CN109575105B - Marine peptide compound and preparation method and application thereof - Google Patents
Marine peptide compound and preparation method and application thereof Download PDFInfo
- Publication number
- CN109575105B CN109575105B CN201811280501.4A CN201811280501A CN109575105B CN 109575105 B CN109575105 B CN 109575105B CN 201811280501 A CN201811280501 A CN 201811280501A CN 109575105 B CN109575105 B CN 109575105B
- Authority
- CN
- China
- Prior art keywords
- compound
- marine peptide
- preparation
- cell
- peptide compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Public Health (AREA)
- Urology & Nephrology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a marine peptide compound. The marine peptide compound has good activity and strong repairing effect on cell damage. In the proliferation, migration and invasion screening experiments of human umbilical vein endothelial cells, the marine peptide compound shows remarkable activity on the cells and has excellent damage repair effect on the human umbilical vein endothelial cells. The marine peptide compound has good application prospect in the aspect of preparing damage repair medicines or ischemic disease medicines.
Description
Technical Field
The invention relates to derivatives derived from marine peptide compounds with novel structures, in particular to marine peptide compounds and a preparation method and application thereof.
Background
The marine natural product Xyloallenoid dA is a representative metabolite separated from marine fungi in the subject group, is abnormal and novel in structure, and contains cyclic tripeptide, alkenyl and cinnamamide in molecules. Among them, cyclic tripeptide has been proved to have the activity of promoting the vascular proliferation of zebra fish. A series of derivatives are designed on the basis of the cyclic tripeptide, wherein the tripeptide consisting of D-Val, L-Lys and L-Ala has remarkable activity.
The biomacromolecule medicine faces the problems of low bioavailability, difficult administration mode and the like in the aspect of pharmacokinetics. Compared with cyclic peptide and macromolecular protein substances, the small molecular marine peptide compound has more advantages in the aspects of preparation process, property, pharmacokinetics and the like, for example, the price is lower, the property is more stable, the tripeptide structure is simple, and the like. Therefore, the tripeptide is expected to become a candidate of a novel small molecular drug in the aspects of treating injury repair and ischemic diseases.
Disclosure of Invention
The invention aims to provide a marine peptide compound. The marine peptide compound has good activity.
The invention also aims to provide a preparation method of the marine peptide compound.
The invention also aims to provide the application of the marine peptide compound in preparing a medicament for repairing damage or a medicament for treating ischemic diseases.
The invention also aims to provide the application of the marine peptide compound in preparing a cell injury repair medicament.
The invention also aims to provide the application of the marine peptide compound in preparing a medicament for promoting cell proliferation.
The invention also aims to provide the application of the marine peptide compound in preparing a medicament for promoting cell migration.
The invention also aims to provide the application of the marine peptide compound in preparing a medicament for promoting cell invasion.
In order to solve the technical problems, the invention adopts the technical scheme that:
marine peptide compounds comprise compounds 1-8, and the structural formula is as follows:
the marine peptide compound provided by the invention belongs to small molecules and contains a simple tripeptide structure. The marine peptide compound has good activity and strong repairing effect on cell damage. Compared with cyclic peptide and macromolecular protein substances, the small molecular marine peptide compound has more advantages in the aspects of preparation process, properties, pharmacokinetics and the like, for example, the price is lower, the properties are more stable, and the like.
The invention also provides a preparation method of the marine peptide compound, which comprises the following steps:
s1, uniformly mixing a first raw material, O-benzotriazole-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N, N' -diisopropylethylamine in DMF (dimethyl formamide) for condensation reaction, terminating the reaction, and treating to obtain a dipeptide compound;
the first raw material is one or more of Cbz-D-Val-OH, H-L-Lys (Boc) -OMe, cbz-D-His (Cbz) -OH and H-L-Arg (Pbf) -OMe;
s2, saponifying the dipeptide compound in the step S1, and carrying out condensation reaction on the dipeptide compound and a second raw material to obtain a tripeptide compound;
the second raw material is one or more of H-L-Ala-OH, H-L-Met-OH, H-L-Phe-OH, H-L-Tyr-OH, H-L-Pro-OH and H-D-Val-OH;
s3, removing the protecting group after saponifying the tripeptide compound in the step S2 to obtain a target product.
Preferably, the reaction in step S1. Is carried out at room temperature for 14-18 h. Adding a first raw material into a round-bottom flask, adding DMF, adding O-benzotriazole-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N, N' -diisopropylethylamine, and stirring at room temperature overnight for 14-18 h. Wherein, the O-benzotriazole-tetramethylurea hexafluorophosphate in the step S1 is used as a carboxyl activating agent, the 1-hydroxybenzotriazole is used as a racemization inhibitor, and N, N' -diisopropylethylamine provides an alkaline condition for the system.
Preferably, the reagent for terminating the reaction in step S1. Is saturated NH 4 Cl。
Preferably, the treatment in step s1. Is extraction, washing, drying, concentration, separation. Extraction with EtOAc (ethyl acetate) and combination of the organic layers followed by saturated NH 4 Cl, saturated brine, anhydrous MgSO 4 Drying, concentrating and performing column chromatography to obtain the dipeptide compound.
Preferably, the saponification conditions in step S2. Are LiOH with THF/H 2 And O. Under LiOH conditions, THF/H 2 And saponifying in O to obtain dipeptide acid, and carrying out condensation reaction on the dipeptide acid and a second raw material to obtain the tripeptide compound. Preferably, the conditions of the condensation reaction in step s2. Are the same as in step s1.
Preferably, in step s3, the protecting group is one or more of a Cbz group, a pbf group, and a Boc group. Firstly, saponifying a tripeptide compound under an alkaline condition to obtain tripeptide acid; then, the target product was obtained by removing the Cbz group by catalytic hydrogenation with 10% Pd-C for three hours at normal temperature and pressure, or removing the Pbf group by reaction with TFA/DCM for two hours, or removing the Boc group under HCl/acetone conditions.
The invention also protects the application of the marine peptide compound in preparing a medicament for repairing damage or a medicament for treating ischemic diseases.
The invention also protects the application of the marine peptide compound in preparing a cell injury repair medicament.
The invention also protects the application of the marine peptide compound in preparing a medicament for promoting cell proliferation.
The invention also protects the application of the marine peptide compound in preparing a medicament for promoting cell migration.
The invention also protects the application of the marine peptide compound in preparing a medicine for promoting cell invasion.
The marine peptide compound has good activity, has strong repairing effect on cell injury, can be used as an inducer for repairing cell injury, and has high bioavailability and simple administration mode. The marine peptide compound can be a candidate of a novel micromolecule drug for treating injury repair and ischemic diseases, and has good application prospect in the aspect of preparing injury repair drugs or ischemic disease drugs.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a marine peptide compound which has good activity, high bioavailability, simple administration mode and strong repairing effect on cell damage. In the proliferation, migration and invasion screening experiments of human umbilical vein endothelial cells, the compound 5, the compound 6 and the compound 7 show obvious activity on the cells and have excellent damage repair effect on the human umbilical vein endothelial cells. The marine peptide compounds have good application prospect in the aspect of preparing injury repair medicines or ischemic disease medicines.
Drawings
FIG. 1 is a structural diagram of compounds 1 to 8.
FIG. 2 shows the effect of compounds 1-8 on human umbilical vein endothelial cell proliferation.
FIG. 3 is a graph of migration of human umbilical vein endothelial cells by compounds 1 and 5-7.
FIG. 4 shows the effect of compounds 1, 5-7 on human umbilical vein endothelial cell migration.
(*p<0.01versus control)
FIG. 5 is the invasion pattern of compounds 1 and 5-7 on human umbilical vein endothelial cells.
FIG. 6 shows the effect of compounds 1 and 5 to 7 on human umbilical vein endothelial cell invasion.
(*p<0.01versus control)
Detailed Description
The present invention will be further described with reference to specific embodiments, but the embodiments of the present invention are not limited thereto. The raw materials in the examples are all commercially available; the reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
EXAMPLE 1 preparation of Compounds 1 to 8
Compound 1:
a100 mL round bottom flask was charged with 2.51g of Cbz-D-Val-OH and 2.96g of H-L-Lys (Boc) -OMe, followed by 40 g of LDMF, followed by 2.4g of HBTU,2.6g of HOBt and 3.0 g of LDIEA, and stirred at room temperature overnight. Addition of saturated NH 4 The reaction was stopped with Cl, extracted with ethyl acetate, the organic layers combined and successively saturated NH 4 And washing Cl and saturated saline solution, drying the washed solution by anhydrous magnesium sulfate, concentrating the dried solution, and performing column chromatography to obtain the dipeptide compound. Under LiOH conditions, THF/H 2 Saponifying in O to obtain dipeptide acid, and reacting with 0.89g H-L-Ala-OH under the same condensation condition to obtain tripeptide compound. The tripeptide compound is saponified under alkaline conditions to give tripeptide acids, which are then catalytically hydrogenated with 10% Pd-C at normal temperature and pressure for three hours to remove the Cbz group, finally giving the desired product.
Nuclear magnetic and mass spectrometric characterization of compound 1:
1 H NMR(500MHz,MeOD)δ8.54(d,J＝8.2Hz,1H),8.37(d,J＝7.3Hz,1H),6.68(td,J＝8.4,5.5Hz,1H),4.21(p,J＝7.4Hz,1H),3.68(d,J＝5.2Hz,1H),2.88(dd,J＝13.1,6.7Hz,2H),2.51(dt,J＝3.6,1.8Hz,7H),2.18–1.98(m,1H),1.73–1.45(m,2H),1.30(s,14H),1.29(d,J＝7.3Hz,5H),0.94(dd,J＝11.8,6.9Hz,6H). 13 C NMR(125MHz,MeOD)δ174.26,171.33,168.10,156.00,77.81,57.78,52.63,47.88,30.35,29.52,28.74,22.94,18.86,17.82,17.52.
MS:m/z 417.5(M + )；HRMS calcd for C 19 H 36 O 6 N 4 :417.5257(M + ),found:417.5228。
compound 2:
Nuclear magnetic and mass spectral characterization of compound 2:
1 H NMR(500MHz,MeOD)δ8.72(s,1H),7.38(s,1H),4.42–4.34(m,1H),4.31(dd,J＝8.7,5.3Hz,1H),4.17(t,J＝7.1Hz,1H),3.25(dd,J＝15.0,6.6Hz,1H),3.00(t,J＝7.2Hz,2H),1.77(dt,J＝13.8,7.4Hz,1H),1.69–1.58(m,1H),1.40(d,J＝7.3Hz,4H). 13 C NMR(125MHz,MeOD)δ174.25,172.15,167.32,161.62,157.30,134.85,128.41,117.50,110.01,53.35,52.24,39.74,31.43,29.22,27.38,26.83,22.58,16.05.
MS:m/z 455.10(M + )；HRMS calcd for C 20 H 35 O 6 N 6 :455.2612(M + ),found:455.2611。
compound 3:
compared with the preparation method of the compound 1, the compound 3 has the difference that 1.49g H-L-Met-OH is used for replacing 0.89g H-L-Ala-OH in the preparation method of the compound 3; the amounts of other raw materials and the operating conditions were the same as those of the preparation method of compound 1.
Nuclear magnetic and mass spectral characterization of compound 3:
1 H NMR(500MHz,MeOD)δ4.59(dd,J＝9.5,4.3Hz,1H),4.34(dd,J＝9.0,5.2Hz,1H),3.82(d,J＝3.6Hz,2H),3.80(d,J＝3.5Hz,3H),3.77(d,J＝7.8Hz,5H),3.73–3.67(m,5H),3.67–3.56(m,6H),3.10–2.98(m,2H),2.63(ddd,J＝13.5,8.5,5.0Hz,1H),2.53(dt,J＝13.5,7.9Hz,1H),2.23–2.13(m,2H),2.09(s,4H),1.96(ddd,J＝14.2,8.8,4.3Hz,1H),1.87(dt,J＝12.7,6.1Hz,1H),1.77–1.63(m,1H),1.45(ddd,J＝55.4,14.3,7.2Hz,16H),1.06(dd,J＝6.8,5.1Hz,7H). 13 CNMR(125MHz,MeOD)δ174.81,174.47,169.67,158.59,79.93,73.03,71.40,65.15,59.87,54.90,52.21,32.76,32.19,31.42,31.18,30.54,28.79,24.25,19.03,18.03,15.11.
MS:m/z 477.05(M + )；HRMS calcd for C 21 H 39 O 6 N 4 S:475.2595(M - ),found:475.2597。
compound 4:
compared with the preparation method of the compound 1, the compound 4 is different in that 1.79g H-L-Phe-OH is used for replacing 0.89g H-L-Ala-OH in the preparation method of the compound 4; the amounts of other raw materials and the operating conditions were the same as those of the preparation method of compound 1.
Compound 4 was a white solid in 37.21% yield.
Nuclear magnetic and mass spectral characterization of compound 4:
1 H NMR(500MHz,MeOD)δ7.32–7.24(m,1H),7.23–7.14(m,1H),4.63(dd,J＝8.2,5.2Hz,1H),4.34(dd,J＝9.1,4.8Hz,1H),3.63(d,J＝6.2Hz,1H),3.19(dd,J＝14.0,5.3Hz,1H),3.02(ddd,J＝8.4,7.4,4.4Hz,1H),2.17(dq,J＝13.6,6.8Hz,1H),1.8(ddd,J＝14.3,10.8,5.9Hz,1H),1.73–1.60(m,1H),1.51–1.32(m,3H),1.05(t,J＝7.4Hz,1H). 13 C NMR(125MHz,MeOD)δ173.14,172.67,168.12,157.18,136.98,128.98,128.05,126.39,78.51,88.49,53.99,53.38,39.66,36.85,31.57,29.99,29.12,27.38,22.84,17.65,16.55.
MS:m/z 493.10(M + )；HRMS calcd for C 25 H 39 O 6 N 4 :491.2875(M - ),found:491.2877。
compound 5:
compared with the preparation method of the compound 1, the compound 5 has the difference that 1.83g H-L-Tyr-OH is used for replacing 0.89g H-L-Ala-OH in the preparation method of the compound 5; the amounts of other raw materials and the operating conditions were the same as those of the preparation method of compound 1.
Nuclear magnetic and mass spectral characterization of compound 5:
1 HNMR(500MHz,MeOD)δ7.07(d,J＝8.5Hz,1H),4.56(dd,J＝8.0,5.1Hz,1H),4.34(dd,J＝9.0,4.5Hz,1H),3.86–3.73(m,9H),3.73-3.67(m,4H),3.66–3.57(m,5H),3.14–2.80(m,2H),2.17(dd,J＝13.4,6.7Hz,1H),1.80(dd,J＝14.3,8.2Hz,1H),1.72–1.61(m,1H),1.05(dd,J＝8.3,7.1Hz,3H). 13 C NMR(125MHz,MeOD)δ155.92,150.16,129.99,114.80,71.63,70.00,63.75,58.50,54.25,53.39,36.11,31.59,29.99,29.13,27.38,22.84,17.66,16.54.
MS:m/z 509.05(M + )；HRMS calcd for C 25 H 39 O 7 N 4 :507.2824(M - ),found:507.2825。
compound 6:
compared with the preparation method of the compound 1, the compound 6 has the difference that 1.15g H-L-Pro-OH is used for replacing 0.89g H-L-Ala-OH in the preparation method of the compound 6; the other raw material amounts and the operation conditions are the same as those of the preparation method of the compound 1.
Nuclear magnetic and mass spectral characterization of compound 6:
1 H NMR(500MHz,MeOD)δ5.05(t,J＝5.5Hz,1H),4.71(dd,J＝8.9,4.9Hz,2H),4.47–4.39(m,2H),4.35(dd,J＝9.7,4.2Hz,1H),3.91(dd,J＝10.8,6.0Hz,2H),3.64(dt,J＝9.5,4.7Hz,4H),3.49(ddd,J＝19.1,11.3,6.0Hz,1H),3.10–2.95(m,4H),2.32–2.24(m,2H),2.22–2.14(m,2H),2.11–2.00(m,5H),1.94(dd,J＝15.0,7.6Hz,1H),1.8(dd,J＝16.2,7.0Hz,2H),1.67(ddd,J＝14.0,11.6,6.8Hz,2H),1.1300.97(m,14H). 13 C NMR(125MHz,MeOD)δ173.93,171.00,167.96,157.19,78.48,59.62,59.22,58.49,58.31,39.50,30.57,29.14,28.83,27.38,24.20,22.74,22.03,17.69,16.54.
MS:m/z 443.05(M + )；HRMS calcd for C 21 H 37 O 6 N 4 :441.2718(M - ),found:441.2720。
compound 7:
Nuclear magnetic and mass spectrometric characterization of compound 7:
1 HNMR(500MHz,MeOD)δ4.58(dd,J＝7.9,6.0Hz,1H),4.32(d,J＝5.6Hz,1H),3.85–3.77(m,2H),3.77(s,1H),3.69(q,J＝5.7Hz,2H),3.63(dd,J＝11.1,5.9Hz,1H),3.26–3.14(m,3H),2.19(dqd,J＝13.7,6.9,4.0Hz,2H),1.94–1.81(m,1H),1.81–1.53(m,4H),1.06(dd,J＝8.6,7.0Hz,7H),0.98(dd,J＝6.8,4.2Hz,8H). 13 C NMR(125MHz,MeOD)δ171.95,168.26,157.24,71.63,70.01,63.75,58.43,57.90,40.56,30.39,30.03,29.42,25.21,16.29,17.60,16.54.
MS:m/z 373.10(M + )；HRMS calcd for C 16 H 33 O 4 N 6 :373.2557(M + ),found:373.2557。
compound 8:
a100 mL round-bottom flask was charged with 1.044g of Compound 1 and Boc group removal was performed under HCl/acetone conditions to give the desired product.
Nuclear magnetic and mass spectral characterization of compound 8:
1 H NMR(400MHz,MeOD)δ4.41(dd,J＝11.8,6.0Hz,1H),3.71(d,J＝6.2Hz,1H),2.92(t,J＝7.5Hz,1H),2.20(dq,J＝13.4,6.6Hz,1H),1.90(tq,J＝14.0,7.4Hz,1H),1.74(ddd,J＝23.2,15.0,7.8Hz,2H),1.54(dd,J＝15.0,7.5Hz,1H),1.39(dd,J＝36.4,19.5Hz,2H),1.16-0.97(m,3H).
MS:m/z 317.20(M + )；HRMS calcd for C 14 H 29 O 4 N 4 :317.2183(M + ),found:317.2178。
EXAMPLE 2 experiment of injury repair Effect of Compounds on human umbilical vein endothelial cells
Materials and methods
1. Human umbilical vein endothelial cell recovery and passage
Rapidly taking out 3 cell freezing tubes from a-80 deg.C refrigerator, immediately placing into a 37 deg.C constant temperature water bath box, rapidly shaking the freezing tubes to accelerate thawing, completely thawing the cell liquid in the freezing tubes within two minutes, sterilizing, transferring to a clean bench, transferring the whole freezing tubes into a 10mL centrifugal tube filled with 8mL10% 2 Culturing in a constant temperature incubator. Taking a well-grown bottle of HUVECs cell culture flask, discarding the supernatant, adding 1mL of 0.25% (v/v) EDTA pancreatin for digestion, slightly wetting the flask, immediately removing the pancreatin solution, and continuously keeping at 37 deg.C for 5% CO 2 Culturing for 4-5 min in a constant temperature incubator, taking out cell bottles, uniformly blowing with 6mL10% 2 Culturing in a constant temperature incubator.
2. Drug treatment and microscopic observation of human umbilical vein endothelial cell proliferation
All compounds were dissolved in DMSO at initial concentrations of 2.5X 10 4 Mu mol/L, 2. Mu.L each time, diluting to the required concentration according to the experimental requirements, the culture medium is maintained by 0.4%1640 of diluent, the DMSO concentration is maintained at 0.2%. The experiment is divided into a control group and an experimental group, and the prepared medicine is prepared for use. Control group added DMSO only 0.2% and maintained the culture solution by 0.4%.
Collecting human umbilical vein endothelial cells in logarithmic growth phase, washing with PBS, digesting with 0.25% trypsin digestion solution, and blowing with 10%1640 culture solutionMaking into cell suspension, counting by cell counting plate, and adjusting cell concentration to 5.0 × 10 4 about/mL. Seeding the cell suspension in 96-well plates at 100. Mu.L per well, 5% CO at 37 ℃ 2 A constant temperature incubator. After 24 hours of culture, the culture medium was discarded, replaced with 0.4% by 100. Mu.L/well of a 0.4% maintenance medium, 37 ℃ 5% CO 2 And (5) carrying out culture pretreatment in a constant-temperature incubator for 24 hours to achieve cell synchronization.
Starving the cells which are synchronized after 24 hours of culture, discarding the original culture solution, adding 100 mu L/hole of each drug to be tested according to the experimental requirements, dividing the cells into 10 concentration groups according to the concentration of each drug to be tested, setting the compound concentration to be 0 mu mol/L, 0.0625 mu mol/L, 0.125 mu mol/L, 0.25 mu mol/L, 0.5 mu mol/L, 1 mu mol/L, 2 mu mol/L, 5 mu mol/L, 10 mu mol/L and 50 mu mol/L, setting 5 compound holes in each group, and then placing the cells at 37 ℃ for 5 mu mol CO 2 The incubation was continued in the incubator for 48 hours.
And taking out the cell pore plate cultured for 48 hours, carefully discarding supernatant in the pore, and slightly washing the cell pore plate for 1-2 times by using PBS buffer solution so as to prevent the effect of the medicine and MTT from influencing the experimental result. Add 10. Mu.L of MTT and 90. Mu.L of 0.4%1640 medium to each well, then place at 37 ℃ 5% CO 2 Continuously culturing in a constant temperature incubator. After 4 hours, the supernatant in the wells was discarded, 100. Mu.L of lysis buffer was added to each well, the crystals in each well were dissolved sufficiently by continuous shaking, and the optical density in each well was measured using a microplate reader (double wavelength 570nm, 630nm).
Cell proliferation rate = [ (actual OD value of well-average OD value of blank well)/average OD value of blank well ]. Times.100%
3. Drug treatment and microscopic observation of human umbilical vein endothelial cell migration
The synchronized cells were taken and the supernatant discarded, and scratched in each well along the bottom in a line perpendicular to the well plate baseline using the tip of a 200 μ L pipette. The well plate is washed 1-2 times by PBS buffer solution to wash off suspended cells, and three fields of view are arbitrarily selected for each well under a 100-fold OLYMPUS inverted microscope and a CCD camera, so as to record the relative distance of each scratch area and mark the scratch area. Add 0.4%1640 medium containing the prepared compound 500. Mu.L/well, 3 duplicate wells per dose group, and take and record the relative distance of the scratched area at 0, 12, 24, 36, 48 hours, respectively.
4. Medicine treatment and microscopic observation of human umbilical vein endothelial cell invasion
The well-grown human umbilical vein endothelial cells are cleaned and digested, and the cell suspension is cultured by using 1 mLDMEM. Counting with cell counting plate and adjusting cell concentration to 1 × 10 5 ～10×10 5 approximately/mL cells were seeded into 24-well transwell top chambers, 200 μ L cell suspension per well, divided into experimental and control groups, with 3 replicate wells per dose group. Each well of the transwell chamber was inoculated with 600. Mu.L of a medium composed of 480. Mu.L of DMEM high-sugar and 120. Mu.L of serum, the compound was added to the upper and lower chambers in such concentrations that the final concentration was 10. Mu. Mol/L, and the mixture was allowed to stand at 37 ℃ for 5% CO 2 The incubation was continued in the incubator for 24 hours.
Gently take out the transwell chamber, discard the supernatant, wet with sterile cotton swab PBS and carefully wipe off the still uninfected cells in the upper chamber; gently placing the chamber into PBS buffer solution by using a forceps to rinse off cells which do not invade the filter membrane, inverting the transwell chamber and naturally drying; fixing the cells in the microporous membrane by 4% paraformaldehyde for 15 minutes, and continuously rinsing the cells by PBS buffer solution for 2 to 3 times, wherein each time takes 5 minutes; staining with hematoxylin, then placing at 37 ℃ 5% CO 2 . The incubation was continued in the incubator for 30 minutes and washed 3-4 times with PBS buffer, each for about 10 minutes. Cells in the microporous membrane lower layer of the transwell chamber were photographed under an inverted microscope and randomly counted 100-fold in three fields per well. The experiment was repeated three times.
Migration proliferation rate = [ (number of test cell migrated through membrane-number of control cell penetrated)/number of control cell penetrated through membrane ]. Times.100%
5. Statistical analysis
The expression mode of experimental data adopts a mean plus or minus standard deviation form (x plus or minus s), the mean comparison between two groups adopts t test, the mean comparison between multiple groups adopts one-factor variance analysis, SPSS 19.0 statistical analysis software is used for data processing, and when P is positive, the data is processed<0.05, the difference between the data is considered to have a systemThe meaning of the study is calculated; the scratch result is obtained by ImageJ analysis; EC (EC) 50 The calculation method is calculated by adopting GraphPadprism 5 software.
(II) results of the experiment
1. Cell proliferation assay
FIG. 2 shows the effect of compounds 1-8 on human umbilical vein endothelial cell proliferation. In the proliferation experiment of human umbilical vein endothelial cells, the 8 compounds were preliminarily screened at different concentrations and different times. Table 1 shows the half maximal Effect Concentrations (EC) of Compounds 1 to 8 50 ). As is clear from the table, compound 5, compound 6 and compound 7 were able to promote cell proliferation, EC 50 Respectively 1.0. Mu. Mol/L, 0.88. Mu. Mol/L.
TABLE 1 half maximal effect concentrations of Compounds 1 to 8
2. Cell migration assay
Table 2 shows the effect of compound 1, compound 5, compound 6 and compound 7 and control Ctrl on cell migration at 0, 12, 24, 36, 48 hours, respectively. With increasing time, cell mobility also increases; the cell mobility was greatly increased with the addition of compound 1, compound 5, compound 6 and compound 7 compared to the control group. The cell mobility of compound 1, compound 5, compound 6 and compound 7 was 80.63%, 58.53%, 80.66%, 60.71%, respectively, indicating that compound 1, compound 5, compound 6 and compound 7 improved cell migration ability.
TABLE 2 Effect of Compounds 1, 5 to 7 on cell migration
3. Cell invasion assay
FIGS. 5 and 6 are graphs and effects of Compound 1, compound 5, compound 6 and Compound 7, respectively, and control Ctrl on human umbilical vein endothelial cell invasion. As can be seen from the figure, compound 1, compound 5, compound 6 and compound 7 improved the invasion ability of cells compared to the control group, and the invasion rates were 53.17%, 49.08%, 47.24% and 56.24%, respectively.
Therefore, the marine peptide compound provided by the invention has the advantages of good activity, high bioavailability, simple administration mode and strong repairing effect on cell damage. Compared with compound 1, the activity of compound 5, compound 6 and compound 7 is better, and the proliferation, migration and invasion capacity of human umbilical vein endothelial cells can be simultaneously improved. The results show that the compound 5, the compound 6 and the compound 7 have remarkable activity and excellent damage repair effect on human umbilical vein endothelial cells. Therefore, the marine peptide compound has good application prospect in the aspect of preparing injury repair medicines or ischemic disease medicines.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (6)
1. Marine peptide compounds characterized by the structural formula:
2. a process for the preparation of marine peptide compounds according to claim 1, comprising the steps of:
s1, uniformly mixing a first raw material, O-benzotriazole-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N, N' -diisopropylethylamine in DMF (dimethyl formamide) for condensation reaction, terminating the reaction, and treating to obtain a dipeptide compound;
the first raw material is Cbz-D-Val-OH and H-L-Lys (Boc) -OMe;
s2, after the dipeptide compound in the step S1 is saponified, carrying out condensation reaction with a second raw material to obtain a tripeptide compound;
the second raw material is H-L-Pro-OH;
s3, removing the protecting group after saponifying the tripeptide compound in the step S2 to obtain a target product.
3. The method of claim 2, wherein the protecting group in step S3 is one or more of a Cbz group, a pbf group and a Boc group.
4. The method according to claim 2, wherein the reagent for terminating the reaction in step S1. Is saturated NH 4 Cl。
5. The process according to claim 2, wherein the saponification in step S2 is carried out under LiOH and THF/H 2 O。
6. The use of a marine peptide compound according to claim 1 for the preparation of a medicament for treating ischemic diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811280501.4A CN109575105B (en) | 2018-10-30 | 2018-10-30 | Marine peptide compound and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811280501.4A CN109575105B (en) | 2018-10-30 | 2018-10-30 | Marine peptide compound and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109575105A CN109575105A (en) | 2019-04-05 |
| CN109575105B true CN109575105B (en) | 2022-10-18 |
Family
ID=65920904
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811280501.4A Active CN109575105B (en) | 2018-10-30 | 2018-10-30 | Marine peptide compound and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109575105B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434648A (en) * | 2008-09-28 | 2009-05-20 | 华东师范大学 | Peptide for inhibiting angiogenesis, and use thereof in angiogenesis medicament preparation |
| CN101979402A (en) * | 2010-10-19 | 2011-02-23 | 中山大学 | Marine cyclic peptide compound and preparation method and use thereof |
| CN102702310A (en) * | 2012-06-08 | 2012-10-03 | 中山大学 | Marine peptides compounds and application of marine peptides compounds to preparation of angiogenesis medicines |
| CN109796522A (en) * | 2019-02-18 | 2019-05-24 | 广州凯瑞腾生物医疗科技有限公司 | 10 Cyclopeptide derivatives of aplysiatoxin and its preparation method and application |
-
2018
- 2018-10-30 CN CN201811280501.4A patent/CN109575105B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434648A (en) * | 2008-09-28 | 2009-05-20 | 华东师范大学 | Peptide for inhibiting angiogenesis, and use thereof in angiogenesis medicament preparation |
| CN101979402A (en) * | 2010-10-19 | 2011-02-23 | 中山大学 | Marine cyclic peptide compound and preparation method and use thereof |
| CN102702310A (en) * | 2012-06-08 | 2012-10-03 | 中山大学 | Marine peptides compounds and application of marine peptides compounds to preparation of angiogenesis medicines |
| CN109796522A (en) * | 2019-02-18 | 2019-05-24 | 广州凯瑞腾生物医疗科技有限公司 | 10 Cyclopeptide derivatives of aplysiatoxin and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| Marine cyclotripeptide X-13 promotes angiogenesis in zebrafish and human endothelial cells via PI3K/Akt/eNOS signaling pathways;Xi-Lin Lu等;《Marine Drugs》;20120630;第10卷(第6期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109575105A (en) | 2019-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106232616B (en) | Amphipathic synthetic antibacterial peptide, its medical composition and its use | |
| CN112625094B (en) | Broad-spectrum coronavirus membrane fusion inhibitor and pharmaceutical application thereof | |
| CN105503627B (en) | A kind of new gossypol Shiff base derivative and preparation method and application | |
| CN109575105B (en) | Marine peptide compound and preparation method and application thereof | |
| CN103421090A (en) | Novel antimicrobial peptide | |
| CN107417772B (en) | Polypeptide HIP-20 capable of antagonizing RNA binding activity of hnRNPU protein and application thereof | |
| CN102656166A (en) | Crystalline salts of methyl 2-((r)-(3-chloro phenyl) ((r)-l-((s)-2-(methylamino)-3-((r)-tetrahydro-2h-pyran-3-yl)propylcarbamoyl) | |
| DE4120327A1 (en) | NEW PEPTIDES, THEIR PREPARATION AND USE | |
| CN102702310B (en) | Marine peptides compounds and application of marine peptides compounds to preparation of angiogenesis medicines | |
| CN112939910B (en) | Santamarine derivative, pharmaceutical composition thereof, preparation method and application thereof | |
| CN112110898B (en) | Synephrine sulfonylation derivative and intermediate, preparation method and application thereof | |
| CN105566447B (en) | The class peptide antagonists and its synthetic method of a kind of apoptosis inhibitory protein and application | |
| CN112521302A (en) | Capsaicin compound CP-X-Y with neuroprotective effect and application thereof | |
| CN114106305A (en) | Preparation method of supramolecular hydrogel factor with uniform and controllable chirality | |
| CN115043891B (en) | Compound and preparation method and application thereof | |
| US5798382A (en) | 2,3-dihydrobenzofuran derivatives, method for producing the same, and use thereof | |
| CN104004052B (en) | Azepine polypeptide compounds and preparation method thereof | |
| CN104497107B (en) | A kind of self-assembled short peptide and its application in three-dimensional cell cultivation | |
| RU2626972C2 (en) | Peptide with treatment action against alzheimer's disease | |
| CN110101841A (en) | Application of the antibacterial peptide Lchamp2-3 in antitumor cell diversion medicaments | |
| CN114957403B (en) | Broad-spectrum coronavirus membrane fusion inhibitor and pharmaceutical application thereof | |
| CN113546176B (en) | Synthesis method of drug specifically used for inhibiting intraocular angiogenesis | |
| AU2021105757A4 (en) | A Bioactive Peptide And Its Synthesis Method | |
| CN110078747B (en) | Novel thiazole drug molecule for hospital disinfection and preparation method thereof | |
| CN111777668B (en) | Marine cyclopeptide Samoamide A-based modified polypeptide, synthetic method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |