Synthesis and initial in vitro biological evaluation of two new zinc-chelating compounds: Comparison with TPEN and PAC-1

Article abstract of DOI:10.1016/j.bmc.2013.06.037

The lipophilic, cell-penetrating zinc chelator N,N,N′,N′,- tetrakis(2-pyridylmethyl) ethylenediamine (TPEN, 1) and the zinc chelating procaspase-activating compound PAC-1 (2) both have been reported to induce apoptosis in various cell types. The relations

Full text of DOI:10.1016/j.bmc.2013.06.037

Bioorganic & Medicinal Chemistry 21 (2013) 5175–5181
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and initial in vitro biological evaluation of two new
zinc-chelating compounds: Comparison with TPEN and PAC-1
O. Alexander H. Åstrand a, Gulzeb Aziz b, Sidra Farzand Ali b, Ragnhild E. Paulsen b, Trond Vidar Hansen a,
Pål Rongved a,
⇑
a School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, P.O. Box 1068, Blindern, N-0316 Oslo, Norway
b School of Pharmacy, Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068, Blindern, N-0316 Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
The lipophilic, cell-penetrating zinc chelator N,N,N0,N0,-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN,
1) and the zinc chelating procaspase-activating compound PAC-1 (2) both have been reported to induce
apoptosis in various cell types. The relationship between apoptosis-inducing ability and zinc affinity (Kd),
have been investigated with two new model compounds, ZnA-DPA (3) and ZnA-Pyr (4), and compared to
that of TPEN and PAC-1. The zinc-chelating o-hydroxybenzylidene moiety in PAC-1 was replaced with a
2,20-dipicoylamine (DPA) unit (ZnA-DPA, 3) and a 4-pyridoxyl unit (ZnA-Pyr, 4), rendering an order of
zinc affinity TPEN > ZnA-Pyr > ZnA-DPA > PAC-1. The compounds were incubated with the rat pheochro-
mocytoma cell line PC12 and cell death was measured in combination with ZnSO4, a caspase-3 inhibitor,
or a ROS scavenger. The model compounds ZnA-DPA (3) and ZnA-Pyr (4) induced cell death at higher con-
centrations as compared to PAC-1 and TPEN, reflecting differences in lipophilicity and thereby cell-pene-
trating ability. Addition of ZnSO4 reduced cell death induced by ZnA-Pyr (4) more than for ZnA-DPA (3).
The ability to induce cell death could be reversed for all compounds using a caspase-3-inhibitor, and most
so for TPEN (1) and ZnA-Pyr (4). Reactive oxygen species (ROS), as monitored using dihydro-rhodamine
(DHR), were involved in cell death induced by all compounds. These results indicate that the Zn-chelators
ZnA-DPA (3) and ZnA-Pyr (4) exercise their apoptosis-inducing effect by mechanisms similar to TPEN (1)
and PAC-1 (2), by chelation of zinc, caspase-3 activation, and ROS production.
Article history:
Received 10 April 2013
Revised 12 June 2013
Accepted 14 June 2013
Available online 26 June 2013
Keywords:
Apoptosis
Caspase-3 activation
PAC-1 analogues
ROS production
Zinc chelators
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
constant (Kd) of 0.26 fM.14 All six nitrogen atoms in TPEN coordi-
nate to zinc in a hexadentate manner.15 The procaspase-activating
The cellular apoptotic mechanism is a tightly regulated signal-
ing cascade that eventually leads to the activation of caspase-3,
the chief executioner caspase in the cell apoptotic pathway.1 Zinc
is the second most abundant transition metal in the human body
and is involved in the catalytic function and structural stability
of over 300 enzymes and proteins.2 Manipulation of the freely
available zinc has been shown to affect a great range of diseases
and conditions,3–5 and the relationship between depletion of labile
zinc and activation of caspase-3 is well described; zinc is an endo-
genic inhibitor of procaspase-3.6–9 The concentration of free zinc
varies in biological tissues depending on zinc buffering capacity.10
In PC12, HeLa, and HT-29 cell lines, as well as in primary cultures
of cardiac myocytes and neurons in vitro, the concentration of free
zinc has been determined to be approximately 5 nM.11 The lipo-
philic, cell-penetrating zinc chelator N,N,N0,N0-tetrakis(2-pyridyl-
methyl) ethylenediamine9,12,13 (TPEN, 1, Fig. 1) has a dissociation
compound PAC-1 (2, Fig. 1), described in 2006 as the first selective
procaspase-3-activating compound,16 has also zinc-chelating prop-
erties (Kd = 42 nM).4 The zinc-chelating property of PAC-1 is attrib-
uted to the o-hydroxybenzylidene-hydrazide moiety.4,17,18
Compounds that activate caspase-3 through mechanisms other
than chelation of zinc have also been reported.19
We hypothesized that introduction of a zinc-chelating function
in PAC-1 resulting in a lower Kd than 42 nM would reflect an in-
creased ability to induce cell death in an in vitro model cell line.
With this in mind, the phenolic moiety in PAC-1 was replaced with
the well described zinc-chelating unit 2,20-dipicolylamine (DPA) in
ZnA-DPA (3, Fig. 1), while keeping the hydrazide moiety intact.
Structural variations of the DPA moiety in combination with a
fourth or fifth donor atom, has been reported to afford lipophilic
and selective zinc chelators,20–22 displaying Kd values in the range
1–10 nM. Next, in ZnA-Pyr (4, Fig. 1), the phenolic moiety in PAC-1
was replaced with a 4-pyridoxyl group.23–26 A 4-pyridoxyl moiety
also comprising the o-hydroxybenzylidene-hydrazide moiety has
an estimated Kd with zinc of approximately 200–300 fM,23 an order
⇑
Corresponding author. Tel.: +47 22855024; fax: +47 22855947.
E-mail address: pal.rongved@farmasi.uio.no (P. Rongved).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.037

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O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 21 (2013) 5175–5181
Figure 1. Overview of the compounds in this study.
of magnitude lower than for ZnA-DPA. Consequently, the antici-
pated order of zinc affinity for the four test substances is TPEN > Z-
nA-Pyr > ZnA-DPA > PAC-1.
PAC-1, while keeping the rest of the molecule intact. However,
these molecular modifications did lead to a significant reduction
in logD7.4 by
a factor of roughly two for ZnA-DPA (3)
As lipophilicity affects cell membrane penetration, we calcu-
lated the logP-values for the model compounds and compared
them to the literature values for TPEN and PAC-1. TPEN is an indu-
cer of apoptosis involving ROS.12 However, it is to our knowledge
not reported whether the apoptotic effects of PAC-1 involves
ROS. Thus, the aims of the present study were twofold: 1) to inves-
tigate the ability of ZnA-DPA (3) and ZnA-Pyr (4) to induce cell
death and caspase-3 activation compared to TPEN (1) and PAC-1
when the zinc-chelating o-hydroxybenzylidene-hydrazide moiety
in PAC-1 was exchanged with two metal-chelating functionalities
with lower Kd. 2) to investigate if cell death induced by TPEN (1),
PAC-1, ZnA-DPA (3) or ZnA-Pyr (4), correlated to other parameters
such as zinc concentration, caspase-3 activation, or ROS
production.
(logD7.4 = 1.40) and ZnA-Pyr (logD7.4 = 1.27) as compared to PAC-
1 (logP = 3.43).27 The increased zinc-binding affinity of ZnA-DPA
(3) and ZnA-Pyr compared to PAC-1, as expressed by reduced Kd,
will therefore be offset by the reduction in lipophilicity, since smal-
ler portion of the chelators will be able to cross the cell membranes
and affect the cell zinc homoeostasis. This is evident by the ob-
served reduction in toxicity. Despite the differences in physical
properties, we sought to investigate the cell toxicity and the mech-
anism of toxicity for all compounds.
2.3.1. Concentration dependant cell death
TPEN (1) and PAC-1 (2) induced significant cell death in PC12
cells; as compared to control in a concentration range 25–
200
(3) and ZnA-Pyr (4) also induced cell death but significant induc-
tion was only observed at higher concentration, 75–200
lM, in a concentration dependent manner (Fig. 3). ZnA-DPA
2. Results and discussion
2.1. Chemistry
lM
(Fig. 3). The negative control, treated only with DMSO, showed
no induction of cell death at any of the concentrations used (not
shown). The EC50 values for TPEN (1) and PAC-1 (2) were calculated
The new zinc chelators were synthesized according to Scheme 1.
All new compounds exhibited spectra in accordance with their
anticipated structures. Purity was analysed using a HPLC system
and all final products were P95% pure (not shown).
to be 38 and 122
lM, respectively, using Graph Pad Prism software.
EC50 values for ZnA-DPA (3) and ZnA-Pyr (4) could not be calcu-
lated because of solubility problems above 200 lM.
2.3.2. Procaspase-3 activation
2.2. Zinc binding
Since PAC-1 is reported to induce toxicity by disrupting the zinc
homeostasis and activation of caspase-3,4 we sought to investigate
the role of caspase-3 activation by all tested compounds. The PC12
cells were cultured in 96 well black plates at cell density 28 ꢀ 104
The zinc-binding ability of ZnA-Pyr (4) was assessed using UV
spectroscopy. ZnA-DPA (3) contained no UV-sensitive chromo-
phore that gave a significant response when treated with zinc
and could therefore not be determined spectrophotometrically.
ZnA-Pyr (4), however, showed a dose-response in absorbance
when treated with zinc (Fig. 2). Literature values20,21 for zinc affin-
ity for analogous compounds were used qualitatively for discus-
sion purposes in the present study.
for 24 h and then exposed to TPEN (1) (25
l
M), PAC-1 (2)
(100 lM), ZnA-DPA (3), or ZnA-Pyr (4) (both 200 lM) for four
hours, concentrations where they exhibited a comparable level of
cell death. Cells treated with DMSO were used as control. Stauro-
sporine 0.5 lM and 1 lM concentrations were used as a positive
control to activate caspase-3. Results presented in Fig. 4 show that
all the compounds activated caspase-3 over the control level, but
less than staurosporine, which gave 6000 and 30,000 RFU/million
2.3. Biological evaluation
cells, using 0.5 or 1 lM, respectively (not shown).
Two new compounds, ZnA-DPA (3) and ZnA-Pyr (4), were syn-
thesized with the aim of comparing them to the reported procas-
pase-3 activators PAC-1 and TPEN (1). The design of the new
compounds included stronger zinc binding motifs as compared to
ZnA-Pyr (4) and ZnA-DPA (3) do produce significant amounts of
caspase-3 relative to control, albeit less than TPEN (1) and PAC-1
(2). It is also here reasonable to assume that lipophilicity affects
these results.

O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 21 (2013) 5175–5181
5177
Scheme 1. Synthesis of PAC-1 analogs ZnA-Pyr (4) and ZnA-DPA (3).
Figure 2. Zinc chelation with ZnA-Pyr (3). Dose dependent response of ZnA-Pyr (50 lM) when treated with 0–50 lM ZnSO4 in Hepes buffer (pH 7.4) is shown. Inset: The
relative increase in absorption at 360 nm and 400 nm in response to added ZnSO4.
2.3.3. Reactive oxygen species
hours, and TPEN (1), PAC-1 (2), and ZnA-Pyr (4), but not ZnA-
DPA (3), showed significantly higher DHR oxidation than vehicle
control at this time-point. None of the compounds showed sig-
nificant difference to the DMSO control after 4 h, indicating a ra-
pid and transient ROS induction. DHE, however, did not detect
any ROS production with any of the compounds (results not
shown).
ROS production could also be a major cause of toxicity, either
up-stream or down-stream of caspase-3 activation, and so the
ability to produce ROS was detected using dihydrorhodamine
(DHR) and dihydroethidium (DHE) as ROS probes. Relative ROS
production using DHR as probe (Fig. 5) was higher as measured
one hour after addition of the compounds compared to four

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O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 21 (2013) 5175–5181
120
100
80
60
40
20
0
A
B
120
*
*
TPEN
*
PAC-1
100
80
*
*
*
*
*
60
*
40
*
*
20
0
Conc (µM) 10
25
50
75
100
200
Conc (µM) 10
25
50
75
100
200
100
C
D
100
ZnA-DPA
80
60
40
20
0
ZnA-Pyr
80
60
40
20
0
*
*
*
*
*
Conc (µM) 10
25
50
75
100
200
Conc (µM) 10
25
50
75
100
200
Figure 3. Induction of cell death in PC12 cells. PC12 cells were treated with TPEN, PAC-1, ZnA-DPA, or ZnA-Pyr. Cell death is expressed as percentage relative to the total
number of cells. The results are average of 4 experiments + S.D. Significant difference is shown as compared to DMSO control (⁄), P < 0.05, one-way ANOVA with Holm-Sidak
post hoc method.
700
600
500
400
300
200
100
0
12
10
8
*/#
*/#
*/#
*
*
*
*
6
*
*
*
4
*
*
2
0
Time
1h 4h 1h 4h 1h 4h 1h 4h 1h 4h 1h 4 h
Contr DMSO TPEN PAC-1 ZnA-DPA ZnA-Pyr
DMSO TPEN PAC-1 ZnA-DPA ZnA-Pyr
Figure 5. Induction of ROS in PC12 cells. PC12 cells were loaded with DHR (0.1
for 30 min, and left untreated or treated with DMSO (vehicle control), TPEN
(25 M), PAC-1 (100 M), ZnA-DPA, or ZnA-Pyr (200 M) for one hour or four hours
lM)
Figure 4. Caspase-3 activity. PC12 cells were treated with TPEN (25
(100 M), ZnA-DPA, or ZnA-Pyr (200 M). Cells treated with DMSO were used as
lM), PAC-1
l
l
l
l
l
followed by reading the slope for ROS production for 30 min. Results are expressed
control. Results are expressed as average of
5 experiments + S.D. Significant
difference as compared to control is shown by ⁄. Significance is P < 0.05, one-way
relative to the untreated control slope at 4 h, as average of 3 experiments S.D.
⁄
#
Significant difference as compared to untreated is shown by and to DMSO by
,
ANOVA with Holm-Sidak method.
P < 0.05, one-way ANOVA with Holm-Sidak post hoc testing.
ZnSO4, caspase-3 inhibitor, or DHR used as ROS scavenger. Cells
were exposed for 24 h and then stained with trypan blue (Fig. 6).
No cell death was observed with the inhibitors alone. TPEN (1)
caused cell death that was significantly reduced by ZnSO4 or cas-
pase-3 inhibitor. For PAC-1 (2) and ZnA-Pyr (4) the addition of
DHR, ZnSO4 and caspase-3 inhibitor gave a significant reduction
of cell death, whereas ZnA-DPA (3) caused cell death that was sig-
nificantly prevented by DHR and caspase-3 inhibitor, but less sig-
nificant by ZnSO4 compared to the other compounds. Different
2.3.4. Reduction in cell death by inhibitors
Since caspase-3 activation and ROS production were detected
with all compounds, it was of interest to determine how addition
of inhibitors would affect the toxicity. Therefore, DHR and cas-
pase-3 inhibitor were used as inhibitors of cell death. ZnSO4 was
used to determine whether an increase in the zinc level could re-
duce cell death. Cells were exposed to TPEN (1) (25
(2) (100 M), ZnA-DPA (3), or ZnA-Pyr (4) (both 200
were treated with compounds alone and in combination with
l
M), PAC-1
l
lM). Cells

O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 21 (2013) 5175–5181
5179
70
60
50
40
30
20
10
0
A
B
70
PAC-1
TPEN
60
50
40
30
20
10
0
*
*
*
*
*
Zinc
C3-I
DHR
Zinc
C3-I
DHR
70
60
50
40
30
20
10
0
C
D
70
60
50
40
30
20
10
0
ZnA-DPA
ZnA-Pyr
*
*
*
*
*
Zinc
C3-I
DHR
Zinc
C3-I
DHR
Figure 6. Inhibition of cell death with zinc, caspase-3 inhibitor, or DHR. PC12 cells were exposed to TPEN (25
lM), PAC-1 (100
l
M), ZnA-DPA, or ZnA-Pyr (200
l
M), alone and
with inhibitors ZnSO4 (zinc, 10 M), caspase-3 inhibitor (C3-I, 1 M), or DHR (1 lM). Cell death was counted as percentage relative to the total number of cells and results are
l
l
expressed as average from 4 experiments + S.D. Results were significant at P < 0.05 (One-way ANOVA with Holm-Sidak post hoc method). Significance was calculated based
on difference compared to DMSO and are showed by (⁄).
levels of reduction in toxicity were seen with caspase-3 inhibitor.
The reduction for 100 M PAC-1 (2) was roughly 33%, but for
200 M ZnA-Pyr (4) it was 66%.
As can be seen when adding DHR to the experiments, ROS is an
important contributor to toxicity for these compounds. The toxic-
ity of PAC-1 (2), ZnA-DPA (3) and ZnA-Pyr (4) was significantly re-
duced with roughly the same factor.
to correlate well with lipophilicity, indicating limited cell pene-
tration for ZnA-DPA (3) and ZnA-Pyr (4). When comparing ZnA-
DPA (3) and ZnA-Pyr (4), the latter was a better zinc chelator
and more sensitive to the zinc level in the cells. However, distur-
bance of zinc homeostasis seems to be the primary mechanism of
toxicity for all four model compounds, since cell death can be re-
duced not only with zinc or caspase inhibitors but also with a
ROS scavenger.
l
l
2.3.5. Mechanism of toxicity
Disruption of the zinc homeostasis seems to be the primary
mechanism of toxicity for all four model compounds. The reduc-
tion in cell death observed by addition of zinc was similar or great-
er than by addition of a caspase-3 inhibitor or a ROS scavenger.
Regarding the new compounds, ZnA-Pyr (4) is a stronger zinc bin-
4. Experimental procedures
4.1. Materials
Caspase-3 fluorometric substrate ((DEVD)2-Rhodamine110)
was purchased from Bachem AG (Switzerland), and caspase-3
inhibitor (Ac-DEVD-cmk) was purchased from Calbiochem (San
Diego, CA). TPEN was purchased from TCI Europe and was used
without further analysis. Dulbecco’s modified Eagle’s medium
(DMEM), horse serum, and fetal bovine serum were purchased
from Gibco (Paislet, Scotland). All other reagents were from Sigma
(St. Louis, USA).
der than ZnA-DPA (3) and the cell toxicity of 200
significantly more reduced upon addition of 10
200 M ZnA-DPA (3). The relative reduction in toxicity upon addi-
lM ZnA-Pyr (4) is
lM ZnSO4 than for
l
tion of zinc to ZnA-Pyr (4) is even larger than for PAC-1 (2), but not
TPEN (1), which has the highest affinity for zinc, and is adminis-
tered as 25 lM. Thus, the added amount of zinc relative to the test
concentrations of the model compounds indicates that the abso-
lute reduction in toxicity correlates with the Kd values for the
compounds.
4.2. Methods
3. Conclusion
4.2.1. Zinc titration
Compounds were diluted from their 10 mM DMSO stock solu-
tions into 3 mL of buffer (50 mM Hepes, 100 mM NaNO3, pH 7.4
The new compounds, ZnA-DPA (3) and ZnA-Pyr (4), induce
procaspase-3 activation, which in turn leads to cell death. They
are less lipophilic than the parent compounds TPEN (1) and
PAC-1 (2), and would therefore presumably not cause significant
neurotoxicity at relevant doses, as recently found for PAC-1.28
They are, however, only mildly toxic to PC12 cells. Toxicity seems
to give a final concentration of 50
lM in quartz cuvettes. Then
10–50 L of a 3 mM ZnSO4 solution in buffer was added to the cuv-
l
ettes in order to make samples with 0–1 equiv of added ZnSO4
(50 mM Hepes, 100 mM NaNO3, pH 7.4). The solutions were mixed
and allowed to equilibrate for 60 min. The absorbance spectra

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O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 21 (2013) 5175–5181
between 260 and 500 nm were then acquired using a spectropho-
tometer (Biochrom Libra S32PC).
Autospec Ultima GC-MS (Micromass Ltd. Manchester, England).
The MS was equipped with an electron ionization (EI) ion source
producing 70 eV electrons. The voltage scan time was 1 s. and
the inter scan delay time was 0.20 s. The mass spectrometer was
tuned to a resolution of 12,000. The ion source temperature was
set to 200 °C and the samples were introduced into the instrument
via a direct insertion probe, which was cooled with water. The soft-
ware used was MassLynx version 4.0. (Waters, Milford, MA, USA).
4.2.2. Biological evaluation and toxicity measurement
PC12 cells (a rat pheochromocytoma cell line) were grown in
DMEM 7emented with 5% (v/v) horse serum, 10% (v/v) fetal bovine
serum, 1% (v/v) penicillin/streptomycin mixture (Gibco) and 1% (v/
v) sodium pyruvate (Gibco). Cells were seeded in 58 mm dishes
(3.3 mL/dish at a density of 350,000 cells/mL). Cultures were main-
tained at 37 °C in a humidified atmosphere containing 5% CO2.
4.3.1. Synthesis of ethyl 2-(4-benzylpiperazin-1-yl)acetate (6)
Ethyl chloroacetate (1.14 mL, 11.0 mmol, 1.1 equiv) in 10 mL
acetone was added to a stirring solution of 1-benzylpiperazine
(5) (1.73 mL, 10.0 mmol, 1.0 equiv) in 10 mL acetone. Sodium
hydrogen carbonate (1.05 g, 12.5 mmol, 1.25 equiv) was then
added to the reaction mixture with the aid of 5 mL acetone. The
suspension was then heated to reflux and left for 22 h at which
point no starting material was visible on TLC (2:1 hexane/EtOAc).
The mixture was filtered and the salt was washed with 3 ꢀ 5 mL
acetone. The pale yellow filtrate was then concentrated under re-
duced pressure to give 2.67 g of a yellow oil. The crude product
was purified by column chromatography (130 g of SiO2 using
1:1–2:1 EtOAc/hexane) affording 2.29 g (87%) of a pale yellow
oil. 1H NMR (400 MHz, CDCl3) d 7.39–7.17 (m, 5H), 4.18 (q,
J = 7.1 Hz, 2H), 3.52 (s, 2H), 3.20 (s, 2H), 2.81–2.36 (m, 8H), 1.26
(t, J = 7.1 Hz, 3H). The spectroscopic data obtained are in accor-
dance with published data.16
4.2.3. Cell death assays
PC12 cells were treated with TPEN (1), PAC-1 (2), ZnA-DPA (3),
or ZnA-Pyr (4) at 10, 25, 50 75, 100, and 200 lM concentrations,
dissolved in DMSO (0.1% final concentration). Cell death was mea-
sured with trypan blue exclusion after 24 h of treatment. Trypan
blue in 0.9% NaCl solution was added 1:4 into the medium and
the cells were incubated for 30 min at 37 °C. Cells excluding the
dye (unstained cells) were scored as viable and the cells stained
with dye (blue cells) were scored as dead. Untreated and DMSO
treated cells were used as control.
4.2.4. Caspase-3 activity assay
For detection of caspase-3 activity cells were treated with TPEN
(1) (10
lM), PAC-1 (2) (100
lM), ZnA-DPA (3) (200 lM), ZnA-Pyr
(4) (200
l
M), or DMSO directly in black 96 well plates for four
hours. Modified RIPA buffer (50 mM Tris–HCl (pH 7.4), 1% Igepal,
0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EGTA) sup-
plemented with 20 lM of caspase-3 fluorometric substrate
(DEVD)2-Rhodamine 110 was added in four wells. Another two
wells were added RIPA buffer supplemented with substrate and
4.3.2. Synthesis of 2-(4-benzylpiperazin-1-yl)acetohydrazide (7)
Hydrazine hydrate (2.22 mL 80%, 36.63 mmol, 3.0 equiv) was
added drop wise to a stirring solution of ethyl 2-(4-benzylpipera-
zin-1-yl)acetate (6) (3.20 g, 12.21 mmol, 1.0 equiv) in 17 mL abso-
lute ethanol. After the addition, the mixture was heated to reflux
and left overnight. The pale yellow solution was concentrated under
reduced pressure and mixed with 20 mL (1:1 brine/water pH >12)
and extracted with 3 ꢀ 15 mL CH2Cl2 followed by 15 mL EtOAc.
The combined organic phases were pooled and dried over MgSO4, fil-
tered and concentrated under reduced pressure to give 3.24 g of a
thick pale oil. It was dissolved in EtOH and left overnight in a 1:15
EtOH/Et2O solution to precipitate needle like white crystals. The
mother liquor was then concentrated, dissolved in EtOH and poured
into Et2O to give a total yield of 2.96 g (97%). 1H NMR (300 MHz,
CDCl3) d 8.12 (s, 1H), 7.40–7.18 (m, 6H), 3.84 (d, J = 4.6 Hz, 2H),
3.51 (s, 2H), 3.07 (s, 2H), 2.64–2.31 (m, 11H). The spectroscopic data
obtained are in accordance with published data.16
1
lM caspase-3 inhibitor Ac-DEVD-cmk (negative control). There
was more than 85% reduction in caspase-3 activity with 1 M cas-
l
pase-3 inhibitor added to the cells or added in the assay. The cell
lysate was incubated for 24 h at 37 °C before fluorescence was
measured at excitation 485 nm and emission 535 nm, using a fluo-
rometer (Perkin–Elmer HTS 7000 Plus. Bio Assay Reader). There
was a gradual increase in fluorescence with time in this period.
This method was adopted from literature.28,29
4.2.5. Reactive oxygen species detection assay
To determine the effects of TPEN (1), PAC-1 (2), ZnA-DPA (3),
and ZnA-Pyr (4) on reactive oxygen species generation, cells were
cultured in black 96 well plates. Cells were incubated with DHR
(0.1
treatment with TPEN (1) (10
(3) (200 M), or ZnA-Pyr (4) (200
l
M) for 30 min at 37 °C, and washed with medium before
M), PAC-1 (2) (100 M), ZnA-DPA
M) for one hour or four hours.
l
l
4.3.3. Synthesis of (E)-2-(4-benzylpiperazin-1-yl)-N0-((3-
hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-
l
l
Medium was replaced with PBS and the plate was measured for
30 min at excitation 485 nm and emission 535 nm, using a fluo-
rometer (Perkin–Elmer HTS 7000 Plus. Bio Assay Reader). The slope
was used in the calculation of ROS production.
yl)methylene)acetohydrazide (4, ZnA-Pyr)
The hydrazide 2-(4-benzylpiperazin-1-yl)acetohydrazide (7)
(25 mg, 1.0 mmol, 1.0 equiv), pyridoxal hydrochloride (22 mg,
0.11 mmol, 1.1 equiv) and sodium carbonate (7 mg, 0.13 mmol,
1.3 equiv) were mixed and dissolved in 2 mL water whereupon
the mixture turned yellow. The mixture was heated to 80 °C and
left for 90 min after which a white solid started precipitating.
The mixture was cooled to room temperature and extracted with
3 ꢀ 2 mL CH2Cl2 (in which the solid dissolved). The combined or-
ganic phases was washed with 3 mL H2O, dried over MgSO4 and
concentrated to a yellow solid affording 12 mg (40%) of the title
compound. 1H NMR (400 MHz, CDCl3) d 11.51 (s, 1H), 10.25 (s,
1H), 8.69 (s, 1H), 7.76 (s, 1H), 7.40–7.17 (m, 5H), 4.70 (s, 2H),
3.54 (s, 2H), 3.16 (s, 2H), 2.55 (m, 11H). 13C NMR (101 MHz, CDCl3)
d 166.5, 151.9, 150.3, 146.3, 138.8, 137.9, 131.3, 129.3, 128.5,
127.4, 120.2, 63.0, 60.95 (d, J = 8.4 Hz), 53.9, 53.0, 19.2. HRMS
(EI) calcd for C21H27N5O3 397.2114, found 397.2099.
4.2.6. Statistical analysis
Statistical differences were analysed by one-way repeated mea-
sures analysis of variance (ANOVA) followed by Holm-Sidak post
hoc test (Sigma Stat software international, Ashburn, VA). A P value
of <0.05 was considered significant.
4.3. Synthesis
PAC-1 was synthesized in our laboratory according to litera-
ture.16 The prepared compounds were identified by 1H, 13C NMR
spectroscopy, and HRMS, and shown to be >95% pure by HPLC anal-
ysis. High resolution mass spectrometry was obtained using an

O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 21 (2013) 5175–5181
5181
4.3.4. Synthesis of ethyl 2-(bis(pyridin-2-ylmethyl)amino)-
acetate (9)
like to thank associate professor Dag Ekeberg at The Norwegian
University of Life Sciences for high resolution mass spectroscopy.
2,20-Dipicolylamine (8) (199 mg, 1.0 mmol, 1.0 equiv) was di-
luted in 1 mL acetonitrile and mixed with sodium carbonate
(75 mg, 1.2 mmol, 1.2 equiv). Ethyl chloroacetate (135 mg,
1.1 mmol, 1.1 equiv) in 1 mL acetonitrile was then added drop wise
to the stirring solution. The reaction was then heated to reflux for
18 h. The reaction mixture was filtered to remove inorganic impu-
rities and the filter cake washed with 3 ꢀ 5 mL diethyl ether. The
combined organic phases were concentrated to give 338 mg of
dark orange oil. The crude product was then purified by column
chromatography (SiO2 column with 10% MeOH in CH2Cl2 as eluent)
to afford 263 mg (quantitative) of a rust red oil. 1H NMR (300 MHz,
CDCl3) d 8.58–8.46 (m, 2H), 7.69–7.60 (m, 2H), 7.56 (d, J = 7.7 Hz,
2H), 7.18–7.09 (m, 2H), 4.21–4.09 (m, 2H), 4.00 (d, J = 6.3 Hz,
4H), 3.48 (dd, J = 6.3, 3.9 Hz, 2H), 1.32–1.18 (m, 3H). 13C NMR
(75 MHz, CDCl3) d 171.4, 159.3, 149.2, 136.6, 123.3, 122.2, 60.6,
60.1, 55.2, 14.4. The spectroscopic data obtained are in accordance
with published data.30
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.037.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
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4.3.5. Synthesis of 2-(4-benzylpiperazin-1-yl)-N0-(2-(bis(pyridin-
2-ylmethyl)amino)acetyl)acetohydrazide (3, ZnA-DPA)
The ester ethyl bis(pyridin-2-ylmethyl)glycinate (9) (522 mg,
1.83 mmol, 1.0 equiv) was dissolved in 2 mL H2O and 2 mL 5 M
KOH solution. The mixture was stirred at room temperature for
60 min and then the pH was adjusted to 7 with 1 M HCl. The solu-
tion was concentrated under reduced pressure to a pale orange
sticky solid and it was washed with 3 ꢀ 10 mL hot absolute etha-
nol. The combined ethanol was then concentrated under reduced
pressure to give a dark red oil which was used without purification
in the next step. 2-(4-Benzylpiperazin-1-yl)acetohydrazide (3)
(64 mg, 0.26 mmol, 1.0 equiv) was mixed with the crude acid from
the previous step (80 mg, 0.27 mmol, 1.03 equiv), N-methyl mor-
pholine (65 lL, 0.57 mmol, 2.2 equiv), HOBT (8 mg, 0.05 mmol,
0.2 equiv), diluted in 1 mL absolute ethanol and cooled to 10 °C.
EDC (60 mg, 0.31 mmol, 1.2 equiv) was then added to the stirring
solution. The solution was then heated to room temperature and
stirred for four hours. The mixture was then concentrated under
reduced pressure, diluted in 5 mL H2O and extracted with
3 ꢀ 5 mL CH2Cl2. The combined organic phases were dried over
MgSO4 and concentrated to give a pale orange oil. The crude prod-
uct was purified by column chromatography (neutral Al2O3, eluent
5–10% MeOH in EtOAc) to afford 179 mg (90%) as a pale orange oil.
1H NMR (300 MHz, CDCl3) d 12.04 (s, 1H), 9.30 (s, 1H), 8.57 (d,
J = 4.7 Hz, 2H), 7.58 (td, J = 7.7, 1.7 Hz, 2H), 7.34–7.18 (m, 7H),
7.13 (dd, J = 6.9, 5.3 Hz, 2H), 3.92 (s, 4H), 3.51 (d, J = 8.4 Hz, 4H),
3.15 (s, 2H), 2.73–2.38 (m, 8H). 13C NMR (75 MHz, CDCl3) d
168.7, 166.7, 158.4, 149.4, 138.1, 136.8, 129.3, 128.3, 127.2,
123.2, 122.5, 63.0, 60.9, 60.3, 57.7, 53.8, 53.2. HRMS (EI) calcd for
C27H33N7O2 487.2696, found 487.2722.
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Acknowledgements
This work was supported by the University of Oslo and Statoil’s
Research Fund. The funding sources had no influence on the study
design or the decision to publish this paper. The authors would also