Radiopharmaceutical products for diagnosis and therapy of adrenal carcinoma

Abstract

A radiopharmaceutical composition is disclosed comprising novel iodometomidate derivatives of formula (I) which bind specifically to adrenal enzymes and which exhibit an improved stability. The compounds of formula (I) are suitable for use in a diagnostic imaging method, e.g. for diagnosis of adrenocortical carcinoma. The compounds of formula (I) are further suitable for use in the treatment of adrenocortical carcinoma, by means of radionuclide therapy.

Claims

1. A method for the diagnosis of a Cyp11B enzyme expressing tumor comprising administering an effective amount of a radiopharmaceutical composition comprising a compound of Formula (I) to a human in need of said diagnosis, wherein Formula (I) has the structure: ##STR00048## wherein A is selected from the group consisting of .sup.123I, .sup.124I, .sup.125I and .sup.131I; R represents ##STR00049## wherein R.sub.1 represents ##STR00050## and wherein R.sub.2 and R.sub.3 together with the N-atom, to which they are connected, form a 4-membered ring, optionally substituted with one or more substituents independently selected from the group consisting of OH, F, CI, I, Br and a linear or branched C.sub.1-C.sub.4 alkyl, optionally substituted with one or more substituents independently selected from the group consisting of OH, F, CI, I and Br; or a pharmaceutically acceptable salt thereof, or solvate thereof.

2. A method for the diagnostic imaging of a disease or disorder comprising administering an effective amount of a radiopharmaceutical composition comprising a compound of Formula (I) to a human in need of diagnostic imaging, and obtaining an image of the human using single photon emission computed tomography, wherein Formula (I) has the structure: ##STR00051## wherein A is selected from the group consisting of .sup.123I, .sup.124I, .sup.125I and .sup.131I; R represents ##STR00052## wherein R.sub.1 represents ##STR00053## and wherein R.sub.2 and R.sub.3 together with the N-atom, to which they are connected, form a 4-membered ring, optionally substituted with one or more substituents independently selected from the group consisting of OH, F, CI, I, Br and a linear or branched C.sub.1-C.sub.4 alkyl, optionally substituted with one or more substituents independently selected from the group consisting of OH, F, CI, I and Br; or a pharmaceutically acceptable salt thereof, or solvate thereof.

3. A method for treating a Cyp11B enzyme expressing tumor comprising administering an effective amount of a radiopharmaceutical composition comprising a compound of Formula (I) to a patient with the Cyp11B enzyme expressing tumor, wherein Formula (I) has the structure: ##STR00054## wherein A is selected from the group consisting of .sup.123I, .sup.124I, .sup.125I and .sup.131I; R represents ##STR00055## wherein R.sub.1 represents ##STR00056## and wherein R.sub.2 and R.sub.3 together with the N-atom, to which they are connected, form a 4-membered ring, optionally substituted with one or more substituents independently selected from the group consisting of OH, F, CI, I, Br and a linear or branched C.sub.1-C.sub.4 alkyl, optionally substituted with one or more substituents independently selected from the group consisting of OH, F, CI, I and Br; and or a pharmaceutically acceptable salt thereof, or solvate thereof.

4. The method of claim 2, wherein the disease or disorder is selected from the group consisting of adrenocortical carcinoma, adrenal adenoma, Conn adenoma and adrenal hyperplasia.

5. The method of claim 1, wherein the method for the diagnosis of a Cyp11B enzyme expressing tumor is by differential diagnosis.

6. The method of claim 1, wherein the Cyp11B enzyme expressing tumor is selected from an adrenal mass.

7. The method of claim 6, wherein the adrenal mass is an adrenocortical carcinoma.

8. The method of claim 1, wherein R.sub.1 is selected from the group consisting of ##STR00057##

9. The method of claim 1, wherein A is .sup.131I.

10. The method of claim 3, wherein the Cyp11B enzyme expressing tumor is an adrenocortical carcinoma.

11. The method of claim 3, wherein R.sub.1 is selected from the group consisting of ##STR00058##

12. The method of claim 3, wherein R.sub.1 represents ##STR00059##

13. The method of claim 3, wherein A is .sup.131I.

14. The method of claim 1, wherein R.sub.1 represents ##STR00060##

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the kinetics of IMTO metabolization.

(2) FIG. 2 shows the uptake of IMTO and (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid ethyl-methyl amide (compound 2a) in adrenal glands in mice in ex vivo experiments.

(3) FIG. 3 shows the uptake of (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid azetinylamide (compound 2e) in adrenal glands in mice in ex vivo experiments.

(4) FIG. 4 shows the in vitro uptake of IMTO in cell experiments.

(5) FIG. 5 shows the in vitro uptake of (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid azetinylamide (compound 2e) in cell experiments.

(6) FIG. 6 shows the biodistribution of IMTO in mice.

(7) FIG. 7 shows the biodistribution of (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid ethyl-methyl amide (compound 2a) in mice.

(8) FIG. 8 shows the biodistribution of (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid azetinylamide (compound 2e) in mice.

(9) FIG. 9 shows the biodistribution of IMTO, (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid azetinylamide (compound 2e), (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid pyrrolidinylamid and (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid ethyl-methyl amide (compound 2a) in adrenals of mice.

(10) The present invention is further illustrated by the following examples.

EXAMPLES

Example 1

1. Synthesis of the Non-Radioactive Standard 2a Via 8 Steps

Step 1: R-(+)-1-(4-Iodophenyl)-ethylamine

(11) ##STR00021##

(12) A solution of 58.6 mL (55.5 g, 458 mmol) R-(+)-1-phenylethylamine in 80 mL 1,2-dichloroethane was cooled in an ice bath, a solution of 80 mL (119 g, 570 mmol) trifluoroacetic acid anhydride in 80 mL 1,2-dichloroethane was added dropwise, and stirring was performed for 90 min at rt. After cooling in an ice bath, 55.5 g (218 mmol) iodine and 100 g (222 mmol) Bis(trifluoracetoxy)iodobenzene were added. The solution was stirred overnight and quenched by 1050 mL of a 10% sodium thiosulfate-solution. After extraction with 1000 mL methylene chloride the organic phase was washed with a saturated solution of sodium bicarbonate and dried over sodium sulfate. The solvent was stripped under reduced pressure and the crude product redissolved in 410 mL of warm diethyl ether and 113 mL hexanes. After cooling in a refrigerator, the product precipitated.

(13) Melting point: 162-4° C.

(14) The intermediate was dissolved in 2100 mL of methanol and 1200 mL 1 N sodium hydroxide and stirred overnight. The solution was concentrated to half of the volume under vacuum and the remaining aqueous solution was extracted with methylene chloride (4×250 mL). After drying over sodium sulfate the solvent was stripped and the product was obtained as a white solid.

(15) Product Characteristics:

(16) Yield 46.0 g (186 mmol, 40.6% over two steps)

(17) Melting point: 102-8° C.

(18) .sup.1H-NMR (CDCl.sub.3): δ=7.61 (d, 2H), 7.03 (d, 2H), 4.01 (q, 1H), 1.87 (bs, 2H), 1.27 (d, 3H)

Step 2: Synthesis of N-(α-Methyl-4-iodobenzyl)-glycine methylester

(19) ##STR00022##

(20) To a solution of 30 g (121 mmol) R-(+)-1-(4-iodophenyl)-ethylamine in 250 mL acetonitrile 25.0 g (201 mmol) dry potassium carbonate and 10.8 mL (13.2 g, 121 mmol) chloroacetic acid methylester were added. The solution was refluxed for 16 h under an atmosphere of argon. After cooling the solution was filtrated and the solvent stripped under vacuum. The crude product was purified by column chromatography (heptane/ethyl acetate/triethylamine 70/30/0.1).

(21) Product Characteristics:

(22) Yellow oil

(23) Yield 27.4 g (85.9 mmol, 71.0%)

(24) Thin-Layer Chromatography (Silica Gel):

(25) R.sub.f R-(+)-1-(4-Iodophenyl)-ethylamine (heptane/ethyl-acetate 80/20)=0.0

(26) R.sub.f N-(α-Methyl-4-iodobenzyl)-glycine methylester (heptane/ethyl acetate 80/20)=0.35

(27) .sup.1H-NMR (CDCl.sub.3): δ=7.62 (d, 2H), 7.12 (d, 2H), 4.20 (q, 2H), 3.81 (q, 1H), 3.38 (q, 2H), 1.94 (s, 1H), 1.43 (d, 3H), 1.29 (t, 3H)

Step 3: Synthesis of N-(α-Methyl-4-iodobenzyl)-N-formylglycine methylester

(28) ##STR00023##

(29) A solution of 30.0 g (94.0 mmol) N-(α-Methyl-4-iodobenzyl)-N-formylglycine methylester and 6.49 mL (7.87 g, 171 mmol) formic acid in 82 mL toluene was refluxed for 3 h. Water was removed by a dean-stark trap; the resulting solution was directly used in the next step.

Step 4: Synthesis of N-(α-Methyl-4-iodobenzyl)-N-formyl-C-formyl-glycine methylester

(30) ##STR00024##

(31) 11.9 mL (11.5 g, 188 mmol) formic acid methylester and 7.66 g (141 mmol) sodium methoxide were added under cooling and stirring was performed for 16 h at rt. Finally the product was extracted as an enolate two times each with 78 mL water and directly used in the next step.

Step 5: Synthesis of N-(α-Methyl-4-iodobenzyl)-2-mercaptoimidazole-5-carboxylic acid methylester

(32) ##STR00025##

(33) The aqueous phase obtained above was diluted with 40 mL methanol and 48 mL conc. HCl. A solution of 18.2 g (188 mmol) potassium thiocyanate in 21 mL water was added and stirring performed for 2 h at 60° C. After cooling to rt the product was extracted two times each with 82 ml tetrachloromethane and directly used in the next step.

Step 6: Synthesis of 4-Iodometomidate

(34) ##STR00026##

(35) A flask containing 289 mL water, 289 mg sodium nitrite and 7.3 mL conc. nitric acid was heated to 35° C. A solution of N-(α-Methyl-4-iodobenzyl)-2-mercaptoimidazole-5-carboxylic acid methylester in 164 mL tetrachloromethane was added dropwise while the temperature was maintained below 40° C. After complete addition, the solution was stirred for 2 h at 40-50° C., cooled to rt and the phases were separated. The aqueous phase was neutralized by careful addition of sodium carbonate and the product extracted with methylene chloride. After stripping the solvents of the combined organic phases the crude product was separated by column chromatography (CH.sub.2Cl.sub.2/MeOH/DEA 97/3/0.1).

(36) Product Characteristics:

(37) Appearance: White solid

(38) Melting point: 72-4° C.

(39) Yield: 13.7 g (38.5 mmol, 40.9%)

(40) Thin-Layer Chromatography (Silica Gel):

(41) R.sub.f 4-Iodometomidate (heptane/ethyl acetate) (80/20)=0.10

(42) .sup.1H-NMR (CDCl.sub.3): δ=9.90 (s, 1H), 8.19 (s, 1H) 7.72 (d, 2H), 7.20 (d, 2H), 6.55 (q, 1H), 3.93 (s, 3H), 2.10 (d, 3H)

Step 7: Synthesis of (R)-1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid

(43) ##STR00027##

(44) A solution of 4 g (11 mmol) Iodometomidate in 75 mL 10% NaOH was refluxed for 5 h. After cooling to rt a pH-value of 2-3 was adjusted by addition of conc. HCl. The precipitating product was filtered off and washed with water. The crude product was suspended in 100 ml toluene and dried by azeotropic distillation using a dean-stark trap. After cooling the solvent was stripped and the product was dried in a desiccator.

(45) Product Characteristics:

(46) Appearance: White solid

(47) Melting point: >250° C. (dec.)

(48) Yield: 3.32 g (9.70 mmol, 88.2%)

(49) Thin-Layer Chromatography (Silica Gel):

(50) R.sub.f (R)-1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid (heptane/ethyl acetate) (80/20)=0.00

(51) .sup.1H-NMR (d.sub.6-DMSO): δ=8.37 (s, 1H), 7.72 (m, 3H), 6.95 (d, 2H), 6.30 (q, 1H), 1.80 (d, 3H)

Step 8: Synthesis of (R)-1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid ethylmethylamide (2a)

(52) ##STR00028##

(53) To a solution of 761 mg (3.0 mmol) iodine in 40 mL methylene chloride 787 mg (3.0 mmol) triphenylphosphine was added, giving the solution a brown-yellow color. Then, 449 mg (6.6 mmol) imidazole was added, changing the color to light yellow. Subsequently, 684 mg (2.0 mmol) (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid was added and the solution was stirred for 5 min at room temperature, and 1.09 mL (740 mg, 12.5 mmol) ethylmethylamine was added and the solution stirred for two days. The solution was diluted with 50 mL chloroform, washed once with 50 mL of a saturated sodium thiosulfate-solution and three times with each 50 mL water and dried over sodium sulfate. After stripping the solvent, the crude product was purified by column chromatography (CH.sub.2Cl.sub.2/CH.sub.3OH 95/5/).

(54) Product Characteristics:

(55) Appearance: Yellow oil

(56) Yield: 293 mg (0.76 mmol, 38.2%)

(57) Thin-Layer Chromatography (Silica Gel):

(58) R.sub.f (R)-1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid ethylmethylamide (CH.sub.2Cl.sub.2/CH.sub.3OH 95/5)=0.20

(59) .sup.1H-NMR (CDCl.sub.3): δ=7.75 (s, 1H), 7.65 (d, 2H), 7.20 (s, 1H), 6.80 (d, 2H), 5.85 (q, 1H), 2.85 (s, 3H), 1.80 (d, 2H), 0.95 (t, 3H)

2. Synthesis of the Labelling Precursor: Synthesis of (R)-1-[1-(4-Trimethylstannyl-phenyl)ethyl]-1H-imidazole-5-carboxylic acid ethylmethylamide

(60) ##STR00029##

(61) To a solution of 293 mg (0.76 mmol) (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid ethylmethylamide in 12 mL toluene 728 mg (2.26 mmol) hexamethylditin, 67 mg (0.055 mmol) Tetrakis-(triphenylphosphine)-palladium (0) and 1.80 mL (1.30 g, 13.0 mmol) triethylamine were added. The solution was refluxed overnight, the solvent stripped and the crude product purified by column chromatography (100% methanol).

(62) Product Characteristics:

(63) Appearance: Brown-yellow oil

(64) Yield: 206 mg (0.49 mmol, 64.5%)

(65) Thin-Layer Chromatography (Silica Gel):

(66) R.sub.f (R)-1-[1-(4-Trimethylstannylphenyl)ethyl]-1H-imidazole-5-carboxylic acid ethylmethylamide (heptane/EtOAc 50/50)=0.05

(67) .sup.1H-NMR (CDCl.sub.3): δ=8.05 (s, 1H), 7.65 (d, 2H), 7.20 (s, 1H), 6.80 (d, 2H), 5.85 (q, 1H), 2.85 (s, 3H), 1.80 (d, 2H), 0.95 (t, 3H), 0.25 (s, 9H)

3. Radiosynthesis of [131I](R)-1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid ethylmethylamide ([131I]2a)

(68) ##STR00030##

(69) The following stock solutions were prepared: An Eppendorf-vial containing 40 μg (R)-1-(1-(4-trimethylstannyl-phenylethyl))-1H-imidazole-5-carboxylic acid ethylmethylamide was taken from the refrigerator (−20° C.) and 30 μL ethanol were pipetted into the vial. Oxidation agent: 15 mg Chloramin-T Trihydrate were dissolved in 10 mL water. HPLC-eluent: methanol, water and ammonia (25%) were mixed 70/30/0.1 (v/v/v) and degassed in the ultrasonic bath. 1 N HCl 1 N NaOH

(70) To the Eppendorf-vial containing the ethanolic solution of (R)-1-(1-(4-trimethylstannyl-phenylethyl))-1H-imidazole-5-carboxylic acid ethylmethylamide a solution of sodium [.sup.131I]iodide was pipetted. The vial was closed and the activity determined by a curiemeter. 6 μL 1 M HCl and 10 μL of the chloramin-T-solution were added and the reaction was allowed to proceed for 3 min at rt. The reaction was quenched by the addition of 7 μL 1 M NaOH. 100 μL of the HPLC-eluent were added and the solution was transferred to the HPLC-system:

(71) Column: Nucleosil 100-7 250×4.6 mm

(72) Eluent: Methanol/water/ammonia (25%) 70/30/0.1 (v/v/v)

(73) Flow: 1.0 ml/min

(74) Detection: UV (254 nm and 230 nm) and radioactivity (NaI(Tl) scintillation detector).

(75) The HPLC-product fraction with a retention time of 7-8 min was collected in a flask and the solvent stripped under reduced pressure. The flask was transferred into a laminar airflow cabinet, the dry tracer dissolved in 3-4 mL PBS/20% ethanol and drawn into a sterile syringe. The solution was passed through a sterile filter (0.22 μm) into a sterile vial.

(76) The tracer was obtained in a radiochemical yield of 90% and a radiochemical purity>98%.

Example 2

Synthesis of (R)-1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide (2e)

(77) ##STR00031##

(78) A solution of 534 μL (558 mg, 4.5 mmol) trimethylphosphite in 24 mL methylene chloride was cooled in an ice bath and was charged with 1.14 g (4.5 mmol) iodine. After complete dissolution of the iodine, 1.55 g (4.5 mmol) (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid and 1.04 mL (759 mg, 7.5 mmol) triethylamine were added. 10 min later, 507 μL (429 mg, 7.5 mmol) azetidine was added and the solution stirred for 3 h at room temperature. The solution was diluted with 100 mL chloroform, washed once with 100 mL of a saturated sodium thiosulfate-solution and three times with each 50 mL water and dried over sodium sulfate. After stripping the solvent, the crude product was purified by column chromatography (CH.sub.2Cl.sub.2/CH.sub.3OH 95/5).

(79) Product Characteristics:

(80) Appearance: Yellow waxy solid

(81) Yield: 770 mg (2.02 mmol, 44.9%)

(82) Thin-Layer Chromatography (Silica Gel):

(83) R.sub.f (R)-1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide (CH.sub.2Cl.sub.2/CH.sub.3OH 95/5)=0.30

(84) .sup.1H-NMR (CDCl.sub.3): δ=7.73 (s, 1H), 7.63 (d, 2H), 7.31 (s, 1H), 6.90 (d, 2H), 6.43 (q, 1H), 4.00-4.40 (m, 4H), 2.20-2.40 (m, 2H), 1.82 (d, 3H)

Step 2b: Synthesis of the Labelling Precursor

Synthesis of (R)-1-[1-(4-Trimethylstannylphenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamid

(85) ##STR00032##

(86) To a solution of 191 mg (0.50 mmol) (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide in 8 mL toluene 479 mg (1.49 mmol) hexamethylditin, 44 mg (0.036 mmol) Tetrakis-(triphenylphosphine)-palladium (0) and 1.18 mL (855 mg, 8.55 mmol) triethylamine were added. The solution was refluxed overnight, the solvent stripped and the crude product purified by column chromatography (100% ethyl acetate).

(87) Product Characteristics:

(88) Appearance: yellow waxy solid

(89) Yield: 125 mg (0.30 mmol, 59.8%)

(90) Thin-Layer Chromatography (Silica Gel):

(91) R.sub.f (R)-1-[1-(4-Trimethylstannylphenyl)ethyl]-1H-imidazole-5-carboxylic acid ethylmethylamide (CH.sub.2Cl.sub.2/CH.sub.3OH 95/5)=0.25

(92) .sup.1H-NMR (CDCl.sub.3): δ=7.72 (s, 1H), 7.43 (d, 2H), 7.25 (s, 1H), 7.19 (d, 2H), 6.43 (q, 1H), 4.00-4.35 (m, 4H), 2.20-2.35 (m, 2H), 1.82 (d, 3H), 0.28 (s, 9H)

Step 3b: Radiosynthesis of [131I](R)-1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide ([131I]2e)

(93) ##STR00033##

(94) The following stock solutions were prepared: An Eppendorf-vial containing 40 μg (R)-1-(1-(4-trimethylstannyl-phenylethyl))-1H-imidazole-5-carboxylic acid azetidinylamide was taken from the refrigerator (−20° C.) and 30 μL ethanol were pipetted into the vial. Oxidation agent: 15 mg Chloramin-T Trihydrate were dissolved in 10 mL water. HPLC-eluent: methanol, water and ammonia (25%) were mixed 70/30/0.1 (v/v/v) and degassed in the ultrasonic bath. 1 N HCl 1 N NaOH

(95) To the Eppendorf-vial containing the ethanolic solution of (R)-1-(1-(4-trimethylstannyl-phenylethyl))-1H-imidazole-5-carboxylic acid ethylmethylamide a solution of sodium [.sup.131I]iodide was pipetted. The vial was closed and the activity determined by a curiemeter. 6 μL 1 M HCl and 10 μL of the chloramin-T-solution were added and the reaction was allowed to proceed for 3 min at rt. The reaction was quenched by the addition of 7 μL 1 M NaOH. 100 μL of the HPLC-eluent were added and the solution was transferred to the HPLC-system:

(96) Column: Nucleosil 100-7 250×4.6 mm

(97) Eluent: Methanol/water/ammonia (25%) 70/30/0.05 (v/v/v)

(98) Flow: 1.0 ml/min

(99) Detection: UV (254 nm and 230 nm) and radioactivity (NaI(Tl) scintillation detector).

(100) The HPLC-product fraction with a retention time of 8-9 min was collected in a flask and the solvent stripped under reduced pressure. The flask was transferred into a laminar airflow cabinet, the dry tracer dissolved in 3-4 mL PBS/20% ethanol and drawn into a sterile syringe. The solution was passed through a sterile filter (0.22 μm) into a sterile vial.

(101) The tracer was obtained in a radiochemical yield of 90% and a radiochemical purity>98%.

Example 3: Evaluation of Specificity for CYB11B1 and CYP11B2 Inhibition In Vitro

(102) Plasmid constructs and transfection: To induce expression of human cytochrome P450 family 11B1 (Cyp11B1) and Cyp11B2 enzymes in Y1 cells, the full-length cDNAs for the proteins were subcloned into the multicloning site of pcDNA3.1 (zeo) (Invitrogen, Eggenstein, Germany). The cDNA fragments were isolated by PCR and digested by EcoRI. The individual fragments were ligated into the linearized vectors digested by EcoRI. Human Cyp11B1 and Cyp11B2 enzymes were expressed in Y1 cells using liposome/lipid-mediated DNA transfection. Purified plasmid DNA was mixed with Lipofectamine (Invitrogen) transfection reagents according to the manufacturer's protocol. To generate a stable Y1-Cyp11B1 and Y1-Cyp11B2 cell line, Y1 cells were transfected with the pcDNA3.1 (zeo)-Cyp11B1 and pcDNA3.1 (zeo)-Cyp11B2 vector, respectively.

(103) Transfected cells were selected with 1000 μg/ml zeocin (Invitrogen). To screen colonies, Western blotting and real-time PCR were used to determine the level of Cyp11B1 and Cyp11B2 expression. Colonies with the highest Cyp11B expression were further tested for their ability to synthesize cortisol or aldosterone from deoxycortisol (RSS) and 11-deoxycorticosterone (DOC), respectively. Experimental protocols were standardized regarding substrate concentrations and incubation periods.

(104) In vitro evaluation of specificity: To evaluate Cyp11B1 and Cyp11B2 inhibition by the inventive compounds, Y1-Cyp11B1 and Y1-Cyp11B2 cells were subcultured on six-well plates (0.5×106 cells per well) in 2 ml culture medium.

(105) The enzyme reaction was started after 24 hours by the addition of 1 ml culture medium containing either 11-deoxycortisol (RSS) or 11-deoxycorticosterone (DOC) as substrate and the corresponding inhibitor. RSS and DOC were dissolved in ethanol to a final test concentration of 1 μM. For determination of IC.sub.50 values, the inhibitors were added to the culture medium at concentrations between 0.6 nM and 60 μM and incubated for 48 h. Y1-Cyp11B1 and Y1-Cyp11B2 cells, which were treated in the same way but without inhibitors, served as controls. As further controls, untransfected Y1 cells were also incubated with RSS and DOC, respectively. Both RSS and DOC were obtained from Sigma (Deisenhofen, Germany).

(106) The results obtained for selected compounds of the invention are presented in the following Tables 1 and 2.

(107) TABLE-US-00001 TABLE 1   compound R.sub.1                         IC.sub.50 11β-hydroxylase [nM]                       IC.sub.50 aldosterone synthase [nM] iodometomidate (IMTO) OCH.sub.3 2.6 ± 1.1 2.0 ± 2.3 2a 3.9 ± 0.6  4.2 ± 0.37 2b 36.6 10.9 ± 2.02 2e 3.16 ± 0.14 1.22 ± 0.08 2g 11.7 5.5 ± 2.2 2h 805 182 2i 0 >1000 15.7 2j —NH2 329 ± 310 1410 ± 2070 2k 336 ± 398 221 ± 168 2l 74.7 3.5 ± 7.5 2m 78.2  147 ± 61.9 2n 1322 390 ± 170

(108) TABLE-US-00002 TABLE 2 R                 IC.sub.50 11β- hydroxylase [nM]                 IC.sub.50 aldosterone synthase [nM] 4a 72.6 1750 6a 10.6 ± 9.2 90.1

(109) As can be seen from the data shown in the above table, the inventive compounds described herein bind selectively to 11β-hydroxylase and aldosterone synthase. Thus, corresponding radiopharmaceutical compositions are suitable for diagnostic and treatment purposes within the living body of a mammal having adrenal glands.

Example 4: Uptake of the Inventive Compounds in Adrenal Glands in Mice in Ex Vivo Experiments

(110) The uptake of IMTO, (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid ethyl-methyl amide (compound 2a) and (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide (compound 2e) in adrenal glands in mice in ex vivo experiments has been examined as follows. Male CD-1 mice were injected i.v. with 1 μCi (37 kBq) IMTO, (R)-1-[1-(4-[.sup.125I]iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid ethyl-methyl amide or (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide (compound 2e). At predefined time points (15 min, 30 min, 120 min, and 240 min) mice were killed (n≥6 per time point) and adrenals were excised and weighed. Radioactivity was measured using a gamma-counter.

(111) The results of this experiment are shown in FIGS. 2 and 3. It can be seen from the results that the uptake of compound 2a is increased compared to the state of the art compound IMTO, especially after 120 and 240 minutes. The uptake of compound 2e is greatly increased compared to IMTO at any time point. After 240 minutes the uptake is increased by a factor of about 10.

(112) This is of great advantage, both for a diagnostic and therapeutic application of the compounds of the present invention. In diagnostic applications the higher uptake provides for an improved visualization of the target tissue. In therapeutic applications higher doses within the tumor might give improved therapeutic outcome of the patients.

Example 5: Cell Uptake Studies

(113) For each experiment 250,000 NCI-H295 cells were incubated with 0.1 MBq of IMTO or (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide (compound 2e) at 37° C. in a humidified atmosphere (5% CO.sub.2). For the blocking experiment, the cells were co-incubated with non-radioactive (0, 0.1 μM, 10 μM, 100 μM) etomidate to monitor the specific uptake of the substances. To stop the tracer uptake aliquots were taken after different time points (0 min, 2 min, 5 min, 10 min and 30 min) from the reaction mix immediately to a 4° C. ice-bath. After 5 min incubation time the solutions was centrifuged. After washing twice with PBS/0.5% Tween 80 the collected washing solution was measured in a gamma counter to determine the total amount in counts per minute (cpm) together with the samples to correct for radioactivity decay. The assay was performed in triplicates. The results of this experiment are shown in FIGS. 4 and 5.

Example 6: Biodistribution of Compound 2a in Mice

(114) The biodistribution of IMTO, (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid ethyl-methyl amide (compound 2a) and (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide (compound 2e) has been assessed in mice as follows. Male CD-1 mice were injected iv with 1 μCi (37 kBq) (R)-1-[1-(4-[.sup.125I]iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid ethyl-methyl amide. At predefined time points (15 min, 30 min, 2 h, and 4 h), mice were killed (n 6 per time point). Blood was collected, and heart, lung, liver, intestine, stomach, spleen, kidneys, adrenals, testes, brain and other organs were excised and weighed. Radioactivity was measured using a gamma-counter.

(115) FIGS. 6-8 show the biodistribution of IMTO, (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazol-5-carboxylic acid ethyl-methyl amide (compound 2a) and (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide (compound 2e), respectively, in different organs and at different time points in mice. It can be seen that the uptake both of compound 2a and compound 2e is predominately in adrenal gland, i.e. that the compounds of the invention show a highly selective and specific binding.