Dopamine D4 ligands and models of receptor activation: 2-(4-pyridin-2-ylpiperazin-1-ylmethyl)-1H-benzimidazole and related heteroarylmethylarylpiperazines exhibit a substituent effect responsible for additional efficacy tuning.

Article abstract of DOI:10.1021/jm0305669

A series of subtype selective dopamine D(4) receptor ligands from the hetroarylmethylphenylpiperazine class have been discovered that exhibit a remarkable structure-activity relationship (SAR), revealing a substituent effect in which regiosubstitution on the terminal arylpiperazine ring can modulate functional or intrinsic activity. Other structure-dependent efficacy studies in the dopamine D(4) field have suggested a critical interaction of the heteroarylmethyl moiety with specific protein microdomains in controlling intrinsic activity. Our studies indicate that for some binding orientations, the phenylpiperazine moiety also plays a key role in determining efficacy. These data also implicate a kinetic or efficiency term, contained within measured functional affinities for agonists, which support a sequential binding and conformational stabilization model for receptor activation. The structural similarity between partial agonist and antagonist, within this subset of ligands, and lack of bioisosterism for this substituent effect are key phenomena for these hypotheses.

Full text of DOI:10.1021/jm0305669

2348
J . Med. Chem. 2004, 47, 2348-2355
Dop a m in e D4 Liga n d s a n d Mod els of Recep tor Activa tion :
2-(4-P yr id in -2-ylp ip er a zin -1-ylm eth yl)-1H-ben zim id a zole a n d Rela ted
Heter oa r ylm eth yla r ylp ip er a zin es Exh ibit a Su bstitu en t Effect Resp on sible for
Ad d ition a l Effica cy Tu n in g
Andrew O. Stewart,* Marlon D. Cowart, Robert B. Moreland, Steve P. Latshaw, Mark A. Matulenko,
Pramila A. Bhatia, Xueqing Wang, J erome F. Daanen, Sherry L. Nelson, Marc A. Terranova,
Marian T. Namovic, Diana L. Donnelly-Roberts, Loan N. Miller, Masaki Nakane, J ames P. Sullivan, and
J orge D. Brioni
Department R4ND, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories,
100 Abbott Park Road, Abbott Park, Illinois 60064-6115
Received November 7, 2003
A series of subtype selective dopamine D4 receptor ligands from the hetroarylmethylphenylpip-
erazine class have been discovered that exhibit a remarkable structure-activity relationship
(SAR), revealing a substituent effect in which regiosubstitution on the terminal arylpiperazine
ring can modulate functional or intrinsic activity. Other structure-dependent efficacy studies
in the dopamine D4 field have suggested a critical interaction of the heteroarylmethyl moiety
with specific protein microdomains in controlling intrinsic activity. Our studies indicate that
for some binding orientations, the phenylpiperazine moiety also plays a key role in determining
efficacy. These data also implicate a kinetic or efficiency term, contained within measured
functional affinities for agonists, which support a sequential binding and conformational
stabilization model for receptor activation. The structural similarity between partial agonist
and antagonist, within this subset of ligands, and lack of bioisosterism for this substituent
effect are key phenomena for these hypotheses.
In tr od u ction
purification and limited detailed characterization of
protein structural morphology or ligand binding orien-
tation that can be obtained through traditional X-ray
or NMR techniques. Ligand recognition site description
and relative binding orientation of given dopamine
receptor ligands are proposed on the basis of eloquent
mutagenesis studies7 and the comparative binding data
using mutants from these studies. Site-directed mu-
tagenesis data can indicate key residues involved in
translation of conformational information through cer-
tain helices to the G protein but do not give detailed
information with regard to ligand binding orientation.
Other general descriptions of ligand binding can be
derived from structure-activity relationship (SAR) data.
The ubiquitous incorporation of piperazine derivatives
within ligands having high affinity for the A-family or
aminergic class of GPCR receptors supports a consistent
binding interaction.8 This interaction would correlate
to the protonated basic amine of endogenous biogenic
amine agonist with a highly conserved aspartic acid
(ASP) in transmembrane-spanning domain 3 (TM3)
(ASP3.32)9 and is one definitive feature of the competitive
recognition site and ligand pharmacophore. In addition
to TM3 for binding, another general structural deter-
minate is TM6, implicated in activation mechanisms.10
This large class or supergenus of arylpiperazines de-
rivatives can therefore be considered as privileged
structures11 for this family of GPCR receptors. The
compounds in this study are from this class.
G-protein-coupled receptors (GPCRs) involved in sec-
ond messenger signaling are one of the most studied
classes of targets for drug discovery.1 They comprise
greater than 1% of the human genome,2 represent the
molecular targets for over 45% of all currently marketed
drugs, and may account for one-third of current phar-
maceutical research.3 Investigations of these proteins
and corresponding high-affinity ligands have probably
contributed more to the conceptualization of pharma-
cological agonism or antagonism than any other class.
Competitive agonists and antagonists for these recep-
tors, utilizing the same recognition site, often reveal a
continuum of ligand efficacy or intrinsic activity. Cur-
rent models of activation mechanisms propose confor-
mational isomers or states of the protein, thereby
affecting the morphology of the recognition site surface
but while still employing common site residues.4 The
realization that receptors can have constitutive intrinsic
activity5 in their native environment, presumably in the
absence of ligand, has complicated the understanding
of protein conformational control of intrinsic activity and
the role of ligand stabilization in this process. Driven
by selective ligand binding or stabilization, conforma-
tional selection models and model variants, incorporat-
ing sequential binding and conformational stabilization,
form the basis for most models of activation involving
ligand mediation.6
The complex structures and associated cellular ma-
chinery of membrane-embedded GPCRs have hindered
The dopamine receptor has been the target for many
therapeutic drug strategies.12 Dopamine D4 antagonists
were pursued as a possible intervention for schizophre-
* To whom correspondence should be addressed. Phone: 847-937-
8566. Fax: 847-938-0072. E-mail: andrew.o.stewart@abbott.com.
10.1021/jm0305669 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/23/2004

Benzimidazole and Arylpiperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2349
Ch a r t 1. Structurally Similar Heteroarylmethylarylpiperazines: Selective D4 Ligands
nia (Chart 1).13 The D4 selective antagonist to advance
Sch em e 1
the farthest in development for this indication, L-745,-
870 (1a ),14 failed to demonstrate clinical efficacy. Much
debate has centered on the true intrinsic activity of 1a
and whether it is indeed a partial agonist,15 thus
highlighting the difficulties in understanding the range
of ligand intrinsic activity or how this measure or aspect
of a ligand’s biochemical profile relates to ligand affinity
or a ligand’s dissociation constant for a given low-energy
conformation of the receptor. Compound 2 has been
reported16 as a “complete antagonist” with regard to
studies on 1a . In the Gmeiner et al. communication,
comparison of efficacy data from mitogenesis functional
assays, D4/D2 selectivities, and mutagenesis data from
D2 and D4 receptors has provided a better understand-
ing of ligand binding orientations and transmembrane
domains involved in receptor activation for ligands 1a
and 2.16
Resu lts
Synthesis of compounds 3a -k were accomplished by
reacting commercially available 2-chloromethylbenz-
imidazole (3) with the appropriate arylpiperazine (a -
k ) in DMF at room temperature in the presence of
triethylamamine (Scheme 1). Arylpiperazines a and
c-k were commercially available. Piperazine derivatve
b, the 3-pyridyl isomer, was prepared by Buchwald
coupling of 3-bromopyridine and piperazine.
Compound 1b was prepared using the procedure
described for the synthesis of 1a , using azaindole,
formaldehyde, and the arylpiperazine derivative in
acetic acid and water, by substituting 2-chlorophe-
nylpiperazine for the 4-chloro derivative.21 Compounds
2 and 4 were prepared as previously described.16,18
D4 efficacy or intrinsic activities of ligands were
measured using recombinant human D4 receptor ex-
pressed in HEK-293 cells also expressing a cotransfected
GRqo5 protein.22 D4 ligand binding affinity was deter-
mined by radioligand competition against [3H]spiper-
one,23 using membranes from the engineered HEK-293
cells. D2 binding affinity was determined by [3H]spip-
erone competition using membranes from human D2
transfected HEK-293 cells.
Our original SAR study began with two sets of
ligands: (a) variation of the benzofused azaheterocyle
of both 1a and 4; (b) analogues with different selected
substituents on the terminal piperazine aryl ring. We
discovered that the novel benzimidazole analogues of
both azaindole 1a or indole 4 produced high-affinity D4
ligands 3f and 3a , respectively. The calcium flux assay
revealed that, like 4, 3a was a potent partial agonist.
Recently, a therapeutic target for selective D4 agonists
has been disclosed.17 We have identified compound 3a ,
a selective D4 agonist, for treatment of male erectile
dysfunction (MED).18 From the intense search for selec-
tive D4 antagonists, a few selective partial D4 agonists,
such as 4, were reported.19 The structural similarity of
1a and 4 stimulated our investigation of this class of
compounds to determine SAR for intrinsic activity at
the D4 subtype and led to the discovery of 3a . As part
of that SAR study, we reported that within this narrow
class of benzimidazoylmethyl-4-arylpiperazines, substi-
tution on the terminal piperazine aryl ring dramatically
influenced intrinsic activity. In this report we examine
this substituent effect in more detail using sets of
monosubstituted congeners related to 3a and contrast
data using binding competition, previous studies on this
class of compounds, and models of GPCR receptor
activation. Both the structural similarity between an-
tagonists and agonist and the wide range of substituents
that produce the observed substituent effect support this
effect as not being related to enthalpy contributions20
to the ∆G of binding or ligand dissociation constant for
a given conformation of the protein. Some conclusions
with regard to ligand binding orientation are suggested;
however, more significantly, a kinetic component or
efficiency terms, reflected in both the observed EC50 and
intrinsic activity of functionally active ligands, which
correlate to stabilization of a transition state between
two conformations are indicated.

2350 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Stewart et al.
Ta ble 1. Human D4 Binding Affinity and Functional Data for
Benzimidazole Analogues 3a -k
Ta ble 2. Intrinsic Activities of Terminal
Monochlorosubstituted Phenylpiperazine Analogues in the hD4
Calcium Flux Assay with Cotransfected GRqo5 Protein
a
compd
EC50
intrinsic activityb
1a
1b
2
2230 ( 1010
10000c
154 ( 56
32 ( 7.4
20 ( 2.9
19 ( 2.8
19 ( 3.6
46 ( 4.1
4
a
aryl
substituent
(AR)
Concentration giving 50% signal, expressed as nM ((SEM, n
b
binding
affinity Ki
% efficacyc
(intrinsic activity)
g 3). % activation relative to 10 µM dopamine ((SEM, n g 3).
a
b
c Highest dose tested.
compd
EC50
3a
3b
3c
3d
3e
3f
3g
3h
3i
2-pyridyl
3-pyridyl
4-pyridyl
2-ClC6H4
3-ClC6H4
4-ClC6H4
C6H5
167 ( 2.4
376 ( 38
>10000
12.4 ( 0.5
ND
NA
1.0 ( 0.1
NA
61.3 ( 3.4
24.9 ( 4.9
4.8 ( 0.8
43.0 ( 5.9
3.8 ( 0.6
4.7 ( 0.7
37.5 ( 4.0
47.5 ( 4.7
5.0 ( 0.4
4.8 ( 0.5
9.9 ( 2.6
Ta ble 3. Human D2/D4 Receptor Subtype Selectivity of
Monochloro-Substituted Phenylpiperazine Analogues
Determined Using [3H]spiperone Binding Competition
8.5 ( 0.6
9.6 ( 0.6
a
a
compd
D4
D2
D2/D4
14.8 ( 3.0 NA
52 ( 5.8
1a
1b
2
3d
3e
3f
0.4 ( 0.05
719 ( 109
2.7 ( 0.5
8.5 ( 0.6
9.6 ( 0.6
14.8 ( 3.0
448 ( 54
>10000
1120
>14
10.1 ( 0.8
5.6 ( 0.5
2-OCH3C6H4 4.3 ( 0.3
1310 ( 583
>10000
489
3-OCH3C6H4 26.5 ( 1.6 NA
4-OCH3C6H4 20.4 ( 0.15 NA
>1176
>1041
>756
3j
3k
>10000
>10000
4-FC6H4
79.6 ( 6.3 NA
a
Competition against [3H]spiperone, expressed as nM ((SEM,
a
Ki value, from binding competition against [3H]spiperone,
b
n
g 3). Concentration giving 50% signal, expressed as nM
c
expressed as nM ((SEM, n g 3).
((SEM, n g 3). NA: not applicable. ND: not determined.
activation relative to 10 µM dopamine ((SEM, n g 3).
%
azaindole series. Human D4 functional data for this
analogue, the reference partial agonist 4, and ligand 2
in our chimeric system are shown in Table 2. The
change of the p-chloro substituent (1a ) to the ortho
position (1b) produces little effect on intrinsic activity.
Ligand 1b is a weak partial agonist similar to 1a
exhibiting only approximately 20% activation at micro-
molar concentrations. Null cell experiments and block-
ade of the calcium flux assay by D4 antagonists confirm
these signals for 1a , 1b and 2 are from interactions with
dopamine D4 receptors (data not shown). The potent
partial agonism observed for 4 agrees well with the
reported profile of this compound.19a Ligand 2 produces
little activation of the D4 receptor, similar to 1a , but
activation occurs at much lower concentrations. This
comparison provides an example of the dual nature and
independent measurement of affinity and efficacy.
D2/D4 selectivity data were used as an additional
probe of ligand-binding orientation. Subtype selectivity
was measured for monochlorophenyl analogues 3d , 3e,
3f, 1a , 1b, and 2 and are shown in Table 3. Ligands
3d -f are D4/D2 selective at the highest concentrations
tested and support similar binding modes for these three
analogues. By comparison of 1a and 1b, compound 1b
has a much weaker affinity at D4, presumably as a
result of changes in ligand interaction of the chlorophe-
nyl moiety. Compound 2 is similar in D4 affinity and
selectivity to 1a , as proposed by the previous overlay
models.16
Shown in Table 1 are EC50 values, percent efficacy, and
corresponding binding affinities for sets of monosubsti-
tuted arylpiperazine congeners related to 3a and 3f.
Of the three pyridyl isomers, only the 2-pyridyl
analogue (3a ) is a potent partial agonist with an EC50
of 12 nM and achieving 61% activation relative to 10
µM dopamine. The 3-pyridyl isomer (3b) has weaker
affinity on the basis of binding data and an appreciable
lower intrinsic activity or the ability to activate the
receptor. The 4-pyridyl isomer (3c) has weak affinity
for the receptor. This data set indicates that a nitrogen
atom in the ortho position of the terminal aryl ring could
play a key role in molecular interaction between the
ligand and receptor. The binding affinities were consis-
tent within the three monosubstituted chlorophenyl
analogues (3d -f), revealing a higher affinity than the
corresponding pyridyl analogues; however, the same
SAR phenomenon for receptor activation is observed.
The o-chloro analogue (3d ) produced a partial 43%
response with a potent EC50 of 1 nM. The EC50 is also
indicative of high affinity but may only appear to
correlate well with the competition binding data for this
ligand. Although the m- and p-chloro substituted ana-
logues (3e, 3f) have similar apparent dissociation
constants, on the basis of radioligand competition, they
lack the ability to induce calcium mobilization. The
three monomethoxy substituted analogues also exhibit
an identical trend. Only the 2-methoxy analogue (3h )
is a potent partial agonist producing approximately 50%
of the response of 10 µM dopamine. The other two
methoxy analogues (3i, 3j) have potent affinity but
cannot function as agonists. The unsubstituted phenyl
analogue (3g) indicates that a hydrogen substituent in
this ortho position is sufficient in favoring an active form
of the receptor. In contrast, a p-fluoro substituent
precludes substantial agonist function (compare 3g to
3k ), consistent with the trend that substituents in the
meta or para position other than hydrogen do not lead
to ligands having agonist function.
Discu ssion
Details of ligand binding orientation obtained through
docking models are one fundamental aspect of under-
standing mechanisms of receptor activation. Compound
2, described as a “complete” antagonist,16 was designed
as part of a study on antagonists related to 1a . The
reported efficacy tuning for ligands 1a and 2 is proposed
by using docking models that overlay the common
p-chlorophenylpiperazine moiety for the two ligands. D2/
D4 selectivity was used as one factor to establish relative
ligand orientation within the recognition site and at-
Ligand 1b, the o-chloro analogue of 1a , was prepared
to test the generality of this ortho effect toward the

Benzimidazole and Arylpiperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2351
tributed to interaction of the phenylpiperazine moiety
with the TM2,3,7 microdomain. Following this model,
the heteroarene subunits of the two ligands then engage
with different vectors into a cluster of residues in the
TM5,6 microdomain affecting different degrees of recep-
tor activation. Compound 1b displays similar weak
agonism or efficacy as 1a (Table 2) and supports a
similar interaction of the azaindole with the TM5,6
microdomain. The generality of the ortho effect seen for
the benzimidazole analogues does not translate to the
chlorophenylazaindole analogues and suggests subtle
differences in binding orientation for the monochlo-
rophenyl derivatives between the azaindole and benz-
imidazole series. The change in chlorosubstitution for
1b as reflected by interaction with the TM2,3,7 micro-
domain produces the observed differences for 1a and 1b
in D2 and D4 affinities. However, the D2/D4 selectivity
data and the efficacy data still support similar general
ligand binding orientation and receptor activation mech-
anism for 1b compared to that proposed for 1a .
F igu r e 1. Two receptor state or conformational control model
of receptor activation: Ri, antagonist or inactive receptor
conformation; Ra , high agonist affinity or active receptor
conformation; L, ligand; G, G-protein; LRa G, initial ligand
bound, active receptor-G protein complex (specific order or
sequence is not implied with regard to ligand, receptor, and
G protein association in formation of bound active receptor
complex); LRa *G, bound transitional complex leading to
receptor activation and G protein subunit release.
ligand correlate to general models of ligand interaction
and receptor activation. Two fundamental concepts or
models6 of receptor activation are conformational selec-
tion and sequential binding-conformational stabiliza-
tion. A general receptor activation model is shown in
Figure 1.
Two-state models of conformational selection are
formed on the basis of equilibrium ligand interaction
with multiple conformational states.25 There are con-
vincing data for an initial conformational selection
process by a ligand between Ri and Ra . This concept is
supported by GTP shift experiments,23,26 the use of cells
that do not contain G proteins that can react with
expressed receptors,27 and data using pertussis toxin,28
which prevent association of specific G proteins and
receptors. It is known that antagonists often recognize
both affinity states (Ri and Ra ) of the receptor as
manifested in biphasic binding curves of agonists versus
radioligand antagonists.23,27a,29 By use of antagonist
radioligands, agonist affinities for both states in D2 and
D4 receptors have been reported in these studies. Full
agonists, having catecholamine structures, display a
significant difference in affinities for the high- and low-
affinity states. Restated, these full agonists have a high
selectivity for the agonist state, as predicted by the
simplest two-state model. By use of binding data from
competition using radioligands of different functional
profiles or conformational selectivity, intrinsic activities
of competing ligands have been successfully predicted.30
This general conformational selection model is also
useful in explaining constitutive activity in the absence
of ligand based on the basal position of the ratio Ra /
Ri. Biphasic binding curves suggest that antagonists
can identify both states. This implies that ligand affinity
for the active state (LRa G) is by itself insufficient for
receptor activation. Introduction of additional confor-
mational selection states has been introduced to explain
these phenomena and expand this model.25c
Sequential binding-conformational stabilization mod-
els more thoroughly encompass concepts of induced fit31
and easily embrace ligand-mediated helical or trans-
membrane domain changes identified using mutagen-
esis data and docking models. Additional ligand-
mediated receptor conformational changes after formation
of LRa G leading to LRa *G and G protein subunit
dissociation mechanistically distinguish agonist binding.
Our three monochlorophenyl analogues (3d -f) have
similar potent single nanomolar binding affinities com-
peting against the antagonist spiperone. These data
together with the structural similarity of these ana-
logues strongly suggest that both the partial agonist 3d
and the antagonist type ligands 3e and 3f are nonselec-
tive with regard to the two affinity states of the D4
The complexity introduced by our study of the benz-
imidazole analogues is centered on an apparent key role
of the terminal arylpiperazine moiety. Substituents in
the ortho position of this ring, possessing a diverse range
of substituent parameters, produce a similar efficacy
effect, while the heteroarene moiety is kept constant.
Much of our data could fit the rationale of Gmeiner et
al., since all ortho-substituted analogues could be argued
to adopt a similar binding orientation, one different from
the meta and para analogues. These two types of
terminal ring orientations would then produce agonism
or antagonism by altering the position of the pendent
benzimidazole engaging with the TM5,6 domain. It is
hard to rationalize why the minimal size of an ortho
lone pair (2-pyridyl, 3a ) or ortho hydrogen (3g) has the
same effect or influence on binding orientation as the
larger ortho substituents in our study, such as the
o-chloro analogue (3d ) or the even larger o-SMe and
o-nitro analogues reported in our previous SAR study.18
Neither polarity nor the electron-withdrawing nature
of the ortho substituent produces modulation of the
generality of this ortho effect. This lack of obvious
Hansch-Mitscher bioisosterism24 for this positional
substituent implies that this substituent is not in close
contact with the protein in the putative “agonist”
binding orientation for these ortho-substituted conge-
ners. This lack of bioisosterism also precludes associat-
ing this effect with typical SAR parameters, such as
electronic effects on the π systems of the aryl rings,
effects on the pKa of the important protonated pipera-
zine nitrogen, or ligand conformational or shape analy-
sis that normally relates enthalpy contributions of
protein-ligand interaction to binding energy. Our three
monochloro analogues have remarkably similar D4
binding affinities and D2/D4 selectivites (Table 3). These
data support similar binding orientations for these three
benzimidazole analogues but are distinct from ana-
logues derived from 1 and 2. The similar affinities also
suggest a lack of close contact with the protein for the
chloro group of meta and para analogues 3e and 3f.
Bound ligand orientation and associated helical do-
main changes provide a basic mechanistic model of
receptor activation. Difficulty remains in understanding
how measured biochemical parameters for a given

2352 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Stewart et al.
receptor in our membrane assays. It should be noted
that data are not presented in this study to support the
assumption that 3e and 3f as well as 3i and 3j are D4
antagonists. In our previous SAR study,18 antagonist
data were given for compounds that are close analogues
to 3a , and recently, 3f and 3j have been reported as D4
receptor antagonists.32
driving force may be difficult to replicate in our equi-
librium binding assays with truncated cellular machin-
ery resulting from membrane preparation. Equilibrium
affinities as measured by binding assays therefore are
constrained by assay conditions and time frames. Pro-
tein-ligand interactions as described by induced fit
models for receptor activation, measuring several changes
or movements of the bound ligand-receptor-G protein,
are more complex. The difficulty in assigning dissocia-
tion constants to ligand interaction in multiconforma-
tional state processes has been documented.26,35
The use of the GRqo5 chimeric protein might be
questioned in our study as providing a tool for rapid
drug discovery but one having little relevance to the true
pharmacology of the D4 receptor. Chimeric systems have
been validated for the closely related D2 receptor.36 The
similarity of our D4 binding data for the antagonist
spiperone22 and agonist dopamine (data not shown) to
literature values with the native signaling Gio suggests
the desired morphology of the recognition site in our
chimeric system relative to the native D4 receptor;
however, a caveat regarding the stoichiometry of the
remaining G protein in our membrane binding system
is valid. The correlation of our D4 receptor activation
data for 216 and the reference compound 419a also
supports the validity of our system in relation to the
native system. Our value for the EC50 of 1a is consider-
ably less potent than some reports,15,16 but it is consis-
tent with the predominant D4 antagonist profile of this
compound. Even considering potential limitations,37 the
engineered system provides a valuable tool for direct
observation of G-protein-linked receptor activation and
understanding the ligand’s role in unified activation
mechanisms.
Theoretically, extensive dose-response competition
curves for antagonist radioligand with a reference full
agonist, having sufficient selectivity between the two
receptor conformations, can reveal a biphasic curve that
may be manipulated by GTP shift experiments. How-
ever, we do not observe a biphasic curve when compet-
ing our partial agonists against the antagonist spiper-
one, and for these partial agonists, it seems more
plausible that a lack of ligand selectivity between the
conformations exists. If ligand affinity selectivity is less
than 10-fold between the two conformations, the pres-
ence of two states would not be detected by a Hill
analysis of binding data.
Although not indicated to be in close contact with the
protein in bound ground states by the lack of influence
on the binding affinities, the m- and p-chloro substitu-
ents for 3e and 3f could create steric interactions with
residues moving to accommodate subsequent changes
in protein conformation. This unfavorable interaction
would thereby raise the activation energy for required
protein movement to gain access to LRa *G. LRa *G can
represent a higher energy species or transition state to
the penultimate conformation leading to subunit dis-
sociation. Whether it is a ground-state conformation or
not, the rationale of protein-ligand interaction contrib-
uting to a ∆G of binding is a unifying thermodynamic
principle. The ortho substituent could favor these
subsequent conformational changes after binding. The
nature of such a positive effect could be in the lowering
of the activation energy to the key transition state as
governed by the Arrhenius equation. This proposal
complicates the understanding of ligand stabilization
correlating to LRa *G, since it is not supported by
known bioisosterism for this substituent. Therefore, the
ortho effect may represent a lower energy pathway
relative to a negative effect or higher activation energies
exhibited by the meta and para analogues. Both pos-
sibilities define kinetic effects for these substituents.
Con clu sion s
Structural similarity and consistent binding affinities
within a set of benzimidazoylmethylarylpiperazine
ligands, which display a range of intrinsic activity,
indicate these ligands have poor selectivity for high- and
low-affinity states of the dopamine D4 receptor. This is
consistent with conformational selection models and the
partial agonism observed for the ortho-substituted
analogues. It is equally consistent with the known
recognition of both states by antagonists, which are
represented by the meta- and para-substituted ana-
logues. The lack of substituent bioisosterism and con-
formational selectivity indicates that kinetic terms
relating activation energies between conformational
changes are significant components of observed affinity.
These changes of the receptor-ligand-G protein com-
plex occur after initial agonist binding. Kinetic param-
eters are prominent in both the observed EC50 and the
intrinsic activity for functional agonist and partial
agonist ligands. A subtle balance in ligand stabilization
of an active ground state(s), yet not introducing activa-
tion barriers between subsequent conformations along
the reaction coordinate of the receptor-ligand complex,
appears to describe receptor activation for this set of
partial agonists. These data support a sequential bind-
ing and conformational stabilization model under ki-
netic control within the cell. Agonists are a class of
ligands for small-molecule drug development that pos-
sess complex combinations of dissociation constants and
Conceptually, incorporation of kinetic parameters or
velocities of formation rates into these models are easily
accommodated. With some analogy to enzyme kinetics,
the velocity of formation for a sequence of bound
conformations can be partially decoupled from the
dissociation constant in that the highest dissociation
constant may not produce the most efficient formation
rate.33 This kinetic term can then be reflected in both
ligand EC50 and intrinsic activity. Agonist or partial
agonist affinity for Ri modulates the concentration of
LRa G ([LRa G] ) [Ro] - [LRi]) and can have a dramatic
effect on the velocity or rate of formation of LRa *G by
reducing the concentration term in the rate equation.
Ligand-driven partitioning of the receptor pool may
also be largely a kinetic process and not a thermody-
namic process, analogous to the Curtin-Hammet prin-
ciple.34 This process can be driven by energy manipu-
lation (phosphorylation) within the cell and by the large
entropy term of the G-protein dissociation. This type of

Benzimidazole and Arylpiperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2353
trated in vacuo. The residue was purified by chromatography
to give 3.6 g (100%) of 1-(3-pyridyl)piperazine as a colorless
oil. By use of this material without further purification, 3b
was prepared by the general procedure (39%). Mp 192-193
kinetic parameters as part of their biochemical profile,
both of which can be manipulated, somewhat indepen-
dently, through SAR strategies.
1
°C. H NMR (DMSO-d6, 300 MHz) δ 2.64 (t, J ) 6 Hz, 4 H),
Exp er im en ta l Section
3.24 (t, J ) 6 Hz, 4 H), 3.79 (s, 2 H), 7.15 (m, 2 H), 7.21 (dd,
J ) 7, 6 Hz, 1 H), 7.30 (m, 1 H), 7.43 (d, J ) 7.5 Hz, 1 H), 7.57
(d, J ) 7.5 Hz, 1 H), 7.98 (dd, J ) 1, 5 Hz, 1 H), 8.28 (d, J )
4.5 Hz, 1 H), 12.31 (brs, 1 H); MS (DCI/NH3) m/z 294 (M +
H)+. Anal. (C17H19N5) C, H, N.
All solvents were of anhydrous reagent grade from com-
mercial sources. Unless otherwise noted, all chemicals and
reagents were obtained commercially and used without puri-
1
fication. The H NMR spectra were obtained at 300 MHz on a
Nicolet/GE QE300 spectrometer. Chemical shifts are reported
in parts per million (ppm) relative to TMS or TSP as an
internal standard. Mass spectra were obtained on a Kratos
MS-50 instrument in DCI/NH3 mode. Elemental analyses were
performed by Robertson Microlit Laboratories, Inc., Madison,
NJ . Flash chromatography was carried out using silica gel 60
(E. Merck, 230-400 mesh) or prepacked 40 mm silica gel
columns from BioTage. Thin-layer chromatography was per-
formed on 250 µM silica-coated glass plates from EM Science.
Samples were analyzed by HPLC-MS-ELSD on an Open
Access Finnigan Navigator/Agilent 1100/Sedere Sedex 75
system using a Phenomenex Luna C8 column (5 µm, 2.1 mm
× 50 mm). The elution system used was a gradient of 10-
100% over 4.5 min at 1.5 mL/min, and the solvent was either
acetonitrile/0.1% aqueous TFA or acetonitrile/10 mM am-
monium acetate. The MS was operated in the +APCI mode.
Melting points were determined on a Buchi 510 melting point
apparatus and are uncorrected.
3-{[4-(2-Ch lor op h en yl)p ip er izin -1yl]m et h yl}-1H -p yr -
r olo[2,3-b]p yr id in e (1b) (35%) (Gla ss). Compound 1b was
prepared by the literature procedure described for 1a by
substituting 2-chlorophenylpiperazine for the 4-chloro deriva-
tive.21 1H NMR (300 MHz, CDCl3) δ 2.88 (m, 2H), 3.10 (m, 2H),
5.25 (s, 2H), 6.52 (d, J ) 3.4 Hz, 2H), 6.96 (m, 1H), 7.03 (m,
1H), 7.09 (dd, J ) 7.8, 4.8 Hz, 1H), 7.20 (m, 1H), 7.32 (dd, J )
7.8, 1.7 Hz, 1H), 7.93 (dd, J ) 7.8, 1.2 Hz, 1H), 8.33 (dd, J )
4.8, 1.4 Hz, 1H); MS (DCI/NH3) m/z 327 (M + H)+. Anal.
(C18H19ClN4) C, H, N.
2-(4-P yr id in -4-ylp ip er a zin -1-ylm eth yl)-1H-ben zim id a -
zole (3c). 3c was purified by reversed-phase HPLC and
isolated as the CF3CO2H salt (28%) (glass). 1H NMR (CD3OD,
300 MHz) δ 2.83 (t, J ) 5 Hz, 4 H), 3.83 (t, J ) 5 Hz, 4 H), 4.2
(s, 2 H), 7.29 (d, J ) 7.8 Hz, 1 H), 7.60 (dd, J ) 6, 3 Hz, 2 H),
7.78 (dd, J ) 6, 3 Hz, 2 H), 8.14 (d, J ) 7.8 Hz, 1 H); MS (DCI/
NH3) m/z 294 (M + H)+. Anal. (C17H19N5‚2.1CF3CO2H‚0.9H2O)
C, H, N. F: calcd, 21.80; found, 21.31.
2-{[4-(3-Ch lor op h en yl)p ip er izin -1yl]m eth yl}-1H-ben z-
im id a zole (3e). 3e was purified by reversed-phase HPLC and
isolated as the CF3CO2H salt (64%) (glass). 1H NMR (CD3OD,
300 MHz) δ 2.92 (t, J ) 5 Hz, 4H), 3.38 (t, J ) 5 Hz, 4H), 4.23
(s, 2H), 6.82 (dd, J ) 7.5, 1.8 Hz, 1H), 6.92 (dd, J ) 8.7, 1.8
Hz, 1H), 6.95 (t, J ) 1.8, 1H), 7.20 (t, J ) 8.7 Hz, 1H), 7.55
(dd, J ) 6, 3 Hz, 2H), 7.76 (dd, J ) 6, 3 Hz, 2H); MS (DCI/
NH3) m/z 327 (M + H)+. Anal. (C18H19ClN4‚1.2CF3CO2H‚
0.3H2O) C, H, F, N.
2-{[4-(4-Ch lor op h en yl)p ip er izin -1yl]m eth yl}-1H-ben z-
im id a zole (3f). 3f was purified by reversed-phase HPLC and
isolated as the CF3CO2H salt (64%) (glass). 1H NMR (CD3OD,
300 MHz) δ 2.93 (t, J ) 5 Hz, 4H), 3.33 (t, J ) 5 Hz, 4H), 4.23
(s, 2H), 6.95 (d, J ) 7.8 Hz, 1H), 7.21 (d, J ) 7.8 Hz, 1H), 7.55
(dd, J ) 6, 3 Hz, 2H), 7.77 (dd, J ) 6, 3 Hz, 2H); MS (DCI/
NH3) m/z 327 (M + H)+. Anal. (C18H19ClN4‚1.3CF3CO2H‚
0.7H2O): C, F, N. H: calcd, 4.49; found, 3.98.
2-{[4-(3-Meth oxyph en yl)piper izin -1yl]m eth yl}-1H-ben z-
im id a zole (3i). 3i was purified by reversed-phase HPLC and
isolated as the CF3CO2H salt (69%) (glass). 1H NMR (CD3OD,
300 MHz) δ 3.00 (t, J ) 5 Hz, 4H), 3.48 (t, J ) 5 Hz, 4H), 3.78
(s, 3H), 4.25 (s, 2H), 6.52 (dd, J ) 7.5, 1.8 Hz, 1H), 6.59 (t, J
) 1.8, 1H), 6.65 (dd, J ) 9, 1.8 Hz, 1H), 7.20 (t, J ) 9.0 Hz,
1H), 7.55 (dd, J ) 6, 3 Hz, 2H), 7.76 (dd, J ) 6, 3 Hz, 2H); MS
(DCI/NH3) m/z 323 (M + H)+. Anal. (C19H22N4O‚1.7CF3CO2H‚
0.4H2O) C, H, F, N.
Com p ou n d s 3b, 3c, 3e, 3f, 3i, 3j, 3k . These compounds
were prepared by the general procedure given for 3a (see
below). Compounds 3d , 3g, and 3h were previously prepared
by a similar method.18
2-(4-P yr id in -2-yl-p ip er a zin -1-ylm eth yl)-1H-ben zim id a -
zole, Dih yd r och lor id e Sa lt (3a ). To a rapidly stirred solu-
tion of 2.5 g (15.4 mmol) of 2-pyridylpiperazine in 10 mL of
1:1 CH3CN/DMF in a large round-bottom flask in a water bath
at 20 °C was added 2.55 g (15.4 mmol) of 2-chloromethylbenz-
imidazole as a powder over 2 min. Triethylamine (3.2 mL, 1.5
equiv) was added, and the reaction mixture was stirred for 24
h. The reaction was processed by adding 250 mL of 0.4 M
aqueous Na2CO3, then extracting the product with 220 mL of
10:1 CH2Cl2/butanol. The organic phase was washed with 300
mL of H2O, dried over Na2SO4, then concentrated in vacuo to
yield a tan powder. Recrystallization from 330 mL of 1:10
ethanol/hexane gave 1.66 g (37%) of the free base as a white
powder. Mp 220-221 °C; 1H NMR (DMSO-d6, 300 MHz) δ 8.09
(dd, J ) 4.5, 1.8 Hz, 1 H), 7.41-7.58 (m, 3 H), 7.14 (m, 2 H),
6.81 (d, J ) 8.7 Hz, 1 H), 6.62 (dd, J ) 6.6, 4.5 Hz, 1 H), 3.77
(s, 2 H), 3.52 (t, J ) 4.5 Hz, 4 H), 2.55 (t, J ) 4.5 Hz, 4 H); MS
m/z 294 (M + H)+.
The crystalline dihydrochloride monohydrate salt was found
to have high aqueous solubility (>65 mg/mL) and was prepared
by addition of 2.4 equiv of 12 M hydrochloric acid to a stirred
suspension of 350 mg of the free base in methanol. The clear
solution obtained deposited large, well-formed crystals, which
were collected by filtration and washed with ethyl acetate and
ethanol. Mp 235-237 °C. Anal. (C17H19N5‚2.0HCl‚1.0H2O), C,
H, N.
2-{[4-(4-Meth oxyph en yl)piper izin -1yl]m eth yl}-1H-ben z-
1
im id a zole (3j) (41%) (Gla ss). H NMR (300 MHz, CDCl3) δ
2.76 (m, 4 H), 3.14 (m, 4 H), 3.77 (s, 3 H), 3.92 (s, 2 H), 6.88
(m, 4 H) 7.26 (dd, J ) 6.10, 3.05 Hz, 2 H), 7.59 (dd, J ) 5.76,
3.05 Hz, 2 H); MS (DCI/NH3) m/z 339 (M + H)+. Anal.
(C19H22N4O) C, H, N.
2-{[4-(4-F lu or op h en yl)p ip er izin -1yl]m eth yl}-1H-ben z-
1
im id a zole (3k ) (12%) (Gla ss). H NMR (300 MHz, CD3OD)
δ 2.71 (m, 4 H), 3.16 (m, 4 H), 3.86 (s, 2 H), 6.95 (d, J ) 6.44
Hz, 4 H), 7.22 (dd, J ) 6.10, 3.05 Hz, 2 H), 7.54 (dd, J ) 5.93,
3.22 Hz, 2 H); MS (DCI/NH3) m/z 311 (M + H)+. Anal. (C18H19
FN4) C, H, N.
-
Hu m a n D4.4 HEK F LIP R Assa y. Human D4.4 was coex-
pressed with GRqo5 in HEK293 cells as described.22 Cells were
plated into 96-well, black-wall/clear-bottom microplates (Bio-
coat, Becton Dickinson, Boston, MA) at 20 000 cells per well.
After 2 days of culture, the culture medium was removed by
aspiration and replaced by 0.1 mL of DPBS (Dulbecco’s
phosphate-buffered saline with D-glucose and sodium pyru-
vate) containing 0.04% Pluronic F-127 and 4 µM Fluo-4,
fluorescent calcium indicator dye. After incubation for 1 h at
room temperature, the cells were washed four times with
DPBS in a plate washer (Molecular Devices). After the final
wash, 150 µL of DPBS was added to each well. Fluorometric
imaging plate reader (FLIPR 384, Molecular Devices) trans-
ferred 50 µL from the compound plate to the cells and recorded
fluorescence reading for 3 min (every second for the first
minute and every 5 s for the next 2 min). The instrument
software normalizes the fluorescent reading to give equivalent
2-(4-P yr id in -3-ylp ip er a zin -1-ylm eth yl)-1H-ben zim id a -
zole (3b). A mixture of piperazine (22.6 g, 260 mmol),
3-bromopyridine (6.7 g, 42 mmol), NaO-t-Bu (5.6 g, 58 mmol),
Pd2dba3 (20 mg, 0.022 mmol), and P(t-Bu)3 (2 mg, 0.012 mmol)
in 25 mL of o-xylene was stirred at ambient temperature for
1 h and then heated at reflux for 4 h. After the mixture cooled,
solids were removed by filtration and the filtrate was concen-

2354 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Stewart et al.
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initial readings at time zero, and all the data were normalized
to the response of 10 µM dopamine.
3[H]sp ip er on e Hu m a n D4.4 Bin d in g Assa y. Human
dopamine D4.4 receptor-transfected HEK-293 cells22 were
cultured in DMEM supplemented with 10% fetal calf serum,
1 mM glutamine, 100 U/mL penicillin and 100 µg/mL strep-
tomycin (Invitrogen, Rockville, MD). For membrane prepara-
tion, the cells were seeded into a Cell Factory (VWR, Plainfield,
NJ ) and the confluent cells were rinsed with PBS and detached
with cell dissociation buffer (Invitrogen). The resulting cell
suspension was centrifuged, and the pellet was homogenized
by a Polytron for 10 s in 50 mM Tris-HCl, pH 7.4. Membrane
aliquots were stored at -80 °C until use.
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R.; J ohnson, D.; Colbry, N.; Rubin, R.; Blackburn, A.; Akunne,
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Dopamine Receptor Subtypes: Synthesis and Receptor Binding
Studies. J . Med. Chem. 1998, 41, 4933-4938. (e) Cha, M. Y.;
Choi, B. C.; Kang, H. K.; Pae, A. N.; Choi, K. I.; Cho, Y. S.; Koh,
H. Y.; Lee, H.-Y.; J ung, D.; Kong, J . Y. Design and Synthesis of
a Piperazinylalkylisoxazole Library for Subtype Selective Dopa-
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A. E.; Knowles, M.; Marwood, R.; Mcallister, G.; Myers, J .; Patel,
S.; Curtis, N.; Kluagowski, J . J .; Leeson, P. D.; Ridgill, M.;
Graham, M.; Matheson, S.; Rathbone, D.; Watt, A. P.; Bristow,
L. J .; Rupniak, N. M. J .; Baskin, E.; Lynch, J . J .; Ragan, C. I.
Biological Profile of L-745,870, a Selective Antagonist with High
Affinity for the Dopamine D4 Receptor. J . Pharmacol. Exp. Ther.
1997, 283, 636-647.
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agents, L-745,870 and U-101958 in HEK293 cells expressing the
human dopamine D4.4 receptor. Br. J . Pharmacol. 1998, 124,
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Efficacy Tunning of Selective Dopamine D4 Receptor Ligands
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piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine (FAUC 213). J .
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(17) (a) Hsieh, G. C.; Hollingsworth, P. R.; Martino, B.; Chang, R.;
Terranova, M. A.; O’Neill, A. B.; Lynch, J . J .; Moreland, R. B.;
Donnelly-Roberts, D.; Kolasa, T.; Mikusa, J . P.; McVey, J . M.;
Marsh, K. C.; Sullivan, J . P.; Brioni, J . D. Central Mechanisms
Regulating Penile Erection in Conscious Rats: The Dopamin-
ergic Systems Related to the Pro-erectile Effect of Apomorphine.
J . Pharmacol. Exp. Ther. 2004, 308, 330-338. (b) Brioni, J . D.;
Moreland, R. B.; Cowart, M.; Hsieh, G. C.; Stewart, A. O.;
Hedlund, P.; Donnelly-Roberts, D. L.; Nakane, M.; Lynch, J .;
Binding assays22 were initiated by addition 250 µL of
membrane to 200 µL of [3H]spiperone (125 Ci/mmol) and were
incubated at room temperature for 2 h. Nonspecific binding
was determined in the presence of 10 µM haloperidol (RBI-
Sigma). The incubation buffer consisted of 50 mM Tris-HCl,
pH 7.4, 5 mM KCl, 120 mM NaCl, 5 mM MgCl2, and 1mM
EDTA. In competition binding studies, agonists or antagonists
were prepared with 0.1% ascorbic acid in the buffer. The final
concentration for [3H]spiperone was 0.1 nM. The reaction was
terminated by rapid filtration through UniFilter-96 GF/B
filers, using a Filtermate harvester (Packard, Meriden, CT).
Filters were washed three times with 1 mL of ice-cold 50 mM
Tris-HCl, pH 7.4. Radioactivity was measured by a TopCount
microplate scintillation counter (Packard, Meriden, CT). Pro-
teins were determined by the BCA protein assay kit (Pierce,
Rockford, IL) using BSA as a standard.
3[H]sp ip er on e Hu m a n D2L Bin d in g Assa y. Cloned hu-
man dopamine D2L receptor cells (hD2L-HEK293)38 were cul-
tured in RPMI medium supplemented with 10% fetal calf
serum, 1 mM glutamine, 100 U/mL penicillin, and 100 µg/mL
streptomycin. For membrane preparation, the cells were
seeded into a Cell Factory (VWR, Plainfield, NJ ) and the
confluent cells were rinsed with PBS and detached with cell
dissociation buffer (Invitrogen/Life Sciences, Rockville, MD).
The resulting cell suspension was centrifuged, and the pellet
was homogenized by Polytron for 10 s in 50 mM Tris-HCl, pH
7.4. Membrane aliquots were stored at -80 °C until use.
Saturation binding assays were conducted with 20 µg of hD2L
membrane using 0.01-2 nM [3H]spiperone (Amersham, Ar-
lington Heights, IL) in 50 mM Tris, pH 7.4, 120 mM NaCl, 5
mM KCl, 2 mM Mg Cl2, and 2 mM CaCl2. Nonspecific binding
was determined in the presence of 10 µM haloperidol. Kd values
determined in this way were 0.09 nM. In competition assays,
concentrations of compounds (0.1-10000 nM) were competed
with 0.1 nM [3H]spiperone. Nonspecific binding was also
determined in the presence of 10 µM haloperidol. After
incubation for 1 h at room temperature, samples were filtered
and counted. Nonlinear regression analysis derived IC50 values
were converted to Ki values using GraphPad Prism software.
Ackn owledgm en t. The authors thank and acknowl-
edge discussions and manuscript review with Professor
Peter Beak of the University of IllinoissUrbana-Cham-
paign, Professor Les Mitscher of the University of
Kansas, and both Dr. Michael J arvis and Dr. Yvonne
Martin of Abbott Laboratories.
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