Multidrug resistance reverting activity and antitumor profile of new phenothiazine derivatives

Article abstract of DOI:10.1016/j.bmc.2008.05.040

A series of easily affordable phenothiazine derivatives bearing a rigid but-2-ynyl amino side chain were synthesized and tested to evaluate the MDR reverting activity and full antitumor profile. Some compounds endowed with remarkable MDR reverting effect were identified, and the most active one (6c) was shown to increase doxorubicin retention in multidrug resistant cells, suggesting a direct interaction with P-glycoprotein. Furthermore, a broad range of cellular activities were observed for different compounds. In particular, the ability of some derivatives to induce antiproliferative effects on resistant cell lines and to interfere with the G₁ phase of the cell cycle, a phase usually not affected by classical antitumor agents, was noted. Moreover, the most cytotoxic compounds of the series were able to induce apoptosis in resistant cell lines, via an atypical pathway of caspase cascade activation, and a synergistic effect in combination with doxorubicin was also found.

Full text of DOI:10.1016/j.bmc.2008.05.040

Bioorganic & Medicinal Chemistry 16 (2008) 6474–6482
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Multidrug resistance reverting activity and antitumor profile of new
phenothiazine derivatives
a,*
b
a
a
c
b,*
Alessandra Bisi , Maria Meli , Silvia Gobbi , Angela Rampa , Manlio Tolomeo , Luisa Dusonchet
a
Dipartimento di Scienze Farmaceutiche, Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Dipartimento di Scienze Farmacologiche ‘‘Pietro Benigno”, Università di Palermo, Palermo, Italy
Centro Interdipartimentale di Ricerche in Oncologia Clinica, Policlinico Universitario di Palermo, Palermo, Italy
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 February 2008
Revised 8 May 2008
Accepted 16 May 2008
Available online 20 May 2008
A series of easily affordable phenothiazine derivatives bearing a rigid but-2-ynyl amino side chain were
synthesized and tested to evaluate the MDR reverting activity and full antitumor profile. Some com-
pounds endowed with remarkable MDR reverting effect were identified, and the most active one (6c)
was shown to increase doxorubicin retention in multidrug resistant cells, suggesting a direct interaction
with P-glycoprotein. Furthermore, a broad range of cellular activities were observed for different com-
pounds. In particular, the ability of some derivatives to induce antiproliferative effects on resistant cell
Keywords:
1
lines and to interfere with the G phase of the cell cycle, a phase usually not affected by classical antitu-
Phenothiazine derivatives
Drug resistance
Anticancer drugs
Apoptosis
mor agents, was noted. Moreover, the most cytotoxic compounds of the series were able to induce apop-
tosis in resistant cell lines, via an atypical pathway of caspase cascade activation, and a synergistic effect
in combination with doxorubicin was also found.
Ó 2008 Elsevier Ltd. All rights reserved.
1
. Introduction
common features for the interaction of ligands with this protein
were defined: protonable nitrogen, aromatic rings, high lipophilic-
1
0,11
Resistance to multiple chemotherapeutic agents is a common
ity, and H-bond interactions.
that, besides the overall lipophilicity of the molecule, weak interac-
tions, such as those produced by the overlapping of orbitals of
aromatic rings, can play an important role in stabilizing the bind-
Moreover, there are indications
and still unsolved clinical problem in the treatment of cancer.
The identification of new experimental strategies involving novel
revertant agents or drugs capable of killing resistant cells is, there-
fore, of utmost interest in cancer research. The phenomenon
known as multidrug resistance (MDR) can arise de novo or after
exposure of cancer cells even to a single drug and is characterized
by resistance to a series of structurally unrelated compounds with
different subcellular targets.1 The best characterized mechanism
contributing to MDR is the overexpression of the energy dependent
efflux pump, P-glycoprotein (P-gp, also known as ABCB1), a
p
1
2,13
ing of MDR reverting agents to P-gp.
Despite extensive studies,
the promise of this field of investigations has not been fulfilled yet
and there are currently no clinically available reversal agents.
Furthermore, it is increasingly recognized that other tumoral
factors can significantly contribute to multidrug resistance such
as failure to undergo apoptosis and/or kinetic properties of neopla-
1
4,15
sias
: in fact, it is well known that the effects of most classical
1
6
1
70 kDa protein belonging to the ATP binding cassette (ABC)
antiproliferative agents are cell cycle dependent. So, with pro-
gresses in understanding the basic molecular mechanisms under-
lying cell cycle regulation and apoptosis and how these processes
are impaired in tumor cells, recent research has been addressed
to identify molecules capable of interfering directly with the spe-
cific intracellular targets involved such as cyclines, CDK, Bcl-2,
superfamily of transporters and capable of preventing drugs, such
as anthracyclines or Vinca alkaloids, to reach effective concentra-
tions within the cells. However, other proteins of the same family
such as the multidrug resistance associated protein (MRP1, also
named ABCC1), the lung resistance protein (LRP), and the breast
cancer resistance protein (BCRP, also named ABCG2) have also
2
1
7,18
IAPs, etc.
3
–5
been implicated.
A large variety of compounds (anticancer
The chemical structure of phenothiazine (PTZ) provides a valu-
able molecular template for the development of agents able to
interact with a wide variety of biological processes, and synthetic
PTZs and/or PTZs-derived agents have proved to be effective in
the treatment of a number of medical conditions with widely dif-
agents, calcium channel blockers, neuroleptics, antiarrhythmics,
antimalarial drugs, antifungal agents)6 with the properties of
inhibiting P-gp were selected by different approaches and limited
–9
1
9
ferent etiologies. In particular, these compounds possess some
significant characteristics which suggest a potential use as antitu-
mor drugs, because they show an inherent cytotoxicity and
*
Corresponding authors. Fax: +39 0512099734 (A.B.).
E-mail addresses: alessandra.bisi@unibo.it (A. Bisi), dusonc@unipa.it
(
L. Dusonchet).
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.040

A. Bisi et al. / Bioorg. Med. Chem. 16 (2008) 6474–6482
6475
structural modifications involving the introduction of a nitrogen
atom that could lead to more potent cytotoxic agents.20 The role
of PTZs as chemosensitizers in multidrug resistance has been
clearly assessed, and several studies have been performed in order
to establish the structural features determining their anti-MDR
ence on the cell cycle and apoptosis induction was evaluated in or-
der to establish the possible mechanism involved.
4
4
. Results
2
1,22
activity.
Usually, structural modifications that increased lipo-
.1. Cytotoxic activity
philicity of PTZs also increased MDR reverting potency, and it has
been found that both the type of substituent in position 2 of the
PTZ ring and the length of the alkyl bridge connecting the ring sys-
tem with the amino group play some role in the reverting activity.
In particular, a four carbon atom side chain was superior to alkyl
bridges of shorter length, and tertiary cyclic amines, mainly piper-
azine, were preferred to primary or secondary ones.2
In order to identify the maximal ‘non-cytotoxic’ concentrations
of each PTZ derivative to be used in combination with DXR for MDR
modulation, all the synthesized compounds were tested for direct
cytotoxicity in a preliminary in vitro assay against two human leu-
kemia cell lines, the HL60 and the CCRF/CEM, and their MDR vari-
ants. The results indicated that most compounds were devoid of
significant antiproliferative effects on the sensitive cell lines at
3,24
In this paper, a series of new PTZ derivatives bearing a rigid side
chain and different tertiary amines were designed and synthesized
in order to assess the MDR reverting effect. The rigid four carbon
atom side chain was selected because it had been successfully
introduced in EDP 42 (Chart 1), a Verapamil analog with an im-
concentrations ranging from 0.1 to 10
tions of 5f and 5h, that were able to kill about 50% of the cells ex-
posed when employed at 10 M (data not shown). A different
lM, with the only excep-
l
profile was observed in resistant leukemia cell lines: several com-
pounds showed significant cytotoxic effects, 5f, 5h, 5a and 6a being
the most active ones (Table 2). Furthermore, for these latter mole-
cules the effect appeared to be concentration dependent, with IC50
values calculated on the HL60R cell line of 1.1, 5.0, 9.5, and 10 lM,
respectively. Thus, these particular compounds showed to induce a
25
proved reverting activity ; we have also recently reported new
MDR modulators bearing the same chain.26 The amino groups were
chosen considering the structure–activity relationship (SAR) stud-
ies previously performed on PTZs and Verapamil itself. Moreover,
the two-step synthesis of the new compounds proved to be simple
and straightforward. In view of the results obtained from the pre-
liminary data on cytotoxicity, the full antitumor profile of PTZ
derivatives was also deeply investigated. The structures of the
compounds are collected in Table 1.
higher cytotoxicity in resistant rather than in sensitive tumor cells
2
7,28
as reported also for other modulators.
For a deeper evaluation
of this interesting cytotoxic behavior, the effect of these com-
pounds on cell distribution in the different cell cycle phases in
the resistant HL60R cells was also measured by flow cytometry.
The results, collected in Table 3, indicated, for all the tested com-
2
. Chemistry
1
pounds, the ability to recruit cells in G , a cell cycle phase usually
The synthesis of compounds 5a–h, 6a–c, 7a–c is depicted in
unaffected by the classic anticancer agents.
Scheme 1. The selected PTZ was alkylated with propargyl bromide
by means of potassium tert-butoxide in DMSO. The final com-
pounds were then obtained via the Mannich reaction, refluxing
the propargyl intermediates 2–4 with the selected amine and form-
aldehyde. The amines which were not commercially available were
prepared by conventional methods. All the final compounds were
4.2. MDR modulating activity
To establish the ability of the compounds to inhibit P-gp activ-
ity, non-cytotoxic concentrations were tested in MDR cell lines to
assess their ability and to restore DXR sensitivity. The results, col-
lected in Table 4, show a different degree of reverting activity by
different compounds. In particular, compounds 5d, 5e, and 6c were
the most effective, reducing the IC50 of DXR by 4-, 6-, and 8-fold,
respectively, in the HL60R cell line. This effect was concentration
1
characterized by H NMR, mass spectra, and elemental analyses.
3
. Biology
The PTZ derivatives were tested for cytotoxicity and, after-
dependent, being the maximum at the dose of 10 lM (data not
wards, for reverting activity in combination with doxorubicin
DXR), employing two hematological tumor cell lines, the HL60
and the CCRF/CEM, and their MDR variants HL60R and CEM/
VBL300. When a significant cytotoxic effect was found, the influ-
shown); overall, the new PTZs were more effective at sensitization
than the reference compound chlorpromazine, even if they were
less effective than Verapamil (Chart 1 and Table 4). Similar results
were obtained in the CEM/VBL300 cell line.
(
O
O
O
O
O
CN
CN
O
N
N
O
O
Verapamil
EDP42
S
N
Cl
N
Chlorpromazine
Chart 1.

6
476
A. Bisi et al. / Bioorg. Med. Chem. 16 (2008) 6474–6482
Table 1
Synthesized compounds
To assess the real involvement of P-gp in reverting activity, the
intracellular accumulation of DXR in sensitive and resistant cell
lines was evaluated, with or without the most active compound
of the series (6c). As shown in Figure 1, this new PTZ derivative
was able to significantly increase the accumulation of DXR in resis-
tant cells, even if the number of resistant cells capable of accumu-
lating the drug (about 70%) did not reach the level observed in the
sensitive cell line (about 100% accumulating cells).
S
N
R
R1
4
.3. Apoptotic effects
Compound
R
R
1
Since some PTZ elicited cytotoxic effects in resistant cells as de-
N
O
O
scribed above, we wondered whether these effects could be as-
cribed to apoptosis induction by the compounds in HL60R cells.
This finding could be relevant because HL60R cells were shown
to be resistant also to apoptosis induction by different stimuli.15
Interestingly, the morphological analysis showed a concentra-
tion-dependent induction of apoptosis by the compounds en-
dowed with a high cytotoxic effect 5a, 5f, and 6a (data not
shown). Hence, further studies have been performed, employing
different caspase inhibitors, to establish the pathways involved in
this phenomenon. As shown in Figure 2, the apoptotic effect was
inhibited by the pancaspase inhibitor Z-VAD, but not by other
inhibitors such as Z-IETD, Z-LEHD, and Z-DEVD (inhibitors of cas-
pase 8, 9, and 3, respectively). This could mean that PTZ derivatives
were able to activate the caspase cascade, but neither the classic
extrinsic (caspase 8-mediated) nor the intrinsic (caspase 9-medi-
ated) pathways seem to be implicated.
5
a
H
N
N
N
5
5
5
b
c
H
H
H
O
N
O
d
N
N
N
N
O
5
5
e
f
H
H
4
.4. Synergistic effects between cytotoxic derivatives and
doxorubicin
N
N
N
O
Furthermore, we tested whether the PTZ compounds that were
found to be endowed with a cytotoxic activity could induce syner-
gistic effects when combined with DXR in resistant cell lines. To this
purpose, the resistant cells were exposed to increasing concentra-
tions of DXR combined with PTZ compounds at concentrations capa-
ble of inducing a moderate cytotoxic effect (less than 50%). As
reported inFigures 3 and 4, the cytotoxic effect of all the associations
tested appeared significantly higher than the predicted values in
both HL60R and CEM/VBL300 cells, demonstrating a synergistic
interaction. However, the combinations elicited an additive effect,
when DXR was employed at very low concentrations. The synergis-
tic effect did not appear to be related to an increase in apoptotic cell
death as shown inFigure 5. On the contrary, most of the treated cells
presented necrotic morphologic alterations (data not shown).
OH
5
5
g
H
H
O
O
N
N
h
O
O
6
6
6
a
b
c
Cl
Cl
Cl
5
. Discussion
N
O
N
The present study describes the identification and activity of a
number of novel compounds derived from phenothiazines de-
signed to selectively circumvent multidrug resistance. From the re-
sults obtained, it can be seen that the new derivatives, tested at
non-cytotoxic concentrations, showed different efficacies as MDR
modulators. The most active compounds appeared to be 5d, 5e
and 6c, which were able to significantly, though only partially, re-
store the sensitivity to DXR in the resistant cell lines. This is in
agreement with the accumulation assay: the resistant cells treated
with PTZ derivatives showed an increase in DXR retention, without
reaching the levels observed in sensitive cell lines. Even if this re-
sult could be due to the fact that further increasing in concentra-
tions was limited by PTZs cytotoxicity, it could also be related to
the activation of resistance pathways independent from P-gp: in-
deed, several literature data indicate multiple mechanisms in-
N
N
O
O
7
7
7
a
b
c
CF
CF
CF
3
3
3
N
N
O
N
1
4,15
volved in MDR development.

A. Bisi et al. / Bioorg. Med. Chem. 16 (2008) 6474–6482
6477
5
S
4
6
3
2
R
7
8
S
N
a
S
N
b
1
N
H
0
R
R
1
9
R1
R = H, 2
R = Cl 3
R = H 5a-h
R = Cl 6a-c
R = CF
3
4
3
R = CF 7a-c
Scheme 1. Reagents and conditions: (a) potassium tert-butoxide, propargyl bromide, DMSO, rt; (b) selected amine, formaldehyde, CuSO
4 2
, EtOH/H O, reflux. R
1
, see Table 1.
Table 2
Table 4
Influence of PTZ derivatives on DXR cytotoxicity in multidrug resistant cells
Cytotoxicity of PTZ derivatives (10
l
M) in resistant HL60R and CEM/VBL300 cellsa
Cytotoxicity (%)
Compound
Modulator
l
M
IC50 DXR (
l
g/ml)a
RRIb
CEM/VBL300
HL60R
CEM/VBL300
HL60R
CEM/VBL300
HL60R
5
5
5
5
5
5
5
5
6
6
6
7
7
7
a
b
c
d
e
f
54
0
25
8
41
0
1
15
2
93
28
53
42
7
7
26
0
None
Chlorpromazine
Verapamil
—
6.0
1.0
b
10
10
1
3.2
0.21
2.8
9.1
1.85
1.5
1.1
5.64
3.9
6.0
2.1
2.5
0.76
2.9
3.7
2.1
0.45
0.09
0.34
0.97
0.69
0.26
0.3
0.41
0.97
0.44
0.63
0.52
0.13
0.6
1.9
28.0
2.1
0.66
3.2
4.0
5.9
1.1
1.5
1.0
2.8
2.4
7.9
2.1
1.6
2.8
2.2
10.7
2.9
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
6
1
1.03
97
45
62
52
9
10
36
13
35
10
10
10
0.1
1
0.1
1
10
10
1
10
1
1.4
3.8
3.3
2.4
1.03
2.3
1.6
1.9
7.7
1.7
1.4
1.3
g
h
a
b
c
a
b
c
24
7
7
7
a
b
c
a
Cells viability was evaluated by the MTS test as described inSection 7; hence, %
/N
 100 where N
is the number of cells in the control.
0.71
0.78
of cytotoxicity was calculated according to the formula 100 À N
is the number of cells in the treated well and N
t
c
t
c
b
a
This compound was tested at 1.0
l
M because of low solubility.
IC50: growth inhibitory concentration 50%.
RRI: resistance reverting index determined as the ratio of the IC50 for DXR alone
b
in resistant cells divided by the IC50 for DXR in the presence of various PTZs.
Table 3
Effect of PTZ derivatives on cell cycle distribution in HL60R cells
a
Treatment
None
G
1
(%)
S (%)
2
G -M (%)
37
54
57
67
64
49
34
32
25
26
13
12
11
8
5
5
5
6
f, 1 lM
h, 5 lM
DXR 0.5 μg/ml
DXR 0.5 μg/ml
a, 10
lM
a, 10
lM
10
6c 10 μM+ DXR 0.5 μg/ml
a
The cells were exposed to the compounds at the indicated concentrations for
4 h and examined by flow cytometry as described in Section 7.
100
75
2
From these figures, it is difficult to draw clear structure–activity
relationships, because some compounds of this series were tested
at different concentrations due to their cytotoxicity, leading to
barely comparable data. According to the previously reported
5
0
5
0
2
3
SAR studies for PTZs, the most interesting compounds were ex-
pected to be 6a, 6c, 7a, and 7c, bearing a Cl or a CF substituent
3
2
in position 2 and showing also the highest logP value. Moreover,
all the derivatives bearing a piperazine amino group were active
as MDR modulators, while the morpholine derivatives 6b and 7b
showed poor activity in this series: this amine led to a decrease
in logP values and, as expected, a consequent decrease in MDR
reverting properties. Unfortunately, compounds 6a, 7a, and 7c
were too cytotoxic to be properly evaluated, but compound 6c, a
HL60
HL60R
Figure 1. Influence of 6c on DXR accumulation in HL60R cells. HL60R cells were
exposed for 1 h to DXR alone (white bar) or to 6c for 1 h followed by a 1-h exposure
to DXR (hatched bar). The black bar shows DXR accumulation in the sensitive HL60
cells exposed to DXR alone. After treatment the cells were washed with cold PBS
and examined by flow cytometry. Fluorescence of cells exposed to 6c alone was
similar to the control.
2
-chlorine PTZ derivative bearing a phenylpiperazine in the side
chain, was able to reduce the IC50 of DXR 8-fold, and can therefore
be considered the most effective of the series as a reverting agent.

6
478
A. Bisi et al. / Bioorg. Med. Chem. 16 (2008) 6474–6482
This effect was independent from the ability of the compounds to re-
5
6
a 20μM
a 20μM
verse resistance, even though P-gp overexpression was considered a
relevant factor. Furthermore, some modulators were reported to in-
duce cytotoxicity by causing ATP depletion in MDR cells.28 In our
work, the ability of the compounds to induce apoptosis in an apopto-
sis resistant cell line was a very relevant finding that still needs to be
fully explained at the molecular level. Finally, it is worth noting that
our results suggest that these latter compounds, although not suit-
able as MDR modulators due to a high cytotoxic activity, could be
successfully combined with DXR inducing synergistic effects. In
our view, this interesting synergism could be partially ascribed to
the ability of PTZs and DXR to act on different phases of the cell cycle
as well as to an increase in intracellular DXR concentration induced
by PTZs with a potentiation of DXR cytotoxicity.
1
00
7
5
2
5
0
5
0
6
. Conclusions
+
Z-IETD + Z-LEHD + Z-DEVD + Z-VAD
A series of readily affordable PTZ derivatives bearing a rigid
Figure 2. Effect of several caspase inhibitors on PTZ induced apoptosis in HL60R
cells. Cells were pretreated with caspase inhibitors for 2 h and then exposed to the
PTZ derivatives at the concentrations indicated for 48 h. Apoptosis was evaluated
morphologically as described in the text.
side chain and different amines were synthesized tested to eval-
uate the MDR reverting activity and antitumor profile. The results
showed that some compounds were endowed with a remarkable
MDR reverting effect, compound 6c being the most interesting.
Furthermore, several molecules, such as 5a, 5f, 5h and 6a, proved
to be capable of inducing cytotoxic effects in resistant cell lines
Particularly interesting results were obtained with the introduc-
tion of
a
N-[2-(3,4-dimethoxyphenyl)ethyl]-N-methyl-amino
1
by interfering with the cell cycle in the G phase, usually not af-
group, the same of Verapamil, on the butynic side chain as in com-
pounds 5a, 6a, and 7a. While it was difficult to evaluate the reverting
properties of these three compounds, they showed remarkable di-
rect cytotoxicity in MDR cell lines, the ability to trigger apoptosis
andasynergisticeffectwithDXR. Similarly, variousauthorsreported
fected by classical antitumor agents. Finally, it was interesting to
note that the cytotoxic compounds demonstrated to trigger apop-
tosis via an atypical pathway of caspase activation and to induce
synergistic effects when combined with DXR in resistant cell
lines.
2
7
that a number of modulators were selectively toxic to MDR cells.
DXR
1
00
7c 10 μM + DXR
7c 10 μM + DXR predicted
7a 10 μM + DXR
7a 10 μM + DXR predicted
5
0
0
0.1
1
10
DXR μg/ml
DXR
5
1
00
f 1 μM + DXR
f 1 μM + DXR predicted
h 1 μM + DXR
h 1 μM + DXR predicted
5
5
5
5
0
0
0.1
1
10
DXR μg/ml
Figure 3. Effects of PTZ derivatives + DXR in HL60R cells. Cells were treated with the PTZ derivatives and after 1 h increasing concentrations of DXR were added. Cytotoxicity
was evaluated after 72 h by MTS. Dashed lines indicate additive effect calculated according to the fractional product method of Webb. Survival values of combinations laying
below the additivity lines represent a synergistic effect.

A. Bisi et al. / Bioorg. Med. Chem. 16 (2008) 6474–6482
6479
100
DXR
5a 10 μM + DXR
5a 10 μM + DXR predicted
7a 10 μM + DXR
50
7a 10 μM + DXR predicted
0
0
.01
0.1
1
DXR μg/ml
100
DXR
5
5
5
f 1 μM + DXR
f 1 μM + DXR predicted
h 1 μM + DXR
50
5
h 1 μM + DXR predicted
0
0
.01
0.1
1
DXR μg/ml
Figure 4. Effects of PTZ derivatives + DXR in CEM/VBL300 cells. Cells were exposed to the PTZ derivatives for 1 h and then increasing concentrations of DXR were added.
Cytotoxicity was evaluated by MTS after 72 h. Dashed lines indicate additive effect calculated according to the fractional product method of Webb. Survival values of
combinations laying below the additivity lines represent a synergistic effect.
pounds were named following IUPAC rules as applied by AUTO-
DXR 10μg/ml
NOM, a PC software for systematic names in organic chemistry,
100
PTZ 10μM
Beilstein-Institut and Springer.
PTZ + DXR
7
.1.1. 10-Prop-2-ynyl-10H-phenothiazine (2)
7
5
2
5
0
5
0
Four grams (0.036 mol) of potassium tert-butoxide were added
to a suspension of phenothiazine (5 g, 0.025 mol) in 15 mL of
DMSO and the mixture was stirred at room temperature for
30 min. The solution obtained was then added dropwise to a solu-
tion of propargyl bromide (4 mL, 0.034 mol) in DMSO, stirred at
room temperature for 3 h and poured into ice. The aqueous solu-
tion was extracted with methylene chloride, which was then
2 4
washed with water, dried with Na SO and evaporated to dryness.
The residue was purified by flash chromatography (cyclohexane/
methylene chloride 9:1), to yield 3.8 g (65%) of 2, mp 80–82 °C
2
9
1
(
ligroin; lit 92 °C). H NMR: d 2.45 (t, J = 3.0 Hz, 1H), 4.50 (d,
5a
6a
J = 3.2 Hz, 2H), 6.90–7.30 (m, 8H arom).
Figure 5. Influence of PTZ derivatives on DXR induced apoptosis in HL60R cells.
Cells were exposed to the PTZ derivatives for 1 h followed by 48 h treatment with
DXR. Apoptosis was evaluated by fluorescence microscopy.
7.1.2. 2-Chloro-10-prop-2-ynyl-10H-phenothiazine (3)
Using the previous procedure, 4.0 g (60%) of 3 were obtained,
3
0
1
mp 79–81 °C (ligroin; lit
112–115 °C). H NMR: d 2.55 (t,
J = 3.1 Hz, 1H), 4.50 (d, J = 3.2 Hz, 2H), 6.95–7.40 (m, 7H arom).
7
7
. Experimental
7
.1.3. 10-Prop-2-ynyl-2-trifluoromethyl-10H-phenothiazine (4)
.1. Chemistry: general methods
Using the previous procedure, 5.3 g (70%) of 4 were obtained,
1
mp 84–86 °C (ligroin). H NMR: d 2.55 (t, J = 3.0 Hz, 1H), 4.50 (d,
J = 3.2 Hz, 2H), 6.95–7.20 (m, 7H arom).
All melting points were determined in open glass capillaries
1
using a Büchi apparatus and are uncorrected. H NMR spectra were
recorded on a Varian Gemini 300 spectrometer in CDCl
3
solutions,
7.2. General procedure for the synthesis of compounds 5a–h,
6a–c, and 7a–c
with Me Si as the internal standard. Mass spectra were recorded
4
on a V.G. 7070 E spectrometer or on a Waters ZQ 4000 apparatus
operating in electrospray (ES) mode. Elemental analyses were
within 0.4% of theoretical value unless otherwise indicated. Com-
A suspension of formaldehyde (0.21 mL, 0.02 mol), the selected
amine (0.02 mol), and 0.05 g of CuSO4 was added to a solution of

6
480
A. Bisi et al. / Bioorg. Med. Chem. 16 (2008) 6474–6482
1
the propynyl derivative (2–4) (0.7 g, 0.02 mol) in 10 mL of water/
235 °C (methanol/diethylether). H NMR (free base): d 2.30 (s, 3H),
ethanol 1:1, and the mixture was refluxed for 24 h. After cooling,
2.50–2.75 (m, 8H), 2.85–2.95 (m, 2H), 3.40 (s, 2H), 4.00 (t, J = 3.8
1
5 mL of ammonia was added and the mixture was extracted with
Hz, 2H), 4.20–4.30 (m, 1H), 4.55 (s, 2H), 6.60–7.25 (m, 12H arom).
diethylether, dried with Na SO , and evaporated to dryness, to give
2
4
33 3 2
ES-MS: m/z 500 (M+1). Anal. (C30H N O S) Calcd: C, 72,11; H,
an oily compound which was purified by flash chromatography.
Where indicated, the oxalate salt was prepared dissolving the com-
pound in ethanol and adding the equivalent amount of oxalic acid.
After stirring for 30 min at rt, the mixture was filtered and diethyl
ether was added until crystallization occurred. The compounds
were obtained with yields ranging from 55% to 70%.
6,66; N, 8,41; found: C, 72.09; H, 6.70; N, 8.40.
7.2.7. 10-(4-Piperidin-1-yl-but-2-ynyl)-10H-phenotiazine (5g)
The compound was purified by flash chromatography (toluene/
acetone 4:1) and converted to the oxalate salt, mp 191–193 °C
1
(methanol/diethylether). H NMR(free base): d 1.35–1.45 (m, 2H),
1
.60 (t, J = 4.2 Hz, 4H), 2.50 (t, J = 4.2 Hz, 4H), 3.35 (t, J = 3.7 Hz,
2H), 4.55 (t, J = 3.7 Hz, 2H), 6.90–7.30 (m, 8H arom). ES-MS: m/z
335 (M+1). Anal. (C21 S) Calcd: C, 75.41; H, 6.63; N, 8.38;
7
1
.2.1. [2-(3,4-Dimethoxyphenyl)ethyl]methyl-(4-phenothiazin-
0-yl-but-2-ynyl)amine (5a)
22 2
H N
The compound was purified by flash chromatography (toluene/
found: C, 75.40; H, 6.65; N, 8.40.
acetone 4:1) and converted to the oxalate salt, mp 185–187 °C
1
(
2
methanol/diethylether). H NMR (free base): d 2.35 (s, 3H),
.65–2.75 (m, 4H), 3.45 (s, 2H), 3.80 (s, 3H), 3.85 (s, 3H), 4.60 (t,
J = 4.2 Hz, 2H), 6.65–7.30 (m, 11H, arom). MS: m/z (rel. abundance)
7.2.8. 10-[4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)
but-2-ynyl]-10H-phenothiazine (5h)
The compound was purified by flash chromatography (petro-
leum ether/ethylacetate 9:1) and crystallized from ethanol, mp
+
4
44 (M , 16.4%), 32 (100%), 266 (70.7%). Anal. (C27
C, 72,94; H, 6,35; N, 6,30; found: C, 72,92; H, 6,36; N, 6,29.
28 2 2
H N O S) Calcd:
1
136–138 °C. H NMR: d 2.90 (d, J = 3.2 Hz, 4H), 3.55 (t, J = 3.4 Hz,
2
H), 3.70 (s, 2H), 3.80 (s, 3H), 3.85 (s, 3H), 4.50 (t, J = 3.4 Hz, 2H),
7
1
.2.2. 10-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]but-2-ynyl}-
0H-phenothiazine (5b)
The compound was purified by flash chromatography (toluene/
6.55–7.35 (m, 10H arom). ES-MS: m/z 443 (M+1). Anal.
(C27H26N O S) Calcd: C, 73.27; H, 5.92; N, 6.33; found: C, 73.25;
H, 5.94; N, 6.30.
2 2
acetone 9:1) and converted to the oxalate salt, mp 137–139 °C
(
methanol/diethylether). 1H NMR (free base): d 2.75–2.85 (m,
7.2.9. [4-(2-Chlorophenothiazin-10-yl)but-2-ynyl]-[2-(3,4-
dimethoxyphenyl)ethyl]methylamine (6a)
The compound was purified by flash chromatography (toluene/
acetone 4:1) and converted to the oxalate salt, mp 165–167 °C
4
6
7
H), 3.15–3.25 (m, 4H), 3.45 (s, 2H), 3.90 (s, 3H), 4.60 (s, 2H),
.90–7.30 (m, 12H arom). MS: m/z (rel. abundance) 441 (M ,
.7%), 44 (100%), 136 (67.7%). Anal. (C27H N OS) Calcd: C, 73.44;
27 3
+
1
H, 6.16; N, 9.52; found: C, 73.42; H, 6.15; N, 9.53.
(methanol/diethylether). H NMR (free base): d 2.45 (s, 3H),
2
3
.65–2.75 (m, 4 H), 3.50 (t, J = 3.2 Hz, 2H), 3.80 (s, 3H), 3.85 (s,
7
.2.3. 10-[4-(4-Phenylpiperazin-1-yl)but-2-ynyl]-10H-
H), 4.50 (t, J = 3.3 Hz, 2H), 6.65–7.20 (m, 10H arom). MS: m/z
phenotiazine (5c)
+
(
(
rel. abundance) 478 (M , 14.3%), 95 (100%), 327 (68.4%). Anal.
C H27ClN O S) Calcd: C, 67.70; H, 5.68; N, 5.85; found: C,
27 2 2
The compound was purified by flash chromatography (toluene/
acetone 4:1) and converted to the oxalate salt, mp 118–120 °C
6
7.72; H, 5.67; N, 5.86.
(
methanol/diethylether). 1H NMR (free base): d 2.65–2.75 (m,
4
2
3
H), 3.15–3.25 (m, 4H), 3.40 (t, J = 4.1 Hz, 2H), 4.55 (t, J = 4.1 Hz,
H), 6.80–7.30 (m, 13H arom). MS: m/z (rel. abundance) 411 (M ,
3.9%), 198 (100%), 56 (20.1%). Anal. (C26H N S) Calcd: C, 75.88;
25 3
7
.2.10. 2-Chloro-10-(4-morpholin-4yl-but-2-ynyl)-10H-
+
phenothiazine (6b)
The compound was purified by flash chromatography (toluene/
H, 6.12; N, 10.21; found: C, 75.90; H, 6.10; N, 10.22.
acetone 4:1) and converted to the oxalate salt, mp 182–184 °C
1
(
methanol/diethylether). H NMR (free base): d 2.55–2.65 (m,
7
.2.4. 4-(4-Phenothiazin-10-yl-but-2-ynyl)piperazine-1-
4
2
3
6
H), 3.40 (d, J = 4.2 Hz, 2H), 3,70–3.80 (m, 4H), 4.55 (d, J = 4.3 Hz,
H), 6.90–7.25 (m, 7H arom). MS: m/z (rel. abundance) 370 (M ,
1.1%), 45 (100%), 232 (92.0%). Anal. (C20H19ClN OS) Calcd: C,
2
4.77; H, 5.16; N, 7.55; found: C, 64.78; H, 5.14; N, 7.56.
carboxylic acid ethyl ester (5d)
+
The compound was purified by flash chromatography (toluene/
acetone 4:1) and converted to the oxalate salt, mp 157–159 °C
(
methanol/diethylether). 1H NMR (free base): d 1.25–1.35 (m,
3
4
H), 2.45–2.55 (m, 4H), 3.40 (t, J = 4.2 Hz, 2H), 3.50–3.65 (m, 4H),
7
1
.2.11. 2-Chloro-10-[4-(4-phenylpiperazin-1-yl)but-2-ynyl]-
0H-phenothiazine (6c)
The compound was purified by flash chromatography (toluene/
.10–4.20 (m, 2H), 4.60 (t, J = 4.2 Hz, 2H), 6.90–7.20 (m, 8H arom).
+
MS: m/z (rel. abundance) 407 (M , 23.0%), 198 (100%), 45 (58.9%).
Anal. (C23
H
25
N
O
3 2
S) Calcd: C, 67.79; H, 6.18; N, 10.31; found: C,
acetone 9:1) and converted to the oxalate salt, mp 101–103 °C
6
7.81; H, 6.20; N, 10.33.
1
(
methanol/diethylether). H NMR (free base): d 2.65–2.75 (m,
4
2
8
H), 3.20–3.35 (m, 4H), 3.40 (t, J = 3.5 Hz, 2H), 4.50 (t, J = 3.5 Hz,
H), 6.90–7.30 (m, 12H arom). MS: m/z (rel. abundance) 445 (M ,
.6%), 32 (100%), 233 (65.6%). Anal. (C26H24ClN S) Calcd: C, 70.02;
3
7
.2.5. 10-[4-(4-Methylpiperazin-1-yl)but-2-ynyl]-10H-
+
phenothiazine (5e)
The compound was purified by flash chromatography (toluene/
H, 5.42; N, 9.42; found: C, 70.00; H, 5.44; N, 9.43.
ethyl acetate 3:2) and converted to the oxalate salt, mp 216–220 °C
1
(
methanol/diethylether). H NMR (free base): d 2.40 (s, 3H), 2.55–
7
.2.12. [2-(3,4-Dimethoxyphenyl)ethyl]methyl-[4-(2-
2
7
1
6
.80 (m, 8H), 3.40 (t, J = 5.4 Hz, 2H), 4.60 (t, J = 5.4 Hz, 2H), 7.00–
.35 (m, 8H arom). MS: m/z (rel. abundance) 349 (M , 16.7%),
98 (100%), 150 (24.9%). Anal. (C21H N S) Calcd: C, 72.17; H,
23 3
.63; N, 12.02; found: C, 72.15; H, 6.60; N, 12.00.
+
trifluoromethyl-phenothiazin-10-yl)but-2-ynyl]amine (7a)
The compound was purified by flash chromatography (tolu-
ene/acetone 4:1) and converted to the oxalate salt, mp 133–
1
1
2
4
35 °C (methanol/diethylether). H NMR: d 2.35 (s, 3H), 2.65–
.75 (m, 4H), 3.50 (t, J = 4.2 Hz, 2H), 3.80 (s, 3H), 3.85 (s, 3H),
.55 (t, J = 4.2 Hz, 2H), 6.65–7.40 (m, 10H arom). MS: m/z (rel.
7
.2.6. 1-[4-(4-Phenothiazin-10-yl-but-2-ynyl)piperazin-1-yl]-3-
p-tolyloxy-propan-2-ol (5f)
The compound was purified by flash chromatography (toluene/
ethylacetate 1:1) and converted to the hydrochloride salt, mp 232–
+
abundance) 512 (M , 16.8%), 361 (100%), 32 (97.4%). Anal.
(
C
28
H
27
F
3
N
2
O
2
S) Calcd: C, 65.61; H, 5.31; N, 5.47; found: C,
6
5.60; H, 5.32; N, 5.45.

A. Bisi et al. / Bioorg. Med. Chem. 16 (2008) 6474–6482
6481
7
1
.2.13. 10-(4-Morpholin-4-yl-but-2-ynyl)-2-trifluoromethyl-
0H-phenothiazine (7b)
that produced 610% inhibition of growth in resistant cells when
employed alone.
The compound was purified by flash chromatography (toluene/
To study the combined effects of DXR plus concentrations of the
cytotoxic PTZs that alone produced 650% growth inhibition, the
acetone 4:1) and converted to the oxalate salt, mp 103–105 °C
31
(
methanol/diethylether). 1H NMR (free base): d 2.55–2.65 (m,
fractional product method of Webb was applied. Experimental
4
2
1
6
H), 3.35 (d, J = 3.5 Hz, 2H), 3.70–3.85 (m, 4H), 4.56 (d, J = 3.5 Hz,
H), 7.00–7.30 (m, 7H arom). MS: m/z (rel. abundance) 404 (M ,
0.6%), 32 (100%), 74 (94.9%). Anal. (C21H F N OS) Calcd: C,
19 3 2
2.36; H, 4.74; N, 6.93; found: C, 62.38; H, 4.75; N, 6.92.
dose–response curves were compared with the theoretical lines
of additivity. These predicted curves were created by calculating
a predicted value (c) for each drug combination according to the
equation c = a  b/100, where a and b indicate cell survival values
with single agents. When the cytotoxic effects of combinations
and the predicted values overlapped, the interaction was consid-
ered additive, while it was considered synergistic when the cyto-
toxic effect was higher than the predicted value.
+
7
.2.14. 10-[4-(4-Pheny-piperazin-1-yl)but-2-ynyl]-2-(trifluoro-
methyl)-10H-phenothiazine (7c)
The compound was purified by flash chromatography (toluene/
acetone 9:1) and converted to the oxalate salt, mp 114–116 °C
Apoptosis was evaluated by fluorescence microscopy according
methanol/diethylether). 1H NMR (free base): d 2.65–2.75 (m,
to the method of Duke and Cohen. After treatment with the drugs
32
(
4
2
5
H), 3.15–3.25 (m, 4H), 3.40 (t, J = 3.4 Hz, 2H), 4.55 (t, J = 3.4 Hz,
H), 6.90–7.40 (m, 12H arom). MS: m/z (rel. abundance) 479 (M ,
for 48 h, cells were centrifuged and the pellet was resuspended in
25 ll of a dye mixture containing acridine orange and ethidium
+
.7%), 45 (100%), 120 (17.1%). Anal. (C27
H
24
F
3
N
3
S) Calcd: C, 67.62;
bromide. Live and apoptotic cells were identified by fluorescence
microscopy. The caspase inhibitors were added 2 h before drug
treatment. Alternatively, apoptosis was evaluated by flow cytome-
try as the percentage of hypodiploid nuclei accumulated in the
H, 5.04; N, 8.76; found: C, 67.62; H, 5.03; N, 8.75.
7
.3. Cell culture and treatment
0 1
sub-G –G
peak after labeling with propidium iodide.33
The human acute promyelocytic cell line HL60, the human lym-
phoblastic leukemia cell line CCRF/CEM, and their MDR variants
HL60R and CEM/VBL300 were employed in the study. HL60R cells
express P-glycoprotein and are about 300-fold resistant to the
cytotoxic effects of the anticancer drug DXR; in addition, this cell
line shows a high degree of resistance to apoptosis induction by
different chemically unrelated agents. CEM/VBL300 cells also ex-
press P-glycoprotein and are about 300-fold resistant to vinblas-
tine. Cells were cultured in RPMI 1640 medium (Gibco, Grand
Island, NY, USA) supplemented with 10% (v/v) heat-inactivated
7.5. Cell cycle analysis
The effects of the compounds on the cell cycle were studied by
flow cytometry. Briefly, the cells were washed once in ice-cold PBS
6
and resuspended at 1 Â 10 /ml in a hypotonic fluorochrome solu-
tion containing propidium iodide (Sigma) 50 lg/ml in 0.1% sodium
citrate plus 0.03% (v/v) Nonidet P-40 (Sigma). After 30 min of incu-
bation in this solution, the samples were filtered through nylon
FCS (Gibco), 100 U/ml penicillin (Gibco), 100
Gibco) and 2 mM of -glutamine (Gibco) at 37 °C in a humidified
% CO atmosphere. DXR was purchased from Sigma–Aldrich
S.r.l. (Milano, Italy). The caspase inhibitors Z-IETD-FMK (Z-Ile-
Glu-Thr-Asp-fluoromethylketone), Z-LEHD-FMK (Z-Leu-Glu
OMe)-His-Asp (OMe)-fluoromethylketone), Z-DEVD-FMK (Z-Asp
OMe)-Glu (OMe)-Val-Asp (OMe)-fluoromethylketone), and Z-
l
g/ml streptomycin
cloth, 40 lm mesh, and their fluorescence was analyzed as sin-
(
5
L
gle-parameter frequency histograms by using a FACSort (Becton
Dickinson, Mountain View, CA, USA). A minimum of 10,000 events
for each sample was collected in list mode. The distribution of cells
in the cell cycle was analyzed with the ModFit LT program (Verity
Software House, Inc.).
2
(
(
VAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone)
were purchased from Alexis Biochemicals (Laufelfingen, Switzer-
land). All the PTZ derivatives were dissolved in DMSO. However,
because of poor water solubility and compound precipitation,
7.6. Determination of DXR accumulation
Intracellular DXR concentrations were evaluated by flow
5
cytometry. In brief, cells at a concentration of 5 Â 10 cells/ml were
PTZ concentrations in samples could not exceed 10
l
M.
exposed to the PTZ for 1 h and then to DXR 0.5 lg/ml for 1 h. Cells
were then washed twice in cold PBS, resuspended in fresh medium
6
7
.4. Cell viability and apoptosis
at a concentration of 1 Â 10 cells/ml and anthracycline fluores-
cence was immediately examined using the FACScan with the
appropriate filter.
Cytotoxicity was assessed by the trypan blue dye exclusion test
or the MTS-assay. In brief, cells were seeded at a density of
6
0
.2 Â 10 cells/ml in 24-well plates and exposed to the modulators
Acknowledgment
and after 1 h to doxorubicin. After 72 h of exposure, aliquots of
cells were harvested, stained with trypan blue and counted by light
This work was supported by a PRIN Project Grant from MIUR,
Italy.
5
microscopy. Alternatively, cells were seeded at a density of 1 Â 10
per milliliter in 96-well microtiter plates, treated as described
above and cell viability was evaluated measuring the reduction
of the tetrazolium compound (MTS) with an assay kit (Promega,
Madison, WI) according to the supplier manual. The absorbance
was recorded using a spectrophotometer at k 595 nm. All samples
were measured in triplicate in at least three independent experi-
ments. Resistance index was calculated as the ratio between the
DXR IC50 (inhibitory concentration 50%) in the resistant cells and
that in the parental sensitive cell line. The resistance reverting
index (RRI) was determined as the ratio of the IC50 for DXR alone
divided by the IC50 for DXR in the presence of the maximal
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