Synthesis and biological characterization of [3H] (2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)-(4-chlorophenyl)-methanone, the first radiolabelled adenosine A1 allosteric enhancer

Article abstract of DOI:10.1016/j.bmcl.2005.11.037

Among the adenosine A₁ allosteric enhancers reported so far, compound (2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)-(4-chlorophenyl)- methanone 1 (named T-62) has shown biological properties similar to those of PD 81,723, the reference A

Full text of DOI:10.1016/j.bmcl.2005.11.037

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1402–1404
Synthesis and biological characterization of [3H]
(2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)-
(4-chlorophenyl)-methanone, the first radiolabelled
adenosine A1 allosteric enhancer
Pier Giovanni Baraldi,a,* Maria Giovanna Pavani,a Edward Leung,b Allan R. Moorman,b
Katia Varani,c Fabrizio Vincenzi,c Pier Andrea Boreac and Romeo Romagnolia
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, 44100 Ferrara, Italy
bKing Pharmaceuticals Research and Development Inc., Cary, NC 27513, USA
`
Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Universita di Ferrara, 44100 Ferrara, Italy
c
Received 12 October 2005; revised 10 November 2005; accepted 10 November 2005
Available online 29 November 2005
Abstract—Among the adenosine A1 allosteric enhancers reported so far, compound (2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-
yl)-(4-chlorophenyl)-methanone 1 (named T-62) has shown biological properties similar to those of PD 81,723, the reference A1 allo-
steric enhancer and it has been more fully pharmacologically investigated. The preparation of the radiolabelled form of compound 1
and its characterization by saturation binding experiments are reported. These studies allowed us to demonstrate for the first time
the existence of a specific, allosteric site on the A1 receptor.
Ó 2005 Elsevier Ltd. All rights reserved.
Adenosine and adenosine A1 agonists reduce hypersensi-
tivity following inflammation and peripheral nerve injury
in models of chronic pain.1 However, the use of such
direct agonists is complicated by a variety of adverse ef-
fects, such as angina pain, atrioventricular block, head-
ache, nausea and retching. One approach to avoid
adverse effects from direct agonists is to enhance the effect
of the endogenous ligand, adenosine, by administering a
positive allosteric modulator of its receptor.2
T-62,8 while Leung has described its use for treating
neuropathic pain in humans.9 T-62 is currently being
investigated as a possible treatment for this disorder.10
The promising properties of this compound prompted
us to design and synthesize the radiolabelled form of
T-62 (2).11
Although several approaches appeared viable, the strat-
egy that ultimately proved successful involved the intro-
duction of the radioisotope into the phenyl ring (Scheme
1). Recognizing that the preparation of the 4-chloro-3,5-
diiodobenzoic ester 3 was known,12 we adopted the
synthetic scheme we have previously used to prepare
similar 2-amino-3-aroylthiophene compounds13 to
One class of molecules reported to act as allosteric
enhancers at the A1 adenosine receptor are the 2-amino-
3-benzoylthiophene derivatives, of which PD 81,723 is
the prototypical example.3
The closely related compound T-62 (1) has shown
biological properties similar to those of PD 81,723.4–7
Baraldi has described the preparation of compound
CF3
O
Keywords: Allosteric enhancer; Adenosine A1 receptor; Radiolabelled
compound.
NH2
S
*
Corresponding author. Tel.: +39 0532 291293; fax: +39 0532
291296; e-mail: baraldi@unife.it
PD 81, 723
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.037

P. G. Baraldi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1402–1404
1403
T-62 was able to increase in a concentration-dependent
way the agonist-specific binding of [3H]CCPA to adeno-
sine A1 receptors in crude membrane preparations from
CHO-A1 cells, rat cortex and human brain, determining
a 51%, 15% and 22% increase of binding in hCHO-A1,
rat cortex and human brain membranes, respectively.
Cl
O
NH2
S
T-62 increased the BMAX of the agonist [3H]CCPA to re-
combinant human A1 adenosine receptors determining a
65%, 15% and 23% BMAX increase of [3H]CCPA binding
in hCHO-A1, rat cortex and human brain membranes,
respectively.
T-62 (1)
obtain derivative 5. Condensation of the ester 3 with the
anion of acetonitrile afforded the requisite b-ketonitrile
(4). The formation of the 2-amino-3-benzoylthiophene
5 was obtained by the base-catalyzed condensation of
cyclohexanone and b-ketonitrile 4.14 In turn, compound
5 was dehalogenated to compound 1 by catalytic
hydrogenolysis and similar to compound 2 by hydroge-
nation in the presence of tritium gas.
T-62 did not increase the binding of the agonists
[3H]CGS 21680 and [125I]AB-MECA on hCHO-A2A
and hCHO-A3 membranes, thus behaving as a selective
allosteric modulator for A1 adenosine receptors.
The binding properties of the radiolabelled T-62 were
investigated by saturation assays performed on both
hCHO-A1 cells and membranes.18 The analysis of the
experimental data19 revealed an affinity value in the
micromolar range (KD = 4.6 1.1 lM) and a high
receptor density (BMAX = 76.5 8.2 pmol/mg of
protein).
The deiodination was therefore the crucial step of the
synthesis. It was performed following a procedure
reported in the literature for tyrosine analogues.15 Deio-
dination was obtained by catalytic hydrogenolysis of
derivative 5 at 50 psi in methanol/dioxane, with 10%
Pd/C as catalyst to give compound 1 in good yield.16
Tritiation of 5 was achieved under the same conditions,
affording [3H]T-62 with an estimated specific activity of
47 Ci/mmol.17
The biological investigation of [3H]T-62 suggested the
presence of an allosteric binding site on adenosine A1
receptor distinct from the active binding site for agonists
and antagonists.
Compound T-62 was tested in saturation binding exper-
iments and in the functional cAMP assay6 and it was
shown to have good activity as an allosteric enhancer.
The development of a new radiolabelled adenosine A1
allosteric enhancer could provide more information
about the allosteric binding site on adenosine A1
receptor.
T62 caused a decrease of cAMP content of 55% at a
concentration of 10 lM in CHO cells expressing the
human A1 receptor.
Acknowledgment
We thank King Pharmaceuticals Research and Develop-
ment for proposal and financial support.
Cl
I
Cl
Cl
I
I
I
I
I
(ii)
(i)
O
References and notes
N
COOMe
C
O
NH2
S
3
1. Lee, Y. W.; Yaksh, T. L. J. Pharmacol. Exp. Ther. 1996,
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4
5
(iii)
2. Christopoulos, A. Nat. Rev. Drug Discov. 2002, 1, 198.
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Mol. Pharmacol. 1990, 38, 950.
Cl
(iv)
Cl 3H
3H
O
5. Van der Klein, P. A. M.; Kouronakis, A. P.; IJzerman, A.
P. J. Med. Chem. 1999, 42, 3629.
NH2
S
6. Baraldi, P. G.; Zaid, A. Z.; Lampronti, I.; Fruttarolo, F.
F.; Pavani, M. G.; Tabrizi, M. A.; Shryock, J. C. S.;
Leung, E.; Romagnoli, R. Bioorg. Med. Chem. Lett. 2000,
10, 1953.
7. Childers, S. R.; Li, X.; Xiao, R.; Eisenach, J. C. J.
Neurochem. 2005, 93, 715.
1 (T-62)
O
NH2
2 ([3H] T-62)
S
Scheme 1. Reagents and conditions: (i) NaH, MeCN; (ii) cyclohexa-
none, S, morpholine, EtOH; (iii) TEA, 10% Pd/C, H2, 50 psi,
overnight, MeOH/dioxane 2:1; (iv) TEA, 10% Pd/C, tritium, 50 psi,
overnight, MeOH/dioxane 2:1.
8. Baraldi, P. G. US Patent 5,939,432, 1997.
9. Leung, E. US Patent 6,248,774, 2002.
10. Pan, H.-L.; Xu, Z.; Leung, E.; Eisenach, J. C. Anesthe-
siology 2001, 95, 416.

1404
P. G. Baraldi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1402–1404
11. Pavani, M. G. Doctoral thesis at University of Ferrara,
Italy, 2004, pp 58–65.
12. Borrows, E. T.; Clayton, J. C.; Hems, B. A. J. Chem. Soc.
1949, 42, 185.
13. Baraldi, P. G.; Romagnoli, R.; Pavani, M. G.; Nunez, M.
C.; Tabrizi, M. A.; Shryock, J. C.; Leung, E.; Moorman,
A. R.; Uluoglu, C.; Iannotta, V.; Merighi, S.; Borea, P. A.
J. Med. Chem. 2003, 46, 794.
14. Gewald, K.; Schinke, E.; Boettcher, H. Chem. Ber 1966,
99, 94.
radioligand [3H]T-62 and non-specific binding is estimated
at each concentration of radioligand. Non-specific binding
was defined as binding in the presence of 20 lM T-62.
Different incubation times (90, 120 and 180 min) and
temperatures (4, 25 and 37 °C) were studied and applied to
two substrates such as hCHO-A1 cells (500.000 cells/
sample) and hCHO-A1 membranes (100 lg of protein/
sample) to investigate the better experimental conditions.
Bound and free radioactivity were separated by filtering
the assay mixture through Whatman GF/B glass-fibre
filters using a Micro-Mate 196 cell harvester (Packard
Instrument Company). To reduce non-specific binding of
[3H]T-62 to glass fibre filter, an antiadsorbent reagent,
such as polyethyleneimine at a concentration of 0.1%, was
used. The filter bound radioactivity was counted on Top
Count Microplate scintillation Counter (efficiency 57%)
with Micro-Scint 20. In ÔcoldÕ saturation binding experi-
ments to hCHOA1 membranes, [3H]T-62 at the concen-
tration 10 nM was incubated for 90 min at 25 °C with
different concentrations of unlabelled ligand T-62 in the
range 1 nM–50 lM. Bound to free radioactivity was
separated by centrifugation, a method adopted when the
affinity of radioligand (KD) to its receptor binding site is
more than 10À8M. See: Yamamura, H. I.; Enna, S. J.;
Kuhar, H. J. In Neurotrasmitter Receptor Binding; Raven
Press: New York, 1985; pp 61–89. In particular, the
samples were centrifuged at 0 °C for 3 min at 12000g in a
Beckman microcentrifuge and the tip of microtube con-
taining the particulate pellet was cut off, transferred to
vials containing 5 ml of Aquassure and counted by
scintillation spectrometry.
´
15. Verschueren, K.; Toth, G.; Tourwe, D.; Lebl, M.; Van
Binst, G.; Hruby, V. Synthesis 1992, 458.
16. Procedure for deiodination reaction: A mixture of 6
(0.313 mmol, 170 mg), TEA (4 equiv, 174 ll) and 10%
Pd/C (200 mg) in MeOH/1,4 dioxane (2:1 v/v, 15 ml) was
reduced by Parr shaker on a high-pressure autoclave
(50 psi, overnight) (TLC: ethyl acetate/petroleum ether
2:8). After filtration over Celite, the solvent was evap-
orated. The residue was dissolved in AcOEt , washed
with water, brine and finally dried over Na2SO4. The
evaporation of the solvent gave the crude product that
was purified by column chromatography. Recrystalliza-
tion from petroleum ether gave compound 1.
(Yield = 80%).
17. The synthesis of [3H]T-62 was performed at Amersham
International (Buckinghamshire, UK) from tritium gas
through a method developed by Nycomed Amersham plc.
The product was purified by high-performance liquid
chromatography using a hexane/ethyl acetate gradient.
The mass spectrum was consistent with the proposed
structure 2 and the non-labelled reference 1.
18. In ÔhotÕ saturation binding experiments, a given receptor
population is incubated with increasing amounts of
19. Munson, P. J.; Rodbard, D. Anal. Biochem. 1980, 107,
220.