Development of 18F-labeled picolinamide probes for PET imaging of malignant melanoma

Article abstract of DOI:10.1021/jm301740k

Melanoma is an aggressive skin cancer with worldwide increasing incidence. Development of positron emission tomography (PET) probes for early detection of melanoma is critical for improving the survival rate of melanoma patients. In this research, ¹⁸F-picolinamide-based PET probes were prepared by direct radiofluorination of the bromopicolinamide precursors using no-carrier-added ¹⁸F-fluoride. The resulting probes, ¹⁸F-1, ¹⁸F-2 and ¹⁸F-3, were then evaluated in vivo by small animal PET imaging and biodistribution studies in C57BL/6 mice bearing B16F10 murine melanoma tumors. Noninvasive small animal PET studies demonstrated excellent tumor imaging contrasts for all probes, while ¹⁸F-2 showed higher tumor to muscle ratios than ¹⁸F-1 and ¹⁸F-3. Furthermore, ¹⁸F-2 demonstrated good in vivo stability as evidenced by the low bone uptake in biodistribution studies. Collectively, these findings suggest ¹⁸F-2 as a highly promising PET probe for translation into clinical detection of melanoma.

Full text of DOI:10.1021/jm301740k

Article
pubs.acs.org/jmc
Development of 18F‑Labeled Picolinamide Probes for PET Imaging of
Malignant Melanoma
Hongguang Liu,† Shuanglong Liu,† Zheng Miao,† Zixin Deng,‡ Baozhong Shen,§ Xuechuan Hong,‡
and Zhen Cheng*,†
†Molecular Imaging Program at Stanford (MIPS), Bio-X Program, Department of Radiology, Stanford University, California,
94305-5344, United States
‡Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University, Ministry of Education, Wuhan University
School of Pharmaceutical Sciences, Wuhan 430071, People’s Republic of China
§Department of Radiology, Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, People’s Republic of China
S
* Supporting Information
ABSTRACT: Melanoma is an aggressive skin cancer with
worldwide increasing incidence. Development of positron
emission tomography (PET) probes for early detection of
melanoma is critical for improving the survival rate of melanoma
patients. In this research, 18F-picolinamide-based PET probes
were prepared by direct radiofluorination of the bromopicolina-
mide precursors using no-carrier-added 18F-fluoride. The resulting
probes, 18F-1, 18F-2 and 18F-3, were then evaluated in vivo by
small animal PET imaging and biodistribution studies in C57BL/
6 mice bearing B16F10 murine melanoma tumors. Noninvasive
small animal PET studies demonstrated excellent tumor imaging
contrasts for all probes, while 18F-2 showed higher tumor to
muscle ratios than 18F-1 and 18F-3. Furthermore, 18F-2 demonstrated good in vivo stability as evidenced by the low bone uptake
in biodistribution studies. Collectively, these findings suggest 18F-2 as a highly promising PET probe for translation into clinical
detection of melanoma.
months earlier than those conventional techniques.7,8 However,
INTRODUCTION
■
because the uptake and cellular retention of 18F-FDG involve
Malignant melanoma is one of the most aggressive and lethal
increased glucose metabolism, which also occurs in many other
cancers and has increasing incidence world wide, especially in the
Caucasian population.1,2 Its strong tendency to metastasize and
tumor types or in surgical wounds, pneumonia, and infection/
inflammation conditions, 18F-FDG lacks high specificity for
the absence of effective treatment for advanced diseases result in
a poor overall survival.3,4 Therefore, development of novel and
melanoma imaging, and false-positive detections often hap-
pen.6,7,9
accurate molecular imaging techniques to detect melanoma at its
earliest stages is critical for improving the survival of patients that
have malignant melanoma.
Over the last a few decades, noninvasive molecular imaging of
malignant melanoma with various modalities has been
extensively studied; these modalities include radionuclide
imaging with positron emission tomography (PET) and single
Nuclear imaging modalities (SPECT and PET) heavily rely on
probes for monitoring specific molecular targets or pathways in
vivo. Nuclear probes with high specificity based on different
targeting or molecular recognition mechanisms help to increase
the effectiveness of PET or SPECT for melanoma detection.
Therefore, many radiolabeled probes for imaging different
molecular targets or processes associated with malignant
photon emission computed tomography (SPECT), magnetic
melanoma have been designed and evaluated for melanoma
resonance imaging (MRI), computed tomography (CT), and
imaging, such as monoclonal antibodies against melanoma-
ultrasound (US) imaging. The higher sensitivity of PET and
SPECT than the traditional imaging methods such as MRI, CT,
associated antigens,10,11 iodoamphetamine,12 α-melanocyte-
stimulating hormone peptides,13−17 or benzamide (BZA)-
and US has attracted more and more research interest on
based compounds.18−20 So far, BZA analogs have been shown
to be among the most promising melanoma targeting agents for
both diagnosis and therapeutic applications. An in vitro cell study
developing nuclear imaging agents for melanoma detection.5
Especially, PET imaging with 18F-fluoro-deoxyglucose (18F-
FDG) has demonstrated much higher sensitivity and specificity
than that obtained by CT, ultrasound, radiography, and liver
function tests and histology or clinical follow-up.6 Furthermore,
18F-FDG PET was shown to detect malignant melanoma up to 6
Received: September 19, 2012
Published: January 9, 2013
© 2013 American Chemical Society
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demonstrated the high binding affinity of BZA analogs to
melanin that is present in melanoma.21 Moreover, a clinical study
using 123I-labeled BZA for detecting malignant melanoma and its
metastases revealed 81% diagnostic sensitivity, 87% accuracy,
and 100% specificity.22 Consequently, 18F-labeled BZA (18F-
FBZA, Figure 1D) has been developed for PET imaging of
Scheme 1. Synthetic Schemes for Preparation of the
Precursors for 18F-Fluorination and 19F Authentic Standards
produced in more than 95% radiochemical purity and free of
the corresponding bromo-precursors, as demonstrated by quality
control analysis (RP-HPLC). The specific activity for these PET
probes was in the range of 100−150 GBq/μmol.
Figure 1. The chemical structures of 18F-1, 18F-2, 18F-3 (18F-MEL050),
and 18F-FBZA.
Small Animal PET Imaging Studies. The tumor-targeting
efficacy and imaging properties of 18F-1 and 18F-2 were evaluated
in B16F10 tumor-bearing mice, and the results were compared
with 18F-3, which was examined in the same tumor model. In
static small animal PET scans, representative coronal images of
B16F10 tumor bearing mice (n = 4) at different times after
intravenous injection of about 3.7 MBq (100 μCi) of 18F-1, 18F-2,
or 18F-3 are shown in Figure 2. B16F10 tumors were clearly
melanoma and its metastases using mice bearing B16F10
melanoma tumors.23,24 It was reported that the B16F10 tumor
uptake at 2 h postinjection (p.i.) reached 5.94 1.83 percentage
injected dose per gram (%ID/g).23 However, the multistep
radiosynthesis of 18F-FBZA could be a bottleneck for its large
production and potential clinical translation.
Recently the pyridine-based precursor 18F-6-fluoro-N-[2-
(diethylamino)ethyl] pyridine-3-carboxamide (18F-MEL050,
Figure 1C) was successfully synthesized in single step with
high radiochemical yield (RCY). This novel PET probe displays
excellent performance for imaging of primary and metastatic
melanoma.25−27 In pigmented melanoma B16-F0 xenografts,
18F-MEL050 exhibits high tumor uptake and tumor-to-back-
ground ratio of approximately 50:1 at 2 h p.i. of the probe. The
excellent in vivo performance of 18F-MEL050, plus its easy
radiosynthesis, encouraged us to design and biologically evaluate
more 18F-MEL050 analogs for melanoma imaging. More
specifically, N-(2-(diethylamino)ethyl)-18F-4-fluoropicolina-
mide (shortened as 18F-1, Figure 1A) and N-(2-(diethylamino)-
ethyl)-18F-5-fluoropicolinamide (shortened as 18F-2, Figure 1B)
were synthesized and compared side-by-side with 18F-MEL050
(shorted as 18F-3, Figure 1C) by small animal PET imaging and
biodistribution studies in B16F10-tumor bearing mice.
Figure 2. Decay-corrected whole-body coronal small animal PET
images of C57BL/6 mice bearing B16F10 murine melanomas from a
static scan at 0.5, 1, and 2 h after injection of 18F-1, 18F-2, and 18F-3.
Tumors are indicated by arrows.
RESULTS
■
Chemistry and Radiochemistry. The authentic 19F-
fluorine compounds (19F-1, 19F-2, and 19F-3), and their
precursors (4, 5, and 6, Scheme 1) for 18F-fluorination were
prepared by condensation of a bromopicolinic acid or
bromoniconitic acid with N,N-diethylethylenediamine (DEED)
via O-(N-succinimidyl)-1,1,3,3-tetramethyl-uronium tetrafluor-
oborate (TSTU) activation in the presence of diisopropylethyl-
amine (DIPEA) (Scheme 1 and Supporting Information). The
azeotropically dried 18F-fluoride can replace the bromo leaving
group in each precursor to make the corresponding product in
one step. Hence, all of the 18F-labeled products were prepared
within 1 h, including reversed-phase high-performance liquid
chromatography (RP-HPLC) purification and product formula-
tion for further biological evaluation. The resultant products, 18F-
1, 18F-2, and 18F-3, were prepared in radiochemical yields of
24.5% 6.7%, 9.5% 1.9%, and 21.5% 15.5% (n = 3, non-
decay-corrected), respectively. All of the 18F probes were
visualized with high tumor-to-background contrast at all time
points from 0.5 to 2 h. The highest uptakes observed in the
kidneys at early time points suggested that these PET probes
were mainly excreted through the renal system. Quantification
analysis of tumors and other major organ activity accumulation in
PET images was done by analyzing the regions of interest (ROI)
that circle the entire organ on the coronal images. The tumor
uptakes of 18F-1 were determined to be 9.52 1.27, 8.97 1.76,
and 9.73 1.41 %ID/g at 0.5, 1, and 2 h. The tumor uptakes of
18F-3 were determined to be 7.54 1.60, 7.94 0.96, and 8.47
1.35 %ID/g at 0.5, 1, and 2 h. 18F-1 and 18F-3 had much lower
tumor uptake compared with 18F-2, which were 12.74 1.70,
16.61
2.60, and 16.87
1.23 %ID/g at 0.5, 1, and 2 h,
respectively (Figure 3A−C).
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Figure 3. Small animal PET images quantification of tumors and major organs at 0.5 h, 1 h, and 2 h after injection of (A) 18F-1, (B) 18F-2, and (C) 18F-3
(∼100 μCi/mouse, n = 4). (D) Time−activity curves of tumor and major organs of C57BL/6 mice bearing B16F10 murine melanoma tumors from 35
min dynamic scans after intravenous injection of 18F-2 (∼100 μCi/mouse, n = 4).
a
Table 1. Biodistributions of 18F-1, 18F-2, and 18F-3 in C57BL/6 Mice Bearing B16F10 Murine Melanoma Tumors at 1 and 2 h p.i.
18F-1
18F-2
18F-3
organ
tumor
1 h
2 h
1 h
2 h
1 h
2 h
b
b
b
b
b
,
c
b
b
b
c
,
,
,
c
c
c
c
c
7.54 1.64
0.33 0.08
0.60 0.07
0.88 0.13
2.07 0.47
1.09 0.87
1.06 0.36
2.24 0.60
0.49 0.14
2.28 0.36
1.34 0.28
0.73 0.16
0.60 0.14
11.29 2.88
8.66 1.42
0.15 0.01
0.37 0.04
0.59 0.08
1.41 0.12
0.31 0.02
0.44 0.02
1.64 0.59
0.24 0.03
0.98 0.04
0.69 0.06
0.63 0.35
0.20 0.03
10.27 3.59
32.45 3.67
15.20 3.37
0.42 0.13
1.17 0.26
1.58 0.33
4.71 1.47
1.78 0.49
1.61 0.48
4.26 2.62
1.28 0.33
2.01 0.47
3.32 1.23
1.30 0.48
1.16 0.29
2.40 0.26
37.47 2.55
16.97 3.28
0.19 0.02
0.59 0.04
0.69 0.07
3.32 0.39
1.13 0.87
0.61 0.04
1.47 0.53
0.42 0.06
0.88 0.21
1.03 0.09
0.68 0.41
0.41 0.04
1.72 0.45
32.83 5.25
9.54 3.44
0.46 0.07
1.54 0.53
1.52 0.43
4.47 2.27
2.88 1.63
3.58 1.69
5.03 1.78
1.56 0.41
3.46 1.26
5.63 1.72
2.37 0.64
1.11 0.18
3.48 0.80
34.00 9.19
9.29 2.49
0.20 0.01
0.40 0.06
0.92 0.13
1.07 0.25
0.56 0.06
0.71 0.07
2.27 0.33
0.41 0.07
0.89 0.53
1.20 0.29
0.71 0.39
0.52 0.08
2.12 0.38
32.23 7.73
blood
heart
b
b
b
b
b
b
c
c
c
lungs
b
b
b
b
b
liver
spleen
pancreas
stomach
brain
b
b
b
b
b
intestine
kidneys
skin
b
b
b
b
b
b
b
b
muscle
bone
b
eyes
35.96 12.17
Uptake Ratio
b
b
b
b
,
c
b
,
,
c
c
c
c
c
c
tumor to blood
tumor to lung
tumor to liver
tumor to muscle
28.32 3.32
44.33 7.35
9.46 1.76
4.16 1.25
27.75 4.24
41.44 8.09
9.94 1.29
3.79 1.41
13.84 2.38
88.25 9.77
24.92 6.56
5.14 1.03
36.79 5.21
20.44 4.34
6.23 0.84
2.24 0.32
11.24 2.76
39.20 4.66
10.49 4.33
7.26 3.33
15.22 3.91
c
b
8.81 2.44
3.85 1.32
13.12 4.15
b
b,
c
c
a
b
Data are expressed as normalized accumulation of activity in %ID/g SD (n = 4). P < 0.05, comparison of biodistribution between 18F-1 and18F-
c
2. P < 0.05, comparison of biodistribution between 18F-2 and 18F-3.
The higher tumor uptake of 18F-2 encouraged us to perform a
35-min dynamic small animal PET scans for this novel probe (n =
4). As shown in Figure 3D, 18F-2 was rapidly cleared from renal
system as determined by ROI analysis of the kidneys. At 5 min
after tail vein injection of 18F-2, radioactivity rapidly accumulated
in kidneys (24.91 5.29 %ID/g) and decreased to 9.44 4.04 %
ID/g at 35 min p.i. In contrast, tumor uptake reached 5.28 1.54
%ID/g at 5 min p.i. and gradually increased to the highest 10.52
1.29 %ID/g at the end of the 35 min dynamic scan. During the
whole dynamic scan frames, low levels of liver and muscle
uptakes were observed.
Biodistribution Studies. We also performed a biodistribu-
tion experiment by direct-sampling tumors and tissues of
interest. The results are shown in Table 1. The B16F10 tumor
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uptakes for 18F-1 were 7.54 1.64 and 8.66 1.42 %ID/g at 1
and 2 h, respectively, while for 18F-3, they were 9.54 3.44 and
9.29 2.49 %ID/g at 1 and 2 h p.i. Both 18F-1 and 18F-3 have
lower uptake than 18F-2, which were 15.20 3.37 %ID/g and
16.97 3.28 %ID/g at 1 and 2 h p.i., respectively. Of note,
because of the high melanin concentration in C57BL/6 mouse
eyes, the uptake in eyes remained at high levels for all three
probes. Interestingly, the bone uptake of 18F-2 (2.40 0.26 and
1.72 0.45 %ID/g at 1 and 2 h, respectively, n = 4) and 18F-3
(3.48 0.80 and 2.12 0.38 %ID/g at 1 and 2 h, respectively, n =
4) were significantly lower than those of 18F-1 (11.29 2.88 and
10.27 3.59 %ID/g at 1 and 2 h, respectively, n = 4) (P < 0.05),
which indicated that 18F-2 and 18F-3 have better in vivo stability
against defluorination. In comparison, 18F-1 showed significant
defluorination in vivo as demonstrated by its high bone uptake.
Moreover, because of the rapid clearance of these 18F-labeled
picolinamide probes from normal nontargeted organs, most of
the tumor-to-normal tissue ratios increase with time. For
example, the tumor to muscle ratio of 18F-2 was 13.84 2.38
and 36.79 5.21 at 1 and 2 h, respectively; while for 18F-3, it was
11.24 2.76 and 15.22 3.91 at 1 and 2 h p.i., respectively
(Table 1).
aromatic substitution,30 some weak nucleophiles, such as water,
amino acid, or proteins, can potentially replace the 18F in these
two positions. In contrast, the relative low reactivity of 3-position
in the pyridine ring increases the relative in vivo stability of 18F-2.
It is noteworthy that the bone uptake of 18F-1 is about 10 times
higher than that of 18F-2 or 18F-3, which suggests 18F in the 4-
position is very unstable (Table 1).
In our previous study, 18F-FBZA was developed as a melanin-
targeting PET probe with a chemical structure similar to that of
18F-2.23 However, 18F-FBZA has a much lower tumor uptake in
the B16F10 melanoma tumors than 18F-2 (5.94 1.83 %ID/g
for 18F-FBZA at 2 h p.i. vs. 16.97 3.28 %ID/g for 18F-2 at 2 h
p.i.). Furthermore, a three-step radiosynthesis of N-succinimidyl
4-18F fluorobenzoate (18F-SFB) is needed before direct coupling
of the 18F-SFB with the amine compound to prepare 18F-FBZA;
thus the total radiosynthesis time is typically more than 3 h,
compared with only 1 h for preparation 18F-1 and 18F-2. In this
study, we also show that 18F-2 is superior to 18F-3 in terms of
tumor uptake, tumor to normal organ ratios (Table 1), and
tumor imaging contrast (Figure 2). Overall, 18F-2 is demon-
strated to be an excellent candidate for translation as a clinical
PET probe for melanoma diagnosis in term of radiosynthesis,
tumor targeting efficiency, and in vivo stability.
DISCUSSION AND CONCLUSIONS
In conclusion, we designed and synthesized two novel PET
probes and a reported PET probe for melanoma diagnosis based
on the picolinamide structure. The small animal PET and
biodistribution studies in murine melanoma xenografts resulted
in excellent tumor imaging contrast using all of these probes.
Especially 18F-2 shows high in vivo stability and favorable
pharmacokinetic properties such as fast clearance from urinal
system and almost background level of uptake for all of the major
organs at 2 h. The high selectivity and specificity of 18F-2, as
evidenced by the high tumor-to-non tumor ratios, highlight that
18F-2 PET has high potential to improve melanoma detection. All
the desirable properties of 18F-2 warrant large scale production
and potential clinical applications of this novel PET probe.
■
18F (t1/2 = 109.7 min; β+, 99%) is an ideal PET radionuclide for
labeling biologically active ligands such as small molecules,
peptides, or small proteins for PET probe development. The
success of 18F-FDG has made PET a powerful tool in cancer
diagnosis, patient stratification, and treatment monitoring of
cancer patients.28 In the development of melanoma-specific
imaging probes other than the generic tumor imaging probes
such as 18F-FDG, 123I-labeled BZA compounds have been
evaluated in melanoma patients with 100% specificity.22 Because
of favorable physical properties of 18F-fluoride, efforts have been
made to develop 18F-labeled BZA (18F-FBZA)23,24 and 18F-
labeled BZA-like 18F-MEL050 (18F-3).27 It has been found that
18F-3 offers high tumor uptakes, fast clearance, and low
background.27 The syntheses of 18F-3 analogs have been
optimized recently; however, in vivo tumor targeting efficacy
was not reported therein.29 To further develop 18F-3 analogs
with improved in vivo performance, we designed and synthesized
18F-1 and 18F-2 herein and compared their in vivo tumor imaging
properties with 18F-3 in the same B16F10 melanoma model.
The authentic standards (1−3) and nonradioactive precursors
(4−6) can be readily prepared in high yields from their
corresponding acids. The compounds 1−6 were fully charac-
terized using nuclear magnetic resonance (NMR) and electro-
spray ionization mass spectrometry (ESI-MS). The direct and
single step 18F-fluorination of the precursors can be easily
accomplished to achieve the targeted PET probes in good
radiochemical yields. Because of the low reactivity of the 3-
position, the RCYs of 18F-2 are lower than the other two
radioligands (P < 0.05). However, the radiosynthesis of 18F-2 can
still be easily automated for large quantity synthesis (3.7−37
GBq) for potential clinical translation, because of the short
synthetic time and the straightforward radiochemistry.
EXPERIMENTAL SECTION
■
General. All chemicals obtained commercially were of analytic grade
and used without further purification. No-carrier-added 18F-fluoride was
obtained from an in-house PETtrace cyclotron (GE Healthcare).
Reversed-phase extraction C18 Sep-Pak cartridges were obtained from
Waters and were pretreated with ethanol and water before use. The
syringe filter and polyethersulfone membranes (pore size, 0.22 μm;
diameter, 13 mm) were obtained from Nalge Nunc International. The
semipreparative RP-HPLC using a Vydac protein and peptide column
(218TP510; 5 μm, 250 mm × 10 mm) was performed on a Dionex 680
chromatography system with a UVD 170U absorbance detector and
model 105S single-channel radiation detector (Carroll & Ramsey
Associates). The recorded data were processed using Chromeleon
version 6.50 software. With a flow rate of 5 mL/min, the mobile phase
was changed from 95% solvent A [0.1% trifluoroacetic acid (TFA) in
water] and 5% B [0.1% TFA in acetonitrile (MeCN)] (0−2 min) to 35%
solvent A and 65% solvent B at 32 min. Analytical RP-HPLC has the
same gradient system except that the flow rate was 1 mL/min with a
Vydac protein and peptide column (218TP510; 5 μm, 250 mm × 4.6
mm). The UV absorbance was monitored at 218 nm, and the
identification of the small molecules was confirmed based on the UV
spectrum acquired using a PDA detector. All synthesized compounds
showed more than 95% purity (RP-HPLC). Small animal PET scans
were performed on a microPET R4 rodent model scanner (Concorde
Microsystems Inc.). The scanner has a computer-controlled bed and
10.8 cm transaxial and 8 cm axial fields of view (FOVs). It has no septa
and operates exclusively in the three-dimensional (3D) list mode.
As expected, 18F-1, 18F-2, and 18F-3 all exhibit high tumor
targeting efficiency, excellent tumor imaging contrasts, and
desirable biodistribution patterns (Figure 2, Table 1). Partic-
ularly, 18F-2 shows significantly lower bone uptake than 18F-1
and 18F-3 (P < 0.05) (Table 1). Because of the high reactivity of
2- and 4-positions of the pyridine ring toward the nucleophilic
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Animals were placed near the center of the FOV of the scanner, where
the highest image resolution and sensitivity are available.
Radiochemistry. An aqueous 18F-fluoride solution (15−30 mCi) was
added to a 10 mL vial containing anhydrous acetonitrile (1 mL), K2.2.2
(15 mg), and K2CO3 (3 mg). The solvent was evaporated under a stream
of argon at 100 °C under vacuum to produce the K18F−K2.2.2 complex.
This azeotropic drying was repeated twice by anhydrous acetonitrile (2
× 1 mL). The bromo-precursor (4, 5, or 6, 5 mg) was dissolved in
anhydrous DMSO (200 μL) and added to the dried K18F−K2.2.2
complex. The reaction was stirred and heated at 110 °C for 10 min
and cooled to room temperature. The mixture was then diluted with 1
mL of 5% acetic acid solution for RP-HPLC purification. The collected
radioactive peak was dried using a rotary evaporator and the radiolabeled
products were reconstituted in phosphate-buffered saline (PBS, 0.1 M,
pH = 7.4) and passed through a 0.22 μm Millipore filter into a sterile vial
for in vitro and in vivo experiments. The radiochemical yields were
calculated based on the obtained radioactive product divided by the
activity loaded into the reaction vessel.
Cell Culture. B16F10 cells were cultured in Dulbecco’s modified
Eagle high-glucose medium (Gibco Life Sciences) supplemented with
10% fetal bovine serum with penicillin and streptomycin. The cells were
regularly maintained in a 37 °C, 5% CO2 humidified incubator.
Animal Biodistribution Studies. Animal procedures were
performed according to a protocol approved by the Stanford University
Institutional Animal Care and Use Committee. All of the animals were
purchased from Charles River Laboratory. Cultured B16F10 cells (∼1.0
× 106) were suspended in PBS and subcutaneously implanted in the
right shoulder of C57BL/6 mice. Tumors were allowed to grow to a size
of 0.5 cm (∼10 day) before use. For biodistribution studies, the tumor-
bearing mice (n = 4 for each group) were injected with about 3.7 MBq
(100 μCi) of 18F-1, 18F-2, or 18F-3 through the tail vein and sacrificed at
1.0 and 2.0 h p.i. Tumor and normal tissues of interest were removed and
weighed, and their radioactivity was measured in a γ-counter. The
radioactivity uptake in the tumor and normal tissues was calculated as %
ID/g.
Small Animal PET Imaging. For dynamic scan, B16F10 tumor-
bearing mice (n = 4) were injected via the tail vein with approximately
3.7 MBq (100 μCi) of 18F-2, and scans (6 × 20 s, 8 × 60 s, 10 × 150 s,
total of 24 frames) were started roughly 2.0 min after the injection of the
probe and continued for 35 min. For static scans, the mice bearing
B16F10 (n = 4 for each probe) tumor xenografts were injected with
about 3.7 MBq (100 μCi) of 18F-1, 18F-2, or 18F-3 via the tail vein. At 0.5,
1, and 2 h p.i., the mice were anesthetized with isoflurane (5% for
induction and 2% for maintenance in 100% O2) using a knock-down
box. With the help of a laser beam attached to the scanner, the mice were
placed in the prone position and near the center of the field of view of the
scanner. The 3-min static scans were then obtained. Images were
reconstructed using two-dimensional ordered subsets expectation
maximization (OSEM) algorithm. No background correction was
performed. ROI (5 pixels for coronal and transaxial slices) were drawn
over the tumor on decay-corrected whole-body coronal images. The
maximum counts per pixel per minute were obtained from the ROI and
converted to counts per milliliter per minute using a calibration
constant. On the basis of the assumption of a tissue density of 1 g/mL,
the ROIs were converted to counts per gram per min. Image ROI-
derived %ID/g values were determined by dividing counts per gram per
minute by injected dose. No attenuation correction was performed.
Statistical Analysis. Quantitative data are expressed as mean SD.
Means were compared using the Student t test. P values of <0.05 were
considered statistically significant.
Chemistry and Radiochemistry. Preparation of 19F-1, 19F-2, and
19F-3 and their bromo-precursors 4, 5, and 6 (Scheme 1 and Supporting
Information) was accomplished using the same protocol. As an example,
19F-1 was synthesized as follows: To a solution of 4-fluoropicolinic acid
(5.0 mg, 35.5 μmol) in 200 μL of N,N-dimethylformamide (DMF) was
added TSTU (10.0 mg, 33.0 μmol) and 20 μL of DIPEA. After
incubating at 60 °C for 3 h, the reaction mixture was cooled to room
temperature, followed by addition of N,N-diethylethylenediamine (7.0
mg, 60 μmol). After 2 h, the mixture was diluted with 1 mL of 5% acetic
acid solution. The product 19F-1 was isolated by semipreparative RP-
HPLC. The collected fractions were combined, and acetonitrile was
removed under reduced pressure. The final product was obtained by
lyophilization.
N-(2-(Diethylamino)ethyl)-4-fluoropicolinamide (19F-1). The prod-
uct was obtained as white powder in the yield of 56% with 98% purity as
determined by RP-HPLC. ESI-MS: m/z 240.3 [M + H]+ (C12H19FN3O,
calculated molecular weight 240.2). 1H NMR (CDCl3, 300 MHz): δ =
8.94 (br, 1H), 8.57 (dd, J = 5.2 Hz, 9.0 Hz, 1H), 7.83 (dd, J = 2.4 Hz, 9.0
Hz, 1H), 7.15 (dd, J = 2.4 Hz, 5.2 Hz, 1H), 3.93 (t, J = 6.1 Hz, 2H), 3.29
(t, J = 5.9 Hz, 2H), 3.22 (q, J = 8.6 Hz, 4H), 1.37 (t, J = 7.1 Hz, 6H). 13C
NMR (CDCl3, 75 MHz): δ = 167.9, 164.6 (d, JC,F = 101.8 Hz), 152.5,
151.2 (d, JC,F = 6.6 Hz), 114.2 (d, JC,F = 16.5 Hz), 110.4 (d, JC,F = 18.7
Hz), 51.2, 47.3, 35.0, 8.4.
N-(2-(Diethylamino)ethyl)-5-fluoropicolinamide (19F-2). The prod-
uct was obtained as white powder in the yield of 75% with 98% purity as
determined by RP-HPLC. ESI-MS: m/z 240.5 [M + H]+ (C12H19FN3O,
calculated molecular weight 240.2). 1H NMR (CDCl3, 300 MHz): δ =
11.35 (br, 1H), 8.41 (d, J = 2.7 Hz, 1H), 8.17 (dd, J = 4.4, 8.2 Hz, 1H),
7.45 (dd, J = 2.7, 8.2 Hz, 1H), 3.88 (t, J = 6.1 Hz, 2H), 3.38−3.08 (m,
6H), 1.36 (t, J = 7.3 Hz, 6H). 13C NMR (CDCl3, 75 MHz): δ = 164.9,
161.5 (d, JC,F = 38.5 Hz), 161.4 (d, JC,F = 261.5 Hz), 145.2 (d, JC,F = 4.5
Hz), 137.3 (d, JC,F = 25.4 Hz), 124.0 (d, JC,F = 5.4 Hz), 51.5, 47.2, 34.8,
8.3.
N-(2-(Diethylamino)ethyl)-6-fluoronicotinamide (19F-3). The
product was obtained as white powder in the yield of 62% with 97%
purity as determined by RP-HPLC. ESI-MS: m/z 240.3 [M + H]+
1
(C12H19FN3O, calculated molecular weight 240.2). H NMR (CDCl3,
300 MHz): δ = 11.00 (br, 1H), 8.80 (d, J = 1.7 Hz, 1H), 8.33 (dd, J = 1.7,
8.5 Hz, 1H), 6.99 (dd, J = 1.7, 8.5 Hz, 1H), 3.82 (t, J = 3.9 Hz, 2H), 3.37
(t, J = 3.9 Hz, 2H), 3.23 (q, J = 7.3 Hz, 2H), 1.35 (t, J = 7.3 Hz, 6H). 13C
NMR (CDCl3, 75 MHz): δ = 167.0, 164.6 (d, JC,F = 137.4 Hz), 148.7 (d,
JC,F = 15.9 Hz), 140.4 (d, JC,F = 9.3 Hz), 127.1 (d, JC,F = 4.4 Hz), 109.5 (d,
JC,F = 37.4 Hz), 52.4, 48.6, 35.8, 8.6.
4-Bromo-N-(2-(diethylamino)ethyl)picolinamide (4). The product
was obtained as white powder in the yield of 82% with 97% purity as
determined by RP-HPLC. ESI-MS: m/z 300.4 [M + H]+
(C12H19BrN3O, calculated molecular weight 300.1). 1H NMR
(CDCl3, 300 MHz): δ = 8.97 (br, 1H), 8.40 (d, J = 5.2 Hz, 1H), 8.28
(d, J = 1.7 Hz, 1H), 7.61 (dd, J = 1.7, 3.2 Hz, 1H), 3.91 (t, J = 3.7 Hz,
2H), 3.39 (t, J = 3.7 Hz, 2H), 3.23 (q, J = 7.1 Hz, 4H), 1.34 (t, J = 7.1 Hz,
6H). 13C NMR (CDCl3, 75 MHz): δ = 164.5, 150.2, 149.5, 134.3, 129.8,
125.8, 51.2, 47.1, 34.9, 8.3.
5-Bromo-N-(2-(diethylamino)ethyl)picolinamide (5). The product
was obtained as white powder in the yield of 90% with 98% purity as
determined by RP-HPLC. ESI-MS: m/z 300.4 [M]+ (C12H19BrN3O,
calculated molecular weight 300.1). 1H NMR (CDCl3, 300 MHz): δ =
8.90 (br, 1H), 8.62 (s, 1H), 8.01−7.95 (m, 1H), 7.61 (dd, J = 1.7, 3.2 Hz,
1H), 3.89 (t, J = 5.8 Hz, 2H), 3.26 (t, J = 5.8 Hz, 2H), 3.25 (q, J = 7.3 Hz,
4H), 1.34 (t, J = 7.3 Hz, 6H). 13C NMR (CDCl3, 75 MHz): δ = 165.1,
149.9, 147.4, 140.0, 124.6, 123.5, 51.4, 47.3, 34.8, 8.3.
6-Bromo-N-(2-(diethylamino)ethyl)nicotinamide (6). The product
was obtained as white powder in the yield of 78% with 97% purity as
determined by RP-HPLC. ESI-MS: m/z 300.2 [M + H]+
(C12H19BrN3O, calculated molecular weight 300.1). 1H NMR
(CDCl3, 300 MHz): δ = 9.38 (br, 1H), 8.92 (s, 1H), 8.08 (d, J = 8.3
Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 3.84 (m, 2H), 3.34 (m, 2H), 3.21 (m,
4H), 1.35 (t, J = 7.1 Hz, 6H). 13C NMR (CDCl3, 75 MHz): δ = 165.8,
150.1, 145.7, 137.2, 128.0, 123.8, 52.3, 48.6, 35.7, 8.
ASSOCIATED CONTENT
■
S
* Supporting Information
RP-HPLC and mass spectrometry data of synthesized
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
899
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Journal of Medicinal Chemistry
Article
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AUTHOR INFORMATION
■
Corresponding Author
*Mailing address: Molecular Imaging Program at Stanford,
Canary Center at Stanford for Cancer Early Detection
Department of Radiology and Bio-X Program, 1201 Welch
Road, Lucas Expansion, P095 Stanford University, Stanford, CA
94305. Phone: 650-723-7866. Fax: 650-736-7925. E-mail:
zcheng@stanford.edu.
Author Contributions
H. Liu and S. Liu contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported, in part, by the Melanoma Research
Alliance, National Cancer Institute (NCI) In Vivo Cellular
Molecular Imaging Center (ICMIC) Grant P50 CA114747, and
the international cooperation projects of the Chinese Ministry of
Science and Technology (Grant 2009DFB30040). The authors
thank the Radiochemistry Facility of Molecular Imaging Program
at Stanford for 18F-fluoride production.
ABBREVIATIONS USED
■
PET, positron emission tomography; BZA, benzamide; %ID/g,
percentage injected dose per gram; 18F-1, N-(2-(diethylamino)-
ethyl)-18F-4-fluoropicolinamide; 18F-2, N-(2-(diethylamino)-
ethyl)-18F-5-fluoropicolinamide; 18F-3(18F-MEL050), 18F-6-flu-
oro-N-[2-(diethylamino)ethyl] pyridine-3-carboxamide; RCYs,
radiochemical yields
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