EP3765488A1 - Procédé de fabrication d'analogue de pthrp - Google Patents
Procédé de fabrication d'analogue de pthrpInfo
- Publication number
- EP3765488A1 EP3765488A1 EP19709065.7A EP19709065A EP3765488A1 EP 3765488 A1 EP3765488 A1 EP 3765488A1 EP 19709065 A EP19709065 A EP 19709065A EP 3765488 A1 EP3765488 A1 EP 3765488A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- leu
- lys
- arg
- fragment
- glu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims description 26
- 238000004519 manufacturing process Methods 0.000 title abstract description 5
- 101150071808 PTHLH gene Proteins 0.000 title 1
- 239000012634 fragment Substances 0.000 claims abstract description 132
- 108010038051 abaloparatide Proteins 0.000 claims abstract description 54
- BVISQZFBLRSESR-XSCWXTNMSA-N abaloparatide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NC(C)(C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](C)N)C(C)C)C1=CN=CN1 BVISQZFBLRSESR-XSCWXTNMSA-N 0.000 claims abstract description 53
- 229950001959 abaloparatide Drugs 0.000 claims abstract description 53
- 239000011347 resin Substances 0.000 claims description 133
- 229920005989 resin Polymers 0.000 claims description 133
- 238000005859 coupling reaction Methods 0.000 claims description 69
- 230000008878 coupling Effects 0.000 claims description 65
- 238000010168 coupling process Methods 0.000 claims description 65
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 54
- 108010033276 Peptide Fragments Proteins 0.000 claims description 53
- 102000007079 Peptide Fragments Human genes 0.000 claims description 53
- 125000006239 protecting group Chemical group 0.000 claims description 34
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 31
- 150000001413 amino acids Chemical class 0.000 claims description 30
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims description 25
- -1 (Benzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate Chemical compound 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 23
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 21
- LCFXLZAXGXOXAP-UHFFFAOYSA-N ethyl 2-cyano-2-hydroxyiminoacetate Chemical compound CCOC(=O)C(=NO)C#N LCFXLZAXGXOXAP-UHFFFAOYSA-N 0.000 claims description 17
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 12
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 6
- 210000004899 c-terminal region Anatomy 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 6
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- 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 claims description 4
- SNUSZUYTMHKCPM-UHFFFAOYSA-N 1-hydroxypyridin-2-one Chemical compound ON1C=CC=CC1=O SNUSZUYTMHKCPM-UHFFFAOYSA-N 0.000 claims description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 2
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims 1
- DJQVPXPEXAWGRE-UHFFFAOYSA-N 2,3-dihydrotriazolo[4,5-b]pyridin-7-one Chemical compound O=C1C=CN=C2NNN=C12 DJQVPXPEXAWGRE-UHFFFAOYSA-N 0.000 claims 1
- 239000000543 intermediate Substances 0.000 abstract description 9
- 108700020797 Parathyroid Hormone-Related Proteins 0.000 abstract description 8
- 102000043299 Parathyroid hormone-related Human genes 0.000 abstract description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 95
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 49
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 43
- 108090000765 processed proteins & peptides Proteins 0.000 description 34
- 230000015572 biosynthetic process Effects 0.000 description 30
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 24
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 24
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 24
- 238000003786 synthesis reaction Methods 0.000 description 24
- 238000011068 loading method Methods 0.000 description 23
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 238000010511 deprotection reaction Methods 0.000 description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- LCFXLZAXGXOXAP-DAXSKMNVSA-N ethyl (2z)-2-cyano-2-hydroxyiminoacetate Chemical compound CCOC(=O)C(=N/O)\C#N LCFXLZAXGXOXAP-DAXSKMNVSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 238000003776 cleavage reaction Methods 0.000 description 11
- 230000007017 scission Effects 0.000 description 11
- 239000000654 additive Substances 0.000 description 9
- 238000009833 condensation Methods 0.000 description 9
- 230000005494 condensation Effects 0.000 description 9
- 239000012535 impurity Substances 0.000 description 9
- 230000000996 additive effect Effects 0.000 description 8
- 125000003277 amino group Chemical group 0.000 description 8
- 238000010647 peptide synthesis reaction Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 description 7
- 230000008961 swelling Effects 0.000 description 7
- XXMYDXUIZKNHDT-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(N=C1)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXMYDXUIZKNHDT-QNGWXLTQSA-N 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 150000002148 esters Chemical class 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- WDGICUODAOGOMO-DHUJRADRSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino)pentanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)CC(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 WDGICUODAOGOMO-DHUJRADRSA-N 0.000 description 5
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 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 4
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 description 4
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 4
- KPFBUSLHFFWMAI-HYRPPVSQSA-N [(8r,9s,10r,13s,14s,17r)-17-acetyl-6-formyl-3-methoxy-10,13-dimethyl-1,2,7,8,9,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-17-yl] acetate Chemical compound C1C[C@@H]2[C@](CCC(OC)=C3)(C)C3=C(C=O)C[C@H]2[C@@H]2CC[C@](OC(C)=O)(C(C)=O)[C@]21C KPFBUSLHFFWMAI-HYRPPVSQSA-N 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 3
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 3
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 description 2
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 description 2
- QVHJQCGUWFKTSE-YFKPBYRVSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical class OC(=O)[C@H](C)NC(=O)OC(C)(C)C QVHJQCGUWFKTSE-YFKPBYRVSA-N 0.000 description 2
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 2
- PAMIQIKDUOTOBW-UHFFFAOYSA-N 1-methylpiperidine Chemical compound CN1CCCCC1 PAMIQIKDUOTOBW-UHFFFAOYSA-N 0.000 description 2
- HOZZVEPRYYCBTO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)-2-methylpropanoic acid Chemical compound C1=CC=C2C(COC(=O)NC(C)(C)C(O)=O)C3=CC=CC=C3C2=C1 HOZZVEPRYYCBTO-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- WNAHIZMDSQCWRP-UHFFFAOYSA-N dodecane-1-thiol Chemical compound CCCCCCCCCCCCS WNAHIZMDSQCWRP-UHFFFAOYSA-N 0.000 description 2
- 150000002431 hydrogen Chemical class 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 150000007530 organic bases Chemical class 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000007086 side reaction Methods 0.000 description 2
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- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 1
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 description 1
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 description 1
- ZOWOHMFPXMYFKJ-WBTWNKCNSA-N (4S)-4-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-aminopropanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-5-oxopentanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-3-carboxypropanoyl]amino]hexanoyl]amino]acetyl]amino]hexanoyl]amino]-3-hydroxypropanoyl]amino]-3-methylpentanoyl]amino]-5-oxopentanoyl]amino]-3-carboxypropanoyl]amino]-4-methylpentanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-methylpentanoyl]amino]propanoyl]amino]-5-[[(2S,3S)-1-[[(2S)-1-[[(2S,3R)-1-[[(1S)-1-carboxyethyl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-5-oxopentanoic acid Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](C)N)C(C)C)[C@@H](C)CC)C1=CN=CN1 ZOWOHMFPXMYFKJ-WBTWNKCNSA-N 0.000 description 1
- SJHPCNCNNSSLPL-CSKARUKUSA-N (4e)-4-(ethoxymethylidene)-2-phenyl-1,3-oxazol-5-one Chemical class O1C(=O)C(=C/OCC)\N=C1C1=CC=CC=C1 SJHPCNCNNSSLPL-CSKARUKUSA-N 0.000 description 1
- BYEAHWXPCBROCE-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoropropan-2-ol Chemical compound FC(F)(F)C(O)C(F)(F)F BYEAHWXPCBROCE-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- DFPYXQYWILNVAU-UHFFFAOYSA-N 1-hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1.C1=CC=C2N(O)N=NC2=C1 DFPYXQYWILNVAU-UHFFFAOYSA-N 0.000 description 1
- AVFZOVWCLRSYKC-UHFFFAOYSA-N 1-methylpyrrolidine Chemical compound CN1CCCC1 AVFZOVWCLRSYKC-UHFFFAOYSA-N 0.000 description 1
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 description 1
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 1
- JWUJQDFVADABEY-UHFFFAOYSA-N 2-methyltetrahydrofuran Chemical compound CC1CCCO1 JWUJQDFVADABEY-UHFFFAOYSA-N 0.000 description 1
- RRRCPCOJPQLWEP-UHFFFAOYSA-N 3-hydroxytriazolo[4,5-b]pyridine Chemical compound C1=CN=C2N(O)N=NC2=C1.C1=CN=C2N(O)N=NC2=C1 RRRCPCOJPQLWEP-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101001135770 Homo sapiens Parathyroid hormone Proteins 0.000 description 1
- 101001135995 Homo sapiens Probable peptidyl-tRNA hydrolase Proteins 0.000 description 1
- NFNVDJGXRFEYTK-YUMQZZPRSA-N Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O NFNVDJGXRFEYTK-YUMQZZPRSA-N 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 1
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 1
- AHVYPIQETPWLSZ-UHFFFAOYSA-N N-methyl-pyrrolidine Natural products CN1CC=CC1 AHVYPIQETPWLSZ-UHFFFAOYSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 102100036893 Parathyroid hormone Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000012317 TBTU Substances 0.000 description 1
- 239000003875 Wang resin Substances 0.000 description 1
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- AOGYCOYQMAVAFD-UHFFFAOYSA-N chlorocarbonic acid Chemical compound OC(Cl)=O AOGYCOYQMAVAFD-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 102000058004 human PTH Human genes 0.000 description 1
- 229910000040 hydrogen fluoride Inorganic materials 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229940126601 medicinal product Drugs 0.000 description 1
- COTNUBDHGSIOTA-UHFFFAOYSA-N meoh methanol Chemical compound OC.OC COTNUBDHGSIOTA-UHFFFAOYSA-N 0.000 description 1
- WOOWBQQQJXZGIE-UHFFFAOYSA-N n-ethyl-n-propan-2-ylpropan-2-amine Chemical compound CCN(C(C)C)C(C)C.CCN(C(C)C)C(C)C WOOWBQQQJXZGIE-UHFFFAOYSA-N 0.000 description 1
- SHDMMLFAFLZUEV-UHFFFAOYSA-N n-methyl-1,1-diphenylmethanamine Chemical compound C=1C=CC=CC=1C(NC)C1=CC=CC=C1 SHDMMLFAFLZUEV-UHFFFAOYSA-N 0.000 description 1
- 108010054971 parathyroid hormone-related protein (1-34) Proteins 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 150000003140 primary amides Chemical class 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- OGBMKVWORPGQRR-UMXFMPSGSA-N teriparatide Chemical class C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)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](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 OGBMKVWORPGQRR-UMXFMPSGSA-N 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 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
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 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
- 125000005500 uronium group Chemical group 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
Definitions
- the present invention relates to a manufacturing process for an analogue of parathyroid hormone related peptide (PTHrP), abaloparatide and its related intermediates.
- PTHrP parathyroid hormone related peptide
- Abaloparatide is an analog of human parathyroid hormone related peptide, PTHrP (1-34), represented by Formula (I):
- the medicinal product containing abaloparatide (marketed under the trade name TYMLOSTM), was approved by the U.S. Food and Drug Administration (FDA) in April 2017 for the treatment of postmenopausal women with osteoporosis at high risk for fracture.
- FDA U.S. Food and Drug Administration
- WO97/02834 describes the synthesis of human PTH (1-34) and PTHrP analogues using Solid Phase Peptide Synthesis (SPPS), which involves step-by-step addition of amino acids which are Boc-protected at the amino terminus.
- SPPS Solid Phase Peptide Synthesis
- This method of peptide synthesis requires repeated deprotection cycles with strong acids such as trifluoroacetic acid (TFA), for the removal of the Boc-protecting group at each addition step.
- TFA trifluoroacetic acid
- the process requires the use of Hydrogen Fluoride (HF) for final cleavage of the peptide from the resin.
- Hydrogen fluoride is a highly toxic and corrosive compound and its use is not recommended in large scale production of pharmaceutical compounds. As an overall consequence, these drawbacks result in an extremely difficult and environment unfriendly process.
- CN 106146648 discloses the synthesis of a truncated abaloparatide analogue (aa 1-33) by using a fragment-based approach consisting of the synthesis of three fragments (aa 1-15, aa 16-23 and aa 24-33) on different resins such as a chloro trltyl resin or a Rink amide resin, and condensation of these fragments using different coupling reagents.
- One of the possible reasons for the low yield as well as the low purity of this approach is the risk of racemization of amino acid residues during the condensation of fragments in solution phase.
- the product obtained that way needs to be purified further due to the presence of various impurities resulting in further yield loss.
- the C-terminal carboxylic group of the fragment has to be activated prior to coupling.
- the activation of C-terminal carboxylic groups of the fragments may however lead to both activated ester and oxazolone formation, which can be easily racemized in the basic conditions (N. L. Benoiton. Chemistry of Peptide Synthesis. Tailor and Francis Group. 2006). Therefore, the crude peptides obtained this way contain impurities corresponding to the isomerized product. Since the isomeric impurities have very similar retention times as the required target peptide, it is very difficult to separate these impurities from the desired target peptide. In summary, the activation step required for sufficient coupling of fragments often results in racemisation and hence the formation of impurities, which are extremely difficult to remove.
- the present invention provides an improved process for the preparation of the peptide abaloparatide which is an analogue of parathyroid hormone related peptide.
- the process of the present invention results in a product with high yield and high purity.
- the invention relates to the preparation of abaloparatide of formula I through a fragment-based convergent synthesis.
- the process for the preparation of abaloparatide comprises the steps of:
- fragment A is a fragment having an achiral amino acid at the C-terminal reactive site
- fragment B is a fragment, optionally bound to a resin at its C-terminal amino acid
- the fragments A and B are protected fragments.
- the invention relates to novel intermediates for the synthesis of abaloparatide wherein the intermediate may be fragment A or fragment B.
- Fragment A is selected from
- fragment B is selected from
- P is a side chain protecting group or hydrogen, independently for each amino acid
- PG is an alpha-amino protecting group or hydrogen
- R’ is a resin or a carboxy protecting group
- R is a resin or NH 2 .
- peptide fragment or“fragment” describe a peptide with a partial abaloparatide amino acid sequence, that can be optionally attached to a resin at its C-terminal amino acid, and that can be protected or unprotected.
- protected fragment or“protected peptide fragment” describe a fragment which bears either a terminal protecting group or side-chain protecting groups, or both of them.
- terminal protecting group refers to a protecting group for the alpha- amino group of an amino acid or of a peptide, or a peptide fragment.
- the type of the terminal protecting group used is depending on the type of resin used and on the protective groups required for side chain protection.
- the terminal protecting group preferably is tert-butyloxycarbonyl (Boc); and wherein fragment B is used, which preferably is linked to a Rink amide resin, the terminal protecting group preferably is 9-fluorenylmethyloxycarbonyl (Fmoc).
- side-chain protecting group is a protecting group for an amino acid side-chain functional group which is not removed when the terminal protecting group for an amino acid is removed.
- side-chain protecting groups are included to protect side chains of amino acids which are particularly reactive or labile, to avoid side reactions and/or branching of the growing molecule.
- acid-labile protecting groups such as tert- butyloxycarbonyl (Boc), tert-butyl (tBu), trityl (Trt), 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl (Pbf) etc.
- the criterion for selecting side-chain protecting groups is that the protecting group should generally be stable to the reagent under the reaction conditions selected for removing the terminal protecting group at each step of the synthesis and should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain.
- Resin is used to describe a solid support suitable to perform peptide synthesis.
- Resin in the present context may be selected from the group comprising Rink amide resin (RAM resin), Rink acid resin, Rink amide MB HA resin, trityl based resin, Sieber resin and Wang resin.
- Preferred resins are the 2-chloro trityl chloride resin (CTC resin), preferably for preparation of fragment A, and the Rink amide resin, preferably for preparation of fragment B and/or preparation of abaloparatide.
- the carboxylic group of a C-terminal amino acid can be attached to a resin.
- it can be in the form of a primary amide (when R is NH 2 ) or it can be protected with a carboxy protecting group such as an ester. In the latter cases, the coupling of these fragments is performed in Liquid Phase Peptide Synthesis.
- the ester is preferably an ester that is stable in basic conditions, i.e. orthogonal to the terminal protecting groups.
- the ester is tert-butyl ester, allyl ester or benzyl ester. More preferably, the ester is tert-butyl ester, which is removable in acidic conditions.
- the present invention in one aspect, relates to the preparation of abaloparatide, represented by formula I:
- Abaloparatide is prepared by fragment-based convergent synthesis, comprising coupling of peptide fragments.
- the process for the preparation of abaloparatide comprises the steps of:
- fragment A has an achiral amino acid at the C-terminal reactive site
- fragment B is optionally bound to a
- the peptide fragments in step a) are protected peptide fragments.
- the peptide fragment A and peptide fragment B can both be prepared by a fragment-based convergent approach starting from shorter peptides, both in SPPS or LPPS.
- peptide fragment A and peptide fragment B can also be prepared using step by step SPPS by coupling of amino acids according to the required sequence to the terminal amino acid residue attached to the resin using a coupling reagent and an activating agent.
- the resin is activated by the removal of the protecting group.
- the activated resin is coupled with the first amino acid wherein the amino acid is protected by a terminal protecting group and optionally a side-chain protecting group. Thereafter the following steps are repeated with different amino acids until the desired fragment, which is attached to the resin, is formed: i) removal of the terminal protecting group of amino acid
- the terminal protecting group may be removed by treatment with a base.
- the base may be an inorganic or organic base.
- the base is an organic base selected from the group consisting of piperidine, piperazine, DBU and diethylamine, preferably piperidine. Even more preferably, the terminal protecting group is removed with 20% piperidine in DMF.
- the coupling of amino acids takes place in the presence of a coupling reagent.
- the coupling reagent may be selected from the group consisting of N,N'-diisopropylcarbodiimide (DIC), N,N’-dicyclohexylcarbodiimide (DCC), (Benzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(7-Aza-lH-benzotriazole-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(lH-benzotriazole-l-yl)- l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N-(3-di mcthylam i nopropyl )-N'- ethylcarbodiimide (EDC
- the coupling reaction is further facilitated by the addition of an additive.
- the additive is selected from the group consisting of l-hydroxybenzotriazole (HOBt), 2-hydroxypyridine N-oxide, N- hydroxysuccinimide, l-hydroxy-7-azabenzotriazole, endo-N-hydroxy-5-norbornene-2,3- dicarboxamide and ethyl-2-cyano-2-hydroxyimino acetate (also known as OxymaPure).
- the reaction is carried out in the presence of ethyl-2-cyano-2-hydroxyiminoacetate.
- step iii) possible unreacted sites after resin loading and after each coupling are blocked (“capped”) to prevent any side reactions and the formation of truncated sequences.
- Capping is achieved by a short treatment of the peptide resin with a large excess of a highly reactive unhindered reagent, which is chosen according to the reactive site to be capped.
- a highly reactive unhindered reagent which is chosen according to the reactive site to be capped.
- the reagent is an acid derivative, such as an anhydride or a carboxyl chloride, in a basic medium.
- acetic anhydride, or benzoyl chloride, and a base, for instance diisopropylethylamine, are used.
- the reagent is an alcohol in a basic medium, such as methanol with diisopropylethylamine.
- a basic medium such as methanol with diisopropylethylamine.
- peptide fragments prepared as described above are optionally cleaved from the resin prior to step a) for the preparation of abaloparatide.
- step a) coupling of a protected peptide fragment A with a protected peptide fragment B takes place to obtain protected abaloparatide or a shorter protected fragment.
- Fragment A is selected from
- Fragment B is selected from
- P are a side-chain protecting groups or hydrogen, independently for each amino acid
- PG is an alpha-amino protecting group or hydrogen
- R’ is a resin or a carboxy protecting group
- R is a resin or NH 2 .
- the coupling of peptide fragments for the synthesis of abaloparatide or a shorter fragment thereof takes place in the presence of a coupling reagent.
- the coupling reagent is selected from the list of coupling reagents reported above for step ii). More preferably, the reaction is carried out in the presence of N,N'-diisopropylcarbodiimide (DIC) .
- DIC N,N'-diisopropylcarbodiimide
- the coupling reaction is further facilitated by the addition of an additive.
- the additive is selected from the list of additives reported above for step ii).
- the reaction is carried out in the presence of ethyl-2-cyano-2-hydroxyimino acetate.
- the coupling reaction may be carried out in the presence of a base, for instance a tertiary amine.
- a base for instance a tertiary amine.
- the base is selected from the group consisting of diisopropylethylamine, triethylamine, N-methylmorpholine and N-methylpiperidine; more preferably, the reaction is carried out in the presence of diisopropylethylamine.
- the coupling of fragments takes place in the presence of a solvent selected from the group consisting of dimethylformamide, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran, N-methylpyrrolidone and N- methylpyrrolidine.
- a solvent selected from the group consisting of dimethylformamide, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran, N-methylpyrrolidone and N- methylpyrrolidine.
- the coupling is carried out in DMF.
- the achiral amino acid at the C-terminal reactive site of fragment A may be a natural or an un natural amino acid.
- the achiral amino acid may be selected from glycine (Gly) and 2- aminoisobutyric acid (Aib).
- step b) abaloparatide or the shorter fragment thereof is deprotected and optionally cleaved from the resin by treatment with an acid; preferably with trifluoroacetic acid.
- an acid preferably with trifluoroacetic acid.
- the cleavage may be performed with a mixture TFA/water/TIPS/dodecanethiol (v/v 94/2.5/1/2.5).
- step c) abaloparatide or the shorter fragment thereof obtained from step b) may be optionally purified by crystallization or chromatographic techniques well known in the art.
- the coupling of peptide fragment A with fragment B, optionally bound to a resin is a promising alternative to step-by-step SPPS to produce the target peptides (i.e. abaloparatide or a shorter fragment thereof) because it reduces the formation rate of truncated sequences and unwanted isomeric forms, which are difficult to separate from the target peptide.
- peptide fragment A is PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH (Fragment 1, (1-12)); and
- R is a resin and P and PG are as defined above.
- the coupling of Fragment 1 with Fragment 2 for the synthesis of abaloparatide takes place in the presence of a coupling reagent and an additive as described above. More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate.
- Fragment 1 and Fragment 2 can be prepared by stepwise SPPS as explained above.
- the fragments can be prepared by a fragment-based convergent approach starting from shorter peptides, both in SPPS or LPPS.
- peptide Fragment 2 (13-34) is obtained by coupling of peptide Fragment 3
- R is a resin and P and PG are as defined above.
- the coupling of Fragment 3 with Fragment 5 for the synthesis of Fragment 2 takes place in the presence of a coupling reagent and an additive, as described above for step a). More preferably, the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino- acetate.
- the coupling being carried out in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate; and subsequent Fmoc deblocking.
- R is a resin and P and PG are as defined above.
- the coupling of Fragment 4 with Fragment 5 for the synthesis of abaloparatide takes place in the presence of a coupling reagent and an additive as described above. More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate.
- Fragment 4 and Fragment 5 can be prepared by stepwise SPPS as explained above.
- the fragments can be prepared by a convergent approach starting from shorter peptides, both in SPPS or LPPS.
- Fragment 4 (1-29) can be obtained by coupling of peptide Fragment 1
- R’, P and PG are as defined above.
- the coupling of Fragment 1 with Fragment 3’ for the synthesis of Fragment 4 takes place in the presence of a coupling reagent and an additive, as described above for step a). More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino- acetate.
- the obtained compound is optionally deprotected and/or cleaved from the resin by treatment with an acid; preferably with trifluoroacetic acid. Subsequently, it is further reacted in a coupling reaction with another fragment or it is purified.
- crude abaloparatide obtained by deprotection and cleavage from the resin is purified by using various methods known in the art.
- the purification is performed by preparative HPLC.
- a solution of the peptide is loaded into an HPLC column with a suitable solid phase, preferably Cl 8 or C8 modified silica, and a suitable aqueous mobile phase containing one or several organic solvents, preferably acetonitrile or methanol or mixtures thereof, is passed through the column. A gradient of the organic solvent is applied, if necessary.
- the peptide with desired purity is collected and optionally lyophilized.
- the peptide sequences of fragments A and B are partial sequences of abaloparatide, which are suitable for the coupling strategy that involves coupling via a site with an achiral amino acid at the C terminus of fragment A.
- Step 1 Preparation of Boc-Ala-Val-Ser(tBu)-Glu(tBu)-His(Trt)-Gln(Trt)-Leu-Leu-His(Trt) ⁇ Asp(tBu)-Lys(Boc)-Gly-OH
- Synthesis of the peptide fragment 1 was carried out by step-by-step SPPS using 2-chloro trltyl chloride resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Gly- OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DMF were added.
- the reaction mixture was stirred for 1 hour, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mL).
- the loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.8 mmol/g.
- the amino acids were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min.
- Fmoc-Lys(Boc)- OH, Fmoc-Asp(7Bu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc- Glu(/Bu)-OH, Fmoc-Ser(7Bu)-OH, Fmoc-Val-OH and Boc-Ala-OH derivatives were used. The unreacted amino groups were capped each cycle with the mixture AC 2 0/DIPEA/DMF (2x30 min).
- the Fmoc deprotection cycles were carried out by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml). After coupling with last Boc-protected amino acid, dry peptide resin was suspended in 4 ml of TFA/DCM 1:99 (v/v) solution and stirred for 15 min. Then the resin was filtered and the solution was neutralized by DIEA. The cleavage was repeated 3 times and the collected fractions were evaporated. The resulted oil was washed with water (3 x 1 ml) and dried in vacuo to obtain a powder that was used in the next step without further purification.
- Synthesis of the peptide fragment 2 was carried out by SPPS using Fmoc-protected Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc group was removed by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml).
- Dry peptide resin from step 3 was suspended in 4 ml of TFA/water/TIPS/dodecanethiol (v/v 94/2.5/1/2.5) and stirred for 4 h. Then the resin was filtered and washed with 2 ml of TFA. The organic solutions were collected and 15 ml of methyl /e/7-butyl ether were added. The solid residue was filtered, washed with methyl /e/7-butyl ether and dried to get crude abaloparatide with an HPLC crude purity of 81%.
- the crude abaloparatide as obtained in step 4 was dissolved in 1 ml of a mixture ACN/water (v/v 2/8) and purified by reversed phase preparative HPLC using C18 Jupiter (Phenomenex) column and water and acetonitrile containing 0.1% of TFA (Eluents A and B, respectively). The following gradient of acetonitrile was used: 10% of Eluent B in 15 min, then from 10% to 40% of Eluent B in 60 min. The fractions containing the product with more than 99% purity were collected and lyophilized to obtain 170 mg of abaloparatide (yield 65%).
- Synthesis of the peptide fragment 5 was carried out by SPPS using Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc protective group was removed by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml).
- Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu- OH, Fmoc-Lys(Boc)-OH were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) for 3 min and coupled to the resin in 60 min.
- the unreacted amino groups were capped each cycle with the mixture Ac 2 0/DIPEA/DCM (2x30 min).
- the Fmoc deprotection cycles were carried out as described above in order to finally obtain the title peptide fragment (coupled to the resin).
- Step 2 Preparation of Fmoc-Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)- Arg(Pbf)- Arg(Pbf)-Glu(tBu)-Leu-Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-OH (3)
- Synthesis of the peptide fragment 3 was carried out by step-by-step SPPS using 2-chloro trltyl chloride resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Aib- OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DMF were added. The reaction mixture was stirred for 1 hour, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mF).
- the loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.4 mmol/g.
- the amino acids were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min.
- Fmoc-Feu-OH, Fmoc-Fys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH protected amino acids were used. The unreacted amino groups were capped each cycle with the mixture AC 2 0/DIPEA/DCM (2x30 min).
- the Fmoc deprotection cycles were carried out by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml). After coupling with last Fmoc- protected amino acid, dry peptide resin was suspended in 4 ml of TFA/DCM 1:99 (v/v) solution and stirred for 15 min. Then the resin was filtered and the solution was neutralized by DIPEA. The cleavage was repeated 3 times and the collected fractions were evaporated. The resulted oil was washed with water (3 x lml) and dried in vacuo (yield 82%).
- Step 4 Purification of Abaloparatide.
- the crude peptide was purified as described in Example 1 to obtain 175 mg of Abaloparatide (Yield 72%).
- Synthesis of the peptide fragment 24-34 was carried out by SPPS using Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc protective group was removed by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml).
- the unreacted amino groups were capped using Ac 2 0/DIPEA/DMF mixture (2x30 min).
- the intermediate Fmoc deprotection cycles were carried out as described above in order to obtain side-chain protected FEKFFAibKFHTA-Rink resin.
- Synthesis of the peptide fragment 16-23 was carried out by SPPS using CTC resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Feu-OH and DIPEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DCM were added. The reaction mixture was stirred for 2 hours, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mF). The loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.6 mmol/g.
- the amino acids were pre-activated by DIC and OxymaPure (three fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min.
- Fmoc-Glu(7Bu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Feu-OH, Fmoc-Asp(7Bu)-OH, Fmoc-Gln(Trt)- OH derivatives were used. In case of Arg the couplings were repeated twice.
- Synthesis of the peptide fragment 1-15 was carried out by SPPS using CTC resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Ile-OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DCM were added. The reaction mixture was stirred for 2 hours, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mL). The loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.5 mmol/g.
- the amino acids were pre-activated by DIC and OxymaPure (three fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min.
- Step 4a Condensation of fragments (16-23) + (24-34) with HBTU/HOBt/DIPEA
- Fmoc deprotection was carried out by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml) to obtain side-chain protected QDLRRRELLEKLLAibKLHTA-Rink resin (fragment 16-34).
- Step 4b Condensation of fragments (16-23) + (24-34) with DIC/Oxyma
- Fmoc deprotection was carried out by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml) to obtain side- chain protected QDFRRREFFEKFFAibKFHTA-Rink resin (fragment 16-34).
- Step 5a Condensation of fragments (1-15) + (16-34) with PyBOP/HOBt/DIPEA
- the cleavage of the peptide was carried out with 2 ml of the mixture TFA / ethanedithiol / thiophenol / phenol / water (80:5:5:5:5 v/v/v/v/v) for 3 h.
- the resin was filtered off, the product was precipitated in 5 ml of diethyl ether, washed twice with diethyl ether and dried to obtain crude abaloparatide with an HPFC purity of 1.58%.
- Step 5b Condensation of fragments (1-15) + (16-34) with DIC/Oxyma.
- the cleavage of the peptide was carried out with 2 ml of the mixture TFA / ethanedithiol / thiophenol / phenol / water (80:5:5:5:5 v/v/v/v/v) for 3 h.
- the resin was filtered off, the product was precipitated in 5 ml of diethyl ether, washed twice with diethyl ether and dried obtain crude abaloparatide with an HPFC purity of 0.64%.
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WO2022093374A1 (fr) * | 2020-10-26 | 2022-05-05 | Purdue Research Foundation | Composés, compositions et méthodes d'utilisation pour le traitement de spondylodèses |
US11806387B1 (en) * | 2022-05-20 | 2023-11-07 | Radius Health, Inc. | Process of making abaloparatide |
CN116023467A (zh) * | 2022-12-28 | 2023-04-28 | 江苏诺泰澳赛诺生物制药股份有限公司 | 一种阿巴帕肽的固相合成方法 |
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