CN116063451B - Synthetic method of capelin - Google Patents
Synthetic method of capelin Download PDFInfo
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- CN116063451B CN116063451B CN202211697497.8A CN202211697497A CN116063451B CN 116063451 B CN116063451 B CN 116063451B CN 202211697497 A CN202211697497 A CN 202211697497A CN 116063451 B CN116063451 B CN 116063451B
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- 241001417902 Mallotus villosus Species 0.000 title abstract description 19
- 238000010189 synthetic method Methods 0.000 title description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 17
- 150000001413 amino acids Chemical class 0.000 claims abstract description 16
- 125000001494 2-propynyl group Chemical group [H]C#CC([H])([H])* 0.000 claims abstract description 13
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 claims abstract description 13
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229960004117 capecitabine Drugs 0.000 claims abstract description 13
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 11
- 230000007017 scission Effects 0.000 claims abstract description 11
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 11
- 238000005859 coupling reaction Methods 0.000 claims abstract description 7
- FHOAKXBXYSJBGX-YFKPBYRVSA-N (2s)-3-hydroxy-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@@H](CO)C(O)=O FHOAKXBXYSJBGX-YFKPBYRVSA-N 0.000 claims abstract description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000008878 coupling Effects 0.000 claims abstract description 6
- 238000010168 coupling process Methods 0.000 claims abstract description 6
- 230000003647 oxidation Effects 0.000 claims abstract description 5
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 32
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 claims description 16
- 101000780028 Homo sapiens Natriuretic peptides A Proteins 0.000 claims description 13
- 238000007363 ring formation reaction Methods 0.000 claims description 13
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 claims description 10
- 229950008486 carperitide Drugs 0.000 claims description 10
- 102000056614 human NPPA Human genes 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 6
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 5
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 4
- 238000007792 addition Methods 0.000 claims description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 9
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract 1
- 229910052799 carbon Inorganic materials 0.000 abstract 1
- 229910052757 nitrogen Inorganic materials 0.000 abstract 1
- YBYIRNPNPLQARY-UHFFFAOYSA-N 1H-indene Chemical compound C1=CC=C2CC=CC2=C1 YBYIRNPNPLQARY-UHFFFAOYSA-N 0.000 description 18
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 14
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 12
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- 229920005989 resin Polymers 0.000 description 10
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 description 8
- -1 fmoc-Phe-OH Chemical compound 0.000 description 8
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 7
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 6
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000005215 recombination Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 238000005336 cracking Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 3
- KJYAFJQCGPUXJY-UMSFTDKQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-(tritylamino)butanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KJYAFJQCGPUXJY-UMSFTDKQSA-N 0.000 description 3
- WDGICUODAOGOMO-DHUJRADRSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino)pentanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)CC(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 WDGICUODAOGOMO-DHUJRADRSA-N 0.000 description 3
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 3
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 3
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 3
- BLAKAEFIFWAFGH-UHFFFAOYSA-N acetyl acetate;pyridine Chemical compound C1=CC=NC=C1.CC(=O)OC(C)=O BLAKAEFIFWAFGH-UHFFFAOYSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000010511 deprotection reaction Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000012982 microporous membrane Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- 125000003396 thiol group Chemical class [H]S* 0.000 description 3
- BPYLRGKEIUPMRJ-QMMMGPOBSA-N (2s)-3-[(2-methylpropan-2-yl)oxy]-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC[C@@H](C(O)=O)NC(=O)OC(C)(C)C BPYLRGKEIUPMRJ-QMMMGPOBSA-N 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- QSECPQCFCWVBKM-UHFFFAOYSA-N 2-iodoethanol Chemical compound OCCI QSECPQCFCWVBKM-UHFFFAOYSA-N 0.000 description 2
- 102000002723 Atrial Natriuretic Factor Human genes 0.000 description 2
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 2
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 2
- 206010007556 Cardiac failure acute Diseases 0.000 description 2
- 206010007558 Cardiac failure chronic Diseases 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 210000002464 muscle smooth vascular Anatomy 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 230000024883 vasodilation Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 108010078321 Guanylate Cyclase Proteins 0.000 description 1
- 102000014469 Guanylate cyclase Human genes 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000036316 preload Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cardiology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a synthesis method of capelin, belonging to the technical field of polypeptide synthesis. The method adopts special amino acid Fmoc-Ile- (propargyl) Gly-OH for 15, 16-position amino acids; the amino acid at the 5,6 positions adopts Boc-Ser (Fmoc-Ser) -OH; and coupling the rest amino acids from the carbon end to the nitrogen end sequentially according to the peptide sequence, synthesizing the full-protection capecitabine peptide by solid phase, and obtaining the capecitabine peptide line peptide after cleavage. Dissolving with pure water, adding PbCl 2 Further cleavage of propargyl, adjustment of pH to alkaline, oxidation of Pb to form disulfide bond, and obtaining capelin. The method improves the purity of the crude peptide, greatly reduces the labor cost and the purification cost, and is beneficial to industrialized amplified production.
Description
Technical Field
The invention relates to the field of polypeptide medicine synthesis, in particular to a method for synthesizing a medicine suitable for treating acute heart failure (including chronic heart failure and severe) diseases.
Background
Capelin peptide sequence:
H-Ser 1 -Leu 2 -Arg 3 -Arg 4 -Ser 5 -Ser 6 -Cys 7 -Phe 8 -Gly 9 -Gly 10 -Arg 11 -Met 12 –Asp 13 -Arg 14 -Ile 15 -Gly 16 -Ala 17 -Gln 18 -Ser 19 -Gly 20 -Leu 21 -Gly 22 -Cys 23 -Asn 24 -Ser 25 -Phe 26 -Arg 27 -Tyr 28 -OH
the indication for capelin is acute heart failure (including exacerbation of chronic heart failure). The pharmacological effect of the carperitide is to stimulate myocardial extension, synthesize by particles in the ventricle, then act on vascular smooth muscle and kidney tissues through the whole body of the coronary artery distribution, and regulate blood pressure and in vivo electrolyte balance. The carperitide is a circulation regulating hormone composed of 28 amino acids and has the functions of vasodilation and diuresis. The vasodilation by capelin is achieved by binding to the ANP (atrial natriuretic peptide) receptor of vascular smooth muscle and by increasing the guanylate cyclase activity, suggesting that cardiac preload can be reduced.
Current methods for the synthesis of capecitabine fall into two broad categories: firstly, gene recombination, and secondly, liquid phase or solid phase synthesis by adopting a Boc route. ZL200510012425.5 discloses a method for producing the capelin by adopting a gene recombination technology, CN102382188 reports a method for preparing the capelin by coupling one by adopting a solid-phase Fmoc route, CA1245635A1 reports that the capelin is obtained by adopting a Boc route for solid-phase synthesis and finally steps such as HF resin cutting, liquid-phase oxidation, HPLC (high-pressure liquid chromatography) purification and the like, CA1245637A1 reports that the capelin is produced by adopting a gene recombination technology, and other patent documents such as EP0440311A1, JP3004169A, US4673732A and the like are all the capelin produced by adopting a gene recombination technology or the capelin is obtained by adopting a Boc route for solid-phase or liquid-phase synthesis. Adopts the gene recombination technology, has complex operation, large investment, low medicine purity and higher technical barriers. The use of liquid or solid phase synthesis by the Boc route requires the use of HF and the use of large amounts of TFA, which results in serious environmental pollution. Meanwhile, the liquid phase method has the defects of long operation period, complicated operation and the like, and is not beneficial to large-scale industrial production.
Disclosure of Invention
The invention aims to solve the problems of more impurities, low purity and yield, high cost, complicated operation steps, excessive waste liquid and adverse industrial production in the existing synthesis process and provides an optimized synthesis method of the capecitabine.
The technical scheme for realizing the aim of the invention is as follows:
a method for synthesizing capelin, which is characterized by comprising the following steps:
(1) The propargyl Fmoc-Ile- (propargyl) Gly-OH is introduced into amino acids 15-16 of the capelin sequence; amino acids 5-6 of the carperitide sequence were Boc-Ser (Fmoc-Ser) -OH;
(2) Sequentially synthesizing amino acids at other sites of the carperitide sequence in solid phase, synthesizing fully-protected carperitide, performing cleavage precipitation, dissolving, and adding PbCl 2 Removing propargyl, regulating pH, and cyclizing to obtain the carperitide.
The further preferable technical scheme of the synthesis method of the carperitide is that in the step (2), the condensing agent used for synthesizing each amino acid is one or more of DIC/HOBt, HBTU/HOBt/DIEA, pyBop/HOBt/DIEA.
According to the synthesis method of the capelin, a further preferable technical scheme is that in the step (2), one or more of DCM, DMF, NMP, DMSO reaction solvents are used.
According to the synthesis method of the capelin, a further preferable technical scheme is that in the step (2), the Fmoc removal reagent is a v/v20% piperidine/DMF solution.
The further preferable technical scheme of the synthesis method of the carperitide is that in the step (2), the adopted cracking reagent is as follows: TFA: TA: EDT: anisole=90:5:3:2.
The further preferable technical scheme of the synthesis method of the capecitabine peptide is that in the step (2), the cyclization method is as follows: dissolving and adding PbCl 2 The pH is adjusted to be alkaline, and disulfide bonds are formed by oxidation.
The further preferable technical scheme of the synthesis method of the carperitide is that in the step (2), the carperitide is dissolved until the concentration is 2g/L.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the capecitabine adopts special protected amino acid: fmoc-Ile- (propargyl) Gly-OH, boc-Ser (Fmoc-Ser) -OH. The propargyl is introduced to improve the ductility of peptide chains, overcome the problem of difficult peptide sequence linkage, and greatly improve the yield of crude peptide; pH is regulated to be weak alkaline by PbCl 2 The propargyl is removed, and simultaneously, disulfide bond oxidation can be carried out, so that the synthesis steps are greatly simplified by a one-pot method, the labor cost and the purification cost are reduced, and the industrial scale-up production is facilitated.
Drawings
FIG. 1 is a chromatogram of a crude peptide obtained in example 1 of the present invention;
FIG. 2 is a chromatogram of a crude peptide obtained in example 2 of the present invention;
FIG. 3 is a chromatogram of a crude peptide obtained in example 3 of the present invention.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1:
preparation of Capellitide peptide resins (sequential Synthesis)
Wang Resin 30.00g (sub=0.42 mmol/g) was weighed into a solid phase reactor and 200 mM DCM swollen Resin was added for 0.5h. The solvent was drained and washed 3 times with DMF. 16.0g Fmoc-Tyr (tBu) -OH, DIC, DMAP, DMF was added and coupled for 3h, after which the solvent was removed and washed three times. Adding Ac 2 O-pyridine end capping, the degree of substitution was determined to be 0.33mmol/g.
30g of Fmoc-Tyr (tBu) -Wang Resin (sub=0.33 mmol/g) was weighed and added to 200 mM DTMF for swelling for 1h. The solvent was removed and a 200mLv/v20% piperidine/DMF solution was added to deprotect the reaction for 15+15min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Arg (Pbf) -OH 21.41g, HOBt4.82g, DIC5.5mL,200 mM LDMF solution was weighed, ice-bath activated for 10 minutes, and the activation temperature was not more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
The above steps were repeated with the amino acid sequences Fmoc-Tyr (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Phe-OH, fmoc-Ser (tBu) -OH, fmoc-Asn (Trt) -OH, fmoc-Cys (Trt) -OH, fmoc-Gly-OH, fmoc-Leu-OH, fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Ala-OH, fmoc-Gly-OH, fmoc-Ile-OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, fmoc-Met-OH, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Phe-OH, fmoc-Cys (Trt) -OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH. After the coupling, 250mL of DMF was added for 3 times, followed by deprotection by addition of 200mLv/v20% piperidine/DMF solution for 15+15min. The mixture was drained and washed 6 times with 250mL of DMF. The DCM and MeOH were alternately washed 3 times each with 400mL each, and the indene test was positive. And (5) drying in vacuum to obtain the carperitide peptide resin.
Cracking:
500mL of cleavage reagent is prepared, wherein the cleavage reagent is TFA: EDT: anisole=90:5:3:2, the full-protection peptide is added under the ice bath condition, the reaction is continued for 2 hours at room temperature, and anhydrous isopropyl ether is added for precipitation after the reaction is finished. The precipitate was centrifuged 4 times, washed with 3000mL of isopropyl ether each. The product obtained after drying was 35.65g of capecitabine peptide.
Cyclization:
19L of purified water was added to the cyclization tank, 38.60g of linear peptide was weighed, added to the cyclization tank and stirred uniformly at a concentration of about 2g/L.
Slowly dripping an iodoethanol solution for cyclization; thiol detection was performed with Ellmans reagent and the negative reaction was completed. Dropping ascorbic acid until the crude peptide solution is colorless, and standing for 1 hour; filtering with 0.45um microporous membrane, collecting filtrate, and sampling and detecting. The crude peptide is quantified by 20.50g of a reference substance, the total yield is 40.50%, and the purity is 41.97%; the chromatogram of the crude peptide is shown in FIG. 1.
Example 2:
preparation of Capelii peptide resin (Boc-Ser (Fmoc-Ser) -OH)
Wang Resin 30.00g (sub=0.42 mmol/g) was weighed into a solid phase reactor and 200 mM DCM swollen Resin was added for 0.5h. The solvent was drained and washed 3 times with DMF. 16.0g Fmoc-Tyr (tBu) -OH, DIC, DMAP, DMF was added and coupled for 3h, after which the solvent was removed and washed three times. Adding Ac 2 O-pyridine end capping, the degree of substitution was determined to be 0.33mmol/g.
30g of Fmoc-Tyr (tBu) -Wang Resin (sub=0.33 mmol/g) was weighed and added to 200 mM DTMF for swelling for 1h. The solvent was removed and a 200mLv/v20% piperidine/DMF solution was added to deprotect the reaction for 15+15min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Arg (Pbf) -OH 21.41g,HOBt4.82g,DIC5.5mL,200mL DMF solution is weighed, and the solution is activated for 10 minutes in an ice bath, wherein the activation temperature is not more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
The above steps were repeated with the amino acid sequences Fmoc-Tyr (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Phe-OH, fmoc-Ser (tBu) -OH, fmoc-Asn (Trt) -OH, fmoc-Cys (Trt) -OH, fmoc-Gly-OH, fmoc-Leu-OH, fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Ala-OH, fmoc-Gly-OH, fmoc-Ile-OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, fmoc-Met-OH, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Phe-OH, fmoc-Cys (Trt) -OH, boc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Phe-OH, fmoc-Cys (Trt) -OH, fmoc-Ser (Tbf) -OH, fmoc-Arg (Pbf) -OH). After the coupling, 250mL of DMF was added for 3 times, followed by deprotection by addition of 200mLv/v20% piperidine/DMF solution for 15+15min. The mixture was drained and washed 6 times with 250mL of DMF. The DCM and MeOH were alternately washed 3 times each with 400mL each, and the indene test was positive. And (5) drying in vacuum to obtain the carperitide peptide resin.
Cracking:
500mL of cleavage reagent is prepared, wherein the cleavage reagent is TFA: EDT: anisole=90:5:3:2, the full-protection peptide is added under the ice bath condition, the reaction is continued for 2 hours at room temperature, and anhydrous isopropyl ether is added for precipitation after the reaction is finished. The precipitate was centrifuged 4 times, washed with 3000mL of isopropyl ether each. The product obtained after drying was namely 37.65g of capelin peptide.
Cyclization:
19L of purified water was added to the cyclization tank, 38.60g of linear peptide was weighed, added to the cyclization tank and stirred uniformly at a concentration of about 2g/L.
Slowly dripping an iodoethanol solution for cyclization; thiol detection was performed with Ellmans reagent and the negative reaction was completed. Dropping ascorbic acid until the crude peptide solution is colorless, and standing for 1 hour; filtering with 0.45um microporous membrane, collecting filtrate, and sampling and detecting. The crude peptide was quantified by the control to 23.50g, overall yield 53.70% and purity 49.80%. The chromatogram of the crude peptide is shown in FIG. 2.
Example 3:
preparation of Capelip peptide resin (Fmoc-Ile- (propargyl) Gly-OH, boc-Ser (Fmoc-Ser) -OH)
Wang Resin 30.00g (sub=0.42 mmol/g) was weighed into a solid phase reactor and 200 mM DCM swollen Resin was added for 0.5h. The solvent was drained and washed 3 times with DMF. 16.0g Fmoc-Tyr (tBu) -OH, DIC, DMAP, DMF was added and coupled for 3h, after which the solvent was removed and washed three times. Adding Ac 2 O-pyridine end capping, the degree of substitution was determined to be 0.33mmol/g.
30g of Fmoc-Tyr (tBu) -Wang Resin (sub=0.33 mmol/g) was weighed and added to 200 mM DTMF for swelling for 1h. The solvent was removed and a 200mLv/v20% piperidine/DMF solution was added to deprotect the reaction for 15+15min. The mixture was drained and washed 6 times with 250mL of DMF. The indene test result is positive. Fmoc-Arg (Pbf) -OH 21.41g,HOBt4.82g,DIC5.5mL,200mL DMF solution is weighed, and the solution is activated for 10 minutes in an ice bath, wherein the activation temperature is not more than 10 ℃. And adding the activated solution into a reactor, reacting for 1h, and draining after the indene detection result is negative. DMF was added and washed 3 times with 200mL each.
The above steps were repeated with the amino acid sequences Fmoc-Tyr (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Phe-OH, fmoc-Ser (tBu) -OH, fmoc-Asn (Trt) -OH, fmoc-Cys (Trt) -OH, fmoc-Gly-OH, fmoc-Leu-OH, fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Ala-OH, fmoc-Ile- (propargyl) Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, fmoc-Met-OH, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Phe-OH, fmoc-Cys (Trt) -OH, boc-Ser (tBu) -OH, fmoc-Ser (Fmoc-OH). After the coupling, 250mL of DMF was added for 3 times, followed by deprotection by addition of 200mLv/v20% piperidine/DMF solution for 15+15min. The mixture was drained and washed 6 times with 250mL of DMF. The DCM and MeOH were alternately washed 3 times each with 400mL each, and the indene test was positive. And (5) drying in vacuum to obtain the carperitide peptide resin.
Cracking:
500mL of cleavage reagent is prepared, wherein the cleavage reagent is TFA: EDT: anisole=90:5:3:2, the full-protection peptide is added under the ice bath condition, the reaction is continued for 2 hours at room temperature, and anhydrous isopropyl ether is added for precipitation after the reaction is finished. The precipitate was centrifuged 4 times, washed with 3000mL of isopropyl ether each. The product obtained after drying was namely 38.65g of capelin peptide.
Cyclization:
19L of purified water was added to the cyclization tank, 38.60g of linear peptide was weighed, added to the cyclization tank and stirred uniformly at a concentration of about 2g/L.
Adding PbCl 2 Removing propargyl, adjusting pH to be alkaline, and oxidizing to form disulfide bonds; thiol detection was performed with Ellmans reagent and the negative reaction was completed. Standing for 1 hour; filtering with 0.45um microporous membrane, collecting filtrate, and sampling and detecting. The crude peptide is quantified by 29.50g through a reference substance, the total yield is 67.50%, and the purity is 76.09%; the chromatogram of the crude peptide is shown in FIG. 3.
Claims (6)
1. A method for synthesizing capecitabine, comprising the steps of: solid phase synthesis of fully protected capecitabine, cleavage precipitation, dissolution, and addition of PbCl 2 Removing propargyl, regulating pH, and cyclizing to obtain the capecitabine; in the solid-phase synthesis of the full-protection carperitide, fmoc-Ile- (propargyl) Gly-OH is adopted in amino acids 15-16 of the carperitide sequence for coupling reaction, and Boc-Ser (Fmoc-Ser) -OH is adopted in amino acids 5-6 of the carperitide sequence for coupling reactionAmino acids at other sites of the peptide are synthesized sequentially.
2. The method of claim 1, wherein the condensing agent used for synthesizing each amino acid is one of DIC/HOBt, HBTU/HOBt/DIEA, pyBop/HOBt/DIEA.
3. The method of claim 1, wherein the reaction solvent for synthesizing the fully protected capecitabine is one of DCM, DMF, NMP, DMSO.
4. The method for synthesizing capecitabine according to claim 1, wherein the cleavage reagent used in the step is: TFA: TA: EDT: anisole=90:5:3:2.
5. The method for synthesizing capecitabine according to claim 1, wherein in the step, the cyclization method is as follows: dissolving and adding PbCl 2 The pH is adjusted to be alkaline, and disulfide bonds are formed by oxidation.
6. The method of claim 5, wherein the step comprises dissolving the carperitide to a concentration of 2g/L.
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