Synthesis of 3-O-Acetylbetulinic and betulonic aldehydes according to svern and the pharmacological activity of related oximes

Full text of DOI:10.1023/A:1020824506140

Pharmaceutical Chemistry Journal
Vol. 36, No. 6, 2002
SYNTHESIS OF 3-O-ACETYLBETULINIC AND BETULONIC ALDEHYDES
ACCORDING TO SVERN AND THE PHARMACOLOGICAL ACTIVITY
OF RELATED OXIMES
1
1
2
1
2
O. B. Flekhter, O. Yu. Ashavina, E. I. Boreko, L. T. Karachurina, N. I. Pavlova,
1
2
1
2
1
N. N. Kabal’nova, O. V. Savinova, F. Z. Galin, S. N. Nikolaeva, F. S. Zarudii,
L. A. Baltina, and G. A. Tolstikov
1
1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 36, No. 6, pp. 21 – 24, June, 2002.
Original article submitted March 28, 2002.
As is known, triterpenoids of the lupane group exhibit a
wide spectrum of biological activity [1 – 3]. Of considerable
interest are lupane aldehydes, the derivatives of which were
reported to possess anti-HIV and antimelanoma properties
the initial compound. The complete conversion of compound
II requires using two molar equivalents of the Svern reagent;
the yield of product III in this case amounts to 95%. The oxi-
dation process conducted at –10°C led to only a 50% conver-
sion of the initial reagents, while the reaction at 0°C yielded
no aldehydes at all.
[
4, 5].
Previously, compounds of the lupane group were oxi-
dized by reagents containing Cr(VI): CrO in acetone [6],
3
The proposed structures of the target aldehydes III and
pyridinium bichromate in a DMF – H O mixture, and
13
1
2
IV were confirmed by the results of IR and C and H NMR
measurements (Tables 1 and 2) and by agreement with the
published data [5, 6]. The NMR spectra of the synthesized
compounds contained no signals of alcohol hydroxy groups,
pyridinium chlorochromate in CH Cl [4, 5]. The carbonyl
2
2
products were obtained with a yield of not greater than 80%,
sometimes in the form of difficultly separable mixtures of ac-
ids and deeper oxidation products [7]. Recently, Evers, et al.
1
3
but displayed signals from the aldehyde (206.4 ppm in
C
[
8] demonstrated that a convenient method for the synthesis
and 9.64 ppm in H NMR) and keto (217.6 ppm in 13C
1
of 3b-[(dimethyl-tert-butylsilyl)oxy]betulinic aldehyde is of-
fered by the oxidation reaction according to Svern.
NMR) groups.
Aldehydes III and IV were used for the synthesis of re-
lated oximes (V – VIII). 3-O-Acetyl-28-oxime-betulin (V)
and 3,28-dioxime-betulin (VI) were obtained with a yield of
In continuation of our previous investigations devoted to
the transformation of lupane triterpenoids [9 – 11], we have
synthesized 3-O-acetylbetulinic aldehyde (III) and betulonic
aldehyde (IV) by oxidizing betulin (I) and its 3-O-acetate (II)
with “activated” DMSO according to Svern.
It was established that the oxidation of triterpenoid I by
one or two molar equivalents of the oxidizing reagent pro-
ceeds in a nonselective manner, leaving unreacted betulin,
with the formation of betulinic and betulonic (IV) aldehydes.
The complete conversion of betulin (I) into betulonic alde-
hyde (IV) requires using four molar equivalents of activated
DMSO; the yield of product IV in this case amounts to 93%
85 – 92% by boiling aldehydes III and IV with hydroxyl-
amine hydrochloride in pyridine. Saponification of the ace-
tate group of oxime V with a 5% KOH solution in methanol
led to 3b-hydroxy-28-oxime-betulin (VII). Dehydration of
oxime VI in acetic anhydride led to 3-oxime- betulonic acid
nitrile (VIII) with a yield of 87%.
It was established that compounds VI – VIII possess pro-
nounced antiviral properties with respect to the type A strain
of influenza virus. This result is in discrepancy with the data
reported in [13], according to which betulin was ineffective
against the influenza virus. In our tests, compound VI was
also capable of suppressing the growth of ECHO-6 and her-
pes simplex virus (HSV), but to a lower extent than it was
with the type A influenza virus (Table 3).
(
Table 1).
The oxidation of 3-O-acetylbetulin (II) at –30°C with
one molar equivalent of activated DMSO to 3-O-acetylbetu-
linic aldehyde (III) proceeds with a very low conversion of
1
Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy
Experiments on the models of mucous membrane dam-
age induced by indomethacin and acetylsalicylic acid (aspi-
rin) showed that compound III produces an antiulcer effect
of Sciences, Ufa, Bashkortostan, Russia.
Research Institute of Epidemiology and Microbiology, Ministry of Public
2
Health of Belarus, Minsk, Belarus.
303
0
091-150X/02/3606-0303$27.00 © 2002 Plenum Publishing Corporation

3
04
O. B. Flekhter et al.
melting points were deter-
mined using a Boetius heating
stage.
2
8
TLC analyses were con-
duced on Silufol plates
(Chemapol, Czech Republic)
eluted with a chloroform–me-
thanol (20 : 1) solvent mixture.
The spots were developed by
(
COCl) /DMSO
2
CH OH
2
CHO
CH Cl , –30 °C
2
2
2
3
R
1
RO
III, IV
I, II
R
I: R = H,
II: R = Ac
III: R
IV: R
1
= AcO, R
+ R = O
2
= H;
treatment
in
a
20%
2
1
phosphotungstic acid solution
in ethanol, followed by
2
1
– 3 min
00 – 120°C. DMSO was puri-
heating
at
NH OH · HCl, pyridine
5%KOH/MeOH
NOH
2
III
90 °Ñ
CH
fied by distillation at a reduced
pressure over freshly calcined
P O . Pyridine was dried and
distilled over KOH. Betulin
and 3-O-acetylbetulin were
synthesized as described in
CH
NOH
2
5
AcO
HO
VII
V
[11, 14], and betulinic alde-
NH OH · HCl, pyridine
Ac O, 100 °C
hyde (used as a TLC marker)
was obtained according to [6].
Reagents for the syntheses
were purchased from Sigma
2
2
IV
90 °Ñ
CH
NOH
CN
(
USA).
HON
HON
VIII
3-O-Acetylbetulinic alde-
VI
hyde (III) was obtained by ox-
idation of 3-O-acetylbetulin
with two molar equivalents of
the Svern reagent. A solution
(
(
Table 4) analogous to that of the reference drug venter
sucralfate).
of 0.19 ml (2.2 mmole) of oxalyl chloride (COCl ) in 5 ml of
2
CH Cl was placed in a three-neck flask equipped with a
2
2
magnetic stirrer, thermometer, two dropping funnels with
pressure compensator, and calcium chloride tubes. One drop-
ping funnel was charged with a solution of DMSO (0.34 ml,
EXPERIMENTAL CHEMICAL PART
The IR absorption spectra were recorded on Specord
M80 and UR-20 spectrophotometers using samples prepared
as suspensions in Vaseline oil. The 13C and H NMR spectra
were measured on a Bruker AM-300 spectrometer (operating
at 75.5 and 300 MHz, respectively) using deuterated chloro-
4
.8 mmole) in 5 ml of CH Cl and the other, with a solution
2 2
of 3-O-acetylbetulin II (0.53 g, 1.0 mmole) in 25 ml of
CH Cl . To the oxalyl chloride solution, cooled down to
1
2
2
–30°C, was gradually (over 5 min) added the DMSO solu-
tion and the mixture was stirred for 10 min. Then, the solu-
tion of II was added dropwise and the mixture was stirred for
form as the solvent and SiMe as the internal standard. The
4
TABLE 1. Yields and Physicochemical Characteristics of Compounds III – VIII
–
1
Compound
Yield, %
M.p., °C
IR spectrum: n, cm
Empirical formula
III
IV
V
95
93
85
85
91
87
173 – 175*
162 – 164
> 260
1735, 1715, 1645, 1485, 1395, 1355, 1085, 1030, 890, 840, 770, 725, 710
1710, 1700, 1650, 1465, 1390, 1095, 930, 815, 765, 710
C
C
C
C
C
C
32
30
32
30
30
30
H
H
H
H
H
H
50
46
O
O
3
2
3350, 1740, 1640, 1455, 1385, 1105, 990, 836, 755, 715
51NO
3
VI
244 – 246
223 – 225
> 260
3240, 1905, 1645, 1616, 1430, 1380, 1120, 1020, 940, 890, 750, 735, 710
3480, 2210, 1655, 1640, 1568, 1385, 1136, 930, 888, 795, 730
2920, 1955, 1688, 1396, 1280, 1105, 908, 855, 765, 735, 705
48 2 2
N O
VII
49NO
2
VIII
46 2
N O
*
Reported m.p., 171 – 176°C [12].

Synthesis of 3-O-Acetylbetulinic and Betulonic Aldehydes According to Svern
305
1
5 min (with TLC monitoring). Finally, 1.4 ml of
tic anhydride was boiled with reflux for 2 h and diluted with
water. The product was extracted with chloroform
(2 ´ 15 ml). The combined extracts were washed sequen-
tially with water, 5% aqueous Na CO solution, and water
triethylamine was added at –30°C and the mixture was
heated to room temperature and diluted with 6 ml of water.
The organic layer was separated and the aqueous layer
washed with CH Cl (20 ml). The organic phases were com-
2
3
again, and dried over Mg SO . Finally, the solvent was evap-
2
2
2
4
bined, washed sequentially with a 5% aqueous HCl solution,
water, 5% aqueous Na CO solution, and water again, and
orated in vacuum to obtain 0.41 g of the target nitrile VIII.
2
3
dried over Na SO . Finally, the solvent was evaporated in
2
4
EXPERIMENTAL PHARMACOLOGICAL PART
vacuum to obtain 0.49 g of the target compound III.
Betulonic aldehyde (IV) was obtained using a proce-
dure analogous to that described above, by oxidizing betulin
The antiviral properties of the synthesized compounds
were experimentally studied using cell cultures inoculated
with A/FPV/Rostock/34(H7N1) influenza virus, type 1 HSV
(
I) with four molar equivalents of the Svern reagent.
General method for the synthesis of 3-O-acetyl-28-
oxime-betulin (V) and 3,28-dioxime-betulin (VI).
(HSV-1), and ECHO-6 virus. The tests were performed by
measuring the plaque reduction on the primary chicken em-
bryo fibroblast culture with FPV and by determining the
cytopathic effect on human rhabdomyosarcoma (HRD) cells
with HSV-1 and ECHO-6 as described in [15]. The drugs
were dissolved in a 10% aqueous ethanol (stock solutions)
and then diluted with the corresponding maintenance me-
dium to a required concentration.
A mixture of 1 mmole of aldehyde III (0.48 g) or IV
(
1
0.44 g) and 0.5 – 1.0 g of hydroxylamine hydrochloride in
0 ml of pyridine was boiled with reflux for 2 h and poured
into a 5% aqueous hydrochloric acid solution. The precipi-
tate was separated by filtration, washed with water, dried,
and recrystallized from a CHCl –MeOH mixture to obtain
3
0
.41 – 0.38 g of the target product.
b-Hydroxy-28-oxime-betulin (VII). To a solution of
.49 g (1 mmole) of oxime V in 15 ml of MeOH was added
The antiviral effect was judged by reduction of the viral
titer in the presence of the synthesized compounds relative to
the control and characterized by calculating the effective
concentration producing a 50% inhibition of the virus growth
(EC ) and the ratio of a maximum permissible drug concen-
3
0
with stirring 10 ml of a 5% KOH solution in methanol. The
mixture was allowed to stand for 6 h and diluted with water.
The precipitate was separated by filtration, washed with wa-
ter, and dried to obtain 0.46 g of compound VII.
5
0
tration (MPC) to EC . The MPC values were determined
5
0
upon 72-h incubation of uninoculated cell cultures.
3
-Oxime-betulonic acid nitrile (VIII). A solution of
The antiulcer activity of dioxime VI was studied on a
group of 36 white mongrel rats weighing 180 – 200 g with
0
.47 g (1 mmole) of 3,28-dioxime-betulin VI in 5 ml of ace-
TABLE 2. NMR Spectra (d, ppm) of Compounds III – VIII (solvent, CDCl3)
1
3
1
Compound
C NMR spectrum: d, ppm
H NMR spectrum: d, ppm
0.84, 0.86, 0.97, 1.00, 1.02 (5s, 15H, 5CH ), 1.00 – 2.00 (m, 24H,
CH, CH ); 1.60 (s, 3H, H-30), 2.04 (s, 3H, OAc); 2.35 – 2.42 (m,
III
IV
V
13.9, 15.6, 15.9, 16.2, 17.9, 18.7, 20.5, 21.1, 23.4, 25.2, 27.7,
3
2
4
1
8.5, 28.9, 29.6, 32.9, 33.9, 36.6, 37.5, 38.2, 38.4, 40.6, 42.3,
5.7, 47.3, 47.8, 50.1, 55.1, 80.6, 109.9 (C-29), 149.4 (C-20),
70.7, 206.4 (C-28)
2
1H, H-19), 4.42 – 4.47 (m, 1H, H-3); 4.60, 4.72 (2bs, 2H, H-29);
9.52 (s, 1H, H-28)
13.8, 15.3, 15.6, 15.6, 18.6, 19.2, 20.6, 25.1, 25.1, 26.3, 28.4,
0.75, 0.79, 0.97, 1.00, 1.10 (5s, 15H, 5CH
3
); 1.00 – 2.00 (m, 24H,
2
4
2
8.7, 29.4, 33.2, 33.7, 36.5, 38.3, 39.2, 39.3, 40.3, 42.2, 46.8,
7.1, 47.5, 49.4, 54.4, 109.8 (C-29), 150.0 (C-20), 206.1 (C-28), H-19); 4.55, 4.68 (2bs, 2H, H-29); 9.64 (s, 1H, H-28)
17.6 (C-3)
CH , CH); 1.63 (s, 3H, H-30); 2.38 (dt, J = 10.0, 4.0, 3.1 Hz, 1H,
2
13.8, 15.3, 15.6, 15.6, 18.6, 19.2, 20.6, 25.1, 25.1, 26.3, 28.4,
0.78, 0.86, 0.99, 1.01, 1.02 (5s, 15H, 5CH
CH, CH ); 1.65 (s, 3H, H-30), 2.03 (s, 3H, OAc); 2.48 – 2.58 (m,
7.1, 47.5, 49.4, 54.4, 109.8 (C-29), 150.0 (C-20), 206.1 (C-28), 1H, H-19); 4.43 – 4.49 (m, 1H, H-3); 4.60, 4.72 (2bs, 2H, H-29);
17.6 (C-3) 7.55 (s, 1H, H-28); 7.58 (bs, 1H, NOH-28)
0.95, 0.98, 1.02, 1.05, 1.12 (5s, 15H, 5CH
CH, CH ); 1.76 (s, 3H, H-30); 2.22 – 2.32 (m, 1H, H-19); 4.62,
3
); 1.00 – 1.95 (m, 24H,
2
4
2
8.7, 29.4, 33.2, 33.7, 36.5, 38.3, 39.2, 39.3, 40.3, 42.2, 46.8,
2
VI
14.6, 15.8, 16.0, 17.2, 19.0, 19.2, 21.1, 22.9, 25.2, 27.3, 27.8,
3
); 1.00 – 2.00 (m, 24H,
2
4
1
7.9, 29.7, 32.4, 33.9, 36.9, 37.2, 38.6, 38.7, 40.3, 42.2, 45.6,
9.3, 49.8, 49.9, 54.4, 110.1(C-29), 149.8(C-20), 155.5(C-28),
67.3(C-3)
2
4.76 (2bs, 2H, H-29); 7.24 (s, 1H, H-28); 7.58 (bs, 1H, NOH-28);
8.21 (bs, 1H, NOH-3)
VII
14.7, 15.3, 15.9, 16.0, 18.2, 19.1, 20.7, 25.0, 25.1, 27.2, 27.7,
0.78, 0.84, 0.99, 1.06, 1.12 (5s, 15H, 5CH
3
); 1.05 – 2.00 (m, 24H,
2
4
1
7.9, 29.6, 32.2, 34.2, 37.1, 38.5, 38.6, 38.8, 40.8, 42.7, 47.8,
9.3, 49.7, 50.3, 55.2, 78.9(C-3), 110.0(C-29), 149.7(C-20),
55.2(C-28)
CH, CH ); 1.68 (s, 3H, H-30); 2.44 – 2.58 (m, 1H, H-19);
4.42 – 4.47 (m, 1H, H-3); 4.60, 4.72 (2bs, 2H, H-29); 7.38 (s, 1H,
H-28); 7.67 (bs, 1H, NOH-28); 8.84 (s, 1H, OH-3)
2
VIII
14.8, 15.8, 15.8, 17.1, 18.0, 19.4, 21.0, 22.1, 25.0, 27.3, 29.1,
0.84, 0.98, 1.02, 1.06, 1.11 (5s, 15H, 5CH
CH, CH ); 1.72 (s, 3H, H-30); 2.32 – 2.41 (m, 1H, H-19); 4.68,
4.77 (2bs, 2H, H-29); 9.38 (bs, 1H, NOH-3)
3
); 1.02 – 2.05 (m, 24H,
2
4
1
9.6, 31.1, 34.0, 35.8, 37.3, 38.6, 40.3, 40.7, 41.3, 42.4, 48.6,
9.1, 50.1, 55.6, 110.9(C-29), 123.4(C-3), 148.1(C-20),
66.9(C-28)
2

3
06
O. B. Flekhter et al.
TABLE 3. Antiviral Properties of Compounds VI – VIII
Influenza A
Compound
HSV-1
ECHO-6
EC50, mM
MPC/EC50
EC50, mM
MPC/EC50
EC50, mM
MPC/EC50
V
25.11
< 1
> 204.79
48.17
< 1
> 200.9
15.38
< 1
VI
5.82 (6.63 ¸ 5.12)
73.26
8.86
6.93
(
(
57.28 ¸ 40.51)
17.81
22.19 ¸ 14.27)
(16.09 ¸ 14.72)
n/s
VII
0.43 (1.00 ¸ 0.19)
31.25
13.51
1.49
n/s
n/s
VIII
131.14
n/s
n/s
n/s
(143.70 ¸ 119.69)
Note: n/s = not studied.
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TABLE 4. Antiulcer Effect of 3,28-dioxime-betulin (VI)
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5
0
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Basic Research (project Nos. 00-03-81174, 01-03-33131,
and 02-03-81007) and by the Belarus Foundation for Basic
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5
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