Synthesis of bioactive 28-hydroxy-3-oxolup-20(29)-en-30-al with antileukemic activity

Article abstract of DOI:10.1080/10286020.2011.640774

An easy and efficient route to partial synthesis of bioactive 28-hydroxy-3-oxolup-20(29)-en-30-al (1), starting from betulinic acid (2), has been developed (eight steps, 44% overall yield). Structures of all the compounds were determined by spectral studies (IR, ¹H, ¹³C NMR, MS, NOESY, COSY, etc.). Compound 1 and the precursors (2, 3, 5, and 7) showed antiproliferative activities against human K562 leukemia, murine WEHI3 leukemia, and murine MEL erythroid progenitor.

Full text of DOI:10.1080/10286020.2011.640774

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Synthesis of bioactive 28-hydroxy-3-
oxolup-20(29)-en-30-al with
antileukemic activity
Pranab Ghosh a , Amitava Mandal a , Joydip Ghosh b , Chiranjib Pal
b & Ashis Kumar Nanda c
a Natural Product and Polymer Chemistry Laboratory, Department
of Chemistry, University of North Bengal, Darjeeling, 734 013,
West Bengal, India
b Cellular Immunology and Experimental Therapeutics Laboratory,
Department of Zoology, West Bengal States University, Barasat,
700 126, 24 PGS (N), West Bengal, India
c Department of Chemistry, University of North Bengal, Darjeeling,
734 013, West Bengal, India
Version of record first published: 14 Dec 2011.
To cite this article: Pranab Ghosh, Amitava Mandal, Joydip Ghosh, Chiranjib Pal & Ashis Kumar
Nanda (2012): Synthesis of bioactive 28-hydroxy-3-oxolup-20(29)-en-30-al with antileukemic
activity, Journal of Asian Natural Products Research, 14:2, 141-153
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Journal of Asian Natural Products Research
Vol. 14, No. 2, February 2012, 141–153
Synthesis of bioactive 28-hydroxy-3-oxolup-20(29)-en-30-al with
antileukemic activity
Pranab Ghosha*, Amitava Mandala, Joydip Ghoshb, Chiranjib Palb and
Ashis Kumar Nandac
aNatural Product and Polymer Chemistry Laboratory, Department of Chemistry, University of North
Bengal, Darjeeling 734 013, West Bengal, India; Cellular Immunology and Experimental
b
Therapeutics Laboratory, Department of Zoology, West Bengal States University, Barasat 700 126,
24 PGS (N), West Bengal, India; cDepartment of Chemistry, University of North Bengal, Darjeeling
734 013, West Bengal, India
(Received 19 August 2011; final version received 10 November 2011)
An easy and efficient route to partial synthesis of bioactive 28-hydroxy-3-oxolup-
20(29)-en-30-al (1), starting from betulinic acid (2), has been developed (eight steps,
44% overall yield). Structures of all the compounds were determined by spectral
studies (IR, 1H, 13C NMR, MS, NOESY, COSY, etc.). Compound 1 and the precursors
(2, 3, 5, and 7) showed antiproliferative activities against human K562 leukemia,
murine WEHI3 leukemia, and murine MEL erythroid progenitor.
Keywords: triterpenoid; partial synthesis; betulinic acid; antileukemic activity
1. Introduction
[10], antidiabetic [11] and so on. There-
fore, it is important to supply those
triterpenoids with novel structures in
sufficient amounts for further biological
testing. And, in this regard, synthesis
rather than isolation from natural sources
is more efficient and also economical.
Leukemia is a type of cancer of the
blood or bone marrow characterized by an
abnormal increase of white blood cells. It
is a broad term covering a spectrum of
diseases. In turn, it is part of the even
broader group of diseases called hemato-
logical neoplasms. In 2000, approximately
256,000 children and adults around the
world developed some form of leukemia,
and 209,000 died from it [12]. About 90%
of all leukemias are diagnosed in adults.
Most forms of leukemia are treated with
pharmaceutical medications, typically
combined into a multi-drug chemotherapy
Triterpenes represent a varied and import-
ant class of natural compounds. Among
these, pentacyclic lupane-type triterpenes
are one of the most significant subclasses
which have shown to possess several
medicinal properties [1,2]. The antitumor
properties of plant extracts comprising
lupane-derived triterpenoids have been
demonstrated during the past 25 years for
their cytostatic activity on various in vitro
and in vivo cancer model systems [3].
Betulinic acid, one of the lupane-derived
triterpenoids, exerts a selective antitumor
activity on cultured human melanoma [3],
neuroblastoma [4,5], malignant brain
tumor [6], and leukemia cells [7]. Other
pharmacological activities of lupane-type
triterpenoids include anti-inflammatory
activity [8], anti-carcinogenic activity [3],
photosynthetic inhibitors [9], anti-HIV
*Corresponding author. Email: pizy12@yahoo.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.640774
http://www.tandfonline.com

142
P. Ghosh et al.
regimen. Some are also treated with
radiation therapy. In some cases, a bone
marrow transplant is useful. All these
treatments are useful but only to a very
limited extent, and the chemotherapeutics
used to date are not specific to the affected
cells, thus causing severe damage to the
body. Despite recent improvements in the
treatment of early-stage disease, leukemia
blast crisis remains a therapeutic chal-
lenge because it is highly refractory to
standard induction chemotherapy, with a
response rate in myeloid blast crisis of
less than 30% [13]. Therefore, develop-
ment of mild selective chemotherapeutics
is a real demand in contemporary medical
sciences.
leukemia HL60 cells and mediate cleavage
of PARP and upregulation of Bax proteins.
Compound
1
(28-hydroxy-3-oxolup-
20(29)-en-30-al) was among the most
cytotoxic substances obtained [22]. They
also investigated the potential effects of 1
on growth inhibition of HL60 cells [17].
According to their results [17], this
compound induced apoptosis in a dose-
dependent manner. The molecular mech-
anism of compound 1 toward cancer cells
is still a subject of continuous investi-
gation and a specific target(s) has yet to be
identified. Therefore, suitable derivatives
of lupane may be considered as a group of
compounds having promising bioactivity
which can be used for further chemical as
well as biological research. Henceforth,
the present demands of this type of rare
(scarce) naturally occurring triterpenoids
are enormous in the contemporary medic-
inal research. Thus, it was felt necessary to
supply this novel triterpenoid 1 in
sufficient amounts for further biological
testing. And, in this regard, synthesis
rather than isolation from natural sources
is more efficient and economical.
Medicinal plants and their phytocon-
stituents have always been a better choice
for leukemia and nutraceuticals have been
proven to have antileukemic activity in
experimental studies [14]. Derivatives of
betulin, basically triterpenoids of lupane
skeleton, are reported to possess signifi-
cant cytotoxicity against a wide variety of
cancer cell lines [15]. In a recent
publication, one such naturally occurring
compound, 28-hydroxy-3-oxolup-20(29)-
en-30-al (1), which has been reported [16]
for the first time from the bark of Acacia
mellifera, showed significant cytotoxicity
against NSCLC-N6 cell line. Sub-
sequently, Chen et al. [17] reported the
presence of 1 in the methanol extract of
Microtropis fokienensis, which has been
reported [17] to induce apoptosis of human
In view of the above and in continu-
ation of our studies toward the chemical
transformations of pentacyclic triter-
penoids [18], we report herein a multistep
protocol for the synthesis of 28-hydroxy-
3-oxolup-20(29)-en-30-al (1) from betuli-
nic acid (2; Figure 1). In vitro, anticancer
activities of all the compounds having
C-30 ZCHO group (1, 2, 3, 5, and 7) are
29
30
OHC
21
19
20
12
26
22
28
18
17
13
14
11
9
25
OH
COOH
1
4
16
2
3
8
10
15
27
7
5
HO
O
6
24
23
1
2
Figure 1. Structures of compounds 1 and 2.

Journal of Asian Natural Products Research
143
also tested against different cell lines.
Derivatization of C-30 methyl group was
accomplished in good yield by SeO2
oxidation in refluxing aqueous dioxan.
This is the first report of synthesis of
compound 1 from 2 and its potent antic-
ancer activity against human K562 leuke-
mia, murine WEHI3 leukemia, and murine
MEL erythroid progenitor cell lines.
(diazomethane) at C-28 to form 28-
carbomethoxy-lup-20(29)-en-3b-ol (3)
almost quantitatively. Lithium aluminum
hydride reduction of 3 in anhydrous THF
gave betulin (4) in 72% yield. Allylic
oxidation of the C-30 methyl was then
carried out with SeO2 in aqueous dioxan
under refluxing condition after protecting
C-3 and C-28 hydroxyl groups as acetate.
The incorporation of formyl group was
1
assigned by IR and NMR (both H and
2. Results and discussion
13C) spectra of compound 6. The IR
spectrum showed peaks at 1732
(ZOCOCH3), 1691 (conjugated alde-
hyde), 1459 and 1369 (gem dimethyl),
and 1244, 1028, 978, 936, and 889
(vCH2) cm21. The molecular formula of
2.1 Chemical part
The sequential steps involved in the
synthesis of compound 1 are illustrated
in Scheme 1. Betulinic acid (2), isolated
from Bischofia javanica, was esterified
OH
COOMe
COOH
a
b
HO
HO
HO
3
2
4
OHC
OAc
e
OAc
d
c
AcO
AcO
5
6
OHC
OHC
OHC
OH
f
OAc
OAc
g
HO
HO
O
7
8
9
OHC
OH
h
O
1
Scheme 1. Partial synthesis of compound 1 from betulinic acid (2). Reagents and conditions: (a)
CH2N2, ether, over night, AcOH (gal.), Na2SO4; (b) LiAlH4, dry THF, 2 h, saturated Na2SO4
solution, ether, Na2SO4; (c) C5H5N, Ac2O, 6 h (1008C) ice-cold H2O, ether, Na2SO4; (d) SeO2, aq.
dioxan, 2 h, ice-cold H2O, ether, Na2SO4; (e) 10% alcoholic KOH, THF, 4 h, ice-cold H2O, ether,
Na2SO4; (f) C5H5N, Ac2O, (5–108C), 8 h, ice-cold H2O, Na2SO4; (g) C5H5N, dry CrO3, overnight,
ice-cold H2O, CH2Cl2, MgSO4; (h) 10% alcoholic KOH, THF, 4 h, ice-cold H2O, ether, Na2SO4.

144
P. Ghosh et al.
compound 6 was assigned as C34H52O5
(Mþ 540.29, analytical calculation % C
1
75.42, % H 9.56). The H NMR spectrum
applied for the formation of the desired
aldehyde 8.
A solution of 7 (350 mg, 0.76 mmol) in
CHCl3 (10 ml) and pyridine (15 ml) was
treated with Ac2O (10 ml, 0.098 mmol) at
very low temperature (5–108C). After the
reaction was over (checked by TLC), the
mixture was diluted with CHCl3 (20 ml),
and then the organic layer was washed with
saturated aq. NaCl (10 ml £ 3), dried over
anhydrous sodium sulfate, and concen-
trated in vacuo. Purification of the reaction
mixture gave compound 8 with mp 260–
2628C. The IR spectrum gave peaks at
3461 (ZOH), 1732 (ZOCOCH3), 1691
(.CvCZCHO), 1459, 1369 (gem
dimethyl), and 1244, 1028, 978, 936, and
890 (vCH2) cm21. The molecular formula
of compound 8 was assigned as C32H50O4
(Mþ 498.35, analytical calculation % C
77.02, % H 10.12). The 1H NMR spectrum
of 8 showed a singlet at dH 9.56 (s, 1H) for
the aldehyde proton. The 13C NMR
spectrum of compound 8 (Table 1)
accounted for all the carbons. Acetylation
of only the C-28 ZOH group was
of compound 6, taken in CDCl3, gave a
singlet at dH 9.56 with an integration of
one proton in addition to five methyls at dH
0.93 (s, 3H), 0.94 (s, 3H), 1.02 (s, 3H),
1.06 (s, 3H), and 1.39 (s, 3H) for lupane
skeleton. Two olefinic protons appeared at
dH (proton chemical shift position) 6.28 (s,
1H) and at dH 5.93 (s, 1H) and two geminal
hydrogens of C-28 each gave a doublet at
dH 4.24 (d, 1H, J ¼ 10.6 Hz) and at dH
3.68 (d, 1H, J ¼ 10.6 Hz). Two acetyl
methyls appeared as two sharp singlets at
dH 2.07 (s, 3H) and at dH 2.03 (s, 3H). In
the proton-decoupled 13C spectrum of 6, a
singlet at dc 194.6 (Table 1) clearly
indicated the presence of a formyl group
at C-30. Careful hydrolysis of 6 gave a
yellowish gummy residue after evapor-
ation of the solvent in vacuo. Purification
of this gummy material over a column of
silica gel gave almost quantitatively a
powdered white solid of compound 7 with
melting point (mp) 274–2768C. The IR
spectrum gave peaks at 3393 (ZOH), 1688
(conjugated aldehyde), 1453, and 1375
(gem dimethyl), and 1029, 942, and 890
(vCH2) cm21. The molecular formula of
compound 7 was assigned as C30H48O3
(Mþ 456.32, analytical calculation % C
1
confirmed by comparison of H and 13C
NMR data of C-3 hydroxy compound and
with the acetate as reported in the literature
[21]. The assigned 13C NMR data of all the
compounds are given in Table 1. Thus, on
the basis of the above spectral data, the
structure of compound 8 was assigned as
28-acetoxy-lup-20(29)-en-3b-ol-30-al.
In the subsequent step, C-3 ZOH group
was converted to ketone 9 (175 mg), using
anhydrous CrO3 in dry pyridine (see
Experimental section) at ambient tempera-
ture. After purification it showed mp 276–
2788C. The exact structure of compound 9
was elucidated by spectroscopic studies.
The IR spectrum gave peaks at 1730
(.CvO) and at 1706, 1696 (CH2
vCZCHO) cm21. The molecular formula
of compound 9 was assigned as C32H48O4
(Mþ 496.34, analytical calculation % C
77.28, % H 9.62). In its 1H NMR spectrum,
compound9gaveasingletatdH 9.56(s, 1H)
for the aldehydic proton. Two olefinic
1
78.66, % H 10.52). Disappearance of H
NMR signals at dH 2.03 (s, 3H) and 2.07 (s,
3H) and the presence of two singlets at dc
78.9 (for C-3) and 60.2 (for C-28) in the
13C NMR spectrum of 7 clearly indicated
the deprotection of both the hydroxyl
groups at C-3 and at C-28 during the
formation of 7 from diacetate (6).
A number of methods [19] were then
applied for the selective oxidation of C-3
secondary hydroxyl group, keeping C-28
primary hydroxyl group intact. None of the
existing methods [19], including the one
developed by Mckillop et al. [20] in 1979,
was found effective in producing the
desired selectivity. Thus, once again the
protection–deprotection method was

Journal of Asian Natural Products Research
145

146
P. Ghosh et al.
protons of C-29 appeared at dH 6.23 (s, 1H)
and5.92(s, 1H). Fortwogeminalprotonsof
juncture, it is relevant to mention that the
shift in 13C signals for the above-
mentioned carbons started appearing in
all the compounds (viz. compounds 6, 7, 8,
and 9) once the SeO2 oxidation step was
carried out. The shifts in the NMR
spectrum and possible epimerization of
the side chain during the SeO2 oxidation
were already reported in the literature from
our laboratory [18] and the existence of
such a conformational isomer is also
documented [23]. However, in the present
case, we could not get the isomeric peaks
for the relevant carbons in the 13C NMR
spectra of 1, 6, 7, 8, and 9 as observed by
the previous workers [18,23]. Therefore,
on the basis of the above spectral analysis,
the structure of compound X has been
assigned as 28-hydroxy-3-oxolup-20(29)-
en-30-al (1) or 10 (Figure 2).
Further confirmation of the stereo-
chemistry at C-19 was settled by carrying
out the 2D NMR techniques on compound
6 (first compound after the SeO2 oxi-
dation) in which the probable stereoche-
mical change would occur. The NOSEY
spectrum of compound 6 gave significant
information about the stereochemistry at
C-19. All nuclear overhauser effect (NOE)
cross-peaks have opposite phase to the
diagonal, indicating that these arose from
positive NOE enhancements as anticipated
for a molecule of this size (Mþ 540.4)
under ambient conditions. H-19 at dH 2.76
(m, 1H) showed strong correlations
(Figure 3) with b-H-12 at dH 1.03 (m,
1H), b-H-13 at dH 1.66 (td, 1H, J ¼ 12.2,
C28 ZH2 group, each appeared as a doublet
at dH 3.83 (d, 1H, J ¼ 10.6 Hz) and 3.38 (d,
1H, J ¼ 10.6 Hz). All the five methyls
appeared between dH 0.75 and 0.95 along
with other peaks of lupane skeleton. The
13C NMR spectrum (Table 1) was also in
good agreement with the proposed struc-
ture of compound 9 (Table 1). Deprotection
of C-28 hydroxyl group finally afforded a
white amorphous solid X (44% overall
yield), mp 288–2908C.
The IR spectrum gave peaks at 3424
(ZOH), 1725 ( .CvO), 1706, 1697
(conjugated aldehyde), 1461, 1380 (gem
dimethyl), and 1163, 987 cm21. The
molecular formula was established as
C30H46O3 by HR-EI-MS (m/z 554.38
[M]þ, calcd. 554.34). In the 1H NMR
spectrum, five tertiary methyl groups
appeared at dH 0.91 (s, 3H), 0.93 (s, 3H),
1.01 (s, 3H), 1.06 (s, 3H), and 1.15 (s, 3H).
Two olefinic C-29 protons appeared at dH
6.91 (s, 1H) and 6.28 (s, 1H). The singlet at
dH 9.52 (s, 1H) was due to the aldehydic
proton of C-30. Two geminal protons of
C28–H2 appeared at dH 3.78 (d, 1H,
J ¼ 10.6 Hz) and 3.36 (d, 1H,
J ¼ 10.6 Hz). The 13C spectrum revealed
the presence of two olefinic carbons at dc
133.6 (C-29) and 156.2 (C-20). The
aldehydic C-30 carbon appeared at dc
194.6. C-3 appeared at dc 218.1 and C-28
appeared at dc 60.2.
Thus, the presence of C-30 aldehyde
and C20ZC29 double bond is obvious from
the above 13C and 1H NMR spectra of
compound X. However, the shift in d
values for carbons C-12, C-13, C-17, C-18,
C-19, and C-21 (Table 1) in compound X in
comparison with that observed for betu-
linic acid [17] and lupeol [18] may be
either because of the isomeric nature of the
attached isopropenyl group of the cyclo-
pentane ring at C-19 of lupane skeleton
[18] or because of the introduction of
C-30 ZCHO group during the SeO2
oxidation of the C-30 methyl [18]. At this
H
OHC
OH
O
10
Figure 2. Epimeric form of compound 1.

Journal of Asian Natural Products Research
147
Therefore, the stereochemistry at C-19
would be the same as 6 for all the
subsequent molecules (7, 8, 9, and 1)
derived from it.
H
R
H
H
H
CH2OAc
CHO
2.2 Antileukemic activity
In this study, compounds were subjected to
cytotoxic assay against human K562
leukemia, murine WEHI3 leukemia, and
murine MEL erythroid progenitor, and the
assays were carried out in three indepen-
dent experiments as per the guidelines of
biosafety committee of West Bengal State
University (Figures 4–6). To determine
whether the compounds had any effect on
cell lines, cell cultures were incubated
with various concentrations of compounds
(dissolved in 0.1% v/v DMSO). DMSO
had no effect on the growth of cell lines at
a final concentration of 0.1% (v/v).
Compounds 1, 2, 3, 5, and 7 had
differential effects on the growth of the
cell lines. The effect of 4 and 6 could not
be checked due to its poor solubility in
DMSO.
AcO
C
R =
CH
2
Figure 3. Key NOESY correlations of
compound 6.
3.6 Hz), and b-H-21 at dH 2.18 (m, 1H).
The NOE observed in the NOESY
spectrum of compound 6 indicated the
involvement of b-H-12 at dH 1.03 (m, 1H)
and b-H-13 at dH 1.66 (td, 1H, J ¼ 12.2,
3.6 Hz) with b-H-21 at dH 2.18 (m, 1H).
These observations may be explained by
considering the H-19 configuration as b as
depicted in Figure 3. Additionally, b-H-13
at dH 1.66 (td, 1H, J ¼ 12.2, 3.6 Hz)
showed positive NOE with b-H-12 at dH
1.03 (m, 1H; Figure 2). From these data, it
can be concluded that the original stereo-
chemistry at C-19 was retained during
SeO2 oxidation on 5 and the shift in
chemical shift values for C-12, C-13,
C-17, C-18, C-19, and C-21 in the 13C
NMR spectrum of subsequent oxidized
products with respect to that for lupane
skeleton is due to the angular dependence
through space effects, such as the aniso-
tropic magnetic susceptibility and/or elec-
tric field effect [16], offered by the
conjugated carbonyl group. The through
space distance is obviously more import-
ant than the number of intervening bonds
since the chemical shift changes are much
smaller for C-13 than for C-12, which is
one bond further away but closer in space
[23]. SeO2 oxidation of compound 5 to 6
may only be considered as the step where
the stereochemical change at C-19 would
have occurred, since in all other sub-
sequent steps no reaction was carried out
that can alter the stereochemistry at C-19.
All the compounds showed potent
activities against the entire cell lines used;
although the activity against murine MEL
erythroid progenitor was not as good as for
other two cell lines (Figure 5). However, 1
was the most cytotoxic against murine
MEL erythroid progenitor. The anti-pro-
liferative effect of all the compounds
helped us to predict some of the struc-
ture–activity relationships. A comparison
of bioassay data between 2 and 3 revealed
that compound 2 was more active than 3
against human K562 leukemia and murine
WEHI3 leukemia. The only difference
between their structures was at C-28.
Compound 2 had a ZCOOH group and 3
had a ZCOOCH3 group, i.e. more polar
grouping is necessary (ZCOOH) to have
high activity. Incorporation of aldehyde
group at C-30 had increased the activity
further. The study also revealed that against
human K562 leukemia and murine WEHI3
leukemia cell lines, 7 showed better

148
P. Ghosh et al.
3
5
7
DMSO
5µg/ml
10µg/ml
DMSO
5µg/ml
10µg/ml
DMSO
5µg/ml
10µg/ml
8
6
4
2
0
8
8
6
4
2
0
6
4
2
0
Day2
Day4
Day6
Day2
Day4
Day6
Day2
Day4
Day6
1
2
DMSO
5µg/ml
10µg/ml
DMSO
5µg/ml
10µg/ml
8
6
4
2
0
8
6
4
2
0
Day2
Day4
Day6
Day2
Day4
Day6
Figure 4. Anti-proliferative effect of 3, 5, 7, 1, and 2 on WEHI3 cells.
activities than 1. A closer look at the
structures of these two compounds
revealed that they had a difference in their
structures only at C-3 and the data indicated
that ZOH group at C-3 was the one which
contributed to higher activity.
3. Experimental
3.1 General experimental procedures
[a]D was measured in Autopol III Auto-
matic Polarimeter. Melting points were
recorded by open capillary method and are
uncorrected. IR spectra were recorded in
3
5
7
35
35
30
25
20
15
10
5
35
30
25
20
15
10
5
DMSO
5µg/ml
10µg/ml
DMSO
5µg/ml
10µg/ml
DMSO
5µg/ml
10µg/ml
30
25
20
15
10
5
0
0
0
Day2
Day4
Day6
Day2
Day4
Day6
Day2
Day4
Day6
1
2
35
35
30
25
20
15
10
5
DMSO
5µg/ml
10µg/ml
30
25
20
15
10
5
DMSO
5µg/ml
10µg/ml
0
0
Day2
Day4
Day6
Day2
Day4
Day6
Figure 5. Anti-proliferative effect of 3, 5, 7, 1, and 2 on MEL cells.

Journal of Asian Natural Products Research
149
3
5
7
DMSO
5µg/ml
10µg/ml
DMSO
5µg/ml
10µg/ml
DMSO
5µg/ml
10µg/ml
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
Day2
Day4
Day6
Day2
Day4
Day6
Day2
Day4
Day6
1
2
DMSO
5µg/ml
10µg/ml
DMSO
5µg/ml
10µg/ml
8
6
4
2
0
8
6
4
2
0
Day2
Day4
Day6
Day2
Day4
Day6
Figure 6. Anti-proliferative effect of 3, 5, 7, 1, and 2 on K562 cells.
Shimadzu 800 FT-IR spectrophotometer
and NMR spectra were recorded in Bruker-
Avance 300 MHz FT NMR spectrometer
located in the Department of Chemistry,
University of North Bengal, India – 734
013. IR spectra were recorded using both
the KBr disk and nujol and the NMR
chemical shift was reported in ppm relative
to 7.20 and 77.0 ppm of CDCl3 solvent as
the standards. 1H spectra were recorded at
300 MHz frequencies and 13C NMR
spectra were recorded at 75.4 MHz fre-
quencies. Coupling constant ‘J’ was
calculated in Hz. Betulinic acid was
isolated from the outer bark of B. javanica
through soxhlet apparatus in toluene. All
the chemicals used were of commercial
grade and were purified prior to their use.
The mass spectra were recorded in 4800
(ABSciex) MALDI-TOF/TOF Tandem
Mass Spectrometer and elemental analyses
were carried out in Vario EL-III from
CDRI, Lucknow, India – 226 001.
conical flask. To this an excess of
diazomethane dissolved in ether at very
low temperature (0–58C) was added
slowly with constant shaking. The whole
reaction sequence was carried out in a
fume cupboard. The resultant yellowish
solution was kept overnight under dark-
ness. After that 2 ml of glacial acetic acid
was added to this to neutralize the traces of
diazomethane. The solution was then
diluted with cold water and extracted
with ether. Evaporation of solvent at
vacuum gave a gummy residue that was
purified over a column of silica gel
(60–120 mesh).
Compound 3 was obtained (2.9 g, 98%)
as a white solid with mp 222–2248C and
[a]D þ 5.0 (CHCl3). The IR spectrum gave
peaks at 3540 (ZOH), at 1733 (ZCOOMe),
and at 1660 and 890 (vCH2) cm21
.
Elemental analysis: found: C, 78.79%, H,
10.52%; calcd for C31H50O3: C, 79.10%, H,
10.71%. 1H NMR dH 0.75 (s, 3H), 0.81 (s,
3H), 0.91 (s, 3H), 0.96 (s, 3H), 1.40 (s, 3H),
1.68 (s, 3H), 2.99 (m, 1H, H-C-3), 3.18 (dd,
1H, J ¼ 5.1, 10.8 Hz, H-3), 3.66 (s, 3H,
ZOMe), 4.59 (s, 1H, H-C-29), and 4.73 (s,
1H, H-C-29). The compound was found
3.2 Methyl betulinate (3)
Three grams (6.58 mmol) of betulinic acid
were dissolved in diethyl ether (100 ml) at
very low temperature (0–58C) in a 250 ml

150
P. Ghosh et al.
identical to an authentic sample (co-TLC,
mixed mp).
washed with 6 N HCl and again with water
till neutral. The residue obtained after
evaporation of the solvent at reduced
pressure was dried and purified over a
column of silica gel (60–120 mesh).
Compound 5 was obtained (1.6 g,
94%) as a white solid with mp 222–
2238C and [a ]D þ 22 (CHCl3; 0.4 g/ml).
The IR spectrum gave peaks at 1735
(ZOCOCH3), 1639, 1459, and 1370 (gem
dimethyl), and 1243, 1027, 979, and 889
(vCH2) cm21. Elemental analysis: found:
C, 77.46%, H, 10.31%; cacld for
C34H54O4: C, 77.52%, H, 10.33%. 1H
NMR dH 0.75 (s, 3H), 0.81 (s, 3H), 0.91 (s,
3H), 0.96 (s, 3H), 1.59 (s, 3H), 1.65 (s,
3H), 2.43 (m, 1H, H-C-18), 3.84 (d, 1H,
J ¼ 11.1 Hz, H-C-28), 4.24 (d, 1H,
J ¼ 11.1 Hz, H-C-28), 4.46 (m, 1H, H-C-
3), and 4.59 (s, 1H), 4.68 (s, 1H). Acetate
peaks appeared at dH 2.04 (s, 6H).
The compound was found identical to an
authentic sample (co-TLC, mixed mp,
etc.).
3.3 Lup-20(29)-en-3b,28-diol (4)
Compound 3 (2.8 g, 5.95 mmol) was added
to 50 ml of dry THF in a 150 ml round-
bottomed flask. The solution was hom-
ogenized and to this LAH (75.28 mg,
1.98 mmol) was added in small lots at low
temperature (10–158C), and the solution
was stirred for 15 min. Stirring was
continued for another 3 h at room tempera-
ture and saturated solution of sodium
sulfate was added dropwise till the excess
LAH was destroyed. The reaction mixture
was poured into 200 ml cold water and
extracted with diethyl ether and dried over
anhydrous sodium sulfate. Evaporation of
solvent gave a residue that was then
purified over a column of silica gel
(60–120 mesh).
Compound 4 was obtained (76%) as a
white solid with mp 256–2578C and
[a]D þ 16 (10% v/v MeOH in CHCl3;
0.35 g/ml). The IR spectrum gave peaks
at 3393 (ZOH), 1453 and 1375 (gem
dimethyl), and 1229, 1029, 942, and 889
3.5 Lup-20(29)-en-3b,28-diyl acetate-
30-al (6)
1
(vCH2) cm21. H NMR dH 0.75 (s, 3H),
Compound 5 (1.5 g, 2.85 mmol) was
dissolved in 10 ml of aqueous dioxan and
20 ml of SeO2 was added to it. The
resultant reaction mixture was refluxed for
2 h and after cooling poured into 100 ml
ice-cold water. A curdy white precipitate
developed. After usual work up with
diethyl ether, it was dried over anhydrous
sodium sulfate and the solvent was
evaporated under reduced pressure. The
yellow gummy residue obtained was
purified over a column of silica gel
(60–120 mesh).
0.81 (s, 3H), 0.91 (s, 3H), 0.96 (s, 3H),
1.37 (s, 3H), 1.68 (s, 3H), 2.43 (m, 1H,
H-C-18), 3.84 (d, 1H, J ¼ 11.1 Hz,
H-C-28), 4.24 (d, 1H, J ¼ 11.1 Hz, H-C-
28), 4.46 (m, 1H, H-C-3), 4.59 (s, 1H),
4.68 (s, 1H). The compound was found
identical to an authentic sample (co-TLC,
mixed mp, etc.).
3.4 Lup-20(29)-en-3b,28-diyl
acetate (5)
Compound 4 (1.8 g, 4.06 mmol) was
dissolved in 50 ml dry pyridine in a
100 ml round-bottomed flask and to this
10 ml acetic anhydride was added. The
reaction mixture was warmed under water
bath for 6 h. After cooling, it was poured
into 150 ml ice-cold water and extracted
with diethyl ether. The ether layer was
Compound 6 was obtained (62%) as a
white solid with mp 246–2488C. IR nmax
:
1732 (ZOCOCH3), 1691 (.CvCZCHO)
1459, 1369 (gem dimethyl), 1244, 1028,
978, 936, 889 (vCH2) cm21. 1H NMR: dH
0.93 (s, 3H), 0.94 (s, 3H), 1.02 (s, 3H), 1.06
(s, 3H), 1.39 (s, 3H), 6.28 (s, 1H, H-C-29),
5.93 (s, 1H, H-C-29), 4.24 (d, 1H,

Journal of Asian Natural Products Research
151
J ¼ 10.6 Hz, H-C-28) and dH 3.68 (d, 1H,
J ¼ 10.6 Hz, H-C-28), 9.56 (s, 1H, alde-
hyde hydrogen at C-30). Two acetyl
methyls appeared at dH 2.07 (s, 3H) and at
2.03 (s, 3H). C-3 hydrogen appeared as a
broad multiplet centeredat dH 4.44 (m, 1H).
Elemental analysis: found: C, 75.42%, H,
9.56%; cacld for C34H52O5: C, 75.51%, H,
9.69%.
ified by column chromatography over
silica gel (60–120 mesh).
Compound 8 was obtained (72%) as a
white solid, mp 260–2628C, IR nmax: 3461
(ZOH), 1732 (ZOCOCH3), 1691 (.
CvCZCHO), 1459, 1369 (gem dimethyl),
1244, 1028, 978, 936, 890 (vCH2) cm21
.
1H NMR dH 0.75 (s, 3H), 0.80 (s, 3H), 0.89
(s, 3H), 0.92 (s, 3H), 0.95 (s, 3H), 2.1 (s, 3H,
acetate methyl), 3.18 (m, 1H, proton at C-
3), 3.38 (d, 1H, J ¼ 10.6 Hz, H-C-28), 3.83
(d, 1H, J ¼ 10.6 Hz, H-C-28), 5.94 (s, 1H,
H-C-29), 6.32 (s, 1H, H-C-29), 9.56 (s, 1H,
aldehyde hydrogen at C-30). Elemental
analysis: found: C, 77.02%, H, 10.12%;
calcd for C32H50O4: C, 77.06%, H, 10.10%.
3.6 Lup-20(29)-en-3b,28-diol-30-al (7)
Compound 6 (900 mg, 1.66 mmol) was
refluxed with 10% (w/v) alcoholic KOH
solution for 4 h. After completion of the
reaction, it was poured into 100 ml ice-
cold water. After usual work up with
diethyl ether, the ether layer was washed
several times with water, dried over
anhydrous sodium sulfate, and the recov-
ered material was purified by column
chromatography over silica gel (60–120
mesh).
3.8 28-Acetoxy-3-oxolup-20(29)-en-30-
al (9)
Compound 8 (150 mg, 0.30 mmol) was
dissolved in dry pyridine (30 ml). Two
hundred and thirty milligrams (2.91 mmol)
of dry CrO3 were added in small lots and
the reaction mixture was kept overnight. It
was then poured into ice-cold water and
the resultant yellowish solid was extracted
with ether, washed with 6 N HCl and again
with water till neutral, and dried over
anhydrous magnesium sulfate to get a
gummy residue. Purification of the gum
over a column of silica gel (60–120 mesh)
yielded compound 9.
Compound 7 was obtained (756 mg,
84%) as a white solid, mp 274–2768C,
IR nmax: 3393 (ZOH), 1688 (.CvCZ
CHO), 1453, 1375 (gem dimethyl), 1029,
942, 890 (vCH2) cm21. 1H NMR: dH 0.75
(s, 3H), 0.81 (s, 3H), 0.95 (s, 3H), 1.00 (s,
3H), 1.38 (s, 3H), 2.62 (m, 1H, hydrogen at
C-3), 3.15 (m, 1H, proton of-OH at C-3),
3.38 (d, 1H, J ¼ 10.6 Hz, H-C-28), 3.83
(d, 1H, J ¼ 10.6 Hz, H-C-28), 5.94 (s, 1H,
H-C-29), 6.32 (s, 1H, H-C-29), 9.56 (s, 1H,
aldehyde hydrogen at C-30). Elemental
analysis: found: C, 78.66%, H, 10.52%;
calcd for C30H48O3: C, 78.90%, H, 10.59%.
Compound 9 was obtained (84 mg,
56%) as a white solid, mp 276–2788C.
IR nmax: 1730 (.CvO), 1706, 1697
(.CvCZCHO), 1461, 1380 (gem
dimethyl), 1244, 1163, 987, 890 (vCH2)
cm21. Elemental analysis: found: C,
77.28%, H, 9.62%; calcd for C32H48O4:
3.7 3b-Hydroxy-28-acetoxy-lup-
20(29)-en-30-al (8)
1
A solution of 7 (350 mg, 0.76 mmol) in
CHCl3 (10 ml) and pyridine (15 ml) was
treated with Ac2O (10 ml, 0.098 mmol) at
very low temperature (0–58C) for 12 h.
The solution was diluted with CHCl3
(20 ml), and then the organic layer was
washed with saturated aq. NaCl
(10 ml £ 3), dried, concentrated, and pur-
C, 77.38%, H, 9.74%. H NMR dH 0.75
(s, 3H), 0.80 (s, 3H), 0.89 (s, 3H), 0.92
(s, 3H), 0.95 (s, 3H), 2.17 (s, 3H, acetate
methyl at C-28) 3.38 (d, 1H, J ¼ 10.6 Hz,
H-C-28), 3.83 (d, 1H, J ¼ 10.6 Hz,
H-C-28), 5.92 (s, 1H, H-C-29), 6.23
(s, 1H, H-C-29), 9.56 (s, 1H, aldehyde
hydrogen at C-30).

152
P. Ghosh et al.
3.9 28-Hydroxy-3-oxolup-20(29)-en-
30-al (1)
supplemented with 1% penicillin–strepto-
mycin, and were incubated at 378C under
5% CO2 atmosphere. In these experiments,
cells were seeded in quadruplicate in 24-
well plates (105 cells/mL) with the com-
pounds (1, 2, 3, 5, and 7) dissolved in
DMSO (0.1% v/v) at various concen-
trations. The effect of 4 and 6 could not
be checked due to its poor solubility in
DMSO. The cells were incubated for 2, 4,
and 6 days. Growth of cells was monitored
by counting the number of live cells
microscopically using Neubauer hemocyt-
ometer by trypan blue exclusion method.
Statistical analyses for all experiments
were carried out by Student’s t-test using
the program SigmaPlot.
Compound 9 (75 mg, 0.15 mmol) was
refluxed with 10% alcoholic KOH solution
for 4 h. After completion of the reaction, it
was poured into 100 ml ice-cold water.
After usual work up with ether, the ether
layer was washed several times with water,
dried over anhydrous magnesium sulfate,
and the recovered material was purified by
column chromatography (silica gel,
60–120 mesh).
Compound 1 was obtained (80.6 mg,
96%) as a white amorphous solid;
[a]D þ 16.3 (MeOH), mp 288–2908C.
IR nmax: 3424 (ZOH), 1725 (.CvO),
1706, 1697 (.CvCZCHO), 1461, 1380
(gem dimethyl), 1163, 987 cm21 1H
.
NMR: dH 0.91 (s, 3H), 0.93(s, 3H), 1.01
(s, 3H), 1.06 (s, 3H), and 1.15 (s, 3H), 6.91
(s, 1H, H-C-29), 6.28 (s, 1H, H-C-29), 3.36
(d, 1H, J ¼ 10.6 Hz, H-C-28), 3.78 (d, 1H,
J ¼ 10.6 Hz, H-C-28), 9.52 (s, 1H, alde-
hyde hydrogen at C-30). 13C NMR spectral
data are depicted in Table 1. HR-EI-MS:
m/z 554.38 [M]þ (calcd for C30H46O3,
554.34). Elemental analysis: found: C,
79.18%, H, 10.21%; calcd for C30H46O3:
C, 79.25%, H, 10.20%.
Acknowledgement
Financial support from UGC, New Delhi, India,
is cordially acknowledged for carrying out the
work.
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