Discovery, optimization, and characterization of novel D2 dopamine receptor selective antagonists

Article abstract of DOI:10.1021/jm500126s

The D2 dopamine receptor (D2 DAR) is one of the most validated drug targets for neuropsychiatric and endocrine disorders. However, clinically approved drugs targeting D2 DAR display poor selectivity between the D2 and other receptors, especially the D3 DA

Full text of DOI:10.1021/jm500126s

Article
pubs.acs.org/jmc
Discovery, Optimization, and Characterization of Novel D2 Dopamine
Receptor Selective Antagonists
Jingbo Xiao,† R. Benjamin Free,‡ Elena Barnaeva,† Jennie L. Conroy,‡ Trevor Doyle,‡ Brittney Miller,‡
Marthe Bryant-Genevier,† Mercedes K. Taylor,† Xin Hu,† Andres E. Dulcey,† Noel Southall,†
́
Marc Ferrer,† Steve Titus,† Wei Zheng,† David R. Sibley,‡ and Juan J. Marugan*,†
†Discovery Innovation, NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes
of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, United States
‡ Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 5625
Fishers Lane, Room 4S-04, MSC-9405, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: The D2 dopamine receptor (D2 DAR) is one of the most validated drug
targets for neuropsychiatric and endocrine disorders. However, clinically approved drugs
targeting D2 DAR display poor selectivity between the D2 and other receptors, especially the
D3 DAR. This lack of selectivity may lead to undesirable side effects. Here we describe the
chemical and pharmacological characterization of a novel D2 DAR antagonist series with
excellent D2 versus D1, D3, D4, and D5 receptor selectivity. The final probe 65 was obtained
through a quantitative high-throughput screening campaign, followed by medicinal chemistry
optimization, to yield a selective molecule with good in vitro physical properties, metabolic
stability, and in vivo pharmacokinetics. The optimized molecule may be a useful in vivo probe for studying D2 DAR signal
modulation and could also serve as a lead compound for the development of D2 DAR-selective druglike molecules for the
treatment of multiple neuropsychiatric and endocrine disorders.
due to off-target cross-GPCR reactivity.9 It would thus be
desirable to develop a class of novel therapeutic agents with
higher selectivity for the D2 DAR.
INTRODUCTION
■
G-protein-coupled receptors (GPCRs) are among the most
intensely investigated drug targets in the pharmaceutical
It should be noted that although the therapeutic potential for
industry.1 Over 40% of all FDA approved drugs target these
more selective D2 DAR antagonists may be enormous, this
important receptor proteins.2 Unfortunately, many of the
approach may also provide a way forward for developing
ligands that are used as drugs or pharmacological tools are not
selective pharmacological probes that provide a better under-
selective and exhibit some unintended activity on nontarget
GPCRs or other proteins.3 Dopamine receptors (DARs)
standing of the dopamine neurocircuitry. Among the D2-like
family of DARs (D2, D3, and D4), only the D4 DAR has ligands
belong to a large superfamily of neurotransmitter and hormone
receptors.4 Five functionally active DARs have been identified
(both agonists and antagonists) that are truly specific versus D2
and D3 DARs.10 This is not surprising given that the D4 DAR is
in the mammalian genome.5 D1-like DARs (D1 and D5) are Gαs
more structurally divergent compared to the D2/D3 DARs. D2
and D3 receptors share 78% homology in their transmembrane
coupled, and D2-like DARs (D2, D3, and D4) are Gαi/o
coupled.4,6 There are two isoforms of the D2 DAR, short and
spanning domains, which harbor the ligand binding sites, and
long (D2S and D2L, respectively), which are derived from
thus, the pharmacologic properties between these two receptor
alternative RNA splicing and vary in the size of their third
subtypes are quite similar.4,11 Therefore, it is very challenging
intracellular loops. The D2L isoform is more prevalent, although
both isoforms appear to be functionally similar.7 Among the
to identify small molecules that can selectively bind to and/or
functionally modulate either D2 or D3 DAR receptor subtypes.
DARs, the D2 DAR is arguably one of the most validated drug
With respect to the D3 DAR, there are several compounds that
targets in neurology and psychiatry. For instance, all receptor-
exhibit good selectivity versus the D4 DAR and moderate−high
selectivity versus the D2 DAR.12 Some of these D3-selective
compounds have been used for in vivo experiments, but the
results have been controversial in many instances.13 In contrast,
to the best of our knowledge, there are only a few series of
compounds that exhibit even moderate selectivity for the D2
based antiparkinsonian drugs work via stimulating the D2 DAR
(although controversy exists for a minor role of the D1 DAR),
whereas all FDA approved antipsychotic agents are antagonists
of this receptor.8 The D2 DAR is also therapeutically targeted in
other disorders such as restless legs syndrome, tardive
dyskinesia, Tourette’s syndrome, and hyperprolactinemia.
Most drugs targeting the D2 DAR (orthosteric agonists and
antagonists) are problematic by being less efficacious than
desired or possessing limiting side effects, most of which are
Received: January 23, 2014
Published: March 25, 2014
This article not subject to U.S. Copyright.
Published 2014 by the American Chemical
Society
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Figure 1. Known chemical series of selective D2 versus D3 DAR antagonists. Superscript “a” indicates that additional information is in Table S15 in
Supporting Information.
Figure 2. (A) Structure of the hit compound 1. (B) Graphical representation of the dose−response curves of 1 in D2 Ca2+ assay (green, AC50
=
0.280 0.012 μM), D2 β-arrestin assay (blue, AC50 = 2.89 0.25 μM), and D3 β-arrestin assay (red, AC50 = 5.76 1.18 μM). (C) Graphical
representation of the dose−response curves of 1 in binding assays for D1 (black), D2 (blue, Ki = 0.30 0.09 μM), D3 (red, Ki = 1.9 0.9 μM), D4
(green), and D5 (brown).
DAR receptor versus the D3 and D4 DARs within the D2-like
DAR subfamily (Figure 1).14 Selective antagonists of the D2
DAR could be useful for the therapy of disorders currently
treated with relatively nonselective D2 blockers, including
Tourette’s syndrome, tardive dyskinesia, Huntington’s chorea,
and schizophrenia. Probably the biggest impact, in terms of
patient population, would be in the treatment of schizophrenia.
Here, we present a novel series of selective small molecule
antagonists of D2 DAR identified from a quantitative high-
throughput screening (qHTS) campaign.14 Optimized lead
compounds in this series, represented by 65, exhibit excellent
D2 versus D3 and D4 DAR selectivity in addition to good
ADME and in vivo pharmacokinetics properties, which make
them good pharmacological tools to perform proof-of-concept
studies using animal models as well as for further development
into druglike molecules.
RESULTS AND DISCUSSION
■
The primary goal of this project was to identify and develop
novel small molecule modulators of the D2 DAR with
selectivity versus the D3 DAR for use as in vitro and in vivo
pharmacological tools and in proof-of-concept experiments in
animal models of neuropsychiatric disease. To this end, we
performed a full qHTS campaign of ∼380 000 molecules of the
NIH Molecular Libraries Small Molecule Repository
(MLSMR). For the initial qHTS screen, we employed a
calcium release assay where cellular coexpression of the D2
DAR with a chimeric Gqi5 protein enabled the receptor to
stimulate Ca2+ mobilization, which was detected through the
excitation of an intracellular fluorescent dye. The hit
compounds were orthogonally validated using a D2 DAR β-
arrestin assay (DiscoveRx) and counterscreened using the same
β-arrestin assay but with the D3 DAR. Compounds that showed
selectivity for D2 versus D3 receptors were further characterized
using radioligand binding assays, and Ki values were determined
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Scheme 1. General Synthetic Route for Analogues Shown in Tables 1−3
Figure 3. (A) Preparative chiral HPLC separation of the original racemic hit 1 and absolute stereochemistry assignment. (B) Single crystal X-ray
structure of the inactive enantiomer ((R)-59).
for each receptor subtype. While multiple modulator chemo-
types were identified within this qHTS project, we specifically
focused here on the identification of a novel antagonist with
selectivity for the D2 receptor. From the primary screen, 2294
compounds were identified with D2 DAR antagonist activity,
although 858 of these were not subsequently confirmed. Of the
remaining, 499 compounds were selected for counterscreening
against the D3 DAR as described above. Several compounds
were identified that exhibited D2 versus D3 DAR selectivity with
the most promising hit compounds being selected for chemical
optimization.
The hit compound 1 (Figure 2A), initially identified from
pilot screens for the project, displayed selectivity for the D2
versus the D3 DAR in a functional assay (Figure 2B) and
selectivity versus other DAR subtypes in radioligand binding
assays (Figure 2C). This molecule bears a benzothiazepine
moiety, a chemical scaffold that is often capable of crossing the
blood−brain barrier (BBB).15 In addition, an interesting aspect
of this scaffold is the lack of basic nitrogen. Almost all known
compounds with affinity to dopaminergic receptors contain a
basic nitrogen in the structure. The lack thereof in this
structural class would thus be of interest to the scientific
community. Furthermore, this molecule has promising physical
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Scheme 2. General Synthetic Routes (A) for Analogues Shown in Table 4, (B) for Analogues Shown in Table 5, and (C) for
Analogues Shown in Table 6
properties rendering it a candidate for further optimization.
Importantly, in functional assays the molecule clearly
demonstrated receptor selectivity, and therefore, we decided
to pursue SAR studies with this molecule to identify improved
D2-receptor selective probes, specifically focusing on D2 versus
D3 receptor selectivity.
In order to identify both potent and selective D2 antagonists,
we employed a two-stage development plan. In the first stage
we used a D2 DAR Ca2+ mobilization assay to drive our SAR
study identifying potent D2 antagonists. Optimized analogues
with good potency were further characterized for receptor
selectivity in the second stage using D2 and D3 β-arrestin
activation functional assays, as well as direct radioligand binding
assays.
According to our general practice, we started our medicinal
chemistry campaign with a resynthesis of the original hit 1
(Scheme 1).16 The activity of the synthesized material was
reconfirmed with slightly better potency than that of the
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a
Table 1. SAR with Modifications on the Benzyl Moiety
a
AC50 SEM (n = 3) is from a calcium accumulation assay.
original qHTS sample (AC50 = 0.280 μM versus 0.560 μM for
the qHTS sample). As the hit compound 1 represents a racemic
mixture, both R- and S-enantiomers were obtained from
preparative chiral HPLC separation of racemic material.
Interestingly, when the enantiomerically pure samples were
evaluated, only one isomer showed activity and, as expected,
had twice the potency than the racemic material. The other
enantiomer was inactive up to 77 μM (Figure 3A). In order to
assign the absolute stereochemistry of the active enantiomer, a
pair of analogues bearing a known second chiral center (R-)
and a heavy atom (Br) were prepared. Fortunately, a single
crystal was obtained for the inactive enantiomer of this pair of
molecules. X-ray diffraction analysis was successfully performed
disclosing that the inactive isomer ((R)-59) had the 5-oxide as
the R-configuration (Figure 3B). Therefore, we concluded that
the active enantiomer has an S-configuration within the
sulfoxide functional group.
saponificated with LiOH to offer the acid 5. The nitro
functional group was reduced using H2 and a mixed catalyst of
10% palladium on carbon and platinum oxide to produce the
amine 6. The intramolecular cyclization was induced by CDI to
provide the benzothiazepine 7. Alkylation of the nitrogen of the
amide or the oxygen of the acid with a variety of alkyl halides
gave intermediate 8. Selective hydrolysis of esters motif
provided the 8-carboxylic acid substituted benzothiazepine 9.
Thioester mono-oxidation with hydrogen peroxide yields
sulfoxides 10 which were coupled with different amines to
offer the final products as shown in Scheme 1. Additionally,
thioester 9c can be overoxidized with MCPBA to produce the
sulfone 11 (Scheme 2A). Curtius rearrangement of the
carboxylic acid 10c with diphenylphosphinylazide provides
the 8-amino substituted benzothiazepine 12 as a key
intermediate for the synthesis of reverse amide analogues 55
and 56 (Scheme 2B). The enantiomerically pure analogues can
be prepared using two routes alternating the chiral separation
and the amide formation as it is shown in Scheme 2C.
With these promising results in hand, we decided to initiate
our medicinal chemistry SAR studies with the aim of evaluating
several distinct areas of the molecule. The synthesis of
analogues is shown in Scheme 1 and Scheme 2. A SNAr
reaction between methyl 4-fluoro-3-nitrobenzoate (2) and
methyl 2-mercaptobenzoate (3) gave intermediate 4 which was
A summary of our medicinal chemistry SAR efforts to
improve the potency of the compounds is disclosed in Tables
1−6. As an initial approach, we conducted this study using
racemic mixtures. The first area evaluated was the 4-
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methoxybenzyl moiety of the molecule starting with the
systematic replacement of functional groups around the benzyl
ring. In doing so, the benzothiazepine moiety and amide linker
were held constant while the substitution pattern and steric and
electronic effects of the substituents were fully explored, as
shown in Table 1. It can be seen that the o-methoxy substituent
was not well tolerated producing a considerable loss of potency
(15, AC50 = 4.46 μM), while the m-methoxy substitution
resulted in an analogue with slightly improved potency (14,
AC50 = 0.112 μM). Removal of all functionality within the
benzyl ring (16, AC50 = 0.445 μM) or addition of one methyl
group was >4-fold more active than the corresponding analogue
35 with a plain phenyl group.
With several improved analogues in hand, SAR studies were
focused on replacements within the benzothiazepine core, as
shown in Table 4. Replacement of the sulfoxide by a thioether
functional group resulted in a 2.5-fold reduction of activity (48,
AC50 = 0.705 μM). Importantly, the corresponding sulfone
analogue was completely inactive (49), in line with our
previous observation that only the enantiomer ((S)-1, AC50
=
0.141 μM) with S-configuration displays antagonism and the
analogue (R)-1 with R-configuration is totally inactive. Other
three-ring system replacements, such as anthracene-9,10-dione
or 2-phenylisoindoline-1,3-dione, led to a complete loss of
activity (50 and 51, inactive).
Next, we examined different linkers as replacement for the
amide functionality as detailed in Table 5. Alkylation of the N−
H led to a complete or major loss in activity (52, inactive; 53,
AC50 = 56.0 μM). Switching of the amide moiety to an ester
group eliminated any activity (54, inactive). Analogues with a
reversed amide moiety were also tolerated displaying
reasonable activity (55, AC50 = 0.705 μM; 56, AC50 = 0.223
μM). After investigation of SAR in all the previously described
areas, a clear SAR picture of this chemical scaffold was
developed and is summarized in Figure 4.
Lastly, 10 selected analogues were subjected to preparative
chiral HPLC in order to obtain enantiomerically pure analogues
for further evaluation and profiling. The activity of the 10 active
enantiomers (S-configuration) is listed in Table 6. Most of the
analogues in this table showed AC50 values lower than 300 nM.
As we expected, the corresponding chiral versions of the three
most potent racemic analogues (41, 44, and 45 in Table 3)
were still ranked as the top three most potent analogues (62,
AC50 = 0.089 μM; 64, AC50 = 0.070 μM; 65, AC50 = 0.070
μM). For further confirmation of the absolutely stereo-
chemistry of our most active compounds a single crystal X-
ray structure of 65 was successfully obtained resulting in the
expected S-configuration (Figure 5).
substituent in the α-methylene functional group (17, AC50
=
0.560 μM) yielded analogues with 2 times lower potencies than
that of the original hit (1, AC50 = 0.280 μM). Interestingly,
neither the electronic nor the steric nature of para substituents
had a significant effect on the activity, as many of them were
generally well tolerated (18−25, AC50 = 0.089−0.509 μM),
with the 4-fluoro compound (22, AC50 = 0.089 μM) being the
most potent analogue. Multisubstituted analogue 26 showed a
6-fold decrease of potency (AC50 = 1.77 μM). Cyclization of
the 3,4-disubstituted methoxy groups restored the activity (27,
AC50 = 0.353 μM). Both removal of a methylene spacer
functional group (phenyl, 28 and 29) and inclusion of an
additional methylene functional group (phenylethylene, 30)
were well tolerated, although the effect was dependent on the
substitution on the aromatic ring (1 versus 29; 16 versus 28).
In parallel to systematic SAR studies of the 4-methoxybenzyl
functional group, we investigated the effect of substituents
within the benzothiazepine amide moiety, as detailed in Table
2. Total removal of the ethyl group led to a loss in activity (31,
Table 2. SAR with Modifications on the Benzothiazepine
a
Amide Moiety
In addition to the activity in the D2 Ca2+ assay, these chiral
analogues were profiled for D2/D3 receptor selectivity along
with the original hit 1, the racemic mixture of the probe
molecule 44 (rac-65), and its separated inactive R-configuration
analogue (R)-65 (Table 7). Because of the lack of a D3 DAR
stably transfected cell line with which to activate the chimeric
G-protein (Gqi5) Ca2+ pathway, we used cell lines designed to
interrogate the β-arrestin pathway to evaluate the D2/D3
functional selectivity. Overall, antagonistic activities in the D2
Ca2+ assay correlated quite well with those observed in the D2
β-arrestin assay; however, a ∼10-fold potency shift between two
assays was noted. The selectivity between D2 and D3 β-arrestin
assays ranged from 2.0- to 22.3-fold. In addition to the
comparison of functional selectivities in β-arrestin assay, we
further evaluated the D2/D3 receptor binding selectivity using a
radioligand displacement assay to determine the Ki values of the
compounds. While we did observe some differences in fold
selectivity between the two methods (possibly due to different
error rates inherent in calculating both values and potential
small differences in functional selectivity among analogues), all
compounds showed D2 DAR selectivity and the lead analogues,
including 44 and 65, consistently displayed greater than 17-fold
selectivity for the D2 DAR using both β-arrestin and radioligand
binding assays. Specifically, by use of radioligand binding, which
directly measures the affinity of test compound for the
receptors, analogue 65 displayed 41-fold selectivity for the D2
compd
R2
Ca2+, AC50 (μM)
31
32
33
34
H
0.887 0.221
0.112 0.025
0.705 0.413
8.87 1.39
Me
n-Pr
Bn
a
AC50 SEM (n = 3) is from a calcium accumulation assay.
AC50 = 0.887 μM). Analogue 32 with methyl substitution gave
a 2.5-fold activity increase. Moreover, the size of the substituent
decreased potency (methyl, 32, AC50 = 0.112 μM; ethyl, 1,
AC50 = 0.280 μM; n-propyl, 33, AC50 = 0.705 μM; benzyl, 34,
AC50 = 8.87 μM). In light of the methyl group being the most
promising substituent for R2, additional analogues that carried a
methyl group as the substituent on the benzoazepine amide are
detailed in Table 3. Other than substituted phenyl and benzyl
rings (35−41), heteroaryl (42−44), heterocyclic (45 and 46),
and aliphatic (47) groups were also evaluated. Most of R1
substituents listed in Table 3 were well tolerated except
analogue 42 containing a 2-pyridylmethyl group. Several potent
analogues with an AC50 of less than 0.1 μM were identified
from this round of SAR investigation (41, AC50 = 0.071 μM;
44, AC50 = 0.089 μM; 45, AC50 = 0.089 μM). It was interesting
to note that activity of analogue 36 with a 4-methoxyphenyl
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a
Table 3. SAR with Combined Modifications on the Benzyl Moiety and Benzothiazepine Amide Moiety
a
AC50 SEM (n = 3) is from a calcium accumulation assay.
a
Table 4. SAR with Modifications on the Benzothiazepine
a
AC50 SEM (n = 3) is from a calcium accumulation assay.
versus the D3 receptor (Table 7 and Supporting Information
Table S15 for comparison to reference compounds). This
compound could thus be used to substantially block the D2
receptor in vivo with minimal effects on the D3 receptor.
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a
excellent PBS aqueous buffer solubility (113.3 μM), good
mouse and human liver microsomal stability with a very
reasonable intrinsic half-lives (T1/2 of 24.7 and 26.4 min,
respectively), promising mouse and human plasma stability, and
good cell permeability. Overall, analogue 65 has an excellent
metabolic profile and physical properties. Therefore, analogue
65 was further evaluated using C57BL/6 mice for in vivo
pharmacokinetics (PK) studies to investigate the organ
distribution of this compound in plasma and brain using an
intraperitoneal (ip) single dose administration of 30 mg/kg
(Figure 6). Analogue 65 displays a plasma half-life of 1.67 h
with a similar value in brain (T1/2 = 1.30 h), demonstrating also
acceptable blood−brain barrier (BBB) penetration. The brain
to plasma ratio was found to be 0.181. The concentration of
analogue 65 in brain reached 4.19 μmol/kg (Cmax) within 15
min. Further in vivo studies, including the displacement of
radiotracers, are warranted for confirmation of this activity.
Table 5. SAR with Modifications on the Left Amide Linker
CONCLUSION
■
In summary, analogue 65 displays the best combination of
overall activity, improved potency, and good physical proper-
ties. Furthermore, the excellent selectivity between D2 and D3
receptors coupled with a promising ADME profile (Table 8)
and encouraging in vivo pharmacokinetics properties (Figure 6,
Supporting Information) led us to declare the lead compound
65 as a selective D2/D3 DAR antagonist.
a
AC50 SEM (n = 3) is from a calcium accumulation assay.
EXPERIMENTAL SECTION
■
Considering ligand potency and D2/D3 selectivity, as
determined using β-arrestin functional assays and radioligand
binding assays, we chose analogue 65 for further in vitro
metabolic profile and in vivo mouse pharmacokinetics (PK)
studies. As shown in Table 8, analogue 65 demonstrated
a. Chemistry. All air or moisture sensitive reactions were
performed under positive pressure of nitrogen with oven-dried
glassware. Anhydrous solvents such as dichloromethane, N,N-
dimethylformamide (DMF), acetonitrile, methanol, and triethylamine
were purchased from Sigma-Aldrich (St. Louis, MO). Preparative
Figure 4. SAR summary.
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a
Table 6. Enantiomerically Pure Version of Selected Analogues
a
AC50 SEM (n = 3) is from a calcium accumulation assay.
Figure 5. X-ray crystal structure of 65. X-ray parameters are listed in the Supporting Information.
purification was performed on a Waters semipreparative HPLC system
(Waters Corp., Milford, MA). The column used was a Phenomenex
Luna C18 (5 μm, 30 mm × 75 mm; Phenomenex, Inc., Torrance, CA)
at a flow rate of 45.0 mL/min. The mobile phase consisted of
acetonitrile and water (each containing 0.1% trifluoroacetic acid). A
gradient of 10−50% acetonitrile over 8 min was used during the
purification. Fraction collection was triggered by UV detection at 220
nM. Analytical analysis was performed on an Agilent LC/MS (Agilent
Technologies, Santa Clara, CA). Method 1 consisted of the following:
a 7 min gradient of 4−100% acetonitrile (containing 0.025%
trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid)
was used with an 8 min run time at a flow rate of 1.0 mL/min. Method
2 consisted of the following: a 3 min gradient of 4−100% acetonitrile
(containing 0.025% trifluoroacetic acid) in water (containing 0.05%
trifluoroacetic acid) was used with a 4.5 min run time at a flow rate of
1.0 mL/min. A Phenomenex Luna C18 column (3 μm, 3 mm × 75
mm) was used at a temperature of 50 °C. Purity determination was
performed using an Agilent diode array detector for both method 1
and method 2. Mass determination was performed using an Agilent
6130 mass spectrometer with electrospray ionization in the positive
mode. 1H NMR spectra were recorded on Varian 400 MHz
spectrometers (Agilent Technologies, Santa Clara, CA). Chemical
shifts are reported in ppm with undeuterated solvent (DMSO at 2.49
ppm) as internal standard for DMSO-d6 solutions. All of the analogues
tested in the biological assays have a purity of greater than 95% based
on both analytical methods. High resolution mass spectra were
recorded on Agilent 6210 time-of-flight (TOF) LCMS system.
Confirmation of molecular formula was accomplished using electro-
spray ionization in the positive mode with the Agilent Masshunter
software (version B.02).
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a
Table 7. Selectivity Profiling of D2 versus D3 for 13 Selected Analogues
a
Blue columns are for D2 receptor, and red columns are for D3 receptor. The asterisk (∗) indicates a value without error and that the curve is
provided in the Supporting Information.
Table 8. Comparison of Physical and Metabolic Properties for Hit Compound 1 and Lead Compound 65
liver microsomal stability
plasma stability (% remaining
after 2 h)
aqueous kinetic solubility
(T1/2 in min)
Caco-2 permeability (10−6, cm/s)
compd
μg/mL
μM
mouse
human
mouse
human
Papp(A−B)
Papp(B−A)
1
11.5
46.5
26.4
35.9
24.7
N/A
26.4
N/A
95.2
N/A
98.4
21.6
18.8
16.2
23.8
65
113.3
Detailed Preparation Procedure for 65 Is Described Below.
Methyl 4-(2-(Methoxycarbonyl)phenylthio)-3-nitrobenzoate (4). A
solution of methyl 4-fluoro-3-nitrobenzoate (2, 12.1 g, 60.9 mmol)
and methyl 2-mercaptobenzoate (3, 9.21 mL, 66.9 mmol) in DMF
(6.00 mL) was treated with Cs2CO3 (19.8 g, 60.9 mmol) at room
temperature. The reaction mixture was stirred at 40 °C for 4 h and
then cooled to room temperature. Ice−water was added to induce the
precipitation. The precipitate was filtered, washed with water, and
dried to give 21.0 g (99%) of the title compound as a yellow solid
which was used directly in the next reaction without further
water, and dried to give 19.1 g (99%) of the title compound as a yellow
solid which was used directly in the next reaction without further
purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 13.53 (br s, 1 H),
13.27 (br s, 1 H), 8.58 (d, J = 1.6 Hz, 1 H), 8.01 (dd, J = 8.6, 2.0 Hz, 1
H), 7.85−7.95 (m, 1 H), 7.48−7.68 (m, 3 H), 7.07 (d, J = 8.6 Hz, 1
H); LCMS tR = 4.73 min, m/z 341.9 [M + Na+]; HRMS (ESI) m/z
calcd for C14H9NNaO6S [M + Na+] 342.0043, found 342.0047.
3-Amino-4-(2-carboxyphenylthio)benzoic Acid (6). A solution of
4-(2-carboxyphenylthio)-3-nitrobenzoic acid (5, 8.70 g, 27.2 mmol) in
MeOH (300 mL) was treated at room temperature with platinum(IV)
oxide (300 mg, 1.32 mmol) and Pd/C (10%, 600 mg, 5.64 mmol). A
balloon containing H2 was connected to the flask, and the reaction
flask was repeatedly evacuated and refilled with H2. After 16 h,
additional Pd/C (10%, 600 mg, 5.64 mmol) was added and the
reaction mixture was stirred under H2 balloon for an additional 32 h.
The reaction mixture was filtered through a pad of Celite and
concentrated to give 7.80 g (99%) of the title compound as a gray-
yellow solid which was used directly in the next reaction without
further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.99 (br
s, 2 H), 7.92 (dd, J = 7.8, 1.6 Hz, 1 H), 7.42 (d, J = 1.6 Hz, 1 H), 7.37
(d, J = 7.8 Hz, 1 H), 7.31−7.36 (m, 1 H), 7.18 (td, J = 7.6, 1.2 Hz, 1
1
purification. H NMR (400 MHz, DMSO-d6) δ ppm 8.63 (d, J =
2.0 Hz, 1 H), 8.04 (dd, J = 8.4, 1.8 Hz, 1 H), 7.87−7.99 (m, 1 H),
7.57−7.77 (m, 3 H), 7.05 (d, J = 8.6 Hz, 1 H), 3.88 (s, 3 H), 3.71 (s, 3
H); LCMS tR = 6.19 min, m/z 365.0 [M + Na+]; HRMS (ESI) m/z
calcd for C16H13NNaO6S [M + Na+] 371.0387, found 371.0393.
4-(2-Carboxyphenylthio)-3-nitrobenzoic Acid (5). A solution of
methyl 4-(2-(methoxycarbonyl)phenylthio)-3-nitrobenzoate (4, 21.0
g, 60.5 mmol) in THF (150 mL) and water (150 mL) was treated at
room temperature with LiOH (14.5 g, 605 mmol). The reaction
mixture was stirred at 60 °C for 2 h. The organic solvent was removed
and the aqueous solution was washed with EtOAc and acidified with 2
N HCl until pH ≈ 2. The yellow precipitate was filtered, washed with
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1
without further purification. H NMR (400 MHz, DMSO-d6) δ ppm
13.31 (br s, 1 H), 7.98 (d, J = 1.2 Hz, 1 H), 7.67−7.76 (m, 2 H),
7.59−7.66 (m, 1 H), 7.47−7.54 (m, 1 H), 7.35−7.45 (m, 2 H), 3.52
(s, 3 H); LCMS tR = 4.76 min, m/z 286.0 [M + H+]; HRMS (ESI) m/
z calcd for C15H12NO3S [M + H+] 286.0532, found 286.0536.
10-Methyl-11-oxo-10,11-dihydrodibenzo[b,f ][1,4]thiazepine-8-
carboxylic Acid 5-Oxide (10b). A suspension of 10-methyl-11-oxo-
10,11-dihydrodibenzo[b,f ][1,4]thiazepine-8-carboxylic acid (9b, 660
mg, 2.31 mmol) in acetic acid (18.8 mL) was treated at room
temperature with H2O2 (5.91 mL, 30%, 57.8 mmol) for 8 h. Upon
completion, the reaction mixture was poured into a cold saturated
solution of Na2S2O3 in water and stirred at room temperature for 3 h.
The mixture was then extracted with 20% of MeOH in dichloro-
methane. The organic layer was separated, dried, and concentrated to
give 595 mg (85%) of the title compound as a white solid containing
1
∼5% of dioxide as a byproduct. H NMR (400 MHz, DMSO-d6) δ
ppm 8.00 (d, J = 1.2 Hz, 1 H), 7.96 (dd, J = 8.2, 1.6 Hz, 1 H), 7.62−
7.76 (m, 4 H), 7.52−7.59 (m, 1 H), 3.53 (s, 3 H); LCMS tR = 4.02
min, m/z 302.0 [M + H+]; HRMS (ESI) m/z calcd for C15H12NO4S
[M + H+] 302.0482, found 302.0486.
Figure 6. Mean plasma and brain concentration−time profiles of 65
after an ip dose of 30 mg/kg in male C57BL/6 mice (N = 3). The
mice appeared less active at 5 min after dosing, and it lasted for about
2 h. The ip dosing solution was prepared in 10% NMP + 20% PEG
400 + 70% of 25% HP-β-CD in water. Full PK parameters are listed in
the Supporting Information.
10-Methyl-11-oxo-10,11-dihydrodibenzo[b,f ][1,4]thiazepine-8-
carboxylic Acid 5-(S)-Oxide ((S)-10b). The enantiomerically pure title
compound was purified to >98% purity using supercritical fluid
chromatography (SFC) preparative systems at Lotus Separations, LLC
(Princeton, NJ, USA). For preparative separation, an IC (2 cm × 15
cm) column was used with an eluent of 40% methanol (0.1% DEA)/
CO2, 100 bar. Flow rate was 60 mL/min, and detection wavelength
was 220 nm. For analytical separation, an IC (15 cm × 0.46 cm)
column was used with an eluent of 40% methanol/CO2, 100 bar. Flow
rate was 3 mL/min, and detection wavelengths were 220 and 280 nm.
Retention time was 3.42 min. Retention time for the R-configuration
enantiomer was 2.40 min. The material was used directly in the next
coupling reaction.
H), 7.13 (dd, J = 8.0, 1.8 Hz, 1 H), 6.61 (dd, J = 8.2, 0.8 Hz, 1 H), 5.40
(br s, 2 H); LCMS tR = 4.25 min, m/z 290.0 [M + H+]; HRMS (ESI)
m/z calcd for C14H12NO4S [M + H+] 290.0482, found 290.0486.
11-Oxo-10,11-dihydrodibenzo[b,f ][1,4]thiazepine-8-carboxylic
Acid (7). A solution of 3-amino-4-(2-carboxyphenylthio)benzoic acid
(6, 4.76 g, 16.5 mmol) in THF (100 mL) was treated at 0 °C with
1,1′-carbonyldiimidazole (CDI) (10.7 g, 65.8 mmol) via several
portions. The reaction mixture was warmed to room temperature and
stirred at room temperature overnight. The reaction mixture was
poured into 140 mL of ice−water containing concentrated HCl (20.0
mL) and stirred for 1 h. The white precipitate was filtered, washed
with water, and dried to give 3.89 g (87%) of the title compound as a
white solid which was used directly in the next reaction without further
purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 13.20 (br s, 1 H),
10.79 (s, 1 H), 7.76 (d, J = 1.2 Hz, 1 H), 7.62−7.70 (m, 3 H), 7.41−
7.57 (m, 3 H); LCMS tR = 4.55 min, m/z 271.9 [M + H+]; HRMS
(ESI) m/z calcd for C14H10NO3S [M + H+] 272.0376, found
272.0376.
10-Methyl-11-oxo-N-(2-(thiophen-2-yl)ethyl)-10,11-
dihydrodibenzo[b,f ][1,4]thiazepine-8-carboxamide 5-(S)-Oxide
(65). A solution of 10-methyl-11-oxo-10,11-dihydrodibenzo[b,f ][1,4]-
thiazepine-8-carboxylic acid 5-(S)-oxide ((S)-10b, 100 mg, 0.332
mmol) in DMF (5.00 mL) was treated at room temperature with
HATU (139 mg, 0.365 mmol) and diisopropylethylamine (0.174 mL,
0.996 mmol) followed by 2-(thiophen-2-yl)ethanamine (84.0 mg,
0.664 mmol). The reaction mixture was stirred at room temperature
for 3 h and poured into ice−water. The white precipitate was filtered
and dried to give a white solid, which was purified via silica gel
chromatography using a gradient of 10−100% of EtOAc in hexanes to
Methyl 10-Methyl-11-oxo-10,11-dihydrodibenzo[b,f ][1,4]-
thiazepine-8-carboxylate (8b). A solution of 11-oxo-10,11-
dihydrodibenzo[b,f ][1,4]thiazepine-8-carboxylic acid (7, 200 mg,
0.74 mmol) in DMF (5.00 mL) was treated at 0 °C with NaH (295
mg, 7.37 mmol). The reaction mixture was warmed to room
temperature and stirred at room temperature for 1 h. Then a solution
of methyl iodide (0.46 mL, 7.37 mmol) in DMF (2.00 mL) was added
dropwise to the mixture. The reaction mixture was stirred at room
temperature for 1.5 h. Water was carefully added, and the aqueous
layer was washed with EtOAc. The aqueous layer was acidified with
HCl to induce the precipitation. The precipitate was filtered, washed,
and dried to give 200 mg (91%) of the title compound as a yellow
solid which was used directly in the next reaction without further
1
give 118 mg (87%) of the title compound as a white solid. H NMR
(400 MHz, DMSO-d6) δ ppm 8.76 (t, J = 5.7 Hz, 1 H), 7.94 (d, J = 2.0
Hz, 1 H), 7.86 (dd, J = 8.2, 2.0 Hz, 1 H), 7.69−7.77 (m, 2 H), 7.67 (d,
J = 8.2 Hz, 2 H), 7.51−7.60 (m, 1 H), 7.31 (dd, J = 5.1, 1.2 Hz, 1 H),
6.92 (dd, J = 5.1, 3.1 Hz, 1 H), 6.83−6.90 (m, 1 H), 3.55 (s, 3 H),
3.43−3.52 (m, 2 H), 3.02 (t, J = 7.0 Hz, 2 H); 13C NMR (400 MHz,
DMSO-d6) δ ppm 165.16, 165.10, 147.66, 145.98, 141.75, 137.87,
137.02, 132.92, 131.69, 131.23, 128.20, 127.39, 126.51, 125.70, 124.57,
124.43, 121.05, 119.40, 41.55, 38.03, 29.49. LCMS retention time:
t1(method 1) = 5.348 min; t2(method 2) = 3.188 min. HRMS (ESI)
m/z (M + H)+ calcd for C21H19N2O3S2 [M + H+] 411.0832, found
411.0831.
1
purification. H NMR (400 MHz, DMSO-d6) δ ppm 8.00 (d, J = 1.2
b. Pharmacology. For the primary screen, a calcium accumulation
assay was developed using a cell line that stably expresses the D2 DAR
under the control of tetracycline (Flp-In T-REx 293, Invitogen), as
well as a stably expressed chimeric G-protein (Gqi5) to allow coupling
of the D2 DAR to calcium release. In this system, D2 DAR expression
is induced by addition of 1 μM tetracycline to the cells prior to the
assay and intracellular Ca2+ release is detected with a specific Ca2+
fluorescent dye. The resting concentration of calcium ions (Ca2+) in
the cytoplasm is normally maintained in the range of 10−100 nM. To
maintain this low concentration, Ca2+ is actively pumped from the
cytosol to the extracellular space and into the endoplasmic reticulum
(ER) and sometimes into the mitochondria. Signaling occurs when the
cell is stimulated to release Ca2+ from intracellular stores. The most
common signaling pathway that increases cytoplasmic calcium
Hz, 1 H), 7.68−7.79 (m, 2 H), 7.59−7.66 (m, 1 H), 7.47−7.54 (m, 1
H), 7.37−7.45 (m, 2 H), 3.83 (s, 3 H), 3.52 (s, 3 H); LCMS tR = 5.59
min, m/z 300.0 [M + H+]; HRMS (ESI) m/z calcd for C16H14NO3S
[M + H+] 300.0689, found 300.0693.
10-Methyl-11-oxo-10,11-dihydrodibenzo[b,f ][1,4]thiazepine-8-
carboxylic Acid (9b). A solution of methyl 10-methyl-11-oxo-10,11-
dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylate (8b, 150 mg, 0.50
mmol) in THF (3.00 mL), MeOH (1.50 mL), and water (0.50 mL)
was treated at room temperature with LiOH (120 mg, 5.01 mmol).
The reaction mixture was stirred at room temperature for 1 h, diluted
with water, and acidified with HCl. The aqueous mixture was extracted
with 20% of MeOH in dichloromethane. The organic layer was
separated, dried, and concentrated to give 140 mg (98%) of the title
compound as a gray solid which was used directly in the next reaction
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concentration is the phospholipase C (PLC) pathway. In the
engineered cell line used for screening, dopamine stimulation of the
D2 DAR activates the chimeric Gqi5 G-protein, which in turn acts on
PLC which hydrolyzes the membrane phospholipid PIP2 to form
inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 diffuses to
the ER, binds to its receptor (IP3 receptor), which is a Ca2+ channel,
and thus releases Ca2+ from the ER to the cytosol. To measure this
cytosolic Ca2+ accumulation, we used the Screen Quest Fluo-8 calcium
assay kit (AAT Bioquest, Sunnyvale, CA). Acetoxymethyl (AM) esters
bound to Fluo-8 dye are nonpolar molecules that easily cross live cell
membranes transporting the dye inside the cell and are then rapidly
hydrolyzed by cellular esterases inside live cells. As Fluo-8 is freed
from AM esters, it binds to Ca2+ and emits a fluorescent signal at 514
nm that escalates with increasing cytosolic Ca2+. To measure this
calcium flux signal, we used the functional drug screening system
(FDSS) (Hamamatsu, Japan), a high throughput screening device,
which allows optical detection of signal transmissions within living
cells in a time-resolving fashion.
Primary qHTS of Sytravon Library and Confirmatory Screen. D2
Ca2+ Screen Assay. The cells were plated in DMEM medium with
high glucose (Gibco, no. 10564), 10% FBS, 1xNEAA, and penicillin/
streptomycin. At 24 h after thawing, selective antibiotics hygromycin B
(10 μg/mL), puromycin (2 μg/mL), and blasticidin (15 μg/mL) were
added to the medium for growing and passaging the cells. Cells were
split and harvested for the experiment at ∼90% confluence with
TrypLE dissociation reagent and seeded at 2100 cells, 3 μL/well in
complete medium without selective antibiotics with added Tet (1 μg/
mL) using a MultiDrop Combi dispenser (Thermo Scientific, Logan,
UT) onto 1536-well tissue culture treated, black-walled, clear bottom
plates (Greiner Bio-One North America). Quest Fluo-8 calcium
reagent was freshly prepared prior to adding to the cells (lyophilized
Fluo-8 dye provided with the kit was resuspended in 200 μL of DMSO
as a 500× stock and stored at −20 °C). Then 2 μL/well of Quest
Fluo-8 calcium dye diluted in HBSS + 10 mM HEPES buffer (for
every 10 mL buffer, 1 mL of 10x quencher provided in the kit and 20
μL of 500x DMSO stock dye were added) was added to cells with a
Multi-Drop Combi (ThermoScientific) and incubated for 45−90 min
in the dark at ambient temperature. Then the plates were introduced
into the FDSS where they were pinned with 23 nL of test compound
or dopamine controls and were read by FDSS in nonstimulated mode
for 180 s for detection of agonists. Compounds were tested at 2 and 10
μM (final concentrations). At that time point, 1 μL/well dopamine at
1 nM (EC20) or 14 nM (EC80) final concentration was added by the
FDSS pipet head followed by 120 s kinetic read for detection/
recording of potentiators or antagonists stimulation. The cell line
HEK293 D2 T-REx used in the primary screen was also used to
confirm the activity of the active compounds selected from the primary
qHTS and synthesized analogues on 1536-well format following the
same protocol as described above for the primary screen. Compounds
were generally tested in either 7-point titration or 12-point titrations
from 5 nM to 77 μM (final concentrations).
Diego, CA). Compounds were generally tested in either 7-point
titration or 12-point titrations from 5 nM to 77 μM (final
concentrations). The cells were incubated with tested compounds
for 90 min at 37 °C in 5% CO2 and stimulated with 14 nM (EC80)
dopamine, after which 1.5 μL/well of DiscoveRx detection reagent was
added with BioRAPTR FRD dispenser. The detection reagent was
prepared by mixing of Galacton Star substrate, Emerald II solution,
and PathHunter buffer (supplied by the assay kit) together at a 1:5:19
proportion just prior to dispensing. The plates were incubated at
ambient temperature for 1 h, and the luminescent signal was read on a
ViewLux plate reader (PerkinElmer, Waltham, MA).
Radioligand Binding Assays. Compounds were tested for differ-
ences in affinity among three dopamine receptor subtypes using
radioligand binding assays. The first assay was to determine the Ki
value of the compounds using the D2 DAR subtype using stable HEK
cell lines expressing the D2L human dopamine receptors (Codex
Biosciences, Gaithersburg, MD). Cells were cultured in Dulbecco’s
modified Eagle medium containing 10% FBS, 1000 units/mL
penicillin, 1000 mg/mL streptomycin, 100 mM sodium pyruvate, 1
μg/mL gentamicin, and 250 mg/mL G418. All cells were maintained
at 37 °C in 5% CO2 and 90% humidity. For radioligand binding assays,
cells were removed mechanically using calcium and magnesium-free
Earle’s balanced salt solution (EBSS(−)). Intact cells were collected by
centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl2
at pH 7.4 in a glass homogenizer. Homogenates were centrifuged at
20000g for 30 min. The membranes were resuspended in EBSS (pH
7.4) and protein concentration was determined using a Bradford assay
according to the manufacturer’s recommendations (Bio-Rad).
Membranes were diluted to 12 μg/250 μL mixture, or 48 μg/mL. It
was determined in preliminary experiments that this protein
concentration gave optimal binding with minimal ligand depletion.
Membrane preparations were incubated for 90 min at room
temperature with various concentrations of radioligand in a reaction
volume of 250 μL of EBSS containing 200 mM sodium metabisulfite.
Nonspecific binding was determined in the presence of 4 μM
(+)-butaclamol. Bound ligand was separated from unbound by
filtration through GF/C filters using a PerkinElmer cell harvester
with ice cold EBSS (four washes) and quantified on a Top-count
(PerkinElmer) after addition of scintillation solution. Saturation
experiments generated a Kd value of 0.2 nM and a Bmax of ∼4200
fmol/mg for [3H]methylspiperone binding to D2 receptors. In order to
determine the affinity of a given compound for a receptor type,
competition-binding assays were performed. For these assays the
reaction mixture was incubated with a single concentration of
radiolabeled ligand (0.2 nM [3H]methylspiperone) and various
concentrations of competing compound. Reactions were incubated,
terminated, and quantified as indicated above. Ki values of compounds
were determined from observed IC50 values using the Cheng−Prussoff
equation.
DiscoveRx D3 β-Arrestin Assay. To determine the functional
selectivity of the compounds for D2 versus D3 receptor antagonism, we
used a D3 PathHunter β-arrestin cell line from DiscoveRx (Fremont,
CA). As for D2, a CHO cell line was engineered to overexpress D3
dopamine receptor (DAR) and fused with a small 42-amino acid
fragment of β-gal called ProLink. In addition, these cells stably express
a fusion protein of β-arrestin and a larger N-terminal deletion mutant
of β-galactosidase (“enzyme acceptor”). When DAR is activated by
dopamine, it stimulates binding of β-arrestin to ProLink-tagged DARs,
and the two complementary parts of β-galactosidase form a functional
enzyme. When substrate (PathHunter detection reagent) is added, β-
galactosidase hydrolyzes it and generates a chemiluminescent signal.
For the 1536-well assay, D3 PathHunter β-arrestin cells were seeded at
2100 cells/well in 3 μL/well medium (DiscoveRx plating reagent 2)
with MultiDrop Combi dispenser (Thermo Scientific, Logan, UT)
onto white tissue culture treated 1536-well Aurora plates (Brooks
Automation, Chelmsford, MA) and allowed to attach overnight at 37
°C in 5% CO2. Next, an amount of 23 nL/well of compound solutions
in DMSO was added with a pin tool transfer (Kalypsis, San Diego,
CA). Compounds were generally tested in either 7-point titration or
12-point titrations from 5 nM to 77 μM (final concentrations). The
DiscoveRx D2 β-Arrestin Assay. For a secondary screen and
selectivity assays, DAR PathHunter β-arrestin GPCR cell lines from
DiscoveRx (Fremont, CA) were used. In the D2 receptor PathHunter
β-arrestin GPCR cell line, the D2 GPCR receptor (DAR) is
overexpressed and fused with a small 42-amino acid fragment of β-
galactosidase called ProLink on a CHO cellular background expressing
a fusion protein of β-arrestin and a larger N-terminal deletion mutant
of β-galactosidase (“enzyme acceptor”). When DAR is activated by
dopamine, it stimulates binding of β-arrestin to the ProLink-tagged
DAR and the two complementary parts of β-galactosidase form a
functional enzyme. When substrate (PathHunter detection reagent) is
added, β-galactosidase hydrolyzes it and generates a chemiluminescent
signal. D2 receptor PathHunter β-arrestin cells were seeded at 2100
cells/well in 3 μL/well medium (DiscoveRx Plating Reagent 2) with
MultiDrop Combi dispenser (Thermo Scientific, Logan, UT) onto
white tissue culture treated 1536-well Aurora plates (Brooks
Automation, Chelmsford, MA) and allowed to attach overnight at
37 °C in 5% CO2. Next, an amount of 23 nL/well of compound
solutions in DMSO was added with a pin tool transfer (Kalypsis, San
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cells were incubated with tested compounds for 90 min at 37 °C in 5%
CO2 and stimulated with 14 nM (EC80) dopamine, after which 1.5
μL/well of DiscoveRx detection reagent was added with BioRAPTR
FRD dispenser. The detection reagent was prepared by mixing of
Galacton Star substrate, Emerald II solution, and PathHunter buffer
(supplied by the assay kit) together at a 1:5:19 proportion just prior to
dispensing. The plates were incubated at ambient temperature for 1 h,
and the luminescent signal was read on ViewLux plate reader
(PerkinElmer, Waltham, MA).
Research (Grants U54MH084681 and R21NS064831 to
D.R.S.) and the Intramural Research Program of the National
Center for Advancing Translational Sciences and National
Institute of Neurological Disorders and Stroke, National
Institutes of Health.
ABBREVIATIONS USED
■
NIH, National Institutes of Health; DAR, dopamine receptor;
GPCR, G-protein-coupled receptor; CNS, central nervous
system; FDA, Food and Drug Administration; RNA,
ribonucleic acid; qHTS, quantitative high-throughput screen-
ing; ADME, absorption, distribution, metabolism, and
excretion; MLSMR, Molecular Libraries Small Molecule
Repository; BBB, blood−brain barrier; AC50, compound
concentration that produces half the maximal compound
activity; HPLC, high-performance liquid chromatography;
SAR, structure−activity relationship; SNAr, nucleophilic
aromatic substitution; CDI, 1,1′-carbonyldiimidazole;
MCPBA, m-chloroperbenzoic acid; PK, pharmacokinetics;
PBS, phosphate buffered saline; ip, intraperitoneal; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; TOF,
time-of-flight; THF, tetrahydrofuran; SFC, supercritical fluid
chromatography; DEA, diethylamine; HATU, 1-[bis-
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate; ER, endoplasmic
reticulum; PLC, phospholipase C; IP3, inositol trisphosphate;
DAG, diacylglycerol; AM, acetoxymethyl; FDSS, functional
drug screening system; DMEM, Dulbecco’s modified Eagle
medium; FBS, fetal bovine serum; HEK, human embryonic
kidney cell; EBSS, Earle’s balanced salt solution
D3 Binding Assay. Compounds were counterscreened for affinity
for the D3 dopamine receptor. This was accomplished by determining
the Ki values for the compounds using stable (HEK293 based) cell
lines expressing the D3 human dopamine receptors (Codex
Biosciences, Gaithersburg, MD). Cells were cultured in Dulbecco’s
modified Eagle medium containing 10% FBS, 1000 units/mL
penicillin, 1000 mg/mL streptomycin, 100 mM sodium pyruvate, 1
μg/mL gentamicin, and 250 mg/mL G418. All cells were maintained
at 37 °C in 5% CO2 and 90% humidity. For radioligand binding assays,
cells were removed mechanically using calcium and magnesium-free
Earle’s balanced salt solution (EBSS(−)). Intact cells were collected by
centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl2
at pH 7.4 in a glass homogenizer. Homogenates were centrifuged at
20000g for 30 min. The membranes were resuspended in EBSS (pH
7.4), and protein concentration was determined using a Bradford assay
according to the manufacturer’s recommendations (Bio-Rad).
Membranes were diluted to 18 μg/250 μL mixture, or 72 μg/mL,
the predetermined optimal protein concentration for binding but
minimal ligand depletion. Membrane preparations were incubated for
90 min at room temperature with various concentrations of
radioligand in a reaction volume of 250 μL EBSS containing 200
mM sodium metabisulfite. Nonspecific binding was determined in the
presence of 4 μM (+)-butaclamol. Bound ligand was separated from
unbound by filtration through GF/C filters using a PerkinElmer cell
harvester with ice cold EBSS (four washes) and quantified on a Top-
count (PerkinElmer) after addition of scintillation solution. Saturation
experiments generated a Kd value of 0.125 nM and a Bmax of ∼600
fmol/mg for [3H]methylspiperone binding to D3 receptors. In order to
determine the affinity of a given compound for a receptor type,
competition-binding assays were performed. For these assays the
reaction mixture was incubated with a single concentration of
radiolabeled ligand (0.5 nM [3H]methylspiperone) and various
concentrations of competing compound. Reactions were incubated,
terminated, and quantified as indicated above. Ki values of compounds
were determined from observed IC50 values using the Cheng−Prusoff
equation.
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■
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and spectral data of all analogues, single
crystal X-ray parameters, and PK parameters for analogue 65.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 301-217-9198. E-mail: maruganj@mail.nih.gov.
■
Notes
The authors declare no competing financial interest.
(8) (a) Kapur, S.; Remington, G. Dopamine D2 receptors and their
role in atypical antipsychotic action: still necessary and may even be
sufficient. Biol. Psychiatry 2001, 50, 873−883. (b) Beaulieu, J.-M.;
Gainetdinov, R. R. The physiology, signaling, and pharmacology of
dopamine receptors. Pharmacol. Rev. 2011, 63, 182−217. (c) Tandon,
R.; Keshavan, M. S.; Nasrallah, H. A. Schizophrenia, “just the facts”:
what we know in 2008: part 1: overview. Schizophr. Res. 2008, 100, 4−
19.
ACKNOWLEDGMENTS
■
We thank Paul Shinn, Danielle van Leer, William Leister, Chris
LeClair, and Heather Baker for assistance with compound
purification, HRMS analysis, and compound management. We
also thank Drs. Arnold L. Rheingold and Curtis Moore at the
University of California, San Diego for the X-ray analysis of
compounds (R)-59 and 65. This research was supported by the
Molecular Libraries Initiative of the NIH Roadmap for Medical
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