Synthesis and antitumor activity of cyclopropane derivatives of betulinic and betulonic acids

Article abstract of DOI:10.1007/s11171-005-0039-z

New cyclopropane derivatives of betulin were synthesized by attachment of dichlorocarbenes or dibromocarbenes to the double bond of betulin diacetate, followed by the deprotection of hydroxyl groups. The betulin cyclopropane derivative was obtained from 20,29-dihydro-20,29-dichloromethylenebetulin by treatment with lithium in tert-butanol. These compounds were converted into the corresponding derivatives of betulonic acid by oxidation with chromium trioxide. The reduction of oxo group with sodium borohydride led to the corresponding derivatives of betulinic acid. 20,29-Dihydro-20,29-dichloromethylenebetulinic acid proved to be the most cytotoxic toward human melanoma of the Colo 38 and Bro lines and human ovarian carcinoma of the CaOv line (IC₅₀ 10 μM). 20,29-Dihydro-20,29-dibromomethylenebetulinic acid inhibited the growth of the Bro melanoma cell line and the CaOv carcinoma cell line practically by 50% at a concentration of 10 μM. The other derivatives of betulinic and betulonic acids were active toward all of the three cancer cell lines at concentrations higher than 10 μM.

Full text of DOI:10.1007/s11171-005-0039-z

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 3, 2005, pp. 286–291. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 320–325.
Original Russian Text Copyright © 2005 by Symon, Veselova, Kaplun, Vlasenkova, Fedorova, Lyutik, Gerasimova, Shvets.
Synthesis and Antitumor Activity of Cyclopropane Derivatives
of Betulinic and Betulonic Acids
1
A. V. Symon*, N. N. Veselova*, A. P. Kaplun*, N. K. Vlasenkova**, G. A. Fedorova*,
A. I. Lyutik*, G. K. Gerasimova**, and V. I. Shvets*
Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 117571 Russia
*
*
* Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe sh. 24, Moscow, 115478 Russia
Received July 19, 2004; in final form, September 30, 2004
Abstract—New cyclopropane derivatives of betulin were synthesized by attachment of dichlorocarbenes or
dibromocarbenes to the double bond of betulin diacetate, followed by the deprotection of hydroxyl groups. The
betulin cyclopropane derivative was obtained from 20,29-dihydro-20,29-dichloromethylenebetulin by treat-
ment with lithium in tert-butanol. These compounds were converted into the corresponding derivatives of bet-
ulonic acid by oxidation with chromium trioxide. The reduction of oxo group with sodium borohydride led to
the corresponding derivatives of betulinic acid. 20,29-Dihydro-20,29-dichloromethylenebetulinic acid proved
to be the most cytotoxic toward human melanoma of the Colo 38 and Bro lines and human ovarian carcinoma
of the CaOv line (IC 10 µM). 20,29-Dihydro-20,29-dibromomethylenebetulinic acid inhibited the growth of
50
the Bro melanoma cell line and the CaOv carcinoma cell line practically by 50% at a concentration of 10 µM.
The other derivatives of betulinic and betulonic acids were active toward all of the three cancer cell lines at con-
centrations higher than 10 µM.
Key words: betulinic acid, betulonic acid, cyclopropane derivatives, melanoma
1
INTRODUCTION
bicity in the area of isopropenyl group can result in the
betulinic acid derivatives with higher antitumor activi-
The number of people suffering from melanoma has
3
2
ties. This problem could be easily solved by the attach-
been increased in the last few decades. An average
lifetime of patients with metastatic melanoma is 6–
1
ment of low-polar groups to the isopropenyl residue, in
particular, cyclopropanation.
0 months [1, 2]. Nowadays, dacarbazin is the most
useful preparation for the treatment of metastatic inop-
erable melanoma. The combinations of dacarbazin with
such agents as carmustin, cisplatin, tamoxifen, inter-
feron α and interleukin-2 are also used. However, the
efficiency of this treatment is low, and, in addition,
most of the drugs are highly toxic [3].
It was demonstrated by antitumor screening of more
then 2500 plant extracts that extract from the Ziziphus
mauritiana Lam (Rhamnaceae) bark exhibits a selective
cytotoxicity toward the human melanoma cells [4]. Bet-
ulinic acid (I) proved to be the active principle. Unlike
the most modern antitumor therapeutic agents, it is low
toxic for human organism and was found to be capable
of inhibiting the growth of human melanoma cells by
the launching the apoptosis mechanism [4].
RESULTS AND DISCUSSION
We chose betulin diacetate (III) as a starting com-
pound (scheme) in order to prevent side reactions of
both hydroxyl groups of betulin (II) at the stage of car-
bene attachment. The synthesis was performed accord-
ing to the Makoshi standard procedures of attachment
of dichlorocyclopropane and dibromocyclopropane to
olefines [5] using sodium trichloroacetate or bromo-
form as a carbene source and TEBA as a catalyst of
interphase transfer. The acetyl groups were removed
from the corresponding cyclopropane derivatives of
betulin diacetate (IV) and (VIII) by alkaline hydrolysis
without further purification to obtain dibromo- and
dichlorocyclopropane derivatives of betulin (V) and
1
Our analysis of the structure–activity relationship (IX). The H NMR resonances from olefin protons at
for a number of triterpenes with antitumor and anti- 4.67 and 4.57 ppm were present in the starting betulin
inflammatory effect demonstrated that an increase in diacetate (III) and absent from both cyclopropane
the volume without a significant decrease in hydropho- derivatives of betulin (V) and (IX).
Compound (XII) was prepared from betulin deriva-
1
Corresponding author; phone:+7 (095) 434-8355; e-mail:
tive (IX) by the treatment with lithium in tert-butanol
alex.kaplun@mtu-net.ru
2
and recrystallization of the raw product from isopro-
Abbreviations: IC , the concentration of tested compound that
5
0
causes death of 50% cells in the culture; TEBA, triethylbenzyl-
ammonium chloride.
3
Data not published.
1
068-1620/05/3103-0286 © 2005 Pleiades Publishing, Inc.

SYNTHESIS AND ANTITUMOR ACTIVITY
CH2
287
2
0
9
CH2
CH2
2
H C
3
H C
3
H C
3
H
19
H
H
OH
OAc
OH
CH3
CH3
H
CH3
CH3
CH3
H
CH3
CH3
CH3
H
28
O
H
H
CH3
H
H
CH3
H
H
CH3
3
HO
AcO
H C
(
II)
(III)
HO
(I)
H C
3
CH3
3
H C
3
Betulinic acid
Br
Br
H C
Cl
Cl
H C
3
3
H
H
OAc
OAc
CH3
CH3
H
CH3
CH3
H
H
H
CH3
H
H
CH3
AcO
AcO
H C
H C
CH3
CH3
3
(IV)
3
(VIII)
Br
Br
H C
Cl
Cl
3
H C
3
H C
3
H
CH3
CH3
H
H
OH
OH
OH
CH3
H
CH3
CH3
H
CH3
CH3
H
H
H
H
H
CH3
H
H
CH3
HO
H C
HO
HO
(V)
(IX)
(XII)
CH3
H C
CH3
3
H C
CH3
3
3
Br
Br
H C
Cl
Cl
H C
H3C
3
3
H
H
H
OH
OH
OH
CH3
CH3
CH3
H
CH3
CH3
CH3
H
CH3
CH3
CH3
H
O
O
O
H
H
CH3
H
H
CH3
H
H
CH3
R
H C
R
H C
3
R
H3C
(
VI) R=O
(X) R=O
(XIII) R=O
3
(VII) R=H, β-OH
(XI) R=H, β-OH
Scheme 1.
(XIV) R=H, β-OH
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

2
88
SYMON et al.
Colo 38
Bro
CaOv
dichloromethylenebetulinic
acid
(XI):
521.5
35
35
+
35
37
+
(
[M( Cl, Cl) – H O] ), 523.5 ([M( Cl, Cl) – H O] ),
2 2
37
Survival, % of the control
00
37
+
and 541.5 ([M( Cl, Cl) – H O] ). The weak signal of
1
2
molecular ion of 20,29-dihydro-20,29-dibromomethyl-
79
81
+
enebetulonic acid (VI) ([M( Br, Br)] 626.5) was
found under the condition of recording of its mass spec-
trum. Molecular ion of 20,29-dihydro-20,29-dibro-
momethylenebetulinic acid (VII) was not observed.
7
5
0
5
0
5
2
The cytotoxic activity of the derivatives of betulinic
and betulonic acids was studied on the cultures of
human tumor cells of different histogenesis: melanoma
(
XIII) (XIV) (X) (XI) (VI) (VII)
Compounds
(
(
the Colo line and the Bro line) and ovarian carcinoma
the CaOv line). The cytotoxic effect was determined
by MTT test [8]. According to the criteria accepted by
the Cancer Research Center of the Russian Academy of
Medical Sciences for screening of substances with a
known molecular mass, the tested compound is consid-
ered to be active when IC ≤ 10 µM. The used cells
Survival of human cancer cells after the action of betulinic
acid derivatives (10 µM).
panol. A multiplet from four protons of cyclopropane
5
0
1
ring appeared in strong field (δ 0.24 ppm) of its H
were normally sensitive toward the cytostatic doxorubi-
NMR spectrum.
cin (IC 0.5 µM).
50
Betulin derivatives (V), (IX), and (XII) were con-
One can see from the results given in the table that
only 20,29-dihydro-20,29-dichloromethylenebetulinic
acid (XI) exhibited the cytotoxic activity toward the
tested cell cultures comparable with the cytotoxicity of
betulinic acid (I). Moreover, derivative (XI) was more
toxic than betulinic acid toward the Bro cell line.
verted into the corresponding cyclopropane derivatives
of betulonic acid (VI), (X), and (XIII) by CrO oxida-
3
tion in acetic acid [6]. The cyclopropane derivatives of
betulinic acid were prepared by the reduction of 3-keto-
group with NaBH . Compounds (VII), (XI), and (XIV)
4
were isolated and separated from 3α-epimers formed
after the reduction by chromatography on silica gel due
to their higher chromatographic mobility in comparison
with the target 3β-epimers [7]. Homogeneity of the syn-
thesized compounds was no less than 98% according to
HPLC. The structure of these compounds was con-
firmed by elemental analyses and mass spectrometry.
For example, signals of molecular ions were present in
mass spectra of 20,29-dihydro-20,29-methylenebetu-
A comparison of the cytotoxicity of synthesized
compounds was made by studying the survival of the
cancer cells at the concentration of 10 µM. The figure
demonstrates that dibromocyclopropane (VII) and
dichlorocyclopropane (XI) derivatives of betulinic acid
are the most active. The survival of the Bro and CaOv
cell lines was 50%. However, dibromoderivative (VII)
exhibited a low effectivity toward the Colo 38 cells,
whereas dichloroderivative (XI) inhibited their growth
by 50%. It has been demonstrated previously that betu-
lonic acid is much active than betulinic acid [9, 10].
However, the cytotoxicity of the synthesized cyclopro-
pane derivatives of betulonic acid toward the tested cell
lines was lower than that of the corresponding deriva-
tives of betulinic acid.
+
lonic acid (ïIII) ([M] 469) and 20,29-dihydro-20,29-
+
methylenebetulinic acid (ïIV) ([M] 471). Signals of
several molecular ions containing chlorine isotopes in
different composition were observed in the mass spec-
trum of 20,29-dihydro-20,29-dichloromethylenebetu-
3
5
35
+
lonic acid (ï): 537.5 ([M( Cl, Cl)] ), 539.5
35
37
+
37
37
+
[
M( Cl, Cl)] , and 541.5 [M( Cl, Cl)] ). Molecular
ions corresponding to the dehydration products were
found in the mass spectrum of 20,29-dihydro-20,29-
Thus, none of the synthesized compounds demon-
strated a cytotoxic activity higher than that of betulinic
acid. However, these results require a refinement,
because solubility of the compounds is very low:
approximately 0.001 mg per ml (~2 µM) in water and
0.01 mg per ml of DMSO. In other words, significant
part of the compounds was probably precipitated dur-
ing the incubation with the cell cultures at their concen-
trations higher than 2 µM, and the synthesized deriva-
tives of betulinic and betulonic acid could not exhibit
their maximal activities. Therefore, further studies
should be undertaken when searching for the solubili-
zation methods of the compounds synthesized.
Cytotoxic activity of the cyclopropane derivatives of betulin-
ic and betulonic acids toward human cancer cells (IC50 µM)
Compound
Cell
(I) (XIII)* (XIV) (X)* (XI)* (VI) (VII)*
Colo 38
Bro
8
>10
>10
>10
>10 >10
>10 >10
>10 >10
10
10
10
>10 >10
>10 >10
>10 >10
>10
9
CaOv
*
The substance has a low solubility in DMSO.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

SYNTHESIS AND ANTITUMOR ACTIVITY
289
EXPERIMENTAL
(60 ml) and methanol (30 ml), mixed with a solution of
NaOH (1.90 g, 47.5 mmol) in water (10 ml), and stirred
at room temperature for 16 h. The reaction mixture was
neutralized with 20% HCl and evaporated to dryness.
The residue was dissolved in chloroform, washed with
water (3 × 100 ml), dried with Na SO , and evaporated
Reagents of domestic production with the quality of
no less than kh. ch. (reagent grade) were used in this
study. Betulin (II) isolated from the birch bark [11] had
mp 256–258°C (mp is 258°C according to literature
11]).
1H NMR spectra were recorded on a Bruker DPX-
00 spectrometer (Germany) in CDCl at working fre-
2
4
[
to dryness. A crystalline substance was obtained; it was
recrystallized from a mixture of dichloromethane and
isopropanol. The betulin derivative (V) was obtained as
3
3
quency of 300 MHz. Chemical shifts (δ, ppm) are given
relative to tetramethylsilane. Mass spectra were regis-
tered on a KRATOS MS890 spectrometer (EI 70 eV,
straw-colored crystals; yield of 3.10 g (53.1%); mp
1
1
62–164°C; R 0.16 (A); H NMR (CDCl ): 3.70 (1 H,
f
3
d, J 10.2 Hz, H28), 3.21 (1 H, d, J 10.2 Hz, H28), 3.18
2
50°C). The optical density was measured on a Multi-
(
0
1 H, m, H3), 2.40 (1 H, m, H19), 1.24, 0.98, 0.97, 0.94,
skan MCC/340 scanning spectrometer (LabSystem,
Finland) at 540 nm. Elemental analyses was obtained
on a CHNS EA 1112 elemental analyzer (Thermo Fini-
gan, Italy). Melting points were determined on a
PTP(M) device (OAO Khimlaborpribor, Russia).
.80, and 0.74 (all 3 H, s, CH ). Found, %: C 60.82, H
3
8
.83. Calc. for ë ç Br O , %: C 60.59, H 8.20.
31
50
2
2
20,29-Dhydro-20,29-dibromomethylenebetulonic
acid (VI). A solution of CrO (1.46 g, 14.6 mmol) in a
3
mixture of water (3 ml) and glacial acetic acid (45 ml)
was added to a solution of betulin derivative (V) (2.00 g,
TLC was carried out on Sorbiton Diol plates
Khromdet-Ekologiya, Russia) in the following chro-
(
3
.25 mmol) in glacial acetic acid (36 ml) at stirring.
matographic systems: (A) chloroform, (B) 10 : 0.2
chloroform–methanol, and (C) 10 : 0.5 chloroform–
methanol. Triterpenes were detected on the plates by
After 10 min, 10% NaCl (100 ml) was added to the
reaction mixture. The reaction products were extracted
with ether (2 × 100 ml). The combined ether extract was
washed with 10% NaCl (4 × 80 ml) and evaporated.
The residue was purified by chromatography on a silica
gel column eluted with a 10 : 0.1 chloroform–acetoni-
trile mixture. Acid (VI) was obtained as a white pow-
5
0% sulfuric acid with the subsequent heating. Silica
gel L 60/100 µm (Merck, Germany) was used for col-
umn chromatography.
The cytotoxicity of derivatives of betulinic and bet-
ulonic acids was examined in the Research Institute of
Experimental Diagnostics and Cancer Therapy of the
Blokhin Cancer Research Center of the Russian Acad-
emy of Medical Sciences on cultures of human cancers
the Colo 38 and Bro melanoma cell lines and ovarian [M( Br, Br)] . Found, %: C 59.01, H 7.40, Br 25.91.
carcinoma).
Calc. for ë31 , %: C 59.43, H 7.40, Br 25.51.
Betulin diacetate (III). Acetic anhydride (170 ml)
20,29-Dihydro-20,29-dibromomethylenebetulinic
der; yield 1.50 g (73.7%); mp 228.5–230.5°C; R 0.18
f
1
(B); H NMR (CDCl ): 1.24, 1.05, 1.00, 0.97, 0.91, and
3
0
.90 (all 3 H, s, CH ); mass spectrum, m/z: 626.5
3
79
81
+
(
ç
46Br O
2 3
was added to betulin (II) (20.0 g, 45.2 mmol) and acid (VII). Sodium borohydride (4 × 24 mg,
heated to the complete dissolution. The reaction mix- 0.63 mmol) was added to a solution of (VI) (0.300 g,
ture was cooled, and the precipitated crystals were fil- 0.480 mmol) in a mixture of methanol (40 ml) and tet-
tered, washed with water to pH 7, dried in a vacuum rahydrofurane (10 ml) for 3 h at stirring. Water (10 ml)
desiccator over P O , and recrystallized from isopro- and 10% HCl (20 ml) were added to the reaction mix-
2
5
panol, to give (III) as white acicular crystals; yield 15.3 g ture. A gel-like precipitate was formed; it was filtered,
(
64.3%); mp 223–224°C (lit. mp 223–224°C [12]); R washed with water, and dried with P O . The reaction
f
2 5
1
product was purified by chromatography on a silica gel
column eluted with 10 : 0.1 chloroform–acetonitrile
0
.59 (Ä); ç NMR (CDCl ): 4.67 (1 H, m, =Cç ), 4.57
3 2
(
1 H, m, =Cç ), 4.44 (1 H, m, H3), 4.22 (1 H, d, J 10.7
2
mixture. Acid (VII) was obtained as a white powder;
Hz, H28), 3.82 (1 H, d, J 10.7 Hz, H28), 2.42 (1 H, m,
H19), 2.05 (3 H, s, CH CO), 2.02 (3 H, s, CH CO),
1
yield 0.250 g (82.9%); mp 202–204°C; R 0.49 (C); H
f
3
3
NMR (CDCl ): 3.17 (1 H, m, H3), 1.24, 0.97, 0.95,
1
.66, 1.00, 0.94, 0.82, 0.82, and 0.81 (all 3 H, s, CH3).
3
0
5
5
.89, 0.80, and 0.74 (all 3 H, s, CH ). Found, %: C
3
2
0,29-Dihydro-20,29-dibromomethylenebetulin
8.99, H 7.68, Br 25.29. Calc. for C H Br O , %: C
3
1
48
2
3
(
V). TEBA (23.7 mg, 0.120 mmol), a solution of NaOH
7.60 g, 190 mmol) in water (20 ml), ethanol (0.1 ml),
9.24, H 7.70, Br 25.43.
(
and bromoform (1.66 ml, 19.0 mmol) were added to a
20.29-Dihydro-20,29-dichloromethylenebetulin
solution of betulin diacetate (III) (5.00 g, 9.5 mmol) at (IX). Sodium trichloracetate (3.67 g, 19.8 mmol) and
stirring. The reaction mixture was stirred (1000 rpm) TEBA (38 mg, 0.2 mmol) were added to a solution of
for 20 h at room temperature, and additional TEBA betulin diacetate (5.20 g, 9.90 mmol) in chloroform
(
1
1
23.7 mg, 0.120 mmol), the solution of NaOH (7.60 g, (10 ml) at room temperature. The reaction mixture was
90 mmol) in water (20 ml), and bromoform (1.66 ml, heated to 80°C and stirred at 1200 rpm for 2 days,
9.0 mmol) were added, and the reaction mixture was cooled to room temperature, twice washed with water,
stirred another 20 h at room temperature, and evapo- and evaporated. The product (5.00 g) was dissolved
rated. The residue was dissolved in a mixture of THF without further purification in a mixture of THF (50 ml)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

2
90
SYMON et al.
and methanol (13 ml), treated with 4 N NaOH (9 ml), 10% HCl (110 ml) and THF (40 ml). The upper organic
and stirred at room temperature for 17 h. The reaction phase was dried with Na SO and evaporated. Recrys-
2
4
mixture was neutralized with 20% HCl to pH 7 and tallization from isopropanol yielded 1.20 g (72.0%) of
1
evaporated to dryness. The obtained crystals were dis-
(
XII) as white crystals; mp 233–234.5; R 0.26 (A); H
f
solved in chloroform (100 ml), washed with water (3 ×
NMR (CDCl ): 3.70 (1 H, d, J 10.7 Hz, H28), 3.18
1 H, d, J 10.7 Hz, H28), 3.14 (1 H, m, H3), 0.98, 0.97,
.94, 0.88, 0.81, and 0.74 (all 3 H, s, CH ), 0.24 (4 H,
m, CH CH ); mass spectrum, m/z: 457 [M] . Found, %:
C 81.37, H 11.52. Calc. for ë ç O , %: C 81.52, H
3
1
00 ml), dried with Na SO , and evaporated. The resi-
2 4
(
0
due was recrystallized from a dichloromethane–isopro-
3
panol mixture to give betulin derivative (IX) as white
+
2
2
crystals; yield 3.50 g (67.3%); mp 201–202.5°C; R
f
1
31 52
2
0
.16 (A); H NMR (CDCl ): 3.71 (1 H, d, J 10.7 Hz,
3
1
1.47.
0,29-Dihydro-20.29-methylenebetulonic
XIII). A solution of CrO (1.97 g, 19.7 mmol) in a
H28), 3.21 (1 H, d, J 10.7 Hz, H28), 3.19 (1 H, m, H3),
2
acid
2
0
.30 (1 H, m, H19), 1.21, 0.99, 0.98, 0.95, 0.81, and
(
3
.74 (all 3 H, s, CH ). Found, %: C 70.63, H 9.54. Calc.
3
mixture of water (4 ml) and glacial acetic acid (61 ml)
for C H Cl O , %: C 70.84, H 9.59.
3
1
50
2
2
was added to a suspension of (XII) (2.00 g, 4.40 mmol)
2
0,29-Dihydro-20,29-dichloromethylenebetulonic in glacial acetic acid (47 ml) at stirring. We observed
acid (X). A solution of CrO (2.90 g, 29.3 mmol) in a the dissolution of betulin derivative (XII).After 10 min,
3
mixture of glacial acetic acid (60 ml) and water (10 ml) the reaction mixture was diluted with 10% NaCl
was added to a stirred solution of betulin derivative (150 ml); extracted with ether (2 × 90 ml); and the
(
IX) (3.40 g, 6.50 mmol) in glacial acetic acid (100 ml) extract was washed with 10% NaCl (4 × 100 ml) and
at room temperature. After 10 minutes, the reaction evaporated. The reaction product was chromatographed
mixture was diluted with 10% NaCl (200 ml) and on a silica gel column eluted with chloroform. Acid
extracted with ether (2 × 130 ml). The extract was (XIII) was obtained as a white powder; yield 1.20 g
1
washed with 10% NaCl (4 × 150 ml) and evaporated. (58.2%); mp 278–280°C; R 0.40 (B); H NMR
f
The reaction product was chromatographed on a silica
gel column eluted with a 10 : 0.5 chloroform–methanol
mixture. Acid (X) was obtained as a white powder;
(
CDCl ): 1.04, 1.00, 0.97, 0.93, 0.91, and 0.89 (all 3 H,
3
s CH ), 0.28 (4 H, m, CH CH ); mass spectrum, m/z:
69 [M] . Found, %: C 79.45, H 10.33. Calc. for
3
2
2
+
4
yield of 2.14 g (61.2%); mp 253–254.5°C; R 0.60 (C);
f
C H O , %: C 79.44, H 10.32.
3
1
48
3
1
H NMR (CDCl ): 1.21, 1.05, 1.00, 0.98, 0.92, and 0.91
3
2
0,29-Dihydro-20.29-methylenebetulinic
acid
(
[
[
all 3 H, s, CH ); mass spectrum, m/z: 537.5
3
(
XIV). NaBH (220 mg, 5.80 mmol) was added in por-
35
35
+
35
37
+
4
M( Cl, Cl)] ,
M( Cl, Cl)] . Found, %: C 69.09, H 8.54. Calc. for
ë ç Cl O , %: C 69.26, H 8.62.
539.5
[M( Cl, Cl)] ,
541.5
tions to a solution of (XIII) (1.00 g, 2.12 mmol) in
methanol (150 ml) for 1 h at stirring. The reaction mix-
ture was diluted with 10% HCl (100 ml) and water
37
37
+
31
46
2
3
2
0,29-Dihydro-20,29-dichloromethylenebetulinic (15 ml). The precipitate was filtered, washed with
acid (XI). NaBH (280 mg, 7.41 mmol) was added in water, and dried in a vacuum desiccator over P O
2 5
. The
4
portions to a stirred solution of (X) (1.50 g, 2.79 mmol) reaction product was chromatographed on a silica gel
in methanol (250 ml) for 1 h. After 1.5 h, the reaction column eluted with a 10 : 0.1 chloroform–methanol
mixture was diluted with 10% HCl (40 ml) and hot mixture. Acid (XIV) was obtained as a white powder;
1
water (100 ml). The gel-like precipitate was formed; it yield 0.920 g (92.2%); mp 282–284°C; R 0.33 (C); H
f
was filtered, washed with water (250 ml), and dried in NMR (CDCl ): 3.16 (1 H, m, H3), 0.98, 0.95, 0.91,
3
a vacuum desiccator over P O . The reaction product
2
5
0.89, 0.82, and 0.74 (all 3 H, s, CH ), 0.26 (4 H, m,
CH CH ); mass spectrum, m/z: 471 [M] . Found, %: C
9.15, H 10.97. Calc. for C H O , %: C 79.10, H
3
was chromatographed on a silica gel column eluted
with a 10 : 0.1 : 0.05 chloroform–methanol–formic acid
mixture. Acid (XI) was obtained as a white powder;
+
2
2
7
1
31 50 3
0.71.
1
yield 1.10 g (73.1%); R 0.39 (C); mp 236–237.5°C; H
f
Determination of cytotoxicity of betulinic acid
NMR (CDCl ): 3.11 (1 H, m, H3), 1.21, 0.97, 0.94,
3
derivatives. Cultures of human melanoma cells of the
MS line (doubling time T = 36 h) and the Bro line (T =
0
.91, 0.80, and 0.73 (all 3 H, s, CH ); mass spectrum,
3
d
d
35
35
+
35
37
m/z: 521.5 [M( Cl, Cl) – H O] , 523.5 [M( Cl, Cl) –
H O] , 525.5 [M( Cl, Cl) – H O] . Found, %: C 68.87,
2
48 h) and a culture of human ovarian carcinoma of the
CaOv line (T = 40 h) were grown as a monolayer on
+
37
37
+
2
2
d
H 9.04, Cl 13.35. Calc. for ë ç Cl O , %: C 69.00, H
31
48
2
3
the DMEM culture medium (Sigma) containing 10%
8
.97, Cl 13.14.
FBS (fetal bovine serum purchased from Gibco DRL),
2
mM of L-glutamine, and 40 µg/ml of gentamicin in
2
0,29-Dihydro-20,29-methylenebetulin (XII). A
the atmosphere of 5% CO and 95% air at 37°C.
2
finely cut lithium (3.20 g, 464 mmol) was added to a
solution of (IX) (1.92 g, 3.65 mmol) in anhydrous tet-
The cell suspension was placed into 96-well plates.
rahydrofurane (20 ml), and anhydrous tert-butanol The tested substances at the concentrations of 10, 5, 1,
(
86 ml) was added dropwise for 1 h at stirring. The 0.5, and 0.1 µM were added to the tumor cultures at the
reaction mixture was stirred for 18 h and diluted with phase of their exponential growth and incubated for
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 3 2005

SYNTHESIS AND ANTITUMOR ACTIVITY
291
7
2 h. The cell survival was determined by the MTT test
4. Pisha, E., Chai, H., Lee, I.S., Chagwedera, T.E., Farn-
sworth, N.R., Cordell, G.A., Beecher, C.W., et al., Nat.
Med., 1995, no. 1, pp. 1046–1051.
5. Dehmlow, E.V. and Dehmlow, S.S., Phase Transfer
Catalysis, Weinheim: Verlag Chemie, 1983. Translated
under the title Mezhfaznyi kataliz, Moscow: Mir, 1987.
[
8]. The formed formazane crystals were dissolved in
DMSO, and optical absorption was measured on the
scanning spectrophotometer at 540 nm. The portion of
surviving cells was determined as percentage of the
ratio of the absorption value of the experimental sam-
ples to that of the control (the cancer cells in the growth
medium without the tested substances).
6
. Le Bang Shon, Kaplun, A.P., Shpilevskii, A.A., Andiya-
Pravdivyi, Yu.E., Alekseeva, S.G., Grigor’ev, V.B., and
Shvets, V.I., Bioorg. Khim., 1998, vol. 24, pp. 787–793.
7
. Symon, A.V., Kaplun, A.P., Vlasenkova, N.K., Gerasi-
mova, G.K., Le Bang Shon, Litvin, E.F., Kozlova, L.M.,
Surkova, E.L., and Shvets, V.I., Bioorg. Khim., 2003,
vol. 29, pp. 202–207.
. Alley, C.M., Scudiero, D.A., and Monks, A., Cancer
Res., 1988, vol. 48, pp. 589–601.
ACKNOWLEDGMENTS
This study was supported by the grant of President
of Russian Federation for the Leading Scientific
Schools (NSh-2329.2003.4), project no. 1781.
8
9
. Kim, D.S., Pezzuto, J.M., and Pisha, E., Bioorg. Med.
Chem. Lett., 1998, vol. 8, pp. 1707–1712.
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