Selective oxidation of betulin for the preparation of betulinic acid, an antitumoral compound

Article abstract of DOI:10.1081/SCC-200034788

This paper describes a semisynthetic approach for the preparation of betulinic acid from betulin. The main step of this synthetic approach is the selective oxidation of primary alcohol function of betulin. This reaction is accomplished with chromic oxide adsorbed on silica gel to obtain betulinal in an adequate yield. Betulinal is then almost quantitatively oxidized to betulinic acid by potassium permanganate action.

Full text of DOI:10.1081/SCC-200034788

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Selective Oxidation of
Betulin for the Preparation of
Betulinic Acid, an Antitumoral
Compound
a
a b
André Pichette , Hongyan Liu
, Christian Roy
b
a
a
,
Steve Tanguay , François Simard & Serge
a
Lavoie
a
Laboratoire LASEVE, Département des
sciences fondamentales, Université du Québec
à Chicoutimi, 555 Boulevard del'Université,
Chicoutimi, Québec, G7H 2B1, Canada
b
Département de génie chimique, Université
Laval, Sainte‐Foy, Québec, Canada
Version of record first published: 10 Jan 2011.
To cite this article: André Pichette, Hongyan Liu, Christian Roy, Steve Tanguay,
François Simard & Serge Lavoie (2004): Selective Oxidation of Betulin for
the Preparation of Betulinic Acid, an Antitumoral Compound, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 34:21, 3925-3937
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SYNTHETIC COMMUNICATIONS
Vol. 34, No. 21, pp. 3925–3937, 2004
Selective Oxidation of Betulin for the
Preparation of Betulinic Acid, an
Antitumoral Compound
1,*
1,2
Andr e´ Pichette, Hongyan Liu, Christian Roy,
2
1
Steve Tanguay, Fran c¸ ois Simard, and
1
1
Serge Lavoie
1
Laboratoire LASEVE, D e´ partement des sciences fondamentales,
Universit e´ du Qu e´ bec a` Chicoutimi, Chicoutimi, Qu e´ bec, Canada
2
D e´ partement de g e´ nie chimique, Universit e´ Laval, Sainte-Foy,
Qu e´ bec, Canada
ABSTRACT
This paper describes a semisynthetic approach for the preparation of
betulinic acid from betulin. The main step of this synthetic approach is
the selective oxidation of primary alcohol function of betulin. This reac-
tion is accomplished with chromic oxide adsorbed on silica gel to obtain
*
Correspondence: Andr e´ Pichette, Laboratoire LASEVE, D e´ partement des sciences
fondamentales, Universit e´ du Qu e´ bec a` Chicoutimi, 555 Boulevard del’Universit e´ ,
Chicoutimi, Qu e´ bec G7H 2B1, Canada; E-mail: andre_pichette@ugac.ca.
3
925
DOI: 10.1081/SCC-200034788
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

3
926
Pichette et al.
betulinal in an adequate yield. Betulinal is then almost quantitatively
oxidized to betulinic acid by potassium permanganate action.
Key Words: Betulinic acid; Chromium oxide; Oxidation; Solid sup-
ported reagent.
INTRODUCTION
Betulinic acid is a lupane type of triterpene possessing several biological
In 1995, interest in this compound heightened when Pisha et al.
[1–8]
activities.
[2]
demonstrated that it selectively attacked melanoma cells. In addition, it was
shown that betulinic acid intervened by a mechanism of apoptosis, a process of
[
2]
cellular disassembly. The high toxicity of betulinic acid towards cancerous
cells and its weak toxicity towards healthy cells show it to be a promising anti-
tumor agent. As a result, this compound was included in a Rapid Access to
Intervention Development (RAID) program by the National Cancer Institute
(
NCI) in order to obtain the authorization to carry out clinical trials on
humans. Preclinical studies require the establishment of efficient production
methods that meet the requirements of this type of study with regard to
product purity, yield, and a few other factors.
Betulinic acid has been found in certain plants but only in small
[9]
amounts. An interesting method of preparation of this substance was
[
10]
achieved by Pezzuto et al.
Their semisynthetic approach involved the
use of the natural product betulin, a lupane type triterpene possessing a
primary and a secondary alcohol function and which is very abundant in the
[
11]
bark of several species of birch trees.
using Jone’s reagent to betulonic acid followed by reduction of the ketone
In this approach, betulin is oxidized
[10]
function with NaBH in order to obtain betulinic acid.
4
Even though these
two reactions give good yields, the reduction step results in the formation
of a mixture of epimers that are difficult to separate. In order to circumvent
this problem, Pezzuto’s group proposed another synthetic route that involves
the use of protecting groups. This synthetic pathway is interesting but
relatively long as it involves five chemical reactions.
One of the aims of our research group is to develop a short, semisynthetic
route to betulinic acid that would avoid the problems of separating epimers
encountered in the approach using Jones reagent. One method involves the
selective oxidation of the primary alcohol function of betulin to obtain
[
12]
the aldehyde betulinal.
acid. Pezzuto et al. tried to selectively oxidize betulin using mild oxidizing
Betulinal is then selectively oxidized to betulinic
[
12]
agents such as PDC and PCC, but without success.
Recently, the selective

Selective Oxidation of Betulin
3927
oxidation of betulin with PDC was reported; however, based on the results, the
[
13]
yields were less than 34%.
In the course of the last few years, several oxidizing reactions using an
[14]
oxidizing agent adsorbed on a solid support were reported in the literature.
In several cases, these reagents showed numerous advantages such as selectiv-
ity and faster rates of reaction. Moreover, the work-up procedures involved in
these heterogeneous reactions are often easier to carry out than those done
[
15]
under homogeneous conditions.
Based on the knowledge acquired from
the several chromium-based oxidizing reagents that have been studied
and considering the low cost of these reagents, a study was undertaken to
evaluate their potential as selective oxidizing reagents of betulin in order to
develop a synthetic pathway to betulinic acid. In this study, we present the
results of our studies on the selective oxidation of betulin using a reagent
adsorbed on solid supports as part of our work on developing a synthesis of
betulinic acid.
RESULTS AND DISCUSSION
A study based on the selective oxidation of primary alcohols with CrO3
adsorbed on SiO (CrO /SiO ) led us to believe that a selective oxidation
2
3
2
[
16]
of betulin could be achieved.
possess selectivity similar to other oxidizing reagents such as PCC, PDC,
Initial experiments showed this reagent to
Swern, TPAP/NMO, and Dess-Martin. A more systematic study was then
undertaken to evaluate the potential of using CrO on a solid support as a
3
selective oxidizing agent. A literature search revealed that a large variety of
adsorbents can be used as a solid support and that the nature of the adsorbent
has an impact on the selectivity and the yield. The influence of the various
solid supports was evaluated by testing common adsorbents under normal
reaction conditions (DCM, RT, etc.). As is shown in Table 1, silica gel
gave the best yield of betulinal and good selectivity under the reaction
conditions shown. Even though only the primary alcohol was oxidized
when using K10 and celite, the yield of betulinal was very small.
Previous reports indicated that the preparation method of the reagent may
[17]
have a significant impact on the selectivity and yield.
Four different
methods of preparation were tested. The first two methods involved grinding
the CrO with the silica in a mortar and activating the reagent for 4 hours at
3
different temperatures (methods A and B). The other two methods consisted
in dissolving the CrO in acetone and adding the silica gel. After evaporating
3
the solvent, the reagent can be used directly (method C) or activated for 4
hours at 1008C (method D). The results of these trials carried out in DCM are
presented in Table 2. The yields of betulinal using the reagents prepared by

3
928
Pichette et al.
Table 1. Effect of the solid support on the oxidation reaction of betulin with adsorbed
CrO as the oxidant.
3
Betulinal
b
yield (2nd
d
reaction )
Betulinal
b
yields
Unreacted
b
betulin
a
c
Solid support
Selectivity
Silica gel
37%
4%
55%
96%
94%
87%
5.26 : 1
—
—
58%
8%
Montmorillonite K10
Celite
Alumina
6%
11%
11%
21%
5.66 : 1
a
Reaction in DCM with 0.23 mmol betulin, 0.45 mmol CrO , 180 mg adsorbent, RT,
3
6
0 min.
Determined by GC.
b
c
Ratio indicating oxidation of primary alcohol vs. secondary alcohol.
d
Total amount expected after oxidation of unreacted betulin from the first reaction
assuming identical yields).
(
methods A and C are similar but much greater than the yields obtained using
the reagents prepared by methods B and D. However, it is important to note
that the amount of unreacted betulin recovered with the reagent prepared
by method A is smaller than the amount that was collected with the reagent
prepared by method C. This latter method results in the formation of fewer
secondary products (Fig. 1) and is thus more selective. The larger amount of
betulin recovered in method C can then be used in a second oxidation reaction
resulting in a higher yield.
Table 2. Influence of the reagent preparation method on the oxidation of betulin with
CrO
3
2
/SiO .
Betulinal
b
yield (2nd
d
reaction )
Betulinal
b
yield
Unreacted
b
betulin
a
c
Method
Selectivity
A
B
C
D
39%
27%
38%
16%
12%
45%
36%
60%
1.67 : 1
1.74 : 1
2.35 : 1
1.67 : 1
44%
39%
52%
26%
a
Reaction in DCM with 0.23 mmol betulin, 1.13 mmol CrO , 452 mg SiO , RT, 90 min.
3
2
b
c
Determined by GC.
Ratio indicating oxidation of primary alcohol vs. secondary alcohol.
d
Total amount expected after oxidation of unreacted betulin from the first reaction
assuming identical yields).
(

Selective Oxidation of Betulin
3929

3
930
Pichette et al.
The yield and selectivity of this type of reaction is also affected by the solvent
[18]
used. The oxidation of betulin was carried out in different solvents using the
reagent prepared by method C. Table 3 shows that the use of moderate to high
polarity solvents (THF-DMSO) gives low yields and poor selectivity. Of the
various solvents tried, toluene gave the best yields and the highest selectivity.
The ratio of CrO to silica gel can also have an effect on the yield and
3
[
19]
selectivity.
1
The best yields and selectivity were obtained with a ratio of
: 10, respectively (Table 4).
Kinetic measurements were recorded during these latter experiments
Fig. 2). These measurements showed that under the best reaction conditions
(
the maximum quantity of betulinal (64%) was reached after 15 min. Under
these same conditions, the lowest amount of nonreacted betulin remaining
was 31% (results not shown). The remaining amount of betulin was recovered
and oxidized in a subsequent reaction.
Finally, betulinal must be quickly purified on alumina because of
its instability on silica gel. Ideally, the isolated betulinal should be used
immediately following this first oxidation reaction. Betulinal is oxidized to
betulinic acid in a yield greater than 85% when using KMnO in acetone at
4
08C. The duration of this reaction should not be extended beyond 10
minutes as numerous secondary products are formed.
To conclude, this paper describes the work carried out to develop a
synthetic approach for the preparation of betulinic acid from betulin. The
key step of this approach involves the selective oxidation of betulin to
betulinal by using chromium oxide adsorbed on silica gel. Among the
3 2
Table 3. Effect of solvent on the oxidation of betulin with the reagent CrO /SiO .
Betulinal
b
yield (2nd
d
reaction )
Betulinal
b
yield
Unreacted
b
betulin
a
c
Solvent
Selectivity
DCM
32%
43%
trace
30%
7%
48%
38%
95%
31%
90%
2.03 : 1
2.74 : 1
—
47%
59%
—
Toluene
DMSO
Acetone
THF
1.26 : 1
2.94 : 1
39%
14%
a
Reaction in solvent with 0.23 mmol betulin, 1.13 mmol CrO , 452 mg SiO , RT,
3
2
1
5 min.
Determined by GC.
b
c
Ratio indicating oxidation of primary alcohol vs. secondary alcohol.
d
Total amount expected after oxidation of unreacted betulin from the first reaction
assuming identical yields).
(

Selective Oxidation of Betulin
3931
3 2
Table 4. Effect of the CrO /SiO ratio on the oxidation reaction of betulin.
Betulinal
b
yield (2nd
d
reaction )
Betulinal
b
yield
Unreacted
b
betulin
a
c
Ratio
Selectivity
1
1
1
: 2
18%
64%
52%
71%
31%
30%
2.45 : 1
13.09 : 1
3.25 : 1
31%
84%
68%
: 10
: 20
a
Reaction in toluene with 0.23 mmol betulin, 0.45 mmol CrO
RT, 60 min.
3 2
, 90, 450 or 900 mg SiO ,
b
Determined by GC.
Ratio indicating oxidation of primary alcohol vs. secondary alcohol.
c
d
Total amount expected after oxidation of unreacted betulin from the first reaction
assuming identical yields).
(
Figure 2. Yields of betulinal as a function of time of oxidation of betulin and various
ratios of CrO /SiO . Reaction in toluene with 0.23 mmol betulin and 0.45 mmol
CrO , RT.
3
2
3

3
932
Pichette et al.
several reaction conditions experimented, the oxidation takes place in good
yield and with good selectivity when toluene is used as solvent and two
equivalents of CrO /SiO are used in a mass ratio of 1 : 10. Betulinal is oxi-
3
2
dized almost quantitatively to betulinic acid with KMnO in acetone at 08C.
4
EXPERIMENTAL SECTION
General Procedures
Alumina (Brockmann I, 150 mesh) and the solvents (toluene, dichloro-
methane, acetone, ethyl acetate, petroleum ether, hexane, and DMSO) were
purchased from Laboratoire MAT and used as received. Silica Gel (230–
˚
400 mesh, 60 A) and TLC plates (SiO layer thickness: 250 mm, 230–400
mesh, 60 A, with F2 fluorescent indicator) were purchased from Silicycle
2
˚
54
inc. The Celite was from Fisher, whereas the Montmorillonite K10 and
Chromium oxide were from Aldrich. NMR spectra were recorded on Bruker
(
300 MHz) instruments and chemical shifts are TMS-referenced.
Isolation of Betulin (2)
Finely ground birch bark (100 g) was macerated under reflux in 1 L of
chloroform. The mixture was filtered and then vacuum-evaporated to obtain
8 g of crude betulin. This extract was purified by silica gel chromatography
using a petroleum ether and ethyl acetate solution as eluent (90 : 10 to 85 : 15).
2
Betulin (17 g) was obtained as a white solid, which was .95% pure by GC and
showed only one spot on TLC (eluent: 15% EtOAc/Hex, R ¼ 0.15). Only
f
signals due to this compound were observed and were essentially identical
[
to the NMR spectrum reported in the literature. . H NMR (300 MHz,
20] 1
CDCl ) d 4.68 (1 H, d, H-29), 4.58 (1 H, d, H-29), 3.82 (1 H, d, J ¼ 7.8 Hz,
3
H-28), 3.33 (1 H, d, J ¼ 7.8 Hz, H-28), 3.19 (1 H, m, H-3), 2.39 (1 H, m,
H-19), 1.04–2.04 (26 H, complex, CH , CH), 1.68, 1.02, 0.98, 0.97, 0.82,
2
þ
0
5
.76 (18 H, all s, 6 ꢀ CH ); EIMS, m/z, 442 (M , 5%), 43 (100), 95 (96),
3
5 (96), 81 (95), 69 (84), 41 (81), 67 (79), 93 (79), 189 (76).
Reagent Preparation
The number of equivalents of the oxidant to be used depends on the
quantity of betulin used and takes into account the ratio of oxidant : adsorbent
of the reagent

Selective Oxidation of Betulin
3933
Method A
The required amount of oxidant was ground in a mortar with the
adsorbent. The resulting powder was activated in an oven at 1008C for 4 h.
Method B
The required amount of oxidant was ground in a mortar with the adsor-
bent. The resulting powder was activated in an oven at 2008C for 4 h.
Method C
The required amount of oxidant was dissolved in acetone (1 g/100 mL)
while maintaining the temperature of the solution between 5–158C
using an ice bath. WARNING! Adding the oxidant to acetone is highly
exothermic and may result in a violent combustion. The adsorbent was
added to the solution and the solvent was evaporated using a rotating
evaporator.
Method D
The required amount of oxidant was dissolved in acetone (1 g/100 mL)
while maintaining the temperature of the solution between 5–158C using an
ice bath. WARNING! Adding the oxidant to acetone is highly exothermic
and may result in a violent combustion. The adsorbent was added to the sol-
ution and the solvent was evaporated using a rotating evaporator. The powder
obtained was activated in an oven at 1008C for 4 h.
Oxidation of Betulin (2)
Betulin (100 mg, 0.23 mmol) was dissolved in a minimum of solvent
roughly 25 mL). The reagent was added to this solution and the resulting het-
(
erogeneous mixture was vigorously stirred for 15 to 60 minutes. The progress
of the reaction was followed by TLC (elution: 15% EtOAc/Hex, detection:
ammonium ceric molybdate). Once the reaction was completed, the solution
was filtered through SiO (3 cm) and rinsed with EtOAc. The filtrate was ana-
2
lyzed by GC-MS. A small quantity of alumina (400 mg) was then added to the
solution and the solvent was evaporated under vacuum. The powder obtained
containing a mixture of oxidized products was purified by chromatography on
alumina using as eluent solutions of EtOAc in hexane (10% to 25%). It was

3
934
Pichette et al.
important to use alumina chromatography as betulinal (3) was unstable on
silica gel.
Betulonal was eluted first as a white solid, which was .95% pure by
GC and showed only one spot on TLC (eluent: 15% EtOAc/Hex,
R ¼ 0.50). Only signals due to this compound were observed and were essen-
f
[21] 1
tially identical to the NMR spectrum reported in the literature.
(
H NMR
300 MHz, CDCl ) d 9.67 (1 H, d, J ¼ 1.7 Hz, H-28), 4.76 (1 H, d,
3
J ¼ 2.2 Hz, H-29), 4.63 (1 H, t, J ¼ 1.8 Hz, H-29), 2.88 (1 H, td, J ¼ 11.2
and 5.8 Hz, H-19), 2.44 (2 H, c, H-2), 1.10–2.12 (25 H, complex, CH and
2
CH), 1.70, 1.07, 1.02, 0.98, 0.95, 0.92 (18 H, all s, 6 ꢀ CH ); EIMS, m/z,
3
þ
4
38 (M , 3%), 55 (100), 41 (81), 81 (76), 67 (72), 79 (67), 69 (66), 43
63), 93 (63), 91 (61).
(
Betulinal was eluted next as a white solid, which was .95% pure by GC
and showed only one spot on TLC (eluent: 15% EtOAc/Hex, R ¼ 0.30).
f
Only signals due to this compound were observed and were essentially iden-
tical to the NMR spectrum recorded in the literature.
[22] 1
H NMR (300 MHz,
CDCl ) d 9.67 (1 H, d, J ¼ 1.5 Hz, H-28), 4.75 (1 H, d, J ¼ 1.8 Hz, H-29),
3
4
.62 (1 H, s, H-29), 3.18 (1 H, dd, J ¼ 10.7 and 5.8 Hz, H-3), 2.86 (1 H, td,
J ¼ 11.1 and 5.8 Hz, H-19), 1.00–2.11 (25 H, complex, CH and CH), 1.69,
2
þ
0
3
1
.97, 0.96, 0.91, 0.81, 0.73 (18 H, all s, 6 ꢀ CH ); EIMS, m/z, 440 (M ,
3
%), 43 (100), 55 (93), 41 (85), 81 (79), 69 (75), 95 (71), 67 (67), 93 (65),
89 (63).
The betulin was eluted with betulone and this fraction was used in a
second oxidation reaction.
Oxidation of Betulinal (3)
Due to its rapid degradation, betulinal was oxidized immediately after its
isolation. This was done by dissolving it (100 mg, 0.23 mmol) in 25 mL of
acetone at 08C followed by addition of 71.4 mg of KMnO (0.45 mmol).
4
The solution was stirred at room temperature for 30 minutes and then
7
5 mL of 10% H SO was added. Betulinic acid was extracted with DCM
2 4
(
then dried with Na SO . The white solid obtained was purified by silica gel
3 ꢀ 50 mL). The organic phase was washed with H O until neutral and
2 4
2
chromatography using a solution of EtOAc in hexane (25%) as eluent. The
purified betulinic acid was analyzed by NMR. Only signals due to this com-
pound were observed and were essentially identical to the NMR spectrum
[
23] 1
recorded in the literature.
H-29), 4.53 (1 H, s, H-29), 3.02 (1 H, c, H-3), 2.33 (1 H, m, H-19), 1.02–
.26 (26 H, complex, CH and CH), 1.66, 0.97, 0.93, 0.91, 0.82, 0.70 (18
H NMR (300 MHz, CDCl ) d 4.68 (1 H, s,
3
2
2
H, all s, 6 ꢀ CH3).

Selective Oxidation of Betulin
3935
GC-MS Analysis
GC-MS analyses were performed with a HP 5890 equipped with a
nonpolar HP-5MS column (30 m ꢀ 0.25 mm ꢀ 0.25 mm). Carrier gas was
helium at 1 mL/min. The temperature program was 1008C for 1 min, then
2
58C/min to 2808C, and 58C/min to 3258C and held constant for 30 min.
Injector and detector temperatures were 2808C and 3258C, respectively. The
compounds were identified by mass spectra using a HP5972 mass spec-
trometer at 70 eV.
The selectivity was calculated as a ratio of oxidation of primary alcohol
betulinal and betulonal) to oxidation of the secondary alcohol (betulone and
(
betulonal). In order to do this, the peak areas from the GC-MS were entered
into the following equation:
SBetulinal þ SBetulonal
Selectivity ¼
SBetulone þ SBetulonal
The yield of the second reaction was calculated by assuming that
unreacted betulin (2) was recuperated and oxidized in a second reaction.
yield2nd reaction ¼ yieldBetulinal þ ðyieldBetulinal ꢀ yieldBetulinÞ
ACKNOWLEDGMENTS
This project was supported by Fonds Qu e´ b e´ cois de la Recherche sur la
Nature et les Technologies (Fonds Forestiers), Scierie Thomas-Louis
Tremblay, and Clinique des Lipides du Complexe Hospitalier de la
Sagamie. We are also grateful to Prof. Fran c¸ ois-Xavier Garneau for his scien-
tific advices and discussions.
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Received in the USA May 12, 2004