99mTc-tricarbonyl labeled agents for cell labeling: Development, biodistribution in normal mice and preliminary in vitro evaluation

Article abstract of DOI:10.1016/j.bmc.2009.10.045

We have developed four ^99mTc(CO)₃-labeled lipophilic tracers as potential radiolabeling agents for cells based on a hexadecyl tail. ^99mTc(CO)₃-hexadecylamino-N,N′-diacetic acid (negatively charged), ^99mTc(CO)₃-hexadecylamino-N-α-picolyl-N′-acetic acid (uncharged), ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine (positively charged), ^99mTc(CO)₃-N-hexadecylaminoethyl-N′-aminoethylamine (positively charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed in mixed blood cells with or without plasma and were compared with ^99mTc-d,l-HMPAO and [¹⁸F]FDG. In plasma-free blood cells, maximum uptake (78%) was obtained for ^99mTc(CO)₃-N-hexadecylaminoethyl-N′-aminoethylamine after 60 min incubation (compared to 55% and 23% for ^99mTc-d,l-HMPAO and [¹⁸F]FDG, respectively) while in plasma-rich medium, ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine was best bound (54%, similar to the binding of ^99mTc-d,l-HMPAO). Biodistribution in normal mice showed mainly hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the least lipophilic agent (^99mTc(CO)₃-N,N′-dipicolylhexadecylamine, log P = 1.3) showed the best cell uptake, there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In view of its comparable cell uptake to well known cell labeling agent ^99mTc-d,l-HMPAO, ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent.

Full text of DOI:10.1016/j.bmc.2009.10.045

Bioorganic & Medicinal Chemistry 18 (2010) 396–402
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
99mTc-tricarbonyl labeled agents for cell labeling: Development,
biodistribution in normal mice and preliminary in vitro evaluation
*
Humphrey Fonge , Lixin Jin, Jan Cleynhens, Guy Bormans, Alfons Verbruggen
Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences, K.U. Leuven, BE-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
We have developed four 99mTc(CO)3-labeled lipophilic tracers as potential radiolabeling agents for cells
Article history:
based on
a
hexadecyl tail. 99mTc(CO)3-hexadecylamino-N,N0-diacetic acid (negatively charged),
Received 3 August 2009
Revised 23 October 2009
Accepted 24 October 2009
Available online 31 October 2009
99mTc(CO)3-hexadecylamino-N-
a
-picolyl-N0-acetic acid (uncharged), 99mTc(CO)3-N,N0-dipicolylhexade-
cylamine (positively charged), 99mTc(CO)3-N-hexadecylaminoethyl-N0-aminoethylamine (positively
charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed
in mixed blood cells with or without plasma and were compared with 99mTc-d,l-HMPAO and [18F]FDG.
In plasma-free blood cells, maximum uptake (78%) was obtained for 99mTc(CO)3-N-hexadecylaminoeth-
yl-N0-aminoethylamine after 60 min incubation (compared to 55% and 23% for 99mTc-d,l-HMPAO and
Keywords:
Cell labeling
99mTc(CO)3
[
18F]FDG, respectively) while in plasma-rich medium, 99mTc(CO)3-N,N0-dipicolylhexadecylamine was best
Lipophilic hexadecyl derivatives
bound (54%, similar to the binding of 99mTc-d,l-HMPAO). Biodistribution in normal mice showed mainly
hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed
good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the
least lipophilic agent (99mTc(CO)3-N,N0-dipicolylhexadecylamine, log P = 1.3) showed the best cell uptake,
there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In
view of its comparable cell uptake to well known cell labeling agent 99mTc-d,l-HMPAO, 99mTc(CO)3-N,N0-
dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
these therapies to finally reach the bedside, questions such as opti-
mal time and route of delivery, their contribution to functional re-
Cell-based therapies hold great promise in the treatment of
many human diseases notably in cardiology, neurology and genet-
ically inheritable diseases.1–4 Tracking of these cells is necessary to
evaluate the efficacy of therapy as well as plan subsequent treat-
ment regimens. In cardiology, radiolabeled stem cells have been
used on numerous occasions to study the migration, distribution
and efficacy of therapy following administration of stem cells.5–7
In some instances labeled cells have been used to understand the
involvement of certain cells or factors in the pathogenesis of dis-
eases. For example, tracking of endothelial progenitor cells labeled
with [18F]-fluorodeoxylglucose ([18F]FDG) has been used to study
their involvement in vascuologenesis and angiogenesis hence their
involvement in tumor growth and development using positron
emission tomography (PET).8 These and many other interesting
preclinical studies on cell-based therapies have led to a growing
number of early phase human studies to demonstrate the potential
usefulness of these cell-based therapies. However, in order for
pair and biodistribution need to be answered. Labeling and
tracking of cells is therefore an invaluable tool in the evaluation
of cell-based therapies.
Indirect radionuclide labeling of cells using reporter genes-re-
porter probe concept provides one of the most elegant approaches
to track in vivo the fate of administered cells. Accumulation of the
reporter probe depends on the expression of a reporter gene prod-
uct which directly correlates with cell viability.9 However, design
of the reporter gene requires extensive molecular biological tech-
niques limiting this application to centers with such facilities. Indi-
rect labeling involving incubation of cells with appropriate
radiolabeling agents remains, however, very popular. Many radio-
pharmaceuticals have been used to radiolabel cells and track their
fate in vivo. Agents for positron emission tomography (PET) such as
[
18F]FDG only permit short tracking of labeled cells due to its short
half-life (t1/2 = 110 min). Recently, copper-64-pyruvaldehyde-
bis(N4-methylthiosemicarbazone) ([64Cu]PTSM, 64Cu, t1/2 = 12.7 h)
has been introduced as a potential cell radiolabeling agent even
though its potential usefulness remains to be fully evaluated.10
Technetium-99m labeled d,l-hexamethylpropylene amine oxime
* Corresponding author at present address: Department of Pharmaceutical
Sciences, University of Toronto, 144 College Street, Toronto ON, Canada M5S 3M2.
Tel.: +1 416 978 6628; fax: +1 416 978 8511.
(
99mTc-d,l-HMPAO, exametazime) is also a well established single
photon emission computed tomography (SPECT) labeling agent.11
E-mail address: humphrey.fonge@utoronto.ca (H. Fonge).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.045

H. Fonge et al. / Bioorg. Med. Chem. 18 (2010) 396–402
397
Recently, a simple method has been described for labeling stem
cells using a lipophilic long-chain ester, hexadecyl-4-[18F]fluor-
obenzoate ([18F]HFB), which is efficiently and quickly absorbed onto
the cellular membranes in a similar fashion as fluorescent dyes used
for cell labeling.12 Absorption onto cell membranes means less tox-
icity to cell’s nuclei resulting from Auger electrons as in the case of
indium-111 labeled cells. The short half-life of fluorine-18 means
these cells cannot be tracked for a long period in addition to the fact
that the use of short-lived cyclotron-based radionuclides is until
now limited to centers with an onsite cyclotron. Labeling with tech-
netium-99m (t1/2 = 6 h) would be an advantage for longer cell track-
þ
ing. Technetium-99m tricarbonyl
(
99mTcðCOÞ3
)
labeling of
biomolecules has the advantage of ease of preparation, efficient
complex formation and inertness of the complex due the d6 metal
core.13 We herein report the synthesis of lipophilic agents using li-
gand precursors with a hexadecyl tail and their labeling with
þ
99mTcðCOÞ3 precursor (Fig. 1A–D). We have studied their biodistri-
Scheme 1. Reaction scheme for the synthesis of hexadecylamino-N,N0-diacetic acid
bution in normal NMRI mice and evaluated their potential for cell
labeling in preliminary in vitro experiments using mixed blood cells.
Uptake in cells was compared with that of 99mTc-d,l-HMPAO and
þ
and labeling with 99mTcðCOÞ3
.
[
18F]FDG, which are well known cell labeling agents.
2. Results and discussion
2.1. Chemistry and radiolabeling
Synthesis and labeling of hexadecylamino-N,N0-diacetic acid
þ
with 99mTcðCOÞ3
(
99mTc(CO)3-hexadecylamino-N,N0-diacetic acid,
compound 3) is shown in Scheme 1. Synthesis of hexadecylami-
no-N,N0-diacetic acid was accomplished in two steps starting from
the alkylation of hexadecylamine with methyl bromoacetate fol-
lowed by base hydrolysis in an overall yield of 75%. Radiolabeling
þ
with the 99mTcðCOÞ3 precursor (pH 7.4) was accomplished by
incubating compound 2 with the precursor at 70 °C for 20 min.
An overall radiolabeling yield of 97% was obtained resulting in a
negatively charged complex (Fig. 1A, compound 3).
99mTc(CO)3-hexadecylamino-N- -picolyl-N0-acetic acid (com-
a
pound 8) was prepared in four steps as shown in Scheme 2 by
alkylation of -picolylamine with methyl bromoacetate followed
by monoester hydrolysis and in situ activation of the monomethyl
ester (
-picolyl-N-methylacetyl-N0-acetic acid) with benzotria-
zole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro-
phosphate (BOP) in the presence of hexadecylamine. Hydrolysis
yielded the final product hexadecylamino-N-
-picolyl-N0-acetic
a
a
Scheme 2. Reaction scheme for the synthesis of hexadecylamino-N-
a
-picolyl-N0-
þ
acetic acid and labeling with 99mTcðCOÞ3
.
a
99mTc(CO)3-N,N0-dipicolylhexadecylamine (compound 10) was
synthesized as shown in Scheme 3 (overall chemical yield for the
precursor was 12%). Radiolabeling (pH 7.4 at 70 °C for 20 min)
acid. Radiolabeling (pH 7.4 at 70 °C for 20 min) yield of 95% was
obtained resulting in a neutral complex (Fig. 1B, compound 8).
Figure 1. Proposed structure of (A) 99mTc(CO)3-hexadecylamino-N,N0-diacetic acid
(compound 3), (B) 99mTc(CO)3-hexadecylamino-N- -picolyl-N0-acetic acid (com-
a
pound 8), (C) 99mTc(CO)3-N,N0-dipicolylhexadecylamine (compound 10) and (D)
99mTc(CO)3-N-hexadecylaminoethyl-N0-aminoethylamine (compound 12).
Scheme 3. Reaction scheme for the synthesis of N,N0-dipicolylhexadecylamine and
þ
labeling with 99mTcðCOÞ3
.

398
H. Fonge et al. / Bioorg. Med. Chem. 18 (2010) 396–402
lipophilicity, it was necessary in this preliminary study to correlate
lipophilicity (log P) of the radioligands with their uptake in cells.
Octanol–buffer partition coefficient values provide a more reliable
measure of the relative lipophilicity. Log P values (mean of five
determinations standard deviation, SD) of the tracers ranged from
1.3 to 2.5. The compounds were ranked according to increasing log P
values ( standard deviation, SD): Compound 10 (1.3 0.04) < com-
pound 3 (2.17 0.34) < compound 12 (2.22 0.31) < compound 8
(2.50 0.21). The log P of [18F]HFB was not determined by Ma
et al.12 but is probably in the 2.5–3 range being a lipophilic ester,14
while log P of 99mTc-d,l-HMPAO is 1.9.15
2.3. Preliminary in vitro cell labeling
Scheme 4. Reaction scheme for the synthesis of N-hexadecylaminoethyl-N0-
Figure 3 shows that all the radiolabeled compounds were well
bound in plasma-free mixed blood cells after 60 min. Uptake of
the radiolabeled compounds in blood cells after an incubation per-
iod of 60 min (Fig. 3) in PBS (plasma-free mixed blood cells) with
mixed blood cells was of the order: compound 12 (positively
charged, 78%) > compound 8 (uncharged, 77%) > compound 10
þ
aminoethylamine and labeling with 99mTcðCOÞ3
.
yield: P90% was achieved. 99mTc(CO)3-N-hexadecylaminoethyl-N0-
aminoethylamine (compound 12) was prepared in a one step aky-
lation reaction of diethylenetriamine with 1-bromohexadecane.
Radiolabeling (pH 11 at 70 °C for 20 min) yield of 99% was achieved
(Scheme 4). Compounds 10 and 12 resulted in positively charged
complexes (Fig. 1C and D). The structures of the precursors and fi-
nal products were confirmed by high resolution mass spectroscopy
(HRMS) using different standards and 1HNMR. Stability study of
HPLC-purified compounds 3, 8, 10 and 12 was examined after re-
injection of the compounds on RP-HPLC over a 24 h period at rt.
All compounds were >97% stable during this period.
Figure 2A–C shows reversed-phase (RP)-HPLC chromatograms
of the crude radiolabeling mixture of compounds 3, 8, 10 and 12,
respectively. Compounds 8, 10, and 12 have identical retention
times (tR ꢀ10.1 min) on RP-HPLC while compound 3 (tR ꢀ8 min)
was least retained. Lipophilicity of the HPLC-purified compounds
was determined by their octanol–buffer partition coefficients.
(positively charged, 69%) > compound
3 (negatively charged,
55%). Cell uptake of all radiolabeled compounds was better than
that of [18F]FDG. When incubated with mixed blood cells contain-
ing plasma (Fig. 4), 54% of compound 10 was bound to blood cells
(compared to 55% binding of 99mTc-d,l-HMPAO) while compounds
3 and 8 were more associated with plasma. The amount of the radi-
olabeled compound bound to blood cells after an incubation period
of 60 min (in mixed blood cells containing plasma) was of the or-
der: compound 10 (54%) > compound 12 (46%) > compound 8
(35%) > compound 3 (24%). The order indicates that the positively
charged compound 10 had the best uptake in cells while the neg-
atively charged compound 3 was more associated with plasma.
High uptake in blood cells in plasma-rich medium means better
uptake of the radiolabeled agent in vivo and less transchelation
or association with plasma proteins. Although the least lipophilic
(compound 10, log P = 1.3) was the most bound to the cells, a clear
relationship between lipophilicity and tracer uptake in the cells
could not be established. Uptake of compound 10 was similar to
99mTc-d,l-HMPAO (Fig. 4), a widely used cell labeling compound.
2.2. Octanol–buffer partition coefficient
Since the labeling of cells with [18F]HFB was hypothesized to be
due to absorption of the agent to cell membrane as a result of its
Figure 2. RP-HPLC chromatograms of crude labeling mixture of (A) compounds 3, (B) compound 8, (C) compound 10 and (D) compound 12 (see text for HPLC condition).

H. Fonge et al. / Bioorg. Med. Chem. 18 (2010) 396–402
399
8 were more rapidly cleared. Negligible stomach uptake is an indi-
cation of the in vivo stability of the agents.
3. Conclusions
In vitro radiolabeling using mixed blood cells served as a useful
tool to evaluate the affinity of these lipophilic agents for blood cells
or lipoproteins. If a tracer with high association with plasma pro-
tein is used to radiolabel cells, the radiolabel will be rapidly lost
in vivo. On the other hand, a tracer with high uptake in blood cells
in a plasma-rich blood cell environment has good in vivo stability
when used to radiolabel cells. The results show that compound 10
(
9mTc(CO)3-N,N0-dipicolylhexadecylamine) is potentially the most
stable for in vivo tracking of radiolabeled cells: compound
10 > compound 12 > compound 8 > compound 3. Compound 10
shows more promising results as a potential cell labeling agent.
There was no relationship between the lipophilicity and labeling
efficiency of the radiolabeled compounds. Compound 10 shall be
further evaluated for its ability to label stem cells.
Figure 3. Percentage of tracer uptake after incubation in plasma-free mixed blood
cells for 60 min. In plasma-free mixed blood cells, all compounds were bound with
a maximum uptake obtained for positively charged compound 12, (99mTc-d,l-
HMPAO, 55%) while [18F]FDG (23%) were less taken-up.
4. Experimental
4.1. Reagents
Hexadecylamine, benzotriazole-1-yl-oxy-tris-(dimethylamino)-
phosphonium hexafluorophosphate (BOP), diethylenetriamine,
picolylchloride and triethylamine were from Sigma Aldrich (Stein-
heim, Germany); methyl bromoacetate was from Janssen Chimica
(Geel, Belgium); 2-chloromethylpyridine, bromohexadecane and
all solvents were obtained from Acros Organics (Geel, Belgium)
and were used as purchased. Generator eluate containing Na99mT-
cO4 was obtained from an Ultratechnekow generator (Tyco Health-
care, Petten, The Netherlands). The Isolink kit used for preparing
Figure 4. Percentage of tracer uptake after incubation in mixed blood cells with
plasma for 60 min. Compound 10 had the highest uptake (54%) while compounds 3
(35%) and 8 (24%) were more associated with plasma. In comparison 99mTc-d,l-
HMPAO and [18F]FDG had 55% and 18% uptake, respectively.
þ
99mTcðCOÞ3 was a generous gift from Tyco Healthcare.
4.2. Instrumentations
1H NMR spectra were acquired with a Gemini 300 MHz spec-
trometer (Varian, Palo Alto, CA, USA). Chemical shifts are reported
in parts per million (ppm) relative to TMS (d = 0). Coupling con-
stants are reported in hertz (Hz). Normal-phase column chroma-
tography was performed using silica gel (silica 63–200, 60 Å, MP
Biomedicals, Eschwege, Germany). High-performance liquid chro-
matography (HPLC) analysis was performed on a LaChrom Elite
HPLC system (Hitachi, Darmstadt, Germany) using an XTerra-RP
In comparison, [18F]FDG had 23% and 18% uptake in plasma-free
and plasma-rich mixed blood cells, respectively. Twenty-five per-
cent of [18F]HFB was bound to mesenchymal stem cells 30 min
after incubation with the compound in PBS.12
2.4. Biodistribution in normal mice
C18 column (5
l
m, 4.6 mm ꢁ 250 mm; Waters, Milford, USA). The
Biodistribution results in normal NMRI mice of compounds 3, 8,
10 or 12 at 10 and 60 min post injection (p.i.) are shown in Table 1
expressed as percentage of injected dose per gram (%I.D./g) of tis-
sue. As was observed after systemic administration of [18F]HFB la-
beled mesenchymal stem cells,12 the radiolabeled compounds
were excreted mainly via the hepatobiliary pathway. 99mTc(CO)3-
N,N0-dipicolylhexadecylamine (compound 10) had the highest liver
uptake at 60 min p.i. (38.8%I.D./g). Except for compound 8 which
showed fast hepatobiliary clearance (36.1%I.D./g and 6.7%I.D./g at
10 min and 60 min, respectively), all the other agents showed a
rather slow hepatobiliary clearance at 10 and 60 min p.i. Lung up-
take of all tracers was high. If used to track stem cells targeting the
myocardium such a high lung uptake might obscure visualization
of the heart at early time points. However, lung uptake decreases
rapidly at 60 min p.i. making it favorable for imaging at 60 min
p.i. and at later time points. Renal clearance was generally low.
Blood clearance was fast with maximally 7.9%I.D./g at 60 min p.i.
(compound 12). Higher blood activity for compounds 10 and 12 re-
flects the higher affinity for blood cells and plasma proteins,
respectively, that was observed in vitro while compounds 3 and
compounds were eluted from the column using an isocratic mix-
ture of 0.05 M ammonium acetate buffer pH 6.8/ethanol (25/75
v/v) at a flow rate of 0.9 mL/min over 30 min. The column effluent
was monitored using a UV detector set at 254 nm, and the output
signal was acquired on a Laura Lite system (Lablogic, Sheffield, UK).
For analysis of radiolabeled compounds, the HPLC eluate was led
over a 3 in NaI(Tl) scintillation detector connected to a single-
channel analyzer. Radioactivity counting for biodistribution stud-
ies was done using an automated system with a gamma counter
(3 in NaI(Tl) well crystal) coupled to a multichannel analyzer in a
sample changer (Wallac, 1480 Wizard 300, Turku, Finland). The re-
sults were corrected for background radiation and physical decay
during counting. Accurate mass measurement was performed by
co-infusion with a 10 lg/mL solution of a reference as an internal
calibration standard on a time-of-flight mass spectrometer (LCT,
Micromass, Manchester, UK) equipped with an electrospray ioniza-
tion (ESI) interface, operated in positive (ES+) or negative (ESꢂ)
mode. Samples were infused in acetonitrile/water using a Harvard
22 syringe pump (Harvard instruments, Massachusetts, USA).

400
H. Fonge et al. / Bioorg. Med. Chem. 18 (2010) 396–402
Table 1
Biodistribution in normal NMRI mice of compounds 3, 8, 10 or 12 at 10 and 60 min post injection (p.i.) expressed as % injected dose (I.D.)/gram (standard deviation, n = 4 per time
point)
Organ
%I.D./g (SD) at 10 min p.i.
%I.D./g (SD) at 60 min p.i.
3
8
10
12
3
8
10
12
Kidneys
Liver
Spleen + pancreas
Lungs
Heart
Intestines + feces
Stomach
Cerebrum
Cerebellum
Blood
9.5 (1.9)
18.8 (1.7)
6.6 (0.8)
24.2 (3.1)
5.8 (1.1)
1.8 (0.4)
0.9 (0.4)
0.7 (0.2)
1.3 (0.3)
15.5 (2.1)
3.5 (0.7)
2.6 (0.6)
36.0 (2.4)
17.9 (1.5)
18.3 (4.6)
2.9 (0.8)
0.4 (0.0)
0.6 (0.3)
0.1 (0.1)
0.3 (0.1)
6.9 (1.5)
2.5 (0.2)
30.5 (3.7)
29.3 (16.1)
11.4 (1.8)
2.7 (0.9)
0.3 (0.0)
0.3 (0.1)
0.2 (0.0)
0.3 (0.0)
9.9 (0.9)
6.6 (0.9)
30.6 (4.4)
6.2 (1.1)
12.0 (2.9)
4.3 (0.8)
5.6 (1.1)
1.5 (1.2)
0.1 (0.0)
0.1 (0.0)
1.7 (0.4)
6.2 (1.3)
4.5 (2.8)
38.8 (3.7)
15.4 (2.3)
11.7 (5.0)
2.2 (0.5)
0.8 (0.2)
0.8 (0.2)
0.1 (0.0)
0.2 (0.0)
5.6 (1.3)
3.5 (0.3)
28.9 (2.3)
12.1 (0.6)
8.5 (0.8)
2.7 (0.3)
0.8 (0.1)
0.4 (0.0)
0.1 (0.0)
0.2 (0.0)
7.9 (0.5)
36.1 (3.0)
21.9 (2.8)
43.9 (12)
3.8 (0.3)
0.5 (0.1)
0.4 (0.1)
0.2 (0.0)
0.4 (0.1)
7.5 (1.5)
6.7 (1.5)
3.8 (0.8)
10.3 (2.4)
1.8 (0.3)
8.1 (2.0)
3.6 (1.4)
0.3 (0.1)
0.5 (0.1)
1.3 (0.3)
%I.D./g: percentage of injected dose per gram tissue, SD: Standard deviation. Compound 3: 99mTc(CO)3-hexadecylamino-N,N0-diacetic acid, compound 8: 99mTc(CO)3-hex-
adecylamino-N-
-picolyl-N0-acetic acid, compound 10: 99mTc(CO)3-N,N0-dipicolylhexadecylamine, compound 12: 99mTc(CO)3-N-hexadecylaminoethyl-N0-aminoethylamine.
a
Acquisition and processing of data was done using Masslynx soft-
ware (Micromass, version 3.5). All animal experiments were con-
ducted with the approval of the institutional ethical committee
for conduct of experiments on animals.
4.3.4. a
-Picolyl-N-methylacetyl-N0-acetic acid (5)
Saponification of one methyl ester of compound 4 (2.52 g,
10 mmol) was done in a mixture of 1 M NaOH (10 mL) and
MeOH/H2O (1:1, 20 mL) at 50 °C for 4 h. After cooling to rt, the
reaction mixture was evaporated in vacuo with MeOH
(4 ꢁ 10 mL). The crude dry reaction mixture was applied on a silica
gel column and washed with EtOAc/NEt3 (95:5 v/v) to remove
residual compound 4. The monomethyl ester (compound 5) was
eluted with EtOAc/MeOH/NEt3 (10:10:0.5 v/v). Yield: 2.0 g (83%).
HRMS theoretical C11H15N2O4 [M+H]+ 239.2483, found 239.2408.
1H NMR (CDCl3): d (ppm) 3.3 (s, 2H, N–CH2–COOH); 3.4 (s, 2H,
N–CH2–COOCH3); 3.6 (s, COOCH3), 3.9 (s, 2H, CH2–N–CH2); 7.1
(dt, 1H, PyrH); 7.3 (d, 1H, PyrH); 7.6 (dt, 1H, PyrH); 8.5 (d, 1H,
PyrH).
4.3. Chemistry
4.3.1. Hexadecylamino-N,N0-dimethyl acetate (1)
Hexadecylamine (238 mg, 0.99 mmol) was dissolved in metha-
nol (50 mL) under N2. Triethylamine (NEt3, 0.41 mL, 3 mmol) was
added and the mixture was stirred at rt for 30 min. Methyl bromo-
acetate (0.19 mL, 2 mmol) was added drop wise after which the
solution was refluxed for 96 h and then cooled to rt. Water
(50 mL) and CH2Cl2 (50 mL) were added and the organic layer
was separated. Additionally, the aqueous layer was twice washed
with CH2Cl2 and the combined extracts was dried over MgSO4
and evaporated under reduced pressure. The resulting oil was puri-
fied by column chromatography on silica gel with CH2Cl2/MeOH
(95:5 v/v) as eluting solvent to give a clear oil. Yield: 290 mg
(75%). HRMS theoretical C22H44NO4 [M+H]+: 386.5899, found:
386.5889. 1H NMR (CDCl3): d (ppm) 0.9 (t, 3H, CH3–CH2–CH2);
1.3 (m, 28H, (CH2)14), 2.3 (q, 2H, CH2–CH2–N); 3.5 (s, 4H, OC–
CH2–N–CH2–CO); 3.7 (s, 6H, COOCH3).
4.3.5. Hexadecylamino-N-a
-picolyl-N0-acetic acid (7)
The monomethyl ester (compound 5, 0.4 mmol, 95 mg) was dis-
solved in THF (10 mL). HOBt (0.4 mmol, 54 mg), hexadecylamine
(0.52 mmol, 127 mg) and benzotriazole-1-yl-oxy-tris-(dimethyl-
amino)-phosphonium hexafluorophosphate (BOP, 0.6 mmol,
228 mg) were added, respectively. The reaction mixture was stir-
red at rt overnight. Analysis by TLC (MeOH/EtOAc/NEt3, 10:10:0.5
v/v) revealed that the reaction was incomplete. An additional
amount of BOP (0.6 mmol, 228 mg) was added and the reaction
mixture was further stirred at rt for 5 days. The precipitate was fil-
tered, washed with THF and dried in vacuo. The product was puri-
fied by column chromatography on silica gel with CH2Cl2/MeOH
(97:3 v/v) to give a white solid (compound 6). Yield: 70 mg
(38%). HRMS: theoretical C27H48N3O3 [M+H]+ 462.6894, found
462.6796. 1H NMR (CDCl3): d (ppm) 0.9 (t, 3H, CH3–CH2); 1.2 (m,
26H, (CH3)13); 1.5 (m, 2H, CH2); 3.2 (m, 2H, NH–CH2); 3.4 (d, 4H
2ꢁCH2–N); 3.7 (s, 3H, COOCH3); 3.9 (s, 2H, N–CH2–Pyr); 7.2
(dDD, PyrH); 7.25 (d, PyrH); 7.6 (dt, 1H, PyrH); 8.2 (m, NH–CO);
8.6 (d, 1H, PyrH).
4.3.2. Hexadecylamino-N,N0-diacetic acid (2)
Compound 1 was treated with 1 M NaOH (2 mL) and EtOH
(2 mL) under reflux for 1 h. After cooling to rt, water (20 mL) was
added followed by acidification to pH 2 with 1 N HCl. The precipi-
tate formed was filtered off, washed with water and dried in vacuo
at 60 °C to give a white powder. Yield: 270 mg (100%). HRMS the-
oretical C20H40NO4 [M+H]+ 358.5367, found 358.5314. 1H NMR
(DMSO-d6): d (ppm) 0.9 (t, 3H, CH3–CH2–CH2); 1.2 (m, 28H,
(CH2)14)); 2.4 (m, 2H, CH2–CH2–N); 3.4 (s, 4H, OC–CH2–N–CH2–CO).
4.3.3.
a
-Picolyl-N,N0-dimethylacetate (4)
Hydrolysis of compound 6 in 1 M NaOH gave compound 7 as a
white powder. HRMS C26H46N3O3 [M+H]+ theoretical 448.6623,
found 448.6634.
Picolylchloride (7.7 mL, 75 mmol) was dissolved in MeOH
(100 mL) and NEt3 (25 mL) was added. Methyl bromoacetate
(15 mL, 150 mmol) was added drop wise and the reaction mix-
ture was stirred at 60 °C overnight. After cooling to rt, solution
was filtered and the organic solvent was removed in vacuo. The
residue was taken up with ethyl acetate, washed with H2O
(3 ꢁ 100 mL), dried over MgSO4 and purified by column chroma-
tography on silica gel with hexane/EtOAc/NEt3, (30:10:1 v/v) as
eluting solvents. Yield: 8.5 g (43%). HRMS theoretical C12H15N2O4-
Na [M+Na]+ 275.10, found 275.32. 1H NMR (CDCl3): d (ppm) 3.4
(s, 4H, N–CH2–COO); 3.6 (s, 6H, COOCH3); 3.9 (s, 2H, N–CH2–
Pyr); 7.1 (dt, 1H, PyrH); 7.3 (d, 1H, PyrH); 7.6 (dt, 1H, PyrH);
8.5 (d, 1H, PyrH).
4.3.6. N,N0-Dipicolylhexadecylamine (9)
Hexadecylamine (1.15 g, 4.8 mmol) and 2-chloromethylpyri-
dine (1.57 g, 9.6 mmol) were suspended in a mixture of water/
MeOH (1:1 v/v, 60 mL). 5 M NaOH was added until pH 10. The
pH was kept constant (at 10) by addition of 5 M NaOH. The mixture
was stirred overnight at rt, evaporated and suspended in a mixture
of water/EtOAc (1:1 v/v). The organic layer was separated, dried
over MgSO4 and concentrated in vacuo. The resulting clear oil
was purified by column chromatography on silica gel using hex-
ane/EtOAc/NEt3 (75:20:5 v/v) as eluting solvent. Yield: 270 mg

H. Fonge et al. / Bioorg. Med. Chem. 18 (2010) 396–402
401
(12%). HRMS theoretical C28H46N3 [M+H]+ 424.6863, found
424.6848. 1H NMR (CDCl3): d (ppm) 0.9 (t, 3H, CH3–CH2–CH2);
1.2 (d, 28H, (CH2)14); 1.5 (m, 2H, CH2–CH2–N); 3.8 (s, 4H, CH2–
N–CH2); 7.3 (dt, 2H, PyrH); 7.5 (d, 2H, PyrH), 7.6 (dt, 2H, PyrH);
8.5 (dd, 2H, PyrH).
erTM (containing 7.2 mg K2EDTA; Beckton Dickinson, Franklin Lakes,
USA) and subsequently transferred to a 50-mL conical falcon tube.
Five-mL blood samples were transferred into 15-mL conical falcon
tubes and centrifuged at 3000 rpm (1837 g) for 5 min, to separate
plasma. Plasma was carefully transferred into a 15-mL conical fal-
con tube. To the mixed blood cells (plasma-free) from 5 mL blood
was added HPLC-purified compounds 3, 8, 10, 12, 99mTc-d,l-
HMPAO, or [18F]FDG followed by gentle mixing. After 15 min of
incubation at rt in plasma-free blood cells, the cells were centri-
fuged for 5 min and the activity in the cells and supernatant frac-
tions was counted in a gamma counter. This was immediately
followed by twice washing with PBS, pH 7.4. The total time for this
uptake phase was 30 min, after which, the cells were fractioned
into two to which was added an equal volume of saline or plasma
followed by 60 min incubation at rt. Afterwards, the cells were
centrifuged for 5 min and the supernatant (saline or plasma) was
carefully separated followed by two PBS washes. Radioactivity in
the supernatant (saline or plasma) and the cells was counted in a
gamma counter. The percentage of radiolabeled compounds bound
to the cells was calculated by dividing the counts (decay corrected)
in the cells at time (t) by the total counts (cells + supernatant) at
time (t) ꢁ 100.
4.3.7. N-Hexadecylaminoethyl-N0-aminoethylamine (11)
Diethylenetriamine (DETA, 21.6 mL, 200 mmol) and 1-bromo-
hexadecane (15 mL, 50 mmol) were heated at 180 °C during
3.5 h. After cooling, the precipitate was filtered and the filtrate
was distilled to remove unreacted DETA (60–100 °C, 0.1 mmHg).
The residue containing the final product and some side products
was used as such for labeling experiments. HRMS theoretical
C20H45N3 [M+H]+ 327.5922, found 327.5931.
4.4. Radiolabeling with technetium-99m
þ
99mTcðCOÞ3ðH2OÞ3 precursor. The technetium-99m tricarbonyl
precursor was prepared using an Isolink kit by adding 1.1–
2.5 GBq of Na99mTcO4 in 1 mL saline to the kit followed by heating
at 100 °C for 20 min. After cooling, the solution was neutralized
with 1 M HCl and then adjusted to desired pH with 1 M NaOH.
99mTc(CO)3-hexadecylamino-N,N0-diacetic acid (compound 3),
99mTc(CO)3-hexadecylamino-N- -picolyl-N0-acetic acid (compound
a
4.7. Biodistribution in mice
8), 99mTc(CO)3-N,N0-dipicolylhexadecylamine (compound 10) and
99mTc(CO)3-N-hexadecylaminoethyl-N0-aminoethylamine (compound
12). To a 10-mL labeling vial was added 1 mg of compounds 2, 7,
9 or 11 dissolved in 0.2 mL of ethanol. The vial was purged with
Mice were anesthetized with isoflurane (2%) in oxygen at a flow
rate of 1 L/min and then injected via a tail vein with 74 kBq of
HPLC-pure compound 3, 8, 10 or 12 in 0.1 mL of saline. They were
sacrificed by decapitation under anesthesia at 10 and 60 min p.i.
(n = 4 mice per time point). The organs were dissected and
weighed in tared tubes and radioactivity in all organs was counted
in a gamma counter. Corrections were made for background radi-
ation and physical decay during counting. Activity in the organs
was expressed as % I.D./organ and % I.D./g of organ. Activity in
blood was calculated on the assumption that blood constitutes
7% of total body weight.
þ
nitrogen, and 0.2–1.1 GBq of 99mTcðCOÞ3ðH2OÞ3 precursor (0.2–
0.5 mL, pH 7.4 or 11 (for compound 11)) was added. The mixture
was heated at 70 °C for 20 min followed by cooling to rt. The crude
labeling reaction mixture was analyzed and purified by RP-HPLC as
described above.
99mTc-d,l-HMPAO. d,l-HMPAO was synthesized as described by
Troutner and Volkert16 Radiolabeling of d,l-HMPAO was performed
in a 10-mL glass vial containing 1 mg of d,l-HMPAO in a mixture of
100
adding 7
lL ethanol and 900 lL saline (0.9% NaCl) and consecutively
g SnCl2.2H2O in 3.5 l
L 0.05 M HCl and 400 MBq Na99mT-
Acknowledgments
l
cO4 in 1 mL saline followed by rt incubation17
thesized at our PET radiochemistry laboratory.
[
18F]FDG was syn-
The authors would like to thank Peter Vermaelen for invaluable
contribution to the study. Financial support for the project was ob-
tained from grants awarded by Geconcerteerde Onderzoeksactie
(GOA) of the Flemish Government, FWO-Vlaanderen Grant G.
0257.05 and in part by the EC-FP6-project DiMI, LSHB-CT-2005-
512146.
4.5. Octanol–buffer partition coefficient
To 50 lL (74 kBq) of a solution of HPLC-purified radiolabeled
compound (3, 8, 10 or 12) in a test vial was added 2 mL of 1-octa-
nol and 2 mL of 0.025 M phosphate buffer pH 7.4. The vial was vor-
texed at rt for 2 min and then centrifuged (Centrifuge 4226, Analis,
References and notes
Gent, Belgium) at 1700 g for 10 min. Approximately 50
lL of the 1-
1. Lindvall, O.; Kokaia, Z.; Martinez-Serrano, A. Nat. Med. 2004, 10, S42.
2. Lindvall, O.; Bjorklund, A. NeuroRx 2004, 1, 382.
octanol phase and 500 L of the phosphate buffer phase were
l
3. Chen, S. L.; Fang, W. W.; Ye, F.; Liu, Y. H.; Qian, J.; Shan, S. J.; Zhang, J. J.;
Chunhua, R. Z.; Liao, L. M.; Lin, S.; Sun, J. P. Am. J. Cardiol. 2004, 94, 92.
4. Sueblinvong, V.; Suratt, B. T.; Weiss, D. J. Clin. Chest Med. 2007, 28, 361.
5. Kraitchman, D. L.; Tatsumi, M.; Gilson, W. D.; Ishimori, T.; Kedziorek, D.;
Walczak, P.; Segars, W. P.; Chen, H. H.; Fritzges, D.; Izbudak, I.; Young, R. G.;
Marcelino, M.; Pittenger, M. F.; Solaiyappan, M.; Boston, R. C.; Tsui, B. M.; Wahl,
R. L.; Bulte, J. W. Circulation 2005, 112, 1451.
6. Chin, B. B.; Nakamoto, Y.; Bulte, J. W.; Pittenger, M. F.; Wahl, R.; Kraitchman, D.
L. Nucl. Med. Commun. 2003, 24, 1149.
7. Beeres, S. L.; Bengel, F. M.; Bartunek, J.; Atsma, D. E.; Hill, J. M.; Vanderheyden,
M.; Penicka, M.; Schalij, M. J.; Wijns, W.; Bax, J. J. J. Am. Coll. Cardiol. 2007, 49,
1137.
pipetted and weighed into separate tared test tubes with adequate
care to avoid cross contamination between the phases. The volume
of fluid pipetted was calculated by dividing the net weight of the
fluid by its density. The radioactivity of the test tubes was counted
using a 3-inch NaI(Tl) scintillation detector mounted in a sample
changer. Corrections were made for background radiation and
physical decay during counting. The octanol–buffer partition coef-
ficient P was calculated as:
cpm=mL in octanol
P ¼
where cpm ¼ counts per min
8. Tamura, M.; Unno, K.; Yonezawa, S.; Hattori, K.; Nakashima, E.; Tsukada, H.;
Nakajima, M.; Oku, N. Life Sci. 2004, 75, 575.
cpm=mL in buffer
9. Wu, J. C.; Inubushi, M.; Sundaresan, G.; Schelbert, H. R.; Gambhir, S. S.
Circulation 2002, 106, 180.
10. Adonai, N.; Nguyen, K. N.; Walsh, J.; Iyer, M.; Toyokuni, T.; Phelps, M. E.;
McCarthy, T.; McCarthy, D. W.; Gambhir, S. S. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 3030.
11. Barbash, I. M.; Chouraqui, P.; Baron, J.; Feinberg, M. S.; Etzion, S.; Tessone, A.;
Miller, L.; Guetta, E.; Zipori, D.; Kedes, L. H.; Kloner, R. A.; Leor, J. Circulation
2003, 108, 863.
4.6. Preliminary in vitro cell labeling
Preliminary in vitro labeling of tracer was performed in mixed
blood cells by incubating the agents in mixed blood cells with or
without plasma for 60 min. Blood was collected into a BD vacutain-

402
H. Fonge et al. / Bioorg. Med. Chem. 18 (2010) 396–402
12. Ma, B.; Hankenson, K. D.; Dennis, J. E.; Caplan, A. I.; Goldstein, S. A.; Kilbourn,
M. R. Nucl. Med. Biol. 2005, 32, 701.
15. Koslowsky, I. L.; Brake, S. E.; Bitner, S. J. J. Nucl. Med. Technol. 2001, 29,
197.
13. Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A. P.; Abram, U.; Kaden, T. A. J. Am.
Chem. Soc. 1998, 120, 7987.
14. Bormans, G.; Sherman, P.; Snyder, S. E.; Kilbourn, M. R. Nucl. Med. Biol. 1996, 23,
513.
16. Troutner, D. E.; Volkert, W. A. Eur. Pat. Appl. EP 0123504, 1984.
17. Vanderghinste, D.; Van Eeckhoudt, M.; Terwinghe, C.; Mortelmans, L.;
Bormans, G. M.; Verbruggen, A. M.; Vanbilloen, H. P. J. Pharm. Biomed. Anal.
2003, 32, 679.