Allosteric modulators of the adenosine A1 receptor: Synthesis and pharmacological evaluation of 4-substituted 2-amino-3-benzoylthiophenes

Article abstract of DOI:10.1021/jm9002582

A series of 4-substituted 2-amino-3-benzoylthiophenes was screened using a functional assay of A1ARmediated phosphorylation of ERK1/2 in intact CHO cells to identify both potential agonistic effects as well the ability to allosterically modulate the activ

Full text of DOI:10.1021/jm9002582

J. Med. Chem. 2009, 52, 4543–4547 4543
DOI: 10.1021/jm9002582
Allosteric Modulators of the Adenosine A1 Receptor: Synthesis and Pharmacological Evaluation
of 4-Substituted 2-Amino-3-benzoylthiophenes
Luigi Aurelio,† Celine Valant,†, ‡ Bernard L. Flynn,† Patrick M. Sexton,†, ‡ Arthur Christopoulos,*,†, ‡ and Peter J. Scammells*,†
†Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville VIC
3052, Australia, and ‡Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology,
Monash University, Clayton VIC 3800, Australia
Received March 2, 2009
A series of 4-substituted 2-amino-3-benzoylthiophenes was screened using a functional assay of A1AR-
mediated phosphorylation of ERK1/2 in intact CHO cells to identify both potential agonistic effects as
well the ability to allosterically modulate the activity of the orthosteric agonist, R-PIA. More detailed
concentration-response experiments were subsequently performed on two compounds (9a and 9o)
utilizing both the ERK1/2 assay as well as a second assay of [35S]GTPγS binding to activated G proteins.
Introduction
substantial detail and were found to be effective AEs
(Figure 1). PD 81,723 (1)5 was the most potent, selective, and
efficacious enhancer with the best ratio of enhancer/antago-
nistic activity. This research also highlighted the importance of
the 2-amino and 3-carbonyl moieties as crucial substituents
whose modification or removal greatly diminished enhancing
activity. Following this work, a number of researchers in the
field have probed the structure-activity relationships (SAR)
of the 2-amino-3-carbonylthiophene core as the basis for the
development of new and improved allosteric enhancers of the
A1AR. In 1999, van der Klein et al.7 synthesized and evaluated
LUF 5484 (4), an analog of PD 117,975 (3), and found it to be
2.4 times more potent than compound 1 while showing
comparable antagonistic activity. An extension of this theme
by Baraldi et al.8 yielded the 3-naphthoyl derivatives 5a-e with
improved potency relative to 1 for increasing radiolabeled
agonist binding to both human and rat A1ARs.
Allosteric modulation of the multimeric ion-channel GA-
BAAreceptor is a validated therapeutic approach that underlies
the well-known mode of action of the benzodiazepines,1 which
act specifically at sites that are topographically distinct from
the endogenous (orthosteric) agonist binding sites. Over the
last two decades, the relatively unexplored concept of allosteric
modulation of G protein-coupled receptors (GPCRsa) has
also grown in interest to the point where it now represents one
of the most exciting areas in modern drug discovery.2 One
GPCR system that is of particular interest with respect to
allosteric modulation is the agonist, adenosine, and its cognate
receptors.3,4 Adenosine is an important tissue-protective agent
that is released during ischemia, hypoxia, or inflammation to
interact with multiple subtypes of GPCRs that can regulate a
variety of effectors including adenylate cyclase and the ERK1/
2 MAP kinases. Efforts to selectively target these receptors
with modified adenosine analogs have resulted in therapeutics
that are limited by side effects due to their ubiquitous nature
and poor receptor subtype selectivity. The potential therapeu-
tic applications of allosteric enhancers (AEs) at the adenosine
A1 receptor (A1AR) are far more enticing than the classical
agonist/orthosteric interactions alone, the idea being that the
AE binds to a site that shows greater sequence divergence
between adenosine receptor subtypes and also preferentially
acts in the presence of elevated levels of endogenous adenosine.
Thus, this approach can lead to greater selectivity in action as a
consequence of both site- and receptor-specific modulation.
In 1990, Bruns et al.5,6 reported that 2-amino-3-aroylthio-
phenes acted as AEs of the A1AR by enhancing the binding
of N6-cyclopentyladenosine, although at higher concentra-
tions of modulator, antagonistic effects were also observed.
In that body of work, three compounds (1-3) were tested in
Tranberg et al.9 further investigated the structure-activity
relationships of 2-amino-3-aroylthiophenes. In that study, the
target compounds were screened using a radioligand dissocia-
tion kinetic assay that measured the ability of the candidate AEs
to stabilize an orthosteric agonist-receptor-G-protein ternary
complex. This research identified a number of 3-aroyl groups
that support allosteric activity, and also suggested the presence
of a second region in the allosteric binding site that interacts
constructively with alkyl substituents in the 4- and 5-positions.
*To whom correspondence should be addressed. For A.C.: phone,
+61 (0)3 9905 3817; fax, +61 (0)3 9905 5953; E-mail, Arthur.
Christopoulos@med.monash.edu.au. For P.J.S.: phone, +61 (0)3
9903 9542; fax, +61 (0)3 9903 9583; E-mail, Peter.Scammells@pharm.
monash.edu.au.
a Abbreviations: AE, allosteric enhancer; AR, adenosine receptor;
GPCR, G protein-coupled receptor; ERK1/2, extracellular signal-regu-
lated kinases 1 and 2.
Figure 1
r
2009 American Chemical Society
Published on Web 06/10/2009
pubs.acs.org/jmc

4544 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Aurelio et al.
Scheme 1a
Figure 2
Lu.tjens et al.10 synthesized a series of 4,5-disubstituted
2-amino-3-benzoylthiophenes and found that compound 7
(Figure 2) was effective and more potent than compound 1 in
the kinetic binding assay described above. This was the first
example of a 5-bromothiophene with AE activity equal to or
greater than compound 1. This finding prompted a broader
study of the structure-activity relationships of the 5-position
of compound 7.11 A series of 5-phenyl moieties with both
electron donating and withdrawing activity were prepared
and evaluated as A1AR allosteric enhancers. The 5-phenyl
analog 8 (R2 = H) was the most potent of the series. However,
the precursor with no substituent in the 5-position (compound
9a) proved to be the most efficacious. The current study
involved the synthesis of analogs of 9a, with attention to the
3- and 4-positions, incorporating different substituents on the
phenyl rings and the use of cell-based assays of A1AR-
mediated signaling in order to validate the functional allos-
teric properties of 9a and its analogs as well as obtaining novel
insights into their modes of action.
a (i) TiCl4, pyridine, CH2Cl2; (ii) sulphur, Et2NH, THF.
20-chloroacetophenone produced only trace amounts (as de-
termined by TLC and NMR) and 20-bromoacetophenone did
not provide any Knoevenagel product 12. This was also the
case for 20-trifluoromethylacetophenone and other 20-substi-
tuted acetophenones larger than chlorine. Thiophenes 9a-p
and 13a-p were isolated and purified by silica gel chroma-
tography and recrystallized from an appropriate solvent.
The yield of the Knoevenagel-Gewald sequence was
modest at best (ranging from 3 to 40%), although adequate
amounts of the desired products were obtained for pharma-
cological evaluation. Improved methodology was sought in
order to facilitate the scale-up and further evaluation of the
compounds of this type. We have recently found that yields
can be improved by using phenacyl bromide or chloride
rather than acetophenone and modified conditions in the
Gewald step (NaSH was used instead of elemental sulfur and
an organic base). In a representative example, compound 9b
was synthesized in this fashion and twice the yield (60%) of
that previously achieved from acetophenone was obtained.
Biological Activity. To assess the biological activity of the
putative AEs, compounds were initially screened using an
AlphaScreen plate-based assay of A1AR-mediated phos-
phorylation of ERK1/2 (pERK1/2) in intact CHO cells.16
For each compound, two concentrations (3 and 10 μM) were
tested alone and against an EC50 concentration of the
orthosteric agonist, R-PIA. Under these conditions, an
increase in functional response represents either an allosteric
enhancement of R-PIA function or intrinsic agonism of the
test compound (or both). An EC50 concentration of R-PIA
was chosen to allow for the detection of either positive of
negative allosteric modulators with limited degrees of co-
operativity.18 It should be noted that the use of only two
concentrations of orthosteric agonist in these initial screens
may fail to detect compounds with more complex profiles of
A1 receptor modulation (e.g., mixed allosteric/orthosteric
modes), but the emphasis of the current study was to follow-
up only on compounds that were unambiguously acting as
potentiators over the concentration ranges utilized. As in-
dicated in Table 1 and Figure 3B, a range of compounds were
identified that demonstrated a substantial functional poten-
tiation of the ability of R-PIA to promote A1AR-mediated
ERK1/2 phosphorylation. In addition, some compounds
clearly displayed agonistic activity in their own right
(Figure 3A). The most robust degree of potentiation exhibited
by a number of the compounds, including 9a, resulted
in approximately 90% or more of the maximum attainable
Results and Discussion
Chemistry. The targeted 3- and 4-substituted 2-aminothio-
phenes were prepared from the appropriately substituted
acetophenone 10 and benzoylacetonitrile 11 via a two-step
procedure that involved a Knoevenagel condensation fol-
lowed by a Gewald reaction (Scheme 1). A one-pot Gewald
procedure was not employed in this study as it is well
documented that aryl ketones are unreactive in this proce-
dure12 and that the common method for preparing these
thiophenes is via the two-step technique by initially forming
the R,β-unsaturated nitriles 12 under Knoevenagel conden-
sation conditions ( β-alanine, AcOH, benzene, or toluene
reflux) and then cyclizing to the thiophene with elemental
sulfur and base.13,14 This approach utilizes the classical
azeotropic removal of water in the condensation step and
in larger scale is quite versatile, whereas in our hands, this
technique fails to produce adequate yields on a smaller scale.
Therefore, the modified version of the condensation step
developed by Lehnert,15 which utilizes titanium(IV) chlor-
ide/pyridine system, provided low to moderate yields of
R,β-unsaturated nitriles 12 that were subsequently cyclized
to the thiophenes 9a-p and 13a-p. Attempts to drive the
condensation step to completion by increasing the amount of
titanium(IV) chloride resulted in lower yields and in some
cases total consumption of acetophenones was observed with-
out sign of intermediate 12, as revealed by TLC and NMR.
It was found that 1 equivalent of titanium(IV) chloride was
sufficient to allow formation of 12, and in the case of forming
9a, utilizingone equivalent of titanium(IV)chloride compared
to two equivalents11 improved the yield by 7% after recrys-
tallization from isopropanol. 20-Substituted acetophenones
were submitted for Knoevenagel condensation, and it was
found that the 20-fluoroacetophenone did condense, whereas

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4545
Table 1. Effect of Test Compounds on A1AR-Mediated Stimulation of
ERK1/2 Phosphorylation in Intact CHO FlpIn Cells in the Presence of
an EC50 Concentration of R-PIA
reference compound 9a and the novel compound 9o, as both
compounds exhibited strong functional potentiation in the
initial screens. As shown in Figure 4, increasing concentra-
tions of either compound caused a significant potentiation of
the potency of R-PIA for mediating either ERK1/2 phos-
phorylation (Figure 4a) or [35S]GTPγS binding to activated
GR protein subunits (Figure 4b). In agreement with the
results of the initial ERK1/2-based screens, a substantial
degree of R-PIA-independent receptor activation was also
noted in both assays in response to the modulators, confirm-
ing that the AEs also behaved as allosteric (partial) agonists
in their own right; similar agonistic effects of A1AR AEs
have been noted in the past17 and are consistent with the
notion that part of the mechanism of action of these com-
pounds is to stabilize an active receptor-G protein ternary
complex. Because complete concentration-response pro-
files were determined for 9a and 9o using the two functional
assays, we were also able to apply a novel operational model
of allosterism18,19 (see Supporting Information) to the data
to derive quantitative estimates of the affinity of each
modulator for the allosteric site on the unoccupied A1AR
(expressed as a pKB; negative logarithm of the dissociation
constant) as well as a measure of the cooperativity (Log Rβ)
exerted on the actions of R-PIA by the modulators. The
latter parameter reflects the magnitude of the allosteric effect
of the modulator on R-PIA affinity (R) and R-PIA signaling
efficacy ( β); under the present experimental conditions,
individual estimates of these parameters could not be
obtained, but the composite cooperativity factor, Rβ, was
determined as a measure of the overall allosteric effect. For
the ERK1/2 experiments, the pKB of 9a was estimated as
6.37 ( 0.16 and Log Rβ as 0.38 ( 0.07 (i.e., Rβ = 2.4-fold
positive cooperativity), whereas for the [35S]GTPγS assay,
the values were pKB = 5.96 ( 0.17 and Log Rβ = 0.84 ( 0.07
(i.e., Rβ = 6.9-fold positive cooperativity). For compound
9o, the parameters were pKB = 5.22 ( 0.07 and Log Rβ
as 1.06 ( 0.08 (i.e., Rβ = 11.5-fold positive cooperativity)
for the stimulation of ERK1/2 phosphorylation and pKB =
5.82 ( 0.27 and Log Rβ = 1.42 ( 0.16 (i.e., Rβ = 26.3-fold
positive cooperativity) for the [35S]GTPγS assay. There was
no significant difference between the two pKB values ( p >
0.05, unpaired t test) for either AE between signaling assays
as would be expected because the parameter reflects the
affinity of the modulator for the allosteric site on the
unoccupied A1AR and should be independent of the assay
used to determine the value. In addition, there was no signifi-
cant difference ( p > 0.05, unpaired t test) between the esti-
mates of positive cooperativity exerted on the signaling of
R-PIA by 9o between the two different functional assays,
indicating that the degree of allosteric potentiation is similar
irrespective of which signaling pathway is investigated. How-
ever, there was a significant difference ( p < 0.05, unpaired
t test) between the estimates of positive cooperativity exerted
on the signaling of R-PIA by 9a between the two differ-
ent functional assays. This suggests that 9a may display
functional selectivity with respect to the degree of allosteric
potentiation it can exert on a given signaling pathway. Such
“stimulus trafficking” by orthosteric compounds is a phe-
nomenon that has gained significant recognition in the
GPCR field in recent years,20 and it is quite possible that
allosteric modulators can also engender this property by
promoting unique receptor conformations that differentially
affect one signaling pathway relative to another.19
structure
activitya
no.
R0
R
3 μM
10 μM
9a
9b
H
H
3-CF3Ph
Ph
2-FPh
83 ( 3
49 ( 1
55 ( 1
51 ( 1
51 ( 1
45 ( 1
46 ( 1
67 ( 9
46 ( 1
51 ( 4
47 ( 2
50 ( 1
41 ( 10
52 ( 4
83 ( 7
55 ( 1
64 ( 8
51 ( 3
70 ( 3
76 ( 6
62 ( 2
58 ( 1
50 ( 1
62 ( 9
45 ( 1
54 ( 8
53 ( 1
47 ( 5
58 ( 3
51 ( 3
69 ( 9
54 ( 4
59 ( 1
48 ( 2
68 ( 3
52 ( 2
60 ( 5
52 ( 3
51 ( 5
76 ( 15
49 ( 2
50 ( 1
50 ( 1
47 ( 2
34 ( 17
52 ( 3
93 ( 8
73 ( 2
73 ( 8
64 ( 11
87 ( 7
90 ( 10
61 ( 10
75 ( 18
50 ( 1
69 ( 2
46 ( 4
61 ( 8
42 ( 7
52 ( 4
60 ( 6
55 ( 3
74 ( 7
67 ( 24
9c
9d
H
H
3-FPh
4-FPh
9e
H
9f
9g
H
3-ClPh
4-ClPh
H
9h
9i
H
3-BrPh
4-BrPh
4-IPh
H
9j
9k
H
H
3-NO2Ph
4-NO2Ph
3-OCH3Ph
4-OCH3Ph
3,5-CF3Ph
2-Naphth
3-CF3Ph
Ph
9l
H
9m
9n
H
H
9o
9p
H
H
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
2-FPh
3-FPh
4-FPh
3-ClPh
4-ClPh
3-BrPh
4-BrPh
4-IPh
3-NO2Ph
4-NO2Ph
3-OCH3Ph
4-OCH3Ph
3,5-CF3Ph
2-Naphth
a Effect of two different concentrations (3 and 10 μM) of substi-
tuted (2-amino-4-phenylthiophen-3-yl)(phenyl)methanones on A1AR-
mediated stimulation of ERK1/2 phosphorylation in intact CHO FlpIn
cells, in the presence of an EC50 concentration of R-PIA (determined on
the same day as each assay). Data represent the mean ( standard
deviation of two experiments conducted in triplicate.
R-PIA response by a concentration of agonist that normally
yields 50% of the maximum response (Table 1, Figure 3). In
general, the series that possessed a 4-chlorobenzoyl group
inthe3-positionof the thiophene (compounds13a-p) showed
slightly higher activity than the corresponding 4-benzoyl
series (9a-p). However, there were exceptions to this trend.
In both series, 3-trifluoromethylphenyl (9a, 13a) and 3,5-di-
trifluoromethylphenyl (9o, 13o) substitution of the 4-position
supported robust activity. In the 4-benzoyl series, 2-fluoro-
phenyl, 4-fluorophenyl, 3-bromophenyl, and 2-naphthyl
substitution (compounds 9c, 9e, 9h, and 9p, respectively)
all augmented the R-PIA response from 50% (in the absence
of compound) to g60% of the maximum attainable
R-PIA response. A wider range of analogs achieved this
level of activity in the 4-chlorobenzoyl series, namely com-
pounds 13a-13f, 13h, 13j, 13m, 13o, and 13p. In addition
to those substituents described above, phenyl, 3-fluoro-
phenyl, 3-chlorophenyl, 4-iodophenyl, and 3-methoxyphenyl
also augmented the R-PIA response to g60% of the max-
imum attainable R-PIA response. A similar trend for activity
was also seen for basal responses to the compounds.
To further validate the results of the initial functional
screen, more detailed experiments were performed using

4546 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Aurelio et al.
Figure 3. Effect of two different concentrations (3 μM, left bar; 10 μM, right bar) of novel 4-substituted 2-aminothiophenes on A1AR-
mediated stimulation of ERK1/2 phosphorylation in intact CHO FlpIn cells, in the 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 experiments conducted in triplicate.
Figure 4. Effect of compounds 9a or 9o on (A) R-PIA mediated stimulation of ERK1/2 phosphorylation in intact CHO FlpIn cells and (B)
R-PIA mediated binding of [35S]GTPγS (100 pM) to activated G proteins in membranes from CHO FlpIn cells stably expressing the human
A1AR. Data points represent the mean ( SEM of three experiments conducted in triplicate. Curves drawn through the points represent the best
global fit of an operational model of allosterism to the data sets.
Conclusion
by targeting an allosteric site on the A1AR, some of these
compounds may also display signaling pathway bias via
allosterically engendering functional selectivity in the actions
of orthosteric agonists at the same receptor.
In summary, a series of 4-substituted 2-amino-3-ben-
zoylthiophenes and 2-amino-3-(4-chlorobenzoyl)thiophenes
were prepared using a two-step sequence involving a Knoe-
venagel condensation and Gewald reaction. An initial screen
supplemented by more comprehensive functional assays iden-
tified and validated novel 4-substituted 2-aminothiophenes as
potent functional AEs of A1AR-mediated signaling. In addi-
tion to a potential for exhibiting greater selectivity of action
Experimental Section
General Procedure for the Preparation of Compounds 9a-p
and 13a-p (Method A). The appropriate benzoylacetonitrile 11
(3 mmol) and acetophenone 10 (3.3 mmol) were dissolved in
dry dichloromethane (12 mL) in a two-neck flask in an N2

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4547
atmosphere and cooled to 0 °C with an ice bath. To the cooled
solution was added dropwise neat TiCl4 (3.3 mmol, 362 μL).
After approximately 10-30 min, dry pyridine (215 μL) was
added dropwise and the ice bath was removed. After 1 h, a
further aliquot of dry pyridine (644 μL) was added dropwise and
left to stir at room temperature overnight. The mixture was
diluted with 2 M HCl (30 mL) and the organic phase separated.
The aqueous phase was extracted with CH2Cl2 (2 ꢀ 30 mL), and
the combined organics were washed with water and then brine,
dried over MgSO4, filtered, and concentrated to a resin. The
resin was taken up in THF (6 mL), and elemental sulfur
(3.3 mmol, 106 mg) was added, followed by Et2NH (610 μL),
and stirred at room temperature for 18 h. The mixture was
diluted with ethyl acetate and washed with water (ꢀ2) and then
brine, dried over MgSO4, filtered, and concentrated to a resin.
The resin was chromatographed on silica gel eluting with
10-30% ethyl acetate, petroleum ether (40-60 °C), providing
after concentration of the appropriate fractions a solid that was
recrystallized from isopropyl alcohol. Compounds 9k-l and
13k-l were recrystallized from ethyl acetate, and compounds
9o and 13o were recrystallized from petroleum ether (40-60 °C).
Analytical HPLC was conducted on a Waters 2690 instrument
with 996-diode array detector (chromatograms show UV ab-
sorbance at 254 nm) on a Phenomenex C8 (5 μm, 150 ꢀ 4.6 mm2)
column. All compounds were of g98% purity.
Alternative Procedure for the Preparation of Compound 9b
(Method B). Benzoylacetonitrile 11 (0.94 mmol) and phenacyl
bromide (1.03 mmol) were dissolved in dry CH2Cl2 (4 mL) in a
two-necked flask fitted with a rubber septum and nitrogen inlet.
The mixture was cooled with an ice-water bath, and neat TiCl4
(206 μL) was added dropwise. After 0.5 h, dry pyridine (67 μL) was
added dropwise and the ice-water bath was removed. After
stirring a further 1 h, another aliquot of dry pyridine (200 μL)
was added dropwise and the reaction mixture left to stir overnight.
The reaction mixture was partitioned between 2 M HCl (50 mL)
and CH2Cl2 (20 mL). The phases were separated, and the aqueous
layer was extracted with DCM (2 ꢀ 20 mL). The combined organic
phases were washed with water and finally brine, dried (MgSO4),
filtered, and concentrated under reduced pressure to give the E/Z
mixture of olefins as an amber resin or oil. The crude mixtures were
used directly in the next reaction without purification.
The E/Z mixture was dissolved in EtOH (5 mL) and cooled to
-78 °C (MeOH/dry ice) and a solution of NaSH (2.0-
2.2 mmol) in EtOH (8 mL) was added dropwise over 30-60 s.
The reaction mixture was left to stir while the MeOH/dry ice-
bath was kept in place, but no further additions of dry ice were
made. The MeOH/dry ice-bath slowly warmed up to 10 °C
(approximately 1 h), and a small amount of precipitate was
observed. The mixture was diluted with CH2Cl2 and while
stirring 2 M HCl (20 mL) was added slowly with gas evolution
occurring (CAUTION! H2S is generated, which is highly toxic!).
The organic layer was separated and the aqueous layer extracted
with CH2Cl2. The aqueous layer was treated with house hold
bleach to oxidize residual H2S. The combined organics are dried
(MgSO4), filtered and concentrated under reduced pressure in
a fume cupboard, to a solid. The solid is recrystallized with
isopropyl alcohol, providing 9b as a yellow powder (157 mg,
60% yield). This material was identical in all respects to 9b as
synthesized via method A above.
Supporting Information Available: Full details of the product
purity and characterization and the pharmacological evalua-
tion. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Acknowledgment. This research was supported by Discovery
Grant DP0558184 of the Australian Research Council and Pro-
gram Grant 519461 of the National Health and Medical Research
Council (NHMRC) of Australia. A.C. is a Senior Research
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Note Added after ASAP Publication. This paper was
published June 10, 2009 with an error in Figure 1. The revised
version was published on June 15, 2009.