B. Cosimelli, S. Taliani, et al.
MED
2508C. After cooling to room temperature, the mixture was
poured into a large volume of ice water, and the solid mass was
collected by filtration. The crude product was treated with boiling
MeOH for 2 h, and charcoal was added. After filtration, the MeOH
solution was cooled to precipitate the desired products. Yields,
melting points, and spectral data are reported in the Supporting
Information.
NH), 7.30–7.21 (m, 2H, system AB), 2.79–2.73 (m, 2H, CH2), 2.60–
2.54 (m, 2H, CH2), 1.97 (s, 3H, CH3), 1.75–1.68 ppm (m, 4H, 2ꢂCH2).
2-Amino-3-nitro-5,6,7,8-tetrahydronaphthalene 31 and 2-amino-
1-nitro-5,6,7,8-tetrahydronaphthalene 32: A 1:1 (v/v) solution of
H2SO4/H2O (6 mL) was added slowly at room temperature to a
stirred suspension of acetamide 29 or 30 in 10 mL of EtOH. Follow-
ing this addition, the reaction mixture was heated at reflux for 1 h
(TLC, petroleum ether/EtOAc, 3:1 v/v), then cooled to room tem-
perature. The crude product was poured into ice water (120 mL),
and the pH was adjusted to 8.0 with 30% NH4OH. The resulting
yellow–orange precipitate was removed by filtration and treated
with 10% HCl (20 mL) until boiling. Compounds 31 (90%) and 32
(65%) were obtained as pure solids by filtration, and were purified
by recrystallization from EtOH.
General procedure 3 for the synthesis of 4a, b, e, f, and 5a, b,
e, f:
A mixture of isoquinoline-3-carboxylic acid 26 (0.50 g,
2.9 mmol) or isoquinoline-1-carboxylic acid 27 (0.50 g, 2.9 mmol),
combined with the appropriate diamine (8, 12, 13, 22, 2.9 mmol)
and polyphosphoric acid (17.5 g) was stirred for 4 h at 2508C. After
cooling to room temperature, the mixture was poured into a large
volume of ice water, and the pH was adjusted to 8.0 with 30%
NH4OH. The solid mass was collected by filtration, washed with
water, and dried. Treatment with boiling toluene furnished an ex-
tract which, after evaporation, yielded target compounds with the
desired degree of purity. Yields, melting points, and spectral data
are reported in the Supporting Information.
2-Amino-3-nitro-5,6,7,8-tetrahydronaphthalene 31: mp: 124.8–
1
125.68C (lit. [29] 124.5–1268C); H NMR ([D6]DMSO): d=7.67 (s, 1H,
H-4), 7.11 (brs, 2H, NH2), 6.69 (s, 1H, H-1), 2.66–2.60 (m, 4H, 2ꢂ
CH2), 1.69–1.62 ppm (m, 4H, 2ꢂCH2).
2-Amino-1-nitro-5,6,7,8-tetrahydronaphthalene 32: mp: 96.3–
97.48C (lit. [29] 94–968C); 1H NMR ([D6]DMSO): d=6.98 (m, 1H, part
A of the system AB), 6.72 (m, 1H, part B of the system AB), 5.87
(brs, 2H, NH2), 2.61–2.54 (m, 4H, 2ꢂCH2), 1.70–1.60 ppm (m, 4H,
2ꢂCH2).
5,6,7,8-Tetrahydronaphthalene-2,3-diamine 20 and 5,6,7,8-tetra-
hydronaphthalene-1,2-diamine (21): A solution of 2-amino-3-
nitro-5,6,7,8-tetrahydronaphthalene 31 (0.95 g, 4.0 mmol) or 2-
amino-1-nitro-5,6,7,8-tetrahydronaphthalene 32 (0.95 g, 4.0 mmol)
and tin(II) chloride dihydrate (4.55 g, 20.0 mmol) in EtOAc (65 mL)
was stirred at reflux for 2 h. After cooling, the reaction mixture was
poured into ice water (200 mL). While stirring, the pH was adjusted
to 8.0 with saturated sodium bicarbonate solution, and the result-
ing precipitate was removed by filtration. The aqueous phase was
separated and extracted with EtOAc (4ꢂ50 mL). The combined or-
ganic phases were dried with Na2SO4 and evaporated to dryness to
give the desired compounds 20 (88%) or 21 (91%), respectively,
which were recrystallized from EtOH.
Biology
Adenosine receptor binding assay
Materials: [3H]DPCPX, [3H]NECA, and [125I]AB-MECA were obtained
from DuPont–NEN (Boston, MA, USA). ADA was obtained from
Sigma–Aldrich (St. Louis, MO, USA). All other reagents were from
standard commercial 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).[32]
5,6,7,8-Tetrahydronaphthalene-2,3-diamine (20): mp: 135.3–
136.28C (lit. [29] 135–1368C); 1H NMR ([D6]DMSO): d=6.18 (s, 2H,
H-1 and H-4), 4.15 (brs, 4H, 2ꢂNH2), 2.48–2.42 (m, 4H, 2ꢂCH2),
1.65–1.59 ppm (m, 4H, 2ꢂCH2).
Human A1 adenosine receptors: Aliquots of membranes (50 mg pro-
tein) obtained from A1 CHO cells were incubated at 258C for
180 min in 500 mL T1 buffer (50 mm Tris-HCl, 2 mm MgCl2, 2 UmLꢁ1
ADA, pH 7.4) containing [3H]DPCPX (3 nm) and six different concen-
trations of the newly synthesized compounds. Nonspecific binding
was determined in the presence of 50 mm RPIA.[26] The dissociation
constant (Kd) for [3H]DPCPX in A1 CHO cell membranes was 3 nm.
5,6,7,8-Tetrahydronaphthalene-1,2-diamine (21): mp: 85.8–
86.78C (lit. [29] 84–858C); 1H NMR ([D6]DMSO): d=6.34 (m, 1H, part
A of the system AB), 6.14 (m, 1H, part B of the system AB), 4.16
(brs, 2H, NH2), 4.05 (brs, 2H, NH2), 2.53 (pt, 2H, CH2), 2.36 (pt, 2H,
CH2), 1.75–1.70 (m, 2H, CH2), 1.69–1.56 ppm (m, 2H, CH2).
N-(3-Nitro-5,6,7,8-tetrahydronaphthalen-2-yl)acetamide (29) and
N-(1-nitro-5,6,7,8-tetrahydronaphthalen-2-yl)acetamide (30):
A
Human A2A adenosine receptors: Aliquots of cell membranes (80 mg
protein) were incubated at 258C for 90 min in 500 mL T2 buffer
(50 mm Tris-HCl, 2 mm MgCl2, 2 UmLꢁ1 ADA, pH 7.4) in the pres-
ence of 30 nm of [3H]NECA and six different concentrations of the
newly synthesized compounds. Nonspecific binding was deter-
mined in the presence of 100 mm NECA.[26] The dissociation con-
stant (Kd) for [3H]NECA in A2A CHO cell membranes was 30 nm.
1:1 (v/v) solution of HNO3 (65%) and acetic anhydride (2.4 mL) was
added dropwise to a stirred suspension of 2-amino-5,6,7,8,-tetrahy-
dronaphthalene 28 (13.6 mmol) in acetic anhydride (2.6 mL), main-
taining a temperature below 408C. Following this addition, the
mixture was stirred for 1 h and then treated with ice water (80 mL)
to give a yellow precipitate, which was removed by filtration under
vacuum, washed with petroleum ether, and dried over NaOH. The
crude residue was purified by flash chromatography (petroleum
ether/EtOAc, 3:1!2:1 v/v) to yield compounds 29 and 30 in 36%
and 26% yields, respectively. Both 29 and 30 were purified by re-
crystallization from EtOH.
Human A3 adenosine receptors: Aliquots of cell membranes (40 mg
protein) were incubated at 258C for 90 min in 100 mL T3 buffer
(50 mm Tris-HCl, 10 mm MgCl2, 1 mm EDTA, 2 UmLꢁ1 ADA, pH 7.4)
in the presence of 1.4 nm [125I]ABMECA and six different concentra-
tions of the newly synthesized compounds. Nonspecific binding
was determined in the presence of 50 mm R-PIA.[26] The dissociation
constant (Kd) for [125I]AB-MECA in A3 CHO cell membranes was
1.4 nm.
N-(3-Nitro-5,6,7,8-tetrahydronaphthalen-2-yl)acetamide 29: mp:
134.1–135.08C (lit. [30] 133–1358C); 1H NMR ([D6]DMSO): d=10.06
(s, 1H, NH), 7.67 (s, 1H, H-4), 7.33 (s, 1H, H-3), 2.80–2.74 (m, 4H, 2ꢂ
CH2), 2.03 (s, 3H, CH3), 1.76–1.70 ppm (m, 4H, 2ꢂCH2).
All compounds were dissolved in DMSO and diluted with assay
buffer to the final concentration, the amount of DMSO never ex-
ceeding 2%. Percentage inhibition values for specific radiolabeled
N-(1-Nitro-5,6,7,8-tetrahydronaphthalen-2-yl)acetamide 30: mp:
1
129.2–129.98C (lit. [31] 1278C); H NMR ([D6]DMSO): d=9.79 (s, 1H,
1916
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ChemMedChem 2011, 6, 1909 – 1918