Epimerization of Hydroxyl Group in Lupan Series Triterpenoids

Article abstract of DOI:10.1023/A:1023220701797

Two methods of obtaining 3α-betulinic acid and related compounds from their 3β-epimers were studied: the reaction of bimolecular substitution and the stereoselective reduction of 3-ketoderivatives. The substitution of acyloxy by formyloxy group in 3-O-tosyllupeol or of the belulin hydroxyl by benzoyloxy group resulted only in Δ^{2, 3}-elimination products, with none of the expected products of bimolecular substitution being found. The catalytic hydrogenation of betulonic acid over Raney nickel resulted only in reduction of the isopropenyl double bond, whereas the use of 5% Ru/C gave a 60:40 mixture of epimers of dihydrobetulinic acid. Practically the same mixture of betulinic acid epimers was obtained when reducing betulonic acid with L-Selectride. The cytotoxic activity of 3α-betulinic acid increased toward the Bro melanoma cells and decreased toward the MS melanoma cells.

Full text of DOI:10.1023/A:1023220701797

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 2, 2003, pp. 185–189. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 2, 2003, pp. 208–213.
Original Russian Text Copyright © 2003 by Symon, Kaplun, Vlasenkova, Gerasimova, Le Bang Shon, Litvin, Kozlova, Surkova, Shvets.
Epimerization of Hydroxyl Group in Lupan Series Triterpenoids
1
A. V. Symon*, A. P. Kaplun*, N. K. Vlasenkova**, G. K. Gerasimova**, Le Bang Shon*,
E. F. Litvin***, L. M. Kozlova***, E. L. Surkova*, and V. I. Shvets*
*Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 119571 Russia
**Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe sh. 24, Moscow, 115478 Russia
***Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119992 Russia
Received January 14, 2002; in final form, March 26, 2002
Abstract—Two methods of obtaining 3α-betulinic acid and related compounds from their 3β-epimers were
studied: the reaction of bimolecular substitution and the stereoselective reduction of 3-ketoderivatives. The sub-
stitution of acyloxy by formyloxy group in 3-é-tosyllupeol or of the belulin hydroxyl by benzoyloxy group
resulted only in ∆2, 3-elimination products, with none of the expected products of bimolecular substitution being
found. The catalytic hydrogenation of betulonic acid over Raney nickel resulted only in reduction of the isopro-
penyl double bond, whereas the use of 5% Ru/C gave a 60 : 40 mixture of epimers of dihydrobetulinic acid.
Practically the same mixture of betulinic acid epimers was obtained when reducing betulonic acid with L-Selec-
tride. The cytotoxic activity of 3α-betulinic acid increased toward the Bro melanoma cells and decreased toward
the MS melanoma cells.
Key words: betulinic acid, epimerization, melanoma
1
INTRODUCTION
development of a convenient method of the 3α-betu-
linic acid (Xa) preparation. At present, the only work
[9] has been published in which the authors isolated
3-epibetulinic acid by the extraction of the bark of
Picramnia pentandra sw. (0.8 g from 0.9 kg of the
bark) that grows in the United States.
The growth of melanoma morbidity has been 300%
during the last 40 years [1]. Cytostatics (dacarbazin and
cisplatin) and cytokines (α2-interferon and interleukin-
2) used for the melanoma treatment do not allow com-
plete recovery [2]. In addition, the known anticancer
cytostatics are very toxic and, during their use, resis-
tance of the tumors under treatment to the drugs often
arise, which reduces their efficiency. Therefore, the
search for new compounds displaying anticancer activ-
ity remains topical.
It was found in 1995 that betulinic acid (Xb) selec-
tively initiates apoptosis of human melanoma cells [3].
It inhibits the growth of tumor cells three times as effec-
tively as dacarbazin and simultaneously displays a low
in vivo toxicity. Some derivatives of betulinic acid also
exhibit a high anti-HIV activity [4–6].
We showed that cytotoxicity of betulonic acid (VII)
toward the melanoma MS cells is 18 times higher than
that of betulinic acid (Xb) [7]. Similar results were
obtained in Illinois University [8].
Our analysis of the structure–activity relationship in
a number of triterpenes demonstrated that their biolog-
ical activity as a rule increases in the order: 3β-
hydroxy–3-keto–3-α-hydroxy compounds. We hypoth-
esized that the antimelanoma activity for betulinic acid
derivatives would follow the same regularity and 3α-
betulinic acid would display a higher activity. The goal
of this work is the test of this hypothesis. This required
RESULTS AND DISCUSSION
We studied two approaches for the preparation of
3α-betulinic acid (Xa): (1) the epimerization of the 3β
series compounds and (2) the reduction of betulonic
acid (VII). Two reactions, which were effective in the
case of cholestane-3β-ol, were checked for the epimer-
ization: the substitution of formyloxy for tosyloxy
group [10] and the substitution of benzoyloxy for
hydroxy group using the Mitsunobu reaction [11]. In
our case, both methods gave only ∆2, 3-elimination prod-
ucts (Scheme 1 and Table 1): 3-é-tosyllupeol (III) gave
3-deoxy-2,3-dehydrolupeol (VI) and betulin (I),
3-deoxy-2,3-dehydrobetulin (IV) and 3-deoxy-2,3-
dehydrobetulin benzoate (V). 1H NMR spectra of these
compounds exhibited the resonances of protons at an
extra double bond (δ 5.39–5.28 ppm) and the absence
of C3 proton resonances. In the spectrum of (V), the
resonances of the CH2O methylene protons shifted
downfield by 0.65 and 0.75 ppm in comparison with the
corresponding resonances in starting betulin (I).
Unlike the compounds of cholestane series, the ring
A in the lupan compounds contains two heminal methyl
group at C4. The sterical and electronic effects resulted
from this structural peculiarity favor elimination reac-
1
Please send correspondence to phone, +7 (095) 434-8355 or
e-mail, alex.kaplun@mtu-net.ru.
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186
SYMON et al.
29CH2
H33C0 20
29CH2
H33C0 20
19
19
H
H
R2
R3
H3C
CH3
H
H3C
CH3
H
28
28
2
3
2
3
H
4
H
CH3
H
4
H
CH3
R1O
H3C CH3
(I) R1 = H, R2 = OH
H3C
CH3
(IV) R3 = OH
(V) R3 = OBz
(VI) R3 = H
(II) R1 = H, R2 = H
(III) R1 = Ts, R2 = H
Scheme 1.
tions and hinder the reactions of bimolecular substitu-
We succeeded to obtain epibetulinic acid (Xa) using
tion. In this connection, any variants of bimolecular sterically hindered lithium tri-sec-butylborohydride (L-
Selectride). According to 1H NMR spectrum, the reduc-
tion of betulonic acid with this reagent results in a 62 :
38 mixture of epimers (Xa) and (Xb) (Scheme 2 and
Table 1). Note that the chromatographic mobilities of
epibetulinic (Xa) and betulonic (VII) acids are close,
whereas the difference in the mobilities of betulinic
acid epimers (Xa) and (Xb) is rather significant.
substitution at position 3 of lupans seem to be unprom-
ising.
The catalytic hydrogenation of betulonic acid (VII)
over Raney nickel up to the temperature of 50°ë and
the pressure of 50 atm resulted only in the reduction of
the double bond of isopropenyl group and the forma-
tion of 20,29-dihydrobetulonic acid (VIII). Two char-
acteristic doublets of methyl protons at C30 (0.88 ppm)
and C29 (0.76 ppm) are observed in the 1H NMR spec-
trum of this compound. At the same time, the resonance
of the C3 proton was not found. The use of a more
active catalyst, 5% Ru/C, led to a mixture of α- and β-
epimers of 20,29-dihydrobetulinic acid (IXa and IXb)
Cytotoxic activity of epimers of betulinic acid (Xa)
and (Xb) was studied in human tumor cell cultures of
various histogeneses, namely, melanomas Bro and MS
and ovary carcinoma CaOv. The cytotoxic effect was
determined using the MTT test [12]. The cells used
were conventionally sensitive to doxorubicin, a cyto-
static agent (IC50 0.1–0.5 µM).
1
at a ratio of 60 : 40 according to H NMR spectrum
(Scheme 2 and Table 1). The presence of a characteris-
tic signal of hydroxy group in its IR spectrum
(3430 cm–1) confirmed the reduction of the betulonic
acid keto group. In addition, the resonances from the
isopropyl group protons and 3-protons corresponding
Our results (Table 2) show that betulinic acid (Xb)
displays cytotoxic properties toward two cell cultures,
MS and CaOv (IC50 5 µM) and is not toxic toward mel-
anoma Bro cells (IC50 > 10 µM). Epibetulinic acid (Xa)
to α- and β-epimers (at 3.29 and 3.15 ppm) appeared in did not exhibit an expected rise in cytotoxicity (IC50 >
the 1H NMR spectrum of the compound.
10 µM). However, epimer (Xa) demonstrated a low
Table 1. Epimerization of hydroxyl group in triterpenes of lupane series
Starting
Reaction conditions
compounds
Reaction products
Yield, %
(I)
Ph3P, PhCO2H, EtCO2N=NCO2Et/THF
20°C/10 h
(IV)
44
16
17
95
45
29
38
19
(V)
(III)
DMF
78°C/23 h
(VI)
(VII)
H2/Ni(Raney)/iPr
H2/5% Ru/C/MeOH
50°C/50 atm
80°C/80 atm
(VIII)
(IXa)
(IXb)
(Xa)
(Xb)
L-Selectride/íHF
–80°C/5 h
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 2 2003

EPIMERIZATION OF HYDROXYL GROUP
187
R2
29CH2
H33C0 20
H33C0 20
19
19
H
H
OH
OH
H3C
CH3
H
H3C
CH3
H
28
28
O
O
H
H
CH3
H
H
CH3
3
3
R1
H3C
O
CH3
(VIII) R1 = O, R2 = H, CH3
H3C
CH3
(VII)
(IXa) R1 = H, α-OH, R2 = H, CH3
(IXb) R1 = H, β-OH, R2 = H, CH3
(Xa) R1 = H, α-OH, R2 = CH2
(Xb) R1 = H, β-OH, R2 = CH2
Scheme 2.
cytotoxicity toward all the three tumor cell cultures, compounds. Column chromatography was performed
whose survival was 70–75% (Table 3).
on Silica gel L 60/100 µ (Chemapol, Czech Republic).
Thus, we did not find the expected increase in cyto-
toxicity of epibetulinic acid (Xa) toward human mela-
noma MS cells in comparison with the cytotoxic activ-
ity of betulinic acid (Xb). However, the spectrum of
biological activity of epimer (Xa) somewhat differs
from that of (Xb), since it displays cytotoxic properties
toward melanoma Bro cells, which are insensitive to
betulinic acid.
Betulin (I) and lupeol (II) were isolated from a birch
bark [14]. Betulonic acid (VII) was obtained by the
procedure in [7].
A comparative cytotoxicity of epimers of betulinic
acid was studied in the Research Institute of Experi-
mental Diagnostics and Therapy of Tumors, Blokhin
Cancer Research Center, Russian Academy of Medical
Sciences, using human melanoma (lines Bro and MS)
and ovary carcinoma (CaOv) cells.
EXPERIMENTAL
An attempt of betulin (I) epimerization by Mit-
sunobu’s reaction. Betulin (I) (0.20 g, 0.45 mmol),
Ph3P (0.26 g, 0.99 mmol), a solution of benzoic acid
(0.12 g, 0.99 mmol) in dry THF (2 ml), and a solution
of diisopropyl azodicarboxylate (0.20 g, 0.99 mmol)
were added under stirring to dry THF (2 ml); the solu-
tion was stirred for 10 h at 20°ë and evaporated. The
residue was chromatographed on a silica gel column
eluted with chloroform to give 3-deoxy-2,3-dehydro-
betulin (IV) (0.083 g, 44%) as an oil and 3-deoxy-
2,3-dehydrobetulin 28-benzoate (V) (0.037 g, 16%) as
an oil.
Diisopropyl azodicarboxylate and L-Selectride
were from Aldrich (United States); other chemicals
were of domestic production of at least the reagent
grade.
1H NMR spectra were registered on a Bruker MSL-
200 spectrometer (Germany) in CDCl3, CD3OD, or
acetone-d6 at a working frequency of 200.13 MHz.
Chemical shifts are given in a δ scale relative to Me4Si.
IR spectra were recorded on a Hitachi 270-30 data pro-
cessor (Japan) within the ν range of 800–3600 cm–1 in
Vaseline oil. Optical absorption was measured on a
scanning Multiskan MCC/340 spectrometer (Lab-
System, Finland) at 540 nm.
1
(IV): Rf 0.50 (A); IR: 3470 (OH), 1660 (C=C); H
NMR (CDCl3): 5.37 (2 H, m, CH=CH), 4.66 (1 H, d,
=ëç2), 4.56 (1 H, d, =ëç2), 3.81 (1 H, m, H28), 3.26
For TLC, Sorbton-Diol plates (Chromdet-Ecologia,
Russia) were used in the following systems: (A) chlo-
roform; (B) 100 : 2 : 0.5 chloroform–methanol–formic
acid; and (C) 40 : 1 : 0.1 chloroform–methanol–30%
ammonia. Triterpenes were detected on the plates using
the anisaldehyde-containing reagent [13]; we modified
its composition as follows: anisaldehyde (1.85 ml),
alcohol (95 ml), H2SO4 (7.5 ml), and acetic acid
(0.75 ml). An alcoholic solution of 2,4-dinitrophenyl-
hydrazine was used for detecting carbonyl-containing
Table 2. Cytotoxicity of betulinic acid epimers relative to
the human tumor cells (IC50, µM)
Cells
Epibetulinic acid (Xa) Betulinic acid (Xb)
CaOv
MS
>10
>10
>10
5
5
Bro
>10
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 2 2003

188
SYMON et al.
Table 3. Viability of the human tumor cells under the action
of epibetulinic and betulinic acids (10 µM) (% of control)
The temperature was then increased to 50°ë, the ç2
pressure remaining constant. After 17 h, the catalyst
was filtered off, and the filtrate was evaporated. The res-
idue was diluted with ether (70 ml) and 10% HCl
(30 ml) in a separating funnel, shaken up to the com-
plete dissolution of crystals, the ether layer was sepa-
rated, washed with water, dried with sodium sulfate,
and evaporated to give dihydrobetulonic acid (VIII) as
colorless crystals; yield 0.450 g (95%); mp 250–253°ë
(lit. mp 251–254°ë [15]); Rf 0.42 (C); IR: 1703 (C=O
in RCOR), 1687 (C=O in COOH); 1H NMR (CDCl3):
1.07 (3 H, s, CH3), 1.03 (3 H, s, CH3), 0.97 (3 H, s,
CH3), 0.93 (3 H, s, CH3), 0.87 (3 H, d, CH3), 0.84 (3 H,
s, CH3), 0.75 (3 H, d, CH3).
Epibetulinic
acid (Xa)
Betulinic
acid (Xb)
Cells
CaOv
MS
75
70
70
52
52
Bro
100
(1 H, m, H28), 2.36 (1 H, m, H19), 1.66 (3 H, s, CH3),
1.23 (3 H, s, CH3), 0.96 (3 H, s, CH3), 0.94 (3 H, s,
CH3), 0.80 (3 H, s, CH3), and 0.74 (3 H, s, CH3).
(V): Rf 0.69 (A); IR: 3030, 3050 (C–H aromatic),
B. Hydrogenation in the presence of 5% Ru/C. A
solution of betulonic acid (VII) (0.20 g, 0.44 mmol) in
methanol (15 ml) was hydrogenated over 5% Ru/C
(0.10 g) at 40 atm and 20°ë. After 22 h, the hydrogen
pressure and the temperature were increased to 70 atm
and 50°ë, respectively. After 7 h, the stirring and heat-
ing were stopped, and the mixture was kept in the auto-
clave for 15 h at 20°ë. Then the hydrogen pressure was
elevated to 80 atm and the temperature to 80°ë. After
4.5 h, the stirring and heating were stopped, and the
mixture was kept in the autoclave for 15 h. The reaction
mixture was washed off from the catalyst, evaporated,
and chromatographed on a silica gel column eluted
with chloroform to give 3α-20,29-dihydrobetulunic
acid (IXa) as colorless crystals; yield 0.090 g (45%);
mp 297–300°ë (lit. mp 298–301°ë [9]); Rf 0.39 (C);
1720 (C=O in PhCOOR), 1650 (C=C), 1590 (C=C aro-
matic), 1280 (C–O in RCOOR); H NMR (CDCl3):
1
8.03 (2 H, m, aromatic protons), 7.50 (3 H, m, aromatic
protons), 5.35 (2 H, m, CH=CH), 4.70 (1 H, d, =ëç2),
4.59 (1 H, d, =ëç2), 4.50 (1 H, d, H28), 4.12 (1 H, d,
H28), 2.38 (1 H, m, H19), 1.66 (3 H, s, CH3), 1.23 (3 H,
s, CH3), 0.96 (3 H, s, CH3), 0.94 (3 H, s, CH3), 0.80 (3
H, s, CH3), and 0.74 (3 H, s, CH3).
3-O-Tosyllupeol (III). Lupeol (II) (0.080 g,
0.19 mmol) and tosyl chloride (0.075 g, 0.39 mmol)
were added to pyridine (0.94 ml) at 20°ë. The mixture
was stirred for 24 h, and water was added until a precip-
itate was formed. The precipitate was filtered, dissolved
in chloroform, washed with 5% HCl (3 × 5 ml), and
dried over Na2SO4. The solution was evaporated, and
the residue was dried over P2O5 in a vacuum to give
1
IR: 3500 (OH in COOH), 1690 (C=O in COOH); H
NMR (CDCl3): 3.31 (1 H, m, H3β), 1.04 (3 H, s, CH3),
0.97 (3 H, s, CH3), 0.93 (3 H, s, CH3), 0.85 (3 H, d,
CH3), 0.84 (3 H, s, CH3), 0.77 (3 H, s, CH3), and 0.74
(3 H, d, CH3). The subsequent elution of the column
gave 3β-20,29-dihydrobetulunic acid (IXb) as colorless
crystals; yield 0.060 g (29%); mp > 310–313°ë (lit. mp
312–314°ë [16]); Rf 0.26 (C); IR: 3500 (OH in
1
(III) as an oil; yield 0.090 g (85%); Rf 0.63 (Ä); H
NMR (CDCl3): 7.34 (4 H, m, aromatic protons), 4.66
(1 H, d, =Cç2), 4.56 (1 H, d, =CH2), 4.18 (1 H, m, H3),
2.31 (1 H, m, H19), 2.21 (3 H, m, CH3Ar), 1.66 (3 H, s,
CH3), 1.23 (3 H, s, CH3), 0.96 (3 H, s, CH3), 0.94 (3 H,
s, CH3), 0.92 (3 H, s, CH3), 0.80 (3 H, s, CH3), and 0.74
(3 H, s, CH3).
COOH), 1690 (CO in COOH); 1H NMR (CDCl3): 3.15
(1 H, m, H3α), 1.06 (3 H, s, CH3), 0.98 (3 H, s, CH3),
0.95 (3 H, s, CH3), 0.85 (3 H, d, CH3), 0.84 (3 H, s,
CH3), 0.78 (3 H, s, CH3), and 0.76 (3 H, d, CH3).
An attempt of 3-O-tosyllupeol (III) epimerization
in DMF. A solution of (III) (0.080 g, 0.14 mmol) in
undistilled DMF (3.2 ml) was heated at 78°ë for 23 h,
evaporated, and chromatographed on a silica gel col-
umn eluted with a 1 : 1 hexane–chloroform mixture to
give 3-deoxy-2,3-dehydrolupeol (VI) as an oil; yield
3a-Betulinic acid (Xa). A 1 M solution of L-Selec-
tride in THF (5.6 ml) was added to a solution of betu-
lonic acid (VII) (0.33 g, 0.73 mmol) in dry THF (10 ml)
at –80°ë under argon. The mixture was stirred for 5 h.
A 2 N NaOH (18 ml) and 38% ç2é2 (4 ml) were added
to the reaction mixture, stirred for 1 h, THF was evapo-
rated, and 10% HCl (15 ml) and ether (20 ml) were
added to the resulting precipitate. The mixture was
stirred until the precipitate was completely dissolved,
and the ether layer was separated and evaporated to
give 254 mg of the residue. It was chromatographed on
a silica gel column eluted with chloroform to give 3α-
betulinic acid (Xa) as colorless crystals; yield 127 mg
(38%); mp 278–281°ë (lit. mp 279–283°ë [9]); Rf 0.38
1
0.010 g (17%); Rf 0.67 (Ä); H NMR (CDCl3): 5.35
(2 H, m, CH=CH), 4.67 (1 H, m, =ëç2), 4.55 (1 H, m,
=ëç2), 2.38 (1 H, m, H19), 1.66 (3 H, s, CH3), 1.23
(3 H, s, CH3), 0.96 (3 H, s, CH3), 0.94 (3 H, s, CH3),
0.91 (3 H, s, CH3),0.80 (3 H, s, CH3), and 0.74 (3 H, s,
CH3).
Catalytic hydrogenation of betulonic acid (VII).
A. Hydrogenation over Raney nickel under pres-
sure. Raney nickel (2 g) was added to a solution of bet-
ulonic acid (VII) sodium salt (0.500 g, 1.05 mmol) in
isopropanol (40 ml), and the reaction mixture was
stirred at 20°ë under H2 pressure of 50 atm for 17 h. (C); 1H NMR (1 : 1 CDCl3–acetone-d6): 4.64 (1 H, m,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 2 2003

EPIMERIZATION OF HYDROXYL GROUP
189
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=CH2), 4.51 (1 H, m, =CH2), 3.29 (1 H, m, H3β), 2.99
(1 H, m, H19), 1.62 (3 H, s, CH3), 0.94 (3 H, s, CH3),
0.88 (3 H, s, CH3), 0.86 (3 H, s, CH3), 0.78 (3 H, s,
CH3), and 0.75 (3 H, s, CH3). The subsequent elution
gave colorless crystals of 3β-betulunic acid (Xb); yield
0.063 g (19%); mp 291–293°ë (lit. mp 291–292°ë
[17]); Rf 0.31 (C); 1H NMR (3 : 1 CDCl3–CD3OD): 4.71
(1 H, m, =CH2), 4.59 (1 H, m, =CH2), 3.15 (1 H, m,
H3α), 3.01 (1 H, m, H19), 1.67 (3 H, s, CH3), 0.96
(3 H, s, CH3), 0.94 (3 H, s, CH3), 0.93 (3 H, s, CH3),
0.81 (3 H, s, CH3), and 0.74 (3 H, s, CH3).
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FBS (embryonic cow serum from Gibco DRL), 2 mM
L-glutamine, and gentamycin (40 µg/ml) in the 5%
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were added to the cell suspensions of the tumor cell cul-
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ACKNOWLEDGMENTS
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The work was supported by the Russian Foundation
for Basic Research, projects nos. 00-04-48363 and
01-04-06098, and Ministry of Higher Education of
Russian Federation, project no. 203.05.04.003.
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