Development of 99mTc-thioflavin-T derivatives for detection of systemic amyloidosis

Article abstract of DOI:10.1002/jlcr.1536

In a search for a ^99mTc-labelled tracer agent for imaging of amyloid plaques in patients with systemic amyloidosis (SA), we have conjugated three bifunctional chelating ligands, namely S-benzyl-mercaptoacetyl-l- aspartyl(tBu)-glycine, HYdrazino

Full text of DOI:10.1002/jlcr.1536

Research Article
Received 20 March 2008,
Revised 21 May 2008,
Accepted 8 July 2008
Published online 27 August 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1536
Development of 99mTc-thioflavin-T derivatives
for detection of systemic amyloidosis
Ã
K. Serdons, T. Verduyckt, J. Cleynhens, G. Bormans, and A. Verbruggen
In a search for a 99mTc-labelled tracer agent for imaging of amyloid plaques in patients with systemic amyloidosis (SA), we
have conjugated three bifunctional chelating ligands, namely S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-glycine, HYdrazino-
NICotinic acid and nitrilotriacetic acid, to the 2-phenylbenzothiazole core of thioflavin-T (ThT), which has known affinity for
amyloid. The compounds were successfully synthesized and labelled with technetium-99m. Their structure was confirmed
by radio-LC-MS analysis. After i.v. injection in normal mice, all three 99mTc-labelled ThT derivatives were excreted almost
exclusively via liver and intestines. This indicates that the new tracer agents have not the required biokinetics for
application as a probe for detection of SA in the abdominal region.
Keywords: amyloid; thioflavin-T; technetium-99m; SPECT; 2-phenylbenzothiazoles
In view of the nearly optimal physical and imaging
characteristics of technetium-99m, the aim of this study was
Introduction
the development of a 99mTc-labelled tracer agent for visualiza-
Amyloidosis is an acquired or hereditary disorder of protein
folding, in which whole or fragments of normally soluble
proteins are deposited extracellularly as abnormal, insoluble
fibrils that accumulate, disrupt the structure and function of
organs and tissues and cause disease.1 In systemic amyloidosis
(SA) the deposits may be present in the parenchyma of the
viscera and of all tissues except the brain as well as in the walls
of blood vessels throughout the body. The depositions consist
of proteins with a typical b-sheet form called amyloid b (Ab).2,3
The most accepted theory concerning the aetiology of the
disease is the amyloid cascade theory. SA is usually fatal,
although the prognosis has been improved by haemodialysis,
kidney, liver and heart transplantation and the increasing
capacity to effectively treat the acquired primary diseases that
are complicated by amyloid deposition. It is, however, essential
that the diagnosis is made as early as possible, if these benefits
are to be recognized.4
Clinical diagnosis of SA is now done by immunohistochemical
staining of tissue sections obtained by invasive biopsy, surgical
resection or autopsy. Congo red still remains the gold standard
in staining Ab.5 Other histological techniques using antibodies,
immunoelectron microscopy and Western blotting give good
results.6 However, histology cannot provide information about
the overall whole body load or distribution of amyloid deposits,
nor does it permit in vivo monitoring of the natural history of
amyloidosis or its response to treatment. Therefore, 123I-labelled
serum amyloid P component has been used to rapidly and
specifically localize amyloid deposits in vivo allowing diagnosis
and quantification of deposits by scintigraphy.7 This technique
tion of SA, based on the structure of thioflavin-T (ThT, Figure
1(A)). ThT has a high affinity for the b-pleated sheet structure of
Ab8 and is a rather polar compound due to the presence of a
quaternary amine. Therefore, radiolabelled derivatives of ThT
could be useful for non-invasive in vivo detection of amyloid
plaques in the human body. Such a radiolabelled compound
should have a relatively low molecular mass and should be
hydrophilic, as the excretion from the body should be fast and
mainly through the kidneys to avoid pronounced uptake in the
abdominal region.
In this study, three bifunctional chelating ligands (BCLs) were
coupled to the 2-phenylbenzothiazole core of ThT.9 In a first
approach, S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-glycine (Fig-
ure 2(A)) was coupled to 2-(40-aminophenyl)-1,3-benzothiazole
(Figure 1(B)) in order to obtain a negatively charged 99mTc
complex.10 It was hypothesized that such a negatively charged
99mTc-labelled ThT derivative could be useful for detection of SA
in view of its polar characteristics, which would favour renal
excretion and decrease uptake in the hepatobiliary system. In a
second approach, the phenylbenzothiazole core was conjugated
to HYdrazinoNICotinic acid (HYNIC) (Figure 2(B)). The HYNIC
chelating group was chosen in view of the favourable behaviour
of 99mTc-HYNIC-octreotide, i.e. high and rapid excretion through
the kidneys and low hepatic uptake.11 In a third approach the
technetium-tricarbonyl labelling method was used to also
Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences,
has 90–100% diagnostic sensitivity and is the only in vivo K.U.Leuven, Herestraat 49 Bus 821, 3000 Leuven, Belgium
method available nowadays. However, this tracer agent is not
*Correspondence to: Kim Serdons, Laboratory for Radiopharmacy, Faculty of
Pharmaceutical Sciences, K.U.Leuven, Herestraat 49 Bus 821, 3000 Leuven,
Belgium.
E-mail: Kim.serdons@pharm.kuleuven.be
available as a licenced radiopharmaceutical, is only in use in a
very few centres and has the disadvantage of its proteinacous
origin with all associated risks.
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K. Serdons et al.
obtain a negatively charged 99mTc-labelled ThT derivative.12 In confirmation with radio-LC-MS and a preliminary biological
this case a conjugate of nitrilotriacetic acid (Figure 2(C)) and 2- evaluation of the new 99mTc-labelled ThT derivatives.
(40-aminophenyl)-1,3-benzothiazole was made and used as the
Tc-binding ligand.
Results and discussion
This paper describes the synthesis of three conjugates of the
2-phenylbenzothiazole core of ThT, deprotection of the
technetium-binding groups, labelling and purification, structure
Synthesis, labelling and structure confirmation
As ThT does not contain sufficient metal-binding donor atoms in
its structure to bind 99mTc, we have attached three BCLs to the
2-phenylbenzothiazole core of ThT.
For preparing 99mTc-2, 2-(40-aminophenyl)-1,3-benzothiazole
was coupled to S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-glycine
to obtain a precursor of a negatively charged 99mTc(V)O complex
(Scheme 1). The BCL was synthesized according to published
data10 and contains an S-benzyl-protected thiol and a tert-butyl-
protected carboxylate in the aspartyl moiety.
+
N
S
N
S
N
NH2
B
A
Figure 1. Structure of (A) thioflavin-T (ThT) and (B) 2-(40-aminophenyl)-1,3-
benzothiazole.
O
O
O
HOOC
COOH
COOH
O
NH
S
HN
HOOC
NH NH2
*
N
*
N
HO
O
B
C
*
A
Figure 2. Bifunctional chelating agents: (A) S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-glycine; (B) HYDrazinoNICotinic acid (HYNIC); and (C) nitrilotriacetic acid. Ã indicates
the site of coupling with the phenylbenzothiazole core of ThT.
NH2
SH
N
S
PPA
NH2
HOOC
NH2
+
180 C, 4 h
1
O
O
O
EDC.HCl
HOBt
CH3CN
NH
NH
O
RT, overnight
O
OH
S
-1
O
O
O
O
COOH
O
NH
NH
NH
S
O
N
N
O
Tc
0.5 M phoshate buffer pH 9
NaKtartrate
O
O
N
S
SnCl2.2H2O
99mTcO4
-
99mTc-2
100 C, 15 min
N
S
N
S
2
Scheme 1. Synthesis and labelling with 99mTc of 2-[40-(S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-glycinamido)phenyl]-1,3-benzothiazole (2).
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Copyright r 2008 John Wiley & Sons, Ltd.
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K. Serdons et al.
Removal of the tert-butyl ester of 2 in a mixture of CH2Cl2/
trifluoroacetic acid (TFA) (5:1 v/v) at room temperature (RT), as
described in literature,10 provided only low yields of the
derivative of 2 with a deprotected carboxylate group. However,
we found that it was not necessary to remove the tert-butyl
protective group prior to the radiolabelling reaction, as the
combination of pH 9 and a heating step of 15 min during the
labelling reaction appeared sufficient for efficient formation of
the 99mTc-2 complex with a deprotected carboxylate. The
labelling yield was 80% (Figure 3(A)).
(A)
The use of liquid chromatography with UV and radiometric
detection in combination with mass spectrometry allows one to
determine which molecular ion mass corresponds to a peak
observed in the radiometric channel. Small amounts of carrier
99Tc (about 1.5 mg in the form of NH499TcO4) were added to the
preparation to be able to determine the mass of the selected
peaks.13–16
(B)
Figure 4(A) shows the single ion mass chromatogram of the
reaction product after labelling of 2 over the mass range
583.406–583.474 Da and Figure 4(B) the radiometric signal of the
radio-LC-MS analysis. The peak at 11.38 min is supposed to be
the intended Tc(V)-oxo complex with deprotected 2 (99mTc-2,
Scheme 1). The relatively large peak at 0.87 min, corresponding
to the void volume of the column, is supposed to be 99mTcO4ꢀ as
a radiochemical impurity because the yield of the carrier-added
labelling reaction was lower than that of the no-carrier-added
procedure. In Figure 4(D), the background subtracted mass
spectrum over the peak with retention time (Rt) 11.38 min
shows a molecular mass of 583 Da, which is in perfect
agreement with the theoretical molecular ion mass for the
supposed structure of 99mTc-2 (Figure 4(C)). This mass is in
accordance with a negatively charged complex in which a
[Tc(V)O]31 core is bound to the deprotonated thiol sulphur
atom and three deprotonated amide nitrogen atoms of the
(C)
Figure 3. RP-HPLC chromatograms of the labelling reaction mixtures of: (A) 2-[40-
(S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-glycinamido)phenyl]-1,3-benzothiazole
(2) with technetium-99m at pH 9; (B) 2-[20-(N-BOC-6-hydrazinonicotinamido-3-
propoxy)-40-amino]phenyl-1,3-benzothiazole (7) with technetium-99m in the
presence of tricine as co-ligand; and (C) 2-[40-(N,N-diacetic acid)-N-acetamidophe-
nyl]-1,3-benzothiazole (8) with [
99mTc(CO)3(OH2)3]1 solution at pH 7. HPLC
conditions: XTerraTM RP C18 column (5 mm, 250 mm ꢁ 4.6 mm) eluted with
gradient mixtures of CH3CN and 0.05 M ammonium acetate (for 2 and 8: 0 min:
0:100 v/v, 20 min: 90:10 v/v, 25 min: 90:10 v/v, 30 min: 0:100 v/v; for 7: 0 min:
0:100 v/v, 15 min: 90:10 v/v, 20 min: 90:10 v/v, 25 min: 0:100 v/v) at a flow rate of
1 ml/min.
(A)
(B)
(C)
(D)
Figure 4. (A) Single ion mass chromatogram (ESꢀ, 583.406–583.474 Da); (B) radiometric chromatogram obtained by LC-MS analysis of 99mTc-2-[40-(S-benzyl-
mercaptoacetyl-l-aspartyl(tBu)-glycinamido)phenyl]-1,3-benzothiazole (99mTc-2); (C) theoretical molecular ion mass of 99mTc-2; and (D) background subtracted mass
spectrum of the peak at 11.38 min. HPLC conditions: XTerraTM MS C18 column (3.5 mm, 50 mm ꢁ 2.1 mm) eluted with gradient mixtures of CH3CN and 0.05 M ammonium
acetate (0 min: 0:100 v/v, 20 min: 90:10 v/v, 25 min: 90:10 v/v, 30 min: 0:100 v/v) at a flow rate of 0.3 ml/min.
J. Label Compd. Radiopharm 2008, 51 357–367
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K. Serdons et al.
mercaptotriamide tetraligand. The presence of a non-coordi- and two equivalents of the HYNIC ligand and yielded the desired
nated carboxylic group of the aspartyl moiety, which is almost conjugate 7. Although 5 also bears an aromatic amine, coupling
fully ionized at pH 7, renders an overall charge of ꢀ2 to 99mTc-2 of the HYNIC precursor with this aromatic amine was not
at physiological pH.
observed. This can be explained in view of the much lower
For preparing 99mTc-7, 2-(20-hydroxy-40-aminophenyl)-1,3- nucleophilic character of the aromatic amine as compared with
benzothiazole was coupled to BOC-protected HYNIC (Scheme the aliphatic amine.
2) via a 3-aminopropyl spacer.
Deprotection of the N-BOC-protected HYNIC moiety in 7 was
99mTc-labelled HYNIC conjugates are known to possess polar performed in an acidic dioxane solution following a described
properties, especially in the presence of a polar co-ligand17 such procedure,19 which yielded the HCl salt of deprotected 7.
as tricine. The spacer precursor N-BOC-3-amino-1-propyl-p- Labelling of HYNIC derivatized compounds with 99mTc requires
tosylate (3) and the HYNIC precursor (succinimidyl-6-N-BOC- the presence of a co-ligand with metal-binding donor atoms, as
hydrazinopyridine-3-carboxylic acid, 6) were synthesized accord- the HYNIC moiety as such does not provide a sufficient number
ing to published data.17,18 To allow conjugation of the HYNIC of donor atoms for the formation of a stable complex with
precursor 6 with 5, the N-BOC protective group on the amine of 99mTc.20 In this study, tricine, ethylene diamine-N,N0-diacetic acid
the spacer of 5 was removed. Initially, this was attempted with a (EDDA) and a mixture of tricine and nicotinic acid have been
mixture of TFA, triisopropylsilane and a few drops of water, but used as different co-ligands. With tricine as the co-ligand, the
analysis of the reaction product by reversed phase high- labelling reaction yielded 70% of 99mTc-tricine-7 (Figure 3(B)).
performance liquid chromatography (RP-HPLC) revealed that Yield of the labelling reaction in the presence of a mixture of
compound 5 was not stable in these conditions. Removal of the tricine and nicotinic acid was 77% and in the presence of EDDA
N-BOC group in the absence of water was more successful and it was 52%. The RP-HPLC chromatograms show that 99mTc-
resulted in a complete deprotection of the amine group of the EDDA-7 (Rt of approximately 15 min) is more lipophilic than
spacer. Deprotected 5 was coupled to the succinimidyl activated 99mTc-tricine/nicotinic acid-7 and 99mTc-tricine-7 (both have an
HYNIC precursor in dioxane with diethylamine (DIEA) as base Rt of approximately 13 min). As the goal was to obtain a more
NH2
HOOC
HO
NH2
+
SH
PPA
110 C, 1 h
N
S
N
S
OTos
H2N
+
H2N
NH BOC
CH3CN/CH3OH (9:1)
NaOCH3
O
OH
5
50 C, overnight
3
4
NH BOC
TFA
TRIS
RT, 15 min
N
S
H2N
N
S
O
H2N
O
NH2
Dioxane
DIEA
NH BOC
NH
O
NH
RT, overnight
N
O
NH BOC
N
O
O
7
NH
N
O
6
Scheme 2. Synthesis of 2-[20-(N-BOC-6-hydrazinonicotinamido-3-propoxy)-40-aminophenyl]-1,3-benzothiazole (7).
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K. Serdons et al.
(A)
(B)
(C)
Figure 5. (A) Single ion mass chromatogram (ES1, 749.084–749.312 Da); (B) radiometric chromatogram obtained by LC-MS analysis of 99mTc-tricine-2-[20-(N-BOC-6-
hydrazinonicotinamido-3-propoxy)-40-amino]phenyl-1,3-benzothiazole (99mTc-tricine-7); and (C) background subtracted mass spectrum of the peak at 10.63 min. HPLC
conditions: XTerraTM MS C18 column (3.5 mm, 50 mm ꢁ 2.1 mm) isocratic eluted with a mixture of CH3CN and 0.05 M ammonium acetate (25:75 v/v) at a flow rate of 0.3 ml/
min.
hydrophilic complex, we did not focus further on the technetium- each separated by two or three atoms. Such donor atoms can
99m complex using EDDA as co-ligand. Although the labelling easily replace the three loosely bound water molecules of the
yield using tricine/nicotinic acid as co-ligand was somewhat
[
99mTc(CO)3(OH2)3]1 precursor, resulting in the formation of two
higher than with tricine alone (77 versus 70%), the labelling 5- (or 6-) rings. The iminodiacetic acid (IDA) chelating group in
procedure is more complex and requires a boiling step. Therefore, compound 8 contains two carboxylate oxygen atoms and one
we only used 99mTc-tricine-7 for further evaluation.
amine nitrogen atom, which function as donor atoms. Their
Figure 5(B) shows the radiometric signal of the radio-LC-MS binding results in a negatively charged 99mTc-tricarbonyl-IDA
analysis of the reaction mixture after labelling of deprotected 7 complex. Labelling of 8 with a 99mTc-tricarbonyl core starting
with 99mTc with tricine as the co-ligand. The peak at 10.63 min is from
a
IsolinkTM (Mallinckrodt Medical B.V., Pettten, The
supposed to be the intended radiolabelled compound 99mTc- Netherlands) kit or using the CO-bubbling method24 yielded in
tricine-7. In Figure 5(C), the background subtracted mass both cases a Tc complex with identical Rt on RP-HPLC. Because
spectrum of the peak eluting at 10.63 min is depicted and the of the alkaline pH of the [99mTc(CO)3(OH2)3]1 solution, it was
mass obtained in this spectrum is 749 Da. Figure 5(A) depicts the necessary to adjust its pH to 7 with 1 M HCl before precursor 8
single ion mass over the mass range 749.084–749.312 Da. There was added to the radioactive solution because of degradation of
has been a lot of speculation and different hypotheses have 8 at higher pH values. The yield of the labelling reaction at pH 7
been proposed about the structure of Tc-HYNIC-co-ligand was 64% (Figure 3(C)).
complexes without a clear conclusion.21 Recent literature
Figure 6(B) shows the radiometric signal of the radio-LC-MS
reports suggest that with tricine as co-ligand, Tc-HYNIC/tricine analysis of the reaction mixture after labelling of 8 with
complexes can contain one or two tricine molecules, depending 99mTc(CO)3. The peak at 12.01 min is supposed to be the
on the presence or not of additional donor atoms at a suitable intended radiolabelled compound 99mTc-8. In Figure 6(C), the
position in the conjugate.22 The radio-LC-MS result of 99mTc- background subtracted mass spectrum shows a mass of 579 Da
tricine-7 (molecular mass of 749 Da) is not in agreement with a for this compound, which corresponds to the theoretical
structure containing two tricine molecules, which would result molecular ion mass for the supposed structure of 99mTc-8
in a mass of 884 Da. The mass found for 99mTc-tricine-7 may (Figure 6(D)). Figure 6(A) depicts the single ion mass chromato-
correspond with the disodium salt of a complex in which a Tc- gram over the mass range 579.523–580.051 Da. These data
ion is bound to one molecule of 7 and one molecule of tricine strongly support the assumption that the peak eluting at
with the loss of seven protons.
12.01 min in the radiometric channel corresponds to a structure
The synthesis of 2-[40-(N,N-diacetic acid)-N-acetamidophenyl]- as shown in Scheme 3. In such a Tc(CO)3 complex, Tc has a
1,3-benzothiazole (8) was carried out starting from nitrilotriace- valence of 11, whereas two protons of the IDA moiety are lost,
tic acid anhydride, which was formed in situ by mixing acetic resulting in an overall anionic complex (charge ꢀ1).
acid anhydride and nitrilotriacetic acid in pyridine23 (Scheme 3).
For 99mTc-labelling of 8 we used the tricarbonyl method in
which [99mTc(CO)3(OH2)3]1 is reacted with a ligand that has in its
Partition coefficients
Depending on the functional groups of the BCL and the
oxidation state of technetium in the Tc complex, the new 99mTc-
structure preferentially three suitable metal-binding donor
atoms (such as an amine nitrogen or a carboxylate oxygen)
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K. Serdons et al.
O
HOOC
N
S
O
H2N
+
O
1
pyridine
100 C, 1 h
O
HOOC
HOOC
N
S
N
HN
8
[
99mTc(CO)3(OH2)3]+
pH 7
70 C, 20 min
-1
O
OC
O
O
OC
Tc
O
N
N
HN
OC
S
O
99mTc-8
Scheme 3. Synthesis and labelling with a 99mTc-tricarbonyl core of 2-[40-(N,N-diaceticacid)-N-acetamidophenyl]-1,3-benzothiazole (8).
(A)
(B)
(C)
(D)
Figure 6. (A) Single ion mass chromatogram (ESꢀ, 579.523–580.051 Da); (B) radiometric chromatogram obtained by LC-MS analysis of 99mTc-2-[40-(N,N-diacetic acid)-N-
acetamidophenyl]-1,3-benzothiazole (99mTc-8); (C) background subtracted mass spectrum of the peak at 12.01 min; and (D) theoretical molecular ion mass of 99mTc-8.
HPLC conditions: XTerraTM MS C18 column (3.5 mm, 50 mm ꢁ 2.1 mm) eluted with gradient mixtures of CH3CN and 0.05 M ammonium acetate (0 min: 0:100 v/v, 20 min:
90:10 v/v, 25 min: 90:10 v/v, 30 min: 0:100 v/v) at a flow rate of 0.3 ml/min.
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K. Serdons et al.
labelled compound is negatively charged or neutral. The new for the 99mTc-labelled phenylbenzothiazoles of this study. The
tracer agents developed for this study should preferentially be non-expected biodistribution results might be due to a rapid
hydrophilic as they have been designed for detection of SA, for metabolism of the compounds.
which renal clearance is an important requirement. The log P
values were found to be ꢀ0.4970.02 for 99mTc-2, which is rather
Experimental section
hydrophilic, 0.8170.01 for 99mTc-tricine-7 and 1.170.02 for
99mTc-8. Only 99mTc-2 has a value in accordance with a relatively
Materials and methods
hydrophilic compound, whereas the log P values of 99mTc-
All reagents and solvents used in synthesis were obtained
from Acros Organics (Geel, Belgium), Aldrich, Fluka or Sigma
(Sigma-Aldrich, Bornem, Belgium) and were used without
further purification. Purification of reaction mixtures was
done by column chromatography using silica gel with a particle
tricine-7 and 99mTc-8 indicate that these two radiolabelled
compounds are rather hydrophobic.
Biodistribution studies
The results of the biodistribution studies of the technetium-
99m-labelled phenylbenzothiazoles 2, 7 and 8 in normal mice at
2 and 60 min p.i. are summarized in Tables 1–3. Although the
nature of the technetium-binding ligand and of the final 99mTc
complexes in these 99mTc-labelled phenylbenzothiazoles is quite
different, their biological behaviour is rather similar.
Surprisingly, none of the compounds is excreted efficiently
through the renal system. The kidney value at 2 min p.i. varies
from 4 to 9% ID. These low values are solely the result of a low
extraction efficiency and not of a rapid transit into the urine as
the urinary activity at 60 min p.i. is almost negligible. The
assumption that the presence of negative charges on the
complex at physiological pH and thus of more polar character-
istics would promote extraction and excretion by the kidneys
was found invalid.
size varying between 0.04 and 0.063 mm (230–400 Mesh)
(MN Kieselgel 60 M, Macherey-Nagel, Du¨ren, Germany) as the
stationary phase. The structure of the synthesized products
was confirmed with 1H-nuclear magnetic resonance (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 tetramethylsilane (d = 0). Coupl-
ing constants are reported in Hertz. Splitting patterns are
defined by s (singlet), d (doublet), dd (double doublet),
t
(triplet) and m (multiplet). Melting points (Mps) were
determined with an IA9100 digital Mp apparatus (Electrother-
mal, Essex, UK) in open capillaries and are reported
uncorrected.
Generator eluate containing 99mTc in the form of pertechne-
tate was obtained from an Ultratechnekow generator (Tyco
Healthcare, Petten, The Netherlands). NH949TcO4 and IsolinkTM
labelling kits were a gift from Tyco Healthcare.
Quantitative determination of radioactivity in samples was done
using an automatic g-counter coupled to a multi-channel analyser
(Wallac 1480 Wizards 300, Wallac, Turku, Finland). The values were
corrected for physical decay and background radiation.
RP-HPLC runs were performed with a system consisting of a
TSP SpectraSeries P4000 quaternary pump (Thermo Separation
Products, San Jose, CA, USA) connected with a TSP SpectraSeries
UV 100 detector (Thermo Separation Products) and a 3-in NaI(Tl)
crystal connected with a Medi-Lab Select SC7II single channel
analyzer (Medi-Lab Select, Mechelen, Belgium). The output
Moreover, the three 99mTc-labelled phenylbenzothiazoles are
efficiently taken up by the liver and excreted into the intestines.
Liver uptake (in % ID) at 2 min p.i. varies from 48% (99mTc-tricine-
7) to 77% (99mTc-8) and decreases at 60 min p.i. to values
between 20% (99mTc-2) and 43% (99mTc-8). The activity in the
intestines at 60 min p.i. varies between 39% (99mTc-tricine-7) and
73%
(
99mTc-2). The most polar complex, i.e. 99mTc-2
(log P = ꢀ0.49), is concentrated most efficiently and rapidly in
the hepatobiliary system (activity in liver1intestines ranging
from 75% at 2 min p.i. to 93% at 60 min p.i.). The clearance from
blood and liver is efficient and rapid for 99mTc-2 and 99mTc-8, but
more than three times faster for 99mTc-2 than for 99mTc-8. The
HYNIC derivative 7, labelled with 99mTc in the presence of
tricine, shows a slower clearance. The general assumption that
polar compounds are extracted from the blood mainly by the
kidneys and lipophilic compounds by the liver is clearly not valid
signal was recorded and analyzed using
acquisition system (Lablogic, Sheffield, UK).
a RaChel data
Radio-HPLC combined with mass spectrometry (radio-LC-MS)
was performed on a system consisting of a Waters Alliance 2690
Table 1. Tissue distribution of 99mTc-2-[40-(S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-glycinamido)phenyl]-1,3-benzothiazole
(
99mTc-2) after i.v. injection in normal mice at 2 and 60 min p.i. (n = 4 at each time point)
% ID7s.d.
% ID/g tissue7s.d.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
Urine
Kidneys
Liver
Intestines
Spleen1pancreas
Lungs
0.170.0
7.670.1
71.771.1
3.371.2
0.470.1
0.670.3
0.370.1
0.470.1
0.0970.03
0.0770.02
8.671.8
0.870.3
2.370.5
20.4711.5
72.7712.6
0.170.0
0.170.1
0.170.0
0.570.4
11.070.5
31.473.2
3.370.6
8.174.4
1.170.2
2.170.6
1.870.6
0.470.1
0.2870.08
0.6370.19
3.170.7
0.270.1
0.470.3
0.470.1
0.670.5
0.2070.10
0.5070.14
0.370.2
Heart
Stomach
Cerebrum
Cerebellum
Blood
0.0670.02
0.0570.02
0.970.4
J. Label Compd. Radiopharm 2008, 51 357–367
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K. Serdons et al.
Table 2. Tissue distribution of 99mTc-tricine-2-[20-(N-BOC-6-hydrazinonicotinamido-3-propoxy)-40-amino]phenyl-1,3-benzothia-
zole (99mTc-tricine-7) after i.v. injection in normal mice (n = 4) at 2 and 60 min p.i. (n = 4 at each time point)
% ID7s.d.
% ID/g tissue7s.d.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
Urine
Kidneys
Liver
Intestines
Spleen1pancreas
Lungs
0.470.3
3.570.3
48.071.8
5.070.4
0.770.1
1.370.3
0.470.1
0.670.2
0.1270.03
0.0770.02
26.171.4
2.070.1
2.670.3
32.673.6
39.473.1
0.370.1
0.970.2
0.270.0
1.070.5
5.470.4
19.372.7
3.970.2
13.372.0
1.670.2
5.170.6
2.570.2
0.970.2
0.3870.07
0.5970.19
9.370.5
0.970.2
3.270.5
1.470.2
1.671.1
0.1770.05
0.2370.06
5.670.6
Heart
Stomach
Cerebrum
Cerebellum
Blood
0.0570.01
0.0370.01
14.970.9
Table 3. Tissue distribution of 99mTc-2-[40-(N,N-diacetic acid)-N-acetamidophenyl]-1,3-benzothiazole (99mTc-8) after i.v.
injection in normal mice (n = 4) at 2 and 60 min p.i. (n = 4 at each time point)
% ID7s.d.
% ID/g tissue7s.d.
2 min p.i.
60 min p.i.
2 min p.i.
60 min p.i.
Urine
Kidneys
Liver
Intestines
Spleen1pancreas
Lungs
0.170.1
8.871.0
77.071.0
2.671.3
0.270.1
0.470.1
0.170.0
0.270.1
0.0270.00
0.0270.01
4.970.9
0.370.1
3.370.2
43.373.0
46.374.9
0.170.1
0.270.0
0.170.0
3.073.8
0.0170.00
0.0170.00
1.670.1
13.070.2
36.571.6
5.170.4
23.471.6
0.770.3
1.770.4
0.770.2
0.470.3
0.0770.02
0.1270.05
1.770.4
0.370.1
0.670.1
0.470.2
5.075.4
0.0370.01
0.0870.06
0.670.0
Heart
Stomach
Cerebrum
Cerebellum
Blood
separation module (Waters, Milford, MA, USA) coupled to a and dried in a vacuum oven. Crystallization from methanol
reverse phase XTerraTM MS C18 3.5 mm column (2.1mmꢁ 50mm) yielded 7.24 g (32 mmol, 64%) of 1 as light yellow crystals.
(Waters). The column eluate was first analyzed by a UV
1H-NMR (DMSO, 200 MHz): d 5.8 (2H, s, NH2); d 6.68 (2H, d, 30-H
spectrometer (Waters 2487 dual wavelength absorbance detec- 50-H); d 7.32 (1H, t, 6-H); d 7.45 (1H, t, 5-H); d 7.76 (2H, d, 20-H 60-
tor), subsequently by radiometric detection consisting of a 3-in H); d 7.89 (1H, d, 4-H); d 7.98 (1H, d, 7-H). Mass: [MꢀH]ꢀ 225
NaI(Tl) crystal coupled to a single channel analyzer (The Nucleus, (calculated: 225). Mp: 147.5–1501C.
Oak Ridge, TE, USA) and finally by a time-of-flight mass
spectrometer (LCT, Micromass, Manchester, UK) equipped with 2-[40-(S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-glycinamido)phe-
nyl]-1,3-benzothiazole (2)
an electrospray ionization (ESI) source. Acquisition and processing
of data were performed with Masslynx software (version 3.5).
Animal studies were performed according to the Belgian code
of practice for the care and use of animals, after approval from
the university ethics committee for animals.
To a solution of S-benzyl mercaptoacetyl-l-aspartyl(tBu)-glycine
(410 mg, 1 mmol) in 10 ml of CH3CN, 1 (226 mg, 1 mmol) was
added together with 1-hydroxybenzotriazole hydrate (HoBt)
(141 mg, 1 mmol) and N-(3-dimethylaminopropyl)-N0-ethylcarbo-
diimide hydrochloride (EDC ꢂ HCl) (192 mg, 1 mmol). The reaction
mixture was stirred at RT overnight and the precipitate was
filtered off and washed with CH3CN. The residual yellow oil was
dissolved in CH2Cl2 and the solution was washed with 10%
NaHCO3 and brine, dried over MgSO4, filtered and evaporated to
dryness. The residue was purified with silica column chromato-
graphy using gradient mixtures of CH2Cl2 and CH3OH (up to 5%)
yielding 80 mg of 2 (0.13 mmol, 13%).
Synthesis of 2-[40-(S-benzyl-mercaptoacetyl-l-aspartyl(tBu)-
glycinamido)phenyl]-1,3-benzothiazole (2)
2-(40-Aminophenyl)-1,3-benzothiazole (1). Method A
2-Aminothiophenol (6.26 g, 50 mmol) and p-aminobenzoic acid
(6.86 g, 50 mmol) were added to 80 g of polyphosphoric acid
and the mixture was stirred at 1801C for 4 h. The reaction
mixture was allowed to cool down to RT and poured into a 10%
(m/V) solution of Na2CO3. The white precipitate was filtered off
1H-NMR (CDCl3): d 1.46 (9H, s, tBu); d 2.69 and 2.88 (2H, 2 ꢁ
dd, CH–COOtBu); d 3.20 (2H, s, CO–CH2–SBz); d 3.76 (2H, s,
S–CH2–Bz); d 4.01 and 4.18 (2H, 2 ꢁ dd, CO–CH2–NH); d 4.65 (1H,
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J. Label Compd. Radiopharm 2008, 51 357–367

K. Serdons et al.
m, CO–CH); d 7.04 (1H, t, CH2–NH–CO); d 7.28 (5H, s, C6H5–CH2); 6-HBe); d 7.87 (2H, d, 4-HBe 7-HBe); d 7.95 (1H, dd, 4-HYNIC); d
d 7.36 (1H, t, 5-H); d 7.48 (1H, t, 6-H); d 7.57 (1H, d, CH–NH–CO); d 8.09 (1H, dd, 6-HPh); d 8.55 (1H, d, 2-HYNIC). Mass: [M1H]1 535
7.80 (2H, d, 20-H 60-H); d 7.87 (1H, d, 7-H); d 8.01 (2H, d, 30-H 50-H); (calculated: 535). Mp: 147.4–149.51C.
d 8.04 (1H, d, 4-H); d 8.72 (1H, s, F-NH–CO). Mass: [M1H]1 619
(calculated: 619). Mp: 141.1–143.61C.
Synthesis of 2-[40-(N,N-diacetic acid)-N-acetamidophenyl]-
1,3-benzothiazole (8)
Synthesis of 2-[20-(N-BOC-6-hydrazinonicotinamido-3-pro-
poxy)-40-amino]phenyl-1,3-benzothiazole (7)
A solution of nitrilotriacetic acid (129 mg, 0.67 mmol) in 1.75 ml
pyridine was heated at 501C for 10 min. Acetic acid anhydride
(0.12 ml, 1.2 mmol) was added and the mixture was heated
2-(20-Hydroxy-40-aminophenyl)-1,3-benzothiazole (4)
further at 1001C for 30 min. The mixture was allowed to cool to
501C and 1 (226 mg, 1 mmol) was added. The mixture was
Compound 4 was synthesized following method A starting from
heated again at 1001C for 1 h and, after cooling, pyridine was
4-aminosalicylic acid (7.70 g, 50 mmol) and 2-aminothiophenol
evaporated under reduced pressure. The pH was adjusted to 9.5
(6.26 g, 50 mmol) and yielding 4 g (17 mmol, 33%) of 4 as a
by the addition of 2 M NH4OH. The mixture was extracted three
slightly yellow solid.
times with diethylether and the organic layer was discarded.
1H-NMR (DMSO): d 5.97 (2H, s, NH2); d 6.18 (1H, d, 30-H); d 6.26
After acidification of the water layer to pH 2 with 6 M HCl, the
(1H, dd, 50-H); d 7.34 (1H, t, 5-H); d 7.45 (1H, t, 6-H); d 7.63 (1H, d,
precipitate was filtered off and crystallized from ethanol/water
60-H); d 7.89 (1H, dd, 7-H); d 8.03 (1H, dd, 4-H); d 11.76 (1H, s, OH).
(50:50 v/v) to yield 50 mg of 8 (0.13 mmol, 19.4%).
Mass: [MꢀH]ꢀ 241 (calculated: 241). Mp: 186.9–1871C.
1H-NMR (DMSO): d 3.51 and 3.55 (6H, 3 ꢁ s, 3 ꢁ CH2); d 7.42
(1H, t, 5-H); d 7.53 (1H, t, 6-H); d 7.69 (1H, s, NHCO); d 7.86 (2H, d,
2-[20-(N-BOC-3-aminopropoxy)-40-aminophenyl]-1,3-benzothiazole
30-H 50-H); d 8.02 (2H, d, 20-H 60-H); d 8.10 (2H, d, 4-H 7-H); d 10.92
(5)
(1H, s, COOH). Mass: [MꢀH]ꢀ 398 (calculated: 398). Mp:
Compound 4 (4 g, 17 mmol) was dissolved in 50 ml of CH3CN/ 183.9–1851C.
CH3OH (9:1 v/v) and 50 mmol of freshly prepared NaOCH3 was
added. To this solution, N-BOC-3-amino-1-propyl-p-tosylate
(3)13 (11.86 g, 36 mmol) dissolved in 50 ml CH3CN/CH3OH
(9:1 v/v) was added dropwise and the mixture was stirred
Deprotection and labelling of 2
overnight at 501C. After cooling down to RT, water was added
and the mixture was extracted three times with CH2Cl2. The
residue was purified with silica column chromatography using
gradient mixtures of CH2Cl2 and CH3OH (up to 2%) to yield
3.32 g of 5 (8.3 mmol, 49% yield).
1H-NMR (CD3OD): d 1.38 (9H, s, tBu); d 2.17 (2H, m,
In a labelling vial were successively mixed 100 ml of a 1 mg/ml
solution of 2 in CH3CN, 500 ml of 0.5 M phosphate buffer pH 9,
250 ml of a 40 mg/ml solution of NaKtartrate in water, 25 ml of a
4 mg/ml solution of SnCl2 ꢂ 2H2O in 0.05 M HCl and about
600 MBq 99mTcOꢀ4 in 1 ml saline. The mixture was heated in a
boiling water bath for 15 min and RP-HPLC was performed after
cooling to RT. The labelling yield was 80%.
CH2–CH2–CH2); d 3.40 (2H, m, CH2–NH); d 4.20 (2H, m, O–CH2);
d 5.92 (2H, s, NH2); d 6.33 (1H, d, 50-H); d 6.35 (1H, d, 30-H); d 7.00
(1H, s, NH–CO); d 7.30 (1H, t, 5-H); d 7.44 (1H, t, 6-H); d 7.88 (1H,
d, 7-H); d 7.98 (1H, d, 4-H); d 8.13 (1H, d, 60-H). Mass: [M1H]1 400
Two-step procedure for deprotection and labelling of 7
(calculated: 400). Mp: 170.2–171.61C.
For deprotection purpose, a solution of 6 mg of 7 in a mixture of
2 ml of dioxane and 2 ml of HCl/dioxane (prepared by bubbling HCl
gas at a moderate rate through dioxane for about 10 min) was
stirred at RT for 4 h after which the precipitate was filtered off and
washed several times with ether. The precipitate (the HCl salt of
deprotected 7) was used for labelling without further purification.
For labelling with 99mTc three different co-ligands were used.
The first attempt (A) used tricine as co-ligand. To a labelling vial
were successively added 100 ml of a 1 mg/ml solution of
deprotected 7 (HCl salt) in ethanol/water (50:50 v/v), 200 ml of
0.5 M phosphate buffer pH 7, 15 ml of a 100 mg/ml solution of
tricine in water, 12.5 ml of a 4 mg/ml solution of SnCl2 ꢂ 2H2O in
0.05 M HCl and about 600 MBq 99mTcOꢀ4 in 1 ml of saline. The
mixture was incubated at RT for 15 min. The second attempt (B)
used EDDA as co-ligand and was performed in the same way as
A, but 5 mg EDDA was used instead of 1.5 mg tricine. The third
attempt (C) used a mixture of tricine and nicotinic acid as co-
ligand and was also performed in the same manner as A, but
15 mg tricine and 2 mg nicotinic acid were used and the mixture
was heated in a boiling water bath for 15 min. RP-HPLC was
performed after cooling to RT. The labelling yield varied from
52% (method B) to 77% (method C).
2-[20-(N-BOC-6-hydrazinonicotinamido-3-propoxy)-40-amino]phe-
nyl-1,3-benzothiazole (7)
A solution of 5 (100 mg, 0.25 mmol) in a mixture of 19.5 ml TFA
and 0.5 ml of triisopropylsilane (TRIS) was stirred at RT for
15 min. TFA was removed under reduced pressure and the
residue was co-evaporated four times with hexane. The
residue was taken up in CH2Cl2 (50 ml). The solution was
extracted twice with 50 ml 10% (m/V) NaHCO3, washed with
brine, dried over MgSO4, filtered and evaporated under
reduced pressure. The residue (100 mg, 0.334 mmol) was
dissolved in 20 ml of dioxane and DIEA (60 ml, 100 mmol) and
succinimidyl-6-BOC-hydrazinopyridine-3-carboxylic acid (6)13
(235 mg, 0.67 mmol) were added. The mixture was stirred
overnight at RT. The organic solvent was evaporated and the
residue was purified by silica column chromatography using
5% CH3OH in CH2Cl2 as the eluent to yield 80 mg of
green–yellow solid 7 (0.15 mmol, 45%).
1H-NMR (CD3OD): d 1.47 (9H, s, tBu); d 2.28 (2H, m,
CH2–CH2–CH2); d 3.71 (2H, m, CH2–NH); d 4.22 (2H, m, O–CH2);
d 6.37 (1H, d, 5-HPh); d 6.40 (1H, d, 3-HPh); d 6.65 (1H, d, 5-
HYNIC); d 6.92 (1H, s, NH–CO); d 7.28 (1H, t, 5-HBe); d 7.42 (1H, t,
J. Label Compd. Radiopharm 2008, 51 357–367
Copyright r 2008 John Wiley & Sons, Ltd.
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K. Serdons et al.
Labelling of 8
the partition coefficient P was calculated using the following
equation:
When using an IsolinkTM kit, about 600 MBq of 99mTcOꢀ4 in a
maximum of 1 ml saline was added to a labelling kit, which was
heated in a boiling water bath for 20 min. The pH of the reaction
mixture was adjusted to 7 with 1 M HCl and 250 ml of this
neutralized solution was added to 100 ml of a 1mg/ml solution of 8
in CH3CN/CH3OH/H2O (3:3:4 v/v). The mixture was heated at 701C
for 20 min and RP-HPLC analysis was performed after cooling to RT.
As an alternative, [99mTc(I)(CO)3(OH2)3]1 was prepared by the
CO-bubbling method.20 Briefly, CO gas was bubbled through a
mixture containing 4.5 mg Na2CO3, 20–40 mg NaBH4 and 20 mg
NaKtartrate. Then, about 600 MBq 99mTcO4ꢀ in a maximum of
1 ml saline was added and the mixture was heated at 751C for
20 min. The pH of the resulting [99mTc(I)(CO)3(OH2)3]1 solution
was adjusted to pH 7 by the addition of approximately 250 ml of
a 1 M HCl solution and 250 ml of this mixture was added to 100 ml
of a 1 mg/ml solution of 8 in CH3CN/CH3OH/H2O (3:3:4 v/v). The
mixture was heated at 701C for 20 min and RP-HPLC analysis was
performed after cooling to RT. The mean labelling yield for both
procedures was 64%.
cpm=ml octanol
P ¼
cpm=ml buffer
with cpm being counts per minute. Experiments were
performed in triplicate.
Biodistribution studies
The peak containing the desired labelled compound was
isolated on RP-HPLC and diluted with saline to a concentration
of 150 kBq/ml. Male NMRI mice were sedated with an
intraperitoneal injection of 0.1 ml Hypnorms (fentanyl citrate
63 mg/ml1fluanisone 2 mg/ml) and injected with 100 ml of the
compound solution via a tail vein in a series of eight mice. The
mice were sacrificed by decapitation at 2 or 60 min p.i. (n = 4 at
each time point). Blood was collected in a tared tube and
weighed. All organs and other body parts were dissected and
weighed and their activity was counted in a g-counter. Results
were corrected for background activity and are expressed as
percentage of the injected dose per organ (% ID) or as
percentage of the injected dose per gram tissue (% ID/g). To
calculate the activity in the blood, blood mass was assumed to
be 7% of the total body mass.
Analysis of 99mTc-labelled compounds with radio-LC-MS
Radio-LC-MS was carried out using an XTerraTM MS C18 column
(3.5 mm, 50 mm ꢁ 2.1 mm, Waters) with gradient mixtures of
CH3CN and 0.05 M ammonium acetate as eluent at a flow rate of
0.3 ml/min.
Conclusion
Three derivatives of the amyloid-binding agent ThT with a
technetium-binding moiety have been successfully synthesized in
sufficient yields and their identity and structure were confirmed.
Deprotection and labelling of the studied compounds with
technetium-99m were done using either direct labelling, exchange
labelling or Tc(CO)3 labelling, providing good labelling yields for all
three compounds as confirmed by RP-HPLC. Results of radio-LC-MS
analysis support the hypothesized structures of two of the
complexes, whereas the structure of the Tc-HYNIC-tricine complex
remains speculative. Despite the fact that adding a negative charge
to the neutral phenylbenzothiazole core will have an unknown
effect on the binding of the derivative to Ab, all compounds were
evaluated in normal mice. The results of the biodistribution studies
of these 99mTc-labelled phenylbenzothiazoles indicate that none of
these tracer agents has ideal biological characteristics. The target-
to-background activity ratio in the abdominal organs would be
unfavourable due to the pronounced uptake in liver and intestines.
Further evaluation should investigate the potential usefulness for
detection of SA in body parts other than the abdomen.
Labelling of 2 and 7 for LC-MS analysis
Deprotection and labelling of 2 and 7 were performed as
described earlier, but to the solution of 99mTcO4ꢀ 0.1 ml of a
solution of 150 mg NH949TcO4 in 10 ml of water (1.5 mg Tc) was
added. In addition, 25 and 50 ml of the 4 mg/ml solution of
SnCl2 ꢂ 2H2O in 0.05 M HCl were used for 2 and 7, respectively.
For 99mTc-7, mass determination was only carried out after
labelling with tricine as co-ligand.
Labelling of 8 for LC-MS analysis
To perform a carrier-added labelling, the amount of reducing
agent in the IsolinkTM kit is too low. To obtain mass data of a
99mTc-tricarbonyl labelled compound, it is therefore necessary to
perform the preparation of the [99mTc(CO)3(OH2)3]1 precursor
via the CO-bubbling method. The amount of NaBH4 was
increased because of the high amount of technetium-(99m1
99) in the reaction mixture. To the solution of 600 MBq 99mTcO4ꢀ
in 0.9 ml saline, 0.1 ml of a solution of 150 mg NH949TcO4 in 10 ml
of water was added (1.5 mg Tc). The rest of the procedure was
followed as described earlier.
Acknowledgement
The authors acknowledge the help of Christelle Terwinghe
(Radiopharmacy, UZLeuven, Leuven, Belgium) for performing
the biodistribution studies and Tyco Healthcare for providing
the IsolinkTM labelling kits.
Partition coefficients
The lipophilicity of the RP-HPLC isolated 99mTc complexes was
determined using a modification of the method described by
Yamauchi and co-workers.25 To a test tube containing 2 ml of 1-
octanol and 2 ml of 0.025 M phosphate buffer pH 7.4, 25 ml of
the RP-HPLC isolated 99mTc complex was added. The test tube
was vortexed at RT for 3 min followed by centrifugation at
2700 g for 10 min. Aliquots of 60 and 500 ml were drawn from
the 1-octanol and buffer phases, respectively, and weighed. The
radioactivity in each aliquot was counted using a g-counter and
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www.jlcr.org