Synthesis and biological evaluation of 11C-labeled β-galactosyl triazoles as potential PET tracers for in vivo LacZ reporter gene imaging

Article abstract of DOI:10.1016/j.bmc.2009.05.056

In our aim to develop LacZ reporter probes with a good retention in LacZ expressing cells, we report the synthesis and preliminary evaluation of two carbon-11 labeled β-galactosyl triazoles 1-(β-d-galactopyranosyl)-4-(p-[¹¹C]methoxyphenyl)-1,2,

Full text of DOI:10.1016/j.bmc.2009.05.056

Bioorganic & Medicinal Chemistry 17 (2009) 5117–5125
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 11C-labeled b-galactosyl triazoles
as potential PET tracers for in vivo LacZ reporter gene imaging
Sofie Celen a, Jan Cleynhens a, Christophe Deroose b, Tjibbe de Groot b, Abdelilah Ibrahimi c,
Rik Gijsbers c, Zeger Debyser c, Luc Mortelmans b, Alfons Verbruggen a, Guy Bormans a,
*
a Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Herestraat 49 bus 821, BE-3000 Leuven, Belgium
b Department of Nuclear Medicine, U.Z. Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium
c Division of Molecular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
In our aim to develop LacZ reporter probes with a good retention in LacZ expressing cells, we report the
Received 12 December 2008
Revised 20 May 2009
Accepted 23 May 2009
Available online 29 May 2009
synthesis and preliminary evaluation of two carbon-11 labeled b-galactosyl triazoles 1-(b-
D
-galactopyr-
anosyl)-4-(p-[11C]methoxyphenyl)-1,2,3-triazole ([11C]-6) and 1-(b-
D
-galactopyranosyl)-4-(6-[11C]meth-
oxynaphthyl)-1,2,3-triazole ([11C]-13). The precursors for the radiolabeling and the non-radioactive
analogues (6 and 13) were synthesized using straightforward ‘click’ chemistry. In vitro incubation exper-
iments of 6 with b-galactosidase in the presence of o-nitrophenyl b-D-galactopyranoside (ONPG) showed
Keywords:
that the triazolic compound was an inhibitor of b-galactosidase activity. Radiolabeling of both precursors
was performed using [11C]methyl iodide as alkylating agent at 70 °C in DMF in the presence of a small
amount of base. The log P values were À0.1 and 1.4, respectively, for [11C]-6 and [11C]-13, the latter there-
fore being a good candidate for increased cellular uptake via passive diffusion. Biodistribution studies in
normal mice showed a good clearance from blood for both tracers. [11C]-6 was mainly cleared via the
renal pathway, while the more lipophilic [11C]-13 was excreted almost exclusively via the hepatobiliary
system. Despite the lipophilicity of [11C]-13, no brain uptake was observed. Reversed phase HPLC analysis
of murine plasma and urine revealed high in vivo stability for both tracers. In vitro evaluation in HEK-
293T cells showed an increased cell uptake for the more lipophilic [11C]-13, however, there was no sta-
tistically higher uptake in LacZ expressing cells compared to control cells.
b-Galactosyl triazole
Carbon-11
PET
Click chemistry
LacZ reporter gene
b-Galactosidase
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
computed tomography (SPECT).4,5 Reporter genes that have been
used for radionuclide imaging can be divided into three major cat-
With the advance of molecular biology, gene expression has
been extensively studied using reporter genes.1 One of the most
widely used reporter genes is the LacZ gene which encodes the
bacterial b-galactosidase (b-gal) enzyme. There are several chro-
mogenic and fluorogenic reporter probes commercially available
for detection of b-gal.2,3 However, these optical-based reporter sys-
tems usually require histochemical staining or spectrophotometric
assays of tissue acquired by invasive sampling or can only be used
to image small animals that are transparent for light. Applications
for in vivo monitoring of LacZ reporter gene expression with these
techniques in larger living animals and humans is not possible due
to the limited tissue penetration of visible light.
egories.6 The first category are reporter genes that lead to the pro-
duction of an enzyme capable of metabolizing and trapping of the
reporter probes. A theoretical advantage of these enzymatic repor-
ter systems is that one molecule of enzyme can trap many mole-
cules of reporter substrate, leading to amplification of the signal
and an increased sensitivity. The most widely studied enzyme-
based reporter system is the herpes simplex virus type 1 thymidine
kinase (HSV1-tk) reporter gene for which radiolabeled substrate-
probes are phosphorylated and trapped in HSV1-tk expressing
cells. The amount of HSV1-tk expressing cells can thus be quanti-
fied in vivo by PET or SPECT.7–9 The second class of reporter genes
are those that encode proteins that act as a receptor for binding
with the reporter probe. Since this is a one-to-one stoichiometric
interaction between one ligand and one receptor, the receptor-
based approach is less sensitive. An example is the human
dopamine-2-receptor (hD2R) in combination with [18F]fluoroeth-
ylspiperone. The third category of reporter genes are transporter
proteins located in the cell membrane that actively pump the radi-
olabeled probes from the extracellular space into the cell. The
Nuclear medicine technology has been applied to monitor
repetitively and quantitatively reporter gene expression in living
systems using specific radiolabeled probes in combination with
positron emission tomography (PET) or single photon emission
* Corresponding author. Tel.: +32 16 330447; fax: +32 16 330449.
E-mail address: guy.bormans@pharm.kuleuven.be (G. Bormans).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.056

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S. Celen et al. / Bioorg. Med. Chem. 17 (2009) 5117–5125
human sodium iodide symporter (hNIS) has been studied as trans-
porter reporter gene in combination with a wide range of iodine iso-
topes. However, the major problem encountered using this reporter
system is that the radioactive iodide is not sequestered, leading
to a rapid efflux of radioactivity out of the hNIS expressing cells.6,10
Concerning the LacZ reporter gene, which is an enzyme-based
reporter system, little progress has been made in the development
of suitable radiolabeled probes. Most problems encountered are
poor cell membrane penetration and inefficient sequestering of
the tracers in the target cells.
sized using reliable and efficient ‘click’ chemistry,21,22 which
offers the ability for rapid generation of combinatorial libraries
for screening of the best inhibitory effect.
In this study we report the synthesis and preliminary evalua-
tion of two carbon-11 labeled b-galactosyl triazoles [11C]-6 and
[
11C]-13, containing respectively, a phenyl and naphthyl moiety
in the aglycon. The naphthylic aglycon makes [11C]-13 more lipo-
philic, allowing to study the effect of increased lipophilicity on cell
membrane penetration and cell uptake. We report the synthesis of
stable and radiolabeled triazoles 6 and 13, their biodistribution in
normal mice and the study of the in vivo stability. Finally a cell up-
take study was carried out in LacZ expressing human embryonic
kidney (293T) cells.
Bormans et al.11 synthesized fluorine-18 labeled lactose using
an enzyme-catalyzed reaction between fluorine-18 labeled glucose
([18F]FDG) and galactose. Biodistribution studies in Rosa-26 mice
(expressing LacZ in almost every tissue) compared to tissue distri-
bution studies in normal control mice revealed that radiolabeled
lactose was unable to cross the cell membrane. In Escherichia coli,
cellular uptake of lactose, which is the natural substrate of b-galac-
tosidase and other galactopyranosides, is mediated by lactose per-
mease.12,13 However, this transporter protein is not expressed in
eukaryotic cells and since lactose is too hydrophilic for cell mem-
brane penetration, no accumulation of radiolabeled lactose in the
studied mammalian cells/tissue was observed.
2. Results and discussion
2.1. Chemistry
The precursors for the radiolabeling (4 and 11) and the non-
radioactive reference compounds (6 and 13) are 4-substituted
triazolyl galactopyranosides that were synthesized using straight-
forward ‘click’ chemistry (Scheme 1). ‘Click’ chemistry is a chemi-
cal philosophy that refers to a set of powerful and selective
reactions that form heteroatom links using spring-loaded reac-
tants. Reactions defined as ‘click’ reactions are high in yield and re-
quire only benign reaction conditions (e.g., water as solvent) with
simple work-up and purification procedures.21,22 The prototype
‘click’ reaction is the Huisgen 1,3-dipolar cycloaddition of azides
with terminal alkynes to afford 1,2,3-triazoles. Further optimiza-
tion by Sharpless and Meldal using Cu(I)-catalysis resulted in high
regioselectivity affording the 1,4-regioisomer exclusively.23,24 The
Cu(I)-catalyzed 1,3-dipolar cycloaddition was used in this study
to synthesize the precursors and cold reference compounds. The
general reaction procedure applied to couple b-galactosyl azide
with the terminal alkynes is depicted in Scheme 1 (Step 1).
Instead of radiolabeled substrates, Kim et al.14 developed a
radiolabeled inhibitor against b-gal activity by introducing iodine-
123/iodine-125 in 2-phenylethyl 1-thio-b-D-galactopyranoside
(PETG). PETG is a commercially available competitive inhibitor
specific against E. coli b-galactosidase. In vivo imaging studies of
[
123I]iodo-PETG injected intravenously in nude mice showed only
a slightly improved visualization of a LacZ expressing tumor versus
a control tumor. The low contrast was partly ascribed to poor
transport across the cell membrane.15 Non-modified
D
-galactose
is avidly transported into mammalian cells and has been proposed
to share a single transport mechanism with -glucose and 2-
deoxy-glucose.16 Although structurally related to
-galactose,
D
D
galactopyranosides derivatized at position C1 (like lactose and
PETG) are not transported by the hexose transport system. Since
in literature no evidence of active transport for C1-derivatized
galactopyranosides is found, cell uptake of these probes is assumed
to be mediated by passive diffusion. This implies that the devel-
oped LacZ tracers should be lipophilic enough to penetrate the cell
membrane by passive diffusion.
The acetyl-protected b-galactosyl azide (2) was synthesized in
two steps according to reported procedures.25,26 First, penta-acet-
ylated b-D-galactose was converted into acetobromo-a-D-galactose
(1). In a second step the bromine was replaced by an azide group
using sodium azide. Inversion of the configuration at the anomeric
site resulted in the formation of the protected b-galactosyl azide
(2), as could be concluded from the large axial-axial coupling of
the anomeric H-1 with H-2 (3JH1–H2 = 8.64 Hz) in the 1H NMR spec-
trum. The alkynes used in this paper were commercially available
except for 6-ethynyl-2-naphthol (9) that was synthesized in three
steps (Scheme 2) starting from 6-bromo-2-naphthol.
In a first step the free hydroxyl function was protected with a
tert-butyldimethylsilyl group to afford compound 7 according to
a patented procedure.27 Next, the bromine was converted into a
trimethylsilyl-ethynyl group using a Sonogashira coupling reaction
of 7 with trimethylsilylacetylene in 1,4-dioxane in the presence of
diisopropylamine and catalytic amounts of PdCl2(PhCN)2, CuI and
tri(tert-butyl)phosphine to obtain compound 8.28 Finally, removal
of the silyl-protecting groups with tetrabutylammonium fluoride
(TBAF) in tetrahydrofuran afforded the desired 6-ethynyl-2-naph-
thol (9).29
For the catalysis of the cycloaddition between acetylated b-
galactosyl azide (2) and the terminal alkynes (Scheme 1, Step 1)
we preferred a mixture of Cu(II)SO4 and a reducing agent (ascorbic
acid) to produce Cu(I) in situ, instead of commercial sources of
Cu(I), for example, CuI or CuBr, which are prone to oxidation. The
reaction was carried out at elevated temperatures using water as
solvent from which the crude reaction product could be easily
recovered by filtration. Recrystallization from 95% ethanol afforded
the desired acetylated cycloadducts, no further purification using
column chromatography was necessary. Finally, deprotection un-
Recently, we have developed fluorine-18 and carbon-11 labeled
phenyl b-D D-galactosyl esters (sub-
-galactopyranosides17 and 1-b-
mitted). Although both tracer classes were good substrates of b-
gal, in vitro evaluation in LacZ expressing HEK-293T (human
embryonic kidney) cells and control HEK cells showed low cell up-
take levels. This low uptake can be partly ascribed to the fact that
these probes were still too hydrophilic for efficient cell entry via
passive diffusion.
In our aim to synthesize LacZ reporter probes with a good reten-
tion in LacZ expressing cells, we have synthesized and evaluated
radiolabeled inhibitors to probe b-galactosidase. In this case, no
radiolabeled hydrolysis products are liberated and the radioactiv-
ity that probes the enzyme will remain localized in the target cells.
From this point of view the inhibitor approach may have some
advantages over the use of substrates of which the radiolabeled
hydrolysis products may not be efficiently retained.
Glycosyl triazoles are nitrogen containing heterocyclic glyco-
conjugates that have been reported as inhibitors of glycosidase
activity.18,19 The triazoles function as rigid linking units that can
mimic the atom placement and electronic properties of a peptide
bond without the same susceptibility to hydrolytic and enzymatic
cleavage, resulting in metabolically stable compounds.20 Perhaps
due in part to their ability to mimic certain aspects of a peptide
bond, many 1,2,3-triazoles possess both anti-viral and anti-bacte-
rial activity.20 Furthermore, these molecules can be easily synthe-

S. Celen et al. / Bioorg. Med. Chem. 17 (2009) 5117–5125
5119
CH2OAc
CH2OAc
N
N
AcO
AcO
O
O
N3
CuSO4 / ascorbic acid
H2O, 70 ºC
N
R
OAc
OAc
R
+
3, 5, 10, 12
OAc
OAc
2
NaOMe, MeOH, RT
2
CH2OH
N 3
N
1
N
HO
O
OH
4
R
OH
R =
4
6
11
13
OH
OH
OCH3
OCH3
Scheme 1. Step 1: Cu(I)-catalyzed 1,3-dipolar cycloaddition of acetyl-protected b-galactosyl azide (2) with various terminal alkynes to afford the acetyl-protected 4-
substituted 1,2,3-triazolyl b-
references (6 and 13).
D-galactopyranosides. Step 2: Deprotection under alkaline reaction conditions resulting in the radiolabeling precursors (4 and 11) and the cold
OH
OSi
TBSCl, imidazole
THF
Br
Br
7
PdCl2(PhCN)2, CuI
tBu3P, HN(i-C3H7)2
Si
dioxane
OSi
OH
TBAF (in THF)
THF, 0 ºC
9
8
Si
Scheme 2. 3-Step-synthesis of 6-ethynyl-2-naphthol (9) starting from 6-bromo-2-naphthol.
der alkaline reaction conditions (Scheme 1, Step 2) afforded the de-
sired 4-substituted triazolyl galactopyranosides: two precursors 4
and 11 and two non-radioactive reference compounds 6 and 13.
High stereo- and regioselectivity was achieved as could be con-
cluded from the NMR spectra. The stereoselectivity was proven
by the large axial–axial coupling of the anomeric H-1 with H-2
(3JH1–H2 = >8 Hz) in the 1H NMR spectra, showing that the galacto-
syl triazoles had the b-configuration. This is a necessary condition
to have affinity for the b-galactosidase enzyme. The regiochemistry
was unambiguously attested by performing a set of 2D-NMR
experiments (COSY, HSQC and HMBC). The 1H–13C HMBC spectrum
showed 3-J cross coupling between the anomeric C-1 and the H-5
of the phenyl ring and between the anomeric H-1 and the C-5 of
the phenyl ring, proving that the 4-substituted triazole (6) was
synthesized regioselectively. Formation of the 5-substituted tria-
zole, which could sterically hinder binding to the active site of
the enzyme, was prevented, presumably due to the presence of
the Cu(I)-catalyst.23
2.2. Radiochemistry
Radiosynthesis of [11C]-6 and [11C]-13 was carried out using
compounds 4 and 11, respectively, as precursors and [11C]methyl
iodide ([11C]MeI) as labeling agent (Scheme 3). [11C]MeI was

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S. Celen et al. / Bioorg. Med. Chem. 17 (2009) 5117–5125
Table 1
synthesized following a reported procedure with some modifica-
tions.30 The labeling was performed in DMF with NaOH as base.
At 70 °C slightly higher yields were obtained compared to room
temperature. Both tracers were purified using semi-preparative
RP-HPLC and the chemical and radiochemical purity was found
to be >99% using analytical HPLC. Co-elution with the authentic
non-radioactive compounds (6 and 13) after co-injection on ana-
lytical RP-HPLC confirmed the identity of the desired tracers.
Biodistribution of [11C]-6 in NMRI mice 2 min and 60 min p.i.
[
11C]-6
% IDa
% ID/gb
2 min
60 min
2 min
60 min
Urine
Kidneys
Liver
Intestines
Spleen + pancreas
Lungs
Heart
Stomach
Brain
Blood
Carcass
3.1 1.2
16.1 3.0
18.7 0.9
6.6 0.4
0.6 0.1
1.5 0.4
0.6 0.1
0.9 0.2
0.1 0.0
27.0 4.4
38.1 3.0
47.5 4.0
4.0 1.5
13.4 2.1
9.7 1.5
0.6 0.3
0.6 0.1
0.2 0.1
0.9 0.4
0.0 0.0
8.1 0.4
19.6 1.7
19.3 1.0
8.7 0.6
2.0 0.3
2.3 0.2
6.2 0.5
2.8 0.2
1.5 0.7
0.5 0.1
8.9 0.1
4.4 1.6
6.3 1.1
3.0 0.8
1.7 0.5
2.0 0.3
1.2 0.2
1.4 0.8
0.3 0.1
2.4 0.2
2.3. In vitro incubation with b-galactosidase
To validate whether the galactosyl triazoles act as inhibitors of
b-galactosidase, some preliminary in vitro incubation experiments
were performed. When a solution of 1 mg/mL of the non-radioac-
tive triazole 6 was incubated with 10 U of b-galactosidase at
37 °C overnight and analyzed using RP-HPLC, no hydrolysis prod-
ucts were detected. The same observation was made when the
radiolabeled compound [11C]-6 was incubated with the enzyme
(10 U). These findings indicate that the triazolic compound is not
cleaved by the enzyme. When the chromogenic substrate ONPG
(1 mg/mL) was incubated under the same conditions for 15 min,
complete conversion into the corresponding nitrophenol was ob-
served. However, when ONPG (1 mg/mL) was added to an incuba-
tion mixture of triazole 6 (1 mg/1 mL) and 10 U b-galactosidase,
the conversion of ONPG was slowed down. After 15 min, only
40% of ONPG was hydrolyzed, after 90 min, the conversion reached
70%. Addition of an excess of ONPG resulted in an immediate yel-
low color formation and HPLC analysis showed complete conver-
sion into the nitrophenol. These incubation experiments indicate
that the triazole binds to the enzyme and that it acts as a compet-
itive inhibitor.
Data are expressed as mean SD; n = 4 per time point; p.i. = post injection.
a
Percentage of injected dose calculated as cpm in organ/total cpm recovered.
Percentage of injected dose per gram tissue.
b
Table 2
Biodistribution of [11C]-13 in NMRI mice 2 min and 60 min p.i.
[
11C]-13
% IDa
% ID/gb
2 min
60 min
2 min
60 min
Urine
Kidneys
Liver
Intestines
Spleen + pancreas
Lungs
Heart
Stomach
Brain
Blood
Carcass
0.1 0.0
5.7 0.7
25.8 3.4
6.1 1.2
0.8 0.2
2.6 1.7
0.9 0.1
0.9 0.1
0.2 0.0
50.9 7.0
34.3 4.6
2.8 1.2
1.0 0.2
7.1 0.4
73.4 4.0
0.4 0.1
0.3 0.1
0.2 0.0
0.4 0.2
0.0 0.0
4.2 1.5
12.2 2.1
7.2 0.6
12.1 1.7
1.6 0.2
2.9 0.4
8.5 3.1
5.1 0.4
1.1 0.3
0.9 0.1
18.6 1.2
1.4 0.4
3.4 0.5
19.8 1.3
1.2 0.4
1.1 0.4
0.8 0.2
0.2 0.1
0.2 0.1
1.6 0.6
2.4. Partition coefficient
Data are expressed as mean SD; n = 4 per time point; p.i. = post injection.
a
Percentage of injected dose calculated as cpm in organ/total cpm recovered.
Percentage of injected dose per gram tissue.
The log partition coefficient (log P) values of [11C]-6 and [11C]-
13 were found to be À0.1 and 1.4, respectively. Since in literature
no mammalian active transporters are described for this kind of
tracers, we assume that uptake in cells and in the brain must occur
by passive diffusion. According to Dischino et al.,31 C-11 labeled
compounds with log P values between 0.9 and 2.5 are able to pass
freely across the blood–brain barrier (BBB). Extrapolating this to
cell membrane penetration, we could postulate that the naphthylic
tracer [11C]-13 could have a more efficient uptake in LacZ express-
ing cells due to its increased lipophilicity. [11C]-6 has a negative
log P value and will presumably be too hydrophilic for good cell
entry.
b
should be less than 650 Da and the compound should be un-
charged.31,32 Both tracers [11C]-6 and [11C]-13 are uncharged at
physiological pH and their molecular mass is 336 Da and 386 Da,
respectively. [11C]-6 has a negative log P value (À0.1) and is prob-
ably too hydrophilic to cross the BBB freely, explaining its low con-
centration in the brain. The naphthylic tracer [11C]-13 on the other
hand has an additional aromatic ring which makes it more lipo-
philic. Despite the presence of the polar sugar moiety, the addition
of the naphthalene ring increases its lipophilicity to a log P value of
1.4. Although [11C]-13 fulfills all the above-mentioned theoretical
requirements, it also shows negligible brain uptake. This implies
that besides the general requirements of lipophilicity (log P esti-
mation), molecular mass and charge, other factors or properties
of the molecule do influence cell membrane penetration. Presum-
ably, the presence of the polar hydroxyl-bearing sugar moiety is
detrimental for BBB penetration of these galactose-derivatives,
irrespective of their overall lipophilicity. Apart from the partition
2.5. Biodistribution in normal mice
The results of the in vivo distribution studies of [11C]-6 and
[
11C]-13 in male NMRI mice at 2 min and 60 min p.i. are shown
in Table 1 and Table 2. As described above, for a reasonable uptake
in the brain via passive diffusion, the log P value of a compound
should be between 0.9 and 2.5. Furthermore, the molecular mass
CH2OH
CH2OH
N
N
N
N
HO
O
HO
O
N
11CH3I , 70 ºC
N
OH
OH
300 µL DMF
5 µL NaOH (1 M)
O11CH3
OH
OH
OH
4
[
11C]- 6
Scheme 3. Radiolabeling of precursor 4 yielding 1-(b-
D
-galactopyranosyl)-4-(p-[11C]methoxyphenyl)-1,2,3-triazole ([11C]-6). The naphthylic tracer 1-(b-
D-galactopyranosyl)-
4-(6-[11C]methoxynaphthyl)-1,2,3-triazole ([11C]-13) was synthesized following the same labeling procedure with compound 11 as precursor.

S. Celen et al. / Bioorg. Med. Chem. 17 (2009) 5117–5125
5121
coefficient, another important factor in the prediction of passive
2.0
1.5
1.0
0.5
0.0
diffusion of a compound across membranes is the overall hydrogen
bond capacity, which can be estimated by calculating the molecu-
lar polar surface area (PSA) of the compound.33 This PSA is defined
as the sum of surface contributions of polar atoms, usually oxy-
gens, nitrogens and attached hydrogens, in a molecule34 and has
been shown to correlate well with BBB penetration.35 Since our
radiolabeled galactose-derivatives have four polar hydroxyl func-
tions, the PSA will be large (130 Å2 compared to e.g., 24 Å2 for eth-
anol)34 and thus the hydrogen bond interaction will probably be
too high to allow efficient passive diffusion of these tracers across
the BBB. Nevertheless, the sugar-hydroxyl functions should be
present because they are essential for binding to the active site
[
[
11C]-13
11C]-6
0
30
60
90
120
Incubation time (min)
Figure 2. Time-dependent uptake levels of [11C]-6 and [11C]-13 in LacZ gene (Ç)-
and VZV-tk gene (s)-transduced 293T cells. Data are expressed as % radioactivity in
the cell fraction and are mean values of triplicate samples.
of the enzyme.36,37 11C]-6 was cleared from blood mainly via the
[
renal pathway with 47.5% of the injected dose (%ID) in the urine
at 60 min p.i. and to a lesser extent via the liver to the intestines.
In accordance to its higher log P value, the more lipophilic [11C]-
13 was excreted via the hepatobiliary system with 73.4% ID in
the intestines. Only a negligible fraction (3% ID) was found in the
urine at 60 min p.i.
are shown in Figure 2. The phenylic tracer ([11C]-6) had a poor cell
uptake: the percentage of radioactivity in the cell fraction did not
exceed 0.4% (Fig. 2, 120 min incubation time point). Compared to
[
11C]-6 and the previously synthesized galactosyl ethers17 and
galactosyl esters (submitted), the more lipophilic napthylic com-
pound [11C]-13 had an increased uptake of radioactivity in the cell
fraction (not normalized to protein content) and the accumulation
in the cells increased as a function of the incubation time from 0.6%
after 30 min of incubation to 1.4% after 120 min of incubation.
However, there was no significant difference in cell uptake be-
tween the LacZ expressing cells and the control cells (Fig. 2). This
can be due to the decreased sensitivity of the inhibitor approach
compared to the use of radiolabeled substrates. According to liter-
2.6. Biostability in normal mice
The metabolic stability was assessed in plasma and urine col-
lected from normal mice 30 min after injection of the tracer using
RP-HPLC. The cold intact compound (6 or 13) was co-injected onto
HPLC to identify the intact parent tracer. An example of the analy-
sis of urine obtained 30 min p.i. of [11C]-6 and of a plasma sample
obtained at 30 min p.i. of [11C]-13 is shown in Figures 1a and b. For
ature, 1-(b-D-galactopyranosyl)-4-phenyl-1,2,3-triazole, which has
a similar structure as compound 6 but without the methoxy group,
[
11C]-6, in plasma as well as in urine, almost 100% of the recovered
has an inhibition constant that is situated in the high micromolar
radioactivity was present as intact tracer. The same result was
found for the plasma analysis of [11C]-13 (metabolite analysis of
a urine sample was not performed), indicating that both tracers
are very stable in vivo.
range (Ki ꢀ 330
l
M).18 PET imaging of gene expression using radi-
olabeled inhibitors is comparable to the use of radiolabeled PET li-
gands for imaging receptor occupancy, in which the interaction
between receptor and ligand also has a 1:1 stoichiometry. How-
ever, to have a good target to background signal, PET imaging of
receptors requires ligands with an affinity in the nanomolar range.
Using ‘click’ chemistry, libraries of different variants of galactosyl
triazoles can be prepared that can be used to identify inhibitors
with higher affinity. Furthermore to increase the signal to noise ra-
tio it could be preferred to synthesize 18F-labeled galactosyl tria-
zoles that, because of their longer half-life compared to 11C-
labeled tracers, allow to perform imaging up to several hours post
injection. By this time, the tracer will probably be better cleared
from cells and tissue that do not express the LacZ gene, resulting
in better in vitro and in vivo contrast.
2.7. In vitro evaluation of the tracers
Both tracers were further evaluated in vitro by assessing their
uptake and accumulation in b-gal expressing and VZV-TK express-
ing (control) HEK-293T cells. The results of these cell experiments
1
3. Conclusion
An efficient and convenient chemical and radiochemical syn-
thesis of two 4-substituted 1,2,3-triazolyl b-D-galactopyranosides
0
0
5
10
15
20
25
30
was developed. The two precursors 4 and 11 and the two non-
radioactive reference compounds 6 and 13 were synthesized in
good yields using a Cu(I)-catalyzed 1,3-dipolar cycloaddition reac-
tion between acetylated b-galactosyl azide and the corresponding
terminal alkynes. Radiolabeling produced [11C]-6 and [11C]-13 in
amounts and purity suitable for PET studies. Both tracers were very
stable in vivo. Cell uptake experiments in LacZ expressing and con-
trol 293T cells, revealed an increased cell uptake for the naphthylic
tracer [11C]-13 compared to the phenylic triazole [11C]-6. However,
no difference in uptake was observed between LacZ expressing
cells and control cells, which is presumably due to the decreased
sensitivity using radiolabeled inhibitors instead of substrates.
Development of lipophilic 11C- and 18F-labeled b-galactosyl tria-
zoles with a higher binding affinity for LacZ may lead to higher cell
uptake ratios and better in vivo imaging contrasts.
Figure 1a. Metabolite analysis of [11C]-6: RP-HPLC radiochromatogram of a urine
sample from a normal mouse collected 30 min p.i. Fractions were collected per 20 s.
1
0
0
5
10
15
20
25
30
35
40
Figure 1b. Metabolite analysis of
[
11C]-13: RP-HPLC radiochromatogram of
a
plasma sample from a normal mouse collected 30 min p.i. Fractions were collected
per minute.

5122
S. Celen et al. / Bioorg. Med. Chem. 17 (2009) 5117–5125
and was used as such for the synthesis of compound 2 without fur-
ther purification.
4. Experimental
4.1. General
4.2.2. 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl azide (2)
4-Ethynylphenylacetate was purchased from GFS chemicals
(Columbus, Ohio, USA). 4-Ethynylanisole was purchased from May-
bridge (Cornwall, UK). All other reagents and solvents were ob-
tained commercially from Acros Organics (Geel, Belgium), Aldrich,
Fluka, Sigma (Sigma–Aldrich, Bornem, Belgium), Merck (Darmstadt,
Germany) or Fischer Bioblock Scientific (Tournai, Belgium) and
used as supplied. For ascending thin layer chromatography (TLC),
pre-coated aluminum backed plates (Silica Gel 60 with fluorescent
indicator, 0.2 mm thickness; Macherey-Nagel, Düren, Germany)
were used and developed using mixtures of ethyl acetate and hep-
tane, ethyl acetate and methanol or acetonitrile and water as mo-
bile phase. After evaporation of the solvent, compounds were
detected under UV light (254 nm). The sugar compounds were
additionally visualized by spraying with an oxidating solution (5%
H2SO4 in ether), followed by heating. 1H NMR spectra were re-
corded on a Bruker AVANCE 300 MHz spectrometer (Bruker AG,
Faellanden, Zwitserland) using CDCl3 or DMSO-d6 as solvent. Chem-
ical shifts are reported in parts per million relative to tetramethyl-
silane (d = 0). Coupling constants are reported in hertz. Splitting
patterns are defined by s (singlet), d (doublet), dd (double doublet),
t (triplet) or m (multiplet). High performance liquid chromatogra-
phy (HPLC) purification and analysis was performed either on a
Merck Hitachi L6200 intelligent pump (Hitachi, Tokyo, Japan) or
on a Waters 600 pump (Waters Corporation, Milford, USA) con-
Compound 2 was prepared from 2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl bromide (1) according to the method of Jarrah-
pour et al.26 Yield: 41% of white crystals.
1H NMR (CDCl3) d 1.99, 2.07, 2.1, 2.17 (12H, 4 Â s, 4 Â CH3CO),
4.03 (1H, t, H-5), 4.16–4.19 (2H, m, 2 Â H-6), 4.62 (1H, d,
3
3
3JH1–H2 = 8.64 Hz, H-1), 5.05 (1H, dd, JH3-H2 = 10.35 Hz, JH3–H4
=
3
3
3.33 Hz, H-3), 5.17 (1H, dd, JH2–H3 = 10.35 Hz, JH2–H1 = 8.64 Hz,
3
H-2), 5.42 (1H, d, JH4–H3 = 3.33 Hz, H-4).
Accurate mass (ESI-MS) for C14H19N3O9 [M+Na]+: found
396.1015, calcd 396.1013.
4.2.3. 1-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-4-(p-
acetoxyphenyl)-1,2,3-triazole (3)
An amount of azide (2) (1.0 g, 2.7 mmol), CuSO4 (10 mg,
0.04 mmol), ascorbic acid (0.1 g, 0.6 mmol) and 4-ethynylpheny-
lacetate (0.43 g, 2.7 mmol) was stirred in water (15 mL) at 70 °C
overnight. The mixture was cooled in an ice bath, the solid was fil-
tered off and subsequently washed with water (10 mL) and meth-
anol (10 mL). Recrystallization from 95% ethanol yielded 1.05 g
(1.97 mmol, 73%) of a pale yellow solid.
1H NMR (CDCl3) d 1.91, 2.04, 2.07, 2.26, 2.33 (15H, 5 Â s,
5 Â CH3CO), 4.15–4.30 (3H, m, H-5; 2 Â H-6), 5.29 (1H, dd,
3
3
3JH3–H2 = 9.75 Hz, JH3–H4 = 3.24 Hz, H-3), 5.59 (1H, d, JH4–H3
=
3
3
3.24 Hz, H-4), 5.64 (1H, t, JH2–H3 = 9.75 Hz, JH2–H1 = 9.75 Hz, H-2),
nected to a UV-spectrometer (Waters 2487 Dual
c absorbance
5.9 (1H, d, JH1–H2 = 9.75 Hz, H-1), 7.19 (2H, d, 3J = 8.3 Hz, Har),
3
detector) set at 254 nm. The output signal was recorded and ana-
lyzed using a RaChel data acquisition system (Lablogic, Sheffield,
UK). For analysis of radiolabeled compounds, after passing through
the UV detector, the HPLC eluate was led over a 2-in. NaI(Tl) scintil-
lation detector connected to a single channel analyzer (Medilab-Se-
lect, Mechelen, Belgium). The radioactivity measurements during
log P determinations, biodistribution studies, in vivo stability anal-
ysis and cell uptake studies were done using an automated gamma
counter equipped with a 3-in. NaI(Tl) well crystal coupled to a
multichannel analyzer (Wallac 1480 Wizard, Wallac, Turku,
Finland). The values were corrected for background radiation and
physical decay during counting. Mass measurements were per-
7.89 (2H, d, 3J = 8.3 Hz, Har), 8.04 (1H, s, H-5 triazole).
Accurate mass (ESI-MS) for C24H27N3O11 [M+Na]+: found
556.1584, calcd 556.1538.
4.2.4. 1-(b-
D-Galactopyranosyl)-4-(p-hydroxyphenyl)-1,2,3-
triazole (4)
To a solution of compound 3 (0.14 g, 0.3 mmol) in methanol
(2 mL) was added dry NaOMe (1.4 mg, 0.03 mmol) and the reaction
mixture was stirred overnight at room temperature. Neutralization
of the solution with Amberlite IR-120 (H+) ion-exchange resin, fol-
lowed by filtration and evaporation of the filtrate to dryness, affor-
ded
a pale yellow solid. This solid was purified by flash
formed on
a time-of-flight spectrometer (LCT, Micromass,
chromatography on silica gel (ethyl acetate/methanol 9:1 v/v) to
afford 32 mg (0.09 mmol, 38%) of a white solid.
Manchester, UK) equipped with an orthogonal electrospray ioniza-
tion (ESI) interface. Samples were infused in acetonitrile/water
mixtures with a Harvard 22 syringe pump (Harvard Apparatus,
Holliston, Massachusetts, USA). Accurate mass determination was
1H NMR (DMSO-d6) d 3.4–3.6 (3H, m, H-5; 2 Â H-6), 3.7–3.79
3
(2H, m, H-3; H-4), 4.08 (1H, t, H-2), 5.47 (1H, d, JH1–H2 = 9.06 Hz,
H-1), 6.81 (2H, d, 3J = 8.31 Hz, Har), 7.68 (2H, d, 3J = 8.31 Hz, Har),
8.52 (1H, s, H-5 triazole).
done by co-infusion with a 10
b- -galactopyranoside (ONPG) in acetonitrile/water as an internal
calibration standard in positive mode (ES+) and a 10 g/mL solution
lg/mL solution of o-nitrophenyl
D
Accurate mass (ESI-MS) for C14H17N3O6 [M+Na]+: found
346.1027, calcd 346.1010. Mp 207–208 °C.
l
of meso-2,3-dibromosuccinic acid in water/acetonitrile as internal
calibration standard in negative mode (ESÀ). For compound 4,
kryptofix 222 was used as internal calibration standard in positive
mode. Acquisition and processing of the data was done with
MASSLYNXTM software (version 3.5, Waters). Melting points (mp) were
determined using an IA9000 digital melting point apparatus
(Electrothermal, Southend-on-Sea, England).
The 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.
4.2.5. 1-(2,3,4,6-Tetra-O-acetyl-b-
methoxyphenyl)-1,2,3-triazole (5)
The title compound was prepared from acetylated b-
D-galactopyranosyl)-4-(p-
D-galacto-
pyranosyl azide (2) and 4-ethynylanisole according to the method
described for the synthesis of compound 3. Yield: 0.57 g (1.1 mmol,
42%) of a fluffy, white solid.
1H NMR (CDCl3) d 1.90, 2.02, 2.05, 2.25 (12H, 4 Â s, 4 Â CH3CO),
3.85 (3H, s, CH3O), 4.15–4.29 (3H, m, H-5; 2 Â H-6), 5.28 (1H, dd,
3
3
3JH3–H2 = 9.9 Hz, JH3–H4 = 3.27 Hz, H-3), 5.57 (1H, d, JH4–H3
=
3
3
4.2. Synthesis
3.27 Hz, H-4), 5.64 (1H, t, JH2–H3 = 9.9 Hz, JH2–H1 = 9.9 Hz, H-2),
5.9 (1H, d, JH1–H2 = 9.9 Hz, H-1), 6.97 (2H, d, 3J = 8.64 Hz, Har),
3
4.2.1. 2,3,4,6-Tetra-O-acetyl-
Compound 1 was prepared from 1,2,3,4,6-penta-O-acetyl-b-
galactopyranose according to the method of Hanessian et al.25
a-
D
-galactopyranosyl bromide (1)
7.78 (2H, d, 3J = 8.64 Hz, Har), 7.97 (1H, s, H-5 triazole).
Accurate mass (ESI-MS) for C23H27N3O10 [M+Na]+: found
528.1608, calcd 528.1589.
D
-

S. Celen et al. / Bioorg. Med. Chem. 17 (2009) 5117–5125
5123
4.2.6. 1-(b-
triazole (6)
D
-Galactopyranosyl)-4-(p-methoxyphenyl)-1,2,3-
gel column that was eluted with n-heptane. Yield: 0.33 g
(1.96 mmol, 98%) of a brown solid.
To a stirring solution of compound 5 (0.13 g, 0.3 mmol) in meth-
anol (2 mL) was added dry NaOMe (1.4 mg, 0.03 mmol). After
30 min, colorless crystals were formed. After stirring overnight at
room temperature, the crystals were filtered off, washed with cold
methanol and dried. Yield: 70 mg (0.21 mmol, 80%).
1H NMR (CDCl3) d 3.1 (1H, s, C„CH), 7.1–7.13 (2H, m, Har), 7.45
(1H, d, 3J = 8.52 Hz, Har), 7.58 (1H, d, 3J = 9.57 Hz, Har), 7.69 (1H, d,
3J = 9.57 Hz, Har), 7.94 (1H, s, Har).
Accurate mass (ESI-MS) for C12H8O [MÀH]À: found 167.0495,
calcd 167.0502.
1H NMR (DMSO-d6) d 3.52–3.58 (3H, m, H-5; 2 Â H-6), 3.73–
3.77 (2H, m, H-3; H-4), 3.79 (3H, s, CH3O), 4.07–4.11 (1H, m, H-
2), 4.71 (t, OH), 4.74 (d, OH), 5.08 (d, OH), 5.28 (d, OH), 5.49 (1H,
4.2.10. 1-(2,3,4,6-Tetra-O-acetyl-b-
hydroxynaphthyl)-1,2,3-triazole (10)
The title compound was prepared from acetylated b-D-galacto-
D-galactopyranosyl)-4-(6-
3
d, JH1–H2 = 9.15 Hz, H-1), 7.02 (2H, d, 3J = 8.8 Hz, Har), 7.82 (2H, d,
3J = 8.8 Hz, Har), 8.64 (1H, s, H-5 triazole).
pyranosyl azide (2) and 6-ethynyl-2-naphthol (9) according to
the method described for the synthesis of compound 3. Yield:
0.22 g (0.4 mmol, 19%) of a yellow solid.
Accurate mass (ESI-MS) for C15H19N3O6 [M+H]+: found
338.1329, calcd 338.1347. Mp 240–241 °C.
1H NMR (CDCl3) d 1.9, 2.04, 2.06, 2.27 (12H, 4 Â s, 4 Â CH3CO),
3
4.2.7. 6-Bromo-2-tert-butyldimethylsilyloxy-naphthalene (7)
The title compound was synthesized according to the meth-
od described in patent WO 2007/02810427 with minor adapta-
tions. To a solution of 6-bromo-2-naphthol (10 g, 44.6 mmol)
in anhydrous DMF (75 mL) was added, under nitrogen atmo-
sphere, imidazole (4.24 g, 62.4 mmol) followed by tert-butyldim-
ethylsilylchloride (TBSCl, 9.36 g, 62.4 mmol). After stirring at
room temperature for 18 h, the reaction mixture was poured
into ice water (300 mL) followed by an extraction with diethyl-
ether (100 mL, 4Â). The combined organic extracts were washed
with brine (100 mL), dried over MgSO4 and the solvent was
removed under vacuo. Yield: 12.25 g (36.3 mmol, 81.3%) of
colorless crystals.
4.21–4.27 (3H, m, H-5; 2 Â H-6), 5.3 (1H, dd, JH3–H2 = 10.23 Hz,
3
3JH3–H4 = 3.33 Hz, H-3), 5.59 (1H, d, JH4–H3 = 3.33 Hz, H-4), 5.67
3
3
(1H, t, JH2–H3 = 9.45 Hz, JH2–H1 = 9.45 Hz, H-2), 5.93 (1H, d,
3JH1–H2 = 9.45 Hz, H-1), 6.24 (s, OH), 7.1–7.14 (2H, m, Har), 7.65
(1H, d, 3J = 8.64 Hz, Har), 7.7 (1H, d, 3J = 8.64 Hz, Har), 7.82 (1H, d,
3J = 8.52 Hz, Har), 8.14 (1H, s, Har), 8.23 (1H, s, H-5 triazole).
Accurate mass (ESI-MS) for C26H26N3O10Na [M+Na]+: found
564.1664, calcd 564.1589.
4.2.11. 1-(b-
D-Galactopyranosyl)-4-(6-hydroxynaphthyl)-1,2,3-
triazole (11)
The title compound was prepared by deprotection of compound
10 according to the method described for the synthesis of com-
pound 4. Yield: 0.03 g (0.08 mmol, 31%) of a pale yellow solid.
1H NMR (DMSO-d6) d 3.52–3.6 (3H, m, H-5; 2 Â H-6), 3.74–3.81
(2H, m, H-3; H-4), 4.12 (1H, m, H-2), 5.52 (1H, d, 3JH1–H2 = 9.12 Hz,
H-1), 7.08–7.11 (2H, m, Har), 7.71 (1H, d, 3J = 8.64 Hz, Har), 7.78 (1H,
d, 3J = 8.64 Hz, Har), 7.89 (1H, d, 3J = 8.52 Hz, Har), 8.3 (1H, s, Har),
8.77 (1H, s, H-5 triazole).
1H NMR (CDCl3) d 0.24 (6H, s, OSi(CH3)2), 1.01 (9H, s, C(CH3)3),
7.09 (1H, d, 3J = 8.82 Hz, Har), 7.15 (1H, s, Har), 7.47 (1H, d,
3J = 8.76 Hz, Har), 7.55 (1H, d, 3J = 8.76 Hz, Har), 7.62 (1H, d,
3J = 8.82 Hz, Har), 7.91 (1H, s, Har).
4.2.8. 6-Trimethylsilyl ethynyl-2-tert-butyldimethylsilyloxy-
naphthalene (8)
Accurate mass (ESI-MS) for C18H19N3O6 [M+Na]+: found
396.1156, calcd 396.1166. Mp 216–217 °C.
Compound 8 was prepared according to the method of Carpita
et al.28 with minor adaptations. To a mixture of bis(benzonitrile)-
palladium(II)chloride (PdCl2(PhCN)2, 0.16 g, 0.4 mmol) and CuI
(54 mg, 0.29 mmol) in 1,4-dioxane (5 mL) was added a solution
of compound 7 (4.82 g, 14.3 mmol) and tri(tert-butyl)phosphine
4.2.12. 1-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-4-(6-
methoxynaphthyl)-1,2,3-triazole (12)
The title compound was prepared from acetylated b-D-galacto-
(214
l
L, 0.86 mmol) in 1,4-dioxane (5 mL), followed by the addi-
pyranosyl azide (2) and 2-ethynyl-6-methoxy-naphthalene
according to the method described for the synthesis of compound
3. Yield: 0.6 g (1.1 mmol, 41%) of a white solid.
tion of trimethylsilylacetylene (2.35 mL, 17.1 mmol) and diisopro-
pylamine (2.4 mL, 17.1 mmol). The resulting mixture was stirred
overnight at room temperature under nitrogen. The black reaction
mixture was diluted with ethyl acetate (100 mL), filtered over Cel-
ite and the filtrate was concentrated under reduced pressure to
give a red oil. This oil was purified by flash column chromatogra-
phy on silica gel with n-heptane as eluent. Yield: 1.2 g (3.4 mmol,
23.7%) of orange crystals.
1H NMR (CDCl3) d 2.03, 2.04, 2.05, 2.26 (12H, 4 Â s, 4 Â CH3CO),
3.94 (3H, s, CH3O), 4.19–4.27 (3H, m, H-5; 2 Â H-6), 5.3 (1H, dd,
3
3
3JH3–H2 = 10.2 Hz, JH3–H4 = 3.21 Hz, H-3), 5.58 (1H, d, JH4–H3
=
3
3
3.21 Hz, H-4), 5.68 (1H, t, JH2–H3 = 9.6 Hz, JH2–H1 = 9.6 Hz, H-2),
3
5.92 (1H, d, JH1–H2 = 9.6 Hz, H-1), 7.15–7.19 (2H, m, Har), 7.79
(2H, d, 3J = 9.24 Hz, Har), 7.91 (1H, d, 3J = 7.5 Hz, Har), 8.13 (1H, s,
Har), 8.3 (1H, s, H-5 triazole).
1H NMR (CDCl3) d 0.25 (6H, s, OSi(CH3)2), 0.27 (9H, s, CSi(CH3)3),
1.01 (9H, s, C(CH3)3), 7.06 (1H, dd, 3J = 8.79 Hz, 4J = 2.4 Hz, Har), 7.13
(1H, d, 4J = 2.4 Hz, Har), 7.43 (1H, d, 3J = 8.79 Hz, Har), 7.62 (1H, d,
3J = 8.97 Hz, Har), 7.66 (1H, d, 3J = 8.97 Hz, Har), 7.91 (1H, s, Har).
Accurate mass (ESI-MS) for C14H19N3O9 [M+Na]+: found
578.1834, calcd 578.1745.
4.2.13. 1-(b-
D-Galactopyranosyl)-4-(6-methoxynaphthyl)-1,2,3-
4.2.9. 6-Ethynyl-2-naphthol (9)
triazole (13)
The title compound was synthesized according to the method of
Pirali et al.29 with minor adaptations. A volume of tetrabutylam-
monium fluoride solution (TBAF, 1 M in THF, 2.35 mL) was added
slowly to a solution of compound 8 (0.71 g, 2 mmol) in THF
(5 mL) at 0 °C and was further stirred at this temperature for
30 min. Next, a saturated solution of NH4Cl (15 mL) was added
and the mixture was extracted with ethyl acetate (15 mL, 3Â).
The combined organic fractions were washed with water (15 mL)
and brine (15 mL) and subsequently dried with MgSO4. An amount
of silica gel was added and the solvent was evaporated. The silica
gel impregnated with the crude product was applied on a silica
The title compound was prepared by deprotection of compound
12 according to the method described for the synthesis of com-
pound 6. Yield: 0.31 g (0.8 mmol, 80%) of a fluffy, white solid.
1H NMR (DMSO-d6) d 3.54–3.63 (3H, m, H-5; 2 Â H-6), 3.77–
3.82 (2H, m, H-3; H-4), 3.89 (3H, s, CH3O), 4.1–4.18 (1H, m, H-2),
4.72–4.76 (m, 2 Â OH), 5.09 (d, OH), 5.32 (d, OH), 5.56 (1H, d,
3JH1–H2 = 9.12 Hz, H-1), 7.2 (1H, d, 3J = 8.88 Hz, Har), 7.34 (1H, s,
Har), 7.88 (2H, d, 3J = 8.61 Hz, Har), 8.01 (1H, d, 3J = 8.52 Hz, Har),
8.4 (1H, s, Har), 8.83 (1H, s, H-5 triazole).
Accurate mass (ESI-MS) for C19H21N3O6 [M+Na]+: found
410.1363, calcd 410.1323. Mp 272–274 °C.

5124
S. Celen et al. / Bioorg. Med. Chem. 17 (2009) 5117–5125
4.3. Radiochemistry
10-lL sample was collected and analyzed for the presence of o-
nitrophenol (hydrolysis product of ONPG) using the same RP-HPLC
method.
4.3.1. Production of [11C]methyl iodide ([11C]MeI) and
radiosynthesis of [11C]-6
Carbon-11 was produced by a 14N(p,
a)
11C nuclear reaction. The
4.4.2. Incubation of [11C]-6 with b-gal
target gas (a mixture of 95% N2 and 5% H2) was irradiated using 18-
MeV protons at a beam current of 25 A for about 30 min, to yield
11C]CH4. The [11C]CH4 was then reacted with vaporous I2 at 650 °C
in a home-built recirculation synthesis module to convert it to
11C]MeI. The resulting volatile [11C]MeI was bubbled with a flow
of helium through a solution of 0.8 mg precursor (4) in 0.3 mL
DMF containing 5 L NaOH 1 M. When the radioactivity in the vial
An aliquot of 0.2 mL HPLC purified [11C]-6 (10 MBq) was added
to 0.4 mL water and 0.2 mL of a HEPES buffered solution. A volume
l
[
of 67
3.2.1.23, 149 U/mg, Fluka) was added (=10 U) and the solution was
incubated for 60 min at 37 °C (n = 1). A 10- L sample was collected
lL of a 1 mg/mL HEPES buffered solution of b-gal (E. coli, E.C.
[
l
and analyzed using the same RP-HPLC method (coupled to radio-
metric detection) as described for the incubation experiment with
non-radioactive triazole.
l
had stabilized, the reaction mixture was heated at 70 °C for 3 min.
Subsequently, unreacted [11C]MeI was evaporated by heating the
mixture at 70 °C for 2 min with a flow of helium. After cooling,
4.5. Partition coefficient (log Pn-octanol/phosphate buffer pH 7.4
)
the resulting mixture was diluted with 1.2 mL water and purified
TM
by semi-preparative HPLC on an XTerra RP C18 column (5
l
m,
An aliquot of 30 lL of RP-HPLC purified tracer solution contain-
7.8 mm x 150 mm; Waters), eluted with 0.1 M ammonium acetate
buffer pH 7.1/ethanol (85:15 v/v) at a flow rate of 2 mL/min. The
radiolabeled compound [11C]-6 eluted after 14 min with a radio-
chemical yield of approximately 51% (relative to starting activity
ing approximately 555 kBq of [11C]-6 was added to a tube contain-
ing 2 mL 0.025 M sodium phosphate buffer pH 7.4 and 2 mL n-
octanol (density = 0.827 g/mL). The tube was vortexed at room
temperature for 2 min followed by centrifugation at 3000 rpm
(1837 g) for 5 min (Eppendorf centrifuge 5810, Eppendorf, West-
of [11C]MeI). Radiochemical purity and specific activity were as-
TM
sayed using HPLC on an analytical XTerra RP C18 column (5
l
m,
bury, USA). Aliquots of 900 lL and 100 lL were drawn from the
4.6 mm  250 mm; Waters) eluted with gradient mixtures of
0.1 M ammonium acetate buffer pH 7.1/acetonitrile (0 min:
90:10 v/v, 20 min: 30:70 v/v, linear gradient) at a flow rate of
1 mL/min. [11C]-6 eluted after 9.5 min and was obtained with an
average specific activity of 118 GBq/lmol at the end of synthesis
(EOS) and a radiochemical purity of >99%.
n-octanol and aqueous phases, respectively, taking care to avoid
cross-contamination between the two phases. The samples were
weighed and their radioactivity was counted using an automated
gamma counter. After correcting for density and mass difference
between the two phases, the partition coefficient (P) was calcu-
lated as [radioactivity (cpm/mL) in n-octanol]/[radioactivity
(cpm/mL) in phosphate buffer pH 7.4]. The experiments were per-
formed at least in triplicate.
4.3.2. Radiosynthesis of [11C]-13 using [11C]MeI
Radiosynthesis of [11C]-13 was performed following the same
The same procedure was followed for [11C]-13 except that the
procedure as described for [11C]-6 with compound 11 as precursor.
volumes were switched: 100
lL was taken from the octanol layer
Purification of the crude radiolabeling mixture was done by semi-
preparative HPLC on an XTerra RP C18 column (5 lm,
and 900 L was taken from the buffer layer.
l
TM
7.8 mm  150 mm; Waters), eluted with 0.1 M ammonium acetate
buffer pH 7.1/ethanol (70:30 v/v) at a flow rate of 2 mL/min. The
radiolabeled compound [11C]-13 eluted after 16 min with a radio-
chemical yield of approximately 48% (relative to starting activity of
4.6. Biodistribution study in normal mice
Biodistribution of both tracers was studied in male NMRI mice
(body mass 35–45 g). These mice were anesthetized with 2.5% iso-
flurane in 100% O2 at a flow rate of 1 L/min. Solutions of [11C]-6 and
[
11C]MeI). The purified tracer was further analyzed by HPLC on an
analytical XTerra RP C18 column (Waters) eluted with gradient
mixtures of 0.1 M ammonium acetate buffer pH 7.1/acetonitrile
(0 min: 90:10 v/v, 20 min: 30:70 v/v, linear gradient) at a flow rate
of 1 mL/min. [11C]-13 eluted after 13.5 min and was obtained with
[
11C]-13 obtained after RP-HPLC purification were diluted using
TM
0.9% NaCl for injection to obtain an ethanol concentration < 10%,
and further to a concentration of about 37 MBq/mL. A volume of
0.1 mL of the diluted purified tracer solution was then injected
via a tail vein in the anesthetized mice and the mice were sacrificed
by decapitation at 2 or 60 min post injection (p.i., n = 4 per time
point). Blood and major organs were collected in tared tubes and
weighed. The radioactivity in blood, organs and other body parts
was counted using a 3-in. NaI(Tl) well counter, corrected for back-
ground radioactivity and expressed as percentage of the injected
dose (% ID) or as percentage of the injected dose per gram tissue
(% ID/g). For the calculation of total radioactivity in blood, blood
mass was assumed to be 7% of the body mass.
an average specific activity of 61 GBq/lmol at the end of synthesis
(EOS) and a radiochemical purity of >99%.
4.4. In vitro incubation experiments with purified b-gal
4.4.1. Incubation of non-radioactive compound 6 with b-gal
An amount of 1 mg of compound 6 was dissolved in 1 mL of a
solution containing HEPES buffer pH 7.0 (120 mM), MgSO4
(4 mM) and NaCl (180 mM). A volume of 67 lL of a 1 mg/mL HEPES
buffered solution of b-gal (E. coli, E.C. 3.2.1.23, 149 U/mg, Fluka)
was added (=10 U) and the solution was incubated at 37 °C
(n = 1). After 60 min of incubation, the mixture was analyzed for
4.7. Biostability in normal mice
the presence of metabolites using RP-HPLC coupled to UV detec-
Metabolic stability of [11C]-6 and [11C]-13 was studied in NMRI
mice by determination of the relative amounts of the parent tracer
and radiolabeled metabolites in plasma and in urine. After intrave-
nous administration of about 18 MBq of [11C]-6 or [11C]-13 into
anesthetized mice (isoflurane), the animals were sacrificed by
decapitation at 10 or 30 min p.i. (n = 2), blood was collected into
TM
tion using an XTerra RP C18 column (5
lm, 4.6 mm  250 mm;
Waters) eluted with a mixture of 30% acetonitrile in 0.1 M ammo-
nium acetate buffer pH 7.1 at a flow rate of 1 mL/min. After 20 min
the percentage acetonitrile was increased to 70% in order to elute
possible lipophilic hydrolysis products. The same HPLC analysis
was performed after incubation overnight. Next, the experiment
was repeated, but 1 mg ONPG in 1 mL HEPES buffered solution
was added to the incubation mixture of compound 6 with 10 U
b-gal at 37 °C. 15 min and 90 min after the addition of ONPG, a
TM
a BD vacutainer (containing lithium heparin; BD, Franklin Lakes,
USA) and stored on ice. The samples were then centrifuged at
3000 rpm (1837g) for 5 min (Eppendorf centrifuge 5810) to sepa-
rate plasma. A volume of 0.5 mL of plasma sample was isolated,

S. Celen et al. / Bioorg. Med. Chem. 17 (2009) 5117–5125
5125
mixed with 15
l
L of a 1 mg/mL solution of authentic non-radioac-
nological Research in industry (IWT), the IDO grant (IDO/02/012)
of the Katholieke Universiteit Leuven, and by the EC-FP6-project
DiMI, LSHB-CT-2005-512146.
tive 6 or 13 and injected onto a Chromolith Performance RP C18 col-
umn (3 mm  100 mm; Merck), that was eluted with gradient
mixtures of 0.05 M ammonium acetate buffer pH 6.8/acetonitrile
(0 min: 100:0 v/v, 4 min: 100:0 v/v, 20 min: 30:70 v/v, 30 min:
10:90 v/v, linear gradient for [11C]-6 and 0 min: 100:0 v/v, 4 min:
100:0 v/v, 25 min: 10:90 v/v, 35 min: 10:90 v/v, linear gradient
for [11C]-13) at a flow rate of 1 mL/min. After passing through an
in-line UV detector, the HPLC-eluate was collected. The first 4-
min eluate, containing proteins of the biological matrix, was col-
lected in one fraction. The rest of the eluate was collected in
0.33-mL fractions (fraction collection each 20 s) for [11C]-6 and in
1-mL fractions for [11C]-13 using an automatic fraction collector.
The radioactivity in all fractions was measured using a gamma
counter. Urine samples were collected from mice sacrificed at
30 min p.i. by manual voiding of the bladder, mixed with authentic
6 and analyzed following the same procedure without any pre-
treatment or work-up. The whole eluate was collected in 0.33-
mL fractions and counted for radioactivity.
References and notes
1. Wood, K. V. Curr. Opin. Biotechnol. 1995, 6, 50.
2. Lin, W. C.; Pretlow, T. P.; Pretlow, T. G., 2d; Culp, L. A. Cancer Res. 1990, 50, 2808.
3. Tung, C.-H.; Zeng, Q.; Shah, K.; Kim, D.-E.; Schellingerhout, D.; Weissleder, R.
Cancer Res. 2004, 64, 1579.
4. Gambhir, S. S.; Barrio, J. R.; Herschman, H. R.; Phelps, M. E. Nucl. Med. Biol. 1999,
26, 481.
5. Herschman, H. R. Crit. Rev. Oncol. Hematol. 2004, 51, 191.
6. Acton, P. D.; Zhou, R. Q. J. Nucl. Med. Mol. Imaging 2005, 49, 349.
7. Gambhir, S. S.; Herschman, H. R.; Cherry, S. R.; Barrio, J. R.; Satyamurthy, N.;
Toyokuni, T.; Phelps, M. E.; Larson, S. M.; Balatoni, J.; Finn, R.; Sadelain, M.;
Tjuvajev, J.; Blasberg, R. Neoplasia 2000, 2, 118.
8. Tjuvajev, J. G.; Doubrovin, M.; Akhurst, T.; Cai, S. D.; Balatoni, J.; Alauddin, M.
M.; Finn, R.; Bornmann, W.; Thaler, H.; Conti, P. S.; Blasberg, R. G. J. Nucl. Med.
2002, 43, 1072.
9. Alauddin, M. M.; Shahinian, A.; Park, R.; Tohme, M.; Fissekis, J. D.; Conti, P. S. J.
Nucl. Med. 2004, 45, 2063.
10. Serganova, I.; Blasberg, R. Nucl. Med. Biol. 2005, 32, 763.
11. Bormans, G.; Verbruggen, A. J. Labelled Compd. Radiopharm. 2001, 44, 417.
12. Sahin-Tòth, M.; Akhoon, K. M.; Runner, J.; Kaback, H. R. Biochemistry 2000, 39,
5097.
4.8. In vitro evaluation of the tracers
13. Abramson, J.; Smirnova, I.; Kasho, V.; Verner, G.; Kaback, H. R.; Iwata, S. Science
2003, 301, 610.
14. Choi, J. H.; Choe, Y. S.; Lee, K. H.; Choi, Y.; Kim, S. E.; Kim, B. T. Carbohydr. Res.
2003, 338, 29.
15. Lee, K.-H.; Byun, S. S.; Choi, J. H.; Paik, J.-Y.; Choe, Y. S.; Kim, B.-T. Eur. J. Nucl.
Med. Mol. Imaging 2004, 31, 433.
16. Plagemann, P. G. W.; Wohlhueter, R. M.; Graff, J.; Erbe, J.; Wilkie, P. J. Biol. Chem.
1981, 256, 2835.
17. Celen, S.; Deroose, C.; de Groot, T.; Chitneni, S. K.; Gijsbers, R.; Debyser, Z.;
Mortelmans, L.; Verbruggen, A.; Bormans, G. Bioconjugate Chem. 2008, 19, 441.
18. Rossi, L. L.; Basu, A. Bioorg. Med. Chem. Lett. 2005, 15, 3596.
19. Périon, R.; Ferriéres, V.; García-Moreno, M. I.; Mellet, C. O.; Duval, R.; García
Fernández, J. M.; Plusquellec, D. Tetrahedron 2005, 61, 9118.
20. Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Eur. J. Org. Chem. 2006, 51.
21. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
22. Moses, J. E.; Moorhouse, A. D. Chem. Soc. Rev. 2007, 36, 1249.
23. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
24. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 7, 3057.
25. Hanessian, S.; Saavedra, O. M.; Mascitti, V.; Marterer, W.; Oehrlein, R.; Mak,
C.-P. Tetrahedron 2001, 57, 3267.
26. Jarrahpour, A. A.; Shekarriz, M.; Taslimi, A. Molecules 2004, 9, 29.
27. Auspex Pharmaceuticals (USA). Novel Therapeutic Agents for the Treatment
of Cancer, Metabolic Diseases and Skin Disorders. WO 2007/028104, 2007;
p 51.
A lentiviral vector (LV) encoding the cDNA of b-gal linked to the
puromycin-N-acetyl-transferase (pac) gene from Streptomyces
alboniger through an encephalomyocarditis virus internal ribosome
entry site (IRES) was produced as described38 and denominated
LV-LacZ-I-P. A similar vector encoding the varicella zoster virus
thymidine kinase (VZV-tk) was used as control (LV-VZV-tk-I-P).
Human embryonic kidney cells (293T) transduced with LV-LacZ-
I-P and with LV-VZV-tk-I-P were maintained in culture in medium
(Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax sup-
plemented with 10% heat-inactivated fetal calf serum) containing
1 lg/mL puromycin. They were plated in triplicate at a density of
200,000 cells per well in 24-well plates. After 24 h, the medium
was discarded and 0.25 mL of fresh culture medium with HPLC-
purified tracer (1.1 MBq per well) was added. The cells were then
incubated at 37 °C in a 5% CO2 atmosphere for time intervals of
30, 60, 90 or 120 min (n = 3 per time point, per vector). Following
incubation and removal of the medium, the cells were washed three
times with 0.4 mL of ice-cold phosphate-buffered saline (PBS). The
cells were then lysed with 0.25 mL of Cell Culture Lysis Reagent 1Â
solution (Promega Corporation, Madison, USA) for 10 min after
which the lysate was collected, followed by a 0.125-mL rinse using
the same solution. The cell fractions (lysate and rinse) as well as the
supernatant fractions (medium and PBS) were collected separately
for each well and the radioactivity was measured using a gamma
counter. This procedure was repeated for each incubation period.
Normalization for protein content was not performed.
28. Carpita, A.; Mannocci, L.; Rossi, R. Eur. J. Org. Chem. 2005, 1859.
29. Pirali, T.; Gatti, S.; Di Brisco, R.; Tacchi, S.; Zaninetti, R.; Brunelli, E.; Massarotti,
A.; Sorba, G.; Luigi Canonico, P.; Moro, L.; Genazzani, A. A.; Cesare Tron, G.;
Billington, R. A. Chem. Med. Chem. 2007, 2, 437 (Supplementary data).
30. Larsen, P.; Ulin, J.; Dahlstrom, K.; Jensen, M. Appl. Radiat. Isot. 1997, 48, 153.
31. Dischino, D. D.; Welch, M. J.; Kilbourn, M. R.; Raichle, M. E. J. Nucl. Med. 1983,
24, 1030.
32. Young, R. C.; Mitchell, R. C.; Brown, T. H.; Ganellin, C. R.; Griffiths, R.; Jones, M.;
Rana, K. K.; Saunders, D.; Smith, I. R.; Sore, N. E.; Wilks, T. J. J. Med. Chem. 1998,
31, 656.
33. Norinder, U.; Haeberlein, M. Adv. Drug Delivery Rev. 2002, 54, 291.
34. Ertl, P.; Rohde, B.; Selzer, P. Novartis Pharma AG, ChemInformatics, Basel,
Switzerland. http://www.daylight.com/meetings/emug00/Ertl/index.html.
35. Clark, D. E. J. Pharm. Sci. 1999, 88, 815.
Acknowledgements
36. Huber, R. E.; Gaunt, M. T. Arch. Biochem. Biophys. 1983, 220, 263.
37. McCarter, J. D.; Adam, M. J.; Withers, S. G. Biochem. J. 1992, 286, 721.
38. Geraerts, M.; Michiels, M.; Baekelandt, V.; Debyser, Z.; Gijsbers, R. J. Gene Med.
2005, 7, 1299.
We thank Peter Vermaelen and Kim Serdons for their help with
the biodistribution studies. This work was supported by SBO grant
(IWT-30 238) of the Flemish Institute supporting Scientific-Tech-