CA1105006A - Peptides - Google Patents
PeptidesInfo
- Publication number
- CA1105006A CA1105006A CA306,411A CA306411A CA1105006A CA 1105006 A CA1105006 A CA 1105006A CA 306411 A CA306411 A CA 306411A CA 1105006 A CA1105006 A CA 1105006A
- Authority
- CA
- Canada
- Prior art keywords
- peptide
- amino acid
- prepared
- formula
- residues
- 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.)
- Expired
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 76
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 19
- 150000001413 amino acids Chemical class 0.000 claims abstract description 55
- 150000003839 salts Chemical class 0.000 claims abstract description 26
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 20
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 18
- 230000007935 neutral effect Effects 0.000 claims abstract description 14
- 125000006239 protecting group Chemical group 0.000 claims abstract description 11
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 9
- 239000001257 hydrogen Substances 0.000 claims abstract description 9
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 7
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims abstract description 4
- 210000004899 c-terminal region Anatomy 0.000 claims abstract 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 74
- 239000000047 product Substances 0.000 claims description 74
- 238000000034 method Methods 0.000 claims description 59
- -1 hydroxy, amino Chemical group 0.000 claims description 48
- 230000008569 process Effects 0.000 claims description 38
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 29
- 239000002253 acid Substances 0.000 claims description 17
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 17
- 230000008878 coupling Effects 0.000 claims description 16
- 238000010168 coupling process Methods 0.000 claims description 16
- 238000005859 coupling reaction Methods 0.000 claims description 16
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 claims description 13
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 12
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 12
- 229960000583 acetic acid Drugs 0.000 claims description 9
- 238000009833 condensation Methods 0.000 claims description 9
- 230000005494 condensation Effects 0.000 claims description 9
- 150000002148 esters Chemical class 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 150000001408 amides Chemical class 0.000 claims description 7
- 238000010511 deprotection reaction Methods 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 125000002072 seryl group Chemical group 0.000 claims description 7
- 239000003054 catalyst Substances 0.000 claims description 6
- 229950003188 isovaleryl diethylamide Drugs 0.000 claims description 6
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 6
- 125000001288 lysyl group Chemical group 0.000 claims description 6
- 125000000405 phenylalanyl group Chemical group 0.000 claims description 6
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims description 6
- 125000002114 valyl group Chemical group 0.000 claims description 6
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 5
- 150000001540 azides Chemical class 0.000 claims description 5
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 5
- 229910000042 hydrogen bromide Inorganic materials 0.000 claims description 5
- 238000005984 hydrogenation reaction Methods 0.000 claims description 5
- AHLPHDHHMVZTML-BYPYZUCNSA-N ornithyl group Chemical group N[C@@H](CCCN)C(=O)O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 claims description 5
- 229910052763 palladium Inorganic materials 0.000 claims description 5
- 125000001980 alanyl group Chemical group 0.000 claims description 4
- 150000008064 anhydrides Chemical class 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 150000007513 acids Chemical class 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 150000001718 carbodiimides Chemical class 0.000 claims description 2
- 238000006482 condensation reaction Methods 0.000 claims description 2
- 239000012362 glacial acetic acid Substances 0.000 claims description 2
- 150000004820 halides Chemical class 0.000 claims description 2
- JBFYUZGYRGXSFL-UHFFFAOYSA-N imidazolide Chemical compound C1=C[N-]C=N1 JBFYUZGYRGXSFL-UHFFFAOYSA-N 0.000 claims description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims description 2
- DYUMLJSJISTVPV-UHFFFAOYSA-N phenyl propanoate Chemical compound CCC(=O)OC1=CC=CC=C1 DYUMLJSJISTVPV-UHFFFAOYSA-N 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 2
- 229940095064 tartrate Drugs 0.000 claims description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims 4
- 230000002194 synthesizing effect Effects 0.000 claims 2
- 150000001860 citric acid derivatives Chemical class 0.000 claims 1
- 239000012467 final product Substances 0.000 claims 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 21
- 238000000586 desensitisation Methods 0.000 abstract description 13
- 229960005486 vaccine Drugs 0.000 abstract description 7
- 206010020751 Hypersensitivity Diseases 0.000 abstract description 3
- 208000026935 allergic disease Diseases 0.000 abstract description 2
- 230000007062 hydrolysis Effects 0.000 abstract description 2
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 2
- 230000007815 allergy Effects 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 152
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 73
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 72
- 239000000243 solution Substances 0.000 description 42
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 36
- 235000001014 amino acid Nutrition 0.000 description 36
- 150000001875 compounds Chemical class 0.000 description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 34
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 32
- CKGCFBNYQJDIGS-LBPRGKRZSA-N (2s)-2-azaniumyl-6-(phenylmethoxycarbonylamino)hexanoate Chemical compound [O-]C(=O)[C@@H]([NH3+])CCCCNC(=O)OCC1=CC=CC=C1 CKGCFBNYQJDIGS-LBPRGKRZSA-N 0.000 description 27
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 241000700159 Rattus Species 0.000 description 21
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 21
- ONOURAAVVKGJNM-SCZZXKLOSA-N (2s,3r)-2-azaniumyl-3-phenylmethoxybutanoate Chemical compound [O-]C(=O)[C@@H]([NH3+])[C@@H](C)OCC1=CC=CC=C1 ONOURAAVVKGJNM-SCZZXKLOSA-N 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 20
- 229960001340 histamine Drugs 0.000 description 16
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 15
- 238000005481 NMR spectroscopy Methods 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 14
- 239000011541 reaction mixture Substances 0.000 description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000000872 buffer Substances 0.000 description 12
- KQSSATDQUYCRGS-UHFFFAOYSA-N methyl glycinate Chemical compound COC(=O)CN KQSSATDQUYCRGS-UHFFFAOYSA-N 0.000 description 11
- 239000007787 solid Substances 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 10
- 238000000354 decomposition reaction Methods 0.000 description 10
- 210000003630 histaminocyte Anatomy 0.000 description 10
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 8
- 210000003491 skin Anatomy 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical group NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 5
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 5
- 239000011651 chromium Substances 0.000 description 5
- 238000002425 crystallisation Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000001953 recrystallisation Methods 0.000 description 5
- RIQBRKVTFBWEDY-RHYQMDGZSA-N Arg-Lys-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RIQBRKVTFBWEDY-RHYQMDGZSA-N 0.000 description 4
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 4
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000013543 active substance Substances 0.000 description 4
- 108010081551 glycylphenylalanine Proteins 0.000 description 4
- 108010033706 glycylserine Proteins 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 150000004702 methyl esters Chemical class 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- FEUPVVCGQLNXNP-IRXDYDNUSA-N Gly-Phe-Phe Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FEUPVVCGQLNXNP-IRXDYDNUSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
- 235000019257 ammonium acetate Nutrition 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000010647 peptide synthesis reaction Methods 0.000 description 3
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 239000010414 supernatant solution Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000006188 syrup Substances 0.000 description 3
- 235000020357 syrup Nutrition 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- FHIAJWBDZVHLAH-YUMQZZPRSA-N Lys-Gly-Ser Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FHIAJWBDZVHLAH-YUMQZZPRSA-N 0.000 description 2
- YKBSXQFZWFXFIB-VOAKCMCISA-N Lys-Thr-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCCCN)C(O)=O YKBSXQFZWFXFIB-VOAKCMCISA-N 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 241001504519 Papio ursinus Species 0.000 description 2
- GKZIWHRNKRBEOH-HOTGVXAUSA-N Phe-Phe Chemical compound C([C@H]([NH3+])C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)C1=CC=CC=C1 GKZIWHRNKRBEOH-HOTGVXAUSA-N 0.000 description 2
- GRVMHFCZUIYNKQ-UFYCRDLUSA-N Phe-Phe-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O GRVMHFCZUIYNKQ-UFYCRDLUSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 125000000649 benzylidene group Chemical group [H]C(=[*])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000003610 charcoal Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229910052804 chromium Inorganic materials 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000003792 electrolyte Substances 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 230000020477 pH reduction Effects 0.000 description 2
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
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- 239000000377 silicon dioxide Substances 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000010626 work up procedure Methods 0.000 description 2
- NHUCANAMPJGMQL-ZDUSSCGKSA-N (2,5-dioxopyrrolidin-1-yl) (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoate Chemical compound C([C@H](NC(=O)OC(C)(C)C)C(=O)ON1C(CCC1=O)=O)C1=CC=CC=C1 NHUCANAMPJGMQL-ZDUSSCGKSA-N 0.000 description 1
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 1
- KPYXMALABCDPGN-HYOZMBHHSA-N (4s)-5-[[(2s)-6-amino-1-[[(2s,3s)-1-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2r)-1-[[2-[[2-[[(1s)-3-amino-1-carboxy-3-oxopropyl]amino]-2-oxoethyl]amino]-2-oxoethyl]amino]-1-oxo-3-sulfanylpropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]a Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN)CC1=CC=C(O)C=C1 KPYXMALABCDPGN-HYOZMBHHSA-N 0.000 description 1
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 description 1
- 101100281516 Caenorhabditis elegans fox-1 gene Proteins 0.000 description 1
- 101100385401 Caenorhabditis elegans kin-10 gene Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 241000518994 Conta Species 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 101800000268 Leader protease Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001024304 Mino Species 0.000 description 1
- 241000353097 Molva molva Species 0.000 description 1
- 101100270435 Mus musculus Arhgef12 gene Proteins 0.000 description 1
- 241001474977 Palla Species 0.000 description 1
- 241000282520 Papio Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- QSKCKTUQPICLSO-AVGNSLFASA-N Pro-Arg-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O QSKCKTUQPICLSO-AVGNSLFASA-N 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 206010040914 Skin reaction Diseases 0.000 description 1
- 101100323865 Xenopus laevis arg1 gene Proteins 0.000 description 1
- 159000000021 acetate salts Chemical class 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- YBCVMFKXIKNREZ-UHFFFAOYSA-N acoh acetic acid Chemical compound CC(O)=O.CC(O)=O YBCVMFKXIKNREZ-UHFFFAOYSA-N 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 238000011685 brown norway rat Methods 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 210000005178 buccal mucosa Anatomy 0.000 description 1
- 125000004744 butyloxycarbonyl group Chemical group 0.000 description 1
- 125000004063 butyryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 125000002668 chloroacetyl group Chemical group ClCC(=O)* 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 229960000265 cromoglicic acid Drugs 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- VLARUOGDXDTHEH-UHFFFAOYSA-L disodium cromoglycate Chemical compound [Na+].[Na+].O1C(C([O-])=O)=CC(=O)C2=C1C=CC=C2OCC(O)COC1=CC=CC2=C1C(=O)C=C(C([O-])=O)O2 VLARUOGDXDTHEH-UHFFFAOYSA-L 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- LHWWETDBWVTKJO-UHFFFAOYSA-N et3n triethylamine Chemical compound CCN(CC)CC.CCN(CC)CC LHWWETDBWVTKJO-UHFFFAOYSA-N 0.000 description 1
- 235000019439 ethyl acetate Nutrition 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 1
- 125000000717 hydrazino group Chemical group [H]N([*])N([H])[H] 0.000 description 1
- 150000002431 hydrogen Chemical group 0.000 description 1
- 238000007327 hydrogenolysis reaction Methods 0.000 description 1
- COQRGFWWJBEXRC-UHFFFAOYSA-N hydron;methyl 2-aminoacetate;chloride Chemical compound Cl.COC(=O)CN COQRGFWWJBEXRC-UHFFFAOYSA-N 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002218 isotachophoresis Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- COTNUBDHGSIOTA-UHFFFAOYSA-N meoh methanol Chemical compound OC.OC COTNUBDHGSIOTA-UHFFFAOYSA-N 0.000 description 1
- UKVIEHSSVKSQBA-UHFFFAOYSA-N methane;palladium Chemical compound C.[Pd] UKVIEHSSVKSQBA-UHFFFAOYSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- CMWTZPSULFXXJA-VIFPVBQESA-N naproxen Chemical group C1=C([C@H](C)C(O)=O)C=CC2=CC(OC)=CC=C21 CMWTZPSULFXXJA-VIFPVBQESA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 125000006503 p-nitrobenzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1[N+]([O-])=O)C([H])([H])* 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000012736 patent blue V Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 108010084572 phenylalanyl-valine Proteins 0.000 description 1
- 125000000612 phthaloyl group Chemical group C(C=1C(C(=O)*)=CC=CC1)(=O)* 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 231100000489 sensitizer Toxicity 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 231100000430 skin reaction Toxicity 0.000 description 1
- 230000035483 skin reaction Effects 0.000 description 1
- PXLIDIMHPNPGMH-UHFFFAOYSA-N sodium chromate Chemical compound [Na+].[Na+].[O-][Cr]([O-])(=O)=O PXLIDIMHPNPGMH-UHFFFAOYSA-N 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-N sulfonic acid Chemical compound OS(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-N 0.000 description 1
- GEVPIWPYWJZSPR-UHFFFAOYSA-N tcpo Chemical compound ClC1=CC(Cl)=CC(Cl)=C1OC(=O)C(=O)OC1=C(Cl)C=C(Cl)C=C1Cl GEVPIWPYWJZSPR-UHFFFAOYSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- IOGXOCVLYRDXLW-UHFFFAOYSA-N tert-butyl nitrite Chemical compound CC(C)(C)ON=O IOGXOCVLYRDXLW-UHFFFAOYSA-N 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
- C07K5/06052—Val-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
- C07K5/06069—Ser-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/32—Modification to prevent enzymatic degradation
Abstract
ABSTRACT
Peptides(6-12 amino acids) and their salts, useful in allergy desensitisation compositions, particularly vaccines, have the formula where R is optional and, if present, is a group resistant to enzyme hydrolysis; R1 is a basic amino acid residue optionally linked to basic and/or neutral nonhydrophobic amino acid residues; R2 is one or more neutral nonhydrophobic amino acid residues; R3 is one or more hydrophobic amino acid residues optionally linked to neutral nonhydrophobic amino acid residues; X is hydrogen or an N-protecting group; and Y is hydroxyl or a C-terminal protecting group. Examples are:
Peptides(6-12 amino acids) and their salts, useful in allergy desensitisation compositions, particularly vaccines, have the formula where R is optional and, if present, is a group resistant to enzyme hydrolysis; R1 is a basic amino acid residue optionally linked to basic and/or neutral nonhydrophobic amino acid residues; R2 is one or more neutral nonhydrophobic amino acid residues; R3 is one or more hydrophobic amino acid residues optionally linked to neutral nonhydrophobic amino acid residues; X is hydrogen or an N-protecting group; and Y is hydroxyl or a C-terminal protecting group. Examples are:
Description
"Peptidesl This invention relates to certain peptides useful for desensitisation therapy, and to desensitisation compositions containing them.
It is well known that many substances are capable of releasing histamine from mast cells. Such release is often due to disruption of the cell membrane, and reagents behaving in this way are said to be non-selective. One of the physiological systems causing release of histamine involves attachment of an antibody to the cell surface.
When the antibody reacts wit~ its appropriate antigen, a process is initiated leading to release o~ histamine. It has been~postulated that such antigen-antibody reaction causes distortion of the antibody molecule, so that a portion of it is brought into contact with the cell sur~ace, thereby inltiating the response. Release of histamine brought about in this fashion does not lnvolve rupture o~
the cell m~mbrane, and is said to be selective (see D. R. S~anworth, "Immedia~e Hypersensitivity", Chapter 8 North Holland Publishing Company London, 1973).
Experimentally, non-selective and selective release of histamine can ba distinguished respectively by the presence or absence either o~ simultaneously released intracellular enæymes, or o~ radloactive chromium previously absorbed by _ I r . - . .: -:, . . :
- - - .: ~. . :- : : . . .
., , , ~ . , . . . . ~ , .. , - . .
w 2 the cell.
We now believe that substances capable of causing selective release o~ histamine may be used in desensitisation therapy of allergic humans.
An object of this invention is to provide a novel class of peptides having this property, and desensitisation compositions, particularly vaccines containing such peptides.
Accordingly the present invention provides a peptide of formula (I).
X - R ~ Rl ~ R2 R3 (I) and salts thereof, consisting of 6 to 12 naturally occurring amino acid residues, wherein R is an optionally present group, capable of confering on a peptide resistance to enzyme breakdown; Rl represents a residue of a basic amino acid, optionally linked to one or more residues of neutral non-hydrophobic amino acids and/or basic amino acids; R2 represents a residue of a neutral non-hydrophobic amino acid, optionally linked to one or more further residues of neutral non-hydrophobic amino acids;
R3 represent.s a residue o a hydrophobic amino acid, optionally linked to one or more residues of neutral non-hydrophobic amino acids and/or hydrophobic amino acids;
X is hydrogen, or a N - pxotecting group; and Y is hydroxyl, or a C - terminal protecting group.
Unless otherwise stated, the amino acids referred to hereafter are in the L- configuration.
When R is present, it is a group capable of confering on a peptide resistance to enzyme breakdown. Examples of suitable groups R are given in ~. Rudinger,"The Design of Peptide Hormone Analogues",Chapter 9 in Drug Design, VoIum2 (II) edited by E. J. Ariens, Academic Press, New York and London, 1971.
Thus suitable examples of R, when present, include prolyl, hydroxyprolyl, the D- ~orm of a common amino acid xesidue, or an amino acid resldue with omission of the terminal amino group.
- .
' ` "~' ~ ' ~' ' Suitable examples of Rl include arginyl, lysyl and oxnithyl and combinations of such residues, optionally with residue(s) of neutral non-hydrophobic amino acids such as threonyl and seryl. Parti~ularly suitable examples of R
include Lys-Thr-Lys, Arg-Lys-Thr Lys and the like.
Normally Rl will consist of 1 to 5 amino acid residues, suitably 3 to 5 residues. Rl will often contain at least two basic amino acid residues and at least one neutral non-hydrophohic amino acid residue.
Suitable examples of neutral non-hydrophobic amino acids R2 include glycyl, alanyl, seryl and threonyl and combinations of such residues. A particularly suitable example of R2 is Gly-Ser-Gly. Preferably R2 consists of 1 to 5 amino acid residues, for example 3 amino acid residues.
Suitable examples of hydrophobic amino acids R3 include residues of amino acids notionally derived from alanine ~-substituted by an aromatic or aliphatic hydxophobic group, such as phenylalanyl~ valyl and leucyl;
and combinations of such residues. Particularly sui~able examples of R3 include Phe-Phe and Phe-Phe-Val-Phe.
Preferably R3 consists of 1 to 4 amino acid residues, for example 2 or 4 residues.
X is hydrogen or a N-protecting group. Suitable exampl~s o~ N-protecting groups X include those conventionally known for this use in peptide chemistry.
Examples of such groups include carboxylic acid groups such as acetyl, chloroacetyl, trifluoroacetyl, butyryl, benzoyl, phenylacetyl, pyridine-carbonyl; or an acid group derived from carbonic acid such as ethoxycarhonyl, benzyloxy-carbonyl, t-butyLoxycarbonyl, biphenylisopropoxycarbonyl, p-methoxy-benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycaxbonyl, p-phenylazoben2yloxycarbonyl, p-(p'-methoxyphenylazo)~benzyloxycarbonyl, t amyloxycarbonyl;
or an acid yroup derived ~rom a sulphonic or p-toluene-sulphonic acid; or other groups such as benzyl, trityl,formyl, phthaloyl, o-nitrophenylsulphenyl, benzylidene or nitro. Preferred N-protecting groups X include t-butyloxy-carbonyl or benzyloxycarbonyl.
.
- ~ : - .
-.
, . - . .
$~i Suitable C- terminal protecting groups Y include ester residues, ~or example residues of Cl 6 alkyl esters such as methoxy, ethoxy and t-buto~y; benzyloxy, ~-nitrobenzyloxy, ~-methoxybenzyloxy; residues of trimethylsilyl esters; and residues of amides, substituted amides (e.g. amides substituted by one or two Cl 6 alkyl groups, or by a Cl 6 acyl group), and hydrazino residues. Preferred groups Y
include hydroxyl and methoxy.
The peptides of the invention have 6 to 12 amino acid residues. Preferably they have 8 to 10 amino acid residues.
One particularly suitable group of peptides is o~
fonmula (II):
X - R - [b-c~d-e~ - [f-g~h] ~ k-l~ - Y (II) wherein X, Y and R are as defined; c and e are lysyl, arginyl or ornithyl; d is threonyL or seryl; b is an optionally present arginyli lysyl or ornithyl; f and h are glycyl or alanyl; g is seryl or threonyl; i and j are phenylalanyl t valyl or leucyl; and k and 1 are optionally present phenylalanyl, valyl or leucyl; and salts thereo.
Preferably in foxmula (II) X is hydrogen and Y is hydroxyl,-NM2 or Cl 4 alkoxy such as methoxy, and,when R is present/ it is prolyl or hydroxyprolyl.
Examples of pep~ides within the scope of the invention are:-Lys Thr hys Gly Ser Gly Phe Phe _ yl Arg Lys Thr Lys Gly Ser Gly Phe Phe ~ Y
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe _ yl Arg Lys Thr ~ys Gly Ser Gly Phe Phe Val Phe ~ yl Pro Arg Lys Thr ~ys Gly Ser Gly Phe Phe _ yl Rro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe _ yl wherein yl is hydroxyl~-N~2 or methoxy.
The peptides o~ this invention may be prepared by methods known in khe art o~ pepkide synthesis comprising coupling the amino acids from which the peptide i~ derived se~uentially to build up khe desired peptide.
:, -' ' ` ' :
.
M~thods of sequential coupling o~ amino acids to formpeptides by forming amide links are well known in the art.
In general the amino acids, provided with protecting groups where necessary, are coupled in the correct order, or 5 smalle~ peptides are combined into larger units. The amide linkage is usually prepared by condensing an amino acid, or peptide, having a protected ~-amino group and a free o.r activated terminal carboxyl group, with an amino acid or peptide with a protected carboxyl group and a free ~-amino group.
Activation of the carboxyl group can be e~fected, for example, by converting the carboxyl group into an acid halide, an azide/ anhydride or imidazolide, or into an activated ester such as the cyanomethyl ester, p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentachlorophenyl est~r, ~-hydroxysuccinimide ester, benztriazole ester.
The most widely used methods of condensation of amino acids or peptldes include the carbodiimide method, the azide method, the anhydride method, and the activated esters method, as described, for example, by Schroder and Lubke in "The Peptides", Volume 1 (1969), (Academic Press).
An alternative method is the solid phase method of Merrifield (J. Am. Chem. Soc., 85, 2149 (1963)).
Any reactive groups in the amino acid or peptide which are not to take part in the condensation reaction should be protected by any of the N-protecting groups or carboxyl protecting groups described above which can be readily removed a~ter the condensation.
. The removal oE the protecting group(s) present in the resultant peptide may be e~fected by an appropriate proceduxe depending upon the kind~s) of the protective group(s). Some t~pical procedures are as follows:
hydrogenation in the presence of palla~ium catalyst (e.g~
palladium carbon, palladium black) for benzyloxycarbonyl, p~nitrobenzyloxycarbonyl, p~bromo-benzyloxycarbonyl, p-phenylazobenzylox~carbonyl, p-(p'-methoxyphenylazo)-benzyloxycarbonyl and trityl groups protecting the amino end; treatment with hydrogen bromide in glacial acetic acid . . .. . .. .. ~ . . . ~
- . , .... . ..
: ,.
- . ': : .: ' :
- . .~ . : .
. - : . ' ' -.:
~;r for benzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-phenyl-azoben~yloxycaxbonyl and t~butyloxycarbonyl groups protecting the amino encl; treatment with metallic sodium in liquid ammonia for benzyloxycarbonyl, p-bromobenzyloxy~
carbonyl and tosyl groups protecting the amino end;
treatment with hydrochloric acid and/or acetic acid for trityl, t-butyloxycarbon~l, formyl and benzylidene groups protecting the amino end; treatment with alkali for methyl, ethyl and benzyl esters protecting the carboxyl end, treatment with acid for methyl, ethyl, benzyl, p-methoxy-benzyl and t-butyl esters protecting the carboxyl end; and hydrogenation in the presence of palladium catalyst for benzyl and p-nitrobenzyl esters protecting the carboxyl end.
Acid addition salts of compounds of formula (I) are included within this invention, for example the salts of pharmaceutically acceptable acids as a hydrohalide, especially the hydrochloride or hydrobromide; or the phosphate, acetate, phenylpropionate, maleate, tartrate and citrate.
The peptides and salts of the present invention may be employed as the active agents in desensitisation vaccines.
Such vaccines are well known to thos~ skilled in the art and comprise a sterile liquid vehicle in which the active agent is dissolved or suspended. If suspended, the particles of active agent should be small enough not to block the orifice of an injection needle. Certain adjuvants such as tyrosine are often included in such vaccine compositions and are believed to provide a support and prolonged slow release of active material in vivo.
Usually a patient receiving treatment with such desensitisation vaccines is administered a number of injection~, spread over a period of weeks or days, each injection conta.ining a higher concentration of active agent than the preceding one. In th~s way the patient is desensitis~d such that his allergic reaction to allergens is reduced or eliminated.
An alternative mode of administration for desensitisation agents is by application to the nasal ~ .
~ ?~
mucosa as a liquid spray or as a dry powder snuff.
Yet another possible route of administration would be by application to the buccal mucosa, again as a li~uid or dry composition.
Accordingly, the present invention includes a pharmaceutical composition adapted for use in desensitisation therapy, comprising a peptide or pharmaceutically acceptable salt of formula (I) together with a pharmaceutically acceptable carrier suitable for parenteral, intra-nasal or buccal administration.
A preferred composition of the invention is a desensiti~ation vaccine.
The compositions o~ the invention may be administered in conventional manner for desensitisation therapy.
The preparation and properties o~ some of the peptides o~ this invention are illustrated by the following examples.
Peptides were synthesised by classical methods o~
peptide synthesis described in the literature of peptide chemistry, for example by means of classical solution synthesis or solid phase peptide synthesis (SPPS), or by use of a combination of these methods.
Where appropriate amino acids refer to the L-con~iguration unless otherwise stated, and the following 25 abbreviations are used:
Boc T~rt-Butyloxycarbonyl BAW Butanol:Acetic Acid:
Bzl Benzyl water Z Benzyloxyca~bonyl CECL3 Chlorofornt ~ (Icarbobenzoxy) MES ~orpholine Ethy].
30 OTcP 2,4,5 l'richlorQphenyl Sulphonic ~cid ester EtAc ~thyl Acetate DMF . Dimethylformamide MeOH Methanol Et3N Triethylamine EtOh Ethanol DCCX Dicyclohexylcarbodiimide -OMe Methyl ester 35 THF Tetxahydro~uxan AcOH Acetic Acid OSu N hydroxysuccinimide HCL Hydrochloric ~cid ester TLC Thin Layer OBz Benzyl ester Chromatography M.D.C. Methylene dichloride M.P~ Melting Point :: . : ' . . . ' : ' . : . . .
.. . .
..
.. . . : :
' ' r~
R~ Ratio of product distance: NMR Nuclear Magnetic solvent front distance, Resonance from point of application T~Fo~ Trifl.uoroacetic acid C Nun~er of grams per lOOmls r ~ p.Tsa p-roluensulphonate [a~ Specific rotation at 25C
using sodium light (the D R-T- Room Temperature line) . . .
- ---: , - ;~, :, ' .
~, , , ; , : . , ~3~jt~
_ 9 _ EX~UPI,E 1: ~he prepar_tion of L~ysThrLy~sGlySerGlxPhePheOMe The octapeptide methyl ester was prepared by a 4+4 fragment condensation strategy, one fragment (I) being prepared by solid phase peptide synthesis (SPPS) 5 (according to SPPS Manual by J. M~ Stewart and J. D. Young Freeman and Company San Francisco 1969) and the other fragment (II) by classical solution synthesis. Combination of I and II gave fully protected octapeptide (III) which on deprotection a~forded the desired product (V).
(I) BOC Lys(Z)Thr(Bzl)Lys(Z)Gly N2H3 This intermediate was prepared by SPPS, employing standard DCCI mediated coupling procedures using a 0.47mM/g glycine substituted Merrlfield Resin. The fully protected tetrapeptide-resin was cleaved by treatment with 100 equivalents of hydrazine hydrate in DMF at room temperature ~or 3 days. Standard work-up gave I in good yield. This was orystallised from EtOH/water and then EtAc; m.pO 132-134C; TLC homogeneous in 9:1 CHC13:
MeOH/I2 stain with Rf 0.44; NMR consistent with structure;
~a]D = -1.3 (C = 1, DMF); amino acid anaIysis required: 1.00 Thr: 1.00 Gly: 2.00 Lys.
ound: 1.00 Thr: 1.05 Gly: 2~01 Lys.
(II) Ser(Bzl)Gly~hePheOMe HCl The tetrapeptide methyl ester hydrochloride was prepared by solution ~ynthesis in ~ix stages.
(i) BOC-Phe.Phe.OMe:
BOC-Phe-OSu (5.25g, 0.0145M) was coupled to Phe.OMe.
HC~ (3.13g,0.0145M) in DMF (25ml) in the presence of 1 equivalent of Et3N (2.03ml) at room temperature o~er 3 days.
The reaction mixture was poured into water (250ml) and the product extraated into EtAc (lOOm~). It was isolated in ~1% (5.00g~ yield and crystallisation from petrol (b.pt.
80-100C) gave a m.p. of 123-124C; ~a3~5= -11.0 (C = 1, DMF).
- - : . .
.- ................. .. , ~ , , ,' , - -, . . ~ . . :
~ii) Phe.PheOOme.HCl:
.
The intermediate (i) (4.65g) was BOC~deprotected using a solution of 2N HCL in EtAc (30ml) over 2 hours at room temperature. The product precipitated from solution in 78~ yield (3.10g) and had m~p. 205C;[~]25_ 43.3 (C = 1, AcOH).
(iii)BOC.Gly.Phe~Phe.OMa:
BOC.Gly.OSu ~2.18g,0.008M) was coupled to (ii)(2.90g, 0.008M) in DMF in the presence of 1 equivalent of Et3N
(1.12ml) at room temperature over 3 days. Similar work-up described for isolation of (i), gave the product in 67%
yield (2.60g~; m.p O 159-161C, after crystallisation from EtAc (40ml) [~]D5= ~9 5 ~C = 1, DMF); amino acid analysis:
required: 1.00 Gly: 2~00 Phe.
found: l.oO Gly: 2.02 Phe.
(iv) GlyphepheoMe~Hcl:
The intermediate (iii) (2~60g) was BOC-deprotected in~
a similar manner to that described for (ii). The product deposited as an oil which was triturated with ether to give a white crystalline solid in almost quantitative yield.
The material was purified further on Sephadex L~2 ~column eluting with water and had m.p. 196-199C; TLC in 9:1 CHC13:
~eOH showed one spot with I2 stain at Rf. 0.22. Amino acid analysis-required: 1.00 Gly: 2.00 Phe.
found: 1.00 Gly: 1.94 Phe.
(v~ BOC.S r_Bzl)GlyPhePheOM~:
BOC.Ser~Bzl)OH ~1.66g, 0.0056M) was coupled to (iv) (2.36g, 0.0056M) in MDC (20ml) at 0C using DCCI
(1~16g, 0.0056M) and Et3N (0.79 ml: 1 equivalent). The reaction mixture was stirred at oC for ~ hour, room temperature for 2 hours, filtered and filtrate evaporated in vacuo. Crystallisation of the residue from EtAc/petrol ~80-100C) afforded a 67~ yield ~2.50g) of product, m.p.
163-167C. TLC in 9:1 CHC13: MeOH ~I2 stain) showed rR~
. ~ ~
.
product at Rf 0.68; [a~D = -13.0 (C - 1, DMF). The NMR
spectrum was consistent with structure. Amino acid analysis:
required: 1.00 Ser: 1.00 Gly: 2.00 Me.
found: 0.94 Ser: 1.12 Gly: 2.00 Phe.
(vi) Ser(Bzl)GlyPhePheOMe.HCl (II):
Intexmediate (v) (1.75g) above was BOC-deprotected in a similar manner to that described for (ii). Addition of ether to the reactio~ mixture gave product as a solid in 96% yield (1.52g), [~ D ~ 21.0 (C - 1, AcOH). It was puri~ied on Sephade LH20 eluting with lM AcOHI []D =21.0 (C = 1, AcOH) TLC examination in 9:1 CHC13: MeOH (I2 stain) showed product (acetate salt) as one spot at Rf 0.27. The NMR spectrum was consistent with structure. Amino acid analysis:
re~uired: 1.00 Ser: 1.00 Gly: 2.00 Phe.
found: 1.00 Ser: 1.11 Gly: 1.92 Phe.
(III)BOC.L~s(Z)Thr(Bzl)Lys(Z)GlySer(Bzl)GlyPhePheOMe Tertiary-butyl nitrite (0.32ml,0.00266M) was added with vigorous stirxing to a solution of (I) (1.60g, 0.00177M) in DMF (30ml) containing 60 equivalents 2N HCL in THF
(5.5ml/ O.OOllM) at -20C. After 30 minutes, (II) (1.05g, 0~00177M) in DMF (Sml) with sufficient Et3N (~.llml) present to neutralise all HCl present, was added, and the reaction mixtuxe stirred for 18 hours at 4C, filtered and filtrate concentrated in vacuo. Addition of cold water gave product which was obtained in 50% yield (1.28g) after crystallisation from EtOH. TLC in 9:1 CHC13:MeOH (I2 s~ain) was homogeneous and showed product at R~ 0.6. M.p. 202-203C; ~~D5= ~5 9 (C = 1, DMF). r~he NMR spectrum was consistent with structure. Amino acid analysis:
required: 2.00 Lys: 2.00 Phe: 2.00 Gly: 1.00 Thr: 1.00 Ser.
found: 2~00 Lys: 1.94 Phe: 2.06 Gly: 0.99 Ser: 0.96 Thr.
(IV) ~ys(Z)Thr(Bzl~Lys(Z)GlySer(Bzl)G~Ph _heOMe.HCl Fully pxotected octapeptide (II) (1.20g) was BOC-deprotected in 2N HCl solution in a 6:14 DME/EtAc solvent ~'Ta~D~
-, ~
.
. . .. . . . . .
.
' ' .: . ,. .. : '' . ~. . : , . :
- 12 ~
mixture (20ml). Prolonged reaction time o 4 hours was used at room temperatuxe and addition of ether deposited product.
Recrystallisation from MeOH/ether gave a 52~ yield (0.60g) of product. TLC in 9:1 CHC13: MeOH (I2 stain) showed one major spot at Rf 0.4; [a]D = 10.1 (C = 1, AcOH). ~mino acid analysis:
required: 1.00 Thr: 1.00 Ssr: 2.00 Gly: 2.00 Phe: 2.00 Lys.
found: 1.00 Thr: 1.00 Ser: 2~17 Gly: 2.19 Phe: 2.32 Lys.
(V) LysThrLysGlySerGlyPhePheOMe Partially protected octapeptide (IV) (O.lOg) was hydrogenated in 85% AcOH (70ml) with 10% Pd/C catalys~
(0.20g) over a steady stream of hydrogen for 20 hours. The mixture was filtered, evaporated in vacuo and residue filtered on Sephadex LH2 ~ eluting with water to give the desired octapeptide methyl ester (V) ~0.03g, 46~ yield).
TLC examination showed 1 spot at Rf 0.2 in 5:2:2 BAW
(t--BuOCl/KI-starch stain) and Rf 0.5 in 5:2:3 BAW
(Ninhydrin stain). Amino acid analysis:
re~uired: 1.00 Ser: 1.00 Thr: 2~00 Gly: 2.00 Phe: 2.00 Lys.
found: 1.00 Ser: 1.03 Thr: 2.02 Gly: 2.06 Phe: 1.98 Lys.
Isotachophoretic examination showed one band in ~95% amount (leading electrolyte lOmM KOH + MES pH 6.0 and terminating electrolyte lOmM ~-alanine and HCl pH 4.23). The NMR 80 HzFT spectrum was consistent with structur~.
It is well known that many substances are capable of releasing histamine from mast cells. Such release is often due to disruption of the cell membrane, and reagents behaving in this way are said to be non-selective. One of the physiological systems causing release of histamine involves attachment of an antibody to the cell surface.
When the antibody reacts wit~ its appropriate antigen, a process is initiated leading to release o~ histamine. It has been~postulated that such antigen-antibody reaction causes distortion of the antibody molecule, so that a portion of it is brought into contact with the cell sur~ace, thereby inltiating the response. Release of histamine brought about in this fashion does not lnvolve rupture o~
the cell m~mbrane, and is said to be selective (see D. R. S~anworth, "Immedia~e Hypersensitivity", Chapter 8 North Holland Publishing Company London, 1973).
Experimentally, non-selective and selective release of histamine can ba distinguished respectively by the presence or absence either o~ simultaneously released intracellular enæymes, or o~ radloactive chromium previously absorbed by _ I r . - . .: -:, . . :
- - - .: ~. . :- : : . . .
., , , ~ . , . . . . ~ , .. , - . .
w 2 the cell.
We now believe that substances capable of causing selective release o~ histamine may be used in desensitisation therapy of allergic humans.
An object of this invention is to provide a novel class of peptides having this property, and desensitisation compositions, particularly vaccines containing such peptides.
Accordingly the present invention provides a peptide of formula (I).
X - R ~ Rl ~ R2 R3 (I) and salts thereof, consisting of 6 to 12 naturally occurring amino acid residues, wherein R is an optionally present group, capable of confering on a peptide resistance to enzyme breakdown; Rl represents a residue of a basic amino acid, optionally linked to one or more residues of neutral non-hydrophobic amino acids and/or basic amino acids; R2 represents a residue of a neutral non-hydrophobic amino acid, optionally linked to one or more further residues of neutral non-hydrophobic amino acids;
R3 represent.s a residue o a hydrophobic amino acid, optionally linked to one or more residues of neutral non-hydrophobic amino acids and/or hydrophobic amino acids;
X is hydrogen, or a N - pxotecting group; and Y is hydroxyl, or a C - terminal protecting group.
Unless otherwise stated, the amino acids referred to hereafter are in the L- configuration.
When R is present, it is a group capable of confering on a peptide resistance to enzyme breakdown. Examples of suitable groups R are given in ~. Rudinger,"The Design of Peptide Hormone Analogues",Chapter 9 in Drug Design, VoIum2 (II) edited by E. J. Ariens, Academic Press, New York and London, 1971.
Thus suitable examples of R, when present, include prolyl, hydroxyprolyl, the D- ~orm of a common amino acid xesidue, or an amino acid resldue with omission of the terminal amino group.
- .
' ` "~' ~ ' ~' ' Suitable examples of Rl include arginyl, lysyl and oxnithyl and combinations of such residues, optionally with residue(s) of neutral non-hydrophobic amino acids such as threonyl and seryl. Parti~ularly suitable examples of R
include Lys-Thr-Lys, Arg-Lys-Thr Lys and the like.
Normally Rl will consist of 1 to 5 amino acid residues, suitably 3 to 5 residues. Rl will often contain at least two basic amino acid residues and at least one neutral non-hydrophohic amino acid residue.
Suitable examples of neutral non-hydrophobic amino acids R2 include glycyl, alanyl, seryl and threonyl and combinations of such residues. A particularly suitable example of R2 is Gly-Ser-Gly. Preferably R2 consists of 1 to 5 amino acid residues, for example 3 amino acid residues.
Suitable examples of hydrophobic amino acids R3 include residues of amino acids notionally derived from alanine ~-substituted by an aromatic or aliphatic hydxophobic group, such as phenylalanyl~ valyl and leucyl;
and combinations of such residues. Particularly sui~able examples of R3 include Phe-Phe and Phe-Phe-Val-Phe.
Preferably R3 consists of 1 to 4 amino acid residues, for example 2 or 4 residues.
X is hydrogen or a N-protecting group. Suitable exampl~s o~ N-protecting groups X include those conventionally known for this use in peptide chemistry.
Examples of such groups include carboxylic acid groups such as acetyl, chloroacetyl, trifluoroacetyl, butyryl, benzoyl, phenylacetyl, pyridine-carbonyl; or an acid group derived from carbonic acid such as ethoxycarhonyl, benzyloxy-carbonyl, t-butyLoxycarbonyl, biphenylisopropoxycarbonyl, p-methoxy-benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycaxbonyl, p-phenylazoben2yloxycarbonyl, p-(p'-methoxyphenylazo)~benzyloxycarbonyl, t amyloxycarbonyl;
or an acid yroup derived ~rom a sulphonic or p-toluene-sulphonic acid; or other groups such as benzyl, trityl,formyl, phthaloyl, o-nitrophenylsulphenyl, benzylidene or nitro. Preferred N-protecting groups X include t-butyloxy-carbonyl or benzyloxycarbonyl.
.
- ~ : - .
-.
, . - . .
$~i Suitable C- terminal protecting groups Y include ester residues, ~or example residues of Cl 6 alkyl esters such as methoxy, ethoxy and t-buto~y; benzyloxy, ~-nitrobenzyloxy, ~-methoxybenzyloxy; residues of trimethylsilyl esters; and residues of amides, substituted amides (e.g. amides substituted by one or two Cl 6 alkyl groups, or by a Cl 6 acyl group), and hydrazino residues. Preferred groups Y
include hydroxyl and methoxy.
The peptides of the invention have 6 to 12 amino acid residues. Preferably they have 8 to 10 amino acid residues.
One particularly suitable group of peptides is o~
fonmula (II):
X - R - [b-c~d-e~ - [f-g~h] ~ k-l~ - Y (II) wherein X, Y and R are as defined; c and e are lysyl, arginyl or ornithyl; d is threonyL or seryl; b is an optionally present arginyli lysyl or ornithyl; f and h are glycyl or alanyl; g is seryl or threonyl; i and j are phenylalanyl t valyl or leucyl; and k and 1 are optionally present phenylalanyl, valyl or leucyl; and salts thereo.
Preferably in foxmula (II) X is hydrogen and Y is hydroxyl,-NM2 or Cl 4 alkoxy such as methoxy, and,when R is present/ it is prolyl or hydroxyprolyl.
Examples of pep~ides within the scope of the invention are:-Lys Thr hys Gly Ser Gly Phe Phe _ yl Arg Lys Thr Lys Gly Ser Gly Phe Phe ~ Y
Lys Thr Lys Gly Ser Gly Phe Phe Val Phe _ yl Arg Lys Thr ~ys Gly Ser Gly Phe Phe Val Phe ~ yl Pro Arg Lys Thr ~ys Gly Ser Gly Phe Phe _ yl Rro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe _ yl wherein yl is hydroxyl~-N~2 or methoxy.
The peptides o~ this invention may be prepared by methods known in khe art o~ pepkide synthesis comprising coupling the amino acids from which the peptide i~ derived se~uentially to build up khe desired peptide.
:, -' ' ` ' :
.
M~thods of sequential coupling o~ amino acids to formpeptides by forming amide links are well known in the art.
In general the amino acids, provided with protecting groups where necessary, are coupled in the correct order, or 5 smalle~ peptides are combined into larger units. The amide linkage is usually prepared by condensing an amino acid, or peptide, having a protected ~-amino group and a free o.r activated terminal carboxyl group, with an amino acid or peptide with a protected carboxyl group and a free ~-amino group.
Activation of the carboxyl group can be e~fected, for example, by converting the carboxyl group into an acid halide, an azide/ anhydride or imidazolide, or into an activated ester such as the cyanomethyl ester, p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentachlorophenyl est~r, ~-hydroxysuccinimide ester, benztriazole ester.
The most widely used methods of condensation of amino acids or peptldes include the carbodiimide method, the azide method, the anhydride method, and the activated esters method, as described, for example, by Schroder and Lubke in "The Peptides", Volume 1 (1969), (Academic Press).
An alternative method is the solid phase method of Merrifield (J. Am. Chem. Soc., 85, 2149 (1963)).
Any reactive groups in the amino acid or peptide which are not to take part in the condensation reaction should be protected by any of the N-protecting groups or carboxyl protecting groups described above which can be readily removed a~ter the condensation.
. The removal oE the protecting group(s) present in the resultant peptide may be e~fected by an appropriate proceduxe depending upon the kind~s) of the protective group(s). Some t~pical procedures are as follows:
hydrogenation in the presence of palla~ium catalyst (e.g~
palladium carbon, palladium black) for benzyloxycarbonyl, p~nitrobenzyloxycarbonyl, p~bromo-benzyloxycarbonyl, p-phenylazobenzylox~carbonyl, p-(p'-methoxyphenylazo)-benzyloxycarbonyl and trityl groups protecting the amino end; treatment with hydrogen bromide in glacial acetic acid . . .. . .. .. ~ . . . ~
- . , .... . ..
: ,.
- . ': : .: ' :
- . .~ . : .
. - : . ' ' -.:
~;r for benzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-phenyl-azoben~yloxycaxbonyl and t~butyloxycarbonyl groups protecting the amino encl; treatment with metallic sodium in liquid ammonia for benzyloxycarbonyl, p-bromobenzyloxy~
carbonyl and tosyl groups protecting the amino end;
treatment with hydrochloric acid and/or acetic acid for trityl, t-butyloxycarbon~l, formyl and benzylidene groups protecting the amino end; treatment with alkali for methyl, ethyl and benzyl esters protecting the carboxyl end, treatment with acid for methyl, ethyl, benzyl, p-methoxy-benzyl and t-butyl esters protecting the carboxyl end; and hydrogenation in the presence of palladium catalyst for benzyl and p-nitrobenzyl esters protecting the carboxyl end.
Acid addition salts of compounds of formula (I) are included within this invention, for example the salts of pharmaceutically acceptable acids as a hydrohalide, especially the hydrochloride or hydrobromide; or the phosphate, acetate, phenylpropionate, maleate, tartrate and citrate.
The peptides and salts of the present invention may be employed as the active agents in desensitisation vaccines.
Such vaccines are well known to thos~ skilled in the art and comprise a sterile liquid vehicle in which the active agent is dissolved or suspended. If suspended, the particles of active agent should be small enough not to block the orifice of an injection needle. Certain adjuvants such as tyrosine are often included in such vaccine compositions and are believed to provide a support and prolonged slow release of active material in vivo.
Usually a patient receiving treatment with such desensitisation vaccines is administered a number of injection~, spread over a period of weeks or days, each injection conta.ining a higher concentration of active agent than the preceding one. In th~s way the patient is desensitis~d such that his allergic reaction to allergens is reduced or eliminated.
An alternative mode of administration for desensitisation agents is by application to the nasal ~ .
~ ?~
mucosa as a liquid spray or as a dry powder snuff.
Yet another possible route of administration would be by application to the buccal mucosa, again as a li~uid or dry composition.
Accordingly, the present invention includes a pharmaceutical composition adapted for use in desensitisation therapy, comprising a peptide or pharmaceutically acceptable salt of formula (I) together with a pharmaceutically acceptable carrier suitable for parenteral, intra-nasal or buccal administration.
A preferred composition of the invention is a desensiti~ation vaccine.
The compositions o~ the invention may be administered in conventional manner for desensitisation therapy.
The preparation and properties o~ some of the peptides o~ this invention are illustrated by the following examples.
Peptides were synthesised by classical methods o~
peptide synthesis described in the literature of peptide chemistry, for example by means of classical solution synthesis or solid phase peptide synthesis (SPPS), or by use of a combination of these methods.
Where appropriate amino acids refer to the L-con~iguration unless otherwise stated, and the following 25 abbreviations are used:
Boc T~rt-Butyloxycarbonyl BAW Butanol:Acetic Acid:
Bzl Benzyl water Z Benzyloxyca~bonyl CECL3 Chlorofornt ~ (Icarbobenzoxy) MES ~orpholine Ethy].
30 OTcP 2,4,5 l'richlorQphenyl Sulphonic ~cid ester EtAc ~thyl Acetate DMF . Dimethylformamide MeOH Methanol Et3N Triethylamine EtOh Ethanol DCCX Dicyclohexylcarbodiimide -OMe Methyl ester 35 THF Tetxahydro~uxan AcOH Acetic Acid OSu N hydroxysuccinimide HCL Hydrochloric ~cid ester TLC Thin Layer OBz Benzyl ester Chromatography M.D.C. Methylene dichloride M.P~ Melting Point :: . : ' . . . ' : ' . : . . .
.. . .
..
.. . . : :
' ' r~
R~ Ratio of product distance: NMR Nuclear Magnetic solvent front distance, Resonance from point of application T~Fo~ Trifl.uoroacetic acid C Nun~er of grams per lOOmls r ~ p.Tsa p-roluensulphonate [a~ Specific rotation at 25C
using sodium light (the D R-T- Room Temperature line) . . .
- ---: , - ;~, :, ' .
~, , , ; , : . , ~3~jt~
_ 9 _ EX~UPI,E 1: ~he prepar_tion of L~ysThrLy~sGlySerGlxPhePheOMe The octapeptide methyl ester was prepared by a 4+4 fragment condensation strategy, one fragment (I) being prepared by solid phase peptide synthesis (SPPS) 5 (according to SPPS Manual by J. M~ Stewart and J. D. Young Freeman and Company San Francisco 1969) and the other fragment (II) by classical solution synthesis. Combination of I and II gave fully protected octapeptide (III) which on deprotection a~forded the desired product (V).
(I) BOC Lys(Z)Thr(Bzl)Lys(Z)Gly N2H3 This intermediate was prepared by SPPS, employing standard DCCI mediated coupling procedures using a 0.47mM/g glycine substituted Merrlfield Resin. The fully protected tetrapeptide-resin was cleaved by treatment with 100 equivalents of hydrazine hydrate in DMF at room temperature ~or 3 days. Standard work-up gave I in good yield. This was orystallised from EtOH/water and then EtAc; m.pO 132-134C; TLC homogeneous in 9:1 CHC13:
MeOH/I2 stain with Rf 0.44; NMR consistent with structure;
~a]D = -1.3 (C = 1, DMF); amino acid anaIysis required: 1.00 Thr: 1.00 Gly: 2.00 Lys.
ound: 1.00 Thr: 1.05 Gly: 2~01 Lys.
(II) Ser(Bzl)Gly~hePheOMe HCl The tetrapeptide methyl ester hydrochloride was prepared by solution ~ynthesis in ~ix stages.
(i) BOC-Phe.Phe.OMe:
BOC-Phe-OSu (5.25g, 0.0145M) was coupled to Phe.OMe.
HC~ (3.13g,0.0145M) in DMF (25ml) in the presence of 1 equivalent of Et3N (2.03ml) at room temperature o~er 3 days.
The reaction mixture was poured into water (250ml) and the product extraated into EtAc (lOOm~). It was isolated in ~1% (5.00g~ yield and crystallisation from petrol (b.pt.
80-100C) gave a m.p. of 123-124C; ~a3~5= -11.0 (C = 1, DMF).
- - : . .
.- ................. .. , ~ , , ,' , - -, . . ~ . . :
~ii) Phe.PheOOme.HCl:
.
The intermediate (i) (4.65g) was BOC~deprotected using a solution of 2N HCL in EtAc (30ml) over 2 hours at room temperature. The product precipitated from solution in 78~ yield (3.10g) and had m~p. 205C;[~]25_ 43.3 (C = 1, AcOH).
(iii)BOC.Gly.Phe~Phe.OMa:
BOC.Gly.OSu ~2.18g,0.008M) was coupled to (ii)(2.90g, 0.008M) in DMF in the presence of 1 equivalent of Et3N
(1.12ml) at room temperature over 3 days. Similar work-up described for isolation of (i), gave the product in 67%
yield (2.60g~; m.p O 159-161C, after crystallisation from EtAc (40ml) [~]D5= ~9 5 ~C = 1, DMF); amino acid analysis:
required: 1.00 Gly: 2~00 Phe.
found: l.oO Gly: 2.02 Phe.
(iv) GlyphepheoMe~Hcl:
The intermediate (iii) (2~60g) was BOC-deprotected in~
a similar manner to that described for (ii). The product deposited as an oil which was triturated with ether to give a white crystalline solid in almost quantitative yield.
The material was purified further on Sephadex L~2 ~column eluting with water and had m.p. 196-199C; TLC in 9:1 CHC13:
~eOH showed one spot with I2 stain at Rf. 0.22. Amino acid analysis-required: 1.00 Gly: 2.00 Phe.
found: 1.00 Gly: 1.94 Phe.
(v~ BOC.S r_Bzl)GlyPhePheOM~:
BOC.Ser~Bzl)OH ~1.66g, 0.0056M) was coupled to (iv) (2.36g, 0.0056M) in MDC (20ml) at 0C using DCCI
(1~16g, 0.0056M) and Et3N (0.79 ml: 1 equivalent). The reaction mixture was stirred at oC for ~ hour, room temperature for 2 hours, filtered and filtrate evaporated in vacuo. Crystallisation of the residue from EtAc/petrol ~80-100C) afforded a 67~ yield ~2.50g) of product, m.p.
163-167C. TLC in 9:1 CHC13: MeOH ~I2 stain) showed rR~
. ~ ~
.
product at Rf 0.68; [a~D = -13.0 (C - 1, DMF). The NMR
spectrum was consistent with structure. Amino acid analysis:
required: 1.00 Ser: 1.00 Gly: 2.00 Me.
found: 0.94 Ser: 1.12 Gly: 2.00 Phe.
(vi) Ser(Bzl)GlyPhePheOMe.HCl (II):
Intexmediate (v) (1.75g) above was BOC-deprotected in a similar manner to that described for (ii). Addition of ether to the reactio~ mixture gave product as a solid in 96% yield (1.52g), [~ D ~ 21.0 (C - 1, AcOH). It was puri~ied on Sephade LH20 eluting with lM AcOHI []D =21.0 (C = 1, AcOH) TLC examination in 9:1 CHC13: MeOH (I2 stain) showed product (acetate salt) as one spot at Rf 0.27. The NMR spectrum was consistent with structure. Amino acid analysis:
re~uired: 1.00 Ser: 1.00 Gly: 2.00 Phe.
found: 1.00 Ser: 1.11 Gly: 1.92 Phe.
(III)BOC.L~s(Z)Thr(Bzl)Lys(Z)GlySer(Bzl)GlyPhePheOMe Tertiary-butyl nitrite (0.32ml,0.00266M) was added with vigorous stirxing to a solution of (I) (1.60g, 0.00177M) in DMF (30ml) containing 60 equivalents 2N HCL in THF
(5.5ml/ O.OOllM) at -20C. After 30 minutes, (II) (1.05g, 0~00177M) in DMF (Sml) with sufficient Et3N (~.llml) present to neutralise all HCl present, was added, and the reaction mixtuxe stirred for 18 hours at 4C, filtered and filtrate concentrated in vacuo. Addition of cold water gave product which was obtained in 50% yield (1.28g) after crystallisation from EtOH. TLC in 9:1 CHC13:MeOH (I2 s~ain) was homogeneous and showed product at R~ 0.6. M.p. 202-203C; ~~D5= ~5 9 (C = 1, DMF). r~he NMR spectrum was consistent with structure. Amino acid analysis:
required: 2.00 Lys: 2.00 Phe: 2.00 Gly: 1.00 Thr: 1.00 Ser.
found: 2~00 Lys: 1.94 Phe: 2.06 Gly: 0.99 Ser: 0.96 Thr.
(IV) ~ys(Z)Thr(Bzl~Lys(Z)GlySer(Bzl)G~Ph _heOMe.HCl Fully pxotected octapeptide (II) (1.20g) was BOC-deprotected in 2N HCl solution in a 6:14 DME/EtAc solvent ~'Ta~D~
-, ~
.
. . .. . . . . .
.
' ' .: . ,. .. : '' . ~. . : , . :
- 12 ~
mixture (20ml). Prolonged reaction time o 4 hours was used at room temperatuxe and addition of ether deposited product.
Recrystallisation from MeOH/ether gave a 52~ yield (0.60g) of product. TLC in 9:1 CHC13: MeOH (I2 stain) showed one major spot at Rf 0.4; [a]D = 10.1 (C = 1, AcOH). ~mino acid analysis:
required: 1.00 Thr: 1.00 Ssr: 2.00 Gly: 2.00 Phe: 2.00 Lys.
found: 1.00 Thr: 1.00 Ser: 2~17 Gly: 2.19 Phe: 2.32 Lys.
(V) LysThrLysGlySerGlyPhePheOMe Partially protected octapeptide (IV) (O.lOg) was hydrogenated in 85% AcOH (70ml) with 10% Pd/C catalys~
(0.20g) over a steady stream of hydrogen for 20 hours. The mixture was filtered, evaporated in vacuo and residue filtered on Sephadex LH2 ~ eluting with water to give the desired octapeptide methyl ester (V) ~0.03g, 46~ yield).
TLC examination showed 1 spot at Rf 0.2 in 5:2:2 BAW
(t--BuOCl/KI-starch stain) and Rf 0.5 in 5:2:3 BAW
(Ninhydrin stain). Amino acid analysis:
re~uired: 1.00 Ser: 1.00 Thr: 2~00 Gly: 2.00 Phe: 2.00 Lys.
found: 1.00 Ser: 1.03 Thr: 2.02 Gly: 2.06 Phe: 1.98 Lys.
Isotachophoretic examination showed one band in ~95% amount (leading electrolyte lOmM KOH + MES pH 6.0 and terminating electrolyte lOmM ~-alanine and HCl pH 4.23). The NMR 80 HzFT spectrum was consistent with structur~.
2~ EXAMPLE 2: The ~reparatio~ of Ar~L~sThrL~sGlvSerGlYPhePheOMe This nonapeptide was prepared by coupling o~ (IV) above with Z.Arg(Z)~.OSu, followed by hydrogenolysis o~ the resultant fully protected nonapeptide.
(i) Z.A~g(Z)2Lys(Z~Thr(Bzl)Lys(Z)GlySer(Bzl)GlyPhePheOMe:
To octapeptide (IV) (0.344g, 0.30025M) above in DMF
(3ml) at 0C was added (1 equivalent) Et3N (0.025g in lml DMF) a~d Z-Arg(Z)20Su (0.17g, 0.00025M in 2ml DMF). The solution was left at 4C for 65 hours, diluted with wat~r (8ml) and tha deposited product filtered off and dried (0.37g, 78% yleld). Crystallisation from DMF/EtOH gave ~ TAq P~
- : :
J
product with m.p. 204-210C (decomposition). TLC
examination in 9:1 CHC13: MeOh (I2 stain) showed on U.V.
visualisation l spot at Rf 0.69. The NMR spectrum was consistent with structure. Amino acid analysis:
required: l.OO Thr: l.Oo Ser: 2.oo Gly: 2.00 Phe: 2.00 Lys:
l.Oo Arg.
found- l.OO Thr: l.oO Sex: 2.10 Gly: 2.01 Phe: 2.o4 Lys:
1.04 Arg.
Fully protected nonapeptide (i) (0.07g) above was dissolved in a minimum amount of DMF and 5 times the volume of AcOH added. The mixture was hydrogenated in the presence o~ 10~ Pd/C catalyst (2.5 times weight of compound) for 19 hours at room tamperature using a steady stream o~
hydrogen. Water was added to give a 15% a~ueous solution and the mixture hydro~enated for a further 3 hours.
Filtration and evaporation in vacuo at 45C gave product as a glassy solidO Purification was per~ormed on a Sephadex~
hH20 column eluting with lM AcOH and pxoduct isolated in 26% yield (0.018g). TLC in 5:3:5 BAW (ninhyrin stain) showed product at Rf O.3~. Amino acid analysis:
re~uired:l.OO Thr l.OO Ser:2.00 Gly:2.00 Phe:2.00 Lys:l.OO Arg.
found: 1.00 Thr:l.Ol Ser:~.07 Gly:2.00 Phe:2~07 Lys:l.OO Arg.
EXAMPLE 3: The preparation of LysThrLysGlySerGlyPhaPheVal _ PheOMe The decapeptide methyl ester was synthesised by a 4~2~4 ~ragment condensation strategy as follows:-BOC.Ser(Bzl)Gly,OSu-~PhePheValPheOMe (VI) ¦ (VIX~
~ (ooupLing and BCC-deprotection) ~ Ser(~zl)~lyPhePheValPheOMe (Pn~xatio~ (VIII) described in Example l) (coupling and selective deprotection) ~ ,~ -Product (example 3) n~
:
- . - .- .
. . -.. . ~ - . - .. . .
. .. . - . ... . .... , ., , ., .,. ~ . .. . . .
.. . . . .... . ...
(VI) BOC.Ser(Bzl)Gly.OSu Prepared in three stages:-(i) BOC.Ser(Bzl)GlyOMe BOC.Ser(Bzl)OH (5.0g, 0.017M) was coupled to Gly.OMe.HCl t2.l3g~ 0.017M) in M.D.C. ~lOOml) at R.T. for
(i) Z.A~g(Z)2Lys(Z~Thr(Bzl)Lys(Z)GlySer(Bzl)GlyPhePheOMe:
To octapeptide (IV) (0.344g, 0.30025M) above in DMF
(3ml) at 0C was added (1 equivalent) Et3N (0.025g in lml DMF) a~d Z-Arg(Z)20Su (0.17g, 0.00025M in 2ml DMF). The solution was left at 4C for 65 hours, diluted with wat~r (8ml) and tha deposited product filtered off and dried (0.37g, 78% yleld). Crystallisation from DMF/EtOH gave ~ TAq P~
- : :
J
product with m.p. 204-210C (decomposition). TLC
examination in 9:1 CHC13: MeOh (I2 stain) showed on U.V.
visualisation l spot at Rf 0.69. The NMR spectrum was consistent with structure. Amino acid analysis:
required: l.OO Thr: l.Oo Ser: 2.oo Gly: 2.00 Phe: 2.00 Lys:
l.Oo Arg.
found- l.OO Thr: l.oO Sex: 2.10 Gly: 2.01 Phe: 2.o4 Lys:
1.04 Arg.
Fully protected nonapeptide (i) (0.07g) above was dissolved in a minimum amount of DMF and 5 times the volume of AcOH added. The mixture was hydrogenated in the presence o~ 10~ Pd/C catalyst (2.5 times weight of compound) for 19 hours at room tamperature using a steady stream o~
hydrogen. Water was added to give a 15% a~ueous solution and the mixture hydro~enated for a further 3 hours.
Filtration and evaporation in vacuo at 45C gave product as a glassy solidO Purification was per~ormed on a Sephadex~
hH20 column eluting with lM AcOH and pxoduct isolated in 26% yield (0.018g). TLC in 5:3:5 BAW (ninhyrin stain) showed product at Rf O.3~. Amino acid analysis:
re~uired:l.OO Thr l.OO Ser:2.00 Gly:2.00 Phe:2.00 Lys:l.OO Arg.
found: 1.00 Thr:l.Ol Ser:~.07 Gly:2.00 Phe:2~07 Lys:l.OO Arg.
EXAMPLE 3: The preparation of LysThrLysGlySerGlyPhaPheVal _ PheOMe The decapeptide methyl ester was synthesised by a 4~2~4 ~ragment condensation strategy as follows:-BOC.Ser(Bzl)Gly,OSu-~PhePheValPheOMe (VI) ¦ (VIX~
~ (ooupLing and BCC-deprotection) ~ Ser(~zl)~lyPhePheValPheOMe (Pn~xatio~ (VIII) described in Example l) (coupling and selective deprotection) ~ ,~ -Product (example 3) n~
:
- . - .- .
. . -.. . ~ - . - .. . .
. .. . - . ... . .... , ., , ., .,. ~ . .. . . .
.. . . . .... . ...
(VI) BOC.Ser(Bzl)Gly.OSu Prepared in three stages:-(i) BOC.Ser(Bzl)GlyOMe BOC.Ser(Bzl)OH (5.0g, 0.017M) was coupled to Gly.OMe.HCl t2.l3g~ 0.017M) in M.D.C. ~lOOml) at R.T. for
3~ hours in the presence of 1 equivalent of Et3N and using DCCI (3.5g, 0.017M~ as the condensing agent. The precipitate was filtered off and the solution washed X 2 with water, aqueous NaHC03, water, dried and evaporated in vacuo to leave an oil (7.lg).
T.L.C. 9:1 CHC13: ~eOH ~I2 stain) showed one spot at R~ 0.64; ta]~5 C= 4 7 (C - 1 M OH) (ii) BOC.Ser ~s~ y~
Compound (i) above (7.0g) was dissolved in dioxan (25ml) and treated with an equal volume of lN NaOH (25ml~
and the solution stirred ~or ~ hour at R.T. N HCl (25ml) was added to a slight excess and the oil that formed extracted into EtAc. The organic layer was back-extracted into NaHC03 and acidified to pH 3.8 with 20% citric acid, extracted with EtAc, the organic layer washed with water, brine, dried and evaporated to leave the product as a syrup t4 0g) T.L.C. 1 1 CHC13/EtO~I (I2 stain) showed product at Rf 0.59.
The NMR spectrum was consistent with structure.
(iii)BOC~Ser(Bz~
Compound (ii) above (4g 0.01135M) was treated with HOSu (1.3g O.OllM) and DCCI (2.34g O.OllM) in dioxan (50ml) at R.T. overnight. The precipitate that formed was ~iltered o~, solvent removed and the product crystallised ~xom I.P.A. (lOOml) in 59% yield (3.00g).
~.L.C. 9:1 CHC13/MeOH (I2 stain) showed one ma~or spot R~ 0.57; M.P. 132 134C; [~25= -1.8 (C = 1, MeOH).
(VII)Ph_Phe_al_heOMe Prepared in six steps:-(i) BOC.ValPheOMe BOC.ValOSu (lO.Og, 0.0328M~ was coupled to PheOMe,Hcl .,:. : ,, , :
.. , , , .. . ,. , . ~ ~ .
. ., .,- ,:: : . . . ~ . ~ , .. ..
- . . . : , .. .. : . ~ . , . ~ :
?~'~
(6.85g 0~0318M) in toluene (2.00ml) at room temperature overnight and in the presence of Et3N (1 equivalent). The mixture was filtered and filtrate washed with lN HC1, satura-ted NaCl solution, dried and evaporated in vacuo to give product (11.31g) as a white crystalline compound in 94%
yield.
T.L.C~ in 9 1 (CHC13:~eOH) (I2 stain) shows one spo~ at Rf 0.77 ~a3D -~27.4 (C=l~ MeOH).
(ii~ ValPheOMe.HCl ,~
Compound (i) (9.25g) was BOC-deprotected in 2N HCl in EtAc (lOOml) for 24 hours at room temperature when the product precipitated. The mix was diluted with dry EtAc and product ~iltered off in 78~ yield (6.0g). The product was finally purified on Sephadex ~H20. M.P. 193-193~5;
T.L.C. 9:1 CHCL3/MeOH (I2 stain) shows one spot at Rf 0.60.
La]D5= 63.3 (C=l, AcOH) ¢iii)BOC.PheValPheOMe ",... . . . .. .
Compound (ii) (5.34g, 0.017M) was coupled to ~OC.PheOSu (6.15g, 0.017M) in 25% D.M.F. in Toluene ~250ml) at room temperature for 65 hours in the presence of Et3N (1 e~uiv.).
The mixture was then filtered, solvent removed in vacuo and the syrup quenched with water. The white precipitated (8.5g) was filtered off and recxystallised from EtAc/80-100 petrol; yield 80%.
T.L.C. 9 1 CHC13/MeOH (I2 stain) shows one spot at Rf 0.69 [a ~ = -31.0 (C = 1 MeOH). NMR consistent with structure.
(iv) PheValPheOMe HCl _ Compound (ili) (6.87g) was BOC-deprotected in 2N ~Cl in EtAc (lOOml) for ~ hours at room temperature when a white solid precipitated ~5.84g) representing 97% yield of product. M.P. 243-245 (decomposition) T.L,C, in 9:1 CHC13:MeOH (I2 stain) shows one spot at ~25 = 8 8 (C = 1, AcOH) ~R~D~ R/~
, . - ~ , , ,, . : ', ~' '' '' :, ' : ` .: : :
- - .
~ 16 -(v) ~4~
BOC.PheOSu (4.30g O.Oll9M) was coupled to compound (iv) (5.5g 0.0119M) in toluene (lOOml) at room temperature for 65 hours in the presence of sufficient D.M.F. to produce solution, ant Et3N (1 equivalent). The solvent was evaporated ln ~acuo and the syrup quenched with water and product filtered off. The product was then triturated with hot ethanol, cooled and collected (6 9 38g, 80~ yield).
M.P. 218~219C
T.L.C. in 9:1 CHCl3:MeOH (I~ stain) shows one spot at Rf 0.62. [~}25= _15.g ~C - 1, D.M.F.) The NMR spectxum-was consistent with structure.
(vi) PhePheValPheOMe Tfa Compound ~) (5g) was BOC-deprotected in T.F.A (25ml) at 0C for ~ hour, and at room temperature for ~ hour~
The solution was then quenched with ether (75ml) and product filtered off (4.48g, 88% yield). M.P. 224-226 (decomposition) T.L C. 9:1 C~C13:MeOH (I2 stain) shows one spot at Rf 0.49.
[a]D5= 10.6 (C = l, AcOH). The NMR spectxum was consistent with structure.
(VIII)Ser(Bzl)GlyPhePheValPhe.OMe Prepated in two stages:-(i) BOC.Ser Bzl)GlyPhePheValPheOMe Intermediate VI (2.37g, 0.00528M) was coupled tointermediate VII (3.62g, 0.00528M~ in toluene (500ml) overnight at room temperature in the presence of Et3N
(0.74ml, 0.00528M). The mixture was washed with water, and solvent evaporated in vacuo. The solid obtained was triturated with water, dried and recrystallized ~rom EtOH
(4.42y, yield 93%).
T.L.C. 9~1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.61.
[]D5 =~13.5 (C = 1 D.M.F.). The NRM spectrum was consistent with structure.
(ii) ~ e _ Compound (i) (2.8g) was BOC~deprotected in T.F.A.
(30ml) for 40 minutes at 0C. The solution was quenched ., ~ .
.: '.. , , , . : . ~ ' .
. , . . . . -:
- . : : , , - , : . . .
.. . . .
~ 17 with ether (200ml) and the precipitated product obtained in quantitative yieldO
T.L.C, 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.2.
M.P. 214~216C (decomposition). ~a]D5 = 3.7 (C = l,AcOH) The NMR spectr~um was consistent with structure.
BOC.Lys(~)Thr(Bzl)~ys(Z)Gly~Ser(Bzl)GlyPhePheVdlPheOMe Peptide I (2.25g) (see Example 1) was coupled to VIII
(2.19g) by the Henzyl-Ruding~r modification of the azide method, as previously described for the octapeptide. The 10 product was recrystallized from EtOH and obtained in (2.5g, 61% yield). M.P. 243-244 .
Amino acid analysis: calculated~ lThr: lSer: 2GLy: lVal:
3Phe: 2Lys found: 1.13Thr: l llSer: 2.15Gly:
25~ l.OOVal: 3.04Phe: 1.98Lys.
~a~D = ~7 5 ~C = 1~ D~M~F~ ~ o ~he NMR spectrum was consistent with structure.
~y~ s~ SerGl~_he~ e _ Ln eLUe The intermediate abo~e ~0018g) was BOC, Z and Bzl-20 deprotected by treatment with 33% HBr in dioxan (5ml) at room temperature for 1 hour when a precipitate formed.
Additional HBr/dioxan (5ml) and water (lml) was then added which effected solution and reaction continued for a fuxther ~ hour. Acetone (50ml) was then added and the solution ~uenched with ether (lOOml). The supernatent was decanted and solid dissolved in water ~7ml) and freeze-dried to give 0.145g product as the txi-hydrobromide salt.
Amino acid analysis: re~uired: lThr: lSer: 2Gly: lVal: 3~he:
2Lys ~ound: 1.o5Thr: l.OOSex: 2~14Gly:
1.02Val: 3.13Ph~ 2.00Lys An ali~uot of product was purified on Sephadexr~LH20 elutin~
with water, to a one~spot material with Rf 0~384 (BAW 5:2:2, ni~hydrin spray).
D ~ m~hi~
. ' . ' ~` ~ ' ~ , . : , , .
EXAMæLE 4: The preparation of LysThrLysGlySerGlyPhePheVal.
PheOH
The decapeptide free acid was synthesised by a 4+2~4 fragment condensaiton strategy as follows:-BOC.Ser(Bzl~GlyOSu ~ PhePheValPheOBz (VI:preparaticn as in (IX) E~le 3) ~ (coupling and BOC-dPprotection~
ZLys(Z~Thr(Bzl)Lys(Z)&ly( ~cp + Ser(Bzl3GlyPhePheValPheOBz (XI) ¦ (X~
~ ~coupling and ccmplete deprotection) Product (example 4) (XX) P ~b~valebeoDz Prepared in six stages:-(i) BOC.ValPheOBz BOC.ValOSu (15.7g, Q.05QM) was coupled to PheOBZ.pTsa (21.35g, 0.050M) in dioxan (200ml) at R.T. for 4~ hours in the presence of 1 e~uivalent of Et3N. The reaction mixtuxe was evaporated at reduced pressure and the resulting residue dissolved in EtAc and the solution washed with water, dried and evaporated in vacuo to leave a crystalline solid (21.3g).T.L.C. 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.93;
25 = -31 8 (C - 1, MeOH).
(ii~ ValPheOBz.HCl Compound (i) (21.3g) was BOC-deprotected in 2N HCl in EtAc (240mL) for 4~ hours at R.T. when the product preclpitated. The mix was diluted with dry e~her and product filtared off in 78~ yield (15.25g). M.P. 180-182;
T.L.C. 9:1 CHC13:MeOh (I~ ~taln) shows one spot at Rf 0.44.
[a]25= 24 4 (C = 1 AcOH) ~iii)BO ValPheOBz Compound ~ii) (15.25g, 0.039~) was coupled to BOC.PhP
OSu ~14.13g, 0.039M) in 50~ dioxan/DMF (450ml) at R.T. for
T.L.C. 9:1 CHC13: ~eOH ~I2 stain) showed one spot at R~ 0.64; ta]~5 C= 4 7 (C - 1 M OH) (ii) BOC.Ser ~s~ y~
Compound (i) above (7.0g) was dissolved in dioxan (25ml) and treated with an equal volume of lN NaOH (25ml~
and the solution stirred ~or ~ hour at R.T. N HCl (25ml) was added to a slight excess and the oil that formed extracted into EtAc. The organic layer was back-extracted into NaHC03 and acidified to pH 3.8 with 20% citric acid, extracted with EtAc, the organic layer washed with water, brine, dried and evaporated to leave the product as a syrup t4 0g) T.L.C. 1 1 CHC13/EtO~I (I2 stain) showed product at Rf 0.59.
The NMR spectrum was consistent with structure.
(iii)BOC~Ser(Bz~
Compound (ii) above (4g 0.01135M) was treated with HOSu (1.3g O.OllM) and DCCI (2.34g O.OllM) in dioxan (50ml) at R.T. overnight. The precipitate that formed was ~iltered o~, solvent removed and the product crystallised ~xom I.P.A. (lOOml) in 59% yield (3.00g).
~.L.C. 9:1 CHC13/MeOH (I2 stain) showed one ma~or spot R~ 0.57; M.P. 132 134C; [~25= -1.8 (C = 1, MeOH).
(VII)Ph_Phe_al_heOMe Prepared in six steps:-(i) BOC.ValPheOMe BOC.ValOSu (lO.Og, 0.0328M~ was coupled to PheOMe,Hcl .,:. : ,, , :
.. , , , .. . ,. , . ~ ~ .
. ., .,- ,:: : . . . ~ . ~ , .. ..
- . . . : , .. .. : . ~ . , . ~ :
?~'~
(6.85g 0~0318M) in toluene (2.00ml) at room temperature overnight and in the presence of Et3N (1 equivalent). The mixture was filtered and filtrate washed with lN HC1, satura-ted NaCl solution, dried and evaporated in vacuo to give product (11.31g) as a white crystalline compound in 94%
yield.
T.L.C~ in 9 1 (CHC13:~eOH) (I2 stain) shows one spo~ at Rf 0.77 ~a3D -~27.4 (C=l~ MeOH).
(ii~ ValPheOMe.HCl ,~
Compound (i) (9.25g) was BOC-deprotected in 2N HCl in EtAc (lOOml) for 24 hours at room temperature when the product precipitated. The mix was diluted with dry EtAc and product ~iltered off in 78~ yield (6.0g). The product was finally purified on Sephadex ~H20. M.P. 193-193~5;
T.L.C. 9:1 CHCL3/MeOH (I2 stain) shows one spot at Rf 0.60.
La]D5= 63.3 (C=l, AcOH) ¢iii)BOC.PheValPheOMe ",... . . . .. .
Compound (ii) (5.34g, 0.017M) was coupled to ~OC.PheOSu (6.15g, 0.017M) in 25% D.M.F. in Toluene ~250ml) at room temperature for 65 hours in the presence of Et3N (1 e~uiv.).
The mixture was then filtered, solvent removed in vacuo and the syrup quenched with water. The white precipitated (8.5g) was filtered off and recxystallised from EtAc/80-100 petrol; yield 80%.
T.L.C. 9 1 CHC13/MeOH (I2 stain) shows one spot at Rf 0.69 [a ~ = -31.0 (C = 1 MeOH). NMR consistent with structure.
(iv) PheValPheOMe HCl _ Compound (ili) (6.87g) was BOC-deprotected in 2N ~Cl in EtAc (lOOml) for ~ hours at room temperature when a white solid precipitated ~5.84g) representing 97% yield of product. M.P. 243-245 (decomposition) T.L,C, in 9:1 CHC13:MeOH (I2 stain) shows one spot at ~25 = 8 8 (C = 1, AcOH) ~R~D~ R/~
, . - ~ , , ,, . : ', ~' '' '' :, ' : ` .: : :
- - .
~ 16 -(v) ~4~
BOC.PheOSu (4.30g O.Oll9M) was coupled to compound (iv) (5.5g 0.0119M) in toluene (lOOml) at room temperature for 65 hours in the presence of sufficient D.M.F. to produce solution, ant Et3N (1 equivalent). The solvent was evaporated ln ~acuo and the syrup quenched with water and product filtered off. The product was then triturated with hot ethanol, cooled and collected (6 9 38g, 80~ yield).
M.P. 218~219C
T.L.C. in 9:1 CHCl3:MeOH (I~ stain) shows one spot at Rf 0.62. [~}25= _15.g ~C - 1, D.M.F.) The NMR spectxum-was consistent with structure.
(vi) PhePheValPheOMe Tfa Compound ~) (5g) was BOC-deprotected in T.F.A (25ml) at 0C for ~ hour, and at room temperature for ~ hour~
The solution was then quenched with ether (75ml) and product filtered off (4.48g, 88% yield). M.P. 224-226 (decomposition) T.L C. 9:1 C~C13:MeOH (I2 stain) shows one spot at Rf 0.49.
[a]D5= 10.6 (C = l, AcOH). The NMR spectxum was consistent with structure.
(VIII)Ser(Bzl)GlyPhePheValPhe.OMe Prepated in two stages:-(i) BOC.Ser Bzl)GlyPhePheValPheOMe Intermediate VI (2.37g, 0.00528M) was coupled tointermediate VII (3.62g, 0.00528M~ in toluene (500ml) overnight at room temperature in the presence of Et3N
(0.74ml, 0.00528M). The mixture was washed with water, and solvent evaporated in vacuo. The solid obtained was triturated with water, dried and recrystallized ~rom EtOH
(4.42y, yield 93%).
T.L.C. 9~1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.61.
[]D5 =~13.5 (C = 1 D.M.F.). The NRM spectrum was consistent with structure.
(ii) ~ e _ Compound (i) (2.8g) was BOC~deprotected in T.F.A.
(30ml) for 40 minutes at 0C. The solution was quenched ., ~ .
.: '.. , , , . : . ~ ' .
. , . . . . -:
- . : : , , - , : . . .
.. . . .
~ 17 with ether (200ml) and the precipitated product obtained in quantitative yieldO
T.L.C, 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.2.
M.P. 214~216C (decomposition). ~a]D5 = 3.7 (C = l,AcOH) The NMR spectr~um was consistent with structure.
BOC.Lys(~)Thr(Bzl)~ys(Z)Gly~Ser(Bzl)GlyPhePheVdlPheOMe Peptide I (2.25g) (see Example 1) was coupled to VIII
(2.19g) by the Henzyl-Ruding~r modification of the azide method, as previously described for the octapeptide. The 10 product was recrystallized from EtOH and obtained in (2.5g, 61% yield). M.P. 243-244 .
Amino acid analysis: calculated~ lThr: lSer: 2GLy: lVal:
3Phe: 2Lys found: 1.13Thr: l llSer: 2.15Gly:
25~ l.OOVal: 3.04Phe: 1.98Lys.
~a~D = ~7 5 ~C = 1~ D~M~F~ ~ o ~he NMR spectrum was consistent with structure.
~y~ s~ SerGl~_he~ e _ Ln eLUe The intermediate abo~e ~0018g) was BOC, Z and Bzl-20 deprotected by treatment with 33% HBr in dioxan (5ml) at room temperature for 1 hour when a precipitate formed.
Additional HBr/dioxan (5ml) and water (lml) was then added which effected solution and reaction continued for a fuxther ~ hour. Acetone (50ml) was then added and the solution ~uenched with ether (lOOml). The supernatent was decanted and solid dissolved in water ~7ml) and freeze-dried to give 0.145g product as the txi-hydrobromide salt.
Amino acid analysis: re~uired: lThr: lSer: 2Gly: lVal: 3~he:
2Lys ~ound: 1.o5Thr: l.OOSex: 2~14Gly:
1.02Val: 3.13Ph~ 2.00Lys An ali~uot of product was purified on Sephadexr~LH20 elutin~
with water, to a one~spot material with Rf 0~384 (BAW 5:2:2, ni~hydrin spray).
D ~ m~hi~
. ' . ' ~` ~ ' ~ , . : , , .
EXAMæLE 4: The preparation of LysThrLysGlySerGlyPhePheVal.
PheOH
The decapeptide free acid was synthesised by a 4+2~4 fragment condensaiton strategy as follows:-BOC.Ser(Bzl~GlyOSu ~ PhePheValPheOBz (VI:preparaticn as in (IX) E~le 3) ~ (coupling and BOC-dPprotection~
ZLys(Z~Thr(Bzl)Lys(Z)&ly( ~cp + Ser(Bzl3GlyPhePheValPheOBz (XI) ¦ (X~
~ ~coupling and ccmplete deprotection) Product (example 4) (XX) P ~b~valebeoDz Prepared in six stages:-(i) BOC.ValPheOBz BOC.ValOSu (15.7g, Q.05QM) was coupled to PheOBZ.pTsa (21.35g, 0.050M) in dioxan (200ml) at R.T. for 4~ hours in the presence of 1 e~uivalent of Et3N. The reaction mixtuxe was evaporated at reduced pressure and the resulting residue dissolved in EtAc and the solution washed with water, dried and evaporated in vacuo to leave a crystalline solid (21.3g).T.L.C. 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.93;
25 = -31 8 (C - 1, MeOH).
(ii~ ValPheOBz.HCl Compound (i) (21.3g) was BOC-deprotected in 2N HCl in EtAc (240mL) for 4~ hours at R.T. when the product preclpitated. The mix was diluted with dry e~her and product filtared off in 78~ yield (15.25g). M.P. 180-182;
T.L.C. 9:1 CHC13:MeOh (I~ ~taln) shows one spot at Rf 0.44.
[a]25= 24 4 (C = 1 AcOH) ~iii)BO ValPheOBz Compound ~ii) (15.25g, 0.039~) was coupled to BOC.PhP
OSu ~14.13g, 0.039M) in 50~ dioxan/DMF (450ml) at R.T. for
4 hours in the presence of Et3N (1 equivalent). The mixture was poured into iced water and the resulting white precipitate ~20.0g) was iltered off and recrystallised ': : - .
- -- . ~ . .. . . . .
from EtAc/40-60~ petrol; yield 85%o M.P. 160-162~o T.L.C. 9:1 C~C13:MeOH (I2 stain) shows one spot at Rf 0.72.
E ]250 -36 0 (C = 1, MeOH).
(iV) rh~ A~
Compound (iii) (20.0g, 0.033M) was BOG-deprotected in 2N HCl in ~tAc ~240ml) for 2 hours at R.T. when a white solid precipitated (15.23g) representins 85~ yield of product~ M.P. 228-229 (decomposition) T.L~C 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.63.
o L ] 25 = -6 9 (c = 1, AcOH)-(v) BOCPhePheValPheOBz BOC.PheOSu (10.26g, 0.0283M) was coupled to compound (i~) (15.23g, 0.0283M) in 5~dioxan/DMF (250ml) at R.T. for 4 hours in the presence o~ Et3N (1 equivalent). The mixture was poured into iced water and the resulting white precipitate filtered o~f and recrystallised from EtAc/40-600 petrol in quantitative yield (21.41g). M.P. 191-193.
T.L.Co 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.72.
[a]D = -12.8 (C - 1, DMF).
(vi) PhePheValPheOBz.HCl Compound (v) (21,15g, 0.028M) was BOC-deprotected in 2N HCl in EtAc (500ML) ~or 2 hours at R.T. The product (17.9g) was precipitated in 92% yield upon addition of dry ether~ M.P. 242 (decomposition).
T.L C 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.74.
~}D = ~5 9 (C = 1, AcOH). The NMR spectrum was consistent w.~th structure.
(.X~ ~ lPheOBz Prepared in two stages~
(~ BOC~Ser(Bzl)GlyPhePh alPheOBz Intermediate (VI~ (4.49g, O.OlOM) was coupled to intermediate (IX) (6.85g, O.OlOM) in 35~ DMF/dioxan (75ml) at R.T. for 4 hours in the presence of Et3N (1 e~uivalent).
The mixture was poured i~to iced water and the precipitated product (9.39g~ recrystallised from methanol in 91% yield.
' M.P. 226-228.
T.L.C 9:1 CHCl :MeOH (I2 stain) shows one spot at Rf 0.74.
[a]D = ~ 13.0 (C = 1, DMF). The F.T~ 'H NMR was consistent with structure.
(ii) ~
Compound (i) (5.0g, 0.0051M) was BOC-deprotected in 2N HCl in EtAc(150ml) for 2 hours at R.T. The product (4.42g) was precipitated in 94% yield upon addition of dry ether. M.P. 232 ~234 (decomposition) T,L C 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.44.
[a~D = ~4 3 (C = 1, AcOH). The NMR was consistent with structure.
(XI) Zh~s~Z)Thr(Bzl ~ (Z)GlyOI'cp Prepared in seven stages:-(i) BOC.Lys(Z~GlyoMe BOC.Lys~Z)OSu (23.85g, 0.050M) was coupled to GlyOMe.
HCl (6.25g, O.OSOM) in 50% dioxan/DMF at R.T. for 4~ hours in the presence of 1 equivalent of Et3N. The reaction mixture was evaporated in vacuo and the residue dissolved in EtAc, The solution was washed, dried, filtered and evaporated to a colourless oil which solidified on standing i~ 89~ yield.
T.L.C. 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.54.
~ii) ~
Compound (i) (20.00g, 0.0443M) was BOC-deprotected in 2N HCl in EtAc (250ml) ~ox 2 hours at R.T. when the product precipitated. The mixture was diluted with dry ether and the product filtered of e in ~6~ yield (15.67g).
M.P. 158-159.
T.L.C. EtAc (I2 stain) showed one spot at Rf 0.52.
~lii) ~
BOC.Thr(Bzl)OH ~6.18g, 0.020M) was coupled to compound (ii) (7.76g, 0.020M) in 30~ DMF/dioxan (75ml) in iced water for 1 hour th~n at R. T . for a further 2 hours in the .
.
, I ~
presence of DCCI (1 equivalent~ and Et3N (1 equivalent).
The reaction mixture was filtered and evaporated in vacuo and the residue purified by silica column chromatography eluting with chlorofoxm. The product was isolated as a colourl~ss solid in 49~ yield. M.P. 135-136.
T.L.C. 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.57.
The F~To 3C NMR was consistent with structure.
(iv) Thr(Bzl~Lys(Z)Gl~OMe.HC1 Compound ~iii) (3.48g, 0.0054M) was BOC-deprotected in 2N HCl in EtAc(lOOml) for 2 hours at R.T~ The product (2.88g) was precipitated in 91% yield upon addition of dry ether. M.P. 100-101.
TcL CO 9;1 CHC13:MeO~ (I2 stain) showed one spot at Rf 0.52.
[~]~ = -13.5 (C = 1, AcOH).
15 (v) ~Ly~
ZLys(Z)OTcp (1.80g, 0.003M) was coupled to compound (iv) (1.72g, 0.003M) in dioxan (45ml) at R.T. for 4 hours in the presence of Et3N (1 equivalent). The product was ~iltered o~f, washed with water and dried in vacuo (1.36g~ 50~ ~ield). ~.P. 185-188.
T,L.C. 9:1 CHC13:MeOH (I2 stain showed one spot at Rf 0.76.
(vi ) Zhys ~ Z ) Thr (Bz 1 ) Lys ( Z j GlyOH
A solution of compound (v) ~0~92g, O.OOlM) in 50%
DMF~methanol was .reated with lN NaOH solution (205ml) and stirred at ~.I'. for 1 hour. Upon acidification the precipitated pxoduct (0.45g) was recrystallised from m~thanol ln 4g% yield. M.P. 171-173.
T.L.C. 2:1 CHC13:MeOH (t.butyl chloro~ormate/NaI - starch spray) show~d one spot at Rf 0 50 [~D = 5.4 (C = 1, AcOH). The F.T.13C NMR was consi~tent with structure.
~vii ~
A solution of TcpO~ (O.lOg~ 0.0005M) and compound (vi) (0.46g, o.ooo5M) in DMF was treated with DCCI (O.llg, 0.0005M) and ~tirred at 5 ~or 1 hour then at R. T .
~5 overnight. The reaction mixture was filtered and the ~, , ' .
,: : ' .
product (0.60g) isolated as a crispy solid upon evaporation in vacuo. M.P. 176-178°.
T.L.C. 9:1 CHCl3:MeOH (I2 stain) showed one spot at Rf 0.71 [.alpha.]?5° = -6.0° (C = 1, AcOH).
Peptide (XI) (0.55g, 0.0005M) was coupled to compound (C) (0.46g, 0.0005M) in DMF at R.T. for 4 hours in the presence of Et3N (1 equivalent). The reaction mixture was poured into iced water and the resulting precipitate filtered off and dried in vacuo. Purification of silica column chromatography, eluting with CHCl3, gave the product (0.92g) in 85% yield.
T.L.C. 9:1 CHCl3:MeOH (I2 stain showed one spot at Rf 0.34.
The F.T.13C NMR was consistent with structure.
The intermediate above (0.76g, 0.0003M) was deprotected by continuous hydrogenation in 85% acetic acid with 1N HCl (1mM) for 18 hours in the presence of 10% Pd/charcoal (0.80g). The product was purified in a Biogel* P2 column eluting with 1M ammonium acetate and subsequently on a CM32* cellulose column eluting with 0.1M ammonium acetate pH5. Final isolation of the product in 23% yield was by lyopholisation.
T.L.C. butanol/acetic acid/water (5:2:2) (ninhydrin spray) showed one spot at Rf 0.22. Amino acid analysis:
calculated: 2Lys: 1Thr: 2Gly: 1Ser: 3Phe: 1Val:
found: 1.80Lys: 0.02The: 2.00Gly: 1.07Ser: 3.24Phe:
0.91Val EXAMPLE 5: The preparation of This decapeptide was synthesised by a 1+1+4+4 fragment condensation strategy as follows:-* Trade mark ~ 23 BOCOArg(N02)0Su ~ Lys(Z)Thr(Bzl)Lys(Z)GlyOMe ~XII3 ~ coupling and BOC-deprotection ZProOSu ~ Arg(N02)Lys(Z)ThL(Bzl)Lys(Z)GlyOMe (XIII) . coupling and hydrolysis ZProArg(N02)Lys(Z)Thr(Bzl)Lys(Z)GlyOH ~ Ser~Bzl)GlyPhePhe~b (VIII) (preparation described Ln Exa~ple 3) coupling and complete deprotection Product (example 5) :
(XII) ~ Prepared in two steps ~rom Thr(Bzl)hys~Z)GlyOMe described in example 4.
(i) ~ ~
BOCoLys(Z)OSu (2.38g, 0.005M) was coupled to Thr(Bzl) Lys(Z)GlyOMe.HCl (2.87g~ 0.005M) in dioxan (60ml) at R.T.
for 4 hours in the presence of Et3N (1 equivalent)~ The reaction mixture was poured into iced water to give the re~uired product (3.80g) as a crystalline white solid in 84% yield. M.P. 103-105.
T.L.C~ 9:1 CHCl3:MeOH (I~ stain) showed one spot at Rf 0.55.
~~D5 = 10.4 (C = 1, AcOH) ~ii) 2$ Compou~d (i)~(3~80g, 0.0042M) was BOC-deprotected ln 2N HCl in EtAc ~lOOml~ for 2 hours at R.T. The product (3. 30g) was precipitated in 93% yield upon addition of dry ether . M. P . 184-186.
T.L C 9:1 CHC13:MeOH (I.2stain) showed one spot at Rf 0.30, Ca]D = 5.6 (C ~ lr AcOH).
(XIII) ~(NO~)Lys(Z)Thr(BzI)~ys(Z)GlyOMe Prepared in ~wo stages:-: BOC~Arg(N02~0Su (1.40g, 0.0033M~ was coupled to compound (XII) (3.30g, 0.004M) in 10~ DMF/dioxan (55ml) at . ~
:
,, ., . , ~ . ~
~ 24 -R.~. ~or 3 hours in the presence of Et3N (1 equivalent).
Unreacted (XII) was filtered off and the reaction mixture poured into iced water, extracted with EtAc to give the product (2.60g) which was recrystallised from IPA in 71~ yield. M.P. 133-135.
T.~ C 9:1 C~C13:MeOH ~I2 stain) showed one spot at Rf 0~42.
[a~D = -6.7 (C - 1, AcOH).
(ii) Ar~(N_~)Lys(Z)Thr(Bzl)Lys(Z)GlyOMe.HCl Compound (i) (2.0g, 0.0018M~ was BOC-deprotected in 2N HCl in EtAc(50ml) for 2 hours at R.T. The product (1.75g) was precipitated in 92% yield upon addition of dry ether.
M~P. 157 (decomposition) T.L.C. 2-1 CHC13.MeOH (I~ stain) showed one spot at Rf 0.53.
[]25= _5 6~ (C - 1, ~OH). ---(XIV)ZProArg(NO~?Lys(Z)Thr(Bzl)L~s(Z)_G~ Prepared in two stages:-(i~ 2ProAr~(NO )Lys(Z)Thr(Bzl)I,ys(Z)GlyOMe ZProOsu (0.57g, 0.0016M) was coupled to compound (XIII) (1.71g, ~.0016M) in 20~ D~/dioxan (30ml) at R.T. for 2 hours in the presence of Et3N (1 equivalent). The reactionmixture was poured into iced water and extracted with EtAc to ~ive the product (l.llg) in 54~ yields.
T ~LoC ~ 9 o 1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.36.
~ii) ZProAr~(NO~)Lys(Z)Thr(Bzl)Lys(z)GlyoH
A solu~ion of compound (i) (0.87g, 0.0007M) in 50% DMF/
mathanol (30ml) was treated with lN NaOH solution (1.7ml) and stirred at R.T. ~or 2 hours. Acidification precipitated the product (0.43g) in 50~ yleld~
T.~.C. 2.1 CHC13:MeOH (I2 stain~ showed one spot at R 0.77~ [a~D5 - -4.1 (C = 1, AcOH). The NMR was consistent with structure.
: . .: , - .- : . . ~ . ::: . : : - : :
. : ~
~ 25 -ZProArg(N02)Lys(Z)Thr(Bzl3Lys~GlySer(Bzl~GlyPhePheOMe Peptide (XIV) 0.~Og, 0.0033M) was coupled to compound (VIII) (0.20g, 0.0034~) in DMF (5ml) in the pre~ence o Et3~ (1 equivalent~, DCCI (0.07g, 000035M) and hydroxybenzotriazole (0.044g, 0.0035M) at 5 for 1 hour then at R.T. or 1 hour. The precipitated urea was filtered of and the required product (0.50g) isolated by pouring the reaction mixtur~ into iced water and isolating by filtration in 88~ yield.
~.h.C. 9:1 CHC13:MeOH (I2 stain) showed one spok at Rf 0.520 The NMR was consistent with strustura~
ProAr~L_~l3~ L~ erClyPhepbeoM~
The in~ermediate above (0.40g, 0.0022M) wa~ deprotected by continuous hy~rogenation in 8S~ acetic acid for 18 hours in the presence of 10% Pd/charcoal catalyst (0.40g). .The product was puri~ied on a Biogel P2 column eluting with water and subsequently on an I,H20 Sephade ~column again with aqueous elution. Final isolation of tha product ln 34% yield was by lyopholisation.
T.L.C. butanol/acetlc acid/water (5:2:2) (ninhydrin spray) showed one spot at Rf 0.34.
~ 7~ P ~ f~
'~
. , .
~: . . . . . .
. - . , .. ~, :
- . .
q~6 Ami~o acid analysis calculated; lPro. lArg: 2Lys: lThr:
2Gly: lSer: 2Phe ~ound: 0.95Pro: O.99Arg: 1~88Lys:
l.OOThr: 2.00Gly: l.OlSer:
1.96Phe~
The octapaptide free acid was ~ynthesised by a 4~4 fra~ment condensation strategy as follows:
BOC.Lys(Z)Thr(Bzl)Lys(Z)GlyOSu + Ser(Bzl)GlyPhePheOBz (XVI) ~XV) ~oDuplin~ and compl~te depr~tec-tiQn) Pro~ ~uct texample 6) (XV) Ser(Bzl)GLyPhePheOBz Prepared in six stages:- . --15 (i) BOCPhePheOBz BOCPheOH (11.88g, 0.045M) was coupled to PheOBz.pTsa.
19.4g, 0.o45M) in MDC (200 ml) at 0 for l hour then at R.T. overnight in the presence of E~3N (l equivalent) and DCCI (1 equivalent). The reaction mixture was filtered ~nd the product (14.92g) isolated in 6~% yield upon evaporation in vacuo and recrystallisation from EtOAc/80-100 petrol (14.92g). M.P. 123.5~124.5.
T~L~Co l:l EtAC~ 80-100 petrol (I2 stain) showed one spot at R~ 0.68. ~]25 = -16.7 ~C - 1I MeOH).
~ii) PhePheOBz.T~a Compound (i) (14.0g, 0.028M) was BOC-deprotected in 50% Tfa in MDC (lOOml) for ~ hour at 0. The solution was quenched with dry ether and the product tl4~23g) filtered off in quantitative yield. M.P. 180 (decomposition) T ~LoC 9 1 CHC}3:MeO~ (I2 stain) showed o.ne spot at Rf 0.73.
~a]D5 = 17.5 (C - lt AcOH).
(iii)BOCGlyPhePhe OBz BOC.GlyOSu (9.6g~ 0.0353M) was coupled to compound ~ii) (18.20g, 0.0353M) in ~oluene, MDC and D~ (125ml) at , , . . . ,, . :
.. , ... . . , -:: . : .
- : .: , . . . :, . ~ ., , ~ , , : :
, . . .
R.T. overnight in the presence of Et3N (1 equivalent).
The reaction mi~ture was evaporated at reduced pressure and the resulting residue dissolved in EtAc, washed, dried and evaporated in vacuo to leave a crystalline solid (19.71g) in quantitative yield. M.P. 127-130.
T.L.C. 9:1 CHCl3MeOH ~I2 s~ain) showed one spot at Rf 0.81.
[ ]250= -17 4 (C = 1, MeOH)~
(iv) Gl~PhePheO~z.Tfa Compound (iii) (19.3g, 0.0345M) was BOC-deprotected in 50~ Tfa in MDC (130ml) fox 1~ hours at O . The solution was quenched with ether and the product (17.79g) filtared of~ in 90~ yield.
T.L,C. 9.1 CHC13:MeO~ (I2 stain) showed single spot at Rf 0.35. [a]25 - 6.0 ~C ~ 1 AcOH) (v) BOC.Ser (Bzl)Gl~PhePheOBz BOC.Ser(Bzl)OSu (11.03g, 0.0282M) was coupled to compound (iv) (16.13g, 0.0282M) in 15% DMF/toluene (350ml) at R.T. overnight in the presence of Et3N (1. equivalent).
The reaction mixture was evaporated at reduced pressure and the resul~ing residue dissolved in EtAc, washed, dried and evaporated in vacuo to give the product (14.88g) in 72% yield upon recrystallisation from EtAc/petrol.
M.P. 149-151.
T.L~C. 3:1 CHCl :MeOH ~I2 stain) showed a single spot at Rf 0.65. [a~D - -11.2 (C = 1, MeOH~.
(vi) Ser(Bzl)GlyPhePheOBz Compound (v) (14.45g, 0.0196M) was BOC-deprotected in ~0~ Tfa in MDC (140ml) for 1 hour at 0. The solution was quenched with ether and the product (13.1g) filtered off in 8g% yield. M.P. 185-187 (decomposition).
T.L.C. 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.43.
[~25~_ 10.1 (C - 1, AcOH).
5XVI)BOC,Ly~Z)~hr(Bzl)L~s(Z)GL~OSu Prepared in four stages from BOC.Thr(Bzl)Lys(Z)GlyOMe described in example 5.
.
- .~ . .
. . ' `~` , ' :
- ' :
(i) Thr (Bzl) Lys (Z ) GlyOMe .Tfa BOC.Thr(Bzl)Lys(Z)GlyOMe (2.87g, 0.0045M~ was BOC-deprotected in 5G% Tfa in MDC (5~ml) for 1 hour at O .
The solution was quenched with ether and the product (2,10g) filtered off in 72% yield~
T~L ~C o 9 1 CHC13:MeOH (I2 s*ain) showed one spot at Rf 0.26.
(ii) BOC~-Lys(æ)Thr(Bzl)Lys(Z)GlyOMe BOC~Lys (Z)OSu (2.00g, 0.003M) was coupled to compound (i) (1.45g, 0.003M) in 10~ DMF/toluene at R~T. overnight.
The reaction mixture was evaporated at reduced pressure and the resulting residue dissolved in EtAc~ washed, dried and evaporated in vacuo to give the product (2.04y) in 74~ yield upon recrystallisation from EtAc/petrol.
M.P~ 117-119.
T.L.C~ 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.60.
(iii)BOC.Lys(Z)Thr(Bzl)Lys_Z)GlyO~
A solution o~ compound (ii) (l.9~g, 0.0022M) in DMSO
(30ml) was treated with lN NaOH solution (1~ equivalents) and stirred at R.T. for 1 hour. Acidi~ication gave the product (1.82g) in quantitative yield.
T.L.C. 5:1 CHC13:MeO~ (I2 stain) showed compound just above baseline.
(iv) BOC .Lys (Z ) Thr_Bæl) Lys (Z ) GlyOSu ~OSu (0.24g, 0.002M~ was oupled to compound (iii) ~1.82g, 0.002M) in dioxan (25ml) at R.T~ ~or 4 hours in the presence of DCCI (1 e~uivalent). The reaction mixture was filtered and the ~iLtxate evaporated at reduced pressure.
Recrystallisation o~ the residue ~rom EtOH gave the product (0.30g) in 15~ yield. M.P. 118-122.
T ~L . C . 9 :1 CHC13:MeOH (I2 stain) showed one spot at R~ 0.50.
[~]D = ~ 4 (C = 1~ DMF). The NMR was consistent with structure.
BOC. ~ Bzl)hys(Z)Gl~r(Bzl)Gly~ePheOBz Peptida (XVI~ (0.22g, 0.0023M) was coupled to compound -. . : - , . : . . . .
... . : .... :, ~
.. . .
. . . . . .
~XV) ~0017g, 0.0023M) in 5% DMF/toluene (21ml) at R.T.
overnight in the presence of Et3N (1 equivalent). The reaction mixture was evaporated at reduced pressure and the residue recrystallised from EtOH to give the product (0.29g)
- -- . ~ . .. . . . .
from EtAc/40-60~ petrol; yield 85%o M.P. 160-162~o T.L.C. 9:1 C~C13:MeOH (I2 stain) shows one spot at Rf 0.72.
E ]250 -36 0 (C = 1, MeOH).
(iV) rh~ A~
Compound (iii) (20.0g, 0.033M) was BOG-deprotected in 2N HCl in ~tAc ~240ml) for 2 hours at R.T. when a white solid precipitated (15.23g) representins 85~ yield of product~ M.P. 228-229 (decomposition) T.L~C 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.63.
o L ] 25 = -6 9 (c = 1, AcOH)-(v) BOCPhePheValPheOBz BOC.PheOSu (10.26g, 0.0283M) was coupled to compound (i~) (15.23g, 0.0283M) in 5~dioxan/DMF (250ml) at R.T. for 4 hours in the presence o~ Et3N (1 equivalent). The mixture was poured into iced water and the resulting white precipitate filtered o~f and recrystallised from EtAc/40-600 petrol in quantitative yield (21.41g). M.P. 191-193.
T.L.Co 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.72.
[a]D = -12.8 (C - 1, DMF).
(vi) PhePheValPheOBz.HCl Compound (v) (21,15g, 0.028M) was BOC-deprotected in 2N HCl in EtAc (500ML) ~or 2 hours at R.T. The product (17.9g) was precipitated in 92% yield upon addition of dry ether~ M.P. 242 (decomposition).
T.L C 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.74.
~}D = ~5 9 (C = 1, AcOH). The NMR spectrum was consistent w.~th structure.
(.X~ ~ lPheOBz Prepared in two stages~
(~ BOC~Ser(Bzl)GlyPhePh alPheOBz Intermediate (VI~ (4.49g, O.OlOM) was coupled to intermediate (IX) (6.85g, O.OlOM) in 35~ DMF/dioxan (75ml) at R.T. for 4 hours in the presence of Et3N (1 e~uivalent).
The mixture was poured i~to iced water and the precipitated product (9.39g~ recrystallised from methanol in 91% yield.
' M.P. 226-228.
T.L.C 9:1 CHCl :MeOH (I2 stain) shows one spot at Rf 0.74.
[a]D = ~ 13.0 (C = 1, DMF). The F.T~ 'H NMR was consistent with structure.
(ii) ~
Compound (i) (5.0g, 0.0051M) was BOC-deprotected in 2N HCl in EtAc(150ml) for 2 hours at R.T. The product (4.42g) was precipitated in 94% yield upon addition of dry ether. M.P. 232 ~234 (decomposition) T,L C 9:1 CHC13:MeOH (I2 stain) shows one spot at Rf 0.44.
[a~D = ~4 3 (C = 1, AcOH). The NMR was consistent with structure.
(XI) Zh~s~Z)Thr(Bzl ~ (Z)GlyOI'cp Prepared in seven stages:-(i) BOC.Lys(Z~GlyoMe BOC.Lys~Z)OSu (23.85g, 0.050M) was coupled to GlyOMe.
HCl (6.25g, O.OSOM) in 50% dioxan/DMF at R.T. for 4~ hours in the presence of 1 equivalent of Et3N. The reaction mixture was evaporated in vacuo and the residue dissolved in EtAc, The solution was washed, dried, filtered and evaporated to a colourless oil which solidified on standing i~ 89~ yield.
T.L.C. 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.54.
~ii) ~
Compound (i) (20.00g, 0.0443M) was BOC-deprotected in 2N HCl in EtAc (250ml) ~ox 2 hours at R.T. when the product precipitated. The mixture was diluted with dry ether and the product filtered of e in ~6~ yield (15.67g).
M.P. 158-159.
T.L.C. EtAc (I2 stain) showed one spot at Rf 0.52.
~lii) ~
BOC.Thr(Bzl)OH ~6.18g, 0.020M) was coupled to compound (ii) (7.76g, 0.020M) in 30~ DMF/dioxan (75ml) in iced water for 1 hour th~n at R. T . for a further 2 hours in the .
.
, I ~
presence of DCCI (1 equivalent~ and Et3N (1 equivalent).
The reaction mixture was filtered and evaporated in vacuo and the residue purified by silica column chromatography eluting with chlorofoxm. The product was isolated as a colourl~ss solid in 49~ yield. M.P. 135-136.
T.L.C. 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.57.
The F~To 3C NMR was consistent with structure.
(iv) Thr(Bzl~Lys(Z)Gl~OMe.HC1 Compound ~iii) (3.48g, 0.0054M) was BOC-deprotected in 2N HCl in EtAc(lOOml) for 2 hours at R.T~ The product (2.88g) was precipitated in 91% yield upon addition of dry ether. M.P. 100-101.
TcL CO 9;1 CHC13:MeO~ (I2 stain) showed one spot at Rf 0.52.
[~]~ = -13.5 (C = 1, AcOH).
15 (v) ~Ly~
ZLys(Z)OTcp (1.80g, 0.003M) was coupled to compound (iv) (1.72g, 0.003M) in dioxan (45ml) at R.T. for 4 hours in the presence of Et3N (1 equivalent). The product was ~iltered o~f, washed with water and dried in vacuo (1.36g~ 50~ ~ield). ~.P. 185-188.
T,L.C. 9:1 CHC13:MeOH (I2 stain showed one spot at Rf 0.76.
(vi ) Zhys ~ Z ) Thr (Bz 1 ) Lys ( Z j GlyOH
A solution of compound (v) ~0~92g, O.OOlM) in 50%
DMF~methanol was .reated with lN NaOH solution (205ml) and stirred at ~.I'. for 1 hour. Upon acidification the precipitated pxoduct (0.45g) was recrystallised from m~thanol ln 4g% yield. M.P. 171-173.
T.L.C. 2:1 CHC13:MeOH (t.butyl chloro~ormate/NaI - starch spray) show~d one spot at Rf 0 50 [~D = 5.4 (C = 1, AcOH). The F.T.13C NMR was consi~tent with structure.
~vii ~
A solution of TcpO~ (O.lOg~ 0.0005M) and compound (vi) (0.46g, o.ooo5M) in DMF was treated with DCCI (O.llg, 0.0005M) and ~tirred at 5 ~or 1 hour then at R. T .
~5 overnight. The reaction mixture was filtered and the ~, , ' .
,: : ' .
product (0.60g) isolated as a crispy solid upon evaporation in vacuo. M.P. 176-178°.
T.L.C. 9:1 CHCl3:MeOH (I2 stain) showed one spot at Rf 0.71 [.alpha.]?5° = -6.0° (C = 1, AcOH).
Peptide (XI) (0.55g, 0.0005M) was coupled to compound (C) (0.46g, 0.0005M) in DMF at R.T. for 4 hours in the presence of Et3N (1 equivalent). The reaction mixture was poured into iced water and the resulting precipitate filtered off and dried in vacuo. Purification of silica column chromatography, eluting with CHCl3, gave the product (0.92g) in 85% yield.
T.L.C. 9:1 CHCl3:MeOH (I2 stain showed one spot at Rf 0.34.
The F.T.13C NMR was consistent with structure.
The intermediate above (0.76g, 0.0003M) was deprotected by continuous hydrogenation in 85% acetic acid with 1N HCl (1mM) for 18 hours in the presence of 10% Pd/charcoal (0.80g). The product was purified in a Biogel* P2 column eluting with 1M ammonium acetate and subsequently on a CM32* cellulose column eluting with 0.1M ammonium acetate pH5. Final isolation of the product in 23% yield was by lyopholisation.
T.L.C. butanol/acetic acid/water (5:2:2) (ninhydrin spray) showed one spot at Rf 0.22. Amino acid analysis:
calculated: 2Lys: 1Thr: 2Gly: 1Ser: 3Phe: 1Val:
found: 1.80Lys: 0.02The: 2.00Gly: 1.07Ser: 3.24Phe:
0.91Val EXAMPLE 5: The preparation of This decapeptide was synthesised by a 1+1+4+4 fragment condensation strategy as follows:-* Trade mark ~ 23 BOCOArg(N02)0Su ~ Lys(Z)Thr(Bzl)Lys(Z)GlyOMe ~XII3 ~ coupling and BOC-deprotection ZProOSu ~ Arg(N02)Lys(Z)ThL(Bzl)Lys(Z)GlyOMe (XIII) . coupling and hydrolysis ZProArg(N02)Lys(Z)Thr(Bzl)Lys(Z)GlyOH ~ Ser~Bzl)GlyPhePhe~b (VIII) (preparation described Ln Exa~ple 3) coupling and complete deprotection Product (example 5) :
(XII) ~ Prepared in two steps ~rom Thr(Bzl)hys~Z)GlyOMe described in example 4.
(i) ~ ~
BOCoLys(Z)OSu (2.38g, 0.005M) was coupled to Thr(Bzl) Lys(Z)GlyOMe.HCl (2.87g~ 0.005M) in dioxan (60ml) at R.T.
for 4 hours in the presence of Et3N (1 equivalent)~ The reaction mixture was poured into iced water to give the re~uired product (3.80g) as a crystalline white solid in 84% yield. M.P. 103-105.
T.L.C~ 9:1 CHCl3:MeOH (I~ stain) showed one spot at Rf 0.55.
~~D5 = 10.4 (C = 1, AcOH) ~ii) 2$ Compou~d (i)~(3~80g, 0.0042M) was BOC-deprotected ln 2N HCl in EtAc ~lOOml~ for 2 hours at R.T. The product (3. 30g) was precipitated in 93% yield upon addition of dry ether . M. P . 184-186.
T.L C 9:1 CHC13:MeOH (I.2stain) showed one spot at Rf 0.30, Ca]D = 5.6 (C ~ lr AcOH).
(XIII) ~(NO~)Lys(Z)Thr(BzI)~ys(Z)GlyOMe Prepared in ~wo stages:-: BOC~Arg(N02~0Su (1.40g, 0.0033M~ was coupled to compound (XII) (3.30g, 0.004M) in 10~ DMF/dioxan (55ml) at . ~
:
,, ., . , ~ . ~
~ 24 -R.~. ~or 3 hours in the presence of Et3N (1 equivalent).
Unreacted (XII) was filtered off and the reaction mixture poured into iced water, extracted with EtAc to give the product (2.60g) which was recrystallised from IPA in 71~ yield. M.P. 133-135.
T.~ C 9:1 C~C13:MeOH ~I2 stain) showed one spot at Rf 0~42.
[a~D = -6.7 (C - 1, AcOH).
(ii) Ar~(N_~)Lys(Z)Thr(Bzl)Lys(Z)GlyOMe.HCl Compound (i) (2.0g, 0.0018M~ was BOC-deprotected in 2N HCl in EtAc(50ml) for 2 hours at R.T. The product (1.75g) was precipitated in 92% yield upon addition of dry ether.
M~P. 157 (decomposition) T.L.C. 2-1 CHC13.MeOH (I~ stain) showed one spot at Rf 0.53.
[]25= _5 6~ (C - 1, ~OH). ---(XIV)ZProArg(NO~?Lys(Z)Thr(Bzl)L~s(Z)_G~ Prepared in two stages:-(i~ 2ProAr~(NO )Lys(Z)Thr(Bzl)I,ys(Z)GlyOMe ZProOsu (0.57g, 0.0016M) was coupled to compound (XIII) (1.71g, ~.0016M) in 20~ D~/dioxan (30ml) at R.T. for 2 hours in the presence of Et3N (1 equivalent). The reactionmixture was poured into iced water and extracted with EtAc to ~ive the product (l.llg) in 54~ yields.
T ~LoC ~ 9 o 1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.36.
~ii) ZProAr~(NO~)Lys(Z)Thr(Bzl)Lys(z)GlyoH
A solu~ion of compound (i) (0.87g, 0.0007M) in 50% DMF/
mathanol (30ml) was treated with lN NaOH solution (1.7ml) and stirred at R.T. ~or 2 hours. Acidification precipitated the product (0.43g) in 50~ yleld~
T.~.C. 2.1 CHC13:MeOH (I2 stain~ showed one spot at R 0.77~ [a~D5 - -4.1 (C = 1, AcOH). The NMR was consistent with structure.
: . .: , - .- : . . ~ . ::: . : : - : :
. : ~
~ 25 -ZProArg(N02)Lys(Z)Thr(Bzl3Lys~GlySer(Bzl~GlyPhePheOMe Peptide (XIV) 0.~Og, 0.0033M) was coupled to compound (VIII) (0.20g, 0.0034~) in DMF (5ml) in the pre~ence o Et3~ (1 equivalent~, DCCI (0.07g, 000035M) and hydroxybenzotriazole (0.044g, 0.0035M) at 5 for 1 hour then at R.T. or 1 hour. The precipitated urea was filtered of and the required product (0.50g) isolated by pouring the reaction mixtur~ into iced water and isolating by filtration in 88~ yield.
~.h.C. 9:1 CHC13:MeOH (I2 stain) showed one spok at Rf 0.520 The NMR was consistent with strustura~
ProAr~L_~l3~ L~ erClyPhepbeoM~
The in~ermediate above (0.40g, 0.0022M) wa~ deprotected by continuous hy~rogenation in 8S~ acetic acid for 18 hours in the presence of 10% Pd/charcoal catalyst (0.40g). .The product was puri~ied on a Biogel P2 column eluting with water and subsequently on an I,H20 Sephade ~column again with aqueous elution. Final isolation of tha product ln 34% yield was by lyopholisation.
T.L.C. butanol/acetlc acid/water (5:2:2) (ninhydrin spray) showed one spot at Rf 0.34.
~ 7~ P ~ f~
'~
. , .
~: . . . . . .
. - . , .. ~, :
- . .
q~6 Ami~o acid analysis calculated; lPro. lArg: 2Lys: lThr:
2Gly: lSer: 2Phe ~ound: 0.95Pro: O.99Arg: 1~88Lys:
l.OOThr: 2.00Gly: l.OlSer:
1.96Phe~
The octapaptide free acid was ~ynthesised by a 4~4 fra~ment condensation strategy as follows:
BOC.Lys(Z)Thr(Bzl)Lys(Z)GlyOSu + Ser(Bzl)GlyPhePheOBz (XVI) ~XV) ~oDuplin~ and compl~te depr~tec-tiQn) Pro~ ~uct texample 6) (XV) Ser(Bzl)GLyPhePheOBz Prepared in six stages:- . --15 (i) BOCPhePheOBz BOCPheOH (11.88g, 0.045M) was coupled to PheOBz.pTsa.
19.4g, 0.o45M) in MDC (200 ml) at 0 for l hour then at R.T. overnight in the presence of E~3N (l equivalent) and DCCI (1 equivalent). The reaction mixture was filtered ~nd the product (14.92g) isolated in 6~% yield upon evaporation in vacuo and recrystallisation from EtOAc/80-100 petrol (14.92g). M.P. 123.5~124.5.
T~L~Co l:l EtAC~ 80-100 petrol (I2 stain) showed one spot at R~ 0.68. ~]25 = -16.7 ~C - 1I MeOH).
~ii) PhePheOBz.T~a Compound (i) (14.0g, 0.028M) was BOC-deprotected in 50% Tfa in MDC (lOOml) for ~ hour at 0. The solution was quenched with dry ether and the product tl4~23g) filtered off in quantitative yield. M.P. 180 (decomposition) T ~LoC 9 1 CHC}3:MeO~ (I2 stain) showed o.ne spot at Rf 0.73.
~a]D5 = 17.5 (C - lt AcOH).
(iii)BOCGlyPhePhe OBz BOC.GlyOSu (9.6g~ 0.0353M) was coupled to compound ~ii) (18.20g, 0.0353M) in ~oluene, MDC and D~ (125ml) at , , . . . ,, . :
.. , ... . . , -:: . : .
- : .: , . . . :, . ~ ., , ~ , , : :
, . . .
R.T. overnight in the presence of Et3N (1 equivalent).
The reaction mi~ture was evaporated at reduced pressure and the resulting residue dissolved in EtAc, washed, dried and evaporated in vacuo to leave a crystalline solid (19.71g) in quantitative yield. M.P. 127-130.
T.L.C. 9:1 CHCl3MeOH ~I2 s~ain) showed one spot at Rf 0.81.
[ ]250= -17 4 (C = 1, MeOH)~
(iv) Gl~PhePheO~z.Tfa Compound (iii) (19.3g, 0.0345M) was BOC-deprotected in 50~ Tfa in MDC (130ml) fox 1~ hours at O . The solution was quenched with ether and the product (17.79g) filtared of~ in 90~ yield.
T.L,C. 9.1 CHC13:MeO~ (I2 stain) showed single spot at Rf 0.35. [a]25 - 6.0 ~C ~ 1 AcOH) (v) BOC.Ser (Bzl)Gl~PhePheOBz BOC.Ser(Bzl)OSu (11.03g, 0.0282M) was coupled to compound (iv) (16.13g, 0.0282M) in 15% DMF/toluene (350ml) at R.T. overnight in the presence of Et3N (1. equivalent).
The reaction mixture was evaporated at reduced pressure and the resul~ing residue dissolved in EtAc, washed, dried and evaporated in vacuo to give the product (14.88g) in 72% yield upon recrystallisation from EtAc/petrol.
M.P. 149-151.
T.L~C. 3:1 CHCl :MeOH ~I2 stain) showed a single spot at Rf 0.65. [a~D - -11.2 (C = 1, MeOH~.
(vi) Ser(Bzl)GlyPhePheOBz Compound (v) (14.45g, 0.0196M) was BOC-deprotected in ~0~ Tfa in MDC (140ml) for 1 hour at 0. The solution was quenched with ether and the product (13.1g) filtered off in 8g% yield. M.P. 185-187 (decomposition).
T.L.C. 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.43.
[~25~_ 10.1 (C - 1, AcOH).
5XVI)BOC,Ly~Z)~hr(Bzl)L~s(Z)GL~OSu Prepared in four stages from BOC.Thr(Bzl)Lys(Z)GlyOMe described in example 5.
.
- .~ . .
. . ' `~` , ' :
- ' :
(i) Thr (Bzl) Lys (Z ) GlyOMe .Tfa BOC.Thr(Bzl)Lys(Z)GlyOMe (2.87g, 0.0045M~ was BOC-deprotected in 5G% Tfa in MDC (5~ml) for 1 hour at O .
The solution was quenched with ether and the product (2,10g) filtered off in 72% yield~
T~L ~C o 9 1 CHC13:MeOH (I2 s*ain) showed one spot at Rf 0.26.
(ii) BOC~-Lys(æ)Thr(Bzl)Lys(Z)GlyOMe BOC~Lys (Z)OSu (2.00g, 0.003M) was coupled to compound (i) (1.45g, 0.003M) in 10~ DMF/toluene at R~T. overnight.
The reaction mixture was evaporated at reduced pressure and the resulting residue dissolved in EtAc~ washed, dried and evaporated in vacuo to give the product (2.04y) in 74~ yield upon recrystallisation from EtAc/petrol.
M.P~ 117-119.
T.L.C~ 9:1 CHC13:MeOH (I2 stain) showed one spot at Rf 0.60.
(iii)BOC.Lys(Z)Thr(Bzl)Lys_Z)GlyO~
A solution o~ compound (ii) (l.9~g, 0.0022M) in DMSO
(30ml) was treated with lN NaOH solution (1~ equivalents) and stirred at R.T. for 1 hour. Acidi~ication gave the product (1.82g) in quantitative yield.
T.L.C. 5:1 CHC13:MeO~ (I2 stain) showed compound just above baseline.
(iv) BOC .Lys (Z ) Thr_Bæl) Lys (Z ) GlyOSu ~OSu (0.24g, 0.002M~ was oupled to compound (iii) ~1.82g, 0.002M) in dioxan (25ml) at R.T~ ~or 4 hours in the presence of DCCI (1 e~uivalent). The reaction mixture was filtered and the ~iLtxate evaporated at reduced pressure.
Recrystallisation o~ the residue ~rom EtOH gave the product (0.30g) in 15~ yield. M.P. 118-122.
T ~L . C . 9 :1 CHC13:MeOH (I2 stain) showed one spot at R~ 0.50.
[~]D = ~ 4 (C = 1~ DMF). The NMR was consistent with structure.
BOC. ~ Bzl)hys(Z)Gl~r(Bzl)Gly~ePheOBz Peptida (XVI~ (0.22g, 0.0023M) was coupled to compound -. . : - , . : . . . .
... . : .... :, ~
.. . .
. . . . . .
~XV) ~0017g, 0.0023M) in 5% DMF/toluene (21ml) at R.T.
overnight in the presence of Et3N (1 equivalent). The reaction mixture was evaporated at reduced pressure and the residue recrystallised from EtOH to give the product (0.29g)
5 in 85% yield. M.P. 195-199.
T.L.C. 9:1 CHC13:MeOH ~I2 stain3 showed on spot at Rf 0.67.
The NMR was consistent with structure.
LysThrLysGl~serGly-p-hepheoH
The intermediate above ~0.25g, 0.0017M) was dissolved in Tfa ~lOml) and deprotected by bubbling through HBr at R.T. for 1 hour. The solution was quenched with dry ether and the product dried in vacuo over P2O5 and KOH. The product was purified on a Biogel P~ column eluting with water and subse~uently on a CM32~cellulose column eluting with a linear ionic strength gradient of ammonium acetate pH5 which gave separation of the free acid and some benzyl ester contaminant. Final isolation of the product was by lyopholisation.
T.1.C. butanol/acetic acid/water (5:2:2) ~ninhydrin spxay) showed one spot at R~ Oc20. Amino acid analysis:
calculated: 2 ~ys: lThr; 2Gly: lSer: 2Phe ~ound: 1.77~ys: 0.96Thx: 2.00Gly: 1.06Ser: 2.10Phe L38199~ e~ ty Biologlcal results obta~ned for Example 1-6 in three different assay systems are presented in Tables 1 and 2.
As is apparent from Table 1, the peptides were capable of releasing histamine selectively from rat mast cells in vitro, and producing histamine release e~fects in rat and baboon skin ln vivo. In the latter case in particular (primate tissue) activity was unusually high.
Table 2 demonstrates cross-desensitisation in rat mast cells in vitro between the peptides of Example 3 and an antigen.
~ 7~P-P ~
, . - . : :
~ 30 --able 1 Histamine Release Effects of Synthetic Peptides (Examples 1-6) _ _ . _~ ~ __ _ __ ___ _____~_ PEP~L'IDE 5YST~M Ex~sf ACrIV~Y LDHl~51CSr f ._ _ ~., . _ __ _ , ~ _ Lysl~rLysGlySer- Rat M~st Cells 7 7.5X10 6~ None at 50~hibition GlyPhePheC~qe m vitrol lo-6~ by Intal (ExarrQle No. 1) ~ . tlO-4M) . _ , . ~ .~ .. _ .
Rat ~in 2 l.OX10-4~1 in v~i.vo2 . , .,~ ._ _ ........ ... _ _ _. _ _ .~
Babocn Ski~ 2 5 . OX10 7M
ixl vivo3 ~ ~ , . . _ . ~
ArgL~smrLysGly- Rat ~st Cells 7 1.5X10 7M
SerGlyPhePh~a in ~7itro (Exanple No. 2) __ . - .. - ~ ., .. . _ . .. ~ . _ __ Rat S}~ 2 l.OX10 M
~ vivo . .. __ _. ,. .. , _ , _ ... . ~
Babo~ Skill 2 1. OXlG M
in vivo3 ---_ --~ . _ ~
Lys m rLysGly~exÇly~ Rat Mast Cells 10 l.OX10 M None at PhePheValPheCMe in vitrol 10 4M
(Example No. 3) _ _ peptide ~ . . ~T _ _ ____ _ _ . __ ~___ _ __~ _____ , I . _ __ . _ Ra.t ~kin 5 l.OX10 5M
~vivo2 ,_.. _- - . __ _ _ .__ Babocn 5kin 14 l.OX10 M P.artial vivo3 Inhibition by A~tihistamine or Ihtal-like d~
_____~__ ~ _ _ . _ _ _ . _ _ _ . _ TT
LysIhr~ysGlySer- Rat ~ast Cells 2 3.3XlO 5M
GlyPhePheValPhe in ~ltro (Example No~ 4) _ , : _ _ . __ _ __ ~ T _ ~ . _ _ .__T _ : . _ _ . _ .
Rat ~n 4 l.OX10-4~$
inv~v~ :, __ _ _ .. ._ . ~
, .:
' . ' ', . : ,, .
.
, -- 31 ~
Tahle l (contq) ~ . . ~
PEPTIDE SYSTEM No of AcTr~y LDH~SlCr IA~U~ovtltyQn ~ _ ~ __ _ _ __ __ __e _-_ B~on Skin 2 l.OXlO M
i~ vivo3 ~ _ ~ _ I ~ . r . __ _ . :
Pn~ ysThrLysGly; Rat Mast Cells 2 9.2XlO 6M
SerGlyPhePheCMb in vitro ~E~ple No. 5) - . . - ._ . . ~. . _ _ .
Rat Skin 4 l.OX10-~
i viyo2 ~, . _ . , _ ~
B~xKn 5kin 2 loQXlO M
VlV~3 ~__ _ LysThrLysGly Rat Mast Ce11s 4 loOX10 M
SerGl~PhePhe in vitro (Exa~ple No. 6) ~_ _ _. ___ , _ .
Rat 5kin 4 l.OX10 in vivo2 _._ . ~ ,. . _.
B~xx~ Skin 3 l.OXlO 5M
in vivo3 . ' ' _ _ _ _.. _ _ _ A . -- ---- I .. _ ~
1. The average concentration of peptide to give approximately 50%
release of available histamine from rat mast cells in a number of experiments.
2. The average conaentration of peptide to give an end point in in~radernal titrations ~n rat skin in a number of expeximents.
3. The average concentration of peptide to give an end point in intradernal ti~rations in baboon skin in a number of exp~arimeAts .
' ~, - 32 ~
Table 2 Cross Desensitisation betw~en Antlgc_L ~_~BI~ 7 (PeE tide of Example 3 ) n the Rat Mast Cell System ~ . . . . _ .
Total ~sensitiser 1 Desen~ ;itisati~ ~ Steps 4 Challenge histamne . _ . ~ _ ~ . n .~. . _ /. . __ ~ ___ Bu:Efer Bu~fer Buf~er Buf~er :!3uffer B~ fer 4 Buffer Buffer Buf fer Buffer(10~ 3M) 47 ~uffer Buffer Buffer Buffer Xt~ l~ml 56 ~____ _ ~ . . .~ . __ , . . . . ~ . _ BRL 21547 5XlO 8M 10-7M 5XlO 7M lO 6M Buffer 6 5Xl -8 10-7M 5X10 7M lo~6M ~ 21547 6 5X10 M 10--7M 5X10--7M lo~6M XC~ l~g/ml 17 __ ~ ~ ,,~,.... ~ ... ~
O.C05 (,u~/ml) 0.01 0~05 0.1 Bui~er 6 0 . 005 ~l~g/ml 0 . 01 0 . 05 0 . l tlo_5M) 6 0.005(~g/ml) 0.01 O.OS 0.1 }~OA l,ug/ml 6 ~ ~ ... _ , . __ _ . __ . ... ,._ . .
Metho.ds " , . . ~
(a) (l) Histamine~ (2~ cr51 and (3) Lactic Dehvdroaenase Release from Rat ~3R~ a~Mast Ceils ~ t Mast Cell in v _ tes~
.
Mast cells, derived from the peritoneal washings of three male, outbred Wistar rates (250-300g), were puri~ied by the procedure according to Cooper and S~anworth (Preparati~e Biochem. 4~2), 105, 1975).
The purified ceIls were washed twice in Dulbecco's incomplete (i.e. fxee ~rom mineral salts) bufer and then resuspended in Dulbecco~s medi~un to the re~uired volume.
In a typical experiment~ sufficient cells were available for 30 duplicate challenges~ i.e. 60 samples and in this case the resuspension volume employed was 6.1 mls, 0~1 ml o~ cell suspension were taken ~or estimating the cell count.
. , , . . , ~ . .
.: . .......... , .,,: . .~ .. . . : . . ..
, - , . . -: . - . . :. :
_ (1) Histamlne release:
One third of the cell suspension was employed. To 0.9 ml duplicate ali~uots of challenge solution, prepared in complete Dulbecco's medium and prewarmed to 37C, was added 0.1 ml of cell suspension. The solutions were then shaken gently, and allowed to incubate for 5 minutes at 37C. The reaction tubes wers then quickly removed from the incubator and placed in an ice bath. Supernatants were then separated from the cell population following centrifugation for 3 minutes at lOOO r~p.m. The cell residues were then treated with 2 mls of 0.4N perchloric acid and allowed to stand or appr~ximately 30 minutes at ambient temperature. The precipitated protein was removed by centrifugation and the supernatant solutions set aside for histamine analysis~ The original supernatant solutions w0re treated with 1.0 ml of 0.8N perchlorate and then treated in a similar manner to the cell residues.
Histamine was measured by the method according to Evans, Lewis and Thompson (Life Sci~nces, 12, 327, 1973) using a Technicon Auto-analyser. Histamine release was calculated as a percentage of total histamine available in each challenge solution.
(2) Cr51 release:
One third of the cell suspension was employed. To approximately 2.0 ml o~ cell suspension in Dulbecco's medium was added 0.1 ml of a solutlon of Cr labelled sodium chromate. Approximately 50-lOO~Cl Cr5l was employed (specific activity: 300-500 ~Ci/mg Cr). The cells were allowed to stand for 30 minutes at ambient temperature and then excess chromium was removed by washing the cells three times in Dulbecco' 5 buffer. The cell pellet was finally resuspended in the same buffer and 0.1 ml of cell swspension was then added to 0.9 ml of each challenge solution, prewarmed to 37Co After 5 minutes'incubation the cell suspensions were removed from the water bath and the supernants separated by centrifugation. Activity present in the whole recovered supernatants was measured using a .
. . . . .
, ,, . . - , ~ -. . ~ . - - , . :
- 34 ~
Tracer Laborakor~ Spectromatic y counter. The percentage of Cr released was assessed in relation to the values obtained for the positive and negative control solutions.
(3) LDH measurement:
one third of the cell suspension was employed~ The incubation procedure was identical to that described above and carried out simultaneously until the challenge solution supernatants were separated from the cell residues. Lactic dehydrogenase activity was then estimated directly in the supernatant solutions by the method according to Johnson and Erdos (Proc. Soc. Exp. Biol. Med. 142. 1252. 1973).
To 0.5 ml o supernatant was added 0.5 ml of NAD (lmM in o.2M Tris buffer, pH 8.5)~ 0.5 ml of this solution was then taken and treated with 50~1 of lactic acid (50mM in 0~2M Tris buffer, pH 8.5); as control, 50~1 of 0.2M Tris bufer (pH 8.5) was added to a second aliquot (0.5 ml) of the NAD solution. The solutions were incubated at ambient temperature for 20 minutes and the fluorescence emission was then measured. The excitation and emission wave leng~hs used were 340 and 460nm respectively. All measurements were carried out using a Baird Atomic automatic spectrofluorimeter (Fluoripoint). The LDH
activity was assessed in terms of the increase of fluorescence over control due to N~DH fonnation ~ollowing lactate addition. The percentage of LDH released was assessed in relation to the fluorescence intensity obtained in the positiva control challenge solution supernatan~s ~i.e. Triton X 100 challenge).
(b) Skin Test Method _ _ _ Skin tests were carrled out in the shaved backs of animals ~rats and bahoons~ immediately after intravenous injection of pon~amine sky blue (5~) in aqueous sodium chlorid~ solution (0.9%) at a dose of 0.1 ml per kilogram of body welght in the case of rats and 5 ml per anim~l in the case of baboons.
.
. . ; - . - .. . - :.- . . , :,:.. , .. . : . . :
, : . . . ,. :,, ., , ,:
: ,. . .. . .. : : :
. .: - , , ,., ,' : ' -Peptide in aqueous sodium chloride solution (0.9~), or saline control, were injected intradernally in 0.05 ml or 0.10 ml volumes. Skin reactions were read 20 minutes after intradernal challenge.
(c) Cross Desensitisation in the in vitro rat mast cell system between anti~en and peptide Brown Norway rats were immunised intraperitoneally with lOO~g of ovalbumen (XOA) in lmg 'alum'. On day 27, peritoneal ~ast cells were removed, bulked and washed.
Aliquots of cells were desensitised by the addition of 4 x 5 minute incubations with various XOA or peptide concen~rations or buffer alo~e. The cells were then submitted to an optimal histamine releasing challenge of peptide, XOA, or challenged with buffer alone.
~-r~
:
.
. ~ .. .. - .. , . . , .
... .. . - ... ~
. ; . . . . . .
.. . . . ...
.. , . ., ..... , -. .. . ~ . . . . ~ . . .
T.L.C. 9:1 CHC13:MeOH ~I2 stain3 showed on spot at Rf 0.67.
The NMR was consistent with structure.
LysThrLysGl~serGly-p-hepheoH
The intermediate above ~0.25g, 0.0017M) was dissolved in Tfa ~lOml) and deprotected by bubbling through HBr at R.T. for 1 hour. The solution was quenched with dry ether and the product dried in vacuo over P2O5 and KOH. The product was purified on a Biogel P~ column eluting with water and subse~uently on a CM32~cellulose column eluting with a linear ionic strength gradient of ammonium acetate pH5 which gave separation of the free acid and some benzyl ester contaminant. Final isolation of the product was by lyopholisation.
T.1.C. butanol/acetic acid/water (5:2:2) ~ninhydrin spxay) showed one spot at R~ Oc20. Amino acid analysis:
calculated: 2 ~ys: lThr; 2Gly: lSer: 2Phe ~ound: 1.77~ys: 0.96Thx: 2.00Gly: 1.06Ser: 2.10Phe L38199~ e~ ty Biologlcal results obta~ned for Example 1-6 in three different assay systems are presented in Tables 1 and 2.
As is apparent from Table 1, the peptides were capable of releasing histamine selectively from rat mast cells in vitro, and producing histamine release e~fects in rat and baboon skin ln vivo. In the latter case in particular (primate tissue) activity was unusually high.
Table 2 demonstrates cross-desensitisation in rat mast cells in vitro between the peptides of Example 3 and an antigen.
~ 7~P-P ~
, . - . : :
~ 30 --able 1 Histamine Release Effects of Synthetic Peptides (Examples 1-6) _ _ . _~ ~ __ _ __ ___ _____~_ PEP~L'IDE 5YST~M Ex~sf ACrIV~Y LDHl~51CSr f ._ _ ~., . _ __ _ , ~ _ Lysl~rLysGlySer- Rat M~st Cells 7 7.5X10 6~ None at 50~hibition GlyPhePheC~qe m vitrol lo-6~ by Intal (ExarrQle No. 1) ~ . tlO-4M) . _ , . ~ .~ .. _ .
Rat ~in 2 l.OX10-4~1 in v~i.vo2 . , .,~ ._ _ ........ ... _ _ _. _ _ .~
Babocn Ski~ 2 5 . OX10 7M
ixl vivo3 ~ ~ , . . _ . ~
ArgL~smrLysGly- Rat ~st Cells 7 1.5X10 7M
SerGlyPhePh~a in ~7itro (Exanple No. 2) __ . - .. - ~ ., .. . _ . .. ~ . _ __ Rat S}~ 2 l.OX10 M
~ vivo . .. __ _. ,. .. , _ , _ ... . ~
Babo~ Skill 2 1. OXlG M
in vivo3 ---_ --~ . _ ~
Lys m rLysGly~exÇly~ Rat Mast Cells 10 l.OX10 M None at PhePheValPheCMe in vitrol 10 4M
(Example No. 3) _ _ peptide ~ . . ~T _ _ ____ _ _ . __ ~___ _ __~ _____ , I . _ __ . _ Ra.t ~kin 5 l.OX10 5M
~vivo2 ,_.. _- - . __ _ _ .__ Babocn 5kin 14 l.OX10 M P.artial vivo3 Inhibition by A~tihistamine or Ihtal-like d~
_____~__ ~ _ _ . _ _ _ . _ _ _ . _ TT
LysIhr~ysGlySer- Rat ~ast Cells 2 3.3XlO 5M
GlyPhePheValPhe in ~ltro (Example No~ 4) _ , : _ _ . __ _ __ ~ T _ ~ . _ _ .__T _ : . _ _ . _ .
Rat ~n 4 l.OX10-4~$
inv~v~ :, __ _ _ .. ._ . ~
, .:
' . ' ', . : ,, .
.
, -- 31 ~
Tahle l (contq) ~ . . ~
PEPTIDE SYSTEM No of AcTr~y LDH~SlCr IA~U~ovtltyQn ~ _ ~ __ _ _ __ __ __e _-_ B~on Skin 2 l.OXlO M
i~ vivo3 ~ _ ~ _ I ~ . r . __ _ . :
Pn~ ysThrLysGly; Rat Mast Cells 2 9.2XlO 6M
SerGlyPhePheCMb in vitro ~E~ple No. 5) - . . - ._ . . ~. . _ _ .
Rat Skin 4 l.OX10-~
i viyo2 ~, . _ . , _ ~
B~xKn 5kin 2 loQXlO M
VlV~3 ~__ _ LysThrLysGly Rat Mast Ce11s 4 loOX10 M
SerGl~PhePhe in vitro (Exa~ple No. 6) ~_ _ _. ___ , _ .
Rat 5kin 4 l.OX10 in vivo2 _._ . ~ ,. . _.
B~xx~ Skin 3 l.OXlO 5M
in vivo3 . ' ' _ _ _ _.. _ _ _ A . -- ---- I .. _ ~
1. The average concentration of peptide to give approximately 50%
release of available histamine from rat mast cells in a number of experiments.
2. The average conaentration of peptide to give an end point in in~radernal titrations ~n rat skin in a number of expeximents.
3. The average concentration of peptide to give an end point in intradernal ti~rations in baboon skin in a number of exp~arimeAts .
' ~, - 32 ~
Table 2 Cross Desensitisation betw~en Antlgc_L ~_~BI~ 7 (PeE tide of Example 3 ) n the Rat Mast Cell System ~ . . . . _ .
Total ~sensitiser 1 Desen~ ;itisati~ ~ Steps 4 Challenge histamne . _ . ~ _ ~ . n .~. . _ /. . __ ~ ___ Bu:Efer Bu~fer Buf~er Buf~er :!3uffer B~ fer 4 Buffer Buffer Buf fer Buffer(10~ 3M) 47 ~uffer Buffer Buffer Buffer Xt~ l~ml 56 ~____ _ ~ . . .~ . __ , . . . . ~ . _ BRL 21547 5XlO 8M 10-7M 5XlO 7M lO 6M Buffer 6 5Xl -8 10-7M 5X10 7M lo~6M ~ 21547 6 5X10 M 10--7M 5X10--7M lo~6M XC~ l~g/ml 17 __ ~ ~ ,,~,.... ~ ... ~
O.C05 (,u~/ml) 0.01 0~05 0.1 Bui~er 6 0 . 005 ~l~g/ml 0 . 01 0 . 05 0 . l tlo_5M) 6 0.005(~g/ml) 0.01 O.OS 0.1 }~OA l,ug/ml 6 ~ ~ ... _ , . __ _ . __ . ... ,._ . .
Metho.ds " , . . ~
(a) (l) Histamine~ (2~ cr51 and (3) Lactic Dehvdroaenase Release from Rat ~3R~ a~Mast Ceils ~ t Mast Cell in v _ tes~
.
Mast cells, derived from the peritoneal washings of three male, outbred Wistar rates (250-300g), were puri~ied by the procedure according to Cooper and S~anworth (Preparati~e Biochem. 4~2), 105, 1975).
The purified ceIls were washed twice in Dulbecco's incomplete (i.e. fxee ~rom mineral salts) bufer and then resuspended in Dulbecco~s medi~un to the re~uired volume.
In a typical experiment~ sufficient cells were available for 30 duplicate challenges~ i.e. 60 samples and in this case the resuspension volume employed was 6.1 mls, 0~1 ml o~ cell suspension were taken ~or estimating the cell count.
. , , . . , ~ . .
.: . .......... , .,,: . .~ .. . . : . . ..
, - , . . -: . - . . :. :
_ (1) Histamlne release:
One third of the cell suspension was employed. To 0.9 ml duplicate ali~uots of challenge solution, prepared in complete Dulbecco's medium and prewarmed to 37C, was added 0.1 ml of cell suspension. The solutions were then shaken gently, and allowed to incubate for 5 minutes at 37C. The reaction tubes wers then quickly removed from the incubator and placed in an ice bath. Supernatants were then separated from the cell population following centrifugation for 3 minutes at lOOO r~p.m. The cell residues were then treated with 2 mls of 0.4N perchloric acid and allowed to stand or appr~ximately 30 minutes at ambient temperature. The precipitated protein was removed by centrifugation and the supernatant solutions set aside for histamine analysis~ The original supernatant solutions w0re treated with 1.0 ml of 0.8N perchlorate and then treated in a similar manner to the cell residues.
Histamine was measured by the method according to Evans, Lewis and Thompson (Life Sci~nces, 12, 327, 1973) using a Technicon Auto-analyser. Histamine release was calculated as a percentage of total histamine available in each challenge solution.
(2) Cr51 release:
One third of the cell suspension was employed. To approximately 2.0 ml o~ cell suspension in Dulbecco's medium was added 0.1 ml of a solutlon of Cr labelled sodium chromate. Approximately 50-lOO~Cl Cr5l was employed (specific activity: 300-500 ~Ci/mg Cr). The cells were allowed to stand for 30 minutes at ambient temperature and then excess chromium was removed by washing the cells three times in Dulbecco' 5 buffer. The cell pellet was finally resuspended in the same buffer and 0.1 ml of cell swspension was then added to 0.9 ml of each challenge solution, prewarmed to 37Co After 5 minutes'incubation the cell suspensions were removed from the water bath and the supernants separated by centrifugation. Activity present in the whole recovered supernatants was measured using a .
. . . . .
, ,, . . - , ~ -. . ~ . - - , . :
- 34 ~
Tracer Laborakor~ Spectromatic y counter. The percentage of Cr released was assessed in relation to the values obtained for the positive and negative control solutions.
(3) LDH measurement:
one third of the cell suspension was employed~ The incubation procedure was identical to that described above and carried out simultaneously until the challenge solution supernatants were separated from the cell residues. Lactic dehydrogenase activity was then estimated directly in the supernatant solutions by the method according to Johnson and Erdos (Proc. Soc. Exp. Biol. Med. 142. 1252. 1973).
To 0.5 ml o supernatant was added 0.5 ml of NAD (lmM in o.2M Tris buffer, pH 8.5)~ 0.5 ml of this solution was then taken and treated with 50~1 of lactic acid (50mM in 0~2M Tris buffer, pH 8.5); as control, 50~1 of 0.2M Tris bufer (pH 8.5) was added to a second aliquot (0.5 ml) of the NAD solution. The solutions were incubated at ambient temperature for 20 minutes and the fluorescence emission was then measured. The excitation and emission wave leng~hs used were 340 and 460nm respectively. All measurements were carried out using a Baird Atomic automatic spectrofluorimeter (Fluoripoint). The LDH
activity was assessed in terms of the increase of fluorescence over control due to N~DH fonnation ~ollowing lactate addition. The percentage of LDH released was assessed in relation to the fluorescence intensity obtained in the positiva control challenge solution supernatan~s ~i.e. Triton X 100 challenge).
(b) Skin Test Method _ _ _ Skin tests were carrled out in the shaved backs of animals ~rats and bahoons~ immediately after intravenous injection of pon~amine sky blue (5~) in aqueous sodium chlorid~ solution (0.9%) at a dose of 0.1 ml per kilogram of body welght in the case of rats and 5 ml per anim~l in the case of baboons.
.
. . ; - . - .. . - :.- . . , :,:.. , .. . : . . :
, : . . . ,. :,, ., , ,:
: ,. . .. . .. : : :
. .: - , , ,., ,' : ' -Peptide in aqueous sodium chloride solution (0.9~), or saline control, were injected intradernally in 0.05 ml or 0.10 ml volumes. Skin reactions were read 20 minutes after intradernal challenge.
(c) Cross Desensitisation in the in vitro rat mast cell system between anti~en and peptide Brown Norway rats were immunised intraperitoneally with lOO~g of ovalbumen (XOA) in lmg 'alum'. On day 27, peritoneal ~ast cells were removed, bulked and washed.
Aliquots of cells were desensitised by the addition of 4 x 5 minute incubations with various XOA or peptide concen~rations or buffer alo~e. The cells were then submitted to an optimal histamine releasing challenge of peptide, XOA, or challenged with buffer alone.
~-r~
:
.
. ~ .. .. - .. , . . , .
... .. . - ... ~
. ; . . . . . .
.. . . . ...
.. , . ., ..... , -. .. . ~ . . . . ~ . . .
Claims (36)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of a peptide or a salt thereof characterized by containing a sequence of 6 to 12 naturally occurring amino acid residues ?R1-R2-R3?, wherein, R1 consîsts of a residue of a basic amino acid, op-tionally linked to one or more residues of a neutral non-hydrophobic amino acid or to one or more further residues of a baslc amino acid or optionally linked to one or more of both residues;
R2 consists of one or more residues of a neutral non-hydrophobic amino acid; and R3 consists of a residue of a hydrophobic amino acid, optionally linked to one or more residues of a neutral non-hydrophobic amino acid or to one or more further residues of a hydrophobic amino acid or optionally linked to one or more of both residues;
the said basic amino acid resldues are selected from arginyl, lysyl and ornithyl;
the said neutral non-hydrophobic amino acid residues are selected from glycyl, alanyl, seryl and threonyl; and the said hydrophobic amino acid residues are selected from phenylalanyl, valyl and leucyl;
said peptide having the formula (I):
X-R?R1-R2-R3?Y (I) wherein the sequence of ?R1-R2-R3?- is as defined and;
X is hydrogen, or a N-protecting group;
Y is hydroxy, or a C-terminal protecting group; and R is an optionally present group, capable of confer-ing on a peptide resistance to enzyme breakdown, which process comprises the sequential coupling of the amino acids from which the peptide is derived.
R2 consists of one or more residues of a neutral non-hydrophobic amino acid; and R3 consists of a residue of a hydrophobic amino acid, optionally linked to one or more residues of a neutral non-hydrophobic amino acid or to one or more further residues of a hydrophobic amino acid or optionally linked to one or more of both residues;
the said basic amino acid resldues are selected from arginyl, lysyl and ornithyl;
the said neutral non-hydrophobic amino acid residues are selected from glycyl, alanyl, seryl and threonyl; and the said hydrophobic amino acid residues are selected from phenylalanyl, valyl and leucyl;
said peptide having the formula (I):
X-R?R1-R2-R3?Y (I) wherein the sequence of ?R1-R2-R3?- is as defined and;
X is hydrogen, or a N-protecting group;
Y is hydroxy, or a C-terminal protecting group; and R is an optionally present group, capable of confer-ing on a peptide resistance to enzyme breakdown, which process comprises the sequential coupling of the amino acids from which the peptide is derived.
2. A process as claimed in claim 1 wherein the peptide or a pharmaceutically acceptable salt thereof is prepared.
3. A process as cla.imed in claim 1 wherein a peptide of the formula (I) wherein R is not present, X is hydrogen and Y is hydroxy, amino or methoxy is prepared.
4. A process as claimed in claim 1 wherein the peptide contains a sequence of 8 to 10 naturally occurring amino acid residues ?R1-R2-R3? is prepared.
5. A process as claimed in claim 1 wherein a peptide of the formula (I) wherein R is not present, X is hydrogen and Y is hydroxy, amino or methoxy is prepared and the peptide contains a sequence of 8 to 10 naturally occurring amino acid residues ?R1-R2-R3? is prepared.
6. A process as claimed in claim 5 wherein R3 only con-tains hydrophobic amino acid residues is prepared.
7. A process as claimed in claim 1 wherein the peptide has the structure X-R-[b-c-d-e]-[f-g-h]-[i-j-k-l]-Y
wherein X, Y and R are as defined in claim 1; c and e are lysyl, arginyl or ornithyl; d is threonyl or seryl; b is an optionally present arginyl, lysyl or ornithyl; f and h are glycyl or alanyl; g is seryl or threonyl; and i and j are phenylalanyl, valyl or leucyl; and k and l are optionally present phenylalanyl, valyl or leucyl; or a salt thereof is prepared.
wherein X, Y and R are as defined in claim 1; c and e are lysyl, arginyl or ornithyl; d is threonyl or seryl; b is an optionally present arginyl, lysyl or ornithyl; f and h are glycyl or alanyl; g is seryl or threonyl; and i and j are phenylalanyl, valyl or leucyl; and k and l are optionally present phenylalanyl, valyl or leucyl; or a salt thereof is prepared.
8. A process as claimed in claim 1 wherein a peptide selected from:
wherein Y1 is hydroxyl, -NH2 or methoxy, or a salt thereof is prepared.
wherein Y1 is hydroxyl, -NH2 or methoxy, or a salt thereof is prepared.
9. A process as claimed in claim 7 wherein the peptide or a pharmaceutically acceptable salt thereof is prepared.
10. A process as claimed in claim 8 wherein the peptide or a pharmaceutically acceptable salt thereof is prepared.
11. A process for the preparation of a peptide of the formula which comprises synthesizing the peptide by a 4+2+4 fragment condensation strategy consisting of coupling and BOC -deprotecting and to obtain which is then coupled with and deprotected, and isolating the final product.
12. A process as claimed in claim 1 wherein the peptide of the formula or a pharmaceutically acceptable salt thereof is prepared.
13. A process as claimed in claim 1 wherein the peptide of the formula or a pharmaceutically acceptable salt thereof is prepared.
14. A process for the preparation of a peptide of the formula which comprises synthesizing by a 4+2+4 fragment condensa-tion strategy consisting of coupling and BOC-deprotecting and to obtain , which is then coupled with to obtain , from which, on selective deprotection, the product is isolated.
15. A process as claimed in claim 1, wherein the peptide of the formula or a pharmaceutically acceptable salt thereof is prepared.
16. A peptide of the formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof whenever pre-pared by the process of claim 2 or an obvious chemical equivalent thereof.
17. A peptide of the formula X-R-[b-c-d-e]-[f-g-h]-[i-j-k-l]-Y
wherein the symbols are defined as in claim 7 or a pharmaceutically acceptable salt thereof whenever prepared by the process of claim 9 or an obvious chemical equivalent thereof.
wherein the symbols are defined as in claim 7 or a pharmaceutically acceptable salt thereof whenever prepared by the process of claim 9 or an obvious chemical equivalent thereof.
18. A peptide selected from the peptides of the formulae as defined in claim 8 or a pharmaceutically acceptalbe salt thereof whenever prepared by the process of claim 10 or an obvious chemical equivalent thereof.
19. A peptide of the formula defined in claim 11 whenever prepared by the process of claim 11 or an obvious chemical equivalent thereof.
20. A peptide of the formula defined in claim 12 or a pharmaceutically acceptable salt thereof whenever prepared by the process of claim 12 or an obvious chemical equivalent thereof.
21. A peptide of the formula defined in claim 13 or a pharmaceutically acceptable salt thereof whenever prepared by the process of claim 13 or an obvious chemical equivalent thereof.
22. A peptide of the formula defined in claim 14 whenever prepared by the process of claim 14 or an obvious chemical equivalent thereof.
23. A peptide of the formula defined in claim 15 or a pharmaceutically acceptable salt thereof whenever prepared by the process of claim 15 or an obvious chemical equivalent thereof.
24. A process as claimed in claim 1 wherein the sequential coupling of amino acids to form amide links con-sists of providing the amino acids with protecting groups, where necessary and coupling the acids in the correct order.
25. A process as claimed in claim 1 wherein the sequential coupling of amino acids to form amide links con-sists of combining smaller peptides into larger units.
26. A process as claimed in claim 24 wherein the amide links are formed by condensing an amino acid, or peptide, having a protected .alpha.-amino group and a free or activated terminal carboxyl group, with an amino acid or peptide with a protected carboxyl group and a free .alpha.-amino group.
27. A process as claimed in claim 25 wherein the amide links are formed by condensing an amino acid, or peptide, having a protected .alpha.-amino group and a free or activated terminal carboxyl group, with an amino acid or peptide with a protected carboxyl group and a free .alpha.-amino group.
28. A process as claimed in claim 26 or 27 wherein activation of the carboxyl group is effected by converting the carboxyl group into an acid halide, an azide, an anhy-dride, an imidazolide, or an activated ester.
29. A process as claimed in claim 26 or 27 wherein activation of the carboxyl group is effected by converting the carboxyl group into an activated ester selected from cyanomethyl ester, p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester or benztriazole ester.
30. A process as claimed in claim 26 or 27 wherein the condensation of amino acids or peptides is carried out by the carbodiimide method, the azide method, the anhydride method, the activated esters method or the solid phase method.
31. A process as claimed in claim 26 or 27 wherein the reactive groups in the amino acid or peptide which are not to take part in the condensation reaction are protected by N-protecting groups or carboxyl protecting groups.
32. A process as claimed in claim 26 or 27 wherein the protecting groups present in the resultant peptide are re-moved by one of the following procedures:
a) hydrogenation in the presence of palladium catalyst;
b) treatment with hydrogen bromide in glacial acetic acid;
c) treatment with metallic sodium in liquid ammonia;
d) treatment with hydrochloric acid or acetic acid or both acids;
e) treatment with alkali;
f) treatment with acid; and g) hydrogenation in the presence of palladium catalyst.
a) hydrogenation in the presence of palladium catalyst;
b) treatment with hydrogen bromide in glacial acetic acid;
c) treatment with metallic sodium in liquid ammonia;
d) treatment with hydrochloric acid or acetic acid or both acids;
e) treatment with alkali;
f) treatment with acid; and g) hydrogenation in the presence of palladium catalyst.
33. A process as claimed in claim 2 wherein the pharmace-utically acceptalbe salts are selected from hydrohalide, phosphate, acetate, phenylpropionate, maleate, tartrate and citrate salts.
34. A process as claimed in claim 33 wherein the salts are the hydrochloride or hydrobromide salts.
35. A peptide of the formula (I) as defined in claim 1 in the form of the pharmaceutically acceptable salts defined in claim 33 whenever prepared by the process of claim 33 or an obvious chemical equivalent thereof.
36. A peptide of the formula (I) as defined in claim 1 in the form of the hydrochloride or hydrobromide salts whenever prepared by the process of claim 34 or an obvious chemical equivalent thereof.
Applications Claiming Priority (2)
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GB27140/77 | 1977-06-29 |
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CA1105006A true CA1105006A (en) | 1981-07-14 |
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ID=10254870
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EP (1) | EP0000252B1 (en) |
JP (1) | JPS5416402A (en) |
AU (1) | AU522641B2 (en) |
CA (1) | CA1105006A (en) |
DE (1) | DE2861593D1 (en) |
DK (1) | DK292778A (en) |
ES (1) | ES471243A1 (en) |
IE (1) | IE47105B1 (en) |
IL (1) | IL54967A (en) |
IT (1) | IT7850095A0 (en) |
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JPS56119178U (en) * | 1980-02-13 | 1981-09-11 | ||
US4683292A (en) * | 1983-08-12 | 1987-07-28 | Immunetech, Inc. | Immunotherapeutic polypeptide agents which bind to lymphocyte immunoglobulin FC receptors |
US4752601A (en) * | 1983-08-12 | 1988-06-21 | Immunetech Pharmaceuticals | Method of blocking immune complex binding to immunoglobulin FC receptors |
JPS62500025A (en) * | 1984-08-10 | 1987-01-08 | イミユーンテツク・フアーマシユーテイカルズ | Polypeptide agents for immunotherapy |
US5955076A (en) * | 1989-06-15 | 1999-09-21 | Peptide Therapeutics Limited | Immunoactive peptides and antibodies and their use in anti-allergy treatment |
GB8913737D0 (en) * | 1989-06-15 | 1989-08-02 | Univ Birmingham | A novel anti-allergy treatment |
GB9422294D0 (en) * | 1994-11-04 | 1994-12-21 | Peptide Therapeutics Ltd | Peptides for anti-allergy treatment |
US6262017B1 (en) | 1996-01-16 | 2001-07-17 | Research Corporation Technologies, Inc. | Peptides for altering osteoblast adhesion |
DE69718150T3 (en) | 1996-03-01 | 2009-03-05 | Novartis Ag | PEPTIDE IMMUNOGENES FOR THE PROTECTION AGAINST THE TREATMENT OF ALLERGIES |
US6573372B2 (en) * | 1999-01-07 | 2003-06-03 | Heska Corporation | Feline immunoglobulin E molecules and compositions there of |
RU2309144C2 (en) | 2005-03-25 | 2007-10-27 | Общество С Ограниченной Ответственностью "Фарминтерпрайсез" | Phenyl-containing n-acylamine derivatives, method for production thereof, pharmaceutical composition and uses thereof as anti-inflammatory and analgesic agents |
US7425020B2 (en) | 2006-04-25 | 2008-09-16 | Mats Lindkvist | Steering wheel suspension system |
ES2304223B2 (en) | 2007-03-08 | 2009-05-01 | Universidad De Sevilla | NEW CANDIES WITH ELEVATE CONTAINED IN PREBIOTIC OLIGOSACARIDS, PREPARATION AND USE PROCEDURE. |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NL6501206A (en) * | 1964-02-12 | 1965-08-13 | ||
NL6509727A (en) * | 1965-07-28 | 1967-01-30 | ||
US3832337A (en) * | 1970-07-28 | 1974-08-27 | Squibb & Sons Inc | Peptide enzyme inhibitors |
JPS5648502B2 (en) * | 1972-01-17 | 1981-11-16 | ||
US3864481A (en) * | 1972-12-14 | 1975-02-04 | St Lukes Hospital | Anti disease producing synthetic material for the prevention suppression and diagnosis of multiple sclerosis and method of treatment therefor |
US3987014A (en) * | 1974-01-10 | 1976-10-19 | Becton, Dickinson And Company | Secretin intermediates and derivatives |
US4113858A (en) * | 1975-01-20 | 1978-09-12 | St. Luke's Hospital | Novel compounds, compositions and methods of their use |
DE2649146A1 (en) * | 1975-10-29 | 1977-05-12 | Parke Davis & Co | NONAPEPTIDES |
SE436645C (en) * | 1976-04-29 | 1996-07-04 | Bonnierfoeretagen Ab | Antigenically active polypeptide which can be used in cancer diagnosis and in the production of antibodies |
US4059693A (en) * | 1976-06-11 | 1977-11-22 | University Patents, Inc. | Analgesic action of substance P |
US4087419A (en) * | 1976-11-05 | 1978-05-02 | Parke, Davis & Company | Heptapeptides and methods for their production |
-
1978
- 1978-06-15 EP EP78300046A patent/EP0000252B1/en not_active Expired
- 1978-06-15 DE DE7878300046T patent/DE2861593D1/en not_active Expired
- 1978-06-21 NZ NZ187638A patent/NZ187638A/en unknown
- 1978-06-21 IL IL54967A patent/IL54967A/en unknown
- 1978-06-28 IE IE1290/78A patent/IE47105B1/en unknown
- 1978-06-28 CA CA306,411A patent/CA1105006A/en not_active Expired
- 1978-06-28 ZA ZA00783699A patent/ZA783699B/en unknown
- 1978-06-28 DK DK292778A patent/DK292778A/en not_active Application Discontinuation
- 1978-06-28 ES ES471243A patent/ES471243A1/en not_active Expired
- 1978-06-29 AU AU37628/78A patent/AU522641B2/en not_active Expired
- 1978-06-29 JP JP7920678A patent/JPS5416402A/en active Pending
- 1978-06-29 IT IT7850095A patent/IT7850095A0/en unknown
- 1978-06-29 US US05/920,151 patent/US4223016A/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
DK292778A (en) | 1978-12-30 |
US4223016A (en) | 1980-09-16 |
IE781290L (en) | 1978-12-29 |
ES471243A1 (en) | 1979-10-01 |
EP0000252A1 (en) | 1979-01-10 |
AU3762878A (en) | 1980-01-03 |
IL54967A0 (en) | 1978-08-31 |
IL54967A (en) | 1982-03-31 |
NZ187638A (en) | 1981-07-13 |
AU522641B2 (en) | 1982-06-17 |
JPS5416402A (en) | 1979-02-07 |
DE2861593D1 (en) | 1982-03-11 |
ZA783699B (en) | 1979-06-27 |
IE47105B1 (en) | 1983-12-28 |
IT7850095A0 (en) | 1978-06-29 |
EP0000252B1 (en) | 1982-02-03 |
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