CN103641894B - A kind of preparation method treating the polypeptide drugs of hypercortisolism - Google Patents
A kind of preparation method treating the polypeptide drugs of hypercortisolism Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 49
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 34
- 208000014311 Cushing syndrome Diseases 0.000 title abstract description 10
- 208000037171 Hypercorticoidism Diseases 0.000 title abstract description 10
- 201000005255 adrenal gland hyperfunction Diseases 0.000 title abstract description 10
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 10
- 239000003814 drug Substances 0.000 title abstract description 6
- 229920001184 polypeptide Polymers 0.000 title abstract description 5
- 229940079593 drug Drugs 0.000 title abstract description 4
- 239000011347 resin Substances 0.000 claims abstract description 131
- 229920005989 resin Polymers 0.000 claims abstract description 131
- 108700017947 pasireotide Proteins 0.000 claims abstract description 81
- 229960005415 pasireotide Drugs 0.000 claims abstract description 74
- 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 claims abstract description 70
- 230000008878 coupling Effects 0.000 claims abstract description 62
- 238000010168 coupling process Methods 0.000 claims abstract description 62
- 238000005859 coupling reaction Methods 0.000 claims abstract description 62
- ZPGDWQNBZYOZTI-UHFFFAOYSA-N 1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)C1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-UHFFFAOYSA-N 0.000 claims abstract description 40
- 230000000903 blocking effect Effects 0.000 claims abstract description 18
- KHXOBMIHCYCHQB-QFIPXVFZSA-N prop-2-enyl (2s)-6-amino-2-(9h-fluoren-9-ylmethoxycarbonylamino)hexanoate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCN)C(=O)OCC=C)C3=CC=CC=C3C2=C1 KHXOBMIHCYCHQB-QFIPXVFZSA-N 0.000 claims abstract description 16
- 238000005336 cracking Methods 0.000 claims abstract description 15
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 68
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 41
- 238000006467 substitution reaction Methods 0.000 claims description 24
- 239000007822 coupling agent Substances 0.000 claims description 18
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 16
- 239000007790 solid phase Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- PARWUHTVGZSQPD-UHFFFAOYSA-N phenylsilane Chemical compound [SiH3]C1=CC=CC=C1 PARWUHTVGZSQPD-UHFFFAOYSA-N 0.000 claims description 10
- PCJHOCNJLMFYCV-NRFANRHFSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-2-phenylacetic acid Chemical compound C1([C@H](NC(=O)OCC2C3=CC=CC=C3C3=CC=CC=C32)C(=O)O)=CC=CC=C1 PCJHOCNJLMFYCV-NRFANRHFSA-N 0.000 claims description 9
- 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 claims description 9
- 239000012317 TBTU Substances 0.000 claims description 9
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 claims description 9
- 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 claims description 8
- 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 claims description 8
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 7
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 7
- 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 6
- 238000002156 mixing Methods 0.000 claims description 5
- 101100030361 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) pph-3 gene Proteins 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 114
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 abstract description 40
- 238000000034 method Methods 0.000 abstract description 30
- 150000001413 amino acids Chemical class 0.000 abstract description 20
- 230000015572 biosynthetic process Effects 0.000 abstract description 8
- 238000003786 synthesis reaction Methods 0.000 abstract description 8
- 125000006239 protecting group Chemical group 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 60
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 36
- 239000000243 solution Substances 0.000 description 34
- 239000003960 organic solvent Substances 0.000 description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 18
- 230000004913 activation Effects 0.000 description 17
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 15
- 239000005457 ice water Substances 0.000 description 14
- 238000003756 stirring Methods 0.000 description 13
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 11
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 11
- 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 8
- 238000003746 solid phase reaction Methods 0.000 description 8
- 238000010671 solid-state reaction Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 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 description 7
- 239000012071 phase Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 150000003053 piperidines Chemical class 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000007363 ring formation reaction Methods 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 229960000890 hydrocortisone Drugs 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- -1 9-fluorenylmethyloxycarbonyl Boc Tertbutyloxycarbonyl Chemical group 0.000 description 3
- 239000000055 Corticotropin-Releasing Hormone Substances 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 238000010504 bond cleavage reaction Methods 0.000 description 3
- 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 3
- 229960000258 corticotropin Drugs 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 108050001286 Somatostatin Receptor Proteins 0.000 description 2
- 102000011096 Somatostatin receptor Human genes 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000002101 lytic effect Effects 0.000 description 2
- 229960004219 pasireotide diaspartate Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- SWZCTMTWRHEBIN-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(O)C=C1 SWZCTMTWRHEBIN-QFIPXVFZSA-N 0.000 description 1
- AVQQQNCBBIEMEU-UHFFFAOYSA-N 1,1,3,3-tetramethylurea Chemical compound CN(C)C(=O)N(C)C AVQQQNCBBIEMEU-UHFFFAOYSA-N 0.000 description 1
- 206010000599 Acromegaly Diseases 0.000 description 1
- 208000010067 Pituitary ACTH Hypersecretion Diseases 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 208000020627 Pituitary-dependent Cushing syndrome Diseases 0.000 description 1
- 208000035977 Rare disease Diseases 0.000 description 1
- 102400000336 Thyrotropin-releasing hormone Human genes 0.000 description 1
- 101800004623 Thyrotropin-releasing hormone Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001343 alkyl silanes Chemical group 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- MQYQOVYIJOLTNX-UHFFFAOYSA-N dichloromethane;n,n-dimethylformamide Chemical compound ClCCl.CN(C)C=O MQYQOVYIJOLTNX-UHFFFAOYSA-N 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- PEECTLLHENGOKU-UHFFFAOYSA-N n,n-dimethylpyridin-4-amine Chemical compound CN(C)C1=CC=NC=C1.CN(C)C1=CC=NC=C1 PEECTLLHENGOKU-UHFFFAOYSA-N 0.000 description 1
- VWBWQOUWDOULQN-UHFFFAOYSA-N nmp n-methylpyrrolidone Chemical compound CN1CCCC1=O.CN1CCCC1=O VWBWQOUWDOULQN-UHFFFAOYSA-N 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 229940000673 orphan drug Drugs 0.000 description 1
- 239000002859 orphan drug Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 208000010916 pituitary tumor Diseases 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940050423 signifor Drugs 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 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 1
- 229940075620 somatostatin analogue Drugs 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- JBWKIWSBJXDJDT-UHFFFAOYSA-N triphenylmethyl chloride Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 JBWKIWSBJXDJDT-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention belongs to field of pharmaceutical chemistry technology, disclose a kind of preparation method treating the polypeptide drugs of hypercortisolism, is a kind of preparation method of SOM230 specifically.Described preparation method take Fmoc-Lys-OAll as raw material; the first coupling of the method for pure solid phase synthesis is adopted to obtain Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll, then elder generation and Boc-NH-C under the prerequisite not removing Fmoc protecting group
2h
4-NH-COOH coupling synthesis side chain amino acid, then the amino acid of coupling Fmoc protection successively, after finally removing the blocking group-All of C end, cyclisation obtains SOM230 resin cracking purifying and obtains SOM230.Compared with prior art, preparation method's raw material of the present invention is easy to get, and reactions steps is few, the yield of SOM230 is high, the scale operation that is suitable for SOM230, and obtained SOM230 purity is high.
Description
Technical field
The invention belongs to field of pharmaceutical chemistry technology, be specifically related to a kind of preparation method treating the polypeptide drugs of hypercortisolism, particularly relate to a kind of preparation method of SOM230.
Background technology
Hypercortisolism is a kind of rare disease jeopardizing patients ' lives, thyroliberin (ACTH) is caused to be secreted in a large number because pituitary tumor stimulates, thus stimulate suprarenal gland growth and secrete a large amount of hydrocortisone, the symptoms such as body weight increases (especially face and neck), skin easily abrades, facial fine hair is heavy, muscle and bone dies down, elevation of blood pressure that cause patient to occur.Ocal resection is the effective ways for the treatment of hypercortisolism, but inoperable patient, what there is no approval before this specially controls medicine.The medicine of current many treatment hypercortisolisms all belongs to over adaptation card and uses.
SOM230, English pasireotide diaspartate by name, its structure is:
SOM230 is manufactured by Novartis Pharma Schweiz AG, is a kind of somatostatin analogue that can be combined with polyceptor, has high-bond with somatostatin receptor sst1 ~ 3 and sst5.SOM230 can suppress GHI, the secretion of GF-I and ACTH, points out it may be used for the treatment of acromegaly and hypercortisolism.After wherein SOM230 and somatostatin receptor are given and closed, the release of ACTH can be stoped, thus reduce the cortisol levels in blood, alleviate the symptom of hypercortisolism.Clinical study proves, by 900 μ g SOM230, has the cortisol levels in the patient urine of 41% at least to reduce by 50%; By 600 μ g SOM230, the cortisol levels in the patient urine of 34% is had at least to reduce by 50%.U.S. food Drug Administration (FDA) is used for the treatment of not by hypercortisolism (Cushing ' s disease) patient of operative treatment in approval on November 14th, 2012 new " Orphan drug " Signifor (SOM230, pasireotide diaspartate) injection liquid of knowing clearly.Therefore SOM230 has very high pharmaceutical use and wide market outlook.
Publication number is the synthesis technique disclosing SOM230 in the patent of CN1446229A, by first preparing special material Fmoc-Pro (4-OCO-NH-CH2-CH2-NH-BOC)-OH, then solid phase method is adopted to carry out the synthesis of chain, last cracking, carry out cyclisation in the liquid phase, finally obtain SOM230.But aforesaid method needs pre-synthesis special material Fmoc-Pro (4-OCO-NH-CH2-CH2-NH-BOC)-OH, then synthesized by solid liquid phase combining method, there is reactions steps many, technique is loaded down with trivial details, special material is not easy to obtain, and is unfavorable for the shortcomings such as scale operation.
Summary of the invention
In view of this, the scale operation the object of the invention is the defect for prior art, provide a kind of preparation method of SOM230, described preparation method's step yield that is few, SOM230 is high, being suitable for SOM230, simultaneously obtained SOM230 purity is high.
For realizing object of the present invention, the present invention adopts following technical scheme:
A preparation method for SOM230, comprising:
Step 1: by Fmoc-Lys-OAll and solid phase carrier coupling, obtains Fmoc-Lys (Resin)-OAll;
Step 2:Fmoc-Lys (Resin)-OAll is coupling Fmoc-D-Trp (Boc)-OH, Fmoc-Phg-OH and Fmoc-Pro (4-OH)-OH one by one, obtains Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll;
Step 3:Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (Resin)-OAll and Boc-NH-C
2h
4-NH-COOH coupling obtains Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll;
Step 4:Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll successively coupling Fmoc-Phe-OH and Fmoc-Tyr (Bzl)-OH obtain NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll, after removing the blocking group-All of C end, cyclisation obtains SOM230 resin;
Step 5: SOM230 pitch shake solution obtains the thick peptide of SOM230;
Step 6: the thick peptide purification of SOM230 obtains SOM230.
Preferably, solid phase carrier described in step 1 is Trt Resin or 2-CTC Resin.
Preferably, the substitution degree of Fmoc-Lys described in step 1 (Resin)-OAll is 0.4 ~ 0.6mmol/g.
Preferably, the coupling agent of coupling described in step 1 is diisopropylethylamine.
Preferably, the coupling agent of coupling described in step 2 is DIC/HOBt.
Preferably, be DIC/HOBt/DMAP with the coupling agent of Boc-NH-C2H4-NH-COOH coupling described in step 3.
Preferably, the coupling agent of coupling successively described in step 4 is DIC/HOBt.
Preferably, step 4 removes the reagent of the blocking group-All of C end is Pd (PPh3) 4/ phenyl silane.
Preferably, the cyclizing agent of cyclisation described in step 4 is the one in HBTU/HOBt/DIPEA, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.
Preferably, the cracking agent of cracking described in step 5 is the mixing solutions of TFA, TIS and water.
First the solid phase carrier coupling of Fmoc-Lys-OAll and suitable substitution degree is obtained Fmoc-Lys (Resin)-OAll by the preparation method of SOM230 of the present invention; coupling Fmoc-D-Trp (Boc)-OH, Fmoc-Phg-OH and Fmoc-Pro (4-OH)-OH obtain Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll one by one afterwards, then Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll elder generation and Boc-NH-C under the prerequisite not removing Fmoc protecting group
2h
4-NH-COOH coupling synthesis side chain amino acid, then coupling Fmoc-Phe-OH and Fmoc-Tyr (Bzl)-OH obtains NH successively
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll, after finally removing the blocking group-All of C end, cyclisation obtains SOM230 resin, and cracking obtains the thick peptide of SOM230, and purifying obtains SOM230 sterling.Compared with prior art, preparation method of the present invention take Fmoc-Lys-OAll as raw material, the method of pure solid phase synthesis is adopted to prepare SOM230, raw material is easy to get, the scale operation that reactions steps is few, the yield of SOM230 is high, be suitable for SOM230, the impurity that the side chain amino acid method that solid phase coupling obtains SOM230 resin again of simultaneously first synthesizing SOM230 produces is few, and obtained SOM230 purity is high.
Embodiment
The embodiment of the invention discloses a kind of preparation method of SOM230.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope method as herein described is changed or suitably change with combination, realize and apply the technology of the present invention.
For realizing object of the present invention, the present invention adopts following technical scheme:
A preparation method for SOM230, comprising:
Step 1: by Fmoc-Lys-OAll and solid phase carrier coupling, obtains Fmoc-Lys (Resin)-OAll;
Step 2:Fmoc-Lys (Resin)-OAll is coupling Fmoc-D-Trp (Boc)-OH, Fmoc-Phg-OH and Fmoc-Pro (4-OH)-OH one by one, obtains Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll;
Step 3:Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (Resin)-OAll and Boc-NH-C
2h
4-NH-COOH coupling obtains Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll;
Step 4:Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll successively coupling Fmoc-Phe-OH and Fmoc-Tyr (Bzl)-OH obtain NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll, after removing the blocking group-All of C end, cyclisation obtains SOM230 resin;
Step 5: SOM230 pitch shake solution obtains the thick peptide of SOM230;
Step 6: the thick peptide purification of SOM230 obtains SOM230.
The preparation method of SOM230 of the present invention adopts Fmoc/tBu synthesis strategy, first the solid phase carrier coupling of Fmoc-Lys-OAll and suitable substitution degree is obtained Fmoc-Lys (Resin)-OAll, coupling Fmoc-D-Trp (Boc)-OH one by one afterwards, Fmoc-Phg-OH and Fmoc-Pro (4-OH)-OH obtains Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll, then Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll elder generation and Boc-NH-C under the prerequisite not removing Fmoc protecting group
2h
4-NH-COOH coupling synthesis side chain amino acid, then coupling Fmoc-Phe-OH and Fmoc-Tyr (Bzl)-OH obtains NH successively
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll, after finally removing the blocking group-All of C end, cyclisation obtains SOM230 resin, and cracking obtains the thick peptide of SOM230, and purifying obtains SOM230 sterling.
First Fmoc-Lys-OAll and solid phase carrier coupling are obtained Fmoc-Lys (Resin)-OAll by preparation method's step 1 of the present invention.
Wherein, preferably, solid phase carrier described in step 1 is Trt Resin or 2-CTC Resin, can reduce resin cost.
Further, the initial substitution degree of solid phase carrier described in step 1 is preferably 0.4mmol/g ~ 1.0mmol/g.Be more preferably 1.0mmol/g.The substitution degree that described coupling obtains Fmoc-Lys (Resin)-OAll is preferably 0.4 ~ 0.6mmol/g.Being 0.4mmol/g in certain embodiments, is 0.5mmol/g in certain embodiments, is 0.6mmol/g in certain embodiments.
Usually coupling agent is needed, with activated amino acid in the amino acid of Fmoc blocking group and the process of solid phase carrier coupling.Preferably, the coupling agent of coupling described in step 1 is diisopropylethylamine (DIPEA).
Concrete, described Fmoc-Lys-OAll organic solvent dissolution, joins in solid state reaction post after adding DIPEA activation, carry out linked reaction with the solid phase carrier of prior organic solvent dissolution under ice bath.
As preferably, the organic solvent of described dissolving Fmoc-Lys-OAll and solid phase carrier is DMF.
As preferably, the condition of linked reaction is room temperature reaction 60min.
Linked reaction described in above-mentioned steps 1 needs after terminating to carry out purifying to reaction solution, is separated and obtains Fmoc-Lys (Resin)-OAll.Described purification process concrete grammar is closed for adding anhydrous methanol in reaction solution, and successively with DMF and DCM washing, methyl alcohol shrinks to be drained, and obtains Fmoc-Lys (Resin)-OAll resin.
Preparation method's step 2 of the present invention adopts the mode of coupling one by one Fmoc-D-Trp (Boc)-OH, Fmoc-Phg-OH and Fmoc-Pro (4-OH)-OH to be coupled on Fmoc-Lys (Resin)-OAll by polypeptide solid-state reaction method and obtains Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll.
Wherein, the coupling agent that described in step 2, coupling mode uses one by one is preferably DIC/HOBt.
Further, described in above-mentioned coupling agent, DIC and HOBt mol ratio is preferably 1:1.
Described in step 2, coupling agent consumption is preferably excessive.In certain embodiments, the mol ratio of the described amino acid with Fmoc blocking group and coupling agent is 1:1.2.Namely described is 1:1.2:1.2 with the amino acid of Fmoc blocking group, the mol ratio of DIC and HOBt.
Further, also comprise before often walking coupling in coupling mode one by one described in step 2 and remove Fmoc step.In certain embodiments, the reagent removing Fmoc described in is DBLK(piperidines: DMF=1:4).
Concrete; amino acid Fmoc-D-Trp (the Boc)-OH of described Fmoc protection first mixes with HOBt; dissolve afterwards and add DIC activation in organic solvent under ice bath, then remove Fmoc-Lys (the Resin)-OAll that Fmoc protects carry out linked reaction with prior organic solvent dissolution.Fmoc-Phg-OH and Fmoc-Pro (the 4-OH)-OH according to said method coupling one by one of Fmoc protection.
As preferably, the amino acid of described dissolving Fmoc protection and the organic solvent of Fmoc-Lys (Resin)-OAll are DMF.
As preferably, described in remove Fmoc for add DBLK solution room temperature stirring reaction with organic solvent dissolution Fmoc-Lys (Resin)-OAll.Be more preferably, add DBLK solution room temperature stirring reaction 5min, drain, repeat to add DBLK solution once, stirring at room temperature reaction 7min.
As preferably, described in above-mentioned steps 2, the condition of linked reaction is room temperature reaction 60min.
Linked reaction described in above-mentioned steps 2 detects with ninhydrin method and judges each linked reaction terminal.If resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.
Preparation method's step 3 of the present invention under the prerequisite not removing Fmoc protecting group, Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll elder generation and Boc-NH-C
2h
4-NH-COOH coupling synthesis side chain amino acid, obtains Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll.
Wherein, described in step 3, the coupling agent of coupling is preferably DIC/HOBt/DMAP.
Further, described in above-mentioned coupling agent, DIC, HOBt and DMAP mol ratio is preferably 1.2:1.2:1.In certain embodiments, described Boc-NH-C2H4-NH-COOH, DIC, HOBt and DMAP mol ratio are preferably 1:1.2:1.2:1.
Concrete, Fmoc-Pro described in step 3 (4-OH)-Phg-D-Trp (Boc)-Lys (Resin)-OAll elder generation and Boc-NH-C
2h
4-NH-COOH linked reaction is specially described Boc-NH-C
2h
4-NH-COOH first mixes with HOBt and DMAP, dissolve afterwards and add DIC activation in organic solvent under ice bath, Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (the CTC)-OAll then obtained with step 2 carries out linked reaction and obtains Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll.
As preferably, described organic solvent is DMF.
As preferably, described in above-mentioned steps 3, the condition of linked reaction is room temperature reaction 12h.
Preparation method's step 4 of the present invention is first at Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll successively coupling Fmoc-Phe-OH and Fmoc-Tyr (Bzl)-OH obtain NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll.
Wherein, the coupling agent of coupling successively described in step 4 is for being preferably DIC/HOBt.
As preferably, DIC and HOBt mol ratio described in above-mentioned coupling agent is 1:1.In certain embodiments, the mol ratio of the described amino acid with Fmoc blocking group, DIC and HOBt is 1:1.2:1.2.
Further, described in step 4, coupling removes Fmoc step with also comprising before often walking coupling in the amino acid whose reaction of Fmoc blocking group successively.In certain embodiments, the reagent removing Fmoc described in is DBLK(piperidines: DMF=1:4).
Concrete; described in step 4, coupling is that the amino acid Fmoc-Phe-OH that described Fmoc protects first mixes with HOBt with the amino acid of Fmoc blocking group successively; dissolve afterwards and add DIC activation in organic solvent under ice bath, then with the Fmoc-Pro (4-OCO-NH-C removing Fmoc in advance and protect
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll(i.e. Pro (4-OCO-NH-C2H4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll) carry out linked reaction.Fmoc-Tyr (the Bzl)-OH according to said method coupling of Fmoc protection.
As preferably, the organic solvent of coupling successively described in above-mentioned steps 4 is DMF.
As preferably, described in above-mentioned steps 4, the condition of the linked reaction of coupling is successively room temperature reaction 2h.
Further, described in above-mentioned steps 4, the Fmoc that removes of coupling is successively the resin protected with organic solvent dissolution Fmoc, adds DBLK solution room temperature stirring reaction.Be more preferably, add DBLK solution room temperature stirring reaction 5min, drain, repeat to add DBLK solution once, stirring at room temperature reaction 7min.
Further, described in above-mentioned steps 4, linked reaction judges each linked reaction terminal with ninhydrin method detection successively.If resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.
Preparation method's step 4 of the present invention is obtaining NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc) remove the blocking group-All of C end after-Phg-D-Trp (Boc)-Lys (Resin)-OAll after cyclisation obtain SOM230 resin.
Wherein, the reagent removing the blocking group-All of C end described in step 4 is preferably Pd (PPh3) 4/ phenyl silane.
Further, Pd (PPh described in the above-mentioned reagent removing the blocking group-All of C end
3)
4be 1:100 with the mol ratio of phenyl silane.
Preferably, the cyclizing agent of cyclisation described in step 4 is the one in HBTU/HOBt/DIPEA, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.
Further, PyBOP(or HBTU or TBTU described in above-mentioned cyclizing agent), the mol ratio of HOBt and DIPEA is 1:1.2:2.Namely in certain embodiments, the mol ratio of PyBOP, HOBt and DIPEA is 1:1.2:2.In certain embodiments, the mol ratio of HBTU, HOBt and DIPEA is 1:1.2:2.In certain embodiments, the mol ratio of TBTU, HOBt and DIPEA is 1:1.2:2.
In certain embodiments, described NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-O, PyBOP/HBTU/TBTU, HOBt and DIPEA mol ratio be 1:1:1.2:2.
Concrete, removing cyclisation after the blocking group-All of C end described in step 4 is NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll linear peptides resin dissolves in organic solvent, add Pd (PPh subsequently
3)
4blocking group-the All removing C and hold is reacted with phenyl silane.Separately get one in PyBOP, HBTU or TBTU and HOBt to dissolve and add DIPEA activation, then with the above-mentioned resin (NH removing All and protect under ice bath in organic solvent
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-O) mix and carry out cyclization.
As preferably, the organic solvent removing All protection and cyclisation described in above-mentioned steps 4 is DCM, DMF or NMP.In certain embodiments, the organic solvent removing All protection is DCM, and the organic solvent of cyclisation is DMF.In certain embodiments, the organic solvent removing All protection is DCM, and the organic solvent of cyclisation is NMP.
As preferably, the condition removing the reaction of All protection described in above-mentioned steps 4 is NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll linear peptides resin first reacts 3 minutes with phenyl silane, then adds Pd (PPh
3)
4room temperature reaction 45 minutes.
As preferably, described in above-mentioned steps 4, the condition of cyclization is room temperature reaction 2h.
Further, cyclization described in above-mentioned steps 4 detects with ninhydrin method and judges reaction end.If resin water white transparency.
Further, cyclization described in above-mentioned steps 4 needs after terminating to carry out purifying to reaction solution, is separated and obtains SOM230 resin.Described purification process concrete grammar is that cyclization terminates rear organic solvent washing, and methyl alcohol shrinks drains and obtain SOM230 resin.As preferably, described organic solvent is DMF.
SOM230 pitch shake solution is obtained the thick peptide of SOM230 by preparation method's step 5 of the present invention.
Wherein, cracking agent described in step 5 is preferably the mixing solutions of TFA, TIS and water.Be more preferably TFA, TIS and water volume ratio is the mixing solutions of 95:5:5.
Further, the add-on of cracking agent described in step 5 is preferably pressed mL/g and is calculated, and the ratio of cracking agent and peptide resin is 10:1.Namely every 1g peptide resin adds the above-mentioned cracking agent of 10mL.
Further, described in step 5, crack reacting condition is preferably stirring at room temperature 2h.
In some embodiments, scission reaction described in above-mentioned steps 3 needs after terminating to carry out purifying to reaction solution, is separated and obtains the thick peptide of SOM230.Described purification process concrete grammar is that reaction solution sand core funnel filters, and collect filtrate, resin washs with a small amount of TFA again, concentrating under reduced pressure after merging filtrate, add freezing anhydrous diethyl ether precipitation, collecting precipitation anhydrous diethyl ether washs 3 times, and vacuum-drying obtains the thick peptide of SOM230.Wherein TFA washing can wash out, the product be adsorbed on resin of remnants to reduce the loss as far as possible.
The SOM230 purifying crude that cracking obtains by preparation method's step 6 of the present invention obtains SOM230 sterling, and wherein said purifying is preferably RPLC purifying.
RPLC, English name reversed phase high performance liquidchromatography, is called for short, RP-HPLC, the liquid chromatographic system be made up of non-polar stationary phase and polarity moving phase.It is just in time contrary with the liquid chromatographic system (normal-phase chromatography) be made up of polar stationary phase and low-pole moving phase.RP-HPLC is the topmost clastotype of current liquid chromatography, almost can be used for all organic separation and purification that can be dissolved in polarity or weak polar solvent.The SOM230 crude product that step 5 cracking obtains by preparation method of the present invention adopts reversed-phased high performace liquid chromatographic purifying to obtain SOM230 sterling.
Preferably, described reversed-phased high performace liquid chromatographic is specially: with anti-phase octadecylsilane or eight alkyl silane bonded silica gels for stationary phase, after the SOM230 crude product solution loading that cracking obtains, purify with 0.2%TFA/ acetonitrile mobile phase, collects object peak cut.
Further, after RPLC purifying, freeze-drying obtains SOM230.
In order to understand the present invention further, below in conjunction with embodiment, the present invention is described in detail.
The implication of the abbreviation used in specification sheets and claims is listed in the following table:
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| Boc | Tertbutyloxycarbonyl |
| NMP | N-Methyl pyrrolidone |
| DBLK | 20% hexahydropyridine/DMF solution |
| DIPEA | DIPEA |
| DMAP | 4-dimethylamino pyridine |
| TBTU | O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid |
| CTC Resin | 2-chlorine trityl chloride resin |
| Trt Resin | Trityl resin |
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| DIC | DIC |
| HOBt | I-hydroxybenzotriazole |
| HBTU | Benzotriazole-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester |
| PyBOP | Phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus |
| TBTU | O-benzotriazole-N, N, N, N-tetramethylurea (TMU) Tetrafluoroboric acid ester |
| DMF | DMF |
| DCM | Methylene dichloride |
| TFA | Trifluoroacetic acid |
| TIS | Tri isopropyl silane |
| Pd(PPh 3) 4 | Four (triphenyl) phosphine palladium |
Embodiment one: substitution degree is the preparation of Fmoc-Lys (the CTC)-OAll of 0.4mmol/g
Take the 2-CTC resin 100g(100mmol that substitution degree is 1.0mmol/g), join in solid state reaction post, 2 times are washed with DMF, after 30 minutes, 19.6g(48mmol is got with DMF swellable resins) Fmoc-Lys-OAll 200mL DMF dissolves, and adds 9.3g(72mmol under ice-water bath) after DIPEA activation, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 6.2g(48mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 81mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Lys (CTC)-OAll resin 115.7g, detection substitution degree is 0.409mmol/g.
Embodiment two: substitution degree is the preparation of Fmoc-Lys (the CTC)-OAll of 0.5mmol/g
Take the 2-CTC resin 100g(100mmol that substitution degree is 1.0mmol/g), join in solid state reaction post, 2 times are washed with DMF, after 30 minutes, 24.5g(60mmol is got with DMF swellable resins) Fmoc-Lys-OAll 200mL DMF dissolves, and adds 11.6g(90mmol under ice-water bath) after DIPEA activation, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 7.7g(60mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 81mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Lys (CTC)-OAll resin 119.8g, detection substitution degree is 0.512mmol/g.
Embodiment three: substitution degree is the preparation of Fmoc-Lys (the CTC)-OAll of 0.6mmol/g
Take the 2-CTC resin 100g(100mmol that substitution degree is 1.0mmol/g), join in solid state reaction post, 2 times are washed with DMF, after 30 minutes, 29.4g(72mmol is got with DMF swellable resins) Fmoc-Lys-OAll 200mL DMF dissolves, and adds 13.9g(108mmol under ice-water bath) after DIPEA activation, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 9.3g(72mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 81mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Lys (CTC)-OAll resin 126.5g, detection substitution degree is 0.603mmol/g.
Embodiment four: substitution degree is the preparation of Fmoc-Lys (the Trt)-OAll of 0.4mmol/g
Take the Trt resin 100g(100mmol that substitution degree is 1.0mmol/g), join in solid state reaction post, 2 times are washed with DMF, after 30 minutes, 19.6g(48mmol is got with DMF swellable resins) Fmoc-Lys-OAll 200mL DMF dissolves, and adds 9.3g(72mmol under ice-water bath) after DIPEA activation, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 6.2g(48mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 81mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Lys (Trt)-OAll resin 116.3g, detection substitution degree is 0.401mmol/g.
Embodiment five: substitution degree is the preparation of Fmoc-Lys (the Trt)-OAll of 0.5mmol/g
Take the Trt resin 100g(100mmol that substitution degree is 1.0mmol/g), join in solid state reaction post, 2 times are washed with DMF, after 30 minutes, 24.5g(60mmol is got with DMF swellable resins) Fmoc-Lys-OAll 200mL DMF dissolves, and adds 11.6g(90mmol under ice-water bath) after DIPEA activation, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 7.7g(60mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 81mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Lys (Trt)-OAll resin 118.6g, detection substitution degree is 0.504mmol/g.
Embodiment six: substitution degree is the preparation of Fmoc-Lys (the Trt)-OAll of 0.6mmol/g
Take the Trt resin 100g(100mmol that substitution degree is 1.0mmol/g), join in solid state reaction post, 2 times are washed with DMF, after 30 minutes, 29.4g(72mmol is got with DMF swellable resins) Fmoc-Lys-OAll 200mL DMF dissolves, and adds 13.9g(108mmol under ice-water bath) after DIPEA activation, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 9.3g(72mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 81mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol shrinks to be drained, and obtain Fmoc-Lys (Trt)-OAll resin 126.3g, detection substitution degree is 0.608mmol/g.
The preparation of embodiment seven: Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll
Fmoc-Lys (CTC)-OAll resin 97.7g(50mmol in Example two), wash 2 times with DMF, after 30 minutes, drain DMF with DMF swellable resins.Add DBLK(piperidines: DMF=1:4) solution is about 300mL for removing Fmoc, stirring at room temperature reaction 5min, drains, reaction repeated once, reaction 7min.Then 6 times are washed with DMF.Take 79.0g(150mmol) Fmoc-D-Trp (Boc)-OH, 24.3g(180mmol) HOBt, dissolve with 200mL DMF, under ice-water bath, add 22.7g(180mmol) DIC activation after, add room temperature reaction 2h in resin.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; coupling Fmoc-Phg-OH and Fmoc-Pro (4-OH)-OH successively; do not need to remove N after the complete Fmoc-Pro of coupling (4-OH)-OH and hold Fmoc, obtain Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll.
Embodiment eight: Pro (4-OCO-NH-C
2h
4-NH-Boc) preparation of-Phg-D-Trp (Boc)-Lys (CTC)-OAll
Get 36.0g(150mmol) Boc-NH-C
2h
4-NH-COOH, 24.3g(180mmol) HOBt, 1.8g(15mmol) DMAP, dissolve with 250mL DMF, under ice bath, add 22.7g(180mmol) DIC activation after, add in the resin of embodiment seven and react 12h.Drain reaction solution, wash 3 times with DMF.Add DBLK(piperidines: DMF=1:4) solution is about 300mL for removing Fmoc, stirring at room temperature reaction 5min, drains, reaction repeated once, reaction 7min.Then 6 times are washed with DMF.Namely Pro (4-OCO-NH-C is obtained
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll.
Embodiment nine: NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc) preparation of-Phg-D-Trp (Boc)-Lys (Resin)-OAll
Take 58.1(150mmol) Fmoc-Phe-OH, 24.3g(180mmol) HOBt, with 200mLDMF dissolve, add 22.7g(180mmol under ice-water bath) DIC activation after, add room temperature reaction 2h in the resin of embodiment eight.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, coupling Fmoc-Tyr (Bzl)-OH, removes Fmoc protecting group, and DMF washs 6 times, and methyl alcohol shrinks to be drained, and obtains NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll peptide resin.
The preparation of embodiment ten: Pasireotide peptide resin
Get NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll linear peptides resin (50mmol), after 30 minutes, drains swelling solution with DCM swellable resins.Rejoin 50mL methylene dichloride, then add 123.1mL phenyl silane (1000mmol), react 3 minutes, then add 7.0g Pd (PPh
3)
4(10mmol), room temperature reaction 45 minutes.
Take PyBOP26.0g(50mmol), HOBt8.1g(60mmol), dissolve with 200mL DMF, under ice-water bath, add 17.4mLDIPEA(100mmol) and activate 3 minutes, add reaction column and react 2 hours, detect with ninhydrin method and judge reaction end.Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Pasireotide peptide resin 155.6g.
The preparation of embodiment 11: Pasireotide peptide resin
Get NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll linear peptides resin (50mmol), after 30 minutes, drains swelling solution with DCM swellable resins.Rejoin 50mL methylene dichloride, then add 123.1mL phenyl silane (1000mmol), react 3 minutes, then add 7.0g Pd (PPh
3)
4(10mmol), room temperature reaction 45 minutes.
Take HBTU19.0g(50mmol), HOBt8.1g(60mmol), dissolve with 200mL NMP, under ice-water bath, add 17.4mLDIPEA(100mmol) and activate 3 minutes, add reaction column and react 2 hours, detect with ninhydrin method and judge reaction end.Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Pasireotide peptide resin 153.9g.
The preparation of embodiment 12: Pasireotide peptide resin
Get NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll linear peptides resin (50mmol), after 30 minutes, drains swelling solution with DCM swellable resins.Rejoin 50mL methylene dichloride, then add 123.1mL phenyl silane (1000mmol), react 3 minutes, then add 7.0g Pd (PPh
3)
4(10mmol), room temperature reaction 45 minutes.
Take TBTU16.1g(50mmol), HOBt8.1g(60mmol), dissolve with 200mL NMP, under ice-water bath, add 17.4mLDIPEA(100mmol) and activate 3 minutes, add reaction column and react 2 hours, detect with ninhydrin method and judge reaction end.Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Pasireotide peptide resin 156.3g.
The preparation of the thick peptide of embodiment 13: Pasireotide
In Example ten to ten two, the peptide resin of preparation is placed in scission reaction wherein, adds lytic reagent (TFA:TIS: water=90:5:5(V/V) with the ratio of 10mL/g resin), stirring at room temperature 2h.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide of Pasireotide, and weight yield is 95.1% ~ 99.6%.HPLC purity is greater than 50%.Single mixing is less than 5%.
Embodiment 14: adopt Trt resin-made for the thick peptide of Pasireotide
Fmoc-Lys (Trt)-OAll resin 99.2g(50mmol in Example five), wash 2 times with DMF, after 30 minutes, drain DMF with DMF swellable resins.Add DBLK(piperidines: DMF=1:4) solution is about 300mL for removing Fmoc, stirring at room temperature reaction 5min, drains, reaction repeated once, reaction 7min.Then 6 times are washed with DMF.
Take 79.0g(150mmol) Fmoc-D-Trp (Boc)-OH, 24.3g(180mmol) HOBt, dissolve with 200mL DMF, under ice-water bath, add 22.7g(180mmol) DIC activation after, add room temperature reaction 2h in resin.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, coupling Fmoc-Phg-OH and Fmoc-Pro (4-OH)-OH successively, do not need to remove N after the complete Fmoc-Pro of coupling (4-OH)-OH and hold Fmoc.Get 36.0g(150mmol) Boc-NH-C
2h
4-NH-COOH, 24.3g(180mmol) HOBt, 1.8g(15mmol) DMAP, dissolve with 250mL DMF, under ice bath, add 22.7g(180mmol) DIC activation after, add in the resin of embodiment seven and react 12h.Drain reaction solution, wash 3 times with DMF.Add DBLK(piperidines: DMF=1:4) solution is about 300mL for removing Fmoc, stirring at room temperature reaction 5min, drains, reaction repeated once, reaction 7min.Then 6 times are washed with DMF.Take 58.1(150mmol) Fmoc-Phe-OH, 24.3g(180mmol) HOBt, dissolve with 200mL DMF, under ice-water bath, add 22.7g(180mmol) DIC activation after, add room temperature reaction 2h in the resin of embodiment eight.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, coupling Fmoc-Tyr (Bzl)-OH, removes Fmoc protecting group, and DMF washs 6 times.Add 50mL methylene dichloride, then add 123.1mL phenyl silane (1000mmol), react 3 minutes, then add 7.0g Pd (PPh
3)
4(10mmol), room temperature reaction 45 minutes.Take PyBOP26.0g(50mmol), HOBt8.1g(60mmol), dissolve with 200mL DMF, under ice-water bath, add 17.4m LDIPEA(100mmol) and activate 3 minutes, add reaction column and react 2 hours, detect with ninhydrin method and judge reaction end.Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Pasireotide peptide resin 162.8g
Get peptide resin and be placed in scission reaction wherein, add lytic reagent (TFA:TIS: water=90:5:5(V/V) with the ratio of 10mL/g resin), stirring at room temperature 2h.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide of Pasireotide, and weight yield is 97.8%.HPLC purity 53.2%.Maximum list assorted 4.1%.
The preparation of embodiment 15: Pasireotide essence peptide
To take in embodiment ten three to ten four after any 30.0g Pasireotide thick peptide 1500mL water dissolution, adopt Waters2545RP-HPLC system, wavelength 230nm, chromatographic column is the anti-phase C18 post of 50 × 250mm, conventional 0.2%TFA/ acetonitrile mobile phase purifying, collect object peak cut, obtain purity and be greater than 99.0% smart peptide.Rotary evaporation concentrates, and freeze-drying obtains Pasireotide essence peptide 10.2g, and RP-HPLC purity is greater than 99.2%.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (10)
1. a preparation method for SOM230, is characterized in that, comprising:
Step 1: by Fmoc-Lys-OAll and solid phase carrier coupling, obtains Fmoc-Lys (Resin)-OAll;
Step 2:Fmoc-Lys (Resin)-OAll is coupling Fmoc-D-Trp (Boc)-OH, Fmoc-Phg-OH and Fmoc-Pro (4-OH)-OH one by one, obtains Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (CTC)-OAll;
Step 3:Fmoc-Pro (4-OH)-Phg-D-Trp (Boc)-Lys (Resin)-OAll and Boc-NH-C
2h
4-NH-COOH coupling obtains Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll;
Step 4:Fmoc-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (CTC)-OAll successively coupling Fmoc-Phe-OH and Fmoc-Tyr (Bzl)-OH obtain NH
2-Tyr (Bzl)-Phe-Pro (4-OCO-NH-C
2h
4-NH-Boc)-Phg-D-Trp (Boc)-Lys (Resin)-OAll, after removing the blocking group-All of C end, cyclisation obtains SOM230 resin;
Step 5: SOM230 pitch shake solution obtains the thick peptide of SOM230;
Step 6: the thick peptide purification of SOM230 obtains SOM230.
2. preparation method according to claim 1, it is characterized in that, solid phase carrier described in step 1 is Trt Resin or 2-CTC Resin.
3. preparation method according to claim 1, it is characterized in that, the substitution degree of Fmoc-Lys described in step 1 (Resin)-OAll is 0.4 ~ 0.6mmol/g.
4. preparation method according to claim 1, it is characterized in that, the coupling agent of coupling described in step 1 is diisopropylethylamine.
5. preparation method according to claim 1, it is characterized in that, the coupling agent of coupling described in step 2 is DIC/HOBt.
6. preparation method according to claim 1, is characterized in that, with Boc-NH-C described in step 3
2h
4the coupling agent of-NH-COOH coupling is DIC/HOBt/DMAP.
7. preparation method according to claim 1, it is characterized in that, the coupling agent of coupling successively described in step 4 is DIC/HOBt.
8. preparation method according to claim 1, is characterized in that, the reagent that step 4 removes the blocking group-All of C end is Pd (PPh3) 4/ phenyl silane.
9. preparation method according to claim 1, it is characterized in that, the cyclizing agent of cyclisation described in step 4 is the one in HBTU/HOBt/DIPEA, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.
10. preparation method according to claim 1, it is characterized in that, the cracking agent of cracking described in step 5 is the mixing solutions of TFA, TIS and water.
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| CN104447962B (en) * | 2014-12-29 | 2018-07-17 | 成都圣诺生物科技股份有限公司 | A method of synthesis parritide |
| WO2016207912A1 (en) * | 2015-06-22 | 2016-12-29 | Biophore India Pharmaceuticals Pvt. Ltd. | Novel process for the preparation of pasireotide |
| CN113461775A (en) * | 2021-08-23 | 2021-10-01 | 成都诺和晟泰生物科技有限公司 | Preparation method of polypeptide compound |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1446229A (en) * | 2000-08-01 | 2003-10-01 | 诺瓦提斯公司 | Somatostatin analogues |
| WO2005014624A2 (en) * | 2003-08-08 | 2005-02-17 | Novartis Ag | Preparation of somatostatin peptides |
| CN101883785A (en) * | 2007-12-03 | 2010-11-10 | 意大利法尔马科有限公司 | New non-selective somatostatin analogues |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1446229A (en) * | 2000-08-01 | 2003-10-01 | 诺瓦提斯公司 | Somatostatin analogues |
| WO2005014624A2 (en) * | 2003-08-08 | 2005-02-17 | Novartis Ag | Preparation of somatostatin peptides |
| CN1832962A (en) * | 2003-08-08 | 2006-09-13 | 诺瓦提斯公司 | Preparation of somatostatin peptides |
| CN101883785A (en) * | 2007-12-03 | 2010-11-10 | 意大利法尔马科有限公司 | New non-selective somatostatin analogues |
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