CA2105872A1 - Macrocyclic thioether ligands and their use as intermediates for binding ions to substrates - Google Patents
Macrocyclic thioether ligands and their use as intermediates for binding ions to substratesInfo
- Publication number
- CA2105872A1 CA2105872A1 CA 2105872 CA2105872A CA2105872A1 CA 2105872 A1 CA2105872 A1 CA 2105872A1 CA 2105872 CA2105872 CA 2105872 CA 2105872 A CA2105872 A CA 2105872A CA 2105872 A1 CA2105872 A1 CA 2105872A1
- Authority
- CA
- Canada
- Prior art keywords
- ligand
- group
- ligands
- extended
- substituted
- 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.)
- Abandoned
Links
- 239000000758 substrate Substances 0.000 title claims abstract description 16
- 239000003446 ligand Substances 0.000 title claims description 56
- 230000027455 binding Effects 0.000 title claims description 8
- 150000003568 thioethers Chemical class 0.000 title description 7
- 239000000543 intermediate Substances 0.000 title description 4
- 150000002500 ions Chemical class 0.000 title description 3
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 12
- 229910052711 selenium Inorganic materials 0.000 claims abstract description 9
- 229910052714 tellurium Inorganic materials 0.000 claims abstract description 9
- 125000005647 linker group Chemical group 0.000 claims abstract description 8
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 6
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 4
- 125000000547 substituted alkyl group Chemical group 0.000 claims abstract description 3
- 229910052751 metal Inorganic materials 0.000 claims description 16
- 239000002184 metal Substances 0.000 claims description 16
- 150000004696 coordination complex Chemical class 0.000 claims description 4
- 125000002768 hydroxyalkyl group Chemical group 0.000 claims description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- 125000004183 alkoxy alkyl group Chemical group 0.000 claims description 3
- 125000005127 aryl alkoxy alkyl group Chemical group 0.000 claims description 3
- 125000005160 aryl oxy alkyl group Chemical group 0.000 claims description 3
- 238000001959 radiotherapy Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 15
- 108090000623 proteins and genes Proteins 0.000 abstract description 15
- 150000001875 compounds Chemical class 0.000 abstract description 12
- 239000013522 chelant Substances 0.000 abstract description 8
- 238000002560 therapeutic procedure Methods 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- PQNPKQVPJAHPSB-UHFFFAOYSA-N 1,4,7-trithionane Chemical compound C1CSCCSCCS1 PQNPKQVPJAHPSB-UHFFFAOYSA-N 0.000 abstract description 2
- 229910052785 arsenic Inorganic materials 0.000 abstract description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 17
- 239000000243 solution Substances 0.000 description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 239000002738 chelating agent Substances 0.000 description 8
- -1 cyano- Chemical class 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 6
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 6
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 6
- 150000002148 esters Chemical class 0.000 description 6
- 150000002739 metals Chemical class 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 125000004429 atom Chemical group 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002523 gelfiltration Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 230000001588 bifunctional effect Effects 0.000 description 4
- 239000010949 copper Substances 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 238000005227 gel permeation chromatography Methods 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 229910052753 mercury Inorganic materials 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000005864 Sulphur Substances 0.000 description 3
- VWQVUPCCIRVNHF-OUBTZVSYSA-N Yttrium-90 Chemical compound [90Y] VWQVUPCCIRVNHF-OUBTZVSYSA-N 0.000 description 3
- KCXMKQUNVWSEMD-UHFFFAOYSA-N benzyl chloride Chemical compound ClCC1=CC=CC=C1 KCXMKQUNVWSEMD-UHFFFAOYSA-N 0.000 description 3
- 229940073608 benzyl chloride Drugs 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 150000002678 macrocyclic compounds Chemical class 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 238000004452 microanalysis Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- XFNJYAKDBJUJAJ-UHFFFAOYSA-N 1,2-dibromopropane Chemical compound CC(Br)CBr XFNJYAKDBJUJAJ-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 241000534944 Thia Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 230000001815 facial effect Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000011287 therapeutic dose Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- PCHJSUWPFVWCPO-AHCXROLUSA-N (193au)gold Chemical compound [193Au] PCHJSUWPFVWCPO-AHCXROLUSA-N 0.000 description 1
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 1
- OSUBRKJCBHXEAK-UHFFFAOYSA-N 1,4,7-trithiecane Chemical compound C1CSCCSCCSC1 OSUBRKJCBHXEAK-UHFFFAOYSA-N 0.000 description 1
- UDKGXKYEWBGQCG-UHFFFAOYSA-N 1-bromo-3-(chloromethyl)benzene Chemical compound ClCC1=CC=CC(Br)=C1 UDKGXKYEWBGQCG-UHFFFAOYSA-N 0.000 description 1
- QVHWHOCGIAYOSX-UHFFFAOYSA-N 1-bromo-4-(2,3-dibromopropoxymethyl)benzene Chemical group BrCC(Br)COCC1=CC=C(Br)C=C1 QVHWHOCGIAYOSX-UHFFFAOYSA-N 0.000 description 1
- WUAPFZMCVAUBPE-NJFSPNSNSA-N 188Re Chemical compound [188Re] WUAPFZMCVAUBPE-NJFSPNSNSA-N 0.000 description 1
- QWVCIORZLNBIIC-UHFFFAOYSA-N 2,3-dibromopropan-1-ol Chemical compound OCC(Br)CBr QWVCIORZLNBIIC-UHFFFAOYSA-N 0.000 description 1
- KSJBMDCFYZKAFH-UHFFFAOYSA-N 2-(2-sulfanylethylsulfanyl)ethanethiol Chemical compound SCCSCCS KSJBMDCFYZKAFH-UHFFFAOYSA-N 0.000 description 1
- JUIKUQOUMZUFQT-UHFFFAOYSA-N 2-bromoacetamide Chemical compound NC(=O)CBr JUIKUQOUMZUFQT-UHFFFAOYSA-N 0.000 description 1
- BAFSCBZTLCVTIE-UHFFFAOYSA-N 4-(2,3-dibromopropoxymethyl)benzoic acid Chemical group OC(=O)C1=CC=C(COCC(Br)CBr)C=C1 BAFSCBZTLCVTIE-UHFFFAOYSA-N 0.000 description 1
- LOQLDQJTSMKBJU-UHFFFAOYSA-N 4-(chloromethyl)benzonitrile Chemical compound ClCC1=CC=C(C#N)C=C1 LOQLDQJTSMKBJU-UHFFFAOYSA-N 0.000 description 1
- CONKBQPVFMXDOV-QHCPKHFHSA-N 6-[(5S)-5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-2-oxo-1,3-oxazolidin-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C[C@H]1CN(C(O1)=O)C1=CC2=C(NC(O2)=O)C=C1 CONKBQPVFMXDOV-QHCPKHFHSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- MZRVEZGGRBJDDB-UHFFFAOYSA-N N-Butyllithium Chemical compound [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 description 1
- 241001163743 Perlodes Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- GKLVYJBZJHMRIY-OUBTZVSYSA-N Technetium-99 Chemical compound [99Tc] GKLVYJBZJHMRIY-OUBTZVSYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000003190 augmentative effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 150000007942 carboxylates Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- HRYZWHHZPQKTII-UHFFFAOYSA-N chloroethane Chemical compound CCCl HRYZWHHZPQKTII-UHFFFAOYSA-N 0.000 description 1
- 239000002026 chloroform extract Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 150000004699 copper complex Chemical class 0.000 description 1
- 229910001497 copper(II) tetrafluoroborate Inorganic materials 0.000 description 1
- RYGMFSIKBFXOCR-YPZZEJLDSA-N copper-62 Chemical compound [62Cu] RYGMFSIKBFXOCR-YPZZEJLDSA-N 0.000 description 1
- RYGMFSIKBFXOCR-IGMARMGPSA-N copper-64 Chemical compound [64Cu] RYGMFSIKBFXOCR-IGMARMGPSA-N 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- KBYOBAICCHNMNJ-UHFFFAOYSA-L diperchloryloxymercury Chemical compound [Hg+2].[O-]Cl(=O)(=O)=O.[O-]Cl(=O)(=O)=O KBYOBAICCHNMNJ-UHFFFAOYSA-L 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229960003750 ethyl chloride Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- XMBWDFGMSWQBCA-RNFDNDRNSA-M iodine-131(1-) Chemical compound [131I-] XMBWDFGMSWQBCA-RNFDNDRNSA-M 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Substances [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000009206 nuclear medicine Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- 238000002600 positron emission tomography Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000001226 reprecipitation Methods 0.000 description 1
- WUAPFZMCVAUBPE-IGMARMGPSA-N rhenium-186 Chemical compound [186Re] WUAPFZMCVAUBPE-IGMARMGPSA-N 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229940056501 technetium 99m Drugs 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- QEMXHQIAXOOASZ-UHFFFAOYSA-N tetramethylammonium Chemical class C[N+](C)(C)C QEMXHQIAXOOASZ-UHFFFAOYSA-N 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium atom Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D341/00—Heterocyclic compounds containing rings having three or more sulfur atoms as the only ring hetero atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
- C07F9/6578—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and sulfur atoms with or without oxygen atoms, as ring hetero atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
1,4,7-Trithiacyclononane (9S3) and related compounds of formula (I), where one T is S or Se or Te, another T is S or Se or Te and the third T is S, Se, Te, >PX or >AsX, X being a univalent atom or group, and where A1 is -C2H4- or (a), or -C3H6-, A2 is -C2H4- or (a), and A3 is -C2H3R- or (b), or except when A1 is -C3H6-, -C3H5R-, where R is alkyl or substituted alkyl or, if the third T contains P or As, R may also be H, may be substituted with a linker group which can bind to a substrate such as a protein and which itself retains co-ordinating functionality to chelate a radionuclide for diagnosis or therapy.
Description
MACROCYCLIC ~HIOETHER LIGANDS AND THEIR USE AS INTERMEDIATES
FOR BINDING IONS TO SUBSTRATES
This invention relates to certain macrocyclic thioether ligands and their use as intermediates for binding ions to substrates, especially the attachment of radionuclides to biologically active substrates such as immunoglobulins.
05 For a variety of uses, both diagnostic and therapeutic, for -example in tracing biological processes or applying radiation to a desired part of the body, there is a need for the synthesis of bifunctSonal chelates whlch would trap metallic radlonuclides and themselves be capable of reaction to covalently bond to the biologically active molecule of interest e.g. an ~mmunoglobulin or be reactable with functions on proteins (e.g. lysine residues> or ~ :
with an anti-tumour monoclonal antibody.
Not all radionuclides are equally suitable for such purposes, as will be explained below. --The potential value of radiolabelled antibodies for in vivo localisation of tumours has been recognised since the discovery of the hybrtdoma techn~que for raising monoclonal antibodies in 1976. -Methods using radio10dine as the labell~ng nuclide (I-123 for gamma camera imaging, and I-131 for both imaging and radloimmunotherapy) have more recently been augmented by the use of chelate-conjugated antibod1es labelled w1th rad~ometals. The most wldely investigated example of thls method for lmaglng purposes employs 1ndium-111 chelated to a derlvatlve of dlethylenetrlamlnepentaacetic acid (DTPA). These procedures have been the sub~ect of considerable Z5 clinical evaluation and pharmacokinetic analysis.
- Desp1te the great activity and interest in this important medical f~eld, the performance of current reagents is far from optimal. For example, radioiodine labelled antibodies are known to lose a large portion of their label ~as free iodide) in vivo. This results in the need for blocking of the thyro~d gland, leads to ima~e d~gradation and causes dosimetric problems when therapeutic i ~, . ~ ~ . . .
. . .
W o 92/16520 PCT/GB92/~0492 21~8~2 2 doses are being considered. A characteristic of indium-lll and yttrium-90 labelled antibodies is high non-specific uptake of radioactivity in the liver spleen and bone marrow. This leads to excessive radiation doses to these organs if beta-emitting nuclides 05 such as yttrium-90 are adminlstered for radiotherapy and restricts the diagnostic usefulness of indium-labelled antibodies in cases where liver metastates may be present. -Indium and yttrium the radiometal ions receiving most attentlon at present are hard Lew~s acids ~i.e. they are lO complexed most strongly by ligands contaln~ng hard donors such as oxygen and n~trogen). Consequently the chelates employed have been hard donors such as DTPA and EDTA. ~hile these undoubtedly have high association constants for nuclides such as In-lll or Y-90 in vitro human serum contains hard cations (Ca2~ Mg2+ Fe3+
lS Zn2~ K+ and Na~) in much h~gher concentrations than the radiolabel and these compete with the radionuclide for the chelate. Also present in serum are a number of ~ron-sequestering proteins which have high affinities for In3 and Y3+ and are therefore able to abstract the nuclide from the antibody. In 20 addition at physiological pH a kinetic destab~lisat~on ar~ses in that these chelates are readily protonated further diminish~ng their effectiveness as radionuclide-binding chelates. The resulting detachment of label from the antibody leads to its accumulation in the l~ver and other organs.
These difficulties may be circumvented by use of radionuclides of soft metals (i.e. those that are complexed most strongly by ligands containing soft donors such as sulphur and phosphorus also known as Class B metals). Indeed a number of such nuclides are available which have radlological properties suitable for 30 exploitation in nuclear medicine for both diagnosis and therapy of tumours. A selection is presented in Table l below together with their relevant properties.
. : - , .
.
21 0 ~ 8 7 2 Table 1 Nuclide tY(h) y-energy ~-energy Available (keV) (MeV) specific (mCi/mg) Ag-lll 178 340 250 1.0 nca Cu-62 0.16 ~+ _ nca Cu-64 13 ~+ 0.6 30~
Cu-67 62 185 92 0.5 nca Au-193 19 256 186 _ nca Au-198 58 412 87 1.2 20~
Au-l99 77 159 208 0.4 nca Hg-197 65 77 _ nca Hg-203 1128 279 0.2 2*
Tc-99m 6 141 _ ca Re-186 91 137 1.07 0.93 5~ -Re-188 17 155 2.12 nca Mo-99 66 217 219 245 1.2 nca Ru-97 70 2t6 325 _ nca , . . . ,. . '. .'."
.
Higher activities may be available.
nca: no carrier added Note that the half-llves are such that the radiatlon dose need not be excesslve whlle the gamma-emission energies are within the range suitable for detection by gamma-cameras. Gamma em~tters with suitable emission energies may be used for diagnostic imaging.
05 Copper-64 and copper-62 are positron emitters of potential value in positron emission tomography. Several of the ~-emitters are turrently available at specific activities sufficiently high to administer therapeutic doses with reasonable amounts of antibody (e.g. lmCi/mg antibody). Moreover some pairs of isotopes of the same element are available e.g. gold-193 and gold-l99 of which ::
,, ' ' .
w o 92/16520 PCT/GB92/00492 ~os~72 ., .
one could be used for imaging and the other for therapy using the same antibody-chelate conjugate. Thus, the target specif~city of the therapeutic dose could be reliably predicted by imaging. The combination of technetium-99m and rhenium-188 or rhenium 186 (both 05 of which metals, in their lower oxidation states, may be described as soft ) may also offer this advantage because of the close chemical similarity between the two elements. Beta emitters and those gamma emitters which also emit cell-killing secondary radiation (e.g. Auger electrons) may be used in cancer therapy.
Certain ligands containing soft donors such as oxygen or sulphur have been ava~lable for many years. Thus the preparation of 1,4,7-trithiacyclononane ~9S3) is descr~bed by Sellmann et al., Angewandte Chemie, Int. Ed., 23, No. 10, 1984, 807-808, while a compound of similar structure in which the ethylene bridges have fused benzene rings, i.e. 2,3,7,8,12,13-hexamethoxytribenzo-~1,4,7]-trithiacyclononatriene, is described by Weiss et al., Z. Naturforsch., B. Anorg. Chem., Org. Chem., 1979, 34B (3), 448-SO.
The cyclically symmetrical nine-membered ring compound 9S3 is a powerful thioether ligand that is uniquely well suited to facial-mode tridentate coordination through the sulphur atoms.
It forms stable complexes w~th many transition and main group metals, especlally soft ones, in many cases ~mparting unusual structural and electronic properties. The homologue 1,4,7-trithia-cyclodecane (lOS3) shows similar properties in lesser measure (Grant ~ al., Inorg. Chem., 1989, ~, 4128-4132).
The 9S3 complexes have unusual and useful properties such as h~gh stabiiity (thioethers are typical1y relatively weakly coordinating ligands), high coordination numbers, access to unusual ox~dat~on states and high electron self-exchange rates.
The affinities of these structurally specialised chelates for the hard metal ions which occur in plasma are low, however, suggesting that soft -metal-chelate combinations of this type would be well protected in vivo from cross reaction with plasma proteins and metal ions. In additlon, the basicity of thioether sulphur is extremely low so that there would be no interference due to protonation at physiological pH.
..... ,.. . . . . . ~ . . ~ -, ': ' ' ''. . ' .,', . .' , . . ; , :
. ~ . . . ,:: - - - .
. . . - .
W O 92/16~20 PCT/GB92/00492 21~872 ,~
However, the ligands of 9S3 type described in the literature are not 1ntrinsically suitable for complexatlon with biologically active substrates.
The present invention provides llgands of formula I
Al - T~
\A3 - T' (I) 05 where one T is S or Se or Te, another T is S or Se or Te and the third T is S, Se, Te, >PX or >AsX, X being a univalent atom or group, and where Al is -C2H4- or ~ or -C3H6-~
A2 j5 -C2H4- or ~
and A is -C2H3R- or ~R or except when Al is -C3H6-, -C3H5R-, where R is alkyl or substituted alkyl and is preferably selected fro0 hydroxyalkyl, alkoxyalkyl, aryloxyalkyl and arylalkoxyalkyl groups which groups may be substituted, or, if the third T contains P or As, R may also be H.
Preferably each T is S or two T s are S and the third is the group ,PX. Preferably A1 and A2 are -C2H4-. X may suitably be a hydrogen atom or an alkyl or phenyl group, which groups may be substituted. R is preferably a group capable of further der~vat1sation and thus ls preferably hydroxyalkyl, alkoxyalkyl, aryloxyalkyl or arylalkoxyalkyl optionally further substituted, for example by halo-, carboxy- or cyano-.
While not wishing to be bound by any particular theory, it is considered that the properties of 9S3 ligands may derive (at least in part) from their predisposition to facial coordination as a result of their preferred endodentate conformation. Thus, according to one preferred aspect of the invention, the inventors have conceived the notion of further improving these ligands by substituting (9-lO)S3 compounds in the thia crown in such a way as to provide ': :
,: . ; . .
.,~ . ' , .
.
. ~.
w o 92/16520 PCT/GB92/00492 210~7 ~
(i) a phosphine donor in place of a thioether donor and/or (ii) a further co-ordinatlng group to satlsfy the vacant co-ordination slte of four-co-ordinate metal lons or a further tridentate crown to satlsfy slx-co-ordlnate metal lons.
05 One aspect of the inventlon therefore comprlses ligands of formula I above in which one T is `PX, where X ls preferably a ::
group, such as phenyl, which may be further derlvatlsed by the introduction of at least one substituent, preferably an ortho : :
substituent, which is a donor group or atom cr second macrocycle so ~-as to extend the co-ordinatlon. Thus, for example, compounds such as the 1,4-d~thla,7-phospha~P-mono-orthosubstituted-phenyl)-cyclononanes, which have the ortho substttuent polnt~ng towards the sulphur atoms, are postulated as being useful because a ligand substltuted onto that ortho site wlll assist a tetrahedral complexation of a metal in co-operation with the S, S and P.
A synthetic route to such a compound, wh~ch not only exhibits an additional donor group but also a llnker group as described below, is as follows:
Br~----N BuLi Li~-C--N P~3 Cl_p~tN
SMe SMe ~l SMe ~ ¦NaBH3CN
Br Br HS~ S
P~CN -- H2P~CN
HS~J SMe SMe s3 SMe \ HOEt/HCl ~S~p_~NH~
S~J SMe . .
.
. ~
w O 92/16520 PCT/GB92/00492 _ 7 _ 2 I 0 ~ 3 7 2 Such extended ligands with or without a fourth (or three more) ligand(s) with a co-ordinating activity directed generally on the -axis of symmetry of the thia crown and with such linker groups as may be expedient may be bound to a protein or other biologically 05 active molecule or moiety enabling the latter to be tracked through the body or enabling tumours to be located and/or treated by radioimmunolocalisation. `
Metal complexes of such macrocyclic thiaphosph~nes which have been found to exhibit surprisingly high stability are also included with~n the invention. Synthesis of such metal complexes and single crystal X-ray structure determinatlon of the cyclic ligand shows that it is capable of facial tridentate coordination and suggests that i-t will ligate more strongly than 9S3 in that the phosphine donor of the non-facially coordinating ligand binds to the metal in preference to the thioethers.
It has surprisingly been found that such derivatisation of compounds of the 9S3 ring structure and variations thereon such as the replacement of a sulphur atom by a higher valent element such as phosphorus allows retention of the unusual and useful co-ord~nation chemistry of the 9S3 ring structure.
Therefore the invention further includes extended ligands in which at least one univalent atom or group of a ligand of formùla I
has been replaced by a moiety extending the co-ordinating functionality of the ligand of formula I and/or a 11nking moiety capable of facil~tat~ng blndlnq to a substrate.
Preferred extended 1igands according to the invent~on may be represented by replacing that atom or group by (i) a moiety derived ~ -from formula I as set forth above or (ii) a linker group which can bind to a substrate the linker group optionally add~tionally falling within the definition of said moiety and/or having co-ordinating functionality such as carboxyl or amine or more preferably phosphine or even more preferably thiol or thloether.
., , , .
21~872 Exampl es of linker groups are:
O I ~ t- 0- N ~ ~nd - 0 1 ~ ~NH2+
such groups being linked for example to a ligand of formula I where R is hydroxyalkyl.
Alternatively, where one T is `PX or ~AsX, X may su~tably 05 incorporate additionally a donor group or atom or a second macrocycle so as to extend the co-ordinating functionality of the ligand.
Compounds based on 9S3 with such a linker group (even made other than via ligands as set forth above) are w~thin the scope of the invention.
The ligands and extended ligands according to the inventton may find appllcation as chelators for reac$1On with other reagents at the point of use or as intermediates ~n the preparation of chelators and ready-l~nked ~helated reagents for use in diagnosis or therapy.
Thus, the molecules set forth above can find application as chelators, which may be sold and used to bind metals such as radlonucl~des to substrates, e.g. 1n k1ts for labelling antlbodies. The molecules may be sold as chelates, l.e. as ready-made metal complexes, which the user may then optionally -attach to a substrate. Alternatively, the molecules may be sold bound to a substrate, ready for the user then optionally to complex a metal therein. The molecule according to the invention can lastly be a metal complex bound to a substrate.
The invention therefore includes ligands and extended ligands as defined above when complexed with either a radionuclide, a biologically act~ve substrate, such as an immunoglobul~n, or both.
~ The invention further includes a kit suitable for use in i rad~odiagnosis or radiotherapy includlng a ligand, extended l~gand :
.
.; : ~
i: . ... .
w O 92/16520 PCT/GB92/00492 9 21 05~72 or a complex thereof. Examples of reactive linking groups and substrate targets are shown in the following Table 2.
Table 2 - METHODS FOR CHELATE CON~UGATION
For CHELATE = R R-X + IgG-T ~ R-XT-IgG
REACTIVE GROUP (X) TARGET RESIDUE (T) SITE SPECIFIC?
.
RCH2-C=O
~0 H2N-CH2-I9G No l-butyl-C=O . .
Anhydrlde .....
RCH2-C~-O o ~ ~ H2N-CH2-IgG No NHS Ester . -. .-6~ - ' :
~NH2 Cl H2N-CH2-IgG No ~
Imldate Ester : :
.: ;., ' RCH2-C-O CHO-IgG (From `NH-NH2 Oxldlsed sugar Yes Carboxylic Hydraz~de Groups) . : ' RCH2-NH-C.O HS-IgG (From :.
~CH2-Br Reduced S-S Bonds) Yes ~ .
Bromoacetamide ~
_ _ ~.
~
RCH2-N `~ HS-IgG (From O ~ Reduced S-S Bonds~ Yes N-alkyl malelm1de . ' . ' ' : . '.
: , - .-.
W o 92/16520 PCTtGB92/00492 2~ ,rl 2 Both the anhydride and N-hydroxysucclnlmlde (NHS) methods have previously been used for correspondlng purposes but share a number of disadvantages. They react primarily wlth lyslne resldues on the protein molecule and so there is no control over the site at whlch 05 conjugatlon takes place.
The three other methods ln the table all employ slte- speclfic groups. Each wlll react only at s~tes dlstant from the ant~gen binding slte conferrlng advantages of fully-reta~ned immunoreactivity ~mproved in vivo blod~stribution and pharmacoklnetlcs and h~gh avallable speclflc radloactlvlty.
The inventlon wlll now be descrlbed by way of example.
Example 1 - PreDaratlon of llgands A to E
FOR BINDING IONS TO SUBSTRATES
This invention relates to certain macrocyclic thioether ligands and their use as intermediates for binding ions to substrates, especially the attachment of radionuclides to biologically active substrates such as immunoglobulins.
05 For a variety of uses, both diagnostic and therapeutic, for -example in tracing biological processes or applying radiation to a desired part of the body, there is a need for the synthesis of bifunctSonal chelates whlch would trap metallic radlonuclides and themselves be capable of reaction to covalently bond to the biologically active molecule of interest e.g. an ~mmunoglobulin or be reactable with functions on proteins (e.g. lysine residues> or ~ :
with an anti-tumour monoclonal antibody.
Not all radionuclides are equally suitable for such purposes, as will be explained below. --The potential value of radiolabelled antibodies for in vivo localisation of tumours has been recognised since the discovery of the hybrtdoma techn~que for raising monoclonal antibodies in 1976. -Methods using radio10dine as the labell~ng nuclide (I-123 for gamma camera imaging, and I-131 for both imaging and radloimmunotherapy) have more recently been augmented by the use of chelate-conjugated antibod1es labelled w1th rad~ometals. The most wldely investigated example of thls method for lmaglng purposes employs 1ndium-111 chelated to a derlvatlve of dlethylenetrlamlnepentaacetic acid (DTPA). These procedures have been the sub~ect of considerable Z5 clinical evaluation and pharmacokinetic analysis.
- Desp1te the great activity and interest in this important medical f~eld, the performance of current reagents is far from optimal. For example, radioiodine labelled antibodies are known to lose a large portion of their label ~as free iodide) in vivo. This results in the need for blocking of the thyro~d gland, leads to ima~e d~gradation and causes dosimetric problems when therapeutic i ~, . ~ ~ . . .
. . .
W o 92/16520 PCT/GB92/~0492 21~8~2 2 doses are being considered. A characteristic of indium-lll and yttrium-90 labelled antibodies is high non-specific uptake of radioactivity in the liver spleen and bone marrow. This leads to excessive radiation doses to these organs if beta-emitting nuclides 05 such as yttrium-90 are adminlstered for radiotherapy and restricts the diagnostic usefulness of indium-labelled antibodies in cases where liver metastates may be present. -Indium and yttrium the radiometal ions receiving most attentlon at present are hard Lew~s acids ~i.e. they are lO complexed most strongly by ligands contaln~ng hard donors such as oxygen and n~trogen). Consequently the chelates employed have been hard donors such as DTPA and EDTA. ~hile these undoubtedly have high association constants for nuclides such as In-lll or Y-90 in vitro human serum contains hard cations (Ca2~ Mg2+ Fe3+
lS Zn2~ K+ and Na~) in much h~gher concentrations than the radiolabel and these compete with the radionuclide for the chelate. Also present in serum are a number of ~ron-sequestering proteins which have high affinities for In3 and Y3+ and are therefore able to abstract the nuclide from the antibody. In 20 addition at physiological pH a kinetic destab~lisat~on ar~ses in that these chelates are readily protonated further diminish~ng their effectiveness as radionuclide-binding chelates. The resulting detachment of label from the antibody leads to its accumulation in the l~ver and other organs.
These difficulties may be circumvented by use of radionuclides of soft metals (i.e. those that are complexed most strongly by ligands containing soft donors such as sulphur and phosphorus also known as Class B metals). Indeed a number of such nuclides are available which have radlological properties suitable for 30 exploitation in nuclear medicine for both diagnosis and therapy of tumours. A selection is presented in Table l below together with their relevant properties.
. : - , .
.
21 0 ~ 8 7 2 Table 1 Nuclide tY(h) y-energy ~-energy Available (keV) (MeV) specific (mCi/mg) Ag-lll 178 340 250 1.0 nca Cu-62 0.16 ~+ _ nca Cu-64 13 ~+ 0.6 30~
Cu-67 62 185 92 0.5 nca Au-193 19 256 186 _ nca Au-198 58 412 87 1.2 20~
Au-l99 77 159 208 0.4 nca Hg-197 65 77 _ nca Hg-203 1128 279 0.2 2*
Tc-99m 6 141 _ ca Re-186 91 137 1.07 0.93 5~ -Re-188 17 155 2.12 nca Mo-99 66 217 219 245 1.2 nca Ru-97 70 2t6 325 _ nca , . . . ,. . '. .'."
.
Higher activities may be available.
nca: no carrier added Note that the half-llves are such that the radiatlon dose need not be excesslve whlle the gamma-emission energies are within the range suitable for detection by gamma-cameras. Gamma em~tters with suitable emission energies may be used for diagnostic imaging.
05 Copper-64 and copper-62 are positron emitters of potential value in positron emission tomography. Several of the ~-emitters are turrently available at specific activities sufficiently high to administer therapeutic doses with reasonable amounts of antibody (e.g. lmCi/mg antibody). Moreover some pairs of isotopes of the same element are available e.g. gold-193 and gold-l99 of which ::
,, ' ' .
w o 92/16520 PCT/GB92/00492 ~os~72 ., .
one could be used for imaging and the other for therapy using the same antibody-chelate conjugate. Thus, the target specif~city of the therapeutic dose could be reliably predicted by imaging. The combination of technetium-99m and rhenium-188 or rhenium 186 (both 05 of which metals, in their lower oxidation states, may be described as soft ) may also offer this advantage because of the close chemical similarity between the two elements. Beta emitters and those gamma emitters which also emit cell-killing secondary radiation (e.g. Auger electrons) may be used in cancer therapy.
Certain ligands containing soft donors such as oxygen or sulphur have been ava~lable for many years. Thus the preparation of 1,4,7-trithiacyclononane ~9S3) is descr~bed by Sellmann et al., Angewandte Chemie, Int. Ed., 23, No. 10, 1984, 807-808, while a compound of similar structure in which the ethylene bridges have fused benzene rings, i.e. 2,3,7,8,12,13-hexamethoxytribenzo-~1,4,7]-trithiacyclononatriene, is described by Weiss et al., Z. Naturforsch., B. Anorg. Chem., Org. Chem., 1979, 34B (3), 448-SO.
The cyclically symmetrical nine-membered ring compound 9S3 is a powerful thioether ligand that is uniquely well suited to facial-mode tridentate coordination through the sulphur atoms.
It forms stable complexes w~th many transition and main group metals, especlally soft ones, in many cases ~mparting unusual structural and electronic properties. The homologue 1,4,7-trithia-cyclodecane (lOS3) shows similar properties in lesser measure (Grant ~ al., Inorg. Chem., 1989, ~, 4128-4132).
The 9S3 complexes have unusual and useful properties such as h~gh stabiiity (thioethers are typical1y relatively weakly coordinating ligands), high coordination numbers, access to unusual ox~dat~on states and high electron self-exchange rates.
The affinities of these structurally specialised chelates for the hard metal ions which occur in plasma are low, however, suggesting that soft -metal-chelate combinations of this type would be well protected in vivo from cross reaction with plasma proteins and metal ions. In additlon, the basicity of thioether sulphur is extremely low so that there would be no interference due to protonation at physiological pH.
..... ,.. . . . . . ~ . . ~ -, ': ' ' ''. . ' .,', . .' , . . ; , :
. ~ . . . ,:: - - - .
. . . - .
W O 92/16~20 PCT/GB92/00492 21~872 ,~
However, the ligands of 9S3 type described in the literature are not 1ntrinsically suitable for complexatlon with biologically active substrates.
The present invention provides llgands of formula I
Al - T~
\A3 - T' (I) 05 where one T is S or Se or Te, another T is S or Se or Te and the third T is S, Se, Te, >PX or >AsX, X being a univalent atom or group, and where Al is -C2H4- or ~ or -C3H6-~
A2 j5 -C2H4- or ~
and A is -C2H3R- or ~R or except when Al is -C3H6-, -C3H5R-, where R is alkyl or substituted alkyl and is preferably selected fro0 hydroxyalkyl, alkoxyalkyl, aryloxyalkyl and arylalkoxyalkyl groups which groups may be substituted, or, if the third T contains P or As, R may also be H.
Preferably each T is S or two T s are S and the third is the group ,PX. Preferably A1 and A2 are -C2H4-. X may suitably be a hydrogen atom or an alkyl or phenyl group, which groups may be substituted. R is preferably a group capable of further der~vat1sation and thus ls preferably hydroxyalkyl, alkoxyalkyl, aryloxyalkyl or arylalkoxyalkyl optionally further substituted, for example by halo-, carboxy- or cyano-.
While not wishing to be bound by any particular theory, it is considered that the properties of 9S3 ligands may derive (at least in part) from their predisposition to facial coordination as a result of their preferred endodentate conformation. Thus, according to one preferred aspect of the invention, the inventors have conceived the notion of further improving these ligands by substituting (9-lO)S3 compounds in the thia crown in such a way as to provide ': :
,: . ; . .
.,~ . ' , .
.
. ~.
w o 92/16520 PCT/GB92/00492 210~7 ~
(i) a phosphine donor in place of a thioether donor and/or (ii) a further co-ordinatlng group to satlsfy the vacant co-ordination slte of four-co-ordinate metal lons or a further tridentate crown to satlsfy slx-co-ordlnate metal lons.
05 One aspect of the inventlon therefore comprlses ligands of formula I above in which one T is `PX, where X ls preferably a ::
group, such as phenyl, which may be further derlvatlsed by the introduction of at least one substituent, preferably an ortho : :
substituent, which is a donor group or atom cr second macrocycle so ~-as to extend the co-ordinatlon. Thus, for example, compounds such as the 1,4-d~thla,7-phospha~P-mono-orthosubstituted-phenyl)-cyclononanes, which have the ortho substttuent polnt~ng towards the sulphur atoms, are postulated as being useful because a ligand substltuted onto that ortho site wlll assist a tetrahedral complexation of a metal in co-operation with the S, S and P.
A synthetic route to such a compound, wh~ch not only exhibits an additional donor group but also a llnker group as described below, is as follows:
Br~----N BuLi Li~-C--N P~3 Cl_p~tN
SMe SMe ~l SMe ~ ¦NaBH3CN
Br Br HS~ S
P~CN -- H2P~CN
HS~J SMe SMe s3 SMe \ HOEt/HCl ~S~p_~NH~
S~J SMe . .
.
. ~
w O 92/16520 PCT/GB92/00492 _ 7 _ 2 I 0 ~ 3 7 2 Such extended ligands with or without a fourth (or three more) ligand(s) with a co-ordinating activity directed generally on the -axis of symmetry of the thia crown and with such linker groups as may be expedient may be bound to a protein or other biologically 05 active molecule or moiety enabling the latter to be tracked through the body or enabling tumours to be located and/or treated by radioimmunolocalisation. `
Metal complexes of such macrocyclic thiaphosph~nes which have been found to exhibit surprisingly high stability are also included with~n the invention. Synthesis of such metal complexes and single crystal X-ray structure determinatlon of the cyclic ligand shows that it is capable of facial tridentate coordination and suggests that i-t will ligate more strongly than 9S3 in that the phosphine donor of the non-facially coordinating ligand binds to the metal in preference to the thioethers.
It has surprisingly been found that such derivatisation of compounds of the 9S3 ring structure and variations thereon such as the replacement of a sulphur atom by a higher valent element such as phosphorus allows retention of the unusual and useful co-ord~nation chemistry of the 9S3 ring structure.
Therefore the invention further includes extended ligands in which at least one univalent atom or group of a ligand of formùla I
has been replaced by a moiety extending the co-ordinating functionality of the ligand of formula I and/or a 11nking moiety capable of facil~tat~ng blndlnq to a substrate.
Preferred extended 1igands according to the invent~on may be represented by replacing that atom or group by (i) a moiety derived ~ -from formula I as set forth above or (ii) a linker group which can bind to a substrate the linker group optionally add~tionally falling within the definition of said moiety and/or having co-ordinating functionality such as carboxyl or amine or more preferably phosphine or even more preferably thiol or thloether.
., , , .
21~872 Exampl es of linker groups are:
O I ~ t- 0- N ~ ~nd - 0 1 ~ ~NH2+
such groups being linked for example to a ligand of formula I where R is hydroxyalkyl.
Alternatively, where one T is `PX or ~AsX, X may su~tably 05 incorporate additionally a donor group or atom or a second macrocycle so as to extend the co-ordinating functionality of the ligand.
Compounds based on 9S3 with such a linker group (even made other than via ligands as set forth above) are w~thin the scope of the invention.
The ligands and extended ligands according to the inventton may find appllcation as chelators for reac$1On with other reagents at the point of use or as intermediates ~n the preparation of chelators and ready-l~nked ~helated reagents for use in diagnosis or therapy.
Thus, the molecules set forth above can find application as chelators, which may be sold and used to bind metals such as radlonucl~des to substrates, e.g. 1n k1ts for labelling antlbodies. The molecules may be sold as chelates, l.e. as ready-made metal complexes, which the user may then optionally -attach to a substrate. Alternatively, the molecules may be sold bound to a substrate, ready for the user then optionally to complex a metal therein. The molecule according to the invention can lastly be a metal complex bound to a substrate.
The invention therefore includes ligands and extended ligands as defined above when complexed with either a radionuclide, a biologically act~ve substrate, such as an immunoglobul~n, or both.
~ The invention further includes a kit suitable for use in i rad~odiagnosis or radiotherapy includlng a ligand, extended l~gand :
.
.; : ~
i: . ... .
w O 92/16520 PCT/GB92/00492 9 21 05~72 or a complex thereof. Examples of reactive linking groups and substrate targets are shown in the following Table 2.
Table 2 - METHODS FOR CHELATE CON~UGATION
For CHELATE = R R-X + IgG-T ~ R-XT-IgG
REACTIVE GROUP (X) TARGET RESIDUE (T) SITE SPECIFIC?
.
RCH2-C=O
~0 H2N-CH2-I9G No l-butyl-C=O . .
Anhydrlde .....
RCH2-C~-O o ~ ~ H2N-CH2-IgG No NHS Ester . -. .-6~ - ' :
~NH2 Cl H2N-CH2-IgG No ~
Imldate Ester : :
.: ;., ' RCH2-C-O CHO-IgG (From `NH-NH2 Oxldlsed sugar Yes Carboxylic Hydraz~de Groups) . : ' RCH2-NH-C.O HS-IgG (From :.
~CH2-Br Reduced S-S Bonds) Yes ~ .
Bromoacetamide ~
_ _ ~.
~
RCH2-N `~ HS-IgG (From O ~ Reduced S-S Bonds~ Yes N-alkyl malelm1de . ' . ' ' : . '.
: , - .-.
W o 92/16520 PCTtGB92/00492 2~ ,rl 2 Both the anhydride and N-hydroxysucclnlmlde (NHS) methods have previously been used for correspondlng purposes but share a number of disadvantages. They react primarily wlth lyslne resldues on the protein molecule and so there is no control over the site at whlch 05 conjugatlon takes place.
The three other methods ln the table all employ slte- speclfic groups. Each wlll react only at s~tes dlstant from the ant~gen binding slte conferrlng advantages of fully-reta~ned immunoreactivity ~mproved in vivo blod~stribution and pharmacoklnetlcs and h~gh avallable speclflc radloactlvlty.
The inventlon wlll now be descrlbed by way of example.
Example 1 - PreDaratlon of llgands A to E
3-thiapentane-1 5-dlthlol HS(C2H4)S(C2H4)SH was complexed wlth a carbonyl Mo(C0)3 to form a template complex tMo(C0)3~SCH2CH2SCH2CH2S)]2 . A solutlon of thls (prepared in sltu as a blstetramethylammonium salt) in acetonltrile was treated w~th racemlc CH2BrCHBrR to generate the correspondlng tr~th~amacrocyclic complex. The macrocycle ltself was displaced from the complex ~n DMS0 (d~methylsulphox~de) solution by add~t~on of tNMe4~2tSCH2CH2SCH2CH2S~. In all cases a ma~or by-product was 1 4-d~th~acyclohexane removed by slllca gel chromatography.
R was as follows:
L~gand A: -CH3 (yleld 21X~
Llgand B: -CH20H (yleld 24%) Llgand C: -CH20CH2-phenyl (yleld 6X) L~gand D: -CH20CH2-parabromophenyl (yield 20X) Llgand E: -CH20CH2-parabenzoate (y~eld 8X) ~he react~on scheme may be dep~cted as S ¦ / 1 Br ~r S S
to~ l ~ co ~
:
.:~. -: : ... . .
, ~: . . , . : , :
.
' ~ ' . '' ' ~ . .
. . . .
~ , ', ' ' :
. 2lass72 The coordination chemlstry of L~gand A with Fe(II), Ni(II), Cu(II), Ag(I) and Hg(II), and of Ligand C with Fe(II), has surprisingly been found to be closely analogous to the parent (unsubstituted) 9S3 complexes, as regards stoichiometry, electronic 05 spectra, stability, co-ordination number and electrochemical behaviour. ~ -Representative Ligands were synthesised in detail as follows:-Preparation of Liaand B, viz 9S3-CH20H - -Molybdenum hexacarbonyl, Mo(C0~6, 0.02 mol, and acetonitrile ~ --(60 mL) were heated together to reflux under a dinitrogen atmosphere with stirring for 2 hours. The resulting solution of Mo(C0)3(MeCN)3 was transferred anaeroblcally to a flask containing the bis(tetramethylammonium) salt of 3-thiapentane-1,5-dithiol (~NMe4]2[SCH2CH2SCH2CH2S], 0.02 mol). The resulting suspension was lS stirred for 24h at room temperature, after which a solution of racemic 1,2-dibromopropan-3-ol (0.02 mol) in acetonitrile (10 ml) was added. After stirring for 2h the solvent was removed in vacuo and the residue treated with tNMe4]2tSCH2CH2SCH2CH25], (0.02 mol) in dimethylsulphoxide (S0 ml); After stirring at room temperature under dinitrogen for 24h the mlxture was extracted with diethyl ether (4 x 25ml) and the combined extracts evaporated to dryness.
The residue was dissolved in chloroform and chromatographed on silica gel using chloroform as eluent. Pure 9S3-CH20H (as a vlscous oil which crystallised on prolonged vacuum drying, 24% yield) was obtained by evaporation of the relevant fractions.
PreDaration of Licand D. 9S3-CH20CH2C6H4-Br Thts compound was prepared by a method analogous to that for B (9S3-CH20H), substituting 1,2-dibromo-3-(4 -bromobenzyloxy) propane for 2,3-dibromopropane. It was purified by s~l~ca gel chromatography as described for C (later). Yield 20%.
PreDaration of Liaand E, 9S3-CH20CH2C6H4-4-COOH
This compound was prepared by a method analogous to that for -B (9S3-CH20H), substituting 1,2-dibromo-3-(4 -carboxybenzyloxy)-propane for 2,3-dibromopropane. It was purified as follows. The DMS0 reaction solution was diluted with water (200 ml) and the -.
., -, . , , , .... . . ... .. ~ .. ., . . ... . , - .-.. , .. , .-. . ~.. .. . . -. . . .. .
2~58~ 2 mixture extracted with chloroform. The chloroform extracts were washed with water, dried over MgS04 and evaporated to dryness. The residue was extracted into 10% sodium hydroxide solution, the extract washed wlth chloroform and acidif1ed wlth concentrated 05 hydrochloric acid, resulting in precipitation of a solid. This was dissolved ln ethanol, the solut10n filtered and treated with an equal volume of iced water, and the resulting precipitate collected and dissolved in chloroform. This solution was dried over MgS04 and evaporated to dryness leaving the product as a white powder.
Yield 8%.
Example 2 - PreDaration of Ligands C. F and G
The above synthesis can be developed, building on Ligand B.
This Ligand may be reacted with the bromide RlBr according to the reaction scheme OH R~r Rl may be as follows:
Ligand C (_ Ligand C abovej: -CH2-phenyl (yield 87X) Ligand F: -CH2-paracyanophenyl (yield 43%) L1gand G: -CH2-meta-monobromo-phenyl ~yield 57X) These ligands were syntheslsed ln detall as follows:-PreDaration of Liaand C, 9S3-CH20CH2Ph Ligand B (1 mmol) was reacted with sodium hydride (1 mmol) in dry dimethylformamide (5 ml) followed by addition of benzyl chloride (1 mmol) and stirring at room temperature for 3 days.
25 ml of water was then added and the resulting mixture extracted with diethyl ether and purified by silica gel chromatography using toluene followed by 20% dichloromethane in toluene, then dichloromethane, as eluant. Yield 877.
' .
.
. ;~ . .. . . . , ~
W O 92/16520 pcr/GB92/oo492 ~1~58'72 Preparation of L~aand F, 9S3-CH20CH2C6H4-4-CN
This compound was prepared by a method analogous to that for C, subst~tutlng 4-cyanobenzyl chloride for benzyl chloride. The product was pur~fled by s~lica gel chromatography also as described 05 for C. Yield 43%.
PreDaration of Liqand G. 9S3-CH20CH2C6H4-3-Br Th~s compound was prepared by a method analogous to that for C, subst~tuting 3-bromobenzyl chlor~de for benzyl chloride. The product was purif~ed by s~llca gel chromatography as described for C. Y~eld 57X
ExamDle 3 - PreDaration of l-phenvl-l-phosDha-4 7-d~thiacvclo-nonane(9PhPS2) ~sJ~
Caes~um carbonate (0.013 mol) was st~rred in dry dimethylformam~de (250 ml) at 70C under a din~trogen atmosphere.
1,2-d~chloroethane (0.01 mol) ~n DMF (50 ml) and 3-phenyl-3-phosphapentane-1,5-d~th~ol (prepared by a l~terature method, 0.01 mol) ~n DMF (50 ml) were added dropwise, simultaneously and at the same rate (a per~staltlc pump was used to ma~ntaln the equal and constant rate of addltlon at 4.5 mlh~l). After completlon of the addlt~on the mlxture was st~rred for a further llh at 70C, --then coc~led to room temperature. The solvent was removed in vacuo -and the res~due extracted w~th diethyl ether (30 ml). The extract was washed w~th water (2 x 30 ml) and drled over MgS04. The solvent was removed and the residue chromatographed on silica gel, ~ -elut~ng first with dichloromethane/chloroform 1:1, then toluene.
The product was obtained by removal of solvent from the relevant fract~ons in 37X y~eld as a colourless, v~scous o~l wh~ch - crystalllsed below room temperature.
,.
-.' .: ,.,. . . . . .. . ~,... . . .
- ~, . .. . . .. . .
w o 92/16~20 PCT/GB92/00492 2 ~ ~ 5 87 2 _ 14 -Example 4 - Pre~aration of Metal ~om~lexes of 9PhPS2 Mercurv (II) perchlorate complex with 9PhPS2 l-phenyl-l-phospha-4,7-dithiacyclononane (L) (60mg, 0.23 mmole) was dissolved in acetonitrile (3ml), and stirred at room 05 temperature while mercury (II) perchlorate (50mg, Q.ll mmole) was added. The solution was stirred for 1 hour, then ether was slowly added until the solution turned cloudy (about lOml). The product came out of solution as white crystals on refrigeration.
67mg of ~9PhPS2)2Hg(C104)2 (61%) were obtained.
10 Microanalysis Found C 31.53%, H 3.33%
C24H34C12H908P254 requ~res C 31.60X, H 3.76X
IR (mull, cm~l) 1095(s), 1080(s), 930(s), 875, 830, 820, 810, 765, 745, 725, 700, 695, 635.
Nickel (II) tetrafluoroborate complex with 9PhPS2 Nickel (II) tetrafluoroborate hexahydrate (0.03579, 0.0105 mmole) in ethanol (2ml) was added to a stirred solution of L (0.055g, 0.215 mmole) in ethanol (12ml). A pea-green colour formed at once. The solution was left at 0C for 1 hour, and the resulting green complex was filtered off, washed with ether and 20 dried under vacuum. 0.059g (67%) of (9PhPS2)2Ni(BF4)2.H20 was obtained.
Microanalysis Found C 38.14%, H 4.39%
C24H35B2F8N'Ol/2P2s4 requ~res C 38.22X, H 4.68X
Copper (II) tetrafluoroborate comDlex with 9PhPS2 L (64.5mg, 0.25 mmole) was stirred in ether ~lml), and ethanol ~lOml) added. Copper ~II) tetrafluoroborate ~28.5mg, 0.12 mmole) in ethanol (2ml) was added, and the red product was stirred for 10 min. All attempts to remove the fine solid by filtration failed.
The mixture was centrifuged, and the supernatant decanted off. The 30 red solid was shaken with ether (12ml) and again centrifuged. The solvent was poured off and the product dried under high vacuum.
45mg of (9PhPS2)2Cu(BF4)2 (51%) were obtained.
Microanalysis Found C 38.23%, H 4.27%
C24H34B2CUFgP2S4 requ~res C 38.44~, H 4.57X
35 IR (mull, cm~l) 1325, 1095~s), 1060(s), 1045(s), 1000, 930, 885, 820, 810, 760, 725, 700, 525, 49~.
'.
s, ;
. . ,, ... ,, , , . . . . , :
w o 92/16520 PCT/GB92/00492 21 ~872 CoD~er ~I) hexafluoroDhosDhate complex wlth 9PhPS2 L (120mg, 0.468 mmole) was st~rred ln acetonitrile (4ml~.
Cu(CH3CN)4PF6 (82mg, 0.22 mmole) was added to the solutlon which became very fa~nt plnk in colour as the copper complex dissolved.
OS Ether (20ml) was added, the solution lost its colour and became cloudy. The solution was left overn~ght at 4C to give off-white crystals. These were flltered off and drled under vacuum to yield 134mg (80Z) of (9PhPS2)2CuPF6.CH3CN.
Microanalys~s Found C 40.74X, H 4.58%, N 1.81X
C26H37CuFNP3S4 requlres C 40.96X, H 4.89X, N 1.84%
IR ~mull, cm~l) 1410, 840~s), 745~s), 695, SS0, 480.
The llgand structure was determlned by slngle crystal X-ray to conf1rm the structure Cu(9PhPS2)2~ as hav~ng tetrahedral coord~nation w~th the copper bound by one fac~ally tridentate lS 119and and a slngle phosph~ne donor from the second l~gand. The structures is shown in F~gure 1.
~amDle S - PreDaratlon of extended 9S3 liaand~
1. N-hYdroxvsulDhosucc~nlm~de ester. sod1um salt ~ ~ ~ 0 - N
Llgand E ~see Example 1) ~51mg, O.lS mmol), d~cyclohexyl-carbodl1mlde (31mg, 0.16 mmol), and N-hydroxysulphosuccln~mlde, sodlum salt ~32.5mg, O.lS mmol) were st1rred 1n dry dtmethyl-formam~de ~lml) under dry d~nltrogen at room temperature for 48h.
A whlte sol~d was preclp~tated (dlcyclohexylurea) and was removed by flltration. The f11trate was diluted with lSml d1ethylether, 25- result~ng in format~on of a gummy solid wh~ch was collected and freed of solvent under high vacuum. Infra red absorption ~cm~l):
3500 (br), 2900, 2840, 1760, 1730, 1655, 1615, 1600, 1405, 1355, 1225 (br), 1095, 1070, 1035, 985.
w o 92/16520 PCT/GB92/00492 2~5872 The product was dissolved in dimethylsulphoxide (DMS0) to a concentration of lOmg/ml for use ~n protein labelling, and stored below 0C. (Hereafter the product is identified as bifunctional chelator solution ).
05 2. N-hvdroxvsuccinimide ester Ligand E (0.1039, 0.3 mmol), dicyclohexylcarbodiimide (0.0629, 0.3 mmol), and N-hydroxysuccimide (0.062g, 0.3 mmol) were stirred at 4C in dry tetrahydrofuran (3ml~ under dry dinitrogen for 3h.
The wh~te prec~p~tate (dicyclohexyl urea) that formed was removed after overnight refr~geratlon at 4C, by filtrat on, and the filtrate dried under vacuum. The IR spectrum of th~s material showed the presence of impur~ties (dicyclohexylcarbodiim~de) wh~ch were partially removed by reprecipitation from chloroform (2ml) by addition of cyclohexane (20ml) followed by decantation of the supernatant and drying the oily residue under vacuum. IR: 2920, 2845, 1795, 1765, 1735, 1620, 1605, 1570, 1530, 1445, 1410, 1359. ~ :
Example 6 - PreDaration ofl abelled chelate - protein coniuaate Demonstration of the preparat~on and stability of the conjugate is descr~bed as follcws with reference to Figures 2 to 7 wherein: ~ -F~gure 2 gives the results for gel filtrat~on of rabbit IgG
incubated with 197Hg pre-chelated with the bifunctional chelator (9S3-act~vated ester): radloactivity and absorbance elut~on profile;
Flgure 3 shows gel filtration of rabblt IgG incubated with 197Hg pre-chelated wlth ~ntermediate E ~9S3-carboxylate):
radloact~v~ty and absorbance elut~on profile;
Figures 4 and 5 show gel f~ltration of 197Hg-labelled rabbit IgG incubated wtth whole human serum for 24h: rad~oactivity and absorbance elution profiles respectively; and Figures 6 and 7 show gel filtration of 197Hg-labelled rabbit IgG ln PBS: radioactivity and absorbance profiles respectively.
Bifunctional chelator solution (prepared as described in Example 5-1) (3.62~1) was diluted to 46~1 with DMS0 and the solution added to 2~1 aqueous carrier-free 197HgC12 (37 MBq/ml, Medgenix plc). After 2 minutes this solution was added to rabbit IgG (Sigma, 2mg in iml phosphate buffered sal~ne, PBS).
.. . .
, . . . ,, .. : ...... , ~. ... ~ .
.
w o 92/16520 PCTIGB92/00492 21058~2 - 17 _ After incubation at room temperature for lh, the sample was chromatographed on a sephadex G25M gel filtration column (Pharmacia PD10) eluting with PBS. 30 fractions of 1.4ml were collected. The bulk of the radioactivity (52-93%) eluted with the 05 protein fractions (4ml). The results are shown in Figure 2. As a control, the ligand E (which contains a carboxylate group instead of the activated ester), was employed in place of the bifunctional chelator. This was expected to bind mercury ions but to be incapable of reacting with the protein. In this case, less than 5X
of the radioactivity eluted as an identifiable protein peak. The results are shown in Figure 3. In a second control, the radioactive mercuric chloride was incubated with protein in the absence of any chelator; in this case 32X of the activity was eluted with protein, indicating significant non-specific binding.
Thus, the chelating moiety binds mercury sufficiently strongly to suppress non-specific binding, while the activated ester functionality causes efficient binding to protein.
The above results indicate that the chelated mercury is stable towards transfer of mercury ions to non-specific protein binding sites on rabbit IgG. The types of co-ordinating functional groups available under these circumstances are broadly typical of the b~olog~cal milieu. To further define the stability under biological conditions, the conjugate was incubated wtth human serum, and with bovine serum albumin which is known to be capable of b~nding class b metals (e.g. copper).
Stabll~tv to bov~ne serum album~n Rabbit IgG labelled with 203Hg by the procedure described above, taken from the most radioactive IgG fraction obtained from the gel filtration step, was incubated with an excess of bovine serum albumin (lml of lmg!ml) for lh at 37C. The sample was then loaded onto a protein-A-derivatised affinity column (Pharmacia :
HiTrap lml) and eluted with pH 8.9 NaCl/glycine buffer ~6ml) followed by pH4 citrate buffer (7ml). 13 x lml fractions were collected and assayed for protein, by measuring abs~rbance at 280nm, and for radioactivity. 90X of the radioactivity eluted with the ,~
, :
.
; . , ~ . , , . , ~ , .
, .. : . ~ . .:
WO 92/16520 PCr/GB92/00492 21 ~72 IgG fraction (compared to 94% in the control in which labelled IgG
was incubated alone). Thus transfer of radioactivlty to albumin was slight (of the order of 4%) after the first hour. However, the loss may become more significant after longer periods.
05 Stabilitv to human serum -Rabbit IgG labelled with 197Hg by the procedure described above(O.lml) was added to human serum (2ml) obtained by clotting whole blood, and lncubated for 24h at 37C under sterile cond~tions. A
sample of the serum was then loaded onto a 300 x 8mm gel filtration column ~LKB GlasPak, TSK G3000SW) and eluted with phosphate buffered salirie. Absorbance at 280nm was monitored and 0.5ml fractions were collected and counted. Results are shown in Figure 5. Although the-A280 trace showed several peaks, the radioactivity was confined to a single well defined peak as shown in Figure 4. A control in which labelled IgG was incubated alone showed an identical eluate radioactivity profile as shown in Figures 6 and 7. These results thus fail to demonstrate any transfer of radioactivity to serum components that are separable from IgG and so do not reveal any ma~or instability over the time perlod studied.
R was as follows:
L~gand A: -CH3 (yleld 21X~
Llgand B: -CH20H (yleld 24%) Llgand C: -CH20CH2-phenyl (yleld 6X) L~gand D: -CH20CH2-parabromophenyl (yield 20X) Llgand E: -CH20CH2-parabenzoate (y~eld 8X) ~he react~on scheme may be dep~cted as S ¦ / 1 Br ~r S S
to~ l ~ co ~
:
.:~. -: : ... . .
, ~: . . , . : , :
.
' ~ ' . '' ' ~ . .
. . . .
~ , ', ' ' :
. 2lass72 The coordination chemlstry of L~gand A with Fe(II), Ni(II), Cu(II), Ag(I) and Hg(II), and of Ligand C with Fe(II), has surprisingly been found to be closely analogous to the parent (unsubstituted) 9S3 complexes, as regards stoichiometry, electronic 05 spectra, stability, co-ordination number and electrochemical behaviour. ~ -Representative Ligands were synthesised in detail as follows:-Preparation of Liaand B, viz 9S3-CH20H - -Molybdenum hexacarbonyl, Mo(C0~6, 0.02 mol, and acetonitrile ~ --(60 mL) were heated together to reflux under a dinitrogen atmosphere with stirring for 2 hours. The resulting solution of Mo(C0)3(MeCN)3 was transferred anaeroblcally to a flask containing the bis(tetramethylammonium) salt of 3-thiapentane-1,5-dithiol (~NMe4]2[SCH2CH2SCH2CH2S], 0.02 mol). The resulting suspension was lS stirred for 24h at room temperature, after which a solution of racemic 1,2-dibromopropan-3-ol (0.02 mol) in acetonitrile (10 ml) was added. After stirring for 2h the solvent was removed in vacuo and the residue treated with tNMe4]2tSCH2CH2SCH2CH25], (0.02 mol) in dimethylsulphoxide (S0 ml); After stirring at room temperature under dinitrogen for 24h the mlxture was extracted with diethyl ether (4 x 25ml) and the combined extracts evaporated to dryness.
The residue was dissolved in chloroform and chromatographed on silica gel using chloroform as eluent. Pure 9S3-CH20H (as a vlscous oil which crystallised on prolonged vacuum drying, 24% yield) was obtained by evaporation of the relevant fractions.
PreDaration of Licand D. 9S3-CH20CH2C6H4-Br Thts compound was prepared by a method analogous to that for B (9S3-CH20H), substituting 1,2-dibromo-3-(4 -bromobenzyloxy) propane for 2,3-dibromopropane. It was purified by s~l~ca gel chromatography as described for C (later). Yield 20%.
PreDaration of Liaand E, 9S3-CH20CH2C6H4-4-COOH
This compound was prepared by a method analogous to that for -B (9S3-CH20H), substituting 1,2-dibromo-3-(4 -carboxybenzyloxy)-propane for 2,3-dibromopropane. It was purified as follows. The DMS0 reaction solution was diluted with water (200 ml) and the -.
., -, . , , , .... . . ... .. ~ .. ., . . ... . , - .-.. , .. , .-. . ~.. .. . . -. . . .. .
2~58~ 2 mixture extracted with chloroform. The chloroform extracts were washed with water, dried over MgS04 and evaporated to dryness. The residue was extracted into 10% sodium hydroxide solution, the extract washed wlth chloroform and acidif1ed wlth concentrated 05 hydrochloric acid, resulting in precipitation of a solid. This was dissolved ln ethanol, the solut10n filtered and treated with an equal volume of iced water, and the resulting precipitate collected and dissolved in chloroform. This solution was dried over MgS04 and evaporated to dryness leaving the product as a white powder.
Yield 8%.
Example 2 - PreDaration of Ligands C. F and G
The above synthesis can be developed, building on Ligand B.
This Ligand may be reacted with the bromide RlBr according to the reaction scheme OH R~r Rl may be as follows:
Ligand C (_ Ligand C abovej: -CH2-phenyl (yield 87X) Ligand F: -CH2-paracyanophenyl (yield 43%) L1gand G: -CH2-meta-monobromo-phenyl ~yield 57X) These ligands were syntheslsed ln detall as follows:-PreDaration of Liaand C, 9S3-CH20CH2Ph Ligand B (1 mmol) was reacted with sodium hydride (1 mmol) in dry dimethylformamide (5 ml) followed by addition of benzyl chloride (1 mmol) and stirring at room temperature for 3 days.
25 ml of water was then added and the resulting mixture extracted with diethyl ether and purified by silica gel chromatography using toluene followed by 20% dichloromethane in toluene, then dichloromethane, as eluant. Yield 877.
' .
.
. ;~ . .. . . . , ~
W O 92/16520 pcr/GB92/oo492 ~1~58'72 Preparation of L~aand F, 9S3-CH20CH2C6H4-4-CN
This compound was prepared by a method analogous to that for C, subst~tutlng 4-cyanobenzyl chloride for benzyl chloride. The product was pur~fled by s~lica gel chromatography also as described 05 for C. Yield 43%.
PreDaration of Liqand G. 9S3-CH20CH2C6H4-3-Br Th~s compound was prepared by a method analogous to that for C, subst~tuting 3-bromobenzyl chlor~de for benzyl chloride. The product was purif~ed by s~llca gel chromatography as described for C. Y~eld 57X
ExamDle 3 - PreDaration of l-phenvl-l-phosDha-4 7-d~thiacvclo-nonane(9PhPS2) ~sJ~
Caes~um carbonate (0.013 mol) was st~rred in dry dimethylformam~de (250 ml) at 70C under a din~trogen atmosphere.
1,2-d~chloroethane (0.01 mol) ~n DMF (50 ml) and 3-phenyl-3-phosphapentane-1,5-d~th~ol (prepared by a l~terature method, 0.01 mol) ~n DMF (50 ml) were added dropwise, simultaneously and at the same rate (a per~staltlc pump was used to ma~ntaln the equal and constant rate of addltlon at 4.5 mlh~l). After completlon of the addlt~on the mlxture was st~rred for a further llh at 70C, --then coc~led to room temperature. The solvent was removed in vacuo -and the res~due extracted w~th diethyl ether (30 ml). The extract was washed w~th water (2 x 30 ml) and drled over MgS04. The solvent was removed and the residue chromatographed on silica gel, ~ -elut~ng first with dichloromethane/chloroform 1:1, then toluene.
The product was obtained by removal of solvent from the relevant fract~ons in 37X y~eld as a colourless, v~scous o~l wh~ch - crystalllsed below room temperature.
,.
-.' .: ,.,. . . . . .. . ~,... . . .
- ~, . .. . . .. . .
w o 92/16~20 PCT/GB92/00492 2 ~ ~ 5 87 2 _ 14 -Example 4 - Pre~aration of Metal ~om~lexes of 9PhPS2 Mercurv (II) perchlorate complex with 9PhPS2 l-phenyl-l-phospha-4,7-dithiacyclononane (L) (60mg, 0.23 mmole) was dissolved in acetonitrile (3ml), and stirred at room 05 temperature while mercury (II) perchlorate (50mg, Q.ll mmole) was added. The solution was stirred for 1 hour, then ether was slowly added until the solution turned cloudy (about lOml). The product came out of solution as white crystals on refrigeration.
67mg of ~9PhPS2)2Hg(C104)2 (61%) were obtained.
10 Microanalysis Found C 31.53%, H 3.33%
C24H34C12H908P254 requ~res C 31.60X, H 3.76X
IR (mull, cm~l) 1095(s), 1080(s), 930(s), 875, 830, 820, 810, 765, 745, 725, 700, 695, 635.
Nickel (II) tetrafluoroborate complex with 9PhPS2 Nickel (II) tetrafluoroborate hexahydrate (0.03579, 0.0105 mmole) in ethanol (2ml) was added to a stirred solution of L (0.055g, 0.215 mmole) in ethanol (12ml). A pea-green colour formed at once. The solution was left at 0C for 1 hour, and the resulting green complex was filtered off, washed with ether and 20 dried under vacuum. 0.059g (67%) of (9PhPS2)2Ni(BF4)2.H20 was obtained.
Microanalysis Found C 38.14%, H 4.39%
C24H35B2F8N'Ol/2P2s4 requ~res C 38.22X, H 4.68X
Copper (II) tetrafluoroborate comDlex with 9PhPS2 L (64.5mg, 0.25 mmole) was stirred in ether ~lml), and ethanol ~lOml) added. Copper ~II) tetrafluoroborate ~28.5mg, 0.12 mmole) in ethanol (2ml) was added, and the red product was stirred for 10 min. All attempts to remove the fine solid by filtration failed.
The mixture was centrifuged, and the supernatant decanted off. The 30 red solid was shaken with ether (12ml) and again centrifuged. The solvent was poured off and the product dried under high vacuum.
45mg of (9PhPS2)2Cu(BF4)2 (51%) were obtained.
Microanalysis Found C 38.23%, H 4.27%
C24H34B2CUFgP2S4 requ~res C 38.44~, H 4.57X
35 IR (mull, cm~l) 1325, 1095~s), 1060(s), 1045(s), 1000, 930, 885, 820, 810, 760, 725, 700, 525, 49~.
'.
s, ;
. . ,, ... ,, , , . . . . , :
w o 92/16520 PCT/GB92/00492 21 ~872 CoD~er ~I) hexafluoroDhosDhate complex wlth 9PhPS2 L (120mg, 0.468 mmole) was st~rred ln acetonitrile (4ml~.
Cu(CH3CN)4PF6 (82mg, 0.22 mmole) was added to the solutlon which became very fa~nt plnk in colour as the copper complex dissolved.
OS Ether (20ml) was added, the solution lost its colour and became cloudy. The solution was left overn~ght at 4C to give off-white crystals. These were flltered off and drled under vacuum to yield 134mg (80Z) of (9PhPS2)2CuPF6.CH3CN.
Microanalys~s Found C 40.74X, H 4.58%, N 1.81X
C26H37CuFNP3S4 requlres C 40.96X, H 4.89X, N 1.84%
IR ~mull, cm~l) 1410, 840~s), 745~s), 695, SS0, 480.
The llgand structure was determlned by slngle crystal X-ray to conf1rm the structure Cu(9PhPS2)2~ as hav~ng tetrahedral coord~nation w~th the copper bound by one fac~ally tridentate lS 119and and a slngle phosph~ne donor from the second l~gand. The structures is shown in F~gure 1.
~amDle S - PreDaratlon of extended 9S3 liaand~
1. N-hYdroxvsulDhosucc~nlm~de ester. sod1um salt ~ ~ ~ 0 - N
Llgand E ~see Example 1) ~51mg, O.lS mmol), d~cyclohexyl-carbodl1mlde (31mg, 0.16 mmol), and N-hydroxysulphosuccln~mlde, sodlum salt ~32.5mg, O.lS mmol) were st1rred 1n dry dtmethyl-formam~de ~lml) under dry d~nltrogen at room temperature for 48h.
A whlte sol~d was preclp~tated (dlcyclohexylurea) and was removed by flltration. The f11trate was diluted with lSml d1ethylether, 25- result~ng in format~on of a gummy solid wh~ch was collected and freed of solvent under high vacuum. Infra red absorption ~cm~l):
3500 (br), 2900, 2840, 1760, 1730, 1655, 1615, 1600, 1405, 1355, 1225 (br), 1095, 1070, 1035, 985.
w o 92/16520 PCT/GB92/00492 2~5872 The product was dissolved in dimethylsulphoxide (DMS0) to a concentration of lOmg/ml for use ~n protein labelling, and stored below 0C. (Hereafter the product is identified as bifunctional chelator solution ).
05 2. N-hvdroxvsuccinimide ester Ligand E (0.1039, 0.3 mmol), dicyclohexylcarbodiimide (0.0629, 0.3 mmol), and N-hydroxysuccimide (0.062g, 0.3 mmol) were stirred at 4C in dry tetrahydrofuran (3ml~ under dry dinitrogen for 3h.
The wh~te prec~p~tate (dicyclohexyl urea) that formed was removed after overnight refr~geratlon at 4C, by filtrat on, and the filtrate dried under vacuum. The IR spectrum of th~s material showed the presence of impur~ties (dicyclohexylcarbodiim~de) wh~ch were partially removed by reprecipitation from chloroform (2ml) by addition of cyclohexane (20ml) followed by decantation of the supernatant and drying the oily residue under vacuum. IR: 2920, 2845, 1795, 1765, 1735, 1620, 1605, 1570, 1530, 1445, 1410, 1359. ~ :
Example 6 - PreDaration ofl abelled chelate - protein coniuaate Demonstration of the preparat~on and stability of the conjugate is descr~bed as follcws with reference to Figures 2 to 7 wherein: ~ -F~gure 2 gives the results for gel filtrat~on of rabbit IgG
incubated with 197Hg pre-chelated with the bifunctional chelator (9S3-act~vated ester): radloactivity and absorbance elut~on profile;
Flgure 3 shows gel filtration of rabblt IgG incubated with 197Hg pre-chelated wlth ~ntermediate E ~9S3-carboxylate):
radloact~v~ty and absorbance elut~on profile;
Figures 4 and 5 show gel f~ltration of 197Hg-labelled rabbit IgG incubated wtth whole human serum for 24h: rad~oactivity and absorbance elution profiles respectively; and Figures 6 and 7 show gel filtration of 197Hg-labelled rabbit IgG ln PBS: radioactivity and absorbance profiles respectively.
Bifunctional chelator solution (prepared as described in Example 5-1) (3.62~1) was diluted to 46~1 with DMS0 and the solution added to 2~1 aqueous carrier-free 197HgC12 (37 MBq/ml, Medgenix plc). After 2 minutes this solution was added to rabbit IgG (Sigma, 2mg in iml phosphate buffered sal~ne, PBS).
.. . .
, . . . ,, .. : ...... , ~. ... ~ .
.
w o 92/16520 PCTIGB92/00492 21058~2 - 17 _ After incubation at room temperature for lh, the sample was chromatographed on a sephadex G25M gel filtration column (Pharmacia PD10) eluting with PBS. 30 fractions of 1.4ml were collected. The bulk of the radioactivity (52-93%) eluted with the 05 protein fractions (4ml). The results are shown in Figure 2. As a control, the ligand E (which contains a carboxylate group instead of the activated ester), was employed in place of the bifunctional chelator. This was expected to bind mercury ions but to be incapable of reacting with the protein. In this case, less than 5X
of the radioactivity eluted as an identifiable protein peak. The results are shown in Figure 3. In a second control, the radioactive mercuric chloride was incubated with protein in the absence of any chelator; in this case 32X of the activity was eluted with protein, indicating significant non-specific binding.
Thus, the chelating moiety binds mercury sufficiently strongly to suppress non-specific binding, while the activated ester functionality causes efficient binding to protein.
The above results indicate that the chelated mercury is stable towards transfer of mercury ions to non-specific protein binding sites on rabbit IgG. The types of co-ordinating functional groups available under these circumstances are broadly typical of the b~olog~cal milieu. To further define the stability under biological conditions, the conjugate was incubated wtth human serum, and with bovine serum albumin which is known to be capable of b~nding class b metals (e.g. copper).
Stabll~tv to bov~ne serum album~n Rabbit IgG labelled with 203Hg by the procedure described above, taken from the most radioactive IgG fraction obtained from the gel filtration step, was incubated with an excess of bovine serum albumin (lml of lmg!ml) for lh at 37C. The sample was then loaded onto a protein-A-derivatised affinity column (Pharmacia :
HiTrap lml) and eluted with pH 8.9 NaCl/glycine buffer ~6ml) followed by pH4 citrate buffer (7ml). 13 x lml fractions were collected and assayed for protein, by measuring abs~rbance at 280nm, and for radioactivity. 90X of the radioactivity eluted with the ,~
, :
.
; . , ~ . , , . , ~ , .
, .. : . ~ . .:
WO 92/16520 PCr/GB92/00492 21 ~72 IgG fraction (compared to 94% in the control in which labelled IgG
was incubated alone). Thus transfer of radioactivlty to albumin was slight (of the order of 4%) after the first hour. However, the loss may become more significant after longer periods.
05 Stabilitv to human serum -Rabbit IgG labelled with 197Hg by the procedure described above(O.lml) was added to human serum (2ml) obtained by clotting whole blood, and lncubated for 24h at 37C under sterile cond~tions. A
sample of the serum was then loaded onto a 300 x 8mm gel filtration column ~LKB GlasPak, TSK G3000SW) and eluted with phosphate buffered salirie. Absorbance at 280nm was monitored and 0.5ml fractions were collected and counted. Results are shown in Figure 5. Although the-A280 trace showed several peaks, the radioactivity was confined to a single well defined peak as shown in Figure 4. A control in which labelled IgG was incubated alone showed an identical eluate radioactivity profile as shown in Figures 6 and 7. These results thus fail to demonstrate any transfer of radioactivity to serum components that are separable from IgG and so do not reveal any ma~or instability over the time perlod studied.
Claims (12)
1. A ligand of formula I
(I) where one T is S or Se or Te, another T is S or Se or Te and the third T is S, Se, Te, >PX or >AsX, X being a univalent atom or group, and where Al is -C2H4- or or -C3H6-, A2 is -C2H4- or and A3 is -C2H3R- or or except when A1 is -C3H6-, -C3H5R-, where R is alkyl or substituted alkyl or, if the third T contains P
or As, R may also be H.
(I) where one T is S or Se or Te, another T is S or Se or Te and the third T is S, Se, Te, >PX or >AsX, X being a univalent atom or group, and where Al is -C2H4- or or -C3H6-, A2 is -C2H4- or and A3 is -C2H3R- or or except when A1 is -C3H6-, -C3H5R-, where R is alkyl or substituted alkyl or, if the third T contains P
or As, R may also be H.
2. A ligand according to claim 1 wherein R is selected from hydroxyalkyl, alkoxyalkyl, aryloxyalkyl and arylalkoxyalkyl groups which may be further substituted.
3. A ligand according to claim 1 or 2 wherein A1 and A2 are -C2H4-.
4. A ligand according to any one of claims 1 to 3 wherein each T
is S.
is S.
5. A ligand according to any one of claims 1 to 3 wherein two T's and S and the third is the group .
6. A ligand according to claim 5 wherein X is a phenyl group which may be substituted.
7. A ligand according to claim 6 wherein X is ortho-substituted.
8. An extended ligand comprising a ligand in accordance with any one of the preceding claims in which at least one univalent atom or group thereof has been replaced by a moiety extending the co-ordinating functionality of the ligand and/or a linking moiety capable of facilitating binding to a substrate.
9. A metal complex of a ligand or extended ligand according to any one of the preceding claims.
10. A metal complex according to claim 9 wherein the metal is a radionuclide.
11. A ligand extended ligand or metal complex in accordance with any one of the preceding claims when complexed with a biologically active substrate.
12. A kit suitable for use in radiodiagnosis or radiotherapy including a ligand, extended ligand or complex in accordance with any one of the preceding claims.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9105898.2 | 1991-03-20 | ||
GB919105898A GB9105898D0 (en) | 1991-03-20 | 1991-03-20 | Compounds useful as intermediates for binding ions to substrates |
GB919125882A GB9125882D0 (en) | 1991-12-05 | 1991-12-05 | Cyclic ligands |
GB9125882.2 | 1991-12-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2105872A1 true CA2105872A1 (en) | 1992-09-21 |
Family
ID=26298609
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA 2105872 Abandoned CA2105872A1 (en) | 1991-03-20 | 1992-03-19 | Macrocyclic thioether ligands and their use as intermediates for binding ions to substrates |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0576492A1 (en) |
JP (1) | JPH06505975A (en) |
CA (1) | CA2105872A1 (en) |
GB (1) | GB2253850B (en) |
WO (1) | WO1992016520A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5879657A (en) * | 1993-03-30 | 1999-03-09 | The Dupont Merck Pharmaceutical Company | Radiolabeled platelet GPIIb/IIIa receptor antagonists as imaging agents for the diagnosis of thromboembolic disorders |
WO1999014192A1 (en) * | 1997-09-18 | 1999-03-25 | Pierce Chemical Company | Sulfo-n-hydroxy succinimide and method of preparation |
US5892057A (en) * | 1997-09-18 | 1999-04-06 | Pierce Chemical Company | Preparation of sulfo-N-hydroxysuccinimide salts |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4782013A (en) * | 1987-07-23 | 1988-11-01 | Eastman Kodak Company | Photographic element containing a macrocyclic ether compound |
-
1992
- 1992-03-19 GB GB9205992A patent/GB2253850B/en not_active Expired - Fee Related
- 1992-03-19 JP JP4506056A patent/JPH06505975A/en active Pending
- 1992-03-19 WO PCT/GB1992/000492 patent/WO1992016520A1/en not_active Application Discontinuation
- 1992-03-19 EP EP19920906587 patent/EP0576492A1/en not_active Withdrawn
- 1992-03-19 CA CA 2105872 patent/CA2105872A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP0576492A1 (en) | 1994-01-05 |
GB2253850A (en) | 1992-09-23 |
WO1992016520A1 (en) | 1992-10-01 |
JPH06505975A (en) | 1994-07-07 |
GB9205992D0 (en) | 1992-04-29 |
GB2253850B (en) | 1994-11-09 |
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