Microwave-assisted synthesis of thieno[2,3-c]pyridine derivatives as a new series of allosteric enhancers at the adenosine A1 receptor

Article abstract of DOI:10.1016/j.bmcl.2006.08.041

The microwave-assisted aromatization method has been used for the synthesis of a series of novel thieno[2,3-c]pyridines. This rapid method produces compounds in good yield within minutes in comparison with conventional heating method. The synthesized mole

Full text of DOI:10.1016/j.bmcl.2006.08.041

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5530–5533
Microwave-assisted synthesis of thieno[2,3-c]pyridine derivatives as
a new series of allosteric enhancers at the adenosine A1 receptor
a,
b
Romeo Romagnoli,a,* Pier Giovanni Baraldi, Allan R. Moorman,
*
Maria Antonietta Iaconinoto,a Maria Dora Carrion,a Carlota Lopez Cara,a
Mojgan Aghazadeh Tabrizi,a Delia Preti,a Francesca Fruttarolo,a Stephen P. Baker,c
Katia Varanid and Pier Andrea Boread
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, 44100 Ferrara, Italy
bKing Pharmaceuticals Research and Development Inc., Cary, NC, USA
cDepartment of Pharmacology and Therapeutics, University of Florida, USA
`
Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Universita di Ferrara, 44100 Ferrara, Italy
d
Received 3 July 2006; revised 7 August 2006; accepted 8 August 2006
Available online 22 August 2006
Abstract—The microwave-assisted aromatization method has been used for the synthesis of a series of novel thieno[2,3-c]pyridines.
This rapid method produces compounds in good yield within minutes in comparison with conventional heating method. The syn-
thesized molecules have been evaluated as a potential new series of allosteric enhancers acting at the adenosine A1 receptor. In a
functional assay, one compound (3h) showed activity comparable with that of reference compound PD 81,723.
Ó 2006 Elsevier Ltd. All rights reserved.
Allosteric effects are observed when there are interac-
tions between two binding processes that occur simulta-
neously or sequentially: the binding of one ligand affects
the binding of another ligand. The flexible nature of
interactions between receptors and various allosteric
modulators, together with the potential for subtype
selectivity, makes allosteric sites attractive for therapeu-
tic intervention.1 2-Amino-3-benzoyl thiophenes are
allosteric enhancers acting at the adenosine A1 recep-
tor.2,3 Bruns and coworkers reported that this class of
derivatives are capable of enhancing both the binding
and activity of reference A1 receptor agonists, such as
N6-cyclopentyladenosine (CPA), at the adenosine A1
receptor. This effect is manifested as a slowing of the
rate of dissociation of the agonist from the receptor.4
Bruns also reported that these compounds were capable
of acting as competitive antagonists at the same recep-
tor, usually at higher concentrations.5 Therefore, the
concentration range where these compounds can en-
hance the effects of agonists is limited. Among the com-
pounds tested by Bruns, PD 81,723 (2-amino-4,5-
dimethylthien-3-yl)-[3-(trifluoromethyl)phenyl]-metha-
none (1) represents a specific and selective allosteric
enhancer of agonist binding to the A1 receptor, with
the best ratio of enhancement to antagonistic action at
this receptor.6
PD 81,723 is selective for adenosine A1 receptor, having
no effects on other adenosine receptor subtypes or on
other classes of receptors. While the exact molecular
mechanism(s) through which PD 81,723 exerts its allo-
steric actions remains unknown, the available data indi-
cate that PD 81,723 functions to stabilize a high affinity
or agonist-preferring state of the A1 receptor.7
To study the role of various substitutions on the phenyl
ring and the importance of the 4,5-dimethyl group on
the thienyl ring, several research groups5,8 have described
the synthesis and biological evaluation of compounds
useful as potent, yet selective allosteric enhancers of the
adenosine A1 receptor. The 2-amino and 3-benzoyl
groups were found to be crucial for the activity. It was
evident from previous SAR studies that substitution on
the benzoyl moiety at the 3-position of the thiophene ring
with electron-withdrawing substituents, such as chlorine
Keywords: Microwave; A1-adenosine receptor; Allosteric enhancer.
*
Corresponding authors. Tel.: +39 0532 291290; fax: +39 0532 291296
(R.R.); tel.: +39 0532 291293; fax: +39 0532 291296 (P.G.P); e-mail
addresses: rmr@unife.it; baraldi@unife.it
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.041

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5530–5533
5531
R
S
and trifluoromethyl, resulted in higher enhancement
R
O
O
activity. Substitution at the 4-position of the thiophene
ring with simple alkyl or aryl groups increased activi-
ty,2–4 while bulky 5-alkyl substitution had little or no
beneficial effect on activity, with the exception of a small
series of thiophene 5-bromo derivatives.8e In addition,
several studies indicated that the addition of a fused ring
on the thiophene provided improved allosteric enhancer
activity. Among these compounds, 2-amino-3-aroyl-
4,5,6,7-tetrahydro[2,3-c]pyridine derivatives with general
formula 2 were considered worthy of further attention. In
the series of molecules which bear the 3-(trifluorometh-
yl)benzoyl substituent of PD 81,723, N6-benzyl substitu-
tion (derivative 2a) was beneficial, while its removal (2b)
or substitution with a methyl (2c) was detrimental for the
enhancer activity.5,8a
O
b, c
a
R
NHAc
NH2
HN
N
CN
S
Boc
4a-j
6a-j
5a-j
a, R=C6H5
b, R=p-Cl-C6H5
c, R=o-Cl-C6H5
d, R=m,p-Cl2-C6H4
e, R=p-Br-C6H5
f, R=p-Me-C6H5
g, R=p-OMe-C6H5
h, R=m-CF3C6H5
i, R=1-naphthyl
j, R=2-thienyl
d
R
R
O
O
e
NHAc
N
NH2
S
N
S
7a-j
3a-j
Scheme 1. Reagents and conditions: (a) N-Boc-4-piperidone, S8,
morpholine, EtOH, 70 °C for 1 h then 1 h at rt; (b) AcCl, pyridine,
DCM, rt, 2 h; (c) TFA, DCM. 2 h, rt; (d) MnO2 toluene, 120 °C,
5 min, microwave (CEM, 300 W, ramp time 2 min); (e) NaOH, EtOH,
reflux, 1 h.
In this article, we report the synthesis and evaluation as
allosteric enhancers at the adenosine A1 receptor of a
novel class of ligands with general formula 3, based on
the 2-amino-thieno[2,3-c]pyridines’ molecular skeleton,
that contains different aroyl groups at the 3-position.
For compounds 3b–h, the substituents on the benzoyl
group were identified which further improved activity
(e.g., trifluoromethyl, bromo, chloro, methyl, and meth-
oxy).3,4,8 Replacing the aroyl for a naphthoyl moiety
also yielded potent enhancers.2,3 By the synthesis of 3-
(1-naphthoyl) derivative 3i, we seek to examine if the
receptor environment neighboring the benzoyl binding
site is lipophilic. Finally, we prepared 3j to further ex-
plore the effect of the replacement of the phenyl at the
3-position with an isosteric/isoelectronic 2-thienyl
group.
(MW) furnished the thieno[2,3-c]pyridines 7a–j. In com-
parison with conventional (thermal) heating, microwave
heating reduced the reaction time (3 h vs. 5 min, respec-
tively), although we did not notice any yield improve-
ment (53–65%). These latter derivatives were
transformed by saponification into the final products
3a–j.
The effects of compounds 3a–j were determined on for-
skolin-stimulated cAMP accumulation in the presence
of an A1-adenosine agonist (CPA) in Chinese hamster
ovary (CHO) cells expressing the cloned human adeno-
sine A1 receptor (hA1AR). Activation of these receptors
causes an inhibition of the activity of adenylyl cyclase
and a reduction of cAMP content of CHO cells.12 The
results are shown in Table 1.
Compounds 3a–j were obtained by an oxidative aroma-
tization procedure under microwave irradiation, starting
from the corresponding 4,5,6,7-tetrahydrothieno[2,3-
c]pyridine derivatives.
Allosteric enhancement was measured as the ability of
compounds 3a–j at four different concentrations (0.01,
0.1, 1, and 10 lM) to further reduce the cAMP content
of CHO:hA1 cells. The reference compound for compar-
ison was PD 81,723. As shown in Table 1, PD 81,723
CF3
CF3
R
S
O
O
O
NH2
N
N
R
NH2
NH2
S
3
S
Table 1. Effect of compounds 3a–j on cAMP cell content in CHO cells
expressing human adenosine A1 receptor
1, PD 81,723
2a, R=Bn
2b, R=H
2c, R=Me
R=substitued and unsubstituted
phenyl, 1-naphthyl, 2-thienyl.
Compound
cAMP content as percent of control
(mean SEM)a
The thieno[2,3-c]pyridine system was previously pre-
pared in low yield by reaction of 3-bromo-4-cyanometh-
ylpyridine with phenylisothiocyanates and sodium
hydride.9 Thieno[2,3-c]pyridine analogs with general
structure 3 were synthesized by a five-step synthesis de-
scribed in Scheme 1. Bicyclic N6-tert-butoxycarbonyl
(Boc) protected 2-amino-3-aroyl-4,5,6,7-tetrahydrothie-
no[b]pyridines 5a–j were obtained by the Gewald reac-
tion10 applied to b-ketonitriles 4a–j11 and N-Boc-4-
piperidone. Acetylation of the amino group using acetyl
chloride followed by removal of the N6-Boc protecting
group with trifluoroacetic acid (TFA) afforded the
4,5,6,7-tetrahydro[2,3-c]pyridine derivatives 6a–j. The
subsequent aromatization by treatment with manganese
dioxide (MnO2) in toluene under microwave irradiation
Concentration of compounds
0.01 lM
0.1 lM
1 lM
10 lM
3a
3b
91
104
8
6
89 10
90 11
96 10
100
114 10
8
102
98
4
5
8
5
9
9
6
7
5
3
3c
3d
103 11
97
103
84
5
5
6
6
6
6
5
8
3
111
105
104
121
94
8
7
4
5
9
4
8
9
2
113
97
5
6
9
108
89
3e
3f
3g
102
107
97
107
90
95 10
3h
3i
101
5
4
6
3
95
82
63
66
113
111
97
112
104
93
107
107
81
3j
PD 81,723
98
52
a The results are the average of six experiments at each of four con-
centrations of tested compound.

5532
R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5530–5533
inhibited forskolin-stimulated cAMP accumulation in a
concentration-dependent manner from 1 to 10 lM with
a maximum inhibition of 48%. The pyridine ring fused
to the 2-amino-3-aroyl thiophene ring system led to a
significant loss of activity. Thus, these compounds gen-
erally have no potency or efficacy as allosteric enhancers
through the concentration range tested. Only one com-
pound of this series (3h) exhibited a concentration-de-
pendent activity comparable with that of the reference
compound, PD 81,723, at all tested concentrations, with
37% inhibition at 10 lM. Replacing the benzoyl with a
bulky 1-naphthoyl yielded compound (3i), active only
at 10 lM.
from this series (3h) was shown to have activity as an
allosteric enhancer and thus the thieno[2,3-c]pyridine
nucleus could be useful as a precursor for the synthesis
of further compounds.
Acknowledgment
We thank King Pharmaceuticals Research and Develop-
ment, Inc. for financial support.
References and notes
1. Christopoulos, A.; Kennakin, T. Pharmacol. Rev. 2002,
54, 323.
2. Gao, Z. G.; Kim, S.-K.; Ijzerman, A. P.; Jacobson, K. A.
Mini-Rev. Med. Chem. 2005, 5, 545.
3. Baraldi, P. G.; Moorman, A. R.; Tabrizi, M. A.; Pavani,
M. G.; Romagnoli, R. Exp. Opin. Ther. Pat. 2004, 14, 71.
4. Bruns, R. F.; Fergus, J. H. Mol. Pharmacol. 1990, 38, 939.
5. Bruns, R. F.; Fergus, J. H.; Coughenour, L. L.; Court-
land, G. G.; Pugsley, T. A.; Dodd, J. H.; Tinney, F. J.
Mol. Pharmacol. 1990, 38, 950.
Since many known allosteric enhancers are also adeno-
sine A1 receptor antagonists at some (usually higher)
concentration, the fact that most of the synthesized
compounds did not show a greater efficacy than did
10 lM PD 81,723 could be explained by possible antag-
onist properties. Compounds 3a–j were tested at a con-
centration of 10 lM for their ability to displace the
binding of [3H]DPCPX, [3H]ZM 241385, and
[3H]MRE-3008F20 to the ligand binding site of CHO:-
hA1, CHO:hA2A, and CHO:hA3 adenosine receptors
(AR), respectively (Table 2).13
6. Kourounakis, A.; Visser, C.; DE Groote, M.; Ijzerman, A.
P. Biochem. Pharmacol. 2001, 61, 137.
7. Bhattacharya, S.; Linden, J. Biochem. Biophys. Acta 1995,
1265, 15.
The prototype enhancer, PD 81,723, did not inhibit the
binding of a radiolabeled antagonist to A1 and A2A
receptors, but it reduced by 21% the binding of
[3H]MRE-3008F20 to the A3 receptor. None of the
selected compounds inhibited binding at the hA2AAR
and hA3AR, but some derivatives (3b, 3f, and 3j) inhib-
ited binding to the hA1 AR to some extent (35–50%).
For the only compound of this series active as an alloste-
ric enhancer (3h), it was possible to achieve a good sep-
aration between enhancing activity and binding to the
orthosteric site, being devoid of antagonist activity on
A1, A2A, and A3 receptors.
8. (a) Baraldi, P. G.; Zaid, A. N.; Lampronti, I.; Fruttarolo,
F.; Pavani, M. G.; Tabrizi, M. A.; Shryock, J. C.; Leung,
E.; Romagnoli, R. Bioorg. Med. Chem. Lett. 2000, 10,
1953; (b) Van der Klein, P. A. M.; Kourounakis, A. P.;
IJzerman, A. P. J. Med. Chem. 1999, 42, 3629; (c)
Kourounakis, A. P.; Visser, C.; de Groote, M.; IJzerman,
A. P. Drug Dev. Res. 2000, 51, 207; (d) Tranberg, C. E.;
Zickgraf, A.; Giunta, B. N.; Luetjens, H.; Figler, H.;
Murphree, L. J.; Falke, R.; Fleischer, H.; Linden, J.;
Scammells, P. J.; Olsson, R. A. J. Med. Chem. 2002, 45,
382; (e) Lutjens, H.; Zickgraf, A.; Fingler, H.; Linden, J.;
¨
Olsson, R. A.; Scammels, P. J. J. Med. Chem. 2003, 46,
1870; (f) Nikolakopoulos, G.; Figler, H.; Linden, J.;
Scammells, P. J. Bioorg. Med. Chem. 2006, 14, 2358.
9. Bremner, D. H.; Dunn, A. D.; Wilson, K. A.; Sturrock, K.
R.; Wishart, G. Synthesis 1998, 1095.
In conclusion, we have reported a new and general
methodology for the construction of a series of 2-ami-
no-3-aroyl-thieno[2,3-c]pyridines as potential allosteric
enhancer at the A1-adenosine receptor. This route is
convenient for its simplicity, availability of starting
materials, and good yields obtained. One compound
10. Gewald, K.; Schinke, E.; Bo¨ettcher, H. Chem. Ber. 1966,
99, 94.
11. For the synthesis of 4a–h, see Ref. 8b, 4i: (a) Baraldi, P.
G.; Romagnoli, R.; Pavani, M. G.; Nunez, M. C.; Tabrizi,
M. T.; Shryock, J. C.; Leung, E.; Moorman, A. R.;
Uluoghu, C.; Iannotta, V.; Merighi, S.; Borea, P. A. J.
Med. Chem. 2003, 46, 794; 4j: (b) Baraldi, P. G.; Pavani,
M. G.; Shryock, J. C.; Iannotta, V.; Merighi, S.; Borea, P.
A.; Moorman, A. R.; Romagnoli, R. Eur. J. Med. Chem.
2004, 39, 855.
12. A stock solution (10 mM) of each compound was
prepared in DMSO at the initial time of use and stored
at À20 °C. Chinese hamster ovary (CHO) cells expressing
recombinant human A1-adenosine receptors were used to
test A1-adenosine receptor allosteric enhancers. These cells
expressed the A1 receptor at a density of approximately
5 pmol/mg cellular protein. Cells were grown in 48-well
culture plates with Ham’s F12 culture medium, fetal bovine
serum, and antibiotic G-418 for 48–72 h (1 day preconflu-
ent). To begin an experiment, the culture medium was
removed from each well, and warm Hanks’ balanced salt
solution (HBSS) was added. This wash solution was
removed after 6 min at 37 °C and replaced with fresh
HBSS solution that included adenosine deaminase
Table 2. Percent Inhibition activity of enhancer compounds 3a–j
a
b
c
Compound
hA1
hA2A
hA3
3a
3b
0
50
0
0
5
0
0
0
5
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
3c
3d
0
0
0
3e
0
0
3f
3g
45
0
0
0
3h
3i
0
0
0
0
3j
PD 81,723
35
0
0
21
Inhibition activity was expressed as percent displacement value
( STD, n = 3) of 1 nM of [3H]DPCPXa, [3H]ZM 241385b or [3H]MRE
3008F20c by 10 lM of tested compound.

R. Romagnoli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5530–5533
5533
(0.5 U/ml) and 20 lM rolipram with or without 1 lM
forskolin, 0.03 nM CPA, and varying concentrations of
T-compounds or PD 81,723. After a 6-min incubation at
37 °C, the incubation solution was aspirated, and HCl
(0.5 ml, 50 mM) was added to each well to terminate drug
action. The cAMP content in each well was determined by a
radioimmunoassay. Briefly, a standard curve was prepared
in duplicate with tubes containing 100 ll cAMP (0.001–
10 pmol) in 50 mM HCl. The 48-well plates containing the
samples were gently agitated to mix the contents of the
wells. A 5 ll aliquot was then transferred from each well to
test tubes containing 100 ll of 50 mM HCl. Each sample
was then acetylated by the addition of 4.5 ll triethylamine
and acetic anhydride solution (3.5:1), and immediately
vortexed. A 10 ll aliquot of [125I]-ScAMP-TME containing
20,000 cpms was added, followed by 100 ll cAMP antibody
in a solution of 50 mM Na-acetate buffer (pH 4.75)
containing 0.125% BSA. The tubes were then incubated
either at room temperature for 2 h or at 4 °C overnight.
Following the incubation, 50 ll hydroxyapatite in a 1:1
suspension with water was added to each tube and
incubated for 10 min at room temperature. Using a Brandel
cell harvester, samples were filtered and rinsed 3 times with
ice-cold 10 mM Tris buffer, pH 7.0. The filters were placed
in vials and [125I]-ScAMP-TME bound was measured using
a Beckman Gamma Counter. In each experiment (3 plates,
48 wells per plate), the response to the tested compounds
and PD 81,723 was determined in quadruplicate at
concentrations of 0.01, 0.1, 1.0, and 10 lM. Additionally,
each plate included controls: basal, 1 lM forskolin, 1 lM
forskolin with 100 nM CPA, and 1 lM forskolin with
0.03 nM CPA. Each experiment was repeated at least 3
times. The data were analyzed using Microsoft Excel and
graphed using GraphPad Prism. The results are expressed
as the means SEM.
13. Competition experiments of 1 nM [3H]DPCPX to CHO:-
hA1 membranes were performed incubating membranes
(100 lg of protein/assay) at 25 °C for 150 min. Competi-
tion experiments were performed in duplicate in a final
volume of 250 ll in test tubes containing 50 lM Tris–HCl
buffer, pH 7.4, and 100 ll of membranes and at least six to
eight different concentrations of the tested compounds.
Non-specific binding was defined as the binding in the
presence of 1 lM DPCPX and was about 25% of total
binding. Competition experiments of 2 nM [3H]ZM241385
to CHO:hA2A membranes were performed incubating
membranes (100 lg of protein/assay) at 4 °C for 60 min.
Competition experiments were performed in duplicate in a
final volume of 250 ll in test tubes containing 50 lM Tris–
HCl buffer, 10 lM MgCl2, pH 7.4, and 100 ll of
membranes, and at least six to eight different concentra-
tions of the tested compounds. Non-specific binding was
defined as the binding in the presence of 1 lM ZM241385
and was about 30% of total binding. Competition exper-
iments of 2 nM [3H]MRE-3008F20 to CHO:hA3 mem-
branes were performed incubating membranes (100 lg of
protein/assay) at 4 °C for 150 min. Competition experi-
ments were performed in duplicate in a final volume of
250 ll in test tubes containing 50 lM Tris–HCl buffer,
10 lM MgCl2, 1 mM EDTA, pH 7.4, and 100 ll of
membranes, and at least six to eight different concentra-
tions of the tested compounds. Non-specific binding was
defined as the binding in the presence of 1 lM MRE-
3008F20 and was about 30% of total binding.