Long-chain aminoalcohol and diamine derivatives induce apoptosis through a caspase-3 dependent pathway

Article abstract of DOI:10.1016/S0960-894X(02)00476-6

A number of long chain diamines and aminoalcohols and several of their alkyl, acyl and carbamoyl derivatives, have been synthesized and evaluated for their apoptotic activities using the Jurkat cell line. Apoptosis was measured by flow cytometry and the best results were found for the aminoalcohols displaying either a free alcohol or an amine with at least, one free hydrogen atom. The apoptotic pathway was mediated by a disruption of the mitochondria transmembrane potential and caspase-3 activation, inducing DNA fragmentation at the phase G₁/S of the cell cycle.

Full text of DOI:10.1016/S0960-894X(02)00476-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2621–2626
Long-Chain Aminoalcohol and Diamine Derivatives Induce
Apoptosis through a Caspase-3 Dependent Pathway
Esther del Olmo,a,* Antonio Macho,b Mario Alves,a Jose L. Lopez,a Fadwa el Banoua,b
Eduardo Munozb and Arturo San Felicianoa
a
´
Departamento Quımica Farmaceutica, Facultad de Farmacia, Univ. de Salamanca, 37007-Salamanca, Spain
´
Departamento Biologıa Celular, Fisiologıa e Inmunol, Fac. Medicina, Avda. Menendez Pidal s/n, 14004 Cordoba, Spain
b
´
´
´
Received 5 February 2002; revised 8 May 2002; accepted 17 June 2002
Abstract—A number of long chain diamines and aminoalcohols and several of their alkyl, acyl and carbamoyl derivatives, have
been synthesized and evaluated for their apoptotic activities using the Jurkat cell line. Apoptosis was measured by flow cytometry
and the best results were found for the aminoalcohols displaying either a free alcohol or an amine with at least, one free hydrogen
atom. The apoptotic pathway was mediated by a disruption of the mitochondria transmembrane potential and caspase-3 activation,
inducing DNA fragmentation at the phase G1/S of the cell cycle. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
hyperproduce reactive oxygen species (ROS) which may
contribute to the cell death process. It has been shown
that these ROS are derived from the complex III of the
mitochondrial respiratory chain.6 Although less clear,
there are also other ways of apoptosis described, that
did not imply the mitochondria directly, for example via
granzime B or the direct activation of signaling cas-
pases, such as caspase-8, that leads to the direct activa-
tion of executor caspases without the requirement of
cyt-c release and apoptosome formation, even though a
ÁÉm breakdown occurs secondarily.7 Also, in some
cases, ROS production can precede and cause the ÁÉm
breakdown and the later cell degradation. These ROS
are mainly of extramitochondrial origin.8
Apoptosis, or programmed cell death (PCD), is a nat-
ural form of cell death controlled by a constitutively
expressed machinery that induces condensation of
nucleoplasm and cytoplasm, blebbing of cytoplasmic
membranes and fragmentation of the cell into ‘apopto-
tic bodies’, which are rapidly recognized and eliminated
by adjacent cells.1 According to current understanding,
morphological and biochemical alterations in nuclear
and chromatin structures of cells undergoing apoptosis
are controlled mainly by the mitochondria.2 Thus, a
breakdown of the mitochondrial transmembrane
potential (ÁÉm) is an invariant feature of early apopto-
sis that precedes DNA strandbreak, regardless the cell
type and the apoptotic stimuli.3 ÁÉm disrupture medi-
ated the release of cytochrome-c and the (so-called)
apoptotic inducing factor (AIF), which can mediate
nuclear fragmentation.4 The release of cytochrome-c
(cyt-c) into the cytoplasm results in formation of the
apoptosome, leading to activation of final executor cas-
pases, such as caspase-3, responsible for the morpholo-
gical and biochemical features of apoptosis, through the
activation of multiple cytoplasmic and nuclear
enzymes.5 After the initial ÁÉm dissipation, cells
Compounds inducing apoptosis in cancer cells, con-
stitute a promising way to develop new anticancer
drugs. We can find several biological activities reported
in the literature for lipidic aminoalcohols an diamines.
Among them, their anti-inflammatory activity,9 their
cytotoxicity for neoplastic cells10 and their binding to
DNA in concentration levels similar to that shown by
doxorubicin,11 are the most relevant aspects in relation
with the research described in this paper, which
attempts to establish the mechanism of cytotoxicity for
this type of compounds. Thus, we now report the results
of our studies on the apoptosis induced by several long
chain aminoalcohol and diamine derivatives in tumoral
cells. The apoptotic pathway induced by the compounds
*Corresponding author. Tel.: +34-923-294500x1825; fax: +34-923-
294515; e-mail: olmo@usal.es
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00476-6

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E. del Olmo et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2621–2626
assayed is mediated in all the cases through the disrup-
tion of the mitochondrial permeability transition and
the activation of caspase-3.
To evaluate Ácm and ROS generation cells were incu-
bated with 20 nM DiOC6(3),17 which accumulates in the
mitochondria as a function of its potential and 2 mM
dihydroethidine (HE), fluorescent after oxidation fol-
lowed by cytofluorimetric analysis.
Chemistry
To determine both DNA strand breaks (apoptosis) and
cell cycle, cells were subjected to paraformaldehyde fix-
ation and TUNEL staining, followed by counterstain
with propidium iodide (PI) and a treatment with
RNAse as described.18 Caspase-3 activity was studied
by incubating 30 mg of cytoplasmic proteins from cell
lysates with 15 mg of the specific caspase-3 substrate Ac-
DEVD-AMC, following the manufacturer’s indica-
tions.19
The synthetic pathways are summarized in Schemes 1
and 2 and the compounds prepared are shown in Tables
1–3. Details of the reactions, experimental conditions
and characterization of the products, will be reported in
a complete publication. As a representative preparation,
Boc-aminoacid 1, prepared from diethyl 2-acet-
amidomalonate and 1-bromotetradecane,12 was trans-
formed into a mixed anhydride, and reduced by NaBH4
to give Boc-aminoalcohol13 2c (R1=R3=H, R2=Boc).
The alcohol group was protected as benzyl ether (2b),
and the amine properly functionalized to give secondary
amines. Treatment with 1.2 equivalents of ethyl bro-
mide, n-hexyl bromide or ethyl bromoacetate yielded
compounds 2f, 2h and 2l respectively, and with one
mole of glutaric or succinic anhydrides provided com-
pounds 2n and 2o, respectively. The tertiary amine
derivative 2q was obtained by treatment with 2.2
equivalents of the alkylating reagent. Elimination of the
benzyl group by hydrogenolysis gave the free alcohols
2a, 2e, 2g, 2k, 2m and 2p. Compound 2l, by saponifica-
tion with 10% KOH/MeOH, gave the free acid 2j and
its debenzylation led to the unprotected aminoalcohol
2i.
Results and Discussion
In preliminary experiments, using combined cytofluori-
metric techniques to discriminate both necrosis and
apoptosis (data not shown), we determined that cell
death induced in Jurkat cells by the compounds tested
was due to apoptosis, in the range of the concentrations
studied.
To study structure–activity relationships for our com-
pounds, the apoptosis induced by them in this cell line
after 24 h of stimulation was measured by cell cycle
analysis after PI staining of DNA in untreated or trea-
ted cells. The percentage of hypodiploidy was used as
indicator of cell death by apoptosis (cells with frag-
mented DNA). The results are indicated in Tables 1–3
as IC50 values, calculated from experiments with
increased concentrations of the compounds from 0.1 to
100 mM.
Diamines 3 were prepared from the aminoalcohol 2c by
mesylation, substitution with sodium azide and reduc-
tion with NaBH4 in the presence of Pd/C, to give the
Boc-diamine 3b,14 which through alkylation/acylation
as above indicated, followed (or not) by Boc-deprotec-
tion, gave the diamines and diamine derivatives 3. Acy-
lation of compound 2c with heptanoyl chloride or
palmitoyl chloride provided the corresponding ester
derivatives, which after Boc deprotection and new acy-
lation gave compounds 2r, 2s and 2t.
It can be seen from data in Table 1, that all three types
of compounds, primary (2) and secondary (4) amino-
alcohols and ethylenediamines (3), contain representa-
tive substances inducing apoptosis.
Aminoalcohols 4, were obtained from 1-hexadecene by
epoxidation with m-chloroperbenzoic acid and sub-
sequent opening of the oxirane ring by ammonium
hydroxide, or cyclohexyl or benzylamines.
Within the series of primary aminoalcohols 2, it can be
noted, that the acylation of the amino group (2a, 2b/2m,
2n) and the diacylation of both the amino and the alco-
hol functions (2r, 2s, 2t) either decreased or abolished
the apoptotic activity, respectively. On the other hand,
monoalkylation of the amino group left the potency
practically unchanged (2a/2e), while dialkylation (2a/2p)
led to the most potent compound of this series. Related
to the benzylation of the hydroxyl group, it influences
differently the apoptotic capacity of these compounds
depending on the degree of substitution of the amine. A
small increase of the potency or no influence is observed
Biological Assays
Jurkat cells (T-lymphoma cells) were used for all biolo-
gical determinations.15 The percentage of cells under-
going
apoptosis
was
determined
by
flow
cytofluorimetric analysis of the cell cycle.16
Scheme 1. Synthesis of lipidic aminoalcohols and diamines and their derivatives (2 and 3). R=(CH2)13CH3; (i) (a) EtOOCCl/N-methylmorpholine/
THF; (b) NaBH4/MeOH; (c) BnCl/NaH/DMF; (d) HCl/THF/argon; (ii) EtBr (1.1 or 2.2 equiv) or EtOOCCH2Br (1.1 equiv)/Et3N/DMF; (iii)
glutaric anhydride/EtOAc; (iv) H2/Pd/C/AcOH; (v) 10% KOH/MeOH; (vi) (a) MsCl/Et3N/CH2Cl2; (b) NaN3/DMF; (c) NaBH4/MeOH and Pd/C/
AcOH; (vii) EtBr (1.1 or 2.2 equiv) or EtOOCCH2Br (1.1 or 2.2 equiv)/Et3N/DMF; (viii) n-C6H13COCl or n-C15H31COCl/Et2O.

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2621–2626
2623
The trisubstituted diamines are practically inactive (3m,
3q) and the most potent apoptotic compounds contain
one (3b) or two (3d, 3k) substituents on the nitrogen
atoms. Interestingly and, in contrast with the case of
aminoalcohols, carbamoylation (acylation) of the
amino group attached to a secondary carbon atom in
the aliphatic chain, seems to enhance (3a/3b) rather than
decrease (2a/2c) or remove (2r, 2s, 2t) the activity.
Scheme 2. Synthesis of lipidic aminoalcohols 4: R=(CH2)13CH3; (i)
m-CPBA/CH2Cl2/NaHCO3; (ii) H2N-R1.
Table 1. Structures of aminoalcohols 2 and their apoptosis induction
values
To investigate the apoptotic pathway induced by these
compounds, we selected for further studies one repre-
sentative compound from each group, namely the
aminoalcohols 2a and 4b and the diamine 3a. Jurkat
cells were treated with increasing concentrations of
these compounds for 24 h and the percentage of sub-
diploid cells (DNA fragmented cells) determined by PI
staining. Values are meansꢀSE of three independent
experiments (Fig. 1).
2
R1
R2
R3
IC50 mM
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
H
Bn
H
Bn
H
Bn
H
Bn
H
Bn
H
Bn
H
Bn
Bn
H
H
Boc
Boc
Et
Et
Hex
Hex
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Et
Et
H
H
H
7
3
28
nt
6
6
6
Next, we investigated the possible role of the mito-
chondria in the apoptotic pathway induced by these
compounds. Jurkat cells were treated for 6 h with a dose
of each compound that represents the IC50 for apoptosis
induction, and the loss of Ácm detected by flow cyto-
metry. We show in Figure 2A a representative experi-
ment carried out with compound 2a, inducing a clear
loss in the mitochondrial transmembrane potential
(53% of the cells) compared to untreated cells (<1%).
Interestingly, 22% of the cells having low DiOC6(3)
staining, presented also a significant increase in the
amounts of intracellular ROS.
nt
nt
3
nt
nt
24
>50
30
1
20
na
na
na
CH2COOH
CH2COOH
CH2COOEt
CH2COOEt
CO(CH2)3COOH
CO(CH2)3COOH
CO(CH2)2COOH
Et
H
Bn
Et
C6H13CO
C6H13CO
C14H29CO
C6H13CO
C14H29CO
C14H29CO
s
t
R=–(CH2)13CH3; nt=not tested; na=not active.
Taking into account that the dissipation of mitochon-
dria transmembrane potential precedes the DNA frag-
mentation (data not shown), we could assume that the
mitochondria plays a key role in the apoptotic pathway
induced by aminoalcohols and diamines. Thus, pre-
treatment of Jurkat cells with rotenone, a good inhibitor
of the complex I of the mitochondrial respiratory
chain,22 clearly reduced the percentage of (HE!Eth)high
cells, showing that the ROS generation induced by this
compounds are from a mitochondrial source (Fig. 2A).
These results are in agreement with observations indi-
cating that, in most cases of apoptosis, ROS are origi-
nated at complex III of the mitochondria.6 It seems
likely that the mechanism of the pre-apoptotic ÁÉm
dissipation is mediated by so-called ‘permeability tran-
sition (PT) pores’, a step that can be transiently blocked
by Cyclosporin A (CsA), which binds to cyclophillin-D
present in the mitochondria pores and abrogates the PT
opening mediated by some apoptotic stimuli.23 This
holds true also for the ÁÉm breakdown induced by the
compound 2a, since, as it is shown in Figure 2A, pre-
treatment of Jurkat cells with CsA prevented the
aminoalcohol-induced ÁÉm disruption.
in the cases of the free amine (2a/2b) or monoalkyl-
amine (2e/2f), respectively, but a 20-fold decrease is
provoked in the case of dialkylamine (2p/2q). These
results mean that, at least, one of the three labile
exchangeable hydrogen atoms of the molecule should be
retained to maintain the apoptotic activity.
In the cases of secondary aminoalcohols 4 and diamines
3 (Tables 2 and 3) the number of structural variations
covered are fairly lesser than those that could be possi-
ble by changing the substituents on the two heteroatoms,
thus a complete structure–activity analysis cannot be
performed. Nevertheless, both types of compounds seem
to display levels of activity similar to those of primary
aminoalcohols and also to share a similar behaviour.
Table 2. Structures of aminoalcohols 4 and their apoptosis induction
values
The caspase family consists of postaspartate-cleaving
cysteine proteases that have been shown to be required
for apoptosis in a number of experimental systems. The
loss of ÁÉm is followed by the release of cyt-c from the
mitochondria, formation of the apoptosome and acti-
vation of the executor caspase-3. To study the activa-
tion of this caspase in a direct way, we incubated the
4
R1
R2
IC50 mM
a
b
c
H
c-C6H11
Bn
H
H
H
nt
6
27
R=–(CH2)13CH3; nt, not tested; na, not active.

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E. del Olmo et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2621–2626
Table 3. Structures of diamines 3 and their apoptosis induction values
3
R1
R2
R3
IC50 mM
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
H
H
H
H
H
H
H
H
H
H
Et
Et
H
H
Et
Et
H
Boc
H
Boc
H
Boc
H
Boc
H
Boc
H
Boc
55
6
nt
6
CH2COOH
CH2COOH
CH2COOEt
CH2COOEt
CH2(CH2)3COOH
CH2(CH2)3COOH
Et
31
nt
na
28
55
18
7
21
na
nt
nt
32
>60
Et
Et
Et
CH2(CH2)3COOH
CH2COOH
CH2COOH
CH2COOEt
CH2COOEt
CH2COOH
CH2COOH
CH2COOEt
CH2COOEt
H
Boc
H
Boc
R=–(CH2)13CH3; nt, not tested; na, not active.
cells with the representative compounds 2a, 3a and 4b
for 6 h, a time at which the ÁÉm dissipation is evident
(Fig. 2A), and the activity of caspase-3 was determined
by a highly specific and sensitive fluorometric method.
Fig. 2B shows that the treatment of Jurkat cells with
these compounds led to the activation of this caspase in
a specific manner, since this activation was inhibited by
the caspase-3 specific inhibitor zDEVD.fmk as it is
shown for compound 2a.
Finally, to examine whether the aminoalcohol-induced
apoptosis in Jurkat cells is dependent on a specific phase
of the cell cycle, we performed double staining experi-
ments with PI and FITC-dUTP as described above.
With this technique the phase of the cell cycle when
Figure 1. Induction of apoptosis by aminoalcohols 2a and 3a and
diamine 4b.
Figure 2. (A) Aminoalcohol 2a induce ÁÉm disruption and ROS generation in Jurkat cells.20 (B) Aminoalcohols and diamines activate caspase-3 in
Jurkat cells.21

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2621–2626
2625
Figure 3. Cell cycle dependency for apoptosis induced by aminoalcohol 2a (10 mM) on Jurkat cells.24
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DNA fragmentation occurs can be established. Figure 3
shows that in Jurkat cells treated for 24 h with com-
pound 2a, most of the DNA fragmentation occurs in
the transition of the G1/S phase of the cell cycle. Thus,
apoptosis induced by this compound will be primarily
directed to growing cells, a fact which converts these
type of structures into interesting lead compounds for
the development of new anticancer drugs.
Acknowledgements
This work was supported by Junta de Castilla y Leon
(Grant N.: SA-25/00B) to E.O., and by a grant from the
Ministerio de Ciencia y Tecnologia 1FD97–0683-C05–
03 to E.M., and performed under the auspices of the
CYTED Program (Programa Iberoamericano de Cien-
cia y Tecnologıa para el Desarrollo), Subprogram X.
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15. Otherwise indicated, all reagents were from Sigma Che-
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16. For that, ethanol fixed cells were subjected to RNA
digestion and PI staining. With this method, low molecular
weight DNA leaks from the ethanol-fixed apoptotic cells and
the subsequent staining allows the determination of the per-
centage of subdiploid cells (sub-G0/G1 fraction).
17. 3,30-Dihexyloxacarbocyanine iodide, (Molecular Probes,
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fluorometer (Hitachi F-2500 model, Hitachi Ltd., Tokyo,
Japan) with an excitation wavelength of 380 nm and an emission

2626
E. del Olmo et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2621–2626
wavelength range of 400–550 nm. Data were collected as the
integer of relative fluorescence intensity minus the background
fluorescence. As a control, the cell-permeable caspase-3 inhi-
bitor zDEVD-fmk (Bachem, Bubendorf, Switzerland) was used.
20. Cells were pretreated or not for 1 h with either 10 mM
cyclosporin A (CsA) or 100 mM rotenone, followed by incu-
bation with 10 mM of compound 2a for 6 h, and then stained
with the potential sensitive dye DiOC6(3) and the ROS-oxid-
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obtained in biparametric histograms delimited by four com-
partments, namely, ÁÉhmigh (normal cells, bottom-right com-
partment); ÁÉmlow (bottom-left); (HE->Eth)high (ROS
generating cells, top-right), and (HE->Eth)high/ÁÉmhlow (pre-
apoptotic cells, top-left compartment). Results are representa-
tive of five independent experiments.
3 substrate Ac-DEVD-AMC for 1 h at 37 ꢁC. Results repre-
sent the integer of relative fluorescence intensity from the
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cence. The activation of caspase-3 by compounds 2a was
inhibited in the presence of zDEVD-fmk (20 mM). Values are
meansꢀSE of three independent experiments.
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then cell cycle: X-axis, G0/G1, S and G2/M phases separated
by vertical lines and the DNA strand breaks (Y-axis) analysed
by flow cytometry. Results are representative of three different
experiments.
21. Cells were treated with the IC50 apoptotic concentrations
of compounds 2a, 3a, and 4b for 6 h, lysed and 30 mg of pro-
tein from cell extracts were incubated with the specific caspase-