Synthesis of A-seco derivatives of betulinic acid with cytotoxic activity

Article abstract of DOI:10.1021/np049938m

In this study, the relationships between the chemical structure and cytotoxic activity of betulinic acid (1) derivatives were investigated. Eight lupane derivatives (1-8), one of them new (6), five diosphenols (9-13), four of them new (10-13), two new norderivatives (14 and 15), five seco derivatives (16-20), four of them new (16, 17, 19, and 20), and three new seco-anhydrides (21-23) were synthesized from 1, and their activities were compared with the activities of known compounds. The effects of substitution on the A-ring and esterification of the carboxyl group in position 28 on cytotoxicity were of special interest. Significant cytotoxic activity against the T-lymphoblastic leukemia cell line CEM was found in diosphenols 9 and 13 (TCS₅₀ 4 and 5 μmol/L) and seco-anhydrides 22 and 23 (TCS₅₀ 7 and 6 μmol/L). All compounds were also tested on cancer cell lines HT 29, K562, K562 Tax, and PC-3, and these confirmed activity of diosphenols 9, 10, and 11 and anhydride 22. Diosphenols, as the most promising group of derivatives, were further tested on four more lines (A 549, DU 145, MCF 7, SK-Mel2).

Full text of DOI:10.1021/np049938m

1100
J . Nat. Prod. 2004, 67, 1100-1105
Syn th esis of A-Seco Der iva tives of Betu lin ic Acid w ith Cytotoxic Activity
Milan Urban,† J an Sarek,*,† J iri Klinot,† Gabriela Korinkova,‡ and Marian Hajduch‡
Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague,
Hlavova 8, 128 43 Prague 2, Czech Republic, and Laboratory of Experimental Medicine, Department of Pediatrics,
Faculty of Medicine, Palacky University and Faculty Hospital in Olomouc, Puskinova 6, 775 20 Olomouc, Czech Republic
Received February 3, 2004
In this study, the relationships between the chemical structure and cytotoxic activity of betulinic acid (1)
derivatives were investigated. Eight lupane derivatives (1-8), one of them new (6), five diosphenols (9-
13), four of them new (10-13), two new norderivatives (14 and 15), five seco derivatives (16-20), four of
them new (16, 17, 19, and 20), and three new seco-anhydrides (21-23) were synthesized from 1, and
their activities were compared with the activities of known compounds. The effects of substitution on the
A-ring and esterification of the carboxyl group in position 28 on cytotoxicity were of special interest.
Significant cytotoxic activity against the T-lymphoblastic leukemia cell line CEM was found in diosphenols
9 and 13 (TCS50 4 and 5 µmol/L) and seco-anhydrides 22 and 23 (TCS50 7 and 6 µmol/L). All compounds
were also tested on cancer cell lines HT 29, K562, K562 Tax, and PC-3, and these confirmed activity of
diosphenols 9, 10, and 11 and anhydride 22. Diosphenols, as the most promising group of derivatives,
were further tested on four more lines (A 549, DU 145, MCF 7, SK-Mel2).
Triterpenes are widespread in living organisms, and a
number of biologically active derivatives were found in this
group of compounds. This fact has created an interest in
new derivatives with structures similar to known active
natural compounds prepared either by organic synthesis1
or by microbial transformations.2,3 Betulinic acid (1) is a
pentacyclic triterpene, which occurs in the bark of Platanus
acerifolius and many other plant sources.4 Betulinic acid
(1) is a compound with anti-HIV5,6 activity, cytotoxicity,7
and antitumor8 properties. The cytotoxic activity on human
melanoma (MEL-2)9 and lung carcinoma10 (A549) cell lines
was the first to be reported, and subsequent research
showed that amides of 1 had even higher activity than the
free acid, not only on human melanoma (MEL-2) and lung
carcinoma (A549) but also on neuroblastoma,11 medullo-
blastoma,11 glioblastoma,11 ovarian (OVCAR-3),12 colon
(HCT-15),12 and central nervous system carcinoma (XF498)12
cell lines. High in vivo activities on MEL-2,13 the above-
mentioned neuroectodermal tumors,13,14 and Evings sar-
coma14 have also been observed. In more recent studies,15-17
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-28.18 In contrast, all alkyl-esters were found to be
inactive.15,16 Moreover, hydrogenation of the 20(29) double
bond caused significant increase in the cytotoxicity on HeLa
and OVCAR-3 cell lines.16 Analogous oxidation of the 3â-
hydroxy group of 1 afforded derivatives with higher activ-
ity, and substitution with an amino group gave derivatives
with properties similar to 1. In contrast, acylation of the
3â-hydroxy group caused a decrease in cytotoxicity.
Despite the plenthora of recent research in this area, the
influence of derivatization of the A-ring in betulinic acid
(1) on cytotoxicity has not been studied in detail (with the
exception of C-3 derivatives). In this paper, we describe
both the synthesis and structure-activity relationships in
a group of 14 new (6, 10-17, 19-23) and 9 known (1-5,
7-9, 18) compounds, derivatives of betulinic acid (1), which
were modified in the A-ring.18
Resu lts a n d Discu ssion
3â-Hydroxylup-20(29)-en-28-oic acid (1) was extracted
from the bark of plane trees, Platanus acerifolius (collected
in the Czech Republic).
The seco derivatives 14-23 were prepared using three
oxidative steps. Oxidation of acid 1 and ester 2 with CrO3
in DMF18 afforded ketones 7 and 8. Further oxidation was
performed by introducing air into the tert-butyl alcohol
solution of ketones 7 and 8 in the presence of potassium
2-methyl-2-propoxide.18 Two types of products resulted
from the reaction; the major products were diosphenols 9
and 10, and the minor ones were lactols 14 and 15.
Compounds 9 and 10 predominated by optimizing time for
the oxidation (30-40 min). Prolonging the oxidation time
to 48 h, under the same reaction conditions, gave lactols
14 and 15 as the major products. Using pure oxygen
instead of air afforded lactols 14 and 15 in similar yields
in 2 h. Subsequent treatment of diosphenol 9 with dimethyl
sulfate in the presence of KOH afforded methyl ether 13,
and treatment of 9 with acetic anhydride in pyridine gave
acetate 11 in high yields. Diosphenols 9 and 10 were then
cleaved with a solution of hydrogen peroxide and KOH18
in refluxing MeOH. The reactions afforded cleaved seco
derivatives 16 and 17. Reduction of triacid 16 with LAH
in refluxing THF afforded triol 19, and its treatment with
acetic anhydride gave triacetate 20. Treatment of A-seco
derivatives 16 and 17 with acetic anhydride gave seven-
membered cyclic anhydrides 21-23. These anhydrides are
not readily cleaved with water. Anhydride 23 alone lost
an acetyl group from position 28. During the acetylation
of acids with a free C-28-carbonyl group, 1 and 16,
formation of anhydrides 5 and 23 as byproducts were
observed. Although these anhydrides were somewhat un-
stable, HPLC and crystallization afforded pure compounds
5 and 23. A small amount of anhydride 6 was obtained
after the acetylation of 150 g of betulinic acid (1), and this
was stable owing to steric hindrance in the neighborhood
of the anhydride functional group.Our cytotoxicity study
was consistent with the literature.15-17 Both acetylation of
the 3â-OH and esterification of the carboxyl on C-17 caused
a significant decrease in cytotoxicity (e.g., compounds 2,
3, 4, and 8). Betulinic acid (1) was active against CEM
T-lymphoblastic leukemia and SK-Mel2 cell lines, but less
* To whom correspondence should be addressed. Tel: +420221951332.
Fax: +420221951332. E-mail: jan.sarek@volny.cz.
† Charles University, Prague.
‡ Palacky´ University, Olomouc.
10.1021/np049938m CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/23/2004

A-Seco Derivatives of Betulinic Acid
J ournal of Natural Products, 2004, Vol. 67, No. 7 1101
Sch em e 1. Preparation of Diosphenois
Sch em e 2. Oxidative Cleavage of Diosphenols and Further
Ta ble 1. Cytotoxicity of A-Seco Derivatives of Betulinic Acid
Derivatization
against HT 29, K562, K562 Tax, and PC-3 Cell Lines
IC50 (µmol/La)
compound
CEM
HT 29
K562
K562 Tax
PC-3
1
27
150
16
200
22
240
17
89
61
250
24
110
173
17
250
6
56
71
250
18
88
104
6
239
6
8
5
60
11
81
119
145
77
104
9
112
65
250
19
108
242
17
91
109
250
17
2
3
4
5
93
6
187
15
7
8
250
4
250
11
250
10
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
23
6
8
10
20
7
16
11
91
46
19
58
65
219
88
99
11
25
98
5
10
20
96
81
79
250
250
113
250
20
85
116
169
63
250
75
76
11
245
49
250
101
7
12
29
12
89
6
a
The lowest concentration that kills 50% of tumor cells.
active in other lines we used. The R-diketones (diosphenols)
9-13 were more active than betulinic acid (1), even if they
were transformed to enol ether 13. The diosphenols showed
activity in a number of cancer cell lines. In contrast, the
activities of triacid 16 and triol 19 were surprisingly low.
This may have been due to very low solubility of these
compounds in water and organic solvents. Esterification
and acetylation of these compounds did not lead to any
active compounds either. In contrast, easily cleavable
anhydrides 22 and 23 had the highest activities on CEM
among all derivatives synthesized (TCS50 7 µmol/L).
Derivatization of 1 in the A-ring afforded derivatives with
higher cytotoxicity on CEM cell lines than 1 and betulonic
acid (7) used as standards. This led us to investigate all
compounds on four other cancer cell lines (Table 1) and
the most promising on four more lines (Table 2). In
conclusion, the cytotoxicity of new derivatives is not limited
to the CEM line, since similar results were obtained on
the following cell lines: HT 29, K562, K562 Tax, PC-3, A
549, DU 145, MCF 7, SK Mel2 (Tables 1, 2). Several
compounds were not tested on the broader range of cell
lines, mostly because of their poor availability in amounts
large enough for these tests.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined using a Kofler block and are uncorrected. Optical

1102 J ournal of Natural Products, 2004, Vol. 67, No. 7
Urban et al.
Ta ble 2. Cytotoxic Activity of Betulinic Acid (1), Betulonic
Acid (7), and Selected Compounds (9, 10, and 11) on A 549, DU
145, MCF 7, and SK-Mel2 Cell Lines
°C for 3 h. The solution was then cooled to 5 °C for 3 days.
White crystals of 5 were removed from the solution by filtration
under reduced pressure and washed with ethyl acetate and
EtOH, and the filtrate was collected and yielded other com-
pounds as described below. Crystallization from butanone
afforded white crystals of the anhydride of 3â-acetoxylup-20-
(29)-en-28-oic acid and acetic acid (5) (40 g, 23% yield): Rf 0.59;
IC50 (µmol/L (n )4))
compound
A 549
DU 145
MCF 7
SK-Mel2
betulinic acid (1)
betulonic acid (7)
9
10
11
146
15
10
11
11
196
36
23
9
143
29
14
6
21
26
23
11
24
mp 177-178 °C; [R]25 +11° (c 0.42); IR (CHCl3) νmax 1811,
D
1
1720vb, 1643 cm-1; H NMR (CHCl3) δ 0.83, 0.84, 0.85, 0.96,
0.97, 1.69 (18H, all s, 6 × CH3), 1.90-2.02 (2H, m), 2.04 (3H,
s, 3-OAc), 2.23 (3H, s, 28-COOAc), 2.19-2.31 (1H, m), 2.97 (1H,
td, J (H-19â, H-18R) ) 11.2 Hz, J (H-19â, H-21R) ) 11.2 Hz,
J (H-19â, H-21â) ) 4.8 Hz, H-19 â), 4.47 (1H, m, ∑J ) 16.3
Hz, H-3R) 4.62 (1H, m, H-29 pro-E), 4.74 (1H, bd, J ) 2.1 Hz,
H-29 pro-Z); EIMS m/z 540 [M]+ (0.2), 499 (1), 453 (15), 438
(5), 395 (5), 249 (4), 232 (5), 215 (5), 203 (10), 189 (38), 43 (100);
anal C 75.73%, H 9.58%, calcd for C34H52O5, C 75.51%, H
9.69%.
18
17
rotations were measured using CHCl3 solutions (unless oth-
erwise stated) on an Autopol III (Rudolph Research, Flanders,
NJ ) polarimeter. NMR spectra were recorded on a Varian
UNITY INOVA 400 instrument (1H NMR spectra at 399.95
MHz) using CDCl3 solutions (unless otherwise stated), with
SiMe4 as an internal standard. EIMS were 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/z > 50. TLC was carried out using silica
gel 60 F254, and detection was by spraying with 10% aqueous
H2SO4 and heating to 150-200 °C. Column chromatography
was performed using silica gel 60 (Merck 7734). The HPLC
system consisted of a Gilson high-pressure pump (model 361),
a Rheodyne injection valve, preparative column (25 × 250 mm)
with filling silica gel (Biospher 7 µm), differential-refracto-
metrical detector (Laboratorni pristroje, Praha, CZ) connected
with a PC (software Chromulan), and a Gilson automatic
fraction collector (model 246). A mixture of ethyl acetate and
hexane was used as the mobile phase, its composition specified
in each experiment. TLC was carried out on Kieselgel 60 F254
plates (Merck) using toluene/diethyl ether, 5:1, unless other-
wise stated.
Workup refers to pouring the reaction mixture into H2O,
extraction of the product with Et2O, and washing the organic
layer successively with H2O, dilute aqueous HCl, H2O, satu-
rated aqueous NaHCO3, and again H2O, followed by drying
over MgSO4, filtration, and evaporation of the filtrate under
reduced pressure. Analytical samples were dried over P2O5
under diminished pressure.
Potassium tert-butoxide, tert-butyl alcohol, dimethyl sulfate,
dimethylformamide (DMF), tetrahydrofurane, lithium alumi-
num hydride, and chromium(VI) oxide were purchased from
Sigma-Aldrich Company.
Decomposition of anhydride 5 using chromatography over
silica gel eluted with toluene was observed, and this yielded
acetate 4.
The above-mentioned filtrate was worked up in the usual
manner. The obtained 150 g of crude brown powder was then
chromatographed over silica gel (1 kg); two compounds (4 and
6) were obtained: 3â-a cetoxylu p -20(29)-en -28-oic a cid (4)
was the major chromatography product. Crystallization from
MeOH afforded white crystals of 4 (50 g, 31% yield): Rf 0.51;
mp 277-278 °C; [R]25D +22° (c 0.49).20 The minor product was
3â-a cetoxylu p -20(29)-en -28-oic a n h yd r id e (6) (3 g, 2%): Rf
0.59; mp 256-257 °C; [R]25D +6° (c 0.70); IR (CHCl3) νmax 1798,
1723, 1643 cm-1 1H NMR (CHCl3) δ 0.83, 0.84, 0.85, 0.97,
;
(30H, all s, 10 × CH3), 1.69 (6H, s, H-30), 1.93-2.03 (4H, m),
2.04 (6H, s, 2 × Ac), 2.15-2.30 (4H, m), 3.01 (2H, td, J (H-19â,
H-18R) ) 11.4 Hz, J (H-19â, H-21R) ) 11.4 Hz, J (H-19â, H-21â)
) 5.1 Hz, 2 × H-19â), 4.47 (2H, m, ∑J ) 16.2 Hz 2 × H-3R),
4.62 (2H, m, 2 × H-29 pro-E), 4.74 (2H, bd, J ) 2.3 Hz, 2 ×
H-29 pro-Z); EIMS m/z 979 [M]+ (not found), 498 (2), 483 (1),
453 (14), 438 (14), 423 (4), 393 (19), 377 (2), 351 (3), 249 (7),
233 (10), 203 (47), 189 (100); anal C 78.31%, H 10.13%, calcd
for C64H98O7, C 78.48%, H 10.08%.
3-Oxolu p -20(29)-en -28-oic Acid (7). Chromium(VI) oxide
(15.0 g, 150.1 mmol) and sulfuric acid (1 mL, 98%) were added
to a solution of acid 1 (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
H2O, filtered, and washed with H2O. Column chromatography
of crude material 7 (14.1 g) over silica gel (300 g) eluted with
CHCl3 afforded ketone 7 (10.7 g, 71% yield): Rf 0.39; mp 250-
254 °C (MeOH), [R]25 +32° (c 0.37).3
D
3â-Hyd r oxylu p -20(29)-en -28-oic a cid (1). The bark of
plane trees Platanus acerifolius (5 kg) was extracted with
MeOH (15 L, twice). Collected 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
Meth yl 3-Oxolu p -20(29)-en -28-oa te (8). Sodium dichro-
mate (37.0 g, 124.2 mmol) and sodium acetate (8.0 g) were
added to a vigorously stirred solution of crude 2 (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 usual manner. Column chromatography of the crude
product (30.5 g) over silica gel (300 g) eluted with toluene
afforded keto ester 8 (16.1 g, 49% yield): Rf 0.57; mp 161-
165 °C (MeOH); RD +28° (c 0.41).21
Diosp h en ols 9 a n d 10. Each ketone (7 and 8) (17.6 mmol)
was dissolved in a mixture of potassium tert-butoxide (72 g)
in tert-butyl alcohol (700 mL). Air was constantly introduced
into the vigorously stirred solution at 40 °C for 40 min. The
reaction mixture was then poured into dilute HCl and worked
up in the usual manner. The crude product was chromato-
graphed over silica gel (800 g) eluted with toluene/diethyl ether
(15:1) and crystallized from diethyl ether to give compounds
9 and 10 in 75% and 85% yields.
needles (50 g, 1% yield): Rf 0.16; mp 304-305 °C; [R]25 +8°
D
19
(c 0.37); IR (CHCl3) νmax 1720vb, 1643 cm-1
.
Met h yl 3â-H yd r oxylu p -20(29)-en -28-oa t e (2). Diazo-
methane (50 mmol) in diethyl ether was added to a solution
of 1 (5 g, 10.9 mmol) in CHCl3 (400 mL). Organic solvents were
evaporated under vacuum, and the crude product was chro-
matographed over silica gel (50 g) eluted with toluene and then
crystallized from MeOH to afford 2 (4.5 g, 87% yield): Rf 0.25;
mp 224-228 °C; [R]25 +3° (c 0.36); IR (CHCl3) νmax 1720vb,
D
19
1643 cm-1
.
Meth yl 3â-Acetoxylu p -20(29)-en -28-oa te (3). Acetic an-
hydride (6 mL, 60 mmol) was added to a suspension of methyl
ester 2 (15 g, 31.9 mmol) in pyridine (3 mL). The mixture was
worked up after 6 h. The crude product was chromatographed
over silica gel (150 g) eluted with toluene and then crystallized
from MeOH to afford 3 (14 g, 86% yield): Rf 0.65; mp 203-
2-Hyd r oxy-3-oxolu p a -1,20(29)-d ien -28-oic a cid (9): Rf
0.45; mp 203-205 °C (diethyl ether); [R]25 +13° (c 0.75); IR
D
206 °C; [R]25D +17° (c 0.35); IR (CHCl3) νmax 1811, 1720vb, 1643
(CHCl3) νmax 1730 sh, 1696, 1669, 1644 cm-1; 1H NMR δ 0.98,
1.01, 1.10, 1.13, 1.20 (15H, all s, 5 × CH3), 1.70 m (3H, s, H-30),
1.74-1.82 (1H, dm, J ) 13.0 Hz), 1.94-2.06 (2H, m), 2.24 (1H,
ddd, J ) 12.8, 11.6, 3.7 Hz), 2.30 (1H, dt, J ) 12.5, 3.2, 3.2
Hz), 3.02 (1H, td, J (H-19â, H-18R) ) 10.8 Hz, J (H-19â, H-21R)
20
cm-1
.
Acetyla tion of 1. Acetic anhydride (60 mL, 0.6 mol) was
added to a suspension of 150 g of crude betulinic acid (1) in
pyridine (30 mL). The mixture was vigorously stirred at 100

A-Seco Derivatives of Betulinic Acid
J ournal of Natural Products, 2004, Vol. 67, No. 7 1103
) 10.8 Hz, J (H-19â, H-21â) ) 4.8 Hz, H-19â), 4.64 (1H, m,
H-29 pro-E), 4.76 (1H, bd, J ) 2.3 Hz, (H-29 pro-Z), 5.91 (1H,
bs, OH), 6.44 (1H, s, H-1); EIMS m/z 468 [M]+ (43), 453 (2),
422 (22), 407 (5), 379 (3), 340 (23), 295 (5), 269 (12), 248 (7),
235 (15), 215 (100); anal. C 76.99%, H 9.22%, calcd for
g) eluted with toluene/diethyl ether (3:1) to give acid 14 (0.8
g, 53% yield): Rf 0.08; mp 274-276 °C (MeOH); [R]25 -116°
D
(c 0.02); IR (CHCl3) νmax 3280 vb, 1693, 1643 cm-1; 1H NMR δ
0.99, 1.03, 1.18, 1.25, 1.26 (15H, all bs, 5 × CH3), 1.60 (1H, t,
J
) 11, 11 Hz, H-18R), 1.69 (3H, s, H-30), 3.00 (1H, td,
C
30H44O4, C 76.88%, H 9.46%.5
Met h yl
J (H-19â, H-18R) ) 10.8 Hz, J (H-19â, H-21R) ) 10.8 Hz,
J (H-19â, H-21â) ) 4.2 Hz, H-19â), 4.59 (1H, m, H-29 pro-E),
4.72 (1H, bd, J ) 2.1, H-29 pro-Z), 5.22 (1H, s, H-1â); EIMS
m/z 472 [M]+ (1), 454 (2), 444 (7), 426 (26), 411 (14), 397 (37),
385 (11), 367 (8), 357 (27), 339 (28), 327 (15), 316 (18), 301 (8),
259 (79), 248 (17), 213 (18), 201 (26), 189 (49), 55 (100); anal.
C 73.81%, H 9.24%, calcd for C29H44O5, C 73.69%, H 9.38%.
Met h yl 2-Oxa -1r-h yd r oxy-3-oxolu p -20(29)en -28-oa t e
(15). Diosphenol 10 (1.5 g, 3.1 mmol) was oxidized analogously
as described above. The crude product was chromatographed
over silica gel (100 g) eluted with toluene/ether (10:1) to give
lactol 15 (0.9 g, 60% yield): Rf 0.25; mp 143-146 °C (MeOH);
2-h yd r oxy-3-oxolu p a -1,20(29)-d ien -28-oa t e
(10): Rf 0.60; mp 122-124 °C (diethyl ether); RD +3° (c 0.70);
IR (CHCl3) νmax 1720 sh, 1643 cm-1; 1H NMR δ 0.96, 0.98, 1.10,
1.12, 1.20 (15H, all s, 5 × CH3), 1.69 (3H, m, H-30), 1.74-1.82
(1H, dm, J ) 13.3), 1.84-1.98 (2H, m), 2.20-2.30 (2H, m), 3.00
(1H, td, J (H-19â, H-18R) ) 11.1 Hz, J (H-19â, H-21R) ) 11.1
Hz, J (H-19â, H-21â) ) 4.5 Hz, H-19â), 3.68 (1H, s, OCH3), 4.62
(1H, m, H-29 pro-E), 4.75 (1H, bd, J ) 2.3, H-29 pro-Z), 5.88
(1H, s, OH), 6.44 (1H, s, H-1); EIMS m/z 482 [M]+ (91), 467
(4), 450 (6), 422 (50), 407 (9), 354 (18), 273 (19), 235 (12), 215
(100); anal. C 76.90%, H 9.88%, calcd for C31H46O4, C 77.14%,
H 9.61%.
[R]25 +13° (c 0.33); IR (CHCl3) νmax 3200-3550, 1720, 1642
D
1
2-Acetoxy-3-oxolu pa -1,20(29)-d ien -28-oic Acid (11). Ace-
tic anhydride (3 mL, 30 mmol) and DMAP (0.3 g, 2.42 mmol)
were added to a solution of 9 (1.0 g, 2.1 mmol) in pyridine (5
mL). The reaction mixture was worked up after 15 h. Chro-
matography on silica gel (150 g) eluted with toluene/diethyl
ether (25:1) afforded acetate 11 (0.77 g, 71% yield): Rf 0.32;
cm-1; H NMR δ 0.96, 1.00, 1.01, 1.19, 1.26, (15H, all s, 5 ×
CH3), 1.60 (1H, t, J (H-13â, H-18R) ) 12.0 Hz, J (H-18R, H-19â)
) 12.0 Hz, H-18R), 1.68 (3H, s, H-30), 2.98 (1H, td, J (H-19â,
H-18R) ) 11.2 Hz, J (H-19â, H-21R) ) 11.2 Hz, J (H-19â, H-21â)
) 4.4 Hz, H-19â), 3.67 (3H, s, OCH3), 4.59 m, 1 H, (H-29 pro-
E); 4.73 bd, 1H, J ) 2.4 (H-29 pro-Z); 5.30 s, 1H, (H-1â). EIMS
m/z 486 [M]+ (5), 457 (14), 440 (8), 427 (11), 411 (6), 397 (24),
371 (18), 339 (47), 311 (9), 273 (86), 247 (18), 213 (22), 201
(37), 187 (78), 175 (94), 121 (100); anal C 74.15%, H 9.42%,
calcd for C30H46O5, C 74.04%, H 9.53%.
mp 149-151 °C (lyophyllizate from benzene); [R]25 +33° (c
D
1.30); IR (CHCl3) νmax 1758, 1686, 1644 cm-1; 1H NMR δ 1.00,
1.02, 1.11, 1.13, 1.19 (15H, all s, 5 × CH3), 1.69 (3H, m, H-30),
1.79 (1H, m), 1.95-2.06 (2H, m), 2.19 (3H, s, Ac), 2.20-2.33
(2H, m), 3.02 (1H, td, J (H-19â, H-18R) ) 10.7 Hz, J (H-19â,
H-21R) ) 10.7 Hz, J (H-19â, H-21â) ) 4.9 Hz, H-19â), 4.63 (1H,
m, H-29 pro-E), 4.75 (1H, bd, J ) 2.3 Hz, H-29 pro-Z), 6.75
(1H, s, H-1); EIMS m/z 510 [M]+ (3), 468 (30), 450 (8), 422
(10), 407 (2), 340 (2), 315 (3), 43 (100); anal C 75.42%, H 9.00%,
calcd for C32H46O5, C 75.26%, H 9.08%.
Using oxygen for the reaction instead of air allowed us to
reduce the reaction time from 48 to 2 h without marked
influence on yield.
P r ep a r a tion of 2,3-Seco Der iva tives 16-18. A solution
of each diosphenol (9 and 10) (4.5 mmol) in a mixture of KOH
(6.5 g) and MeOH (350 mL) was heated under reflux, and
hydrogen peroxide (35 mL, 30%) was added during 100 min.
The solution was then poured into cold H2O, and the product
was extracted with ethyl acetate (2 × 100 mL). The product
was chromatographed over silica gel (200 g) eluted with CHCl3/
ethyl acetate/acetic acid (100:15:1) and crystallized to give seco-
acids 16 and 17 in 35% and 80% yields, respectively.
2,3-Secolu p -20(29)en -2,3,28-t r ioic a cid (16): Rf 0.22
(CHCl3/EtOAc/AcOH 10:2.5:0.15); mp 276-277 °C (MeOH/
CHCl3); [R]25D +10° (c 0.22); IR (CHCl3) νmax 2600-3400, 1725,
Meth yl 2-Acetoxy-3-oxolu p a -1,20(29)-d ien -28-oa te (12).
Acetic anhydride (1 mL, 10 mmol) and DMAP (0.1 g, 0.81
mmol) were added to a solution of diosphenol 10 (300 mg, 0.6
mmol) in pyridine (2 mL). The reaction mixture was worked
up after 15 h. HPLC in a 10% mixture of ethyl acetate in
hexane afforded acetate 12 (150 mg, 45% yield): Rf 0.51; mp
95-97 °C (MeOH); [R]25 +43° (c 0.35); IR (CHCl3) νmax 1759,
D
1
1720, 1682, 1643 cm-1; H NMR δ 0.98, 0.99, 1.12, 1.13, 1.19
(15H, all s, 5 × CH3), 1.69 (3H, s, H-30), 2.19 (3H, s, OAc),
2.23-2.40 (2H), 3.00 (1H, td, J (H-19â, H-18R) ) 11.1 Hz, J (H-
19â, H-21R) ) 11.1 Hz, J (H-19â, H-21â) ) 4.7 Hz (H-19â), 3.68
(3H, s, OCH3), 4.61 (1H, m, H-29 pro-E), 4.74 (1H, bd, J )
2.3, H-29 pro-Z), 6.75 (1H, s, H-1); EIMS m/z 524 [M]+ (29),
482 (40), 464 (39), 422 (31), 354 (6), 328 (11), 273 (100), 235
(14), 213 (64); anal. C 75.32%, H 9.46%, calcd for C33H48O5, C
75.53%, H 9.22%.
1
1643 cm-1; H NMR (CDCl3/CD3OD) δ 0.93, 0.95, 1.00, 1.23,
1.24 (15H, all s, 5 × CH3), 1.67 (3H, m, H-30), 1.89-2.00 (2H,
m), 2.20-2.31 (2H, m), 2.38-2.45 (2H, m), 2.46 (2H, s, H-1R,
H-1â), 3.00 (1H, td, J (H-19â, H-18R) ) 10.8 Hz, J (H-19â,
H-21R) ) 10.8 Hz, J (H-19â, H-21â) ) 4.4 Hz, H-19â), 4.57 (1H,
m, H-29 pro-E), 4.73 (1H, bd, J ) 2.3 Hz, H-29 pro-Z); EIMS
m/z 502 [M]+ (1), 484 (3), 456 (17), 442 (12), 415 (13), 397 (24),
379 (7), 369 (100), 355 (32), 351 (16), 309 (8), 261 (20), 248
(17), 233 (16), 215 (35), 201 (34), 189 (46); anal C 71.92%, H
9.07%, calcd for C30H46O6, C 71.68%, H 9.22%.
Meth yl 2-Meth oxy-3-oxolu pa-1,20(29)-dien -28-oate (13).
Dimethyl sulfate (2 g, 16 mmol) was added to a vigorously
stirred solution of diosphenol 9 (1 g, 2.1 mmol) in a solution of
KOH (0.5 g, 8.8 mmol) in a mixture of dioxane (20 mL) and
H2O (10 mL). The mixture was then refluxed for 1 h and
worked up in the usual manner. The crude product was
crystallized from MeOH to give compound 13 (0.95 g, 87%
28-Meth yl ester of 2,3-secolu p -20(29)en -2,3,28-tr ioic
a cid (17): Rf 0.25 (CHCl3/EtOAc/AcOH 10:2.5:0.15); mp 132-
135 °C (hexane/diethyl ether); [R]25D +23° (c 0.31); IR (CHCl3)
ν
max 2400-3400, 1718, 1690, 1642 cm-1; 1H NMR (CDCl3/CD3-
yield): Rf 0.36; mp 105-107 °C (MeOH); [R]25 +46° (c 0.57);
OD, 50 °C) δ 0.92, 0.93, 0.96, 1.17, 1.25 (15H, all s, 5 × CH3),
1.68 (3H, s, H-30), 1.82-1.93 (2H), 2.14-2.26 (2H, m), 2.47
(1H, d, J (H-1R, H-1â) ) 19.6 Hz, H-1R), 2.64 (1H, d, J (H-1â,
H-1R) ) 19.6 Hz, H-1â), 2.99 (1H, td, J (H-19â, H-18R) ) 11.0
Hz, J (H-19â, H-21R) ) 11.0 Hz, J (H-19â, H-21â) ) 4.4 Hz,
H-19â), 3.66 (3H, s, OCH3), 4.60 (1H, m, H-29 pro-E), 4.73 (1H,
bd, J ) 2.3, H-29 pro-Z); EIMS m/z 516 [M]+ (2), 498 (11), 470
(8), 456 (23), 439 (12), 429 (24), 411 (9), 397 (10), 384 (8), 369
(93), 351 (14), 275 (59), 262 (41), 249 (24), 215 (48), 201 (59),
189 (100); anal C 71.99%, H 9.42%, calcd for C31H48O6, C
72.06%, H 9.36%.
D
IR (CHCl3) νmax 1713, 1674, 1642, 1621 cm-1; 1H NMR δ 0.99,
0.99, 1.07, 1.19, 1.15, (15H, all s, 5 × CH3), 1.71 (3H, m, H-30),
1.76-1.85 (1H), 1.85-1.98 (2H), 2.24-2.34 (2H), 3.02 (1H, td,
J (H-19â, H-18R) ) 11.0 Hz, J (H-19â, H-21R) ) 11.0 Hz, J (H-
19â, H-21â) ) 4.7 Hz (H-19â), 3.55 (3H, s, 2-OCH3), 3.68 (3H,
s, 28-OCH3), 4.63 (1H, m, H-29 pro-E), 4.76 (1H, bd, J ) 2.3,
H-29 pro-Z), 6.05 (1H, s, H-1); EIMS m/z 496 [M]+ (97), 481
(4), 464 (2), 436 (24), 421 (5), 354 (17), 329 (3), 295 (6), 273
(47), 258 (6), 235 (5), 213 (62), 165 (100); anal. C 77.51%, H
9.62%, calcd for C32H48O4, C 77.38%, H 9.74%.
2-Oxa -1r-h yd r oxy-3-oxolu p -20(29)en -28-oic Acid (14).
Diosphenol 9 (1.5 g, 3.3 mmol) was dissolved in a solution of
potassium tert-butoxide (15 g) in tert-butyl alcohol (140 mL).
Air was constantly introduced into the vigorously stirred
solution at 40 °C for 48 h. The reaction mixture was then
poured into dilute HCl and worked up in the usual manner.
The crude product was chromatographed over silica gel (100
Tr im eth yl 2,3-Secolu p -20(29)en -2,3,28-tr ioa te (18). An
excess of diazomethane in diethyl ether was added to a solution
of triacid 16 (200 mg, 0.4 mmol) in CHCl3 (10 mL). The
solvents were removed in vacuo, and the crude product was
then chromatographed over silica gel (10 g) eluted with toluene
to afford trimethyl ester 18 (150 mg, 69% yield): Rf 0.56; mp
186-188 °C (MeOH); [R]25 -8° (c 0.31); IR (CHCl3) νmax
D

1104 J ournal of Natural Products, 2004, Vol. 67, No. 7
Urban et al.
1720vb, 1641 cm-1; 1H NMR δ 0.89, 0.92, 0.99, 1.22, 1.23 (15H,
all s, 5 × CH3), 1.69 (3H, m, H-30), 1.82-1.94 (2H, m), 2.18-
2.27 (3H, m), 2.36-2.42 (2H, m), 3.00 (1H, td, J (H-19â, H-18R)
) 11.0 Hz, J (H-19â, H-21R) ) 11.0 Hz, J (H-19â, H-21â) ) 4.7
Hz, H-19â), 3.60, 3.64, 3.66 (9H, all s, COOCH3), 4.59 (1H, m,
H-29 pro-E), 4.73 (1H, bd, J ) 2.4, H-29 pro-Z); EIMS m/z 544
[M]+ (1), 513 (1), 485 (5), 471 (16), 443 (9), 411 (7), 383 (33),
369 (64), 187 (24), 169 (100); anal. C 72.94%, H 9.46%, calcd
for C33H52O6, C 72.76%, H 9.62%.22
439 (22), 423 (10), 416 (18), 395 (11), 384 (25), 325 (21), 262
(41), 201 (32), 187 (60), 55 (100); anal. C 74.50%, H 9.47%,
calcd for C31H46O5, C 74.66%, H 9.30%.
An h yd r id e of 2,3-a n h yd r id e of 2,3-secolu p -20(29)-en -
2,3,28-tr ioic a cid a n d a cetic a cid (23): Rf 0.63; mp 152-
154 °C (diethyl ether/hexane); [R]25D +41° (c 0.50); IR (CHCl3)
ν
max 3526, 1809, 1796, 1753, 1702, 1643 cm-1; 1H NMR δ 0.99,
1.00, 1.05, 1.26, 1.37 (15H, all s, 5 × CH3), 1.69 (3H, m, H-30),
1.74-1.82 (2H, m), 1.90-2.04 (m, 2H), 2.21 (1H, d, J ) 13.7
Hz, H-1a), 2.23 (3H, s, Ac), 2.21-2.36 (2H, m), 2.86 (1H, d, J
) 13.9 Hz, H-1b), 2.97 (1H, td, J (H-19â, H-18R) ) 11.1 Hz,
J (H-19â, H-21R) ) 11.1 Hz, J (H-19â, H-21â) ) 4.6 Hz, H-19â),
4.64 (1H, m, H-29 pro-E), 4.74 (1H, bd, J ) 2.0 Hz, H-29 pro-
Z); EIMS m/z 526 [M]+ (1), 498 (2), 484 (6), 466 (3), 456 (6),
438 (29), 423 (5), 395 (7), 371 (5), 325 (8), 248 (15), 201 (17),
187 (24), 43 (100); anal. C 72.69%, H 9.08%, calcd for C32H46O6,
C 72.97%, H 8.80%.
2,3-Secolu p -20(29)-en -2,3,28-tr iol (19). LAH (1 g, 26.4
mmol) was added to a solution of triacid 16 (500 mg, 1 mmol)
in tetrahydrofuran (30 mL), and the mixture was refluxed for
5 h. Ethyl acetate (50 mL) was slowly added after 5 h to
decompose residual LAH, and the mixture was poured into
cold H2O and worked up as usual. The crude product was
chromatographed over silica gel (50 g) eluted with diethyl ether
and crystallized to afford triol 19 (350 mg, 76% yield): Rf 0.13
(CHCl3/EtOAc/AcOH 10:2.5:0.15); mp 234-235 °C (hexane/
diethyl ether); [R]25D +32° (c 0.25); IR (CHCl3) νmax 3626, 1641
Cell Lin es. Cell lines A 549, DU 145, HT 29, K562, MCF
7, PC-3, and SK-Mel2 were all purchased from the American
Tissue Culture Collection (ATTC). Paclitaxel-resistant subline
of K562 cells was kindly provided by Dr. J . Dummont
(University of Lyon, France). The human T-lymphoblastic
leukemia cell line, CEM, was used for routine screening of
compounds. To prove a common mechanism of action, selected
compounds, which showed activity in the screening assay, were
tested in a panel of cell lines. These lines were from different
species and of various histogenetic origin, and they possess
various alterations in their cell cycle-regulatory proteins and
hormone receptor status (Tables 1 and 2). The cells were
maintained in Nunc/Corning 80 cm2 plastic tissue culture
flasks and cultured in cell culture medium (DMEM/RPMI 1640
with 5 g/L glucose, 2 mM glutamine, 100 U/mL penicillin, 100
µg/mL streptomycin, 10% fetal calf serum, and NaHCO3).
Cytotoxicity Assa y. Cell suspensions were prepared and
diluted according to the particular cell type and the expected
target cell density (2500-30 000 cells/well based on cell growth
characteristics). Cells were added by pipet (80 µL) into 96-
well microtiter plates. Inoculates were allowed a preincubation
period of 24 h at 37 °C and 5% CO2 for stabilization. Four-fold
dilutions, in 20 µL aliquots, of the intended test concentration
were added at time zero to the microtiter plate wells. All tested
compounds were dissolved in 10% DMSO, and concentrations
were examined in duplicate. Incubation of the cells with the
test compounds lasted for 72 h at 37 °C, in a 5% CO2
atmosphere at 100% humidity. At the end of the incubation
period, the cells were assayed using MTT. Aliquots (10 µL) 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 µL/
well of 10% aqueous SDS (pH ) 5.5), followed by a further
incubation at 37 °C overnight. The optical density (OD) was
measured at 540 nm with a Labsystem iEMS Reader MF.
Tumor cell survival (TCS) was calculated using the following
equation: TCS ) (ODdrug-exposed well/mean ODcontrol wells) × 100%.
The TCS50 value, the drug concentration lethal to 50% of the
tumor cells, was calculated from appropriate dose-response
curves.
1
cm-1; H NMR δ 0.93, 0.94, 0.97, 1.06, 1.08 (15H, all s, 5 ×
CH3), 1.69 (3H, s, H-30), 2.40 (1H, td, J (H-19â, H-18R) ) 10.9
Hz, J (H-19â, H-21R) ) 10.9 Hz, J (H-19â, H-21â) ) 5.9 Hz,
H-19â), 3.30 (1H, bd, J ) 10.8 Hz), 3.37 (1H, d, J ) 11.1 Hz),
3.43 (1H, d, J ) 11.1 Hz), 3.56 (1H, m), 3.74-3.82 (2H), 4.58
(1H, m, H-29 pro-E), 4.68 (1H, bd, J ) 2.4, H-29 pro-Z); EIMS
m/z 460 [M]+ (1), 442 (2), 429 (4), 411 (4), 399 (1), 383 (2), 369
(5), 357 (3), 245 (12), 215 (13), 189 (19), 81 (100); anal. C 78.46,
H 11.19%, calcd for C30H52O3, C 78.21%, H 11.38%.
2,3-Secolu p -20(29)-en -2,3,28-tr iyl Tr ia ceta te (20). Ace-
tic anhydride (2 mL, 19.6 mmol) was added to a solution of
triol 19 (80 mg, 0.17 mmol) in pyridine. The reaction mixture
was worked up after 12 h, and the crude product was
chromatographed using HPLC eluted with 15% ethyl acetate
in hexane and lyophyllized to afford acetate 20 (50 mg, 43%
yield): Rf 0.38; mp less than +50 °C (tert-butyl alcohol); [R]25
D
+17° (c 0.45); IR (CHCl3) νmax 1729, 1642 cm-1; 1H NMR δ 0.95,
0.97, 1.05, 1.13, 1.27 (15H, all s, 5 × CH3), 1.68 (3H, s, H-30),
2.03, 2.07, 2.09 (9H, all s, 3 × Ac), 2.45 (1H, td, J (H-19â,
H-18R) ) 11.1 Hz, J (H-19â, H-21R) ) 11.1 Hz, J (H-19â, H-21â)
) 5.9 Hz, H-19â), 3.74 (1H, d, J ) 10.8 Hz), 3.86 (1H, d, J )
11.1 Hz), 4.02 (1H, d, J ) 10.8 Hz), 4.04-4.10 (1H), 4.20-
4.28 (2H), 4.59 (1H, m, H-29 pro-E), 4.69 (1H, bd, J ) 2.4 Hz,
H-29 pro-Z); EIMS m/z 586 [M]+ (1), 526 (3), 513 (1), 466 (5),
411 (5), 356 (8), 287 (7), 255 (5), 189 (14), 43 (100); anal. C
73.36%, H 10.14%, calcd for C36H58O6, C 73.68%, H 9.96%.
Rea ction of Seco Der iva tives w ith Acetic An h yd r id e.
Acetic anhydride (2 mL, 19.6 mmol) was added to a solution
of each seco derivative 16 or 17 (250 mg, 0.5 mmol) in pyridine
(3 mL). After 10 h, the mixture was poured into cold H2O and
worked up. HPLC in 15% ethyl acetate in hexane afforded two
products (when 16 was the reactant). The first fraction was
anhydride 23 (120 mg, 50% yield), and the second fraction was
anhydride 21 (60 mg, 23% yield). Both were crystallized from
hexane/diethyl ether. Reaction with 17 afforded only 22 (102
mg, 40% yield).
2,3-An h ydr ide of 2,3-secolu p-20(29)-en -2,3,28-tr ioic acid
(21): Rf 0.40; mp 242-244 °C (diethyl ether/hexane); [R]25
D
+42° (c 0.25); IR (CHCl3) νmax 3515, 1797, 1753, 1696, 1643
1
cm-1; H NMR δ 0.97, 0.99, 1.04, 1.26, 1.37 (15H, all s, 5 ×
Ack n ow led gm en t. This study was supported in part by
the Ministry of Education of the Czech Republic (MSM
113100001), which paid for instrumental equipment, the Czech
Science Foundation (203/00/1549), which paid for refilling of
HPLC column and all material for TLC, and the Czech Science
Foundation (203/03D152), from which the chemicals and all
other material support were paid. Biological testing was
supported by the Czech Science Foundation (301/03/1570). We
are grateful to I. Tislerova for measurement of NMR spectra
and S. Hilgard and M. Kvasnica for measurement of IR spectra
and MS. Special thanks to B. Sperlichova for measurement of
optical rotations.
CH3), 1.70 (3H, m, H-30), 1.74-1.82 (2H, m), 1.95-2.03 (2H,
m), 2.21 (1H, d, J ) 13.7 Hz, H-1a), 2.86 (1H, d, J ) 13.7 Hz,
H-1b), 3.00 (1H, m, H-19â), 4.63 (1H, m, H-29 pro-E), 4.74 (1H,
bd, J ) 2.4 Hz, H-29 pro-Z); EIMS m/z 484 [M]+ (3), 466 (2),
456 (12), 438 (15), 423 (6), 413 (3), 384 (8), 371 (6), 325 (13),
301 (8), 248 (42), 233 (15), 201 (37), 187 (42), 69 (100); anal. C
74.22%, H 9.27%, calcd for C30H44O5, C 74.34%, H 9.15%.
2,3-An h yd r id e of 28-m eth yl ester of 2,3-secolu p -20(29)-
en -2,3,28-tr ioic a cid (22): Rf 0.67; mp 206-208 °C (diethyl
ether/hexane); [R]25D +43° (c 0.60); IR (CHCl3) νmax 1797, 1754,
1
1720, 1643 cm-1; H NMR δ 0.95, 0.98, 1.04, 1.26, 1.37 (15H,
all s, 5 × CH3), 1.69 (3H, m, H-30), 1.73-1.81 (2H, m), 1.84-
1.93 (2H, m), 2.21 (1H, d, J ) 13.7 Hz, H-1a), 2.20-2.28 (2H,
m), 2.85 (1H, d, J ) 13.7 Hz, H-1b), 2.99 (1H, m, H-19â), 3.67
(3H, s, OCH3), 4.62 (1H, m, H-29 pro-E), 4.73 (1H, bd, J ) 2.3
Hz, H-29 pro-Z); EIMS m/z 498 [M]+ (35), 483 (3), 470 (11),
Su p p or tin g In for m a tion Ava ila ble: Tables of 13C NMR data for
compounds 1-23 and 1H NMR data for compound 16. This material
is available free of charge via the Internet at http://pubs.acs.org.

A-Seco Derivatives of Betulinic Acid
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