Anticancer effect of A-ring or/and C-ring modified oleanolic acid derivatives on KB, MCF-7 and HeLa cell lines

Article abstract of DOI:10.1039/c2ob06923g

New A-ring or/and C-ring modified methyl oleanolate derivatives were prepared. New simple method of synthesis of 3,12-diketone (3) from methyl oleanonate (2) was worked out. The obtained new compounds were tested for cytotoxic activity on KB, MCF-7 and HeLa cell lines. The derivatives had acetoxy, oxo or hydroxyimino function at the C-3 position and in some cases oxo, hydroxyimino or acyloxyimino group at the C-12 position. Almost all of the compounds showed strong cytotoxic activity, higher than unchanged oleanolic acid. The most active substances turned out to be the derivatives with acyloxyimino function, especially 4 and 8d. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob06923g

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Volume 10 | Number 11 | 21 March 2012 | Pages 2177–2340
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Barbara Bednarczyk-Cwynar et al.
Anticancer effect of A-ring or/and C-ring modified oleanolic acid derivatives on
KB, MCF-7 and HeLa cell lines

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COMMUNICATION
Anticancer effect of A-ring or/and C-ring modified oleanolic acid derivatives
on KB, MCF-7 and HeLa cell lines
Barbara Bednarczyk–Cwynar,*a Lucjusz Zaprutko,a Piotr Ruszkowskib and Bogusław Hładońb
Received 15th November 2011, Accepted 5th December 2011
DOI: 10.1039/c2ob06923g
relationship analyses. One of the most popular structures within
this group is the oleanane skeleton with oleanolic acid as a repre-
sentative system. This acid is widely distributed in the plant
kingdom, both in the form of free compound and as numerous
glycosides. It has been reported to have a broad spectrum of
pharmacological activities, including hepatoprotective, antitumor,
cardiovascular or immunomodulatory action.2 Many derivatives
of oleanolic acid have been synthesized for a variety of other
biological activities.3–9 For example, it has been reported that
amino acid derivatives of oleanolic acid (OA) of different types
show the inhibitory activity on the formation of osteoclast-like
multi-nucleated cells (OCLs),3 complement fixation inhibitory4
and antidiabetic activity.5 Acylated derivatives of oleanolic acid
also turned out to complement fixation inhibitors,4 act as anti-
diabetic5 as well as anti-HIV agents,6 its esters acted as inhibitors
of cancer cell apoptosis7 and some derivatives with seven-mem-
bered lactam system are good transdermal transport promoters.8
Huand et al. have found that oleanonic acid inhibits significantly
the growth of cancer cells derived from different tissues.9
Some of the above mentioned reports, concerning oleanolic
acid derivatives as potential anticancer agents prompted us to
embark on an investigation to modify the structure to strengthen
the cytotoxic activity. Since the discovery of anticancer activity
of oleanolic acid coming from natural sources, interest in syn-
thesis of new compounds based on this triterpene acid, which is
easy to obtain, has considerably increased in view of design and
production of potential bioactive semi-synthetic compounds. In
fact, many authors10 have reported that modifications of oleano-
lic acid at the C-3, C-12 and C-28 positions can provide a
number of potentially important derivatives with potent anti-
cancer activity.
New A-ring or/and C-ring modified methyl oleanolate
derivatives were prepared. New simple method of synthesis
of 3,12-diketone (3) from methyl oleanonate (2) was worked
out. The obtained new compounds were tested for cytotoxic
activity on KB, MCF-7 and HeLa cell lines. The derivatives
had acetoxy, oxo or hydroxyimino function at the C-3 pos-
ition and in some cases oxo, hydroxyimino or acyloxyimino
group at the C-12 position. Almost all of the compounds
showed strong cytotoxic activity, higher than unchanged olea-
nolic acid. The most active substances turned out to be the
derivatives with acyloxyimino function, especially 4 and 8d.
Despite the progress in technology and medicine, cancer diseases
are still the leading cause of death in the world and they pose a
great problem for doctors, because the index for cancer cure is
still not satisfying and cancer treatment is a challenge. The main
reason for this situation is the ability of cancer cells to develop
various types of resistance mechanisms, among them the
expression of natural or chemically induced multidrug resistance
(MDR) and the lack of sensitivity to proapoptotic stimulants.
Clinically MDR is characterized by the lack of response to
several structurally unrelated drugs acting according to different
types of mechanisms. Because of the complexity and versatility
of cellular MDR mechanisms, many difficulties are encountered
in anticancer drug synthesis and cancer therapy.
As cancer diseases are now one of the most frequent cause of
death all over the world, new antitumor agents have been con-
tinuously developed. Much attention has been paid to natural
products, which have become one of the most important and
promising sources of new substances for cytotoxic drug develop-
ment. It is estimated that about 60% of the antitumor drugs
approved for use by regulatory agencies are of natural origin.1
Pentacyclic triterpenoids make a class of pharmacologically
active and structurally rich natural products with molecules
susceptible to further modifications and structure–activity
To explore the effects of the C-3 and C-12 substituents on
cytotoxic activity, we converted the C-3 hydroxyl group into
acetoxy, oxo and hydroxyimino function or/and the C-12–C-13
double bond within methyl oleanolate molecule to oxo, hydro-
xyimino or acyloxyimino group.
The starting material for our syntheses was oleanolic acid iso-
lated from by-products formed during the production of ethano-
lic extract from fresh mistletoe herb (Viscum alba). The purity of
the obtained triterpene was proven on the basis of spectral data
which were in accordance with literature.11 The spectral data of
the product obtained after methylation with dimethyl sulphate
were also in agreement with the literature.12
aDepartment of Organic Chemistry, Faculty of Pharmacy, Poznan
University of Medical Sciences, Grunwaldzka Str. 6, 60–780 Poznan,
Poland. E-mail: bcwynar@ump.edu.pl; Fax: +48-61-854-66-80;
Tel: +48-61-854-66-74
bDepartment of Pharmacology, Faculty of Pharmacy, Poznan University
of Medical Sciences, Rokietnicka Str. 5, 60–806 Poznan, Poland.
E-mail: pruszkowski@gmail.com
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Scheme 1 Synthesis of A- or/and C-ring modified oleanolic acid derivatives.
To obtain new derivatives with A-ring or/and C-ring modifi-
cations, the set of transformations presented in Scheme 1 was
applied. Some of the characteristic carbon atoms within methyl
oleanolate (1) in Scheme 1 are numbered. In order to introduce
the additional carbonyl group in the C-ring, oleanolic acid was
first methylated to its methyl ester (1) and the obtained product
was then oxidized with Jones’ reagent in acetone at room temp-
erature, on the basis of the published procedure.13 It is known
from literature protocol that the C-12–C-13 double bond of
acetyl methyl oleanolate can be oxidizied with m-CPBA in
methylene chloride at room temperature14 and the compound
with C-12 oxo function is obtained as one of the products. It is
also known,15 that the oxidation of C-3 ketoderivative of oleano-
lic acid ester or other triterpenes with the use of m-chloroperben-
zoic acid (m-CPBA), in the presence of Li2CO3, known as
Baeyer–Villiger oxidation, leads to a compound with enlarged
A-ring containing an additional oxygen atom (A-ring ε-lactone)
with the unchanged C-12–C-13 double bond.
Taking under consideration the above mentioned results of
oleanolic acid derivatives reactions, the oxidation of methyl
3-oxooleanolate oxidation with m-CPBAwithout lithium carbon-
ate or any other additives was performed.16
The obtained mixture of products was subjected to column
chromatography. The oxidation of the C-12–C-13 double bond
of 3-O-acetyl methyl oleanolate (5) first led to a compound with
C-12–C-13 epoxy function14 which was subsequently converted
to an appropriate 12-oxocompound 6 with the use of an iron(III)-
picolinate complex.
The application of column chromatography with a column
filled with silica gel allowed us to eliminate the necessity of the
additional use of chemicals. In this process silica gel acted as an
acid catalyst and the opening of the epoxide ring of the inter-
mediate took place. The appropriate 3,12-diketone (3) was
obtained with a yield of above 80%.
The structure of product 3, which was received straight from
compound 2, with the appliance of the new, simple method, was
verified by spectral analysis and the obtained spectral data were
in agreement with those from the literature.17 The obtained 3,12-
dioxocompound 3 was subjected to the reaction with hydroxyla-
mine hydrochloride in ethanol, on the basis of the procedure
known for other oxotriterpenoids.18 According to the spectral
data, the only group that interacted with the above reagent was
the 3-oxo function and 3-oxime-12-oxoderivative of methyl
oleanolate (4), so far unknown in triterpene subject matter, was
obtained as the only product.19
The second series of derivatives, originated also from methyl
oleanolate (1), was synthesized following the reaction sequence
shown in Scheme 1. Methyl ester of oleanolic acid (1) was
acetylated with a 10-fold excess of acetic anhydride in anhy-
drous pyridine20 to give product 5. Then it was oxidized with
m-CPBA in methylene chloride14 and after that chromatographed
on SiO2. The deprotection of the hydroxyl group of the resulting
12-ketone (6) by boiling the triterpene in 5% ethanolic NaOH
gave a 3-hydroxy-12-oxocompound, which was oxidized with
Jones’ reagent giving only one product: 3,12-dioxoderivative
(3), identical with that obtained from the earlier transformation
2202 | Org. Biomol. Chem., 2012, 10, 2201–2205
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Table 1 The anticancer activity of oleanolic acid derivatives with
A-ring or/and C-ring modifications
to oleanolic acid (OA) and inactive against the MCF-7 cell line.
The replacement of the 3-acetoxy group in 12-ketone 6 with oxo
function (diketone 3) led to a compound which was about 2.5-
fold more active against KB cells (IC50 6.19 μM) and against
HeLa (IC50 5.03 μM) cells than OA. The transformation of the
C-3 oxo group of product 3 into a hydroxyimine function
(monooxime 4) led to a compound which was suspected to be
much more active, as some of the biological tests26 have shown
that compounds having hydroxyimino group within the molecule
of triterpene belong to a class of the most active triterpenic
species. In fact, the oximation of diketone 3 to monooxime 4
caused significant intensification of cytotoxic activity against all
of the tested cell lines: ketoxime 4 was almost 9-fold more active
against KB and MCF-7 (IC50 1.74 and 1.60 μM, respectively)
and 6.5-fold more active against HeLa cells (IC50 1.80 μM), in
comparison to OA. When the ketone function at the C-3 of com-
pound 3 was replaced with an acetoxy group and the second
ketone group, at C-12, was transformed into a hydroxyimine
(product 7), the new compound turned out to be 7-fold more
active against the KB cell line (IC50 2.06 μM) and almost 9-fold
more active against the HeLa cell line (IC50 1.34 μM) than the
starting material, OA. The introduction of an acetyl group
instead of the proton within the C-12 hydroxyimino function
(newly obtained compound 8a) led to a compound which was
inactive against all of the tested cell lines—the IC50 value
exceeded 15.00 μM. This compound possessed two acetoxy
groups within the molecule and it is probable that the 12-acetyl-
oxyimino function weakens the anticancer activity of the
obtained compound 8a. In the molecules of new products 8b–8e,
which also had 3-acetoxy function, the 12-acetyloxyimino group
was modified (8b, 8c, 8e) or replaced with the 12-propionyloxyi-
mino function (8d). The replacement of the 12-acetyloxyimino
group with the chloroacetyloxyimino function made the new
compound 8b about 1.6-fold more active against KB cells with
comparison to mother OA and at the same time the product 8b
was about 1.3-fold more active than OA against MCF-7 and
HeLa cell lines. The compound 8c, with a bromoacetyl function
instead of a hydroxyimino proton was about 3–4-fold more
active against KB, MCF and HeLa cell lines in comparison to
OA. The acylation of the 12-oxime group of compound 7 with
simple propionic acid resulted in significant intensification of
anticancer activity, especially against KB cells: the propionyl-
oxyimino compound 8d was about 21-fold more active against
the above mentioned cell line (IC50 0.72 μM) than OA and about
6.5-fold more active against MCF-7 and HeLa cells (IC50 2.13
and 1.87 μM, respectively). The compound with an o-nitroben-
zoyl function instead of a hydroxyimino proton (8e) turned out
to be about 1.5-fold more active against all of the tested cell
lines in comparison to the reference compound.
IC50 values (× 10−2, μM L−1
)
Comp.
no.
KB (carcinoma
nasopharynx)
MCF-7 (breast
cancer)
HeLa (cervical
cancer)
3
4
6
7
8a
8b
8c
8d
8e
OAa
6.19 0.12
1.74 0.04
8.51 0.13
2.06 0.07
>15.00
9.42 0.46
4.90 0.37
0.72 0.04
10.10 0.40
14.93 0.13
>15.00
1.60 0.08
>15.00
11.27 0.09
>15.00
7.26 0.33
3.76 0.33
2.13 0.05
9.28 0.32
13.95 0.09
5.03 0.17
1.80 0.02
7.38 0.13
1.34 0.06
>15.00
9.19 029
4.41 0.36
1.87 0.09
9.84 0.82
11.82 0.04
a Reference compound: oleanolic acid (OA)
of ketone 2. 12-Oxoderivative 6 was also exposed to hydroxy-
lamine hydrochloride in ethanol according to a known method.18
The spectral data of the obtained product 7, also so far unknown
in triterpene subject matter, confirmed the expected structure of
this compound.21 In the presented NMR spectral data of the all
newly obtained compounds only the most characteristic signals
are included.
The hydroxy group within hydroxyimino function of com-
pound 7 was acylated with carboxylic acids. The reaction was
performed in dioxane in the presence of DCC (dicyclohexylcar-
bodiimide) at room temperature. Simple aliphatic or aromatic
carboxylic acids were applied as acylating agents. After
30–60 min of stirring the acylation was complete as TLC control
shown.22 The spectral data confirmed the expected structure of
the products obtained.23
The biological tests were performed for two known com-
pounds: diketone 3 and 12-monoketone 6 as well as for seven
new compounds: 3-oxime-12-ketone 4, 12-monooxime 7 and for
acylated 12-monooximes 8a–8e. As known from literature data24
the introduction of propionoxy group into the molecule of triter-
pene can improve the pharmacological activity of the new sub-
stance, so the compound 8d, with propionoxyimino function at
the C-12 position was expected to be the most active against the
tested cell lines.
The results of these tests were converted to the values sum-
marized in Table 1 with the usage of the formula known from lit-
erature protocol.25
Unmodified oleanolic acid (OA) was applied as a reference
compound with IC50 values for KB, MCF-7 and HeLa cell lines
of 14.93, 13.95 and 11.82 μM, respectively (see Table 1). The
IC50 values for the tested oleanolic acid derivatives 3, 4, 6, 7 and
8a–8e ranged from 0.72 to 10.10 μM for KB cells (apart from
8a, which was inactive, IC50 > 15.00 μM), from 1.60 to
11.27 μM (except for 3, 6 and 8a with IC50 > 15.00 μM) for
MCF-7 cells and from 1.34 to 9.84 μM (also apart from 8a,
which was inactive, IC50 > 15.00 μM) for HeLa cells. Analysis
of the chemical structures of the tested compounds and their
anticancer activity has shown a strong correlation between them.
The derivative 6, with a 3-O-acetyl group and oxo function at
the C-12 position was about 1.5-fold more active against KB and
HeLa cells (IC50 8.51 and 7.38 μM, respectively) in comparison
In the biological experiments, KB and HeLa cells were cul-
tured in RPMI 1640 media, MCF-7 cells were cultured in
D-MEM media. Both media were supplemented with 10% fetal
bovine serum, 1% L-glutamine and 1% penicillin/streptomycine
solution. The cell lines were maintained at 37 °C and 5% CO2 in
an incubator. The optimal plating density of cell lines was deter-
mined to be 5 × 104 cells.
The protein-staining sulforhodamine B (SRB, Sigma-Aldrich)
microculture colorimetric assay, developed by the National
Cancer Institute for in vitro antitumor screening, was used in this
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 2201–2205 | 2203

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13 M. S. Ali, M. Jahangir, S. S. ul Hussan and M. I. Choudhary, Phytochem-
istry, 2002, 60, 295.
14 I. Okamoto, T. Takeya, Y. Kagawa and E. Kotani, Chem. Pharm. Bull.,
2000, 48, 120.
15 e.g. (a) S. Qian, H. Li, Y. Chen, W. Zhang, S. Yang and Y. Wu, J. Nat.
Prod., 2010, 73, 1743; (b) C. S. Graebin, H. Verli, J. A. Guimarães and
J. Braz, Chem. Soc., 2010, 21, 1595.
16 The synthesis was performed as follows: To a solution of triterpene 2
(940 mg, 2.0 mmol) in dried CH2Cl2 (15 mL), a solution of a 2-fold
amount (690 mg) of m-CPBA in dried CH2Cl2 (10 mL) was added and
the resulting solution was left in darkness at room temperature overnight.
After that it was washed successively with 5% solutions of FeSO4,
Na2CO3, HCl and finally water. The organic solution was dried, filtered,
evaporated to dryness and the resulted residue was chromatographed on
silica gel. Compound 3: yield: 710 mg (73.4%), mp.: 158 °C (C6H6),
white needles
17 T. Honda, B. A. V. Rounds, L. Bore, H. J. Finlay, F. G. Jr. Favaloro,
N. Suh, Y. Wang, M. B. Sporn and G. W. Gribble, J. Med. Chem., 2000,
43, 4233.
18 Ch. Ma, N. Nakamura and M. Hattori, Chem. Pharm. Bull., 2000, 48,
1681.
study to estimate the cell number by providing a sensitive index
of total cellular protein content, being linear to cell density. The
monolayer cell culture was trypsinized and the cell count was
adjusted to 5 × 104 cells. To each well of a 96-well microtiter
plate, 0.1 mL of the diluted cell suspension (approximately
10 000 cells) was added. After 24 h, when a partial monolayer
was formed, the supernatant was washed out and 100 μL of six
different compound concentrations (0.1 μM, 0.2 μM, 1.0 μM,
2.0 μM, 10.0 μM, 20.0 μM) were added to the cells in microtiter
plates. The compounds investigated were dissolved in DMSO
(20 μL) and the content of DMSO did not exceed 0.1%, the con-
centration was found to be nontoxic to the cell lines. The cells
were exposed to compounds for 72 h. After this time, 25 μL of
50% trichloroacetic acid was added to the wells and the plates
were incubated for 1 h at 4 °C. Then the plates were washed out
with the distilled water to remove traces of medium and then
dried in air. The air-dried plates were stained with 100 μL SRB
and kept for 30 min at room temperature. The unbound dye was
removed by rapidly washing with 1% acetic acid and then air
dried overnight. The optical density was read at 490 nm.27 All
cytotoxicity experiments were performed three times and the
values presented in Table 1 are the mean values. Cell survival
was measured as the percentage absorbance compared to the
control (non-treated cells).
19 Compound 4: yield: 850 mg (85.2%); mp.: 194–195°C (ethanol), white
needles; mol. mass: 499.73; IR (KBr; ν, cm−1): 3410 (OH, N-OH); 1720
(CvO, C̲O̲
OCH3); 1705 (CvO, C-12); 930 (N-O, N-OH); 1H NMR
(300 MHz, CDCl3; δ, ppm): 9.04 (1H, s/br./, N-OH); 3.73 (3H, s,
COOCH3); 2.79 (1H, dt, J = 3.4 and 11.6 Hz, C18–H); 2.62 (1H, d, J =
4.1 Hz, C13–H); 1.16; 1.06; 0.99; 0.97; 0.97; 0.92; 0.90 (21H, singlets, 7
× CH3); 13C NMR (75 MHz, CDCl3; δ, ppm): 211.4 (C-12); 178.4
(C̲O̲OCH3); 166.3 (C-3); 51.8 (COOC̲H̲3); 48.1 (C-17); 43.3 (C-13);
̲
32.7 (C-18); DEPT: 8 × CH3, 10 × CH2, 4 × CH; EIMS (m/z): 499.3
(30.9%) M+˙
The cytotoxic activity of semi-synthetic oleanane-type deriva-
tives has been investigated. The overall results suggest that some
of the new oleanolates can effectively inhibit the growth of KB,
MCF-7 and HeLa cancer cell lines at microgram concentrations
and could be promising new anticancer agents. The hydroxy-
imino derivatives were shown to be more potent anticancer
agents in comparison to their mother compounds. The substi-
tution of a hydrogen atom within a hydroxyimino function with
propionyl group, led to a further significant intensification of
anticancer activity. The introduction of an electron withdrawing
substituent into the acyloxyimino group on the C-12 atom led to
a moderate increase in anticancer activity.
20 T. Honda, H. J. Finlay, G. W. Gribble, N. Suh and M. B. Sporn, Bioorg.
Med. Chem. Lett., 1997, 7, 1623.
21 For compound 7: yield 848 mg (78.0%); mp.: 196–198.5 °C (ethanol),
white needles; mol. mass: 543.79; IR (KBr; ν, cm−1): 3430 (OH, N-OH);
1730 and 1710 (CvO, CH3COO and COOCH3); 905 (N-O, N-OH); 1H
NMR (300 MHz, CDCl3; δ, ppm): 8.05 (1H, s/br/, N-OH); 4.47 (1H, dt,
J = 5.7 and 10.5 Hz, C3-H); 3.67 (3H, s, COOCH3); 2.85 (1H, dt, J = 3.3
and 11.0 Hz; C18-H); 2.52 (1H, d, J = 3.7 Hz; C13-H); 2.04 (3H, s,
CH3COO); 0.91; 0.91; 0.88; 0.87; 0.85; 0.85; 0.81 (7 × 3H, 7 × s, 7 ×
CH3); 13C NMR (75 MHz, CDCl3; δ, ppm): 178.5 (C-28); 170.8
(CH3C̲O̲O); 159.8 (C-12); 80.7 (C-3); 51.8 (COOC̲H3̲ ); 48.1 (C-17);
̲
43.3 (C-13); 32.7 (C-18); 21.3 (C̲H̲3COO); EIMS (m/z): 543.4 (49.6)
̲
M+˙; Anal. calcd for C33H53NO5 (%): C = 72.89; H = 9.82; N = 2.58;
found: C = 72.53; H = 9.86; N = 2.85
22 The synthesis was performed as follows (general method): To a stirred at
room temperature solution of compound 7 (544 mg, 1.0 mmol) in
dioxane (9 mL) 1.2 mmol of carboxylic acid and 1.5 mmol of DCC was
added. The stirring was continued at room temperature for about 30 min.
The resulting precipitate was filtered, washed with dioxane and the filtrate
was poured into a 5-fold volume of water slightly acidified with HCl. The
obtained precipitate was filtered, washed with water, dissolved in ethanol
and reprecipitated with water. The white solid was filtered, washed with
water and dried. Yield >90%
Notes and references
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23 For compound 8a: yield: 562 mg (95.2%); mp.: 120–125 °C (precipt.
with water from ethanolic solution), white powder; mol. mass: 585.82; IR
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(KBr; ν, cm−1): 1765 (CvO, CH3C
̲
̲
̲O̲OCH3);
1710 (CvO, CH3C̲O̲
̲
̲
–
̲
̲
̲
̲
̲
̲
̲O̲
7 L. Chen, Y. Zhang, X. Kong, S. Penga and J. Tian, Bioorg. Med. Chem.
Lett., 2007, 17, 2979.
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10 e.g. (a) S. Gupta, K. Kalani, M. Saxena, S. K. Srivastava, S. K. Agrawal,
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2005, 15, 2215.
̲
̲
̲
̲
̲
̲H3̲ COO); DEPT: 10 × CH3, 10 × CH2, 5 × CH;
̲
̲
EIMS (m/z): 585.5 (0.5%) M+˙. For compound 8b: yield: 600 mg
(96.8%); mp.: 118–124 °C (precipt. with water from ethanolic solution),
white powder; mol. mass: 620.27; IR (KBr; ν, cm−1): 1750 (CvO,
ClCH2C
NMR (300 MHz, CDCl3; δ, ppm): 4.47 (1H, dd, J = 4.4 and 11.4 Hz,
C3–H); 4.34 (2H, d, J = 2.7 Hz, ClCH2COO); 3.69 (3H, s, COOCH3);
2.73 (1H, d, J = 3.9 Hz, C18–H); 2.05 (3H, s, C
13.0 Hz, C13–H); 0.96, 0.94, 0.93, 0.88, 0.86, 0.85 × 2 (21H, singlets, 7
× CH3); 13C NMR (δ, ppm): 178.4 (COOCH3); 170.9 (CH3C
O); 168.2
(C-12); 166.7 (ClCH2C ON); 80.3 (C-3); 51.8 (COOCH3); 47.9 (C-17);
̲O̲ON); 1735 (CvO, C̲O̲OCH3); 1715 (CvO, CH3C̲O̲
O); 1H
̲
̲
̲
̲H3̲ COO); 1.94 (1H, d, J =
̲
̲O̲
11 C. Y. Hung and G. C. Yen, LWT–Food Sci. Technol., 2001, 34, 306.
12 K. G. Lewis and D. J. Tucker, Aust. J. Chem., 1983, 36, 2297.
̲
̲
̲ ̲
̲
2204 | Org. Biomol. Chem., 2012, 10, 2201–2205
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44.2 (C-13); 32.5 (C-18); 33.1 (ClC
̲
̲
COON); 21.3 (C
̲
̲
COO); DEPT:
̲
(C̲
̲
COO); DEPT: 10 × CH3, 11 × CH2, 5 × CH; EIMS (m/z): 599.3
̲
̲
9 × CH3, 11 × CH2, 5 × CH; MS (m/z): 620.7 (3.0%) M+˙. For compound
8c: yield: 610 mg (92.5%); mp.: 110–116 °C (precipt. with water from
ethanolic solution), white powder; mol. mass: 664.72; IR (KBr; ν, cm−1):
(2.7%) M+˙. For compound 8e: yield: 647 mg (93.8%); mp.: 164–169 °C
(precipt. with water from ethanolic solution), white powder; mol. mass:
692.89; IR (KBr; ν, cm−1): 3030 (C-H, Ar); 1750 (CvO, o-NO2-Ar-
1740 (CvO, BrCH2C
CH3C
O); 1H NMR (300 MHz, CDCl3; δ, ppm): 4.47 (1H, dd, J = 4.7
and 11.4 Hz, C3–H); 4.08 (2H, d, J = 0.6 Hz, BrCH2COO); 3.69 (3H, s,
COOCH3); 2.73 (1H, d, J = 3.9 Hz, C18–H); 2.3 (1H, dd, J = 2.4 and
10.7 Hz, C13–H); 2.05 (3H, s, CH3COO); 0.96, 0.95, 0.93, 0.88, 0.86,
0.85 × 2 (21H, singlets, 7 × CH3); 13C NMR (75 MHz, CDCl3; δ, ppm):
178.4 (COOCH3); 170.9 (CH3C O); 168.3 (C-12); 166.2 (BrCH2-
ON); 80.3 (C-3); 51.8 (COOCH3); 47.9 (C-17); 44.2 (C-13); 32.5
H2
O
̲
̲
̲
C̲O̲ON); 1725 (CvO, C̲O̲OCH3); 1710 (CvO, CH3C̲O̲O); 1520 and
̲
̲
1340 (NO2); 1H NMR (300 MHz, CDCl3; δ, ppm): 8.06 (1H, dd, J = 1.2
and 7.9 Hz) and 7.97 (1H, dd, J = 0.9 and 8.1) and, 7.84–7.62 (2H, m)
Ar-H; 4.48 (1H, dd, J = 5.1 and 11.5 Hz, C3–H); 3.70 (3H, s, COOC
2.67 (1H, d, J = 3.9 Hz, C18–H); 1.92(1H, dd, J = 6.8 and 10.2 Hz, C13
H); 2.05 (3H, s, C 3COO); 0.92, 0.88, 0.86, 0.85, 0.84 × 2, 0.73 (21H,
singlets, 7 × CH3); 13C NMR (75 MHz, CDCl3; δ, ppm): 178.5
(C OCH3); 170.9 (CH3C O); 169.2 (C-12); 156.7 (o-NO2-Ar-C ON);
147.5, 134.3, 133.8, 133.3, 130.8, 123.8 (o-NO2-A -COON); 80.4 (C-3);
51.8 (COOC 3); 48.0 (C-17); 44.3 (C-13); 32.4 (C-18); 21.3
̲H3̲ );
̲
–
̲H̲
̲
C
O̲
̲
̲
̲
̲
̲O̲
(C-18); 25.5 (BrC
̲
̲
̲r̲
̲
̲H̲
̲
(C̲H3̲ COO); DEPT: 9 × CH3, 10 × CH2, 9 × CH; EIMS (m/z): 692.8
̲
(0.9%) M+˙
(CvO, CH3CH2C
CH3C
O); 1H NMR (300 MHz, CDCl3; δ, ppm): 4.47 (1H, dd, J = 4.9
and 11.3 Hz, C3–H); 3.69 (3H, s, COOCH3); 2.79 (1H, d, J = 3.8 Hz,
̲
̲
C
̲
OCH3); 1710 (CvO,
24 R. Mukherjee, M. Jaggi, M. J. A. Siddiqui, S. K. Srivastava,
P. Rajendran, A. Vardhan and A. C. Burman, Bioorg. Med. Chem. Lett.,
2004, 14, 4087.
25 V. Vichai and K. Kirtikara, Nat. Protoc., 2006, 1, 1112.
26 A. Paszel, L. Zaprutko, B. Bednarczyk–Cwynar, J. Hofmann and
M. Rybczyńska, Oncologie, 2005, 28 (Suppl. 2), 44.
27 P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J. T. Warren, H. Bokesch, S. Kenney and M. R. Boyd, J. Natl. Cancer
Inst., 1990, 82, 1107.
̲O̲
̲
̲
C
C
18–H); 2.58–2.51 (2H, m, CH3C
̲ ̲ CH2COON); 0.97,
13–H); 2.05 (3H, s, CH3COO); 1.20 (3H, s, CH3
̲
H2
̲
COON); 2.18 (1H, d, J = 13.4 Hz,
̲
̲
̲
̲
(75 MHz, CDCl3; δ, ppm): 178.5 (C
170.9 (CH3C O); 166.2 (C-12); 80.3 (C-3); 51.8 (COOC
(C-17); 44.0 (C-13); 32.5 (C-18); 26.6 (CH3CH2COON); 21.3
̲
̲
̲O̲ON);
̲
̲
̲ ̲
̲
̲
̲
̲
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 2201–2205 | 2205