Structure-activity relationships of 2-amino-3-aroyl-4-[(4-arylpiperazin-1- yl)methyl]thiophenes. Part 2: Probing the influence of diverse substituents at the phenyl of the arylpiperazine moiety on allosteric enhancer activity at the A1 adenosine receptor

Article abstract of DOI:10.1016/j.bmc.2011.11.044

In a preliminary article, we reported the potent allosteric enhancer activity at the A₁ adenosine receptor of a small series of 2-amino-3-(4-chlorobenzoyl)-4-[4-(aryl)piperazin-1-yl)methyl]thiophene derivatives bearing electron-withdrawing or electron-releasing groups at the para-position of the phenylpiperazine moiety. In the present study, we report the development of the compounds previously studied by modifying both the number and position of substituents on the phenylpiperazine moiety, aimed at establishing a structure-activity relationship identifying additional compounds with improved activity. The nature and the position of substituents on the phenyl ring tethered to the piperazine seemed to exert a fundamental influence on the allosteric enhancer activity, with the 3,4-difluoro 4i, 3-chloro-4-fluoro 4o, and 4-trifluoromethoxy 4ak derivatives being the most active compounds in binding (saturation and competition experiments) and functional cAMP studies. This study shows that it is also possible to obtain a good separation between allosteric enhancement and antagonistic activity at the A₁ adenosine receptor.

Full text of DOI:10.1016/j.bmc.2011.11.044

Bioorganic & Medicinal Chemistry 20 (2012) 996–1007
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of 2-amino-3-aroyl-4-[(4-arylpiperazin-1-yl)m-
ethyl]thiophenes. Part 2: Probing the influence of diverse substituents at the
phenyl of the arylpiperazine moiety on allosteric enhancer activity at the A1
adenosine receptor
Romeo Romagnoli a, , Pier Giovanni Baraldi a, , Maria Dora Carrion a, Carlota Lopez Cara a,
Olga Cruz-Lopez a, Maria Kimatrai Salvador a, Delia Preti a, Mojgan Aghazadeh Tabrizi a,
John C. Shryock b, Allan R. Moorman c, Fabrizio Vincenzi d, Katia Varani d, Pier Andrea Borea d
⇑
⇑
a Dipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 17–19, Università di Ferrara, 44121 Ferrara, Italy
b Department of Medicine, University of Florida, Gainesville, FL, United States
c King Pharmaceuticals Inc., Research and Development, 4000 CentreGreen Way, Suite 300, Cary, NC, United States
d Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Università di Ferrara, Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In a preliminary article, we reported the potent allosteric enhancer activity at the A1 adenosine receptor
of a small series of 2-amino-3-(4-chlorobenzoyl)-4-[4-(aryl)piperazin-1-yl)methyl]thiophene derivatives
bearing electron-withdrawing or electron-releasing groups at the para-position of the phenylpiperazine
moiety. In the present study, we report the development of the compounds previously studied by mod-
ifying both the number and position of substituents on the phenylpiperazine moiety, aimed at establish-
ing a structure–activity relationship identifying additional compounds with improved activity.
The nature and the position of substituents on the phenyl ring tethered to the piperazine seemed to
exert a fundamental influence on the allosteric enhancer activity, with the 3,4-difluoro 4i, 3-chloro-4-flu-
oro 4o, and 4-trifluoromethoxy 4ak derivatives being the most active compounds in binding (saturation
and competition experiments) and functional cAMP studies. This study shows that it is also possible to
obtain a good separation between allosteric enhancement and antagonistic activity at the A1 adenosine
receptor.
Received 7 September 2011
Revised 19 October 2011
Accepted 20 November 2011
Available online 1 December 2011
Keywords:
A1 adenosine receptor
Allosteric enhancer
G protein-coupled receptors
2-Amino-3-benzoylthiophene
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
mechanisms such as ischemic preconditioning (IPC) or modulating
the actions of inflammatory cells, as in reperfusion injury.
Adenosine is an endogenous nucleoside modulator able to
mediate several effects through activation of four G-protein cou-
pled adenosine receptors (ARs) named as A1, A2A, A2B and A3.1 In
particular A1 and A3ARs are coupled to Gi proteins mediating a de-
crease of cAMP production whilst A2A and A2BARs are linked to Gs
proteins and increase cAMP accumulation. The adenosine A1AR is
highly and widely expressed in the Central Nervous System
(CNS) with high levels in brain, cortex, cerebellum and hippocam-
pus, but also in other tissues such as fat cells, bladder and heart.
The A1ARs modulate the activity of the CNS mediating anticonvul-
sant and anxiolytic effects and playing a role in adenosine-
mediated analgesia.2 Adenosine accumulates rapidly at sites of
tissue hypoxia or injury exerting protective effects through
Due to their ubiquitous presence, the ability to selectively mod-
ulate A1ARs within specific target tissues may be of great therapeu-
tic importance. The side effects induced by the A1 activation limit
the clinical application of A1 agonists. A more controlled and selec-
tive ‘tuning’ action is feasible through the allosteric modulation of
G protein-coupled receptors (GPCRs).3 ‘Allosteric’ refers to binding
sites that are different from the ‘orthosteric’ primary substrate or
ligand binding site, to which the binding of modulators results in
conformation changes that might profoundly influence GPCR
function.4
The binding of an allosteric enhancer (AE) to its site causes con-
formational changes in the A1AR, slowing the dissociation of
endogenous agonist adenosine from the orthosteric site.4 By the
screening of chemical libraries, Bruns and colleagues identified
various 2-amino-3-benzoylthiophene derivatives capable of slow-
ing the dissociation of the agonist N6-cyclopentyladenosine (CPA)
from the A1AR. They also reported that these compounds were
⇑
Corresponding authors. Tel.: +39 (0) 532455303; fax: +39 (0) 532455953 (R.R.);
tel.: +39 (0) 532455293; fax: +39 (0) 532455953 (P.G.B.).
E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.044

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
997
capable of acting as competitive antagonists at the same receptor
subtype at higher concentrations.6,7 Further investigations
have shown that this class of molecules is able to act as AEs at
the A1AR, both in binding and in vitro functional assays.7 Among
the tested compounds, PD 81,723 (2-amino-4,5-dimethylthien-3-
yl)-[3-(trifluoromethyl)phenyl]-methanone (compound 1) was
pharmacologically studied and recognized as the first specific
and selective AE of the A1AR.7–9
Since the structure of the allosteric site of the A1ARs has not
been resolved at the atomic level and intrigued by the peculiarity
of the PD 81,723 structure, several groups started a molecular
modulation of this ligand to elucidate the binding site of the 2-ami-
no-3-aroyl-thiophene moiety.10–19 These studies highlighted that
the 2-amino group is essential for activity. In addition, electron-
withdrawing substituents, such as chloro and trifluoromethyl, at
either the meta or para-position on the benzoyl moiety at the 3-po-
sition of the thiophene ring greatly increased enhancement activ-
ity. A range of alkyl and aryl groups in the 4-and 5-positions of
the thiophene ring also favored allosteric enhancing activity.
Among the synthesized compounds, derivative 2 {T-62, (2-ami-
no-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)(4-chlorophenyl)meth-
anone, was taken into Phase II clinical trials by King Pharmaceutical
for the treatment of neuropathic pain.20
moieties attached to the 4-position of the thiophene ring by a
methylene unit (Chart 1).21 In detail, we have evaluated the effect
on AE activity by electron-withdrawing substituents such as F, Cl,
CN and CF3, (compounds 3e, 3f, 3i and 3j, respectively) and elec-
tron-donating groups (CH3 and 3,4-methylenedioxy for 3g and
3h, respectively) introduced as substituents on the N-phenylpiper-
azine moiety. The results obtained with compounds 3a–l make it
possible to isolate in the phenylpiperazine moiety a region that
is critical for interaction with the allosteric site of the A1ARs, and
whose manipulation can greatly improve enhancer activity. It
may be possible that the phenyl ring of the phenylpiperazine moi-
ety contributes to activity with a p–p interaction (such as charge-
transfer), interacting with a lipophilic pocket. This was confirmed
by replacement of the phenyl moiety with electron-deficient het-
eroaromatic pyridin-2-yl (3k) or pyrimidin-2-yl (3l) rings, which
incorporate one or two basic nitrogens in the critical region of
binding, causing a reduction of the AE activity.
For these reasons, derivatives 3a–l were the starting point for
achieving potent and efficacious compounds with reduced compet-
itive antagonistic activity that is not required for the enhancement
at A1ARs. The nature of group at the para-position of the phenylpip-
erazine moiety seemed to exert an important influence on AE activ-
ity, although only a limited series of substituents were considered to
investigate their effect. Therefore, in the current study we have
investigated the structural modification of the phenyl piperazine
moiety at the 4-position of the 2-amino-3-(4-chlorobenzoyl)-
In a preliminary article, we have reported the AE activity at the
A1ARs of a series of 2-amino-3-(4-chlorobenzoyl)thiophene deriv-
atives 3a–l, with various alkyl- (3a–b) or aryl- (3c–l) piperazine
3a; R=CH3
Cl
CF3
Cl
3b; R=C6H11
3c; R=C6H5
3d; R=C6H5CH2
3e; R=4-F-C6H4
O
3f; R=4-Cl-C6H4
O
O
3g; R=4-CH3-C6H4
R
N
N
3h; R=3,4-methylenedioxyphenyl
3i; R=4-CN-C6H4
NH2
NH2
1, PD 81,723
NH2
S
S
S
3j; R=4-CF3-C6H4
2, T 62
3a-l
3k; R=2-pyridinyl
3l; R=2-pyrimidinyl
R
R=4-Cl or 3-CF3
Ar=phenyl substituted with
EWG or ERG or pyridinyl
X=N for 4a-am and 4ao-aq
X=CH for 4an
O
Ar
X
N
NH2
S
4
Ar=4-Cl-C6H4COCH2, R=4-Cl, 4u
Ar=3-Cl-C6H4, R=4-Cl, 4v
Ar=(CH3)2CH, R=4-Cl, 4a
Ar=cyclopentyl, R=4-Cl, 4b
Ar=cycloheptyl, R=4-Cl, 4c
Ar=1-naphthyl, R=4-Cl, 4d
Ar=2-Cl-C6H4, R=4-Cl, 4w
Ar=2,6-diCl-C6H3, R=4-Cl, 4x
Ar=2,5-diCl-C6H3, R=4-Cl, 4y
Ar=3,4-diCl-C6H3, R=4-Cl, 4z
Ar=2,4-diCl-C6H3, R=4-Cl, 4aa
Ar=2,3-diCl-C6H3, R=4-Cl, 4ab
Ar=3,5-diCl-C6H3, R=4-Cl, 4ac
Ar=2-Cl,4-F-C6H3, R=4-Cl, 4ad
Ar=2-F,4-Cl-C6H3, R=4-Cl, 4ae
Ar=4-CH3O-C6H4, R=4-Cl, 4af
Ar=4-NO2-C6H4, R=4-Cl, 4ag
Ar=3-CF3-C6H4, R=4-Cl, 4ah
Ar=2-CF3-C6H4, R=4-Cl, 4ai
Ar=4-Cl,3-CF3-C6H3, R=4-Cl, 4aj
Ar=4-CF3O-C6H4, R=4-Cl, 4ak
Ar=3-pyridinyl, R=4-Cl, 4al
Ar=4-F-C6H4CH2, R=4-Cl, 4e
Ar=3-F-C6H4, R=4-Cl, 4f
Ar=2-F-C6H4, R=4-Cl, 4g
Ar=2,4-diF-C6H3, R=4-Cl, 4h
Ar=3,4-diF-C6H3, R=4-Cl, 4i
Ar=3,5-diF-C6H3, R=4-Cl, 4j
Ar=2,6-diF-C6H3, R=4-Cl, 4k
Ar=2,3-diF-C6H3, R=4-Cl, 4l
Ar=2,5-diF-C6H3, R=4-Cl, 4m
Ar=2,4,6-triF-C6H2, R=4-Cl, 4n
Ar=3-Cl,4-F-C6H3, R=4-Cl, 4o
Ar=4-Cl-C6H4CO, R=4-Cl, 4p
Ar=4-Cl-C6H4CH2, R=4-Cl, 4q
Ar=4-Cl-C6H4(CH2)2, R=4-Cl, 4r
Ar=4-Cl-C6H4(CH2)3, R=4-Cl, 4s
Ar=4-Cl-C6H4CH2CO, R=4-Cl, 4t
Ar=4-pyridinyl, R=4-Cl, 4am
Ar=4-Cl-C6H4, R=4-Cl, 4an
Ar=4-CF3-C6H4, R=3-CF3, 4ao
Ar=3-F-C6H4, R=3-CF3, 4ap
Ar=2,6-diF-C6H4, R=3-CF3, 4aq
Chart 1. 2-Amino-3-aroyl thiophene derivatives, allosteric modulators for A1 adenosine receptor.

998
R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
thiophene scaffold, by the synthesis of a wide series of compounds
with one or more electron withdrawing substituents (mainly, but
not exclusively, F, Cl and CF3) in all five positions of the phenyl ring
linked to the piperazine moiety. By the synthesis of compounds 4e
and 4p–u, we have also investigated the effect on the AE activity
due to the presence of a linker (one, two or three methylene units
for compounds 4e–q, 4r and 4s, respectively), carbonyl for 4p, acetyl
and ‘reversed acetyl’ for 4t and 4u, between the aromatic ring and
the N1-nitrogen of the piperazine ring.
of 7a–aq with ethanolic hydrazine afforded the final compounds
4a–aq.
3. Results and discussion
3.1. Functional assays
Functional assays were performed using CHO cells stably trans-
fected with the recombinant human A1ARs In these experiments
the ability to reduce the cAMP levels in hA1CHO cells by the novel
compounds 4a–aq and by the reference compound (PD 81,723) at
2. Chemistry
four different concentrations (0.01, 0.1, 1 and 10 lM) was mea-
The approach taken for the preparation of compounds 4a–aq is
shown in Scheme 1. Preparation of the studied compounds was done
according to literature methods for the derivatives 3a–l.21 The 4-
bromomethyl-5-bromo thiophene analogs 5a and 5b were used as
common intermediates for the synthesis of final compounds 4a–
an and 4ao–aq, respectively. Derivatives 5a and 5b were coupled
with the appropriate N-alkyl/arylpiperazines or p-chlorophenylpi-
peridine (2 equiv) in dichloromethane to afford the derivatives
6a–aq in good yields. Dehalogenation by catalytic hydrogenolysis
using 10% Pd/C furnished the 5-unsubstituted thiophenes 7a–aq,
with the exception of 7ag. This latter compound was obtained by
the condensation of 8 (prepared in poor yield by the chemoselective
dehalogenation of 5a) with 1-(4-nitrophenyl)piperazine. Treatment
sured (Table 1). The effect of each tested compound on cAMP pro-
duction was reported as a percentage in comparison with the
controls (100%). Novel compounds with the potential role of AEs
were able to activate human A1ARs showing a decrease of the
cAMP production in hA1CHO cells.
Among the forty-three synthesized compounds, thirteen com-
pounds (4a–d, 4g, 4t, 4w–x, 4ag, 4al–am, 4ao and 4aq) were less
active than PD 81,723. The remaining derivatives decreased the
percentage of cAMP production more than (from 70% to 82% for
4e–f, 4h–o, 4q–s, 4v, 4aa–ae, 4ah and 4ak) or comparable ( 4p,
4u, 4y–z, 4af, 4ai–aj, 4an and 4ap) to PD 81,723 at the highest con-
centration tested (10 lM). At 10-fold lower concentrations (1 lM),
derivatives 4f, 4h–o, 4q, 4y–ac, 4ae, 4ah, 4aj and 4ak cause a
Cl
O
Br
O
N
S
8
O
a
b for 5a
R
R
R
b
a
O
O
O
Br
Ar
X
N
Ar
X
N
O
O
O
Br
Br
N
N
S
N
S
S
O
O
O
7a-aq
5a, R=4-Cl
6a-aq
5b, R=3-CF3
c
Ar=(CH3)2CH, 4, 6-7a
4 6-7b
4 6-7u
Ar=4-Cl-C6H4COCH2,
,
Ar=cyclopentyl,
,
Ar=3-Cl-C6H4, 4, 6-7v
R
Ar=cycloheptyl, 4, 6-7c
4 6-7w
,
Ar=2-Cl-C6H4,
4 6-7d
Ar=1-naphthyl,
,
Ar=2,6-diCl-C6H3, 4, 6-7x
4 6-7e
Ar=4-F-C6H4CH2,
,
4 6-7y
Ar=2,5-diCl-C6H3,
Ar=3,4-diCl-C6H3,
,
,
O
Ar=3-F-C6H4, 4, 6-7f and 4, 6-7ap
4 6-7z
Ar
X
N
4 6-7g
Ar=2-F-C6H4,
,
Ar=2,4-diCl-C6H3, 4, 6-7aa
Ar=2,4-diF-C6H3, 4, 6-7h
4 6-7ab
Ar=2,3-diCl-C6H3,
,
NH2
S
4a-aq
4 6-7i
Ar=3,4-diF-C6H3,
Ar=3,5-diF-C6H3,
,
Ar=3,5-diCl-C6H3, 4, 6-7ac
4 6-7j
,
4 6-7ad
Ar=2-Cl,4-F-C6H3,
Ar=2-F,4-Cl-C6H3,
,
,
Ar=2,6-diF-C6H3, 4, 6-7k and 4, 6-7aq
4 6-7ae
4 6-7a-an
R=4-Cl for
,
4 6-7l
Ar=2,3-diF-C6H3,
,
Ar=4-CH3O-C6H4, 4, 6-7af
4 6-7ao-aq
,
R=3-CF3 for
Ar=2,5-diF-C6H3, 4, 6-7m
4 7ag
Ar=4-NO2-C6H4,
,
X=N for 4, 6-7a-am and 4, 6-7ao-aq
4 6-7n
Ar=2,4,6-triF-C6H2,
Ar=3-Cl,4-F-C6H3,
,
Ar=3-CF3-C6H4, 4, 6-7ah
4 6-7an
X=CH for
,
4 6-7o
,
4 6-7ai
Ar=2-CF3-C6H4,
,
Ar=4-Cl-C6H4CO, 4, 6-7p
4 6-7aj
Ar=4-Cl,3-CF3-C6H3,
,
4 6-7q
Ar=4-Cl-C6H4CH2,
,
Ar=4-CF3O-C6H4, 4, 6-7ak
Ar=4-Cl-C6H4(CH2)2, 4, 6-7r
4 6-7al
Ar=3-pyridinyl,
,
4 6-7s
Ar=4-Cl-C6H4(CH2)3,
Ar=4-Cl-C6H4CH2CO,
,
Ar=4-pyridinyl, 4, 6-7am
4 6-7t
,
4 6-7an
Ar=4-Cl-C6H4,
Ar=4-CF3-C6H4,
,
4 6-7ao
,
Scheme 1. Reagents and conditions: (a) N-Alkyl/aryl piperazine or p-chlorophenylpiperidine, K2CO3, CH2Cl2, +4 °C for 30 min then rt for 2 h; (b) H2, 10% Pd/C, DMF, rt; (c)
NH2NH2, EtOH, rx.

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
999
Table 1
4d) caused a decrease of enhancement. Because other substitution
patterns that increased lipophilicity (i.e., compounds 4i, 4j, and
4ac) without significantly increasing the steric size of the aryl moi-
ety did not show this decrease in enhancement, we suggest that
the detrimental effect of the naphthyl moiety is primarily associ-
ated with the increase in ring size. Among the isomeric pyridine
derivatives 3k, 4al and 4am, the pyridin-2-yl derivative 3k was
more active than the pyridin-3-yl and pyridin-4-yl analogs 4al
Effect of compounds 3b–f and 3j–k, 4a–aq and PD 81,723 in cAMP assays on hA1CHO
cells
Compound
% Change in cAMP production (mean SEM)a
concentration of compounds
0.01
l
M
0.1
l
M
1.0
lM
10 lM
PD 81,723
3b21
3c21
3d21
3e21
3f21
3j21
3k21
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
À8.0 0.5
+5.3 0.5
À4.7 0.1
+3.2 0.4
+3.5 0.7
+10 4.2
À10 1.7
+9.4 1.4
À1.0 0.1
+9.1 1.2
+2.9 0.5
+8.7 1.1
+2.2 0.3
À18 2.0
+2.1 0.7
0.0 0.0
À3.2 0.6
À13 2.6
À12 1.7
À13 1.6
+3.1 0.2
+4.2 0.3
+4.2 0.5
À6.2 0.7
À2.2 0.4
+1.2 0.4
0.0 0.0
+2.1 0.2
À5.0 0.6
À3.1 0.3
À2.1 0.0
+15 1.5
+13 1.2
À1.2 0.0
+2.0 0.0
+1.0 0.0
À1.0 0.0
+6.1 0.0
À9.2 0.6
À1.2 0.0
À12 1.3
À8.2 0.6
0.0 0.0
À3.4 0.6
+3.8 0.7
À26 1.5
+4.6 0.2
À19 3.4
À48 14
À53 5.8
À5.6 1.6
+3.6 0.2
+2.1 0.4
+13 4.2
0.0 0.0
À3.9 0.7
À37 5.9
À1.0 0.1
À17 2.8
À34 4.3
À5.0 1.3
À23 4.2
À5.2 1.3
À13 2.2
À3.9 0.1
À21 2.3
À10 1.4
+4.2 0.3
À3.1 0.7
À3.0 0.3
0.0 0.0
+1.0 0.0
À20 1.6
+2.0 0.7
0.0 0.0
À6.2 0.5
À13 1.4
À11 1.4
+3.2 0.1
À22 7.2
À6.2 0.2
À9.4 0.4
0.0 0.0
À31 6.2
À13 1.4
À62 11
À30 11
À63 4.9
À65 4.9
À84 7.9
À46 14
+7.3 0.7
+10 2.3
+3.7 0.5
+2.9 0.1
À34 11
À86 5.8
À9.2 0.8
À66 3.7
À78 2.8
À66 10
À65 3.6
À66 10
À59 4.1
À56 3.2
À60 8.3
À34 2.8
À43 1.7
À32 5.2
À30 5.2
À8.1 0.4
À25 5.2
À59 4.1
À5.2 0.1
À2.2 0.4
À42 2.9
À60 8.2
À54 5.2
À57 6.4
À69 4.2
À39 3.2
À57 1.2
À15 1.1
À8.3 0.7
À72 0.2
À39 7.2
À47 5.2
À51 3.2
À2.7 0.7
À8.3 1.5
À24 3.2
À27 5.0
À35 3.3
+1.3 0.0
À57 2.3
À52 3.9
À78 4.8
À78 4.9
À77 2.8
À78 2.8
À87 5.9
À69 1.8
À7.4 1.2
À30 7.2
À25 9.1
À21 8.1
À82 3.9
À82 2,7
À15 2.2
À82 2.9
À87 2.8
À79 4.8
À80 1.9
À79 4.3
À80 3.9
À78 3.9
À78 6.7
À60 4.7
À82 2.9
À76 3.8
À76 5.2
À42 3.2
À62 6.2
À77 3.2
À18 2.6
À47 1.2
À62 5.2
À62 1.8
À74 1.2
À77 3.5
À74 3.5
À70 2.3
À81 3.2
À52 4.1
À17 1.0
À80 3.3
À60 4.2
À60 3.3
À74 10
À23 2.0
À15 3.1
À61 4.3
À47 4.1
À62 4.2
+4.0 0.5
and 4am, respectively, at concentrations of 10 nM–1 lM.
Several electron-withdrawing substituents (Cl, F and CF3) at the
ortho, meta and para positions of the phenyl moiety linked to the
piperazine were investigated. As reported previously, replacement
of the phenyl (3c) with a benzyl group (3d) was detrimental to the
allosteric enhancement activity at 0.01, 0.1 and 1 lM concentra-
tions, though the two compounds appeared equivalent at the high-
est concentration. Starting from the 4-fluorophenyl derivative 3e,
the 4-fluorobenzyl derivative (4e) displayed a similar pattern, with
3e and 4e appearing to have similar efficacies at a concentration of
10
the meta (derivative 4f), increased the reduction of the cAMP level
at the concentrations of 1, 0.1 and 0.01 M, while 3e and 4f
showed comparable activity at the higher concentration (10 M).
lM. Shifting the fluorine atom from the para position of 3e to
l
l
The meta-fluorine derivative 4f was found one of the most po-
tent derivatives at any concentration, showing the same potency
both at 1 and 10 lM of concentration, being able to reduce the
4o
4p
4q
4r
4s
4t
4u
4v
cAMP level of 82%. Moreover this compound did not increase the
content of cAMP of hA1CHO cells at any concentration. In contrast,
a single ortho-fluoro substitution (4g) is resolutely unfavorable. In
fact, compound 4g is almost inactive as an allosteric enhancer.
Derivative 4f showed a profile comparable to that of the para-tri-
fluoromethyl derivative 3j, the most active compound of the previ-
ously published series. As with the three fluorophenyl isomers, the
para- and meta-chloro derivatives 3f and 4v, respectively, showed
4w
4x
4y
4z
comparable activities at the higher concentrations (1 and 10 lM),
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
4ai
4aj
4ak
4al
4am
4an
4ao
4ap
4aq
which was reduced at lower concentrations (10 and 100 nM). Sim-
ilarly, the 2-chloro derivative (4w) was inactive, indicating that the
mono-substitution at the ortho position is adverse for activity.
Starting from the para-chlorophenyl piperazine derivative 3f, by
the addition of one-, two- or three-methylene units between the
p-chlorophenyl and the N4-nitrogen of the piperazine (4q, 4r and
À13 1.5
À30 4.2
À7.5 0.4
À12 1.4
À13 1.7
+3.4 0.8
+1.4 0.0
À2.3 0.5
À11 4.0
À12 1.5
4s, respectively), a similar efficacy was observed at the 10
lM con-
À2.5 0.4
centration. At 1 M, the activity decreased to increase the length of
l
0.0 0.0
the alkyl chain, suggesting that the introduction of a spacer be-
tween the phenyl and piperazine was detrimental for the activity.
Moreover, the replacement of the alkyl spacer with a different lin-
ker, such as carbonyl, acetyl and ‘reversed’ acetyl (compounds 4p,
4t and 4u, respectively) led to a substantial decline in activity at
any concentration relative to 3f. The presence of a basic nitrogen
in the piperazine ring was important for the activity. In fact, its
replacement with the isosteric carbon, to furnish the piperdine
analog 4an, has a detrimental effect on AE activity at each concen-
tration tested.
+3.2 0.9
À4.0 0.3
0.0 0.0
À12 3.2
À11 2.5
+2.1 0.7
+1.3
0
a
The results are the average of four experiments at each of four concentrations of
tested compounds.
With the aim of verifying if the presence of a second fluorine
atom on the phenyl ring would lead to an increase of activity,
the difluorophenyl derivatives 4h–m were synthesized. All of these
latter compounds maintain an AE activity comparable to those of
the mono-fluoro substituted derivatives 3e and 4f at 1 and
greater than 42% decrease of cAMP accumulation (31% for PD,
81,723), appearing to be considerably more active than PD
81,723; while compounds4e, 4p, 4r–s, 4ad, 4ai and 4ap were com-
parable to PD 81,723. In addition, the compounds 4f, 4j–k, 4o, 4v,
4ac and 4ah caused significantly greater inhibition of cAMP pro-
10 lM. Among the compounds with two fluorine atoms, derivative
duction than PD 81,723 at 0.1 lM concentration.
4i in which the fluorines are adjacent to each other at the 3- and 4-
positions on the phenyl ring exhibited the highest activity at any
concentration. Starting from the 2,6-difluoroderivative 4k, the
incorporation of an additional fluorine at the 4-position, furnishing
the 2,4,6-trifluoro derivative 4n, was tolerated at 1 and 10 lM.
Replacement of the meta-fluoro substituent in compound 4i (3,4-
The N-isopropyl, N-cyclopropyl and N-cyclohepthyl derivatives
(compounds 4a–c) appeared less active as an allosteric enhancer
than the reference compound PD 81,723 at all concentrations
tested, and were less active than the N-cyclohexyl analog (3b) or
the N-phenyl analog (3c). A further increase in lipophilicity and
size of the ring linked to the N-4 position of the piperazine, by
the replacement of phenyl ring in 3c with a naphthyl (compound
diF) with a more bulky Cl (3-Cl, 4-F, derivative 4o) increased the

1000
R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
inhibition of cAMP production at all concentrations, resulting
in one of the most active compounds of the whole series, along
with 4i.
to displace the radioligands from the orthosteric site as suggested
from competition binding experiments.22
Based upon these results, the addition of a second chlorine atom
to the 4-chloro derivative (3f) at different positions of the phenyl
ring was investigated, affording the di-chlorinated derivatives
4x–ac. The di-chloro derivatives 4y–ac maintained the same activ-
3.3. Effect on enhancers on A1 AR binding parameters
Saturation binding experiments of the selective adenosine A1
agonist [3H]CCPA and A1 antagonist [3H]DPCPX were performed
to verify if the novel compounds modified the A1ARs binding
parameters. From these experiments, A1AR affinity (KD) and density
(Bmax) were evaluated in the presence and absence of the examined
compounds (PD 81,723, 3a–l and 4a–aq at a concentration of
ity of para- and meta-chloro derivatives 3f and 4v at 1 and 10 lM
concentrations, while the 2,6-dichloro derivative (4x) was found to
be the least active of the series. Staring from the 2,4-dichloro-
phenyl derivative 4aa, replacement of ortho-chloro moiety with a
fluorine to furnish the 2-F, 4-Cl analog 4ae, retained the activity.
From this latter compound, exchange each other the substituents
on the phenylpiperazine moiety, to yield the 2-Cl, 4-F analog
4ad, reduced slightly the enhancement at A1ARs. Replacement of
chlorine in 3f with a more electron-withdrawing and less lipophilic
nitro group (derivative 4ag) was detrimental to activity at all
concentrations.
10 lM). No differences were found in affinity values (KD) derived
from saturation binding experiments as reported in Table 2. From
the receptor density calculated in the presence and in the absence
of enhancers, Bmax shift was evaluated (Table 2, column A).
[3H]CCPA competition binding experiments were also performed,
with the aim of verifying the specific in the absence and in the pres-
ence of examined enhancers (Bound shift). In [3H]CCPA saturation
binding experiments, the reference compound PD 81,723 induced
a Bmax shift to human A1ARs of 1.3-fold. Under the same experimen-
tal conditions, compounds 4e–f, 4h–t, 4v, 4x–af, 4ah–ak and 4an–
ap were significantly more potent than PD 81,723. The derivatives
4h, 4i–k, 4o, 4ak and 4ap were the most active compounds, causing
a Bmax shift of 6.1-, 7.2-, 6.2-, 6.3-, 7.0-, 6.8- and 6.0-fold, respec-
tively. In [3H]DPCPX saturation binding experiments, the tested
compounds did not modify either affinity or receptor density in
comparison to control conditions (Table 2, column B). Interestingly,
the presence of enhancers mediated an increase in Bmax shift de-
rived from [3H]CCPA saturation binding experiments, suggesting
the ability of these novel compounds to mediate a shift from the
ground state (R) to the activated state (R⁄) of the A1ARs. Bmax values
from [3H]DPCPX saturation binding experiments were generally
greater than the Bmax values obtained from [3H]CCPA saturation
binding experiments, suggesting that DPCPX as a typical adenosine
antagonist, was able to label both the R and R⁄ states of the A1ARs.
Table 2 also reports the derived apparent affinity (Ki) values for
CCPA (column C), based on a one-state model of analysis, in the ab-
sence and in the presence of tested enhancers. This table shows
that the CCPA shift, representing the ratio of apparent Ki values
in the absence and in the presence of the tested compounds at
Among the previously published para-substituted phenyl piper-
azine derivatives 3a–l, the strong electron-withdrawing trifluoro-
methyl group led to the most potent compound of the whole
series (analog 3j). Shifting the trifluoromethyl moiety from the
para- to meta-position (4ah) maintained the activity at 10
with slightly reduced activity at lower concentrations (1 and
0.1 M). It was confirmed that mono-substitution of the phenyl
lM,
l
ring at the 2-position is unfavorable. In fact, the ortho-CF3
compound 4ai showed diminished activity with respect to the
para- and meta-isomers (3j and 4ah, respectively), though not as
pronounced as with the corresponding 2-fluoro- and 2-chloro
derivatives. Staring from the meta-trifluoromethyl derivative 4ah,
the introduction of
a less hydrophobic electron-withdrawing
chlorine group to furnish the 4-Cl, 3-CF3 derivative 4aj, caused a
reduction in the enhancement activity.
The replacement of the para-trifluoromethyl group of 3j with a
less lipophilic and electron-releasing trifluoromethoxy moiety
(compound 4ak), maintained the enhancement at the higher
concentration of 10 lM, while the activity decreased at the lower
concentrations with respect to 3j.
The beneficial role of electron-withdrawing substituents was
confirmed by the synthesis of the derivative 4af, characterized by
the presence of electron-releasing methoxy moiety on the para-po-
sition of phenyl ring. This compound was less active as an AE than
the unsubstituted phenylpiperazine derivative (3c) at all concen-
trations tested.
Comparing compounds characterized by the presence of the
same phenylpiperazine moiety at the 4-position of the thiophene
ring (4f vs 4ap, 4k vs 4aq and 3j vs 4ao), replacement of 4-chloro-
benzoyl group at the 3-position of the thiophene with a 3-(trifluo-
romethyl)benzoyl moiety reduced allosteric enhancer activity.
10 lM concentration. In the hA1CHO membranes, by using
[3H]DPCPX as radioligand, the Ki value of CCPA was
15.1 1.6 nM. Interestingly, a significant decrease in the apparent
Ki value was observed in the presence of the putative allosteric
enhancers suggesting an increase in the number of the high affinity
binding sites. In the presence of PD 81,723, the apparent affinity of
CCPA increased 1.7-fold. The CCPA affinity data in the presence of
the derivatives4e–f, 4h–o, 4q–r, 4v, 4x–af, 4ah–ak and 4an–ap re-
veal that the displacement curves are shifted left, suggesting lower
Ki values for CCPA. In particular, the largest affinity shift was ob-
served for compounds 4i, 4o and 4ak. These molecules decreased
the apparent Ki values of CCPA approximately of 5.5-, 5.4- and
5.2-fold, respectively (Table 2). Compounds 4h, 4j–k, 4ac–ae,
4ah, 4aj and 4ap caused a similar shift of the apparent CCPA affin-
ity, ranging from four to fivefold, while derivatives 4e–f, 4m–n, 4q,
4r, 4v–z, 4aa–ab,4ae, 4ai and 4an–ao afforded an apparent in-
crease ranging from three to fourfold. Thus, the enhancers were
able to mediate a shift of the A1ARs towards the high affinity state,
as suggested from the increase of the CCPA affinity expressed as Ki
values (Table 2).
3.2. Antagonistic activity
The ability of compounds 4a–aq to displace the binding of
[3H]DPCPX, [3H]ZM241385 and [3H]MRE3008F20 to human A1,
A2A and A3ARs were evaluated in CHO cells. The prototype enhan-
cer PD 81,723 did not inhibit the binding of the radiolabeled antag-
onists to A1 and A2AARs, but at 10
binding of [3H]MRE 3008F20 to A3ARs. None of the examined com-
pounds (10 M) significantly inhibited the specific binding of the
lM, it reduced by 21% the
l
radioligands to A1, A2A and A3ARs, reaching a very low percentage
of inhibition (see Table 1 Supplementary data). Binding and func-
tional experiments demonstrated that it was possible to achieve
a good separation between enhancing activity and the binding to
the orthosteric site. Twenty compounds ( 4e–f, 4h–j, 4l, 4m–o,
4q–s, 4u–v, 4y, 4aa, 4ac–ae and 4ak) were more active than PD
81,723 in the enhancing activity, and at the same time were unable
In Figure 1, we have shown the effects of the allosteric modula-
tors PD 81,723, 3j, 4i and 4o at 10 l
M concentration in [3H]CCPA
saturation binding experiments on A1AR binding parameters such
as affinity and density. In Figure 2A, representative binding curves
showing the displacement of [3H]DPCPX by different concentra-
tions of CCPA alone and in the presence of PD81,723, 3j, 4i and

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
1001
Table 2
Saturation binding assays in hA1 CHO membranes obtained by using [3H]CCPA (A) and [3H]DPCPX (B) as radioligands. Modulation of enhancers (10
l
M) on CCPA affinity (Ki) and
CCPA (Ki) shift obtained from [3H]DPCPX competition binding experiments (C)
Compound
(A) [3H]CCPA
(B) [3H]DPCPX
(C) [3H]DPCPX
saturation binding experiments
saturation binding experiments
competition binding experiments
KD (nM) Bmax (fmol/mg
protein)
Bmax shift (fold of
increase)
KD (nM) Bmax (fmol/mg
protein)
Bmax shift (fold of
increase)
CCPA Ki
(nM)
CCPA Ki shift (fold of
increase)
PD 81,723
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
4y
4z
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
4ai
4aj
4ak
4al
4am
4an
4ao
4ap
4aq
1.1 0.1
1.0 0.1
1.1 0.1
1.0 0.1
1.1 0.1
1.1 0.1 1906 184
1.0 0.1 2472 264
1.1 0.1
1.1 0.1 3142 312
1.2 0.1 3708 384
1.1 0.1 3193 329
1.2 0.1 3245 324
1.1 0.1 2472 238
1.2 0.1 2421 226
1.1 0.1 2318 227
1.1 0.1 3605 376
1.0 0.1
1.0 0.1 2318 216
1.1 0.1 1582 147
1.1 0.1
1.0 0.1
1.1 0.1
1.1 0.1 2266 231
1.0 0.1 618 57
1.0 0.1 1700 167
1.1 0.1 1648 172
1.1 0.1 2369 242
1.0 0.1 2266 214
1.1 0.1 2163 189
1.1 0.1 2987 285
1.0 0.1 2678 273
1.0 0.1 2730 286
1.0 0.1 1670 181
670 52
1.3 0.1
1.1 0.1
1.1 0.1
1.1 0.1
1.1 0.1
3.7 0.3
4.8 0.4
1.1 0.1
6.1 0.6
7.2 0.6
6.2 0.6
6.3 0.6
4.8 0.4
4.7 0.4
4.5 0.5
7.0 0.6
1.6 0.1
4.5 0.4
3.1 0.3
1.6 0.2
1.5 0.1
1.4 0.1
4.4 0.4
1.2 0.1
3.3 0.3
3.2 0.3
4.6 0.5
4.4 0.4
4.2 0.3
5.8 0.6
5.2 0.3
5.3 0.5
3.3 0.3
1.1 0.1
5.3 0.5
4.4 0.3
5.7 0.6
6.8 0.6
1.3 0.1
1.1 0.1
3.9 0.3
4.4 0.4
6.0 0.6
1.1 0.1
1.8 0.2 3340 372
1.8 0.1 3089 286
1.6 0.2 3106 346
1.6 0.1 3009 281
1.9 0.1 3024 369
1.6 0.1 3145 275
1.6 0.2 3364 333
1.7 0.2 3341 328
1.7 0.1 3465 322
1.7 0.2 3652 412
1.8 0.1 3524 367
1.7 0.1 3588 394
1.9 0.1 3263 323
1.6 0.2 3498 363
1.7 0.1 3361 277
1.8 0.1 3678 358
1.8 0.2 3076 366
1.9 0.2 3355 393
1.7 0.2 3263 372
1.9 0.1 3054 295
1.9 0.1 3052 378
2.0 0.2 2996 257
1.8 0.1 3226 304
1.6 0.1 2987 246
1.7 0.1 3111 258
1.6 0.2 3154 352
2.0 0.2 3264 296
1.9 0.1 3441 425
1.9 0.2 3274 294
1.8 0.1 3266 316
1.6 0.2 3630 304
1.9 0.1 3426 342
2.0 0.1 3153 311
1.6 0.2 3102 266
1.7 0.1 3408 346
1.8 0.2 3479 319
1.8 0.2 3394 314
1.9 0.1 3588 264
1.8 0.2 3021 293
1.8 0.1 3112 278
1.7 0.1 3321 259
1.9 0.2 3332 286
2.0 0.2 3589 411
1.6 0.1 3022 297
1.0 0.1
0.9 0.1
0.9 0.1
0.9 0.1
0.9 0.1
0.9 0.1
1.0 0.1
1.0 0.1
1.0 0.1
1.1 0.1
1.1 0.1
1.1 0.1
1.0 0.1
1.0 0.1
1.0 0.1
1.1 0.1
0.9 0.1
1.0 0.1
1.0 0.1
0.9 0.1
0.9 0.1
0.9 0.1
1.0 0.1
0.9 0.1
0.9 0.1
0.9 0.1
1.0 0.1
1.0 0.1
1.0 0.1
1.0 0.1
1.1 0.1
1.0 0.1
0.9 0.1
0.9 0.1
1.0 0.1
1.0 0.1
1.0 0.1
1.1 0.1
0.9 0.1
0.9 0.1
1.0 0.1
1.0 0.1
1.1 0.1
0.9 0.1
9.2 0.8
1.7 0.2
1.2 0.1
1.3 0.1
1.2 0.1
1.3 0.1
3.1 0.3
3.9 0.4
1.4 0.1
4.8 0.5
5.5 0.4
4.8 0.5
5.0 0.4
4.0 0.3
3.8 0.3
3.6 0.3
5.5 0.5
1.6 0.2
3.7 0.3
3.4 0.3
1.6 0.2
1.5 0.1
1.4 0.1
3.7 0.3
1.4 0.1
2.9 0.3
2.8 0.3
3.8 0.4
3.7 0.4
3.5 0.4
4.5 0.4
4.2 0.4
4.3 0.4
2.8 0.2
4.2 0.4
4.3 0.4
3.6 0.3
4.5 0.5
5.3 0.5
1.4 0.1
1.2 0.1
3.3 0.3
3.4 0.3
4.7 0.5
1.2 0.1
567 53
567 59
567 47
567 42
12.6 1.3
11.7 1.2
13.1 0.1
11.6 1.1
4.9 0.4
4.0 0.3
11.3 1.1
3.2 0.3
2.8 0.3
3.2 0.3
3.1 0.2
3.9 0.4
4.1 0.4
4.3 0.5
2.8 0.3
9.4 0.5
4.2 0.4
4.5 0.4
9.4 0.9
10.1 1.2
11.0 1.1
4.2 0.4
11.1 1.1
5.4 0.5
5.6 0.6
4.1 0.3
4.2 0.4
4.4 0.4
3.4 0.3
3.7 0.3
3.6 0.4
5.5 0.6
3.7 0.4
3.6 0.3
4.3 0.3
3.4 0.3
2.9 0.3
10.8 1.0
12.6 1.2
4.7 0.5
4.6 0.5
3.3 0.3
12.6 1.3
585 52
824 85
824 71
773 68
721 70
1.1 0.1
567 49
1.2 0.1 2730 273
1.2 0.1 2266 263
1.2 0.1 2936 318
1.1 0.1 3502 341
1.1 0.1
1.0 0.1
1.2 0.1 2009 193
1.1 0.1 2266 237
1.1 0.1 3090 314
670 63
567 52
1.2 0.1
567 59
The results are mean values ( SEM) of three independent experiments.
(A) = KD (nM), Bmax (fmol/mg protein) and Bmax shift obtained in [3H]CCPA saturation binding experiments performed in the absence (KD = 1.1 0.1 nM, Bmax = 512 43 fmol/
mg protein) or in the presence of 10 M enhancers.
(B) = KD (nM), Bmax (fmol/mg protein) and Bmax shift obtained in [3H]DPCPX saturation binding experiments performed in the absence (KD = 1.7 0.2 nM,
Bmax = 3350 320 fmol/mg protein) and in the presence of 10 M enhancers.
(C) = Ki values of CCPA in the presence of 10 M tested compounds and CCPA shift = Ki(CCPA)/Ki(CCPA + 10
l
l
l
lM enhancers) where the Ki of CCPA was 15.4 1.7 nM.
4o at 10
l
M concentration. Figure 2B shows a very high correlation
3,4-difluoro-4i, 3-chloro,4-fluoro-4o, and 4-(trifluoromethoxy)-
4ak derivatives being the most active compounds in binding (sat-
uration and displacement experiments) and functional cAMP stud-
ies. In competition binding experiments, the Ki values of CCPA in
the presence of compounds 4i, 4o and 4ak was decreased approx-
imately 5.5, 5.4 and 5.2-fold, respectively. The results clearly show
that moving a single fluorine and chlorine atom from the para to
the ortho-position of the phenyl ring was detrimental for activity
(i.e., 3e and 3f vs 4g and 4w, respectively), while the 2,6-difluoro
derivative 4k was equipotent to 3e. This presumably reflects the
importance of the relative position in space between the pipera-
zine and phenyl rings, and suggests that a detrimental ortho-effect,
as indicated by compound 4g and 4w, can be overcome by the
number and position of other substituents on the aryl moiety, as
shown by compound 4k. Moreover, the relative size of the
detrimental substituent at the ortho-position and the positive
of the binding data for the examined compounds, expressed as
Bmax shift from saturation binding experiments and CCPA shift
from competition binding experiments.
4. Conclusions
In conclusion, as an extension of the series of arylpiperazine
derivatives 3a–l previously synthesized, we have now reported
the results of a systematic modification of the lead molecules 3e,
3f and 3j aimed at defining the interplay between structure and
activity in the new (4a–aq) and old (3a–l) synthesized compounds.
Of the 43 newly synthesized molecules, only nine compounds
showed AE activity lower than that PD 81,723: seven derivatives
showed activity at least as high as that of PD 81,723, with 27 ana-
logs having substantially higher activity, among these the

1002
R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
substituent at an alternative position may influence the magnitude
A
4000
3000
2000
1000
0
of the overall effect, as seen with compounds 4ad and 4ae (2-
chloro-4-fluoro vs 2-fluoro-4-chloro, respectively). In contrast,
substitution in the meta-position (F for 4f and Cl for 4v) gave re-
sults comparable to that of the para-position. The effects of com-
pounds 4a–aq on functional assays (cAMP content) were
consistent with their behavior in binding studies.
Control
+ PD 81,723
+ 3j
+ 4o
+ 4i
5. Experimental section
5.1. Chemistry
0
5
10
15
20
[3H]CCPA free (nM)
5.1.1. Materials and methods
4000
3000
2000
1000
0
B
Control
+ PD 81,723
+ 3j
+ 4o
+ 4i
4-Chlorobenzoylacetonitrile and 3-trifluorobenzoylacetonitrile
are commercially available. All aryl piperazines were commercially
available or were synthesized as described in the literature (see
Supplementary data).
1H NMR spectra were recorded on a Bruker AC 200 spectrome-
ter. Chemical shifts (d) are given in ppm relative to tetramethylsil-
ane as internal standard, and the spectra were recorded in
appropriate deuterated solvents indicated in the procedure. All
products reported showed 1H NMR spectra in agreement with
the assigned structures. Positive-ion electrospray ionization (ESI)
mass spectra were recorded on a double-focusing Finnigan MAT
95 instrument with BE geometry. Melting points (mp) were deter-
mined on a Buchi-Tottoli apparatus and are uncorrected. Elemental
analyses were conducted by the Microanalytical Laboratory of the
Chemistry Department of the University of Ferrara and were per-
formed on a Yanagimoto MT-5 CHN recorder analyzer. Results
were within 0.4% of the theoretical values. All reactions were per-
formed under an inert atmosphere of dry nitrogen, unless other-
wise described. Standard syringe techniques were applied for
transferring dry solvents. Reaction courses and product mixtures
were routinely monitored by TLC on silica gel (precoated F254
Merck plates) and visualized with aqueous KMnO4. Flash chroma-
tography was performed using 230–400 mesh silica gel and the
solvent system indicated in the procedure. All commercially avail-
able compounds were used without further purification. Organic
solutions were dried over anhydrous Na2SO4. Dichloromethane
(DCM) and DMF were distilled from calcium chloride and stored
over molecular sieves (3 Å). In high-pressure hydrogenation exper-
iments, a Parr shaker on a high-pressure autoclave was used.
0
1000
2000
3000
4000
Bound
Figure 1. [3H]-CCPA saturation binding curves to human A1ARs (A). In control
conditions, KD value was 1.1 0.1 nM and the Bmax was 515 47 fmol/mg protein.
In the presence of novel enhancers (10 lM), KD values were similar to those
obtained in controls and Bmax values were reported in Table 2. Scatchard plots of the
same experimental data (B). Values are the means and vertical lines are the SEM of
three separate experiments as described in experimental procedures.
A
100
CCPA
+ PD 81,723
+ 3j
+ 4o
+ 4i
80
60
40
20
0
-11
-10
-9
-8
-7
-6
-5
5.2. General procedure (A) for the synthesis of compounds
6a–aq
log CCPA concentration (M)
B
To a stirred solution of compound 5a–b (2 mmol) in dry DCM
(10 mL) was added K2CO3 (1.1 equiv, 2.2 mmol, 304 mg). The mix-
ture was cooled with a bath of ice/water, and then the appropriate
N-substituted piperazine (2 equiv, 4 mmol), dissolved in DCM
(2 mL), was added slowly over 30 min. The mixture was then stir-
red at room temperature for two hours, diluted with DCM (10 mL),
washed with water (10 mL) and then with brine (10 mL). The or-
ganic layer was dried (Na2SO4), filtered and concentrated in vacuo
to give a brown residue that was purified by column chromatogra-
phy to furnish the derivatives 6a–aq.
8
6
4
2
0
r = 0.991
p < 0.01
n = 56
0
2
4
6
8
5.3. General procedure (B) for the synthesis of compounds
(7a–aq)
Bmax shift (fold of increase)
Figure 2. Inhibition curves of specific [3H]-DPCPX binding to hA1ARs of CCPA in the
absence and in the presence of novel enhancers (10 M) values are the means and
l
A solution of piperazine derivative 6a–aq (2 mmol) in DMF
(20 mL), containing Et3N (0.3 mL, 2 mmol, 1 equiv) was hydroge-
nated over 120 mg of 10% Pd/C at 60 psi for 3 h. The catalyst was
removed by filtration, the filtrate concentrated, and the residue
dissolved in DCM (20 mL), washed with water (5 mL), brine
vertical lines are the SEM of three separate experiments as described in
experimental procedures (A). Correlation between CCPA Ki shift calculated by
using the affinity of the agonist in the absence and in the presence of novel
enhancers (10
experiments in the absence and in the presence of novel enhancers (10
l
M) and Bmax shift obtained from [3H]-CCPA saturation binding
l
M) (B).

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
1003
(5 mL), dried (Na2SO4). After filtering, the solvent was removed in
5.4.6. {2-Amino-4-[(4-(3-fluorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4f)
vacuo to obtain
chromatography.
a residue that was purified by column
Following the general procedure (C), derivative 4f was purified
by column chromatography (EtOAc/DCM 2:8 as eluent). Yield: 48%.
Yellow solid, mp 150–152 °C. 1H NMR (CDCl3): d: 2.02 (t, J = 4.8 Hz,
4H), 2.93 (t, J = 4.8 Hz, 4H), 3.00 (s, 2H), 6.08 (s, 2H), 6.13 (s, 1H),
6.45 (t, J = 7.0 Hz, 1H), 6.53 (m, 2H), 7.17 (q, J = 7.6 Hz, 1H), 7.40
(d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H). MS (ESI):
[M+1]+ = 430.1. Anal. (C22H21ClFN3OS): C, H, N.
5.4. General procedure (C) for the synthesis of compounds
(4a–aq)
A stirred suspension of thiophene derivatives 7a–aq (0.5 mmol)
and 100% hydrazine monohydrate (1.2 equiv, 0.6 mmol, 29 ll) in
abs ethanol (10 mL) was refluxed for 3 h, then the resulting solu-
tion was kept at room temperature for 1 h. The solvent was evap-
orated and the residue was partitioned between EtOAc (10 mL) and
water (5 mL). The separated organic phase was washed with brine
(2 mL), dried, then concentrated in vacuo to obtain a residue that
was purified by column chromatography to give the desired prod-
ucts 4a–aq.
5.4.7. {2-Amino-4-[(4-(2-fluorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4g)
Following the general procedure (C), derivative 4g was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 48%.
Yellow solid, mp 102–104 °C. 1H NMR (CDCl3) d: 2.04 (t, J = 5.2 Hz,
4H), 2.86 (t, J = 5.2 Hz, 4H), 3.02 (s, 2H), 6.04 (s, 2H), 6.12 (s, 1H),
6.74 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H), 6.86 (t, J = 7.6 Hz,
1H), 6.92 (t, J = 7.6 Hz, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.54 (d,
J = 8.2 Hz, 2H). MS (ESI): [M+1]+ = 430.2. Anal. (C22H21ClF2N3OS):
C, H, N.
5.4.1. [2-Amino-4-((4-(isopropyl)piperazin-1-yl)methyl)
thiophen-3-yl](4-chlorophenyl) methanone (4a)
Following the general procedure (C), derivative 4a was purified
by column chromatography (EtOAc/MeOH 3:7). Yield: 39%. Yellow
oil. 1H NMR (CDCl3) d: 1.02 (m, 6H), 1.99 (t, J = 5.2 Hz, 4H), 2.35 (t,
J = 5.2 Hz, 4H), 2.57 (m, 1H), 2.93 (s, 2H), 6.05 (br s, 2H), 6.10 (s,
1H), 7.36 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H). MS (ESI):
[M+1]+ = 378.1. Anal. (C19H24ClN3OS): C, H, N.
5.4.8. {2-Amino-4-[(4-(2,4-difluorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4h)
Following the general procedure (C), derivative 4h was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 51%.
Yellow solid, mp 170–172 °C. 1H NMR (CDCl3) d: 2.04 (t, J = 4.8 Hz,
4H), 2.77 (t, J = 4.8 Hz, 4H), 3.00 (s, 2H), 6.06 (br s, 2H), 6.13 (s, 1H),
6.80 (m, 3H), 7.35 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H). MS
(ESI): [M+1]+ = 448.1. Anal. (C22H20ClF2N3OS): C, H, N.
5.4.2. [2-Amino-4-((4-(cyclopentyl)piperazin-1-yl)methyl)
thiophen-3-yl](4-chlorophenyl)-methanone (4b)
Following the general procedure (C), derivative 4b was purified
by column chromatography (EtOAc/MeOH 6:4 as eluent). Yield:
36%. Yellow solid, mp 95–97 °C. 1H NMR (CDCl3) d: 1.16 (m, 4H),
1.48 (m, 5H), 1.82 (t, J = 5.2 Hz, 4H), 2.79 (s, 2H), 2.85 (t,
J = 5.2 Hz, 4H), 5.72 (br s, 2H), 6.90 (s, 1H), 7.32 (d, J = 7.0 Hz,
2H), 8.00 (d, J = 7.0 Hz, 2H). MS (ESI): [M+1]+ = 404.2. Anal.
(C21H26ClN3OS): C, H, N
5.4.9. {2-Amino-4-[(4-(3,4-difluorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4i)
Following the general procedure (C), derivative 4i was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 48%.
Yellow solid, mp 143–145 °C. 1H NMR (CDCl3) d: 2.01 (t, J = 4.8 Hz,
4H), 2.84 (t, J = 4.8 Hz, 4H), 3.00 (s, 2H), 6.07 (br s, 2H), 6.13 (s, 1H),
6.52 (m, 2H), 7.02 (m, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.53 (d,
J = 8.2 Hz, 2H). MS (ESI): [M+1]+ = 448.2. Anal. (C22H20ClF2N3OS):
C, H, N.
5.4.3. [2-Amino-4-((4-(cycloheptyl)piperazin-1-yl)methyl)
thiophen-3-yl](4-chlorophenyl) methanone (4c)
Following the general procedure (C), derivative 4c was purified
by column chromatography (EtOAc/MeOH 7:3 as eluent). Yield:
38%. Yellow oil. 1H NMR (CDCl3) d: 1.26 (m, 12H), 1.79 (t,
J = 5.2 Hz, 4H), 2.09 (t, J = 5.2 Hz, 4H), 2.81 (m, 1H), 3.03 (s, 2H),
6.03 (br s, 2H), 6.09 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.45 (d,
J = 8.8 Hz, 2H). MS (ESI): [M+1]+ = 432.3. Anal. (C23H30ClN3OS): C,
H, N.
5.4.10. {2-Amino-4-[(4-(3,5-difluorophenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4j)
Following the general procedure (C), derivative 4j was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 48%.
Yellow solid, mp 162–163 °C. 1H NMR (CDCl3) d: 2.00 (t, J = 4.8 Hz,
4H), 2.93 (t, J = 4.8 Hz, 4H), 3.00 (s, 2H), 6.10 (br s, 2H), 6.13 (s, 1H),
6.26 (m, 3H), 7.38 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H). MS
(ESI): [M+1]+ = 448.1. Anal. (C22H20ClF2N3OS): C, H, N.
5.4.4. [2-Amino-4-((4-(naphthalen-1-yl)piperazin-1-yl)methyl)
thiophen-3-yl](4-chlorophenyl) methanone (4d)
Following the general procedure (C), derivative 4d was purified
by column chromatography (EtOAc/DCM 2:8 as eluent). Yield: 46%.
Brown solid, mp 178 °C. 1H NMR (CDCl3) d: 2.42 (t, J = 5.4 Hz, 4H),
2.54 (t, J = 5.4 Hz, 4H), 3.03 (s, 2H), 6.04 (s, 2H), 6.11 (s, 1H), 6.93 (d,
J = 7.2 Hz, 2H), 7.06 (d, J = 7.2 Hz, 2H), 7.33 (m, 3H), 7.56 (d,
J = 7.8 Hz, 2H), 7.87 (d, J = 7.8 Hz, 2H). MS (ESI): [M+1]+ = 462.2.
Anal. (C26H24ClN3OS): C, H, N.
5.4.11. {2-Amino-4-[(4-(2,6-difluorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4k)
Following the general procedure (C), derivative 4k was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 47%,
Yellow oil. 1H NMR (CDCl3) d: 1.99 (t, J = 4.8 Hz, 4H), 2.95 (t,
J = 4.8 Hz, 4H), 2.98 (s, 2H), 6.08 (br s, 2H), 6.13 (s, 1H), 6.82 (m,
3H), 7.36 (d, J = 6.6 Hz, 2H), 7.58 (d, J = 6.6 Hz, 2H). MS (ESI):
[M+1]+ = 448.1. Anal. (C22H20ClF2N3OS): C, H, N.
5.4.5. {2-Amino-4-[(4-(4-fluorobenzyl)piperazin-1-yl)methyl]
thiophen-3-yl}(4-chlorophenyl) methanone (4e)
Following the general procedure (C), derivative 4e was purified
by column chromatography (MeOH/DCM 0.5:9.5 as eluent). Yield:
53%. Yellow solid, mp 230–231 °C. 1H NMR (CDCl3) d: 1.55 (t,
J = 4.6 Hz, 4H), 1.88 (t, J = 4.6 Hz, 4H), 2.92 (s, 2H), 2.92 (s, 2H),
6.02 (br s, 2H), 6.09 (s, 1H), 6.96 (t, J = 8.8 Hz, 2H), 7.21 (dd,
J = 8.8 and 5.6 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz,
2H). MS (ESI): [M+1]+ = 444.1. Anal. (C23H23ClFN3OS): C, H, N.
5.4.12. {2-Amino-4-[(4-(2,3-difluorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4l)
Following the general procedure (C), derivative 4l was purified
by column chromatography (EtOAc/DCM 0.5:9.5 as eluent). Yield:
52%. Yellow solid, mp 138 °C. 1H NMR (CDCl3) d: 1.99 (t,
J = 4.8 Hz, 4H), 2.78 (t, J = 4.8 Hz, 4H), 2.95 (s, 2H), 6.00 (br s, 2H),
6.07 (s, 1H), 6.54 (t, J = 7.6 Hz, 1H), 6.67 (d, J = 7.8 Hz, 1H), 6.85

1004
R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
(t, J = 8.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H). MS
(s, 2H), 6.02 (br s, 2H), 6.09 (s, 1H), 7.10 (d, J = 8.6 Hz, 2H), 7.20
(d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H).
MS (ESI): [M+1]+ = 488.2. Anal. (C25H27Cl2N3OS): C, H, N.
(ESI): [M+1]+ = 448.2. Anal. (C22H20ClF2N3OS): C, H, N.
5.4.13. {2-Amino-4-[(4-(2,5-difluorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4m)
Following the general procedure (C), derivative 4m was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 49%.
Yellow solid, mp 62–63 °C. 1H NMR (CDCl3) d: 1.99 (t, J = 4.8 Hz,
4H), 2.95 (t, J = 4.8 Hz, 4H), 2.98 (s, 2H), 6.08 (br s, 2H), 6.13 (s,
1H), 6.82 (m, 3H), 7.36 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H).
MS (ESI): [M+1]+ = 448.3. Anal. (C22H20ClF2N3OS): C, H, N.
5.4.20. 1-{4-[5-Amino-4-(4-chlorobenzoyl)-thiophen-3-
ylmethyl]-piperazin-1-yl}-2-(4-chlorophenyl)-ethanone (4t)
Following the general procedure (C), derivative 4t was purified
by column chromatography (EtOAc-DCM 2–8 as eluent). Yield:
46%. Yellow solid, mp 60–61 °C. 1H NMR (CDCl3) d: 1.73 (t,
J = 4.8 Hz, 2H), 1.78 (t, J = 4.8 Hz, 2H), 2.93 (s, 2H), 3.19 (t,
J = 5.2 Hz, 2H), 3.64 (t, J = 5.2 Hz, 2H), 3.59 (s, 2H), 6.06 (s, 1H),
6.09 (s, 2H), 7.10 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.39
(d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H). MS (ESI): [M]+ = 487.1.
Anal. (C24H23Cl2N3OS): C, H, N.
5.4.14. {2-Amino-4-[(4-(2,4,6-trifluorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4n)
Following the general procedure (C), derivative 4n was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 48%.
Yellow solid, mp 153–155 °C. 1H NMR (CDCl3) d: 1.98 (t, J = 4.6 Hz,
4H), 2.87 (t, J = 4.8 Hz, 4H), 2.98 (s, 2H), 6.08 (br s, 2H), 6.13 (s, 1H),
6.58 (t, J = 9.0 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz,
2H). MS (ESI): [M+1]+ = 466.2. Anal. (C22H19ClF3N3OS): C, H, N.
5.4.21. 2-{4-[5-Amino-4-(4-chlorobenzoyl)-thiophen-3-
ylmethyl]-piperazin-1-yl}-1-(4-chlorophenyl)-ethanone (4u)
Following the general procedure (C), derivative 4u was purified
by column chromatography (EtOAc-MeOH 9.5–0.5 as eluent).
Yield: 46%. Yellow solid, mp 77–78 °C. 1H NMR (CDCl3) d: 2.73 (t,
J = 4.8 Hz, 4H), 3.42 (t, J = 4.8 Hz, 4H), 3.56 (s, 2H), 3.72 (s, 2H),
6.06 (br s, 2H), 6.08 (s, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.35 (d,
J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H). MS
(ESI): [M+1]+ = 488.1. Anal. (C24H23Cl2N3OS): C, H, N.
5.4.15. {2-Amino-4-[(4-(3-chloro-4-fluorophenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4o)
Following the general procedure (C), derivative 4o was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 56%.
Yellow solid, mp 161–163 °C. 1H NMR (CDCl3) d: 2.01 (t, J = 4.6 Hz,
4H), 2.85 (t, J = 4.6 Hz, 4H), 3.00 (s, 2H), 6.07 (br s, 2H), 6.13 (s, 1H),
6.68 (m, 1H), 6.81 (dd, J = 6.4 and 3.0 Hz, 1H), 6.93 (t, J = 8.8 Hz,
1H), 7.38 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H). MS (ESI):
[M+1]+ = 464.2. Anal. (C22H20Cl2FN3OS): C, H, N.
5.4.22. {2-Amino-4-[(4-(3-chlorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4v)
Following the general procedure (C), derivative 4v was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 51%.
Yellow solid, mp 197–199 °C. 1H NMR (CDCl3) d: 2.01 (t, J = 5.2 Hz,
4H), 2.93 (t, J = 5.2 Hz, 4H), 3.00 (s, 2H), 6.08 (br s, 2H), 6.13 (s, 1H),
6.68 (d, J = 7.6 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.81 (s, 1H), 7.12 (t,
J = 8.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H). MS
(ESI): [M+1]+ = 446.3. Anal. (C22H21Cl2N3OS): C, H, N.
5.4.16. {2-Amino-4-[(4-(4-chlorobenzoyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4p)
Following the general procedure (C), derivative 4p was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 54%.
Yellow solid, mp 185–186 °C. 1H NMR (CDCl3) d: 1.83 (t, J = 4.8 Hz,
2H), 1.92 (t, J = 4.8 Hz, 2H), 2.99 (s, 2H), 3.15 (t, J = 5.0 Hz, 2H), 3.49
(t, J = 5.0 Hz, 2H), 6.08 (s, 1H), 6.10 (s, 2H), 7.24 (d, J = 8.2 Hz, 2H),
7.28 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz,
2H). MS (ESI): [M]+ = 473.1. Anal. (C23H21Cl2N3OS): C, H, N.
5.4.23. {2-Amino-4-[(4-(2-chlorophenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4w)
Following the general procedure (C), derivative 4w was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 44%.
Yellow solid, mp 115–117 °C. 1H NMR (CDCl3) d: 2.04 (t, J = 5.2 Hz,
4H), 2.78 (t, J = 5.2 Hz, 4H), 3.10 (s, 2H), 6.06 (br s, 2H), 6.11 (s, 1H),
6.60 (d, J = 7.4 Hz, 1H), 6.72 (d, J = 7.4 Hz, 1H), 6.80 (t, J = 7.4 Hz,,
1H), 7.02 (t, J = 7.4 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.52 (d,
J = 8.2 Hz, 2H). MS (ESI): [M+1]+ = 446.0. Anal. (C22H21Cl2N3OS): C,
H, N.
5.4.17. {2-Amino-4-[(4-(4-chlorobenzyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4q)
Following the general procedure (C), derivative 4q was purified
by column chromatography (EtOAc/DCM 1:1 as eluent). Yield: 53%.
Yellow solid, mp 65–67 °C. 1H NMR (CDCl3) d: 1.88 (t, J = 4.6 Hz,
4H), 2.21 (m, 4H), 2.92 (s, 2H), 3.36 (s, 2H), 6.02 (br s, 2H), 6.09
(s, 1H), 7.24 (m, 4H), 7.34 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz,
2H). MS (ESI): [M+1]+ = 460.2. Anal. (C23H23Cl2N3OS): C, H, N.
5.4.24. {2-Amino-4-[(4-(2,6-dichlorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4x)
Following the general procedure (C), derivative 4x was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 42%.
Yellow solid, mp 128–130 °C. 1H NMR (CDCl3) d: 2.02 (t, J = 4.8 Hz,
4H), 2.82 (t, J = 4.6 Hz, 4H), 3.02 (s, 2H), 6.05 (br s, 2H), 6.09 (s, 1H),
7.01 (d, J = 8.6 Hz, 1H), 7.17 (t, J = 8.6 Hz, 1H), 7.29 (d, J = 2.6 Hz,
1H), 7.34 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H). MS (ESI):
[M+1]+ = 480.2. Anal. (C22H20Cl3N3OS): C, H, N.
5.4.18. (2-Amino-4-{(4-[2-(4-chlorophenyl)ethyl]piperazin-1-
yl)methyl}thiophen-3-yl)(4-chlorophenyl) methanone (4r)
Following the general procedure (C), derivative 4r was purified
by column chromatography (DCM/MeOH 9.5:0.5 as eluent). Yield:
48%. Yellow oil. 1H NMR (CDCl3) d: 1.92 (t, J = 4.6 Hz, 4H), 2.27 (m,
4H), 2.47 (t, J = 7.2 Hz, 2H), 2.67 (t, J = 7.2 Hz, 2H), 2.95 (s, 2H), 6.09
(br s, 3H), 7.08 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.36 (d,
J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H). MS (ESI): [M+1]+ = 474.1.
Anal. (C24H25Cl2N3OS): C, H, N.
5.4.25. {2-Amino-4-[(4-(2,5-dichlorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4y)
Following the general procedure (C), derivative 4y was purified
by column chromatography (EtOAc/DCM 0.5:9.5 as eluent). Yield:
49%. Yellow solid, mp 78–80 °C. 1H NMR (CDCl3) d: 2.07 (t,
J = 4.6 Hz, 4H), 2.81 (t, J = 4.6 Hz, 4H), 3.03 (s, 2H), 6.08 (br s, 2H),
6.15 (s, 1H), 6.93 (s, 1H), 7.24 (d, J = 9.2 Hz, 2H), 7.39 (d,
J = 8.6 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H). MS (ESI): [M+1]+ = 480.4.
Anal. (C22H20Cl3N3OS): C, H, N.
5.4.19. (2-Amino-4-{(4-[2-(4-chlorophenyl)propyl]piperazin-1-
yl)methyl}thiophen-3-yl)(4-chlorophenyl)methanone (4s)
Following the general procedure (C), derivative 4s was purified
by column chromatography (DCM/MeOH 9.5:0.5 as eluent). Yield:
48%. Yellow solid, mp 60–61 °C. 1H NMR (CDCl3) d: 1.62 (m, 2H),
1.90 (t, J = 4.8 Hz, 4H), 2.24 (m, 4H), 2.58 (t, J = 4.8 Hz, 4H), 2.95

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
1005
5.4.26. {2-Amino-4-[(4-(3,4-dichlorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4z)
Following the general procedure (C), derivative 4z was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 41%.
Yellow solid, mp 102–104 °C. 1H NMR (CDCl3) d: 2.04 (t, J = 4.6 Hz,
4H), 2.90 (t, J = 4.6 Hz, 4H), 3.00 (s, 2H), 6.08 (br s, 2H), 6.13 (s, 1H),
6.66 (dd, J = 9.0 and 2.8 Hz, 1H), 6.87 (d, J = 2.8 Hz, 1H), 7.21 (s, 1H),
7.40 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H). MS (ESI):
[M+1]+ = 480.0. Anal. (C22H20Cl3N3OS): C, H, N.
1H), 6.82 (d, J = 10.2 Hz, 2H), 6.86 (d, J = 10.2 Hz, 2H), 7.36 (d,
J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H). MS (ESI): [M+1]+ = 442.2.
Anal. (C23H24ClN3OS): C, H, N.
5.4.33. {2-Amino-4-[(4-(4-nitrophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ag)
Following the general procedure (C), derivative 4ag was puri-
fied by column chromatography (EtOAc/DCM 1:9 as eluent). Yield:
43%. Yellow solid, mp 110–112 °C. 1H NMR (CDCl3) d: 2.46 (t,
J = 5.4 Hz, 4H), 2.84 (t, J = 5.4 Hz, 4H), 3.03 (s, 2H), 6.21 (br s, 2H),
6.24 (s, 1H), 6.81 (d, J = 9.6 Hz, 2H), 7.46 (d, J = 9.2 Hz, 2H), 7.65
(d, J = 9.6 Hz, 2H), 8.13 (d, J = 9.2 Hz, 2H). MS (ESI):
[M+1]+ = 457.5. Anal. (C22H21ClN4O3S): C, H, N.
5.4.27. {2-Amino-4-[(4-(2,4-dichlorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4aa)
Following the general procedure (C), derivative 4aa was puri-
fied by column chromatography (EtOAc/DCM 1:9 as eluent). Yield:
44%. Yellow solid, mp 142–143 °C. 1H NMR (CDCl3) d: 2.04 (t,
J = 4.6 Hz, 4H), 2.76 (t, J = 4.6 Hz, 4H), 3.01 (s, 2H), 6.05 (br s, 2H),
6.14 (s, 1H), 6.88 (d, J = 8.6 Hz, 1H), 7.14 (dd, J = 11 and 2.6 Hz,
1H), 7.31 (d, J = 2.6 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.56 (d,
J = 8.8 Hz, 2H). MS (ESI): [M+1]+ = 480.1. Anal. (C22H20Cl3N3OS): C,
H, N.
5.4.34. {2-Amino-4-[(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ah)
Following the general procedure (C), derivative 4ah was puri-
fied by column chromatography (EtOAc/DCM 0.5:9.5 as eluent).
Yield: 48%. Yellow solid, mp 167–169 °C. 1H NMR (CDCl3) d: 2.04
(t, J = 5.2 Hz, 4H), 2.97 (t, J = 5.2 Hz, 4H), 3.00 (s, 2H), 6.07 (br s,
2H), 6.14 (s, 1H), 7.01 (m, 3H), 7.26 (t, J = 9.6 Hz, 1H), 7.38 (d,
J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H). MS (ESI): [M+1]+ = 480.1.
Anal. (C23H21ClN4O3S): C, H, N.
5.4.28. {2-Amino-4-[(4-(2,3-dichlorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ab)
Following the general procedure (C), derivative 4ab was puri-
fied by column chromatography (EtOAc/DCM 1:9 as eluent). Yield:
47%. Yellow oil. 1H NMR (CDCl3) d: 2.05 (t, J = 4.4 Hz, 4H), 2.78 (t,
J = 4.4 Hz, 4H), 3.01 (s, 2H), 6.07 (br s, 2H), 6.14 (s, 1H), 6.89 (d,
J = 4.8 Hz, 1H), 7.11 (d, J = 5.2 Hz, 2H), 7.43 (t, J = 7.0 Hz, 2H), 7.58
(d, J = 8.6 Hz, 2H). MS (ESI): [M+1]+ = 480.2. Anal. (C22H20Cl3N3OS):
C, H, N.
5.4.35. {2-Amino-4-[(4-(2-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ai)
Following the general procedure (C), derivative 4ai was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 52%.
Yellow oil. 1H NMR (CDCl3) d: 2.01 (t, J = 4.8 Hz, 4H), 2.67 (t,
J = 5.2 Hz, 4H), 3.00 (s, 2H), 6.05 (br s, 2H), 6.13 (s, 1H), 7.12 (t,
J = 8.4 Hz, 1H), 7.18 (m, 2H), 7.36 (d, J = 8.8 Hz, 2H), 7.44 (m, 1H),
7.57 (d, J = 8.8 Hz, 2H). MS (ESI): [M+1]+ = 480.2. Anal.
(C23H21ClF3N3OS): C, H, N.
5.4.29. {2-Amino-4-[(4-(3,5-dichlorophenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ac)
Following the general procedure (C), derivative 4ac was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 44%.
Yellow solid, mp 185–187 °C. 1H NMR (CDCl3) d: 1.99 (t, J = 4.8 Hz,
4H), 2.92 (t, J = 4.8 Hz, 4H), 2.99 (s, 2H), 6.09 (br s, 2H), 6.13 (s, 1H),
6.64 (s, 2H), 6.76 (s, 1H), 7.38 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz,
2H). MS MS (ESI): [M+1]+ = 480.1. Anal. (C22H20Cl3N3OS): C, H, N.
5.4.36. {2-Amino-4-[(4-(4-chloro-3-(trifluoromethyl)
phenyl)piperazin-1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)
methanone (4aj)
Following the general procedure (C), derivative 4aj was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 44%.
Yellow solid, mp 141–143 °C. 1H NMR (CDCl3) d: 1.96 (t, J = 4.4 Hz,
4H), 2.87 (t, J = 4.4 Hz, 4H), 2.94 (s, 2H), 6.01 (br s, 2H), 6.07 (s, 1H),
6.78 (dd, J = 8.2 and 2.8 Hz, 1H), 7.00 (s, 1H), 7.19 (d, J = 8.2 Hz, 1H),
7.33 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H). MS (ESI):
[M+1]+ = 514.1. Anal. (C23H20Cl2F3N3OS): C, H, N.
5.4.30. {2-Amino-4-[(4-(2-chloro-4-fluorophenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ad)
Following the general procedure (C), derivative 4ad was puri-
fied by column chromatography (EtOAc/DCM 1:9 as eluent). Yield:
51%. Yellow solid, mp 59–61 °C. 1H NMR (CDCl3) d: 2.06 (t,
J = 4.6 Hz, 4H), 2.75 (t, J = 4.6 Hz, 4H), 3.02 (s, 2H), 6.07 (br s, 2H),
6.15 (s, 1H), 6.92 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 1H), 6.93
(t, J = 8.8 Hz, 1H), 7.38 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H).
MS (ESI): [M+1]+ = 464.1. Anal. (C22H20Cl2FN3OS): C, H, N.
5.4.37. {2-Amino-4-[(4-(4-(trifluoromethoxy)phenylpiperazin-
1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ak)
Following the general procedure (C), derivative 4ak was puri-
fied by column chromatography (EtOAc/DCM 1:9 as eluent). Yield:
40%. Yellow solid, mp 151–153 °C. 1H NMR (CDCl3) d: 2.04 (t,
J = 4.8 Hz, 4H), 2.92 (t, J = 4.8 Hz, 4H), 3.01 (s, 2H), 6.09 (br s, 2H),
6.14 (s, 1H), 6.80 (d, J = 8.6 Hz, 1H), 7.07 (d, J = 8.6 Hz, 2H), 7.38
(d, J = 7.8 Hz, 2H), 7.56 (d, J = 7.8 Hz, 2H). MS (ESI):
[M+1]+ = 496.4. Anal. (C23H21ClF3N3O2S): C, H, N.
5.4.31. {2-Amino-4-[(4-(2-fluoro-4-chlorophenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ae)
Following the general procedure (C), derivative 4ae was purified
by column chromatography (EtOAc/DCM 1:9 as eluent). Yield: 54%.
Yellow solid, mp 161–163 °C. 1H NMR (CDCl3) d: 2.04 (t, J = 4.8 Hz,
4H), 2.80 (t, J = 4.8 Hz, 4H), 3.01 (s, 2H), 6.07 (br s, 2H), 6.14 (s, 1H),
6.78 (t, J = 7.6 Hz, 1H), 7.01 (m, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.55 (d,
J = 8.6 Hz, 2H). MS (ESI): [M+1]+ = 464.4. Anal. (C22H20Cl2FN3OS): C,
H, N.
5.4.38. [2-Amino-4-(4-(pyridin-3-yl)piperazin-1-yl)methyl)
thiophen-3-yl](4-chlorophenyl) methanone (4al)
Following the general procedure (C), derivative 4al was purified
by column chromatography (EtOAc/MeOH 6:4 as eluent). Yield:
34%. Yellow solid, mp 101–103 °C. 1H NMR (CDCl3) d: 2.01 (t,
J = 4.8 Hz, 4H), 2.84 (t, J = 4.8 Hz, 4H), 3.00 (s, 2H), 6.09 (br s, 2H),
6.13 (s, 1H), 6.52 (d, J = 7.8 Hz, 1H), 6.78 (m, 1H), 7.42 (d,
J = 8.6 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 8.07 (s, 1H), 8.24 (d,
J = 9.0 Hz, 1H). MS (ESI): [M+1]+ = 413.1. Anal. (C21H21ClN4OS): C,
H, N.
5.4.32. {2-Amino-4-[(4-(4-(methoxy)phenyl)piperazin-1-yl)
methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4af)
Following the general procedure (C), derivative 4af was purified
by column chromatography (EtOAc/DCM 2:8 as eluent). Yield: 38%.
Yellow oil. 1H NMR (CDCl3) d: 2.04 (t, J = 5.4 Hz, 4H), 2.83 (t,
J = 5.4 Hz, 4H), 3.10 (s, 2H), 3.75 (s, 3H), 6.08 (br s, 2H), 6.14 (s,

1006
R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
5.4.39. [2-Amino-4-((4-(pyridin-4-yl)piperazin-1-
grown to a confluent monolayer. To begin an experiment, growth
medium was removed from the culture plates and cells were
washed once with Hanks’ buffered saline solution. The wash solu-
tion was then removed and replaced with fresh Hanks’ solution
yl)methyl)thiophen-3-yl](4-chlorophenyl) methanone (4am)
Following the general procedure (C), derivative 4am was puri-
fied by column chromatography (EtOAc/MeOH 7:3 as eluent).
Yield: 38%. Yellow solid, mp 86–88 °C. 1H NMR (CDCl3) d: 2.01 (t,
J = 5.2 Hz, 4H), 3.00 (s, 2H), 3.24 (t, J = 5.2 Hz, 4H), 6.09 (br s, 2H),
6.12 (s, 1H), 6.72 (d, J = 5.6 Hz, 2H), 6.87 (d, J = 5.6 Hz, 2H), 8.06
(d, J = 7.4 Hz, 2H), 8.19 (d, J = 7.4 Hz, 2H). MS (ESI):
[M+1]+ = 413.2. Anal. (C21H21ClN4OS): C, H, N.
containing forskolin (1 lM), rolipram (20 lM), CPA (0.01 nM),
adenosine deaminase (2 U/mL), and the allosteric enhancer to be
tested. Forskolin was used to stimulate the activity of adenylyl cy-
clase, rolipram to inhibit cAMP phosphodiesterase, adenosine
deaminase to degrade endogenous adenosine, and CPA to cause a
small increase of the number of activated adenosine receptors.
After 6 min of incubation at 36 °C in the presence of drugs, the
incubation solution was removed and hydrochloric acid (final con-
centration, 50 mM) was added to cells to terminate drug action.
The content of cAMP in acidified extracts of cells was determined
by radioimmunoassay as previously described.5 Because the mag-
nitude of the effects of allosteric enhancers on hA1CHO cells chan-
ged subtly with passage number and differed slightly among
different aliquots of cells, the action of tested compounds and
the action of the reference compound PD 81,723 were assayed in
each experiment. Allosteric enhancement was measured as the ac-
tion of a test compound at different concentrations (0.01, 0.1, 1 and
5.4.40. {2-Amino-4-[(4-(4-chlorophenyl)piperidin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl) methanone (4an)
Following the general procedure (C), derivative 4an was puri-
fied by column chromatography (EtOAc/DCM 1:9 as eluent). Yield:
45%. Yellow oil. 1H NMR (CDCl3) d: 1.67 (m, 4H), 2.27 (m, 1H), 2.54
(t, J = 5.4 Hz, 4H), 3.04 (s, 2H), 6.09 (s, 2H), 6.16 (s, 1H), 7.33 (s, 4H),
7.39 (d, J = 8.6 Hz, 2H), 7.51 (t, J = 8.6 Hz, 2H). MS (ESI):
[M+1]+ = 445.2. Anal. (C23H22Cl2N2OS): C, H, N.
5.4.41. {2-Amino-4-[(4-(4-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(3-
(trifluoromethyl)phenyl)methanone (4ao)
10 lM) to reduce the cAMP content of hA1CHO cells in the pres-
Following the general procedure (C), derivative 4ao was puri-
fied by column chromatography (EtOAc/DCM 0.25:9.75 as eluent).
Yield: 52%. Yellow solid, mp: 143–145 °C. 1H NMR (CDCl3) d: 1.95
(t, J = 4.8 Hz, 4H), 2.94 (s, 2H), 2.98 (t, J = 4.8 Hz, 4H), 6.14 (s, 1H),
6.28 (br s, 2H), 6.82 (t, J = 8.8 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H),
7.54 (t, J = 8.0 Hz, 1H), 7.74 (m, 2H), 7.85 (s, 1H). MS (ESI):
[M+1]+ = 514.5. Anal. (C25H22F6N2OS): C, H, N.
ence of CPA (0.05–0.1 nM) which causes a slight reduction of cAMP
content by activation of A1ARs. Allosteric enhancement of the ac-
tion of CPA causes a further reduction of the cAMP content of hA1-
CHO cells. Because the spontaneous activity of adenosine receptors
in hA1CHO cells causes an inhibition of adenylyl cyclase activity
even in the absence of an agonist,23 antagonists of adenosine
receptors increase cAMP content of cells. Therefore, compounds
that increased cAMP content of cells in this study were provision-
ally identified as A1 adenosine receptor antagonists.
5.4.42. {2-Amino-4-[(4-(3-fluorophenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(3-
(trifluoromethyl)phenyl)methanone (4ap)
5.5.2. Saturation and competition binding experiments
5.5.2.1. Materials.
Following the general procedure (C), derivative 4ap was puri-
fied by column chromatography (EtOAc/DCM 1:9 as eluent). Yield:
55%. Yellow solid, mp: 100–102 °C. 1H NMR (CDCl3) d: 1.94 (t,
J = 4.8 Hz, 4H), 2.90 (t, J = 4.8 Hz, 4H), 2.93 (s, 2H), 6.14 (s, 1H),
6.29 (br s, 2H), 6.49 (m, 2H), 6.57 (d, J = 9.6 Hz, 1H), 7.15 (q,
J = 7.2 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.74 (m, 2H), 7.84 (s, 1H).
MS (ESI): [M+1]+ = 464.4. Anal. (C24H22F4N2OS): C, H, N.
[3H]DPCPX ([3H]1,3-dipropyl-8-cyclopen-
tyl-xanthine; specific activity, 120 Ci/mmol) and [3H]CCPA
([3H]2-chloro-N6-cyclopentyladenosine; specific activity, 55 Ci/
mmol) were obtained from Perkin Elmer (Boston, MA); [3H]ZM
241385 ([3H](4-(2-[7-amino-2-(2-furil)[1,2.4]triazolo[2,3-a][1,3,5]
triazin-5-ylamino]ethyl)phenol); specific activity, 17 Ci/mmol)
was obtained from Biotrend (Cologne, Germany); [3H]MRE
3008F20 ([3H]5-N-(4-methoxyphenylcarbamoyl)amino-8-propyl-
2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; specific
activity, 67 Ci/mmol) was obtained from Amersham International
(Buckinghamshire, UK). DPCPX (1,3-dipropyl-8-cyclopentyl-xan-
5.4.43. {2-Amino-4-[(4-(2,6-difluorophenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(3-
(trifluoromethyl)phenyl)methanone (4aq)
Following the general procedure (C), derivative 4aq was puri-
fied by column chromatography (EtOAc/DCM 0.5:9.5 as eluent).
Yield: 62%. Yellow solid, mp: 163–165 °C. 1H NMR (CDCl3) d: 1.92
(t, J = 4.8 Hz, 4H), 2.89 (t, J = 4.8 Hz, 4H), 3.02 (s, 2H), 6.73 (s, 1H),
6.84 (m, 5H), 7.34 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.84
(d, J = 7.8 Hz, 1H), 8.01 (s, 1H). MS (ESI): [M+1]+ = 482.1. Anal.
(C24H21F5N2OS): C, H, N.
thine), R-PIA ((R)-N6-(
L-2-Phenylisopropyl)adenosine) and CPA
(N6-cyclopentyladenosine) were obtained from Sigma (St. Louis,
MO, USA). All other reagents were of analytical grade and obtained
from commercial sources.
5.5.2.2. Membrane preparation from CHO cells transfected
with hA1, hA2A and hA3ARs.
The hA1CHO, hA2ACHO and hA3-
CHO cells were grown adherently and maintained in Dulbecco’s
modified Eagle’s medium with nutrient mixture F12, containing
5.5. Biology experiments
10% fetal calf serum, penicillin (100 U/mL), streptomycin (100
lg/
5.5.1. Cyclic AMP accumulation in CHO cells
mL), -glutamine (2 mM), geneticine (G418) 0.2 mg/mL at 37 °C
L
Chinese hamster ovary cells expressing human recombinant A1
adenosine receptors (hA1CHO cells) at a density of approximately
8000 fmol/mg protein were prepared as previously described5
and aliquots of these cells at low passage numbers were frozen
and stored in liquid nitrogen. Upon the arrival of a group of com-
pounds for testing in the laboratory, an aliquot of cells was re-
moved from liquid nitrogen storage and grown in Ham’s F-12
culture medium with 10% fetal bovine serum and 0.5 mg/mL of
antibiotic G-418.23 Cells were passaged thrice weekly and aliquots
of cells were placed into 12-well culture plates with culture med-
ium, serum, and antibiotic for 48 h, by which time the cells had
in 5% CO2/95% air.24 Cells were splitted two or three times weekly
at a ratio of 1:5. For membrane preparation the culture medium
was removed, the cells were washed with PBS and scraped off
T75 flasks in ice-cold hypotonic buffer (5 mM Tris–HCl, 2 mM
EDTA, pH 7.4). The cell suspension was homogenized with Polytron
and the homogenate was spun for 10 min at 1,000Âg. The superna-
tant was then centrifuged for 30 min at 100,000Âg. The membrane
pellet was resuspended in 50 mM Tris–HCl buffer pH 7.4 for A1ARs,
50 mM Tris–HCl buffer pH 7.4, 10 mM MgCl2 for A2AARs, 50 mM
Tris–HCl buffer pH 7.4, 10 mM MgCl2, 1 mM EDTA for A3ARs and
incubated with 3 UI/mL of adenosine deaminase for 30 min at

R. Romagnoli et al. / Bioorg. Med. Chem. 20 (2012) 996–1007
1007
37 °C. The protein concentration was determined according to a
Supplementary data
Bio-Rad method (Bradford, 1976) with bovine albumin as a
standard reference.
Supplementary data (data on antagonist activity of compounds
PD 81,723, 3a–l and 4a–aq, synthetic procedure for the prepara-
tion of compound 5b, detailed characterization of compounds
6a–aq, 7a–aq and 8. Elemental analyses of compounds 4a–aq)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2011.11.044.
5.5.2.3.
membranes.
[3H]CCPA
Saturation binding experiments of [3H]CCPA
Binding
Experiments
in
hA1CHO
(0.05–20 nM) to hA1CHO membranes were performed in triplicate
at 25 °C for 90 min in 50 mM Tris–HCl, pH 7.4, in the absence and
presence of the tested compounds at the final concentration of
10
presence of 1
l
M.14 Non specific binding was defined as binding in the
References and notes
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11. 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.
12. 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.
Competition binding experiments were carried out in triplicate
in a final volume of 250
Tris–HCl, pH 7.4 and 100
six to eight different concentrations of the tested compounds in
the range from 1 nM to 10
M for 90 min at 25 °C.15 Non specific
binding was defined as binding in the presence of 1 M R-PIA.
l
l
l containing 1 nM [3H]CCPA, 50 mM
l of diluted membranes and at least
l
l
5.5.2.4. [3H]DPCPX Binding experiments in hA1CHO
membranes.
Saturation binding experiments of [3H]DPCPX
(0.05–20 nM) to hA1CHO membranes were performed in triplicate
at 25 °C for 90 min in 50 mM Tris–HCl, pH 7.4, in the absence and
presence of the tested compounds at the final concentration of
10
presence of 1
l
M.14 Non specific binding was defined as binding in the
lM DPCPX.
Competition binding experiments of 1 nM [3H]DPCPX were per-
formed in triplicate in 50 mM Tris–HCl, pH 7.4, for 90 min at 25 °C.
The effect of different tested compounds at the concentration
10 lM on CCPA curve (0.01 nM–1 lM) was investigated. Non spe-
cific binding was defined as binding in the presence of 1
l
M DPCPX.
13. Luetjens, H.; Zickgraf, A.; Figler, H.; Linden, J.; Olsson, R. A.; Scammells, P. J. J.
Med. Chem. 2003, 46, 1870.
14. Baraldi, P. G.; Romagnoli, R.; Pavani, M. G.; Nuñez, 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.
5.5.2.5. Assay of adenosine antagonist activity.
A1, A2A and
A3 AR competition binding experiments were performed using
1 nM [3H]DPCPX,25 1 nM [3H]ZM 24138526 and 2 nM [3H]MRE
3008F2027 as radioligands, respectively. Membrane suspensions
were incubated in 50 mM Tris–HCl, pH 7.4, at 25 °C for 120 min,
in 50 mM Tris–HCl, 10 mM MgCl2, pH 7.4, at 4 °C for 60 min, and
in 50 mM Tris–HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4 at 4 °C for
120 min to study A1, A2A and A3ARs, respectively. Non specific bind-
15. Baraldi, P. G.; Pavani, M. G.; Shryock, J. C.; Moorman, A. R.; Iannotta, V.; Borea,
P. A.; Romagnoli, R. Eur. J. Med. Chem. 2004, 39, 855.
16. (a) Baraldi, P. G.; Iaconinoto, M. A.; Moorman, A. R.; Carrion, M. D.; Cara, C. L.;
Preti, D.; López, O. C.; Fruttarolo, F.; Tabrizi, M. A.; Romagnoli, R. Mini-Rev. Med.
Chem. 2007, 7, 559; (b) Romagnoli, R.; Baraldi, P. G.; Aghazadeh Tabrizi, M.;
Gessi, S.; Borea, P. A.; Merighi, S. Curr. Med. Chem. 2010, 17, 3488.
17. (a) Aurelio, L.; Figler, H.; Flynn, B. L.; Linden, J.; Scammells, P. J. Bioorg. Med.
Chem. 2008, 16, 1319; (b) Aurelio, L.; Christopoulos, A.; Flynn, B. L.; Scammells,
P. J.; Sexton, P. M. Bioorg. Med. Chem. Lett. 2011, 21, 3704; (c) Aurelio, L.; Valant,
C.; Sexton, P. M.; Christopoulos, A.; Scammells, P. J. J. Med. Chem. 2009, 52,
4543; (d) Aurelio, L.; Valant, C.; Figler, H.; Flynn, B. L.; Linden, J.; Sexton, P. M.;
Christopoulos, A.; Scammells, P. J. Bioorg. Med. Chem. 2009, 17, 7353.
18. Kourounakis, A. P.; van der Klein, P. A. M.; IJzerman, A. P. Drug Dev. Res. 2000,
49, 227.
19. Nikolakopoulos, G.; Linden, J.; Figler, H.; Scammells, P. J. Bioorg. Med. Chem.
2006, 14, 2358.
20. For a complete pharmacological characterization of T-62 see: Childers, S. R.; Li,
X.; Xiao, R.; Eisanach, J. C. J. Neurochem. 2005, 93, 715.
21. Romagnoli, R.; Baraldi, P. G.; Carrion, M. D.; Lopez-Cara, C.; Cruz-Lopez, O.;
Iaconinoto, M. A.; Preti, D.; Shryock, J. C.; Moorman, A. R.; Vincenzi, F.; Varani,
K.; Borea, P. A. J. Med. Chem. 2008, 51, 5875.
ing was defined as the binding in the presence of 1 lM DPCPX or ZM
241385 or MRE 3008F20 for A1, A2A and A3ARs, respectively. Inhibi-
tion was expressed as percentage of control specific binding (100%).
Test agents were dissolved in DMSO and added to the assay from a
100-fold concentrated solution in DMSO. Control incubations also
contained 1% DMSO. Bound and free radioactivity were separated
by filtering the assay mixture through Whatman GF/B glass fiber fil-
ters using a Brandel cell harvester (Brandel Instruments, Unterföh-
ring, Germany). The filter bound radioactivity was counted by
Scintillation Counter Packard Tri Carb 2810 TR (Perkin Elmer).
5.6. Data analysis
22. In our experiments, the reference compound PD 81,723 (at a concentration of
10
CHO cells. For the same reference compound, Bruns (Ref. 7) showed a Ki value
of 11
M obtained in competition binding experiments by using [3H]DPCPX as
l
M) did not inhibit [3H]DPCPX binding to human A1 receptors transfected in
Saturation and competition binding experiments were analyzed
with the program LIGAND28 which performed weighted, non-lin-
ear, least squares curve fitting program. Inhibitory binding con-
stants, Ki, were also calculated from the IC50 values according to
the Cheng and Prusoff equation Ki = IC50/(1 + [C⁄]/KD⁄), where
[C⁄] is the concentration of the radioligand and KD⁄ its dissociation
constant.29 All experimental data are expressed as mean stan-
dard error of the mean (S.E.M.) of three or four independent exper-
iments performed in duplicate.
l
radioligand on rat membranes. Furthermore, data performed on CHO-K1cells
stably expressing the human A1 receptors (Ref. 12) reported an inhibition of
[3H]DPCPX binding to human A1 receptors by PD 81,723 only of 42 7%, when
tested at 100 lM. We speculate that species differences in affinity binding of
PD 81,723 may explain the discrepancy between the data.
23. Shryock, J. C.; Ozeck, M. J.; Belardinelli, L. Mol. Pharmacol. 1998, 53, 886.
24. Klotz, K. N.; Hessling, J.; Hegler, J.; Owman, C.; Kull, B.; Fredholm, B. B.; Lohse,
M. J. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1998, 357, 1.
25. Borea, P. A.; Dalpiaz, A.; Varani, K.; Gessi, S.; Gilli, G. Life Sci. 1996, 59, 1373.
26. Borea, P. A.; Dalpiaz, A.; Varani, K.; Gessi, S.; Gilli, G. Biochem. Pharmacol. 1995,
49, 461.
27. Varani, K.; Merighi, S.; Gessi, S.; Klotz, K. N.; Leung, E.; Baraldi, P. G.; Cacciari,
B.; Romagnoli, R.; Spalluto, G.; Borea, P. A. Mol. Pharmacol. 2000, 57, 968.
28. Munson, P. J.; Rodbard, D. Anal. Biochem. 1980, 107, 220.
29. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 1, 3099.
Acknowledgments
We wish to thank Dr. Alberto Casolari and Valerio Forlani for
technical assistance.