W. Xie et al. / Bioorg. Med. Chem. 8 (2000) 699±706
705
� 1
Reaction of 1 with dimethyl acetylenedicarboxylate to
yield (8). To compound 1 (50 mg, 0.13 mmol) dissolved
in 5 mL methanol, was added 1.5 equiv of dimethyl
acetylenedicarboxylate with stirring at rt overnight.
Concentration and washing the residue with petroleum
ether:ethyl acetate (3:1) gave compound 8 as a light
5-dien-20-one:14 IR (n cm ) 3350 (NH), 1690 (CO);
1
H NMR (CDCl ): d 0.78 (s, 3H), 0.98 (s, 3H), 0.98 (s,
3
3H), 2.02 (s, 3H), 2.44 (m, 2H), 5.96 (m, 1H), 6.18 (m,
+
1H); FABMS m/z (relative intensity): 300 (M +1,100),
+
285 (M +1-CH , 20), 243 (15). 6-Aza-pregnan-3, 5-
3
dien-20-one upon reduction with 10% Pd/C in glacial
14
1
yellow gum (60 mg, 0.11 mmol, 85%). H NMR
acetic acid yielded 6-aza-pregnan-20-one (14) : IR (n
�
1
1
(
J=5.4 Hz, 3H), 0.94 (s, 3H), 2.61 (m, 1H), 2.96 (m, 1H),
CDCl ): d 0.68 (s, 3H), 0.85 (d, J=5.4 Hz, 6H), 0.89 (d,
cm ) 3350 (NH), 1701 (CO); H NMR (CDCl ): d
3
3
0.77 (s, 3H), 0.90 (s, 3H), 2.04 (s, 3H), 2.12 (t, J=12.0
Hz, 1H), 2.44 ( t, J=10.2 Hz, 1H), 2.98 (dd, J=2.2, 6.9
2
4
2
.96 (m, 1H), 3.47 (m, 2H), 3.62 (s, 3H), 3.86 (s, 3H),
+
.94 (s, 1H); EIMS m/z (relative intensity): 531 (M ,
13
Hz, 1H); C NMR: 12.30, 13.77, 20.68, 21.72. 23.24,
24.15, 25.84, 28.64, 31.83, 35.99, 36.67, 36.81, 39.22,
44.67, 52.41, 54.22, 54.29, 63.86, 65.81, 210.10; EIMS
+
5), 516 (M -CH ), 499 (95), 412 (100); anal. (C H
3
32 54
.
NO 0.5H O) C, H, N.
2
5
+
+
m/z (relative intensity): 303 (M , 90), 288 (M -CH ,
3
Reaction of 1 with diethyl acetylenedicarboxylate to
yield (9). Reaction of 1 with diethyl acetylenedicarboxyl-
40), 246 (100); HRMS calcd for C H NO 303.2562,
33
found 303.2541.
20
ate as described above gave 9 as a yellow gum (80%).
1
H NMR (CDCl ): d 0.65 (s, 3H), 0.83 (d, J=5.4 Hz,
Similarly, as for the described preparation of 3b-hy-
droxy-6-aza-cholestane, 1 gave initially the acyl azide
which upon Curtius rearrangement in benzene, then
treatment with acid in acetone and ®nally hydrogena-
tion (10% Pt/C) in glacial AcOH gave an epimeric
3
6H), 0.88 (d, J=5.4 Hz, 3H), 0.93 (s, 3H), 1.21 (t,
J=7.2 Hz, 3H), 1.33 (t, J=7.2 Hz, 3H), 2.54 (dd,
J=12.6 Hz, J=10.8 Hz, 1H), 2.96 (m, 1H), 3.46 (m,
2H), 4.04 (q, 2H), 4.30 (q, 2H), 4.92 (s, 1H); FABMS
m/z (relative intensity): 560 (M +1, 50), 390 (100);
+
mixture (5:1) of 3b, 20-dihydroxy-6-aza-pregnane (15):
+
1
HRMS calcd for C H NO (M +1) 560.4315, found
3
H NMR (CDCl ): d 0.81 (s, 3H), 0.89 (s, 3H), 1.04 (d,
4
58
5
3
.
60.4253; anal. (C H NO 0.6H O) C, H, N.
5
J=6.0 Hz, 3H), 2.18 (m, 1H), 2.80 (br, 1H), 2.88 (dd,
J=2.2, 6.9 Hz, 1H), 3.60 (m, 2H); CIMS m/z (relative
3
4
58
5
2
+
+
Reaction of 3ꢀ-hydroxy-6-aza-cholestane (1) with cyclic
anhydrides to give amidoacids 10±13. General proce-
dure: A solution of 3b-hydroxy-6-aza-cholestane, 1 (50
mg) and 1.0 molar equiv of the anhydride in 5 mL DMF
was stirred at room temperature overnight. The ami-
doacid products were obtained by evaporation of the
solvent followed by washing the residue with ether or
chloroform and ®ltration. In all cases the products were
intensity): 322 (M +1, 10), 304 (M +1-H O, 80), 288
2
(100); HRMS calcd for C H NO 321.2668, found
35
20
2
321.2648.
Biological methods
The test cells were obtained from American Type Cul-
ture Collection (ATCC, Rockville, MD, USA). All the
cells were maintained in Dulbecco-modi®ed Eagle's
medium (DMEM; Sigma) supplemented with 10% fetal
bovine serum (FBS; Gibco, Grand Island, NY, USA).
obtained as amorphous solids. Compound 13 from
1
phthalic anhydride: H NMR (CDCl ): d 0.57 (s, 3H),
3
0.84 (d, J=6.6 Hz, 9 H), 0.98 (s, 3H), 3.25 (m, 1H), 4.97
(
FABMS m/z (relative intensity): 538 (M +1, 100), 390
m, 1H), 7.49 (m, 2H), 8.01 (m, 2H), 8.88 (br, 1H);
+
PI-PLCg in vitro inhibition assay was measured using
1,17
bovine brain PI-PLCg as previously described.
Anti-
proliferative assay was measured over 7 days as pre-
(
5
40); HRMS calcd for C H NO 538.3896, found
3
4
52
4
viously described.1
,17
.
38.3895; anal. (C H NO 0.6CHCl ) C, H, N. Com-
34 52 4 3
1
pound 10 from succinic anhydride: H NMR (CDCl ): d
3
0
3
3
.65 (s, 3H), 0.83 (d, J=6.6 Hz, 6H), 0.86 (d, J=6.6 Hz,
The in vitro cytotoxicity assays were carried out at the
National Cancer Institute. Details of the assay proce-
H), 0.89 (s, 3H), 2.96 (d, J=15 Hz, 2H), 3.40 (m, 1H),
+
.76 (m, 1H); FABMS m/z (relative intensity): 490 (M
20
dures have been reported previously. In vitro anti-
tumor activity was tested against MCF-7 human breast
cancer xenografts in the scid mouse. Groups of 8 mice
+
+
C H NO (M +1) 490.3856, found 490.3867. Com-
pound 11 from maleic anhydride: H NMR (CDCl ): d
1, 100), 472 (M -H O), 390 (25); HRMS calcd for
+
2
3
0
52
4
1
6
were transplanted sc with 2Â10 MCF-7 human breast
3
0.65 (s, 3H), 0.83 (d, J=6.6 Hz, 9 H), 0.87 (s, 3H), 2.70
cancer cells. Treatment with compound 1 was begun ip
on day 4 and continued daily for 14 days at 1 and 2 mg/
kg. Control mice received vehicle alone. All the mice
receiving 2 mg/kg compound 1 died by day 12. In
Figure 6 the upper panel shows tumor volume and the
lower panel body weight. Each point is the mean of 8
mice and the bars are S.E. *Indicates that it is sig-
ni®cantly dierent from the vehicle treated control.
Details of the determination procedures have been
(
(
br, 2H), 3.30 (m, 1H), 3.76 (m, 1H), 6.28 (br, 2H), 8.16
br, 1H), 8.70 (br, 1H); FABMS m/z (relative intensity):
+
488 (M +1, 5), 390 (100); HRMS calcd for C H
NO (M +1) 488.3740, found 488.3766. Compound 12
from glutaric anhydride: H NMR (CDCl ): d 0.63 (s,
3
0
3
30 50
+
4
1
3
H), 0.82 (d, J=6.6 Hz, 6H), 0.86 (d, J=6.6 Hz, 3H),
.90 (s, 3H), 2.89 (d, J=23.7 Hz, 2H), 3.38 (m, 1H),
.69 (m, 1H). 8.00 (br, 1H); FABMS m/z (relative
+
+
21
intensity): 504 (M +1, 15), 486 (M -H O), 390 (100).
2
reported previously.
6
-Aza-pregnan-20-one (14) and 3ꢀ, 20-dihydroxy-6-aza-
In vitro binding assay
pregnane (15). Following the same procedures used in
the preparations of 6-aza-cholestane (3), pregnenolone
acetate was converted initially into 6-aza-pregnan-3,
The eect of compound 1 on the adhesion of malignant
HT-1080 cells to matrigel proteins was examined using