CN101270145A - Preparation technique for GHGKHKNK octapeptide and medicine use - Google Patents
Preparation technique for GHGKHKNK octapeptide and medicine use Download PDFInfo
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Abstract
The present invention discloses a method for preparing GHGKHKNK octapeptide, in which, 2-chlorine-triphenylmethyl resin, triphenylmethyl resin, 4-methyl triphenylmethyl resin or 4-methoxy triphenylmethyl resin are used as starting material which is orderly linked with aminophenols with protective groups by a method of solid phase synthesis to obtain protective octapeptide resin, meanwhile, Fmoc protective groups are orderly removed, TBTU or HBTU and HOBT are used as condensing agents to have peptide synthesis reaction, the removal of side chain protecting groups and peptide cleavage are synchronously carried out, and therefore the crude product of the octapeptide compound is obtained; the crude product is separated and purified by a C18 or C8 column, and after refrigeration and drying, the finished product is produced. The present invention also provides the application of the GHGKHKNK octapeptide as a main active ingredient in the preparation of anti-tumour drugs.
Description
Technical field
The invention provides a kind of preparation technology of GHGKHKNK octapeptide, the invention also discloses its medicinal use aspect anti metastasis, belong to field of polypeptide medicine technology in biochemistry.
Background technology
In recent years, along with going deep into of research, polypeptide causes people's attention gradually to the restraining effect of tumor growth and transfer.Chinese scholars is by methods such as extraction from animals and plants, phage peptide library screening and chemosynthesis, obtain some and had the micromolecule polypeptide of anti-tumor activity, these little peptides can be at the growth of tumour cell, the different links of development, the infiltration and the transfer of specific killing, inhibition tumour cell.Discovery 5 peptide YIGSR (tyrosine-Isoleucine-glycine-Serine-arginine) such as nineteen ninety Alino can make lung cancer metastasis ability drop (Alino SF, Unda F J, Perez-Yarza G.Laminin surface binding sites and metastatic potential of 3LLtumor cells, increased by indomethacin[J] .Biochem Biophys ResCommun, 1990,167 (2): 731-738.).Antineoplastic micromolecule polypeptide molecular weight is little, active high, the tumor cell line of multi-medicine resistance is also had good inhibition activity.Micromolecule polypeptide administration in many ways is difficult for causing immune response simultaneously, and the production cost of artificial chemosynthesis is lower, and these are indicating that all the small molecules natineoplaston has good application prospects at clinicing aspect.
Recently, discover that the zone of being rich in Histidine in people's high-molecular-weight kininogen the 5th structural domain can optionally suppress human umbilical vein and the epithelial propagation of capillary vessel and suppress the formation of mouse cornea neovascularity, also can be as anti-adhesive albumen and VN competition, combine with urokinase receptor, and then suppress epithelial invasion and attack and adhesion.
Summary of the invention
The present invention discloses a kind of preparation technology of GHGKHKNK octapeptide, has process stabilizing, and the raw and auxiliary material convenient sources is with short production cycle, the characteristics that production cost is low.
The invention also discloses the application of GHGKHKNK octapeptide in the preparation antitumor drug.
The GHGKHKNK octapeptide that the present invention relates to has following primary structure:
Gly-His-Gly-Lys-His-Lys-Asn-Lys
The concrete preparation technology of GHGKHKNK octapeptide of the present invention is as follows:
With 2-chloro-trityl resin, 4-methyl trityl resin, 4-methoxyl group trityl resin or trityl resin is starting raw material; method by solid phase synthesis connects the amino acid with blocking group successively; obtain protection octapeptide resin; slough the Fmoc-blocking group therebetween successively; with TBTU or HBTU and HOBT is that condensing agent connects reactive polypeptide; take off the side chain protected group synchronously and cut peptide, obtain this octapeptide compound crude product.Through C18 (or C8) column separating purification, after the lyophilize, make elaboration again.
According to the present invention, connect amino acid successively with blocking group, obtain protection octapeptide resin, the concrete steps of sloughing the Fmoc-blocking group therebetween successively comprise:
(1) preparation Fmoc-Lys (Boc)-resin
Will be with 2-chloro-trityl resin, 4-methyl trityl resin, 4-methoxyl group trityl resin or trityl resin, soaked 10-60 minute with DMF, make the abundant swelling of resin, add DIPEA or DMPA and Fmoc-Lys (Boc)-OH, 10～15 ℃ were reacted 1～24 hour;
Add 10～50 ℃ of reactions of methyl alcohol 1～20 hour again, nitrogen dries up, and resin washs with DMF, obtains Fmoc-Lys (Boc)-resin;
Said resin replacement amount is: 0.3-1.5mmol/g; Particle diameter is 100～400 orders;
The mole number of Fmoc-Lys (Boc)-OH is 2～5 times of resin;
The mole number of DIPEA or DMPA is 2～20 times of resin;
The add-on of reagent of raising one's hat is that benchmark is 5-20ml/g with the weight of resin;
The mole coefficient of methyl alcohol is 2～20 times of resin;
(2) preparation Fmoc-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Lys of step (1) (Boc)-resin, add the reagent of raising one's hat, 10～50 ℃ were reacted 10～60 minutes, and dried up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Asn (Trt)-OH, TBTU/HBTU and HOBT, 10～50 ℃ were reacted 1～5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Asn (Trt)-Lys (Boc)-resin;
The said reagent of raising one's hat is: PIP: DMF=1: 2～5, and volume ratio;
The add-on of reagent of raising one's hat is that benchmark is 5-20ml/g with the weight of resin;
In the mixture: the mole coefficient of Fmoc-Asn (Trt)-OH is 2～5 times of resin;
The mole coefficient of TBTU/HBTU and HOBT is 2～5 times of resin;
The said peptide reagent that connects is: NMM: DMF=1: 5～15, and volume ratio;
Connect in the peptide reagent, the mole coefficient of Fmoc-Asn (Trt)-OH is 2～5 times of resin;
" TBTU/HBTU and HOBT " refers to: the mixture of TBTU and HOBT, or the mixture of HBTU and HOBT;
(3) preparation Fmoc-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Asn of step (2) (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-Lys (Boc)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-Lys (Boc)-OH is 2～5 times of resin.
The same step of all the other operations and processing condition (2);
(4) preparation Fmoc-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Lys of step (3) (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-His (Trt)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-His (Trt)-OH is 2～5 times of resin.
The same step of all the other operations and processing condition (2);
(5) preparation Fmoc-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-His of step (4) (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-Lys (Boc)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Fmoc-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-Lys (Boc)-OH is 2～5 times of resin.
The same step of all the other operations and processing condition (2);
(6) preparation Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Lys of step (5) (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-Gly-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-Gly-OH is 2～5 times of resin.
The same step of all the other operations and processing condition (2);
(7) preparation Fmoc-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Gly-Lys of step (6) (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-His (Trt)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-His (Trt)-OH is 2～5 times of resin.
The same step of all the other operations and processing condition (2);
(8) preparation Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-His of step (7) (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Boc-Gly-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Boc-Gly-OH is 2～5 times of resin.
The same step of all the other operations and processing condition (2);
(9) preparation Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc) is in step (8) Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add to meet and be chilled to 10～20 ℃ the peptide reagent of cutting, 10～50 ℃ were reacted 1～5 hour, pressure reducing and steaming desolventizes, add ether sedimentation, the collecting precipitation thing is washed with ether, P
2O
5Vacuum-drying obtains crude product;
Said component of cutting peptide reagent is: TFA/EDT/HO/TIS=90-95/2-5/1-5, volume ratio;
According to optimized technical scheme of the present invention, separation and purification comprises the steps:
Crude product is dissolved in the ammonium acetate, filters, filtrate is through C18 (or C8) column purification, moving phase: 0.1MNH
4AC: acetonitrile (7.5: 2.5); Mobile speed is 100～650ml/min; The detection wavelength is: 280nm; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph, the sample peak desalts after merging, and freeze-drying obtains finished product, and total recovery is 30～40%.
Operational path of the present invention has following positively effect: take to cut peptide reagent (TFA/EDT/TIS) and cut peptide, connect peptide yield height (per step connects the peptide yield and is 〉=99%), the method that adds the ether sedimentation crude product, avoid using poisonous reagents such as hydrogen fluoride, pollution in three fens is few, and improves yield, adopt C18 (or C8) column separating purification, avoid using TFA to carry out purifying, reduce the three wastes, per step connects the peptide yield all more than 98%; Total recovery is 25～35%.Possess the large-scale production ability, process stabilizing, the raw and auxiliary material convenient sources, with short production cycle, production cost is low, and the three wastes are few, the yield height, steady quality,
The present invention prepares antitumor drug with the GHGKHKNK octapeptide as main active ingredient, has obvious inhibition metastases activity, and it can be used for treating the relevant disease of kinds of tumors.
Below experiment shows that the GHGKHKNK octapeptide is to the treatment for cancer effect:
One, the GHGKHKNK octapeptide is to the vitro inhibition effect of mouse melanin tumor cell B16-F10
According to people such as Buttke ([1] Buttke TU, Mc Cubrgy JA, Owen TC.Use of an aqeous tetrazolium/JImmunol Methods, 1993,157:233-240.) described MTS colorimetry detects the vitro inhibition effect of GHGKHKNK octapeptide to mouse melanin tumor cell B16-F10, observes the vitro inhibition effect of GHGKHKNK octapeptide to mouse melanin tumor cell B16-F10.
The mouse melanin tumor cell B16-F10 single cell suspension that this test is taken the logarithm vegetative period contains 10%FBSIMDM and adjusts cell concn to 2 * 10
5/ ml is inoculated in 96 orifice plates with every hole 100 μ l, and 37 ℃, 5%CO2 are hatched 24h.Abandon supernatant, every hole adds the octapeptide 100 μ l of serum-free IMDM preparation, and the medicine final concentration is respectively 10
-4, 10
-5, 10
-6, 10
-7, 10
-8Mol/L is a control group with serum-free IMDM.Establish 4 multiple holes for every group.37 ℃, 5%CO
2Cultivate 24h, 48h respectively.The centrifugal supernatant that goes adds MTS/PMS liquid 40 μ l, 37 ℃, 5%CO
2Hatch 4h.The centrifugal supernatant that goes, mixing are surveyed absorbancy (A) value in the 490nm place.Be calculated as follows cell inhibitory rate.Cell inhibitory rate=(1-administration group A value/control group A value) * 100%.Octapeptide drug level among the present invention is 10
-4Mol/L～10
-8Mol/L in external generation to mouse melanin tumor cell B16-F10 growth-inhibiting in various degree.The concentration of GHGKHKNK octapeptide is 10
-4During mol/L, can significantly suppress the growth of mouse melanin tumor cell B16-F10 behind interaction in vitro 24h and 48h, concrete outcome is as follows:
The GHGKHKNK octapeptide is to the vitro inhibition effect of mouse melanin tumor cell B16-F10
Relatively has significant difference (P＜0.05) with control group
Two, the external influence of GHGKHKNK octapeptide to tumour cell clonality and invasive ability
According to people (Neoplasia.2006 November such as Li Liu, 8 (11): 917-924) described method is finished plate clone and is formed experiment and cell invasion experiment, observes the external influence to tumour cell clonality and invasive ability of octapeptide of the present invention.
The method that plate clone forms experiment is that 3 groups of cells with logarithmic phase become the individual cells suspension with the 2.5g/L tryptic digestion respectively, and makes the gradient doubling dilution, and in 24 orifice plates, every group of cell inoculated 6 holes by 50 cell inoculations in every hole.In static cultivation 2-3 week, when appearring in culture plate, macroscopic clone stops cultivating.PBS adds fixedly 15min of pure methyl alcohol 1mL after cleaning, the dyeing of Jim Sa liquid.Culture plate is placed clone's number of counting under the low multiple of microscope greater than 50 cells, by formula calculate cloning efficiency.Cloning efficiency (%)=clone's number/inoculation number * 100%.
The method of cell invasion experiment is to be the upper surface that 0.5g/L Matrige1 artificial substratum glue 20 μ L are laid on the poly-carbon ester microporous membrane (aperture 8 μ m) of Transwell invasion and attack cell with concentration, puts 37 ℃ of 30min and makes it gather into gel.Add respectively in the last chamber of Transwell and digested the resuspended cell 100 μ L (1 * 10 that respectively organize
8/ L), add 600 μ L in the following chamber and contain chemokine serum-free RPMI-1640 substratum, take out after cultivating 24h, PBS cleans, cotton swab is removed confluent monolayer cells on the filter membrane, to invade and be attached at the cell fixation and the dyeing of Jim Sa liquid of microporous membrane lower floor, the microscopically direct viewing is passed the cell count of film.Count 5 visuals field at random, count the cell count of passing 8 μ m micropores in each visual field.Relative number with the invasion and attack cell is represented the invasion by tumor cells ability.GHGKHKNK octapeptide of the present invention external to tumour cell clonality and invasive ability to influence the result as follows:
Various dose GHGKHKNK octapeptide all has restraining effect to clone's formation of mouse melanin tumor cell B16-F10, and comparing with control group all has significant difference (P＜0.05); Octapeptide GHGKHKNK compound 10 wherein
-4Mol/L dosage inhibition rate of tumor cell reaches 56.81% (see Table 1, Fig. 1).
The restraining effect result that table 1 GHGKHKNK octapeptide forms mouse melanin tumor cell B16-F10 clone
10
-4Mol/L and 10
-5Mol/LGHGKHKNK can significantly suppress the invasion and attack of mouse melanin tumor cell B16-F10 behind interaction in vitro 48h, wear theca cell number and control group and relatively have significant difference (P＜0.05) (see Table 2, Fig. 2).
The invasion and attack restraining effect of table 2 GHGKHKNK octapeptide mouse melanin tumor cell B16-F10
From shown in the above data as can be seen, octapeptide among the present invention has the obvious suppression effect to clonality and the invasive ability of mouse melanin tumor cell B16-F10, and this restraining effect is dose-dependently, and octapeptide of the present invention has improved the clonality of vitro inhibition mouse melanin tumor cell B16-F10 and the activity of invasive ability significantly.
Three, the external influence of tumour cell being sticked ability of GHGKHKNK octapeptide
According to people (Neoplasia.2006 November such as Li Liu, 8 (11): 917-924) described immunohistochemical methods method detects the influence to LN-R, ICAM-1, E-cadhesion expression, observes the external influence of tumour cell being sticked ability of GHGKHKNK octapeptide of the present invention.
Experiment uses immunocytochemistry SP test kit (LN-R, ICAM-1, E-cadhesion) to carry out.The mouse melanin tumor cell B16-F10 that at first will be in logarithmic phase is digested to single cell suspension, and adjusting cell concn is 1 * 10
5Ready slide is placed in 24 well culture plates, cell suspension is dripped to little slide, when treating that the cell stand density reaches 75% saturation ratio, discard the every hole of substratum and add the serum-free RPMI-1640 substratum 0.9ml that contains different pharmaceutical concentration, control group adds 1ml serum-free RPMI-1640 substratum, 37 ℃, 5%CO
2Incubator is hatched 48h, PBS cleans 3 times then, with the fixing 10min of methyl alcohol, finish again with PBS develop a film dry for 1 time after, be fixed on the slide glass with neutral gum, 3% hydrogen peroxide is hatched 8min, and PBS cleans 3 times, drips normal goats serum working fluid, incubated at room 15min discards, drip LN-R, ICAM-1, E-cadhesion antibody working fluid respectively, place 1h for 37 ℃, PBS cleans 3 times, the DAB after washing that develops the color, the Mayer haematoxylin redyeing is got five visuals field at random at microscopically then and is write down 100 cells respectively, the record positive cell number.Octapeptide among the present invention to the expression of LN-R, ICAM-1, three kinds of factors of E-cadhesion to influence the result as follows:
Table 3:GHGKHKNK octapeptide is to the result that influences of mouse melanin tumor cell B16-F10LN-R, ICAM-1, E-cadhesion expression
From shown in the above data as can be seen, octapeptide among the present invention can be reduced LN-R, ICAM-1, the E-cadhesion expression at mouse melanin tumor cell B16-F10,, this downward modulation effect demonstrates octapeptide of the present invention and has improved the activity that vitro inhibition mouse melanin tumor cell B16-F10 sticks ability significantly.
Four, the GHGKHKNK octapeptide is to the therapeutic action of mouse melanin tumor cell B16-F10 artificial lung metastasis model
According to people such as Han Rui chief editor (cancer therapy drug research and experiment technology. Beijing: combined publication society of Beijing Medical University of China Concord Medical Science University, 1997:368～369.) described method is finished the therapeutic action experiment of octapeptide to melanochrome tumour B16-F10 cell artificial lung metastasis model, observes the therapeutic action of GHGKHKNK octapeptide of the present invention to mouse melanin tumor cell B16-F10 artificial lung metastasis model.
Briefly, the mouse melanin tumor cell B16-F10 in vegetative period that at first takes the logarithm, Hanks liquid is adjusted cell concn to 1 * 10
6/ ml, C57BL/6J mouse tail vein injection, every 0.2ml.Be divided into physiological saline group (0.2ml/d) at random, GHGKHKNK octapeptide dosage I organizes [500 μ g (kgd)
-1], GHGKHKNK octapeptide dosage II organizes [50 μ g (kgd)
-1], GHGKHKNK octapeptide dosage III organizes [5 μ g (kgd)
-1].Time Nikkei abdominal injection is respectively organized medicine behind the tumor inoculation, once a day, and successive administration 20 days.Finish the back in administration and take off cervical vertebra execution mouse next day, get lung, Bouin is liquid-solid fixed, and dissecting microscope is counting pulmonary metastases kitchen range number down, and animal lung tissue is carried out paraffin section, is calculated as follows inhibiting rate.
Inhibiting rate=(1-administration group mean transferred knurl kitchen range number/physiological saline group mean transferred knurl kitchen range number) * 100%.
GHGKHKNK octapeptide among the present invention is as follows to the therapeutic action result of melanochrome tumour B16-F10 cell artificial lung metastasis model:
The therapeutic action that table 4:GHGKHKNK octapeptide shifts mouse melanin tumor cell B16-F10 artificial lung
*: compare P＜0.05# with control group: compare P＜0.05 with ring phosphamide group
The pathological observation result: microscopically, physiological saline group pulmonary metastases tissue is nido, sheet piece, is distributed in around the blood vessel or the pleura place more, is multiple, is dispersed in distribution, and area is bigger, knurl bolt in the accidental blood vessel.It is in the majority that administration group pulmonary metastases tissue is spherical shape, and multidigit is in pipe week or pleura place, but area is less, even by several cellularities, area the greater, visible central authorities or the sheet necrosis of off normal place.Above-mentioned two groups of oncocyte forms size basically identical.See Fig. 3.
Five, the GHGKHKNK octapeptide is to the influence of transplantability ascitic type liver cancer H22 mouse survival time.
According to people such as Han Rui chief editor (cancer therapy drug research and experiment technology. Beijing: combined publication society of Beijing Medical University of China Concord Medical Science University, 1997:368～369.) described method is finished the influence of GHGKHKNK octapeptide to solid tumor growth-inhibiting effect of H22 hepatic ascites knurl mice with tumor and survival time, observes the influence of GHGKHKNK octapeptide of the present invention to transplantability ascitic type liver cancer H22 mouse survival time.
6-8 days ascitic type liver cancer H22 tumor-bearing mice is got in test under aseptic condition, put to death fixing, with the tincture of iodine and alcohol disinfecting operating station skin, cut then, pass abdominal muscle with asepsis injector and extract ascites, with the dilution of Hanks liquid, 1200rpm, after the 10min washed twice, adjusting cell count with Hanks liquid is 1 * 10
7The cell suspension of/ml, except that the normal control group, other the every mouse abdominal injection of each treated animal ascites diluent 0.1ml.Basic, normal, high dosage group of octapeptide and physiological saline group are in tumor inoculation the first five day beginning administration, endoxan (CPA) positive controls begins administration next day behind tumor inoculation, each is organized in the physiological saline that medicine all is dissolved in 0.2ml, intraperitoneal injection, every day 1 time, continuous 60 days, perhaps when animal dead, finish.24hr puts to death the half mouse after art time administration, and every treated animal is observed the generalized case of animal day by day after administration, record death time, existence fate, calculating increase in life span.Calculate increase in life span as follows: increase in life span=(experimental group The average survival time fate-control group The average survival time fate)/control group The average survival time fate * 100%.Pluck knurl and weigh, judge tumor killing effect with tumour inhibiting rate (TGI),
The tumour inhibiting rate calculation formula is as follows:
TGI (%)=(control group knurl weight-experimental group knurl is heavy)/control group knurl heavy * 100%.
Test repeats 3 times, and the result is the mean value of 3 tests.And by flow cytometry ascites is detected, GHGKHKNK octapeptide of the present invention to transplantability ascitic type liver cancer H22 mouse survival time to influence the result as follows: normal control group mouse to experimental observation finishes 60 days, still all survivals.The normal control group has been compared significant difference (P＜0.05) with the physiological saline control group, the endoxan group can obviously prolong ascitic type liver cancer H22 mouse survival time, compared significant difference (P＜0.05) with the physiological saline control group, each administration group of octapeptide all can obviously prolong the survival time of ascitic type liver cancer H22 mouse, has compared significant difference (P＜0.05) with the physiological saline control group.The increase in life span of H22 mouse was 60.25% when the octapeptide dosage was 5ug/kg, and increase in life span and dosage present certain dose-effect relationship (seeing Table).Curative effect was the most remarkable when wherein dosage was 50ug/kg, reached 60.25%.Curative effect descended on the contrary to some extent when dosage was 500ug/kg.60 days serving as to observe the time limit, survival time is greater than thinking long-term surviving in 60 days.The endoxan group is not good at the phase survival mice, and each group of octapeptide all has the long-term surviving mouse.By flow cytometry ascites of each group mouse detect is found in the visible significantly phenomena of apoptosis of administration group simultaneously, especially with middle low dose group the most obviously (as Fig. 4, shown in 5).
Concrete outcome is as follows:
Table 5:GHGKHKNK octapeptide is to the influence of transplantability ascitic type liver cancer H22 mouse survival time
* compare (P＜0.05=with the normal control group
Table 6:GHGKHKNK octapeptide is to the apoptosis rate of transplantability ascitic type liver cancer H22 mouse ascites
Compare P＜0.05 with the normal control group
According to known method in the medicine industry, pharmaceutically acceptable carrier of GHGKHKNK octapeptide and one or more or vehicle are mixed mutually, make the pharmaceutical composition that is suitable for the various different dosage forms of administration outside gi tract or gi tract.Pharmaceutically acceptable carrier comprises avirulent and activeconstituents is not had the prescription auxiliary of solid, semisolid or liquid filling agent, thinner, capsule lapping or any kind of interference effect.Administrations such as obtained pharmaceutical composition can be outside gi tract, oral and part.
The outer approach of gi tract comprises in intravenously, intramuscular, body cavity (as abdominal cavity, thoracic cavity or myelencephalon chamber) and the tissue lumen (as nasal cavity, oral cavity, rectum, vagina or uterine cavity), and route of administration such as intracutaneous, subcutaneous or intraocular.For through the gi tract external administration, GHGKHKNK octapeptide of the present invention can be dissolved in and make solution or suspension in water, salt solution, dextrose solution, ethanol or the oil.Employed carrier also can comprise other compositions such as sanitas, suspensoid, solubilizing agent, buffer reagent.When compound (peptide) during, also it can be dissolved in the cerebrospinal fluid with the intrathecal route administration.
For oral or mucosal, the GHGKHKNK octapeptide can be made solid or liquid preparation that capsule, pill, tablet, the agent of speeding, powder agent, suspension agent or emulsion form.Can make carriers such as water, ethylene glycol, oil, alcohol, flavouring agent, sanitas, tinting material, suspension agent or ancillary component prepare oral liquid formulation (as solution, suspension and the agent of speeding); Or use carrier such as starch, sugar, granulating agent, solid diluent, lubricant, binding agent, disintegrating agent or auxiliary to prepare solid-state oral preparations (as powder agent, tablet, suppository and capsule).In case of necessity, can use microcapsule or liposome activeconstituents, making can be by the stabilization formulations (as referring to international monopoly WO 86/11698) of gi tract or hemato encephalic barrier.
The dosage of pharmaceutical composition of the present invention was generally 5 to 500 μ g/kg/ days, and can divide administration several times every day with the sub-doses form.But definite dosage should be according to the character of disease to be treated, disease severity, patient's age and the general situation of health, and factor such as route of administration is determined by the clinician.
Above-mentioned experiment in vitro shows that tentatively GHGKHKNK octapeptide of the present invention is to the clonality of tumour cell, and invasive ability and the ability of sticking have the obvious suppression effect.
GHGKHKNK compound of the present invention or its pharmaceutical composition can be used for preventing or treat former or the various solid tumors or the hemopoietic system cell neoplasm disease that shift.When being used for the prophylaxis of tumours transfer, can use separately or with tumor radiotherapy or chemotherapy combined.For example can behind the solid tumor radiotherapy, unite and use peptide compounds of the present invention and one or more conventional tumor chemotherapeutic drugs.Perhaps,, continue to use peptide compounds of the present invention through after operation, radiotherapy and the chemotherapy, removing the small metastases kitchen range that may exist in the body, or stable and suppress any remaining primary tumo(u)r.
Description of drawings
The restraining effect result that Fig. 1, GHGKHKNK octapeptide form mouse melanin tumor cell B16-F10 clone;
The invasion and attack restraining effect of Fig. 2, GHGKHKNK octapeptide mouse melanin tumor cell B16-F10;
Fig. 3, lung metastatic tumour histopathology form (HE * 40);
A：Lung?metastatic?lesion?treated?with?50μg/kg/d?peptide?compoundGHGKHKNK；
B：Lung?metastatic?lesion?treated?with?500μg/kg/d?peptide?compoundGHGKHKNK；
C：Lung?metastatic?lesion?treated?with?normal?saline.
Fig. 4, low dose group cell cycle analysis figure;
Fig. 5, physiological saline group cell cycle analysis figure;
Fig. 6.
Embodiment
For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood that it is to explaination of the present invention but not to any type of restriction of the present invention.
Embodiment 1:
Peptide chain Gly-His-Gly-Lys-His-Lys-Asn-Lys's is synthetic
1, synthetic peptide chain
(1) preparation Fmoc-Lys (Boc)-resin
Get 2-chloro-trityl resin 50 grams, soaked 30 minutes with DNF, make the abundant swelling of resin, add 44mlDIPEA, 46.9g Fmoc-Lys (Boc)-OH reacted 3 hours, added 50ml methyl alcohol again and continued reaction 3 hours.Nitrogen dries up, and resin washs three times with DMF.Obtain Fmoc-Lys (Boc)-resin;
(2) preparation Fmoc-Asn (Trt)-Lys (Boc)-resin:
Add the 800ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen blows the elimination hexahydropyridine, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
Adding Fmoc-Asn (Trt)-OH (MV:596.7,200mmol) 119.9g, TBTU (NW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NNM 44.4ml (MW:101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
Add the 500ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen dries up, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
(3) preparation Fmoc-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Lys (Boc)-OH (MV:468.5,200mmol) 93.7g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
Add the 500ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen dries up, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
(4) preparation Fmoc-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-His (Trt)-OH (MV:619.7,200mmol) 123.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(5) preparation Fmoc-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Lys (Boc)-0H (MV:468.5,200mmol) 93.7g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(6) preparation Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Gly-OH (MV:297.3,200mmol) 59.5g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(7) preparation Fmoc-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-His (Trt)-OH (MV:619.7,200mmol) 123.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(8) preparation Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Boc-Gly-OH (MV:175.2,200mmol) 35.4g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
Again with anhydrous methanol washing three times.After draining, it is dry to put into vacuum drier, weighs, and gets the about 90g of octapeptide resin.(MW=906.2 43mmol), connects the peptide total recovery and is about 86%.
2, cut peptide
Get 88.9g octapeptide resin transfer in eggplant-shape bottle, cold cut adds down cuts peptide reagent: (TFA/ water/dithioglycol/thioanisole=650ml/17ml/17m/34ml), 25 ℃ were stirred 2 hours.Filter, drain, filtrate adds the anhydrous diethyl ether precipitation, and filtering collecting precipitation must about 35g octapeptide crude product.
3, purifying
Filtrate is in batches through the C18 column purification, moving phase: 0.1MNH
4AC: acetonitrile (7.5: 2.5); The speed that flows is 380ml/min; The detection wavelength is: 280nm; Desalt after the sample peak merges, freeze-drying, approximately 13.9g white loose block finished product (MW:906.2,15.3mmol); Yield 30.6%.Follow the tracks of the needed effluent liquid of collection with liquid chromatograph
Embodiment 2:
1, synthetic peptide chain
(1) preparation Fmoc-Lys (Boc)-resin
Get 4-methyl trityl resin 50 grams, soaked 30 minutes with DMF, make the abundant swelling of resin, add 44mlDIPEA, 46.9g Fmoc-Lys (Boc)-OH reacted 3 hours, added 50ml methyl alcohol again and continued reaction 3 hours.Nitrogen dries up, and resin washs three times with DMF.Obtain Fmoc-Lys (Boc)-resin;
(2) preparation Fmoc-Asn (Trt)-Lys (Boc)-resin:
Add the 800ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen blows the elimination hexahydropyridine, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
Adding Fmoc-Asn (Trt)-OH (MV:596.7,200mmol) 119.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
Add the 500ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen dries up, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
(3) preparation Fmoc-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Lys (Boc)-OH (MV:468.5,200mmol) 93.7g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
Add the 500ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen dries up, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
(4) preparation Fmoc-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-His (Trt)-OH (MV:619.7,200mmol) 123.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(5) preparation Fmoc-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Lys (Boc)-OH (MV:468.5,200mmol) 93.7g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(6) preparation Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Gly-OH (MV:297.3,200mmol) 59.5g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(7) preparation Fmoc-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-His (Trt)-OH (MV:619.7,200mmol) 123.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(8) preparation Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Boc-Gly-OH (MV:175.2,200mmol) 35.4g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
Again with anhydrous methanol washing three times.After draining, it is dry to put into vacuum drier, weighs, and gets the about 92g of octapeptide resin.(MW=906.2 43mmol), connects the peptide total recovery and is about 88%.
2, cut peptide
Get 92.1g octapeptide resin transfer in eggplant-shape bottle, cold cut adds down cuts peptide reagent: (TFA/ water/dithioglycol/thioanisole=650ml/17ml/17m/34ml), 25 ℃ were stirred 2 hours.Filter, drain, filtrate adds the anhydrous diethyl ether precipitation, and filtering collecting precipitation must about 37g octapeptide crude product.
3, purifying
Filtrate is in batches through the C18 column purification, moving phase: 0.1MNH
4AC: acetonitrile (7.5: 2.5); The speed that flows is 380ml/min; The detection wavelength is: 280nm; Desalt after the sample peak merges, freeze-drying, approximately 14.5g white loose block finished product (MW:906.2,15.3mmol); Yield 32.0%.Follow the tracks of the needed effluent liquid of collection with liquid chromatograph.
Embodiment 3:
1, synthetic peptide chain
(1) preparation Fmoc-Lys (Boc)-resin
Get 4-methoxyl group trityl resin 50 grams, soaked 30 minutes with DMF, make the abundant swelling of resin, add 44mlDIPEA, 46.9g Fmoc-Lys (Boc)-OH reacted 3 hours, added 50ml methyl alcohol again and continued reaction 3 hours.Nitrogen dries up, and resin washs three times with DMF.Obtain Fmoc-Lys (Boc)-resin;
(2) preparation Fmoc-Asn (Trt)-Lys (Boc)-resin:
Add the 800ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen blows the elimination hexahydropyridine, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
Adding Fmoc-Asn (Trt)-OH (MV:596.7,200mmol) 119.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
Add the 500ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen dries up, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
(3) preparation Fmoc-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Lys (Boc)-OH (MV:468.5,200mmol) 93.7g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
Add the 500ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen dries up, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
(4) preparation Fmoc-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-His (Trt)-OH (MV:619.7,200mmol) 123.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(5) preparation Fmoc-Lys (Boc)-Hi s (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Lys (Boc)-OH (MV:468.5,200mmol) 93.7g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(6) preparation Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Gly-OH (MV:297.3,200mmol) 59.5g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(7) preparation Fmoc-His (Trt)-Gl y-Lys (Boc)-Hi s (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-His (Trt)-OH (MV:619.7,200mmol) 123.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(8) preparation Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Boc-Gly-OH (MV:175.2,200mmol) 35.4g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
Again with anhydrous methanol washing three times.After draining, it is dry to put into vacuum drier, weighs, and gets the about 88.0g of octapeptide resin.(MW=906.2 43mmol), connects the peptide total recovery and is about 84%.
2, cut peptide
Get 88.0g octapeptide resin transfer in eggplant-shape bottle, cold cut adds down cuts peptide reagent: (TFA/ water/dithioglycol/thioanisole=650ml/17ml/17m/34ml), 25 ℃ were stirred 2 hours.Filter, drain, filtrate adds the anhydrous diethyl ether precipitation, and filtering collecting precipitation must about 34.5g octapeptide crude product.
3, purifying
Filtrate is in batches through the C18 column purification, moving phase: 0.1MNH
4AC: acetonitrile (7.5: 2.5); The speed that flows is 380ml/min; The detection wavelength is: 280nm; Desalt after the sample peak merges, freeze-drying, approximately 13.2g white loose block finished product (MW:906.2,15.3mmol); Yield 29.2%.Follow the tracks of the needed effluent liquid of collection with liquid chromatograph.
Embodiment 4:
1, synthetic peptide chain
(1) preparation Fmoc-Lys (Boc)-resin
Get basic trityl resin 50 grams, soaked 30 minutes with DMF, make the abundant swelling of resin, add 44ml DIPEA, 46.9g Fmoc-Lys (Boc)-OH reacted 3 hours, added 50ml methyl alcohol again and continued reaction 3 hours.Nitrogen dries up, and resin washs three times with DMF.Obtain Fmoc-Lys (Boc)-resin;
(2) preparation Fmoc-Asn (Trt)-Lys (Boc)-resin:
Add the 800ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen blows the elimination hexahydropyridine, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
Adding Fmoc-Asn (Trt)-OH (MV:596.7,200mmol) 119.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
Add the 500ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen dries up, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
(3) preparation Fmoc-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Lys (Boc)-OH (MV:468.5,200mmol) 93.7g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
Add the 500ml volumetric concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes, nitrogen dries up, and with DMF, anhydrous methanol, DMF washing three times, nitrogen dries up respectively.
(4) preparation Fmoc-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-His (Trt)-OH (MV:619.7,200mmol) 123.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(5) preparation Fmoc-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Lys (Boc)-OH (MV:468.5,200mmol) 93.7g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(6) preparation Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-Gly-OH (MV:297.3,200mmol) 59.5g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(7) preparation Fmoc-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Fmoc-His (Trt)-OH (MV:619.7,200mmol) 123.9g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
(8) preparation Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the resin of the above-mentioned reaction of raising one's hat, add Boc-Gly-OH (MV:175.2,200mmol) 35.4g, TBTU (MW:32l, 200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW:101.2), 400mlDMF, the same step of concrete grammar (3).
Again with anhydrous methanol washing three times.After draining, it is dry to put into vacuum drier, weighs, and gets the about 91.5g of octapeptide resin.(MW=906.2 43mmol), connects the peptide total recovery and is about 87%.
2, cut peptide
Get 91.5g octapeptide resin transfer in eggplant-shape bottle, cold cut adds down cuts peptide reagent: (TFA/ water/dithioglycol/thioanisole=650ml/17ml/17m/34ml), 25 ℃ were stirred 2 hours.Filter, drain, filtrate adds the anhydrous diethyl ether precipitation, and filtering collecting precipitation must about 36.6g octapeptide crude product.
4, purifying
Filtrate is in batches through the C18 column purification, moving phase: 0.1MNH
4AC: acetonitrile (7.5: 2.5); The speed that flows is 380ml/min; The detection wavelength is: 280nm; Desalt after the sample peak merges, freeze-drying, approximately 14.3g white loose block finished product (MW:906.2,15.3mmol); Yield 31.4%.Follow the tracks of the needed effluent liquid of collection with liquid chromatograph.
Claims (3)
1. the preparation technology of a GHGKHKNK octapeptide, it is characterized in that: making resin, 4-methyl trityl resin or 4-methoxyl group trityl resin with 2-chloro-trityl resin, trityl resin is starting raw material, method by solid phase synthesis connects the amino acid with blocking group successively, obtain protection octapeptide resin, slough the Fmoc-blocking group therebetween successively, with TBTU or HBTU and HOBT is that condensing agent connects reactive polypeptide, take off the side chain protected group synchronously and cut peptide, obtain this octapeptide compound crude product; Through C18 or C8 column separating purification, after the lyophilize, make elaboration again;
2. preparation technology according to claim 1 comprises the steps:
(1) preparation Fmoc-Lys (Boc)-resin
Will be with 2-chloro-trityl resin, 4-methyl trityl resin, 4-methoxyl group trityl resin or trityl resin, soaked 10-60 minute with DMF, make the abundant swelling of resin, add DIPEA or DMPA and Fmoc-Lys (Boc)-OH, 10～15 ℃ were reacted 1～24 hour;
Add 10～50 ℃ of reactions of methyl alcohol 1～20 hour again, nitrogen dries up, and resin washs with DMF, obtains Fmoc-Lys (Boc)-resin;
Said resin replacement amount is: 0.3-1.5mmol/g; Particle diameter is 100～400 orders;
The mole number of Fmoc-Lys (Boc)-OH is 2～5 times of resin;
The mole number of DIPEA or DMPA is 2～20 times of resin;
The add-on of reagent of raising one's hat is that benchmark is 5-20ml/g with the weight of resin;
The mole coefficient of methyl alcohol is 2～20 times of resin;
(2) preparation Fmoc-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Lys of step (1) (Boc)-resin, add the reagent of raising one's hat, 10～50 ℃ were reacted 10～60 minutes, and dried up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Asn (Trt)-OH, TBTU/HBTU and HOBT, 10～50 ℃ were reacted 1～5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Asn (Trt)-Lys (Boc)-resin;
The said reagent of raising one's hat is: PIP: DMF=1: 2～5, and volume ratio;
The add-on of reagent of raising one's hat is that benchmark is 5-20ml/g with the weight of resin;
In the mixture: the mole coefficient of Fmoc-Asn (Trt)-OH is 2～5 times of resin;
The mole coefficient of TBTU/HBTU and HOBT is 2～5 times of resin;
The said peptide reagent that connects is: NMM: DMF=1: 5～15, and volume ratio;
Connect in the peptide reagent, the mole coefficient of Fmoc-Asn (Trt)-OH is 2～5 times of resin;
" TBTU/HBTU and HOBT " refers to: the mixture of TBTU and HOBT, or the mixture of HBTU and HOBT;
(3) preparation Fmoc-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Asn of step (2) (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up; Add with the mixture that meets peptide reagent dissolved Fmoc-Lys (Boc)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-Lys (Boc)-OH is 2～5 times of resin;
The same step of all the other operations and processing condition (2);
(4) preparation Fmoc-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Lys of step (3) (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up; Add with the mixture that meets peptide reagent dissolved Fmoc-His (Trt)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-His (Trt)-OH is 2～5 times of resin;
The same step of all the other operations and processing condition (2);
(5) preparation Fmoc-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-His of step (4) (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up; Add with the mixture that meets peptide reagent dissolved Fmoc-Lys (Boc)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Fmoc-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-Lys (Boc)-OH is 2～5 times of resin;
The same step of all the other operations and processing condition (2);
(6) preparation Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Lys of step (5) (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up; Add with the mixture that meets peptide reagent dissolved Fmoc-Gly-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-Gly-OH is 2～5 times of resin;
The same step of all the other operations and processing condition (2);
(7) preparation Fmoc-His (Trt)-Gly-Lys (Boc)-His (Trt)-Ly s (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-Gly-Lys of step (6) (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up; Add with the mixture that meets peptide reagent dissolved Fmoc-His (Trt)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Fmoc-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Fmoc-His (Trt)-OH is 2～5 times of resin;
The same step of all the other operations and processing condition (2);
(8) preparation Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin:
In the Fmoc-His of step (7) (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up; Add with the mixture that meets peptide reagent dissolved Boc-Gly-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin;
In the mixture: the mole coefficient of Boc-Gly-OH is 2～5 times of resin;
The same step of all the other operations and processing condition (2);
(9) preparation Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)
In step (8) Boc-Gly-His (Trt)-Gly-Lys (Boc)-His (Trt)-Lys (Boc)-Asn (Trt)-Lys (Boc)-resin, add to meet and be chilled to 10～20 ℃ the peptide reagent of cutting, 10～50 ℃ were reacted 1～5 hour, pressure reducing and steaming desolventizes, add ether sedimentation, the collecting precipitation thing is washed with ether, P
2O
5Vacuum-drying obtains crude product;
Said component of cutting peptide reagent is: TFA/EDT/HO/TIS=90-95/2-5/1-5, volume ratio.
3, the application of GHGKHKNK octapeptide in the preparation antitumor drug.
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| CN2008100506855A CN101270145B (en) | 2008-05-07 | 2008-05-07 | Preparation technique for GHGKHKNK octapeptide and medicine use |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102406921A (en) * | 2011-11-16 | 2012-04-11 | 北华大学 | Medical application of GHGKHKNK octopeptide with anti-tumor synergetic effect on 5-fluorouracil |
| WO2020006922A1 (en) * | 2018-07-06 | 2020-01-09 | 泰安市启航生物科技有限公司 | Synthetic peptide sp4 and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526725A (en) * | 2003-09-23 | 2004-09-08 | �Ϻ���ͨ��ѧ | Antitumor polypeptide and its application |
| CN100427502C (en) * | 2005-06-09 | 2008-10-22 | 南京大学 | Antineoplastic oligopeptide and its preparation method and application |
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2008
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102406921A (en) * | 2011-11-16 | 2012-04-11 | 北华大学 | Medical application of GHGKHKNK octopeptide with anti-tumor synergetic effect on 5-fluorouracil |
| CN102406921B (en) * | 2011-11-16 | 2013-11-13 | 北华大学 | Medical application of GHGKHKNK octopeptide with anti-tumor synergetic effect on 5-fluorouracil |
| WO2020006922A1 (en) * | 2018-07-06 | 2020-01-09 | 泰安市启航生物科技有限公司 | Synthetic peptide sp4 and use thereof |
| US11939399B2 (en) | 2018-07-06 | 2024-03-26 | Taian City Qihang Biotechnology Co. | Synthetic peptide sp4 and use thereof |
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