BACKGROUND
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  and can be divided into two subfamilies, d1-like  and d2-like  receptors, according to their sequence homologies, biochemical properties, and pharmacologic profiles  <cit> .
d1-like receptor stimulation activates adenylyl cyclase  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  <cit> .
dopamine receptors are clinically important drug targets for the treatment of disorders such as parkinson's disease and schizophrenia  <cit> .
blockade of dopamine d <dig> receptors is the main feature of antipsychotic action.
typical antipsychotics like the first generation d <dig> receptor antagonists haloperidol or chlorpromazine can cause therapy-limiting extrapyramidal-motor side effects .
second generation  antipsychotics are serotonin/dopamine antagonists with no or low eps at doses showing antipsychotic activity and have significantly greater affinity for 5ht2a than for d <dig> receptors  <cit> .
this serotonin-dopamine ratio could contribute to atypicality  <cit>  but further investigations are needed to define the precise mechanism of atypical antipsychotics.
however, antipsychotic activity is not only the result of d <dig> and 5ht2a receptor blockade but an inhibitory/modulating effect on various dopamine and serotonin  and further receptors  <cit> .
within the heterogeneous group of atypical antipsychotics, only clozapine exhibits effects against treatment-resistant schizophrenia  <cit> .
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 d <dig> antagonism alone has not turned out as an effective antipsychotic principle  <cit> .
le <dig>  an indolobenzacezine  has previously been characterized  <cit>  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 d <dig>  d2l, and d <dig> receptors by a previously published functional calcium assay  <cit> .
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 le <dig>  at all human dopamine and 5ht2a receptors, to test whether data from the calcium assay initially used for screening of le300-derived compounds  <cit>  correlate with other assays measuring functional activation of gpcrs , 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  and  measurements and -gtpγs-binding.
indeed, dibenzazecine compounds with a previously not available receptor profile  were found.
3d-qsar studies were performed resulting in qsar models allowing further rational ligand design at a molecular level.
RESULTS
receptor expression and characterization
homologous radioligand competition binding experiments were performed to determine the receptor expression levels  and binding affinities  of the used radioligands.
average bmax and kd values for each receptor are shown in table  <dig>  all kd values except for 5ht2a receptors were 3-6-fold higher than those found in the literature .
this effect could be attributed to the use of isotonic krebs-hepes-buffer ph  <dig>  in this study instead of the widely used tris-hcl buffer ph  <dig>  in the literature.
figure  <dig> shows as an example the buffer-dependent inhibition by le <dig> of sch <dig> binding to d <dig> receptor membranes.
using krebs-hepes instead of tris-hcl buffer yielded ~4-fold higher ki values of le <dig>  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 .
a) data taken from  <cit>
sch <dig> was used for d1-like, spiperone for d2-like and 5ht2a receptors in homologous competition experiments.
data are mean ± sem, n ≥  <dig>
radioligand binding studies
binding affinities of the compounds le <dig>  le <dig>  le <dig>  le <dig>  le <dig>  le <dig>  and le <dig>  were estimated at recombinant dopamine and 5ht2a receptors in cell membrane preparations.
further compounds used for 3d-qsar analysis of d <dig> receptor ligands  were tested at d <dig> receptors only due to limited availability.
for the sake of comparison, haloperidol as a classical antipsychotic, clozapine as an atypical antipsychotic, and le <dig> were included as reference compounds.
figure  <dig> shows the radioligand displacement curves of the most potent hd <dig> and hd2l ligands le <dig> and le <dig> at hd <dig> , hd2l , and 5ht2a  receptors.
pki values are displayed in table  <dig>  and table  <dig> .
a) displacement of radioligand was < 30% at  <dig> μm
haloperidol, clozapine, and le compounds were characterized at dopamine and h5ht2a receptors.
sch <dig> was used for hd1-like and spiperone for hd2-like and h5ht2a receptors.
displayed are pki values ± sem, n ≥  <dig>
displayed are pki values ± sem, n ≥  <dig>
all compounds showed similar affinities at hd <dig> and hd <dig> receptors.
the mono-hydroxylated le <dig> turned out as the most potent compound at hd1/hd <dig> receptors with pki values of  <dig>  and  <dig> , respectively, followed by the bis-hydroxylated le <dig> which is 3-10-fold less potent than le <dig>  replacement of the hydroxy- by methoxy-substituents resulting in le <dig> dramatically decreased the affinity at all tested receptors.
an increase of the size of the nitrogen substituent  further decreased the affinity at all tested receptors.
except le <dig> and le <dig>  all other compounds possessed up to 33fold  higher affinities for d1-like than for d2-like receptors .
among d2-like receptors, all compounds – except le <dig> – showed the highest affinity at hd2l and lower affinities at hd <dig> and hd <dig>  receptors similar to the profile of haloperidol at d2-like receptors.
however, different to haloperidol which shows a strong d <dig> over d <dig> selectivity, le compounds  show selectivity for d <dig> over d <dig>  removal of the hydroxy-group of le <dig> yielding le <dig> resulted in a dramatic loss of d <dig> over d <dig> selectivity, and left le <dig> as the most potent compound at hd2l and hd <dig> receptors with pki values of  <dig>  and  <dig> , respectively.
bioisosteric replacement of one benzene residue in le <dig> by thiophene gave le <dig> showing a similar receptor profile as le <dig> but with reduced affinity at all tested receptors.
le <dig> displayed a receptor profile within the d2-like receptors which is unique among the tested le compounds.
within the d2-like receptors, le <dig> reached the highest affinity at hd <dig>  , a slightly lower affinity at hd2l , and the lowest affinity at hd <dig> receptors .
the d2-like receptor affinity pattern of le <dig> is thus similar to clozapine .
in contrast to clozapine which appeared ~ equipotent at d1/d <dig> receptors in all of our test systems, le <dig> shows 25fold selectivity for d <dig> over d <dig>  le <dig> displayed higher affinities than le <dig> at all dopamine receptors except hd2l where both compounds are ~ equipotent.
all compounds except le <dig> showed the highest affinities among all tested receptors at 5ht2a.
the most potent compound at 5ht2a was le <dig> with an affinity in the subnanomolar range followed by le <dig> in the low nanomolar range.
le <dig>  le <dig>  le <dig>  le <dig>  le <dig>  and le <dig> achieved ki-d2/ki-5ht2a selectivity ratios >  <dig>
functional studies  at hd <dig> and hd2l receptors
for functional studies, hd <dig> 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  and  in intact hek <dig> cells, and -gtpγs binding in hek <dig> cell membranes were estimated.
table  <dig> shows ec <dig> and ic <dig> values of standard ligands at d <dig> and d2l receptors.
the effects of le compounds on -gtpγs binding were determined in the presence of agonist.
at hd <dig> receptors the full agonist dihydrexidine  <cit>  was used for -gtpγs binding experiments instead of skf <dig> which was used in camp and ca2+ studies.
in membrane preparations from hek293-hd <dig> cells, dihydrexidine showed a significantly higher increase in -gtpγs binding than skf <dig> .
dihydrexidine gave an ec <dig> of  <dig>  ±  <dig>  nm .
a difference between dihydrexidine and skf <dig> was not observed in intracellular  and  measurements , and thus skf <dig> was used in ca2+ and camp studies.
the ec <dig> of quinpirole at hd2l receptors was estimated as  <dig> ±  <dig>  nm .
all le compounds except le <dig> showed an inhibition of -gtpγs binding between  <dig> and 40% .
data shown are ec50/ic <dig> values in nm ± sem, n ≥  <dig>
none of the tested compounds  showed any agonist effect in functional studies .
all test compounds inhibited agonist-stimulated effects on intracellular  and  and on -gtpγs binding at d <dig> or d2l receptors, respectively, in a concentration-dependent manner.
le <dig> in ca2+ studies and le <dig> in all functional assays achieved ≤ 50% inhibitory activity at  <dig> μm.
concentration-inhibition curves of the most potent novel compounds at d <dig> and d2l receptors, le <dig> and le <dig>  are displayed in figure  <dig>  apparent functional pki values  derived from inhibition experiments of all compounds in camp, ca2+, and -gtpγs studies are presented in table  <dig>  when comparing pki values of one compound from camp, ca2+, and -gtpγs studies, differences may occur :  <dig> ; pki :  <dig> ) but also good accordance was observed .
the rank orders of potency of the tested compounds at d <dig> and d2l receptors, respectively, remained similar for the three functional assays: the most potent compound at d <dig> receptors in all three functional assays  and in binding  is le <dig> whereas the weakest compounds are le <dig> and le <dig>  at d2l receptors, le <dig>  le <dig>  and le <dig> are the most potent compounds after haloperidol whereas again, le <dig> and le <dig> are the weakest .
le <dig> has a 25-fold selectivity for d <dig> over d2l receptors based on binding .
this d <dig> preference was lost in camp and -gtpγs experiments  but a certain d <dig> preference  was retained in ca2+ studies .
haloperidol showing a 100-fold d2l over d <dig> selectivity in binding  retained this 100-fold d2l selectivity in -gtpγs experiments but showed an increased d2l selectivity in camp and ca2+ studies .
le <dig> which displayed an only moderate d <dig> selectivity in binding  became d2l selective in camp and ca2+ studies but was ~ equipotent in -gtpγs binding.
these results show that camp and ca2+ studies uprate the potency of compounds at d2l compared to d <dig> receptors .
a) inhibitory activity was ≤ 50% at  <dig> μm.
concentration-effect curves were obtained with hd <dig> and hd2l receptors.
data shown are apparent pki values  ± sem, n ≥  <dig>
statistical comparison of functional and binding data at d <dig> and d2l receptors
the multiple intercorrelation and thus the equality of the results obtained by binding and the three functional assays at d <dig> and d2l receptors, respectively, was determined by principal component analysis .
results of the pca comparing the four test systems  are displayed in table  <dig>  the first extracted principal component  for d <dig> receptors described  <dig> % of the total variance among the four pki variables  with factor loadings >  <dig>   leaving an eigenvalue of only  <dig>  for the second pc.
for d2l receptors, the first extracted pc explained  <dig> % of the total variance among the four pki variables  leaving an eigenvalue of only  <dig>  for the second pc.
following the idea that a pc with an eigenvalue of <<  <dig> has no legitimacy for the description of the total variance  <cit> , the pca results indicate a significant multiple correlation among the four variables for d <dig> and d2l receptors, respectively.
nature of antagonism of le compounds at d <dig> and d <dig> receptors
next, the nature of antagonism of le compounds at d <dig> and d2l receptors was tested by clark analysis  <cit> .
since le <dig> was the most potent compound at d <dig> and le <dig> the most potent at d2l receptors , le <dig> and le <dig> were chosen as representatives to undergo functional analysis for competitive antagonism.
in the presence of increasing concentrations of le <dig> and le <dig>  parallel rightward shifts of the agonist concentration-effect curves in the ca2+ assay were observed without loss of maximum efficacy at hd <dig> and hd2l receptors .
the rightward shift of the concentration-effect curves of the agonist was analyzed with non-linear regression analysis according to lew and angus  <cit> .
data were fitted to equations  and  .
an f-test showed no significant difference , thus equation  with a hill slope of  <dig> was the preferred model and used to obtain pkb values.
results for le <dig> at hd <dig> and hd2l receptors are presented in figures 6a and 6b.
inserts show the clark plots  which yielded straight lines at both receptor subtypes.
pkb values were calculated as: hd1: pkb le <dig> =  <dig>  ±  <dig> ; pkb le <dig> =  <dig>  ±  <dig> ; hd2l: pkb le <dig> =  <dig>  ±  <dig> ; pkb le <dig> =  <dig>  ±  <dig> .
pkb values of le <dig> and le <dig> derived from non-linear clark analysis show no significant difference to those derived from schild analysis  <cit>  .
both functional analyses  give thus evidence for a competitive antagonistic behaviour of le <dig> and le <dig> at d <dig> and d2l receptors.
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-d <dig> and 5ht2a receptors among the examined compounds, multiple intercorrelations of binding affinity values  as well as binding selectivity values  were investigated in two separate pca's.
pca has already successfully been applied to define similar and deviating responses among biological data   <cit> .
le <dig> was excluded from both pca's because precise pki values were missing at hd <dig>  and h5ht2a receptors .
in the first pca, eight compounds were examined  for their affinity in six test systems .
the pca resulted in two pc's from which the first extracted  <dig> % of the total variance among the eight pki variables, and the second extracted  <dig> %.
the factor loadings of the eight variables  are listed in table  <dig> and show that the eight compounds define three subgroups of dopamine/5ht2a ligands: 1) clozapine, le <dig>  le <dig>  le <dig>  and le <dig> with factor loadings contributing to the first pc of >  <dig> ; 2) haloperidol in the second pc with a factor loading of - <dig> ; 3) le <dig> and le <dig> in the second pc with opposite direction to haloperidol  indicating that le <dig> and le <dig> display an affinity profile opposite to that of haloperidol.
for the second pca, for each of the eight compounds, log  values  were calculated for all possible  <dig> receptor affinity ratios  using the data from table  <dig>  the resulting log  data matrix contains selectivity information for each of the compounds.
results of this second  pca were basically identical to results from the first  pca .
the first extracted pc explained  <dig> % of the total variance among the eight variables  values), and the second pc extracted  <dig> % of the total variance.
the second  pca discovered the same three subgroups of dopamine/5ht2a ligands as did the first pca: 1) clozapine, le <dig>  le <dig>  le <dig>  and le <dig> with factor loadings contributing to the first pc of >  <dig>  ; 2) haloperidol in the second pc with a factor loading of - <dig> ; 3) le <dig> and le <dig> in the second pc with opposite direction to haloperidol .
thus, regardless of using affinity information  or selectivity information ) 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.
the first pca is using pki values from table  <dig> , the second log  values .
log  values  were calculated for all possible  <dig> receptor affinity ratios using data from table  <dig>  displayed are factor loadings for the first two pc's after varimax rotation.
3d-qsar
since the main feature of the le compounds is their d <dig> selectivity, a 3d-qsar pharmacophore model for the d <dig> receptor was establish using the  <dig> compounds shown in figure  <dig> and their d1-pki values from table  <dig> and  <dig>  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/d <dig> selective antagonist -2b-sch <dig>  was taken as a pharmacophore template.
-2b-sch <dig> is a benzonaphthazepine, a rigid analogue of sch <dig>  thus limiting the number of possible conformations   <cit> .
unfortunately, -2b-sch <dig> 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  <dig> compounds from figure  <dig>  essential pharmacophore features of -2b-sch <dig> are the two aromatic rings and the basic nitrogen  while the hydroxyl group served as an optional h-donor/acceptor feature .
results of the alignment of the final models of the le compounds are shown in figure  <dig>  the aromatic residues and basic nitrogen atoms remain the main pharmacophore features.
crossvalidation results  for the final models for comfa and comsia both using steric and electrostatic fields are displayed in table  <dig>  and show crossvalidation parameters q <dig> of  <dig>  for comfa and  <dig>  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 .
the test showed a high stability of the models presented in figure  <dig> with a mean q <dig> of  <dig>   for the combined steric and electrostatic field in comfa and a mean q <dig> of  <dig>   in comsia.
the distribution of the q <dig> values for this validation is shown in figure  <dig>
* sdep: standard error of prediction
the models are displayed in figure  <dig> using steric and electrostatic fields for both comfa and comsia.
discussion
among a group of new azecine compounds, this study has revealed two dibenzacezines  with potent activity at dopamine and 5ht2a receptors displaying a novel receptor profile at d1-d <dig> and 5ht2a receptors.
compounds were evaluated in binding studies at d1-d <dig> and 5ht2a receptors and functionally  at d <dig> and d2l receptors, representative for the two subgroups of gs  and gi  coupled dopamine receptors.
pca revealed the equivalence of functional and binding pki values  even though binding, camp, ca2+, and -gtpγs assays differ strongly in the applied conditions  and used endpoints .
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 ~ <dig> in camp, ~ <dig> in ca2+, and ~ <dig> in -gtpγs and binding studies but the rank order of potency remains almost unchanged .
mottola et al.
<cit>  have introduced the term "functional selectivity" to propose that depending on the experimental  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 d <dig> and d2l receptor expression in this study  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 sch <dig> and spiperone in this study and in the literature  and for the ~ <dig> – <dig> -fold differences in the affinity of le <dig> in this and a previous study  <cit> .
further, affinities in this study were tested at recombinantly expressed receptors in hek <dig> cell membranes in krebs-hepes-buffer whereas the previous study used cho cell membranes in a tris-mg2+-buffer  <cit> .
as was shown in figure  <dig>  different buffers can result in significantly different affinity of a ligand.
le <dig> and le <dig> are competitive antagonists as was shown by clark analysis .
pkb values of le <dig> and le <dig> derived from these functional analyses are in accordance with pki values derived from inhibition curves .
statistical analysis  of binding affinity data  and binding selectivity data  resulted in three groups of ligands: first: haloperidol; second: clozapine, le <dig>  le <dig>  le <dig>  and le420; and – interestingly – a third group: containing le <dig> and le <dig> .
the most potent compounds in group  <dig> and group  <dig> are le <dig> and le <dig>  le <dig> has a similar affinity profile as clozapine except the lower potency of le <dig> at the hd <dig>  receptor .
in contrast, le <dig> has a 25-fold selectivity for d <dig> 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  <dig> 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, le <dig>  le <dig>  le <dig>  le <dig>  and 3) le <dig> and le <dig> .
thus, instead of six receptors and  <dig> ki ratios, a reduction to three receptors  and two ki ratios is sufficient to obtain the same clustering of compounds.
a) value is calculated from  <cit> .
b) value is calculated from  <cit> .
c) value is calculated from  <cit> .
further, data for sch <dig> and rmi- <dig> are calculated from literature data.
ki values used are derived from radioligand binding studies.
meltzer et al.
suggested the use of d1/d2l and d2l/5ht2a ratios to allow a clustering of antipsychotics into typical and atypical compounds  <cit> .
however, instead of meltzer et al.
who calculated pki ratio values which are imprecise in defining selectivity , ki ratios  or log  values  were calculated in this study.
ki ratios recalculated from data of meltzer et al.
<cit>  and ki ratios from this study were no more different than 3-fold .
le <dig>  le <dig>  le <dig>  le <dig>  and le <dig> achieved ki-d2/ki-5ht2a selectivity ratios >  <dig> which may suggest an atypical behaviour of these compounds according to meltzer et al.
<cit> .
however, so far there are no in vivo behavioural studies underlying an antipsychotic effect of the le compounds.
the third group of ligands, le <dig> and le <dig>  differ from le <dig> by a 15-20-fold increase in d <dig> selectivity .
rmi- <dig> has very similar d1/d <dig> and d2/5ht2a ki ratios as le <dig> and le <dig>  and was shown to exert antipsychotic effects  <cit> .
a further increase in d <dig> selectivity over d <dig>  e.g., compound sch <dig> , results in a complete loss of antipsychotic activity  <cit> .
therefore, le <dig> and le <dig> 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   <cit> .
hydroxylated versus non-hydroxylated dibenzacezines differ in their affinity and selectivity profiles  and define  <dig> separate groups.
monohydroxylation  results in higher potency than bis-hydroxylated compounds .
abolishing the h-donor properties by exchanging hydroxyl by methoxy groups was detrimental to the potency .
binding data of all compounds in figure  <dig> have been used to establish a valid 3d-qsar pharmacophore model for d <dig> receptors .
the resulting model shows excellent q <dig> values for crossvalidation results and random groups pls tests for both, comfa and comsia  excluding a chance correlation.
the pharmacophore model is thus a solid basis for further improvement of dopamine receptor ligands.
CONCLUSIONS
in conclusion, this study has revealed two compounds, the dibenzacezines le <dig> and le <dig> with a novel dopamine/5ht2a receptor profile.
le <dig> and le <dig> differ in their d1/d2l selectivity.
le <dig> clusters in one group with the atypical antipsychotic clozapine but has a different d2-like receptor profile  than clozapine .
le <dig> clusters in a separate group from clozapine/le <dig> and from haloperidol and shows increased d <dig> selectivity similar to the experimental compound rmi- <dig> which displayed antipsychotic activity  <cit> .
an antipsychotic activity of le <dig> and le <dig> in in vivo studies still needs to be shown.
further, a validated 3d-qsar pharmacophore model for d <dig> antagonists is 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 and can be divided into two subfamilies, d1-like and d2-like receptors, according to their sequence homologies, biochemical properties, and pharmacologic profiles. d1-like receptor stimulation activates adenylyl cyclase 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 d receptors is the main feature of antipsychotic action. typical antipsychotics like the first generation d receptor antagonists haloperidol or chlorpromazine can cause therapy-limiting extrapyramidal-motor side effects. second generation antipsychotics are serotonin/dopamine antagonists with no or low eps at doses showing antipsychotic activity and have significantly greater affinity for 5ht2a than for d receptors. this serotonin-dopamine ratio could contribute to atypicality but further investigations are needed to define the precise mechanism of atypical antipsychotics. however, antipsychotic activity is not only the result of d and 5ht2a receptor blockade but an inhibitory/modulating effect on various dopamine and serotonin 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 d antagonism alone has not turned out as an effective antipsychotic principle. le an indolobenzacezine 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 d d2l, and d receptors by a previously published functional calcium assay. 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 le 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, 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 and measurements and -gtpγs-binding. indeed, dibenzazecine compounds with a previously not available receptor profile were found. 3d-qsar studies were performed resulting in qsar models allowing further rational ligand design at a molecular level.  receptor expression and characterization homologous radioligand competition binding experiments were performed to determine the receptor expression levels and binding affinities of the used radioligands. average bmax and kd values for each receptor are shown in table all kd values except for 5ht2a receptors were 3-6-fold higher than those found in the literature. this effect could be attributed to the use of isotonic krebs-hepes-buffer ph in this study instead of the widely used tris-hcl buffer ph in the literature. figure shows as an example the buffer-dependent inhibition by le of sch binding to d receptor membranes. using krebs-hepes instead of tris-hcl buffer yielded ~4-fold higher ki values of le 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. a) data taken from sch was used for d1-like, spiperone for d2-like and 5ht2a receptors in homologous competition experiments. data are mean ± sem, n ≥ radioligand binding studies binding affinities of the compounds le le le le le le and le were estimated at recombinant dopamine and 5ht2a receptors in cell membrane preparations. further compounds used for 3d-qsar analysis of d receptor ligands were tested at d receptors only due to limited availability. for the sake of comparison, haloperidol as a classical antipsychotic, clozapine as an atypical antipsychotic, and le were included as reference compounds. figure shows the radioligand displacement curves of the most potent hd and hd2l ligands le and le at hd, hd2l, and 5ht2a receptors. pki values are displayed in table and table. a) displacement of radioligand was < 30% at μm haloperidol, clozapine, and le compounds were characterized at dopamine and h5ht2a receptors. sch was used for hd1-like and spiperone for hd2-like and h5ht2a receptors. displayed are pki values ± sem, n ≥ displayed are pki values ± sem, n ≥ all compounds showed similar affinities at hd and hd receptors. the mono-hydroxylated le turned out as the most potent compound at hd1/hd receptors with pki values of and, respectively, followed by the bis-hydroxylated le which is 3-10-fold less potent than le replacement of the hydroxy- by methoxy-substituents resulting in le dramatically decreased the affinity at all tested receptors. an increase of the size of the nitrogen substituent further decreased the affinity at all tested receptors. except le and le all other compounds possessed up to 33fold higher affinities for d1-like than for d2-like receptors. among d2-like receptors, all compounds – except le – showed the highest affinity at hd2l and lower affinities at hd and hd receptors similar to the profile of haloperidol at d2-like receptors. however, different to haloperidol which shows a strong d over d selectivity, le compounds show selectivity for d over d removal of the hydroxy-group of le yielding le resulted in a dramatic loss of d over d selectivity, and left le as the most potent compound at hd2l and hd receptors with pki values of and, respectively. bioisosteric replacement of one benzene residue in le by thiophene gave le showing a similar receptor profile as le but with reduced affinity at all tested receptors. le displayed a receptor profile within the d2-like receptors which is unique among the tested le compounds. within the d2-like receptors, le reached the highest affinity at hd, a slightly lower affinity at hd2l, and the lowest affinity at hd receptors. the d2-like receptor affinity pattern of le is thus similar to clozapine. in contrast to clozapine which appeared ~ equipotent at d1/d receptors in all of our test systems, le shows 25fold selectivity for d over d le displayed higher affinities than le at all dopamine receptors except hd2l where both compounds are ~ equipotent. all compounds except le showed the highest affinities among all tested receptors at 5ht2a. the most potent compound at 5ht2a was le with an affinity in the subnanomolar range followed by le in the low nanomolar range. le le le le le and le achieved ki-d2/ki-5ht2a selectivity ratios > functional studies at hd and hd2l receptors for functional studies, hd 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 and in intact hek cells, and -gtpγs binding in hek cell membranes were estimated. table shows ec and ic values of standard ligands at d and d2l receptors. the effects of le compounds on -gtpγs binding were determined in the presence of agonist. at hd receptors the full agonist dihydrexidine was used for -gtpγs binding experiments instead of skf which was used in camp and ca2+ studies. in membrane preparations from hek293-hd cells, dihydrexidine showed a significantly higher increase in -gtpγs binding than skf. dihydrexidine gave an ec of ± nm. a difference between dihydrexidine and skf was not observed in intracellular and measurements, and thus skf was used in ca2+ and camp studies. the ec of quinpirole at hd2l receptors was estimated as ± nm. all le compounds except le showed an inhibition of -gtpγs binding between and 40%. data shown are ec50/ic values in nm ± sem, n ≥ none of the tested compounds showed any agonist effect in functional studies. all test compounds inhibited agonist-stimulated effects on intracellular and and on -gtpγs binding at d or d2l receptors, respectively, in a concentration-dependent manner. le in ca2+ studies and le in all functional assays achieved ≤ 50% inhibitory activity at μm. concentration-inhibition curves of the most potent novel compounds at d and d2l receptors, le and le are displayed in figure apparent functional pki values derived from inhibition experiments of all compounds in camp, ca2+, and -gtpγs studies are presented in table when comparing pki values of one compound from camp, ca2+, and -gtpγs studies, differences may occur:; pki: ) but also good accordance was observed. the rank orders of potency of the tested compounds at d and d2l receptors, respectively, remained similar for the three functional assays: the most potent compound at d receptors in all three functional assays and in binding is le whereas the weakest compounds are le and le at d2l receptors, le le and le are the most potent compounds after haloperidol whereas again, le and le are the weakest. le has a 25-fold selectivity for d over d2l receptors based on binding. this d preference was lost in camp and -gtpγs experiments but a certain d preference was retained in ca2+ studies. haloperidol showing a 100-fold d2l over d selectivity in binding retained this 100-fold d2l selectivity in -gtpγs experiments but showed an increased d2l selectivity in camp and ca2+ studies. le which displayed an only moderate d selectivity in binding became d2l selective in camp and ca2+ studies but was ~ equipotent in -gtpγs binding. these results show that camp and ca2+ studies uprate the potency of compounds at d2l compared to d receptors. a) inhibitory activity was ≤ 50% at μm. concentration-effect curves were obtained with hd and hd2l receptors. data shown are apparent pki values ± sem, n ≥ statistical comparison of functional and binding data at d and d2l receptors the multiple intercorrelation and thus the equality of the results obtained by binding and the three functional assays at d and d2l receptors, respectively, was determined by principal component analysis. results of the pca comparing the four test systems are displayed in table the first extracted principal component for d receptors described % of the total variance among the four pki variables with factor loadings > leaving an eigenvalue of only for the second pc. for d2l receptors, the first extracted pc explained % of the total variance among the four pki variables leaving an eigenvalue of only for the second pc. following the idea that a pc with an eigenvalue of << has no legitimacy for the description of the total variance, the pca results indicate a significant multiple correlation among the four variables for d and d2l receptors, respectively. nature of antagonism of le compounds at d and d receptors next, the nature of antagonism of le compounds at d and d2l receptors was tested by clark analysis. since le was the most potent compound at d and le the most potent at d2l receptors, le and le were chosen as representatives to undergo functional analysis for competitive antagonism. in the presence of increasing concentrations of le and le parallel rightward shifts of the agonist concentration-effect curves in the ca2+ assay were observed without loss of maximum efficacy at hd and hd2l receptors. 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 and. an f-test showed no significant difference, thus equation with a hill slope of was the preferred model and used to obtain pkb values. results for le at hd and hd2l receptors are presented in figures 6a and 6b. inserts show the clark plots which yielded straight lines at both receptor subtypes. pkb values were calculated as: hd1: pkb le = ±; pkb le = ±; hd2l: pkb le = ±; pkb le = ±. pkb values of le and le derived from non-linear clark analysis show no significant difference to those derived from schild analysis. both functional analyses give thus evidence for a competitive antagonistic behaviour of le and le at d and d2l receptors. 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-d and 5ht2a receptors among the examined compounds, multiple intercorrelations of binding affinity values as well as binding selectivity values were investigated in two separate pca's. pca has already successfully been applied to define similar and deviating responses among biological data. le was excluded from both pca's because precise pki values were missing at hd and h5ht2a receptors. in the first pca, eight compounds were examined for their affinity in six test systems. the pca resulted in two pc's from which the first extracted % of the total variance among the eight pki variables, and the second extracted %. the factor loadings of the eight variables are listed in table and show that the eight compounds define three subgroups of dopamine/5ht2a ligands: 1) clozapine, le le le and le with factor loadings contributing to the first pc of >; 2) haloperidol in the second pc with a factor loading of -; 3) le and le in the second pc with opposite direction to haloperidol indicating that le and le display an affinity profile opposite to that of haloperidol. for the second pca, for each of the eight compounds, log values were calculated for all possible receptor affinity ratios using the data from table the resulting log data matrix contains selectivity information for each of the compounds. results of this second pca were basically identical to results from the first pca. the first extracted pc explained % of the total variance among the eight variables values), and the second pc extracted % of the total variance. the second pca discovered the same three subgroups of dopamine/5ht2a ligands as did the first pca: 1) clozapine, le le le and le with factor loadings contributing to the first pc of >; 2) haloperidol in the second pc with a factor loading of -; 3) le and le in the second pc with opposite direction to haloperidol. thus, regardless of using affinity information or selectivity information ) 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. the first pca is using pki values from table, the second log values. log values were calculated for all possible receptor affinity ratios using data from table displayed are factor loadings for the first two pc's after varimax rotation. 3d-qsar since the main feature of the le compounds is their d selectivity, a 3d-qsar pharmacophore model for the d receptor was establish using the compounds shown in figure and their d1-pki values from table and 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/d selective antagonist -2b-sch was taken as a pharmacophore template. -2b-sch is a benzonaphthazepine, a rigid analogue of sch thus limiting the number of possible conformations. unfortunately, -2b-sch 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 compounds from figure essential pharmacophore features of -2b-sch are the two aromatic rings and the basic nitrogen while the hydroxyl group served as an optional h-donor/acceptor feature. results of the alignment of the final models of the le compounds are shown in figure the aromatic residues and basic nitrogen atoms remain the main pharmacophore features. crossvalidation results for the final models for comfa and comsia both using steric and electrostatic fields are displayed in table and show crossvalidation parameters q of for comfa and 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. the test showed a high stability of the models presented in figure with a mean q of for the combined steric and electrostatic field in comfa and a mean q of in comsia. the distribution of the q values for this validation is shown in figure * sdep: standard error of prediction the models are displayed in figure using steric and electrostatic fields for both comfa and comsia. discussion among a group of new azecine compounds, this study has revealed two dibenzacezines with potent activity at dopamine and 5ht2a receptors displaying a novel receptor profile at d1-d and 5ht2a receptors. compounds were evaluated in binding studies at d1-d and 5ht2a receptors and functionally at d and d2l receptors, representative for the two subgroups of gs and gi coupled dopamine receptors. pca revealed the equivalence of functional and binding pki values even though binding, camp, ca2+, and -gtpγs assays differ strongly in the applied conditions and used endpoints. 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 ~ in camp, ~ in ca2+, and ~ in -gtpγs and binding studies but the rank order of potency remains almost unchanged. mottola et al. have introduced the term "functional selectivity" to propose that depending on the experimental 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 d and d2l receptor expression in this study 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 sch and spiperone in this study and in the literature and for the ~ – -fold differences in the affinity of le in this and a previous study. further, affinities in this study were tested at recombinantly expressed receptors in hek cell membranes in krebs-hepes-buffer whereas the previous study used cho cell membranes in a tris-mg2+-buffer. as was shown in figure different buffers can result in significantly different affinity of a ligand. le and le are competitive antagonists as was shown by clark analysis. pkb values of le and le derived from these functional analyses are in accordance with pki values derived from inhibition curves. statistical analysis of binding affinity data and binding selectivity data resulted in three groups of ligands: first: haloperidol; second: clozapine, le le le and le420; and – interestingly – a third group: containing le and le. the most potent compounds in group and group are le and le le has a similar affinity profile as clozapine except the lower potency of le at the hd receptor. in contrast, le has a 25-fold selectivity for d 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 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, le le le le and 3) le and le. thus, instead of six receptors and ki ratios, a reduction to three receptors and two ki ratios is sufficient to obtain the same clustering of compounds. a) value is calculated from. b) value is calculated from. c) value is calculated from. further, data for sch and rmi- are calculated from literature data. ki values used are derived from radioligand binding studies. meltzer et al. suggested the use of d1/d2l and d2l/5ht2a ratios to allow a clustering of antipsychotics into typical and atypical compounds. however, instead of meltzer et al. who calculated pki ratio values which are imprecise in defining selectivity, ki ratios or log values 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. le le le le and le achieved ki-d2/ki-5ht2a selectivity ratios > 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, le and le differ from le by a 15-20-fold increase in d selectivity. rmi- has very similar d1/d and d2/5ht2a ki ratios as le and le and was shown to exert antipsychotic effects. a further increase in d selectivity over d e.g., compound sch, results in a complete loss of antipsychotic activity. therefore, le and le 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. hydroxylated versus non-hydroxylated dibenzacezines differ in their affinity and selectivity profiles and define separate groups. monohydroxylation results in higher potency than bis-hydroxylated compounds. abolishing the h-donor properties by exchanging hydroxyl by methoxy groups was detrimental to the potency. binding data of all compounds in figure have been used to establish a valid 3d-qsar pharmacophore model for d receptors. the resulting model shows excellent q values for crossvalidation results and random groups pls tests for both, comfa and comsia 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 le and le with a novel dopamine/5ht2a receptor profile. le and le differ in their d1/d2l selectivity. le clusters in one group with the atypical antipsychotic clozapine but has a different d2-like receptor profile than clozapine. le clusters in a separate group from clozapine/le and from haloperidol and shows increased d selectivity similar to the experimental compound rmi- which displayed antipsychotic activity. an antipsychotic activity of le and le in in vivo studies still needs to be shown. further, a validated 3d-qsar pharmacophore model for d antagonists is presented. 
regardless of using affinity data or selectivity data ), 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. the purpose of this study was to characterize in vitro a series of novel azecine-type compounds at human dopamine d1-d and 5ht2a receptors and to assign them to different classes according to their dopamine/5ht2a receptor profile. the monohydroxylated dibenzacezine le clusters in a separate group from clozapine/le and from haloperidol and shows increased d selectivity. the most potent dibenzazecine clustering in the same group as clozapine was the non-hydroxylated le which shows a slightly different d2-like receptor profile than clozapine. reducing the number of ki ratios used for principal component analysis from to two obtained the same three groups of compounds. in conclusion, two compounds with a novel dopamine/5ht2a receptor profile, le and le with some differences in their respective d1/d receptor affinities including a validated pharmacophore-based 3d-qsar model for d antagonists are presented.
 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-d and 5ht2a receptors and to assign them to different classes according to their dopamine/5ht2a receptor profile.  regardless of using affinity data or selectivity data ), 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 to two obtained the same three groups of compounds. the most potent dibenzazecine clustering in the same group as clozapine was the non-hydroxylated le which shows a slightly different d2-like receptor profile than clozapine. the monohydroxylated dibenzacezine le clusters in a separate group from clozapine/le and from haloperidol and shows increased d selectivity.  in conclusion, two compounds with a novel dopamine/5ht2a receptor profile, le and le with some differences in their respective d1/d receptor affinities including a validated pharmacophore-based 3d-qsar model for d antagonists are presented. 