Synthesis and pharmacological activity of 20-keto-29-norlupane derivatives

Article abstract of DOI:10.1007/s10600-006-0016-7

New 20-oxo- and hydroxyimino-derivatives of betulin that exhibit immunotropic and antiviral activities were synthesized. 2005 Springer Science+Business Media, Inc.

Full text of DOI:10.1007/s10600-006-0016-7

Chemistry of Natural Compounds, Vol. 41, No. 6, 2005
SYNTHESIS AND PHARMACOLOGICAL ACTIVITY OF
20-KETO-29-NORLUPANE DERIVATIVES
O. B. Flekhter,1 G. V. Giniyatullina,1 F. Z. Galin,1
N. Zh. Baschenko,1 N. S. Makara,1 F. S. Zarudii,1
E. I. Boreko,2 O. V. Savinova,2 N. I. Pavlova,2
Z. A. Starikova,3 and G. A. Tolstikov4
UDC 547.824:542.91:548.737
New20-oxo- and hydroxyimino-derivatives of betulin that exhibit immunotropic and antiviral activities were
synthesized.
Key words: triterpenoids, ozonolysis, oximes, antiviral activity, immunotropic activity.
In continuation of our studies on the synthesis of new triterpene derivatives with valuable pharmacological activity[1-
5], wesynthesized betulin 29-norlup-20-ketones and their hydroxyiminoderivatives and studied theantiviralandimmunotropic
activties of several compounds.
Ozonation of 3β,28-di-O-acetylbetulin (1) and its 30-bromo derivative (2) produced the corresponding 20-ketones 3
and 4 in yields of 73 and 65%, respectively. The structures of the compounds were established using NMR spectroscopy. Thus,
13
the C NMR spectra of 3 and 4 lacked signals for the C-20(29) double bonds typical of starting acetates 1 and 2 but contained
signals for a C-20 carbonyl at δ 211 ppm. The PMR of 3 and 4 exhibited a weak-field shift for the signals of H-30 to δ 2.11-
2.16 ppm. 3β,28-Di-O-acetyl-29-norlup-20-one (3) crystallized from ethanol as colorless needles. This enabled its structure
to be confirmed by an x-ray structure analysis (XSA) (Fig. 1). Boiling 3 and 4 with hydroxylamine in pyridine produced in
13
quantitative yield the corresponding oximes 5 and 6, the C NMR spectra of which gave signals for C-20 at δ 162.3 ppm and
the PMR spectra of which had broad signals for the NOH group at δ 7.27 and 9.20 ppm.
R
R
H
H
H
CH OAc
2
H
CH OAc
2
a, b
H
H
AcO
AcO
1, 2
1, 3, 5:R = H;2, 4, 6:R = Br;3, 4: X = O;5, 6: X = NOH
a. O , CH Cl , -60° C, Zn/AcOH;b. NH OH HCl, pyridine, 115°C, 2h
3 - 6
H
H
3
2
2
2
A study of the antiviral activities of 2-5 toward flu and Type I herpes simplex viruses showed that only oxime 5
possessed weak ability to suppress reproduction of flu virus. Ketone 4 had a comparable degree of activity toward herpes virus
whereas the 30-bromo derivative of 3β,28-di-O-acetylbetulin (2) was even less active (Table 1). Despite the low activities of
the studied compounds, the results confirm that derivatives of lupane triterpenoids can exhibit antiviral properties toward flu
virus, which we first established [6].
1) Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, 450054, Ufa, pr. Oktyabrya,
71, e-mail: obf@anrb.ru; 2) Scientific Research Institute of Epidemiology and Microbiology, Ministry of Health of
Belarus, Minsk; 3) N. A. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
Moscow; 4) N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Division, Russian Academy of Sciences.
Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 582-584, November-December, 2005. Original article submitted
August 5, 2005.
0009-3130/05/4106-0706 ©2005 Springer Science+Business Media, Inc.
706

TABLE 1. Antiviral Activity of Betulin Derivatives
Flu virus
Herpes virus
EC50 (I95)/EC90 (I95)
Compound
EC50
MTC/EC50
MTC/EC90
MTC/EC50
MTC/EC90
(I95)/EC90(I95) µM
µM
2
3
4
5
>627.26
277.98/5072.32
87.91/202.73
5.94/31.76
<1
<1
180.02
3.48
1.21
6.38
1.43
18.6
2.36
<1
0.64
0.03
0.89
0.25
7.31
1.37
55.85/248.66
2.09/16.54
86.84
<1
______
MTC is the maximum tolerated concentration; EC50, the average effective concentration, EC90, the concentration for 90%
suppression of virus multiplication; I95, the confidence range at 95% probability.
TABLE 2. Effect of Betulin Derivatives on Functional Activity of the Immune System of White Mice
Body mass
Spleen mass
Thymus mass
Antibody titer, lg2
Dose,
Compound
initial,
on day of
absolute,
mg
relative, absolute, relative,
mg/kg
hemagglutinin hemolysine
g
sacrifice, g
%
mg
%
1
5
50
50
50
50
20.0±0.6
21.0±1.2
20.7±0.9
21.7±0.9
21.0±1.1
20.5±0.5 201.0±13.8 0.98±0.10 30.9±2.3 0.15±0.08 6.5±0.35
9.70±0.1
10.0±0.1
11.0±0.01
10.2±0.1
9.3±0.2
20.8±0.7 191.0±27.0 0.91±0.14 33.0±3.1 0.15±0.02
21.5±0.8 298.0±27.0 1.38±0.16 30.5±3.2 0.1±0.01
21.5±0.8 291.6±19.7 1.35±0.18 34.1±3.5 0.15±0.02
22.5±0.8 198.0±11.5 0.88±0.04 38.5±2.0 0.17±0.09
6.0±0.3
7.3±0.5
7.0±0.3
5.4±0.2
Betulinic acid
Hydroxymethyluracil
Control
C 3 4
O 1
C 3 3
C 6
O 2
C 2 6
C 8
C 7
C 9
C 1 5
C 1 4
C 2 8
C 2 3
C 4
C 5
C 2 5
C 1 6
C 1 3
C 1 8
O 4
C 1 0
C 1
C 1 7
C 3 2
C 3
C 11
C 2 2
C 2 1
C 3 0
C 3 1
O 5
C 2
C 2 7
C 1 2
C 1 9
C 2 0
O 3
Fig. 1. Structure of 3β,28-di-O-acetyl-29-norlup-20-one (3).
EXPERIMENTAL
13
PMR and C NMR spectra were recorded on a Bruker AM-300 spectrometer (300 and 75.5 MHz, respectively) in
CDCl with TMS internal standard. Melting points were determined on a Boetius microstage; optical density, on a
3
Perkin—Elmer 241 MC polarimeter in a 1-dm tube. TLC was performed on Silufol (Chemapol, Czech Rep.) plates using
CHCl :CH OH(25:1). Compoundsweredevelopedwith phosphotungsticacidsolution (5%) in ethanolwith subsequent heating
3
3
at 100-120°C for 2-3 min. 1 and 2 were prepared as before [7, 8].
707

3β,28-Di-O-acetyl-29-norlup-20-one (3). Ozonated oxygen was passed through a solution of 1 (2 mmol, 0.9 g) in
CH Cl (50 mL) at -60°C until ozone broke through. The temperature was adjusted to 0°C. Glacial AcOH (10 mL) and zinc
2
2
dust (1 g) were added. The solution was stirred at 0°C for 1 h. The zinc dust was filtered off. The organic layer was washed
with saturated Na CO solution (3 × 20 mL) and water (3 × 25 mL), dried over Na SO , and evaporated in vacuo (water
2
3
2
4
aspirator). The solid was chromatographed over a column of Al O with elution by CHCl . Yield 0.59 g (73%), white needles,
2
3
3
R 0.55, mp 188-190°C, lit. [9] mp 190-191°C, C H O (MW 528.769).
f
33 52
5
PMR spectrum (δ, ppm, J/Hz): 0.81, 0.82, 0.94, 0.96, 1.00 (15H, 5s, 5CH ), 1.00-2.00 (24H, m, CH , CH), 1.99 and
3
2
2.03 (6H, 2s, 2OAc), 2.11 (3H, s, H-30), 2.57-2.64 (1H, m, H-19), 3.77 and 4.23 (2H, both d, J = 11, H-28), 4.43 (1H, dd,
J = 5, 9, H-3).
13
C NMR spectrum (δ, ppm): 14.4, 15.8. 16.0, 16.3, 18.0, 20.6, 20.8, 21.1 (CH CO), 23.6, 26.8. 27.0, 27.4, 27.8, 29.2,
3
29.5, 33.0, 34.3, 36.3, 36.9, 37.6, 38.2, 40.7, 42.5, 46.2, 49.2, 51.5, 55.2, 62.4 (C28), 80.6 (C3), 170.7 and 171.2 (CH CO),
3
211.2 (C20).
XSA of 3. Colorless needlelike crystals C H O (MW = 528.75) were orthorhombic, at 293 K a = 12.626(3),
33 52
5
3
3
b = 15.258(3), c = 15.987(3) Å, V = 3080(1) Å , space group P2 2 2 , Z = 4, d
= 1.140 g/cm . A data set of reflections was
calc
1 1 1
obtained on a Enraf—Nonius CAD4 diffractometer at 293 K (λ Mo Kα-radiation, 2θ
= 49°) from a single crystal of
max
dimensions 0.40 × 0.30 × 0.25 mm. Equivalent reflections were averaged to produce 5366 independent reflections
[R(int) = 0.0222] that were used to solve and refine the structure. The structure was solved by direct methods and refined
anisotropically (H atoms were placed at positions calculated geometrically and refined isotropicallyusing a rocker model) over
2
F
. The final agreement factors were R1 = 0.0514 [calculated over F for 2749 reflections with I > 2σ(I)], wR2 = 0.1389
hkl
hkl
2
(calculated over F
for all 5366 reflections), GOOF = 1.014, 343 refined parameters. All calculations were performed using
hkl
the SHELXTL PLUS 5 programs. Atomic coordinates and temperature factors were deposited in the Cambridge
Crystallographic Database (CCDC 277178).
3β,28-Di-O-acetyl-29-norlup-30-bromo-20-one (4) was prepared analogouslyto 3 from 2 (1 g). Yield 0.65 g (65%),
20
yellow compound, R 0.39, mp 93-94°C, [α]
-11.3° (c 0.013, CHCl ), C H BrO (MW 607.665).
3 33 51 5
f
D
PMR spectrum (δ, ppm, J/Hz): 0.95, 0.97, 0.98, 0.99, 1.04 (15H, 5s, 5CH ), 1.00-1.90 (24H, m, CH , CH), 2.02 and
3
2
2.05 (6H, 2s, 2OAc), 2.16 (2H, s, H-30), 2.60 (1H, m, H-19), 3.75 and 4.18 (2H, both d, J = 11, H-28), 4.43 (1H, dd, J = 6, 9.9,
H-3).
13
C NMR spectrum (δ, ppm): 14.6, 15.9, 16.0, 16.4, 18.1, 20.7, 20.9, 21.2 (CH CO), 23.6, 26.9, 27.1, 27.4, 27.9, 29.3,
3
29.5, 33.9 (C30), 34.4, 36.4, 37.0, 37.7, 38.3, 40.7, 42.5, 46.3, 49.3, 50.1, 51.6, 55.3, 62.4 (C28), 80.7 (C3), 170.7, 171.3
(CH CO), 211.4 (C20).
3
3β,28-Di-O-acetyl-29-norlup-20-oxime (5). A solution of 3 (1 mmol, 0.46 g) in anhydrous pyridine (30 mL) was
treated with NH OH·HCl (7 mmol, 0.5 g), refluxed for 2 h, cooled, and poured into HCl solution (150 mL, 5%). The resulting
2
solid was filtered off, washed with water, and dried. Yield 0.39 g (85%), light yellow compound, R 0.30, mp 133-135°C,
f
20
[α]
+21.4° (c 0.02, CHCl ), C H NO (MW 543.783).
3 33 53 5
D
PMR spectrum (δ, ppm, J/Hz): 0.81, 0.82, 0.94, 0.96, 1.00 (15H, 5s, 5CH ), 1.00-1.90 (24H, m, CH , CH), 1.81 (3H,
3
2
s, H-30), 1.97, 2.01 (6H, 2s, 2OAc), 2.58-2.67 (1H, m, H-19), 3.77, 4.23 (2H, both d, J = 11, H-28), 4.43 (1H, dd, J = 5, 9, H-3),
9.20 (1H, br.s, NOH).
13
C NMR spectrum (δ, ppm): 10.8, 14.4, 15.8, 16.0, 16.3, 18.0, 20.5, 20.8, 21.2 (CH CO), 23.5, 25.2, 26.8, 27.2, 27.8,
3
29.6, 33.9, 34.5, 36.7, 36.9, 37.6, 38.2, 40.7, 42.5, 44.9, 46.0, 49.1, 49.9, 55.2 (C5), 62.4 (C28), 80.7 (C3), 162.3 (C20), 170.9,
171.3 (CH CO).
3
3β,28-Di-O-acetyl-29-norlup-30-bromo-20-oxime (6) was prepared analogously to 5 from 4 (0.46 g). Yield 0.41 g
20
(89%), R 0.51, mp 112-115°C, [α]
+17.6° (c 0.07, CHCl ), C H BrNO (MW 622.68).
3 33 52 5
f
D
PMR spectrum (δ, ppm, J/Hz): 0.90, 0.96, 0.98, 1.03 (15H, 4s, 5CH ), 1.10-2.00 (24H, m, CH , CH), 1.82 (2H, s,
3
2
H-30), 2.05, 2.07 (6H, 2s, 2OAc), 2.58 (1H, m, H-19), 3.81, 4.27 (2H, both d, J = 11, H-28), 4.45 (1H, dd, J = 6, 9.9, H-3), 7.27
(1H, br.s, NOH).
13
C NMR (δ, ppm): 10.8, 14.6, 16.0, 16.1, 16.5, 18.2, 20.7, 21.0, 21.3, 23.7, 25.4, 26.9, 27.3, 27.9, 29.7, 34.1, 34.6,
36.9, 37.0, 37.8, 38.4, 40.9, 42.6, 45.3, 46.2, 49.1, 50.1, 55.4, 62.6 (C28), 80.9 (C3), 162.5 (C20), 171.0, 171.4 (CH CO).
3
Antiviral properties were determined in experiments on cell cultures with A/FPV/Rostock/34 (H7N1) flu virus and
herpes simplex virus type I (HSV-I). The studies were carried out by reduction of plaques in culture of primarychicken embryo
fibroblasts with FPV and estimation of the cytopathic effect on culture of human rhabdomyosarcoma cells with HSV-I by the
708

literature method [3]. The studied compounds were dissolved beforehand in ethanol (10%) and then in the support medium
until the required concentrations were attained. The criterion for antiviral activity was a reduction of the virus titer in the
presence of the studied compounds compared with the control. The concentration for 50% (average effective concentration,
EC ) and 90% (EC ) suppression ofvirus multiplication and the ratios ofthe maximal tolerated concentration (MTC) to EC
50
90
50
and EC was alsocalculated. The MTC ofthe compounds for uninfected cell cultures were determined after 72 h ofincubation.
90
Table 1 lists the results.
The immunotropic activity of 1, 5, and betulinic acid were estimated from the titers of circulating antibodies
(hemolysine and hemagglutinin) [10]. Sheep erythrocytes (SE) were used as a thymus-dependent antigen. Immunization was
carried out by i.p. injection to white mice of the optimal (108 cells) dose of SE in 0.2 mL per 20 g of body mass. The reaction
was estimated using antibodytiters on the 7th dayafter immunization byrecording the masses ofthe bodyand lymphatic organs
(thymus, spleen). The studied compounds were administered per os during 7 d beginning with the first day of immunization.
A single dose of the tested compounds did not exceed 50 mg/kg. The controls were animals that received oral
hydroxymethyluracil (50 mg/kg) and water during 7 d after immunization with SE. Table 2 lists the results.
ACKNOWLEDGMENT
The work was supported financially by the RFBR (projects No. 02-03-81007 and 05-03-32832) and the BFBR (project
No. B02R-016). OBF thanks the Foundation for Assistance of Domestic Science (program "Young Science Candidates").
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