Antipsychotics are divided into typical and atypical compounds based on clinical efficacy and side effects. The purpose of this study was to characterize in vitro a series of novel azecine-type compounds at human dopamine D1-D5 and 5HT2A receptors and to assign them to different classes according to their dopamine/5HT2A receptor profile.
Regardless of using affinity data (pKi values at D1-D5 and 5HT2A) or selectivity data (15 log (Ki ratios)), principal component analysis with azecine-type compounds, haloperidol, and clozapine revealed three groups of dopamine/5HT2A ligands: 1) haloperidol; 2) clozapine plus four azecine-type compounds; 3) two hydroxylated dibenzazecines. Reducing the number of Ki ratios used for principal component analysis from 15 to two (the D1/D2 and D2/5HT2A Ki ratios) obtained the same three groups of compounds. The most potent dibenzazecine clustering in the same group as clozapine was the non-hydroxylated LE410 which shows a slightly different D2-like receptor profile (D2L > D3 > D4.4) than clozapine (D4.4 > D2L > D3). The monohydroxylated dibenzacezine LE404 clusters in a separate group from clozapine/LE410 and from haloperidol and shows increased D1 selectivity.
In conclusion, two compounds with a novel dopamine/5HT2A receptor profile, LE404 and LE410, with some differences in their respective D1/D2 receptor affinities including a validated pharmacophore-based 3D-QSAR model for D1 antagonists are presented.
Dopamine is an important neurotransmitter in the mammalian CNS which has influence on physiological, behavioural and neuroendocrine functions, mediated through receptors on the cell surface. Five different dopamine receptor subtypes have been cloned and characterized. They belong to the super-family of G protein-coupled receptors (GPCR) and can be divided into two subfamilies, D1-like (D1, D5) and D2-like (D2, D3, D4) receptors, according to their sequence homologies, biochemical properties, and pharmacologic profiles . D1-like receptor stimulation activates adenylyl cyclase (AC) via coupling to stimulatory G protein Gαs/Gαolf subunits leading to an increase in intracellular cAMP concentrations. In contrast, D2-like receptors are Gαi/Gαo linked and inhibit AC activity . Dopamine receptors are clinically important drug targets for the treatment of disorders such as Parkinson's disease and schizophrenia . Blockade of dopamine D2 receptors is the main feature of antipsychotic action. Typical antipsychotics like the first generation D2 receptor antagonists haloperidol or chlorpromazine can cause therapy-limiting extrapyramidal-motor side effects (EPS). Second generation (atypical) antipsychotics are serotonin/dopamine antagonists with no or low EPS at doses showing antipsychotic activity and have significantly greater affinity for 5HT2A than for D2 receptors . This serotonin-dopamine ratio could contribute to atypicality [5-7] but further investigations are needed to define the precise mechanism of atypical antipsychotics. However, antipsychotic activity is not only the result of D2 and 5HT2A receptor blockade but an inhibitory/modulating effect on various dopamine and serotonin (D1, D2, D3, D4, 5HT1A, 5HT1D, 5HT2A, 5HT2C) and further receptors . Within the heterogeneous group of atypical antipsychotics, only clozapine exhibits effects against treatment-resistant schizophrenia . Responsible for this net effect among atypical antipsychotics may be the moderate affinity of clozapine at various receptor subtypes, especially at D1-receptors. A dysfunction in D1-receptor modulation in the prefrontal cortex contributes to the negative symptoms and cognitive deficits observed in schizophrenia. However, selective D1 antagonism alone has not turned out as an effective antipsychotic principle [9,10].
LE300, an indolobenzacezine (figure 1) has previously been characterized  and shows a binding profile similar to that of clozapine, however with a greater affinity for D1- than D2-like receptors. A series of LE300-derived compounds was recently synthesized and screened at dopamine D1, D2L, and D5 receptors by a previously published functional calcium assay [12,13]. The aim of the current study was to investigate the comprehensive binding and functional receptor profile of the most active of the dibenzazecine derivatives of LE300 (LE400, LE401, LE403, LE404, LE410, and LE420, figure 1) at all human dopamine and 5HT2A receptors, to test whether data from the calcium assay initially used for screening of LE300-derived compounds  correlate with other assays measuring functional activation of GPCRs (cAMP, [35S]-GTPγS), and to establish a 3D-QSAR pharmacophore model of these ligands. Heterologous competition binding experiments were carried out at recombinantly expressed human dopamine and 5HT2A receptors, and obtained data were compared with functional data from intracellular [cAMP] and [Ca2+] measurements and [35S]-GTPγS-binding. Indeed, dibenzazecine compounds with a previously not available receptor profile (increased antagonist activity at D1-like and 5HT2A receptors) were found. 3D-QSAR studies were performed resulting in QSAR models allowing further rational ligand design at a molecular level.
Figure 1. Structural formulas of the indolobenzazecine LE300, SCH23390, and a series of ten derived compounds.
Receptor expression and characterization
Homologous radioligand competition binding experiments were performed to determine the receptor expression levels (Bmax) and binding affinities (Kd) of the used radioligands. Average Bmax and Kd values for each receptor are shown in table 1. All Kd values except for 5HT2A receptors were 3-6-fold higher than those found in the literature (table 1, [14-17]). 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 Ki 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 Kd ratios among the receptor subtypes using Krebs-HEPES buffer were equal to literature data using TRIS-HCl (not shown).
Table 1. Characterization of recombinantly expressed human dopamine and h5HT2A receptors in HEK293 cell membrane preparations
Figure 2. Buffer-dependent differences in hD1 receptor potencies of LE300 in competition binding. Inhibition by LE300 of the binding of 0.2 nM [3H]SCH23390 to hD1 receptor expressing HEK293 cell membranes using Krebs-HEPES buffer pH 7.4 (●) or TRIS-HCl pH 7.4 (○), respectively. Hill slopes were not different from unity. Nonspecific binding was determined with 1 μM SCH23390, and was less than 7%. Data shown are mean ± SEM, n = 3. Ki (Krebs-HEPES): 10.0 ± 1.15 nM; Ki (TRIS-HCl): 2.36 ± 0.13 nM.
Radioligand binding studies
Binding affinities of the compounds LE300, LE400, LE401, LE403, LE404, LE410, and LE420 (figure 1) were estimated at recombinant dopamine and 5HT2A 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 hD2L ligands LE404 and LE410 at hD1 (A), hD2L (B), and 5HT2A (C) receptors. pKi values are displayed in table 2 (LE300, LE400, LE401, LE403, LE404, LE410, and LE420) and table 3 (AHAD11, B157, LERU301, SH3).
Figure 3. Heterologous competition binding curves of LE404 (■) and LE410 (●) at hD1 (A), hD2L (B), and 5HT2A (C) receptors. Data shown are the means ± SEM of specific binding of at least four determinations assayed in triplicate. A. 0.2 nM [3H]SCH23390 was used for hD1 receptors. Nonspecific binding was determined with 1 μM LE300. B. 0.1 nM [3H]spiperone was used for hD2L receptors. Nonspecific binding was determined with 1 μM haloperidol. C. 0.1 nM [3H]spiperone was used for h5HT2A receptors. Nonspecific binding was determined with 1 μM ketanserin.
Table 2. Characterization of compounds by heterologous competition binding
Table 3. Characterization of AHAD11, B157, LERU301, and SH3 at hD1 receptors used for 3D-QSAR analysis.
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 pKi 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 hD2L and lower affinities at hD3 and hD4.4 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 hD2L and hD3 receptors with pKi 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 hD4.4 (pKi: 7.23), a slightly lower affinity at hD2L (pKi: 7.10), and the lowest affinity at hD3 receptors (pKi: 6.73). The D2-like receptor affinity pattern of LE404 is thus similar to clozapine (D4.4 ≥ D2L > 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 hD2L where both compounds are ~ equipotent. All compounds except LE401 showed the highest affinities among all tested receptors at 5HT2A. The most potent compound at 5HT2A 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 Ki-D2/Ki-5HT2A selectivity ratios > 7.
Functional studies (cAMP, Ca2+ and [35S]-GTPγS binding) at hD1 and hD2L receptors
For functional studies, hD1 and hD2L 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 D2L receptors. The effects of LE compounds on [35S]-GTPγS binding were determined in the presence of agonist. At hD1 receptors the full agonist dihydrexidine  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 hD2L 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).
Table 4. EC50 and IC50 values of reference compounds at D1 and D2L receptors in functional studies.
Figure 4. hD1 receptor stimulation of [35S]-GTPγS-binding by dihydrexidine (●) and SKF38393 (○). Both agonists were used in HEK293 cell membranes recombinantly expressing hD1 receptors in the presence of 1 μM GDP. Data shown are means ± SEM of at least three experiments.
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 D2L 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 D2L receptors, LE404 and LE410, are displayed in figure 5. Apparent functional pKi values (pKi app) derived from inhibition experiments of all compounds in cAMP, Ca2+, and [35S]-GTPγS studies are presented in table 5. When comparing pKi values of one compound from cAMP, Ca2+, and [35S]-GTPγS studies, differences may occur (e.g., clozapine at D1 receptors: pKi(cAMP): 6.46; pKi ([35S]-GTPγS): 7.47) but also good accordance was observed (e.g., LE404 at D1 receptors: pKi values between 7.95 and 8.20). The rank orders of potency of the tested compounds at D1 and D2L 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 D2L 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 D2L receptors based on binding (table 2). This D1 preference was lost in cAMP and [35S]-GTPγS experiments (LE404 is ~ equipotent at D1 and D2L) but a certain D1 preference (3-fold) was retained in Ca2+ studies (table 5). Haloperidol showing a 100-fold D2L over D1 selectivity in binding (table 2) retained this 100-fold D2L selectivity in [35S]-GTPγS experiments but showed an increased D2L selectivity in cAMP and Ca2+ studies (> 1000-fold). LE410 which displayed an only moderate D1 selectivity in binding (~2-fold, table 2) became D2L 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 D2L compared to D1 receptors (tables 2 and 5).
Table 5. Inhibitory potencies of the LE compounds on agonist-induced effects on [cAMP]i, [Ca2+]i, and [35S]-GTPγS binding
Figure 5. Functional characterisation of LE404 (■) and LE410 (●) at hD1 (A, B, C) and hD2L receptors (D, E, F). A Inhibition by LE404 and LE410 of 100 nM SKF38393-stimulated accumulation of intracellular [cAMP]. Data shown are means ± SEM of at least four determinations assayed in triplicate. B Inhibition by LE404 and LE410 of 100 nM SKF38393-stimulated increase in intracellular [Ca2+]. Data shown are means ± SEM of at least four determinations assayed in triplicate. C Inhibition by LE404 and LE410 of G-protein activation obtained by 1 μM dihydrexidine-stimulation. Data shown are means ± SEM of two independent experiments assayed in duplicate. D Inhibition by LE404 and LE410 of 100 nM quinpirole-stimulated decrease of intracellular [cAMP] in the presence of 10 μM forskolin. Data shown are means ± SEM of at least four determinations assayed in triplicate. E Inhibition by LE404 and LE410 of 30 nM quinpirole-stimulated increase in intracellular [Ca2+]. Data shown are means ± SEM of at least four determinations assayed in triplicate. F Inhibition by LE404 and LE410 of G-protein activation obtained by 10 μM quinpirole-stimulation. Data shown are means of two independent experiments assayed in duplicate.
Statistical comparison of functional and binding data at D1 and D2L receptors
The multiple intercorrelation and thus the equality of the results obtained by binding and the three functional assays at D1 and D2L 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 pKi 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 D2L receptors, the first extracted PC explained 97.5% of the total variance among the four pKi 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 , the PCA results indicate a significant multiple correlation among the four variables for D1 and D2L receptors, respectively.
Table 6. Factor loadings of the four variables used in principal component analysis
Nature of antagonism of LE compounds at D1 and D2 receptors
Next, the nature of antagonism of LE compounds at D1 and D2L receptors was tested by Clark analysis . Since LE404 was the most potent compound at D1 and LE410 the most potent at D2L 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 hD2L 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 . 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 pKb values. Results for LE404 at hD1 and hD2L 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] + Kb) which yielded straight lines at both receptor subtypes. pKb values were calculated as: hD1: pKb LE404 = 8.09 ± 0.15; pKb LE410 = 7.69 ± 0.13; hD2L: pKb LE404 = 7.61 ± 0.10; pKb LE410 = 8.05 ± 0.11. pKb values of LE404 and LE410 derived from non-linear Clark analysis show no significant difference to those derived from Schild analysis  (data not shown). Both functional analyses (Schild, Clark) give thus evidence for a competitive antagonistic behaviour of LE404 and LE410 at D1 and D2L receptors.
Figure 6. Functional analysis of the antagonist effect of LE404 at hD1 and hD2L receptors. The analysis was carried out by measuring the attenuation by LE404 of the agonist-induced increase in intracellular [Ca2+] in HEK293 cells recombinantly expressing hD1 and hD2L receptors, respectively. Dashed lines show 95% confidence intervals. SKF38393 was used as agonist at hD1 receptors, quinpirole at hD2L. All slopes were not significantly different from unity. Presented data are means ± SEM from at least three independent experiments each with at least threereplicates. A, B. Clark analysis of LE404 at hD1 (A) and hD2L receptors (B). Inserts show Clark plots.
Statistical analysis of binding affinities and selectivities at dopamine and 5HT2A receptors
In order to perform a statistically valid test for the discovery of ligands with differing affinity profiles at dopamine D1-D5 and 5HT2A receptors among the examined compounds, multiple intercorrelations of binding affinity values (pKi, table 2) as well as binding selectivity values [log (Ki ratio) = log (Ki Receptor 1/Ki 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) [22,23]. LE401 was excluded from both PCA's because precise pKi values were missing at hD4.4 and h5HT2A 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 5HT2A receptors). The PCA resulted in two PC's from which the first extracted 80.5% of the total variance among the eight pKi 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/5HT2A 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 (Ki ratio) values [= log (Ki Receptor 1/Ki Receptor 2)] were calculated for all possible 15 receptor affinity ratios (D1/D2L, D1/D3, D1/D4.4, D1/D5, D1/5HT2A, D2L/D3, D2L/D4.4, D2L/D5, D2L/5HT2A, D3/D4.4, D3/D5, D3/5HT2A, D4.4/D5, D4.4/5HT2A, D5/5HT2A) using the data from table 2. The resulting log (Ki 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 (Ki ratio) values), and the second PC extracted 15.4% of the total variance. The second ("selectivity") PCA discovered the same three subgroups of dopamine/5HT2A 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 (pKi) or selectivity information (log (Ki ratio)) for PCA, the same three subgroups of dopamine/5HT2A ligands were discriminated. The agreeing results from both PCA's underline that the statistical analysis of binding affinities and selectivities at dopamine and 5HT2A receptors did not create chance correlations.
Table 7. PCA results of affinity and selectivity data at dopamine and 5HT2A receptors
3D-QSAR (CoMFA/CoMSIA studies)
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-pKi 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) . 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.
Figure 7. 3D model of (-)-2b-SCH39166 with H-donor/acceptor and aromatic features. This model was used as pharmacophore-template for the LE compounds.
Figure 8. Alignment of the final 3D-QSAR models of the LE compounds.
Figure 9. Validation of the final alignment models using the random groups PLS method ("leave-many-out"). A. CoMFA field. B. CoMSIA field.
Table 8. Crossvalidation results for the final alignment models of the LE compounds
Among a group of new azecine compounds, this study has revealed two dibenzacezines (LE404 and LE410) with potent activity at dopamine and 5HT2A receptors displaying a novel receptor profile at D1-D5 and 5HT2A receptors. Compounds were evaluated in binding studies at D1-D5 and 5HT2A receptors and functionally (cAMP, Ca2+, [35S]-GTPγS) at D1 and D2L receptors, representative for the two subgroups of Gs (D1-like) and Gi (D2-like) coupled dopamine receptors. PCA revealed the equivalence of functional and binding pKi values (table 6) even though binding, cAMP, Ca2+, and [35S]-GTPγS assays differ strongly in the applied conditions (equilibrium: binding, cAMP, [35S]-GTPγS; non-equilibrium: Ca2+) and used endpoints (competition binding, G protein activation, second [cAMP] and "third" [Ca2+] messenger generation). A comparison of pKi values of one compound in the four different assays thus leads to differences, e.g., Ki ratios of haloperidol at D1/D2L receptors are ~1200 in cAMP, ~2500 in Ca2+, and ~100 in [35S]-GTPγS and binding studies but the rank order of potency remains almost unchanged (tables 2 and 5). Mottola et al.  have introduced the term "functional selectivity" to propose that depending on the experimental (buffer, equilibrium) and cellular conditions regarding receptor and G protein expression, a mixture of agonist/partial agonist and/or antagonist actions are likely. The ~2-fold difference in D1 and D2L receptor expression in this study (table 1) may thus contribute to differences in pKi values observed in functional and binding studies. The same reasons may serve as an explanation for differences in the Kd values of SCH23390 and spiperone in this study and in the literature (table 1) and for the ~1.4–5.5-fold differences in the affinity of LE300 in this and a previous study . Further, affinities in this study were tested at recombinantly expressed receptors in HEK293 cell membranes in Krebs-HEPES-buffer whereas the previous study used CHO cell membranes in a Tris-Mg2+-buffer . As was shown in figure 2, different buffers can result in significantly different affinity of a ligand.
LE404 and LE410 are competitive antagonists as was shown by Clark analysis (figure 6). pKb values of LE404 and LE410 derived from these functional analyses are in accordance with pKi values derived from inhibition curves (tables 5 and 2). Statistical analysis (PCA) of binding affinity data (pKi values, table 2) and binding selectivity data [log (Ki ratio) values, calculated from table 2] resulted in three groups of ligands: first: haloperidol; second: clozapine, LE300, LE400, LE410, and LE420; and – interestingly – a third group: containing LE403 and LE404 (table 7). The most potent compounds in group 2 and group 3 are LE410 and LE404. LE410 has a similar affinity profile as clozapine except the lower potency of LE410 at the hD4.4 receptor (table 2). In contrast, LE404 has a 25-fold selectivity for D1 over D2L receptors and thus a novel dopamine/5HT2A receptor profile. Interestingly, if instead of all Ki ratio values which have been used for the PCA in table 7 only the D1/D2L and D2L/5HT2A ratios of all compounds were used for clustering, the same three groups were found: 1) haloperidol, 2) clozapine, LE400, LE410, LE420, LE300, and 3) LE403 and LE404 (table 9). Thus, instead of six receptors and 15 Ki ratios, a reduction to three receptors (D1, D2L, 5HT2A) and two Ki ratios is sufficient to obtain the same clustering of compounds.
Table 9. Ki ratio values (Ki-D1/Ki-D2 and Ki-D2/Ki-5HT2A) of all test compounds except LE401
Meltzer et al. suggested the use of D1/D2L and D2L/5HT2A ratios to allow a clustering of antipsychotics into typical and atypical compounds [5-7]. However, instead of Meltzer et al. who calculated pKi ratio values which are imprecise in defining selectivity (same selectivity may result in different pKi ratios depending on the potency), Ki ratios (table 9) or log (Ki ratio) values (for PCA in table 7) were calculated in this study. Ki ratios recalculated from data of Meltzer et al.  and Ki ratios from this study were no more different than 3-fold (table 9). LE300, LE403, LE404, LE410, and LE420 achieved Ki-D2/Ki-5HT2A selectivity ratios > 7 which may suggest an atypical behaviour of these compounds according to Meltzer et al. . However, so far there are no in vivo behavioural studies underlying an antipsychotic effect of the LE compounds. The third group of ligands, LE403 and LE404, differ from LE410 by a 15-20-fold increase in D1 selectivity (table 9). RMI-81582 has very similar D1/D2 and D2/5HT2A Ki ratios as LE403 and LE404 (table 9) and was shown to exert antipsychotic effects . A further increase in D1 selectivity over D2, e.g., compound SCH23390 (table 9), results in a complete loss of antipsychotic activity [5,9,10]. Therefore, LE403 and LE404 might display an antipsychotic effect which however needs to be proven in in vivo studies. Only in vivo studies take into account the complexity of neuropsychiatric diseases including expression, distribution, and regulation of multiple receptors as well as adaptive processes.
This study confirmed recent findings that an increase in the size of the residue of the azecine nitrogen is detrimental to the affinity at dopamine/5HT2A receptors (table 2) . Hydroxylated versus non-hydroxylated dibenzacezines differ in their affinity and selectivity profiles (LE410, LE404, table 2) and define 2 separate groups. Monohydroxylation (LE404) results in higher potency than bis-hydroxylated compounds (LE403). Abolishing the H-donor properties by exchanging hydroxyl by methoxy groups was detrimental to the potency (LE400 versus LE403). Binding data of all compounds in figure 1 have been used to establish a valid 3D-QSAR pharmacophore model for D1 receptors (figure 8). The resulting model shows excellent q2 values for crossvalidation results and random groups PLS tests for both, CoMFA and CoMSIA (figure 9) excluding a chance correlation. The pharmacophore model is thus a solid basis for further improvement of dopamine receptor ligands.
In conclusion, this study has revealed two compounds, the dibenzacezines LE410 and LE404 with a novel dopamine/5HT2A receptor profile. LE404 and LE410 differ in their D1/D2L selectivity. LE410 clusters in one group with the atypical antipsychotic clozapine but has a different D2-like receptor profile (hD2L > hD3 > hD4.4) than clozapine (hD4.4 > hD2L > hD3). LE404 clusters in a separate group from clozapine/LE410 and from haloperidol and shows increased D1 selectivity similar to the experimental compound RMI-81582 which displayed antipsychotic activity . An antipsychotic activity of LE404 and LE410 in in vivo studies still needs to be shown. Further, a validated 3D-QSAR pharmacophore model for D1 antagonists is presented.
LE300, 400, 401, 403, 404, 410, and 420 were synthesized according to methods previously published [11,13]. [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)  and a pcDNA3.1+ construct containing cDNA coding for the h5HT2A receptor was obtained from the UMR cDNA resource center . All other reagents were supplied by Sigma Chemicals unless otherwise stated.
HEK293 cells stably expressing hD1, hD2L, or hD5 dopamine receptors were established as previously described [11,29]. Stable cell lines of HEK293 cells (ATCC, Rockville, MD, USA) were generated by transfecting the plasmids coding for hD3 and h5HT2A 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 D4.4 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  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  with gamma immunoglobulin as standard.
Radioligand binding experiments
The equilibrium dissociation constants Kd of the radioligands used ([3H]SCH23390 for hD1-like, [3H]spiperone for hD2L-like and h5HT2A receptors) were determined in homologous competition binding experiments and receptor densities of the respective dopamine receptor cell membrane preparations (Bmax values) were calculated using the DeBlasi equation . 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 5HT2A receptors) as described previously . 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 5HT2A 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 hD2L-like receptors and 1 μM ketanserin for h5HT2A receptors.
Estimation of [35S]-GTPγS-binding in HEK293 membranes
Cell membranes (for hD1: total protein amount ~16 μg, ~1.5 pmol receptor/mg protein; for hD2L: 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 hD2L) 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+] in HEK293 cells
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) . 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; hD2L: 30 nM quinpirole).
Measurement of changes in intracellular [cAMP] in HEK293 cells
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 hD2L 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 hD2L, 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; hD2L: 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 antagonism
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 hD2L receptors. At least four antagonist concentrations were used. Functional data were used for nonlinear regression analysis according to Clark . 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), pKb is the negative decadic logarithm of the antagonist dissociation constant, n the Hill coefficient, and log c the difference between the pKb 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 Ki from radioligand binding competition experiments were calculated from IC50 values using the Cheng-Prusoff equation . Apparent functional Ki values were calculated according to the following equation adapted from Cheng and Prusoff :
Ki = 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 hD2L 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).
3D-QSAR (CoMFA/CoMSIA) studies
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.
CHO, Chinese hamster ovary; CoMFA, comparative molecular field analysis; CoMSIA, comparative molecular similarity indices analysis; HEK, human embryonic kidney; PC, principal component; PCA, principal component analysis; RMI 81582, 2-chloro-11-(3-dimethylaminopropylidene)morphanthridine; SCH23390, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; (-)-2b-SCH39166, (-)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo [d]naptho-(2,1-b)-azepine; SKF38393; (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol.
AH established recombinant cell lines, carried out functional measurements and radioligand binding studies, and performed data evaluation. Further, AH drafted the manuscript. MWeigt carried out the 3D-QSAR studies. MWiese provided intellectual input and critical interpretation of the data. BH carried out calcium measurements. JL provided the LE compounds. MUK carried out principal component analyses and finalized the manuscript for publication. All authors read and approved the final manuscript.
The authors wish to thank Dr. Carsten Tober, Reutlingen, for performing [35S]-GTPγS-binding. Source of funding for all authors were their respective budgets from the Universities. There was no influence/role of the University on this study, the writing of the manuscript, or decisions to submit the manuscript for publication.
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