Synthesis and characterization of novel 2-amino-3-benzoylthiophene derivatives as biased allosteric agonists and modulators of the adenosine A 1 receptor

Article abstract of DOI:10.1021/jm201600e

A series of novel 2-amino-3-benzoylthiophenes (2A3BTs) were screened using a functional assay of A₁R mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in intact CHO cells to identify potential agonistic effects as well as the ability to allosterically modulate the activity of the orthosteric agonist, R-PIA. Two derivatives, 8h and 8i, differing only in terms of the absence or presence of an electron-withdrawing group on the benzoyl moiety of the 2A3BT scaffold, were identified as biased allosteric agonists and positive allosteric modulators of agonist function at the adenosine A₁ receptor (A₁R) in two different functional assays. Our findings indicate that subtle structural variations can promote functionally distinct receptor conformational states.

Full text of DOI:10.1021/jm201600e

Article
pubs.acs.org/jmc
Synthesis and Characterization of Novel 2-Amino-3-
benzoylthiophene Derivatives as Biased Allosteric Agonists and
Modulators of the Adenosine A1 Receptor
Celine Valant,† Luigi Aurelio,‡ Shane M. Devine,‡ Trent D. Ashton,‡ Jonathan M. White,§
Patrick M. Sexton,† Arthur Christopoulos,*,† and Peter J. Scammells*,‡
†Drug Discovery Biology and Department of Pharmacology and ‡Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences,
Monash University, 381 Royal Parade, Parkville VIC 3052, Australia
§School of Chemistry and the Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne VIC
3010, Australia
ABSTRACT: A series of novel 2-amino-3-benzoylthiophenes (2A3BTs) were screened using a
functional assay of A1R mediated phosphorylation of extracellular signal-regulated kinases 1 and 2
(ERK1/2) in intact CHO cells to identify potential agonistic effects as well as the ability to allosterically
modulate the activity of the orthosteric agonist, R-PIA. Two derivatives, 8h and 8i, differing only in terms
of the absence or presence of an electron-withdrawing group on the benzoyl moiety of the 2A3BT
scaffold, were identified as biased allosteric agonists and positive allosteric modulators of agonist function
at the adenosine A1 receptor (A1R) in two different functional assays. Our findings indicate that subtle
structural variations can promote functionally distinct receptor conformational states.
that these compounds are able to stabilize A1Rs in more than
one active state and may thus promote stimulus bias even in the
absence of orthosteric agonist.6,20,21 For example, we recently
found that the well-characterized 2A3BT, T62 (3), as well as
two novel modulators/agonists, VCP520 (4) and VCP333 (5)
(Figure 1B), exhibited stimulus bias toward cAMP inhibition
compared to pERK1/2, whereas the orthosteric agonist, R-PIA,
was strongly biased toward the latter pathway.
INTRODUCTION
■
G protein-coupled receptors (GPCRs) are the largest single
class of drug targets,1 and it is now well recognized that most, if
not all, GPCRs possess topographically distinct allosteric
binding sites that present attractive opportunities for the
development of more targeted therapeutics.2−5 The A1R was
one of the first GPCRs for which positive allosteric modulators
of agonist function were described.6,7 Despite the early
identification of the 2A3BT scaffold as a key feature of
allosteric modulators of A1R and the numerous structure−
activity relationships (SAR) subsequently disclosed, improve-
ments in the potency and degree of positive allosteric
modulation over and above the original series described by
Bruns and colleagues have been relatively modest.8−17
An alternative approach to achieving selectivity of signaling at
GPCRs is to pursue ligands that preferentially stabilize distinct
subsets of receptor conformations to the relative exclusion of
others. This phenomenon has been dubbed “stimulus bias” or
“functional selectivity”18,19 and is associated with ligand-
directed signaling outcomes manifested as changes in rank
orders of potency and or maximal effects relative to a reference
(e.g., the endogenous) agonist. Allosteric modulators may
impose stimulus bias in the actions of orthosteric ligands by
differentially modulating some pathways relative to others.
Indeed, we have demonstrated this to be the case with the
2A3BTs compounds 1 and 2 (Figure 1A), which differentially
modulated the effects of the orthosteric agonist, R-PIA, in
mediating ERK1/2 phosphorylation (pERK1/2) relative to
[35S]GTPγS binding to activated G proteins.15 Interestingly,
many 2A3BTs that show some degree of allosteric potentiation
of A1R agonist function also display direct agonist properties on
their own (i.e., in the absence of orthosteric ligand), suggesting
In both cases, the compounds under comparison possessed a
2-amino group and the same substituent in the 3-position [3-
benzoyl group or 3-(4-chlorobenzoyl)], with the point of
divergence being in the 4/5 positions of the thiophene core.
However, in numerous earlier studies, the nature of the 3-
benzoyl group has been shown to have a profound effect on
allosteric activity. Examples of substituents that impart
favorable activity include the 3-(3-trifluoromethyl)benzoyl
present in PD81,723, the 3-(3,4-dichlorobenzoyl) in LUF
5484 as well as the 3-(4-chlorobenzoyl) in T62.7,8 Most
recently, we have shown that an analogue of T62 with a 3-(4-
methylbenzoyl) substituent acted as both an allosteric enhancer
and an allosteric agonist. Herein, we report our investigations
on the influence of further substitution on the 3-benzoyl group
of allosteric modulation and biased signaling. More specifically,
we have targeted analogues of compounds 1 and 2 that possess
a 4-phenyl with electronegative substituents, namely (3-
trifluoromethyl) or 4-(3,5-bistrifluoromethyl), as this has
proven to be an important feature for engendering intrinsic
agonist activity. A test set of the most interesting 3-benzoyl
groups from previous studies (i.e., 4-Cl, 3,4-Cl, 3-CF3, and 4-
Received: November 28, 2011
Published: February 8, 2012
© 2012 American Chemical Society
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Figure 1. Structure of allosteric ligands mentioned in this study. (A) Allosteric modulators that imposed biased signaling in the actions of the
orthosteric agonist, R-PIA.21 (B) Previously characterized A1R allosteric modulators that promote stimulus bias in the absence of orthosteric
agonist.21
Me) has been used to probe the influence of this group on
allosteric activity and downstream cell signaling. We find that
even simple substitutions can lead to marked changes in the
signaling bias of the A1R when occupied by allosteric agonists
alone and in the presence of a cobound orthosteric agonist.
Interestingly, compound 8h was found to dimerize upon
standing in CDCl3. The structure of the dimer was confirmed
by X-ray crystallography (Figure 2). This dimerization resulted
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of the 2-amino-3-benzoylthio-
phenes 8a−q was performed via either a two-step or one-pot
microwave-assisted Gewald procedure (Scheme 1). The two-
a
Scheme 1
Figure 2. A thermal ellipsopid plot for the dimer 8h. The 8-membered
ring adopts a tub conformation, with a local 2-fold axis of symmetry
orthogonal to the plane defined by the atoms N1, N2, C13, and C33
in the 8-membered ring, the 2-fold symmetry is broken only by the
orientations of the trifluoromethylphenyl substituents.
from the condensation of the 2-amino moiety of one molecule
with the 3-benzoyl group of another. We have only previously
observed dimerization of this type under acidic conditions
(refluxing in 0.5 M HCl for 7 h).22 A stability study was
conducted in order to determine if this dimerization might be
occurring in DMSO stock solutions upon storage. However, 8h
was found to be stable in DMSO (over a 4 week period), which
suggested that the dimerization observed previously was
promoted by traces of acid in the CDCl3.
Pharmacology. To assess the biological activity of the
novel series of compounds, we initially screened all compounds
using a plate-based assay of A1R-mediated pERK1/2 in intact
CHO cells.14−17 For each compound, two concentrations (3
and 10 μM) were tested alone to assess intrinsic agonism, and
against an EC50 concentration of the orthosteric agonist R-PIA,
to assess enhancement or inhibition of the A1R agonist activity.
Most compounds, being analogues of 1 and 2, displayed a high
degree of agonism on their own, as indicated in Figure 3A. It
has already been observed previously that the presence of a 3-
trifluoromethyl or 3,5-bis(trifluoromethyl) group on the phenyl
in the 4-position of the thiophene ring induces strong agonist
a
Method A: (i) CH2Cl2, TiCl4, pyridine; (ii) THF, S8, Et2NH. Method
B: S8, Et2NH, microwave.
step approach (method A) initially involved a titanium(IV)
chloride mediated Knoevenagel condensation to give an
intermediate alkene that subsequently underwent ring closing
under basic conditions in the presence of elemental sulfur to
furnish the desired 2A3BTs. In the one-pot procedure (method
B), the benzoylacetonitrile, acetophenone or propiophenone,
sulfur, and base (Et2NH) were heated at 130 °C for 1 h in a
microwave reactor. In both cases, the crude thiophenes were
purified via silica gel chromatography.
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agonists for the pERK1/2 pathway in their own right. At the
level of cAMP inhibition (Figure 4C,D), both 2A3BTs again
enhanced the potency of the orthosteric agonist, R-PIA, but
only 8h retained appreciable allosteric agonist activity.
To quantify these properties, we applied an operational
model of agonism and allosterism to the data to derive
estimates of the following three parameters: the affinity of each
modulator for the allosteric site on the unoccupied A1R
(quantified as the negative logarithm of the dissociation
constant, pKB), a measure of the operational efficacy of each
modulator at the A1R (quantified as the logarithm, logτB), and a
measure of the cooperativity of each modulator on R-PIA
potency (quantified as a logarithm of the composite
cooperative effects on both binding affinity and signaling
efficacy, logαβ). The results of this analysis are shown in Table
2.
For compound 8h, the affinity estimates were similar
between pERK1/2 and cAMP assays but significantly higher
than the pKB values estimated for 8i at the corresponding
pathways (Table 2). This suggests that the presence of the 4-Cl
on the benzoyl group of the 2A3BT scaffold was detrimental to
the interaction of the ligand with the allosteric binding site in
the A1R. In terms of the magnitude of the allosteric effect of
each 2A3BT on R-PIA signaling, 8h exhibited similar degrees of
cooperativity with R-PIA between pERK1/2 (αβ = 4.1) and
cAMP inhibition (αβ = 3.2), while the values for the effect of 8i
were higher, αβ = 12 and αβ = 18 for pERK1/2 and cAMP
inhibition, respectively. This suggests that the presence of a 4-
chloro on the benzoyl group of the 2A3BT scaffold increases
the allosteric interaction with R-PIA, even though it reduces the
affinity of the modulator at the A1R. It also highlights the fact
that these two parameters are not correlated, thus justifying
why they should be separately determined in order to better
understand underlying SAR of allosteric ligands.
When comparing the intrinsic agonist properties of each of
the allosteric ligands, 8h exhibited similar efficacy values for
pERK1/2 (τB = 0.63) and cAMP (τB = 0.59), whereas 8i
showed greater efficacy in pERK1/2 (τB = 2.0) than cAMP
inhibition (τB = 0.06). Given that the potency (as defined by
the EC50) of the prototypical orthosteric agonist, R-PIA, is 100-
fold greater for signaling to pERK1/2 (pEC50 = 9.64 0.04)
relative to cAMP inhibition (pEC50 = 7.73 0.03), the lack of
change in the efficacy of 8h between the same two pathways is
particularly striking and indicative that the allosteric agonist is
biased relative to the orthosteric agonist. This is more evident
in Figure 5A, where the percentage responses to equimolar
concentrations of each agonist between the two pathways are
plotted against each other in the form of a “bias plot”.21,23 For
comparative purposes, we also generated the bias plot for our
previously published compound 4,15 which demonstrated an
even more marked bias toward cAMP accumulation over
ERK1/2 phosphorylation than that of 8h. In contrast, 8i
followed the pathway preferences of R-PIA, suggesting that this
allosteric agonist is not biased relative to the orthosteric
agonist. This does not mean that either of these latter agents
are “non-biased”, only that their coupling preferences are
distinct from those of 8h.
Figure 3. Effect of two different concentrations (3 μM, white bar; 10
μM, black bar) of novel 2-amino-3-benzoylthiophenes on A1R-
mediated stimulation of ERK1/2 phosphorylation in intact CHO
FlpIn cells, in absence (A) or presence (B) of an EC50 concentration
of R-PIA (determined on the same day as each assay). Data represent
the mean standard deviation of two to three experiments conducted
in triplicate.
properties of the 2A3BT derivatives.15 The capacity of these
compounds to potentiate R-PIA and A1R-mediated ERK1/2
phosphorylation was also screened at two concentrations (3
and 10 μM) (Figure 3B and Table 1). Pleasingly, the
combination of substituents known to promote allosteric
activity in the 3- and 4-positions afforded a series of
compounds that demonstrated substantial functional potentia-
tion of R-PIA/A1R mediated increases in ERK1/2 phosphor-
ylation. Even at the lowest test concentration (3 μM), 13 of the
17 new 2A3BTs afforded greater than 80% of the maximum
attainable R-PIA response by a concentration of agonist that
normally yields 50% of the maximal response. Five of these
compounds produced over 90% of the maximum attainable R-
PIA response at this concentration (Table 1).
To further investigate the results of this initial screen, more
detailed experiments were performed using the most efficacious
allosteric modulator identified in this series of compounds, 8i,
as well as its closest analogue, 8h, differing only in terms of the
absence of an electron-withdrawing group, 4-Cl, on the benzoyl
moiety of the 2A3BT scaffold. The biological activity of these
two 2A3BT derivatives was assessed in both pERK1/2 and
cAMP inhibition assays performed in recombinant CHO-FlpIn
cells stably expressing the human A1R. As shown in parts A and
B of Figure 4, increasing concentrations of either compound
caused a significant enhancement of the potency of R-PIA for
mediating pERK1/2, indicative of positive allosteric modulation
of the orthosteric agonist. Concomitantly, a substantial degree
of R-PIA-independent receptor activation was also noted for
both novel 2A3BT derivatives, as previously observed in the
initial functional screen, indicating that they were allosteric
To statistically quantify the bias of our 2A3BT derivatives
compared to R-PIA, we determined “bias factors” for each of
the agonists using a method based on the operational model of
agonism; see Experimental Section. Specifically, we have shown
previously24,25 that the ratio of τB/KB for a given agonist at a
signal pathway (referred to as the “transduction ratio”, TR),
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Table 1. Effect of Test Compounds on A1R-Mediated Stimulation of ERK1/2 Phosphorylation in Intact CHO Cells in the
Presence of an EC50 Concentration of R-PIA
structure
Y
activity
R
Z
3 μM
10 μM
n
115
415
8a
H
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
H
83
3
8
6
2
2
59
1
8
5
2
3
2
2
2
3
3
H
H
H
H
4-Cl
64
88
93
85
73
88
94
83
3,4-Cl
3-CF3
4-CH3
8b
8c
215
8d
8e
8f
H
H
H
H
H
3,5-CF3
3,5-CF3
3,5-CF3
3,5-CF3
3,5-CF3
H
83
69
90
90
86
7
9
1
2
4
93
74
90
84
87
8
7
2
8
4
2
2
3
3
3
4-Cl
3,4-Cl
3-CF3
4-CH3
8g
8h
8i
CH3
CH3
CH3
CH3
CH3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
H
77
94
73
90
86
6
3
3
1
3
73
94
75
86
90
3
4
3
1
3
3
3
2
2
3
4-Cl
8j
3,4-Cl
3-CF3
4-CH3
8k
8l
8m
8n
8o
8p
8q
CH3
CH3
CH3
CH3
CH3
3,5-CF3
3,5-CF3
3,5-CF3
3,5-CF3
3,5-CF3
H
83
84
78
84
92
9
2
5
3
1
95
88
84
84
3
4
2
3
2
2
2
2
2
4-Cl
3,4-Cl
3-CF3
4-CH3
80 10
once normalized to that of a reference agonist, can be used to
derive a bias factor as a quantitative measure of signal pathway
bias. If the test agonist (i.e., allosteric ligand) and reference
agonist (i.e., R-PIA) activate the two pathways via a common
receptor conformation, the bias factor should be 1.0,
irrespective of differences in response amplification between
pathways. In contrast, significant deviation of the bias factors
from 1.0 indicates the involvement of distinct conformations
for the different agonists in mediating the various pathways. As
shown in Table 3 and Figure 5B, 8h displayed a significant 45-
fold bias toward cAMP relative to pERK1/2 when compared to
the signaling preferences of R-PIA; in contrast, the bias toward
cAMP signaling of 8i was only 3.5-fold. This is not to say the R-
PIA and 8i do not display signaling bias, but for the pathways
studied herein, their coupling preferences individually at the
A1R track with one another, whereas those of 8h clearly
diverge. Collectively, these results suggest that the nature of the
binding locus used by the different ligands, i.e., orthosteric
versus allosteric site, is unlikely to be the sole determinant of
the ability of a given agonist to engender stimulus-bias.
pERK1/2 and cAMP inhibition pathways. However, this does
not hold for 8i because it is clearly less efficacious at inhibiting
cAMP relative to promoting pERK1/2 yet has similar robust
positive cooperativities with R-PIA at both pathways; if
anything, the positive cooperativity is greater at cAMP
inhibition, the pathway associated with the least direct allosteric
agonism (Table 2). This finding highlights two important
points. First, it is necessary to postulate the existence of more
than a single active receptor state to accommodate the data.
Second, although 8i, on its own, appears to bias receptor
signaling in manner that is similar to that of R-PIA, the
cobinding of the modulator and R-PIA to the A1R promotes a
functionally distinct state, an example of allosteric modulator-
engendered functional selectivity.26−29 In contrast, the opposite
appears to be the case with 8h, i.e., as an allosteric agonist, this
compound is biased, but as an allosteric modulator of R-PIA, it
retains similar preferences across pathways.
CONCLUSIONS
■
In summary, two novel 2A3BT derivatives, differing only with
regards to the absence (8h) or presence (8i) of a halogen atom
in the 4-position of the benzoylthiophene ring, promote
functionally biased states of the adenosine A1R. In comparison
to the orthosteric agonist, R-PIA, 8h (alone) was biased as an
allosteric agonist toward cAMP accumulation over ERK1/2
phosphorylation, whereas 8i showed minimal bias. In contrast,
when combined with R-PIA, 8h allosterically modulated the
According to a simple two-state model of receptor activation,
it would be expected that the degree of positive allosteric
enhancement should correlate with the degree of direct
allosteric agonism displayed by a given modulator.26,27 The
behavior of 8h when combined with R-PIA is certainly in
accord with this hypothesis, given that it displayed similar
degrees of agonism (τB) and cooperativity (αβ) between
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Figure 4. Positive allosteric modulation of R-PIA-mediated response by 2A3BTs. Effects of 8h (A,C) or 8i (B,D) on R-PIA-mediated ERK1/2
phosphorylation (A−B), or cAMP inhibition (C−D). Data points represent the mean SE obtained from three to five experiments conducted in
duplicate. Curves drawn through the data represent the fit of an operational model of allosterism.
Table 2. Operational Model Parameters for the Functional Allosteric Interaction between R-PIA and Modulators at the A1R
(Parameter Values Represent the Mean SEM from Three Experiments Performed in Duplicate)
a
b
Negative logarithm of the equilibrium dissociation constant of modulators (pKb). Logarithm of the operational efficacy parameter of the 2A3BTs
c
as allosteric agonists. Antilogarithm shown in parentheses. Logarithm of the product of the binding (α) and activation (β) cooperativity factors
between R-PIA and modulators. Antilogarithm shown in parentheses.
1
Biotage Initiator microwave synthesizer. All H NMR and 13C NMR
activity of the orthosteric agonist at the two pathways in a
nonbiased manner, whereas the combination of 8i and R-PIA
spectra were recorded on a Bruker Avance III 400 Ultrashield Plus
spectrometer at 400.13 and 100.62 MHz, respectively. Thin-layer
resulted in the generation of pathway-biased allosteric
chromatography was conducted on 0.2 mm plates using Merck silica
modulation. Collectively, these results highlight how a GPCR
gel 60 F254. Column chromatography was achieved using Merck silica
bound with both an allosteric modulator and an orthosteric
gel 60 (particle size 0.063−0.200 μm, 70−230 mesh) and eluent
agonist should be viewed as a unique protein state that differs
percentages are described in volume (%v/v). High resolution mass
from those promoted by either orthosteric or allosteric agonist
alone. Furthermore, 8h and 8i represent novel tools with which
to further probe the mechanistic basis of biased signaling at the
A1R.
spectra (HR-ESI) were obtained on a Waters LCT Premier XE (TOF)
using electrospray ionization. Compound purity was analyzed on an
Agilent 1200 series LCMS system employing a RP-HPLC
Phenomenex column (Luna 5 μm C8(2), 50 mm × 4.60 mm ID) at
30 °C, with a photodiode array detector (214/254 nm) coupled
EXPERIMENTAL SECTION
directly to an electrospray ionization source and a single quadrupole
mass analyzer (Agilent 6120 Quadrupole MS). Chromatograms show
UV absorbance at 254 nm. The following buffers were used; buffer A
99.9% H2O, 0.1% formic acid; buffer B 99.9% CH3CN, 0.1% formic
acid. The following gradient was used with a flow rate of 0.5 mL/min
and total run time of 12 min; 0−4 min 95% buffer A and 5% buffer B,
■
Chemistry. All reagents and anhydrous DMF were purchased from
Aldrich and used without further purification. LR grade methanol,
petroleum ether (40−60 °C), ethyl acetate, diethyl ether, and
dichloromethane were purchased from Merck and were used without
further purification. All microwave reactions were performed in a
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Figure 5. A1R orthosteric and allosteric ligand signaling bias alone (A,B) or upon coadministration (C,D). (A,C,D) Bias plots of the responses
corresponding to equimolar concentratons of the indicated ligands for ERK1/2 phosphorylation and cAMP inhibition assays. Data for compound 4
replotted from ref 21. (B) Bias factors, ΔΔlog(τ/KA), determined in both ERK1/2 phosphorylation and cAMP inhibition assays. *** indicates P <
0.001 relative to value for R-PIA.
Table 3. Estimation of Bias Factors for R-PIA and Selected 2A3BTs at the A1R, Determined in Both ERK1/2 Phosphorylation
a
and cAMP Inhibition Assays
ERK1/2 phosphorylation
cAMP inhibition
ligand
logTR
logTRn
logTR
logTRn
LogBiasFactor [cAMP-pERK1/2] (bias factor)
R-PIA
4
9.55 0.04
4.86 0.29
5.89 0.04
5.64 0.03
0.00 0.06
−4.69 0.29
−3.66 0.05
−3.91 0.05
7.71 0.06
5.78 0.15
5.70 0.07
4.34 0.28
0.00 0.08
−1.93 0.16
−2.01 0.09
−3.37 0.29
0.00 0.10 (1.00)
2.76 0.33 (575)
1.65 0.11 (45)
0.54 0.30 (3.5)
8h
8i
a
LogTR is the log of the “transduction ratio” (τA/KA or τB/KB), and logTRn values are normalized to the logTR for R-PIA. LogBF[cAMP-pERK1/2]
represent the logarithm of the bias factor for cAMP inhibition pathway compared to pERK1/2 pathway; antilogarithms of the bias factor (BF) are
shown in parentheses. Values represent the mean SEM from three experiments performed in duplicate. For compound 4, these values were
derived from our prior study.21
4−7 min 0% buffer A and 100% buffer B, 7−12 min 95% buffer A and
5% buffer B. Mass spectra were acquired in positive and negative ion
mode with a scan range of 0−1000 m/z at 5 V. All compounds were of
≥95% purity.
added followed by diethylamine (0.654 mL) and stirred at
room temperature for 5 h. The mixture was diluted with ether
(40 mL) and washed with water (3 × 20 mL) and finally with
brine (10 mL). The organic layer was dried (MgSO4), filtered,
and then concentrated to a resin that is chromatographed on
silica gel with CH2Cl2.
Method B. The appropriate benzoylacetonitrile 7 (1.26 mmol) and
appropriate acetophenone or propiophenone 6 (2.56 mmol, 2.0
equiv), S8 (46 mg, 1.44 mmol, 1.1 equiv), and Et2NH (26 μL, 0.25
mmol, 0.2 equiv) were heated at 130 °C for 1 h via microwave
irradiation. The crude reaction mixture was purified by column
chromatography using 4:1 petroleum spirits:EtOAc.
(2-Amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(3,4-
dichlorophenyl)methanone (8a). Method B. Compound 8a was
isolated as a yellow oil (181 mg, 31%). 1H NMR (400 MHz, CDCl3) δ
7.32−7.27 (m, 1H), 7.23−7.19 (m, 3H), 7.18−7.16 (m, 1H), 7.15 (dd,
J = 8.2, 1.9 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 6.90 (s, 2H), 6.22 (s,
1H). 13C NMR (101 MHz, CDCl3) δ 189.7, 167.4, 139.8, 139.7, 137.9,
134.8, 131.8, 131.6 (d, J = 1.1 Hz), 130.9, 130.4 (q, J = 32.3 Hz),
General Procedure for the Synthesis of 8a−q. Method A.
The acetophenone or propiophenone 6 (0.5 g, 2.47 mmol) and
appropriate nitrile 7 (2.47 mmol) were dissolved in dry CH2Cl2
(16 mL) in an N2 atmosphere and cooled to 0 °C with an ice
bath. To the cooled solution was added neat titanium(IV)
chloride (0.271 mL, 2.47 mmol) dropwise. After stirring on the
ice bath for 0.5 h, pyridine (0.181 mL) was added dropwise and
left to stir a further hour. Then another aliquot of pyridine
(0.503 mL) was added dropwise and the ice bath removed, and
the mixture was left to stir overnight. The mixture was diluted
with CH2Cl2 (30 mL) and washed with 1 M HCl (30 mL). The
organic was then washed with water (3 × 20 mL) and finally
with brine (10 mL). The organic layer was dried (MgSO4),
filtered, and then concentrated to a resin that was taken up in
THF (5 mL) and elemental sulfur (0.087 g, 2.71 mmol) was
2372
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129.8, 128.7, 127.61, 125.4 (q, J = 3.8 Hz), 123.8 (q, J = 272.6 Hz),
123.6 (q, J = 3.7 Hz), 114.3, 106.8. HR-ESMS calcd for
C18H11Cl2F3NOS+ (M + H) 415.9885, found 415.9878.
Hz), 118.0, 116.1, 13.4. HR-ESMS calcd for C19H15F3NOS+ (M + H)
362.0821, found 362.0838.
(2-Amino-5-methyl-4-(3-(trifluoromethyl)phenyl)thiophen-3-
yl)(4-chlorophenyl)methanone (8i). Method A. Compound 8i was
(2-Amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(3-
1
trifluoromethylphenyl)methanone (8b). Method A. Compound 8b
isolated as a yellow resin (342 mg, 35% yield). H NMR (400 MHz,
1
was isolated as a yellow oil (251 mg, 34%). H NMR (400 MHz,
CDCl3) δ 7.25 (d, J = 7.2 Hz, 1H, ArH), 7.21−6.97 (m, 5H, ArH),
6.96−6.82 (m, 2H, ArH), 6.44 (bs, 2H, NH2), 2.15 (s, 3H, CH3). 13
C
CDCl3) δ 7.53 (d, J = 7.7 Hz, 1H), 7.41 (s, 1H), 7.35 (dd, J = 7.8, 0.6
Hz, 1H), 7.22−7.08 (m, 5H), 6.96 (s, 2H), 6.22 (s, 1H). 13C NMR
(101 MHz, CDCl3) δ 190.8, 167.5, 140.8, 139.9, 137.7, 131.7 (d, J =
0.9 Hz), 131.6 (d, J = 1.0 Hz), 130.3 (q, J = 32.3 Hz), 129.8 (q, J =
32.8 Hz), 128.6, 128.5, 127.1 (q, J = 3.6 Hz), 125.6 (q, J = 3.6 Hz),
125.5 (q, J = 3.6 Hz), 123.8 (q, J = 272.5 Hz), 123.6 (q, J = 272.5 Hz),
123.5 (q, J = 3.7 Hz), 114.2, 106.9. HR-ESMS calcd for
C19H12F6NOS+ (M + H) 416.0538, found 416.0537.
NMR (101 MHz, CDCl3) δ 191.3, 164.5, 138.6, 137.1, 136.2, 134.2,
133.3, 130.1 (q, J = 32.2 Hz), 129.7, 128.3, 127.6, 127.1 (q, J = 3.8
Hz), 123.9 (q, J = 272.5 Hz), 123.1 (q, J = 3.7 Hz), 118.2, 115.9, 13.4.
HR-ESMS calcd for C19H14ClF3NOS+ (M + H) 396.0431, found
396.0439.
(2-Amino-5-methyl-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)-
(3,4-dichlorophenyl)methanone (8j). Method B. Compound 8j was
1
(2-Amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(p-tolyl)-
methanone (8c). Method B. Compound 8c was isolated as a yellow
amorphous solid (109 mg, 16%) after recrystallization from a mixture
isolated as an orange oil (137 mg, 25% yield). H NMR (CDCl3, 400
MHz) δ 7.28−7.21 (m, 2H), 7.15 (app d, J = 7.2, 1H), 7.10 (app t, J =
1
1.2, 2H), 7.06 (app d, J = 5.2, 2H), 6.83 (br s, 2H), 2.16 (s, 3H). H
1
of 2-propanol, water, and acetone. H NMR (400 MHz, CDCl3) δ
NMR (400 MHz, CDCl3) δ 7.29−7.20 (m, 2H), 7.18−7.14 (m, 1H),
7.12−7.09 (m, 2H), 7.05 (m, 2H), 6.84 (s, 2H), 2.16 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 189.6, 165.1, 139.9, 137.1, 134.2, 134.0,
133.2, 131.5, 130.5, 130.3 (q, J = 32.5 Hz), 129.8, 128.5, 127.3, 126.8
(q, J = 3.8 Hz), 123.9 (q, J = 272.6 Hz), 123.3 (q, J = 3.7 Hz), 118.5,
115.6, 13.4. HR-ESMS calcd for C19H13Cl2F3NOS+ (M + H)
430.0042, found 430.0059.
(2-Amino-5-methyl-4-(3-(trifluoromethyl)phenyl)thiophen-3-
yl)(3-(trifluoromethyl)phenyl)methanone (8k). Method B. Com-
pound 8k was isolated as a yellow oil (123 mg, 24%). 1H NMR
(400 MHz, CDCl3) δ 7.41 (d, J = 7.7 Hz, 1H), 7.35−7.28 (m, 2H),
7.19−7.08 (m, 5H), 6.88 (s, 2H), 2.16 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 190.9, 165.1, 141.1, 136.9, 134.0, 133.3 (d, J = 1.1 Hz),
131.4 (d, J = 1.0 Hz), 130.2 (q, J = 32.3 Hz), 129.6 (q, J = 32.6 Hz),
128.5, 128.4, 126.9 (q, J = 3.8 Hz), 126.6 (q, J = 3.6 Hz), 125.1 (q, J =
3.6 Hz), 123.8 (q, J = 272.5 Hz), 123.7 (q, J = 272.5 Hz), 123.3 (q, J =
3.7 Hz), 118.5, 115.6, 13.4. HR-ESMS calcd for C20H14F6NOS+ (M +
H) 430.0695, found 430.0714.
(2-Amino-5-methyl-4-(3-(trifluoromethyl)phenyl)thiophen-3-
yl)(4-tolyl)methanone (8l). Method B. Compound 8l was isolated as a
yellow oil (36 mg, 5%). 1H NMR (400 MHz, CDCl3) δ 7.20−7.16 (m,
1H), 7.13 (s, 1H), 7.11−7.08 (m, 2H), 7.06 (d, J = 8.1 Hz, 2H), 6.72
(d, J = 7.8 Hz, 2H), 6.62 (s, 2H), 2.16 (s, 3H), 2.14 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 192.9, 163.4, 140.6, 137.4 (d, J = 2.2 Hz), 134.7,
133.3, 129.8 (q, J = 32.2 Hz), 128.6, 128.1, 128.0, 127.1 (q, J = 3.8
Hz), 124.1 (q, J = 272.8 Hz), 122.7 (q, J = 3.7 Hz), 117.9, 116.5, 21.3,
13.4. HR-ESMS calcd for C20H17F3NOS+ (M + H) 376.0977, found
376.0975.
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-methylthiophen-
3-yl)(phenyl)methanone (8m). Method B. Compound 8m was
isolated as a yellow gum (66 mg, 15%). 1H NMR (400 MHz,
CDCl3) δ 7.41 (s, 1H), 7.34 (s, 2H), 7.15−7.10 (m, 2H), 7.09−7.03
(m, 1H), 6.98−6.91 (m, 2H), 6.79 (s, 2H), 2.16 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 192.5, 164.4, 140.1, 138.7, 133.1, 130.9 (q, J =
33.3 Hz), 130.4, 130.2 (d, J = 2.6 Hz), 128.2, 127.7, 123.2 (q, J = 272.7
Hz), 120.0 (dt, J = 7.6, 3.7 Hz), 119.0, 115.6, 13.3. HR-ESMS calcd for
C20H14F6NOS+ (M + H) 430.0695, found 430.0704.
7.23−7.07 (m, 6H), 6.77 (d, J = 7.8 Hz, 2H), 6.61 (s, 2H), 6.22 (s,
1H), 2.15 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 192.8, 165.7,
141.3, 140.6, 138.3, 137.2, 131.6 (d, J = 1.1 Hz), 130.1 (q, J = 32.2
Hz), 129.0, 128.3, 128.2, 125.6 (q, J = 3.9 Hz), 124.0 (q, J = 272.3
Hz), 123.0 (q, J = 3.8 Hz), 115.2, 106.4, 21.4. HR-ESMS calcd for
C19H15F3NOS+ (M + H) 362.0821, found 362.0831.
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-
chlorophenyl)methanone (8d).15 Method A. Compound 8d was
1
isolated as a yellow solid. H NMR (400 MHz, CDCl3) δ 7.54 (bs,
1H), 7.42 (bs, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H),
6.83 (bs, 2H), 6.31 (s, 1H). 13C NMR (75.4 MHz, CDCl3) δ 190.9,
167.0, 139.4, 139.3, 138.2, 137.1, 131.2 (q, J = 33.4 Hz), 129.9, 128.5,
127.9, 123.0 (q, J = 272.8 Hz), 120.2, 114.2, 107.6. HR-ESMS calcd for
C19H11ClF6NOS+ (M + H) 450.0149, found 450.0163.
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(3,4-
dichlorophenyl)methanone (8e). Method B. Compound 8e was
isolated as a yellow oil (159 mg, 23%). 1H NMR (400 MHz, CDCl3) δ
7.55 (s, 1H), 7.42 (s, 2H), 7.19−7.09 (m, 3H), 7.01 (s, 2H), 6.29 (s,
1H). 13C NMR (101 MHz, CDCl3) δ 189.3, 167.8, 139.5, 139.2, 138.2,
135.2, 132.1, 131.5 (q, J = 33.5 Hz), 130.8, 130.1, 128.4 (d, J = 2.7
Hz), 127.5, 123.0 (q, J = 272.9 Hz), 120.5 (dt, J = 7.5, 3.8 Hz), 113.9,
108.0. HR-ESMS calcd for C20H9F9NOS− (M − H) 481.9613, found
481.9629.
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(3-
trifluoromethylphenyl)methanone (8f). Method B. Compound 8f
was isolated as a yellow oil (81 mg, 12%). 1H NMR (400 MHz,
CDCl3) δ 7.55 (d, J = 7.7 Hz, 1H), 7.46 (s, 1H), 7.43−7.35 (m, 4H),
7.25−7.19 (m, 1H), 6.98 (s, 2H), 6.31 (s, 1H). 13C NMR (101 MHz,
CDCl3) δ 190.4, 167.7, 140.6, 139.0, 138.3, 131.7 (d, J = 0.7 Hz),
131.5 (q, J = 33.4 Hz), 130.2 (q, J = 33.2 Hz), 128.8, 128.4 (d, J = 2.7
Hz), 127.4 (q, J = 3.5 Hz), 125.5 (q, J = 3.5 Hz), 123.4 (q, J = 272.6
Hz), 123.0 (q, J = 272.8 Hz), 120.5 (dt, J = 7.6, 3.7 Hz), 113.9, 108.1.
HR-ESMS calcd for C20H9F9NOS− (M − H) 482.0267, found
482.0278.
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-
tolyl)methanone (8g). Method B. Compound 8g was isolated as a
yellow solid (102 mg, 13%) after recrystallization from a mixture of 2-
propanol, water, and acetone. 1H NMR (400 MHz, CDCl3) δ 7.45 (s,
1H), 7.40 (s, 2H), 7.14−7.08 (m, 2H), 6.77 (m, 4H), 6.28 (s, 1H),
2.15 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 192.4, 166.3, 141.6,
139.6, 139.0, 137.1, 131.0 (q, J = 33.3 Hz), 128.8, 128.6 (d, J = 2.7
Hz), 128.4, 123.2 (q, J = 272.8 Hz), 119.9 (dt, J = 7.6, 3.8 Hz), 114.8,
107.4, 21.3. HR-ESMS calcd for C20H14F6NOS+ (M + H) 430.0695,
found 430.0705.
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-methylthiophen-
3-yl)(4-chlorophenyl)methanone (8n). Method B. Compound 8n
1
was isolated as an orange oil (151 mg, 26%). H NMR (CDCl3, 400
MHz) δ 7.51 (s, 1H), 7.33 (s, 2H), 7.08−7.02 (m, 2H), 6.94−6.88 (m,
2H), 6.83 (br s, 2H), 2.17 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
191.0, 164.7, 138.6, 138.5, 136.6, 132.8, 131.2 (q, J = 33.3 Hz), 130.2
(d, J = 2.5 Hz), 129.6, 127.9, 123.1 (q, J = 272.8 Hz), 120.1 (dt, J =
7.4, 3.7 Hz), 119.2, 115.7, 13.3. HR-ESMS calcd for C20H13ClF6NOS+
(M + H) 464.0305, found 464.0304.
(2-Amino-5-methyl-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)-
(phenyl)methanone (8h). Method A. Compound 8h was isolated as a
yellow resin, which slowly solidified upon standing and was
recrystallized from 2-propanol (587 mg, 66% yield); mp 129−131
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-methylthiophen-
3-yl)(3,4-dichlorophenyl)methanone (8o). Method B. Compound 8o
1
1
was isolated as an orange oil (160 mg, 25%). H NMR (CDCl3, 400
°C. H NMR (400 MHz, CDCl3) δ 7.22−7.12 (m, 4H, ArH), 7.15−
MHz) δ 7.53 (s, 1H), 7.36 (s, 2H), 7.11−7.01 (m, 3H), 6.92 (br s,
2H), 2.17 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 189.3, 165.5,
139.8, 138.5, 134.6, 132.5, 131.9, 131.4 (q, J = 33.1 Hz), 130.3, 130.0,
129.9 (d, J = 2.7 Hz), 127.2, 123.1 (q, J = 272.7 Hz), 120.3 (dt, J = 7.5,
7.01 (m, 3H, ArH), 6.99−6.89 (m, 2H, ArH), 6.67 (bs, 2H, NH2),
2.16 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 192.8, 164.0,
140.2, 137.3, 134.5, 133.3, 130.2, 129.9 (d, J = 32.1 Hz), 128.4, 128.1,
127.4, 127.1 (q, J = 3.7 Hz), 124.0 (q, J = 272.4 Hz), 123.0 (q, J = 3.5
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3.8 Hz), 119.6, 115.2, 13.4. HR-ESMS calcd for C20H12Cl2F6NOS+ (M
+ H) 497.9915, found 497.9933.
mixed with the activated lysate mixture in a ratio of 6:5 (v/v),
respectively, in a 384-well opaque Optiplate. Plates were incubated in
the dark at 37 °C for 1 h before the fluorescence signal was measured
by use of a Fusion plate reader (PerkinElmer Life and Analytical
Sciences) with standard AlphaScreen settings. Data were normalized
to the maximal response elicited by 10 μM R-PIA.21
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-methylthiophen-
3-yl)(3-(trifluoromethyl)phenyl)methanone (8p). Method B. Com-
1
pound 8p was isolated as an orange oil (167 mg, 27%). H NMR
(CDCl3, 400 MHz) δ 7.43−7.40 (m, 2H), 7.34−7.32 (m, 4H), 7.16 (t,
J = 7.6, 1H), 6.92 (br s, 2H), 2.18 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 190.4, 165.4, 141.0, 138.3, 132.5, 131.4 (d, J = 0.9 Hz),
131.3 (q, J = 33.3 Hz), 130.1 (q, J = 33.5 Hz), 130.0 (d, J = 2.7 Hz),
128.6, 126.9 (q, J = 3.6 Hz), 125.0 (q, J = 3.8 Hz), 123.5 (q, J = 272.5
Hz), 123.0 (q, J = 272.8 Hz), 120.3 (dt, J = 7.6, 3.7 Hz), 119.7, 115.3,
13.4. HR-ESMS calcd for C21H13F9NOS+ (M + H) 498.0569, found
498.0573.
(2-Amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-methylthiophen-
3-yl)((4-tolyl)methanone (8q). Method B. Compound 8q was isolated
as a yellow oil (121 mg, 22%). 1H NMR (CDCl3, 400 MHz) δ 7.42 (s,
1H), 7.32 (s, 2H), 7.01 (d, J = 7.9 Hz, 2H), 6.76−6.64 (m, 4H), 2.16
(s, 3H), 2.13 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 192.6, 163.8,
140.8, 138.9, 137.3, 133.3, 130.9 (q, J = 33.2 Hz), 130.2 (d, J = 2.6
Hz), 128.4, 128.3, 123.2 (q, J = 272.6 Hz), 119.7 (dt, J = 7.7, 3.9 Hz),
118.9, 116.2, 21.2, 13.3. HR-ESMS calcd for C21H16F6NOS+ (M + H)
444.0851, found 444.0869.
Crystallography. Intensity data for the dimer of compound 8h
was collected with an Oxford Diffraction Sapphire CCD diffractometer
using Cu Kα radiation (graphite crystal monochromator λ = 1.54184
Å). The temperature during data collection was maintained at
130.0(1) using an Oxford cooling device. Data were reduced and
corrected for absorption.30 The structures were solved by direct
methods and difference Fourier synthesis.31 Thermal ellipsoid plots
were generated using the program ORTEP-332 integrated within the
WINGX33 suite of programs.
Crystal Data for the 8h dimer. C38H24F6N2S2, M = 686.71, T =
130.0(1) K, λ = 1.54184, monoclinic, space group P1, a = 8.4415(11),
b = 13.141(2), c = 15.552(2) Å, a = 102.37(1)°, β = 99.43(1)°, γ =
106.33(1)°, V = 1570.2(4) Å3, Z = 2, Dc = 1.452 mg M−3, μ(Cu Kα)
2.128 mm−1, F(000) = 704, crystal size 0.34 × 0.10 × 0.02 mm3, 9065
reflections measured, 5266 independent reflections (Rint = 0.082), the
final R was 0.0581 [I > 2σ(I)] and wR(F2) was 0.1485 (all data).
Biology. Materials. Dulbecco’s modified Eagle’s medium
and hygromycin B were purchased from Invitrogen (Carlsbad,
CA). Fetal bovine serum (FBS) was purchased from
ThermoTrace (Melbourne, VIC, Australia). Adenosine deam-
inase, derived from calf intestine, was purchased from Roche
(Basel, Switzerland). The Sure-Fire cellular ERK1/2 assay kits
were a generous gift from TGR BioSciences (Adelaide, SA,
Australia). AlphaScreen reagents for ERK1/2 and cAMP assays
were from PerkinElmer Life and Analytical Sciences. All other
reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Cell Culture. Flp-In CHO cells stably expressing adenosine A1
receptors (CHO A1R) were cultured at 37 °C in 5% CO2 in DMEM
supplemented with 5% (v/v) FBS, 16 mM HEPES (Stewart et al.,
2009).
cAMP Accumulation Assay. CHO A1R cells were plated into 96-
well plates and cultured overnight at 37 °C in 5% CO2. Cells were
washed with PBS and cultured overnight in serum-free media. Thirty
minutes before assaying, the culture medium was replaced with phenol
red-free DMEM with 0.1% bovine serum albumin (BSA), 1 U·mL−1
ADA, and 500 μM 3-isobutyl-1-methylxanthine, and incubated at 37
°C in 5% CO2. Cells were treated with 10 μM forskolin, R-PIA, and/or
allosteric modulators and incubated for 30 min at 37 °C in 5% CO2 as
per the manufacturer’s instructions. Medium was aspirated, and cells
were lysed in lysis buffer (dH2O, 0.3% Tween 20, 5 mM HEPES, 0.1%
BSA). Lysates were transferred to a 384-well plate, and mixtures of
lysis buffer/donor bead-conjugated anti-cAMP antibody and lysis
buffer/biotinylated cAMP/acceptor bead-conjugated streptavidin were
added to the lysates according to the PerkinElmer cAMP Alphascreen
protocol. Plates were incubated in the dark at room temperature
overnight before the fluorescence signal was measured by use of a
Fusion plate reader (PerkinElmer Life and Analytical Sciences) by use
of standard AlphaScreen settings. Data were normalized to the
response elicited by 10 μM forskolin at the same time point.21
Data Analysis. Computerized nonlinear regression was performed
using Prism 5.03 (GraphPad Software, San Diego, CA). For whole-cell
functional ligand combination studies, the interaction between the
orthosteric agonist R-PIA and the allosteric ligands 8h and 8i was
fitted to the operational model of allosterism and agonism15,28 to
derive functional estimates of modulator affinity, intrinsic efficacy, and
the composite cooperativity on affinity and efficacy together.
Quantitative measures of functional selectivity between agonists for
different signaling assays were estimated using an operational model of
agonism.24,25 This analysis yielded a value of τB/KB for each allosteric
agonist, which was then normalized to the τA/KA value of R-PIA as
follows: ΔLog(τB/KB) = Log(τB/KB)Allo − Log(τA/KA)R‑PIA. Sub-
sequently, the actual bias of each allosteric agonist for the different
signaling pathways was determined by statistically evaluating the
ΔLog(τB/KB) values between the pathways. The ligand bias of an
agonist for one pathway, j1, over another, j2, is given as: Bias =
10ΔΔLog(τ /K )
where ΔΔLog(τB/KB)j1−j2 = ΔLog(τB/KB)j1 −
B
B j1−j2
ΔLog(τB/KB)j2 = LogBF. To account for the propagation of error
associated with the determination of composite parameters, the
following equation was used: Pooled SEM = ((SEM1)2
+
(SEM2)2)1/2
.
AUTHOR INFORMATION
■
Corresponding Author
*For P.J.S.: phone, +61 (0)3 9903 9542; E-mail, Peter.
Scammells@monash.edu For A.C.: phone, +61 (0)3 9903
9067; E-mail, Arthur.Christopoulos@monash.edu
Extracellular Signal-Regulated Kinase 1/2 Phosphorylation
Assays. Initial ERK1/2 phosphorylation time course experiments
were performed to determine the time at which ERK1/2
phosphorylation was maximal after stimulation by each ligand. Cells
were seeded into transparent 96-well plates at 30000 cells per well and
grown for over 8 h. Cells were then washed once with phosphate-
buffered saline (PBS) and incubated in serum-free DMEM at 37 °C
overnight to allow FBS-stimulated phosphorylated ERK1/2 levels to
subside. Before stimulation, cells were treated with 1 U·mL−1 ADA for
30 min. Cells were then stimulated with allosteric modulator for 5 min
prior to the addition of R-PIA for a further 5 min at 37 °C in 5% CO2.
For all experiments, 3% (v/v) FBS was used as a positive control, and
vehicle controls were also performed. The reaction was terminated by
removal of drugs and lysis of cells with 100 μL of SureFire lysis buffer
(TGR Biosciences). The lysates were agitated for 1−2 min and were
diluted at a ratio of 4:1 (v/v) lysate/Surefire activation buffer in a total
volume of 50 μL. Under low-light conditions, a 1:240 (v/v) dilution of
AlphaScreen beads/Surefire reaction buffer was prepared and this was
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by Discovery grant DP110100687
of the Australian Research Council and Program Grant 519461
of the National Health and Medical Research Council
(NHMRC) of Australia. A.C. is a Senior, and PMS a Principal
Research Fellow of the NHMRC. We are grateful to Drs.
Michael Crouch and Ron Osmond, TGR Biosciences, Adelaide,
for generously providing the ERK SureFire Alphascreen kit
reagents.
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Journal of Medicinal Chemistry
Article
(17) Aurelio, L.; Christopoulos, A.; Flynn, B. L.; Scammells, P. J.;
Sexton, P. M.; Valant, C. The synthesis and biological evaluation of 2-
amino-4,5,6,7,8,9-hexahydrocycloocta[b]thiophenes as allosteric mod-
ulators of the A1 adenosine receptor. Bioorg. Med. Chem. Lett. 2011, 21,
3704−3707.
(18) Urban, J. D.; Clarke, W. P.; von Zastrow, M.; Nichols, D. E.;
Kobilka, B.; Weinstein, H.; Javitch, J. A.; Roth, B. L.; Christopoulos, A.;
Sexton, P. M.; Miller, K. J.; Spedding, M.; Mailman, R. B. Functional
selectivity and classical concepts of quantitative pharmacology. J.
Pharmacol. Exp. Ther. 2007, 320, 1−13.
(19) Stallaert, W.; Christopoulos, A.; Bouvier, M. Ligand functional
selectivity and quantitative pharmacology at G protein-coupled
receptors. Expert Opin. Drug Discovery 2011, 6, 811−825.
(20) Bhattacharya, S.; Linden, J. The allosteric enhancer, PD 81,723,
stabilizes human A1 adenosine receptor coupling to G proteins.
Biochim. Biophys. Acta 1995, 1265, 15−21.
ABBREVIATIONS USED
■
2A3BT, 2-amino-3-benzoylthiophenes; A1R, A1 adenosine
receptor; GPCR, G protein-coupled receptor; ERK1/2,
extracellular signal-regulated kinases 1 and 2; DMEM,
Dulbecco’s Modified Eagle Medium; FBS, fetal bovine serum;
R-PIA, N6-(R-phenylisopropyl)adenosine
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■
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