Homologous radioligand competition binding experiments were performed to determine the receptor expression levels (B_max) and binding affinities (K_d) of the used radioligands. Average B_max and K_d values for each receptor are shown in table 1. All K_d values except for 5HT_2A receptors were 3-6-fold higher than those found in the literature (table 1, 14151617). This effect could be attributed to the use of isotonic Krebs-HEPES-buffer pH 7.4 in this study instead of the widely used TRIS-HCl buffer pH 7.4 in the literature. Figure 2 shows as an example the buffer-dependent inhibition by LE300 of [³H]SCH23390 binding to D₁ receptor membranes. Using Krebs-HEPES instead of TRIS-HCl buffer yielded ~4-fold higher K_i values of LE300 (figure 2) but allowed a better comparison of functional and binding data. A buffer-dependent change of affinity was also observed with the test compounds. However, the K_d ratios among the receptor subtypes using Krebs-HEPES buffer were equal to literature data using TRIS-HCl (not shown).

Binding affinities of the compounds LE300, LE400, LE401, LE403, LE404, LE410, and LE420 (figure 1) were estimated at recombinant dopamine and 5HT_2A receptors in cell membrane preparations. Further compounds used for 3D-QSAR analysis of D₁ receptor ligands (AHAD11, B157, LERU301, SH3, figure 1) were tested at D₁ receptors only due to limited availability. For the sake of comparison, haloperidol as a classical antipsychotic, clozapine as an atypical antipsychotic, and LE300 were included as reference compounds. Figure 3 shows the radioligand displacement curves of the most potent hD₁ and hD_2L ligands LE404 and LE410 at hD₁ (A), hD_2L (B), and 5HT_2A (C) receptors. pK_i values are displayed in table 2 (LE300, LE400, LE401, LE403, LE404, LE410, and LE420) and table 3 (AHAD11, B157, LERU301, SH3).

All compounds showed similar affinities at hD₁ and hD₅ receptors. The mono-hydroxylated LE404 turned out as the most potent compound at hD₁/hD₅ receptors with pK_i values of 8.47 and 8.53, respectively, followed by the bis-hydroxylated LE403 which is 3-10-fold less potent than LE404. Replacement of the hydroxy- by methoxy-substituents resulting in LE400 dramatically decreased the affinity at all tested receptors. An increase of the size of the nitrogen substituent (allyl group of LE401) further decreased the affinity at all tested receptors. Except LE400 and LE401, all other compounds possessed up to 33fold (LE403, LE404) higher affinities for D₁-like than for D₂-like receptors (table 2). Among D₂-like receptors, all compounds – except LE404 – showed the highest affinity at hD_2L and lower affinities at hD₃ and hD_4.4 receptors similar to the profile of haloperidol at D₂-like receptors. However, different to haloperidol which shows a strong D₂ over D₁ selectivity, LE compounds (except LE400 and LE401) show selectivity for D₁ over D₂. Removal of the hydroxy-group of LE404 yielding LE410 resulted in a dramatic loss of D₁ over D₂ selectivity, and left LE410 as the most potent compound at hD_2L and hD₃ receptors with pK_i values of 7.54 and 6.86, respectively. Bioisosteric replacement of one benzene residue in LE410 by thiophene gave LE420 showing a similar receptor profile as LE410 but with reduced affinity at all tested receptors. LE404 displayed a receptor profile within the D₂-like receptors which is unique among the tested LE compounds. Within the D₂-like receptors, LE404 reached the highest affinity at hD_4.4 (pK_i: 7.23), a slightly lower affinity at hD_2L (pK_i: 7.10), and the lowest affinity at hD₃ receptors (pK_i: 6.73). The D₂-like receptor affinity pattern of LE404 is thus similar to clozapine (D_4.4 ≥ D_2L > D₃). In contrast to clozapine which appeared ~ equipotent at D₁/D₂ receptors in all of our test systems, LE404 shows 25fold selectivity for D₁ over D₂. LE404 displayed higher affinities than LE300 at all dopamine receptors except hD_2L where both compounds are ~ equipotent. All compounds except LE401 showed the highest affinities among all tested receptors at 5HT_2A. The most potent compound at 5HT_2A was LE300 with an affinity in the subnanomolar range followed by LE404 in the low nanomolar range. LE300, LE400, LE403, LE404, LE410, and LE420 achieved K_i-D2/K_i-5HT2A selectivity ratios > 7.

For functional studies, hD₁ and hD_2L receptors were chosen as characteristic representatives of each of the two dopamine receptor subtype groups allowing a comparison of functional and binding data. The inhibition by LE compounds of agonist-induced changes in intracellular [cAMP] and [Ca²⁺] in intact HEK293 cells, and [³⁵S]-GTPγS binding in HEK293 cell membranes were estimated. Table 4 shows EC₅₀ and IC₅₀ values of standard ligands at D₁ and D_2L receptors. The effects of LE compounds on [³⁵S]-GTPγS binding were determined in the presence of agonist. At hD₁ receptors the full agonist dihydrexidine 18 was used for [³⁵S]-GTPγS binding experiments instead of SKF38393 which was used in cAMP and Ca²⁺ studies. In membrane preparations from HEK293-hD₁ cells, dihydrexidine showed a significantly higher increase in [³⁵S]-GTPγS binding than SKF38393 (figure 4). Dihydrexidine gave an EC₅₀ of 43.8 ± 8.23 nM (hD₁, figure 4). A difference between dihydrexidine and SKF38393 was not observed in intracellular [Ca²⁺] and [cAMP] measurements (data not shown), and thus SKF38393 was used in Ca²⁺ and cAMP studies. The EC₅₀ of quinpirole at hD_2L receptors was estimated as 437 ± 93.1 nM (data not shown). All LE compounds except LE401 showed an inhibition of [³⁵S]-GTPγS binding between 25 and 40% (not shown).

None of the tested compounds (neither LE compounds nor reference compounds haloperidol or clozapine) showed any agonist effect in functional studies (data not shown). All test compounds inhibited agonist-stimulated effects on intracellular [cAMP] and [Ca²⁺] and on [³⁵S]-GTPγS binding at D₁ or D_2L receptors, respectively, in a concentration-dependent manner. LE400 in Ca²⁺ studies and LE401 in all functional assays achieved ≤ 50% inhibitory activity at 10 μM. Concentration-inhibition curves of the most potent novel compounds at D₁ and D_2L receptors, LE404 and LE410, are displayed in figure 5. Apparent functional pK_i values (pK_{i app}) derived from inhibition experiments of all compounds in cAMP, Ca²⁺, and [³⁵S]-GTPγS studies are presented in table 5. When comparing pK_i values of one compound from cAMP, Ca²⁺, and [³⁵S]-GTPγS studies, differences may occur (e.g., clozapine at D₁ receptors: pK_i(cAMP): 6.46; pK_i ([³⁵S]-GTPγS): 7.47) but also good accordance was observed (e.g., LE404 at D₁ receptors: pK_i values between 7.95 and 8.20). The rank orders of potency of the tested compounds at D₁ and D_2L receptors, respectively, remained similar for the three functional assays: the most potent compound at D₁ receptors in all three functional assays (table 5) and in binding (table 2) is LE404 whereas the weakest compounds are LE400 and LE401. At D_2L receptors, LE300, LE410, and LE404 are the most potent compounds after haloperidol whereas again, LE400 and LE401 are the weakest (binding: table 2, functional assays: table 5). LE404 has a 25-fold selectivity for D₁ over D_2L receptors based on binding (table 2). This D₁ preference was lost in cAMP and [³⁵S]-GTPγS experiments (LE404 is ~ equipotent at D₁ and D_2L) but a certain D₁ preference (3-fold) was retained in Ca²⁺ studies (table 5). Haloperidol showing a 100-fold D_2L over D₁ selectivity in binding (table 2) retained this 100-fold D_2L selectivity in [³⁵S]-GTPγS experiments but showed an increased D_2L selectivity in cAMP and Ca²⁺ studies (> 1000-fold). LE410 which displayed an only moderate D₁ selectivity in binding (~2-fold, table 2) became D_2L selective in cAMP and Ca²⁺ studies but was ~ equipotent in [³⁵S]-GTPγS binding. These results show that cAMP and Ca²⁺ studies uprate the potency of compounds at D_2L compared to D₁ receptors (tables 2 and 5).

The multiple intercorrelation and thus the equality of the results obtained by binding and the three functional assays at D₁ and D_2L receptors, respectively, was determined by principal component analysis (PCA). Results of the PCA comparing the four test systems (factor loadings) are displayed in table 6. The first extracted principal component (PC) for D₁ receptors described 89.8% of the total variance among the four pK_i variables (cAMP, Ca²⁺, [³⁵S]-GTPγS, and binding) with factor loadings > 0.91 (table 6) leaving an eigenvalue of only 0.237 for the second PC. For D_2L receptors, the first extracted PC explained 97.5% of the total variance among the four pK_i variables (factor loadings > 0.98, table 6) leaving an eigenvalue of only 0.050 for the second PC. Following the idea that a PC with an eigenvalue of << 1 has no legitimacy for the description of the total variance 19, the PCA results indicate a significant multiple correlation among the four variables for D₁ and D_2L receptors, respectively.

Next, the nature of antagonism of LE compounds at D₁ and D_2L receptors was tested by Clark analysis 20. Since LE404 was the most potent compound at D₁ and LE410 the most potent at D_2L receptors (binding, table 2), LE404 and LE410 were chosen as representatives to undergo functional analysis for competitive antagonism. In the presence of increasing concentrations of LE404 and LE410, parallel rightward shifts of the agonist concentration-effect curves in the Ca²⁺ assay were observed without loss of maximum efficacy at hD₁ and hD_2L receptors (data not shown). The rightward shift of the concentration-effect curves of the agonist was analyzed with non-linear regression analysis according to Lew and Angus 20. Data were fitted to equations (1) and (2) (see methods). An F-Test showed no significant difference (p > 0.2), thus equation (2) with a Hill slope of 1 was the preferred model and used to obtain pK_b values. Results for LE404 at hD₁ and hD_2L receptors are presented in figures 6A and 6B. Inserts show the Clark plots (mean log EC₅₀ values of the agonist concentration-effect curves plotted against log ([LE404] + K_b) which yielded straight lines at both receptor subtypes. pK_b values were calculated as: hD₁: pK_{b LE404} = 8.09 ± 0.15; pK_{b LE410} = 7.69 ± 0.13; hD_2L: pK_{b LE404} = 7.61 ± 0.10; pK_{b LE410} = 8.05 ± 0.11. pK_b values of LE404 and LE410 derived from non-linear Clark analysis show no significant difference to those derived from Schild analysis 21 (data not shown). Both functional analyses (Schild, Clark) give thus evidence for a competitive antagonistic behaviour of LE404 and LE410 at D₁ and D_2L receptors.

In order to perform a statistically valid test for the discovery of ligands with differing affinity profiles at dopamine D₁-D₅ and 5HT_2A receptors among the examined compounds, multiple intercorrelations of binding affinity values (pK_i, table 2) as well as binding selectivity values [log (K_i ratio) = log (K_{i Receptor 1}/K_{i Receptor 2})] were investigated in two separate PCA's. PCA has already successfully been applied to define similar and deviating responses among biological data (variables) 2223. LE401 was excluded from both PCA's because precise pK_i values were missing at hD_4.4 and h5HT_2A receptors (table 2). In the first PCA, eight compounds were examined (haloperidol, clozapine, LE300, LE400, LE403, LE404, LE410, and LE420) for their affinity in six test systems (D₁-D₅ and 5HT_2A receptors). The PCA resulted in two PC's from which the first extracted 80.5% of the total variance among the eight pK_i variables, and the second extracted 11.4%. The factor loadings of the eight variables (compounds) are listed in table 7 and show that the eight compounds define three subgroups of dopamine/5HT_2A ligands: 1) clozapine, LE300, LE400, LE410, and LE420 with factor loadings contributing to the first PC of > 0.739; 2) haloperidol in the second PC with a factor loading of -0.923; 3) LE403 and LE404 in the second PC with opposite direction to haloperidol (factor loadings 0.868 and 0.886) indicating that LE403 and LE404 display an affinity profile opposite to that of haloperidol. For the second PCA, for each of the eight compounds, log (K_i ratio) values [= log (K_{i Receptor 1}/K_{i Receptor 2})] were calculated for all possible 15 receptor affinity ratios (D₁/D_2L, D₁/D₃, D₁/D_4.4, D₁/D₅, D₁/5HT_2A, D_2L/D₃, D_2L/D_4.4, D_2L/D₅, D_2L/5HT_2A, D₃/D_4.4, D₃/D₅, D₃/5HT_2A, D_4.4/D₅, D_4.4/5HT_2A, D₅/5HT_2A) using the data from table 2. The resulting log (K_i ratio) data matrix contains selectivity information for each of the compounds. Results of this second ("selectivity") PCA were basically identical to results from the first ("affinity") PCA (table 7). The first extracted PC explained 74.8% of the total variance among the eight variables (log (K_i ratio) values), and the second PC extracted 15.4% of the total variance. The second ("selectivity") PCA discovered the same three subgroups of dopamine/5HT_2A ligands as did the first PCA: 1) clozapine, LE300, LE400, LE410, and LE420 with factor loadings contributing to the first PC of > 0.780 (table 7); 2) haloperidol in the second PC with a factor loading of -0.901; 3) LE403 and LE404 in the second PC with opposite direction to haloperidol (factor loadings 0.933 and 0.893). Thus, regardless of using affinity information (pK_i) or selectivity information (log (K_i ratio)) for PCA, the same three subgroups of dopamine/5HT_2A ligands were discriminated. The agreeing results from both PCA's underline that the statistical analysis of binding affinities and selectivities at dopamine and 5HT_2A receptors did not create chance correlations.

Since the main feature of the LE compounds is their D₁ selectivity, a 3D-QSAR pharmacophore model for the D₁ receptor was establish using the 12 compounds shown in figure 1 and their D₁-pK_i values from table 2 and 3. For a successful CoMFA/CoMSIA study, it is crucial to find an appropriate alignment of the examined compounds. It is not necessary that all compounds possess the bioactive conformation but it is useful that the compounds adopt a relative conformation and position to each other as they would bind to the receptor. The D₁/D₅ selective antagonist (-)-2b-SCH39166 (ecopipam) was taken as a pharmacophore template. (-)-2b-SCH39166 is a benzonaphthazepine, a rigid analogue of SCH23390, thus limiting the number of possible conformations (figure 7) 24. Unfortunately, (-)-2b-SCH39166 was not available to us for testing, and was thus not used for the final QSAR-analysis. However, due to its rigid nature, it was helpful to find a good starting point for selecting conformations and alignments of the 12 compounds from figure 1. Essential pharmacophore features of (-)-2b-SCH39166 are the two aromatic rings and the basic nitrogen (hydrogen acceptor) while the hydroxyl group served as an optional H-donor/acceptor feature (figure 7). Results of the alignment of the final models of the LE compounds are shown in figure 8. The aromatic residues and basic nitrogen atoms remain the main pharmacophore features. Crossvalidation results (leave-one-out) for the final models for CoMFA and CoMSIA both using steric and electrostatic fields are displayed in table 8, and show crossvalidation parameters q² of 0.82 for CoMFA and 0.88 for CoMSIA. To prove that these models were not a result of a chance correlation, a stability test was performed using the random groups PLS method ("leave-many-out"). The test showed a high stability of the models presented in figure 8 with a mean q² of 0.76 (SD 0.10) for the combined steric and electrostatic field in CoMFA and a mean q² of 0.81 (SD 0.12) in CoMSIA. The distribution of the q² values for this validation is shown in figure 9.

LE300, 400, 401, 403, 404, 410, and 420 were synthesized according to methods previously published 1113. [³H]SCH23390 (66.0 Ci/mmol), [³H]spiperone (118 Ci/mmol), and [³⁵S]-GTPγS were obtained from Amersham Biosciences (Buckinghamshire, UK). SKF38393 was purchased from TOCRIS (Bristol, U.K.). A pRc/CMV vector construct for hD₃ receptors was kindly provided by Dr. P. Sokoloff (Paris, France) 27 and a pcDNA3.1+ construct containing cDNA coding for the h5HT_2A receptor was obtained from the UMR cDNA resource center 28. All other reagents were supplied by Sigma Chemicals unless otherwise stated.

HEK293 cells stably expressing hD₁, hD_2L, or hD₅ dopamine receptors were established as previously described 1129. Stable cell lines of HEK293 cells (ATCC, Rockville, MD, USA) were generated by transfecting the plasmids coding for hD₃ and h5HT_2A using polyfect^® transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions and were selected using G-418 (400 μg/ml medium). All stably transfected cell lines were grown in Dulbecco's modified Eagle Medium Nutrient Mixture F-12 Ham (DMEM/F12 1:1 mixture) containing 10% fetal bovine serum, 100 μg/ml streptomycine, 100 U/ml penicillin G, 5 mM L-glutamine, and 200 μg/ml active G-418. The human D_4.4 receptor was stably expressed in CHO cells (kindly provided by Dr. van Tol, Toronto, Canada) and grown in Ham F12 medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, 100 U/ml penicillin G, 1 mM L-glutamine, and 200 μg/ml active G-418. Cells were incubated at 37°C in a humidified atmosphere under 5% CO₂.

Confluent 145 mm tissue culture dishes (Greiner Bio-One, Frickenhausen, Germany) of HEK293 or CHO cells were harvested by scraping, resuspended in ice-cold Krebs-HEPES buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 4.2 mM NaHCO₃, 11.7 mM D-Glucose, 1.3 mM CaCl₂, 10 mM HEPES, pH 7.4), and disrupted using a Polytron homogenizer on ice (Kinematica AG, Basel, Switzerland). After centrifugation at 40,000 × g at 2°C, the supernatant was discarded, and pellet was washed twice with ice-cold Krebs-HEPES buffer. Eventually, the pellet was resuspended in the appropriate binding buffer (see below) and stored in aliquots at -80°C until use for radioligand binding. The method of Bradford 30 was used to determine the protein content of membrane preparations with bovine serum albumin as standard.

For [³⁵S]-GTPγS-binding, cell pellets were resuspended in 10 mM Tris-HCl/1 mM EDTA, pH 7.4, homogenized in a glass-teflon homogenizer and centrifuged for 15 min (40,000 × g, 4°C). Supernatant was discarded, and pellet was washed twice with ice-cold Tris-HCl/EDTA buffer and finally resuspended in 50 mM Tris-HCl, 4 mM MgCl₂, 1 mM EDTA, 250 mM sucrose, pH 7.4, and stored at -80°C. The protein content was determined according to the Bradford method 30 with gamma immunoglobulin as standard.

The equilibrium dissociation constants K_d of the radioligands used ([³H]SCH23390 for hD₁-like, [³H]spiperone for hD_2L-like and h5HT_2A receptors) were determined in homologous competition binding experiments and receptor densities of the respective dopamine receptor cell membrane preparations (B_max values) were calculated using the DeBlasi equation 31. Heterologous competition binding experiments were performed in Krebs-HEPES buffer in a final volume of 1.1 ml at 26°C for 2 h (D₁-like receptors) or 3 h (D₂-like receptors and 5HT_2A receptors) as described previously 11. Cell membranes (total protein amount ~90 μg/tube) were incubated with 0.2 nM final [³H]SCH23390 (D₁-like receptors) or with 0.1 nM final [³H]spiperone (D₂-like receptors and 5HT_2A receptors) and competing drugs. The assay was terminated by rapid filtration of 1 ml through polyethylene imine pretreated (0.2%) glass fiber filters (Schleicher und Schuell, Dassel, Germany), followed by two washes with ice-cold distilled water. Filters were soaked in 5 ml of scintillation fluid for at least 12 h and bound radioactivity was determined by liquid scintillation counting. Nonspecific binding of [³H]SCH23390 was determined in the presence of 1 μM LE300, nonspecific binding of [³H]spiperone in the presence of 1 μM haloperidol for hD_2L-like receptors and 1 μM ketanserin for h5HT_2A receptors.

Cell membranes (for hD₁: total protein amount ~16 μg, ~1.5 pmol receptor/mg protein; for hD_2L: total protein amount ~16 μg, ~0.3 pmol receptor/mg protein) were incubated with test compounds, 1 μM GDP, agonist (1 μM dihydrexidine for hD₁, 10 μM quinpirole for hD_2L) and 100 pM [³⁵S]-GTPγS in microplates in a total volume of 200 μl assay buffer (20 mM HEPES-NaOH (pH 7.4), 100 mM NaCl, 3 mM MgCl₂). Plates were incubated for 60 min at 30°C. Reaction was terminated by rapid vacuum filtration through GF/C filter plates (PerkinElmer), and filter plates were washed four times with 200 μl Tris-HCl (pH 7.4). Radioactivity retained on the filter plates was counted in a microplate counter (Microbeta, PerkinElmer).

Measurement of changes in intracellular [Ca²⁺] was performed as previously described using a NOVOstar microplate reader with a built-in pipetor (BMG LabTech, Offenburg, Germany) 11. HEK293 cells expressing the respective dopamine receptor were loaded with 3 μM Oregon Green 488 BAPTA-1/AM (Molecular Probes, Eugene, OR) for 1 h at 25°C in Krebs-HEPES buffer containing 1% Pluronic F-127. Then, cells were rinsed three times with Krebs-HEPES buffer containing 0.5% bovine serum albumin, diluted, and evenly plated into 96 well plates (Greiner, Frickenhausen, Germany) at a density of ~35,000 cells/well. Microplates were kept at 37°C. Fluorescence intensity was measured at 520 nm (bandwidth 35 nm) for 5 s at 0.4 s intervals to monitore baseline. Buffer alone or test compounds dissolved in buffer were then injected into separate wells, and fluorescence intensity was monitored at 520 nm (bandwidth 35 nm) for 25 s at 0.4 s intervals. Excitation wavelength was 485 nm (bandwidth 12 nm). Concentration-inhibition curves in the presence of the test compounds were obtained by pre-incubating the cells with the compounds for 30 min at 37°C prior to injection of agonist (hD₁: 100 nM SKF38393; hD_2L: 30 nM quinpirole).

Intracellular [cAMP] levels were estimated by using a cAMP reporter gene assay. pCRE-Luc Cis-Reporter plasmid (Path Detect^® CRE Cis-Reporting System, Stratagene, La Jolla, CA) coding for the firefly luciferase under the control of a cAMP response element was transiently transfected in HEK293 cells stably expressing the hD₁ or hD_2L receptor. 24 h after transfection, cells were reseeded in poly-L-lysine-coated (Biochrom, Berlin, Germany) white 96-well plates with clear bottom (Greiner, Frickenhausen, Germany) at a density of ~25,000 cells/well. Microplates were incubated for 48 h at 37°C and 5% CO₂ before using the cells for adenylyl cyclase stimulation or inhibition experiments. Cells were then exposed to increasing concentrations of test compounds dissolved in serum-free and phenol red-free medium and incubated for 3 h at 37°C and 5% CO₂. In case of hD_2L, 10 μM forskolin was added. Antagonistic activity was tested by pre-incubation of test compounds for 30 min at 37°C and 5% CO₂ prior to the addition of agonist (hD₁: 100 nM SKF38393; hD_2L: 100 nM quinpirole plus 10 μM forskolin) for 3 h. Incubation was terminated by adding 100 μl of cell lysis buffer (8 mM tricine, 1 mM dithiothreitol, 2 mM EDTA, 5 % Triton^® X-100, pH 7.8) for 20 min at 4°C. Luciferase activities were measured with the LUMIstar microplate reader (BMG LabTech, Offenburg, Germany). After monitoring the baseline for 0.3 s, 100 μl of luciferase assay reagent (30 mM tricine, 0.5 mM ATP, 10 mM MgSO₄, 0.5 mM EDTA, 10 mM dithiothreitol, 0.5 mM coenzyme A, 0.5 mM D-luciferin, pH 7.8) was added and luminescence was measured at 25°C for 12.7 s at 0.1 s intervals. Luminescence was corrected by subtracting baseline levels.

Functional analysis of the antagonist effect of LE404 and LE410 was carried out by measuring the attenuation by LE404 or LE410 of the agonist-induced increase in intracellular [Ca²⁺] in HEK293 cells recombinantly expressing hD₁ or hD_2L receptors. At least four antagonist concentrations were used. Functional data were used for nonlinear regression analysis according to Clark 20. The pEC₅₀ values of the agonist curves were plotted against the concentration of test compounds LE404 or LE410 and analyzed by non-linear regression curve fitting using the following equations:

(1) pEC₅₀ = -log ([B]ⁿ + 10^-pKb) - log c

(2) pEC₅₀ = -log ([B] + 10^-pKb) - log c

where [B] is the concentration of antagonist (LE404 or LE410), pK_b is the negative decadic logarithm of the antagonist dissociation constant, n the Hill coefficient, and log c the difference between the pK_b and the pEC₅₀ value of the agonist concentration-response curve in absence of the antagonist. Fits to equations (1) and (2) were compared by an F-test.

Radioligand-binding and functional data (measurement of intracellular [Ca²⁺], [cAMP], and [³⁵S]GTPγS binding) were analyzed by fitting the pooled data from at least three experiments (each with three replicates) to the four parameter logistic equation using Prism software 3.0 from GraphPad (GraphPad Software; San Diego, CA, USA). Competition-binding experiments were fitted best to a one-site binding model. Inhibition constants K_i from radioligand binding competition experiments were calculated from IC₅₀ values using the Cheng-Prusoff equation 32. Apparent functional K_i values were calculated according to the following equation adapted from Cheng and Prusoff 32:

K_i = IC₅₀/(1+L/EC₅₀)

where IC₅₀ is the inhibitory concentration of the antagonist to block by 50% the agonist effect, EC₅₀ is the effective concentration 50% of the used agonist (i.e., SKF38393 for hD₁, and quinpirole for hD_2L receptors), and L is the molar concentration of the used agonist. Data (data points in figures and numbers in tables) are given as mean ± SEM of at least three independent experiments each performed with triplicates unless otherwise stated. Statistical analyses including principal component analysis were performed using SPSS (version 12.0.1 for Windows).

All calculations were carried out on an x86-compatible PC running SuSE-Linux 9.2. For molecular modelling, SYBYL 7.0 (Tripos Inc., St. Louis, Missouri, USA) and MOE 2004.03 for Linux (Chemical Computing Group Inc., Montreal, Quebec, Canada) were used. Conformational clustering was done using MATLAB Release 13 for Linux (The MathWorks Inc., Natick, MA, USA). Conformational analyses of all 12 compounds from figure 1 were done using a repeated molecular dynamics based simulated annealing approach as implemented in SYBYL 7.0. MMFF94 served as the force field with distance dependent electrostatics. A molecule was heated up to 1000 K within 2000 fs, held at this temperature for 2000 fs and annealed to 0 K for 10000 fs using an exponential annealing function. By applying this procedure, a total of 100 conformations were sampled during 100 cycles to account for conformational flexibility and to find the most likely conformations occurring most often in the resulting pool. This was done for both configurations of the protonated nitrogen atom because molecular mechanics is not able to switch configurations. All conformations were then optimized with the semi-empirical quantum mechanics method AM1 as implemented in MOPAC 6 from SYBYL and further compared using the SYBYL MATCH algorithm. Subsequently, a MATLAB clustering algorithm was used to extract the most divergent conformations using the root mean square (RMS) values of the comparison and the AM1 heat of formation. The most diverse and most often represented conformations of each compound were selected and overlaid with the pharmacophore resulting from the rigid ligand (-)-2b-SCH-39166 using the program MOE. The best 2–4 matched alignments per compound were selected for the CoMFA/CoMSIA study upon minimum RMS criteria and visual examination. These conformations were transferred to a SYBYL database and used as an initial alignment for the CoMFA/CoMSIA study. During an automated procedure, all possible combinations were tested on the CoMFA and CoMSIA combined steric/electrostatic fields with partial least squares analysis (PLS). In subsequent PLS analyses, the alignment was refined and the CoMFA/CoMSIA models were optimized. To prove that these models were not a result of a chance correlation, a stability test was performed using the random groups PLS method. Within this method, cross-validation was done with groups of more than one compound, which were excluded earlier during the model-building regression. Unlike the leave-one-out cross-validation, these groups are selected on a random basis and instead of twelve cross-validation groups, only five were used. Because of the random selection of the group members, this cross-validation was repeated a hundred times.