Proof of concept study for designed multiple ligands targeting the dopamine D2, serotonin 5-HT2A, and muscarinic M1 acetylcholine receptors

Article abstract of DOI:10.1021/jm5013243

Herein we describe the hybridization of a benzoxazinone M₁ scaffold with D₂ privileged structures derived from putative and clinically relevant antipsychotics to develop designed multiple ligands. The M₁ mAChR is an attractive target for the cognitive deficits in key CNS disorders. Moreover, activity at D₂ and 5-HT_2A receptors has proven useful for antipsychotic efficacy. We identified 9 which retained functional activity at the target M₁ mAChR and D₂R and demonstrated high affinity for the 5-HT_2AR.

Full text of DOI:10.1021/jm5013243

Brief Article
pubs.acs.org/jmc
Proof of Concept Study for Designed Multiple Ligands Targeting the
Dopamine D2, Serotonin 5‑HT2A, and Muscarinic M1 Acetylcholine
Receptors
Monika Szabo,†,‡ Herman D. Lim,‡ Carmen Klein Herenbrink,‡ Arthur Christopoulos,‡ J. Robert Lane,*,‡
and Ben Capuano*,†
‡
†Medicinal Chemistry, and Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal
Parade, Parkville 3052, Victoria, Australia
S
* Supporting Information
ABSTRACT: Herein we describe the hybridization of a
benzoxazinone M1 scaffold with D2 privileged structures
derived from putative and clinically relevant antipsychotics to
develop designed multiple ligands. The M1 mAChR is an
attractive target for the cognitive deficits in key CNS disorders.
Moreover, activity at D2 and 5-HT2A receptors has proven
useful for antipsychotic efficacy. We identified 9 which retained
functional activity at the target M1 mAChR and D2R and
demonstrated high affinity for the 5-HT2AR.
HT2AR).2,3 Indeed the favorable polypharmacology that is
observed with many atypical antipsychotics such as clozapine
INTRODUCTION
■
All antipsychotic drugs currently on the market for the
treatment of schizophrenia antagonize the dopamine D2
receptor (D2R), a member of the G protein-coupled receptor
(GPCR) family. They are effective in alleviating the positive
was achieved through serendipitous discovery rather than by a
rational drug design process.4 A newer class of clinically used
antipsychotics are the D2R partial agonists, of which
aripiprazole (3, Figure 1) is the most commonly prescribed
symptoms of schizophrenia (hallucinations, delusions) which
antipsychotic in the U.S. for the treatment of schizophrenia and
are postulated to arise from hyperdopaminergia in the
other CNS disorders.5 Partial agonists act to stabilize
mesolimibic pathway of the brain.1 Antipsychotics such as
ziprasidone (1) and risperidone (2, Figure 1) have also been
dopaminergic signaling rather than exert the complete
inhibition associated with D2R antagonists.6 Since the success
shown to cause improvements in negative symptoms (social
of aripiprazole, other D2R partial agonists have emerged such as
cariprazine7 and brexpiprazole,8 which both have the 2,3-
withdrawal, lack of motivation), an effect attributed to their
favorable polypharmacology and in particular their action as
high affinity antagonists at the serotonin 5-HT2A receptor (5-
dichlorophenylpiperazine motif present in 3. Another D2R
partial agonist bifeprunox (4) exhibits a structurally more
diverse heterocycle attached to the piperazine and incorporates
a hydrophobic and unfunctionalized biphenyl substituent.
There are also high affinity D2R partial agonists such as 5
that contain a 2-methoxy substituted phenylpiperazine motif
rather than 2,3-dichlorophenylpiperazine.9 However, no current
antipsychotic drug addresses the cognitive deficits associated
with schizophrenia, which is an equally important component
of the etiology of schizophrenia.
There is evidence to support that targeting the M1
muscarinic acetylcholine receptor (M1 mAChR), also belonging
to the GPCR family, improves the cognitive deficits of patients
suffering from schizophrenia and other CNS disorders.10,11
However, selective targeting of this receptor remains a
challenge due to the high conservation of the orthosteric site
across the mAChR receptor family, and activity at other
Figure 1. Antipsychotics: ziprasidone (1), risperidone (2), aripiprazole
(3), bifeprunox (4), and a high affinity D2 partial agonist (5).
Received: August 29, 2014
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
receptors in this family is associated with limiting side effects.12
Of interest, M1 allosteric agonists have particularly gained a
great deal of research focus.13 Ligands that act at an allosteric
site (a topographically distinct site to the orthosteric site) offer
the added benefit of possibly being subtype selective, as the
residues are less conserved in an allosteric site versus an
orthosteric site.14 Sams et al. used this rationale to generate the
putative M1 allosteric agonist LuAE51090 (6, Figure 2) that has
Figure 2. Structure of 6 highlighting common dopaminergic D2
structural characteristics: a motif containing features of common
privileged structures, namely, an aryl system and ionizable nitrogen
atom, an aliphatic spacer/linker, and a heterocyclic group.
Figure 3. Merged DMLs (7−11) derived from combining the
propylbenzoxazinone moiety from 6 with D2 privileged structures
(colored segments) from parent ligands 1−5.
a favorable M1 profile (EC50 = 61 nM; intracellular Ca2+
mobilization assay) but exhibits poor binding affinity at the
D2R and the 5-HT2AR (Ki ≈ 1 μM).15 We found this surprising,
as 6 displays some key pharmacophoric features of many D2R
ligands, both antagonists and partial agonists as highlighted in
Figure 2 and in an earlier publication.16
RESULTS AND DISCUSSION
■
Chemistry. The syntheses of all DMLs and the reference
M1 ligand (6) are outlined in Scheme 1. This initially required
Therefore, utilizing 6 as our primary scaffold, we envisioned
that we could design in an enhanced D2R binding profile using
the designed multiple ligand (DML) approach described by
Morphy that takes two separate pharmacophores with distinct
pharmacology and integrates them into one molecule that has
the attributes of both parent molecules. In this approach the
degree of integration is systematically increased until the
structure becomes merged and essentially more druglike.17 We
made use of privileged structures from known D2R ligands that
covered three distinct classes (Figure 3): (1) phenylpiperazines
which are known to be important motifs for D2R affinity and
functional activity; our previous work demonstrated that
compounds incorporating the 2,3-dichlorophenylpiperazine
and 2-methoxyphenylpiperazine scaffolds are useful in the
design of antagonists and partial agonists for the D2R;16 (2)
using privileged structures from two distinct antipsychotics
(ziprasidone and risperidone) that have a piperazine or
piperidine moiety followed by similar structural heterocycles;18
(3) using a privileged structure from a partial agonist
(bifeprunox) which is unique compared to the 2,3-dichloro-
and 2-methoxyphenylpiperazine family.
As such, rather than embarking on a screening program to
identify ligands that display the desired pharmacology for two
or more receptors required for activity, we hoped to achieve
this polypharmcology through the rational combination of
distinct pharmacophores.19 To characterize the ligands, we
pharmacologically evaluated them in radioligand binding assays
for all three receptors (D2R, M1 mAChR, and 5-HT2AR). To
evaluate the ligands in functional assays, we tested them in
ERK1/2 phosphorylation assays for the D2R, intracellular Ca2+
mobilization assays for the M1 mAChR and IP1 accumulation
assays for the 5-HT2AR.
a
Scheme 1. Synthesis of DMLs (7−11) and 6
a
Reagents and conditions: (a) dibromopropane, NaH (60%), DMF,
N2, rt, 67%; (b) privileged structures, DIPEA, NaI, CH3CN, reflux, 5 h
or overnight, 17−75%; (c) 4-N-Boc-aminopiperidine, K2CO3,
CH3CN, 80 °C, 62%; (d) TFA, DCM, rt, overnight, 73%; (e) 2-
phenylacetyl chloride, Et3N, THF, N2, 0 °C → rt, 1.5 h, 82%.
the installation of a three-carbon atom spacer to the precursor
benzoxazinone (12). This reaction was performed under
alkaline conditions using 60% sodium hydride and a large
excess of 1,3-dibromopropane to give the key intermediate 13.
Rapid diversification with 13 and the required privileged
structures, as illustrated in Figure 3, furnished the target DMLs
(7−11) in yields of 17−75%. The chemical synthesis of 6
(which is also commercially available) followed similar
conditions to that previously published.15 Compound 13 was
refluxed under basic conditions with 4-N-Boc-aminopiperidine
to afford 14 in respectable yield. The final step was achieved by
removal of the Boc protecting group (15) and subsequent
B
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Brief Article
a
Table 1. Binding and Functional Data at the D2R, M1 mAChR, and 5-HT2AR for All DMLs
a
b
Data are the mean of three to four experiments SEM performed in duplicate. n/a = compound not active. Binding affinity values are obtained
using [3H]raclopride (D2L-FlpIn CHO whole cells) or [3H]NMS (M1 mAChR-FlpIn CHO whole cells) or [3H]ketanserin (5-HT2A-FlpIn CHO cell
membranes). Value of functional affinity (KB) obtained from interaction studies with varying concentrations of dopamine in an assay measuring
levels of pERK1/2. Data are fit to the Gaddum−Schild analysis. Emax is the percentage of maximal activity relative to the maximal activity of ACh in
the intracellular calcium mobilization assay. Value of functional affinity (KB) obtained from interaction studies with varying concentrations of
c
d
e
serotonin in an IP1 accumulation assay.
reaction with 2-phenylacetyl chloride to generate the target 6 in
82% yield.
affinity of 11 may result from being the only heterocyclic
moiety that contains a H-bond donor capable of hydrogen
bond interactions with residues such as serines present in
transmembrane domain 5 in the orthosteric binding site.
For functional pERK1/2 assays, all ligands were initially
tested in time-course assays (data not shown), upon which we
identified ligands 7−10 to be antagonists at the D2R and 11 as
an agonist. Importantly, this latter result is consistent with the
partial agonist action of bifeprunox; the ligand from which the
7-(piperazin-1-yl)benzo[d]oxazol-2(3H)-one moiety was de-
rived.
To determine the functional affinity (pKB) of each of the
antagonists, we performed interaction studies using varying
concentrations of dopamine. The functional affinities correlated
well with the binding affinities obtained. Compound 11,
derived from merging the privileged structure of 4 with 6, was a
potent agonist at the D2R in pERK1/2 and cAMP signaling
assays (Table 2; 0.64 nM and 96 pM, respectively). We
calculated a bias factor for 11 relative to the full agonist
ropinirole (Supporting Information Table 1 and Figure 1) and
found that 11 displays a similar bias toward cAMP over
pERK1/2 to that previously determined for aripiprazole (fold
bias of 448 and 102, respectively).16 The results from Table 1
show that the D2 profile of the ligands are significantly
Pharmacology. D2R Binding and Functional Character-
ization of DMLs. The synthesized DMLs were pharmacolog-
ically characterized in radioligand binding and functional
ERK1/2 phosphorylation (pERK1/2) assays for the D2R.
The pERK assay provides a robust readout for D2R activation
and is medium throughput, thereby allowing efficient screening
of a number of compounds. The results of these studies are
summarized in Table 1. Compound 6 demonstrated the
weakest binding affinity (pKi), consistent with previously
published results.15 Compounds 10 and 11 (consisting of the
motifs from risperidone (2) and bifeprunox (4), respectively)
exhibited the highest binding affinities (Ki of 3.2 and 1.7 nM,
respectively) for the D2R overall. This result is consistent with
the high affinity of the parent compounds 2 and 4.20
Indeed the incorporation of D2R privileged structures
conferred a significant increase in D2R affinity for all
compounds as compared to 6 apart from the incorporation
of the 2,3-dichlorophenylpiperazine moiety (7). The high
binding affinity of 10 at the D2R may be attributed to its
bicyclic system of greater molecular size as opposed to 8
(phenylpiperazine) and its piperidine moiety compared to the
piperazine of 8 and 9. Furthermore, the equally high binding
C
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Journal of Medicinal Chemistry
Brief Article
a
of these privileged structures into the M1 mAChR/D2R DML
and tested the ligands in a radioligand binding assay to
determine their affinities for this receptor (Table 1). Consistent
with the literature,15 the M1 mAChR agonist 6 demonstrated
poor binding to the 5-HT2AR (Ki ≈ 1 μM). Both phenyl-
piperazine analogues 7 and 8 showed no notable enhancements
in affinity as compared to 6. Of note, DMLs 9 and 10 show a
strong binding affinity for the 5-HT2AR (Ki of 5.8 and 2.7 nM,
respectively). Compound 11, which as mentioned previously
has a slightly different heterocycle following the piperazine, had
a diminished binding affinity similar to its M1 mAChR binding
profile. As 9 and 10 maintain their D2/5-HT2A binding profiles,
it is evident that both privileged structures exhibit versatility for
use in the design of ligands with favorable polypharmacology.
The compounds were also tested in an IP1 functional assay at
the 5-HT2AR. When tested in the absence of serotonin
(Supporting Information Figure 2), all compounds showed
no activation of the receptor except for compound 7. As such
compounds 8−11 are all antagonists at the serotonin receptor.
In agreement with the binding data for the 5-HT2AR, 6
demonstrated the weakest functional affinity. DMLs containing
the ziprasidone and risperidone privileged structures both
showed the highest functional affinities with 10 displaying an
11-fold increase over 9 (p < 0.05). As mentioned in the
Introduction, there is considerable evidence that antagonism at
the 5-HT2AR is useful for antipsychotics; therefore, all DMLs,
excluding 7, are useful starting compounds for further SAR
studies and optimization.
Compound 9 was our most promising candidate, as it
displayed activity at all three receptor targets. Despite showing
strong affinities for the D2R and 5-HT2AR (Table 1; Ki of 17.7
and 5.8 nM, respectively), there was a significant reduction in
potency at the M1 mAChR (EC50 = 1.04 μM, Emax = 64%).
However, we have shown that we can use a merged DML
approach utilizing D2R privileged structures to confer a D2R
pharmacological profile to a putative M1 mAChR allosteric
agonist. In addition to this, privileged structures derived from
parent structures with known D2/5-HT2A receptor binding
profiles were maintained, thus validating their usefulness in a
DML approach. It should be noted that it was difficult to
maintain activity at the M1 mAChR, as only one structure was
able to show any noteworthy agonism. It is therefore possible
that key elements in the structure of 6 that account for its M1
mAChR allosteric agonist profile were lost as a result of the
integration process. As such, new structural analogues may look
at incorporating greater elements of the original structure. To
expand on this work, it may also be useful to incorporate other
benzoxazinones or heterocyclic compounds similar to this
scaffold as a way to optimize and possibly enhance the M1
mAChR profile. Additionally, other selective M1 mAChR
agonists22,23 may be explored for optimization as a DML.
Table 2. Profiling of 11 in Functional Assays at the D2R
ERK1/2 phosphorylation
cAMP
pEC50 SEM
pEC50 SEM
b
b
compd
(EC50, nM)
Emax
SEM
3
(EC50, nM)
Emax
97
SEM
2
11
9.20 0.29
(0.64)
26
10.02 0.13
(0.096)
a
Data are the mean of four to six experiments SEM performed in
duplicate. Data are generated via concentration−response curves.
Emax is the percentage of maximal activity relative to the maximal
b
activity of FBS (pERK1/2) or ropinirole (cAMP).
enhanced with the incorporation of the privileged structures
and compounds from all three classes (phenylpiperazines,
antipsychotics, and partial agonists) with the benzoxazinone
scaffold from 6 at the D2R.
M1 mAChR Binding and Functional Characterization of
DMLs. As previously indicated, we tested compounds in
radioligand binding and functional assays at the M1 mAChR
(Table 1). All compounds displaced [3H]NMS at the M1
mAChR with relatively weak inhibitory potencies (IC50), with
7 displaying the highest inhibitory potency (IC50= 1.9 μM), as
compared to 6 (IC50 = 8.5 μM, p < 0.05). As a functional assay
for the M1 mAChR, we used an intracellular Ca2+ mobilization
assay as a measure of coupling to Gq pathways. Only DML 9
showed activity at the M1 mAChR, displaying a diminished
potency (EC50 =1.04 μM) as compared to 6 (EC50 = 129 nM)
equating to an 8-fold loss in potency. The maximal stimulation
(Emax) of 9 was also reduced to 64% compared to 6 which
demonstrated an Emax of 101% (defined by the maximal effect
of ACh) consistent with an action as a partial agonist. The
subtle differences in the D2R privileged structures used could
account for their loss in M1 mAChR activity. For example the
M1 mAChR may not accommodate the more linear orientation
of the phenylpiperazine analogues 7 and 8 as compared to a
more flexible structure present in 6. For the antipsychotics, the
ziprasidone and risperidone privileged structures reveal slightly
different heterocycles (benzoisothiazole vs benzoisoxazole), in
addition to the absence of a fluorine substituent on the
aromatic ring, perhaps making 9 more favorable for the M1
mAChR than 10, as a fluorine atom is powerfully electron-
withdrawing and subsequently deactivates an aromatic system.
This makes it partially positive in nature and complementary to
relatively electron rich and activated aryl systems of amino acids
in a receptor such as tryptophan, tyrosine, and phenylalanine.
The results also suggest that it is unclear whether a piperazine
or piperidine is more ideally suited for activity at the M1
mAChR and perhaps the functionality before and after the six-
membered ring containing ionizable nitrogen at physiological
pH is more detrimental for activity. Compound 11, as
compared to similar structures 9 and 10, was connected to
the flexible piperazine through the phenyl ring as opposed to
the five-membered ring being directly connected to the
piperazine or piperidine, which possibly affected the binding
of the ligand at the M1 mAChR and therefore attributed to its
loss of activity.
CONCLUSION
■
In terms of antipsychotic action, engaging multiple targets has
become useful in the drug design process to address the
numerous symptom domains of schizophrenia. The DML
approach offers a way to selectively design in polypharmacology
by using privileged structures that are known to be advanta-
geous toward the targets of interest. Ligands in this study were
designed to be D2 antagonists or partial agonists for the positive
symptoms, M1 mAChR allosteric agonists for the cognitive
deficits, and antagonists at the 5-HT2AR to address the negative
symptoms and reduce the occurrence of extrapyramidal side
5-HT2AR Binding and Functional Characterization of
DMLs. There is substantial literature evidence that antagonism
of the 5-HT2AR is important for the therapeutic efficacy of
atypical antipyschotics such as clozapine.21 In addition, 9 and
10 stem from antipsychotics 1 and 2, respectively, that display
high affinity for the 5-HT2AR. Therefore, we deemed it prudent
to investigate if this attribute was maintained upon integration
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Journal of Medicinal Chemistry
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effects. The privileged structures covered phenylpiperazines in
addition to mixed piperazine/piperidine heterocyclic com-
pounds derived from antipsychotics or a clinically developed
compound. The final DMLs (7−11) were generally well-
tolerated at the D2R and 5-HT2AR but are in need of further
development at the M1 mAChR. Despite this, we identified 9,
incorporating a privileged structure derived from the
antipsychotic ziprasidone, that retained strong binding and
functional activity at the D2R and 5-HT2AR and weak partial
agonism at the M1 mAChR. Compound 9 represents a useful
starting point for further optimization to improve its M1
mAChR profile.
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*J.R.L.: phone, +613 9903 9095; fax, +613 9903 9581; e-mail,
rob.lane@monash.edu.
■
*B.C.: phone, +61 3 9903 9556; fax, +61 3 9903 9581; e-mail,
ben.capuano@monash.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
EXPERIMENTAL SECTION
Chemistry. All solvents and chemicals were purchased from
standard suppliers and were used without any further purification. H
■
This research was supported by Project Grant APP1049564
and Program Grant APP1055134 of the National Health and
Medical Research Council (NHMRC). A.C. is a Principal
Research Fellow (NHMRC). J.R.L. is a R.D. Wright Biomedical
Career Development Fellow (APP1052304, NHMRC) and a
Larkin’s Fellow (Monash University, Australia). M.S. acknowl-
edges an Australian Postgraduate Award. C.K.H. acknowledges
a Monash Graduate Scholarship.
1
NMR and 13C NMR spectra were acquired at 400.13 and 100.62 MHz,
respectively, on a Bruker Advance III 400 MHz UltrashieldPlus NMR
spectrometer using TOPSPIN 2.1 software. Chemical shifts (δ) for all
1H spectra are reported in parts per million (ppm) using
tetramethylsilane (TMS, 0 ppm) as the reference. The data for all
spectra are reported in the following format: chemical shift (δ),
(multiplicity, coupling constants J (Hz), integral), where the
multiplicity is defined as s = singlet, d = doublet, t = triplet, q =
quartet, p = pentet, st = sextet, and m = multiplet. For 13C NMR
ABBREVIATIONS USED
■
ERK, extracellular signal-regulated kinase; Boc, tert-butylox-
ycarbonyl; cAMP, cyclic adenosine monophosphate; ACh,
acetylcholine
spectra C = quaternary carbon, CH = methine carbon, CH2
=
methylene carbon, and CH3 = methyl carbon. The purity and
retention time of final products were determined on an Agilent 1260
Infinity analytical reverse-phase HPLC system fitted with a Poroshell
120 SB-C18 4.6 mm × 100 mm 2.7 μm column. The HPLC operates
on Agilent OpenLAB CDS, revision C.01.04, software. Solvent A is
water + 0.1% TFA, and solvent B is acetonitrile + 0.1% TFA. Samples
were run using a gradient method (5−100% solvent B over 10 min).
The purities of all compounds are ≥95%. Thin layer chromatography
(TLC) was carried out routinely on silica gel 60F254 precoated plates
(0.25 mm, Merck). Flash column chromatography was carried out
using Merck silica gel 60, 230−400 mesh ASTM.
General Procedure for the Synthesis of Merged DMLs.
Compound 13 (1 equiv) was dissolved in CH3CN (10 mL). NaI (1
equiv), DIPEA (1−2 equiv), and the required amine (1 equiv) were
added and heated at reflux for 5−6 h. After this time, the CH3CN was
removed in vacuo and the resulting residue dissolved in ethyl acetate
(20 mL). Aqueous K2CO3 (1 M, 20 mL) was added and the product
further extracted with ethyl acetate (2 × 20 mL). The organic layers
were combined and washed with water (20 mL) and brine (20 mL),
dried over anhydrous Na2SO4, filtered, and evaporated to dryness to
give the crude product. Purification via column chromatography
(petroleum spirits/ethyl acetate 1:1) gave the pure product.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis, characterization, and pharmacology for all com-
pounds; bias calculations for 11 at the D2R and compounds
tested in the absence of serotonin at the 5-HT2AR. This
E
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DOI: 10.1021/jm5013243
J. Med. Chem. XXXX, XXX, XXX−XXX