Synthesis and antiviral activity of lupane triterpenoids with modified cycle e

Article abstract of DOI:10.1134/S1068162007060088

A reductive transformation of the peroxide products of ozonolysis of derivatives of 3β-O-acetyl-22(17→28)-abeo-lupa-17(28),20(29)-diene and the subsequent intramolecular ketalization led to a compound with a trioxane fragment. This is a new approach to a

Full text of DOI:10.1134/S1068162007060088

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 6, pp. 584–588. © Pleiades Publishing, Inc., 2007.
Original Russian Text © O.B. Flekhter, N.I. Medvedeva, O.S. Kukovinets, L.V. Spirikhin, E.G. Galkin, F.Z. Galin, D.G. Golovanov, N.I. Pavlova, O.V. Savinova, E.I. Boreko,
G.A. Tolstikov, 2007, published in Bioorganicheskaya Khimiya, 2007, Vol. 33, No. 6, pp. 629–634.
Synthesis and Antiviral Activity of Lupane Triterpenoids
with Modified Cycle E
O. B. Flekhtera,1, N. I. Medvedevaa, O. S. Kukovinetsa, L. V. Spirikhina, E. G. Galkina,
F. Z. Galina, D. G. Golovanovb, N. I. Pavlovac, O. V. Savinovac,
E. I. Borekoc, and G. A. Tolstikovd
a Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences,
pr. Oktyabrya 71, Ufa, 450054 Bashkortostan, Russia
b Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
ul. Vavilova 28, Moscow, 117813 Russia
c State Research Institute of Epidemiology and Microbiology, Minsk, Belarus
d Vorozhtsov Institute of Organic Chemistry, Siberian Division, Russian Academy of Sciences,
pr. Akademika Lavrent’eva 9, Novosibirsk, 630090 Russia
Received October 30, 2006; in final form, March 6, 2007
Abstract—A reductive transformation of the peroxide products of ozonolysis of derivatives of 3β-O-acetyl-
22(17
28)-abeo-lupa-17(28),20(29)-diene and the subsequent intramolecular ketalization led to a com-
pound with a trioxane fragment. This is a new approach to a skeletal modification of triterpenoid cycle E. An
activity of the synthesized compounds was found toward the viruses of type A influenza and herpes simplex.
Key words: lupane triterpenoids, ozonolysis, antiviral activity
DOI: 10.1134/S1068162007060088
INTRODUCTION
onstrated that 18-oxo-19,20,21,29,30-pentanoracid (I)
exhibits a high anticancer activity [1]. Glycoside of the
E-seco-ursane type (II) isolated from the Ilex crenata
exhibits a strong antiallergenic effect. Its antiinflamma-
tory activity is ten times higher than that of glycyrrhizic
Modification of cycle E in triterpenoids is a prospec-
tive approach to the synthesis of compounds with a pro-
nounced pharmacological activity.2 Oxidation of the
18(19)-lupene triterpenoids with ruthenium oxide dem- acid [2].
O
O
H
O
OH
CH2OAc
COOH
H
HO
O
AcO
O
OH
(I)
OH
(II)
RESULTS AND DISCUSSION
Derivatives of 22(17 28)-abeo-lupa-
17(28),20(29)-diene, easily preparable from betulin,
are interesting subjects for skeletal transformations in
triterpenoid cycle E. For example, 3β-é-acetyl-
1
2
Corresponding author; phone/fax: (3472) 35-6066; e-mail:
obf@anrb.ru.
Abbreviations: MTC, maximum tolerated concentration; HSV,
herpes simplex virus; PFU, plague-forming unit; TCID, tissue
cytopathogenic infecting dose.
584

SYNTHESIS AND ANTIVIRAL ACTIVITY
585
22(17
28)-abeo-lupa-17(28),20(29)-diene (IV) was from X-ray data (figure). Resonances from protons and
synthesized from 3β-é-acetylbetulin (III) by heating at carbon atoms of compound (IV) in its NMR spectra
100°ë in pyridine in the presence of POCl3 according to were completely attributed using two-dimensional
spectroscopy and CH-correlation (CH-CORR).
Deacetylation of (IV) was achieved in methanol in the
presence of 5% KOH, and 3β-hydroxyderivative (V)
was obtained in 73% yield.
the method described in [3] (scheme). The target prod-
uct (IV) was formed in 73% yield and crystallized as
needles of 1–1.5 cm in length. Its structure followed
O
20
H
H
b
H
H
CH2OH
CH2OR
H
H
AcO
AcO
(III)
(VII) R = H
H
H
c
(VIII) R = Ts
a
d
28
29
22
21
O
H
O
30
20
20
19
20
O
H
H
18O
28
28
17
b
b
17
H
17
H
H
H
H
H
.
3
AcO
RO
AcO
H
H
H
(IX)
(VI)
(IV) R = Ac
(V) R = H
e
CH Cl /methanol, –60°C, zinc/acetic acid or Me S;
3
Conditions: (a) POCl /C H N, 2 h, 100°C; (b) O ,
2
2
2
3
5
5
(c) p-
KOH/methanol.
TosCl, C H N, 48 h, 20°C; (d) NaOAc, 2 h, 100°C; (e) 5%
5
5
Scheme.
We tried to carry out an oxidative cleavage of the obtained by ozonolysis of 20-oxo-22(17
28)-abeo-
endocyclic bond of acetate (IV) to prepare E-seco- 29-norlup-17(28)-ene (IX), which was prepared
derivative, but a nontrivial product was synthesized by from3β-é-acetylbetulin by ozonolysis of norketone
exhaustive ozonolysis in the CH2Cl2–methanol solution (VII), followed by solvolysis of tosylate (VIII) by the
at −60°ë. Compound (VI) with molecular mass of method [4]. As expected, the ozonolysis of ketone (IX)
500.345 with a composition of ë31ç48é5 according to with the subsequent reduction of the peroxide products
its mass spectrum was obtained. Resonances at δ 104.5,
94.8, and 105.5 were observed in its 13C NMR spec-
trum. They correspond to the carbon atoms connected
to oxygen atoms (–C17–O–C28–O–C20–O–). Reso-
C29
C20
nance from H28 looked as a doublet at 5.41 ppm
é2
é1
C2
(J 6.2 Hz) in the 1H NMR spectrum. These results indi-
cate the presence of a ketal fragment in ozonolysis
product (VI). Formation of compound (VI) from ace-
tate (IV) can be explained by spontaneous intramolec-
ular cyclization with the predominant formation of
zwitter ion and carbonyl functional groups at C17 and
C20 atoms.
C32
C31
C11
C12
C19
C1
C10
C30
C13
C3
C25
C21
C27
C14
C9
C8
C26
C18
C17
C4
C22
C28
C23
C5
C6 C24
C15
C7
C16
We tried to confirm the structure of product (VI) by
elucidation of the character of derivatives that could be
Structure
of
3β-é-acetyl-22(17
28)-abeo-lupa-
17(28),20(29)-diene (IV).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

586
FLEKHTER et al.
Antiviral activity of triterpenoids (IV)–(VI)
FPV
HSV-1
Compound
EC50 (I95), µM
MTC/EC50
MTC/EC90
EC50 (I95), µM
EC90 (I95), µM
MTC/EC50
MTC/EC90
EC90 (I95), µM
(IV)
(V)
164.50 (249.82–108.30)
1371.10 (2082.37–902.78)
33.80 (58.06–19.68)
2.52
0.3
>207.15
12.77 (14.75–11.05)
>799.44
<1
1.81
0.36
14.23
1.69
19.19
9.67
<1
169.29 (290.78–98.55)
56.18 (79.06–39.91)
(VI)
472.61 (665.17–335.78)
Note: I is a confidence interval.
95
resulted in the product whose physicochemical and
EXPERIMENTAL
spectral characteristics were identical to those of (VI).
TLC was carried out on Silufol plates (Chemapol,
Czech Republic) in 20 : 1 of chloroform–methanol
mixture. The substance spots were detected by the
treatment with 5% phosphotungstic acid solution in
ethanol with the subsequent heating at 100–120°ë for
2–3 min. 1ç and 13C NMR spectra (δ, ppm, J, Hz) were
recorded on a Bruker AM-300 spectrometer (300 and
75.5 MHz, respectively) in CDCl3 using SiMe4 as an
internal standard. Optical rotation was measured on a
Perkin-Elmer 241 MC polarimeter in a tube of 1 dm in
length. Mass spectra were recorded on a ThermoFinni-
gan MAT 95 XP spectrometer. Mps were determined on
a Boetius device. An Ozon-2K ozonator was used for
the ozonation. 3β-é-Acetylbetulin (III) was prepared
by the procedure [10].
This result is not anomalous and probably arises
from the original structure of the starting compound,
which contains an isopropylidenecyclohexene frag-
ment. The ozonolysis of naphthalene, 3-carene, and a
number of steroids is known to give such nontrivial
products [5–7]. Note that compounds with such frag-
ments occur in nature. For example, the tetranortriter-
penoid fragmalin, belonging to melecyanins of the
Melia plant family, has a similar structure, and can
exhibit anticancer properties.
Thus, a compound with a trioxane fragment was
prepared by reductive conversion of the peroxide prod-
ucts of ozonolysis of the derivatives of 3β-é-acetyl-
3b-é-Acetyl-22(17
28)-abeo-lupa-17(28),20(29)-
22(17
28)-abeo-lupa-17(28),20(29)-diene as
a
diene (IV). Freshly distilled POCl3 (4 ml) was added to
a solution of 3β-é-acetylbetulin (III) (0.43 g, 1 mmol)
in anhydrous pyridine (10 ml). The reaction mixture
was heated with a backflow condenser on a water bath
for 2 h and carefully poured out on ice (50 g). The pre-
cipitate was filtered, washed with water, and dried. The
product was purified on a column with Al2O3 eluted
with benzene. Compound (IV) was obtained as white
needle crystals of 1–1,5-cm in length in yield of 0.31 g
result of intramolecular ketalization. This is a new
approach to a skeletal modification of cycle E in lupane
triterpenoids.
The results of studies of antiviral activity of triterpe-
noids (IV)–(VI) are given in the table. Among the stud-
ied compounds, 3β-hydroxy-22(17
28)-abeo-lupa-
17(28),20(29)-diene (V) had the highest activity
against the herpes simplex virus. The decrease in the
virus titer in the presence of (V) taken at the maximum
tolerate concentration (245.1 µM) and at a lower con-
centration close to MTC proved to be 1.6 log
(73%); Rf 0.75, mp 200°ë; [α]2D0 –0.35° (c 2, CH2Cl2);
1H NMR: 0.75 (1 H, m, H5), 0.79 (3 H, s, H26), 0.80 (3
H, s, H23), 0.83 (3 H, s, H25), 0.90 (3 H, s, H24), 0.95–
0.97 (2 H, m, H1a and H15a), 1.00 (3 H, s, H27), 1.65–
1.67 (2 H, m, H1b and H15b), 1.05 and 1.40 (2 H, m,
H11), 1.30–1.37 (7 H, m, H2a, H7, H9, H18, and H22),
1.45–1.55 (5 H, m, H2b, H6, and H12), 1.70 (3 H, s,
H30), 1,82–1.85 (2 H, m, H21), 1.95–1.98 and 2.15–
2.17 (2H, both m, H16), 2.00–2.05 (2 H, m, H13 and
H19), 2.02 (3 H, s, OAc), 4.65 and 4.72 (2 H, both d, J
0.8, H29), 4.45 (1 H, dd, J 5.5 and 10.5, H3), 5.30 (1 H,
t, J 4.0, H28); 13C NMR: 14.8 (C25), 15.8 (C27), 16.3
(C26), 16.4 (C30), 18.2 (C6), 21.3 (C11), 21.8
(éëéëç3), 22.3 (C24), 23.5 (C2), 23.7 (C12), 26.5
(C22), 27.8 (C21), 27.9 (C23), 32.7 (C15), 33.7 (C7),
*
TCID50//ml. The MTC/EC50 and MTC/EC90 values for
(V) indicate a sufficiently wide range of nontoxic con-
centrations exhibiting the antiviral effect. Compounds
(IV) and (VI) were inactive toward HSV-1. Com-
pounds (IV)–(VI) similarly inhibited the reproduction
of the FPV influenza virus, but only (VI) at the concen-
trations of 799.4 (MTC) and 399.7 µM decreased the
virus titer by the value more than 1 log PFU/ml. These
results confirms the possibility of inhibition of the
influenza virus reproduction by the betulin derivatives,
which has earlier been demonstrated [8, 9].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

SYNTHESIS AND ANTIVIRAL ACTIVITY
587
34.1 (C16), 37.0 (C10), 37.8 (C4), 38.5 (C1), 40.9 (C8), flask. The solution was cooled to 0°ë, diluted with gla-
40.9 (C19), 42.3 (C14), 44.0 (C13), 46.1 (C18), 50,4 cial acetic acid (10 ml), and zinc powder (1 g) was
(C9), 55.4 (C5), 80.9 (C3), 108.9 (C29), 118.3 (C28), added. The reaction mixture was stirred for 1 h, and the
141.5 (C17), 150.6 (C20), 170.8 (éëéëç3).
zinc powder was filtered off. The filtrate was washed
with the saturated solution of Na2CO3 (3 × 20 ml) and
water (3 × 20 ml), dried with Na2SO4, and purified on a
column with Al2O3 eluted with chloroform. 3β-é-
Acetyl-28-hydroxy-20-oxo-29-norlupane (VII) was
obtained as a white substance in yield 0.77 g (82%); Rf
X-Ray analysis of (IV): crystals (C32H50O2, å
466.72) are monoclinic, a 13.061(4), b 6.579 (1), and c
31.284 (9) Å, β = 90.65(3)°, V = 2688.1(13) Å3 at 120 K,
spatial group ë2, Z 4 (Z' = 1), dcalc = 1.153 g/cm–3,
µ(MoKα) = 0.69 cm–1, F(000) = 1032. Intensities of
8106 reflections were measured at 120 K on a Smart
1000 CCD diffractometer (λ(MoKα) = 0.71072 Å, ω-
0.48; mp 141–143°ë; [α]2D0 +0.02° (c 1.0, CH2Cl2); 1H
NMR: 0.84, 0.85, 1.00, and 1.01 (15 H, all s, 5 ëç3),
1.10–2.00 (m, ëç2, CH), 2.04 (3 H, s, OAc), 2.16 (3 H,
s, H30), 3.25 and 3.78 (2 H, both d, J 11.0, H28), 4.46
(1 H, dd, J 5.8 and 10.5, H3). Found, %: C 79.67, H
10.08. Calc. for C 10.08. C31H48O3 (år 468.717), %: C
79.46, H 10.36. p-TosCl (1.30 g) was added to a solu-
tion of (VII) (0.94 g, 2 mmol) in anhydrous pyridine
(50 ml). The reaction mixture was stirred for 48 h at
room temperature and poured out into 5% solution of
HCl (150 ml). The precipitate was filtered, washed, and
dried. 3β-é-Acetyl-20-oxo-29-norlup-28-O-tosylate
(VIII) was obtained as a yellow substance in yield 0.83
scans, 2θ < 47°), 3544 Independent reflections (Rint
=
0.0308) were used for further decoding and refinement
of the structure. The experimental data were processed
and averaged using the SAINT Plus program complex
[11]. The account for absorption was carried out
semiempirically according to equivalent reflections by
the SADABS program [12]. The structure was decoded
by the direct method and sequential syntheses of elec-
tron density. All the atoms, except for hydrogen atoms,
were localized from difference syntheses of the elec-
tron density. Positions of the hydrogen atoms were geo-
metrically calculated. The refinement was carried out
g (88%); Rf 0.88; mp 134–136°ë; [α]2D0 +12.5° (c 2.0,
according to F2hkl in anisotropic approximation for non-
CH2Cl2); 1H NMR: 0.78, 0.83, 0.87, and 0.95 (15 H, all
s, 5 ëç3), 1.00–2.00 (m, ëç2 and CH), 2.05 (3 H, s,
OAc), 2.13 (3 H, s, H30), 3.65 and 4.00 (2 H, both d,
J 95, H28), 4.40–4.50 (1H, m, H3), 7.35 and 7.80 (4H,
both d, aromatic H). Found, %: C 71.15, H 8.55, S 5.27.
Calc. for C38H56O6S (år 626.888), %: C 70.89, H 8.68,
S 5.11. NaOAc (2.8 g) was added to a solution of (VIII)
(1.25 g, 2 mmol) in acetic acid (100 ml). The reaction
mixture was refluxed for 2 h and poured into cool water
(200 ml). The precipitate was filtered, washed with
water, dried, and purified by a column chromatography
on Al2O3 with a chloroform elution. Compound (IX)
was obtained as white crystals; yield 1.12 g (90%); Rf
hydrogen atoms, positions of the hydrogen atoms were
fixed. The final value of reliability factors was R1 =
0.0624 (calculated according to Fhkl for 2959 reflec-
tions with I > 2s(I)), wR2 = 0.1690, the number of
refined parameters was 314, and GOF = 1.062. The cal-
culations were carried out using the SHELXTL 5.10
program complex [13]. Coordinates of the atoms and
temperature factors were deposited in the Cambridge
Bank of Structural Data (CCDC 290282;
http://www.ccdc.cam.ac.uk/products/csd/request/).
3b-Hydroxy-22(17
28)-abeo-lupa-17(28),20(29)-
diene (V). A 5% solution of KOH in methanol (5 ml)
was added to a solution of (IV) (0.77 g, 2 mmol) in
methanol (10 ml) and stirred for 2 h. The reaction mix-
ture was poured into 5% HCl (50 ml). The precipitate
was filtered, washed with water, dried, and purified on
a column withAl2O3 eluted with chloroform. Yield of
0.75; mp 121–123°ë; [α]2D0 +6.7° (c 3.0, CH2Cl2); H
1
NMR: 0.82, 0.85, 0.92, and 1.04 (15 H, all s, 5 ëç3),
1.10–2.00 (m, ëç2 and CH), 2.07 (3 H, s, OAc), 2.17 (3
H, s, H30), 4.47 (1 H, dd, J 5.5 and 10.5, H3), 5.38 (2
H, broadened s, H28); 13C NMR: 14.5, 15.7, 16.3, 16.4,
18.1, 21.2, 21.3, 23.6, 24.5, 26.8, 27.7, 27.9, 28.4, 32.2,
32.9, 34.0, 36.9, 37.7, 38.4, 38.5, 40.7, 41.9, 46.0, 50.2,
53.5, 55.3, 80.8 (C3), 117.9 (C28), 140.6 (C17), 170.9,
211.5 (C20). Found, %: C 79.67, H 10.05. Calc. for C
10.05. C31H48O3 (år 468.717), %: C 79.46, H 10.36.
(V) was 0.56 g (73%); Rf 0.48; mp 175–177°ë; [α]2D0
–
18° (c 2, CH2Cl2); 1H NMR: 0.83, 0.85, 0.86, 0.94, and
1.04 (15 H, all s, ëç3), 1.10–1.90 (25 H, m, ëç2, CH),
1.76 (3 H, s, H30), 3.20–3.30 (1 H, m, H3), 4.65 and
4.73 (2 H both broadened s, H29), 5.35 (2 H, broadened
s, H28); 13C NMR: 14.8, 15.3, 15.8, 16.2, 18.2, 21.2,
21.9, 22.3, 23.5, 26.2, 27.3, 27.7, 28.0, 32.7, 33.7, 34.1,
37.1, 38.7, 40.8, 40.9, 42.2, 43.9, 46.0, 50.4, 55.3, 78.9
(C3), 108.8 (C29), 118.4 (C28), 141.4 (C17), 150.6
(C20). Found, %: C 87.18, H 9.12. Calc. for C 9.12.
C30H48O (Mr 520.884), %: C 87.62, H 9.31.
(4R)-10-Acetyloxy-4,5,9,9,13,24-hexamethyl-
23,25,26-trioxaheptacyclo[20.3.1.01,18.04,17.05,14
.
08,13. 019,24]hexacosane (VI). a. Excess of ozonated
oxygen was passed through a solution of (VI)
(2 mmol) in a 1 : 1 dichloromethane–methanol mix-
ture (50 ml) at – 60°ë. The reaction was monitored by
TLC. After the completeness of reaction, the reaction
mixture was heated to 0°ë. The peroxide products were
reduced by two methods. The first method consisted in
3b-é-Acetyl-20-oxo-22(17
28)-abeo-29-norlup-
17(28)-ene (IX). Ozonated oxygen was passed through
a solution of 3β-é-acetylbetulin (III) (0.94 g, 2 mmol)
in CH2Cl2 (50 ml) until ozone began to exit out of a
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

588
FLEKHTER et al.
the treatment of the reaction mixture with zinc powder incubation with the uninfected cell culture for 72 h at
37°ë.
(1 g, 0.0154 g-atoms) in glacial acetic acid (10 ml)
under stirring at 0°ë for 1 h. The zinc powder was fil-
tered off, and the filtrate was washed with a saturated
solution of Na2CO3 (3 × 20 ml) and water (3 × 20 ml),
dried with Na2SO4, and evaporated in a vacuum.
According to the second method, Me2S (0.2 ml, 0.17 g,
2.75 mmol) was added, and the reaction mixture was
stirred for 2 h at room temperature and filtered. The fil-
trate was washed with 5% NaCl (3 × 20 ml) and water
(3 × 20 ml), dried with Na2SO4, and evaporated in a
vacuum. The residues obtained by both methods were
purified on columns with Al2O3 eluted with chloro-
form. Compound (VI) was obtained as a white sub-
stance in yield of 0.60 g (71%); Rf 0.40; mp 159°ë;
ACKNOWLEDGMENTS
This study was supported by the Russian Founda-
tion for Basic Research, project no. 05-03-32832 and
by the grant of the President of Russian Federation for
Support of Young Scientists and Leading Scientific
Schools, project no. NSh-1589.2006.3, and by the
UNESCO grant (IBSP/4-VN-01).
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1
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Antiviral properties of compounds (IV)–(VI)
were studied on a cell culture with the A/FPV/Ros-
tock/34 (H7N1) influenza virus and with HSV-1, IC
strain, as described previously [14, 15]. Antiviral activ-
ity toward the influenza virusA was studied on a culture
of fibroblasts of chicken embryos by the method of
plague reduction. Studies with HSV-1 were carried out
on a cell line of human rabdomyosarcoma (RD). The
result was evaluated according to a development delay
of the cytopathogenic effect of the virus. The studied
substances were preliminarily dissolved in 10% etha-
nol, and serial twofold dilutions in a supporting
medium (medium 199, Sigma, United States) were pre-
pared. A decrease in the virus titer in comparison with
control, 50% and 90% effective concentrations (EC50
and EC90) of the studied substances, and the ratio of
their MTC to their EC50 (MTC/ES50) and to EC90
(MTC/EC90, the selectivity index) were calculated as
quantitative criteria of the observed antiviral effect.
MTCs of the compounds were determined after the
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007