Dimethylaminopyridine derivatives of lupane triterpenoids are potent disruptors of mitochondrial structure and function

Article abstract of DOI:10.1016/j.bmc.2010.06.075

Development of mitochondrially-targeted drugs is receiving increasing attention because of the central roles these organelles play in energy production, reactive oxygen generation, and regulation of cell death pathways. Previous studies have demonstrated that both natural and synthetic triterpenoids can disrupt mitochondrial structure and function. In this study, we tested the ability of a number of dimethylaminopyridine (DMAP) derivatives of lupane triterpenoids to target mitochochondria in two human melanoma cell lines and an untransformed normal fibroblast line. These compounds induced a striking fragmentation and depolarization of the mitochondrial network, along with an inhibition of cell proliferation. A range of potencies among these compounds was noted, which was correlated with the number, position, and orientation of the DMAP groups. Overall, the extent of proliferation inhibition mirrored the effectiveness of mitochondrial disruption. Thus, DMAP derivatives of lupane triterpenoids can be potent mitochondrial perturbants that appear to suppress cell growth primarily via their mitochondrial effects.

Full text of DOI:10.1016/j.bmc.2010.06.075

Bioorganic & Medicinal Chemistry 18 (2010) 6080–6088
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dimethylaminopyridine derivatives of lupane triterpenoids are potent
disruptors of mitochondrial structure and function
Jon Holy a, , Oksana Kolomitsyna , Dmytro Krasutsky , Paulo J. Oliveira , Edward Perkins ,
b
b
c
d
*
b
Pavel A. Krasutsky
a
Department of Anatomy, Microbiology and Pathology, University of Minnesota School of Medicine, Duluth, USA
Natural Resources Research Institute, University of Minnesota, Duluth, USA
Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal
b
c
d
Mercer School of Medicine, Savannah, GA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Development of mitochondrially-targeted drugs is receiving increasing attention because of the central
roles these organelles play in energy production, reactive oxygen generation, and regulation of cell death
pathways. Previous studies have demonstrated that both natural and synthetic triterpenoids can disrupt
mitochondrial structure and function. In this study, we tested the ability of a number of dimethylamino-
pyridine (DMAP) derivatives of lupane triterpenoids to target mitochochondria in two human melanoma
cell lines and an untransformed normal fibroblast line. These compounds induced a striking fragmenta-
tion and depolarization of the mitochondrial network, along with an inhibition of cell proliferation.
A range of potencies among these compounds was noted, which was correlated with the number, posi-
tion, and orientation of the DMAP groups. Overall, the extent of proliferation inhibition mirrored the
effectiveness of mitochondrial disruption. Thus, DMAP derivatives of lupane triterpenoids can be potent
mitochondrial perturbants that appear to suppress cell growth primarily via their mitochondrial effects.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 8 May 2010
Revised 16 June 2010
Accepted 18 June 2010
Available online 1 July 2010
Keywords:
Lupane triterpenoid
Dimethylaminopyridine
Mitochondria
Cell proliferation
Melanoma
1
. Introduction
Natural products have played major roles in the development of
peutic agents. The activity of CDDO and imidazolide, amide, and
dinitrile derivatives on the behavior of a variety of solid tumors
and leukemias has been documented, and a methyl ester of CDDO
1
1
medically useful drugs. Triterpenoids are a very large and interest-
ing family of compounds, many members of which exhibit biolog-
ical activity in mammalian cells, including cytotoxic, antitumor,
is in clinical trials.
The activities of a number of signaling networks have been
shown to be altered by triterpenoids, including Nf B, NRF2, TGFb,
j
1,2
11–16
antiviral, antifungal, and anti-inflammatory effects.
Potential
ERK, STAT, Akt, mTOR, PPAR
c
, and AMPK pathways.
Induction
uses include developing triterpenoids as anti-cancer drugs, both
as chemopreventatives and as chemotherapeutics. Early interest
in this area was generated by the discovery that the lupane triter-
penoid betulinic acid (BA) displays a notable level of discrimina-
tion in promoting apoptosis of melanoma cells.3 Subsequent
studies showed that BA also exhibits activity against glioma, ovar-
ian carcinoma, small-cell and non-small cell lung carcinomas, and
cervical carcinoma cell lines.4 Interestingly, cells of neuroecto-
dermal origin appear to be especially sensitive to this pentacyclic
of apoptosis or autophagy is frequently reported in studies of
triterpenoid action, but chemoprotective effects have also been
reported. Notably, the net effects of a number of triterpenoids
appear to be centered around disruption of mitochondrial function.
Events associated with these compounds include generation of
reactive oxygen species (ROS), both pro- and anti-oxidant func-
tions and disruption of redox status, calcium deregulation, a
decrease in mitochondrial membrane potential, and cytochrome
–6
6
,11
C release.
From a chemotherapeutic perspective, actions that
7
–9
triterpenoid.
Oleanane, ursane, and a number of other types of
target mitochondria are of particular interest as they could be of
use in promoting apoptosis in malignant cells.
1
0
triterpenoids are also being investigated as anti-cancer agents,
and increasing attention is being given toward the production of
synthetic triterpenoid derivatives. In particular, substantial efforts
have been invested in developing 2-cyano-3,12-dioxooleana-
Mitochondria are recognized to play pivotal roles in transducing
or amplifying signals to cell death pathways, and are thus attrac-
tive cancer chemotherapeutic targets. Multiple subgroups of trit-
erpenoids (including lupane, oleane and ursane triterpenoids)
have been reported to perturb mitochondria, but relatively little
information is available on actual mechanisms of action, or how
specific functional groups affect mitochondrial dynamics. As part
1
,9(11)-dien-28-oic (CDDO) derivatives of oleanolic acid as thera-
*
Corresponding author. Tel.: +1 218 726 8885.
E-mail address: jholy@d.umn.edu (J. Holy).
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.075

J. Holy et al. / Bioorg. Med. Chem. 18 (2010) 6080–6088
6081
NaClO ,
2
TEMPO,
DMFA,
NaH PO ,
2
4
t-BuOH,
1
.1 A; 3.4V
CH Cl
2
2
CH OH 50 oC,
2
CHO
COOH
HO
HO
Scheme 1. Synthesis of betulinic acid (18).
HO
1
7
18
1
6
of our efforts in examining a number of natural and semi-synthetic
derivatives of lupane triterpenoids for bioactivity, we found that
quaternary salt (QUAT) dimethylaminopyridine (DMAP) deriva-
tives of pentacyclic triterpenes dramatically alter mitochondrial
structure and function and suppress cell proliferation. Structure–
activity studies indicate that the mitochondrial effects of these
compounds are not simply a consequence of the presence of DMAP
groups, but are dependent on the specific numbers and orienta-
tions of these groups. These findings reveal that DMAP derivatives
of lupane triterpenoids constitute a novel category of mitochon-
drial poisons that may have utility as research and biomedical
tools.
ing hydroxy groups at C3- and C28-carbons with the chloroacids
and bromoacids shown in Scheme 2. As a result, corresponding
C3 and C28 chloro and bromoacylates were produced as precursors
for DMAP derivatives. Bromination with 2,2 -azobisisobutyronitrile
4
in CCl was performed for subsequent synthesis of C28 quaternary
salts of DMAP. It is necessary to note that betulinic acid (2) may not
be selectively brominated with AIBN, because very low solubility
4
in CCl . Therefore this process was provided through intermediate
acylation of betulinic acid (2) with trifluoroacetic acid. Then, 3-tri-
fluoroacetate of betulinic acid was selectively brominated at
C29-carbon. Subsequent soft hydrolysis of this trifluoroacetate
with 5% NaOH in THF at rt yielded to 30-bromobetulinic acid as
a precursor for corresponding DMAP derivative of betulinic acid
0
(
18). All tested DMAP derivatives have been characterized with
H and C NMR spectra and HRMS-analyses.
Fifteen lupane triterpenoid–dimethylaminopyridinium (LT–
2
2
. Results and discussion
1
13
.1. Chemistry
DMAP) derivatives (Fig. 1) of betulin and betulinic acid were pro-
duced by the Scheme 2. The LT–DMAP compounds 1–15 were pre-
pared as stock solutions in dimethylsulfoxide (DMSO), and added
to culture medium, such that the final concentration of DMSO
never exceeded 0.2%. Control groups received vehicle (DMSO) only,
corresponding to the highest concentration used (0.2%). Synthe-
sized compounds and their precursors revealed a remarkable level
Birch bark lupane triterpenoids betulin (16) and betulinic acid
(
18) have been chosen as basic natural precursors for synthesis of
1
dimethylaminopyridinium QUATS. Betulin with 99% + purity was
isolated from the extract of outer birch bark of Betula papyrifera—
the North American commercial birch tree—in accordance with a
previously developed procedure.17 Betulinic acid (18) was synthe-
1
9
20
1
8
of anti-bacterial
studies.
and anti-proliferative activity in previous
sized by a recently developed two step process (Scheme 1). The
first step is the selective N-oxyl-mediated electrooxidation of
betulin (16) into betulinic aldehyde (17) with TEMPO in dimethyl-
formamide. The betulin aldehyde was subsequently converted to
2
.2. Cell lines and cell culture
2 2
betulinic acid with NaClO or KClO . These methods are proposed
WM3211 radial-phase and WM793 vertical-phase human mela-
as the most convenient for betulinic acid synthesis in the laboratory
as well as for industrial scales. Pilot tests of these methods have been
successfully conducted in our laboratory in a 50-L apparatus which
was equipped with graphite (anode) and copper (cathode)
electrodes.
noma cells were the kind gifts of Dr. Meinhard Herlyn (WISTAR
Inst.). BJ fibroblasts were obtained from ATCC. Cells were cultured
in high-glucose Dulbecco’s modified Eagles medium (DMEM) con-
taining 8% Fetal Clone III (HyClone, Logan, UT), 3.5% horse serum
Gibco) and 5 mM Glutamax (Invitrogen). Cells were passaged and
prepared for experiments using standard trypsin-EDTA methods.
(
Betulin (16) and betulinic acid (18) were converted to DMAP
derivatives (1–15) through intermediate acylation of correspond-
1
2
3
2
3 2
. (CF CO) O
. NBS,AIBN,CCl
1. BrCOCH
2
Br
2
4
. NaOH,H O,THF
or ClCOCH Cl, DMAA
2
2. NBS, AIBN, CCl
. DMAP, DMAA
4
1
3. DMAP, DMAA
COOH
2, 3
1
. BrCOCH
2
Br,DMAA
HO
2
. DMAP, DMAA
6
1
. ClCO(CH )nCHRCl
2
1
2
3
. BrCOCH Br or ClCOCH Cl
2 2
n=0-2; R=H,CH
3
. Al(i-PrO) , i-PrOH
3
2
or BrCO(CH )nCHRCl
. DMAP, DMAA
n=0,1,3; R=H,C H
8,10
2
5
2. DMAP, DMAA
1
. ClCOCH(CH )Cl
CH OH
2
9, 11, 12, 13, 14, 15
3
3
3
or BrCOCH(CH )Br
1.ClCHCH COCl, CHCl , DMAA
2. DMAP, DMAA
3
2. NBS,AIBN, CCl
4
3. DMAP, DMAA
HO
7
4, 5
Scheme 2. Synthesis of LT–DMAPs (1–15).

6
082
J. Holy et al. / Bioorg. Med. Chem. 18 (2010) 6080–6088
weak mitochondrial perturbants, and have little noticeable effect
on either mitochondrial polarization or morphology. Compounds
–10 are intermediate in strength, and can be roughly ordered
6
based on strength of mitochondrial effects (6 < 10 < 8 < 9 < 7). In
general, the radial-phase WM3211 melanoma cells are more sensi-
tive to these compounds than the WM793 vertical-phase cells.
Mitochondria of both cell lines exposed to 4 lg/ml of 6 remain
strongly polarized, but WM3211 mitochondria begin to round up
and appear more spherical. Interestingly, some WM793 cells trea-
ted with this concentration of 6 appear brighter than controls, sug-
gesting some hyperpolarization of mitochondrial membranes may
be occurring in this cell line. Compound 10 reduces mitochondrial
polarization in both cell lines, with a relatively stronger effect on
WM3211s. In addition, cytoplasmic vacuolization occurs in
WM793 cells, and co-localization studies demonstrate that many
of these vacuoles are derived from mitochondria, based on TMRM
fluorescence (Fig. 3). Little vacuolization is apparent in WM3211
cells treated with 4 lg/ml of 10. In both cell lines, compounds 8
and 9 clearly depolarize mitochondria, with the greatest effects
again appearing in WM3211 cells. Mitochondria are extensively
fragmented, and depolarization is almost complete at this concen-
tration and timepoint of compound 7. In addition, extensive cyto-
plasmic vacuolization occurs in both cell lines, with the most
prominent effects displayed by WM793 cells. Compounds 11–15
result in essentially complete mitochondrial fragmentation and
Figure 1. The 15 LT–DMAP compounds synthesized by Schemes 1 and 2.
depolarization in both cell lines at concentrations under 4 lg/ml
2
.3. Proliferation assays
within a few hours of treatment. Some cytoplasmic vacuolization
is occasionally present, but occurs to a much more limited extent
than with 7 and 10.
Sulforhodamine B assays were carried out essentially as de-
scribed by Skehan et al.21 Cells were seeded in 48-well plates
20,000 cells/ml) and allowed to recover for 24 h prior to addition
of LT–DMAPs. Serial 1:1 dilutions of compounds from 2 mg/ml stock
solutions in DMSO, spanning from 0.25 to 4 g/ml were added, and
The fragmentation and vesiculation of mitochondria induced by
LT–DMAPs resembles the ‘thread-grain’ transition described by
(
22
Skulachev et al. Fragmentation of the mitochondrial network
l
has been shown to result from treatment with respiratory inhibi-
plates subsequently incubated for 72 h. Vehicle-only control wells
received 0.2% DMSO, which corresponded to the amount delivered
with the highest concentration of compound tested (4 lg/ml). Plates
were gently rinsed twice with PBS, and then cells were fixed over-
tors, uncouplers, and oxidative stress, and involves the dynamin-
23–25
related GTPase DRP-1.
In general, conditions that compromise
mitochondrial membrane potential induce fragmentation and
26,27
vesiculation of the mitochondrial network.
Indirect drug
night with freezer-temperature (ꢀ10 °C) absolute methanol and
actions can also lead to mitochondrial network fragmentation, as
evidenced by mitochondrial vesiculation caused by ouabain, which
1
% acetic acid. Fixative was decanted and the plates allowed to air
dry prior to the addition of 0.5% SRB in 1% acetic acid. Cells were
stained for 30 min at rt, and then gently rinsed three times with
1
1
23
is an inhibitor of plasma membrane Na/K-ATPase. In this case,
mitochondrial effects of ouabain were reported to be cell-type
dependent, occurring in CV-1, but not HeLa cells. We found that
the strong LT–DMAPs induce mitochondrial network fragmenta-
tion in every type of cell we have so far examined, which includes
over ten normal and malignant cell lines of mouse, human, and tel-
eost origin (data not shown), suggesting that mitochondria are a
direct target of these compounds.
% acetic acid and air dried. SRB was eluted from the cells with
0 mM Tris, pH 10, and the absorbance at 540 nm measured with
a Synergy (Bio-Tek) multiplate reader.
2
.4. Effects of LT–DMAPs on mitochondrial structure and
function
Changes in mitochondrial TMRM fluorescence intensity after
LT–DMAP exposure were quantitated using flow cytometry meth-
ods. Treatment of cells under conditions comparable to the fluores-
Tetramethylrhodamine methylester (TMRM) and related probes
have been used extensively for detecting changes in mitochondrial
membrane polarization, as well as displaying the morphology of
polarized mitochondria. In this study, TMRM labeling of intact cells
demonstrates that a number of differences exist in the mitochon-
drial networks of the WM3211 radial-phase and WM793 verti-
cal-phase melanoma cell lines. WM3211 cells generally display
dense masses of short mitochondria, whereas vertical-phase
WM793 cells exhibit a more diffuse network of elongated mito-
chondria (Figs. 2 and 3). In addition, the overall intensity of mito-
chondrial labeling is somewhat less intense in WM793 cells. Initial
screening of a number of lupane triterpenoid–dimethylaminopyri-
dine conjugates (LT–DMAPs) in these cell lines revealed that mito-
chondrial structure and polarization were radically altered by
cence microscope studies (4 lg/ml LT–DMAP for 6 h) demonstrates
that compounds 1–6 do not significantly reduce mitochondrial
TMRM labeling intensity (Fig. 4). In fact, some of these compounds
appear to slightly increase TMRM signal strength in WM793 cells,
suggesting that mitochondrial membranes perhaps become
slightly hyperpolarized in the presence of these compounds. Com-
pounds 8 and 10 significantly reduce mitochondrial polarization in
WM3211 cells, but not WM793 cells. Compounds 7 and 9 reduce
TMRM fluorescence in both cell lines, with stronger effects again
in the WM3211 cells. Compounds 11–15 significantly reduce
TMRM fluorescence in both cell lines. In some experiments, the
extent of mitochondrial depolarization was compared to that ob-
some of these compounds. At up to 4
lg/ml, the highest concentra-
tained with 20 lM of the ionophore carbonyl cyanide-p-trif-
tion tested, the effects of LT–DMAPs range from no discernable ef-
fects, to complete vesiculation and depolarization of mitochondria,
depending on the compound (Fig. 2). Compounds 1–5 are very
luoromethoxyphenylhydrazone (FCCP). The values of the strong
LT–DMAPs were consistently at or below the FCCP value (not
shown). It should be noted that a reduction in fluorescence inten-

J. Holy et al. / Bioorg. Med. Chem. 18 (2010) 6080–6088
6083
Figure 2. Morphology and mitochondrial polarization of WM3211 and WM793 cells treated with representative LT–DMAPS at 4
lg/ml for 5 h, and then labeled with TMRM.
The same field of view for each sample is shown by both phase contrast and epifluorescence microscopy. Note that compound 3 (representative of the weak group) does not
markedly influence cell or mitochondrial morphology or mitochondrial membrane polarization. The intermediate strength compounds 6–10 show a range of effects on
mitochondria at this concentration and time point, with significant loss of mitochondrial membrane polarization with the strongest compounds of this group (8, 9 and 7). The
strong group all resulted in fragmentation and essentially complete depolarization of mitochondria, represented by 13. All fluorescence images were photographed and
rendered using identical camera settings and software methods to allow for direct comparison of fluorescence labeling strength. Bar (top right image) represents 20 lm.
sity measured by flow cytometry to about 20–30% of control cells
corresponded to an essentially complete lack of mitochondrial
fluorescence by epifluorescence microscopy. In general, there was
excellent agreement between the mitochondrial fluorescence val-
ues measured by flow cytometry and the fluorescence intensity ob-
served by microscopy (compare Figs. 2 and 4).
2.5. Effects of LT–DMAPs on cell proliferation
Dose–response sulforhodamine B (SRB) assays were used to test
for effects of LT–DMAPs on cell proliferation. Similar to the effects
on mitochondrial polarization, the effects of LT–DMAPs on cell pro-
liferation could be grouped into strong, intermediate, and weak ef-
Based on the strength of mitochondrial effects (as well as effects
on proliferation, see below), the 15 DMAP derivatives of triterpe-
noids can be grouped into three general categories: (i) 1–5 are very
weak mitochondrial perturbants; (ii) 6–10 are intermediate in
strength; and (iii) 11–15 are very strong, resulting in fragmenta-
tion and depolarization of mitochondria at concentrations under
fects. Representative of the weak group, up to 4 lg/ml of
compound 3 exhibited little effect on cell proliferation over three
days of treatment (Fig. 5). Compound 6 was the weakest of the
intermediate group, and inhibited proliferation to between 40%
and 50% of controls at 4
this group strongly inhibited proliferation at concentrations over
g/ml. Members of the strong group of compounds inhibited pro-
liferation at concentrations over 0.5 g/ml. Notably, members of the
lg/ml after three days; other members of
4
l
g/ml. Interestingly, both the weak and strong groups exhibit
2 l
similar effects on both melanoma cell lines tested, whereas some
members of the intermediate group (ii) exhibit some specificity,
with the radial melanoma cell line WM3211 displaying greater
sensitivity than the vertical WM793 cell line. In this group, the
compounds 7, 8 and 10 showed the largest differences in effects
between the two cell lines.
l
intermediate group once again showed distinct effects between the
cell lines, with the radial-phase WM3211 cells being more sensitive
to growth inhibition than the vertical-phase WM793 cells. In some
experiments, a non-transformed cell line (BJ foreskin fibroblasts)
was included to compare with the melanoma cell lines. Again,

6
084
J. Holy et al. / Bioorg. Med. Chem. 18 (2010) 6080–6088
Figure 3. Effects of representative LT–DMAPs on WM793 mitochondrial structure and polarization. (A–C) Confocal microscopy of cells double-labeled with Hoechst 33342 to
label nuclei (blue) and TMRM to label polarized mitochondria (red). (A) DMSO control; (B) cells treated with 10 at 4 g/ml for 5 h; (C) cells treated with 15 at 4 g/ml for 5 h.
Note the distinct fragmentation of mitochondria with both compounds, and the almost complete loss of membrane polarization with 15. (D–F) phase contrast and
epifluorescence microscopy of the same cell, treated with 10 at 4 g/ml for 5 h. In this higher magnification micrograph, the phase contrast image shown in D was colorized
l
l
l
blue in F. The red TMRM signal was colorized yellow (E), and the two overlaid in F to better distinguish between TMRM-negative (blue in F) and TMRM-positive (yellow in F)
vacuoles. This demonstrates that the vacuoles visible by phase contrast microscopy (arrows in D) are comprised of both labeled (red arrows) and unlabeled (white arrows)
subpopulations. The labeled vacuoles presumably represent swollen mitochondria, based on the residual TMRM labeling. The unlabeled vacuoles could represent either more
damaged and completely depolarized mitochondria, or, alternatively, non-mitochondrial structures such as swollen cisternae of the endoplasmic reticulum. A–C are the same
magnification; bar in C represents 10 lm. D–F are the same magnification; bar in F represents 5 lm.
cell proliferation suggests that the effect of these compounds is pri-
marily mitochondrial, and that the inhibition of proliferation
results from perturbation of mitochondrial structure and function.
To examine this possibility more closely, TMRM fluorescence was
plotted against the ED50 proliferation values (Fig. 6). For both cell
lines, correlation of compounds with proliferation ED50s under
4 lg/ml with mitochondrial TMRM fluorescence intensity dis-
played a Pearson coefficient of 0.854. This coefficient was 0.767
for the WM3211s, and 0.899 for the WM793s. From the plot, four
points deviated somewhat from the trendline, and included com-
pound 7 for WM793 cells, 8 for WM3211 cells, and 10 for both
WM793 and WM3211 cells. Without these four data points, the
Pearson coefficient rises to 0.966. Interestingly, these four points
all belong to the intermediate group of LT–DMAPs. Overall, the
strong correlation between mitochondrial TMRM fluorescence
intensity and proliferative capacity suggest that the effects of these
compounds on cell proliferation are primarily due to their disrup-
tive effects on mitochondria. However, the fact that the com-
pounds that best distinguish between radial and vertical cell
lines also tend to lie furthest from the trendline raises the possibil-
ity that the ability of these compounds to differentially target the
radial and vertical melanoma cell lines is due to additional, non-
mitochondrial affects.
Figure 4. Quantitation of mitochondrial polarization (TMRM fluorescence) by flow
cytometry. Cells were treated with compounds 1–15 at 4 g/ml for 5 h, and
l
subsequently labeled with TMRM and analyzed by flow cytometry. Fluorescence
values are expressed as percent control (DMSO only vehicle control) for each cell
line, which was set to 100%. Note that the progressive loss of mitochondrial
polarization (reduction in TMRM fluorescence intensity) is more accentuated in the
WM3211 cell line. The values between 20% and 40% for both cell lines exhibited by
compounds 11–15 correspond to an essentially complete lack of visually-detectable
labeling by epifluorescence microscopy (see Fig. 2). Values reflect the mean of three
independent experiments; error bars indicate standard error of the mean.
differences in cell behavior were noted in the intermediate group.
For example, BJ fibroblasts were more sensitive to 10 than WM793
cells, but less sensitive than WM3211 cells (Fig. 5). Similar to both
melanoma cell lines, the weak group of LT–DMAPs exhibited little
effect on BJ cell proliferation (not shown), and the strong group
inhibited BJ growth to about the same extent as both melanoma lines
Examination of cultures by phase contrast microscopy during
the drug treatment experiments revealed a notable level of cell
death in cultures treated with higher concentrations of the strong
group of compounds 11–15 after a few days of exposure (not
shown). In these wells, the inhibition of proliferation clearly in-
cluded cell death as a factor. However, little or no cell death was
evident in cultures treated with lower concentrations of the strong
group, or with the medium and weak groups over the time course
studied (three days of treatment). Therefore, these compounds are
also able to inhibit proliferation by slowing or arresting the cell cy-
cle. Mitochondrial effects could explain both cell cycle inhibitions
due to depletion of ATP levels, as well as induction of cell death
by the intrinsic pathway of apoptosis. Triterpenoids reported to
lead to a reduction in intracellular ATP levels include dehy-
(Fig. 5). The ED50 concentrations (the concentration of compound
thatinhibitscell proliferationto50%ofthe vehicle-onlycontrolcells),
calculated from the dose–response graphs, are listed in Table 1.
2
.6. Correlation between mitochondrial depolarization and cell
proliferation
The similarity of effects of the three groups (weak, intermediate,
and strong) of LT–DMAPs on both mitochondrial polarization and
1
6,28,29
droeburicoic acid, toosendanin, and avicin D.
Avicin D has

J. Holy et al. / Bioorg. Med. Chem. 18 (2010) 6080–6088
6085
Figure 5. SRB dose–response curves for cell proliferation in the presence of LT–DMAPs. Values are shown as percent of controls (DMSO vehicle only; set to 100%) after three
days of treatment. Representative compounds are shown, spanning effects from little inhibition of proliferation 3 to significant effects at less than 1 g/ml (10, 8, 9 and 15).
l
For 10 and 15, effects on the normal (untransformed) BJ fibroblast cell line are also shown. In general, the largest differences of effects on proliferation based on cell type were
exhibited by the intermediate strength compounds 6–10.
Table 1
ED50 values for LT–DMAP compounds
Compound
WM3211
g/ml)
WM3211
M)
WM793
g/ml)
WM793
(lM)
(
l
(
l
(
l
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
>4
>4
>4
>4
>6.1
>4.4
>4.7
>3.5
>3.8
2.6
0.8
0.6
0.7
0.3
>4
>4
>4
>4
>4
4
1.7
1.7
1.3
1.2
0.7
0.5
0.4
0.4
0.4
>6.1
>4.4
>4.7
>3.5
>3.8
5.7
2.6
2.5
1.3
1.9
>4
1.8
0.5
0.4
0.7
0.2
0.5
0.3
0.4
0.4
0.4
1
1
1
1
1
1
0.6
0.3
0.5
0.4
0.8
0.5
0.5
0.4
Figure 6. Graph plotting inhibition of proliferation (ED50, X-axis) versus mitochon-
drial polarization (TMRM fluorescence, Y-axis). ED50 values were from Table 1, and
mitochondrial fluorescence values were from Fig. 4. Pearson coefficients were
calculated using Microsoft Excel. Compounds for the four points that lie somewhat
off the trendline are: point 1 = 7; point 2 = 10; point 3 = 8; point 4 = 10. In general,
there is very good agreement between loss of mitochondrial fluorescence and
inhibition of cell proliferation.
0.5
0.5
Values represent the concentration (both in lg/ml and lM) required to inhibit cell
proliferation to 50% of the control value over three days of culture after addition of
compound. Values were derived from graphs representing the mean of three
independent experiments.
(
BA) has been shown to exert direct effects on mitochondria,
increasing mitochondrial membrane permeability and triggering
the permeability pore transition. BA causes mitochondrial depo-
been shown to activate the AMPK pathway,16 which is involved in
coordinating cellular energy charge with growth and prolifera-
37
larization and apoptotic cell death, which can be blocked by over-
expression of Bcl-2 or bongkrekic acid, and thus is suggested to
3
0
tion. Conversely, some triterpenoids have also been reported to
enhance ATP levels and protect mitochondria from chemical in-
3
7,38
involve activation of the permabilization pore.
BA increases
3
1,32
sult.
It is likely that the effects of triterpenoids on cellular en-
ROS production, which appears to be upstream of pore formation
6
,7,38,39
ergy charge depend upon the specific features of the triterpenoid,
the type of cell, and the metabolic state of the cell. In addition to
affecting cell proliferation rates through effects on energy metabo-
lism, some triterpenoids are associated with alterations in the
and initiation of apoptosis.
Interestingly, it has been sug-
gested that some compounds may lead to mitochondrial permeabi-
lization through unregulated mechanisms, including the formation
of membrane aggregates that form hydrophilic channels,40 and evi-
dence for the ability of the synthetic triterpenoid CDDO to act in
expression patterns of cell cycle regulatory proteins such as cyc-
lins, MYC, p21 and p27.1
1,15,33–36
The extent to which these effects
41
this way has been reported. Preliminary experiments indicate
on cell cycle regulatory proteins are direct or indirect remain un-
clear at present.
Cell proliferation can also be reduced by upregulating cell death
pathways, and mitochondria play central roles in the intrinsic
pathway of apoptosis. The pentacyclic triterpenoid betulinic acid
that co-treatment with cyclosporin A does not reduce mitochon-
drial network fragmentation induced by the strong group of LT–
DMAPs (data not shown), raising the possibility that these com-
pounds are also able to act at least in part via non-regulated pore
formation.

6
086
J. Holy et al. / Bioorg. Med. Chem. 18 (2010) 6080–6088
2
.7. Structure–activity relationships in LT–DMAP mitochondrial
present. The weak group included BA derivatives possessing one,
disruption
two and three DMAP groups; however, the strongest compounds
all contained two DMAP groups. Interestingly, the compounds that
best distinguished between the radial and vertical phase mela-
noma cell lines were all mono-DMAPs. Although some differences
in responses were observed between the cell lines (especially
among the mono-DMAPs), a more meaningful demonstration of
bioselectivity will require the comparison of larger numbers of cell
lines and tumor types in both in vitro and in vivo settings.
Organic ions can penetrate biological membranes if the charge
is delocalized or screened by hydrophobic moieties, and the polar-
ized nature of mitochondria can accumulate high concentrations of
charged molecules that are membrane permeable.4 In particular, a
2
variety of lipophilic cations have been shown to be concentrated
effectively by mitochondria.4
3,44
In this study, triterpenoids deriv-
atized with one, two, or three DMAP groups were compared in
their ability to disrupt mitochondria and suppress cell prolifera-
tion, and a few notable patterns emerged. The strongest com-
pounds all possessed two DMAP groups, but those with three
DMAP groups were generally not as effective. Mono-DMAP com-
pounds exhibited a range of efficacies, and some of these displayed
different potencies with respect to the radial and vertical mela-
noma cell lines. Preliminary screening of a number of DMAP com-
pounds with a linear aliphatic chain substituted for the pentacyclic
triterpenoid core indicates that these molecules are not very effec-
tive in either disrupting mitochondrial structure or inhibiting cell
proliferation. This suggests that the triterpenoid portion is a critical
element in the LT–DMAP anti-mitochondrial actions. It is currently
unclear precisely how the triterpenoid skeleton is functioning, but
it is likely that its lipophilicity contributes to the membrane perme-
ability of these molecules. It is possible that the presence of two
dimethylaminopiridinium groups anchors LT–DMAPs to mitochon-
drial membranes more effectively than a single group, whereas
three such charged groups begins to inhibit the ability of these mol-
ecules to penetrate membranes, resulting in reduced potency. Along
with the tri-DMAPs, di-DMAP molecules with C28-carboxylic acid
derivatives are also less effective, again perhaps due to reduced
membrane permeability. It is noteworthy that the potent di-DMAP
compounds 11 and 12 had their dimethylaminopiridinioacetoxy
groups at C28- and b-C3 positions, on the same (b) side of the
triterpenoid molecule plane. Such a stereochemistry could facilitate
membrane interactions of both active coordination sites, presum-
ably leading to more effective membrane depolarization and
subsequent mitochondrial fragmentation. To test for possible con-
tributions of counter-ions to compound activity, compound pairs
4
. Experiments
Melting points were determined on an Original Mel-Temp Lab-
1
oratory devices INC apparatus (USA). The H NMR spectra were re-
corded with a Varian 500 MHz, the deuterated solvents indicated
were used. Chemical shifts are reported in parts per million
(
ppm) relative to TMS. TLC was run on the silica gel coated alumi-
num sheets (Silica Gel 60 GF254, E. Merk, Germany) and visualized
in UV light (254 nm). All chemicals and solvents were purchased
from Aldrich Chemical or Acros Organics and dried by standard
procedure.
4
4
.1. Synthesis
.1.1. O-acylation of betulin (16) and betulinic acid (18)
The following chloro- and bromocarboxylic acids chlorides or
bromides have been used as a reagents for O-acylation: Reagent
(
(
20 mmol) was added to a solution of betulin or betulinic acid
5–10 mmol) in 30–50 ml of N,N-dimethylacetamide (DMA). Reac-
tion mixture was stirred for 24 h at rt. Benzene (70–100 ml) was
added to reaction mixture, washed with 10% KHCO
(2 ꢁ 15 ml)
and water (3 ꢁ 10 ml), dried over Na SO and evaporated under re-
duced pressure. The residue was purified on silica with hexane/
Et O (4:1). Yielded white crystals (95%) were subjected to the reac-
tion of quaternization with DMAP.
3
2
4
2
4
.1.2. Bromination of O-acetylated LT
NBS (20 mmol) and ABIN (1 mmol) were added to the solution
4
of O-acetylated LT (3 mmol) in 80 ml of dry CCl . Reaction mixture
2
and 3, and 14 and 15 were synthesized so that they differed only
was refluxed for 20 min, stirred at rt for 8 h and filtered. Solvent
was removed in vacuum. Residue was crystallized from 2-propa-
nol, yielded 83–86% off-white crystals.
ꢀ
ꢀ
in the counter ion (Br or Cl ) present. Little or no difference in
compound activity was noted within these pairs, suggesting that
the effects on proliferation and mitochondrial structure and func-
tion are independent of the type of counter-ion.
4
.1.3. Quaternization with DMAP
To a solution of O-acylated and/or brominated LT (3 mmol) in
High concentrations of cations in mitochondria have previously
been shown to lead to an increase in the permeability of the inner
mitochondrial membrane, non-specifically inhibit mitochondrial
DMA (15 ml) was added in one portion DMAP (6–20 mmol). Reac-
tion mixture was stirred for 10 h at 60 °C. After cooling to rt diethyl
ether (20–30 ml) was added, precipitate was separated, washed
with diethyl ether 2 ꢁ 15 ml and dried in vacuum.
4
3
enzymes, reduce ATP levels, and facilitate cell death pathways.
The higher apparent polarization state of WM3211 mitochondria
based on TMRM labeling intensity) raises the possibility that they
(
may accumulate mono- and di-DMAPs to a higher degree than
WM793s, which could explain their greater sensitivity to some of
these compounds.
4.1.4. Characteristics of synthesized LT QUATS (1–15)
4.1.4.1. 3b-hydroxy-30-(4-dimethylaminopyridinio)lup-20-en-
1
2
8-oic acid bromide (1). H NMR (500 MHz, CD
3
OD): d = 8.11
3
. Conclusion
(d, 2H), 7.02 (d, 2H), 4.78 (s, 1H), 4.71 (s, 1H), 3.27 (s, 6H), 3.11
m, 1H), 2.37 (t, 1H), 2.23 (d, 1H), 2.0–0.68 (m, 42H). Mp 192–
(
+
+
Dimethylaminopyrimidine derivatives of lupane triterpenoids
193 °C. HRMS (ESI ) MH : 657.3628 (calcd mass for
þ
(
LT–DMAPs) can be potent disruptors of mitochondrial structure
C
55
H82Br
3
N
6
O4 : 657.3625).
and function. LT–DMAPs can also inhibit cell proliferation, and of
the compounds tested in this study, their ability to inhibit prolifer-
ation is strongly correlated with their ability to disrupt mitochon-
dria. The 15 compounds examined in this study fall into three
general categories of weak, intermediate, and strong, based on
their potency and ability to exert distinct effects on different cell
lines. Structure–activity data indicates that the potency of these
compounds is not simply related to the number of DMAP groups
4.1.4.2. 3b-[2-(4-Dimethylaminopyridinio)acetoxy]-30-(4-dim-
1
ethylaminopyridinio)lup-20-en-28-oic acid dibromide (2).
H
NMR (500 MHz, CD OD): d = 8.1 (d, 4H), 7.03 (d, 4H), 5.11 (m,
3
2H), 4.79 (s, 1H), 4.69 (s, 1H), 4.57 (m, 1H), 4.5 (m, 2H), 3.28 (s,
12H), 2.9 (m, 1H), 2.4 (m, 1H), 2.23 (d, 1H), 2.04–0.53 (m, 38H).
+
+
Mp 105–106 °C. HRMS (ESI ) MH : 899.3683 (calcd mass for
þ
C
46
H69Br
2
N
4
O4 : 899.3680).

J. Holy et al. / Bioorg. Med. Chem. 18 (2010) 6080–6088
6087
4
.1.4.3. 3b-[2-(4-Dimethylaminopyridinio)acetoxy]-30-(4-dim-
4.1.4.12. 3b,28-Di[2-(4-dimethylaminopyridinio)butyryloxy]lup-
20-en dibromide (12 ). H NMR (500 MHz, CDCl ): d = 8.23 (d,
3
1
ethylaminopyridinio)lup-20-en-28-oic acid bromide chloride
1
(
(
3
3). H NMR (500 MHz, CD
m, 2H), 4.79 (s, 1H), 4.69 (s, 1H), 4.57 (m, 1H), 4.51 (m, 2H),
.28 (s, 12H), 2.92 (m, 1H), 2.42 (m, 1H), 2.25 (d, 1H), 2.07–0.57
3
OD): d = 8.2 (d, 4H), 7.1 (d, 4H), 5.11
4H), 7.06 (d, 4H), 5.17 (dt, 1H), 5.01 (dd, 1H), 4.69 (d, 1H), 4.58 (d,
1H), 4.49 (d, 1H), 4.0 (d, 1H), 3.29 (s, 12H), 2.39 (m, 3H), 2.19 (m,
+
+
2H), 2–0.75 (m, 49H). Mp 157–158 °C. HRMS (ESI ) MH :
+
+
þ
(
m, 38H). Mp 93–94 °C. HRMS (ESI ) MH : 855.4188 (calcd mass
983.4616 (calcd mass for C55
H81Br
2
N
4
O4 : 983.4619).
þ
for C46
H
69BrClN
4
O4 : 855.4185).
4
.1.4.13. 3b,28-Di[2-(4-dimethylaminopyridinio)propanyloxy]lup-
1
3
20-en dichloride (13). H NMR (500 MHz, CD OD): d = 8.22 (d, 4H),
4
.1.4.4. 3b,28-Di[2-(4-dimethylaminopyridinio)acetoxy]-30-(4-
1
7.03 (d, 4H), 5.33 (dd, 2H), 4.71 (s, 1H), 4.6 (s, 1H), 4.55 (dd, 1H), 4.45
d, 1H), 4.0 (d, 1H), 3.28 (s, 12H), 2.46 (m, 1H), 2–0.75 (m, 48H). HRMS
dimethylaminopyridinio)lup-20-en tribromide (4).
H NMR
(
(
(
1
500 MHz, CD
s, 1H), 4.65 (s, 1H), 4.55 (m, 1H), 4.5 (d, 1H), 4.0 (d, 1H), 3.29 (s,
2H), 3.28 (s, 6H), 2.4 (m, 1H), 2–0.75 (m, 41H). Mp 123–124 °C.
3
OD): d = 8.16 (d, 6H), 7.06 (d, 6H), 5.18 (m, 4H), 4.82
+
+
þ
(ESI ) MH : 867.5318 (calcd mass for C50
2 4
H77Cl N O4 : 867.5316).
+
+
þ
4.1.4.14. 3b,28-Di[2-(4-dimethylaminopyridinio)acetoxy]lup-20-
3 6
HRMS (ESI ) MH : 1127.3946 (calcd mass for C55H82Br N O4 :
1
1
en dibromide (14). H NMR (500 MHz, CD
3
OD): d = 8.11 (d, 4H),
.02 (d, 4H), 5.11 (m, 4H), 4.71 (s, 1H), 4.6 (s, 1H), 4.56 (m, 1H),
.51 (d, 1H), 4.01 (d, 1H), 3.28 (s, 12H), 2.47 (m, 1H), 2.07–0.82
127.3942).
7
4
(
4
.1.4.5. 3b,28-Di[2-(4-dimethylaminopyridinio)acetoxy]-30-(4-
+
+
m, 42H). Mp 227–228 °C. HRMS (ESI ) MH : 927.3991 (calcd mass
dimethylaminopyridinio)lup-20-en 29-bromide dichloride
þ
for C48
H73Br
2
N
4
O4 : 927.3993).
1
(
5). H NMR (500 MHz, CD
3
OD): d = 8.14 (d, 6H), 7.04 (d, 6H),
.14 (m, 4H), 4.81 (s, 1H), 4.6 (s, 1H), 4.59 (m, 1H), 4.5 (d, 1H),
.96 (d, 1H), 3.28 (s, 12H), 3.27 (s, 6H), 2.4 (m, 1H), 2–0.75 (m,
5
3
4
4
.1.4.15. 3b,28-Di[2-(4-dimethylaminopyridinio)acetoxy]lup-20-
1
en dichloride (15). H NMR (300 MHz, CD
3
OD): d = 8.14 (d, 4H),
.04 (d, 4H), 5.14 (m, 4H), 4.72 (s, 1H), 4.6 (s, 1H), 4.57 (m, 1H),
.52 (d, 1H), 4.02 (d, 1H), 3.28 (s, 12H), 2.47 (m, 1H), 2.07–0.82
+
+
1H). Mp 120–121 °C. HRMS (ESI ) MH : 1039.4955 (calcd mass
7
4
þ
for C55
H
82BrCl
2
N
6
O4 : 1039,4952).
+
+
(m, 42H). Mp 226–227 °C. HRMS (ESI ) MH : 839.5005 (calcd mass
4.1.4.6. 3b-[2-(4-Dimethylaminopyridinio)acetoxy]lup-20-en-28-
þ
for C48
H73Cl
2
N
4
O4 : 839.5003).
1
oic acid bromide (6). H NMR (500 MHz, CD
.03 (d, 2H), 5.12 (m, 2H), 4.7 (s, 1H), 4.6 (s, 1H), 4.55 (m, 1H), 3.28
s, 6H), 3.04 (m, 1H), 2.35 (t, 1H), 2.23 (d, 1H), 2.07–0.75 (m, 41H).
3
OD): d = 8.12 (d, 2H),
7
(
4
.2. Mitochondrial labeling
+
+
Mp 292–293 °C. HRMS (ESI ) MH : 699.3729 (calcd mass for
C
For microscopy, cells were plated on glass-bottom cell culture
dishes (Mat-Tek Inc.) and allowed to grow until approximately
0% confluent. Compounds were all added at a final concentration
of 4 g/ml, and cells incubated for 5 h in a cell culture incubator.
þ
39
H60Br
3
N
2
O4 : 699.3731).
3
4
.1.4.7. 3b-Hydroxy-28-[2-(4-dimethylaminopyridinio)propanyl-
l
1
oxy]lup-20-en-28-ol chloride (7). H NMR (500 MHz, CD
d = 8.24 (d, 2H), 7.04 (d, 2H), 5.35 (dd, 1H), 4.71 (s, 1H), 4.6 (s,
H), 4.5 (dd, 1H), 3.99 (dd, 1H), 3.28 (s, 6H), 3.11 (dd, 1H), 2.44
3
OD):
Vehicle-only controls received an equivalent amount of dimethyl-
sulfoxide (DMSO) only, which corresponded to a concentration of
0.2%. Tetramethylrhodamine methylester (TMRM; Sigma Chem.
Co.) was added to a final concentration of 100 nM, and cells incu-
bated further for 60 min. Culture medium/TMRM was then aspi-
rated and replaced with PBS containing 5% Fetal Clone III, and
dishes immediately examined and photographed on an inverted
Nikon TE-2000 epifluorescence microscope equipped with an Cool-
snap ES camera (Photometrics).
For flow cytometry, cells were suspended in 0.5 ml aliquots of
culture medium at a density of 30,000 cells/ml. Compound addi-
tion and TMRM labeling was carried out as described for micros-
copy, above, with the exception that after the TMRM incubation
period, cells were immediately analyzed by flow cytometry with-
out medium replacement. TMRM fluorescence intensity was mea-
sured using the FL2 channel of a Becton-Dickenson FACScalibur,
and plotted against size (forward scatter). Mean fluorescence
intensity was obtained from identically-sized gated regions.
1
+
+
(
m, 1H), 2–0.69 (m, 46H). Mp 222–223 °C. HRMS (ESI ) MH :
þ
6
55.4604 (calcd mass for C40
H64ClN
2
O3 : 655.4600).
4.1.4.8. 3b-[2-(4-Dimethylaminopyridinio)acetoxy]lup-20-en-28-
1
ol bromide (8). H NMR (500 MHz, CDCl
2
3
3
): d = 8.12 (d, 2H), 7.03 (d,
H), 5.1 (m, 1H), 4.68 (s, 1H), 4.61 (d, 1H), 4.56 (s, 1H), 3.73 (d, 1H),
.28 (s, 6H), 2.44 (m, 1H), 2.07–0.75 (m, 45H). Mp 292–293 °C. HRMS
+
+
þ
(
ESI ) MH : 685.3935 (calcd mass for C39
H62BrN
2
O3 : 685.3938).
4
2
4
4
.1.4.9. 3b,28-Di[4-(4-dimethylaminopyridinio)butyryloxy]lup-
1
3
0-en dibromide (9). H NMR (500 MHz, CD OD): d = 8.17 (d,
H), 7.01 (d, 4H), 4.69 (d, 1H), 4.57 (d, 1H), 4.4 (d, 1H), 4.23 (m,
H), 3.8 (d, 1H), 3.28 (s, 12H), 2.42 (m, 5H), 2.15 (m, 4H), 2–0.75
+
+
(
m, 43H). Mp 149–150 °C. HRMS (ESI ) MH : 983.4621 (calcd mass
þ
for C52
H
81Br
2
N
4
O4 : 983.4619).
Acknowledgements
4
2
7
3
2
C
.1.4.10. 3b-[2-(4-Dimethylaminopyridinio)acetoxy]lup-20-en-
8-ol chloride (10). H NMR (500 MHz, CDCl ): d = 8.12 (d, 2H),
3
1
This study was supported by a grant from the Whiteside Insti-
tute for Clinical Research to J.H. and P.A.K., a research grant from
the University of Minnesota—Duluth to P.A.K., and a research grant
from the Portuguese Foundation of Science and Technology to
P.J.O. (PTDC/QUI/101409/2008).
.03 (d, 2H), 5.1 (m, 2H), 4.68 (s, 1H), 4.57 (m, 1H), 4.55 (s, 1H),
.72 (d, 1H), 3.28 (s, 6H), 2.44 (m, 1H), 2.07–0.82 (m, 44H). Mp
+
+
89–290 °C. HRMS (ESI ) MH : 641.4445 (calcd mass for
þ
39
H62Br
3
N
2
O3 : 641.4443).
4
.1.4.11. 3b,28-Di[3-(4-dimethylaminopyridinio)propionyloxy]-
References and notes
1
lup-20-en dichloride (11). H NMR (500 MHz, CD
3
OD): d = 8.21
d, 4H), 6.99 (d, 4H), 4.69 (d, 1H), 4.57 (d, 1H), 4.46 (m, 4H), 4.4
d, 1H), 3.8 (d, 1H), 3.28 (s, 12H), 2.99 (m, 4H), 2.42 (m, 1H), 2–
(
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H77Cl
2
N
4
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