CN103965299A - Cyclic pentapeptide as well as synthetic method and application thereof - Google Patents
Cyclic pentapeptide as well as synthetic method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of organic chemical synthesis, and discloses cyclic pentapeptide with a novel structure, as well as a synthetic method and application thereof. The cyclic pentapeptide with the novel structure is cyclo-(N-Me-D-Phe-D-Leu-N-Me-D-Leu-D-Leu-L-Leu), the molecular formula is C35H57N5O5, and the structural formula is shown in the description. The cyclic pentapeptide adopts D-Leu, L-Leu, D-Phe and D-Leu benzyl ester tosilate as raw materials, a tripeptide midbody and a dipeptide midbody are respectively prepared through a proper technology, and a cyclization locus is selected at the amido linkage position adjacent to D and L configurations, so that the cyclization efficiency is effectively increased. The cyclic pentapeptide has the advantages of simple synthetic process, low raw material cost, mild reaction conditions, high purity and easy industrial production, and can fully meet the requirements of medical experiments and clinical application.
Description
Technical field
The invention belongs to organic chemical synthesis field, be specifically related to a kind of ring pentapeptide and synthetic method and application.
Background technology
Cyclic peptide compound is widespread in nature.Plant, animal, marine organisms, microorganism, bacterium and the germ etc. such as low contain micro-cyclic peptide, although the content of these cyclic peptide is low, when their specific structure and pharmacological action, terrestrial organism is incomparable.Coupling reagent compounds has significant pharmacology stability and potent property, toxicological effect is relatively little, the difficult and complicated cases such as anti-curing cancers, acquired immune deficiency syndrome (AIDS), cardiovascular and cerebrovascular disease, senile disease are had to unique effect, become one of main direction of developing new drug, specifics.
Because cyclic peptide lacks free N end and C end, can form restricted conformation.In general, compare with corresponding linear peptides, it has better metabolic stability in vivo, and resistance to enzymolysis ability is stronger.Many research is verified, from the guide structure peptide of straight chain changes cyclic peptide into, makes original biological activity improve tens times to several ten thousand times.(FairlieD.P., Abbenante G., March D.R., Curr.Med.Chem., 1995,2,654-686; Bruce J, Aungstand Hirosi Saitoh, Pharmaceutical Research, 1996,13 (1), 114-119; Dae-Yeon Suh, YongChul Kim, Young-Hwa Kang.J.Nat.Prod., 1997,60,265-269; Bogdanowich-Knipp S.J., Jois D.S.S., Siahaan T.J.J.Pept.Res., 1999,53,523-529; DermocCox, ToshiakiAoki, JiroSeki, Yukio Motoyama, keizo YoshidaMed.Res.Rev., 1994,14 (2), 195-228.) in view of the plurality of advantages of cyclic peptide, in recent years the study hotspot of polypeptide has been transferred to the synthetic and biological assessment of cyclic peptide.
A kind of Novel ring pentapeptide (Xu W.J. that the red algae emulsus silk joint algae (Galaxaura filamentosa) that Galaxamide Shi Ben seminar gathers from Chinese Xisha Islands of South China Sea Yongxing Island, separation obtains, Liao X.J., Xu S.H., Organic Letters, 2008,10 (20), 4569-4572.), anti tumor activity in vitro experiment shows that this compound is to human hepatoma cell strain HepG
2and Bel-7402, human breast cancer cell strain MCF-7, the kinds of tumor cells such as human cervical carcinoma cell lines Hela have certain cytotoxicity, this compound induction liver cancer cell Bel-7402 apoptosis mechanism has been carried out to preliminary study simultaneously, result shows that Galaxamide is expected to become the lead compound of new type anticancer medicine, but the natural product with better activity that natural origin separation obtains is extremely a small amount of, can not meet people's needs, therefore further explore its structure activity relationship, further investigate its pharmacological action, Galaxamide is carried out structural modification and synthesizes its analogue seeming particularly important.Galaxamide analogue and intermediate thereof are carried out to anti tumor activity in vitro screening, and contrast with Galaxamide, to developing the lead compound of new type antineoplastic medicine.
Summary of the invention
The weak point existing in order to solve prior art, primary and foremost purpose of the present invention is to provide a kind of ring pentapeptide.
Another object of the present invention is to provide the synthetic method of above-mentioned ring pentapeptide.
A further object of the present invention is to provide the application of above-mentioned ring pentapeptide in preparing cancer therapy drug.
The object of the invention is achieved through the following technical solutions:
A ring pentapeptide, its chemical name is: ring (N-methyl D-phenylalanyl-D-leucyl-N-methyl D-leucyl-D-leucyl-L-leucyl); English abbreviation expression formula is: cyclo-(N-Me-D-Phe-D-Leu-N-Me-D-Leu-D-Leu-L-Leu); Molecular formula is C
35h
57n
5o
5; Structural formula is as follows:
The synthetic method of above-mentioned ring pentapeptide, specifically comprises following synthesis step:
(1) by L-Leu, D-Leu and D-phenylalanine respectively with tert-Butyl dicarbonate (Boc
2o) carry out aminoterminal protective reaction, obtain tertbutyloxycarbonyl-L-Leu (Boc-L-Leu-OH), tertbutyloxycarbonyl-D-Leu (Boc-D-Leu-OH) and tertbutyloxycarbonyl-D-phenylalanine (Boc-D-Phe-OH);
(2) tertbutyloxycarbonyl-D-Leu step (1) being obtained and tertbutyloxycarbonyl-D-phenylalanine carry out methylation reaction with methyl iodide respectively, obtain tertbutyloxycarbonyl-N-methyl D-leucine and tertbutyloxycarbonyl-N-methyl D-phenylalanine;
(3) tertbutyloxycarbonyl-N-methyl D-leucine step (2) being obtained and tertbutyloxycarbonyl-N-methyl D-phenylalanine carry out condensation reaction with D-Leu benzyl ester tosilate respectively, obtain tertbutyloxycarbonyl-N-methyl D-leucyl-D-Leu benzyl ester two ends and protect two PEPDs
1(Boc-N-Me-D-Leu-D-Leu-OBn) and tertbutyloxycarbonyl-N-methyl D-phenylalanyl-D-Leu benzyl ester two ends protect two PEPDs
2(Boc-N-Me-D-Phe-D-Leu-OBn);
(4) two PEPDs that step (3) obtained
2remove after tertbutyloxycarbonyl protection, the tertbutyloxycarbonyl-L-Leu obtaining with step (1) carries out condensation reaction and obtains tertbutyloxycarbonyl-L-leucyl-N-methyl D-phenylalanyl-D-Leu benzyl ester two ends protection tripeptides T (Boc-L-Leu-N-Me-D-Phe-D-Leu-OBn);
(5) two PEPDs that step (3) obtained
1remove tertbutyloxycarbonyl protection, the tripeptides T that step (4) is obtained removes benzyl protection, then both is carried out to condensation reaction and obtains tertbutyloxycarbonyl-L-leucyl-N-methyl D-phenylalanyl-D-leucyl-N-methyl D-leucyl-D-Leu benzyl ester two ends protection line style pentapeptide (Boc-L-Leu-N-Me-D-Phe-D-Leu-N-Me-D-Leu-D-Leu-OBn);
(6) linear pentapeptide step (5) being obtained removes respectively tertbutyloxycarbonyl protection and benzyl protection, carries out ring-closure reaction and obtain target product ring (N-methyl D-phenylalanyl-D-leucyl-N-methyl D-leucyl-D-leucyl-L-leucyl) pentapeptide (cyclo-(N-Me-D-Phe-D-Leu-N-Me-D-Leu-D-Leu-L-Leu)) under the effect that mixes condensation reagent.
In the synthetic method of above-mentioned ring pentapeptide, described in step (1), the concrete operation step of aminoterminal protective reaction is: under nitrogen protection, in round-bottomed flask, add successively tetrahydrofuran (THF) (THF, 150mL), H
2o (100mL) and 1.0mol/L NaOH solution (150mL), cooling disposable L-Leu (50mmol), D-Leu (50mol) or the D-phenylalanine (50mmol) of adding of ice-water bath, stirring and dissolving is even, then the disposable Boc that adds
2o (60mol, 1.2eq), after continuing to stir 10min under ice-water bath, slowly rise to room temperature, regulating pH is 10～10.5, and stirring is spent the night, remove under reduced pressure after solvent, with anhydrous diethyl ether extraction, water under ice-water bath cooling and with 1mol/L hcl acidifying to pH=2, be then extracted with ethyl acetate 3 times, merge organic phase and use saturated common salt water washing, anhydrous MgSO
4after dry, filter, finally steam solvent, obtain respectively tertbutyloxycarbonyl-L-Leu, tertbutyloxycarbonyl-D-Leu or tertbutyloxycarbonyl-D-phenylalanine.
In the synthetic method of above-mentioned ring pentapeptide, described in step (2), the concrete operation step of methylation reaction is: tertbutyloxycarbonyl-D-Leu or tertbutyloxycarbonyl-D-phenylalanine are dissolved in to tetrahydrofuran (THF) (THF, 100mL) in solution, add methyl iodide (5eq) and sodium hydride (3eq), reaction 1-2 days, ice-water bath downhill reaction system adds ethyl acetate (100mL) and water (20mL), remove organic solvent under reduced pressure, twice of the aqueous solution use anhydrous diethyl ether obtaining and water washing, merge water under ice-water bath with hcl acidifying to pH=2～3, ethyl acetate extraction 3 times, merge organic phase, then distinguish water, 5%Na
2s
2o
3the aqueous solution and saturated common salt water washing, anhydrous sodium sulfate drying, filters, and steaming desolventizes, and oil pump is dry, obtains respectively tertbutyloxycarbonyl-N-methyl D-leucine or tertbutyloxycarbonyl-N-methyl D-phenylalanine.
In the synthetic method of above-mentioned ring pentapeptide, step (3), (4), (5) condensation reaction described in refers at condensation reagent 3-(diethoxy phosphoryl oxy)-1, 2, under the existence of 3-phentriazine-4-ketone (DEPBT) and diisopropylethylamine (DIEA), treat that accordingly condenses carries out dehydrating condensation for two kinds, its concrete operation step is: under nitrogen protection, the condenses for the treatment of of tertbutyloxycarbonyl protection is dissolved in tetrahydrofuran (THF), under ice-water bath, add successively DEPBT (1.5eq) and DIEA (3eq), after reaction 10～15min, add benzyl protection compound, it is to be dissolved that rear with DIEA, to regulate pH be 7～8, naturally rise to room temperature, stirring is spent the night, after complete by thin-layer chromatography TLC detection raw material consumption, remove solvent under reduced pressure, silica gel column chromatography, obtain condensation product,
In the synthetic method of above-mentioned ring pentapeptide, described in step (4), (5), (6), remove tertbutyloxycarbonyl protection trifluoroacetic acid (TFA) and methylene dichloride (CH that to refer in volume ratio be 1:4
2cl
2) mixed solution in remove the corresponding tertbutyloxycarbonyl for the treatment of in de-thing, its concrete operation step is: the de-thing for the treatment of of tertbutyloxycarbonyl protection is dissolved in to CH
2cl
2in, under nitrogen protection, add methyl-phenoxide (2eq), under ice bath, stir, drip TFA, react after 4～6 hours, remove solvent under reduced pressure, then add CH
2cl
2, removing under reduced pressure, repetitive operation is removed TFA three times, and oil pump is drying to obtain the product that removes tertbutyloxycarbonyl protection.
In the synthetic method of above-mentioned ring pentapeptide, step (5), (6) described in, remove the palladium carbon hydrogenolysis reducing that benzyl protection refers to that the quality percentage composition with palladium is 10% and remove the corresponding benzyl for the treatment of in de-thing, its concrete operation step is: the de-thing for the treatment of of benzyl protection is dissolved in ethyl acetate, under nitrogen protection, add the 10%Pd/C (mass ratio) that treats de-1/3 times of amount of amount, under air-proof condition, pass into the hydrogen reducing that pressure is 0.1Mpa, stirring reaction under room temperature, TLC follows the tracks of, react 4～6 hours, stop passing into hydrogen, filter, reclaim Pd/C, remove solvent under reduced pressure, dry, obtain removing the product of benzyl protection.
In the synthetic method of above-mentioned ring pentapeptide; described in step (6), ring-closure reaction refers to benzotriazole-N at O-; N; N'; N'-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), 2-(7-azo benzotriazole)-N; N; N'; N'-tetramethyl-urea phosphofluoric acid ester (HATU), 3-(diethoxy phosphoryl oxy)-1; 2; under the existence of 3-phentriazine-4-ketone (DEPBT) and diisopropylethylamine (DIEA), carry out cyclization, its concrete operation step is: the linear pentapeptide that removes tertbutyloxycarbonyl protection and benzyl protection is dissolved in to the THF-CH that volume ratio is 2:1:2
2cl
2-CH
3in CN mixing solutions, nitrogen protection under room temperature, adds HATU successively, DEPBT and TBTU, and successive reaction 4～6 days, removes solvent under reduced pressure, uses CH
2cl
2after dissolving, use successively saturated NaHCO
3the aqueous solution and water washing, anhydrous MgSO
4dry, concentrated, with reversed-phase HPLC (Agilent Eclipse ZORBAX SB-C18,5
μm, 9.4 * 250nm) separation obtains encircling pentapeptide product.
Above-mentioned ring pentapeptide adopts mtt assay to study its anti tumor activity in vitro, it detects principle: in viable cell plastosome, succinodehydrogenase can be reduced to water-fast bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) exogenous MTT and is deposited in cell, and dead cell there is no this function.Add dimethyl sulfoxide (DMSO) (DMSO) to dissolve first a ceremonial jade-ladle, used in libation, by microplate reader, at 570nm wavelength place, survey its absorbance, can indirectly reflect viable cell quantity.Its concrete operation step is:
(1) inoculating cell: collect logarithmic phase cell, adjust concentration of cell suspension, be inoculated in 96 orifice plates every pore volume 100 μ l (marginal pore is filled with aseptic PBS) with 1000～10000, every hole cell;
(2) culturing cell: at 5%CO
2, in the cell culture incubator of 37 ℃, hatch, dosing in morning next day, every hole adds the sample to be tested of 100 μ l different concns, establishes 3 multiple holes;
(3) colour generation: cultivate 72 hours, every hole adds 20 μ l MTT solution, and (5mg/mL, with PBS preparation, pH=7.4), continue to hatch 4h, stop cultivating, careful suction abandoned culture supernatant in hole, every hole adds 150 μ l DMSO, and decolorization swinging table vibration 10min, fully dissolves crystallisate;
(4) colorimetric: select 570nm wavelength, measure the absorbance in each hole in microplate reader, record result, calculate cell survival rate, take the time as X-coordinate, light absorption value is that ordinate zou is drawn cell growth curve, and tries to achieve half-inhibition concentration (IC simultaneously
50).
Above-mentioned cyclic pentapeptide compound adopts the two methods of dying of Annexin V-FITC/PI to detect it to HepG 2 cell apoptosis mode, and this method can be distinguished viable cell, viable apoptotic cell and dead cell.Its concrete operation step is as follows:
(1) inoculating cell: collect logarithmic phase cell, adjust concentration of cell suspension, be inoculated in 6 orifice plates every pore volume 2mL with 1000000, every hole cell;
(2) culturing cell: at 5%CO
2, in the cell culture incubator of 37 ℃, hatch, dosing in morning next day, every hole adds the sample to be tested of 1mL different concns, cultivates 72 hours;
(3) collecting cell, comprises the cell swimming on substratum, and 2000 leave the heart 5 minutes, abandon supernatant, and with the resuspended sample presentation of 1mL PBS, normal components cell separates 200 μ l volumes and do not dye;
(4) centrifugal: 1000 revs/min, 5min, abandons supernatant;
(5) resuspended: to add 200 μ l Binding Buffer damping fluids to mix;
(6) dyeing: add 5 μ l Annexin V-FITC, room temperature lucifuge is hatched 10 minutes;
(7) centrifugal: 1000 revs/min, 5min, abandons supernatant;
(8) resuspended: to add 200 μ l Binding Buffer damping fluids to mix;
(9) dyeing: add 5 μ l PI, directly use cells were tested by flow cytometry result.
Compared with prior art, tool of the present invention has the following advantages and beneficial effect:
(1) ring pentapeptide of the present invention is synthetic, and Guan Huan site is chosen in D, amido linkage position that L configuration is adjacent, has avoided containing the place's cyclization of N-methyl nitrosourea key, thereby has effectively improved cyclization efficiency, and cyclization yield can reach 65.5%;
(2) the ring pentapeptide that prepared by the present invention has advantages of that purity is high, can fully meet the needs of medical experiment and clinical use, has very high medical value;
(3) to adopt lower-cost leucine and D-phenylalanine be synthesis material to ring pentapeptide of the present invention, and synthesis technique is simple, and reaction conditions is gentle, is easy to industrialization, has wide market outlook.
(4) activity experiment shows, compares with Galaxamide, and ring pentapeptide of the present invention has better anti-tumor activity, and normal cell and tumour cell are demonstrated to good selectivity.
Accompanying drawing explanation
Fig. 1 is the building-up reactions equation of ring pentapeptide of the present invention;
Fig. 2 is the flow cytometry figure of Galaxamide and ring pentapeptide PX of the present invention, the flow cytometry figure that figure four width figure are at the middle and upper levels Galaxamide, and four width figure of lower floor are the flow cytometry figure of ring pentapeptide PX of the present invention; In figure, Q1 refers to physical abuse, and Q2 is non-viable non-apoptotic cell, and Q3 is viable cell, and Q4 is viable apoptotic cell; Percentage ratio in figure is the mass concentration of medicine; In figure 0,3,6,12,24 be indicated concentration (unit: μ g/mL) respectively.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
The equation of the building-up reactions of the ring pentapeptide of the present invention that embodiment 1-10 records as shown in Figure 1.
Synthetic tertbutyloxycarbonyl-the L-Leu (Boc-L-Leu-OH) of embodiment 1
Under nitrogen protection, in round-bottomed flask, add successively tetrahydrofuran (THF) (THF, 150mL), H
2o (100mL) and 1.0mol/L NaOH solution (150mL), the cooling disposable L-Leu (50mmol, 6.55g) that adds of ice-water bath, stirring and dissolving is even, then the disposable Boc that adds
2o (60mol, 13.40g), after continuing to stir 10min under ice-water bath, slowly rise to room temperature, regulating pH is 10～10.5, and stirring is spent the night, remove under reduced pressure after solvent, with anhydrous diethyl ether extraction, water under ice-water bath cooling and with 1mol/L hcl acidifying to pH=2, be then extracted with ethyl acetate 3 times, merge organic phase and use saturated common salt water washing, anhydrous MgSO
4after dry, filter, finally steam solvent, obtain white powder tertbutyloxycarbonyl-L-Leu, productive rate 98.7%.
Synthetic tertbutyloxycarbonyl-the D-Leu (Boc-D-Leu-OH) of embodiment 2
Repeat the operation of embodiment 1, difference is to replace L-Leu with D-Leu, thereby makes white powder tertbutyloxycarbonyl-D-Leu, productive rate 99.2%.
Synthetic tertbutyloxycarbonyl-the D-phenylalanine (Boc-D-Phe-OH) of embodiment 3
Repeat the operation of embodiment 1, difference is to replace L-Leu with D-phenylalanine, thereby makes transparent oily matter tertbutyloxycarbonyl-D-phenylalanine, productive rate 98.5%.
Synthetic tertbutyloxycarbonyl-N-methyl D-the leucine (Boc-N-Me-D-Leu-OH) of embodiment 4
Tertbutyloxycarbonyl-D-Leu (20mol that embodiment 2 is obtained, 4.62g) be dissolved in tetrahydrofuran (THF) (THF, 100mL) in solution, add methyl iodide (100mol, 6.3mL) and sodium hydride (60mol, 2.4g), reaction 1-2 days, ice-water bath downhill reaction system adds ethyl acetate (100mL) and water (20mL), remove organic solvent under reduced pressure, the anhydrous diethyl ether for the aqueous solution (60mL) obtaining and water (100mL) washed twice, merge water under ice-water bath with hcl acidifying to pH=2～3, ethyl acetate extraction (100mL * 3), merge organic phase, then distinguish water (150mL), 5%Na
2s
2o
3the aqueous solution (100mL * 3) and saturated aqueous common salt (100mL) washing, anhydrous sodium sulfate drying, filters, and steaming desolventizes, and oil pump is dry, obtains transparent oily matter tertbutyloxycarbonyl-N-methyl D-leucine, productive rate 89.2%.Synthetic tertbutyloxycarbonyl-N-methyl D-the phenylalanine (Boc-N-Me-D-Phe-OH) of embodiment 5
Repeat the operation of embodiment 4, difference is that the tertbutyloxycarbonyl-D-phenylalanine obtaining with embodiment 3 replaces tertbutyloxycarbonyl-D-Leu, thereby makes transparent oily matter tertbutyloxycarbonyl-N-methyl D-phenylalanine, productive rate 87.2%.
The synthetic Boc-N-Me-D-Leu-D-Leu-OBn of embodiment 6
Under nitrogen protection; getting the Boc-N-Me-D-Leu-OH1.47g that embodiment 4 obtains is dissolved in 10mL THF; under ice-water bath, add successively 2.70g DEPBT and 1.6ml DIEA, after reaction 10min, add 2.60g D-Leu benzyl ester tosilate; to be dissolvedly with DIEA, regulate pH=7～8 afterwards; naturally rise to room temperature, stirring is spent the night, and TLC follows the tracks of and reacts to the disappearance of raw material point; remove solvent under reduced pressure, silica gel column chromatography (eluent V
sherwood oil: V
acetone=25:1), obtain white crystal shape tertbutyloxycarbonyl-N-methyl D-leucyl-D-Leu benzyl ester two ends and protect two PEPDs
1(Boc-N-Me-D-Leu-D-Leu-OBn) 2.52g, yield is 93.6%.
The synthetic Boc-N-Me-D-Phe-D-Leu-OBn of embodiment 7
Under nitrogen protection; getting the Boc-N-Me-D-Phe-OH1.67g that embodiment 5 obtains is dissolved in 10mL THF; under ice-water bath, add successively 2.70g DEPBT and 1.6ml DIEA, after reaction 10min, add 2.60g D-Leu benzyl ester tosilate; to be dissolvedly with DIEA, regulate pH=7～8 afterwards; naturally rise to room temperature, stirring is spent the night, and TLC follows the tracks of and reacts to the disappearance of raw material point; remove solvent under reduced pressure, silica gel column chromatography (eluent V
sherwood oil: V
acetone=25:1), obtain white crystal shape tertbutyloxycarbonyl-N-methyl D-phenylalanyl-D-Leu benzyl ester two ends and protect two PEPDs
2(Boc-N-Me-D-Phe-D-Leu-OBn) 2.68g, yield is 92.5%.
The synthetic Boc-L-Leu-N-Me-D-Phe-D-Leu-OBn of embodiment 8
Get the Boc-N-Me-D-Phe-D-Leu-OBn2.41g that embodiment 7 obtains and be dissolved in 20mL CH
2cl
2in, under nitrogen protection, add 1.05ml methyl-phenoxide, under ice bath, stir, drip 5ml TFA, react after 4 hours, remove solvent under reduced pressure, then add 20mL CH
2cl
2, remove under reduced pressure, repetitive operation is removed TFA three times, oil pump is drying to obtain the N-methyl D-phenylalanyl-D-Leu benzyl ester (H-N-Me-D-Phe-D-Leu-OBn) that removes tertbutyloxycarbonyl protection, be dissolved in 5mL THF standby, under nitrogen protection, 1.67g embodiment 1 is obtained to Boc-L-Leu-OH to be dissolved in 10mL THF, under ice-water bath, add successively 2.24g DEPBT and 1.3ml DIEA, after reaction 10min, add standby H-N-Me-D-Phe-D-Leu-OBn THF solution, with DIEA, regulate pH=7～8, naturally rise to room temperature, stirring is spent the night, with TLC, follow the tracks of and react to the disappearance of raw material point, remove solvent under reduced pressure, silica gel column chromatography (eluent V
sherwood oil: V
acetone=25:1), obtain transparent oily tertbutyloxycarbonyl-L-leucyl-N-methyl D-phenylalanyl-D-Leu benzyl ester two ends protection tripeptides T (Boc-L-Leu-N-Me-D-Phe-D-Leu-OBn) 2.56g, two step yields are 85.3%.
The synthetic Boc-L-Leu-N-Me-D-Phe-D-Leu-N-Me-D-Leu-D-Leu-OBn of embodiment 9
Get protection two PEPDs that embodiment 6 obtains
12.24g, removes tertbutyloxycarbonyl protection by embodiment 8, obtains N-methyl D-leucyl-D-Leu benzyl ester (H-N-Me-D-Leu-D-Leu-OBn) and be dissolved in 5mL THF standby; Getting the protection tripeptides T2.38g that embodiment 8 obtains is dissolved in 20mL ethyl acetate, under nitrogen protection, add 0.8g10%Pd/C, under air tight condition, pass into the hydrogen reducing that pressure is 0.1MPa, stirring reaction under room temperature, TLC follows the tracks of, react 4～6 hours, stop passing into hydrogen, filter, remove solvent under reduced pressure, be dried, obtain removing tertbutyloxycarbonyl-L-leucyl-N-methyl D-phenylalanyl-D-Leu (Boc-L-Leu-N-Me-D-Phe-D-Leu-OH) of benzyl protection; Under nitrogen protection; the Boc-L-Leu-N-Me-D-Phe-D-Leu-OH obtaining is dissolved in 10mL THF; under ice-water bath, add successively 1.80g DEPBT and 1.1ml DIEA, after reaction 10min, add standby H-N-Me-D-Leu-D-Leu-OBn THF solution; with DIEA, regulate pH=7～8; naturally rise to room temperature, stirring is spent the night, and with TLC, follows the tracks of and reacts to the disappearance of raw material point; remove solvent under reduced pressure, silica gel column chromatography (eluent V
sherwood oil: V
acetone=25:1), obtain transparent oily two ends protection line style pentapeptide (Boc-L-Leu-N-Me-D-Phe-D-Leu-N-Me-D-Leu-D-Leu-OBn) 2.72g, three step yields are 81.4%.
The synthetic cyclo-(N-Me-D-Phe-D-Leu-N-Me-D-Leu-D-Leu-L-Leu) of embodiment 10
Getting the two ends that embodiment 9 obtains protects linear pentapeptide 0.4277g to be dissolved in 8mL CH
2cl
2in, under nitrogen protection, add 0.1ml methyl-phenoxide, under ice bath, stir, slowly drip 2ml TFA, react after 4 hours, remove solvent under reduced pressure, then add 10mL CH
2cl
2, removing under reduced pressure, repetitive operation is removed TFA three times, and oil pump is drying to obtain the linear pentapeptide that removes tertbutyloxycarbonyl protection; The linear pentapeptide that removes tertbutyloxycarbonyl protection is dissolved in 10mL ethyl acetate, under nitrogen protection, add 0.14g10%Pd/C, under air tight condition, pass into the hydrogen reducing that pressure is 0.1MPa, stirring reaction under room temperature, TLC follows the tracks of, react 4～6 hours, stop passing into hydrogen, filter, remove solvent under reduced pressure, be dried, obtain removing the linear pentapeptide of benzyl protection; The above-mentioned linear pentapeptide that removes benzyl protection is dissolved in to the THF-CH that 120mL volume ratio is 2:1:2
2cl
2-CH
3in CN mixing solutions, nitrogen protection under room temperature, adds 0.1426gHATU, 0.1122g DEPBT and 0.1204g TBTU successively, and successive reaction 4～6 days, removes solvent under reduced pressure, uses CH
2cl
2after dissolving, use successively saturated NaHCO
3the aqueous solution and water washing, anhydrous MgSO
4dry, concentrated, with reversed-phase HPLC (Agilent Eclipse ZORBAX SB-C18,5 μ m, 9.4 * 250nm) separation obtains white needles ring pentapeptide crystal c yclo-(N-Me-D-Phe-D-Leu-N-Me-D-Leu-D-Leu-L-Leu) 205.6mg, productive rate 65.5%, purity reaches more than 99%.The structural characterization data of products therefrom are as follows, confirm as structural formula compound ring as follows (N-methyl D-phenylalanyl-D-leucyl-N-methyl D-leucyl-D-leucyl-L-leucyl):
1H-NMR(500MHZ，CDCl3)，δ(ppm)：
7.89(s，1H)，7.21(m，5H)，6.70(m，1H)，5.53(m，1H)，5.09(m，1H)，4.82(d，J＝10.0Hz，1H)，4.77(m，1H)，4.31(m，1H)，3.67(dd，J＝5.0Hz，5.0Hz，1H)，3.56(ddd，J＝5.0Hz，5.0Hz，5.0Hz，1H)，3.34(m，1H)，3.13(m，2H)，2.98(m，2H)，2.81(m，1H)，2.68(d，J＝5.0Hz，1H)，2.27(m，1H)，1.85(m，4H)，1.55(m，7H)，0.90(m，18H)，0.70(m，6H)；
13C-NMR(125MHZ，CDCl3)，δ(ppm)：
173.4，172.9，172.1，171.4，168.5，138.8，129.5(2C)，128.8，128.7，127，68.36(2C)，59.37，55.37，47.66，47.3，41.7，36.0(3C)，30.8(2C)，25.4(3C)，23.0(3C)，22.6(3C)，22.03(C)。
Embodiment 11 cell cultures
The tumor cell line that the present invention uses comprises HepG2 cell lines, human colon cancer cell strain SW480, brain glioblastoma cell strain U87, human breast cancer cell MCF-7 and human normal liver cell L 02.In substratum (DMEM), add foetal calf serum (FBS, 10%), Streptomycin sulphate and penicillin dual anti-(1%).Culture condition: 37 ℃, 5%CO
2; Relative humidity: 95%.
(1) experiment pre-treatment: the consumptive material after needed every high pressure is placed in Bechtop, sprays experiment table top with alcohol watering can, and closes after working light is opened ultra violet lamp 30min and start experimental implementation;
(2) recovery cell: cryopreservation tube is taken out from liquid nitrogen, drop into immediately in 37 ℃ of water-baths, slightly shake.After liquid all melts, take out specking 75% alcohol and be placed in Bechtop.Then cell suspending liquid is drawn onto in 15mL centrifuge tube, with substratum, cryopreservation tube is washed one time, sticking under cell on wall all washes, add 5mL substratum, 2000 leave the heart 3 minutes.Then abandon supernatant, add 1mL substratum suspension cell, be drawn onto in the 7.5cm plastic culture bottle that 5mL substratum is housed, all around shake gently, the cell in culturing bottle is uniformly distributed.Finally mark cell category, date and cultivator etc., be placed on CO
2in incubator, cultivate, every day, observation of cell growing state, changed substratum according to its growth conditions, when cell growth state is stable, and while being logarithmic phase, can be used for experiment.
Embodiment 12 anti tumor activity in vitro researchs
Ring pentapeptide and Galaxamide adopt mtt assay to study its anti tumor activity in vitro, and concrete operation step is:
(1) inoculating cell: collect logarithmic phase cell, adjust concentration of cell suspension, be inoculated in 96 orifice plates every pore volume 100 μ l (marginal pore is filled with aseptic PBS) with 1000～10000, every hole cell;
(2) culturing cell: at 5%CO
2, in the cell culture incubator of 37 ℃, hatch, dosing in morning next day, every hole adds the sample to be tested of 100 μ l different concns, establishes 5 multiple holes;
(3) colour generation: cultivate 72 hours, every hole adds 20 μ l MTT solution, and (5mg/mL, with PBS preparation, pH=7.4), continue to hatch 4h, stop cultivating, careful suction abandoned culture supernatant in hole, every hole adds 150 μ l DMSO, and decolorization swinging table vibration 10min, fully dissolves crystallisate;
(4) colorimetric: select 570nm wavelength, measure the absorbance in each hole in microplate reader, record result, calculate cell survival rate, and try to achieve half-inhibition concentration (IC
50/ μ g/mL).
The experiment of table 1 anti tumor activity in vitro
| HepG2 | SW480 | U87 | MCF-7 | L02 | |
| Galaxamide | 4.63 | 43.82 | 10.61 | 14.09 | 60.08 |
| PX | 1.46 | 5.1 | 1.85 | 4.18 | 50.68 |
Adopt mtt assay to study the anti tumor activity in vitro of ring pentapeptide of the present invention and Galaxamide, result is as shown in table 1.Selected HepG2 cell lines, human colon cancer cell strain SW480, brain glioblastoma cell strain U87, human breast cancer cell MCF-7 and a kind of Human normal hepatocyte strain L02 to study their activity, drug treating time is all 72h.As can be seen from the table, ring pentapeptide of the present invention has good restraining effect, its IC to hepatoma cell strain HepG2, human colon cancer cell strain SW480, brain glioblastoma cell strain U87
50value is respectively 1.46 μ g/mL, 5.1 μ g/mL and 1.85 μ g/mL; The anti-tumor activity of ring pentapeptide of the present invention is better than Galaxamide, and both are little more a lot of than tumour cell to the toxicity of normal cell L02; Ring pentapeptide of the present invention demonstrates good selectivity to normal cell and tumour cell, aspect the treatment of tumour and prevention, is showing good application prospect.
The apoptotic mensuration research of embodiment 13
The research of cyclic pentapeptide compound to HepG 2 cell apoptosis mode, is characterized in that adopting the two methods of dying of Annexin V-FITC/PI to detect apoptosis, and this method can be distinguished viable cell, viable apoptotic cell and dead cell.Its concrete operation step is as follows:
(1) inoculating cell: collect logarithmic phase cell, adjust concentration of cell suspension, be inoculated in 6 orifice plates every pore volume 2mL with 1000000, every hole cell;
(2) culturing cell: at 5%CO
2, in the cell culture incubator of 37 ℃, hatch, dosing in morning next day, every hole adds the sample to be tested of 1mL different concns, cultivates 72 hours;
(3) collecting cell, comprises the cell swimming on substratum, and 2000 leave the heart 5 minutes, abandon supernatant, and with the resuspended sample presentation of 1mL PBS, normal components cell separates 200 μ l volumes and do not dye;
(4) centrifugal: 1000 revs/min, 5min, abandons supernatant;
(5) resuspended: to add 200 μ l Binding Buffer damping fluids to mix;
(6) dyeing: add 5 μ l Annexin V-FITC, room temperature lucifuge is hatched 10 minutes;
(7) centrifugal: 1000 revs/min, 5min, abandons supernatant;
(8) resuspended: to add 200 μ l Binding Buffer damping fluids to mix;
(9) dyeing: add 5 μ l PI, directly use cells were tested by flow cytometry result.
As shown in Figure 2, result shows measured result, and along with the increase of drug level, apoptosis rate also increases gradually.In addition, the energy force rate Galaxamide of ring pentapeptide cell death inducing of the present invention is stronger, and under same concentrations, the effect of ring pentapeptide of the present invention is stronger.Result shows, ring pentapeptide of the present invention has the ability of better cell death inducing.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (9)
1. encircle a pentapeptide, it is characterized in that: described ring pentapeptide has following structural formula:
2. a kind of synthetic method of encircling pentapeptide claimed in claim 1, is characterized in that comprising following synthesis step:
(1) L-Leu, D-Leu and D-phenylalanine are carried out to aminoterminal protective reaction with tert-Butyl dicarbonate respectively, obtain respectively tertbutyloxycarbonyl-L-Leu, tertbutyloxycarbonyl-D-Leu and tertbutyloxycarbonyl-D-phenylalanine;
(2) tertbutyloxycarbonyl-D-Leu step (1) being obtained and tertbutyloxycarbonyl-D-phenylalanine carry out methylation reaction with methyl iodide respectively, obtain respectively tertbutyloxycarbonyl-N-methyl D-leucine and tertbutyloxycarbonyl-N-methyl D-phenylalanine;
(3) tertbutyloxycarbonyl-N-methyl D-leucine step (2) being obtained and tertbutyloxycarbonyl-N-methyl D-phenylalanine carry out condensation reaction with D-Leu benzyl ester tosilate respectively, obtain tertbutyloxycarbonyl-N-methyl D-leucyl-D-Leu benzyl ester two ends and protect two PEPDs
1and two PEPDs are protected at tertbutyloxycarbonyl-N-methyl D-phenylalanyl-D-Leu benzyl ester two ends
2;
(4) two PEPDs that step (3) obtained
2remove after tertbutyloxycarbonyl protection, the tertbutyloxycarbonyl-L-Leu obtaining with step (1) carries out condensation reaction and obtains tertbutyloxycarbonyl-L-leucyl-N-methyl D-phenylalanyl-D-Leu benzyl ester two ends protection tripeptides T;
(5) two PEPDs that step (3) obtained
1remove tertbutyloxycarbonyl protection, the tripeptides T that step (4) is obtained removes benzyl protection, then both is carried out to condensation reaction and obtains tertbutyloxycarbonyl-L-leucyl-N-methyl D-phenylalanyl-D-leucyl-N-methyl D-leucyl-D-Leu benzyl ester two ends protection line style pentapeptide;
(6) linear pentapeptide step (5) being obtained removes respectively tertbutyloxycarbonyl protection and benzyl protection, carries out ring-closure reaction and obtain target product ring (N-methyl D-phenylalanyl-D-leucyl-N-methyl D-leucyl-D-leucyl-L-leucyl) pentapeptide under the effect that mixes condensation reagent.
3. a kind of synthetic method of encircling pentapeptide according to claim 2, is characterized in that: described in step (1), the concrete operation step of aminoterminal protective reaction is: under nitrogen protection, in round-bottomed flask, add successively tetrahydrofuran (THF), H
2o and 1.0mol/L NaOH solution, cooling disposable L-Leu, D-Leu or the D-phenylalanine of adding of ice-water bath, stirring and dissolving is even, then the disposable tert-Butyl dicarbonate that adds, after continuing to stir 10min under ice-water bath, rise to room temperature, regulating pH is 10～10.5, stirring is spent the night, remove under reduced pressure after solvent, with anhydrous diethyl ether extraction, water under ice-water bath cooling and with 1mol/L hcl acidifying to pH=2, be then extracted with ethyl acetate 3 times, merge organic phase and use saturated common salt water washing, anhydrous MgSO
4after dry, filter, finally steam solvent, obtain respectively tertbutyloxycarbonyl-L-Leu, tertbutyloxycarbonyl-D-Leu or tertbutyloxycarbonyl-D-phenylalanine.
4. a kind of synthetic method of encircling pentapeptide according to claim 2, it is characterized in that: described in step (2), the concrete operation step of methylation reaction is: tertbutyloxycarbonyl-D-Leu or tertbutyloxycarbonyl-D-phenylalanine are dissolved in tetrahydrofuran solution, add methyl iodide and sodium hydride, reaction 1-2 days, ice-water bath downhill reaction system adds ethyl acetate and water, remove organic solvent under reduced pressure, twice of the aqueous solution use anhydrous diethyl ether obtaining and water washing, merge water under ice-water bath with hcl acidifying to pH=2～3, ethyl acetate extraction 3 times, merge organic phase, then distinguish water, massfraction 5%Na
2s
2o
3the aqueous solution and saturated common salt water washing, anhydrous sodium sulfate drying, filters, and steaming desolventizes, and oil pump is dry, obtains respectively tertbutyloxycarbonyl-N-methyl D-leucine or tertbutyloxycarbonyl-N-methyl D-phenylalanine.
5. a kind of synthetic method of encircling pentapeptide according to claim 2, it is characterized in that: step (3), (4), (5) condensation reaction described in refers at condensation reagent 3-(diethoxy phosphoryl oxy)-1, 2, under the existence of 3-phentriazine-4-ketone and diisopropylethylamine, treat that accordingly condenses carries out dehydrating condensation for two kinds, its concrete operation step is: under nitrogen protection, the condenses for the treatment of of tertbutyloxycarbonyl protection is dissolved in tetrahydrofuran (THF), under ice-water bath, add successively 3-(diethoxy phosphoryl oxy)-1, 2, 3-phentriazine-4-ketone and diisopropylethylamine, after reaction 10～15min, add benzyl protection compound, it is to be dissolved that rear with diisopropylethylamine, to regulate pH be 7～8, naturally rise to room temperature, stirring is spent the night, after complete by thin-layer chromatography TLC detection raw material consumption, remove solvent under reduced pressure, silica gel column chromatography, obtain condensation product.
6. a kind of synthetic method of encircling pentapeptide according to claim 2, it is characterized in that: step (4), (5), (6) described in, remove in the mixed solution of the tertbutyloxycarbonyl protection trifluoroacetic acid that to refer in volume ratio be 1:4 and methylene dichloride and remove the corresponding tertbutyloxycarbonyl for the treatment of in de-thing, its concrete operation step is: the de-thing for the treatment of of tertbutyloxycarbonyl protection is dissolved in methylene dichloride, under nitrogen protection, add methyl-phenoxide, under ice bath, stir, drip trifluoroacetic acid, react after 4～6 hours, remove solvent under reduced pressure, add again methylene dichloride, remove under reduced pressure, repetitive operation is removed trifluoroacetic acid three times, oil pump is drying to obtain the product that removes tertbutyloxycarbonyl protection.
7. a kind of synthetic method of encircling pentapeptide according to claim 2, it is characterized in that: step (5), (6) described in, remove the Pd/C hydrogenolysis reducing that benzyl protection refers to that the quality percentage composition with palladium is 10% and remove the corresponding benzyl for the treatment of in de-thing, its concrete operation step is: the de-thing for the treatment of of benzyl protection is dissolved in ethyl acetate, under nitrogen protection, add the 10%Pd/C that treats de-amount 1/3, under air-proof condition, pass into the hydrogen reducing that pressure is 0.1Mpa, stirring reaction under room temperature, TLC follows the tracks of, react 4～6 hours, stop passing into hydrogen, filter, reclaim Pd/C, remove solvent under reduced pressure, dry, obtain removing the product of benzyl protection.
8. a kind of synthetic method of encircling pentapeptide according to claim 2; it is characterized in that: described in step (6), ring-closure reaction refers to benzotriazole-N at O-; N; N'; N'-tetramethyl-urea Tetrafluoroboric acid ester, 2-(7-azo benzotriazole)-N; N; N'; N'-tetramethyl-urea phosphofluoric acid ester, 3-(diethoxy phosphoryl oxy)-1; 2; under the existence of 3-phentriazine-4-ketone and diisopropylethylamine, carry out cyclization, its concrete operation step is: the linear pentapeptide that removes tertbutyloxycarbonyl protection and benzyl protection is dissolved in to the THF-CH that volume ratio is 2:1:2
2cl
2-CH
3in CN mixing solutions, nitrogen protection under room temperature, adds 2-(7-azo benzotriazole)-N successively; N, N', N'-tetramethyl-urea phosphofluoric acid ester; 3-(diethoxy phosphoryl oxy)-1,2,3-phentriazine-4-ketone and O-benzotriazole-N; N; N', N'-tetramethyl-urea Tetrafluoroboric acid ester, successive reaction 4～6 days; remove solvent under reduced pressure, use CH
2cl
2after dissolving, use successively saturated NaHCO
3the aqueous solution and water washing, anhydrous MgSO
4dry, concentrated, with reversed-phase HPLC separation, obtain encircling pentapeptide product.
9. a kind of ring pentapeptide claimed in claim 1 is being prepared medicines resistant to liver cancer, the application in inhibitor against colon carcinoma cells medicine or anti-cerebral glioma medicine.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188231A (en) * | 2015-05-25 | 2016-12-07 | 中国医学科学院药物研究所 | The synthesis of pasireotide pentapeptide intermediate and application |
| CN106188231B (en) * | 2015-05-25 | 2021-10-22 | 中国医学科学院药物研究所 | Synthesis and application of pasireotide pentapeptide intermediate |
| WO2018096484A1 (en) * | 2016-11-25 | 2018-05-31 | Siegfried (Nantong) Pharmaceuticals Co. Ltd. | Process for the production of n-boc-2-amino-3,3-dimethylbutyric acid |
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| US10822304B2 (en) | 2016-11-25 | 2020-11-03 | Siegfried (Nantong) Pharmaceuticals Co. Ltd. | Process for the production of N-Boc-2-amino-3,3-dimethylbutyric acid |
| CN107936084A (en) * | 2017-11-01 | 2018-04-20 | 十堰市太和医院 | A kind of production method for the CAP23145 polypeptides based on targeting FMRP for treating glioma |
| CN113667079A (en) * | 2021-07-19 | 2021-11-19 | 江苏师范大学 | Preparation method of carbon-carbon double bond bridged chiral porous organic polymer |
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