Antimycobacterial activity of cinnamate-based esters of the triterpenes betulinic, oleanolic and ursolic acids

Article abstract of DOI:10.1248/cpb.56.194

Betulinic acid, oleanolic acid and ursolic acid have been modified at the C-3 position to cinnamate-based esters and in vitro antimycobacterial activity against Mycobacterium tuberculosis H₃₇Ra has been determined. The results indicated that mo

Full text of DOI:10.1248/cpb.56.194

194
Notes
Chem. Pharm. Bull. 56(2) 194—198 (2008)
Vol. 56, No. 2
Antimycobacterial Activity of Cinnamate-Based Esters of the Triterpenes
Betulinic, Oleanolic and Ursolic Acids
Tanud TANACHATCHAIRATANA,a John Barnard BREMNER,b Ratchanaporn CHOKCHAISIRI,a and
,a
*
Apichart SUKSAMRARN
a Department of Chemistry, Faculty of Science, Ramkhamhaeng University; Bangkapi, Bangkok 10240, Thailand: and
b School of Chemistry, University of Wollongong; Wollongong, NSW 2522, Australia.
Received August 3, 2007; accepted November 9, 2007; published online November 16, 2007
Betulinic acid, oleanolic acid and ursolic acid have been modified at the C-3 position to cinnamate-based es-
ters and in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra has been determined. The
results indicated that modification of the parent structures of betulinic acid, oleanolic acid and ursolic acid to the
p-coumarate and, in the case of the latter two triterpenes, the ferulate ester analogues resulted in high antimy-
cobacterial activity. Structure–activity relationships within the lupane, oleanane and ursane analogues and be-
tween these triterpenes are discussed.
Key words triterpene ester; antimycobacterial activity; structure–activity relationship; betulinic acid; oleanolic acid; ursolic
acid
Tuberculosis is the leading cause of mortality among all of interest to investigate whether such an ester moiety would
infectious diseases worldwide and is responsible for two mil- also increase antimycobacterial activity with respect to the
lion deaths annually.1) The situation has recently been com- triterpenes 1—3. In order to study the relationships between
plicated by the human immunodeficiency virus (HIV) pan- the structure of the ester moiety and antimycobacterial activ-
demic and the increased prevalence of multi-drug resistant ity in triterpenes, other substituted cinnamate ester analogues
strains of Mycobacterium tuberculosis.2,3) The recent in- were also prepared for biological evaluation. This present
crease in the number of multi-drug resistant clinical isolates paper describes the in vitro antimycobacterial activity of the
of M. tuberculosis has created an urgent need for the evolu- triterpenes 1—3 and their C-3 substituted cinnamate ester
tion of new antituberculosis therapeutics.4,5) The structural analogues.
modification of natural products is one potential strategy for
The chemical modifications described in this paper were
the development of new antitubercular drugs which are dif- focused on the introduction of the cinnamoyl and substituted
ferent from the drugs currently used. In this context, triter- cinnamoyl groups at the C-3 hydroxyl group of betulinic acid
penes and derivatives are of particular interest.
(1), oleanolic acid (2) and ursolic acid (3). The ester moieties
Triterpenes exist abundantly in the plant kingdom. This selected for this study were the unsubstituted cinnamate, p-
class of natural products and their derivatives have been re- coumarate, the acetate and methyl ether derivatives of the p-
ported to have interesting biological activities, such as anti- coumarate esters, the ferulate (4-hydroxy-3-methoxycinna-
HIV,6,7) antiplasmodial8,9) and cytotoxic activities.10,11) Previ- mate) esters and their acetate derivatives, the caffeate (3,4-di-
ous investigations have shown that triterpenes also exhibit hydroxycinnamate) esters and their acetate derivatives, and
moderate to high in vitro antimycobacterial activity against the p-chlorocinnamate esters. The last group of esters were
M. tuberculosis.12—14) It was reported12) that betulinic acid (1) selected as representatives of non-oxygenated substituted
was more active than its C-3 epimer, epi-betulinic acid. cinnamate esters. The esters were obtained by treatment
Oleanolic acid (2) was 4-fold less active than its C-3 epimer, of the triterpenes 1—3 with the appropriate acid chloride
3-epioleanolic acid12,15) whereas the acetate derivative of 2 in the presence of 4-N,N-dimethylaminopyridine (DMAP).
was as active as its parent compound 2.16) The 3-keto ana- Treatment of 1 with cinnamoyl, p-acetoxycinnamoyl, p-
logue, oleanonic acid, exhibited an equivalent MIC to that of methoxycinnamoyl, 4-O-acetylferuloyl, 3,4-di-O-acetylcaf-
3-epi-oleanolic acid.15) The presence of an ester function at feoyl, and p-chlorocinnamoyl chlorides (the latter five acid
C-22 of 2 resulted in a 2-fold decrease in activity.17) Addition chlorides being prepared by reaction of the corresponding
of the hydroxylic functionality at the C-19, and C-2 and C-19 carboxylic acids with oxalyl chloride) in benzene in the pres-
positions of ursolic acid (3) to give pomolic acid and tormen- ence of DMAP furnished the cinnamate esters 4—9 in 59—
tic acid, resulted in an equivalent or a 2-fold decrease in an- 97% yields (Chart 1). Treatment of 2 with the foregoing acid
timycobacterial activity, respectively.12,15) The C-3 epimer of chlorides gave the corresponding cinnamate esters 10—15 in
the triterpene 3, 3-epi-ursolic acid, exhibited relatively high 62—81% yields (Chart 2). The cinnamate esters 16—21
activity.18) Modifications of triterpene rings also affected were similarly prepared from 3 in 60—87% yields (Chart 2).
their antimycobacterial activity.19,20) The results observed Deacetylation of the p-acetoxycinnamate esters 5, 11 and 17,
among the various groups of triterpenes are rather conflict- the 4-O-acetylferulate esters 7, 13 and 19, and the 3,4-di-O-
ing, thus making it difficult to predict structural requirements acetylcaffeate esters 8, 14 and 20 with 10% aqueous K2CO3
for antimycobacterial activity. We have discovered, however, afforded the corresponding p-coumarate esters 5a, 11a and
that the presence of a p-coumarate moiety at the C-2 hy- 17a, the ferulate esters 7a, 13a and 19a, and the caffeate es-
droxyl group of alphitolic acid (2a-hydroxybetulinic acid) ters 8a, 14a and 20a in 67—96% yields. All synthetic com-
resulted in increased antimycobacterial activity.21) It was thus pounds were purified by column chromatography and their
∗ To whom correspondence should be addressed. e-mail: apichart@ram1.ru.ac.th
© 2008 Pharmaceutical Society of Japan

February 2008
195
Table 1. Antimycobacterial Activity (in Vitro) of the Triterpenes 1—3 and
Their Cinnamate-Based Ester Analogues
Triterpene group
Compound
MICa,b) (mg/ml)
Betulinic acid group
1
4
5
5a
6
50
ꢀ200
12.5
6.25
ꢀ200
200
7
7a
8
200
200
8a
9
200
ꢀ200
Oleanolic acid group
2
10
50
50
Chart 1. Synthesis of Cinnamate-Based Esters of Betulinic Acid (1)
11
11a
12
12.5
6.25
200
Reagents and conditions: (i) RCOCl/DMAP/benzene, 60 °C, 0.5—1 h; (ii) 10%
K2CO3/MeOH, 30 min.
13
50
13a
14
14a
15
12.5
12.5
200
ꢀ200
Ursolic acid group
3
16
17
12.5
ꢀ200
25
17a
18
6.25
100
19
19a
20
3.13
3.13
100
20a
21
200
ꢀ200
a) MIC at ꢀ200 mg/ml is regarded as inactive. b) The standard drugs, kanamycin
sulfate, isoniazid and rifampicin, showed MIC values of 2.5, 0.06 and 0.004 mg/ml, re-
spectively.
ester analogue 7a and its acetate derivative 7 as well as the
caffeate ester analogue 8a and its acetate derivative 8 were
only weakly active (MICs 200 mg/ml).
Chart 2. Synthesis of Cinnamate-Based Esters of Oleanolic Acid (2) and
Ursolic Acid (3)
In the oleanolic acid (2) group, almost all compounds ex-
hibited antimycobacterial activity, including the cinnamate
ester 10 which exhibited equal activity (MIC 50 mg/ml) to
Reagents and conditions: (i) RCOCl/DMAP/benzene, 60 °C, 0.5—1 h; (ii) 10%
K2CO3/MeOH, 30 min.
1
structures were established by spectroscopic (IR, H-NMR that of the parent triterpene 2. The p-coumarate ester 11a
and MS) analysis (see Experimental).
showed the highest activity of the group, with an MIC of
All cinnamate esters of betulinic acid (1), oleanolic acid 6.25 mg/ml. The corresponding acetate 11 was 2-fold less ac-
(2) and ursolic acid (3) were screened for their in vitro an- tive than the parent p-hydroxy analogue 11a. Methylation of
timycobacterial activity against M. tuberculosis H37Ra by the the parent compound 11a to the corresponding methyl ether
Microplate Alamar Blue Assay (MABA)22) for the determi- analogue 12 resulted in sharp decrease in antimycobacterial
nation of MIC, which is defined as the minimum concentra- activity. The p-chlorocinnamoyl analogue 15 was the only in-
tion of compound required to exhibit 90% inhibition of active analogue of the group. The ferulate ester analogue 13a
bacterial growth; MICs of the compounds are reported in was 2-fold less active than the p-coumarate ester 11a, the
Table 1. The known active antimycobacterial compounds most active analogue of the oleanane group. The 4-O-acetyl
kanamycin sulfate, isoniazid and rifampicin were included in analogue 13 was 4-fold less active than the ferulate ester 13a.
the assays as controls and reference points. Their MIC In contrast to the ferulate ester 13a and its acetate derivative
values are 2.5, 0.06 and 0.004 mg/ml (4.29, 0.44, 0.005 mM), 13, the acetate derivative of the caffeate ester (compound 14)
respectively.
Betulinic acid (1) exhibited low antimycobacterial activity pound 14a was only weakly active (MIC 200 mg/ml).
with an MIC of 50 mg/ml. The cinnamoyl, p-methoxycin- In the ursolic acid (3) group, the parent compound 3 ex-
was as active as the ferulate ester 13a, while the parent com-
namoyl and p-chlorocinnamoyl analogues 4, 6 and 9 were in- hibited antimycobacterial activity (MIC 12.5 mg/ml), whereas
active in this assay. The p-coumaroyl analogue 5a, on the its cinnamoyl and p-chlorocinnamoyl analogues 16 and 21
other hand, showed very good activity with an MIC of were inactive. The p-coumarate ester 17a was highly active,
6.25 mg/ml. The corresponding acetate derivative 5 was 2- with an MIC value of 6.25 mg/ml. The corresponding acetate
fold less active than its parent compound 5a. The ferulate and methyl ether derivatives, 17 and 18, showed lower activ-

196
Vol. 56, No. 2
ity with MICs of 25 and 100 mg/ml respectively. Both the fer- antimycobacterial drug, although an esterase-resistant ester
ulate ester 19a and its acetate 19 were highly active with bioisosteric replacement would probably need to be intro-
equal MICs of 3.13 mg/ml. However, the corresponding caf- duced. The presence of the ferulate moiety resulted in a 4-
feate ester 20a and its acetate 20 were much less active, with fold increase in activity of compound 2. In contrast to this,
respective MICs of 200 and 100 mg/ml.
the introduction of the ferulate ester in the triterpene 1
The results indicated that the presence of unsubstituted caused a 4-fold decrease in antimycobacterial activity. In all
cinnamate ester functionality in the lupane and ursane triter- three triterpene groups, the p-chloro group decreased the ac-
penes (1, 3 respectively) lead to inactive compounds, while tivity of the cinnamate esters. The unsubstituted cinnamate
no improvement in the antimycobacterial activity was seen and its p-methoxyl and p-chloro analogues decreased the ac-
with respect to the oleanane triterpene parent (2). Acetyla- tivity. Within the cinnamate esters tested, the p-hydroxyl
tion of the hydroxyl group of the p-coumarate moiety re- group contributed to high antimycobacterial activity, while
sulted in a 2- to 4-fold decrease in activity whereas methyla- methylation and acetylation of the phenolic hydroxyl group,
tion of the p-coumarate hydroxyl group led to a sharp de- with the exception of the caffeate esters, decreased activity.
crease in activity. The presence of the p-chloro group re-
Experimental
sulted in inactivity or did not improve antimycobacterial ac-
tivity of the triterpene esters. The presence of an extra
General Procedures Melting points were determined using a Gal-
lenkamp melting point apparatus and were uncorrected. IR spectra were
methoxyl group at the 3-position of the p-coumarate moiety
to yield the ferulate ester gave rise to varying effects: almost
complete loss of activity was observed in the betulinic acid
group, whereas a 2-fold decrease and increase in activity
were noted for the oleanolic acid and ursolic acid group, re-
spectively. The ferulate analogue 19a and its acetate 19
showed very high antimycobacterial activity, both with MICs
of 3.13 mg/ml (4.64, 4.95 mM respectively). Their MIC values
were comparable to the reference drug kanamycin sulfate
(MIC 4.29 mM), though isoniazid and rifampicin (MIC 0.44,
0.005 mM, respectively) are considerably more active.
recorded in KBr on a Perkin-Elmer FT-IR Spectrum BX spectrophotometer.
1H-NMR spectra were recorded on a Bruker AVANCE 400 spectrometer.
Mass spectra were obtained using a Finnigan LC-Q mass spectrometer. High
resolution mass spectra were obtained using a Finnigan MAT 90, a Bruker
micrOTOF and a Micromass Q-Tof 2 mass spectrometer. Column chro-
matography and TLC were carried out using Merck silica gel 60 (ꢁ0.063
mm) and precoated silica gel 60 F254 plates, respectively. Betulinic acid (1)
was isolated from Ziziphus cambodiana21) whereas oleanolic acid (2) and ur-
solic acid (3) were purchased from Sigma-Aldrich, Inc. Unless indicated
otherwise, TLC of the triterpenes and analogues was run with CH2Cl2–
MeOH (80 : 1) as developing solvent and compounds were detected under
UV light (254 nm) and by spraying with anisaldehyde–H2SO4 reagent fol-
lowed by heating. Solvent ratios were all v/v. All purified synthetic ana-
1
logues were homogeneous on TLC, and H-NMR spectra indicated ꢀ95%
Among the three triterpene groups, ursolic acid (3) was
the most active compound; it was 4-fold more active than
both compounds 1 and 2. The p-coumarate esters of each of
these parents (compounds 5a, 11a, 17a) exhibited the same
antimycobacterial activity (MICs 6.25 mg/ml). The ferulate
ester 19a of ursolic acid (3) was 4-fold more active than the
ferulate ester 13a of oleanolic acid (2). Rather surprisingly,
the ferulate ester 7a of betulinic acid (1) was only weakly ac-
tive (MIC 200 mg/ml). The weak activity of the ferulate ester
of betulinic acid was, in fact, not unusual, since it has also
been observed in other biological evaluations. For example, it
has been reported6) that winchic acid (22), the C-27 ferulate
ester analogue of betulinic acid (1), was inactive in an anti-
plasmodial evaluation, while messagenic acid B (23), the C-
27 p-coumarate ester analogue of betulinic acid (1), was
moderately active (IC50 3.8 mg/ml) in this assay. It is also
worth noting that, while having a 3,4-dioxygenated cin-
namoyl moiety as in the ferulate ester analogues 19a and 19,
the caffeate ester analogues 20a and 20 were much less ac-
tive. This finding implied that the methyl ether function at
the 3-position of the ferulate moiety contributed to high
antimycobacterial activity in the ursolic acid group.
The results indicated that introduction of the p-coumarate
moiety at the C-3 position of betulinic acid (1), oleanolic
acid (2) and ursolic acid (3) resulted in an 8-fold increase in
antimycobacterial activity of the parent triterpenes 1 and 2,
and a 2-fold increase in this activity of the triterpene 3. Intro-
duction of a methoxyl group at the 3-position of the p-
coumarate moiety to give the ferulate moiety caused further
2-fold increase in activity in the ursane group. Thus the pres-
ence of the ferulate ester group at the 3-hydroxyl position of
ursolic acid (3) to afford 19a resulted in a 4-fold increase in
antimycobacterial activity. The compound 19a constitutes an
interesting potential lead for the development of a new
purity. The Rf values for compounds 1, 2 and 3 are 0.28, 0.20 and 0.24, re-
spectively. References to the literature are given for known synthesized com-
pounds.
Procedure for the Preparation of Cinnamate Ester Analogues of
Triterpenes To a stirred triterpene (0.03 mmol) solution in dry benzene
(2 ml) was added 4—8 eq of the cinnamic acid chloride and 4—5 eq of
DMAP and the solution was stirred at 60 °C for 0.5—1 h. Except for the
commercially available cinnamoyl chloride, all the other acid chlorides were
prepared by reacting the corresponding carboxylic acids with oxalyl chloride
at reflux for 2 h; the remaining oxalyl chloride was distilled off and the
residue was dissolved in dry benzene (1 ml). After the completion of the es-
terification, water was added and the solution mixture was extracted three
times with EtOAc. The combined organic layer was washed with water, and
then dried over anhydrous Na2SO4. The solvent was then evaporated and
the residue chromatographed on a column by elution with CH2Cl2–MeOH
(80 : 2).
3-O-(E)-Cinnamoylbetulinic Acid (4): 97% yield from compound 1;
amorphous solid from MeOH, mp ꢀ300 °C (lit.23) 320 °C); Rfꢂ0.54; IR
(KBr) cmꢃ1: 3247, 2941, 2869, 1725, 1696, 1450, 1296, 1191, 1005, 982;
1H-NMR data (CDCl3) were consistent with the reported values23); ES-MS
m/z: 609 [MꢄNa]ꢄ.
3-O-(E)-Cinnamoyloleanolic Acid (10): 72% yield from compound 2;
amorphous solid from MeOH, mp 251—253 °C; Rfꢂ0.42; IR (KBr) cmꢃ1
:
3614, 2945, 2870, 1713, 1693, 1462, 1450, 1365, 1305, 1280, 1203, 1173,
1144; 1H-NMR (400 MHz, CDCl3ꢄ4 drops CD3OD) d: 0.76, 0.90, 0.90,
0.91, 0.92, 0.96, 1.13 (each 3H, each s, CH3), 2.80 (1H, br d, Jꢂ10.2 Hz, H-
18), 4.63 (1H, dd, Jꢂ8.5, 7.4 Hz, H-3), 5.27 (1H, br s, H-12), 6.43 (1H, d,
Jꢂ16.0 Hz, H-2ꢅ), 7.36 (3H, br s, H-3ꢆ—H-5ꢆ), 7.51 (2H, br s, H-2ꢆ and H-
6ꢆ), 7.65 (1H, d, Jꢂ16.0 Hz, H-3ꢅ); ES-MS m/z: 609 [MꢄNa]ꢄ; HR-FAB-
MS (ꢃve) m/z: 585.3942 [MꢃH]ꢃ (Calcd for C39H54O4ꢃH: 585.3944).
3-O-(E)-Cinnamoylursolic Acid (16): 78% yield from compound 3; amor-
phous solid from MeOH, mp 255—257 °C (lit.23) 258 °C); Rfꢂ0.50; IR
(KBr) cmꢃ1: 3500, 2971, 2926, 1709, 1693, 1638, 1450, 1303, 1280, 1204,
1
1172, 1002, 767; H-NMR data (CDCl3) were consistent with the reported
values23); ES-MS m/z: 609 [MꢄNa]ꢄ.
3-O-(E)-p-Acetoxycinnamoylbetulinic Acid (5): 61% yield from com-
pound 1; amorphous solid from MeOH, mp 141—143 °C; Rfꢂ0.48; IR
(KBr) cmꢃ1: 3301, 2946, 1734, 1700, 1636, 1508, 1458, 1374, 1246, 1201,
1166, 1014, 980; 1H-NMR (400 MHz, CDCl3) d: 0.81, 0.82, 0.86, 0.91, 0.95
(each 3H, each s, CH3), 1.67 (3H, s, CH3-30), 2.29 (3H, s, OAc), 2.98 (1H,

February 2008
197
m, H-19), 4.45 (1H, m, H-3), 4.59 (1H, br s, H-29a), 4.72 (1H, br s, H-29b),
6.37 (1H, d, Jꢂ16.0 Hz, H-2ꢅ), 7.10 (2H, d, Jꢂ8.0 Hz, H-3ꢆ and H-5ꢆ), 7.52
(2H, d, Jꢂ8.0 Hz, H-2ꢆ and H-6ꢆ), 7.61 (1H, d, Jꢂ16.0 Hz, H-3ꢅ); ES-MS
m/z: 667 [MꢄNa]ꢄ; HR-ES-TOF-MS (ꢃve): m/z 643.3987 [MꢃH]ꢃ (Calcd
for C41H56O6-H: 643.4004).
H-2ꢅ), 7.18 (1H, d, Jꢂ8.4 Hz, H-5ꢆ), 7.34 (1H, d, Jꢂ1.8 Hz, H-2ꢆ), 7.38 (1H,
dd, Jꢂ8.4, 1.8 Hz, H-6ꢆ), 7.56 (1H, d, Jꢂ16.0 Hz, H-3ꢅ); HR-ES-TOF-MS
(ꢄve) m/z: 725.4029 [MꢄNa]ꢄ (Calcd for C43H58O8ꢄNa: 725.4023).
3-O-(E)-(3,4-Di-O-acetylcaffeoyl)oleanolic Acid (14): 62% yield from
compound 2; powder from MeOH, mp 211—213 °C; Rfꢂ0.31; IR (KBr)
cmꢃ1: 3429, 2944, 1780, 1695, 1640, 1505, 1462, 1367, 1204, 1179, 1110,
1028, 997; 1H-NMR (400 MHz, CDCl3) d: 0.77, 0.89, 0.89, 0.91, 0.92, 0.95,
1.13 (each 3H, each s, CH3), 2.27 (3H, s, OAc), 2.28 (3H, s, OAc), 2.81 (1H,
br d, Jꢂ10.1 Hz, H-18), 4.61 (1H, dd, Jꢂ8.6, 7.4 Hz, H-3), 5.28 (1H, br s, H-
12), 6.36 (1H, d, Jꢂ16.0 Hz, H-2ꢅ), 7.19 (1H, d, Jꢂ8.4 Hz, H-5ꢆ), 7.34 (1H,
d, Jꢂ1.8 Hz, H-2ꢆ), 7.38 (1H, dd, Jꢂ8.4, 1.8 Hz, H-6ꢆ), 7.57 (1H, d,
Jꢂ16.0 Hz, H-3ꢅ); HR-ES-TOF-MS (ꢄve) m/z: 725.4030 [MꢄNa]ꢄ (Calcd
for C43H58O8ꢄNa: 725.4023).
3-O-(E)-(3,4-Di-O-acetylcaffeoyl)ursolic Acid (20): 60% yield from com-
pound 3; amorphous solid; Rfꢂ0.40; IR (KBr) cmꢃ1: 3452, 2930, 1777,
1693, 1636, 1503, 1457, 1372, 1205, 1111, 1045, 1017, 902; 1H-NMR
(400 MHz, CDCl3) d: 0.80, 0.88, 0.90, 0.96, 1.07 (each 3H, each s, CH3),
0.87 (3H, d, Jꢂ6.1 Hz, H-29), 0.95 (3H, d, partially obscured signal, H-30),
2.14 (1H, br d, Jꢂ11.6 Hz, H-18), 2.28 (2ꢇ3H, s, 2ꢇOAc), 4.61 (1H, dd,
Jꢂ8.9, 6.9 Hz, H-3), 5.32 (1H, m, H-12), 6.36 (1H, d, Jꢂ16.0 Hz, H-2ꢅ),
7.20 (1H, d, Jꢂ8.4 Hz, H-5ꢆ), 7.36 (1H, br s, H-2ꢆ), 7.38 (1H, dd, Jꢂ8.4,
1.9 Hz, H-6ꢆ), 7.57 (1H, d, Jꢂ16.0 Hz, H-3ꢅ); HR-ES-TOF-MS (ꢄve) m/z:
725.4029 [MꢄNa]ꢄ (Calcd for C43H58O8ꢄNa: 725.4023).
3-O-(E)-p-Acetoxycinnamoyloleanolic Acid (11): 62% yield from com-
pound 2; needles from MeOH, mp 269—271 °C; Rfꢂ0.36; IR (KBr) cmꢃ1
:
3289, 2945, 2855, 1769, 1700, 1634, 1507, 1459, 1368, 1307, 1280, 1203,
1
1166, 1016, 912, 836; H-NMR data (CDCl3) were consistent with the re-
ported values24); ES-MS m/z: 667 [MꢄNa]ꢄ.
3-O-(E)-p-Acetoxycinnamoylursolic Acid (17): 87% yield from com-
pound 3; amorphous solid from MeOH, mp ꢀ300 °C; Rfꢂ0.42; IR (KBr)
cmꢃ1: 3420, 2926, 1769, 1691, 1678, 1508, 1462, 1371, 1313, 1277, 1248,
1
1205, 1167, 1017, 833; H-NMR data (CDCl3) were consistent with the re-
ported values25); ES-MS m/z: 667 [MꢄNa]ꢄ.
3-O-(E)-p-Methoxycinnamoylbetulinic Acid (6): 96% yield from com-
pound 1; amorphous solid from CH2Cl2–MeOH, mp ꢀ300 °C (lit.23)
320 °C); Rfꢂ0.55; IR (KBr) cmꢃ1: 3217, 2942, 1725, 1686, 1665, 1638,
1604, 1513, 1459, 1304, 1252, 1172, 1035, 977, 826; 1H-NMR data (CDCl3)
were consistent with the reported values23,26); ES-MS m/z: 615 [MꢃH]ꢃ.
3-O-(E)-p-Methoxycinnamoyloleanolic Acid (12): 81% yield from com-
pound 2; amorphous solid from CH2Cl2–MeOH, mp 292—293 °C; Rfꢂ0.45;
IR (KBr) cmꢃ1: 3356, 2930, 2854, 1707, 1636, 1604, 1513, 1451, 1253,
1170, 1020; 1H-NMR (400 MHz, CDCl3ꢄ4 drops CD3OD) d: 0.80, 0.89,
0.90, 0.92, 0.92, 0.94, 1.14 (each s, each 3H, CH3), 2.81 (1H, d, Jꢂ10.2 Hz,
H-18), 3.82 (3H, s, OCH3), 4.61 (1H, t, Jꢂ8.0 Hz, H-3), 5.30 (1H, br s, H-
12), 6.29 (1H, d, Jꢂ15.9 Hz, H-2ꢅ), 6.88 (2H, d, Jꢂ8.7 Hz, H-3ꢆ and H-5ꢆ),
7.46 (2H, d, Jꢂ8.7 Hz, H-2ꢆ and H-6ꢆ), 7.60 (1H, d, Jꢂ15.9 Hz, H-3ꢅ); ES-
MS m/z: 615 [MꢃH]ꢃ; HR-ES-TOF-MS (ꢃve): m/z: 615.4059 [MꢃH]ꢃ
(Calcd for C40H56O5ꢃH: 615.4055).
3-O-(E)-p-Methoxycinnamoylursolic Acid (18): 86% yield from com-
pound 3; amorphous solid from CH2Cl2–MeOH, mp 283—284 °C (lit.23)
267 °C); Rfꢂ0.51; IR (KBr) cmꢃ1: 3622, 2929, 2855, 1707, 1636, 1604,
1513, 1253, 1170, 1024; 1H-NMR data (CDCl3) were consistent with the re-
ported values23); ES-MS m/z: 615 [MꢃH]ꢃ.
3-O-(E)-(4-O-Acetylferuloyl)betulinic Acid (7): 59% yield from com-
pound 1; amorphous solid from MeOH, mp 187—188 °C; Rfꢂ0.45; IR
(KBr) cmꢃ1: 3394, 2946, 1768, 1705, 1687, 1638, 1600, 1513, 1453, 1375,
1259, 1192, 1173, 1155, 1033, 979, 883; 1H-NMR (400 MHz, CDCl3) d:
0.80 (1H, d, Jꢂ4.9 Hz, H-5), 0.84, 0.85, 0.88, 0.91, 0.94 (each 3H, each s,
CH3), 1.66 (3H, s, CH3-30), 2.29 (3H, s, OAc), 2.97 (1H, m, H-19), 3.83
(3H, s, OCH3), 4.57 (1H, br s, H-3), 4.58 (1H, br s, H-29a), 4.70 (1H, br s,
H-29b), 6.34 (1H, d, Jꢂ16.0 Hz, H-2ꢅ), 7.01 (1H, d, Jꢂ8.0 Hz, H-5ꢆ), 7.07
(1H, br s, H-2ꢆ), 7.08 (1H, br d, Jꢂ8.0 Hz, H-6ꢆ), 7.57 (1H, d, Jꢂ16.0 Hz, H-
3ꢅ); ES-MS m/z: 697 [MꢄNa]ꢄ; HR-ES-TOF-MS (ꢃve) m/z: 673.4121
[MꢃH]ꢃ (Calcd for C42H56O6ꢃH: 673.4110).
3-O-(E)-(4-O-Acetylferuloyl)oleanolic Acid (13): 68% yield from com-
pound 2; amorphous solid from MeOH, mp 176—177 °C; Rfꢂ0.32; IR
(KBr) cmꢃ1: 3310, 2946, 2870, 1771, 1700, 1637, 1601, 1509, 1466, 1368,
1259, 1198, 1157, 1123, 1011, 902; 1H-NMR (400 MHz, CDCl3) d: 0.75,
0.890, 0.894, 0.91, 0.92, 0.95, 1.13 (each 3H, each s, CH3), 2.30 (3H, s,
OAc), 2.81 (1H, dd, Jꢂ10.1, 3.4 Hz, H-18), 3.84 (3H, s, OCH3), 4.62 (1H, t,
Jꢂ8.0 Hz, H-3), 5.27 (1H, br s, H-12), 6.36 (1H, d, Jꢂ16.0 Hz, H-2ꢅ), 7.02
(1H, d, Jꢂ7.9 Hz, H-5ꢆ), 7.08 (1H, br s, H-2ꢆ), 7.09 (1H, br d, Jꢂ8.4 Hz, H-
6ꢆ), 7.59 (1H, d, Jꢂ16.0 Hz, H-3ꢅ); HR-FAB-MS (ꢃve) m/z: 673.4108
[MꢃH]ꢃ (Calcd for C42H58O7ꢃH: 673.4104).
3-O-(E)-(4-O-Acetylferuloyl)ursolic Acid (19): 63% yield from com-
pound 3; amorphous solid from MeOH, mp 202—204 °C; Rfꢂ0.39; IR
(KBr) cmꢃ1: 3427, 2926, 1772, 1686, 1654, 1636, 1560, 1508, 1458, 1260,
1199, 1034; 1H-NMR (400 MHz, CDCl3) d: 0.80, 0.91, 0.93, 0.98, 1.09
(each 3H, each s, CH3), 0.87 (3H, d, Jꢂ6.1 Hz, H-29), 0.94 (3H, d, partially
obscured signal, H-30), 2.19 (1H, dd, Jꢂ10.1, 3.4 Hz, H-18), 2.32 (3H, s,
OAc), 3.86 (3H, s, OCH3), 4.64 (1H, t, Jꢂ8.1 Hz, H-3), 5.26 (1H, br s, H-
12), 6.37 (1H, d, Jꢂ15.9 Hz, H-2ꢅ), 7.04 (1H, d, Jꢂ7.9 Hz, H-5ꢆ), 7.10 (1H,
br s, H-2ꢆ), 7.11 (1H, br d, Jꢂ7.9 Hz, H-6ꢆ), 7.61 (1H, d, Jꢂ15.9 Hz, H-3ꢅ);
HR-FAB-MS (ꢃve) m/z: 673.4105 [MꢃH]ꢃ (Calcd for C42H58O7ꢃH:
673.4104).
3-O-(E)-p-Chlorocinnamoylbetulinic Acid (9): 67% yield from compound
1; amorphous solid from MeOH, mp ꢀ300 °C; Rfꢂ0.54; IR (KBr) cmꢃ1
:
3232, 2939, 1725, 1642, 1492, 1462, 1328, 1305, 1196, 1014, 981; 1H-NMR
(400 MHz, CDCl3ꢄ4 drops CD3OD) d: 0.84, 0.85, 0.87, 0.92, 0.95 (each
3H, each s, CH3), 1.66 (3H, s, CH3-30), 2.96 (1H, m, H-19), 4.57 (1H, m, H-
3), 4.58 (1H, br s, H-29a), 4.70 (1H, br s, H-29b), 6.38 (1H, d, Jꢂ16.0 Hz,
H-2ꢅ), 7.32 (2H, d, Jꢂ8.4 Hz, H-3ꢆ and H-5ꢆ), 7.43 (2H, d, Jꢂ8.4 Hz, H-2ꢆ
and H-6ꢆ), 7.56 (1H, d, Jꢂ16.0 Hz, H-3ꢅ); ES-MS m/z: 643 [MꢄNa]ꢄ; HR-
ES-TOF-MS (ꢃve) m/z: 655.3319 [MꢄCl]ꢃ (Calcd for C39H53ClO4ꢄCl:
655.3326).
3-O-(E)-p-Chlorocinnamoyloleanolic Acid (15): 65% yield from com-
pound 2; amorphous solid from CH2Cl2–MeOH, mp ꢀ300 °C; Rfꢂ0.44; IR
(KBr) cmꢃ1: 3461, 2942, 1716, 1689, 1639, 1491, 1465, 1303, 1274, 1202,
1176, 1093, 1010; 1H-NMR (400 MHz, CDCl3ꢄ4 drops CD3OD) d: 0.73,
0.84, 0.84, 0.86, 0.86, 0.89, 1.08 (each 3H, each s, CH3), 4.56 (1H, t,
Jꢂ7.8 Hz, H-3), 5.22 (1H, br s, H-12), 6.35 (1H, d, Jꢂ16.0 Hz, H-2ꢅ), 7.29
(2H, d, Jꢂ8.3 Hz, H-3ꢆ and H-5ꢆ), 7.40 (2H, d, Jꢂ8.3 Hz, H-2ꢆ and H-6ꢆ),
7.54 (1H, d, Jꢂ16.0 Hz, H-3ꢅ); ES-MS m/z: 643 [MꢄNa]ꢄ; HR-ES-TOF-
MS (ꢃve) m/z: 655.3328 [MꢄCl]ꢃ (Calcd for C39H53ClO4ꢄCl: 655.3326).
3-O-(E)-p-Chlorocinnamoylursolic Acid (21): 66% yield from compound
3; amorphous solid from CH2Cl2–MeOH, mp ꢀ300 °C; Rfꢂ0.51; IR (KBr)
cmꢃ1: 3404, 2927, 1716, 1696, 1637, 1458, 1274, 1172, 1093, 1014, 826;
1H-NMR (400 MHz, CDCl3) d: 0.78, 0.89, 0.91, 0.97, 1.07 (each 3H, each s,
CH3), 0.85 (3H, d, Jꢂ6.2 Hz, CH3-29), 0.93 (3H, d, partially obscured sig-
nal, CH3-30), 2.18 (1H, d, Jꢂ11.0 Hz, H-18), 4.61 (1H, t, Jꢂ7.8 Hz, H-3),
5.24 (1H, m, H-12), 6.39 (1H, d, Jꢂ16.0 Hz, H-2ꢅ), 7.33 (2H, d, Jꢂ8.5 Hz,
H-3ꢆ and H-5ꢆ), 7.44 (2H, d, Jꢂ8.5 Hz, H-2ꢆ and H-6ꢆ), 7.59 (1H, d,
Jꢂ16.0 Hz, H-3ꢅ); ES-MS m/z: 643 [MꢄNa]ꢄ; HR-ES-TOF-MS (ꢃve) m/z:
655.3308 [MꢄCl]ꢃ (Calcd for C39H53ClO4ꢄCl: 655.3326).
Procedure for the Deacetylation of Acetoxycinnamic Acid Esters of
Triterpenes To a stirred solution of the acetoxycinnamic acid ester (0.02
mmol) in MeOH (1 ml) was added excess 10% aqueous K2CO3 (0.5 ml) and
the solution was stirred at ambient temperature for 30 min. Water (20 ml)
was added and the solution mixture was acidified with 5% aqueous HCl and
extracted three times with EtOAc (3ꢇ20 ml) and the combined organic
phase was then washed with water (1ꢇ20 ml). The organic layer was dried
over anhydrous Na2SO4; the solvent was then evaporated and the residue
chromatographed by elution with CH2Cl2–MeOH (80 : 2).
3-O-(E)-p-Coumaroylbetulinic Acid (5a): 92% yield from compound 5;
amorphous solid from MeOH, mp 188—189 °C; Rfꢂ0.25; IR (KBr) cmꢃ1
:
3422, 2945, 1696, 1606, 1515, 1458, 1169, 1020, 668; 1H-NMR data
(CDCl3ꢄ4 drops CD3OD) were consistent with the reported values27); ES-
MS m/z: 625 [MꢄNa]ꢄ.
3-O-(E)-p-Coumaroyloleanolic Acid (11a): 89% yield from compound
11; amorphous solid from MeOH, mp ꢀ300 °C; Rfꢂ0.18; IR (KBr) cmꢃ1
:
3-O-(E)-(3,4-Di-O-acetylcaffeoyl)betulinic Acid (8): 66% yield from
compound 1; powder from MeOH, mp 218—220 °C; Rfꢂ0.44; IR (KBr)
cmꢃ1: 3443, 2929, 1776, 1688, 1640, 1504, 1454, 1379, 1205, 1176, 1111,
1016, 902; 1H-NMR (400 MHz, CDCl3) d: 0.80 (1H, obscured signal, H-5),
0.86, 0.86, 0.88, 0.93, 0.97 (each 3H, each s, CH3), 1.68 (3H, s, CH3-30),
2.28 (3H, s, OAc), 2.29 (3H, s, OAc), 2.98 (1H, m, H-19), 4.58 (1H, m, H-
3), 4.60 (1H, br s, H-29a), 4.72 (1H, br s, H-29b), 6.35 (1H, d, Jꢂ16.0 Hz,
3348, 2949, 1706, 1606, 1516, 1458, 1270, 1181, 1010; 1H-NMR data
(CDCl3ꢄ4 drops CD3OD) were consistent with the reported values24); ES-
MS m/z: 625 [MꢄNa]ꢄ.
p-Coumaroylursolic Acid (17a): 96% yield from compound 17; amor-
phous solid from MeOH, mp ꢀ300 °C; Rfꢂ0.23; IR (KBr) cmꢃ1: 3348,
2925, 1707, 1690, 1606, 1516, 1458, 1371, 1277, 1168, 1100, 1017, 826;

198
Vol. 56, No. 2
1H-NMR data (CDCl3ꢄ4 drops CD3OD) were consistent with the reported
values25,27,28); ES-MS m/z: 625 [MꢄNa]ꢄ.
3) Shah N. S., Wright A., Bai G.-H., Barrera L., Boulahbal F., Martin-
Casabona N., Drobniewski F., Gilpin C., Havelková M., Lepe R.,
Lumb R., Metchock B., Portaels F., Rodrigues M. F., Rüsch-Gerdes S.,
Deun A. V., Vincent V., Laserson K., Wells C., Cegielski J. P., Emerg.
Infect. Dis., 13, 380—387 (2007).
4) Warner D. F., Mizrahi V., Clin. Microbiol. Rev., 19, 558—570 (2006).
5) Zhang Y., Annu. Rev. Pharmacol. Toxicol., 45, 529—564 (2005).
6) Sun I.-C., Wang H.-K., Kashiwada Y., Shen J.-K., Cosentino L. M.,
Chen C.-H., Yang L.-M., Lee K.-H., J. Med. Chem., 41, 4648—4657
(1998).
7) Zhu Y.-M., Shen J.-K., Wang H.-K., Cosentino L.-M., Lee K.-H.,
Bioorg. Med. Chem. Lett., 11, 3115—3118 (2001).
8) Suksamrarn A., Tanachatchairatana T., Kanokmedhakul S., J.
Ethnopharmacol., 88, 275—277 (2003).
9) Ziegler H. L., Franzyk H., Sairafianpour M., Tabatabai M., Tehrani M.
D., Bagherzadeh K., Hägerstrand H., Staerk D., Jaroszewski J. W.,
Bioorg. Med. Chem., 12, 119—127 (2004).
3-O-(E)-Feruloylbetulinic Acid (Lawsonic Acid) (7a): 96% yield from
compound 7; amorphous solid from MeOH, mp 289—291 °C (lit.29) 299—
300 °C); Rfꢂ0.32; IR (KBr) cmꢃ1: 3532, 2940, 1702, 1638, 1606, 1515,
1
1464, 1429, 1376, 1319, 1267, 1207, 1169, 1157, 1036, 974, 887, 817; H-
NMR data (CDCl3) were consistent with the reported values30); ES-MS m/z:
655 [MꢄNa]ꢄ.
3-O-(E)-Feruloyloleanolic Acid (Scaphopetalumate) (13a): 80% yield
from compound 13; amorphous solid from MeOH, mp 160—162 °C (lit.31)
amorphous powder); Rfꢂ0.20; IR (KBr) cmꢃ1: 3420, 2928, 1702, 1516,
1458, 1267, 1169, 1035; 1H-NMR data (CDCl3) were consistent with the re-
ported values31); ES-MS m/z: 655 [MꢄNa]ꢄ.
3-O-(E)-Feruloylursolic Acid (19a): 71% yield from compound 19; amor-
phous solid from MeOH, mp 176—178 °C; Rfꢂ0.31; IR (KBr) cmꢃ1: 3420,
2926, 1701, 1636, 1595, 1515, 1458, 1379, 1268, 1170, 1034; 1H-NMR
(400 MHz, CDCl3) d: 0.78, 0.90, 0.92, 0.97, 1.07 (each 3H, each s, CH3),
0.85 and 0.93 (each 3H, d, Jꢂ6.2 Hz and d, Jꢂca. 7.0 Hz, CH3-29 and CH3- 10) Liu J., J. Ethnopharmacol., 49, 57—68 (1995).
30), 2.17 (1H, br d, Jꢂ11.3 Hz, H-18), 3.91 (3H, s, OCH3), 4.62 (1H, dd, 11) Baglin I., Mitiane-Offer A.-C., Nour M., Tan K., Cavé C., Lacaille-
Jꢂ8.7, 7.2 Hz, H-3), 5.24 (1H, br s, H-12), 6.27 (1H, d, Jꢂ15.9 Hz, H-2ꢅ),
6.89 (1H, d, Jꢂ8.2 Hz, H-5ꢆ), 7.02 (1H, br s, H-2ꢆ), 7.05 (1H, br d, 12) Cantrell C. L., Franzblau S. G., Fischer N. H., Planta Med., 67, 685—
Jꢂ8.2 Hz, H-6ꢆ), 7.57 (1H, d, Jꢂ15.9 Hz, H-3ꢅ); ES-MS m/z: 655
694 (2001).
[MꢄNa]ꢄ; HR-FAB-MS (ꢃve) m/z: 631.3998 [MꢃH]ꢃ (Calcd for 13) Copp B. R., Pearce A. N., Nat. Prod. Rep., 24, 278—297 (2007).
Dubois M.-A., Mini Rev. Med. Chem., 3, 525—539 (2003).
C40H56O6ꢃH: 631.3999).
14) Okunade A. L., Elvin-Lewis M. P. F., Lewis W. H., Phytochemistry,
65, 1017—1032 (2004).
3-O-(E)-Caffeoylbetulinic Acid (8a): 79% yield from compound 8; amor-
phous solid from EtOAc, mp ꢀ300 °C (lit.32) ꢀ300 °C); Rfꢂ0.04; Rfꢂ0.40 15) Wächter G. A., Valcic S., Flagg M. L., Franzblau S. G., Montenegro
(CH2Cl2–MeOH, 25 : 1); IR (KBr) cmꢃ1: 3436, 2943, 1687, 1645, 1620,
G., Suarez E., Timmermann B. N., Phytomedicine, 6, 341—345
(1999).
16) Kanokmedhakul K., Kanokmedhakul S., Phatchana R., J. Ethnophar-
macol., 100, 284—288 (2005).
17) Jiménez-Arellanes A., Meckes M., Torres J., Luna-Herrera J., J.
Ethnopharmacol., 111, 202—205 (2007).
1
1531, 1450, 1352, 1281, 1218, 1175, 1121, 1043, 975, 850, 817; H-NMR
data (CDCl3ꢄ3 drops CD3OD) were consistent with the reported values32)
;
HR-ES-TOF-MS (ꢄve) m/z: 641.3818 [MꢄNa]ꢄ (Calcd for C39H54O6ꢄNa:
641.3812).
3-O-(E)-Caffeoyloleanolic Acid (14a): 74% yield from compound 14;
powder from EtOAc, mp ꢀ300 °C (lit.33) ꢀ300 °C); Rfꢂ0.02; Rfꢂ0.29 18) Woldemichael G. M., Franzblau S. G., Zhang F., Wang Y., Timmer-
(CH2Cl2–MeOH, 25 : 1); IR (KBr) cmꢃ1: 3434, 2945, 1687, 1645, 1619,
1600, 1531, 1450, 1352, 1326, 1280, 1217, 1175, 1120, 1031, 975, 850,
mann B. N., Planta Med., 69, 628—631 (2003).
19) Nareeboon P., Kraus W., Beifuss U., Conrad J., Klaiber I., Sut-
thivaiyakit S., Tetrahedron, 62, 5519—5526 (2006).
1
817; H-NMR data (CDCl3ꢄ3 drops CD3OD) were consistent with the re-
ported values33); HR-ES-TOF-MS (ꢄve) m/z: 641.3818 [MꢄNa]ꢄ (Calcd 20) Rojas R., Caviedes L., Aponte J. C., Vaisberg A. J., Lewis W. H.,
for C39H54O6ꢄNa: 641.3812).
Lamas G., Sarasara C., Gilman R. H., Hammond G. B., J. Nat. Prod.,
69, 845—846 (2006).
3-O-(E)-Caffeoylursolic Acid (20a): 67% yield from compound 20; amor-
phous solid, mp ꢀ300 °C (lit.34,35) 324—326 °C); Rfꢂ0.03; Rfꢂ0.33 21) Suksamrarn S., Panseeta P., Kunchanawatta S., Distaporn T., Ruktas-
(CH2Cl2–MeOH, 25 : 1); IR (KBr) cmꢃ1: 3435, 2928, 1687, 1645, 1619,
ing S., Suksamrarn A., Chem. Pharm. Bull., 54, 535—537 (2006).
22) Collins L. A., Franzblau S. G., Antimicrob. Agents Chemother., 41,
1004—1009 (1997).
23) Baglin I., Poumaroux A., Nour M., Tan K., Mitaine-Offer A. C.,
Lacaille-Dubois M. A., Chauffert B., Cavé C., J. Enz. Inhibit. Med.
Chem., 18, 111—117 (2003).
1
1532, 1450, 1376, 1280, 1218, 1175, 1121, 975, 817; H-NMR (400 MHz,
CDCl3) d: 0.78, 0.85, 0.88, 0.95, 1.05 (each 3H, each s, CH3), 0.82 and 0.89
(each 3H, d, Jꢂ6.4 Hz and d, Jꢂca. 7.0 Hz, CH3-29 and CH3-30), 4.57 (1H,
dd, Jꢂ8.6, 7.2 Hz, H-3), 5.21 (1H, br s, H-12), 6.18 (1H, d, Jꢂ16.0 Hz, H-
2ꢅ), 6.78 (1H, d, Jꢂ8.1 Hz, H-5ꢆ), 6.91 (1H, br d, Jꢂ8.1 Hz, H-6ꢆ), 7.00 (1H,
br s, H-2ꢆ), 7.48 (1H, d, Jꢂ16.0 Hz, H-3ꢅ); HR-ES-TOF-MS (ꢄve) m/z: 24) Takahashi H., Iuchi M., Fujita Y., Minami H., Fukuyama Y., Phyto-
641.3818 [MꢄNa]ꢄ (Calcd for C39H54O6ꢄNa: 641.3812).
chemistry, 51, 543—550 (1999).
Antimycobacterial Assay Antimycobacterial activity was assessed 25) Jahan N., Malik A., Afza N., Choudhary M. I., Shahzad-ul-Hassan S.,
against Mycobacterium tuberculosis H37Ra using the Microplate Alamar
Z. Naturforsch. B: Chem. Sci., 55, 1206—1210 (2000).
Blue Assay.22) This testing was undertaken by the National Center for Ge- 26) Jagadeesh S. G., David Krupadanam G. L., Srimannarayana G., J.
netic Engineering and Biotechnology, Thailand. In our system, the standard
Agric. Food Chem., 46, 2797—2799 (1998).
drugs, kanamycin sulfate, isoniazid and rifampicin showed MIC values of 27) David J. P., David M. M. J. M., Guedes M. L. S., Quim. Nova, 27, 62—
2.5, 0.06 and 0.004 mg/ml, respectively. The assay results are presented in
Table 1.
65 (2004).
28) Murphy B. T., MacKinnon S. L., Yan X., Hammond G. B., Vaisberg A.
J., Neto C. C., J. Agric. Food Chem., 51, 3541—3545 (2003).
Acknowledgments This work was supported by the Royal Golden Ju- 29) Chien N. Q., Hung N. V., Santarsiero B. N., Mesecar A. D., Cuong N.
bilee Ph.D. Program of The Thailand Research Fund. Support from the Re-
search Team Strengthening Grant of the National Center for Genetic Engi-
M., Soejarto D. D., Pezzuto J. M., Fong H. H. S., Tan G. T., J. Nat.
Prod., 67, 994—998 (2004).
neering and Biotechnology is gratefully acknowledged. We are grateful to 30) Siddiqui B. S., Nadeem Kardar M., Phytochemistry, 58, 1195—1198
the Center for Innovation in Chemistry: Postgraduate Education and Re-
search Program in Chemistry for partial support. We are indebted to Dr. Su-
nunta Wangkarn, Department of Chemistry, Chiang Mai University, Chiang
Mai, and Mr. Nitirat Chimnoi, Chulabhorn Research Institute, Bangkok, for
(2001).
31) Vardamides J. C., Azebaze A. G. B., Nkengfack A. E., Van Heerden F.
R., Fomum Z. T., Ngando T. M., Conrad J., Vogler B., Kraus W., Phy-
tochemistry, 62, 647—650 (2003).
recording the high resolution mass spectra. We also thank Ramkhamhaeng 32) Tinto W. F., Blair L. C., Alli A., Reynolds W. F., McLean S., J. Nat.
University and the University of Wollongong for support.
Prod., 55, 395—398 (1992).
33) Fuchino H., Satoh T., Tanaka N., Chem. Pharm. Bull., 43, 1937—1942
(1995).
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