Isotopically marked quinoline derivatives as adenosin A3 receptor ligands

4-Methoxy-N-(6-[ 125 I]iodo4-benzylamino-3-cyanoquinolin-2-yl)benzamide

In the general formula (I) R 1 stands for phenyl group, R 2 stands for 4-methoxyphenyl group.

a.) To 8 μl of 0.1 mg/ml methanolic solution of 4-methoxy-N-(6-tributylstannyl-4-benzylamino-3-cyanoquinolin-2-yl)benzamide are added 25 μl (1.1 nmol) of 1% (v/v) methanolic solution of trifluoroacetic acid and 70 MBq [ 125 I]NaI solution (21 μl). To the reaction mixture 5 μl (2 nmol) of 0.1 mg/ml aqueous solution of chloramine-T is added and the mixture is stirred at room temperature for 15 minutes. After the incubation period the reaction is stopped by the addition of 7 μl (3.5 nmol) of 0.1 mg/ml sodium pyrosulfite solution and the product is immediately purified by using RP-HPLC method, applying UV and radioactivity detection. By this manner 31 MBq of the title compound is obtained (molar activity 81.4 GBq/mmol), radiochemical purity >95%. The purified product is stored in methanol-water (0.1% TFA) 3:1 mixture (activity concentration: 29 MBq/ml).

b.) 4-Methoxy-N-(6-tributylstannyl-4-benzylamino-3-cyanoquinolin-2-yl)benzamide.

0.33 g of 4-methoxy-N-(6-iodo-4-benzylamino-3-cyanoquinolin-2-yl)benzamide is dissolved in 5 ml of N-methyl-2-pyrrolidone and to the solution 240 mg of potassium acetate, 50 mg of tetrakis(tri(o-tolyl)phosphine)palladium(0) and 0.6 ml of hexabutyl distannane are added. The reaction mixture is stirred at room temperature under argon atmosphere for 16 hours, then it is poured onto 20 ml of water and extracted with 2×20 ml of toluene. The united toluene phase is dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue is chromatographed on a silicagel coloumn using chloroform-ethyl acetate (10:0.5) mixture as eluent. After evaporation of the pure fractions 250 mg of the title compound is obtained.

1 H-NMR (CDCl 3 ) δ 0.9 (m, 9H), 1.1 (M, 6H), 1.3 (m, 6H), 1.55 (m, 6H), 3.8 (s, 3H), 5.08 (d, 2H), 7.06 (d, 2H), 7.2-7.4 (m, 5H), 7.55 (d, 2H), 7.9-8.1 (m, 3H), 8.66 (m, 1H), 10.7 (s, 1H).

4-Methoxy-N-(6-[ 125 I]iodo-4-[2-thienylmethylamino]-3-cyanoquinolin-2-yl)benzamide

In the general formula (I) R 1 stands for thienyl group, R 2 stands for 4-methoxyphenyl group.

a.) To 8 μl (1.1 nmol) of 0.1 mg/ml methanolic solution of 4-methoxy-N-(6-tributylstannyl-4-[2-thienylmethylamino]-3-cyanoquinolin-2-yl)benzamide are added 25 μl (1.1 nmol) of 1% (v/v) methanolic solution of trifluoroacetic acid and 70 MBq [ 125 I]NaI solution (21 μl). To the reaction mixture 5 μl (2 nmol) of 0.1 mg/ml aqueous solution of chloramine-T is added and the mixture is stirred at room temperature for 15 minutes. After the incubation period the reaction is stopped by the addition of 7 μl (3.5 nmol) of 0.1 mg/ml sodium pyrosulfite solution and the product is immediately purified by RP-HPLC method, while applying UV and radioactivity detection. By this manner 28 MBq of the title compound is obtained (molar activity 81.4 GBq/mmol), radiochemical purity >95%. The purified product is stored in methanol-water (0.1% TFA) 3:1 solvent mixture (activity concentration: 28 MBq/ml).

b.) 4-Methoxy-N-(6-tributylstannyl-4-[2-thienylmethylamino]-3-cyanoquinolin-2-yl)benzamide.

0.3 g 4-Methoxy-N-(6-iodo-4-[2-thienylmethylamino]-3-cyanoquinolin-2-yl)benzamide is dissolved in 5 ml of N-methyl-2-pyrrolidone and to the solution 240 mg of potassium acetate, 50 mg of tetrakis(tri(o-tolyl)phosphine)palladium(0) and 0.6 ml of hexabutyl distannane are added. The reaction mixture is stirred at room temperature under argon atmosphere for 16 hours, then it is poured onto 20 ml of water and extracted with 2×20 ml of toluene. The united toluene phase is dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue is chromatographed on a silicagel coloumn using chloroform-ethyl acetate (10:0.5) mixture as eluent. By evaporating the pure fractions 235 mg of the title compound is obtained.

1 H-NMR (CDCl 3 ) δ 0.9 (m, 9H), 1.1 (M, 6H), 1.3 (m, 6H), 1.55 (m, 6H), 3.85 (s, 3H), 5.1 (d, 2H), 6.9-7.17 (m, 4H), 7.43-7.54 (m, 2H), 8.03 (m, 3H), 8.72-8.82 (m, 2H), 10.86 (s, 1H).

A./ Biological Methods

Human Adenozin A 1 Receptor Binding

Preparing membrane suspension: collect ovarium cells of cloned Chinese hamster expressing human A 1 receptors (further: CHO-hA 1 ), wash them three times with PBS, centrifuge (1000×g 10 min.) and homogenize (B.Braun Potter S) at 1500/min rotation speed. Buffer: 50 mM Tris HCl, pH 7,4. Centrifuge this homogenized mixture (43.000 g, 10 min), suspense the pellet in the above buffer with adjustment of the protein concentration to 5 mg/mL (Bradford method) and complete with 2 U/mL ADA.

Binding protocol: incubate CHO-hA 1 membrane preparation (50 μg protein content), in the presence of the test compound and 10 nM [ 3 H]CCPA (2-chloro-N 6 -cyclopenthyl-adenosine) (80.000 dpm) in incubation buffer (50 mM Tris HCl, pH 7.4, 2 U/mL adenosine deaminase). The non-specific binding is defined in the presence of 10 μM R-PIA (N 6 -[L-2-phenylisopropyl]adenosine) in a total volume of 100 μL for 3 hr at room temperature. Filter over Whatman GF/B glass fibre filters (presoaked in 0.5% polyethylimine for 3 hours), wash 4× with 1 mL ice-cold 50 mM Tris HCl (pH 7.4) on 96-well Brandel Cell Harvester. Detection of activity: in 96-well plate in the presence of HiSafe-3 cocktail in beta-counter (1450 Microbeta, Wallac). Inhibition [%]=100−((activity in the presence of test compound−non-specific activity)/(total activity−non-specific activity))*100

Human Adenosine A 2a Receptor Binding

Incubate 7 μg of membranes (human A 2 , adenosine receptors transfected into HEK-293 cells, source: Receptor Biology, Inc.), in the presence of the test compound and 20 nM [ 3 H]CGS-21680 (2-[p-(2-carbonylethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine) (200.000 dpm) in incubation buffer (50 mM Tris HCl, 10 mM MgCl 2 , 1 mM EDTA, 2 U/mL adenosine deaminase, pH 7.4). The non-specific binding is defined in the presence of 100 μg NECA (5′-N-ethylcarboxamido-adenosine) in a total volume of 100 μl for 90 min at room temperature. Filter in vacuum over Whatman GF/B glass fibre filters (presoaked for 3 hours in 0.5% polyethylimine), wash 4× with 1 mL ice-cold buffer (50 mM Tris HCl, 10 mM MgCl 2 , 1 mM EDTA, 0.9% NaCl, pH 7.4) on 96-well Brandel Cell Harvester. Detection of activity: in beta-counter (1450 Microbeta, Wallac) in the presence of 200 μL HiSafe-3 cocktail. Inhibition [%]=100−((activity in the presence of test compound−non-specific activity)/(total activity−non-specific activity))*100.

Human Adenosine A 2b Receptor Binding

Binding protocol: incubate 20.8 μg of membranes (human A 2b adenosine receptors transfected into HEK-293 cells, source: Receptor Biology, Inc.), in the presence of the test compound and 32.4 nM [ 3 H]DPCPX (8-cyclopenthyl-1,3-dipropylxanthine) (800.000 dpm) in incubation buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM EDTA, 0.1 mM benzamidine, 2 U/mL adenosine deaminase, pH 6.5). The non-specific binding is defined in the presence of 100 μM NECA (5′-N-ethylcarboxamido-adenosine) in a total volume of 100 μL for 30 min at room temperature. Filter under 25 Hgmm vacuum over Whatman GF/C glass fibre filters (presoaked in 0.5% polyethylimine for 3 hours), wash 4× with 1 mL ice-cold 50 mM Tris HCl (pH 6.5) on 96-well Brandel Cell Harvester. Detection of activity: in beta-counter (1450 Microbeta, Wallac) in the presence of 200 μL of HiSafe-3 cocktail. Inhibition [%]=100−((activity in the presence of test compound−non-specific activity)/(total activity−non-specific activity))*100

Human Adenosine A 3 Receptor Binding

Preparing membrane suspension: collect ovarium cells of cloned Chinese hamster expressing human A 3 receptors (further: CHO-hA 3 ), wash them three times with PBS, centrifuge (1000×g 10 min.) and homogenize (B.Braun Potter S) at 1500/min rotation speed. Buffer: 50 mM Tris, 10 mM MgCl 2 , 1 mM EDTA, pH 8.0. Centrifuge this homogenized mixture (43.000 g, 10 min), suspense the pellet in the above buffer with adjustment of the protein concentration to 0.1 mg/mL (Bradford method) and complete with 2 U/mL ADA.

Receptor Binding in the Presence of [ 125 I]AB-MECA:

Incubate the CHO-hA 3 membrane preparation (protein content 2 μg) in the presence of the test compound and 0.5 nM [ 125 I]AB-MECA (4-amino-3-iodo-benzyl-5′-N-methylcarboxamide-adenosine) (100.000 cpm) in incubation buffer (50 mM Tris, 10 mM MgCl 2 , 1 mM EDTA, 2 U/mL adenosine deaminase, pH 8.0). The non-specific radioligand binding is defined in the presence of 100 μM R-PIA (N 6 -[L-2-phenylisopropyl]adenosine) in a total volume of 50 μL for 60 min at room temperature. Filter under 25 Hgmm vacuum over Whatman GF/C glass fibre filters (presoaked in 0.5% polyethylimine for 3 hours), wash 4× with 1 mL ice-cold buffer (50 mM Tris, 10 mM MgCl 2 , 1 mM EDTA, pH 8) on 96-well Brandel Cell Harvester. Detection of radioactivity: in gamma-counter (1470 Wizard, Wallac). Inhibition [%]=100−((activity in the presence of test compound−non-specific activity)/(total activity−non-specific activity))*100.

Receptor Binding in the Presence of the Radioactive Iodine-Containing Compound of Example 2/a:

Incubate the CHO-hA 3 membrane preparation (protein content 4 μg) in the presence of the test compound and 0.5 nM of the compound containing radioactive iodine (100.000 cpm), described in Example 2/a, in incubation buffer (50 mM Tris, pH 8.0, 10 mM MgCl 2 , 1 mM EDTA, 0.08% CHAPS, 0.5% BSA, 2 U/mL adenosine deaminase). The non-specific radioligand binding is defined in the presence of 100 μM R-PIA (N 6 -[L-2-phenylisopropyl]adenosine) in a total volume of 50 μL for 60 min at room temperature. Keep the isotop preparation and the reaction mixture in polyethylene tube to decrease adsorption. Filter under 25 Hgmm vacuum over Whatman GF/C glass fibre filters (presoaked in 0.5% polyethylimine for 3 hours), wash 4× with 1 mL ice-cold buffer (50 mM Tris (pH 8), 10 mM MgCl 2 , 1 mM EDTA, 0.08% CHAPS, 0.25% BSA) on 96-well Brandel Cell Harvester. Detection of radioactivity: in gamma-counter (1470 Wizard, Wallac). Inhibition [%]=100−((activity in the presence of test compound−non-specific activity)/(total activity−non-specific activity))*100.

B./ Biological Results

I Affinity of the Un-Labeled Iodine-Containing Compound Given in Example 2/b as Starting Material (i.e. the Un-Labeled Analogue of the New Radioligand Given in Example 2/a) to the Adenosine Receptor Sub-Types in the Presence of Known Radioligands

The affinity of the starting compound of Example 2/b to the adenosine A 3 receptor (K i =1.5 nM) exhibits at least thousand-fold selectivity compared to the other adenosine receptor sub-types (Table 1).

II/A Investigation of the New Radioactive Iodine-Containing Compound of Example 2/a on the Human Adenosine A 3 Receptor by Scatchard Analysis Used for the Characterisation of Radioligands

On the basis of radioisotop saturation curves, by Scatchard analysis (G. Scatchard, Ann. N. Y. Acad. Sci. 51:660, 1949) the dissociation constant (K D ) of the new radioligand of Example 2/a on CHO-hA 3 membrane preparation is determined. In the investigated concentration range (0.156 nM-10 nM) the radioligand binds to only one binding place in the presence of a membrane preparate containing 4 μg of protein. The value of K D was found to be 4 nM, the maximal binding capacity 985 femtomol/1 mg protein (see FIG. 1 ).

FIG. 1 : Scatchard saturation curve of the new radioligand of Example 2/a in the presence of CHO-hA 3 membrane preparation

II/B Comparison of a Known Adenosine A 3 Radioligand with the New Radioligand of Example 2/a on the Basis of the Affinity Values of Reference compounds, in the Presence of 4 μg CHO-hA 3 Preparate

Knowing the K D values, by the Cheng-Prusoff equation (Y. J. Cheng and W. H. Prusoff, Biochem. Pharmacol. 22:3099, 1973) the K i constants of the investigated reference compounds and that of the starting compound of Example 2/b (i.e. of the un-labeled analogue of the new radioligand of Example 2/a) were calculated on the basis of the IC 50 values. The reference compounds exhibited similar affinity values in the presence of the known and of the new radoligands, proving the suitability of the radioligand of Example 2/a. The un-labeled analogue of the new radioligand exhibited a K i value nearly equal to the K D value of the isotop-labeled form (4.0 nM and 1.3 nM), also proving the specific binding of the new radioligand (see Table 2).