CN118047836A - Preparation method of goserelin impurity E - Google Patents
Preparation method of goserelin impurity E Download PDFInfo
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- CN118047836A CN118047836A CN202410078587.1A CN202410078587A CN118047836A CN 118047836 A CN118047836 A CN 118047836A CN 202410078587 A CN202410078587 A CN 202410078587A CN 118047836 A CN118047836 A CN 118047836A
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- 229960002913 goserelin Drugs 0.000 title claims abstract description 35
- 108010069236 Goserelin Proteins 0.000 title claims abstract description 34
- BLCLNMBMMGCOAS-URPVMXJPSA-N Goserelin Chemical compound C([C@@H](C(=O)N[C@H](COC(C)(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(=O)NNC(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 BLCLNMBMMGCOAS-URPVMXJPSA-N 0.000 title claims abstract description 34
- 239000012535 impurity Substances 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 239000011347 resin Substances 0.000 claims abstract description 89
- 229920005989 resin Polymers 0.000 claims abstract description 89
- 239000012634 fragment Substances 0.000 claims abstract description 59
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 39
- 239000012043 crude product Substances 0.000 claims abstract description 11
- 238000006698 hydrazinolysis reaction Methods 0.000 claims abstract description 11
- 238000004108 freeze drying Methods 0.000 claims abstract description 7
- 239000000047 product Substances 0.000 claims abstract description 6
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims abstract description 4
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims abstract description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 41
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 40
- 238000000034 method Methods 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 14
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 14
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 230000008878 coupling Effects 0.000 claims description 8
- 238000010168 coupling process Methods 0.000 claims description 8
- 238000005859 coupling reaction Methods 0.000 claims description 8
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- BYEAHWXPCBROCE-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoropropan-2-ol Chemical compound FC(F)(F)C(O)C(F)(F)F BYEAHWXPCBROCE-UHFFFAOYSA-N 0.000 claims description 5
- 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 4
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 230000007017 scission Effects 0.000 claims description 4
- 239000007790 solid phase Substances 0.000 claims description 4
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 3
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 3
- 239000012046 mixed solvent Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 claims description 2
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 2
- 239000007821 HATU Substances 0.000 claims description 2
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 11
- 238000003786 synthesis reaction Methods 0.000 abstract description 11
- 229920001184 polypeptide Polymers 0.000 abstract description 4
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 4
- 238000006243 chemical reaction Methods 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000002253 acid Substances 0.000 description 9
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 5
- 125000000734 D-serino group Chemical group [H]N([H])[C@@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 125000006239 protecting group Chemical group 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine group Chemical group NC(=N)N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 108700012941 GNRH1 Proteins 0.000 description 1
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 229930182833 estradiol Natural products 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- RXMHIKWOZKQXCJ-SFHVURJKSA-N fmoc-arg(no2)-oh Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N[N+]([O-])=O)N)C(O)=O)C3=CC=CC=C3C2=C1 RXMHIKWOZKQXCJ-SFHVURJKSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine group Chemical group NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 108010004034 stable plasma protein solution Proteins 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
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- 229960003604 testosterone Drugs 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method of goserelin impurity E, which belongs to the technical field of polypeptide synthesis, and comprises the following steps: first, a first fragment heptapeptide Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-OH is synthesized. And (3) connecting Arg by using Boc-Pro-PAM resin as a carrier to obtain a second fragment peptide resin. And (3) connecting the first fragment heptapeptide to the second fragment peptide resin to obtain a third fragment peptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-PAM resin. And carrying out hydrazinolysis on the third fragment peptide resin by using a hydrazine hydrate solution to obtain a third fragment goserelin impurity E crude product, and purifying and freeze-drying to obtain a goserelin impurity E finished product. The preparation method disclosed by the invention is simple and convenient in steps, safe to operate, high in synthesis yield and higher in purity of the obtained goserelin impurity E.
Description
Technical Field
The invention relates to the field of polypeptide medicine impurity preparation, and relates to goserelin, in particular to a preparation method of goserelin impurity E, which is simple in step, safe in operation and high in synthesis yield.
Background
Goserelin (Goserelin) is a synthetic analog of luteinizing hormone releasing hormone with a decapeptide structure, which has the sequence: pyr-His-Trp-Ser-Tyr-D-Ser (tBu) -Leu-Arg-Pro-Azagly-NH 2, molecular formula: c 59H84N18O14, molecular weight: 1269.4.
Long-term administration of goserelin inhibits secretion of luteinizing hormone from the pituitary gland, thereby causing a decrease in serum testosterone in men and serum estradiol in women, and thus atrophy of hormone-sensitive tumors. Can be used for hormone therapy of prostate cancer and pre-menopausal and perimenopausal breast cancer, and also for the treatment of endometriosis, such as pain relief and reduction of endometrial damage.
Chinese name: goserelin impurity E, english name: des-9-L-pro line-goserelin, peptide sequence: glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-NH- 2, molecular weight: 1226.4, molecular formula: c 58H83N17O13 CAS 147688-42-4.
The structural formula is as follows:
Goserelin impurity E has obvious characteristics: the C-terminal of the first peptide chain contains a hydrazine structure R-NH-NH 2, and the side chain of the second DSer is provided with tertiary butyl protection. These two characteristics result in the fact that the impurity is difficult to synthesize by the traditional Fmoc step-by-step solid phase synthesis strategy, and the synthesis difficulty is great.
Few reports of the synthesis of this impurity are currently available.
The method adopted by the Chinese patent CN110128505A is that a fragment Pyr-His-Trp-Ser-Tyr-D-Ser (tBu) -Leu-Arg-Pro-OH is synthesized firstly, then coupled with Boc-NH-NH 2 under a coupling agent to obtain a crude product of goserelin impurity E, side chain protection is removed under the condition of trifluoroacetic acid, and a pure product of the goserelin impurity E is obtained after purification and freeze-drying. Fmoc-Arg (NO 2) -OH is used in the synthesis of the method, the catalytic hydrogenation is needed to reduce the nitro, the operation is complex, and the hydrogenation process has a certain danger. Furthermore, the above method retains tBu on DSer by selectively cleaving the remaining protecting groups by adjusting the acid concentration, and the acid concentration and reaction time are difficult to control. If the acid concentration is too high or the reaction time is too long, the tBu on DSer is very easy to fall off, so that the purity of the target compound is reduced.
Disclosure of Invention
In view of the above, the invention provides a preparation method of goserelin impurity E, which has the advantages of simple steps, safe operation and high synthesis yield, the invention adopts a fragment method to synthesize a first fragment heptapeptide and a second fragment peptide resin, then the first fragment heptapeptide and the second fragment peptide resin are coupled, then the first fragment heptapeptide and the second fragment peptide resin are subjected to hydrazinolysis to obtain a Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-NH 2 crude product, and then the crude product of goserelin impurity E is obtained through purification and freeze-drying.
The invention adopts the fragment method to synthesize, which not only makes the operation of the first fragment heptapeptide simple and convenient and has higher purity, but also can effectively avoid the problem of low purity of crude products caused by that the guanidine group of the Arg side chain of the second fragment peptide resin is not protected and the subsequent amino acid is gradually connected upwards. Meanwhile, the second fragment peptide resin is based on Boc-Pro-PAM resin, so that the problem of low synthesis yield caused by the fact that peptide chains fall off from the resin when the Boc is removed by acid is avoided, and subsequent hydrazinolysis is facilitated.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a preparation method of goserelin impurity E, which comprises the following steps:
a) Using CTC resin, firstly, synthesizing a first fragment heptapeptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-CTC in a solid phase, and cutting to obtain a first fragment heptapeptide Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-OH;
b) Removing Boc by using Boc-Pro-PAM resin, and then carrying out solid phase synthesis to connect Boc-Arg-OH to obtain Boc-Arg-Pro-PAM resin, and carrying out acidolysis to remove Boc to obtain second fragment peptide resin H 2 N-Arg-Pro-PAM resin;
c) Coupling the first fragment heptapeptide obtained in the step a) to the second fragment peptide resin obtained in the step b) through solid phase synthesis to obtain a third fragment peptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-PAM resin;
d) And c), carrying out hydrazinolysis on the third fragment peptide resin obtained in the step c) to obtain a crude product of Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-NH- 2, and purifying and freeze-drying to obtain a finished product of goserelin impurity E.
In the technical scheme, the method synthesizes the first fragment heptapeptide by adopting a fragment method, so that the operation is simple and convenient, the purity is higher, and the problem of low crude product purity caused by unprotected Arg side chain guanidino of the second fragment peptide resin when the subsequent amino acid is gradually connected upwards can be effectively avoided. Meanwhile, the second fragment peptide resin is based on Boc-Pro-PAM resin, so that the problem of low synthesis yield caused by the fact that peptide chains fall off from the resin when the Boc is removed by acid is avoided, and subsequent hydrazinolysis is facilitated.
As a preferred embodiment of the present invention, in step a), the amino acid used in the ligation of the first heptapeptide resin is Fmoc-Leu-OH, fmoc-DSer (tBu) -OH, fmoc-Tyr-OH, fmoc-Ser-OH, fmoc-Trp-OH, fmoc-His-OH and H-Pyr-OH.
In the technical scheme, the amino acid is directly connected with other amino acids except for the DSer side chain with tBu protecting groups, and the side chain of the amino acid is not provided with the protecting groups, so that the problem of impurity increase caused by selective removal of the side chain protecting groups under the strong acid condition is avoided.
In a preferred embodiment of the present invention, in step a), the cleavage of the first-stage heptapeptide resin uses a hexafluoroisopropanol/dichloromethane system or a trifluoroethanol/acetic acid/dichloromethane system.
In a preferred embodiment of the present invention, in step a), the cleavage of the first-stage heptapeptide resin uses a 30% volume fraction hexafluoroisopropanol/dichloromethane solution.
In the technical scheme, the solvent can be directly evaporated by a rotary evaporator after cutting is finished and then put into the step 3) for use.
As a preferred embodiment of the invention, in step b), the reagent used in removing Boc is a mixture of trifluoroacetic acid and dichloromethane.
As a preferred embodiment of the invention, in step b), the reagent used in removing Boc is a 50% volume fraction trifluoroacetic acid/dichloromethane mixture.
In the technical scheme, boc is directly removed on the solid phase resin, and the next reaction can be carried out after washing and alkali neutralization, so that the operation is simple and convenient.
As a preferred embodiment of the present invention, the coupling reagent used in the solid phase synthesis strategy is a mixture of HOBt and DIC, or a mixture of HOAt and DIC, or a mixture of PyAop and a base, or a mixture of PyBop and a base, or a mixture of HBTU and a base, or a mixture of HATU and a base.
As a preferred embodiment of the present invention, the coupling reagent used in the solid phase synthesis strategy is a mixture of HOBt and DIC.
In a preferred scheme of the invention, in the step d), the reagent used for hydrazinolysis is a hydrazine hydrate solution with the mass fraction of 85%, and the solvent used is a mixed solvent of 1, 4-dioxane and DMF.
Compared with the prior art, the invention has the following beneficial effects:
1) According to the invention, the first fragment heptapeptide Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-OH is synthesized in advance by using CTC resin, and the mature polypeptide solid phase synthesis (SPPS) method is adopted, so that the synthesized first fragment heptapeptide is simple and convenient to operate and has higher purity. Meanwhile, the adoption of the heptapeptide fragment can avoid the problem of low crude product purity caused by the fact that the guanidine group on the Arg side chain of the second fragment peptide resin is unprotected and the subsequent amino acid is gradually connected upwards.
2) The invention adopts Boc-Pro-PAM resin, on one hand, the resin structure is stable to acid, and the problem of low synthesis yield caused by the fact that peptide chains fall off from the resin when the Boc is removed by acid is avoided. On the other hand, the Boc-Pro-PAM resin can be adopted to facilitate hydrazinolysis so as to obtain the target compound.
Drawings
FIG. 1 is a synthetic flow chart of the present invention.
FIG. 2 is an HPLC chromatogram of example 1.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. 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.
In the present invention, the raw materials and reagents or equipment used are commercially available.
Referring to fig. 1, the invention provides a preparation method of goserelin impurity E, which comprises the following steps:
a) Using CTC resin, firstly, synthesizing a first fragment heptapeptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-CTC in a solid phase, and cutting to obtain a first fragment heptapeptide Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-OH;
b) And (3) removing the Boc by using the Boc-Pro-PAM resin, and performing solid-phase synthesis to connect the Boc-Arg-OH to obtain the Boc-Arg-Pro-PAM resin. Removing Boc by acidolysis to obtain second fragment peptide resin H 2 N-Arg-Pro-PAM resin;
c) Coupling the first fragment heptapeptide obtained in the step a) to the second fragment peptide resin obtained in the step b) through solid phase synthesis to obtain a third fragment peptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-PAM resin;
d) Hydrazinolysis of the third fragment peptide resin obtained in step c) to obtain
And (3) purifying and freeze-drying the crude Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-NH- 2 to obtain a goserelin impurity E finished product.
In the present invention, "%" refers to volume fractions unless otherwise specified.
Example 1
The embodiment discloses a preparation method of goserelin impurity E, which comprises the following steps:
a) A blank CTC resin (50 mmol,50.0 g) with a degree of substitution of 1.0mmol/g was weighed into a solid phase synthesis reactor, fmoc-Leu-OH (100 mmol,35.3 g) was added, and DCM was added three times the resin volume, and diisopropylethylamine DIEA (200 mmol,33 mL) was added under stirring; after 2.5h of reaction, 50mL of methanol is added for end capping; the reaction solution was withdrawn, washed three times with DMF, washed three times with methanol and dried to give Fmoc-Leu-CTC resin, the degree of substitution was found to be 0.5mmol/g.
Swelling Fmoc-Leu-CTC resin (10 mmol,20 g) with DMF for more than 2h, adding a 20% piperidine DMF solution with 1.0 times of the resin volume for reaction for 5min, extracting the deprotected solution, adding a 20% piperidine DMF solution with 1.0 times of the resin volume for reaction for 15min, and extracting the deprotected solution; the resin layer was washed 8 times with 1.0 times the resin volume of DMF and ninhydrin detected as positive.
Fmoc-DSer (tBu) -OH (30 mmol,11.5 g), HOBt (30 mmol,4.0 g) was weighed, dissolved in DMF, and activated by adding DIC (30 mmol,4.6 mL) under ice water bath for 10-15min; and (3) adding the activated reaction liquid into a reactor, and reacting at 15-25 ℃ until ninhydrin detection is negative. After the reaction, the reaction mixture was dried, and DMF was added to wash the resin 3 times. The steps of removing Fmoc protection and adding corresponding protected amino acid or protected polypeptide coupling are repeated, fmoc-Leu-OH, fmoc-DSer (tBu) -OH, fmoc-Tyr-OH, fmoc-Ser-OH, fmoc-Trp-OH, fmoc-His-OH, H-Pyr-OH are sequentially used from the C end to the N end according to the first fragment heptapeptide sequence, and the preparation of the first fragment heptapeptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-CTC resin is completed.
The first fragment heptapeptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-CTC resin completed previously was cleaved using 30% hexafluoroisopropanol/dichloromethane solution, and the resulting solution was spin distilled to give the first fragment heptapeptide Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-OH.
B) Weighing Boc-Pro-PAM (1 mmol,1.25 g) resin with the substitution degree of 0.8mmol/g, placing the Boc-Pro-PAM resin into a solid phase synthesis reactor, adding 3.0 times of resin volume DMF to swell for more than 2 hours, pumping off the DMF, adding 50% trifluoroacetic acid/dichloromethane solution, reacting for 30min to remove Boc, washing the resin 5 times by using DCM, and washing the resin layer to be alkaline by using 5% NMM/DMF; ninhydrin detection was positive.
Boc-Arg-OH (3 mmol,0.82 g), HOBt (3 mmol,0.405 g) were weighed and dissolved in DMF, DIC (3 mmol, 0.4635 mL) was added under ice water bath to activate for 10-15min, the activated reaction solution was added into the reactor, and the reaction was carried out at 20-40 ℃ until ninhydrin detection was negative. After the reaction, the reaction mixture was dried, and DMF was added to wash the resin 3 times. Repeating the steps of removing Boc protection and neutralizing with alkali to obtain a second fragment peptide resin H 2 N-Arg-Pro-PAM resin.
C) The first heptapeptide Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-OH (3 mmol,2.87 g), HOBt (3 mmol,0.405 g) was weighed, dissolved in DMF, and activated by adding DIC (3 mmol, 0.463 mL) under ice water bath for 10-15min; and (3) adding the activated reaction liquid into a reactor, and reacting at 20-40 ℃ until ninhydrin detection is negative. And after the reaction is finished, pumping out the reaction solution, adding DMF (dimethyl formamide) to wash the resin for 3 times, and washing the resin with methanol for 3 times to obtain the third fragment peptide resin.
D) The third fragment peptide resin was added to 10mL of 1, 4-dioxane: adding 1mL of hydrazine hydrate solution with the mass fraction of 85% into a mixed solvent of DMF (V: V) =2:1, oscillating for 4 hours, filtering and collecting filtrate, spin-drying to obtain a crude goserelin impurity E, purifying and freeze-drying to obtain a finished goserelin impurity E product.
The crude product yield is: 97%. Referring to fig. 2, the hplc purity was 61.92%.
Therefore, the invention adopts the fragment method to synthesize, which not only ensures that the first fragment heptapeptide is simple and convenient to operate and has higher purity, but also can effectively avoid the problem of low purity of crude products caused by unprotected Arg side chain guanidino of the second fragment peptide resin when the subsequent amino acid is gradually connected upwards. Meanwhile, the second fragment peptide resin is based on Boc-Pro-PAM resin, so that the problem of low synthesis yield caused by the fact that peptide chains fall off from the resin when the Boc is removed by acid is avoided, and subsequent hydrazinolysis is facilitated.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (9)
1. A process for the preparation of goserelin impurity E, characterized in that it comprises the following steps:
a) Using CTC resin, firstly, synthesizing a first fragment heptapeptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-CTC in a solid phase, and cutting to obtain a first fragment heptapeptide Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-OH;
b) Removing Boc by using Boc-Pro-PAM resin, and then carrying out solid phase synthesis to connect Boc-Arg-OH to obtain Boc-Arg-Pro-PAM resin, and carrying out acidolysis to remove Boc to obtain second fragment peptide resin H 2 N-Arg-Pro-PAM resin;
c) Coupling the first fragment heptapeptide obtained in the step a) to the second fragment peptide resin obtained in the step b) through solid phase synthesis to obtain a third fragment peptide resin Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-PAM resin;
d) And c), carrying out hydrazinolysis on the third fragment peptide resin obtained in the step c) to obtain a crude product of Glp-His-Trp-Ser-Tyr-DSer (tBu) -Leu-Arg-Pro-NH- 2, and purifying and freeze-drying to obtain a finished product of goserelin impurity E.
2. The process for the preparation of goserelin impurity E according to claim 1, wherein in step a) the amino acids used for the attachment of the first-stage heptapeptide resin are Fmoc-Leu-OH, fmoc-DSer (tBu) -OH, fmoc-Tyr-OH, fmoc-Ser-OH, fmoc-Trp-OH, fmoc-His-OH and H-Pyr-OH.
3. The process for the preparation of goserelin impurity E according to claim 1, wherein in step a) the cleavage of the first-stage heptapeptide resin uses a hexafluoroisopropanol/dichloromethane system or a trifluoroethanol/acetic acid/dichloromethane system.
4. A process for the preparation of goserelin impurity E, according to claim 1 or 3, wherein in step a) the cleavage of the first-stage heptapeptide resin uses a 30% hexafluoroisopropanol/dichloromethane solution.
5. A process for the preparation of goserelin impurity E according to claim 1, wherein in step b) the reagent used in removing Boc is a mixture of trifluoroacetic acid and dichloromethane.
6. Process for the preparation of goserelin impurity E according to claim 1 or 5, wherein in step b) the reagent used for removing Boc is a 50% trifluoroacetic acid/dichloromethane mixture.
7. The process for the preparation of goserelin impurity E according to claim 1, wherein the coupling reagent used in the solid phase synthesis strategy is a mixture of HOBt and DIC or a mixture of HOAt and DIC, or PyAop and a base or PyBop and a base, or HBTU and a base or HATU and a base.
8. The process for the preparation of goserelin impurity E according to claim 1 or 7, wherein the coupling reagent used in the solid phase synthesis strategy is a mixture of HOBt and DIC.
9. The process for preparing goserelin impurity E of claim 1, wherein in step d), the reagent used for hydrazinolysis is 85% hydrazine hydrate solution and the solvent used is a mixed solvent of 1, 4-dioxane and DMF.
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