Semisynthetic Ursolic Acid Fluorolactone Derivatives Inhibit Growth with Induction of p21waf1 and Induce Apoptosis with Upregulation of NOXA and Downregulation of c-FLIP in Cancer Cells

Article abstract of DOI:10.1002/cmdc.201200282

A series of ursolic acid ((1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10-hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid) derivatives with a 12-fluoro-13,28β-lactone moiety were synthesized using the electrophilic fluorination reagent Selectfluor. The antiproliferative effects of these novel compounds were evaluated in AsPC-1 pancreatic cancer cells, and the structure-activity relationships (SARs) were evaluated. Of the compounds synthesized, ursolic acid derivatives carrying a heterocyclic ring, such as imidazole or methylimidazole, and cyanoenones were among the more potent inhibitors of AsPC-1 pancreatic cancer cell growth. 2-Cyano-3-oxo-12α-fluoro-urs-1-en-13,28β-olide, compound 20, was the most effective inhibitor with IC₅₀ values of 0.7, 0.9 and 1.8μM in pancreatic cancer cell lines AsPC-1, MIA PaCa-2 and PANC-1, respectively. This compound also exhibited better antiproliferative activities against breast (MCF7), prostate (PC-3), hepatocellular (Hep G2) and lung (A549) cancer cell lines, with IC₅₀ values lower than 1μM. The mechanism of action by which these compounds exert their biological effect was evaluated in AsPC-1 cells using the most potent inhibitor synthesized, compound 20. At 1μM, the cell cycle arrested at the G1 phase with upregulation of p21^waf1. Apoptosis was induced at an inhibitor concentration of 8μM with upregulation of NOXA and downregulation of c-FLIP. These data indicate that fluorolactone derivatives of ursolic acid have improved antiproliferative activity, acting through arrest of the cell cycle and induction of apoptosis.

Full text of DOI:10.1002/cmdc.201200282

MED
DOI: 10.1002/cmdc.201200282
Semisynthetic Ursolic Acid Fluorolactone Derivatives
Inhibit Growth with Induction of p21waf1 and Induce
Apoptosis with Upregulation of NOXA and
Downregulation of c-FLIP in Cancer Cells
Ana S. Leal,[a, b, c] Rui Wang,[c] Jorge A. R. Salvador,*[a, b] and Yongkui Jing*[c]
A
series of ursolic acid ((1S,2R,4aS,6aR,6aS,6bR,8aR,10-
creatic cancer cell lines AsPC-1, MIA PaCa-2 and PANC-1, re-
spectively. This compound also exhibited better antiprolifera-
tive activities against breast (MCF7), prostate (PC-3), hepatocel-
lular (Hep G2) and lung (A549) cancer cell lines, with IC50
values lower than 1 mm. The mechanism of action by which
these compounds exert their biological effect was evaluated in
AsPC-1 cells using the most potent inhibitor synthesized, com-
pound 20. At 1 mm, the cell cycle arrested at the G1 phase
with upregulation of p21waf1. Apoptosis was induced at an in-
hibitor concentration of 8 mm with upregulation of NOXA and
downregulation of c-FLIP. These data indicate that fluorolac-
tone derivatives of ursolic acid have improved antiproliferative
activity, acting through arrest of the cell cycle and induction of
apoptosis.
S,12aR,14bS)-10-hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-
2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-
carboxylic acid) derivatives with a 12-fluoro-13,28b-lactone
moiety were synthesized using the electrophilic fluorination re-
agent Selectfluor. The antiproliferative effects of these novel
compounds were evaluated in AsPC-1 pancreatic cancer cells,
and the structure–activity relationships (SARs) were evaluated.
Of the compounds synthesized, ursolic acid derivatives carry-
ing a heterocyclic ring, such as imidazole or methylimidazole,
and cyanoenones were among the more potent inhibitors of
AsPC-1 pancreatic cancer cell growth. 2-Cyano-3-oxo-12a-
fluoro-urs-1-en-13,28b-olide, compound 20, was the most ef-
fective inhibitor with IC50 values of 0.7, 0.9 and 1.8 mm in pan-
Introduction
Ursolic acid (1) is a pentacyclic triterpenoid present in plants,
vegetables and fruits,[1] which has been found to have many
biological properties, such as anti-inflammatory,[2] antidiabet-
ic,[3] cardioprotective,[4,5] hepactoprotective,[6] antitumoral and
chemopreventive[7–9] activities. The antitumoral and chemopre-
ventive activities of ursolic acid (1) are due to induction of
apoptosis,[10–14] inhibition of differentiation,[15] invasion,[16,17]
promotion/progression,[18] and angiogenesis,[19,20] promotion of
chemosensitization,[21] and induction of cell cycle arrest.[11,22]
Several groups have studied ursolic acid (1) aiming to in-
crease its antitumor activity. The structural modifications inves-
tigated so far are mostly on the alcohol group at position C3,
and on the carboxylic acid group at position C28.[23–29] Amides
and esters are the most common semisynthetic derivatives of
ursolic acid (1). Other groups have also designed and tested
amide derivatives in several cancer cell lines.[24,26,30] Tu et al.
synthesized a series of new ursolic acid ester and anhydride
derivatives.[31] Ma et al. designed a series of straightforward
esters with improved cytotoxic activity in leukemia, stomach,
liver and cervix cancer cell lines.[27] Despite the various semi-
synthetic ursolic acid derivatives already described, new deriva-
tives still need to be designed and synthesized in order to im-
prove the cytotoxic activity of ursolic acid (1) further.
use has increased from less than 2% to around 20% since
1970.[34,35] Fluorinated organic compounds can be achieved
using nucleophilic, electrophilic and radical forms of fluorine
reagents.[36] Fluorine is a small and highly electronegative
atom, and its presence in key positions of a biologically active
molecule has the potential to improve the metabolic and
chemical stability, membrane permeability and protein binding
affinity of the agent.[34,37–40] 5-Fluorouracil was the first synthet-
ic fluorinated chemotherapeutic compound, which is widely
used for cancer treatment.[34,41,42] A fluorine substituent is often
[a] A. S. Leal, Prof. J. A. R. Salvador
Laboratory of Pharmaceutical Chemistry
Faculty of Pharmacy, University of Coimbra
Polꢀ das CiÞncias da Safflde
Azinhaga de Santa Comba, 3000-548 Coimbra (Portugal)
E-mail: salvador@ci.uc.pt
[b] A. S. Leal, Prof. J. A. R. Salvador
CNC-Center for Neuroscience and Cell Biology
University of Coimbra
3000-517 Coimbra (Portugal)
[c] A. S. Leal, Dr. R. Wang, Prof. Y. Jing
Division of Hematology/Oncology
Tisch Cancer Institute, Mount Sinai School of Medicine
One Gustave L. Levy Place, New York, 10029 NY (USA)
E-mail: yongkui.jing@mssm.edu
Fluorine is the least abundant halide in natural compounds
and few living cells can metabolize it.[32,33] The presence of a flu-
orine substituent in pharmaceutical compounds currently in
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200282.
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found in other currently used anticancer drugs, including top-
oisomerase inhibitor gemcitabine[43–45] and antiandrogen agent
flutamide.[33,46] This led us to develop fluorinated ursolic acid
(1) derivatives as an attempt to further improve its antitumor
activity.
Compounds 11, 18 and 19 were synthesized by reaction of
1, 2 and 3, respectively, with Selectfluor at 808C in a mixture
of nitrometane and dioxane, with the insertion of the b-fluo-
rine at C12 and 13,28b-lactone, with yields above 45%
(Schemes 1 and 2).
Fluorine was introduced into the C ring of ursolic acid (1),
and a series of ursane fluorolactone derivatives were synthe-
sized
using
1-chloromethyl-4-fluoro-1,4-diazoniabicyclo-
[2.2.2]octane bis(tetrafluoroborate) (Selectfluor), an electrophil-
ic fluorinating reagent. New fluorinated ursolic acid (1) deriva-
tives with esters, carbamates, and a carbonyl group at position
C3, and a cyano group at position C2 were prepared. Imidazole,
methylimidazole and triazole rings in conjugation with an un-
saturated a,b ketone in the A ring of the ursane fluorolactone
backbone were also synthesized. The antiproliferative effects
of these new semisynthetic compounds were tested in a pan-
creatic cancer cell line, AsPC-1, and the structure–activity rela-
tionships (SARs) were analyzed. The most active compounds
were further tested for their antiproliferative activity in other
cancer cell lines. According to our previous experience with
semisynthetic triterpenoids,[47,48] 2-Cyano-3-oxo-12a-fluoro-urs-
1-en-13,28b-olide (20) was selected as a model compound to
explore the mechanism of action in AsPC-1, MIA PaCa-2 and
PANC-1 cells. Our data indicate that compound 20 is a potent
inducer of cyclin p21waf1, which is correlated with cell cycle
arrest in G1 phase. Compound 20 at high concentrations in-
duced apoptosis with downregulation of c-FLIP and upregula-
tion of NOXA. These new ursane fluorolactone compounds
with novel mechanism of action should have therapeutic ef-
fects in solid cancers.
Results and Discussion
Scheme 1. Reagents and conditions: a) Selectfluor, dioxane, nitromethane,
808C, 24 h, 88%; b) Jones reagent, acetone, ice, 22%; c) CDI or CBMI, THF,
N2, reflux, 13: 3 h 47%, 14: 8 h 49%; d) Anhydride, DMAP, THF, RT, 15: 1 h
31%, 16: 8 h 50%, 17: 1 h 28%.
Chemistry
Selectfluor, an electrophilic fluorination reagent, was used to
introduce a fluorine atom into ursolic acid (1) and derivatives.
Selectfluor is a crystalline white solid that is stable at high tem-
peratures and is easy to work with because the byproducts are
usually easily removed with aqueous workup.[49] There are sev-
eral papers describing the reaction of alkenes with Selectfluor,
usually in the presence of a nucleophilic donor.[50,51] The sol-
vent, the temperature, and the nucleophilic donor can influ-
ence the efficiency and the outcome (number of isomers and
byproducts) of this reaction. The optimal conditions for fluori-
nation of ursane-type substrates with Selectfluor were ach-
ieved using a mixture of two inert solvents, dioxane and nitro-
methane, at 808C.
The fluorination of ursane-type triterpenoids, when per-
formed in a reaction solution without a nucleophilic donor,
allows the synthesis of a fluorolactone. The free acid behaves
like the nucleophilic donor, losing the proton and promoting
cyclization to the carbon C13 in the ursane backbone. This reac-
tion is not possible in ursane-type compounds where the acid
is protected, however, as the free acid alone does not react
with Selectfluor.[52]
Scheme 2. Reagents and conditions: a) Acetic anhydride, DMAP, THF, RT, 1 h,
92%; b) Phthalic anhydride, DMAP, pyridine, 1158C, 23.5 h, 90%; c) Select-
fluor, dioxane, nitromethane, 808C, 18: 24 h 43%, 19: 23 h, 62%.
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The choice of performing the fluorination before the Jones
oxidation, formation of carbamates and esterification in com-
pounds 12, 13–14 and 15-17, respectively, was due to the fact
that some of these functionalities can also be fluorinated,
giving a mixture of diverse fluorinated compounds. Com-
pounds 18 and 19 were fluorinated after esterification since
this synthetic route proceeds with better yields. The new fluo-
rolactones achieved are chemically stable and allow perfor-
mance of several post-fluorination modifications using acidic
and basic conditions (Scheme 1).
Previously, our group found that introduction of heterocyclic
rings into betulin and betulinic acid increased their antiprolifer-
ative activities.[48,53,54] Ursolic acid (1) has several points in the
structure that are accessible for these types of chemical modifi-
cations. The introductions of a heterocyclic ring in ursolic acid
fluorolactones are achieved with good yields. The preparation
of the new derivatives with the fluorolactone and a heterocyclic
ring, compounds 13–14 and 23–25, was made by the dissolu-
tion of the respective fluorolactone (11 or 22) in tetrahydrofur-
an (THF), heating at reflux under an inert atmosphere, and
then adding 1,1’-carbonyldiimidazole (CDI), 1,1’-carbonylbis(2’-
methylimidazole) (CBMI) or 1,1’-carbonyl-di(1,2,4-triazole)
(CDT), to give imidazole, methylimidazole and triazole deriva-
tives, respectively (Schemes 1 and 4). The reaction of alcohols
Honda et al. first described the introduction of a cyano
group at the C2 carbon of ursolic acid (1).[29] We performed
a similar reaction on ursolic acid (1) with additional introduc-
tion of the fluorolactone in the ursane backbone (Scheme 3).
Despite optimization of the process, this reaction gives a mix-
ture of a and b-isomers, in which compound 21, the b-isomer
of fluorolactone, is the major reaction product, with determina-
tion by NMR and separation by flash column chromatography.
Scheme 4. Reagents and conditions: a) Ethyl formate, NaOMe, benzene, RT,
4.5 h, 67%; b) CDI or CBMI or CDT, THF, N2, reflux, 23: 4h 65%, 24: 4.5 h,
37%, 25: 5 h, 33%.
with these reagents (CDI, CBMI and CDT) is described to give
N-alkylimidazoles[55,56] or imidazole carboxylic esthers (carba-
mates),[53,54,57] depending on the alcohol and reaction condi-
tions used. Compound 11 with a secondary alcohol at position
C3 affords carbamates 13 and 14 (Scheme 1). Compound 22,
with a vinyl alcohol at position C2 gives the N-alkylimidazoles
23–25 (Scheme 4).
Full structural elucidation of these fluorolactones was made
by means of infrared (IR) spectroscopy, 1D and 2D nuclear
magnetic resonance (NMR) spectroscopy, and mass spectrome-
try (MS). In IR spectra, a CꢀF stretching vibration typically ap-
pears between 1000–1400 cmꢀ1, which in these compounds
coincides with other bands, such as CꢀO and CꢀH for example.
The geminal proton of the b-fluorine on the carbon C12 gives
a double triplet or a double quartet at a d value of 4.85–
4.98 ppm, with a coupling constant of around 62 Hz. The a-
isomer also gives a double triplet slightly upfield of the b-
isomer (d=4.42-4.51 ppm), with a larger coupling constant of
Scheme 3. Reagents and conditions: a) CH3I, K2CO3, DMF, RT, N2, 3 h, 93%;
b) Jones reagent, acetone, ice, 93%; c) Ethyl formate, benzene, NaOMe, RT,
1 h, 94%; d) NH2OH·HCl, H2O, EtOH, reflux, 1 h, 96%; e) NaOMe, Et2O, MeOH,
08C!RT, 1 h, 86%; f) DDQ, benzene, reflux, 4 h, 88%; g) LiI, DMF, reflux,
20.5 h, 64%; h) Selectfluor, dioxane, nitromethane, 808C, 2.5 h, 20: 13%, 21:
37%.
1
around 67 Hz. These differences between the H NMR spectra
allow discrimination between a and b isomers. The NOESY
spectra of the fluorolactones further confirmed the stereo-
chemistry; for example, in compound 21, proton H12 corre-
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lates with proton H9 and the methyl group at C27,
indicating a b configuration of fluorine (see the
Supporting Information).
Table 2. The inhibitory activities (IC50) of ursolic acid fluorolactones 11–19 in the pancre-
atic cancer cell line, AsPC-1.
The proton of the exocyclic double bond at C2
1
of compounds 22–25 appears in the H NMR spec-
trum as a peak at 8.62–7.66 ppm, depending on
the substituent group present. The imidazole
group of compounds 13 and 23 has three specific
protons that appear in the 1H NMR at d values
greater than 7 ppm. For compounds 14 and 24,
the methylimidazole group has only two protons
Compd
Ring A
IC50[a] [mm] Compd
18.4ꢁ0.09 16
Ring A
IC50[a] [mm]
1.2ꢁ0.01
1
in the H NMR between 6.7–7.4 ppm, and in com-
11
pound 25, the triazole group also has two protons
that appear at d values of greater than 8 ppm.
In the 13C NMR spectra of compounds bearing
a b-fluorolactone, the carbon C12 appears as a dou-
blet at a d value between 88.5–89.6 ppm, with
a coupling constant of around 180 Hz. Carbon C13
also appears as a doublet with d values of 91.5–
91.9 ppm, with a smaller coupling constant of
around 14 Hz. 13C NMR resonances for carbons C18,
C14, C11 and C9 of the ursane b-fluorolactone also
appear as doublets. The 13C NMR spectra of com-
pound 20 exhibits some differences from com-
pounds with a b-fluorolactone: carbon C12 appears
at downfield (d=96 ppm) with a coupling con-
stant of 189 Hz, and carbon C13 presents as a singlet
at 90 ppm, which indicate the a configuration.
12
13
6.0ꢁ0.07 17
9.5ꢁ0.1 18
2.2ꢁ0.1
13.4ꢁ0.1
14
15
6.1ꢁ0.04 19
10.0ꢁ0.07
18.0ꢁ0.06
[a] AsPC-1 cells were treated with various concentration of test compound for 72 h. The
antiproliferative effects were determined by using an MTT assay, and the IC50 was calcu-
lated from the results. Data represent the meanꢁSE of three independent experiments.
Biological activity
Compounds 16 and 17 were made by esterification with
Compound 11 has a similar antiproliferative activity against
AsPC-1 cells when compared to ursolic acid (1). However, com-
pounds 18 and 19 with introduction of the fluorolactone
moiety have increased antiproliferative activities compared
with compounds 2 and 3, respectively (Tables 1 and 2; Fig-
ure 1a). Several additional modifications were performed at C3
of the ursane fluorolactone to yield compounds 12–19. Oxida-
tion of the hydroxy group was reported to increase the activity
of triterpenoid compounds, however, compound 12 only ex-
hibited slightly improved antiproliferative activity compared
with compound 11. Carbamates 13 and 14 have an increase of
two- and threefold in the antiproliferative activity against
AsPC-1 cancer cells, when compared with 11 (Table 2).
benzoic and butyric anhydrides, respectively. This process gen-
erated some of the more active compounds, with 15- and 8-
fold increase in antiproliferative activity, compared with com-
pound 11 (Table 2 and Figure 1b).
It has been previously found that introduction of a cyano
group at C2 and a carbonyl group at C11 significantly improved
ursolic acid (1) cytotoxicity.[28] We applied a similar chemical
procedure in the A ring, as previously described,[29] and synthe-
sized cyanoenones 20 and 21 (Scheme 3). Compounds 20 and
21 are more effective than the previously synthesized com-
pounds 9 and 10 in the antiproliferative assay using AsPC-
1 cells (Tables 1 and 3), having a 19-fold improvement in anti-
proliferative activity when compared with ursolic acid (1) (Fig-
ure 1b). The intermediates of compounds 20 and 21 are less
active, suggesting that addition of a fluorolactone moiety fur-
ther improves the antiproliferative activity of triterpenoids
bearing a cyanoenone in the A ring.
Table 1. The inhibitory activities (IC50) of ursolic acid intermediates 1–6, 9
and 10 in the pancreatic cancer cell line, AsPC-1.
With the intent of exploring the effects of introducing differ-
ent substituents at the C2 position of the fluorolactone struc-
ture, we synthesized compound 22, with a vinyl alcohol in that
position. The substitution of the alcohol group of compound
22 for a heterocyclic ring, imidazole or methylimidazole, af-
forded compounds 23 and 24, respectively, with improved an-
tiproliferative effects in AsPC-1 cells, reaching an improvement
of 17-fold compared with compound 11 (Figure 1b). N-Alkyli-
midazoles 23 and 24 have similar activities against AsPC-1 cell
Compd
IC50[a] [mm]
Compd
IC50[a] [mm]
>100
1
2
3
4
12.6ꢁ0.03
71.0ꢁ0.1
42.3ꢁ0.02
17.7ꢁ0.06
5
6
9
10
7.2ꢁ0.02
2.0ꢁ0.1
15.9ꢁ0.2
[a] AsPC-1 cells were treated with various concentration of test com-
pound for 72 h. The antiproliferative effects were determined by using an
MTT assay, and the IC50 was calculated from the results. Data represent
the meanꢁSE of three independent experiments.
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growth (less than onefold) as those of
compounds 20 and 21 (Table 2). The anti-
proliferative effects of compounds 20, 21,
and 23–25 were determined in two more
pancreatic cancer cell lines. MIA PaCa-2
and PANC-1 cells were less sensitive than
AsPC-1 cells to these compounds, with
higher IC50 values (Table 3).
The antiproliferative activities of com-
pounds 20, 21, 23 and 25 were also tested
in MCF7, PC-3, Hep G2 and A549 cell lines.
Among these compounds, compound 20,
with a cyanoenone in the A ring conjugat-
ed with the fluorolactone, is the most
active in all tested cell lines, with IC50
values lower than 1 mm (Table 4).
The introduction of an electron-with-
drawing group at C2 of the ursolic acid (1)
to give compounds 20 and 21 amplified
the antitumor activity of these compounds,
allowing nucleophilic attack by the thiol or
other nucleophiles at C1, which in turns ac-
quires an increased electrophilic nature
due to the substituent at C2.[29] The pres-
ence of a fluorolactone group might fur-
ther increase the ability of these com-
pounds to interact with the potential tar-
gets. Overall, modification by introducing
a fluorolactone (compounds 20 and 21) in-
stead of a methoxy group at C28 has im-
proved antiproliferative activities of more
than fourfold compared with compound 9
(with a cyano group at C2), and more than
19-fold compared with ursolic acid (1)
(without a cyano group at C2) in AsPC-
1 cells (Figure 1b).
Since compound 20 is the most active
against cell growth, it was selected as
a model compound to study the mecha-
nism by which it exerts the antiproliferative
activity in AsPC-1 cells. Compound 20 ar-
rested cell cycle at the G1 phase with 1 mm
(Figure 2a,b) and increased the population
of cells in sub-G1 phase at increased con-
centrations (Figure 2c). These data suggest
Figure 1. Structure–activity relationships (SARs) among sev-
eral synthetic derivatives of ursolic acid (1). The comparison
was made based on the IC50 value of each compound in in-
hibiting AsPC-1 cell growth. a) Compound 18 is fivefold
more potent than 2, and compound 19 is twofold more
potent than 3. b) Compound 9 is fivefold more potent than
ursolic acid (1). Compounds 20 and 21 are 19-fold more
potent than ursolic acid (1), fourfold more potent than 9,
and 26-fold more potent than 11. Compounds 23 and 24
are 11-fold more potent than ursolic acid (1), twofold more
potent than 9, and 17-fold more potent than 11. Compound
16 is 11-fold more potent than ursolic acid (1), and 15-fold
more potent than 11. Compound 17 is sixfold more potent
than ursolic acid (1) and 8-fold more potent than 11.
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by compound 20 at 1 mm, it suggests that p21waf1 is a primary
target of compound 20. Increased concentrations of com-
pound 20 did not further increase the levels of p21waf1, but de-
creased the levels of c-FLIP (Figure 3a). Ursolic acid (1) has
been shown to decrease the levels of c-FLIP as a mechanism
to potentiate TRAIL-induced apoptosis in HCT116 human colon
adenocarcinoma cell line.[12] These data suggest that the down-
regulation of c-FLIP could be the cause of the observed apop-
totic effects.
Table 3. The inhibitory activities (IC50) of ursolic acid fluorolactones 20–
25 in three pancreatic cancer cell lines.
IC50[a] [mm]
Compd Ring A
R
AsPC-1
MIA PaCa 2
PANC-1
We observed evidence of apoptosis induction by compound
20 at 8 mm, which was not only accompanied by downregula-
tion of c-FLIP, but also by upregulation of NOXA (Figure 3a).
NOXA is the BH3 protein blocking the antiapoptotic protein
Mcl-1. Unlike other triterpenoid derivatives, compound 20 did
not decrease the levels of Mcl-1, suggesting a different mecha-
nism of action for this new compound.[21] The upregulation of
p21waf1 and NOXA, and the downregulation of c-FLIP after
treatment with compound 20 were also observed in MIA PaCa-
2 and PANC-1 cells (Figure 3b), suggesting that p21waf1, NOXA
and c-FLIP proteins might be the targets of these fluorolactone
derivatives.
20
0.7ꢁ0.05
0.9ꢁ0.01
1.8ꢁ0.04
21
0.5ꢁ0.08
2.1ꢁ0.1
14.3ꢁ0.03
22
23
11.0ꢁ0.05
1.1ꢁ0.02
ND
ND
6.0ꢁ0.03
4.6ꢁ0.03
24
25
1.0ꢁ0.05 28.1ꢁ0.02 21.4ꢁ0.06
7.5ꢁ0.07 10.2ꢁ0.02 10.1ꢁ0.02
Conclusions
A series of novel ursolic acid fluorolactone derivatives (11–25)
was synthesized, and 2-cyano-3-oxo-12a-fluoro-urs-1-en-
13,28b-olide (20) was found to be the most effective inhibitor
of cancer cell growth. Compound 20 arrests the cell cycle in
the G1 phase and induces apoptosis with upregulation of
NOXA and p21waf1 and downregulation of c-FLIP. The mecha-
nisms of increasing NOXA and p21waf1 levels and decreasing c-
FLIP levels are worthy of further investigating. Given their ac-
tivity and potentially novel mode of action, these ursolic acid
fluorolactones represent good leads for anticancer drug dis-
covery.
[a] AsPC-1, MIA PaCa 2 and PANC-1 cells were treated with various con-
centration of test compound for 72 h. The antiproliferative effects were
determined by using an MTT assay, and the IC50 was calculated from the
results. Data represent the meanꢁSE of three independent experiments.
ND: not determined.
Table 4. The inhibitory activities (IC50) of ursolic acid fluorolactones in
breast (MCF7), prostate (PC-3), hepatic (Hep G2) and lung (A549) cancer
cell lines.
Experimental Section
IC50[a] [mm]
IR spectra were recorded in JASCO FT/IR-420. 1H, 13C, DEPT-135,
HMQC, HMBC, COESY and NOESY spectra were recorded on
a Bruker Avance III 400 MHz spectrometer. The chemical shifts (d)
were recorded in parts per million (ppm) using the CHCl3 residual
solvent peaks at d=7.26 ppm (1H) and d=77.00 ppm (13C) as inter-
nal standards, and coupling constants (J) are given in hertz (Hz).
Low-resolution mass spectrometry (MS) was obtained using a Finni-
gan Polaris QGC/MS Benchtop Ion Trap spectrometer with a direct
insertion probe, and elemental analyses were performed on an An-
alyzer Elemental Carlo Erba 1108 by chromatographic combustion.
Melting points (mp) were determined using a BUCHI melting point
B-540 apparatus and are uncorrected. For thin-layer chromatogra-
phy (TLC) analysis, Kiesel gel 60HF254/60G plates were used, and
flash column chromatography (FCC) was performed using Kiesel
gel 60 (230–400 mesh, Merck).
Compd
MCF7
PC-3
Hep G2
A549
20
21
23
25
0.7ꢁ0.2
0.9ꢁ0.01
2.0ꢁ0.03
4.0ꢁ0.03
5.6ꢁ0.02
0.5ꢁ0.12
3.0ꢁ0.11
6.8ꢁ0.07
10.6ꢁ0.14
0.9ꢁ0.05
10.0ꢁ0.05
9.5ꢁ0.08
16.2ꢁ0.03
2.2ꢁ0.11
2.8ꢁ0.04
7.0ꢁ0.05
[a] MCF7, PC-3, Hep G2 and A549 cells were treated with various concen-
tration of test compound for 72 h. The antiproliferative effects were de-
termined by using an MTT assay, and the IC50 was calculated from the re-
sults. Data represent the meanꢁSE of three independent experiments.
that this compound has dual effects of arresting cell cycle pro-
gression and inducing apoptosis (Figure 2b,c).
We tested the effects of compound 20 on the levels of cell-
cycle-regulating and apoptosis-related proteins at concentra-
tions of 1–8 mm. Compound 20 significantly increased the level
of p21waf1 at 1 mm, which correlates with cell cycle arrest in the
G1 phase. Since other proteins were not significantly regulated
Ursolic acid (1), Selectfuor, dimethylaminopyridine (DMAP), fluoro-
acetic anhydride, benzoic anhydride, butyric anhydride, acetic an-
hydride, phthalic anhydride, ethyl formate, 2,3-dichloro-5,6-dicya-
nobenzoquinone (DDQ), sodium methoxide (NaOMe), lithium
iodide, tetrahydrofuran (THF), dioxane, nitromethane, dimethylfor-
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Ursolic Acid Fluorolactone Derivatives
MED
80.92 ppm (C3); IR (film CHCl3): n˜ =
2926.5, 1735.6, 1691.3 1460.8 cmꢀ1
;
MS (EI, 70 eV): m/z (%): 497.82 (1)
[M]+, 437.89 (23), 93.76 (37), 91.78
(50), 89.79 (100), 81.87 (22), 79.88
(83), 77.90 (73), 75.89 (34), 67.87
(23), 65.86 (62); Anal. calcd for
C32H50O4: C 77.06, H 10.10, O 12.83
found: C 77.46, H 10.05, O 12.49.
3b-Phthaloxy-urs-12-en-28-oic-
acid (3):
A stirred mixture of
1 (200 mg 0.44 mmol) in pyridine
(5 mL) at 1158C was treated with
DMAP (53.77 mg 1 mmol) and
phthalic anhydride (651.73 mg,
0.25 mmol). After 23.5 h, the reac-
tion mixture was diluted with ice
(50 mL), and the aqueous phase
was extracted with CHCl3 (3ꢁ
40 mL). The organic phase was
washed with water (3ꢁ40 mL),
dried over Na2SO4, filtered and
concentrated to dryness, to afford
a solid (240 mg, 90%); mp: 198.3–
1
205.18C; H NMR (400 MHz, CDCl3):
d=7.89 (d, J=6.92 Hz, 1H), 7.72
(d, J=6.85 Hz, 1H), 7.59 (m, J=
26.69 Hz, 2H), 5.24 (s, 1H, H12),
4.75 (dd, J=14.88 Hz, 1H, H3), 1.09
(s, 3H), 0.94 (s, 6H), 0.93 (s, 3H),
0.88 (s, 3H), 0.85 (s, 3H), 0.78 ppm
(s, 3H); 13C NMR (100 MHz, CDCl3):
d=183.93 (C28), 171.62 (OCOC),
167.93 (COOH), 137.95 (C13),
131.59, 131.53, 131.02, 130.65,
129.61, 128.83, 125.68 (C12),
82.902 ppm (C3); IR (film CHCl3):
n˜ =3064.3, 2926.5, 1716.3, 1691.3,
1457.0 cmꢀ1; MS (EI, 70 eV): m/z
(%): 604.97 (1) [M]+, 247.72 (52),
202.77 (50), 188.87 (20), 148.90
(27), 133.19 (100), 118.96 (24),
105.10 (30), 90.94 (23); Anal. calcd
for C38H52O6.H2O: C 73.28, H 8.74, O
17.98, found: C 73.68, H 8.52, O
17.8.
Figure 2. Cell cycle arrest and induction of apoptosis by compound 20 in AsPC-1 cells. a) Cell cycle analysis of
AsPC-1 cells treated with compound 20. b) Representative FACS analyses of the G1 phase arrest of AsPC-1 cells
treated with compound 20 at 1 mm for 24 h. c) Representative FACS analyses of induction of the sub-G1 phase
(apoptotic cells) of AsPC-1 cells treated with compound 20 at 8 mm for 24 h. The cell cycle was determined by
FACS after staining with propidium iodide (PI).
mamide (DMF), benzene, 1,1’-carbonyldiimidazole (CDI), 1,1’-car-
bonylbis(2’-methylimidazole) (CBMI), and 1,1’-carbonyl-di(1,2,4-tria-
Methyl 3b-hydroxy-urs-12-en-28-oate (4): Prepared from 1 accord-
ing to a previously described method[53] to give 4 as a white solid
(44 mg, 93%); mp: 170.7–172.08C; 1H NMR (400 MHz, CDCl3): d=
5.23 (t, J=7.32 Hz, 1H, H12), 3.59 (s, 3H, COOCH3), 3.20 (dd, J=
15.83 Hz, 1H, H3), 1.07 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H), 0.91 (s,
3H), 0.85 (d, J=6.48 Hz, 3H), 0.77 (s, 3H), 0.73 ppm (s, 3H),
13C NMR (100 MHz, CDCl3): d=178.05 (C28), 138.12 (C13), 125.55
(C12), 79.03 ppm (C3); IR (film CHCl3): n˜ =3434.6, 2926.5, 1718.3,
1645.9, 1457.0 cmꢀ1; MS (EI, 70 eV): m/z (%): 471.04 (7) [M]+, 261.99
(53), 207.08 (21), 203.07 (89), 202.15 (20), 189.19 (28), 134.11 (38),
133.21 (100), 119.13 (26), 91.22 (21); Anal. calcd for
C31H50O3·0.5hexane: C 79.48, H 11.18, O 9.34, found: C 79.68, H
11.31, O 9.01.
zole) (CDT) were purchased from Sigma–Aldrich. Solvents used in
the workups were purchased from VWR Portugal and were of ana-
lytical grade. KHCO3, NaCl, NaHCO3, Na2SO3, and hydroxylamine
chlorhydrate were purchased from Merck Co. All solvents used in
reactions were first purified and dried according to literature pro-
cedures.
The purity of all compounds tested for biological activity was de-
termined to be great than 98% by NMR and elemental analysis.
3b-Acetoxy-urs-12-en-28-oic acid (2): Prepared from ursolic acid
(1) according to a previously described method[26] to give 2 as
1
a white solid (98 mg, 92%); mp: 287.4–290.88C; H NMR (400 MHz,
CDCl3): d=5.21 (t, J=6.54 Hz, 1H), 4.47 (dd, J=15.99 Hz, 1H), 2.16
(d, J=11.51 Hz, 1H), 2.02 (s, 3H), 1.05 (s, 3H), 0.94 (s, 3H), 0.84 (s,
6H), 0.83 (s, 3H), 0.74 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
183.84 (C28), 171.03 (OCOCH3), 137.92 (C13), 125.71 (C12),
Methyl 3-oxours-12-en-28-oate (5): Prepared from 4 according to
a previously described method[29] to give 5 as a white solid
1
(250 mg, 93%); mp: 196.3–197.28C; H NMR (400 MHz, CDCl3): d=
ChemMedChem 0000, 00, 1 – 13
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J. A. R. Salvador, Y. Jing, et al.
MED
Methyl
2-cyano-3-hydroxy-urs-
2,12-dien-28-oate (8): Prepared
from 7 according to a previously
described method[29] to give 8 as
a white solid (221 mg, 86%); mp:
136.4–140.38C; 1H NMR (400 MHz,
CDCl3): d=5.28 (s, 1H), 3.61 ppm
(s, 3H); IR (film CHCl3): n˜ =2947.7,
1869.6, 1719.2, 1457.9 cmꢀ1; MS (EI,
70 eV): m/z (%): 494.2 (9) [M+H]+,
262.0 (34), 249.1 (23), 203.02 (62),
189.05 (54), 133.1 (100), 119.13
(32), 105.09 (22), 91.13 (27).
Methyl
dien-28-oate (9): Prepared from 8
according to previously de-
2-cyano-3-oxours-1,12-
a
scribed method[29] to give 9 as
a yellow solid (170 mg, 88%); mp:
134.7–137.58C; 1H NMR (400 MHz,
CDCl3): d=7.75 (s, 1H, H1), 5.33 (s,
1H, H12), 3.62 (s, 3H), 1.22 (s, 3H),
1.20 (s, 3H), 1.14 (s, 3H), 1.10 (s,
3H), 0.95 (d, J=5.87 Hz, 3H), 0.87
(d, J=6.43 Hz, 3H), 0.85 ppm (s,
3H); IR (film CHCl3): n˜ =2947.7,
Figure 3. Regulation of apoptosis and cell-cycle-related proteins in pancreatic cancer cells by compound 20.
a) AsPC-1 cells treated with compound 20 at the indicated concentrations for 24 h. b) MIA PaCa-2 and PANC-
1 cells treated with 8 mm of compound 20 for 24 h. The relative levels of each indicated protein were determined
by Western blot analysis. GAPDH was used as a loading control.
2872.5,
1721.2,
1685.5,
1457.0 cmꢀ1; MS (EI, 70 eV): m/z
(%): 492.17 (7) [M+H]+, 203.05
(35), 189.08 (58), 187.12 (20),
5.26 (s, 1H, H12), 3.61 (s, 3H), 1.08 (s, 6H), 1.04 (s, 6H), 0.94 (d, J=
5.73 Hz, 3H), 0.86 (d, J=6.33 Hz, 3H), 0.79 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=217.70 (C3), 177.99 (C28), 138.29 (C13),
125.33 ppm (C12); IR (film CHCl3): n˜ =2946.7, 2872.5, 1725.0,
1704.8, 1456.0 cmꢀ1; MS (EI, 70 eV): m/z (%): 470.48 (27) [M+H]+,
469.47 (94) [M]+, 467.99 (12) [M]ꢀ1, 264.09 (51), 262.48 (100), 203.81
(25), 202.52 (26), 201.52 (23), 134.34 (25), 133.12 (64).
133.11 (100), 119.13 (46), 117.13 (20), 105.13 (28), 91.11 (33); Anal.
calcd for C32H45NO3: C 78.17, H 9.22, N 2.85, O 9.76, found: C 77.82,
H 9.23, N 2.86, O 10.09.
2-Cyano-3-oxours-1,12-dien-28-oic acid (10): Prepared from 9 ac-
cording to a previously described method[29] to give 10 as a solid
(86 mg, 64%); mp: 213.7–217.68C; 1H NMR (400 MHz, CDCl3): d=
7.76 (s, 1H), 5.32 (s, 1H), 1.22 (s, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 1.11
(s, 3H), 0.96 (d, J=5.90 Hz, 3H), 0.88 ppm (s, 6H); 13C NMR
(100 MHz, CDCl3): d=198.02 (C3), 183.07 (C28), 169.98 (C1), 138.87
(C13), 124.27 (C12), 114.91, 113.91 ppm; IR (film CHCl3): n˜ =2926.5,
2870.5, 1652.2, 1609.3, 1456.0 cmꢀ1; MS (EI, 70 eV): m/z (%): 477.84
(1) [M+H]+, 247.85 (64), 218.84 (25), 202.86 (70), 188.92 (27),
186.92 (22), 132.93 (100), 118.94 (33), 104.95 (29), 90.91 (31); Anal.
calcd for C31H43NO3: C 77.95, H 9.07, N 2.93, O 10.05, found: C
77.89, H 9.47, N 2.94, O 9.70.
Methyl 2-hydroxymethylene-3-oxours-12-en-28-oate (6): Pre-
pared from 5 according to a previously described method[29] to
1
give 6 as a white solid (111 mg, 94%); mp: 127.9–131.78C; H NMR
(400 MHz, CDCl3): d=14.99 (s, 1H, CHOH), 8.57 (s, 1H, CHOH), 5.30
(s, 1H, H12), 3.61 (s, 3H), 1.19 (s, 3H), 1.11 (s, 3H), 1.09 (s, 3H), 0.94
(d, J=5.98 Hz, 3H), 0.91 (s, 3H), 0.87 (d, J=6.39 Hz, 3H), 0.81 ppm
(s, 3H); 13C NMR (100 MHz, CDCl3): d=190.84 (C3), 188.17 (CHOH),
178.00 (C28), 138.23 (C13), 125.35 (C12), 105.79 ppm (C2); IR (film
CHCl3): n˜ =3441.4, 2924.5, 2872.5, 1725.0, 1634.4 cmꢀ1; MS (EI,
70 eV): m/z (%): 497.40 (30) [M]+, 263.63 (40), 262.47 (99), 249.52
(35), 233.44 (52), 204.71 (31), 203.41 (95), 189.41 (45), 187.33 (25),
134.35 (48), 133.23 (100), 119.23 (27); Anal. calcd for
C32H48O4·0.25H2O: C 76.91, H 9.75, O 13.34 found: C 76.63, H 10.20,
O 13.17.
3b-ol-12b-Fluoro-urs-13,28b-olide (11):
A stirred solution of
1 (300 mg 0.66 mmol) in NO2Me (6 mL) and dioxane (4 mL) at 808C
was treated with Selectfluor (701.43 mg 1.98 mmol). After 24 h, the
reaction mixture was diluted with water (60 mL), the aqueous
phase was extracted with Et2O (3ꢁ55 mL). The resulting organic
phases were washed with 10% aq NaCl (3ꢁ60 mL), dried over
Na2SO4, filtered and concentrated to dryness. The crude solid was
purified by flash column chromatography (hexane/EtOAc, 90:10!
30:70) to afford a white solid (11) (276 mg, 88%); mp: 316.8–
317.68C; 1H NMR (400 MHz, CDCl3): d=4.85 (dt, J=62.84 Hz, 1H,
H12), 3.19 (dd, J=16.18 Hz, 1H, H3), 1.20 (s, 3H), 1.19 (s, 3H), 1.13
(d, J=6.26 Hz, 3H), 0.97 (s, 6H), 0.91 (s, 3H), 0.76 ppm (s, 3H)
ppm; 13C NMR (100 MHz, CDCl3): d=178.88 (C28), 91.97 (J=
14.24 Hz, C13), 88.70 (J=185.75 Hz, C12), 78.68 (C3), 55.32, 52.55
(J=3.58 Hz), 49.18 (J=9.078 Hz), 45.19, 43.90 (J=2.83 Hz), 42.53,
39.53, 39.03, 38.84, 38.40, 37.12, 33.95, 31.34, 30.71, 27.92, 27.62,
27.22, 25.37 (J=19.72 Hz), 22.39, 19.39, 18.50, 17.65, 17.22, 16.65,
16.46, 15.26 ppm; IR (film CHCl3): n˜ =3337.2, 2927.4, 2872.5, 1765.5,
Methyl isoxazolo[4,5-b]urs-12-en-28-oate (7): Prepared from 6 ac-
cording to a previously described method[29] to give 7 as a white
1
solid (312 mg, 96%); mp: 119.9–120.08C; H NMR (400 MHz, CDCl3):
d=7.97 (s, 1H), 5.30 (s, 1H, H12), 3.61 (s, 3H), 1.30 (s, 3H), 1.21 (s,
3H), 1.10 (s, 3H), 0.94 (d, J=5.95 Hz, 3H), 0.89 (s, 3H), 0.87 (d, J=
6.52 Hz, 3H), 0.80 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
177.08 (C28), 173.03 (C3), 150.15 (CHNO), 138.23 (C13), 125.25
(C12), 108.83 ppm (C2); IR (film CHCl3): n˜ =2925.5, 2869.6, 1723.1,
1483.0, 1457.0 cmꢀ1; MS (EI, 70 eV): m/z (%): 494.22 (14) [M+H]+,
262.03 (32), 203.09 (67), 189.07 (41), 133.13 (100), 119.15 (34),
105.17 (24), 91.16 (26).
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Ursolic Acid Fluorolactone Derivatives
MED
1459.8 cmꢀ1; MS (EI, 70 eV): m/z (%): 475.12 (8) [M+H]+, 455.3 (93),
246.24 (89), 207.32 (66), 189.36 (100), 187.37 (77), 119.22 (76); Anal.
calcd for C30H47FO3: C 75.91, H 9.98, found: C 75.52, H 10.18.
1457.9, 1395.3, 1373.1, 1294.0 cmꢀ1; MS (EI, 70 eV): m/z (%): 583.2
(6) [M]+, 190.1 (53), 189.1 (99), 187.2 (55), 147.1 (57), 145.1 (48),
133.1 (65), 127.1 (54), 121.1 (60), 119.1 (100), 109.1 (74), 107.1 (84),
105.1 (77), 95.1 (79), 93.1 (64), 91.1 (78); Anal. calcd for
C35H51FN2O4: C 72.13, H 8.82, N 4.81, found: C 71.97, H 8.56, N 4.73.
3-Oxo-12b-fluoro-urs-13,28b-olide (12): Prepared from 11 accord-
ing to a previously described method.[29] Purification of the crude
by flash column chromatography (hexanes/EtOAc, 80:20!65:35)
afforded 12 as a white solid (389 mg, 22%); mp: 327.3–329.28C;
1H NMR (400 MHz, CDCl3): d=4.87 (dq, J=62.47 Hz, 1H), 1.25 (s,
3H), 1.21 (s, 3H), 1.14 (d, J=6.31 Hz, 3H), 1.08 (s, 3H), 1.04 (s, 3H),
1.02 (s, 3H), 0.96 ppm (d, J=5.56 Hz, 3H); 13C NMR (100 MHz,
CDCl3): d=216.97 (C3), 178.75 (C28), 91.91 (J=14.27 Hz, C13),
89.49 (J=185.77 Hz, C12), 54.83, 52.57, 48.48 (J=9.33 Hz), 47.28,
45.19, 44.02 (J=3.09 Hz), 42.40, 39.79, 39.55, 38.51, 36.85, 33.89,
33.37, 31.33, 30.73, 27.64, 26.58, 25.75 (J=19.71 Hz), 22.39, 20.97,
19.40, 18.99, 18.17, 17.09, 16.47, 16.42 ppm; IR (film CHCl3): n˜ =
2928.4, 1771.3, 1704.8, 1457.9 cmꢀ1; MS (EI, 70 eV): m/z (%): 473.03
(1) [M]+, 245.97 (67), 205.02 (65), 210.03 (79), 187.03 (82), 163.04
(62), 119.03 (100), 107.02 (64), 105.03 (58), 91 (71), 78.98 (75); Anal.
calcd for C30H45FO3·0.5EtOAc: C 74.38, H 9.56, found: C 74.16, H
9.61.
3b-Trifluoroacetoxy-12b-fluoro-urs-13,28b-olide (15):
A stirred
mixture of 11 (200 mg 0.42 mmol) in THF (5 mL) at room tempera-
ture was treated with DMAP (10% of the mass of 11) and triflouro-
acetic anhydride (0.58 mL 4.2 mmol). After 1 h, the reaction mixture
was acidified with 10% aq HCl (50 mL), and the aqueous phase
was extracted with Et2O (3ꢁ40 mL). The organic phase was
washed with saturated aq NaHCO3 (3ꢁ40 mL) and water (3ꢁ
40 mL), dried over Na2SO4, filtered and concentrated to dryness to
afford a solid (205.8 mg). The residue was purified by flash column
chromatography (hexanes/EtOAc, 95:5!75:25) to afford 15 as
1
a white solid (74 mg, 31%); mp: 280.2–282.28C; H NMR (400 MHz,
CDCl3): d=4.87 (dd, J=62.64 Hz, 1H, H12), 4.68 (dd, J=16.16 Hz,
1H, H3), 1.26 (s, 3H), 1.23 (s, 3H), 1.21 (s, 3H), 1.15 (d, J=6.19 Hz,
3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.91 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=178.82 (C28), 91.84 (J=14.52 Hz, C13), 89.53 (J=
185.90 Hz, C12), 85.78 (C3), 55.30, 52.58, 49.10 (J=9.24 Hz), 45.21,
43.95 (J=3.16 Hz), 42.58, 39.57, 38.57, 38.46, 38.09, 37.06, 33.85,
31.37, 30.75, 29.70, 27.77, 27.68, 25.47 (J=19.59 Hz), 23.15, 22.41,
19.44, 18.56, 17.52, 17.24, 16.75, 16.54, 16.19 ppm; IR (film CHCl3):
n˜ =2953.5, 2927.4, 2873.4, 1777.1, 1459.8 cmꢀ1; MS (EI, 70 eV): m/z
(%): 570.84 (2) [M]+, 245.98 (54), 201.05 (65), 189.08 (71), 187.1 (87),
145.16 (51), 119.16 (100), 107.15 (69), 105.15 (61), 93.16 (62), 91.23
(92), 79.17 (81), 77.16 (53), 67.14 (54); Anal. calcd for C31H43F4O4: C
67.35, H 8.12, found: C 67.74, H 8.46.
3b-(1H-Imidazole-1-carbonyloxy)-12b-fluoro-urs-13,28b-olide
(13): A stirred solution of 11 (300 mg 0.63 mmol) in THF (6.5 mL)
under N2 at 708C was treated with CDI (204.31 mg 1.26 mmol).
After 3 h, the reaction mixture was diluted with water (60 mL), and
the aqueous phase was extracted with EtOAc (3ꢁ50 mL). The re-
sulting organic phases were washed with 10% aq NaCl (3ꢁ50 mL),
dried over Na2SO4, filtered and concentrated to dryness. The crude
residue was purified by flash column chromatography (hexanes/
EtOAc, 70:30!55:45) to afford 13 as a white solid (168 mg, 47%);
1
mp: 285.9–286.88C; H NMR (400 MHz, CDCl3): d=8.13 (s, 1H), 7.40
3b-benzoxy-12b-fluoro-urs-13,28b-olide (16): A stirred mixture of
11 (200 mg 0.42 mmol) in THF (5 mL) at room temperature was
treated with DMAP (10% of the mass of 11) and benzoic anhydride
(475.08 mg 2.1 mmol). After 8 h, the workup was performed as for
15. Purification by flash column chromatography (hexanes/EtOAc,
85:15!75:25) afforded 16 as a white solid (122 mg, 50%); mp:
319.2–322.68C; 1H NMR (400 MHz, CDCl3): d=8.04 (d, J=7.53 Hz,
2H), 7.55 (t, J=14.59 Hz, 1H), 7.44 (t, J=15.16 Hz, 2H), 4.89 (dt, J=
62.70 Hz, 1H, H12), 4.73 (dd, J=15.80 Hz, 1H, H3), 1.24 (s, 3H), 1.22
(s, 3H), 1.16 (d, J=6.18 Hz, 3H), 1.02 (s, 3H), 0.99 (d, J=5.33 Hz,
6H), 0.94 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=178.88 (C28),
166.25 (OCO), 132.80, 130.78, 129.50 (2C), 128.33 (2C), 91.95 (J=
14.25 Hz, C13), 89.64 (J=185.93 Hz, C12), 81.09 (C3), 55.48, 52.58
(J=3.33 Hz), 49.12 (J=9.08 Hz), 45.20, 43.93 (J=2.77 Hz), 42.58,
39.55, 38.74, 38.43, 38.20, 37.11, 33.90, 31.36, 30.73, 28.06, 27.65,
25.45 (J=19.18 Hz), 23.61, 22.42, 19.43, 18.56, 17.58, 17.24, 16.75,
16.71, 16.54 ppm; IR (film CHCl3): n˜ =3064.3, 2030.3, 2872.5, 1785.8,
1715.4, 1599.7, 1452.1 cmꢀ1; MS (EI, 70 eV): m/z (%): 578.87 (1) [M]+
, 245.90 (32), 201.97 (23), 201.01 (25), 190.04 (35), 189.07 (60),
187.12 (33), 119.07 (29), 106.98 (27), 105.21 (100), 91.14 (22), 79.07
(21), 77.1 (49); Anal. calcd for C37H51FO4: C, 76.78, H 8.88, found: C
76.92, H 9.09.
(s, 1H), 7.07 (s, 1H), 4.87 (dt, J=45.82 Hz, 1H, H12), 4.68 (dd, J=
15.87 Hz, 1H, H3), 1.22 (s, 3H, C26), 1.21 (s, 3H, C27), 1.14 (d, J=
6.20 Hz, 3H, C29), 0.98 (s, 3H, C25), 0.96 (s, 9H, C23 or C24 or C30),
0.86 ppm (d, J=8.75 Hz, 1H, H5); 13C NMR (100 MHz, CDCl3): d=
178.15 (C28), 148.46 (COO), 136.93 (Cimidazole), 130.48 (Cimidazole),
117.00 (Cimidazole), 91.80 (J=14.31 Hz, C13), 89.49 (J=185.86 Hz,
C12), 85.84 (C3), 55.32 (C5), 52.55 (J=3.34 Hz, C18), 49.06 (J=
9.38 Hz, C9), 45.17 (C17), 43.94 (C14), 42.54 (C7), 39.53 (C20), 38.57,
38.40 (C19), 38.19 (C4), 37.03 (C10), 33.82 (C7), 31.33, 30.70, 28.02
(C23 or C24 or C30), 27.63 (C15), 25.44 (J=19.38 Hz), 23.45, 22.38,
19.39 (C25), 18.53 (C26), 17.50, 17.21 (C27), 16.69 (C23 or C24 or
C30), 16.58 (C23 or C24 or C30), 16.50 ppm (C29); IR (film CHCl3):
n˜ =3136.7, 2976.6, 2876.3, 1767.4, 1758.8, 1524.5, 1470.5, 1395.3,
1319.1, 1288.2, 1240.0 cmꢀ1; MS (EI, 70 eV): m/z (%): 569.2 (8) [M]+,
189.2 (89), 187.2 (62), 133.2 (64), 119.2 (100), 107.2 (75), 105.2 (78),
95.2 (75), 93.2 (62), 91.2 (78), 79.15 (76); Anal. calcd for
C34H49FN2O4.C 71.80, H 8.68, N 4.93, found: C 72.06, H 8.58, N 4.96.
3b-(2’-Methyl-1H-imidazole-carbonyloxy)-12b-fluoro-urs-13,28b-
olide (14): A stirred solution of 11 (300 mg 0.63 mmol) in THF
(4 mL) under N2 at 708C was treated with CBMI (239.66 mg
1.26 mmol). After 8 h, the workup was performed as for 13. Purifi-
cation by flash column chromatography (hexanes/EtOAc, 70:30!
50:50) afforded 14 as a white solid (180 mg, 49%); mp: 293.1–
293.68C; 1H NMR (400 MHz, CDCl3): d=7.33 (s, 1H), 6.86 (s, 1H),
4.87 (dt, J=45.78 Hz, 1H), 4.66 (dd, J=15.70 Hz, 1H), 2.65 (s, 3H),
1.23 (s, 3H), 1.21 (s, 3H), 1.15 (d, J=6. 31 Hz, 3H), 0.98 (s, 3H),
0.96 ppm (s, 9H); 13C NMR (100 MHz, CDCl3): d=178.77, 149.37,
147.88, 127.64, 117.98, 91.83 (J=14.21 Hz), 89.51 (J=186.13 Hz),
85.54, 55.37, 52.60, 52.56, 49.13, 49.04, 45.18, 43.96, 43.93, 42.56,
39.55, 38.64, 38.42, 38.14, 37.06, 33.84, 31.35, 30.72, 28.05, 27.65,
25.56, 25.36, 23.53, 22.40, 19.40, 18.54, 17.53, 17.23, 16.80, 16.79,
16.70, 16.52 ppm; IR (film CHCl3): n˜ =2933.2, 2878.2, 1769.4, 1758.8,
3b-Butyroxy-12b-fluoro-urs-13,28b-olide (17): A stirred mixture of
11 (200 mg 0.42 mmol) in THF (5 mL) at room temperature was
treated with DMAP (10% of the mass of 11) and butyric anhydride
(0.34 mL 2.1 mmol). After 1 h, the workup was performed as for 15.
Purification by flash column chromatography (hexanes/EtOAc,
85:15!80:20) afforded 17 as a white solid (67 mg, 28%); mp:
251.4–2558C; 1H NMR (400 MHz, CDCl3): d=4.86 (dt, J=62.73 Hz,
1H, H12), 4.49 (dd, J=16.28 Hz, 1H, H3), 1.21 (s, 3H), 1.20 (s, 3H),
1.14 (d, J=6.22 Hz, 3H), 0.97 (s, 3H), 0.94 (d, J=3.49 Hz, 6H),
0.85 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=178.87 (C28),
173.49 (OCO), 91.94 (J=14.16 Hz, C13), 89.64 (J=185.88 Hz, C12),
ChemMedChem 0000, 00, 1 – 13
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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J. A. R. Salvador, Y. Jing, et al.
MED
80.13 (C3), 55.41, 52.56 (J=3.47 Hz), 49.10 (J=9.28 Hz), 45.19,
43.90 (J=2.90 Hz), 42.55, 39.55, 38.71, 38.42, 37.83, 37.06, 36.69,
33.89, 31.36, 30.73, 27.91, 27.64, 25.41 (J=19.09 Hz), 23.59, 22.41,
19.42, 18.59, 18.53, 17.55, 17.22, 16.72, 16.53, 16.47, 13.70 ppm; IR
Compound 20 (light-yellow solid); mp: 264.9–268.68C; 1H NMR
(400 MHz, CDCl3): d=7.81 (s, 1H, H1), 4.51 (dt, J=66.17 Hz, 1H,
H12), 1.30 (d, J=7.26 Hz, 3H), 1.25 (s, 3H), 1.22 (s, 3H), 1.20 (s, 3H),
1.16 (s, 3H), 0.98 ppm (t, J=9.45 Hz, 6H); 13C NMR (100 MHz,
CDCl3): d=197.72 (C3), 177.51 (C28), 168.79 (C1), 114.69, 114.61,
96.11 (J=189.19 Hz, C12), 90.38 (C13), 53.00, 45.264 45.08 (J=
9.305 Hz), 44.68, 43.46 (J=17.41 Hz), 42.35 (J=9.78 Hz), 41.04,
40.93, 39.04, 37.34, 34.87, 31.67, 29.10, 27.396, 25.94 (J=25.20 Hz),
25.74, 22.73 (J=3.35 Hz), 21.81, 20.94, 20.49, 20.34, 20.07, 19.46,
(film CHCl3): n˜ =2964.1, 2933.2, 2874.4, 1772.3, 1729.8, 1458.9 cmꢀ1
;
MS (EI, 70 eV): m/z (%): 544.25 (1) [M]+, 201.04 (43), 190.08 (53),
189.12 (100), 187.14 (56), 120.01 (36), 119.11 (51), 93.18 (38), 91.2
(44), 79.35 (42); Anal. calcd for C34H53FO4: C, 74.96, H 9.81, found: C
75.36, H 9.70.
19.11 ppm; IR (film CHCl3): n˜ =2926.5, 1740.4, 1685.5, 1457.9 cmꢀ1
;
MS (EI, 70 eV): m/z (%): 495.91 (4) [M]+, 123.06 (70), 122.04 (92),
121.06 (45), 119.1 (48), 107.09 (100), 105.1 (46), 91.13 (60), 81.11
(57), 79.09 (66), 67.05 (46); Anal. calcd for C31H42FNO3: C, 75.12, H
8.54, N 2.83, found: C 74.77, H 8.94, N 2.94.
3b-Acetoxy-12b-fluoro-urs-13,28b-olide (18): A stirred solution of
2 (150 mg, 0.3 mmol) in NO2Me (3 mL) and dioxane (2 mL) at 808C
was treated with Selectfluor (318.8 mg, 0.9 mmol). After 24 h, the
workup was performed as for 11. Purification by flash column chro-
matography (hexanes/EtOAc, 90:10!75:25) afforded 18 as a white
1
Compound 21 (white solid); mp: 365.5–366.88C; H NMR (400 MHz,
1
solid (67 mg, 43%); mp: 280.2–281.68C; H NMR (400 MHz, CDCl3):
CDCl3): d=7.82 (s, 1H), 4.94 (dq, J=62.15 Hz, 1H, H12), 1.31 (s, 3H,
C26), 1.25 (s, 3H, C29), 1.22 (d, J=3.13 Hz, 3H, C27), 1.20 (s, 3H,
C25), 1.15 (s, 6H, C23 and C24), 0.98 ppm (d, J=5.71 Hz, 3H, C30);
13C NMR (100 MHz, CDCl3): d=197.70 (C3), 178.38 (C28), 169.04
(C1), 114.70 (CN), 114.41 (C2), 91.52 (J=14.49 Hz, C13), 88.52 (J=
187.42 Hz, C12), 52.71 (C5), 52.61 (J=3.16 Hz, C18), 45.14, 45.05
(C4), 44.17 (J=2.72 Hz, C17), 43.48 (C8), 42.43 (J=9.85 Hz, C9)
40.40 (C10), 39.53, 38.39, 33.06, 31.25, 30.67, 29.67, 27.69 (C25),
27.64, 25.64 (J=20.53 Hz), 21.29, 19.38 (C30), 18.91 (C26 and C29),
18.25, 17.00 (C27), 16.53 ppm (C23 or C24); IR (film CHCl3): n˜ =
2926.5, 1767.4, 1684.5, 1457.9 cmꢀ1; MS (EI, 70 eV): m/z (%): 496.15
(6) [M]+, 189.11 (42), 187.17 (56), 163.2 (53), 119.2 (100), 107.18
(51), 91.21 (53), 79.2 (49); Anal. calcd for C31H42FNO3: C 75.12, H
8.54, N 2.83, found: C 75.04, H 8,93, N 2.95.
d=4.86 (dt, J=62.76 Hz, 1H, H12), 4.48 (dd, J=16.35 Hz, 1H, H3),
2.05 (s, 3H), 1.25 (s, 3H), 1.21 (s, 3H), 1.20 (s, 3H), 1.14 (d, J=
6.26 Hz, 3H), 0.96 (d, J=12.52 Hz, 3H), 0.85 ppm (s, 6H); 13C NMR
(100 MHz, CDCl3): d=178.88 (C28), 170.98 (OCO), 91.93 (J=
14.19 Hz, C13), 89.64 (J=185.74 Hz, C12), 80.48 (C3), 55.42, 52.57
(J=3.39 Hz), 49.12 (J=9.22 Hz), 45.20, 43.91 (J=2.26 Hz), 42.56,
39.55, 38.73, 38.42, 37.80, 37.06, 33.89, 31.36, 30.73, 27.89, 27.64,
25.42 (J=19.20 Hz), 23.54, 22.41, 21.26, 19.42, 18.54, 17.56, 17.21,
16.74, 16.53, 16.41 ppm; IR (film CHCl3): n˜ =2928.4, 1767.4, 1729.8,
1457.9 cmꢀ1; MS (EI, 70 eV): m/z (%): 516.91 (5) [M]+, 497.04 (66),
190.12 (51), 189.14 (100), 187.19 (72), 119.13 (64), 107.11 (62),
105.35 (55), 93.36 (55), 91.03 (57), 79.13 (67); Anal. calcd for
C32H49FO4: C 74.38, H 9.56, found: C 74.78, H 9.40.
2-Hydroxymethylene-3-oxo-12b-fluoro-urs-13,28b-olide (22): Pre-
pared from 12 according to a previously described method.[29] Pu-
rification of the crude solid (154.5 mg 0.33 mmol) by flash column
chromatography (hexanes/EtOAc, 80:20!70:30) afforded pure 22
as a white solid (111 mg, 67%); mp: 272.2–274.28C; 1H NMR
(400 MHz, CDCl3): d=14.89 (s, 1H), 8.62 (s, 1H), 4.89 (dq, J=
62.70 Hz, 1H, H12), 1.27 (s, 3H), 1.22 (s, 3H), 1.19 (s, 3H), 1.14 (d,
J=6.30 Hz, 3H), 1.11 (s, 3H), 0.97 (d, J=5. 63 Hz, 3H), 0.92 ppm (s,
3H); 13C NMR (100 MHz, CDCl3): d=189.87 (C3), 188.84 (CHOH),
178.79 (C28), 105.21 (C2), 91.93 (J=14.19 Hz, C13), 89.50 (J=
185.98 Hz, C12), 52.56 (J=3.52 Hz), 51.98, 47.29 (J=9.44 Hz), 45.21,
44.04 (J=2.83 Hz), 42.21, 40.18, 39.97, 39.52, 38.55, 36.53, 33.06,
31.30, 30.72, 28.19, 27.61, 25.76 (J=19.43 Hz), 22.36, 20.55, 19.39,
18.77, 17.99, 17.03, 16.41, 15.65 ppm; IR (film CHCl3): n˜ =344.2,
2930.3, 1771.3, 1636.3, 1587.1, 1457.9 cmꢀ1; MS (EI, 70 eV): m/z (%):
500.79 (2) [M]+, 245.72 (79), 232.77 (73), 200.8 (68), 186.81 (100),
172.83 (60), 144.85 (56), 118.86 (82), 106.85 (67), 104.87 (58), 90.83
(64), 78.8 (60); Anal. calcd for C31H45FO4: C 74.36, H 9.06, found: C
73.97, H 8.81.
3b-Phthaloxy-12b-fluoro-urs-13,28b-olide (19): A stirred solution
of 3 (304.8 mg 0.5 mmol) in nitromethane (3 mL) and dioxane
(2 mL) at 808C was treated with Selectfluor (531.39 mg 1.5 mmol).
After 23 h, the workup was performed as for 11. Purification by
flash column chromatography (hexanes/EtOAc, 50:50!35:65) gave
19 as a white solid (153 mg, 62%); mp: 241.9–243.68C; 1H NMR
(400 MHz, CDCl3): d=7.89 (d, J=6.92 Hz, 1H), 7.72 (d, J=6.85 Hz,
1H), 7.59 (quintet, J=26.69 Hz, 2H), 4.88 (dt, J=62.80 Hz, 1H,
H12), 4.76 (dd, J=16.04 Hz, 1H, H3), 1.21 (s, 6H), 1.15 (d, J=
6.24 Hz, 3H), 0.97 (d, J=8.35 Hz, 6H), 0.93 (s, 3H), 0.89 ppm (s,
3H); 13C NMR (100 MHz, CDCl3): d=178.96 (C28), 171.75, 167.77,
133.35, 131.99, 130.89, 130.27, 129.85, 128.94, 91.98 (J=14.25 Hz,
C13), 89.62 (J=185.85 Hz, C12), 82.59 (C3), 55.53, 52.58 (J=
2.74 Hz), 49.09 (J=9.03 Hz), 45.21, 43.91 (J=2.56 Hz), 42.56, 39.55,
38.75, 38.42, 38.05, 37.07, 33.88, 31.35, 30.72, 27.98, 27.64, 25.44
(J=19.48 Hz), 22.94, 22.40, 19.42, 18.53, 17.54, 17.23, 16.68,
16.55 ppm (2C); IR (film CHCl3): n˜ =2929.3, 1770.3, 1723.1,
1457.9 cmꢀ1; MS (EI, 70 eV): m/z (%): 623.12 (2) [M+H]+, 245.87
(100), 201 (50), 188.96 (93), 187.02 (64), 148.95 (57), 118.96 (55);
Anal. calcd for C38H51FO6: C 73.28, H 8.25, found: C 73.19, H 8.34.
2-(1H-Imidazol-1-yl)-methylene-3-oxo-12b-fluoro-urs-13,28b-
olide (23): A stirred solution of 22 (240 mg, 0.48 mmol) in THF
(3 mL) under N2 at 708C was treated with CDI (155.66 mg,
0.96 mmol). After 4 h, the workup was performed as for 13. Purifi-
cation by flash column chromatography (hexanes/EtOAc, 50:50!
30:70) gave 23 as a white solid (206 mg, 63%); mp: 316.4–
321.28C; 1H NMR (400 MHz, CDCl3): d=7.86 (s, 1H), 7.71 (s, 1H),
7.24 (s, 1H), 7.20 (s, 1H), 4.96 (dq, J=62.31 Hz, 1H, H12), 3.01 (d,
J=15.83 Hz, 1H), 2.30 (d, J=15.76 Hz, 1H), 1.28 (s, 3H), 1.25 (s,
3H), 1.18 (s, 6H), 1.13 (s, 3H), 0.99 (s, 3H), 0.92 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=206.00 (C3), 178.65 (C28), 130.78,
130.02, 129.81, 123.30, 119.61, 91.81 (J=14.26 Hz, C13), 89.25 (J=
186.24 Hz, C12), 52.60 (J=2.99 Hz), 52.22, 46.95 (J=9.38 Hz), 45.19,
44.09 (J=2.57 Hz), 43.63, 42.18, 39.53, 38.62, 36.27, 32.80, 31.30,
2-Cyano-3-oxo-12a-fluoro-urs-1-en-13,28b-olide (20) and 2-
cyano-3-oxo-12b-fluoro-urs-1-en-13,28b-olide (21): A stirred solu-
tion of 10 (178.6 mg, 0.38 mmol) in nitromethane (6 mL) and diox-
ane (4 mL) at 808C was treated with Selectfluor (404.24 mg,
1.14 mmol). After 2.5 h, the workup was performed as for 11. Purifi-
cation of the crude by flash column chromatography (hexanes/
EtOAc, 80:20!60:40) afforded pure 21 (40 mg, 21%) and two
mixed fractions. Further purification of the mixture fractions (hex-
anes/EtOAc, 75:25!60:40) gave additional pure 21 (30 mg, 16%)
and a final mixed fraction. Purification of this last mixed fraction by
flash column chromatography (hexanes/EtOAc, 75:25!65:35) af-
forded 20 (24 mg, 13%).
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ChemMedChem 0000, 00, 1 – 13
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Ursolic Acid Fluorolactone Derivatives
MED
30.73, 29.53, 27.57, 26.02 (J=19.68 Hz), 22.65, 22.34, 22.04, 19.62,
19.38, 17.65, 16.93, 16.51 ppm (2C); IR (film CHCl3): n˜ =3114.5,
2929.3, 1771.3, 1685.5, 1609.3, 1518.7, 1487.8 cmꢀ1; MS (EI, 70 eV):
m/z (%): 550.5 (8) [M]+, 297.1 (47), 285.09 (100), 187.01 (44), 132.97
(38), 118.97 (45), 104.94 (50), 90.94 (46), 78.9 (47), 68.95 (79); Anal.
calcd for C34H47FN2O3: C 74.15, H 8.60, N 5.09, found: C 73.90, H
8.99, N 4.96.
caspase-8 and p21waf1 from BD Biosciences (San Jose, USA), for c-
FLIP from Alexis Biochemicals (Plymouth Meeting, USA), to Mcl-
1 from Santa Cruz Biotechology, Inc. (Santa Cruz, USA), for cas-
pase 9, cleaved caspase 9, caspase 3, cleaved caspase 3 and BcL-xL
from Cell Signaling, for NOXA from Abcam.
Cell culture: Cancer AsPC-1, MIA PaCa-2, PANC-1, MCF7, PC-3, Hep
G2 and A549 cells were cultured in RPMI 1640 supplemented with
10% fetal bovine serum (FBS).
2-(2’-Methyl-1H-imidazol-1-yl)-methylene-3-oxo-12b-flouro-urs-
13,28b-olide (24): A stirred solution of 22 (240 mg 0.48 mmol) in
THF (3 mL) under N2 at 708C was treated with CBMI (182.6 mg
0.96 mmol). After 4.5 h, the workup was performed as for 13. Purifi-
cation by flash column chromatography (hexanes/EtOAc, 45:55!
25:75) afforded 24 as a white solid (100 mg, 37%); mp: 321.2–
Determination of cell growth inhibition: AsPC-1, MIA PaCa-2, PANC-
1, MCF7, PC-3, Hep G2 and A549 cells were plated with 1.0ꢁ103
cells/well in 96-well plates in 100 mL medium. Test compounds
(100 mL) at various concentrations were added 24 h after seeding,
and the cells were cultured for 3 days. MTT (0.5 mgmLꢀ1, 50 mL)
was added to each well, and the plates were incubated for 4 h. Su-
pernatant was removed, and the precipitated formazan was dis-
solved in DMSO (100 mL). The density of absorbance was measured
at 570 nm on a multiple plate reader. Concentrations that inhibited
cell growth by 50% (IC50) were determined by nonlinear regression
with GraphPad Prism software version 5.0 (GraphPad Software,
Inc., San Diego, USA).
1
325.68C; H NMR (400 MHz, CDCl3): d=7.66 (s, 1H, C2-CH), 7.16 (s,
1H, H4’), 7.03 (s, 1H, H5’), 4.93 (dq, J=62.33 Hz, 1H, H12), 2.97 (d,
J=15.83 Hz, 1H, H1), 2.48 (s, 3H, CH3 imidazole), 1.26 (s, 3H, C26),
1.24 (s, 3H, C27), 1.16 (s, 6H, C23 or C24 or C29), 1.13 (s, 3H, C23
or C24), 0.97 (s, 3H, C30), 0.90 ppm (s, 3H, C25); 13C NMR
(100 MHz, CDCl3): d=206.08 (C3), 178.59 (C28), 147.37 (C2’), 130.67
(C2-CH), 128.76 (C5’), 123.05 (C2), 117.97 (C4’), 91.74 (J=14.41 Hz,
C13), 89.33 (J=186.24 Hz, C12), 52.57 (J=3.34 Hz, C18), 52.40 (C5),
47.08 (J=9.41 Hz, C9), 45.24 (C17), 45.16 (C4), 44.06 (J=2.74 Hz,
C14), 42.87 (C1), 42.21 (C8), 39.51 (C20), 38.57 (C19), 36.32 (C10),
32.83, 31.27, 30.69, 29.45 (C23 or C24), 27.56, 25.90 (J=19.64 Hz),
22.33, 22.15 (C23 or C24), 19.59, 19.36 (C30), 17.69 (C26), 16.94
(C27), 16.48 (C29), 16.41 (C25), 13.66 ppm (CH3 imidazole); IR (film
CHCl3): n˜ =3110.6, 2929.3, 1770.3, 1684.5, 1603.5, 1539.9 cmꢀ1; MS
(EI, 70 eV): m/z (%): 564.06 (10) [M]+, 300.09 (21), 299.06 (100),
189.96 (81), 187.06 (21), 119.03 (20), 107.01 (22), 91 (22), 82.99 (38),
78.98 (20); Anal. calcd for C35H49FN2O3: C, 74.43, H 8.74, F 3.36, N
4.96, O 8.50, found: C 74.54, H 8.88, N 5.14.
Cell cycle analysis: Cell cycle was assessed by flow cytometry using
a fluorescence-activated cell sorter (FACS). AsPC-1 cells were treat-
ed with different concentrations of compound 20 for 24 h, and
then fixed with ice-cold 70% EtOH at a density of 1ꢁ106 cellsmLꢀ1
.
The cells were treated with PI/RNase solution according to the
manufacture’s protocol. DNA content was quantitated by flow cy-
tometry (Becton–Dickinson, San Jose, USA) with an excitation
wavelength of 488 nm and an emission wavelength of 625 nm.
Western blot analysis: Protein extracts of cells treated with com-
pound 20 at various concentrations for 24 h were prepared with
RIPA lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 0.1% sodium dode-
cyl sulfate (SDS), 1% NP-40, 0.5% sodium deoxycholate, 1 mm phe-
nylmethylsulfonyl fluoride (PMSF), 100 mm leupeptin, and 2 mgmLꢀ1
aprotinin, pH 8.0). These protein extracts were separated on 8% or
12% SDS-polyacrylamide gel and transferred to nitrocellulose
membranes. The membranes were stained with 0.2% Ponceau S
red to assure equal protein loading and transfer. After blocking
with 5% nonfat milk for 30 min, the membranes were incubated
with a primary specific antibody overnight at 48C. The membranes
were then washed three times with TBS-tween 1% buffer and incu-
bated for 1.5 h with the appropriate secondary antibody in 1%
nonfat milk. After incubation, the membranes were washed with
TBS-tween 1% buffer. The immunocomplexes were visualized
using enhanced chemiluminescence western blotting detection re-
agent (Amersahm Biosciences, England, UK).
2-(4H-Triazol-4-yl)-methylene-3-oxo-12b-fluoro-urs-13,28b-olide
(25): A stirred solution of 22 (250 mg 0.50 mmol) in THF (3 mL)
under N2 at 708C was treated with CDT (246.2 mg 1.5 mmol). After
5 h, the workup was performed as for 13. Purification by flash
column chromatography (hexanes/EtOAc, 70:30!60:40) afforded
25 as a white solid (145 mg, 33%); mp: 209.2–212.18C; 1H NMR
(400 MHz, CDCl3): d=8.39 (s, 1H), 8.12 (s, 1H), 7.81 (s, 1H), 4.98
(dq, J=62.17 Hz, 1H), 3.59 (d, J=17.43 Hz, 1H), 2.39 (d, J=
17.26 Hz, 1H), 1.27 (s, 3H), 1.24 (s, 3H), 1.18 (s, 6H), 1.13 (s, 3H),
0.98 (s, 3H), 0.90 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
206.56 (C3), 178.71 (C28), 153.03, 146.27, 128.29, 124.84, 91.92 (J=
14.37 Hz, C13), 89.48 (J=185.96 Hz, C12), 52.60 (J=3.51 Hz), 52.48,
46.80 (J=9.59 Hz), 45.28, 45.20, 44.11, 43.72, 42.15, 39.54, 38.61,
35.98, 32.85, 31.31, 30.73, 29.45, 27.59, 25.92 (J=19.45 Hz), 22.36,
22.15, 19.65, 19.39, 17.66, 16.94, 16.50 ppm (2C); IR (film CHCl3):
n˜ =2929.3, 2870.5, 1771.3, 1688.4, 1616.1, 1509.0, 1457.9 cmꢀ1; MS
(EI, 70 eV): m/z (%): 552.14 (6) [M]+, 201.01 (57), 187.02 (84), 173.04
(58), 145.04 (69), 133.04 (60), 119.04 (100), 107.03 (78), 105.04 (82),
Acknowledgements
91.02
(96),
78.99
(80);
Anal.
calcd
for
C33H46FN3O3·0.5hexane·0.5EtOAc: C 71.44, H 8.99, N 6.58, found: C
71.24, H 9.04, N 6.38.
Ana S. Leal is supported by the Fundażo para a CiÞncia e Tecno-
logia (SFRH/BD/41566/2007). Jorge A. R. Salvador is supported by
the Universidade de Coimbra (Portugal). Yongkui Jing is support-
ed by the Samuel Waxman Cancer Research Foundation (New
York, USA). NMR data were obtained at the Nuclear Magnetic
Resonance Laboratory of the Coimbra Chemistry Centre, Universi-
dade de Coimbra (http://www.nmrccc.uc.pt), supported in part
by grant REEQ/481/QUI/2006 from the FCT, POCI-2010 and
FEDER, Portugal. Elemental analysis was conducted at the Centro
de Apoio Científico e Tecnolꢀxico µ Investigaciꢀn (CACTI) of the
Universidade de Vigo (Spain).
Biologic activity assays
Reagents: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) powder and dimethylsulfoxide (DMSO) were obtained
from Sigma–Aldrich (St. Louis, USA). Test compounds were dis-
solved in DMSO to create stock solutions that were stored at
ꢀ808C. Working solutions were prepared in cell culture medium.
Antibodies for poly-(ADP-ribose)-polymerase (PARP) was obtained
from Boehringer–Ingelheim (Mannheim-Waldhof, Germany), for
ChemMedChem 0000, 00, 1 – 13
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J. A. R. Salvador, Y. Jing, et al.
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Keywords: anticancer agents · apoptosis · fluorolactones ·
p21waf1 · triterpenoids · ursolic acids
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Published online on && &&, 0000
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ChemMedChem 0000, 00, 1 – 13
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FULL PAPERS
Semisynthetic derivatives of ursolic
acid were synthesized and evaluated for
their antiproliferative activity in AsPC-
1 pancreatic cancer cells. Introduction
of a fluorolactone moiety improved the
antiproliferative activity, and preliminary
evaluation revealed a novel mode of
action.
A. S. Leal, R. Wang, J. A. R. Salvador,*
Y. Jing*
&& – &&
Semisynthetic Ursolic Acid
Fluorolactone Derivatives Inhibit
Growth with Induction of p21waf1 and
Induce Apoptosis with Upregulation
of NOXA and Downregulation of c-
FLIP in Cancer Cells
ChemMedChem 0000, 00, 1 – 13
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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&
ÞÞ
These are not the final page numbers!