7-Nitrobenzofurazan (NBD) derivatives of 5′-N-ethylcarboxamidoadenosine (NECA) as new fluorescent probes for human A3 adenosine receptors

Article abstract of DOI:10.1016/S0960-894X(01)00610-2

New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N⁶ position of NECA, of NBD-moieties with linear alkyl spacers of increasing length, proved to possess a high affinity and selectivity for the A₃ subtype expressed in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A₃-AR, permitted visualization and localization of this human receptor subtype, showing its potential suitability for internalization and trafficking studies in living cells.

Full text of DOI:10.1016/S0960-894X(01)00610-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 3023–3026
7-Nitrobenzofurazan (NBD) Derivatives of
-N-Ethylcarboxamidoadenosine (NECA) as NewFluorescent
0
5
Probes for Human A Adenosine Receptors
3
a,
b
a
c
Marco Macchia, * Francesca Salvetti, Simone Bertini, Valeria Di Bussolo,
f
a
d
e
Lisa Gattuso, Marco Gesi, Mahmoud Hamdan, Karl-Norbert Klotz,
b
b
a
a
Teresina Laragione, Antonio Lucacchini, Filippo Minutolo, Susanna Nencetti,
a
b
b
Chiara Papi, Daniela Tuscano and Claudia Martini
aDipartimento di Scienze Farmaceutiche, Universit a` di Pisa, via Bonanno 6, 56126 Pisa, Italy
bDipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, via Bonanno 6, 56126 Pisa, Italy
cDipartimento di Chimica Bioorganica, Universita` di Pisa, via Bonanno 33, 56126 Pisa, Italy
dDipartimento Morfologia Umana e Biologia Applicata, via Roma 55, 56126 Pisa, Italy
eGlaxoSmithKline Medicines Research Center, via Fleming 4, 37134 Verona, Italy
fInstitut fu¨r Pharmakologie und Toxikologie, Universita¨t, Wu¨rzburg, Germany
Received 21 June 2001; revised 4 September 2001; accepted 6 September 2001
6
Abstract—New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N position ofNECA, ofNBD-
moieties with linear alkyl spacers ofincreasing length, proved to possess a high affinity and selectivity fo r the A
in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A -AR, permitted visual-
3
subtype expressed
3
ization and localization ofthis human receptor subtype, showing its potential suitability fo r internalization and trafficking studies in
living cells. # 2001 Elsevier Science Ltd. All rights reserved.
Adenosine is an autacoid that exerts its physiological
actions through specific cell surface receptors. The
pharmacological, behavioral and binding properties of
adenosine receptors (ARs) have been extensively stud-
able to be further visualized in living cells by means of
confocal microscopy. Past attempts to develop AR
ligands for this purpose were made by K. A. Jacobson,
using new macromolecular probes in which the avidin–
1
4
ied. By comparison, much less is known about the
biotin technology was applied.
intracellular routing and addressing ofARs either
before or after agonist binding. Yet an understanding of
the cellular regulation ofthis class ofreceptors is of
importance, since it is involved in the mechanism of
receptor desensitization, resensitization and down reg-
2
,3
ulation. Unfortunately, the main limitation of recep-
tor internalization and trafficking studies is related to
the protocol, which requires fixing ofthe cells and pro-
hibits a direct correlation between receptor activation,
trafficking and desensitization in living cells. One way to
overcome this obstacle would be to probe the ligand–
receptor complex with a fluorescently labeled agonist
We have recently developed and characterized com-
pounds oftype 1 as fluorescent ligands for ARs. These
6
probes were obtained by the insertion, in the N position of
NECA, a potent adenosine agonist, ofdansylaminoalkyl
*Corresponding author. Tel.: +39-050-500209; fax: +39-050-40517;
e-mail: mmacchia@farm.unipi.it
0
960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00610-2

3024
M. Macchia et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3023–3026
8
moieties with alkyl spacers ofincreasing linear aliphatic
5
base, affording NBD-substituted purines 7a–e. Final
chain length (from 3 to 12 carbon atoms). Although
the compounds have been successfully employed to
deprotection ofthe acetonide group present in the sugar
portion of 7a–e was achieved by acid hydrolysis with a
1 N aqueous solution ofhydrochloric acid, affording,
after column chromatography purification, the target
visualize A ARs in rat cerebellar cortex by fluorescence
1
microscopy, their use is restricted by the short fluor-
ophore excitation wavelength (340 nm) at which many
cells and tissues autofluoresce. To overcome these prob-
lems, we thought it conceivable to prepare new fluor-
escent ligands, in which the dansyl group ofcompounds
oftype 1 has been replaced by a longer excitation
wavelength fluorophore such as 7-nitrobenzofurazan
9
adenosine fluorescent probes 2a–e.
Radioligand Binding Assays
The affinity offluorescent probes 2a–e for A , A and
1
2a
(
NBD, Abs 465 nm, Em 535 nm).
A ARs was assessed by measuring their ability to dis-
3
3
10,11
3
12,13
place [ H]DPCPX,
[ H]CGS 21680,
and
[
125I]AB-MECA binding to membranes derived from
14
Herein, we describe the development and characteriza-
tion offluorescent compounds 2a–e and their utility in
visualizing adenosine receptors.
human cerebral cortex, human striatum and CHO
1
5
16
cells expressing human A ARs, respectively. The
3
results ofthese tests are shown in Table 1. Compounds
2
a–e generally showed a good binding affinity for the A3
receptor subtype; in particular, fluorescent probes 2c–e
with n varying from 6 to 10) seemed to be the most
Chemistry
(
Compounds 2a–e were prepared as outlined in Scheme 1.
interesting ones, with K values for the A receptor ran-
i
ging from 7.4 to 28.1 nM.
3
0
6
Treatment of6-chloropurine-5 -ribouronamide 4 with
the appropriate mono N-Boc-protected diamine 3a–e
afforded purine intermediates 5a–e, which were purified
In contrast, all the compounds synthesized possess a
low affinity for the A receptor subtype, especially in the
7
by column chromatography. Compounds 3a–c were
1
commercially available, whereas compounds 3d and 3e
were prepared by mono-Boc protection of1,8-diamino-
octane and 1,10-diaminodecane, respectively. Sub-
sequent deprotection ofthe N-Boc group present in 5a–e
with trifluoroacetic acid in CH Cl afforded free pri-
case ofcompounds 2a–c (with n varying from 2 to 6)
whose K values were higher than 10 mM.
i
Also, in the case ofthe A 2a receptor subtype, the affi-
nities ofcompounds 2a–e were modest, with K values in
2
2
i
mary amines 6a–e which were directly used in the next
step. The condensation between purines 6a–e and 7-
nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl) was
run in anhydrous THF, in the presence of2,6-di- t-butyl-
the micromolar range. Among all the compounds tes-
ted, fluorescent probe 2d (n=8) proved to be the most
active and selective one for the A receptor subtype,
3
with an affinity about 450 and 680 times higher than
that for the A and A receptor subtypes, respectively.
4
-methylpyridine (DTBMP) as the ‘acid scavenger’
1
2
ꢀ
Scheme 1. Reagents and conditions: (i) triethylamine, abs EtOH, 80 C, 24 h; yields from 32 to 94%; (ii) CF
ꢀ
3
2 2
COOH/CH Cl (1:1), rt, 2 h;
ꢀ
(
iii) DTBMP, an THF, 70 C, 48 h; yields from 21 to 42%, two steps; (iv) 1 N HCl, 60 C, 6 h; yields from 25 to 58%.

M. Macchia et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3023–3026
3025
Table 1. Affinities ofderivatives 2a–e at human A
sine receptor subtypes
1
, A2a and A
3
adeno-
insertion ofNBD with alkyl spacers ofincreasing car-
6
bon chain length in the N position ofNECA. This
structural modification confers to 2a–e a pharmacologi-
cal profile which is different from NECA. These deriva-
tives generally possess a high affinity and selectivity for
i 3
K (A
, nM) a,d
Compd
n
K
(A
1
, nM)a,b
K
i
(A2a, nM) a,c
i
2a
2b
2c
2d
2e
2
4
6
8
10
>10,000
>10,000
>10,000
6750ꢁ1021
>10,000
6083ꢁ912
5096ꢁ764
6100ꢁ915
15.1ꢁ3.42
55.8ꢁ3.44
381ꢁ85.7
23.6ꢁ3.25
7.44ꢁ2.38
28.1ꢁ3.80
8.22ꢁ2.14
the A receptor subtype expressed in CHO cells, with no
3
significant affinity for A or A human ARs.
2a
1
3476ꢁ521
6350ꢁ952
200ꢁ45.3
NECA
All the alkyl spacers allow an efficient and selective
interaction ofthese types ofstructures with A recep-
tors, although the longer ones (2c–e, n=6–10) seem to
3
aBinding data were computer-analyzed by non-linear least squares
analysis (GraphPad Prism Softwares, San Diego, CA, USA). IC50
values were determined and converted to K (inhibition constant)
i
values by the Cheng and Prusoff equation.17 Values represent the
be preferred.
meansꢁSE ofthree experiments.
Compound 2d proved to be the most active and selec-
tive for human A ARs and it was then used in fluores-
b
c
3
10,11
Displacement of[ H]DPCPX from human cortical membranes.
3
3
12,13
Displacement of[ H]CGS 21680 from human striatal membranes.
125
cence microscopy. Real-time visualization ofbinding of
the fluorescent compound was successfully achieved in
CHO cells transfected with human A ARs. The fluor-
d
receptor14
Displacement of[
I]AB-MECA from the human A
3
expressed in CHO cells.15
3
escent staining ofthe plasma membrane was almost
completely inhibited by an excess ofNECA, demon-
strating the specificity ofthe interaction with A 3 ARs.
In conclusion, compound 2d can be considered as a new
selective fluorescent probe for human A receptor sub-
3
type. This new probe, possessing a 7-nitrobenzofurazyl
fluorophore which is excited by visible light (Abs 465
nm) is suitable to visualize and localize human A ARs
3
in cells. This property ofcompound 2d makes it a
potentially useful probe for receptor internalization and
trafficking studies in living cells.
3
Figure 1. Fluorescent labeling studies ofhuman A ARs in CHO cells.
CHO cells were cultured on coverslips and incubated with compound
ꢀ
2
d (500 nM) for 120 min at 22 C in the (a) absence or (b) presence of
References and Notes
500 mM ofNECA. At the end ofthe incubation time, the cells were
washed, and A
Magnification ꢂ335.
3
ARs were localized by fluorescence microscopy.
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Compound 2d, the most active and selective one for the
A receptor subtype, was incubated for 2 h with CHO
3
1
5
cells expressing human A ARs.
3
This incubation time was established based on pre-
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1
7
0
1
4
3
. For example, compound 5d: H NMR (CDCl , 200 MHz) d
.91 (t, 3H, J=7.3 Hz), 1.38 (s, 3H), 1.44 (s, 9H), 1.62 (s, 3H),
.27–2.80 (m, 12H), 3.04–3.21 (m, 4H), 3.49–3.62 (m, 2H),
.52 (bs, 1H, NH), 4.71 (d, 1H, J=1.5 Hz), 5.30–5.40 (m, 2H),
1
8
microscope. At a concentration about 50 times higher
than the K value (necessary to obtain a consistent,
i
5.86 (bs, 1H, NH), 6.02 (d, 1H, J=2.5 Hz), 7.19 (bs, 1H, NH),
+
+
reproducible labeling), compound 2d (500 nM) selec-
tively stained almost exclusively the membrane surface
ofCHO cells expressing A 3 ARs (see Fig. 1a). Binding
was completely prevented in the presence ofthe adeno-
sine agonist NECA (500 mM) (see Fig. 1b).
7.79 (s, 1H), 8.32 (s, 1H); MS (FAB ) m/e 576 (M+H) .
1
8. For example, compound 7d: H NMR (CDCl
.91 (t, 3H, J=7.4 Hz), 1.39 (s, 3H), 1.63 (s, 3H), 1.39–1.98
m, 12H), 3.07–3.21 (m, 2H), 3.43–3.74 (m, 4H), 4.71 (s, 1H),
3
, 200 MHz) d
0
(
5
6
.29–5.36 (m, 2H), 6.02–6.05 (m, 1H), 6.18 (d, 1H, J=8.8 Hz),
.33 (bs, 1H, NH), 7.83 (s, 1H), 8.32 (s, 1H), 8.51 (d, 1H,
+
+
J=8.3 Hz); MS (FAB ) m/e 639 (M+H) .
1
9
. For example, compound 2d: H NMR (CD OD, 200 MHz)
3
d 0.62 (t, 3H, J=7.1 Hz), 1.16–1.76 (m, 12H), 3.29–3.63 (m,
H), 4.48 (s, 1H), 5.14–5.21 (m, 2H), 6.02 (d, 1H, J=7.9 Hz),
6.34 (d, 1H, J=8.3 Hz), 8.14 (s, 1H), 8.21 (s, 1H), 8.52 (d, 1H,
Discussion and Conclusions
6
We synthesized compounds 2a–e as a new class of
fluorescent derivatives. These were obtained by the
+
+
J=8.5 Hz); MS (FAB ) m/e 599 (M+H) .

3026
M. Macchia et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3023–3026
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5
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1
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