Y. Kamano et al./Bioorg. Med. Chem. 6 (1998) 1103±1115
1105
hydrazide (8) as a colorless prism, mp 255±258 ꢀC, [a]d
1.6, 22-H); 13C NMR(CDCl3) d 16.8 (C-18), 18.8 (C-19),
59.7 (C-17), 73.6 (C-15), 115.4 (C-23), 121.8 (C-20), 124.3
(C-1), 128.0 (C-2), 146.7 (C-22), 149.7 (C-21), 154.4 (C-4),
161.9 (C-24), 167.6 (C-5), 186.0 (C-3); Anal. calcd. for
C24H26O4: C,76.16; H, 6.93. Found: C, 75.89; H, 7.07.
8.3ꢀ (c 1.0, CHCl3); m/z 600(M)+; lmax(MeOH)/nm
1
297 (log e 3.40); nmax(KBr)/cm
3600, 3355, 1740,
1678, 1655, 1552, 1240, 970, 835, 797; 1H NMR (CDCl3)
d 0.70 (3H, s, 18-CH3), 0.95 (3H, s, 19-CH3), 2.17 (1H, m,
17-H), 1.46 (9H, s, COOC(CH3)3), 2.53 and 2.70 (4H,
each m, OCO(CH2)2CO), 5.10 (1H, bs, 3-H), 6.27 (1H, d,
J=9.8, 23-H), 6.59 (1H, bs, 3-OCO(CH2)2CONHNH-
COOC(CH3)3), 7.23 (1H, d, J=1.5, 21-H), 7.76 (1H, bs,
3-OCO(CH2)2CONHNHCOOC(CH3)3), 7.84 (1H, dd,
J=9.8, 2.9, 22-H); 13C NMR(CDCl3) d 16.6 (C-18),
21.3 (C-19), 28.1 (COOC(CH3)3), 53.8 (C-17), 71.1
(C-3), 81.9 (COOC(CH3)3), 85.4 (C-14), 115.3 (C-23),
122.7 (C-20), 146.8 (C-22), 148.6 (C-21), 155.4
(3-OCO(CH2)2CONHNHCOOC(CH3)3), 162.5 (C-24),
171.4 (3-OCO(CH2)2CONHNHCOOC(CH3)3), 172.3
(3-OCO(CH2)2CONHNHCOOC(CH3)3); Anal. calcd
for C33H48N2O8: C, 65.97; H, 8.05; N, 4.66. Found: C,
66.03, H, 7.92, N, 4.63.
Desacetyl-cinobufagin 3-acetate-16-succinate (48). A mix-
ture of 47 (500 mg),1 succinic anhydride (500 mg) and
pyridine (10 mL) was re¯uxed for 4 h. The mixture was
poured into ice-water and extracted with CHCl3. After
the removal of solvent, the residue (553 mg) was chro-
matographed on a silica gel column with n-hexane-acet-
one (3:1). Recrystallization from n-hexane-acetone gave
495 mg of colorless needles of 48: [a]d +10.7ꢀ (c 1.0,
CHCl3); m/z 542(M)+; lmax(MeCN)/nm 295 (log e
1
3.38); nmax(KBr)/cm
3400±3100, 1740, 1720, 1700,
1645, 1545, 1250, 1235, 958, 894, 790; 1H NMR (CDCl3)
d 0.83 (3H, s, 18-CH3), 1.00 (3H, s, 19-CH3), 2.07 (3H,
s, 3-OCOCH3), 2.55 (4H, m, 2ÂCH2 of succinate), 2.81
(1H, d, J=9.5, 17-H), 3.68 (1H, d, J=1.5, 15-H), 5.10
(1H, bs, 3-H), 5.53 (1H, dd, J=9.5, 1.5, 16-H), 6.25
(1H, d, J=10.2, 23-H), 7.19 (1H, d, J=3.0, 21-H), 7.95
(1H. dd, J=10.2, 3.0, 22-H); 13C NMR (CDCl3) d 17.2
(C-18), 23.7 (C-19), 50.4 (C-17), 59.5 (C-15), 70.2 (C-3),
72.3 (C-14), 75.1 (C-16), 114.1 (C-23), 116.2 (C-20),
148.3 (C-22), 151.4 (C-21), 162.3 (C-24), 170.7 (3-
OCOCH3), 171.5 (16-OCO(CH2)2COOH), 175.5 (16-
OCO(CH2)2COOH); Anal. calcd for C30H38O9: C,66.40;
H, 7.06. Found: C, 66.44; H, 7.02.
14b,15ꢀ-Epoxy-3-oxo-bufa-1,20,22-trienolide(Á1-3-oxo-
resibufogenin) (27) and 14ꢀ, 15ꢀ-epoxy-3-oxo-bufa-
1,4,20,22-tetraenolide(Á1,4-3-oxo-resibufogenin) (28). A
mixture of 3-oxo-resibufogenin (26)12 (1.25 g, 3.27 mmol)
and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(DDQ)
(816 mg, 3.6 mmol) in dioxane (30 mL) was re¯uxed for
4 h. The reaction mixture was mixed with silica gel
(10 g), which was dried and placed on a silica gel column
(700Â30 mm). The column was eluted with n-hexane-
acetone (4:1) to give four major fractions. 27 was
isolated from the second fraction, which was further
puri®ed by preparative TLC. Compound 27: colorless
needles, mp 221±223 ꢀC, [a]d +70ꢀ (c 0.83, CHCl3); m/z
3-Oxo-14ꢁ-artebufogenin (56). To a solution of 14a-
artebufogenin (55)15 (50 mg) in acetic acid (3 mL), a
solution of chromic acid (10 mg) in acetic acid (1 mL)
was added with stirring. The oxidation was conducted
at 15±18 ꢀC for 4 h. After decomposition of the excess
reagent with MeOH (0.25 mL, with stirring), the mixture
was poured into ice-water and extracted with CHCl3.
The extract was washed with water and concentrated to
dryness. Recrystallization of the crude product from
MeOH gave 37 mg of 3-oxo-14a-artebufogenin (56) as a
colorless needles: mp 210±213 ꢀC, [a]d +47.6ꢀ (c 1.0,
380 (M)+; lmax(MeOH)/nm 297 (log e 2.42), 223 (log e
1
2.99); nmax(KBr)/cm
2963, 2936, 2872, 1742, 1723,
1696, 1680, 1538, 1124, 952, 825, 830, 785; 1H
NMR(CDCl3) d 0.75 (3H, s, 18-CH3), 1.16 (3H, s, 19-
CH3), 2.45 (1H, m, 17-H), 3.46 (1H, s, 15-H), 5.94(1H,
d, J=10.2, 2-H), 6.18 (1H, dd, J=9.7, 0.9, 23-H), 6.78
(1H, d, J=10.2, 1-H), 7.17 (1H, dd, J=2.7, 0.9, 21-H),
7.70 (1H, dd , J=9.7, 2.8, 22-H); 13C NMR (CDCl3) d
16.8 (C-18), 20.7 (C-19), 59.8 (C-17), 73.8 (C-15), 115.4
(C-23), 122.0 (C-20), 127.7 (C-1, C-2), 146.8 (C-22),
149.7 (C-21), 160.1 (C-24), 200.1 (C-3); Anal. calcd for
C24H28O4: C,75.76; H, 7.42. Found: C, 75.98; H, 7.35.
CHCl3); m/z 382 (M)+ for C24H30O4; lmax(MeCN)/nm
1
298 (log e 3.70); nmax(KBr)/cm
1639, 1541, 1450, 1386, 1322, 1249, 1227, 1204, 1123,
2933, 2855, 1742,
1
990, 950, 833, 789; H NMR (CDCl3) d 0.67 (3H, s, 18-
CH3), 1.05 (3H, s, 19-CH3), 1.82 (1H, m, 14-H), 2.79
(1H, m, 17-H), 6.34 (1H, d, J=1.0, 23-H), 7.27 (1H, dd,
J=12.7, 2.9, 22-H), 7.33(1H, m, 21-H); 13C NMR
(CDCl3) d 13.9 (C-18), 20.3 (C-19), 64.8 (C-14), 115.8
(C-20), 116.1 (C-23), 144.1 (C-22), 149.1 (C-21), 161.4
(C-24), 212.4 (C-3), 212.5 (C-15). Anal. calcd for
C24H30O4: C,75.36; H, 7.91. Found: C, 75.57; H, 7.95.
The fourth fraction was dried and the residue was
recrystallized from acetone-hexane to give 170 mg
(14%) of tetraenolide (28) as a colorless needles, [a]d
+20.0ꢀ (c 1.0, CHCl3); m/z 378 (M)+; lmax(MeOH)/
nm 298 (log e 3.73), 236 (log e 4.19); nmax(KBr)/cmx1
2940, 2871, 1746, 1722, 1662, 1642, 1538, 1124, 950, 832,
785; 1H NMR(CDCl3) d 0.86 (3H, s, 18-CH3), 1.26 (3H,
s, 19-CH3), 2.17 (1H, m, 17-H), 3.46 (1H, s, 15-H), 6.35
to 6.0 (3H, m, 23-H, 2-H, 4-H), 7.05 (1H, d, J=10.0, 1-H),
7.25 (1H, dd, J=1.6, 0.9, 21-H), 7.75 (1H, dd, J=9.9,
Digitoxigenin 3-methylsuberate (67). To a solution of
digitoxigenin 3-suberate (66) (100 mg) in Et2O (10 mL),
a solution of CH2N2 (ca. 6 mg) in Et2O (15 mL) was