Synthesis and evaluation of a 99mTc-BAT-phenylbenzothiazole conjugate as a potential in vivo tracer for visualization of amyloid β

Article abstract of DOI:10.1016/j.bmcl.2007.09.055

We have conjugated S,S′-bis-trityl-N-BOC-N′-acetic acid-1,2-ethylenedicysteamine, a protected bis-amino-bis-thiol (BAT) tetraligand, with 2-(4′-aminophenyl)-1,3-benzothiazole, a derivative of thioflavin-T with known affinity for amyloid. The conjugate was

Full text of DOI:10.1016/j.bmcl.2007.09.055

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6086–6090
99m
Synthesis and evaluation of a Tc-BAT-phenylbenzothiazole
conjugate as a potential in vivo tracer for visualization of amyloid b
a,
a
a
a
b
*
K. Serdons, T. Verduyckt, J. Cleynhens, C. Terwinghe, L. Mortelmans,
a
a
G. Bormans and A. Verbruggen
a
Laboratory for Radiopharmacy, K.U.Leuven, B-3000 Leuven, Belgium
b
Nuclear Medicine, U.Z.Gasthuisberg, B-3000 Leuven, Belgium
Received 5 July 2007; revised 13 September 2007; accepted 13 September 2007
Available online 18 September 2007
0
0
Abstract—We have conjugated S,S -bis-trityl-N-BOC-N -acetic acid-1,2-ethylenedicysteamine, a protected bis-amino-bis-thiol
0
(
BAT) tetraligand, with 2-(4 -aminophenyl)-1,3-benzothiazole, a derivative of thioflavin-T with known affinity for amyloid. The
Tc by heating of the protected precursor in diluted hydrochloric acid followed by neutral-
9
9m
conjugate was efficiently labelled with
ization and heating in the presence of
9
9m
99m
Tc-tartrate. It was demonstrated that the
Tc-BAT-phenylbenzothiazole conjugate binds
in vitro to amyloid b present in postmortem brain slices of Alzheimer’s patients. Despite its high lipophilicity and neutral character,
the radiolabelled conjugate did not cross the blood–brain barrier to a sufficient degree and therefore is not useful for detection of
9
9m
Alzheimer’s disease. Further evaluation of this
loid plaques in peripheral amyloidosis.
Tc-labelled tracer agent could elucidate its potential usefulness to visualize amy-
ꢀ
2007 Elsevier Ltd. All rights reserved.
The increasing incidence of dementia, in particular Alz-
heimer’s disease (AD), is at the basis of a continuing
search for a radioactive tracer which allows to definitely
diagnose the disease in vivo in an early stage. AD is
characterized by the presence of extensive, extracellular
deposits of amyloid b (Ab) in cortical brain tissue and
blood vessels together with so-called neurofibrillar tan-
sive in vivo imaging and diagnosis of AD in an early
stage and follow up of treatment. Such a radiotracer
should have high affinity for the Ab plaque deposits
and a good blood–brain barrier (BBB) passage. To cross
the BBB, the compound should have a neutral charac-
ter, a partition coefficient between 1 and 2.5 and a
7
molecular mass not exceeding 600.
1
,2
gles which are found intracellularly. The tangles con-
sist of the microtubule-binding protein tau in a specific
paired helical and straight filament form. The formation
of Ab probably leads to an abnormal cleavage of the tau
protein, causing intracellular assemblage of tau into
pathological filaments ending up with neuronal cell
Systemic or peripheral amyloidosis (PA) is characterized
by the presence of deposited proteins with the same
properties, namely highly b-pleated Ab plaques with a
high propensity to aggregate, in liver, heart, kidneys,
joints, etc. So, radiolabelled compounds binding to
Ab may also be used to visualize the plaques in PA.
1
3
,4
death and dementia.
now done by neuro-psychological tests but yields only
Clinical diagnosis of AD is
5
indirect information. Immunohistochemical staining
In the past years, radiolabelled derivatives of several of
the higher mentioned dyes have been proposed and eval-
uated as potential diagnostic tracer agent for this pur-
of brain tissue with polyaromatic dyes such as Congo
red, chrysamine G, thioflavin-T (ThT, Fig. 1), thiofla-
vin-S, or silver can provide direct information but only
8
99m
pose. Our group studied a
Tc-labelled conjugate of
6
postmortem. Therefore it would be useful to have a
radiolabelled tracer agent which would allow non-inva-
+
N
Keywords: Amyloid b; Thioflavin-T; 2-Phenylbenzothiazoles; Techne-
tium-99m.
N
S
*
Corresponding author. Tel.: +32 16330441; fax: +32 16330449;
e-mail: kim.serdons@pharm.kuleuven.be
Figure 1. Structure of thioflavin-T.
0
960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.055

K. Serdons et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6086–6090
6087
chrysamine G with a monoamide–monoaminedithiol
9
BOC
BOC
N
N
COOEt
,10
N
HN
(
MAMA) chelating ligand.
However, brain uptake
a
of this radiolabelled agent was minimal, probably be-
cause of its large size and ionized character at physiolog-
ical pH. The most promising of the reported compounds
S
S
S
S
Tr
Tr
Tr
Tr
1
2
1
25
are uncharged derivatives of thioflavin-T such as
1
I-
b
1
TZDM (Fig. 2A) and the carbon-11 labelled 2-phen-
ylbenzothiazoles BTA (Fig. 2B) and 6-OH-BTA, also
1
2
BOC
known as Pittsburgh Compound-B or PIB (Fig. 2C).
The latter has already been tested in several clinical
studies and was shown to allow clear differentiation be-
NH
2
N
S
N
S
COOH
+
HOOC
c
NH
2
SH
1
3
tween AD and control subjects.
Tr
Tr
3
1
1
Although the first clinical results using [ C]PIB seem to
be very promising, its routine clinical use is limited be-
cause of the short half-life of carbon-11 and the high
cost of production (cyclotron) and imaging (PET cam-
era). Therefore, great effort has been made to develop
N
NH
2
d
S
4
N
S
BOC
NH
9
9m
Tc-labelled tracers for in vivo detection of amyloid
Tc has attractive nuclear-physical proper-
N
N
9
9m
O
b plaques.
ties and is continuously available in the hospital at a rel-
atively low cost. However, contrary to the labelling of a
Tc needs a
5
S
S
Tr
Tr
e,f
1
1
compound with C, the transition metal
99m
chelating structure to become stably bound to an organ-
ic molecule.
N
S
NH
N
9
O
N
S
The aim of this study was to synthesize a neutral techne-
0
O
9m
Tc
tium-99m labelled derivative of ThT, namely 2-(4 -ami-
nophenyl)-1,3-benzothiazole conjugated to
6
S
a
bis-
amine-bis-thiol (BAT) ligand, and to study its biological
characteristics. Synthesis of the BAT type bifunctional
2
Scheme 1. Reagents and conditions: (a) DIEA, BrCH COOEt,
CH
reflux, overnight; (c) polyphosphoric acid, 180 ꢁC, 4 h; (d) EDCIÆHCl,
2 2 2
Cl , room temperature (rt), overnight; (b) H O/EtOH, NaOHsat,
0
chelating agent S,S -bis-trityl-N-BOC-1,2-ethylenedicy-
steamine (Scheme 1, 1) was done according to published
HOBt, CH Cl
2
2
, rt, overnight; (e) HCl 0.5 M, 20 min, 100 ꢁC; (f)
9
9m
ꢀ
1
4
NaKtartrate buffer, pH 7, SnCl Æ2H O,
TcO4 , 15 min, 100 ꢁC.
2
2
data. The thiol groups were protected with triphe-
nylmethyl (trityl) groups to prevent oxidation (e.g.,
disulfide formation) and one amine was protected with
a tert-butoxycarbonyl (BOC) group to allow introduc-
tion of a single ethyl acetate substituent on the other
amine. The reaction with ethyl bromoacetate was done
in CH Cl with N,N-diisopropylethylamine (DIEA) as
cal coupling reagents used in peptide chemistry.
Although the reagents were added in excess and a long
reaction time was used, only a moderate yield of 23%
1
9
was obtained for precursor 5. No attempts were made
to optimize the reaction because only low amounts of
the metal complexing ligand are required for labelling
with technetium-99m.
2
2
1
5
base and provided 2 in 37.5% yield. In the next step,
the ethyl ester was removed with NaOH in H O/EtOH
2
1
6
under reflux, yielding the free acid 3 (50% yield). 2-
0
17
(
according to a method described by Shi and coworkers
4 -aminophenyl)-1,3-benzothiazole 4 was synthesized
8
In precursor 5, the two thiol groups are protected with a
trityl group and the secondary amine with a BOC group.
Removal of thiol protective trityl groups is usually done
with trifluoroacetic acid and triisopropylsilane in an in-
ert solvent. This procedure also removes the N-BOC
protective group and allows performing deprotection
1
and was obtained in 64% yield after crystallization. Cou-
pling of the aromatic amine of the phenylbenzothiazole
with the carboxylic acid group of the protected BAT li-
gand was performed in the presence of 1-(3-dimethyl-
9
9m
aminopropyl)-3-ethyl
(
carbodiimide
hydrochloride
and labelling with
Tc as a one-pot reaction. Based
2
0
EDCIÆHCl) and hydroxybenzotriazole (HOBt), classi-
on our previous work, however, deprotection in aque-
ous medium using heating in the presence of hydrochlo-
ric acid was preferred. The latter method generates a low
amount of the fully deprotected precursor, while the ma-
jor part is converted to the mono-S-trityl or di-S-trityl
form without N-BOC group as shown by mass spec-
trometry (MS) analysis. Such partial deprotection ap-
R2
N
N
R3
R1
S
pears to be sufficient to allow successful exchange
labelling of the BAT chelating structure with
A. R1=125I, R =R =CH
(
125
I-TZDM)
2
3
3
1
99m
Tc.
1
1
2
3
B. R = H R =H, R = CH (BTA)
,
3
Such labelling can be done by heating in a boiling water
bath in the presence of stannous ions and tartrate as a
weak chelating agent (the formation of an intermediate
weak Tc(V)O–tartrate complex prevents the formation
1
1
1
2
3
C. R = OH R =H, R = CH3 (6-OH-BTA, PIB)
,
Figure 2. Structures of some proposed tracers for visualization of
amyloid b plaques.

6088
K. Serdons et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6086–6090
9
9m
18.74
of
with
TcO in colloidal form). The efficient labelling
2
100
%
9
9m
a
b
Tc can be explained by the fact that only nano-
9
9m
molar amounts of
400–800 MBq) and that low amount of fully deprotec-
ted ligand is sufficient for chelation of the few nano-
Tc are present in the used activities
(
13.19
9
9m
0
grams of
Tc. In addition, previous studies have
shown that upon binding of technetium to one or more
ligand atoms, the remaining protecting groups are
cleaved off, apparently by the ‘chelate effect’. The mean
yield of the labelling reaction was 86% and the different
products present in the labelling reaction mixture could
be efficiently separated by reversed phase high perfor-
1.64
1
00
%
0
6.82
2.95
18.78
99m
6
Tc-2.4
6
8
.81
9.99
Time
30.00
2
1,22
10.00
20.00
mance liquid chromatography (RP-HPLC) (Fig. 3).
+
Figure 4. (a) Single ion mass chromatogram (ES , 559.172–
559.322 Da) and (b) = radiometric chromatogram of 6.
Identification of the peak eluting at 15.3 min was done
by comparing its retention time (t ) with that of the
R
9
9m
complex formed between deprotected 3 and Tc
Tc-BAT-CH -COOH). The presence of this com-
9
9m
ing at 18.78 min can be assumed to have with high
probability the structure as depicted in 6 (Scheme 1).
In the radiometric signal, the peak at 18.78 min corre-
(
plex as a radiochemical impurity is due to the presence
2
of a low amount of BAT-CH -COOH as an impurity
2
9
9m
sponds to 6, while
volume at 1.64 min. Peaks at 2.95 and 6.82 min corre-
Tc-tartrate elutes with the void
after heating of 5 in acidic conditions, and the fact that
this small molecule can bind
9
9m
Tc more efficiently than
9
9m
spond to pertechnetate and
Tc-BAT-CH -COOH,
2
the more sterically hindered 5. The yield of the labelling
reaction was, however, sufficient for further biological
evaluation of 6.
respectively. The radiochemical yield of 6 in carrier-
added preparations was lower as compared to prepara-
tions using no-carrier-added technetium-99m (Fig. 3).
9
9m
Tc la-
Due to the tiny amounts of technetium in a
The molecular ion mass found for 6 (559.2 Da) is in
agreement with a Tc(V)O-complex of a BAT chelating
belled preparation (typically in the nano- to picomolar
range), it is difficult to obtain useful mass spectra of
technetium-99m labelled compounds in no-carrier-
added form. In order to be able to determine the mass
of 6, we increased the amount of technetium in the
radiolabelling mixture by addition of technetium-99 in
the form of ammonium pertechnetate to obtain a car-
rier-added form. Technetium-99 emits b-rays and has a
3
+
structure of 5. Upon complexation of the [Tc(V)O]
core, both thiols and one amine of the BAT ligand lose
3
+
a proton. Three positive charges of the [Tc(V)O] core
are balanced by the loss of these three protons and a
neutral complex is thus formed. The partition coefficient
2
4
was determined using a described procedure and
found to be 83.28 ± 2.35 (logP = 1.92 ± 0.012), which
is in a good range for passive BBB diffusion. As its
molecular mass does not exceed 600 Da, 6 has in princi-
ple favorable characteristics which allow passage
through the BBB.
5
half-life of 2.14 · 10 year, so care should be taken when
9
9
Tc is used to prevent contamination. Figure 4b depicts
2
3
the radiometric signal of the radio-LC–MS analysis of
carrier-added 6, which shows the presence of different
radioactive compounds. Figure 5a shows the back-
ground subtracted mass spectrum of the peak with tR
9
9m
The affinity of
ied by incubation of postmortem brain slices of AD pa-
Tc-labelled 6 for amyloid b was stud-
1
8.78 min (summed mass spectra obtained prior and
after the peak at 18.78 min subtracted from the summed
mass spectrum over the peak at 18.78 min), showing a
single molecular ion mass of 559.2326 Da, which corre-
sponds to the theoretical ion mass of 6 (Fig. 5b;
tients with 6 in the absence or in the presence of ThT
2
5,26
(1 lM).
Figure 6a shows the visualization of Ab pla-
ques in the brain cortex (red areas). In Figure 6b, the
binding of 6 on an adjacent slice was decreased in the
presence of ThT (1 lM) showing that both molecules
have the same binding sites.
5
59.0112 Da). Figure 4a shows the single ion mass chro-
matogram over the mass range 559.172–559.322 Da. As
the peak on this single ion mass chromatogram has both
an identical retention time and shape as the peak ob-
served in the radiometric channel, the Tc-complex elut-
2
5
Biodistribution of 6 was studied in normal mice, which
were sacrificed at 2 min or 60 min post-injection (pi).
The results are shown in Table 1. At 2 min pi, the per-
centage of injected dose (ID) in the cerebrum and the
cerebellum was only 0.09% and 0.11%, respectively.
Activity in both organs decreased to 0.03% ID at
6
0 min pi, showing that brain uptake was minimal.
Therefore the compound is not useful for detection of
amyloid plaques in the living AD brain.
These findings are in agreement with those of similar at-
Tc-labelled N S -complexes for
2 2
Figure 3. RP-HPLC chromatogram of the reaction mixture after
9
9
9m
9m
99m
tempts to develop
imaging amyloid plaques in brain. One of the reasons
labelling of 5 with
Tc. Radiolabelled 6 elutes at 25 min and
Tc-
2
7
BAT-CH -COOH at 15.3 min.
2

K. Serdons et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6086–6090
6089
5
59.2326
1
1
00
%
0
a
1
12.608187.0086 280.0093
559.0112
00
%
b
187.3428 280.5106
560.0140
0
1
m/z
1000
00
200
300
400
500
600
700
800
900
Figure 5. (a) Background subtracted mass spectrum of the chromatogram peak at 18.78 min (Fig. 4a) and (b) theoretical molecular ion mass of 6.
9
9m
Further evaluation of this
Tc-labelled tracer agent is
required to investigate its potential usefulness to visual-
ize amyloid plaques in peripheral amyloidosis.
References and notes
1
. Ghiso, J.; Frangione, B. Adv. Drug Delivery Rev. 2002, 54,
539.
. Selkoe, D. J. Physiol. Rev. 2001, 81, 741.
1
2
3
. Gamblin, T. C.; Chen, F.; Zambrano, A.; Abraha, A.;
Lagalwar, S.; Guillozet, A. L.; Lu, M.; Fu, Y.; Garcia-
Sierra, F.; LaPointe, N.; Miller, R.; Berry, R. W.; Binder,
L. I.; Cryns, V. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
1
0032.
Figure 6. (a) Visualization of Ab in postmortem brain slices of AD
patients after incubation with 6 and (b) in the presence of ThT (1 lM).
4
. Rapoport, M.; Dawson, H. N.; Binder, L. I.; Vitek, M. P.;
Ferreira, A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6364.
. Friedland, R. P. Neurology 1993, 43, S45.
. Vallet, P. G.; Guntern, R.; Hof, P. R.; Golaz, J.;
Delacourte, A.; Robakis, N. K.; Bouras, C. Acta Neuro-
pathol. 1992, 83, 170.
5
6
Table 1. Biodistribution of 6 in normal mice at 2 min and 60 min pi
(
n = 4)
%
ID
2 min pi
60 min pi
7. Dischino, D. D.; Welch, M. J.; Kilbourn, M. R.; Raichle,
M. E. J. Nucl. Med. 1983, 24, 1030.
Kidneys
Liver
Lungs
4.4
32.6
1.8
2.2
19.2
0.4
8
. Wu, C.; Pike, V. W.; Wang, Y. Curr. Top. Dev. Biol. 2005,
0, 171.
7
. Dezutter, N. A.; de Groot, T. J.; Busson, R. H.; Janssen,
9
Intestines
Blood
5.1
24.2
5.7
G. A.; Verbruggen, A. J. Labelled Compd. Radiopharm.
1
18.8
0.09
0.11
999, 42, 309.
Cerebrum
Cerebellum
0.03
0.03
1
0. Dezutter, N. A.; Dorn, R. J.; de Groot, T. J.; Bormans,
G.; Verbruggen, A. Eur. J. Nucl. Med. 1999, 26, 1392.
1. Zhuang, Z. P.; Kung, M.-P.; Hou, C.; Skovronsky, D.;
Gur, T. L.; Trojanowski, V. M. Y.; Kung, H. F. J. Med.
Chem. 2001, 44, 1905.
1
9
9m
for the poor passage of the studied
ylbenzothiazole through the BBB might be its low
in vivo stability. We studied the percentage of intact tra-
cer in plasma of normal mice (n = 4) and found that at
min pi already 85% of the compound is converted into
a polar radiolabelled metabolite, so only a small per-
centage of the intact tracer remains available for brain
uptake. Surprisingly, the same percentage of intact tra-
cer was found in plasma at 10, 30, and 60 min pi, indi-
cating a very rapid initial metabolism but no further
degradation of the residual intact fraction.
Tc-labelled phen-
1
1
1
2. Klunk, W. E.; Wang, Y.; Huang, G.; Debnath, M. L.;
Holt, D. P.; Mathis, C. A. Life Sci. 2001, 69, 1471.
3. Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.;
Mathis, C. Ann. Neurol. 2004, 55, 306.
2
8
2
4. Bormans, G.; Cleynhens, B.; de Groot, T. J.; Mortelmans,
L.; Moretti, J.-L.; Verbruggen, A. J. Labelled Compd.
Radiopharm. 2003, 46, 575.
1
15. Compound 2: H NMR (CDCl ): d 1.3 (3H, t); d 1.38 (9H,
3
s); d 2.27–2.58 (8H, m); d 2.90–3.04 (6H, m); d 4.1 (2H, q);
+
d 7.1–7.4 (30H, m). Mass: [M+H] 851 (calcd: 851).
1
6. Compound 3: H NMR (CDCl
1
1
3
): d 1.35 (9H, s); d 2.25–
99m
2.32 (8H, m); d 2.81–2.97 (6H, m); d 7.16–7.42 (30H, m).
Mass: [M+H] 823 (calcd: 823).
In summary, the synthesis of the newly developed Tc-
0
2-(4 -aminophenyl)-1,3-benzothiazole]-BAT conjugate
+
[
1
7. Compound 4: H NMR (DMSO): d 5.8 (2H, s); d 6.68
was successful and its structure was confirmed using
radio-LC–MS. It was demonstrated that the new radio-
labelled conjugate binds in vitro to amyloid b. Despite
its high lipophilicity and neutral character, the radiola-
belled conjugate did not cross the BBB to a sufficient de-
gree and thus is not useful for in vivo detection of AD.
(
7
2
2H, d); d 7.32 (1H, t); d 7.45 (1H, t); d 7.76 (2H, d); d
ꢀ
.89 (1H, d); d 7.98 (1H, d). Mass: [MꢀH] 225 (calcd:
25).
1
8. Shi, D.-F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J.;
Lelieveld, P.; Fichtner, I.; Stevens, M. F. G. J. Med.
Chem. 1996, 39, 3375.

6090
K. Serdons et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6086–6090
1
9. Compound 5: H NMR (CDCl
1
2
2
3
): d 1.38 (9H, s); d 2.27–
USA) and then analyzed using a time-of-flight mass
spectrometer (LCT, Micromass, Manchester, England)
equipped with an electrospray ionization (ESI) source.
Acquisition and processing of data was performed with
Masslynx software (version 3.5).
2.58 (8H, m); d 2.97 (6H, m); d 7.1–7.6 (36H, m); d 7.95
+
(
1H, d); d 8.05 (1H, d). Mass: [M+H] : 1031 (calcd: 1031).
0. Cleynhens, B.; Bormans, G.; Vanbilloen, H.; Vanderghin-
ste, D.; Kieffer, D.; de Groot, T.; Verbruggen, A.
Tetrahedron Lett. 2003, 44, 2597.
1. Compound 6: A stock solution of 5 (1 mg/ml in acetoni-
trile) was prepared and also a labelling buffer solution
consisting of a mixture of 5 ml 0.5 M phosphate buffer, pH
24. Yamauchi, H.; Takahashi, J.; Seri, S.; Kawashima, H.;
Koike, H.; Kato-Azuma, M. In Technetium and rhenium in
chemistry and nuclear medicine; Nicolini, M., Bandoli, G.,
Mazzi, U., Eds.; Verona, Italy: Cortina International,
1989; Vol. 3, pp 475–502.
7
(
, 2.5 ml 0.1 M disodium ethylenediaminetetraacetate
Na EDTA), and 2.5 ml NaKtartrate solution (40 mg/
ml). A mixture of 200 ll of the stock solution and 60 ll of
.5 M HCl in a labelling vial was heated in a boiling water
bath for 20 min. After cooling, 15 ll SnCl Æ2H O solution
2
25. All animal experiments and the use of AD brain slices
were approved by the local Ethical Committee.
0
26. Paraffinized postmortem brain sections of AD patients
were incubated for 1 h with radiolabelled HPLC-purified 6
in the absence or presence of ThT (1 lM) after deparaff-
inization (toluene 3 · 3 min, ethanol 3 · 3 min). After
rinsing three times with 40% ethanol and once with water,
the sections were allowed to dry at room temperature and
exposed overnight to a high-performance storage phos-
phor screen (Packard, Meriden, CT, USA) which was
2
2
(
tion, and 1 ml generator eluate containing 400–800 MBq
20 mg/5 ml 0.05 M HCl), 200 ll of labelling buffer solu-
99m
ꢀ
TcO4 were added. The mixture was heated again in
of
a boiling water bath for 15 min. After cooling to room
temperature, RP-HPLC analysis was performed.
TM
2
2
2. RP-HPLC analysis of 6 was carried out using an Xterra
RP C18 column (5 lm, 250 mm · 4.6 mm, Waters, Mil-
TM
analyzed in a phosphor imaging system (Cyclone ,
ford, MA, USA) with gradient mixtures of CH CN and
3
Packard) using Packard Optiquant software (Packard).
Non-specific binding was determined in the presence of
1 lM ThT.
0.05 M ammonium acetate as eluent at a flow rate of 1 ml/
min.
3. Combined liquid chromatography and mass spectrometry
27. Zhuang, Z.-P.; Kung, M.-P.; Hou, C.; Ploessl, K.; Kung,
H. F. Nucl. Med. Biol. 2005, 32, 171.
(
LC–MS) was performed on a system consisting of a
Waters Alliance 2690 separation module (Waters) coupled
28. Mice were injected iv with intact HPLC-purified radiola-
belled 6 and sacrificed by decapitation at 2, 10, 30 or
60 min pi. Blood was collected and centrifuged. Plasma
TM
to a reverse phase Xterra MS C18 3.5 lm column
(
mixture of CH CN and 0.05 M ammonium acetate at a
2.1 mm · 50 mm, Waters). The eluent was a gradient
TM
3
was injected on a Chromolith column (Merck, Darms-
tadt, Germany), eluted with a gradient mixture of 0.05 M
NH OAc and CH CN at a flow rate of 1 ml/min, and the
flow rate of 0.3 ml/min. The eluate was monitored for UV-
absorbance (Waters 2487 Dual wavelength absorbance
detector) and radioactivity (3-in. NaI(Tl)-crystal coupled
to a single channel analyzer, The Nucleus, Oak Ridge, TE,
4
3
HPLC eluate was collected in 1 ml fractions of which the
radioactivity was measured using a gamma counter.