Subtype-Selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A2A Receptor

Article abstract of DOI:10.1021/acs.jmedchem.9b01856

Among class A G protein-coupled receptors (GPCR), the human adenosine A_2A receptor (hA_2AAR) remains an attractive drug target. However, translation of A_2AAR ligands into the clinic has proved challenging and an improved un

Full text of DOI:10.1021/acs.jmedchem.9b01856

Subscriber access provided by BIU Pharmacie | Faculté de Pharmacie, Université Paris V
Article
Subtype-selective Fluorescent Ligands as Pharmacological
Research Tools for the Human Adenosine A2A Receptor
Eleonora Comeo, Nicholas D. Kindon, Mark Soave, Leigh A. Stoddart,
Laura Kilpatrick, Peter J. Scammells, Stephen John Hill, and Barrie Kellam
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01856 • Publication Date (Web): 30 Dec 2019
Downloaded from pubs.acs.org on December 30, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Subtype-selective Fluorescent Ligands as
Pharmacological Research Tools for the Human
Adenosine A Receptor.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
A
Eleonora Comeo1,3,4; Nicholas D. Kindon : Mark Soave ; Leigh A. Stoddart ; Laura E.
1,3
2,3
2,3
Kilpatrick ; Peter J. Scammells ; Stephen J. Hill and Barrie Kellam1,3 *
2
,3
4
2,3*
.
1
Division of Biomolecular Sciences and Medicinal Chemistry, School of Pharmacy, Centre for
Biomolecular Sciences, University of Nottingham, NG7 2RD, UK.
2
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University
of Nottingham, NG7 2UH, UK.
3
Centre of Membrane Proteins and Receptors (COMPARE) University of Birmingham and
University of Nottingham, The Midlands, NG7 2UH, UK.
4
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381
Royal Parade, Parkville, Victoria 3052 Australia
KEYWORDS: Adenosine A2A receptor, fluorescent antagonist, BODIPY, GPCR, NanoBRET,
preladenant, imaging, FRET
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
Among class A G protein-coupled receptors (GPCR), the human adenosine A2A receptor
hA AR) remains an attractive drug target. However, translation of A AR ligands into the
(
2
A
2A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
clinic has proved challenging and an improved understanding of A AR pharmacology could
2A
promote development of more efficacious therapies. Subtype-selective fluorescent probes would
allow detailed real-time pharmacological investigations both in vitro and in vivo. In the present
study, two families of fluorescent probes were designed around the known hA AR selective
2
A
antagonist preladenant (SCH 420814). Both families of fluorescent antagonists retained affinity
at the hA AR, selectivity over all other adenosine receptor subtypes and allowed clear
2
A
visualization of specific receptor localization through confocal imaging. Furthermore, the
AlexaFluor647-labelled conjugate allowed measurement of ligand binding affinities of unlabeled
hA AR antagonists using a bioluminescence resonance energy transfer (NanoBRET) assay. The
2
A
fluorescent ligands developed here can therefore be applied to a range of fluorescence-based
techniques to further interrogate hA AR pharmacology and signaling.
2A
INTRODUCTION
The adenosine receptors belong to class A of the G protein-coupled receptor (GPCR)
superfamily with the latter accounting for about 30% of the molecular targets for currently
1
marketed drugs. The pharmacological profile of the adenosine receptors is defined by the
function of four adenosine receptor subtypes- A , A , A and A - each of which possess
1
2A
2B
3
2
distinct signaling and pharmacological properties . Among this family, the adenosine A
2
A
receptor (A AR) represents an attractive drug target which has been the subject of intensive
2A
medicinal chemistry research over the last forty years.3,4 The A ARs signal primarily through
2
A
ACS Paragon Plus Environment
2

Page 3 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
5
G proteins in the periphery but can also signal through G in the striatum. Upon receptor
S
olf
activation, both G and G -coupled receptors act by stimulating adenylyl cyclase leading to an
s
olf
increase of intracellular cAMP levels, and subsequent activation of a series of downstream
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
signaling cascades. Over thirty X-ray crystal structures for the hA AR have been solved in
2
A
complex with a range of molecules, providing useful insights into the structural framework of
this membrane protein and helped the rational design and development of selective hA AR
2
A
ligands7–11 for the treatment of several diseases including cardiovascular, inflammatory and CNS
disorders.12–14 More recently blockade of the A AR pathway has generated renewed interest
2
A
because of the role of adenosinergic signaling in cancer immunotherapy.15 In vitro studies and
animal models have shown that adenosine-mediated activation of the high affinity A ARs
2
A
expressed on the surface of immune cells suppresses the anti-tumor response in the host,
facilitating cancer growth and dissemination.16,17 Moreover, the expression of A ARs correlates
2A
with the malignancy of the tumor, suggesting these membrane-bound proteins might be
considered as a prognostic biomarker for the severity of neoplastic disease.18 A number of
small-molecule A AR antagonists are currently being explored as novel therapeutics to inhibit
2
A
this adenosine-mediated suppression of the immune responses in patients with solid
malignancies, including non-small cell lung cancer, breast cancer and prostate carcinoma (Figure
1
).
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Small molecule hA AR antagonists currently investigated as single agents, or in
2
A
combination with monoclonal antibodies, in patients with solid tumors.
To date only two molecules targeting the A AR have reached the market: Regadenoson
2
A
Lexiscan, Astellas Pharma) a pharmacological stress agent for myocardial perfusion injury12,19
(

and Istradefylline (NOURIANZ , Kyowa Kirin Inc.), a xanthine-based hA AR antagonist
2
A
which has reached the market in Japan for treating the symptoms of Parkinson’s Disease
PD)20,21 However, despite the many therapeutic opportunities associated with the A AR,
(
2
A
translation of adenosine ligands from the bench into the clinic has been hampered by a lack of
understanding of the tissue and cell-type specific receptor pharmacology leading to on-target side
effects.13 Additional knowledge on the function of the A AR could be achieved through real-
2
A
time monitoring of the localization and function of the receptor at native expression levels in
specific human cell types in both health and disease, with the aim of developing more selective
and efficacious therapies. Fluorescent ligands therefore represent useful tools to achieve this aim
ACS Paragon Plus Environment
4

Page 5 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
as they offer the ability to study the target receptor at endogenous expression levels, in real-time
and, importantly, in unmodified living cells.22
Fluorescent ligands for the ARs have been successfully developed and have allowed various
aspects of their pharmacology, such as the signaling23,24 and dynamics25,26, to be investigated.
For example, the BODIPY 630/650-X-labelled xanthine amine congener (XAC), developed in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
our laboratories, allowed the quantification of specific A AR-antagonist complexes at the single
1
2
6
molecule level in living cells using fluorescence correlation spectroscopy (FCS). However,
intracellular accumulation of the ligand observed over extended incubation times through
confocal microscopy, as well as the promiscuous nature of the probe in binding other adenosine
receptors, restricted its use to recombinant cells. Receptor-subtype selectivity is particularly
important when probing the A AR pharmacology in unmodified, endogenously expressing
2
A
tissues and cells, as other adenosine receptor subtypes are often co-expressed.2,12 Jacobson and
co-workers pioneered the development of fluorescent probes selective for the hA AR, including
2
A
fluorescent analogues of the A AR selective antagonist SCH442416 labelled with an
2
A
2
7
AlexaFluor488 dye, that have been used successfully for fluorescence polarization and flow
2
8
cytometry assays. However, the utility of these probes for bioimaging studies has not yet been
reported. This suggests there still remains the need to develop additional selective hA AR
2
A
probes, optimized for imaging studies that would allow specific visualization of the hA AR at
2
A
the single cell level in healthy and diseased systems.
In the present study, we sought to expand the current pharmacological toolkit for the hA AR
2
A
by generating a more versatile suite of fluorescent probes with regard to their photochemical and
physicochemical properties and areas of potential application. Here we report the design,
synthesis and pharmacological evaluation of a novel series of subtype-selective fluorescent
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ligands (12-17) for the human adenosine hA AR receptor based upon preladenant (a high
2
A
2
9
29
affinity (hA AR K = 1.0 nM) and highly selective (hA , hA and hA K > 1000 nM)
2
A
i
1
2B
3
i
hA AR antagonist) which are amenable to use in a variety of fluorescence-based techniques.
2
A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
These A AR selective fluorescent ligands will be valuable tools for the wider scientific
2
A
community for both in vitro and in vivo applications and high-resolution imaging studies into the
role of A AR in health and disease.
2A
RESULTS AND DISCUSSION
Design Strategy. Our synthetic strategy commenced by exploring the existing structure-
activity-relationships (SAR)4,30–32 of the selected parent drug preladenant in binding to the
A AR to identify the optimal position of the molecule that would be relatively insensitive to
2
A
structural modifications and allow fluorophore attachment without affecting the original
pharmacological properties of the ligand. This preliminary screening was complemented through
in silico studies which were undertaken to rationalize the possible binding interactions
established between the parent ligand and the hA AR (Figure 2).
2
A
ACS Paragon Plus Environment
6

Page 7 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Molecular Docking of preladenant to the crystal structure of the hA AR (PDB=4EIY)
2
a
executed with Schrödinger’s Glide (Schrödinger release 2018-3). The images were generated
with PyMOL (version 2.2.0). Preladenant is shown in spheres colored magenta. The GPCR is
shown in ribbon colored cyan. The key residues are labelled and shown in sticks. A zoomed view
of preladenant (shown in licorice) in the binding pocket is depicted in the box with hydrogen
bonding interactions depicted as dashed lines.
Molecular docking simulations of preladenant to the 1.8 angstrom resolution crystal structure
9
(
PDB = 4EIY) of the hA AR revealed the core pyrazolo[4,3-e][1,2,3]triazolo[1,5-c]pyrimidin-
2A
5-amine (PTP) scaffold interacts with key receptor residues in an equivalent fashion to the
antagonist ZM241385, including polar contacts with Asn253 and Glu169 and a stacking
7
,9
interaction with Phe168 . The side chain of preladenant adopted a conformation extending
towards extracellular loop 3 (ECL3) and, therefore, to a more solvent-exposed region of the
GPCR. These results were further validated by previously reported SAR studies which showed
that modifications at the extended methoxyethylether moiety were well tolerated.4,30,31,33 Thus, it
seemed logical to exploit this side chain of preladenant as a flexible point to explore
linker/fluorophore elaboration. We hypothesized that an amino alkyl spacer would provide
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
sufficient spatial separation between the fluorophore and the orthosteric binding moiety. When
designing fluorescent probes the introduction of a spacer between the parent pharmacophore and
the fluorophore is often included in the design strategy and the nature and the length of the linker
and the fluorophore itself can significantly influence the final pharmacological profile of the
fluorescent conjugate.34,35 Structural modifications must be performed at specific positions which
can be easily tolerated and do not cause significant impact upon pharmacological activity.
Therefore, we undertook modification of the methoxy tail of the parent compound preladenant
by conjugation with an amino alkyl spacer, leading to the corresponding amino functionalized
congener amenable to conjugation with a series of commercially available fluorophores (Scheme
1). This novel series of fluorescent ligands included probes featuring the 4,4-difluoro-4-bora-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
a,4a-diaza-s-indacene (BODIPY) dyes alongside water-soluble dyes such as sulfonated
cyanine-5 (sulfo-Cy5) and AlexaFluor647 fluorophores. These probes were selected on the basis
of several considerations. For example, the use of fluorophores with long-wavelength
excitation/emission spectra is preferred as it minimizes the degree of overlap with background
autofluorescence in living cells.23,35–38 The BODIPY fluorophores embody the first choice for
fluorescent derivatization given their stability and photochemical properties.39 The 6-(((4,4-
difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid
(BODIPY630/650) is often the favored among this family of dyes due to its advantageous
spectral profile (excitation 630 nm, emission 650 nm). It has been previously suggested that
given the lipophilic nature of the probe, the BODIPY moiety is likely to reside within the
membrane bilayer. Indeed, this fluorophore has been shown to be heavily quenched in aqueous
solution, therefore the optimal location would be in a lipid environment as this reduces
background fluorescence from non-bound ligand which is particularly advantageous for imaging
ACS Paragon Plus Environment
8

Page 9 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
34
studies. However, the intrinsic lipophilic nature of these dyes may limit their applicability in
vivo due to the reduced stability in the bloodstream as well as limited detection after IV
4
0
administration. One possible strategy to overcome these issues is by employing water-soluble
fluorophores such as those belonging to the Cy5 and AlexaFluor families of dyes. These water-
soluble dyes also possess good photochemical properties and have therefore been particularly
advantageous for the development of in vivo probes such as in cancer imaging.41
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of the Fluorescent hA AR Antagonists. Based on the information obtained from the
2
A
molecular docking studies, a novel series of fluorescent antagonists for the hA AR receptor
2
A
were synthesized featuring BODIPY-, Cy5- and AF647-based probes. The general route to the
desired probes is illustrated in Scheme 1.
The synthesis comprised ten steps to a common precursor from which six different fluorescent
4
2
analogues were generated. Firstly, a one pot procedure reported by Kuo et al. for the synthesis
of intermediate 4 was followed. This involved reaction of commercially available 2-amino-4,6-
diclhoropyrimidine-5-carbaldehyde (1) with 2-furoic hydrazide in anhydrous THF in the
presence of Et N under reflux for 2h to obtain 2 via nucleophilic aromatic substitution (S Ar).
3
N
Once the desired product was formed, solvent was removed in vacuo and a solution of 2-
hydroxyethylhydrazine in a mixture of CH CN/H O was added to the crude product to displace
3
2
the chlorine at the 2-position through S Ar and afforded compound 3 after ring rearrangement.
N
Thereafter, 3 was cyclised in the presence of a catalytic amount of ZnBr and an excess of
2
POCl3.
a
Scheme 1. Synthesis of the Fluorescent hA AR Antagonists .
2
A
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions a) 2-furoic hydrazide, NEt
3
, dry THF, reflux b) 2- hydroxyethylhydrazine, CH
, 80°C for 5h, 11% over three steps d) TsCl, DIPEA, DMAP 5%mol, DCM, 86% e) CbzCl,
CO , CH CN, 100°C for 6h, 40% g) H2, Pd/C, EtOH, 82% h) 4, DIPEA, DMF, 80°C, 20%
i) 4N HCl in dioxane, RT, quantitative j) 12-14-15-16-17 Fluorophore-NHS, DIPEA, DMF, RT,97% -quantitative;
3 BODIPY carboxylic acid, HATU, DIPEA, DMF, 96%.
3 2
CN/H O,
3 2
reflux c) POCl , ZnBr
K
2
CO
3
, DMF, 66% f) K
2
3
3
1
4
4
This dehydrative cyclization occurred with concomitant Dimroth rearrangement to afford
1
intermediate 4 in a disappointing overall yield (11% over three steps). Mass spectroscopy and H
ACS Paragon Plus Environment
10

Page 11 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
NMR showed that the desired product afforded was a 2:1 mixture of chloride and bromide
+
+
1
derivatives: one single peak at t =2.50, M+H = 304 (4 ) and M+H = 348 (4 ); in the H NMR
R
a
b
analysis the peaks characteristic of the aromatic region of the spectra were in common to both.
Conversely, the peaks characteristic of the alkyl halide tail appeared in the spectra with different
chemical shifts for both bromide and chloride derivatives: peaks at 4.66 ppm (t, J=6.2 Hz, 1H, -
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH Br), 4.60 ppm (t, J=5.9 Hz, 2H, -CH Cl), 4.09 ppm (t, J=5.9 Hz, 2H, -CH CH Cl), 3.94 ppm
2
2
2
2
(
t, J=6.2 Hz, 1H, -CH CH Br).
2
2
Subsequently, synthesis of the amino functionalized tail of the final molecule started by
activating the hydroxyl group of commercially available 5 with tosyl chloride in DCM to afford
6. Under basic conditions, commercially available 1-(4-hydroxyphenyl) piperazine 7 was
protected at the free secondary amino group with benzyl chloroformate to afford the
corresponding benzylcarbamate-protected piperazine derivative 8 with reasonable yield (66%).
Under basic conditions, nucleophilic displacement of the tosyl group of 6 by the phenolic
hydroxyl of 8 afforded the di-protected intermediate 9. This allowed selective removal of the two
protecting groups employing different conditions: hydrogenolysis of the benzylcarbamate group
in the presence of 10% palladium-on-carbon (Pd/C) gave the corresponding free secondary
amine in quantitative yield. Under basic conditions, substitution of the primary ethyl halide tail
of compound 4 by the nucleophilic piperazine nitrogen afforded compound 10 which underwent
acidolytic Boc deprotection to generate the corresponding amine 11 as its di-HCl salt. Finally,
11 was coupled with the appropriate commercially available BODIPY, Cy5 and AF647
succinimidyl esters and to a BODIPY 630/650 carboxylic acid (13) synthesized in our
laboratory45 to afford a focused library of six fluorescent analogues (12-17).
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The novel fluorescent conjugates were isolated and purified by reverse-phase high
performance liquid chromatography (RP-HPLC). The final purity of the fluorescent ligands was
confirmed by analytical RP-HPLC with dual wavelength detection and were determined as being
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0

96% homogenous. Additionally, the chemical identity of probes (12-17) was confirmed by
High Resolution Mass Spectrometry (HRMS) (TOF ES+ and ES-).
Pharmacological Evaluation of the Fluorescent hA AR Antagonists. The novel preladenant-
2
A
based fluorescent ligands were assessed in a variety of pharmacological assays. Firstly, the novel
compounds were validated through a Bioluminescence Resonance Energy Transfer (BRET)46,47
ligand binding assay, a proximity assay (<10 nm) that exploits the energy transfer between the
4
8
donor bioluminescent protein nanoluciferase (NanoLuc) tagged to the receptor of interest at its
N-terminus (Nluc-A AR) and an acceptor fluorescent molecule. For all of the fluorescent
2A
ligands used in the present study, a clear saturable component of specific binding could be
detected (Table 1 and Figure 3) to the hA AR that was associated with low levels of non-
2
A
specific binding across all of the concentrations of fluorescent ligands measured.
ACS Paragon Plus Environment
12

Page 13 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. NanoBRET saturation binding curves measured in HEK293G cells transiently
expressing the NanoLuc- hA AR for a) 12, b) 13, c) 14, d) 15, e) 16 and f) 17 in the presence
2
A
(open circles) and absence (closed circles) of 1M preladenant. The graphs depicted are
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
representative of n = 5 (a, d, e and f) or n = 4 (b and c) separate experiments. Each data point
represents the mean value  SEM performed in triplicate in a single experiment.
The novel fluorescent conjugates were able to bind to the hA AR with good affinity. However,
2
A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the affinity values measured for the water-soluble probes were slightly lower than the BODIPY
conjugates (Table 1). A possible explanation of the differences in binding affinity values among
the two groups of fluorescent ligands could be attributed to the fluorophores employed,
considering they constituted the only structural difference between the compounds. It is possible
that the boron dipyrromethene scaffold of the BODIPY-labelled hA AR fluorescent antagonists
2A
2
8
engages in a cooperative fashion with key residues in an additional pocket of the receptor ,
thereby contributing to increase the binding affinity of the ligands. Conversely, due to the more
hydrophilic nature of the cyanine5 and AF647-bearing probes it is possible that these dyes are
likely to be extending towards the extracellular medium without engaging to key residues. This
hypothesis is consistent with previous work from our laboratories in which it has been
demonstrated that the selective A fluorescent conjugate bearing a Cy5 moiety displayed 300-
3
fold lower affinity compared to the corresponding BODIPY630/650 conjugate with the same
pharmacophore and linker, suggesting that the BODIPY fluorophores directly contribute in
enhancing the binding affinity of ligands.23,28,35
Differences in the BRET ratio observed can be attributed to a variety of factors (Figure 3). For
example, when using green-emitting ligands (excitation 503 nm, emission 512 nm) a greater
contribution from the NanoLuc emission spectra (emission peak 462 nm) is detected in the green
acceptor emission channel (535 nm), leading to an increase in the BRET ratio. Conversely, with
red-shifted ligands the contribution of the NanoLuc emission is markedly reduced in the red
46
acceptor channel and this leads to a reduction in the background BRET ratio. The distance and
ACS Paragon Plus Environment
14

Page 15 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the specific orientation of the acceptor fluorophore with respect to the donor NanoLuc can also
influence the efficiency of the energy transfer between the two species and contribute to the
4
9
absolute differences in the BRET ratio observed .
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Binding Affinities of the novel Fluorescent Ligands determined in whole
HEK293G cells transiently expressing the NanoLuc-hA AR.
2
A
a
Compound
pK (LogM)
n
5
4
4
5
5
5
D
1
1
2 (BY630/650-X)
3 (BY630/650)
7.39  0.05
7.39  0.02
7.77  0.06
7.65  0.10
7.08  0.09
7.22  0.05
14 (BY-FL-X)
15 (BY-FL)
16 (Sulfo-Cy5)
17 (AF647)
a
pK value was calculated from the negative logarithm of the equilibrium dissociation constant
D
(
K ) determined from saturation ligand binding assay using increasing concentration of labelled
D
ligand in the absence or presence (to determine non-specific binding) of unlabeled ligand
preladenant (1μM). Data are expressed as mean  SEM of n experiments, where each experiment
was performed in triplicate.
Achieving A AR selectivity represents one of the major objectives of our study, especially
2
A
when the fluorescent ligands are being developed with the intention of being applied to a variety
of fluorescence-based techniques in different tissues and animal models endogenously
expressing other adenosine receptor subtypes. The NanoBRET saturation ligand binding assay
allowed us to determine the binding affinity of each fluorescent A AR antagonist in HEK293
2
A
cells stably expressing either the NanoLuc-A , NanoLuc-A or the NanoLuc-A ARs. The
1
2B
3
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
results revealed that no specific binding of the fluorescent ligands was detectable at the A , A
1
2B
and A AR subtypes at concentrations tested (Figure 4).
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Specific binding of the novel fluorescent ligands at each subtype of adenosine
receptor. The selectivity profiling of compound 12 a), 13 b), 14 c), 15 d), 16 e) and 17 f) is
depicted by showing only the specific binding at each adenosine receptor subtype. For each
compound the specific binding at each adenosine receptor was obtained by subtracting the non-
specific binding component from the total binding derived from saturation binding experiments
ACS Paragon Plus Environment
16

Page 17 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
see Figure 1 and Table 1). Non-specific binding was defined in the presence of 1 μM
preladenant for NanoLuc-A AR, 10 μM DPCPX for NanoLuc-A , 10 μM MRS1220 for
2
A
1
NanoLuc-A and 10 μM PSB603 for NanoLuc-A , respectively. The graphs shown above are
3
2B
representative of n=4 (c and d) and n=5 (a, b, e and f) experiments and the data are mean  SEM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of triplicate determinations in a single representative experiment.
Given the higher affinity displayed at the A AR, the BODIPY-labelled ligands were selected
2
A
to assess whether the novel synthesized probes were able to retain the functional antagonistic
ability of the parent compound preladenant. This was performed using a previously described
CRE-SPAP reporter gene assay50 in CHO cells expressing the human adenosine hA AR
2A
receptors. All the BODIPY-labelled ligands were able to inhibit adenosine-5-N-
ethylcarboxamide (NECA)-mediated increase in SPAP production in a competitive fashion,
causing a rightward shift of the NECA response curve as shown in Figure 5. From these data, it
was possible to perform Schild regression analysis and estimate the affinity of each compound as
shown in Table 2.
Table 2. pA values of the BODIPY-labeled Fluorescent Ligands.
2
a
compound
hA AR pA
n
7
6
5
6
2A
2
†
1
1
1
1
2 (BY630/650-X)
3 (BY630/650)
4 (BY-FL-X)
5 (BY-FL)
9.31 0.38
8.61 0.30
7.91 0.26
8.40 0.26†
a
Affinity of the BODIPY-labelled Fluorescent Ligands. Ability for these ligands to inhibit the
NECA-stimulated CRE-SPAP response in CHO cells expressing the hA AR. Data are mean 
2
A
SEM for n separate experiments performed in triplicate (statistical significance was defined as p
†
< 0.05 unpaired t-test comparing 12 and 14. p < 0.05 unpaired t-test comparing the binding
affinity of 12 and 15 measured in CRE-SPAP assay and the NanoBRET assay).
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Inhibition of NECA-stimulated SPAP-secretion by the antagonists 12 (a), 13 (b), 14
(c) and 15 (d). CHO CRE-SPAP cells expressing the human A receptor were stimulated with a
2
A
concentration range of the agonist NECA in the absence and presence of the fluorescent
antagonist of interest (1 nM, 10 nM, 100 nM for 12 and 15; 10 nM, 100 nM, 1000 nM for 13 and
1
4). Data have been expressed as a percentage of the SPAP production in response to serum-free
media (0%) and to 1M forskolin (FSK, 100%). The data shown represent the combined mean 
SEM of n=7 (a), n=6 (b and d) and n=5 (c) experiments, where each experiment was performed
in triplicate.
ACS Paragon Plus Environment
18

Page 19 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The fluorescent ligands largely retained their functional antagonism as compared to the parent
compound preladenant. Moreover, when comparing fluorescent ligands bearing a short linker
(see Scheme 1) no significant difference in the measured functional affinity was observed (p >
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.05, comparing compounds 13 and 15, unpaired t-test). Conversely, when comparing
fluorescent ligands containing a longer linker the BODIPY 630/650 conferred 20-fold increase in
affinity to the congener (p < 0.05, comparing compounds 12 and 14, unpaired t-test). In this
pharmacological assessment, compound 12 was the most potent of the series (pA = 9.31  0.38,
2
Figure 5A and Table 2). Interestingly, the affinity values measured via the NanoBRET saturation
ligand binding assay (Table 1) were significantly lower compared to the affinity values obtained
in CRE-SPAP gene-reporter assay (Table 2). The discrepancy in the affinity values is most likely
due to the different host cell type and species (hamster versus human) employed in the two
assays (CHO cells for CRE-SPAP and HEK293G cells for NanoBRET), the assay configuration
(e.g. the influence of the furimazine substrate for NanoLuc) as well as the influence of the tag
used in the NanoBRET experiments (transiently expressed NanoLuc-hA AR for NanoBRET
2
A
5
1
and stably expressed wild-type hA AR for CRE-SPAP). However, it is important to note that
2
A
a small reduction in binding affinity of the fluorescent probe is not necessarily detrimental as
long as the probe binds to the targeted receptor with sufficient specificity.52
Measuring the binding kinetics of a drug candidate is becoming extremely relevant considering
the emerging concept that the rates at which a drug binds to its molecular target (k ) and
on
dissociates from it (k ) may be considered more valuable parameters for predicting the
off
effectiveness of a drug candidate in vivo.53,54 In this regard, the BODIPY630/650-X-labelled
analogue 12 was selected to be screened for its kinetic profile through a time-resolved
fluorescence energy transfer (TR-FRET)55–58 assay in membrane preparations from SNAP-
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tagged-hA AR-HEK293 cells (Figure 6). TR-FRET is a methodology which exploits proteins
2
A
labeled with lanthanides (e.g. terbium) as donor fluorophores. Lanthanides display a long
emission lifetime which allows for a time delay of the measurements between the excitation and
the fluorescence of the acceptor fluorophore. This increases the sensitivity of the assay due to a
reduction of the short-lived background fluorescence.52,55,58 The TR-FRET technology allowed
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
us to monitor the binding kinetics of 12 by measuring the observed association constant (K ) at
obs
four different concentrations of fluorescent ligand over a period of 1 h. The specific FRET signal
was obtained by subtracting the non-specific signal (determined in the presence of 10 M
6
ZM241385) from the total signal at each time point. The kinetic parameters k = 2.4  0.3 x 10
on
-1
-1
-1
M min , k = 0.029  0.004 min , pK = 7.91  0.06 and the residence time (1/ k ) Tr =
off
D
off
35.36  4.11 min, n = 5 were then calculated by globally fitting the resulting FRET data. This
study revealed that fluorescent conjugate 12 has a relatively rapid on-rate but once bound
dissociates slowly. In addition, from the 60 min time point we were also able to plot an
equilibrium saturation curve of 12 and directly measure the equilibrium dissociation constant
(
K = 15.47  1.92 nM, Figure S2) which was comparable to the value obtained from the kinetic
D
analysis fit.
ACS Paragon Plus Environment
20

Page 21 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. TR-FRET association kinetic curve of specific 12 binding to terbium-labelled SNAP-
o
tagged-hA AR-HEK293 cell membranes at 37 C. Cell membranes were treated with the
2
A
indicated concentration of fluorescent ligand and the FRET signal was monitored every 30 s for
h. Non-specific binding was determined in the presence of 10 M ZM241385. The graph
1
presented is a representative of n = 5 experiments performed in triplicate.
Interestingly we found that the binding affinity pK of 12 measured in this kinetic study (7.91
D

0.06) in HEK293G cell membranes was similar to the pK (7.39  0.05) measured in the same
D
cells in NanoBRET experiments. These data suggest that the large difference in observed binding
affinity for 12 between NanoBRET experiments in HEK293G cells and CRE-SPAP functional
experiments in CHO-K1 cells is largely due to the host cellular environment. Having established
that the novel fluorescent probes retain functional activity and binding affinity at the hA AR we
2A
next evaluated their utility for confocal microscope imaging studies. Ideally, the novel
fluorescent ligands should bind specifically to the A AR receptors on the cell surface and show
2
A
both low levels of nonspecific binding and diffusion into the cell cytosol. From the BODIPY-
labelled probes, we selected compounds 12 (red-emitting) and 15 (green-emitting) (Scheme 1)
and determined whether the selected compounds were suitable for visualization of receptors in
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
live cells. To this end confocal microscopy was conducted on HEK293 cells expressing the
SNAP-tagged hA AR (Figure 7). The images captured with the confocal microscope revealed
2
A
that both compounds 12 and 15 bound specifically to the SNAP-tagged hA AR shown by the
2
A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
high level of fluorescence localized at the cell membrane. Pre-incubation with 1 M of unlabeled
antagonist ZM241385 produced a significant reduction of the fluorescence intensity and very
little intracellular fluorescence was observed. This demonstrated that binding of both compounds
was specific at the SNAP-tagged hA AR as it was surmounted by high concentration of
2
A
unlabeled hA AR antagonist.
2
A
ACS Paragon Plus Environment
22

Page 23 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Live cell confocal imaging of 12 (10 nM) and 15 (30 nM) binding to SNAP-tagged
hA AR HEK293 cells in the absence or presence of 1 μM of unlabelled antagonist ZM241385.
2
A
o
Cells were incubated at 37 C with BG-AlexaFluor488 when imaging red-shifted ligands (12) or
BG-AlexaFluor647 to label the SNAP-tag on the N-terminus of the receptor when imaging
green-shifted ligands (15) (middle frames). Images are representative of images captured in three
separate experiments (scale bar 20 μm).
We also investigated whether the water-soluble hA AR fluorescent ligands were able to allow
2
A
receptor visualization through confocal imaging. The results revealed that probes 16 and 17 bind
to the SNAP-tagged hA AR as demonstrated by the high degree of fluorescence observed at the
2
A
cell membranes (Figure 8). Most importantly, the binding of the two ligands to the A AR was
2
A
highly specific as pre-incubation with 1 M ZM241385 completely inhibited the binding of each
fluorescent ligand. Very little non-specific binding was detected and rewardingly, no measurable
uptake of these ligands into in the cytosol could be seen.
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
24

Page 25 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 8. Live cell confocal imaging of 16 (30 nM) and 17 (30 nM) binding to SNAP-tagged
o
hA AR expressed in HEK293 cells. Cells were incubated 30 min at 37 C with BG-
2
A
AlexaFluor488 prior incubation in the absence or presence of 1 M ZM241385. Subsequently,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
cells were incubated with 30 nM fluorescent ligand for 30 min at 37 C prior to imaging. The
images generated are from one single experiment and are representative of three different
experiments. The images were all captured using the same settings for laser power and detector
gain. Scale bars are 50 m.
Compound 17 was selected for further studies. In particular, the utility of AlexaFluor647-
bearing probe 17 in measuring the binding affinities of unlabelled hA AR antagonists was
2A
investigated using the NanoBRET competition ligand binding assay. HEK293 cells transiently
expressing the NanoLuc-hA AR were incubated with 17 (50 nM) and increasing concentrations
2
A
of the hA AR antagonists ZM241385, SCH58261, SCH442416 and MRS1334 a selective
2A
adenosine A AR antagonist which was used as negative control. A concentration dependent
3
decrease of the BRET signal was observed for the A AR selective compounds, allowing the
2
A
binding affinities of the unlabeled antagonists to be measured (Figure 9 and Table 3). The values
obtained through fluorescent binding assays were comparable to the reported literature values
2
4,59
(
R = 0.97, Figure S1) measured in radioligand binding experiments.
No concentration-
dependent decrease of the BRET signal was observed in the presence of the selective A AR
3
antagonist MRS1224 confirming the specificity of the binding of the probe 17 to the A AR.
2A
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 9. Displacement of 17 (50 nM) by unlabeled hA AR antagonists ZM241385,
2
A
SCH58261, SCH442416 and hA AR antagonist MRS1334 in HEK293G cells transiently
3
expressing the Nanoluc-hA AR. Blue and red bars represent the total BRET signal of 17 in
2
A
absence or presence of ZM241385 (10 M) respectively. The decrease in BRET ratio was
monitored as a function of increasing concentration of unlabeled antagonist. Each data point
represents mean value  SEM of triplicate determinations in a single representative experiment
of n = 4 experiments.
Table 3. Binding affinities (pK ) of target hA AR antagonists calculated from Cheng-
i
2A
Prusoff analysis of the data displayed in Figure 9.
a
Compound
ZM241385
SCH58261
SCH442416
pK (BRET binding assay)
pK (literature).
i
i
8.80 b
8.30 c
8.38 d
8.84  0.09
7.92  0.10
7.90  0.10
a
b, c
Reported binding affinities of target hA AR antagonists.
Values determined in
2
A
radioligand binding experiments in CHO-hA2A and HEK293-hA2A cells using [H3]
SCH58261.59,60 d Values determined in HEK293-hA2A using the radiolabelled agonist [ H] CGS
3
ACS Paragon Plus Environment
26

Page 27 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
0
2
1680. The data measured through NanoBRET competition ligand binding assay are means 
SEM of n = 4 experiments performed in triplicate.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
In the present study, we have reported the successful design, development and
pharmacological evaluation of a novel series of hA AR fluorescent antagonists, encompassing
2
A
BODIPY-bearing probes and water soluble Cy5-labelled and AF647-labelled probes, based on
the high affinity and selective preladenant scaffold. In particular, derivatization of the amino
functionalized congener 11 with two families of fluorescent tags led to six hA AR fluorescent
2
A
antagonists with retained affinity and, most importantly, selectivity at the A AR subtype. Both
2
A
families of compounds allowed visualization of A ARs expressed in live cells as demonstrated
2A
by the high fluorescence intensity at the cell membranes. The binding of these fluorescent
ligands could be surmounted by a high concentration of ZM241385 confirming that the binding
of the probes was specific. Very little non-specific binding was observed for the water-soluble
fluorescent antagonists (Figure 8). Within the water-soluble family of probes, fluorescent
conjugate 17 was selected to successfully monitor ligand binding of reported hA AR
2
A
antagonists (Figure 9 and Table 3).
The novel hA AR fluorescent ligands developed can be broadly applied in a series of
2
A
fluorescence-based techniques to study the signaling and dynamics of the adenosine A AR
2
A
receptors in their native cellular environment spanning from in vitro to in vivo applications. Most
importantly we believe that these ligands in combination with the NanoBRET approach and
bioimaging studies can help to shed light on the complexity of the A AR signaling in different
2
A
biological elements and during disease progression. This ultimately should provide useful
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
insights concerning specific A AR-engagement in several disease phenotypes, including
2
A
cancer.13,61,62
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Chemistry: Materials and Methods
Chemicals and solvents of analytical and HPLC grade were purchased from commercial
suppliers and used without further purification. BODIPY630/650-X-SE, BODIPY-FL-X-SE, and
BODIPY-FL-SE were purchased from Molecular Probes (Thermo Fisher Scientific).
AlexaFluor647 NHS ester was purchased from Thermo Fischer Scientific and Sulfo-Cyanine5
NHS ester from Lumiprobe. All reactions were carried out at ambient temperature unless
otherwise stated. Reactions were monitored by thin-layer chromatography on commercially
2
54
available silica pre-coated aluminium-backed plates (Merck Kieselgel 60 F ). Visualisation was
under UV light (254 nm and 366 nm), followed by staining with ninhydrin or KMnO dips. Flash
4
column chromatography was performed using silica gel 60, 230-400 mesh particle size (Sigma
1
Aldrich). NMR spectra were recorded on a Bruker-AV 400. H NMR spectra were recorded at
1
3
13
1
4
00.13 MHz and C NMR spectra at 101.62 MHz. All C NMR are H broadband decoupled.
Solvents used for NMR analysis (reference peaks listed) were CDCl supplied by Cambridge
3
Isotope Laboratories Inc., (δH = 7.26 ppm, δC = 77.16) Chemical shifts (δ) are recorded in parts
per million (ppm) and coupling constants are recorded in Hz. The following abbreviations are
used to described signal shapes and multiplicities; singlet (s), doublet (d), triplet (t), quadruplet
(q), broad (br), dd (doublet of doublets), ddd (double doublet of doublets), dtd (double triplet of
doublets) and multiplet (m). Processing of the NMR data was carried out using the NMR
software Topspin. LC-MS spectra were recorded on a Shimadzu UFLCXR system coupled to an
ACS Paragon Plus Environment
28

Page 29 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Applied Biosystems API2000 and visualised at 254 nm (channel 1) and 220 nm (channel 2). LC-
MS was carried out using a Phenomenex Gemini-NX C18 110A, column (50 mm × 2 mm x 3
μm) at a flow rate 0.5 mL/min over a 5 min period (Method A). All high resolution mass spectra
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(HRMS) were recorded on a Bruker microTOF mass spectrometer using MS electrospray
ionization operating in positive ion mode. RP-HPLC was performed on a Waters 515 LC system
and monitored using a Waters 996 photodiode array detector at wavelengths between 190 and
8
00 nm. Spectra were analysed using Millenium 32 software. Semi-preparative HPLC was
performed using YMC-Pack C8 column (150 mm × 10 mm × 5 μm) at a flow rate of 5.0 mL/min
using a gradient method of 20-70% B over 21 minutes (Solvent A = 0.01% formic acid in H O,
2
solvent B = 0.01% formic acid in CH CN (Method B)). Analytical RP-HPLC was performed
3
using a YMC-Pack C8 column (150 mm × 4.6 mm × 5 μm) and a Phenomenex Gemini NX-C18
column (250 mm × 4.6 mm × 5 μm) at a flow rate of 1.0 mL/min. Final products were one single
peak and >98% pure. The retention time of the final product is reported using a gradient method
of 10-90% solvent B in solvent A over 30 min. (Solvent A = 0.01% formic acid in H O, solvent
2
B = 0.01% formic acid in CH CN (Method C)).
3
General Procedure 1: Synthesis of Fluorescent Conjugates. The amine congener (as the di-
HCl salt) was dissolved in DMF (0.5 mL), treated with DIPEA (0.6 L 3.7 mmol, 4 eq.) and then
reacted with BODIPY630/650–X-NHS (0.5 mg, 1 eq.), BODIPY-FL-X-NHS (1 eq., 0.5 mg),
BODIPY-FL-NHS (1 eq., 0.5 mg), Sulfo-Cyanine5-NHS ester (0.8 mg, 1 eq.), AlexaFluor647-
NHS (0.8 mg, 1 eq.) and HATU (1.7 mg, 1 eq.) prior reacting with BODIPY630/650-carboxylic
acid (2 mg). The resulting solution was stirred at RT under the exclusion of light for 1-5 h. Upon
completion of the reaction monitored by LC-MS, the solution was concentrated to dryness and
ACS Paragon Plus Environment
29