Influence of esterification and modification of A-ring in a group of lupane acids on their cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2005.07.011

The aim of this work was to find an optimal ester group for preparation of lupane derivatives connecting high cytotoxicity with good chemical and pharmacological properties. Activities of methyl-, pivaloyloxymethyl- (Pom-), and acetoxymethyl- (Acm-) esters were compared with the activity of free acids. Although the methyl- and Pom-esters were generally less active than free acids, some Acm-esters had cytotoxicity similar to or even better than the starting compounds. Cytotoxic activity was measured in five cancer cell lines.

Full text of DOI:10.1016/j.bmc.2005.07.011

Bioorganic & Medicinal Chemistry 13 (2005) 5527–5535
Influence of esterification and modification of A-ring
in a group of lupane acids on their cytotoxicity
a
Milan Urban, Jan Sarek, Iva Tislerova, Petr Dzubak and Marian Hajduch
a,
a
b
b
*
a
Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague,
Hlavova 8, 128 43 Prague 2, Czech Republic
b
Laboratory of Experimental Medicine, Departments of Pediatrics and Oncology, Faculty of Medicine,
Palacky University in Olomouc, Puskinova 6, 775 20 Olomouc, Czech Republic
Received 3 March 2005; revised 28 June 2005; accepted 1 July 2005
Abstract—The aim of this work was to find an optimal ester group for preparation of lupane derivatives connecting high cytotox-
icity with good chemical and pharmacological properties. Activities of methyl-, pivaloyloxymethyl- (Pom-), and acetoxymethyl-
(
Acm-) esters were compared with the activity of free acids. Although the methyl- and Pom-esters were generally less active than
free acids, some Acm-esters had cytotoxicity similar to or even better than the starting compounds. Cytotoxic activity was measured
in five cancer cell lines.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1. Introduction
trast, acylation of the 3b-hydroxy group caused a de-
crease in cytotoxicity.
Betulinic acid (1a) is a pentacyclic triterpene, which oc-
curs, for example, in the bark of Platanus hispanica. Bet-
Although the alkyl esters of betulinic acid (1a) were
found to be inactive, they dispose of better chemical
and pharmacological properties. Esters are usually more
soluble than free acids in all organic solvents, which is a
significant advantage for both the chemical preparation
and purification. The main aim of this work was to find
an ester group that has optimal properties and does not
cause a decrease in cytotoxic activity. This can be per-
formed using esters, which are easily cleavable by non-
specific intracellular esterases. Moreover, our study
was performed using Pom- and Acm-esteric groups,
which were previously shown to better penetrate cellular
membranes and to release free compounds after intracel-
1
ulinic acid (1a) is a compound with anti-HIV activity,
3
2
cytotoxicity, and anti-tumor properties. The cytotoxic
4
activity on human melanoma (MEL-2) and lung carci-
5
noma (A549) cell lines was the first to be reported and
subsequent research showed that this activity is not lim-
ited to those cell lines, since similar results were obtained
on malignant cells of different histogenetic origin,
though with a certain selectivity against neuroectoder-
6
–10
mal tumors. In more recent studies,
the cytotoxic
activity of certain betulinic acid derivatives has been
investigated. Their activity was usually associated with
a free carboxylic or a carbonylic group in position C-
8
2
8. In contrast, all alkyl esters were found to be inac-
6
lular enzymatic deprotection, as demonstrated on peni-
11,12
,7
tive. Moreover, hydrogenation of the 20(29) double
bond caused a significant increase in the cytotoxicity
cillins. Methyl ester and free acids were used as
standards for comparison of the biological activity.
7
to HeLa and OVCAR-3 cell lines. Analogous oxidation
of the 3b-hydroxy group of 1a afforded derivatives with
higher activity and substitution with an amino group
gave derivatives with properties similar to 1a. In con-
2
. Chemistry
Betulinic acid 1a was extracted from the bark of a plane
tree, P. hispanica (collected in the Czech Republic).
Keywords: Betulinic acid; Esters; Cytotoxicity; Diosphenols; Seco de-
rivatives; Cleavage; Oxidation.
Thirty-two compounds (1b–1h, 2a–2d, 3a–3f, 4a–4g, 5a–
5e, and 6a–6c), betulinic acid analogues, were prepared
from 1a as shown in Schemes 1 and 2. Esters 1b–1d were
*
Corresponding author. Tel./fax: +420 221 951 332; e-mail:
jan.sarek@volny.cz
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.011

5528
M. Urban et al. / Bioorg. Med. Chem. 13 (2005) 5527–5535
Scheme 1. Derivatization of betulinic acid 1a, betulonic acid 1e, and diosphenol 3a. Reagents and conditions: (a) Isopropenyl acetate, TsCl, reflux;
b) CrO , DMF, rt; (c) O , ether, rt; (e) PomCl, MeCN, CH Cl , DBU, rt; (f) AcmBr, MeCN, CH Cl
, t-BuOK, t-BuOK, 400 ꢁC, 40 min; (d) CH
DBU, rt; (g) Me SO , KOH, dioxane, reflux; (h) H , KOH, MeOH, refl.
(
3
2
2
N
2
2
2
2
2
,
2
4
2 2
O
dimethyl sulfate in the presence of KOH afforded methyl
ether 3e. A reaction of 5a and 5c with acetic anhydride
in pyridine gave compounds 5b, 5d, and 5e. Diosphenols
3
a and 3b were then cleaved with a solution of hydrogen
9
peroxide and KOH in refluxing MeOH. The reactions
afforded cleaved secoderivatives 4a and 4b. Methyl es-
ters 4c and 6a were prepared by the reaction of acids
4
CHCl . Pom esters 4d, 4f, and 6b were prepared by alcy-
a and 5a with diazomethane in diethylether and
3
lation of free acids 4a, 4e, and 5a with PomCl and DBU
in CHCl and MeCN. Acm-esters 4e, 4g, and 6c were
2
Scheme 2. Derivatization of oxidized derivatives of betulinic acid 1a.
Reagents and conditions: (a) O
, t-BuOK, t-BuOK, 400 ꢁC, 48 h; (b)
CH , ether, rt; (c) PomCl, MeCN, CH Cl , DBU, rt; (d) AcmBr,
MeCN, CH Cl , DBU, rt; (e) Ac O, pyridine rt.
prepared by alcylation of free acids 4a, 4b, and 5a with
AcmBr and DBU in CHCl and MeCN.
2
2
2
N
2
2
2
2
2
2
3. Results and discussion
obtained by alcylation of 1a (1b: CH N in diethyl ether/
2
2
CHCl ; 1c: PomCl with DBU in CH Cl and MeCN; 1d:
3
Eight lupane derivatives 1a–1h, four of them new (1c,
1d, 1g, and 1h), four new enol acetates (2a–2d), six dio-
sphenols 3a–3f, three of them new (3c, 3d, and 3f), seven
secoderivatives 4a–4g, four of them new (4d–4g), five
lactols (5a–5e), three of them new (5c–5e), and three
new aldehydes (6a–6c) were synthesized from 1a. All
new compounds were characterized using spectral and
physical data.
2
2
AcmBr with DBU in CH Cl and MeCN). Previously
2
2
9
published procedure was used for the preparation of
oxidized compounds. Oxidation of acid 1a with CrO3
in DMF afforded ketone 1e. Esters 1f–1h were prepared
in the same manner as described for compounds 1b–1d.
Enol acetate 2a was prepared by treatment of 1e with
isopropenyl acetate in presence of TsOH and than alcy-
lated in same conditions as described above to give es-
ters 2b–2d. Further oxidation of 1e and 1f was
performed by introducing air 40 min into the tert-butyl
alcohol solution of each ketone in presence of potassium
Methyl-, Pom-, and Acm-esters were synthesized from
terpenic acids to compare their structure–activity rela-
tionships. Also, acetates were prepared in derivatives 5.
9
,13
2
-methyl-2-propoxide.
8
Our results correspond to previously published lit.
Two types of products resulted from the reaction, the
major products were diosphenols 3a and 3b. Com-
pounds 5a and 5b were obtained as by-products, when
the reaction time was longer than 1.5 h. Pure com-
pounds 5a and 5b were obtained by further oxidation
of diosphenols 3a and 3b in the same conditions as they
showing that esterification of the C-28 carboxyl group
with alkyl ester (methyl) decreases in vitro the cytotoxic-
ity majority of the synthesized derivatives. Similar effect
was obtained with Pom esters in our study (Table 1).
Compounds 1b, 1c, 1f, 1g, 2b, 2c, 5b, and 5d are less ac-
tive than corresponding free acids 1a, 1e, 2a, and 5a.
Also activity of acetate 5d was even lower than free lac-
tol 5a and methyl ester5b.
9
were prepared, when the reaction time was 48 h. After
the alcylation of 3a in usual conditions, esters 3c and
3
d were obtained. In contrast to all previous alcylation,
double-alcylated product 3f was also obtained (35%
yield). Subsequent treatment of diosphenol 3a with
Acm esters 1d, 1h, 2d, and 6c, however, showed higher
activity than both of the previously mentioned ester

M. Urban et al. / Bioorg. Med. Chem. 13 (2005) 5527–5535
Table 1. Cytotoxic activity of compounds 1a–6c against A 549, DU
5529
tion of free acids, preserving their biological activities.
Although the cytotoxic activity of some of the deriva-
tives presented here was in the high micromolar ranges,
several compounds recruited from the group of diosphe-
nols 3 showed interesting anti-cancer activity in vitro,
and deserve further investigation. An influence of A-ring
modification on the cytotoxicity of derivatives prepared
here was also studied. The most active compounds were
found among diosphenols 3a–3f with a conjugated
dicarbonylic system on the A-ring, which seems to be
a more efficient modification than esterification. Among
more oxidized compounds, the secoacids and esters 4a–
4g were less active than starting material. Also, the lac-
tols 5a–5e and aldehydes 6a–6c are not very promising
anti-cancer agents. Protecting the 3-oxogroup in betuli-
nic acid 1e was not successful either.
1
45, MCF 7, K562, and K562-Tax cell lines
a
IC50 (lmol/L)
Compound A 549 DU 145 MCF 7 K562 K562-Tax
1
1
1
1
1
1
1
1
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
6
6
6
a
b
c
d
e
f
146
184
>250
83
196
110
>250
71
143
120
>250
96
56
71
112
65
>250
83
>250
34
17
15
36
29
6
>250
>250
19
>250
>250
12
>250
>250
24
239
250
15
>250
>250
29
g
h
a
b
c
d
a
b
c
d
e
f
40
79
100
>250
>250
225
14
49
34
>250
>250
112
10
>250
>250
153
23
>250
>250
80
>250
>250
95
4
60
11
25
11
9
6
230
69
178
50
203
47
48
35
36
5. Materials and methods
140
10
23
18
12
10
>250
>250
99
>250
>250
85
>250
>250
71
>250
145
77
240
>250
75
5.1. Chemicals
a
b
c
d
e
f
Potassium tert-butoxide, tert-butyl alcohol, dimethyl
sulphate, isopropenyl acetate, DBU, PomCl, and
AcmBr were purchased from Sigma–Aldrich company.
184
>250
110
110
149
93
113
>250
97
107
>250
88
104
>250
75
76
>250
47
45
66
74
41
5.2. Cell lines
g
a
b
c
d
e
a
b
c
119
107
124
17
119
126
130
21
110
107
250
32
67
121
91
Cell lines A 549, DU 145, MCF 7, and K562 were pur-
chased from the American Tissue Culture Collection
(ATTC). Paclitaxel-resistant subline of K562 cells (K-
562-Tax) was prepared and characterized in our labora-
115
20
82
>250
197
>250
207
167
206
41
243
107
243
>250
215
178
135
>250
188
108
>250
186
248
155
76
1
7
206
210
179
tories. The cells were maintained in Nunc/Corning
8
2
0 cm plastic tissue culture flasks and cultured in cell
culture medium (DMEM/RPMI 1640 with 5 g/L glu-
cose, 2 mM glutamine, 100 U/mL penicillin, 100 lg/mL
streptomycin, 10% fetal calf serum, and NaHCO3).
Value >250 lmol/L means that compound is not active at maximum
tested concentration.
a
The concentration lethal to 50% of tumor cells. mean value from 3 to
4 independent experiments with standard deviation <20% of the
average.
5.3. Cytotoxic MTT assay
Cell suspensions were prepared and diluted according to
the particular cell type and the expected target cell den-
sity (2500–30,000 cells/well based on cell growth charac-
teristics). Cells were added by pipette (80 lL) into 96-
well microtiter plates. Inoculates were allowed a pre-in-
types. Cytotoxicity was similar to free acids, and more-
over, in the case of compound 1d, even higher than the
corresponding acid 1a.
cubation period of 24 h at 37 ꢁC and 5% CO for stabil-
2
In contrast with these facts, general structure–activity
relationship was not confirmed in groups of derivatives
ization. Fourfold dilutions, in 20-lL aliquots, of the
intended test concentration were added at time zero to
the microtiter plate wells. All test compound (dissolved
in 10 lL of 10% DMSO) concentrations were examined
in duplicate. Incubation of the cells with the test com-
3
a–3f, 4a–4g, and 6a–6c. This is probably due to wider
modification in the A-ring which affects mutual interac-
tion and accessability of ester groups to esterases, and
their activity can be caused by this modification. The
most interesting is the activity of diosphenols 3b and
pounds lasted for 72 h at 37 ꢁC, in a 5% CO atmo-
2
sphere at 100% humidity. At the end of the incubation
period, the cells were assayed using MTT. Aliquots
3
e which are active as methyl esters themselves.
(10 lL) of the MTT stock solution were pipetted into
each well and incubated for a further 1–4 h. After this
incubation period formazan produced was dissolved
by the addition of 100 lL/well of 10% aq SDS (pH
5.5), followed by a further incubation at 37 ꢁC over-
night. The optical density (OD) was measured at
540 nm with a Labsystem iEMS Reader MF. Tumor cell
survival (TCS) was calculated using the following equa-
tion: TCS = (OD of drug-exposed well/OD of control
4
. Conclusion
The cytotoxicity of a large group of triterpenoid esters
was studied in five cancer cell lines. It was found that
methyl and Pom esters are inactive; however, Acm esters
have activity similar to the starting acids (Table 1). Acm
esters can be used as a suitable ester group for protec-

5530
M. Urban et al. / Bioorg. Med. Chem. 13 (2005) 5527–5535
wells) · 100%. The TCS value, the drug concentration
acid in 9 mL CH Cl and 3 mL MeCN. The reaction
2 2
mixture was worked-up after 10–12 h. Crude product
was than purified by chromatography on silica gel
5
0
(in lmol/L) lethal to 50% of the tumor cells, was calcu-
lated from appropriate dose–response curves.
(30 g) eluted with toluene/diethyl ether (5:1) and crystal-
lized from methanol.
6. Experimental
6
.1.4. Betulinic acid (1a). Bark of the plane tree P. hispa-
6
.1. General experimental procedures
nica (5 kg) was extracted with MeOH (15 L, twice). Col-
lected extracts were evaporated and the residual crude
betulinic acid (1) (150 g) was then purified by several
crystallizations from MeOH (70 mL/g) to give white
Melting points were determined using a Kofler block
and are uncorrected. Optical rotations were measured
using CHCl solutions (unless otherwise stated) on an
Autopol III (Rudolph Research, Flanders, NJ) polarim-
eter. NMR spectra were recorded on a Varian UNITY
INOVA 400 instrument ( H NMR spectra at
needles (50 g, 1% yield): mp 304–305 ꢁC. [a] +8ꢁ (c
3
D
À1
14
0.37). IR m (CHCl ) cm : 1720vb, 1643.
3
1
6.1.5. Methyl 3b-hydroxylup-20(29)-en-28-oate (1b). Dia-
zomethane (50 mmol) in diethylether was added to a
solution of betulinic acid (1) (5 g, 10.9 mmol) in CHCl3
(400 mL). Organic solvents were evaporated in vacuum
and crude product was chromatographed over silica
gel (50 g) eluted with toluene and then crystallized from
3
stated), with SiMe as an internal standard. EIMS were
99.95 MHz) using CDCl solutions (unless otherwise
3
4
recorded on an INCOS 50 (Finigan MAT) spectrometer
at 70 eV and an ion source temperature of 150 ꢁC. The
samples were introduced from a direct exposure probe
at a heating rate of 10 mA/s. Relative abundances stated
are related to the most abundant ion in the region of m/
methanol to afford methyl ester 2 (4.5 g, 87% yield): mp
224–228 ꢁC. [a] +3ꢁ (c 0.36). IR m (CHCl ) cm
1720vb, 1643.
À1
:
D
3
1
4
z > 50. TLC was carried out using silica gel 60 F254
detection was by spraying with 10% aq H SO and heat-
,
2
4
ing to 150–200 ꢁC. Column chromatography was per-
formed using silica gel 60 (Merck 7734). The HPLC
system consisted of a High Pressure Pump Gilson (mod-
el 361), and Rheodyne Injections Valve, preparative col-
umn (25 · 250 mm) with silica gel fillings (Biospher
6.1.6. Pivaloyloxymethyl 3b-hydroxylup-20(29)-en-28-
oate (1c). 1.5 g (3.3 mmol) of 1a was treated with PomCl
in the general manner to give white needles of 1c (1.4 g,
75% yield): mp 155–158 ꢁC (methanol). [a] +6ꢁ (c
D
À1
1
0.34). IR m (CHCl ) cm : 1748, 1721 sh, 1642.
H
3
7
lm), Differential-Refractometrical Detector (Laborat-
NMR d 0.76, 0.82, 0.92, 0.96 (15H, each s, 5 · CH ),
3
orni pristroje, Praha, CZ) connected with PC (software
Chromulan) and Automatic Fraction Collector Gilson
1.22 (9H, s, 3 · CH Pom), 1.68 (3H, m, H-30), 1.80–
3
1.93 (2H, m), 2.20–2.28 (2H, m), 2.97 (1H, td, J(H-19b,
H-18a) = 11.0 Hz, J(H-19b, H-21a) = 11.0 Hz, J(H-
19b, H-21b) = 4.8 Hz, H-19b), 3.18 (1H, dd, J(H-3a, H-
2b) = 11.2 Hz, J(H-3a, H-2a) = 5.0 Hz, H-3a), 4.60
(
model 246). A mixture of ethyl acetate and hexane
was used as the mobile phase, its composition specified
for each experiment separately.
(1H, m, H-29 pro-E), 4.73 (1H, d, J = 2.3 Hz, H-29 pro-
Work-up refers to pouring the reaction mixture into
H O, extraction of the product with Et O, and washing
the organic layer successively with H O, dilute aq HCl,
Z), 5.75 (1H, d, J(H-31a, H-31b) = 5.43 Hz, H-31a),
5.79 (1H, d, J(H-31b, H-31a) = 5.5 Hz, H-31b). MS m/z
(relative intensity): 570 (6, M ), 552 (1), 540 (0.1), 455
2
2
+
2
H O, saturated aq NaHCO , and again H O, followed
2
by drying over MgSO , filtration, and evaporation of
4
(8), 438 (15), 423 (4), 410 (50), 395 (9), 362 (11), 332 (3),
320 (4), 271 (4), 247 (8), 234 (10), 230 (10), 220 (24), 207
(67), 189 (100). Anal. Calcd for C H O : C, 75.75%;
3
2
the filtrate under reduced pressure. Analytical samples
were dried over P O under diminished pressure.
3
6
58
5
H, 10.24%. Found C, 75.73%; H, 10.77%.
2
5
6.1.1. General procedure for the preparation of methyl
esters. Each acid was dissolved in chloroform and
6.1.7. Acetoxymethyl 3b-hydroxylup-20(29)-en-28-oate
(1d). 1.6 g (3.5 mmol) of acid 1a was treated with AcmBr
in the general manner to give needles of 1d (1.4 g, 75%
CH N in diethyl ether was added since the development
2
2
of N gas stopped. Methyl ester was than purified over
2
silica gel eluted with toluene and crystallized from
methanol.
yield): mp 186–189 ꢁC (methanol). [a] +12ꢁ (c 0.35).
D
À1
1
IR m (CHCl ) cm : 1752b, 1642. H NMR d 0.75,
3
0.82, 0.92, 0.96, (15H, all s, 5 · CH ), 1.68 (3H, s, H-
3
3
0), 1.85–1.95 (2H, m), 2.10 (3H, s, CH Acm), 2.18
3
6
0
.1.2. General procedure for a preparation of Pom esters.
.50 mL (3.33 mmol) of PomCl was added to a solution
(1H, td, J = 13.1 Hz, J = 13.1 Hz, J = 3.8 Hz), 2.26
1 2 3
(1H, m), 3.00 (1H, td, J(H-19b, H-18a) = 11.4 Hz),
J(H-19b, H-21a) = 11.4 Hz, J(H-19b, H-21b) = 5.1 Hz,
(H-19b), 3.18 (1H, m, RJ = 16.3 Hz, (H-3a), 4.61 (1H,
m, H-29 pro-E), 4.47 (1H, bd, J = 2.5 Hz, H-29 pro-Z),
5.71 (1H, d, J(H-31a, H-31b) = 6.8 Hz, H-31a), 5.80
(1H, d, J(H-31b, H-31a) = 6.8 Hz, H-31a). MS m/z (rel-
of 0.52 mL (3.42 mmol) of DBU and 3.2 mmol of each
acid in 9 mL CH Cl and 3 mL MeCN. The reaction
mixture was worked-up after 10–12 h. Crude product
was than purified by chromatography on silica gel
2
2
(30 g) eluted with toluene and crystallized from
methanol.
+
ative intensity): 528 (19, M ), 510 (5), 456 (5), 438 (9),
4
23 (4), 410 (35), 395 (8), 320 (32), 306 (6), 248 (9),
6
.1.3. General procedure for a preparation of Acm esters.
.51 mL (3.33 mmol) of AcmBr was added to a solution
235 (14), 220 (26), 207 (68), 189 (100). Anal. Calcd for
C H O : C, 74.96%; H, 9.91%. Found C, 74.95%; H,
10.00%.
0
of 0.52 mL (3.42 mmol) of DBU and 3.2 mmol of each
3
3
52
5

M. Urban et al. / Bioorg. Med. Chem. 13 (2005) 5527–5535
5531
6
(
.1.8. 3-Oxolup-20(29)-en-28-oic acid (1e). Chromium
VI) oxide (15.0 g, 150.1 mmol) and sulfuric acid (1 mL,
1e (3 g, 6.6 mmol) in isopropenyl acetate (150 mL). The
solution was refluxed for 9 h and 50 mL of solvents was
distilled off during that time. Then the reaction mixture
was worked-up and crude product was purified by chro-
matography over silica gel (50 g) eluted with (toluene/
diethylether 20:1) to afford 2a (1.8 g, 55% yield): mp
98%) was added to a solution of acid 1a (15.0 g,
32.8 mmol) in DMF (300 mL). The reaction mixture
was stirred for 12 h at room temperature. The product
was precipitated by pouring into vigorously stirred copi-
ous water, filtered, and washed with water. A column
chromatography of crude ketone 7 (14.1 g) over silica
gel (300 g) eluted with chloroform afforded ketone 7
261–264 ꢁC (methanol). [a] +30ꢁ (c 0.30). IR m
D
À1
1
(CHCl ) cm : 1752 sh, 1740, 1695, 1642. H NMR d
3
0.89, 0.94, 0.97, 0.98, 1.00 (15H, all s, 5 · CH ), 1.70
3
(
+
10.7 g, 71% yield): mp 250–254 ꢁC (methanol). [a]
(3H, m, H-30), 2.14 (3H, s, OAc), 3.02 (1H, td, J(H-
19b, H-18a) = 10.7 Hz, J(H-19b, H-21a) = 10.7 Hz,
J(H-19b, H-21b) = 4.8 Hz, H-19b), 4.62 (1H, m, H-29
pro-E), 4.75 (1H, bd, J = 2.3 Hz, H-29 pro-Z), 5.13 (1H,
dd, J(H-2, H-1a) = 6.7 Hz, J(H-2, H-1b) = 2.0 Hz, H-
D
1
5
32ꢁ (c 0.37).
6
.1.9. Methyl 3-oxolup-20(29)-en-28-oate (1f). Sodium
dichromate (37.0 g, 124.2 mmol) and sodium acetate
8.0 g) was added to a vigorously stirred solution of
+
(
2). MS m/z (relative intensity): 496 (7, M ), 481 (1), 454
crude methyl ester 1b (33.0 g, 70.2 mmol) in a mixture
of dioxane (750 mL), glacial acetic acid (250 mL), and
acetic anhydride (100 mL). The solution was then stirred
for 28 h at room temperature and worked-up in the usu-
al manner. Column chromatography of crude product
(100), 439 (5), 408 (8), 393 (3), 248 (6), 235 (6), 218 (8),
205 (36), 189 (24), 175 (16), 161 (10), 147 (16), 135 (26),
119 (38), 105 (38). Anal. Calcd for C H O : C,
3
2
48
4
77.38%; H, 9.74%. Found C, 77.73%; H, 9.55%.
1
afforded ketoester 1f (16.1 g, 49% yield): R 0.57. mp
f (30.5 g) over silica gel (300 g) eluted with toluene
6.1.13. Methyl 3b-acetoxylup-2,20(29)-ene-28-oate (2b).
One hundred milligram (0.2 mmol) of 2a was treated
with CH N using general procedure which yielded crys-
f
1
6
1
61–165 ꢁC (methanol). [a]D +28ꢁ (c 0.41).
2
2
tals of 2b (70 mg, 68%): mp 234–238 ꢁC (methanol). [a]
D
À1
6
.1.10. Pivaloyloxymethyl 3-oxolup-20(29)-en-28-oate
1g). 1.5 g (3.3 mmol) of acid 1e was treated with PomCl
+35ꢁ (c 0.35). IR m (CHCl ) cm : 1753, 1721, 1689,
3
1
(
in the general manner to give needles of 1g (1.04 g, 56%
1642. H NMR d 0.90, 0.93, 0.95, 0.96, 1.00 (15H, all
s, 5 · CH ), 1.69 (3H, m, H-30), 2.14 (3H, s, OAc),
3
yield): mp. 133–138 ꢁC (methanol). [a] +25ꢁ (c 0.40).
3.00 (1H, td, J(H-19b, H-18a) = 11.1 Hz, J(H-19b, H-
21a) = 11.1 Hz, J(H-19b, H-21b) = 4.6 Hz, H-19b),
D
À1
1
IR m (CHCl ) cm : 1748, 1700, 1643. H NMR d
3
0
CH -Pom), 1.68 (3H, s, H-30), 1.82–1.94 (2H), 2.23–
.93, 0.97, 1.02 1.07 (15H, all s, 3 · CH ), 1.22 (9H, s,
3.67 (3H, s, OCH ), 4.60 (1H, m, H-29 pro-E), 4.74
(1H, bd, J = 2.3 Hz, H-29 pro-Z), 5.13 (1H, dd, J(H-2,
3
3
3
2
1
.32 (2H, m), 2.35–2.54 (2H, m), 2.97 (1H, td, J(H-
9b, H-18a) = 11.1 Hz, J(H-19b, H-21a) = 11.1 Hz,
H-1a) = 6.7 Hz, J(H-2, H-1b) = 2.0 Hz, H-2). MS m/z
(relative intensity): 510 (25, M ), 495 (4), 468 (100),
+
J(H-19b, H-21b) = 4.7 Hz, H-19b), 4.61 (1H, m, H-29
pro-E), 4.73 (1H, bd, J = 2.3 Hz, H-29 pro-Z), 5.76
450 (7), 435 (5), 409 (13), 354 (5), 262 (32), 249 (32),
233 (12), 218 (25), 205 (82), 189 (88), 175 (46), 175
(46), 161 (21), 147 (30), 133 (46), 119 (73), 105 (70).
Anal. Calcd for C H O : C, 77.60%; H, 9.87%. Found
(1H, d, J(H-31a, H-31b) = 5.3 Hz, H-31a), 5.80 (1H,
d, J(H-31b, H-31a) = 5.5 Hz, H-31b). MS m/z (relative
3
3
50
4
+
intensity): 568 (9, M ), 538 (4), 454 (12), 436 (17), 421
C, 77.63%; H, 10.03%.
(
(
2), 408 (100), 393 (8), 245 (10), 230 (7), 218 (23), 205
37), 189 (50). Anal. Calcd for C H O : C, 76.01%;
6.1.14. Pivaloyloxymethyl 3b-acetoxylup-2,20(29)-ene-28-
oate (2c). Compound 2c was prepared from 2a (100 mg,
3
6
56
5
H, 9.92%. Found C, 75.83%; H, 9.77%.
0.2 mmol) using general procedure, which yielded crystals
6
.1.11. Acetoxymethyl 3-oxolup-20(29)-en-28-oate (1h).
of 2c (70 mg, 57%): mp 180–183 ꢁC (methanol). [a] +31ꢁ
D
À1
1
Two gram (4.4 mmol) of 1e was treated with AcmBr in the
general manner to give white needles of 1h (1.54 g, 66%):
(c 0.34). IR m (CHCl ) cm : 1749b, 1692, 1642. H NMR
3
d 0.90, 0.93, 0.96, 1.00 (15H, all s, 5 · CH ), 1.22 (9H, s,
3
mp 86–91 ꢁC (methanol). [a] +31ꢁ (c 0.32). IR m
CH -Pom), 1.68 (3H, s, H-30), 2.14 (3H, s, OAc), 2.98
D
3
À1
1
(
CHCl ) cm : 1750b, 1699, 1643. H NMR d 0.93,
(1H, td, J(H-19b, H-18a) = 11.0 Hz, J(H-19b, H-
21a) = 11.0 Hz, J(H-19b, H-21b) = 4.6 Hz, H-19b), 4.60
(1H, m, H-29 pro-E), 4.74 (1H, m, H-29 pro-Z), 5.13
(1H, dd, J(H-2, H-1a) = 6.7 Hz, J(H-2, H-1b) = 2.0 Hz,
H-2), 5.76 (1H, d, J(H-31a, H-31b) = 5.5 Hz, H-31a),
5.80 (1H, d, J(H-31b, H-31a) = 5.4 Hz, H-31b). MS m/z
3
0
H-30), 1.84–1.96 (3H, m), 2.10 (3H, s, CH Acm), 2.18–
.96, 0.98, 1.02, 1.07, (15H, all s, 5 · CH ), 1.68 (3H, s,
3
3
2
.31 (2H, m), 2.35–2.55 (2H, m), 3.00 (1H, td, J(H-19b,
H-18a) = 11.3 Hz, J(H-19b, H-21a) = 11.3 Hz, J(H-
9b, H-21b) = 4.7 Hz, H-19b), 4.61 (1H, m, H-29 pro-
E), 4.74 (1H, m, H-29 pro-Z), 5.72 (1H, d, J(H-31a, H-
1
+
(relative intensity): 610 (10, M ), 568 (86), 495 (7), 478
3
3
1b) = 5.5 Hz, H-31a), 5.81 (1H, d, J(H-31b, H-
1a) = 5.5 Hz, H-31b). MS m/z (relative intensity): 526
(6), 453 (100), 436 (16), 408 (67), 393 (10), 355 (6), 311
(14), 247 (13), 231 (13), 218 (18), 205 (58), 189 (34), 175
(36), 161 (29), 147 (29), 135 (56), 119 (76), 105 (80). Anal.
Calcd for C H O : C, 74.71%; H, 9.57%. Found C,
+
(
23, M ), 511 (2), 496 (3), 483 (0.5), 466 (0.5), 454 (21),
4
3
2
7
46 (10), 436 (23), 408 (100), 393 (11), 365 (8), 354 (10),
20 (45), 307 (8), 259 (10), 248 (19), 235 (26), 218 (31),
05 (63), 189 (87). Anal. Calcd for C H O : C,
3
8
58
6
74.55%; H, 9.63%.
3
3
50
5
5.25%; H, 9.57%. Found C, 75.20%; H, 9.44%.
6.1.15. Acetoxymethyl 3b-acetoxylup-2,20(29)-ene-28-
oate (2d). Compound 2d was prepared from 2a (500 mg,
1 mmol) using general procedure, which yielded crystals
of 2c (360 mg, 63%): mp 141–144 ꢁC (methanol). [a]D
6.1.12. 3b-Acetoxylup-2,20(29)-ene-28-oic acid (2a).
TsOH (0.5 g) was added to a solution of betulonic acid

5532
M. Urban et al. / Bioorg. Med. Chem. 13 (2005) 5527–5535
À1
1
+
38ꢁ (c 0.31). IR m (CHCl ) cm : 1753b, 1690, 1642. H
(26), 340 (37), 320 (29), 269 (19). Anal. Calcd for
C H O : C, 73.30%; H, 8.95%. Found C, 73.37%; H,
9.06%.
3
NMR d 0.90, 0.93, 0.95, 0.97, 1.00 (15H, all s, 5 · CH ),
.69 (3H, s, H-30), 2.10 (3H, s, CH Acm), 3.01 (1H, m,
3
33 48
6
1
3
RJ = 26.9 Hz, H-19b), 4.61 (1H, m, H-29 pro-E), 4.74
1H, dm, H-29 pro-Z), 5.13 (1H, dd, J(H-2, H-
a) = 6.6 Hz, J(H-2, H-1b) = 2.0 Hz, H-2), 5.72 (1H, d,
J(H-31a, H-31b) = 5.5 Hz, H-31a), 5.80 (1H, d, J(H-
1b, H-31a) = 5.6 Hz, H-31b). MS m/z (relative intensi-
(
1
6.1.20. Methyl 2-methoxy-3-oxolupa-1,20(29)-dien-28-
oate (3e). KOH (0.5 g, 8.8 mmol) in water (10 mL) was
added to a mixture of diosphenol 3a (1 g, 2.1 mmol) in
dioxan (20 mL) and dimethylsulfate (2 g, 16 mmol).
The mixture was then refluxed for 1 h and worked-up.
Crude product was crystallized from methanol to give
compound 3e (0.95 g, 87% yield): mp 105–107 ꢁC (meth-
3
+
ty): 568 (14, M ), 553 (0.5), 538 (0.2), 526 (100), 496 (7),
78 (4), 453 (50), 436 (12), 408 (49), 393 (8), 356 (5), 320
4
(
(
16), 247 (14), 235 (23), 218 (24), 205 (71), 189 (71), 175
42), 161 (29), 147 (39), 135 (57), 119 (89), 105 (92). Anal.
À1
anol). [a] +46ꢁ (c 0.57). IR m (CHCl ) cm : 1713, 1674,
D
3
9
Calcd for C H O : C, 73.91%; H, 9.21%. Found C,
3
1642, 1621.
5
52
6
7
4.02%; H, 9.17%.
6
.1.21. Pivaloyloxymethyl 2-pivaloyloxy-3-oxolupa-
6
.1.16. 2-Hydroxy-3-oxolupa-1,20(29)-dien-28-oic acid
3a). This product was obtained by oxidation of 1e
1,20(29)-dien-28-oate (3f). 0.3 g (0.7 mmol) of 3a was
treated with PomCl in the general manner to give white
oil of 3c (0.2 g, 52% yield): mp 103–105 ꢁC (methanol).
(
(
(
10 g, 22 mmol) with air in a mixture of tert-butyl alcohol
900 mL) and tert-butoxide (10 g, 88 mmol) for 40 min
À1
[a] +34ꢁ (c 0.80). IR m (CHCl ) cm : 1747, 1682,
D
3
9
1
according to lit. This yielded 3a (4.1 g, 82%): mp 204–
2
1644. H NMR d 0.97, 1.01, 1.12, 1.15, 1.19 (15H, all
s, 5 · CH ), 1.22 (9H, s, 3 · CH Pom), 1.29 (9H, s,
05 ꢁC (diethylether). [a]D +12ꢁ (c 0.56). IR m
3
3
À1
9
(
CHCl ) cm : 1730 sh, 1696, 1669, 1643.
3 · CH Piv-2), 1.68 (3H, m, H-30), 1.76–1.95 (3H, m),
3
3
2
.34–2.36 (2H, m), 2.98 (1H, td, J(H-19b, H-
6.1.17. Methyl 2-hydroxy-3-oxolupa-1,20(29)-dien-28-
oate (3b). This product was obtained by oxidation of 1f
18a) = 11.2 Hz, J(H-19b, H-21a) = 11.2 Hz, J(H-19b,
H-21b) = 4.8 Hz, H-19 b), 4.61 (1H, m, H-29 pro-E),
4.74 (1H, bd, J = 2.3 Hz, H-29 pro-Z), 5.76 (1H, d,
J(H-31a, H-31b) = 5.4 Hz, H-31a), 5.80 (1H, d, J(H-
31b, H-31a) = 5.4 Hz, H-31b), 6.71 (1H, s, H-1). MS
(
(
6 g, 13 mmol) with air in a mixture of tert-butyl alcohol
450 mL) and tert-butoxide (6 g, 53 mmol) for 40 min
9
according to lit. This yielded 3b (4.5 g, 75%): mp 119–
1
+
22 ꢁC (diethylether). [a]D +3ꢁ (c 0.64). IR
m
m/z (relative intensity): 666 (2, M ), 636 (1), 565 (3),
553 (5), 535 (2), 507 (31), 469 (13), 451 (5), 421 (16),
À1
9
(
CHCl ) cm : 1720 sh, 1643.
3
238 (100). Anal. Calcd for C H O : C, 73.89%; H,
41 62 7
9.37%. Found C, 73.75%; H, 9.25%.
6
.1.18. Pivaloyloxymethyl 2-hydroxy-3-oxolupa-1,20(29)-
dien-28-oate (3c). 0.3 g (0.7 mmol) of 3a was treated with
PomCl in the general manner to give white crystals of 3c
6.1.22. Preparation of 2,3 secoderivatives 4a and 4b. A
solution of each diosphenol 9, 10 (4.5 mmol) in a mixture
of KOH (6.5 g) and methanol (350 mL) was heated under
reflux and hydrogen peroxide (35 mL, 30%) was added
during 100 min. The solution was then worked-up and
chromatographed over silica gel (200 g), eluted with chlo-
roform/ethyl acetate/acetic acid (100:15:1) and crystal-
lized to give secoacids 16 and 17 in 35%, respectively,
80% yields.
(
0.2 g, 52% yield): mp 108–111 ꢁC (methanol). [a] +37ꢁ
D
À1
(
c 0.65). IR m (CHCl ) cm : 1796, 1747vb, 1668, 1644.
3
1
H NMR d 0.96, 0.99, 1.10, 1.12, 1.20 (15H, all s,
5
2
2
· CH ), 1.22 (9H, s, CH -Pom), 1.68 (3H, m, H-30),
3
3
.99 (1H, td, J(H-19b, H-18a) = 11.0 Hz, J(H-19b, H-
1a) = 11.0 Hz, J(H-19b, H-21b) = 4.9 Hz, H-19b), 4.63
(
1H, m, H-29 pro-E), 4.74 (1H, bd, J = 2.0 Hz, H-29
pro-Z), 5.76 (1H, d, J(H-31a, H-31b) = 5.6 Hz, H-31a),
.80 (1H, d, J(H-31b, H-31a) = 5.4 Hz, H-31b), 5.90
1H, s, OH), 6.44 (1H, s, H-1). MS m/z (relative intensity):
5
(
6.1.23. 2,3-Secolup-20(29)en-2,3,28-trioic acid (4a). Mp
276–277 ꢁC (methanol/chloroform). [a] +10ꢁ (c 0.31).
+
5
(
82 (3, M ), 552 (7), 468 (14), 450 (6), 423 (41), 407 (2), 340
9), 238 (61), 215 (100). Anal. Calcd for C H O : C,
D
À1
9
IR m (CHCl ) cm : 2600-3400, 1725, 1643.
3
3
6
54
6
7
4.19%; H, 9.34%. Found C, 74.01%; H, 9.22%.
6
.1.24. 28-Methyl ester of 2,3-secolup-20(29)en-2,3,28-
6.1.19. Acetoxymethyl 2-hydroxy-3-oxolupa-1,20(29)-
dien-28-oate (3d). 0.3 g (0.7 mmol) of 3a was treated
trioic acid (4b). Mp 132–135 ꢁC (hexane/diethylether).
À1
[a] +23ꢁ (c 0.27). IR m (CHCl ) cm : 2400–3400, 1718,
D
3
9
with AcmBr in the general manner to give white oil of
3
1690, 1642.
d (0.17 g, 48% yield): mp 65–68 ꢁC (methanol). [a]
D
À1
1
+
13ꢁ (c 0.42). IR m (CHCl ) cm : 1748, 1644.
H
6.1.25. Trimethyl 2,3-secolup-20(29)en-2,3,28 trioate
(4c). Two hundred milligram (0.4 mmol) of 4a was treat-
ed with CH N using general procedure which yielded
3
NMR d 0.96, 0.99, 1.10, 1.12, 1.20 (15H, all s,
5
2
1
· CH ), 1.69 (3H, m, H-30), 2.11 (3H, s, CH Acm),
3
3
2
2
.17–2.30 (2H, m), 3.01 (1H, td, J(H-19b, H-
8a) = 11.0 Hz, J(H-19b, H-21a) = 11.0 Hz, J(H-19b,
crystals of 4c (150 mg, 69% yield): mp 188–190 ꢁC
À1
(methanol). [a] À8ꢁ (c 0.44). IR m (CHCl ) cm
:
D
3
9
H-21b) = 5.0 Hz, H-19 b), 4.63 (1H, m, H-29 pro-E),
.75 (1H, bd, J = 2.3 Hz, H-29 pro-Z), 5.73 (1H, d,
J(H-31a, H-31b) = 5.5 Hz, H-31a), 5.81 (1H, d, J(H-
1b, H-31a) = 5.5 Hz, H-31b), 5.90 (1H, bs, OH), 6.44
1720vb, 1641.
4
6.1.26. Tripivaloyloxymethyl 2,3-secolup-20(29)en-2,3,28
trioate (4d). One hundred fifty milligram (0.3 mmol) of
4a was treated with PomCl using general procedure
which yielded crystalls of 4d (140 mg, 56% yield): mp
3
+
(
1H, s, H-1). MS m/z (relative intensity): 540 (25, M ),
26 (5), 480 (14), 468 (100), 450 (27), 422 (79), 408
5

M. Urban et al. / Bioorg. Med. Chem. 13 (2005) 5527–5535
5533
8
0–82 ꢁC (methanol). [a]
+15ꢁ (c 0.26). IR
m
(19), 439 (13), 411 (39), 383 (15), 369 (100), 351 (46),
273 (29), 247 (19), 239 (29), 215 (13), 201 (32), 189
(34), 175 (34). Anal. Calcd for C H O : C, 69.32%;
D
1
À1
(
CHCl ) cm : 1749, 1643. H NMR d 0.91, 0.92, 0.98
3
(
(
1
9H, all s, 3 · CH ), 1.20 (9H, s, 3 · CH Pom-2), 1.20
3
3
43 68 10
9H, s, 3 · CH Pom-3), 1.21 (9H, s, 3 · CH Pom-28),
H, 9.20%. Found C, 69.30%; H, 9.17%.
3
3
.23, 1.25 (6H, all s, 2 · CH ), 1.66 (3H, s, H-30),
.78–1.95 (2H, m), 2.18–2.30 (2H, m), 2.33–2.41 (2H.
3
1
6.1.29. 28-Methyl 2,3-diacetoxymethyl 2,3-secolup-
20(29)en-2,3,28 trioate (4g). Three hundred milligram
(0.6 mmol) of 4b was treated with PomCl using general
m), 2.41 (1H, d, J(H-1a, H-1b) = 18.5 Hz, H-1a), 2.50
1H, d, J(H-1b, H-1a) = 18.5 Hz, H-1b), 2.97 (1H, td,
J(H-19b, H-18a) = 11.1 Hz, J(H-19b, H-
1a) = 11.1 Hz, J(H-19b, H-21b) = 4.6 Hz, H-19b),
(
procedure to give crystals of 4g (200 mg, 52% yield): mp
126–127 ꢁC, [a]D +6.0ꢁ (c 0.34). IR m (CHCl ) cm
À1
2
4
2
:
3
1
.58 (1H, m, H-29 pro-E), 4.72 (1H, bd, J = 2.3 Hz, H-
9 pro-Z), 5.62 (1H, d, J = 5.4 Hz), 5.70 (1H, d,
1761b, 1722, 1642. H NMR d 0.92, 0.98, 1.25, (15H,
all s, 5 · CH ), 1.68 (3H, m, H-30), 1.83-1.93 (2H, m),
3
J = 5.5 Hz), 5.75 (1H, d, J = 5.3 Hz), 5.78 (1H, d,
J = 5.5 Hz), 5.79 (1H, d, J = 5.5 Hz), 5.82 (1H, d,
J = 5.5 Hz, 3 · CH Pom). MS m/z (relative intensity):
8
6
5
2.09, 2.11 (6H, all s, 2 · CH -Acm), 2.19-2.30 (3H, m),
3
2.37 (1H, m), 2.39 (1H, d, J(H-1a, H-1b) = 18.2 Hz,
H-1a), 2.53 (1H, d, J = 18.0 Hz, H-1b), 2.99 (1H, m,
2
+
44 (1, M ), 799 (0.9), 712 (9.4), 684 (40), 670 (10),
53 (16), 642 (6), 599 (6), 571 (16), 553 (20), 523 (15),
11 (28), 483 (100), 469 (32), 439 (48), 397 (41), 351
H-19b), 3.66 (3H, s, OCH ), 4.58 (1H, m, H-29 pro-E),
3
4.73 (1H, bd, J = 2.3 Hz, H-29 pro-Z), 5.65 (1H, d,
J = 5.5 Hz), 5.72 (1H, d, J = 5.7 Hz), 5.74 (1H, d,
(
1
62), 309 (34), 215 (24), 189 (41), 172 (33), 163 (21),
45 (21), 133 (27), 107 (38). Anal. Calcd for
C H O : C, 68.22%; H, 9.06%. Found C, 69.99%;
J = 5.8 Hz), 5.76 (1H, d, J = 5.5 Hz, 2 · CH Acm).
2
+
MS m/z (relative intensity): 660 (2, M ), 600 (5), 528
(20), 500 (31), 471 (27), 441 (48), 411 (48), 383 (19),
369 (100), 351 (67), 309 (19), 273 (43), 253 (43), 227
4
8
76 12
H, 8.97%.
(70), 201 (57), 187 (80), 176 (77), 135 (46), 121 (75).
Anal. Calcd for C H O : C, 67.25%; H, 8.54%.
Found C, 67.36%; H, 8.63%.
6
.1.27. Triacetoxymethyl 2,3-secolup-20(29)en-2,3,28 tri-
3
7
56 10
oate (4e). Two hundred milligram (0.4 mmol) of 4a was
treated with PomCl using general procedure and chro-
matographed on HPLC (phase 25) to give oil of 4e
6.1.30. 1b-Hydroxy-2-oxa-3-oxolup-20(29)en-28-oic acid
(5a). This product was obtained by oxidation of 3a (1 g,
2.2 mmol) with air in a mixture of tert-butyl alcohol
(100 mL) and tert-butoxide (6 g, 53 mmol) for 48 h
(
130 mg, 49% yield): mp < 20 ꢁC. [a] +12ꢁ (c 0.59).
D
À1
1
IR m (CHCl ) cm : 1761b, 1642. H NMR d 0.92,
3
0
.92, 0.99, 1.25, 1.25, (15H, all s, 5 · CH ), 1.67 (3H,
3
9
m, H-30), 1.82-1.94 (2H, m), 2.09 (3H, s, Ac), 2.10
3H, s, Ac), 2.11 (3H, s, Ac), 2.38 (1H, d, J = 18.2 Hz,
H-1a), 2.53 (1H, d, J = 17.9 Hz, H-1b), 3.00 (1H, td,
J(H-19b, H-18a) = 11.1 Hz, J(H-19b, H-
1a) = 11.1 Hz, J(H-19b, H-21b) = 4.8 Hz, H-19b),
according to lit. This yielded 5a (0.5 g, 50% yield): mp
275–276 ꢁC (methanol). [a] À116ꢁ (c 0.02). IR m
D
(
À1
9
(CHCl ) cm : 3280 vb, 1693, 1643.
3
2
4
2
6.1.31. Methyl 1b-hydroxy-2-oxa-2-oxolup-20(29)en-28-
oate (5b). This product was obtained by oxidation of 3b
(1 g, 2.1 mmol) with air in a mixture of tert-butyl alco-
hol (100 mL) and tert-butoxide (6 g, 53 mmol) for 48 h
.59 (1H, m, H-29 pro-E), 4.73 (1H, bd, J = 2.3 Hz, H-
9 pro-Z), 5.66 (1H, d, J = 5.5 Hz), 5.71 (1H, d,
J = 5.5 Hz), 5.72 (1H, d, J = 5.7 Hz), 5.75 (1H, d,
J = 5.6 Hz), 5.76 (1H, d, J = 5.7 Hz), 5.80 (1H, d,
9
according to lit. This yielded 5b (0.7 g, 70% yield): mp
J = 5.5 Hz, 3 · CH Acm). MS m/z (relative intensity):
144–145 ꢁC (methanol). [a] +14ꢁ (c 0.56). IR m
2
D
+
18 (not found, M ), 628 (9), 600 (36), 586 (16), 558
À
(CHCl ) cm 1: 3200–3550, 1720, 1642.
3
9
7
(
(
1
9), 529 (19), 511 (12), 469 (19), 441 (7), 427 (41), 411
15), 397 (40), 369 (19), 351 (37), 331 (20), 309 (18),
89 (35). Anal. Calcd for C H O : C, 65.16%; H,
39 58 12
6.1.32. Reaction of lactol 5a with acetic anhydride in
pyridine. Ten milliliter of acetic anhydride was added to
a solution of both: 5a (300 mg, 0.6 mmol) in pyridine
8
.13%. Found C, 65.53%; H, 8.27%.
(10 mL), and 5b (300 mg, 0.6 mmol) in pyridine
6
2
.1.28. 28-Methyl 2,3-dipivaloyloxymethyl 2,3-secolup-
0(29)en-2,3,28 trioate (4f). Two hundred milligram
(10 mL). The mixture was stirred at room temperature
for 3 days and worked up. Crude products were than
purified by HPLC in phase 10 to give 5c and 5e (from
5a) and 5d (from 5b).
(0.4 mmol) of 4b was treated with PomCl using general
procedure and chromatographed on HPLC (phase 10)
to give crystals of 4e (150 mg, 51% yield): mp 113–
À1
1
1
3
3
15 ꢁC. [a] +3ꢁ (c 0.35). IR m (CHCl ) cm : 1749,
6.1.33. 1b-Acetoxy-2-oxa-3-oxolup-20(29)en-28-oic acid
(5c). It was obtained 150 mg (46% yield): mp 258–259 ꢁC
D
3
1
723sh, 1642. H NMR d 0.91, 0.91, 0.98, (9H, all s,
· CH ), 1.20 (9H, s, 3 · CH Pom), 1.21 (9H, s,
À1
(methanol). [a] +51ꢁ (c 0.36). IR m (CHCl ) cm : 1773,
3
3
D
3
1
· CH Pom), 1.23, 1.25, (6H, all s, 2 · CH ), 1.67
1739, 1695, 1643. H NMR d 0.98, 0.99, 1.06, 1.22, 1.30
3
3
(3H, m, H-30), 2.41 (1H, d, J = 18.5 Hz, H-1a), 2.55
(1H, d, J = 18.5 Hz, H-1b), 3.00 (1H, m, H-19b), 4.57
(1H, m, H-29 pro-E), 4.72 (1H, bd, J = 2.4 Hz, H-29
(15H, all s, 5 · CH ), 1.68 (3H, m, H-30), 1.95–2.04 (2H,
3
m), 2.16 (3H, s, OAc), 2.22 (1H, ddd, J = 13.0 Hz,
J = 12.0 Hz, J = 3.7 Hz), 2.29 (1H, dt, J = 12.5 Hz,
1
2
3
1
pro-Z), 5.62 (1H, d, J = 5.3 Hz), 5.71 (1H, d,
J = 5.5 Hz), 5.78 (1H, d, J = 5.7 Hz), 5.82 (1H, d,
J = 3.0 Hz, J = 3.0 Hz), 2.97 (1H, td, J(H-19b, H-
2 3
18a) = 11.2 Hz, J(H-19b, H-21a) = 11.2 Hz, J(H-19b,
H-21b) = 4.6 Hz, H-19b), 4.63 (1H, m, H-29 pro-E),
4.73 (1H, bd, J = 2.3 Hz, H-29 pro-Z), 6.20 (1H, s, H-
J = 5.2 Hz, 2 · CH Pom), MS m/z (relative intensity):
2
+
44 (1, M ), 744 (1), 729 (0.5), 714 (0.6), 684 (3), 630
1), 612 (1), 583 (84), 570 (18), 542 (23), 483 (18), 471
7
(
+
1a). MS m/z (relative intensity): 514 (not found, M ),

5534
M. Urban et al. / Bioorg. Med. Chem. 13 (2005) 5527–5535
4
79 (1), 455 (20), 439 (10), 426 (8), 408 (28), 393 (14), 382
to give crystals of 6b (185 mg, 42% yield): mp 107–
112 ꢁC (methanol). [a]D À14ꢁ (c 0.68). IR
(
(
16), 367 (18), 356 (12), 340 (23), 321 (11), 311 (8), 259
15), 241 (41), 220 (38), 201 (35), 187 (100). Anal. Calcd
m
À1
1
(CHCl ) cm : 1746sh, 1705, 1643. H NMR d 0.96,
3
for C H O : C, 72.34%; H, 9.01%. Found C, 72.21%;
3
H, 9.17%.
1.02, 1.05, 1.07, 1.15 (15H, all s, 5 · CH ), 1.21 (9H, s,
1
46
6
3
3 · CH Pom), 1.21 (9H, s, 3 · CH Pom), 1.66 (3H,
3
3
m, H-30), 1.79 (1H, dd, J = 12.7 Hz, J = 3.1 Hz),
1
2
6
.1.34. Acetyl 1b-acetoxy-2-oxa-3-oxolup-20(29)en-28-
oate (5e). It was obtained 90 mg (25% yield): mp 258–
59 ꢁC (not crystallized). [a]D +48ꢁ (c 0.21). IR m
1.82–1.95 (2H, m), 2.10 (1H, dd, J1 = 12.5 Hz,
J2 = 2.1 Hz), 2.20–2.34 (2H, m), 2.94 (1H, td, J(H-19b,
H-18a) = 11.1 Hz, J(H-19b, H-21a) = 11.1 Hz, J(H-
19b, H-21b) = 4.5 Hz, H-19b), 4.58 (1H, m, H-29 pro-
E), 4.70 (1H, bd, J = 2.1 Hz, H-29 pro-Z), 5.66 (1H, d,
J = 5.4 Hz), 5.75 (1H, d, J = 5.3 Hz), 5.75 (1H, d,
2
À1
1
(
CHCl ) cm : 1812, 1772, 1741, 1697, 1643. H NMR d
3
0
.97, 1.02, 1.07, 1.23, 1.29 (15H, all s, 5 · CH ), 1.67
3
(
2
3H, m, H-30), 1.92–2.04 (2H, m), 2.15 (3H, s, 1-OAc),
.23 (3H, s, 28-OAc), 2.23 (1H, m), 2.31 (1H, ddd,
J = 13.1 Hz, J = 11.7 Hz, J = 3.7 Hz), 2.94 (1H, td,
J = 5.5 Hz), 5.78 (1H, d, J = 5.3 Hz, 2 · CH Pom),
2
9.22 (1H, s, H-1). MS m/z (relative intensity): 700 (not
found, M ), 673 (1), 643 (1), 601 (1), 573 (1), 543 (2),
1
2
3
+
J(H-19b, H-18a) = 10.4 Hz, J(H-19b, H-21a) = 10.4 Hz,
J(H-19b, H-21b) = 4.6 Hz, H-19b), 4.64 (1H, m, H-29
pro-E), 4.73 (1H, bd, J = 2.1 Hz, H-29 pro-Z), 6.20 (1H,
s, H-1a). MS m/z (relative intensity): 556 (not found,
513 (5), 485 (3), 458 (6), 440 (13), 413 (35), 259 (10),
213 (12), 187 (85), 139 (79), 109 (100). Anal. Calcd for
C H O : C, 70.25%; H, 9.20%. Found C, 70.41%; H,
4
1
64
9
+
M ), 515 (1), 497 (1), 469 (2), 455 (4), 439 (1), 408 (10),
9.33%.
3
2
93 (3), 382 (3), 367 (3), 356 (1), 340 (2), 321 (1), 311 (3),
41 (17), 220 (19), 201 (23), 187 (42), 175 (38), 107 (100).
6.1.38.
Diacetoxymethyl
1-oxo-1,3-seco-2-norlup-
Anal. Calcd for C H O : C, 71.19%; H, 8.69%. Found
7
C, 71.27%; H, 8.77%.
20(29)en-3,28-dioate (6c). Three hundred milligram
(0.6 mmol) of 5a was treated with AcmBr using general
procedure and chromatographed on HPLC in phase 25
to give crystals of 6c (145 mg, 37% yield): mp 40–41 ꢁC
3
3
48
6
.1.35. Methyl 1b-acetoxy-3-oxo-2-oxalup-20(29)en-28-
oate (5d). It was obtained 5d (150 mg, 45% yield): mp
01–203 ꢁC (methanol). [a]D +35ꢁ (c 0.29). IR m
(methanol/pentane). [a]
À14ꢁ (c 0.36). IR
m
D
À1
1
2
(CHCl ) cm : 1754sh, 1643. H NMR d 0.96, 1.03,
3
À1
1
(
CHCl ) cm : 1770sh, 1738, 1723, 1643. H NMR d
1.05, 1.08, 1.17 (15H, all s, 5 · CH ), 1.66 (3H, m, H-
3
3
0
.96, 0.97, 1.06, 1.23, 1.29 (15H, all s, 5 · CH ), 1.67
30), 1.79 (1H, dd, J = 12.9 Hz, J = 3.1 Hz), 1.84–1.94
3
1
2
(
3H, m, H-30), 2.15 (3H, s, OAc), 2.20–2.28 (3H, m),
(2H, m), 2.10, 2.11 (3H, all s, 2 · CH Acm) 2.20–2.29
3
2
2
3
.96 (1H, td, J(H-19b, H-18a) = 10.8 Hz, J(H-19b, H-
1a) = 10.8 Hz, J(H-19b, H-21b) = 4.4 Hz, H-19b),
(2H, m), 2.97 (1H, td, J(H-19b, H-18a) = 11.2 Hz,
J(H-19b, H-21a) = 11.2 Hz, J(H-19b, H-21b) = 4.8 Hz,
H-19b), 4.59 (1H, m, H-29 pro-E), 4.70 (1H, bd,
J = 2.2 Hz, H-29 pro-Z), 5.65 (1H, d, J = 5.6 Hz), 5.71
.67 (3H, s, OCH ), 4.61 (1H, m, H-29 pro-E), 4.72
3
(
MS m/z (relative intensity): 528 (2, M ), 468 (99), 453
1H, bd, J = 2.3 Hz, H-29 pro-Z), 6.20 (1H, s, H-1a).
+
(2H, d, J = 5.7 Hz), 5.79 (1H, d, J = 5.5 Hz, 2 · CH
Acm), 9.16 (1H, s, H-1). MS m/z (relative intensity):
2
(
(
(
14), 446 (6), 436 (12), 425 (9), 409 (46), 396 (27), 381
23), 365 (12), 354 (45), 339 (9), 321 (18), 305 (10), 273
12), 255 (13), 247 (24), 241 (27), 220 (39), 201 (37),
+
616 (not found, M ), 588 (1), 516 (2), 500 (4), 471 (6),
427 (2), 409 (5), 339 (47), 259 (58), 213 (49), 201 (60),
187 (100). Anal. Calcd for C H O : C, 68.16%; H,
8.50%. Found C, 68.19%; H, 8.44%.
1
87 (100), 175 (62), 149 (49), 133 (57), 119 (70), 107
70). Anal. Calcd for C H O : C, 72.69%; H, 9.15%.
3
5
52
9
(
Found C, 72.59%; H, 8.97%.
3
2
48
6
6.1.36. Dimethyl 1-oxo-1,3-seco-2-norlup-20(29)en-3,28-
dioate (6a). Three hundred milligram (0.6 mmol) of 5a
Acknowledgments
was treated with CH N using general procedure and
2
This study was supported in part by the Ministry of Edu-
cation of the Czech Republic (MSM 6198959216), which
paid for instrumental equipment, and the Czech Science
Foundation (203/03/D152), which paid for the chemicals
and all other material support. Biological testing was sup-
ported by the Czech Science Foundation (301/03/1570).
Repair parts for HPLC and steam generator were paid
from MPO project (FT-TA/027). All deuterosolvents
for NMR spectra measurement were paid from the Czech
Science Foundation 203/05/P025. We are grateful to Sta-
nislav Hilgard and Miroslav Kvasnica for measurement
of IR spectra. Special thanks to Bohunka Sperlichova
for measurement of optical rotations.
2
chromatographed on HPLC in phase 10 to give crystals
of 6a (100 mg, 31% yield): mp 175–177 ꢁC (methanol).
À1
[
1
a] À26ꢁ (c 0.48). IR m (CHCl ) cm : 1743, 1719,
D
3
1
643. H NMR d 0.95, 1.03, 1.04, 1.07, 1.17 (15H, all
s, 5 · CH ), 1.61 (1H, t, J = 11.5 Hz, J = 11.5 Hz, H-
3
1
2
1
1
8a), 1.66 (3H, m, H-30), 2.96 (1H, td, J(H-19b, H-
8a) = 11.1 Hz, J(H-19b, H-21a) = 11.1 Hz, J(H-19b,
H-21b) = 4.6 Hz, H-19b), 3.59 (3H, s, OCH ), 3.66
3
(
bd, J = 2.0 Hz, H-29 pro-Z), 9.13 (1H, s, H-1). MS m/z
3H, s, OCH ), 4.58 (1H, m, H-29 pro-E), 4.71 (1H,
3
+
(
(
relative intensity): 500 (6, M ), 457 (1), 441 (3), 411
5), 175 (45), 102 (70). Anal. Calcd for C H O : C,
3
1
48
5
7
4.36%; H, 9.66%. Found C, 74.55%; H, 9.71%.
6
2
.1.37. Dipivaloyloxymethyl 1-oxo-1,3-seco-2-norlup-
0(29)en-3,28-dioate (6b). Three hundred milligram
References and notes
(0.6 mmol) of 5a was treated with PomCl using general
procedure and chromatographed on HPLC in phase 15
1. Evers, M.; Poujade, C.; Soler, F.; Ribeill, Y.; James, C.;
Lelicevre, Y.; Gueguen, J. C.; Reisdorf, D.; Morize, I.;

M. Urban et al. / Bioorg. Med. Chem. 13 (2005) 5527–5535
5535
Pauwels, R.; De Clercq, E.; Henin, Y.; Bousseau, A.;
Mayaux, J. F.; Le Pecq, J. B.; Dereu, N. J. Med. Chem.
10. Kvasnica, M.; Sarek, J.; Klinotova, E.; Dzubak, P.;
Hajduch, M. Bioorg. Med. Chem. 2005, 13, 3447–3454.
11. Sarek, J.; Klinot, J.; Brazinova, S.; Dzubak, P.; Klinotova, E.;
Noskova, V.; Krecek, V.; Korinkova, J.; Thomson, J. O.;
Janostakova, J.; Wang, S.; Parsons, S.; Fischer, P. M.; Zhelev,
N. Z.; Hajduch, M. J. Med. Chem. 2003, 46, 5402–5415.
12. Setti, E. L.; Mascretti, O. A. J. Chem. Soc., Perkin Trans.
1988, 1, 2059–2060.
1
996, 39, 1056–1068.
2
. Costantini, P.; Jacotot, E.; Decaudin, D.; Kroemer, G. J.
Natl. Cancer Inst. 2000, 92, 1042–1053.
3
. Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W. W.;
Fong, H. H. S.; Kinghorn, A. D.; Brown, D. M.; Wani,
M. C.; Wall, M. E.; Hieken, T. J.; Gupta, T. K. D.;
Pezzuto, J. M. Nat. Med. 1995, 1, 1046–1051.
. Jeong, H. J.; Chai, H. B.; Park, S. Y.; Kim, D. S. H. L.
Bioorg. Med. Chem. Lett. 1999, 9, 1201–1204.
. Fulda, S.; Jeremias, I.; Pietsch, T.; Debatin, K. M. Int. J.
Cancer 1999, 82, 435–441.
. Hajduch, M.; Sarek, J. PCT Intl. Patent Appl. Publ. WO
13. Endova, M.; Klinotova, E.; Sejbal, J.; Maca, B.; Klinot,
J.; Protiva, J. Collect. Czech. Chem. Commun. 1994, 59,
1420–1429.
4
5
6
7
8
9
14. Zagi, A.; Okamura, N.; Haraguchi, Y.; Noda, K.;
Nishioka, I. Chem. Pharm. Bull. 1978, 26, 1798–1801.
15. Akihisa, T.; Takamine, Y.; Yoshizumi, K.; Tokuda, H.;
Kimura, Y.; Ukiya, M.; Nakahara, T.; Yokochi, T.;
Ichiishi, E.; Nishino, H. J. Nat. Prod. 2002, 65, 278–282.
16. Kashiwada, Y.; Chiyo, J.; Ikeshiro, Y.; Nagao, T.; Okabe,
H.; Cosentino, L. M.; Fowke, K.; Morris-Natschke, S. L.;
Lee, K.-H. Chem. Pharm. Bull. 2000, 48, 1387–1390.
17. Noskova, V.; Dzubak, P.; Kuzmina, G.; Ludkova, A.;
Stehlik, D.; Trojanec, R.; Janostakova, A.; Korinkova,
G.; Mihal, V.; Hajduch, M. Neoplasma 2002, 49, 418–425.
0
1/90046, 2001.
. Kim, J. Y.; Koo, H. M.; Kim, D. S. H. L. . Bioorg. Med.
Chem. Lett. 2001, 11, 2405–2408.
. Hata, K.; Hori, K.; Takahashi, S. J. Nat. Prod. 2001, 65,
645–648.
. Urban, M.; Sarek, J.; Klinot, J.; Korinkova, G.; Hajduch,
M. J. Nat. Prod. 2004, 67, 1100–1105.