Betulin-derived compounds as inhibitors of alphavirus replication

Article abstract of DOI:10.1021/np9003245

This paper describes inhibition of Semliki Forest virus (SFV) replication by synthetic derivatives of naturally occurring triterpenoid betulin (1). Chemical modifications were made to OH groups at C-3 and C-28 and to the C-20-C-29 double bond. A set of heterocyclic betulin derivatives was also assayed. A free or acetylated OH group at C-3 was identified as an important structural contributor for anti-SFV activity, 3,28-di-O-acetylbetulin (4) being the most potent derivative (IC₅₀ value 9.1 μM). Betulinic acid (13), 28-O-tetrahydropyranylbetulin (17), and a triazolidine derivative (41) were also shown to inhibit Sindbis virus, with IC₅₀ values of 0.5, 1.9, and 6.1 μM, respectively. The latter three compounds also had significant synergistic effects against SFV when combined with 3′-amino-3′-deoxyadenosine. In contrast to previous work on other viruses, the antiviral activity of 13 was mapped to take place in virus replication phase. The efficacy was also shown to be independent of external guanosine supplementation.

Full text of DOI:10.1021/np9003245

J. Nat. Prod. 2009, 72, 1917–1926
1917
Betulin-Derived Compounds as Inhibitors of Alphavirus Replication
Leena Pohjala,†,‡,⊥ Sami Alakurtti,§,| Tero Ahola,‡ Jari Yli-Kauhaluoma,§ and Pa¨ivi Tammela*,†
Centre for Drug Research, Faculty of Pharmacy, P.O. Box 56, FI-00014 UniVersity of Helsinki, Finland, Institute of Biotechnology, P.O. Box
56, FI-00014 UniVersity of Helsinki, Finland, DiVision of Pharmaceutical Chemistry, Faculty of Pharmacy, P.O. Box 56, FI-00014 UniVersity
of Helsinki, Finland, DiVision of Pharmaceutical Biology, Faculty of Pharmacy, P.O. Box 56, FI-00014 UniVersity of Helsinki, Finland, and
Technical Research Centre of Finland, VTT, P.O. Box 1000, FI-02044 VTT (Espoo), Finland
ReceiVed May 29, 2009
This paper describes inhibition of Semliki Forest virus (SFV) replication by synthetic derivatives of naturally occurring
triterpenoid betulin (1). Chemical modifications were made to OH groups at C-3 and C-28 and to the C-20-C-29
double bond. A set of heterocyclic betulin derivatives was also assayed. A free or acetylated OH group at C-3 was
identified as an important structural contributor for anti-SFV activity, 3,28-di-O-acetylbetulin (4) being the most potent
derivative (IC50 value 9.1 µM). Betulinic acid (13), 28-O-tetrahydropyranylbetulin (17), and a triazolidine derivative
(41) were also shown to inhibit Sindbis virus, with IC50 values of 0.5, 1.9, and 6.1 µM, respectively. The latter three
compounds also had significant synergistic effects against SFV when combined with 3′-amino-3′-deoxyadenosine. In
contrast to previous work on other viruses, the antiviral activity of 13 was mapped to take place in virus replication
phase. The efficacy was also shown to be independent of external guanosine supplementation.
The genus AlphaVirus consists of enveloped viruses with a single-
stranded positive-sense RNA genome of approximately 11.5
kilobases. These widely distributed viruses infect avian and
mammalian hosts, spreading in nature by using Aedes sp. mosqui-
toes as vectors. In vertebrate cells, the infection is acute and
cytopathic; most of the amplification occurs in small rodents,
whereas humans and other larger mammals are usually dead-end
hosts.1 One of the most prominent human epidemics caused by
alphaviruses was the recent Chikungunya outbreak, which occurred
at different sites surrounding the Indian Ocean in 2006 and involved
more than 1.5 million cases.2 In 2007, an outbreak of 205 confirmed
cases in northern Italy was also reported, raising awareness of the
potential for rapid transmission of tropical arthropod-borne diseases
to temperate areas.3,4 Chikungunya and other alphaviruses found
on the Eurasian and African continents primarily cause polyarthritis,
accompanied by rash-like symptoms and myalgia.5 In contrast,
viruses of the same genus found on the American continents, such
as Western, Eastern, and Venezuelan equine encephalitis viruses,
are primarily associated with small epidemics of encephalitis in
both humans and domestic animals.6 Even though alphaviruses are
considered a potential cause of both economic loss and human
suffering and mortality, currently available pharmacotherapy for
alphavirus-borne diseases is limited to relatively inefficient ribavirin
and interferon combinations and to symptomatic relief.
and C-28, betulin derivatives have been investigated for a variety
of applications. Betulin by itself is quite inactive in pharmaceutical
applications; however, it can be oxidized with the Jones’ reagent
(CrO3/aq. H2SO4) to betulonic acid. Betulonic acid, in turn, can be
reduced with NaBH4 selectively to betulinic acid,14 which is an
important and pharmaceutically more active precursor for further
modifications. The chemistry and therapeutic potential of betulin-
derived compounds have been most widely studied for use against
certain cancers and human immunodeficiency virus type 1 (HIV-
1), and different betulin derivatives are currently undergoing clinical
trials for both indications.15 In anti-HIV therapy, two separate
mechanisms of action have been proposed, involving both early
and late stages in the virus infection cycle. A C-3-substituted
betulinic acid derivative, bevirimat [3-O-(3′,3′-dimethylsuccinyl)-
betulinic acid], has been shown to inhibit HIV-1 maturation by a
previously undescribed mechanism, i.e., by blocking the processing
of Gag polyprotein between the capsid and p2 spacer sequences
and leading to aberrant maturation and decreased infectivity of the
virions.16 Phase II clinical trials with bevirimat were positively
reported in 2007, indicating favorable pharmacokinetics and
preliminary data on efficacy in patients with HIV.17,18 On the other
hand, the C-28-substituted aminoalkyl betulin derivatives ICH9564
and A43-D inhibit HIV-1 entry by targeting the V3 loop of HIV
gp120.19
Betulin 1 (lup-20(29)-ene-3ꢀ,28-diol), a pentacyclic, lupane-type
triterpene, is a major constituent of the bark of white birches (Betula
sp.) that are found in abundance in northern temperate zones. A
more water-soluble compound, betulinic acid, is also present in birch
bark in minor quantities (0.3% of dry weight in B. pendula7).
However, the distribution of these compounds in nature is not
limited to this genus but covers a variety of plant species, including
well-known medicinal plants on most continents.8-10 The spectrum
of naturally occurring betulin-related compounds also includes
betulonic acid, 3-O-sulfates,11 28-O-glycosides, and esters such as
nicotinates and caffeates.12,13
Beyond antiretroviral therapy, reports on the antiviral properties
of betulin derivatives mainly involve the effects of naturally
occurring derivatives on DNA viruses. As such, betulin alone and
in combination with aciclovir has been reported to inhibit Herpes
simplex virus types I and II (HSV I and II), showing approximately
10-fold increased sensitivity to HSV-I when compared to HSV-
II.20 Betulinic acid and betulonic acid are also active against HSV,
as well as against influenza A and ECHO-6 picornavirus. Betulinic
acid was reported to be more potent in the two former cases and
betulonic acid in the latter case.21 Furthermore, the naturally
occurring 3-epi-betulinic acid 3-O-sulfate was recently demonstrated
to inhibit HSV, influenza A, and respiratory syncytial virus (RSV).22
A small set of synthetic C-3- and C-28-substituted betulin deriva-
tives has also been assayed against HSV and influenza A,
emphasizing the potential role of C-28-substitution in antiviral
activity.23
Due to the ease of isolation in large quantities and accessibility
for chemical modification of the hydroxy groups at positions C-3
* Corresponding author. Tel: +358 9 191 59628. Fax: +358 9 191 59138.
E-mail: paivi.tammela@helsinki.fi.
† Centre for Drug Research, University of Helsinki.
‡ Institute of Biotechnology, University of Helsinki.
§ Division of Pharmaceutical Chemistry, University of Helsinki.
⊥ Division of Pharmaceutical Biology, University of Helsinki.
| Technical Research Centre of Finland.
In the present study, anti-alphaviral properties of 51 betulin
derivatives were assayed against Semliki Forest virus (SFV), which
is an extensively studied member of the AlphaVirus genus. Another
10.1021/np9003245 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/19/2009

1918 Journal of Natural Products, 2009, Vol. 72, No. 11
Scheme 1. Synthesis of Compounds 2-6a
Pohjala et al.
a Conditions: (a) Ac2O (1.05 equiv), DMAP, py, CH2Cl2, rt, 22 h, 45%; (b) PCC, CH2Cl2, rt, 24 h, 57%; (c) Ac2O (6 equiv), DMAP, py, CH2Cl2, rt, 17 h, 97%;
(d) HBr, Ac2O, AcOH, PhMe, rt, 21 days, 42%; (e) mCPBA, Na2CO3, CHCl3, rt, 2 h, 65%. DMAP ) 4-(dimethylamino)pyridine; py ) pyridine; PCC ) pyridinium
chlorochromate; mCPBA ) 3-chloroperoxybenzoic acid.
Scheme 2. Synthesis of Compounds 7-10a
a Conditions: (a) DEAD, PPh3, 3,3-dimethylglutarimide, THF, 0 °C f rt, 24 h, 31%; (b) Ac2O, DMAP, py, CH2Cl2, rt, 22 h, 81%; (c) H2, 5% Pd/C, THF-MeOH
(1:2), rt, 22 h, 99%; (d) H2CrO4, acetone, rt, 20 h, 31%. DEAD ) diethyl azodicarboxylate; THF ) tetrahydrofuran.
member, Sindbis virus (SIN), was also shown to be sensitive to
selected betulin-derived compounds. The anti-SFV activity of
betulinic acid was mapped into the replication phase of the virus,
and the derivatives of different structural subgroups were shown
to exhibit strong synergism in SFV inhibition when combined with
3′-amino-3′-deoxyadenosine.
Synthesis of Betulin Derivatives. Compound 2, 28-O-acetyl-
betulin, was obtained in moderate yield (45%) by treating 1 with
acetic anhydride in the presence of DMAP and pyridine in CH2Cl2
(Scheme 1). Subsequent oxidation of 2 with PCC in CH2Cl2
afforded 28-O-acetyl-3-oxobetulin (3) in 57% yield.26 3,28-Di-O-
acetylbetulin (4), in turn, was obtained in excellent (97%) yield by
treating 1 with excess acetic anhydride. Treatment of 4 with HBr
in toluene caused the migration of the C-20-C-29 double bond of
4 to the C-18-C-19 position, giving 3,28-di-O-acetyllup-18-ene
(5) in 42% yield.27-29 The C-18-C-19 double bond of 5 was
epoxidized with mCPBA in CHCl3 to provide the intermediate 6
in 65% yield.
3-Deoxy-2,3-didehydrobetulin (7) was prepared in 31% yield by
treating 1 with a mixture of DEAD, PPh3, and 3,3-dimethylgluta-
rimide in THF30 (Scheme 2). Subsequent acetylation of 7 gave
3-deoxy-2,3-didehydro-28-O-acetylbetulin (8) in 81% yield. Di-
hydrobetulin (9) was obtained in 99% yield after the catalytic
hydrogenation of 1 using Pd/C as a catalyst. Subsequent oxidation
with the Jones reagent in acetone produced the target compound,
dihydrobetulonic acid (10), in 31% yield.
Results and Discussion
To date, only a limited number of organic small molecules have
been found to inhibit alphavirus replication, and most of the existing
reports concern nucleoside analogues with often nonoptimal
selectivity indices (see ref 24 for review). However, the need for
wider structural diversity among the inhibitors of these viruses has
been emphasized by the recent epidemic outbreaks. Natural products
have often been proven invaluable in the search for novel
antimicrobial agents. In the context of alphavirus inhibitors, a seco-
pregnane steroid and steroidal glycosides were recently investigated
as inhibitors of SFV subgenomic RNA production.25 The current
study elucidates the effects of lupane-type triterpenoids on SFV.
Even though betulin-derived compounds are known for their various
antimicrobial and antineoplastic properties, their effectiveness on
RNA viruses remained uncharacterized.
Oxidation of 1 with the Jones reagent in acetone afforded
betulonic acid (11)14 in 44% yield (Scheme 3). Subsequent

Betulin-DeriVed Compounds
Journal of Natural Products, 2009, Vol. 72, No. 11 1919
Scheme 3. Synthesis of Compounds 12-16a
a Conditions: (a) H2CrO4, acetone, 0 °C f rt, 21 h, 44%; (b) (i) (COCl)2, CH2Cl2, rt, 22 h, 85%, (ii) vanillin, DMAP, py, 40 °C, 21 h, 20%; (c) NaBH4, i-PrOH,
rt, 2.5 h, 82%; (d) TMSCHN2, PhMe-MeOH (3:2), rt, 40 min, 89% 14, 66% 15; (e) L-aspartic acid dimethyl ester hydrochloride, TEA, CH2Cl2, rt, 19 h, 42%. TMS
) trimethylsilyl; TEA ) triethylamine.
Scheme 4. Synthesis of Compounds 17-20a
a Conditions: (a) DHP, PPTS, CH2Cl2, rt, 2 days, 30%; (b) Ac2O, DMAP, py, CH2Cl2, rt, 20 h, 95%; (c) PPTS, EtOH, rt, 14 days, 94%; (d) CH3SO2Cl, TEA,
CH2Cl2, 0 °C, 2 h, 99%. DHP ) 3,4-dihydro-2H-pyran; PPTS ) pyridinium p-toluenesulfonate.
treatment of 11 with oxalyl chloride in CH2Cl2 gave betulonoyl
chloride,31 which was immediately allowed to react with vanillin
in the presence of DMAP in pyridine to produce vanillyl betulonate
(12) in 20% yield. Reduction of 11 with NaBH4 in 2-propanol gave
betulinic acid (13) in 82% yield,32 which was subsequently
methylated with TMSCHN2 in PhMe-MeOH to give 14 in 89%
yield.33 Similarly, treatment of 11 with TMSCHN2 in PhMe-MeOH
produced methyl betulonate (15) in 66% yield. Treatment of 11, in
turn, with oxalyl chloride in CH2Cl2 followed by L-aspartic acid
dimethyl ester in the presence of TEA in CH2Cl2 gave the
corresponding L-aspartyl amide of betulonic acid (16) in 42%
yield.31
Treatment of 1 with PPTS and DHP in CH2Cl2 produced a
diastereomeric mixture of the corresponding tetrahydropyranyl ether
(17) in 30% yield (Scheme 4). The THP-protected betulin was
subsequently acetylated to give 18 in excellent 95% yield. Removal
of the THP group with PPTS in EtOH produced 3-O-acetylbetulin
(19) in 94% yield.29 Subsequent treatment of 19 with CH3SO2Cl
in the presence of TEA in CH2Cl2 gave 3-O-acetyl-28-O-mesyl-
betulin (20) in 99% yield.
Betulin (1) was oxidized with PCC (6 equiv) in CH2Cl2 to give
betulonic aldehyde (21) in 82% yield (Scheme 5).34 When a smaller
molar amount of PCC (1.8 equiv) was used, a 3:1 mixture of 21
and betulin aldehyde (22) was produced. Part of the mixture was

1920 Journal of Natural Products, 2009, Vol. 72, No. 11
Scheme 5. Synthesis of Compounds 21-26a
Pohjala et al.
a Conditions: (a) PCC (6 equiv), CH2Cl2, rt, 1 h, 82%; (b) PCC (1.8 equiv), CH2Cl2, rt, 40 min, 22:21 (1:3); (c) NH2OH· HCl, py-EtOH (1:3), 100 °C, 18 h, 10%
23, 33% 24; (d) Ac2O, 120 °C, 2 h, 34% 25, 46% 26.
separated by SiO2 column chromatography, and 22 was isolated in
18% yield. The rest of the mixture was treated with an excess of
hydroxylamine hydrochloride in pyridine-EtOH to produce oximes
23 and 24 in 10% and 33% yields, respectively.35 Separate treatment
of 23 and 24 with neat acetic anhydride at 120 °C gave nitriles 25
and 26 in 34% and 46% yields, respectively.
heterocycles (34-43) with acetyl R2 groups in moderate (16% to
62%) yields after the urazoles were oxidized to the corresponding
urazines with iodobenzene diacetate in situ.39 For the synthesis of
heterocycles (44-51) with different R2 ester groups, a mixture of
3,28-di-O-acetyllupa-12,18-diene and 3,28-di-O-acetyllupa-18,21-
diene was treated with NaOH in THF-MeOH to remove the acetyl
groups. Subsequent acylation with various acyl chlorides yielded
a mixture of dienes (44-51) with new R2 ester groups. Synthesis
of betulin heterocycloadducts will be described in detail elsewhere.
Inhibition of SFV by Betulin Derivatives. The primary screen
of 51 betulin-derived compounds against SFV, combined with a
counterscreen for Huh-7 cell viability, was run in order to determine
the tentative inhibitory potential of each derivative. A relatively
high test concentration (50 µM) was selected for the primary screen
with the added intention of tracking weakly active derivatives for
structure-activity comparisons. The results of the primary screen
were expressed as surviving fractions (remaining percentages of
viral replication or cell viability) after exposure to each compound.
These data were used to divide the derivatives according to their
properties into the following four clusters: cluster 1, selective and
efficient antiviral activity (compounds yielding <20% remaining
viral replication and >80% cell viability); cluster 2a, moderate but
selective antiviral activity (remaining viral replication <50% and
cell viability >80%); cluster 2b, efficient but moderately selective
antiviral activity (remaining viral replication <20% and cell viability
between 50% and 80%); cluster 3, antivirally inactive derivatives
and compounds with unacceptable cytotoxicity (virus replication
>50%/cell viability <50%).
The last series of the synthetic triterpenoids commenced by
treating ethyl chrysanthemate with NaOH in THF-MeOH to
produce chrysanthemic acid (91% yield), which was subsequently
allowed to react with oxalyl chloride in CH2Cl2 to give chrysan-
themoyl chloride in 81% yield (Scheme 6). Chrysanthemoyl
chloride was reacted with 1 to produce a 1:3 mixture of cis- and
trans-28-O-chrysanthemoylbetulin (27) in 63% yield. Treatment of
carvacrol in the presence of chloroacetic acid and NaOH in water
gave carvacryloxyacetic acid (45% yield),36 which was reacted with
1 in PhMe using titanium(IV) isopropoxide as an esterification
catalyst to produce betulinyl 28-carboxymethoxycarvacrolate (28)
in 55% yield. A mixture of 1, levulinic acid, and PPTS was reacted
in PhMe to produce 3,28-di-O-levulinoylbetulin (29) in 23% yield.
Treatment of 1 with nicotinic acid in the presence of DCC and
DMAP in CH2Cl2 gave 28-O-nicotinoylbetulin (30) in 31% yield.
Cinnamic acid was treated with thionyl chloride to produce
cinnamoyl chloride, which was treated immediately with 1 to give
28-O-cinnamoylbetulin (31) in 21% yield. N-Acetylanthranilic acid
was treated with oxalyl chloride to produce N-acetylanthraniloyl
chloride, which was treated immediately with 1 to give 28-O-(N-
acetylanthraniloyl)betulin (32) in 25% yield. Finally, betulin 1 was
treated with t-BuOK in THF followed by addition of methyl
bromoacetate to give 28-O-bromoacetylbetulin (33) in 15% yield.
For the synthesis of heterocyclic betulin derivatives 34-51
(Scheme 7, Table 1), 3,28-di-O-acetyl-18,19-epoxylupane (6) was
treated with PPTS in PhMe to give a mixture (4:1) of conjugated
dienes, 3,28-di-O-acetyllupa-12,18-diene and 3,28-di-O-acetyllupa-
18,21-diene, in 68% yield.37 Reactions of 4-phenyl- or 4-methyl-
1,2,4-triazoline-3,5-dione or reactions of various 4-substituted
urazoles38 with a mixture of dienes gave the corresponding
Table 2 presents the results of the primary screen, listing the
antiviral and cell viability surviving fractions and the corresponding
cluster number for each compound. Compounds in cluster 1
represent the best lead candidates and were thus selected for further
evaluation by dose-response experiments. The anti-SFV IC50 (50%
inhibitory concentration) values for this set of compounds, derived
from the fitting of data into a sigmoidal dose-response curve model,

Betulin-DeriVed Compounds
Journal of Natural Products, 2009, Vol. 72, No. 11 1921
Scheme 6. Synthesis of Compounds 27-33a
a Conditions: (a) (i) ethyl chrysanthemate, NaOH, MeOH-THF (2:1), 80 °C, 4 h, 91%, (ii) chrysanthemic acid, (COCl)2, CH2Cl2, rt, 6 h, 81%, (iii) chrysanthemoyl
chloride, DMAP, py, 40 °C, 48 h, 63%; (b) (i) carvacrol, chloroacetic acid, NaOH, ∆, 3 h, 45%, (ii) carvacryloxyacetic acid, Ti(OPr-i)4, PhMe, ∆, 6 h, 55%; (c)
levulinic acid, PPTS, PhMe, 175 °C, 23 h, 23%; (d) nicotinic acid, DCC, DMAP, CH2Cl2, rt, 23 h, 31%; (e) (i) cinnamic acid, SOCl2, 40 °C, 2 h, 92%, (ii) cinnamoyl
chloride, DMAP, pyridine, 40 °C, 22 h, 21%; (f) (i) N-acetylanthranilic acid, (COCl)2, rt, 3 days, 99%, (ii) N-acetylanthraniloyl chloride, DMAP, py, 40 °C, 40 h, 25%;
(g) t-BuOK, methyl bromoacetate, THF, 75 °C, 10 min, 15%. DCC ) N,N′-dicyclohexylcarbodiimide.
Scheme 7. Synthesis of Compounds 34-51a
Table 1. Heterocyclic Betulin Derivatives 34-51
.
a Synthesis of heterocyclic betulin derivatives 34-51 will be described in
detail elsewhere.
compound
R1
R2
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
3-MeO-Ph
1,3-dioxol-5-yl
indan-5-yl
4-F-Ph
3-NO2-Ph
3-Cl-Ph
PhCH2
Ph
n-Bu
Et
Ph
Me
Me
Me
Me
Me
Ph
t-Bu
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
H
COEt
COPr
COi-Pr
COcHex
COPh
are shown in Table 3. As discussed in more detail in the
structure-activity relationship section, the values range from 9.1
µM (compound 4) to 48.5 µM (compound 3). For comparison, a
standard SFV inhibitor, ribavirin, has an IC50 value of 95 µM in
the assay. In an extended cytotoxicity analysis at 500 µM
concentration performed on all cluster 1 compounds, only betulonic
aldehyde (21) affected the surviving fraction of Huh-7 cells (cell
viability 52% after 24 h exposure). For other cluster 1 compounds,
this high concentration, which is close to the solubility limits, was
well tolerated (cell viability values >80%).
Structure-Activity Relationships. The primary screening
data and the results of the potency analysis were used to examine
the structural determinants for the anti-alphaviral activity of betulin
derivatives and to study the chemical space of antivirally active
betulin-derived compounds. Betulin (1) inhibited SFV replication
with an IC50 value of 45.5 µM. Removal of the C-20-C-29 double
bond yielded compound 9, which failed to show antiviral activity.
Both betulinic acid (13) and betulonic acid (11) yielded improved
antiviral potency compared to 1 (IC50 values 14.6 and 13.3 µM
and p values in Student’s t test <0.05 in both cases). Oxidation of
COPh
COcHex
the OH moieties also compensated for the loss of double-bond
exclusion activity, even though the potency of the 20-29-saturated
dihydrobetulonic acid (10) remained inferior to its unsaturated
counterpart 11. Removal or oxidation of the secondary OH group
at C-3 disturbed the anti-SFV activity, as demonstrated by the

1922 Journal of Natural Products, 2009, Vol. 72, No. 11
Pohjala et al.
Table 2. Antiviral (AV) and Cytotoxic Effects (CV) of
Betulin-Derived Compounds
Table 3. IC50 Values of Betulin-Derived Compounds against
Semliki Forest Virus (SFV)a
virus
replication (%)
cell
viability (%)
compound
IC50 µM (pIC50)
compound
cluster
1
45.5 (-4.34 ( 0.18)
12.1 (-4.92 ( 0.16)
48.5 (-4.32 ( 0.15)
9.1 (-5.04 ( 0.26)
43.2 (-4.37 ( 0.20)
13.3 (-4.94 ( 0.45)
30.6 (-4.52 ( 0.23)
13.3 (-4.88 ( 0.15)
14.6 (-4.84 ( 0.28)
17.2 (-4.76 ( 0.24)
24.7 (-4.61 ( 0.15)
24.2 (-4.62 ( 0.25)
38.3 (-4.42 ( 0.28)
22.9 (-4.64 ( 0.16)
22.1 (-4.65 ( 0.15)
35.9 (-4.44 ( 0.12)
19.7 (-4.71 ( 0.13)
37.9 (-4.42 ( 0.20)
30.1 (-4.52 ( 0.18)
95.1 (-4.02 ( 0.27)
1
2
3
4
5
6
7
8
7
3
83
87
83
97
87
92
85
95
81
102
89
89
123
1
83
30
92
91
86
99
90
0
80
82
85
69
90
103
109
94
96
94
11
93
92
93
95
108
95
101
87
81
40
85
75
93
106
97
108
88
83
1
1
1
2a
1
1
3
3
3
1
1
2a
1
3
3
3
1
1
1
2a
1
3
3
3
3
3
3
3
3
2a
3
3
3
3
3
3
2
3
4
5
11
21
15
16
121
98
95
14
13
43
18
68
71
40
11
10
3
6
10
11
13
17
18
19
21
38
39
40
41
42
46
ribavirin
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
46
8
1
a Dose-response experiments were performed using a luminometric
anti-SFV assay (see Supporting Information) using serial dilutions of
each derivative and fitting the data into sigmoidal dose-response
curves; values are means ( SD (n ) 6).
121
55
73
60
95
80
74
43
111
82
125
121
97
81
64
3
of 9.1 µM. Migration of the terminal double bond from C-20-C-
29 to C-18-C-19 [3,28-di-O-acetyllup-18-ene (5)] reduced the
antiviral activity to 43 µM. Further introduction of 18-19-epoxide
resulted in equally effective and potent activity (compound 6; IC50
value 13 µM), when compared to 4. However, acetylation of 1 at
C-3 as such (derivative 19) or when combined with 28-O-
tetrahydropyranyl derivatization in 18, as well as addition of the
28-O-tetrahydropyranyl (THP) moiety alone (17), yielded cluster
1 compounds 17, 18, and 19 with IC50 values of 17.2, 24.7, and
24.2 µM, respectively. On the other hand, mesylation of the 3-O-
acylation product at C-28 resulted in only moderately active
compound 20, scoring into cluster 2a with surviving viral fraction
of 46% (Table 2). Introducing a nitrile moiety at C-28 led to even
more diminished antiviral activity (26 in cluster 3 and 25 in cluster
2a).
As indicated by the lack of antiviral activity of 3-deoxy and
3-oxime derivatives, maintaining the secondary OH group at C-3
contributed to the anti-SFV activity of the betulin-derived com-
pounds. However, the IC50 values for derivatives with free versus
acetylated C-3 OH groups did differ from each other, 3,28-di-O-
acetylbetulin (9) being the most potent derivative. Thus, the
influence of C-28 substitution on anti-SFV activity was further
examined with C-3-unmodified derivatives. Unfortunately, the
activity of betulin C-28 esters of naturally occurring and biologically
active terpenoid or aromatic carboxylic acids was poor. Esters of
betulin and chrysanthemic acid 27, carvacryloxyacetic acid 28,
N-acetylanthranilic acid 32, or cinnamic acid 31 as well as the 3,28-
dilevulinate of betulin (29) had no detectable antiviral activity,
whereas the 28-nicotinate of betulin (30) was moderately active,
scoring into cluster 2a (Table 2). The antiviral activity of cytotoxic
28-O-bromoacetylbetulin (33) was poor. 28-Vanillinyl betulonate
(12) scored into cluster 2a, whereas the L-aspartyl amide of betulonic
acid (16) was cytotoxic.
3
1
1
1
1
1
3
3
1
1
13
19
24
73
21
18
24
44
22
50
40
2b
1
2a
2a
2a
2a
2a
The data present results from the primary screen of betulin-derived
compounds in anti-SFV and ATP cell viability assays (see the
Supporting Information for experimental details). The numbers represent
surviving fractions (the remaining percentages of viral replication or cell
viability) in each assay. All experiments were made in triplicate using a
concentration of 50 µM of each compound.
inactive 3-deoxy-2,3-didehydro derivatives 7 and 8, as well as the
inactivity of oxime derivative 23. Interestingly, betulonic aldehyde
(21) demonstrated selective antiviral activity in the primary screen,
whereas betulin aldehyde (22) had an inverse activity profile.
However, in the extended cytotoxicity assay at higher concentration,
22 also had indications of host cell toxicity.
Conversely to the inactive methyl esters of both betulinic acid
and betulonic acid (compounds 14 and 15, respectively), 28-O-
acetylbetulin (2) was a potent SFV inhibitor (IC50 value 12.1 µM).
The inhibitory capacity was retained, yet with loss in potency, in
the presence of the individually inactivating 28-O-acetyl-3-oxo-
betulin (3). Acetylation of both the C-3 and C-28 hydroxy groups
in 1 to yield 4 improved the antiviral activity, yielding an IC50 value
In addition to the C-3- and C-28-modified derivatives, a set of
heterocyclic compounds was synthesized by the [4+2] cycloaddition
reaction between N-substituted 1,2,4-triazolidine-3,5-diones (ura-
zines) and 3ꢀ,28-diacyloxylupa-12,18-dienes (34-51; see Table 1).
The heterocycloadducts with 4-n-butyl and 4-ethyl substituents, 42
and 43, were active against SFV, even though the 4-ethyl derivative
43 also affected Huh-7 cell viability (Table 2). The heterocycload-
duct with phenyl at N-4 (compound 41) inhibited SFV, with an
IC50 value of approximately 20 µM, and a switch to the N-4 benzyl
group was accompanied by a slight loss in potency (derivative 40

Betulin-DeriVed Compounds
Journal of Natural Products, 2009, Vol. 72, No. 11 1923
IC50 value 36 µM). Derivatives having an electron-withdrawing
group on the aromatic ring, 3-chlorophenyl (39) and 3-nitrophenyl
(38), were selective and potent SFV inhibitors, having IC50 values
of 22 and 23 µM, respectively (Table 3). However, 4-fluorophenyl-
substituted 37 had poor activity. Derivatives having an electron-
donating group in the aromatic ring, 3-methoxyphenyl (34), 1,3-
dioxol-5-yl (35), and indan-5-yl (36), had no activity against SFV.
However, further removal of the acetyl groups from the antivi-
rally active heterocycle 41 resulted in loss of activity (compound
44), but substitution of the acetyl groups by benzoyl at both
positions (50) yielded a moderately active compound (cluster 2a;
Table 2). In addition, the 4-methyl-1,2,4-triazoline-3,5-dione adducts
47, 48, and 49, combined with either isopropanoyl, cyclohexanoyl,
or phenyl 3,28-diesters of betulin, scored into cluster 2a. Indication
of cytotoxicity was observed in the case of the corresponding
propanoyl 3,28-diester (45). Derivate 51, with bulky substituents
(tert-butyl group in N-4, cyclohexanoyl groups in C-3, and hydroxy
groups in C-28), scored into cluster 2a. However, the most efficient
inhibition of SFV among this subset of heterocycles was achieved
with a cycloadduct between 4-methylurazine and 3,28-di-O-
butyrylbetulin-12,18-diene (46). This compound scored into cluster
1 in the primary screen and yielded an IC50 value of 30 µM.
Inhibition of SIN by Betulin-Derived Compounds. Sindbis
virus (SIN), the causative agent of Pogosta disease (also known as
Carelian fever), is another alphavirus that is widely distributed over
the European, Asian, and African continents. SIN and SFV represent
separate clusters in the alphavirus phylogenetic tree, generated by
comparing E1 glycoprotein sequences. SIN and SFV also fall into
different serocomplexes according to antibody cross-reactivity.
However, the replicase proteins are relatively highly conserved
within the genus.1
Three betulin derivatives from different structural subclasses
showing antiviral potency and selectivity, i.e., 13, 17, and 41, were
assayed for inhibitory potency against SIN using a radiometric RNA
labeling assay. The dose-response curves presented in Figure 1
demonstrate the sensitivity of SIN toward these compounds; the
IC50 values extracted from the data were 0.5 µM (pIC50 -6.34 (
0.09), 1.9 µM (pIC50 -5.72 ( 0.10), and 6.1 µM (pIC50 -5.21 (
0.16) for 13, 17, and 41, respectively. In all three cases, the studied
SIN strain exhibited greater sensitivity toward the derivatives
compared to SFV.
Synergism Studies. Antiviral therapy is often conducted as a
combination of multiple drugs targeting different sites in virus
replication.40 Parallel administration of inhibitors with different
molecular targets is considered beneficial in terms of improved
efficacy and/or prevention of resistance. Previously we reported
the anti-alphaviral efficacy of several modified nucleosides, includ-
ing 3′-amino-3′-deoxyadenosine (3′-NH-3′-dAdo) with an IC50 value
of 18 µM in the reporter gene assay.23 The inhibition of the virus
life cycle by nucleoside analogues is likely to take place during
the replication phase, targeting viral polymerases or components
of cellular nucleoside metabolism, whereas betulin-derived com-
pounds have been related to a variety of antiviral mechanisms
mainly in the early and late stages of the virus life cycle (see below).
Thus, we investigated the possibility of synergistic inhibition of
SFV by 3′-NH-3′-dAdo together with derivatives 13, 17, and 41.
The IC50 values for each interaction partner were determined by
using varying concentrations of 3′-NH-3′-dAdo and test compound
in combination (see Experimental Section and Supporting Informa-
tion for experimental details). The strong Loewe synergism that
was demonstrated in these experiments is visualized by the bending
of the isobolograms below the additivity-indicating diagonal line
in Figure 2. Calculation of Berembel interaction indices for
individual combinations indicated that the most intense Loewe
synergism was achieved when 5 µM 3′-NH-3′-dAdo was combined
with low or moderate concentrations of each betulin derivative. At
this nucleoside concentration, 0.08 µM and 0.4 µM 13 yielded
Figure 1. Dose-dependent activity of (a) betulinic acid (13), (b)
28-O-tetrahydropyranylbetulin (17), and (c) 4-phenyl-substituted
betulin heterocycle 41 against Sindbis virus (SIN). The dose-response
experiments were performed using the radiometric RNA labeling
assay (see Supporting Information) using serial dilutions ranging
from 16 pM to 50 µM. Nonlinear regression was used to fit the
data into sigmoidal dose-response curves; values are means ( SD
(n ) 4).
interaction index values of I ) 0.28 and 0.25, respectively. For 0.4
µM 28-O-tetrahydropyranylbetulin (17), an I value of 0.24 was
obtained, whereas combining 5 µM nucleoside with the heterocycle
41 resulted in the most intense synergism at 2 µM (I ) 0.16).
Increasing compound 17 and 41 concentrations closer to their IC50
values gave moderate or strong synergism (I values at 10 µM
concentration of betulin derivatives with different 3′-NH-3′-dAdo
concentration ranged from 0.25 to 0.47), whereas similar conditions
with 13 yielded additive rather than synergistic inhibition (I values
from 0.52 to 1.26). Complete tables of interaction indices for all
three compounds with 3′-NH-3′-dAdo, as well as the equations used
to calculate the indices, are provided as part of the Supporting
Information.
Mapping of the Target Site for Anti-SFV Activity. Since
the antiviral effects of betulin derivatives are associated with a range

1924 Journal of Natural Products, 2009, Vol. 72, No. 11
Pohjala et al.
Figure 3. Effect of administration time on antiviral effect of betulin-
derived compounds 13, 17, and 41 against SFV. Each of the
compounds was present in high-multiplicity infected cultures (5
PFU/cell) either throughout the experiment (0-5 h), during virus
adsorption (0-1 h), at 1-5 h, or at 2-5 h. The results represent
the surviving virus fraction, determined as remaining luciferase
reporter gene activity in each sample at 5 h; values are means (
SD (n ) 4).
administration to 2 h led to attenuated efficacy, yet the response
was still detectable in the case of 13.
Even though physicochemical and kinetic features, such as
hydrophobicity of the triterpenoids and nucleoside phosphorylation,
may affect the interpretation, the entry phase does not appear to be
the target of the reported anti-alphaviral activity. In the case of
betulinic acid, the activity is associated with the early replication
phase, whereas the two other betulin-derived compounds give less
obvious results. The end point in the experimental setup is in the
translation and processing of viral polyprotein (into which the Rluc
gene is inserted; see ref 24). Thus, inhibitors of viral maturation
would give seemingly negative results in this particular setup. On
the other hand, our previous work indicated that the sensitivity of
an in vitro antiviral assay is highly dependent on the infection
multiplicity used (L. Pohjala, unpublished results). However,
repeating the experiment with a higher concentration (200 µM)
yielded similar results, as shown in Figure 3, at 50 µM. Yet the
effect of inferior potency cannot be completely ruled out by this
means since the target site for the anti-alphaviral activity of 17
and 41 may lie in steps of the virus life cycle occurring after the
production of nonstructural polyproteins. Each of the replication
phases consists of several substages, which are typically inhibited
by different sets of chemical agents.40
As betulin-derived compounds are known to have a wide
spectrum of antimicrobial, anti-inflammatory, and antineoplastic
effects,15 these findings could be reconciled by a general underlying
mechanism, such as interference with cellular nucleoside metabo-
lism. Indeed, certain classical inhibitors of RNA virus replication
that also share anti-inflammatory properties (e.g., ribavirin and
mycophenolic acid) exert their action via depletion of cellular GTP
pools.41,42 This mode of action is characterized by the loss of
inhibitory effect when the cultures are supplemented with external
guanosine to compensate for depletion in cellular guanosine
biosynthesis. However, the betulin-derived compounds 13, 17, and
41 maintained their antiviral activity in the presence of 50 µg/mL
(177 µM) guanosine supplementation also (data not shown),
implying that any contribution of this mechanism to the observed
anti-SFV activity is minor at most. The existence of more specific
virus-related targets is also supported by the distinct structure-activity
relationships reported for different pharmacological uses.15
The study of antiviral mechanisms of betulin-derived compounds
has involved several molecular targets. For relatively simple,
naturally occurring compounds such as betulin and betulinic acid,
the proposed targets include HIV-1 reverse transcriptase,43 HIV
gp41,44 and severe acute respiratory syndrome coronavirus (SARS-
CoV) 3CL protease.45 Recent work on the SARS protease implies
that modulation of a single molecular target may not correlate with
Figure 2. Synergistic activities of (a) betulinic acid (13), (b) 28-
O-tetrahydropyranylbetulin (17), and (c) 4-phenyl-substituted betulin
heterocycle 41 in combination with 3′-amino-3′-deoxyadenosine
against SFV presented as isobolograms. The synergism studies were
performed by titrating different concentrations (from 0.5 to 50 µM)
of 3′-amino-3′-deoxyadenosine against the serial dilutions of each
betulin derivative (from 80 pM to 50 µM). D1/Dm1 and D2/Dm2
values were derived from the data according to ref 48. Ratios were
calculated for IC50 values of each compound alone and in the
presence of different concentrations of 3′-amino-3′-deoxyadenosine
in the combination. The diagonal line in each figure presents a
visualization of Loewe additive effects.
of target sites, an administration time experiment using a high-
multiplicity infection of SFV-Rluc was applied in order to gain
preliminary information on the target site of the reported anti-SFV
activity. Compounds 13, 17, and 41 were administered into cell
cultures at different time points in conditions where the majority
of cells were infected at once. The time scale of this experiment
represents a single virus replication cycle, as the luciferase reporter
gene was expressed by the translation of viral nonstructural proteins,
and the readout was thus taken at 4.5 h (see the Supporting
Information for experimental details). As illustrated in Figure 3,
none of the three betulin derivatives showed antiviral efficacy when
present in the cultures only at the time of viral adsorption (0-1 h).
The same was also observed for the 3′-amino-3′-deoxyadenosine
that was used for comparison. On the other hand, delivery of the
compounds just after the removal of the viral inocula (at 1 h) yielded
inhibition comparable to the effect obtained when the agent was
present throughout the experiment. Furthermore, postponing the

Betulin-DeriVed Compounds
Journal of Natural Products, 2009, Vol. 72, No. 11 1925
in vivo antiviral efficacy: Wen and co-workers45 conclude that both
betulinic acid and betulonic acid inhibit SARS-CoV replication,
but only betulinic acid inhibits 3CL-purified 3C protease. Previous
work on the effects of triterpenoid drugs on HSV also supported
the hypothesis that the antiviral efficacy of such agents may be a
combination of different activities in cell culture, rather than directly
associated with any specific phase in the virus life cycle.46 Such
findings may put into question the usefulness of the compounds
from a rational drug design viewpoint, but may reflect nature’s
strategy for sustained bioactivity. On the other hand, chemically
modified betulin derivatives, such as those in anti-HIV drug
discovery, are thought to exert their activity via more limited sets
of targets, which is also supported by the generation of resistant
HIV strains via point mutations.47,48 The results from the current
screen elucidate a pattern in which most of the relatively simple
derivatives inhibit SFV replication, whereas, among the more
complex structures, the antiviral activity is not ubiquitous but shared
only by certain structural subclasses. On the other hand, betulinic
acid (13) is distinguished from the two derivatives (17 and 41) on
the basis of its behavior in the administration time experiments.
Betulin-derived compounds form a family of natural compounds
that, along with their synthetic derivatives, have a broad spectrum
of antineoplastic and antimicrobial activities. The present results,
together with prior reports on SARS-CoV and ECHO-6 picornavi-
rus, provide evidence for the sensitivity of positive-stranded RNA
viruses toward betulin-derived compounds. The inhibitory activity
against Semliki Forest virus and Sindbis virus, together with the
lack of early signs of toxicity, raise hopes about the therapeutic
potential of betulin-derived compounds used against these pathogens
either alone or in combination with other antiviral therapy.
assistance. Dr. S. Koskimies and Dr. I. Aumu¨ller are also thanked for
their valuable discussions.
Supporting Information Available: Experimental procedures (de-
tailed chemical and bioactivity screening methods) and characterization
data are available free of charge via the Internet at http://pubs.acs.org.
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Bioassays. Full-length infectious cDNA clones of SFV and SIN49,50
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NP9003245