Article

Cite This: J. Med. Chem. 2019, 62, 2638−2650 pubs.acs.org/jmc

Remarkable Brain Penetration of Cyclopentadienyl M(CO)3+ (M =

99m
Tc, Re) Derivatives of Benzothiazole and Benzimidazole Paves the
Way for Their Application as Diagnostic, with Single-Photon-
Emission Computed Tomography (SPECT), and Therapeutic Agents
for Alzheimer’s Disease
Marina Sagnou,† Barbara Mavroidi,† Antonio Shegani,‡ Maria Paravatou-Petsotas,‡
Catherine Raptopoulou,§ Vassilis Psycharis,§ Ioannis Pirmettis,‡ Minas S. Papadopoulos,‡
and Maria Pelecanou*,†
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†
Institutes of Biosciences & Applications, ‡Nuclear & Radiological Sciences & Technology, Energy & Safety, and §Nanoscience and
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Nanotechnology, National Center for Scientific Research “Demokritos”, 15310 Athens, Greece
*
S Supporting Information

ABSTRACT: The synthesis and evaluation of three novel

99m
Tc complexes (99mTc-1−3) and their corresponding Re
complexes (Re-1−3), in which the phenyl ring of 2-
phenylbenzothiazole or 2-phenylbenzimidazole is replaced by
the cyclopentadienyl tricarbonyl [Cp99mTc(CO)3] core, are
reported. Both 99mTc and Re complexes were prepared from
the corresponding ferrocenyl derivatives, and the Re
complexes were fully characterized by elemental analysis,
spectroscopic methods, and X-ray crystallography. The complexes exhibit effective in vitro binding to β-amyloid (Aβ) plaques
and fibrils, inhibit Aβ fibril formation, and significantly reduce Aβ-induced cytotoxicity and reactive oxygen species production
in neuronal cell cultures. The brain uptake of the 99mTc complexes ranges between 7.94 and 3.99% ID/g at 2 min p.i., being the
highest recorded for potential 99mTc Aβ plaque imaging probes in mice. Powered by their high brain uptake, the complexes
represent strong theranostic candidates against Alzheimer’s disease combining single-photon-emission computed tomography
diagnostic (99mTc complexes) and antiamyloid therapeutic (Re complexes) potential.

■ INTRODUCTION
Penetration across the blood−brain barrier (BBB) is the
bottleneck in brain drug development with more than 98% of
all small-molecule drugs, and ∼100% of all large-molecule
drugs, not crossing BBB.1 Alzheimer’s disease (AD) is among
the many central nervous system (CNS) disorders for which
drug discovery is limited by effective CNS drug delivery.2 AD
therapeutic strategies focusing on the β-amyloid (Aβ), like
inhibition of Aβ production, inhibition of Aβ aggregation and
deposition, or Aβ immunotherapy, have failed in clinical trials
partly due to limited transport across the BBB.3 AD diagnostic
strategies focusing on imaging of Aβ plaques have produced
three 18F radioagents in clinical use today, 18F-florbetapir, 18F-
flutametamol, and 18F-florbetaben, which cross the BBB and
allow in vivo imaging of Aβ burden with positron emission
tomography (PET).4,5 However, systematic efforts to circum-
vent the drawbacks of high cost and limited availability
associated with PET imaging6 by developing a 99mTc
radiodiagnostic for in vivo imaging of Aβ plaques with
single-photon-emission computed tomography (SPECT)
have not yet met with success. Many potential 99mTc agents
have been synthesized that fulfill the in vitro evaluation
requirements of an AD imaging probe, namely, efficient
radiolabeling and stability, affinity for Aβ fibrils, and selectivity
and binding affinity for Aβ plaques on postmortem brain tissue
but, nonetheless, fail the prerequisite for adequate in vivo brain
penetration.7−9
In this work, a new type of 99mTc complex is presented in
which the phenyl moiety of 2-phenylbenzothiazole has been
replaced by the cyclopentadienyl tricarbonyl [Cp99mTc(CO)3]
core (99mTc-1, Scheme 1). 2-Arylbenzothiazole is the classical
Aβ binding structure originally developed as the neutral
analogue of thioflavin T (ThT), the histological dye used in
the clinic for ex vivo visualization of Aβ plaques in AD brain.10
As a result, the 2-arylbenzothiazole scaffold has been
extensively used in the investigations for a 99mTc radio-
diagnostic for AD, as the pharmacophore to confer binding
affinity to Aβ plaques.7−9,11 On the other hand, the
[Cp99mTc(CO)3] core possesses properties favorable for BBB
penetration, such as small size, low molecular weight (MW),
lack of charge, moderate lipophilicity, and high stability12 and
Received: December 13, 2018
Published: February 15, 2019

© 2019 American Chemical Society 2638 DOI: 10.1021/acs.jmedchem.8b01949

J. Med. Chem. 2019, 62, 2638−2650
Journal of Medicinal Chemistry Article

99m
Scheme 1. Chemical Structures of Complexes Tc-1−3 and Re-1−3

Scheme 2. Synthetic Route to Ferrocenyl Derivatives Fe-1−3 and to Complexes Re-1−3 and 99m
Tc-1−3a

a
Reagents and conditions: (a) ethanol, reflux, 8 h for Fe-1; ethanol, NaHSO3, reflux, 2 h for Fe-2; Fe-2, CH3I, acetone−water, NaOH, 1 h, room
temperature (rt) for Fe-3; (b) [Re(CO)3Br3][NEt4]2, dimethylformamide (DMF)−0.1 N HCl, 160 °C, 2 h; (c) Na[99mTcO4], Mn(CO)5Br,
DMF−H2O, 110 °C, 90 min.

it has been repeatedly employed in the search for a 99mTc Aβ
imaging agent both through the integrated13 and the
conjugated approach.14−16 In the 99mTc-1 complex, replace-
ment of the phenyl ring of 2-phenylbenzothiazole by
[Cp99mTc(CO)3] resulted in a compact lipophilic 99mTc
agent that maintains affinity for Aβ fibrils and plaques, while
exhibiting an unprecedented for a 99mTc complex uptake in
mouse brain of 7.94% ID/g at 2 min.
The design was extended to include the corresponding
complexes with benzimidazole (99mTc-2) and N-methylbenzi-
midazole (99mTc-3) in place of benzothiazole (Scheme 1) that
display equally impressive properties, with respective brain
uptakes of 3.99 and 5.36% ID/g at 2 min. The analogous
isostructural Re complexes Re-1−3 were also synthesized to
serve, according to the usual practice,17 as nonradioactive
surrogates in structure identification and in vitro biological
evaluation experiments. Nonetheless, the interesting properties
that the Re-1−3 complexes displayed against Aβ aggregation
and toxicity led us to extend the spectrum of potential
application of the new complexes from AD diagnostics (99mTc-
1−3) to AD therapeutics (Re-1−3).
spectroscopies as well as electrospray ionization (ESI) mass
spectrometry and elemental analysis. The UV absorbance and
fluorescence properties of the Re complexes (Figure S4,
Supporting Information) (Re-1: Absmax = 305 nm, Fluemmi =
362 nm, Re-2: Absmax = 304 nm, Fluemmi = 379 nm, Re-3:
Absmax = 300 nm, Fluemmi = 350 nm) are similar to those of the
lead pharmacophore 2-phenylbenzothiazole,20 indicating that
the extended conjugation is maintained in the complexes. X-
ray crystallographic analysis was possible for Fe-1 and Re-1
and showed that indeed the Cp ring lies on the same plane
with the benzothiazole moiety. The Re-1−3 complexes are
soluble in CH2Cl2, dimethyl sulfoxide (DMSO), and ethanol;
sparingly soluble in ether, hexane, and water; and stable in
organic solvents for a period as witnessed by high-performance
liquid chromatography (HPLC) and NMR.
Description of the Structures. Labeled plots of the
molecular structures of Fe-1 and Re-1 are presented in Figures
1 and 2, respectively; important crystallographic data for Fe-1
and Re-1 are listed in Table 1, whereas bond distances and
angles are listed in Tables S3 and S4, Supporting Information.

■ RESULTS
Synthesis of Re Complexes. The Re-1−3 complexes
In addition, in Figures S5−S8 (Supporting Information), a

were synthesized from the corresponding ferrocenyl derivatives

of benzothiazole (Fe-1), benzimidazole (Fe-2), and N-
methylbenzimidazole (Fe-3) (Scheme 2).18,19 The ferrocenyl
derivatives Fe-1 and Fe-2 were obtained from the
condensation of ferrocenyl aldehyde with 2-aminobenzenethiol
and benzene-1,2-diamine, respectively, whereas Fe-3 was
obtained from methylation of Fe-2 with methyl iodide. The
reaction of Fe-1−3 with the rhenium precursor [Re(CO)3Br3]-
[NEt4]2 under vigorous conditions resulted in the formation of
the complexes Re-1−3 in yields ranging from 59 to 72%.
The ferrocenyl derivatives Fe-1−3 and the rhenium
complexes Re-1−3 were characterized by IR and NMR Figure 1. Oak Ridge thermal ellipsoid plot (ORTEP) of compound
(Figures S1−S3; Tables S1 and S2, Supporting Information) Fe-1 with the thermal ellipsoids presented at a level of 50%.

2639 DOI: 10.1021/acs.jmedchem.8b01949

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Journal of Medicinal Chemistry Article

carbons of the Cp2 ring is 2.041 Å, in conformity with previous

Figure 2. ORTEP of complex Re-1 with the thermal ellipsoids
presented at a level of 50%.
detailed presentation of the packing diagrams based on
intermolecular interactions is given for both Fe-1 and Re-1.
Compound Fe-1 crystallizes in the P21/c space group, and
the asymmetric unit of the unit cell contains one molecule with
the cyclopentadienyl (Cp) rings in eclipsed geometry21 and
not in staggered.22 Both Cp1 (C1,..., C5) and Cp2 (C6,...,
C10) rings are essentially planar with 0.001 and 0.003 root-
mean-square (rms) deviations of fitted atoms, respectively, and
also parallel to each other as the dihedral angle between the
mean planes is 0.3(1)°. The Cg1···Fe1 and Cg2···Fe1 distances
(Cg1 and Cg2 are the centroids of Cp1 and Cp2 rings,
respectively) are equal to 1.6558(2) and 1.6395(2) Å,
respectively, and the Cg1−Fe1−Cg2 angle takes the value of
179.18°. All of the above values fall in the range of previous
studies.23 The average C−C bond distance for the Cp1 ring is
1.416 Å and that of the Cp2 one is slightly larger, 1.429 Å. The
average distance of the Fe1 atom to the carbons of the Cp1
ring is 2.046 Å, whereas the corresponding distance to the
studies.21−23 The angle around the bond C10−C11 of the
benzothiazole part of the molecule defined by the atoms C11,
C12, C13, C14, C15, C16, C17, N1, S1 and the cyclo-
pentadienyl ring defined by the atoms C6,..., C10 is 10.08(8)°.
Complex Re-1 crystallizes in the P1̅ space group, and the
asymmetric unit of the unit cell contains one molecule. The
coordination environment about Re1 is described as piano-
stool-type with the three carbonyls in facial arrangement at
approximately 90° to each other (Table S4, Supporting
Information) and the Cp unit occupying the opposite facial
plane of the pseudo-octahedral coordination sphere.15 The Cp
ring (C4,..., C8) is essentially planar with 0.010 rms deviations
of fitted atoms. The Cg1···Re1 distance (Cg1 stands for the
centroid of the Cp ring) is equal to 1.9630(4) Å. The average
C−C bond distance for the Cp ring is 1.414 Å, whereas the
average distance of the Re1 atom to the carbons of the Cp ring
is 2.302 Å. The angle around the bond C8−C9 of the
benzothiazole part with the cyclopentadienyl ring is 6.9(6)°,
rendering the molecule essentially flat.
Synthesis of 99mTc Complexes. The 99mTc-1−3 com-
plexes were also synthesized from the corresponding ferrocenyl
derivatives Fe-1−3 through two different methods. The first
method employs the widely used fac-[99mTc(H2O)3(CO)3]+
precursor (yield 50%), and the second one employs Mn-
(CO)5Br and Na[99mTcO4] according to the double ligand
transfer (DLT) reaction, first introduced by Katzenellenbogen
et al. for the synthesis of [CpM(CO)3] (M = Re, 99mTc)
complexes from ferrocenyl precursors (yield 96%).24,25 It
should be noted that high yields were also obtained by our
group through the DLT reaction during the synthesis of a
[Cp99mTc(CO)3] complex conjugated to phenylbenzothiazole
through an amide bond (95% compared with 35% through the
fac-[99mTc(H2O)3(CO)3]+ precursor).16 The 99mTc complexes
were characterized by comparative HPLC analysis using the

Table 1. Crystallographic Data for Fe-1 and Re-1b

Fe-1 Re-1
formula C17H13FeNS C15H8NO3ReS
Fw 319.19 468.48
T (K) 180 293
radiation Mo Kα Mo Kα
crystal system monoclinic triclinic
space group P21/c P1̅
a (Å) 9.0869 (3) 7.0230 (4)
b (Å) 13.6081 (3) 8.4824 (5)
c (Å) 10.9798 (3) 12.4549 (7)
α (deg) 90.00 84.290 (2)
β (deg) 102.520 (1) 82.139 (2)
γ (deg) 90.00 77.940 (2)
volume (Å3) 1325.43 (6) 716.84 (7)
Z 4 2
D (calcd) (Mg/m3) 1.600 2.170
abs. coef., μ (mm−1) 1.28 8.63
GOF on F2 1.02 1.09
measured/unique/reflections with I > 2σ(I) 37 691/2882/2687 13 413/3114/2767
Rint 0.029 0.075
R1/wR2 (total)a 0.0266/0.0638 0.0559/0.1354
R1/wR2 [for I > 2σ(I)]a 0.0246/0.0626 0.0506/0.1293

a
R1 = ∑(|Fo| − |Fc|)/∑(|Fo|) and wR2 = {∑[w(Fo2 − Fc2)2]/∑[w(Fo2)2]}1/2. bw = 1/[σ2(Fo2) + (αP)2 + bP] and P = [Fo2 + 2Fc2]/3: α = 0.0357, b
= 0.7911 for Fe-1; α = 0.0623, b = 2.3528 for Re-1.

2640 DOI: 10.1021/acs.jmedchem.8b01949

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Figure 3. Confocal fluorescence images (λexc 370 nm, λem 580 nm) of consecutive brain slices from an autopsy-confirmed AD patient stained with
complexes Re-2 (A) and Re-3 (B) and with ThS (C) (0.5 mg/mL) as a positive control. The scale bar corresponds to 50 μm.

Figure 4. Inhibition curves of complexes Re-1 (A), Re-2 (B), and Re-3 (C) for the binding of the corresponding 99mTc-1, 99mTc-2, and 99mTc-3
complexes to Aβ42 aggregates. Error bars represent standard deviation (n = 3).
99m 99m 99m
Table 2. Biodistribution of Radioactivity (% ID/g) after Injection of Complexes Tc-1, Tc-2, and Tc-3 in Healthy
Swiss Albino Mice (n = 4)a
99m 99m 99m
Tc-1 Tc-2 Tc-3
organ 2 min 15 min 90 min 2 min 15 min 90 min 2 min 15 min 90 min
blood 2.90 ± 0.73 1.59 ± 0.23 0.47 ± 0.01 3.38 ± 0.67 1.86 ± 0.13 0.48 ± 0.12 1.82 ± 0.74 0.41 ± 0.08 0.14 ± 0.01
liver 11.30 ± 1.79 28.22 ± 1.82 17.11 ± 1.25 13.97 ± 2.13 30.66 ± 3.96 18.08 ± 1.34 4.46 ± 0.43 10.68 ± 1.69 5.30 ± 0.58
heart 23.81 ± 5.39 4.93 ± 0.34 0.69 ± 0.13 7.53 ± 1.28 1.95 ± 0.26 0.22 ± 0.05 9.43 ± 2.28 1.73 ± 0.07 0.25 ± 0.06
kidneys 15.54 ± 4.30 13.61 ± 1.96 13.91 ± 1.34 9.70 ± 0.92 5.04 ± 0.53 1.57 ± 0.20 8.42 ± 1.48 8.99 ± 2.55 7.42 ± 1.09
stomach 2.87 ± 1.22 7.83 ± 2.57 15.26 ± 3.88 1.63 ± 0.44 3.17 ± 0.43 4.63 ± 0.56 1.29 ± 0.31 3.57 ± 0.98 3.18 ± 1.70
intestines 2.81 ± 0.95 15.70 ± 0.95 15.42 ± 2.41 3.06 ± 0.08 14.80 ± 0.25 23.94 ± 4.27 2.01 ± 0.55 11.89 ± 0.16 22.16 ± 1.53
spleen 2.95 ± 0.89 3.32 ± 0.30 0.67 ± 0.08 2.63 ± 0.40 1.01 ± 0.09 0.17 ± 0.03 1.69 ± 0.45 0.64 ± 0.04 0.16 ± 0.05
muscle 4.12 ± 0.34 2.76 ± 0.12 0.83 ± 0.34 5.23 ± 0.79 1.50 ± 0.19 0.15 ± 0.03 4.10 ± 0.39 1.34 ± 0.28 0.32 ± 0.06
lungs 22.61 ± 5.47 17.07 ± 3.06 8.48 ± 1.18 7.09 ± 0.38 2.44 ± 0.39 0.45 ± 0.13 9.10 ± 1.93 3.77 ± 1.48 1.23 ± 0.30
pancreas 6.99 ± 0.97 4.24 ± 0.35 0.73 ± 0.19 5.99 ± 0.62 1.96 ± 0.25 0.21 ± 0.02 5.35 ± 1.21 1.17 ± 0.12 0.38 ± 0.09
brain 7.94 ± 1.46 3.70 ± 0.40 0.20 ± 0.03 3.99 ± 0.60 0.75 ± 0.11 0.04 ± 0.01 5.36 ± 0.65 0.85 ± 0.05 0.09 ± 0.02
a
Data expressed as the % of the injected dose per gram of wet tissue (% ID/g) ± the standard deviation of the mean.

corresponding Re complexes as reference compounds (Figure
S9, Supporting Information), and they were clearly separated
by HPLC from their corresponding ferrocenyl precursor. Their
lipophilicity was determined by measuring their partition
coefficient (P) in n-octanol/phosphate buffer (0.1 M, pH 7.4)
and resulted in log Poct/water values of 2.52 ± 0.14, 1.84 ± 0.17,
and 1.50 ± 0.12, respectively. Complexes 99mTc-1−3 were
stable (>95%) in their preparation medium for at least 6 h and
in challenge experiments against histidine and cysteine (Table
S5, Supporting Information). Moreover, all 99mTc complexes
were found very stable (99%) in rat plasma for at least 90 min
(Figure S10, Supporting Information).
Biological Evaluation. In Vitro Affinity for Aβ Plaques
and Aβ Fibrils. The affinity of complexes Re-1−3 for Aβ
plaques was evaluated with confocal microscopy in post-
mortem fixed brain sections from an AD patient using a
standard staining procedure.26,27 Figure 3 shows the
2641
fluorescence microscope images obtained in consecutive
brain slices with Re-2 and Re-3 and with the Aβ binding
histological dye thioflavin S (ThS) as a positive control.
Analogous images obtained with Re-1 are shown in Figure
S11, Supporting Information. All three complexes bind
selectively to the Aβ plaques, allowing the clear visualization
of both diffused and dense core ones and producing images of
analogous quality to those of ThS.
The complexes were also found to interact with mature Aβ
fibrils through in vitro competition binding assays between the
stable Re complexes and their radioactive 99mTc counterparts,
conducted according to published procedures.10,16 As shown in
Figure 4 for Re-1, Re-2, and Re-3, the presence of increasing
concentrations of the Re complexes inhibited the binding of
the corresponding 99mTc complexes (99mTc-1, 99mTc-2, and
99m
Tc-3, respectively), which were employed as radioligands.
The Ki values of 65.8 ± 21.3, 7.0 ± 2.9, and 5.7 ± 2.9 nM
DOI: 10.1021/acs.jmedchem.8b01949
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Journal of Medicinal Chemistry
Figure 5. Graphical representation of the biodistribution data (% ID/g) for the 99mTc-1, 99mTc-2, and 99mTc-3 complexes in healthy Swiss albino
mice.
calculated from the inhibition data for Re-1, Re-2, and Re-3
respectively, show strong affinity of the complexes for Aβ42
aggregates. Especially, the Ki values obtained for Re-2 and Re-
3 are the lowest reported for potential Aβ SPECT imaging
agents bearing the [CpM(CO)3] (M = Re, 99mTc) core and
among the lowest reported for complexes of the [MO]3+ (M =
Re, 99mTc) core.8
Biodistribution in Healthy Mice. The affinity displayed by
complexes Re-1−3 for both Aβ plaques and fibrils prompted
us to proceed with biodistribution experiments in healthy mice.
The biodistribution results are shown in Table 2 where an
impressive and unprecedented, for 99mTc complexes, brain
uptake is presented amounting to 7.94 ± 1.46, 3.99 ± 0.60,
and 5.36 ± 0.65% ID/g at 2 min for 99mTc-1, 99mTc-2, and
99m
Tc-3, respectively. A graphical representation of the
biodistribution data of the 99mTc complexes is presented in
Figure 5.
The brain uptake of 99mTc-1 is comparable to that of 18F-
florbetapir (7.33% ID/g at 2 min)28 and substantially higher
than that of 18F-florbetaben (4.77% ID/g at 2 min),29 the U.S.
Food and Drug Administration approved [18F] imaging agents
in clinical use. Overall, the brain uptake of 99mTc-1−3 far
exceeds all values reported for potential 99mTc Aβ imaging
2642
Article
probes in healthy mice,8 the highest uptakes at 2 min being
1.21% ID/g for complexes of the [99mTc(CO)3]+ core30 and
2.11% ID/g for complexes of the [99mTcO]3+ core.31 The high
brain penetration noted for 99mTc-1−3 was accompanied by
fast blood clearance and almost complete washout at 90 min
(Table 2), resulting in brain2min/brain90min clearance ratios of
39.7, 99.8, and 59.6, respectively, which indicate lack of
retention in brain tissue, an advantageous feature for clear
imaging and one of the prerequisites for Aβ imaging probes.7,8
Biodistribution of 99mTc-1 in Transgenic (Tg) 5×FAD
Mice. Biodistribution experiments of the 99mTc-1 complex
were conducted at 15 min with Tg 5×FAD mice (n = 4) as
well as with their wild-type littermates and are reported in
Table S6, Supporting Information. The % ID/g at 15 min is
3.90 ± 0.19, substantially higher than the corresponding % ID/
g of 2.68 ± 0.06 found for the wild-type littermates. In view of
the similar characteristics of the Tg and wt mice in terms of
weight, age, and genetic background, the difference in uptake
of 99mTc-1 is attributed to the presence of pathological AD
features in the Tg 5×FAD mice and especially to the presence
of Aβ plaques. The results are in agreement with literature data
in which a similar increase in uptake is typically observed for
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Figure 6. Fluorescence spectra of ThT (5 μM in phosphate-buffered saline (PBS), 10 mM, pH 7.4) added in Aβ42 solutions incubated for 15 days
in the presence or absence of Re complexes (Aβ42/complexes, 1:1 (A) and 1:2 (B) ratios). Fluorescence was monitored after excitation at 440 nm.
Representative spectra from n = 3 independent experiments are presented.

the Tg mice32−35 and taken as a sign of increased interaction/ hippocampal cells by the MTT assay, following the literature
binding of the tracer with the Aβ plaques. procedure.37,38 As shown in Figure 7, exposure of primary
Staining of brain slices from the Tg 5×FAD revealed an
abundance of Aβ plaques (Figure S12, Supporting Informa-
tion).
Activity of Re-1−3 against Aβ Aggregation and Toxicity.
The high brain penetration and favorable pharmacokinetics of
the 99mTc-1−3 complexes, combined with the displayed
stability in serum and relatively low toxicity as determined
from the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium
bromide (MTT) assay (Figure S13, Supporting Information),
prompted their investigation for possible therapeutic action
against AD. As a first step, the study of their potential to inhibit
the aggregation of Aβ42, which is considered to play a major
role in AD neurotoxicity,36 was attempted with circular
dichroism (CD) spectropolarimetry, a method widely
employed to monitor the aggregation of Aβ and its interactions
with potential aggregation inhibitors. However, the strong CD
absorption of the complexes, attributed to the asymmetric
environment of the [CpRe(CO) 3 ] core (Figure S14,
Supporting Information), precluded the study of the
aggregation process of Aβ (50 μM) at the 1:1 and 1:2 Aβ/
complex ratios usually employed in our CD studies. The CD Figure 7. Effect of 1 and 2 μM of complexes Re-1, Re-2, and Re-3 on
spectra of the Re-1−3 complexes remained noisy even at the the cytotoxicity of 1 μM Aβ42 in primary mouse hippocampal
neurons after 24 h of incubation at 37 °C. Cell viability was assessed
concentration of 6 μM, which is the lowest limit for
using the MTT reduction assay (n = 3 independent experiments, each
monitoring Aβ interactions under our experimental conditions. one performed in six replicates). The data are presented as mean ±
As a result, the complexes were incubated with Aβ at 1:1 and standard error of the mean (SEM), *p ≤ 0.05, **p ≤ 0.01, ***p ≤
1:2 Aβ/complex ratios and only the solution of plain Aβ, which 0.001, and not significant (ns) > 0.05 compared with Aβ42 (1 μM)
served as control, was monitored with CD (Figure S15, treatment and #p < 0.01, ##p < 0.01, and ###p < 0.001 compared with
Supporting Information). When the aggregation of plain Aβ control (untreated cells).
was complete, as witnessed by CD, the thioflavin T (ThT)
fluorescence test, that detects mature Aβ fibrils, was performed hippocampal neurons to plain Aβ42 (1 μM) reduces cell
on all Aβ/complex solutions. The results are presented in viability to 50% of untreated cells, a reduction which is in
Figure 6 and show great reduction in the amount of typical Aβ accordance with values reported in the literature for the
fibrils formed in the presence of the complexes compared to toxicity of Aβ42 in primary neurons.39 It is clear from Figure 7
that in the solutions of plain Aβ. As can be seen in Figure 6B, that the presence of complexes Re-1−3 in the Aβ solution at 1
the reduction is more pronounced at the higher Aβ/complex and 2 μM concentration raises cell viability, reaching almost
ratio of 1:2. The results of the ThT test clearly show that 95% at the 2 μM concentration. The significant rescue effects
interaction of the complexes with Aβ takes place and alters the of 72, 79, and 81% obtained for complexes Re-1, Re-2, and Re-
normal aggregation course of Aβ to toxic fibrils. 3, respectively, at the 1:2 Aβ/complex ratio indicate that the
The potential of the rhenium complexes Re-1−3 to rescue complexes have neuroprotective properties against Aβ-induced
cells from Aβ toxicity was subsequently examined in primary toxicity. In view of the inhibitory action of the Re-1−3
2643 DOI: 10.1021/acs.jmedchem.8b01949
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Journal of Medicinal Chemistry
complexes against formation of toxic Aβ fibrils, as revealed in
the ThT assay (Figure 6), it is plausible that Re-1−3 exert
their neuroprotective action through modulation of the
aggregation pathway of Aβ.
Finally, based on the fact that the pathology of AD is
associated with oxidative stress and the production of reactive
oxygen species (ROS),40 complexes Re-1, Re-2, and Re-3 were
evaluated for their antioxidant potential. As shown in Figure 8,
Figure 8. Effect of 1 and 2 μM of complexes Re-1, Re-2, and Re-3 on
Aβ42 (1 μM)-induced ROS generation in primary mouse hippo-
campal neurons after 24 h of incubation at 37 °C. ROS levels were
measured by the 2’,7’-dichlorofluorescein (DCF) fluorescence assay
(n = 3 independent experiments, each one performed in six
replicates). The data are presented as mean ± SEM, *p ≤ 0.05,
**p ≤ 0.01, ***p ≤ 0.001, and not significant (ns) > 0.05 compared
with Aβ42 (1 μM) treatment and #p < 0.01, ##p < 0.01, and ###p <
0.001 compared with control (untreated cells).
exposure of primary hippocampal cells to 1 μM Aβ40 for 24 h
results in a 10-fold increase of ROS production compared to
that from untreated cells, a value in agreement with literature
data.41 The presence of Re-1−3 in the Aβ solution at 1:1 and
1:2 Aβ/complex ratios attenuates the production of ROS 55.2,
63.2, and 68.1%, respectively, indicating a significant
antioxidant activity for Re-1−3, comparable to that demon-
strated in the literature by known antioxidants.42 As both
soluble and fibrillar aggregates of Aβ have been implicated in
ROS production and oxidative stress in the AD brain,43 the
potential of Re-1−3 to interact with Aβ and alter its
aggregation course (Figure 6) may be very well related to
their antioxidant action.
■ DISCUSSION
In this work, the synthesis of new agents in which the
[CpM(CO)3] (M = Re, 99mTc) moiety has replaced the phenyl
ring of 2-arylbenzothiazole, the classical Aβ binding
structure,11 to generate stable, compact, lipophilic agents
with exquisite brain uptake that preserve the affinity of 2-
arylbenzothiazole for Aβ plaques and Aβ fibrils is reported.
Replacement of a phenyl ring by the [CpRe(CO)3]
(cyrhetrenyl) moiety was pioneered by Jaouen and co-workers
through the synthesis of cyrhetrenyl derivatives of OH-
tamoxifen that retained the high affinity for the estrogen
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receptor.44 In subsequent works, the [CpM(CO)3] moiety (M
= Re and/or 99mTc) has replaced a phenyl group in
chalcones13,45 and benzamides,46 as well as a 1,2,3,4-
tetrahydro-naphthalene group in PB28, a selective sigma-2
receptor agonist,47 resulting in bioactive derivatives. In this
work, the new agents preserve main structural features of 2-
arylbenzothiazole, such as planarity, length, neutrality, and
chemical properties, like fluorescence and affinity for Aβ
plaques, whereas the compactness of the structure and added
lipophilicity provided by the [CpMCO3] (M = Re, 99mTc)
core44 obviously contributes to their impressive brain uptake.
The results support the notion that piano-stool-structured
compounds can topologically mimic phenyl rings in
pharmaceuticals and contribute to the establishment of the
[CpM(CO)3] core as an effective tool in the development of
targeted radiotracers.48,49
The new 99mTc agents 99mTc-1−3 have ideal properties to
develop into SPECT diagnostic tools for AD. They meet the
initial prerequisites for Aβ imaging probes, as presented in the
2014 review by Cui et al.,7 as (1) they present high binding
affinity for synthetic Aβ42 fibrils with Ki values ranging from
65.8 nM for 99mTc-1 down to 5.7 nM for 99mTc-3, (2) they are
stable in their preparation medium, in cell media, and in
plasma with essentially no metabolism in 90 min, a fact that
implies in vivo metabolic stability, (3) they are efficiently
synthesized from the corresponding ferrocenyl precursors with
radiochemical yields of >98%, (4) they selectively label Aβ
plaques in vitro in postmortem human brain as revealed by
fluorescence microscopy, (5) they present impressive brain
penetration ranging from 7.94% ID/g for 99mTc-1 to 3.99%
ID/g for Tc-2 and excellent washout kinetics with a brain2min/
brain90min ratio reaching 99.8 for 99mTc-2. Additionally,
complex 99mTc-1 administered in Tg 5×FAD mice shows
significantly (45%) increased brain uptake compared to that in
wild-type littermates, within the range observed in the
literature for analogous studies.32−35 Overall, the complexes
99m
Tc-1−3 are the first reported 99mTc agents that combine
important key features for a SPECT imaging agent and,
therefore, are excellent candidates for further biological
evaluation as AD diagnostic probes.
The brain uptake values observed for complexes 99mTc-1−3
far exceed those reported in the literature for other neutral and
lipophilic [Cp99mTc(CO)3] complexes designed as potential
Aβ imaging probes, summarized in a recent review.8 The
reported [Cp99mTc(CO)3] complexes are designed through
the conjugated approach in which the Aβ plaque binding
pharmacophore (arylbenzothiazole14,50,51 or stilbene-like52) is
joined to the [Cp99mTc(CO)3] core through alkyl linkers,
suggesting that the compactness of the structure and
consequent low MW, that characterizes complexes 99mTc-1
to 99mTc-3, are crucial factors for BBB penetration. In support
of this conclusion is the fact that complexes mimicking the
chalcone structure designed in an integrated approach similar
to ours through replacement of a phenyl ring by the
[Cp99mTc(CO)3] core present high brain uptake as well.13
Besides the favorable characteristics displayed by 99mTc-1−3
in terms of high brain uptake and fast washout kinetics, the
interesting properties of the corresponding Re-1−3 complexes
against Aβ aggregation and toxicity, in combination with their
high BBB penetration anticipated on the basis of the results of
their 99mTc counterparts, suggest a potential role for Re-1−3 as
therapeutic tools against AD. Inhibition of the aggregation of
Aβ, to either soluble oligomers or insoluble fibrils, is one of the
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Journal of Medicinal Chemistry
therapeutic strategies against AD, and a great number of small
molecules of various structures are known to interact with Aβ
and inhibit its aggregation;53,54 however, the issue of their
delivery to the brain is the main obstacle to their further
development as AD drugs. Overall, BBB penetration represents
one of the greatest challenges for drug delivery to the CNS and
one of the main reasons why clinical trials for AD drugs
fail.55,56 Based on two recent reviews that summarize the
existing data on metal complexes targeting Aβ,57,58 the Re-1−3
complexes appear to be among the very few modulators of Aβ
aggregation with demonstrated potential to cross the BBB in
vivo. This rare property in combination with their relatively
low toxicity advocates the exploration of these complexes in
the quest for AD therapeutic agents targeting Aβ oligomeriza-
tion or fibrilization and possibly other processes of neuro-
degeneration associated with AD.
Based on the above, the 99mTc-1−3 and the corresponding
Re-1−3 complexes capable of labeling and simultaneously
inhibiting the formation of Aβ fibrils form a theranostic pair for
AD, as diagnostic and targeted therapeutic potential are
combined in a single chemical entity synthesized through a
common precursor. Development of theranostics for AD is an
emerging field aiming to streamline the drug-discovery process,
with small molecules integrating imaging and therapeutic
functions exhibiting the most promising properties.59,60 The
prototype theranostic application of the 99mTc/Re pair
addresses the diagnosis and treatment of tumors, with 99mTc
being optimal for diagnostic imaging and the β-emitting 186Re
and 188Re isotopes of rhenium being suitable for radio-
therapy.61 Complexes 99mTc-1−3 and Re-1−3 offer a
promising starting point for developing novel theranostic
tools against brain tumors. Furthermore, the biological
properties (anticancer, antibacterial, antifungal, etc.) of both
the benzazole and the 2-arylbenzazole scaffold present in the
complexes broaden the spectrum of their potential application
to CNS diseases (patent pending).
In conclusion, complexes 99mTc-1−3 and the corresponding
Re-1−3 represent strong candidates in the AD drug develop-
ment field to face the unmet needs for a widely available
SPECT imaging probe, as well as for an Aβ aggregation
inhibitor. Moreover, the high brain uptake of the complexes, in
combination with their inherent pharmacophoric nature,
advocates their investigation as pharmaceutical agents of
CNS diseases.
■
EXPERIMENTAL SECTION
General Information. Warning! 99mTc is a γ emitter (t1/2 = 6.02
h; Ec = 140 keV) and requires special radioprotective measures during
handling.
All reagents used in the synthesis were purchased from Sigma-
Aldrich, Alfa Aesar, and Acros Organics and were used without further
purification. Aβ42 (>95% pure) was purchased from Anaspec. The
media/agents for the cultures of primary neuronal cells and human
glioblastoma cell line were purchased from Thermo Fisher Scientific.
The MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazo-
lium bromide) was bought from AppliChem (Germany), whereas
2′,7′-dichlorofluorescin diacetate (DCFH-DA), thioflavin T, and
thioflavin S were bought from Sigma-Aldrich (Germany). The CO gas
was purchased from Air Liquide (Greece) in a cylinder. All reactions
sensitive to oxygen or moisture were carried out under a nitrogen
atmosphere.
NMR spectra were obtained in DMSO-d6 at 25 °C on Bruker
Avance DRX 500 MHz (1H at 500.13 MHz and 13C at 125.77 MHz).
Tetramethylsilane (TMS) was used as the internal standard.
Assignment of 1H and 13C chemical shifts was based on the combined
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analysis of a series of 1H−1H and 1H−13C correlation experiments
recorded using standard pulse sequences from the Bruker library. The
IR spectra were recorded using a PerkinElmer Spectrum 100
spectrometer with a universal attenuated total reflection accessory
(PerkinElmer) over the range 4000−200 cm−1. The high-resolution
electrospray mass spectra were recorded in the range of m/z 250−
1400 on a TSQ 7000 Finnigan MAT (Scientific Instrument Services).
For the mass spectrometric studies, samples were dissolved in DMSO
and the resulting solution was supplied to the electrospray capillary
through a syringe pump. Fluorescent staining observation was
performed with a Leica TCS SP8 MP (Wetzlar, Germany) confocal
microscope equipped with an IR MaiTai DeepSee Ti:Sapphire laser
(Spectra-Physics, Santa Clara, CA) for multiphoton applications.
Images were acquired with LAS X software (Leica Microsystems CMS
GmbH, Wetzlar, Germany) and are presented without any processing.
An automated well-type γ counter [NaI(Tl)] 3 in. crystal, Packard
Minaxi Auto-γ 5000 Series γ Counter (Canberra Packard Instrument
Co., IL) calibrated for 99mTc was used for radioactivity measurements.
Elemental analysis for C, H, and N was conducted on a PerkinElmer
2400 automatic elemental analyzer (PerkinElmer).
High-performance liquid chromatography (HPLC) analysis was
performed with a Waters 600E Chromatography System coupled to
both a Waters 486 UV detector (for Re complexes) and a GABI γ-
detector from Raytest (for 99mTc complexes). Separations were
achieved on a C-18 reverse phase column (250 mm × 4 mm, 5 μm)
eluted with a binary gradient system at a 1 mL/min flow rate. Mobile
phase A was methanol containing 0.1% trifluoroacetic acid, while
mobile phase B was water containing 0.01% trifluoroacetic acid. The
elution gradient was 0−1 min 5% A (95% B), followed by a linear
gradient to 85% A (15% B) in 9 min; this composition was held for
another 20 min. After a column wash with 95% A (5% B) for 5 min,
the column was re-equilibrated by applying the initial conditions 5% A
(95% B) for 10 min prior to the next injection.
Postmortem brain tissue from an autopsy-confirmed AD case was
obtained through the University of Manchester of the Greater
Manchester Neurosciences Centre and was cut in 6 μm slices using a
Leica RM2135 rotary microtome and brain sections from mice were
cut in 10 μm slices using a Leica VT1000S vibratome. The Swiss
Albino and C57BL/6 mice as well as the Wistar rats and the
transgenic (Tg) AD mouse model (5×FAD) that were used in vitro
and in vivo biological evaluation experiments were obtained from the
breeding facilities of the Institute of Bioscience & Applications, NCSR
“Demokritos”. All the biodistribution studies were carried out in
compliance with the Presidential Decree 56/2013 (published in the
Official Government Gazette of Greece 106 A/30-4-2013) that has
transposed the EU Directive 2010/63 on the protection of animals
used for scientific purposes. U87MG cells (human glioblastoma−
astrocytoma, epithelial-like cell line) is listed in the database of
commonly misidentified cell lines maintained by ICLAC. The cell line
is from the cell bank of the Laboratory of Radiobiology, Institute of
Nuclear & Radiological Sciences and Technology, Energy & Safety,
NCSR “Demokritos” and has not been authenticated. The U87MG
cell line was free of mycoplasma contamination, as judged visually
under microscope observation and by regular 4′,6-diamidino-2-
phenylindole staining of the cell cultures. Na[99mTcO4] was eluted
from a 99Mo/99mTc generator (Mallinckrodt, Petten, Netherlands)
using 0.9% saline (3700−7400 MBq, 100−200 mCi/10 mL). The CO
gas was purchased from Air Liquide (Greece) in a cylinder. The fac-
[ 99mTc(CO)3(H2O)3]+ precursor was prepared as previously
described. Briefly, a vial containing 5.5 mg of NaBH4, 4.4 mg of
Na2CO3, and 10 mg of Na−K tartrate was purged with CO gas, and
then a solution of Na99mTcO4 (1.0 mL, ∼740 MBq) was added. The
vial was heated at 85 °C for 30 min. After cooling, the pH was
adjusted to 7.0 with 1 N HCl, and a sample was analyzed by HPLC
(yield >95%). fac-[NEt4]2[Re(CO)3Br3] was prepared according to a
published procedure.62
Synthesis. Benzo[d]thiazole-2-ferrocene (Fe-1). The ferrocenyl
precursors Fe-1 was synthesized according to a published procedure
with slight modifications. 18 Briefly, a mixture of ferrocene
carboxaldehyde (1.1 g, 5 mmol) and 2-aminothiophenol (600 μL,
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Journal of Medicinal Chemistry
5.5 mmol, 1.1 equiv) in ethanol (40 mL) was refluxed overnight. The
reaction mixture was allowed to cool to room temperature and a
reddish-brown solid precipitated which after flash column chromatog-
raphy on silica gel (eluent: hexane/ethyl acetate 98:2) gave 1.46 g of
the desired product. Yield 92%. IR (cm−1) 498, 483. The 1H and 13C
NMR data are given in Table S1, Supporting Information. Anal. Calcd
for C17H13FeNS: C: 63.97%, H: 4.11%, N: 4.39%. Found: C: 63.58%,
H: 4.32%, N: 4.25%. HPLC: tR = 17.3 min. Crystals suitable for X-ray
crystallography were obtained by slow evaporation from a mixture of
CH2Cl2/CH3OH 3:1.
Benzo[d]imidazole-2-ferrocene (Fe-2). The ferrocenyl precursors
Fe-2 was synthesized according to a published procedure with slight
modifications.19 Briefly, ferrocene carboxaldehyde (0.76 g, 4 mmol)
and sodium bisulfite (1.44 g, 1.4 mmol) were dissolved in a mixture of
absolute ethanol and water (20 mL, 1:1) and the solution was refluxed
for 3 h. After cooling to room temperature, o-phenylenediamine (0.4
g, 4 mmol, 1 equiv) was added and the reaction mixture was refluxed
for additional 2 h. The mixture was allowed to cool to room
temperature and then further down to 0 °C in an ice-water bath. The
precipitate formed was filtered, washed successively with ice-cold
water and cold absolute ethanol, air dried and subjected to flash
column chromatography on silica gel (eluent: dichloromethane/ethyl
acetate 96:4) to afford 0.75 g of the final product as a red solid. Yield
62%. IR (cm−1) 488, 477. 1H and 13C NMR data are given in Table
S1, Supporting Information. Anal. Calcd for C17H14FeN2: C: 67.58%,
H: 4.67%, N: 9.27%. Found: C: 67.35%, H: 4.82%, N: 9.11%. HPLC:
tR = 14.5 min.
1-Methylbenzo[d]imidazole-2-ferrocene (Fe-3). To a solution of
NaOH (0.54 g, 13.4 mmol) in water (1 mL), a solution of Fe-2 (0.54
g, 1.8 mmol) in acetone (7.5 mL) was added at 0 °C and the mixture
was stirred for 5 min. Subsequently, methyl iodide (145 μL, 2.3
mmol) was added dropwise. The reaction mixture was stirred for 1 h
at room temperature. Acetone was evaporated in vacuo and the
reaction mixture was diluted with water (50 mL) and extracted with
CH2Cl2 (3 × 30 mL). The organic phase was dried over Na2SO4,
filtered and the filtrate was concentrated under vacuum. The resulting
oil was purified by flash column chromatography on silica gel
(dichloromethane/ethyl acetate 96:4) to afford 0.54 g of the product.
Yield 95%. IR (cm−1) 488, 478. The 1H and 13C NMR data are given
in Table S1, Supporting Information. Anal. Calcd for C18H16FeN2: C:
68.38%, H: 5.10%, N: 8.86%. Found: C: 68.12%, H: 5.35%, N: 8.92%.
HPLC: tR = 15.5 min.
Benzo[d]thiazole-2-(cyclopentadienyl)tricarbonylrhenium (Re-
1). To a solution of [Re(CO)3Br3][NEt4]2 (35 mg, 0.047 mmol)
and the corresponding ferrocenyl derivatives Fe-1 (32 mg, 0.098
mmol) in DMF (2.5 mL), a solution of 0.1 N HCl (1.5 mL) was
added in a 5 mL glass vial. The mixture was purged with argon for 5
min and the vial was sealed with a Teflon cap. The reaction mixture
was vigorously stirred for 2 h at 160 °C. After cooling, the cap was
carefully removed and the solution diluted with dichloromethane (10
mL). The mixture was washed with water (3 × 10 mL), and the
organic phase was dried over Na2SO4, filtered and evaporated in
vacuo. The crude product was purified by flash column chromatog-
raphy on silica gel (eluent hexane/ethyl acetate 95:5) to afford the
desired Re(CO3)Cp complex as a beige powder. Yield 13 mg (59%).
IR (cm−1) 2020, 1920. 1H and 13C NMR are given in Table S2,
Supporting Information. High-resolution mass spectrometry (HRMS)
(ESI) [M + H]+ calcd for C15H9NO3S187Re, 469.9861; found,
469.9852. Anal. Calcd for C15H8NO3ReS: C: 38.46%, H: 1.72%, N:
2.99%. Found: C: 38.28%, H: 1.78%, N: 2.77%. HPLC: tR = 16.4 min.
Crystals suitable for X-ray crystallography were obtained by slow
evaporation from a mixture of CH2Cl2/CH3OH 3:1.
Benzo[d]imidazole-2-(cyclopentadienyl)tricarbonylrhenium (Re-
2). Re-2 was synthesized from the corresponding ferrocenyl derivative
Fe-2 following the procedure given for Re-1. The crude product was
purified by flash column chromatography on silica gel (eluent
dichloromethane/ethyl acetate 90:10) to afford Re-2 as a beige
powder. Yield 15 mg (70%). IR (cm−1) 2024, 1909. 1H and 13C NMR
are given in Table S2, Supporting Information. HRMS (ESI) [M +
H]+ calcd for C15H10N2O3187Re, 453.0169; found, 453.0246. Anal.
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Calcd for C15H9N2O3Re: C: 39.91%, H: 2.01%, N: 6.21%. Found: C:
39.78%, H: 1.89%, N: 6.08%. HPLC: tR = 14.6 min.
1-Methylbenzo[d]imidazole-2-(cyclopentadienyl)-
tricarbonylrhenium (Re-3). Re-3 was synthesized from the
corresponding ferrocenyl derivative Fe-3 following the procedure
given for Re-1. The crude product was purified by flash column
chromatography on silica gel (eluent hexane/ethyl acetate70:30) to
afford the Re-3 as a pale yellow powder. Yield 16 mg (72%). IR
(cm−1) 2013, 1897. 1H and 13C NMR are given in Table S2,
Supporting Information. HRMS (ESI) [M + H]+ calcd for
C16H12N2O3187Re, 467.0403, found, 467.0405. Anal. Calcd for
C16H11N2O3Re: C: 41.29%, H: 2.38%, N: 6.02%. Found: C:
40.99%, H: 2.54%, N: 5.90%. HPLC: tR = 15.8 min.
X-ray Crystallography. Important crystallographic data for
compounds Fe-1 and Re-1 are listed in Table 1. A crystal of Fe-1
(0.12 × 0.26 × 0.70 mm3) was taken from the mother liquor and
immediately cooled to −93 °C. A crystal of Re-1 was glued with
epoxy on a fiber and the crystal data were collected at room
temperature. Diffraction measurements were made on a Rigaku R-
AXIS SPIDER Image Plate diffractometer using graphite mono-
chromated Mo Kα radiation. Data collection (ω-scans) and
processing (cell refinement, data reduction, and numerical absorption
correction) were performed using the CrystalClear program pack-
age.63 The structures were solved by direct methods using SHELXS-
9764 and refined by full-matrix least-squares methods on F2 with
SHELXL2014/6.65 Further experimental crystallographic details for
Fe-1: 2θmax = 54.00°; 233 parameters refined; (Δ/σ)max = 0.016;
(Δρ)max/(Δρ)min = 0.862/−0.188 e/Å3. Further experimental
crystallographic details for Re-1: 2θmax = 54.00°; 190 parameters
refined; (Δ/σ)max = 0.004; (Δρ)max/(Δρ)min = 1.552/−2.180 e/Å3.
The hydrogen atoms of Fe-1 were located from difference Fourier
maps and were refined isotropically and those of Re-1 were
introduced at calculated positions and were refined as riding on
bonded atoms. All non-hydrogen atoms for both complexes were
refined anisotropically. The structures were drawn with the Diamond
3 program package.66
99m
Tc Labeling. The 99mTc complexes 99mTc-1, 99mTc-2, 99mTc-3,
were prepared from the corresponding ferrocenyl derivatives Fe-1, Fe-
2, F-3 by two methods, one involving a ligand exchange reaction with
the fac-[99mTc(H2O)3(CO)3]+ precursor, and the second a double
ligand transfer (DLT) reaction. For the ligand exchange reaction, each
ferrocenyl derivative (1.0 mg) in 0.5 mL of dimethylformamide was
added to the fac-[99mTc(H2O)3(CO)3]+ precursor (0.1 mL, 74 MBq).
Subsequent heating in an oil bath under nitrogen flow at 120 °C for 3
h afforded the respective 99mTc complex with radiochemical yield of
approx. 50% as shown by HPLC. For the DLT reaction, each
ferrocenyl derivative (1.0 mg) and Mn(CO)5Br (1.0 mg) were
dissolved in 0.5 mL of dimethylformamide in a 10 mL glass tube, and
0.1 mL Na[99mTcO4] (74 MBq) saline solution was added. The vial
was sealed and purged with nitrogen for 2 min and heated in an oil
bath at 110 °C for 90 min to afford the corresponding 99mTc complex
with quantitative radiochemical yield of 98% as shown by HPLC. In
both methods, the identity of the 99mTc complexes was established by
comparative HPLC using the analogous Re complexes as a reference
(Figure S9 (Supporting Information), tR 16.8 min for 99mTc-1, tR 14.9
min for 99mTc-2 and tR 16.2 min for 99mTc-3). The retention time of
each 99mTc complex was sufficiently different from that of the
corresponding ferrocenyl precursor allowing their separation on
HPLC through careful collection of the radioactive peak to
completely exclude the ferrocenyl precursor.
Stability of 99mTc Complexes. For the stability studies against
cysteine and histidine each of the HPLC-purified 99mTc complexes
(0.5 mL, approx. 10 MBq) was mixed with a solution of histidine or
cysteine (2 mM) in 0.1 M PBS, pH 7.4 (0.5 mL). The mixtures were
incubated at 37 °C and analyzed by HPLC after 1, 3, and 6 h (Table
S5). The 99mTc complexes showed high resistance to the presence of
histidine or cysteine even after incubation for 6 h (>95% intact
complex). For the stability studies of 99mTc-1−3 in rat plasma in vitro,
blood from male Wistar rats was collected in heparinized tubes,
centrifuged at 2000g at room temperature for 10 min, and plasma was
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Journal of Medicinal Chemistry
collected. An aliquot of each of the 99mTc complexes (50 μL, 1 MBq),
isolated by HPLC, was mixed with 450 μL of rat plasma and the final
mixture was incubated at 37 °C. After 15 min and 90 min, 250 μL
aliquots of the mixture were withdrawn and mixed with 500 μL of
absolute ethanol, followed by centrifugation at 14 000 rpm for 30 min.
The supernatant was separated from the pellet, and the radioactivity
was measured on a γ-counter. The percentage of 99mTc complex
binding to plasma proteins was calculated as
pellet radioactivity
% protein binding = × 100
pellet radioactivity + supernatant
The stability of the complexes was estimated by radio-reversed-phase
(RP)-HPLC analysis of the supernatants. The supernatant was filtered
with a membrane filter (0.22 μm), and 500 μL of the solution was
injected into the HPLC system. For all 99mTc preparations, the
recovery of radioactivity from the RP-HPLC column after the
injections was monitored and was found to be quantitative.
Determination of Partition Coefficient of 99mTc Complexes.
The partition coefficient of 99mTc complexes was determined by the
shake-flask method as previously reported.67 In a centrifuge tube,
containing 1.5 mL of n-octanol and 1.5 mL of phosphate-buffered
saline (PBS, 0.1 M, pH 7.4), 50 μL of each of the 99mTc complex
solution (1 MBq) isolated by HPLC was added, and the mixture was
vortexed for 1 min and finally centrifuged at 5000 rpm for 10 min.
The radioactivity of three aliquots (0.2 mL) of both 1-octanol and
PBS phases was counted in a γ-counter. The partition coefficient was
calculated as the mean value of each cpm/mL of octanol layer divided
by that of the buffer (Poct/water) and was expressed as log Poct/water. A
sample (0.5 mL) from the n-octanol layer was subsequently
repartitioned in octanol/buffer until constant values were obtained.
This was usually achieved with the third repartition.
Biodistribution Studies of 99mTc Complexes. Biodistribution
studies were performed according to the previously reported
procedure with small modifications27 using healthy Swiss albino
(male, 5 week old, 20−30 g) mice. Three groups of mice (four
animals per group) were injected in the tail vein with HPLC-purified
99m
Tc complexes (70 kBq in 0.1 mL, ethanol/saline 1:9). The mice
were sacrificed at various time points (2, 15, 90 min) post injection.
The organs of interest were excised and weighed, and the radioactivity
was recorded in an automatic γ-counter. The stomach and intestines
were not emptied of food contents prior to radioactivity measure-
ments. The percentage of injected dose per organ (% ID/organ) was
calculated by comparison of sample radioactivity to that of standard
solutions that contained 10% of the injected dose. The percentage of
injected dose per gram (% ID/g) was calculated by dividing the %
ID/organ by the weight of the organ or tissue. The calculation for
blood and muscle was based upon measured activity, sample weight,
and body composition data according to which blood comprises 7%
and muscle 43% of body weight.67 Analogous biodistribution studies
at 15 min p.i. took place with Tg 5×FAD mice (four animals, 11
month old, 20−30 g) as well as with their corresponding wild-type
littermates.
In Vitro Binding of Re Complexes to Aβ Plaques.
Deparaffinization of AD patient sections (6 μm thick) from temporal
cortex mounted on glass slides was carried out in xylene (2 × 5 min),
followed by tissue rehydration (soaking for 3 min successively in 100,
80, 60, 0% ethanol/water v/v). The slides were subsequently washed
for 10 min in running tap water and then incubated in phosphate-
buffered saline (PBS) solution (0.1 M, pH 7.4) for 30 min. Brain
tissue from Tg mice were fixed in 4% paraformaldehyde in PBS at 4
°C. Brains were cut into 10 μm sagittal sections from the genu of the
corpus callosum to the most caudal hippocampus using a vibratome,
and sections were washed in PBS following a published procedure.68
The tissue preparations were treated with Re complexes as well as
thioflavin S (ThS, for confirmation of Aβ plaque localization)
solutions (0.5 mg/mL) in dimethyl sulfoxide (DMSO) for 1 h. The
sections were finally washed with 40% ethanol for 2 min, and the
observation was performed using fluorescence confocal microscopy.
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Preparation of Aβ Stocks and Solutions. Aβ42 was gently
dissolved without vortexing in type 1 (Milli-Q) water to prepare a 100
μM stock solution for CD, ThT, and competitive binding studies and
10 μM for cell viability and ROS measurements. For the CD and ThT
studies, solutions of Aβ42 in phosphate-buffered saline (PBS, 10 mM,
pH 7.4) were prepared by adding equal volumes of PBS to aliquots of
the stock to achieve a final concentration of 50 μM. Appropriate
amounts of the Re complexes (stock concentration of 10 mM in
DMSO) were added to the Aβ42 solutions to achieve final
concentrations of 50 and 100 μM.
Binding Studies Using Aβ42 Fibrillar Aggregates. Solutions
of Aβ42 (50 μM) were allowed to age for 15 days to form Aβ fibrils as
shown by CD and the ThT assay to be used in the competitive
binding studies. The studies were performed in 1 mL solutions in PBS
in 12 mm × 75 mm borosilicate glass tubes according to a published
procedure with modifications.10 Each 1 mL solution contained
appropriate volumes from a 10 mM stock solution of each Re complex
(Re-1, Re-2, and Re-3) to achieve concentrations ranging between
10−4 and 10−10 M, 50 nM of the previously prepared Aβ42 fibrils and
7 kBq of the corresponding 99mTc complex (99mTc-1, 99mTc-2, and
99m
Tc-3, respectively). The solutions were incubated for 3 h at room
temperature and the bound radioactivity was separated from the free
by Millex-GV syringe filters (0.22 μm, Millipore) followed by 2 × 2
mL washes with PBS at room temperature. Filters containing the
bound 99mTc complexes were measured for radioactivity in a γ-
counter. Binding studies were performed in triplicate. The data were
analyzed using GraphPad Prism software with the IC50 value
calculated using a one-site competitive binding linear regression.69
The Ki value was calculated using the Cheng−Prusoff equation70 Ki =
IC50/(1 + [radioligand]/Kd). In the case of homologous competitive
binding, we assume that the Re and 99mTc complexes bind to Aβ with
identical affinities so that Kd and Ki are the same and the above
equation is simplified to Ki = IC50 − [radioligand]. As the
[radioligand] was calculated in all experiments to be in the range
10−12−10−13 M, orders of magnitude smaller than the IC50 value,
essentially Ki = IC50.
Circular Dichroism Measurements. Circular dichroism (CD)
studies with Aβ42 were performed for a time period of 15 days to
monitor the secondary structural changes of Aβ42 that lead to the
formation of Aβ fibrils. During this period, the solutions remained at
the incubator at 33 °C without stirring. CD spectra were recorded on
a JASCO J-715 spectropolarimeter (Jasco Co., Japan), at 33 °C in the
range of 190−260 nm with a 1 mm path length quartz cuvette. Each
spectrum was the average of three scans at a speed of 100 nm/min
and a resolution of 0.5 nm. The analysis of the CD data was
performed using OriginPro 9 program.
Thioflavin T Assay. For the ThT assay, the complexes Re-1−3
(50 μM) were incubated at 33 °C with Aβ in PBS (10 mM, pH 7.4)
at 1:1 and 1:2 Aβ/complex ratios. The solution of plain Aβ that
served as control was monitored over time with CD, whereas the
monitoring of the solutions of Aβ with the complexes was not possible
due to the high absorbance of the complexes. After 15 days, 100 μL of
the aged (15 days) 50 μM solutions of Aβ42 (with or without Re
complexes) was diluted to half with PBS to obtain 200 μL of Aβ
solution (25 μM). To these solutions, a stock solution of ThT in PBS
(10 mM, pH 7.4) was added to achieve a final ThT concentration of 5
μM. The mixture was well agitated with pipetting, and immediately
after this, its fluorescence was recorded after excitation at 440 nm
(EM slit, 2.5 nm; photomultiplier tube voltage, 700 V; and response,
0.4 s) with a Hitachi F-2500 spectrofluorometer. The analysis of the
fluorescence data was performed with OriginPro 9 program.
Cell Cultures. Hippocampal neuronal cultures were obtained from
postnatal day 0−3 (P0−3) mice, which were euthanized, and their
hippocampi were isolated. Briefly, after being dissected, each
hippocampus was enzymatically digested with 0.25% trypsin for 20
min at 37 °C. The hippocampi were then rinsed in 10 mL of
Hibernate-A medium containing 10% (v/v) heat-inactivated fetal
bovine serum (FBS) to remove any traces of serum. After proper
trituration, the cells were seeded on poly-D-lysine 96-well plates at a
density of 2 × 104 per well. Cultures were maintained in Neurobasal-
DOI: 10.1021/acs.jmedchem.8b01949
J. Med. Chem. 2019, 62, 2638−2650
Journal of Medicinal Chemistry
A medium containing 2% B-27 supplement, 0.5 mM GlutaMAX, and
1% penicillin/streptomycin at 37 °C and 5% CO2, until proper
differentiation. Half of the medium was replaced twice a week. After 7
days of incubation in culture well plates, the primary hippocampal
neurons were used for cell rescue from Aβ toxicity and ROS
measurements.
Stability of Re Complexes in Cell Culture Medium. The
stability of Re complexes in cell culture medium at 37 °C was checked
by HPLC to ensure that complexes are not subjected to any potential
decomposition in our experimental conditions. Complexes were
incubated in cell culture medium (described above) at 37 °C, and
after 24 h, an aliquot was removed, centrifuged at 1000 rpm for 10
min in a refrigerated centrifuge, and examined by HPLC.
Cell Rescue from Aβ Toxicity. For the cell viability studies in the
presence of Aβ, the old medium was removed and 90 μL of fresh
medium was added in each well of the 96-well plate. Solutions (10
μL) of Aβ42 (10 μM) in PBS in the absence or presence of the Re
complexes (1:1 and 1:2 ratios of Aβ/complexes) preincubated for 1
day at 37 °C were added to individual wells (final Aβ concentration, 1
μM). It should be noted that, according to our previous studies, 1 day
preincubation of the Aβ42 solutions was necessary in order for the
peptide to exert maximum neurotoxicity. Cell viability was determined
with the standard MTT assay. After 24 h exposure of cells to the Aβ42
solutions, 100 μL of a 0.5 mg/mL stock solution of MTT in
Neurobasal-A was added to each well of primary hippocampal
neurons followed by a 3 h incubation at 37 °C. The medium was
removed, the crystals formed in intact cells were solubilized in
DMSO, and the absorbance was measured at 540 nm (Tecan well
plate reader). Results were expressed as the percentage of reduced
MTT, assuming the absorbance of control cells to be 100%, and are
the mean of three independent experiments with six replicate wells for
each condition. Graphs were analyzed using GraphPad Prism 5.0
software. In each run, the effect of solutions of plain Aβ42 and each of
Re-1−3 complexes was independently checked to serve as internal
standard.
In Vitro Cytotoxicity of Re Complexes with MTT Assay. The
cytotoxicity of the Re complexes was assessed against U87MG cells
(human glioblastoma−astrocytoma, epithelial-like cell line). The
U87MG cells were grown in Dulbecco’s modified Eagle’s medium,
growth medium of pH 7.4, supplemented with 10% FBS, 100 U/mL
penicillin, 2 mM glutamine, and 100 μg/mL of streptomycin. Cell
cultures were maintained in flasks and were grown at 37 °C in a
humidified atmosphere of 5% CO2 in air. Subconfluent cells were
detached using a 0.05% (w/v) trypsin−0.25% (w/v) ethylenediami-
netetraacetic acid solution, and the subcultivation ratio was 1:2−1:5.
In vitro cytotoxicity of Re-1−3 complexes was determined by the
MTT colorimetric assay. Cells were seeded in 96-well plates (4 × 103
cells/well in 100 μL of culture medium) and grown overnight at 37
°C in a 5% CO2 incubator. Exponentially growing cells were
incubated for 72 h with various concentrations ranging between
10−3 and 10−8 M of each complexes, and the final DMSO
concentration never exceeded 0.2%. The medium was then removed
and replaced with 100 μL of MTT solution (1 mg/mL). After a 4 h
incubation, the solution was aspirated, formazan crystals were
solubilized in 100 μL of DMSO, and absorbance was recorded at
540 nm (Tecan well plate reader).
Intracellular ROS Measurements. Primary hippocampal neu-
rons were treated with Aβ42 solutions following the procedure
described in the Cell Rescue from Aβ Toxicity section. After
incubation for 24 h, cells were washed with PBS and incubated with
10 μM DCFH-DA for 30 min at 37 °C in the incubator with 5% CO2
The fluorescence intensity (relative fluorescence unit) of DCF was
determined using a Tecan fluorescence well plate reader at the
excitation wavelength of 485 nm and emission wavelength of 528 nm.
ROS levels are presented as arbitrary fluorescence units. Control
groups consisted of cells incubated with medium only and plain Aβ42
or plain Re complexes.
Statistical Analysis. Data in all assays are the mean of at least
three independent experiments. In hippocampal neuronal cultures, the
statistical significance of changes in different groups was evaluated by
2648
Article
one-way analysis of variance and Student’s t-tests, using GraphPad
Prism 5.0 software. For each experiment, data are expressed as the
mean ± standard error of the mean (SEM), *p ≤ 0.05, **p ≤ 0.01,
***p ≤ 0.001, and not significant (ns) > 0.05 compared with Aβ42 (1
μM) treatment and #p < 0.01, ##p < 0.01, and ###p < 0.001 compared
with control (untreated cells).
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b01949.
Indicative IR and NMR spectra and 1H and 13C NMR
chemical shifts for Fe-1−3 and Re-1−3; electronic
spectra for Re-1−3; geometric parameters for Fe-1, Re-
1; intermolecular interactions in the structures of Fe-1
and Re-1; HPLC chromatograms of Re and 99mTc
complexes; stability studies of 99mTc and Re complexes;
biodistribution of 99mTc-1 in Tg and wt mice; Aβ plaque
staining with Re-1; MTT studies with Re-1−3; CD
spectra of Re-1−3; and CD spectra of the aggregation of
Aβ42 (PDF)
Molecular formula strings for Fe-1−3, Re-1−3, and
99m
Tc-1−3 (CSV)
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: pelmar@bio.demokritos.gr.
ORCID
Minas S. Papadopoulos: 0000-0002-1107-027X
Maria Pelecanou: 0000-0002-7669-2424
Author Contributions
M.S., B.M., A.S., I.P., M.S.P., and M.P. designed the
compounds, executed the experiments, interpreted the results,
and wrote the manuscript; C.R. and V.P. performed the X-ray
analysis and wrote the corresponding sections; and M.P.-P.
provided cell lines and supervised the biological evaluation
experiments involving cells.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors acknowledge support of this work by the projects
“Target Identification and Development of Novel Approaches
for Health and Environmental Applications” (MIS 5002514),
which is implemented under the Action for the Strategic
Development on the Research and Technological Sectors, and
“A Greek Research Infrastructure for Visualizing and
Monitoring Fundamental Biological Processes (BioImaging-
GR)” (MIS 5002755), which is implemented under the Action
“Reinforcement of the Research and Innovation Infra-
structure”. Both projects are funded by the Operational
Programme “Competitiveness, Entrepreneurship and Innova-
tion” (NSRF 2014-2020) and co-financed by “Greece and the
European Union (European Regional Development Fund)”.
M.P. acknowledges financial support by HYGEIA Hospital,
Athens, Greece, and B.M. gratefully acknowledges financial
support by Stavros Niarchos Foundation (SNF) through
implementation of the program of Industrial Fellowships at
NCSR “Demokritos”.
DOI: 10.1021/acs.jmedchem.8b01949
J. Med. Chem. 2019, 62, 2638−2650
Journal of Medicinal Chemistry
■
ABBREVIATIONS
AD, Alzheimer’s disease; BBB, blood−brain barrier; CNS,
central nervous system; DCFH-DA, 2′,7′-dichlorodihydro-
fluorescein diacetate; FBS, fetal bovine serum; MTT, 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; PET,
positron emission tomography; SPECT, single-photon-emis-
sion computed tomography; ThT, thioflavin T; ThS, thioflavin
S hydroxy-4′-R-phenyl)benzothiazole nanoparticles and fluorescence
■
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DOI: 10.1021/acs.jmedchem.8b01949
J. Med. Chem. 2019, 62, 2638−2650