Lupane triterpenes and derivatives with antiviral activity

Article abstract of DOI:10.1016/S0960-894X(03)00714-5

Betulin and betulinic acid have been modified at the C-3 and C-28 positions and the antiviral activity of derivatives has been evaluated in vitro. It was found that simple modifications of the parent structure of lupane triterpenes produced highly effective agents against influenza A and herpes simplex type 1 viruses.

Full text of DOI:10.1016/S0960-894X(03)00714-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3549–3552
Lupane Triterpenes and Derivatives with Antiviral Activity
a,
a
a
b
b
L. A. Baltina, * O. B. Flekhter, L. R. Nigmatullina, E. I. Boreko, N. I. Pavlova,
b
b
a
S. N. Nikolaeva, O. V. Savinova and G. A. Tolstikov
aInstitute of Organic Chemistry, Ufa Research Center of RAS, 71, prospect Oktyabrya, 450054 Ufa, Russia
bBelarussian Research Institute for Epidemiology and Microbiology, 4, K. Zetkin str., 220050 Minsk, Belarus
Received 3 March 2003; revised 24 April 2003; accepted 4 July 2003
Abstract—Betulin and betulinic acid have been modified at the C-3 and C-28 positions and the antiviral activity of derivatives has
been evaluated in vitro. It was found that simple modifications of the parent structure of lupane triterpenes produced highly effec-
tive agents against influenza A and herpes simplex type 1 viruses.
#
2003 Elsevier Ltd. All rights reserved.
Introduction
herpes simplex type I viruses in vitro. It was envisioned
that a series of simple modifications of the triterpenoids
parent structures could produce a number of potentially
important new derivatives with antiviral activity.
Betulin 1 and betulinic acid (BA) 3 are pentacyclic tri-
terpenes of lupane type of natural origin isolated from
various plants. The content of betulin in the outer bark
of white birches trees widespread in the northern lati-
tudes of the world, varies between 10 and 35% depend-
ing on the growth conditions, age, season and so on.
BA was originally extracted from the tropical tree,
Ziziphus mauritiama Lam (Rhamnaceae) and from the
leaves of Syzigium claviflorum and Aerva javanica
Materials and Methods
1
Betulin 1 was extracted from the birch bark of the
Betula pendula Roth collected in Ufa, Russia with 2-
propanol–water mixture (9:1, v/v) according to ref 16.
Betulonic acid 2 was prepared from betulin 90–92% by
2
,3
(Amaranthaceae). BA can be easily prepared from an
available bioprecursor, betulin, by a two-step synthetic
4
using Jones’ oxidation (CrO /H SO /acetone). BA 3
2
3
4
4
process. BA showed selective cytotoxicity against
human melanoma, neuroectodermal and malignant
was synthesized from 2 by NaBH reduction in 2-pro-
4
panol (92% yield) and recrystallized in hot methanol to
ꢁ
5
,6
17
ꢁ
brain tumor cell lines, induced apoptosis in human
7
pure 3b isomer with mp 290–291 C. Lit.: 291–292 C.
neuroblastoma cell lines. BA and its derivatives have
been identified as a potent and selective HIV-1
Antiviral activity was tested in cell cultures with influ-
enza A/FPV/Rostock/34 (H7N1) and herpes simplex
2
,8ꢀ11
inhibitor.
Specific inhibitors of HIV-1 with a new
1
8,19
mode of action were found among a series of $-undec-
anoic amides of BA. It was revealed that this class of
compounds interfere with HIV-1 entry in the cells at a
type 1 (HSV-1, strain 1C) viruses as described early.
Study of the antiviral activity against influenza A virus
was carried out in chicken embryo cells (CEC) by
plaque reduction assay method. Investigations of anti-
viral activity against HSV-1 were carried out by inhibi-
1
0
posbinding step. The esterification of betulin and BA
0
0
0
0
with 3 ,3 -dimethylglutaryl and 3 ,3 -dimethylsuccinyl
groups potentiated the anti-HIV activity of com-
tion
of
viral
cytopathic
effect
(CPE)
in
9
,12,13
pounds. Amino acid conjugates of BA are of
interest as inhibitors of human melanoma and fibro-
sarcome. Betulin and BA have antiviral properties
also against herpes simplex viruse type 1. This paper
describes the synthesis and antiviral evaluation of new
betulin and BA derivatives against influenza A and
rhabdomyosarcoma (RD) cell line. The compounds tes-
ted were predissolved in 10% ethanol and then were
prepared in consecutive 2-fold dilutions in maintenance
medium (medium 199, Sigma Chemical Co).
1
4
1
5
A virus titer reduction in comparison with control, 50%
effective concentration (EC ), and maximum nontoxic
5
0
concentration (MNTC) to EC50 ratio of tested sub-
stances were calculated as antiviral activity criteria.
*Corresponding author. Fax: +7-3472-355288; e-mail: baltina@anrb.ru
0
960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00714-5

3550
L. A. Baltina et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3549–3552
MNTC of compounds were determined in non-infected
ꢁ
chloride according to refs 10 and 11. Hydrazides 6a and
6b were prepared by the reaction of acid chlorides 5a
cell cultures after 72 h incubation at 37 C. The results
of experiments are given in Table 1.
ꢁ
and 5b with NH NH in ether solution at 0ꢂ5 C with
2
2
80 and 90% yields, respectively.
Isocyanates 7a and 7b were obtained from acid chlor-
ides 5a and 5b (1 mmol) and NaN (125 mg) by mixing
in acetone solution for 6 h at 20–22 C. The reaction of
Chemical Modification ofLupane Triterpenes
3
ꢁ
Betulonic acid 2 was converted into 3-oxime 4 (90%) by
refluxing for 2 h in a solution of hydroxylamine hydro-
isocyanates 7a and 7b with primary amines and l-amino
acids methyl esters gave good yields (84–88%) of 28-N -
ꢁ
0
chloride in pyridine. Mp 223–225 C. Acid chlorides 5a
and 5b were obtained in 95 and 92% yields, respectively,
by treatment of 2 and 4 in dry benzene with oxalyl
alkylureides 8a–8d (Scheme 1). Products were purified by
chromatography on Al O . Structures of all synthesized
2
3
Scheme 1.

L. A. Baltina et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3549–3552
3551
compounds were confirmed by IR, H and 13C NMR
spectra.
1
Table 1. Antiviral activity of betulinic acid and related compounds
Compd
Influenza A
HSV-1
MNTC/EC50
Acid chloride 5a (1 mmol) was reacted with amines
EC50 (mM)
MNTC/EC50
EC50
2.5
8.2
81.6
(
hexylamine, octadecylamine, colamine) (1.6 mmol) in
dry refluxing benzene or dioxane giving amides 9a–9c
Scheme 2) (67–74% yields). Amino acids conjugates
0a–10d were synthesized by the reaction of acid chlor-
2
3
4
5.7
>219.0
2.2
9.6
<1
43.9
53.3
1.3
21.3
<1
<1
454.0
11.5
298.5
67.6
177.0
<1
1.4
2.5
<1
<1
(
1
392.2
NT
6
6
7
8
8
8
8
9
9
1
1
1
1
1
a
b
a
a
b
c
d
b
NT
10.0
ide 5a (1 mmol) with l-amino acids (valine, leucine,
methionine) methyl esters hydrochlorides (1.3 mmol) in
chloroform. Yields of conjugates were 79–85%. The
reaction of acid chloride 5a with liquid ammonia in
chloroform resulted in amide 11 with 89% yield
2.0
25.2
<1
<1
>51.6
>221.4
0.2
>221.4
>85.7
5.0
>67.0
3.0
14.6
2.2
0.6
1.4
27.1
2.3
35.2
4.6
<1
<1
3.4
1.2
(
Scheme 2).
55.5
c
>754.7
>234.4
13.3
>377.4
662.8
141.8
1373.2
>666.0
220.4
0a
0b
0c
0d
1
Biological Activity
72.3
14.2
0.7
It was shown by different groups that the introduction
of the CONH function in the molecule of BA resulted in
11.9
1333.3
<1
1
0,11
increase of anti-HIV activity. Lupane triterpenes
and derivatives were evaluated against influenza A and
NT, not tested.
Scheme 2.

3552
L. A. Baltina et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3549–3552
herpes simplex type 1 viruses in vitro (Table 1). Com-
pounds 4, 11 were active against influenza A virus. 3-
Oxime 4 showed a significantly enhanced activity (EC50
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.2 mM) and a remarkably high MNTC/EC50 ratio of
5
3
by a CONH -group the activity was further improved.
92.2. When the C-28 carboxyl group was substituted
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2
8-amide 11 showed on EC50 of 0.7 mM and was char-
acterized by the widest range of active nontoxic con-
centrations (MNTC/EC =1333.3). Ureide 8d was
5
0
effective as influenza A virus inhibitor but its activity
was less expressed. BA was not active in such experi-
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against HSV-1: EC50 0.2, 2.2, 0.6 and 1.4, respectively
9
(Table 1). Their activity was more pronounced than the
activity of lupane acids 2 and 3. Compounds 8a and 8c,
which both contain the NHCONH group, exhibited the
most potent anti-HSV-1 activity and had the widest
range of MNTC/EC50 ratio (454.0 and 298.5). Com-
pound 9b containing a long 28-amide chain showed a
high inhibition of HSV-1 (EC50 1.4, MNTC/
EC =177.0). Betulonic and betulinic acids were less
active as compared with derivatives 8a, 8c, 8d and 9b as
HSV-1 inhibitors. It is obvious that the addition of
CONH or NHCONH groups at position 28 of lupane
acids enhances their antiviral activity against influenza
A and HSV-1 viruses.
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Acknowledgements
1
5
1
5. Carlson, R.M.; Krasutsky, P.A.; Karim, R.-U. US Patent
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6. Flekhter, O. B.; Nigmatullina, L. R.; Baltina, L. A.; Kar-
This research was supported by the Russian Fund for
Basic Research (grants No. 00-03-81174, No. 01-03-
33131, No. 02-03-81007) and Belarussian Fund for
Basic Research (grants No. B99P-031, No. B02R-016).
achurina, L. T.; Galin, F. Z.; Zarudyi, F. S.; Tolstikov, G. A.;
Boreko, E. I.; Pavlova, N. I.; Nikolaeva, S. N.; Savinova, O. V.
Chim.-Pharm. J. 2002, 36, 26.
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