Synthesis and evaluation of F-18 labeled pyrido[3,2-B]pyrazine derivative as a potential imaging agent for non-small-cell lung cancer

Article abstract of DOI:10.1002/bkcs.10335

Pyridopyrazine derivatives have been known as Wnt-2/β-catenin signaling pathway inhibitors. Wnt-2 overexpression may be involved in non-small-cell lung cancer (NSCLC). A novel 2-(4-[¹⁸ F]fluorobutoxy)-3-(phenylethynyl)pyrido[3,2-b]pyrazine was prepared to demonstrate the feasibility of NSCLC imaging agent by uptake of Wnt-2 protein. It was synthesized with tosylated precursor using [¹⁸F]fluoride in radiochemical yield of 44-48%. In cellular uptake evaluation, H460 and H1299, Wnt-2 expressed cancer cell lines, showed 2.5-folds higher cellular uptake than that of MCF10A as a control.

Full text of DOI:10.1002/bkcs.10335

Article
DOI: 10.1002/bkcs.10335
BULLETIN OF THE
J. H. Park et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Evaluation of F-18 Labeled Pyrido[3,2-B]pyrazine
Derivative as a Potential Imaging Agent for Non-Small-Cell Lung Cancer
Jeong Hoon Park,†,k Heejung Kim,‡,k Dong-Yeon Kim,§ Seung Dae Yang,† Min Goo Hur,†
Sang Wook Kim,¶ and Kook Hyun Yu¶,
*
†Radiation Instrumentation Research Division, Korea Atomic Energy Research Institute,
Jeongeup 580-185, Korea
‡Department of Chemistry, Dongguk University-Seoul, Seoul 100-715, Korea
§Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun 519-763, Korea
¶Department of Advanced Materials Chemistry, Dongguk University-Gyeongju, Gyeongju 780-714, Korea.
*E-mail: yukook@dgu.ac.kr
Received September 3, 2014, Accepted March 7, 2015, Published online June 29, 2015
Pyridopyrazine derivatives have been known as Wnt-2/β-catenin signaling pathway inhibitors. Wnt-2 over-
expression may be involved in non-small-cell lung cancer (NSCLC). A novel 2-(4-[18F]fluorobutoxy)-3-(phe-
nylethynyl)pyrido[3,2-b]pyrazine was prepared to demonstrate the feasibility of NSCLC imaging agent
by uptake of Wnt-2 protein. It was synthesized with tosylated precursor using [18F]fluoride in radiochemical
yield of 44–48%. In cellular uptake evaluation, H460 and H1299, Wnt-2 expressed cancer cell lines, showed
2.5-folds higher cellular uptake than that of MCF10A as a control.
Keywords: Wnt-2 inhibitors, Non-small-cell lung cancer, Pyridopyrazine derivatives,
[18F]fluoride, Imaging agent
Introduction
techniques such as positron emission tomography (PET).
Recently, Gong et al. screened 1434 compounds representing
20 000 structurally diverse and heterocyclic molecules such
asbenzopyrans,oxazoles,andthiazoles,andidentified13deri-
vatives of 2,3,6-trisubstituted pyrazine derivatives which
promised to inhibit the Wnt-2/β-catenin pathway and cell pro-
liferation. Among them 3-arylethynyl-substituted pyrido[2,3-
b]pyrazine derivatives were shown to have an excellent
potency (IC50 = 0.1–0.5 μM) for the inhibition of the Wnt-2/
β-catenin pathway of NSCLC.17 F-18 has been widely used
as a PET tracer for cancer diagnosis because its half-life
(110 min) allows for more effective synthesis and scanning
than other tracers.18 In this study, we report the preparation
of 18F-labeled pyrido[3,2-b]pyrazine and the feasibility of
labeling of Wnt-2/β-catenin pathway inhibitors as a radiotra-
cer for PET imaging.
Wnt proteins constitute a family of highly conserved glyco-
proteins that play multiple roles in the development and pro-
gression of disease. Wnt signaling is one of the key signaling
pathways that regulate cell proliferation, differentiation, and
morphogenesis.1,2 The role of Wnt signaling in cancer was
suggested by the discovery of Wnt-1 as an integration site
ofmouse mammary tumor virus(MMTV) inmouse mammary
carcinoma.3 The overexpression of Wnt (e.g., Wnt-1 and Wnt-
2) that signals through β-catenin promotes the transformation
of mouse mammary epithelial C57MG cells.4,5 There is
increasing evidence that the Wnt signaling pathway is
involved in tumor development and/or progression.6–8 Muta-
tions in β-catenin, adenomatosis polyposis coli (APC), and
Axin have been found in human colon cancers, suggesting that
constitutive activation of the Wnt canonical pathway contri-
butes to human carcinogenesis.9 For instance, the overexpres-
sion of Wnt-2 protein has frequently been reported in human
colorectal cancer and gastric cancer.10 Moreover, You et al.
analyzed the expression of Wnt-2 protein in human non-
small-cell lung cancer (NSCLC) cells by Western blotting,
and these results suggested that Wnt-2 overexpression may
be involved in human NSCLC.11 Moreover, overexpression
of the Wnt signaling pathway has been reported in other
human cancers including colon and lung carcinoma,12–16
which makes it a potential target for therapeutic intervention
and diagnosis based on noninvasive medical imaging
Experimental
All commercial reagents and solvents were purchased from
Sigma-Aldrich (Munich, Germany) or Merck (Darmstadt,
Germany) and analytical grade reagents were used without
further purification. The 1H and 13C NMR spectra were
recorded on a JEOL ECA-500 FT-NMR spectrometer
(Tokyo, Japan). All chemical shifts were reported on the
ppm scale with tetramethylsilane as an internal standard. Mass
spectra were recorded on a JEOL JMS-AX505WA spectrom-
eter. Molecular mass of synthesized compounds were meas-
ured by fast atom bombardment (FAB) methods. Gravity
column chromatography was performed using Merck silica
k These authors contributed equally to this work.
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F-18 Labeled Pyridopyrazine Derivative as a Lung Cancer Imaging Agent
KOREAN CHEMICAL SOCIETY
gel 60 (70–230 mesh, the America Society for Testing &
Methods). Thin-layer chromatography (TLC) was performed
on Merck silica gel 60 F254 glass plates and was visualized
by UV light. Purification was achieved by high-performance
liquid chromatography (HPLC; SP930D pump, UV730D UV
detector; Young-Lin Inc., Seoul, Korea,) and a FC-3200
high-energy gamma detector (Bioscan, Washington, DC,
USA) was used to measure the radioactivity. UV detection
wavelength of 254 nm was used for all experiments.
Semi-preparative HPLC columns (Phenomenex Luna, C18,
10 × 250 mm) and analytical HPLC columns (Phenomenex
Gemini, 5 μm, C18, 250 × 4.6 mm) were used for the separa-
tion and determination. A CRC-712MH radioisotope calibra-
tor (Capintec Instruments, Ramsey, NJ, USA) was used
for the radioactivity measurements. Analysis of radioactivity
was performed using a 1480 WIZARD 3 gamma counter
(Perkin Elmer, Waltham, MA, USA). No-carrier-added (n.c.
a) [18F]fluoride was produced on a GE PET trace cyclotron
(16.4 MeV) by irradiation of [18O] H2O water target.
vacuum, and extracted with a mixture of EtOAc and water.
The organic layer was separated, washed with water, and dried
over MgSO4. After the removal ofthe solventbyarotary evap-
orator, the residue was purified by column chromatography
using (EtOAc: n-hexane = 1:4) to achieve compound 4 as a
1
yellow solid with 68% yield: mp 224 ꢀC; H-NMR (500
MHz, CDCl3): δ 9.18 (dd, J = 4.2 and 2.0 Hz, 1H), 8.36 (dd,
J = 8.3 and 2.0 Hz, 1H), 7.75 (dd, J = 8.5 and 4.2 Hz, 1H),
7.70–7.73 (m, 2H), 7.42–7.49 (m, 3H); 13C NMR (126
MHz, CDCl3): δ 154.85, 149.48, 148.43, 140.03, 137.13,
136.32, 132.75, 130.54, 128.74, 126.15, 121.01, 99.29,
85.26; MS (FAB) m/z 266, (M++H); HRMS of C15H9ClN3
266.0485 (calculated) and 266.0482 (observed).
Synthesis of 4-(3-(phenylethynyl)pyrido[3,2-b]pyrazin-2-
yloxy)butan-1-ol (5). About 3 g of compound 4 (11.3 mmol)
and 6.3 g of potassium carbonate (45.2 mmol) were
dissolved in 15 mL of DMF. The mixture was heated at
100 ꢀC for 8 h and extracted with ETOAc. The organic layer
was separated, washed with water, and dried over MgSO4.
After the removal of the solvent using a rotary evaporator,
the residue was purified by SiO2 column chromatography
(CH2Cl2: Methanol = 95:5) to obtain compound 5 (67%):
Synthesis of Pyrido[3,2-b]pyrazine-2,3(1H,4H)-dione (2).
The mixture of 5.0 g of pyridine-2,3-diamine (1, 45.8 mmol)
and 4.5 g of oxalic acid (50.0 mmol) in 50 mL of 3 N HCl
was refluxed for 24 h. The resulting mixture was filtered,
washed with cold water, and dried under vacuum. The result-
ant product, pyrido[3,2-b]pyrazine-2,3(1H,4H)-dione (2),
1
mp 212 ꢀC; H-NMR (500 MHz, CDCl3): δ 8.95 (dd, J =
4.2 and 2.0 Hz, 1H), 8.14 (dd, J = 8.3 and 2.0 Hz, 1H),
7.67–7.70 (m, 2H), 7.58 (dd, J = 8.2 and 4.2 Hz, 1H),
7.39–7.44 (m, 3H), 4.61 (t, J = 6.3 Hz, 2H), 3.82 (t, J = 6.3
Hz, 2H), 2.47 (quintet, J = 6.3 Hz, 2H), 1.87 (quintet, J =
6.3 Hz, 2H); 13C NMR (126 MHz, CDCl3): δ 157.14,
150.41, 147.99, 136.00, 135.72, 135.01, 132.59, 130.01,
128.58, 125.18, 121.44, 97.72, 84.94, 67.76, 62.18, 29.45,
25.33; MS (FAB) m/z 320, (M++H); HRMS of C19H18N3O2
320.1399 (calculated) and 320.1401 (observed).
1
was obtained in yield (93%): mp > 300 ꢀC; H-NMR (500
MHz, DMSO-d6): δ 12.30 (s, 1H), 11.94 (s, 1H), 8.03 (dd,
J = 4.9 and 1.7 Hz, 1H), 8.03 (dd, J = 7.9 and 1.7 Hz, 1H),
8.03 (dd, J = 8.0 and 4.9 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6): δ 156.47, 155.35, 142.54, 139.71, 122.83,
122.29, 119.31; MS (FAB) m/z 164, (M++H); high
resolution mass spectroscopy (HRMS) of C7H6N3O2
164.0460 (calculated) and 164.0458 (observed).
Synthesis of 4-(3-(phenylethynyl)pyrido[3,2-b]pyrazin-2-
yloxy)butyl-4-methylbenzenesulfonate (6). About 2 g of
compound 5 (6.3 mmol) was dissolved in 15 mL of dry
dichloromethane (DCM), and the reaction mixture was cooled
to 0 ꢀC. To this mixture, 4.4 mL of TEA (31.5 mmol) and 1.8 g
of 4-methylbenzene-1-sulfonyl chloride (9.5 mmol) were
added. The solution was then allowed to warm to room tem-
perature and stirred for additional 8 h. The mixture was con-
centrated under reduced pressure to remove the solvent. The
residue was purified by SiO2 column chromatography
(EtOAc: n-hexane = 1:1) to obtain 4-(3-(phenylethynyl)pyr-
ido[3,2-b]pyrazin-2-yloxy)butyl-4-methylbenzenesulfonate
(6) in yield of 37% as a dark green solid: mp 267–270 ꢀC; 1H-
NMR (500 MHz, CDCl3): δ 8.96 (dd, J = 4.2 and 2.0 Hz, 1H),
8.14 (dd, J = 8.3 and 1.7 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H),
7.64–7.68 (m, 2H), 7.58 (dd, J = 8.2 and 4.2 Hz, 1H),
7.40–7.47 (m, 3H), 7.29 (d, J = 8.0 Hz, 2H), 4.53 (t, J = 6.0
Hz, 2H), 4.17 (q, J = 6.0 Hz, 2H), 2.40 (S, 3H), 1.95–2.00
(m, 4H); 13C NMR (126 MHz, CDCl3): δ 156.87, 150.58,
148.02, 144.93, 139.18, 135.96, 134.97, 133.04, 132.67,
130.18, 129.94, 128.74, 127.91, 125.31, 121.51, 97.98,
84.84, 70.06, 66.76, 25.84, 24.93, 21.70; MS (FAB) m/z
474, (M++H); HRMS of C26H24N3O4S 474.1488 (calculated)
and 474.1487 (observed).
Synthesis of 2,3-dichloropyrido[3,2-b]pyrazine (3). About
5 g of 2 (30.7 mmol) was dissolved in 50 mL of phosphorus
oxychloride (POCl3). The mixture was refluxed for 24 h and
concentrated under vacuum until the complete removal of
POCl3 was achieved. The residue was poured into iced-water
and extracted with ethyl acetate (EtOAc). The combined
extracts were washed with saturated NaHCO3 and brine solu-
tion, followed by drying to obtain white powder of 2,3-
dichloropyrido[3,2-b]pyrazine (3) in yield of 81%: mp >
1
280 ꢀC; H-NMR (500 MHz, CDCl3): δ 9.16 (dd, J = 4.3
and 1.8 Hz, 1H), 8.40 (dd, J = 8.3 and 1.7 Hz, 1H), 7.77 (dd,
J = 8.3 and 4.3 Hz, 1H); 13C NMR (126 MHz, CDCl3): δ
155.05, 148.94, 148.86, 146.78, 137.31, 136.17, 126.41;
MS (FAB) m/z 200, (M++H); HRMS of C7H4Cl2N3
199.9782 (calculated) and 199.9786 (observed).
Synthesis of 2-chloro-3-(phenylethynyl)pyrido[3,2-b]pyr-
azine (4). To a stirred solution of 4.5 g of compound 3
(22.5 mmol) in 10 mL of acetonitrile, 2.5 mL of phenylacety-
lene (23.0 mmol), 25 mL of triethylamine (TEA; 180 mmol),
500 mg of palladium(II) acetate (2.2 mmol), 419 mg of copper
iodide (2.2 mmol), and 577 mg of triphenylphosphine (2.2
mmol) were added, and the mixture was heated up to 80 ꢀC
for 3 h. The resulting mixture was concentrated, dried under
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F-18 Labeled Pyridopyrazine Derivative as a Lung Cancer Imaging Agent
KOREAN CHEMICAL SOCIETY
Synthesis of 2-(4-fluorobutoxy)-3-(phenylethynyl)pyrido
[3,2-b]pyrazine (7). To a solution of 500 mg of compound
6 (1.1 mmol) in 20 mL of dry acetonitrile, 350 mg of tetrabu-
tylammonium fluoride (1.1 mmol) was added, and the result-
ant mixture was refluxed for 2 h. The mixture was
concentrated under reduced pressure by a rotary evaporator.
The crude product was purified by SiO2 column chromatogra-
phy (EtOAc: n-hexane = 1:3) to obtain a pale yellow solid
2-(4-fluorobutoxy)-3-(phenylethynyl)pyrido[3,2-b]pyrazine
In Vitro Cell Uptake Studies. H460, H1299, AGS, and
HepG2 cells were grown in RPMI-1640 media supplemented
with 10% fetal bovine serum (FBS), 1% penicillin–streptomy-
cin, and 1% L-glutamine. MCF10A cells were cultured
in a (1:1) mixture of Dulbecco's modified Eagle's medium
and F12 medium (DMEM-F12) supplemented with 5% horse
serum, hydrocortisone (0.5 μg/mL), insulin (10 μg/mL), epi-
dermal growth factor (20 ng/mL), and penicillin–
streptomycin (100 μg/mL each). The cells were maintained
at 37 ꢀC in a 5% CO2 atmosphere. Uptake of the radiotracer
into cells was determined as follows. The cells were treated
with the radiotracer (0.74 MBq) at 37 ꢀC for 0.5, 1, and 2 h,
and then the medium was collected, and the cells were washed
with cold PBS (3 × 1 mL). The cell suspensions and the super-
natant fractions were collected separately for each individual,
and the radioactivity was measured using a gamma counter.
Experiments were performed with triplicate samples. The data
are expressed as the normalized accumulation of the probe (in
dpm cells/dpm medium/μg total protein) SD.
1
(7) in yield of 48%: mp 278–280 ꢀC; H-NMR (500 MHz,
CDCl3): δ 8.96 (dd, J = 4.2 and 2.0 Hz, 1H), 8.15 (dd, J =
8.3 and 1.7 Hz, 1H), 7.66–7.69 (m, 2H), 7.54–7.59 (m, 1H),
7.36–7.45 (m, 3H), 4.66 (dt, J = 47.2 and 6.0 Hz, 2H), 4.65
(q, J = 6.0 Hz, 2H), 1.97–2.11 (m, 4H); 13C NMR (126
MHz, CDCl3): δ157.12, 150.72, 148.30, 136.05, 135.68,
135.02, 132.67, 130.08, 128.68, 125.22, 121.68, 97.73,
84.93, 84.38, 67.26, 27.32, 25.63; MS (FAB) m/z 322,
(M++H); HRMS of C19H17 FN3O 322.1356 (calculated),
322.1360 (observed).
Radiosynthesis of 2-(4-[18F]fluorobutoxy)-3-(phenylethy-
nyl)pyrido[3,2-b]pyrazine ([18F]7). Aqueous [18F]fluoride
was added to a reaction vial containing 7.5 mg of Kryptofix
2.2.2 and 1.5 mg of K2CO3 dissolved in an acetonitrile–water
mixture (1 mL, 9:1[v/v]). The solvent was removed by pur-
ging with gentle nitrogen gas at 100 ꢀC, and the residue was
dried by azeotropic distillation with acetonitrile thrice (31
mL). To this resultant anhydrous residue, 2 mg of prepared
solution of 4-(3-(phenylethynyl)pyrido[3,2-b]pyrazin-2-
yloxy)butyl-4-methylbenzenesulfonate (6) in 0.8 mL of ace-
tonitrile was added. The reaction mixture was heated at
80 ꢀC by stirring for 5 min. The solution was cooled and
injected into a semi-preparative HPLC column system for
purification (mobile phase; acetonitrile:water = 50:50, flow
rate; 3 mL/min, 254 nm, tR; 15.6 min). For the identification
of the 18F-labeled product the collected HPLC fraction was
co-injected with cold compound 7. [18F]7 was dried, made iso-
tonic with sodium chloride, and passed through a 0.20-μm
membrane filter into a sterile vial for in vitro and in vivo
experiments.
Biodistribution Studies in Mice. Biodistribution studies
of [18F]7 was determined in BALB/c mice. Mice were injected
intravenously into the tail vein with 7.4 MBq/animal
(18–20 g, Orient, Kyunggido, Korea) of [18F]7. The animals
were sacrificed after 60 and 120 min, and the selected organs
were subsequently removed (n = 3 at each time point). The
blood, heart, lungs, liver, spleen, stomach, intestine, kidney,
pancreas, muscle, and bone were collected and weighed,
and radioactivity was determined in a γ-counter. Data are
expressed as the percentage of the injected dose per gram of
tissue (%ID/g).
Results and Discussion
2-(4-Fluorobutoxy)-3-(phenylethynyl)pyrido[3,2-b]pyrazine
(7) and precursor (6) were synthesized as illustrated in
Scheme 1. Initially, treatment of 2,3-diaminopyridine (1) with
oxalic acid in the presence of 3 N HCl provided pyrido[3,2-b]
pyrazine-2,3(1H,4H)-dione (2, 93%), which was then chlori-
nated using POCl3 to achieve 2,3-dichloropyrido[3,2-b]pyra-
zine (3, 81%) and compound 3 was coupled with
phenylacetylene under Sonogashira coupling conditions to
In Vitro Stability. The in vitro stability of [18F]7 was
assessed by incubation 0.37 MBq of radioactivity in
1 mL of human serum for 4 h at 37 ꢀC. Proteins were precipi-
tated by adding 2 mL of acetonitrile to the serum sample.
The samples were analyzed by radio-TLC (EtOAc: n-hexane,
1:1).
Partition Coefficient Determination. The log P was meas-
ured by mixing 0.37 MBq of the tracer with 3 mL of 1-octanol
and saline in a test tube. The solution was mixed for 10 min at
room temperature, followed by centrifugation at 3000 rpm for
5 min. After the separation of the two layers, 1 mL of aliquots
of the 1-octanol and aqueous layers were counted on a gamma
counter. Samples from the 1-octanol and aqueous layers were
re-partitioned until consistent values were obtained. The
experiment was performed in triplicate. The partition coeffi-
cient was calculated from the ratio (in cpm/mL) of 1-octanol
to that of saline.
form
2-chloro-3-(phenylethynyl)pyrido[3,2-b]pyrazine
2-(4-Hydroxybutoxy)-3-(phenylethynyl)pyrido
(4).19–22
[3,2-b]pyrazine (5) was obtained by the reaction of 4 and
1,4-butanediol under potassium carbonate in 67% yield. Com-
pound 5 was reacted with 4-methylbenzene-1-sulfonyl chlo-
ride in DCM and TEA to produce 4-(3-(phenylethynyl)
pyrido[3,2-b]pyrazin-2-yloxy)butyl-4-
methylbenzenesulfonate (6, 37%). The reference compound,
2-(4-fluorobutoxy)-3-(phenylethynyl)pyrido[3,2-b]pyrazine
(7) was prepared from the tosylated precursor (6), using tetra-
butylammonium fluoride in acetonitrile at 80 ꢀC (48%). All
compounds synthesized were fully characterized by spectro-
scopic methods.
Radiolabeling of the tosylated precursor 6 was performed
by using the mixture of K18F/Kryptofix 222 in anhydrous
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F-18 Labeled Pyridopyrazine Derivative as a Lung Cancer Imaging Agent
KOREAN CHEMICAL SOCIETY
Scheme 1. Synthesis of 2-(4-fluorobutoxy)-3-(phenylethynyl)pyrido[3,2-b]pyrazine (7).
Scheme 2. Synthesis of 2-(4-[18F]fluorobutoxy)-3-(phenylethynyl)pyrido[3,2-b]pyrazine ([18F]7).
Figure 1. HPLC chromatogram of compound 7 with (a) [18F]7 and (b) purified [18F]7.
acetonitrile at 80 ꢀC for 5 min (Scheme 2). The cooled reaction
mixture was passed through Sep-pak® Plus silica cartridges
(Waters) to remove free 18F−. The crude product was purified
by reverse phase HPLC as shown in Figure 1. The entire label-
ing procedure, including the purification step, completed
within 60 min. Compound 7 and [18F]7 were co-injected into
the radio-HPLC system for comparison (Figure 1(a)). The
product [18F]7(B) was collected at 15 min after injection.
The radiochemical yield was 44–48% (decay corrected,
Scheme 2) in a specific radioactivity of 16.7 GBq/μmol at
the end of synthesis (EOS). The radiochemical purity was
more than 98% using the analytical HPLC system.
TLC developed with EtOAc and n-hexane (1:1, v/v). After
development, the chromatographic strips were scanned on
TLC scanner. The percentage of the remaining [18F]7 (Rf =
0.40–0.45) was more than 95%, which indicates the relatively
good in vitro stability of the radiotracer. The measured log P
value of 1.495 0.003 suggests that [18F]7 is a slightly lipo-
philic compound.
The cellular uptake of [18F]7 are shown in Figure 2. These
values were calculated for H460 (large-cell lung cancer),
H1299 (NSCLC), AGS (gastric adenocarcinoma), HepG2
(hepatocellular carcinoma), and MCF10A (normal breast epi-
thelial) cells at 0.5, 1, and 2 h. H460 and H1299 cell lines have
been known to express Wnt-2 protein from human NSCLC
cell lines.11 MCF10A cell line was used as a negative control.
[18F]7 (0.37 MBq/100 μL) was incubated with human
serum (1.0 mL) for 4 h at 37 ꢀC and then analyzed by radio-
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F-18 Labeled Pyridopyrazine Derivative as a Lung Cancer Imaging Agent
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Similar cellular uptake tendencies were observed for the
H460, H1299, AGS, and HepG2 cell lines. The uptake of
the H460 cells increased in a time dependent manner. The
maximum uptakes were observed at 60 min except for the
H460 cells. The uptake value of the H460 cells increased from
1.12 0.17 at 0.5 h to 1.99 0.27 at 2 h. The uptake value of
the H1299, AGS, and HepG2 cell lines increased from 1.20
0.35, 1.11 0.05, and 1.11 0.20 to 2.35 0.53, 2.93 0.41,
and 2.00 0.18 at 0.5 and 1 h, respectively. The uptake values
of the H460 and H1299 cells were approximately 2.5-fold
higher than that of the MCF10A cell at 2 h. The result suggests
that potential Wnt-2 inhibitor, 2-(4-[18F]fluorobutoxy)-3-
(phenylethynyl)pyrido[3,2-b]pyrazine ([18F]7) has a possibil-
ity as NSCLC radiotracer by Wnt-2 targeting.
physiological uptake and thus potentially sufficient contrast
between specific uptake to tumor and surrounding normal tis-
sue. The main distributions were found in the order of intes-
tine, liver, stomach, and lung, while other organs showed
relatively low accumulations. Higher uptakes in the intestine
and liver compared with the kidneys indicated the predomi-
nance of hepatic and intestinal excretion route.
Conclusion
We have successfully synthesized 2-(4-[18F]fluorobutoxy)-3-
(phenylethynyl)pyrido[3,2-b]pyrazine ([18F]7) as a potential
Wnt-2/β-catenin expressed tumor imaging agent. The radio-
synthesis method of this novel compound proved to be highly
reproducible in specific activity of 16.7 GBq/μmol and in
good radiochemical purity in sufficient quantities to
permit in vitro and in vivo evaluations. In cellular uptake
study, [18F]7 showed 2.5-fold higher uptake for Wnt-2
expressed cell lines (H460 and H1299) compared with the
Biodistribution of [18F]7 in BALB/c mice was measured at
60 and 120 min after injection (IV) (n = 3 at each time point) as
shown in Figure 3. Nonspecific organ distribution and excre-
tion were investigated in normal mice by performing ex vivo
organ distribution experiments in order to ensure low
Figure 2. The cell uptake of [18F]7 in H460, H1299, AG, HepG2, and MCF10A cells at 0.5, 1.0, and 2.0 h (n = 3 at each time point).
Figure 3. Biodistribution in mice of [18F]7 at 60 and 120 min after IV injection (n = 3 at each time point).
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F-18 Labeled Pyridopyrazine Derivative as a Lung Cancer Imaging Agent
KOREAN CHEMICAL SOCIETY
control (MCF10A). These results demonstrate that [18F]7
exhibits promising characteristics for Wnt-2 expressed tumor
imaging.
11. L. You, B. He, Z. Xu, K. Uematsu, J. Mazieres, I. Mikami,
N. Reguart, T. W. Moody, J. Kitajewski, F. McCormick,
D. M. Jablons, Oncogene 2004, 23, 6170.
12. S. M. Van, J. Randall, A. Sergew, L. M. Williams, M. Tennis,
R. A. Winn, Transl. Res. 2008, 151, 175.
13. M. Konigshoff, O. Eickelberg, Am. J. Respir. Cell Mol. Biol.
2010, 42, 21.
14. J. K. Park, J. H. Song, T. C. He, S. W. Nam, J. Y. Lee, W. S. Park,
Neoplasma 2009, 56, 119.
Acknowledgments. The research was supported by Nuclear
R&D Program (2012 M2A2A7035 and 2010-0025899) and
the Converging Research Center Program (2013K000339)
through the Ministry of Science, ICT and Future Planning.
15. X. X. Cheng, Z. C. Wang, X. Y. Chen, Y. Sun, Q. Y. Kong, J. Liu,
H. Li, Cancer Lett. 2005, 223, 339.
References
16. O. Watanabe, H. Imamura, T. Shimizu, J. Kinoshita, T. Okabe,
A. Hirano, K. Yoshimatsu, S. Konno, M. Aiba, K. Ogawa,
Anticancer Res. 2004, 24, 3851.
1. R. T. Moon, J. D. Brown, M. Torres, TrendsGenet. 1997, 13, 157.
2. R. Nusse, Trends Genet. 1999, 15, 1.
3. R. Nusse, H. E. Varmus, Cell 1982, 31, 99.
4. A. Blasband, B. Schryver, J. Papkoff, Oncogene 1992,
7, 153.
5. H. Shimizu, M. A. Julius, M. Giarre, Z. Zheng, A. M. Brown,
J. Kitajewski, Cell Growth Differ. 1997, 8, 1349.
6. M. Bienz, H. Clevers, Cell 2000, 103, 311.
7. M. Peifer, P. Polakis, Science 2000, 287, 1606.
8. P. Polakis, Genes Dev. 2000, 14, 1837.
17. S. B. Lee, Y. I. Park, M. S. Dong, Y. D. Gong, Bioorg. Med.
Chem. Lett. 2010, 20, 5900.
18. K. H. Yu, J. H. Park, S. D. Yang, Bull. Korean Chem. Soc. 2004,
25, 506.
19. E. Negishi, L. Anastasia, Chem. Rev. 2003, 103, 1979.
20. G. Rossi, S. Alijagic, D. Schoeler, M. Schmitt, H. Graf,
B. M. Czarnetzki, D. Wallach, Adv. Exp. Med. Biol. 1995,
378, 129.
9. S. M. Uthoff, M. R. Eichenberger, T. L. McAuliffe,
C. J. Hamilton, S. Galandiuk, Mol. Carcinog. 2001, 31, 56.
10. M. Katoh, Int. J. Oncol. 2001, 19, 1003.
21. K. Sonogashira, Y. Tohda, N. Hagihara, Tetrahedron Lett. 1975,
17, 4467.
22. K. Sonogashira, J. Organomet. Chem. 2002, 653, 46.
Bull. Korean Chem. Soc. 2015, Vol. 36, 1778–1783
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