| Assay Method Information | |
| | radioligand competition assay |
| Description: | A well established radioligand competition assay was carried out to evaluate binding affinity of the test compounds and were compared with that of the reference agent (S)-5-OH-DPAT (Table 3). Binding affinity to rat DA D2 and D3 receptors expressed in HEK-293 cells was determined as described by us previously. Table 3 lists the binding data of new compounds. Compounds (±)-90a-c, which incorporate racemic 2-aminothiazole head group and a piperazine ring connected to the different positions of the carbazole ring, exhibited high affinity for D3 and low to moderate affinity for D2 receptors. When the positions of attachment are at carbon 2 and 3 of the carbazole moiety for compounds 90a and 90b, respectively, both the compounds displayed low affinity for D2 and high affinity for the D3 receptors with high selectivity (Ki, D2=902 nM, D3=6.18 nM, D2/D3=146 and D2=612 nM, D3=3.12 nM, D2/D3=196 for 90a and 90b, respectively). Interestingly, covalent attachment at position 4 of the carbazole ring dramatically improved the affinity for D2 while that for D3 receptor remained the same (Ki, D2=76.9 nM, D3=7.8 nM, D2/D3=9.86 for 90c). This indicated highest tolerance of the 4-substituted carbazole derivative for interaction with the D2 and D3 receptors. As expected, we observed a 2-4-fold improvement in binding affinity when enantiomerically pure aminothiazole moiety was attached to the carbazole as in (−)-91a and (−)-91b compared to their racemic counterparts (Ki, D2=504 nM, D3=3.94 nM, D2/D3=128 and D2=135 nM, D3=3.80 nM, D2/D3=35 for (−)-91a and (−)-91b respectively). However, for (−)-91c we did not observe much difference from its racemic version.Next, the effect of bioisosteric replacement of the aminothiazole moiety with aminotetraline functionality on the receptor binding of target compounds was evaluated. In corroboration with our previous results, aminotetraline substituted compounds (±)-94a-c and (−)-95a-c exhibited high affinity at both D2 and D3 receptors. For instance, the aminotetraline analogue (−)-95a has been found to have very high affinity for D2 while displaying subnanomolar affinity for D3 receptor compared to the corresponding thiazolidium counterpart (−)-91a (Ki, D2=71.2 nM, D3=0.40 nM, D2/D3=177 for (−)-95a vs D2=504 nM, D3=3.94 nM, D2/D3=128 for (−)-91a). Among the three enantiomerically pure isomers (−)-95a-c, which differ only in the substitution positions at the carbazole moiety, positions 2, 3 and 4, showed variable binding affinity at both D2 and D3 receptors (Ki, D2=71.2 nM, D3=0.40 nM for (−)-95a (D2=61.6 nM, D3=1.94 nM for (−)-95b and D2=16.9 nM, D3=0.36 nM for (−)-95c). As discussed before, substitution at the 4-position of the carbazole aromatic ring resulted in compounds 90c, (−)-91c, 94c and (−)-95c) with better D2/D3 binding affinities in comparison to other isomeric analogues with compound (−)-95c exhibiting the highest affinity among all the molecules, underscoring the importance of positional attachment to the carbazole ring. Finally, the binding affinities were evaluated for another series of compounds in which the piperazine ring of the agonist fragment was appended directly to the carbazole nitrogen atom through a methylene linker. As shown in Table 3, enantiomeric compound (−)-101 displayed relatively higher binding affinity at D2 and comparable affinity at D3 receptor with moderate selectivity compared to the racemic compound (±)-100 (Ki, D2=435 nM, D3=6.60 nM, D2/D3=65.9 and D2=82.6 nM, D3=7.18 nM, D2/D3=12 for 100 and (−)-101, respectively). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |