Auxiliary-directed oxidation of ursolic acid by 'Ru'-porphyrins: Chemical modulation of cytotoxicity against tumor cell lines

Article abstract of DOI:10.1016/j.tetlet.2012.01.107

Derivatization of ursolic acid, one of the natural ursene-type pentacyclic triterpenes, was investigated by the oxidation with dioxoruthenium(VI) tetraphenylporphyrins. Oxidation selectivity on the terpene structure was modulated by the auxiliaries introd

Full text of DOI:10.1016/j.tetlet.2012.01.107

Tetrahedron Letters 53 (2012) 1756–1759
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Auxiliary-directed oxidation of ursolic acid by ‘Ru’-porphyrins: chemical
modulation of cytotoxicity against tumor cell lines
Katsunori Tanaka a, , Kishor Mazumder a, , Eric R.O. Siwu a, Satoshi Nozaki b, Yasuyoshi Watanabe b,
⇑
Koichi Fukase a,
⇑
a Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka-shi, Osaka 560-0043, Japan
b RIKEN Center for Molecular Imaging Science, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe-shi, Hyogo 650-0047, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Derivatization of ursolic acid, one of the natural ursene-type pentacyclic triterpenes, was investigated by
the oxidation with dioxoruthenium(VI) tetraphenylporphyrins. Oxidation selectivity on the terpene
structure was modulated by the auxiliaries introduced on the tetraphenylporphyrin. Chemical oxidation
of the ursolic acid affected the cytotoxic activity against tumor cell lines.
Received 21 December 2011
Revised 20 January 2012
Accepted 23 January 2012
Available online 1 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Ursolic acid
Dioxoruthenium(VI) tetraphenylporphyrin
Biomimetic oxidation
Cytochrome P450
Cytotoxicity
Natural products are the most consistently successful source of
biologically diverse compounds, and especially, the plant continu-
ously provides a large bank of rich, complex, and highly varied
structures.1 Many higher plants particularly contain the various
hydroxylated terpene metabolites through the action of the cyto-
chrome P4502,3; the resulting structural variants are responsible
for showing the diverse range of the biological activities.4 We have
investigated to isolate the natural products from the extracts of
Bangladeshi medicinal plant, Saurauja roxburghii, a higher plant
indigenous to South East Asia and some parts of North America.
By using the conventional extraction procedures, we have isolated
the several ursene-type pentacyclic triterpenes, including ursolic
acid 1,5 corosolic acid 2, 24-hydroxyl corosolic acid 3, and
7,24-dihydroxyl ursolic acid 4, from the leaves of the plant
(Fig. 1).6 Interestingly, despite these four compounds share the
same ursene-type pentacyclic triterpene skeleton, only ursolic acid
position and numbers of hydroxyls on the terpene structure affect
the activity and selectivity to the cancer cell lines.
Further trials on isolating the other oxidized ursene-type natu-
ral products from this plant, in pursuit of enhancing the cytotoxic-
ity and/or modulating the tumor selectivity, however, were not
feasible, because so many oxidized terpenes, which were similar
CO2H
CO2H
HO
HO
HO
2 (corosolic acid)
C6 (+), A431 (+)
1 (ursolic acid)
C6 (+), A431 (-)
1 and corosolic acid 2 showed the cytotoxicity at 10–100 lM con-
centrations (Fig. 1). While the corosolic acid 2 showed the cytotox-
icity against both C6 rat glioma and A431 human skin carcinoma
cell lines, the ursolic acid 1 exhibited the selective cytotoxicity
against the C6 glioma cell. These results clearly showed that the
CO2H
CO2H
HO
HO
HO
OH
⇑
OH
OH
Corresponding authors. Tel.: +81 6 6850 5391; fax: +81 6 6850 5419 (K.T.);
4
3
tel.: +81 6 6850 5388; fax: +81 6 6850 5419 (K.F.).
E-mail addresses: ktzenori@chem.sci.osaka-u.ac.jp (K. Tanaka), koichi@chem.
sci.osaka-u.ac.jp (K. Fukase)
C6 (-), A431 (-)
C6 (-), A431 (-)
Figure 1. Natural ursene-type triterpenes from Saurauja roxburghii and their
Assistant Professor in Department of Pharmacy, University of Science and
Technology Chittagong (USTC), Bangladesh.
cytotoxic activities against C6 rat glioma and A431 human skin carcinoma cell
lines at 10–100 lM.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.01.107

K. Tanaka et al. / Tetrahedron Letters 53 (2012) 1756–1759
1757
in physical and chemical properties, could not be separated one an-
other even by the repeated HPLC. We therefore decided to chal-
lenge in diversifying the ursene-type triterpenes by chemical
oxidation for further SAR studies. Herein, we report the auxiliary-
directed oxidation of the ursolic acid 1 by the dioxoruthenium(VI)
tetraphenylporphyrins. Distribution of the oxidized products was
affected by the auxiliaries on the porphyrin. Furthermore, the
chemical oxidation of the ursolic acid gave this natural product
an additional cytotoxic activity against A431 tumor cell line.
Although the selective oxidation of the hydrocarbons at the late
stage of the synthesis is the recent trend in natural products syn-
thesis,7 it still remains the challenging task to date, due to the scar-
city of reagents that efficiently and selectively oxidize the C–H
bonds, such as of the hydrophobic terpene skeleton 1. On the other
hand, biomimetic oxidation8 using the porphyrin derivatives,2a,9
that is, the mimic of cytochrome P450, is intriguing opportunity
for diversifying the core-structure of ursolic acid 1; in fact, the oxi-
dation of ursolic acid derivatives by ‘Fe’-porphyrin has previously
been examined by Konoike and co-workers.10 While the sub-
strate-directed C–H oxidation trials of the terpene or the steroid
natural products, as exemplified by the pioneering work of Breslow
and co-workers,11 have been reported,12 the chemical and site-
selectivities, which could be affected by the auxiliary on the oxi-
dant structures, that is, oxidation efficiency of the C–H, O–H, or
olefins at the different positions of the target substrates, has not
been much investigated. We therefore performed the oxidation
of 1 by using the seven ‘Ru’-porphyrins A–G (Scheme 1) which con-
tain the acid and the various chiral and non-chiral amides, in antic-
ipating that the additional auxiliaries might affect the oxidation
efficiency and selectivity. We used the tetraphenylporphyrin13
throughout this work, since the acid-functionalized tetraphenyl-
porphyrin, that is, 5 (Scheme 1), is available from the commercial
source14 and this porphyrin can easily be handled for the chemical
transformations, namely, easily derivatized and purified by silica
gel chromatography without the stacking problems.13
steps. Trans-dioxoruthenium(VI) tetraphenylporphyrins A–G were
then obtained in quantitative yield by oxidizing mCPBA in CH2Cl2,16
which were used immediately for the oxidation of 1 after rapid sil-
ica-gel chromatography and characterization by MALDI-TOF MS.
Unfortunately, the standard oxidation conditions using catalytic
or equimolar amounts of the dioxoruthenium(VI) porphyrins A–G
failed to give the desired oxidized products, but the starting ursolic
acid 1 was completely recovered. Since TLC analysis of the reaction
mixture detected the decomposition of the dioxoruthenium com-
plexes, we instead applied the in situ oxidation protocol using
more stable porphyrins a–g (Scheme 1),17 of which protocol was
successfully utilized by Nagano and co-workers for oxidizing the
steroids.18 Thus, under the optimized conditions (Table 1), the
ursolic acid 1 was treated with 0.03 equiv of porphyrins a–g in
the presence of 2,6-dichloropyridine N-oxide as the co-oxidant
(3 equiv) and 47% HBr in DMF at 50 °C for 3 days, providing the
variously oxidized products, of which spots on TLC were clearly
distinguished with one another; these are the lactone 6,19 C11-hy-
droxy 7,10,20 C11-ketone 8,21 dienes 9 and 10,22 and C3-ketone
11,23 which could easily be isolated by preparative TLC on silica
gel (Table 1). All of the compounds 6–11 were the known natural
and/or chemically derivatized compounds and their structures
were unambiguously determined by comparing with the literature
data (data shown in Supplementary data). While the compound 11
was obtained by the direct oxidation of the C3-hydroxyl group of 1,
the compounds 6–10 could be derived by the allylic oxidation at
C11. We also obtained the small amounts of the other oxidized
products, which might be derived from the oxidation of the unac-
tivated C–H bonds on the terpene skeleton, but enough amounts
could not be obtained to determine the structures.
Interestingly, the distribution of the oxidized products 6–11
was apparently affected by the auxiliaries of the ‘Ru’-porphyrins
a–g. Thus, the parent ‘Ru’-porphyrin a gave the lactone 6 (25%)
and the
entry 1), but the production of 7 was significantly decreased by
the acid derivative (4%), instead equally producing the
a-hydroxylated 7 (35%) as two major products (Table 1,
‘Ru’-tetraphenylporphyrin derivatives A–G were prepared as
shown in Scheme 1. Acid 5 obtained from the commercially available
methyl ester13 was activated as the succinimidyl ester in 86%, which
was reacted with the various amines to give the corresponding amides.
Ruthenium was incorporated by reacting with Ru3(CO)12 in hot deca-
lin,15 providing the dioxoruthenium precursors a–g in 20–30% for two
b
other derivatives 8–10 (each about 10%, entry 2); the introduction
of the acid group on the metalloporphyrin might cause the
interaction with the substrate 1, for example, with the carboxylic
acid or the hydroxy groups in 1, hence alter the oxidation
selectivity.
O
1)
HO
N
EDC / CH2Cl2, rt
86 %
NH
N
N
NH
N
N
O
R
R
CO2H
HN
HN
2) amines / CH2Cl2, rt
5
O
CO
N
N
Ru N
Ru N
Ru3(CO)12
mCPBA
CH2Cl2, rt
quant
R
decalin, reflux
20-30 % (2 steps)
N
N
N
N O
a-g
A-G
R =
O
O
O
O
O
O
(R)
(S)
(R)
(S)
H
OH
N
H
N
N
H
N
N
Hd,D
e,E
H f,F
g,G
H
a,A
b,B
c,C
Scheme 1. Synthesis of ‘Ru’-tetraphenylporphyrins.

1758
K. Tanaka et al. / Tetrahedron Letters 53 (2012) 1756–1759
Table 1
Oxidation of ursolic acid by ‘Ru’-porphyrin derivatives. Signs (+) and (À) represent the cytotoxic activities against C6 rat glioma and A431 human skin carcinoma cell lines
at 100
l
M
O
O
13
11
11
HO
11
O
CO2H
CO2H
CO
NRu N
R
Cl
HO
N
N
HO
N
O
Cl
HO
11
a-g
8: C6 (-), A431 (-)
7: C6 (-), A431 (-)
CO2H
6: C6 (+), A431 (+)
(0.03 eq)
(3 eq)
HO
47% HBr, 50 oC, DMF, 72h
1: C6 (+), A431 (-)
CO2H
HO
CO2H
CO2H
3
HO
O
9: C6 (-), A431 (-)
10: C6 (-), A431 (-)
11: C6 (-), A431 (-)
Entry
Porphyrin catalyst
Product yields (%)
1 (Recovery)
25
6
7
8
9
10
11
1
2
H a
25
35
5
ND
ND
ND
O
35
35
4
5
8
5
10
8
ND
10
b
OH
O
O
O
O
O
3
4
5
6
80
ND
ND
ND
c
N
H
d
ND
50
75
90
ND
ND
ND
ND
70
30
5
5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
(R)
(R)
(S)
(S)
N
H
e
g
10
5
N
H
f
N
H
7
10
ND
N
H
ND, Not detected.
On the other hand, while the (R)-isomers d and e gave the
-hydroxylated 7 as the major product (entries 4 and 5), the reactivity
against both A431 and C6 glioma cell lines at relatively higher
concentration, that is, at 100 M (Fig. 2); therefore the oxidation
a
l
of the corresponding (S)-oxidants f and g was significantly reduced
(entries 6 and 7). For both cases, the oxidation efficiency was retarded
by using the sterically more demanding 1-naphthyl derivatives e and g
(the starting 1 was recovered in 50% and 90%, respectively). Mean-
while, the ketone 11 was obtained only when the indane derivative
c was utilized (entry 3). This is the first observation that the auxiliaries
on metalloporphyrin-based oxidants a–g gave profound effects on the
oxidation reactivity and selectivity. Namely, the even small functional
groups at the side chain of the tertaphenylporphyrins, that is,
acid, amides, or chirality, could recognize the triterpene structure 1,
and exhibited the match and/or mismatch combinations for the
dioxoruthenium-catalyzed oxidation.
at C11 and C13 positions of ursolic acid 1, gave the ursolic acid
an additional cytotoxic activity against A431 tumor cell. As envi-
sioned, the modulation of the cytotoxic activity by the chemical
oxidation was thus achieved.
In conclusion, we could control the cytotoxic activity of the nat-
ural ursolic acid 1 against the tumor cells, by simply modifying the
oxidation states on the core terpene structure. In this research, the
chemical oxidation of natural terpenoids by ‘Ru’-tetraphenylpor-
phyrins was also developed; oxidation outcome could dramatically
be affected by slightly modifying the porphyrin side chain. Oxida-
tion results described in this Letter therefore could give a new direc-
tion in designing the porphyrin-based as well as the other oxidants.
It is noted that the chemically oxidized products 6–11 were all nat-
urally occurring compounds, but were difficult to isolate from the S.
roxburghii as investigated previously,6 thus the chemical protocol
can be complementary to the natural products isolation.
Finally, the cytotoxic activity4e of the chemically oxidized
derivatives was tested against C6 rat glioma and A431 human
skin carcinoma cell lines (Fig. 2 and Table 1).6 Out of the six
compounds 6–11, cytotoxicity was observed only for lactone 6

K. Tanaka et al. / Tetrahedron Letters 53 (2012) 1756–1759
1759
Figure 2. Cytotoxic effects of lactone 6 on C6 rat glioma and A431 human skin carcinoma cell lines. Cells were treated with the 100
lM (cell culture media) of the compound
at 37 °C and the morphological changes were observed by phase contrasts microscopy (200Â) over 48 h. (a) upper: control (media); lower: lactone 6 against C6 glioma. (b)
upper: control (media); lower: 6 against A431.
5. (a) Cheung, H.; Yan, T. Aust. J. Chem. 1972, 25, 2003–2012; (b) Doddrell, D. M.;
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This work was supported in part by Grants-in-Aid for Scientific
Research No. 19681024 and 19651095 from the Japan Society for
the Promotion of Science, Global COE program, Osaka University,
New Energy and Industrial Technology Development Organization
(NEDO, project ID: 07A01014a). We are thankful to Mr. Hiroshi
Adachi for NMR measurement.
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Supplementary data
Supplementary data (experimental procedures, characteriza-
tion data, and the representative 1H NMR spectra of porphyrins
a–g and oxidized products 6–11) associated with this article can
be found, in the online version, at doi:10.1016/j.tetlet.2012.01.107.
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