Download A Library of Cobalt (III) Schiff Base Complexes for pH

A Library of Cobalt (III) Schiff Base Complexes for pH-Triggered Protein
Inhibition
By Viktorie Reichova
Faculty Adviser: Professor Thomas Meade
Mentor: Dr. Marie Heffern
Abstract
Cobalt(III) Schiff base (Co(III)-sb) complexes inhibit histidine-containing proteins by
interfering with structural or active sites. Through inhibition of proteins relevant to diseases
processes such as cancer metastasis, Co(III)-sb complexes can be developed for therapeutic
approaches. The Meade lab is developing pH-responsive, self-reporting Co(III)-sb
complexes. Here, a library of Co(III)-sb complexes with modifications to the equatorial
ligand was developed in an effort to enhance stability while retaining pH lability. Electronwithdrawing (chloride and phenyl) substituents were added to the parent Co(III)-sb
complexes, termed Co-Acacen, to form Co-ClAcacen, Co-Benacen, and Co-ClBenacen.
These complexes were compared to the parent compound through fluorescence studies.
Although differences were seen between the compounds, solubility issues prevented a deeper
analysis of the results. The complexes with phenyl substituents had a decreased pHdependent lability while the complexes with chloride substituents exhibited greater stability
under neutral conditions.
Introduction
Following the discovery
of cisplatin as an anti-cancer
drug in the late 20th century,
metal complexes have been
gaining popularity as therapeutics. The Meade lab has extensively investigated cobalt(III)
Schiff base complexes (Co(III)-sb) as inhibitors of proteins involved in disease progression
(1-6). The complex contains two labile axial ligands and a tetradentate ligand,
acetylacetonatoethylenediimine (acacen), in the equatorial plain (Figure 1). Co-sb complexes
bind to histidine (His) residues through a dissociative exchange mechanism of the labile axial
ligands (Figure 1) (1-5, 7). His-coordination disrupts protein structure and inhibits function
(Figure 2).
Recent
research
has
shown
that
Co(III)-sb complexes can inhibit two proteins
involved in cancer metastasis: 1) Snail zinc
finger transcription factors and 2) matrix
metalloproteinases. Cancer metastasis is
responsible for >90% of cancer-related deaths, making these proteins highly attractive drug
targets (2, 5-6, 8). Snail proteins regulate a pathway known as the epithelial-to-mesenchymal
transition (EMT) whereby cancer cells lose their adhesion properties and adopt migratory and
invasive behavior (9). Matrix metalloproteinases (MMPs) are endopeptidases that cleave
extracellular matrix proteins, facilitating cancer cell invasion into the bloodstream (10). Both
of these proteins contain zinc-dependent sites that utilize histidine residues to coordinate to
the zinc(II) ion. In the case of Snail proteins, the zinc(II) stabilizes the structure required for
gene regulation, and Co(III)-sb coordination displaces the Zn(II) and disrupts this structure
(Figure 2) (4). In the case of MMPs, zinc(II) facilitates their catalytic function, and Co(III)-sb
coordination to the active site blocks enzyme activity (11). Targeted inhibition can be
achieved by incorporating protein-specific molecules to the acacen equatorial ligand (6).
The goal of this research is to increase control of protein inhibition by developing a
class of self-reporting, pH-responsive Co-sb complexes using a prodrug strategy. Prodrugs
are inactive forms of a drug that activate in the presence of an internal trigger (such as a
change in light, temperature, or chemical composition) (12-13). Prodrugs increase the
efficacy and minimize undesirable side effects of parent drugs by reducing the necessary
dosage and ensuring that the drug activates in the locale of its target (12-13). The use of pH
as a prodrug trigger is applicable in two scenarios: 1) the low pH environment of tumor
microenvironments for selective activation in cancer tissues and 2) the low pH of endosomes
for intracellular activation to reduce off-target binding with extracellular and plasma proteins,
a common challenge with metal-based therapies (14).
To achieve pH-dependent protein inhibition, axial ligands that are inert under neutral
pH but dissociate under decreased pH are incorporated into the Co(III)-sb complex.
Dissociation exposes coordination sites for His binding within the target proteins. The first
generation activatable cobalt complex, termed Co-Acacen (Figure 5), contained axial ligands
that included an imidazole moiety and a coumarin
fluorophore (C3Im) (Figure 3). pH-dependent dissociation
was conferred by the imidadazole moiety of C3Im. The
imidazoles exhibited tight coordination to the cobalt(III)
center under neutral aqueous conditions, rendering the complex inactive. In low pH
environment, protonation of the imidazole nitrogens reduced binding affinity of the axial
ligands, promoting ligand exchange with solvent (water) ligands. The coumarin fluorophore
served to report on ligand dissociation. When coordinated to the Co(III) center, C3Im showed
reduced fluorescence due to quenching by the cobalt(III) center. Fluorescence was restored
upon
ligand
dissociation.
Thus,
fluorescence intensity could be used to
monitor
ligand
dissociation
and
activation of the complex (Figure 4).
While ligand dissociation of CoAcacen exhibited the desired pHdependent dissociation, in the presence
of a competing imidazole molecule
(resembling a His residue) under neutral pH, substitution of the axial ligands in Co-Acacen
by the competing imidazole was observed to occur in part. Consequently, in its unmodified
form, Co-Acacen would be expected to prematurely activate at neutral pH in biological
environments by coordinating to non-target molecules containing His residues. Thus, efforts
must be directed to enhance the stability of the complex in neutral pH.
The acacen ligand in Co-Acacen contains proton (hydrogen) and methyl substituents.
These substituents can be altered to influence the electropositivity of the Co(III) center and
therefore axial ligand stability (15). We hypothesized that altering the substituents of the
acacen ligand may confer the desired stability to the “inactive” state of the Co(III)-sb
complexes (16).
The electron-withdrawing substituents are expected to decrease the electron density of
the electropositive cobalt to tighten coordination of the electron-donating axial ligands. This
has been demonstrated in computational modeling of Co(III)-sb complexes (15). To this end,
electron-withdrawing groups, chloride
and phenyl, were chosen as potential
stability-enhancing substituents. Three
new cobalt complexes derived from the
parent Co-Acacen were synthesized with
these substituents: Co-ClAcacen, CoBenacen, and Co-ClBenacen (Figure 5).
Successful synthesis and purification
were
verified
by nuclear
magnetic
resonance (NMR spectroscopy), and
characterized
both
for
their
pH-
dependent lability and stability in neutral conditions.
Materials and Methods
Synthesis of Equatorial Ligands (Acacen, ClAcacen, Benacen, ClBenacen)
These syntheses were done according to literature procedures in references 17-21.
To synthesize Acacen, 2,4-pentadione was cooled to 0°C in 30% v/v ethanol (EtOH).
Ethylenediamine was added dropwise while stirring. The solution was brought to room
temperature, washed with cold ether, and filtered to produce a white powder (yield=16.6%).
To synthesize the equatorial ligand of Co-ClAcacen, ClAcacen, Acacen was combined
with N-chlorosuccinimide (NClS) in dichloromethane (DCM) and stirred on ice for 20 min.
The reaction mixture was concentrated by rotary evaporation, recrystallized in water, and
filtered to obtain an off-white powder (yield=30.2%).
To synthesize the equatorial ligand of Co-Benacen, Benacen, 1-phenyl-1,3-butanedione
was dissolved in EtOH and cooled to 0°C. Ethylenediamine in EtOH was added dropwise
while stirring. The solution was warmed to room temperature and dried by rotary
evaporation. Product was purified via recrystallization in hot tolune to yield white and shiny
crystals (yield=77.0%).
To synthesize the equatorial ligand of Co-ClBenacen, ClBenacen, Benacen was
combined with NClS in DCM and stirred on ice for 20 min. The mixture was dried by rotary
evaporation and recrystallized in acetone to yield a pastel yellow powder (yield=67.6%).
Synthesis of Axial Ligand, C3Im
Coumarin-3-carboxylic
acid,
dicyclohexylcarbodiimide
(DCC),
and
N-
hydroxysuccinimide (NHS) were dissolved in dry dimethylformamide (DMF) and stirred for
2 h under N2. Histamine in DMF/H2O was added and the reaction was left stirring overnight
under N2. The resulting solution was dried by rotary evaporation, filtered and purified by
high-performance liquid chromatography (HPLC). After collection and evaporation of the
HPLC fractions, the product was resuspended in water and lyophilized to yield a white
flocculent powder. (yield=84.0%).
Synthesis of Cobalt Complexes
C3Im, CoBr2*XH2O, and the corresponding equatorial ligand (Acacen, ClAcacen,
Benacen, or ClBenacen) were dissolved in methanol (MeOH). N,N-Diisopropylethylamine
(DIEA) was added and the reaction was left stirring overnight open to air. The solution was
concentrated by rotary evaporation and the product purified by ether precipitation from
methanol and ethanol. (Co-Acacen: brown powder, yield=33.3%; Co-ClAcacen: light brown
powder, yield=53.9%; Co-Benacen: orange-brown powder, yield=62.1%; Co-ClBenacen:
light brown powder, yield=60.5%).
Characterization of pH-dependent Lability
The pH-dependent lability of the complexes in phosphate buffer (PB, 20 mM, pH 8.0)
was assessed by monitoring fluorescence with HCl titration. 5 mM solutions of the cobalt
complexes were prepared in MeOH. 10μL of each MeOH stock was added to 490μL PB and
0, 3, 7, 7.25, 7.5, 7.75, 8 or 10 μL of 1M HCl were added. The solutions were incubated at
37°C for 1 h and fluorescence intensities were measured using a Synergy 4 High Throughput
Fluorimeter with excitation at 334nm and emission at 410nm. The pH of each sample was
measured with a Symphony pH probe. To determine if fluorescence intensity changes could
be primarily attributed to decrease in pH and not an increase in the ionic strength of the
solution, fluorescence intensities were measured using similar conditions, but titrating NaCl
in the place of HCl.
Characterization- Stability
Stability of the complexes at neutral pH in 100% MeOH in the presence of a competing
imidazole ligand was assessed with fluorescence intensity measurements. 5mM solutions of
the complexes were prepared in MeOH. 0, 0.5, 1, or 4 equivalents of a competing ligand
(4MeIm) were added to 10μL of the methanolic cobalt solutions. The resultant samples were
diluted to a volume of 500μL with MeOH and incubated at 37°C for 1 h. Fluorescence
intensity measurements were subsequently acquired as described for the pH-dependent
lability studies on Synergy 4 High Throughput Fluorimeter.
Results and Discussion
The pH-dependent lability
of the cobalt complexes was
investigated
by
evaluating
fluorescence intensity with HCl
titrations (Figure 6). Despite
altering the substituents on the
equatorial
ligand,
the
Figure 6- HCl titration curves of the four cobalt complexes.
pH-
dependent lability of the axial
ligands is retained for the three new complexes. This suggests that changing the electron
density does not affect the mechanism by which the C3Im axial ligand can dissociate, namely
through protonation of the imidazole moiety. The midpoints of the titration curves
(Fluorescence Intensity vs. pH) represent the pH when half of the axial imidazoles have been
protonated and presumably dissociated from the cobalt complex. For an effective prodrug, an
optimal midpoint would reside in a biologically relevant pH range (between a pH of 4 and 6).
The Co-Acacen and Co-ClAcacen exhibit similar midpoints at pH ~4.5. The midpoint for the
Co-Benacen complex is shifted to slightly more acidic values (pH 3-4). However, at the pH
range tested, a plateau at low pH was not observed, complicating determination of exact
midpoint. Additionally, it should be noted that the Co-Benacen complex exhibited slight
insolubility in the various solvent combinations tested. These solubility issues were
exacerbated with the Co-ClBenacen complex. This may explain the low fluorescence
intensity values observed for the complex. The solubility issue precludes determination of the
midpoint for this complex under the experimental conditions discussed above. Nonetheless,
in comparing the behavior of Co-Acacen, Co-Clacacen, and Co-Benacen, these results
suggest that introduction of a phenyl group may affect the pH range at which axial ligand
lability is observed whereas introduction of a chloride group elicits minimal effect to this
parameter. The complexes exhibited no change in fluorescence with NaCl titrations,
indicating that the observed effects result from changes in pH rather than ionic strength.
Given
the
retained
pH-dependent
behavior of the complexes, the stability of the
complexes at neutral pH in the presence of a
competing imidazole ligand was evaluated
through titration of 4MeIm, a small molecule
model
of
His
residues.
Increase
in
fluorescence intensity with 4MeIm titrations
corresponds to stability of the complex (higher increase in fluorescence relative to the control
with no 4MeIm corresponds to lower stability). At pH 7.4, the presence of the electronwithdrawing substituents appears to alter stability of the cobalt complexes to ligand exchange
(Figure 7). In comparing the complexes, Co-Acacen exhibited greatest reactivity to 4MeIm
competition (fluorescence intensity increased by a factor of 3.7), and thus the lowest stability.
Co-ClAcacen and Co-ClBenacen exhibited the lowest increases in fluorescence (a factor of
2.4), and thus highest stabilities; interestingly, despite the presence of a phenyl group on only
one of the complexes, the behavior of these two complexes was very similar. Co-Benacen
fluorescence increased by a factor of 3.0, suggesting that its stability is higher than CoAcacen but lower than Co-ClAcacen and Co-ClBenacen.
These results imply that the addition of a chloride substituent induced greater effect on
the stability of the complexes than addition of a phenyl group. Previous computational studies
have shown that substitutions made at the ortho/para position have a greater effect on the
thermodynamic and kinetic properties of the molecule, properties that ultimately affect both
stability and lability (15). However, in these studies, the chloride groups are incorporated
meta to both oxygen and nitrogen while the phenyl group is ortho to the oxygen and para to
the nitrogen. Thus, the experimental data deviates from the results predicted by computation
if the position of the substituent were the only influencing factor to stability. Our data suggest
that electronic differences between the substituents may affect stability to a greater degree
than the positions of the substituents of the equatorial backbone.
Conclusions and Future Work
The studies described sought to develop a next generation activatable cobalt complex
through improving stability at a neutral pH while retaining pH-dependent lability at a
biologically relevant range. Three new complexes were synthesized with varying electronwithdrawing substituents. The complexes differed in their stability and lability from the
parent complex. Interestingly, introduction of phenyl groups showed greater effect on the pHdependent lability of the complex than chloride substituents, whereas chloride substituents
conferred greater stability to the complex in the presence of a competing imidazole than the
phenyl group at neutral pH.
A major limitation of this study was the solubility of the complexes. The Co-ClBenacen
in particular was partially insoluble in both phosphate buffer and MeOH, while the CoBenacen and Co-ClAcacen were slightly insoluble in phosphate buffer. Therefore, although
interesting differences were observed between the complexes, the results may be influenced
by differences of solubility. One means by which this challenge was circumvented was by
performing the stability studies in MeOH. However, the behavior of the complexes in
methanol serves only as proof-of-concept and cannot be directly applied to a biological
system. Nevertheless, these results illustrate design principles that can be applied to new
generations of activatable cobalt complexes. Future work will explore different fluorophores
for the axial ligands and more soluble electron-withdrawing substituents to the equatorial
ligand to render the systems biologically relevant.
Another approach to improve stability of Co(III) Schiff base prodrugs that is currently
under investigation is focusing on altering the axial rather than equatorial ligand. A strategy
is being employed in which a bridging linker is incorporated between two imidazoles to
produce a bidentate axial ligand for the complexes. Due to entropic effects, the complex
shows enhanced stability compared to the parent Co-Acacen. This approach will be
developed further, and it may be combined with alterations to the equatorial ligand (guided
by these studies) to produce cobalt complexes with high stability that are activated under
disease-relevant external triggers.
Acknowledgments
I thank Professor Thomas Meade for the opportunity to take part in research in his lab. I also
thank Dr. Marie Heffern, my mentor, for her invaluable guidance, support and advice
throughout my research. I am grateful for the entire Meade lab for their discussions and
assistance with my project. I also wish to acknowledge the WCAS Summer Research Grant,
the Undergraduate Research Grant, and the CLP Summer Fellowship for providing funding
for these studies.
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