Highly potent 5-aminotetrahydropyrazolopyridines: Enantioselective dopamine D3 receptor binding, functional selectivity, and analysis of receptor-ligand interactions

ARTICLE

pubs.acs.org/jmc

Highly Potent 5-Aminotetrahydropyrazolopyridines: Enantioselective

Dopamine D₃Receptor Binding, Functional Selectivity, and Analysis of

Receptor-Ligand Interactions

Nuska Tschammer,^†Jan Elsner,^†Angela Goetz,^†Katharina Ehrlich,^†Stefan Schuster,^†Miriam Ruberg,^†

Julia K€uhhorn,^†Dawn Thompson,^‡Jennifer Whistler,^‡Harald H€ubner,^†and Peter Gmeiner*^,†

^†Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, D-91052 Erlangen,

Germany

^‡Ernest Gallo Clinic and Research Center and Department of Neurology, University of California—San Francisco, Emeryville, California

94608, United States

S Supporting Information

b

ABSTRACT: Heterocyclic dopamine surrogates of types 5 and

7 were synthesized and investigated for their dopaminergic

properties. The enantiomerically pure biphenylcarboxamide

(S)-5a displayed an outstanding K_iof 27 pM at the agonist-

labeled D₃receptor and signiﬁcant selectivity over the D₂

subtype. Measurement of [³⁵S]GTPγS incorporation in the presence of a coexpressed PTX-insensitive G_R0-1subunit indicated

highly eﬃcient G-protein coupling. Comparison of ligand eﬃcacy data from cAMP accumulation and [³H]thymidine incorporation

experiments revealed that ligand biased signaling is exerted by the test compound (S)-5a. Starting from the D₃crystal structure, a

combination of homology modeling and site directed mutagenesis gave valuable insights into the binding mode and the

intermolecular origins of stereospeciﬁc receptor recognition. According to these data, the superior aﬃnity of the eutomer 5a is

caused by the favorable binding energy that results from interaction between the ligand’s central ammonium unit and the aspartate

residue in position 3.32 of the receptor.

’ INTRODUCTION

derivative 2 were reported as selective D₃partial agonists

(Chart 1).^12-14We have shown that ferrocene analogues of 2

behave as neutral antagonists at the dopamine receptors D₃

and D₄and as partial agonists at the D₂subtype (intrinsic

activity of 57%, EC₅₀= 2.5 nM).¹⁵Incorporating enyne-

derived headgroups as nonaromatic catechol bioisosteres,

we could furthermore demonstrate that N-butyl-4-biphenyl-

carboxamides exhibit ligand biased signaling (functional

selectivity) when compared to their dipropylamine substi-

tuted congeners.¹⁶The long-chain analogues have been more

eﬃcacious in the D₃receptor mediated [³H]thymidine in-

corporation (mitogenesis) compared to a substantially lower

eﬃcacy in the inhibition of cAMP accumulation.¹⁶

Dopamine receptors are phylogenetically classiﬁed into D₁

and D₅as well as D₂, D₃, and D₄subtypes coupling to the G

proteins G_sand G_i/0, respectively.¹Substantial attenuation of

dopaminergic neurotransmission caused by gradual loss of

dopaminergic neurons in the pars compacta of the substantia

nigra leads to chronic progressive Parkinson’s disease.²A com-

mon pharmacotherapy used for the treatment of Parkinson’s

disease is the dopamine precursor levodopa (^L-dopa).^2,3Long-

term use of ^L-dopa often results in a reduced treatment eﬃcacy,

motor ﬂuctuations with dyskinesia, and psychological distur-

bances including hallucinations.⁴Dopamine receptor agonists

that target D₂and D₃dopamine receptors have been successfully

used to replace dopamine. Some neuroprotective eﬀects have

been reported for D₃-preferring agonists during the early stages

of Parkinson’s disease.^5-7Because of the lack of highly selective

D₂and D₃receptor agonists, it is not clear if subtype speciﬁc

stimulation may be advantageous over simultaneous D₂/D₃

receptor activation. The question, which pattern of intrinsic

activities may be the most beneﬁcial for the treatment of

Parkinson’s disease, remains.

As an extension of recent investigations,^11,17,18we have

studied a formal structural hybridization of dopaminergic dipro-

pylamines of type 1 with partial agonists of type 2 leading to

molecular probes of type 3. To design novel dopaminergic

chemotypes displaying potentially higher binding aﬃnity, we

envisaged a start from our previously described 5-aminotetrahy-

dropyrazolopyridines 4¹⁹and the tricyclic ergoline analogues 6 as

lead structures and transformed them into the novel target

compounds of types 5 and 7, respectively. Herein, we describe

Complementing the classical D₂/D₃agonists of type 1

representing conformationally restricted dopamine analogues

and heterocyclic bioisosteres thereof,^8-11N-butyl-4-biphenyl-

carboxamido substituted phenylpiperazines including the

Received: December 23, 2010

Published: March 09, 2011

r

2011 American Chemical Society

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Chart 1. Structural Hybridization of Dopaminergic

Dipropylamines and Arenecarboxamides

Scheme 2^a

^aReagents and conditions: (a) NCS, CHCl₃, reﬂux, 16 h (69%); (b)

POCl₃, DMF, 90 °C, 2 h (65%).

Scheme 3^a

Scheme 1^a

^aReagents and conditions: (a) CH₃NO₂, CH₃NH₂HCl, CH₃COOK,

3

MeOH, room temp, 3 days (80%); (b) (1) NaBH₃(CN), MeOH, 0 °C,

40 min (82%); (2) POCl₃, DMF, 0 °C, 15 min, room temp, 15 min, H₂O/

DMF, 0 °C, 15 min, Et₃N, MeOH, room temp, 15 min (51%); (c) (1)

NaBH₄, MeOH, room temp, 40 min (81%); (2) tin powder, acetic acid,

EtOH, room temp, 3 days (65%); (d) (1) propionaldehyde, NaCNBH₃,

MeOH, 0 °C, 45 min, (53%); (2) 4-(4-phenylbenzoylamino)butyralde-

hyde, NaBH(OAc)₃, 1,2-dichloroethane, room temp, 18 h (87%).

’ RESULTS AND DISCUSSION

Synthesis. Fused pyrazole derivatives have been described as

excellent molecular scaffolds in dopaminergic bi- and tricyclic

ergoline analogues.^18,19,21,22Efficient synthetic pathways to the

fused pyrazole derivatives of types 5 and 7 were elaborated.

Starting from N-amino-4-benzyloxypyridinium mesitylene sulfo-

nate 8, we followed our previously described protocol involving

1,3-dipolar cycloaddition with methyl propiolate under oxidative

conditions, simultaneous cleavage of the benzyl ether function,

removal of the carboxylic ester group, catalytic hydrogenation,

activation of the secondary alcohol, nucleophilic displacement

with sodium azide, and subsequent reduction to afford the

primary amine 9 in racemic form (Scheme 1). To prepare the

pure enantiomers (R)-9 and (S)-9, the primary amino group of 9

was reacted with (-)-menthyl chloroformate, resulting in a

mixture of the diastereomeric carbamates that were chromato-

graphically separated and cleaved under hydrolytic conditions.

N-Monopropylation of 9 was accomplished upon reaction of 9,

(R)-9 and (S)-9, with propionaldehyde and NaBH₃CN at 0 °C

^aReagents and conditions: (a) ref 19; (b) propionaldehyde, NaBH₃CN,

MeOH, 0 °C, 45 min (68%); (c) 4-(4-phenylbenzoylamino)butyral-

dehyde, NaBH(OAc)₃, 1,2-dichloroethane, room temp, 18 h (87%).

the chemical synthesis of these “long chain analogues”, receptor

binding studies, and functional activity proﬁles in assays measur-

ing the D_2longand D₃receptor mediated incorporation of

[³⁵S]GTPγS, inhibition of cAMP accumulation, and stimulation

of [³H]thymidine incorporation (mitogenesis). Additionally, we

investigated the ability of novel compounds to induce the D_2long

receptor internalization. The phenylpiperazine 2 and the pure

enantiomers of the dipropylamine substituted heterocycle 4

served as reference agents. To better understand the binding

mode of our test compounds, mutagenesis experiments and

20

docking studies based on the recent crystal structure of D₃

were performed.

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when a reaction time of 45 min was crucial to success. The thus

formed secondary amines 10, (S)-10 and (R)-10, were reduc-

tively alkylated with 4-(4-phenylbenzoylamino)butyraldehyde²³

in the presence of NaBH(OAc)₃to give rise to the test

compounds 5a, (R)-5a and (S)-5a, respectively. Preparative

HPLC employing a chiral column gave an alternative access to

(R)-5a and (S)-5a via separation of racemic material. To

investigate if the biphenylcarboxamides of type 5 follow the

same SAR rules as the 5-dipropylamino-4,5,6,7-tetrahydropyra-

zolo[1,5-a]pyridines (4), chlorination and formylation in posi-

tion 3 were intended. Thus, treatment of 5a with N-chlorosucci-

nimide in refluxing chloroform afforded the 3-chloro derivative

5b in 69% yield. Employing Vilsmeier reaction conditions, the

tetrahydropyrazolopyridine 5a was transformed into the fused

pyrazole carbaldehyde 5c (Scheme 2).

For the preparation of the tricyclic ergoline analogue 6, we

recently established a multistep reaction sequence involving a

Dieckmann type condensation and an electrophilic amination as

the key reaction steps. For the development of a more practical

approach, we envisioned taking advantage of the heterocyclic

building block 13, which we currently used for the synthesis and

SAR studies of selective dopamine D₄receptor ligands

(Scheme 3).²⁴Knoevenagel type condensation reaction of the

carbaldehyde 13 with nitromethane gave the nitrooleﬁn 14.

Subsequent sodium borohydride promoted reduction of the

CdC double bond resulted in formation of the saturated

synthetic intermediate in 82% yield. As the key reaction step,

phosphorus oxychloride promoted Vilsmeier formylation fol-

lowed by cyclocondensation aﬀorded the periannelated system

15 in 51% yield. Consecutive reductions of CdC double bond

and the nitro group using sodium borohydride and tin powder

aﬀorded the primary amine 16. Finally, reductive N-propylation

and sodium triacetoxyborohydride promoted N-alkylation with

4,4-phenylbenzoylaminobutylcarbaldehyde gave rise to the ﬁnal

product 7.

compete with [³H]spiperone for the cloned human dopamine

receptor subtypes D_2long, D_2short, D₃, and D_4.4stably expressed in

Chinese hamster ovary (CHO) cells. D₁receptor affinities were

determined utilizing porcine striatal membranes and the D₁

selective radioligand [³H]SCH 23390. Our initial investigations

evaluated binding properties associated with the fused pyrazole

5a as our primary target molecule. The biphenylcarboxamide

derived molecular appendage, formally ligated to one of the N-

propyl groups of the lead compound 4, led to a substantial

increase of D₃affinity (racemic 5a, K_i= 0.71 nM,). Binding

affinity for the subtypes D_2long, D_2short, and D₄increased only

moderately (K_iof 150, 77, and 59 nM, respectively) compared to

that of dipropylamine 4. Comparison of the binding affinities

indicates that the formal elongation of the propyl chain leads to

an increase of D₃receptor selectivity, which proved to be greater

than 2 orders of magnitude. In accordance with our recent

observations on the dipropylamines of type 4,¹⁹the introduction

of a chlorine substituent into position 3 of the heterocyclic

system did not significantly affect the ligand binding properties

(K_i= 0.59 for 5b). In contrast, the attachment of a formyl group

led to an approximately 50-fold attenuation of receptor recogni-

tion for the carbaldehyde 5c. These data indicated that com-

pounds of types 4 and 5 might interact with the D₃receptor

binding site in a similar mode. The tricyclic pyrazole derivative 7

revealed highly potent and selective D₃receptor binding with

selectivity patterns that were similar to that of the bicyclic

congener of type 5. Only poor to moderate D₁and R₁receptor

affinity was observed for the test compounds. This was in

contrast to the phenylpiperazine derived reference agent 2

showing a K_iof 5.6 nM for the R₁adrenoreceptor (Table 1).

Because D₂, D₃, and D₄receptors showed substantial diﬀer-

entiation between the two enantiomers of the dipropylamine 4

with ratios of >70, we investigated detailed binding properties of

the optical antipodes (R)-5a and (S)-5a. Determination of

binding aﬃnity to the high aﬃnity state of D_2longand D₃

receptors facilitates an adequate measurement of subtype

selectivity.²⁵Therefore, we performed displacement experiments

with (S)-4, (S)-5a, and (R)-5a at the high aﬃnity binding site of

the D_2longand D₃receptor which was selectively labeled with the

agonist radioligand [³H]7-OH-DPAT. The radioligand displace-

ment assays were performed in a sodium free buﬀer supplemen-

ted with MgCl₂(5 mM). At the D₃receptor, K_ivalues were 14,

Ligand Binding. To evaluate the biological impact of an

arenecarboxamide derived molecular appendage, radioligand

displacement assays were done. The binding affinity and selec-

tivity profiles of target compounds of types 5 and 7 were

compared with those of the core structure 4 and the phenylpi-

perazine derived reference agent 2 (Table 1). The binding data

were generated by measuring the ability of the test compounds to

Table 1. Receptor Binding Data for 2, 4, 5a-c, and 7 Employing Porcine D₁Receptor, the Human Subtypes D_2long, D_2short, D₃,

and D_4.4and the Porcine R₁Receptor

K_i( SEM (nM)^a

[³H]spiperone

[³H]SCH 23390

pD₁

[³H]prazosin

compd

hD_2long

48 ( 6.8

hD_2short

42 ( 2.4

hD₃

hD_4.4

pR₁

2

570 ( 26

0.22 ( 0.010

31 ( 4.8

27 ( 4.4

5.6 ( 0.84

5100 ( 920^b

2900 ( 470^b

520 ( 2.5

(S)-4

(R)-4

5a

53000 ( 17000^b

68000 ( 11000^b

6800 ( 430

410 ( 88

270 ( 51

380 ( 21

17000 ( 4700

59 ( 9.9

55000 ( 13000

150 ( 14

53000 ( 15000

77 ( 25

2100 ( 370

0.71 ( 0.076

0.59 ( 0.088

39 ( 6.9

5b

3600 ( 450

100 ( 15

43 ( 11

190 ( 36

6300 ( 840

230 ( 60

260 ( 26

5c

23000 ( 5300

1800 ( 210

8400 ( 2300

270 ( 42

4700 ( 950

240 ( 89

3800 ( 1900

86 ( 5.5

7

0.55 ( 0.10

^aK_i( standard error of mean derived from three to nine experiments each done in triplicate. ^bK_i( standard deviation derived from two individual

experiments each done in triplicate.

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Table 2. Characterization of the High Aﬃnity Binding Site for (S)-4, (R)-4, (S)-5a, and (R)-5a Employing the Human Subtypes

D_2longand D₃Receptor

K_i( SEM (nM)^a

receptor

D_2long

(S)-4

(R)-4

(S)-5a

(R)-5a

[³H]spiperone

Hill Slope (n_H)

[³H]7-OH-DPAT

[³H]spiperone

Hill Slope (n_H)

[³H]7-OH-DPAT

410 ( 88

55000 ( 13000

-0.90 ( 0.067

57000 ( 34000^b

2100 ( 370

5.3 ( 0.55

300 ( 42

-0.51 ( 0.018

4.6 ( 0.65^b

31 ( 4.8

-0.51 ( 0.006

1.0 ( 0.24

-0.65 ( 0.075

49 ( 12^b

D₃

0.19 ( 0.024

-0.81 ( 0.033

0.027 ( 0.0044

15 ( 3.8

-0.93 ( 0.10

14 ( 4.3

-0.84 ( 0.079

4300 ( 1800^b

-0.84 ( 0.049

6.3 ( 1.2

^aK_i( standard error of mean derived from three to eight experiments each done in triplicate. ^bK_i( standard deviation derived from two individual

experiments each done in triplicate.

Figure 1. D_2longand D₃receptor mediated [³⁵S]GTPγS incorporation in the presence of PTX-insensitive GR_o1or GR_i2subunits as a matter of receptor

activation. The assay was performed on membrane preparations of transiently transfected HEK cells expressing given dopamine receptor and G_R

protein. Normalized curves from three to six experiments, each performed in eight replicates, are shown. The error bars represent the SEM.

0.027, and 6.3 nM for (S)-4, (S)-5a, and (R)-5a, respectively. An

exchange of one propyl unit of (S)-4 (K_i= 14 nM) by the

carboxamidobutyl function of (S)-5a caused a 520-fold increase

of binding with an outstanding K_iof 0.027 nM (Table 2). At the

D_2longreceptor, K_ivalues were 4.6, 1.0, and 49 nM for (S)-4, (S)-

5a, and (R)-5a, respectively. By employment of these values to

determine subtype selectivity, the reference dipropylamine 4

preferentially recognized D₂(3-fold). In contrast, the long chain

derivatives (S)-5a and (R)-5a showed a 37- and 8-fold D₃

selectivity over D_2longreceptor. Eudismic ratios of 49 and 230

were calculated for the subtypes D_2longand D₃, respectively,

indicating that the receptor ligand interactions of 5a strongly

depend on the absolute stereochemistry of the enantiomers.

Functional Characterization. To investigate the impact of

structural hybridization on the activity profiles of our newly

synthesized compounds at the D_2longand D₃receptors, we

measured the [³⁵S]GTPγS incorporation in the presence of

different pertussis toxin (PTX) insensitive GR subunits, the

inhibition of adenylyl cyclase, and the stimulation of

[³H]thymidine incorporation. We also investigated the influ-

ence of our test compounds on dopamine receptor internaliza-

tion in a biotin protection assay and in antibody-feeding

experiments.

(S)-5a displayed substantial ligand eﬃcacy. The EC₅₀values of

(S)-5a were 26 and 370 nM at the D_2long/GR_o1and D_2long/GR_i2

membrane preparations, respectively (Figure 1B and Figure 1C).

Despite signiﬁcant absolute diﬀerence in the EC₅₀values be-

tween assays performed on the D_2long/GR_o1and D_2long/GR_i2

membrane preparations, a comparison relative to the reference

quinpirole indicated that (S)-5a behaved similarly in both assays.

To put this observation in terms of numbers, the operational

model of agonism²⁸was applied to quantify the relative ability of

a ligand to produce a response in a given system. We used this

model previously to analyze ligand biased signaling at the D_2long

receptor.²⁹The operational model of agonism enables the

description and quantiﬁcation of agonist bias for a selected

signaling pathway in terms of aﬃnity and eﬃcacy.^30,31In this

model, the aﬃnity (K_A) describes an equilibrium dissociation

constant of an agonist-receptor complex and the transduction

coeﬃcient (τ) is a parameter encompassing both the eﬃcacy of

an agonist and the sensitivity of a system. The value of Δ log(τ/

K_A) relative to a chosen standard (e.g., full agonist like

quinpirole) for a system is calculated to quantify the relative

ability of each agonist to produce a response. The diﬀerence of

the Δ log(τ/K_A) between selected pathways yields ΔΔ log(τ/

K_A), which describes agonist bias or functional selectivity of a

given ligand. The quantiﬁcation of relative ability of (S)-5a to

produce agonistic response (Δ log(τ/K_A), relative to quinpirole)

gave values of 11 and 17 at D_2long/GR_o1and D_2long/GR_i2

membrane preparations, respectively (Supporting Information).

These comparable Δ log(τ/K_A) values implied that (S)-5a is not

functionally selective between GR_o1or GR_i2. Functional

Preferential coupling of the D_2longreceptor with GR_o1and

GR_i2subunits has been described.^26,27Thus, we intended to

measure D_2longreceptor mediated incorporation of [³⁵S]GTPγS

in the presence of PTX-insensitive GR_o1or GR_i2subunits. By use

of this system, the reference compound 2 behaved as

an antagonist. In contrast, our dopaminergic test compound

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Figure 2. Intrinsic activity of the compounds 2, (S)-4, (S)-5a, and (R)-5a at the D_2longand D₃receptors measured by inhibition of cAMP accumulation

and stimulation of [³H]thymidine incorporation (mitogenesis assay). Both assays were performed on whole CHO cells, stably expressing given

dopamine receptor. The cAMP production was stimulated with 20 μM forskolin (the D_2longreceptor) and 2 μM forskolin (the D₃receptor). Normalized

curves from three to six experiments, each performed in triplicate, are shown. The error bars represent the SEM.

selectivity at the level of GR subunits coupling was reported for

propylnorapomophine (NPA), dinapsoline, and S-(3)-PPP.^32,33

Because D₃couples almost exclusively with the GR_o1

subunit,²⁶[³⁵S]GTPγS incorporation was measured in the

presence of a coexpressed PTX-insensitive GR_o1subunit. There-

by, the reference compound 2 behaved as an antagonist

(Figure 1A) while formal exchange of the phenylpiperazine

moiety by the heterocyclic dipropylamine of type 4 yielded the

superpotent agonist (S)-5a with an EC₅₀of 0.056 nM . The

optical antipode (R)-5a behaved also as a full agonist but with

910-fold reduced potency (EC₅₀= 51 nM).

To further characterize the functional properties of our highly

potent agonist (S)-5a, D_2longand D₃receptor mediated inhibi-

tion of cAMP accumulation and the stimulation of [³H]-

thymidine incorporation were measured in CHO cells expressing

the corresponding receptor. For the D₃receptor, we reported

that coupling of enyne units with N-butyl-4-biphenylcarboxa-

mides resulted in a signiﬁcantly biased functional outcome.¹⁶

The enyne unit alone did not demonstrate any functional

selectivity between the selected pathways. When coupled with

N-butyl-4-biphenylcarboxamide, a strong ligand bias was ob-

served. This long chain compound strongly preferred the D₃

receptor mediated stimulation of [³H]thymidine incorporation,

where it behaved as a full agonist. Simultaneously it behaved only

as weak partial agonists in the inhibition of adenylyl cyclase.¹⁶

For the D_2longreceptor, we reported very recently that subtle

modiﬁcations of the phenylpiperazine moiety signiﬁcantly

modulate ligand biased signaling (functional selectivity).²⁹Here,

the reference compound 2 induced signiﬁcant ligand biased

signaling at the D_2longreceptor, behaving as an antagonist in

the inhibition of cAMP accumulation and as a partial agonist

(EC₅₀= 12 nM) in the stimulation of [³H]thymidine incorpora-

tion (Figure 2A, Figure 2B). This is in accordance with our

previous work, where we described the phenylpiperazine 2 as a

functionally selective ligand, strictly preferring the ERK1/2

pathway over cAMP signaling.²⁹The test compounds (S)-4

and (R)-5a were full agonists in both pathways investigated

but with a decreased potency compared with quinpirole

(Figure 2A, Figure 2B). The use of operational model of agonism

demonstrated that (S)-4 does not exhibit any ligand bias.

Compound (R)-5a showed some ligand bias (ΔΔ log(τ/K_A)),

preferring the stimulation of [³H]thymidine incorporation 80-

fold over the inhibition of cAMP accumulation. The ability of the

full agonist (S)-5a to inhibit cAMP accumulation (EC₅₀= 110

nM, Figure 2A) was weaker than that of quinpirole, whereas its

ability to stimulate [³H]thymidine incorporation (EC₅₀= 0.51

nM) was greater than that of the reference. According to the

operational model of agonism, biased signaling of (S)-5a resulted

in a 170-fold preference for the stimulation of [³H]thymidine

incorporation over the inhibition of cAMP accumulation.

At the D₃receptor with the exception of (S)-5a all compounds

displayed weaker agonist properties than quinpirole (Δ log(τ/

K_A) values negative) and no substantial ligand biased signaling

could be observed (Figure 2C and Figure 2D). Compared to

quinpirole for compound (S)-5a a 7-fold better agonism was

indicated in its ability to inhibit cAMP accumulation (Figure 2B).

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Figure 3. Ligand induced D_2longreceptor internalization. (A) FLAG-tagged D_2longreceptors expressed stably in HEK293 cells were incubated with M1

antibody followed by treatment with 10 μM 2 (upper panel, left), 10 μM 5a (upper panel, right), 10 μM quinpirole (lower panel, right) or left untreated

(lower panel, left). Treatment with quinpirole or 5a potently induced D_2longreceptor internalization (indicated with arrows). No internalization was

observed after incubation with 2. (B) HEK 293 cells stably expressing the FLAG-tagged D_2longreceptor were surface biotinylated (lane 1) and then

stripped (lane 2), incubated with 10 μM quinpirole (lane 3), left untreated (lane 4), incubated with 10 μM 5a (lane 5) or with 2 for 30 min (lane 6). The

bar chart represents the mean recovery of surface biotinylated receptors (relative to 30 min of stimulation with 10 μM quinpirole).

Because of the fact that (S)-5a stimulated [³H]thymidine

incorporation (EC₅₀= 0.0028 nM) with a 820-fold greater

potency than quinpirole (Figure 2D), this yields about 140-fold

functional selectivity of (S)-5a for the stimulation of [³H]thymi-

dine incorporation over the inhibition of cAMP accumulation

according to the operational model. One of the most potent

reported D₃receptor agonist (S)-N-(4-((2-amino-4,5,6,7-

tetrahydrobenzo[d]thiazol-6-yl)(propyl)amino)butyl)-2-naph-

thamide with a modest D₂/D₃selectivity and a binding aﬃnity of

0.043 nM on the D₃receptor was unfortunately tested only in a

mouse model, making the comparison of functional data not

possible.³⁴

To investigate the ability of racemic 5a to promote D_2long

receptor endocytosis, antibody feeding experiments were per-

formed. Therefore, live cells expressing the extracellular FLAG-

tag D_2longreceptor were incubated with the M1 antibody

directed against the FLAG epitope to label the mature pool of

receptors at the plasma membrane.³⁵Cells were then left

untreated or treated with quinpirole (positive control), 2 or 5a

at 10 μM, because in a typical antibody feeding experiment, cells

are stimulated with saturating concentrations of compounds.^35,36

Both quinpirole and 5a induce receptor internalization indicated

by translocation of the receptor from the plasma membrane to

the cytosol (Figure 3A). To quantify the internalization rate of

the D_2longreceptor after stimulation with the diﬀerent ligands, a

biotin protection/degradation assay, which measures internaliza-

tion of biotinylated receptors by inaccessibility to a membrane

impermeable reducing agent, was used. No internalization for the

phenylpiperazine 2 but a signiﬁcant endocytosis in response to

quinpirole and 5a could be observed, which is in good agreement

with the results of the antibody feeding experiment. By use of this

quantitative assay, it is possible to conclude that the internalized

receptor pool for quinpirole is similar to that of 5a (Figure 3B).

In contrast to the D_2longreceptor, the D₃subtype does not

undergo agonist-mediated internalization.^36,37In accordance

with these reports, we have not observed any change in the

receptor distribution upon the stimulation of the D₃receptor

with agonists quinpirole and 5a.

Mutagenesis of the D₃Receptor. Our previous SAR studies

on phenylpiperazines combining chemical synthesis, molecular

modeling, and site-directed mutagenesis demonstrated that

Asp3.32 and discrete amino acids interacting with the dopamine

simulating headgroup (e.g., Ser5.42, Ser5.46, Phe6.51, Phe6.52,

and His6.55) and with an affinity generating lipophilic appen-

dage (e.g., Val2.61, Leu2.64, Phe3.28, and Val3.29) importantly

modulate the binding of phenylpiperazines.³⁸These residues also

serve for the intermolecular recognition of the antagonist eticlo-

pride as evident from the D₃receptor crystal structure.²⁰

To investigate if the tetrahydropyrazolopyridines of types 4

and 5 utilize the same contacting amino acids as the phenylpi-

perazine 2, diagnostic residues were mutated to determine their

inﬂuence on binding aﬃnity. The residue Asp3.32, which inter-

acts with the ligands’ cationic ammonium center,^16,20,38was

mutated to glutamate.³⁸The aﬃnity of (S)-5a dropped drasti-

cally (11000-fold) underscoring the important contribution of

Asp3.32 to the binding aﬃnity of the extremely well aligned

ligand (Table 3). The aﬃnity of the compounds (S)-4 and (R)-

5a with poorer starting aﬃnities was reduced only up to 74-fold.

The conserved serines Ser5.42, Ser5.43, and Ser5.46 in TM5

represent an important dopamine receptor microdomain that is

involved in the hydrogen bonding with the catechol hydroxyl

groups of dopamine.^39-41Ser5.42 was described as the impor-

tant residue in the D₃receptor for the interaction with dopamine-

like agonists.³⁹As evident from the crystal structure, Ser5.42

interacts with the antagonist eticlopride.²⁰While the aﬃnities of

(S)-5a, (S)-4, and (R)-5a were attenuated 30-fold, 5.6-fold, and

13-fold, respectively, for Ser5.42, the mutation Ser5.46Ala had no

signiﬁcant inﬂuence on the aﬃnity of the compounds. This was

expected because our test compounds exhibit only one polar

function in their headgroups, which is obviously tied up in an

interaction with Ser5.42. The mutation His6.55Ala led to an

increase in aﬃnity of the 1,4-disubstituted phenypiperazine 2,

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Table 3. Binding Data of Compounds 2, (S)-4, (S)-5a, and (R)-5a at the D₃Wild Type and Mutant Receptors

K_i( SEM (nM)^a

receptor

2

(S)-4

150 ( 30

(S)-5a

(R)-5a

19 ( 3.8

D₃wt

0.90 ( 0.22

0.076 ( 0.0059

D₃D3.32E

D₃V2.61F

D₃S5.42A

D₃S5.46A

D₃H6.55A

790 ( 81 (880)

180 ( 5.0 (200)

2.8 ( 0.30 (3.1)

0.16 ( 0.010 (0.18)

0.58 ( 0.060 (0.64)

8100 ( 2000 (54)

420 ( 78 (2.8)

840 ( 100 (5.6)

45 ( 6.0 (0.30)

420 ( 73 (2.8)

850 ( 280 (11000)

170 ( 17 (2200)

2.3 ( 0.52 (30)

1400 ( 760 (74)

3700 ( 1100 (190)

240 ( 80 (13)

0.058 ( 0.0059 (0.76)

0.98 ( 0.079 (12)

15 ( 0.58 (0.79)

43 ( 6.7 (2.3)

^aData derived from competition binding experiments with [³H]spiperone at D₃wild type and mutant receptors transiently expressed in HEK 293 cells.

Values are given as mean K_i( SEM of 3-12 independent experiments each done in triplicate. Fold changes in aﬃnity compared to wild type are

indicated in parentheses.

which was more distinct for our previously characterized

phenylpiperazines.³⁸On the other hand the aﬃnities of the

dipropylamine (S)-4 and its derivatives (S)-5a and (R)-5a

decreased at the His6.55Ala receptor (3-, 12-, and 2-fold,

respectively). The activation of 7TM receptors encompasses a

movement of transmembrane helices in particular of TM6 in a

rigid-body fashion making vertical “see-saw” movement toward

TM3 and resulting in a closing around the main ligand-binding

pocket at the extracellular side and an opening at the intracellular

side for G-protein binding.^42-45We speculate that during the

activation and the transition of the receptor into the high aﬃnity

state (coupled with trimeric G-proteins) agonists favorably

interact with His6.55 whereas repulsive interactions perturb

conformational changes of antagonist bound complexes. Appar-

ently, the mutation His6.55Ala nulliﬁes beneﬁcial interactions

between His6.55 and the agonists of types 4 and 5 that take place

during receptor activation.

To observe binding interactions of the ligands’ lipophilic

appendages, Val2.61 was mutated by the more sterically demand-

ing phenylalanine.^20,38,46-48The mutation Val2.61Phe substan-

tially attenuated ligand binding of the long chain derivatives 2,

(S)-5, and (R)-5 leading to a 200-, 2200-, and 190-fold reduction

of aﬃnity, respectively. On the other hand, D₃recognition of the

dipropylamine (S)-4 was only slightly inﬂuenced. These data

indicate that the introduction of the sterically more demanding

phenylalanine at position 2.61 hinders the binding of long chain

ligands at the D₃receptor, thus restricting the optimal length of

synthetic ligands and contributing to D₂/D₃/D₄selectivity. The

identiﬁcation of a critical ligand length that induces a clash with

the mutation Val2.61Phe will give valuable insights into the

determinants for D₂, D₃, D₄subtype selectivity and thus for a

rational drug design.

Ligand Docking and Analysis. To better understand the

binding modes of our test compounds and the results of the

mutagenesis experiments at the D₃receptor, ligand docking and

a careful analysis of the obtained complexes were performed. The

tetrahydropyrazolopyridines (R)-5a and (S)-5a and the refer-

ence phenylpiperazine 2 were docked into the crystal structure of

the recently published dopamine D₃receptor by means of

AUTODOCK4.^20,49-51Ligands were subjected in protonated

form at the basic amine to enable the formation of the essential

charge reinforced hydrogen bond between ligand and Asp3.32 in

TM3. The protonation of the tertiary amine of (R)-5a and (S)-5a

resulted in two diastereoisomers, which bound differently to the

D₃receptor structure with respect of the orientation of the

propyl moiety. The (R)-N isomer of (R)-5a and (S)-5a formed a

receptor-ligand complex in which the propyl group points

toward TM7 enclosed by Asp3.32, Trp6.48, and Thr7.39. In

contrast, the propyl moiety of the (S)-N isomer was positioned

close to TM3 surrounded by Leu3.29, Asp3.32, and Val3.33.

According to earlier studies on the binding mode of dopamine

receptor agonists, the propyl moiety binds to a “propyl cleft” that

extends from Asp3.32 of TM3 toward TM2 and TM7.⁵²Only the

(R)-N isomer of (R)-5a and (S)-5a possesses the possibility to

occupy this microdomain and was therefore chosen for further

analysis.

Docking of the phenylpiperazine 2resulted in docking poses that

were similar to receptor-ligand complexes of related phenylpiper-

azine derivatives.³⁸On the basis of DrugScore^onlinescoring values⁵³

and experimental data, a ﬁnal complex was selected and subjected

to energy minimization. Docking of (R)-5a and (S)-5a resulted in

two ligand-receptor complexes in each case, which showed a

distinct position and orientation of the tetrahydropyrazolopyridine

moiety. All four complexes were submitted to energy minimization.

On the basis of a careful analysis of the complexes and recent D₃

mutagenesis data, the receptor-ligand complexes presented in

Figure 4 were chosen for further investigations. Conserved serine

residues located at positions 5.42, 5.43, and 5.46 in TM5 play an

important role in the binding dopamine-like agonists and the

dopamine receptor activation.^39-41The side chain conformation

of Ser5.42 enabled formation of a hydrogen bond with the lone pair

of the tetrahydropyrazolopyridine moiety of (R)-5a and (S)-5a. In

accordance with the crystal structure of the β₂-adrenergic receptor

bound to the agonist FAUC 50,⁵⁴the side chain of Ser5.43 was

rearranged before energy minimization, yielding a conformation

where it points into the binding pocket.

As expected from recent crystal structures, analysis of the ﬁnal

receptor-ligand complex models showed a polar interaction

between Asp3.32 and the cationic ammonium group. The

tetrahydropyrazolopyridine head groups of (R)-5a and (S)-5a

(Figure 4B) formed hydrophobic interactions with the amino

acids residues Val3.33, Ser5.46, Phe6.51, Phe6.52, and His6.55.

An analogous binding mode was observed for the phenylpiper-

azine moiety of our reference compound 2 (Figure 4A); the

methoxy group was located in proximity to His6.55. This is

diﬀerent for the tetrahydropyrazololpyridine headgroup of (R)-

5a and (S)-5a, which is more distant to this residue. These results

are in agreement with our mutagenesis data, when a higher

susceptibility indicated diﬀerential binding properties of phenyl-

piperazines and (R)-5a/(S)-5a.³⁸In the region of TM5 we

observed that our reference compound 2 does not form hydro-

gen bonds to serine residues whereas the endocyclic nitrogen of

the tetrahydropyrazolopyridine moiety of (R)-5a and (S)-5a

forms a hydrogen bond interaction with Ser5.42, but not with

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Journal of Medicinal Chemistry

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Figure 4. Reference ligand 2 (A) and test compounds (R)-5a and (S)-5a (B and C) docked into the crystal structure of the dopamine D3 receptor.

Residues involved in ligand binding and the ligands are represented as sticks. The D3 receptor backbone is represented as yellow ribbons. 2 is indicated in

violet. (R)-5a is colored in green, and (S)-5a is shown in orange. Residues involved in mutagenesis experiments are presented in blue.

displacement assays using [³H]spiperone. As a molecular probe

Table 4. Inﬂuence of the Mutation Val2.61Phe in the D₃

to determine the critical length of ligands, the achiral aminoin-

dane molecular scaﬀold was chosen. Aminoindane derivatives are

easily accessible and were previously described as high aﬃnity

agonists for D₂-like receptors.^55,56Starting from the 2-N,N-

dipropylaminoindane⁵⁵core structure (17), the length of the

appendage was gradually increased to reach the size of the N-

butylbiphenylcarboxamide substituted aminoindane⁵⁶21 as

depicted in Table 4.

Receptor on the Binding Aﬃnity of the Ligands 17-21

Depending on the Size of Their Lipophilic Appendage

After characterization of the molecular probes 17-21 toward

diagnostic D₃mutants (Supporting Information) binding aﬃ-

nities of the 2-dipropylaminoindane 17 and its methyl (18), butyl

(19), phenyl (20), and biphenyl (21) carboxamidomethyl sub-

stituted derivatives were tested at D₃wild type compared to the

Val2.61Phe mutant (Table 4). The mutation Val2.61Phe had no

inﬂuence on the binding aﬃnity of 2-dipropylaminoindane (17)

and the methylcarboxamidomethyl substituted derivative 18.

Further elongation of the appendage by formal exchange of the

methyl residue by a butyl group at the carboxamide caused

signiﬁcant susceptibility of the test compound 19 toward the

Val2.61Phe mutation, resulting in a 17-fold reduction of binding

aﬃnity. Evaluation of the benzamide 20 indicated that a sterically

more demanding phenyl substituent exerts substantial repulsion

with the phenylalanine residue in position 2.61, leading to an

almost 100-fold increase of the K_i. Finally, the biphenylcarbox-

amide 21 also displayed strongly impaired binding (230-fold

reduction) that was comparable to the susceptibility of the

biphenylcarboxamides of type 2 and 5. Overall, however, the

major interactions between our long chain ligands and the amino

acid residue in position 2.61 of the D₃receptor appear to

originate in the substituent that is directly bound to the CO

group of the aminocarbonyl function.

K_i( SEM (nM)^a

susceptibility^b

D₃wt D₃V2.61F K_i^mut/K_i^wt

compd

R

17

H

230 ( 87 350 ( 87

1.5

0.8

17

18 methylcarbonylaminomethyl 410 ( 65 330 ( 57

19 butylcarbonylaminomethyl 44 ( 11 760 ( 89

20 benzylcarbonylaminomethyl 2.7 ( 0.6 250 ( 40

21 biphenylcarbonylaminomethyl 1.0 ( 0.2 230 ( 48

93

230

^aBinding data were determined in competition binding experiments

with [³H]spiperone at the D₃wild type and the D₃Val2.61Phe mutant

receptors transiently expressed in HEK 293 cells. Values given are mean

K_i( SEM of three to eight independent experiments each done in

triplicate; ^bSusceptibility of the mutation on ligand binding expressed by

the ratio K_i^mut/K_i^wt, changes in aﬃnity compared to wild type.

Ser5.43. These results were also supported by our mutagenesis

experiments.

Receptor binding studies revealed a 250-fold better aﬃnity of

the eutomer (S)-5a to the D₃receptor compared to the distomer

(R)-5a. According to our mutagenesis experiments at D₃,

Asp3.32Glu exchange led to a drastic decrease in aﬃnity of

(S)-5a, whereas (R)-5a reacted less sensitively to this mutation.

The comparison of the docking poses (Figure 4C) showed that

eutomer and distomer of 5a diﬀer in position and orientation of

the ammonium center, the propyl moiety and the chain linking

the biphenylcarboxamide moiety. Diﬀerent binding energies

resulting from the distinct orientations would explain the re-

duced D₃aﬃnity and decreased sensitivity of (R)-5a toward

Asp3.32Glu D₃mutation.

The N-butylbiphenylcarboxamide appendage of all three

ligands is in proximity to a microdomain that consists of residues

at position 2.61, 2.64, 3.28, and 3.29 at TM2 and TM3, which is

known to be responsible for D₂/D₃/D₄selectivity.^20,38,46-48

To better understand these interactions, a series of com-

pounds was synthesized and investigated, in which we gradually

elongated the chain length, and was tested in the radioligand

’ CONCLUSIONS

To learn more about the structural origins of the diﬀerential

activity proﬁles, we could show that a formal structural hybridiza-

tion of dopaminergic dipropylamines of type 1 with lipophilic

appendages leads to novel dopaminergic chemotypes of types 5

and 7. The biphenylcarboxamide (S)-5a displayed an outstand-

ing K_iof 27 pM at the agonist-labeled D₃receptor and signiﬁcant

selectivity over the D₂subtype. Measurement of [³⁵S]GTPγS

incorporation in the presence of a coexpressed PTX-insensitive

GR_o1subunit indicated highly eﬃcient G-protein coupling.

Comparison of ligand eﬃcacy data from cAMP accumulation

and [³H]thymidine incorporation experiments revealed that the

test compound (S)-5a displays ligand biased signaling with a

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Journal of Medicinal Chemistry

ARTICLE

signiﬁcant preference for [³H]thymidine incorporation. This is

in accordance to our recent observations with enyne-derived

dopaminergics.¹⁶

Hadleigh, U.K.) at a flow of 0.5 mL/min of isopropanol/hexane 1/1:

t_R=22.8minfor(R)-5aand t_R=20.6minfor(S)-5a. IR: 3320, 2950, 2930,

1640, 1550, 1490, 1300, 700 cm^-1. ¹H NMR (CDCl₃, 360 MHz) δ0.89 (t,

J = 7.5 Hz, 3H), 1.41-1.72 (m, 6H), 1.92-2.04 (m, 1H), 2.11-2.17 (m,

1H), 2.47 (t, J = 7.5 Hz, 2H), 2.56 (t, J = 7.1 Hz, 2H), 2.68 (dd, J = 16.0,

11.0 Hz, 1H), 2.96 (dd, J = 16.0, 4.6 Hz, 1H), 3.05-3.13 (m, 1H), 3.47-

3.53 (m, 2H), 4.04 (ddd, J= 12.7, 12.3, 4.9 Hz, 1H), 4.38 (ddd, J= 12.7, 5.6,

2.2 Hz, 1H), 5.96 (d, J = 1.8 Hz, 1H), 6.29 (br s, 1H), 7.36-7.49 (m, 4H),

7.59-7.67 (m, 4H,), 7.83 (br d, J = 8.5 Hz, 2H). EIMS m/z 430. Anal.

(C₂₇H₃₄N₄O) C, H, N. (R)-5a: [R]²_D⁰36.8° (c 0.5, CHCl₃). (S)-5a: [R]²_D⁰

-36.3° (c 0.5, CHCl₃).

Starting from the D₃crystal structure,²⁰a combination of

homology modeling and site directed mutagenesis gave valuable

insights into the binding mode and the intermolecular origins of

stereospeciﬁc receptor recognition. The concept of biased ligands

and functional selectivity at the 7TM receptors raises the possi-

bility of developing chemical probes and drugs with precisely

deﬁned properties.⁵⁷Mutagenesis data indicate that the eutomer

can better take advantage from the charge-enforced hydrogen

bond with the carboxylate function of Asp3.32. Thus, the favor-

able binding energy that results from the interaction between the

ligand’s central ammonium unit and the aspartate residue in

position 3.32 obviously controls stereospeciﬁcity of binding.

Recent progress in the structural biology of the 7TM receptors

provided important clues about the agonist-bound states of the

β2 receptor.^54,58,59The crystal structure of the 7TM receptor

cocrystallized with both enantiomers of a very potent agonist like

5a could provide most desirable structural information about the

stereospeciﬁcity of binding.

N-(4-(4-Phenylbenzoylamino)butyl)-N-propyl-5-amino-3-

chloro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine (5b). To a

solution of 5a (16 mg, 0.037 mmol) in CHCl₃(4 mL) was added N-

chlorosuccinimide (10 mg, 0.075 mmol). The mixture was refluxed for

16 h, evaporated, and the residue was purified by flash chromatography

(CH₂Cl₂/MeOH 95/5) to give 5b (12 mg, 69%) as a colorless oil. IR:

3320, 2930, 1640, 1550, 1490, 1350, 1010, 750 cm^-1. ¹H NMR(CDCl₃,

360 MHz) δ 0.89 (t, J = 7.3 Hz, 3H), 1.42-1.72 (m, 6H), 1.91-2.03 (m,

1H), 2.09-2.16 (m, 1H), 2.45-2.59 (m, 5H), 2.90 (dd, J = 16.3, 5.2 Hz,

1H), 3.05-3.13 (m, 1H), 3.48-3.53 (m, 2H), 4.01 (ddd, J = 12.7, 12.3,

4.6 Hz, 1H), 4.32 (ddd, J = 12.7, 5.4, 2.3 Hz, 1H), 6.25 (br s, 1H), 7.37 (s,

1H), 7.37-7.41 (m, 1H), 7.43-7.48 (m, 2H), 7.59-7.67 (m, 4H), 7.83

(br d, J = 8.6 Hz, 2H). MS m/z 464. Anal. (C₂₇H₃₃ClN₄O) C, H, N.

N-(4-(4-Phenylbenzoylamino)butyl)-N-propyl-5-amino-4,

5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carbaldehyde

(5c). To a solution of 5a (20 mg, 0.047 mmol) in DMF (2 mL), POCl₃

(16 mg, 0.1 mmol) was added. The mixture was stirred at 90 °C for 2 h.

Then H₂O and 2 N NaOH were added. The mixture was extracted with

CHCl₃, and the organic layer was dried (MgSO₄) and evaporated. The

residue was purified by flash chromatography (CH₂Cl₂/MeOH 97/3)

to give 5c (14 mg, 65%) as a colorless oil. IR: 3340, 2930, 1640, 1540,

1510, 1440, 1070, 750 cm^-1. ¹H NMR (CDCl₃, 360 MHz) δ 0.89 (t, J =

7.4 Hz, 3H), 1.42-1.72 (m, 6H), 1.97-2.09 (m, 1H), 2.15-2.20 (m,

1H), 2.48 (t, J = 7.4 Hz, 2H), 2.58 (t, J = 7.0 Hz, 2H), 2.89 (dd, J = 17.4,

10.3 Hz, 1H), 3.10-3.17 (m, 1H), 3.33 (dd, J = 17.4, 5.3 Hz, 1H), 3.47-

3.53 (m, 2H), 4.10 (ddd, J = 13.3, 12.7, 5.4 Hz, 1H), 4.39 (ddd, J = 13.3,

5.3, 2.5 Hz, 1H), 6.25 (br s, 1H), 7.36-7.48 (m, 3H), 7.59-7.67 (m,

4H), 7.83 (br d, J = 8.4 Hz, 2H), 7.87 (s, 1H), 9.84 (s, 1H). MS m/z 458.

Anal. (C₂₈H₃₄N₄O) C, H, N.

’ EXPERIMENTAL SECTION

Chemistry. All reactions were carried out under nitrogen atmos-

phere. Dry solvents and reagents were commercial quality and used as

purchased. MS were run on a Finnigan MAT TSQ 700 spectrometer by

EI (70 eV) with solid inlet or with an ion-trap mass with APCI

ionization. HR-EIMS were run on a Finnigan MAT 8200 using peak

Peak-Matching (M/ΔM = 10000). NMR spectra were obtained on a

Bruker Avance 360 or a Bruker Avance 600 spectrometer relative to

TMS in the solvents indicated (J in Hz). IR spectra were performed on a

Jasco FT/IR 410 spectrometer using a film of substance on a NaCl pill.

Purification by flash chromatography was performed using silica gel 60 if

not stated otherwise. TLC analyses were performed using Merck 60

F254 aluminum sheets and analyzed by UV light (254 nm) or by

spraying with ninhydrin reagent. Analytical HPLC/MS was performed

on Agilent 1100 HPLC systems employing a VWL detector connected

to a Bruker Esquire 2000. A Zorbax SB-C18 (4.6 mm i.d. ꢀ 150 mm, 5

μm) was used as the column and run with a flow rate of 0.5 mL/min

(eluent methanol/water/0.1 N formic acid, 10% methanol for 3 min to

100% methanol in 15 min, 100% for 6 min to 10% in 3 min, 10% for 3

min). CHN elementary analyses were performed at the Chair of Organic

Chemistry, Friedrich Alexander University Erlangen-N€urnberg. HPLC

was run with MeOH (eluent I) and 0.1% aqueous formic acid (eluent II)

and the following gradient: MeOH 10% for 3 min ascending to 100% in

15 min, 100% for 6 min. Flow rate was 0.5 mL/min, and λ = 254 nm. All

SAR compounds were determined to be >95% pure.

N-(4-(4-Phenylbenzoylamino)butyl)-N-propyl-5-amino-4,5,

6,7-tetrahydropyrazolo[1,5-a]pyridine (5a), (R)-N-(4-(4-Phen-

ylbenzoylamino)butyl)-N-propyl-5-amino-4,5,6,7-tetrahydro-

pyrazolo[1,5-a]pyridine ((R)-5a), and (S)-N-(4-(4-Phenylben-

zoylamino)butyl)-N-propyl-5-amino-4,5,6,7-tetrahydropyra-

zolo[1,5-a]pyridine ((S)-5a). To a solution of 10, (R)-10, or (S)-10

(17 mg, 0.095 mmol) in 1,2-dichlorethane (2 mL) was added 4-(4-

phenylbenzoylamino)butyraldehyde²³(25 mg, 0.095 mmol) and sodium

triacetoxyborohydride (30 mg, 0.14 mmol). The solution was stirred at

room temperature for 18 h and extracted with saturated NaHCO₃. The

organic layer was dried (MgSO₄) and evaporated, and the residue was

purified by flash chromatography to give pure 5a, (R)-5a, or (S)-5a,

respectively (35 mg, 85%), as a colorless oil. Alternatively, enantiopure

(R)-5a and (S)-5a could be obtained by chiral resolution of racemic 5a

employing HPLC separation with a Chiralcel OD column (Dycel,

N-(4-(4-Phenylbenzoylamino)butyl)-N-propyl-4-amino-4,

5-dihydro-3H-pyrazolo[4,5,1-i,j]quinoline (7). (1) To a solution

of 16 (40 mg, 0.23 mmol) in MeOH (5.5 mL) was added propionic

aldehyde (135 mg, 2.32 mmol) and sodium cyanoborohydride (29 mg,

0.46 mmol). After the mixture was stirred for 45 min at 0 °C, aqueous

HCl (1 N, 2 mL) and saturated NaHCO₃(6 mL) were added. After

addition of diethyl ether, the organic layer was dried (MgSO₄) and

evaporated and the residue was purified by flash chromatography

(CH₂Cl₂/MeOH 97/3) to give pure 4-propylamino-4,5-dihydro-3H-

pyrazolo[4,5,1-i,j]quinoline (26 mg, 53%) as a colorless oil.²¹

(2) To a solution of 4-propylamino-4,5-dihydro-3H-pyrazolo[4,5,1-i,

j]quinoline (20 mg, 0.093 mmol) in 1,2-dichloroethane (2 mL), 4-(4-

phenylbenzoylamino)butyraldehyde²³(25 mg, 0.093 mmol) and so-

dium triacetoxyborohydride (30 mg, 0.14 mmol) were added. The

mixture was stirred at room temperature for 18 h. Then saturated

NaHCO₃and CH₂Cl₂were added. The organic layer was dried

(MgSO₄) and evaporated and the residue was puriﬁed by ﬂash chro-

matography (CH₂Cl₂/MeOH 95/5) to give 7 (38 mg, 87%) as a

1

colorless oil. IR: 3315, 2930, 1640, 1550, 1070, 750 cm^-1. H NMR

(CDCl₃, 360 MHz) δ 0.90 (t, J = 7.4 Hz, 3H), 1.43-1.75 (m, 6H), 2.54

(t, J = 7.3 Hz, 2H), 2.64 (t, J = 6.8 Hz, 2H), 2.79-3.02 (m, 4H), 3.21-

3.30 (m, 1H), 3.48-3.53 (m, 2H), 6.32 (br s, 1H), 6.60 (dd, J = 6.8, 6.6

Hz, 1H), 6.67 (d, J = 6.6 Hz, 1H), 7.35-7.40 (m, 1H), 7.43-7.48 (m,

2H), 7.58-7.61 (m, 2H), 7.62 (br d, J = 8.4 Hz, 2H), 7.67 (s, 1H, H-2),

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7.82 (br d, J = 8.4 Hz, 2H), 8.17 (d, J = 6.8 Hz, 1H). MS m/z 466. Anal.

(C₃₀H₃₄N₄O) C, H, N.

powder (300 mg) and acetic acid (3 mL). The mixture was stirred at

room temperature for 3 days. Then it was ﬁltered with Celite and

evaporated. After addition of water, aqueous NaOH (20%), and

CH₂Cl₂, the organic layer was extracted with saturated NaCl solution.

The organic layer was dried (MgSO₄) and evaporated and the residue

was puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 8/2) to give

pure 16²¹(140 mg, 65%) as a colorless oil.

5-Propylamino-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine

(10), (R)-5-Propylamino-4,5,6,7-tetrahydropyrazolo[1,5-a]

pyridine ((R)-10), and (S)-5-Propylamino-4,5,6,7-tetrahy

dropyrazolo[1,5-a]pyridine ((S)-10). A mixture of 9, (R)-9 or

(S)-9 (20 mg, 0.15 mmol), sodium cyanoborohydride (18.6 mg, 0.29

mmol), propionic aldehyde (106 μL, 1.5 mmol), and MeOH (4 mL) was

stirred at 0 °C for 45 min. After addition of HCl (1 N, 3 mL) the mixture

was basified with aqueous NaOH (2 N). After addition of diethyl ether

the organic layer was dried (MgSO₄), evaporated and the residue was

purified by flash chromatography (CH₂Cl₂/MeOH 9/1) to give pure 10,

(R)-10 or (S)-10 (17 mg, 65%), as a colorless oil. IR: 2960, 2920, 2780,

2730, 1440, 1320, 710 cm^-1. ¹H NMR (CDCl₃, 360 MHz) δ 0.87 (t, J =

7.4 Hz, 3H), 1.40-1.53 (m, 2H), 1.88-2.02 (m, 1H), 2.09-2.15 (m,

1H), 2.46 (t, J = 7.4 Hz, 2H), 2.65 (dd, J = 15.4, 11.0 Hz, 1H), 2.92 (dd,

J = 15.4, 4.7 Hz, 1H), 3.01-3.15 (m, 1H), 4.02 (ddd, J = 12.9, 12.2Hz, 4.5

Hz, 1H), 4.35 (ddd, J = 12.9, 5.6, 2.3 Hz, 1H), 5.96 (br d, J = 1.8 Hz, 1H),

7.42 (d, J = 1.8 Hz, 1H). MS m/z 179. Anal. (C₁₀H₁₇N₃) C, H, N. (R)-9:

[R]²_D⁰47.4° (c 0.5, CHCl₃). (S)-9: [R]_D²⁰-47.1° (c 0.5, CHCl₃).

4-(2-Nitrovinyl)pyrazolo[1,5-a]pyridine (14). To a mixture of

13 (590 mg, 4.0 mmol) in MeOH (6.5 mL) were added nitromethane

(0.24 mL, 4.4 mmol), methylammonium chloride (100 mg), and

potassium acetate (100 mg). The mixture was stirred for 3 days at room

temperature. Then the precipitate was filtered and dried to leave pure 14

(610 mg, 80%) as a colorless solid (mp 162 °C). IR: 2360, 1640, 1510,

1330, 1190, 97 ss, 760 cm^-1. ¹H NMR (CDCl₃, 360 MHz) δ 6.74-6.75

(br d, J = 2.5 Hz, 1H), 6.72 (dd, J = 7.1, 7.1 Hz, 1H), 7.45 (d, J = 7.1 Hz,

1H), 7.76 (d, 13.8 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H), 8.16 (d, J = 13.8 Hz,

1H), 8.61-8.63 (br d, J = 7.1 Hz, 1H). MS m/z 189. Anal. (C₉H₇N₃O₂)

C, H, N.

4-Nitro-5H-pyrazolo[4,5,1-i,j]quinoline (15). (1) To a solu-

tion of 14 (2.0 g, 11 mmol) in MeOH (110 mL) was added sodium

cyanoborohydride (1.3 g, 34 mmol) within 20 min. After being stirred

for 20 min, the mixture was acidified to pH 5 by acidic acid at 0 °C. After

evaporation CH₂Cl₂and H₂O were added to the residue. The organic

layer was dried (MgSO₄) and evaporated and the residue was purified by

flash chromatography (hexane/EtOAc 2/1) to give pure 4-(2-

nitroethyl)pyrazolo[1,5-a]pyridine (1.7 g, 82%).

(2) To DMF (1 mL) was slowly added POCl₃(88 μL, 0.96 mmol)

under N₂atmosphere. Then a solution of 4-(2-nitroethyl)pyrazolo[1,5-

a]pyridine (160 mg, 0.83 mmol) in DMF (1 mL) was slowly added at

0 °C. The mixture was allowed to warm to room temperature and stirred

for further 15 min. Then H₂O (0.17 mL, 9.2 mmol) and DMF (0.70 mL)

were added. After 15 min a mixture of triethylamine (1.2 mL, 8.4 mmol)

and MeOH (3.3 mL) was added at 0 °C. After 15 min saturated citric

acid and ethyl acetate were added. The organic layer was dried (MgSO₄)

and evaporated and the residue was puriﬁed by ﬂash chromatography

(EtOAc/hexane 2/8) to give pure 15 (86 mg, 51%) as a colorless solid

(mp 202 °C). IR: 3354, 1634, 1588, 1463, 955 cm^-1. ¹H NMR (CDCl₃,

360 MHz) δ 4.53 (br s, 2H), 6.88 (dd, J = 7.1, 6.7 Hz, 1H), 7.05 (br d, J =

7.1 Hz, 1H), 7.94 (s, 1H), 8.12 (br s, 1H), 8.22 (br d, J = 6.7 Hz, 1H). MS

m/z 201. Anal. (C₁₀H₇N₃O₂) C, H, N.

N-[(N⁰-Indan-2-yl-N⁰-propyl)-4-aminobutyl]methylcarbox

amide (18). A solution of (N-indan-2-yl-N-propyl)butane-1,4-diamine

(103 mg, 0.42 mmol) (synthesized as previously described⁵⁶) in CHCl₃

(10 mL) was cooled to 0 °C before triethylamine (100 μL, 0.72 mmol)

and acetyl chloride (50 μL, 0.70 mmol) were added. Then the mixture

was allowed to warm to room temperature and stirred for 3 h before

another equivalent of acetyl chloride (50 μL, 0.70 mmol) was added.

The mixture was stirred for a further 2 h. Then aqueous NaHCO₃was

added. The aqueous layer was extracted with CH₂Cl₂, and the combined

organic layers were dried (Mg₂SO₄) and evaporated. The residue was

purified by flash chromatography (CH₂Cl₂/MeOH 9/1 þ 1% NEt₂Me)

to give 18 as a brown oil (113 mg, 93%). IR 3297, 2956, 2935, 2870,

1

1651, 1558, 1460, 1369, 1297, 744, 638 cm^-1. H NMR (360 MHz,

CDCl₃) δ 0.89 (t, J = 7.4 Hz, 3H), 1.43-1.60 (m, 6H), 1.96 (s, 3H),

2.47-2.59 (m, 4H), 2.90 (dd, J = 15.4, 8.6 Hz, 2H), 3.03 (dd, J = 15.3,

7.7 Hz, 2H), 3.26 (m, 2H), 3.68 (m, 1H), 5.99 (m, 1H), 7.11-7.20 (m,

4H). ¹³C NMR (90 MHz, CDCl₃) δ 12.0, 19.9, 23.4, 25.0, 27.8, 36.4,

39.6, 51.0, 53.3, 62.9, 124.5, 126.4, 141.7, 169.9. HR-EIMS calcd m/z for

C₁₈H₂₈N₂O, 288.2202; found 288.2202.

N-[(N⁰-Indan-2-yl-N⁰-propyl)-4-aminobutyl]butylcarbox

amide (19). Compound 19 was prepared according to the protocol of

18 using a solution of (N-indan-2-yl-N-propyl)butane-1,4-diamine (146

mg, 0.59 mmol) (synthesized as previously described⁵⁶) and triethyla-

mine (150 μL, 1.1 mmol) and butyrylchloride (100 μL, 0.96 mmol).

Purification by flash chromatography (hexane/EtOAc 1/2 þ 0.5%

NEt₂Me) gave 19 as a brown oil (46 mg, 24%). IR 3293, 3072, 2958,

1

2933, 2871, 1644, 1552, 1459, 1074, 742 cm^-1. H NMR (360 MHz,

CDCl₃) δ 0.88 (t, J = 7.3 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H), 1.43-1.55

(m, 4H), 1.60-1.71 (m, 4H), 2.12 (t, J = 7.5 Hz, 2H), 2.46-2.56 (m,

4H), 2.87 (dd, J = 15.4, 8.7 Hz, 2H), 3.01 (dd, J = 15.2, 7.7 Hz, 2H), 3.27

(m, 2H), 3.66 (m, 1H), 5.79 (m, 1H), 7.09-7.20 (m, 4H). ¹³C NMR

(90 MHz, CDCl₃) δ 12.0, 13.8, 19.2, 20.1, 25.0, 27.9, 36.5, 38.8, 39.4,

51.0, 53.4, 63.0, 124.4, 126.3, 141.8, 172.8. HR-EIMS calcd m/z for

C₂₀H₃₂N₂O, 316.2515; found 316.2515.

N-[(N⁰-Indan-2-yl-N⁰-propyl)-4-aminobutyl]phenylcarbo-

xamide (20). Compound 20 was prepared according to the protocol of

18 using a solution of (N-indan-2-yl-N-propyl)butane-1,4-diamine (100

mg, 0.41 mmol) (synthesized as previously described⁵⁶) and triethyla-

mine (170 μL, 1.2 mmol) and benzoylchloride (50 μL, 0.41 mmol).

Purification by flash chromatography (hexane/EtOAc 1/4 þ 0.5%

TEA) gave 20 as a colorless oil (45 mg, 24%). IR 3320, 2934, 2869,

1638, 1577, 1543, 1489, 1308, 1075 cm^-1. ¹H NMR (360 MHz, CDCl₃)

δ 0.87 (t, J = 7.4 Hz, 3H), 1.49 (m, 2H), 1.72-1.55 (m, 4H), 2.52 (m,

2H), 2.58 (m, 2H), 2.87 (dd, J = 15.4, 8.6 Hz, 2H), 3.00 (dd, J = 15.2, 7.7

Hz, 2H), 3.48 (m, 2H), 3.68 (m, 1H), 6.51 (m, 1H), 7.09-7.19 (m, 4H),

7.35-7.50 (m, 3H), 7.71-7.86 (m, 2H). ¹³C NMR (150 MHz, CDCl₃)

δ 12.0, 19.9, 25.1, 27.8, 36.4, 40.1, 51.0, 53.4, 63.0, 124.4, 126.3, 126.9,

128.5, 131.2, 125.0, 141.8, 167.7. HR-EIMS calcd m/z for C₂₃H₃₀N₂O,

350.2358; found 350.2358.

4-Amino-4,5-dihydro-3H-pyrazolo[4,5,1-i,j]quinoline

(16). (1) To a solution of 15 (440 mg, 2.2 mmol) in MeOH (35 mL)

was added sodium borohydride (1.3 g, 33 mmol) over 20 min. After

further 20 min the mixture was acidified to pH 5 by acetic acid at 0 °C.

Then the mixture was evaporated, and H₂O and CH₂Cl₂were added to

the residue. The organic layer was dried (MgSO₄) and evaporated and

the residue was purified by flash chromatography (hexane/EtOAc 2/1)

to give pure 4-nitro-4,5-dihydro-3H-pyrazolo[4,5,1-i,j]quinoline (360

mg, 81%) as a colorless solid.

Residue Numbering Scheme. For reasons of clarity amino acids

positions are numbered according to Ballesteros and Weinstein.⁶⁰

Residues are numbered consecutively as [TMx].n relative to the most

conserved residues within each TM, which is designed as [TMx].50.

Cell Culture and Transfection. Human embryonic kidney

(HEK) 293 cells (American Type Culture Collection) were grown in

DMEM/F-12 supplemented with 10% FBS, 2 mM ^L-glutamine, 1%

penicillin-streptomycin. The cells were transiently transfected with the

(2) To

a solution of 4-nitro-4,5-dihydro-3H-pyrazolo[4,5,1-i,

j]quinoline (250 mg, 1.2 mmol) in EtOH (12 mL) were added tin

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corresponding cDNA by the CaHPO₄method or using the TransIT-293

transfection reagent (Mirus Bio Corporation, Madison, WI). N-Term-

inal FLAG-tagged dopamine D_2longreceptor constructs⁶¹were stably

expressed in HEK 293 cells. For generation of stable monoclonal cell

lines single colonies were chosen, propagated in the presence of

selection-containing media (200 μg/mL Zeocin), and characterized

for the expression of FLAG-D_2longreceptor. The generation and

maintenance of CHO cell stably expressing D_2long, D_2short, D₃, and

D_4.4receptor was described previously.^56,62-64

Site Directed Mutagenesis. The D₃receptor wild type and

D₃D3.32E, D₃V2.61F, D₃L2.64F, D₃FV3.28,3.29LM, and D₃H6.55A

mutants were generated as described previously.^16,38The D₃S5.42A,

D₃S5.46A, D₃F6.52W, D₃Y7.43A, and D₃Y7.43F were constructed

using polymerase chain reaction (PCR) with the oligonucleotides

(Supporting Information) bearing the desired mutation. The inserts

bearing the desired mutations were cloned in the wild type vector using

HindIII/BspEI (for the TM5 mutants), EcoRI/BspEI (for the TM6 and

TM7 mutants). The entire coding region of the D₃receptor clones was

sequenced to ensure that the correct mutation was introduced and to

confirm the absence of unwanted mutations.

homogenate buffer (50 mM Tris-HCl, 5 mM EDTA, 1.5 mM CaCl₂,

5 mM MgCl₂, 5 mM KCl, 120 mM NaCl, pH 7.4) and subsequently

lysed with an Ultraturrax. After additional centrifugation at 50000g, the

membranes were resuspended in binding buffer (50 mM Tris, 1 mM

EDTA, 5 mM MgCl₂, 100 μg/mL bacitracin, 5 μg/mL soybean trypsin

inhibitor) and homogenized 10 times with a glass-Teflon homogenizer

at 4 °C. The homogenized membranes were shock-frozen in liquid

nitrogen and stored at -80 °C. The protein concentration was

determined with the Lowry method⁶⁷using bovine serum albumin as

a standard.

Radioligand Displacement Studies on the Membrane

Preparations of Transiently Transfected Cells. The radioligand

[³H]spiperone was used to determine the K_dand B_maxvalues for the

membrane preparations of transiently transfected HEK 293 cells expres-

sing the D₃wild type and mutant receptors from saturation experiments,

and the K_ivalues for the compounds were obtained by competition

experiments.⁶⁵Briefly, the assays were carried out in the 96-well plates at a

protein concentration of 20-200 μg/mL in a final volume of 200 μL. After

incubation for 1 h at 37 °C the assay was stopped by filtration through

Whatman GF/B filters presoaked with 0.3% polyethylenimine. The filters

were rinsed 5 times with ice cold Tris-NaCl buffer. After 3 h of drying at

60 °C filters were sealed with melt-on scintillator sheets MeltiLex B/HS

(PerkinElmer, Rodgau, Germany) and the filter-bound radioactivity was

measured in a MicroBeta TriLux liquid scintillator counter (PerkinElmer,

Rodgau, Germany). Three to six experiments per compound were

performed with each concentration in triplicate.

Radioligand Displacement Studies on the Wild Type

Receptors. Receptor binding studies were carried out as described.⁶⁵

In brief, competition binding experiments with the human D_2long

,

D_2short, D₃, and D_4.4receptors were run on the membrane preparations

from CHO cells stably expressing the corresponding receptor and

[³H]spiperone (specific activity of 84 Ci/mmol, PerkinElmer, Rodgau,

Germany) at final concentrations of 0.1-0.5 nM according to the

individual K_Dvalues. Membranes, radioligand, and test compound were

incubated for 60 min at 37 °C in binding buffer (50 mM Tris-HCl, 1 mM

EDTA, 5 mM MgCl₂, 0.1 mM DL-dithiothreitol, 100 μg/mL bacitracin, 5

μg/mL soybean trypsin inhibitor; pH 7.4). The K_Dvalues were 0.036-

[³⁵S]GTPγS Incorporation Assay. The [³⁵S]GTPγS binding

assay was performed on membrane preparations of transiently trans-

fected HEK293 cells that expressed the corresponding dopamine

receptor and the pertussin toxin insensitive GR protein (D_2long

þ

GR_o, D_2longþ GR_i2, or D₃þ GR_o). The receptor expression was

determined in saturation experiments (1500 ( 50, 4900 ( 440, and

1100 ( 90 fmol/mg protein, respectively). The assay was carried out in

96-well plates at the final volume of 200 μL. The incubation buffer

0.19, 0.070-0.24, 0.060-0.36, and 0.11-0.50 nM for the D_2long

,

D_2short, D₃, and D₄receptor, respectively. The corresponding B_max

values were in the range of 600-1600 fmol/mg for D_2long, 1100-2500

fmol/mg for D_2short, 1200-4200 fmol/mg for D₃, and 800-1600

fmol/mg for D₄receptor, respectively. The assays were carried out in

96-well plates at protein concentrations of 2-20 μg/mL. Competition

binding experiments at the high affinity binding site of the D_2longand the

D₃receptor were done using preparations from CHO cells stably

expressing the corresponding receptor (protein concentrations of 200

and 2 μg/well for D_2longand D₃, respectively) and [³H]7-OH-DPAT

contained 20 mM Hepes, 10 mM MgCl₂6H₂0,100 mM NaCl, and 70

3

mg/L saponin (pH 7.4).⁶⁸Membranes (30-50 μg/mL of membrane

protein), compounds, and 10 μM GDP were preincubated in the

absence of [³⁵S]GTPγS for 30 min at 37 or 30 °C (for D_{2 L}þ GR_i2).

After the addition of 0.10 nM [³⁵S]GTPγS membranes were incubated

for an additional 30 min at 37 °C or 75 min at 30 °C (for D_{2 L}þ GR_i2).

Longer incubation period for the D_{2 L}þ GR_i2pair is necessary because

both the dissociation of GDP from GR_i2and the association of

[³⁵S]GTPγS to GR_i2are 2-3 times slower than for GR_o.⁶⁹Incubation

was terminated by filtration through Whatman GF/B filters soaked with

ice cold PBS. The filter-bound radioactivity was measured as described

above. Three to four experiments per compound were performed with

each concentration in eight replicates.

(specific activity of 50 Ci/mmol for D₃and 161 Ci/mmol for D_2long

;

custom synthesis of [³H](R)-(þ)-7-OH-DPAT by Biotrend, Cologne,

Germany) at final concentrations of 1.0 and 0.5 nM for D_2longand D₃,

respectively. D_2longcompetition was done in 24-well plates at a final

volume of 500 μL considering K_Dof 1.2-1.4 nM and B_maxof 145-260

fmol/mg while for D₃binding a K_Dof 0.66 nM and a B_maxof 6000

fmol/mg was used. The D₁and R1 receptor binding experiments were

performed with homogenates prepared from porcine cerebral cortex as

described.⁶⁶Assays were run with membranes at protein concentrations

per well of 40-60 μg/mL and radioligand concentrations of 0.2 nM

[³H]prazosin and 0.5 nM [³H]SCH 23390 with K_Dof 0.050-0.16 nM

for the R1 receptor and 0.56-0.95 nM for the D₁receptor. Protein

concentration was established by the method of Lowry⁶⁷using bovine

serum albumin as a standard.

Adenylyl Cyclase Inhibition Assay. Bioluminescence based

cAMP-Glo assay (Promega, Mannheim, Germany) was performed

according to the manufacturer’s instructions. Briefly, CHO cells expres-

sing D_2longand D₃wild type receptor were seeded into a white half-area

96-well plate (5000 cells/well) 24 h prior to the assay. On the day of the

assay cells were first briefly washed with phosphate buffered saline (PBS,

pH 7.4) to remove traces of serum and incubated with various

concentrations of compounds in the presence of 20 μM (for D_2long

)

Membrane Preparations of Transiently Transfected HEK293

Cells. HEK 293 cells transiently transfected with the corresponding

cDNA as indicated by CaHPO₄method or using TransIT-293 transfec-

tion reagent (Mirus Corporation) were harvested 48 h after transfection.

The medium was aspirated. Cells were washed once with the ice cold

phosphate buffered saline (PBS, pH 7.4) and detached with the harvest

buffer (10 mM Tris-HCl, 0.5 mM EDTA, 5.4 mM KCl, 140 mM NaCl,

pH 7.4). The cells were scraped, collected in a centrifuge tube, and spun

at 220g for 8 min. The resulting pellet was resuspended in 5 mL of the

or 2 μM (for D₃) forskolin in serum-free medium that contained 500 μM

IBMX and 100 μM Ro 20-1724 at pH 7.4. After 15 min of incubation at

25 °C the cells were lysed with cAMP-Glo lysis buffer. After lysis the

kinase reaction was performed with a reaction buffer containing PKA. At

the end of the kinase reaction an equal volume of Kinase-Glo reagent was

added. The plates were read with a luminescence protocol on a

microplate reader Victor³V (Perkin-Elmer, Rodgau, Germany). The

experiments were performed three to nine times per compound with

each concentration in triplicate.

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ARTICLE

[³H]Thymidine Incorporation Assay. Determination of ligand

efficacy of representative compounds was carried out by measuring the

incorporation of [³H]thymidine into proliferating cells after stimulation

with the test compound as described in the literature.^70,71For this assay

D₃expressing CHO dhfr^-cells or D_2longexpressing CHO10001 cells

have been incubated with 0.02 μCi [³H]thymidine per well (specific

activity 25 μCi/mmol, Biotrend, Cologne, Germany) for 2 h. Dose-

response curves of 6-11 experiments have been normalized and pooled

to get a mean curve from which the EC₅₀value and the maximum

intrinsic activity of each compound could be compared to the effects of

the full agonist quinpirole.

Data Analysis. The resulting competition curves of the receptor

binding experiments and activity assays were analyzed by nonlinear

regression using the algorithms in PRISM 5.0 (GraphPad Software, San

Diego, CA). Competition curves were fitted to the sigmoid curve by

nonlinear regression analysis in which the log EC₅₀and the Hill

coefficient were free parameters. EC₅₀values were transformed to K_i

values according to the equation of Cheng and Prusoff.⁷²For the

receptor binding experiments (number of experiments g3, regardless

of number of replicates), standard error of mean was calculated. In the

case were only two experiments were performed (because of poor

binding affinity of a compound), standard deviation was calculated. The

data obtained from functional assay were normalized (using reference

agonist quinpirole to set 0% and 100%) and pooled, and the 95%

confidential interval for EC₅₀values and efficacy were estimated. Dose-

response curves of activity assays were fitted to the operational model

developed by Black and Leff²⁸to obtain an estimate of the transducer

constant τ:

Biotin Protection/Degradation Assay. HEK 293 cells stably

expressing the N-terminally FLAG tagged D_2longreceptor were grown to

80% confluency in poly-^D-lysine pretreated 10 cm plates and subjected

to the biotin protection/degradation assay protocol as described.⁷⁴Cells

were left untreated or stimulated for 30 min with 10 μM quinpirole, 10

μM 2, or 10 μM 5a. Briefly, cells were treated with 3 μg/mL disulfide-

cleavable biotin (Pierce) for 30 min at 4 °C. Cells were then washed in

PBS and placed in prewarmed medium for 15 min before treatment with

ligand (or no treatment) for 30 min. Concurrent with ligand treatment,

100% and strip plates remained at 4 °C. After ligand treatment, plates

were washed with PBS and remaining cell surface-biotinylated receptors

were stripped in 50 mM glutathione, 0.3 M NaCl, 75 mM NaOH, 1%

fetal bovine serum at 4 °C for 30 min. Cells were quenched with PBS

containing 50 mM iodoacetamide and 1% bovine serum albumin and

then lysed in 0.1% Triton X-100, 150 mM NaCl, 25 mM KCl, 10 mM

Tris-HCl, pH 7.4, with protease inhibitors (Roche Applied Science,

Basel, Switzerland). Cleared lysates were incubated with M2 anti-FLAG

affinity resin (Sigma) overnight at 4 °C, washed extensively, and

deglycosylated with PNGase F (New England Biolabs) for 2 h at

37 °C. Samples were denatured in SDS sample buffer with no reducing

agent added, resolved by SDS-PAGE using 8-16% Tris-glycine precast

gels (Invitrogen), transferred to the PVDF membrane, overlaid with

streptavidin (Vectastain ABC immunoperoxidase reagent, Vector La-

boratories, Burlingame, CA), and finally developed with enhanced

chemiluminescence reagent (Invitrogen). For quantification, data gen-

erated on three independent blots were analyzed using ImageJ software,

where the nontreatment was designated as 0% and treatment with

quinpirole for 30 min was designated as 100% signal.

E_mτⁿ½Aⁿꢁ

Docking Procedure. The recently published crystal structure of

the dopamine D₃receptor²⁰was used for docking investigations.

Compounds 2, (R)-5a, and (S)-5a were geometry optimized by means

of Gaussian 98 at the 6-31** level.⁷⁵A formal charge of þ1 was attributed

to the ligands because the tertiary amine of (R)-5a and (S)-5a and the

piperazine nitrogen connected to the aliphatic chain of 2 are assumed to

be protonated at physiological pH. The protonation of (R)-5a and (S)-

5a resulted in two stereoisomers.

The ligands were then docked into the crystal structure of the

dopamine D₃receptor²⁰using the program AUTODOCK4.^49-51To

completely enclose the binding site crevice, which is deﬁned by the

extracellular parts of TM2, TM3, TM5, TM6, and TM7, a grid of 70

points in each of the x, y, and z directions and a grid spacing of 0.375 Å

were applied. The AUTODOCK Lamarckian genetic algorithm with a

randomly selected starting position was used to generate 50 docking

conformations of each compound that were clustered manually and

scored by DrugScore^online, applying the knowledge-based DrugScore^CSD

scoring function.⁵³On the basis of this scoring, the best conformation of

each cluster was selected. A ﬁnal selection was made according to

experimental data (site-directed mutagenesis and radioligand displace-

ment studies).

Analysis of the docking poses of the two stereoisomers of (R)-5a and

(S)-5a, which were generated by the protonation of the tertiary amine,

showed that only the R-enantiomer possesses the possibility to position

the propyl moiety in the “propyl cleft”, which has been proposed to

extend from TM3 toward TM2 and TM7.⁵²Therefore, the R-enantio-

mer of (R)-5a and (S)-5a was chosen for further analysis. In addition,

docking of compounds (R)-5a and (S)-5a resulted in two ligand-

receptor complexes in each case, which showed distinct position and

orientation of the tetrahydropyrazolopyridine moiety. All four com-

plexes were considered for further analysis.

After the selection of the ﬁnal docking pose, the receptor-ligand

complexes of 2, (R)-5a, and (S)-5a were subjected to energy minimiza-

tion using the SANDER classic module of AMBER 10.⁷⁶For the energy

minimization of (R)-5a and (S)-5a, the side chain conformation of

Ser5.43 was rearranged in accordance with the crystal structure of the

E ¼

ð1Þ

n

ðK_Aþ ½AꢁÞ þ τⁿ½Aꢁ

with E denoting the effect, E_mthe maximum possible effect, [A] the

agonist concentration, and K_Athe dissociation constant of the agonist-

receptor complex. The transducer constant τ is a parameter describing

the signal transduction efficiency of the system, and the intrinsic efficacy

of the agonist is estimated from the fit of the data. Quinpirole was used as

the reference full agonist, defining zero and a maximal (100%) response

of the system. The curves of all agonists had a Hill coefficient of unity or

close to unity. The log(τ/K_A) values were calculated, which depicted the

“strength” of a given agonist to activate a defined pathway in a defined

system.^31,73The Δ log(τ/K_A) values were calculated as

Δlogðτ=K_AÞ_{agonist=reference}¼ logðτ=K_AÞ_agonist- logðτ=K_AÞ_reference

ð2Þ

to compare various agonists within signaling pathway. The difference of

the Δ log(τ/K_A) between selected pathways yields ΔΔ log(τ/K_A),

which describes agonist bias or functional selectivity of a given ligand.

Immunocytochemistry. The antibody-feeding immunocyto-

chemistry experiments were performed as previously described.⁷⁴

Briefly, cells stably expressing the FLAG-tagged D_2longreceptor (or

the HA-tagged D₃receptor) were grown on coverslips pretreated with

gelatin to ∼50% confluency. Live cells were fed M1 antibody (Sigma)

directed against the FLAG epitope or HA-11 antibody directed against

the FLAG epitope (Covance) (1:1000, 30 min) and then incubated with

ligands (10 μM, 30 min) or left untreated. Quinpirole (10 μM, 30 min),

2 (10 μM), and 5a (10 μM) were used. Cells were then fixed with 4%

formaldehyde in PBS, pH 7.4. After fixation cells were permeabilized in

BLOTTO (3% milk, 0.1% Triton X, 1 mM CaCl₂, 50 mM Tris-HCl,

pH 7.5) and stained with Alexa Fluor 594 goat anti-mouse IgG2b or

AlexaFluor 594 goat anti-mouse IgG1 antibody (Invitrogen, 1:500, 45

min). The coverslips were mounted using mounting media from

Vectashield. Mounted sections were examined by using LSM 510 laser

confocal microscope (Zeiss).

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ARTICLE

β₂-adrenergic receptor bound to the agonist FAUC 50.⁵⁴The side chain

conformation of Ser5.43 enabled a hydrogen bond interaction with the

tetrahydropyrazolopyridine moiety in addition to the interaction be-

tween Ser5.42 and this moiety. Then 500 cycles of steepest descent

minimization were applied, followed by 4500 cycles of conjugate

gradient minimization. All calculations were carried out in a water box

with periodic boundary conditions and a nonbonded cutoﬀ of 8.0 Å. The

all atom force ﬁeld ﬀ99SB was applied.⁷⁷Parametrization of the

compounds was accomplished with antechamber. The charges for the

ligand atoms were calculated using Gaussian 98, with a 6-31** basis set,

and then the RESP procedure was applied as described in the

literature.^75,78

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’ ASSOCIATED CONTENT

S

Supporting Information. Experimental data, results of

b

functional assays and site directed mutagenesis, binding data, and

results from operational model. This material is available free of

charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: þ49(9131)852-9383. Fax. þ49(9131)852-2585. E-mail:

peter.gmeiner@medchem.uni-erlangen.de.

’ ACKNOWLEDGMENT

We thank Prof. Graeme Milligan (University of Glasgow, U.K.)

for providing us with the cDNA for GR_o1or GR_i2proteins, and we

thank Raquel Ortega for the synthesis of a molecular probe.

’ ABBREVIATIONS USED

7TM, seven transmembrane; TM, transmembrane; SCH 23390,

7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol;

spiperone, 8-[4-(4-ﬂuorophenyl)-4-oxo-butyl]-1-phenyl-1,3, 8-

triazaspiro[4.5]decan-4-one; WAY100635, N-[2-[4-(2-meth-

oxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecar

boxamide; prazosin, 2-[4-(2-furoyl)piperazin-1-yl]-6,7-dimeth

oxyquinazolin-4-amine; EDTA, ethylenediaminetetraacetic acid; 7-

OH-DPAT, 7-hydroxy-2-(N,N-di-n-propylamino)tetralin; IBMX,

3-isobutyl-1-methylxanthine; Ro 20-1724, [4-(3-butoxy-4-methox-

ybenzyl)imidazoline; CHO, Chinese hamster ovary; HEK, human

embryonic kidney; FBS, fetal bovine serum; PBS, phosphate

buﬀered saline; ELISA, enzyme-linked immunosorbent assay;

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electro-

phoresis; PVDF, polyvinylidene ﬂuoride; EC₅₀, half maximal eﬀec-

tive concentration; PKA, protein kinase A; MAPK, mitogene-

activated protein kinase; ERK1/2, extracellular regulated kinase 1/

2; SAR, structure-activity relationships; NMR, nuclear magnetic

resonance; MHz, megahertz; LC/MS, liquid chromatography/

mass spectrometry; APC, atmospheric pressure chemical; TLC,

thin layer chromatography; HRMS, high resolution mass spectro-

metry;HPLC, high performance liquid chromatography;EIMS,

electron ionization mass spectrometry; DMF, dimethylformamide

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dx.doi.org/10.1021/jm101639t |J. Med. Chem. 2011, 54, 2477–2491

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Highly potent 5-aminotetrahydropyrazolopyridines: Enantioselective dopamine D3 receptor binding, functional selectivity, and analysis of receptor-ligand interactions

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