Lantadenes and their esters as potential antitumor agents

Article abstract of DOI:10.1021/np800167x

Lantadenes are pentacyclic triterpenoids of the weed Lantana camara. Five new lantadenes (14-18) and their methyl esters (20-24) were synthesized, characterized, and screened for cytotoxicity against four human cancer cell lines. The parent compound (1) and the four most active compounds (15, 16, 21, and 22) were further studied for their in vivo tumor inhibitory potential on squamous cell carcinogenesis in Swiss albino mice induced by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol- 13-acetate (TPA). These results were supported by histopathological studies and discussed in terms of structure-activity relationships. The results inferred the importance of the groups attached to C-22 and C-17 in relation to the antitumor activity of these compounds.

Full text of DOI:10.1021/np800167x

1222
J. Nat. Prod. 2008, 71, 1222–1227
Lantadenes and Their Esters as Potential Antitumor Agents
M. Sharma,† P. D. Sharma,*,† and M. P. Bansal‡
UniVersity Institute of Pharmaceutical Sciences, Panjab UniVersity, Chandigarh-160014, India, and Department of Biophysics, Panjab
UniVersity, Chandigarh-160014, India
ReceiVed March 17, 2008
Lantadenes are pentacyclic triterpenoids of the weed Lantana camara. Five new lantadenes (14-18) and their methyl
esters (20-24) were synthesized, characterized, and screened for cytotoxicity against four human cancer cell lines. The
parent compound (1) and the four most active compounds (15, 16, 21, and 22) were further studied for their in ViVo
tumor inhibitory potential on squamous cell carcinogenesis in Swiss albino mice induced by 7,12-dimethylbenz[a]an-
thracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). These results were supported by
histopathological studies and discussed in terms of structure-activity relationships. The results inferred the importance
of the groups attached to C-22 and C-17 in relation to the antitumor activity of these compounds.
Lantana camara L. (Verbenaceae) is one of the most noxious
weeds that grows wild in tropical and subtropical parts of the
world.1 It provides a huge amount of biomass that is of interest to
exploit for natural products in drug research.2 Some pentacyclic
triterpenoids are known to have antibacterial, anti-inflammatory,
antitumor, or anti-AIDS activity.3,4 Lantadenes (1-4) isolated from
L. camara belong to the oleanane series, which have attracted
considerable interest mainly because of their toxicity and antitumor
activity. Compounds 1 and 2 were found to inhibit Epstein-Barr
virus activation in Raji cells induced by 12-O-tetradecanoylphorbol-
13 acetate (TPA) and also exhibited tumor inhibitory activity in a
two-stage carcinogenesis model in mice.5,6 We have carried out
studies on lantana and found that leaf extract showed a chemopre-
ventive effect on DMBA-induced squamous cell carcinogenesis in
Swiss albino mice.7 It was also found that compound 1 induces
apoptosis in human leukemia HL-60 cells.8 These compounds differ
in the structure of the group attached to C-22, and there are
indications that the structural variations play an important role in
pharmacological activities of these compounds. We have reported
the importance of structural variations involving C-22 and C-17 in
the antitumor activity of lantadene A congeners.9 In this paper, we
report the synthesis of five new lantadenes (14-18) and their methyl
esters (20-24), along with their cytotoxicity against four human
cancer cell lines. The parent compound 1 and four most active
compounds (15, 16, 21, and 22) were studied for their in ViVo tumor
inhibitory potential on two-stage squamous cell carcinogenesis in
Swiss albino mice, induced by 7,12-dimethylbenz[a]anthracene
(DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate
(TPA).
protected, followed by acylation, and finally the protecting group
was removed to yield different lantadene analogues. The protecting
group was selected on the basis of its ease, both in synthesis and
cleavage under mild conditions without disturbing the ester at C-22.
The protecting group was also selected for its selectivity toward
the carboxy group at C-17. The carboxy group of 5 was protected
by diphenyldiazomethane, which was prepared by treating ben-
zophenone hydrazone with yellow mercury oxide.10,11 Compound
5 was treated with diphenyldiazomethane to obtain 8 in 30% yield,
which was treated with various acyl chlorides, or anhydrides, to
obtain diphenylmethyl esters (9-13). The protecting group was
removed by treating with anisole and trifluroacetic acid (1:4) to
obtain the corresponding lantadenes (14-18). Compound 5 was
also treated with acetic anhydride and benzoyl chloride to yield 14
and 18, respectively. This reaction failed with other acyl chlorides
and anhydrides, which may be due to the formation of a mixed
anhydride (6) and finally ꢀ-lactone (7).17
It has been reported that esterification of C-17 carboxy-
triterpenoids with MeOH showed enhancement of biological
activity.12 Thus, it was thought worthwhile to prepare methyl esters
of synthesized lantadenes (20-24). For this purpose, instead of
using the synthesized lantadenes (14-18) for esterification, an
alternative route was used involving the preparation of 19 by treating
5 with diazomethane, followed by acylation with acyl chlorides or
anhydrides in the presence of pyridine to obtain the corresponding
compounds (20-24). This route involved fewer steps (Scheme 1,
route c) and gave better yields. All structures were confirmed by
1
using spectroscopic techniques and elemental analysis. H NMR
spectra of the diphenylmethyl esters showed a methyl singlet at
unusually high field (δ 0.4). This chemical shift was the result of
anisotropic shielding by the phenyl rings of the diphenylmethyl
ester group. Hence, the high signal was assigned to the C-26 methyl
group. The one proton singlet (COOCH) of diphenylmethyl ester
appeared in the range δ 6.1-6.8. The free acids showed singlets
for seven tertiary methyl groups in the range δ 0.83-1.18. The
12-H signal appeared in the range δ 5.30-5.41 as a triplet.
Similarly, 22-H and 18-H signals appeared in the ranges δ
5.07-5.16 and 3.02-3.08 as a triplet and double doublet, respec-
tively. Analyses of 13C NMR showed signals characteristic for C-28
carbonyl carbons and C-22 attached to OH groups in the ranges δ
180.1-181.4 and 75.4-77.7, respectively. The spectra also showed
a downfield shift of the C-3 keto groups, which resonated in the
range δ 214.3-219.2. The mass spectra of these compounds showed
base peaks at m/z 452, which resulted from loss of the corresponding
acid radical from the molecular ion. Loss of ketene from the
molecular ion resulted in a peak at m/z 470. The RDA fragmentation
Results and Discussion
Five new lantadenes (14-18) and their methyl esters (20-24)
were synthesized as shown in Scheme 1. The crude lantadene
fraction was isolated from the dried leaves of lantana and
hydrolyzed to 5 in 45% yield. The carboxylic group of 5 was
* Corresponding author. E-mail: pritamdevsharma@hotmail.com. Phone:
+91 172-2534117. Fax: +91 172-2541142.
† University Institute of Pharmaceutical Sciences.
‡ Department of Biophysics.
10.1021/np800167x CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/14/2008

Lantadenes as Potential Antitumor Agents
Journal of Natural Products, 2008, Vol. 71, No. 7 1223
Scheme 1. Sequence of Steps Involved in the Synthesis of Lantadenes (14-18) and Their Methyl Esters (20-24)
Table 1. In Vitro Cytotoxicity of Lantadene Ester Analogues
against Human Cancer Cell Lines (IC50 in µg/mL)
There was a significant decrease in cytotoxicity with branching of
the side chain. Introduction of a phenyl ring in the side chain further
decreased the cytotoxicity. The C-17 methyl esters all were more
cytotoxic than the corresponding free acids, with 22 showing the
best activity (IC50 19.3-22.4 µg/mL) among the synthesized
compounds. The cytotoxicity of 22 was comparable to that of 21
(IC50 22.4-28.4 µg/mL). Similarly, 23 and 24 were more cytotoxic
than their corresponding free acids.
compound
HL 60
19.8 ( 0.21a 23.3 ( 0.08b 21.3 ( 0.05b
<100 >100 >100
HeLa
colon 502713 lung A-549
1
5
21.8 ( 0.21a
>100
14
15
16
17
18
19
20
21
22
23
24
38.5 ( 0.05a 37.3 ( 0.15a 34.4 ( 0.020b 36.8 ( 0.08
26.4 ( 0.17b 28.6 ( 0.05a 25.4 ( 0.05a
21.6 ( 0.120a 22.8 ( 0.08b 25.2 ( 0.15c
29.6 ( 0.14b
27.1 ( 0.05c
41.2 ( 0.020a 43.6 ( 0.21c 37.6 ( 0.21c 44.2 ( 0.21a
Carcinogenesis is a multistep process involving the sequential
phases of initiation, promotion, and progression. Chemoprevention
is one desirable strategy to reverse, arrest, or inhibit carcinogen-
esis.13 The mouse skin carcinogenesis model is often used to study
the genetic and biological changes associated with chemical
initiation of lesions and their subsequent transition to squamous
cell carcinoma. DMBA is a polycyclic aromatic hydrocarbon
capable of inducing skin lesions when applied repeatedly on mouse
skin.14 The primary bioactivation of DMBA occurs via formation
of DMBA-3,4-dihydrodiol. DMBA-diol-epoxide has been suggested
to be the ultimate carcinogen responsible for inducing chronic
inflammation, ROS production, and oxidative damage of DNA
resulting in the transformation of normal cells to tumor cells.15
When mouse skin is repeatedly exposed to DMBA, the Langerhans
cells will be depleted and local immunosuppression takes place.16
This leads to the induction of tumors on the skin of the mice. TPA
50.8 ( 0.08a 46.3 ( 0.08a 51.2 ( 0.020b >100
70.6 ( 0.05a >100
72.4 ( 0.05
84.2 ( 0.05
36.8 ( 0.05c
28.4 ( 0.21c
22.4 ( 0.05c
42.2 ( 0.08a
>100
37.3 ( 0.15b 36.2 ( 0.05c 34.6 ( 0.08a
24.2 ( 0.21b 26.4 ( 0.08c 22.4 ( 0.08a
19.3 ( 0.08a 20.1 ( 0.21a 21.1 ( 0.05c
39.5 ( 0.05b 41.1 ( 0.21b 38.1 ( 0.05a
48.0 ( 0.05a 50.7 ( 0.05b >100
paclitaxeld <2
>3
<2
<2
a P < 0.05. b P < 0.01. c P > 0.001. d Positive control.
gave a major peak at m/z 206. The methyl ester derivatives showed
a base peak at m/z 466.
The derivatives were studied for cytotoxicity against four human
cancer cell lines (HL-60, HeLa, colon 502713, and lung A-549),
and the results are summarized in Table 1. Compound 20 showed
cytotoxicity in the range 34.4-38.4 µg/mL. Cytotoxicity increased
as the length of the side chain increased from acetoxy to propoxy.

1224 Journal of Natural Products, 2008, Vol. 71, No. 7
Sharma et al.
tetramethylsilane was used as internal standard. Mass spectra were obtained
with Micromass 70-VSE mass spectrometer at 70 eV using electron
ionization (EI). Elemental analysis of compounds was within (0.04% of
the theoretical values. All solvents were freshly distilled and dried prior
to use according to standard procedures. All chemicals were purchased
from SD Fine Chemicals, Qualigen, and Loba Chemicals. DMBA and
TPA were purchased from Sigma Chemicals Ltd.
Plant Material. Leaves of Lantana camara were collected in
September from Palampur (HP), India. The leaves were shade-dried
and powdered. A voucher specimen (LC; 097 UIPS) was deposited in
the Herbarium at the University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh, India.
Animals. Female Swiss albino mice (6 weeks old) weighing 18-22
g were obtained from the Central Animal House of Panjab University.
Animals were kept in the Departmental Animal House with a controlled
temperature of 23 ( 5 °C, 60 ( 5% humidity, and a 12 h light/dark
cycle. They were fed a basal diet and water. The mice were acclimated
for 1 week before experimentation.
Extraction and Isolation of Compounds 1-4. Compounds 1-4
were isolated from lantana leaf powder, and compound 5 was prepared
by basic hydrolysis as reported previously.17
Diphenylmethyl 22ꢀ-hydroxy-3-oxoolean-12-en-28-onate (8). In
a 250 mL two-necked flask, 0.1 g (0.21 mmol) of 22ꢀ-hydroxy-3-
oxoolean-12-en-28-oic acid (5) was dissolved in 20 mL of CHCl3. The
solution was stirred at 40 °C, and diphenyldiazomethane in CHCl3 was
added dropwise until the purple color of the reagent was no longer
evident. The solvent was removed under reduced pressure, and the
residue was chromatographed on a silica gel G column (20 g, 100-200
mesh) using CHCl3 and a CHCl3-MeOH (9.5-0.5) mixture as eluting
solvents. The desired product-enriched fractions were pooled, and the
solvent was removed in Vacuo to yield 8 as white crystals (0.04 g,
29.5%): mp 230 °C; IR (KBr) νmax 3520 (OH), 3313 (aromatic) 1740
(CdO, ester), 1690 (CdO, keto) cm-1; 1H NMR (CDCl3, 300 MHz) δ
0.4 (3H, s, C-26-CH3), 0.90 (3H, s, CH3), 0.97 (3H, s, CH3), 1.02 (3H,
s, CH3), 1.06 (3H, s, CH3), 1.12 (6H, s, 2 × CH3), 3.05 (1H, dd, J )
14.0; 3.7 Hz, C-18-H), 4.0 (1H, t, J ) 3 Hz, C-22-H), 5.4 (1H, t, J )
3.4 Hz, C-12-H), 6.8 (1H, s, COOCH), 7.4-8.1 (10H, m, Ar-H).
General Procedure for Synthesis of Diphenylmethyl 22ꢀ-Acy-
loxyoleanonates (9-13). Compound 8 was stirred with different
acylating agents in the presence of pyridine at room temperature, and
the reactions were monitored on TLC (hexane-ethyl acetate; 18.5-1.5).
The reaction mixtures were diluted with water (10 mL), stirred for 1 h,
adjusted to pH 2 by adding HCl, and extracted with CHCl3 (2 × 5
mL). The organic layer was dried over anhydrous Na2SO4, and the
solvent was removed under reduced pressure to yield compounds 9-13
as oily residues.
is a phorbol ester, which was isolated from croton oil. This is an
active constitutent of croton oil, responsible for the promotion of
tumors.
Compounds 15, 16, 21, and 22 were selected for two-stage
carcinogenesis studies. Squamous cell carcinogenesis was induced
in Swiss albino mice (LACCA/female) by DMBA and promoted
by TPA. The onset of papillomas was observed in the fourth week
in the DMBA/TPA-treated mice and found to be 36.3%. There was
a gradual rise in the incidence of tumors that reached 100% during
the eighth week. The incidence of DMBA/TPA-induced papillomas
was delayed for 3 weeks by 15 and 16 and 4 weeks by 21 and 22.
The animals treated with 16 showed a significant decrease in the
incidence of cancer (17.2% vs 100%) at the end of 20 weeks as
compared with the DMBA/TPA-treated group (P > 0.001). The
overall papilloma incidence in those treated with 15, 21, and 22
was 19.6% (P > 0.001, P > 0.01), 17.9% (P > 0.001, P > 0.05),
and 12.4% (P > 0.001, P > 0.05), respectively, in comparison with
the DMBA/TPA-treated group. The parent compound (1) group
showed 18.1% incidence of tumors and a delay of three weeks.
The survival rate decreased significantly in DMBA/TPA-treated
mice as compared with the vehicle-treated group (30% vs 80%).
Survival rates with compound-treated animals were significantly
higher in comparison to the DMBA/TPA-treated group (P > 0.01,
P > 0.05). The 15-, 17-, 21-, and 22-treated groups showed similar
survival rates (80%). The average body weight of DMBA/TPA-
treated mice decreased significantly. However, there was slight
increase in the average body weight of 15-, 17-, 21-, and 22-treated
mice at the termination of the study (P > 0.01, P > 0.05).
Histopathological examination of the depilated back of mice
revealed normal skin and the presence of subcutaneous tissue in
acetone-treated mice. Twice weekly application of DMBA for 8
weeks on depilated mice induced well-differentiated squamous cell
carcinoma with formation of keratin pearls. There was marked
infiltration of cancer cells in the underlying dermis. The skin section
of mice treated with synthesized compounds showed hyperplastic
papillomatous lesions with no evidence of infiltration or cytological
atypia.
The pretreatment of animals with derivatives could be associated
with their ability to interfere with the initiation of carcinogenesis,
which is a relatively rapid process, and the continuous treatment
after TPA with the promotion, which is a slow process. These agents
also increased the survival time of the animals. Slight weight gain
in 15-, 16-, 21-, and 22-treated mice could be a result of recovery
from the effect of DMBA/TPA or, alternatively, with better
papilloma control.
Diphenylmethyl 22ꢀ-acetoxy-3-oxoolean-12-en-28-oate (9): oil
(0.04 g, 31.1%); IR (KBr) ν´max 3037 (aromatic), 1742 (CdO, ester),
1
1682 (CdO, keto) cm-1; H NMR (CDCl3, 300 MHz) δ 0.4 (3H, s,
C-26-CH3), 0.91 (3H, s, CH3), 0.98 (3H, s, CH3), 1.03 (3H, s, CH3),
1.05 (3H, s, CH3), 1.10 (6H, s, 2 × CH3), 2.09 (3H, s, OCOCH3) 3.05
(1H, dd, J ) 14.0; 3.7 Hz, C-18-H), 5.0 (1H, t, J ) 3 Hz, C-22-H), 5.4
(1H, t, J ) 3.4 Hz, C-12-H), 6.6 (1H, s, COOCH), 7.4-8.0 (10H, m,
Ar-H).
The cytotoxicity profile of these derivatives showed that, with
removal of the ester at C-22, there was significant decrease in the
activity. Compound 5 was inactive. Methylation of the C-17
carboxyl group resulted in some activity. Compound 19 showed
better cytotoxicity than 5, and similarly methyl esters 20-24 were
more cytotoxic than 14-18. This effect may be attributed to
increased liphophilicity and better bioavailability. An increase in
length of the ester side chain at C-22 gave an increase in the activity,
and branching decreased the activity. An aromatic ester at C-22
also resulted in decreased activity. These results indicate the
importance of the C-22 and C-17 positions in the antitumor activity
of these compounds.
Diphenylmethyl 22ꢀ-propoxy-3-oxoolean-12-en-28-oate (10): oil
(0.017 g, 45.7%); IR (KBr) νmax 3042 (aromatic), 1738 (CdO, ester),
1
1685 (CdO, keto) cm-1; H NMR (CDCl3, 300 MHz) δ 0.4 (3H, s,
C-26-CH3), 0.91 (3H, s, CH3), 0.97 (3H, s, CH3), 1.04 (3H, s, CH3),
1.08 (3H, s, CH3), 1.14 (6H, s, 2 × CH3), 1.73 (3H, t, J ) 7.1 Hz,
-OCOCH2CH3,), 3.04 (1H, dd, J ) 14.2; 3.7 Hz, C-18-H), 3.61 (2H,
q, J ) 7.1 Hz, -OCOCH2CH3,), 5.9 (1H, t, J ) 3 Hz, C-22-H), 5.6
(1H, t, J ) 3.4 Hz, C-12-H), 6.5 (1H, s, COOCH), 7.5-8.2 (10H, m,
Ar-H).
Diphenylmethyl 22ꢀ-butyrloxy-3-oxoolean-12-en-28-oate (11): oil
Experimental Section
(0.04 g, 36%); IR (KBr) νmax 3061 (aromatic), 1735 (CdO, ester), 1689
1
(CdO, keto) cm-1; H NMR (CDCl3, 300 MHz) δ 0.4 (3H, s, C-26-
General Experimental Procedures. Melting points were determined
on a Buchi melting point apparatus and are uncorrected. For thin-layer
chromatography (TLC), glass plates coated with silica gel G (E. Merck)
were used. The TLC plates were activated at 110 °C for 30 min and
visualized by exposure to iodine vapors. Glass columns of appropriate
sizes were used. Silica gel (60-120 mesh) was used as adsorbent. IR
spectra were recorded on a Perkin-Elmer 882 spectrometer using potassium
CH3), 0.84 (3H, s, CH3), 0.90 (3H, s, CH3), 0.96 (3H, t, J ) 7.1 Hz,
-OCOCH2CH2CH3), 1.04 (3H, s, CH3), 1.09 (3H, s, CH3), 1.17 (6H, s,
2 × CH3), 1.72 (2H, sextet, J ) 7.1 Hz, -OCOCH2CH2CH3), 2.25
(2H, t, J ) 7.1 Hz, -OCOCH2CH2CH3), 3.04 (1H, dd, J ) 14.1; 3.7
Hz, C-18-H), 5.1 (1H, t, J ) 3 Hz, C-22-H), 5.3 (1H, t, J ) 3.4 Hz,
C-12-H), 6.1 (1H, s, -COOCH), 7.5-8.2 (10H, m, Ar-H).
1
bromide pellets. H NMR and 13C NMR spectra were recorded with a
Diphenylmethyl 22ꢀ-isobutyrloxy-3-oxoolean-12-en-28-oate (12):
oil (0.05 g, 45%); IR (KBr) νmax 3062 (aromatic), 1745 (CdO, ester),
Bruker AC 300F 300 MHz spectrometer using CDCl3 as solvent, and

Lantadenes as Potential Antitumor Agents
Journal of Natural Products, 2008, Vol. 71, No. 7 1225
1
1690 (CdO, keto) cm-1; H NMR (CDCl3, 300 MHz) δ 0.4 (3H, s,
J ) 7.2 Hz, -OCOCH2CH2CH3), 2.23 (2H, t, J ) 7.2 Hz,
-OCOCH2CH2CH3,), 3.03 (1H, dd, J ) 14.2; 3.08 Hz, C-18-H), 5.10
(1H, t, J ) 3 Hz, C-22-H), 5.30 (1H, t, J ) 3.4 Hz, C-12-H); 13C
NMR (75 MHz) δ 37.2 (C-1), 33.4 (C-2), 217.9 (C-3), 46.3 (C-4),
55.3 (C-5), 19.1 (C-6), 32.6 (C-7), 39.6 (C-8), 46.2 (C-9), 36.4 (C-
10), 24.4 (C-11), 121.2 (C-12), 143.3 (C-13), 42.2 (C-14), 27.7 (C-
15), 23.6 (C-16), 52.1 (C-17), 39.2 (C-18), 45.5 (C-19), 30.3 (C-20),
38.1 (C-21), 76.9 (C-22), 26.5 (C-23), 21.3 (C-24), 15.1 (C-25), 16.6
(C-26), 25.5 (C-27), 180.1 (C-28), 33.6 (C-29), 26.8 (C-30), 172.0 (C-
1′), 36.1 (C-2′), 18.1 (C-3′), 13.4 (C-4′); EMIS m/z 540 [M+], 525
(10), 522 (17), 497 (14), 496 (22), 452 (100), 469 (22), 435 (8), 409,
(10) 407 (3), 334 (19), 319 (42), 206 (34), 203 (22); anal. C34H52O5 C,
75.51%; H, 9.69%; found C, 75.56%; H, 9.70%.
C-26-CH3), 0.84 (3H, s, CH3), 0.90 (3H, s, CH3),1.04 (3H, s, CH3),
1.09 (3H, s, CH3), 1.17 (6H, s, 2 × CH3), 1.20 (6H, d, J ) 7.4 Hz, 2
× -OCOCH(CH3)2,), 2.58 (1H, septet, J ) 7.4 Hz, -OCOCH(CH3)2),
3.04 (1H, dd, J ) 14.1; 3.7 Hz, C-18-H), 5.1 (1H, t, J ) 3 Hz, C-22-
H), 5.3 (1H, t, J ) 3.4 Hz, C-12-H), 6.3 (1H, s, -COOCH), 7.4-8.0
(10H, m, Ar-H).
Diphenylmethyl 22ꢀ-benzyloxy-3-oxoolean-12-en-28-oate (13): oil
(0.05 g, 34.6%); IR (KBr) νmax 3043 (aromatic), 1738 (CdO, ester),
1
1693 (CdO, keto) cm-1; H NMR(CDCl3, 300 MHz) δ 0.4 (3H, s,
C-26-CH3), 0.85 (3H, s, CH3), 0.90 (3H, s, CH3), 1.04 (3H, s, CH3),
1.08 (3H, s, CH3), 1.18 (6H, s, 2 × CH3), 3.04 (1H, dd, J ) 14.1; 3.6
Hz, C-18-H), 5.2 (1H, t, J ) 3 Hz, C-22-H), 5.3 (1H, t, J ) 3.4 Hz,
C-12-H), 6.1 (1H, s, -COOCH), 7.6-8.1 (15H, m, Ar-H).
General Procedure for Synthesis of 22ꢀ-Acyloxy-3-oxoolean-12-
en-28-oic Acids (14-18). Diphenylmethyl 22ꢀ-acyloxy-3-oxoolean-
12-en-28-oate (0.25 g, 0.37 mmol), anisole, and trifluroacetic acid were
stirred vigorously for 5 min at room temperature. The solvent was
removed under reduced pressure, and 10 mL of light petroleum was
added to the residue. The solid product was collected by filtration,
washed with light petroleum, and recrystallized from MeOH and H2O
to give compounds 14-18.
22ꢀ-Isobutyrloxy-3-oxoolean-12-en-28-oic acid (17): white crystals
(0.11 g, 53.1%); mp 199 °C; IR (KBr) νmax 3485 (COOH), 1738 (CdO,
ester),1734 (CdO, COOH), 1688 (CdO, keto) cm-1; 1H NMR (CDCl3,
300 MHz) δ 0.84 (3H, s, C-26-CH3), 0.90 (3H, s, CH3), 1.01 (6H, s,
2 × CH3), 1.04 (3H, s, CH3), 1.09 (3H, s, CH3), 1.17 (3H, s, CH3),
1.20 (6H, d, J ) 7.5 Hz, 2 × -OCOCH(CH3)2,), 2.58 (1H, septet, J )
7.5 Hz,-OCOCH(CH3)2,), 3.02 (1H, dd, J ) 14.3; 3.8 Hz, C-18-H),
5.13 (1H, t, J ) 3 Hz, C-22-H), 5.32 (1H, t, J ) 3.3 Hz, C-12-H); 13
C
NMR (75 MHz) δ 37.9 (C-1), 33.3 (C-2), 217.2 (C-3), 46.4 (C-4),
55.1 (C-5), 19.4 (C-6), 32.2 (C-7), 39.6 (C-8), 46.4 (C-9), 36.5 (C-
10), 24.2 (C-11), 121.2 (C-12), 143.3 (C-13), 42.2 (C-14), 27.7 (C-
15), 23.6 (C-16), 52.2 (C-17), 39.1 (C-18), 45.3 (C-19), 30.1 (C-20),
38.2 (C-21), 77.1 (C-22), 26.5 (C-23), 21.3 (C-24), 15.1 (C-25), 16.7
(C-26), 25.5 (C-27), 180.2 (C-28), 33.8 (C-29), 26.6 (C-30), 174.5 (C-
1′), 38.5 (C-2′), 17.4 (C-3′ and C-4′); EMIS m/z 540 [M+], 525 (12),
522 (21), 452 (100), 469 (36), 334 (34), 319 (31), 248 (12), 246 (15),
231 (16), 206 (27), 203 (20); anal. C34H52O5 C, 75.51%; H, 9.69%;
found C, 75.56%; H, 9.70%.
22ꢀ-Acetoxy-3-oxoolean-12-en-28-oic acid (14): white crystals
(0.12 g, 63.5%); mp 296 °C; IR (KBr) νmax 3303 (COOH), 1743 (CdO,
ester), 1721 (CdO, COOH), 1688 (C) O, keto) cm-1; 1H NMR (CDCl3,
300 MHz) δ 0.83 (3H, s, C-26-CH3), 0.90 (3H, s, CH3), 1.04 (6H, s,
2 × CH3), 1.06 (3H, s, CH3), 1.09 (3H, s, CH3), 1.18 (3H, s, CH3)
1.94 (3H, s, -OCOCH3) 3.03 (1H, dd, J ) 14.2; 3.08 Hz, C-18-H),
5.07 (1H, t, J ) 3 Hz, C-22-H), 5.37 (1H, t, J ) 3.4 Hz, C-12-H); 13
C
NMR (75 MHz) δ 39.3 (C-1), 34.2 (C-2), 214.3 (C-3), 47.5 (C-4),
55.4 (C-5), 19.5 (C-6), 32.3 (C-7), 39.2 (C-8), 46.9 (C-9), 36.8 (C-
10), 24.4 (C-11), 121.2 (C-12), 143.3 (C-13), 42.0 (C-14), 27.7 (C-
15), 23.6 (C-16), 52.2 (C-17), 39.1 (C-18), 45.8 (C-19), 30.1 (C-20),
38.1 (C-21), 75.4 (C-22), 26.5 (C-23), 21.5 (C-24), 15.1 (C-25), 16.7
(C-26), 25.8 (C-27), 180.2 (C-28), 33.8 (C-29), 26.8 (C-30), 166.2 (C-
1′), 15.3 (C-2′); EMIS m/z 512 [M+], 494 (16), 469 (32), 468 (40),
452 (100), 306 (36), 291 (25), 206 (20), 203 (18); anal. C32H48O5 C,
74.96%; H, 9.44%; found C, 74.96%; H, 9.47%.
22ꢀ-Benzoyloxy-3-oxoolean-12-en-28-oic acid (18): brown crystals
(0.13 g, 59.0%); mp 237 °C; IR (KBr) νmax 3478 (COOH), 1735 (CdO,
ester),1738 (CdO, COOH), 1693 (CdO, keto) cm-1; 1H NMR (CDCl3,
300 MHz) δ 0.85 (3H, s, C-26-CH3), 0.90 (3H, s, CH3), 1.02 (6H, s,
2 × CH3), 1.04 (3H, s, CH3), 1.08 (3H, s, CH3), 1.18 (3H, s, CH3),
3.06 (1H, dd, J ) 14.3; 3.8 Hz, C-18-H), 5.16 (1H, t, J ) 3 Hz, C-22-
H), 5.36 (1H, t, J ) 3.4 Hz, C-12-H), 7.4-8.0 (15H, m, Ar-H); 13C
NMR (75 MHz) δ 39.5 (C-1), 32.3 (C-2), 219.2 (C-3), 47.4 (C-4),
55.6 (C-5), 19.7 (C-6), 32.7 (C-7), 39.9 (C-8), 46.7 (C-9), 35.5 (C-
10), 24.2 (C-11), 121.9 (C-12), 143.8 (C-13), 42.7 (C-14), 27.9 (C-
15), 23.9 (C-16), 52.7 (C-17), 39.8 (C-18), 45.7 (C-19), 31.4 (C-20),
37.2 (C-21), 77.7 (C-22), 26.9 (C-23), 21.8 (C-24), 15.7 (C-25), 16.9
(C-26), 25.9 (C-27), 181.4 (C-28), 33.9 (C-29), 26.9 (C-30), 167.0 (C-
1′), 130.5 (C-2′′), 129.7 (C-3′ and C-7′′), 128.8 (C-4′ and C-6′), 132.8
(C-5′); EMIS m/z 574 [M+], 548 (12), 546 (32), 532 (27), 530 (22),
523 (20), 470 (8), 469 (27), 452 (100), 278 (29), 353 (24), 248 (13),
246 (19), 231 (15), 206 (16), 203 (21); anal. C37H50O5 C, 77.31%; H,
9.77%; found C, 77.31%; H, 8.78%.
Compound 18 was prepared by stirring a solution of 22ꢀ-hydroxy-
3-oxoolean-12-en-28-oic acid (0.12 g, 0.25mmol) in 2.4 mL of pyridine
and 1.480 g (1.3 mL, 1.5 mmol) of benzoyl chloride for 4.5 h at room
temperature. Progress of the reaction was monitored by TLC. The
reaction mixture was diluted with 10 mL of H2O, acidified with HCl,
and extracted with ether (3 × 10 mL). The combined organic layer
was washed with H2O (5 mL) and dried over anhydrous Na2SO4, and
the solvent was removed by distillation. The residue was recrystallized
from a MeOH and H2O mixture to afford 18 (0.08 g, 54.5%), mp 237
°C.
Compound 14 was also prepared by stirring a solution of 0.12 g
(0.25 mmol) of 22ꢀ-hydroxy-3-oxoolean-12-en-28-oic acid in 2 mL
of pyridine and 1 mL (1.079 g, 1.1 mmol) of acetic anhydride overnight
at room temperature using magnetic stirring. The progress of the
reaction was monitored by TLC. The reaction mixture was diluted with
10 mL of H2O, acidified with HCl, and extracted with ether (3 × 10
mL). The combined organic layer was washed with 5 mL of H2O and
dried over anhydrous Na2SO4, and the solvent was removed under
reduced pressure. The residue obtained was recrystallized from a MeOH
and H2O mixture to afford white crystals of 14 (0.05 g, 38.2%), mp
296 °C. The spectroscopic and elemental data were identical with 14
reported above.
22ꢀ-Propoxy-3-oxoolean-12-en-28-oic acid (15): white crystals
(0.15 g, 77.3%); mp 274-276 °C; IR (KBr) νmax 3437 (COOH), 1747
(CdO, ester), 1726 (CdO, COOH), 1691 (CdO, keto) cm-1; 1H NMR
(CDCl3, 300 MHz) δ 0.86 (3H, s, C-26-CH3), 0.91 (3H, s, CH3), 1.02
(6H, s, 2 × CH3), 1.04 (3H, s, CH3), 1.06 (3H, s, CH3), 1.19 (3H, s,
CH3) 1.70 (3H, t, J ) 7.2 Hz, -OCOCH2CH3,) 3.03 (1H, dd, J )
14.0; 3.08 Hz, C-18-H), 3.64 (2H, q, J ) 7.2 Hz, -OCOCH2CH3,),
5.11 (1H, t, J ) 3 Hz, C-22-H), 5.41 (1H, t, J ) 3.4 Hz, C-12-H); 13
C
NMR (75 MHz) δ 36.3 (C-1), 34.4 (C-2), 216.6 (C-3), 46.3 (C-4),
55.4 (C-5), 19.3 (C-6), 32.5 (C-7), 39.1 (C-8), 46.5 (C-9), 36.7 (C-
10), 24.1 (C-11), 121.6 (C-12), 143.2 (C-13), 42.3 (C-14), 27.8 (C-
15), 23.5 (C-16), 52.3 (C-17), 39.4 (C-18), 45.7 (C-19), 30.3 (C-20),
38.3 (C-21), 76.5 (C-22), 26.2 (C-23), 21.4 (C-24), 15.3 (C-25), 16.8
(C-26), 25.9 (C-27), 180.3 (C-28), 33.8 (C-29), 26.9 (C-30), 172.2 (C-
1′), 26.7 (C-2′), 9.1 (C-3′); EMIS m/z 526 [M+], 511 (27), 498 (10),
497 (13), 484 (21), 482 (33), 469 (37), 452 (100), 296 (12), 320 (34),
305 (10), 248 (16), 246 (19), 206 (21), 203 (17); anal. C33H50O5 C,
75.25%; H, 9.57%; found C, 75.21%; H, 9.59%.
22ꢀ-Butyrloxy-3-oxoolean-12-en-28-oic acid (16): white crystals
(0.15 g, 73.4%); mp 241-243 °C; IR (KBr) νmax 3411 (COOH), 1752
(CdO, ester), 1731 (CdO, COOH), 1686 (CdO, keto) cm-1; 1H NMR
(CDCl3, 300 MHz) δ 0.86 (3H, s, C-26-CH3), 0.91 (3H, s, CH3), 0.95
(3H, t, J ) 7.2 Hz, -OCOCH2CH2CH3), 1.02 (6H, s, 2 × CH3), 1.04
(3H, s, CH3), 1.09 (3H, s, CH3), 1.17 (3H, s, CH3), 1.71 (2H, sextet,
Methyl 22ꢀ-hydroxy-3-oxoolean-12-en-28-onate (19). Excess ethe-
real diazomethane was added to 0.15 g (0.32 mmol) of 5, and the
reaction mixture was kept overnight. Excess diazomethane was neutral-
ized by adding acetic acid. The solvent was removed under reduced
pressure, and the residue was crystallized from MeOH to obtain 19 as
white crystals (0.12 g, 77.6%): mp 179-181 °C; IR (KBr) νmax 3595
(OH), 2911 (aliphatic), 1711 (CdO, ester), 1684 (CdO, keto) cm-1
;
1H NMR (CDCl3, 300 MHz) δ 0.85 (3H,s, CH3), 0.89 (3H, s, CH3),
1.04 (6H, s, 2 × CH3), 1.09 (3H, s, CH3), 1.11 (3H, s, CH3), 1.15 (3H,
s, CH3), 3.01 (1H, dd, J ) 14.2; 3.7 Hz, C-18-H), 3.59 (3H, s,
-COOCH3), 3.71 (1H, t, J ) 3 Hz, C-22- H), 4.81 (1H, broad, C-22-
OH, exchanged with D2O), 5.28 (1H, t, J ) 3.5 Hz, C-12-H); 13C NMR
(75 MHz) δ 39.4 (C-1), 34.2 (C-2), 217.4 (C-3), 47.5 (C-4), 55.4 (C-
5), 19.5 (C-6), 32.1 (C-7), 39.2 (C-8), 46.9 (C-9), 36.6 (C-10), 24.3
(C-11), 121.2 (C-12), 143.3 (C-13), 42.0 (C-14), 27.7 (C-15), 23.6 (C-
16), 52.1 (C-17), 39.1 (C-18), 45.8 (C-19), 30.1 (C-20), 38.1 (C-21),

1226 Journal of Natural Products, 2008, Vol. 71, No. 7
Sharma et al.
76.6 (C-22), 26.5 (C-23), 21.5 (C-24), 15.1 (C-25), 16.7 (C-26), 25.8
(C-27), 180.5 (C-28), 33.8 (C-29), 26.8 (C-30), 51.1 (COOCH3); EMIS
m/z 484 [M+], 466 (100), 409 (21), 407 (15), 248 (31), 246 (17), 203,
(26) 201 (11); anal. C31H48O4 C, 76.82%; H, 9.98%; found C, 76.82%;
H, 9.99%.
General Procedure for Synthesis of Methyl 22ꢀ-acyloxyoleanon-
ates (20-24). A solution of 19 (0.15 g, 0.31 mmol) was stirred with
pyridine and the appropriate acylating agents. Progress of reactions
was monitored by TLC. The reaction mixtures were diluted with 10
mL of H2O, acidified with HCl, and extracted with ether (3 × 10 mL).
The organic layer was dried over anhydrous Na2SO4, and solvent was
removed under reduced pressure. The residue obtained was recrystal-
lized from a MeOH-H2O mixture to afford 20-24.
s, -COOCH3), 5.20 (1H, t, J ) 3 Hz, C-22-H), 5.49 (1H, t, J ) 3.5
Hz, C-12-H); 13C NMR (75 MHz) δ 38.9 (C-1), 34.6 (C-2), 217.6 (C-
3), 47.4 (C-4), 55.5 (C-5), 19.6 (C-6), 32.3 (C-7), 39.4 (C-8), 47.8
(C-9), 36.5 (C-10), 24.8 (C-11), 121.4 (C-12),143.6 (C-13), 42.7 (C-
14), 28.7 (C-15), 24.1 (C-16), 52.7 (C-17), 39.9 (C-18), 46.7 (C-19),
30.9 (C-20), 38.7 (C-21), 78.3 (C-22), 26.7 (C-23), 21.7 (C-24), 16.6
(C-25), 16.9 (C-26), 25.8 (C-27), 180.9 (C-28), 34.5 (C-29), 27.4 (C-
30), 51.8 (COOCH3), 166.1 (C-1′), 35.8 (C-2′), 17.4 (C-3′), 17.4 C-4′);
EMIS m/z 554 [M+], 526 (12), 511 (26), 483 (13), 466 (100), 348
(12), 333 (17), 248 (7), 206 (16), 203 (19); anal. C35H54O5 C, 75.77%;
H, 9.81%; found C, 75.79%; H, 9.84%.
Methyl 22ꢀ-benzyloxy-3-oxoolean-12-en-28-oate (24): white crys-
tals (0.12 g, 61.7%); mp 186-188 °C; IR (KBr) νmax 3433 (aromatic),
2956 (aliphatic), 1740 (CdO, ester), 1709 (CdO, ester), 1702 (CdO,
Methyl 22ꢀ-acetoxy-3-oxoolean-12-en-28-oate (20): white crystals
(0.10 g, 61.3%); mp 216-218 °C; IR (KBr) νmax 2950 (aliphatic), 1746
1
keto) cm-1; H NMR (CDCl3, 300 MHz) δ 0.85 (3H, s, CH3), 0.89
1
(CdO, ester), 1715 (CdO, ester), 1689 (CdO, keto) cm-1; H NMR
(3H, s, CH3), 1.04 (6H, s, 2 × CH3), 1.09 (3H, s, CH3), 1.12 (3H, s,
CH3), 1.17 (3H, s, CH3), 3.04 (1H, dd, J ) 14.3; 3.8 Hz, C-18-H),
4.26 (3H, s, -COOCH3), 5.07 (1H, t, J ) 3 Hz, C-22-H), 5.46 (1H, t,
J ) 3.5 Hz, C-12-H), 7.2-8.1 (5H, m, Ar-H); 13C NMR (75 MHz) δ
39.5 (C-1), 34.9 (C-2), 219.6 (C-3), 47.5 (C-4), 55.4 (C-5), 19.9 (C-
6), 32.8 (C-7), 39.6 (C-8), 48.3 (C-9), 36.6 (C-10), 24.9 (C-11), 121.7
(C-12), 143.8 (C-13), 42.9 (C-14), 28.8 (C-15), 24.4 (C-16), 52.8 (C-
17), 39.9 (C-18), 46.9 (C-19), 30.7 (C-20), 38.9 (C-21), 78.6 (C-22),
27.5 (C-23), 21.9 (C-24), 16.7 (C-25), 17.4 (C-26), 25.9 (C-27), 181.5
(C-28), 34.8 (C-29), 27.4 (C-30), 51.8 (COOCH3), 167.2 (C-1′), 122.3,
122.9, 129.1, 130.2, 133.5 (C-Ph); EMIS m/z 588 [M+], 560 (14), 557
(13), 511 (21), 483 (18), 466 (100), 292 (18), 367 (23), 246 (15), 206
(37), 203 (19); anal. C38H52O5 C, 77.51%; H, 8.90%; found C, 77.56%;
H, 8.92%.
(CDCl3, 300 MHz) δ 0.85 (3H, s, CH3), 0.89 (3H, s, CH3), 1.04 (6H,
s, 2 × CH3), 1.09 (3H, s, CH3), 1.11 (3H, s, CH3), 1.15 (3H, s, CH3),
2.09 (3H, s, -OCOCH3) 3.05 (1H, dd, J ) 14.2; 3.7 Hz,C-18-H), 3.78
(3H, s, -COOCH3), 5.10 (1H, t, J ) 3 Hz, C-22- H), 5.39 (1H, t, J )
3.5 Hz, C-12-H); 13C NMR (75 MHz) δ 39.7 (C-1), 34.6 (C-2), 217.8
(C-3), 47.8 (C-4), 55.7 (C-5), 19.3 (C-6), 32.6 (C-7), 39.5 (C-8), 47.2
(C-9), 36.6 (C-10), 24.3 (C-11), 121.6 (C-12), 143.8 (C-13), 42.3 (C-
14), 27.9 (C-15), 23.7 (C-16), 52.1 (C-17), 39.4 (C-18), 46.1 (C-19),
30.4 (C-20), 38.2 (C-21), 77.6 (C-22), 26.8 (C-23), 21.6 (C-24), 15.8
(C-25), 16.6 (C-26), 26.1 (C-27), 180.1 (C-28), 34.1 (C-29), 26.8 (C-
30), 51.4 (COOCH3), 166.1 (C-1′), 15.3 (C-2′); EMIS m/z 526 [M+],
511 (11), 483 (25), 466 (100), 435 (8), 409 (10), 320 (24), 305 (14),
231 (44), 206 (22), 203 (13); anal. C33H50O5 C, 75.25%; H, 9.57%;
found C, 75.27%; H, 9.59%.
Cell Culture and MTT Assay. Four cell lines (HL-60, HeLa, colon
502713, and A-549) were obtained from NCCS, Pune, India, and
maintained in RPMI medium supplemented with 10% fetal bovine
serum, 100 µg/mL streptomycin, and 100 IU/mL penicillin. Cells were
incubated at 37 °C in a humidified atmosphere of 5% CO2 for 48 h for
comparison of cytotoxicity between compounds. The cell lines (2 ×
10-4/0.1 mL well) were treated with serial dilutions of compounds in
96-well culture plates (Costar) for 48 h. During the last 4 h, cells were
reacted with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo-
lium bromide) at 37 °C for a colorimetric MTT-based cytotoxicity assay.
The reaction product, formazan, was extracted with DMSO, and the
absorbance was read at 540 nm.18 Data represent the mean values and
standard deviation of triplicate assays in at least one experiment.
In ViWo Antitumor Activity. Squamous cell carcinogenesis was
induced in Swiss albino mice (LACCA/female) according to the
established method.19 Animal care and handling was done according
to the guidelines set by the World Health Organization (WHO), Geneva,
Switerzerland, and the Indian National Science Academy (INSA), New
Delhi, India. Depilatory cream was used to remove hairs from the back
of mice. The animals were left for 2 days and divided into 17 groups.
Animals in group I (n ) 10) were treated with 100 µL of vehicle
(acetone). The acetone was topically applied on the depilated back of
each mouse twice weekly for 20 weeks. The animals of group II (n )
10) were topically treated with DMBA (100 nmol/100 µL of acetone)
on the depilated back of each mouse for 2 weeks and promoted with
twice weekly applications of TPA (1.7 nmol/100 µL of acetone) for
the next 18 weeks. The animals of groups III-VII (n ) 10) were orally
treated with test compounds suspended in water and carboxymethyl
cellulose. Treatments started 1 week before DMBA application and
continued for 20 weeks thereafter. Body weight, incidence of skin
papillomas, and number of animals that survived after the 20-week
treatment period were recorded. Body weight, number of deaths, and
papillomas were recorded at weekly intervals. Only those papillomas
that persisted for 2 weeks or more were taken into consideration for
final evaluation of the data.
Methyl 22ꢀ-propoxy-3-oxoolean-12-en-28-oate (21): white crystals
(0.12 g, 71.7%); mp 231 °C; IR (KBr) νmax 2959 (aliphatic), 1742
(CdO, ester), 1713 (CdO, 17 ester), 1693 (CdO, keto) cm-1; 1H NMR
(CDCl3, 300 MHz) δ 0.85 (3H, s, CH3), 0.89 (3H, s, CH3), 1.04 (6H,
s, 2 × CH3), 1.09 (3H, s, CH3), 1.11 (3H, s, CH3), 1.14 (3H, t J ) 7.1
Hz, -OCOCH2 CH3,)1.15 (3H, s, CH3), 2.21 (2H, q, J ) 7.1 Hz,
-OCOCH2CH3,), 3.05 (1H, dd, J ) 14.1; 3.7 Hz, C-18-H), 3.77 (3H,
s, -COOCH3), 5.10 (1H, t, J ) 3 Hz, C-22- H), 5.39 (1H, t, J ) 3.5
Hz, C-12-H); 13C NMR (75 MHz) δ 39.8 (C-1), 34.7 (C-2), 217.2 (C-
3), 47.6 (C-4), 55.8 (C-5), 19.7 (C-6), 32.7 (C-7), 39.6 (C-8), 47.4
(C-9), 36.7 (C-10), 24.6 (C-11), 121.7 (C-12), 143.9 (C-13), 42.6 (C-
14), 28.2 (C-15), 23.8 (C-16), 52.4 (C-17), 39.5 (C-18), 46.3 (C-19),
30.5 (C-20), 38.3 (C-21), 77.9 (C-22), 26.5 (C-23), 21.5 (C-24), 15.9
(C-25), 16.5 (C-26), 26.3 (C-27), 180.8 (C-28), 34.3 (C-29), 26.9 (C-
30), 51.5 (COOCH3), 166.3 (C-1′), 26.7 (C-2′), 9.1 (C-3′); EMIS m/z
540 [M+], 525 (16), 512 (16), 511 (12), 483 (24), 466 (100), 334 (17),
319 (27), 248 (12), 246 (16), 203 (14); anal. C34H52O5 C, 75.51%; H,
9.69%; found C, 75.53%; H, 9.71%.
Methyl 22ꢀ-butyrloxy-3-oxoolean-12-en-28-oate (22): white crys-
tals (0.017 g, 34.9%); mp 192-194 °C; IR (KBr) νmax 2962 (aliphatic),
1
1737 (CdO, ester), 1714 (CdO, ester), 1696 (CdO, keto) cm-1; H
NMR (CDCl3, 300 MHz) δ 0.84 (3H, s, CH3), 0.89 (3H, s, CH3), 1.04
(6H, s, 2 × CH3), 1.09 (3H, s, CH3), 1.11 (3H, s, CH3), 1.15 (3H, s,
CH3), 1.32 (3H, t, J ) 7.2 Hz, -OCOCH2CH2CH3,), 1.46 (2H, sextet,
J ) 7.2 Hz, -OCOCH2CH2CH3,), 2.33 (2H, t, J ) 7.2 Hz, -OCOCH2-
CH2CH3,), 3.03 (1H, dd, J ) 14.0; 3.7 Hz, C-18-H), 3.87 (3H, s,
COOCH3), 5.04 (1H, t, J ) 3 Hz, C-22-H), 5.38 (1H, t, J ) 3.5 Hz,
C-12-H); 13C NMR (75 MHz) δ 38.9 (C-1), 34.5 (C-2), 217.1 (C-3),
47.9 (C-4), 55.3 (C-5), 19.6 (C-6), 32.2 (C-7), 39.3 (C-8), 47.7 (C-9),
36.6 (C-10), 24.7 (C-11), 121.3 (C-12), 143.5 (C-13), 42.8 (C-14), 28.5
(C-15), 23.9 (C-16), 52.6 (C-17), 39.7 (C-18), 46.5 (C-19), 30.7 (C-
20), 38.6 (C-21), 78.2 (C-22), 27.1 (C-23), 21.7 (C-24), 16.2 (C-25),
16.7 (C-26), 26.6 (C-27), 181.2 (C-28), 34.5 (C-29), 27.3 (C-30), 51.7
(COOCH3), 166.1 (C-1′), 36.1 (C-2′), 18.2 (C-3′), 13.4 (C-4′); EMIS
m/z 554 [M+], 526 (10), 511 (22), 510 (19), 483 (21), 466 (100), 348
(17), 333 (23), 248 (13), 231 (7), 206 (12), 203 (15); anal. C35H54O5
C, 75.77%; H, 9.81%; found C, 75.75%; H, 9.82%.
Histology. The incidence of skin lesions, tumors per mouse, body
weight of mice, and number of mice that survived the 20-week period
were recorded. The body weight, number of deaths, and papillomas
appearing on depilated skin were recorded at weekly intervals. Only
those papillomas that persisted for 2 weeks or more were considered
for final evaluation of the data. The skin nodules were excised and
fixed in Zenker, routinely processed, and embedded in paraffin. Sections
(7 µm thick) were stained with hematoxylin and eosin and examined
under a light microscope to carry out histopathology.
Methyl 22ꢀ-isobutyrloxy-3-oxoolean-12-en-28-oate (23): white
crystals (0.08 g, 41.1%); mp 163 °C; IR (KBr) νmax 2958 (aliphatic),
1
1739 (CdO, ester), 1721 (CdO, ester), 1698 (CdO, keto) cm-1; H
NMR (CDCl3, 300 MHz) δ 0.86 (3H, s, CH3), 0.89 (3H, s, CH3), 1.03
(6H, s, 2 × CH3), 1.09 (3H, s, CH3), 1.10 (3H, s, CH3), 1.15 (3H, s,
CH3), 1.25 (6H, d, J ) 7.0 Hz, 2 × -OCOCH(CH3)2,), 2.61 (1H, m,
-OCOCH(CH3)2) 3.05 (1H, dd, J ) 14.1; 3.5 Hz, C-18-H), 3.80 (3H,

Lantadenes as Potential Antitumor Agents
Journal of Natural Products, 2008, Vol. 71, No. 7 1227
(9) Sharma, M.; Sharma, P. D.; Bansal, M. P.; Singh, J. Chem. BiodiVersity
2007, 4, 932–939.
(10) Furniss, A. S.; Hannaford, A. J.; Smith, P. W. G.; Ta Tatchell, A. R.
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Acknowledgment. We thank the Indian Council of Medical
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