New 3-O-acyl betulinic acids from Strychnos vanprukii Craib

Article abstract of DOI:10.1021/np030469i

Three new betulinic acid derivatives, 3β-O-trans-feruloylbetulinic acid (1), 3β-O-cis-feruloylbetulinic acid (2), and 3β-O-cis- coumaroylbetulinic acid (4), along with two known triterpenes, 3β-O-trans-coumaroylbetulinic acid (3) and ursolic acid (6) were isolated from the leaves and twigs of Strychnos vanprukii Craib. All isolates showed moderate anti-HIV activity with IC₅₀ values ranging from 3 to 7 μg/mL (5 to 15 μM) in an indicator cell line for HIV infectivity. The structures of the new isolates were elucidated by spectroscopic techniques including 1D and 2D NMR spectroscopy. In addition, the structure of 1 was confirmed by X-ray crystallography.

Full text of DOI:10.1021/np030469i

994
J . Nat. Prod. 2004, 67, 994-998
New 3-O-Acyl Betu lin ic Acid s fr om Str ych n os va n pr u kii Cr a ib
Nguyen Quyet Chien,† Nguyen Van Hung,† Bernard D. Santarsiero,‡ Andrew D. Mesecar,‡
Nguyen Manh Cuong,§ D. Doel Soejarto, J ohn M. Pezzuto,| Harry H. S. Fong, and Ghee T. Tan*,
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and
Pharmacognosy (M/ C 877), College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street,
Chicago, Illinois 60612, The Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, Illinois 60607,
Institute of Chemistry, National Center for Science and Technology, Nghia Do, Tu Liem, Hanoi, Vietnam, and
Cuc Phuong National Park, Nho Quan District, Ninh Binh Province, Vietnam
Received October 23, 2003
Three new betulinic acid derivatives, 3â-O-trans-feruloylbetulinic acid (1), 3â-O-cis-feruloylbetulinic acid
(2), and 3â-O-cis-coumaroylbetulinic acid (4), along with two known triterpenes, 3â-O-trans-coumaroyl-
betulinic acid (3) and ursolic acid (6) were isolated from the leaves and twigs of Strychnos vanprukii
Craib. All isolates showed moderate anti-HIV activity with IC50 values ranging from 3 to 7 µg/mL (5 to
15 µM) in an indicator cell line for HIV infectivity. The structures of the new isolates were elucidated by
spectroscopic techniques including 1D and 2D NMR spectroscopy. In addition, the structure of 1 was
confirmed by X-ray crystallography.
Our International Cooperative Biodiversity Group (ICBG)
project was established in collaboration with institutions
in the United States, Vietnam, and Laos under the auspices
of the Fogarty International Center, NIH.1 As part of the
antiviral drug discovery program initiated under this
ICBG, Strychnos vanprukii Craib (Loganiaceae) collected
at the Cuc Phuong National Park in Vietnam was found
to exhibit anti-HIV activity. Bioassay-guided fractionation
of the MeOH extract of the dried leaves and twigs of the
plant led to the isolation of five triterpenes from the CHCl3-
soluble fraction. Four of the isolates were determined to
be betulinic acid derivatives, three of which are new [3â-
O-trans-feruloylbetulinic acid (1), 3â-O-cis-feruloylbetulinic
acid (2), and 3â-O-cis-coumaroylbetulinic acid (4)]. The
fourth triterpene ester was identified as 3â-O-trans-cou-
maroylbetulinic acid (3), and the free triterpene was
established to be ursolic acid (6). The bioassay-directed
isolation, structure elucidation, and biological activity of
these compounds are reported herein.
Resu lts a n d Discu ssion
Compound 1 was obtained as colorless needles and was
shown to have a molecular formula of C40H56O6 according
to high-resolution electrospray ionization mass spectrom-
etry (HRESIMS). The IR spectrum of 1 exhibited absorp-
tion bands for hydroxyl (3531 and 3393 cm-1), ester
carbonyl (1694 cm-1), conjugated olefin (1627 cm-1), and
aromatic functionalities (1514 cm-1). The NMR spectra
suggest a structure with 40 carbon atoms composed of a
triterpene and a phenylpropane moiety. The acyl moiety
was recognized as trans-feruloyl through its 1H NMR
signals at δ 7.42, 7.31, and 7.24 (two protons with ortho-
coupling, J ) 8 Hz, and one isolated proton), which is
characteristic for a 1,2,4-trisubstituted benzene ring, coupled
* To whom correspondence should be addressed. Tel: (312) 413-2725.
Fax: (312) 413-5894. E-mail: ghee@uic.edu.
† Institute of Chemistry, Hanoi.
‡ The Center for Pharmaceutical Biotechnology, University of Illinois.
§ Cuc Phuong National Park.
Program for Collaborative Research in the Pharmaceutical Sciences,
with signals at δ 6.77 (d, J ) 15.8 Hz) and 8.05 (d, J )
15.8 Hz) for a trans-oriented double bond conjugated. The
attachment of the methoxy group to C-6′ was evident by
University of Illinois.
| Current address: Schools of Pharmacy, Nursing, and Health Sciences,
Purdue University, 575 Stadium Mall Dr., West Lafayette, IN 47907-2091.
10.1021/np030469i CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/26/2004

3-O-Acyl Betulinic Acids from Strychnos vanprukii
J ournal of Natural Products, 2004, Vol. 67, No. 6 995
Ta ble 1. 1H NMR Data of Compounds 1-4 (300 MHz,
pyridine-d5, J in Hz)
Ta ble 2. 13C NMR Data of Compounds 1-4 (75 MHz,
pyridine-d5)
H
1
2
3
4
C
1
2
3
4
H-1a
H-1b
H-2a
H-2b
H-3
1.60
1.85
1.58
0.94
1.80
1.72
1.62
1.00
1.83
1.60
0.96
1.82
1.71
4.82
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
38.62t
24.32t
80.52d
38.27s
55.70d
18.48t
38.61t
24.19t
80.58d
38.16s
55.69d
18.47t
38.64t
24.31t
80.56d
38.15s
55.72d
18.50t
38.63t
24.14t
80.67d
38.12s
55.71d
18.48t
4.92, dd
(4.5, 11.3)
0.83
4.83, m
4.89, dd
(4.8, 11.8)
0.83
1.45
1.33
1.38
1.37
1.37
1.20
H-5
0.82
1.45
1.35
1.47
1.33
1.35
1.19
1.94
1.20
2.73
1.88
1.29
2.64
0.82
1.44
1.35
1.35
1.32
1.37
1.19
1.92
34.62t
34.63t
34.65t
34.62t
H-6a
H-6b
H2-7
H-9
H-11a 1.38
H-11b
H-12a 1.9
H-12b 1.2
H-13
H-15a 1.85
H-15b 1.25
H-16a 2.65
H-16b 1.60
H-18
H-19
1.44
41.07s
50.71d
37.33s
21.18t
41.06s
50.72d
37.32s
21.18t
41.09s
50.74d
37.35s
21.20t
41.07s
50.71d
37.33s
21.18t
C-9
1.4
1.37
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-7′
C-8′
C-9′
6′-OMe
26.02t
26.02t
26.04t
26.01t
38.55d
42.85s
30.25t
38.56d
42.85s
30.26t
38.58d
42.87s
30.24t
38.55d
42.84s
30.24t
1.93
2.73
2.73
1.85
1.26
2.65
1.58
1.78
3.53
2.25
2.72
1.82
1.26
2.62
1.59
1.76
3.52
2.25
1.55
2.25
32.85t
32.87t
32.87t
32.85t
56.61s
49.73d
47.78d
151.29s
31.20t
56.63s
49.73d
47.78d
151.30s
31.20t
56.63s
49.76d
47.78d
151.29s
31.22t
56.61s
49.75d
47.76d
151.27s
31.21t
1.8
3.54
1.80
3.54
2.25
37.59t
37.60t
37.59t
37.57t
H-21a 2.25
H-21b 1.60
H2-22 2.25
28.15q
16.96q
16.31q
16.36q
14.89q
178.84s
110.01t
19.46q
167.31s
115.96d
145.36d
126.62s
111.43d
149.02s
151.11s
116.85d
123.9d
55.88q
28.12q
16.85q
16.29q
16.35q
14.88q
178.86s
110.01t
19.46q
166.78s
116.59d
144.53d
127.04s
115.24d
148.11s
150.35s
116.12d
126.89d
55.89q
28.16q
16.95q
16.31q
16.37q
14.90q
178.84s
109.98t
19.47q
167.29s
115.85d
144.97d
126.24s
130.70d
116.87d
161.46s
116.87d
130.70d
28.13q
16.86q
16.27q
16.35q
14.88q
178.81s
109.96t
19.46q
166.65s
116.87d
143.80d
126.70s
133.64d
115.97d
160.55s
115.97d
133.64d
1.55
2.26
2.25
Me-23 0.97, s
Me-24 0.95, s
Me-25 0.80, s
Me-26 1.04, s
Me-27 1.10, s
H-29a 4.95, br s
H-29b 4.78, br s
Me-30 1.81, s
H-2′
H-3′
H-5′
H-6′
H-8′
H-9′
0.94, s
0.90, s
0.77, s
1.03, s
1.09, s
4.95, br s
4.78, br s
1.80, s
0.97, s
0.97, s
0.81, s
1.05, s
1.10, s
4.95, br s
4.78, br s
1.81, s
0.93, s
0.88, s
0.77, s
1.05, s
1.10, s
4.95, br s
4.77, br s
1.79, s
6.77, d (15.8) 6.05, d (12.9) 6.70, d (15.9) 6.02, d (12.8)
8.05, d (15.8) 7.01, d (13.0) 8.02, d (15.9) 6.98, d (12.9)
7.42, br s
8.34, br s
7.65, d (8.5) 8.09, d (8.6)
7.18, d (8.5) 7.18, d (8.6)
7.24, d (8.0) 7.23, d (9.0) 7.18, d (8.5) 7.18, d (8.6)
7.31, d (8.1) 7.52, d (9.0) 7.65, d (8.5) 8.09, d (8.6)
6′-OMe 3.80, s
3.90, s
the HMBC correlation of δH 3.80 (OMe) and δC 149.02 (C-
6′) as well as by the ROESY correlation between δH 3.80
and δH 7.42 (H-5′). Characteristic UV absorption at 327
nm and the fragments at m/z 616.4283 (loss of Me),
437.3713 (loss of the acyl moiety), and 175.0618 (loss of
the triterpene moiety) confirmed this partial structure.
After assigning signals to the feruloyl moiety, the remain-
ing NMR signals of 1 indicated the presence of a pentacyclic
triterpene bearing six quaternary methyl, one carboxylic
acid (δC 178.84), one oxymethine (δH 4.92) linked to the
feruloyl moiety, and one isopropenyl group attached to a
five-membered ring, with δH 4.78, 4.95 assigned to the
vinylic methylene, δH 1.81 for the attached methyl, and δH
3.54 for the allylic carbinyl proton. This pattern suggested
a lupan skeleton. Comparison of the NMR data (Tables 1
and 2) with those published for betulinic acid (5),2 coupled
with connectivity studies of 2D NMR spectral data, con-
solidated the above findings and further defined 1 as 3â-
O-trans-feruloylbetulinic acid. The 3â-configuration of the
feruloyl moiety was deduced from the H-3 proton signal
pattern at δH 4.92 (dd, J ) 4.5, 11.3 Hz) and from the effect
of the acyl substituent upon chemical shifts of 2-CH2, 23-
CH3, and 24-CH3 as compared to those of betulinic acid
(5) (Tables 1 and 2). This was further confirmed by HMBC
correlations between δH 4.92 and δC 16.96 (C-24), 28.15 (C-
23), and 38.27 (C-4) as well as by the ROESY correlation
between δH 4.92 and 0.83 (H-5) and 0.97 (H3-23). As
additional confirming evidence, compound 1 was subjected
to alkaline hydrolysis to yield betulinic acid (5) and trans-
ferulic acid, which proved to be identical with authentic
samples with respect to physical and spectroscopic proper-
ties.
In addition, a colorless crystal of 1 was selected for X-ray
analysis. The X-ray data confirmed the structure of 1 and
further revealed that in the crystal structure there are four
distinguishable rotamers, derived from two independent
units (Figure 1). The first unit consists of the major rotamer
A1 and the minor rotamer A2 and has a rotamer disorder
ratio of 63.1%:36.9% around the C3′-C4′ single bond. The
second unit consists of the major rotamer B1 and the minor
rotamer B2 and has a rotamer disorder ratio of 50.9%:
49.1% around the C1′-C2′ single bond. Analysis of the
torsional angles of these rotamers (Table 3) led to the
conclusion that in the crystal the preferred conformation
of 1 along the C(1′)-C(2′) single bond is the s-trans
conformation, and along the C(3′)-C(4′) bond the skew
conformation, with the bulky half of the benzene ring close
to the olefinic double bond, is the more favored one.
Compound 2 was obtained as white powder and was
shown by HRESIMS to have the same molecular formula,
C40H56O6, as well as the same MS fragments as 1. The
NMR spectral data of 2 closely resembled those of 1, except
for the signals assignable to the aromatic acyl moiety.
Differences in the UV absorption with a hypsochromic shift
of 2 nm, along with the appearance of two neighboring
olefinic proton signals at δH 6.05 and 7.01 with a coupling
constant of 12.9 Hz instead of 15.8 as in the case of 1, led
to the deduction that 2 was the cis-isomer of 1. Analysis of

996 J ournal of Natural Products, 2004, Vol. 67, No. 6
Chien et al.
Ta ble 4. Anti-HIV Activity and Cytotoxicity of Compounds
1-6
HOG.R5
CEM-SS/MTS
a
b
CC50 IC50
CC50 % cytoprotection
compound
(µM) (µM) SIc (µM) @ nontoxic conc
1
2
3
13.4
22.2 11.1
14.3
15.9
16.7
5.1
3
2
3
2
5
1
<1.6 toxic
<1.6 toxic
<1.7 toxic
<1.7 toxic
5.6
8.0
3.1
4
5d
6
5.9
2.0 µMe (SI ) 3)
15.4 14.4
21.9 22% @ 2.7 µM
Nt40 0.06 µM
Nt40 0.36 µM
Nt40 0.31 µM
Nt40 0.03 µM
delavirdine mesylate Nt40f 0.02
3TC
calanolide A
nelfinavir
Nt40
Nt40
Nt40
1.20
0.07
0.09
a
b
CC50 ) concentration mediating 50% cytotoxicity. IC50
)
concentration mediating 50% antiviral response. c SI ) selectivity
d
index. Betulinic acid was isolated previously from Vatica cinerea
King.7 e ED50, 50% cytoprotective activity against HIV-1IIIB cyto-
pathicity in vitro in µM (SI). f Nt40 ) nontoxic @ 40 µg/mL.
F igu r e 1. ORTEP drawing of the four rotamers of 1 showing two
independent units in the crystal structure (top: rotamers A1 and A2;
bottom: rotamers B1 and B2).
in the HOG.R5 reporter cell line at a concentration of 20
µg/mL with no observable toxicity (100% cell viability).
Consequently, all major fractions and pure isolates from
the plant were assayed for anti-HIV activity and cytotox-
icity according to established protocols.5 Compounds iso-
lated as a result of this bioactivity-guided fractionation
strategy and their activities are listed in Table 4. Anti-
HIV IC50 values of 5-11 µM were obtained yielding SI
(Selectivity Index) values of 2-3, consistent with that
published for other 3-O-acyl derivatives of betulinic acid
such as acetyl, benzoyl, crotonyl, sulfonyl, and succinyl.6
The isolates also exhibited toxicity levels in the HOG.R5
cell line (CC50 ) 13-22 µM) that are in agreement with
literature values for similar compounds tested in H9
lymphocytes.
Data of the parent compound, betulinic acid (5), was also
included for comparison purposes. Betulinic acid (5) was
reported to have an SI value of 9.3 (CC50 13 µM/IC50 1.4
µM) in H9 lymphocytes.6 Previously, bioassay-guided frac-
tionation led us to isolate betulinic acid from Vatica cinerea
King.7 The compound had an SI value of 5 (CC50 16.6 µM/
IC50 3.1 µM) in the HOG.R5 assay. In addition, several
standard anti-HIV agents were also tested in the HOG.R5
system for quality control purposes. The IC50 values of
these positive control compounds were observed to be very
similar to that reported in the literature using either H9
or CEM-SS cells (Table 4).
Ta ble 3. Torsional Angles along the C1′-C2′ and the C3′-C4′
Single Bonds of the Four Rotamers of 1 in Its Crystal Structure
torsional angle
A1
-2.02° -12.60°
177.36° 165.28°
145.73° -21.99°
147.39° -21.25°
A2
B1
B2
7.18°
O3-C1′-C2′-C3′
O1′-C1′-C2′-C3′
167.44°
-8.93° -171.64°
C2′-C3′-C4′-C5′ -32.62°
-20.35°
153.69°
C2′-C3′-C4′-C9′
158.02°
the 1D and 2D NMR spectra finally defined 3 as 3â-O-cis-
feruloylbetulinic acid.
Compound 3 was obtained as colorless needles and was
evaluated to be the known 3â-O-trans-coumaroylbetulinic
1
acid.3 As H NMR assignments for 3 are incomplete, and
since 13C NMR, melting point, and optical rotation data
are not available, they are presented in the Experimental
Section and in Tables 1 and 2.
Compound 4 was obtained as a white powder and was
shown to have a molecular formula of C39H54O5 according
to HRESIMS, suggesting it to be an isomer of 3 and an
analogue of 1 and 2. A comparison of the NMR spectral
data (Tables 1 and 2) of 4 with those of 1, 2, and 3
confirmed this observation and showed a cis/trans relation-
ship between 4 and 3. This led to the conclusion that 4
was 3â-O-cis-coumaroylbetulinic acid.
1H NMR and reversed-phase HPLC have shown that
under normal storage conditions at room temperature and
protection from light the trans-isomers 1 and 3 are stable,
while the cis-compounds 2 and 4 slowly underwent cis-
trans isomerization. Samples of 2 and 4 were initially
obtained in a pure state as confirmed by 1H NMR and
HPLC. Two and a half years of storage resulted in a
mixture of 2 parts cis- and 1 part of the corresponding
All pure compounds obtained were further tested in the
CEM-SS/MTS cytoprotection assay utilizing HIV-1IIIB (2 ng
p24/mL and MOI ) 0.4). Reagents required for this virus-
induced cytopathic effect (CPE)-inhibition assay were
provided by the CellTiter 96 AQueous One Solution Cell
Proliferation Assay kit (Promega Corporation, Madison,
WI). The procedure measures CEM-SS cell viability and
is based on the reduction of the tetrazolium dye, 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium (MTS), by mitochondrial enzymes
of viable host cells into MTS formazan.8
In all cases (except for ursolic acid, 6), the compounds
isolated were found to be more toxic to CEM-SS cells than
to HOG.R5. Minimal (<20%) cytoprotection was observed
for all compounds tested at nontoxic concentrations. The
exceedingly toxic nature of the betulinic acid derivatives
precluded the accurate assessment of their protective
effects against virus-induced cytopathogenicity in CEM-
SS cells. On the other hand, the antiproliferative effect of
betulinic acid (5) on normal cells such as peripheral blood
mononuclear cells (PBMCs) is reported to be mild.9 There-
1
trans-isomer when the compounds were examined by H
NMR. It has also been observed that pyridine, which was
used as solvent for NMR analyses, increased this rate of
conversion. Compound 2 showed a conversion rate of 50%
in approximately 3 months when stored in pyridine-d5.
However, the trans-isomers 1 and 3 remained pure with
prolonged storage, regardless of whether they were stored
dry or in pyridine-d5.
Ursolic acid (6) was obtained as a white powder, and its
identity was deduced by comparison of its spectroscopic
data with literature information.4
During the initial screen, the total methanol extract of
Strychnos vanprukii completely inhibited HIV-1 replication

3-O-Acyl Betulinic Acids from Strychnos vanprukii
J ournal of Natural Products, 2004, Vol. 67, No. 6 997
Ta ble 5. Cytotoxic Activities of Compounds 1-5 in Cultured
the herbaria of CPNP, Institute of Ecology and Biological
Resources (National Center for Science and Technology, Ha-
noi), and the Field Museum of Natural History (Chicago, IL).
An ti-HIV Assa y in HOG.R5 Cells. Anti-HIV and cytotox-
icity assays were performed in parallel utilizing the green
fluorescent protein (GFP)-based HOG‚R5 reporter cell line that
was constructed and developed specifically for quantitating
HIV-1 infectivity. The system was validated and adapted as a
moderately high-throughput procedure for screening natural
products for anti-HIV activity in our laboratory.5 Briefly,
cultures in microtiter wells were infected with HIV-1IIIB (2.5
ng/mL p24) in the presence of plant extracts, after which the
fluorescence output was measured at the end of 4 days. Virus
was omitted from parallel cultures treated with identical
concentrations of plant extracts in order to monitor changes
in cellular viability by a combination of microscopic and
fluorometric measurements.
Cellsa,b
compound
Lu1 Col-2 KB LNCaP HUVEC Mel2
1
2
3
4
8.5
15.7
12.1
11.0
5.4
7.4
6.6
6.0
2.5
5.4
5.5
4.3
3.2
5.2
5.7
3.2
27.4
9.5
6.2
5.3
6.8
28.7
3.8
3.8
4.2
3.8
1.3
betulinic acid (5) 40.6 43.9 35.7
a
b
Results are expressed as ED50 values (µM). Key to cell lines
used: Lu1 ) human lung cancer; Col-2 ) human colon cancer;
KB ) human oral epidermoid carcinoma; LNCaP ) human
hormone-dependent prostate cancer; HUVEC ) human umbilical
vein endothelial cell; Mel2 ) human melanoma.
fore, in the interest of clinical relevance, the compounds
are scheduled to be evaluated against HIV-1IIIB in phyto-
hemagglutinin (PHA)-stimulated PBMCs.
In h ibition of HIV-1-In d u ced Cytop a th ic Effect (CP E)
in CEM-SS Cells. CellTiter 96 AQueous One Solution Cell
Proliferation kit (Promega Corporation, Madison, WI) is
employed for the assessment of antiviral activity based on the
protective effect of compounds or extracts on HIV-induced
cytopathic effects and cell death.8 The CellTiter 96 AQueous One
Reagent contains a novel tetrazolium compound [3-(4,5-dim-
ethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-
nyl)-2H-tetrazolium, inner salt; MTS(b)] and an electron
coupling reagent (phenazine ethosulfate; PES). PES has
enhanced chemical stability, which allows it to be combined
with MTS to form a convenient “one solution” assay format.
On the day preceding the assay, cells are split 1:2 to ensure
they are in exponential growth phase at the time of infection.
On the day of the experiment cells are washed, pelleted, and
resuspended at (2-3) × 104 cells/mL in RPMI 1640 containing
10% heat-inactivated FBS, 4 mM L-glutamine, 100 units/mL
penicillin, and 100 µg/mL streptomycin. Extracts or compounds
are diluted in complete RPMI1640. Test agents (50 µL) are
added to cells (100 µL) in cell culture plates. Virus (50 µL) is
then added to test wells and virus controls. The amount of
virus to be added to each well will be the amount determined
to give complete cell killing at 6 days post infection, i.e.,
maximum CPE is observed in untreated virus control cultures.
Approximately 2 ng p24/mL virus (MOI ∼0.4) is routinely used.
Fresh medium is added to cell control wells, toxicity control
wells, and test agent colorimetric control wells. After 6 days
of incubation at 37 °C in a humidified 5% CO2 incubator, 40
µL of MTS/PES solution is added to each well and the plate is
reincubated for 4 h at 37 °C. The final culture concentrations
of MTS and PES are 317 µg/mL and 50 µM, respectively. At
the end of the incubation period, adhesive plate sealers are
used to seal plates. Sealed plates are inverted several times
to mix the soluble MTS formazan product. The plate is read
spectrophotometrically at 490/650 nm with a PowerWave 200
plate reader (Bio-tek Instruments, Inc.) equipped with KC-
junior software for data acquisition and analysis. The optical
density (OD) value of each culture is a function of the amount
of formazan produced which is proportional to the number of
viable cells. Data computed include % cytoprotection (% CPE
reduction), % cell viabilty, IC50, and CC50. Further testing is
performed on active compounds using a five-concentration
dose-response format.
Numerous synthetic betulinic acid derivatives have been
produced in an attempt to enhance its activity.10 This has
resulted in compounds that have shown effects in virion
assembly or budding,11 or fusion with the host cell mem-
brane.12,13 These findings attest to the importance of
continued investigation of betulinic acid derivatives as new
antiviral agents with novel mechanisms of action.
The toxicity of these betulinic acid derivatives to CEM-
SS and HOG.R5 cells prompted us to extend the evaluation
to include a panel of six cancer cell lines (Lu1, Col-2, KB,
LNCaP, HUVEC, and Mel2). Assays were performed ac-
cording to protocols established in our laboratories.14,15
Cytotoxicity data for the six cancer cell lines tested are
presented in Table 5. At an ED50 value of 1.3 µM, betulinic
acid (5) demonstrated selective cytotoxicity against cul-
tured human melanoma cells consistent with data reported
in the literature.15 Cytotoxicity obtained with the other cell
lines was 20-30-fold less pronounced than that observed
with Mel2. Unlike their parent 5, compounds 1-4 were less
active against Mel2, demonstrating ED50 values of ap-
proximately 3.8 µM (Table 5). In addition, no selectivity
was apparent across the panel of cancer cell lines; only a
2-4-fold difference was observed between activity in Mel2
and the cytotoxic effect seen in the least susceptible cell
line, Lu1.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured with a Perkin-Elmer model 241 polarimeter.
IR spectra were recorded on a J asco FT/IR-410 spectrometer
as a film on a KBr plate. 1D and 2D NMR spectra were
recorded on a Bruker Avance DPX-300 MHz spectrometer.
Chemical shifts (δ) are expressed in ppm with reference to
internal TMS. All NMR experiments were obtained by using
standard pulse sequences supplied by the vendor. Column
chromatography was carried out on silica gel (230-400 mesh,
Whatman). Thin-layer chromatography was performed on
Whatman plates coated with 0.25 mm layers of silica gel 60.
High-resolution electrospray ionization mass spectra (HRES-
IMS) and MS/MS spectra were recorded on a Q-ToF-2 hybrid
quadrupole/time-of-flight mass spectrometer in the negative
mode. X-ray crystallographic data were collected on a Bruker
APEX CCD area detector at 100(1) K. Pure samples of
betulinic acid and trans-ferulic acid were obtained from
Indofine Chemical Company, Inc., Somerville, NJ , and Sigma-
Aldrich, St. Louis, MO, respectively.
Cytotoxicity Assa y. Compounds were evaluated for cyto-
toxicity against a panel of human cancer cell lines that
comprised Lu1, Col-2, LNCaP, HUVEC, Mel2, and KB cells.
Assays involving Lu1, Col-2, LNCaP, and KB cell lines utilized
established protocols,14,15 while HUVEC were propagated and
assayed in more specialized medium. HUVEC were purchased
and grown in media and components supplied in the EGM-2
BulletKit (Cambrex Bio Science Walkersville, Inc., MD) with
2% fetal bovine serum (FBS). The HUVEC line constitutes a
test system to identify samples with potential antiangiogenic
activity.
P la n t Ma ter ia l. The initial collection of leaf and twig
samples of Strychnos vanprukii Craib (Loganiaceae) was made
at the Cuc Phuong National Park (CPNP), with voucher
herbarium specimens represented by living collections in the
park. A larger amount of the plant sample (5.5 kg) was
subsequently re-collected at CPNP for complete isolation work.
Voucher specimens of both collections are on deposit at both
Extr a ction a n d Isola tion . The dried, milled leaves and
twigs (5.5 kg) of S. vanprukii were extracted with MeOH and
defatted with n-hexane. The methanolic solution was subse-
quently evaporated under vacuum to a syrup and partitioned

998 J ournal of Natural Products, 2004, Vol. 67, No. 6
Chien et al.
sequentially with petroleum ether, CHCl3, and MeOH. The
CHCl3 extract (24.4 g) was dissolved in MeOH, filtered, and
chromatographed over a silica gel column (1000 g), which was
developed by gradient elution with CHCl3 and increasing
concentrations of MeOH to afford 68 fractions. The fractions
were pooled into four well-resolved groups based on the
presence of antiviral activity. Group 1 (fractions 13-14, 663
mg) was chromatographed over a Sephadex LH-20 column and
eluted with MeOH to afford an active fraction (56.4 mg), which
was then subjected to repeated column chromatography over
silica gel and eluted with hexane/acetone (8:2) to yield com-
pound 2 (8.2 mg). Similar workup of group 2 (fractions 15-
18, 663 mg) led to compound 1 (39.8 mg). Group 3 (fractions
30-41, 966 mg), on treatment with CHCl3, gave a precipitate,
which was recrystallized from a mixture of CHCl3/MeOH to
afford ursolic acid (6) (19.1 mg). The CHCl3-soluble portion
was chromatographed as described above to afford compound
4 (30.4 mg). Group 4 (fractions 47-51, 2.486 g) was subjected
to Sephadex LH-20 chromatography and eluted with MeOH
to yield an active fraction (179.2 mg). This active fraction was
sequentially chromatographed over silica gel using CHCl3/
MeOH (9:1) and then rechromatographed using hexane/
acetone (7:3) to yield 3 (31.2 mg).
1.224 g/cm3, λ ) 0.71073 Å, µ(Mo KR) ) 0.080 mm-1, and
F(000) ) 2744. A hemisphere of 33 429 reflections yielded
12 097 unique, averaged observations to 2θ ) 56.08°; 5687
have intensities greater than 2σ. The structure was refined
2
by full-matrix least-squares on F with SHELXL18 using all
data to R ) 0.112, R(I > 2σI) ) 0.054, GOF ) 0.765. ORTEP
was used to generate Figure 1.19
Ack n ow led gm en t. Sample collections, taxonomic identi-
fication of samples, bioassay-guided chemical isolation, and
structure elucidation work presented in this paper were made
under a grant administered by the Fogarty International
Center, NIH (Grant 1 UO1-TW01015-01), as part of an
International Cooperative Biodiversity Group (ICBG) program.
Permits in the collection of samples of S. vanprukii and its
export from Vietnam were granted by the Ministry of Agri-
culture and Rural Development, Hanoi, and from the Cuc
Phuong National Park, Vietnam. The authors are grateful to
the staff of the Cuc Phuong National Park for assistance with
the re-collection of S. vanprukii samples used in the present
study. The authors also wish to acknowledge Dr. D. Nicolic,
Mass Spectrometry Lab, Department of Medicinal Chemistry,
College of Pharmacy, UIC, for the acquisition of the MS data,
and the AIDS Research and Reference Reagent Program,
NIAID, NIH, for the supply of reagents critical to this study.
3â-O-tr a n s-F er u loylbetu lin ic a cid (1): colorless needles
(hexane/acetone), mp 299-300 °C; [R]20D +27.0° (c 0.33, EtOH);
UV (EtOH) λmax (log ꢀ) 236 (4.20), 302 (sh) (4.28), 327 (4.45)
nm; IR νmax (film) 3531, 3393, 2845, 1694, 1627, 1514, 1268,
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallographic data
for compound 1. This material is available free of charge via the
Internet at http://pubs.acs.org.
1175, 756 cm-1 1H (pyridine-d5, 300 MHz) and 13C NMR
;
(pyridine-d5, 75 MHz) data, see Tables 1 and 2; HRESIMS m/z
631.3996 [M - H]- (calcd for C40H55O6, 631.3999); HRESIMS
(MS/MS mode, 50 eV) m/z 616.4283, 437.3713, 175.0618.
3â-O-cis-F er u loylbetu lin ic a cid (2): white powder (hex-
Refer en ces a n d Notes
(1) Soejarto, D. D.; Gyllenhaal, C.; Regalado, J . C.; Pezzuto, J . M.; Fong,
H. H. S.; Tan, G. T.; Hiep, N. T.; Xuan, L. T., Binh, D. Q.; Hung, N.
V.; Bich, T. Q.; Thin, N. N.; Loc, P. K.; Vu, B. M.; Southavong, B. H.;
Sydara, K.; Bouamanivong, S.; O’Neill, M. J .; Lewis, J .; Xie, X.;
Dietzman, G. Pharm. Biol. 1999, 37 (Suppl), 100-113.
(2) Sholichin, M.; Yamasaki, K.; Kasai, R.; Tanaka, O. Chem. Pharm.
Bull. 1980, 28, 1006-1008.
ane/acetone), mp 155-160 °C; [R]20 +16.1° (c 0.37, EtOH);
D
UV (EtOH) λmax (log ꢀ) 232 (3.91), 304 (sh) (3.97), 325 (4.06)
nm; IR νmax (film) 3533, 3414, 2942, 1695, 1595, 1514, 1173,
980, 758 cm-1 1H (pyridine-d5, 300 MHz) and 13C NMR
;
(pyridine-d5, 75 MHz) data, see Tables 1 and 2; HRESIMS m/z
631.3975 [M - H]- (calcd for C40H55O6, 631.3999); HRESIMS
(MS/MS mode, 50 eV) m/z 616.4181, 437.3713, 175.0564.
3â-O-tr a n s-Cou m ar oylbetu lin ic acid (3): colorless needles
(hexane/acetone), mp 272-274 °C; [R]20D +32.4° (c 0.28, EtOH);
UV (EtOH) λmax (log ꢀ) 231 (4.03), 303 (sh) (4.28), 314 (4.34)
nm; IR νmax (film) 3333, 2945, 1693, 1605, 1515, 1274, 1169,
(3) Barba, B.; Diaz, J . G.; Herz, W. Phytochemistry 1994, 37, 837-845.
(4) Lee, J . S.; Kim, B. Y.; Lee, H. S.; Ahn, J . S.; Chang Y. S. J . Nat.
Prod. 2000, 63, 753-756.
(5) Hoang, V. D.; Tan, G. T.; Zhang, H. J .; Tamez, P. A.; Hung, N. V.;
Cuong, N. M.; Soejarto, D. D.; Fong, H. H. S.; Pezzuto, J . M.
Phytochemistry 2002, 59, 325-29.
(6) Fujioka, T.; Kashiwada. Y.; Kilkuskie, R. E.; Cosentino, L. M.; Ballas,
L. M.; J iang, J . B.; J anzen, W. P.; Chen, I. S.; Lee, K. H. J . Nat. Prod.
1994, 57, 243-247.
1
831, 757; H (pyridine-d5, 300 MHz) and 13C NMR (pyridine-
(7) Zhang, H. J .; Tan, G. T.; Hoang, V. D.; Hung, N. V.; Cuong, N. M.;
Soejarto, D. D.; Pezzuto, J . M.; Fong, H. H. S. J . Nat. Prod. 2003, 66,
263-268.
d5, 75 MHz) data, see Tables 1 and 2; HRESIMS m/z 601.3827
[M - H]- (calcd for C39H53O5, 601.3893); HRESIMS (MS/MS
mode, 50 eV) m/z 437.3473, 145.0328.
(8) Weislow, O. S.; Kiser, R.; Fine D. L.; Bader, J .; Shoemaker, R. H.;
Boyd, M. R. J . Natl. Cancer Inst. 1989, 8, 577-586.
(9) Zuco, V.; Supino, R.; Righetti, S. C.; Cleris, L.; Marchesi, E.;
Gambacorti-Passerini, C.; Formelli, F. Cancer Lett. 2002, 175, 17-
25.
3â-O-cis-Cou m a r oylbetu lin ic a cid (4): white powder
(hexane/acetone), mp 180-182 °C; [R]20D +18.0° (c 0.65, EtOH);
UV (EtOH) λmax (log ꢀ) 231 (3.93), 302 (sh) (4.18), 312 (4.22)
nm; IR νmax (film) 3352, 3041, 2817, 1693, 1605, 1514, 1168,
(10) Baglin, I.; Mitaine-Offer, A. C.; Nour, M.; Tan, K.; Cave, C.; Lacaille-
Dubois, M. A. Mini Rev. Med. Chem. 2003, 3, 525-539.
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M.; Goto, T.; Sano, K.; Nakashima, H. Antimicrob. Agents Chemother.
2001, 45, 1225-1230.
(12) Mayaux, J . F.; Bousseau, A.; Pauwels, R.; Huet, T.; Henin, Y.; Dereu,
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(14) Likhitwitayawuid, K.; Angerhofer, C. K.; Cordell, G. A.; Pezzuto, J .
M.; Ruangrungsi, N. J . Nat. Prod. 1993, 56, 30-38.
(15) Pisha, E.; Chai, H.; Lee, I.; Chagwedera, T. E.; Farnsworth, N. R.;
Cordell, G. A.; Beecher, C. W. W.; Fong, H. H. S.; Kinghorn, A. D.;
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(16) Farrugia, L. J . J . Appl. Crystallogr. 1999, 32, 837-838.
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979, 758 cm-1 1H (pyridine-d5, 300 MHz) and 13C NMR
;
(pyridine-d5, 75 MHz) data, see Tables 1 and 2; HRESIMS m/z
601.3901 [M - H]- (calcd for C39H53O5, 601.3893); HRESIMS
(MS/MS mode, 50 eV) m/z 437.3472, 145.0328.
Alk a lin e Hyd r olysis of 1. A sample of 1 (9.1 mg) in 10%
methanolic KOH (3 mL) and MeOH (7 mL) was refluxed for 3
h, the solvent evaporated under vacuum, and the residue
neutralized and acidified with 1 N H2SO4 and extracted with
EtOAc (10 mL × 3). The extract was evaporated to dryness
and chromatographed on a silica gel column, eluted with
CHCl3/MeOH (95:5) to yield betulinic acid (5) (3.1 mg) and
trans-ferulic acid (1.8 mg).
X- r a y Cr ysta l Str u ctu r e of 3â-O-tr a n s-F er u loylbetu -
lin ic Acid (1). A colorless crystal with dimensions 0.2 × 0.2
× 0.2 mm was selected for X-ray analysis. Structure analysis
was performed using WinGX,16 and the non-hydrogen atoms
were found using the SIR-92 package.17 Compound 1 crystal-
lized in space group P212121 (No. 19) with a ) 11.332(2) Å, b
) 14.578(3) Å, c ) 41.500(7) Å, V ) 6856(2) Å3, Z ) 8, Dcalc
)
NP030469I