Optimization of labeling dipicolylamine derivative, N,N'-(5-(4-aminobutoxy) -1,3-phenylene)bis(methylene)bis(1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl) methanamine), with three 18F-prosthetic groups as potential imaging agents for metastatic infec

Article abstract of DOI:10.1002/jlcr.2911

The aim of this study was to develop ¹⁸ F-labeled dipicolylamine derivative (compound 1) with three ¹⁸ F-prosthetic groups, ¹⁸ F-NFP, ¹⁸ F-SFB, and ¹⁸ F-FET, which were synthesized with labeling yield

Full text of DOI:10.1002/jlcr.2911

Research Article
Received 9 November 2011,
Revised 9 February 2012,
Accepted 10 February 2012
Published online 14 March 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2911
Optimization of labeling dipicolylamine
derivative, N,N’-(5-(4-aminobutoxy)-1,3-
phenylene)bis(methylene)bis(1-(pyridin-2-yl)-
N-(pyridin-2-ylmethyl)methanamine),
18
with three F-prosthetic groups as
potential imaging agents for metastatic
infectious disease
Junling Li, Brian D. Gray, Koon Y Pak, and Chin K. Ng
a
b
b
a
*
The aim of this study was to develop 18 F-labeled dipicolylamine derivative (compound 1) with three 18 F-prosthetic groups,
18 F-NFP, 18 F-SFB, and 18 F-FET, which were synthesized with labeling yields of 72 Æ 10%, 75 Æ 8%, and 90 Æ 8%, respectively.
Compound 1 was then conjugated with 18 F-NFP, 18 F-SFB, and 18 F-FET, respectively. Factors such as the amount of 1,
reaction temperature, and time were examined to optimize the yields. The optimal labeling conditions were found to be 1
18
(0.1 mg, 0.17 mmol), room temperature, and 10 min for both 18 F-NFP and F-SFB; 1 (4 mg, 6.8 mmol), 100 ꢀC, and 10 min
for 18 F-FET. The total synthesis time, the overall yields, and the average specific activity were 105, 75, and 65 min;
68 Æ 9%, 71 Æ 11%, and 76 Æ13%; 625 Ci/mmol, 853 Ci/mmol, and 3.5 Ci/mmol for 18 F-FP-1,18F-FB-1, and 18 F-FE-1, respec-
tively (n = 5, decay-corrected based on 18F).
Keywords: 18F-prosthetic groups; dipicolylamine derivative; metastatic infectious disease; PET
for localizing infectious foci.11,12 Annexin V, an endogenous
human protein with a high affinity to PS, can be used in vitro to
detect apoptosis. However, radiolabeled annexin V and its deriva-
tives currently have limited in vivo applications due to low target-
Introduction
Non-invasive imaging has significantly augmented the investiga-
tion of various metastatic disease processes caused by infection,
inflammation or cancer.1–3 Since the advent of gallium-67 (67Ga)
citrate for routine infection imaging, many imaging agents have
been developed and evaluated to better localize and detect
areas of metastatic infection within the body.4,5 However, almost
all of the commonly used imaging agents such as 18 F-FDG loca-
lize to areas of inflammation rather than specifically those of
infection, making clinical interpretation difficult and unreliable,
particularly when the infection requires aggressive therapeutic
intervention.6 Therefore, it is critical to develop a more reliable
and more specific imaging agent for metastatic infection.
to-background ratios and high liver and kidney accumulations.13
Bis(zinc(II)–dipicolylamine) (Zn2+–DPA) coordination complexes
are synthetic small molecules that can mimic the apoptosis
sensing function of annexin-V. PSVueW794, a commercially avail-
able fluorescent Zn2+-DPA coordination complex, was shown to
selectively stain the same cells as fluorescently labeled annexin-
V in cell culture.14 Near-infrared fluorescent (NIR) Zn2+-DPA deri-
vatives have been recently developed to effectively target cell
death.15,16 Moreover, several fluorescent dye labeled Zn2+-DPA
derivative probes demonstrated high selectivity to the anionic
surfaces of bacterial infected cells and can be used for in vivo
Biomarkers will be of significant interest if they can be used to
map the molecular features of the infected cells. Phosphatidyl-
serine (PS) is the major anionic phospholipid in the plasma
membrane, which is sequestered almost completely in the mem-
brane inner leaflet. PS is usually externalized to the outer leaflet
of the membrane during cell death, apoptosis, and neutrophil
necrosis.7–9 The appearance of PS on the cell surface is a
common indicator for most types of cell death.10 The resolution
of infection, in part, is associated with neutrophil apoptosis,
leading to necrosis; thus PS can be used as an excellent target
aDepartment of Diagnostic Radiology, University of Louisville, Louisville, KY, USA
bMolecular Targeting Technologies, Inc., West Chester, PA, USA
*Correspondence to: Chin K. Ng, Department of Diagnostic Radiology, University
of Louisville, Louisville, KY, USA.
E-mail: chin.ng@louisville.edu
J. Label Compd. Radiopharm 2012, 55 149–154
Copyright © 2012 John Wiley & Sons, Ltd.

J. Li et al.
imaging of gram positive as well as gram negative bacterial (compound 1) was kindly provided by Molecular Targeting
infection in mice.17–19 Although NIR labeled Zn2+-DPA complexes Technologies, Inc. (West Chester, PA, USA). Sep-Pak Accell plus
have achieved some early success, further applications are lim- QMA plus cartridge and C18 Sep-Pak cartridges were pur-
ited by the disadvantages of fluorescence imaging in sensitivity chased from Waters, Inc. QMA was pretreated with 5 ml 8.4%
and tissue penetration. Positron emission tomography (PET) NaHCO3 and 5 ml water and C18 cartridge with ethanol and
and single photon emission computed tomography (SPECT) ima- water before use.
ging hold great advantages for clinic translational research. Wyffels
Analytical methods. For online radioactivity and UV mea-
et al.20 recently developed 99mTc labeled Zn2+-DPA coordina- surements, the HPLC column was connected to a UV-visible
tion complexes with two different chelators and performed detector (Varian Inc., Walnut Creek, CA, USA) and then to a NaI
the preliminary in vivo evaluation for the detection of cell (Tl) scintillation flow-through detector (Bioscan, Washington
death. Results indicated that selective accumulation in a rat DC, USA), and the data were recorded and processed by Galaxie
model of ischemia-reperfusion injury was detected for two tra- software system (Varian Inc., Walnut Creek, CA, USA) for deter-
cers in the apoptotic area with high target-to-background mination of radiochemical yields. The analytical HPLC column
ratios. Although 99mTc labeled Zn2+-DPA coordination com- used for the identification of standards and purity of radioactive
plexes could successfully detect cell death, the results remains products was Luna mm C18 150 Â 4.6 mm (Phenomenex,
preliminary and the application will be only limited to SPECT Torrance, CA, USA). The mobile phase was 40% CH3CN/0.1%
imaging. PET isotope, 64Cu, was recently used to coordinate TFA, and the flow rate was 1 mL/min. The semi-preparative
with DPA derivative by simply addition of 64Cu directly. Both HPLC column used for purifying radioactive products was Luna
in vitro and in vivo PET studies revealed high U87MG tumor 5 mm C18 250 Â 10.00 mm (Phenomenex, Torrance, CA, USA).
retention at all time points.21 However, high accumulation Flow rate was 5 mL/min. The gradient was 95% solvent A
and retention in the liver were observed as in the case of many [0.1% TFA in water] and 5% B [0.1% TFA in acetonitrile]
other 64Cu labeled tracers.22 Studies showed that compound 1 (0–2 min) to 60% solvent A and 40% solvent B at 20 min, then
is a small molecule and can be modified with various imaging 80% B (22–30 min). UV absorbance was monitored at 254 nm.
probe.17–19 indicating the possibility of labeling 1 with a Mass Spectrometry (MS) analysis was performed using the Mass
short-lived PET isotope such as 18F. Compared with other metal Spectrometry Laboratory at the University of Louisville. 1H NMR
isotopes, 18F (t1/2 = 109.6 min) is currently the most commonly spectra were obtained using a Mercury 800-MHz instrument
used PET isotope with favorable nuclidic properties, for exam- (NMR Instrumentation Facility at the University of Louisville), and
ple, minor disturbance of original biomolecules. The selection the chemical shifts were reported in parts per million on the d scale
of suitable prosthetic groups plays a critical role in the radiola- relative to an internal Tetramethylsilane (TMS) standard.
beling process because 18F cannot be linked directly to the
molecules by nucleophilic substitution.23 In order to obtain
Synthesis of cold reference standards
an ideal prosthetic group with the smallest steric hindrance,
three different 18 F-prosthetic groups, 4-nitrophenyl 2-18 F-
3Synthesis of N-(4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)
fluoropropionate (18 F-NFP), N-succinimidyl 4-18F-fluorobenzo-
phenoxy)butyl)-2-fluoropropanamide (FP-1)
ate (18F-SFB), and 2-18F-Fluoroethyl toslyate (18F-FET), were
2-Fluoropropionic acid (5 mg, 54.3 mmol) in anhydrous CH3CN
selected to label 1, respectively. Because Zn2+-DPA coordina-
(0.5 mL) was added to TSTU (17.6 mg, 58.5 mmol). The reaction
mixture was stirred at room temperature for 0.5 h, and 1 (3 mg,
5.1 mmol) and DIPEA (20 mL, 115 mmol) in DMSO (0.1 mL) were
added. The mixture was further stirred at room temperature
tion complexes can be simply prepared by adding two equiva-
lents of zinc nitrate into one equivalent of 1 for 30 min immediately
before performing any biological experiment; we chose to focus on
optimizing the radiolabeling yields of 18F–1 by different reaction
for 2 h and purified using semi-preparative HPLC. The collected
factors without the Zn complex step.
fractions were lyophilized and analyzed by MS (calculated:
1
661.6, found: 662.4) and H NMR (800 MHz, d6-DMSO), selected
peaks: d 8.50 (d, J = 4.8 Hz, 4H), 7.75 (t, J = 8 Hz, 4H), 7.59
(d, J = 8 Hz, 4H), 7.25–7.30 (m, 4H), 7.09 (s, 1H), 6.80 (s, 2H),
Results and discussion
5.82–6.90 (m, 1H, CF-H).
Experimental
General
Synthesis of N-(4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)
phenoxy)butyl)-4-fluorobenzamide (FB-1).
Chemicals. Reagents and solvents were all commercially avail-
able and used as received, without further purification. Water 4-Fluorobenzoic acid (5 mg, 35.7 mmol) in anhydrous CH3CN
for ion chromatography, acetic acid, trifluoroacetic acid (TFA), (0.5 mL) was added to TSTU (12 mg, 40 mmol). The reaction
anhydrous acetonitrile (CH3CN), anhydrous dimethyl sulfoxide mixture was heated at 100 ꢀC for 10 min, and the solvent was
(DMSO), N,N-Diisopropylethylamine (DIPEA), tetrabutylammonium evaporated. Compound 1 (3 mg, 5.1 mmol) and DIPEA (20 mL,
hydroxide (40 wt%) in water (TBAH), potassium carbonate, O-(N- 115 mmol) in DMSO (0.1 mL) were added, stirred at room
Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate temperature for 2 h and purified by semi-preparative HPLC. The
(TSTU), 2-fluoropropionic acid, 4-fluorobenzoic acid, ethyl 2- collected fractions were lyophilized and analyzed by MS (calcu-
1
bromopropionate, bis(4-nitrophenyl)carbonate, ethyl 4-dimethylamino lated: 709.9, found: 710.3) and H NMR (800 MHz, d6-DMSO): d
benzoate, methyl triflate, ethylene di(p-toluenesulfonate), kryptofix- 8.68 (d, J = 4.8 Hz, 4H), 7.97–7.98 (m, 2H), 7.94 (t, J = 8 Hz, 4H),
2.2.2 (K222) and 1-octanol were purchased from Sigma Aldrich. 7.60 (d, J = 8 Hz, 4H), 7.48 (t, J = 6.4 Hz, 4H), 7.35–7.37 (m, 4H),
2-fluoroethyl tosylate was purchased from ABX (Radeberg, 7.19 (s, 1H), 7.11 (s, 2H), 4.27 (s, 8H), 4.16 (s, 4H), 4.04 (t, J = 6.4Hz,
Germany). N,N’-(5-(4-aminobutoxy)-1,3-phenylene)bis(methylene) 2H), 3.40 (q, J = 6.4 Hz, 2H), 1.82–1.84 (m, 2H), 1.73–1.75 (m, 2H),
bis(1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine)
1.30–1.33 (m, 2H).
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 149–154

J. Li et al.
Synthesis of N-(4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl) at 100 ꢀC for 5 min. After cooling down, 5% acetic acid (8 mL)
phenoxy)butyl)-2-fluoroacetamide (FE-1).
was added and loaded onto a Sep-Pak plus C18 cartridge,
which was then washed with 10 ml water and 10 ml 10%
CH3CN. The product was eluted with 1 mL of CH2Cl2 and dried
with a flow of nitrogen. For the coupling with 1, the dry resi-
due of 18F-SFB was reconstituted in DMSO (0.1 mL), mixed
with 1 (0.1 mg, 0.17 mmol) and DIPEA (20 mL, 115 mmol). The
mixture was stirred at room temperature for 10 min, then
diluted with 5% acetic acid (3 ml) and loaded onto semi-
preparative C18 column.18F-FB-1 was collected at 18.4 min.
2-Fluoroethyl tosylate (9 mg, 41 mmol), 1 (3 mg, 5.1 mmol), and
DIPEA (20 mL, 115 mmol) in DMSO (0.2 mL) were mixed together,
stirred at 100 ꢀC for 30 min and purified by semi-preparative
HPLC. The collected fractions were lyophilized to afford yellow
solid and analyzed by MS (calculated: 647.1, found: 646.6) and
1H NMR (800 MHz, d6-DMSO): d 8.51 (d, J = 4.0 Hz, 4H), 7.76
(t, J = 8 Hz, 4H), 7.60 (d, J = 8 Hz, 4H), 7.28 (t, J = 6.4 Hz, 4H),
7.12–7.14 (m, 1H), 6.83–6.84 (m, 2H), 4.73 (td, J = 42, 4.8 Hz, 1H),
3.97 (t, J = 6.4 Hz, 2H), 3.74 (s, 8H), 3.54 (s, 4H), 3.40–3.42 (m, 2H),
1.56–1.70 (m, 4H).
Preparation of N-(4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)
phenoxy)butyl)-2-[18F]fluoroacetamide (18 F-FE-1)
18 F-FET was prepared following reported procedure with minor
modification.27 For a typical run, ethylene di(p-toluenesulfonate)
(5 mg,13 mmol) was added to dry 18F/K222/K2CO3 residue and
heated at 100 ꢀC for 10 min. After cooling, the reaction mixture
was diluted with 45% CH3CN/water (3 mL) with 0.1% acetic acid
and loaded onto a semi-prep Phenomenex Luna C18 column
running at 5 mL/min with 60% CH3CN/water containing 0.1%
TFA as the mobile phase. The product was collected in multiple
fractions following radioactive peak with a retention time of
9.5 min. The fraction with the maximum radioactivity was then
diluted with water with 0.1% acetic acid (30 mL). The solution
containing the desired product was passed through a Sep-Pak
plus C18 cartridge, and the activity trapped in the cartridge
was washed with water (1 mL). The product was eluted with
dichloromethane (1 mL). The water layer on the top was carefully
removed, and the organic layer with product was gently dried
with nitrogen at room temperature. For the reaction with 1,
dry residue of 18 F-FET was reconstituted in DMSO (0.1 mL),
Radiochemistry
Preparation of N-(4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)
phenoxy)butyl)-2-[18F]-fluoropropanamide (18F-FP-1)
18 F-NFP was prepared following reported procedure with
minor modification.24 For a typical run, 15.6 mCi [18F]-fluoride
was trapped on a QMA cartridge. The activity was eluted to
a
v-vial with 0.6 mL 90% acetonitrile containing K222
(30.0 mg, 80 mmol) and potassium carbonate (5 mg, 36 mmol).
Azeotropic evaporation was performed at 100 ꢀC with a nitro-
gen flow, and drying was repeated by addition of CH3CN
(0.5 mL Â 3). Ethyl 2-bromopropionate (5 mg, 28 mmol) in anhy-
drous CH3CN (0.5 mL) was added and heated at 100 ꢀC for
10 min. Afterwards, TBAH (ꢀ20 mL,31 mmol) in CH3CN (0.5 mL) was
added and heated at 100 C for 5 min. The liquid was carefully
removed with nitrogen and bis-4-nitropenyl carbonate (20 mg,
66 mmol) in dry CH3CN (1 mL) was added and heated at 100 ꢀC
for 10 min. After cooling, the reaction mixture was diluted with
45% CH3CN/water with 0.1% acetic acid (3 mL) and loaded onto
a semi-prep Phenomenex Luna C18 column running at 5 mL/min
with 60% CH3CN/water containing 0.1% TFA as the mobile phase.
The product was collected in multiple fractions following radioac-
tive peak at a retention time of 8.7min. The fraction with the
maximum radioactivity was then diluted with water with 0.1%
acetic acid (30 mL). The solution containing the desired product
was passed through a Sep-Pak plus C18 cartridge. The activity
trapped in the cartridge was washed with water (1mL). The pro-
duct was eluted with dichloromethane (1 mL). The water layer on
the top was carefully removed and the organic layer with product
was gently dried with nitrogen at room temperature. For the cou-
pling with 1, the dry residue of 18F-NFP was typically reconsti-
tuted in DMSO (0.1mL), mixed with 1 (0.1mg, 0.17mmol) and
DIPEA (20 mL, 115 mmol). The mixture was stirred at room tem-
perature for 10 min, then diluted with 5% acetic acid (3mL) and
loaded onto the semi-preparative C18 column. 18F-FP-1 was col-
lected at 16.2 min.
NH2
O
NH
18F
O
O
N
N
N
N
N
N
N
N
N
N
N
N
18F-FP-1
1
18F
O
18F
NH
NH
Preparation of N-(4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)
phenoxy)butyl)-4-[18F]fluorobenzamide (18 F-FB-1)
O
O
18F-SFB was prepared following reported procedure with
minor modification.25,26 Typically, a solution of ethyl 4-
(trimethylammonium triflate) benzoate (5 mg, 31 mmol) in
CH3CN (0.5 mL), which was prepared following reported pro-
cedure,23 was added to 10 mCi of dry 18F/K222/K2CO3 residue
and heated at 100 ꢀC for 10 min. Afterwards, TBAH (20 mL,
31 mmol) in CH3CN (0.5 mL) was added and heated at 100 ꢀC
for 5 min. The liquid was removed with nitrogen and TSTU
(10 mg, 33 mmol) in dry CH3CN (1 mL) was added and heated
N
N
N
N
N
N
N
N
N
N
N
N
18F-FB-1
18F-FE-1
Scheme 1. Chemical structures of 1, 18 F-FP-1, 18 F-FB-1, and 18 F-FE-1.
J. Label Compd. Radiopharm 2012, 55 149–154
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

J. Li et al.
mixed with 1 (4 mg, 6.8 mmol) and DIPEA (20 mL, 115 mmol). The
Three prosthetic groups, 18 F-NFP, 18 F-SFB, and 18 F-FET,
mixture was heated at 100 ꢀC for 10 min. 18 F-FE-1 was collected were selected to conjugate with 1, respectively. 18 F-NFP
at 14.3 min.
was synthesized in a three-step and one-pot reaction with
HPLC purification in the end. The total synthesis time was
about 60 min with an average yield of 72 Æ 10% (decay-
corrected, n = 5). To saponify the ester efficiently in a shorter
time, TBAH (20 mL, 115 mmol) was used instead of high
volume potassium hydroxide as reported in the literature,24
which is less toxic and faster to evaporate the solvent for
the following step. After HPLC purification, 18 F-NFP was pro-
cessed with a C18 cartridge and eluted with 1 ml of CH2Cl2.
Because of the highly volatility and hydrolysis nature of
18 F-NFP, evaporation needs to be performed at room tem-
perature with low stream of gas in a short time (<10 min).
18 F-SFB was synthesized in a three-step and one-pot method
using one C18 cartridge for purification at the end. The total
synthesis time for 18 F-SFB was about 40 min with an average
Results and discussion
Chemistry
The chemical yields for three cold reference standards, FP-1,
FB-1, and FE-1 were 95%, 90%, and 65%, respectively. Structures
of three compounds were confirmed with MS and H NMR. All
these three reference compounds were used to verify the identi-
fication of the corresponding labeled radiotracers by co-injection
in the HPLC analysis.
1
Radiochemistry
Structures of 1 and three 18 F-labeled radiotracers were shown in yield of 75 Æ 8% (decay corrected, n = 5). TBHA was also used
Scheme 1. The reaction schemes for radiosyntheis of 18 F-FP-1, to simplify reaction by avoiding one C18 cartridge purifica-
18 F-FB-1, and 18 F-FE-1 were shown in Scheme 2. Experimental tion step for the acid intermediate,25–27 which reduced the
results indicated that all the injected radioactivity was eluted out reaction time for at least 15 min and avoided at least 20%
of the analytical HPLC column for all the experiments (data not radioactivity loss. 18 F-FET was prepared in one step with
shown); therefore, analytical HPLC can be used to determine the HPLC purification. The synthesis time was about 30 min with
yields and purity. All three labeled tracers correlated well with their an average yield of 90 Æ 8% (decay corrected, n = 5). The spe-
corresponding cold references by co-injection in the HPLC analysis. cific activity for 18 F-NFP, 18 F-SFB, and 18 F-FET was 8.4–13.5 mCi/nmol,
O
O
O
18F-/K222/K2CO3
TBAH, CH3CN
100 0C, 10 min
OEt
O
OEt
CH3CN, 100 0C, 10 min 18F
18F
Br
NBu4
NO2
NO2
NO2
O
O
1, DMSO
O
O
NH
1
O
CH3CN, 100 0C, 10 min
18F
18F
DIPEA, RT, 10 min
18F-NFP)
(
18F-FP-1)
(
18F-/K222/K2CO3
O
O
TBAH, CH3CN
100 0C, 5 min
(H3C)3N
SO3CF3
18F
CH3CN, 100 0C, 10 min
OEt
OEt
O
TSTU
O
O
O
18F
18F
N
0
O- +NBu4
CH CN,
100 C, 5 min
3
O
(
18F-SFB)
O
1, DMSO
18F
NH 1
DIPEA, RT, 10 min
(
18F-FB-1 )
18F-/K222/K2CO3
CH3CN, 100 0C, 10 min
1, DMSO, 10 min
OTs
NH 1
OTs
18F
18F
TsO
0
DIPEA, 100 C
18F-FET)
(
18F-FE-1 )
(
Scheme 2. Reaction schemes for 18 F-FP-1, 18 F-FB-1, and 18 F-FE-1 using three prosthetic groups, 18 F-NFP, 18 F-SFB, and 18 F-FET, respectively.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 149–154

J. Li et al.
100
80
60
40
20
0
12.7–21.5 mCi/nmol, and 22.9–25.8 mCi/nmol, respectively.
Theradiochemical purity for three tracers was > 99.5% as deter-
mined by analytical HPLC.
For the following experiments, factors such as the amount of 1,
reaction time, and reaction temperature were studied to optimize
the labeling yield of 1 using three different prosthetic groups.
18F-NFP
18F-SFB
18F-FET
Effect of the amount of 1 on yield
As shown in Figure 1, the yield with 18 F-NFP was > 90% in the
range of 50 mg–1 mg (0.085 mmol–1.7 mmol), demonstrating the
high reactivity of 18 F-NFP. Lower amount than that was not eval-
uated. For the other two prosthetic groups, the amount of 1
highly affected the yield. For 18 F-SFB, the yield was less than
10% at 50 mg (0.085 mmol) but rapidly increased to over 95% at
=0.1 mg (0.17 mmol), whereas the yield for 18 F-FET was < 30%
even at 1 mg (1.7 mmol) and about 85% at 4 mg (17 mmol).
20
30
40
50
60
70
80
90
100
Reaction temperature (oC)
Figure 2. Effect of reaction temperature (ꢀC) on labeling yield with three prosthetic
groups (n = 3). 1(0.1 mg, 0.17 mmol) was used for 18 F-FP-1 and 18 F-FB-1, and 1(1 mg,
1.7 mmol) for 18 F-FE-1. Reaction time: 10 min.
Effect of reaction time on yield
The yields gradually increased with time for all the three groups
approximately by 10% from 5 to 30 min, and slowly achieved
the plateau (Figure 2). Considering the short half-life of 18F,
10 min was selected as the optimal reaction time for all three
prosthetic groups.
18 F-NFP and 18 F-SFB with 1 involves the formation of a bond
between carboxylic group and amine group that can be success-
fully performed at room temperature in aqueous or organic
solvents in high yields. However, the reaction of 18 F-FET with
1 is an SN2 reaction, which usually requires harsh conditions to
obtain reasonable yield; for example, anhydrous aprotic solvent
and high temperature.
Effect of reaction temperature on yield
The reaction temperature significantly affected the 18 F-FET yield.
When 1 (1 mg,1.7 mmol) was used and reacted for 10 min, only
0%–5% of yields were obtained at 25–80 ꢀC and increased by
~25% at 100 ꢀC. For the other two groups, there was no notice-
able change with temperature (Figure 3).
Results in this study indicated that although slightly more
compound 1 is required for 18 F-SFB than 18 F-NFP in order to
obtain similar range of yields; both prosthetic groups had similar
labeling conditions, reactivity, and labeling yields, whereas
18 F-FET required harsh labeling conditions, such as high tem-
perature, organic solvent, and high amount of 1. The reason
was probably because of the different mechanisms involved in
the reaction of 1 with 18 F-FET. The conjugations of both
Although the coupling reaction of amino group and carboxyl
group can be performed in both organic and aqueous condi-
tions, experimental results showed that the coupling efficiency
in anhydrous organic solvent was much higher than that in
aqueous or organic solvent with some portion of moisture.
In our case, we observed that the coupling is more efficient
in anhydrous organic solvents than aqueous solvents; the
presence of trace amounts of moisture reduced the yields by
about 30%.
In addition, 18 F-FE-1 was not completely separated from origi-
nal compound 1 because of close retention time (<0.5 min). Our
results showed that at least 30% of unreacted 1 was not removed
Labeling yield with different prosthetic groups
100
80
100
80
18F-NFP
18F-SFB
18F-FET
18F-NFP
60
60
18F-SFB
18F-FET
40
20
0
40
20
0
0
1
2
3
4
0
20
40
60
80
100
120
140
DPA (mg)
Reaction time (min)
Figure 1. Effect of amount of 1 on labeling yield with three prosthetic groups
Figure 3. Effect of reaction time on labeling yield with three prosthetic groups
(n = 3). 1 (0.1 mg, 0.17 mmol) and 25 ꢀC were used for 18 F-FP-1 and 18 F-FB-1, and
1 (1 mg, 1.7 mmol) and 100 ꢀC were used for 18 F-FE-1.
(n = 3). 25 ꢀC was used for 18 F-FP-1 and F-FB-1. 100 ꢀC was used for 18 F-FE-1.
18
Reaction time: 10 min.
J. Label Compd. Radiopharm 2012, 55 149–154
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J. Li et al.
from the labeled 1; thus, the specific activity of 18 F-FE-1 was
highly reduced. But the potential advantage for 18 F-FET was the
smaller size of the 18 F-ethyl-group than 18 F-propyl-group and
18 F-benzyl-group, which could potentially bring less steric
hindrance to the original molecule of 1 and thus could better
keep the intactness of 1. Studies have shown that zinc complexa-
tion is necessary to ensure 1 binds to PS with high affinity and
specificity.17–19 Therefore, it is critical to reconstitute 18 F-1 with
Zn2+ prior to performing any biological evaluation.
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Conclusions
Compound 1 was successfully labeled with three different
prosthetic groups. The optimal labeling conditions were 0.1 mg
(0.17 mmol) of 1, room temperature, 10 min for both 18 F-NFP
and 18 F-SFB; 4 mg (6.8mmol) of 1, 100ꢀC and 10 min for 18 F-FET.
The total synthesis time, the overall yields, and the average
specific activity were 105, 75, and 65 min; 68 Æ 9%, 71 Æ 11%,
and 76 Æ13%; 625 Ci/mmol, 853 Ci/mmol, and 3.5 Ci/mmol
for 18 F-FP-1,18 F-FB-1, and 18 F-FE-1, respectively (n = 5, decay-
corrected based on 18 F).
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Acknowledgements
The authors wish to thank Dr. Bo Xu, Ph.D., Assistant Professor
of the Department of Chemistry at the University of Louisville,
1
for the analysis of HNMR.
Annual Meeting, San Antonia.
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Conflict of Interest
[23] S. Guhlke, H. H. Coenen, G. Stocklin. Appl. Radiat. Isot. 1994, 45,
The authors did not report any conflict of interest.
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