4070 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Colotta et al.
1
J ) 8.3 Hz), 7.54-7.61 (m, 2H, ar), 7.86 (d, 1H, ar, J ) 7.5 Hz),
8.03 (d, 2H, ar, J ) 8.3 Hz), 8.21 (d, 1H, ar, J ) 8.6 Hz), 9.64 (s,
1H, H-1), 10.24 (s, 1H, NH); IR 3380, 1674. Anal. (C19H16N4O)
C, H, N.
30: yield 95%; mp 226-228 °C (ethanol); H NMR (DMSO-
d6) 2.32 (s, 3H, CH3), 4.60 (d, 2H, CH2, J ) 5.5 Hz), 7.28-7.52
(m, 9H, ar), 7.77 (d, 1H, J ) 8.1 Hz), 8.03 (s, 1H, ar), 8.14 (d, 2H,
7.1 Hz), 9.27 (s, 1H, NH), 9.67 (s, 1H, H-9), 10.50 (t, 1H, NH, J
) 5.5 Hz); IR 3420, 1686. Anal. (C25H21N5O) C, H, N.
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16: yield 81%; mp 196-199 °C (2-ethoxyethanol); H NMR
1
31: yield 68%; mp 224-226 °C (2-methoxyethanol); H NMR
(DMSO-d6) 2.40 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 7.19 (d, 2H,
ar, J ) 8.9 Hz), 7.55-7.60 (m, 2H, ar), 7.85 (d, 1H, ar, J ) 7.6
Hz), 8.05 (d, 2H, ar, J ) 8.8 Hz), 8.20 (d, 1H, ar, J ) 7.0 Hz),
9.57 (s, 1H, H-1), 10.24 ((s, 1H, NH); IR 3252, 1670. Anal.
(C19H16N4O2) C, H, N.
(DMSO-d6) 2. 41(s, 3H, CH3), 4.61 (d, 2H, CH2, J ) 5.7 Hz),
7.29-7.51 (m, 9H, ar), 7.77 (d, 1H, ar, J ) 7.7 Hz), 8.08-8.15
(m, 3H, ar), 9.24 (s, 1H, NH), 9.65 (s, 1H, H-9), 10.50 (t, 1H, NH,
J ) 5.7 Hz); IR 3423, 1687. Anal. (C25H21N5O) C, H, N.
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1
32: yield 78%; mp 245-246 °C (2-methoxyethanol); H NMR
18: yield 83%; mp 219-220 °C (2-methoxyethanol); H NMR
(DMSO-d6) 3.87 (s, 3H, CH3), 4.61 (d, 2H, CH2, J ) 5.7 Hz),
7.20 (d, 1H, ar, J ) 9.1 Hz), 7.27-7.55 (m, 8H, ar), 7.77 (d, 1H,
ar, J ) 8.3 Hz), 8.14 (m, 3H, ar), 9.18 (s, 1H, NH), 9.58 (s, 1H,
H-9), 10.50 (t, 1H, NH, J ) 5.7 Hz); IR 3419, 1687. Anal.
(C25H21N5O2) C, H, N.
(CDCl3) 2.59 (s, 3H, CH3), 7.43-7.75 (m, 8H, ar), 7.79 (d, 1H, ar,
J ) 7.4 Hz), 7.92 (s, 1H, ar), 7.97 (d, 1H, ar, J ) 7.6 Hz), 8.65 (s,
2H, ar), 8.67 (s, 1H, H-1); IR 3620, 1620. Anal. (C24H18N4O) C,
H, N.
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19: yield 88%; mp 163-165 °C (2-methoxyethanol); H NMR
General Procedure for the Synthesis of 4-Dibenzoylamino-
2-aryl-2H-pyrazolo[3,4-c]quinolines (33-36). A solution of ben-
zoyl chloride (5 mmol) in anhydrous methylene chloride (3 mL)
was dropwise added to a suspension of the 4-amino derivatives
8-11 (2.5 mmol) in anhydrous methylene chloride (7 mL) and
anhydrous pyridine (25 mmol). The mixture was stirred at room
temperature for about 24 h. Evaporation of the solvent at reduced
pressure afforded a residue that was treated with water (4-5 mL),
collected by filtration, and recrystallized.
(CDCl3) 2.49 (s, 3H, CH3), 7.32-7.60 (m, 8H, ar), 7.92 (d, 2H, ar,
J ) 8.1 Hz), 7.96 (s, 1H, ar, J ) 7.8 Hz), 8.41-8.59 (m, 2H, ar),
8.69 (s, 1H, H-1); IR 3620, 1626. Anal. (C24H18N4O) C, H, N.
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20: yield 69%; mp 203-204 °C (2-methoxyethanol); H NMR
(CDCl3) 3.93 (s, 3H, CH3), 7.09 (d, 2H, ar, J ) 8.9 Hz), 7.43-
7.52 (m, 6H, ar), 7.93-7.95 (m, 3H, ar), 8.49-8.52 (m, 2H, ar),
8.62 (s, 1H, H-1); IR 3629, 1626. Anal. (C24H18N4O2) C, H, N.
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22: yield 84%; mp 184-186 °C (ethanol); H NMR (DMSO-
d6) 2.48 (s, 3H, CH3), 4.06 (s, 2H, CH2), 7.26-7.61 (m, 9H, ar),
7.87 (d, 1H, ar, J ) 6.9 Hz), 7.92 (d, 1H, ar, J ) 7.9 Hz), 7.98 (s,
1H, ar), 8.19-8.23 (m, 1H, ar), 9.68 (s, 1H, H-1), 10.55 (s, 1H,
NH); IR 3366, 1673. Anal. (C25H20N4O) C, H, N.
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33: yield 72%; mp 224-226 °C (2-ethoxyethanol); H NMR
(DMSO-d6) 7.44-7.69 (m, 11H, ar), 7.78 (d, 1H, ar, J ) 7.7 Hz),
7.88 (d, 4H, ar, J ) 7.3 Hz), 8.02 (d, 2H, ar, J ) 7.8 Hz), 8.29 (d,
1H, ar, J ) 7.7 Hz), 9.81 (s, 1H, H-1); IR 1689. Anal. (C30H20N4O2)
C, H, N.
1
23: yield 80%; mp 170-172 °C (2-methoxyethanol); H NMR
(DMSO-d6) 2.42 (s, 3H, CH3), 4.04 (s, 2H, CH2), 7.26-7.29 (m,
1H, ar), 7.36 (t, 2H, ar, J ) 7.5 Hz), 7.44-7.48 (m, 4H, ar), 7.57-
7.59 (m, 2H, ar), 7.87 (d, 1H, ar, J ) 8.8 Hz), 8.02 (d, 2H, ar, J )
8.3 Hz), 8.22 (d, 1H, ar, J ) 6.5 Hz), 9.66 (s, 1H, H-1), 10.55 (s,
1H, NH); IR 3372, 1678. Anal. (C25H20N4O) C, H, N.
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34: yield 77%; mp 207-208 °C (ethanol); H NMR (DMSO-
d6) 2.42 (s, 3H, CH3), 7.31 (d, 1H, ar, J ) 7.0 Hz), 7.44-7.56 (m,
8H, ar), 7.68 (t, 1H, ar, J ) 7.1 Hz), 7.76-7.88 (m, 7H, ar), 8.28
(d, 1H, ar, J ) 7.8 Hz), 9.77 (s, 1H, H-1); IR 1707, 1687. Anal.
(C31H22N4O2) C, H, N.
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24: yield 87%; mp 185-187 °C (2-ethoxyethanol); H NMR
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35: yield 65%; mp 241-243 °C (2-methoxyethanol); H NMR
(DMSO-d6) 3.87 (s, 3H, CH3), 4.05 (s, 2H, CH2), 7.21 (d, 2H, ar,
J ) 9.0 Hz), 7.29 (m, 1H, ar), 7.36 (t, 2H, ar, J ) 7.4 Hz), 7.45 (d,
2H, ar, J ) 7.3 Hz), 7.55-7.61 (m, 2H, ar), 7.88-7.86 (m, 1H,
ar), 8.04 (d, 2H, ar, J ) 8.9 Hz), 8.20-8.22 (m, 1H, ar), 9.59 (s,
1H, H-1), 10.53 (s, 1H, NH); IR 3371, 1667. Anal. (C25H20N4O2)
C, H, N.
(DMSO-d6) 2.33 (s, 3H, CH3), 7.42-7.47 (m, 6H, ar), 759-7.53
(m, 3H, ar), 7.67 (t, 1H, ar, J ) 7.6 Hz), 7.77 (d, 1H, ar, J ) 8.3
Hz), 7.86-7.91 (m, 6H, ar), 8.28 (d, 1H, ar, J ) 7.6 Hz), 9.74 (s,
1H, H-9); IR 1706, 1684. Anal. (C31H22N4O2) C, H, N.
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36: yield 68%; mp 247-250 °C (2-methoxyethanol); H NMR
(DMSO-d6) 3.84 (s, 3H, CH3), 7.17 (d, 2H, ar, J ) 9.0 Hz), 7.45
(t, 4H, ar, J ) 7.7 Hz), 7.53-7.59 (m, 3H, ar), 7.67 (t, 1H, ar, J )
7.52 Hz), 7.77 (d, 1H, J ) 7.7 Hz), 7.87 (d, 4H, ar, J ) 8.2 Hz),
7.93 (d, 2H, ar, J ) 7.6 Hz), 8.26 (d, 1H, J ) 7.7 Hz), 9.68 (s, 1H,
H-1); IR 1706, 1683. Anal. (C31H22N4O3) C, H, N.
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25: yield 44%; mp 208-210 °C (2-ethoxyethanol); H NMR
(DMSO-d6) 5.74 (br s, 1H, CH), 7.27-7.40 (m, 6H, ar), 7.49-
7.71 (m, 7H, ar), 7.69 (t, 2H, ar, J ) 7.7 Hz), 7.88-7.90 (m, 1H,
ar), 8.13 (d, 2H, ar, J ) 8.0 Hz), 8.23-8.25 (m, 1H, ar), 9.71 (s,
1H, H-9), 10.91 (s, 1H, NH); IR 3278, 1667. Anal. (C30H22N4O)
C, H, N.
(B) Biochemistry. Human A1, A2A, and A3 Receptor Binding.
Displacement of [3H]DPCPX and [3H]NECA from, respectively,
hA1 and hA2A ARs, stably expressed in CHO cells, was performed
as previously described.43 Displacement of [125I]AB-MECA from
hA3 ARs, stably expressed in CHO cells, was performed as reported
in ref 20.
Rat A1 and A2A Receptor Binding. Rat brain cortex and striatum
were dissected from male Wistar rats. Membrane preparation was
carried out as previosly reported for the bovine membrane prepara-
tion.44 The A1 binding assays were performed in triplicate by
incubating aliquots of brain cortex membranes (40-50 µg of
protein) at 25 °C for 180 min in 0.5 mL of binding buffer (50 mM
Tris/HCl, 2 mM MgCl2 pH ) 7.7) containing 0.2-0.5 nM [3H]-
DPCPX. Nonspecific binding was defined in presence of 20 µM
R-PIA. Incubation was terminated by rapid filtration through
Whatman GF/C glass microfiber filters and washing twice with 4
mL of ice-cold buffer. KD value on brain cortex was 0.51 nM.45
Binding assays at the rA2A AR were performed as previously
described.44
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26: yield 80%; mp 220-221 °C (2-ethoxyethanol); H NMR
(DMSO-d6) 2.46 (s, 3H, CH3), 5.75 (br s, 1H, CH), 7.27-7.40 (m,
7H, ar), 7.49-7.61 (m, 7H, ar), 7.88-7.94 (m, 2H,ar), 7.97 (s, 1H,
ar), 8.23-8.24 (m, 1H, ar), 9.69 (s, 1H, H-9), 10.89 (s, 1H, NH);
IR 3367, 1700. Anal. (C31H24N4O) C, H, N.
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27: yield 44%; mp 215-218 °C (2-ethoxyethanol); H NMR
(DMSO-d6) 2.43 (s, 3H, CH3), 5.72 (br s, 1H, CH), 7.27-7.31 (m,
2H, ar), 7.38 (t, 4H, ar, J ) 7.6 Hz), 7.49 (d, 6H, ar, J ) 7.8 Hz),
7.57-7.62 (m, 2H, ar), 7.87-7.90 (m, 1H, ar), 8.02 (d, 2H, ar, J
) 8.0 Hz), 8.21-8.23 (m, 1H, ar), 9.67 (s, 1H, H-9), 10.94 (br s,
1H, NH); IR 3284, 1681. Anal. (C31H24N4O) C, H, N.
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28: yield 70%; mp 211-213 °C (2-ethoxyethanol); H NMR
(DMSO-d6) 3.88 (s, 3H, CH3), 5.74 (br s, 1H, CH), 7.17-7.14 (m,
8H, ar), 7.49 (d, 4H, ar, J ) 7.4 Hz), 7.56-7.62 (m, 2H, ar), 7.87-
7.90 (m, 1H, ar), 8.02 (d, 2H, ar, J ) 9 Hz), 8.21-8.24 (m, 1H,
ar), 9.60 (s, 1H, H-9), 10.89 (s, 1H, NH); IR 3318, 1675. Anal.
(C31H24N4O2) C, H, N.
General Procedure for the Synthesis of 4-Benzylureido-2-
aryl-2H-pyrazolo[3,4-c]quinolines (30-32). Benzyl isocyanate
(1.5 mmol) was added to a suspension of the 4-amino derivatives
9-11 (1 mmol) in anhydrous tetrahydrofuran (20 mL). The mixture
was refluxed for 24-48 h under nitrogen atmosphere. The resulting
solid was collected, washed with diethyl ether, and recrystallized.
Rat A3 Receptor Binding. Binding of [3H]-(R)-PIA (37 Ci/
mmol) to rat testis membranes was measured in the presence of
DPCPX (150 nM) as previously described.46,47 Briefly, fresh
testicular tissue from Wistar rats was dissected free of epididymis,
and membranes were prepared as described.46 Rat testis membranes
(0.1-0.2 mg of protein) and [3H]-(R)-PIA 4 nM were incubated in