Identification of a potent activator of Akt phosphorylation from a novel series of phenolic, picolinic, pyridino, and hydroxamic zinc(II) complexes

Article abstract of DOI:10.1007/s00775-010-0716-0

The discovery of small-molecule modulators of signaling pathways is currently a particularly active area of research. We aimed at developing unprecedented metal-based activators of Akt signaling which can potentially find applications as tools for regulat

Full text of DOI:10.1007/s00775-010-0716-0

J Biol Inorg Chem (2011) 16:195–208
DOI 10.1007/s00775-010-0716-0
ORIGINAL PAPER
Identification of a potent activator of Akt phosphorylation
from a novel series of phenolic, picolinic, pyridino,
and hydroxamic zinc(II) complexes
•
Savvas N. Georgiades Lok Hang Mak Inmaculada Angurell
•
•
•
Evelyn Rosivatz M. Firouz Mohd Mustapa Christoulla Polychroni
•
•
•
Rudiger Woscholski Ramon Vilar
Received: 7 July 2010 / Accepted: 7 October 2010 / Published online: 23 October 2010
Ó SBIC 2010
Abstract The discovery of small-molecule modulators of
signaling pathways is currently a particularly active area of
research. We aimed at developing unprecedented metal-
based activators of Akt signaling which can potentially find
applications as tools for regulating glucose metabolism
downstream of Akt or serve as lead structures for devel-
oping antidiabetic drugs. In this context, a highly diverse
library of 11 new zinc(II) complexes with phenolic, pi-
colinic, pyridino, and hydroxamic ligands, all containing
features beneficial for medicinal purposes, was prepared
and screened in an assay that detected levels of phospho-
Akt in lysates from NIH3T3 cells after treatment with the
compounds. The complexes featuring hydroxamic ligands
were found to be the most prominent activators of Akt
among the molecules prepared, with the most efficient
compound acting at submicromolar concentrations. Further
characterization revealed that this compound induces
phosphorylation of the Akt downstream effector glycogen
synthase kinase 3b, but does not act as an inhibitor of
tyrosine phosphatases or PTEN.
Introduction
Small molecules with the ability to modulate cellular sig-
naling have the potential to act as spatial and temporal
perturbagens of biological processes, and thus constitute a
reversible, practical, easily tuned, and readily accessible
alternative to genetic mutations in our study of biological
systems [1, 2]. Using chemical tools to interfere with
processes that involve protein phosphorylation, for exam-
ple, has a high likelihood of leading to significant biolog-
ical consequences (both structural and functional) [3], since
it is now well established that in all living systems phos-
phorylation serves as a universal signal that marks the
activation or deactivation of enzymes and signaling cas-
cades [4–8].
A particularly appealing target for modulation is the
serine/threonine kinase Akt. The activation of Akt (Fig. 1)
is due to the phosphorylation of two sites (Thr-308 and Ser-
473) by protein kinases that are controlled by phosphoin-
ositide 3-kinase (PI3K) [9]. PI3K is activated by upstream
signals such as insulin binding to its receptor. Numerous
reports have linked Akt phosphorylation to critical bio-
logical events such as cell growth, proliferation, and sur-
vival [10], thus rendering it medicinally significant for
cancer [11] and neurological diseases [12] among others.
Akt activation has also been implicated in cellular glucose
uptake and utilization; consequently, Akt is also a highly
relevant target in the development of antidiabetic drugs
[13–15].
Keywords Zinc ꢀ Hydroxamic acid ꢀ Phosphorylation ꢀ
Akt ꢀ Phosphoinositide 3-kinase
S. N. Georgiades ꢀ I. Angurell ꢀ M. Firouz Mohd Mustapa ꢀ
C. Polychroni ꢀ R. Vilar (&)
Department of Chemistry,
Imperial College London,
Exhibition Road,
London SW7 2AZ, UK
e-mail: r.vilar@imperial.ac.uk
Identification of new small molecules that make it
possible to regulate at will the phosphorylation levels of
Akt might yield control over some of these downstream
processes. With a focus on the diabetes implications of Akt
activation, we have previously shown that a series of
vanadium(IV) and vanadium(V) complexes with picolinic
L. H. Mak ꢀ E. Rosivatz ꢀ R. Woscholski
Division of Cell and Molecular Biology,
Imperial College London,
Exhibition Road,
London SW7 2AZ, UK
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Results and discussion
Synthesis of ligands and complexes
As part of our effort to discover new and effective metal-
based activators of Akt signaling, we prepared an extended
series of zinc(II) complexes, which would be inclusive of a
diverse set of ligands. The compound design had to meet
certain requirements: the complexes should exhibit distinct
coordination geometries and 3D shapes; they should exhibit
various patterns of hydrogen-bond donors and acceptors;
they should include pharmaceutically beneficial functional-
ities; they should have good aqueous solubility and mem-
brane permeability; and they should be readily synthesized.
We chose to investigate four major classes of ligands, namely
phenolic, picolinic (similar to the ones we previously eval-
uated with vanadium), pyridino, and hydroxamic. The
functional groups present in these ligands were deemed
compatible with all of the above-mentioned criteria, on the
basis of their performance in a multitude of previous phar-
maceutical applications. Eleven complexes were synthesized
in total, all of which are novel compounds.
Fig. 1 Simplified representation of the biochemical pathways that
connect the insulin receptor (IR), phosphoinositide 3-kinase (PI3K),
and PTEN with Akt and its downstream effector glycogen synthase
kinase 3b (GSK-3b). PI(4,5)P2 phosphatidylinositol 4,5-bisphosphate,
PI(3,4,5)P3 phosphatidylinositol 3,4,5-trisphosphate
ligands were able to induce Akt phosphorylation, acting by
inhibiting PTEN phosphatase, an enzyme upstream of Akt
that directly counteracts the function of PI3K [13]. In
parallel, other laboratories have suggested that protein
tyrosine phosphatases (PTP) are possible targets for com-
plexes of vanadium [16–20]. Recent studies by Sakurai
et al. describing metal-based small molecules with the
ability to enhance Akt phosphorylation levels have
involved zinc(II) as the metal center [21, 22]. Most of these
reports have suggested that zinc(II) complexes carry out
their effects in a PI3K-dependent mode [23, 24], and that
relatively high concentrations of complexes are needed to
achieve significant Akt phosphorylation enhancements.
Encouraged by the findings described above and given
the relatively limited number of zinc(II) compound classes
evaluated as Akt activators so far, we investigated a
broader range of zinc(II) compound families, with the aim
of identifying novel and more potent small molecules that
can find applications as chemical perturbagens of Akt
signaling and glucose metabolism. Zinc(II) favorably
exhibits low toxicity (especially in comparison with
vanadium) as well as the ability to afford complexes with a
variety of coordination geometries, spanning from tetrac-
oordination to hexacoordination. These properties were
taken into account in the design of our compounds, along
with the inclusion of new phenolic, picolinic, pyridino, and
hydroxamic ligands, which we considered likely to con-
tribute favorable properties in terms of functional and
structural diversity, hydrogen-bonding ability, aqueous
solubility, membrane transport, and pharmacokinetics. The
synthesis of 11 new zinc(II) complexes (Fig. 2) and their
biological evaluation leading to identification of a potent
Akt activator is described herein.
The first group of complexes (compounds 1–4,
Scheme 1) includes polydentate Schiff base ligands (or
their reduced counterparts) derived from salicyl precursors,
and these complexes were formed using Zn(OAc)2 as the
metal source. Compounds 1 and 2 exhibit an O–N–O
coordination pattern in their tridentate ligand. Compound 1
was obtained by condensation of glycine at the 2-aldehyde
position of 2,4-di(formyl)phenol in the presence of the
metal, followed by a second condensation of the remaining
aldehyde moiety to an ethanolamine unit and reduction.
Complex 2 was obtained in one step by reacting an
a-amino acid, homocysteic acid, with a similar salicyl
aldehyde that features a sulfonic acid substituent at the
4-position. The polar functionalities chosen in these
compounds confer the desired hydrophilicity and hydro-
gen-bonding ability to the complexes, both beneficial for
the aqueous solubility of the compounds and their potential
to interact with biomolecules. As suggested by our
study and in agreement with a reported crystal structure of
1a [25], complexes 1 and 2 were isolated in a hydrated
state and in aqueous solution can potentially reach
hexacoordination through further hydration. The fact that
zinc(II) has no specific preference for a fixed geometry
allows water to occupy free coordination sites, which is
likely the case for other complexes in this study as well.
Ligands for the synthesis of 3 and 4 were prepared from the
same aromatic precursor as for 2. In the case of 3 (which
has a ligand-to-metal stoichiometry of 2:1) the organic
precursor was modified via reductive amination with n-
propylamine to afford the N–O bidentate amine ligand 3a
prior to complexation. The ligand in 4 (which exhibits an
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J Biol Inorg Chem (2011) 16:195–208
197
Fig. 2 Proposed structures of
all zinc(II) coordination
compounds prepared in this
study
H2
N
Cl
H
SO3Na
SO3Na
OH
H
N
H
O
Zn
N
O
O
N
SO3Na
O
N
Zn
Zn
H2O
H2O
H2O
H2O
O
O
H
O
O
NaO3S
3
2
1
O
OH2
O
OH2
O
O
NaO3S
SO3Na
H
N
Zn
N
Zn
N
N
H
N
N
H2O
O
H
H
5
4
O
H2N
2
2
H
H
H
N
H
O
N
N
N
N
Zn
N
N
H2O
N
O
H
S
n
N
O
H2O
O
N
O
Zn
N
Zn
2
OH2
2
O
N
S
O
SO4
OH2
O
H
SO4
N
N
H
N
n
H
N
H
H
NH2
6: n=3
7: n=4
9
8
O
O
NH2
S
OH2
OH2
N
CF3
H
O
O
O
O
O
HN
HN
Zn
Zn
NH
NH
O
O
O
H
N
11
10
F3C
H2N
S
O
O
O–N–N–O coordination pattern) links two precursor mol-
ecules via double imine formation upon their condensation
with ethylenediamine. The complex likely has distorted
pyramidal geometry, as has been demonstrated previously
for other zinc(II)–salen complexes [26].
compounds 6b and 7b (ligands for 6 and 7, respectively),
the amine components reacting with 2-formylpyridine
contained an ethyl urea moiety. In the synthesis of 8,
propylamine was reacted with 5-nitro-2-formylpyridine,
and the nitro/imine intermediate 8a was reduced to the
amine/amine ligand 8b. Complexation to obtain complexes
6–8 was achieved with ZnSO4. In the case of 9, the zwit-
terionic amine taurine was reacted with 2-formylpyridine
under basic conditions, followed by complexation with
ZnCl2 in the same pot. Although this subset of molecules
also offers extensive hydrogen-bonding patterns, they have
larger steric requirements compared with some of the
asymmetric members of the series.
Another bidentate N–O ligand was derived from
5-aminopicolinic acid (Scheme 2). Although this specific
complex has not been previously reported, it should be
noted that zinc(II) complexes with other derivatives of
picolinic acid have been studied previously as insulin mi-
metics [27–29]. The amine handle was initially modified to
afford benzylamine 5a [30] to explore the impact of this
bulky moiety on the biological activity of the correspond-
ing zinc(II) complex. Complex 5 was prepared by reacting
the ligand with zinc sulfate under basic conditions.
Hydroxamic complexes 10 and 11 were generated as
shown in Scheme 4. A benzoic acid functionalized at the
4-position with an amine handle (either directly attached to
the ring or as an aminomethyl moiety, respectively) was
A group of four ligands were derived from 2-formyl-
pyridines (Scheme 3) via imine formation. In the case of
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J Biol Inorg Chem (2011) 16:195–208
Scheme 1 Synthesis of
OH
H2
Cl
i)
H2N
MeOH/r.t./16 h
phenolic zinc(II) complexes 1–4
(yields 1 99%, 2 54%, 3 70%, 4
95%). r.t. room temperature
N
CHO
OH
CHO
OH
ii) MeOH/NaBH4/
0 oC-r.t./1-2 h
H
H
iii) H2O/HCl/r.t./30 min
O
CHO
N
O
N
Zn
Zn
H2O
H2O
H2O
O
O
O
O
H2O
1a
1
i)
SO3H
CO2H
SO3Na
SO3Na
H2N
H2O/70 °C/1.5 h
H
O
N
CHO
SO3Na
ii) H2O/Zn(OAc)2/
NaOH/70 °C/2 h
Zn
ONa
H2O
O
O
H2O
2
SO3Na
H
SO3Na
SO3Na
i)
H2N
N
MeOH/Zn(OAc)2/
50 °C/16 h
MeOH/r.t./3 h
O
H
Zn
N
O
N
ii) NaBH4/0 oC/1 h
then r.t./12 h
CHO
ONa
ONa
3a
3
H
NaO3S
i)
NH2
OH2
SO3Na
H2N
O
O
Zn
NaO3S
SO3Na
MeOH/r.t./1 h
ii) Zn(OAc)2/
50 °C/16 h
N
N
CHO
H
H
ONa
4
O
Scheme 2 Synthesis of
picolinic zinc(II) complex 5
(yield 34%). DMF
H
H2O/EtOH/DMF/
ZnSO4/NaOH/r.t./3 h
O
H2N
OH2
N
H
N
Zn
N
N
dimethylformamide
N
OH
H
N
CO2H
H2O
N
O
O
5a
5
O
converted to its corresponding methyl ester using known
procedures [31, 32]. A critical step in ligand preparation was
the transformation of these esters to hydroxamic acids using
hydroxylamine sulfate under basic aqueous conditions.
Conversion of methyl ester 10a to hydroxamic acid 10b was
performed in one step [33] leaving the free amine intact,
whereas 11a underwent modification with trifluoromethane
sulfonic anhydride to form sulfonamide 11b, prior to its
conversion to the desired hydroxamic acid 11c. Ligands 10b
and 11c afforded (2:1 ligand-to-metal) complexes upon
reaction with Zn(OAc)2. Hydroxamates are considered
excellent chelators for metal ion transport through cell
membranes [34], whereas both the hydroxamate [34] and the
sulfonamide [35] functionalities are of high medicinal rel-
evance and are present in many bioactive compounds.
Biological evaluation of the effect of compounds
on the insulin/PI3K/PTEN/Akt pathway
Zinc complexes induce Akt activation
The zinc(II) ion and some of its complexes have been
reported in the past to elicit activation of Akt in several cell
types [23, 36–39]. The activation of Akt in these cases
occurs via the PI3K route and can be monitored by
detecting the level of phosphorylation of residue Thr-308
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199
2
H
N
n
H
N
H
H2N
H
N
H
N
N
O
H2O/MeOH/
ZnSO4 /r.t./2 h
6a
: n=3
7a: n=4
N
H
n
2
H2O
nN
O
N
SO4
Zn
H
H
OH2
O
MeOH/
-10 oC-r.t./16 h
N
N
N
N
CHO
n
6b
N
H
N
: n=3
N
O
6
: n=3
7: n=4
7b: n=4
H
H
2
H2N
MeOH/NaBH4/
10% Pd-C/
5 oC/1-1.5 h
H2N
MeOH/H2O/
ZnSO4/r.t./3 h
N
Zn
N
O2N
H2N
H2O
N
H
O2N
2
SO4
N
MeOH/r.t./5 h
H
N
N
N
CHO
OH2
H
N
HN
8b
8a
8
NH2
i)
SO3
H3N
H
O
N
O
S
DMF/Et3N/r.t./30 min
ii) ZnCl2/110 oC/4 h
N
O
Zn
O
N
CHO
N
S
O
O
N
H
9
Scheme 3 Synthesis of pyridino zinc(II) complexes 6–9 (yields 6 83%, 7 51%, 8 30%, 9 20%)
Scheme 4 Synthesis of
hydroxamic zinc(II) complexes
10 and 11 (yields 10 92%, 11
84%). DCM dichloromethane,
Tf2O trifluoromethane sulfonic
anhydride
OH
NH2
O
OH
MeOH/H2O/
Zn(OAc)2/
65 oC/12 h
O
OMe
O
NH
OH2
O
O
HN
Zn
NH
O
O
O
NH2
NH2
10a
NH2
10b
H2N
10
OH
NH
(NH2OH)2.H2SO4/
Na2SO3/NaOH/
H2O/45 oC/16 h
O
OMe
O
OMe
O
OH
DCM/Et3N/Tf2O/
-78 oC-0oC/3-4 h
O O
S
O O
11c
S
11a
11b
N
H
CF3
N
CF3
NH2
NH2
H
O O
S
EtOH/H2O/
OH2
N
CF3
H
NaOH/Zn(OAc)2/
80 oC/16 h
O
O
O
HN
Zn
NH
O
H
N
F3C
11
S
O O
or residue Ser-473 of Akt. To examine whether the new
zinc complexes prepared in this study induce Akt activa-
tion, NIH3T3 cells were treated with a 10 lM concentra-
tion of each compound, followed by light stimulation with
0.04 lM insulin. Western blots were run from the cell
lysates and phospho-Akt was quantified using a phospho-
Akt (Ser-473) antibody. Total Akt was also quantified as
a loading control. Figure 3 summarizes these results.
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2 µM insulin
0.04 µM insulin
spectrophotometer from the absorbance at 590 nm. As can
be seen in Fig. 4, compound 11 shows cytotoxic effects on
NIH3T3 cells at concentrations equal to or higher than
100 lM. At the 10 lM concentration, which was used in
the initial screen of all 11 compounds (Fig. 3), compound
11 has no cytotoxic effect on the cells.
A
Phospho-Akt
Ser473
Akt
To determine the minimum concentration of compound
11 required to induce Akt activation, NIH3T3 cells were
treated with various concentrations of the complex. As can
be seen in Fig. 5a, Akt activation commenced at a 500 nM
concentration of compound 11. Moreover, when probing
the compound-treated cell lysates for glycogen synthase
kinase 3b (GSK-3b) phosphorylation at Ser-9, we found
that 11 was also able to stimulate GSK-3b phosphorylation
with similar potency (Fig. 5a). GSK-3b, a key enzyme in
High
insulin insulin
Low
1
2
3
4
5
2 µM insulin
0.04 µM insulin
B
Phospho-Akt
Ser473
Akt
High
insulin insulin
Low
6
7
8
9
10
11
Fig. 3 a Compounds 1–5 and b compounds 6–11. NIH3T3 cells were
starved overnight, incubated with 10 lM compound (or vehicle) for
15 min, followed by insulin stimulation as indicated. Cells were
collected and analyzed by western blotting using phospho-Akt (Ser-
473) and Akt antibodies. The blots were quantified using ImageJ. The
percentage of activation of Akt (Akt phosphorylation normalized for
loading using the Akt blotting intensities) is shown. Low insulin
stimulation (0.04 lM) was set to 0% and high insulin stimulation
(2 lM) was set to 100%, respectively.The results (bar chart) are
presented as the mean the standard deviation of three independent
experiments
Fig. 4 Cytotoxicity of compound 11. NIH3T3 cells were treated with
up to 1 mM compound 11. The results are presented as the
mean the standard deviation (n = 4)
A positive control (stimulation with high insulin concen-
tration, 2 lM) and a negative control (low insulin con-
centration equal to the concentration used to activate the
insulin receptor, 0.04 lM) were included for comparison.
All compounds, with the exception of 1–3, whose effect is
negligible, appear to activate Akt by elevating phospho-
Akt levels to different extents. Although most of the
compounds exhibit Akt activation to around 50% of that of
the positive control, the two compounds derived from hy-
droxamic acid ligands (10 and 11) stand out, as they appear
to perform equally effectively as or considerably better
than the high insulin activation control, respectively.
Compound 11, as the most promising Akt activator, was
submitted to further investigation. First, we determined the
cytotoxicity of this compound with the 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay on NIH3T3 cells [40]. MTT is reduced to formazan
by living cells, and this reduction can be quantified using a
Fig. 5 a Activation of Akt and the downstream element GSK-3b
(Ser-9) with compound 11 at various concentrations. Starved cells
were incubated with 100, 250, and 500 nM compound 11 for 15 min,
followed by stimulation of insulin at a concentration of 0.04 lM.
b Compound 11 does not increase phosphorylation levels of p44 and
p42 mitogen-activated protein kinase (MAPK). Same cell lysates
(from a) were probed with a phospho-p44/phospho-p42 MAPK (Thr-
202/Tyr-204) antibody
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J Biol Inorg Chem (2011) 16:195–208
201
the regulation of glucose metabolism, is phosphorylated at
Ser-9 by Akt, suggesting that the activation of Akt induced
by the zinc complexes is propagated downstream of Akt to
enhance glycogen synthesis. For comparison, the effect of
compound 11 on the mitogen-activated protein kinase
(MAPK) pathway was investigated by probing cellular
extracts for phosphorylation of p42 and p44 MAPKs. As
shown in Fig. 5b, compound 11 fails to activate p44 and
p42 MAPKs at a concentration of 500 nM, implying that
compound 11 might be a useful chemical tool to probe
glucose metabolism and the signaling controlling it.
suggests they do not act directly at the insulin receptor level,
but rather between the receptor and PI3K. Taking these
observations together, we suggest that the zinc compounds
induce Akt activation in a PI3K-dependent fashion.
Zinc-complex-induced Akt activation is not via inhibition
of PTPs or PTEN
The PI3K/Akt cascade is modulated by phosphatases of the
PTP family and PTEN. PTPs oppose the signaling medi-
ated by receptor tyrosine kinases by removing tyrosine
phosphorylation, whereas PTEN directly antagonizes PI3K
by dephosphorylating phosphatidylinositol 3,4,5-trisphos-
phate, the product of PI3K activity. As both PTPs and
PTEN are upstream of Akt, their phosphatase activity can
influence the level of Akt activation. Indeed, it has been
found that inhibition of PTPs and PTEN will increase the
cellular activity of Akt [42, 43].
Zinc-complex-induced Akt activation is PI3K-dependent
To investigate whether the observed Akt phosphorylation is
PI3K-mediated, we studied the effects of compound 11 on
Akt phosphorylation levels in the presence of LY-294002, a
known PI3K inhibitor [41]. If the observed Akt activation is
PI3K-dependent, the presence of LY-294002 would be
expected to abolish the activation induced by the zinc
compound. Indeed, use of LY-294002 abrogated the phos-
phorylation of Akt induced by compound 11 as shown in
Fig. 6. To further confirm the hypothesis that the zinc-
complex-mediated activation occurs via PI3K, we exam-
ined whether any of the prepared complexes can induce Akt
activation without any insulin stimulation. Notably, when
there is complete deprivation of insulin, the compounds are
unable to induce Akt phosphorylation (Fig. 7), which
Zinc(II) has been reported to have the ability to inhibit
phosphatases such as k-phosphoprotein phosphatase [44],
PTP 1B [45], as well as PTEN [24]. To investigate whether
the observed Akt activation was due to inhibition of PTEN
and PTPs, the two most promising Akt-activating com-
pounds (hydroxamic subset; 10 and 11) were evaluated as
PTEN and PTP-b inhibitors in vitro. As shown in Fig. 8,
the inhibition of PTP-b and PTEN by compounds 10 and
11 revealed a significantly higher potency toward PTP-b,
but failed to demonstrate the nanomolar potency achieved
for Akt phosphorylation in cellulo. A similar picture was
observed for other representative members of the series
(results not shown).
Conclusion
In summary, a series of 11 novel zinc(II) complexes incor-
porating medicinally relevant functionalities were prepared
based on phenolic, picolinic, pyridino, and hydroxamic
ligands. Screening of the compounds for their potential to
upregulate Akt phosphorylation in cellulo revealed a sul-
fonamide-containing hydroxamic complex as a potent acti-
vator of Akt at submicromolar concentrations. This lead
structure can be potentially used as a chemical tool in dia-
betes research, owing to the fact that apart from activating
Akt signaling, its effect is propagated downstream of Akt
toward GSK-3b, an important regulator of glycogen syn-
thesis/glucose metabolism. Our findings suggest that the
observed effects on Akt phosphorylation by the hydroxamic
zinc complexes, although PI3K-mediated, are not due to
the inhibition of the tumor-suppressor phosphatase PTEN or
PTPs. This signifies a departure from other zinc and
vanadium complexes that have been shown to cause Akt
activation by inhibiting these phosphatases.
Fig. 6 Effects of compound 11 on Akt phosphorylation with and
without the PI3K inhibitor LY-294002
Fig. 7 Phospho-Akt (Ser-473) blots after cells were treated either
with the zinc complexes without any insulin stimulation or with
insulin (2 lM). The loading control was performed using the
detection of total Akt
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Fig. 8 Inhibition of PTEN and protein tyrosine phosphatase b (PTP-
b) with zinc(II) complexes 10 and 11 using 3-O-methylfluorescein
phosphate (OMFP) and p-nitrophenyl phosphate (p-NPP) as sub-
strates. Recombinant PTEN and PTP-b were preincubated with either
compound 10 or compound 11 at various concentrations at room
temperature for 10 min. Reactions were initialized by adding 200 lM
OMFP for PTEN and 1 mM p-NPP for PTP-b. The IC50 in the
presence of compound 10 was calculated to be 116 25 lM for
PTEN and 42 7 lM for PTP-b. The IC50 of compound 11 was
determined to be 77 5 lM for PTEN and 19 1 lM for PTP-b.
The results are presented as the mean the standard deviation of
three independent experiments
Future research will be directed toward elucidating the
exact mechanism of action of hydroxamic zinc complexes
on Akt activation, as well as the development of improved
Akt activators based on evolution of this novel template.
NaBH4 (0.026 g, 0.70 mmol, 2 equiv) was added dropwise.
The mixture was stirred initially at 0 °C for 1 h, then
allowed to warm up to room temperature. It was then fil-
tered and the yellow precipitate collected was washed with
methanol and dried to afford 43% of reductive amination
product (0.053 g, 0.15 mmol). This compound was then
dissolved in 10 mL of water to afford a clear solution.
Aqueous 0.6 M HCl (0.25 mL, 0.15 mmol, 1 equiv) was
added to the solution and the mixture was stirred briefly at
room temperature, before the solvent was evaporated to
dryness. The solid residue obtained was washed with die-
thyl ether to afford complex 1 in nearly quantitative yield
(0.058 g, 0.15 mmol). 1H-NMR (D2O, 4.79 ppm) d (ppm):
3.07 (t, 2H, J = 5.4 Hz), 3.75 (t, 2H, J = 5.4 Hz), 4.06 (s,
2H), 4.08 (s, 2H), 6.69 (bd, 1H, J = 8.6 Hz), 7.24 (m, 2H),
8.26 (s, 1H). 13C-NMR (D2O) d (ppm): 48.1, 50.4, 56.7,
56.8, 119.5, 122.4, 126.0, 135.2, 137.6, 149.9, 168.9. Ele-
mental analysis: calculated for C12H19ClN2O6Zn: C 37.13,
H 4.93, N 7.22; found: C 36.57, H 4.12, N 6.96.
Materials and methods
General procedures
1
The purity of the organic chemicals was verified by H-
1
NMR spectroscopy. H-NMR and 13C-NMR spectra were
recorded with a Bruker Avance 400 MHz Ultrashield NMR
spectrometer. Infrared spectra were recorded with a Perkin
Elmer Fourier transform infrared spectrometer. Electro-
spray ionization (ESI) mass spectra were recorded with a
Bruker Daltronics Esquire 3000 spectrometer. Elemental
analyses were performed by either London Metropolitan
University or the University of Cambridge. The following
compounds were prepared following literature procedures:
1a [25], 5a [30], 10a [31], 10b [33], and 11a [32].
Synthesis of 2
Synthesis
Racemic homocysteic acid (0.150 g, 0.82 mmol, 1 equiv)
was added to 5 mL of water and the resulting mixture was
Synthesis of 1
heated at 70 °C.
A solution of 3-formyl-4-hydro-
xybenzenesulfonic acid disodium salt (0.201 g, 0.82 mmol,
1 equiv) in 5 mL of water was then added and the mixture
Intermediate 1a (0.107 g, 0.35 mmol, 1 equiv) was sus-
pended in 45 mL of methanol. Ethanolamine (42 lL,
0.70 mmol, 2 equiv) was added and the mixture was stirred
at room temperature overnight. The resulting solution was
then cooled to 0 °C and 2 mL of a methanolic solution of
was stirred at 70 °C for 90 min.
A solution of
Zn(OAc)2ꢀ2H2O (0.223 g, 1.02 mmol, 1.24 equiv) in 10 mL
of water was finally added. During this last addition, the pH
of the solution was adjusted to about 7 with aqueous NaOH
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203
Synthesis of 4
(0.5 M). The solution was stirred at 70 °C for a further 2 h.
The solvent was then evaporated to dryness and a yellow-
orange residue was obtained. Successive recrystallizations
in water/methanol yielded compound 2 as a white solid in
54% yield (0.217 g, 0.44 mmol). 1H-NMR (D2O,
4.79 ppm) d (ppm): 2.25 (m, 2H), 2.86 (m, 2H), 4.03 (t,
1H, J = 6.2 Hz), 6.72 (d, 1H, J1 = 9.0 Hz), 7.57 (dd, 1H,
J1 = 9.0 Hz, J2 = 2.5 Hz), 7.66 (d, 1H, J2 = 2.5 Hz),
8.38 (s, 1H). 13C-NMR (D2O) d (ppm): 29.9, 46.8, 66.3,
118.4, 122.2, 128.5, 130.9, 133.9, 169.7, 171.1, 179.1.
Elemental analysis: calculated for C11H13NNa2O11S2Znꢀ
2H2O: C 24.16, H 3.16, N 2.56; found: C 24.42, H 3.33, N
2.54.
A solution of ethylenediamine (0.027 mL, 0.41 mmol, 0.5
equiv) in 2 mL of methanol was added to a solution of 3-
formyl-4-hydroxybenzenesulfonic acid disodium salt
(0.202 g, 0.82 mmol, 1 equiv) in 40 mL of methanol and
the reaction mixture was stirred at room temperature for
1 h. Zn(OAc)2ꢀ2H2O (0.090 g, 0.41 mmol, 0.5 equiv) was
then added and the mixture was heated at 50 °C overnight.
It was subsequently filtered, and the white residue collected
was washed with cold methanol and dried under a vacuum
1
to afford 4 in 95% yield (0.216 g, 0.39 mmol). H-NMR
(D2O, 4.79 ppm) d (ppm): 3.79 (s, 4H), 6.80 (d, 2H,
J1 = 8.9 Hz), 7.55 (dd, 2H, J1 = 8.9 Hz, J2 = 2.5 Hz),
7.64 (d, 2H, J2 = 2.5 Hz), 8.43 (s, 2H). 13C-NMR (D2O) d
(ppm): 55.8, 119.2, 122.6, 128.3, 130.0, 133.1, 168.1,
170.8. Elemental analysis: calculated for C16H14N2Na2
O9S2Znꢀ0.5H2O: C 34.15, H 2.69, N 4.98; found: C 34.11,
H 2.72, N 4.89.
Synthesis of 3a
3-Formyl-4-hydroxybenzenesulfonic acid disodium salt
(0.300 g, 1.22 mmol, 1 equiv) was dissolved in 40 mL of
methanol, and propylamine (0.121 mL, 1.47 mmol, 1.2
equiv) was added. The solution was stirred at room
temperature for 3 h, at which point an aliquot was
obtained to verify by NMR that the imine intermediate
had formed. The solution was then cooled in an ice-bath
and NaBH4 (0.185 g, 4.88 mmol, 4 equiv) was added.
The mixture was stirred at 0 °C for 1 h and at room
temperature overnight. A few drops of water were added
and then the solvent was evaporated to dryness. The
residue was redissolved in methanol and insoluble
impurities were removed by filtration. The filtrate was
concentrated under a vacuum and diethyl ether was
added to precipitate the product. Compound 3a was
Synthesis of 5
A solution of ZnSO4ꢀ7H2O (0.050 g, 0.175 mmol, 0.5
equiv) in 1.5 mL of water was added to a solution of 5a
(0.080 g, 0.35 mmol, 1 equiv) in 3 mL of ethanol/dime-
thylformamide (1:1). Aqueous NaOH (0.5 M, 0.7 mL,
0.35 mmol, 1 equiv) was added and the mixture was stirred
at room temperature for 3 h. The resulting precipitate was
then filtered off, washed with water, and dried under a
vacuum to furnish 34% of complex
5 (0.033 g,
0.06 mmol). 1H-NMR [dimethyl sulfoxide (DMSO)-d6,
2.50 ppm] d (ppm): 4.34 (d, 4H, J = 5.2 Hz), 7.12 (d, 2H,
J = 7.2 Hz), 7.24 (m, 2H), 7.32 (m, 8H), 7.45 (bs, 2H),
7.85 (d, 2H, J = 8.8 Hz), 7.94 (bs, 2H). Elemental analy-
sis: calculated for C26H26N4O6Zn: C 56.17, H 4.71, N
10.08; found: C 56.18, H 4.60, N 10.14.
obtained as
a pink solid in 72% yield (0.255 g,
1
0.88 mmol). H-NMR (D2O, 4.79 ppm) d (ppm): 0.85 (t,
3H, J = 7.4 Hz), 1.58 (bs, 2H), 2.88 (t, 2H, J = 7.4 Hz),
4.02 (s, 2H), 6.55 (d, 1H, J = 8.6 Hz), 7.45 (m, 2H).
This ligand was used for the synthesis of 3 without any
further purification.
Synthesis of 6a
Synthesis of 3
Ethyl isocyanate (0.29 mL, 3.66 mmol, 1 equiv) was
slowly added to a cooled solution (-10 °C) of 1,3-diami-
nopropane (0.305 mL, 3.66 mmol, 1 equiv) in 35 mL of
CH2Cl2. The mixture was then stirred overnight at room
temperature, resulting in precipitation of the desired urea
product (6a). This solid was collected by filtration. Addi-
tionally, the filtrate was dried under a vacuum to yield an
oily residue, which upon treatment with diethyl ether
afforded more of the product. The combined product was
washed with diethyl ether and dried. Overall, 63% of 6a
To a solution of 3a (0.100 g, 0.35 mmol, 1 equiv) in 10 mL of
methanol was added a solution of Zn(OAc)2ꢀ2H2O (0.038 g,
0.175 mmol, 0.5 equiv) in 5 mL of methanol and the resulting
mixture was stirred at 50 °C overnight. The solvent was
evaporated to dryness and complex 3 was obtained as a yel-
low residue. It was further purified by recrystallization in
methanol/diethyl ether to afford 70% (0.081 g, 0.12 mmol) of
3. 1H-NMR (D2O, 4.79 ppm) d (ppm): 0.86 (t, 6H,
J = 7.1 Hz), 1.59 (bs, 4H), 2.90 (bt, 4H, J = 6.7 Hz), 4.06 (s,
4H), 6.63 (d, 2H, J = 9.2 Hz), 7.59 (m, 4H). Also present was
1 equiv of NaOAc impurity: 1.82 (s, 3H). Elemental analysis:
calculated for C20H26N2Na2O8S2ZnꢀNaOAc: C 38.86, H
4.30, N 4.12; found: C 38.77, H 4.41, N 3.98.
1
was obtained (0.335 g, 2.31 mmol). H-NMR (DMSO-d6,
2.50 ppm) d (ppm): 0.95 (t, 3H, J = 7.0 Hz), 1.40 (m, 2H),
2.50 (m, 2H), 3.05 (m, 4H), 5.80 (bs, 2H). Liquid chro-
matography–mass spectrometry (LC–MS) (ESI?):
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Synthesis of 7b
calculated for C6H15N3O (M?H) 146.13, found 146.13.
This compound was used in the next step without any
further purification.
This compound was prepared in 99% yield, following a
1
method analogous to that used for the synthesis of 6b. H-
NMR (DMSO-d6, 2.50 ppm) d (ppm): 0.96 (t, 3H,
J = 7.2 Hz), 1.40 (m, 2H), 1.61 (m, 2H), 2.99 (m, 4H),
3.62 (t, 2H, J = 7.2 Hz), 5.71 (t, 1H, J = 5.6 Hz), 5.80 (t,
1H, J = 5.2 Hz), 7.45 (m, 1H), 7.86 (td, 1H, J1 = 7.6 Hz,
J2 = 1.2 Hz), 7.93 (d, 1H, J1 = 7.6 Hz), 8.33 (s, 1H), 8.63
(d, 1H, J = 4.8 Hz). 13C-NMR (DMSO-d6, 39.52 ppm) d
(ppm): 16.2, 28.2, 28.4, 34.5, 60.6, 120.8, 125.5, 137.3,
149.8, 154.6, 158.5, 162.1. Elemental analysis: calculated
for C13H20N4O: C 62.88, H 8.12, N 22.56; found: C 62.96,
H 8.09, N 22.48.
Synthesis of 6b
Pyridine-2-carboxaldehyde (0.10 mL, 1.0 mmol, 1 equiv)
was slowly added (over 10 min) to a cold solution
(-10 °C) of 6a (0.145 g, 1.0 mmol, 1 equiv) in 2 mL of
methanol, and the mixture was stirred overnight at room
temperature. The solvent was then removed under a vac-
uum to yield a white residue, which was washed several
times with diethyl ether and dried to furnish 93% of 6b
1
(0.217 g, 0.93 mmol). H-NMR (DMSO-d6, 2.50 ppm) d
(ppm): 0.97 (t, 3H, J = 7.2 Hz), 1.73 (m, 2H), 2.98 (m,
2H), 3.05 (apparent q, 2H, J = 6.4 Hz), 3.62 (t, 2H,
J = 6.8 Hz), 5.76 (t, 1H, J = 5.6 Hz), 5.86 (t, 1H,
J = 5.6 Hz), 7.45 (m, 1H), 7.86 (td, 1H, J1 = 7.6 Hz,
J2 = 1.2 Hz), 8.34 (s, 1H), 8.63 (d, 1H, J = 4.8 Hz), 8.94
(d, 1H, J1 = 7.6 Hz). 13C-NMR (DMSO-d6, 39.52 ppm) d
(ppm): 16.2, 31.6, 34.5, 37.7, 58.4, 120.9, 125.5, 137.3,
149.8, 154.6, 158.5, 162.4. Elemental analysis: calculated
for C12H18N4O: C 61.52, H 7.74, N 23.91; found: C 61.52,
H 7.64, N 23.84.
Synthesis of 7
This compound was prepared in 51% yield, following a
method analogous to that used for the synthesis of 6. 1H-NMR
(DMSO-d6, 2.50 ppm) d (ppm): 0.93 (t, 6H, J = 7.2 Hz),
1.31 (m, 4H), 1.61 (m, 4H), 2.95 (m, 8H), 3.60 (bs, 4H), 5.93
(bs, 2H), 6.02 (bs, 2H), 7.67 (bs, 2H), 7.97 (d, 2H,
J = 7.6 Hz), 8.10 (bs, 2H), 8.53 (s, 2H), 8.82 (bs, 2H). Ele-
mental analysis: calculated for C26H44N8O8SZnꢀ2.5H2O: C
42.25, H 6.68, N 15.16; found: C 42.13, H 5.89, N 15.16.
Synthesis of 6
Synthesis of 8a
A solution of ZnSO4ꢀ7H2O (0.100 g, 0.35 mmol, 0.5
equiv) in 2 mL of water was added to a solution of 6b
(0.163 g, 0.70 mmol, 1 equiv) in 2 mL of methanol, and
the resulting mixture was stirred at room temperature for
2 h. The solvent was then removed under a vacuum,
yielding complex 6 as a pale-brown solid in 83% yield
A solution of 5-nitropyridine-2-carboxaldehyde (0.386 g,
2.54 mmol, 1 equiv) in 20 mL of dry methanol was added
dropwise to
a solution of propylamine (0.24 mL,
2.92 mmol, 1.15 equiv) in 8 mL of methanol. The mixture
was stirred for 5 h at room temperature and the solvent was
then removed under a vacuum, to afford 8a quantitatively
as a dark oil (0.49 g, 2.54 mmol). 1H-NMR (CDCl3,
7.26 ppm) d (ppm): 1.00 (t, 3H, J = 7.4 Hz), 1.79
(apparent sextet, 2H, J = 7.2 Hz), 3.73 (td, 2H, J1 =
6.9 Hz, J2 = 1.3 Hz), 8.23 (d, 1H, J3 = 8.7 Hz), 8.47 (bs,
1H), 8.53 (dd, 1H, J3 = 8.7 Hz, J4 = 2.4 Hz), 9.47 (d, 1H,
J4 = 2.4 Hz). 13C-NMR (CDCl3, 77.00 ppm) d (ppm):
11.9, 23.8, 63.5, 121.21, 131.6, 144.4, 144.9, 159.2, 159.9.
This compound was used for the next step without any
further purification.
1
(0.193 g, 0.29 mmol). H-NMR (DMSO-d6, 2.50 ppm) d
(ppm): 0.88 (t, 6H, J = 7.2 Hz), 1.74 (m, 4H), 2.86 (m,
4H), 3.01 (apparent quartet, 4H, J = 5.6 Hz), 3.65 (bs,
4H), 5.85 (bs, 2H), 6.17 (bs, 2H), 7.66 (bs, 2H), 7.94
(d, 2H, J = 7.6 Hz), 8.08 (bs, 2H), 8.52 (s, 2H), 8.78
(bs, 2H). Elemental analysis: calculated for C24H40N8O8
SZnꢀH2O: C 42.14, H 6.19, N 16.38; found: C 42.33, H
5.69, N 16.33.
Synthesis of 7a
Synthesis of 8b
This compound was prepared in 28% yield, following a
1
method analogous to that used for the synthesis of 6a. H-
A solution of 8a (0.293 g, 1.52 mmol, 1 equiv) in 25 mL of
dry methanol was mixed with 0.09 g of 10% Pd–C and
cooled to 5 °C. NaBH4 (0.23 g, 6.1 mmol, 4 equiv) was
added portionwise to the mixture over 30 min. The
resulting solution was stirred at the same temperature for
1 h, then allowed to warm up to room temperature. It was
NMR (DMSO-d6, 2.50 ppm) d (ppm): 0.95 (t, 3H,
J = 7.1 Hz), 1.40 (m, 4H), 2.50 (bt, 2H, overlapping with
DMSO peak), 3.00 (m, 4H), 5.71 (bt, 1H), 5.80 (bt, 1H).
LC–MS (ESI?): calculated for C7H17N3O (M?H) 160.15,
found 160.15. This compound was used in the next step
without any further purification.
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205
1
subsequently filtered through Celite and the solvent was
evaporated to dryness. The remaining residue was resus-
pended in ethyl acetate and the organic phase was washed
with water. The aqueous phase was back-extracted with
CHCl3. All organic fractions were dried over MgSO4 and
the solvent was evaporated to dryness to afford 8b as a
a pink powder in 20% yield (0.10 g, 0.2 mmol). H-NMR
(DMSO-d6, 2.50 ppm) d (ppm): 3.03 (bs, 4H), 4.24 (bs,
4H), 7.67 (t, 2H, J = 6.4 Hz), 8.03 (d, 2H, J = 8.0 Hz),
8.17 (bs, 2H), 8.36 (bs, 2H), 8.88 (bs, 2H). 13C-NMR
(DMSO-d6, 39.52 ppm) d (ppm): 49.86, 54.46, 128.94,
141.11, 148.63, 163.60. IR (cm-1): 738, 1,228, 1,530,
1,594, 2,943, 3,235, 3,331. Elemental analysis: calculated
for C16H18N4O6S2Zn: C 39.07, H 3.69, N 11.39; found: C
39.13, H 3.73, N 11.45.
1
yellow oil in 55% yield (0.139 g, 0.84 mmol). H-NMR
(CDCl3, 7.26 ppm) d (ppm): 0.93 (m, 3H), 1.54 (m, 2H),
2.61 (m, 2H), 3.66 (bs, 2H), 3.79 (s, 2H), 6.96 (dd, 1H,
J1 = 8.2 Hz, J2 = 2.8 Hz), 7.09 (d, 1H, J1 = 8.2 Hz),
8.06 (d, 1H, J2 = 2.8 Hz). 1H-NMR (DMSO-d6, 2.50 ppm)
d (ppm): 0.85 (m, 3H), 1.41 (m, 2H), 2.46 (m, 2H), 3.60 (s,
Synthesis of 10
0
2H), 5.13 (bs, 2H), 6.88 0 (dd, 1H, J1 = 8.3 Hz,
4-Amino-N-hydroxybenzamide (10b) (0.20 g, 1.32 mmol,
1 equiv) was transferred to a round-bottom flask and dis-
solved in methanol (13.2 mL). Zn(OAc)2ꢀ2H2O (0.145 g,
0.66 mmol, 0.5 equiv) was added, and the reaction mixture
was refluxed at 65 °C for 12 h, during which a white
precipitate formed. After the mixture had cooled to room
temperature, it was filtered and the solid filtered off was
washed several times with chilled water and dried under a
vacuum. Overall, 92% (0.235 g, 0.61 mmol) of complex
10 was obtained. 1H-NMR (DMSO-d6, 2.50 ppm) d (ppm):
5.50 (s, 4H), 6.53 (d, 4H, J = 8.6 Hz), 7.48 (d, 4H,
J = 8.6 Hz), 11.50 (s, 2H). 1H-NMR (D2O, 4.79 ppm, with
1% formic acid-d2) d (ppm): 7.26 (d, 4H, J = 8.0 Hz), 7.71
(d, 4H, J = 8.0 Hz). LC–MS (ESI?): calculated for
C14H14N4O4Zn (M?H) 367.04, found 367.04. IR (cm-1):
1,504, 1,563, 1,598, 3,018, 3,170, 3,431. Elemental anal-
ysis: calculated for C14H16N4O5Zn: C 43.59, H 4.18, N
14.53; found: C 44.02, H 3.71, N 14.35.
0
J2 = 2.7 Hz), 7.03 (d, 1H, J1 = 8.3 Hz), 7.85 (d, 1H,
J2 = 2.7 Hz). 13C-NMR (CDCl3, 77.00 ppm) d (ppm):
0
11.8, 23.2, 51.5, 54.7, 122.3, 122.6, 136.9, 141.0, 149.9.
13C-NMR (DMSO-d6, 39.52 ppm) d (ppm): 12.3, 22.9,
51.1, 54.4, 121.0, 122.4, 135.8, 143.7, 147.3. This com-
pound was used for the next step without any further
purification.
Synthesis of 8
A solution of ZnSO4ꢀ7H2O (0.121 g, 0.42 mmol, 0.5
equiv) in 10 mL of methanol was added to a solution of 8b
(0.139 g, 0.84 mmol, 1 equiv) in 10 mL of methanol, and
the resulting mixture was stirred at room temperature for
3 h. The solvent was then evaporated to dryness and the
residue was washed with CH2Cl2 and ethyl ether. Recrys-
tallization in methanol/diethyl ether afforded complex 8 as
1
a yellow solid in 30% yield (0.066 g, 0.125 mmol). H-
NMR (DMSO-d6, 2.50 ppm) d (ppm): 0.81 (t, 6H,
J = 7.3 Hz), 1.50 (m, 4H), 2.62 (bs, 4H), 3.80 (bs, 4H),
5.58 (bs, 2H), 5.71 (bs, 4H), 7.18 (bs, 4H), 8.03 (bs, 2H).
13C-NMR (DMSO-d6, 39.52 ppm) d (ppm): 11.8, 20.9,
50.3, 50.4, 123.3, 123.7, 134.7, 142.4, 145.6. LC–MS
(ESI?): calculated for C18H28N6Zn (M?H) 393.18, found
393.18. IR (cm-1): 1,118 (SO42-), 1,506 (m), 1,581 (m),
1,630 (m), 1,735 (br), 2,875 (br), 2,964 (br), 3,229, 3,349.
Elemental analysis: calculated for C18H34N6O6SZnꢀ2H2O:
C 38.33, H 6.79, N 14.90; found: C 38.58, H 6.15, N 14.27.
Synthesis of 11b
Methyl 4-(aminomethyl)benzoate (11a) (1.65 g, 10 mmol,
1 equiv) was transferred to a round-bottom flask, which
was then sealed and purged with nitrogen. Anhydrous
CH2Cl2 (33 mL) was added and the mixture was cooled to
-78 °C. Triethylamine (2.8 mL, 20 mmol, 2 equiv) was
added, followed by dropwise addition of trifluorometh-
anesulfonic anhydride (1.7 mL, 10 mmol, 1 equiv) at -
78 °C. The mixture was stirred for 3–4 h, with the tem-
perature slowly rising to 0 °C. The reaction mixture was
then diluted with CH2Cl2, and the resulting mixture was
washed once with aqueous NH4Cl (saturated), once with
aqueous NaCl (saturated), and once with H2O. The organic
phase was dried over Na2SO4, then filtered and concen-
trated under a vacuum. The sample was applied to a silica
column and eluted with hexane–ethyl acetate (first 2:1,
then 1:1), to afford 11b in 88% yield (2.61 g, 8.8 mmol) as
Synthesis of 9
Pyridine-2-carboxaldehyde (0.19 mL, 2 mmol, 1 equiv)
was dissolved in 50 mL of dimethylformamide in a round-
bottom flask. Taurine (0.25 g, 2 mmol, 1 equiv) was added
to this solution, followed by anhydrous triethylamine
(0.28 ml, 2 mmol, 1 equiv). After the mixture had been
stirred at room temperature to solubilize all components,
ZnCl2 was added (0.14 g, 1 mmol, 0.5 equiv) and the
reaction mixture was heated at 110 °C for 4 h. The
resulting precipitate was filtered and washed to afford 9 as
1
a white solid. H-NMR (CDCl3, 7.26 ppm) d (ppm): 3.92
(s, 3H), 4.51 (s, 2H), 5.43 (bs, 1H), 7.39 (d, 2H,
J = 8.3 Hz), 8.01 (d, 2H, J = 8.3 Hz). 13C-NMR (CDCl3,
77.00 ppm) d (ppm): 47.65, 52.37, 119.62 (CF3 quartet),
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J Biol Inorg Chem (2011) 16:195–208
127.61, 130.03, 130.19, 140.49, 166.89. LC–MS (ESI?):
calculated for C10H10F3NO4S (M?H) 298.04, found
298.04. IR (cm-1): 1,001, 1,018, 1,081, 1,119, 1,150,
1,181, 1,233, 1,289, 1,306, 1,386, 1,445, 1,615, 1,695,
3,229. Elemental analysis: calculated for C10H10F3NO4S:
C 40.41, H 3.39, N 4.71; found C 40.51, H 3.27, N 4.64.
2.50 ppm) d (ppm): 4.27 (s, 4H), 7.35 (d, 4H, J = 7.7 Hz),
7.66 (d, 4H, J = 7.7 Hz), 11.82 (s, 2H). LC–MS (ESI?):
calculated for C18H16F6N4O8S2Zn (M?H) 658.97, found
658.97. IR (cm-1): 874, 918, 1,022, 1,056, 1,149, 1,190,
1,231, 1,372, 1,441, 1,503, 1,565, 1,606, 3,350 (br). Ele-
mental analysis: calculated for C18H18F6N4O9S2ZnꢀH2O: C
31.07, H 2.90, N 8.05; found C 31.00, H 2.40, N 7.63.
Synthesis of 11c
Biological materials and methods
Hydroxylamine sulfate (0.82 g, 5 mmol, 1 equiv) was
transferred to a round-bottom flask containing 5 g of ice. A
solution of NaOH (1.0 g, 25 mmol, 5 equiv) in 5 mL of
water was added to the flask and the mixture was stirred at
room temperature for 5 min. A catalytic amount of Na2SO3
(0.095 g, 0.75 mmol, 0.15 equiv) and methyl 4-
Cell culture and western blot analysis
NIH3T3 fibroblasts were purchased from ATCC. Cells
were grown in Dulbecco’s modified Eagle’s medium
(DMEM) (Sigma) supplemented with 10% (v/v) bovine
calf serum (ATCC) in an atmosphere of 5% CO2 at 37 °C.
For testing the zinc complexes, cells were starved in
DMEM (without serum) for 16 h at 37 °C. Subsequent to
serum deprivation, cells were treated with zinc compounds
for 15 min in DMEM at 37 °C. Following compound
treatment, cells were stimulated with either 2 lM insulin or
0.04 lM insulin for 15 min. A control was included where
cells were neither treated with zinc compounds nor stim-
ulated with insulin. The cells were washed afterwards three
times with ice-cold phosphate-buffered saline and then
lysed in sodium dodecyl sulfate (SDS)–polyacrylamide gel
electrophoresis buffer, containing 150 mM tris(hydroxy-
methyl)aminomethane (Tris–HCl), pH 6.8, 25% glycerol,
5% SDS, 5% b-mercaptoethanol, and 0.01% bromophenol
blue. Cell lysates were separated on a 10% SDS–poly-
acrylamide gel and transferred onto a nitrocellulose
membrane. Membranes were blocked with 5% skimmed
milk in 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH
7.4 (TBS/T) for 1 h at room temperature. Phospho-Akt
(Ser-473) rabbit monoclonal antibody, mass-Akt rabbit
polyclonal antibody, GSK-3b (Ser9) rabbit polyclonal
antibody, and phospho-p44/p42 MAPK (Thr-202/Tyr-204)
rabbit polyclonal antibody (Cell Signaling) were used as
primary antibodies for immunoblotting analysis. Primary
antibody incubation was performed in 5% bovine serum
albumin in TBS/T for 16 h at 4 °C. Blots were washed for
15 min with TBS/T, then incubated with horseradish per-
oxidase conjugated secondary antibody (Bio-Rad) in
blocking buffer for 1 h, and then probed with western
blotting detection reagent (ECL, GE Healthcare).
[(trifluoromethylsulfonamido)methyl]benzoate
(11b)
(1.49 g, 5 mmol, 1 equiv) were then added in solid form,
and the mixture was heated at 45 °C for 16 h. After the
mixture had cooled to room temperature, the pH was
modified to about 5 by dropwise addition of 10% (w/w)
aqueous H2SO4, and the solvent was removed under a
vacuum. Cold methanol was added to the solid residue and
the resulting suspension was vigorously stirred for 15 min
and filtered. The hydroxamic acid, free of most inorganic
impurities, was recovered by drying the methanolic filtrate.
This sample was redissolved in 90:10 CH2Cl2–CH3OH and
applied to a silica column. Elution took place with the same
solvent system (first 90:10, then 85:15) to afford 76%
(1.13 g, 3.8 mmol) of hydroxamic acid 11c as a white
1
solid. H-NMR (DMSO-d6, 2.50 ppm) d (ppm): 4.42 (s,
2H), 7.40 (d, 2H, J = 8.1 Hz), 7.75 (d, 2H, J = 8.1 Hz),
9.04 (s, 1H), 10.02 (s, 1H), 11.22 (s, 1H). 13C-NMR
(DMSO-d6, 39.52 ppm) d (ppm): 46.34, 119.69 (CF3
quartet), 127.21, 127.38, 132.20, 140.22, 163.92. LC–MS
(ESI?): calculated for C9H9F3N2O4S (M?H) 299.03,
found 299.03. IR (cm-1): 1,511, 1,542, 1,577, 1,618, 3,388.
Elemental analysis: calculated for C9H9F3N2O4S: C 36.24,
H 3.04, N 9.39; found C 36.25, H 3.02, N 9.11.
Synthesis of 11
N-Hydroxy-4-[(trifluoromethylsulfonamido)methyl]benzam-
ide 11c (0.63 g, 2.11 mmol, 1 equiv) was transferred to a
round-bottom flask and dissolved in absolute ethanol
(15 mL). A solution of NaOH (0.084 g, 2.11 mmol, 1
equiv) in 5 mL of water was added, and the mixture was
stirred at room temperature for 5 min. Solid
Zn(OAc)2ꢀ2H2O (0.23 g, 1.055 mmol, 0.5 equiv) was then
added, and the reaction was refluxed at 80 °C for 16 h. The
solvent was then removed under a vacuum and the solid
residue was washed extensively with cold water and dried
under a vacuum to afford complex 11 in 84% yield (0.60 g,
0.89 mmol), as a white solid. 1H-NMR (DMSO-d6,
PTEN expression and purification
PTEN was expressed as a glutathione S-transferase (GST)
fusion protein and purified according to methods previ-
ously described [13]. Protein expression was induced in the
Escherichia coli strain XL-1 blue for 24 h using 1 mM
isopropyl b-D-1-thiogalactopyranoside at 23 °C. Cells were
123

J Biol Inorg Chem (2011) 16:195–208
207
Acknowledgments This work was supported by the Leverhulme
Trust, the Lowe Syndrome Trust, Imperial Innovations, and the De-
harvested and stored at -20 °C. The harvested cells were
resuspended in lysis buffer containing 50 mM Tris (pH
7.4), 1% Triton X-100, 10 mM benzamidine hydrochlo-
ride, 100 lg/mL soybean trypsin inhibitor, 1 mM 4-(2-
aminoethyl)benzenesulfonyl fluoride hydrochloride, and
2 mM dithiothreitol (DTT). Lysis was performed by add-
ing lysozyme to the cell suspension at a concentration of
2 mg/mL and sonication. Cell debris was removed by
centrifugation at 18,000g for 1 h at 4 °C. The supernatant
was loaded onto a glutathione Sepharose column, pre-
equilibrated with 50 mM Tris (pH 7.4), 140 mM NaCl, and
2.7 mM KCl. After loading, the column was washed twice
with 50 mM Tris (pH 7.4), 140 mM NaCl, 2.7 mM KCl,
and 2 mM DTT. Another two washes were performed
using the same buffer with 500 mM NaCl. The GST-tag-
ged PTEN was eluted using 20 mM glutathione in 50 mM
Tris (pH 7.4), 250 mM NaCl, 20% glycerol, and 2 mM
DTT. Protein concentration was determined using the
Bradford assay.
´
partament d’Educacio i Universitats de la Generalitat de Catalunya. In
addition, the UK’s Engineering and Physical Sciences Research
Council is thanked for a Leadership Fellowship to R.V.
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