CN113698457B - Preparation method of gonadorelin acetate - Google Patents
Preparation method of gonadorelin acetate Download PDFInfo
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- CN113698457B CN113698457B CN202011320462.3A CN202011320462A CN113698457B CN 113698457 B CN113698457 B CN 113698457B CN 202011320462 A CN202011320462 A CN 202011320462A CN 113698457 B CN113698457 B CN 113698457B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 108700012941 GNRH1 Proteins 0.000 title claims abstract description 37
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 title claims abstract description 36
- NGCGMRBZPXEPOZ-HBBGHHHDSA-N acetic acid;(2s)-n-[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[2-[[(2s)-1-[[(2s)-1-[(2s)-2-[(2-amino-2-oxoethyl)carbamoyl]pyrrolidin-1-yl]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-3-(4-hydroxyphenyl)- Chemical compound CC(O)=O.C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 NGCGMRBZPXEPOZ-HBBGHHHDSA-N 0.000 title claims abstract description 36
- 229960005122 gonadorelin acetate Drugs 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 84
- 238000011282 treatment Methods 0.000 claims abstract description 80
- 239000000178 monomer Substances 0.000 claims abstract description 68
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 58
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- 229920005989 resin Polymers 0.000 claims abstract description 47
- 229920001184 polypeptide Polymers 0.000 claims abstract description 24
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 24
- -1 Boc-amino Chemical group 0.000 claims abstract description 18
- 239000007858 starting material Substances 0.000 claims abstract description 18
- 238000005915 ammonolysis reaction Methods 0.000 claims abstract description 17
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims abstract description 15
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims abstract description 9
- 238000012546 transfer Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 216
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- 238000010168 coupling process Methods 0.000 claims description 65
- 238000005859 coupling reaction Methods 0.000 claims description 65
- 230000008878 coupling Effects 0.000 claims description 63
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 57
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical group C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 43
- 239000000243 solution Substances 0.000 claims description 40
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical group C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 39
- 239000012141 concentrate Substances 0.000 claims description 39
- 101500026183 Homo sapiens Gonadoliberin-1 Proteins 0.000 claims description 37
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- 229960001442 gonadorelin Drugs 0.000 claims description 35
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 31
- 239000007864 aqueous solution Substances 0.000 claims description 23
- 238000001914 filtration Methods 0.000 claims description 22
- 239000002904 solvent Substances 0.000 claims description 22
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 21
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- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 17
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- 238000004128 high performance liquid chromatography Methods 0.000 claims description 17
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 15
- 239000007787 solid Substances 0.000 claims description 15
- MDXGYYOJGPFFJL-QMMMGPOBSA-N N(alpha)-t-butoxycarbonyl-L-leucine Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)OC(C)(C)C MDXGYYOJGPFFJL-QMMMGPOBSA-N 0.000 claims description 14
- 229940024606 amino acid Drugs 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 14
- ZQEBQGAAWMOMAI-ZETCQYMHSA-N (2s)-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)(C)OC(=O)N1CCC[C@H]1C(O)=O ZQEBQGAAWMOMAI-ZETCQYMHSA-N 0.000 claims description 12
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- CNBUSIJNWNXLQQ-NSHDSACASA-N (2s)-3-(4-hydroxyphenyl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CNBUSIJNWNXLQQ-NSHDSACASA-N 0.000 claims description 11
- HSQIYOPBCOPMSS-ZETCQYMHSA-N (2s)-5-(diaminomethylideneamino)-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CCCN=C(N)N HSQIYOPBCOPMSS-ZETCQYMHSA-N 0.000 claims description 10
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- NFVNYBJCJGKVQK-ZDUSSCGKSA-N N-[(Tert-butoxy)carbonyl]-L-tryptophan Chemical compound C1=CC=C2C(C[C@H](NC(=O)OC(C)(C)C)C(O)=O)=CNC2=C1 NFVNYBJCJGKVQK-ZDUSSCGKSA-N 0.000 claims description 7
- 229960002885 histidine Drugs 0.000 claims description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 6
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 19
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- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 8
- 238000013459 approach Methods 0.000 description 8
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
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- 238000005303 weighing Methods 0.000 description 7
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 6
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- 239000008215 water for injection Substances 0.000 description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 239000001632 sodium acetate Substances 0.000 description 5
- 235000017281 sodium acetate Nutrition 0.000 description 5
- WXYGVKADAIJGHB-ZDUSSCGKSA-N (2s)-3-(1h-indol-2-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound C1=CC=C2NC(C[C@H](NC(=O)OC(C)(C)C)C(O)=O)=CC2=C1 WXYGVKADAIJGHB-ZDUSSCGKSA-N 0.000 description 4
- 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 4
- DCLJSEPKYJSEHW-HNNXBMFYSA-N (2s)-3-[1-(4-methylphenyl)sulfonylimidazol-4-yl]-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N1C=C(C[C@H](NC(=O)OC(C)(C)C)C(O)=O)N=C1 DCLJSEPKYJSEHW-HNNXBMFYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
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- 239000012507 Sephadex™ Substances 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 231100000540 amenorrhea Toxicity 0.000 description 2
- 150000003862 amino acid derivatives Chemical class 0.000 description 2
- 229960003589 arginine hydrochloride Drugs 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 2
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- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
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- AHDSRXYHVZECER-UHFFFAOYSA-N 2,4,6-tris[(dimethylamino)methyl]phenol Chemical compound CN(C)CC1=CC(CN(C)C)=C(O)C(CN(C)C)=C1 AHDSRXYHVZECER-UHFFFAOYSA-N 0.000 description 1
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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
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the technical field of medicines, in particular to a preparation method of gonadorelin acetate. The preparation method of the gonadorelin acetate comprises the following steps: (a) To be used forTaking Boc-amino acid derivatives and pGlu as starting materials, gradually grafting peptide, subjecting polypeptide resin to ammonolysis resin removal by polypeptide resin (b), purifying, and performing acetate transfer treatment to obtain gonadorelin acetate; in each step of peptide-binding treatment, the monomer, the first condensing agent, the second condensing agent and
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a preparation method of gonadorelin acetate.
Background
Gonadorelin, chemical name 5' -oxidized prolyl-L-histidyl-L-seryl-L-tyrosyl-glycyl-L-leucyl-L-arginyl-L-prolyl-glycylamine, has the following structural formula:
Gonadorelin is an artificially synthesized gonadotrophin releasing hormone, belongs to peptide compounds, and is decapeptides. Gonadorelin is useful for diagnosing hypothalamic-pituitary-gonadal dysfunction, treating infertility caused by amenorrhea and hypogonadotrophin secretion and polyfollicular ovary, for ovulation promotion to treat infertility caused by hypothalamic amenorrhea, primary ovarian dysfunction, etc.
In the existing method for synthesizing the gonadorelin, china patent application CN105646671A discloses a method for gradient elution and purification by combining an SPE column and a reversed phase chromatographic column, but in the method, a trifluoroacetic acid aqueous solution system and a phosphoric acid aqueous solution system are adopted, trifluoroacetic acid is strong acid and is easy to volatilize and difficult to prepare, phosphoric acid is difficult to remove in a subsequent organic solvent removal process, and the solvent residue risk in bulk drugs is increased; according to the method, acetate conversion is carried out by adopting 0.05% -0.3% of acetic acid aqueous solution, the acetic acid content is low, salt conversion is incomplete, most of acetic acid exists in a free form, the combination is unstable, the acetic acid is easily removed in the later rotary evaporation process, the acetic acid content of the bulk drug is low, and the result is difficult to control and repeat. Chinese patent application CN107176975A discloses a method for synthesizing gonadorelin by solid phase method, the post process is complex, at least 3 freeze drying operations are needed, the freeze drying cost is high, the production period is long, more TFA, HF or TFE with strong corrosiveness is needed, and the waste liquid generated is difficult to treat. In the step of synthesizing the crude gonadorelin peptide by the gonadorelin peptide resin, diethyl ether is adopted for precipitation, formed particles are fine, and centrifugation and filtration are difficult to carry out.
In the process for synthesizing other small molecular polypeptides similar to the gonadorelin in the prior art, the problems of relatively high dosage of amino acid monomers and condensing agents in the peptide grafting process, complex washing process after peptide grafting and large solvent dosage exist, so that the cost of raw materials is high, and the industrialized development of the gonadorelin is limited to a certain extent.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a preparation method of gonadorelin acetate, which aims to solve the technical problems of high cost of raw materials for preparing the gonadorelin acetate and the like in the prior art.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
The preparation method of the gonadorelin acetate comprises the following steps:
(a) To be used for Gradually grafting peptide by taking Boc-amino acid derivative and pGlu as monomers as starting materials, and treating the polypeptide resin/>
(B) Carrying out ammonolysis resin removal on the polypeptide resin, purifying, and then carrying out acetate transfer treatment to obtain the gonadorelin acetate;
The condensing agent adopted in the peptide grafting treatment comprises a first condensing agent and a second condensing agent, wherein the first condensing agent is at least one of HOBT and HOAT, and the second condensing agent comprises DCCI;
In each step of peptide-binding treatment, the monomer, the first condensing agent, the second condensing agent and The mol ratio of (1.5-3) to (1.72-3.45) to 1.
According to the invention, the solid-phase polypeptide is synthesized into the gonadorelin acetate by adopting the Boc method, so that the cost of raw materials and auxiliary materials is reduced and the product yield is ensured and even improved under the condition of reducing the dosage of a monomer and a condensing agent.
In a preferred embodiment of the present invention, in each step of the polypeptide treatment, the monomer, the first condensing agent, the second condensing agent andThe molar ratio of (2) to (2) is 1.5:2:2:1.
In a preferred embodiment of the present invention, the first condensing agent is HOAT. The combination of condensing agents of HOAT and DCCI is preferred.
In a specific embodiment of the present invention, the Boc-amino acid derivative comprises Boc-L-proline, boc-L-arginine, boc-L-leucine, boc-glycine, boc-L-tyrosine, N-BOC-O-t-butyl-L-serine, boc-L-tryptophan and N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine.
In practice, the Boc-amino acid derivative may also be the hydrochloride and/or monohydrate of each Boc-amino acid.
In a specific embodiment of the present invention, each step of the peptide-grafting treatment comprises: deprotection, neutralization of free amino terminal, coupling of the corresponding monomer and washing treatment after coupling;
The coupled washing treatment comprises: the washing treatment was performed using DMF and CH 2Cl2.
In a preferred embodiment of the present invention, the post-coupling wash treatment comprises: washing with DMF 2-3 times and washing with CH 2Cl2 2-4 times.
In the condition of the coupled washing treatment, DMF and CH 2Cl2 are adopted to wash the coupled resin, ethanol is not adopted, the problems of shrinkage, space reduction, steric hindrance increase and the like of the resin caused by ethanol washing are avoided, the washing of other reagents is not facilitated, the required washing procedure is increased, and the process is complicated. The effect is particularly pronounced for amino acid derivative monomers having side chains.
Furthermore, by optimizing the washing conditions, the washing effectiveness can be ensured under the condition of less washing times, the process is simplified, and the yield and purity are ensured and even improved.
In a preferred embodiment of the present invention, the washing treatment after coupling of N-BOC-O-tert-butyl-L-serine, boc-L-tryptophan comprises: the washing was performed 3 times with DMF and 2 times with CH 2Cl2.
In a preferred embodiment of the present invention, the washing treatment after coupling of N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine comprises: the washing was performed 3 times with DMF and 3 times with CH 2Cl2.
In a preferred embodiment of the invention, the washing treatment after coupling pGlu comprises: the washing was performed 3 times with CH 2Cl2, 3 times with DMF and 1 time with CH 2Cl2.
In a preferred embodiment of the invention, the washing treatments after coupling Boc-L-proline, boc-L-arginine, boc-L-leucine, boc-glycine, boc-L-tyrosine, respectively, comprise: the washing was performed 2 times with DMF and 2 times with CH 2Cl2.
In a specific embodiment of the present invention, the method for deprotecting a group comprises: and (3) placing the material to be deprotected in an organic solvent containing HCl/iPrOH, stirring for 40-60 min, pumping, and washing.
In a specific embodiment of the present invention, in the peptide-grafting treatment of respectively grafting Boc-L-proline, boc-L-arginine, boc-L-leucine, boc-glycine, boc-L-tyrosine, N-BOC-O-tert-butyl-L-serine, boc-L-tryptophan, the organic solvent is methylene chloride. Preferably, the volume ratio of the HCl/iPrOH to the dichloromethane is 1:1. Further, in the HCl/iPrOH, the concentration of the HCl is 9-10 mol/L.
In a specific embodiment of the present invention, in the method of deprotecting the peptide-grafting treatment of N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine and pGlu, respectively, the organic solvent comprises methylene chloride and mercaptoethanol. Preferably, the volume ratio of HCl/iPrOH, dichloromethane and mercaptoethanol is 5:4:1. Further, in the HCl/iPrOH, the concentration of the HCl is 9-10 mol/L.
In a specific embodiment of the present invention, the washing treatment in the deprotection process comprises: washing was performed with CH 2Cl2 and DMF. Further, the mixture is washed 3 times by CH 2Cl2 in turn, drained, washed 1 time by DMF and drained.
In a specific embodiment of the invention, the method of neutralizing the free amino terminus comprises: washing with dichloromethane solution containing triethylamine for 3 times, draining, and washing. Further, in the dichloromethane solution containing triethylamine, the volume ratio of the triethylamine to the dichloromethane is 5:95. Further, the washing treatment includes: washing with DMF for 1 time, draining, washing with CH 2Cl2 for 5-6 times until the mixture is neutral, and draining.
In a specific embodiment of the present invention, the method of coupling the corresponding monomers comprises: the monomer and the condensing agent are respectively dissolved by adopting an organic solvent, mixed and activated for 5 to 30 minutes at the temperature of between 0 and 5 ℃, then subjected to solid-liquid separation, the liquid is mixed with the neutralized precursor peptide-resin, and the solvent is pumped down after the reaction is carried out for 12 to 24 hours at the temperature of between 5 and 10 ℃.
In a specific embodiment of the present invention, the ammonolysis method comprises:
(b1) The polypeptide resin is shaken in an alcohol solvent for 18 to 30 hours under the airtight condition of anhydrous liquid ammonia, then filtered, the solid is washed by the alcohol solvent, and the combined filtering and washing liquid is collected;
(b2) Concentrating the filtering and washing liquid to be dry, adding an alcohol solvent to dissolve and then concentrating to be dry, and repeating the dissolving and concentrating to be dry for at least three times to obtain a foam concentrate I;
(b3) Dissolving the concentrate I by using methanol, adding acetone for precipitation, filtering, dissolving a filter cake by using an acetic acid aqueous solution, concentrating to dryness, adding methanol for dissolving, concentrating to dryness, and repeating the operation of adding methanol for dissolving and concentrating to dryness for at least three times to obtain a foam concentrate II; wherein the volume ratio of the methanol to the acetone is 1:4-5; preferably, the ratio of the concentrate I to the methanol to the acetone is 1g to 5mL to (20-25) mL;
(b4) Dissolving the concentrate II by using methanol, adding acetone for precipitation, filtering, washing a filter cake by using acetone for 1 time, washing by using diethyl ether for 2 times, and drying to obtain a crude gonadorelin product; wherein the volume ratio of the methanol to the acetone is 1:1.5-2; preferably, the ratio of the concentrate II, the methanol and the acetone is 1g to 10mL to (15-20) mL.
Wherein, the proportion of the concentrate I, the methanol and the acetone is that the dosage of the methanol is 5mL and the dosage of the acetone is 20-25 mL relative to 1g of the concentrate I; the ratio of concentrate II, methanol and acetone means that the amount of methanol is 10mL and the amount of acetone is 15-20 mL for each 1g of concentrate II.
In a preferred embodiment of the invention, in step (b 3) the volume ratio of methanol to acetone is 1:5, and in step (b 4) the volume ratio of methanol to acetone is 1:2; or in the step (b 3), the volume ratio of the methanol to the acetone is 1:4, and in the step (b 4), the volume ratio of the methanol to the acetone is 1:1.5.
In a preferred embodiment of the invention, the ratio of concentrate I, methanol, acetone is 1 g:5 mL:20 mL; the ratio of concentrate II, methanol and acetone was 1 g:10 mL:15 mL.
In a specific embodiment of the invention, the purification method comprises: CM purification and HPLC purification.
According to the invention, a certain purification method is adopted, and the impurity content is reduced through pre-purification, so that the impurity load capacity in the chromatographic column is reduced, the use times of the chromatographic column such as a C18 column are prolonged, and the product purity is improved.
In a specific embodiment of the invention, reverse phase HPLC method is used to transdetate. Further, the stationary phase of the reverse phase HPLC method trans-acetate is octadecylsilane chemically bonded silica.
In a specific embodiment of the present invention, the method for converting acetate comprises: and diluting the purified gonadorelin intermediate with water, adsorbing the diluted gonadorelin intermediate onto an HPLC preparation column subjected to equilibrium treatment, washing the purified gonadorelin intermediate by adopting an ammonium acetate buffer solution, washing the purified gonadorelin intermediate by adopting an acetonitrile water solution with the volume fraction of 3%, eluting the purified gonadorelin intermediate by adopting an acetonitrile water solution with the volume fraction of 80%, and collecting an eluent with an absorption value.
In a specific embodiment of the invention, the washing volume of the ammonium acetate buffer is 5 to 5.5 column volumes, preferably 5 column volumes; the washing volume of the acetonitrile aqueous solution with the volume fraction of 3% is 10 to 11 times the column volume, preferably 10 times the volume.
Further, the equilibrium treatment was performed on the HPLC preparation column using an aqueous acetonitrile solution with a volume fraction of 3%.
Further, the ammonium acetate buffer is ammonium acetate buffer with pH of 5.0 and concentration of 0.1 mol/L.
In a specific embodiment of the invention, the eluent is concentrated to dryness, dissolved in water and concentrated to dryness, and repeated at least 3 times, to obtain concentrate III.
In the step of transferring acetate, acetonitrile solution with lower concentration (such as volume fraction of 3%) is adopted in the balancing and washing process, so that the adsorption force of target peptide on a column in the process of loading sample can be enhanced, and no flow-through loss is caused in the washing process, so that the single loading quantity can be greatly increased, and the eluting effect is ensured by adopting acetonitrile solution with higher concentration (such as volume fraction of 80%) in the subsequent eluting process. Therefore, the step of the invention reduces the sampling times, ensures the sufficient salt transferring effect and avoids the loss caused by incomplete elution.
In a specific embodiment of the present invention, the method further comprises the step of subjecting the material subjected to the trans-acetate treatment to freeze-drying treatment.
In practice, the lyophilization process comprises:
Pre-freezing: the inlet temperature of the heat conduction oil is less than-40 ℃, and the heat preservation is carried out for about 2 hours;
sublimation: the temperature rising speed of the heat conduction oil is about 10 ℃/h, the temperature of the heat conduction oil rises to 38 ℃, and the vacuum degree of the box is not more than 20Pa before process control;
when the temperature of the heat conduction oil reaches about 38 ℃, the heat is preserved for about 18 hours to the drying end point.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, through optimizing an amino acid monomer, a condensing agent, a washing process and the like, the falling-off of-OH is reduced, the generation of side reaction is reduced, the reaction conversion rate is improved, the consumption of the amino acid monomer and the condensing agent is reduced, and the cost of raw materials and auxiliary materials is greatly reduced; meanwhile, the use amount of the organic solvent and the generation of organic waste liquid are reduced by about 40%, and the purchase cost of the organic solvent and the treatment cost of the waste liquid are reduced;
(2) In the preparation method of the gonadorelin acetate, the washing process conditions after the coupling step of the butt peptide are improved, so that the product yield is ensured and even improved under the condition of simplifying the washing process; meanwhile, the purity of the product is greatly improved by optimizing the solvent consumption in the ammonolysis step; in addition, the salt transferring process condition is further explored, and the sample loading quantity is improved while the yield is ensured.
Detailed Description
The technical solution of the present invention will be clearly and completely described in conjunction with the specific embodiments, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative only and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the description of "N-fold volume" of a certain solvent class in the present invention, N refers to a numerical value or a numerical range of a positive number, and as in the description of the present invention, "5-fold volume" of methanol, "5-fold volume" of an aqueous acetic acid solution with a volume fraction of 50%, 10-fold volume "of methanol," 15-fold volume "of acetone," 20-fold volume "of acetone," 25-fold volume "of acetone," 50-fold volume "of acetone," 1-2-fold volume "of water for injection, etc., the representative meaning refers to: the amount of a certain solvent used was N X mL (mL represents volume unit) relative to the mass Xg (g represents mass unit) of the corresponding solid matter.
Taking "the foamy concentrate I was weighed, dissolved in 5 volumes of methanol, precipitated in 20 volumes of acetone, filtered, and the cake was dissolved in 5 volumes of aqueous acetic acid with a volume fraction of 50%) as described in ③ in step (11) of example 1, the following description was made: means that for every 1g of concentrate I, the amount of methanol is 5mL and the amount of acetone is 20mL; the amount of the aqueous acetic acid solution having a volume fraction of 50% was 5mL per 1g of the cake.
The preparation method of the gonadorelin acetate comprises the following steps:
(a) To be used for Gradually grafting peptide by taking Boc-amino acid derivative and pGlu as monomers as starting materials, and treating the polypeptide resin/>
(B) Carrying out ammonolysis resin removal on the polypeptide resin, purifying, and then carrying out acetate transfer treatment to obtain the gonadorelin acetate;
The condensing agent adopted in the peptide grafting treatment comprises a first condensing agent and a second condensing agent, wherein the first condensing agent is at least one of HOBT and HOAT, and the second condensing agent comprises DCCI;
In each step of peptide-binding treatment, the monomer, the first condensing agent, the second condensing agent and The mol ratio of (1.5-3) to (1.72-3.45) to 1.
/>Refers to polystyrene resin.
According to the invention, the solid-phase polypeptide is synthesized into the gonadorelin acetate by adopting the Boc method, so that the cost of raw materials and auxiliary materials is reduced and the product yield is ensured and even improved under the condition of reducing the dosage of a monomer and a condensing agent.
In actual operation, pressIn the order of raw materials/>The amino acid monomer structures containing Pro, arg, leu, gly, tyr, ser, trp, his and pGlu are sequentially connected, and the raw material of each peptide connection step is precursor peptide-resin obtained after peptide connection in the previous step.
In each step of the polypeptide treatment, as in the various embodiments, the monomer, the first condensing agent, the second condensing agent, andThe molar ratio of (2) may be 1.5:1.72:1.72:1, 1.5:12:2:1, 3:3.45:3.45:1, etc.
In a preferred embodiment of the present invention, in each step of the polypeptide treatment, the monomer, the first condensing agent, the second condensing agent andThe molar ratio of (2) to (2) is 1.5:2:2:1.
Wherein,The number of moles of Boc-Gly bound to the resin is calculated.
The condensing agent may be HOBT and DCCI, or both HOBT and DCCI, as in various embodiments. In a preferred embodiment of the present invention, the first condensing agent is HOAT. Preferably, a combination of condensing agents of HOAT and DCCI is used.
In a specific embodiment of the present invention, the Boc-amino acid derivative comprises Boc-L-proline, boc-L-arginine, boc-L-leucine, boc-glycine, boc-L-tyrosine, N-BOC-O-t-butyl-L-serine, boc-L-tryptophan and N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine.
In practice, the Boc-amino acid derivative may also be the hydrochloride and/or monohydrate of each Boc-amino acid. For example, boc-L-arginine may also be Boc-L-arginine hydrochloride (Boc-Arg-HCl), boc-L-arginine hydrochloride monohydrate (Boc-Arg-HCl.H 2 O), and Boc-L-leucine may also be Boc-L-leucine monohydrate (Boc-Leu.H 2 O).
In a specific embodiment of the present invention, each step of the peptide-grafting treatment comprises: deprotection, neutralization of free amino terminal, coupling of the corresponding monomer and washing treatment after coupling;
The coupled washing treatment comprises: the washing treatment was performed using DMF and CH 2Cl2.
Wherein the coupling of the corresponding monomers refers toIn the order of raw materials/>The amino acid monomer structure containing Pro, arg, leu, gly, tyr, ser, trp, his and pGlu is connected on the upper part in sequence.
In a preferred embodiment of the present invention, the post-coupling wash treatment comprises: washing with DMF 2-3 times and washing with CH 2Cl2 2-4 times.
In the condition of the coupled washing treatment, DMF and CH 2Cl2 are adopted to wash the coupled resin, ethanol is not adopted, the problems of shrinkage, space reduction, steric hindrance increase and the like of the resin caused by ethanol washing are avoided, the washing of other reagents is not facilitated, the required washing procedure is increased, and the process is complicated. The effect is particularly pronounced for amino acid derivative monomers having side chains.
In a specific embodiment of the present invention, the amount of solvent used per wash in the peptide-receiving treatment step is 2000 to 2200mL/300mmol. Specifically, the amount of the solvent used per 300mmol of the starting material is 2000 to 2200mL per washing.
Furthermore, by optimizing the washing conditions, the washing effectiveness can be ensured under the condition of less washing times, the process is simplified, and the yield and purity are ensured and even improved.
In a preferred embodiment of the present invention, the washing treatment after coupling of N-BOC-O-tert-butyl-L-serine, boc-L-tryptophan comprises: the washing was performed 3 times with DMF and 2 times with CH 2Cl2.
In a preferred embodiment of the present invention, the washing treatment after coupling of N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine comprises: the washing was performed 3 times with DMF and 3 times with CH 2Cl2.
In a preferred embodiment of the invention, the washing treatment after coupling pGlu comprises: the washing was performed 3 times with CH 2Cl2, 3 times with DMF and 1 time with CH 2Cl2.
In a preferred embodiment of the invention, the washing treatments after coupling Boc-L-proline, boc-L-arginine, boc-L-leucine, boc-glycine, boc-L-tyrosine, respectively, comprise: the washing was performed 2 times with DMF and 2 times with CH 2Cl2.
In a specific embodiment of the present invention, the method for deprotecting a group comprises: and (3) placing the material to be deprotected in an organic solvent containing HCl/iPrOH, stirring for 40-60 min, pumping, and washing. Wherein the material to be deprotected is a precursor peptide-resin material obtained after the previous step of peptide grafting.
In a specific embodiment of the present invention, in the peptide-grafting treatment of respectively grafting Boc-L-proline, boc-L-arginine, boc-L-leucine, boc-glycine, boc-L-tyrosine, N-BOC-O-tert-butyl-L-serine, boc-L-tryptophan, the organic solvent is methylene chloride. Preferably, the volume ratio of the HCl/iPrOH to the dichloromethane is 1:1. Further, in the HCl/iPrOH, the concentration of the HCl is 9-10 mol/L.
In a specific embodiment of the present invention, in the method of deprotecting the peptide-grafting treatment of N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine and pGlu, respectively, the organic solvent comprises methylene chloride and mercaptoethanol. Preferably, the volume ratio of HCl/iPrOH, dichloromethane and mercaptoethanol is 5:4:1. Further, in the HCl/iPrOH, the concentration of the HCl is 9-10 mol/L.
In a specific embodiment of the present invention, the washing treatment in the deprotection process comprises: washing was performed with CH 2Cl2 and DMF. Further, the mixture is washed 3 times by CH 2Cl2 in turn, drained, washed 1 time by DMF and drained.
In a specific embodiment of the invention, the method of neutralizing the free amino terminus comprises: washing with dichloromethane solution containing triethylamine for 3 times, draining, and washing. Further, in the dichloromethane solution containing triethylamine, the volume ratio of the triethylamine to the dichloromethane is 5:95. Further, the washing treatment includes: washing with DMF for 1 time, draining, washing with CH 2Cl2 for 5-6 times until the mixture is neutral, and draining.
In a specific embodiment of the present invention, the method of coupling the corresponding monomers comprises: the monomer and the condensing agent are respectively dissolved by adopting an organic solvent, mixed and activated for 5 to 30 minutes at the temperature of between 0 and 5 ℃, then subjected to solid-liquid separation, the liquid is mixed with the neutralized precursor peptide-resin, and the solvent is pumped down after the reaction is carried out for 12 to 24 hours at the temperature of between 5 and 10 ℃. Further, the organic solvent includes dichloromethane.
In a specific embodiment of the present invention, the starting materialsThe preparation method of (2) comprises the following steps:
(1) The Boc-Gly and Cs 2CO3 react in the solution to obtain Boc-Gly-Cs;
(2) Boc-Gly-Cs and chloromethyl resin After reaction in solution, post-treatment to obtain/>
Further, in the step (1), the mass ratio of Boc-Gly to Cs 2CO3 is 1: (1.15-1.25); in step (2), boc-Gly-Cs and chloromethyl resinThe mol ratio of (1-1.5) to 1. Chloromethyl resin/>The number of moles of (2) is calculated as the amount of chlorine substitution.
In a specific embodiment of the present invention, the ammonolysis method comprises:
(b1) The polypeptide resin is shaken in an alcohol solvent for 18 to 30 hours under the airtight condition of anhydrous liquid ammonia, then filtered, the solid is washed by the alcohol solvent, and the combined filtering and washing liquid is collected;
(b2) Concentrating the filtering and washing liquid to be dry, adding an alcohol solvent to dissolve and then concentrating to be dry, and repeating the dissolving and concentrating to be dry for at least three times to obtain a foam concentrate I;
(b3) Dissolving the concentrate I by using methanol, adding acetone for precipitation, filtering, dissolving a filter cake by using an acetic acid aqueous solution, concentrating to dryness, adding methanol for dissolving, concentrating to dryness, and repeating the operation of adding methanol for dissolving and concentrating to dryness for at least three times to obtain a foam concentrate II; wherein the volume ratio of the methanol to the acetone is 1:4-5; preferably, the ratio of the concentrate I to the methanol to the acetone is 1g to 5mL to (20-25) mL;
(b4) Dissolving the concentrate II by using methanol, adding acetone for precipitation, filtering, washing a filter cake by using acetone for 1 time, washing by using diethyl ether for 2 times, and drying to obtain a crude gonadorelin product; wherein the volume ratio of the methanol to the acetone is 1:1.5-2; preferably, the ratio of the concentrate II, the methanol and the acetone is 1g to 10mL to (15-20) mL.
In a specific embodiment of the present invention, the alcoholic solvent comprises methanol.
In a preferred embodiment of the invention, in step (b 3) the volume ratio of methanol to acetone is 1:5, and in step (b 4) the volume ratio of methanol to acetone is 1:2; or in the step (b 3), the volume ratio of the methanol to the acetone is 1:4; in step (b 4), the volume ratio of methanol to acetone is 1:1.5.
In a preferred embodiment of the invention, the ratio of concentrate I, methanol, acetone is 1 g:5 mL:20 mL; the ratio of concentrate II, methanol and acetone was 1 g:10 mL:15 mL.
In practice, the conditions of the concentration may be: concentrating under reduced pressure at 40-45 ℃. Further, the vacuum degree of the reduced pressure concentration is-0.08 MPa to-0.1 MPa.
The invention can obviously improve the purity and content of the crude product while ensuring the yield by further optimizing the proportion of the dissolved and precipitated methanol and acetone in the ammonolysis process.
In a specific embodiment of the invention, the purification method comprises: CM purification and HPLC purification.
In practice, the method of CM purification comprises: adopting a CM-Sephadex C25 chromatographic column, after equilibrium treatment, loading, washing with a washing solution until the absorbance value is less than 0.2 after loading is completed, then eluting with an eluent, determining the absorbance value of the eluting effluent at 280nm, when the absorbance value OD 280 is increased to be more than or equal to 0.4, starting to collect the eluting effluent, and stopping collecting when the absorbance value is reduced to be less than or equal to 0.4; wherein the washing solution is 0.05mol/L sodium acetate aqueous solution with pH=6.0, and the eluent is 0.5mol/L sodium acetate aqueous solution with pH=6.0. Further, the balancing process includes: the solution was washed to neutrality with purified water and equilibrated with 0.05mol/L sodium acetate solution (buffer) at ph=6.0 until the effluent pH and conductivity values were substantially consistent with the buffer. Further, dissolving the crude gonadorelin product with 0.05mol/L sodium acetate aqueous solution with pH=6.0, passing through a 0.45 μm membrane, and collecting a sample solution for loading; wherein the concentration of the crude gonadorelin product in the sample solution is about 1g/100mL; the loading was 0.05mmol/mL of chromatographic column.
In practice, the method of HPLC purification includes: passing the elution effluent collected by CM purification through a 0.45 mu m membrane, then loading the elution effluent on a balanced preparation type HPLC column, performing gradient elution, and collecting and combining the elution effluent with the purity of the gonadorelin intermediate being more than or equal to 97%; wherein, the mobile phase A of the gradient elution is an aqueous solution containing 0.5 percent of acetic acid and 5 percent of acetonitrile by volume, and the mobile phase B is an aqueous solution containing 0.5 percent of acetic acid and 80 percent of acetonitrile by volume, so as to ensure separation and purification.
In a specific embodiment of the invention, reverse phase HPLC method is used to transdetate. Further, the stationary phase of the reverse phase HPLC method trans-acetate is octadecylsilane chemically bonded silica.
In a specific embodiment of the present invention, the method for converting acetate comprises: the purified gonadorelin intermediate is diluted by water and then is adsorbed on an HPLC preparation column after balance treatment, and after being washed by an ammonium acetate buffer solution, the purified gonadorelin intermediate is washed by an acetonitrile water solution with the volume fraction of 3 percent, and then is eluted by an acetonitrile water solution with the volume fraction of 80 percent, and eluent with an absorption value is collected.
In a specific embodiment of the invention, the washing volume of the ammonium acetate buffer is 5 to 5.5 column volumes, preferably 5 column volumes; the washing volume of the acetonitrile aqueous solution with the volume fraction of 3% is 10 to 11 times the column volume, preferably 10 times the volume.
Further, the equilibrium treatment was performed on the HPLC preparation column using an aqueous acetonitrile solution with a volume fraction of 3%.
Further, the ammonium acetate buffer is ammonium acetate buffer with pH of 5.0 and concentration of 0.1 mol/L.
According to the invention, ammonium acetate is adopted for acetate conversion, and the competitive combination mode of ammonium acetate is utilized to convert all the ammonium acetate into diacetate, so that the combination fastness is improved.
In a specific embodiment of the invention, the eluent is concentrated to dryness, dissolved in water and concentrated to dryness, and repeated at least 3 times, to obtain concentrate III.
In a specific embodiment of the present invention, the method further comprises the step of subjecting the material subjected to the trans-acetate treatment to freeze-drying treatment.
In practice, the lyophilization process comprises:
Pre-freezing: the inlet temperature of the heat conduction oil is less than-40 ℃, and the heat preservation is carried out for about 2 hours;
sublimation: the temperature rising speed of the heat conduction oil is about 10 ℃/h, the temperature of the heat conduction oil rises to 38 ℃, and the vacuum degree of the box is not more than 20Pa before process control;
when the temperature of the heat conduction oil reaches about 38 ℃, the heat is preserved for about 18 hours to the drying end point.
Example 1
The embodiment provides a preparation method of gonadorelin acetate, which comprises the following steps:
(1) Is prepared from
① 86.7G of Boc-Gly is weighed and placed in a container, and about 300mL of methanol is added for dissolution, so as to obtain a methanol solution of Boc-Gly; 104.7g of Cs 2CO3 is weighed and placed in another container, and about 300mL of water is added for dissolution, so as to obtain an aqueous solution of Cs 2CO3; slowly adding the Cs 2CO3 aqueous solution into the Boc-Gly methanol solution, and slightly shaking by hand until the reaction is complete, thus obtaining colorless and transparent Boc-Gly-Cs solution with pH of 7.0-7.5.
② Concentrating the Boc-Gly-Cs solution obtained in the step ① in a water bath at 40-50 ℃ under reduced pressure, and evaporating the solvent to obtain a solid; about 150mL of methanol is added to the solid, the mixture is evaporated to dryness under reduced pressure, and the steps are repeated twice (the step of evaporating after adding the methanol) to obtain a solid substance; and (3) drying the solid substance in a P 2O5 dryer in vacuum to constant weight to obtain a Boc-Gly-Cs dried product.
③ According toIs calculated, 330mmol of chloromethyl polystyrene resin/>, is weighed(Cas No. 55844-94-5) in a 3L three-necked flask; dissolving the dried Boc-Gly-Cs obtained in the step ② in about 2100mL of DMF to obtain a DMF solution of Boc-Gly-Cs; adding a DMF solution of Boc-Gly-Cs into the three-necked flask; placing the three-necked bottle in a water bath kettle with the temperature of 50 ℃ and stirring at constant temperature for reaction for 48 hours; after the reaction is finished, filtering, washing the solid resin with DMF 3 times (about 750mL of DMF is used each time), washing the solid resin with purified water until no Cl - is generated (AgNO 3 reagent detection: the washed purified water is added with silver nitrate in a dropwise manner, the solution is slightly swayed and colorless and transparent), washing the solid resin with ethanol 4 times (about 300mL of ethanol is used each time), and washing the solid resin with methanol 2 times (about 300mL of methanol is used each time); drying the washed resin in an oven at 40-50 ℃, and then vacuum drying in a P 2O5 dryer until the weight is constant to obtain/>And (5) drying the product.
(2) Dipeptide-grafted
① Deprotection: 300mmol of Boc-Gly combined on the resin was weighed outAnd (5) placing the dried product into a 10L reaction kettle, washing for 2-3 times by CH 2Cl2, and pumping. About 780mL of 9-10 mol/L HCl/iPrOH and about 780mL of CH 2Cl2 are added, the mixture is stirred for 40min and then pumped, and the solid is washed 3 times by CH 2Cl2 and then is pumped 1 time by DMF.
② Neutralization of the free amino terminus: adding triethylamine/CH 2Cl2 with the volume ratio of 5:95 into the material obtained in the step ①, washing for 3 times, and draining; washing with DMF for 1 time, draining, washing with CH 2Cl2 for 5-6 times until the mixture is neutral, and draining.
③ Coupling monomer Boc-Pro-OH: weighing 97g (450 mmol) of Boc-Pro-OH, 81g (600 mmol) of HOAT and 124g (600 mmol) of DCCI, respectively dissolving Boc-Pro-OH, HOAT and DCCI by using DMF, uniformly mixing and reacting for 5-30 min at the temperature of 0-5 ℃; after the reaction is finished, liquid is collected after filtration, the liquid is added into the resin material treated in the step ②, the bottle mouth is closed, the solvent is pumped out after the reaction is carried out for 18 hours at the temperature of 5 to 10 ℃, and the coupled resin material is obtained.
④ Washing treatment after coupling: washing the coupled resin material obtained in the step ③ with DMF for 2 times, washing with CH 2Cl2 for 2 times, and draining to obtain
(3) Tripeptide grafting
The reference dipeptide approach differs in that:
Step ① deprotection starting materials are all those prepared as described above
Step ③ coupling monomer was Boc-Arg-HCl.H2 2 O, weighing Boc-Arg-HCl.H2 2 O148 g (450 mmol), HOAT 81g (600 mmol), DCCI 124g (600 mmol); the monomer is not limited thereto, and 140g (450 mmol) of Boc-Arg-HCl may be weighed;
step ④ finally obtains
(4) Tetrapeptides
The reference dipeptide approach differs in that:
Step ① deprotection starting materials are all those prepared as described above
Step ③ coupling monomer was Boc-Leu.H2 2 O, weighing Boc-Leu.H2 2 O112g (450 mmol), HOAT 81g (600 mmol), DCCI 124g (600 mmol); the monomer is not limited to this, and Boc-Leu 105g (450 mmol) may be weighed;
step ④ finally obtains
(5) Grafted pentapeptide
The reference dipeptide approach differs in that:
Step ① deprotection starting materials are all those prepared as described above
Step ③ the coupling monomer is Boc-Gly, and weighing 79g (450 mmol), 81g (600 mmol) of HOAT and 124g (600 mmol) of DCCI;
step ④ finally obtains
(6) Hexapeptide grafting
The reference dipeptide approach differs in that:
Step ① deprotection starting materials are all those prepared as described above
Step ③ the coupling monomer was Boc-Tyr, and Boc-Tyr 127g (450 mmol), HOAT 81g (600 mmol), DCCI 124g (600 mmol) were weighed;
step ④ finally obtains
(7) Heptapeptide
The reference dipeptide approach differs in that:
Step ① deprotection starting materials are all those prepared as described above
Step ③ coupling monomer was Boc-Ser (tBu) -OH, and weighing 118g (450 mmol) of Boc-Ser (tBu) -OH, 81g (600 mmol) of HOAT, 124g (600 mmol) of DCCI;
step ④ washing the coupled resin material obtained in step ③ with DMF for 3 times, washing with CH 2Cl2 for 2 times, and draining to obtain
(8) Octapeptide grafting
The reference dipeptide approach differs in that:
Step ① deprotection starting materials are all those prepared as described above And the stirring time of the deprotection reaction is 60min;
Step ③ the coupling monomer was Boc-Trp, and 137g (450 mmol) of Boc-Trp, 81g (600 mmol) of HOAT, and 124g (600 mmol) of DCCI were weighed;
step ④ washing the coupled resin material obtained in step ③ with DMF for 3 times, washing with CH 2Cl2 for 2 times, and draining to obtain
(9) Grafted nonapeptide
The reference dipeptide approach differs in that:
Step ① deprotection starting materials are all those prepared as described above And the deprotection system is as follows: about 780mL of 9-10 mol/L HCl/iPrOH, about 624mL of CH 2Cl2 and 156mL of mercaptoethanol, and the stirring time of deprotection reaction is 60min;
Step ③ the coupling monomer was Boc-His (Tos), and 184g (450 mmol), HOAT 81g (600 mmol) and DCCI 124g (600 mmol) of Boc-His (Tos) were weighed;
Step ④ washing the coupled resin material obtained in step ③ with DMF for 3 times, washing with CH 2Cl2 for 3 times, and draining to obtain
(10) Grafted decapeptide
The reference dipeptide approach differs in that:
Step ① deprotection starting materials are all those prepared as described above And the deprotection system is as follows: about 780mL of 9-10 mol/L HCl/iPrOH, about 624mL of CH 2Cl2 and 156mL of mercaptoethanol, and the stirring time of deprotection reaction is 60min;
Step ③ the coupling monomer is pGlu, and 58g (450 mmol), 81g (600 mmol) HOAT and 124g (600 mmol) DCCI of pGlu are weighed;
Step ④ the coupled resin material obtained in step ③ was washed 3 times with CH 2Cl2, 3 times with DMF and 1 time with CH 2Cl2, and then dried. Drying the solid material in a drying oven at 40-50 deg.c for over 0.5 hr, and drying in a P 2O5 drying oven for over 12 hr to obtain polypeptide resin
(11) Ammonolysis
① The polypeptide resin prepared in the step (10) is preparedPlacing in a cracking bottle, adding 3000mL of methanol, adding 4500mL of anhydrous liquid ammonia, sealing, shaking at room temperature for 24h, opening the cracking bottle under cooling condition, filtering, washing solid resin material with methanol for 4 times, and combining filtering washing solutions;
② Concentrating the filtrate obtained in the step ① at 40-45 ℃ under reduced pressure (vacuum degree is minus 0.08MPa to minus 0.1 MPa) until the filtrate is dry, adding methanol for dissolving, concentrating under reduced pressure (vacuum degree is minus 0.08MPa to minus 0.1 MPa) until the filtrate is dry, and repeating the steps for three times to obtain foam concentrate I;
③ Weighing the foam concentrate I, dissolving with 5 times of methanol, precipitating with 20 times of acetone, filtering, dissolving a filter cake with 5 times of acetic acid aqueous solution with the volume fraction of 50%, concentrating under reduced pressure at 40-45 ℃ to dryness (the vacuum degree is minus 0.08MPa to minus 0.1 MPa), dissolving with methanol, concentrating under reduced pressure (the vacuum degree is minus 0.08MPa to minus 0.1 MPa) to dryness, and repeating for at least three times to obtain the foam concentrate II.
④ Weighing the foam concentrate II, dissolving with 10 times of methanol, precipitating with 15 times of acetone, filtering, washing the filter cake with acetone for 1 time, washing with diethyl ether for 2 times, and pumping; and (3) drying the solid obtained by pumping in vacuum to obtain a crude product of the gonadorelin. The filtrate produced by filtration in step ③ and step ④ can be recycled, and the above steps ③ and ④ are repeated to obtain the crude gonadorelin product.
(12) CM purification
① Column selection: the filler is a pretreatment chromatographic column of CM-Sephadex C25; a pretreatment chromatographic column of a suitable volume is selected according to a loading of 0.05mmol/mL, for example, when a crude gonadorelin product of 100mmol is added, a pretreatment chromatographic column of about 2000mL is selected.
② Chromatography column balancing: the mixture was washed with purified water to neutrality and equilibrated with 0.05mol/L sodium acetate buffer at pH=6.0 until the effluent pH and conductivity values were substantially consistent with the buffer for use.
③ Loading and processing: dissolving the crude gonadorelin product obtained in the step (11) with 0.05mol/L sodium acetate buffer solution with pH=6.0, passing through a 0.45 μm membrane, and loading onto a chromatographic column; after the sample is applied, washing the sample with a washing solution (pH=6.0 sodium acetate buffer solution with the concentration of 0.05 mol/L) until the absorbance value is less than 0.2, eluting the sample with an eluent (pH=6.0 sodium acetate aqueous solution with the concentration of 0.5 mol/L), measuring the absorbance value of the eluting effluent at 280nm, and when the absorbance value OD 280 is increased to be more than or equal to 0.4, starting to collect the eluting effluent, and stopping collecting when the absorbance value is reduced to be less than or equal to 0.4.
(13) HPLC purification
① Column balance: the preparative HPLC column (4L reverse phase C18 liquid chromatography column) was equilibrated with mobile phase A at a flow rate of 400mL/min and equilibrated to baseline for further use.
② Loading: after passing the elution effluent collected in step (12) through a 0.45 μm membrane, the column is equilibrated in step ①.
③ Gradient elution: gradient elution is carried out by adopting a mobile phase A and a mobile phase B; mobile phase a was an aqueous solution containing 0.5% by volume of acetic acid and 5% by volume of acetonitrile, and mobile phase B was an aqueous solution containing 0.5% by volume of acetic acid and 80% by volume of acetonitrile.
④ And (3) collecting: and collecting main peak solution in sections, detecting by an HPLC method, wherein the part with the purity of more than or equal to 97.0% is qualified, recovering the unqualified part, purifying by HPLC again, combining all the parts with the purity of more than or equal to 97.0%, and uniformly mixing to obtain the gonadorelin intermediate.
(14) Salt transfer-concentration
① Column balance: the preparative HPLC column (4L reverse phase C18 liquid chromatography column) was equilibrated with an aqueous solution containing 3% acetonitrile by volume until baseline stabilized for use.
② Diluting the gonadorelin intermediate obtained in the step (13) with 1-2 times of water for injection, adsorbing the diluted gonadorelin intermediate on an HPLC preparation column subjected to balanced treatment in the step ①, washing 5 times of column volume by adopting an ammonium acetate buffer solution with pH of 5.0 and 0.1mol/L, washing 10 times of column volume by adopting an acetonitrile water solution with volume fraction of 3%, eluting by adopting an acetonitrile water solution with volume fraction of 80%, and collecting eluent with an absorption value; concentrating the collected eluent at 40-45 ℃ under reduced pressure (vacuum degree is minus 0.08MPa to minus 0.1 MPa) until the eluent is dry, adding a proper amount of water for injection for dissolving, concentrating the eluent again until the eluent is dry, and repeating the process for at least 3 times to obtain a concentrate.
(15) And (3) freeze-drying: dissolving the concentrate obtained in step (14) with appropriate amount of water for injection, filtering with two filters of 0.22 μm in series, and filtering into sterilized container under partial A-level layer flow. Finally controlling the concentration of the filtered collection liquid to be 8% -10%; pouring the filtered collection liquid into a freeze-drying disc subjected to sterilization treatment, freeze-drying in a freeze-drying box subjected to sterilization treatment, and discharging to obtain a sterile gonadorelin acetate bulk drug (pharmaceutical grade);
The freeze-drying process conditions are as follows: prefreezing-heat conducting oil inlet temperature is less than-40 ℃, and preserving heat for about 2 hours; the heating speed of sublimation-heat conduction oil is about 10 ℃/h, the temperature of the heat conduction oil rises to 38 ℃, and the vacuum degree of the box is not more than 20Pa before process control; and when the temperature of the heat conduction oil reaches about 38 ℃, preserving the heat for about 18 hours to the drying end point.
Example 2
This example refers to the preparation method of example 1, differing only in: in the process from dipeptide to decapeptide, each corresponding coupling monomer, HOAT, DCCI and starting materialThe molar ratio of the dry products is different; the method comprises the following steps:
Starting materials 300Mmol of dried product;
and (3) dipeptide: coupling monomers Boc-Pro-OH 193.5g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol);
And (3) tripeptide: coupling monomers Boc-Arg-HCl.H2 2 O295.5 g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol);
receiving tetrapeptides: coupling monomers Boc-Leu.H 2 O225.0 g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol);
And (3) grafting pentapeptide: coupling monomer Boc-Gly 157.5g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol);
Grafting hexapeptide: coupling monomer Boc-Tyr 253.5g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol);
And (3) seven-peptide grafting: coupling monomer Boc-Ser (tBu) -OH 235.5g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol);
Receiving octapeptide: coupling monomers Boc-Trp 274.5g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol);
And (3) nine peptide: coupling monomer Boc-His (Tos) 369.0g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol);
And (3) grafting decapeptide: coupling monomer pGlu 115.5g (900 mmol), HOAT 139.7g (1035 mmol), DCCI 213.0g (1035 mmol).
Example 3
This example refers to the preparation method of example 1, differing only in: in the process from dipeptide to decapeptide, each corresponding coupling monomer, HOAT, DCCI and starting materialThe molar ratio of the dry products is different; the method comprises the following steps:
Starting materials 300Mmol of dried product;
And (3) dipeptide: coupling monomers Boc-Pro-OH 97g (450 mmol), HOAT 70g (517 mmol), DCCI 107g (517 mmol);
And (3) tripeptide: coupling monomers Boc-Arg-HCl.H 2 O148 g (450 mmol), HOAT 70g (517 mmol) and DCCI 107g (517 mmol);
Receiving tetrapeptides: coupling monomers Boc-Leu.H 2 O112 g (450 mmol), HOAT 70g (517 mmol), DCCI 107g (517 mmol);
And (3) grafting pentapeptide: coupling monomer Boc-Gly 79g (450 mmol), HOAT 70g (517 mmol) and DCCI 107g (517 mmol);
grafting hexapeptide: coupling monomer Boc-Tyr 127g (900 mmol), HOAT 70g (517 mmol), DCCI 107g (517 mmol);
And (3) seven-peptide grafting: coupling monomer Boc-Ser (tBu) -OH 118g (450 mmol), HOAT 70g (517 mmol), DCCI 107g (517 mmol);
Receiving octapeptide: coupling monomer Boc-Trp 137g (450 mmol), HOAT 70g (517 mmol) and DCCI 107g (517 mmol);
And (3) nine peptide: coupling monomer Boc-His (Tos) 184g (450 mmol), HOAT 70g (517 mmol), DCCI 107g (517 mmol);
And (3) grafting decapeptide: 58g (450 mmol) of coupling monomer pGlu, 70g (517 mmol) of HOAT and 107g (517 mmol) of DCCI.
Example 4
This example refers to the preparation method of example 2, differing only in:
condensing agent is HOBT and DCCI, and each peptide grafting step is carried out according to monomer, HOBT, DCCI and DCCI The molar ratio of (3) to (3.45) to (1) is that of the mixture;
the washing treatment conditions after coupling in each peptide treatment were different, and specifically were as follows:
the washing treatments after the coupling of the dipeptide to the nonapeptide were all: washing with CH 2Cl2 for 3 times, washing with absolute ethyl alcohol for 4 times, washing with DMF for 2 times, and washing with CH 2Cl2 for 3 times in sequence, and draining;
The washing treatment after the coupling of the decapeptide is as follows: washing 3 times by adopting CH 2Cl2, washing 4 times by adopting absolute ethyl alcohol, washing 2 times by adopting DMF, washing 2-3 times by adopting CH 2Cl2, washing 4-5 times by adopting methanol, and then pumping.
Example 5
This example refers to the preparation method of example 1, differing only in:
in the ammonolysis step ③ of the step (11), 5 times of methanol is dissolved, 25 times of acetone is precipitated, and the rest operations are the same;
In step ④ of ammonolysis in step (11), 10 volumes of methanol is dissolved, 20 volumes of acetone is precipitated, and the rest of the operation is the same.
Example 6
This example refers to the preparation method of example 1, differing only in:
In the ammonolysis step ③ of the step (11), 5 times of methanol is dissolved, 50 times of acetone is precipitated, and the rest operations are the same;
in step ④ of ammonolysis in step (11), 10 volumes of methanol is dissolved, 50 volumes of acetone is precipitated, and the rest of the operation is the same.
Example 7
This example refers to the preparation method of example 1, differing only in:
(14) Salt transfer-concentration
① Column balance: the preparative HPLC column (4L reversed phase C18 liquid chromatography column) was equilibrated with an aqueous solution containing 0.5% acetic acid by volume and 5% acetonitrile by volume until baseline stabilized for use.
② Diluting the gonadorelin intermediate obtained in the step (13) with 1-2 times volume of water for injection, then adsorbing the diluted gonadorelin intermediate onto an HPLC preparation column subjected to balanced treatment in the step ①, washing the mixture by adopting an ammonium acetate buffer solution with the pH of 5.0 and 0.1mol/L, washing the mixture by adopting an aqueous solution containing 0.5 percent of acetic acid and 5 percent of acetonitrile by volume, eluting the mixture by adopting an aqueous solution containing 0.5 percent of acetic acid and 40 percent of acetonitrile by volume, and collecting eluent with an absorption value; concentrating the collected eluent at 40-45 ℃ under reduced pressure (vacuum degree is minus 0.08MPa to minus 0.1 MPa) until the eluent is dry, adding a proper amount of water for injection for dissolving, concentrating the eluent again until the eluent is dry, and repeating the process for at least 3 times to obtain a concentrate.
Example 8
This example refers to the preparation method of example 1, differing only in:
condensing agent is HOAT and DCCI, and each peptide grafting step is carried out according to monomer, HOBT, DCCI and DCCI The molar ratio of (2) to (1) is 1.5:2:2:1;
the washing treatment conditions after coupling in each peptide treatment were different, and specifically were as follows:
the washing treatments after the coupling of the dipeptide to the nonapeptide were all: washing with CH 2Cl2 for 3 times, washing with absolute ethyl alcohol for 4 times, washing with DMF for 2 times, and washing with CH 2Cl2 for 3 times in sequence, and draining;
The washing treatment after the coupling of the decapeptide is as follows: washing 3 times by adopting CH 2Cl2, washing 4 times by adopting absolute ethyl alcohol, washing 2 times by adopting DMF, washing 2-3 times by adopting CH 2Cl2, washing 4-5 times by adopting methanol, and then pumping.
Experimental example 1
In order to comparatively illustrate the process difference of the preparation method of the gonadorelin acetate according to the present invention, the yield and/or purity of the preparation method of the gonadorelin acetate according to various embodiments are tested from various aspects. The test results are shown in tables 1 to 3.
TABLE 1 influence of the amount of monomers and condensing Agents used in the various peptide grafting steps on the polypeptide resin obtained after the decapeptidation
As can be seen from the peptide grafting process conditions in examples 1 to 4, when HOBT is used as the first condensing agent and before the washing treatment conditions are not optimized, the coupling monomer, the first condensing agent and the second condensing agent are used in large amounts, and the cost is high, and the yield of the polypeptide resin is low [ i.e., (800 g/900 g) ×100% =88.9% ]; according to the invention, a large number of creative experiments show that when the first condensing agent is HOAT, the dosage of monomers can be reduced, and the reaction impurities are reduced, and the specific washing treatment condition is further adopted on the basis, so that the reaction efficiency is improved, the yield of polypeptide resin is also remarkably improved, and the dosage of coupling monomers, the first condensing agent and the second condensing agent is reduced to 1mol per 1molWhen only 1.5mol of the coupling monomer, 2mol of the first condensing agent, and 2mol of the second condensing agent are used, the yield is 96.3% or higher [ i.e., (867 g/900 g) ×100% =96.3% ]. Therefore, by adopting specific condensing agent and washing treatment conditions after coupling, the yield of the polypeptide resin can be improved under the condition of obviously reducing the dosage of the coupling monomer and the condensing agent, the cost of raw materials and auxiliary materials and the cost of solvents used for washing are greatly reduced, and the process is simplified.
TABLE 2 influence of specific Process of ammonolysis step on crude purity, content and yield of gonadorelin
/>
Wherein, the purity of the crude product is as follows: detecting the crude product by HPLC, wherein the ratio of the peak area of the target peptide to the total peak area in the spectrum (the sum of the areas of all peaks in the HPLC) is the purity of the crude product (in the HPLC detection process, not all substances are detected and peak is output); the purity of the crude product mainly reflects the relation between the target peptide and related impurities (such as other peptide chains of non-target peptide and the like);
Crude product content: the ratio of the mass of the target peptide in the crude product to the total mass of the crude product; the content of the crude product mainly represents the content of target peptide in the crude product.
From the ammonolysis process conditions of example 1, example 5 and example 6, it is known that by adopting a specific ammonolysis process to dissolve the precipitation conditions, the purity of the crude product can be significantly improved while ensuring the yield, and the amount of the organic solvent (methanol and acetone) used is relatively small.
TABLE 3 influence of salt transfer-concentration step specific Process on Single sample Loading
| Numbering device | Single sample loading |
| Example 1 | About 90g |
| Example 7 | About 30g |
From the salt transfer-concentration process conditions of examples 1 and 7, the invention can avoid the loss caused by flow-through in the processes of loading and washing, improve the loading amount and improve the treatment efficiency by improving the salt transfer-concentration step process conditions.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (15)
1. The preparation method of the gonadorelin acetate is characterized by comprising the following steps of:
(a) To be used for Gradually grafting peptide by taking Boc-amino acid derivative and pGlu as monomers as starting materials, and treating the polypeptide resin/>
(B) Carrying out ammonolysis resin removal on the polypeptide resin, purifying, and then carrying out acetate transfer treatment to obtain the gonadorelin acetate;
The condensing agent adopted in the peptide grafting treatment comprises a first condensing agent and a second condensing agent, wherein the first condensing agent is HOAT, and the second condensing agent is DCCI;
In each step of peptide-binding treatment, the monomer, the first condensing agent, the second condensing agent and The molar ratio of (1.5-3):1.72-3.45:1.72-3.45:1;
each step of peptide grafting treatment comprises the following steps: deprotection, neutralization of free amino terminal, coupling of the corresponding monomer and washing treatment after coupling; the coupled washing treatment comprises: washing with DMF for 2-3 times and CH 2Cl2 for 2-4 times;
The ammonolysis method comprises the following steps:
(b1) The polypeptide resin is shaken in an alcohol solvent for 18 to 30 hours under the airtight condition of anhydrous liquid ammonia, then filtered, the solid is washed by the alcohol solvent, and the combined filtering and washing liquid is collected;
(b2) Concentrating the filtering and washing liquid to be dry, adding an alcohol solvent to dissolve and then concentrating to be dry, and repeating the dissolving and concentrating to be dry for at least three times to obtain a foam concentrate I;
(b3) Dissolving the concentrate I by using methanol, adding acetone for precipitation, filtering, dissolving a filter cake by using an acetic acid aqueous solution, concentrating to dryness, adding methanol for dissolving, concentrating to dryness, and repeating the operation of adding methanol for dissolving and concentrating to dryness for at least three times to obtain a foam concentrate II; wherein the volume ratio of the methanol to the acetone is 1: (4-5);
(b4) Dissolving the concentrate II by using methanol, adding acetone for precipitation, filtering, washing a filter cake by using acetone for 1 time, washing by using diethyl ether for 2 times, and drying to obtain a crude gonadorelin product; wherein the volume ratio of the methanol to the acetone is 1: (1.5-2).
2. The method for producing gonadorelin acetate according to claim 1, wherein in each step of peptide-grafting treatment, the monomer, the first condensing agent, the second condensing agent andThe molar ratio of (2) to (2) is 1.5 to 1.
3. The process for the preparation of gonadorelin acetate according to claim 1, characterized in that the washing treatments after coupling of N-BOC-O-t-butyl-L-serine, BOC-L-tryptophan respectively comprise: the washing was performed 3 times with DMF and 2 times with CH 2Cl2.
4. The process for the preparation of gonadorelin acetate according to claim 1, characterized in that the washing treatment after coupling of N-BOC-N (imidazole) - (4-methylbenzenesulfonyl) -L-histidine comprises: the washing was performed 3 times with DMF and 3 times with CH 2Cl2.
5. The process for the preparation of gonadorelin acetate according to claim 1, characterized in that the washing treatment after coupling pGlu comprises: the washing was performed 3 times with CH 2Cl2, 3 times with DMF and 1 time with CH 2Cl2.
6. The process for the preparation of gonadorelin acetate according to claim 1, characterized in that the washing treatments after coupling Boc-L-proline, boc-L-arginine, boc-L-leucine, boc-glycine, boc-L-tyrosine respectively comprise: the washing was performed 2 times with DMF and 2 times with CH 2Cl2.
7. The process for producing gonadorelin acetate according to claim 1, wherein in step (b 3), the ratio of concentrate i, methanol and acetone is 1 g/5 mL/20 to 25 mL.
8. The process for producing gonadorelin acetate according to claim 1, wherein in step (b 4), the ratio of concentrate ii, methanol and acetone is 1 g/10 mL/15 to 20 mL.
9. The process for the preparation of gonadorelin acetate according to claim 1, characterized in that in step (b 3) the volume ratio of methanol to acetone is 1:5; in step (b 4), the volume ratio of methanol to acetone is 1:2;
Or in step (b 3), the volume ratio of methanol to acetone is 1:4; in step (b 4), the volume ratio of methanol to acetone is 1:1.5.
10. The process for the preparation of gonadorelin acetate according to claim 1, characterized in that in step (b 3) the ratio of concentrate i, methanol, acetone is 1 g/5 mL/20 mL; in the step (b 4), the ratio of the concentrate II, methanol and acetone was 1 g/10 mL/15 mL.
11. The process for the preparation of gonadorelin acetate according to claim 1, characterized in that the reverse phase HPLC method is used for trans-acetate.
12. The process for the preparation of gonadorelin acetate according to claim 11, characterized in that the process for the trans-acetate comprises: and diluting the purified gonadorelin intermediate with water, adsorbing the diluted gonadorelin intermediate onto an HPLC preparation column subjected to equilibrium treatment, washing the purified gonadorelin intermediate by adopting an ammonium acetate buffer solution, washing the purified gonadorelin intermediate by adopting an acetonitrile water solution with the volume fraction of 3%, eluting the purified gonadorelin intermediate by adopting an acetonitrile water solution with the volume fraction of 80%, and collecting an eluent with an absorption value.
13. The process for the preparation of gonadorelin acetate according to claim 12, characterized in that the washing volume of the ammonium acetate buffer is 5-5.5 column volumes; the washing volume of the acetonitrile water solution with the volume fraction of 3 percent is 10 to 11 times of the column volume.
14. The process for the preparation of gonadorelin acetate according to claim 12, characterized in that the ammonium acetate buffer is an ammonium acetate buffer having a pH of 5.0 and a concentration of 0.1 mol/L.
15. The process for the preparation of gonadorelin acetate according to claim 12, characterized in that it further comprises the step of freeze-drying said trans-acetate treated substance.
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