Arch. Pharm. Chem. Life Sci. 2010, 10, 631–638
Pyrazolobenzotriazinone Derivatives as COX Inhibitors
637
CH3), 4.22 (q, 2H, CH2), 6.60 (s, 2H, NH2, exchangeable), 7.49–8.52
(a set of signals, 8H, aromatic protons), 10.80 (s, 1H, NH,
exchangeable). Anal. calcd. for C18H16ClN5O3: C, 56.04; H, 4.18;
N, 18.15; found: C, 56.37; H, 4.22; N, 18.33.
cooled (ice-bath, 0–58C) and under magnetic stirring an aqueous
solution of potassium nitrite (7.0 mmol) was added. After the
complete addition, the solution was left on a magnetic stirrer for
45 min, and then was poured in cold water. The solid which
separated out was collected and recrystallized from ethanol.
Ethyl 5-(2-amino-4-chlorobenzamido)-1-(pyridin-2-yl)-1H-
5-(4-Oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-2-yl-1H-
pyrazole-4-carboxylate 15c
Yield: 60%; m. p.: 208–2098C (ethanol); IR (Nujol) cmꢁ1: 3479–
3368 (NH and NH2), 1711–1670 (CO); 1H-NMR (DMSO-d6) d: 1.22
(t, 3H, CH3), 4.22 (q, 2H, CH2), 6.50 (s, 2H, NH2, exchangeable),
7.78–8.47 (a set of signals, 8H, aromatic protons), 10.70 (s, 1H, NH,
exchangeable). Anal. calcd. for C18H16ClN5O3: C, 56.04; H, 4.18; N,
18.15; found: C, 56.22; H, 4.15; N, 18.26.
pyrazole-4-carboxylic acid 18a
Yield: 55%; m. p.: 205–2068C (ethanol); IR (Nujol) cmꢁ1: 1714 (CO);
1H-NMR (DMSO-d6) d: 7.31–8.46 (a set of signals, 9H, aromatic
protons), 13.15 (s, 1H, COOH, exchangeable). Anal. calcd.
for C16H10N6O3: C, 57.49; H, 3.02; N, 25.14; found: C, 57.35; H,
3.17; N, 25.26.
Synthesis of ethyl 5-(6-R1-7-R2-4-oxo-1,2,3-benzotriazin-
5-(6-Chloro-4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-
3(4H)-yl)-1-pyridin-2-yl-1H-pyrazole-4-carboxylate 16a–c
To a magnetically stirred, cold solution (ice-bath, 0–58C) of ethyl
5-(2-amino-5-R1-4-R2-benzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-
carboxylate 15a–c (3 mmol) in acetic acid (60 mL), an aqueous
solution of potassium nitrite (7.0 mmol) was added dropwise.
Stirring was continued for 2 h at 0–58C and then the solution
was warmed to room temperature. The solid which separated out
after the addition of cold water (100 mL) was collected and
recrystallized.
2-yl-1H-pyrazole-4-carboxylic acid 18b
Yield: 50%; m. p.: 215–2168C (ethanol); IR (Nujol) cmꢁ1: 1708 (CO);
1H-NMR (DMSO-d6) d: 7.33–8.56 (a set of signals, 9H, aromatic
protons), 13.24 (s, 1H, COOH, exchangeable). Anal. calcd.
for C16H9ClN6O3: C, 52.12; H, 2.46; N, 22.79; found: C, 52.05;
H, 2.53; N, 22.64.
5-(7-Chloro-4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-
2-yl-1H-pyrazole-4-carboxylic acid 18c
Yield: 58%; m. p.: 230–2318C (ethanol); IR (Nujol) cmꢁ1: 1710,
1684 (CO); 1H-NMR (DMSO-d6) d: 7.35–8.45 (a set of signals, 9H,
aromatic), 13.39 (s, 1H, COOH, exchangeable). Anal. calcd.
for C16H9ClN6O3: C, 52.12; H, 2.46; N, 22.79; found: C, 52.23;
H, 2.41; N, 22.67.
Ethyl 5-(4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-pyridin-2-yl-
1H-pyrazole-4-carboxylate 16a
Yield: 75%; m. p.: 140–1418C (ethanol); IR (Nujol) cmꢁ1: 1718–
1700 (CO); 1H-NMR (DMSO-d6) d: 1.09 (t, 3H, CH3), 4.20 (q, 2H, CH2),
7.10–8.43 (a set of signals, 9H, aromatic protons). Anal. calcd. for
C18H14N6O3: C, 59.67; H, 3.89; N, 23.19; found: C, 59.78; H, 3.87;
N, 26.16.
Biology
The reference compounds indomethacin and NS398 were pur-
chased from Cayman Chemical, Ann Arbor, MI, USA (Cat. No.
70270 and 70590, respectively). Male Wistar rats (Catholic
University School of Medicine, Rome, Italy), weighting 200–
300 g, were used. They were kept four per cage and maintained
at a temperature of 23 ꢀ 1.58C, with a relative humidity of
65 ꢀ 2%. The animals were exposed to 12 h of light (06.00
a.m. to 06.00 p.m.) followed by 12 h of darkness, and they had
free accessed to food and water. Compounds 16a, 18a, and 4 were
dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich S.r.l.,
Milano, Italy) and diluted to working concentrations in incu-
bation media. IL-1b was obtained from Boehringer (Mannheim,
Germany). The cytokine was dissolved in phosphate-buffered
saline containing 0.1% BSA and further diluted in incubation
medium. All drugs tested did not interfere with the PGE2 assay.
Ethyl 5-(6-chloro-4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-
pyridin-2-yl-1H-pyrazole-4-carboxylate 16b
Yield: 60%; m. p.: 161–1628C (ethanol); IR (Nujol) cmꢁ1: 1710 (CO);
1H-NMR (DMSO-d6) d: 1.03 (t, 3H, CH3), 4.14 (q, 2H, CH2), 7.36–8.58
(a set of signals, 8H, aromatic protons). Anal. calcd. for
C18H13ClN6O3: C, 54.49; H, 3.30; N, 21.18; found: C, 54.36; H,
3.34; N, 21.24.
Ethyl 5-(7-chloro-4-oxo-1,2,3-benzotriazin-3(4H)-yl)-1-
pyridin-2-yl-1H-pyrazole-4-carboxylate 16c
Yield: 65%; m. p.: 105–1068C (ethanol); IR (Nujol) cmꢁ1: 1715 (CO);
1H-NMR (DMSO-d6) d: 1.03 (t, 3H, CH3), 4.15 (q, 2H, CH2), 7.36–8.54
(a set of signals, 8H, aromatic protons). Anal. calcd. for
C18H13ClN6O3: C, 54.49; H, 3.30; N, 21.18; found: C, 54.52; H,
3.27; N, 21.28.
COX1/COX2 inhibition
Ovine COX-1(Cat. No. 60100), COX-2 (Cat. No. 60120), arachidonic
acid (Cat. No. 90010) as well as the reference inhibitors NS398
(Cat. No. 70590) and indomethacin (Cat. No. 70270) were pur-
chased from Cayman Chemical. Porcine hematine (Cat. No.
H3281), luminol (Cat. No. A4685), and gelatine (Cat. No.
G7765) were received from Sigma Aldrich S.r.l.
Chemiluminescence measurements were performed in 96-
well white polystyrene microtiter plates using a microtiter-plate
luminometer.
Synthesis of 5-(6-R1-7-R2-4-oxo-1,2,3-benzotriazin-3(4H)-
yl)-1-pyridin-2-yl-1H-pyrazole-4-carboxylic acid 18a–c
Ethyl
5-(2-amino-5-R1-4-R2-benzamido)-1-(pyridin-2-yl)-1H-pyra-
zole-4-carboxylates 15a–c (3 mmol) dissolved in ethanol
(20 mL) were treated with an aqueous solution of sodium hydrox-
ide 4% (30 mL) and refluxed for 15 min. The mixture was then
reduced to a small volume, cooled, and acidified at pH ¼ 5.0
with aqueous HCl. The product which precipitated was collected
and dissolved in acetic acid (60 mL). The obtained mixture was
Briefly, COX-1 or COX-2 (50 U/mL in 0.1 M PBS, pH 6.5, with
1 mg/mL gelatine), hematine (6 mM in 0.1 M PBS, pH 6.5), and
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