Highly potent and selective fluorescent antagonists of the human adenosine A3 receptor based on the 1,2,4-triazolo[4,3-a]quinoxalin-1-one scaffold

Article abstract of DOI:10.1021/jm201722y

The adenosine-A₃ receptor (A₃AR) is a G protein-coupled receptor that shows promise as a therapeutic target for cancer, glaucoma, and various autoimmune inflammatory disorders, and as such, there is a need for molecular probes to study this receptor. Here, we report a series of fluorescent ligands containing different linkers and fluorophores based around a 1,2,4-triazolo[4,3-a]quinoxalin-1-one antagonist. One of these conjugates (19) displayed high affinity for the A₃AR (pK_D = 9.36 ± 0.12) and is >650-fold selective over other adenosine receptor subtypes. Confocal microscopy revealed clear, displaceable membrane labeling of CHO-A ₃ cells with 19, with no detectable labeling of CHO-A₁ cells under identical conditions. This fluorescent ligand was also able to specifically label the A₃AR in HEK293T cells containing a mixed adenosine receptor population. The subtype specificity, along with its excellent imaging properties, make 19 an ideal tool for studying A₃AR distribution and organization, particularly in the presence of other adenosine receptor subtypes.

Full text of DOI:10.1021/jm201722y

Article
pubs.acs.org/jmc
Highly Potent and Selective Fluorescent Antagonists of the Human
Adenosine A3 Receptor Based on the 1,2,4-Triazolo[4,3-a]quinoxalin-
1-one Scaffold
Andrea J. Vernall,† Leigh A. Stoddart,‡ Stephen J. Briddon,‡ Stephen J. Hill,*,‡ and Barrie Kellam*,†
†School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, United Kingdom
‡Institute of Cell Signalling, School of Biomedical Science, Queen's Medical Centre, University of Nottingham, United Kingdom
ABSTRACT: The adenosine-A3 receptor (A3AR) is a G protein-
coupled receptor that shows promise as a therapeutic target for cancer,
glaucoma, and various autoimmune inflammatory disorders, and as
such, there is a need for molecular probes to study this receptor. Here,
we report a series of fluorescent ligands containing different linkers and
fluorophores based around a 1,2,4-triazolo[4,3-a]quinoxalin-1-one
antagonist. One of these conjugates (19) displayed high affinity for
the A3AR (pKD = 9.36 0.12) and is >650-fold selective over other
adenosine receptor subtypes. Confocal microscopy revealed clear,
displaceable membrane labeling of CHO-A3 cells with 19, with no
detectable labeling of CHO-A1 cells under identical conditions. This
fluorescent ligand was also able to specifically label the A3AR in
HEK293T cells containing a mixed adenosine receptor population. The
subtype specificity, along with its excellent imaging properties, make 19 an ideal tool for studying A3AR distribution and
organization, particularly in the presence of other adenosine receptor subtypes.
particular, for colon cancer, the A3AR has been proposed as a
diagnostic marker for tumorigenesis and disease progression.11
With the increasing importance of the A3AR as a therapeutic
target comes a requirement for biological tools to study this
receptor. To fill this need, herein, we report a highly potent and
selective fluorescent antagonist (19) that can specifically bind
to the A3AR in a cell line that endogenously expresses multiple
AR subtypes.
Using fluorescence as a means to study GPCRs allows access
to a large range of pharmacological techniques that can capture
dynamic processes involving unmodified receptors in live
cells.12 In particular, fluorescently labeled ligands that act as an
agonist or antagonist have been developed to target many
GPCRs.12,13 Visualization of a GPCR using a fluorescent ligand
is a very sensitive technique, and measurements can be made at
the single cell level; therefore, large numbers of cells are not
required. Through the use of confocal microscopy, a fluorescent
ligand can give real-time spatial information about dynamic
protein−protein interactions, trafficking, localized expression
level, and the mobility of the receptor. This is important as
GPCRs are not uniformly distributed on the cell membrane but
are organized into microdomains such as membrane rafts and
caveolae.14 Information about receptor mobility and its
conformation can also be studied, for example, by using
fluorescent correlation spectroscopy.15 Fluorescent ligands also
offer advantages over radioligands in competitive binding
INTRODUCTION
■
G protein-coupled receptors (GPCRs) are the largest family of
transmembrane signaling proteins in the human genome and
are estimated to be the target of around 40% of all currently
marketed drugs. Structurally, GPCRs consist of seven trans-
membrane spanning helices linked via intra- and extracellular
loop regions, and their cell signaling is critical to a vast array of
physiological processes.1 Within the GPCR superfamily, there
are four adenosine receptors (AR) that bind and are activated
by the purine nucleotide. There are four characterized AR
subtypes, the A1AR, A2AAR, A2BAR, and the larger A3AR. The
A3AR is the least characterized of the receptor subtypes and is
more commonly found in peripheral tissues such as lung and
liver, yet also in immune cells such as mast cells and
neutrophils.2
The A3AR is increasingly being implicated in many biological
processes and diseases, registering this receptor as a therapeutic
target worthy of further study,3,4 with both agonist and
antagonist A3AR ligands showing potential as therapeutic
agents and diagnostic tools.5 For example, A3AR agonists have
been evaluated for the treatment of autoimmune inflammatory
diseases such as rheumatoid arthritis, psoriasis, and dry-eye
disease6 and for liver cancer.7 A3AR activation by a subtype-
selective agonist has also been shown to have a cardioprotective
effect in ischemic myocardium.8 Receptor inactivation by an
A3AR antagonist is being considered as a treatment for
glaucoma.9 Several tumor cell lines have been shown to
contain elevated levels of A3ARs, with the expression level
thought to correlate to the tumor stage and severity.10 In
Received: December 21, 2011
Published: January 25, 2012
© 2012 American Chemical Society
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assays,16 as there are health risks and legal and disposal costs
associated with radioactivity. In addition, there is currently no
commercially available radiolabeled antagonist for the A3AR.
There are examples of fluorescently labeled ligands that have
been developed as biological probes for the ARs.17,18 Xanthine-
based antagonists have been functionalized as fluorescent
ligands19,20 but were poorly selective between the AR subtypes.
There are several examples of fluorescent antagonists that are
selective for the A2AAR,21,22 but here, we report the first
example of a fluorescent antagonist that is selective for the
human A3AR. To study the A3AR in primary human cell lines, it
is essential that a molecular probe is exquisitely subtype
selective, as nearly every mammalian cell expresses one or more
of the AR subtypes.
There are several factors that must be considered when
appending a fluorophore to a relatively small pharmacophore,
as opposed to a peptide or protein. Both the linker type and
length that separates the pharmacophore from the fluorophore
and the fluorophore itself can affect the pharmacology of the
final conjugate.20 The linker must be attached to the
pharmacophore in a position that is relatively insensitive to
structural modification and that can tolerate bulky substituents.
For a GPCR probe, the physiochemical properties of the final
conjugate must be considered to minimize nonspecific
membrane binding and/or prevent high intracellular ligand
accumulation. Additionally, from an imaging perspective, the
choice of fluorophore based on the adsorption/emission profile
should be matched to the end biological application of the
fluorescent ligand. In this study, we selected fluorophores that
emit at a wavelength of 550−650 nm, as this does not overlap
with cellular autofluorescence and allows colocalization studies
with receptors tagged with fluorescent proteins (for example,
YFP and GFP) to be carried out. The boron dipyrromethene
scaffold has proven particularly useful in many biological
imaging studies due to the excellent stability and photophysical
properties of this fluorophore.23
common intermediate. Our goal therefore was not to further
optimize the quinoxalin-1-one scaffold as an antagonist but
rather to produce a fluorescent ligand that displayed a
maintained potency and selectivity profile to the parent drug
molecule.
RESULTS AND DISCUSSION
■
Synthesis. Drawing upon previous successes, we sought to
develop congeners equipped with both short and long linker
variants of differing physiochemical properties. For the linear
hydrocarbon and polyether variants, monoprotected diamines 4
and 5 were acylated with succinic anhydride to afford amides 6
and 7 in excellent yields (Scheme 1). Quinoxalin-1-one 125 was
then coupled to either 6 or 7 in the presence of 2-(1H-7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronoium hexafluoro-
phosphate (HATU)/1-hydroxy-7-azabenzotriazole (HOAt) in
DMF to afford the protected congeners 10 and 11, respectively.
tert-Butyl carbamate (Boc)-Gly-OH (8) was coupled to 1 to
afford 12, while coupling the quinoxalin-1-one with benzyl
carbamate (CBZ)-protected 4-amino benzoic acid (9)
generated 13. The Boc protecting groups of 10, 11, and 12
were subsequently removed using trifluoroacetic acid to give
14, 15, and 16, respectively. The Cbz protecting group of 13
was removed by hydrogenolysis using 10% palladium-on-
carbon and hydrogen to afford the 4-amino benzoyl derivative
17.
The quinoxalin-1-one congeners (14−17) were then coupled
to a range of commercially available fluorescent probes
(Scheme 2). 6-(((4,4-Difluoro-5-(2-thienyl)-4-bora-3a,4a-
diaza-s-indacene-3-yl)styryloxy)acetyl)amino hexanoic acid
(BODIPY-X-630/650)-succinimidyl ester (SE) was reacted
with amines 14 and 15 to give conjugates 19 and 18,
respectively. The polyether congener (14) was additionally
coupled to 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-
diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid
(BODIPY-X-TR)-SE, 3H-indolium,2-[5-[1-[6-[(2,5-dioxo-1-
pyrrolidinyl)oxy]-6-oxohexyl]-1,3-dihydro-3,3-dimethyl-5-
sulfo-2H-indol-2 ylidene]-1,3-pentadien-1-yl]-1-ethyl-3,3-di-
methyl-5-sulfopotassium salt (Cy5-X)-SE, 6-(tetramethylrhod-
amine-5-(and-6)-carboxamido)hexanoic acid (TAMRA-5/6-X)-
SE, and 5-TAMRA-polyethylene glycol (PEG)-SE to afford the
fluorescent derivatives 20−23. Attempts to couple the
succinimidyl activated fluorophores to either 16 or 17 were
regrettably unsuccessful, with both of these two congeners
suffering from very poor organic solvent solubility; however,
the extended linkers housed on congeners 14 and 15 helped
obviate this solubility issue. All fluorescent ligands were initially
isolated and purified using preparative reversed-phase high-
pressure liquid chromatography (RP-HPLC); their final purity
was confirmed using RP-HPLC with dual wavelength detection
with the compound identity subsequently confirmed by HRMS
(TOF ES+). The purities of all compounds subsequently tested
in biological systems were determined as being ≥95%.
A variety of chemical structures have been discovered that
show selective A3AR antagonism.24 We chose trizoloquinoxalin-
1-one 1,25 a selective A3AR antagonist, as the pharmacophore
starting point for congener assembly (Figure 1). Lenzi and co-
Figure 1. Previously reported 1,2,4-triazolo[4,3-a]quinoxalin-1-one
derivatives.25,26
workers have shown that a 4-methoxy group on the 2-phenyl
ring and acylation of the 4-amino group instill a degree of A3AR
subtype selectivity to this compound family, for example, the 4-
benzamido (2) (Ki = 2.9 0.13 nM) and 4-acetamido (3) (Ki
= 35.7 2.40 nM) derivatives (Figure 1).26 In addition, further
structure−activity relationships have demonstrated that re-
ceptor affinity can be maintained when bulky functional groups
are added to the 4-amino group of 1.27 Consequently, acylation
of this 4-amino group was chosen as the method for linker
attachment, meaning 1 could be readily synthesized as a
Pharmacology: Functional Studies. To initially address
whether this novel series of fluorescent ligands were able to
antagonize agonist-stimulated responses at the A3AR, we
evaluated their capacity to attenuate functional activity in
Chinese hamster ovary (CHO) cells, which expressed both the
target receptor alongside a six cyclic adenosine monophosphate
(cAMP) response element (6 CRE) promoter driving the
expression of a human-secreted placental alkaline phosphatase
reporter gene.28 All of the quinoxalin-1-one congeners,
alongside their respective fluorescent conjugates, were able to
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a
Scheme 1. Synthesis of Quinoxalin-1-one Congeners
a
Reagents and conditions: (i) Succinic anhydride, CHCl3, 0 °C and then 12 h room temperature, 82−95%. (ii) HATU, HOAt, N,N-
diisopropylethylamine, DMF, carboxylic acid (6, 7, 8, or 9), 18 h, 10−67%. (iii) For 10−12, trifluoroacetic acid, CH2Cl2, 1 h, quantitative; for 13,
10% Pd/C, H2, MeOH/H2O/acetic acid, 3 h, quantitative.
antagonize the adenosine-5-N-ethylcarboxamide (NECA)-
mediated agonist response at the human A3AR (Table 1),
and of the fluorescent ligands tested, 19 and 20 displayed the
highest affinity. Comparison of the literature binding affinities
of the known antagonists 1−3 to our novel linker-modified
ligands revealed that acyl extension from the 4-amino position
of 1 was an amenable strategy for congener assembly (Table 1).
The polyether variant 14 (pKD = 7.68 0.25; Table 1) proved
to be the worst of the linker-modified compounds, yet still
maintained a binding affinity similar to the native drug (1)
(pKD = 7.43 0.13).25 The binding affinity of aniline 17 was
reduced 40-fold as compared to the benzoyl equivalent (2),
demonstrating that introduction of a p-amino group is not
advantageous as a solitary chemical maneuver. The two most
potent congeners were glycyl derivative 16 (pKD = 8.84 0.16;
Table 1) and aminooctanoyl derivative 15 (pKD = 9.48 0.16;
Table 1). The former displayed a small affinity enhancement
over the simple acetamido analogue 3 and a similar affinity to 2,
whereas the latter displayed a greater affinity than both 3 and 2.
The binding affinities of 18−23 varied about 300-fold,
demonstrating that the fluorophore exerts a major influence on
the final pharmacology of the conjugate. We have previously
established how the fluorophore component of both xanthine
amine congener (XAC)- and NECA-based conjugates greatly
influences the ultimate binding affinities at the A1AR.20 The
C8-linked conjugate 18, which contained the BODIPY 630/
650-X moiety, displayed a moderate affinity at the A3AR (pKD
= 7.78 0.16); however, this represented a 40-fold reduction
in affinity when compared to the precursor congener 15. Five
fluorescent ligands (19−23) were synthesized from PEG-linked
14, of which the BODIPY 630/650-X variant (19) showed
excellent affinity (pKD = 9.36 0.12) at the A3AR, acting as a
competitive antagonist (Figure 2a). This was extremely
rewarding, since this was an increase on the already high
affinity of the benchmark quinoxalin-1-one 2 and suggested that
the BODIPY 630/650 scaffold must be contributing in a
favorable way to the binding affinity of 19, as it was significantly
better than the PEG-linked congener 14. However, the
interplay of contributing factors is complicated; if it were
simply the BODIPY 630/650 that was responsible for the
increase in affinity, then one would not unreasonably expect the
binding affinity of C8-linked 18 to also be better than the
corresponding congener 15. There is a 40-fold difference in
binding affinity between conjugates 18 and 19 that differ only
by the replacement of two methylenes with an ether oxygen,
indicating that the linker is also contributing to the overall
conjugates pharmacology. Comparing the congener−conjugate
pairs, there is an 80-fold increase in affinity going from the
PEG-linker compound 14 to the corresponding fluorescent
conjugate 19, while from the analogous C8-linker 15 to C8
fluorescent conjugate 18, there is a overall 40-fold decrease in
binding affinity.
PEG-linked conjugate 20, which contained the BODIPY-TR
fluorophore, also acted competitively as a high-affinity
antagonist at the A3AR (pKD = 8.47 0.16, Figure 2b). The
only structural difference between 19 and 20 is that the former
contains an ethenyl spacer between the borondipyrromethane
scaffold and the connecting phenyl group; yet, this imparts an
8-fold difference in activity. This would suggest that the
BODIPY component of 18, 19, and 20 is likely engaging in
specific interactions with the A3AR and is not simply acting as a
membrane anchor or protruding unbound into the extracellular
environment. Replacing the BODIPY 630/650 of 19 with a
Cy5 fluorophore to give 21 resulted in a 300-fold reduction in
A3AR affinity. This reiterates the large contribution that the
fluorophore has to conjugate binding, as 19 and 21 contain the
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a
Scheme 2. Synthesis of Fluorescent A3AR Antagonists
a
Reagents and conditions: (i) Fluorophore-SE, N,N-diisopropylethylamine, DMF, 22−60% yield after RP-HPLC purification.
Table 1. Binding Affinities of 1,2,4-Triazolo[4,3-
a]quinoxalin-1-one Derivatives at Human A3AR
TAMRA and the quinoxalin-1-one core improved overall
binding.
The two highest affinity fluorescent conjugates 19 and 20
were also assayed for their ability to antagonize the A1 and A2B
ARs (Table 2 and Figure 2). Affinity values at the A1AR were
measured in CHO cells expressing the human form of the
A1AR receptor, where an agonist-stimulated increase in
intracellular calcium was measured, and the ability of a single
concentration of 19 or 20 to shift these NECA concentration
response curves was analyzed. As with the A3AR, both 19 and
20 acted as competitive A1AR antagonists but with appreciably
lower affinities (Figure 2c,d). This revealed that 19 has a 650-
fold increase in affinity and 20 a 50-fold higher affinity at the
A3AR over the A1AR. Affinity values at the A2BAR were
measured in human embryonic kidney 293 (HEK293) cells that
endogenously express the human A2BAR and coexpressed the
Glosensor cAMP biosensor. HEK293 cells were again
stimulated with the AR agonist NECA, and the resulting
increase in cAMP was measured. The shifts in the agonist
concentration−response curves in the presence of 19 or 20
were used to estimate the affinity at the A2BAR. At relatively
high concentrations (1 mM), both 19 and 20 were unable to
cause a significant shift in the NECA concentration response
curve, indicating very low affinity at the A2B AR.
a
compd
hA3AR pKD
n
14
15
16
17
18
19
20
21
22
23
7.68 0.25
9.48 0.16
8.84 0.16
7.42 0.23
7.78 0.16
9.36 0.12
8.47 0.16
6.83 0.05
7.53 0.11
8.25 0.16
9
3
6
8
4
6
4
3
3
3
a
pKD values were obtained from Gaddum shifts of FSK-stimulated
CRE-SPAP responses to NECA in CHO cells expressing the human
A3AR. Values are means SEMs for n separate experiments.
same pharmacophore and linker. Conjugate 22, which
contained the charged TAMRA moiety, showed similar activity
to precursor 14 and displayed significantly higher affinity than
the charged Cy5 conjugate 21. To study the effect of moving
the charged fluorophore component of the ligand further away
from the quinoxalin-1-one core, the extended PEG-linked 23
was synthesized. Conjugate 23 was 5-fold more active than 22,
indicating that in this case an increased distance between the
Confocal Microscopy. While an excellent binding affinity
and A3AR subtype selectivity is a key prerequisite for a high-
quality fluorescent ligand, this does not necessarily translate
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Figure 2. Functional analysis of fluorescent ligands 19 and 20 at A3AR and A1AR. (a and b) A3AR-expressing CRE-SPAP cells were stimulated with 1
■
●
▲
▼
μM FSK and increasing concentrations of the agonist NECA ( ). The ability of 10 nM ( ), 100 nM ( ), and 1 μM ( ) 19 (a) or 20 (b) to inhibit
the NECA response was analyzed. Data are normalized to basal (in the absence of FSK) and 1 μM FSK SPAP production. (c and d) A1AR-
●
expressing CHO cells were stimulated with increasing concentrations of NECA, and the resulting increase in calcium was measured ( ). The ability
of 1 μM ( ) 19 (c) and 20 (d) to inhibit the NECA response was analyzed. The data shown represent the mean SEM of eight (a and c), four (b),
■
and six (d) experiments performed in triplicate.
Table 2. Binding Affinities of Fluorescent 1,2,4-Triazolo[4,3-
a]quinoxalin-1-one Conjugates 19 and 20 at Human A3, A1,
and A2B ARs
furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acet-
amide (MRS1220), a nonfluorescent selective A3AR antagonist,
the specific-membrane fluorescence was substantially reduced
in cells treated with 19, although low levels of membrane-
localized fluorescence was still present in cells treated with 20,
indicating that the majority of the membrane fluorescence
observed is specific labeling of the A3AR. The ability of 19 and
20 to bind to CHO-A1 cells was also examined using confocal
microscopy, to see if the difference in measured binding affinity
at A1AR is reflected in the degree of observed ligand−receptor
binding. Low levels of fluorescence were observed when CHO-
A1 cells were treated with 25 nM (Figure 3a) and even up to
100 nM (data not shown) of 19, and there was very little
membrane localization of the fluorescence. Pretreatment of the
CHO-A1 cells with the nonfluorescent A1AR selective
antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) before
addition of 19 had little effect on the location and level of the
observed fluorescence. This indicated that, at these concen-
trations, binding of 19 to the A1AR was negligible. In line with
the functional selectivity data, treatment of CHO-A1 cells with
25 nM 20 (Figure 3b) resulted in a small amount of localized
membrane fluorescence, which could only be partially displaced
when the cells were pretreated with the A1AR antagonist
DPCPX. This suggests that 20 has some nonspecific binding to
CHO cell membranes, but in cells expressing A3AR, the levels
of specific binding are high enough to allow it to be easily
distinguished from nonspecific binding.
a
b
c
compd
hA3AR pKD
hA1AR pKD
hA2BAR pKD
n
19
20
9.36 0.12
8.47 0.16
6.56 0.1
>6
>6
6
4
6.78 0.13
a
Measured by the shift of NECA-mediated inhibition of FSK-
stimulated CRE-SPAP response in CHO cells expressing the human
b
A3AR. Measured by inhibition of increases in calcium induced by
c
NECA in CHO cells expressing the human A1AR. Measured by the
inhibition of increase in cAMP induced by NECA as determined by a
cAMP biosensor in HEK293 cells that endogenously express the
human A2BAR.
into an effective biological probe for optical-based experiments.
The physiochemical and photochemical properties of the ligand
must also be suitable; the final conjugate must bind to the
A3AR with little or no nonspecific membrane binding and
should not diffuse into the cell cytosol in large amounts.
Fluorescent ligands 19 and 20 were examined using confocal
microscopy for their ability to bind to CHO cells stably
expressing either the human A3AR or A1AR (Figure 3).
The confocal microscope images of fluorescent ligands 19
and 20 with CHO-A3 cells (Figure 3a,b) show localized
membrane fluorescence and very little intracellular fluores-
cence. When cells were pretreated with N-[9-chloro-2-(2-
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Figure 3. Visualization of the binding of (a) 19 and (b) 20 on CHO cells expressing A3AR (left-hand panels) or A1AR (right-hand panels). In each
case, the upper row of images shows a confocal image, while the bottom row shows the transmitted light image of the same field of cells. In all
conditions, cells were incubated for 30 min at 37 °C in the presence or absence of 100 nM of either MRS1220 or DPCPX followed by a 10 min
incubation with a 25 nM concentration of the required fluorescent conjugate. Single equatorial confocal images were then obtained in the continued
presence of the fluorescent ligand and/or unlabeled antagonist. For each fluorescent ligand, images were obtained using identical settings for laser
power and detector offset and gain, as described in the Experimental Section. Images shown are from a single experiment representative of 3−5
performed.
Selectivity of fluorescent probes for the A3AR is particularly
important when attempting to observe and measure the
receptor in native systems, where it is often expressed along
with the A2A or A2BAR subtypes. We therefore investigated the
ability of 19 to bind specifically to the A3AR in the presence of
A2B receptors using HEK293 cells, which express the A2BAR
endogenously.29 A mixed population of these cells transfected
to express the human A3AR, tagged on its N terminus with the
sig-SNAP-tag,30 were incubated with 19 (5 nM) in the presence
and absence of the A3AR antagonist MRS1220 (Figure 4).
As depicted in Figure 4, clear membrane binding of 19 was
seen in HEK293 cells that expressed the sigSNAP-A3AR. Cells
in the same field of view that did not express the receptor
(examples indicated in Figure 4 by the black arrow) showed no
membrane labeling, indicating specific labeling of the A3AR in
these experiments. Such nonexpressing cells did, however, show
some intracellular accumulation of ligand. Specificity of labeling
was further confirmed in cells that had been pre-exposed to the
nonfluorescent, high-affinity, A3AR-selective antagonist,
MRS1220 (100 nM), where membrane binding from A3AR-
expressing cells was clearly displaced. Similar experiments using
the A2BAR-selective antagonist 8-[4-[4-(4-chlorophenzyl)-
piperazide-1-sulfonyl)phenyl]]-1-propylxanthine (PSB603)
(100 nM) indicated no displacement of the binding of 19
from cells expressing the A3AR or any changes in binding in
cells not expressing the A3AR (i.e., only the A2BAR). Together,
these results indicate that 19 will be of particular use in labeling
the endogenous A3AR in native tissues or cells, where
coexpression of A2BAR is a complicating factor.
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Figure 4. Binding of 19 to HEK293 cells expressing the sigSNAP-A3AR. A mixed population of HEK293 cells expressing the SNAP-tagged A3AR
was incubated with 19 (5 nM, 10 min, room temperature) in the absence (top) or presence of MRS1220 (middle) or PSB603 (bottom; both 100
nM, 30 min, 37 °C). Single equatorial images were taken showing the BG-AlexaFluor488-labeled A3AR (green) and 19 (red). Colocalized pixels of
ligand and A3AR (as determined by colocalizaiton analysis) are shown in white, as well as the fluorescent image overlaid onto a transmitted light
image of the same cells. The black arrow indicates example cells in which there is little or no A3AR expression. Data shown are representative of
images taken in four independent experiments, with all images taken using identical settings for laser power, gain, and offset in both channels.
that designing a fluorescent conjugate with greater potency
than the parent pharmacophore is an achievable outcome. At
this juncture, molecular modeling studies have not yet been
undertaken, as we believe binding interactions between the
large and highly flexible 19 and an A3AR homology model itself
based on either rhodopsin32 or the recently published
A2AAR33−35 crystal structures may not be very informative,
especially beyond the proposed quinoxalin-1-one binding
pocket.27
Fluorescent conjugate 19 could, however, be used in the
wider scientific community to identify and study the A3AR in
live cells that are involved in biological processes of interest.
For example, 19 could be used to study the change in
expression levels of A3AR in disease states such as certain
cancers, colocalization with other fluorescently tagged receptor
types, and enable visualization of cell−cell interfaces where the
A3AR may be involved in cell signaling. In addition to an A3AR
identification tool, 19 could also be used in fluorescent
correlation spectroscopy experiments or as a fluorescence
ligand in a competitive binding assay to replace the more
traditional radioligand-based approach.
CONCLUSIONS
■
In this study, we have shown that the quinoxalin-1-one-based
A3AR antagonist 1 can be modified to include a linker and
fluorophore, and for conjugate 19, results in a fluorescent
molecule displaying enhanced A3AR binding affinity when
compared to the native drug molecule, while maintaining
excellent A3AR subtype selectivity. As with our previously
reported fluorescent ligands, the BODIPY moiety proved to be
the superior fluorophore among our triazoloquinoxalin-1-one
conjugates 18−23. On the basis of selectivity and structure−
activity relationships of the pharmacophore-linkers as compared
to the final conjugates, the BODIPY moiety of 19 may be
favorably interacting with different receptor residues as
compared to the presumably orthosteric binding pocket
occupied by the quinoxalin-1-one core. This could account
for the very high A3AR affinity of 19. A similar hypothesis has
previously been proposed by Tahtaoui et al., who identified
Rhodamine and BODIPY-labeled pirenzepine antagonists of
the human muscarinic M1 receptor.31 They suggested that there
are different binding sites within the muscarinic M1 receptor
that accommodate the fluorophore and pirenzepine, with
kinetic analysis revealing that fluorescent ligand dissociation is
sensitive to treatment with a known allosteric modulator.31 The
possibility that 19 is interacting via a binding site extended from
the classic orthosteric pocket is intriguing and certainly
warrants further investigation. This study further demonstrates
EXPERIMENTAL SECTION
■
Chemistry: Materials and Methods. Chemicals and solvents of
an analytical grade were purchased from commercial suppliers and
used without further purification. BODIPY-630/650-X-SE, BODIPY-
1777
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Journal of Medicinal Chemistry
Article
TR-X-SE, and TAMRA5/6-X-SE were purchased from Molecular
Probes (Invitrogen, United Kingdom). Cy5-X-SE was purchased from
GE Healthcare UK Ltd. 5-TAMRA-PEG-SE was purchased from
Cambridge Bioscience. Unless otherwise stated, reactions were carried
out at ambient temperature. Reactions were monitored by thin-layer
chromatography on commercially available precoated aluminum-
backed plates (Merck Kieselgel 60 F254). Visualization was by
examination under UV light (254 and 366 nm) or staining with
KMnO4 dip. Flash chromatography was performed using Merck
Kieselgel 60, 230−400 mesh (Merck KgaA, Darmstadt, Germany) on a
Biotage Flashmaster II system. NMR spectra were recorded on a
(m, 2H, ArH), 7.87 (m, 2H, ArH), 10.15 (s, 1H, NH), 12.66 (br s, 1H,
CO2H).
General Procedure B: Synthesis of 4-Amido-2-(4-methox-
yphenyl)-1,2,4-triazolo[4,3-a]quinoxalin-1-one Derivatives
(10−13). The appropriate carboxylic acid (2 equiv), HATU (2
equiv), HOAt (2 equiv), and DIPEA (4 equiv) were dissolved in DMF
(1 mL). This solution was added to a solution of 4-amino-2-(4-
methoxyphenyl)-1,2,4-triazolo[4,3-a]quinoxalin-1-one (1)25 (1 equiv)
in DMF (2 mL). The mixture was stirred at 50 °C for 6 h and then
treated with a solution of the carboxylic acid (2 equiv), HATU (2
equiv), HOAt (2 equiv), and DIPEA (4 equiv) in DMF (1 mL). The
mixture was stirred at 50 °C for a further 12 h. Evaporation of the
solvent under reduced pressure gave a residue that was purified using
silica flash column chromatography, eluting with 1−8% MeOH/DCM.
tert-Butyl N-2-{2-[2-(3-{[2-(4-Methoxyphenyl)-1-oxo-1H,2H-
[1,2,4]triazolo[4,3-a]quinoxalin-4-yl]carbamoyl}propanamido)-
ethoxy]ethoxy}ethylcarbamate (10). 4-Amino-2-(4-methoxyphenyl)-
1,2,4- triazolo[4,3-a]quinoxalin-1-one (1) (0.3 mmol) was treated
twice with a solution of 14-diaza-17,17-dimethyl-4,15-dioxo-8,11,16-
trioxaoctadecanoic acid (6) (0.6 mmol), HATU (0.6 mmol), HOAt
(0.6 mmol), and DIPEA (1.2 mmol) to afford the title compound (16
1
Bruker-AV 400, H spectra were recorded at 400.13 MHz, and 13C
NMR spectra were recorded at 101.62 MHz. Solvents used for NMR
analysis (reference peaks listed) were DMSO-d6 [(CHD2)2SO at δH
2.50 ppm, (CD3)2SO at 39.52 ppm] and CDCl3 (CHCl3 at δH 7.26
ppm, CDCl3 at 77.16). Chemical shifts (δ) are recorded in parts per
million (ppm). Coupling constants (J) are recorded in Hz, and the
significant multiplicities are described by singlet (s), doublet (d),
triplet (t), quadruplet (q), broad (br), and multiplet (m). Spectra were
assigned using appropriate COSY, DEPT, HSQC, and HMBC
sequences. High-resolution mass spectra (HRMS)−time-of-flight,
electrospray (TOF ES ) were recorded on a Waters 2795 separation
module/micromass LCT platform. RP-HPLC was performed using a
Waters 2767 sample manager and Waters 2525 binary gradient module
and visualized at 254 and 366 nm with a Waters 2487 dual wavelength
absorbance detector. Spectra were analyzed using MassLynx.
Preparative RP-HPLC was performed using a Phenomenex Luna
C8(2) 11A Axia F column (150 mm × 30 mm × 10 μm) at a flow rate
of 20 mL/min. Semipreparative RP-HPLC was performed using a
YMC-Pack C8 column (150 mm × 10 mm × 5 μm) at a flow rate of 3
mL/min. Analytical RP-HPLC was performed using a YMC-Pack C8
column (150 mm × 4.6 mm × 5 μm) at a flow rate of 1.0 mL/min.
Analytical RP-HPLC was used to confirm that all final products were
>95% pure. The retention time (Rt) of the final pure product is
reported using an analytical RP-HPLC method of 0−2 min 10%
solvent B in solvent A followed by a 2−25 min gradient of 10−90%
solvent B in solvent A (solvent A = 0.05% TFA in H2O, solvent B =
0.05% TFA in 9:1 v:v CH3CN:H2O).
1
mg, 0.03 mmol, 10% yield) as a pale green solid. H NMR (DMSO-
d6): δ 1.36 (s, 9H, C(CH3)3), 2.45−2.50 (m, 4H, CH2), 2.91 (t, 2H, J
= 7.0 Hz, CH2), 3.10 (m, 2H, NHCH2), 3.22 (m, 2H, NHCH2),
3.33−3.43 (m, 6H, CH2), 3.82 (s, 3H, OCH3), 6.76 (br m, 1H,
NHCH2), 7.12 (m, 2H, ArH phenyl), 7.49 (m, 1H, ArH quinoxaline),
7.56 (m, 1H, ArH quinoxaline), 7.69 (d, 1H, J = 7.8 Hz, ArH
quinoxaline), 7.95−7.99 (m, 3H, NHCH2, ArH phenyl), 8.70 (d, 1H, J
= 8.1 Hz, ArH quinoxaline), 10.56 (s, 1H, NH). 13C NMR (DMSO-
d6): δ 28.22, 28.77, 29.71, 32.07, 38.63, 51.26, 55.43, 69.15, 69.46,
69.55, 77.58, 114.24, 114.31, 121.63, 125.52, 126.44, 127.60, 127.78,
130.19, 130.86, 133.85, 141.13, 148.08, 155.59, 157.78, 171.23, 171.50.
HRMS calculated for C31H40N7O8 (M + H)+, 638.2938; found,
638.2930. Analytical RP-HPLC Rt = 18.10 min.
tert-Butyl N-8-(3-{[2-(4-Methoxyphenyl)-1-oxo-1H,2H-[1,2,4]-
triazolo[4,3-a]quinoxalin-4-yl]carbamoyl}propanamido)-
octylcarbamate (11). 4-Amino-2-(4-methoxyphenyl)-1,2,4- triazolo-
[4,3-a]quinoxalin-1-one (1) (0.35 mmol) was treated twice with a
solution of 5,14-diaza-17,17-dimethyl-4,15-dioxo-16-oxaoctadecanoic
acid (7) (0.8 mmol), HATU (0.8 mmol), HOAt (0.8 mmol), and
DIPEA (1.2 mmol) to give 11 (45 mg, 0.07 mmol, 20% yield) as an
tert-Butyl 2-(2-(2-Aminoethoxy)ethoxy)ethylcarbamate (4).
Compound 4 was synthesized according to the literature.36
1
off-white solid. H NMR (DMSO-d6): δ 1.21−1.24 (m, 8H, CH2),
tert-Butyl 8-Aminooctylcarbamate (5). Compound 5 was
synthesized according to the literature.37
1.33−1.36 (m, 13H, CH2, C(CH3)3), 2.44 (t, 2H, J = 7.9 Hz, CH2),
2.85−2.92 (m, 4H, CH2), 3.04 (m, 2H, CH2), 3.82 (s, 3H, OCH3),
6.74 (br m, 1H, NHCH2), 7.12 (m, 2H, ArH phenyl), 7.50 (m, 1H,
ArH quinoxaline), 7.56 (m, 1H, ArH quinoxaline), 7.69 (d, 1H, J = 7.8
Hz, ArH quinoxaline), 7.86 (m, 1H, NHCH2), 7.96 (m, 2H, ArH
phenyl), 8.71 (d, 1H, J = 8.1 Hz, ArH quinoxaline), 10.57 (s, 1H,
NH). 13C NMR (DMSO-d6): δ 28.25, 28.68, 28.74, 29.13, 29.44,
29.81, 32.16, 38.56, 39.73*, 55.41, 77.24, 114.26, 114.30, 121.69,
125.52, 126.43, 127.59, 127.76, 130.18, 130.85, 133.84, 141.13, 148.07,
155.54, 157.76, 170.84, 171.51. *Underneath the DMSO-d6 solvent
peak, the chemical shift derived from HSQC spectrum. HRMS
calculated for C33H44N7O6 (M + H)+, 634.3353; found, 634.3347.
tert-Butyl N-([2-(4-Methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo-
[4,3-a]quinoxalin-4-yl]carbamoylmethyl)carbamate (12). 4-Amino-
2-(4-methoxyphenyl)-1,2,4- triazolo[4,3-a]quinoxalin-1-one (1) (0.06
mmol) was treated twice with a solution of Boc-Gly-OH (8) (0.12
mmol), HATU (0.12 mmol), HOAt (0.12 mmol), and DIPEA (0.24
mmol) to give 12 (17 mg, 0.04 mmol, yield 67%) as a pale yellow
General Procedure A: Synthesis of 6 and 7 via Acylation
with Succinic Anhydride. To the monoprotected amine 4 or 5 (4−
7 mmol) in chloroform (20 mL) at 0 °C was added succinic anhydride
(1 equiv, 4−7 mmol). The solution was warmed to ambient
temperature and stirred for 12 h. The solvent was evaporated under
reduced pressure to afford the desired product.
5,14-Diaza-17,17-dimethyl-4,15-dioxo-8,11,16-trioxaoctadeca-
noic Acid (6). tert-Butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate
1
(4) gave 6 (1.14 g, 3.3 mmol, 82% yield) as a colorless oil. H NMR
(CDCl3): δ 1.43 (s, 9H, C(CH3)3), 2.50 (m, 2H, CH2), 2.67 (m, 2H,
CH2), 3.32 (m, 2H, CH2), 3.54 (m, 2H, CH2), 3.51−3.56 (m, 4H,
CH2), 3.60−3.61 (m, 4H, CH2), 6.92 (br s, 1H, NH), 7.44 (br s, 1H,
NH). C15H27N2O7 (M − H)− calculated, 347.1818; found, 347.1887.
5,14-Diaza-17,17-dimethyl-4,15-dioxo-16-oxaoctadecanoic Acid
(7). tert-Butyl 8-aminooctylcarbamate (5) (6.7 mmol) gave 7 (2.29 g,
1
6.7 mmol, quantitative) as a white solid. H NMR (DMSO-d6): δ
1
1.21−1.24 (m, 8H, CH2), 1.35−1.37 (m, 13H, CH2, C(CH3)3), 2.28
(m, 2H, CH2), 2.40 (m, 2H, CH2), 2.89 (m, 2H, CH2), 3.00 (m, 2H,
CH2), 6.73 (m, 1H, NH), 7.77 (m, 1H, NH).
solid. H NMR (DMSO-d6): δ 1.38 (s, 9H, C(CH3)3), 3.81 (s, 3H,
OCH3), 4.08 (d, 2H, J = 6.3 Hz, NHCH2), 7.12 (m, 2H, ArH phenyl),
7.19 (m, 1H, NHCH2), 7.50 (m, 1H, ArH quinoxaline), 7.57 (m, 1H,
ArH quinoxaline), 7.72 (m, 1H, ArH quinoxaline), 7.95 (d, 2H, J = 9.0
Hz, ArH phenyl), 8.70 (d, 1H, J = 8.0 Hz, ArH quinoxaline), 10.58 (s,
1H, NH). 13C NMR (DMSO-d6): 28.21, 51.60, 55.44, 78.23, 114.32,
121.71, 125.56, 126.48, 127.68, 127.80, 130.14, 130.82, 133.75, 140.93,
148.09, 155.83, 157.82, 169.40, 170.88. HRMS calculated for
C23H25N6O5 (M + H)+, 465.1886; found, 465.1901.
4-(Benzyloxycarbonylamino)benzoic Acid (9). To a solution of 4-
aminobenzoic acid (1.5 mmol) and sodium bicarbonate (13 mmol) in
water (5 mL) and dioxane (5 mL) was added benzyl chloroformate
(1.5 mmol). The mixture was stirred for 4 h, water (2 mL) was added,
and the mixture was stirred for 12 h. The solution was acidified to pH
3 with 1 M HCl, and the precipitate was filtered, washed, and dried to
1
give 9 (375 mg, 1.4 mmol, 93% yield) as a white solid. H NMR
Benzyl N-(4-[2-(4-Methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo-
[4,3-a]quinoxalin-4-yl]carbamoylphenyl)carbamate (13). 4-Amino-
(DMSO-d6): δ 5.18 (s, 2H, OCH2), 7.35−7.45 (m, 5H, ArH), 7.58
1778
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2-(4-methoxyphenyl)-1,2,4- triazolo[4,3-a]quinoxalin-1-one (1) (0.06
{4-[(E)-2-(4,4-Difluoro-4,4a-dihydro-5-(thiophen-2-yl)-4-bora-
3a,4a-diaza-s-indacene-3-yl)ethenyl]phenoxy}acetamido)-
hexanamido]ethoxy}ethoxy)ethyl]succinamide (19). BODIPY-630/
650-SE (2.0 mg, 3.0 μmol) gave 19 (0.9 mg, 0.8 μmol, 28% yield) as a
dark blue solid. C55H58BF2N10O9S (M + H)+, 1083.4170; found,
1083.4175. Analytical RP-HPLC Rt = 22.30 min.
mmol) was treated twice with
a solution of 4-
(benzyloxycarbonylamino)benzoic acid (9) (0.12 mmol), HATU
(0.12 mmol), HOAt (0.12 mmol), and DIPEA (0.24 mmol) to give
a pale yellow solid (30 mg) that contained the desired acylated
product 13 along with starting material 1 (9:1 by analytical RP-
HPLC). This material was therefore further purified by preparative
N-[2-(4-Methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-4-yl]-N′-(2-{2-[2-(6-{2-[4-(4,4-difluoro-4,4a-dihydro-5-
(thiophen-2-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy]-
acetamido}hexanamido)ethoxy]ethoxy}ethyl)succinamide (20).
BODIPY-TR-SE (2.0 mg, 3.1 μmol) gave 20 (1.0 mg, 0.9 μmol,
29% yield) as a dark red solid. C53H56BF2N10O9S (M + H)+,
1057.4014; found, 1057.3986. Analytical RP-HPLC Rt = 21.97 min.
N-[2-(4-Methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-4-yl]-N′-[2-(2-{2-[6-(2-2-[(1E,3E)-5-[(2E)-1-ethyl-3,3-di-
methyl-5-sulfo-2,3-dihydro-1H-indol-2-ylidene]penta-1,3-dien-1-
yl]-1-{5-[(2-{2-[2-(3-{[2-(4-methoxyphenyl)-1-oxo-1H,2H-[1,2,4]-
triazolo[4,3-a]quinoxalin-4-yl]carbamoyl}propanamido)ethoxy]-
ethoxy}ethyl)carbamoyl]pentyl}-3,3-dimethyl-3H-indol-1-ium-6-sul-
fonate (21). Cy5-X-SE (1.0 mg, 1.3 μmol) gave 21 (0.7 mg, 0.8 μmol,
60% yield) as a bright blue solid. C59H70N9O13S2 (M + H)+,
1177.4534; found, 1177.4570. Analytical RP-HPLC Rt = 14.94 min.
2-[6-(Dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl]-5(4)-
({5-[(2-{2-[2-(3-{[2-(4-methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo-
1
RP-HPLC, to afford 13 (8 mg, 24% yield) as a pale yellow solid. H
NMR (DMSO-d6): δ 3.79 (s, 3H, OCH3), 5.20 (s, 2H, OCH2), 7.09
(d, 2H, J = 9.4 Hz, ArH phenyl), 7.36−7.47 (m, 5H, ArH Cbz), 7.54
(m, 1H, ArH quinoxaline), 7.62−7.66 (m, 3H, ArH quinoxaline,
phenyl), 7.78 (d, 1H, J = 8.1 Hz, ArH quinoxaline), 7.88 (d, 2H, J =
8.7 Hz, ArH phenyl), 8.00 (d, 1H, J = 8.7 Hz, ArH quinoxaline), 10.20
(s, 1H, NH), 11.00 (s, 1H, NH). 13C NMR (DMSO-d6): δ 55.41,
66.12, 114.37, 117.32, 121.83, 126.08, 126.60, 126.71, 128.17, 18.24,
128.50, 129.68, 130.06, 131.72, 133.69, 136.34, 142.84, 143.19, 148.07,
153.24, 157.83, 165.61, 170.26, 170.89. HRMS calculated for
C31H25N6O5 (M + H)+, 561.1886; found, 561.1880. Analytical RP-
HPLC Rt = 18.45 min.
General Procedure C: Synthesis of 14−16. To a solution of the
Boc-protected compound 10, 11, or 12 in dichloromethane (1 mL)
was added trifluoroacetic acid (1 mL), and the mixture was stirred for
1 h. The solvent was removed under reduced pressure to give the
product (14, 15, or 16) as the trifluoroacetate salt in quantitative yield,
which was then used without further purification.
[4,3-a]quinoxalin-4-yl]carbamoyl}propanamido)-ethoxy]ethoxy}-
isomers
ethyl)carbamoyl]pentyl}carbamoyl)benzoate*mixed
(22).
TAMRA 5/6-X-SE (1.0 mg, 1.6 μmol) gave 22 (0.6 mg, 0.6 μmol,
38% yield) as a pink solid. An analytical RP-HPLC trace of the crude
reaction mixture revealed only one peak that corresponded to the mass
of 22. This peak was collected by semipreparative RP-HPLC and
analyzed by analytical RP-HPLC, which revealed only one symmetrical
peak. Therefore, it was assumed that the purified 22 (0.6 mg) was a
mixture of the 5- and 6-isomers. C57H64N10O11 (M + H)+, 1064.4756;
found, 1064.5231. Analytical RP-HPLC Rt = 16.42 min.
2-[6-(Dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl]-5-
((53-((2-(4-methoxyphenyl)-1-oxo-1,2-dihydro-[1,2,4]triazolo[4,3-
a ] q u i n o x a l i n - 4 - y l ) a m i n o ) - 3 9 , 5 0 , 5 3 - t r i o x o -
3,6,9,12,15,18,21,24,27,30,33,36,43,46-tetradecaoxa-40,49-
diazatripentacontyl)carbamoyl)benzoate (23). 5-TAMRA-PEG-SE
(1.0 mg, 0.9 μmol) gave 23 (0.3 mg, 0.2 μmol, 22% yield) as a pink
solid. C78H106N10O23 (M + 2H)+, 775.3711; found, 775.3694.
Analytical RP-HPLC Rt = 16.53 min.
General Pharmacology Methods: Generation of Constructs
and Cell Lines Used and Cell Culture. CHO cells stably expressing
a cAMP response element-secreted placental alkaline phosphatase
(CRE-SPAP) reporter gene were transfected with cDNA encoding the
human A3AR using Lipofectamine (Invitrogen, Paisley, United
Kingdom) according to the manufacturer's instructions. Cells were
cultured under selection pressure in medium containing 1 mg/mL
G418, and a clonal cell line was generated. These cells were used in the
CRE-SPAP assay and for imaging. CHO cells stably expressing the
A1AR were as previously described38 and used for the calcium flux
assays and imaging. HEK293 cells stably expressing the Glosensor
cAMP biosensor (Promega, United States) were used in the Glosensor
cAMP assay. The sigSNAP-A3AR construct was generated as follows;
cDNA encoding the full-length human A3AR, which did not contain a
methionine start codon, was fused in frame with cDNA encoding a cell
membrane localization signal sequence and the SNAP-tag (New
England Biolabs, Ipswich, MA). DNA sequencing was carried out to
confirm the sequence of the construct. To obtain a mixed population
of sigSNAP-A3AR-expressing cells, HEK293 cells were grown to 80%
confluency in 75 cm2 tissue culture flasks and then transfected with the
sig-SNAP-A3AR construct (10 mg/flask) in Optimem again using
Lipofectamine according to the manufacturer's instructions. Success-
fully transfected cells were initially selected using medium containing
0.8 mg/mL Geneticin and subsequently cultured in 0.2 mg/mL
Geneticin to maintain selection pressure. The CHO cell lines were
maintained in Dulbecco's modified Eagle's medium (DMEM) nutrient
mix F12 (DMEM/F12) supplemented with 10% fetal calf serum and 2
mM L-glutamine. The HEK293 cell lines were maintained in DMEM
containing 10% fetal calf serum and 2 mM L-glutamine. All cell lines
N-{2-[2-(2-Aminoethoxy)ethoxy]ethyl}-N′-[2-(4-methoxyphenyl)-
1-oxo-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl]succinamide (14). tert-
Butyl N-2-{2-[2-(3-{[2-(4-methoxyphenyl)-1-oxo-1H,2H-[1,2,4]-
triazolo[4,3-a]quinoxalin-4-yl]carbamoyl}propanamido)ethoxy]-
ethoxy}ethylcarbamate (10) (16 mg, 0.02 mmol) gave 14 (14 mg,
quant.) as a pale green solid. C26H32N7O6 (M + H)+, 538.2414; found,
538.2403. Analytical RP-HPLC Rt = 13.69 min.
N-(8-Aminooctyl)-N′-[2-(4-methoxyphenyl)-1-oxo-1H,2H-[1,2,4]-
triazolo[4,3-a]quinoxalin-4-yl]succinamide (15). tert-Butyl N-8-(3-
{[2-(4-methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-4-yl]carbamoyl}propanamido)octylcarbamate (11) (6 mg,
0.009 mmol) gave 15 (5 mg, quant.) as a pale yellow solid.
C28H36N7O4 (M + H)+, 534.2829; found, 534.2819. Analytical RP-
HPLC Rt = 15.60 min.
2-Amino-N-[2-(4-methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo-
[4,3-a]quinoxalin-4-yl]acetamide (16). tert-Butyl N-([2-(4-methox-
yphenyl)-1-oxo-1H,2H-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl]-
carbamoylmethyl)carbamate (12) (5 mg, 0.01 mmol) gave 16 (4 mg,
quant.) as a pale yellow solid. C18H17N6O3 (M + H)+, 365.1362;
found, 365.1365. Analytical RP-HPLC Rt = 13.37 min.
4-Amino-N-[2-(4-methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo-
[4,3-a]quinoxalin-4-yl]benzamide (17). A mixture of 13 and 1 (9:1
by RP-HPLC) (approximately 0.04 mmol of 13) was dissolved in
methanol/water/acetic acid (2 mL, 9:0.9:0.1) and 10% palladium-on-
carbon (2 mg) was added. The flask was evacuated and filled with
hydrogen (balloon) and stirred for 3 h. The reaction mixture was
filtered through Celite, and the filtrate solvent was removed under
reduced pressure to give a yellow solid (15 mg) that contained desired
product 17 along with the starting impurity 1. Two milligrams of this
material was purified by semipreparative RP-HPLC to afford 17 (0.5
mg, 25%) as a pale yellow solid. HRMS calculated for C23H19N6O3 (M
+ H)+, 427.1519; found, 427.1519. Analytical RP-HPLC Rt = 18.65
min.
General Procedure D: Synthesis of 18−23. A solution of amine
14 or 15 (2 equiv), DIPEA (4 equiv), and the fluorophore-SE (1
equiv) was stirred in DMF (1 mL) with the exclusion of light for 12 h.
The solvent was removed under reduced pressure, and the residue was
purified by semipreparative RP-HPLC.
N-[2-(4-Methoxyphenyl)-1-oxo-1H,2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-4-yl]-N′-{8-[6-(2-{4-[(E)-2-(4,4-difluoro-4,4a-dihydro-5-
(thiophen-2-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)ethenyl]-
phenoxy}acetamido)hexanamido]octyl}succinamide (18). BODI-
PY-630/650-SE (1.0 mg, 1.5 μmol) gave 18 (0.8 mg, 0.8 μmol, 75%
yield) as a dark blue solid. C57H62BF2N10O7S (M + H)+, 1079.4585;
found, 1079.4498. Analytical RP-HPLC Rt = 24.53 min.
1779
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Journal of Medicinal Chemistry
Article
were maintained at 37 °C in a humidified atmosphere of air/CO2
(19:1).
For experiments on HEK293-SNAP-A3AR cells, growth medium
was removed, and cells were incubated in full growth medium
containing 0.5 mM BG-AlexaFluor488 (New England Biolabs) for 15
min at 37 °C in a humidified atmosphere of air/CO2 (19:1). Cells
were subsequently washed three times in HBS, before incubation in
the presence or absence of 100nM MRS1220 or PSB603 for 30 min at
37 °C. Cells were then incubated with 5 nM 19 for 10 min at room
temperature, and confocal images were obtained as for CHO cells,
except using dual excitation with 488 (BG-AlexaFluor488) and 633 nm
(BODIPY 630/650) lasers. Light was directed to sample using a 488/
561/633 primary dichroic, and emission split through an NFT565
secondary dichroic to two detection channels through BP500-530IR
and BP650-710IR emission filters. The pinhole diameter was set to 1
Airy unit for 633 nm excitation, and that for the 488 nm channel was
adjusted to match the slice depth. Again, laser powers and detector
gains and offset were kept constant within individual experiments.
Colocalization analysis was performed in Zeiss AIM4.2 software.
In both cases, images are presented as representative of an
individual experiment with matched conditions. Linear adjustments to
image brightness and contrast have been applied equally across all
comparative images using Zeiss AIM4.2 software to prepare images for
presentation.
CRE-SPAP Gene Transcription Assay. A3AR CRE-SPAP cells
were grown to confluence in clear 96-well plates. Cells were serum
starved for 18 h prior to experiment. On the day of experiment, fresh
serum-free medium (DMEM/F12 supplemented with 2 mM L-
glutamine) was added to the cells, and three concentrations of test
compound were added to the appropriate wells, and cells were
incubated for 30 min at 37 °C/5% CO2. After 30 min, increasing
concentrations of the agonist NECA were added to the cells, and the
cells were incubated for a further 30 min at 37 °C/5% CO2, and then,
1 μM forskolin (FSK) was added to stimulate cAMP production
within the cells. Cells were then incubated for a further 5 h. Following
the 5 h incubation, all medium was removed from the cells, 40 μL of
fresh serum-free medium was added to each well, and cells were
incubated for a further 1 h at 37 °C/5% CO2. The plates were then
incubated at 65 °C for 30 min to destroy any endogenous alkaline
phosphatases. Plates were cooled to room temperature, and 100 μL of
5 mM 4-nitrophenyl phosphate in diethanolamine-containing buffer
[10% (v/v) diethanolamine, 280 mM NaCl, 500 μM MgCl2, pH 9.85]
was added to each well; the plates were then incubated at 37 °C for 20
min. The absorbance at 405 nm was measured using a Dynex MRX
plate reader (Chelmsford, MA).
Data Analysis. All data were fitted using Prism5 (GraphPad
Software, San Diego, CA). For the CRE-SPAP gene transcription
assay, NECA concentration−response curves in the absence and
presence of increasing concentrations of test compounds were globally
fit to the following interaction model in Prism5.
Calcium Mobilization Assay. CHO-A1AR cells35 were grown to
confluence in black-walled, clear-bottom 96-well plates. On the day of
the experiment, medium was removed from each well and replaced
with 100 μL of HEPES-buffered saline solution (HBSS; 25 mM
HEPES, 10 mM glucose, 146 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2
mM sodium pyruvate, 1.3 mM CaCl2, and 1.5 mM NaHCO3)
supplemented with 2.5 mM probenecid, 2.3 μM Fluo-4AM, and
0.023% Pluronic F-127. Cells were incubated with Fluo-4-containing
buffer for at least 45 min at 37 °C in the dark. Cells were then washed
twice in HBSS, fresh HBSS with 2.5 mM probenecid with or without
antagonist was added to each well, and cells were incubated for a
further 30 min at 37 °C in the dark. Plates were then loaded onto a
multiwell fluormetric imaging plate reader (FlexStation; Molecular
Devices, Sunnyvale, CA), and the fluorescence was measured
(excitation, 485 nm; emission, 520 nm) every 1.52 s for 200 s. Either
HBSS or HBSS with the required concentration of NECA was added
at 15 s.
Glosensor cAMP Assay. HEK293 Glonsensor cells were grown to
confluence in white-walled, clear-bottom 96-well plates coated with
poly-L-lysine to allow the cells to adhere to the plate. On the day of the
assay, medium was removed from each well and replaced with HBSS
containing 3% v/v Glosensor cAMP reagent. Equilibration of the
Glosensor reagent within the cells was achieved by incubating the cells
for 2 h at 37 °C. Where required, antagonists were added after 1.5 h.
The required concentration of NECA was added after 2 h, and
luminescence was read on an EnVision plate reader (PerkinElmer,
Waltham, MA) every 2 min for 60 min. The maximal luminescence
read was used for analysis.
Confocal Microscopy. Cells were grown to approximately 80%
confluency on 8-well Labtek chambered coverglasses (Nunc Nalgene)
in their appropriate growth medium. For HEK293 cells, plates were
coated with poly-D-lysine prior to seeding of cells. For CHO cell
experiments, cells were washed twice in Hank's balanced salt solution
(HBS) and then incubated in the presence or absence of 100 nM
MRS1220 (CHO-A3AR CRE-SPAP cells) or 100 nM DPCPX (CHO-
A1AR cells) for 30 min at 37 °C. Cells were then incubated with 19 or
20 at the required concentration for 10 min at room temperature,
prior to collection of single equatorial confocal images. Images were
obtained on a Zeiss LSM510 Meta NLO confocal microscope using a
40× c-Apochromat 1.2NA water-immersion objective. For 19, images
were collected using 633 nm excitation, a 488/561/633 dichroic and
emission collected through a 650LP filter. For 20, 561 nm excitation
was used with the same dichroic, with emission collected using a
LP575 filter. In each case, a pinhole diameter of 1 Airy Unit was used,
and laser power, gain, and offset were kept the same for samples within
each experiment.
E
× [A]
max
response =
⎛
⎞
⎛
⎞
⎟
⎠
[B]
[A] + EC × ⎜1 +
⎜
⎝
⎟
⎠
50
K
⎝
D
where Emax is the maximal response, EC50 is the molar concentration of
NECA [A] in the absence of test compound required to generate a
response that is 50% of Emax, [B] is the concentration of test
compound, and KD is the antagonist equilibrium dissociation constant.
For the calcium mobilization and Glosensor experiments, estimated
affinity values (pKD) were calculated from the shift of the agonist
concentration−response curves elicited in the presence of 1 μM
antagonist using the following equation:
[B]
DR = 1 +
K
D
where DR (dose ratio) is the ratio of the antagonist concentration
required to stimulate an identical response in the presence and absence
of antagonist, [B]. Where there was no significant shift in the agonist
concentration−response curve in the presence of 1 μM antagonist, a
pKD of >6 is quoted.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +44-115-8230082. Fax: +44-115-8230081. E-mail:
stephen.hill@nottingham.ac.uk (S.J.H.). Tel: +44-115-
9513026. Fax: +44-115-9513412. E-mail: barrie.kellam@
nottingham.ac.uk (B.K.).
Notes
The authors declare the following competing financial inter-
est(s): B.K and S.J.H. are founding directors of the University
of Nottingham spin-off company CellAura Technologies Ltd.
ACKNOWLEDGMENTS
This work was supported by the Medical Research Council, UK
(Grant No. G0800006).
■
ABBREVIATIONS USED
AR, adenosine receptor; Boc, tert-butyl carbamate; BODIPY-X-
630/650, 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-
■
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Journal of Medicinal Chemistry
Article
adenosine receptors in human colorectal cancer is reflected in
indacene-3-yl)styryloxy)acetyl)amino hexanoic acid; BODIPY-
X-TR, 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-
indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid; BODIPY,
bordifluoropyrromethene; cAMP, cyclic adenosine mono-
phosphate; CBZ, benzyl carbamate; CHO, Chinese hamster
ovary; CRE-SPAP, cyclic AMP response element promoted
secreted placental alkaline phosphatase; Cy5-X, 3H-indolium,2-
[5-[1-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]-1,3-dihy-
dro-3,3-dimethyl-5-sulfo-2H-indol-2 ylidene]-1,3-pentadien-1-
yl]-1-ethyl-3,3-dimethyl-5-sulfopotassium salt; DPCPX, 8-cy-
clopentyl-1,3-dipropylxanthine; FSK, forskolin; GPCR, G
protein-coupled receptor; HATU, 2-(1H-7-azabenzotriazol-1-
yl)-1,1,3,3-tetramethyluronoium hexafluorophosphate;
HEK293, human embryonic kidney 293; HOAt, 1-hydroxy-7-
azabenzotriazole; MRS1220, N-[9-chloro-2-(2-furanyl)[1,2,4]-
triazolo[1,5-c]quinazolin-5-yl]benzene acetamide; NECA, ad-
enosine-5-N-ethylcarboxamide; PEG, polyethylene glycol;
PSB603, 8-[4-[4-(4-chlorophenzyl)piperazide-1-sulfonyl)-
phenyl]]-1-propylxanthine; RP-HPLC, reversed-phase high
pressure liquid chromatography; SE, succinimidyl ester;
TAMRA-5/6-X, 6-(tetramethylrhodamine-5-(and-6)-
carboxamido)hexanoic acid; XAC, xanthine amine congener
peripheral blood cells. Clin. Cancer Res. 2004, 10, 5895−5901.
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