ACS Chemical Neuroscience
Research Article
(
s, 3H), 0.77 (d, J = 6.6 Hz, 3H); 13C NMR (126 MHz, CDCl ) δ
3
1
4
2
74.73, 169.59, 140.69, 121.29, 71.57, 56.54, 51.32, 50.87, 49.91,
5.54, 42.12, 39.32, 37.15, 36.44, 31.91, 31.79, 31.49, 28.59, 25.91,
5.00, 24.73, 20.88, 19.65, 19.31, 18.35, 14.04; MS (ESI, m/z) = 509.5
+
[
M + 23] ; HRMS (ESI): calcd for C H N O Na, 509.3719; found,
30
50
2
3
5
09.3709.
(3S,10R,13S)-N-(+)-(1-(tert-Butylamino)-3-methyl-1-oxobutan-2-
yl)-3-hydroxy-N,10,13-trimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-17-carboxamide
10b). [α]2 = +78.750 (c 0.160, acetone-d ); H NMR (400 MHz,
0
1
(
D
6
acetone-d ) δ 6.15 (s, 1H), 5.35 (d, J = 5.2 Hz, 1H), 4.42 (d, J = 10.7
6
Hz, 1H), 3.58−3.46 (m, 1H), 2.98 (s, 3H), 2.77 (t, J = 8.9 Hz, 1H),
2
3
.37−2.15 (m, 5H), 1.28 (s, 10H), 1.00 (s, 3H), 0.95 (d, J = 6.4 Hz,
H), 0.84 (d, J = 6.6 Hz, 3H), 0.72 (s, 3H); 13C NMR (126 MHz,
CDCl ) δ 175.07, 169.57, 140.66, 121.32, 71.57, 56.63, 51.46, 50.88,
3
4
9.97, 45.35, 42.14, 38.93, 37.17, 36.44, 31.90, 31.77, 31.51, 28.57,
2
5.33, 25.29, 24.67, 21.00, 19.67, 19.29, 18.83, 13.68; MS (ESI, m/z) =
+
5
09.5 [M + 23] ; HRMS (ESI): calcd for C H N O Na, 509.3719;
30
50
2
3
found, 509.3706.
(
3S,10R,13S)-N-Benzyl-N-(−)-(1-(tert-butylamino)-3-methyl-1-ox-
obutan-2-yl)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,-
4,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-
1
2
0
1
carboxamide (11a). [α] = −88.333 (c 0.180, CHCl ); H NMR
D
3
(
400 MHz, CDCl ) δ 7.40−7.09 (m, 6H), 5.32 (s, 1H), 4.95 (d, J =
3
1
2
4
7.9 Hz, 1H), 4.64 (d, J = 18.5 Hz, 1H), 4.22 (s, 1H), 3.49 (s, 1H),
.63 (t, J = 9.0 Hz, 1H), 2.34−2.11 (m, 3H), 1.30 (s, 9H), 1.00 (s,
H), 0.90 (d, J = 6.4 Hz, 3H), 0.86 (s, 3H), 0.71 (d, J = 6.0 Hz, 3H);
13
C NMR (126 MHz, CDCl ) δ 176.54, 170.21, 140.77, 138.03,
3
1
4
2
28.55, 127.20, 126.39, 121.37, 71.64, 56.40, 52.58, 51.00, 49.92,
5.73, 42.21, 38.92, 37.23, 36.54, 31.90, 31.81, 31.58, 28.64, 27.06,
6.70, 24.80, 20.86, 19.93, 19.86, 19.40, 14.19; MS (EI, m/z) = 562
+
(M ); HRMS (EI): calcd for C H N O , 562.4134; found, 562.4131.
36
54
2
3
(
3S,10R,13S)-N-Benzyl-N-(1-(tert-butylamino)-3-methyl-1-oxobu-
tan-2-yl)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,-
4,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-
1
2
0
1
carboxamide (11b). [α] = −28.095 (c 0.210, CHCl ); H NMR
D
3
(
400 MHz, CDCl ) δ 7.32−7.13 (m, 3H), 7.05 (d, J = 7.6 Hz, 2H),
3
6
1
0
.28 (s, 1H), 5.31 (s, 1H), 4.73 (dd, J = 54.3, 17.3 Hz, 2H), 4.47 (s,
H), 3.50 (s, 1H), 2.53 (t, J = 8.9 Hz, 1H), 1.20 (s, 9H), 1.00 (s, 3H),
1
3
.94 (d, J = 6.3 Hz, 3H), 0.88 (d, J = 6.3 Hz, 3H), 0.77 (s, 3H);
C
Figure 4. Compound 18b inhibits LPS-induced NO production in
microglial cells. Microglial cells were preincubated with compound
NMR (126 MHz, CDCl ) δ 176.31, 169.05, 140.69, 138.24, 128.44,
3
1
3
2
26.89, 125.92, 121.45, 71.68, 56.51, 52.83, 51.14, 49.96, 45.70, 42.23,
1
8b for 30 min prior to LPS (0.1 μg/mL) or LPS (0.1 μg/mL)/IFN-γ
9.16, 37.27, 36.54, 31.93, 31.79, 31.61, 28.54, 26.99, 26.36, 24.73,
(
(
50 U/mL) stimulation: BV-2 microglia (A), primary microglia culture
B) HAPI microglia (C), and primary astrocytes (D). The nitrite levels
+
1.10, 19.74, 19.39, 19.17, 14.07; MS (EI, m/z) = 562 (M ); HRMS
(
EI): calcd for C H N O , 562.4134; found, 562.4142.
in the cell culture medium were detected using Griess reagents (upper
half of each panel). MTT assay was performed to assess cell viability
36 54 2 3
(
3S,10R,13S)-N-(+)-(1-(tert-Butylamino)-1-oxo-3-phenylpropan-
2
1
-yl)-3-hydroxy-N,10,13-trimethyl-2,3,4,7,8,9,10,11,12,13,14,-
5,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-car-
(
lower half of each panel). Data shown are the mean ± SD for each
group (n = 3) and are representative of four independent experiments.
Asterisks denote a significant difference from groups receiving LPS or
LPS/IFN-γ treatment alone (*, p < 0.05; **, p < 0.01).
20
1
boxamide (12a). [α]D = +79.335 (c 0.155, CHCl ); H NMR (300
3
MHz, CDCl ) δ 7.29−7.06 (m, 5H), 6.29 (s, 1H), 5.40 (dd, J = 11.0,
3
5
1
(
.6 Hz, 1H), 5.34−5.27 (m, 1H), 3.60−3.40 (m, 1H), 3.17 (dd, J =
5.1, 5.7 Hz, 1H), 2.87 (s, 3H), 2.58 (t, J = 8.8 Hz, 1H), 2.34−2.09
m, 3H), 1.29 (s, 10H), 0.97 (s, 3H), 0.37 (s, 3H); 13C NMR (126
(
3S,10R,13S)-N-(R)-(1-(tert-Butylamino)-3-methyl-1-oxobutan-2-
MHz, CDCl ) δ 175.28, 169.94, 140.78, 137.43, 129.09, 128.51,
yl)-3-hydroxy-N,10,13-trimethyl-2,3,4,7,8,9,10,11,12,13,14,15-do-
3
1
3
26.41, 121.38, 71.70, 57.80, 56.69, 51.48, 50.98, 50.01, 45.15, 42.23,
decahydro-1H-cyclopenta[a]phenanthrene-17-carboxamide (9b).
α] = −249.375 (c 0.160, CHCl ); H NMR (400 MHz, CDCl )
δ 6.26 (s, 1H), 5.87 (s, 1H), 5.38 (s, 1H), 4.42 (d, J = 10.6 Hz, 1H),
20
1
7.75, 37.22, 36.48, 33.52, 31.97, 31.86, 31.59, 31.39, 28.71, 25.04,
[
D
3
3
+
24.65, 20.73, 19.36, 12.98; MS (EI, m/z) = 534 (M ); HRMS (EI):
3
3
.62−3.43 (m, 1H), 2.96 (s, 3H), 1.30 (s, 9H), 1.12 (s, 3H), 1.05 (s,
calcd for C34H N O , 534.3821; found, 534.3825.
50 2 3
13
H), 0.97 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.4 Hz, 3H); C NMR
(3S,10R,13S)-N-(−)-(1-(tert-Butylamino)-1-oxo-3-phenylpropan-
-yl)-3-hydroxy-N,10,13-trimethyl-2,3,4,7,8,9,10,11,12,13,-
4,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-
2
1
(
126 MHz, CDCl ) δ 170.87, 169.22, 148.50, 141.23, 132.58, 121.16,
3
7
3
1
4
1.65, 62.70, 56.63, 50.97, 50.54, 48.70, 42.28, 37.21, 36.75, 34.07,
2.78, 32.42, 31.59, 30.20, 28.68, 25.19, 20.65, 19.84, 19.35, 18.54,
20
1
carboxamide (12b). [α] = −108.889 (c 0.090, CHCl ); H NMR
D
3
+
(300 MHz, CDCl ) δ 7.32−7.11 (m, 5H), 6.03 (s, 1H), 5.47−5.21 (m,
3
6.71; MS (EI, m/z) = 484 (M ); HRMS (EI): calcd for C H N O ,
30
48
2
3
2
2
4
H), 3.59−3.40 (m, 1H), 3.28 (dd, J = 14.5, 8.1 Hz, 1H), 2.96 (s, 3H),
.89 (dd, J = 14.1, 9.3 Hz, 1H), 2.60 (t, J = 9.3 Hz, 1H), 2.37−1.92 (m,
H), 1.28 (s, 9H), 1.01 (s, 3H), 0.78 (s, 3H); 13C NMR (126 MHz,
84.3665; found, 484.3668.
(
3S,10R,13S)-N-(−)-(1-(tert-Butylamino)-3-methyl-1-oxobutan-2-
yl)-3-hydroxy-N,10,13-trimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-17-carboxamide
CDCl ) δ 174.72, 169.62, 140.75, 137.51, 128.95, 128.36, 126.41,
3
(
10a). [α]2 = −130.588 (c 0.085, CHCl ); H NMR (400 MHz,
CDCl ) δ 6.03 (s, 1H), 5.35 (d, J = 5.0 Hz, 1H), 4.52 (d, J = 10.9 Hz,
0
D
1
121.42, 71.69, 57.10, 56.64, 51.30, 51.01, 50.03, 45.51, 42.23, 39.44,
37.26, 36.54, 33.37, 31.98, 31.88, 31.60, 30.96, 28.68, 25.94, 24.76,
3
3
+
1
H), 3.59−3.45 (m, 1H), 2.99 (s, 3H), 2.73 (t, J = 8.8 Hz, 1H), 2.35−
21.01, 19.42, 14.17; MS (EI, m/z) = 534 (M ); HRMS (EI): calcd for
2
.13 (m, 5H), 1.30 (s, 9H), 1.01 (s, 3H), 0.94 (d, J = 6.4 Hz, 3H), 0.78
C H N O , 534.3821; found, 534.3827.
34
50
2
3
F
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX