Subtle Modifications to the Indole-2-carboxamide Motif of the Negative Allosteric Modulator N-((trans)-4-(2-(7-Cyano-3,4-dihydroisoquinolin-2(1 H)-yl)ethyl)cyclohexyl)-1 H-indole-2-carboxamide (SB269652) Yield Dramatic Changes in Pharmacological Activity at the Dopamine D2 Receptor

Article abstract of DOI:10.1021/acs.jmedchem.8b00192

SB269652 (1) is a negative allosteric modulator of the dopamine D₂ receptor. Herein, we present the design, synthesis, and pharmacological evaluation of "second generation" analogues of 1 whereby subtle modifications to the indole-2-carboxamide motif confer dramatic changes in functional affinity (5000-fold increase), cooperativity (100-fold increase), and a novel action to modulate dopamine efficacy. Thus, structural changes to this region of 1 allows the generation of a novel set of analogues with distinct pharmacological properties.

Full text of DOI:10.1021/acs.jmedchem.8b00192

Subscriber access provided by READING UNIV
Brief Article
Subtle modifications to the indole-2-carboxamide motif of the negative
allosteric modulator N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide (SB269652) yield dramatic
2
changes in pharmacological activity at the dopamine D receptor
Anitha Kopinathan, Christopher J. Draper-Joyce, Monika
Szabo, Peter J. Scammells, J. Robert Lane, and Ben Capuano
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00192 • Publication Date (Web): 11 Jun 2018
Downloaded from http://pubs.acs.org on June 12, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Subtle modifications to the indole-2-carboxamide motif of the
negative allosteric modulator N-((trans)-4-(2-(7-cyano-3,4-
dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-
carboxamide (SB269652) yield dramatic changes in pharma-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cological activity at the dopamine D receptor
2
†
‡
†
†
‡*
Anitha Kopinathan , Christopher DraperꢀJoyce , Monika Szabo , Peter J. Scammells , J. Robert Lane
†*
and Ben Capuano
†
‡
Medicinal Chemistry, Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381
Royal Parade, Parkville, Victoria, 3052 Australia.
ABSTRACT: SB269652 (1) is a negative allosteric modulator of the dopamine D receptor. Herein, we present the design, syntheꢀ
2
sis and pharmacological evaluation of ‘second generation’ analogues of 1 whereby subtle modifications to the indoleꢀ2ꢀ
carboxamide motif confer dramatic changes in functional affinity (5000ꢀfold increase), cooperativity (100ꢀfold increase), and a
novel action to modulate dopamine efficacy. Thus structural changes to this region of 1 allows the generation of a novel set of anaꢀ
logues
with
distinct
pharmacological
properties.
INTRODUCTION
SB269652 (1) was recently identified as the first small molꢀ
ecule NAM of the D R displaying an affinity of 776 nM and
2
The dopamine D receptor (D R) is a member of the class A
2
2
1
moderate negative cooperativity with dopamine (αβ = 0.06,
G proteinꢀcoupled receptor (GPCR) family. To date, drug
discovery at this target has primarily focused on the developꢀ
ment of ligands that compete for the orthosteric site of the D R
where dopamine binds. Such drugs are used in the clinic to
treat disorders including schizophrenia (antagonists) and Parꢀ
kinson’s disease (agonists). Although orthosteric D R antiꢀ
conferring a maximal 17ꢀfold decrease in dopamine potenꢀ
5,6
cy). 1 was shown to bind the receptor in a bitopic manner
2
whereby the 1,2,3,4ꢀtetrahydroisoquinolineꢀ7ꢀcarbonitrile
7CNꢀTHIQ) “head” group occupies the orthosteric site and
the cyclohexylene “linker” and the indoleꢀ2ꢀcarboxamide
tail” extend into a secondary pocket formed by residues from
5,6
the extracellular ends of transmembrane helices 1, 2 and 7.
Further SAR investigations of 1 were subsequently conducted
to determine the structural determinants of affinity and negaꢀ
(
2
“
psychotics are effective at treating the positive symptoms of
schizophrenia, use of these drugs is associated with significant
2
sideꢀeffects. First generation or typical antipsychotics (e.g.
haloperidol and chlorpromazine), are associated with extrapyꢀ
ramidal sideꢀeffects (EPS). In contrast, second generation anꢀ
tipsychotics (exemplified by clozapine) display a lower risk of
EPS, but are known to cause hematological (agranulocytosis),
tive cooperativity at the D R. Modifications to the 7CNꢀTHIQ
2
head, the linker and the indoleꢀ2ꢀcarboxamide tail were examꢀ
ined for their impact upon affinity and negative cooperativity
7
2,3
at the D R (Figure 1). The results of this study showed that
2
metabolic and cardiovascular sideꢀeffects.
head groups with small substituents at the 7ꢀposition were
necessary to maintain the moderate negative cooperativity of
, whilst replacement of the cyclohexylene linker with
More recently, GPCR research has extended towards the deꢀ
velopment of allosteric modulators that interact with a topoꢀ
graphically distinct binding site to that occupied by the endogꢀ
enous ligand. Allosteric modulators may provide improved
receptor subꢀtype selectivity compared to orthosteric drugs. In
addition, unlike orthosteric ligands, because allosteric modulaꢀ
tors can allow the endogenous neurotransmitter to bind the
receptor, they may maintain the spatioꢀtemporal profile of
physiological signaling and instead modulate the magnitude of
this signal. Finally, allosteric modulators have a saturable efꢀ
fect determined by their cooperativity with the endogenous
ligands, thus a negative allosteric modulator (NAM) that disꢀ
plays moderate cooperativity with dopamine may act to parꢀ
1
polymethylene linkers of varying lengths was observed to
cause linker length dependent changes to allosteric modulaꢀ
tion. Alterations to the indoleꢀ2ꢀcarboxamide tail, however,
proved a key determinant of negative cooperativity. Replaceꢀ
ment of the bicyclic indole with smaller aromatic and nonꢀ
aromatic heterocyclic moieties (i.e. pyrrole and proline, reꢀ
spectively) led to a change in pharmacology from the weak
negative cooperativity to apparently competitive pharmacoloꢀ
gy. Additionally, a derivative of 1 that lacked the indolic NH
as a hydrogen bond donor displayed apparently competitive
behavior, suggesting that this group is necessary for NAM
action. Indeed, mutagenesis and molecular modeling studies,
in addition to the above SAR study provided evidence of a
hydrogen bond interaction between the indolic NH and
tially antagonize the D R, relieving the positive symptoms of
the disease without causing complete receptor blockade which
is associated with the EPS of typical antipsychotics.
2
4
2.65
Glu95 (where superscript numbering refers to Ballesterosꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 8
8
Weinstein nomenclature) and that this interaction was an imꢀ
portant determinant of the affinity and negative cooperativity
versatile primary amine intermediate (7) and its precursors
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were synthesized following the procedures outlined in Lane et
6
6
of these compounds. Of particular note, substitution of the
al. Following Boc deꢀprotection, two differing amide couꢀ
indoleꢀ2ꢀcarboxamide with a 7ꢀazaindoleꢀ2ꢀcarboxamide (2,
Figure 1) led to the highest affinity analogue of 1 to date with
pling reagents 1ꢀ[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀ
triazolo[4,5ꢀb]pyridinium 3ꢀoxid hexafluorophosphate, HATU
3
0ꢀfold higher affinity (K = 23.4 nM) and a similar level of
or
(benzotriazolꢀ1ꢀyloxy)tris(dimethylamino)phosphonium
B
7
negative cooperativity (αβ = 0.038).
hexafluorophosphate, BOP) were used in order to attach the
required “tail” moieties since some carboxylic acids were unꢀ
reactive in the presence of a variety of amide coupling reaꢀ
gents (e.g. HCTU, HBTU, EDC). HATU in the presence of
DIPEA and dry DMF, however, facilitated the majority of
amide couplings. The fluoroꢀ (8a-d) and methoxyꢀsubstituted
(9a-d) derivatives of SB269652 were successfully synthesized
using HATU and BOP, respectively, in good yields (30ꢀ57%
& 47ꢀ74%). Given the improved functional affinity of 2 in our
previous SAR investigation, a nitrogen walk comprising of the
various azaindoleꢀ2ꢀcarboxylic acids, and the corresponding
pyrazoloꢀ and imidazoꢀpyridineꢀ2ꢀcarboxylic acids was underꢀ
taken. Upon attempting these couplings however, only 10a-c
were successfully synthesized. The remaining 2ꢀsubstituted
carboxylic acids generally favored selfꢀdimerisation over couꢀ
pling to the primary amine (7) (see supporting information),
and in some cases, no reaction was observed. To enable a
more thorough investigation of the biological impact of isoꢀ
steric replacement of the CH for N, the corresponding 3ꢀ
carboxylic acid substituted derivatives of indole, azaindoles,
pyrazoloꢀpyridine and imidazoꢀpyridine (11a-g) were successꢀ
fully synthesized in respectable yields (12ꢀ51%).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. The chemical structure of SB269652 (1) highlighting its
orthosteric head, linker and allosteric tail sections, and the chemiꢀ
cal structure of the 7ꢀazaindoleꢀ2ꢀcarboxamide analogue (2) that
resulted from our initial SAR investigation, which exhibited a
relative 30ꢀfold increase in functional affinity whilst maintaining
robust negative cooperativity with dopamine.
Pharmacology. In order to determine the effect of the synꢀ
thesized compounds upon the action of the neurotransmitter
dopamine, all compounds were tested in an assay measuring
phosphorylation of ERK1/2 (pERK1/2) through activation of
the long isoform of the D R (D R) expressed in FlpIn CHO
Given that our initial SAR investigation determined the inꢀ
doleꢀ2ꢀcarboxamide motif to be an integral structural determiꢀ
nant in maintaining the NAM properties of 1 and the magniꢀ
tude of this allosteric effect, a further SAR study focused priꢀ
marily on the effects of modifications to the indoleꢀ2ꢀ
carboxamide motif of SB269652 was conducted. In making
modifications to this specific region of 1, the aim was to deꢀ
velop a suite of analogues that displayed varying degrees of
negative cooperativity in order to further our understanding of
2
2L
cells. All final compounds (8a ꢀ11g) were assessed for their
ability to antagonize the action of increasing concentrations of
dopamine.An example of these data is presented in Figure 2.
The data was fitted with a derivation of the operational model
of allosterism to derive values of functional affinity (K ) and
B
cooperativity with dopamine (αβ), where α is negative cooperꢀ
ativity with dopamine binding (that manifests in a limited conꢀ
centrationꢀdependent rightward shift in curves), and β is the
modulation of dopamine efficacy (that, depending upon the
level of receptor reserve, may manifest in both a limited a
concentrationꢀdependent decrease in potency and/or a depresꢀ
allosterism at the D R. The SAR study began with a prelimiꢀ
2
nary investigation into the effects of electronꢀdonating and
electronꢀwithdrawing substituents on negative cooperativity
through attachment of either a methoxy or fluorine substituent,
respectively, at positions 4ꢀ7 of the indole ring. To further
explore various azaindoleꢀ2ꢀcarboxamide tail motifs, a nitroꢀ
gen walk around the benzo component of the indole ring of 1
was planned since 2 showed a 30ꢀfold enhancement in affinity
6,9
sion in Emax). The data were also fit to a GaddumꢀSchild
model of competitive antagonism to derive values of K and a
B
10
Schild slope. The data for each compound were analyzed
using both models and the best fit was determined by an Fꢀ
test. Note that, as such, the description of compounds being
either allosteric or competitive within this study is an operaꢀ
tional rather than mechanistic one. Thus, using this approach
we cannot distinguish between competitive behavior and a
ligand that displays very high negative cooperativity such that
a saturable effect is not observed within the concentration
range of the ligand that was used. These data reported (Table 1
and Figure 2) are presented as logarithms to allow for statistiꢀ
and maintained negative cooperativity at the D R. Additionalꢀ
2
ly, various azaindoleꢀ3ꢀcarboxylic acids were investigated to
complement the azaindoleꢀ2ꢀcarboxamide series and enabled
us to determine the effect of repositioning the carboxylic acid
functionality (and therefore resulting carboxamide) from the
2
ꢀ to the 3ꢀposition. Finally, a thienopyrrole analogue was also
synthesized representing an isosteric replacement for the inꢀ
dole ring system of 1.
11
cal comparison. Values of Log αβ < 0 signify negative coopꢀ
RESULTS AND DISCUSSION
erativity.
Chemistry. Scheme 1 depicts the synthetic route employed
to furnish analogues of 1 containing differing “tail” moieties.
The synthesis of all compounds generally followed previously
established methods devised for the synthesis of 1 and associꢀ
6,7
ated analogues from our initial SAR investigations. The
ACS Paragon Plus Environment

Page 3 of 8
Journal of Medicinal Chemistry
Scheme 1. Synthesis of the varying indole/azaindole-
fluorine substituent at the 4ꢀ or 7ꢀposition demonstrated negaꢀ
a
1
2
3
4
5
6
7
8
9
carboxamide derivatives of 1 (8a-d, 9a-d, 10a-c, 11a-g)
tive cooperativity whilst analogues containing a fluorine subꢀ
stituent at the 5ꢀ and 6ꢀ position displayed apparently competiꢀ
tive pharmacology. In contrast, we observed NAM profiles for
8
b and 8c despite the fact that their small fragment counterꢀ
parts displayed competitive pharmacology. These results sugꢀ
gest that the SAR of these extended compounds does not
match that of the isopropylꢀ1Hꢀindoleꢀ2ꢀcarboxamide fragꢀ
ments in terms of apparent allosteric versus competitive beꢀ
havior. As suggested by our previous study, such differences
may relate to the role of the head and linker moieties conferꢀ
ring a distinct orientation of this motif within the allosteric
binding site and/or additional roles for the head and tail group
in dictating an allosteric versus an apparently competitive proꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12
file. In contrast to the fluorinated derivatives, the methoxy
derivatives (9b-d) demonstrated that a ring activating substituꢀ
ent conferred behavior best fit by a competitive model (Table
1
1
) with significant improvements in functional affinity (9b;
11ꢀfold, 9c; 31ꢀfold and 9d; 15ꢀfold), although in all three
cases the Schild slope was below the value of unity expected
for a competitive antagonist. However, the exception to this
rule, compound 9a (4ꢀOMe, KB = 617 nM, αβ = 0.30) apꢀ
peared to act as a NAM with weak negative cooperativity (5ꢀ
fold less than 1) and similar functional affinity to 1. We specuꢀ
late that the methoxy substituent at the 4 position (9a) may
confer a distinct orientation of the indole motif within the alloꢀ
steric pocket as compared to substitutions at the other posiꢀ
tions. This distinct orientation may in turn lead to the distinct
pharmacology observed. Based on this series of compounds, it
is apparent that electronic effects may influence NAM activity
a
Reagents and conditions: (a) EtI, K2CO3, ACN, overnight 50 ᵒC,
quantitative; (b) 1 M DIBALH in toluene, ꢀ78 ᵒC, 1 h, quantitaꢀ
tive; (c) NaBH(OAc) , 1,2ꢀDCE, rt, 16−24 h, 80%; (d) TFA,
DCM, base extraction, 84%ꢀquantitative; (e) HATU or BOP,
DCM or dry DMF, DIPEA, rt, 3ꢀ24 h, 5ꢀ74%.
3
at the D R, whereby the electronꢀwithdrawing fluorine substitꢀ
2
uent appeared more favorable for NAM pharmacology whilst
the electronꢀdonating methoxyꢀcontaining derivatives apꢀ
peared to confer competitive pharmacology. Another interestꢀ
ing observation from this series of analogues was that the adꢀ
dition of either substituent caused significant increases in
functional affinity for the majority of the analogues. This findꢀ
ing is also significant as it suggests that the tail moieties of this
series make a significant contribution to the affinity of these
compounds. While this is in contrast to the findings from our
initial findings with 1, whereby the orthosteric head group was
shown to be the predominant driver of affinity, it extends the
observations of our previous manuscript in which relatively
subtle changes to this motif conferred significant increases in
Initially, we were interested in evaluating the impact of elecꢀ
tronic effects arising from simple aryl substituents given that
replacement of the indoleꢀ2ꢀcarboxamide tail of 1 with the
electronꢀdeficient 7ꢀazaindoleꢀ2ꢀcarboxamide (2) resulted in
the most potent analogue of our initial SAR investigation. We
synthesized a series of compounds containing fluorineꢀ
substituted and methoxyꢀsubstituted indoleꢀ2ꢀcarboxamide
tails (8a-8d and 9a-9d, respectively; Table 1) to test the efꢀ
fects of electronꢀwithdrawing and electronꢀdonating substituꢀ
ents on affinity and negative cooperativity. Upon pharmacoꢀ
logical evaluation, it was evident that the electronꢀ
withdrawing fluorine substituent was beneficial for NAM acꢀ
tivity at the D R. 8a (4ꢀF, K = 47 nM, αβ = 0.03, 32ꢀfold atꢀ
8,12
affinity as exemplified by 2.
2
B
Based on the marked improvement in functional affinity obꢀ
served for 2 (30ꢀfold) compared to 1, we undertook a nitrogen
walk around the sixꢀmembered ring of the indole tail. This
type of scaffoldꢀhopping is a commonly applied medicinal
chemistry strategy for molecular backbone replacements (in
our instance, replacing the carbon at various positions of the
benzo portion of the indole ring with nitrogen). It is a drug
design strategy that is often utilised in the development of
novel compounds with increased potency and altered physicoꢀ
tenuation of dopamine potency) was observed to have virtually
identical pharmacological characteristics as 2, with a 30ꢀfold
improvement in functional affinity relative to 1. 8b (5ꢀF, K =
B
6
.8 nM, αβ = 0.016) and 8d (7ꢀF, K = 4.5 nM, αβ = 0.016)
B
were observed to have significant improvements in functional
affinity (110ꢀ and 194ꢀfold, respectively) and a moderate inꢀ
crease in negative cooperativity (3ꢀfold) compared to 1. In
contrast, 8c (6ꢀF, K = 219 nM, αβ = 0.065) maintained funcꢀ
B
13
tional affinity and negative cooperativity similar to that of 1.
This suggests that whilst incorporation of a fluorine substituꢀ
ent at the 4, 5 and 7 position of the indoleꢀ2ꢀcarboxamide tail
conferred increases in affinity, fluorine substitution at the 6
position had no effect compared to 1 (Table 1). It is also interꢀ
esting to compare these results with those of a previous study
conducted on a novel class of indoleꢀ2ꢀcarboxamide small
chemical characteristics (Table 1). Due to the synthetic inꢀ
tractability of the imidazopyridineꢀ and a number of the azainꢀ
doleꢀ2ꢀcarboxylic acids, only 10a-c were able to be syntheꢀ
sized in appreciable quantities. Nonetheless, we observed
dramatic increases in functional affinity and negative cooperaꢀ
tivity upon moving the nitrogen from the 7ꢀposition (2) to the
6ꢀposition (10a) of the azaindole motif. 10a (K = 2.7 nM, αβ
B
1
2
molecule allosteric modulators for the D R. Within this study
2
= 0.0006) was observed to have a functional affinity that was
the Nꢀisopropylꢀ1Hꢀindoleꢀ2ꢀcarboxamide compounds with a
2
60ꢀ and 8ꢀfold greater than 1 and 2, respectively. Furtherꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
more, 10a is the NAM with the highest detectable negative played a similar effect upon dopamine affinity and efficacy
Page 4 of 8
1
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cooperativity that we have identified to date (causing a 1800ꢀ
fold maximal decrease in dopamine potency), 100ꢀfold and
67ꢀfold greater than that of 1 and 2, respectively. We must
acknowledge, however, that the action of a ligand with such
high negative cooperativity may be indistinguishable comꢀ
pared to that of a competitive antagonist in an in vivo setting.
Indeed, it is also possible that other derivatives that we have
classified as competitive in our in vitro experiments may inꢀ
stead display even higher negative cooperativity such that they
appear competitive. The pyrazolopyridineꢀ2ꢀcarboxamide
but displayed much lower affinity for the D R. Thus, this
2
finding expands the allosteric behavior of bitopic SB269652
derivatives at the D R. However, it should be noted that obꢀ
2
servations of decreases in Emax are not, on their own, concluꢀ
sive evidence of an allosteric mode of action. Such effects can
also be caused by a competitive antagonist with a slow rate of
dissociation in conditions of hemiꢀequilibrium. As such furꢀ
ther experimental evidence is required to definitively describe
the pharmacology of this intriguing ligand.
15
Finally, we observed that moderate negative cooperativity
was maintained despite some analogues (e.g. 10b and 11b)
lacking the indolic NH. This is in contrast to our previous
SAR study in which we found that all derivatives of 1 that
lacked this motif as a hydrogen bond donor displayed apparꢀ
ently competitive pharmacology with dopamine. Furthermore,
our mutagenesis studies and molecular modeling studies led us
to propose that the indolic NH formed a hydrogen bond interꢀ
action with E95 residue at the top of TM2. Thus this study
suggests that such a hydrogen bond is not necessary for alloꢀ
steric pharmacology. This conclusion is in agreement with our
mutagenesis studies that revealed that mutation of E95
alanine did not result in a competitive mode of action for 1 but
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
derivative, 10b (K = 165 nM, αβ = 0.03) displayed a 5ꢀfold
B
improvement in functional affinity compared to 1, but mainꢀ
tained negative cooperativity similar to that of 2 despite a
comparative 16ꢀfold loss in functional affinity. In contrast to
7
this, the thienopyrroleꢀ2ꢀcarboxamide derivative, 10c (K = 87
B
nM) displayed apparently competitive antagonism at the D R,
2L
suggesting that isosteric replacement of the benzene ring of
the indole motif with thiophene may be detrimental to the
moderate NAM activity displayed by 1 (Table 1).
2.65
The attachment point to the scaffold was altered from the 2ꢀ
position to the 3ꢀposition in order to conduct a more compreꢀ
hensive investigation into the effects of a nitrogenꢀcontaining
scaffold hop around the sixꢀmembered ring of the indole tail.
Alteration of the substitution point of the carboxylic acid funcꢀ
tionality resolved the incidence of selfꢀdimerisation that was
observed with some of the arylꢀ2ꢀ carboxylic acids. Conseꢀ
quently, the pyrazolopyridineꢀ, imidazopyridineꢀ and regioiꢀ
someric azaindoleꢀ3ꢀcarboxamides were successfully syntheꢀ
2.65
to
instead decreased both the affinity and the degree of negative
7
cooperativity.
sized (11a-g, Table
carboxamide variant of 1 (11a, K = 63 nM), was best fit by a
model of competitive antagonist pharmacology but displayed a
1 and Figure 2). The indoleꢀ3ꢀ
B
1
2ꢀfold improvement in functional affinity compared to 1. 11c
(
K = 40 nM), unlike its 2ꢀcarboxamide analogue (10b), disꢀ
B
played a 4ꢀfold improvement in functional affinity compared
to 1 but acted in an apparently competitive manner. The 7ꢀ
azaindoleꢀ3ꢀcarboxamide derivative, 11d (K = 34 nM, αβ =
0.03), maintained similar values of negative cooperativity and
B
functional affinity as compared to 2. Compound 11e (K = 10
B
nM, αβ = 0.003) displayed an 8ꢀfold loss in functional affinity
and a 6ꢀfold loss in negative cooperativity compared to its 2ꢀ
carboxamide derivative, 10a. The 5ꢀazaindoleꢀ3ꢀcarboxamide
variant, 11f (K = 3.5 nM) (Figure 2B), displayed a functional
B
affinity similar to that of 10a but was best fit by a competitive
mode of action (Table 1). Finally, the imidazopyridineꢀ3ꢀ
carboxamide analogue, despite lacking an indolic NH (11b; KB
=
24 nM, αβ = 0.005), displayed NAM activity similar to 11e.
Of particular note, the 4ꢀazaindoleꢀ3ꢀcarboxamide derivaꢀ
tive (11g) displayed a novel pharmacological profile compared
to the other analogues in this series. Not only was 11g obꢀ
served to cause a limited dextral shift in dopamine potency in
a similar fashion to our previous analogues, but it effected a
significant limited depression in the Emax of dopamine (Figure
2). 11g (KB = 0.148 nM, α = 0.05, β = 0.16) is, to our
knowledge, the first drugꢀlike D R NAM to display an apparꢀ
ent subꢀnanomolar functional affinity for the D R (~5000ꢀ
fold improvement relative to 1) and acts to modulate both doꢀ
pamine potency (21ꢀfold maximal decrease) as observed for 1,
but also exerts an additional effect upon dopamine efficacy (6ꢀ
Figure 2: A scaffold hop strategy, replacing an aromatic carbon
with nitrogen at different positions of the indoleꢀ3ꢀcarboxamide
motif yielded analogues with distinct pharmacological profiles in
an assay measuring ERK1/2 phosphorylation as a readout of
2
2
L
D LR activation. Compound 11b (A) was best fit by an allosteric
2
model and causes a limited dextral shift of dopamine potency.
This is distinct from the action of 11f which causes a limitless
shift and was best fit by a competitive model (B). (C) Compound
11g causes a decrease in dopamine potency and Emax indicative of
an additional modulatory effect upon dopamine efficacy.
fold maximal decrease). A similar profile was observed for
SB269652 analogues at the dopamine D receptor. Furtherꢀ
more, a series of D R NAMs based upon the indoleꢀ2ꢀ
carboxamide scaffold but lacking the 7CNꢀTHIQ motif disꢀ
14
3
2
ACS Paragon Plus Environment

Page 5 of 8
Journal of Medicinal Chemistry
Table 1. Pharmacological evaluation of the second generation SB269652 analogues through modifications of the indole-2-
carboxamide tail
1
2
3
4
5
6
7
8
9
b
b
a
Logαβ ± SEM
Schild
Slope
± SEM
a
Logαβ ± SEM
(αβ)
[Fold-shift]
pK ± SEM
pK ± SEM
(K nM)
B,
Schild Slope
± SEM
B
B
#
X
(αβ)
Fold-shift]
#
X
c
(
K nM)
B,
f
c
f
[
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ
1.23 ± 0.14
(0.059)
ꢀ3.26 ± 0.09
(0.00055)*
[1800×]
6.11±0.02
776)
e
8.57 ± 0.09
(2.7)
e
1
2
n/a
10a
10b
n/a
(
[17×]
ꢀ
1.42 ± 0.09
(0.038)*
ꢀ1.52 ± 0.09
(0.030)
7.63 ± 0.10
(23)*
e
6.78 ± 0.12
(165)
e
n/a
n/a
[26×]
[33×]
ꢀ1.50 ± 0.19
(0.032)
7
.33 ± 0.24
47)*
e
7.06 ± 0.10
(87)*
b
8
a
n/a
10c
11a
n/a
1.04 ± 0.04
1.01 ± 0.06
(
[32×]
ꢀ
1.80 ± 0.22
(0.016)*
8.17 ± 0.29
6.8)*
e
7.20 ± 0.15
(63)*
b
8
b
n/a
n/a
(
[63×]
ꢀ1.19 ± 0.29
(0.065)
ꢀ2.31 ± 0.12
(0.0048)
[210×]
6
8
.66 ± 0.39
219)
e
7.62 ± 0.12
(24)
e
8c
n/a
11b
11c
n/a
(
[15×]
ꢀ
1.79 ± 0.18
(0.016)*
[62×]
.35 ± 0.29
4.5)*
e
7.39 ± 0.21
(40)
b
8d
n/a
n/a
0.71 ± 0.07
(
ꢀ0.52 ± 0.11
(0.30)*
ꢀ1.50 ± 0.09
(0.032)
6
.21 ± 0.06
617)
e
7.47 ± 0.11
(34)*
e
9a
n/a
11d
n/a
(
[3×]
[32×]
ꢀ2.44 ± 0.15
8
7
7
.70 ± 0.32
d
8.00 ± 0.11
(10)
e
9
b
n/a
n/a
n/a
0.54 ± 0.05 11e
0.69 ± 0.04 11f
(0.0036)
n/a
(2.0)*
[275×]
.60 ± 0.17
25)*
d
d
8.46 ± 0.21
(3.5)
b
9c
n/a
0.90 ± 0.04
(
Logα ± SEM
Logβ ± SEM
(β)
[Fold-shift]
.29± 0.13
51)*
pK ± SEM
B
9d
1.28 ± 0.09
#
1
X
(α)
Fold-shift]
(
(K nM)
B,
f
f
[
ꢀ1.32 ± 0.14
ꢀ0.80 ± 0.08
(0.16)
9
.83 ± 0.16
(0.148)
1g
(0.048)
[21×]
[6×]
a
b
Estimate of the negative logarithm of the equilibrium dissociation constant, the logarithm of the net cooperativity factor with dopamine
and Schild slope, determined in an ERK1/2 phosphorylation assay measuring dopamine activation of the D R expressed in FlpIn CHO
cells. Data represents the mean ± SEM of three experiments conducted in duplicate; n/a: data were best fit by a competitive model of acꢀ
tion therefore no cooperativity factor was derived. n/a: data were best fit by an allosteric model therefore a Schild slope was not deterꢀ
mined; Foldꢀshift denotes the maximal fold attenuation of dopamine potency in the presence of the test compound. (*) Statistically differꢀ
ent from the corresponding parameters for
c
2L
d
e
f
1
(p
<
0.05, one way ANOVA with Dunnett’s postꢀhoc test.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 8
More recent studies have revealed that derivatives of
SB269652 that display apparently competitive behavior are
also sensitive to this mutation. Thus a hydrogen bond interꢀ
affinity and efficacy of dopamine. Thus this compound disꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
plays a distinct action at the D R compared to other NAM
2
16
derivatives and therefore warrants further investigation. In
general, we find that modifications to the tail motif of 1 proꢀ
vides an approach to modulate both affinity and negative coꢀ
operativity, and that relatively subtle changes to this motif
result in quite dramatic differences in pharmacology.
action between the indolic NH of 1 and the side chain of
2.65
E95
appears to determine the degree of cooperativity but
not whether 1 and its derivatives display allosteric versus
competitive pharmacology. Furthermore, molecular dynamics
2
.65
simulations have provided evidence that the E95 to alanine
mutation changes the shape of the D R secondary pocket and
EXPERIMENTAL
2
that this can modulate the allosteric properties of SB269652
Refer to supporting information for details pertaining to the
synthesis, purification, structural characterization and pharmaꢀ
cological evaluation of target compounds 8a-d, 9a-d, 10a-c
and 11a-g. Representatives from the two coupling strategies
employed to afford the target compounds are presented.
17
that does not require a direct interaction with the ligand.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
However, we should also note that even in the above derivaꢀ
tives, the amide NH of 1 and its derivatives may also be able
2
.65
to form an interaction with E95 . Future studies using inforꢀ
mation from the recently determined crystal structure of the
18
2-(2-((trans)-4-(1H-Pyrrolo[3,2-b]pyridine-3-
carboxamido)cyclohexyl)ethyl)-7-cyano-1,2,3,4-
D R will provide further insight into such interaction.
2
tetrahydroisoquinolin-2-ium 2,2,2-trifluoroacetate (11g)
(Method A). 2ꢀ(2ꢀ((trans)ꢀ4ꢀAminocyclohexyl)ethyl)ꢀ1,2,3,4ꢀ
tetrahydroisoquinolineꢀ7ꢀcarbonitrile (7, 114 mg, 402 µmol, 1
eq.), 1Hꢀpyrrolo[3,2ꢀb]pyridineꢀ3ꢀcarboxylic acid (78.3 mg,
483 µmol, 1.2 eq.) and HATU (184 mg, 483 µmol, 1.2 eq.)
were stirred in a minimal volume of anhydrous DMF (3ꢀ4
mL). To this was added an excess of DIPEA (140 µL, 804
µmol, 2 eq.) and the reaction was left to stir overnight and
ceased upon complete consumption of the amine (LCMS). A
CONCLUSIONS
In our previous SAR investigation of 1, we determined the
indoleꢀ2ꢀcarboxamide tail to be a key structural determinant of
NAM activity at the D R. In particular, incorporation of a seꢀ
2
cond nitrogen atom within the indoleꢀ2ꢀcarboxamide tail of 1
in our initial SAR investigation led to the discovery of our
highest affinity analogue (2). Based on these findings, in the
present study we paid particular attention to the influence of
electronic effects upon affinity and cooperativity. A series of
derivatives substituted with either a fluorine or methoxy subꢀ
stituent at the various positions around the benzo portion of
the indole tail were synthesized. The fluoroꢀsubstituted derivaꢀ
tives (8aꢀd) demonstrated significant increases in functional
affinity and modulated cooperativity based on the positioning
of the fluorine. All fluoroꢀsubstituted compounds displayed
moderate negative cooperativity from a level that was equivaꢀ
lent to 1 up to a 4ꢀfold increase. Electronꢀdonating methoxyꢀ
substituted derivatives (9aꢀd), in contrast, displayed both an
increase in affinity relative to 1 and apparent competitive
mixture of sat. NaHCO and water (1:1, 30ꢀ40 mL) was added
3
to the reaction and stirring continued for a further 30 min. The
resulting precipitate was filtered, washed then purified via
preparative HPLC to yield the product as an offꢀwhite solid
1
(30.0 mg, 14%). H NMR (TFA salt) (CD OD) δ 8.70 – 8.56
3
(m, 3H), 7.76 (dd, J = 8.2, 5.9 Hz, 1H), 7.69 ꢀ 7.60 (m, 2H),
7
.50 ꢀ 7.43 (m, 1H), 4.68 – 4.32 (m, 2H), 3.94 (m, 1H), 3.60
(
(
(
m, 2H), 3.43 – 3.32 (m, 2H), 3.30 – 3.23 (m, 1H), 2.13 ꢀ 2.14
m, 2H), 1.98 ꢀ 1.90 (m, 2H), 1.86 – 1.75 (m, 2H), 1.53 – 1.40
13
m, 4H), 1.34 – 1.14 (m, 2H). C NMR (CD OD) δ 162.4 (C),
3
1
36.8 (C), 135.9 (CH), 135.8 (C), 135.6 (CH), 132.9 (C),
131.3 (CH), 130.5 (CH), 129.7 (CH), 129.6 (C), 128.7 (CH),
17.8 (CH), 117.7 (C), 110.8 (C), 106.9 (C), 54.7 (CH ), 52.0
pharmacology at the D R with the exception of 9a which disꢀ
2
played similar functional affinity and reduced negative coopꢀ
erativity (5ꢀfold). The positioning of the second nitrogen atom
within the tail as well as the attachment point of the bicyclic
aryl motif to the linker unit (either 2ꢀ or 3ꢀsubstituted) influꢀ
enced both affinity and, in particular, negative cooperativity
The effects of incorporating a second nitrogen atom at differꢀ
ent positions within the indole tail were studied via a scaffold
hop. The introduction of a second nitrogen was observed to
increase the functional affinity of all compounds within the 2ꢀ
substituted series relative to 1. It was particularly interesting to
note that the level of negative cooperativity was markedly
modulated by the position of the nitrogen within the 6ꢀ
membered ring of the indole tail in a similar fashion to the
fluorineꢀsubstituted analogues. For example, moving the niꢀ
trogen from the 7ꢀ(2) to the 6ꢀposition (10a) caused a dramatic
1
2
(
CH ), 49.2 (CH ), 48.3 (CH), 34.6 (CH), 32.0 (CH ), 31.3
2
2
2
(CH ), 30.6 (CH ), 25.4 (CH ). HPLC: t 4.14 min, >95%
2
2
2
R
purity (214 and 254 nm). HRMS (m/z): C H N O requires
26
30
5
+
[M+H] 428.2445; found 428.2449.
N-((trans)-4-(2-(7-Cyano-3,4-dihydroisoquinolin-2(1H)-
yl)ethyl)cyclohexyl)-4-methoxy-1H-indole-2-carboxamide
(9a) (Method B). To a solution of 4ꢀmethoxyꢀ1Hꢀindoleꢀ2ꢀ
carboxylic acid (35 mg, 0.18 mmol, 1 eq.) in DMF (5 mL) was
added DIPEA (1.2 eq.) under N at rt. BOP (1.1 eq.) was then
2
added, and the reaction left to stir for 5ꢀ10 min. The amine 7
(1.2 eq.) was then added slowly, and after 16 h the solvent was
removed in vacuo and the resulting residue partitioned beꢀ
tween DCM (20 mL) and sat. NaHCO (30 mL). The organic
3
phase was removed and the aqueous phase was further exꢀ
tracted with 3 × 10 mL portions of DCM. The organic layers
were pooled, washed with water (30 mL), brine (30 mL), dried
over anhydrous Na SO , filtered and concentrated to give the
(70ꢀfold) increase in negative cooperativity. The repositioning
of the attachment point of the indoleꢀcarboxamide tail from
the 2ꢀ position to the 3ꢀposition yielded a compound that disꢀ
played 10ꢀfold higher affinity than 1 but with apparently comꢀ
petitive pharmacology with dopamine. Interestingly, a similar
scaffoldꢀhop strategy to incorporate a second nitrogen at difꢀ
ferent positions yielded derivatives that displayed a range of
different properties; from those that acted in an apparently
competitive manner with dopamine to those that acted as
NAMs. Of particular note 11g was found to have subꢀ
2
4
crude product. To remove excess HMPA, the crude product
was dissolved in EtOAc and washed with 3 × 20 mL portions
of 2 M brine solution. The EtOAc layer was dried over anhyꢀ
drous Na SO , filtered and concentrated in vacuo and the reꢀ
2
4
sulting residue was purified by column chromatography (2:98
MeOH:CHCl ) to give the title compound as a beige solid (47
3
1
mg, 56%). H NMR (d ꢀDMSO) δ 11.50 (d, J = 1.7 Hz, 1H)
6
nanomolar affinity for the D R and acted to decrease the poꢀ
2
8
.13 (d, J = 8.1 Hz, 1H), 7.57 – 7.56 (m, 2H), 7.31 (d, J = 8.4
tency and maximal effect (Emax) of dopamine. Such an action
may be consistent with a NAM that acts to modulate both the
Hz, 1H), 7.24 (m, 1H), 7.09 – 6.99 (m, 2H), 6.49 (d, J = 7.4
ACS Paragon Plus Environment

Page 7 of 8
Journal of Medicinal Chemistry
Hz, 1H), 3.87 (s, 3H), 3.74 (m, 1H), 3.58 (s, 2H), 2.88 (m,
H), 2.67 (m, 2H), 2.48 (m, 2H), 1.88 – 1.79 (m, 4H), 1.45
(dd, J = 14.4, 6.8 Hz, 2H), 1.34 – 1.28 (m, 3H), 1.06 (m, 2H).
In Methods in Neurosciences, Stuart, C. S., Ed. Academic Press:
1995; Vol. Volume 25, pp 366ꢀ428.
9) Leach, K.; Sexton, P. M.; Christopoulos, A. Allosteric GPCR
modulators: taking advantage of permissive receptor pharmacology.
Trends Pharmacol. Sci. 2007, 28, 382ꢀ389.
1
2
3
4
5
6
7
8
9
2
(
1
3
C NMR (d ꢀDMSO) δ 160.1 (C), 153.6 (C), 140.6 (C), 137.7
6
(
(
(
(
(
C), 136.7 (C), 130.6 (C), 130.4 (CH), 129.7 (CH), 129.5
CH), 124.1 (CH), 119.1 (C), 118.1 (C), 108.2 (C), 105.4
CH), 100.1 (CH), 99.2 (CH), 55.3 (CH ), 55.0 (CH ), 54.8
(
10) Schild, H.O. Drug antagonism and pAx. Pharmacol. Rev. 1957,
9, 242ꢀ246.
2
3
(11) Christopoulos, A. Assessing the distribution of parameters in
models of ligandꢀreceptor interaction: to log or not to log. Trends
Pharmacol. Sci. 1998, 19, 351ꢀ357.
(12) Mistry, S. N.; Shonberg, J.; DraperꢀJoyce, C. J.; Klein Herenꢀ
brink, C.; Michino, M.; Shi, L.; Christopoulos, A.; Capuano, B.;
Scammells, P. J.; Lane, J. R. Discovery of a novel class of negative
CH ), 49.8 (CH ), 48.2 (CH), 34.7 (CH), 33.6 (CH ), 32.3
2
2
2
CH ), 31.7 (CH ), 28.9 (CH ). HPLC: t 6.32 min, >99% puriꢀ
2
2
2
R
+
ty. HRMS (m/z): C H N O requires [M+H] 457.2604;
2
8
33
4
2
found 457.2614.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
allosteric modulator of the dopamine D
tion of a bitopic ligand. J. Med. Chem. 2015, 58, 6819ꢀ6843.
13) Tung, Y. S.; Coumar, M. S.; Wu, Y. S.; Shiao, H. Y.; Chang, J.
Y.; Liou, J. P.; Shukla, P.; Chang, C. W.; Chang, C. Y.; Kuo, C. C.;
Yeh, T. K.; Lin, C. Y.; Wu, J. S.; Wu, S. Y.; Liao, C. C.; Hsieh, H. P.
Scaffoldꢀhopping strategy: synthesis and biological evaluation of 5,6ꢀ
fused bicyclic heteroaromatics to identify orally bioavailable antiꢀ
cancer agents. J. Med. Chem. 2011, 54, 3076ꢀ3080.
2
receptor through fragmentaꢀ
AUTHOR INFORMATION
Corresponding Author
(
*
B.C.: phone, +61 3 9903 9556; fax, +61 3 9903 9581; eꢀmail,
ben.capuano@monash.edu.
J.R.L.: phone, +613 9903 9095; fax, +613 9903 9581; eꢀmail,
*
rob.lane@monash.edu.
(
14) Kumar, V.; Moritz, A. E.; Keck, T. M.; Bonifazi, A.; Ellenꢀ
ACKNOWLEDGMENT
berger, M. P.; Sibley, C. D.; Free, R. B.; Shi, L.; Lane, J. R.; Sibley,
D. R.; Newman, A. H. Synthesis and pharmacological characterizaꢀ
tion of novel transꢀcyclopropylmethylꢀlinked bivalent ligands that
exhibit selectivity and allosteric pharmacology at the dopamine D3
receptor (D3R). J. Med. Chem. 2017, 60, 1478ꢀ1494.
This research was supported by Project Grant 1049564 of the Nationꢀ
al Health and Medical Research Council (NHMRC). AK gratefully
acknowledges the Faculty of Pharmacy and Pharmaceutical Sciences,
Monash University for financial support.
(15) Kenakin, T. Allosteric modulators: the new generation of recepꢀ
tor antagonist. Mol. Interv. 2004, 4, 222ꢀ229.
ABBREVIATIONS USED
allosteric cooperativity, αβ; functional affinity, K
extracellular signalꢀregulated kinase 1/2, pERK1/2.
(
16) DraperꢀJoyce, C. J.; Michino, M.; Verma, R. K.; Herenbrink, C.
B
; phosphorylated
K.; Shonberg, J.; Kopinathan, A.; Scammells, P. J.; Capuano, B.;
Thal, D. M.; Javitch, J. A.; Christopoulos, A.; Shi, L.; Lane, J. R. The
structural determinants of the bitopic binding mode of a negative
allosteric modulator of the dopamine D2 receptor. Biochem. Pharmaꢀ
col. 2018, 148, 315ꢀ328.
(17) Verma, R. K.; Abramyan, A. M.; Michino, M.; Free, R. B.; Siꢀ
bley, D. R.; Javitch, J. A.; Lane, J. R.; Shi, L. The E2.65A mutation
disrupts dynamic binding poses of SB269652 at the dopamine D2 and
D3 receptors. PLOS Comput. Biol. 2018, 14, e1005948.
ASSOCIATED CONTENT
Supporting Information. Synthesis and characterization for all final
compounds including Molecular Formula Strings. This material is
available free of charge via the Internet at http://pubs.acs.org.
(
18) Wang, S.; Che, T.; Levit, A.; Shoichet, B. K.; Wacker, D.; Roth,
REFERENCES
B. L. Structure of the D2 dopamine receptor bound to the atypical
antipsychotic drug risperidone. Nature 2018, 555, 269ꢀ273.
(
1) Beaulieu, J. M.; Gainetdinov, R. R. The physiology, signaling, and
pharmacology of dopamine receptors. Pharmacol. Rev. 2011, 63, 182ꢀ
17.
2) Seeman, P. Targeting the dopamine D
Expert Opin. Ther. Targets 2006, 10, 515ꢀ531.
3) De Hert, M.; Detraux, J.; van Winkel, R.; Yu, W.; Correll, C. U.
2
(
TOC Graphic
2
receptor in schizophrenia.
(
Metabolic and cardiovascular adverse effects associated with antipsyꢀ
chotic drugs. Nat. Rev. Endocrinol. 2012, 8, 114ꢀ126.
(
4) Rossi, M.; Fasciani, I.; Marampon, F.; Maggio, R.; Scarselli, M.
The first negative allosteric modulator for dopamine D and D recepꢀ
tors SB269652 may lead to a new generation of antipsychotic drugs.
Mol. Pharmacol. 2017, 91, 586ꢀ594.
(5) Silvano, E.; Millan, M. J.; Mannoury la Cour, C.; Han, Y.; Duan,
L.; Griffin, S. A.; Luedtke, R. R.; Aloisi, G.; Rossi, M.; Zazzeroni, F.;
Javitch, J. A.; Maggio, R. The tetrahydroisoquinoline derivative
2
3
3 2
SB269,652 is an allosteric antagonist at dopamine D and D recepꢀ
tors. Mol. Pharmacol. 2010, 78, 925ꢀ934.
(6) Lane, J. R.; Donthamsetti, P.; Shonberg, J.; DraperꢀJoyce, C. J.;
Dentry, S.; Michino, M.; Shi, L.; Lopez, L.; Scammells, P. J.; Capuꢀ
ano, B.; Sexton, P. M.; Javitch, J. A.; Christopoulos, A. A new mechꢀ
anism of allostery in a G proteinꢀcoupled receptor dimer. Nat. Chem.
Biol. 2014, 10, 745ꢀ752.
(7) Shonberg, J.; DraperꢀJoyce, C.; Mistry, S. N.; Christopoulos, A.;
Scammells, P. J.; Lane, J. R.; Capuano, B. Structure–activity study of
Nꢀ((trans)ꢀ4ꢀ(2ꢀ(7ꢀcyanoꢀ3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀ
yl)ethyl)cyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (SB269652), a bitopic
ligand that acts as a negative allosteric modulator of the dopamine D
receptor. J. Med. Chem. 2015, 58, 5287ꢀ5307.
(8) Ballesteros, J. A.; Weinstein, H. [19] Integrated Methods for the
Construction of ThreeꢀDimensional Models and Computational Probꢀ
ing of StructureꢀFunction Relations in G ProteinꢀCoupled Receptors.
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 8
Table of Contents Graphic
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Subtle modifications to the indole-2-carboxamide motif of the negative allosteric
modulator N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-
1
H-indole-2-carboxamide (SB269652) yield dramatic changes in pharmacological
activity at the dopamine D receptor
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
ACS Paragon Plus Environment