A gallium(III) Schiff base-curcumin complex that binds to amyloid-β plaques

Article abstract of DOI:10.1016/j.jinorgbio.2016.02.029

Gallium-68 is a positron-emitting isotope that can be used in positron-emission tomography imaging agents. Alzheimer's disease is associated with the formation of plaques in the brain primarily comprised of aggregates of a 42 amino acid protein called amyloid-β. With the goal of synthesising charge neutral, low molecular weight, lipophilic gallium complexes with the potential to cross the blood–brain barrier and bind to Aβ plaques we have used an ancillary tetradentate N₂O₂ Schiff base ligand and the β-diketone curcumin as a bidentate ligand to give a six-coordinate Ga^3?+ complex. The tetradentate Schiff base ligand adopts the cis-β configuration with deprotonated curcumin acting as a bidentate ligand. The complex binds to amyloid-β plaques in human brain tissue and it is possible that extension of this chemistry to positron-emitting gallium-68 could provide useful imaging agents for Alzheimer's disease.

Full text of DOI:10.1016/j.jinorgbio.2016.02.029

A gallium(III) Schiff Base-curcumin complex that binds to amyloid-β plaques
Jaclyn L. Lange, David J. Hayne, Peter Roselt, Catriona A. McLean,
Jonathan M. White, Paul S. Donnelly
PII:
DOI:
Reference:
S0162-0134(16)30056-3
doi: 10.1016/j.jinorgbio.2016.02.029
JIB 9943
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
12 November 2015
18 February 2016
25 February 2016
Please cite this article as: Jaclyn L. Lange, David J. Hayne, Peter Roselt, Catriona
A. McLean, Jonathan M. White, Paul S. Donnelly, A gallium(III) Schiff Base-curcumin
complex that binds to amyloid-β plaques, Journal of Inorganic Biochemistry (2016), doi:
1
0.1016/j.jinorgbio.2016.02.029
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ACCEPTED MANUSCRIPT
A Gallium(III) Schiff Base-Curcumin Complex That
Binds to Amyloid- Plaques
1
1
2
3
1
Jaclyn L. Lange , David J. Hayne , Peter Roselt , Catriona A. McLean , Jonathan M. White and
*1
Paul S. Donnelly
1
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of
Melbourne, Melbourne, 3010, Victoria, Australia.
2
Research Division, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria,
Australia.
3
Department of Anatomical Pathology, The Alfred Hospital, Victoria 3181, Australia
Highlights
-
-
A neutral gallium complex with curcumin acting as a bidentate ligand has been prepared.
The complex binds to amyloid-beta plaques present in human Alzheimer’s disease brains.
Keywords
Gallium(III) complexes; Gallium radiopharmaceuticals; Alzheimer's disease; Diagnostic imaging;
Positron-emission tomography; Amyloid-beta
Abstract
Gallium-68 is a positron-emitting isotope that can be used in positron-emission tomography
imaging agents. Alzheimer's disease is associated with the formation of plaques in the brain
primarily comprised of aggregates of a 42 amino acid protein called amyloid-. With the goal of
synthesizing charge neutral, low molecular weight, lipophilic gallium complexes with the potential
to cross the blood-brain barrier and bind to A plaques we have used an ancillary tetradentate N O
2
2
3
+
Schiff base ligand and the β-diketone curcumin as a bidentate ligand to give a six-coordinate Ga
1

ACCEPTED MANUSCRIPT
complex. The tetradentate Schiff base ligand adopts the cis- configuration with deprotonated
curcumin acting as a bidentate ligand. The complex binds to amyloid- plaques in human brain
tissue and it is possible that extension of this chemistry to positron-emitting gallium-68 could
provide useful imaging agents for Alzheimer's disease.
Introduction
Alzheimer’s disease (AD), the most common form of neurodegenerative dementia, is
characterised by the presence of extracellular amyloid plaques and intracellular neurofibrillary
tangles in the brain.[1] A major constituent of the extracellular amyloid plaques is an aggregated
peptide called amyloid-β (Aβ), a 39-43 amino acid peptide derived from the amyloid precursor
protein (APP).[2-5] 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.[6] However, oligomers and plaques are thought to be in equilibrium. The
Neurofibrillary Tangles (NFT) consist of a hyper-phosphorylated form of a microtubule-associated
protein called tau.[7]
Definitive diagnosis of AD has traditionally relied on post-mortem histological analysis but
in the last decade diagnostic imaging of A plaque burden in patients has become possible using
positron emission tomography (PET) and radiolabeled tracers that bind to A plaques. These new
imaging agents have provided valuable insights into the role of A plaques to cognitive impairment
and are assisting in re-defining the requirements for clinical diagnosis.[8, 9] One of the first tracers
used for A imaging in humans was a benzothiazole derivative radiolabelled with positron-emitting
1
1
carbon-11, C-PIB (Pittsburgh compound B) (Fig. 1).[10-12] Selected benzothiazole derivatives
are known to bind to A fibrils and plaques and this interaction is mediated by a combination of
hydrophobic and hydrogen bonding interactions. Stilbene and styrylpyridine derivatives are
structurally related to benzothiazoles and also exhibit selective binding to A plaques.[13] A group
of stilbene and styrylpyridine derivatives have been identified as having potential to be of use in the
2

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differential diagnosis of AD from other conditions. Efforts focusing on preparing stilbene or
styrylpyridine derivatives radiolabelled with positron emitting fluorine-18 have culminated with the
1
8
recent FDA approval of F-AV45 (florbetapir).[14-16] The synthesis of compounds radiolabelled
1
8
with F often relies on covalent modification of an organic molecule with the radionuclide that is
produced by a cyclotron. In principle the simple and rapid incorporation of a positron-emitting
metallic isotope into a ligand designed to bind to A plaques is an attractive alternative to covalent
1
8
bond formation with F. Positron-emitting radionuclides of potential interest in this context include
6
1
62
64
68
Cu, Cu, Cu and Ga.[17-22] Relatively recent developments have led to availability of
6
8
68
68
68
positron-emitting Ga from a Ge/ Ga generator. The parent nuclide, Ge, possesses a half-life of
6
8
2
71 days and the generators can provide sufficient quantities of Ga for up to one year resulting in
a relatively inexpensive and reliable source of a positron-emitting radionuclide. The favorable
6
8
decay properties of Ga and a half-life of 68 minutes mean that the radionuclide is attractive for
imaging applications based on peptide-targeted systems and this has been pursued with
6
8
considerable success.[23-33] To date there has been less focus on developing Ga labeled
lipophilic small molecules suitable for brain imaging.[34, 35]
Curcumin (curcH = 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a
component of the Indian spice turmeric that has a long history of use in traditional medicine.
Sustained interest in the broad pharmacological activity of curcumin has led to several clinical trials
investigating the compounds potential as an anticancer agent and as an anti-inflammatory.
Curcumin is also known to bind to A plaques presumably through similar interactions to the
structurally similar Congo Red and substituted stilbene derivatives.[36, 37] As a natural β-diketone
curcumin is a good ligand for metal ions and a large number of metal complexes of curcumin have
99m
been prepared including complexes with the [ Tc(CO) ] core that have potential application in
3
single photon emission tomography (SPECT) imaging.[38-40] The [Ga(curc)] complex has been
6
8
3+
prepared and this lead us to investigate the potential of radiolabeled Ga curcumin complexes as
the basis of new A plaque imaging agents.[41] During the preparation of this manuscript the
3

ACCEPTED MANUSCRIPT
6
8
+
synthesis of complexes proposed to be [ Ga(curc) ] was reported and these complexes
2
demonstrated some affinity for A1-40 fibrils.[42] Herein we described the synthesis of a charge
3
+
neutral lipophilic Ga complex that binds to A plaques in human brains. Our approach employs
an ancillary tetradentate N O Schiff base ligand and the β-diketone curcumin as a bidentate ligand
2
2
3
+
to give a six-coordinate Ga complex.
Experimental
All reagents and solvents were obtained from standard commercial sources and unless otherwise
stated were used as received. Commercial curcumin was purified by chromatography.[41]
Elemental analysis for C, H and N were carried out by Chemical & Microanalytical Services
(CMAS) Pty. Ltd. Belmont, Vic and Microanalytical Unit, Australian National University, ACT.
1
H-NMR spectra were recorded with a Varian FT-NMR 500 spectrometer (Varian, California USA)
1
3
at 500 MHz. C-NMR spectra were recorded with a Varian FT-NMR 400 spectrometer (Varian,
California USA) and acquired at 101 MHz. All NMR spectra were recorded at 298˚K. The reported
chemical shifts (in parts per million) are referenced relative to residual solvent signal. Mass spectra
were recorded on an Agilent 6510 ESI-TOF LC/MS Mass Spectrometer (Agilent, California USA).
Absorbance spectra were obtained on a Shimadzu UV-1650 PC spectrophotometer (Shimadzu,
Sydney NSW) and emission spectra were obtained on a Varian CAREY Eclipse fluorescence
spectrophotometer (Varian, California USA).
Syntheses
N,N-bis(2-ethoxysalicylidene)-1,3-diaminopropane (H L): A mixture of 3-ethoxysalicylaldehyde
2
(0.60 g, 3.6 mmol) and 1,3-diaminopropane (0.13 g, 1. 80 mmol) in methanol (10 mL) was heated
at reflux for 30 minutes. A yellow precipitate formed which was collected by filtration, washed with
cold methanol (2 x 5 mL) and diethyl ether (2 x 5 mL) and dried in vacuo to give yellow crystals of
+
+
H L (0.50 g, 1.35 mmol, 75%); HR-MS: [m/z C H N O + H ] m/z 371.196 (experimental),
2
21 26
2
4
4

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1
3
7
6
6
1
71.190 (calculated). H NMR (400 MHz, DMSO-d ) δ 13.68 (s, 2H, -OH), 8.56 (s, 2H, N=CH),
6
.00 (d, J = 7.9 Hz, 4H, ArH), 6.77 (m, 2H, ArH), 4.02 (q, J = 7.0 Hz, 4H, -CH -CH ), 3.68 (t, J =
2
3
.7 Hz, 4H, -CH -CH -CH -), 2.01 (quintet, J = 6.8 Hz, 2H, -CH -CH -CH ), 1.32 (t, J = 7.0 Hz,
2
2
2
2
2
2-
1
13
H, -CH -CH ). { H} C NMR (101 MHz; DMSO-d ) 166.4, 151.9, 147.1, 123.3, 118.5, 117.7,
2
3
6
16.1, 64.0, 55.4, 31.5, 14.8.
[
GaL(OH )(OHCH )]NO : A mixture of H L (1.19 g, 3.2 mmol) and Ga(NO ) .xH O (1.09 g, 3.0
2 3 3 2 3 3 2
mmol, assuming x = 6) in methanol (20 mL) was heated at reflux for 1.5 hours. A yellow precipitate
was formed which was collected by filtration, washed with cold methanol (10 mL) and diethyl ether
(
20 mL) and dried in vacuo to give [GaL(solvent) ]NO (1.14 g, approx. 2.1 mmol, ~ 71%,
2
3
3
-1
-1
assuming solvent = H O), λ (DMF)/nm 369 (ε/dm cm mol 51,330), λ (DMF)/nm 498 (λ
2
max
em
ex
+
1
3
72); HR-MS: [m/z C H GaN O ] m/z 437.098 (experimental), 437.099 (calculated); H NMR
21 24 2 4
(
400 MHz; DMSO-d ) δ 8.42 (s, 2H, N=CH), 6.99 (m, 4H, ArH), 6.61 (m, 2H, ArH), 4.15 (q, J =
6
7
.0 Hz, 4H, CH -CH ), 3.93-3.90 (m, 4H, N-CH , 1.99-1.98 (m, 2H, CH -CH -CH ), 1.30 (t, J = 7.0
2 3 2 2 2 2
1
13
Hz, 6H, CH -CH ); { H} C NMR (101 MHz; DMSO-d ) δ 170.3, 158.9, 150.0, 126.8, 120.4,
2
3
6
1
18.5, 115.1, 64.8, 60.5, 36.3, 28.1, 25.2, 15.3. Crystals of sufficient quality for structure
determination were grown by exchange of vapours between diethyl ether and a solution of the
complex in ethanol and were revealed by X-ray crystallography to be [GaL(OH )(OHCH )]NO .
2
3
3
[
Ga(curc) ]: Minor modifications to published procedures.[41] A mixture of curcumin (0.88 g, 2.39
3
mmol), Ga(NO ) .xH O (0.34 g, 0.93 mmol, assuming x = 6) and triethylamine (0.16 mL) in
3
3
2
methanol (25 mL) was heated at reflux for 1 hour. The mixture was allowed to cool to ambient
temperature. A red-orange precipitate was isolated using a centrifuge and the supernatant discarded.
The red-orange solid was washed with cold methanol (10 mL) and diethyl ether (2 x 10 mL) and
3
dried in vacuo to give [Ga(curc) ] (0.44 g, 0.38 mmol, 41%); λmax(DMF)/nm 305, 432, 455.5 (ε/dm
3
-1
-1
1
cm mol 17,710, 128,870, 107,350), λ (DMF)/nm 520 (λ 458); H NMR (500 MHz; DMSO-
em
ex.
5

ACCEPTED MANUSCRIPT
d6): δ 9.54 (s, 6H, OH), 7.42 (m, 6H, ArH), 7.23 (m, 6H, ArH), 7.04 (m, 6H, CH=CH), 6.75 (m,
1
13
1
1
2H, ArH), 5.97 (s, 3H, CH), 3.78 (s, 18H, O-CH ). { H} C NMR (101 MHz; DMSO-d ): δ 183.2,
3
6
48.9, 148.0, 139.8, 126.6, 124.8, 122.7, 115.7, 111.0, 55.6, 55.5
[
GaL(curc)]: A mixture of H L (0.10 g, 0.27 mmol) and [Ga(curc) ] (0.16 g, 0.14 mmol) in
2 3
methanol (5 mL) was heated at reflux for 3 hours. The mixture was allowed to cool to ambient
temperature. An orange precipitate formed was collected by filtration, washed with cold methanol
(2 x 5 mL) and dried in vacuo to give [GaL(curc)] (0.05 g, 0.06 mmol, 43%). λmax(DMF)/nm 433.5,
3
-1
-1
4
57 (ε/dm cm mol 54,480, 51,710), λ (DMF)/nm 515 (λ 424). A sample for microanalysis
em ex.
was recrystalized from a concentrated mixture of the complex dissolved in dimethylsulfoxide (left
for several days), calc for C H GaN O : C, 62.62; H, 5.38; N 3.48%. Found: C, 62.4; H, 5.7; N,
4
2
43
2
10
1
3
.4%; H NMR (500 MHz; DMSO-d ): δ 9.59-9.50 (br s, 2H, OH), 8.47 (s, 1H, N-CH), 8.23 (s, 1H,
6
N-CH), 7.47 (d, J = 15.5 Hz, 1H, ArH), 7.29 (d, J = 1.8 Hz, 1H, ArH), 7.11 (m, 1H, ArH), 6.99-
6
3
.94 (m, 2H, ArH), 6.90 (m, 1H, ArH), 6.83-6.79 (m, 3H, ArH (2H) CH=CH (1H)), 6.76-6.65 (m,
H, ArH), 6.54-6.39 (m, ArH), 6.29 (m, 1H, CH=CH), 5.69-5.66 (m, 1H, C-CH-C), 4.19 (m, 1H,
CH -CHH-CH ), 4.09-3.95 (m, 4H, 2 x CH -CH ), 3.83 (s, 3H, OCH ), 3.80 (s, 3H, OCH ), 3.51
2
2
2
3
3
3
(
m, 1H, CH -CHH-CH ), 2.27-2.25 (m, 2H, CH -CH -CH ), 1.86 (m, 2H, CH -CH -CH ), 1.21-
2 2 2 2 2 2 2 2
1
13
1
1
1
1
.16 (m, 3H, CH -CH ), 1.07-1.03 (m, 3H, CH -CH ). { H} C NMR (101 MHz; DMSO-d ): δ
2 3 2 3 6
83.3, 181.9, 168.9, 168.1, 161.7, 160.2, 150.4, 149.7, 148.97, 148.78, 148.0, 147.7, 140.7, 139.6,
26.84, 126.65, 126.53, 126.2, 124.6, 123.4, 122.8, 122.5, 122.2, 121.4, 119.0, 118.0, 117.6, 115.6,
13.6, 111.24, 111.19, 102.9, 65.1, 64.0, 59.7, 57.8, 55.70, 55.52, 30.6, 15.17, 15.08. Crystals
suitable for analysis by x-ray crystallography were grown from a solution of the complex dissolved
in dimethyl sulfoxide.
Crystallography
Intensity data for [GaL(OH )(OHCH CH )]NO and [GaL(curc)] were acquired using either Mo-
2
2
3
3
-
6

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Oxford Cryosystems cooling device. The structures were solved by direct methods and difference
Fourier synthesis.[43] Thermal ellipsoid plots were generated using the program ORTEP-3
integrated within the WINGX suite of programs.[44]
Crystal data for [GaL(OH )(OHCH CH )]NO : C H N O Ga = 564.24, T = 130.0 K, = 1.7107,
2
2
3
3
23 32
3
9
triclinic, space group P -1 , a = 12.4587(8), b = 13.6613(11), c = 14.9608(12) Å,= 86.004(7)°
3
-3

= 82.486(6)°  = 76.117(6) V = 2448.8(3) Å , Z = 4, Z- = 2, D = 1.530 Mg M (Mo-K)
c
-
1
3
1
3
.182 mm , F(000) = 1176, crystal size 0.45 x 0.23 x 0.09 mm , 17916 reflections measured 
=
max
0.02° , 12285 independent reflections [R(int) = 0.0559], the final R was 0.1017 [I > 2(I) 6451
2
data] and wR(F ) was 0.2857 (all data). CCDC 1436486.
Crystal data for [GaL(curc)]: (C H N O Ga).(DMSO).(3(H O), M = 936.67, T = 130.0 K, =
4
2
42
2
10
2
1
8
.54184, triclinic, space group P-1, a = 11.6419(5)), b = 12.2119(5), c = 16.0212(7)) Å, =
3
-3
1.251(4)° = 83.506(4)° = 77.765(3)° V = 2192.37(17) Å , Z = 2, D = 1.419 Mg M (Cu-
c
-1
3
K) 1.893 mm , F(000) = 982, crystal size 0.30 x 0.21 x 0.02 mm , 13663 reflections measured
max = 76.94° , 8762 independent reflections [R(int) = 0.0345], the final R was 0.0600 [I > 2(I)
2
7
349 data] and wR(F ) was 0.1804 (all data). CCDC 1436487.
Assessment of plaque binding in human tissue - Paraffin preserved brain tissue blocks were
provided by the Victoria Brain Bank Network. Brain tissue was collected at autopsy. The National
Neural Tissue Resource Centre performed sourcing and preparation of human brain tissue. AD
pathologic diagnosis was made according to standard National Institute on Aging-Reagan Institute
criteria. Determination of age matched human control (HC) cases was subject to the above criteria.
The AD and HC brain tissues sections (5 μM) were first recovered from paraffin (xylene, 4 × 2
min) followed by rehydration (soaking in a series of 100%, 90%, 70%, and 0% v/v ethanol/water
baths). The hydrated tissue sections were washed in phosphate buffer saline (PBS, 5 min).
Autofluorescence of the tissue was quenched using potassium permanganate (0.25% in PBS, 20
min) and washing with PBS (2 × 2 min). The now brown coloured sections were washed with
potassium metabisulfite and oxalic acid (1% in PBS) until the brown colour was removed followed
by washing with PBS (3 × 2 min). The sections were blocked with bovine serum albumin (2% BSA
in PBS, pH 7.0, 10 min) and then treated with [GaL(curc)] (50 μM in 15% v/v DMSO/PBS, 1 hour).
7

ACCEPTED MANUSCRIPT
The sections were treated with BSA again to remove any [GaL(curc)] non-specifically bound to the
tissue. Finally, the sections were washed with PBS (3 × 2 min), deionised water, and mounted with
non-fluorescent mounting media (Dako). Fluorescence images were visualized using a Leica
(
Bannockburn, IL) DM1RB microscope (λ = 359 nm, λ = 461 nm). The A plaques in every
ex
em
second section of brain tissue (5 μM serial sections) were identified by treatment with primary
antibody to Aβ (1E8) and visualization with standard a peroxidase-based visualization kit
(
DAKO).[20]
Electronic spectroscopy
The UV/visible spectra of [Ga(L)(curc)] and [Ga(L)(OH )(OHCH CH )] (
+
) were measured
2
2
3
in 1% bovine serum albumin (w/w) in 15:85 DMSO:phosphate buffered saline pH 7.4. Absorbance
spectra were taken immediately after mixing then the periodically up to 2 hours using a Shimadzu
UV-1650PC UV/visible spectrophotometer and capped quartz cuvettes.
Results and Discussion
Neutral gallium complexes of tripodal hexadentate Schiff base ligands such as 1,1,1-tris((5-
methoxysalicylaldimino)methyl)ethane have been investigated as myocardial perfusion imaging
agents but in general do not cross the blood-brain barrier to allow assessment of cerebral
perfusion.[45-47] Lipophilic monocationic gallium(III) complexes formed with hexadentate N O
4
2
Schiff base ligands have been investigated as myocardial perfusion imaging agents and as substrates
of P-glycoprotein for imaging multi-drug resistance.[48-50] [51-53]
In this manuscript we report using a tetradentate N O Schiff base ligand, N,N-bis (2-
2
2
ethoxysalicylidene)-1,2-diaminopropane (H L), derived from the condensation of 1,3-
2
diaminopropane and 3-ethoxysalicylaldehyde as supporting ancillary ligands in the formation of
neutral octahedral gallium(III) complexes. The ethoxy substituents ortho- to the phenol group were
incorporated to increase lipophilicity and hopefully increase blood-brain barrier permeability as our
previous work demonstrated that the presence of the ethoxy substituent in cobalt(III) Schiff base
8

ACCEPTED MANUSCRIPT
complexes increased cell membrane permeability in neuronal-like SH-SY5Y cells.[54] When
tetradentate N O Schiff base ligands are used to form 6-coordinate metal complexes it is possible
2
2
for the ligand to adopt three different configurations, trans(2), cis-, and cis-α.[55, 56] If the
remaining two coordination sites of a six-coordinate metal ion are provided by monodentate ligands
the trans configuration is often encountered but the cis- configuration is required if the remaining
two coordination sites are provided by a bidentate ligand.
Reaction of H2L with gallium nitrate in ethanol allowed isolation of
+
[
GaL(OH )(OHCH CH )] as yellow crystals of the nitrate salt with two monodentate solvent
2
2
3
ligands situated trans to each other (Scheme 1).
+
Scheme 1. The synthesis of [GaL(OH )(OHCH CH )] and [GaL(curc)].
2
2
3
3
+
Analysis of the crystals by X-ray crystallography revealed the Ga is in a distorted octahedral
3
+
environment with the Ga-N and Ga-O(phenolate) bonds similar to previous reported Ga structures
with Schiff base ligands (Figure 1).[57] The Ga(1)-O(1) and Ga(1)-O(3) bond lengths to the oxygen
2
of the Schiff base L are 1.898(5) Å and 1.917(5) Å respectively. The Ga-N
bonds are 2.067(6)
imine
Å and 2.050(7) Å.
9

ACCEPTED MANUSCRIPT
+
Figure 1. ORTEP representation of [GaL(OH )(OHCH CH )] (ellipsoids are at 30% probability
2
2
3
level). Hydrogen atoms attached to carbon omitted for clarity.
+
3+
Reactions of [GaL(OH )(OHCH CH )] with curcumin in an attempt to form a Ga complex with
2
2
3
the Schiff base in the cis- configuration to accommodate the bidentate curcumin ligand were
+
unsuccessful. This contrasts with our experience with [Co(salen)] complexes (salen = N,N-
bis(salicylidene)-1,2-diaminothane), with the ligand in the trans configuration, where the ligand
could be forced to adopt a cis- configuration when the complexes were reacted with
acetylacetonate (acac).[54] The reaction of three equivalents of curcH with gallium nitrate allows
the isolation of neutral tris-bidentate complex [Ga(curc)].[41] The reaction of [Ga(curc) ] with H L
3
2
1
in ethanol allowed isolation of neutral [GaL(curc)] in good yields. The H NMR spectrum of
GaL(curc)] reveals that the tetradentate Schiff-base ligand has adopted the cis- configuration with
[
two separate resonances due to the two imine hydrogen atoms at δ 8.23 ppm and 8.47 ppm when
compared to the single resonance at 8.61 ppm in the C2 symmetric complex
+
[
GaL(HOCH CH )(OH )] . Two singlets for each methoxy functional groups of the coordinated
2
3
2
and deprotonated curcumin are at δ 3.83 ppm and 3.80 ppm.
Crystals of [GaL(curc)] suitable for X-ray crystallography were grown from a solution of the
complex dissolved in dimethyl sulfoxide (Figure 2). The gallium(III) ion is an distorted octahedral
1
0

ACCEPTED MANUSCRIPT
environment with the tetradentate ligand in the cis- conformation combining with the curcumin
anion to provide an N O donor environment in a racemic mixture Δ and Λ enantiomers. The bond
2
4
angle of the N(1)-Ga(1)-N(2) six-membered chelate ring is 86 ˚ and the gallium to phenolic oxygen
atoms bond angle, O(1)-Ga-O(3), is 93 ˚. In [Ga(L)curc] the metal ion-phenolate bond lengths, Ga-
O(1) and Ga(1)-O(3) are 1.932(2) Å and 1.924(2) Å respectively and are longer than comparable
+
bonds when the ligand adopts the trans(2) configuration in [GaL(OH )OHCH CH ] . The Ga-
2
2
3
O(curcumin) bond lengths are 1.975(2) and 1.991(2). The bond lengths and angles are comparable
to those found in of [Ga(salpn)(acac)] (salpn = N,N-bis(salicylidene)-1,2-diaminopropane) that was
reported recently.[58]
Figure 2. ORTEP plot of [GaL(curc)] (ellipsoids are at the 30% probability level). Solvent
(
dimethyl sulfoxide and water), the minor component atoms of the disordered methoxyphenol ring
C22-C27, O7, O8, and C44) and protons attached to carbon have been omitted for clarity.
(
In the solid state the molecules of [GaL(curc)] form centrosymmetric dimers of enantiomers
through π-π
the terminal aromatic rings of the curcumin ligand is 13.2(4)° while the distances between the
atoms of one plane to the second aromatic ring range from 3.27(3) – 3.90(3) Å (Figure 3).
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Figure 3. Representation of the centrosymmetric dimers found in the X-ray crystal structure of
[
GaL(curc)] formed through π-π interactions of the curcumin ligand.
The stability of [Ga(L)(curc)] in bovine serum albumin was assessed by electronic
spectroscopy. The UV/visible spectrum of [Ga(L)(curc)] (50 mM) in an aqueous solution (15:85
DMSO:phosphate buffered saline, pH 7.4) with 1% serum (w/w) featured the expected broad
absorption at λ = 430 nm characteristic of curcumin but also an absorption at λ = 295 nm. There
was a small reduction (16 %) in the intensity of this band over a two hour incubation period (chosen
to be approximately twice the half-life of gallium-68) suggesting the complex was sufficiently
stable in serum to warrant further more detailed investigations. The electronic spectrum of
+
[
Ga(L)(OH )(OHCH CH )] had a similar absorption at λ = 295 nm but underwent significant
2
2
3
changes within the first five minutes of being incubated with bovine serum albumin possibly due to
rapid exchange of the monodentate solvent ligands with accessible donor atoms on the protein.
Curcumin is fluorescent (λmax = 522 nm, λ = 453 nm) and [GaL(curc)] is also fluorescent
ex
with a small hypsochromic shift in the emission wavelength (λmax = 508 (λ = 424 nm) and this
ex
fluorescence can be used to characterise the binding of the complex to A plaques. The ability of
[
GaL(curc)] to bind to A plaques was assessed in post-mortem human frontal cortex tissue from a
subject diagnosed with clinical Alzheimer’s disease. The brain tissue (5 μm serial sections) was pre-
treated with bovine serum albumin to minimise non-selective binding and the tissues were treated
with a solution of [Ga(L)(curc)] (50 μM). The localization of [GaL(curc)] on the treated brain tissue
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was measured by epi-fluorescent microscopy (λ = 359 nm, λ = 461 nm) and compared to the
ex
em
contiguous section of tissue stained with an Aβ antibody (1E8) to identify plaques revealing
significant co-localisation of the complex with A plaques (Figure 4). The Aβ plaques are typically
4
0−60 μm in diameter so 5 μm serial sections should largely include features of the same Aβ
plaque.[59] The complex was not retained in samples of human brain tissue from age-matched
control subjects suggesting some degree of specificity for A plaques.
Figure 4. a) Sections of frontal cortex brain tissue from Alzheimer's disease subjects treated with
[
Ga(L)(curc)] and imaged with epi-fluorescent microscopy (λ = 359 nm, λ = 461 nm). b) Image
ex em
of contiguous section of brain tissue stained with Aβ selective antibody (1E8).
Concluding Remarks
In aiming to develop charge neutral, low molecular weight, lipophilic complexes with the potential
to cross the blood-brain barrier and bind to A plaques we have employed an ancillary tetradentate
N O Schiff base ligand and the β-diketone curcumin as a bidentate ligand to give a six-coordinate
2
2
3
+
Ga complex. The tetradentate Schiff base ligand adopts the cis- configuration with the
deprotonated curcumin ligand acting as a bidentate ligand. Promisingly the complex binds to A
plaques in human brain tissue. Given that the synthesis of hexadentate Schiff-base gallium-68
6
8
complexes often use [ Ga(acac) ] as a convenient starting material and the recent report of the
3
+
preparation of [Ga(curc) ] complexes it appears feasible that the approach presented here could be
2
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68
3+
translated to Ga .[42, 49] Further studies are required to develop radiolabeling procedures, assess
the stability of the complexes in vivo and their ability to cross the blood-brain barrier.
Dedication
In memory of Professor Graeme Hanson and his contributions to Bioinorganic Chemistry and
Electron Paramagnetic Resonance
Acknowledgements
The Australian Research Council for funding this research. The Victorian Brain Bank for provision
of human brain tissue. PSD is and ARC Future Fellow.
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Graphical abstract
TOC Synopsis graphic and text:
A gallium(III) complexes that binds to amyloid- plaques associated with Alzheimer's disease has
been prepared. The complex features a tetradentate N O Schiff base as an ancillary ligand and the
2
2
3
+
β-diketone curcumin as a bidentate ligand to give a six-coordinate Ga complex.
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Highlights
-
-
A neutral gallium complex with curcumin acting as a bidentate ligand has been prepared.
The complex binds to amyloid-beta plaques present in human Alzheimer’s disease brains.
2
0