128
L.C. Baratto et al. / European Journal of Medicinal Chemistry 68 (2013) 121e131
00 0
9.09 (1H, d, 2.5 Hz, H3 ), 9.12 (1H, d, 2.5 Hz, H3 ), 11.05 (1H, s, e
NH ),11.13 (1H, s, eNH ). C NMR (50 MHz, CDCl ) d 14.6 (C27),15.0
3
product was purified by column chromatography (CH
give the corresponding benzyl ester (94.5 mg, yield 90%).
A solution of the benzyl ester was prepared in ethanol (5 mL), a
solution of 2,4-DNPH (0.10 g 2,4-DNPH, 0.5 mmol; 0.5 mL H SO
.75 mL distilled H O) was added and the mixture was stirred for
5 h at room temperature. Water (20 mL) was added and the
mixture was extracted with CH Cl
(3 ꢂ 20 mL). The crude extract
was fractionated by column chromatography (n-hexane:CH Cl
0:30 and 50:50), producing compound 7 (colorless resin, 67.4 mg,
2
Cl
2
100%) to
0
0
0
13
(C25), 15.7 (C26), 15.9 (C30), 19.6 (C6), 20.6 (C2), 21.3 (C11), 23.7
(C24), 26.9 (C12), 28.8 (C23), 29.2 (C15), 29.7 (C21), 31.9 (C16), 33.8
(C7), 36.8 (C22), 37.1 (C10), 38.1 (C13), 38.3 (C1), 40.7 (C8), 42.2 (C4),
42.5 (C14), 48.0 (C19), 49.3 (C18), 49.7 (C9), 55.1 (C5), 56.2 (C17),
2
4
;
0
1
2
00 0 0 00 00 0
116.3 (C6 ), 116.4 (C6 ), 123.6 (C3 , C3 ), 128.9 (C2 ), 129.1 (C2 ),
2
2
0
00
00
0
00
2
2
,
129.9 (C5 ), 130.0 (C5 ),137.5 (C4 ), 137.6 (C4 ), 145.2 (C1 ), 145.5
0
7
(C1 ),161.4 (C20), 167.0 (C3), 181.3 (C28).
yield 71.3%). UVevis (MeOHeCH
3
CN)
l
max 354, 371 nm. MS
Compound 10. UVevis (MeOHeCH
3
CN)
l
max 360 nm. MS
ꢃ
1
ꢃ
1
[
(
M ꢃ H] m/z 723.5, C43
H
56
N
4
O
6
. H NMR (200 MHz, CDCl 0.81
3
)
d
[M ꢃ H] m/z 635.5, C35
H
48
N
4
O
7
. H NMR (200 MHz, CDCl ) d
3
0.93
3H, s, H26), 0.90 (3H, s, H25), 0.94 (3H, s, H27), 1.15 (3H, s, H24),
(3H, s, H25), 0.98 (3H, s, H26), 1.02 (3H, s, H27), 1.15 (3H, s, H24),
0
1.27 (3H, s, H23), 1.68 (3H, s, H30), 4.60 (1H, s, H29), 4.73 (1H, s,
1.28 (3H, s, H23), 2.20 (3H, s, H30), 7.93 (1H, d, J 9.6 Hz, H6 ), 8.29
00
0
0
H29), 5.12 (1H, d, J ¼ 12.2 Hz, H7 , eOCH
2
ePh), 5.20 (1H, d,
(1H, dd, J 2.5 Hz, 9.6 Hz, H5 ), 9.12 (1H, d, 2.5 Hz, H3 ), 11.17 (1H, s, e
0
0
00 00
0
13
J ¼ 12.2 Hz, H7 , eOCH
2
ePh), 7.36 (5H, m, H2 -6 ), 7.94 (1H, d, J
NH ). C NMR (50 MHz, CDCl ) d 14.6 (C27), 15.6 (C25), 15.9 (C26),
3
0
0
9
.6 Hz, H6 ), 8.28 (1H, dd, J 2.5 Hz, 9.6 Hz, H5 ), 9.12 (1H, d, J 2.5 Hz,
H3 ), 11.17 (1H, s, eNH). C NMR (50 MHz, CDCl
19.6 (C6), 20.7 (C2), 21.3 (C11), 23.7 (C24), 27.2 (C12), 28.2 (C21),
28.8 (C23), 29.7 (C15), 30.1 (C30), 31.4 (C16), 33.6 (C7), 36.7 (C22),
37.0 (C10), 37.5 (C13), 38.3 (C1), 40.6 (C8), 42.2 (C4), 42.3 (C14), 49.0
0
13
3
)
d
14.6 (C27), 15.6
(
(
(
(
(
C25), 15.7 (C26), 19.4 (C30), 19.7 (C6), 20.7 (C2), 21.3 (C11), 23.8
0
C24), 25.5 (C12), 28.8 (C23), 29.7 (C15), 30.6 (C21), 32.1 (C16), 33.7
C7), 36.9 (C22), 37.0 (C10), 38.2 (C13), 38.4 (C1), 40.7 (C8), 42.2
C4), 42.5 (C14), 46.9 (C19), 49.4 (C18), 49.8 (C9), 55.2 (C5), 56.5
(C18), 49.6 (C9), 51.1 (C19), 55.0 (C5), 56.2 (C17), 116.4 (C6 ), 123.6
0
0
0
0
0
(C3 ),128.9 (C2 ),129.9 (C5 ),137.5 (C4 ),145.6 (C1 ),167.2 (C3),181.7
(C28), 212.1 (C20).
00 0 0
2
C17), 65.8 (eOCH eAr, 7 ), 109.7 (C29), 116.4 (C6 ), 123.6 (C3 ),
0
0
00
00
0
00
00
0
1
28.1 (C4 ), 128.2 (C2 , C6 ), 128.5 (C3 , C5 ), 128.9 (C2 ), 129.9
3.10. Preparation of 3-hydroxy-20-oxo-29-norlupan-28-oic acid
(11)
0
00
0
(
1
C5 ), 136.5 (C1 ), 137.4 (C4 ), 145.6 (C1 ), 150.4 (C20), 167.4 (C3),
75.8 (C28).
Compound 1 (1.03 g, 2.25 mmol) was dissolved in dioxane:H
(35:5), OsO (85 mg, 0.33 mmol) was added and the mixture was
stirred for 1 h. Then NaIO (5 g, 23.4 mmol) was added, in small
2
O
3.8. Preparation of 3,20-dioxo-29-norlupan-28-oic acid (8)
4
4
Compound 2 (0.30 g, 0.66 mmol) was dissolved in dioxane:H
2
O
portions, during 3 h and the mixture was stirred for 3 days at room
temperature. Ethyl acetate (100 mL) was added, and the mixture
(
10.5:1.5), OsO
was stirred for 1 h. NaIO
4
(15 mg, 0.059 mmol) was added and the mixture
(1.5 g, 7.01 mmol) was added, in small
4
was washed with H
separated, dried and the solvent evaporated. The residue was then
2
O (3 ꢂ 100 mL). The organic phase was
portions, during 3 h and the mixture was stirred for 3 days at room
temperature. Ethyl acetate was added and the mixture was washed
fractionated by flash chromatography using mixtures of
with H
evaporated and the residue purified by column chromatography
CH Cl 100%). Compound 8 (151 mg, yield 50.3%) was obtained as a
white-grayish solid, m.p. 230e233 C. MS [2M þ Na ꢃ 2H] m/z
2
O (3 ꢂ 50 mL). The organic phase was separated, the solvent
CH
2 2
Cl :MeOH and heptane:ethyl acetate as mobile phases. Com-
pound 11 was isolated (840 mg, yield 81.3%) as a grayish solid, m.p.
ꢀ
ꢃ
1
(
2
2
275e277 C. MS [2M ꢃ H] m/z 914.4, C29
(200 MHz, CDCl 0.78 (3H, s, H24), 0.81 (3H, s, H25),
þ Pyr-d
0.95 (3H, s, H26), 0.99 (3H, s, H23), 1.02 (3H, s, H27), 2.18 (3H, s,
H
46
O
4
.
H NMR
ꢀ
ꢃ
3
5
) d
1
9
33.3, C29
3H, s, H26),1.01 (3H, s, H27),1.02 (3H, s, H24),1.07 (3H, s, H23), 2.19
3H, s, H30). 13C NMR (50 MHz, CDCl
14.6 (C27), 15.7 (C25), 16.0
44 4 3
H O . H NMR (200 MHz, CDCl ) d 0.91 (3H, s, H25), 0.95
13
(
(
(
(
(
(
(
H30). C NMR (50 MHz, CDCl
3
þ Pyr-d
5
) d 14.7 (C27), 15.5 (C24),
3
)
d
16.0 (C25), 16.1 (C26), 18.3 (C6), 20.9 (C11), 27.4 (C2, C12), 28.1
(C23), 28.5 (C21), 29.8 (C15), 30.1 (C30), 31.9 (C16), 34.3 (C7), 36.9
(C22), 37.2 (C10), 37.4 (C13), 38.8 (C1), 39.0 (C4), 40.6 (C8), 42.3
(C14), 49.4 (C18), 50.5 (C9), 51.5 (C19), 55.4 (C5), 56.2 (C17), 78.5
(C3), 179.0 (C28), 212.8 (C20).
C26), 19.6 (C6), 21.0 (C24), 21.4 (C11), 26.8 (C23), 27.2 (C12), 28.2
C21), 29.7 (C15), 30.1 (C30), 31.4 (C16), 33.5 (C7), 34.0 (C2), 36.7
C22), 36.9 (C10), 37.6 (C13), 39.5 (C1), 40.5 (C8), 42.3 (C14), 47.3
C4), 49.0 (C18), 49.7 (C9), 51.2 (C19), 54.7 (C5), 56.2 (C17), 181.9
C28), 212.2 (C20), 218.3 (C3).
3.11. Preparation of 3-hydroxy-20-[(2,4-dinitrophenyl)hydrazono]-
3.9. Preparation of 3,20-[(2,4-dinitrophenyl)hydrazono]lupan-28-
29-norlupan-28-oic acid (12)
oic acid (9) and 3-[(2,4-initrophenyl)hydrazono]-20-oxolupan-28-
oic acid (10)
A solution of 11 (192 mg, 0.42 mmol) was prepared in ethanol
(
H
10 mL), a solution of 2,4-DNPH (0.25 g 2,4-DNPH, 1.26 mmol; 1 mL
SO ; 1.5 mL distilled H O) was added and the mixture was stirred
for 15 h, at room temperature. Water (20 mL) was added and the
mixture was extracted with CH Cl
(4 ꢂ 25 mL). The organic phase
was dried, the solvent evaporated and the residue was fractionated
by column chromatography using CH Cl :MeOH as mobile phase.
A solution of 8 (126 mg, 0.27 mmol) in ethanol (6.5 mL), a so-
2
4
2
lution of 2,4-DNPH (0.13 g 2,4-DNPH, 0.65 mmol; 0.65 mL H
mL distilled H O) was added and the mixture was stirred for 15 h
at room temperature. Water (20 mL) was added and the mixture
was extracted with CH Cl
(3 ꢂ 20 mL). The organic phase was
separated, the solvent evaporated and the residue was fractionated
by column chromatography (CH Cl 100%; CH Cl :MeOH, 99:1)
2 4
SO ,
1
2
2
2
2
2
2
2
Compound 12 (140.8 mg, yield 73.3%) was obtained as an orange
ꢀ
2
2
2
2
solid, m.p. 285e288 C. UVevis (MeOHeCH
3
CN)
l
max 355, 372 nm.
H NMR (200 MHz,
0.79 (3H, s, H24), 0.80 (3H, s, H25), 0.99 (3H, s,
H26), 1.00 (3H, s, H23), 1.07 (3H, s, H27), 2.04 (3H, s, H30), 7.99 (1H,
ꢃ
1
producing compounds 9 (orange solid, 53.1 mg, yield 42.1%, m.p.
MS [M ꢃ H] m/z 637.5, C35
50 4 7
H N O .
ꢀ
2
1
92e295 C) and 10 (dark orange solid, 48.3 mg, yield 38.3%, m.p.
CDCl
3
þ Pyr-d
5
) d
ꢀ
63e165 C).
0
0
0
0
Compound 9. UVevis (MeOHeCH
3
CN)
l
max 360 nm. MS
d, J 9.6 Hz, H6 ), 8.29 (1H, dd, J 2.5 Hz, 9.6 Hz, H5 ), 9.09 (1H, d,
ꢃ
1
00
00 13
[
(
(
(
M ꢃ H] m/z 815.0, C41
H
52
N
8
O
10. H NMR (200 MHz, CDCl
3
)
d
0.93
2.5 Hz, H3 ), 11.04 (1H, s, eNH ). C NMR (50 MHz, CDCl
3
þ Pyr-d
5
)
3H, s, H25),1.02 (3H, s, H26),1.07 (3H, s, H27),1.14 (3H, s, H24),1.28
d
14.6 (C27, C30), 15.6 (C25), 16.1 (C24, C26),18.4 (C6), 21.0 (C11),
0
00
3H, s, H23), 2.07 (3H, s, H30), 7.94 (2H, t, J 9.7 Hz, H6 ,H6 ), 8.26
26.9 (C21), 27.4 (C2), 28.1 (C23), 29.3 (C12), 29.8 (C15), 32.4 (C16),
34.4 (C7), 37.0 (C22), 37.2 (C10), 37.9 (C13), 38.9 (C1), 39.0 (C4), 40.8
0
00
1H, dd, J 2.5 Hz, 5.5 Hz, H5 ), 8.31 (1H, dd, J 2.5 Hz, 5.5 Hz, H5 ),