4868 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Moreira et al.
m), 3.05 (1H, d, J ) 5.1 Hz), 3.07 (1H, d, J ) 5.1 Hz), 4.67
(1H, d, J ) 14.5 Hz), 4.73 (1H, d, J ) 14.5 Hz), 6.02 (1H, s),
7.29-7.40 (5H, m); δ 13C NMR 8.83, 25.32, 25.36, 46.91, 44.41,
62.29, 78.42, 128.87, 128.93, 129.15, 134.38, 153.77, 166.41,
173.40; ESI-MS m/z 334 (MNH4)+; Anal. (C17H20N2O4) C, H,
N.
79.62, 128.59, 129.12, 129.45, 130.38, 135.12, 135.63, 138.08,
154.15, 166.41, 171.88; ESI-MS m/z 475 (MNH4+). Anal.
(C23H24N2O6S) C, H, N.
3,3-Diethyl-N-[3-(4-methoxyphenyl)oxazolidin-2,4-dion-
5-yl]-4-(phenylsulfonyl)azetidin-2-one (6e). Major diaste-
reomer; recrystallized from acetone-light petroleum; white
crystals (33%); mp 139-141 °C; νmax (film) 2974, 1826, 1755,
1789, 1515, 1368, 1333, 1254 cm-1; δ 1H NMR 0.76 (3H, t, J )
7.4 Hz), 0.97 (3H, t, J ) 7.5 Hz), 1.49 (1H, dq, J ) 7.3, 15 Hz),
1.68 (1H, dq, J ) 7.3, 14.8 Hz), 1.85 (1H, dq, J ) 7.3, 14.8
Hz), 2.00 (1H, dq, J ) 7.3, 14.8 Hz), 3.84 (3H, s), 4.72 (1H, s),
6.20 (1H, s), 7.00 (2H, d, J ) 9.1 Hz), 7.37 (2H, d, J ) 9.1 Hz),
7.63 (2H, t, J ) 7.4 Hz), 7.74 (1H, t, J ) 7.4 Hz), 8.02 (2H, d,
J ) 7.4 Hz); δ 13C NMR 7.98, 8.61, 19.65, 24.68, 55.56, 67.14,
77.16, 78.14, 114.73, 122.92, 127.45, 129.43, 129.75, 135.11,
135.53, 152.59, 160.18, 165.48, 171.53; ESI-MS m/z 490
(MNH4+); Anal. (C23H24N2O7S) C, H, N. Minor diastereomer;
white crystals (17%); mp 89-91 °C; νmax (film) 2971, 1824,
1785, 1757, 1515, 1331, 1303, 1254 cm-1; δ 1H NMR 0.82 (3H,
t, J ) 7.4 Hz), 1.0 (3H, t, J ) 7.5 Hz), 1.63-1.75 (2H, m), 1.89
(1H, dq, J ) 7.3, 14.7 Hz), 2.22 (1H, dq, J ) 7.3, 14.8 Hz),
3.81 (3H, s), 4.69 (1H, s), 5.58 (1H, s), 6.98 (2H, d, J ) 9.2
Hz), 7.37 (2H, d, J ) 9.2 Hz), 7.66 (2H, t, J ) 7.3 Hz), 7.76
(1H, t, J ) 7.3 Hz), 8.06 (2H, d, J ) 7.3 Hz); δ 13C NMR 8.48,
9.05, 20.17, 24.93, 55.95, 67.41, 78.15, 78.89, 115.15, 123.33,
127.90, 129.76, 130.25, 135.56, 137.41, 160.74, 166.3, 172.22;
ESI-MS m/z (%) 490 (MNH4+); Anal. (C23H24N2O7S) C, H, N.
3,3-Diethyl-N-[1-ethoxycarbonyl-1-(2-methoxycarbo-
nylphenyl)amino-carbonyloxy]-methyl-4-(phenylsulfo-
nyl)azetidin-2-one (5f). Major diastereomer; recrystallized
from diethyl ether-light petroleum; white crystals (11%); mp
117-119 °C; νmax (film) 3264, 2974, 1790, 1747, 1693, 1532,
1269, 1205 cm-1; δ 1H NMR 1.05 (3H, t, J ) 7.3 Hz), 1.09 (3H,
t, J ) 7.4 Hz), 1.13 (3H, t, J ) 7.2 Hz), 1.79 (1H, dq, J ) 7.3,
14.7 Hz), 1.98 (1H, dq, J ) 7.3, 14.6 Hz), 2.10 (1H, dq, J )
7.3, 14.9 Hz), 2.48 (1H, dq, J ) 7.3, 14.8 Hz), 3.96 (3H, s),
3.86 (1H, dq, J ) 7.2, 10.6 Hz), 4.01 (1H, dq, J ) 7.2, 10.5
Hz), 4.93 (1H, s), 6.45 (1H, s), 7.05-8.41 (9H, m), 10.85
(1H, brs); δ 13C NMR 8.66, 8.91, 13.87, 20.59, 24.59, 52.45,
62.79, 68.48, 70.55, 77.42, 114.93, 119.03, 122.32, 129.28,
129.40, 130.90, 134.25, 134.74, 138.36, 140.87, 150.78, 164.10,
168.65, 170.97; ESI-MS m/z 564 (MNH4+); Anal. (C26H30N2O9S)
C, H, N.
4-(1,3-Benzoxazol-2-ylthio)-3,3-diethyl-N-[3-(4-meth-
ylphenyl)oxazolidin-2,4-dion-5-yl]azetidin-2-one (6b). The
two diastereomers (70:30 mixture) coeluted in two consecutive
columns; light yellow crystals (63%); mp 89-96 °C; νmax (film)
2973, 1831, 1788, 1758, 1408, 1356 cm-1; δ 1H NMR 0.83 and
0.83 (3H, 2 × t, J ) 7.5 Hz), 1.15 and 1.20 (3H, 2 × t, J ) 7.3
Hz), 1.42-2.07 (4H, m), 2.33 and 2.35 (3H, 2 × s), 5.59 and
5.76 (1H, 2 × s), 6.50 and 6.53 (1H, 2 × s), 7.16-7.84 (8H, m);
δ
13C NMR 7.94, 8.02, 8.62, 8.66, 20.65, 21.16, 21.34, 24.11,
24.46, 67.50, 67.94, 72.40, 72.75, 78.00, 78.62, 110.71, 110.79,
112.50, 112.70, 124.99, 125.04, 125.31, 125.65, 125.74, 127.34,
127.44, 128.20, 130.13, 130.18, 130.22, 139.90, 146.71, 146.85,
152.15, 164.96, 171.49, 179.96; ESI-MS m/z 466 (MH+); Anal.
(C24H23N3O5S) C, H, N.
N-[(1-Benzylaminocarbonyloxy-1-ethoxycarbonyl)-
methyl]-3,3-diethyl-4-phenoxyazetidin-2-one (5c). Only
one diastereomer was isolated; colorless oil (17%); νmax (film)
3365, 2971, 1781, 1763, 1736, 1493, 1224 cm-1; δ 1H NMR
1.00 (3H, t, J ) 7.6 Hz), 1.03 (3H, t, J ) 7.6 Hz), 1.26 (3H, t,
J ) 7.2 Hz), 1.68 (1H, dq, J ) 7.3, 14.7 Hz), 1.75-1.85 (2H,
m), 1.92 (1H, dq, J ) 7.4, 14.9 Hz), 4.13-4.44 (2H, m), 4.34
(1H, d, J ) 15 Hz), 4.41 (1H, d, J ) 15 Hz), 5.23 (1H, t, J )
5.6 Hz), 5.50 (1H, s), 6.33 (1H, s), 6.86 (2H, d, J ) 7.9 Hz),
7.03 (1H, t, J ) 7.4 Hz), 7.26-7.36 (7H, m); δ 13C NMR 8.84,
9.24, 14.12, 22.21, 23.97, 45.38, 62.61, 65.44, 71.73, 87.09,
116.74, 122.97, 127.81, 127.90, 128.93, 129.94, 158.83, 154.09,
165.19, 170.85; ESI-MS m/z (%) 455 (MH)+; Anal. (C25H30N2O6)
C, H, N.
N-(3-Benzyloxazolidin-2,4-dion-5-yl)-3,3-diethyl-4-phe-
noxyazetidin-2-one (6c). Major diastereomer; white crystals
(26%); mp 88-90 °C; νmax (film) 2971, 1824, 1783, 1752, 1440,
1376, 1344, 1223 cm-1; δ 1H NMR 1.03 (3H, t, J ) 7.5 Hz),
1.71 (1H, dq, J ) 7.3, 14.8 Hz), 1.78-1.88 (2H, m), 1.95 (1H,
dq, J ) 7.4, 15 Hz), 4.43 (2H, s), 5.54 (1H, s), 5.88 (1H, s),
6.83 (2H, d, J ) 7.5 Hz), 7.06 (1H, t, J ) 7.5 Hz), 7.16-7.34
(7H, m); δ 13C NMR 8.79, 9.13, 21.76, 23.95, 44.36, 65.95, 77.65,
87.06, 116.98, 123.63, 128.75, 128.83, 129.05, 130.28, 134.03,
156.12, 153.38, 166.06, 171.44; ESI-MS m/z 409 (MH+). Anal.
(C23H24N2O5) C, H, N. Minor diastereomer; light yellow oil
(17%); νmax (film) 2970, 1834, 1781, 1753, 1492, 1376, 1345,
Minor diastereomer; white crystals (8%); mp 115-117 °C;
νmax (film) 3263, 2973, 1789, 1748, 1531, 1452, 1268, 1206 cm-1
;
1
δ H NMR 0.89 (3H, t, J ) 7.3 Hz), 1.05 (3H, t, J ) 7.5 Hz),
1.26 (3H, t, J ) 7.2 Hz), 1.69 (1H, dq, J ) 7.5, 15.1 Hz), 1.81-
1.97 (2H, m), 2.38 (1H, dq, J ) 7.3, 14.7 Hz), 3.95 (3H, s), 4.24
(2H, 2 × dq, J ) 7.2, 9.9 Hz), 4.63 (1H, s), 5.66 (1H, s), 7.05-
8.33 (9H, m), 10.75 (1H, brs); δ 13C NMR 8.35, 8.73, 13.90,
19.98, 24.39, 52.43, 62.92, 67.30, 74.01, 78.51, 115.19, 118.97,
122.35, 128.81, 129.61, 130.95, 134.57, 134.62, 138.05, 140.69,
150.77, 164.14, 168.25, 170.97; ESI-MS m/z 564 (MNH4+);
Anal. (C26H30N2O9S) C, H, N.
1
1223 cm-1; δ H NMR 1.02 (3H, t, J ) 7.5 Hz), 1.03 (3H, t, J
) 7.5 Hz), 1.72 (1H, dq, J ) 7.4, 14.8 Hz), 1.78-1.88 (2H,
m), 1.96 (1H, dq, J ) 7.4, 15.1 Hz), 4.36 (1H, d, J ) 14.5 Hz),
4.44 (1H, d, J ) 14.5 Hz), 5.57 (1H, s), 5.88 (1H, s), 6.82
(2H, d, J ) 7.5 Hz), 7.05 (1H, t, J ) 7.5 Hz), 7.18-7.31 (7H,
m); δ 13C NMR 8.56, 8.88, 21.66, 23.64, 44.00, 65.47, 76.92,
87.74, 116.14, 116.75, 123.24, 128.73, 128.83, 130.07, 133.83,
156.05, 153.34, 166.06, 171.23; ESI-MS m/z 409 (MH+); Anal.
(C23H24N2O5) C, H, N.
HLE Inhibition Studies. Assays were performed at 25 °C
in 0.1 M pH 7.2 HEPES buffer. The substrate used was MeO-
Suc-Ala-Ala-Pro-Val-p-NA, and stock solutions were made up
in DMSO. Enzyme activity was monitored by following the
appearance of the p-nitroaniline product at 410 nm. Reactions
were started by addition of inhibitor stock solution in DMSO
(20 µL) to the incubation solution containing HLE (10 µL from
a 20 µM stock solution in pH 5.0 acetate buffer) and HEPES
buffer (970 µL). At various time intervals the HLE activity
was assayed by diluting 100 µL aliquots of the incubation
solution into 880 µL of 0.1 M pH 7.2 HEPES buffer and 20 µL
of the substrate 50 mM stock solution. Initial rates were
monitored over a period of 60 s, and the gradients of the slopes
obtained were used as a measure of enzyme activity. In all
cases, assays of control incubations were performed at the
same time as inhibitor incubations. Pseudo-first-order rate
constants of inactivation, kobs, were obtained from plots of %
activity (v/v0) against time, where v is the initial rate at time
t and v0 is the initial rate of the control incubation. In the same
experimental conditions, the Km value for the hydrolysis of
N-(3-Benzyloxazolidin-2,4-dion-5-yl)-3,3-diethyl-4-
(phenylsulfonyl)azetidin-2-one (6d). Major diastereomer;
white crystals (27%); mp 54-56 °C; νmax (film) 2973, 1827,
1789, 1751, 1443, 1334, 1155 cm-1; δ 1H NMR 0.70 (3H, t, J )
7.4 Hz), 0.89 (3H, t, J ) 7.5 Hz), 1.49 (1H, dq, J ) 7.3, 15 Hz),
1.59 (1H, dq, J ) 7.4, 14.9 Hz), 1.78 (1H, dq, J ) 7.4, 14.9
Hz), 2.04 (1H, dq, J ) 7.3, 14.8 Hz), 4.54 (1H, d, J ) 14.5 Hz),
4.55 (1H, s), 4.60 (1H, d, J ) 14.3 Hz), 5.60 (1H, s), 7.23-7.85
(10H, m); δ 13C NMR 8.70, 9.28, 20.31, 25.33, 45.15, 68.13,
78.05, 78.37, 129.23, 129.48, 129.68, 129.88, 130.37, 134.47,
135.59, 137.37, 153.80, 166.46, 172.11; ESI-MS m/z 475
(MNH4+); Anal. (C23H24N2O6S) C, H, N.
Minor diastereomer; white crystals (13%); mp 170-173 °C;
νmax (film) 2973, 1826, 1788, 1752, 1443, 1333, 1155 cm-1; δ
1H NMR 0.76 (3H, t, J ) 7.4 Hz), 0.93 (3H, t, J ) 7.5 Hz),
1.62 (1H, dq, J ) 7.3, 14.8 Hz), 1.77 (2H, m), 2.24 (1H, dq, J
) 7.3, 14.6 Hz), 4.53 (1H, d, J ) 15.4 Hz), 4.64 (1H, d, J )
15.2 Hz), 4.93 (1H, s), 5.17 (1H, s), 7.25-7.96 (10H, m); δ 13C
NMR (CD3CN) 8.06, 8.49, 20.14, 24.77, 44.19, 67.50, 77.36,