C. Loncle et al. / Tetrahedron 63 (2007) 12968–12974
12973
5
3
3
0.41, 47.84, 46.06, 42.69, 41.76, 39.45, 39.03, 37.91, 36.82,
6.68, 36.10, 35.81, 35.71, 32.79, 31.79, 31.32, 29.94, 27.96,
4.60, 23.77, 23.60, 22.76, 22.49, 21.11, 18.59, 11.70, 11.65.
d¼71.06, 56.15, 54.67, 51.77, 50.65, 47.96, 46.09, 42.71,
42.10, 39.49, 39.12, 36.84, 36.74, 36.15, 35.86, 35.75,
33.46, 31.94, 31.50, 30.27, 29.80, 29.54, 29.48, 29.40,
28.11, 28.00, 27.40, 26.80, 23.80, 22.80, 22.54, 21.16,
+
+
MS (ESI ) m/z 489.5 (100%, [M+H] ). C H N O calcd C
3
7
3 64 2
+
8.51, H 12.78, N 5.55; found C 78.51, H 12.79, N 6.10.
18.63, 11.74, 11.69. MS (ESI ) m/z 559.5 (100%,
[M+H] ). C H N O calcd C 79.37, H 12.97, N 4.87; found
C 79.37, H 12.92, N 4.82.
+
3
8 74 2
4
6
.4.7. 7b-(1,6-Diaminohexane)-3b-hydroxycholestane
g. Purification by column chromatography (silica gel;
CH Cl /MeOH/NH OH (32%), 7:3:1) afforded a pale yel-
2
low solid in 33% yield. H NMR (300 MHz, CDCl3):
4.4.12. 7b-(1,12-Diaminododecane)-3b-hydroxycholes-
tane 6l. Purification by column chromatography (silica
gel; CH Cl /MeOH/NH OH(32%), 7:3:1) afforded a pale
2
4
1
d¼4.70–4.18 (m, 1H), 3.62–3.50 (m, 1H), 2.72–0.62 (m,
2
2
4
1
3
1
6
5
3
2
2
0H). C NMR (75 MHz, CDCl ): d¼71.06, 56.13, 54.85,
yellow viscous oil in 44% yield. H NMR (300 MHz,
CDCl ): d¼2.62–0.57 (m, 74H). C NMR (75 MHz,
3
1
3
0.54, 47.85, 46.06, 42.71, 41.75, 39.46, 39.03, 36.70,
6.12, 35.81, 35.73, 32.74, 31.75, 31.33, 29.97, 28.87,
8.09, 27.98, 27.16, 26.60, 23.82, 23.59, 22.78, 22.52,
1.43, 21.13, 18.60, 11.72, 11.68. MS (ESI ) m/z 503.6
100%, [M+H] ). C H N O calcd C 78.70, H 12.82, N
3
3
CDCl ): d¼70.55, 56.02, 54.54, 50.54, 49.64, 47.96,
3
45.99, 42.58, 42.02, 39.38, 39.00, 37.96, 36.76, 36.63,
36.02, 35.75, 35.63, 33.58, 31.81, 31.40, 30.21, 29.44,
29.34, 27.99, 27.87, 27.38, 26.73, 23.67, 23.50, 22.68,
+
+
(
4 66 2
+
5
.40; found C 78.71, H 12.84, N 5.44.
22.42, 21.03, 18.51, 11.62, 11.56. MS (ESI ) m/z 587.5
(100%, [M+H] ). C H N O calcd C 79.67, H 13.04, N
4.65; found C 79.64, H 13.12, N 4.59.
+
4
0 78 2
4
.4.8. 7b-(1,7-Diaminoheptane)-3b-hydroxycholestane
h. Purification by column chromatography (silica gel;
6
CH Cl /MeOH/NH OH (32%), 7:3:1) afforded a pale yel-
low solid in 37% yield. H NMR (300 MHz, CDCl ):
3
4.4.13. 7b-(Spermino)-3b-hydroxycholestane 6m. Purifi-
cation by column chromatography (silica gel; CH Cl /
2
2
4
1
2
2
1
3
d¼3.62–3.55 (m, 1H), 2.67–0.61 (m, 63H). C NMR
MeOH/NH OH (32%), 7:3:1) afforded a pale yellow solid
4
1
(
75 MHz, CDCl ): d¼70.97, 66.13, 59.66, 50.66, 47.89,
in 45% yield. H NMR (300 MHz, CDCl ): d¼3.51–0.59
3
3
1
3
4
3
2
2
6.14, 42.67, 42.14, 39.46, 39.09, 38.12, 36.90, 36.73,
6.11, 35.83, 35.71, 33.56, 31.96, 31.39, 30.32, 29.29,
8.07, 27.87, 27.37, 26.90, 26.79, 23.76, 23.57, 22.77,
(m, 72H). C NMR (75 MHz, CDCl ): d¼70.38, 56.07,
3
54.76, 50.72, 49.99, 49.81, 48.87, 47.73, 46.71, 46.15,
42.51, 40.36, 39.32, 38.93, 38.05, 36.90, 36.81, 35.99,
35.74, 35.57, 33.42, 31.76, 31.34, 30.17, 27.93, 27.83,
23.66, 23.45, 22.65, 22.39, 21.01, 18.48, 11.60, 11.56.
+
2.51, 21.13, 18.60, 11.72, 11.68. MS (ESI ) m/z 517.5
+
(
100%, [M+H] ). C H N O calcd C 78.86, H 12.86, N
3
5 68 2
+
+
5
.26; found C 78.87, H 12.92, N 5.26.
MS (ESI ) m/z 589.7 (100%, [M+H] ). C H N O
38 76 4
calcd C 75.43, H 12.66, N 9.26; found C 75.39, H 12.59,
N 9.20.
4
.4.9. 7b-(1,8-Diaminooctane)-3b-hydroxycholestane 6i.
Purification by column chromatography (silica gel;
CH Cl /MeOH/NH OH (32%), 7:3:1) afforded a pale yel-
low solid in 34% yield. H NMR (300 MHz, CDCl ):
4.5. Determination of minimal inhibitory concentrations
2
2
4
1
3
1
3
d¼3.62–3.55 (m, 1H), 2.68–0.61 (m, 65H). C NMR
Antimicrobial activity of the compounds was studied by
determination of minimal inhibitory concentration (MIC)
according to the NCCLS guidelines M7-A2 using the micro-
broth dilution methods. All the strains were issued from the
Institut Pasteur collection (Paris). The yeast was grown
(
75 MHz, CDCl ): d¼70.96, 56.12, 54.68, 50.61, 50.34,
3
4
3
2
2
5
1
7.76, 46.09, 42.68, 41.96, 39.46, 39.08, 38.09, 36.86,
6.69, 36.12, 35.83, 35.71, 33.32, 31.89, 31.35, 30.15,
9.45, 29.27, 28.07, 27.96, 27.27, 26.78, 23.77, 23.58,
+
ꢁ
ꢁ
2.76, 22.51, 21.13, 18.60, 11.71, 11.67. MS (ESI ) m/z
31.5 (100%, [M+H] ). C H N O calcd C 79.05, H
2.90, N 5.12; found C 79.25, H 12.62, N 5.14.
overnight at 28 C (S. cerevisiae CIP 28383) or 37 C
(C. albicans CIP 1180–79) in YPD broth. The bacterial
strains were grown on trypticase soy agar (Becton Dickin-
+
3
6 70 2
ꢁ
son) at 37 C for 24 h (E. coli CIP 54127, S. aureus CIP
4
6
.4.10. 7b-(1,10-Diaminononane)-3b-hydroxycholestane
j. Purification by column chromatography (silica gel;
4.83, E. faecalis CIP103015) in LB broth for E. coli and S.
aureus or BHI broth for S. faecalis. Inocula were prepared
in TCE (tryptone 0.1%, NaCl 8%, wt/vol) by adjusting the
turbidity at 623 nm to obtain 1–3ꢂ10 CFU/mL.
CH Cl /MeOH/NH OH(32%), 7:3:1) afforded a pale yellow
2
viscous oil in 39% yield. H NMR (300 MHz, CDCl ):
3
2
4
1
5
1
3
d¼3.63–3.56 (m, 1H), 2.70–0.62 (m, 67H). C NMR
(
75 MHz, CDCl ): d¼70.86, 56.15, 54.70, 50.64, 47.89,
Antimicrobial activities of the compounds were determined
by using a broth microdilution method performed in sterile
96-well microplates. All compounds were solubilized in
methanol at a concentration of 5 mg/mL and were trans-
ferred to each microplate well (in all cases concentrations
of the desired molecules in methanol do not exceed 2% of
the total proportion), in order to obtain a 2-fold serial dilu-
tion in 100 mL of broth and 100 mL of inocula containing
3
4
3
2
2
6.09, 42.69, 41.79, 39.48, 39.11, 38.10, 36.86, 36.74,
6.14, 35.84, 35.73, 32.90, 31.98, 31.45, 30.25, 29.52,
9.33, 28.09, 27.98, 27.33, 26.76, 23.79, 23.61, 22.79,
+
2.52, 21.14, 18.62, 11.73, 11.68. MS (ESI ) m/z 545.4
+
(
100%, [M+H] ). C H N O calcd C 79.22, H 12.94, N
3
7 72 2
4
.99; found C 79.25, H 12.92, N 5.02.
5
4
.4.11. 7b-(1,10-Diaminodecane)-3b-hydroxycholestane
k. Purification by column chromatography (silica gel;
2–6ꢂ10 CFU of each bacteria and yeast were added to
6
CH Cl /MeOH/NH OH (32%), 7:3:1) afforded a pale
each well. A number of wells were reserved for positive con-
trols, inoculum viability, and solvent effect. After 24 or 48 h
incubation, growth was assayed by absorbance measurement
at 623 nm with an IEMS Labsystem automatic plate reader.
2
2
4
1
yellow solid in 25% yield. H NMR (300 MHz, CDCl ):
3
d¼2.69–0.63 (m, 70H). C NMR (75 MHz, CDCl3):
1
3