Synthesis and evaluation of two uncharged 99mTc-labeled derivatives of thioflavin-T as potential tracer agents for fibrillar brain amyloid

Article abstract of DOI:10.1002/jlcr.1592

Thioflavin-T is a fluorescent dye for in vitro detection of fibrillar amyloid b, a protein found in the brain of patients suffering from Alzheimer's disease. We synthesized and biologically evaluated two uncharged ^99mTc-labeled derivatives of thioflavin-T. The precursors for labeling were synthesized by coupling an S,S′-bis-triphenylmethyl-N-tert- butoxycarbonyl bis-amino-bis-thiol tetradentate ligand via a propoxy spacer to 2-(4′-aminophenyl)-1,3-benzothiazole at the 6-position or the 2′-position. Deprotection and labeling with ^99mTc were done via a one-pot procedure (15% yield) after which the labeled compound was isolated by high performance liquid chromatography (LC). LC in combination with mass spectrometry (MS) was used for identity confirmation of the labeled compounds. Results of electrophoresis and log P determination supported the assumption that the radiolabeled compounds could cross the blood-brain barrier by passive diffusion. However, in normal mice both compounds showed a low brain uptake 2 min post injection. They were mainly excreted through the hepatobiliary system, with some accumulation in the stomach. Sixty minutes after intravenous injection, 37% of the ^99mTc-activity in the blood corresponded to the original compound. In view of the low brain uptake, it is concluded that the studied ^99mTc-labeled derivatives of thioflavin-T are not suitable as tracer agents for in vivo visualization of amyloid in brain. Copyright

Full text of DOI:10.1002/jlcr.1592

Research Article
Received 12 November 2008,
Revised 22 December 2008,
Accepted 27 January 2009
Published online 13 March 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1592
Synthesis and evaluation of two uncharged
99mTc-labeled derivatives of thioflavin-T
as potential tracer agents for fibrillar
brain amyloid
Ã
K. Serdons,a D. Vanderghinste,b M. Van Eeckhoudt,a J. Cleynhens,a
T. de Groot,b G. Bormans,a and A. Verbruggena
Thioflavin-T is a fluorescent dye for in vitro detection of fibrillar amyloid b, a protein found in the brain of patients suffering
from Alzheimer’s disease. We synthesized and biologically evaluated two uncharged 99mTc-labeled derivatives of
thioflavin-T. The precursors for labeling were synthesized by coupling an S,S0-bis-triphenylmethyl-N-tert-butoxycarbonyl
bis-amino-bis-thiol tetradentate ligand via a propoxy spacer to 2-(40-aminophenyl)-1,3-benzothiazole at the 6-position or
the 20-position. Deprotection and labeling with 99mTc were done via a one-pot procedure (15% yield) after which the labeled
compound was isolated by high performance liquid chromatography (LC). LC in combination with mass spectrometry (MS)
was used for identity confirmation of the labeled compounds. Results of electrophoresis and log P determination supported
the assumption that the radiolabeled compounds could cross the blood–brain barrier by passive diffusion. However, in
normal mice both compounds showed a low brain uptake 2 min post injection. They were mainly excreted through the
hepatobiliary system, with some accumulation in the stomach. Sixty minutes after intravenous injection, 37% of the
99mTc-activity in the blood corresponded to the original compound. In view of the low brain uptake, it is concluded
that the studied 99mTc-labeled derivatives of thioflavin-T are not suitable as tracer agents for in vivo visualization of
amyloid in brain.
Keywords: amyloid; thioflavin-T; technetium-99m; SPECT; 2-phenylbenzothiazoles
promising, as human studies in AD patients and healthy controls
show that there is a marked retention of tracer agent in the
Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder, which
typically strikes the older age groups. As life expectancy of the
brain regions rich in fibrillar Ab in AD patients as compared to
the control subjects.6
population increases, an increasing number of people suffer
from AD, placing a heavy burden on society for the health care
of these patients. Nowadays, the diagnosis of AD is done by
clinical assessment of the symptoms such as memory loss and
by exclusion of systemic diseases and other brain diseases.1
However, this kind of diagnosis is only a ‘probable’ diagnosis.
Definite diagnosis is done by (immuno)histochemical staining of
post mortem brain tissue.2 Histochemical dyes, such as silver
(Bielschowsky technique), Congo red, chrysamine G, thioflavin-S
or thioflavin-T (ThT), bind specifically to fibrillar amyloid b (Ab), a
protein that is found in senile brain plaques in persons suffering
from AD. Fibrillar Ab may be a useful biomarker in the
differential diagnosis of AD from other dementias and in the
follow-up of future therapies directed against the Ab deposition
in the brain.
In view of the promising results obtained with radiolabeled
phenylbenzothiazoles and the nearly optimal characteristics of
technetium-99m as radionuclide for nuclear imaging purposes,
several neutral 99mTc-labeled compounds were already devel-
oped and evaluated for their biological characteristics. Our
group first investigated a 99mTc-labeled MAMA-derivative of
chrysamine G.7 However, brain uptake of this radiolabeled agent
was minimal, probably because of its large size and ionized
character at physiological pH. The group of Kung studied
biphenyl derivatives containing N2S2 (BAT)-chelating groups.8
One of these neutral and lipophilic 99mTc-labeled complexes
showed good blood–brain barrier (BBB) penetration (1.18% ID
aLaboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences,
K.U.Leuven, Herestraat 49, Box 821, 3000 Leuven, Belgium
Several uncharged radiolabeled phenylbenzothiazoles based
on the structure of thioflavin-T have already been reported,
including the iodine-125 labeled compounds 125I-TZDM3 and
2-(30-[125I]iodo-40-aminophenyl)-6-hydroxybenzothiazole4 and
the carbon-11 labeled BAT-derivatives.5 Especially 6-OH-BTA-1,
also known as Pittsburgh Compound-B or PIB, seems to be
bRadiopharmacy, U.Z. Gasthuisberg, Leuven, Belgium
*Correspondence to: K. Serdons, Laboratory for Radiopharmacy, Faculty of
Pharmaceutical Sciences, K.U.Leuven, Herestraat 49, Box 821, 3000 Leuven,
Belgium.
E-mail: Kim.serdons@pharm.kuleuven.be
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Copyright r 2009 John Wiley & Sons, Ltd.

K. Serdons et al.
2 min p.i.) with a reasonable brain wash-out in normal mice quent purification and identification (Figure 2). The log P value
(Figure 1(A)). This complex, however, labeled amyloid deposits of the compounds was determined and a biodistribution and
in transgenic APP mouse brain sections, but not in human AD stability study in normal mice was performed.
brain sections. Our group also developed a 99mTc-labeled BAT-
conjugate of 2-(40-aminophenyl)-1,3-benzothiazole that binds
in vitro to amyloid b (Figure 1(B)).9 Despite its high lipophilicity
and absence of charge, this radiolabeled conjugate did not cross
the BBB in a sufficient way (0.1% ID 2 min p.i.) and thus is not
useful for in vivo detection of AD. Recently, the group of Chen
reported a 99mTc-MAMA-BTA complex in which the chelator
MAMA was conjugated with 2-(40-aminophenyl)-1,3-benzothiazole
through a 5-carbon alkyl chain (Figure 1(C)).10 They demonstrated
that this 99mTc-MAMA-BTA complex, which showed brain uptake
in normal mice (1.34% ID/g), binds to Ab aggregates in brain
sections of both transgenic APP mouse and an AD patient.
Here, we report the synthesis of two uncharged derivatives of
2-(40-aminophenyl)-1,3-benzothiazole coupled via a linker at
position 6 or 20 to S,S’-bis-trityl-N-BOC protected BAT ligand,
together with their deprotection, labeling with 99mTc, subse-
Results and discussion
Chemistry
As 2-(40-aminophenyl)-1,3-benzothiazoles do not have the
potential to chelate technetium-99m, we conjugated them with
an S,S’-bis-trityl-N-BOC protected bis-amino-bis-thiol (BAT)
tetradentate ligand via a propoxy linker. Depending on the
intended position of derivatization of the phenylbenzothiazole
(6-position or 20-position), two different synthetic pathways
have been followed. The preparation of 2-(40-aminophenyl)-6-
hydroxy-1,3-benzothiazole was based on the method described
by Shi et al.,11 but some modifications were introduced in
several steps of the synthesis to improve the yields or the purity
of the reaction products. A schematic representation of the
reaction pathway to obtain the conjugation of the phenylben-
zothiazole at position 6 with the protected BAT ligand is shown
in Scheme 1. In the first step, p-anisidine and 4-nitrobenzoyl
chloride were reacted in boiling pyridine to yield N-40-
methoxyphenyl-4-nitrobenzamide (1). According to literature,
H
O
N
N
S
S
Tc
1
should then be reacted with Lawesson’s reagent in
N
(a)
hexamethylphosphoramide, but we were not successful in
producing N-40-methoxyphenyl-4-nitrothiobenzamide (2) fol-
lowing this method. Changing the solvent to 1,4-dioxane and
refluxing the reaction for 3 h resulted in a yield of 93% of 2 after
recrystallization from methanol. Cyclization by Jacobson’s
method with the oxidizing agent potassium ferricyanide in
alkaline solution produced 2-(40-nitrophenyl)-6-methoxy-1,3-
benzothiazole (3) in a yield of 70%. To convert 3 into 2-(40-
aminophenyl)-6-hydroxy-1,3-benzothiazole (5), the nitro group
was first reduced to an amine using stannous chloride in ethanol
and the methoxy group was then demethylated to a hydroxyl
group using boron tribromide. Performing the reduction and
demethylation reactions in the reverse order yielded a reaction
mixture from which it was difficult to isolate 5. For the
preparation of 2-(40-aminophenyl)-6-(300-p-tosyloxypropoxy)-1,3-
benzothiazole (6), 1,3-propanediol di-p-tosylate was reacted
with 5 in the presence of sodium methanolate. A two-fold
excess of 1,3-propanediol di-p-tosylate was used to avoid
reaction at both tosyl groups and this was further inhibited by
adding 5 slowly to the solution of 1,3-propanediol di-p-tosylate.
Final coupling of the S,S’-bis-trityl-N-BOC protected BAT to 6 was
done in DMF in the presence of diisopropylethylamine as a base
to sufficiently deprotonate the unprotected amine of the BAT
precursor. A reaction time of 24 h and a temperature of
501C were necessary to obtain 2-(40-aminophenyl)-6-f300-
[N-(N0-triphenylmethylmercaptoethyl-N0-tert-butoxycarbonyl)-
aminoethyl-N-triphenylmethylmercaptoethyl]aminopropoxyg-1,
3-benzothiazole (7) in a sufficient yield. Rising the temperature to
701C had a negative influence on the yield, as qualitatively and
quantitatively more impurities were formed. Several purification
techniques, including silica gel column chromatography and
preparative TLC were tried, but none of them allowed to separate
the desired products from their impurities. Finally, purification was
done with normal phase high performance liquid chromatogra-
phy (HPLC) on a semi-preparative column. However, we were not
able to completely eliminate in this way the S,S’-bis-trityl-N-BOC
protected BAT ligand from the desired conjugate. Reversed
H
N
S
N
N
N
S
O
O
Tc
(b)
S
O
O
N
N
O
S
NH2
Tc
N
S
(c)
S
Figure 1. Structures of reported potential 99mTc-labelled tracer agents for imaging
of fibrillar amyloid b.
N
NH2
S
N
S
N
S
O
O
Tc
99mTc-7
N
NH2
S
N
S
N
O
O
99mTc-10
Tc
S
Figure 2. Structures of 99mTc-labelled tracer agents 7 and 10.
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 227–235

K. Serdons et al.
NO2
NO2
NH2
NO2
H
N
H
N
i
ii
+
O
S
2
CH3O
1
CH3O
COCl
OCH3
iii
N
S
N
N
v
iv
CH3O
NH2
NH2
NO2
5
S
HO
S
CH3O
vii
3
4
vi
N
S
N
S
NH2
NH2
BOC
O
N
N
O
6
7
OTs
S
S
BOC = tert-butyloxycarbonyl
Tr = triphenylmethyl = trityl
Ts = tosyl
Tr
Tr
Scheme 1. Synthetic pathway to 6-f300-[N-(N0-triphenylmethylmercaptoethyl-N0-tert-butoxycarbonyl)aminoethyl-N-triphenylmethylmercaptoethyl]aminopropoxyg-2-(40-
aminophenyl)-1,3-benzothiazole (7). Reagents and conditions: (i) pyridine, reflux; (ii) Lawesson’s reagent, 1,4-dioxane, N2, reflux; (iii) K3Fe(CN)6, EtOH, 901C; (iv)
SnCl2 ꢀ 2H2O, EtOH, reflux; (v) BBr3, CH2Cl2; (vi) 1,3-propanediol di-p-tosylate, NaOMe, MeCN/MeOH 9:1 V/V; (vii) S,S’-bis-trityl-N-BOC-BAT, DIEA, DMF, 501C.
NH2
N
i
+
HOOC
NH2
NH2
S
SH
HO
HO
8
ii
N
N
S
iii
NH2
N
NH2
S
BOC
N
OTs
O
O
9
10
Ts = tosyl
S
S
Tr
Tr
BOC = tert-butyloxycarbonyl
Tr = triphenylmethyl = trityl
Scheme 2. Synthetic pathway to 2-f20-[300-(N-(N0-triphenylmethylmercaptoethyl-N0-tert-butoxycarbonyl)aminoethyl-N-triphenylmethylmercaptoethyl)aminopropoxy]-40-
aminophenyl}-1,3-benzothiazole (10). Reagents and conditions: (i) polyphosphoric acid, 1801C; (ii) 1,3-propanediol di-p-tosylate, NaOMe, MeCN/MeOH 9:1 V/V; (iii) S,S’-bis-
trityl-N-BOC-BAT, DIEA, DMF, 501C.
phase-HPLC (RP-HPLC) showed that still 1.5% of this reagent was triphenylmethylmercaptoethyl-N0-tert-butoxycarbonyl)aminoethyl-
present in the final product. Evidently, this S,S’-bis-trityl-N-BOC N-triphenylmethylmercaptoethyl)aminopropoxy]-40-aminophenylg-
protected BAT interferes with the radiolabeling, as it is also 1,3-benzothiazole (10) (1.5% as determined by RP-HPLC).
capable to form a complex with Tc after deprotection.
Compounds 7 and 10 were purified using RP-HPLC resulting
Introduction of the Tc-binding side chain on the 20-position of in sufficient amounts to perform labeling with 99mTc. Owing to
the aminophenyl part was done by reaction of 1,3-propanediol the difficult purification, melting point determinations and
di-p-tosylate with 2-(20-hydroxy-40-aminophenyl)-1,3-benzothia- nuclear magnetic resonance (NMR) spectra of these compounds
zole (8), which was obtained by reaction of 2-aminothiophenol could not be performed.
with 4-aminosalicylic acid in polyphosphoric acid at 1801C
(Scheme 2). After introduction of the propoxy linker, 2-[20-(300-p-
tosyloxypropoxy)-40-amino]-1,3-benzothiazole (9) was coupled
Labeling and characterization
with S,S’-bis-trityl-N-BOC protected BAT and subsequent pur-
ification of the reaction mixture was done analogously as
described for 7. There was also contamination with S,S’-bis-trityl-
N-BOC protected BAT ligand in purified 2-f20-[300-(N-(N0-
For the radiolabeling of 7 and 10, a one-pot procedure was
developed. The deprotection of the precursor and consecutive
radiolabeling of the deprotected compound was performed in
the same vial without purification between the two steps.12 In
J. Label Compd. Radiopharm 2009, 52 227–235
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K. Serdons et al.
order to obtain enough deprotected compound for radiolabel- TcO-tartrate. In addition, the phenylbenzothiazole linked via a
ing, 50 ml 0.5 M HCl was added to a solution of the protected propoxy spacer to the BAT tetradentate ligand constitutes a
compound (7 or 10) in EtOH and the mixture was heated in a serious steric hindrance, which slows down the complexation
boiling water bath for 20 min. Liquid chromatography–mass kinetics, also a reason to apply an exchange labeling instead of a
spectrometry (LC–MS) analysis of the reaction mixture after direct labeling. We obtained the highest labeling yield (15%)
deprotection of 10 showed that the BOC-group was easily when the amounts of tartrate and SnCl2 in the reaction mixture
removed, while it was much harder to remove the two trityl were 800 and 50 mg, respectively. The remainder of the 99mTc-
groups. Figure (3.1) shows a typical HPLC-chromatogram of the activity was found under the form of colloidal 99mTcO2, 99mTcO4ꢁ,
reaction mixture after deprotection of 10, with at 1.95 min the 99mTc-tartrate and 99mTc-BAT. The ratios of these side products
fully deprotected compound with both thiols oxidized forming could be influenced by changing the amounts of SnCl2 and
an intramolecular disulfide bridge (accurate MS ES1 m/z tartrate, but changing these parameters did not increase the
[M1H]1 461.1468 Da, 6.5 ppm relative error, Figure (3.4)), at yield of the desired product. Although S,S’-bis-trityl-N-BOC
12.07 min a peak corresponding to the compound without the protected BAT was only present in amounts as low as 1.5% in
BOC group but with the two trityl groups still present (accurate the purified precursors, 99mTc-BAT was present as a 6% impurity
MS ES1 m/z [M1H]1 947.3823, 2.4 ppm relative error, on the radiometric signal of the labeling mixtures. This is
Figure (3.3)) and at 14.54 min the original fully protected probably due to a more efficient deprotection and/or a better
compound 10, (Figure (3.2)). No compound without the BOC ability to form a complex with technetium in view of the
and with only one trityl group was found, except after direct absence of steric hindrance. Analysis using LC in combination
injection of the reaction mixture into the mass spectrometer with mass spectrometry (LC–MS) of the mixture obtained after
without prior neutralization. In these conditions, also the fully labeling of 10 with eluate enriched in 99Tc (because of the
deprotected compound was found in the free-thiol form. This difficult detection of the tracer amounts of technetium in 99mTc-
suggests that neutralizing the reaction mixture results in an labelled preparations) showed that the main radiometric peak,
oxidation of the thiols, either in the reaction vessel or in the eluting at 9.63 min (Figure 4(c)), is with high probability the
LC–MS system, with the completely deprotected compound desired compound, as the background subtracted mass
forming an intramolecular disulfide bridge and the compound spectrum summed over this peak shows a single peak with a
missing the BOC and one trityl group forming a compound with mass close to the theoretical mass of the desired compound
unknown structure. Apparently, only a small percentage of the (Figure 4(b)). Performing an accurate mass assessment of the
precursor is fully deprotected and immediately available for spectrum revealed that this peak has an accurate mass of
complexation of reduced Tc. The fraction of compound missing 575.0370 Da (Figure 5(c)), or an error of only 9.6 ppm compared
the BOC and one trityl group can, however, also be further to the theoretical accurate mass of 575.0425 Da (Figure 5(a)).
deprotected during the metal complexation process but this The peak eluting at 6.40 min corresponds to 99mTc-BAT, as we
requires sufficient energy (heating) and time, which necessitates observed a single peak in the background subtracted mass
the temporary binding of reduced Tc in a weak complex such as spectrum summed over the radiometric peak at 6.39 min, with a
1047.0929
12.07
100
100
1
2
a
b
N
S
1048.1100
NH2
N
BOC
O
N
S
%
%
S
a
c
Tr
Tr
-1
0
5.00
10.00
15.00
20.00
200
400
600
800
1000
1200
Time
m/z
947.0384
461.0071
100
100
3
4
c
b
948.0635
N
S
N
NH2
N
NH2
H
H
S
%
%
O
N
S
O
N
S
N
S
S
Tr
Tr
0
0
200
400
600
800
m/z
1000
1200
200
400
600
800
1000
1200
m/z
Figure 3. LC-MS analysis of 10 after deprotection. (1): UV chromatogram (365 nm); (2, 3 and 4): Background subtracted mass spectra summed over the different peaks. The
spectra are not accurate mass spectra as the signal of the lock mass is lost when background substracted mass spectra are used.
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K. Serdons et al.
6.39
292.886 – 292.957 Da
a
7.02
1.23
8.37
7.87
1.03
1.88
12.09
11.47
9.17
8.62
4.13
9.69
574.900 – 574.993 Da
b
99mTc-10
9.63
99mTc-BAT
Radiometric signal
c
6.40
1.00
2.00
3.00
4.00
5.00
6.00
Time
7.00
8.00
9.00
10.00
11.00
12.00
Figure 4. LC–MS analysis of the reaction mixture after labelling of 10 with 99mTc spiked with 99Tc (after Seppaks Light C18 purification). (a) Ion mass chromatogram
(range 292.886–292.957 Da) corresponding to the molecular ion mass of 99mTc-BAT; (b) ion mass chromatogram (range 574.900–574.993 Da) corresponding to the
molecular ion mass of 99mTc-10; (c) radiometric signal.
575.0425
292.9599
100
100
a
b
Theoretical mass spectra
%
%
0
0
292.9659
575.0370
100
100
Experimental mass spectra
%
c
d
%
0
0
287
288
289
290
291
292
m/z
293
294
295
296
297
570
571
572
573
574
575
m/z
576
577
578
579
580
Figure 5. Accurate mass spectra of 99mTc-10 (left) and 99mTc-BAT (right). The top spectra (a and b) show the theoretical mass spectra of the labelled compounds. The
bottom spectra (c and d) the obtained mass spectra with kryptofixs222 as lock mass.
mass close to theoretical mass (Figure 4(a)). The observed isolated peaks were collected in a vial containing a solution of
accurate mass was 292.9659 (Figure 5(d)), or an error of ascorbic acid and polysorbate 80. The purpose of the ascorbic
20.5ppm compared to the theoretical accurate mass of acid was to prevent re-oxidation of the oxotechnetium(V) to
292.9599 (Figure 5(b)). We found similar results for 99mTc-7, also pertechnetate while the polysorbate 80 was added to prevent
supporting the assumed structure of the labeled compounds.
sticking of the compound to the wall of the vial after
evaporation of the organic solvent prior to injection in mice.
The results of the biodistribution studies of 99mTc-7 and 99mTc-
10 in healthy mice at 2 and 60 min p.i. are shown in Table 1. The
first requirement for a suitable tracer agent for detection of
amyloid plaques in brain is the ability to cross the BBB. As both
compounds show a negligible brain uptake, this important
prerequisite is not fulfilled. The reason for their limited passage
over the BBB is probably the relatively high molecular mass of
574 Da rather than an insuitable lipophilic character. Excretion of
the compounds proceeds mainly through the hepatobiliary
pathway and to a lesser extent through the renal pathway, which
Biological evaluation
The 1-octanol/buffer partition coefficient of 99mTc-7 and
99mTc-10 was found to be 160.8732.8 (log P = 2.2070.10) and
115.578.3 (log P = 2.0670.03), respectively. These values are
within the optimal range for passive diffusion over the BBB.13 In
electrophoresis experiments with 99mTc-7 and 99mTc-10, 88% of
the applied radioactivity remained at the application point,
indicating that the compounds are uncharged at physiological
pH. For biodistribution studies in normal mice, the RP-HPLC-
J. Label Compd. Radiopharm 2009, 52 227–235
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K. Serdons et al.
Table 1. Tissue distribution expressed as percentage of
the injected dose (% ID) and percentage of the injected dose
per gram tissue (% ID/g) after i.v. injection of 99mTc-7 and
99mTc-10 in normal mice at 2 and 60 min p.i. (n = 4 at each
time point)
(Sigma-Aldrich, Bornem, Belgium) and were used without
further purification. MgSO4 was used as drying agent. pH values
of non-radioactive solutions were measured with a P600 pH-
meter (Consort, Turnhout, Belgium) equipped with a glass
electrode, whereas pH values of radioactive solutions were
measured using pH strips (universal indicator pH 0–14, Merck,
Darmstadt, Germany). Evaporation of organic solvents under
reduced pressure was done with a Bu¨chi Rotovapor (Bu¨chi,
Flawil, Switzerland). Column purification was done using silica
gel with a particle size varying between 0.04 and 0.063 mm
(230–400 mesh) (MN Kieselgel 60 M, Macherey Nagel, Du¨ren,
Germany). Thin layer chromatography (TLC) was carried out
using precoated silica TLC plates (DC-Alufolien-Kieselgel, Fluka,
Buchs, Switzerland). The structure of the synthesized products
was confirmed with 1H-NMR spectroscopy on a Gemini 200 MHz
spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts are
reported as d-values (parts per million) relative to tetramethylsi-
lane (d = 0). Coupling constants are reported in Hertz. Splitting
patterns are defined by s (singlet), d (doublet), dd (double
doublet), t (triplet) and m (multiplet). For melting point (mp)
determination, an Electrothermal IA9000 digital melting point
apparatus was used (Electrothermal, Southend-on-Sea, UK).
Generator eluate containing 99mTc in the form of sodium
pertechnetate was obtained from an Ultratechnekow generator
(Tyco Healthcare, Petten, The Netherlands). NH949TcO4 was a gift
from Tyco Healthcare.
% ID7s.d.
% ID/g7s.d.
99mTc-7
2 min p.i. 60 min p.i. 2 min p.i.
60 min p.i.
Urine
Kidneys
Liver
Intestines
Stomach
Cerebrum
0.270.1
6.970.9
37.074.7 34.477.8
6.971.3 32.678.5
1.670.9
0.270.0
0.970.5
2.970.5
–
–
10.070.9
16.272.6
–
4.270.7
4.670.3
–
5.870.5
0.170.0
0.070.0
8.871.3
–
–
0.570.1
0.870.2
9.172.5
0.370.1
0.470.2
3.370.2
Cerebellum 0.070.0
Blood
25.377.7
99mTc-10
Urine
Kidneys
Liver
Intestines
Stomach
Cerebrum
0.270.2
5.171.7
1.070.6
4.270.8
–
–
7.772.5
7.771.6
14.474.8
–
43.374.9 30.7710.9 18.874.3
5.471.9 44.275.2
1.370.4
0.170.0
–
–
4.771.1
0.170.0
0.070.0
4.171.6
–
0.270.1
0.470.0
8.471.9
0.270.2
0.370.4
1.670.4
Quantitative determination of radioactivity in samples was done
using an automatic g counter coupled to a multi-channel analyzer
(Wallac 1480 Wizards 300, Wallac, Turku, Finland). The values were
corrected for physical decay and background radiation.
Cerebellum 0.070.0
Blood 21.274.3
LC–MS was performed on a system consisting of a Waters
Alliance 2690 separation module (Waters, Milford, MA, USA)
containing an Xterras MS C18 3.5 mm (2.1 mm ꢂ 50 mm)
RP-HPLC column (Waters). The mobile phase consisted of a
mixture of acetonitrile (MeCN, A) and 0.1% ammonium formate
(B) with either a gradient a (from 50 A:50 B V/V to 100 A:0 B V/V in
10 min followed by 10 min 100% A at a flow rate of 300 ml/min)
for the unlabeled compounds or a gradient b (0 A:100 B V/V–100
A:0 B V/V in 10 min followed by 10 min 100% A at a flow rate of
300 ml/min) for the radiolabeled compounds. The eluate was first
led through a UV-spectrometer (Waters 2487 Dual l absorbance
detector) and subsequently over a 3 in NaI(Tl)-crystal coupled to
a single channel analyzer (The Nucleus, Oak Ridge, TE, USA) for
radiometric detection before it was fed into the ESI source of the
time-of-flight mass spectrometer (LCT, Micromass, Manchester,
UK). Accurate mass determination was done by co-injection of a
kryptofixs 222-solution as an internal calibration mass when
electrospray ionization was in positive mode and by co-injection
of a meso-2,3-dibromosuccinic acid solution when electrospray
ionization was in negative mode. Acquisition and processing of
the data were done with Masslynx software (Version 3.5).
is rather logic in view of the lipophilic nature of the compounds.
The relatively high uptake in the stomach, 60 min p.i., suggests a
partial re-oxidation of the complexed technetium into pertechne-
tate in vivo. In view of the faster blood clearance of 99mTc-10, a
biostability study in normal mice was performed. Metabolism of
99mTc-10 leads to formation of both hydrophilic 99mTc-labeled
compounds with unknown structures as 99mTcO4ꢁ. To separate the
99mTc-labeled hydrophilic compounds from 99mTcOꢁ4 , a TLC-
system with acetone as mobile phase was used. The relative
amounts of 99mTc-labeled compounds in blood at 60min p.i.
calculated from both the TLC-data and RP-HPLC data are about
37% original intact compound, 33% pertechnetate and 30% other
99mTc-labeled hydrophilic metabolites. RP-HPLC analysis of the
urine showed the presence of hydrophilic radiolabeled metabo-
lites, which was confirmed by TLC-analysis. RP-HPLC analysis of
the bile shows that most activity is found under the form of 99mTc-
labeled hydrophilic metabolites. TLC analysis of the bile is more
difficult to interpret, as the results from paper chromatography
eluted with acetone are not consistent with the results from paper
chromatography eluted with saline. It may be hypothesized that
some metabolites are bound to bile salts, which do not migrate
with acetone or saline. As the results of the biodistribution study
of 99mTc-7 are similar to that of 99mTc-10, it can be assumed that
the metabolism of 99mTc-7 will also be similar.
HPLC was done with
a system consisting of a TSP
SpectraSeries P4000 pump (Thermo Separations Products, San
Jose, CA, USA) connected to a TSP SpectraSeries UV100 detector
and a 3 in NaI(Tl) crystal connected to a Medi-Lab Select SC7II
single channel analyzer (Medi-Lab Select, Mechelen, Belgium).
Data were collected with a RaChel data acquisition system
(Version 1.49, Lablogic, Sheffield, UK).
Experimental
Gas chromatography was performed on a DI 200 gas
chromatograph (Delsi Instruments, Suresnes, France) with a
Porapaks QS 80/100 column (Alltech, Deerfield, IL, USA) of
180 cm ꢂ 0.25 in.
Materials and methods
All reagents and solvents used in synthesis were purchased
from Acros Organics (Geel, Belgium), Aldrich, Fluka or Sigma
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 227–235

K. Serdons et al.
Synthesis of 2-(40-aminophenyl)-6-f300-[N-(N0-triphenylmethyl-
mercaptoethyl-N0-tert-butoxycarbonyl)aminoethyl-N-triphenyl-
methylmercaptoethyl]aminopropoxyg-1,3-benzothiazole (7)
The organic layer was washed two times with 250 ml water and
then with 250 ml brine, dried over MgSO4 and filtered. The
filtrate was concentrated by vacuum evaporation to leave a
residue of 4 (0.49 mmol, 12.58 g, 98.1%), which was used in
subsequent reactions without further purification.
N-40-methoxyphenyl-4-nitrobenzamide (1)
1H-NMR (DMSO-d6, 200 MHz): d 3.83 (3H, s, OCH3); d 6.67 (2H,
d, 3J = 8.0 Hz, 30-H 50-H); d 7.06 (1H, d, 3J = 8.8 Hz, 5-H); d 7.60 (1H,
A solution of p-anisidine (24.63 g, 0.2 mol) and 4-nitrobenzoyl
chloride (37.11 g, 0.2 mol) in 250 ml pyridine was refluxed for 2 h.
After cooling to room temperature, the mixture was poured into
1.5 l of cold water and the precipitate was filtered off, washed
with water and dried in a vacuum oven. Crystallization from
methanol (MeOH) yielded 0.169 mol (46.02 g, 84.5%) of 1 as
yellow-green needles.
3
s, 7-H); d 7.71 (2H, d, J = 8.2 Hz, 20-H 60-H); d 7.80 (1H, d, 3
J = 8.8 Hz, 4-H). Accurate MS ES1 m/z [M1H]1 257.0767
(calculated for C14H13N2OS 257.0743). Mp: 142–1441C.
2-(40-Aminophenyl)-6-hydroxy-1,3-benzothiazole (5)
A dispersion of 4 (12.82 g, 50 mmol) in 180 ml of CH2Cl2 under
nitrogen atmosphere was cooled to ꢁ701C in an acetone/solid
CO2 bath. Over a period of 1 h, BBr3 (0.106 mol, 106 ml of a 1 M
solution in CH2Cl2) was added slowly, after which the mixture
was stirred for another hour at ꢁ701C. The suspension was
allowed to warm up to room temperature and was then stirred
for 12 h. The suspension was again cooled to ꢁ701C and MeOH
was slowly added until no more gas evolved. After warming up
to room temperature, 400 ml of 2 M NaOH was added. The
aqueous layer was separated and neutralized with 3 M HCl (final
pH = 7.5). The precipitate formed was filtered off and dried in a
vacuum oven to yield 36.07 mmol of 5 (8.74 g, 72%) as a
brownish powder.
1H-NMR (DMSO-d6, 200 MHz): d 3.75 (3H, s, OCH3); d 6.94 (2H,
3
3
d, J = 8.8 Hz, 30-H 50-H); d 7.67 (2H, d, J = 8.8 Hz, 20-H 60-H); d
3
3
8.16 (2H, d, J = 8.8 Hz, 3-H 5-H); d 8.35 (2H, d, J = 8.8 Hz, 2-H 6-
H). Accurate MS ESꢁ m/z [MꢁH]ꢁ 271.0724 (calculated for
C14H11N2O4 271.0724). Mp: 196–1981C.
N-40-methoxyphenyl-4-nitrothiobenzamide (2)
A solution of 1 (54.46g, 0.2 mol) and 2,4-bis(4-methoxyphenyl)-
1,3-dithia-2,4-diphosphetane-2,4-disulfide (Lawesson’s reagent,
44.49 g, 0.11mol) in 350 ml 1,4-dioxane was refluxed for 3 h, after
which it was cooled to room temperature and poured into 1.5 l of
cold water. The precipitate was filtered off, washed with water
and dried in a vacuum oven. After recrystallization from MeOH,
0.187 mol (53.80g, 93.3%) of 2 was obtained as orange-red flakes.
1H-NMR (DMSO-d6, 200 MHz): d 3.78 (3H, s, OCH3); d 7.01 (2H,
3
1H-NMR (DMSO-d6, 200 MHz): d 6.67 (2H, d, J = 8.4 Hz, 30-H
50-H); d 6.92 (1H, d, 3J = 8.8 Hz, 5-H); d 7.32 (1H, s, 7-H); d 7.68 (2H,
3
3
d, J = 8.8 Hz, 20-H 60-H); d 7.71 (1H, d, J = 8.8 Hz, 4-H). Accurate
MS ES1 m/z [M1H]1 243.0619 (calculated for C13H11N2OS
243.0587). Mp : 262–2631C.
3
3
d, J = 8.8 Hz, 30-H 50-H); d 7.75 (2H, d, J = 8.8 Hz, 20-H 60-H); d
3
3
7.98 (2H, d, J = 8.4 Hz, 3-H 5-H); d 8.29 (2H, d, J = 8.8 Hz, 2-H 6-
H). Accurate MS ESꢁ m/z [MꢁH]ꢁ 287.0495 (calculated for
C14H11N2O3S 287.0496). Mp: 174.5–175.51C.
2-(40-Aminophenyl)-6-(300-p-tosyloxypropoxy)-1,3-benzothiazole (6)
To a dispersion of 5 (1.817g, 7.5 mmol) in 15ml of a mixture of
acetonitrile (MeCN) and MeOH (9:1V/V) was added Na (190mg,
8.25mmol) dissolved in a mixture of 3 ml MeOH and 12ml MeCN.
The resulting mixture was added to a dispersion of 1,3-
propanediol di-p-tosylate (768mg, 15mmol) in 75ml of a mixture
of MeCN and MeOH (9:1V/V) and the reaction mixture was
heated to 501C for 12h. The formed suspension was filtered off
and the solvent removed from the filtrate by vacuum evapora-
tion. The residue was purified by silica gel column chromato-
graphy with a mixture of CH2Cl2 and MeOH (99:1 V/V) as eluent to
obtain 6 (896mg, 1.97mmol, 26.3%) as a yellow-brown powder.
1H-NMR (CDCl3, 200 MHz): d 2.14 (2H, m, CH2CH2CH2); d 2.30
(3H, s, CH3); d 3.98 (2H, t, OCH2CH2CH2); d 4.27 (2H, t, CH2CH2OTs);
d 6.73 (2H, d, 3J = 8.8 Hz, 30-H 50-H); d 6.87 (1H, d, 3J = 8.8 Hz, 5-H); d
7.16 (1H, s, 7-H); d 7.20 (2H, d, 3J = 8.0 Hz, 200-H 600-H); d 7.75 (2H, d,
3J = 7.6 Hz, 300-H 500-H); d 7.83 (1H, d, 3J = 9.0 Hz, 4-H); d 7.84 (2H, d,
3J = 8.8 Hz, 20-H 60-H). Accurate MS ES1 m/z [M1H]1 455.1067
(calculated for C23H23N2O4S2 455.1094). Mp: 129.5–130.51C.
2-(40-Nitrophenyl)-6-methoxy-1,3-benzothiazole (3)
To a solution of potassium ferricyanide (210.7 g, 0.64 mol) in
400 ml water at 901C was slowly added over a period of 2 h a
solution of 2 (45.70 g, 0.16 mol) in a mixture of 50 ml ethanol
(EtOH) and 300 ml 10% NaOH. After the addition, the obtained
suspension was stirred for 2 h at 901C and then cooled in an ice
bath to 41C. The precipitate was filtered off, washed with water
and dried in a vacuum oven. The dry residue was dispersed in 3 l
of a mixture of CH2Cl2 and EtOH (75:25 V/V). The dispersion was
filtered off and the filtrate was concentrated by vacuum
evaporation. The residue was purified on a silica gel column
with ethyl acetate/hexane 50:50 V/V as the eluent. After
recrystallization from EtOH/ethyl acetate 50:50 V/V, 0.113 mol
(32.35 g, 70.5%) of 3 was obtained as yellow needles.
1H-NMR (DMSO-d6, 200 MHz): d 3.92 (3H, s, OCH3); d 7.16 (1H,
3
3
d, J = 9.4 Hz, 5-H); d 7.38 (1H, s, 7-H); d 8.00 (1H, d, J = 9.0 Hz,
4-H); d 8.21 (2H, d, 3J = 8.4 Hz, 20-H 60-H); d 8.34 (2H, d, 3J = 8.8 Hz,
30-H 50-H). Accurate MS ES1 m/z [M1H]1 287.0488 (calculated
for C14H10N2O3S 287.0485). Mp: 212.5–2141C.
2-(40-aminophenyl)-6-f300-[N-(N0-triphenylmethylmercaptoethyl-N0-
tert-butoxycarbonyl)aminoethyl-N-triphenylmethylmercaptoethyl]-
aminopropoxyg-1,3-benzothiazole (7)
2-(40-Aminophenyl)-6-methoxy-1,3-benzothiazole (4)
To a solution of 6 (114 mg, 0.25 mmol) and N,N-diisopropylethy-
lamine (DIEA, 32 mg, 0.25 mmol) in 1 ml N,N-dimethylformamide
(DMF) was added S,S’-bis-triphenylmethyl-N-(tert-butoxycarbo-
nyl)-1,2-ethylenedicysteamine (S,S’-bis-trityl-N-BOC-BAT, 191 mg,
0.25 mmol) in 0.5 ml DMF.14,15 The mixture was stirred for 24 h at
501C. Purification was done with HPLC using a HS Hyper Prep
A dispersion of 3 (14.32 g, 50 mmol) and SnCl2 ꢀ 2H2O (33.84 g,
150 mmol) in 400 ml EtOH was refluxed under nitrogen for 4 h.
After cooling to room temperature, the EtOH was removed by
vacuum evaporation. The residue was dispersed in 1.5 l ethyl
acetate and the mixture was extracted with 500 ml 2 M NaOH.
J. Label Compd. Radiopharm 2009, 52 227–235
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

K. Serdons et al.
˚
100 A silica 8 mm column (10 mm ꢂ 250 mm, Alltech) and a heated in a boiling water bath for 10 min, cooled to room
mixture of CH2Cl2 and MeOH (99:1 V/V) as eluent at a flow rate of temperature, diluted with 2 ml water and passed over a
3 ml/min. Accurate MS ES1 m/z [M1H]1 1047.4365 (calculated Seppaks Light C18 cartridge (Waters), conditioned with 5 ml
for C65H67N4O3S3 1047.4370).
EtOH and 5 ml H2O. The cartridge was washed with 3 ml H2O
and the labeled compounds were eluted with 1 ml MeCN. This
Synthesis of 2-f20-[300-(N-(N0-triphenylmethylmercaptoethyl- eluate was concentrated by passing a gentle flow of nitrogen
N0-tert-butoxycarbonyl)aminoethyl-N-triphenylmethylmer-
while heating the solution to 401C until approximately 300 ml
solution was left. Before RP-HPLC purification, the mixture was
diluted with 300 ml 0.1% ammonium acetate solution. Purifica-
captoethyl)aminopropoxy]-40-aminophenylg-1,3-benzothia-
zole (10)
tion using RP-HPLC was done on a Hypersil BDS C18 5 mm
column (4.6 mm ꢂ 250 mm, Alltech) with a mixture of MeCN and
0.1% ammonium acetate (50:50 V/V) as the mobile phase at a
2-(20-Hydroxy-40-aminophenyl)-1,3-benzothiazole (8)
A dispersion of 4-aminosalicylic acid (1.534 g, 10 mmol) in 50 g
polyphosphoric acid was heated to 1801C and 2-aminothiophe-
nol (1.252 g, 10 mmol) was added. After stirring for 4 h at 1801C,
the solution was cooled to room temperature and the pH was
adjusted to 5.0 using a 10% Na2CO3 solution. The precipitate
was filtered off, washed with water, dried in a vacuum oven and
then chromatographed on silica gel using CH2Cl2/hexane
(80:20 V/V) as eluent. 1.79 g of 8 was obtained as a brownish
flow rate of 1 ml/min. For mass spectrometry a mixture of
100 ml generator eluate and 100 ml of an 83 mM solution of
NH949TcO4 was used instead of 200 ml generator eluate. Radio-
LC–MS was carried out using an Xterra C18 column (3.5 mm,
50 mm ꢂ 2.1 mm, Waters) with gradient mixtures of
MeCN and 0.05 M ammonium formate as eluent at a flow rate
of 0.3 ml/min.
powder (7.42 mmol, 74.2%).
1H-NMR (DMSO-d6, 200 MHz): d 6.16 (1H, s, 30-H); d 6.24 (1H, d,
Partition coefficient
3J = 8.8 Hz, 50-H); d 7.32 (1H, dd, 3J = 7.2 Hz, 3J = 7.8 Hz, 6-H); d
The lipophilicity of the RP-HPLC-isolated 99mTc-complexes was
3
3
3
7.45 (1H, dd, J = 7.45, J = 7.0 Hz, 5-H); d 7.61 (1H, d, J = 8.4 Hz,
60-H); d 7.87 (1H, d, 3J = 7.8 Hz, 7-H); d 8.01 (1H, d, 3J = 7.6 Hz, 4-H).
Accurate MS ES1 m/z [M1H]1 243.0601 (calculated for
C13H11N2OS 243.0587). Mp: 201–2031C.
determined using a modification of the method described by
Yamauchi et al.16 A 25 ml aliquot of the RP-HPLC isolated 99mTc-
complex was added to a test tube containing 3 ml of 1-octanol
and 3 ml of 0.025 M phosphate buffer pH 7.4. The test tube was
vortexed at room temperature for 2 min and then centrifuged at
2700g for 10 min. A 100 ml aliquot was taken from the 1-octanol
phase and a 900 ml aliquot from the aqueous phase, taking care
to avoid cross contamination between the phases and weighed.
The radioactivity of the aliquots was counted using an
automatic g counter and the partition coefficient P was
calculated using the following equation:
2-[20-(300-p-Tosyloxypropoxy)-40-amino]-1,3-benzothiazole (9)
The same procedure as for the synthesis of 6 was used starting
from 1.21 g (5 mmol) 8. Compound 9 was obtained with a yield
of 64.4%.
1H-NMR (CDCl3, 200 MHz): d 2.17 (3H, s, CH3); d 2.31 (2H, m,
CH2CH2CH2); d 4.01 (2H, t, OCH2CH2CH2); d 4.43 (2H, t,
3
CH2CH2OTs); d 6.16 (1H, s, 30-H); d 6.40 (1H, d, J = 8.0 Hz, 50-H);
cpm=ml octanol
P ¼
3
3
d 6.98 (2H, d, J = 8.2 Hz, 300-H 500-H); d 7.31 (1H, dd, J = 8.4 Hz,
cpm=ml buffer
3J = 6.8 Hz, 6-H); d 7.45 (1H, dd, 3J = 7.0 Hz, 3J = 6.8 Hz, 5-H); d 7.64
3
3
(2H, d, J = 8.4 Hz, 200-H 600-H); d 7.81 (1H, d, J = 7.8 Hz, 60-H); d
7.99 (1H, d, 3J = 7.6 Hz, 7-H); d 8.30 (1H, d, 3J = 8.4 Hz, 4-H).
Accurate MS ES1 m/z [M1H]1 455.1064 (calculated for
C23H23N2O4S2 455.1094). Mp: 130.5–1321C.
with cpm = counts per minute.
Experiments were performed at least in triplicate.
Electrophoresis
A 5 ml aliquot of the RP-HPLC-purified compounds was applied
on a paper strip (3.5 cm ꢂ 13 cm, Whatman 1 chromatography
paper, Whatman, Maidstone, England) wetted with a mixture of
0.025 M phosphate buffer pH 7.4 and methanol (50:50 V/V).
Electrophoresis was performed during 15 min using the
described mixture as the electrolyte solution and an applied
voltage of 300 V. As reference for migration, 5 ml of a 2 mM
solution of thioflavin-T was spotted on the same paper. After
drying, the paper was cut into 0.5 cm strips and the activity on
each strip was counted using an automatic g counter.
2-f20-[300-(N-(N0-triphenylmethylmercaptoethyl-N0-tert-butoxycar-
bonyl)aminoethyl-N-triphenylmethylmercaptoethyl)aminopropoxy]-
40-aminophenylg-1,3-benzothiazole (10)
The same procedure as for the preparation of 7 was used
starting from 228 mg (0.5 mmol) 9. Purification was done with
˚
HPLC using a HS Hyper Prep 100 A silica 8 mm column
(10 mm ꢂ 250 mm, Alltech) and a mixture of CH2Cl2 and MeOH
(99:1 V/V) as eluent at a flow rate of 3 ml/min. Accurate MS ES1
m/z [M1H]1 1047.4314 (calculated for C65H67N4O3S3 1047.4370).
Radiolabeling
Biological evaluation in mice
To a vial containing 200 ml of a 1 mg/ml solution of 7 or 10 in
EtOH was added 50 ml of 0.5 M HCl. The mixture was heated in a
boiling water bath for 20 min. After cooling to room tempera-
ture were added consecutively 50 ml 0.5 M NaOH, 30 ml 0.5 M
phosphate buffer pH 6.0, 20 ml of a 40 mg/ml aqueous solution
of sodium potassium tartrate, 200 ml generator eluate
(370–740 MBq 99mTc) and 5 ml of a freshly prepared 10 mg/ml
solution of SnCl2 ꢀ 2H2O in 0.05 M HCl. The suspension was
The peak containing the desired labeled compound was isolated
using RP-HPLC and collected in a vial containing 250 ml of
a 1 mg/ml solution of ascorbic acid in saline and 250 ml of a
10 mg/ml solution of polysorbate 80 in water. The organic
solvent from the HPLC-eluate was removed at 401C with a
stream of nitrogen for 20 min. The residual aqueous solution
was diluted with saline to a concentration of 370 kBq/ml. Gas
chromatography was used to verify that the concentration of
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 227–235

K. Serdons et al.
acetonitrile was below 0.1%. Male NMRI mice were sedated by with the radioactive label at the 6-position was partially
intraperitoneal injection of 0.75 ml of a 1 to 4 dilution of reoxidized in vivo and partially transformed into more hydro-
Hypnorms (fentanyl citrate 63 mg/ml1fluanisone 2 mg/ml after philic compounds. A similar result can be assumed for the
dilution, Janssen-Cilag, Beerse, Belgium). Each of the eight mice compound conjugated with 99mTc-BAT at the 20-position.
was injected via a tail vein with a 0.1 ml aliquot (37 kBq) of one
of the 99mTc-complexes. The mice were sacrificed by decapita- Acknowledgement
tion at 2 or 60 min post injection (p.i.; four mice at each time
point). Blood was collected in tared tubes and weighed. All
organs and other body parts were dissected and weighed and
their activity was counted using an automatic g counter. Results
were corrected for background activity and are expressed as
percentage of the injected dose (% ID), which is the sum of the
activities in all organs except the activity in the tail, and as
percentage of the injected dose per gram tissue (% ID/g). For
calculation of the total activity in blood, blood mass was
assumed to be 7% of the body mass.17
For biostability studies, a mouse was injected with 1.85 MBq
of 99mTc-10 and sacrificed by decapitation 60 min p.i. Blood was
collected and centrifuged at 2700g for 10 min. The plasma was
transferred to a tube and mixed with an equal volume of MeCN.
The tube was centrifuged again at 2700g for 10 min and the
supernatant was analyzed by RP-HPLC (Hypersil BDS C18 5 mm
column (4.6 mm ꢂ 250 mm) as stationary phase; MeCN/0.1%
ammonium acetate (50:50 V/V) as mobile phase at a flow rate of
1 ml/min). In addition, the urine and bile were analyzed on HPLC
using the same conditions. The HPLC-eluate was collected in
1 ml aliquots and their radioactivity was counted using an
automatic g counter. TLC analysis was done by applying a drop
of plasma, urine or bile on Whatman 1 chromatography paper
and eluting the paper strip in a TLC-tank with either saline or
acetone as elution solvent. After elution, the TLC-strips were cut
2 cm above the application point and the activity of the upper
and lower parts was counted using an automatic g counter.
The authors want to acknowledge the help of Christelle
Terwinghe (Radiopharmacy, UZLeuven, Leuven, Belgium) for
performing the biodistributionstudies and Tyco Healthcare
(Petten, The Netherlands) for providing the IsolinkTM
labeling kits.
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Two 40-aminophenylbenzothiazoles have been derivatized at
the 6 or 20-position with an S,S’-bis-trityl-N-BOC protected BAT
group via
a propoxy linker. After deprotection of the
compounds, they were successfully labeled with 99mTc. Analysis
of the technetium-99 enriched reaction mixture on LC–MS
showed that the main peak in the chromatogram is the
proposed radiolabeled compound. Impurities in the reaction
mixture were 99mTcO4ꢁ, 99mTcO2, 99mTc-tartrate and 99mTc-BAT.
Log P determination and electrophoresis proved that the
compounds are uncharged and have potential to cross the
BBB. Biological evaluation in healthy mice showed however a
low uptake in the brain, which renders these compounds
inappropriate for visualization of Ab plaques in the brain of AD
patients. The reason for the low brain uptake is probably the
high molecular mass, a drawback that will remain a challenge for
the future development of technetium-99m labeled tracers
using the bifunctional ligand approach. In mice, the compound
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