S. Taliani et al. / European Journal of Medicinal Chemistry 69 (2013) 331e337
335
H-30); 7.62e7.58 (m, 2H, H-7 and H-50); 7.38e7.23 (m, 7H, H-5, H-6,
HeAr); 6.82 (m, 1H, H-40); 5.56 (s, 2H, CH2). 13C NMR (100 MHz,
2 units/mL ADA, pH 7.4) in the presence of 30 nM of [3H]NECA and
six different concentrations of the newly synthesized compounds.
DMSO-d6,
d
ppm): 185.3; 180.3; 150.2; 149.4; 140.8; 136.7; 136.5;
Non-specific binding was determined in the presence of 100 mM R-
128.7; 127.8; 127.3; 126.1; 123.9; 123.8; 123.2; 121.5; 113.3; 111.8;
111.2; 49.9. Anal. Calcd. for C21H15NO3 (%): C, 76.58; H, 4.59; N, 4.25.
Found: C, 77.29; H, 4.81; N, 4.07.
PIA [35]. The dissociation constant (Kd) of [3H]NECA in hA2A CHO
cell membranes was 30 nM.
6.2.4. Human A3 adenosine receptors
6.1.2.3. 1-(1-Benzyl-1H-indol-3-yl)-2-(thiophen-2-yl)ethane-1,2-
dione (8c). Ethyl acetate:petroleum ether 1:2 v/v as eluent. Yield
12%, mp 102.3 ꢂC dec. (n-hexane). 1H NMR (400 MHz, DMSO-d6,
Aliquots of cell membranes (30
90 min in 100
m
g) were incubated at 25 ꢂC for
m
L of T3 buffer (50 mM TriseHCl, 10 mM MgCl2, 1 mM
EDTA, 2 units/mL ADA, pH 7.4) in the presence of 1.4 nM [125I]AB-
MECA and six different concentrations of the newly synthesized
compounds. Non-specific binding was determined in the presence
d
ppm): 8.61 (s, 1H, H-2); 8.26 (d, 1H, H-4); 8.23 (d,1H, J ¼ 1.5 Hz, H-
50); 7.99 (d, 1H, H-30); 7.61 (d, 1H, H-7); 7.36e7.25 (m, 8H, H-5, H-6,
H-40, HeAr); 5.57 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6,
of 50
m
M R-PIA [35]. The dissociation constant (Kd) of [125I]AB-
d
ppm): 185.3; 185.0; 141.0; 138.8; 138.0; 137.0; 136.7; 136.5; 129.2;
MECA in hA3 CHO cell membranes was 1.4 nM.
128.7; 127.8; 127.3; 126.2; 124.0; 123.3; 121.6; 111.8; 111.2; 49.9.
Anal. Calcd. for C21H15NO2S (%): C, 73.02; H, 4.38; N, 4.06. Found: C,
74.12; H, 4.61; N, 3.91.
All compounds were routinely dissolved in DMSO and diluted
with assay buffer to the final concentration, where the amount of
DMSO never exceeded 2%. Percentage inhibition values of specific
radiolabelled ligand binding at 1e10
means ꢀ SEM of at least three determinations.
mM concentration are
6.1.3. 1H NMR and elemental analyses of compounds 9a,b
6.1.3.1. N,1-Dibenzyl-1H-indole-3-carboxamide
(400 MHz, DMSO-d6,
(9a). 1H
NMR
d
ppm): 8.50 (t exch., 1H, J ¼ 5.7 Hz, NHeCH2);
6.2.5. Measurement of cyclic AMP levels on human A1, and A2B AR-
transfected CHO cells
8.19 (bs, 2H, AreH); 7.56e7.52 (m, 2H, AreH); 7.35e7.14 (m, 11H,
AreH); 5.47 (s, 2H, 1-CH2Ph); 4.47 (d, 2H, J ¼ 5.7 Hz, NHCH2Ph).
Anal. Calcd. for C23H20N2O (%): C, 81.15; H, 5.92; N, 8.23. Found: C,
81.29; H, 5.81; N, 8.44.
Intracellular cyclic AMP (cAMP) levels were measured using a
competitive protein binding method [50]. CHO cells, expressing
recombinant human ARs, were harvested by trypsinization. After
centrifugation and resuspension in medium, cells (w30,000) were
plated in 24-well plates in 0.5 mL of medium. After 24 h, the me-
dium was removed, and the cells were incubated at 37 ꢂC for 15 min
with 0.5 mL of Dulbecco’s Modified Eagle Medium (DMEM) in the
presence of adenosine deaminase (ADA) (1 U/mL) and the phos-
6.1.3.2. 1-Benzyl-N-phenyl-1H-indole-3-carboxamide (9b). 1H NMR
(400 MHz, DMSO-d6,
d ppm): 9.81 (s exch., 1H, NH); 8.42 (s, 1H, H-
2); 8.24e8.20 (m, 1H, H-4); 7.79e7.75 (m, 2H, AreH); 7.60e7.55 (m,
1H, AreH); 7.33e7.00 (m, 10H, AreH); 5.53 (s, 2H, 1-CH2Ph). Anal.
Calcd. for C22H18N2O (%): C, 80.96; H, 5.56; N, 8.58. Found: C, 81.15;
H, 5.47; N, 8.39.
phodiesterase inhibitor Ro20-1724 (20 mM). The pharmacological
profile of the compounds towards A2B ARs was evaluated by
assessing their ability to modulate NECA-mediated accumulation of
cAMP. The antagonist profile of the compounds towards A1 ARs was
evaluated by assessing their ability to counteract NECA-mediated
6.1.4. Synthesis of 1-benzyl-1H-indole (15)
Indole 10 (1.7 g, 14.5 mmol) was added to a stirred solution of
NaOH (1.2 g, 30 mmol) in DMSO (5 mL); the mixture was stirred for
15 min. After this time benzylchloride was added drop wise to the
cooled (15 ꢂC) mixture. The reaction mixture was stirred for 3 h,
added with ice/water (200 mL) to give a solid that was collected by
filtration and washed with water until neutral pH. Yield 80%, mp
41e42 ꢂC (lit. [47]: mp 41e43 ꢂC; lit. [48]: mp 43 ꢂC).
inhibition of cAMP accumulation in the presence of 1
mM for-
skolin, as non-selective adenylate cyclase (AC) activator. Cells were
incubated in the reaction medium (15 min at 37 ꢂC) with different
concentrations of the target compound (1 nMe10 mM) and then
were treated with the agonist.
Following incubation, the reaction was terminated by the
removal of the medium and the addition of 0.4 N HCl. After 30 min,
lysates were neutralized with 4 N KOH, and the suspension was
centrifuged at 800 rpm for 5 min. For the determination of cAMP
production, bovine adrenal cAMP binding protein was incubated
6.2. Biology
6.2.1. Adenosine receptor binding assay. Materials
[3H]DPCPX, [3H]NECA, and
[
125I]AB-MECA were obtained
with [3H]cAMP (2 nM) and 50
(0e48 pmol) at 0 ꢂC for 150 min in a total volume of 300
m
l of cell lysate or cAMP standard
l. Bound
from DuPont-NEN (Boston, MA). ADA was from Sigma Chemical
Co. (St. Louis, MO). All other reagents were from standard com-
mercial sources and of the highest commercially available
grade. CHO cells stably expressing human A1, A2A, and A3 ARs
were kindly supplied by Prof. K.N. Klotz, Wurzburg University,
Germany [49].
m
radioactivity was separated by rapid filtration through GF/C
glass fiber filters and washed twice with 4 mL 50 mM Tris/HCl,
pH 7.4. The radioactivity was measured by liquid scintillation
spectrometry.
6.2.6. Data analysis
6.2.2. Human A1 adenosine receptors
All binding and functional data were analyzed using the non-
linear regression curve fitting program GraphPad, version 5.0.
EC50, IC50 and Ki values were directly obtained from the dose
response curves. All values obtained are mean values of at least
three different experiments each performed in duplicate.
Aliquots of cell membranes (30 mg proteins) were incubated at
25 ꢂC for 180 min in 500
mL of T1 buffer (50 mM TriseHCl, 2 mM
MgCl2, 2 units/mL ADA, pH 7.4) containing [3H]DPCPX (3 nM) and
six different concentrations of the newly synthesized compounds.
Non-specific binding was determined in the presence of 50 mM R-
PIA [35]. The dissociation constant (Kd) of [3H]DPCPX in hA1 CHO
Acknowledgments
cell membranes was 3 nM.
This work was financially supported by MIUR (PRIN 2007, PRIN
2008). We thank Prof. Dr. Karl-Norbert Klotz for his generous gift of
transfected CHO cells expressing human A1, A2A, A2B and A3
receptors.
6.2.3. Human A2A adenosine receptors
Aliquots of cell membranes (30
90 min in 500 L of T2 buffer (50 mM TriseHCl, 2 mM MgCl2,
m
g) were incubated at 25 ꢂC for
m