Synthesis of betulinic acid from betulin extract and study of the antiviral and antiulcer activity of some related terpenoids

Full text of DOI:10.1023/A:1021844705853

Pharmaceutical Chemistry Journal
Vol. 36, No. 9, 2002
SYNTHESIS OF BETULINIC ACID FROM BETULIN EXTRACT
AND STUDY OF THE ANTIVIRAL AND ANTIULCER ACTIVITY
OF SOME RELATED TERPENOIDS
O. B. Flekhter,1 L. R. Nigmatullina,1 L. A. Baltina,1 L. T. Karachurina,1
F. Z. Galin,1 F. S. Zarudii,1 G. A. Tolstikov,1 E. I. Boreko,2 N. I. Pavlova,2
S. N. Nikolaeva,2 and O. V. Savinova2
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 36, No. 9, pp. 26 – 28, September, 2002.
Original article submitted May 21, 2002.
tained up to 90% of betulin and about 5% of lupeol (IV).
Betulonic acid (III) formed as a result of this oxidation pro-
cess was isolated from the reaction mixture in the form of a
potassium salt (V) with a yield of 82%, which allowed us to
exclude the stage of intermediate product purification by col-
umn chromatography [10]. Upon acidification of an ethanol
solution of the potassium salt V with a 5% hydrochloric acid
solution, betulonic acid (III) of 95% purity was isolated with
a yield of 85%. The physicochemical parameters of the prod-
uct corresponded to the published data [2, 10, 11]. The total
yield of betulonic acid (III) calculated for the initial betulin
extract was 67%. Column chromatography of the organic
fraction yielded 3-keto-lupeol (VI) with a yield of 1.7%.
3b-Hydroxy-lup-20(29)-en-(28)-oic (betulinic) acid (I)
was isolated at the beginning of the 20th century and origi-
nally called gratiolone [1]. In the past decade, some special
biological properties of this triterpenoid has drawn the atten-
tion of researchers. In 1994, betulinic acid was found to ex-
hibit anti-HIV-1 activity in H9 lymphocyte cell culture [2].
The most promising inhibitor of HIV-1 replication is
3-O-(3¢,3¢-dimethylsuccinate) of betulinic acid [3]. In 1995,
it was established that betulinic acid is capable of selectively
inhibiting the growth of malignant human melanoma [4]. In
addition, betulinic acid and its derivatives possess antibacte-
rial, antimalarial, antiinflammatory, bile-expelling, and other
properties [5 – 7].
Since the content of betulinic acid in plants is very low,
isolation of this compound from raw plant material is poorly
profitable. A more effective method of obtaining betulinic
acid is synthesis from betulin (II) and betulonic acid (III).
The first synthesis of betulinic acid was suggested by
Ruzicka et al. [8]. This original procedure involved (i) pro-
tection of the hydroxy groups of II by acetylation, (ii) selec-
tive hydrolysis of the acetyl group at C-28, (iii) oxidation of
the oxymethyl group to carboxy group, and (iv) removal of
the protective acetyl group at C-3. The total yield of betulinic
acid from this process did not exceed 30%.
In the late 1990s, betulinic acid was synthesized (I) in
two steps by oxidizing betulin (II) with Jones reagent or
chromic anhydride in acetic acid, followed by selective oxi-
dation of the resulting betulonic acid (III) with NaBH4
[9, 10], whereby the total yield of the target compound
reached up to 60%. We used Jones reagent to oxidize the ex-
tract obtained by treating birch bark with aqueous
isopropanol in an autoclave at 80°C. The initial extract con-
R3
R1
2
R
I – VI
I: R1 = H, R2 = OH, R3 = COOH;
II: R1 = H, R2 = OH, R3 = CH2OH;
III: R1 + R2 = O, R3 = COOH;
IV: R1 = H, R2 = OH; R3 = CH3;
V: R1 + R2 = O; R3 = COOK;
VI: R1 + R2 = O; R3 = CH3
O
O
1
Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy
O
of Sciences, Ufa, Bashkortostan, Russia.
2
VII
Research Institute of Epidemiology and Microbiology, Ministry of Public
Health of Belarus, Minsk, Belarus.
484
0091-150X/02/3609-0484$27.00 © 2002 Plenum Publishing Corporation

Synthesis of Betulinic Acid from Betulin Extract and Study of the Antiviral
485
Stereoselectivity of the process of betulonic acid (III) re-
duction with NaBH4 depends on the solvent (isopropanol,
dioxane, ethanol – dioxane [12], chloroform – methanol
[13]) (Table 1). It was found that this reduction reaction pro-
ceeds with quantitative yield and the stereoselectivity is vir-
tually independent of the process temperature. The
room-temperature reduction of betulonic acid in propanol
yields betulinic acid (I) with a 97/3 ratio of 3b-OH and
3a-OH epimers (according to 1H NMR data). In
tetrahydrofuran at 0°C, the 3b-OH/3a-OH selectivity ratio
was 95/5 [14]. When the process of reduction in isopropanol
is performed with LiBH4, the proportion of 3b-OH epimer
decreases to 80%. A single recrystallization of the reaction
product from methanol yields pure 3b-OH epimer.
As is known, both betulin (I) and betulinic acid (II)
isomerize into derivatives of the oleanane series with a yield
not exceeding 80% [15 – 18]. By conducting the process in
trifluoroacetic acid, we obtained 28-oxo-allobetulone (VII)
from betulonic acid (III) for 10 min with a yield of 95%.
Investigation of the antiviral properties of compound VII
in a cell culture showed evidence of inhibition of the growth
of influenza type A virus. At the same time, compound VII
was inactive with respect to herpes simplex virus (HSV-1),
and ECHO-6 enterovirus (Table 2). It was also found that
compounds III and VII possess antiinflammatory properties
(Table 3).
Betulin extract (II). A mixture (9 : 1, v/v) of crushed
birch bark (500 g) with aqueous 2-propanol (3 liters) was
treated for 2 h in an autoclave at 80°C and filtered hot, after
which the filtrate was kept overnight at +5°C. The precipitate
was separated by filtration, washed on the filter with cold
isopropanol (until clarification of the washing medium) and
then with cold water, and dried to obtain 117 g (23.4%) of an
extract containing ~90% of betulin (II) and ~5% of lupeol
(HPLC data).
Betulonic acid (III). To a solution of 100 g of betulin
extract in acetone (3 liters) cooled to 5 – 10°C was added
130 ml of freshly prepared Jones solution and the mixture
was stirred for 3 h at 20 – 22°C. Then the solvent was evapo-
rated to dryness in vacuum and the residue was diluted with
water (1 liter). The precipitate was separated by filtration,
washed on the filter with water, and dried to obtain 90 g
(90%) of technical-purity betulonic acid (III). For purifica-
tion, the dry product was dissolved in 300 ml of benzene.
The solution was filtered through a ~5-mm-thick layer of
aluminum oxide and the eluate was treated with a 10% aque-
ous KOH solution until complete precipitation of salt V. The
precipitate was separated by filtration, washed with 100 ml
of cooled benzene, and dried to obtain 73.8 g (82%) of salt V.
This salt was dissolved in 300 ml of methanol or ethanol and
the solution was poured into 1 liter of a 15% aqueous hydro-
chloric acid solution. The precipitate of betulonic acid (III)
was filtered, washed with water, and dried to obtain 67.2 g
(91%) of ~95% purity (HPLC data); Rf , 0.55; m.p.,
246 – 248°C (reported m.p., 247 – 249°C [11]).
EXPERIMENTAL CHEMICAL PART
Benzene solution was was treated with water
(2 ´ 300 ml), dried over CaCl2, and evaporated in vacuum.
The residue was chromatographed on an aluminum oxide
column eluted with benzene to obtain 3-keto-lupeol (VI)
with a yield of 1.7 g (1.7% of the initial extract); Rf, 0.83;
TLC analyses were conduced on Silufol plates
(Chemapol, Czech Republic) eluted with
a chloro-
form–methanol (20 : 1) solvent mixture. The spots were de-
veloped by treatment in a 20% phosphotungstic acid solution
in ethanol, followed by 2 – 3 min heating at 100 – 120°C.
The 13C and 1H NMR spectra were measured on a Bruker
AM-300 spectrometer (operating at 75.5 and 300 MHz, re-
spectively) using SiMe4 as the internal standard. The meting
points were determined using a Boetius heating stage. HPLC
analyses were performed on a DuPont chromatograph using
a procedure described in [19]. The data of elemental analyses
agree with the results of calculations according to the empiri-
cal formulas.
1
m.p., 141 – 143°C; H NMR spectrum (d, ppm): 0.76, 0.82,
0.87, 0.90, 0.92, 0.97 (18H, 6CH3), 1.10 – 2.00 (m, CH2,
CH), 1.69 (s, 3H, CH3), 2.27 – 2.36 (m, 1H, H-19), 4.61 and
4.75 (broad signals, 1H, H-29); 13C NMR spectrum (d, pm):
14.7; 15.8; 16.2; 18.3; 19.3 (H-30); 19.4; 20.9; 21.5; 25.6;
27.1; 29.5; 30.8; 32.0; 33.3; 34.0; 36.9; 37.1; 38.2; 39.5;
40.6; 42.4; 46.9; 47.2; 49.1; 50.3; 54.6; 55.0; 109.6 (C29);
150.6 (C20); 218.0 (C3).
Betulinic acid (I) was obtained by reduction of acid III
at 0 or 20 – 22°C.
TABLE 1. Stereoselectivity of the Reaction of Betulonic Acid Re-
duction under Various Conditions (3b-OH/3a-OH Ratio)
TABLE 2. Antiviral Properties of 28-Oxo-allobetulone (VII)
Temperature
Solvent
Influenza A
HSV-1
ECHO-6
Com-
pound
20 – 22 °C
0 °C
EC50
(I95), mM
EC50
EC50
(I95), mM
MPC/EC50
MPC/EC50
MPC/EC50
(I95), mM
2-Propanol
97/3
93/7
93/7
95/5
96/4
93/7
94/6
95/5
Dioxane
VII
28.13
15.64
> 439.86
< 1
> 219.93
< 1
Ethanol – dioxane
Chloroform –methanol
Notes: MTC = maximum tolerated concentration; EC50 = mean ef-
fective concentration; I95 = 95% confidence probability interval.

486
O. B. Flekhter et al.
(a) To a solution of 0.47 g (1 mmole) of betulonic acid
(e) The procedure is conducted similarly to experiment
(a), using 50 mg (2.3 mmole) of LiBH4 instead of NaBH4;
(III) in 20 ml of 2-propanol was gradually (over 10 min)
added with stirring 50 mg (1.3 mmole) of NaBH4. The mix-
ture was stirred for 2 h and diluted with 30 ml of a 10%
aqueous hydrochloric acid solution. The precipitate of
betulinic acid (I) was filtered, washed with water, and dried
to obtain compound I; Rf, 0.28; m.p., 290 – 292°C (reported
m.p., 291 – 292°C [11]). Process at 0°C: yield, 0.43 g (95%);
1H NMR spectrum (d, ppm): 0.78, 0.81, 0.86, 0.95, 1.01
(15H, 5CH3), 1.10 – 2.00 (m, CH2, CH), 1.69 (s, 3H, CH3),
2.04 – 2.12 (m, 1H, H-13), 2.17 – 2.32 (m, 1H, H-19), 3.15
(m, 0.97H, 3-Ha), 3.40 (m, 0.03H, 3-Hb), 4.61 and 4.75 (bs,
1
yield, 0.42 g (93%); H NMR spectrum (d, ppm): 3.20 (m,
0.80H, 3-Ha), 3.41 (m, 0.20H, 3-Hb).
28-Oxo-allobetulone (VII). To a solution of 0.47 g
(1 mmole) of betulonic acid (III) in 10 ml of chloroform was
added with stirring 2 ml of trifluoroacetic acid and the mix-
ture was kept for 8 min (TLC monitoring). Then the mixture
was neutralized with a 15% aqueous sodium carbonate solu-
tion, washed with water, and dried over MgSO4. The solvent
was evaporated at a reduced pressure (water jet pump) to ob-
tain 0, 43 g (95%) of compound VII in the form of white
1
1H, H-29). Process at 20 – 22°C: yield, 0.42 g (92%); H
1
crystals; m.p., > 300°C; H NMR spectrum (d, ppm): 0.86,
NMR spectrum (d, ppm): 3.15 (m, 0.96H, 3-Ha), 3.40 (m,
0.93, 0.94, 1.01, 1.05 (7CH3), 1.15 – 2.00 (m, CH2, CH),
3.90 (s, 1H, H-19); 13C NMR spectrum (d, ppm): 13.6; 15.3;
16.4; 19.5; 21.0; 21.4; 24.0; 25.5; 26.5; 26.7; 27.9; 28.7;
31.9; 32.3; 33.0; 33.5; 34.0; 36.1; 37.0; 39.8; 40.0; 40.5;
46.1; 46.7; 47.3; 50.6; 55.0; 85.9; 179.8; 217.9.
0.04H, 3-Hb).
(b) To a solution of 0.47 g (1 mmole) of betulonic acid
(III) in 10 ml of dioxane was added 0.15 g (4 mmole) of
NaBH4. The mixture was stirred for 1.5 h and then treated as
in experiment (a). Process at 0°C: yield, 0.42 g (92%); H
NMR spectrum (d, ppm): 3.13 (m, 0.93H, 3-Ha), 3.40 (m,
1
0.07H, 3-Hb).
EXPERIMENTAL PHARMACOLOGICAL PART
(c) The procedure is conducted in 10 ml of an etha-
nol – dioxane (1 : 1, v/v) mixture similarly to experiment
(b). Process at 0°C: yield, 0.41 g (90%); H NMR spectrum
(d, ppm): 3.13 (m, 0.94H, 3-Ha), 3.37 (m, 0.06H, 3-Hb).
Process at 20 – 22°C: yield, 0.42 g (93%); H NMR spec-
The antiviral properties of compound VII were experi-
mentally studied using cell cultures inoculated with
A/FPV/Rostock/34(H7N1) influenza virus, type 1 HSV
(HSV-1), and ECHO-6 virus. The tests were performed by
measuring the plaque reduction on primary chicken embryo
fibroblast culture with FPV and by determining the
cytopathic effect on human rhabdomiosarcoma (HRD) cells
with HSV-1 and ECHO-6 as described in [20]. Compound
VII was dissolved in a 10% aqueous ethanol (stock solution)
and then diluted with the corresponding maintenance me-
dium to the required concentration.
The antiviral effect was judged by reduction of the viral
titer in the presence of compound VII relative to control and
characterized by the effective concentration producing a 50%
inhibition of the virus growth (EC50) and by the ratio of the
maximum tolerated drug concentration (MTC) to EC50. The
MPC values were determined upon 72-h incubation of
uninoculated cell cultures.
1
1
trum (d, ppm): 3.15 (m, 0.93H, 3-Ha), 3.40 (m, 0.07H,
3-Hb).
(d) The procedure is conducted in 20 ml of an chloro-
form – methanol (1 : 1, v/v) mixture similarly to experiment
(a), with 10 mg (0.3 mmole) NaBH4 added every hour. Then
the reaction mixture was acidified by adding 10 ml of a 10%
aqueous hydrochloric acid solution. The organic layer was
separated and the aqueous layer was extracted with chloro-
form (2 ´ 15 ml). The organic layers were combined, treated
with water (2 ´ 30 ml), dried over Na2SO4 and evaporated in
vacuum. Process at 20 – 22°C: yield, 0.40 g (88%); 1H NMR
spectrum (d, ppm): 3.18 (m, 0.95H, 3-Ha), 3.37 (m, 0.05H,
3-Hb).
The antiulcer activity of compound VII and betulonic
acid (III) was studied on a group of 48 white mongrel rats
weighing 180 – 200 g with the models of mucous membrane
damage induced by indomethacin (20 mg/kg, i.p.) and
TABLE 3. Antiulcer Effect of Betulonic Acid (III) and 28-Oxo-
allobetulone (VII)
acetylsalicylic acid (2 ´ 150 mg/kg, i.g.). The compounds
studied in a dose of 50 mg/kg (0.01LD50) and the reference
drug venter (20 mg/kg) were introduced perorally 1 h before
the model ulcer induction. The test animals were killed 24 h
after indomethacin or aspirin administration. The stomachs
were extracted, cut along the lesser curvature, washed, and
characterized with respect to the state of the mucous mem-
brane. The antiulcer activity was judged by the decrease in
the number of damaged sites [21].
Number of damaged sites induced by
Dose,
Compound (n = 6)
mg/kg
Indomethacin
Aspirin
12.3 ± 1.4a
19.6 ± 5.52
12.0 ± 0.68a
28.75 ± 2.73
16.0 ± 0.84b
18.6 ± 1.89
12.5 ± 0.82c
20.0 ± 1.26
III
50
50
20
–
VII
Venter
Control
Notes: differences from control are reliable for a p < 0.001,
b p < 0.05, c p < 0.02.

Synthesis of Betulinic Acid from Betulin Extract and Study of the Antiviral
487
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ACKNOWLEDGMENTS
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This study was supported by the Russian Foundation for
Basic Research (project Nos. 01-03-33131 and 02-03-81007)
and by the Belarus Foundation for Basic Research (project
No. B02R-016).
13. S. Sturm, R. R. Gil, H. B. Chai, et al., J. Nat. Prod., 59,
658 – 663 (1998).
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