CN110759972A - Preparation method of atosiban - Google Patents
Preparation method of atosiban Download PDFInfo
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- CN110759972A CN110759972A CN201911063433.0A CN201911063433A CN110759972A CN 110759972 A CN110759972 A CN 110759972A CN 201911063433 A CN201911063433 A CN 201911063433A CN 110759972 A CN110759972 A CN 110759972A
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- atosiban
- resin
- preparation
- product
- linear peptide
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- 108700007535 atosiban Proteins 0.000 title claims abstract description 81
- 229960002403 atosiban Drugs 0.000 title claims abstract description 79
- VWXRQYYUEIYXCZ-OBIMUBPZSA-N atosiban Chemical compound C1=CC(OCC)=CC=C1C[C@@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CCCN)C(=O)NCC(N)=O)CSSCCC(=O)N1 VWXRQYYUEIYXCZ-OBIMUBPZSA-N 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 150000001413 amino acids Chemical class 0.000 claims abstract description 21
- 229920005989 resin Polymers 0.000 claims description 40
- 239000011347 resin Substances 0.000 claims description 40
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 21
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 16
- 239000012043 crude product Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 10
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 9
- 229920003180 amino resin Polymers 0.000 claims description 9
- 229910052740 iodine Inorganic materials 0.000 claims description 9
- 239000011630 iodine Substances 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007800 oxidant agent Substances 0.000 claims description 6
- 230000001590 oxidative effect Effects 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 5
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000007363 ring formation reaction Methods 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 125000006239 protecting group Chemical group 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 125000002849 D-tyrosine group Chemical group [H]N([H])[C@@]([H])(C(=O)[*])C([H])([H])C1=C([H])C([H])=C(O[H])C([H])=C1[H] 0.000 claims 1
- 230000001681 protective effect Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 33
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 28
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- 239000012071 phase Substances 0.000 description 18
- 229940024606 amino acid Drugs 0.000 description 15
- 235000001014 amino acid Nutrition 0.000 description 15
- 238000000746 purification Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 238000010828 elution Methods 0.000 description 10
- 150000003839 salts Chemical group 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- -1 9-fluorenylmethoxycarbonyl group Chemical group 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- RMYPEYHEPIZYDJ-SNVBAGLBSA-N (2r)-2-azaniumyl-3-(4-ethoxyphenyl)propanoate Chemical compound CCOC1=CC=C(C[C@@H](N)C(O)=O)C=C1 RMYPEYHEPIZYDJ-SNVBAGLBSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008602 contraction Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000012047 saturated solution Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 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 1
- XUKUVROJKPSLLU-XMMPIXPASA-N (2r)-3-(4-ethoxyphenyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC(OCC)=CC=C1C[C@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 XUKUVROJKPSLLU-XMMPIXPASA-N 0.000 description 1
- JOOIZTMAHNLNHE-NRFANRHFSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 JOOIZTMAHNLNHE-NRFANRHFSA-N 0.000 description 1
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- ZPGDWQNBZYOZTI-UHFFFAOYSA-N 1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)C1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-UHFFFAOYSA-N 0.000 description 1
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 1
- YVOOPGWEIRIUOX-UHFFFAOYSA-N 2-azanyl-3-sulfanyl-propanoic acid Chemical compound SCC(N)C(O)=O.SCC(N)C(O)=O YVOOPGWEIRIUOX-UHFFFAOYSA-N 0.000 description 1
- IIVWHGMLFGNMOW-UHFFFAOYSA-N 2-methylpropane Chemical compound C[C](C)C IIVWHGMLFGNMOW-UHFFFAOYSA-N 0.000 description 1
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- OUYCCCASQSFEME-MRVPVSSYSA-N D-tyrosine Chemical compound OC(=O)[C@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-MRVPVSSYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101500028587 Homo sapiens Oxytocin Proteins 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 102000004279 Oxytocin receptors Human genes 0.000 description 1
- 108090000876 Oxytocin receptors Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 1
- 102000004136 Vasopressin Receptors Human genes 0.000 description 1
- 108090000643 Vasopressin Receptors Proteins 0.000 description 1
- 108010004977 Vasopressins Proteins 0.000 description 1
- 102000002852 Vasopressins Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- OHSJPLSEQNCRLW-UHFFFAOYSA-N triphenylmethyl radical Chemical compound C1=CC=CC=C1[C](C=1C=CC=CC=1)C1=CC=CC=C1 OHSJPLSEQNCRLW-UHFFFAOYSA-N 0.000 description 1
- 229960003726 vasopressin Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a preparation method of atosiban, which adopts special protective amino acid segments: mpr (R) -D-Tyr (Et) improves the purity and yield of products in large-scale preparation.
Description
Technical Field
The invention relates to the field of medicine synthesis, and particularly relates to a preparation method of atosiban.
Background
Atosiban (Atosiban) is a synthetic peptide substance that produces competitive inhibition of human oxytocin at the receptor level. The results of animal experiments of rats and guinea pigs show that the product can reduce the contraction frequency and tension of uterus and inhibit the contraction of uterus after being combined with oxytocin receptor. The product also binds to vasopressin receptor and inhibits the action of vasopressin.
Atosiban has the following structure:
regarding the preparation method of atosiban, the common method in the prior art at home at present is Fmoc solid phase synthesis, which adopts amino resin as the starting carrier resin, sequentially accesses protective amino acid, and the obtained atosiban iodine is oxidized and then cracked to obtain the atosiban. However, the above-mentioned conventional processes are insufficient in terms of purity of crude peptide and total yield.
In addition, the product has the structure of D-Tyr (Et), and Fmoc-D-Tyr (Et) is easy to generate racemization reaction in the peptide splicing process to generate Tyr (Et)2]-atosiban impurity, the impurity being in combination with atosibanTosiban is similar in polarity and is difficult to completely remove through purification, thereby affecting the quality of the product.
Disclosure of Invention
The invention provides a preparation method of atosiban, aiming at preparing high-purity atosiban, and in order to realize the aim, the invention provides the following technical scheme:
the invention provides a preparation method of atosiban, which comprises the following steps: the method comprises the following steps of adopting amino resin as starting resin, synthesizing atosiban linear peptide resin in a solid phase manner, carrying out acidolysis on the atosiban linear peptide resin to obtain the atosiban linear peptide, dissolving the atosiban linear peptide by adopting an acetic acid solution, then dropwise adding iodine while stirring until complete cyclization is carried out to obtain a crude atosiban product, and purifying the crude atosiban product to obtain a pure product.
Wherein the synthetic process of the atosiban resin uses the following special protected amino acid fragment Mpr (R) -D-Tyr (Et) besides other conventional protected amino acids.
Atosiban resin was:
mpr (R) -D-Tyr (Et) -Ile-Thr (tBu) -Asn (Trt) -Cys (Trt) -Pro-Orn (Boc) -Gly-amino resin
In the preparation method of atosiban, the amino resin has an amino substitution value of 0.3 to 1.5mmol/g resin, and the preferable substitution value is 0.5 to 1.0mmol/g resin.
In the preparation method of atosiban, the amino resin is one of Rink MBHA resin, Rink Amide resin or Rink Amide AM resin, and Rink Amide MBHA resin is preferred.
In the preparation method of atosiban, the dosage of Fmoc-protected amino acid or protected amino acid fragment is 1.2-6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.
In a preferred embodiment of the present invention, the atosiban resin is subjected to acidolysis while removing the resin and the side chain protecting groups to obtain a crude atosiban linear peptide.
Further, an acidolysis agent adopted in the acidolysis of the atosiban resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water, and the mixture ratio of the mixed solvent is as follows: the TFA ratio is 80-95% (V/V), the EDT ratio is 1-10% (V/V), and the balance is water. The preferred formulation is 89-91% TFA, 4-6% EDT, and the balance water. Preferably, the mixture ratio is 90%, EDT 5% and the balance of water.
The dosage of the acidolysis agent is 4-15 ml of acidolysis agent required by one gram of atosiban resin, and preferably 9-11 ml of acidolysis agent required by one gram of atosiban resin. The time for cracking by using the acidolysis agent is 1-5 hours, preferably 2 hours at room temperature.
Further, the crude product of the atosiban linear peptide is dissolved by acetic acid, and oxidized and cyclized by an oxidizing agent after being filtered to obtain a solution of the crude atosiban. The volume percentage concentration of the acetic acid is 20-40%, and the preferred concentration is 30%. The oxidant is iodine or H2O2Or DMSO, preferably iodine. The oxidant is added in a titration mode, and the oxidant is stopped adding when the oxidation end point is reached.
Further, the atosiban crude product is purified by high performance liquid chromatography and freeze-dried to obtain a pure atosiban product, and the specific method comprises the following steps:
purifying by high performance liquid chromatography, wherein the chromatographic filler for purification is 10 μm reversed phase C18, a mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, a chromatographic column with flow rate of 77mm x 250mm is 90mL/min, a gradient system is adopted for elution, purification is performed by circulating sample injection, a crude product solution is sampled on the chromatographic column, the mobile phase elution is started, a main peak is collected, acetonitrile is evaporated, and then the crude product solution is filtered by a 0.45 μm filter membrane to obtain an atosiban purified intermediate concentrated solution;
performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic packing for purification is reversed phase C18 with 10 μm, and the flow rate of 77mm × 250mm chromatographic column is 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns with different specifications); adopting gradient elution and circulation sample loading method, loading sample in chromatographic column, starting mobile phase elution, collecting atlas, observing change of absorbance, collecting main peak of salt exchange and analyzing liquid phase to detect purity, combining main peak solution of salt exchange, concentrating under reduced pressure to obtain atosiban acetic acid aqueous solution, and freeze-drying to obtain pure atosiban.
Detailed Description
The invention discloses a method for synthesizing atosiban, which can be realized by appropriately improving process parameters by a person skilled in the art according to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made and the techniques of the present invention employed without departing from the spirit and scope of the invention.
In the specific embodiment of the present invention, the Chinese meanings corresponding to the English abbreviations used in the application documents are shown in Table 1.
TABLE 1 English abbreviation definitions
| English abbreviation | Name of Chinese | English abbreviation | Name of Chinese |
| Fmoc | 9-fluorenylmethoxycarbonyl group | Acm | Acetamidomethyl |
| tBu | Tert-butyl radical | Trt | Trityl radical |
| Boc | Tert-butyloxycarbonyl radical | Asn | Asparagine and its use |
| Gly | Glycine | Thr | Threonine |
| Orn | Ornithine | Ile | Isoleucine |
| Pro | Proline | D-Tyr(Et) | O-ethyl-D-tyrosine |
| Cys | Cysteine | Mpr | Mercaptopropionic acid |
The invention is further illustrated by the following examples.
Example 1: synthesis of atosiban peptide resin
Atosiban resin was:
mpr (R) -D-Tyr (Et) -Ile-Thr (tBu) -Asn (Trt) -Cys (Trt) -Pro-Orn (Boc) -Gly-amino resin
Rink Amide MBHA resin is used as initial resin, and is coupled with protected amino acid shown in table 2 in sequence through Fmoc protection removal and coupling reaction to prepare atosiban resin. The protected amino acids or fragments corresponding to the protected amino acids used in this example are shown below:
TABLE 2
| The peptide sequence n ═ | Protected amino acids |
| 1 | Fmoc-Gly-OH |
| 2 | Fmoc-Orn(Boc) |
| 3 | Fmoc-Pro |
| 4 | Fmoc-Cys(Trt) |
| 5 | Fmoc-Asn(Trt) |
| 6 | Fmoc-Thr(tBu) |
| 7 | Fmoc-Ile |
| 8 | Mpr(R)-D-Tyr(Et) |
1. Introduction of the 1 st protected amino acid
Dissolving 0.15mol of the 1 st protected amino acid and 0.15mol of HOBt in a proper amount of DMF; and adding 0.15mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.
0.05mol of Rink Amide MBHA resin (the substitution value is about 0.5mmol/g) is taken to be deprotected by 20 percent PIP/DMF solution for 25 minutes, and the resin after Fmoc removal is obtained by washing and filtering.
And adding the activated 1 st protected amino acid solution into the Fmoc-removed resin, performing coupling reaction for 120-300 minutes, and filtering and washing to obtain the resin containing 1 protected amino acid.
2. 2-8 protective amino acids or fragments are inoculated
And sequentially inoculating the corresponding 1 st to 8 th protected amino acids or fragments by the same method to obtain the atosiban peptide resin.
Example 2: preparation of crude atosiban
Taking 0.025mol of atosiban linear peptide resin prepared in example 1, adding a mixed acidolysis solution consisting of 85 volume percent of TFA, 7.5 volume percent of EDT and 7.5 volume percent of water for acidolysis (10 mL of the mixed acidolysis solution per gram of atosiban resin), uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitation with anhydrous ether for 3 times, and drying under reduced pressure at 35-45 ℃ to obtain the white-like powder.
Dissolving the obtained off-white powder by using a 20% acetic acid solution to prepare a solution of about 3mg/ml, dropwise adding an iodine/ethanol saturated solution while stirring until complete cyclization is achieved, and carrying out reduced pressure concentration at 35-40 ℃ to obtain a concentrated solution of an atosiban crude product, wherein the purity of the crude product is 87.6%.
Example 3: preparation of crude atosiban
Taking 0.025mol of atosiban linear peptide resin prepared in example 1, adding a mixed acidolysis solution consisting of 90 volume percent TFA, 5 volume percent EDT and 5 volume percent water for acidolysis (the mixed acidolysis solution is 10 mL/g of atosiban resin), uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, and drying under reduced pressure at 35-45 ℃ to obtain the white-like powder.
Dissolving the obtained off-white powder by using 30% acetic acid solution to prepare a solution of about 3mg/ml, dropwise adding an iodine/ethanol saturated solution while stirring until complete cyclization is achieved, and concentrating under reduced pressure at 35-40 ℃ to obtain a concentrated solution of an atosiban crude product, wherein the purity of the crude product is 89.4%.
Example 4: purification of crude atosiban
Taking the crude atosiban obtained in the example 3, filtering the solution by a 0.45-micron microporous membrane, and purifying for later use;
purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is 10 μm reversed phase C18, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column of 77mm x 250mm is 90mL/min, eluting by a gradient system, circularly injecting for purification, sampling a crude product solution in the chromatographic column, starting the mobile phase for elution, collecting a main peak, and evaporating acetonitrile to obtain an atosiban purified intermediate concentrated solution;
performing salt exchange by high performance liquid chromatography, wherein a mobile phase system is 1% acetic acid/water solution-acetonitrile, a chromatographic filler for purification is reverse phase C18 with the diameter of 10 mu m, the flow rate of a chromatographic column with the diameter of 77mm 250mm is 90mL/min, gradient elution and a circular sample loading method are adopted, the sample is loaded in the chromatographic column, the mobile phase elution is started, a map is collected, the change of the absorbance is observed, a main salt exchange peak is collected and the purity is detected by an analytical liquid phase, the main salt exchange peak solutions are combined, reduced pressure concentration is performed to obtain an atosiban acetic acid water solution, freeze drying is performed to obtain 15.2g of atosiban pure product, the purity is 99.7%, the maximum single impurity is 0.06%, the total yield is 61.1%, [ Et ]2]Atosiban was not detected. The molecular weight was 994.2 (100% M + H).
Example 5: purification of crude atosiban
Taking the crude product of atosiban obtained in the embodiment 4, filtering the solution by a 0.45-micron microporous membrane, and purifying for later use;
purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is 10 μm reversed phase C18, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column of 77mm x 250mm is 90mL/min, eluting by a gradient system, circularly injecting for purification, sampling a crude product solution in the chromatographic column, starting the mobile phase for elution, collecting a main peak, and evaporating acetonitrile to obtain an atosiban purified intermediate concentrated solution;
performing salt exchange by high performance liquid chromatography, wherein a mobile phase system is 1% acetic acid/water solution-acetonitrile, a chromatographic filler for purification is reverse phase C18 with the diameter of 10 mu m, the flow rate of a chromatographic column with the diameter of 77mm 250mm is 90mL/min, gradient elution and a circular sample loading method are adopted, the chromatographic column is loaded, the mobile phase elution is started, a map is collected, the change of the absorbance is observed, a main salt exchange peak is collected and the purity is detected by analyzing a liquid phase, the main salt exchange peak solutions are combined, reduced pressure concentration is performed to obtain an atosiban acetic acid water solution, freeze drying is performed to obtain 15.9g of atosiban pure product, the purity is 99.8%, the maximum single impurity is 0.04%, the total yield is 63.9%, [ Et ], (Tyr)2]Atosiban was not detected. The molecular weight was 994.2 (100% M + H).
The embodiment shows that the purity of the product obtained by the method provided by the invention is more than 99.5%, and the single impurity is less than 0.15%, so that the product quality is improved, and the method has wide practical value and application prospect.
Claims (8)
1. A method for preparing atosiban is characterized by comprising the following steps: adopting amino resin as initial resin, synthesizing atosiban linear peptide resin in a solid phase, obtaining atosiban linear peptide after acidolysis of the atosiban linear peptide resin, dissolving the atosiban linear peptide by adopting an acetic acid solution, then dropwise adding iodine while stirring until complete cyclization to obtain a crude atosiban product, and obtaining a pure product after purifying the crude atosiban product:
2. a process for the preparation of atosiban according to claim 1, wherein: when Mpr at position 1 and D-Tyr at position 2 are grafted together, the corresponding protected amino acid is Mpr (R) -D-Tyr (Et).
3. A process for preparing atosiban according to claim 1, wherein the amino resin has an amino substitution value of 0.3 to 1.5mmol/g resin, preferably a substitution value of 0.5 to 1.0mmol/g resin.
4. A process for preparing atosiban according to claim 1, wherein the amino resin is one of Rink MBHA resin, Rink Amide resin or Rink Amide AM resin, preferably Rink Amide MBHA resin.
5. A process for the preparation of atosiban according to claim 1, wherein: the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2-6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.
6. A process for the preparation of atosiban according to any one of claims 1 to 5, wherein: and (3) carrying out acidolysis on the atosiban peptide resin, and simultaneously removing the resin and a side chain protecting group to obtain a crude product of the atosiban linear peptide.
7. A process for the preparation of atosiban according to claim 1, wherein: dissolving the atosiban linear peptide crude product by using acetic acid, filtering, and oxidizing and cyclizing by using an oxidizing agent to obtain an atosiban crude product solution. The volume percentage concentration of the acetic acid is 20-40%, and the preferred concentration is 30%. The oxidant is iodine or H2O2Or DMSO, preferably iodine.
8. A process for the preparation of atosiban according to claim 1, wherein: and purifying the atosiban crude product by high performance liquid chromatography and freeze-drying to obtain a pure atosiban product.
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| CN114685614A (en) * | 2020-12-30 | 2022-07-01 | 湖北健翔生物制药有限公司 | Solid-phase synthesis method of atosiban |
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