A phenylbenzothiazole conjugate with the tricarbonyl fac-[M(I)(CO) 3]+ (M = Re, 99Tc, 99mTc) core for imaging of β-Amyloid plaques

Article abstract of DOI:10.1002/ejic.201200450

The 2-(4'-aminophenyl)-6-methylbenzothiazole that is known to display affinity and specificity toward the amyloid plaques of Alzheimer's disease (AD) has been joined to the tricarbonyl [M(CO)₃NNO] chelate (M = Re, ⁹⁹Tc, and ^99mTc) through a five-carbon linker chain to generate the neutral complex 1 (namely, Re-1 for M = Re; ⁹⁹Tc-1 for M = ⁹⁹Tc; and ^99mTc-1 for M = ^99mTc) with the aim of developing a single-photon emission computed tomography (SPECT) radiodiagnostic agent for AD. Re-1 was characterized by spectroscopic methods and X-ray crystallography, whereas the detailed NMR spectroscopic analysis of ⁹⁹Tc-1 demonstrated its structural similarity to Re-1. Complexes Re-1 and ⁹⁹Tc-1 display selective binding affinity for amyloid plaques as evidenced by fluorescence spectroscopy, whereas the biodistribution data of ^99mTc-1-characterized by relatively low brain uptake, fast clearance from brain and blood, and in vivo stability-are considered encouraging for further elaboration on the structural features of 1 in the direction of increased brain uptake.

Full text of DOI:10.1002/ejic.201200450

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DOI: 10.1002/ejic.201200450
A Phenylbenzothiazole Conjugate with the Tricarbonyl fac-[M(I)(CO)3]+
99
(M = Re, Tc, 99mTc) Core for Imaging of β-Amyloid Plaques
Marina Sagnou,[a] Stamatia Tzanopoulou,[a] Catherine P. Raptopoulou,[b]
Vassilis Psycharis,[b] Henrik Braband,[c] Roger Alberto,[c] Ioannis C. Pirmettis,[d]
Minas Papadopoulos,[d] and Maria Pelecanou*[a]
Keywords: Medicinal chemistry / Imaging agents / Rhenium / Technetium / Alzheimer’s disease
The 2-(4Ј-aminophenyl)-6-methylbenzothiazole that is
known to display affinity and specificity toward the amyloid
plaques of Alzheimer’s disease (AD) has been joined to the
tricarbonyl [M(CO)3NNO] chelate (M = Re, 99Tc, and 99mTc)
through a five-carbon linker chain to generate the neutral
complex 1 (namely, Re-1 for M = Re; 99Tc-1 for M = 99Tc; and
99mTc-1 for M = 99mTc) with the aim of developing a single-
photon emission computed tomography (SPECT) radiodiag-
nostic agent for AD. Re-1 was characterized by spectroscopic
methods and X-ray crystallography, whereas the detailed
NMR spectroscopic analysis of 99Tc-1 demonstrated its struc-
tural similarity to Re-1. Complexes Re-1 and 99Tc-1 display
selective binding affinity for amyloid plaques as evidenced
by fluorescence spectroscopy, whereas the biodistribution
data of 99mTc-1Ϫcharacterized by relatively low brain up-
take, fast clearance from brain and blood, and in vivo sta-
bilityϪare considered encouraging for further elaboration on
the structural features of 1 in the direction of increased brain
uptake.
Introduction
Aβ plaques in living brain tissue[7–9] and demonstrated the
potential utility of these agents. The most widely studied
PET amyloid imaging agent is the 2-phenylbenzothiazole
derivative 2-(4Ј-[11C]methylaminophenyl)-6-hydroxybenzo-
thiazole (Pittsburgh compound-B, [11C]PiB), a neutral de-
rivative of the amyloid binding dye thioflavin-T (Scheme 1)
that is employed in clinical practice. The histological and
The development of specific positron emission tomo-
graphy (PET) and/or single-photon emission computed
tomography (SPECT) imaging agents for in vivo imaging
of β-amyloid (Aβ) plaques of Alzheimer’s disease (AD) re-
presents an active area in radiopharmaceutical design.[1–3]
Aβ plaques are one of the signature lesions of the AD brain
that consist of fibrillar deposits of Aβ peptide,[4,5] and their
in vivo visualization might therefore contribute to the defi-
nite and early diagnosis of AD. Furthermore, the spatially
detailed information provided by radioimaging on the ex-
tent and propagation of pathology in the living brain could
contribute to neuropathogenesis studies of AD and in the
evaluation of the efficacy of antiamyloid therapies currently
under investigation.[6]
Clinical trials in AD patients with several PET imaging
agents labeled with either 11C or 18F resulted in imaging of
[a] Institute of Biology, National Centre for Scientific Research
“Demokritos”,
15310 Athens, Greece
Fax: +30-210-6511767
E-mail: pelmar@bio.demokritos.gr
Homepage: http://bio.demokritos.gr/index.php?option=com_
content&view=article&id=39&Itemid=49&lang=en
[b] Institute of Materials Science, National Centre for Scientific
Research “Demokritos”,
15310 Athens, Greece
[c] Department of Inorganic Chemistry, University of Zürich,
Winterthurerstrasse 191, 8053 Zürich, Switzerland
[d] Institute of Radioisotopes
& Radiodiagnostic Products,
National Centre for Scientific Research “Demokritos”,
15310 Athens, Greece
Scheme 1. Structures of thioflavin T, Pittsburg compound B (PiB),
and complex 1.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201200450.
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M. Pelecanou et al.
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biochemical specificity of PiB binding across different re- and enhanced affinity for amyloid in vitro and in
gions of the AD brain was demonstrated by the direct cor- vivo.[8,20,21] In the final ligand 4 (which will be used in the
relation of in vivo [11C]PiB retention with region-matched form of its ethyl ester 4Ј), compound 2 is joined through a
quantitative analyses of Aβ plaques in the same sub- pentanoyl linker to the metal-chelating moiety. The affinity
ject.[2,8,10] However, due to the short half-life of 11C and specificity of both 2 and 4Ј for amyloid plaques was
(20 min), the availability of [11C]PiB is limited to on-site confirmed through in vitro staining experiments on brain
cyclotrons and sophisticated radiochemistry laboratories, tissue (Figure S1 in the Supporting Information). The pur-
and as a result, its structural analogue that contains the pose of the five-carbon linker chain is to alleviate the steric
longer half-life (110 min) radioisotope 18F-fluorine has hindrance imposed by the bulkiness of the metal core and
been introduced.[11] [18F]3Ј-FPiB (18F-Flutemetamol) per- to allow interaction of the phenylbenzothiazole pharmaco-
forms similarly to [11C]PiB, provides a wider accessibility phore with amyloid plaques. It should be mentioned that in
for clinical and research use, and is currently in Phase III the case of the corresponding complex that bears a two-
clinical trials.[12] The successful course of the PiB com- carbon linker chain,[22] no binding to the amyloid plaques
pound indicates that small-molecule amyloid imaging was observed, a fact attributed to steric effects that are im-
agents can be developed through structural modifications posed by the bulkiness of the metal core.
of thioflavin T.
Because PET scanners and the infrastructure for hand-
ling PET isotopes are only available to a limited number
of modern hospitals, considerable effort has been directed
toward the development of agents for imaging of Aβ pla-
ques with SPECT, which is more widely used in routine
diagnostic practice. Even though progress in developing im-
aging agents that target Aβ plaques is less advanced for
SPECT than for PET, a variety of probes labeled with the
SPECT isotopes 123I, 125I, or 99mTc have been prepared and
evaluated in in vitro and in vivo experiments.[13,14] In par-
ticular, the promising results obtained with PET-labeled PiB
and the nearly optimal characteristics of technetium-99m
as a radionuclide for nuclear imaging[15] have led to the syn-
thesis of a number of complexes of 99mTcO(V)3+, or of its
congener ReO(V)3+, with tetradentate S2N2 or SN3 chela-
tors carrying the 2-phenylbenzothiazole pharmacophore
either through a linker[16–19] or integrated into the chelate
moiety.[19] These 99mTcO(V)3+ complexes showed affinity
for Aβ plaques in vitro; however, their unfavorable in vivo
pharmacokinetics in normal miceϪcharacterized either by
low brain uptake or by slow washoutϪhas precluded their
clinical testing,[13] thus suggesting that additional molecular
modifications are required to improve their in vivo proper-
ties. Within the framework of the ongoing investigation of
the development of a 99mTc-based SPECT amyloid imaging
agent, the synthesis of a new 2-phenylbenzothiazole com-
+
plex with the M(CO)3 tricarbonyl core (1; Scheme 1) with
M = Re, 99Tc, and 99mTc is herein reported along with its
initial biological evaluation.
Scheme 2. Synthetic pathways leading to ligand 4. The atom num-
bering is the one used for the NMR spectroscopic assignments pre-
Results and Discussion
sented in Table 1.
Design of Complex 1
In the design of complex 1, 2-(4Ј-aminophenyl)-6-methyl-
The organometallic fac-[99mTc(I)(CO)3]+ core was se-
benzothiazole (2, Scheme 2) was employed as the pharma- lected as an alternative to the 99mTcO(V)3+ core employed
cophore to render affinity for amyloid plaques. Compound so far in the literature for labeling of amyloid plaques be-
2 is a neutral derivative of thioflavin-T in which the methyl cause it forms kinetically stable complexes with high spe-
group on the heterocyclic nitrogen has been removed with cific radioactivity and it is readily available through the Iso-
consequent elimination of the positive charge from the link® kit in a routine radiopharmaceutical environment.[23]
benzothiazole ring. This removal is known to produce Even though the 99mTc-tricarbonyl agents as a class are not
phenylbenzothiazole derivatives with increased lipophilicity characterized by efficient crossing of the blood/brain bar-
2
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Imaging of β-Amyloid Plaques
rier, a number of complexes in the literature with triden- This approach led to the isolation of pure 4Ј after a single
tate[24–26] or bidentate[27] ligands have shown adequate flash chromatography column and at a significantly in-
crossing to justify the utilization of the 99mTc-tricarbonyl creased reaction yield (51% overall). A ligand-exchange re-
core in the development of potential amyloid plaque im- action between 4Ј and the organometallic (NEt4)2[ReBr3-
aging agents. The tridentate NNO ligand[15] picolylamine (CO)3] precursor[21] in the presence of sodium hydroxide
monoacetic acid (PAMA; Scheme 2) was selected as the generated complex Re-1 in high yield. Complex Re-1 was
chelator for the 99mTc(CO)3+ core because it leads to stable, crystallized from CH3OH/water and was fully characterized
neutral complexes that display in vivo stability.[28–30] As is by elemental analysis, spectroscopic techniques, and X-ray
the usual practice in technetium radiochemistry,[31] the crystallography.
stable Re analogue was synthesized first for characteriza-
Complex 99Tc-1 was synthesized in a similar way to its
tion and in vitro evaluation purposes. Chemistry was sub- Re analogue by treating (NEt4)2[99TcCl3(CO)3] with ligand
sequently transferred at the pseudostable 99Tc and at the 4Ј in MeOH.
metastable 99mTc level.
The infrared spectra of complexes Re-1 and 99Tc-1 show
strong bands at 2016, 1910, 1871, and at 2035, 1931, and
1915 cm–1, respectively, which are attributed to stretching
vibrations of the tricarbonyl fac-[M(CO)3]+ unit. In ad-
Synthetic Procedure for Re-1 and 99Tc-1
For the synthesis of ligand 4Ј (Scheme 2), the published dition, the complexes exhibit strong broad absorptions in
procedure[32,33] involves acylation of the aromatic amine of the 1600–1650 cm–1 region that correspond to asymmetrical
2 by 5-bromopentanoyl chloride to generate the bromide C=O stretching vibrations of the amide and the coordi-
3, followed by nucleophilic attack of the nitrogen of the nated carboxylate groups.
secondary amine of the ethyl ester of PAMA to generate
1
The assignments of the H and 13C chemical shifts for
ligand 4Ј in the form of its ethyl ester 4Ј. This synthetic ligand 4Ј and complexes Re-1 and 99Tc-1 proceeded as de-
scheme led to a fairly impure product that required repeti- scribed in detail for related N-derivatized PAMA complexes
tive flash chromatography separations for its purification with the tricarbonyl fac-[Re(CO)3]+ core[28,29] and are pre-
(yield Ͻ 20%). To improve the procedure, a stepwise syn- sented in Table 1. NMR spectroscopic data for the corre-
thetic approach was employed in which the N-pentanoyl- sponding free acid ligand 4 are given in Table S1 of the
bromide 3 initially reacted with 2-(methylamino)pyridine to Supporting Information. Relative to ligand 4, the spectra
yield 5 and subsequently, without purification, ethyl bromo- of Re-1 and 99Tc-1 display distinct features that indicate the
acetate was used to attack the secondary amine formed. coordination of ligand 4 around the metal core, namely: 1)
1
Table 1. H and 13C chemical shifts [ppm] for ligand 4Ј and complexes Re-1 and 99Tc-1 in [D6]DMSO at 25 °C. The numbering of the
atoms is shown in Scheme 2.
4Ј
Re-1
99Tc-1
4Ј
Re-1
99Tc-1
H-1
H-2
H-3
H-4
H-6
H-7
H-9
H-10
H-11
H-12
H-15/H-19
H-16/H-18
H-22
H-23
H-25
H-27
NH
8.46
7.23
7.75
7.48
3.84
3.39
2.62
1.46
1.59
2.32
7.77
8.00
7.89
7.34
7.90
2.45
8.76
7.57
8.14
8.69
7.56
8.06
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
148.64
122.10
136.44
122.53
159.47
59.71
54.44
170.84
53.19
151.97
125.84
140.52
123.91
159.61
67.37
60.81
178.81
69.12
151.80
125.21
140.09
123.68
158.86
66.55
61.61
177.01
68.15
7.71
7.65
4.77/4.53
3.82/3.41
3.57/3.49
1.83/1.77
1.67
2.45
7.80
8.01
7.90
7.34
7.91
4.53/4.45
3.72/3.20
3.44
1.77
1.66
2.44
7.80
8.01
7.89
7.34
7.90
2.45
10.27
C-10
C-11
C-12
C-13
C-14
C-15/C-19
C-16/C-18
C-17
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
Et
26.66
22.66
36.24
23.99
22.17
35.99
23.70
21.98
35.72
171.64
141.96
119.16
127.78
127.43
165.88
151.77
122.04
127.98
134.96
121.75
134.38
21.02
171.34
141.94
119.31
127.85
127.57
165.93
151.80
122.16
128.05
135.04
121.80
134.43
21.07
171.09
141.97
119.26
127.85
127.55
165.93
151.90
122.15
128.05
135.04
121.80
134.43
21.07
2.45
10.27
10.16
4.07, 1.17
Et
59.61, 14.11
CϵO
197.24
197.76
197.87
not
detectable
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downfield shifts of the H-1 to H-4 protons of the pyridine rings in the coordination sphere (i.e., Re–N–C–C–N and
moiety by δ = 0.2–0.6 ppm and of the corresponding C-1 Re–O–C–C–N). The former adopts the envelope configura-
to C-4 carbon atoms by δ = 1.3–4.6 ppm, 2) downfield tion with C8 being 0.59 Å out of the best mean plane of the
shifts up to δ = 16 ppm of the C-6, C-7, C-8, and C-9 car- remaining four atoms. The latter is planar with the largest
bon atoms that are directly attached to the coordinating deviation from the mean plane being ca. 0.1 Å for C10. The
NNO atoms, 3) chemical-shift differentiation of the geminal Re–N2 bond length is slightly longer than the Re–N1, as
protons on C-6, C-7, and C-9 that appear as doublet peaks expected on account of the sp3 character of the former with
with average downfield shifts of δ = 0.6–1 ppm (see Fig- respect to the sp2-hybridization of the latter. The Re–C2
ure S2 in the Supporting Information). The chemical shifts bond length is slightly shorter than the other two Re–CO
of the protons and carbon atoms of the phenylbenzothi- bond lengths on account of the trans effect in the apical
azole moiety that are distant from the metal do not show position. In the crystal lattice, the rhenium complexes form
any appreciable changes upon coordination.
dimers on account of the strong hydrogen bonding between
1
Comparison of the H chemical shifts between the Re-1 the amide NH group and the coordinated carboxylato oxy-
and 99Tc-1 complexes show a small metal dependence for gen atom of a neighboring complex [N3···O4 2.916 Å
the protons close to the coordination sphere, with Re reso- (1 – x, 1 – y, –z), H3N···O4 2.050 Å, N3–H3N···O4 168.3°].
nances being more deshielded and ΔδH(Re,99Tc) ranging
Table 2. Selected bond lengths [Å] and angles [°] for Re-1.
from 0.01 to 0.2 ppm. With the exception of C-7, carbon
atoms follow the same trend with ΔδC(Re,99Tc) ranging
Bond lengths
from 0.2 to 1.8 ppm. Chemical-shift differentiation of the
geminal protons on C-6 and C-7 is smaller for 99Tc-1 than
for Re-1 and it becomes nonexistent for protons on C-9 and
C-10 of the linker chain, thereby possibly indicating higher
chelate mobility for the 99Tc-1 complex than for Re-1. The
Re–C1
Re–C2
Re–C3
1.910(9)
1.888(9)
1.928(7)
Re–N1
Re–N2
Re–O4
2.183(6)
2.227(5)
2.125(5)
Angles
C2–Re–C1
87.9(3)
90.1(3)
87.9(3)
174.9(2)
94.8(3)
94.3(2)
95.7(3)
173.2(2)
C3–Re–N1
O4–Re–N1
C2–Re–N2
C1–Re–N2
C3–Re–N2
O4–Re–N2
N1–Re–N2
97.8(2)
81.2(2)
96.5(2)
97.0(2)
172.0(2)
79.0(2)
76.9(2)
Re-1/99Tc-1 comparison data are in agreement with the C2–Re–C3
trends reported for ReO(V)3+ and TcO(V)3+ homologous
C1–Re–C3
C2–Re–O4
C1–Re–O4
C3–Re–O4
C2–Re–N1
C1–Re–N1
complexes with diaminedithiols.[34]
X-ray Crystallography of Re-1
Compound Re-1 crystallizes in the triclinic space group
¯
P1. The asymmetric unit contains one neutral rhenium
Affinity of Re-1 and 99Tc-1 for Amyloid Plaques
The binding affinity of Re-1 for Aβ plaques was tested
complex and is partially occupied by water and methanol
solvate molecules. The molecular structure of Re-1 is given
in Figure 1, and selected bond lengths and angles are listed with in vitro staining experiments with human AD brain
in Table 2. The coordination geometry about rhenium is tissue and observation under a fluorescence microscope.
distorted octahedral and consists of the fac-[Re(CO)3]+ The phenylbenzothiazole moiety is fluorescent,[35] and com-
moiety and the NNO donor atom set of the tridentate li- plex Re-1 retains the fluorescent properties with an ab-
gand. The NNO donor atom set consists of a pyridine ni- sorbance maximum at 329 nm and an emission maximum
trogen, a tertiary amine nitrogen, and a carboxylato oxygen at 378 nm (Figure S3 in the Supporting Information). Fig-
atom. The apical positions of the octahedron are occupied ure 2 shows microscope images of the stained brain tissue
by the carboxylato oxygen of the tridentate ligand and one in which it is clear that complex Re-1 clearly labels plaques
of the carbonyl groups. Rhenium lies almost in the equato- with blue fluorescence. The results of staining of adjacent
rial plane defined by the two remaining carbonyl groups plaques with the histological dye thioflavin S, which is clini-
and the two coordinated nitrogen atoms of the tridentate cally used for the detection of amyloid plaques (Figure 2,
ligand (displacement 0.09 Å). There are two five-membered B, D), demonstrate that Re-1 labels plaques in a similar way
Figure 1. Partially labeled plot of Re-1 with ellipsoids drawn at the 30% probability level. Hydrogen atoms (except that on N3) are
omitted for clarity.
4
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Imaging of β-Amyloid Plaques
to thioflavin S. Similarly, complex 99Tc-1 showed also the acterized complexes Re-1 and 99Tc-1 (Figure 3). The 99mTc-
ability of in vitro labeling amyloid plaques (Figure S4 in the 1 complex was stable in its preparation reaction mixture for
Supporting Information). These staining results, in combi- a period of at least 6 h at room temperature and was also
nation with the lack of staining when a shorter acetamide found stable against the histidine and cysteine challenge for
linker was employed,[22] confirmed the hypothesis that in the same period of time.
this type of complexes the phenylbenzothiazole pharmaco-
The lipophilicity of the 99mTc-1 complex was evaluated
phore has to be distanced from the metal core to interact according to a literature procedure[36] by the determination
with amyloid plaques.
of its partition coefficient (P) in n-octanol/0.1 m phosphate
buffer (pH 7.4), which resulted in a logPoct/buffer value of
1.69. This logPoct/buffer value falls within the range of 1–2.5
considered ideal for potential brain-imaging agents.[37]
Biodistribution of 99mTc-1
Biodistribution studies of 99mTc-1 were carried out in
healthy Swiss albino mice, and the results at 1, 15, and
60 min p.i. are presented in Table 4. The radioactivity mea-
sured in the brain tissue at 1 min is 0.69% ID/g (percentage
of injected dose of radioactivity per gram of tissue), and at
15 min it is washed out, quickly dropping to 0.05% ID/g.
The radioactivity rapidly clears from the blood and reaches
0.65 and 0.24% ID/g at 15 and 60 min, respectively. The
low stomach and spleen values are indicative of the lack of
in vivo oxidation of technetium to pertechnetate or Tc col-
loid. Excretion of radioactivity occurred mainly through
the hepatobiliary system.
Figure 2. Fluorescence microscopy images of slices of Alzheimer’s-
diseased brain stained with (A, C) complex Re-1 and (B, D) thio-
flavin S. The magnification of the lens is indicated on each picture.
Slices A/B, C/D are adjacent to allow for comparison of the stain-
The biodistribution data for 99mTc-1 compare favorably
to those that exist in the literature for related complexes
ing pattern between complex Re-1 (blue fluoresence, max at with the phenylbenzothiazole moiety. Specifically, a poten-
tial 99mTcO–monoamine–monoamide
(
99mTcO–MAMA)
378 nm) and thioflavin S (yellow-green fluoresence, max at
550 nm). It is clear from the photos that complex Re-1 stains
amyloid plaques in a similar way to thioflavin S.
tracer conjugated to pheynylbenzothiazole through a five-
carbon alkoxy chain,[18] despite its high initial brain uptake
(1.34% ID/g at 2 min p.i.), is associated with high blood
background (4.43% ID/g at 60 min), which is unfavorable
for imaging applications. Other potential 99mTcO–bis-
amine-bis-thiol (99mTcO–BAT) complexes conjugated to
phenylbenzothiazole through either alkoxy[16] or amide[17]
linker chains show low brain uptake between 0.1 and
0.2% ID/organ at 2 min accompanied by low in vivo sta-
bility and the generation of pertechnetate and hydrophilic
metabolites that result in high levels of radioactivity in the
blood (5.7–8.8% ID/organ at 60 min) and in the stomach
(4.7–5.8% ID/organ at 60 min) that preclude their further
investigation. Complex 99mTc-1 lacks the negative charac-
teristics (high blood background, reoxidation to pertechnet-
ate) of the other potential phenylbenzothiazole tracers in
the literature, and even though its % ID brain uptake in
normal mice is relatively low, this percentage might increase
in pathological AD mice in which binding to amyloid pla-
ques and retention of radioactivity is anticipated. Overall,
the biodistribution results justify the further improvement
of the structural characteristics of 99mTc-1 with an aim
towards increased brain uptake. Overall, the biodistribution
results justify the further improvement of the structural
characteristics of 99mTc-1 (e.g., by replacing the pentanoyl
by a butyl linker chain, or replacing the amide functionality
of the linker with amine) according to increased brain up-
take.[37,38]
Synthesis and Characterization of 99mTc-1
Complex 99mTc-1 was prepared by a ligand-exchange re-
action that employed the fac-[99mTc(CO)3(H2O)3]+ precur-
sor as described previously[32,33] in a radiochemical yield of
Ͼ95%. The identity of complex 99mTc-1 was established by
comparative HPLC analysis using samples of the well-char-
Figure 3. Comparative reverse-phase HPLC chromatograms. UV
detection at 254 nm: (A) complex Re-1 and (B) complex 99Tc-1.
Radiometric detection: (C) complex 99mTc-1.
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NMR spectroscopic chemical shifts was based on two-dimensional
1H,1H and 1H,13C correlation experiments (COSY, NOESY,
HSQC, HMBC) were recorded using standard pulse sequences. The
Conclusion
Complex 1, in which the tricarbonyl [M(CO)3NNO] che-
late is joined to the 2-(4Ј-aminophenyl)-6-methylbenzothi-
azole according to the pendant approach, was successfully
synthesized and fully characterized at the macroscopic (Re-
1 and 99Tc-1) and the non-carrier-added technetium (99mTc-
1
chemical shifts for H and 13C are reported in ppm (δ) relative to
tetramethylsilane (TMS).
The starting pharamcophore 2-(4Ј-aminophenyl)-6-methylbenzo-
thiazole (2) as well as the precursors (NEt4)2[ReBr3(CO)3], (NEt4)2-
1) level. To the best of our knowledge, the X-ray crystallo- [99TcCl3(CO)3] and fac-[99mTc(CO)3(H2O)3]+ were prepared accord-
ing to published procedures.[21,33,39]
graphic structure of complex Re-1 is the first example of a
complex with a pendant phenylbenzothiazole pharmaco-
phore to be presented in the literature. The NMR spectro-
scopic study of Re-1 and 99Tc-1 clearly demonstrates their
anticipated structural similarity and provides comparative
Re/Tc data of use in spectral assignments of similar
[M(CO)3NNO] (M = Re, 99Tc) complexes.
5-Bromo-N-[4-(6-methylbenzothiazol-2-yl)phenyl]pentanamide (3):
5-Bromopentanoyl bromide (1.37 g, 6.86 mmol) was added drop-
wise and under nitrogen to
a stirred solution of 2 (1.5 g,
6.14 mmol), and triethylamine (0.95 g, 9.25 mmol) in dry CH2Cl2
(10 mL) at 0 °C. The reaction mixture was stirred for 30 min at
0 °C and then for 2.5 h at room temperature under nitrogen. The
precipitate that was formed during the reaction was filtered,
washed with cold dichloromethane, and air-dried to afford product
3 as pale yellow powder that was purified by flash chromatography
Both Re-1 and 99Tc-1 selectively label amyloid plaques in
vitro with blue fluorescence, thus demonstrating that the
binding affinity of the 2-(4Ј-aminophenyl)-6-methylbenzo-
thiazole pharmacophore is maintained in the complexes.
These results further demonstrate that analogous Re/Tc
complexes interact with amyloid plaques in a similar way,
thereby expanding the range of similar Re/Tc properties de-
scribed in the literature.
1
(1% methanol/chloroform); yield 83%. H NMR (500 MHz, [D6]-
DMSO): δ = 10.23, 8.01, 7.90, 7.89, 7.78, 7.34, 7.23, 3.57, 2.54,
2.40, 1.87, 1.74; 171.2, 165.8, 151.7, 141.8, 134.9, 134.3, 127.9,
127.7, 127.4, 122.0, 121.7, 119.1, 60.9, 35.3, 34.7, 31.6, 23.5,
20.9 ppm. C19H19BrN2OS (403.34): calcd. C 56.58, H 4.75, N 6.95;
found C 56.80, H 4.78, N 6.92.
The 99mTc-1 radioactive analogue displays in vitro sta-
bility and ideal lipophilicity for brain uptake. Its biodistri-
bution data in healthy mice – characterized by relatively low
initial brain uptake, fast clearance from the brain and the
blood, and lack of in vivo re-oxidation – are overall judged
as positive and prompt for further elaboration on this
99mTc–tricarbonyl NNO system, as bearing potential to
generate agents for imaging of amyloid plaques.
N-[4-(6-Methylbenzothiazol-2-yl)phenyl]-5-[(pyridin-2-ylmethyl)-
amino]pentanamide (5): A solution of the bromide 3 (2.5 g,
6.2 mmol) in toluene (30 mL) was treated with a fivefold excess
amount of 2-(aminomethyl)pyridine (3.35 g, 31 mmol) under reflux
conditions overnight. The reaction mixture was filtered while hot
and, upon cooling, the resulting precipitate was washed with ice-
cold 1% Na2CO3. The product was used without further purifica-
tion; yield 75%. 1H NMR (500 MHz, [D6]DMSO): δ = 10.25, 8.62,
8.01, 7.90, 7.89, 7.88, 7.87, 7.85, 7.78, 7.51, 7.4, 7.34, 7.39, 4.32,
4.19, 3.00, 2.45, 2.42, 1.66; 179.8, 169.0, 161.3, 151.4, 148.6, 139.6,
138.5, 135.7, 134.1, 129.0, 126.6, 121.3, 124.1, 121.5, 120.9, 119.7,
52.5, 48.8, 38.0, 29.5, 23.0, 20.8 ppm.
Experimental Section
Materials and Methods: Reagents and solvents were purchased
from Aldrich Chemical Co or Fluka Chemical Co and used with-
out further purification. Na99mTcO4 was obtained in physiological
saline as commercial 99Mo/99mTc generator eluate (Cis Inter-
national). Flash column chromatography was performed with
Merck silica gel 60. C, H, and N analysis was performed with a
Perkin–Elmer 2400 automatic elemental analyzer. High-perform-
ance liquid chromatography (HPLC) analysis was performed with
a Waters 600E Chromatography System coupled to both a Waters
991 photodiode array detector (UV trace for Re and 99Tc com-
plexes) and a GABI gamma detector from Raytest (γ trace for
99mTc complexes). Solvents used in chromatographic analysis were
HPLC grade. Separations were achieved with a Macherey–Nagel
Nucleosil C18 (10 μ, 250ϫ4.6 mm) column eluted with a binary
gradient system at a 1.0 mLmin–1 flow rate. Mobile phase A was
0.1% trifluoroacetic acid (TFA) in methanol, whereas mobile phase
B was 0.1% TFA in water. The elution profile was: 0 to 1 min 0%
of phase A followed by a linear gradient to 75% of phase A in
9 min; this composition was held for another 20 min. After a col-
umn wash with 95% of phase A for 5 min, the column was re-
equilibrated by applying the initial conditions (0% of phase A) for
15 min prior to the next injection. IR spectra were recorded in the
range 4000–500 cm–1 with a Perkin–Elmer 1600 FTIR spectropho-
tometer as KBr pellets and with a Nicolet 6700 ATR-IR from
Thermo Scientific. NMR spectra were acquired at 25 °C in [D6]-
DMSO with a 500 MHz Bruker DRX-Avance spectrometer (1H at
Ethyl N-[4-(Benzothiazol-2-yl)phenylaminocarbonylbutyl]-N-(pyr-
idin-2-ylmethyl)aminoacetate (4Ј): Ethyl bromoacetate (1.4 g,
9 mmol) was added to a stirred solution of 5 (1.25 g, 3 mmol) and
triethylamine (1.13 g, 12 mmol) in dry THF (40 mL) under nitro-
gen. The reaction mixture was heated at reflux for 16 h. It was then
cooled to room temperature, filtered, and the solvent was evapo-
rated to dryness. The residue was purified by means of flash col-
umn chromatography (hexane/ethyl acetate, 7:3 to 5:5) to afford the
1
pure product as an off-white solid; yield 68%. H and 13C NMR
spectroscopic chemical shifts are given in Table 1. C29H32N4O3S
(516.66): calcd. C 67.42, H 6.24, N 10.84; found C 67.24, H 6.32,
N 10.97.
Complex Re-1: A mixture of ligand 4Ј (100 mg, 0.2 mmol), the pre-
cursor [NEt4]2[ReBr3(CO)3] (154 mg, 0.2 mmol) in acetonitrile
(5 mL), and NaOH (2 mL, 0.1 n, 0.2 mmol) was heated to reflux
for 4 h. Upon standing at room temperature a pale yellowish crys-
talline product precipitated and was collected by filtration; yield
75%. t : 19.2 min. IR: ν = 2016, 1909, 1868, 1651 cm–1. Crystals
˜
R
suitable for X-ray analysis were isolated by slow evaporation from
a mixture of methanol/water. 1H and 13C NMR spectroscopic
chemical shifts are given in Table 1. C30H27N4O6ReS (757.83):
calcd. C 47.55, H 3.59, N 7.39; found C 47.74, H 3.62, N 7.42.
Complex 99Tc-1: A mixture of ligand 4Ј (41 mg, 0.08 mmol) and
(NEt4)2[99TcCl3(CO)3] (25 mg, 0.05 mmol) in MeOH (6 mL) was
heated to reflux for 8 h. Upon standing at room temperature, a
1
500.13 MHz and 13C at 125.77 MHz). Assignment of H and 13C
6
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Imaging of β-Amyloid Plaques
pale yellowish crystalline product precipitated and was collected by
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
filtration; yield 86%. t : 19.4 min. IR: ν = 2035, 1931, 1915, 1649
˜
R
cm–1. H and 13C NMR spectroscopic chemical shifts are given in
1
In vitro Binding of Re-1 and 99Tc-1 to Amyloid Plaques: Postmortem
brain tissue from an autopsy-confirmed AD case was obtained
through the University of Manchester of the Greater Manchester
Neurosciences Centre. Serial sections (6 μm thick) of paraffin-em-
bedded blocks from temporal cortex mounted on albumin-coated
glass slides were used for staining. Tissue sections were deparaf-
finized with two 5 min washes in xylene, two 3 min washes in 100%
ethanol, 5 min washes in 80% ethanol/H2O, 5 min washes in 60%
ethanol/H2O, running tap water for 10 min, and then incubated in
phosphate buffer solution (PBS; 1.3 m NaCl, 27 mm KCl, 81 mm
Na2HPO4, 14.7 mm KH2PO4, pH 7) for 30 min. The tissue prepara-
tions were treated with Re-1 and 99Tc-1 solutions in [D6]DMSO
(approximately 1 mgmL–1) for 1 h. The sections were finally
washed with 40% ethanol for 2 min, followed by rinsing with tap
water for 30 s. Fluorescent observation was performed with a Zeiss
Axioplan2 microscope (lens magnification as specified in each im-
age) equipped with DAPI filter set.
Table 1.
X-ray Structure Determination of Re-1: A crystal with approximate
dimensions 0.12ϫ0.14ϫ0.45 mm was taken from the mother
liquor and immediately cooled to –93 °C. Diffraction measure-
ments were made with a Rigaku R-AXIS SPIDER Image Plate
diffractometer using graphite-monochromated Cu-Kα radiation.
Data collection (ω-scans) and processing (cell refinement, data re-
duction, and empirical absorption correction) were performed with
the CrystalClear program package.[40] The structure was solved by
direct methods using SHELXS-97 and refined by full-matrix least-
squares techniques on F2 with SHELXL-97.[41] Important crystal-
lographic data are listed in Table 3. Further experimental crystallo-
graphic details for Re-1: 2θmax = 130°; reflections collected/unique/
used, 20390/5002 (Rint = 0.0548)/5002; 468 parameters refined;
(Δ/σ)max = 0.004; (Δρ)max/(Δρ)min = 1.441/–1.126 eÅ–3; R1/wR2 (for
all data), 0.0488/0.1180. Hydrogen atoms were located by difference
maps and were refined isotropically or were introduced at calcu-
lated positions as riding on bonded atoms. All non-hydrogen atoms
were refined anisotropically, except for the water and methanol
solvate molecules, which were refined isotropically with occupation
factors fixed to 0.4 and 0.3, respectively.
Synthesis of 99mTc-1: A freshly prepared solution (400 μL) of the
fac-[99mTc(CO)3(H2O)3]+ precursor (pH 7) was added to a vial that
contained a solution of 4 (50 μg) in acetonitrile (100 μL). The vial
was sealed, flushed with N2, and heated for 20 min at 70 °C. HPLC
analysis demonstrated the formation of a single complex (tR
=
19.7 min, radiochemical yield Ͼ95%) that was stable for at least
6 h. The radioactivity recovery of the HPLC column was moni-
tored and found to be quantitative. The identity of complex 99mTc-
1 was established by comparative HPLC using samples of the well-
characterized complexes Re-1 and 99Tc-1 (Figure 3).
Table 3. Summary of crystal, intensity collection, and refinement
data for Re-1.
Re-1·0.4H2O·0.3CH3OH
Empirical formula
Mr
C30.3H31N4O6.7ReS
776.65
Determination of Partition Coefficient of 99mTc-1: The partition co-
efficient of 99mTc-1 was determined by the shake-flask method.[36]
The HPLC-purified 99mTc complex (10 μL) was mixed with 1-oc-
tanol (1.5 mL) and phosphate buffer (1.5 mL, 0.1 m, pH 7.4) in a
centrifuge tube. The mixture was vortexed at room temperature for
1 min and finally centrifuged at 5000 rpm for 5 min. Three samples
(0.2 mL each) were drawn both from the octanol and the aqueous
layer and counted in a γ counter. The experiment was run in tripli-
cate. The partition coefficient (logPoct/buffer) was calculated by di-
viding the cpm/mL(oct) to cpm/mL(buffer), and the logPoct/buffer was
found to be 1.69.
Stability of 99mTc-1 against Cysteine and Histidine: Aliquots of
99mTc-1 (70 μL) were added to a 1 mm cysteine or a 1 mm histidine
solution (630 μL) in PBS, pH 7.4. The samples were incubated at
37°C and analyzed by HPLC after 1, 3, and 6 h. Complex 99mTc-
1 remained unaffected by the presence of cysteine or histidine.
T [°C]
–93
λ (Cu-Kα)
Space group
a [Å]
b [Å]
c [Å]
α [°]
β [°]
1.54178
¯
P1
11.3996(2)
11.7390(2)
12.7464(2)
80.435(1)
67.914(1)
86.331(1)
1558.58(5)
2
γ [°]
V [Å3]
Z
Dcalcd. [Mgm–3]
μ [mm–1]
F(000)
1.655
8.680
771
1.068
Goodness-of-fit on F2
R indices
R1 = 0.0440[a], wR2 = 0.1121[a]
[a] For 4494 reflections with IϾ2σ(I).
CCDC-875335 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Biodistribution Studies of 99mTc-1: All the biodistribution studies
were carried out in compliance with the national laws related to
Table 4. Biodistribution results of 99mTc-1 in healthy mice (n = 5).
% ID/g
15 min
1 min
1 min
60 min
AVG
AVG
STD
AVG
STD
AVG
STD
STD
Blood
Liver
Heart
Kidney
Stomach
Intestine
Spleen
Muscle
Lungs
Brain
15.03
15.61
9.20
11.48
1.32
1.34
2.07
1.56
15.19
0.69
2.49
0.37
1.89
1.09
0.29
0.27
0.10
0.22
2.57
0.08
0.65
29.36
1.56
12.30
0.92
10.91
0.60
0.49
2.90
0.05
0.09
3.27
0.22
3.15
0.46
1.92
0.11
0.05
0.47
0.01
0.24
15.52
0.65
3.56
0.87
17.54
0.35
0.24
1.30
0.02
0.08
1.62
0.25
0.68
0.16
2.98
0.14
0.04
0.26
0.01
27.23
26.58
0.91
3.44
0.44
3.98
1.02
17.31
3.11
0.28
3.87
3.48
0.07
0.27
0.04
0.64
0.11
1.59
0.42
0.03
1.27
43.82
0.18
4.17
0.40
28.98
0.17
5.92
0.65
0.02
0.14
1.02
0.02
0.71
0.10
2.69
0.03
0.32
0.07
0.00
0.48
25.35
0.08
1.27
0.32
43.92
0.08
2.94
0.27
0.01
0.11
6.22
0.03
0.17
0.11
4.66
0.02
0.24
0.03
0.00
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M. Pelecanou et al.
FULL PAPER
the conduct of animal experimentation. HPLC-purified 99mTc-1 (1–
2 μCi in 0.1 mL methanol/saline 10:90) was injected through the
tail vein in three groups of five Swiss Albino mice each [male,
(26Ϯ3) g]. The animals were sacrificed by cardiectomy under slight
ether anaesthesia at predetermined time intervals (1, 15, and
60 min). The organs of interest were excised, weighed, and the
radioactivity counted in an automatic γ counter. The stomach and
intestines were not emptied of food contents prior to radioactivity
measurements. The percentage of injected dose per organ (% ID/
organ) was calculated by comparison of sample radioactivity to
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 were based upon measured ac-
tivity, sample weight, and body composition data, considering that
blood comprises 7% and muscle 43% of body weight. The biodis-
tribution data are presented in Table 4.
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Received: May 3, 2012
Published Online: ᭿
8
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Job/Unit: I20450
/KAP1
Date: 08-08-12 16:35:09
Pages: 9
Imaging of β-Amyloid Plaques
Imaging Agents
The fac-[M(NNO)(CO)3] complex (M =
Re, 99Tc, and 99mTc) conjugated to
6-methyl-2-(4Ј-aminophenyl)benzothiazole
was synthesized, completely characterized,
and biologically evaluated in vitro and in
vivo as a potential amyloid imaging agent.
M. Sagnou, S. Tzanopoulou,
C. P. Raptopoulou, V. Psycharis,
H. Braband, R. Alberto, I. C. Pirmettis,
M. Papadopoulos, M. Pelecanou* ..... 1–9
A Phenylbenzothiazole Conjugate with the
Tricarbonyl fac-[M(I)(CO)3]+ (M = Re,
99Tc, 99mTc) Core for Imaging of β-Amyl-
oid Plaques
Keywords: Medicinal chemistry / Imaging
agents / Rhenium / Technetium / Alzhei-
mer’s disease
Eur. J. Inorg. Chem. 0000, 0–0
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