Dopamine/serotonin receptor ligands. Part VIII[1]:the dopamine receptor antagonist LE300 - modelled...

Original article

Dopamine/serotonin receptor ligands. Part VIII[1]:the dopamine receptor antagonist LE300 - modelled and X-ray structure

plus further pharmacological characterization,including serotonin receptor binding, biogenic amine transporter testing

and in vivo testings

Michael Decker a, Klaus-Jürgen Schleifer b, Martin Nieger c, Jochen Lehmann a,*a Lehrstuhl für Pharmazeutische/Medizinische Chemie, Institut für Pharmazie, Friedrich-Schiller-Universität Jena,

Philosophenweg 14, 07743 Jena, Germanyb BASF AG, GVC/C - A030, D-Ludwigshafen, Germany

c Institut für Anorganische Chemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany

Received 18 August 2003; received in revised form 23 January 2004; accepted 5 February 2004

Available online 21 April 2004

Abstract

-LE300, a benz[d]indolo[2,3-g]azecine with nanomolar affinities to the hD1 receptor family, has been further pharmacologicallycharacterized and its modelled structure was compared to its X-ray structure in order to explain NMR data, that was not in accordance to theX-ray structure. Moderate affinity at the hD3 receptor was determined, nanomolar affinities were found at the 5-HT2A and 5-HT2C receptors,micromolar affinity at the 5-HT1A receptor using binding assays. Functional studies indicate moderate antagonist activity at the 5-HT2A site .No activity was found at dopamine, serotonin and norepinephrine transporters. These results suggested the use of LE300 for cocaine addictiontreatment. High activities were found using in vivo testing: LE300 suppressed spontanous locomotor activity with an ID50 of 1.24 mg/kg andattenuated locomotor activity induced by 20 mg/kg cocaine with an AD50 of 1.50 mg/kg. It failed to substitute for the discriminative stimuluseffects produced by cocaine.© 2004 Elsevier SAS. All rights reserved.

Keywords: LE300; Dopamine antagonists; Serotonin antagonists; Cocaine addiction treatment; Low energy conformations; Azecines; X-ray

1. Introduction

The compound LE300 [2], which is 7-methyl-6,7,8,9,14,15-hexahydro-5H-benz[d]indolo[2,3-g]azecine,has been characterized both by radioligand binding assaysand functional testings using cAMP and [Ca2+] as a verypotent antagonist for the human dopamine receptors (D1,D2L, D4.4 and D5) with nanomolar affinities and a 10- to20-fold selectivity for D1 over D2L [3].

This publication deals with two topics: On the one hand,the X-ray structures of both LE300·HCl and its unprotonatedform LE300 could be obtained. A remarkable difference inthe chemical shifts of the respective N-methyl groups arises(13C: d = 44.11 ppm and 46.84 ppm / 1H: 2.75 ppm and

1.99 ppm for LE300·HCl and for LE300, respectively),which cannot be explained out of the X-ray structures. Inorder to find out, how the structure in the lattice differs fromthe one in solution, molecular modelling was applied. Butapart from this fairly obvious reason, LE300 served as a leadfor a large number of analogs [1,4,5], binding data of whichhas been determined: therefore explaining and identifyingthe lowest energy conformations, which are reflected by thespectroscopic data, are important for further modelings ofanalogs, e.g. to find out, which syntheses of new compoundsmight be worth pursuing. Furthermore, correlating the bind-ing affinities of analogs with the modelled structures mightgive some suggestions, how interaction with the receptor canbe understood.

On the other hand (the second topic), intensive pharmaco-logical evaluation of the lead LE300 was pursued. Chemi-cally, LE300 can be regarded as a hybrid of both dopamine

* Corresponding author.E-mail address: [email protected] (J. Lehmann).

European Journal of Medicinal Chemistry 39 (2004) 481–489

www.elsevier.com/locate/ejmech

© 2004 Elsevier SAS. All rights reserved.doi:10.1016/j.ejmech.2004.02.001

(more precisely phenyl ethylamine) and serotonin (moreprecisely tryptamine) resembling parts of a molecule (seeFig. 1). Out of this reason, and because of the assumption,that a combination of affinities at several receptors, e.g.dopamine and 5-HT2A might be beneficial for the avoidanceof extrapyramidal side effects of neuroleptic drugs [6],LE300 is regarded as a lead structure for new compoundswith neuroleptic activity. A more thorough investigation intothe pharmacological profile of this lead structure was neces-sary, therefore, apart from completing the pharmacologicalprofile at the dopamine receptor, i.e. determining the affinityto the D3 receptor, measuring the affinities andagonistic/antagonistic properties at serotonin receptors isvery important in order to see, if the compound in factpossesses affinities to dopamine and serotonin receptors. Inthe case, that no binding of LE300 to the transporter proteinsof biogenic amines, which might be possible because of theobvious resemblance of structures, can be observed, anotherpossible use of LE300 -apart from treatment of psychosis- ispossible: it might also be useful for the treatment of cocaineaddiction. Cocaine blocks the mono amine carriers in thenervous system. Especially the blocking of the dopaminecarrier (i.e. an indirect dopamine agonism) is made respon-sible for the psychic addiction to cocaine. Therefore LE300′sbinding profile made it interesting for investigation of aninteraction with the rewarding effects of cocaine. For thesereasons, in vivo testing for assessing, if LE300 is a stimulantor a depressant, were performed within the NIH CocaineTreatment Discovery Program. Being a depressant in thelocomotor activity assay is an additional indication for thiscompound interacting with dopamine receptors in vivo.

2. Results and Discussion

2.1. Molecular Modelling

The X-ray structures of both LE300 and LE300·HCl indi-cate the N-methyl groups in an equatorial position of theazecine ring outside the anisotropic regions of the aromaticbenzene and indole rings (see Figs. 2 and 3). Nuclear mag-netic resonance analyses, however, indicate chemical shiftsfor the N-methyl carbon atom (13C NMR) and the connectedprotons (1H NMR) in a range that is not in accordance withthis observation. Therefore, a molecular modelling study wascarried out with the aim to investigate molecular flexibility of

these compounds in order to find an explanation for theconflictive results.

High-temperature molecular dynamic simulations (MDS)were performed for the free base and the protonated form tocheck the corresponding flexibility of the azecine rings. Asimulated annealing protocol was used to yield an ensembleof 30 low energy conformers for each derivative. In order toenhance the significance of the results, two different forcefields (cvff [7] and Amber [8,9]) were applied. To avoidartificial effects caused by the lack of shielding solvent mol-ecules in course of the simulation, formal and atomic chargeswere omitted for all molecular mechanics simulations.

Animation of the trajectories verifies the chosen startingtemperature of 1,500 K to be clearly sufficient to surmountall energetic barriers (maxima). Additionally, the relativelyflat run of the curve indicating the annealing period from1,500 to 0 K confirms the anticipated slow relaxation (and nosplat cooling) of all sampled conformers (see Fig. 4).

Analysis of all archived conformations was accomplishedon two different levels. First, the potential energy of allconformers was calculated and energetically identical con-formers were clustered into common energetic families (F)starting with the lowest energy conformation to be desig-nated as F1. Second, all structures within one family weresuperimposed indicating them to be identical, reflected or atleast very similar. Closer examination of the latter ones

H2N

OH

HO

NH2NH

HO

NCH3

NH

LE300

Fig. 1. LE300 as a formal structural hybrid of dopamine and serotoninwithout hydroxy groups.

Fig. 2. X-ray structure of LE300·HCl (crystallizing with one mol of ethanol)

Fig. 3. X-ray structure of LE300 (crystallizing with one mol of ethanol)

482 M. Decker et al. / European Journal of Medicinal Chemistry 39 (2004) 481–489

adduced the geometry of the basic nitrogen atom, which isnot entirely pyramidal but flattened to be responsible for theobserved differences.

Analysis of the results derived from theoretical calcula-tions indicates that independent of the applied force field orthe protonation state the energetically most favourable con-formation is not identical with the experimental X-ray struc-ture. The differences may be clarified by comparing thedistances between the alicyclic nitrogen atom and the aro-matic ring systems (see Table 1). The force fields privilege asandwich-like orientation of the amine group wrapped by theindole and the benzene ring (see Fig. 5). The X-ray structure,also found as a low energy conformation (F2 or F3) in thismolecular mechanics study, is energetically less favourablecompared to the sandwich-like conformation (DE 1.6 -2.2 kcal/mol). For a fitting of both X-ray structures see Fig. 6.

In the next step, one member of each family was semi-empirically geometry-optimized using the AM1 algorithm[10]. In case of the basic compound (LE300) the geometry ofthe minimized conformation is scarcely altered. However,one trend may be observed. The distance between the aminegroup and the aromatic rings is generally shortened (up to18 pm). More relevant, however, is a comparison of theestimated energies. While the force fields prefer thesandwich-like conformation (DE ≈ -2 kcal/mol) the quantummechanical calculation inverts this proportion and favoursthe X-ray structure conformation up to 3.0 kcal/mol (seeTable 1). This may be explained by the fundamental concep-tion of all quantum mechanical methods to optimize theelectron (or charge) distribution. Thus, the atomic orbitals ofthe amine and the p-electrons of the aromatic rings senseeach other and may be combined in an optimal fashion. Thiselectronic interaction was intentionally prevented during theforce field calculations by omitting atomic charges.

In case of the cationic LE300·HCl both calculationsfavour the sandwich-like conformation by 1.7 (ff) and5.0 kcal/mol (AM1), respectively. This is evidently in con-trast to the X-ray crystallographic investigation indicating forboth derivatives the extended conformation. One possibleexplanation for this finding is the presence of ethanol (LE300and LE300·HCl) and the chloride anion (LE300·HCl) in thecrystal lattice of the unit cells forming stabilizing hydrogenbond networks. In the unit cell of LE300 the hydroxy func-tion of ethanol is linking the indolic NH function of onemolecule with the free electron pair of the basic amine of asecond LE300 molecule. This polar interaction combinedwith hydrophobic stacking of the aromatic systems clearlyfavours the extended orientation of the azecine ring. A

B

2000

Temp[K]

0 100 400 700 1000 1300

Time [ps] A

Fig. 4. Graphical analysis of the simulated annealing protocol following theentire MDS (A) and focussed on two individual annealing and heatingperiods (B).

Table 1Results derived from molecular mechanics (ff) and semi-empirical calcula-tions (AM1 [8]) for LE300 and LE300·HCl

familya (ff) DEpot.b distance Ac distance Bc

(# ofconformers)

ff AM1 ff / AM1 (X-ray) ff / AM1 (X-ray)

F1cvff (6) 0 3.0 512 / 503 418 / 402F2cvff (5) 1.6 0 566 / 569 (565) 517 / 499 (507)F1Amb (11) 0 0.8 518 / 516 422 / 426F3Amb (2) 2.2 0 569 / 569 (565) 506 / 499 (507)F1H+/cvff (7) 0 0 520 / 494 418 / 405F2H+/cvff (4) 1.7 5.0 568 / 566 (550) 517 / 508 (512)F1H+/Amb (8) 0 0 514 / 494 409 / 405F3H+/Amb (1) 1.7 5.0 553 / 566 (550) 518 / 508 (512)

a energetically most favourable force field (ff) conformers derived fromMDS in the cvff [5] and the Amber (Amb) force field [6,7] are grouped intofamily 1 (F1), the following ones in F2, and so on. For clarity, only thosefamilies comprising the X-ray conformation (i.e. F2 or F3) are comparedwith F1. Results obtained for the protonated derivative LE300·HCl areindicated with an H+ (e.g. F1H+/cvff).

b DE values in kcal/mol yielded by comparing the lowest energy confor-mers (set to 0) with energetically less favourable families.

c distance (pm) from the centroids of the indolic phenyl (A) and thebenzene ring (B) to the alicyclic nitrogen atom.

Fig. 5. Orthogonal view of the superimposed sandwich-like (dark) andX-ray analogue extended conformation (grey, in ball and sticks) of LE300yielded by molecular mechanics calculations. Hydrogen atoms are omittedfor clarity.

Fig. 6. Fitting of the X-ray structures of LE300 (drawn through line) andLE300·HCl (dotted line).

483M. Decker et al. / European Journal of Medicinal Chemistry 39 (2004) 481–489

sandwich-like conformation would prevent the H-bond be-tween the amine and the ethanolic OH group.

For the protonated LE300·HCl the chloride counteriondisplaces the oxygen atom of the alcohol and takes in itsplace as H-bond acceptor, for H7, H14 of the second LE300molecule and H1e of ethanol (see Figs. 2 and 3). This allowsa perfect antiparallel orientation of two indole rings andincorporation of the benzene rings into the concave azecinecycles. Once again, a sandwich-like conformation woulddisturb this stabilizing intermolecular linkage.

Summarizing the results of the theoretical investigations,force field calculations without consideration of chargesfavour optimal steric complementarity to yield compact,globular-shaped structures. Since this is a well-known prop-erty of lipophilic solute molecules in hydrophilic solvate(and vice versa), the results obtained by NMR analyses couldbe explained in that way. Explicit consideration of atomiccharges in the course of quantum chemical calculations altersthe conditions. Electron-rich centres being the aromatic ringsand the basic nitrogen are pushed away, whereas oppositecharged functions (p-electrons and the ammonium) approachin an optimal fashion. This explains the inverted findingsobtained for the potential energy of LE300 in the force field(sandwich-like) and the semi-empiric calculation (extended).For the cationic LE300·HCl steric contributions privilege thesandwich conformation. Additional electronic features in-crease the tendency to form a compact sandwich form(DE = 5 kcal/mol).

The special conditions in the crystal lattice incorporatingethanol and partly the chloride anion (i.e. LE300·HCl) allowa perfect hydrogen bonding network if the azecine ring has anextended orientation. In the NMR experiments this orderedarrangement is disrupted by the shielding effects of the sol-vent molecules (DMSO). Solute-solute interactions in thisdilution are very rare and the tremendous amount of solventmolecules ordered in more or less organized clusters forcethe solute molecules to optimize attractive and minimizerepulsive inter- and intramolecular interactions. In the spe-cial case considered in this study, optimization of intramo-lecular interactions could explain the non-typical chemicalshifts (d = 1.99 ppm) of the N-methyl groups of the azecinestructures. In a sandwich- (or globular-) like arrangement,the particular methyl groups are positioned in the anisotropicregion of the indole and the benzene ring leading to theobserved shift by approximately 0.76 ppm.

Apart from the explanation of the concrete NMR data, weare able to model analogs, which appropriately reflect theexperimental data. Correlation with the binding data can nowbe made possible.

2.2. Pharmacology

As already reported [3], LE 300 shows nanomolar affintiesto human dopamine receptors with a 10-20-fold selectivity tothe D1 subtype family over the D2 subtype family, includingthe D3 receptor (K0.5 = 86.9 ± 20.2 nM) (see Table 2). It also

shows nanomolar affinities to 5-HT2A and 5-HT2C receptorsusing binding studies (K0.5 = 11.9 ± 2.9 nM andK0.5 = 36.1 ± 3.3 nM, respectively) (see Table 2), in afunctional assay at the 5-HT2A site it was even more potent(Ke = 3.83 ± 0.28 nM, pA2 = 8.35 ± 0.03 nM) (see Table 3),therefore our concept to combine structural elements of bothdopamine and serotonin within one molecule containing aconstrained heterocyclic system to get high affinities at bothreceptor families turned out to be right. No relevant bindingto dopamine, serotonin and norepinephrine transporters wasfound (see Table 4). The resulting receptogram made LE300attractive as a potentially suitable compound for treatment ofwithdrawal symptoms caused by cocaine addiction.

LE300 was tested in vivo for spontanous locomotor activ-ity and for attenuation of of locomotor activity induced bycocaine. Fig. 7 shows average horizontal activity counts/10 min as a function of time (top graph) and dose (bottomgraph), 20 min following LE300 pretreatment. The period0-30 min was selected for analysis of dose-response data,because this is the time period in which cocaine producedmaximal effects. The mean average horizontal activitycounts/10 min for this 30-min period were fit to a linearfunction of lg dose of the descending portion of the doseeffect curve (0.1 to 10 mg/kg dose range). The ID50 (doseproducing 1/2 maximal depressant activity, where maximaldepression = 0 counts/30 min) was calculated 1.24 mg/kg. Aone-way analysis of variance conducted on log10 horizontalactivity counts/10 min for the 0-30 min time period indicated

Table 2Binding affinity for LE300 to dopamine D3 receptor and serotonin receptors

Receptor radioligand K0.5 ± SEM [nM] Hill Slope ± SEM5-HT1A [3H]8-OH-DPAT 1247.2 ± 132.5 not determined because

of low affinty5-HT2A [3H]Ketanserin 11.9 ± 2.9 0.81 ± 0.065-HT2C [3H]Mesulergine 36.1 ± 3.3 1.17 ± 0.11D3 [3H]YM-09151-2 86.9 ± 20.2 0.80 ± 0.03

Table 3Functional assay result for LE300 at the 5-HT2A receptor

Receptor Antagonist5-HT2A Ke ± SEM [nM] pA2 ± SEM Slope ± SEMn

86.89 ± 8.35 ± 0.03 -1.09 ± 0.03 5

Table 4Effects of LE300 on HEK-hDAT, HEK-hSERT and HEK-hNET cells

HEK-hDAT cells LE300 Cocaine[125I]RTI-55 Binding KI [nM] > 10000 472 ± 46Hill coefficient -1.09 ± 0.04[3H]Dopamine Uptake IC50 [nM] 343 ± 31HEK-hSERT cells[125I]RTI-55 Binding KI [nM] > 10000 496 ± 33Hill coefficient -1.03 ± 0.08[3H]Serotonin Uptake IC50 [nM] 344 ± 35HEK-hNET cells[125I]RTI-55 Binding KI [nM] > 10000 2710 ± 260Hill coefficient -0.76 ± 0.05[3H]Norepinephrine Uptake IC50 [nM] 275 ± 30


a significant overall effect F(5, 42) = 54.58, p<0.001;planned comparisons (a priori contrast) against the vehiclecontrol showed a significant difference for 3 and 10 mg/kg(all ps<0.05 denoted on Fig. 7 with an asterik). Fig. 8 givesthe graphic representation according to cocaine interaction.Dose range was 1 to 10 mg/kg, and the AD50 (dose attenuat-ing cocaine-induced stimulation by 50%) was calculated tobe 1.50 mg/kg. [The ordinate value for the AD50 was calcu-lated using the mean of the vehicle plus 0.9% saline (vehicle)group as the minimum value, and the mean of the vehicleplus 20 mg/kg cocaine group as the maximum value.] Aone-way analysis of variance conducted on lg horizontalactivity counts/10 min for the 0-30 min time period indicateda significant overall effect F(4, 35) = 15.52, p<0.001;planned comparisons (a priori contrast) against the cocainegroup showed a significant difference for vehicle, 3 and10 mg/kg (all ps<0.05 denoted on Fig. 8 with an asterik).

LE300 was also tested for substitution for the discrimina-tive stimulus effects produced by cocaine. Total session:Within the dose range of 0.5 to 25 mg/kg, LE300 failed tosubstitute for the discriminative stimulus effects produced by10 mg/kg cocaine. Response rate was decreased following10 and 25 mg/kg, with the maximum effect (13% of vehiclecontrol) following 25 mg/kg of LE300. A one-way, repeatedmeasures analysis of variance conducted on response rate forthe total session indicated a significant overall effect

F(6,30) = 7.35, p<.001; planned comparisons (a priori con-trast) against the vehicle control indicated a significant dif-ference for the 10 and 25 mg/kg doses (all ps<.05 denoted onFig. 9 with an asterisk). First reinforcer: The results for thefirst reinforcer measure were in general accordance with thetotal session data. Response rate was decreased to 18% ofvehicle control following 25 mg/kg of LE300. A one-way,repeated measures analysis of variance conducted on re-sponse rate for the first reinforcer failed to indicate a signifi-cant overall effect F(6,30) = 1.93, p = .108; planned compari-sons (a priori contrast) against the vehicle control indicated asignificant difference for the 25 mg/kg dose (all ps<.05denoted on Fig. 10 with an asterisk). Other observations:Four of six rats failed to complete the first fixed ratio whentested following 25 mg/kg of LE300. No unusual effectswere observed following any dose of LE300. Because ofLE300 being a potent depressant in the mouse locomotoractivity assay, and because it did not generalize to cocaine inthis assay, it will next be tested at the NIH in the rat drugdiscrimination antagonism assay.

LE300 seems to be an interesting lead structure for neuro-leptic drugs, but our new results strongly suggest LE300 alsoas a lead for cocaine addiction treatment. Several structuralmodifications both at the aromatic ring systems and theheterocyclic ring system have been made for further im-provement and are currently under pharmacological investi-

10 20 30 40 50 600

1000

2000

3000

4000

5000 VEHICLE 1 mg/kg

0.1 mg/kg 3 mg/kg

0.3 mg/kg 10 mg/kg

LE300 VS VEHICLE

AST

NU

OC

NO IT

ALU

BM

TIME INTERVAL (min)

0 2 4 6 8 100

500

1000

1500

2000

2500

3000

3500

4000

*

ID50 = 1.24 mg/kg

*0-30 min

01/ST

NU

OC E

GA

RE

VA

NIM 0 3

RE

VO

NIM

LE300 (mg/kg)

Fig. 7. Effect of LE300 on horizontal activity counts/10 min.Upper figure: Time course of effect.Lower figure: Dose response 0-30 min after 20 min pretreatment.* p<0.05 Compared with 0 dose.

10 20 30 40 50 600

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000LE300 VS COCAINE

VEHICLE COC + 1 mg/kg COC (20 mg/kg) COC + 3 mg/kg

COC + 10 mg/kg

STN

UO

C N

OIT ALU B

MA

TIME INTERVAL (min)

VEH COC 1 3 100

1000

2000

3000

4000

5000

6000

7000

8000

9000

*

*

AD 50 = 1.50 mg/kg

*

0-30 min

NIM 03

REVO

NIM 01 /ST

NU

OC E

GAR E VA

COC + LE300 (mg/kg)

Fig. 8. Interaction between LE300 and cocaine on locomotor activity.Upper figure: Time course of effect.Lower figure: Dose response 0-30 min after 20 min pretreatment.* p<0.05 Compared with cocaine alone.


gation. Because of its promising properties in the CocaineTreatment Discovery Program (CTDP), further in vivo test-ings (rat drug discrimination antagonism assay) are currentlyperformed.

3. Experimental protocols

3.1. Molecular Modelling

A theoretical study was performed in order to find areasonable explanation for the obvious discrepancy of thechemical shifts of the N-methyl groups of LE300 andLE300·HCl compared to the X-ray crystallographic data.

High-temperature molecular dynamics simulations (MDS)were carried out with the Discover module of the MSI soft-ware package [7] to generate a multitude of different startingconformers for a further, more detailed investigation. Usingtwo different force fields (cvff [5] and Amber [8,9]), a simu-lated annealing protocol was applied for the free base LE300and the protonated form (LE300·HCl). MDS were initializedat a temperature of 1500 K for a period of 100 picoseconds(ps). The sampling procedure was started at 1500 K for 20 psfollowed by a freezing period of 20 ps cooling the tempera-ture slowly down to 0 K. The yielded structure was archived.

Ctrl 0.1 1 10 10

0.0

0.5

1.0

1.5

2.0

*

*

dnoceS re p ses no ps eR

Dose of LE300 (mg/kg)

Ctrl 0.1 1 10 10

0

20

40

60

80

10

n=5n=4

Vehicle Cocaine(10mg/ kg)LE300

Total Session

)%( gnidnop se

R r eveL -eni a coC


Fig. 9. Total Session: The upper panel shows the mean (±SEM) percentageof responses emitted on the cocaine-appropriate lever during the total ses-sion as a function of dose, for doses with three or more rats completing thefirst fixed ratio. The lower panel shows the mean response rate (±SEM) as afunction of dose for all subjects tested. The sample size is equal to six at alldata points except where noted. To the left of the axis break, control (Ctrl)data are shown for the vehicle (2% methylcellulose) and for the training doseof cocaine (10 mg/kg). Data for the substitution study of LE300 for thetraining dose of cocaine are shown to the right of the axis break.*p<0.05 compared with vehicle control.

Ctrl 0.1 1 10 10

0.0

0.5

1.0

1.5

*

Vehicle Cocaine(10mg/kg)

LE300

d noc eS rep se snopse

R


Ctrl 0.1 1 10 10

0

20

40

60

80

10

n=5

n=4

First Reinforcer

reveL-eni acoC

)%( gn idno pse

R


Fig. 10. First Reinforcer: The upper panel shows the mean (±SEM) percen-tage of responses emitted on the cocaine-appropriate lever for the firstreinforcer as a function of dose, for doses with three or more rats completingthe first fixed ratio. The lower panel shows the mean response rate (±SEM)as a function of dose for all subjects tested. The sample size is equal to six atall data points except where noted. To the left of the axis break, control (Ctrl)data are shown for the vehicle (2% methylcellulose) and for the training doseof cocaine (10 mg/kg). Data for the substitution study of LE300 for thetraining dose of cocaine are shown to the right of the axis break.*p<0.05 compared with vehicle control.


This protocol was repeated 30 times for each derivative (intotal 1.2 ns) to gain 30 low energy conformations. Subse-quently geometry-optimization was accomplished in the cor-responding force field until a convergence criterion of0.01 kcal/Å was reached. Superposition of all relaxed con-formers over the six carbon atoms of the benzene ring pro-vided clusters of common conformational families. The low-est energy conformers of the energetically most favourablefamilies were subjected to charge-considering semi-empirical AM 1 [10] minimizations with the AMPAC/MOPAC module of Insight II [7].

All computations were carried out on SGI Indigo2 R10000and SGI Indy R4600 workstations.

3.2. Pharmacology

5-HT1A(Binding): HA7 (human receptor) are grown toconfluence in DMEM containing 10% fetal bovine serum(FBS), 0.05% penicillin-streptomycin, and 400 µg/mL ofG418. The cells are scraped from 100 x 20 mm plates andcentrifuged at 500 x g for 5 minutes. The pellet is homog-enized in 50 mM Tris-HCl (pH = 7.7), with a polytron,centrifuged at 27,000 x g, and resuspended at 10 mgprotein/mL in the same buffer. The homogenate is the storedat –70°C in 1 mL aliquots. The thawed cells are washed onceand resuspended at 10 mg protein/80mL in 25 mM Tris-HClcontaining 100 µM ascorbic acid and 10 µM nialamide atpH = 7.4. The assay is performed in triplicate in a 96-wellplate. 100 µL of test compound or buffer and 0.8 mL of cellhomogenate (0.1 mg protein/well) are added to 100 µL of[3H]8-OH-DPAT (0.5 nM final concentration). Nonspecificbinding is determined with 1.0 µM dihydroergotamine. Theplates are incubated at 25°C for 60 min and then filtered. Theincubation is terminated by rapid filtration through glassfiber filter plates on a Tomtec cell harvester. The filters arewashed four times with ice-cold 50 mM Tris-HCl (pH = 7.7),dried overnight, and bagged with 10 mL scintillation cocktailbefore counting for two minutes on a Wallac Betaplate1205 liquid scintillation counter.

5-HT2A(Binding): NIH-3T3-GF6 cells (rat receptor) aregrown as described for the HA7 cells. On the day of theexperiment the cells are thawed, resuspended in 50 mMTris-HCl (pH = 7.7), and centriguged at 27,000 x g for12 minutes. The pellet is then resuspended at 1 mgprotein/80 mL in 25 mM Tris-HCl (pH = 7.7), and 0.8 mL ofcell homogenate (0.01 mg/protein/well) is added to wellscontaining 100 µL of the test drug or buffer and 100 µL of[3H]ketanserin (0.4 nM final concentration). Nonspecificbinding is determined with 1.0 µM ketanserin.

5-HT2C(Binding): NIH-3T3-PB cells (rat receptor) aregrown as described for the HA7 cells. The final pellet isresuspended at 3 mg protein/80 mL in 50 mM Tris-HCl(pH = 7.7), 4 mM CaCl2, 10 µM pargyline, and 0.1% ascorbicacid. Wells containing 100 µL of the test drug or buffer,100 µL of [3H]mesulergine (0.4 nM final concentration), and0.8 mL of cell homogenate (0.03 mg protein/well) are incu-

bated at 25°C for 60 minutes. Nonspecific binding is deter-mined with 10 µM of mesulergine.

D3: CHOp-cells (human receptors) are grown to conflu-ence in a-minimal essential medium (a-MEM) containing10% fetal bovine serum (FBS), 0.05% penicillin-streptomycin, and 600 µg/mL of G418, according to theprocedures described for HA7 cells. The final pellet is resus-pended at 1 mg protein/80 mL in 50 mM Tris,containing120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 4 mM MgCl2, and1 mM EDTA (pH = 7.4). Wells containing 100 µL of the testdrug or buffer, 100 µL of [3H]YM-09151-2 (0.21 nM finalconcentration), and 0.8 mL of cell homogenate (0.01 mgprotein/well) are incubated at 25°C for 60 minutes. Nonspe-cific binding is determined with 1 µM chlorpromazine.

5-HT2A (Receptor assay on rat aorta spiral): male albinoWistar rats (200-300 g body weight) are sacrificed and theiraortas quickly removed, cleaned, and cut into a spiral. Thespiral is mounted in an 8 mL water-jacketed tissue bathcontaining Krebs-hydrogencarbonate solution (118 mMNaCl, 2.5 mM CaCl2, 4.7 mM KCl, 25 mM NaHCO3,1.2 mM KH2PO4, 1.2 mM MgSO4, and 11.5 mM glucose).The spiral is incubated with oxygenated (5% CO2 in oxygen)Krebs solution at 37°C under 2.0 g tension for 30 minutes inthe presence of 500 µM pargyline and 10 µM benextraminetetrahydrochloride [11]. Excess unreacted pargyline and ben-etrexamine are removed from the bath by flushing the systemseveral times with Krebs solution. For standardization pur-poses, the spiral is cumulatively contracted with increasingconcentrations of 5-HT in the presence of 0.1 mM ascorbicacid. The 5-HT-induced contractions are recorded using anisometric transducer (Metrigram) coupled to a Gould multi-channel polygraph. Compounds, that do not produce a con-traction on their own are tested for 5-HT2A antagonist activ-ity. The test compound is incubated with the spiral for60 minutes, and the 5-HT standard curve is repeated in thepresence of the drug. Antagonist activities are calculated foreach tissue from full concentration-response curves obtainedbefore and after addition of a single antagonist concentration.At least three different concentrations are used, and only oneantagonist concentration is tested on each tissue. pA2 valuesare determined from Schild plots using a statistical analysisprogramme developed by B. Eynon (SRI International) [12].

Biogenic amine transporter binding: Unknowns areweighed and a 10 mM stock solution in DMSO is preparedout of them. Subsequent dilutions are made in assay buffer,achieving a final concentration of 0.1%. Pipetting is con-ducted using a Biomek 2000 robotic work station. HEK293cells expressing hDAT, hSERT or hNET inserts are grown to80% confluence on 150 mm diameter tissue culture dishesand serve as the tissue source. Cell membranes ar prepared asfollows. Medium is poured off the plate, and the plate iswashed with 10 mL of Ca- and Mg-free PBS. Lysis buffer(10 mL; 2 mM HEPES with 1 mM EDTA) is added. After10 min, cells are scrapped from plates, poured into centrifugetubes, and centrifuged 30,000 x g for 20 min. The supernatantfluid is removed, and the pellet is resuspended in 12-32 mL of


0.32 M sucrose using a Polytron at setting 7 for 10s. Thesuspension volume depends on the density of binding siteswithin a cell line and is chosen to reflect binding of 10% orless of the total radioactivity. For the assays, each assay tubecontains 50 µL of membrane preparation (about 10-15 mg ofprotein), 25 µL of unknown or buffer (Krebs-HEPES,pH = 7.4; 122 mM NaCl, 2.5 mM CaCl2, 1.2 mM MgSO4,10 µM pargyline, 100 µM tropolone, 0.2% glucose and0.02% ascorbic acid, buffered with 25 mM HEPES), 25 µL of[125I]RTI-55 (40-80 pM final concentration) and additionalbuffer sufficient to bring up the final volume to 250 µL.Membranes are preincubated with unknowns for 10 minutesprior to the addition of the [125I]RTI-55. The assay tubes areincubated at 25°C for 90 minues. Binding is terminated byfiltration over GF/C filters using a Tomtec 96-well cell har-vester. Filters are washed for six seconds with ice-cold saline.Scintillation cocktail is added to each square and radioactiv-ity remaining on the filter is determined using a Wallac c- orb-plate reader. Specific binding is defined as the difference inbinding observed in the presence and absence of 5 µM mazin-dol (HEK-hDAT and HEK-hNET) or 5 µM imipramine(HEK-hSERT). Three independent competition experimentsare conducted with duplicate determinations. GraphPADPrism is used to analyze the ensuing data, with IC50 valuesconverted to KI values using the Cheng-Prusoff equation.

Locomotor activity: A dose response study of LE300-induced locomotor depression was conducted using40 Digiscan locomotor activity testing chambers (40.5 x40.5 x 30.5 cm) housed in sets of two, within sound-attenuating chambers.A panel of infrared-beams (16 cm) andcorresponding photodetectors were located in the horizontaldirection along the sides of each activity chamber. A 7.5-Wincandescent light above each chamber provided dim illumi-nation. Fans provided an 80-dB ambient noise level withinthe chamber. Separate groups of 8 non-habituated maleSwiss-Webster mice (Hsd:ND4, aged 2-3 months) were in-jected via the intraperitoneal (IP) route with either vehicleactivity testing. Just prior to placement in the apparatus, allmice received a saline injection. In all studies, horizontalactivity (interruption of photocell beams) was measured for1 h within 10-min periods. Testing was conducted with onemouse per activity chamber. The cocaine interaction studywas also conducted using the above described chambers.Twenty minutes following IP vehicle or LE300 injections (1,3 or 10 mg/kg), groups of 8 non-habituated mice were in-jected with either 0.9% saline or 20 mg/kg cocaine IP andplaced in the Digiscan apparatus for a 1 h session.

Test of substitution for the discriminative stimulus effectsof cocaine: LE300 was tested for its ability to substitute forthe discriminative stimulus effects of cocaine (10 mg/kg) inrats. Testing began December 18, 2002 and ended May 19,2003. Experiments were conducted according to the standardoperating protocol for CTDP (Cocaine Treatment DiscoveryProgram by the NIH) drug discrimination testing in rats(SOP, March 28, 1997). Six male Sprague-Dawley rats weretrained to discriminate cocaine (10 mg/kg) from saline using

a two-lever choice methodology. Food was available as areinforcer under a fixed ratio 10 schedule when respondingoccurred on the injection appropriate lever. All tests occurredin standard, commercially available chambers (CoulbournInstruments), using 45 mg food pellets (Bioserve) as rein-forcers. Training sessions occurred in a double alternatingfashion, and tests were conducted between pairs of identicaltraining sessions (i.e., between either two saline or two co-caine training sessions). Tests occurred only if, in the twopreceding training sessions, subjects met the criteria of emit-ting 85% of responses on the injection correct lever for boththe first reinforcer (first fixed ratio) and the total session. Testsessions lasted for twenty minutes, or until twenty reinforc-ers had been obtained. Doses of the test compound for whichfewer than three rats completed the first fixed ratio were notconsidered in the characterization of discriminative stimuluseffects. Intraperitoneal injections (1 ml/kg) of LE300, or itsvehicle (2% methylcellulose), occurred 30 minutes prior tothe start of the test session. A starting dose for LE300 of0.5 mg/kg was determined based upon data supplied by theproject officer, and a dose range of 0.5 to 25 mg/kg wasexamined. This range included doses that were inactive tothose that had biological activity as evidenced by a decreasein response rate to 13% of vehicle control.

3.3. X-ray analysis

Preparation of crystals: Crystals of LE300 can be directlyobtained by recrystallising LE300 [2] from anhydrous etha-nol.

LE300·HCl is prepared by dissolving 100mg (0.35 mmol)of LE300 in 130 mL of anhydrous diethyl ether and bubblinggasous HCl through this solution. After a short while aturbidity is formed and the reaction is discontinued. Lettingthis solution stand for a further 40 minutes completes pre-cipitation. The solid is filtered and dried. To obtain crystals,the solid is dissolved in anhydrous ethanol, which is allowedto evaporate slowly at 4°C.

Crystallographic data for the structures reported in thispaper have been deposited with the Cambridge Crystallo-graphic Data Centre as supplementary publication numberCCDC-199342 (LE300) and CCDC-198889 (LE300·HCl).

Acknowledgements

The pharmacological testings of compound LE300, whichwere done by NIDA (NIDA Contract N01DA-7-8076), isvery gratefully acknowledged.

We also wish to thank S. Lankow and D.R. Rudolf forpreparing crystals of LE300 and LE300·HCl, respectively.

References

[1] M. Decker, J. Lehmann, Arch. Pharm. Pharm. Med. Chem. 336 (2003)466–476.


[2] T. Witt, F.J. Hock, J. Lehmann, J. Med. Chem. 43 (2000) 2079–2081.[3] M.U. Kassack, B. Höfgen, M. Decker, N. Eckstein, J. Lehmann,

Naunyn-Schmiedeberg′s Arch. Pharmacol. 366 (2002) 543–555.[4] H. El-Subbagh, T. Wittig, M. Decker, S. Elz, M. Nieger, J. Lehmann,

Arch. Pharm. Pharm. Med. Chem. 9 (2002) 443–448.[5] T. Wittig, C. Enzensperger, J. Lehmann, Heterocycles 60 (2003)

887–898.[6] B. Capuano, I.T. Crosby, E.J. Lloyd, Curr. Med. Chem. 9 (2002)

521–548.[7] INSIGHT II/DISVOVER 2.9.7, Biosym/MSI, San Diego, CA, U.S.A.

[8] S.J. Weiner, P.A. Kollman, D.A. Case, U.C. Singh, C. Ghio,G. Algona, S. Profeta Jr, P. Weiner, J. Amer. Chem. Soc. 106 (1984)765–784.

[9] S.J. Weiner, P.A. Kollman, D.T. Nguyen, D.A. Case, J. Comput.Chem. 7 (1986) 230–252.

[10] M.J.S. Dewar, E.G. Zoebisch, E.F. Healy, J.J.P. Stewart, J. Am. Chem.Soc. 107 (1985) 3902–3909.

[11] J. Marin, M. Salaices, B. Gomez, S. Lluch, J. Pharm. Pharmac. 33(1981) 715–719.

[12] H.O. Schild, J. Pharmacol. Chemother. 4 (1949) 277–280.


Dopamine/serotonin receptor ligands. Part VIII[1]:the dopamine receptor antagonist LE300 - modelled...

Documents

Transcript of Dopamine/serotonin receptor ligands. Part VIII[1]:the dopamine receptor antagonist LE300 - modelled...