CA1118409A - Steroid derivatives and their use in radioimmunoassays - Google Patents
Steroid derivatives and their use in radioimmunoassaysInfo
- Publication number
- CA1118409A CA1118409A CA000308300A CA308300A CA1118409A CA 1118409 A CA1118409 A CA 1118409A CA 000308300 A CA000308300 A CA 000308300A CA 308300 A CA308300 A CA 308300A CA 1118409 A CA1118409 A CA 1118409A
- Authority
- CA
- Canada
- Prior art keywords
- steroid
- compound
- accordance
- hydroxy
- beta
- 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
- 238000003127 radioimmunoassay Methods 0.000 title claims abstract description 41
- 150000003431 steroids Chemical class 0.000 title claims abstract description 35
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 18
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 18
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 16
- 239000001257 hydrogen Substances 0.000 claims abstract description 16
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 15
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 11
- KVUXYQHEESDGIJ-UHFFFAOYSA-N 10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthrene-3,16-diol Chemical compound C1CC2CC(O)CCC2(C)C2C1C1CC(O)CC1(C)CC2 KVUXYQHEESDGIJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims description 39
- 238000000034 method Methods 0.000 claims description 33
- -1 steroid compound Chemical class 0.000 claims description 32
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 10
- XMBWDFGMSWQBCA-YPZZEJLDSA-N iodane Chemical compound [125IH] XMBWDFGMSWQBCA-YPZZEJLDSA-N 0.000 claims description 7
- 229940044173 iodine-125 Drugs 0.000 claims description 7
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 6
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 claims description 4
- 125000004203 4-hydroxyphenyl group Chemical group [H]OC1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 claims description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 4
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 claims description 4
- 239000000700 radioactive tracer Substances 0.000 claims description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 3
- 125000002252 acyl group Chemical group 0.000 claims description 3
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 claims description 3
- 229960000890 hydrocortisone Drugs 0.000 claims description 3
- DNXHEGUUPJUMQT-UHFFFAOYSA-N (+)-estrone Natural products OC1=CC=C2C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 DNXHEGUUPJUMQT-UHFFFAOYSA-N 0.000 claims description 2
- PROQIPRRNZUXQM-UHFFFAOYSA-N (16alpha,17betaOH)-Estra-1,3,5(10)-triene-3,16,17-triol Natural products OC1=CC=C2C3CCC(C)(C(C(O)C4)O)C4C3CCC2=C1 PROQIPRRNZUXQM-UHFFFAOYSA-N 0.000 claims description 2
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 claims description 2
- GCKMFJBGXUYNAG-UHFFFAOYSA-N 17alpha-methyltestosterone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C)(O)C1(C)CC2 GCKMFJBGXUYNAG-UHFFFAOYSA-N 0.000 claims description 2
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims description 2
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 claims description 2
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 claims description 2
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 claims description 2
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 claims description 2
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 claims description 2
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 claims description 2
- DNXHEGUUPJUMQT-CBZIJGRNSA-N Estrone Chemical compound OC1=CC=C2[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 DNXHEGUUPJUMQT-CBZIJGRNSA-N 0.000 claims description 2
- MUQNGPZZQDCDFT-JNQJZLCISA-N Halcinonide Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H]3OC(C)(C)O[C@@]3(C(=O)CCl)[C@@]1(C)C[C@@H]2O MUQNGPZZQDCDFT-JNQJZLCISA-N 0.000 claims description 2
- GCKMFJBGXUYNAG-HLXURNFRSA-N Methyltestosterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@](C)(O)[C@@]1(C)CC2 GCKMFJBGXUYNAG-HLXURNFRSA-N 0.000 claims description 2
- XZTUSOXSLKTKJQ-UHFFFAOYSA-N Uzarigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C1(O)CCC2C1=CC(=O)OC1 XZTUSOXSLKTKJQ-UHFFFAOYSA-N 0.000 claims description 2
- 229960002478 aldosterone Drugs 0.000 claims description 2
- 229960002537 betamethasone Drugs 0.000 claims description 2
- UREBDLICKHMUKA-DVTGEIKXSA-N betamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-DVTGEIKXSA-N 0.000 claims description 2
- 229960004311 betamethasone valerate Drugs 0.000 claims description 2
- SNHRLVCMMWUAJD-SUYDQAKGSA-N betamethasone valerate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)CO)(OC(=O)CCCC)[C@@]1(C)C[C@@H]2O SNHRLVCMMWUAJD-SUYDQAKGSA-N 0.000 claims description 2
- 235000012000 cholesterol Nutrition 0.000 claims description 2
- 229940107161 cholesterol Drugs 0.000 claims description 2
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 claims description 2
- 229960004544 cortisone Drugs 0.000 claims description 2
- 229960003957 dexamethasone Drugs 0.000 claims description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims description 2
- XZTUSOXSLKTKJQ-CESUGQOBSA-N digitoxigenin Chemical compound C1([C@H]2CC[C@]3(O)[C@H]4[C@@H]([C@]5(CC[C@H](O)C[C@H]5CC4)C)CC[C@@]32C)=CC(=O)OC1 XZTUSOXSLKTKJQ-CESUGQOBSA-N 0.000 claims description 2
- 229960005309 estradiol Drugs 0.000 claims description 2
- 229930182833 estradiol Natural products 0.000 claims description 2
- 229960001348 estriol Drugs 0.000 claims description 2
- PROQIPRRNZUXQM-ZXXIGWHRSA-N estriol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H]([C@H](O)C4)O)[C@@H]4[C@@H]3CCC2=C1 PROQIPRRNZUXQM-ZXXIGWHRSA-N 0.000 claims description 2
- 229960003399 estrone Drugs 0.000 claims description 2
- 229960002383 halcinonide Drugs 0.000 claims description 2
- 229960004584 methylprednisolone Drugs 0.000 claims description 2
- 229960001566 methyltestosterone Drugs 0.000 claims description 2
- 229960005205 prednisolone Drugs 0.000 claims description 2
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 claims description 2
- 229960003604 testosterone Drugs 0.000 claims description 2
- 229960005294 triamcinolone Drugs 0.000 claims description 2
- GFNANZIMVAIWHM-OBYCQNJPSA-N triamcinolone Chemical compound O=C1C=C[C@]2(C)[C@@]3(F)[C@@H](O)C[C@](C)([C@@]([C@H](O)C4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 GFNANZIMVAIWHM-OBYCQNJPSA-N 0.000 claims description 2
- 229960002117 triamcinolone acetonide Drugs 0.000 claims description 2
- YNDXUCZADRHECN-JNQJZLCISA-N triamcinolone acetonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H]3OC(C)(C)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O YNDXUCZADRHECN-JNQJZLCISA-N 0.000 claims description 2
- 125000001589 carboacyl group Chemical group 0.000 claims 2
- 238000002372 labelling Methods 0.000 claims 2
- BFZHCUBIASXHPK-QJSKAATBSA-N 11alpha-hydroxyprogesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)C[C@H]2O BFZHCUBIASXHPK-QJSKAATBSA-N 0.000 claims 1
- VHRSUDSXCMQTMA-PJHHCJLFSA-N 6alpha-methylprednisolone Chemical compound C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)CO)CC[C@H]21 VHRSUDSXCMQTMA-PJHHCJLFSA-N 0.000 claims 1
- ORNBQBCIOKFOEO-YQUGOWONSA-N Pregnenolone Natural products O=C(C)[C@@H]1[C@@]2(C)[C@H]([C@H]3[C@@H]([C@]4(C)C(=CC3)C[C@@H](O)CC4)CC2)CC1 ORNBQBCIOKFOEO-YQUGOWONSA-N 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- ORNBQBCIOKFOEO-QGVNFLHTSA-N pregnenolone Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 ORNBQBCIOKFOEO-QGVNFLHTSA-N 0.000 claims 1
- 229960000249 pregnenolone Drugs 0.000 claims 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 63
- 239000000243 solution Substances 0.000 description 43
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 33
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 32
- 239000000203 mixture Substances 0.000 description 26
- 239000000427 antigen Substances 0.000 description 25
- 102000036639 antigens Human genes 0.000 description 25
- 108091007433 antigens Proteins 0.000 description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 24
- 238000002844 melting Methods 0.000 description 24
- 230000008018 melting Effects 0.000 description 24
- 229940093499 ethyl acetate Drugs 0.000 description 21
- 235000019439 ethyl acetate Nutrition 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 19
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 239000012267 brine Substances 0.000 description 15
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 15
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 14
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical class O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 description 11
- MLIREBYILWEBDM-UHFFFAOYSA-N cyanoacetic acid Chemical compound OC(=O)CC#N MLIREBYILWEBDM-UHFFFAOYSA-N 0.000 description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 9
- 239000000741 silica gel Substances 0.000 description 9
- 229910002027 silica gel Inorganic materials 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethanethiol Chemical compound CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 8
- 229960004756 ethanol Drugs 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 8
- 238000012746 preparative thin layer chromatography Methods 0.000 description 8
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 7
- LJLKATTUJLTMDE-UHFFFAOYSA-N 3-(4-methoxyphenyl)pentanedioic acid Chemical compound COC1=CC=C(C(CC(O)=O)CC(O)=O)C=C1 LJLKATTUJLTMDE-UHFFFAOYSA-N 0.000 description 6
- OYOAENCCDKJRIT-UHFFFAOYSA-N [4-(2,6-dioxooxan-4-yl)phenyl] acetate Chemical compound C1=CC(OC(=O)C)=CC=C1C1CC(=O)OC(=O)C1 OYOAENCCDKJRIT-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 6
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- XTHFKEDIFFGKHM-UHFFFAOYSA-N Dimethoxyethane Chemical compound COCCOC XTHFKEDIFFGKHM-UHFFFAOYSA-N 0.000 description 5
- 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 description 5
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- 150000002310 glutaric acid derivatives Chemical class 0.000 description 5
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- INZWPLIECFXWSH-UHFFFAOYSA-N 3-(4-hydroxyphenyl)pentanedioic acid Chemical compound OC(=O)CC(CC(O)=O)C1=CC=C(O)C=C1 INZWPLIECFXWSH-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
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- 239000007864 aqueous solution Substances 0.000 description 4
- JIUWTCXNUNHEGP-GJHPUSIBSA-N cardenolide Chemical compound C1([C@H]2CC[C@@H]3[C@H]4[C@@H]([C@]5(CCCCC5CC4)C)CC[C@@]32C)=CC(=O)OC1 JIUWTCXNUNHEGP-GJHPUSIBSA-N 0.000 description 4
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- 238000011161 development Methods 0.000 description 4
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- 229940093495 ethanethiol Drugs 0.000 description 4
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- 239000002243 precursor Substances 0.000 description 4
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- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 3
- YMRKZYYFHSKJDK-UHFFFAOYSA-N 3-[(4-hydroxyphenyl)methyl]pentanedioic acid Chemical compound OC(=O)CC(CC(O)=O)CC1=CC=C(O)C=C1 YMRKZYYFHSKJDK-UHFFFAOYSA-N 0.000 description 3
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 3
- OQNGCCWBHLEQFN-UHFFFAOYSA-N chloroform;hexane Chemical compound ClC(Cl)Cl.CCCCCC OQNGCCWBHLEQFN-UHFFFAOYSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000004821 distillation Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- HQVHAFYINLCOPG-UHFFFAOYSA-N 3-(4-methoxy-3-methylphenyl)pentanedioic acid Chemical compound COC1=CC=C(C(CC(O)=O)CC(O)=O)C=C1C HQVHAFYINLCOPG-UHFFFAOYSA-N 0.000 description 2
- FCOTUTZZWMCHKK-UHFFFAOYSA-N 3-[(4-methoxyphenyl)methyl]pentanedioic acid Chemical compound COC1=CC=C(CC(CC(O)=O)CC(O)=O)C=C1 FCOTUTZZWMCHKK-UHFFFAOYSA-N 0.000 description 2
- JYVLEKUVHGMSFG-UHFFFAOYSA-N 3-[2-(4-methoxyphenyl)ethyl]pentanedioic acid Chemical compound COC1=CC=C(CCC(CC(O)=O)CC(O)=O)C=C1 JYVLEKUVHGMSFG-UHFFFAOYSA-N 0.000 description 2
- NOVCTEYUCHXVMM-UHFFFAOYSA-N 5-(4'-Methoxyphenyl)pentanal Natural products COC1=CC=C(CCCCC=O)C=C1 NOVCTEYUCHXVMM-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-NJFSPNSNSA-N Carbon-14 Chemical compound [14C] OKTJSMMVPCPJKN-NJFSPNSNSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 2
- GTPCLNIINQFZEI-UHFFFAOYSA-N [4-[2-(2,6-dioxooxan-4-yl)ethyl]phenyl] acetate Chemical compound C1=CC(OC(=O)C)=CC=C1CCC1CC(=O)OC(=O)C1 GTPCLNIINQFZEI-UHFFFAOYSA-N 0.000 description 2
- 150000001325 aldosterones Chemical class 0.000 description 2
- 125000005002 aryl methyl group Chemical group 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 2
- SKCNIGRBPJIUBQ-UHFFFAOYSA-N chloroform;ethyl acetate Chemical compound ClC(Cl)Cl.CCOC(C)=O SKCNIGRBPJIUBQ-UHFFFAOYSA-N 0.000 description 2
- CSTBBLHIGMTEAZ-UHFFFAOYSA-N chloroform;ethyl acetate;hexane Chemical compound ClC(Cl)Cl.CCCCCC.CCOC(C)=O CSTBBLHIGMTEAZ-UHFFFAOYSA-N 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 235000008504 concentrate Nutrition 0.000 description 2
- 230000017858 demethylation Effects 0.000 description 2
- 238000010520 demethylation reaction Methods 0.000 description 2
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 2
- 229940093858 ethyl acetoacetate Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 150000007530 organic bases Chemical class 0.000 description 2
- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 229910052722 tritium Inorganic materials 0.000 description 2
- ODIGIKRIUKFKHP-UHFFFAOYSA-N (n-propan-2-yloxycarbonylanilino) acetate Chemical compound CC(C)OC(=O)N(OC(C)=O)C1=CC=CC=C1 ODIGIKRIUKFKHP-UHFFFAOYSA-N 0.000 description 1
- BHKKSKOHRFHHIN-MRVPVSSYSA-N 1-[[2-[(1R)-1-aminoethyl]-4-chlorophenyl]methyl]-2-sulfanylidene-5H-pyrrolo[3,2-d]pyrimidin-4-one Chemical compound N[C@H](C)C1=C(CN2C(NC(C3=C2C=CN3)=O)=S)C=CC(=C1)Cl BHKKSKOHRFHHIN-MRVPVSSYSA-N 0.000 description 1
- NRIVMXXOUOBRAG-UHFFFAOYSA-N 2-(4-methoxyphenyl)acetaldehyde Chemical compound COC1=CC=C(CC=O)C=C1 NRIVMXXOUOBRAG-UHFFFAOYSA-N 0.000 description 1
- RXQBFWGJLQFGPE-UHFFFAOYSA-N 3-(4-hydroxy-3-methylphenyl)pentanedioic acid Chemical group CC1=CC(C(CC(O)=O)CC(O)=O)=CC=C1O RXQBFWGJLQFGPE-UHFFFAOYSA-N 0.000 description 1
- ZOXCMZXXNOSBHU-UHFFFAOYSA-N 3-(4-methoxyphenyl)propanal Chemical compound COC1=CC=C(CCC=O)C=C1 ZOXCMZXXNOSBHU-UHFFFAOYSA-N 0.000 description 1
- JCXFTVFMZPOWJZ-UHFFFAOYSA-N 3-[2-(4-hydroxyphenyl)ethyl]pentanedioic acid Chemical compound OC(=O)CC(CC(O)=O)CCC1=CC=C(O)C=C1 JCXFTVFMZPOWJZ-UHFFFAOYSA-N 0.000 description 1
- CIVSDAZZSGAFFX-UHFFFAOYSA-N 4-(4-methoxyphenyl)butanal Chemical compound COC1=CC=C(CCCC=O)C=C1 CIVSDAZZSGAFFX-UHFFFAOYSA-N 0.000 description 1
- MYLBIQHZWFWSMH-UHFFFAOYSA-N 4-methoxy-3-methylbenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1C MYLBIQHZWFWSMH-UHFFFAOYSA-N 0.000 description 1
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 229910014033 C-OH Inorganic materials 0.000 description 1
- 101100378101 Caenorhabditis briggsae ace-4 gene Proteins 0.000 description 1
- 229910014570 C—OH Inorganic materials 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- FQISKWAFAHGMGT-SGJOWKDISA-M Methylprednisolone sodium succinate Chemical compound [Na+].C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)COC(=O)CCC([O-])=O)CC[C@H]21 FQISKWAFAHGMGT-SGJOWKDISA-M 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 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
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 125000003668 acetyloxy group Chemical group [H]C([H])([H])C(=O)O[*] 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- RTEXIPZMMDUXMR-UHFFFAOYSA-N benzene;ethyl acetate Chemical compound CCOC(C)=O.C1=CC=CC=C1 RTEXIPZMMDUXMR-UHFFFAOYSA-N 0.000 description 1
- SYWUAPJQKHSVPQ-UHFFFAOYSA-N benzene;ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O.C1=CC=CC=C1 SYWUAPJQKHSVPQ-UHFFFAOYSA-N 0.000 description 1
- 229960004217 benzyl alcohol Drugs 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- XTEGARKTQYYJKE-UHFFFAOYSA-N chloric acid Chemical compound OCl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-N 0.000 description 1
- 229940005991 chloric acid Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 150000001885 cortisol derivatives Chemical class 0.000 description 1
- SPWVRYZQLGQKGK-UHFFFAOYSA-N dichloromethane;hexane Chemical compound ClCCl.CCCCCC SPWVRYZQLGQKGK-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012259 ether extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 150000002431 hydrogen Chemical group 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 229960002899 hydroxyprogesterone Drugs 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960004719 nandrolone Drugs 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000003880 polar aprotic solvent Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 229960001479 tosylchloramide sodium Drugs 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
- C07C51/353—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by isomerisation; by change of size of the carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
- C07C51/367—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by introduction of functional groups containing oxygen only in singly bound form
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J19/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 by a lactone ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
- C07J5/0046—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa
- C07J5/0053—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa not substituted in position 16
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Urology & Nephrology (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Endocrinology (AREA)
- Microbiology (AREA)
- Steroid Compounds (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
ABSTRACT Radiolabeled steroid derivatives having the formula wherein St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmunoassay: R is hydrogen or alkyl of 1 to 3 car-bon atoms; n is 0, 1, 2, 3, or 4 and the asterisk (*) indicates tagging with a radioisotope, are useful as tracers in radioimmunoassays.
Description
RB50a ~STEl~OID DERIV~TIVES AND THEIR USE
IN RADIOIMMUNOASSAYS
s The measurement of various substances by the use of radioimmunoassay techniaues has achieved widespread acceptance in recent years. Yalow and Berson, In Vitro Procedures With Radioisotopes in Medicine, International Atomic Energy Agency, Vienna (1970) pgs. 455 et seq., express the principle of radioimmunoassay in the follow-ing terms:
"Unlabelled antigen in unknown samples competes against labelled antigen ("tracer") for binding to antibody and thereby diminishes the binding of labelled antigen. The degree of competitive inhibition observed in unknown samples is com-pared with that obtained in known standard solutions for determination of concentration of antigen in unknowns."
Radioimmunoassay tests require a specific anti-body, a radioisotope-labeled (hereinafter referred to as "radio-labeled") antigen, a pure sample of the anti-gen to be measured to serve as a reference standard, and means for the separation of free antigen from antibody-bound antigen. Radioimmunoassays follow the basic prin-ciple of saturation analysis, i.e., competition between labeled and unlabeled antigen for a fixed number of antibody binding sites.
RB5Oa When radiolabeled antigen, unlabeled antigen, and anti-body are brought together, the amount of radio-labeled antigen bound to antibody and the amount of radiolabeled antigen ramaining unbound (free) has a direct relationship to the amount of unlabeled antigen present when a given amount of antibody is present.
Thus, by using a constant amount of antibody and radiolabeled antigen, and using known concentrations of unlabeled antigen, a standard (calibration) curve can be plotted showing antigen concentration versus the amount of radiolabeled antigen bound or versus radlo-labeled antigen unbound, or versus a ratio of the two measurements. The concentration of antigen in an un-known sample can be read from the standard curve by determining the amount of bound or free radiolabeled antigen (or ratio of the two measurements) resulting when the unknown sample is mixed with the amount of radio-labeled antigen and antibody used to prepare the curve.
In all radioimmunoassay procedures, it is necessary to provide means for separating the bound from the free labeled tracer material. Many widely varied procedures have been developed and used; exemplary procedures are electrophoresis; chromatography; ion exchange;
adsorption to dextran coated charcoal, talc, or aelu-lose; and a number of solid-phase antibody techniques.
The term "antigen", as used in the field of radio-immunoassays, may cover substances of limited immuno-genicity (ability to generate antibodies). In those cases where the ~ubstance to be measured is of limited immu-nogencity, the substance can be coupled with an immu-nogenic carrier, usually a protein, to increase its 34~9 RB50a immunogenicity. A substance that is nonimmunogenic, but requires immunogenicity when linked with a carrier is referred to as a "hapten".
Radioimmunoassay techniques have been used to de-termine the concentration in body fluids of variousendogenous and exogenous steroids. In the development of radioimmunoassays for the various steroids, the preparation of a radiolabeled antigen is of primary concern. Possible radioisotope lables are tritium, car-bon-14, iodine-125, iodine-131, and others. However, because tritium and carbon-14 must be counted by liquid scintillation ta time-consuming and expensive process), iodine-125 and iodine-131 are more desirable. For reasons well-recognized in the art (e.g.,half-life, radiation hazard, counting efficiency and others) iodine-125 has become the radioisotope of choice for use in steroid radioimmunoassays.
The chemical structure of steroids is such that it is generally not possible to radioiodinate them directly.
It is necessary, therefore, to utilize as a precursor of the radiolabeled antigen a derivative of the steroid to be assayed which is readily iodinated. In choosing or developing such a derivative, the primary concern is the affinity of the derivative for the antibodies of the steroid to be assayed; the affinity of the deri-vative for the anti-bodies should, of course, be as close to the affinity of the steroid for the antibodies as possible.
Compounds having the formula RBSOa O
Il 11 St-O-C-CH2- ~ H-CH2-C-O~
~C~2)n S
_ ~ J
R
OH
are readily tagged with a radioisotope and can be used (when radiolabeled) as a tracer in radioimmunoassay procedures fox the determination of steroid levels in a body fluid. In formula I, and throughout the specifi-cation, R is hydrogen or an alkyl group of 1 to 3 carbon atoms; n is 0, 1, 2, 3, or 4 and St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containting deriva-tive of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmunoassay.
Exemplary of hydroxy steroids intended for radio-immunoassay which may be modified structurally as shown in formula I are: cholesterol, cortisol, cortisone, corticosterone, prednisolone, methylprednisolone, tri-amcinolone, betamethasone, dexamethasone, triamcinoloneacetonide, betamethasone valerate, halcinonide, al-dosterone, estrone, estradiol, estriol, testosterone, l9-nortestosterone, methyltestosterone, and pregnenono-lone. Exemplary of hydroxy containing derivatives of a steroid intended for radioimmunoassay (which deriva-tives have a strong affinity for the antibodies of the steroid intended for radioimmunoassay) are digoxigenin, .
lllS409 RB5Oa digitoxigenin and ll~-hydroxyprogesterone.
The steroids of formula I are prepared from the precursor hydroxy steroid having the formula St-OH
and a glutaric anhydride derivative having the formula II O
Rl 0 ~3 (C~2)n C
In formula II, and throughout the specification, Rl is an alkanoyl group having 2 to 6 carbon atoms, acetyl being the preferred group.
The anhydrides of Formula II are novel compounds, and as such constitute an integral part of this inven-tion. They can be prepared by first reacting a 4-methoxyphenylaliphatic aldehyde having the formula III
R
CH30 ~ (CH2)n CH
with at least 2 molar equivalents of cyanoacetic acid in the presence of a base (e.g., sodium hydroxide) to yield, on acid hydrolysis, a compound having the formula RB50a IV R C
C~l30 _ ~3 CH2- -OH
An alternative preparation for the compound of formula IV wherein n is O and R is hydrogen, i.e., 3-(4-methoxy-phenyl)glutaric acid, is disclosed by Smith et al., J.A.C.S., 72, 1877 (1950). In that procedure, anisalde-hyde is condensed with ethyl acetoacetate in the pre-sence of piperidine to give ethyl anisal-bis-acetoace-tate. Cleavage of this product to give the desired 3-(4-methoxyphenyl)glutaric acid can be accomplished with boiling alcoholic sodium hydroxide solution.
Demethylation of the glutaric acid derivatives of formula IV results in glutaric acid derivatives having the formula V O
R
HO ~ (CH2)n C~
and can be accomplished by following one of the several procedures known in the art for the demethylation of aryl methyl etherq. One such procedure, described by Feutrill et al., Aust. J. Chem., 25, 1719 (1972), in-volves the treatment of the aryl methyl ether with thioethoxide ion (readily prepared in situ from ethane-thiol and sodium hydride) in a polar aprotic solvent, RB5Oa preferably dimethylformamide.
'I'he phenolic hydroxy group of a compound of formula V can be protected with an alkanoyl group using art-recognized procedures. One such procedure comprises reacting the glutaric acid derivative with the appro-priate acid anhydride (acetic anhydride is preferred).
The preferred method of preparing a glutaric anhydride derivative of formula II from the glutaric acid deriva-tive of formula V is to combine the conversion of the acid to anhydride and the protection of the phenolic hydroxy group into a single step. When the Rl protecting group is acetyl, this would involve heating a glutaric acid derivative of formula V in acetic anhydride.
The reaction of a steroid precursor having the lS formula St-OH and a glutaric anhydride derivative of formula II to yield a steroid having the formula St-O-C-CH -CH-CH -C-OH
(CH2)n R
O-Rl can be run in the presence of an organic base. Exem-plary organic bases are nitrogen containing hetero-cyclics, e.g., pyridine, and tertiary amines, e.g., triethylamine. The reaction will preferably be run at an elevated temperature.
In those instances wherein the steroid precursor has more than one hydroxy substitutent, it will be possi-ble to monoacylate the steroid with a glutanic anhy-dride derivative of formula II because of the varying ~118409 RB5Oa reactivites of the steroid's hydroxyl substituents. If it is desirable to acylate a hydroxy substituent other than the most reactive one, conventional blocking techniques should be used to protect the more reactive substituents.
~r~al of the phenolic hydroxyl protecting group in a compound of formula VI yields the corresponding pro-duct of formula I.
The compounds of formula I can be labeled ("tagged") 10 with a radioisotope, preferably iodine-125 or iodine-131, and most preferably iodine-125, using procedures well known in the artl to yield a radiolabeled hapten having the formula O O
VII St O C CH2 CH C 2 C
(CH2)n ~, R /
OH
The asterisk (*) in formula VII indicates tagging with a radioisotope. Exemplary of the methods known in the art is the method of Hunter and Greenwood; see Nature, 194:495 (1962). The radiolabeled compounds of formula VII form an integral part of this invention.
The radiolabeled compounds of formula VII can be used as tracers in radioimmunoassay procedures following the general principles set forth hereinabove. Exemplary detailed procedures are described in Jaffe et al., "Methods of Hormone Radioimmunoassay", Academic Press, New York (1974) and Berson et al., "Methods in Investigative and Diagnostic Endocrinology", Vol. 3 on "Steroid Hormones", North Holland, Amsterdam (1975).
RB50a The radiolabeled compounds of this invention are parti-cularly useful as reagents in the automated radioimmu-noassay system of Brooker et al. disclosed in United States patent 4,022,577 issued May 10, 1977.
The following examples are specific embodiments of this invention.
Example 1 (3~,5~,12~)-3-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]
-12,14-dihydroxycard-20(22)-enolide A) 3-(4-Methoxyphenyl)glutaric acid A mixture of ~-anisaldehyde (27.2g), ethyl aceto-acetate (52.1g) and piperidine (4 ml) in 95% ethanol (10 ml) is stirred at room temperature for 5.0 hours while a solid forms. The solid is isolated by filtration, washed with 25% ethanol and crystallized from 95% etha-nol to afford ethyl 2,2'-(4-methoxybenzal)-bls-aceto-acetate (31.4g), melting point 138-141C. The filtrate on dilution with an equal amount of water gives a solid which is crystallized from 95% ethanol to afford another crop of material (8.5g), melting point 137-142C.
A mixture of ethyl 2,2'-(4-methoxybenzal(-bis-acetoacetate (30g), ethanol (450 ml) and 50~ sodium hydroxide (450g) is refluxed vigorously for 1.0 hour.
Water (150 ml) is added and most of the ethanol is re-moved by distillation in vacuo. The concentrate is acidified with concentrated hydrochloric acid and is extracted with ethyl acetate. The ethyl acetate solution is wa~hed with brine, dried, evaporated, and the residue is crystallized from benzene-methanol to afford 3.3g of 3-(4-methoxyphenyl)glutaric acid, melting point 147-150C.
1~1350a B) 3-(4-Hydroxyphenyl)glutaric acid To a stirred suspension of 57% sodium hydride-paraffin (6.45g), in dry dimethylformamide (70 ml) is slowly added ethanethiol (11.89 ml) in dry dimethyl-- S formamide (20 ml). After stirring the resultant slurry for 15 minutes, a salution of 3-(4-methoxyphenyl)glu-taric acid (3.0g) in dry dimethylformamide (20 ml) is added. The slurry is heated in a bath at 165C for 5.0 hours and most of the solvent is removed by distillation ln vacuo. The residue is diluted with water, acidified with concentrated hydrochloric acid and extracted twice with ether (the extracts are discarded). The solution is saturated with sodium chloride and extracted with ethyl acetate. The ethyl acetate solution is washed once with brine, dried and the residue crystallized from chloro-form-hexane to afford 2.3g of 3-(4-hydroxyphenyl)-glutaric acid, melting point 168-170C.
C) 3-(4-Acetyloxyphenyl)glutaric anhydride A solution of 3-(4-hydroxyphenyl)glutaric acid (800 mg) in acetic anhydride (15 ml) is heated at 100C
for 2.5 hours and evaporated to dryness in vacuo. The residual solid is crystallized from chloroform-hexane to afford 600 mg of 3-(4-acetyloxyphenyl)glutaric an-hydride, melting point 140-143C.
D) 12~-(Acetyloxy)-3~,5~)-3-[4carboxy-3-[4-(acetyloxy)-phenyl]-l-oxobutoxy]-14-hydroxycard-20(22)-enolide A solution of digoxigenin-12-acetate (130 mg) and 3-(4-acetyloxyphenyl)glutaric anhydride (286 mg) in dry pyridine (4.0 ml) i8 heated under nitrogen in a bath at 120C for 12 hours. Water (0.5 ml) is added and after 5 minutes, the mixture is evaporated in vacuo. The residue is dissolved in chloroform, washed with 10% hydrochloric 11184(~9 RB50a acid and brine, dried and evaporated. This residue is subjected to preparative thin-layer chromatography (tlcl on a silica gel plate (2.0 X 200 X 200 mm) using chloro-formmethanol (9:1) for development to afford 110 mg of the title compound as an amorphous solid, melting point 118-135C.
E) (3~,5~,12~)-3-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-12,14-dlhydroxycard-2o(22)-enolide To a solution of 12~-(acetyloxy)-(3~,5~)-3-[4-carboxy-3-[4-(acetyloxy)phenyl]-1-oxobutoxy]-14-hydroxy-card-20 (22)-enolide (68 mg) in methanol (3.0 ml) is added a solution of potassium carbonate (83 mg) in water (1.0 ml) and the mixture is stirred at room temp rature for 2.5 hours. The solution is then acidified with 5%
hydrochloric acid and the methanol is evaporated ln vacuo.
The residue is diluted with water (10 ml) and extracted with ethyl acetate. The ethyl acetate solution is wash-ed with brine, dried over anhydrous magnesium sulfate, evaporated and the residue subjected to preparative tlc on silica gel plates (1 X 200 X 200 mm) using chloro-form-methanol (3:1) for development to afford 22 mg of the title compound as an amorphous solid, melting point 110-118C (melting completely to liquid at 144C).
Example 2 (3~,5~,12~)-3-[4-Carboxy-3-[(4-hydroxyphenyl)methyl]-1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide A) 3-1(4-Methoxyphenyl)methyl]glutaric acid To a solution of cyanoacetic acid (3.57g), in water ~118409 RB50a 20 ml is added a solution of sodium hydroxide (2.08g) in water (20ml). The solution is diluted with glyme (70 ml), 4-methoxyphenyl acetaldehyde (3.0g) is added and the mixture is left at room temperature for 6.0 hours. The glyme is evaporated in vacuo, and the resi-due is mixed with I0% hydrochloric acid (200 ml) and re-fluxed for 6.0 hours. After cooling, the mixture is extracted with ethyl acetate. The ethyl acetate ex-tract is washed once with brine, dried, evaporated and the residue subjected to chromatography on a column of silica gel using chloroform-ethyl acetate mixtures for elution to afford 2.2g of 3-[(4-methoxyphenyl) methyl]glutaric acid. Crystallization from a benzene-ethyl acetate-hexane mixture gives a specimen having a melting point 109-110C.
B) 3-[(4-HydroxYphenyl)methyl]glutaric acid To a stirred suspension of 57% sodium hydride-paraffin (1.4g) in dry dimethylformamide (30 ml) is added dropwise a solution of ethanethiol (4.0 ml~ in dry dimethylformamide (10 ml). After 15 minutes, a solution of 3-[(4-methoxyphenyl)methyl]glutaric acid (800 mg) in dry dimethylformamide (20 ml) is added.
The resulting slurry is heated in a bath at 165C for 20 hours and evaporated in vacuo. The residue is acidi-fied with 20~ hydrochloric acid, extracted with etherand the extracts are discarded. The aqueous solution is saturated with salt and extracted with ethyl acetate.
The extracts are combined, washed once with brine, dried, evaporated and the residue crystallized from a mixture of ethyl acetate-chloroform-hexane to give 600 mg of 3-[(4-hydroxyphenyl)methyl]glutaric acid, melting point 117-119C.
.
.
RBSOa C) 3-[[(4-Acetyloxy)phenyl]methyl]glutaric anhydride 3-[(4-Hydroxyphenyl)methyl]glutaric acid (500 mg) in acetic anhydride (15 ml) is heated in a bath at 120C
for 2 hours. The solution is then evaporated to dry-ness in vacuo and the residue triturate~ with chloro-form~exane to afford 490 mg of the title compound, melting point 88-89C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[4-carboxy-3-[[4-(acetyl-oxy)-phenyl]methyl]-l-oxobutoxy]-14-hydroxycard-20(22)-enolide A solution of digoxigenin-12-acetate (216 mg) and 3-[[(4-acetyloxy)phenyl]methyl]glutaric anhydride (500 mg) in dry pyridine (6.0 ml) is refluxed for 20 hours.
Most of the pyridin~ is removed by distillation in vacuo and the residue is diluted with water (20 ml) and aci-dified with concentrated hydrochloric acid. The mix-ture is extracted with ethyl acetate, the extract washed twice with small amounts of brine, dried, evaporated and the residue subjected to preparative thin-layer chromato-graphy (as in Example ID) to isolate 323 mg of 12B-(acetyloxy)-(3~,5~)-3-[4-carboxy-3-[[4-(acetyloxy) pheyny]methyl]-l-oxobutoxy]-14-hydroxycard-20(22)-enolide, melting point 70-7gC.
E) (3B,5B,12~)-[4-Carboxy-3-[(4-hydroxyphenyl)methyl]
1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide A mixture of 12B-(acetyloxy)-(3~,5B)-3-[4-carboxy-3-[[4-(acetyloxy)phenyl]methyl]-1-oxobutoxy]-14-hydroxy-card-20(22)-enolide (160 mg), methanol (20 ml), water (7.0 ml) and potassium carbonate (207 mg) is stirred at 15C for 2.5 hours. The mixture is acidified with concentrated hydrochloric acid and most of the methanol is evaporated ln vacuo at room temperature. The concen-trate is extracted with ethyl acetate, the extract washed ~184~g RB50a with small amounts of brine, dried (MgSO4anhydrous) evaporated and the residue subjected to a preparative thin-layer chromatography on silica gel plates (as in Example lE) to afford 55 mg of (3~,5~,12~)-[4-carboxy-3-[(4-hydroxyphenyl)methyl]-1-oxobutoxy]-12,14-dihydroxy-card-20(22)-enolide, melting point 155-165C.
Example 3 (3~,5~,12~)-3-[[3-tCarboxymethyl)-5-(4-hydroxyphenyl)-1-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide A) 3-[2-(4-Methoxyphenyl)ethyl]glutaric acid To a solution of cyanoacetic acid (850 mg) in water (5.0 ml) is added a solution of sodium hydroxide (440 mg) in water (10 ml). The solution is diluted with glyme (15 ml) and 3-(4-methoxyphenyl)propionaldehyde (820 mg) is added. The solution is kept at room tempera-ture for 10 hours and then evaporated to remove the glyme.
To the residue is added 10% hydrochloric acid (50 ml) and the mixtureis thereafter refluxed for 6 hours with stirring. It is then cooled and extracted with ethyl acetate. The extract is washed with brine, dried over anhydrous magnesium sulfate and evaporated. The resi-due is subjected to chromatography over silica gel (15 g)to isolate in chloroform-ethyl acetate (80/20) fraction, 600 mg of the title compound. Crystallization from ethyl acetate-benzene gives a specimen having a melting point of 98-100C.
B) 3-[2-(4-Hydroxyphenyl)ethyl]glutaric acid To a suspension of 57% sodium hydride-paraffin (2.2g) in dry dimethylformamide (70 ml) in an atmosphere 34()9 - RB50a of nitrogen is slowly added ethanethiol (4.0 ml). The mixture is stirred at room temperature until a clear solution results. To this solution is added a solution of 3-[2-(4-methoxyphenyl)ethyl]-glutaric acid (1.2 g) in dry dimethylformamide (10 ml) and the mixture is heated with stirring in a bath at 165C for 6.0 hours.
The mixture is then evaporated to dryness ln vacuo and the resulting residue is dissolved in water (50 ml), extracted with ether (two 50 ml portions) and the ether extract is discarded. The aqueous solution is acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The ethyl acetate solution is washed with brine, dried with anhydrous magnesium sulfate, and evapor-ated to a residue. Crystallization of this from chloro-form-hexane affords l.Og of the title compound, melting point 142-144C.
C) 3-[2-[4-Acetyloxy)phenyl]ethyl]glutaric anhydride A solution of 3-~2-(4-hydroxyphenyl)ethyl]glutaric acid (600 mg) in acetic anhydride (15 ml) is heated in a bath at 120C for 2.0 hours. It is evaporated in vacuo and the residue is crystallized from dichlorometh-ane-hexane to afford 495 mg of 3-[2-[4-(acetyloxy)phenyl]-ethyl]glutaric anhydride, melting point 97-99C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[[5-[4-(acetyloxy)phenyl]
-3-(carboxymethyl)-1-oxopentyl]oxycard-20(22)-enolide A solution of digoxigenin-12-acetate (200 mg) and 3-[2-[4-(acetyloxy)phenyl]ethyl]glutaric anhydride (470 mg) in dry pyridine (8.0 ml) is refluxed for 20 hours. The pyridine is evaporated ln vacuo, the residue diluted with water (25 ml), acidified with concentrated ,,~
34()g RBSOa hydrochloric acid and extracted with chloroform. The chloroform solution is washed with small amounts of brine, dried, evaporated and purified by preparative thin-layer chromatography (as in Example ID) to isolate 253 mg of 12~-(acetyloxy)-(3~,5~)-3-1[5-[4-(acetyloxy(phen-yl]-3-(carboxymethyl)-1-oxopentyl]-oxy]card-20(22)-enolide.
E) (3~,5~,12~)-3-[[3-(Carboxymethyl)-5-(4-hydroxyphen-yl)-l-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide.
A solution of 12~-(acetyloxy)-(3~,5~)-3-[[5-[4-(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxopentyl]oxy]
card-20(22)-enolide (230 mg) in a mixture of methanol (10 ml) and water (2.5 ml) containing potassium carbonate (210 mg) is stirred at 10 to 15C for 2.5 hours. It is then acidified with concentrated hydrochloric acid.
Isolation and purification of the product as described in Example lE yields 44 mg of (3~,5~,12~)-3-[[3-(carbo-xymethyl)-5-(4-hydroxy-phenyl)-1-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide, melting point 126-134C.
Example 4 (3~,5~,12~)-3-1[3-(carboxymethyl)-6-(4-hydroxyphenyl)-1-oxohexyl]oxy]-12,14-dihydroxycard-20(22)-enolide A) 3-[3-(4-Methoxyphenyl)propyl]glutaric acid 4-(4-Methoxyphenyl)butyraldehyde (5.34 g) is re-acted with cyanoacetic acid (5.15 g) and sodium hydroxide (2.74 g) in a mixture of glyme (50 ml) and water (60 ml).
The mixture is treated with hydrochloric acid and pro-cessed as described in Example 2A to afford the title co~xNnd (2.lg) which on crystallization from ethyl ace-4~)9 RB5Oa tate-chloroform-hexane had a melting poin~ of 71-73C.
B) 3-[3-(4-Hydroxyphenyl)propyl]glutaric acid 3-[3-(4-Methoxyphenyl)propyl]glutaric acid (2.0g) is demethylated by the procedure described in Example 5 lB to afford, after cyrstallization of the product from ethyl acetate-chloroform-hexane, 1.2 g of 3-[3-(4-hydro-xyphenyl)-propyl3glutaric acid, melting point 107-108C.
C) 3-[3-14-(Acetyloxy)phenyl]propyl]glutaric anhydride 3-[3-(4-Hydroxyphenyl)propyllglutaric acid (533 10 mg) is reacted with acetic anhydride as described in Example lC to afford 3-[3-~4-(acetyloxy)phenyl]propyl]
glutaric anhydride, melting point 55-57C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[[6-[4-~acetyloxy)phenyl]
3-(carboxymethyl)-1-oxohexyl]oxy]card-20(22)-enolide A mixture of digoxigenin-12-acetate (216 mg) and 3-[13-(4-acetyloxy)phenyl]propyl]glutaric anhydride (610 mg) in dry pyridine (9.0 ml) is reacted for 18 hours and the product is isolated and purified as described in Example lD to afford 245 mg of 12B~acetyloxy)-(3B,5B)-3-20 [[6-[4-(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxohexyl]
- oxy]card-20(22)-~nolide, melting point 98-113C.
E) (3B,5B,12~)-3-[[3-~Carboxymethyl)-6-(4-hydroxy-phenyl)-l-oxohexyl]oxy]-12,14-dihydroxycard-20(22) -enolide A mixture of 12~-(acetyloxy) (3B,5~)-3-[[6-~4-(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxohexyl]oxy]
card-20(22)-enolide (150 mg) and potassium carbonate (172 mg) is reacted in a mixture of methanol (5.0 ml) and water (2 ml). The mixture is processed and purified 30 as described in Example lE to afford 58 mg of (3B,5B, 12B)-3-[[3-(carboxymethyl)-6-(4-hydroxyphenyl)-1-oxo-hexyl]oxy]-12,14-dihydroxycard-20(22)-enolide, melting point 115-128C.
409 RB50a Example 5 (3~',5~,12p,)-3-[[3-(Carboxymethyl)-7-(4-hydroxyphenyl) -1-oxoheptyl]oxy-12,14-dihydroxycard-20~22)-enolide Following the procedure of Example 4, but sub-stituting 5-(4-methoxyphenyl)valeraldehyde for 4-(4-methoxyphenyl)-butyraldehyde yields the title compound.
Detailed Procedure for Radioiodination of Digoxigenin Derivatives Sodium radioiodide (I125) aqueous solution (10 ml;
approximately 6mCi) is added to a reaction vial contain-15 ing a methanolic solution of (3~,5~,12~)-3-~4-carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-12,14-dihydroxyca~d-20 (22)-enolide (10 ml 1 mg/ml). The vial is stoppered and a chloramine T solution (25 ml; 2 mg/ml in 0.5M
phosphate buffer, pH 7.5) is injected through the stoppër 20 The vial is mixed well by shaking and allowed to stand for five minutes. Sodium metabisulfite solution (20 ml;
3 mg/ml in 0.5M phosphate buffer, pH 7.5) is injected through the stopper to quench the reaction.
The reaction mixture is applied to a 40 ml Sephadex 25 G-10 column and eluted with tris acetate buffer (0.05M, pH 6.5). Forty-four drops per tube are collected with the aid of a fraction collector. Free iodide comes off around tube #38 and the iodinated product elutes off in two fractions, tubes 110 through 130 (fraction I) and tubes 30 131 through 150 (fraction II?. Fraction II is found to be superior and is used in radioimmunoassays.
* Trade Mark B~
~ 4~9 RB50a Example 6 21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-11~,18-epoxy-18-hydroxypregn-4-ene-3,20-dione ~) 21-L3-t4-Acetyloxyphenyl)-4-carboxy-1-oxobutoxy]
-11~ 18-e~oxv-18-hvdroxv~regn-4-ene-3,20-dione Aldosterone (100 mg) is refluxed in dry pyridine 10 (6.0 ml) with 3-(4-acetyloxyphenyl)glutaric anhydride (300 mg) for 2.0 hours. The solution is then cooled to room temperature, water is added and after standing for a few minutes evaporated in vacuo. The residue is dis-solved in ethyl acetate (20 ml), washed with 15% hydro-15 chloric acid (6.0 ml) and brine, dried and evaporated toafford 408 mg of a gum. A thin-layer chromatography examination of this material shows the presence of three steroidal products. It is then subjected to preparative thin-layer chromatography on two 2.0 mm silica gel plates 20 (with two developments of the plates with chloroform-methanol (9:1) to isolate 135 mg of the title compound, melting point 145-160C with consistent spectral data.
B) 21-~4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]~
18-epoxy-18-hydroxypregn-4-ene-3,20-dione A solution of 21-[3-(4-acetyloxyphenyl)-4-carboxy-l-oxobutoxy]~ ,18-epoxy-18-hydroxypregn-4-ene-3,20-dione (130 mg) in anhydrous methanol (15 ml) containing triethylamine (0.2 ml) is allowed to stand at room temp-erature for 24 hours. The methanol is then evaporated 30 in vacuo, the residue is diluted with water (5.0 ml), acidified with 10~ hydrochloric acid and extracted with ethyl acetate (three 5.0 ml portions). The ethyl acetate ~8409 RB50a solution is washed with brine, dried over anhydrous magnesium sulfate, evaporated and the residue is subject-ed to preparative thin-layer chromatography on silica gel plates (using chloroform-methanol, 9:1 for develop-5 ment) to isolate 83 mg of the title compound, meltingpoint 145-152Cr with consistent spectral data.
Detailed Procedure for Radioiodination of Aldosterone Derivative . . .
To a solution of 21-[4-Carboxy-3-(4-hydroxyphenyl)-30 1-oxobutoxy]-11~,18-epoxy-18-hydroxypregn-4-ene-3,20-dione (5.0,ug) in dioxane-0.5M borate buffer (1:9, 50 ul) at pH 8.5 are added successively 20 ~1 of aqueous solutions of sodium radioiodide (I125, 4mCi) and freshly prepared 111~3409 RB5Oa chloramine-T (80 ~g). After 90 seconds, the reaction is stopped by the addition of sodium bisulfite (80,ug) in water (20 ~1). The solution is applied on a 5 X 20 cm silica gel plate which is developed with chloroform-5 methanol (9:1). The band of radioiodinated product islocated using a scanner and is isolated by extraction with ethanol. The concentrated ethanol solution is stored at 15C.
Example 7 21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-11~,17-dihydroxypregn-4-ene-3,20-dione A) 21-[3-(4-Acetyloxyphenyl)-4-carboxy-1-oxobutoxy-11~,17-dihydroxypregn-4-ene-3,20-dione A solution of cortisol (346 mg) and 3-(4-acetyloxy-phenyl)glutaric anhydride (496 mg) in dry pyridine (5.0 ml) is refluxed for 40 minutes. The pyridine is evapor-ated in vacuo, the residue is diluted with ethylacetate 20 (30 ml), washed with 15% hydrochloric acid (10 ml) and brine, dried over anhydrous magnesium sulfate and evapor-ated to yield 840 mg of a powder. Examination of the NMR spectrum and TLC behavior of this material shows that it is a 1:1 mixture of the title compound and 3-25 (4-acetyloxyphenyl)glutaric acid.
B) 21-[4-Carboxy-3-(4-hydroxyphenyl)l-oxobutoxy]-11~, 17- dihyroxypregn-4-ene-3,20-dione The mixture obtained above (700 mg) is dissolved in anhydrous methanol (30 ml) containing triethyamine 30 (1.0 ml) and the solution is refluxed for 5.0 hours.
The solution is cooled, acidified with 10~ hydrochloric acid and most of the methanol is evaporated in vacuo.
The resulting slurry is extracted with ethyl acetate, RBSOa the ethyl acetate solution is washed with brine, dried, and evaporated. The residue is subjected to pre-parative thin-layer chromatography on two 2.0 mm silica gel plates u3ing chloroform-methanol (9:1) for develop-5 ment and the major band is isolated with chloroform-methanol (8:2) to afford 300 mg of the title compound, melting point 98-110C.
Description for Radioiodination of Cortisol Derivative 21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-llr~,17-dihydroxypregn-4-ene-3,20-dione is radiolabeled with I125 using the procedure described above for the 15 radioiodination of aldosterone derivatives.
Example 8 (3r"5r~,12f~)-3-[4-Carboxy-3-(4-hydroxy-3- methylphenyl) 20 -1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide A) 3-(4-Methoxy-3-methylphenyl)glutaric acid A mixture of 4-methoxy-3-methylbenzaldehyde (100 mmole), cyanoacetic acid (210 mmole) and potassium 25 hydroxide (250 mmole( in water SOO ml) is stirred at room temperature for 20 hours. The resulting solution is acidified with concentrated hydrochloric acid (100 ml) and the mixture is refluxed for 60 hours. After Gooling, it is saturated with sodium chloride and extracted with 30 ethyl acetate to afford the title compound.
.~.
RBSOa ;
B) 3-(4-Elydroxy-3-methylphenyl)glutaric acid Followin~ the procedure described in Example lB, but substituting 3-(4-methoxy-3-methylphenyl)glutaric acid for 3-(4-methoxyphenyl)glutaric acid, yields the 5 title compound.
C) 3-[4-(Acetyloxy)-3-methylphenyl)]glutaric anhydride Following the procedure described in Example lC, but substituting 3-(4-hydroxy-3-methylphenyl)glutaric acid for 3-(4-hydroxyphenyl)glutaric acid, yields the 10 title compound.
D) 12~-(Acetyloxy)-(3~,5~)-3-[4-carboxy-3-[4-(acetyl-oxy)-3-methylphenyl]-1-oxobutoxy]-14-hydroxycard -20(22)-enolide Following the procedure described in Example lD, but substituting 3-~4-(acetyloxy)-3-methylphenyl)]glu-taric anhydride for 3-(4-acetyloxyphenyl)glutaric anhy-dride, yields the title compound.
; E) (3~,5~,12~)-3-t4-Carboxy-3-](4-hydroxy-3-methyl-phenyl)-l-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide Following the procedure described in Example lE, but substituting 12~-(acetyloxy)-(3~,5~)-3-t4-carboxy -3-[4-(acetyloxy)-3-methylphenyl]-1-oxobutoxy]-14-hy-droxycard-20(22)-enolide for 12~-(acetyloxy)-(3~,5~)-3-5 [4-carboxy-3-[4-(acetyloxy)phenyl]-1-oxobutoxy]-14-hy-droxycard-20(22)-enolide, yields the title compound.
Examples 9-26 Following the procedure described in Example 6, but ~ubstituting the steroid listed in Column I for al-111~3409 RB50a do~terone and the anhydride reagent listed in ColumnII for 3-(4-acetyloxyphenyl)glutaric anhydride, yields the product listed in Column III.
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IN RADIOIMMUNOASSAYS
s The measurement of various substances by the use of radioimmunoassay techniaues has achieved widespread acceptance in recent years. Yalow and Berson, In Vitro Procedures With Radioisotopes in Medicine, International Atomic Energy Agency, Vienna (1970) pgs. 455 et seq., express the principle of radioimmunoassay in the follow-ing terms:
"Unlabelled antigen in unknown samples competes against labelled antigen ("tracer") for binding to antibody and thereby diminishes the binding of labelled antigen. The degree of competitive inhibition observed in unknown samples is com-pared with that obtained in known standard solutions for determination of concentration of antigen in unknowns."
Radioimmunoassay tests require a specific anti-body, a radioisotope-labeled (hereinafter referred to as "radio-labeled") antigen, a pure sample of the anti-gen to be measured to serve as a reference standard, and means for the separation of free antigen from antibody-bound antigen. Radioimmunoassays follow the basic prin-ciple of saturation analysis, i.e., competition between labeled and unlabeled antigen for a fixed number of antibody binding sites.
RB5Oa When radiolabeled antigen, unlabeled antigen, and anti-body are brought together, the amount of radio-labeled antigen bound to antibody and the amount of radiolabeled antigen ramaining unbound (free) has a direct relationship to the amount of unlabeled antigen present when a given amount of antibody is present.
Thus, by using a constant amount of antibody and radiolabeled antigen, and using known concentrations of unlabeled antigen, a standard (calibration) curve can be plotted showing antigen concentration versus the amount of radiolabeled antigen bound or versus radlo-labeled antigen unbound, or versus a ratio of the two measurements. The concentration of antigen in an un-known sample can be read from the standard curve by determining the amount of bound or free radiolabeled antigen (or ratio of the two measurements) resulting when the unknown sample is mixed with the amount of radio-labeled antigen and antibody used to prepare the curve.
In all radioimmunoassay procedures, it is necessary to provide means for separating the bound from the free labeled tracer material. Many widely varied procedures have been developed and used; exemplary procedures are electrophoresis; chromatography; ion exchange;
adsorption to dextran coated charcoal, talc, or aelu-lose; and a number of solid-phase antibody techniques.
The term "antigen", as used in the field of radio-immunoassays, may cover substances of limited immuno-genicity (ability to generate antibodies). In those cases where the ~ubstance to be measured is of limited immu-nogencity, the substance can be coupled with an immu-nogenic carrier, usually a protein, to increase its 34~9 RB50a immunogenicity. A substance that is nonimmunogenic, but requires immunogenicity when linked with a carrier is referred to as a "hapten".
Radioimmunoassay techniques have been used to de-termine the concentration in body fluids of variousendogenous and exogenous steroids. In the development of radioimmunoassays for the various steroids, the preparation of a radiolabeled antigen is of primary concern. Possible radioisotope lables are tritium, car-bon-14, iodine-125, iodine-131, and others. However, because tritium and carbon-14 must be counted by liquid scintillation ta time-consuming and expensive process), iodine-125 and iodine-131 are more desirable. For reasons well-recognized in the art (e.g.,half-life, radiation hazard, counting efficiency and others) iodine-125 has become the radioisotope of choice for use in steroid radioimmunoassays.
The chemical structure of steroids is such that it is generally not possible to radioiodinate them directly.
It is necessary, therefore, to utilize as a precursor of the radiolabeled antigen a derivative of the steroid to be assayed which is readily iodinated. In choosing or developing such a derivative, the primary concern is the affinity of the derivative for the antibodies of the steroid to be assayed; the affinity of the deri-vative for the anti-bodies should, of course, be as close to the affinity of the steroid for the antibodies as possible.
Compounds having the formula RBSOa O
Il 11 St-O-C-CH2- ~ H-CH2-C-O~
~C~2)n S
_ ~ J
R
OH
are readily tagged with a radioisotope and can be used (when radiolabeled) as a tracer in radioimmunoassay procedures fox the determination of steroid levels in a body fluid. In formula I, and throughout the specifi-cation, R is hydrogen or an alkyl group of 1 to 3 carbon atoms; n is 0, 1, 2, 3, or 4 and St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containting deriva-tive of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmunoassay.
Exemplary of hydroxy steroids intended for radio-immunoassay which may be modified structurally as shown in formula I are: cholesterol, cortisol, cortisone, corticosterone, prednisolone, methylprednisolone, tri-amcinolone, betamethasone, dexamethasone, triamcinoloneacetonide, betamethasone valerate, halcinonide, al-dosterone, estrone, estradiol, estriol, testosterone, l9-nortestosterone, methyltestosterone, and pregnenono-lone. Exemplary of hydroxy containing derivatives of a steroid intended for radioimmunoassay (which deriva-tives have a strong affinity for the antibodies of the steroid intended for radioimmunoassay) are digoxigenin, .
lllS409 RB5Oa digitoxigenin and ll~-hydroxyprogesterone.
The steroids of formula I are prepared from the precursor hydroxy steroid having the formula St-OH
and a glutaric anhydride derivative having the formula II O
Rl 0 ~3 (C~2)n C
In formula II, and throughout the specification, Rl is an alkanoyl group having 2 to 6 carbon atoms, acetyl being the preferred group.
The anhydrides of Formula II are novel compounds, and as such constitute an integral part of this inven-tion. They can be prepared by first reacting a 4-methoxyphenylaliphatic aldehyde having the formula III
R
CH30 ~ (CH2)n CH
with at least 2 molar equivalents of cyanoacetic acid in the presence of a base (e.g., sodium hydroxide) to yield, on acid hydrolysis, a compound having the formula RB50a IV R C
C~l30 _ ~3 CH2- -OH
An alternative preparation for the compound of formula IV wherein n is O and R is hydrogen, i.e., 3-(4-methoxy-phenyl)glutaric acid, is disclosed by Smith et al., J.A.C.S., 72, 1877 (1950). In that procedure, anisalde-hyde is condensed with ethyl acetoacetate in the pre-sence of piperidine to give ethyl anisal-bis-acetoace-tate. Cleavage of this product to give the desired 3-(4-methoxyphenyl)glutaric acid can be accomplished with boiling alcoholic sodium hydroxide solution.
Demethylation of the glutaric acid derivatives of formula IV results in glutaric acid derivatives having the formula V O
R
HO ~ (CH2)n C~
and can be accomplished by following one of the several procedures known in the art for the demethylation of aryl methyl etherq. One such procedure, described by Feutrill et al., Aust. J. Chem., 25, 1719 (1972), in-volves the treatment of the aryl methyl ether with thioethoxide ion (readily prepared in situ from ethane-thiol and sodium hydride) in a polar aprotic solvent, RB5Oa preferably dimethylformamide.
'I'he phenolic hydroxy group of a compound of formula V can be protected with an alkanoyl group using art-recognized procedures. One such procedure comprises reacting the glutaric acid derivative with the appro-priate acid anhydride (acetic anhydride is preferred).
The preferred method of preparing a glutaric anhydride derivative of formula II from the glutaric acid deriva-tive of formula V is to combine the conversion of the acid to anhydride and the protection of the phenolic hydroxy group into a single step. When the Rl protecting group is acetyl, this would involve heating a glutaric acid derivative of formula V in acetic anhydride.
The reaction of a steroid precursor having the lS formula St-OH and a glutaric anhydride derivative of formula II to yield a steroid having the formula St-O-C-CH -CH-CH -C-OH
(CH2)n R
O-Rl can be run in the presence of an organic base. Exem-plary organic bases are nitrogen containing hetero-cyclics, e.g., pyridine, and tertiary amines, e.g., triethylamine. The reaction will preferably be run at an elevated temperature.
In those instances wherein the steroid precursor has more than one hydroxy substitutent, it will be possi-ble to monoacylate the steroid with a glutanic anhy-dride derivative of formula II because of the varying ~118409 RB5Oa reactivites of the steroid's hydroxyl substituents. If it is desirable to acylate a hydroxy substituent other than the most reactive one, conventional blocking techniques should be used to protect the more reactive substituents.
~r~al of the phenolic hydroxyl protecting group in a compound of formula VI yields the corresponding pro-duct of formula I.
The compounds of formula I can be labeled ("tagged") 10 with a radioisotope, preferably iodine-125 or iodine-131, and most preferably iodine-125, using procedures well known in the artl to yield a radiolabeled hapten having the formula O O
VII St O C CH2 CH C 2 C
(CH2)n ~, R /
OH
The asterisk (*) in formula VII indicates tagging with a radioisotope. Exemplary of the methods known in the art is the method of Hunter and Greenwood; see Nature, 194:495 (1962). The radiolabeled compounds of formula VII form an integral part of this invention.
The radiolabeled compounds of formula VII can be used as tracers in radioimmunoassay procedures following the general principles set forth hereinabove. Exemplary detailed procedures are described in Jaffe et al., "Methods of Hormone Radioimmunoassay", Academic Press, New York (1974) and Berson et al., "Methods in Investigative and Diagnostic Endocrinology", Vol. 3 on "Steroid Hormones", North Holland, Amsterdam (1975).
RB50a The radiolabeled compounds of this invention are parti-cularly useful as reagents in the automated radioimmu-noassay system of Brooker et al. disclosed in United States patent 4,022,577 issued May 10, 1977.
The following examples are specific embodiments of this invention.
Example 1 (3~,5~,12~)-3-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]
-12,14-dihydroxycard-20(22)-enolide A) 3-(4-Methoxyphenyl)glutaric acid A mixture of ~-anisaldehyde (27.2g), ethyl aceto-acetate (52.1g) and piperidine (4 ml) in 95% ethanol (10 ml) is stirred at room temperature for 5.0 hours while a solid forms. The solid is isolated by filtration, washed with 25% ethanol and crystallized from 95% etha-nol to afford ethyl 2,2'-(4-methoxybenzal)-bls-aceto-acetate (31.4g), melting point 138-141C. The filtrate on dilution with an equal amount of water gives a solid which is crystallized from 95% ethanol to afford another crop of material (8.5g), melting point 137-142C.
A mixture of ethyl 2,2'-(4-methoxybenzal(-bis-acetoacetate (30g), ethanol (450 ml) and 50~ sodium hydroxide (450g) is refluxed vigorously for 1.0 hour.
Water (150 ml) is added and most of the ethanol is re-moved by distillation in vacuo. The concentrate is acidified with concentrated hydrochloric acid and is extracted with ethyl acetate. The ethyl acetate solution is wa~hed with brine, dried, evaporated, and the residue is crystallized from benzene-methanol to afford 3.3g of 3-(4-methoxyphenyl)glutaric acid, melting point 147-150C.
1~1350a B) 3-(4-Hydroxyphenyl)glutaric acid To a stirred suspension of 57% sodium hydride-paraffin (6.45g), in dry dimethylformamide (70 ml) is slowly added ethanethiol (11.89 ml) in dry dimethyl-- S formamide (20 ml). After stirring the resultant slurry for 15 minutes, a salution of 3-(4-methoxyphenyl)glu-taric acid (3.0g) in dry dimethylformamide (20 ml) is added. The slurry is heated in a bath at 165C for 5.0 hours and most of the solvent is removed by distillation ln vacuo. The residue is diluted with water, acidified with concentrated hydrochloric acid and extracted twice with ether (the extracts are discarded). The solution is saturated with sodium chloride and extracted with ethyl acetate. The ethyl acetate solution is washed once with brine, dried and the residue crystallized from chloro-form-hexane to afford 2.3g of 3-(4-hydroxyphenyl)-glutaric acid, melting point 168-170C.
C) 3-(4-Acetyloxyphenyl)glutaric anhydride A solution of 3-(4-hydroxyphenyl)glutaric acid (800 mg) in acetic anhydride (15 ml) is heated at 100C
for 2.5 hours and evaporated to dryness in vacuo. The residual solid is crystallized from chloroform-hexane to afford 600 mg of 3-(4-acetyloxyphenyl)glutaric an-hydride, melting point 140-143C.
D) 12~-(Acetyloxy)-3~,5~)-3-[4carboxy-3-[4-(acetyloxy)-phenyl]-l-oxobutoxy]-14-hydroxycard-20(22)-enolide A solution of digoxigenin-12-acetate (130 mg) and 3-(4-acetyloxyphenyl)glutaric anhydride (286 mg) in dry pyridine (4.0 ml) i8 heated under nitrogen in a bath at 120C for 12 hours. Water (0.5 ml) is added and after 5 minutes, the mixture is evaporated in vacuo. The residue is dissolved in chloroform, washed with 10% hydrochloric 11184(~9 RB50a acid and brine, dried and evaporated. This residue is subjected to preparative thin-layer chromatography (tlcl on a silica gel plate (2.0 X 200 X 200 mm) using chloro-formmethanol (9:1) for development to afford 110 mg of the title compound as an amorphous solid, melting point 118-135C.
E) (3~,5~,12~)-3-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-12,14-dlhydroxycard-2o(22)-enolide To a solution of 12~-(acetyloxy)-(3~,5~)-3-[4-carboxy-3-[4-(acetyloxy)phenyl]-1-oxobutoxy]-14-hydroxy-card-20 (22)-enolide (68 mg) in methanol (3.0 ml) is added a solution of potassium carbonate (83 mg) in water (1.0 ml) and the mixture is stirred at room temp rature for 2.5 hours. The solution is then acidified with 5%
hydrochloric acid and the methanol is evaporated ln vacuo.
The residue is diluted with water (10 ml) and extracted with ethyl acetate. The ethyl acetate solution is wash-ed with brine, dried over anhydrous magnesium sulfate, evaporated and the residue subjected to preparative tlc on silica gel plates (1 X 200 X 200 mm) using chloro-form-methanol (3:1) for development to afford 22 mg of the title compound as an amorphous solid, melting point 110-118C (melting completely to liquid at 144C).
Example 2 (3~,5~,12~)-3-[4-Carboxy-3-[(4-hydroxyphenyl)methyl]-1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide A) 3-1(4-Methoxyphenyl)methyl]glutaric acid To a solution of cyanoacetic acid (3.57g), in water ~118409 RB50a 20 ml is added a solution of sodium hydroxide (2.08g) in water (20ml). The solution is diluted with glyme (70 ml), 4-methoxyphenyl acetaldehyde (3.0g) is added and the mixture is left at room temperature for 6.0 hours. The glyme is evaporated in vacuo, and the resi-due is mixed with I0% hydrochloric acid (200 ml) and re-fluxed for 6.0 hours. After cooling, the mixture is extracted with ethyl acetate. The ethyl acetate ex-tract is washed once with brine, dried, evaporated and the residue subjected to chromatography on a column of silica gel using chloroform-ethyl acetate mixtures for elution to afford 2.2g of 3-[(4-methoxyphenyl) methyl]glutaric acid. Crystallization from a benzene-ethyl acetate-hexane mixture gives a specimen having a melting point 109-110C.
B) 3-[(4-HydroxYphenyl)methyl]glutaric acid To a stirred suspension of 57% sodium hydride-paraffin (1.4g) in dry dimethylformamide (30 ml) is added dropwise a solution of ethanethiol (4.0 ml~ in dry dimethylformamide (10 ml). After 15 minutes, a solution of 3-[(4-methoxyphenyl)methyl]glutaric acid (800 mg) in dry dimethylformamide (20 ml) is added.
The resulting slurry is heated in a bath at 165C for 20 hours and evaporated in vacuo. The residue is acidi-fied with 20~ hydrochloric acid, extracted with etherand the extracts are discarded. The aqueous solution is saturated with salt and extracted with ethyl acetate.
The extracts are combined, washed once with brine, dried, evaporated and the residue crystallized from a mixture of ethyl acetate-chloroform-hexane to give 600 mg of 3-[(4-hydroxyphenyl)methyl]glutaric acid, melting point 117-119C.
.
.
RBSOa C) 3-[[(4-Acetyloxy)phenyl]methyl]glutaric anhydride 3-[(4-Hydroxyphenyl)methyl]glutaric acid (500 mg) in acetic anhydride (15 ml) is heated in a bath at 120C
for 2 hours. The solution is then evaporated to dry-ness in vacuo and the residue triturate~ with chloro-form~exane to afford 490 mg of the title compound, melting point 88-89C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[4-carboxy-3-[[4-(acetyl-oxy)-phenyl]methyl]-l-oxobutoxy]-14-hydroxycard-20(22)-enolide A solution of digoxigenin-12-acetate (216 mg) and 3-[[(4-acetyloxy)phenyl]methyl]glutaric anhydride (500 mg) in dry pyridine (6.0 ml) is refluxed for 20 hours.
Most of the pyridin~ is removed by distillation in vacuo and the residue is diluted with water (20 ml) and aci-dified with concentrated hydrochloric acid. The mix-ture is extracted with ethyl acetate, the extract washed twice with small amounts of brine, dried, evaporated and the residue subjected to preparative thin-layer chromato-graphy (as in Example ID) to isolate 323 mg of 12B-(acetyloxy)-(3~,5~)-3-[4-carboxy-3-[[4-(acetyloxy) pheyny]methyl]-l-oxobutoxy]-14-hydroxycard-20(22)-enolide, melting point 70-7gC.
E) (3B,5B,12~)-[4-Carboxy-3-[(4-hydroxyphenyl)methyl]
1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide A mixture of 12B-(acetyloxy)-(3~,5B)-3-[4-carboxy-3-[[4-(acetyloxy)phenyl]methyl]-1-oxobutoxy]-14-hydroxy-card-20(22)-enolide (160 mg), methanol (20 ml), water (7.0 ml) and potassium carbonate (207 mg) is stirred at 15C for 2.5 hours. The mixture is acidified with concentrated hydrochloric acid and most of the methanol is evaporated ln vacuo at room temperature. The concen-trate is extracted with ethyl acetate, the extract washed ~184~g RB50a with small amounts of brine, dried (MgSO4anhydrous) evaporated and the residue subjected to a preparative thin-layer chromatography on silica gel plates (as in Example lE) to afford 55 mg of (3~,5~,12~)-[4-carboxy-3-[(4-hydroxyphenyl)methyl]-1-oxobutoxy]-12,14-dihydroxy-card-20(22)-enolide, melting point 155-165C.
Example 3 (3~,5~,12~)-3-[[3-tCarboxymethyl)-5-(4-hydroxyphenyl)-1-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide A) 3-[2-(4-Methoxyphenyl)ethyl]glutaric acid To a solution of cyanoacetic acid (850 mg) in water (5.0 ml) is added a solution of sodium hydroxide (440 mg) in water (10 ml). The solution is diluted with glyme (15 ml) and 3-(4-methoxyphenyl)propionaldehyde (820 mg) is added. The solution is kept at room tempera-ture for 10 hours and then evaporated to remove the glyme.
To the residue is added 10% hydrochloric acid (50 ml) and the mixtureis thereafter refluxed for 6 hours with stirring. It is then cooled and extracted with ethyl acetate. The extract is washed with brine, dried over anhydrous magnesium sulfate and evaporated. The resi-due is subjected to chromatography over silica gel (15 g)to isolate in chloroform-ethyl acetate (80/20) fraction, 600 mg of the title compound. Crystallization from ethyl acetate-benzene gives a specimen having a melting point of 98-100C.
B) 3-[2-(4-Hydroxyphenyl)ethyl]glutaric acid To a suspension of 57% sodium hydride-paraffin (2.2g) in dry dimethylformamide (70 ml) in an atmosphere 34()9 - RB50a of nitrogen is slowly added ethanethiol (4.0 ml). The mixture is stirred at room temperature until a clear solution results. To this solution is added a solution of 3-[2-(4-methoxyphenyl)ethyl]-glutaric acid (1.2 g) in dry dimethylformamide (10 ml) and the mixture is heated with stirring in a bath at 165C for 6.0 hours.
The mixture is then evaporated to dryness ln vacuo and the resulting residue is dissolved in water (50 ml), extracted with ether (two 50 ml portions) and the ether extract is discarded. The aqueous solution is acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The ethyl acetate solution is washed with brine, dried with anhydrous magnesium sulfate, and evapor-ated to a residue. Crystallization of this from chloro-form-hexane affords l.Og of the title compound, melting point 142-144C.
C) 3-[2-[4-Acetyloxy)phenyl]ethyl]glutaric anhydride A solution of 3-~2-(4-hydroxyphenyl)ethyl]glutaric acid (600 mg) in acetic anhydride (15 ml) is heated in a bath at 120C for 2.0 hours. It is evaporated in vacuo and the residue is crystallized from dichlorometh-ane-hexane to afford 495 mg of 3-[2-[4-(acetyloxy)phenyl]-ethyl]glutaric anhydride, melting point 97-99C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[[5-[4-(acetyloxy)phenyl]
-3-(carboxymethyl)-1-oxopentyl]oxycard-20(22)-enolide A solution of digoxigenin-12-acetate (200 mg) and 3-[2-[4-(acetyloxy)phenyl]ethyl]glutaric anhydride (470 mg) in dry pyridine (8.0 ml) is refluxed for 20 hours. The pyridine is evaporated ln vacuo, the residue diluted with water (25 ml), acidified with concentrated ,,~
34()g RBSOa hydrochloric acid and extracted with chloroform. The chloroform solution is washed with small amounts of brine, dried, evaporated and purified by preparative thin-layer chromatography (as in Example ID) to isolate 253 mg of 12~-(acetyloxy)-(3~,5~)-3-1[5-[4-(acetyloxy(phen-yl]-3-(carboxymethyl)-1-oxopentyl]-oxy]card-20(22)-enolide.
E) (3~,5~,12~)-3-[[3-(Carboxymethyl)-5-(4-hydroxyphen-yl)-l-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide.
A solution of 12~-(acetyloxy)-(3~,5~)-3-[[5-[4-(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxopentyl]oxy]
card-20(22)-enolide (230 mg) in a mixture of methanol (10 ml) and water (2.5 ml) containing potassium carbonate (210 mg) is stirred at 10 to 15C for 2.5 hours. It is then acidified with concentrated hydrochloric acid.
Isolation and purification of the product as described in Example lE yields 44 mg of (3~,5~,12~)-3-[[3-(carbo-xymethyl)-5-(4-hydroxy-phenyl)-1-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide, melting point 126-134C.
Example 4 (3~,5~,12~)-3-1[3-(carboxymethyl)-6-(4-hydroxyphenyl)-1-oxohexyl]oxy]-12,14-dihydroxycard-20(22)-enolide A) 3-[3-(4-Methoxyphenyl)propyl]glutaric acid 4-(4-Methoxyphenyl)butyraldehyde (5.34 g) is re-acted with cyanoacetic acid (5.15 g) and sodium hydroxide (2.74 g) in a mixture of glyme (50 ml) and water (60 ml).
The mixture is treated with hydrochloric acid and pro-cessed as described in Example 2A to afford the title co~xNnd (2.lg) which on crystallization from ethyl ace-4~)9 RB5Oa tate-chloroform-hexane had a melting poin~ of 71-73C.
B) 3-[3-(4-Hydroxyphenyl)propyl]glutaric acid 3-[3-(4-Methoxyphenyl)propyl]glutaric acid (2.0g) is demethylated by the procedure described in Example 5 lB to afford, after cyrstallization of the product from ethyl acetate-chloroform-hexane, 1.2 g of 3-[3-(4-hydro-xyphenyl)-propyl3glutaric acid, melting point 107-108C.
C) 3-[3-14-(Acetyloxy)phenyl]propyl]glutaric anhydride 3-[3-(4-Hydroxyphenyl)propyllglutaric acid (533 10 mg) is reacted with acetic anhydride as described in Example lC to afford 3-[3-~4-(acetyloxy)phenyl]propyl]
glutaric anhydride, melting point 55-57C.
D) 12~-(Acetyloxy)-(3~,5~)-3-[[6-[4-~acetyloxy)phenyl]
3-(carboxymethyl)-1-oxohexyl]oxy]card-20(22)-enolide A mixture of digoxigenin-12-acetate (216 mg) and 3-[13-(4-acetyloxy)phenyl]propyl]glutaric anhydride (610 mg) in dry pyridine (9.0 ml) is reacted for 18 hours and the product is isolated and purified as described in Example lD to afford 245 mg of 12B~acetyloxy)-(3B,5B)-3-20 [[6-[4-(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxohexyl]
- oxy]card-20(22)-~nolide, melting point 98-113C.
E) (3B,5B,12~)-3-[[3-~Carboxymethyl)-6-(4-hydroxy-phenyl)-l-oxohexyl]oxy]-12,14-dihydroxycard-20(22) -enolide A mixture of 12~-(acetyloxy) (3B,5~)-3-[[6-~4-(acetyloxy)phenyl]-3-(carboxymethyl)-1-oxohexyl]oxy]
card-20(22)-enolide (150 mg) and potassium carbonate (172 mg) is reacted in a mixture of methanol (5.0 ml) and water (2 ml). The mixture is processed and purified 30 as described in Example lE to afford 58 mg of (3B,5B, 12B)-3-[[3-(carboxymethyl)-6-(4-hydroxyphenyl)-1-oxo-hexyl]oxy]-12,14-dihydroxycard-20(22)-enolide, melting point 115-128C.
409 RB50a Example 5 (3~',5~,12p,)-3-[[3-(Carboxymethyl)-7-(4-hydroxyphenyl) -1-oxoheptyl]oxy-12,14-dihydroxycard-20~22)-enolide Following the procedure of Example 4, but sub-stituting 5-(4-methoxyphenyl)valeraldehyde for 4-(4-methoxyphenyl)-butyraldehyde yields the title compound.
Detailed Procedure for Radioiodination of Digoxigenin Derivatives Sodium radioiodide (I125) aqueous solution (10 ml;
approximately 6mCi) is added to a reaction vial contain-15 ing a methanolic solution of (3~,5~,12~)-3-~4-carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-12,14-dihydroxyca~d-20 (22)-enolide (10 ml 1 mg/ml). The vial is stoppered and a chloramine T solution (25 ml; 2 mg/ml in 0.5M
phosphate buffer, pH 7.5) is injected through the stoppër 20 The vial is mixed well by shaking and allowed to stand for five minutes. Sodium metabisulfite solution (20 ml;
3 mg/ml in 0.5M phosphate buffer, pH 7.5) is injected through the stopper to quench the reaction.
The reaction mixture is applied to a 40 ml Sephadex 25 G-10 column and eluted with tris acetate buffer (0.05M, pH 6.5). Forty-four drops per tube are collected with the aid of a fraction collector. Free iodide comes off around tube #38 and the iodinated product elutes off in two fractions, tubes 110 through 130 (fraction I) and tubes 30 131 through 150 (fraction II?. Fraction II is found to be superior and is used in radioimmunoassays.
* Trade Mark B~
~ 4~9 RB50a Example 6 21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-11~,18-epoxy-18-hydroxypregn-4-ene-3,20-dione ~) 21-L3-t4-Acetyloxyphenyl)-4-carboxy-1-oxobutoxy]
-11~ 18-e~oxv-18-hvdroxv~regn-4-ene-3,20-dione Aldosterone (100 mg) is refluxed in dry pyridine 10 (6.0 ml) with 3-(4-acetyloxyphenyl)glutaric anhydride (300 mg) for 2.0 hours. The solution is then cooled to room temperature, water is added and after standing for a few minutes evaporated in vacuo. The residue is dis-solved in ethyl acetate (20 ml), washed with 15% hydro-15 chloric acid (6.0 ml) and brine, dried and evaporated toafford 408 mg of a gum. A thin-layer chromatography examination of this material shows the presence of three steroidal products. It is then subjected to preparative thin-layer chromatography on two 2.0 mm silica gel plates 20 (with two developments of the plates with chloroform-methanol (9:1) to isolate 135 mg of the title compound, melting point 145-160C with consistent spectral data.
B) 21-~4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]~
18-epoxy-18-hydroxypregn-4-ene-3,20-dione A solution of 21-[3-(4-acetyloxyphenyl)-4-carboxy-l-oxobutoxy]~ ,18-epoxy-18-hydroxypregn-4-ene-3,20-dione (130 mg) in anhydrous methanol (15 ml) containing triethylamine (0.2 ml) is allowed to stand at room temp-erature for 24 hours. The methanol is then evaporated 30 in vacuo, the residue is diluted with water (5.0 ml), acidified with 10~ hydrochloric acid and extracted with ethyl acetate (three 5.0 ml portions). The ethyl acetate ~8409 RB50a solution is washed with brine, dried over anhydrous magnesium sulfate, evaporated and the residue is subject-ed to preparative thin-layer chromatography on silica gel plates (using chloroform-methanol, 9:1 for develop-5 ment) to isolate 83 mg of the title compound, meltingpoint 145-152Cr with consistent spectral data.
Detailed Procedure for Radioiodination of Aldosterone Derivative . . .
To a solution of 21-[4-Carboxy-3-(4-hydroxyphenyl)-30 1-oxobutoxy]-11~,18-epoxy-18-hydroxypregn-4-ene-3,20-dione (5.0,ug) in dioxane-0.5M borate buffer (1:9, 50 ul) at pH 8.5 are added successively 20 ~1 of aqueous solutions of sodium radioiodide (I125, 4mCi) and freshly prepared 111~3409 RB5Oa chloramine-T (80 ~g). After 90 seconds, the reaction is stopped by the addition of sodium bisulfite (80,ug) in water (20 ~1). The solution is applied on a 5 X 20 cm silica gel plate which is developed with chloroform-5 methanol (9:1). The band of radioiodinated product islocated using a scanner and is isolated by extraction with ethanol. The concentrated ethanol solution is stored at 15C.
Example 7 21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-11~,17-dihydroxypregn-4-ene-3,20-dione A) 21-[3-(4-Acetyloxyphenyl)-4-carboxy-1-oxobutoxy-11~,17-dihydroxypregn-4-ene-3,20-dione A solution of cortisol (346 mg) and 3-(4-acetyloxy-phenyl)glutaric anhydride (496 mg) in dry pyridine (5.0 ml) is refluxed for 40 minutes. The pyridine is evapor-ated in vacuo, the residue is diluted with ethylacetate 20 (30 ml), washed with 15% hydrochloric acid (10 ml) and brine, dried over anhydrous magnesium sulfate and evapor-ated to yield 840 mg of a powder. Examination of the NMR spectrum and TLC behavior of this material shows that it is a 1:1 mixture of the title compound and 3-25 (4-acetyloxyphenyl)glutaric acid.
B) 21-[4-Carboxy-3-(4-hydroxyphenyl)l-oxobutoxy]-11~, 17- dihyroxypregn-4-ene-3,20-dione The mixture obtained above (700 mg) is dissolved in anhydrous methanol (30 ml) containing triethyamine 30 (1.0 ml) and the solution is refluxed for 5.0 hours.
The solution is cooled, acidified with 10~ hydrochloric acid and most of the methanol is evaporated in vacuo.
The resulting slurry is extracted with ethyl acetate, RBSOa the ethyl acetate solution is washed with brine, dried, and evaporated. The residue is subjected to pre-parative thin-layer chromatography on two 2.0 mm silica gel plates u3ing chloroform-methanol (9:1) for develop-5 ment and the major band is isolated with chloroform-methanol (8:2) to afford 300 mg of the title compound, melting point 98-110C.
Description for Radioiodination of Cortisol Derivative 21-[4-Carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-llr~,17-dihydroxypregn-4-ene-3,20-dione is radiolabeled with I125 using the procedure described above for the 15 radioiodination of aldosterone derivatives.
Example 8 (3r"5r~,12f~)-3-[4-Carboxy-3-(4-hydroxy-3- methylphenyl) 20 -1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide A) 3-(4-Methoxy-3-methylphenyl)glutaric acid A mixture of 4-methoxy-3-methylbenzaldehyde (100 mmole), cyanoacetic acid (210 mmole) and potassium 25 hydroxide (250 mmole( in water SOO ml) is stirred at room temperature for 20 hours. The resulting solution is acidified with concentrated hydrochloric acid (100 ml) and the mixture is refluxed for 60 hours. After Gooling, it is saturated with sodium chloride and extracted with 30 ethyl acetate to afford the title compound.
.~.
RBSOa ;
B) 3-(4-Elydroxy-3-methylphenyl)glutaric acid Followin~ the procedure described in Example lB, but substituting 3-(4-methoxy-3-methylphenyl)glutaric acid for 3-(4-methoxyphenyl)glutaric acid, yields the 5 title compound.
C) 3-[4-(Acetyloxy)-3-methylphenyl)]glutaric anhydride Following the procedure described in Example lC, but substituting 3-(4-hydroxy-3-methylphenyl)glutaric acid for 3-(4-hydroxyphenyl)glutaric acid, yields the 10 title compound.
D) 12~-(Acetyloxy)-(3~,5~)-3-[4-carboxy-3-[4-(acetyl-oxy)-3-methylphenyl]-1-oxobutoxy]-14-hydroxycard -20(22)-enolide Following the procedure described in Example lD, but substituting 3-~4-(acetyloxy)-3-methylphenyl)]glu-taric anhydride for 3-(4-acetyloxyphenyl)glutaric anhy-dride, yields the title compound.
; E) (3~,5~,12~)-3-t4-Carboxy-3-](4-hydroxy-3-methyl-phenyl)-l-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide Following the procedure described in Example lE, but substituting 12~-(acetyloxy)-(3~,5~)-3-t4-carboxy -3-[4-(acetyloxy)-3-methylphenyl]-1-oxobutoxy]-14-hy-droxycard-20(22)-enolide for 12~-(acetyloxy)-(3~,5~)-3-5 [4-carboxy-3-[4-(acetyloxy)phenyl]-1-oxobutoxy]-14-hy-droxycard-20(22)-enolide, yields the title compound.
Examples 9-26 Following the procedure described in Example 6, but ~ubstituting the steroid listed in Column I for al-111~3409 RB50a do~terone and the anhydride reagent listed in ColumnII for 3-(4-acetyloxyphenyl)glutaric anhydride, yields the product listed in Column III.
lllB~9 RB50a ~5 ,_/
o ~o I I
o o~ ~ ~ o c o ~ o~ o ~ o x x~ ~ X ~ X ~x :~ X ~ X
o o o ~ I o ~1 o ~o X o c~ o ~
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a 1~ ~ R I ~ _I o s ~1~ R --I I ~ A 'a~1 ~ O --I R
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o ~ ,, ~ ~ ~ ~ o ~ ~ ~ ~ a) a) ~
u ~ ~ u ~ u al u ~ s ~ ~ u Q~ s~a ~ u U~r~ U~rl C)~r~ I ~'~1 U'~1U ~'~1 U'~
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Claims (31)
1. A compound selected from the group consisting of a) a steroid compound having the formula wherein R is hydrogen or an alkyl group of 1 to 3 carbon atoms; n is 0, 1, 2, 3, or 4; and St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmuno-assay;
b) a steroid compound having the formula wherein R is hydrogen or alkyl having 1 to 3 carbon atoms;
n is 0, 1, 2, 3 or 4; the asterisk (*) indicates labeling with a radioisotope; and St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmunoassay;
c) compound having the formula wherein R is hydrogen or an alkyl group having 1 to 3 carbon atoms; R1 is alkanoyl having 2 to 6 carbon atoms and n is 0, 1, 2, 3 or 4;
d) a compound having the formula wherein R is hydrogen or an alkyl group having 1 to 3 carbon atoms and n is 1, 2, 3 or 4; and e) a compound having the formula wherein R is hydrogen or alkyl having 1 to 3 carbon atoms and n is 1, 2, 3 or 4.
b) a steroid compound having the formula wherein R is hydrogen or alkyl having 1 to 3 carbon atoms;
n is 0, 1, 2, 3 or 4; the asterisk (*) indicates labeling with a radioisotope; and St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmunoassay;
c) compound having the formula wherein R is hydrogen or an alkyl group having 1 to 3 carbon atoms; R1 is alkanoyl having 2 to 6 carbon atoms and n is 0, 1, 2, 3 or 4;
d) a compound having the formula wherein R is hydrogen or an alkyl group having 1 to 3 carbon atoms and n is 1, 2, 3 or 4; and e) a compound having the formula wherein R is hydrogen or alkyl having 1 to 3 carbon atoms and n is 1, 2, 3 or 4.
2. A steroid compound having the formula wherein R is hydrogen or an alkyl group of 1 to 3 carbon atoms;
n is 0, 1, 2, 3, or 4; and St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmuno-assay.
n is 0, 1, 2, 3, or 4; and St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmuno-assay.
3. A steroid compound in accordance with claim 2 where-in n is 0.
4. A steroid compound in accordance with claim 2 where-in n is 1.
5. A steroid compound in accordance with claim 2 where-in n is 2.
6. A steroid compound in accordance with claim 2 where-in n is 3.
7. A steroid compound in accordance with claim 2 where-in n is 4.
8. A steroid compound in accordance with claim 2 where-in R is hydrogen.
9. A steroid compound in accordance with claim 2 where-in R is an alkyl group of 1 to 3 carbon atoms.
10. A steroid compound in accordance with claim 2 where-in St is a des-hydroxy derivative of a steroid selected from the group consisting of cholesterol, cortisol, cortisone, corticosterone, prednisolone, methylprednisolone, triamcino-lone, betamethasone, dexamethasone, triamcinolone acetonide, betamethasone valerate, halcinonide, aldosterone, estrone, estradiol, estriol, testosterone, 19-nortesterone, methyl-testosterone, pregnenolone, digoxigenin, digitoxigenin and 11.alpha.-hydroxyprogesterone.
11. The steroid compound in accordance with claim 2, which is (3.beta.,5.beta.,12.beta.)-3-[4-carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide.
12. The steroid compound in accordance with claim 2, which is (3.beta.,5.beta.,12.beta.)-3-[4-carboxy-3-[(4-hydroxyphenyl)methyl]-1-oxobutoxy]-12,14-dihydroxycard-20(22)-enolide.
13. The steroid compound in accordance with claim 2, which is (3.beta.,5.beta.,12.beta.)-3-[[3-(carboxymethyl)-5-(4-hydroxy-phenyl)-1-oxopentyl]oxy]-12,14-dihydroxycard-20(22)-enolide.
14. The steroid compound in accordance with claim 2, which is (3.beta.,5.beta.,12.beta.)-3[[3-(carboxymethyl)-6-(4-hydroxy-phenyl)-1-oxohexyl]oxy]-12,14-dihydroxycard-20(22)-enolide.
15. The steroid compound in accordance with claim 2, which is (3.beta.,5.beta.,12.beta.)-3-[[3-(carboxymethyl)-7-(4-hydroxy-phenyl)-1-oxoheptyl]-12,14-dihydroxycard-20(22)-enolide.
16. The steroid compound in accordance with claim 2, which is 21-[4-carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-11.beta.,18-epoxy-18-hydroxypregn-4-ene-3,20-dione.
17. The steroid compound in accordance with claim 2, which is 21-[4-carboxy-3-(4-hydroxyphenyl)-1-oxobutoxy]-11.beta.,17-dihydroxypregn-4-ene-3,20-dione.
18. A compound having the formula wherein R is hydrogen or an alkyl group having 1 to 3 carbon atoms; R1 is alkanoyl having 2 to 6 carbon atoms and n is 0, 1, 2, 3 or 4.
19. A compound in accordance with claim 18 wherein R1 is acetyl and R is hydrogen.
20. A compound in accordance with claim 18 wherein n is 0.
21. A compound in accordance with claim 18 wherein n is 1.
22. A compound in accordance with claim 18 wherein n is 2.
23. A compound in accordance with claim 18 wherein n is 3.
24. A compound in accordance with claim 18 wherein n is 4.
25. A compound having the formula wherein R is hydrogen or an alkyl group having 1 to 3 carbon atoms and n is 1, 2, 3 or 4.
26. A compound having the formula wherein R is hydrogen or alkyl having 1 to 3 carbon atoms and n is 1, 2, 3 or 4.
27. A steroid compound having the formula wherein R is hydrogen or alkyl having 1 to 3 carbon atoms; n is 0, 1, 2, 3 or 4; the asterisk (*) indicates labeling with a radioisotope; and St is a des-hydroxy steroid moiety of (i) a hydroxy steroid intended for radioimmunoassay or (ii) a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmuno-assay.
28. A steroid compound in accordance with claim 27 wherein the radioisotope label is iodine-125 or iodine-131.
29. A steroid compound in accordance with claim 27 wherein the radioisotope label is iodine-125.
30. A process for preparing a steroid compound which can be readily labeled with a radioisotope and used as a tracer is a radioimmunoassay which comprises:
(a) reacting a hydroxy steroid intended for radio-immunoassay or a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmunoassay, with a compound having the formula wherein R is hydrogen or an alkyl group having 1 to 3 carbon atoms; R1 is an alkanoyl group having 2 to 6 carbon atoms and n is 0, 1, 2, 3, or 4; and (b) saponifying the resultant steroid.
(a) reacting a hydroxy steroid intended for radio-immunoassay or a hydroxy containing derivative of a steroid intended for radioimmunoassay, said derivative having a strong affinity for the antibodies of the steroid intended for radioimmunoassay, with a compound having the formula wherein R is hydrogen or an alkyl group having 1 to 3 carbon atoms; R1 is an alkanoyl group having 2 to 6 carbon atoms and n is 0, 1, 2, 3, or 4; and (b) saponifying the resultant steroid.
31. A process in accordance with claim 30 wherein n is 0.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US82401677A | 1977-08-12 | 1977-08-12 | |
US824,016 | 1977-08-12 | ||
US901,952 | 1978-05-01 | ||
US05/901,952 US4230621A (en) | 1978-05-01 | 1978-05-01 | Steroid derivatives and their use in radioimmunoassays |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1118409A true CA1118409A (en) | 1982-02-16 |
Family
ID=27124779
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000308300A Expired CA1118409A (en) | 1977-08-12 | 1978-07-27 | Steroid derivatives and their use in radioimmunoassays |
Country Status (7)
Country | Link |
---|---|
JP (1) | JPS5436247A (en) |
CA (1) | CA1118409A (en) |
DE (1) | DE2834516A1 (en) |
FR (1) | FR2400035A1 (en) |
GB (2) | GB2002385B (en) |
IT (1) | IT7850710A0 (en) |
NL (1) | NL7808393A (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4323511A (en) * | 1978-05-22 | 1982-04-06 | E. R. Squibb & Sons, Inc. | Steroid derivatives and their use in radioimmunoassays |
US4209614A (en) * | 1978-05-30 | 1980-06-24 | E. R. Squibb & Sons, Inc. | Vitamin B12 derivative suitable for radiolabeling |
US4243608A (en) * | 1979-05-14 | 1981-01-06 | E. R. Squibb & Sons, Inc. | 3-(4-Hydroxyphenyl)pentanedioic acid, monohydrazide, derivatives and analogs |
US4366143A (en) * | 1979-09-24 | 1982-12-28 | Amersham International Public Limited Company | Assay for the free portion of substances in biological fluids |
JPS58194900A (en) * | 1982-05-10 | 1983-11-12 | Shionogi & Co Ltd | 4- or 6-substituted aldosterones, its preparation and method for using the same in immunoassay |
JPS59228097A (en) * | 1983-06-07 | 1984-12-21 | 横河電機株式会社 | Paper thickness profile control apparatus |
JPS61108794A (en) * | 1984-08-08 | 1986-05-27 | インパクト システムズ インコ−ポレ−テツド | Thickness control apparatus and method |
JP2015193545A (en) * | 2014-03-31 | 2015-11-05 | 国立大学法人京都大学 | 2-(3-pyridinyl)-1h-benzimidazole derivative compound, and radioactive pharmaceuticals containing the same |
-
1978
- 1978-07-27 CA CA000308300A patent/CA1118409A/en not_active Expired
- 1978-08-07 DE DE19782834516 patent/DE2834516A1/en not_active Withdrawn
- 1978-08-11 FR FR7823770A patent/FR2400035A1/en active Granted
- 1978-08-11 IT IT7850710A patent/IT7850710A0/en unknown
- 1978-08-11 NL NL787808393A patent/NL7808393A/en not_active Application Discontinuation
- 1978-08-12 JP JP9861778A patent/JPS5436247A/en active Pending
- 1978-08-14 GB GB7833217A patent/GB2002385B/en not_active Expired
- 1978-08-14 GB GB7921396A patent/GB2027425B/en not_active Expired
Also Published As
Publication number | Publication date |
---|---|
GB2027425B (en) | 1982-03-03 |
FR2400035B1 (en) | 1981-02-13 |
GB2002385B (en) | 1982-02-24 |
DE2834516A1 (en) | 1979-02-22 |
GB2027425A (en) | 1980-02-20 |
GB2002385A (en) | 1979-02-21 |
IT7850710A0 (en) | 1978-08-11 |
FR2400035A1 (en) | 1979-03-09 |
JPS5436247A (en) | 1979-03-16 |
NL7808393A (en) | 1979-02-14 |
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