Synthesis and biological activity of 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole derivatives, novel pro-apoptotic mitochondrial targeted agents

Article abstract of DOI:10.1016/j.bmc.2008.10.067

This study reports the synthesis of a number of 1- and 2-phenyl derivatives of the 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole nucleus, which were obtained by the reaction of the versatile 7-substituted 2,3-dihydro-3-hydroxymethylene-4H-1-benzothiopyran-4-o

Full text of DOI:10.1016/j.bmc.2008.10.067

Bioorganic & Medicinal Chemistry 17 (2009) 326–336
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole
derivatives, novel pro-apoptotic mitochondrial targeted agents
b
b
c
L. Dalla Via a, A. M. Marini b, , S. Salerno , C. La Motta , M. Condello ,
*
G. Arancia c, E. Agostinelli d, A. Toninello e
a Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy
b Department of Pharmaceutical Sciences, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
c Department of Technology and Health, Istituto Superiore di Sanità, Rome, Italy
d Department of Biochemical Sciences, Institute of Molecular Biology and Pathology, University of Rome ‘‘La Sapienza” and CNR, Rome, Italy
e Department of Biological Chemistry, University of Padova, Via G. Colombo 3, 35121 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
This study reports the synthesis of a number of 1- and 2-phenyl derivatives of the 1,4-dihydrobenzothio-
pyrano[4,3-c]pyrazole nucleus, which were obtained by the reaction of the versatile 7-substituted
2,3-dihydro-3-hydroxymethylene-4H-1-benzothiopyran-4-ones with hydrazine and substituted phen-
ylhydrazines. The antiproliferative activity of the synthesized compounds was evaluated by an in vitro
assay on human tumor cell lines (HL-60 and HeLa) and showed a significant capacity of the 7-meth-
oxy-substituted benzothiopyrano[4,3-c]pyrazoles 3b–d, carrying the pendant phenyl group in the 1-posi-
tion, to inhibit cell growth. Investigation of the mechanism of action indicated the induction of the
mitochondrial permeability transition (MPT) as the molecular event responsible for the inhibition of cell
growth. This phenomenon is related to the ability of the test compounds to cause a rapid Ca2+-dependent
Received 24 April 2008
Revised 27 October 2008
Accepted 30 October 2008
Available online 5 November 2008
Keywords:
Dihydrobenzothiopyranopyrazole
Antiproliferative activity
Mitochondria
Reactive oxygen species
Permeability transition
and cyclosporin A-sensitive collapse of the transmembrane potential (DW) and matrix swelling. All this
leads to the release of caspase activators, such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF),
which trigger the pro-apoptotic pathway leading to DNA fragmentation.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
the inner transmembrane potential (DW), matrix swelling, and
outer membrane disruption, thus leading to the release of a cas-
pase activator, such as cytochrome c (cyt c), which triggers the
pro-apoptotic pathway leading to DNA fragmentation.
In the development of new anticancer agents, the central role of
mitochondria in mediating programmed cell death has raised a
large amount of interest due to their key implications in many
pathways essential to both the life and death of cells.1,2 These
organelles play a central role in the integration of pro- and anti-
apoptotic stimuli. In addition, direct targeting of mitochondrial
functions appears to be of significant therapeutic relevance, since
it is known that inadequate apoptosis leads to over-proliferation
of cells and that the rapid and continuous growth of tumor cells
is highly energy-dependent.
Some mitochondrial deregulations have been described as the
hallmarks of apoptosis; among them a central role is played by
the induction of the mitochondrial membrane permeability transi-
tion (MPT), a process that leads to an increase in the inner mem-
brane’s permeability to solutes with a molecular mass up to
1500 Da.3 This phenomenon is regulated by the opening of a large
conductance channel known as the permeability transition pore.
Opening of this non-selective pore provokes the dissipation of
In this regard, it has to be emphasized that a primary insult,
such as a DNA adduct, that takes place after chemotherapy can
cause apoptotic cell death. In particular, in the absence of DNA re-
pair, an increase in level of p53 occurs. This pro-apoptotic protein
induces an increase in the level of Bax, a mammalian cell death
protein targeting mitochondrial membranes, which moves to the
mitochondria and causes the release of cyt c. Once in the cyto-
plasm, cyt c interacts with additional proteins to form the so-called
‘apoptosome’. The apoptosome implies the participation, besides
cyt c, of APAF-1 (which binds ATP) and caspase-9, which together
activate another component, pro-caspase-3. This, in turn, induces
the activation of caspase-3, which cleaves and activates DNA frag-
mentation factors (DFF) that subsequently activate endonucleases.
Together, these steps produce the biochemical and morphological
changes in the cell that are collectively recognized as apoptosis.4,5
Interestingly, certain conventional, clinically used anticancer
drugs have shown a direct and in some cases specific action on
mitochondria in addition to their cytosolic or nuclear effects.
Indeed, it is well established that the cardiotoxicity and the
* Corresponding author. Tel.: +39 0502219555; fax: +39 0502219605.
E-mail address: marini@farm.unipi.it (A.M. Marini).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.067

L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
327
occurrence of multidrug resistance (MDR) of Anthracenediones
and Anthracyclines have been attributed to their accumulation in
the mitochondrial lipid membrane and subsequent redox activity
of the quinone moiety.6–10 Some Topoisomerase II-targeting anti-
cancer drugs such as Etoposide or Amsacrine, have demonstrated
the capacity to interact with mitochondrial functions, also with a
mechanism related to the permeability transition pore forma-
tion.11–13 However, most of the current anticancer drugs do not
directly target mitochondria but must first damage other molecu-
lar targets to generate the signals that, subsequently, trigger the
mitochondrial pathway of apoptosis. These observations support
the search for compounds that are able to affect mitochondrial
functions, in order to develop novel anticancer derivatives
endowed with low DNA damaging properties and the ability to
retain efficacy in MDR tumor cells.
As a part of our research program devoted to the preparation
and evaluation of new antiproliferative agents, we extensively
studied several polycyclic chromophores14–16 among which we
recently disclosed that the 2-amino-5H-pirido[30,20:5,6]thiopyr-
ano[4,3-d]pyrimidine (PTP) I17 showed a detectable cytotoxic
activity on two human tumor cell lines (HeLa and HL-60) which
was ascribed to its ability to interfere with mitochondrial
functions.
mitochondria and the occurrence of apoptosis on HeLa cells were
demonstrated. Finally, to correlate the induction of MPT with the
antiproliferative activity, the effect of cyclosporin A (CsA) and the
mitochondrial membrane potential on HeLa cells were
investigated.
2. Results and discussion
2.1. Chemistry
The preparation of the benzothiopyranopyrazoles and the 1-
aryl substituted derivatives was performed following the synthetic
route described in Scheme 1. The starting key 7-methoxy- or 7-
chloro-3-hydroxymethylenebenzothiopyranones (1 and 2) were
obtained reacting, in toluene solution, ethyl formate with the suit-
able 7-methoxy- or 7-chloro-2,3-dihydrobenzo[30,20:5,6]thiopy-
ran-4(4H)-one, which were prepared following
a previously
described procedure.19
The condensation of compounds 1 and 2, containing the reac-
tive methine group adjacent to the C@O function, with hydrazine
or the appropriate phenylhydrazine hydrochlorides afforded the
target series 3a–d and 4a–d.
The physicochemical properties and purity of the final com-
pounds were assessed by TLC and analytical and 1H NMR spectral
data; the resultant assignment values are in accordance with the
structures proposed and with our previously reported results.20,21
The synthetic sequence leading to the 2-phenyl substituted 1,4-
dihydrobenzo thiopyrano[4,3-c]pyrazoles (Scheme 2) involved the
conversion of the 3-hydroxymethylene compound 1 into the inter-
mediate p-toluensulfonate 5, in which the reactive methine func-
tionality was protected as previously reported by us.21 The
subsequent condensation of 5 with the appropriate phenylhydr-
azine hydrochloride, in anhydrous dimethylformamide at room
temperature, involves, as a first step, only the carbonylic function
in the 4-position, affording the intermediate arylhydrazones.21
These intermediates easily cyclize in situ by heating (100 °C) the
reaction mixture, thus affording the desired 2-substituted deriva-
tives 6b–d. The compounds were purified by flash chromatography
and characterized by analytical and spectral data. In particular, the
most discriminating feature of the 1H NMR spectra of compounds
6b–d was a singlet at ꢀ8.3 ppm attributed to the proton in the 3-
position of the pyrazole moiety, while the same proton of the anal-
ogous 1-aryl substituted compounds 3b–d resonated at ꢀ7.6 ppm.
With the aim to identify novel active compounds, we carried
out a number of modifications on the structure of I, first obtaining
the isosteric derivative II (R = NH2)18 which was devoid of any
activity; we then substituted the polar amino group with a phenyl
one, to give II (R = C6H5), which showed an appreciable cytotoxic
activity on HL-60 cell line (IC50 = 7.15 l
M).18
R
NH2
N
S
N
N
S
N
H3CO
N
I
II
R = NH2, C6H5
In this paper, we describe the synthesis of the benzothiopyr-
ano[4,3-d]pyrazoles III, in which the pyrazole moiety gave us the
potential to insert a pendant phenyl ring both in the 1- and in
the 2-position of the chromophore.
2.2. Antiproliferative activity
The antiproliferative activity of the new benzothiopyranopyraz-
ole derivatives was evaluated on two human tumor cell lines, HeLa
(cervix adenocarcinoma cells) and HL-60 (myeloid leukemic cells),
R'
N
N
and was expressed as IC50 values, that is, the concentration (lM) of
the compound causing 50% of cell death with respect to the control
culture (Table 1).
The results indicated the occurrence of a significant antiprolif-
erative effect for derivatives 3b–d, which were characterized by
the presence of a methoxy substituent in position 7 and a pendant
phenyl, p-chlorophenyl or p-methoxyphenyl at the 1-position of
the heterocyclic moiety, respectively. In particular, 3c appeared
to be the most active compound in both cell lines, showing IC50
values in the low micromolar range. Interestingly, the parent com-
pound 3a, lacking the phenyl side group, was unable to exert any
cytotoxic effect; the same effect was also true for the derivatives
6b–d, which differ from 3b–d in the position of the phenyl side
group on the pyrazole ring. Thus, to exert a significant antiprolifer-
ative effect, the benzothiopyranopyrazole moiety requires the
presence of a phenyl group, preferably p-chloro substituted, linked
R
S
III
R = OCH3, Cl
R' = H, C6H5, p-Cl-C6H4, p-OCH3-C6H4
The antiproliferative activity of the new derivatives III was eval-
uated on two human tumor cell lines (HL-60 and HeLa); then, the
ability to induce MPT on isolated rat liver mitochondria via an oxi-
dative stress is discussed. Moreover, the release of cyt c and AIF by

328
L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
X
O
O
S
CHOH
HCOOEt
MeONa
Toluene
X
NHNH2 HCl
N
N
R
S
R
MeOH
1: R = OCH3
2: R = Cl
Δ
R = OCH3
R = Cl
R
S
NH2NH2 HCl
3b-d R = OCH3, 4b-d R = Cl
MeOH
Δ
3b, 4b: X = H
3c, 4c: X = Cl
3d, 4d: X = OCH3
H
N
N
R
S
3a: R = OCH3
4a: R = Cl
Scheme 1. Synthesis of 1-substituted-1,4-dihydrobenzothiopyrano[4,3-c]pyrazoles.
O
S
O
S
CHOH
H3C
CHOSO2
CH3
SO2Cl
H3CO
THF
H3CO
Δ
1
5
DMF
X
NHNH2 HCl
Δ
X
N
N
H3CO
S
6b-d
6b: X = H
6c: X = Cl
6d: X = OCH3
Scheme 2. Synthesis of 2-substituted-1,4-dihydrobenzothiopyrano[4,3-c]pyrazoles.
at the 1-position of the heterocyclic scaffold. Finally, a certain role
seems to be played also by the methoxy substituent in position 7,
indeed, its replacement with a chlorine (compounds 4b–d) abol-
ishes the biological activity.
tion of salmon testes DNA alone (continuous line) and in the pres-
ence of 3c (dashed line) are shown in Figure 1 as a representative
example. In both spectra, a strong negative signal at 260 nm
occurred, which is typical of the macromolecule, while at higher
wavelengths, no further signal was detected.
2.3. Interaction with DNA
Similar behaviors were also obtained for 3b and 3d (spectra not
shown). Because the occurrence of a complexation with the DNA
induces a LD signal at wavelengths higher than 260 nm, that is,
in the spectral region where only the added chromophore can
absorb,22 it has to be concluded that the benzothiopyranopyrazole
moiety was unable to form a complex with the macromolecule.
Thus, the antiproliferative effect exerted by the new derivatives
is attributable to a cellular target other than DNA.
The presence of a heterocyclic chromophore suggested that the
new benzothiopyranopyrazole derivatives 3b–d could exert their
antiproliferative effect because of a complexation with DNA, likely
through an intercalative mode of binding. To verify the occurrence
of an interaction with the macromolecule, flow linear dichroism
(LD) experiments were performed. The spectra of an aqueous solu-

L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
329
Table 1
The results reported in Figure 3A show that the mitochondrial
swelling induced by 3c in the presence of Ca2+ was prevented also
by ADP, the alkylant agent NEM, and the AdNT inhibitor, BKA. All
Cell growth inhibition in the presence of examined compounds.
Compound
IC50 (lM) of cell lines
these inhibitors also completely hindered the collapse of
DW,
HeLa
HL-60
which was always induced by 3c plus Ca2+ (Fig. 3B).
3a
3b
3c
3d
4a
4b
4c
4d
6b
6c
6d
>20
>20
Taking into account that CsA, ADP, and BKA are typical inhibi-
tors of the MPT and NEM is a protective agent against thiol oxida-
tion,23 the results of Figures 2 and 3 lead to the conclusion that 3c
behaves as an inducer of the transition pore opening and that its
effect is most likely due to an oxidative stress provoked together
with Ca2+. This connection can be seen in the results reported in
Figure 4.
17.2 0.2
6.7 0.4
17.8 0.1
>20
>20
>20
>20
>20
>20
>20
12.5 0.3
3.8 0.1
6.6 0.5
>20
>20
>20
>20
>20
>20
>20
Figure 4A shows the histogram regarding the changes in the
redox level of the mitochondrial sulfydryl groups (SH) when the
organelles were treated with 3c and Ca2+, either alone or together.
The results demonstrate that both 3c and Ca2+ when incubated
alone decreased the total content of the SH groups by about 20%,
but when incubated together, the decrease of the SH groups was
doubled. These drops in the SH content account for a correspond-
ing formation of oxidized dithiol groups. The strong inhibition of
thiol oxidation exhibited by the reductant DTE further confirms
that all the observed effects are the result of an oxidative stress.
The results reported in Figure 4B also clarify that the above ob-
served oxidative stress is due to hydrogen peroxide generation
and most likely by its derivatives, in particular, the highly damag-
ing hydroxyl radical which is produced by the interaction with the
respiratory chain.24,25 In fact, as observed in the figure, Ca2+ alone
further incremented H2O2 production by the control (0.1 nmol/mg
protein) up to 0.4 nmol/mg protein, while 3c induced a minor in-
crease of about 0.25 nmol/mg protein. Both agents lead to the gen-
eration of about 0.55 nmol/mg protein. The production of H2O2 by
3c and Ca2+ was most likely attributable to a disorganization of the
membrane phospholipids, resulting in an alteration of ubiquinone
mobility and leading to reactive oxygen species (ROS) produc-
tion.26 In addition to the aforementioned effects, the increase in
H2O2 production in the presence of both agents was due to the loss
of the mitochondrial antioxidant systems, resulting in the so-called
catastrophe redox that accompanies the opening of the pore.27
However, it must be emphasized that the generation of ROS by
Ca2+ was not involved in the oxidation of the critical SH groups
responsible for pore opening, presumably because these ROS were
formed away from the above thiols. Instead, the ROS formed by 3c
2.4. Effects on rat liver mitochondria (RLM)
As it is generally accepted, an important antiproliferative effect
is played by the process of apoptosis, or programmed cell death,
which would have the task of eliminating the abnormal cells. A
prominent role in this process is exhibited by the mitochondria
with the induction of the phenomenon of the inner membrane per-
meability transition (MPT), which provokes release in the cytosol
of some factors, such as cyt c, AIF, and Smac DIABLO. These factors
trigger the caspase-dependent and independent cascade, resulting
in the apoptotic phenotype (for a review, see Ref. 3). In this regard,
the main aim of this study is to evaluate the ability of compound 3c
to induce some effects on the mitochondrial functions ascribable to
MPT induction. As reported in Figure 2A, when RLM, incubated in
standard medium as described in Section 4, were treated with
50 lM 3c in the presence of 30 l
M Ca2+ an apparent decrease of
approximately about 1 U in absorbance at 540 nm, was observed
in the mitochondrial suspension. This event was indicative of the
occurrence of a large-amplitude swelling at the mitochondrial ma-
trix, furthermore, 3c and Ca2+ were completely ineffective when
incubated alone (Fig. 2A).
The colloid-osmotic swelling induced by 3c was accompanied
by a complete collapse of the electrical membrane potential
(DW) (Fig. 2B) and DpH (result not reported), demonstrating that
the compound induces a complete de-energization of the mem-
brane. In both cases, the immunosuppressant CsA fully prevented
the effects of 3c.
0.0000
-0.0005
-0.0010
-0.0015
-0.0020
240
260
280
300
320
340
360
380
400
wavelength (nm)
Figure 1. Linear flow dichroism spectra for DNA alone (continuous line) and in the presence of 3c (dashed line) at [drug]/[DNA] = 0.08. [DNA] = 1.6 ꢁ 10ꢂ3 M.

330
L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
A
A
control
ADP + 3
3c (-Ca2+)
c
BKA + 3
c
Δ
A540 0.1
Δ
A540
0.1
2
2
3c
3c
B
B
180
150
180
150
control
ADP + 3
control
c
BKA + 3
c
CsA + 3
c
ΔΕ
10 mV
ΔΕ
10 mV
2
2
min
min
100
100
3c
50
0
50
0
3c
Figure 3. Effects of different MPT inhibitors on mitochondrial swelling (A) and
collapse (B) induced by 3c. The experimental conditions are as indicated in the
legend of Figure 2. When present, the concentrations were 1 mM ADP, 10 M NEM,
and 6 M BKA. Data are representative of five similar experiments.
DW
Figure 2. Effects of 3c on mitochondrial swelling (A) and
inhibition by CsA. All incubations were carried out as indicated in standard
incubation procedures in the presence of 30
M Ca2+, except where indicated (–
Ca2+). When present, the concentrations were 50
M 3c and 1 M CsA. The assays
were performed at least four times with similar results. (A) A downward deflection
indicates absorbance decrease. (B) T is the electrode potential.
DW collapse (B) and
l
l
l
l
l
D
Ca2+ altered the phospholipid architecture of the outer membrane,
broke the network of electrostatic bindings, which stabilizes both
factors in the intermembrane space, and permitted their release.
However, the released amount was not sufficient to trigger the
apoptotic pathway, as demonstrated by the results reported below.
are strictly close to the critical sulfydryl groups located on AdNT.28
It must be taken into account that 3c required the presence of Ca2+
to open the pore because the cation favors the interaction of cycl-
ophylin D with the pore forming structures which was necessary to
induce this event.29
The correlation between the induction of MPT and apoptosis
was demonstrated by the release of cyt c and AIF, as reported in
Figure 5.
The immunoblot in Figure 5A shows that 3c alone exhibited a
negligible effect, as the control, in inducing cyt c efflux, while
Ca2+ alone induced a consistent efflux of this protein. If present to-
gether both agents provoked the release of a very high amount of
cyt c. Similar results are observable in the immunoblot showing
AIF efflux (Fig. 5B). In conclusion, the release of the pro-apoptotic
factors cyt c and AIF by 3c in the presence of Ca2+ was consistent
with the activation of both the caspase-dependent and caspase-
independent signaling pathways, leading to apoptosis. This was
confirmed by the complete inhibition of the mentioned factors
exhibited by CsA. The observed release of both factors by Ca2+
alone was not due to mitochondrial swelling and outer membrane
rupture as in the case with both agents (see Fig. 2A). Most likely,
2.5. Evaluation of mitochondrial membrane potential on HeLa
cells
In order to get information on the mitochondrial activity of
whole HeLa cells, a flow cytometric study was carried out using
JC-1, a fluorescent dye (Fig. 6). This probe is a monomeric molecule
with green fluorescence (FL1, 530 nm emission after excitation at
488 nm), which is able to selectively enter the mitochondria driven
by mitochondrial membrane potential. This uptake increases the
concentration gradient of JC-1 leading to the formation of aggre-
gates, which show high level of red fluorescence emission (FL2,
590 emission). In mitochondria undergoing a transition from
polarized to depolarized potential JC-1 leaks out of the mitochon-
dria into the cytoplasm as monomers resulting in a decrease of red
fluorescence.
A signal FL2 emission of the control HeLa cells is shown in
Figure 6A. The cells treated with 50 lM 3c showed an evident

L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
331
A
100
80
60
40
20
0
+
)
+
2
2
3c
a
ntrol
+ CsA
+ DTE
c
C
o
-
c
- Ca
(
3c
3
3c
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
B
+
2
+
)
2
3c
Ca
-
control
(-Ca
c
3
Figure 4. Mitochondrial thiol oxidation (A) and H2O2 production (B) induced by 3c.
All incubations were carried out as indicated in standard incubation procedures in
the presence of 30
concentrations were 50
l
M Ca2+, except where indicated (–Ca2+). When present, the
l
M 3c, 1 lM CsA, and 2 mM DTE. The data represent
average mean SD from four independent experiments.
Figure 6. Mitochondrial membrane potential assessed by flow cytometry after JC-1
staining. Results are represented as contour plots. Control HeLa cells (A); cells
treated with 50
lM 3c (B) and with 20 lM H2O2 (C). A representative experiment of
four is reported.
A
Cyt c in
a
b
c
d
e
supernatant ®
mitochondrial membrane depolarization, with a decreasing of the
FL2 signal corresponding to 39.4% of the cellular population
(Fig. 6B), as represented by the alterations of the contour plot.
The cells with depolarized mitochondria are those from the middle
of the quadrant to the lower right corner, as they lose greenish
orange fluorescence (in FL2 channel).
Ca2+
-
-
-
+
-
-
-
+
-
+
+
-
+
+
+
3c
CsA
CsA
B
AIF in
supernatant ®
a
b
c
d
e
Similar variations were also observed when the cells were incu-
bated with exogenous H2O2. HeLa cells treated with a 20
lM exog-
enous hydrogen peroxide solution showed mitochondrial
a
Ca2+
3c
CsACsA
-
-
-
+
-
-
-
+
-
+
+
-
+
+
+
membrane depolarization, corresponding to 20% of the cellular
population (Fig. 6C).
2.6. Determination of apoptosis in HeLa cells
Fig. 5. Release of cytochrome c (cyt c) (A) and apoptosis-inducing factor (AIF) (B)
induced by 3c in the presence of Ca2+ and CsA. The result of the Western blotting of
the supernatant fractions is shown. RLM were incubated in standard incubation
The release of cyt c in the cytoplasm led to the activation of the
caspase cascade. Once activated, caspases become the effectors of
cell death through the apoptotic process.
procedures in the presence of 30
l
M Ca2+, except where indicated. When present,
M CsA.
the concentrations were 50 M 3c and 1
l
l

332
L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
Apoptosis induction in HeLa cells, after treatment with 3c, was
a
b
c
d
e
f
evaluated by using Annexin V labeling (Fig. 7). Using DNA-specific
viability dyes, such as PI, it was possible to distinguish early apop-
totic cells (Annexin V-positive cells), late apoptotic cells (Annexin
V- and PI-positive cells) and dead cells (PI-positive cells). After
treatment with 3c for 24 (Fig. 7B) and 48 (Fig. 7C) hours, an in-
crease in apoptotic HeLa cells was observed, as revealed by Annex-
in V positivity of the early apoptotic fraction (19.7% and 8.5%,
respectively) and late apoptotic fraction (28.2% and 39.0%, respec-
tively) of cells. Moreover, a biochemical hallmark of apoptosis is
the cleavage of chromosomal DNA into nucleosomal units that give
rise to a ‘ladder’ of nucleosomal-sized multimers.30 To evaluate
whether the cell death induced by 3c derived from mitochon-
A
Figure 8. Agarose gel electrophoresis of DNA extracted from HeLa cells. Lane a,
untreated control; lane b, 50 M cisplatin; lane c, 100 M 3c; lane d, pre-treatment
with 5 M CsA for 30 min at 37 °C and then 100 M 3c; lane e, 5 M CsA for 30 min
at 37 °C; lane f, solvent alone.
0.6%
1.2%
4.8%
l
l
l
l
l
93.4%
drial-mediated apoptosis, we examined by gel electrophoresis
the nuclear DNA extracted from HeLa cells treated with 3c alone
and after preincubation with CsA. Cells incubated in the presence
of the well known drug cisplatin were taken as the reference.
Figure 8 shows the typical DNA fragmentation which ensued from
the activation of the apoptotic process for cells incubated with cis-
platin (lane b) and with 3c (lane c). The pre-treatment of HeLa cells
with CsA clearly reduced the effect induced by 3c (lane d).
These results indicate that the cell death provoked by 3c was
due to the initiation of intrinsic death signals that lead to apoptosis
and that this latter process is strictly related to the induction of the
opening of the mitochondrial permeability transition pore.
FL1 Height (annexin V-FITC)
B
28.2%
5.4%
2.7. Cyclosporin A (CsA) antagonizes the antiproliferative effect
on HeLa cells
46.7%
19.7%
To correlate the antiproliferative activity of 3c (see Table 1)
with the effects exerted on the mitochondria, HeLa cells were incu-
bated in the standard medium containing Ca2+ and a cell inhibition
growth assay was performed in the presence of CsA. Figure 9A
shows the results, expressed in terms of the number of viable cells
compared with the control cultures. Incubation of cells for 72 h
FL1 Height (annexin V-FITC)
C
with 6 lM 3c, induced, as expected, a reduction in cell number
of about 50% with respect to the untreated cells (control). Accord-
ing to the literature,31 the pre-treatment with CsA alone (con-
trol + CsA) caused
a certain cytotoxic effect and, indeed, a
11.6%
40.9%
39.0%
reduction in the cell number of about 20% with respect to the un-
treated cells, was observed. After the preincubation with CsA and
the treatment with 3c, approximately 64% of the viable cells were
obtained (3c + CsA). Taking into account the above data, it was con-
cluded that 3c, at a concentration close to the IC50 value, induced a
reduction in cell number of less than 20% after preincubation with
CsA. Thus, CsA significantly reduced the antiproliferative effect of
the test compound. The optical microphotographs shown in Fig-
ure 9B further confirm the above data.
8.5%
FL1 Height (annexin V-FITC)
Figure 7. Dual parameter cytogram of FITC-labeled annexin V versus propidium
iodide (PI) staining. Double-negative, non-apoptotic cells fall in the lower left
quadrant, while early and late apoptosis cells fall in the lower and upper right
3. Conclusions
The synthesis of a new heterocyclic benzothiopyranopyrazole
nucleus, bearing a methoxy (3a) or a chlorine (4a) group in the
quadrant, respectively. HeLa cells were incubated in the presence of 50
(A), 24 (B), and 48 (C) hours before staining.
lM 3c for 0

L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
333
100
80
60
40
20
0
3c
+CsA
control
3c
control+CsA
control
control + CsA
3c
3c + CsA
Figure 9. Effects of CsA on cell growth inhibition induced by 3c evaluated as a percentage of cell number (A) and by optical microphotographs (B). HeLa cells were incubated
for 72 h in the absence or in the presence of 6 M 3c as indicated, without pre-treatment (black bars) and after pre-treatment with 2 M CsA for 30 min (grey bars).
l
l
7-position and a p-substituted phenyl either in the 1- (3b–d and
4b–d) or in the 2-position (6b–d) of the pyrazole moiety, is de-
scribed. Within this series of new derivatives, 3b–d demonstrated
the ability to inhibit cell growth and in particular, 3c exhibited the
higher cytotoxic capacity. These data pointed to the phenyl group
in the 1-position of the pyrazole and the methoxy substituent in
the 7-position of the benzothiopyranopyrazole nucleus as essential
structural requirements for the occurrence of an antiproliferative
effect. Investigation into the action mechanism of 3c highlighted
the absence of interaction with DNA. Interestingly, the new deriv-
ative acts as MPT inducer, causing a large-amplitude swelling of
the mitochondrial matrix and the collapse of the electrical poten-
tial, both completely prevented by CsA, ADP, and BKA. The inhibi-
tory effect of the alkylating agent NEM, along with the decrease in
the total content of the SH groups, that accounts for a correspond-
ing formation of oxidized dithiol groups, indicated that the mito-
chondrial events are the result of oxidative stress. Furthermore,
the detection of an increase in the production of hydrogen perox-
ide reinforced this hypothesis. As a consequence of the opening
of the permeability transition pore, 3c provoked the release of
the pro-apoptotic factors cyt c and AIF from the mitochondria, thus
activating the caspase-dependent and caspase-independent apop-
totic pathways, respectively. The HeLa cells treated with 3c
showed an evident mitochondrial membrane depolarization, simi-
larly to that induced by H2O2 already described in the literature,32
thus demonstrating that the effects measured on isolated mito-
chondria occur also in cells. Moreover, these results in whole cells
confirm the above mentioned proposal that 3c exhibits its effects
by means of an oxidative stress. Phosphatidylserine exposure on
the outer surface of the cytoplasmic membrane of HeLa cells
clearly showed the onset of the apoptotic process and moreover,
the nuclear DNA fragmentation, which characterizes cell death
by apoptosis, was observed in the HeLa cells treated with 3c. The
link between the mitochondrial effects induced by 3c and its anti-
proliferative activity emerges from the ability of CsA to prevent the
cell death. In conclusion, the novel benzothiopyranopyrazole
derivative 3c activated the apoptotic pathway damaging mito-
chondrial functions, and this mechanism accounts for the antipro-
liferative effect on tumor cell lines. It should be noted that the
ability to activate the apoptotic process by directly affecting mito-
chondria, along with the lack of interaction with DNA, represent
interesting properties for the development of anticancer drugs. In

334
L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
particular, the inability to form a complex with the macromolecule
reduces the risk of genotoxicity. Furthermore, the mitochondrial
targeted agents could play a crucial role in the attempt to over-
come the chemoresistance acquired both as a consequence of the
neutralization of pro-apoptotic signals that converge to mitochon-
dria, like the loss of p53 expression, and the upregulation of anti-
apoptotic proteins belonging to the Bcl-2 subfamily. Therefore,
for these reasons, the benzothiopyranopyrazole nucleus consti-
tutes a chromophore that should be further developed in an effort
to obtain new improved anticancer therapies.
ate solution to give crude pyrazoles 3a–d, which were purified by
recrystallization from ethanol, and 4a–d, which were purified by
flash chromatography on
a silica gel column (60/0.040–
0.063 mm) using petroleum ether 60–80 °C/ethyl acetate 8/2 as
the eluting system.
Compound 3a: 38% yield; mp 154–156 °C; ir: 3300, 1600, 1480,
1250, 1100, 1035 cmꢂ1 1H NMR: d 3.76 (s, 3H, OCH3); 4.00 (s, 2H,
;
CH2-S); 6.80 (d, 1H, 8-H J8–9 = 8.4 Hz J8–6 = 2.6 Hz); 6.90 (d, 1H, 6-H
J6–8 = 2.6 Hz); 7.56 (s, 1H, 3-H); 7.69 (d, 1H, 9-H J9–8 = 8.4 Hz). Anal.
Calcd for C11H10N2OS: C, 60.55; H, 4.59; N, 12.84. Found: C, 60.52;
H, 4.60; N, 12.85.
4. Experimental
4.1. Chemistry
Compound 3b: 39% yield; mp 105–108 °C; ir: 1600, 1510, 1280,
1225, 1035 cmꢂ1 1H NMR: d 3.72 (s, 3H, OCH3); 3.98 (s, 2H, CH2-
;
S); 6.59–6.62 (m, 2H, 6-H, 40-H); 7.04 (d, 1H, 8-H J = 2.4 Hz);
7.35–7.39 (m, 2H, 20-H, 60-H); 7.51–7.54 (m, 3H, 30-H, 50-H, 9-H);
7.62 (s, 1H, 3-H). Anal. Calcd for C17H14N2OS: C, 69.39; H, 4.76;
N, 9.52. Found: C, 69.51; H, 4.78; N, 9.47.
4.1.1. Materials and methods
The uncorrected melting points were determined using a Reic-
hert Köfler hot-stage apparatus. IR spectra were obtained on a
NICOLET/AVATAR, 360 FT spectrophotometer by Nujol mulls. 1H
NMR spectra were recorded on a Varian Gemini 200 spectrometer
in dimethyl-d6 sulfoxide solution using TMS as the internal stan-
dard. The coupling constants are given in Hertz. Elemental analyses
were performed by our Analytical Laboratory and were within
0.4%. Magnesium sulfate was used as the drying agent. Evapora-
tions were made in vacuo (rotating evaporator). Analytical TLC
was carried out on Merck 0.2 mm precoated silica gel aluminum
sheets (60 F-254). Reagents, starting materials, and solvents were
purchased from commercial suppliers and used as received.
Compound 3c: 47% yield; mp 127–129 °C; ir: 1600, 1500, 1290,
1225, 1040 cmꢂ1 1H NMR: d 3.73 (s, 3H, OCH3); 3.97 (s, 2H, CH2-
;
S); 6.67 (d, 1H, 8-H J8–9 = 2.4 Hz); 6.69 (s, 1H, 6-H); 7.05 (d, 1H,
9-H J9–8 = 2.4 Hz); 7.40 (d, 2H, 20-H, 60-H J = 8.6 Hz); 7.59 (d, 2H,
30-H, 50-H J = 8.6 Hz); 7.65 (s, 1H, 3-H). Anal. Calcd for
C17H13N2OClS: C, 62.10; H, 3.96; N, 8.52. Found: C, 62.15; H,
3.98; N, 8.53.
Compound 3d: 34% yield; mp 150–152 °C; ir: 1590, 1515, 1290,
1240, 1080, 1050 cmꢂ1 1H NMR: d 3.71 (s, 3H, 7-OCH3); 3.82 (s,
;
3H, 40-OCH3); 3.97 (s, 2H, CH2-S); 6.61 (d, 1H, 8-H J8–9 = 2.4 Hz);
6.64 (s, 1H, 6-H); 7.02 (d, 1H, 9-H J9–8 = 2.4 Hz); 7.06 (d, 2H, 20-H,
60-H J = 8.8 Hz); 7.28 (d, 2H, 30-H 50-H J = 8.8 Hz); 7.56 (s, 1H, 3-
H). Anal. Calcd for C18H16N2O2S: C, 66.66; H, 4.94; N, 8.64. Found:
C, 66.97; H, 4.95; N, 8.62.
4.1.2. 7-Methoxy- and 7-chloro-2,3-dihydro-3-
hydroxymethylene-1-benzothiopyran-4(4H)-one (1) and (2)
A solution of ethyl formate (1.25 mL, 15 mmol) in anhydrous
toluene (6 mL) was added dropwise to freshly prepared sodium
methoxide (0.345 g of sodium, 15 mmol, in 6 mL of absolute meth-
anol) in the same solvent (8 mL). Then a solution of 7-methoxy- or
7-chloro-2,3-dihydro-4H-1-benzothiopyran-4-one, (7.5 mmol) in
anhydrous toluene (10 mL) was added dropwise, with stirring, to
the ice-cooled mixture, under a nitrogen atmosphere. Stirring
was continued at room temperature for 24 h to give the sodium
salt of 1 or 2, which was collected and treated with water. The
solution obtained was acidified with hydrochloric acid to give pure
1 (0.542 g, 48% yield) and 2 (1.546 g, 91% yield). An analytical sam-
ple was obtained by recrystallization from petroleum ether 30–
60 °C.
Compound 4a: 59% yield; mp 113–115 °C; ir: 3175; 1580;
1130 cmꢂ1
;
1H NMR: d 4.05 (s, 2H, CH2-S); 7.25 (d, 1H, 8-H J8–9
=
8.1 Hz); 7.41 (s, 1H, 3-H); 7,65 (s, 1H, 6-H); 7.77 (d, 1H, 9-H J9–8
=
8.1 Hz); 12.96 (s, 1H, NH exch.). Anal. Calcd for C10H7N2ClS: C,
53.93; H, 3.15; N, 12.58. Found: C, 54.04; H, 3.14; N, 12.60.
Compound 4b: 20% yield; mp 98–103 °C; ir: 1595, 1500,
1070 cmꢂ1 1H NMR: d 4.03 (s, 2H, CH2-S); 6.68 (dd, 1H, 8-H J8–9
; =
8.4 Hz J8–6 = 1.2 Hz); 7.08 (dt, 1H, 40-H); 7.36–7.40 (m, 2H, ArH);
7.52–7.58 (m, 4H, ArH); 7.68 (d, 1H, 6-H J6–8 = 1.2 Hz). Anal. Calcd
for C16H11N2ClS: C, 64.32; H, 3.69; N, 9.38. Found: C, 64.37; H, 3.70;
N, 9.39.
Compound 4c: 14% yield; mp 56–62 °C; ir: 1580, 1495,
1100 cmꢂ1 1H NMR: d 4.02 (s, 2H, CH2-S); 6.76 (d, 1H, 8-H J8–9
; =
Compound 1: mp 90–92 °C; ir: 1625, 1590, 1230, 1030 cmꢂ1; 1H
NMR: d 3.81 (s, 3H, OCH3); 3.82 (s, 2H, CH2S); 6.82 (dd, 1H, 6-H J6–5
8.2 Hz); 7.15 (d, 1H, 9-H J9–8 = 8.2 Hz); 7.22 (s, 1H, 3-H); 7.42 (d,
2H, 30-H 50-H J = 8.4 Hz); 7.60 (d, 2H, 20-H 60-H J = 8.4 Hz); 7.71
(s, 1H, 6-H). Anal. Calcd for C16H10N2Cl2S: C, 57.66; H, 3.00; N,
8.41. Found: C, 57.70; H, 3.01; N, 8.43.
=
8.8 Hz J6–8 = 2.4 Hz); 6.89 (d, 1H, 8-H J8–6 = 2.4 Hz); 7.89 (d, 1H, 5-H
J6–5 = 8.8 Hz). Anal. Calcd for C11H10O3S: C, 59.46; H, 4.50. Found: C,
59.64; H, 4.49.
Compound 4d: 16% yield; mp 68–73 °C; ir: 1585, 1510, 1240,
Compound 2: mp 102–104 °C; ir: 1640, 1580, 1105 cmꢂ1
;
1H
=
=
1210 cmꢂ1 1H NMR: d 3.80 (s, 3H, OCH3); 4.12 (s, 2H, CH2-S);
;
NMR: d 3.86 (s, 2H, CH2S); 7.31 (dd, 1H, 6-H J6–5 = 8.5 Hz J6–8
2.1 Hz); 7.50 (d, 1H, 8-H J8–6 = 1.8 Hz); 7.91 (d, 2H, 5-H J6–5
8.6 Hz). Anal. Calcd for C10H7ClO2S: C, 52.98; H, 3.09. Found: C,
52.80; H, 3.08.
7.07 (d, 2H, 30-H 50-H J = 8.6 Hz); 7.29 (d, 1H, 8-H J8–9 = 8.2 Hz);
7.46 (s, 1H, 3-H); 7.76 (d, 2H, 20-H 60-H J = 8.6 Hz); 7.87 (d, 1H, 9-
H J9–8 = 8.2 Hz); 8.31 (s, 1H, 6-H). Anal. Calcd for C17H13N2OSCl:
C, 62.10; H, 3.96; N, 8.52. Found: C, 62.13; H, 3.97; N, 8.51.
4.1.3. 7-Methoxy-1,4-dihydro-benzothiopyrano[4,3-c]pyrazole
(3a), 1-(p-substituted-phenyl)derivatives (3b–d), 7-chloro-1,4-
dihydro-benzothiopyrano[4,3-c]pyrazole (4a) and 1-(p-
substituted-phenyl)derivatives (4b–d)
General procedure: Hydrazine hydrochloride or the required
phenylhydrazine hydrochloride (0.811 mmol) was added to a solu-
tion of 1 or 2 (0.676 mmol) in 15 mL of methanol, the reaction mix-
ture was stirred at room temperature for 24 h and then refluxed for
15 h. After cooling, the yellow solid, if present, was collected and
the solution was evaporated under reduced pressure. The solid
and the residue were washed with an aqueous potassium carbon-
4.1.4. 3-[(p-Methylphenyl)sulfonyl]oxymethylen-2,3-dihydro-
benzothiopyran-4(4H)-one (5)
p-Toluensulfonylchloride (3.960 g, 20.8 mmol) was added to a
solution of 1 (2.310 g, 10.4 mmol) in 40 mL of anhydrous tetrahy-
drofurane in the presence of potassium carbonate (5.740 g,
41.6 mmol). The reaction mixture was stirred at room temperature
for 24 h and then refluxed for 15 h. After cooling, the suspension
was concentrated under reduced pressure and the residue ob-
tained was treated with water and then extracted with chloroform.
The organic layer was dried and evaporated to give crude product 5
which was purified by flash chromatography on a silica gel column

L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
335
(60/0.040–0.063 mm) using petroleum ether 60–80 °C/ethyl ace-
tate 7:3 as the eluting system. 68.8% yield: mp 73–75 °C; ir:
pan blue assay was performed to determine cell viability. Cytotox-
icity data were expressed as IC50 values, that is, the concentration
of the test agent inducing 50% reduction in cell number compared
with the control cultures.
1670, 1480, 1300, 1260, 1230, 1180, 1140, 1090, 1050 cmꢂ1 1H
;
NMR: d 2.43 (s, 3H, CH3); 3.81 (s, 3H, OCH3); 3.90 (s, 2H, CH2S);
6.84 (dd, 1H, 6-H J6–5 = 8.8 Hz J6–8 = 2.6 Hz); 6.90 (d, 1H, 8-H
J8–6 = 2.6 Hz); 7.41 (s, 1H, CHO); 7.54 (d, 2H, 30-H 50-H J = 8.5 Hz);
7.92 (d, 1H, 5-H J5–6 = 8.8 Hz); 7.96 (d, 2H, 20-H 60-H J = 8.5 Hz).
Anal. Calcd for C18H16O5S2: C, 57.45; H, 4.26. Found: C, 57.64; H,
4.27.
For the experiments in the presence of CsA, after incubation for
24 h, the HeLa cells were treated with 2
medium was replaced with an equal volume of fresh medium,
M of test agent was added, and the cells were incubated for an-
lM CsA for 30 min; the
6
l
other 72 h. Cell viability was determined by a trypan blue assay
and expressed as a percentage of the cell number of control cul-
ture. For the microphotographs, cell cultures were washed with
PBS buffer, fixed with 3.7% formaldehyde in PBS, and then exam-
ined under an optical microscope (Leitz DMIRB, Leica).
4.1.5. 7-Methoxy-2-(p-substituted-phenyl)-1,4-dihydro-
benzothiopyrano[4,3-c] pyrazoles (6b–d)
General procedure: The required phenylhydrazine hydrochloride
(2.4 mmol) was added to a solution of 5 (0.688 g, 2.00 mmol) in
10 mL of anhydrous dimethylformamide. The reaction mixture
was stirred at room temperature for 24 h and then refluxed at
100 °C for 15 h. After cooling, the suspension was poured into
water and then extracted with chloroform. The organic layer was
dried and evaporated under reduced pressure to give desired crude
pyrazoles 6b–d, which were purified by flash chromatography on a
silica gel column (60/0.040–0.063 mm) using petroleum ether 60–
80 °C/ethyl acetate 8:2 as the eluting system.
4.2.3. Linear flow dichroism
Linear dichroism (LD) measurements were performed on a Jasco
J500A circular dichroism spectropolarimeter converted for LD and
equipped with an IBM PC and a Jasco J interface.
Linear dichroism is defined as
LDðkÞ ¼ A==ðkÞ ꢂ A
?ðkÞ
where A// and A\ correspond to the absorbances of the sample
when polarized light is oriented parallel or perpendicular to the
flow direction, respectively. The orientation is produced by a device
designed by Wada and Kosawa33 at a shear gradient of 500–
700 rpm and each spectrum was acquired four times.
Salmon testes DNA was purchased from Sigma Chemical Co. (D-
1626). The DNA concentration was determined using an extinction
coefficient of 6600 M(p)ꢂ1 cmꢂ1 at 260 nm. A solution of salmon
testes DNA (1.6 ꢁ 10ꢂ3 M) in ETN buffer (containing 10 mM Tris,
10 mM NaCl, and 1 mM EDTA, pH 7) was used. Spectra were re-
corded at 25 °C at [drug]/[DNA] = 0.00 and 0.08.
Compound 6b: 45% yield; mp 110–115 °C; ir: 1600, 1500, 1300,
1250, 1220, 1035 cmꢂ1 1H NMR: d 3.78 (s, 3H, OCH3); 4.09 (s, 2H,
;
CH2-S); 6.85 (dd, 1H, 8-H J8–9 = 8.5 Hz J8–6 = 2.6 Hz); 6.94 (d, 1H, 6-
H J6–8 = 2.6 Hz); 7.30 (t, 1H, 40-H J = 7.3 Hz); 7.50 (t, 2H, 30-H 50-H
J = 7.6 Hz); 7.83 (d, 1H, 9-H J9–8 = 8.5 Hz); 7.85 (d, 2H, 20-H 60-H
J = 7.6 Hz); 8.37 (s, 1H, 3-H). Anal. Calcd for C17H14N2OS: C,
69.39; H, 4.76; N, 9.52. Found: C, 69.44; H, 4.77; N, 9.51.
Compound 6c: 30% yield; mp 88–93 °C; ir: 1600, 1500, 1245,
1095, 1050 cmꢂ1 1H NMR: d 3.78 (s, 3H, OCH3); 4.08 (s, 2H, CH2-
;
S); 6.84 (dd, 1H, 8-H J8–9 = 8.6 Hz J8–6 = 2.6 Hz); 6.94 (d, 1H, 6-H
J6–8 = 2.6 Hz); 7.56 (d, 2H, 20-H 60-H J = 9.0 Hz); 7.83 (d, 1H, 9-H
J9–8 = 8.6 Hz); 7.88 (d, 2H, 30-H 50-H J = 9.0 Hz); 8.40 (s, 1H, 3-H).
Anal. Calcd for C17H13N2OClS: C, 62.10; H, 3.96; N, 8.52. Found:
C, 62.00; H, 3.98; N, 8.54.
Compound 6d: 31% yield; mp 108–112 °C; ir: 1600, 1510, 1300,
1245, 1050 cmꢂ1; 1H NMR: d 3.78 (s, 3H, OCH3); 3.80 (s, 3H, OCH3);
4.07 (s, 2H, CH2-S); 6.83 (dd, 1H, 8-H J8–9 = 8.6 Hz J8–6 = 2.6 Hz);
6.93 (d, 1H, 6-H J6–8 = 2.6 Hz); 7.06 (d, 2H, 20-H 60-H J = 9.0 Hz);
7.75 (d, 2H, 30-H 50-H J = 9.0 Hz); 7.80 (d, 1H, 9-H J9–8 = 8.6 Hz);
8.25 (s, 1H, 3-H). Anal. Calcd for C18H16N2O2S: C, 66.66; H, 4.94;
N, 8.64. Found: C, 66.73; H, 4.95; N, 8.67.
4.2.4. Mitochondrial isolation and standard incubation
procedures
Rat liver mitochondria (RLM) were isolated by conventional dif-
ferential centrifugation in a buffer containing 250 mM sucrose,
5 mM Hepes (pH 7.4), and 1 mM EGTA34 the EGTA was omitted
from the final washing solution. The protein content was measured
by the biuret method with BSA as a standard.35
The mitochondria (1 mg protein/mL) were incubated in a
water-jacketed cell at 20 °C. The standard medium contained
250 mM sucrose, 10 mM Hepes (pH 7.4), 5 mM succinate, and
1.25 lM rotenone. Variations and/or other additions were given
4.2. Biology
with each experiment.
4.2.1. Cell cultures
4.2.5. Determination of mitochondrial functions
HL-60 (human myeloid leukemic) and HeLa (human cervix ade-
nocarcinoma) cells were grown in RPMI 1640 (Sigma Chemical Co.)
supplemented with 15% heat-inactivated fetal calf serum (Biologi-
cal Industries) and in Nutrient Mixture F-12 [HAM] (Sigma Chem-
ical Co.) supplemented with 10% heat-inactivated fetal calf serum
The membrane potential (DW) was calculated on the basis of
distribution of the lipid-soluble cation tetraphenylphosphonium
(TPP+) measured across the inner membrane using a TPP+-specific
electrode.36 The mitochondrial swelling was determined by mea-
suring the apparent absorbance change of the mitochondrial sus-
(Biological Industries). One hundred U/mL penicillin, 100
l
g/mL
pensions at 540 nm in
a
Kontron Uvikon model 922
streptomycin, and 0.25 g/mL amphotericin B (Sigma Chemical
Co.) were added to both media. The cells were cultured at 37 °C
in moist atmosphere of 5% carbon dioxide in air.
l
spectrophotometer equipped with thermostatic control. The pro-
tein sulfydryl oxidation assay was performed as in Santos et al.37
The production of H2O2 in RLM was measured fluorometrically
by the scopoletin method38 in a 4-8202 spectrofluorometer (Amin-
co-Bowman, Silver Spring, MD).
4.2.2. Inhibition growth assays
HL-60 cells (4 ꢁ 104) were seeded into each well of a 24-well
cell culture plate. After incubation for 24 h, various concentrations
of the test agents were added to the complete medium and incu-
bated for another 72 h. HeLa (4 ꢁ 104) cells were seeded into each
well of a 24-well cell culture plate. After incubation for 24 h, the
medium was replaced with an equal volume of fresh medium,
and various concentrations of the test agents were added. The cells
were then incubated in standard conditions for another 72 h. A try-
4.2.6. Detection of cyt c and AIF release
The mitochondria (1 mg protein/mL) were incubated for 15 min
at 20 °C in standard medium with the appropriate additions. The
reaction mixtures were then centrifuged at 13,000 g for 10 min
at 4 °C to obtain mitochondrial pellets. The supernatant fractions
were concentrated using a PAGEprepTM protein clean-up and
enrichment kit (Pierce, Rockford, IL). Aliquots of 20 lL of the con-

336
L. Dalla Via et al. / Bioorg. Med. Chem. 17 (2009) 326–336
centrated supernatants were subjected to 15% and 10% SDS–PAGE
for cyt c and AIF, respectively, and analyzed by Western blotting
using mouse anti-cyt c and rabbit anti-AIF antibodies (Pharmingen,
San Diego, CA).
transilluminated by UV light and fluorescence emission visualized
using a CCD camera coupled to a Bio-Rad Gel Doc 1000 apparatus.
Acknowledgment
4.2.7. Determination of mitochondrial membrane potential on
whole HeLa cells
This work was supported by grants from MIUR (Research fund
Cofin 2006).
The mitochondrial membrane potential was evaluated by using
the lipophilic cationic probe 5,50,6,60-tetrachloro-1,10,3,30-tetra-
ethylbenzimidazolcarbocyanine iodide (JC-1, Molecular Probes)
according to Cossarizza et al.39 HeLa cells were grown to near con-
fluence, were treated with 50 lM 3c in DMSO and then were incu-
bated for 24 h at 37 °C. In the last 10 min of treatment, JC-1 was
added at a final concentration of 5 ng/ml. As positive controls, HeLa
cells were treated with a 20 lM exogenous hydrogen peroxide
solution (commercial H2O2, Baker Analysed Reagent, J.T. Baker,
Deventer, The Netherlands) for 24 h and then labeled as described
above.
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HeLa cells (5 ꢁ 105) were incubated in standard conditions for
24 h at 37 °C and then, where indicated, incubated for 30 min at
37 °C with 5 lM CsA. The medium was then replaced with an equal
volume of fresh medium and the test agent was added at the indi-
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according to the procedure described by Sambrook et al.41 The iso-
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buffer (Tris 0.09 M, EDTA 2 mM, boric acid 0.09 M, pH 8). The gel
was stained with ethidium bromide solution (1 lg/mL) and then