Copper(II) complexes of hybrid hydroxyquinoline-thiosemicarbazone ligands: GSK3β inhibition due to intracellular delivery of copper

Article abstract of DOI:10.1039/c0dt01176b

Cognitive decline associated with Alzheimer's disease appears to be related to the hyper-phosphorylation of the protein tau as a consequence of increased activity of glycogen synthase kinase 3β (GSK3β), and subsequent formation of neurotoxic neurofibrillary tangles. Abberant metal ion homeostasis, particularly involving copper has been implicitly linked to the pathogenesis of the disease. Increasing intracellular copper concentrations has been found to trigger pathways that result in inhibition of GSK3β. The syntheses and characterisation of tetradentate hybrid hydroxyquinoline-thiosemicarbazone proligands is presented. The ligands form stable complexes with Cu^II where the copper ion is four coordinate and essentially square planar as characterised by single crystal X-ray crystallography. The reduction of the metal ion to Cu^I has been studied by electrochemical techniques and occurs at potentials that permit intracellular reduction. The new complexes show class dependent cell membrane permeability in neuronal-like SH-SY5Y cells with subsequent increases in intracellular copper concentrations. The increased intracellular copper results in a dose-dependent inhibition (phosphorylation) of GSK3β.

Full text of DOI:10.1039/c0dt01176b

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PAPER
Copper(II) complexes of hybrid hydroxyquinoline-thiosemicarbazone ligands:
GSK3b inhibition due to intracellular delivery of copper†
James L. Hickey,a Peter J. Crouch,b,c Sithorn Mey,a Aphrodite Caragounis,b Jonathan M White,a
Anthony R. Whiteb,c and Paul S. Donnelly*a
Received 6th September 2010, Accepted 25th November 2010
DOI: 10.1039/c0dt01176b
Cognitive decline associated with Alzheimer’s disease appears to be related to the
hyper-phosphorylation of the protein tau as a consequence of increased activity of glycogen synthase
kinase 3b (GSK3b), and subsequent formation of neurotoxic neurofibrillary tangles. Abberant metal
ion homeostasis, particularly involving copper has been implicitly linked to the pathogenesis of the
disease. Increasing intracellular copper concentrations has been found to trigger pathways that result in
inhibition of GSK3b. The syntheses and characterisation of tetradentate hybrid hydroxyquinoline-
thiosemicarbazone proligands is presented. The ligands form stable complexes with CuII where the
copper ion is four coordinate and essentially square planar as characterised by single crystal X-ray
crystallography. The reduction of the metal ion to CuI has been studied by electrochemical techniques
and occurs at potentials that permit intracellular reduction. The new complexes show class dependent
cell membrane permeability in neuronal-like SH-SY5Y cells with subsequent increases in intracellular
copper concentrations. The increased intracellular copper results in a dose-dependent inhibition
(phosphorylation) of GSK3b.
from microtubules that consequently lose structural integrity
with concomitant impaired axonal transport and compromised
Introduction
synaptic function.5 Importantly, NFT burden does correlate
with the severity of cognitive impairment and some evidence
supports a key downstream role for tau hyper-phosphorylation in
Ab-mediated neuronal degeneration.6
Alzheimer’s disease (AD) is a progressive neurodegenerative
disease that leads to synaptic failure and neuronal death. These
symptoms can initially manifest as mild forgetfulness but progress
to complete loss of cognition and ultimately death.1 Characteristic
pathological hallmarks of the disease include the presence of ex-
tracellular senile plaques and intracellular neurofibrillary tangles
in the brain. The plaques are comprised of insoluble aggregated
peptide amyloid-b (Ab), a 39–43 amino acid peptide derived from
the amyloid precursor protein (APP).2–4 These amyloid plaques
do not consistently correlate with cognitive impairment and some
argue that smaller soluble oligomeric species are the toxic species
responsible for neuronal death.1
Recent developments have highlighted the importance of metal
ion homeostasis in the development of Alzheimer’s disease and
potential therapeutic strategies have focused on targeting copper
metabolism.7–11 Both Ab and APP are known to bind to copper and
there is evidence that brain Cu metabolism is altered in AD. APP-
knockout mice have elevated levels of Cu in the cerebral cortex,
whilst APP-over-expressing mice have reduced brain Cu levels.12–15
Promising results have been obtained with 8-hydroxyquinoline
derivatives such as clioquinol 1 (Fig. 1a) that are known to
bind CuII and other metal ions as well as hydroxypyridone
derivatives.16–18 The mode of action of these ligands was thought to
involve chelation of metals bound to Ab, resulting in dissolution of
amyloid aggregates. However, it is now thought that the hydrox-
yquinoline derivatives may also act as ionophores by chelating
extracellular Cu to form membrane permeable copper complexes
that activate neuroprotective cell signalling pathways by increasing
cellular metal ion bioavailability.19 Central to this neuroprotective
cell signalling pathway is the inhibition of glycogen synthase kinase
3b (GSK3b), a serine and threonine protein kinase responsible
for certain intracellular phosphorylation reactions. GSK3b is a
constitutively active kinase and is inactivated by phosphorylation
at the serine-9 residue. Importantly, increased GSK3b activity
Neurofibrillary tangles (NFT) consist of
a
hyper-
phosphorylated form of microtubule-associated protein
a
called tau. NFT initiate with the formation of bundles of paired
helical filaments that accumulate in the neuronal cytoplasm.
The hyper-phosphorylation of tau results in its detachment
aSchool of Chemistry and Bio21 Molecular Science and Biotechnology
Institute, University of Melbourne, Parkville, Melbourne, Victoria, 3010,
Australia. E-mail: pauld@unimelb.edu.au
bCentre for Neuroscience and Department of Pathology, University of
Melbourne, Parkville, Melbourne, Victoria, 3010, Australia
cMental Health Research Institute of Victoria, Parkville, Melbourne,
Victoria, 3052, Australia
† CCDC reference numbers 801975–801977. For crystallographic data in
CIF or other electronic format see DOI: 10.1039/c0dt01176b
1338 | Dalton Trans., 2011, 40, 1338–1347
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Fig. 1 a) CuII chelating 8-hydroxyquinoline clioquinol 1, b) bis(thiosemicarbazonato) CuII complex, CuII(gtsm) 2, and c) hybrid hydroxyquino-
line-thiosemicarbazone CuII complexes.
(through decreased phosphorylation at serine-9) has been linked
to tau hyperphosphorylation and NFT formation, and increased
GSK3b activity is induced by exposure to Ab.20 Abnormal GSK3b
activity may also be central to a number of other brain disorders,
and as a consequence inhibition of GSK3b is seen an important
target.21,22
confirmed by reverse phase high pressure liquid chromatography
(RP-HPLC) (Scheme 1).
We recently reported an alternative therapeutic approach using
a stable cell permeable bis(thiosemicarbazonato) CuII complex,
CuII(gtsm) 2 (Fig. 1b) for the controlled intracellular delivery of
Cu that resulted in improved cognition in an animal model of
AD. CuII(gtsm) is a neutral low molecular weight complex that
is membrane permeable. The CuII complex is very stable (Ka =
1018 M) but once the complex enters the reducing environment
encountered in most cells the CuII is susceptible to reduction to
CuI. The ligand has a lower affinity for CuI (Ka ~ 1010–13 M) so the
copper becomes essentially bio-available with transfer of CuI to
copper chaperone proteins with higher affinity for CuI.23 We were
able to show that CuII(gtsm) inhibited GSK3b and decreased tau
phosphorylation in cell culture and animal models of AD.24,25
Stimulated by the success of both hydroxyquinoline and
thiosemicarbazone CuII complexes in inhibiting GSK3b respec-
tively, we have combined both types to prepare two new classes
of tetradentate hybrid hydroxyquinoline-thiosemicarbazone proli-
gands and their CuII complexes (Fig. 1c). The new complexes show
class dependent membrane permeability in a cultured neuronal-
like cell line and subsequent potent phosphorylation (inhibition)
of GSK3b.
Scheme
1
Reagents
and
conditions:
(i)
4-N-substituted-3-
thiosemicarbazide, cat. AcOH, EtOH, rf for 4 h; (ii) Cu(OAc)2·2H2O,
DMF, rt for 4 h.
A crystal of 3 suitable for X-ray crystallography was grown
by evaporation of a methanol solution of the ligand (Fig. 2).
The proligand has crystallised in the E isomeric form with the
thiosemicarbazone functional group extending away from the
aromatic ring and the molecule is essentially planar. The C11–
S1 bond distance, 1.689(2) A, and C10–N3 distance, 1.282(2) A
suggests the “free” ligand is best represented in the tautomeric
form shown in Scheme 1. A H nmr spectrum of 3 in d6-dmso
reveals a single isomer with evidence of H-coupling for the NH–
CH3 and the substituent methyl. The quaternary C S results in a
signal at d 178 ppm in the 13C nmr spectrum whereas the N CH
results in a resonance at d 141.8 ppm.
Results and discussion
˚
˚
Synthesis and characterisation of hybrid
1
hydroxyquinoline-thiosemicarbazone CuII complexes
Tetradentate hybrid hydrazinopyridine-thiosemicarbazones have
been investigated previously as proligands for potential
hypoxia selective CuII radiopharmaceuticals and two hy-
brid hydroxyquinoline-thiosemicarbazonato copper(II) complexes
have been evaluated for their anti-cancer activity in a neuroblas-
toma cell line.26,27 This is the first report outlining the synthesis of
six new hybrid hydroxyquinoline-thiosemicarbazone proligands
and their copper complexes along with their ability to deliver
copper into cells and the subsequent effects on the activity of
GSK3b.
The new proligands 3, 4 and 5 were prepared in good yields by
condensation reactions catalysed by acetic acid between 2-formyl-
8-hydroxyquinoline and a 4-N-substituted-3-thiosemicarbazide in
1
ethanol. All were characterised by H nmr, 13C nmr, ESI mass
Fig. 2 An ORTEP (40% probability) representation of molecular struc-
ture of 3. Select hydrogen atoms have been omitted for clarity.
spectrometry and microanalysis; with the purity of each ligand
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The nmr spectra of 4 and 5 are similar to 3 where appropriate,
and again suggest a single isomer dominates in d6-dmso solution.
RP-HPLC confirmed the purity of the ligands (C18 column (4.6 ¥
150 mm, 5 mm) with a 1 mL min-1 flow rate and UV spectroscopic
detection at 214 nm, 220 nm, and 270 nm. Retention times
(Rt/min) were recorded using a water–acetonitrile gradient elution
method). With the retention times suggesting a systematic increase
in lipophilicity (3, Rt = 9.48 min; 4, Rt = 10.71 min; and 5, Rt =
13.27 min).
The CuII complexes 6, 7, and 8 can be prepared by addition
of CuII(OAc)2·2H2O to a solution of proligand in dimethyl
formamide at ambient temperature (Scheme 1). The immediate
colour change to deep red indicates complexation is very rapid
and the neutral CuII complexes can be readily precipitated by the
addition of water. In each case the ESI mass spectrum reveals a
peak at the expected m/z value corresponding to [CuIIL + H+]
with the expected isotope pattern.
A representation of the molecular structure of the neutral CuII
complex 6 as determined by single crystal X-ray crystallography
is shown in Fig. 3. The CuII is 4-coordinate with the dianionic
tetradentate ligand coordinating through a deprotonated phenolic
oxygen, nitrogen of the substituted quinoline ring, the azamethinic
nitrogen and thiolate sulfur. The coordination geometry about the
copper is distorted square planar with a 5-5-5 (O,N,N,S) chelate
ring system. The N2–Cu–S1 bond angle (85.7(1)◦) is significantly
larger than the N1–Cu–O1 bond angle of 81.9(1)◦. The Cu–
Fig. 4 An ORTEP representation (40% probability) of 8·H2O. Select
hydrogen atoms have been omitted for clarity.
The CuII is essentially 4-coordinate with weak axial interactions
˚
with a sulfur atom of an adjacent molecule Cu–S¢ = 2.975(1) A
˚
and short p–p contacts (ca. 3.5 A) to give infinite chains (Fig. 5).
The relatively unusual coordination mode results in a N1–Cu–N3
bond angle of 89.(1)◦ and a N3–Cu–S1 bond angle of 70.2(1)◦.
The synthesis of the copper complexes of 8-hydroxyquinoline-2-
carboxaldehyde-3-thiosemicarbazone and 8-hydroxyquinoline-2-
carboxaldehyde-N-4,4¢-dimethyl-3-thiosemicarbazone have been
reported recently but this is the first report of the copper complexes
of the N-4-methyl, N-4-ethyl and N-4-phenyl derivatives and the
first structural characterization by X-ray crystallography of copper
complexes of this family of tetradentate ligand.
˚
S bond distance (2.223(1) A) and Cu–N azamethinic distance
II
28
˚
(1.999(4) A) are similar to the analogous distance in Cu (gtsm).
Deprotonation to give the thiosemicarbazonato limb of the ligand
˚
is reflected in the C11–S1 distance of 1.767(5) A, suggesting more
thiolate-like character than the thione-like ‘free’ ligand 3 (C11–
˚
S1 = 1.689(2) A) along with a marginally shorter N3–C11 bond
1
˚
˚
distance of 1.334(6) A than N4–C11 in H2L (1.363(2) A), suggest-
ing more double bond-like character. Crystals of 8 were grown by
evaporation of a saturated solution of the complex in ethanol. A
representation of the molecular structure (Fig. 4) reveals the cop-
per binding to the ligand via the hydrazinic nitrogen, rather than
azamethinic nitrogen, creating a 4-membered N–Cu–S chelate
ring. This binding mode in thiosemicarbazonato metal complexes
is rare but precedents include bis(thiosemicarnazonato)nickel(II)
complexes,29,30 a single bis(thiosemicarnazonato)zinc(II) complex
and thiosemicarbazonato(ruthenium(II)) complexes.31
Fig. 5 A representation of the polymeric molecular array of 8 displaying
˚
short contacts between Cu–S¢ = 2.975(1) A.
The complexes 6–8 have limited solubility in water, so it was
of interest to prepare water-soluble derivatives. The introduction
of a sulfonic acid to the backbone of the hydroxyquinoline para
to the phenolic hydroxyl position improved the solubility of
both the ligand and complex in aqueous environments without
interfering with complexation of CuII. An initial attempt to oxidise
2-methyl-8-hydroxyquinoline-5-sulfonic acid to an aldehyde with
SeO2 failed due to the insolubility of the acid in respective
ethereal solvents. The selective oxidation does proceed cleanly
from the 2-methyl-8-hydroxyquinoline-5-sulfonyl chloride 9 to
the aldehyde 10. Subsequent hydrolysis of the sulfonyl chlo-
ride in an acetone/water mixture (50/50) gave the desired 2-
formyl-8-hydroxyquinoline-5-sulfonic acid 11 in good yield (78%)
(Scheme 2).
The sulfonated proligands 12, 13 and 14 were prepared in an
analogous manner to 3–5, via standard condensation reactions
between 2-formyl-8-hydroxyquinoline-5-sulfonic acid and a 4-N-
substituted-3-thiosemicarbazide in ethanol at ambient tempera-
ture (Scheme 3). An additional acid catalyst was not necessary as
the reactions were found to proceed rapidly and in sufficient yields.
Fig. 3 An ORTEP representation (40% probability) of 6·CH3OH. Select
hydrogen atoms have been omitted for clarity.
The new sulfonated derivatives were characterised by 1H nmr, 13
C
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The CuII complexes of the sulfonated proligands were also
prepared by addition of CuII(CH3CO2)2·H2O to a solution of 12–
14 in dimethyl formamide at ambient temperature (Scheme 3). An
immediate colour change to deep red was observed indicating
rapid complexation. The crude neutral CuII complexes 15–17
were precipitated by the addition of diethyl ether and collected.
On redissolving in water the complexes were readily purified on
a cation-exchange column. Characterisation for each complex
was confirmed by microanalysis and an ESI mass spectrum that
revealed a peak at the expected m/z value corresponding to
[CuIIL + H+] with the expected isotope pattern.
Monitoring the stability of CuII complex 6 with biological
molecules by UV/Vis absorbance
The proligand 3 displays characteristic absorbances in the elec-
tronic spectrum at labs = 315 nm (e = 8.4 ¥ 104 M-1) and 350 nm
(e = 9.85 ¥ 104 M-1) that are also present in the spectra of 4 and 5.
Complexation with CuII to give 6 results in two distinct red shifted
chromophoric absorbances at labs = 326 nm (e = 5.36 ¥ 104 M-1)
and 402 nm (e = 2.71 ¥ 104 M-1) as well as a broad absorbance
centred at labs = 490 nm (e = 6.5 ¥ 103 M-1) characteristic of
ligand to metal charge transfer transition. Complexes 7 and 8
Scheme 2 Reagents and conditions: (i) Chlorosulfonic acid, neat, rt 1 h;
(ii) SeO2, Dioxane, rf 12 h; (iii) H2O, Acetone/H2O, rt 1 h.
display similar spectra. The characteristic absorbance at labs
=
402 nm was chosen to monitor the stability of CuII complex 6 in
the presence of competing biologically relevant ligands L-histidine
and L-cysteine and the biological reducing agent ascorbic acid.
A solution of 6 in an aqueous buffer (pH 7.4) was incubated
in the presence of a 10-fold excess of L-histidine, L-cysteine and
ascorbic acid (30% DMSO/PB, 1 mM, pH 7.4) over 24-hours
at 37 ◦C. During this period minimal change in the absorbance
spectra of CuII complex 6 were detected showing it is likely that the
complexes have sufficient stability in extracellular media to serve
as chaperones for intracellular delivery of Cu.
Electrochemistry
Scheme
3 Reagents and conditions: (i) 4-N-substituted-3-thiosemi-
Measurements of reduction potentials in dimethylformamide
(DMF) or dimethylsulfoxide (DMSO) have proved useful in
predicting the retention of copper inside the cell, with clear
correlations between the reduction potential and likely intracel-
lular reduction.32 The CuII complex of the bis(thiosemicarbazone)
proligand derived from diacetyl, CuII(atsm) = (diacetyl-bis(4-N-
methyl-3-thiosemicarbazonato)copper(II), selectively accumulates
in hypoxic cells and is being investigated as a hypoxia tracer
for positron emission tomography. In comparison, the ligand
derived from glyoxal, CuII(gtsm) 2 (Fig. 1) is easier to reduce than
CuII(atsm) by some 160 mV (CuII(gtsm) Em = -0.44 V compared
with CuII(atsm) Em = -0.60 V vs. SCE).23,33 As a result, the reducing
environment encountered inside most cells is sufficient to reduce
the metal ion in CuII(gtsm) to CuI. The ligand has a lower affinity
for CuI (KD ~ 10-13 M) so the copper becomes essentially bio-
available with transfer of CuI to copper chaperone proteins with
higher affinity for CuI.23 In comparison, CuII(atsm) is harder to
reduce and unless cells are hypoxic the complex remains intact
with the metal ion remaining bound to the ligand.
carbazide, EtOH, rt for 4 h; (ii) Cu(OAc)2·2H2O, DMF, rt for 4 h.
nmr, ESI mass spectrometry and microanalysis; with the purity
of each ligand confirmed by RP-HPLC. The retention times are
significantly lower than observed for 3–5 as a result of the polar
sulfonic acid, but also suggest a systematic increase in lipophilicity
(12, Rt = 6.86 min, 13, Rt = 7.69 min and 14, Rt = 9.67 min).
The 1H nmr spectra of proligands 12–14 in d6-dmso are similar
to that of 3–5 in that a single isomer dominates in solution and
H-coupling is observed for the NH-R to a neighbouring alkyl
substituent. The inductive effect of the sulfonic acid results in a
general (and in some cases significant) shift of ~ 1 ppm downfield
in the hydroxyquinoline aromatic signals. The phenolic signal
observed in 3–5 (~9.83 ppm) is not resolved, instead exchanging
with the sulfonic acid and adventitious water. The absence of
resonances attributed to the quaternary C–OH and C–SO3H
in the 13C nmr spectra of 12–14 also suggests rapid exchange
on the nmr timescale. A signal at d ~178 ppm in the 13C nmr
spectra of 12–14 attributed to C S is similar to that observed for
3–5, whereas a resonance observed at d ~150 ppm assigned as the
The electrochemical properties of the complexes 6–8 were
investigated by cyclic voltammetry. The complexes all undergo
an essentially irreversible reduction at Epc = -0.49 V (vs. SCE) at
N
CH is somewhat downfield (~10 ppm) of the analogous signal
in the spectra of 3–5.
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a glassy carbon electrode which can be attributed to a CuII/CuI
process. The initial reduction is followed by two related oxidative
events at Epa = -0.36 V and 0.05 V. This suggests that the initial
electrochemical change is followed by a chemical change. The
irreversible reduction, CuII/CuI process of 6–8 (Epc = -0.49 V)
is close to that of CuII(gtsm) 2 (Epc = -0.48 V),23 suggesting
that the reducing environment encountered in most cells may be
sufficient to reduce the metal ion to CuI that will dissociate from
the hydroxyquinoline-thiosemicarbazone ligand in the presence of
competing ligands found inside cells.
was also tested as a representative of the complexes that did not
increase intracellular copper.
The effects of complexes 6 and 15 on cell viability was
investigated using two different methods (Fig. 7). The dose-
dependent increase in cellular Cu induced by treatment with
6, correlated with a decrease in cellular capacity to reduce
MTS (5-(3-carboxymethoxyphenyl)-3-(4,5-dimethylthiazol-2-yl)-
2-(4-sulfophenyl)-2H-tetrazolium), indicating decreased activity
of cellular reductases. The water soluble sulfonated analogue,
compound 15, had no effect on the cells ability to reduce MTS,
consistent with the cell-uptake data that shows that the water
soluble compound is not membrane permeable. This diminished
ability to reduce MTS does not necessarily imply a loss of cell
viability and correlations between the MTS assays and cytotoxicity
are to be treated with caution.
Cell biology
Neuronal-like SH-SY5Y cells were treated with the CuII com-
pounds 6–8 and 15–17 at 0, 1, 5 or 10 mM for 1 h; cellular
levels of Cu were then measured by inductively coupled plasma
mass spectrometry (ICP-MS). Data presented in Fig. 6 indicates
that CuII compounds 6–8 are all cell permeable and increase
cellular Cu levels in a dose-dependent manner. By contrast, the
sulfonated variants 15–17 did not increase cellular levels of Cu
indicating these compounds were not cell permeable. As 6–8 all
showed a comparable dose-dependent uptake effect, 6 was chosen
to represent the membrane permeable compounds. Whereas 15
Fig. 7 Relative effects of 6 and 15 on the activity of cellular reductases
(MTS reduction) and LDH release. MTS data are expressed relative to
values obtained for cells treated with 0 mM compound. LDH data are
expressed relative to cells treated with 0.1% (v/v) Triton-X 100 to induce
plasma membrane permeabilisation and complete LDH release.
An alternative plasma membrane permeabilisation assay mea-
sures lactate dehydrogenase (LDH) release. In this assay com-
pound 6 had no effect on LDH release, indicating the compound
did not induce membrane permeabilisation and cell death (Fig. 7).
The MTS reduction results are consistent with previous work de-
scribing that cell viability assays based on endocytosis/exocytosis
mechanisms, such as the MTS assay, can be misleading given
the capacity for Cu-based metal–ligand complexes to alter
endocytosis/exocytosis.34
An analysis of cell lysates following treatment with 6 or 15
revealed that the increase in cellular Cu mediated by 6, correlated
with increasing inhibitory phosphorylation of GSK3b (Fig. 8).
This result is consistent with previous in vitro and in vivo work
using metal–ligand complexes capable of increasing bio-available
Fig. 6 Cu content of cell samples after treating with CuII complexes 6–8
and sulfonated CuII complexes 15–17 at the concentrations shown for 1 h.
Metal ion content is expressed relative to cellular protein.
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Table 1 Crystallographic data
3
6
8
Chemical
formula
M
Crystal system
T/K
C12H12N4OS [C12H10N4OSCu]· [C17H12CuN4OS]·
[CH3OH]
353.88
[H2O]
260.32
Monoclinic
130.0(2)
P21/c
10.394(1)
7.0021(8)
16.770(2)
90
98.544(2)
90
1207.0(2)
4
401.92
Orthorhombic
130.0(2)
P212121
4.0657(2)
14.7702(8)
26.266(1)
90
90
90
1577.3(1)
4
Triclinic
130.0(2)
P1
¯
Space group
˚
a/A
7.9196(7)
9.5097(10)
10.0744(11)
91.711(9)
90.001(8)
111.220(9)
706.93(12)
2
˚
b/A
˚
c/A
a (◦)
b (◦)
g (◦)
3
˚
V/A
Fig. 8 Relative effects of 6 and 15 on levels of total and phosphorylated
forms of GSK3b and Akt. Complex 6 but not 15 induces inhibitory phos-
phorylation of GSK3b (serine-9 residue) and activating phosphorylation
of Akt (serine-473) in a dose-responsive manner.
Z
Independent
refl.
2118
2735
2707
Rint
0.029
0.0388
0.0984
0.033
0.0387
0.1599
0.046
0.0452
0.1067
R(I > 2s(I))
wR(all data)
intracellular Cu.19,24,25,35,36 In comparison, treatment with the water
soluble analogue 15 had no effect on levels of GSK3b consistent
with the lack of uptake indicated by ICP-MS.
the cell lines tested and did not elicit an increase in intracellular
copper concentrations and consequently did not phosphorylate
GSK3b or signalling kinase Akt. Cells operate at an overall
reducing environment and there have been some attempts to
quantify the redox environment of cells.38 It is proposed that
once the complex is inside the cell the metal ion is reduced
andreleasedfrom the hydroxyquinoline-thiosemicarbazoneligand
in the presence of competing CuI ligands found in cells. An
alternative explanation of the observed behaviour is that the
complexes are reduced and the metal ion dissociated from the
ligand at the cell surface or witihin the cell membrane followed
by uptake of CuI into the cell. Both possibilites would lead
to an increase in bioavailable CuI inside the cell that results
in inhibitory phosphorylation of GSK3b.34 The hybrid ligands
present a controlled way of delivering copper ions intracellularly.
This results in inactivation of GSK3b, a major kinase involved
in the hyper-phosphorylation of tau an event that appears central
to the pathological progression of Alzheimer’s disease. We have
shown that increased intracellular Cu has a profound effect on
the activity of GSK3b. This finding is significant, considering
the recent success of therapeutic approaches that modulate Cu
metabolism.
To partly examine the cellular mechanisms leading to the
inhibitory phosphorylation of GSK3b by 6, we also examined
effects of the compound on phosphorylation of the signalling
kinase Akt (Fig. 8). The kinase activity of Akt is activated once it
is phosphorylated at serine-473, and like GSK3b, Akt phosphory-
lation in SH-SY5Y cells increased with increasing concentration
of 6 (Fig. 8). These data indicate the phosphorylation of GSK3b
mediated by 6 may be a downstream consequence of Akt activation
since Akt is widely recognised for its capacity to phosphorylate
GSK3b at serine-9.37
Concluding remarks
A
series of new tetradentate hybrid hydroxyquinoline-
thiosemicarbazone proligands were prepared that doubly depro-
tonate to form neutral CuII complexes. Water-soluble analogues
were prepared by the introduction of a sulfonate functional group
in the 5-position of the hydroxyquinoline. The CuII complexes
were characterised by microanalysis, ESI-MS and electronic
spectroscopy. The molecular structure of two of the CuII com-
plexes were determined by single crystal X-ray crystallography
confirming for the first time a 4-coordinate distorted square planar
(O,N,N,S) coordination environment for the copper(II) atom,
whilst revealing an unusual coordination motif of a 4-membered
N–Cu–S chelate ring for complex 8. The copper(II) complexes
were stable in aqueous buffer solutions to a histidine/cysteine
challenge suggesting that formed complexes are of sufficient
stability for biological applications. Electrochemical measure-
ments using cyclic voltammetry revealed that the complexes
were susceptible to an irreversible reduction attributed to a
CuII/CuI reduction at potentials that could be encountered in
the reducing environment found inside most cells. In the case
of the membrane permeable copper complexes the accumulation
of Cu elicited a dose-dependent inhibitory phosphorylation of
both GSK3b and signalling kinase Akt. The introduction of a
sulfonate functional group allowed the formation of water soluble
analogues but these derivatives were not membrane permeable in
Experimental
Crystallography
Crystals of 3, 6, and 8, respectively were mounted in low
temperature oil then flash cooled to 130 K using an Oxford low
temperature device. Intensity data were collected at 130 K with
an Oxford XCalibur X-ray diffractometer with Sapphire CCD
detector using Cu-Ka radiation (graphite crystal monochromator
˚
l = 1.54184 A) (Table 1). Data were reduced and corrected for
absorption.39 The structures were solved by direct methods and
difference Fourier synthesis using the SHELX suite of programs40
as implemented within the WINGX41 software. Thermal ellipsoid
plots were generated using the program ORTEP-342 integrated
within the WINGX suite of programs.
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1
3JHH = 8.0, 1H, ArH), 7.11 (d, 3JHH = 8.0 Hz, 1H, ArH).13C{ H}
General procedures
NMR (500 MHz; DMSO-d6): d (ppm) 193.5 (O CH), 154.9 (C–
OH), 149.8 (C–C O), 138.1 (C), 137.5 (ArCH), 135.1 (C–SO3H),
128.9 (ArCH), 127.1 (C), 116.9 (ArCH), 110.3 (ArCH).
Syntheses. All reagents and solvents were obtained from
commercial sources (Sigma–Aldrich) and used as received unless
otherwise stated. 2-Formyl-8-hydroxyquinoline43 and 2-methyl-
8-hydroxyquinoline-5-sulfonyl chloride (9)44 were prepared ac-
cording to previously reported procedures. Cation-exchange
Synthesis
of
2-(4-N-substituted-3-thiosemicarbazone)-8-
hydroxyquinolines (3–5). 2-Formyl-8-hydroxyquinoline was
dissolved in dry, degassed ethanol (10 mL) and treated with 1.1
equiv of a suitable 4-N-substituted-3-thiosemicarbazide. A drop
of acetic acid was added, and the reaction was subsequently
refluxed for 4 h. The light yellow, crystalline precipitate that
formed was isolated in good yield (>90%) and purity, washed
with ether (3 ¥ 10 mL) and air-dried.
R
chromatography was performed on DOWEXꢀ 50WX2-200 ion-
exchange resin (H+ form 1.5 cm ¥ 10 cm). Elemental analyses for
C, H, and N were carried out by Chemical & MicroAnalytical
Services Pty. Ltd, Vic. NMR spectra were recorded on a Varian
FT-NMR 500 spectrometer (1H NMR at 499.9 MHz and 13C{ H}
1
NMR at 125.7 MHz) at 298 K and referenced to the internal
solvent residue.45 Mass spectra were recorded on an Agilent 6510-
Q-TOF LC/MS mass spectrometer and calibrated to internal
references.
2-(4-N-Methyl-3-thiosemicarbazone)-8-hydroxyquinoline (3).
4-N-methyl-3-thiosemicarbazide (134 mg, 1.27 mmol) was added
to 2-formyl-8-hydroxyquinoline (200 mg, 1.15 mmol) to give a
light yellow crystalline solid (303.7 mg, 92%). Elem anal Found
(calcd) for C12H12N4OS: C, 55.47 (55.37); H, 4.66 (4.65); N, 21.64
(21.52). 1H NMR (500 MHz; DMSO-d6): d (ppm) 11.91 (m, 1H,
UV/Visible spectroscopy. UV/Vis spectra were recorded on
a Cary 300 Bio UV-Vis spectrophotometer, from 800–200 nm at
0.5 nm data intervals with a 600 nm min-1 scan rate. Solutions of
complex 6 in 30% DMSO/PB (1 mM, pH 7.4) was incubated in
the presence of a 10-fold excess of L-histidine, ascorbic acid and
L-cysteine and monitored over 24-hours at 37 ◦C.
3
N–NH–C S), 9.83 (m, 1H, C–OH), 8.79 (d, JHH = 4.5, 1H,
CH3–NH–C S), 8.41 (d, JHH = 8.7, 1H, ArH), 8.30 (d, JHH
3
3
=
3
8.7, 1H, ArH), 8.26 (s, 1H, N CH), 7.43 (t, JHH = 7.8, 1H,
ArH), 7.38 (dd, 3JHH = 8.1, 4JHH = 1.3, 1H, ArH), 7.10 (dd, 3JHH
=
4
3
High pressure liquid chromatography. Analytical RP-HPLC
traces were acquired using an Agilent 1200 series HPLC system
equipped with a Agilent Zorbax Eclipse XDB-C18 column (4.6 ¥
150 mm, 5 mm) with a 1 ml min-1 flow rate and UV spectroscopic
detection at 214 nm, 220 nm, and 270 nm. Retention times
(Rt/min) were recorded using a gradient elution method of 0–
100% B over 25 min, solution A consisted of water (buffered with
0.1% trifluoroacetic acid) and solution B consisted of acetonitrile
(buffered with 0.1% trifluoroacetic acid).
7.5, JHH = 1.4, 1H, ArH), 3.06 (d, JHH = 4.6 Hz, 3H, CH3).
13C{ H} NMR (500 MHz; DMSO-d6): d (ppm) 178.0 (C S),
1
153.8 (C–OH), 151.9 (C–C N), 141.8 (N CH), 138.1 (C), 136.1
(ArCH), 128.7 (C), 128.0 (ArCH), 118.3 (ArCH), 117.7 (ArCH),
112.1 (ArCH), 30.9 (CH3). MS (ES+): m/z (calcd) 261.0800
(261.0732) {M + H+}. HPLC Rt 9.48 min. Crystals suitable for
single-crystal X-ray diffraction were grown from methanol by
slow evaporation at room temperature.
2-(4-N-Ethyl-3-thiosemicarbazone)-8-hydroxyquinoline (4). 4-
N-ethyl-3-thiosemicarbazide (151 mg, 1.27 mmol) was added to
2-formyl-8-hydroxyquinoline (200 mg, 1.15 mmol) to give a light
yellow crystalline solid (290.1 mg, 92%). Elem anal Found (calcd)
for C13H14N4OS: C, 56.99 (56.91); H, 5.16 (5.14); N, 20.52 (20.42).
Electrochemistry. Cyclic voltammograms were recorded using
an AUTOLAB PGSTAT100 equipped with GPES V4.9 software.
Measurements of the complexes were carried out at approximately
1 ¥ 10-4 M in dimethylformamide with tetrabutylammonium
tetrafluoroborate (1 ¥ 10-2 M) as electrolyte using a glassy carbon
disk (d, 3 mm) working electrode, a Pt wire counter/auxiliary
electrode, and a Ag/Ag+ pseudo reference electrode (silver wire in
CH3CN (Bu4NBF4) (0.1 M) AgNO3 (0.01 M)). Ferrocene was used
as an internal reference (Em(Fc/Fc+) = 0.54 V vs. SCE), where Em
refers to the midpoint between a reversible reductive (Epc) and
oxidative (Epa) couple, given by Em = (Epc+Epa)/2. Irreversible
systems are only given reductive (Epc) and oxidative (Epa) values,
respectively.
1H NMR (500 MHz; DMSO-d6): d (ppm) 11.85 (s, 1H, N–NH–
3
C
C
S), 9.83 (s, 1H, C–OH), 8.84 (t, JHH = 5.9, 1H, CH3–NH–
3
3
S), 8.42 (d, JHH = 8.7, 1H, ArH), 8.30 (d, JHH = 8.7, 1H,
3
ArH), 8.27 (s, 1H, N CH), 7.43 (t, JHH = 7.8, 1H, ArH), 7.39
(dd, 3JHH = 8.2, 4JHH = 1.4, 1H, ArH), 7.10 (dd, 3JHH = 7.5, 4JHH
=
3
1.4, 1H, ArH), 3.67–3.61 (m, 2H, CH2), 1.19 (t, JHH = 7.1 Hz,
3H, CH3). 13C{ H} NMR (500 MHz; DMSO-d6): d (ppm) 177.4
1
(C S), 153.8 (C–OH), 152.3 (C–C N), 142.4 (N CH), 138.7
(C), 136.5 (ArCH), 129.2 (C), 128.5 (ArCH), 118.8 (ArCH), 118.2
(ArCH), 112.6 (ArCH), 38.9 (CH2), 14.9 (CH3). MS (ES+): m/z
(calcd) 275.10 (275.09) {M + H+}. HPLC Rt 10.71 min.
Synthetic procedures
2-Formyl-8-hydroxyquinoline-5-sulfonic acid (11). 2-Methyl-8-
hydroxyquinoline-5-sulfonyl chloride (836 mg, 3.24 mmol) was
dissolved in dry dioxane (40 mL) and refluxed with SeO2 (400 mg,
3.57 mmol) for 12 h. On cooling, the selenium precipitate was
filtered off and the filtrate evaporated to dryness. The residue
was suspended in acetone/water (50/50, 20 mL) and stirred at
room temperature for 1 h before filtration and removal of volatiles
in vacuo gave a crystalline beige solid that was used without further
2-(4-N-Phenyl-3-thiosemicarbazone)-8-hydroxyquinoline
(5).
4-N-Phenyl-3-thiosemicarbazide (212 mg, 1.27 mmol) was added
to 2-formyl-8-hydroxyquinoline (200 mg, 1.15 mmol) to give a
light yellow crystalline solid (369 mg, 90%). Elem anal Found
(calcd) for C17H14N4OS: C, 63.32 (63.33); H, 4.29 (4.38); N, 17.26
1
(17.38). H NMR (500 MHz; DMSO-d6): d (ppm) 12.24 (s, 1H,
C–NH–C S), 10.37 (s, 1H, N–NH–C S), 9.88 (s, 1H, C–OH),
3
8.60 (d, JHH = 8.7, 1H, ArH), 8.38 (s, 1H, N CH), 8.31 (d,
1
purification (641 mg, 78%). H NMR (500 MHz; DMSO-d6): d
3JHH = 8.7, 1H, ArH), 7.57 (d, 3JHH = 7.5 Hz, 2H, ArH), 7.46–7.39
4
3
3
3
(ppm) 10.15 (d, JHH = 0.9, 1H, O CH), 9.30 (dd, JHH = 8.8,
(m, 4H, ArH), 7.25 (t, JHH = 7.4, ArH), 7.12 (dd, JHH = 7.5,
4JHH = 0.8, 1H, ArH), 8.02 (d, JHH = 8.8, 1H, ArH), 7.99 (d,
4JHH = 1.2, 1H, ArH). 13C{ H} NMR (500 MHz; DMSO-d6):
3
1
1344 | Dalton Trans., 2011, 40, 1338–1347
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d (ppm) 176.5 (C S), 153.4 (C–OH), 151.7 (C–C N), 142.9
(N CH), 130.0 (C), 138.2 (C), 136.1 (ArCH), 128.9 (C), 128.2
(ArCH), 128.1 (ArCH), 126.3 (ArCH), 125.6 (ArCH), 118.8
(ArCH), 117.7 (ArCH), 112.1 (ArCH). MS (ES+): m/z (calcd)
323.10 (323.09) {M + H+}. HPLC Rt 13.27 min.
Synthesis of 2-(4-N-substituted-3-thiosemicarbazone)-8-hydro-
xyquinoline-5-sulfonic acids (12–14). 2-Formyl-8-hydroxy-
quinoline-5-sulfonic acid was suspended in ethanol (20 mL)
and treated with 1.1 equiv of a suitable 4-N-substituted-3-
thiosemicarbazide. The reaction was stirred at room temperature
for 12 h under nitrogen. The yellow, crystalline precipitate that
formed was isolated in reasonable yield (>80%) and purity,
washed with ether (3 ¥ 10 mL) and air-dried.
Synthesis of 2-(4-N-substituted-3-thiosemicarbazonato)-8-
quinolinolato copper(II) complexes (6–8). 2-(4-N-substituted-3-
thiosemicarbazone)-8-hydroxyquinoline was dissolved in DMF
(5 mL) and treated with an excess (1.2 equiv) of copper(II)acetate.
The reaction darkened considerably whilst stirring at room
temperature for 4 h. Addition of water (5–10 mL) precipitated a
dark red solid that was collected, washed repeatedly with water (3
¥ 5 mL) and dried in vacuo.
2-(4-N-Methyl-3-thiosemicarbazonato)-8-quinolinolato copper-
(II) (6). 2-(4-N-methyl-3-thiosemicarbazone)-8-hydroxyquino-
line (50 mg, 192 mmol) reacted with Cu(II)(OAc)2·2H2O (46 mg,
230 mmol) to give a dark red solid (57.1 mg, 92%). Elem anal
Found (calcd) for C12H10N4OSCu: C, 44.94 (44.78); H, 3.03 (3.13);
N, 17.39 (17.41). MS (ES+): m/z (calcd) 321.9943 (321.9944)
{M + H+}.
2-(4-N-Methyl-3-thiosemicarbazone)-8-hydroxyquinoline-5-sul-
fonic acid (12). 4-N-methyl-3-thiosemicarbazide (23 mg,
220 mmol) was added to 2-formyl-8-hydroxyquinoline-5-sulfonic
acid (50 mg, 200 mmol) to give a yellow crystalline solid (56 mg,
83%). Elem anal Found (calcd) for C12H12N4O4S2·2H2O: C, 38.88
2-(4-N-Ethyl-3-thiosemicarbazonato)-8-quinolinolato copper(II)
(7). 2-(4-N -ethyl-3-thiosemicarbazone)-8-hydroxyquinoline
(61.8 mg, 225 mmol) reacted with Cu(II)(OAc)2·2H2O (54 mg,
270 mmol) to give a dark red solid (66.4 mg, 89%). Elem anal
Found (calcd) for C13H12N4OSCu: C, 46.55 (46.49); H, 3.70 (3.60);
N, 16.68 (16.68). MS (ES+): m/z (calcd) 336.0099 (336.0100)
{M + H+}.
1
(38.29); H, 4.16 (4.28); N, 14.56 (14.88). H NMR (500 MHz;
DMSO-d6): d (ppm) 12.14 (m, 1H, N–NH–C S), 9.24 (d, 3JHH
9.1, 1H, ArH), 8.89–8.87 (m, 1H, CH3–NH–C S), 8.54 (d, 3JHH
=
=
9.1, 1H, ArH), 8.38 (s, 1H, N CH), 7.88 (d, 3JHH = 8.0, 1H, ArH),
7.08 (d, 3JHH = 8.0, 1H, ArH), 3.08 (d, 3JHH = 4.5 Hz, 3H, CH3).
1
13C{ H} NMR (500 MHz; DMSO-d6): d (ppm) 178.0 (C S),
2-(4-N-Phenyl-3-thiosemicarbazonato)-8-quinolinolato copper-
(II) (8). 2-(4-N-phenyl-3-thiosemicarbazone)-8-hydroxyquino-
line (87.8 mg, 272 mmol) reacted with Cu(II)(OAc)2·2H2O (73 mg,
363 mmol) to give a dark red solid (73.1 mg, 96%). Elem anal
Found (calcd) for C17H12N4OSCu: C, 53.20 (53.18); H, 3.28 (3.15);
N, 14.47 (14.59). MS (ES+): m/z (calcd) 384.0099 (384.0100)
{M + H+}.
152.5 (C–C N), 150.7 (N CH), 137.9 (C), 135.2 (ArCH), 126.9
(C), 125.1 (ArCH), 118.1 (ArCH), 110.9 (ArCH), 31.0 (CH3). MS
(ES+): m/z (calcd) 340.03 (340.03) {M + H+}. HPLC Rt 6.86 min.
2-(4-N-Ethyl-3-thiosemicarbazone)-8-hydroxyquinoline-5-sul-
fonic acid (13). 4-N-ethyl-3-thiosemicarbazide (26 mg, 220 mmol)
was added to 2-formyl-8-hydroxyquinoline-5-sulfonic acid (50 mg,
200 mmol) to give a yellow crystalline solid (64 mg, 91%). Elem
anal Found (calcd) for C13H14N4O4S2·2H2O: C, 39.95 (39.99); H,
Synthesis of 2-(4-N-substituted-3-thiosemicarbazonato)-8-
quinolinolato-5-sulfonic acid copper(II) complexes (15–17).
2-(4-N-substituted-3-thiosemicarbazone)-8-hydroxyquinoline-5-
sulfonic acid was dissolved in DMF (2 mL) and treated with an
excess (1.2 equiv) of copper(II)acetate. The reaction darkened
considerably whilst stirring at room temperature for 4 h. Addition
of ether (5–10 mL) precipitated a brown solid that was collected,
washed with ether (3 ¥ 5 mL) and air-dried. The crude product
was dissolved in a minimal volume of water and passed through a
cation-exchange column to remove excess Cu2+. The product was
obtained as a dark solid after the removal of water.
1
4.48 (4.65); N, 13.31 (14.35). H NMR (500 MHz; DMSO-d6):
3
d (ppm) 12.13 (s, 1H, N–NH–C S), 9.28 (d, JHH = 9.1, 1H,
3
ArH), 8.96–8.93 (m, 1H, CH3–NH–C S), 8.58 (d, JHH = 9.1,
3
1H, ArH), 8.41 (s, 1H, N CH), 7.89 (d, JHH = 8.1, 1H, ArH),
3
7.10 (d, JHH = 8.1, 1H, ArH), 3.68–3.63 (m, 2H, CH2), 1.20 (t,
1
3JHH = 7.1 Hz, 3H, CH3). 13C{ H} NMR (500 MHz; DMSO-d6):
d (ppm) 177.0 (C S), 152.2 (C–C N), 150.5 (N CH), 135.3
(ArCH), 127.0 (C), 125.2 (ArCH), 118.2 (ArCH), 110.8 (ArCH),
38.5 (CH2), 14.4 (CH3). MS (ES+): m/z (calcd) 354.04 (354.05)
{M + H+}. HPLC Rt 7.69 min.
2-(4-N-Phenyl-3-thiosemicarbazone)-8-hydroxyquinoline-5-sul-
fonic acid (14). 4-N-Phenyl-3-thiosemicarbazide (37 mg,
220 mmol) was added to 2-formyl-8-hydroxyquinoline-5-sulfonic
acid (50 mg, 200 mmol) to give an orange crystalline solid (70 mg,
88%). Elem anal Found (calcd) for C17H14N4O4S2: C, 50.64 (50.74);
H, 3.60 (3.51); N, 13.84 (13.92). 1H NMR (500 MHz; DMSO-d6):
d (ppm) 12.44 (s, 1H, C–NH–C S), 10.42 (s, 1H, N–NH–C S),
9.24 (d, 3JHH = 9.1, 1H, ArH), 8.70 (d, 3JHH = 9.1, 1H, ArH), 8.48
(s, 1H, N CH), 7.89 (d, 3JHH = 8.1 Hz, 2H, ArH), 7.58–7.56 (m,
2H, ArH), 7.44–7.40 (m, 2H, ArH), 7.28–7.25 (m, 2H, ArH), 7.08
2-(4-N-Methyl-3-thiosemicarbazonato)-8-quinolinolato-5-sul-
fonic acid copper(II) (15). 2-(4-N-methyl-3-thiosemicarbazone)-
8-hydroxyquinoline-5-sulfonic acid (20 mg, 59 mmol) reacted with
Cu(II)(OAc)2·2H2O (14 mg, 70 mmol) to give a dark solid (20.5 mg,
86%). Elem anal Found (calcd) for C12H10N4O4S2Cu·3H2O: C,
31.51 (31.61); H, 3.64 (3.54); N, 11.74 (12.29). MS (ES+): m/z
(calcd) 401.95 (401.94) {M + H+}.
2-(4-N-Ethyl-3-thiosemicarbazonato)-8-quinolinolato-5-sulfonic
acid
copper(II)
(16). 2-(4-N-ethyl-3-thiosemicarbazone)-8-
1
(d, 3JHH = 8.1, 1H, ArH). 13C{ H} NMR (500 MHz; DMSO-d6): d
hydroxyquinoline-5-sulfonic acid (20 mg, 56 mmol) reacted
(ppm) 176.7 (C S), 152.6 (C–C N), 150.5 (N CH), 138.9 (C),
137.3 (C), 135.2 (ArCH), 128.1 (ArCH), 127.1 (C), 126.4 (ArCH),
125.8 (ArCH), 125.3 (ArCH), 118.5 (ArCH), 110.8 (ArCH). MS
(ES+): m/z (calcd) 403.05 (403.05) {M + H+}. HPLC Rt 9.67 min.
with Cu(II)(OAc)2·2H2O (13.5 mg, 68 mmol) to give
a
dark solid (19.8 mg, 85%). Elem anal Found (calcd) for
C13H12N4O4S2Cu·2H2O: C, 34.43 (34.55); H, 3.49 (3.57); N, 11.69
(12.40). MS (ES+): m/z (calcd) 415.97 (414.96) {M + H+}.
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Dalton Trans., 2011, 40, 1338–1347 | 1345
©

View Article Online
2-(4-N -Phenyl-3-thiosemicarbazonato)-8-quinolinolato-5-sul-
fonic acid copper(II) (17). 2-(4-N-phenyl-3-thiosemicarbazone)-
8-hydroxyquinoline-5-sulfonic acid (20 mg, 54 mmol) reacted with
Cu(II)(OAc)2·2H2O (13 mg, 65 mmol) to give a dark solid (22.3 mg,
87%). Elem anal Found (calcd) for C17H12N4O4S2Cu·5H2O: C,
36.89 (36.85); H, 3.37 (4.00); N, 10.06 (10.11). MS (ES+): m/z
(calcd) 463.97 (463.96) {M + H+}.
Inductively coupled plasma mass spectrometry. Cellular levels
of Cu, Fe, and Zn were determined using inductively coupled mass
spectrometry as previously described.24
Acknowledgements
We thank Assoc. Prof. Kevin Barnham for his pivotal role in
initiating this research. We acknowledge funding from the Aus-
tralian Research Council and National Health Medical Research
Council.
Cell culture conditions and treating with CuII complexes of
hydroxyquinoline-thiosemicarbazone ligands. SH-SY5Y neuron-
like cells were grown in DMEM:F-12 media (Invitrogen) supple-
mented with 10% (v/v) foetal bovine serum, 50 mM Hepes, non-
essential amino acids (Invitrogen), and penicillin/streptomycin
sulfate (Invitrogen). Cultures were maintained at 37 ◦C in a
humidified incubator with 5% (v/v) CO2 and passaged every 4–5
days at a dilution of 1/5. Prior to treating with CuII complexes
of hydroxyquinoline-thiosemicarbazone ligands, cells were seeded
into 6 well plates at 4 ¥ 104 cells cm-2, and allowed to grow for 2
days. For treating, media was aspirated from the cells then replaced
with serum free media containing the treatment compounds at 1,
5 or 10 mM. The compounds were prepared in DMSO at a stock
concentration of 1 mM, 5 mM or 10 mM. Stock solutions were
diluted 1000-fold in the treatment media (final DMSO content =
0.1% (v/v)), and 0.1% (v/v) DMSO was therefore used as the
relevant vehicle control. All treatments were for 1 h at 37 ◦C
in a humidified incubator with 5% (v/v) CO2. After 1 h, cells
were scraped into the existing treatment media then pelleted by
centrifugation (1000 ¥ g, 3 min). For Western blot analyses the
cell pellets were lysed with Cytobuster Protein Extraction reagent
(Novagen) supplemented with EDTA-free protease inhibitor cock-
tail (Roche), phenylmethanesulfonyl fluoride, sodium fluoride,
sodium vanadate, b-glycerophosphate, sodium pyrophosphate,
and deoxyribonucleate 5¢-oligonucleotido-hydrolase. Cell lysates
collected by centrifugation (16,000 ¥ g, 3 min) were normalised for
protein content after measuring aliquots using a protein content
determination kit (Pierce), then strored at -20 ◦C. For metal
content analysis, the cell pellets were washed twice with phosphate-
Notes and references
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3 J. Kang, H. G. Lemaire, A. Unterbeck, J. M. Salbaum, C. L. Masters,
K. H. Grzeschik, G. Multhaup, K. Beyreuther and B. Moeller-Hill,
Nature (London, U. K.), 1987, 325, 733.
4 C. L. Masters, G. Simms, N. A. Weinman, G. Multhaup, B. L.
McDonald and K. Beyreuther, Proc. Natl. Acad. Sci. U. S. A., 1985,
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5 J. Marx, Science (Wash. D. C.), 2007, 316, 1416.
6 H.-C. Huang and Z.-F. Jiang, J Alzheimers Dis, 2009, 16, 15.
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8 K. J. Barnham, V. B. Kenche, G. D. Ciccotosto, D. P. Smith, D. J. Tew,
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Bush, C. L. Masters, A. R. White, J. P. Smith, R. A. Cherny and R.
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9 A. I. Bush, Trends Neurosci., 2003, 26, 207.
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14 K. J. Barnham, W. J. McKinstry, G. Multhaup, D. Galatis, C. J. Morton,
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◦
buffered saline then stored at -20 C after measuring an aliquot
16 P. A. Adlard, R. A. Cherny, D. I. Finkelstein, E. Gautier, E. Robb, M.
Cortes, I. Volitakis, X. Liu, J. P. Smith, K. Perez, K. Laughton, Q. X.
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