Synthetic polyamines activating autophagy: Effects on cancer cell death

Article abstract of DOI:10.1016/j.ejmech.2013.06.044

The ability of symmetrically substituted long chain polymethylene tetramines, methoctramine (1) and its analogs 2-4 to kill cancer cells was studied.We found that an elevated cytotoxicity was correlated with a 12 methylene chain length separating the inne

Full text of DOI:10.1016/j.ejmech.2013.06.044

European Journal of Medicinal Chemistry 67 (2013) 359e366
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthetic polyamines activating autophagy: Effects on cancer cell
death
Anna Minarini a, Maddalena Zini b, Andrea Milelli c, Vincenzo Tumiatti c, Chiara Marchetti a,
Benedetta Nicolini b, Mirella Falconi b, Giovanna Farruggia a, Concettina Cappadone a,
,
Claudio Stefanelli c
*
a Department of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy
b Department of Biomedical and Neuromotor Sciences, University of Bologna, 40126 Bologna, Italy
c Department for Life Quality Studies, University of Bologna, Rimini campus, 47921 Rimini, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The ability of symmetrically substituted long chain polymethylene tetramines, methoctramine (1) and its
analogs 2e4 to kill cancer cells was studied. We found that an elevated cytotoxicity was correlated with a
12 methylene chain length separating the inner amine functions (6-12-6 carbon backbone), together
with the introduction of diphenylethyl moieties on the terminal nitrogen atoms (compound 4) of a
tetramine backbone. Compound 4 triggered dissipation of mitochondrial transmembrane potential and
increased intracellular peroxide levels, leading to a caspase-independent HeLa cell death associated with
a rapid activation of autophagy. The antioxidant N-acetylcysteine inhibited cell death and activation of
autophagy, indicating a link between oxidative stress and autophagy. Autophagy was rapidly triggered
even by tetramines 2 and 3, indicating that is related to their polyamine structure. Autophagy did not
protect HeLa cells against cytotoxicity elicited by compound 4. The present study shows that, by mod-
ifications of the methoctramine structure, it is possible to design polyamine derivatives highly cytotoxic
against tumor cells and that the appropriate design of molecules bearing polyamine-like structures leads
to powerful inducers of autophagy.
Received 31 January 2013
Received in revised form
20 June 2013
Accepted 21 June 2013
Available online 1 July 2013
Keywords:
Polyamine analogs
Autophagy
Oxidative stress
Cancer therapy
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
cancer development [5] and by interfering with polyamine meta-
bolism and functions it is possible to affect cancer cell proliferation
Research in the polyamine field continues to have a great po-
tential for the development of novel therapeutic agents against
proliferative disorders. As pointed out by Casero and Woster [1], in
the course of the last thirty years the research has progressively
moved from the search of specific inhibitors acting on enzymes of
polyamine metabolism toward the design of polyamine analogs
acting as polyamine mimetics or polyamine antimetabolites. Poly-
amines are involved in multiple cellular processes, including those
linked to cell growth and survival such as cell cycle, apoptosis and
autophagy [2e4]. For this reason, polyamines have a central role in
at different levels [6e8].
The polycationic structure of alkylpolyamines, together with the
lipophilic polymethylene chains, allow them to interact with a va-
riety of cellular targets so that the polyamine structure may repre-
sent an universal template for the recognition of many receptor sites
[9,10]. In fact, it was verified that it is possible to modulate both
affinity and selectivity for diverse receptor systems by inserting
different groups onto a polymethylene backbone, as well as appro-
priate spacers separating the amine functions [11,12]. Methoctr-
amine (1), for example, represents the prototype of polymethylene
tetramines acting as muscarinic receptor competitive antagonists
[13]. Regarding the muscarinic receptor activity, a huge study of
structureeactivity relationships on 1-derivatives was performed, in
particular, by modifying the methylene chain length between the
inner nitrogen atoms [9]. Only recently we have reported that the
cytotoxicity of 1 and its analogs is strongly correlated to the meth-
ylene chain length separating the inner nitrogen atoms, increasing
from 5 to 12 units [14], suggesting that cytotoxicity of these
Abbreviations: AO, acridine orange; Atg5, autophagy gene 5; AVOs, acidic ve-
sicular organelles; DCFDA, dichlorofluoresceine diacetate; DFMO,
omethylornithine; DENSPM, DiOC6,
a
-difluor-
N1,N11-diethylnorspermine;
3,30-
dihexyloxacarbocyanine iodide; DMSO, dimethylsulphoxide; NAC, N-acetylcys-
teine; TEM, transmission electron microscopy.
* Corresponding author. Tel.: þ39 51 2091207; fax: þ39 51 2091224.
E-mail address: claudio.stefanelli@unibo.it (C. Stefanelli).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.044

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A. Minarini et al. / European Journal of Medicinal Chemistry 67 (2013) 359e366
methylene chain length and diphenylethyl moieties on terminal
nitrogens, with the aim to investigate a possible additive effect of
these two chemical features that we found to increase cytotoxicity.
Very recently we found that compounds with these chemical fea-
tures act as potent N-methyl-D-aspartate (NMDA) receptor channel
blockers [15].
In the present work we went deeply inside to the mechanism of
cytotoxicity elicited by symmetrically substituted tetramines 1e4.
Interestingly, the investigation of the mechanisms of cell death,
revealed that the cytotoxic effect of 1-derivatives against cancer
cells is associated with activation of autophagy.
2. Results and discussion
2.1. Chemistry
Fig. 1A shows the structure of the tetramine compounds used in
this study, i.e, methoctramine (1); the derivative in which the 2-
methoxybenzyl groups of 1 were replaced with a diphenylethyl
moieties (2); the 1-derivative in which the octamethylene spacer
separating the inner amine functions was replaced with a 12 meth-
ylene chain (3); the newly synthesized derivative combining both
modifications, the inner 12 methylene chain and the terminal
diphenylethyl moieties (4). Compounds 1e3 have been synthesized
as previously reported [16e18]. Compound 4 was synthesized
following the procedure reported in Scheme 1 and was characterized
by IR,1H NMR,13C NMR, mass spectra, and elemental analysis. Briefly,
intermediate 5 was obtained by reacting N-[(benzyloxy)carbonyl]-6-
aminocaproic acid with 1,12-diaminododecane; removal of the N-
(benzyloxy)carbonyl group by acidic hydrolysis gave diamine
diamide 6, which was treated with 2,2-diphenylacetyl chloride to
furnish derivative 7; reduction with borane N-ethyl-N-iso-
propylaniline complex (BACH-EI) in dry THF [19] led to the tetra-
amine compound 4.
Fig. 1. Structure and cytotoxicity of the symmetric polymethylene tetramines 1e4. (A)
Chemical structure of compounds 1e4; in red the structural differences with respect to
compound 1 (methoctramine). (B) Induction of cell death by compounds 1e4 in HL60
cells incubated for 24 h with increasing concentrations of the indicated compounds.
The figure reports means ꢀ S.E.M. of three determinations. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article.)
molecules should be associated to lipophilicity and that appropriate
tetramine backbone can be used for the development of anticancer
drugs. In addition, in preliminary experiments we observed an
increased toxic effect, in comparison to 1, of compound 2, obtained
by replacing the terminal 2-methoxybenzyl groups of 1 with the
more lipophilic diphenylethyl moieties. This last chemical feature
2.2. Cytotoxicity
The cytotoxic activities of 1 and its analogs 2e4 were evaluated
against HeLa cervical carcinoma cells, incubated for 24 h in the
presence of increasing concentration of the analogs (Fig. 1B).
Compounds 2 and 3 elicited a similar moderate toxicity, however,
when their structural determinants of toxicity were combined as in
compound 4, the effect was synergistic and cytotoxicity was
largely increased. Cytotoxicity of 1e4 was also examined against
non-cancerous cells, such as immortalized human chondrocytes
was already reported to confer antitumor activity (mM range) when
mounted on terminal nitrogen atoms of spermine, norspermine and
spermidine [1]. For this reason, we thought of interest to design and
synthesize a new polymethylene tetramine (4) bearing an inner 12
Scheme 1. Procedure for the preparation of compound 4. (i) EtOCOCl, NEt3, dioxane, room temp., 18 h, 84% yield; (ii) a) HBr 33% in CH3COOH, room temp., 4 h, quantitative yield; b)
2 N NaOH, CHCl3, 14 h; (iii) 2,2-diphenylacetyl chloride, NEt3, CH2Cl2, room temp., overnight, 20% yield; (iv) BACH-EI, diglyme, reflux, 9 h, 36% yield.

A. Minarini et al. / European Journal of Medicinal Chemistry 67 (2013) 359e366
361
Table 1
48
24
0
400
A
B
Ctrl
4
Ctrl
4
IC50 values of tetramines against different cells. The data were obtained after
treating the cells with increasing concentration of the indicated compound for 24 h.
Data are means ꢀ S.E.M. of three replicates.
Compound
IC50
HeLa
>100
(mM)
200
0
C28/I2 chondrocytes
Rat thymocytes
1 (Methoctramine)
>100
>100
2
3
4
18 ꢀ 4
60 ꢀ 5
77 ꢀ 6
4.3 ꢀ 0.3
82 ꢀ 6
21 ꢀ 4
>100
100 101 102 103
DCF fluorescence
100 101 102 103
1.6 ꢀ 0.3
6.8 ꢀ 0.5
DiOC6 fluorescence
C-28/I2 and primary rat thymocytes (Table 1). In these non-
cancerous cells, compounds 1e3 exhibited a very low cytotoxicity
and the effect of compound 4 was lower respect to HeLa carcinoma
cells.
The IC50 values of 4 against seven different cancer cell lines,
calculated in experiments similar to that depicted in Fig. 1B, were in
the low micromolar range at 24 h and fell in the high nanomolar
range at 48 h (Table 2). Osteosarcoma cells apparently exhibited the
higher sensitivity to 4, being p53-null Saos-2 the most sensitive
cells among those tested.
*
*
100
*
C
+
NAC
50
0
-
NAC
0
1
2
3
Compound 4 (mM)
2.3. Mechanisms associated with cytotoxicity
*
100
50
0
The mechanisms involved in cell death were investigated in
HeLa cells. We have previously shown that the toxicity of com-
pound 3 is linked to cellular internalization and induction of
oxidative stress [14]. In order to detect whether oxidative stress is
also involved in the cytotoxic effect of compound 4 and if it is
associated to mitochondrial events, we investigated the effects of 4
on the mitochondrial membrane potential (DJm), measured in
intact cells by using the fluorescent probe 3,30-dihexylox-
acarbocyanine iodide (DiOC6). This compound can accumulate and
aggregate into mitochondria, leading to a green fluorescence.
D
Compd 4 : - + - + - + - +
Ctrl
Spm DFMO pH 6.4
Following
Jm collapse, DiOC6 no longer enters mitochondria,
resulting in a decrease in green fluorescence. Fig. 2A shows that a
4 h treatment with 4 caused in the cell population a decrease of
more than 50% in the mean DiOC6 signal (from channel 216 to
channel 98), indicating a marked disruption of DJm. Since the
depolarization of the mitochondrial membrane is associated with
mitochondrial production of ROS, we evaluated the intracellular
oxidative stress elicited by dichlorofluoresceine diacetate (DCFDA),
that is hydrolyzed by intracellular esterases to dichlorohydro-
fluorescein which is oxidized to the highly fluorescent dichloro-
fluorescein (DCF) by cellular peroxides. The treatment of HeLa cells
with compound 4 caused a significant increase in DCF fluorescence
in the cell population (from channel 41 to channel 77, Fig. 2B),
indicating increased peroxide production. The antioxidant N-ace-
tylcysteine (NAC) protected the cells from 4-induced cell death,
showing that oxidative stress is involved in the pathway leading to
Fig. 2. Characteristics of cell death triggered by compound 4 in HeLa cells. (A) Change
in mitochondrial membrane potential as estimated from DiOC6 fluorescence in cells
treated for 3 h with DMSO (control) or 2 mM 4. DJm collapse in 4-treated cells is
evidenced by a decrease in DiOC6 fluorescence histograms. (B) Histogram profiles of
control and 4-treated cells upon DCFDA staining. The increase in the mean fluores-
cence intensity (DCF-positive cells) in cells incubated 3 h in the presence of 2 mM 4,
indicates an increase in intracellular peroxides. (C) Cell viability was measured in cells
treated for 24 h with the indicated concentration of compound 4 in the absence or
presence of 5 mM NAC. Results are means ꢀ S.E.M. of triplicate determinations,
*P < 0.05 vs. cells incubated in the absence of NAC. (D) Cell survival was measured in
cells incubated for 24 h in the absence or presence of compound 4 (2 mM) together
with treatments that could affect its cellular uptake: 0.5 mM spermine (together with
1 mM aminoguanidine to inhibit serum amine oxidases in the medium); 0.1 mM DFMO
(48 h pretreatment); incubation in a medium in which the pH was adjusted to 6.4 with
HCl. Results are mean ꢀ S.E.M. of triplicate determinations, *P < 0.05 vs. control cells.
cell death directly or by influencing the redox status of the cell
(Fig. 2C).
The effect of antioxidants has been examined rarely in the
literature dealing with polyamine analogs. However, a protective
effect of NAC was reported in the case of anticancer agents, such as
N1,N11-diethylnorspermine (DENSPM) [20], mononaphthalimide-
spermidine [21], and polyamine-containing naphthalene diimides
[22]. This protective role of antioxidants needs to be taken in ac-
count in evaluating either the therapeutic potential or the strategy
to decrease toxicity in normal non-tumor cells.
Table 2
IC50 values of compound 4 against cancer cell lines. The data were obtained by
treating the cells with increasing concentration of compound 4 for the indicated
time. Results are means ꢀ S.E.M. of three to four replicates.
Cell line
IC50 (mM)
24 h
48 h
Saos-2 osteosarcoma
U2OS osteosarcoma
HeLa cervical carcinoma
HL60 myeloid leukemia
LOVO colorectal adenocarcinoma
HT29 colon cancer
0.9 ꢀ 0.1
1.1 ꢀ 0.2
1.6 ꢀ 0.3
1.9 ꢀ 0.2
2.0 ꢀ 0.3
2.4 ꢀ 0.2
2.8 ꢀ 0.4
0.3 ꢀ 0.1
0.4 ꢀ 0.1
0.6 ꢀ 0.1
0.7 ꢀ 0.1
0.8 ꢀ 0.2
1.1 ꢀ 0.3
0.9 ꢀ 0.2
Cytotoxicity of 4 was not influenced by addition to the medium
of spermine or by 48 h pre-treatment with the polyamine synthesis
inhibitor,
a-difluoromethylornithine (DFMO), used in order to
obtain polyamine depletion (Fig. 2D), suggesting that the poly-
amine transport system is not significantly involved in the action of
SHSY-5Y neuroblastoma

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A. Minarini et al. / European Journal of Medicinal Chemistry 67 (2013) 359e366
4. In fact, spermine should decrease 4 internalization by competi-
tion, whereas DFMO, an inhibitor of ornithine decarboxylase,
should increase the entry of 4, since polyamine depletion activates
the transport system [23]. Compound 4 could enter by simple
diffusion [24] and/or by endocytosis [25], that may be facilitated by
the lipophilicity of the long polymethylene chains and of the ter-
minal groups. Actually, cell survival of 4-treated cells was signifi-
cantly increased when the cells were incubated in a medium in
which the pH was adjusted to 6.4 with HCl, instead of 7.2, in order
to increase the positive net charge of nitrogen atoms that is ex-
pected to decrease the cross of the plasma membrane by diffusion.
Altogether, these data suggest a picture in which compound 4 en-
ters into the cell by diffusion and triggers an oxidative insult,
possibly by interference with mitochondrial function.
2.4. Compound 4 rapidly activates autophagy
Mitochondrial damage and oxidant production are often asso-
ciated with activation of apoptosis, hence we determined the
activation of caspases acting on the substrate Asp-Glu-Val-Asp
(DEVD), i.e. mainly effector caspases 3 and 7, whose activation is
characteristic of apoptotic cell death [26]. The polyamine analog
DENSPM was used as positive control [20]. The cells were treated
for 24 h with 25 mM DENSPM or 2 mM compound 4 (these treat-
ments reduced cell survival by 50e70%). DENSPM significantly
increased caspase activity (Fig. 3A), as expected. On the contrary,
compound 4 did not activate caspases. Furthermore, following DAPI
staining for nuclear morphology, in 4-treated cells it was not
possible to detect the nuclear characteristics of apoptosis [27], i.e.
chromatin condensation, nuclear fragmentation and/or condensa-
tion (not shown). We can conclude that compound 4 kills HeLa cells
in a caspase-independent manner. Actually, cell survival was not
affected by the caspase inhibitor Z-VADfmk (Fig. 3B). Further, the
effect of 4 was not influenced by necrostatin-1 that can discrimi-
nate necroptosis, a different form of cell death [28].
Autophagy is another emerging process that may be associated
to a programmed form of cell death and can be activated by tumor
chemotherapy [28e30]. The natural polyamine spermidine
reportedly activates autophagy [31], so we verified whether cell
death induced by the tetramine compounds used in this study was
linked to autophagy. Transmission electron microscopy (TEM)
analysis of 4-treated HeLa cells (Fig. 3C) demonstrated the presence
of several vacuoles in the cytoplasm, often containing high-
electron-density substances. At higher magnification these vacu-
oles showed organic material inside, characteristic of the auto-
phagocytosis process during autophagy (Fig. 3C, images IIIeV).
Development of acidic vesicular organelles (AVOs) is a typical
feature of autophagy, that indicates the maturation of autopha-
gosomes. Acridine orange (AO) is a marker of AVOs, that fluoresces
green in the whole cell but it is taken up in acidic compartments
(mainly late autophagosomes), where it fluoresces red. HeLa cells
Fig. 3. Compound 4 does not trigger apoptosis, but activates autophagy in HeLa cells.
(A) Caspase (DEVDase) activity was measured in cells treated for 24 h with 25
m
M
DENSPM or the indicated concentration of compounds 4. Data are means ꢀ S.E.M. of
three replicates, *P < 0.05 vs. control cells. (B) Cytotoxicity of 4 was determined in cells
pretreated for 2 h with Z-VADfmk (50
are means ꢀ S.E.M. of three determinations. (C) Representative transmission electron
micrographs showing the ultrastructure of control HeLa cells (panel I, bar 5 m) and of
cells treated with 2 M 4 for 24 h (panels IIeIV). Many vacuoles are detectable in the
cytoplasm of 4-treated cells (II, bar 5 m). Higher magnification of vacuoles reveals
mM), or with necrostatin-1 (NEC-1, 20 mM). Data
m
m
m
that organic material is detectable inside (III, bar 1
mitochondrion entrapped inside a vacuole.
mm). Panel IV (bar 200 nm) shows a
examined LC3-I and LC3-II levels by Western blotting. As shown in
Fig. 4B, LC3-I was largely converted into LC3-II, whose level was
greatly increased during 3 h following the treatment with com-
pound 4. Again, NAC prevented the processing of LC3-I. LC3 pro-
cessing was evident even in the presence of bafilomycin A (not
shown). This observation, together with the formation of AVOs,
indicates the persistence of the autophagic flux. Altogether, these
data show that 4 rapidly activated autophagy in HeLa cells,
apparently as a consequence of an oxidative event, since it is
blocked by NAC.
Autophagy represents a double-edged sword for cell survival
because it acts as a prosurvival mechanism, mainly in adverse
conditions of cellular stress, but it is generally thought that the
process has a self-limiting character and leads to autophagic cell
death when excessive [29,30]. However, this view is not universally
accepted. Other researchers hypothesized that autophagy is only a
protective mechanism and not cause of cell death [33], so the
precise role of autophagy in cell death is still debated [34]. Actually,
were treated with 4 (2
mM) and incubated for various times,
afterward the cells were stained with AO and analyzed by fluo-
rescence microscopy. As soon as 3 h, it resulted evident the
appearance of orangeered fluorescence in a large number of cells,
indicative of the formation of acidic vesicles (Fig. 4A). The forma-
tion of AVOs was inhibited when the HeLa cells were pretreated
with the antioxidant NAC.
To further obtain a biochemical evidence for autophagy, we
determined the change in the expression level of LC3 protein. LC3
exists in two forms, an 18-kDa cytosolic protein (LC3-I) that during
autophagy is processed to a membrane-bound 16-kDa form con-
jugated with phosphatidylethanolamine (LC3-II). The lipidated
LC3-II increases following activation of the autophagic pathway by
conversion from LC3-I, and is a hallmark of autophagy [32]. We
several reports highlight
a tumor-killing role of autophagy
following anticancer treatment or, at the opposite, a protective role
of autophagy against chemotherapy [28e30]. So we wondered how
4-triggered autophagy can affect cell death. To address this

A. Minarini et al. / European Journal of Medicinal Chemistry 67 (2013) 359e366
363
2.5. Autophagy is activated by other polyamine derivatives
Fig. 5A shows that autophagy was rapidly activated within 3 h
even by compounds 2 and 3 at toxic concentrations (20 M),
m
indicating that induction of autophagy is a common feature of
cytotoxic 1-derivatives and seems to be associated with their
polyamine structure and not with the character of terminal groups.
Also the natural polyamine spermidine has been reported to acti-
vate autophagy [31]. In order to compare the effect of spermidine
with that of compound 4, we treated HeLa cells with increasing
concentrations of spermidine: to observe LC3-I processing, it was
necessary to incubate the cells for at least 24 h with a very high
spermidine concentration (8 mM), that elicits toxic effect [36]. On
the other hand, the polyamine analog DENSPM caused a very
limited LC3-I processing, even at toxic levels. The experiment
depicted in Fig. 5B compares the effect of polyamine compounds at
concentrations that reduce cell survival to 30e50% on LC3-I
processing.
To date, in the field of polyamine analogs, autophagy activation
was reported only in the case of an anticancerenorspermine con-
jugate in which the unique role of the norspermine moiety was to
facilitate the import of the cytotoxic part of the molecule into the
cell by means of the polyamines transport system [37]. Differently,
our data show that the ability of the cytotoxic disubstituted long
chain polymethylene tetramines to activate autophagy is correlated
primarily to their polyamine backbone rather than to the terminal
Fig. 4. Characteristics of autophagy activated by compound
Immunofluorescence microscopy of acridine orange-stained HeLa cells treated for 3 h
with DMSO (ctrl) or 2 M 4. Increase in number of cells with AO accumulating acidic
4 in HeLa cells. (A)
m
vesicular organelles (orangeered fluorescence) in 4-treated cells was inhibited by
pretreatment (2 h) with 5 mM NAC. Similar results were observed in two independent
experiments. (B) Western blot analysis of LC3-I and LC3-II expression in cells treated
with 2 mM 4 for 3 h in the presence or absence of 5 mM NAC. The image is repre-
sentative of multiple independent experiments. (C) Untransfected cells and cells
transfected with Atg5-siRNA or control siRNA were incubated for 24 h in the absence
or presence of 2 mM 4. The extent of cell death and the accumulation of acidic vescicles
were then measured. Values are means ꢀ S.E.M. of triplicate determinations, *P < 0.05.
The blot shows Atg5 expression and is representative of 3 experiments.
question we thought to perform experiments in the presence of
autophagy inhibitors. Experiments done by using pharmacological
inhibitors of signal transduction pathways involved in autophagy
activation, such as inhibitors of class III phosphoinositide 30-kinase
(PI3K) like 3-methyladenine, are widely reported in literature.
Since in our assays we observed that these compounds were highly
toxic by themselves to HeLa cells, hampering their use to study cell
survival, a siRNA approach was used. The cells were transfected
with a siRNA targeting Atg5, characterized as part of an ubiquitin-
like conjugation systems specifically required for autophagy [35],
whose silencing is frequently used to inhibit autophagy. The cells
transfected with Atg5 siRNA showed a level of the Atg5 protein
reduced of about 60%, associated with a significant inhibition of
acidic vesicles accumulation after treatment with 4, when
compared with cells transfected with control siRNA (Fig. 4C). In-
hibition of autophagy apparently resulted in a slight attenuation of
the cytotoxic effect of compound 4, but the difference was not
statistically significant (P ¼ 0.12). Our results suggest that auto-
phagy activation is not the main cause of cell death but, on the
other hand, autophagy does not ever protect HeLa cells from 4-
induced cytotoxicity. We suggest that autophagy is activated as a
defense mechanism in response to the oxidative insult triggered by
4, but the effect of 4 is so rapid and powerful that equally leads to
cell death.
Fig. 5. Activation of autophagy by polyamine compounds in HeLa cells. (A) The cells
were incubated for 3 h in the presence of compounds 2 or 3 (20
mM) afterward,
accumulation of acidic vesicles and LC3 expression/processing were examined. Values
are means ꢀ S.E.M. of triplicate measurements. (B) HeLa cells were incubated for 24 h
in the presence of polyamine compounds at concentrations that reduced cell survival
by 50e70%: compounds 4 (2 mM) or spermidine (Spd, 8 mM) or DENSPM (25 mM); cell
survival and LC3 expression/processing were then examined. Values are as
means ꢀ S.EM. of three determinations.

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A. Minarini et al. / European Journal of Medicinal Chemistry 67 (2013) 359e366
aromatic groups. Possibly, the main role of the terminal sub-
stituents and the methylene chain length of the compounds used in
the present study is to facilitate the internalization and the locali-
zation of the molecule inside the cell.
mixture was stirred for 4 h at room temperature. Ether (300 ml)
was then added, yielding a solid, which was filtered, washed with
ether (3 ꢂ 20 ml) and crystallized from EtOH/i-PrOH to a white
solid. The hydrobromide salt was dissolved in water (30 ml) and the
solution was made basic with 2 N NaOH (40 ml). The precipitated
product was separated by continuous extraction with CHCl3
(200 ml) for 14 h. The organic phase was dried and evaporated to
give compound 6 in quantitative yield, mp 199e202 ꢁC, 1H NMR
2.6. Conclusions
In conclusion, we have shown that, by appropriate modifica-
tions of the methoctramine structure, it is possible to design
polyamine derivatives, highly cytotoxic against tumor cells. The
most potent compound 4 triggers an oxidative insult leading to a
caspase-independent form of cell death associated with the rapid
onset of autophagy. Autophagy is a process for recycling cellular
constituents that can be associated with either cell death or cell
survival and is implicated in several pathological and physiological
processes [38]. In our opinion the most relevant finding of this work
is that the design of polyamine-like structures, bearing appropriate
molecular features, leads to powerful inducers of autophagy.
(200 MHz, CD3OD) d 1.32e1.49 (m, 24H), 1.64e1.70 (m, 8H), 2.23 (t,
4H, J ¼ 7.2), 2.95 (t, 4H, J ¼ 7.5), 3.17 (t, 4H, J ¼ 6.7).
3.1.3. Synthesis of N,N0-(dodecane-1,12-diyl)bis(6-(2,2-
diphenylacetamido)hexanamide) (7)
6 (500 mg,1.17 mmol) in dry CH2Cl2 (10 ml) was added dropwise
to a stirred and cooled (0 ꢁC) solution of 2,2-diphenylacetyl chloride
(590 mg, 2.6 mmol) and Et3N (0.49 ml) in dry CH2Cl2 (20 ml). The
mixture was stirred at room temperature overnight and then
washed with water and NaHCO3 saturated solution. After the
evaporation of the solvent, the crude material was purified by flash
chromatography using as eluent a mixture of CH2Cl2/MeOH (9:1).
3. Materials and methods
20% yield, 1H NMR (200 MHz, DMSO)
d 1.22e1.34 (m, 18H), 1.41e
3.1. Chemistry
1.48 (m, 14H), 1.99 (t, 4H, J ¼ 7.4), 2.94e3.06 (m, 8H), 4.90 (s, 2H),
7.16e7.26 (m, 16H), 7.67e7.72 (m, 2H), 8.21e8.27 (m, 2H).
All the synthesized compounds have a purity of at least 95%
determined by elemental analysis. Uncorrected melting point was
taken in glass capillary tubes on a Buchi SMP-20 apparatus. ESI-MS
spectra were recorded on Perkin Elmer 297 and Waters ZQ 4000. 1H
NMR and 13C NMR were recorded on Varian VRX 200 and 400 in-
struments. Chemical shifts are reported in parts per million (ppm)
relative to peak of tetramethylsilane (TMS) and spin multiplicities
are given as s (singlet), br s (broad singlet), d (doublet), t (triplet), q
(quartet) or m (multiplet). IR spectral data were consistent with the
assigned structures. The elemental analysis was performed with
Perkin Elmer elemental analyzer 2400 CHN. From all new com-
pounds satisfactory elemental analyses were obtained, confirming
>95% purity. Chromatographic separations were performed on
silica gel columns by flash (Kieselgel 40, 0.040 e 0.063 mm, Merck)
column chromatography. Reactions were followed by thin layer
chromatography (TLC) on Merck (0.25 mm) glass-packed precoated
silica gel plates (60 F254) and then visualized in an iodine chamber
or with a UV lamp.
3.1.4. Synthesis of N,N0-(dodecane-1,12-diyl)bis(N6-(2,2-
diphenylethyl)hexane-1,6-diamine) (4)
A solution of 2 M BACH-EI inTHF (1.16 ml) was added dropwise to
a room temperature solution of 7 (237 mg, 2.9 mmol) in dry diglyme
(10 ml) under a stream of dry nitrogen. When the addition was
completed, the reaction mixture was heated at reflux temperature
for 5 h. After cooling at room temperature, excess borane was
destroyed by cautious dropwise addition of water (12 ml) and 6 N
HCl (12 ml). The resulting mixture was then heated at reflux tem-
perature for 4 h. After solvent evaporation, the crude product was
washed with Et2O/EtOH (5:1), filtered and the solid was purified by
flash chromatography using as eluent a mixture of CHCl3/MeOH/
aqueous ammonia solution 33% (from 8:2:0.05 to 7:3:0.4). 36%
yield; 1H NMR (200 MHz, CD3OD)
d 1.28e1.41 (m, 24H), 1.65e1.77
(m,12H), 2.95e3.01 (m, 8H), 3.03e3.07 (m, 4H), 3.77 (d, 4H, J ¼ 4.0),
4.51 (t, 2H, J ¼ 3.9), 7.21e7.26 (m, 4H), 7.29e7.40 (m, 16H); 13C NMR
(100 MHz, CD3OD) d 26.5, 27.1, 27.6, 28.8, 30.3, 30.6, 33.7, 45.7, 48.0,
Compounds 1e3 have been synthesized as previously reported
52.9, 128.6, 129.1, 130.2, 141.7; MS (ESIþ) m/z 760 (M þ H)þ.
[16,17]. The newly designed compound
4 was synthesized
following the general procedure developed by our research group
as follows (see Scheme 1):
3.2. Cell culture, treatments, and data analysis
HeLa, HT29, LOVO, Saos-2, and U2OS cells were cultured in
EMEM containing 10% fetal bovine serum, 1% glutamine, 1% non-
essential amino acids, 1 mM pyruvate, and antibiotics. HL60 and
SH-SY5Y cells [14], and human chondrocytes C-28/I2 [39] were
cultured as previously described. Primary coltures of rat thymo-
cytes were prepared as reported [40]. All cells types were routinely
maintained at 37 ꢁC in a humidified atmosphere containing 5% CO2.
All tested compounds were dissolved in DMSO and added to cell
cultures (0.1% with respect to the total volume) in order to obtain
the required concentration in the medium. Control cells received
the corresponding volume of DMSO.
The presented experiments were performed at least three times.
The results are expressed as means ꢀ S.E.M. for the data obtained in
the indicated numbers of independent experiments. In each
experiment every point was done in duplicate and every point was
assayed in duplicate. In some cases, results obtained in one repre-
sentative experiment are shown. When statistical analysis was
applicable, data were compared by Student t-test. Differences were
considered significant for P < 0.05. Data analysis was performed by
the GraphPad Prism 5 software.
3.1.1. Synthesis of dibenzyl ((dodecane-1,12-diylbis(azanediyl))
bis(6-oxohexane-6,1-diyl))dicarbamate (5)
Ethyl chloroformate (1.56 ml, 16.4 mmol) in dry dioxane (50 ml)
was added dropwise to a stirred and cooled (0 ꢁC) solution of
6-(((benzyloxy)carbonyl)amino)hexanoic acid (4.35 g, 16.4 mmol)
and Et3N (2.29 ml, 16.4 mmol) in dioxane (30 ml), followed after
standing for 30 min by the addition of 1,12-diaminododecane (1.64 g,
8.2 mmol) in dioxane (20 ml). After the mixture was stirred at room
temperature for 18 h, it was poured into water (100 ml), and the
white solid was filtered, washed with NaHCO3 saturated solution,
HCl 2 N, i-PrOH, Et2O and crystallized from DMSO to give the
intermediate 5 as white solid. 84% yield, mp 158e161 ꢁC, 1H NMR
(200 MHz, DMSO)
2.99 (m, 8H), 4.98 (s, 4H), 7.31e7.34 (m, 8H), 8.66e8.68 (m, 2H).
d
1.22e1.45 (m, 32H), 2.00 (t, 4H, J ¼ 7.2), 2.94e
3.1.2. Synthesis of N,N0-(dodecane-1,12-diyl)bis(6-
aminohexanamide) (6)
solution of 30% HBr in acetic acid (43 ml) was added to a solu-
tion of 5 (5.13 g, 7.1 mmol) in acetic acid (70 ml), and the resulting

A. Minarini et al. / European Journal of Medicinal Chemistry 67 (2013) 359e366
365
3.3. Peroxide levels and mitochondrial membrane potential
MIUR, Rome (PRIN projects), and by the University of Bologna (RFO
funding).
Mitochondrial membrane potential (DJm) and intracellular
peroxide levels were measured in the whole cells by fluorescent
probes as described [41]. Fluorescence was analyzed by flow
cytometry with logarithmic amplification. Flow cytometric analysis
was performed with a Beckmann Coulter Epics XL MCL cytometer
(USA) equipped with a 15 mW argon ion laser. To measure DJm,
mitochondria in intact cells were probed with the potential-
sensitive dye DiOC6. After the treatment, cells were incubated
with medium containing 4 nM DiOC6 for 40 min at a cell concen-
tration of 1 ꢂ 106 cell/mL at 37 ꢁC in the dark. Cells were counter-
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