CN114621326B - Parafatide aspartate, intermediate thereof and preparation method thereof - Google Patents
Parafatide aspartate, intermediate thereof and preparation method thereof Download PDFInfo
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- CN114621326B CN114621326B CN202011457300.4A CN202011457300A CN114621326B CN 114621326 B CN114621326 B CN 114621326B CN 202011457300 A CN202011457300 A CN 202011457300A CN 114621326 B CN114621326 B CN 114621326B
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- pasireotide
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- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title abstract description 16
- 229940009098 aspartate Drugs 0.000 title abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 178
- VMZMNAABQBOLAK-DBILLSOUSA-N pasireotide Chemical compound C([C@H]1C(=O)N2C[C@@H](C[C@H]2C(=O)N[C@H](C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@H](C(N[C@@H](CC=2C=CC(OCC=3C=CC=CC=3)=CC=2)C(=O)N1)=O)CCCCN)C=1C=CC=CC=1)OC(=O)NCCN)C1=CC=CC=C1 VMZMNAABQBOLAK-DBILLSOUSA-N 0.000 claims abstract description 130
- 229960005415 pasireotide Drugs 0.000 claims abstract description 114
- 108700017947 pasireotide Proteins 0.000 claims abstract description 114
- 235000003704 aspartic acid Nutrition 0.000 claims abstract description 33
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims abstract description 33
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims abstract description 22
- 150000003839 salts Chemical class 0.000 claims abstract description 18
- 125000006239 protecting group Chemical group 0.000 claims abstract description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 90
- 238000000034 method Methods 0.000 claims description 82
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 52
- 239000000243 solution Substances 0.000 claims description 43
- 239000012071 phase Substances 0.000 claims description 39
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 239000003795 chemical substances by application Substances 0.000 claims description 24
- 239000007864 aqueous solution Substances 0.000 claims description 21
- 238000003776 cleavage reaction Methods 0.000 claims description 20
- 230000007017 scission Effects 0.000 claims description 20
- 239000011347 resin Substances 0.000 claims description 19
- 229920005989 resin Polymers 0.000 claims description 19
- 235000001014 amino acid Nutrition 0.000 claims description 18
- 150000001413 amino acids Chemical class 0.000 claims description 18
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 17
- 239000003960 organic solvent Substances 0.000 claims description 17
- 238000000746 purification Methods 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 15
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 12
- 238000007363 ring formation reaction Methods 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- 238000006467 substitution reaction Methods 0.000 claims description 11
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 claims description 10
- 238000005336 cracking Methods 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 9
- 239000000523 sample Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 7
- 238000004811 liquid chromatography Methods 0.000 claims description 7
- 239000007790 solid phase Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 6
- 238000005755 formation reaction Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- 239000007822 coupling agent Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 16
- 229940024606 amino acid Drugs 0.000 description 15
- 238000006345 epimerization reaction Methods 0.000 description 14
- 238000005406 washing Methods 0.000 description 13
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 12
- 239000012535 impurity Substances 0.000 description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000000275 Adrenocorticotropic Hormone Substances 0.000 description 6
- 102400000739 Corticotropin Human genes 0.000 description 6
- 101800000414 Corticotropin Proteins 0.000 description 6
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 description 6
- 229960000258 corticotropin Drugs 0.000 description 6
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
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- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 238000001291 vacuum drying Methods 0.000 description 5
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 238000000967 suction filtration Methods 0.000 description 4
- 230000008961 swelling Effects 0.000 description 4
- ADOHASQZJSJZBT-AREMUKBSSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[1-[(2-methylpropan-2-yl)oxycarbonyl]indol-3-yl]propanoic acid Chemical compound C12=CC=CC=C2N(C(=O)OC(C)(C)C)C=C1C[C@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ADOHASQZJSJZBT-AREMUKBSSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 229960000890 hydrocortisone Drugs 0.000 description 3
- NEEFMPSSNFRRNC-HQUONIRXSA-N pasireotide aspartate Chemical compound OC(=O)[C@@H](N)CC(O)=O.OC(=O)[C@@H](N)CC(O)=O.C([C@H]1C(=O)N2C[C@@H](C[C@H]2C(=O)N[C@H](C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@H](C(N[C@@H](CC=2C=CC(OCC=3C=CC=CC=3)=CC=2)C(=O)N1)=O)CCCCN)C=1C=CC=CC=1)OC(=O)NCCN)C1=CC=CC=C1 NEEFMPSSNFRRNC-HQUONIRXSA-N 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000005086 pumping Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- REHSJSKPWIOKIJ-LJAQVGFWSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(4-phenylmethoxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(C=C1)=CC=C1OCC1=CC=CC=C1 REHSJSKPWIOKIJ-LJAQVGFWSA-N 0.000 description 2
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 2
- 208000014311 Cushing syndrome Diseases 0.000 description 2
- WEQJQNWXCSUVMA-RYUDHWBXSA-N Phe-Pro Chemical compound C([C@H]([NH3+])C(=O)N1[C@@H](CCC1)C([O-])=O)C1=CC=CC=C1 WEQJQNWXCSUVMA-RYUDHWBXSA-N 0.000 description 2
- 208000010067 Pituitary ACTH Hypersecretion Diseases 0.000 description 2
- 208000020627 Pituitary-dependent Cushing syndrome Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- WLJNZVDCPSBLRP-UHFFFAOYSA-N pamoic acid Chemical compound C1=CC=C2C(CC=3C4=CC=CC=C4C=C(C=3O)C(=O)O)=C(O)C(C(O)=O)=CC2=C1 WLJNZVDCPSBLRP-UHFFFAOYSA-N 0.000 description 2
- -1 pasireotide salts Chemical class 0.000 description 2
- 230000001817 pituitary effect Effects 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical class C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
- 208000003200 Adenoma Diseases 0.000 description 1
- 206010001233 Adenoma benign Diseases 0.000 description 1
- 208000037171 Hypercorticoidism Diseases 0.000 description 1
- MYTOTTSMVMWVJN-STQMWFEESA-N Lys-Tyr Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 MYTOTTSMVMWVJN-STQMWFEESA-N 0.000 description 1
- 208000035977 Rare disease Diseases 0.000 description 1
- 108050001286 Somatostatin Receptor Proteins 0.000 description 1
- 102000011096 Somatostatin receptor Human genes 0.000 description 1
- 201000005255 adrenal gland hyperfunction Diseases 0.000 description 1
- 230000001780 adrenocortical effect Effects 0.000 description 1
- 150000001510 aspartic acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
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- 238000010168 coupling process Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000004179 hypothalamic–pituitary–adrenal axis Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 229960004219 pasireotide diaspartate Drugs 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 201000009395 primary hyperaldosteronism Diseases 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229940050423 signifor Drugs 0.000 description 1
- 229940075620 somatostatin analogue Drugs 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
-
- 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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides an intermediate linear peptide having the following sequence: H-Lys (R) 1 )‑Tyr(4‑Bzl)‑Phe‑(4‑R 2 ‑NH‑C 2 H 4 ‑NH‑CO‑O)Pro‑Phg‑D‑Trp(R 3 )‑OH,R 1 、R 2 、R 3 Is a protecting group. The pasireotide (crude peptide) prepared by the intermediate linear peptide has high purity, and the target purity pasireotide obtained by purifying the pasireotide (crude peptide) has high yield. The pasireotide and the aspartic acid can form bimolecular aspartate, and the salt formation is accurate. The whole preparation method is simple and convenient to operate, quick, low in cost and suitable for large-scale production.
Description
Technical Field
The invention relates to the field of medicine. In particular, the invention relates to a preparation method of pasireotide aspartate, an aspartireotide intermediate and a preparation method thereof.
Background
Cushing's Disease is pituitary adrenocorticotropic hormone (ACTH) adenoma or ACTH cell hyperplasia, hypersecretion of ACTH, leading to adrenocortical hyperplasia, hypercortisolism, leading to a series of metabolic disorders and pathological changes, clinically manifested as Cushing's Syndrome, with hypothalamic-pituitary-adrenal axis dysfunction, an exhaustion Disease, rarely self-alleviating, if not diagnosed in time, high mortality.
Aspartic acid pasireotide (Pasireotide Diaspartate), marketed as sign ifor, is a multi-receptor targeted somatostatin analog capable of binding 4 of the 5 somatostatin receptor (SSTS) subtypes (SST 1,2,3, 5) with high affinity, preventing ACTH and GH release. In cushing's disease treatment, the production of cortisol is reduced by inhibiting ACTH secretion, and the levels of Urinary Free Cortisol (UFC) and serum cortisol are reduced, thereby alleviating disease symptoms. The first time 25 days of 4.2012 was approved by the european union pharmaceutical administration (EMA) for marketing according to the relevant regulations for rare diseases for adult cushing patients who were unable to select pituitary surgery or who were not cured by surgery, and then subsequently were approved for marketing in 48 countries.
The aspartic acid pasireotide is a cyclohexapeptide somatostatin analogue, and the structural formula is shown as follows, and is obtained by salifying pasireotide with aspartic acid. In the prior art, the purification has the problem of low yield, and the total yield of the purification and salifying steps of the pasireotide (crude peptide) is low, so that the preparation cost of the pasireotide is too high.
Therefore, it is necessary to provide a method for improving the yield of purification and salification of pasireotide (crude peptide).
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides an intermediate linear peptide, a preparation method thereof, a method for preparing pasireotide, a purification method of pasireotide and a method for preparing pasireotide salt, wherein the pasireotide (crude peptide) prepared from the intermediate linear peptide has high purity, and the pasireotide (crude peptide) is purified to obtain target pasireotide with high yield. The pasireotide and the aspartic acid can form bimolecular aspartic acid salt, the salt formation is accurate, the TFA residue in the aspartic acid pasireotide is lower than 0.01%, and the whole preparation method is simple and convenient to operate, quick, low in cost and suitable for large-scale production.
In the present inventionIn one aspect, the invention provides an intermediate linear peptide. According to an embodiment of the invention, the intermediate linear peptide has the following sequence: H-Lys (R) 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -OH, wherein R 1 、R 2 、R 3 Is a protecting group. Thus, the purity of the obtained pasireotide (crude peptide) can reach more than 90 percent, and the target purity pasireotide obtained by purifying the pasireotide (crude peptide) has high yield.
According to an embodiment of the invention, R 1 、R 2 、R 3 Each independently selected from Boc, adpoc, iboc, poc, Z (OMe), tmz, cbz or Ddz.
According to an embodiment of the invention, the intermediate linear peptide has the following sequence: H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(Boc)-OH。
In another aspect of the invention, the invention provides a method of preparing the intermediate linear peptide described above. According to an embodiment of the invention, the method comprises: taking Trp as an initial amino acid, connecting the initial amino acid protected by Fmoc to a solid phase carrier, and removing Fmoc protection; the following steps are repeated: adding Fmoc-protected amino acid and coupling agent, removing Fmoc protection to obtain H-Lys (R 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -a solid phase carrier; cleavage of the H-Lys (R 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -a solid support, obtaining said intermediate linear peptide.
In the process of preparing intermediate linear peptide by polypeptide solid phase synthesis method, initial amino acid is designed as Trp, and Trp is adopted 1 -Phg 2 -Hyp 3 -Phe 4 -Tyr 5 -Lys 6 Sequentially (e.g., fmoc-Phg-OH, fmoc- (4-R) coupled to a solid support having an initial amino acid attached thereto 2 -NH-C 2 H 4 -NH-CO-O)Pro-OH、Fmoc-Phe-OH、Fmoc-Tyr(Bzl)-OH、Fmoc-Lys(R 1 ) -OH) to give intermediate linear peptides, which may beThe carboxyl end epimerization reaction of the intermediate linear peptide in the cyclization process is effectively controlled, the epimerization degree is reduced, the purity of the obtained pasireotide (crude peptide) can reach more than 90 percent, and the target purity pasireotide obtained by purifying the pasireotide (crude peptide) has high yield. In addition, the method is simple and convenient to operate, quick, low in cost and suitable for large-scale production.
According to an embodiment of the invention, the solid support is selected from 2-chlorotrityl chloride resins.
According to an embodiment of the present invention, the substitution degree of the 2-chlorotrityl chloride resin is 0.6 to 1.2mmol/g.
According to an embodiment of the invention, the condensing agent is selected from DIC and HOBt.
In yet another aspect of the invention, the invention provides a method of preparing a cyclized peptide. According to an embodiment of the present invention, the intermediate linear peptide described above is subjected to cyclization treatment to obtain the cyclized peptide. As described above, the intermediate linear peptide of the present invention can effectively control the carboxyl end epimerization reaction during cyclization, and reduce the epimerization degree.
According to an embodiment of the present invention, the cyclization treatment includes: step 1): dissolving a cyclizing agent in a first organic solvent to obtain a cyclizing agent solution; step 2): and 3) dissolving the intermediate linear peptide in the first organic solvent to obtain an intermediate linear peptide solution, adding the intermediate linear peptide solution into the cyclizing agent solution obtained in the step 1) to react, and concentrating to obtain the cyclized peptide.
According to an embodiment of the present invention, in the step 2), the concentration of the intermediate linear peptide solution is controlled to be 0.02 to 0.06mmol/L.
According to an embodiment of the present invention, in the step 2), the intermediate linear peptide solution is dropped into the cyclizing reagent solution obtained in the step 1).
According to an embodiment of the present invention, in the step 2), the dropping speed of the intermediate linear peptide solution is controlled to be 2-8 mL/min.
According to an embodiment of the present invention, in the step 2), the dropping temperature and the reaction temperature of the intermediate linear peptide solution are controlled to be 20 to 30 ℃.
According to an embodiment of the invention, in step 1), the first organic solvent is selected from one or more of DMF and DCM, preferably DMF.
In yet another aspect of the invention, the invention provides a method of preparing pasireotide. According to an embodiment of the invention, the method comprises the steps of: preparing a cyclized peptide using the method for preparing a cyclized peptide described above; performing cracking treatment on the cyclized peptide and a cracking agent to obtain a cracking product; mixing the cleavage product with a second organic solvent, purifying and collecting the precipitate to obtain the pasireotide. Thereby, in order to remove the protecting group on the cyclized peptide obtained previously and to purify the product. The purity of the obtained pasireotide can reach more than 90 percent, and the yield is higher.
According to an embodiment of the present invention, the concentration of the cyclized peptide in the mixed solution containing the cyclized peptide and the cleavage agent is controlled to be 40 to 60mmol/L.
According to an embodiment of the invention, the cleavage agent is selected from the group consisting of trifluoroacetic acid, water and methylene chloride.
According to an embodiment of the present invention, the volume ratio of trifluoroacetic acid, water and dichloromethane is (85-95): (4-6): (4-6).
According to an embodiment of the present invention, the second organic solvent is glacial diethyl ether.
According to an embodiment of the invention, the method further comprises: subjecting the pasireotide to a purification treatment as follows: dissolving the pasireotide in an acetonitrile aqueous solution, and filtering to obtain a sample solution; and adding the sample liquid into liquid chromatography, and collecting fractions for a preset time to obtain the purified pasireotide.
According to an embodiment of the present invention, the acetonitrile concentration in the acetonitrile aqueous solution is 20 to 30% by volume.
According to an embodiment of the present invention, the chromatographic column used in the liquid chromatography is a C18 column; the detection wavelength is 220nm; the gradient elution mode is adopted, wherein the mobile phase A is 0.05-0.2% by volume of trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile.
In yet another aspect of the invention, the invention provides a method of preparing a pasireotide salt. According to an embodiment of the invention, the method comprises: obtaining purified pasireotide using the method of preparing pasireotide described above; and carrying out salt formation reaction on the purified pasireotide and acid to obtain pasireotide salt, wherein the acid is aspartic acid or pamoic acid.
According to an embodiment of the present invention, the acid is aspartic acid, that is, specifically, the present invention proposes a method for preparing aspartic acid pasireotide, the salifying treatment comprising: eluting the purified pasireotide in a C18 column with acetonitrile and water, eluting with a first eluent, eluting with acetonitrile and water, and finally performing gradient elution with a second eluent.
According to an embodiment of the present invention, the first eluent includes a phase a and a phase B, the phase a is an aspartic acid aqueous solution, the phase B is acetonitrile, the concentration of aspartic acid in the aspartic acid aqueous solution is 0.05-0.2M, the pH value of the aspartic acid aqueous solution is adjusted to 5-8, the content of the phase a is 80-95% by volume, and the content of the phase B is 5-20% by volume.
According to an embodiment of the invention, the second eluent comprises a phase a and a phase B, the phase a is water and the phase B is acetonitrile.
According to an embodiment of the invention, the method further comprises: after the salification, the resulting solution containing pasireotide salt is freeze-dried.
In a further aspect of the invention, the invention proposes the use of the intermediate linear peptide as described hereinbefore for the preparation of pasireotide or pasireotide salts. As described above, the intermediate linear peptide of the invention can effectively control the carboxyl end epimerization reaction in the cyclization process, reduce the epimerization degree, and the purity of the obtained pasireotide (crude peptide) can reach more than 90 percent, so that the yield of target purity pasireotide obtained by purifying pasireotide (crude peptide) is high, and further, the yield of pasireotide salt is improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
fig. 1 shows a high performance liquid chromatogram of purified pasireotide according to one embodiment of the invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The present invention provides an intermediate linear peptide, a method for producing the same, a method for producing cyclized peptide, a method for producing pasireotide salt, and use of the intermediate linear peptide, which will be described in detail below, respectively.
Intermediate linear peptides
In one aspect of the invention, the invention provides an intermediate linear peptide. According to an embodiment of the invention, the intermediate linear peptide has the following sequence: H-Lys (R) 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -OH, wherein R 1 、R 2 、R 3 Is a protecting group. The inventor finds that the linear peptide of the above sequence has high purity and low racemization rate, the carboxyl end amino acid of the peptide sequence is Trp, the carboxyl end epimerization reaction can be effectively controlled in the cyclization process, the epimerization degree is reduced, the purity of the obtained pasireotide (crude peptide) can reach more than 90%, and the yield of the pasireotide (crude peptide) in the purification process is higher.
According to an embodiment of the invention, R 1 、R 2 、R 3 Each independently selected from Boc, adpoc, iboc, poc, Z (OMe), tmz, cbz or Ddz. The protecting groups can play a role in protecting amino acid in the reaction process, and the protecting groups can be removed as required after the reaction is finished to obtain a target product. The purity of the obtained pasireotide (crude peptide) can reach more than 90 percent, and the target purity pasireotide obtained by purifying the pasireotide (crude peptide) has high yield.
According to an embodiment of the invention, the intermediate linear peptide has the following sequence: H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) 2 H 4 -NH-CO-O) Pro-Phg-D-Trp (Boc) -OH. Thus, the purity of the obtained pasireotide (crude peptide) can reach more than 90 percent, and the target purity pasireotide obtained by purifying the pasireotide (crude peptide) has high yield.
Method for preparing intermediate linear peptide
In another aspect of the invention, the invention provides a method of preparing the intermediate linear peptide described above. According to an embodiment of the invention, the method comprises: taking Trp as an initial amino acid, connecting the initial amino acid protected by Fmoc to a solid phase carrier, and removing Fmoc protection; the following steps are repeated: adding Fmoc-protected amino acid and coupling agent, removing Fmoc protection to obtain H-Lys (R 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -a solid phase carrier; cleavage of the H-Lys (R 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -a solid support, obtaining said intermediate linear peptide.
In the process of preparing intermediate linear peptide by polypeptide solid phase synthesis method, initial amino acid is designed as Trp, and Trp is adopted 1 -Phg 2 -Hyp 3 -Phe 4 -Tyr 5 -Lys 6 The intermediate linear peptide can effectively reduce the occurrence of carboxyl end epimerization in the cyclization process, the purity of the obtained pasireotide (crude peptide) can reach more than 90 percent, and the yield of the target purity pasireotide obtained by purifying the pasireotide (crude peptide) is high. And, the method operatesSimple, convenient and quick operation, low cost and suitability for large-scale production.
According to an embodiment of the invention, the solid support is selected from 2-chlorotrityl chloride resins. Thereby, in order to improve the solid phase synthesis efficiency.
According to the examples of the present invention, the substitution degree of the 2-chlorotrityl chloride resin is 0.6 to 1.2mmol/g. The 2-chlorotrityl chloride resin with substitution degree of 0.6-1.2 mmol/g can effectively improve the synthesis efficiency, and the obtained target polypeptide has high yield and purity.
Method for producing cyclized peptide
In yet another aspect of the invention, the invention provides a method of preparing a cyclized peptide. According to an embodiment of the invention, the method comprises: cyclizing the intermediate linear peptide to obtain cyclized peptide. As described above, the intermediate linear peptide of the invention can effectively control the carboxyl end epimerization reaction in the cyclization process, reduce the epimerization degree, and the purity of the obtained pasireotide (crude peptide) can reach more than 90 percent, and the yield of the target purity pasireotide obtained by purifying the pasireotide (crude peptide) is high.
According to an embodiment of the present invention, the cyclization treatment includes: step 1): dissolving a cyclizing agent in a first organic solvent to obtain a cyclizing agent solution; step 2): dissolving intermediate linear peptide in a first organic solvent to obtain intermediate linear peptide solution, adding the intermediate linear peptide solution into the cyclizing agent solution obtained in the step 1) to react, and concentrating to obtain cyclized peptide. Under the action of a cyclizing agent, the intermediate linear peptide may be cyclized to form a cyclized peptide.
According to an embodiment of the present invention, in step 2), the concentration of the intermediate linear peptide solution is controlled to be 0.02 to 0.06mmol/L. Thus, the cyclizing efficiency can be improved, and the cyclized peptide can be produced in a high yield.
According to an embodiment of the present invention, in step 2), an intermediate linear peptide solution is dropped into the cyclizing reagent solution obtained in step 1). Thus, the cyclizing efficiency can be improved, and the cyclized peptide can be produced in a high yield.
According to an embodiment of the present invention, in step 2), the dropping speed of the intermediate linear peptide solution is controlled to be 2-8 mL/min. Thus, the cyclizing efficiency can be improved, and the cyclized peptide can be produced in a high yield.
According to the embodiment of the invention, in the step 2), the dropping temperature and the reaction temperature of the intermediate linear peptide solution are controlled to be 20-30 ℃.
According to an embodiment of the invention, the first organic solvent is selected from one or more of DMF and DCM, preferably DMF. Thereby, in order to solubilize the cyclizing agent and the intermediate linear peptide.
The features and advantages described above for the intermediate linear peptide are equally applicable to the method for preparing cyclized peptide and are not described here.
Method for preparing pasireotide
In yet another aspect of the invention, the invention provides a method of preparing pasireotide. According to an embodiment of the invention, the method comprises the steps of: preparing a cyclized peptide using the method for preparing a cyclized peptide described above; performing cracking treatment on the cyclized peptide and a cracking agent to obtain a cracking product; mixing the cleavage product with a second organic solvent, purifying and collecting the precipitate to obtain the pasireotide. Removing the protecting group under the action of a cracking agent, and precipitating the target cyclized peptide by using a second organic solvent so as to achieve the aim of purification and collection.
According to the embodiment of the invention, the concentration of the concentrate in the mixed solution containing the cyclized peptide and the cleavage agent is controlled to be 40-60 mmol/L. Thus, the protecting group can be effectively removed.
According to an embodiment of the present invention, the cleavage agent is selected from a mixed solution of trifluoroacetic acid, water and dichloromethane, preferably, the volume ratio of trifluoroacetic acid, water and dichloromethane is (85-95): (4-6): (4-6). Thus, the protecting group can be effectively removed.
According to an embodiment of the invention, the second organic solvent is glacial diethyl ether. Thereby, the cyclized peptide is precipitated.
According to an embodiment of the invention, the method further comprises: subjecting the pasireotide to a purification treatment as follows: dissolving the pasireotide in an acetonitrile aqueous solution, and filtering to obtain a sample solution; adding the sample solution into liquid chromatography, and collecting fraction for a predetermined time to obtain purified pasireotide. Therefore, the pasireotide (crude peptide) is conveniently purified, and the purity of the product is improved.
According to an embodiment of the present invention, the acetonitrile concentration in the acetonitrile aqueous solution is 20 to 30% by volume. Therefore, the pasireotide is convenient to dissolve, further the subsequent sample loading is convenient, and the liquid chromatographic separation is not affected.
According to an embodiment of the invention, the chromatographic column used for liquid chromatography is a C18 column; the detection wavelength is 220nm; the gradient elution mode is adopted, wherein the mobile phase A is 0.05-0.2% by volume of trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile. Therefore, the target pasireotide can be fully separated, the interference of other impurities is avoided, the purity of the collected pasireotide is high, and the yield of the purification process is high.
The features and advantages described above for the method for preparing cyclized peptide are equally applicable to the method for preparing pasireotide, and are not described in detail herein.
Process for preparing pasireotide salts
In yet another aspect of the invention, the invention provides a method of preparing a pasireotide salt. According to an embodiment of the invention, the method comprises: obtaining purified pasireotide using the method of preparing pasireotide described above; and carrying out salt formation reaction on the purified pasireotide and acid to obtain pasireotide salt, wherein the acid is aspartic acid or pamoic acid.
According to an embodiment of the invention, the acid is aspartic acid. Namely, the invention also provides a method for preparing the aspartic acid pasireotide. The salifying treatment comprises the following steps: eluting the purified pasireotide in a C18 column with acetonitrile and water, eluting with a first eluent, eluting with acetonitrile and water, and finally performing gradient elution with a second eluent.
In the preparation of pasireotide by reacting the cyclized peptide with a cleavage agent, trifluoroacetic acid (cleavage agent) forms a salt with the cyclized peptide, and trifluoroacetic acid is toxic in clinical use, which affects the quality of the product. Further, the impurity is removed by eluting with acetonitrile and water, then eluting with a mixture of an aqueous aspartic acid solution and acetonitrile to replace trifluoroacetic acid which forms a salt with the cyclized peptide, and then removing the impurity by eluting with acetonitrile and water. The content of pasireotide salt obtained by the above treatment can be detected by liquid chromatography.
According to an embodiment of the present invention, the first eluent includes a phase a and a phase B, the phase a is an aspartic acid aqueous solution, the phase B is acetonitrile, the concentration of aspartic acid in the aspartic acid aqueous solution is 0.05 to 0.2M, the pH value of the aspartic acid aqueous solution is adjusted to 5 to 8, the content of the phase a is 80 to 95% by volume, the content of the phase B is 5 to 20% by volume, and the second eluent includes a phase a and a phase B, the phase a is water, and the phase B is acetonitrile. Therefore, the trifluoroacetic acid which forms salt with the pasireotide can be fully replaced, the trifluoroacetic acid is almost completely removed, the salt formation is accurate, the salt yield is high, and the uniformity is good.
With the salt formation treatment, trifluoroacetic acid can be almost completely removed, and TFA residues are significantly lower than those required in ICH Q3A below 0.2%. In addition, the content of aspartic acid in the aspartic acid pasireotide is 19.67-20.07 wt%, the uniformity of the product is good, and the salification is accurate.
According to an embodiment of the invention, the method further comprises: after the salification, the resulting solution containing pasireotide salt is freeze-dried. Thereby, the preservation is facilitated.
It should be noted that the features and advantages described above for the method for preparing pasireotide are equally applicable to the method for preparing pasireotide salt, and are not described here again.
Use of the same
In a further aspect of the invention, the invention proposes the use of the intermediate linear peptide as described above for the preparation of pasireotide or pasireotide salts. As described above, the intermediate linear peptide of the invention can effectively control the carboxyl end epimerization reaction in the cyclization process, reduce the epimerization degree, and the purity of the obtained pasireotide (crude peptide) can reach more than 90 percent, so that the yield of target purity pasireotide obtained by purifying pasireotide (crude peptide) is high, and further, the yield of pasireotide salt is improved.
The beneficial effects are that:
the intermediate linear peptide can effectively reduce the occurrence of carboxyl end epimerization during cyclization, the purity of the obtained pasireotide (crude peptide) reaches more than 90 percent, the yield of the purification process of the pasireotide (crude peptide) is high, the salifying yield is high, and the process cost can be further reduced. In the obtained aspartic acid pasireotide, the content of aspartic acid (Asp) is about 20 weight percent, the salification is accurate, and the uniformity of the obtained aspartic acid pasireotide is good. After the pasireotide is salified, the obtained aspartic acid pasireotide has almost no counter ion trifluoroacetic acid residue and almost no acetonitrile residue.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 preparation of Fmoc-D-Trp (Boc) -CTC Resin
Experiment group 1
Weighing 2-Chlorotrityl chloride resin with substitution degree of 0.6-0.8mmol/g, swelling with DCM for 30min, washing with DCM and DMF, and draining. Fmoc-D-Trp (Boc) -OH (2 eq) was weighed out and dissolved in DMF, diisopropylethylamine (DIPEA, 3 eq) was activated for 10min and reacted at room temperature for 60min. Suction filtration, resin is washed with DMF for 3 times in sequence, and methanol liquid is added for sealing reaction for 8h. Washing with DMF for 6 times, shrinking with methanol, then pumping, and vacuum drying to obtain Fmoc-D-Trp (Boc) -CTC resin, wherein the detection substitution degree is 0.585mmol/g.
Experiment group 2
Weighing 2-Chlorotrityl chloride resin with substitution degree of 0.8-1.0mmol/g, swelling with DCM for 30min, washing with DCM and DMF, and draining. Fmoc-D-Trp (Boc) -OH (2 eq) was weighed out and dissolved in DMF, diisopropylethylamine (DIPEA, 3 eq) was activated for 10min and reacted at room temperature for 60min. Suction filtration, resin is washed with DMF for 3 times in sequence, and methanol liquid is added for sealing reaction for 8h. Washing with DMF for 6 times, shrinking with methanol, then pumping, and vacuum drying to obtain Fmoc-D-Trp (Boc) -CTC resin, wherein the detection substitution degree is 0.446mmol/g.
Experiment group 3
Weighing 2-Chlorotrityl chloride resin with substitution degree of 1.0-1.2mmol/g, swelling with DCM for 30min, washing with DCM and DMF, and draining. Fmoc-D-Trp (Boc) -OH (2 eq) was weighed out and dissolved in DMF, diisopropylethylamine (DIPEA, 3 eq) was activated for 10min and reacted at room temperature for 60min. Suction filtration, resin is washed with DMF for 3 times in sequence, and methanol liquid is added for sealing reaction for 8h. Washing with DMF for 6 times, shrinking with methanol, then pumping, and vacuum drying to obtain Fmoc-D-Trp (Boc) -CTC resin, wherein the detection substitution degree is 0.452mmol/g.
Example 2
H-Lys(Boc)-Tyr(4-Bzl)-Phe-(4-Boc-NH-C 2 H 4 Preparation of-NH-CO-O) Pro-Phg-D-Trp (Boc) -CTC Resin
Step 1): fmoc-D-Trp (Boc) -CTC resin with a degree of substitution of 0.585mmoL/g obtained in experimental group 1 of example 1 was washed 3 times with DMF, and after swelling the resin with DMF for 30 minutes, the DMF was drained. The reaction was treated with 20% piperidine/DMF solution for 25min, fmoc was removed, the reaction was drained and the resin was washed 6 times with DMF.
Step 2): fmoc-Phg-OH (3 eq), DIC (3 eq) and HOBt (3 eq) are weighed and dissolved in DMF, then the mixture is added into the resin, the coupling reaction is carried out for 90min at room temperature, the reaction end point is monitored and judged by an ninhydrin method, and if the resin is colorless and transparent, the reaction is complete; and when the resin is developed, the reaction is incomplete, the coupling reaction needs to be continued, and the judgment standard is suitable for detecting and judging the reaction end point by an ninhydrin method in the follow-up content. According to standard operation method, fmoc- (4-Boc-NH-C) is coupled in sequence by adding corresponding amino acid coupling step after Fmoc protection is removed 2 H 4 -NH-CO-O) Pro-OH, fmoc-Phe-OH, fmoc-Tyr (Bzl) -OH, fmoc-Lys (Boc) -OH to give Fmoc-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(Boc)-CTC Resin。
Step 3): finally Fmoc removal is carried out on the resin peptide obtained in the step 2), methanol shrinkage is carried out after washing, the resin peptide is dried in vacuum, and H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) is obtained by weighing 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(Boc)-CTC Resin。
Example 3
H-Lys(Boc)-Tyr(4-Bzl)-Phe-(4-Boc-NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(Boc) Preparation of-OH
H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) prepared in example 2 2 H 4 -NH-CO-O) Pro-Phg-D-Trp (Boc) -CTC Resin was placed in a cleavage reactor and cleavage reagent (1% tfa/dcm=95/5 (V/V)) was added in an ice-water bath at a ratio of 10ml/g Resin, stirred for 30min, and transferred to room temperature and stirred for 1.5h. The filtrate was collected by suction filtration, the resin was washed 3 times with a small amount of 1% TFA, and the filtrates were combined and concentrated to dryness under reduced pressure. The yield was 94.5% and the HPLC purity was 85%. [ MS ]]:(MH+=1366)。
Example 4
Cycle[Lys-Tyr(4-Bzl)-Phe-(4-NH 2 -C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp]Preparation of +TFA
Experiment group 1
PyBop (1.5 eq) and DIEPA (3 eq) were weighed into a suitable amount of anhydrous DMF and added to a reactor, and H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) obtained in example 3 was added 2 H 4 -NH-CO-O) Pro-Phg-D-Trp (Boc) -OH was dissolved in anhydrous DMF and added to the reactor. The reaction was stirred at 25 ℃ until complete, the reaction solution was concentrated to dryness to give the cyclized peptide, the cyclized peptide was transferred to a cleavage reactor, trifluoroacetic acid (TFA)/TIS/water/dcm=85/5/5/5 (V/V) was added in an ice-water bath and the reaction was stirred at room temperature for 2h. 8-14eq of glacial ethyl ether is added for sedimentation for 1h, a large amount of white precipitate is formed, the precipitate is centrifuged for 3 times and collected, and white solid is obtained after vacuum drying, and the white solid is pasireotide (crude peptide) with the HPLC purity of 90.4 percent.
Experiment group 2
PyBop (2 eq) and DIEPA (3 eq) were weighed into a suitable amount of anhydrous DMF and added to a reactor, and H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) obtained in example 3 was added 2 H 4 -NH-CO-O) Pro-Phg-D-Trp (Boc) -OH was dissolved in anhydrous DMF and the linear peptide concentrations were controlled to 0.02, 0.04, 0.06, 0.08mmol/L, respectively, and added to the reactor. The reaction was stirred at 25 ℃ until complete, the reaction solution was concentrated to dryness to give the cyclized peptide, the cyclized peptide was transferred to a cleavage reactor, TFA/TIS/water/dcm=85/5/5/5 (V/V) was added in ice-water bath and the reaction was stirred at room temperature for 2h. Adding 8-14eq of glacial ethyl ether for sedimentation for 1h, and centrifuging the precipitate for 3 timesAnd collected, dried in vacuo to give a white solid, pasireotide (crude peptide), with HPLC purities of pasireotide (crude peptide) at various concentrations of linear peptide as shown in table 1. It can be seen that cyclizing with linear peptide having a concentration of 0.02 to 0.06mmol/L gives a pasireotide (crude peptide) having a high purity, and if the linear peptide concentration is too high, the pasireotide (crude peptide) has a reduced purity.
TABLE 1 Effect of different concentrations of Linear peptide on Paradipeptide (crude peptide) purity
Sequence number | Controlling linear peptide concentration | Paripeptide (crude peptide) purity (%) |
1 | 0.02mmol/L | 91.2 |
2 | 0.04mmol/L | 91.8 |
3 | 0.06mmol/L | 91.3 |
4 | 0.08mmol/L | 90.3 |
Experiment group 3
Weighing PyBop (2 eq) and DIEPA (3 eq) and dissolving in proper amount of anhydrous DMFIn a reactor, H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) obtained in example 3 was used 2 H 4 -NH-CO-O) Pro-Phg-D-Trp (Boc) -OH was dissolved in anhydrous DMF and the linear peptide concentration was controlled at 0.04mmol/L and added to the reactor in two drops with controlled drop rates of 2, 4, 8, 10ml/min. The reaction was stirred at 25 ℃ until complete, the reaction solution was concentrated to dryness to give the cyclized peptide, the cyclized peptide was transferred to a cleavage reactor, TFA/TIS/water/dcm=85/5/5/5 (V/V) was added in ice-water bath and the reaction was stirred at room temperature for 2h. 8-14eq of glacial ethyl ether was added to settle for 1h, a large amount of white precipitate was obtained, the precipitate was centrifuged 3 times and collected, and dried under vacuum to obtain white solid, which was pasireotide (crude peptide), and the HPLC purity of pasireotide (crude peptide) at different dropping speeds was shown in Table 2. It can be seen that the purity of the obtained pasireotide (crude peptide) is relatively high by adopting the dropping speed of 2-8 mL/min, and if the dropping speed is too high, the purity of the pasireotide (crude peptide) is reduced.
TABLE 2 Effect of different dripping speeds on Pardepsipeptide (crude peptide) purity
Experiment group 4
PyBop (2 eq) and DIEPA (3 eq) were weighed into a suitable amount of anhydrous DMF and added to a reactor, and H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) obtained in example 3 was added 2 H 4 -NH-CO-O) Pro-Phg-D-Trp (Boc) -OH was dissolved in anhydrous DMF and the linear peptide concentration was controlled to 0.04mmol/L and added to the reactor in two drops with a controlled drop rate of 4ml/min. The temperature was controlled at 25 ℃, the reaction was stirred until completion, the reaction solution was concentrated to dryness to give a cyclized peptide, the cyclized peptide was transferred to a cleavage reactor, TFA/water/dcm=90/5/5 (V/V) was added in an ice-water bath, and the cyclized peptide concentration was controlled to 40, 50, 60, 80mmol/L, respectively, and the reaction was stirred for 30min. Adding 8-14eq of glacial ethyl ether, settling for 1h, obtaining a large amount of white precipitate, centrifuging the precipitate for 3 times, collecting the precipitate, and vacuum drying to obtain whiteThe color solid, pasireotide (crude peptide), was shown in table 3 for HPLC purity at various cyclized peptide solution concentrations. It can be seen that the concentration of the cyclized peptide solution is 40 to 60mmol/L, and the purity of the obtained pasireotide (crude peptide) is high, and if the concentration is too high, the purity of the pasireotide (crude peptide) is lowered.
TABLE 3 influence of different cyclized peptide solution concentrations on the purity of Pardepsipeptide (crude peptide)
Comparative group 1
The starting amino acids were changed and H-Phg-DTrp (Boc) -Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) was obtained by the methods of experiment 1, example 2 and example 3 in example 1 2 H 4 -NH-CO-O) Pro-OH to obtain the linear peptide H-Phg-DTrp (Boc) -Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) 2 H 4 Cyclizing and deprotecting Pro-OH as a linear peptide according to the method of Experimental group 1 of example 4 to give Cycle [ Phg-D-Trp-Lys-Tyr (4-Bzl) -Phe- (4-NH) 2 -C 2 H 4 -NH-CO-O)Pro]+tfa, 86.3% purity.
Comparative group 2
The starting amino acids were changed and H-Tyr (4-Bzl) -Phe-Pro (4-Boc-NH-C) was obtained by the method of experiment 1, example 2 and example 3 of example 1 2 H 4 -NH-CO-O) -Phg-D-Trp (Boc) -Lys (Boc) -OH, which is susceptible to the production of a missing peptide to give the linear peptide H-Tyr (4-Bzl) -Phe-Pro (4-Boc-NH-C) 2 H 4 -NH-CO-O) -Phg-D-Trp (Boc) -Lys (Boc) -OH cyclized and deprotected as in example 4, experimental group 1, to obtain Cycle [ Tyr (4-Bzl) -Phe-Pro (4-NH) 2 -C 2 H 4 -NH-CO-O)-Phg-D-Trp-Lys]+tfa, purity 70.5%.
Comparative group 3
The starting amino acids were changed and H-Phe-Pro (4-Boc-NH-C) was obtained by the methods of Experimental group 1, example 2 and example 3 of example 1 2 H 4 -NH-CO-O) -Phg-DTrp (Boc) -Lys (Boc) -Tyr (Bzl) -OH to obtain the linear peptide H-Phe-Pro (4-Boc-NH-C) 2 H 4 -NH-CO-O) -Phg-DTrp (Boc) -Lys (Boc) -Tyr (Bzl) -OH cyclized, deprotected as in example 4, experimental group 1, to obtain Cycle [ Phe-Pro (4-NH) 2 -C 2 H 4 -NH-CO-O)-Phg-D-Trp-Lys-Tyr(Bzl)]The purity was 79.4%.
As can be seen from the comparison of Experimental group 1 and comparative groups 1 to 3, when H-Lys (Boc) -Tyr (4-Bzl) -Phe- (4-Boc-NH-C) is used 2 H 4 When the-NH-CO-O) Pro-Phg-D-Trp (Boc) -OH is used as the linear peptide, the purity of the pasireotide (crude peptide) is obviously improved.
TABLE 4 Parietide (crude peptide) purity
EXAMPLE 5 purification of pasireotide
Experiment group 1
Step 1) weighing a proper amount of pasireotide (crude peptide) prepared in the second group (50 mmol/L) of experimental group 4 of example 4, dissolving the pasireotide crude product to 20-30mg/ml with a solution of pure acetonitrile/purified water=25%/75% (V/V) and filtering with an organic filter membrane of 0.45 um.
Step 2) using RP-HPLC system, daisogel C18-10-120A, 50X 250mm, wavelength 220nm, mobile phase: phase A: 0.1% aqueous trifluoroacetic acid/phase B: ACN, gradient elution; and collecting target fractions to obtain the purified pasireotide (chromatogram is shown in figure 1), wherein the yield is 94.9%, the purity is more than 99.5%, and the maximum single impurity is less than 0.1%.
Experiment group 2
The pasireotide (crude peptide) prepared in the experimental group 1 of example 4 is weighed and purified according to the purification method of the experimental group 1 to obtain the purified pasireotide, wherein the yield is 80.1%, the purity is more than 99.5%, and the maximum single impurity is less than 0.1%.
Comparative group 1
The pasireotide (crude peptide) prepared in comparative example 1 of example 4 was weighed and purified according to the purification method of experimental group 1 to obtain purified pasireotide with a yield of 63.4%, a purity of more than 99.5% and a maximum single impurity of less than 0.1%.
Table 5 evaluation of pasireotide after purification
Sequence number | Yield of the purification process | Purity of | Maximum single impurity |
Experiment group 1 | 94.9% | More than 99.5% | Less than 0.1% |
Experiment group 2 | 80.1% | More than 99.5% | Less than 0.1% |
Comparative group 1 | 63.4% | More than 99.5% | Less than 0.1% |
EXAMPLE 6 preparation of Parafatide aspartate
Experiment group 1
Step 1) the pass obtained in example 5 was subjected to an RP-HPLC system of Experimental group 1 in example 5 with 95% H 2 Washing with O/5% ACN for 30min; then eluting with 90% (0.05M Asp aqueous solution, pH adjusted to 5.5)/10% ACN for 40min; then use 95% H 2 Washing with O/5% ACN for 40min; finally 70-10% H 2 O/30Gradient reverse elution of 90% ACN for 60min; salt forming treatment is carried out;
step 2) freeze-drying the salified solution;
and 3) detecting the dissolved residue, salt content and water content of the freeze-dried sample.
The sample after lyophilization was tested for 0.004% TFA, 0.0086% acetonitrile, 20.27% Asp, and 3.646% moisture. The purity is more than 99.5%, the maximum single impurity is less than 0.1%, and the yield is 91.3%.
Experiment group 2
Step 1) the acceptable product obtained in example 5 was treated with 97% H 2 Washing with O/3% ACN for 15min; then eluting with 95% (0.1M Asp aqueous solution, pH adjusted to 6.0)/5% ACN for 40min; then use 97% H 2 Washing with O/3% ACN for 40min, and 70-10% H 2 Gradient reverse elution of 30-90% O/ACN for 60min; salt forming treatment is carried out;
step 2) freeze-drying the salified solution;
and 3) detecting the dissolved residue, salt content and water content of the freeze-dried sample.
The TFA residue of the lyophilized sample was measured to be 0.003%, acetonitrile residue to be 0.005%, asp content to be 20.07% and moisture content to be 3.84%. The purity is more than 99.5%, the maximum single impurity is less than 0.1%, and the yield is 89.9%.
Experiment group 3
Step 1) the acceptable product obtained in example 5 was treated with 98% H 2 Washing with O/2% ACN for 25min; then eluting with 99% (0.2M Asp aqueous solution, pH adjusted to 8.0)/1% ACN for 40min; re-using 98% H 2 Washing with O/2% ACN for 40min, and 70-10% H 2 Gradient reverse elution of 30-90% O/ACN for 60min; salt forming treatment is carried out;
step 2) freeze-drying the salified solution;
and 3) detecting the dissolved residue, salt content and water content of the freeze-dried sample.
The TFA residue of the lyophilized sample was measured to be 0.005%, acetonitrile residue 0.012%, asp content 19.80% and moisture content 4.03%. The purity is more than 99.5%, the maximum single impurity is less than 0.1%, and the yield is 87.6%.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (20)
1. A method of preparing an intermediate linear peptide having the sequence: H-Lys (R) 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -OH; wherein R is 1 、R 2 、R 3 Is a protecting group, comprising:
taking Trp as an initial amino acid, connecting the initial amino acid protected by Fmoc to a solid phase carrier, and removing Fmoc protection;
the following steps are repeated: adding Fmoc-protected amino acid and coupling agent, removing Fmoc protection to obtain H-Lys (R 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -a solid phase carrier;
cleavage of the H-Lys (R 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -solid phase carrier, obtainingThe intermediate linear peptide;
the R is 1 、R 2 、R 3 Boc, adpoc, iboc, poc, Z (OMe), tmz, cbz or Ddz.
2. The method of claim 1, wherein the solid support is selected from the group consisting of 2-chlorotrityl chloride resins.
3. The method according to claim 2, wherein the substitution degree of the 2-chlorotrityl chloride resin is 0.6-1.2 mmol/g.
4. A method of preparing a cyclized peptide comprising:
cyclizing the intermediate linear peptide to obtain the cyclized peptide;
the intermediate linear peptide has the following sequence: H-Lys (R) 1 )-Tyr(4-Bzl)-Phe-(4-R 2 -NH-C 2 H 4 -NH-CO-O)Pro-Phg-D-Trp(R 3 ) -OH; wherein R is 1 、R 2 、R 3 Boc, adpoc, iboc, poc, Z (OMe), tmz, cbz or Ddz;
the cyclization treatment includes:
step 1): dissolving cyclizing agent in an organic solvent to obtain cyclizing agent solution;
step 2): dissolving the intermediate linear peptide in the organic solvent to obtain an intermediate linear peptide solution, adding the intermediate linear peptide solution into the cyclizing agent solution obtained in the step 1) to react, and concentrating to obtain the cyclized peptide.
5. The method according to claim 4, wherein in step 1), the organic solvent is selected from one or more of DMF and DCM.
6. The method according to claim 4, wherein in step 1), the organic solvent is selected from DMF.
7. The method according to claim 4, wherein in the step 2), the concentration of the intermediate linear peptide solution is controlled to be 0.02-0.06 mmol/L.
8. The method according to claim 4, wherein in the step 2), the intermediate linear peptide solution is dropped into the cyclizing reagent solution obtained in the step 1).
9. The method according to claim 4, wherein in the step 2), the dropping speed of the intermediate linear peptide solution is controlled to be 2-8 ml/min.
10. The method according to claim 4, wherein in the step 2), the dropping temperature and the reaction temperature of the intermediate linear peptide solution are controlled to be 20-30 ℃.
11. A method of preparing pasireotide comprising the steps of:
preparing a cyclized peptide using the method for preparing a cyclized peptide of any one of claims 4-10;
performing cracking treatment on the cyclized peptide and a cracking agent to obtain a cracking product;
mixing the cleavage product with an organic solvent, purifying and collecting the precipitate to obtain the pasireotide.
12. The method according to claim 11, wherein the concentration of the cyclized peptide is controlled to 40 to 60mmol/L in the mixed solution containing the cyclized peptide and the cleavage agent.
13. The method of claim 11, wherein the cleavage agent is selected from the group consisting of trifluoroacetic acid, water, and methylene chloride.
14. The method of claim 13, wherein the volume ratio of trifluoroacetic acid, water and dichloromethane is (85-95): (4-6): (4-6).
15. The method of claim 11, wherein the organic solvent is glacial diethyl ether.
16. The method as recited in claim 11, further comprising: subjecting the pasireotide to a purification treatment as follows:
dissolving the pasireotide in an acetonitrile aqueous solution, and filtering to obtain a sample solution;
adding the sample liquid into liquid chromatography, and collecting fractions for a preset time to obtain purified pasireotide;
the acetonitrile concentration in the acetonitrile aqueous solution is 20-30% by volume.
17. The method of claim 11, wherein the chromatographic column used in the liquid chromatography is a C18 column; the detection wavelength is 220nm; and a gradient elution mode is adopted, wherein the mobile phase A is 0.05-0.2% by volume of trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile.
18. A method of preparing a pasireotide salt comprising:
obtaining purified pasireotide by the method of any one of claims 11-17;
and carrying out salt forming reaction on the purified pasireotide and acid to obtain pasireotide salt, wherein the acid is aspartic acid.
19. The method of claim 18, wherein the acid is aspartic acid and the salifying comprises:
eluting the purified pasireotide in a C18 column with acetonitrile and water, eluting with a first eluent, eluting with acetonitrile and water, and performing gradient elution with a second eluent;
the first eluent comprises a phase A and a phase B, wherein the phase A is an aspartic acid aqueous solution, the phase B is acetonitrile, the concentration of aspartic acid in the aspartic acid aqueous solution is 0.05-0.2M, the pH value of the aspartic acid aqueous solution is adjusted to 5-8, the content of the phase A is 80-95% by volume, and the content of the phase B is 5-20% by volume;
the second eluent comprises a phase A and a phase B, wherein the phase A is water, and the phase B is acetonitrile.
20. The method of claim 18, wherein the method further comprises:
after the salt formation reaction, the resulting solution containing pasireotide salt is freeze-dried.
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