New Quaternary Ammonium Oxicam Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5239
F igu r e 7. Proteoglycans synthesis by cultured chondrocytes measured in the cell layer (A) and culture medium (B) after
simultaneous treatment with IL-1â and NSAIDs.
2-Met h yl-4-h yd r oxy-2H-1,2-b en zot h ia zin e-3-ca r boxy-
lic Acid Meth yl Ester 1,1-Dioxid e (4). Steps i, ii, and iii
were performed according to Lombardino et al.7,8 with, respec-
tively, 86%, 68%, and 85% yields. To prepare the tritiated
compound 4, 4-hydroxy-2H-1,2-benzothiazine-3-carboxylic acid
methyl ester 1,1-dioxide, 3 (2 g; 7.84 mmol), is reacted with
[3H]-CH3I (370 MBq; 3.15 TBq/mmol) introduced through a
vacuum ramp and then diluted with CH3I (0.35 mL; 5.6 mmol).
After 24 h of agitation at room temperature, excess CH3I (1.5
mL; 24 mmol) is added and the mixture is agitated again,
leading to 1.80 g (85%) of the tritiated title compound 4;
specific radioactivity 49 MBq/mmol; mp 160-162 °C; Rf 0.7
(A); 1H NMR (CDCl3) δ 2.95 (3H, s, NCH3), 3.96 (3H, s, OCH3),
7.71-8.05 (4H, m, C6H4), 12.05 (1H, s, OH).
CH2N), 2.75 (3H, s, N-CH3), 3.35 (2H, m, NH-CH2), 7.6-8.1
(4H, m, Ph), 8.50 (1H, t, NH), 13.2 (1H, s, OH);13C NMR
(DMSO-d6) δ 26.0 (CH2-CH2-CH2), 35.9 (NH-CH2), 43.6 (N-
CH3), 52.2 (CH2-N), 55.6 (N-(CH3)2), 109.5 ()C-CO), 120.3,
126.3, 129.0, 130.7, 132.4, 134.6 (Ph), 160.5 (-C-OH), 168.3
(CdO); pKa 6.4. Anal. (C15H21N3O4S) C, H, N.
[3-(2-Meth yl-4-h yd r oxy-2H-1,2-ben zoth ia zin e-1,1-d iox-
id e-3-[14C]ca r boxa m id o)p r op yl]tr im eth yla m m on iu m Io-
d id e or P r op oxica m -N+ (6b). The conversion of 1.15 g (3.39
mmol) of 5b to 6b was achieved as described for 6a , giving
1.74 g (80%) of a yellow powder; mp 220-222 °C (dec); Rf 0.10
(B); specific radioactivity 125 MBq/mmol; IR (cm-1) 3274, 1629,
1
1179, 1335; H NMR (DMSO-d6) δ 1.98-2.09 (2H, m, CH2-
CH2-CH2), 2.82 (3H, s, N-CH3), 3.12 (9H, s, +N(CH3)3), 3.35-
3.43 (4H, m, NH-CH2
+
+N-CH2), 7.88-8.02 (4H, m, Ph),
N-(2-P yr idyl)-2-[3H]-m eth yl-4-h ydr oxy-2H-1,2-ben zoth i-
a zin e-3-ca r boxa m id e 1,1-Dioxid e or [3H]-P ir oxica m (5a ).
According to the general procedure,7,8 the [3H]-labeled com-
pound 4 (1.76 g; 6.54 mmol) was reacted with 2-aminopyridine
(0.75 g; 8 mmol) to yield 1.65 g (74%) of [3H]-piroxicam (5a );
specific radioactivity 49 MBq/mmol.; mp 200-202 °C (dec); Rf
8.82 (1H, t, NH), 14.30 (1H, s, OH); 13C NMR (DMSO-d6) δ
22.6 (CH2-CH2-CH2), 35.9 (NH-CH2), 38.7 (N-CH3), 52.2
(+N-(CH3)3), 63.3 (CH2-N+), 111.15 ()C-CO), 124.0, 125.9,
127.9, 132.6, 133.3, 133.9 (Ph), 156.0 (dC-OH), 168.2 (CdO);
solubility 10 g/L (H2O) at 20 °C, 20 g/L (H2O-DMSO 5%) at
20 °C; ESI-MS m/z 354.3 (M+); pKa 5.85; Anal. (C16H24N3O4-
SI‚0.5H2O) C, H, N.
1
0.90 (B); H NMR (CDCl3) δ 2.96 (3H, s, N-CH3), 7.73-7.95
(4H, m, Ph), 7.15, 8.06, 8.28, 8.37 (4 × 1H, mmdd, Pyr), 9.06
(1H, s, NH), 13.30 (1H, s, OH); 13C NMR (CDCl3) δ 39.9 (N-
CH3), 111.5 (dC-CO), 114.3, 120.6, 126.6, 148.1, 150.1 (Pyr),
124.8, 132.5, 133.0, 134.6, 138.4 (Ph), 159.9 (dCOH), 166.9
(CdO); pKa 5.9. Anal. (C15H13N3O4S) C, H, N.
P h a r m a cology. Materials. Collagenase was obtained from
Worthington. Dulbecco’s modified Eagle’s medium (DMEM)
and glutamine were purchased from Gibco (Uxbridge, U.K.).
Foetal calf serum (FCS) was from Seromed. Protease inhibi-
tors, Interleukin-1â (IL-1â) and Indomethacin, were purchased
from Sigma Chemical Co. (Poole, U.K.) and guanidinium
hydrochloride (GuHCl) from Merck (Darmstadt, Germany).
[35S]-Sodium sulfate (carrier-free) in aqueous solution (39 GBq/
mmol) was obtained from Amersham (U.K.). Sprague-Dawley
rats weighing 100-120 g were purchased from Iffa-Credo
(L’Arbresle, France).
In Vitr o Exp er im en ts. (1) Cell Cultures. Articular chon-
drocytes were obtained from the knees of a 1 month-old rabbit
(Fauve de Bourgogne, Elevage Scientifique des Dombes-
France) by enzymatic digestion overnight at 37 °C, with 0.2%
collagenase.12 Cells were cultured in DMEM supplemented
with 10% FCS, 2 mM glutamine, 8 µg/mL gentamycin at 37
°C in a 5% CO2 atmosphere. Experiments were performed on
primary confluent culture to avoid differentiation of the
chondrocytes.
(2-[3H]-Meth yl-4-h yd r oxy-2H-1,2-ben zoth ia zin e-1,1-d i-
oxide-3-car boxam ido)-2-m eth ylpyr idin iu m Iodide or [3H]-
P ir oxica m -N+ (6a ). In a 50 mL tight-stopped flask, 5a (1 g;
3.02 mmol), excess methyl iodide (3 mL) and acetone (30 mL)
were heated at 80 °C for 24 h. After the mixture was cooled,
the pale yellow precipitate 6a was filtered, washed with
acetone, and dried under vacuum. Yield 1.15 g (80%); mp 202-
203 °C; Rf 0.69 (B); specific radioactivity 49 MBq/mmol; IR
1
(cm-1) 3425, 3370, 3006, 1660, 1350, 1172; H NMR (DMSO-
d6) δ 2.79 (3H, s, N-CH3), 4.07 (3H, s, +N-CH3), 5.50 (3H, s,
OH + NH + 0.5 H2O), 7.69-8.06 (4H, m, Ph), 7.28, 8.19, 8.53,
8.91 (4 × 1H, ttdd, Pyr);13C NMR (DMSO- d6) δ 38.6 (N-CH3),
43.7 (+N-CH3), 111.1 (dC-CO), 118.9, 120.1, 143.6, 144.3,
149.7 (Pyr), 123.2, 126.7, 131.8, 132.6, 135.0, 140.3 (Ph), 159.0
(dCOH), 163.1 (CdO); ESI-MS m/z 346.2 (M+); pKa 5.5. Anal.
(C16H16N3O4SI) C, H, N.
N-[3-(Dim eth yla m in o)p r op yl]-2-m eth yl-4-h yd r oxy-2H-
1,2-ben zoth iazin e-3-[14C]car boxam ide 1,1-Dioxide or P r o-
p oxica m (5b). The [14C]-labeled compound 4 (1.72 g, 6.4
mmol) was refluxed for 24 h under argon in the presence of
0.65 g (6.4 mmol) of 3-(dimethylamino)propylamine in 125 mL
of anhydrous xylene. After removal of the solvent, the solid
residue was washed with ethanol to give 5b (1.23 g; 57%) as
a white powder; mp 199-200 °C (dec); Rf 0.25 (B); specific
radioactivity 125 MBq/mmol;1H NMR (DMSO-d6) δ 1.6-1.75
(2H, m, CH2-CH2-CH2), 2.35 (6H, s, N-(CH3)2), 2.50 (2H, t,
(2) Pharmacological Test. Chondrocytes were cultured in 9.6
cm2 Petri dishes, and used at confluence.The pharmacological
experiment was performed in incubating cells with IL-1â(200
pg/dish) in the presence or absence of the drugs dissolved in
dimethyl sulfoxide (DMSO) (final concentration in the medium
0.125% v/v) at 10-4 M in DMEM with 10% FCS.13 The products
and medium were changed every second day. At the fifth
day, 35S-sulfate (5 µCi/dish) was added for 24 h to label the
newly synthesized PGs. The amounts of labeled PGs were
measured in the cell layer and cell culture supernatant as