4. Novel 99mTc-2-arylimidazo[2,1-b]benzothiazole derivatives as SPECT imaging agents

Accepted Manuscript
99m
Novel
Tc-2-arylimidazo[2,1-b]benzothiazole derivatives as SPECT imaging
agents for amyloid-β plaques
Sajjad Molavipordanjani, Saeed Emami, Alireza Mardanshahi, Fereshteh Talebpour
Amiri, Zohreh Noparast, Seyed Jalal Hosseinimehr
PII:
S0223-5234(19)30397-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.069
Reference:
EJMECH 11304
To appear in:
European Journal of Medicinal Chemistry
Received Date: 14 January 2019
Revised Date:
9 April 2019
Accepted Date: 27 April 2019
Please cite this article as: S. Molavipordanjani, S. Emami, A. Mardanshahi, F.T. Amiri, Z. Noparast, S.J.
99m
Hosseinimehr, Novel
Tc-2-arylimidazo[2,1-b]benzothiazole derivatives as SPECT imaging agents
for amyloid-β plaques, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.04.069.
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Novel 99mTc-2-arylimidazo[2,1-b]benzothiazole derivatives as SPECT imaging
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agents for amyloid-β plaques
Sajjad Molavipordanjani1,2, Saeed Emami3, Alireza Mardanshahi4, Fereshteh Talebpour Amiri5, Zohreh
Noparast1, Seyed Jalal Hosseinimehr1*
1
Department of Radiopharmacy, Faculty of Pharmacy, Pharmaceutical Sciences Research Center, Mazandaran
3
Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
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University of Medical Sciences, Sari, Iran
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
Department of Radiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
5
Department of Anatomy, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
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Running title:
99m
Tc-2-arylimidazo[2,1-b]benzothiazole tracers for AD imaging
*Corresponding author: Email: [email protected], [email protected]
Orcid: https://orcid.org/0000-0001-8055-8036
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Abstract
Six novel 2-arylimidazo[2,1-b]benzothiazole (IBT) derivatives were synthesized as potential
tridentate radiotracers for AD imaging purposes. Two of these ligands (6a,b) were successfully
99m
Tc radionuclide at high radiochemical purity using fac-[99mTc(CO)3(H2O)3]+
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labeled with
synthon. [99mTc]7a and [99mTc]7b were evaluated as single photon emission computed
tomography (SPECT) imaging agents for Aβ plaque in AD. [99mTc]7a and [99mTc]7b exhibited
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suitable affinity toward Aβ aggregates with IC50 values of 33.2 and 102.5 nM, respectively. The
IC50 value of these radiotracers depends on the length of the spacer (alkyl chain). In
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biodistribution study, these complexes showed good initial brain uptakes (0.78 and 0.86 % ID/g
at 2 min post-injection) and fast blood clearance. Autoradiography results confirmed that these
small 99mTc complexes (Mw about 600 Da) can bind to Aβ plaque in the brain sections of the rat
AD model. Histopathological staining with Congo red approved the presence of Aβ plaques in
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these brain sections.
tomography
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Key words: Alzheimer’s disease, β-amyloid plaque,
(SPECT)
autoradiography,
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b]benzothiazole
imaging,
99m
2
Tc, single photon emission computed
binding
assay,
2-arylimidazo[2,1-
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1. Introduction
Alzheimer’s disease (AD) is an age-related progressive neurodegenerative disorder which leads
to devastative symptoms such as irreversible cognitive decline, memory loss, disorientation, and
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etc. One of the prominent hallmarks of AD is the formation of misfolded extracellular deposition
of β-amyloid (Aβ) including Aβ(1-40) and Aβ(1-42) peptides. The formation of these
depositions, known as Aβ plaques, is an initial event in the pathology of AD which happens in
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the early stages of this disorder [1, 2]. Likewise, Aβ plaques are the main cause of cerebral
amyloid angiopathy (CAA) when they accumulate in the walls of cerebral capillaries and arteries
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[3]. In the past, the detection of these pathological abnormalities was only possible by post
mortem investigation of AD brains using classic staining reagents for Aβ plaques including
Congo Red (CR) and Thioflavin-T (ThT) [4]. To that end, detection of Aβ deposits with noninvasive techniques including positron emission tomography (PET) and single photon emission
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computed tomography (SPECT) could provide a unique tool for in vivo monitoring of AD
progression in the early stages in patients.
In the past decade, various PET and SPECT probes were designed based on CR and ThT
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structures for AD imaging [5, 6]. The U.S. Food and Drug Administration (FDA) approved a few
of these probes for AD PET imaging, including [18F]GE-067 [7], [18F]BAY94-9172 [8] and
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[18F]AV-45 (Fig. 1a) [9]. Due to the unique characteristics of SPECT imaging with 99mTc-labeled
radiopharmaceuticals such as simplicity, availability and, having favorable physical properties
(t1/2 = 6 h, E = 140 KeV), the development of 99mTc-labeled amyloid imaging agents for AD has
attracted the researchers’ attention [10]. Several
99m
Tc-labeled small molecules including
derivatives of benzothiazole, flavone, chalcone, benzofuran, benzoxazole, biphenyl and,
dibenzylideneacetone were investigated as Aβ plaques imaging agents [5]. The most troublesome
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issue with these AD imaging agents is their low brain uptake; hence finding a 99mTc-labeled AD
imaging agent with proper brain uptake and washout is an urgent need.
Certainly, Pittsburgh compound B ([11C]PiB) and 6-iodo-2-(4-dimethylamino-)phenyl-
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imidazo[1,2-a] pyridine ([123I]IMPY) enabled AD diagnosis (not in the early stages) [11, 12].
Further studies to discover an AD imaging agent in the early stages, and improving
imidazo[2,1-b]benzothiazole (IBT) derivatives (Fig. 1b).
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quantification and monitoring the amyloid burden in the brain led to the introduction of 2-aryl-
The IBT scaffold contains benzothiazole part of PiB and the 2-arylimidazo part of IMPY (Fig.
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1b). This planar electron-rich conjugated heteroaromatic system with suitable lipophilicity value
enables blood-brain barrier (BBB) penetration which is required for in vivo imaging of AD
brains [13]. A few IBT radiotracers have been introduced as PET imaging agents for AD and the
results were quite interesting (Fig 1c) [14-16].
99m
Tc-labeled IBT for AD imaging. Herein we tend
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To our knowledge, this is the first report on
to synthesize a series of imidazo[2,1-b]benzothiazoles (IBTs) with various chelators and spacer
length. The IBT compounds were labeled with
99m
Tc using fac-[99mTc(CO)3(H2O)3]+ synthon,
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then they were tested for their stability, lipophilicity and binding affinities to Aβ aggregates in
vitro. The compounds with the best properties were selected for evaluation of their brain uptake
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kinetics in mice. After creating rat AD model and approving the presence of Aβ plaques in the
brain sections with histopathological staining using Congo red, the affinity of compounds toward
Aβ plaques was investigated by performing autoradiography.
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Figure 1. Compounds with affinity toward β-amyloid plaques: (a) FDA approved probes for AD PET imaging. (b)
2. Results
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strategy for the design of IBT backbone for AD imaging. (c) radioactive IBT derivatives for PET imaging of AD.
2.1. Synthesis and Radiolabeling
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The radioligands were synthesized in several steps as illustrated in Fig. 1. The commercially
available 4′-hydroxyacetophenone was brominated with CuBr2 to give compound 1. In order to
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synthesize compound 2, we applied a direct condescension between 2-aminobenzothiazole and
bromoacetophenone 1 which resulted in good yield (58%) and purity of compound 2. The
phenolic hydroxyl of 2 was reacted with 1-bromo-2-chloroethane or 1,3-dibromopropane in the
presence of K2CO3 in acetonitrile to produce 3a or 3b, respectively.
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O
O
NH2
S
N
Br
80% HO
HO
OH
N
b,
a
58%
N
S
1
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2
c 71-79%
O
n
N
N
O
90-95%
O
e, f
g
S
64%
5c
h, 5a
51-58%
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N
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S
N
4a: n = 2
4b: n = 3
C
N
N
52-58%
S
N
H
N
n
N
92-95%
N
N
N
H
N
n
N
S
N
N
6c: n = 2
6d: n = 3
O
h, 5c
O
N
6a: n = 2
6b: n = 3
O
N3
n
N
n
S
O
N
H
N
N
h, 5b
O
5b
H
N
S
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B
N
72%
g
NH2
H
N
NH2
N
5a
N
3a: n = 2, X = Cl
3b: n = 3, X = Br
NH
O
48%
S
n
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NH2
HCl
X
N
4a: n = 2
4b: n = 3
A
O
d
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S
O
N3
N
N
S
6e: n = 2
6f: n = 3
Figure 2. Synthesis of radioligands: (a) CuBr2, EtOH, 78 °C, 8 h; (b) EtOH, 78 °C, 24 h; (c) 1-Bromo-2chloroethane or 1,3-dibromopropane, K2CO3, CH3CN, 90 °C, 6 h; (d) NaN3, DMF, 4 h; (e) K2CO3, CH3CN, 2 h; (f)
propargyl bromide, K2CO3, CH3CN, 4 h; (g) (1) propargyl bromide, K2CO3, THF, 0 °C, 2 h; (2) stirring at room
temperature, 4 h; (h) Cu(CH3COO)2, sodium ascorbate, THF/tert-butanol/water (2:2:1), 30 min.
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The halide group in 3a,b can be substituted with azide group to provide clickable precursors
4a,b. On the other hand, the N-propargylated derivatives including 5a-c were synthesized from
corresponding amines using propargyl bromide. The azide derivatives (4a,b) and N-
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propargylated amines (5a-c) participated in a copper catalyzed click reaction to form final
radioligands (6a-f). All compounds were characterized by means of NMR and MS as well as
elemental analyses.
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We evaluated the ability of final products (6a-f) for labeling with fac-[99mTc(CO)3(H2O)3]+
which was prepared based on previously reported procedure [17]. Compounds 6a,b were reacted
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with fac-[99mTc(CO)3(H2O)3]+ (Fig. 3) and their initial radiochemical purity (RCP) were 99 and
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97%, while other ligands did not display proper features for labeling (Fig. 4).
Figure 3. Radiolabeling procedure of 6a,b: (a) water, pH 8.5-9, 85 °C, 45 min; (b) NaBH4, Na2CO3, Sodium tartrate
dihydrate, CO gas (1atm), water, 85 °C, 30 min.
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Figure 4. HPLC profiles of 6a, 7a and [99mTc]7a (a), and 6b, 7b and [99mTc]7b (b) were obtained by HPLC
equipped with both UV and NaI detector. HPLC conditions: C18 column 100% solvent A (0.1% TFA in water) at
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the beginning gradually was changed to 100% solvent B (0.1% TFA in acetonitrile) at 30 min. Red and blue
chromatograms are related to cold ligands which were recorded by the UV detector at 254 nm. Green
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chromatograms are related to [99mTc]7a and [99mTc]7b which were recorded by the NaI detector in order to
determine RCP.
Using RP-HPLC the retention times for cold ligands (6a,b) were 18.21 and 19.28 min, while the
retention times for labeled radiotracers ([99mTc]7a and [99mTc]7b) were 21.50 and 22.30 min,
respectively. Further investigation confirmed 6a,b (ester form) under labeling condition,
hydrolyzed to carboxylate form (7a,b) with retention time 3.00 and 4.10 min. The retention times
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of other ligands including 6c-f were 26.48, 27.51, 24.96 and 25.20 min as determined by using
the same eluents (Supplementary Material).
2.2. Stability in saline solution and determination of partition coefficient
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[99mTc]7a and [99mTc]7b were further investigated for their stability in saline solution and
displayed good stability (about 97% at 2 h) (Fig. 5). In addition, the partition coefficient of
[99mTc]7a,b were determined using 1-octanol/water method and Log P was calculated. Both
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radiotracers showed lipophilic nature (Log P values 1.08 ± 0.02 and 1.10 ± 0.06, respectively)
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which demonstrates these radiotracers are suitable for brain imaging (Table 1).
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Figure 5. Stability of [99mTc]7a and [99mTc]7b in saline solution.
Table 1. Log P and Mw values of [99mTc]7a and [99mTc]7b
[99mTc]7a
[99mTc]7b
Log P
1.08 ± 0.02
1.1 ± 0.06
Mw (g/mol)
616.06
630.07
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2.3. In vitro binding assays
For screening the affinity of [99mTc]7a,b toward aggregates of Aβ(1-42), the proper aggregates
were prepared according to a previous procedure [18]. In 1 mL of a mixture containing 1.25
[99mTc]7a or [99mTc]7b (5.2×10-6 M, 510
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µg/mL of Aβ(1-42) aggregates and radiolabeled
kBq/mL), 4.1 µg of BTA-1 (as the competing ligand) can block the binding of these radiotracers
to the Aβ(1-42) aggregates. This binding assay confirmed that 9.58 and 14.02% of total
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[99mTc]7a and [99mTc]7b radioactivity bind to Aβ(1-42) aggregates, respectively. Blocking assay
indicated that BTA-1 reduces the Aβ(1-42) aggregates binding of [99mTc]7a and [99mTc]7b to
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2.30 (76% blocked) and 2.89 (79.4% blocked), respectively, that confirmed selective binding of
these radiotracers to Aβ plaques (Fig. 6). Inhibition assay results indicate that IC50 values of
BTA-1 in the presence of [99mTc]7a and [99mTc]7b are 33.2 and 102.5 nM, respectively (Fig. 7).
In another word, [99mTc]7b affinity toward Aβ(1-42) aggregates is higher than that of [99mTc]7a
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by 3.08 folds.
Figure 6. Binding and blocking assay of [99mTc]7a and [99mTc]7b with Aβ(1−42)aggregates. Values are the mean ±
standard error of the mean for 3 experiments. Black columns represent the Aβ(1−42) aggregate-bound
radioactivities (%) of [99mTc]7a and [99mTc]7b. Gray columns represent the Aβ(1−42) aggregate-bound
radioactivities (%) of [99mTc]7a and [99mTc]7b blocked by BTA-1 (4.1 µg/mL) as competing ligand.
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Figure 7. Half-maximal inhibitory concentration (IC50, nM) for the binding of BTA-1 to Aβ(1−42) in the presence
of [99mTc]7a (a) and [99mTc]7b (b) to Aβ(1−42) aggregates.
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2.4. Animal biodistribution studies
The brain uptake of these radiotracers was performed in normal mice. The initial brain uptake at
2 min post-injection for [99mTc]7a and [99mTc]7b was 0.78 ± 0.07 (Table 2) and 0.86 ± 0.07
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(Table 3), respectively. The ratio of initial normal brain uptake at 2 min to normal brain uptake at
60 min post-injection demonstrates the washout rate of radiotracers from brain tissue. This ratio
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for [99mTc]7a and [99mTc]7b was 8.67 and 7.17, respectively. Furthermore, the ratios of blood to
brain radioactivity at 60 min post-injection are 18.78 and 9.5 respectively, which also confirms
rapid wash out of [99mTc]7a,b. Accordingly, due to the fact that mouse normal brain has no
amyloid plaques depositions, radiotracers are unable to accumulate in brain.
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Table 2. Biodistribution of [99mTc]7a in normal female balb-C mice (n = 3). The values are reported as ID/g%.
2min
5min
10min
30min
60min
120min
Blood
5.3±0.39
4.53±0.3
2.96±0.12
2.53±0.52
1.69±0.13
2.05±0.6
Heart
2.82±1.45
2.94±1.49
3.63±2.24
1.06±0.57
1.14±0.79
0.79±0.4
Lung
12.57±2.82
7.14±2.39
2.51±1.15
1.93±0.65
2.6±0.17
1.07±0.06
S &Ta
0.44±0.05
0.54±0.18
0.63±0.2
0.77±0.58
1.02±0.97
2.42±0.73
Liver
14.13±2.58
15.72±2.56
13.4±2.28
13.73±2.25
10.77±1.72
14.07±0.48
Spleen
4.81±2.29
3.59±1.41
1.32±0.98
1.61±1.18
1.19±0.4
0.79±0.12
Kidney
26.15±3.55
27.77±4.59
23.13±1.9
19.75±1.9
19.16±1.65
Stomach
0.68±0.24
0.6±0.03
0.97±0.41
1.57±0.28
Muscle
1.52±0.11
1.31±0.3
0.86±0.05
0.31±0.27
Bone
1.96±0.65
0.86±0.39
1.26±0.37
0.84±0.27
0.35±0.22
0.4±0.06
Brain
0.78±0.07
0.22±0.03
0.23±0.09
0.1±0.02
0.09±0.03
0.11±0.05
Intestine b
17.69±1.61
21.6±5.58
24.69±4.45
28.19±6.48
25.9±5.28
35.33±2.19
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Organ
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2.08±0.26
2.34±0.58
0.35±0.09
0.47±0.14
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a: salivary gland and thyroid
b: reported as ID% not ID/g%
17.44±0.18
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Table 3. Biodistribution of [99mTc]7b in normal female balb-C mice (n = 3). The values are reported as ID/g%.
2min
5min
10min
30min
60min
120min
Blood
3.79±0.32
2.92±0.42
2.14±0.53
1.14±0.87
1.14±0.13
1.07±0.64
Heart
3.67±1.85
3.81±1.91
4.12±2.17
3.23±1.63
2.24±1.39
1.42±0.72
Lung
9.85±2.14
8.62±0.51
7.67±0.72
4.57±0.44
3.36±0.36
2.68±0.52
S &T
0.62±0.1
Liver
13.79±1.94
Spleen
3.27±1.3
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Organ
1.24±0.13
1.74±0.53
1.83±0.34
1.25±0.02
16.3±0.65
13.53±0.66
12.64±0.36
8.8±1.21
7.28±0.24
2.98±0.46
2.95±1.18
1.72±0.27
1.55±1.23
0.92±0.05
16.84±1.54
13.6±0.67
11.39±0.68
7.19±0.5
3.85±0.3
2.64±0.24
Stomach
0.99±0.25
1.6±0.43
1.9±0.92
1.38±0.2
1.25±0.08
1.21±0.37
Muscle
1.18±0.13
1.11±0.35
1.02±0.26
0.92±0.04
0.89±0.23
0.56±0.07
1.43±0.29
1.54±0.87
1.74±0.99
0.98±0.06
0.84±0.1
0.54±0.06
Brain
0.86±0.07
0.51±0.04
0.28±0.05
0.1±0.02
0.12±0.02
0.05±0
Intestine
5.37±3.51
10.8±3.51
10.75±3.83
16.46±1.58
24.39±2.94
25.8±2.41
Kidney
Bone
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0.73±0.11
a: salivary gland and thyroid
b: reported as ID% not ID/g%
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2.5. Tissue staining and autoradiography findings
The brain sections of the rat Alzheimer model were stained with Congo red (Fig. 8 A1). The
formation of Aβ deposits in the area of Aβ injection with the light microscope appeared as red to
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pink-red and nuclei as dark blue/black while other tissue elements are largely unstained. In
contrast, no apparent staining was observed with Congo red in the brain of normal (Fig. 8 N1)
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and control (Fig. 8 C1) rats while nuclei appears as dark blue/black.
Figure 8. Congo red staining and autoradiography on 10 µm thick axial section of AD rat model. (A1) Congo red
staining of a brain section of the rat AD model. Black arrows show the site of Aβ(1−42) aggregates. (A2 and A3)
autoradiography with [99mTc]7a and [99mTc]7b results in a hot spot (black arrow) which corresponds to the injection
site of Aβ(1−42) aggregates. (A4) Brain section of AD rat model and the site of Aβ(1−42) aggregates injection.
(N1) Congo red staining of a brain section of a normal rat. (N2 and N3) autoradiography with [99mTc]7a and
[99mTc]7b brain sections of normal rat. (N4) Brain section of a normal rat. (C1) Congo red staining of a brain
section of a control rat. (C2 and C3) autoradiography with [99mTc]7a and [99mTc]7b on brain sections of control rat.
(C4) Brain section of control rat.
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In order to investigate the binding ability of [99mTc]7a and [99mTc]7b to Aβ deposits at tracer
dose, in vitro autoradiography was performed on the brain sections of the AD model (Fig. 8 A2
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and A3), normal (Fig. 8 N2 and N3) and control (Fig. 8 C2 and C3) rats. Autoradiography
images of rat Alzheimer model (Fig. 8 A2 and A3) brain section with [99mTc]7a and [99mTc]7b
showed a hot spot at the area of Aβ injection while hot spots were not observed for normal and
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control brain sections (Fig. 8 N2, N3, C2 and C3). The binding of [99mTc]7a and [99mTc]7b at the
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area of Aβ injection were in agreement with the Congo red staining results.
3. Discussion
In this study, we developed 2-arylimidazo[2,1-b]benzothiazole derivatives as SPECT imaging
agents for Aβ plaques. The key step in the formation of these derivatives is the click reaction
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which results in final tridentate ligands capable to react with fac-[99mTc(CO)3(OH2)3]+ by ligand
exchange reactions (fig. 3). Among final ligands, the labeling of 6c-f resulted in highly unstable
compounds; hence we did not investigate them any further. The instability of radiolabeled 6e,f
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([99mTc]6e and [99mTc]6f) is more likely due to the orientation of paired electron on sulfur atom
which leads to weak interaction with fac-[99mTc(CO)3(OH2)3]+. Likewise, the labeling of 6c,d
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([99mTc]6c and [99mTc]6d) results in low RCP, even though they are more stable than [99mTc]6e,f.
The stability of [99mTc]6c,d was not enough to qualify them for more investigations. Finally, the
labeling of 6a,b resulted in [99mTc]7a,b with high RCP (99 and 97% respectively) without any
purification (Fig. 4). [99mTc]7a,b were highly stable (Fig. 5); hence we selected them for further
studies.
Compounds 6a,b are essentially ester which possess two proper dents (a N atom in triazole ring
and a N atom of ethyl glycinate motif) to interact with fac-[99mTc(CO)3(OH2)3]+. The data
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obtained from HPLC suggest that in labeling condition (pH 8.5-9) these esters are hydrolyzed to
their corresponding carboxylates (7a,b) (Fig. 3). Furthermore, previous reports suggest that the
weak Lewis base character of technetium is a potential factor that can cause concomitant
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hydrolysis of the ester functional group [19]. The retention times of 6a,b are 18.21 and 19.28
min, respectively that suggest the lipophilic nature of these compound while the corresponding
carboxylates (7a,b) are more hydrophilic compounds (retention time 3.00 and 4.10 min,
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respectively) (Fig. 4). Hydrophilic nature of 7a,b is due to the negative charge of their
carboxylate functional group. The reaction of 7a and 7b with fac-[99mTc(CO)3(OH2)3]+ results in
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neutral complexes ([99mTc]7a and [99mTc]7b) (Fig. 3); hence [99mTc]7a,b are very lipophilic and
their retention times (21.50 and 22.30 min respectively) differ dramatically in comparison with
7a,b. The Log P also provides more evidence that these complexes possess lipophilic nature
which is required for brain penetration. Another requisite for proper brain penetration is low
(about 600 g/mol, Table 2).
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molecular weight. Having that said, the molecular weight of both [99mTc]7a and [99mTc]7b is low
Stability tests of [99mTc]7a,b in saline solution confirmed their high stability (RCP > 92% after 2
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h); hence we investigated the in vivo and in vitro features of these radiotracers.
The animal biodistribution experiments were conducted in normal balb-C mice to evaluate the
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pharmacokinetics of [99mTc]7a,b complexes in the brain. A biodistribution study provides pivotal
information on the penetration ability of radiotracers to the BBB. The optimal log P value
required for proper BBB penetration is reported to be in the range of 0.1 to 3.5 [20, 21]. The log
P values for [99mTc]7a and [99mTc]7b were 1.08 ± 0.02 and 1.1 ± 0.06, respectively, indicating
that these complexes should penetrate the BBB [5]. As expected, [99mTc]7a,b complexes showed
brain uptake at 2 min post-injection, and their radioactivity in the brain washed out with time.
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The initial brain uptakes of [99mTc]7a and [99mTc]7b were 0.78 and 0.86 % ID/g (at 2 min postinjection). Although the brain uptake values were lower than that of [11C]PiB (7.0% ID/g) [11],
[18F]AV-45 (7.33% ID/g) [9], and [123I]IMPY (2.88% ID/g) [12], they were superior to that of
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some other previously reported 99mTc-labeled tracers for AD imaging (0.03−0.65% ID/g) [22-24].
In comparison with IBTs PET radiotracers (FIBT and CIBT, Fig. 1c) [15, 16], [99mTc]7a and
[99mTc]7b possess considerably lower brain uptake. This is mainly due to the higher molecular
99m
Tc-labeled tracers. Even
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weight and lower lipophilicity which is the major drawback of
though, 99mTc is an ideal radionuclide for SPECT imaging, the formation of a complex between
99m
Tc particularly influences lipophilicity of
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small molecules with
99m
Tc-complex and
consequently alters 99mTc-tracer penetration into the brain. In order to alleviate this problem, the
pervious findings suggested the use of fac-[99mTc(CO)3(OH2)3]+ synthon, which results in higher
lipophilic radiolabeled molecules in comparison with other
99m
PET radiotracers.
Tc cores [25]. In addition, the
Tc-complex results in changing molecular size and structure in comparison to
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formation of a
99m
Researchers consider the ratio of brain uptake at 2 min post-injection to brain uptake at 60 min
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post-injection (brain2min / brain60min) in animal normal brain as an index for evaluating
radioactivity clearance in vivo. The brain2min / brain60min of [99mTc]7a and [99mTc]7b was 8.67 and
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7.17, respectively, indicating that [99mTc]7a,b provided very good profile of radioactivity in the
brain which is superior to many radiotracers reported previously including [18F]AV-45 (brain2min
/ brain60min = 3.89) [26-28]. Due to the high lipophilicity of [99mTc]7a,b, the main excretion
route is hepatobiliary which resulted in high radioactivity in the liver and intestines. The low
uptake in salivary glands, thyroid and stomach tissues suggest high in vivo stability of these
radiotracers.
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For further in vitro binding study of [99mTc]7a,b, BTA-1 was selected as the competing ligand
due to its high affinity and specificity toward Aβ(1-42) aggregates. To that end, BTA-1 even can
block Pittsburgh compound B which is another well-documented competitor ligand for Aβ(1-42)
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aggregates [29, 30]. BTA-1 as the competing ligand inhibits the binding of [99mTc]7a,b to Aβ(142) aggregates in a dose-dependent manner, indicating the affinity of these complexes for Aβ
aggregates. The IC50 values for BTA-1 in the presence of [99mTc]7a and [99mTc]7b were 33.2 and
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102.5 nM, respectively, suggesting that [99mTc]7b displayed higher affinity than [99mTc]7a
toward Aβ(1-42) aggregates [18]. In terms of Aβ(1-42) aggregate bound radioactivity, [99mTc]7b
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is superior to [99mTc]7a (compare 14.02% to 9.58 %). In the presence of BTA-1 bound
radioactivity drops to around 2.30-2.89% which can be regarded as nonspecific Aβ(1-42)
aggregate-bound radioactivity, indicating that nearly all of the radioactivity occupied the specific
binding site of BTA-1 in Aβ aggregates. To that end, the length of the alkyl spacer between
99m
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Tc chelating section and the arylimidazo[2,1-b]benzothiazole backbone plays a pivotal role in
the binding of [99mTc]7a and [99mTc]7b to Aβ(1-42) aggregates. In other word, the larger alkyl
spacer decreases the steric hindrance of the chelation section and increases the affinity toward
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Aβ(1-42) aggregates [25, 31].
Regarding the results of binding affinity in vitro and biodistribution in normal mice, we further
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evaluated the capability of [99mTc]7a,b for binding to β-amyloid plaques in AD rat model. The
results of autoradiography imaging showed a single radioactive spot in the brain tissue. The high
intensity of this spot in comparison with background suggests low nonspecific binding of these
tracers. Furthermore, the radioactivity of [99mTc]7a and [99mTc]7b corresponded with the areas of
Congo red staining. In contrast, normal (received no treatment and no surgery) and control
(received 2% DMSO in PBS after surgery) brain sections displayed no distinguishable
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accumulation of these tracers and no staining with Congo red. With respect to these results, we
suggest that [99mTc]7a,b are capable of binding to Aβ plaques and Aβ aggregates.
In
conclusion,
we
successfully designed
and
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4. Conclusion
synthesized
novel
2-arylimidazo[2,1-
b]benzothiazole tridentate ligands, capable of reacting with fac-[99mTc(CO)3(OH2)3]+ synthon for
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the detection of Aβ plaques in the AD brain. In vitro experiments revealed that [99mTc]7a,b
bound to Aβ aggregates, however, it seems that [99mTc]7b bonds to the aggregates more strongly
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than [99mTc]7a. Both of these complexes clearly labeled Aβ plaques in sections of brain tissue
from rat AD model. Moreover, [99mTc]7a,b penetrated to BBB and rapidly washed out from the
normal mice brain after injection. The results of the present study can provide useful information
for further investigation of
99m
Tc-labeled imidazo[2,1-b]benzothiazole derivatives as potential
5. Experimental
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5.1. General information
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candidates for the imaging of β-amyloid plaques in the brain.
All the reagents and solvents were commercially available and purchased from Sigma-Aldrich or
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Merck companies. Aβ(1-42) peptide was purchased from AnaSpec Co. (USA). BTA-1 [2-(4'methylaminophenyl)benzothiazole] was provided from Sigma Co. (USA). The materials were
used without further purification unless otherwise mentioned. The completion of reactions was
checked by TLC using pre-coated silica gel 60 F254 aluminum sheets. The UV lamp (254 nm)
was used for TLC visualization and detection of spots. Melting points were determined in open
capillary tubes using Bibby Stuart Scientific SMP3 apparatus (Stuart Scientific, Stone, UK) and
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the uncorrected values are reported. The 1H NMR and
13
C NMR spectra were recorded using
Bruker 400 or 500 spectrometers and chemical shifts are expressed as δ (ppm). Multiplicity was
defined as singlet (s), doublet (d), triplet (t), or multiplet (m) and coupling constants are reported
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in Hertz (Hz). The mass spectra of compounds were obtained using a HP 5937 Mass Selective
Detector (Agilent Technologies, CA, USA). Sodium pertechnetate was eluted from a 99Mo/99mTc
radionuclide generator (Pars Isotope, Tehran, Iran). The radioactivity of in vitro and in vivo
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experiments was measured using a NaI(Tl) detector equipped gamma counter system (Delshid,
Tehran, Iran).
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High-performance liquid chromatography (HPLC) was performed with a Knauer HPLC system
(analytical reverse phase HPLC (RP-HPLC), Germany) equipped with a precolumn and
Eurospher 100–5 C18, 4.6 × 250 mm column. The RP-HPLC analyses of radiolabeled
compounds were performed on Lablogic radioactivity gamma detector and analyzed with Laura
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image analysis software. A solvent system consisting of 0.1% TFA in water (solvent A) and
0.1% TFA in acetonitrile (solvent B) was administered as the eluent. A gradient with solvents A
and B was run as follows: 0-5 min, 100% A; 5–30 min, 100% A to 100% B total time of 30 min.
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In order to determine initial RCP an extra 10 min of 100% B was used. All solvents were filtered
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and degassed earlier entering the column and delivered at a flow rate of 1.0 mL/min.
5.2. Chemistry
5.2.1. Preparation of 2-bromo-4′-hydroxyacetophenone (1)
Compound 1 was synthesized according to the previously reported procedure with a few
modifications [32]. Briefly, to a stirred solution of 4′-hydroxyacetophenone (5.0 g, 36.7 mmol) in
ethanol, CuBr2 (16.4 g, 73.4 mmol) was added at room temperature. The mixture was refluxed
and the reaction progress was monitored with TLC; after 8 h the reaction was completed.
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Afterward, the mixture was cooled down to room temperature, the white precipitate was filtered
off and the remaining solution was concentrated under reduced pressure. The residue was
dissolved in ethyl acetate (50 mL) and washed with water (3 × 50 mL). The organic phase was
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dried (with Na2SO4) and evaporated under vacuum. The crude product was obtained as a pale
yellow solid that was recrystallized from diethyl ether and n-hexane (3:1), to give pure
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compound 1 (6.36 g, 80%).
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5.2.2. Preparation of 4-(benzo[d]imidazo[2,1-b]thiazol-2-yl)phenol (2)
Compound 2 was synthesized according to the previously reported procedure with some
modifications [13]. A solution of 2-aminobenzothiazole (1.12 g, 7.42 mmol) in ethanol (30 mL)
was dropwise added to the solution of compound 1 (1.6 g, 7.42 mmol) in ethanol (40 mL) at
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room temperature. Then, the reaction mixture was stirred at room temperature for 2 h and
subsequently refluxed for 24 h. After cooling to room temperature, the off-white precipitated
solid was filtrated, washed with ice-cold ethanol and dried at room temperature. The obtained
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compound 2 (1.15 g) was used without further purification. Yield 58%; mp 288-289 °C; 1H
NMR (DMSO-d6, 400 MHz) δ: 9.55 (brs, 1H, OH), 8.58 (s, 1H, H-3 benzoimidazothiazole), 8.01
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(d, 1H, J = 7.6 Hz, H-8 benzoimidazothiazole), 7.95 (d, 1H, J = 8.0 Hz, H-5
benzoimidazothiazole), 7.68 (d, 2H, J = 7.6 Hz, H-2 and H-6 Ar), 7.55 (t, 1H, J = 7.2 Hz, H-6
benzoimidazothiazole), 7.40 (t, 1H, J = 7.2 Hz, H-7 benzoimidazothiazole), 6.83 (d, 2H, J = 8.0
Hz, H-3 and H-5 Ar). MS (m/z, %): 266 (M+, 100), 237 (7), 150 (5), 133 (8). Anal. Calcd for
C15H10N2OS: C, 67.65; H, 3.78; N, 10.52. Found: C, 67.71; H, 3.81; N, 10.36.
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5.2.3. Synthesis of 2-(4-(2-chloroethoxy)phenyl)benzo[d]imidazo[2,1-b]thiazole (3a)
A mixture of compound 2 (0.5 g, 1.88 mmol), 1-bromo-2-chloroethane (0.32 mL, 3.8 mmol) and
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potassium carbonate (0.53 g, 3.8 mmol) in acetonitrile (40 mL) was refluxed for 6 h. Then, the
mixture was cooled down to room temperature. After filtration, the remaining solution was
evaporated and purified by column chromatography using n-hexane-ethyl acetate (3:1) as eluent,
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yielded 0.49g, 79%.; mp 102-103 °C; 1H NMR (CDCl3, 400 MHz) δ: 7.92 (s, 1H, H-3
benzoimidazothiazole), 7.83 (d, 2H, J = 8.8 Hz, H-2 and H-6 phenyl), 7.72 (d, 1H, J = 8.0 Hz, H-
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8 benzoimidazothiazole), 7.62 (d, 1H, J = 7.6 Hz, H-5 benzoimidazothiazole), 7.48 (dt, 1H, J =
8.0 and 0.8 Hz, H-6 benzoimidazothiazole), 7.36 (dt, 1H, J = 8.0 and 1.2 Hz, H-7
benzoimidazothiazole), 7.01 (d, 2H, J = 8.8 Hz, H-3 and H-5 phenyl), 4.30 (t, 2H, J = 6.0 Hz,
OCH2), 3.86 (t, 2H, J = 6.0 Hz, CH2Cl). MS (m/z, %): 330 (M+2, 35), 328 (M+, 100), 266 (100),
8.39.
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134 (9). Anal. Calcd for C17H13ClN2OS: C, 62.10; H, 3.99; N, 8.52. Found: C, 62.21; H, 3.91; N,
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5.2.4. Synthesis of 2-(4-(3-bromopropoxy)phenyl)benzo[d]imidazo[2,1-b]thiazole (3b)
A mixture of compound 2 (0.5 g, 1.88 mmol), 1,3-dibromopropane (0.39 mL, 3.8 mmol) and
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potassium carbonate (0.53 g, 3.8 mmol) in acetonitrile (40 mL) was refluxed for 6 h. Then, the
mixture was cooled down to room temperature and filtrated. The remaining solution was
evaporated and purified by column chromatography using n-hexane- ethyl acetate (3:1) as eluent,
yielded 0.52 g, 71%.; mp 110-112 °C; 1H NMR (CDCl3, 400 MHz) δ: 7.91 (s, 1H, H-3
benzoimidazothiazole), 7.82 (d, 2H, J = 9.2 Hz, H-2 and H-6 phenyl), 7.72 (dd, 1H, J = 8.0 and
0.4 Hz, H-8 benzoimidazothiazole), 7.62 (dd, 1H, J = 8.0 and 0.4 Hz, H-5
benzoimidazothiazole), 7.47 (dt, 1H, J = 8.0 and 0.8 Hz, H-6 benzoimidazothiazole), 7.36 (dt,
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1H, J = 8.0 and 1.2 Hz, H-7 benzoimidazothiazole), 6.99 (d, 2H, J = 8.8 Hz, H-3 and H-5
phenyl), 4.17 (t, 2H, J = 6.0 Hz, OCH2), 3.65 (t, 2H, J = 6.4 Hz, CH2Br), 2.37 (p, 2H, J = 6.0 Hz,
-CH2-). MS (m/z, %): 390 (M+2, 73), 388 (M+, 72), 306 (33), 266 (100), 237 (48), 134 (11).
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Anal. Calcd for C18H15BrN2OS: C, 55.82; H, 3.90; N, 7.23. Found: C, 55.98; H, 3.79; N, 7.21.
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5.2.5. Synthesis of 2-(4-(2-azidoethoxy)phenyl)benzo[d]imidazo[2,1-b]thiazole (4a)
A mixture of 3a (0.4 g, 1.22 mmol) and NaN3 (0.32 g, 4.9 mmol) in DMF (20 mL) was heated at
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90 °C for 4 h. After cooling to room temperature, the reaction mixture was poured in to ice cold
water (100 mL) and maintained at 4 °C for 24 h. The product was separated as pinkish white
solid and purified via column chromatography, using n-hexane-ethyl acetate (3:1) as eluent,
yielded 0.39 g, 95%; mp 105-106 °C; 1H NMR (CDCl3, 400 MHz) δ: 7.92 (s, 1H, H-3
benzoimidazothiazole), 7.84 (d, 2H, J = 8.4 Hz, H-2 and H-6 phenyl), 7.73 (d, 1H, J = 8.0 Hz, H-
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8 benzoimidazothiazole), 7.63 (d, 1H, J = 8.0 Hz, H-5 benzoimidazothiazole), 7.48 (dt, 1H, J =
8.0 and 0.8 Hz, H-6 benzoimidazothiazole), 7.37 (dt, 1H, J = 8.0 and 0.8 Hz, H-7
benzoimidazothiazole), 7.01 (d, 2H, J = 8.8 Hz, H-3 and H-5 phenyl), 4.23 (t, 2H, J = 4.8 Hz,
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OCH2), 3.64 (t, 2H, J = 4.8 Hz, CH2N3). MS (m/z, %): 335 (M+, 30), 307 (50), 266 (100), 237
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(46), 118 (20). Anal. Calcd for C17H13N5OS: C, 60.88; H, 3.91; N, 20.88. Found: C, 61.01; H,
3.95; N, 20.74.
5.2.6. Synthesis of 2-(4-(3-azidopropoxy)phenyl)benzo[d]imidazo[2,1-b]thiazole (4b)
A mixture of 3b (0.4 g, 1.03 mmol) and NaN3 (0.32 g, 4.9 mmol) in DMF (20 mL) was heated at
90 °C for 4 h. After cooling to room temperature, the reaction mixture was poured in to ice cold
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water (100 mL) and maintained in 4 °C for 24 h. The product was separated as pinkish white
solid and purified via column chromatography, using n-hexane-ethyl acetate (3:1) as eluent,
yielded 0.325g, 90%; mp 106-108 °C; 1H NMR (CDCl3, 400 MHz) δ: 7.91 (s, 1H, H-3
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benzoimidazothiazole), 7.82 (d, 2H, J = 8.8 Hz, H-2 and H-6 phenyl), 7.73 (d, 1H, J = 8.0 Hz, H8 benzoimidazothiazole), 7.62 (d, 1H, J = 8.0 Hz, H-5 benzoimidazothiazole), 7.48 (dt, 1H, J =
8.0 and 1.2 Hz, H-6 benzoimidazothiazole), 7.36 (dt, 1H, J = 8.0 and 1.2 Hz, H-7
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benzoimidazothiazole), 6.98 (d, 2H, J = 8.8 Hz, H-3 and H-5 phenyl), 4.12 (t, 2H, J = 6.0 Hz,
OCH2), 3.57 (t, 2H, J = 6.4 Hz, CH2N3), 2.10 (p, 2H, J = 6.4 Hz, -CH2-). MS (m/z, %): 349.1
Found: C, 61.89; H, 4.26; N, 19.97.
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(M+, 4), 266 (100), 237 (25), 133 (9). Anal. Calcd for C18H15N5OS: C, 61.87; H, 4.33; N, 20.04.
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5.2.7. Synthesis of N-propargylethylglycinate (ethyl prop-2-yn-1-ylglycinate) (5a)
Compound 5a was prepared according to the previously reported procedure with some
modifications [33]. Ethyl glycinate hydrochloride (678 mg, 4.85 mmol) and potassium carbonate
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(1.3 g, 9.7 mmol) were added to acetonitrile (30 mL) and the mixture was stirred for 2 h at room
temperature. Then the mixture was cool down to 0 °C and a solution of propargyl bromide (577
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mg, 4.85 mmol) in acetonitrile (30 mL) was dropwise added to the mixture during 2 h.
Afterward, the mixture reached room temperature and was stirred for 4 h. Finally, the mixture
was filtered and the obtained solution was concentrated under reduced pressure resulting in oily
product. The crude product was purified via column chromatography, using n-hexane-ethyl
acetate (3:5) as eluent, yielded yellowish brown oil (0.328 g, 48%). 1H NMR (CDCl3, 400 MHz)
δ: 4.77 ( s, 1H, NH), 4.21 (q, 2H, J = 7.4 Hz, CH2O), 3.51 (s, 2H, -NH-CH2-CO), 3.49 (d, 2H, J
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= 2.4 Hz, -C-CH2-NH-), 2.25 (t, 1H, J = 2.4 Hz, -CH acetylene), 1.29 (t, 1H, J = 7.4 Hz, -CH3).
13
C NMR (CDCl3, 400 MHz) δ: 14.20 (-CH3 ethyl), 37.65 (-CH2- prop-1-yne), 49.22 (-CH2-
glycine), 60.91 (-CH2- ethyl), 72.01 (-CH prop-1-yne), 81.13 (-C- prop-1-yne), 171.92 (CO
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glycine).
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5.2.8. Synthesis of N-propargylpicolylamine (N-(pyridin-2-ylmethyl)prop-2-yn-1-amine)
(5b)
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Compound 5b was synthesized according to the reported procedure with some modifications
[34]. Picolylamine (500 mg, 4.62 mmol) and potassium carbonate (1.3 g, 9.7 mmol) were added
to THF (30 mL) and the mixture was cool down to 0 °C. A solution of propargyl bromide (574
mg, 4.85 mmol) in THF (30 mL) was dropwise added to the mixture during 2 h. Afterward, the
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mixture reached room temperature and was stirred for 6 h. Finally, the mixture was filtered and
the obtained solution was concentrated under reduced pressure to give some yellowish brown oil.
The crude product was purified via flash column chromatography, using CHCl3 and MeOH (9:1)
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as eluent, yielded 0.485 g of 5b, 72%. 1H NMR (CDCl3, 400 MHz) δ: 8.50 (d, 1H, J = 4.0 Hz, H6 pyridine), 7.59 (dt, 1H, J = 7.6 and 1.6 Hz, H-4 pyridine), 7.38 (d, 1H, J = 8.0 Hz, H-3
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pyridine), 7.11 (t, 1H, J = 6.8 Hz, H-5 pyridine), 3.79 (s, 2H, -NH-CH2-pyridine), 3.42 (d, 2H, J
= 2.4 Hz, CH2 propargyl), 2.20 (t, 1H, J = 2.4 Hz, acetylene).
5.2.9. Synthesis of N-propargylthiophenemethylamine (N-(thiophen-2-ylmethyl)prop-2-yn1-amine) (5c)
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The synthesis and purification procedure of 5c was performed exactly like 5b. Thiophen-2ylmethanamine (566 mg, 5.0 mmol), potassium carbonate (1.4 g, 10 mmol) and propargyl
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bromide (595 mg, 5 mmol) were used and final yield was 0.483 g, 64%.
5.3. General procedure for the preparation of final compounds using click reaction
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In order to carry out click reaction, the azide derivatives (4a,b) and N-propargylated amines (5ac) were dissolved in 5 mL of THF/tert-butanol/water (2:2:1) (solution A). Then, 0.1 mL of
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freshly prepared sodium ascorbate solution (30 mg/mL) was added to the solution A, followed
by adding 0.1 mL of Cu(CH3COO)2 solution (10 mg/mL). The solution was stirred at room
temperature for 30 min and the completion of the reaction was confirmed by TLC. The products
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were purified by TLC using 8% MeOH in CHCl3 as eluent.
5.3.1. Ethyl ((1-(2-(4-(benzo[d]imidazo[2,1-b]thiazol-2-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-
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yl)methyl)glycinate (6a)
According to the general procedure, compound 4a (100 mg, 0.3 mmol) and 5a (42 mg, 0.3
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mmol) were used for click reaction and final yield was 73 mg, 51%; mp 97-99 °C; 1H NMR
(DMSO-d6, 400 MHz) δ: 8.69 (s, 1H, H-3 benzoimidazothiazole), 8.15 (s, 1H, triazole), 8.07 (d,
1H, J = 7.6 Hz, H-8 benzoimidazothiazole), 7.99 (d, 1H, J = 8.0 Hz, H-5 benzoimidazothiazole),
7.81 (d, 2H, J = 8.8 Hz, H-2 and H-6 phenyl), 7.61 (t, 1H, J = 7.4 Hz, H-6
benzoimidazothiazole), 7.47 (t, 1H, J = 7.4 Hz, H-7 benzoimidazothiazole), 7.04 (d, 2H, J = 8.8
Hz, H-3 and H-5 phenyl), 4.83 (t, 2H, J = 4.8 Hz, -OCH2-), 4.50 (t, 2H, J = 4.8 Hz, -CH2-
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triazole), 4.12 (q, 2H, J = 7.2 Hz, CH2 ethyl), 3.89 (s, 2H, triazole-CH2-N), 3.33 (s, 2H, N-CH2CO), 1.22 (t, 3H, J = 7.2 Hz, CH3).
13
C-NMR (DMSO-d6, 400 MHz) δ: 14.56 (CH3), 47.95
(triazole-CH2-NH-), 49.82 (CH2N linker), 53.36 (N-CH2 ethylglycinate), 60.43 (CH2
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ethylglycinate), 66.77 (OCH2 linker), 108.50 (C3 benzoimidazothiazole), 113.67 (C5
benzoimidazothiazole), 115.31 (C3 and C5 phenyl), 124.74 (C8 benzoimidazothiazole), 127.09
(C6 benzoimidazothiazole), 125.42 (C7 benzoimidazothiazole), 127.57 (C2 and C6 phenyl),
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129.54 (C1 phenyl), 132.27 (C5 triazole), 132.27 (C8a benzoimidazothiazole), 139.00 (C4a
benzoimidazothiazole), 143.97 (C2 benzoimidazothiazole), 146.59 (C9a benzoimidazothiazole),
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147.58 (C4 triazole), 157.69 (C4 phenyl), 170.89 (CO). MS (m/z, %): 476 (M+, 5.7), 389 (10),
316 (26), 293 (14), 266 (100), 207 (67), 149 (31), 85 (27), 57 (100). Anal. Calcd for
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C24H24N6O3S: C, 60.49; H, 5.08; N, 17.64. Found: C, 60.77; H, 5.12; N, 17.55.
5.3.2. Ethyl ((1-(3-(4-(benzo[d]imidazo[2,1-b]thiazol-2-yl)phenoxy)propyl)-1H-1,2,3-triazol4-yl)methyl)glycinate (6b)
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According to the general procedure, 4b (100 mg, 0.28 mmol) and 5a (40 mg, 0.28 mmol) were
used for click reaction and final yield was 80 mg, 58%; mp 99-101 °C; 1H NMR (DMSO-d6, 400
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C
MHz) δ: 8.66 (s, 1H, H-3 benzoimidazothiazole), 8.06 (s, 1H, triazole), 8.02 (d, 1H, J = 8.0 Hz,
H-8 benzoimidazothiazole), 7.96 (d, 1H, J = 7.6 Hz, H-5 benzoimidazothiazole), 7.78 (d, 2H, J =
8.4 Hz, H-2 and H-6 phenyl), 7.57 (t, 1H, J = 7.6 Hz, H-6 benzoimidazothiazole), 7.42 (t, 1H, J
= 7.6 Hz, H-7 benzoimidazothiazole), 6.98 (d, 2H, J = 8.4 Hz, H-3 and H-5 phenyl), 4.54 (t, 2H,
J = 6.8 Hz, -OCH2-), 4.06 (q, 2H, J = 6.8 Hz, CH2 ethyl), 4.01 (t, 2H, J = 4.8 Hz, -CH2-triazole),
3.79 (s, 2H, triazole-CH2-N), 3.22 (s, 2H, N-CH2-CO), 2.30 (p, 2H, J = 6.4 Hz, -CH2-), 1.17 (t,
26
ACCEPTED MANUSCRIPT
3H, J = 6.8 Hz, -CH3).
13
C-NMR (DMSO-d6, 400 MHz) δ: 14.53 (CH3), 29.98 (CH2 linker),
46.99 (triazole-CH2-NH-), 48.23 (CH2N linker), 53.49 (N-CH2 ethylglycinate), 60.49 (CH2
ethylglycinate), 65.02 (OCH2 linker), 108.43 (C3 benzoimidazothiazole), 113.69 (C5
RI
PT
benzoimidazothiazole), 115.19 (C3 and C5 phenyl), 124.60 (C8 benzoimidazothiazole), 125.47
(C7 benzoimidazothiazole), 126.45 (C2 and C6 phenyl), 127.14 (C6 benzoimidazothiazole),
129.56 (C1 phenyl), 132.32 (C5 triazole), 133.00 (C8a benzoimidazothiazole), 143.72 (C2
SC
benzoimidazothiazole), 146.75 (C9a benzoimidazothiazole), 147.12 (C4 triazole), 158.19 (C4
phenyl), 170.52 (CO). MS (m/z, %): 490 (M+, 5), 403 (6), 307 (22), 266 (53), 237 (6), 207 (21),
M
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150 (48), 56 (100). Anal Calcd. for C25H26N6O3S: C, 61.21; H, 5.34; N, 17.13. Found: C, 61.22;
H, 5.30; N, 17.01.
5.3.3.
1-(1-(2-(4-(benzo[d]imidazo[2,1-b]thiazol-2-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)-
TE
D
N-(pyridin-2-ylmethyl)methanamine (6c)
According to the general procedure, compound 4a (100 mg, 0.3 mmol) and 5b (44 mg, 0.3
mmol) were used for click reaction and final yield was 84 mg, 58%; mp 112-114 °C. 1H NMR
EP
(DMSO-d6, 400 MHz) δ: 8.68 (s, 1H, H-3 benzoimidazothiazole), 8.51 (d, 1H, J = 4.4 Hz, H-6
AC
C
pyridine), 8.10 (s, 1H, triazole), 8.04 (d, 1H, J = 7.6 Hz, H-8 benzoimidazothiazole), 7.96 (d, 1H,
J = 8.0 Hz, H-5 benzoimidazothiazole), 7.79 (d, 2H, J = 8.8 Hz, H-2 and H-6 phenyl), 7.76 (t,
1H, J = 7.6 Hz, H-6 benzoimidazothiazole), 7.58 (t, 1H, J = 7.2 Hz, H-7 benzoimidazothiazole),
7.36-7.52 (m, 2H, H-3 and H-4 pyridine), 7.19 (t, 1H, J = 9.2 Hz, H-5 pyridine), 7.02 (d, 2H, J =
8.8 Hz, H-3 and H-5 phenyl), 4.78 (t, 2H, J = 5.2 Hz, -OCH2-), 4.45 (t, 2H, J = 5.2 Hz, -CH2triazole), 3.85 (s, 2H, -CH2-pyridine), 3.82 (s, 2H, triazole-CH2-N).
13
C-NMR (DMSO-d6, 400
MHz) δ: 46.88 (triazole-CH2-NH-), 50.01 (CH2N linker), 53.91 (N-CH2 pyridine), 67.41 (OCH2
27
ACCEPTED MANUSCRIPT
linker), 108.63 (C3 benzoimidazothiazole), 113.65 (C5 benzoimidazothiazole), 115.21 (C3 and
C5 phenyl), 122.42 (C4 pyridine), 122.48 (C6 pyridine), 123.52 (C8 benzoimidazothiazole),
127.13 (C6 benzoimidazothiazole), 125.47 (C7 benzoimidazothiazole), 126.44 (C2 and C6
RI
PT
phenyl), 127.21 (C5 pyridine), 129.54 (C1 phenyl), 132.30 (C5 triazole), 132.00 (C8a
benzoimidazothiazole), 137.5 (C4a benzoimidazothiazole), 136.96 (C3 pyridine), 146.74 (C9a
benzoimidazothiazole), 149.23 (C4 triazole), 158.19 (C4 phenyl), 159.81 (C1 pyridine). MS
SC
(m/z, %): 481 (M+, 7), 389 (18), 316 (98), 266 (100), 191 (25), 150 (30), 129 (13), 85 (20), 57
(25). Anal. Calcd for C26H23N7OS: C, 64.85; H, 4.81; N, 20.36. Found: C, 65.01; H, 4.79; N,
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20.23.
5.3.4. 1-(1-(3-(4-(benzo[d]imidazo[2,1-b]thiazol-2-yl)phenoxy)propyl)-1H-1,2,3-triazol-4-yl)N-(pyridin-2-ylmethyl)methanamine) (6d)
TE
D
According to the general procedure, 4b (100 mg, 0.28 mmol) and 5b (41 mg, 0.28 mmol) were
used for click reaction and final yield was 73 mg, 52%; mp 113-115 °C. 1H NMR (DMSO-d6,
400 MHz) δ: 8.67 (s, 1H, H-3 benzoimidazothiazole), 8.51 (d, 1H, J = 4.0 Hz, H-6 pyridine),
EP
8.06 (s, 1H, triazole), 8.04 (d, 1H, H-8 benzoimidazothiazole), 7.97 (d, 1H, J = 8.0 Hz, H-5
AC
C
benzoimidazothiazole), 7.80 (d, 2H, J = 8.8 Hz, H-2 and H-6 phenyl), 7.75 (dt, 1H, J = 7.6 and
1.6 Hz, H-6 benzoimidazothiazole), 7.58 (t, 1H, J = 7.2 Hz, H-7 benzoimidazothiazole), 7.44 (d,
1H, J = 8.0 Hz, H-3 pyridine), 7.43 (t, 1H, J = 8.0 Hz, H-4 pyridine), 7.26 (t, 1H, J = 8.0 Hz, H-5
pyridine), 7.01 (d, 2H, J = 8.8 Hz, H-3 and H-5 phenyl), 4.51 (t, 2H, J = 7.2 Hz, -OCH2-), 4.02
(t, 2H, J = 6.0 Hz, -CH2-triazole), 3.84 (s, 2H, -CH2-pyridine), 3.81 (s, 2H, triazole-CH2-N), 2.31
(p, 2H, J = 6.4 Hz, -CH2-).13C-NMR (DMSO-d6, 400 MHz) δ: 29.48 (CH2 linker), 46.07
(triazole-CH2-NH-), 47.03 (CH2N linker), 54.33 (N-CH2 pyridine), 64.93 (OCH2 linker), 108.37
28
ACCEPTED MANUSCRIPT
(C3 benzoimidazothiazole), 113.65 (C5 benzoimidazothiazole), 115.21 (C3 and C5 phenyl),
121.31 (C4 pyridine), 124.91 (C8 benzoimidazothiazole), 125.46 (C7 benzoimidazothiazole),
125.87 (C6 pyridine), 126.45 (C2 and C6 phenyl), 127.12 (C6 benzoimidazothiazole), 127.24
RI
PT
(C5 pyridine), 129.54 (C1 phenyl), 132.29 (C5 triazole), 131.64 (C8a benzoimidazothiazole),
137.39 (C8a benzoimidazothiazole), 146.73 (C9a benzoimidazothiazole), 147.11 (C4 triazole),
147.56 (C3 pyridine), 158.16 (C4 phenyl), 161.56 (C1 pyridine). MS (m/z, %): 495 (M+, 8),
SC
403.3 (28), 316 (38), 266 (100), 191 (17), 150 (72), 123 (19), 85 (34), 57 (20). Anal. Calcd for
5.3.5.
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C27H25N7OS: C, 65.43; H, 5.08; N, 19.78. Found: C, 65.58; H, 5.23; N, 19.67.
1-(1-(2-(4-(benzo[d]imidazo[2,1-b]thiazol-2-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)-
N-(thiophen-2-ylmethyl)methanamine (6e)
TE
D
According to the general procedure, compound 4a (100 mg, 0.3 mmol) and 5c (50 mg, 0.3
mmol) were used for click reaction and final yield was 138 mg, 95%; mp 107-109 °C; 1H NMR
(DMSO-d6, 400 MHz) δ: 8.76 (s, 1H, H-3 benzoimidazothiazole), 8.14 (s, 1H, triazole), 8.11 (d,
EP
1H, J = 8.0 Hz, H-8 benzoimidazothiazole), 8.04 (d, 1H, J = 8.0 Hz, H-5 benzoimidazothiazole),
7.87 (d, 2H, J = 8.8 Hz, H-2 and H-6 phenyl), 7.65 (t, 1H, J = 7.6 Hz, H-6
AC
C
benzoimidazothiazole), 7.51 (t, 1H, J = 7.6 Hz, H-7 benzoimidazothiazole), 7.46 (t, 1H, J = 3.2
Hz, H-4 thiophene), 7.09 (d, 2H, J = 8.8 Hz, H-3 and H-5 phenyl), 7.04 (d, 2H, J = 3.2 Hz, H-3
and H-5 thiophene), 4.85 (t, 2H, J = 4.8 Hz, OCH2), 4.52 (t, 2H, J = 4.8 Hz, -CH2-triazole), 3.96
(s, 2H, -CH2-thiophene), 3.84 (s, 2H, triazole-CH2-N). 13C-NMR (DMSO-d6, 400 MHz) δ: 46.86
(triazole-CH2-NH-), 49.46 (CH2N linker), 53.56 (N-CH2 pyridine), 66.76 (OCH2 linker), 108.52
(C3 benzoimidazothiazole), 113.67 (C5 benzoimidazothiazole), 115.36 (C3 and C5 phenyl),
124.04 (C8 benzoimidazothiazole), 125.47 (C7 benzoimidazothiazole), 125.88 (C3 thiophene),
29
ACCEPTED MANUSCRIPT
126.45 (C2 and C6 phenyl), 127.14 (C4 and C5 thiophene), 127.57 (C6 benzoimidazothiazole),
129.55 (C1 phenyl), 131.51 (C5 triazole), 132.28 (C8a benzoimidazothiazole), 137.55 (C8a
benzoimidazothiazole), 143.8 (C4 triazole), 145.75 (C1 thiophene),
146.51 (C9a
RI
PT
benzoimidazothiazole), 146.91 (C2 benzoimidazothiazole), 157.69 (C4 phenyl). MS (m/z, %):
486 (M+, 8), 403 (8), 266 (100), 237 (7), 207 (6), 150 (35), 121 (14), 93 (60). Anal. Calcd for
SC
C25H22N6OS2: C, 61.71; H, 4.56; N, 17.27. Found: C, 61.77; H, 4.58; N, 17.14.
5.3.6. 1-(1-(3-(4-(benzo[d]imidazo[2,1-b]thiazol-2-yl)phenoxy)propyl)-1H-1,2,3-triazol-4-yl)-
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N-(thiophen-2-ylmethyl)methanamine) (6f)
According to the general procedure, 4b (100 mg, 0.28 mmol) and 5c (42 mg, 0.28 mmol) were
used for click reaction and final yield was 129 mg, 92%; mp 107-108 °C; 1H NMR (DMSO-d6,
400 MHz) δ: 8.67 (s, 1H, H-3 benzoimidazothiazole), 8.04 (d, 1H, J = 8.0 Hz, H-8
8.03
(s,
1H,
triazole),
TE
D
benzoimidazothiazole),
7.97
(d,
1H,
J
=
8.0
Hz,
H-5
benzoimidazothiazole), 7.80 (d, 2H, J = 8.8 Hz, H-2 and H-6 phenyl), 7.58 (dt, 1H, J = 7.2 and
0.8 Hz, H-6 benzoimidazothiazole), 7.43 (dt, 1H, J = 7.2 and 0.8 Hz, H-7 benzoimidazothiazole),
EP
7.36-7.43 (m, 1H, H-4 thiophene), 7.01 (d, 2H, J = 8.8 Hz, H-3 and H-5 phenyl), 6.91-7.00 (m,
AC
C
2H, H-3 and H-5 thiophene), 4.55 (t, 2H, J = 7.2 Hz, OCH2), 4.03 (t, 2H, J = 6.0 Hz, -CH2triazole), 3.91 (s, 2H, -CH2-thiophene), 3.78 (s, 2H, triazole-CH2-N), 2.31 (p, 2H, J = 6.4 Hz, CH2-).13C-NMR (DMSO-d6, 400 MHz) δ: 29.49 (CH2 linker), 46.86 (triazole-CH2-NH-), 47.17
(CH2N
linker),
55.01
(N-CH2
thiophene),
64.96
(OCH2
linker),
108.34
(C3
benzoimidazothiazole), 113.64 (C5 benzoimidazothiazole), 115.20 (C3 and C5 phenyl), 123.41
(C8 benzoimidazothiazole), 125.13 (C7 benzoimidazothiazole), 125.45 (C2 and C6 phenyl),
126.45 (C4 and C5 thiophene), 127.11 (C6 benzoimidazothiazole), 127.22 (C3 thiophene),
30
ACCEPTED MANUSCRIPT
129.55 (C1 phenyl), 132.30 (C5 triazole), 130.90 (C8a benzoimidazothiazole), 137.7 (C8a
benzoimidazothiazole), 146.22 (C1 thiophene), 144.5 (C2 benzoimidazothiazole), 146.75 (C9a
benzoimidazothiazole), 147.11 (C4 triazole), 158.19 (C4 phenyl). MS (m/z, %): 500 (M+, 15),
RI
PT
403 (33), 266 (55), 237 (4), 207 (4), 150 (18), 121 (30), 93 (100). Anal. Calcd for C26H24N6OS2:
5.4. Radiolabeling procedure
5.4.1. Preparation of fac-[99mTc(CO)3(H2O)3]+
SC
C, 62.38; H, 4.83; N, 16.79. Found: C, 62.22; H, 4.89; N, 16.88.
99m
TcO4- was performed based on
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The preparation of fac-[99mTc(CO)3(H2O)3]+ precursor from
the previously published protocol [35]. Briefly, NaBH4 (as the reducing agent, 6 mg), sodium
tartrate dihydrate (as the transfer ligand, 15 mg), Na2CO3 (5.5 mg) and 1.2 mL water were added
to a tightly sealed glass vial with inlet and outlet for CO gas. Then, the vial was flushed with CO
heated to 85 °C,
99m
TE
D
gas to remove any dissolved oxygen for 5 min at room temperature. Finally, the solution was
TcO4- (20 mCi) was added to the solution and the pressure of CO gas was
adjusted to 1 atm. After 30 min, the gas flow was stopped and the vial was allowed to cool down
EP
to room temperature. The pH of the fac-[99mTc(CO)3(H2O)3]+ precursor was set at 8.5 using a
AC
C
neutralizing solution composed of 180 µL HCl 1N and 100 µL phosphate buffer 1M.
5.4.2. Radiolabeling of IBT ligands
Stock solution (5.2×10-3 mol/L) of every ligand (6a-f) was prepared in phosphate-buffered saline
(PBS), pH 7.4/EtOH (1:1). A solution of fac-[99mTc(CO)3(OH2)3]+ ( 100 µL, 40-60 MBq) was
added to the relevant ligand (25 µL) and diluted with PBS (125 µl, pH 7.4) to give final
concentration 5.2×10-4. The final pH of reaction solutions was set at 8.5 and the solutions were
31
ACCEPTED MANUSCRIPT
heated for 45 min at 85 °C. The radiochemical purity (RCP) of fac-[99mTc(CO)3(H2O)3]+,
[99mTc]7a and [99mTc]7b was determined by a RP-HPLC.
RI
PT
5.4.3. Stability in saline solution
Radiolabeled ligands (100 µL, ~20 MBq) were diluted with 900 µL normal saline; and the
resulted solutions were incubated at 37 °C. The saline stability of each radiolabeled ligand was
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5.4.4. Log P determination
SC
assessed at 0, 120, and 240 min after incubation with the RP-HPLC to determine the RCP.
In order to determine the partition coefficients of [99mTc]7a and [99mTc]7b a mixture of 1-octanol
(1000 µL) and PBS buffer (950 µL, pH 7.4) was prepared. Then, 50 µL of the radiotracer (~10
MBq) was added to the mixture and vortexed for 15 min. Afterward, the mixture was centrifuged
TE
D
(15 min, 7000 rpm). Aliquots (50 µL) from the 1-octanol and PBS phases were placed into two
test tubes for measuring the radioactivity by the gamma counter. The Log P was calculated
EP
according to the equation: Log P = log[(count 1-octanol)/(count PBS)].
5.5. Biological distribution in normal mice
AC
C
In mice biodistribution studies, [99mTc]7a or [99mTc]7b (100 µL, ~ 25MBq) was diluted with
2400 µL normal saline. Aliquots (100 µL, ~1MBq) from the diluted radiotracers were injected
into mouse through the tail vein. The mice (n = 3 for each time point) were sacrificed at 2, 5, 10,
30, 60 and 120 min post-injection using an intraperitoneal injection of ketamine/xylazine (Sigma,
St. Louis, MO, USA). The organs and tissues of interest were removed, weighed and counted
using a gamma counting system. The uptake values were expressed as a percentage of the
32
ACCEPTED MANUSCRIPT
injected activity per gram of tissue or organ (%ID/g). For the intestines, the amount of activity
was calculated as %ID for the whole sample.
RI
PT
5.6. Inhibition assay
5.6.1 Preparation of Amyloid β aggregations
Amyloid β aggregations were obtained by dissolving solid form of Aβ(1−42) (0.25 mg/ml) in
SC
PBS (pH 7.4). The solution was incubated (at 37 °C) for 42-48 h with constant shaking.
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5.6.2. Binding assay using Aβ aggregates in solution
A mixture of Aβ(1−42) aggregates (50 µL with final conc., 1.25 µg/mL), 50 µL of [99mTc]7a or
[99mTc]7b complex (final conc., 510 kBq/mL), and 900 µL of 30% EtOH were incubated at
room temperature for 3 h. Then the mixture was filtered through Whatman GF/B filters, and the
gamma counter.
99m
Tc-IBT complex was measured using a
TE
D
radioactivity of the filters containing the bound
EP
5.6.3. Inhibition assay using Aβ Aggregates in solution (IC50 determination)
A mixture of Aβ(1−42) aggregates (50 µL with final conc., 1.25 µg/mL), 50 µL of [99mTc]7a or
AC
C
[99mTc]7b complex (final conc. 5.2×10-6, 510 kBq/mL), and 50 µL of BTA-1 as competing
ligand (4.1×10-4 to 4.0×10-10 M diluted serially in 30% EtOH), and 850 µL of 30% EtOH (final
volume of 1.0 mL) was incubated at 37°C for 3 h (n = 3 for each concentration), and the bound
and the free radioactivity were separated by Whatman GF/B filters, followed by washing with
PBS buffer 3 times. Filters containing the bound radioactivity were assayed for radioactivity in a
33
ACCEPTED MANUSCRIPT
gamma counter. Values for the half-maximal inhibitory concentration (IC50) were determined
RI
PT
from displacement curves using GraphPad Prism 6.0.
5.7. Autoradiography study
SC
5.7.1. Preparation of Amyloid β oligomers for injection to rats’ brain
Amyloid β oligomers (AβO) were prepared according to previously reported studies.[36, 37]
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Briefly, Aβ(1−42) peptide was dissolved in ice-cold 1,1,1,3,3,3-hexafluro-2-propanol (HFIP, 250
µL) to make a solution with final concentration of 1 mg/mL. The solution was vortexed and
aliquoted into microcentrifuge tubes with 25 µL each. Tubes were air-dried on ice for 15 min and
then lyophilized for 1 h. Resulting peptide was stored at -80 oC until use. Prior to surgery,
o
TE
D
peptides were re-dissolved in 5 µL of anhydrous dimethyl sulfoxide, sonicated for 10 min at 37
C, and 10 mM phosphate buffered saline (PBS, pH 7.4, 45 µL) was added to make a final
concentration of 0.5 mg/mL. Immediately after adding PBS, peptide solution was incubated at 4
o
EP
C for 24 h.
AC
C
5.7.2. Preparation of rats Alzheimer model
Rats were anesthetized using an intraperitoneal injection of ketamine/xylazine (1 µL/g body
weight) and fixed onto a stereotaxic apparatus (Stoelting, USA). Body temperature was
maintained at 37 oC during the surgery. Nine male Wistar rats aged 3 months (body weights:
250-380 g) were used. Two cannulas were inserted at the flowing coordinates: bregma: -3.8 mm;
medline: ± 2.0 mm; depth: 2.8 mm. After a recovery period of 7 days, six rats were received 5
34
ACCEPTED MANUSCRIPT
µL (2.5 µg) of AβO solution every other day for 3 weeks. Likewise, the control group (3 rats)
5.7.3. Histological assay and Autoradiography
RI
PT
was received only 5 µL of 2% DMSO in PBS at the same manner to AD model rats.
For the preparation of rats’ brains for Congo red staining and autoradiography, the rats have
overdosed with an intraperitoneal injection of ketamine/xylazine, their brains were removed and
SC
fixed with 10% paraformaldehyde for 24 hours. The brains were cut to smaller sections and
treated with graded alcohols for 30 min and xylene (2 × 30 min). Then the sections were
M
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U
paraffin-embedded and 10 µm thick sections were obtained. The brain sections of rat Alzheimer
model and control rats were deparaffinized in xylene (3 × 2 min) and 100% ethanol (2 × 5 min).
Then, the sections were hydrated to water using 90% ethanol (1 × 2 min), 70% ethanol (1 × 2
5.7.4. Congo red staining
TE
D
min) and running Water (1 × 2 min).
The hydrated sections were stained in congo red solution (5 mg/mL in 50% ethanol) for 15-20
EP
minutes. Then the samples were rinsed in distilled water and differentiate quickly (5-10 dips) in
an alkaline alcohol solution (5 mg of NaOH in 1 mL of 50% ethanol). After rinsing in tap water
AC
C
for 1 minute, the sections were counterstain with Gill's hematoxylin for 30 seconds and rinsed in
tap water for 2 minutes. Finally, the sections were dehydrated through 95% alcohol (2 × 3 min)
and 100% alcohol (2 × 3 min) and mount with resinous mounting medium. Then, slides were
observed by a histologist blindly under an optical microscope (Olympus, Tokyo, Japan).
5.7.5. Autoradiography
35
ACCEPTED MANUSCRIPT
The hydrated brain sections including normal (n = 3), control (n = 3) and AD model (n =3) were
incubated in PBS (0.2 M, pH 7.4) for 30 min. The sections were incubated with radiolabeled
[99mTc]7a or [99mTc]7b (510 KBq /100 µL) for 60 min at room temperature. Then, they were
RI
PT
washed in 40% EtOH before being rinsed with water for 2 min. After drying, the sections were
placed on primax dental x-ray films (RDX-58 E soft ISO Film size 2 (31× 41 mm) ISO Film
Class E) for 24 hours. Afterward, the films were exposed to RRD-10 developer solution (1 min)
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Conflicts of interest
SC
and RRF-10 fixer solution (1 min).
The authors declared no conflict of interest.
Acknowledgment
TE
D
This work was supported by the Iran National Science Foundation (INSF) (grant number
95836340) and Mazandaran University of Medical Sciences, Sari, Iran. This research was the
Medical Sciences.
AC
C
References
EP
subject of the PhD thesis of Sajjad molavipordanjani as a student of Mazandaran University of
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Research Highlights
99m
Tc-radiolabeled 2-arylimidazo[2,1-b]benzothiazole derivatives were synthesized.
[99mTc]7a,b showed moderate brain uptake and fast washout.
[99mTc]7a,b can penetrate to blood brain barrier.
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[99mTc]7a,b have affinity toward β-amyloid aggregates.
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[99mTc]7b showed excellent plaque labeling on the brain section of rat Alzheimer model.