatographic retention behavior, so that the novel cyclic peptide compound is obtained.

The invention realizes the total synthesis of the compound by using solid-phase synthesis, liquid-phase cyclization and final-stage ozonization strategies, and provides quantitative guarantee for subsequent research.

The compound of the invention shows strong inhibitory activity to four tumor cells of MCF-7, HeLa, NCI-H460 and PC9, wherein the compound 1 has IC (integrated Circuit) on the four tumor cells₅₀The values are respectively 3.4, 6.2, 7.1 and 12 mu M, the activity of the compound is equivalent to that of a positive drug cis-platinum, and the IC is very low in toxicity to normal cell H9C2₅₀>100 mu M, and the research on action mechanism finds that the compound 1 is a potential antagonist of RXR alpha, inhibits the proliferation of cancer cells and promotes the apoptosis through a PI3K/AKT signal pathway. Analysis of structure-activity relationship, Kyn and guanidyl are pharmacophores of the compound, and the action targets and the pharmacophores are verified through a molecular docking experiment. Therefore, the compound can be used for preparing antitumor drugs.

The compound of the invention has simple preparation method and obvious tumor cell inhibiting activity. The invention provides a new lead compound for researching and developing new anti-tumor drugs, provides a new method for quickly identifying and directionally tracking the active ingredients of trace cyclic peptide cytotoxic, and provides a new idea for synthesizing the cyclic peptide compound. Provides scientific basis for developing and utilizing marine medicinal resources in China.

Drawings

FIG. 1 is a scheme of the discovery of natural Compound 1;

FIG. 2 is a MS/MS fragment detection scheme for native Compound 1;

FIG. 3 is a high-grade Marfey's analysis of native Compound 1 by LC-MS;

FIG. 4 shows the synthesis and the nature of compound 1¹H NMR(DMSO-d₆600MHz) spectral contrast plot;

FIG. 5 shows the synthesis and the natural of compound 1¹³C NMR(DMSO-d₆150MHz) spectral contrast plot;

FIG. 6 is a graph comparing the MS/MS fragments of Compound 1, both synthetic and natural;

FIG. 7 is a graph of the induction of HeLa apoptosis by various concentrations of Compound 1;

FIG. 8 is a graph of the induction of HeLa cell cycle arrest by various concentrations of Compound 1;

figure 9 is a graph of the effect of compound 1 on RXR α and western blot analysis;

fig. 10 is a graph of the results of molecular docking of compound 1 with RXR α.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1 discovery and extraction of Natural Compound 1 (C) from sponges₄₅H₆₁N₁₁O₈)

From the mass spectrum cracking mechanism of peptide compounds, it is found that secondary fragment ions formed after the cyclic peptide is subjected to collision induced dissociation are often lost certain neutral moleculesFragments result from these neutral molecules being mostly the basic unit constituting the cyclic peptide-each amino acid residue. According to this feature, secondary MS analysis revealed that kynurenine-containing cyclic peptides could be rapidly searched by parent ions with poor mass of 190Da between fragment ions. Scanning crude fractions of Phakellia fusca (Axinellidae) Phakellia fusca (Phakellia) belonging to Halichondridae (Halichondridae) Axinellidae (Axinellidae) of Demospongiae (Demospongiae)) collected from the territorial waters near Hsisha Islands of China using neutral loss mass spectrometry, wherein the parent ion was found to be M/z884.48[ M + H]⁺As shown in fig. 1, on the basis of obtaining fragment ion information and chromatographic retention behavior, a mass spectrum is combined to guide a preparation liquid phase to separate the fragment ion information and the chromatographic retention behavior, so as to obtain a novel cyclic peptide compound, namely compound 1.

The application of the neutral loss scanning mass spectrometry in the screening of the compound is as follows:

1) determination of consensus neutral fragments:

and (3) combining the secondary mass spectrum containing the kynurenine cyclic peptide and the structure of the kynurenine residue, determining the mass of the neutral fragment to be 190Da, and setting the mass difference of the neutral loss scanning mode to be 190 Da.

2) Optimization of "neutral loss scan":

optimizing input voltage, collision energy and the like to obtain high-responsivity fragment ion information as much as possible and ensure that parent ions can be found, wherein the parent ions are 1/3 of the responsivity of the fragment ions; and setting a screening standard, and setting the mass difference to be 190Da as a standard to ensure that the ions with the specific mass difference pass through.

3) Optimizing the pretreatment conditions of the sample:

performing Sephadex LH-20 gel column chromatography with 50% CH₂Cl₂-MeOH eluent elution and mass spectrometric localization tracking of the major molecular weight compounds; after reversed phase medium pressure column chromatography and mass spectrum guided full-preparation high liquid phase chromatography, impurity components are effectively removed, and cyclic peptide components are enriched.

4) Optimization of liquid phase conditions:

in order to realize baseline separation of the obtained specific components, reduce inter-ion interference, facilitate multi-stage ion structure analysis and directional acquisition of the specific components, determine the optimal linear gradient condition and avoid the co-outflow phenomenon as much as possible.

5) And (3) secondary mass spectrum verification:

neutral loss of 190Da parent ion M/z884.48[ M + H ] was detected in the crude fraction of cyclic peptides]⁺And the mass difference of 190Da can be found in the secondary fragment ions after the secondary fragment ions are subjected to the verification of the ion scanning.

6) Preparing and separating by mass spectrum guidance:

on the basis of obtaining fragment ion information and chromatographic retention behavior, M/z884.48[ M + H ] is combined with mass spectrum guide preparation liquid phase]⁺And (5) separating.

The specific steps for extracting the natural compound 1 from the sponge are as follows:

pulverizing Phakellia fusca (dry weight 1.0kg), percolating with 95% ethanol, mixing extractive solutions, concentrating under reduced pressure, recovering ethanol, and concentrating to obtain extractive solution.

The extract was extracted three times with equal volume of ethyl acetate, and the ethyl acetate layers were combined and concentrated to give an ethyl acetate layer extract (34.5 g). Suspending the ethyl acetate layer extract in 90% methanol water, extracting with petroleum ether of the same volume for three times, combining the extracts, and concentrating under reduced pressure to obtain petroleum ether layer (22.5 g). The 90% aqueous layer of methanol was diluted to 60% aqueous methanol, extracted three times with an equal volume of dichloromethane, the extracts combined and concentrated under reduced pressure to give a dichloromethane layer (3.5 g).

Extracting the above dichloromethane extract with 3.5g, performing Sephadex LH-20 gel column chromatography, and purifying with 50% CH₂Cl₂MeOH eluent elution and enrichment of the large molecular weight compounds to 2.1g using mass spectrometric localization tracking. Separating by ODS medium pressure column chromatography with MeOH/H₂Eluting with O gradient (10% -100%, 450min), and performing mass spectrum tracking and positioning analysis to obtain fine fraction containing cyclic peptide compounds with large molecular weight; finally, the fraction is separated by mass-guided semi-preparative high performance liquid chromatography (50-60% methanol-water, flow rate 5.0mL/min, molecular ion detection 884.5 under positive ion mode) to obtain the productA compound 1 is disclosed.

The physicochemical properties and nuclear magnetic resonance data of the natural compound 1 prepared by the above steps are as follows:

compound 1: a pale yellow amorphous powder; [ alpha ] to]25D-71.0(c 0.10,MeOH)；UV(MeOH)λmax(logε)204(4.34)nm；IR(ATR)νmax 3326,2959,2930,2874,1616,1525,1446,1348,1245,1203,1162,1099,1042,748,701,544,484cm-1；HRESIMS m/z 884.4788[M+H]⁺(calcd for C₄₅H₆₁N₁₁O₈,884.4783)。

The data of the nuclear magnetic resonance spectrum are shown in Table 1.

Table 1: NMR spectra data for Compound 1 (DMSO-d6)

^aSequential NOEs.^bOverlapping signals.

From the TOCSY spectra, a 6 amino acid spin-coupled system, comprising one phenylalanine, one valine, one arginine and three prolines, can be determined and verified by the HMBC spectra. The remaining signals demonstrate the presence of the unusual amino acid kynurenine. 1 ketocarbonyl signal δ C199.0 (Kyn- γ -CO) and 1 SP2 hybridized aprotic carbon δ C151.4 (Kyn-C-6) were present in the 13C NMR spectrum; in the 1H NMR spectrum δ H7.64 (2H, d, J ═ 9.0Hz, Kyn-NH, Kyn-H-2) and 6.79(1H, d, J ═ 8.4Hz, Kyn-H-5) are doublets, δ H6.57 (1H, d, J ═ 7.7Hz, Kyn-H-3) and δ H7.27 (1H, d, J ═ 7.2Hz, Kyn-H-4) are triplets, suggesting the presence of an ortho-substituted benzene ring. In addition, the amino acid is determined to be kynurenine according to relevant signals of Kyn-H beta a, Kyn-H beta b and Kyn-H-2 and carbonyl (delta C199.0) in an HMBC spectrum.

The order of the linkage of 7 amino acid residues was determined by analysis of HMBC and ROESY-related signals and ESI-MS/MS. The HMBC related signals Arg-NH/Kyn-CO, Kyn-NH/Phe-CO, Phe-NH/Pro1-CO and Val-NH/Pro3-CO determine the presence of the structural fragments Pro1-Phe-Kyn-Arg and Pro 3-Val. The structure of the cyclic peptide is determined to be cyclo- (Pro1-Phe-Kyn-Arg-Pro2-Pro3-Val) by combining ROESY related signals Arg-H alpha/Pro 2-H delta, Pro2-H alpha/Pro 3-H alpha and Val-H alpha/Pro 1-H delta. And 5, discovering a fragment ion peak of the b series by ESI-QTOF-MS/MS mass spectrum analysis: m/

z

728, 538, 391, 294 and 195, corresponding to parent ions, which sequentially lose fragment ion peaks of neutral molecules such as Arg, Kyn, Phe, Pro and Val; accordingly, y is the fragment ion peak: m/

z

787, 690, 591, 494, 347 and 157, corresponding to the loss of Pro, Val, Pro, Phe and Kyn in sequence of the parent ion, confirm the NMR structural resolution of the cyclic peptide (fig. 2).

The absolute configuration of each amino acid residue was determined by the advanced Marfey's method (fig. 3). The compound (0.1mg) of the present invention was hydrolyzed with 6N hydrochloric acid at 110 ℃ for 6 hours, and then derivatized with L-FDLA, the corresponding L-amino acid standard was derivatized with D/L-FDLA, the obtained derivative was analyzed by UPLC-ESI-QTOF-MS, and the absolute configuration was determined by comparing the retention time of each amino acid derivative in the sample with that of the standard derivative. The retention times of the amino acid standard derivatives were as follows:_L-FDLA-_L-Pro 14.33min，_D-FDLA-_L-Pro 15.95min；_D-FDLA-_L-Arg 9.53min,_L-FDLA-_L-Arg 10.03min；_L-FDLA-_L-Kyn 16.67min，_D-FDLA-_L-Kyn 18.01min；_L-FDLA-_L-Phe 17.24min，_D-FDLA-_L-Phe 18.71min；_L-FDLA-_L-Val 16.06min，_D-FDLA-_Lval 18.28 min. The analysis result shows that all the amino acid residues in the cyclic heptapeptide are_L-configuration.

Example 2 preparation of Compound 1 of the present invention by Synthesis

Synthetic route to Compound 1

The synthesis steps are as follows:

loading a first amino acid into a 2-CTC resin: the 2-CTC resin (100mg, loading: 1.0mmol/g) was swollen for 20 minutes in a disposable vessel (TORIVQ) containing 2mL of anhydrous DCM. A solution of Fmoc-Pro-OH (2.0 equiv.) and DIEA (4.0 equiv.) in DCM was added and the reaction vessel was shaken at room temperature for 1 hour on a vortex. To the reaction mixture was added 200 μ L MeOH, and the resin was spun for 15 minutes. The resin was filtered and washed with dry DCM (3mL × 5 times, 1 min/time), 1: 1DCM/MeOH (v/v) (3mL × 5 times, 1 min/time) and MeOH (3mL × 2 times, 1 min/time).

Fmoc deprotection: fmoc deprotection was performed at room temperature using 3mL of 20% piperidine in DMF for 20 min, and the resin was washed with DMF (3 mL. times.2, 1 min/time).

Peptide coupling: each of the required Fmoc amino acids (3 equivalents), HATU (3 equivalents) and DIEA (6 equivalents) was gently vortexed with the resin in DMF at room temperature for 1 hour, then the resin was washed with DMF (3 mL. times.5, 1 min/time).

Cracking: the resin was filtered and treated with 3mL of 20% (v/v) HFIP lysis solution in dry DCM for 1 hour, and then the procedure was repeated for 30 minutes. After filtration, the resulting cleavage solutions were combined, concentrated in vacuo, and purified by HPLC to give side chain-protected linear peptide 1a (8.7 mg).

Macrocyclization: a solution of linear peptide 1a (1 eq) in DCM was added to EDCI (3 eq), HOAt (3 eq) and DIEA (3 eq) and the solution was stirred at rt for 16h, then the solvent was removed in vacuo.

Side chain deprotection: into cyclic peptides containing protecting groups1mL of cleavage mixture (TFA: TIS: H) was added₂O95/2.5/2.5, v/v/v), stirred for 3h and the reaction monitored by LC-MS. The solution was concentrated in vacuo and then purified by HPLC to give the precursor cyclic peptide 1b (4.8mg, yield 55%).

Ozone oxidation: precursor peptide 1b (4.8mg, 0.005mmol) was dissolved in 0.5mL MeOH, cooled to-78 deg.C, and O was added using an ozone generator₃The reaction was bubbled for 5 minutes. Dimethyl sulfide (40 μ L) was added to the reaction at-78 deg.C and the reaction mixture was allowed to warm to room temperature over 1 hour. The reaction mixture was concentrated in vacuo and then dissolved in 0.5mL of TFA/H₂O (1/1, v/v) and stirring at room temperature for 1h gave 4.6mg of the compound of the invention in 95% yield over 3 steps.

By comparing LC-MS/MS and NMR data, it was demonstrated that Compound 1 of the present invention was synthesized in exact match with that found in nature (FIGS. 4-6).

EXAMPLE 3 in vitro Activity assay of Compound 1 of the present invention

Cytotoxic activity assay: compound 1 of the present invention was tested for cytotoxic activity against six tumor cells (MCF-7, HeLa, NCI-H460, SW480, PC9 and HepG2) and one normal cell (H9C 2). The sample is dissolved by DMSO and stored at low temperature, the concentration of the DMSO in the final system is controlled within the range without influencing the detection activity, and the dilution ratio is 1-100 mu g/mL working concentration. Taking cells in logarithmic growth phase to prepare single cell suspension 1X 10⁶one/mL, add the suspension to a 96-well plate, 100 μ L per well. At 5% CO₂After culturing for 24 hours in an incubator at 37 ℃, the tested drugs with various concentrations are respectively added to ensure that the final concentrations are respectively 100, 50, 25, 12.5 and 6.25 mu g/mL, each sample is provided with 3 multiple holes, a negative control is an isometric culture medium and the corresponding DMSO concentration is a solvent control to eliminate the influence of DMSO on the cell growth, and a positive control drug is cisplatin. At 5% CO₂After culturing at 37 ℃ for 72 hours in an incubator, 10. mu.L of CKK8 solution was added to each well, and after culturing for 4 hours, the absorbance (OD value) of each well was measured at 450 nm. Determining percent inhibition by linear regression method, and calculating IC₅₀The value is obtained. Typically, each sample was provided with multiple wells (n-3) during the test, and the results were obtainedIs expressed as Standard Deviation (SD). Half-effective inhibitory concentration IC of compound 1 on different cells₅₀The values are shown in Table 2.

Table 2: cytotoxic Activity of Compound 1

As can be seen from Table 2, Compound 1 has cytotoxic activity against four tumor cells, MCF-7, HeLa, NCI-H460 and PC9, IC₅₀The values are respectively 3.4, 6.2, 7.1 and 12 mu M, the efficacy is equivalent to that of the positive drug cis-platinum, and the toxicity to normal cells H9C2 is very low IC₅₀>100 μ M. The compound is a potential antitumor drug, and provides a new lead compound for developing a new antitumor drug.

Detecting apoptosis by flow cytometry: taking HeLa cells in logarithmic growth phase, digesting with 0.25% pancreatin to obtain single cell suspension, blowing uniformly, counting at 1 × 10⁶Inoculating to 6-well plate at density of 2mL cell suspension per well, mixing at 37 deg.C with 5% CO₂Culturing in a constant temperature incubator for 24 h. The experimental group was added with test sample solution (5, 10, 20. mu.M) and the blank group was added with DMSO (0.1%), and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in a constant temperature incubator for 48 h. Trypsinized, the cells were collected, transferred to a centrifuge tube and centrifuged (1000rpm, 5min) to discard the supernatant. Adding 500. mu.L Binding Solution to gently resuspend the cells to make 1X 10⁶cells/mL of cell suspension. Adding 5 mu L Annexin V and FITC conjugate, mixing gently, and incubating at room temperature in dark for 15 min; then adding 5 mu L of PI Solution, incubating for 15min in the dark at room temperature, and detecting by using a flow cytometer, wherein Annexin V and FITC are used for detecting in a first channel, and PI is used for detecting in a second channel. Data are presented as mean ± Standard Deviation (SD), statistical analysis using SPSS, ANOVA for inter-group comparisons if normal distribution is met, and Dunnett's t for inter-group comparisons.

From the results of the measurement in fig. 7, it can be seen that compound 1 of the present invention induces HeLa cell apoptosis in a concentration-dependent manner.

Detecting the cell cycle by using a flow cytometry: digesting stably grown HeLa cells to prepare single cell suspension at a ratio of 1 × 10⁶The density of each well was inoculated in 6-well plates at 2 mL/well, 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator. The cells were harvested by trypsinization followed by centrifugation (1000rpm, 5min) of the cells by treatment with different concentrations of fuscasin E (5, 10, 20. mu.M) for 24 h. The supernatant was discarded, 1mL of 70% ethanol precooled at 4 ℃ was added, and the mixture was fixed in a refrigerator at 4 ℃ overnight. Centrifugation (5 min at 1000 rpm) discarded the supernatant, and centrifugation was performed after 1mL of PBS wash for resuspension. The supernatant was discarded, 500. mu.L of PI/RNAse was added to each tube, incubated at room temperature for 15min, filtered and detected by flow cytometry. Data are presented as mean ± Standard Deviation (SD), statistical analysis using SPSS, ANOVA for inter-group comparisons if normal distribution is met, and Dunnett's t for inter-group comparisons. The compound 1 of the invention induces HeLa cell cycle arrest in G in a concentration-dependent manner₂the/M phase (FIG. 8).

RXR alpha double reporter gene detection experiment: 293T cells were plated on 10cm cell culture dishes and plasmids were transfected when 80% fusion was achieved (pG5luc: pBIND-RXR A-LBD: lipo2000 ═ 10. mu.g: 3. mu.g: 20. mu.L). At 37 ℃ with 5% CO₂Culturing in incubator for 12 hr, digesting with 0.25% pancreatin to obtain single cell suspension, blowing uniformly, counting, and counting at 1 × 10⁴The density of each well was inoculated into a 96-well plate and the culture was continued for 12 h. Cells were treated with 9-cis in the experimental group in combination with 20 μ M samples, cells were treated with 9-cis alone in the control group, with 2 replicate wells per group, and incubation continued for 12 h. Cells were collected, lysed, and the cell supernatant, firefly luciferase assay reagent, and Renilla luciferase assay buffer were equilibrated at room temperature. Adding a firefly Luciferase detection reagent, uniformly mixing, and detecting on a luminescence detector to obtain the reading of Luciferase; adding a Renilla luciferase detection reagent, uniformly mixing, detecting on a luminescence detector to obtain the reading of the Rellina, and calculating the relative activity of the firefly luciferase.

In RXR alpha double-reporter gene detection, the compound disclosed by the invention and 9-cis-RA can be used together to obviously inhibit the transcriptional activation of 9-cis-RA on RXR-alpha when the concentration of a sample is 20 mu M, so that the compound is suggested to be a potential small molecular compound interacting with RXR-alpha (figure 9A).

Western blot analysis: culturing 3-5 generation MCF-7 and HeLa cells, digesting with 0.25% trypsin to obtain suspension, and culturing at a concentration of 1 × 10⁶Density per well was seeded in 6 well plates, 2ml per well. Mixing at 37 deg.C with 5% CO₂Culturing in a constant temperature incubator for 24 h. Test samples (10, 20. mu.M) and DMSO (0.1%) were added to the experimental group and the blank was placed at 37 ℃ in 5% CO₂Culturing in an incubator for 24 h. The supernatant was discarded, the plate was placed on ice, washed 2 times with pre-chilled PBS, 100. mu.L of cell lysate (containing PMSF) was added to each well, and the plate was repeatedly shaken and lysed on ice for 3 min. Centrifuging the cell debris and lysate at 12000rpm at 4 deg.C for 5min, sucking the upper protein from the centrifuge tube, packaging, and removing insoluble protein lysate. Protein concentration was determined using the BCA protein concentration assay kit, and the supernatant volume was measured and 1/5 volumes of SDS-PAGE protein loading buffer (5X) were added and stored in a 100 ℃ water bath for 5min at-20 ℃. The protein was electrophoresed in 5% polyacrylamide gel SDS-PAGE, transferred to PVDF membrane, rinsed in TBST, then placed in 5% skimmed milk powder/TBST, shaken slowly on a decolouring shaker, and blocked at room temperature for 1 h. The blocking solution was discarded, the PVDF membrane was washed on a decolorizing shaker by TBST, diluted with primary antibody (1:1000) and incubated overnight in a refrigerator at 4 ℃. The primary antibody incubation was recovered, and the PVDF membrane was rinsed on a TBST shaker, diluted with a secondary antibody (1:5000), and incubated on a shaker at room temperature for 1 hour with slow shaking. The secondary antibody was recovered and rinsed with TBST. And (3) placing the PVDF membrane on a membrane sweeping instrument after the color developing agent develops color, and sweeping the membrane after adjusting parameters.

As shown in FIG. 9, compound 1 of the present invention can significantly inhibit the expression of PI3K catalytic subunits p85 and p-AKT, and can increase the cleavage of PARP protein, indicating that the present compound dose-dependently inhibits the activation of AKT in MCF-7 and HeLa cells, and promotes apoptosis. The above results suggest that the compounds of the present invention may inhibit cell growth through RXR-alpha mediated PI3K/AKT signaling pathway.

Molecular docking assay. In the Discovery Studio 2.5 software, the structure of the compounds of the invention was mapped as ligands and PDB (PDB code: 3NSQ) files were downloaded from the Protein Bank as acceptors. Compounds of the invention were scored by docking into the 3NSQ pocket using default settings according to the LibDock standard protocol for Discovery Studio 2.5. The optimal binding profile of the compounds of the invention was visualized in the Discovery Studio Visualizer client (fig. 10).

EXAMPLES 4-8 Synthesis of Compound 2-6

The compounds 2-6 in the following table 3 are synthesized according to the method of the embodiment 2. Wherein compound 5 and compound 6 are linear peptides, without a macrocyclization step.

Further experiments show that the compounds 2-6 also show better inhibitory activity on tumor cells.

TABLE 3 Compounds 2 to 6

Claims

1. A peptide compound is characterized in that the compound is a compound 1

2. A process for the preparation of a peptidic compound according to claim 1, wherein the peptidic compound is extracted and isolated from Phakellia fusca.

3. The method of claim 2, wherein the peptidic compound is compound 1, and the method steps are specifically as follows:

(b) extraction and separation: extracting the extracting solution for 3-5 times by using equal volume of ethyl acetate, combining ethyl acetate layers, concentrating to obtain an extract, suspending the extract in 80-90% methanol-water, and extracting for 3-5 times by using equal volume of petroleum ether; diluting a 80-90% methanol-water layer to 50-60% methanol-water, extracting for 3-5 times by using dichloromethane with the same volume, combining extract liquor, and concentrating under reduced pressure to obtain a dichloromethane extraction part;

(c) separation and enrichment: subjecting the dichloromethane extract to Sephadex LH-20 gel column chromatography with 50% CH₂Cl₂-MeOH eluent elution and enrichment of high molecular weight compounds by mass spectrometric localization tracking; separating by ODS medium pressure column chromatography with 10% -100% MeOH/H₂Performing gradient elution for 450min, and performing mass spectrum tracking and positioning analysis to obtain fine fractions of cyclic peptide compounds with large molecular weight;

(d) screening for the target compound: screening target compounds by neutral loss scanning mass spectrometry, and detecting parent ion M/z884.48[ M + H ] with neutral loss of 190Da in fraction]⁺Performing ion scanning verification on the fragment ions, and finding out the mass difference of 190Da in the secondary fragment ions so as to obtain the fragment ion information and the chromatographic retention behavior;

4. A process for preparing a peptidic compound according to claim 1, comprising the steps of:

(A) the first amino acid, Fmoc-protected proline, was loaded into 2-CTC resin according to the amino acid sequence on the peptide chain: swelling the 2-CTC resin, adding proline and DIEA protected by Fmoc, and washing to remove unreacted raw materials after complete reaction;

(B) fmoc deprotection: performing Fmoc deprotection by using a DMF (dimethyl formamide) solution containing 15-30% piperidine at room temperature, and washing the resin by using DMF;

(C) peptide coupling: according to the amino acid sequence on the peptide chain, the rest amino acids are coupled through the following steps in sequence: the Fmoc-protected amino acids, HATU and DIEA were gently vortexed with the resin in DMF at room temperature, then the resin was washed with DMF; the side chains of arginine and tryptophan in the rest of the amino acids are respectively protected by Pbf group and Boc group;

(D) fmoc deprotection: performing Fmoc deprotection by using a DMF (dimethyl formamide) solution containing 15-30% piperidine at room temperature, and washing the resin by using DMF;

(F) macrocyclization: adding EDCI, HOAt and DIEA into the solution of the linear peptide in DCM, stirring at room temperature for reaction, closing the ring, and then removing the solvent in vacuum;

(G) side chain deprotection: adding a cleavage mixture into the cyclic peptide containing the protecting group for deprotection, and purifying after complete reaction to obtain a precursor peptide; the cracking mixture contains 90-95% of TFA, 2.5-5% of TIS and 2.5-5% of H in percentage by volume₂O；

(H) Ozone oxidation: dissolving the precursor peptide in MeOH, cooling to-78 deg.C, and passing O through an ozone generator₃Blowing and reacting for 4-8 minutes; adding dimethyl sulfide into the reaction at-78 ℃, and heating the reaction mixture to room temperature within 0.8-1.2 hours; the reaction mixture was concentrated in vacuo, then dissolved in TFA and H₂And stirring the mixture of O for 0.8 to 1.2 hours at room temperature to obtain the target compound.

5. The method of claim 4, wherein the peptidic compound is compound 1, and the method is as follows:

the method specifically comprises the following steps:

(A) loading the first amino acid, Fmoc-protected proline, into 2-CTC resin: swelling the 2-CTC resin in a disposable container filled with anhydrous DCM for 15-30 minutes; adding 1.5-2.5 equivalents of Fmoc-protected proline and 3-5 equivalents of DIEA (diethylhexyl Ether-methyl Ether) DCM solution, and shaking the reaction container in a vortex at room temperature for 0.8-1.5 hours; adding MeOH into the reaction mixture, and rotating the resin for 10-20 minutes; the resin was filtered and washed with DCM, DCM/MeOH and MeOH;

(B) fmoc deprotection: performing Fmoc deprotection for 15-30 minutes by using a DMF (dimethyl formamide) solution containing 15-30% of piperidine at room temperature, and then washing the resin by using DMF;

(C) peptide coupling: according to the amino acid sequence on the peptide chain, the rest amino acids are coupled through the following steps in sequence: 2-4 equivalents of Fmoc-protected amino acid, 2-4 equivalents of HATU 2-4 equivalents and 5-7 equivalents of DIEA are stirred with resin at room temperature in DMF for 0.8-1.2 hours in a gentle vortex manner, and then the resin is washed by DMF; the side chains of arginine and tryptophan in the rest of the amino acids are respectively protected by Pbf group and Boc group;

(D) fmoc deprotection: performing Fmoc deprotection for 15-30 minutes by using a DMF (dimethyl formamide) solution containing 15-30% of piperidine at room temperature, and then washing the resin by using DMF;

(E) cracking: filtering the resin, treating the resin with 3mL of 20% v/v HFIP cracking solution in anhydrous DCM for 0.8-1.5 hours, and then repeating the step for 30 minutes; after filtration, the resulting lysates were combined, concentrated in vacuo, and purified by HPLC to give side chain-protected linear peptide 1 a;

(F) macrocyclization: a solution of linear peptide 1a 1 equivalent in DCM was added EDCI 3 equivalent, HOAt 3 equivalent and DIEA3 equivalent, the solution was stirred at room temperature for 16h, then the solvent was removed in vacuo;

(G) side chain deprotection: to the protecting group-containing cyclic peptide was added the cleavage mixture TFA: and (3) TIS: h₂O95/2.5/2.5, v/v/v, stirred for 3h and the reaction monitored by LC-MS; the solution was concentrated in vacuo and then purified by HPLC to give the precursor peptide 1 b;

(H) ozone oxidation: precursor peptide 1b was dissolved in MeOH, cooled to-78 deg.C, and O was added using an ozone generator₃The reaction was bubbled for 5 minutes; dimethyl sulfide was added to the reaction at-78 ℃ and the reaction mixture was allowed to warm to room temperature over 1 hour; the reaction mixture was concentrated under vacuumThen dissolved in TFA/H₂O, and stirring at room temperature for 1h to obtain the target compound 1.

6. The use of a peptide compound according to claim 1 for the preparation of an anti-tumor medicament, wherein said tumor is breast cancer, cervical cancer, large cell lung cancer, lung adenocarcinoma.