Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

Article abstract of DOI:10.1016/j.bmc.2018.10.039

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs. Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.

Full text of DOI:10.1016/j.bmc.2018.10.039

Accepted Manuscript
Novel fluorescent triazinobenzimidazole derivatives as probes for labelling hu-
man A1 and A2B adenosine receptor subtypes
Elisabetta Barresi, Chiara Giacomelli, Simona Daniele, Ilaria Tonazzini, Marco
Robello, Silvia Salerno, Ilaria Piano, Barbara Cosimelli, Giovanni Greco,
Federico Da Settimo, Claudia Martini, Maria Letizia Trincavelli, Sabrina Taliani
PII:
S0968-0896(18)31368-3
https://doi.org/10.1016/j.bmc.2018.10.039
BMC 14598
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 July 2018
19 October 2018
29 October 2018
Please cite this article as: Barresi, E., Giacomelli, C., Daniele, S., Tonazzini, I., Robello, M., Salerno, S., Piano, I.,
Cosimelli, B., Greco, G., Da Settimo, F., Martini, C., Trincavelli, M.L., Taliani, S., Novel fluorescent
triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes, Bioorganic
& Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.039
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Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human
A1 and A2B adenosine receptor subtypes.
Elisabetta Barresia,†, Chiara Giacomellia,†, Simona Danielea, Ilaria Tonazzinib, Marco
Robelloc, Silvia Salernoa, Ilaria Pianoa, Barbara Cosimellid, Giovanni Grecod, Federico
Da Settimoa, Claudia Martinia, Maria Letizia Trincavellia,*, Sabrina Taliania
† These Authors contributed equally.
aDipartimento di Farmacia, Università di Pisa, Via Bonanno 6, I-56126 Pisa, Italy
bIstituto Nanoscienze-CNR @NEST (National Enterprise for NanoScience and
NanoTechnology), Piazza San Silvestro 12, Pisa, Italy
cNIDDK (National Institute of Diabetes and Digestive and Kidney Diseases), NIH, 8
Center Drive, Bethesda, MD 20814
dDipartimento di Farmacia, Università di Napoli “Federico II”, Via Domenico
Montesano 49, 80131 Naples, Italy.
* Email: maria.trincavelli@unipi.it; Tel: +39 050 2219523
Keywords: Adenosine receptors; fluorescent probes; triazinobenzimidazole derivatives;
mesenchymal stem cells; A2B AR subtype.
1

Abstract
The expression levels and the subcellular localization of adenosine receptors (ARs) are
affected in several pathological conditions as a consequence of changes in adenosine
release and metabolism. In this respect, labelled probes able to monitor the AR
expression could be a useful tool to investigate different pathological conditions.
Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD)
group linked to the N1 (1,2) or N10 (3,4) nitrogen of a triazinobenzimidazole scaffold,
were synthesized. The compounds were biologically evaluated as fluorescent probes for
labelling A1 and A2B AR subtypes in bone marrow-derived mesenchymal stem cells
(BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized
compounds towards the different AR subtypes was determined. The probe 3 revealed a
higher affinity to A1 and A2B ARs, showing interesting spectroscopic properties, and it
was selected as the most suitable candidate to label both AR subtypes in
undifferentiated MSCs.
Florescence confocal microscopy showed that compound 3 significantly labelled ARs
on cell membranes and the fluorescence signal was decreased by the cell pre-incubation
with the A1 AR and A2B AR selective agonists, R-PIA and BAY 60-6583, respectively,
thus confirming the specificity of the obtained signal. In conclusion, compound 3 could
represent a useful tool to investigate the expression pattern of both A1 and A2B ARs in
different pathological and physiological processes. Furthermore, these results provide
an important basis for the design of new and more selective derivatives able to monitor
the expression and localization of each different ARs in several tissues and living cells.
2

1. Introduction
In last decades, the use of fluorescent probes for studying membrane G-coupled protein
receptors (GPCRs) has gained great importance. Given the recent improvements in
imaging technologies, fluorescent probes have become attractive alternatives to
radioligands, which are inevitably associated with peculiar drawbacks, such as high
costs and risks related to handling radioactive materials.1,2
Ideally, a fluorescent probe should retain the pharmacological profile of the parent
unlabelled ligand so as to guarantee optimal conditions for binding studies, including
cellular localization and real-time monitoring of processes triggered by ligand-receptor
interactions in living cells (i.e. internalization, trafficking, sequestration and recycling,
identification of ligand binding site).1,2
Various fluorescent probes have been developed over the past few years for
characterizing adenosine receptors (ARs), and their use in fluorescence resonance
energy transfer (FRET), confocal analysis and flow cytometry (FCM) has been proven
feasible.3-5
The nucleoside adenosine plays an important role in many physiological and
pathological conditions through activation of specific GPCRs, classified as A1, A2A,
A2B, and A3 ARs.6,7 The binding of adenosine to ARs activate G protein-dependent
signalling pathways, such as adenylate cyclase (A1, A2A, A2B and A3 ARs), calcium or
potassium channels (A1 AR), phospholipase C (A1, A2B and A3 ARs) and phospholipase
D (A3 AR),6,7 cascade of mitogen-activated protein (MAP) kinases (A1, A2A and A2B
ARs).8-10
Over the past decades, we have been working on several classes of ligands binding with
high affinity and selectively to various AR subtypes, most of them characterized by an
antagonist efficacy profile.11-21These classes share common structural features
consisting in an aromatic polycyclic nitrogen-containing core bearing mostly lipophilic
substituents and, less often, groups capable to make hydrogen bonds. In some cases,
different substitution pattern on the same scaffold yielded compounds with switched
selectivity from one receptor subtype to a different one.11-13,17,18 Among the ARs-
targeting scaffolds, the triazinobenzimidazole system (general structure I, Chart 1),
originally used for the identification of ligands for the central benzodiazepine receptor,22
was then employed to obtain potent and selective A1 AR antagonists.11,12 Subsequently,
potent triazinobenzimidazole-based A2B AR antagonists were disclosed with scarce or
moderate affinity for A3 and A1 ARs, their degree of A2B/A2A AR selectivity being
3

strictly dependent on the nature of the aryl group at the 3-position of the scaffold.18
More recently, based on this chemotype, a novel carbon-11 labelled tracer was
synthesized and tested by positron emission tomography (PET) to evaluate its
pharmacokinetics in vivo, and to ascertain its potential use for A2B AR imaging.23 As a
result, we identified a probe with a high chemical stability, a good pharmacokinetic
profile, and the ability to image A2B AR. Such a compound represents the first A2B AR
targeting PET tracer disclosed so far.23 Taken together, all these findings supported the
potential usefulness of the triazinobenzimidazole scaffold I (Chart 1) in the design of
novel ARs ligands suitable as molecular probes for imaging experiments.
Recent evidences indicate that adenosine plays a crucial role in mesenchymal stem cell
(MSC) biology by controlling the fates of these cells.24 MSCs are multipotent stem cells
that can differentiate into osteoblasts and adipocytes.24 It has been reported that MSCs
express various AR subtypes,24,25 whose expression differently varies during the
differentiation process.24
Specifically, in human bone marrow-derived (BM)-MSCs, A2B AR is the dominant
receptor promoting osteoblast differentiation, as demonstrated by an increase in alkaline
phosphatase activity Runx2 expression.24-26 D'Alimonte et al.27 have demonstrated that
activation of A1 AR promotes osteogenic differentiation of human dental pulp-derived
MSCs, similarly to what observed upon A2B AR activation. According to Eisenstein, A.
et al.,28 A1 and A2A ARs are implicated in the regulation of BM-MSC differentiation to
adipocytes.
Although the role of ARs in lineage-specific differentiation of MSCs is widely
accepted, the specific involvement of A1 and A2B ARs in the different phases of
differentiation and the related underlying mechanisms are still unclear.
The present paper describes our studies aimed at developing novel
triazinobenzimidazole fluorescent ligands of human ARs as suitable tools to monitor the
evolution of AR expression during early stage of BM-MSC differentiation to
osteoblasts. Specifically, herein we report the synthesis, the spectroscopic properties
and the biological evaluation of novel probes 1-4, bearing the fluorescent moiety linked
to the N1 (compounds 1,2) or N10 (compounds 3,4) nitrogen (Chart 1) of the
triazinobenzimidazole scaffold. The position and the length of the spacer chain were
modified to modulate conformational flexibility required to self-adaptation of the
molecules into the receptor binding site. The well-known 7-nitrobenzofurazan (NBD)
group was selected as the fluorophore for its small sizes. Moreover, thanks to the
4

solvatochromic properties of NBD, NBD-containing compounds typically exhibit a low
quantum yield in aqueous solutions, but they become highly fluorescent in nonpolar
solvents or when bound to membranes as well as to protein hydrophobic clefts.29 This
fluorophore was successfully employed by our group for developing highly performant
fluorescent probes specifically targeting the translocator protein (TSPO).30-32 Indeed, a
NBD-containing TSPO ligand has been successfully used to detect the TSPO
mitochondrial localization in cultured Drosophila S2 cells.33
Chart 1. Structures of triazinobenzimidazole ARs ligands I11,12,18 and fluorescent
probes 1-4.
2. Results and Discussion
2.1. Chemistry
The target compounds 1,2 and 3,4 were synthesized using the triazinobenzimidazoles 9
and 10 as reactants, respectively, which in turn were prepared starting from compound
8; this last product was obtained according to the experimental procedure described in
our previous work (Scheme 1).22 Briefly, 2-hydrazinobenzimidazole (6) was synthesized
from hydrazine monohydrate and 2-chlorobenzimidazole (5) and then reacted with
phenylglyoxylic acid in absolute ethanol to give the intermediate 7, which was finally
cyclized by refluxing for 12 hours in glacial acetic acid, yielding compound 8.22
Due to the tautomeric equilibrium involving 8, treatment of this compound with sodium
hydride and ethyl bromoacetate induced the alkylation at the N1 or N10, affording a
mixture of the regioisomer derivatives 9 (yield 51%) and 10 (yield 22%), respectively,
which were finally separated and purified by flash chromatography (Scheme 1).
5

Scheme 1
Reagents and conditions: i) hydrazine monohydrate, 180 °C, 5h; ii) phenylglyoxylic acid, absolute
ethanol, 80 °C, 2h; iii) glacial acetic acid, 120 °C, 12h; iv) NaH, ethyl bromoacetate, anhydrous DMF,
room temperature, overnight.
1
13
Because of the high similarity of the H-NMR and C-NMR spectra (see Experimental
Section) of regioisomers 9 and 10, these structures were unequivocally attributed by the
synthesis of compound 10 following the procedure reported in Scheme 2, and
subsequent HPLC analysis. The commercial 2-chlorobenzimidazole (5) was firstly N-
alkylated to compound 11 with ethyl bromoacetate and sodium hydride. Subsequent
treatment with hydrazine monohydrate furnished derivative 12, which was directly
reacted with phenylglyoxylic acid in absolute ethanol. In these experimental conditions,
we directly obtained the cyclized compound 10. HPLC analysis, performed with the
mobile phase delivered at isocratic flow and consisting of acetonitrile (70%) and water
(30%) and a flow rate of 1.0 mL/min, revealed a retention time for compound 10 of 5.6
min, with a purity degree > 95%. HPLC analysis repeated in the same conditions for
regioisomers 9 and 10, both in mixture and after separation by flash chromatography,
revealed different retention times for compounds 9 and 10 (8.3 min and 5.6 min,
respectively), allowing their structural assignment (chromatograms are reported in Fig.
S1, S2, S3, S4, Supplementary Data).
6

Scheme 2
Reagents and conditions: i) NaH, ethyl bromoacetate, anhydrous DMF, room temperature, 3h; ii)
hydrazine monohydrate, 180 °C, 5h; iii) phenylglyoxylic acid, absolute ethanol, 80 °C, 2h.
The synthetic procedure for the obtainment of N1- and N10-substituted derivatives (1,2
and 3,4, respectively) is outlined in Scheme 3.
Scheme 3
Reagents and conditions: i) N-Boc-1,4-butanediamine or N-Boc-1,6-hexanediamine, 80 °C, 3h; ii)
trifluoroacetic acid, DCM, room temperature, 2h; iii) NBD-Cl, NEt3, anhydrous DMF, room temperature,
overnight.
7

Compound 9 and 10 were condensed with N-Boc-1,4-butanediamine and N-Boc-1,6-
hexanediamine in a Pyrex capped tube at 80 °C for 3 hours to obtain derivatives 13-16.
Simple deprotection of the BOC group with trifluoroacetic acid in DCM gave the amino
derivatives 17-20, which were then reacted with the commercial 4-chloro-7-
nitrobenzofurazan (NBD-Cl) in anhydrous DMF, in the presence of triethylamine,
yielding the targets fluorescent probes 1-4, finally purified by flash-chromatography.
2.2. Binding profile of compounds 1-4 to adenosine receptors.
The affinities of 1-4 for human A1 and A2A ARs were evaluated in competition binding
experiments by assessing their abilities to displace [3H]8-cyclopentyl-1,3-
dipropylxanthine ([3H]DPCPX) and [3H]5′-N-ethylcarboxamideadenosine ([3H]NECA)
binding from transfected CHO cells, respectively. The experiments were performed as
described elsewhere.34 The ability of 1-4 to bind to A3 AR was evaluated through
functional assays.21 The binding data of 1-4 at the human A1, A2A, and A3 ARs are
summarized in Table 1, together with those of the parent compound 8,18 DPCPX,
NECA, and Cl-IBMECA reported as references.
8

Table 1. Binding of the triazinobenzimidazole derivatives 8, 1-4 at human A1, A2A and
A3 ARs.a
hA1
hA2A
hA3
IC50 (nM)a,e
cpd
n
Ki (nM)a,b
Ki (nM)a,c
Ki (nM)a,d
8f
1
2
3
4
-
728 ± 38
> 10,000
> 10,000
1380 ±120
> 10,000
0.5 ± 0.03
14 ± 4
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
337 ± 28
16 ± 3
> 1,000
-
4
6
4
6
-
> 10,000
-
> 10,000
-
-
> 10,000
> 10,000
-
-
-
DPCPX
 1,000
73  5
0.22  0.02
NECA
890 ± 61
401 ± 25
CI-IBMECA
aThe Ki and IC50 values are the means ± SEM of at least three determinations derived from an
b
iterative curve-fitting procedure (Prism program, GraphPad, San Diego, CA). Displacement of
specific [3H]DPCPX binding in membranes obtained from hA1 AR stably expressed in CHO
3
c
cells. Displacement of specific [ H]NECA binding in membranes obtained from hA2A AR
stably expressed in CHO cells. dDisplacement of specific [125I]AB-MECA binding in
e
membranes obtained from hA3 AR stably expressed in CHO cells. The IC50 values correspond
to the concentrations reducing by 50% the effect of the selective A3 AR agonist Cl-IB-MECA.
fData taken from ref. 18.
None of the new compounds displayed any significant binding affinity for A1, A2A and
A3 ARs, with the exception of 3 that showed micromolar affinity for A1 AR (Ki 1380
nM). Functional cAMP assays demonstrated that 3 behaves as antagonist at A1 AR (Fig.
1A). This compound was able to counteract the NECA-mediated inhibition of cAMP
accumulation in human A1 AR-transfected CHO cells in a concentration-dependent
manner, with an IC50 value comparable with the Ki value measured in the binding assay
(IC50 1112 nM).
9

B
A
CHO hA1
CHO hA2B
100
80
60
40
20
100
80
60
40
20
0
-12
-10
-8
-6
-4
-12
-10
-8
-6
-4
Log [3]
Log[3]
Figure 1. Evaluation of the antagonist profile of 3 towards the A1 and A2B AR subtypes.
A) A1 AR transfected CHO cells were treated with 1 µM forskolin and 10 nM NECA in
the absence or presence of different concentrations of the tested compound (1 nM-10
µM). Data are expressed as the percentage of cAMP intracellular levels with respect to
forskolin, which was set to 100%, and represent the mean ± SEM of at least three
different experiments. Each experiment was performed in duplicate. B) A2B AR-
transfected CHO cells were treated with EC50 NECA concentration (100 nM) in the
absence or presence of different concentrations (0.01 nM–10 μM) of the tested
compound. After 15 min incubation, the reaction was stopped and the intracellular
cAMP levels were quantified. The results are expressed as the percentage of NECA-
stimulated cAMP levels, which was set to 100%. The data represent the mean ± SEM of
at least three different experiments, each performed in duplicate.
Compounds 1-4 were then evaluated for their ability to modulate the increase in cAMP
levels, either alone or in the presence of an EC50 concentration of the agonist NECA in
human A2B AR-transfected CHO cells (Table 2). When tested alone at a 10 µM
concentration, all the compounds did not significantly increase cAMP levels,
demonstrating a lack of A2B AR agonist activity. Compounds 1 and 3 significantly
counteracted the NECA-mediated increase in cAMP, suggesting that they may act as
A2B AR antagonists (Table 2).
10

Table 2. Effects of triazinobenzimidazole derivatives 1-4 on cAMP production in CHO
cells expressing human A2B AR.a
% cAMP production
(vs agonist maximal effect, 100%)b
cpd
hA2B AR
– agonist
3.4 ± 1.2
5.1 ± 2.1
1.2 ± 0.8
1.7 ± 1.4
+ agonist
66.3 ± 4.5*
101.0 ± 7.4
20.7 ± 7.5***
89.6 ± 6.5
1
2
3
4
a Each compound was tested at 10 µM concentration on cAMP production in CHO cells
expressing human A2B ARs (see Biology section). Each compound was tested alone or
b
in the presence of 100 nM of NECA. Data are expressed as percentage of cAMP
production versus agonist maximal effect (100%). All data represent the mean ± SEM of
at least three different experiments each performed in duplicate. *P<0.05, ***P<0.001
vs agonist alone.
Next, the potency of 1 and 3 for AR2B AR was assessed. Compound 1 did not entirely
counteract the NECA-induced cAMP production; thus, its IC50 value in this experiment
could not be determined (>10,000 nM). Conversely, compound 3 completely
counteracted the NECA-mediated increase of cAMP accumulation in human A2B AR-
transfected CHO cells, in a concentration-dependent manner (Fig. 1B), with an IC50
value in the micromolar range (IC50 1350 nM). The presence of the NBD fluorophore
significantly affected 3 potency towards A2B AR with respect to its parent compound 8
(IC50 1.40 nM).18 These results suggested that the side chain bearing the NBD moiety is
hosted in a lipophilic pocket of limited dimensions, such as to allow a moderate
interaction only to the shortest linker at 10-position.
Given the ability of 3 to bind to A1 and A2B ARs in functional assays, this compound
was investigated for its spectroscopic properties to evaluate its potential to visualize
ligand-receptor interactions.
2.3. Spectroscopic properties of the fluorescent probe 3.
The UV and the emission spectra of 3 were measured to investigate the spectroscopic
properties of this compound as a fluorescent probe and how these might be affected by
the chemical environment. Compound 3, dissolved in dimethyl sulfoxide (DMSO), was
11

diluted to a final concentration of 10 µM in different assay solutions (from aqueous to
60% v/v dioxane-water phosphate-buffered saline (PBS)). The percentage of DMSO did
not exceed 1% of the final assay volume solution.
Fig. 2A shows the absorption spectra of 3. In an aqueous solution, the absorption
spectrum of this compound is characterized by two absorption maxima at 366 and 478
nm with extinction coefficients of ε = 10286 M−1 cm−1 and ε = 8857 M−1 cm−1,
respectively. When 3 was dissolved in the PBS solution containing 20% of dioxane, the
wavelengths of the absorption maxima remained nearly unchanged, but the spectrum
was characterized by an increase in both extinction coefficients; this effect was much
more evident at the longer wavelength. A further increase in hydrophobicity of the
medium (40% dioxane/water (v/v) PBS or 60% dioxane/water (v/v) PBS) caused a
slight increase in the extinction coefficients.
Fig. 2B shows the emission spectra of 3. As expected, the decrease in polarity of the
environment (from aqueous buffer to 60% dioxane in PBS) induced a 10-fold increase
in quantum yield (Table 3). The maximum wavelength of emission is in the range of
538-542 nm for all the assay solutions. The same trend was also observed when the
emission of compound 3 was recorded at 5 µM concentration (Figure S5,
Supplementary Data).
12

Figure 2. Absorption spectra of 3. A) Acquisition of absorption spectra of 3 (10 μM) in
solutions varying from aqueous to 60% dioxane-PBS (v/v) B) The influence of the
polarity of the medium on the fluorescence of 3 was investigated by addition of dioxane
(indicated in % v/v) in PBS measured at a λ max of 366 nm.
13

Table 3. Quantum yield (Φ) of compound 3 in solutions varying from aqueous to 60%
dioxane-PBS.
Solution
PBS
Φa
0.0090
0.0124
0.0710
0.1352
PBS/Dioxane 80/20
PBS/Dioxane 60/40
PBS/Dioxane 40/60
acompared to the quinine bisulfate (in 0.1 N H2SO4, Φ=0.55)
2.4. Fluorescent labelling of human BM-MSCs using compound 3.
Compound 3 was employed to visualize ligand-receptor interactions in undifferentiated
BM-MSCs (Fig. 3). As previously reported, these cells express both A2B and A1 ARs,25
so representing an effective cellular model to investigate the potential of 3 as a probe to
visualize both the above mentioned AR subtypes.
The incubation of BM-MSCs with 3 resulted in a clear labelling of the cell (Fig. 3A,B),
which was concentration-dependent. When the NBD was used alone, it was not able to
bind BM-MSCs (Fig. S6). The co-labelling experiment with a specific membrane
fluorescent dye (DiD) evidenced the AR localization on the BM-MSCs membrane (Fig.
3C). A substantial proportion of this binding was prevented by pre-incubation with the
A1 AR selective agonist R-PIA (100 nM, 10 min, 37 °C) and the A2B AR agonist BAY
60-6583 (100 nM, 10 min, 37 °C) (Fig. 4B,C, Fig. S7). R-PIA alone significantly
counteracted the specific binding of 3, thus reducing the fluorescence of labelled BM-
MSCs (Fig. 4A,B). Similarly, BAY 60-6583 alone significantly decreased the labelling
with 3 (Fig. 4A,B). The simultaneous occupation of the A1 and A2B AR binding sites
caused a significant decrease of the fluorescent emission of 3 (Fig. 4A,B),
demonstrating the specificity of the binding of 3 to A1 and A2B ARs.
14

A
3 (10 µM)
DAPI
DAPI
MERGE
3 (50 µM)
MERGE
B
C
2.0
1.5
1.0
0.5
0.0
M
M
µ
µ
0
0
1
5
3 (50 µM)
DiD
MERGE
Figure 3. Fluorescence staining of BM-MSCs. A) Representative images of BM-MSCs
treated with different concentrations of 3 (in green). Samples were fixed and imaged.
Nuclei were counterstained with DAPI (blue). Scale bar = 250 µm. Two independent
experiments were performed in duplicate and five images were taken randomly for each
sample. B) The mean intensity values of fluorescence were quantified. The data
represent the mean ± SEM of five images with at least 25 cells. C) Representative
images of BM-MSCs treated with 3 (in green) and DiD (in red). Samples were fixed
and imaged. Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm.
15

A
3 (50 µM)
DAPI
MERGE
CTRL
R-PIA
BAY60-6583
R-PIA + BAY60-6583
B
1.5
**
1.0
0.5
0.0
***
#
***
)
(
3
8
3
8
A
I
M
µ
P
5
5
-
6
-
6
0
-
R
5
(
0
6
0
+
6
)
3
Y
Y
M
A
A
µ
B
B
0
+
+
5
)
A
I
M
3
P
µ
-
0
R
5
(
+
)
3
M
µ
0
5
(
3
Figure 4. Fluorescent probe displacement in BM-MSCs. A) Representative images of
BM-MSCs treated with: 3 (green, 50 µM), 3 + R-PIA (100 nM), 3 + BAY60-6583 (100
nM), 3 + R-PIA and BAY60-6583. Samples were fixed and imaged. Nuclei were
counterstained with DAPI (blue). Scale bar = 250 µm. Two independent experiments
were performed in duplicate and five images were taken randomly for each sample. B)
The mean intensity values of fluorescence were quantified. The data represent the mean
± SEM of five images with at least 25 cells. The significance of the differences was
determined by one-way ANOVA post hoc Bonferroni ** P ≤ 0.01, *** P ≤ 0.001 vs. 3;
# P ≤ 0.05 vs. BAY alone.
16

3. Conclusions
In this study four green-emitting fluorescent triazinobenzimidazole derivatives were
synthesized and characterized for their capability to interact with ARs. Among these,
compound 3 demonstrated to act as A1 and A2B AR antagonist with similar binding
affinity values for the two receptors. The results obtained from the staining of BM-
MSCs with the fluorescent antagonist 3 resulted in clear labelling of the cell membrane,
with a large proportion of this binding prevented by pre-incubation with the non-
fluorescent R-PIA and BAY60-6583 as selective A1 and A2B AR agonists, respectively.
Although further studies are needed to increase the selectivity of our
triazinobenzimidazole derivatives for each AR subtype, these compounds could be
useful to monitor the expression and localization of each AR subtype in several tissues
and living cells in different physiological and pathological processes, such as MSC
differentiation to osteoblasts and adipocytes.
4. Experimental Section
4.1. Chemistry.
4.1.1. General
The uncorrected melting points were determined using a Reichert Köfler hot-stage
apparatus. NMR spectra were obtained on a Bruker AVANCE 400 or on Varian Gemini
200 spectrometer. The coupling constants are given in Hertz. Magnesium sulfate was
used as the drying agent. Evaporations were made in vacuo (rotating evaporator).
Analytical TLC have been carried out on Merck 0.2 mm precoated silica gel aluminum
sheets (60 F-254). Silica gel 60 (230–400 mesh) was used for column chromatography.
Purity of the target inhibitors 1-4 was determined, using a Shimadzu LC-20AD SP
liquid chromatograph equipped with a DDA Detector ( = 254 nm) using a column C18
(250 mm x 4.6 mm, 5 m, Shim-pack)). The mobile phase, delivered at isocratic flow,
consisted of acetonitrile (60%) and water (40%) and a flow rate of 1.0 mL/min. All the
compounds showed percent purity values ≥ 95%. Fluorescence spectra were obtained by
EnSight PerkinElmer. Absorption spectra were acquired by Victor Wallac 2 (Perkin
Elmer).
Reagents, starting materials, and solvents were purchased from commercial suppliers
and used as received. The following intermediates were obtained according to methods
17

previously
benzo[d]imidazol-2-yl)hydrazono)-2-phenylacetic
phenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 8.22
described:
2-hydrazinyl-1H-benzo[d]imidazole
6,
(Z)-2-(2-(1H-
and 3-
acid
7
4.1.2. N-(4-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)butyl)-2-(4-oxo-3-
phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-1(4H)-yl)acetamide 1. 4-Chloro-7-
nitrobenzofurazan (0.056 g, 0.28 mmol) dissolved in 1 mL of anhydrous DMF was
added dropwise to a stirred solution of derivative 17 (0.109 g, 0.28 mmol) in 10 mL of
the same solvent, cooled at 0 °C, followed by addition of a solution of triethylamine
(0.04 mL, 0.30 mmol). The reaction mixture was stirred at room temperature overnight
and then concentrated in vacuo. The residue was taken up with 0 °C water and extracted
with CH2Cl2 (3 × 40 mL). The combined organic phases were washed with brine, dried
(MgSO4), filtered, and concentrated in vacuo. Purification by column chromatography
on silica gel (ethyl acetate as eluent) provided the target compound 1 as a yellow-
brownish solid. Yield: 54%; m.p. = 182-184 °C; 1H-NMR (DMSO-d6, ppm): 1.51-1.58
(m, 2H); 1.67-1.74 (m, 2H); 3.18-3.22 (m, 2H); 3.41-3.46 (m, 2H); 5.12 (s, 2H); 6.31 (d,
1H, J = 8.0 Hz); 7.32-7.37 (m, 1H); 7.44-7.53 (m, 4H); 7.66 (d, 1H, J = 8.0 Hz); 8.03-
8.06 (m, 2H); 8.26 (bs, 1H, exch D2O); 8.31-8.34 (m, 2H); 9.46 (bs, 1H, exch D2O).
13C-NMR (DMSO-d6, ppm): 25.29; 26.89; 38.54; 43.40; 56.19; 99.47; 115.29; 118.38;
120.96; 122.63; 127.01; 128.68 (2C); 128.85 (2C); 128.91; 129.95; 132.69; 137.84;
138.07; 141.78; 146.64; 152.44; 166.24. Anal. Calcd. for C27H23N9O5 (%): C, 58.59; H,
4.19; N, 22.77. Found: C, 58.41; H, 4.30; N, 22.80.
4.1.3. N-(6-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexyl)-2-(4-oxo-3-
phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-1(4H)-yl)acetamide 2.
Compound 2 was obtained from derivative 18 (0.117 g, 0.28 mmol), according to the
procedure described from amine 17. Yield: 49%; m.p. = 123-125 °C; 1H-NMR (DMSO-
d6, ppm): 1.29-1.33 (m, 4H); 1.38-1.45 (m, 2H); 1.60-1.64 (m, 2H); 3.10-3.15 (m, 2H);
3.39-3.43 (m, 2H); 5.11 (s, 2H); 6.32 (d, 1H, J = 8.0 Hz); 7.36-7.40 (m, 1H); 7.48-7.54
(m, 4H); 7.72 (d, 1H, J = 7.6 Hz); 8.03-8.05 (m, 2H); 8.19 (bs, 1H, exch D2O); 8.36 (d,
1H, J = 8.0 Hz); 8.45 (d, 1H, J = 8.0 Hz); 9.50 (bs, 1H, exch D2O).13C-NMR (DMSO-
d6, ppm): 26.28; 26.44; 27.94; 29.31; 39.06; 43.65; 56.00; 99.41; 115.30; 118.47;
120.88; 122.66; 127.09; 128.69 (2C); 128.86 (2C); 128.94; 129.95; 132.70; 137.89;
18

138.28; 141.82; 146.72; 152.49; 166.06. Anal. Calcd. for C29H27N9O5 (%): C, 59.89; H,
4.68; N, 21.68. Found: C, 59.76; H, 4.74; N, 21.71.
4.1.4. N-(4-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)butyl)-2-(4-oxo-3-
phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-10(4H)-yl)acetamide 3.
Compound 3 was obtained from derivative 19 (0.109 g, 0.28 mmol), according to the
procedure described from amine 17. Yield: 58%; m.p. = 253-255 °C; 1H-NMR (DMSO-
d6, ppm): 1.53-1.56 (m, 2H); 1.68-1.71 (m, 2H); 3.16-3.21 (m, 2H), 3.45-3.49 (m, 2H);
5.11 (s, 2H); 6.39 (d, 1H, J = 8.0 Hz); 7.44-7.50 (m, 3H); 7.62-7.66 (m, 1H); 7.74 (d,
1H, J = 8.4 Hz); 7.96 (s, 1H); 8.15-8.17 (m, 2H); 8.35 (bs, 1H, exch D2O); 8.44 (d, 1H,
J = 9.2 Hz); 8.52 (d, 1H, J = 8.0 Hz); 9.53 (bs, 1H, exch D2O). 13C-NMR (DMSO-d6,
ppm): 24.42; 26.91; 38.62; 45.17; 46.02; 99.55; 110.97; 116.79; 120.99; 123.47;
124.91; 128.19; 128.48 (2C); 128.53 (2C); 129.37; 132.16; 134.50; 138.42; 145.60;
147.17; 150.40; 165.96. Anal. Calcd. for C27H23N9O5 (%): C, 58.59; H, 4.19; N, 22.77.
Found: C, 58.45; H, 4.21; N, 22.86.
4.1.5. N-(6-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexyl)-2-(4-oxo-3-
phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-10(4H)-yl)acetamide 4.
Compound 4 was obtained from derivative 20 (0.117 g, 0.28 mmol), according to the
procedure described from amine 17. Yield: 48%; m.p. = 211-213 °C; 1H-NMR (DMSO-
d6, ppm): 1.30-1.37 (m, 4H); 1.43-1.46 (m, 2H); 1.62-1.67 (m, 2H); 3.10-3.15 (m, 2H);
3.41-3.45 (m, 2H); 5.10 (s, 2H); 6.34 (d, 1H, J = 8.0 Hz); 7.43-7.50 (m, 4H); 7.63-7.67
(m, 1H); 7.74 (d, 1H, J = 8.0 Hz); 8.16-8.18 (m, 2H); 8.31 (bs, 1H, exch D2O); 8.44 (d,
13
1H, J = 8.8 Hz); 8.51 (d, 1H, J = 8.0 Hz); 9.51 (bs, 1H, exch D2O). C-NMR (DMSO-
d6, ppm): 26.27; 26.44; 28.01; 29.28; 39.11; 43.70; 45.19; 99.44; 110.99; 116.77;
120.86; 123.47; 124.92; 128.18; 128.47 (2C); 128.51 (2C); 129.34; 132.17; 134.55;
138.30; 145.57; 147.18; 150.41; 165.80. Anal. Calcd. for C29H27N9O5 (%): C, 59.89; H,
4.68; N, 21.68. Found: C, 59.78; H, 4.75; N, 21.70.
4.1.6. Ethyl 2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-(4H)-yl)acetate
derivatives 9 and 10 (from Scheme 1).
Sodium hydride suspension (60% suspension in paraffin oil) (0.120 g, 3.00 mmol) was
added in small portions to a solution of 3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-
4(10H)-one 8 (0.787 g, 3.00 mmol) in 10 mL of anhydrous DMF, cooled at 0 °C. After
19

the development of H2 (about 1h), ethyl bromoacetate (0.33 mL, 3.00 mmol), dissolved
in 2 mL of the same solvent, was added dropwise. The reaction mixture was maintained
under stirring at room temperature for 24h (TLC analysis). The solution was dripped
into ice and once the ice dissolved, the formed precipitate was collected by filtration and
purified by flash chromatography (toluene:acetonitrile = 9:1 as eluent).
Ethyl 2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-1(4H)-yl)acetate 9.
1
Yield: 51%; m.p. = 198-200 °C; H-NMR (DMSO-d6, ppm): 1.25 (t, 3H, J = 7.2 Hz);
4.23 (q, 2H, J = 7.2 Hz); 5.38 (s, 2H); 7.40-7.44 (m, 1H); 7.52-7.57 (m, 4H); 7.76 (d,
13
1H, J = 8.0 Hz); 8.04-8.06 (m, 2H); 8.38 (d, 1H, J = 8.0 Hz). C-NMR (DMSO-d6,
ppm): 14.47; 54.60; 62.11; 115.44; 118.60; 122.91 (2C); 127.23; 128.72 (2C); 128.97
(2C); 130.11; 132.50; 138.11; 141.72; 146.39; 152.14; 167.74.
Ethyl 2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-10(4H)-yl)acetate 10.
1
Yield: 22%; m.p. = 168-170 °C; H-NMR (DMSO-d6, ppm): 1.26 (t, 3H, J = 7.0 Hz);
4.23 (q, 2H, J = 7.2 Hz); 5.39 (s, 2H); 7.48-7.54 (m, 4H); 7.66-7.70 (m, 1H); 7.85 (d,
13
1H, J = 8.0 Hz); 8.17-8.20 (m, 2H); 8.52 (d, 1H, J = 8.0 Hz). C-NMR (DMSO-d6,
ppm): 14.50; 43.90; 62.15; 111.06; 116.84; 123.73; 124.81; 128.31; 128.56 (2C);
128.64 (2C); 129.48; 131.68; 134.50; 146.20; 146.98; 150.36; 167.66.
4.1.7. Ethyl
2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-10(4H)-
yl)acetate 10 (from Scheme 2).
Compound 11 (0.400 g, 1.70 mmol) was heated at 180 °C in a Pyrex capped tube with
0.81 mL of hydrazine monohydrate (17.0 mmol) for 5 h. After the mixture was cooled,
the precipitate, constituted by the 2-(2-hydrazinyl-1H-benzo[d]imidazol-1-yl)acetate 12,
was collected, dissolved in 10 mL of absolute ethanol and directly reacted added with
phenyloxoacetic acid (0.255 g, 1.70 mmol), due to its scarce stability. The resulting
mixture was refluxed for 5 h. After cooling, the solvent was removed under reduced
pressure and the crude product was purified by column chromatography on silica gel
(toluene:acetonitrile = 9:1 as eluent), furnishing 10 with low yield (15%).
4.1.8. Ethyl 2-(2-chloro-1H-benzo[d]imidazol-1-yl)acetate 11.
Compound 11 was obtained from commercial 2-chlorobenzimidazole 5 (0.500 g, 3.30
mmol), sodium hydride suspension (60% suspension in paraffin oil) (0.132 g, 3.30
mmol) and ethyl bromoacetate (0.38 mL, 3.30 mmol), according to the procedure
1
described for derivatives 9 and 10. Yield: 97%; m.p. = 120-122 °C; H-NMR (DMSO-
20

d6, ppm): 1.23 (t, 3H, J = 7.2 Hz); 4.20 (q, 2H, J = 7.2 Hz); 5.27 (s, 2H); 7.26-7.34 (m,
2H); 7.62-7.65 (m, 2H).
4.1.9. tert-Butyl(4-(2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-1(4H)-
yl)acetamido)butyl)carbamate 13.
Compound 9 (0.209 g, 0.60 mmol) was heated at 80 °C in a Pyrex capped tube with N-
Boc-1,4-butanediamine (0.226 g, 1.20 mmol) for 3h. The precipitate was dissolved with
CH2Cl2 and washed with diluted (10%) HCl. After drying with MgSO4, the
dichloromethane solution was evaporated under reduced pressure, yielding the crude
product that was purified by flash-chromatography (ethyl acetate:hexane = 5:5 as
1
eluent). Yield: 79%; m.p. = 200-202 °C; H-NMR (DMSO-d6, ppm): 1.37-1.54 (m,
13H); 2.89-2.93 (m, 2H); 3.08-3.11 (m, 2H); 5.10 (s, 2H); 6.77 (bs, 1H, exch D2O);
7.47-7.54 (m, 4H); 7.64-7.68 (m, 1H); 7.72-7.74 (m, 1H); 8.19-8.21 (m, 2H); 8.31 (bs,
1H, exch D2O); 8.53 (d, 1H, J = 8.0 Hz).
4.1.10. tert-Butyl(6-(2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-1(4H)-
yl)acetamido)hexyl)carbamate 14.
Compound 14 was obtained from ester 9 (0.209 g, 0.60 mmol) and N-Boc-1,6-
hexanediamine (0.260 g, 1.20 mmol), according to the procedure described for amide
1
13. Yield: 81%; m.p. = 205-207 °C; H-NMR (DMSO-d6, ppm): 1.22-1.24 (m, 4H);
1.36-1.44 (m, 13H); 2.85-2.92 (m, 2H); 3.09-3.10 (m, 2H); 5.10 (s, 2H); 6.75 (bs, 1H,
exch D2O); 7.40-7.45 (m, 1H); 7.51-7.58 (m, 4H); 7.74-7.76 (m, 1H); 8.03-8.06 (m,
2H); 8.16 (bs, 1H, exch D2O); 8.40 (d, 1H, J = 7.6 Hz).
4.1.11. tert-Butyl(4-(2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-10(4H)-
yl)acetamido)butyl)carbamate 15.
Compound 15 was obtained from ester 10 (0.209 g, 0.60 mmol) and N-Boc-1,4-
butanediamine (0.226 g, 1.20 mmol), according to the procedure described for amide
1
13. Yield: 70%; m.p. = 175-177 °C; H-NMR (DMSO-d6, ppm): 1.33-1.44 (m, 13H);
2.89-2.92 (m, 2H); 3.07-3.08 (m, 2H); 5.10 (s, 2H); 6.78 (bs, 1H, exch D2O); 7.46-7.53
(m, 4H); 7.64-7.68 (m, 1H); 7.72-7.74 (m, 1H); 8.19-8.21 (m, 2H); 8.32 (bs, 1H, exch
D2O); 8.53 (d, 1H, J = 8.0 Hz).
21

4.1.12. tert-Butyl(6-(2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-10(4H)-
yl)acetamido)hexyl)carbamate 16.
Compound 16 was obtained from ester 10 (0.209 g, 0.60 mmol) and N-Boc-1,6-
hexanediamine (0.260 g, 1.20 mmol), according to the procedure described for amide
1
13. Yield: 74%; m.p. = 185-187 °C; H-NMR (DMSO-d6, ppm): 1.21-1.26 (m, 4H);
1.32-1.43 (m, 13H); 2.85-2.90 (m, 2H); 3.07-3.10 (m, 2H); 5.10 (s, 2H); 6.76 (bs, 1H,
exch D2O); 7.47-7.54 (m, 4H); 7.64-7.68 (m, 1H); 7.72-7.72 (m, 1H); 8.19-8.21 (m,
2H); 8.30 (bs, 1H, exch D2O); 8.52 (d, 1H, J = 8.0 Hz).
4.1.13. N-(4-Aminobutyl)-2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-
1(4H)-yl)acetamide 17.
Trifluoroacetic acid (0.239 g, 2.10 mmol) was added to a stirred solution of derivative
13 (0.103 g, 0.21 mmol) dissolved in 10 mL of CH2Cl2. The reaction mixture was
stirred at room temperature until the disappearance of the starting material (TLC
analysis). After stirring at room temperature for 3 h, the reaction mixture was
concentrated in vacuo. The solid residue was taken up with water, cooled in an ice bath,
treated with a 3 M solution of NaOH, and extracted with CH2Cl2 (3 × 40 mL). The
combined organic phases were washed with brine, dried (MgSO4), filtered, and
concentrated in vacuo to yield the title compound. Yield: 88%; m.p. = 144-146 °C; 1H-
NMR (DMSO-d6, ppm): 1.46-1.50 (m, 4H); 2.71-2.74 (m, 2H); 3.12-3.13 (m, 2H); 5.11
(s, 2H); 7.42-7.45 (m, 1H); 7.52-7.59 (m, 4H); 7.75-7.77 (m, 1H); 8.04-8.06 (m, 2H);
8.26 (bs, 1H, exch D2O); 8.41 (d, 1H, J = 8.0 Hz).
4.1.14. N-(6-Aminohexyl)-2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-
1(4H)-yl)acetamide 18.
Compound 18 was obtained from derivative 14 (0.109 g, 0.21 mmol), according to the
1
procedure described for amine 17. Yield: 84%; m.p. = 178-180 °C; H-NMR (DMSO-
d6, ppm): 1.36-1.40 (m, 8H); 2.67-2.69 (m, 2H); 3.07-3.09 (m, 2H); 5.09 (s, 2H); 7.40-
7.42 (m, 1H); 7.51-7.56 (m, 4H); 7.72-7.73 (m, 1H); 7.99-8.01 (m, 2H); 8.14 (bs, 1H,
exch D2O); 8.37-8.40 (m, 1H).
4.1.15. N-(4-Aminobutyl)-2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-
10(4H)-yl)acetamide 19.
22

Compound 19 was obtained from derivative 15 (0.103 g, 0.21 mmol), according to the
1
procedure described for amine 17. Yield: 87%; m.p. = 199-201 °C; H-NMR (DMSO-
d6, ppm): 1.32-1.46 (m, 4H); 2.51-2.54 (m, 2H); 3.08-3.13 (m, 2H); 5.10 (s, 2H); 7.48-
7.54 (m, 4H); 7.65-7.69 (m, 1H); 7.72-7.75 (m, 1H); 8.19-8.21 (m, 2H); 8.33 (bs, 1H,
exch D2O); 8.53 (d, 1H, J = 8.0 Hz).
4.1.16. N-(6-Aminohexyl)-2-(4-oxo-3-phenylbenzo[4,5]imidazo[2,1-c][1,2,4]triazin-
10(4H)-yl)acetamide 20.
Compound 20 was obtained from derivative 16 (0.109 g, 0.21 mmol), according to the
1
procedure described for amine 17. Yield: 83%; m.p. = 158-160 °C; H-NMR (DMSO-
d6, ppm): 1.31-1.50 (m, 8H); 2.52-2.55 (m, 2H); 3.06-3.09 (m, 2H); 5.08 (s, 2H); 7.46-
7.53 (m, 4H); 7.63-7.65 (m, 2H); 8.12-8.14 (m, 2H); 8.28 (bs, 1H, exch D2O); 8.50 (d,
1H, J = 8.0 Hz).
4.2. Determination of the Fluorescence Quantum Yield.
The quantum yield of the probe (x) was determined according to equation:
where the subscripts ST and X denote standard and test compound, respectively, Φ is
the quantum yield, D is the area under the fluorescence emission spectra, A is the
absorbance at the excitation wavelength, and η is the refractive index of the solvent
used. Quinine bisulfate (Φf = 0.55 in 0.1 N H2SO4) is used as reference standard.
4.3. Biology.
4.3.1. Adenosine Receptor Binding Assay. Materials. [3H]DPCPX and [3H]NECA were
obtained from DuPont-NEN (Boston, MA). ADA was from Sigma Chemical Co. (St.
Louis, MO). All other reagents were from commercial sources and of the highest
commercially available grade. CHO cells stably expressing human A1, A2A, A3 and A2B
ARs were kindly supplied by Prof. K. N. Klotz, Wurzburg University, Germany.
4.3.2. Human A1 Adenosine Receptors. Competition binding experiments were
performed as previously reported (J. Med. Chem. 2012, 55, 1490−1499).18 Briefly,
membranes of A1 CHO cells were incubated at 25 °C for 180 min in 500 μL of buffer
23

containing [3H]DPCPX (3 nM) and six different concentrations of the newly
synthesized compounds. Nonspecific binding was determined in the presence of 50 μM
RPIA. The dissociation constant (Kd) of [3H]DPCPX in A1 CHO cell membranes was 3
nM.
4.3.3. Human A2A Adenosine Receptors. Competition binding experiments were
performed as previously reported.18 Briefly, membranes of A2A CHO cells were
incubated at 25 °C for 90 min in 500 μL of buffer in the presence of [3H]NECA (30 nM)
and six different concentrations of the newly synthesized compounds. Nonspecific
binding was determined in the presence of 100 μM NECA. The dissociation constant
(Kd) of [3H]NECA in A2A CHO cell membranes was 30 nM.
4.3.4. Measurement of Cyclic AMP Levels in A1 AR, A3 AR or A2B AR CHO.
Intracellular cyclic AMP (cAMP) levels were measured using a competitive protein
binding method, as previously reported.18 A1 AR, A3 AR or A2B AR CHO cells were
seed at a density of 48.000 cell/well in a 24 multiwell plate. After 48 h, the medium was
removed, and the cells were incubated at 37 °C for 15 min with 0.5 mL of Dulbecco’s
Modified Eagle Medium (DMEM) in the presence of ADA (1U/mL) and the
phosphodiesterase inhibitor Ro20-1724 (20 μM).
The agonist efficacy profile of the compounds at ARs was evaluated by assessing their
ability to modulate intracellular cAMP levels in the absence of standard agonists; for the
A1 AR and A3 AR, 1 μM forskolin a non-selective activator of adenylate cyclase were
used. The antagonism profile of the new compounds was evaluated by assessing their
ability to inhibit 100 nM NECA-mediated accumulation of cAMP for A2B AR CHO
cells; 10 nM NECA for A1 AR CHO cells and 1 nM Cl-IBMECA for A3 AR CHO cells.
The new compounds were incubated 10 min before the addiction of the agonists. Then,
cells were incubated in the reaction medium for 15 min at 37 °C. The reaction was
stopped by the removal of the medium and the addition of 0.4 N HCl. After 30 min,
lysates were neutralized with 4 N KOH, and the suspension was centrifuged at 800g for
5 min. For the determination of cAMP production, bovine adrenal cAMP binding
protein was incubated with [3H]cAMP (2 nM) and 50 μL of cell lysate or cAMP
standard (0−16 pmol) at 0 °C for 150 min in a total volume of 300 μL. Bound
radioactivity was separated by rapid filtration through GF/C glass fiber filters and
24

washed twice with 4 mL of 50 mM Tris/HCl, pH 7.4. The radioactivity was measured
by liquid scintillation spectrometry.
4.3.5. Cell cultures
Human bone marrow derived MSCs were purchased by Lonza (Milan, Italy). Cells were
cultured in normal growth medium (MSCGM, Lonza), plated (5.000 cells/cm2) in 75-
cm2 flasks and incubated at 37 °C in 5% CO2 and 95% air. The medium was changed to
remove nonadherent cells every 3 to 4 days, and the cells were used at passages 2 to 5.
4.3.6. Immunofluorescent analysis.
MSCs were seeded at 10.000 cell/cm2 in chamber slide (BD Biosciences, San Jose, CA,
USA). Cells were treated with different concentrations of 3 for 10 min. In displacement
experiments, R-PIA or BAY60-6583 for 10 min, then 3 was added for additional 10
min. Cell membranes were stained with DiD (10 µM, V-22887, ThermoFisher) for 20
min. After treatment, cells were fixed in 2% paraformaldehyde in 0.1 M phosphate
buffer, washed three times with PBS, rinsed. After washing, nuclei were stained with
DAPI (1:5000 dilution; Sigma–Aldrich) for 10 min. Then slides were covered with
Vectashield (Vector Laboratories, Burlingame, CA, USA). Confocal images were
acquired using a laser scanning confocal microscope TCS SP2 (Leica Microsystems,
Germany) with a 40x oil immersion objective, at 1024x1024 pixel resolution. Each
confocal image was a z-series, each averaged two times, and was chosen to cover the
entire region of interest from top to bottom. The resulting z-stack was processed by
Image J software (NIH) into a single image using “z-project” and “max intensity”
options. Images were obtained with a Nikon Ni-E microscope, using a 20× objective
with 1.45 NA and a recommended pinhole size of less than 1.0 micrometer, and
equipped with digital camera Nikon Mod.DS-Ri2. Exposure time was set at 300 ms
(DAPI) and 2 s (3) with the lamp intensity at 25%. The microscope, lamp, and camera
settings were kept constant throughout this study. For presentation purposes, pictures
were exported in TIFF format and processed with Adobe Photoshop, for adjustments of
brightness and contrast. Quantification of the mean fluorescence intensity in selected
regions was carried out by using the Fiji software as previously reported.35 In brief,
compound 3 signal was quantified as follows: the area covered by each cell was first
manually selected as a region of interest (ROI), then the ROI was applied to the
correspondent compound 3 positive image and the intensity was measured by the
25

ImageJ “Measure” tool (option “RawIntDen”). Then, the values were normalized to the
mean Area and background fluorescence intensity were subtracted. Finally, the values
were reported as Cell intensity (a.u.).
4.3.7. Statistical analysis.
The Graph-Pad Prism program (GraphPad Software Inc., San Diego, CA) was used for
data analysis and graphic presentation. All data are the mean ± SEM. Statistical analysis
was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hot
analysis. EC50 values were reported as mean of the values obtained in at least three
independent experiments performed in duplicate ± SEM. P ≤ 0.05 was considered
statistically significant.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGMENTS
We thank Prof. Claudia Gargini for her help in the acquisition and analysis of
immunofluorescence images.
FUNDINGS
This work was supported by University of Pisa (PRA-project), and by the Italian
Ministry of Education (Project of National Research Interest PRIN 2015,
2015E8EMCM_007).
26

References
1.
Leopoldo M, Lacivita E, Berardi F, Perrone R. Developments in fluorescent probes for
receptor research. Drug Discov Today. 2009;14(13-14): 706-712.
2.
receptor interactions in single living cells. Meth Mol Biol. 2011;746: 211-236.
3. Duroux R, Ciancetta A, Mannes P, et al. Bitopic fluorescent antagonists of the A2A
Briddon SJ, Kellam B, Hill SJ. Design and use of fluorescent ligands to study ligand-
adenosine receptor based on pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine
functionalized congeners. MedChemComm. 2017;8(8): 1659-1667.
4.
Baker JG, Middleton R, Adams L, et al. Influence of fluorophore and linker composition
on the pharmacology of fluorescent adenosine A1 receptor ligands. Br J Pharmacol.
2010;159(4): 772-786.
5.
Köse M, Gollos S, Karcz T, et al. Fluorescent-labeled selective adenosine A2B receptor
antagonist enables competition binding assay by flow cytometry. J Med Chem. 2018;61:4301-
4316.
6.
Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, Linden J. International Union of
Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev.
2001;53(4): 527-552.
7.
Fredholm BB, IJzerman AP, Jacobson KA, Linden J, Muller CE. International Union of
Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine
receptors--an update. Pharmacol Rev. 2011;63(1): 1-34.
8.
Br J Pharmacol. 2008;153 Suppl 1: S184-190.
9. Langemeijer EV, Verzijl D, Dekker SJ, Ijzerman AP. Functional selectivity of adenosine
A1 receptor ligands? Purinergic Signal. 2013;9(1): 91-100.
10. Giacomelli C, Daniele S, Romei C, et al. The A2B Adenosine Receptor Modulates the
Zezula J, Freissmuth M. The A(2A)-adenosine receptor: a GPCR with unique features?
Epithelial- Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway
Activation in Human Epithelial Lung Cells. Front Pharmacol. 2018;9: 54.
11.
Da Settimo F, Primofiore G, Taliani S, et al. 3-Aryl[1,2,4]triazino[4,3-a]benzimidazol-
4(10H)-ones: A New Class of Selective A1 Adenosine Receptor Antagonists. J Med Chem.
2001;44(3): 316-327.
12.
Da Settimo F, Primofiore G, Taliani S, et al. A1 adenosine receptor antagonists, 3-
aryl[1,2,4]triazino[4,3-a]benzimidazol-4-(10H)-ones (ATBIs) and N-alkyl and N-acyl-(7-
substituted-2-phenylimidazo[1,2-a][1,3,5]triazin-4-yl)amines (ITAs): Different recognition of
bovine and human binding sites. Drug Dev Res. 2004;63(1): 1-7.
13.
Novellino E, Cosimelli B, Ehlardo M, et al. 2-(Benzimidazol-2-yl)quinoxalines: A Novel
Class of Selective Antagonists at Human A1 and A3 Adenosine Receptors Designed by 3D
Database Searching. J Med Chem. 2005;48(26): 8253-8260.
14.
Da Settimo F, Primofiore G, Taliani S, et al. 5-Amino-2-phenyl[1,2,3]triazolo[1,2-
a][1,2,4]benzotriazin-1-one: A Versatile Scaffold To Obtain Potent and Selective A3 Adenosine
Receptor Antagonists. J Med Chem. 2007;50(23): 5676-5684.
15.
Cosimelli B, Greco G, Ehlardo M, et al. Derivatives of 4-Amino-6-hydroxy-2-
mercaptopyrimidine as Novel, Potent, and Selective A3 Adenosine Receptor Antagonists. J
Med Chem. 2008;51(6): 1764-1770.
16.
Taliani S, La Motta C, Mugnaini L, et al. Novel N2-substituted pyrazolo[3,4-d]pyrimidine
adenosine A3 receptor antagonists: Inhibition of A3-mediated human glioblastoma cell
proliferation. J Med Chem. 2010;53(10): 3954-3963.
17.
Cosimelli B, Taliani S, Greco G, et al. Derivatives of Benzimidazol-2-ylquinoline and
Benzimidazol-2-ylisoquinoline as Selective A1 Adenosine Receptor Antagonists with Stimulant
Activity on Human Colon Motility. ChemMedChem. 2011;6(10): 1909-1918.
27

18.
Taliani S, Pugliesi I, Barresi E, et al. 3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-
one: A Novel Template for the Design of Highly Selective A2B Adenosine Receptor Antagonists.
J Med Chem. 2012;55(4): 1490-1499.
19.
Scatena A, Fornai F, Trincavelli ML, et al. 3-(Fur-2-yl)-10-(2-phenylethyl)-
[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one, a Novel Adenosine Receptor Antagonist with
A2A-Mediated Neuroprotective Effects. ACS Chem Neurosci. 2011;2(9): 526-535.
20.
Rossi D, Nasti R, Marra A, et al. Enantiomeric 4-Acylamino-6-alkyloxy-2
Alkylthiopyrimidines As Potential A3 Adenosine Receptor Antagonists: HPLC Chiral Resolution
and Absolute Configuration Assignment by a Full Set of Chiroptical Spectroscopy. Chirality.
2016;28(5): 434-440.
21.
Cosimelli B, Greco G, Laneri S, et al. Studies on enantioselectivity of chiral 4-
acetylamino-6-alkyloxy-2-alkylthiopyrimidines acting as antagonists of the human A3
adenosine receptor. Med Chem Commun,. 2018;9: 81-86.
22.
Primofiore G, Da Settimo F, Taliani S, et al. 3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-
4(10H)-ones: Tricyclic Heteroaromatic Derivatives as a New Class of Benzodiazepine Receptor
Ligands. J Med Chem. 2000;43(1): 96-102.
23.
Petroni D, Giacomelli C, Taliani S, et al. Toward PET imaging of A2B adenosine
receptors: a carbon-11 labeled triazinobenzimidazole tracer. Nucl Med Biol. 2016;43(5): 309-
317.
24.
Gharibi B, Abraham AA, Ham J, Evans BA. Adenosine receptor subtype expression and
activation influence the differentiation of mesenchymal stem cells to osteoblasts and
adipocytes. J Bone Miner Res. 2011;26(9): 2112-2124.
25.
Costa MA, Barbosa A, Neto E, et al. On the role of subtype selective adenosine
receptor agonists during proliferation and osteogenic differentiation of human primary bone
marrow stromal cells. J Cell Physiol. 2011;226(5): 1353-1366.
26.
Trincavelli ML, Daniele S, Giacomelli C, et al. Osteoblast differentiation and survival: A
role for A2B adenosine receptor allosteric modulators. Biochim Biophys Acta, Mol Cell Res.
2014;1843(12): 2957-2966.
27.
D'Alimonte I, Nargi E, Lannutti A, et al. Adenosine A1 receptor stimulation enhances
osteogenic differentiation of human dental pulp-derived mesenchymal stem cells via WNT
signaling. Stem Cell Res. 2013;11(1): 611-624.
28.
adenosine receptors. J Cell Physiol. 2014;229(4): 414-421.
29. Uchiyama S, Santa T, Okiyama N, Fukushima T, Imai K. Fluorogenic and fluorescent
labeling reagents with a benzofurazan skeleton. Biomed Chromatogr. 2001;15(5): 295-318.
30. Taliani S, Simorini F, Sergianni V, et al. New fluorescent 2-phenylindolglyoxylamide
Eisenstein A, Ravid K. G protein-coupled receptors and adipogenesis: a focus on
derivatives as probes targeting the peripheral-type benzodiazepine receptor: design, synthesis,
and biological evaluation. J Med Chem. 2007;50(2): 404-407.
31.
Taliani S, Da Pozzo E, Bellandi M, et al. Novel irreversible fluorescent probes targeting
the 18 kDa translocator protein: synthesis and biological characterization. J Med Chem.
2010;53(10): 4085-4093.
32.
Milite C, Barresi E, Da Pozzo E, et al. Exploiting the 4-Phenylquinazoline Scaffold for the
Development of High Affinity Fluorescent Probes for the Translocator Protein (TSPO). J Med
Chem. 2017;60(18): 7897-7909.
33.
Lin R, Angelin A, Da Settimo F, et.al. Genetic analysis of dTSPO, an outer mitochondrial
membrane protein, reveals its functions in apoptosis, longevity, and Ab42-induced
neurodegeneration. Aging Cell. 2014;13(3):507-518.
34.
Cosimelli B, Greco G, Laneri S, et al. 4-amino-6-alkyloxy-2-alkylthiopyrimidine
derivatives as novel non-nucleoside agonists for the adenosine A1 receptor. Chem Biol Drug
Des. 2016;88(5): 724-729.
35.
Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for
biological-image analysis. Nat methods. 2012;9(7):676-682.
28

Supplementary Data
Analysis Report
<Sample Information>
Sample Name
Sample ID
: Compound 9
:
:
Data Filename
Method Filename : AcCN70-H2O30.lcm
Batch Filename
Vial #
:
: 1-1
Sample Type
: Unknown
Injection Volume : 1 uL
Date Acquired
: 09/05/2018 15.15.09
Acquired by
Processed by
: HPLC
: HPLC
Date Processed : 16/05/2018 12.25.06
<Chromatogram>
mAU
PDA Multi 1 254nm,4nm
1500
1000
500
0
0,0
2,5
5,0
7,5
10,0
12,5
15,0
17,5
20,0
min
<Peak Table>
PDA Ch1 254nm
Peak# Ret. Time
Area%
1
2
8,351
11,438
99,946
0,054
Total
100,000
Figure S1 Chromatogram of 9
29