Development of 99mTc-labeled trivalent isonitrile radiotracer for folate receptor imaging

Article abstract of DOI:10.1016/j.bmc.2019.04.013

Folate receptors (FR) are frequently overexpressed in a wide variety of human cancers. The aim of this study was to develop a trivalent ^99mTc(CO)₃-labeled folate radiotracer containing isonitrile (CN-R) as the coordinating ligand for

Full text of DOI:10.1016/j.bmc.2019.04.013

Accepted Manuscript
Development of 99mTc-labeled trivalent isonitrile radiotracer for folate receptor
imaging
Nadeem Ahmed Lodhi, Ji Yong Park, Mi Kyung Hong, Young Joo Kim, Yun-
Sang Lee, Gi Jeong Cheon, Jae Min Jeong
PII:
S0968-0896(19)30052-5
https://doi.org/10.1016/j.bmc.2019.04.013
BMC 14867
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 January 2019
4 April 2019
6 April 2019
Please cite this article as: Lodhi, N.A., Park, J.Y., Hong, M.K., Kim, Y.J., Lee, Y-S., Cheon, G.J., Jeong, J.M.,
Development of 99mTc-labeled trivalent isonitrile radiotracer for folate receptor imaging, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.04.013
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Development of 99mTc-labeled trivalent isonitrile radiotracer for folate receptor imaging
Nadeem Ahmed Lodhia,d, Ji Yong Parka,b,c, Mi Kyung Honga,c, Young Joo Kima,c, Yun-Sang Leea,b,c, Gi
Jeong Cheona, Jae Min Jeong*a,b,c
a Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Republic of
Korea
b Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Republic of
Korea
c Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
d Isotope Production Division, Pakistan Institute of Nuclear Science & Technology (PINSTECH), P. O,
Nilore, Islamabad
*Corresponding author: Jae Min Jeong, Ph.D.
Jae Min Jeong: Department of Nuclear Medicine, Seoul National University College of Medicine, 101
Daehak-ro, Jongno-gu, Seoul, 110-744, Korea
Tel: +82-2-2072-3805, Fax: 82-2-766-9083, E-mail: jmjng@snu.ac.kr
Conflict of interest : None declared
1

Abstract
Folate receptors (FR) are frequently overexpressed in a wide variety of human cancers. The aim of this
study was to develop a trivalent 99mTc(CO)3-labeled folate radiotracer containing isonitrile (CN-R) as the
coordinating ligand for FR target imaging. [99mTc]Tc-10 was HPLC purified (>98% chemical purity) and
evaluated in vitro and in vivo as a potential agent for targeting FR-positive KB cells. [99mTc]Tc-10 is a
hydrophilic compound with partition coefficient of −2.90 ± 0.13 that showed high binding affinity (0.04 ±
0.002 nM) in vitro. High accumulation and retention of [99mTc]Tc-10 (5.32 ± 2.99% ID/g) was observed
in mice with KB tumors at 4 h after injection through the tail vein, which was significantly inhibited by
co-injection of free folic acid (FA). SPECT (single photon emission tomography)/CT results were in
accordance with biodistribution data at all time points.
Keywords: Trivalent probe; Technetium-99m; Folic acid; isocyanide; tricarbonyl
2

1. Introduction
Folate receptor (FR) is a membrane-associated glycoprotein and is overexpressed on the surface of
various cancer cells (e.g., ovarian, breast, colorectal, endometrial, and renal carcinomas).1,2 FR shows
high binding affinity (Kd = 10−9 ) to folic acid (FA) and its derivatives.3,4 FR is small (MW = 441 Da), low
cost, and non-immunogenic and is compatible with aqueous and organic solvents. Moreover, it is stable
over a broad range of temperature and pH values and is compatible in easy conjugation synthetic
protocols.5,6 FA and its derivatives are internalized into a cell by an FR-mediated endocytosis pathway.7
All of these features make FA an excellent candidate to deliver diagnostic and therapeutic agents
selectively to tumor cells that express FR.
Over the past decade, several folate-conjugated radiopharmaceuticals have been radiolabeled with
68Ga, 18F, 64Cu, 111In, and 99mTc as positron emission tomography (PET) and single photon emission
tomography (SPECT) imaging agents.1,3,8-12 Among them, technetium-99m (99mTc) is the most suitable
and widely used radionuclide for imaging owing to its ideal physical characteristics (t1/2 = 6 h; Eγ = 140
keV) and easy availability.13,14 Recently, the low valent organometallic species [99mTc][Tc(OH2)3(CO)3]+
developed by Alberto et al has attracted much attention for SPECT application owing to its small size,
versatile chelation chemistry, chemical inertness, and thermodynamic stability. Moreover, it can be easily
prepared using a commercially available aqueous-based kit.15,16 In [99mTc][Tc(OH2)3(CO)3]+ synthon,
three coordination sites occupied by water molecules can be easily replaced by suitable monodentate,
bidentate, or tridentate ligands in an aqueous solution.17,18
It has been previously demonstrated that multimeric arginine–glycine–aspartate (RGD) peptides used
for targeting approach on a molecule increase integrin αvβ3 binding affinities and targeting efficiencies
over the corresponding monomer, resulting in enhanced uptake by the tumor.19,20 Various approaches
have been reported to exploit multivalent scaffolds for the construction of molecular imaging probes.21,22
Recently, Zhang et al. reported a multimeric 99mTc-labeled polyamidoamino (PAMAM) dendrimer–FA
conjugate utilizing 2-hydrazionnicotinic acid (HYNIC) as the bifunctional chelator.22-24 The multivalent
3

ligand binds to the receptor with high avidity and affinity, thereby serving as a powerful inhibitor.
However, these approaches required multistep synthesis and a time-consuming purification process,
which are not suitable for large-scale production as needed for clinical application. Moreover, the slow
clearance of radioactivity from non-target tissues yields undesired tumor-to-background contrast.25,26
Encouraged by the multimerization concept, in this study, we synthesized trivalent 99mTc-labeled
folate conjugate containing isonitrile (CN-R) as the coordinating ligand, [99mTc][Tc(CO)3(CN-FA)3]+
([99mTc]Tc-10), for FR targeting and evaluated the effects of probe properties on diagnostic efficacy.
[
99mTc][Tc(OH2)3(CO)3]+ was coordinated with the CN-R group in a monodentate fashion, with the metal-
to-chelator ratio of 1:3.27,28 Although a number of multimeric constructions of folate-based conjugates
have been reported in the literature, to the best of our knowledge, there is no previous report on the
synthesis of [99mTc]Tc-10 for SPECT imaging of FR-expressing tumors.
2. Results
2.1. Synthesis
A detailed description of the chemical synthesis of a CN-FA (8) is outlined in Scheme 1. N-(2-
aminoethyl)FA (EDA-FA) (3) was synthesized according to a method reported previously.29 Briefly, FA
(1) was reacted with N-Boc-ethylenediamine in the presence of N,N′-dicyclohexylcarbodiimide (DCC) to
obtain γ-conjugate 2 with high selectivity (>95%). The Boc group was removed by TFA to obtain 3 with
90% overall yield. The precursor ligand (8) was prepared by reacting compound 7 with compound 3 in
the presence of triethylamine. Purification by semi-preparative HPLC yielded title compound 8 with >98%
purity. Intermediates and 8 were characterized by 1H NMR and ESI-MS.
Scheme 1. Synthetic procedure for CN-FA (8) synthesis. Reagents (a) N-Boc-ethylenediamine, DCC, and
DMSO at room temperature for 24 h; (b) TFA at room temperature for 3 h; (c) formic acid, acetic
anhydride, at 95°C for 3 h; (d) 2,3,5,6-tetrafluorophenol, DCC, and DMF at room temperature for 24 h; (e)
triphosgene, triethylamine at 0 °C for 1.5 h; (f) 7, TEA at room temperature for 1.5 h.
4

2.2.Radiolabeling and in vitro serum stability
[
99mTc][Tc(H2O)3(CO)3]+ core was prepared with >99% radiochemical purity using the IsoLink kit.
Folate [99mTc]Tc-10 was prepared with high labeling efficiency and stability. Both labeling efficiency and
radiochemical purity were higher than 99%, as determined by radio-HPLC (Figure 1) and radio-ITLC/SG
(Supplementary Figure S1). The retention times for [99mTc][Tc(H2O)3(CO)3]+ and [99mTc]Tc-10 were 4.7
and 14.5 min, respectively. In radio-TLC 99mTc moved with solvent front (Rf: 0.8-1.0), Rf of
[
99mTc][Tc(OH2)3(CO)3]+ was 0.5-0.6, and Rf of [99mTc]Tc-10 radiofolate was 0.1-0.2. Hydrolyzed
technetium species that will remain at the origin of radio-ITLC/SG were not detected (Supplementary
Figure S1). Folate [99mTc]Tc-10 showed high stability in serum, and no sign of decomposition of the
radiotracer was observed over a period of 6 h (Figure 2).
5

Figure 1. Radio-HPLC profile of [99mTc][Tc(H2O)3(CO)3]+ (upper) and radio-HPLC purified [99mTc]Tc-
10 (lower).
1 0 0
9 0
8 0
7 0
6 0
5 0
0
2
4
6
T im e (h )
Figure 2. Stability of [99mTc]Tc-10 in human serum over time shown as percent radiochemical purity
determined by Radio-ITLC/SG.
2.3.Radioactive saturation binding studies
Saturation binding analysis was conducted to determine the binding affinity of [99mTc]Tc-10
expressed on FR-positive KB cells. The saturation-binding curve is depicted in Figure 3. It was observed
that [99mTc]Tc-10 bound to FR-positive KB cells with high affinity. The estimated Kd value was 0.04 ±
0.002 nM, as determined by nonlinear regression. Moreover, the competitive blocking effect of free FA
6

was examined on FR-positive KB cells, which clearly indicated that [99mTc]Tc-10 could specifically
target FR.
0 .0 0 8
N S B nM
TB nM
S B nM
0 .0 0 6
0 .0 0 4
0 .0 0 2
0 .0 0 0
0 .0 0
0 .0 2
0 .0 4
0 .0 6
L iga n d nM
Figure 3. Saturation binding curve of [99mTc]Tc-10 on KB tumor cells. NSB is Nonspecific binding, TB
is total binding and SB is specific binding. KB cells were used.
2.4.Partition coefficient
The partition coefficient (log P) was measured to determine the hydrophilicity of radiolabeled folate-
conjugate [99mTc]Tc-10. The experiment revealed octanol/water partition coefficient of [99mTc]Tc-10 to be
−2.90 ± 0.13, representing its high hydrophilicity.
2.5.Biodistribution study
Results of biodistribution study of [99mTc]Tc-10 in KB tumor-bearing mice are shown in Table 1. As
demonstrated, the radiotracer [99mTc]Tc-10 showed significant uptake by the tumor. The tumor uptake 2 h
post-injection (p.i.) was 4.02 ± 0.80% ID/g, which subsequently increased to 5.32 ± 2.99% ID/g at 4 h p.i..
The tumor uptake significantly reduced to 0.55 ± 0.21% ID/g after 4 h by co-injection of free FA,
indicating highly specific uptake mediated by FR. Furthermore, the highest accumulation of [99mTc]Tc-10
7

was observed in the kidney (64.7 ± 0.99% ID/g) due to high FR expression in proximal tubule cells and
potential renal excretion level, which were significantly reduced by co-administration of free FA.
Moreover, high uptake in the intestine was also observed, which may be because of the lipophilic
character of the tricarbonyl core.
Table 1. Biodistribution of [99mTc]Tc-10 in KB tumor-bearing mice
[
99mTc]Tc-10
Blockade
Organ
2 h
4 h
2 h
4 h
Blood
Muscle
Tumor
Heart
0.29 ± 0.04
1.31 ± 0.20
4.02 ± 0.80
2.07 ± 0.30
1.24 ± 0.06
5.14 ± 1.79
0.33 ± 0.07
7.94 ± 7.80
19.87 ± 8.46
49.54 ± 4.13
0.72 ± 0.05
2.30 ± 0.21
3.13 ± 0.38
0.15 ± 0.02
1.06 ± 0.07
5.32 ± 2.99
1.73 ± 0.11
0.86 ± 0.16
4.26 ± 0.84
0.30 ± 0.04
2.70 ± 1.76
20.51 ± 7.66
64.67 ± 0.99
0.68 ± 0.07
1.97 ± 0.21
3.40 ± 1.00
0.22 ± 0.02
0.10 ± 0.01
0.61 ± 0.03
0.09 ± 0.01
0.33 ± 0.05
2.79 ± 0.58
0.13 ± 0.01
1.99 ± 0.85
0.07 ± 0.04
0.08 ± 0.05
0.55 ± 0.21
0.06 ± 0.02
0.13 ± 0.04
2.39 ± 0.00
0.08 ± 0.01
1.17 ± 0.68
Lung
Liver
Spleen
Stomach
Intestine
Kidney
Bone
21.99 ± 0.85 25.94 ± 1.28
3.34 ± 1.16
0.28 ± 0.09
0.29 ± 0.02
1.21 ± 0.27
8.75 ± 9.32
0.18 ± 0.04
0.17 ± 0.07
1.08 ± 0.64
Thyroid
Tail
*Results are expressed as percentage injected dose per gram (%ID/g) ± SD (n = 4), for blockade studies
[
99mTc]Tc-10 was coinjected with free FA (100 g)
2.6.SPECT Imaging
Results of the imaging studies of [99mTc]Tc-10 in KB tumor-bearing mice are shown in Figure 4.
Significant uptake in the tumor was observed at all time points, and relatively high uptake in the kidney
and intestine was also observed. However, when the blocking experiment was performed in mice by co-
injection of 100 µg free FA, kidney and tumor uptake decreased significantly, suggesting that radiotracer
8

localization in these tissues was mediated by FR. The imaging results were in accordance with results of
biodistribution data in mice.
Figure 4. SPECT/CT imaging of xenografted KB tumor in nude mice (a) Tumor-bearing mice received
[
99mTc]Tc-10 (b) Blocking study with co-injection of FA (100 µg). Image were prepared using
Invivoscope postprocessing software. A Gauss reconstruction filter was applied to the SPECT images.
The SPECT/CT images are presented with the scale adjusted to allow visualization of the most important
organs and tissues.
3. Discussion
FR targeting plays an important role in tumor detection by assisting in nuclear medicine imaging. A
radiolabeled low molecular weight folate conjugate offers the advantage of high tumor-to-background
contrast and faster blood clearance kinetics. Although many promising candidates have been developed
9

over the past decades, only a few have been evaluated for clinical use in patients.11,30 Compared with
other radioisotopes, excellent nuclear characteristics and easy availability of 99mTc makes it a suitable
candidate for SPECT imaging. It has been demonstrated that the multimer approach for a targeting
molecule increases the binding affinity and targeting efficiency by several orders magnitude compared to
that by monomer analogue.19,24 Keeping in view the multimer approach for a targeting molecule, we
successfully synthesized a short chain trivalent CN-R functionalized folate [99mTc]Tc-10 without
significantly influencing the pharmacokinetics of the targeting molecule. Based on this design, we
envisioned that three FA entities would bind to FRs on the cell surface; thus, weak ligand–receptor
interactions would be enhanced. If simultaneous binding of three FA on FRs is difficult, then the FA
concentration is still locally enriched in the vicinity of neighboring FRs, resulting in high binding affinity.
[
99mTc]Tc-10 was prepared using the IsoLink Kit with high radiochemical purity. Figure 1 shows that
the labeling efficacy and radiochemical purity was >99% and no tracer side-product was observed during
the labeling procedure. To verify the structure of [99mTc]Tc-10 complex, macroscale reaction was also
performed with cold [Re(CO)3(H2O)3]+, a 99mTc analogue, for standard chemical characterization (Scheme
2). Comparative HPLC profiles of 99mTc and coldRe were similar (Figure S2), indicating in situ trivalency
of the targeting vector. Stability of the complex is of critical importance in the design of a metal
radiopharmaceutical. In vitro stability of [99mTc]Tc-10 was tested in human serum over a period of 6 h,
and there was no obvious sign of decomposition, suggesting that the complex is highly stable and
chemically inert. This high stability is attributed to the stable complexation of low valent 99mTc(I) with
CN-R chelator.31 The low Log P value of [99mTc]Tc-10 (-2.90 ± 0.13) showed it hydrophilic characteristic
and measured Log P value is in the range reported by others.24,32
10

Scheme 2. Preparation and structure of 99mTc and Re folate conjugates (a) [Re(CO)3(H2O)3]Br, 100 °C, 3
h (b) [99mTc][Tc(CO)3(H2O)3]+, 100 °C, 30 min
Saturation binding analysis of the radiotracer in KB cells revealed its high affinity for FR. Figure 3
shows the saturation binding curve and estimated Kd value (0.04 ± 0.002 nM) determined by nonlinear
regression. Trivalent [99mTc]Tc-10 displayed an order of magnitude of 10-fold higher binding affinity than
that reported by Mueller et al.,33 demonstrating that multivalency on a targeting scaffold increases binding
affinity than that with a monomer. In the present study, biodistribution and imaging studies were tested at
2 and 4 h. In many biodistribution studies of folate radiotracers, these time points are determined as the
most favorable, thus, allowing comparison with other folate radiotracers. In vivo study revealed
significant uptake and prolonged retention in the KB tumor tissue (5.32% ID/g at 4 h p.i.) compared with
that by other reported monomeric 99mTc-labeled folate radiotracers (Table 2).33-35 99mTc]Tc-10 was
[
rapidly cleared from blood and showed high tumor-to-blood ratio (34.7% ID/g at 4 h p.i.). Furthermore,
high accumulation of radioactivity was observed in the kidney, which is a common feature of folate-based
radiotracers due to the overexpression of FR in the proximal tubule of the kidney. The other possible
reason for high kidney uptake is due to the hydrophilic nature of [99mTc]Tc-10 which makes the kidney
primary excretion pathway. In blocking study, complete inhibition of radiotracer uptake in FR-positive
11

tumor and organs expressing FR was observed, indicating the specific binding of the radiotracer.
Surprisingly, a significant portion of radioactivity was accumulated in the intestine, which may be
attributed to the hepatobiliary excretion route of [99mTc]Tc-10.
In consistent with our results, increased tumor uptakes were demonstrated with dimeric and tetrameric
FA derivatives in previous studies (Table 2).23, 24 However, 99mTc-HYNIC-D2-FA4 multivalent radio-
conjugate showed higher kidney accumulation (90.05% ID/g) compared to our results (64.67% ID/g) at 4
h p.i.. In vivo SPECT/CT imaging studies were also conducted to further evaluate the radiotracer in KB
tumor-bearing nude mice at 2 and 4 h p.i.. [99mTc]Tc-10 showed high tumor uptake and prolonged
retention. SPECT imaging results were in accordance with biodistribution results in tumor-bearing mice.
12

Table 2. Comparison of different radiofolate radiotracers with that of present study
% ID/g at 4 h p.i.
Radio-folate conjugates
Blood
Muscle
Tumor
Kidney
Liver
Ref.
34
99mTc(CO)3-DTPA-FA
0.37 ± 0.02 2.40 ± 0.10
0.04 ± 0.00 0.54 ± 0.05
3.3 ± 0.2
47.0 ± 5.0
7.60 ± 0.50
36
99mTc–PAMA-FA
2.33 ± 0.36 18.48 ± 0.72 2.37 ± 2.85
4.84 ± 0.88 27.33 ± 3.61 2.95 ± 1.02
2.9 ± 0.8
2.9 ± 0.9
32
99mTc-Click-FA
99mTc-DTPA-FA
0.12 ± 0.04 0.82 ± 0.16
0.19 ± 0.05 0.70 ± 0.14
0.03 ± 0.02 0.71 ± 0.26
35
35
24
21.0 ± 3.0
25.0 ± 6.0
1.05 ± 0.30
0.64 ± 0.23
111In-DTPA-FA
99mTc-HYNIC-D1-FA2
0.66 ± 0.19 0.69 ± 0.04 10.16 ± 1.16 56.69 ± 3.12 1.01 ± 0.11
23
99mTc-HYNIC-D2-FA4
0.87 ± 0.21 0.42 ± 0.07
8.44 ± 2.01 90.05 ± 5.18 1.44 ± 0.18
Present study
0.15 ± 0.02 1.06 ± 0.07
5.32 ± 2.99 64.67 ± 0.99 5.14 ± 1.79
4. Conclusion
In this study, we reported an FR-targeting short chain CN-FA conjugate labeled with
[
99mTc][Tc(CO)3(H2O)3]+as a potential SPECT imaging agent. In vitro results showed that [99mTc]Tc-10
undergoes FR-mediated uptake by KB cells. In vivo studies in mice bearing KB cell tumor xenografts
revealed that the radiotracer selectively accumulated in tissues containing high concentration of FR. FR
targeted the trimeric [99mTc]Tc-10 radiopharmaceutical and showed higher accumulation and retention
compared to that with a monomeric radiopharmaceutical. However, non-targeted tissue uptake hinders the
potential application of [99mTc]Tc-10 in the clinical field.
5. Experimental
5.1.General
All commercially available chemicals were of analytical grade and were used without further
purification. FA, N-Boc-ethylenediamine, DCC, anhydrous dimethyl sulfoxide (DMSO), trimethylamine
(TEA), formic acid, acetic anhydride, N,N-dimethylformamide (DMF), and trifluoroacetic acid (TFA)
were purchased from Sigma Aldrich, Korea. 2,3,5,6-tetrafluorophenol and triphosgene were obtained
13

from Tokyo Chemical Industry Co., Ltd. Japan. FR-positive KB cells (human cervical carcinoma cell line,
subclone of HeLa cells) were obtained from the Korean Cell Line Bank (Seoul, Korea). [99mTc]NaTcO4
was eluted from a 99Mo/99mTc generator using saline obtained from Unitech, Korea. The radioactive
precursor [99mTc][Tc(H2O)3(CO)3]+ was prepared using the IsoLink kit obtained from Center for
Radiopharmaceutical Science, Paul Scherrer Institute, Villigen, Switzerland. Thin layer chromatography
(TLC) was performed using silica plates (Silica gel 60 F254, Merck Ltd., Germany) developed with
methanol and 1 N HCl (99:1, v/v). Preparative and analytical HPLC were performed using XTerra RP18
10 µm (10 mm × 250 mm) and XTerra RP18 3.5 µm (4.6 mm × 100 mm) columns (Waters Co. Milford,
MA, U.S.A.), respectively. Different HPLC systems used for preparative and analytical studies are
detailed in supporting information. Radio-TLC was quantitated using Bio-Scan AR-2000 System imaging
scanner (Bioscan, WI, U.S.A.). Mass spectra were recorded on Thermo Fisher Scientific LTQ velos
Instrument using the positive ionization mode. Matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) was performed using Voyager DE-STR (Applied Biosystems, U.S.A.). 1H-NMR spectra
were recorded on an AL 300 FT NMR spectrometer (300 MHz for 1H; Jeol, Tokyo, Japan) with the
corresponding solvent signals as the internal standard. Chemical shifts were reported as parts per million
(ppm) relative to the solvent as residual peak. The following abbreviations are used in the experimental
section for the description of 1H-NMR spectra: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m),
doublet of doublets (dd), and bs (broad singlet). Micro-SPECT/CT imaging was performed on a
NanoSPECT/CT device.
5.2.Animal model
Tumors were xenografted in nude mice using KB tumor cells. Approximately, 1 × 106 cultured KB
cells were suspended in phosphate buffered saline (PBS) and subcutaneously implanted in the right thigh
of mice. Mice were maintained on a folate-free diet (3–4 weeks) before biodistribution and imaging
studies. All animal experiments were performed at the Seoul National University Hospital, Seoul, Korea,
14

which is fully accredited by AAALAC International (2007, Association for Assessment and Accreditation
of Laboratory Animal Care International).
5.3.Chemical synthesis
N-(2-aminoethyl) FA (EDA-FA) (3). Compound 3 was prepared according to a reported method with
little modification29. Briefly, FA (500 mg, 1.1 mmol) dissolved in anhydrous DMSO (20 mL) was added
to N-Boc-ethylenediamine (181 mg, 1.1 mmol,) and DCC (374 mg, 1.8 mmol) in DMSO (5 mL). The
reaction mixture was stirred vigorously for 24 h under nitrogen atmosphere at room temperature. N,N′-
dicyclohexylurea (DCU) was filtered, and the filtrate was poured into cold diethyl ether (50 mL). The
precipitate formed was filtered and washed several times with diethyl ether to obtain compound 2 (175
mg, 0.5 mmol) as a yellow solid.
Anhydrous TFA (1 ml) was added to 2 (175 mg, 0.47 mmol) and stirred at room temperature for 3 h.
After removing TFA in vacuo, the residue was washed several times with methanol to remove traces of
TFA and washed with cold diethyl ether several times to afford compound 3 as a yellow solid (quant).
3-formamidopropanoic acid (5). Compound 4 (5 g, 48.5 mmol) was dissolved in formic acid (40 mL)
and acetic anhydride (25 mL) was added dropwise. The reaction mixture was heated under reflux at 95°C
for 3 h. The solvent was removed in vacuum, and the residue was solidified by adding cold diethyl ether.
Purification was achieved by silica gel chromatography (CH2Cl2:methanol, 4:1) to afford compound 5 as
a white solid (3 g, 55%). 1H-NMR (300 MHz, D2O): δ 7.98 (s, 1H), 3.28 (t, 2H), 2.39 (t, 2H).
2,3,5,6-tetrafluorophenyl 3-formamidopropanoate (6). A mixture of 3 (500 mg, 4.27 mmol) and
2,3,5,6-tetrafluorophenol (780 mg, 4.7 mmol) in anhydrous DMF (10 mL) was cooled to 0°C. DCC in
DMF (3 ml) was added dropwise over a period of 10 min. The reaction mixture was stirred in an ice bath
for 10 min, followed by stirring at room temperature for 24 h. Dicycyclohexyl was removed by filtration,
and the filtrate was concentrated under vacuum and purified by silica gel chromatography (ethyl
15

acetate:hexane, 3:2, v/v) to obtain compound 6 as a white solid (680 mg, 60%). 1H-NMR (300 MHz,
CDCl3): δ 8.17 (s, 1H) 6.25 (bs, 1H), 7.04 (m, 1H), 3.68 (q, 2H), 2.95 (t, 2H).
2,3,5,6-tetrafluorophenyl 3-isocyanopropanoate (7). TEA (2.7 mL, 19.25 mmol) was added to an
ice-cold solution of 6 (2 g, 7.7 mmol) in anhydrous DCM (50 ml) and stirred for 10 min. Triphosgene
(913 mg, 3.1 mmol) was added dropwise over a period of 30 min with constant stirring under nitrogen
atmosphere for 1.5 h at 0°C. After completion of the reaction, DCM (20 ml) was added, and the organic
layer was washed once with saturated NaHCO3 (50 mL), twice with distilled water (50 mL), and, finally,
once with brine (20 mL). The separated organic matter was dried over Na2SO4 and concentrated under
reduced pressure. The matter was purified by silica gel column chromatography (hexane:ethyl acetate, 4:1,
v/v) to yield 7 as a light yellow solid (1.2 g, 60%). 1H NMR (300 MHz, CDCl3): δ 6.99 (m, 1H), 3.77 (t,
2H), 3.07 (t, 2H).
CN-ethylenediamine-folate (8). Compound 3 (40 mg, 83 µmol) and TEA (207 µmol, 37 µl) were
dissolved in anhydrous DMSO (1 ml), and stirred at room temperature for 10 min. 2,2,3,3-
tetrafluoropropanol (TFP)-activated CN-R (7) (23 mg, 83 µmol) was added dropwise and stirred at room
temperature in an inert atmosphere for 1.5 h. After completion of the reaction, the product was
precipitated by addition of cold diethyl ether. The product was purified by preparative HPLC System 1
and lyophilized to obtained precursor 8 as a yellow solid (14 mg, 30% ). 1H NMR (300 MHz, DMSO-d6)
δ 8.63 (s, 1H), 7.60 (m, 2H), 6.92–6.87 (m, 2H), 4.46 (s, 2H), 4.24 (t, 2H), 4.12 (t, 1H), 3.66–3.60 (m,
4H), 2.22 (t, 2H), 2.14–2.08 (m, 2H), 1.87 (td, 2H). Electrospray ionization–mass spectrometry (ESI–MS)
m/z [M+H]+ 565.
[
coldRe(CO)3 (CN-FA)3] (9). The cold rhenium compound [Re(CO)3(H2O)3]OTf was synthesized
according to a previously reported method (Scheme 2).37 Precursor 8 (11 mg, 19 µmol) was dissolved in
deionized water (3 ml) and 0.1 M solution of [Re(CO)3(H2O)3]OTf (20 µl, 1.6 µmol) was added. The
reaction mixture was heated at 90°C for 3 h. The resulting mixture was concentrated in a rotary
16

evaporator and purified by preparative reversed phase (RP)-HPLC (System 1) to afford compound 9 as a
yellow solid (11 mg, 30% ). [M]+ 1963.
5.4.Radiolabeling
[
99mTc][Tc(H2O)3(CO)3]+. [99mTc][Tc(H2O)3(CO)3] was prepared according to a reported method with
slight modification. Briefly, 2.9 mg of sodium tetraborate decahydrate, 7.8 mg of sodium carbonate, 9.0
mg of potassium sodium tartrate tetrahydrate, and 4.5 mg of disodium boranocarbonate, in a kit, were
−
added to 1 mL of [99mTc]TcO4 (370 MBq) in a sealed vial. The vial was heated at 95°C for 30 min on
heating block, and the pH was adjusted to 6.5 by adding 200 µL of phosphate buffer (1 M, pH 7.2) and
HCl (1 M) at a ratio of 1:3 (v/v). Radiochemical purity was determined by HPLC System 2.
[
99mTc(CO)3(L)3]+ (10). Freshly prepared solution of fac-[99mTc][Tc(H2O)3(CO)3]+ (500 µL, 37 MBq)
was added to a vial containing solution 8 dissolved in distilled water (100 µg/500 µL). The vial was then
heated at 95°C on a heating block for 30 min. Radiochemical purity was determined by radio-HPLC
System 2 and radio-TLC.
5.5.Stability studies in serum
The in vitro stability of [99mTc]Tc-10 was tested in human serum. In brief, HPLC-purified [99mTc]Tc-
10 310 Ci (50 µl, 11.4 MBq) was incubated with human serum (250 µl) at 37°C in an incubator with
gentle shaking. After 1, 3, and 6 h, an aliquot of the solution was eluted, and radiochemical purity was
determined by radio-TLC/SG chromatography.
5.6.Determination of partition coefficient
The partition coefficient of [99mTc]Tc-10 was determined by measuring the activity partitioning
between 1-octanol and PBS (0.05 M, pH 7.4) under strict equilibrium conditions. Briefly, [99mTc]Tc-10
was purified by HPLC, the solvent was removed using a rotary evaporator, and the residue obtained was
dissolved in 50 mM solution of phosphate buffer (pH 7.4) to a concentration of 370 kBq/mL. One
17

hundred microliter of [99mTc]Tc-10 solution was diluted with 2.9 mL of phosphate buffer and 3 mL of 1-
octanol. The mixture was vortexed for 5 min and then centrifuged at 3300 rpm for 5 min. The counts in
0.2-mL samples of both organic and inorganic layers were determined using an automatic gamma
scintillation counter. The measurement was repeated three times. The partition coefficient (P) was
calculated using the following equation P = (cpm in octanol − cpm in background)/(cpm in buffer − cpm
in background), where cpm denotes counts per minute.
5.7.In vitro binding affinity
FR-positive KB cells were cultured as monolayers in folate-free culture medium RPMI 1640
(FFRPMI) at 37°C in a humidified atmosphere with 5% CO2. FFRPMI was supplemented with 10% fetal
bovine serum, 4.5 g/L D-glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g/L sodium
bicarbonate. KB cells (1 × 105 cells/well in 1 mL) were plated in a 24-well flat-bottom plate and allowed
to form adherent monolayers for 24 h. The medium in each well was then replaced with fresh medium
containing increasing concentrations of [99mTc]Tc-10 in serial dilution and incubated for 1 h at 37°C.
After 1 h, the media were aspirated, and the cells were washed twice by dispersal in fresh medium.
Sodium dodecyl sulfate in PBS was added to each well and gently mixed to dissolve cells and transferred
to 4-ml plastic tubes. The radioactivity of each sample was counted using a gamma counter (Cobra II
automated gamma-counter), using reference counts from samples containing the total amount of original
radioactivity. Non-specific binding was determined in the presence of excess free FA (250 µM). Specific
binding was calculated by subtracting non-specific bound radioactivity from that of total binding.
Dissociation constant (Kd) was calculated by non-linear regression using GraphPad Prism 7 (GraphPad
Software Inc., San Diego, CA, U.S.A.) using one site binding equation.
5.8.Biodistribution study
The biodistribution of [99mTc]Tc-10 in KB tumor-bearing BALB/c nude mice (22–25 g) was evaluated.
All mice were maintained on a folate-free diet for 3 to 4 weeks before biodistribution study. One hundred
microliter of HPLC-purified [99mTc]Tc-10 (37 kBq) was administered via the lateral tail vein in each
18

mouse. The mice (n = 4) were sacrificed 2 and 4 h post-injection. The required tissues and organs were
excised, weighed, and counted in a γ-counter. To confirm the specific uptake of [99mTc]Tc-10, mice were
also injected with free FA (100 µg/mice) as the blocking agent. The percentage of the injected dose per
gram (% ID/g) was calculated by counting all tissues in a γ-counter using stored sample of the injection
solution as the standard to estimate the total dose injected per mice. Values were expressed as mean ± SD
for four animals.
5.9.SPECT/CT imaging study:
SPECT/CT images were captured 2 and 4 h after administration of HPLC-purified [99mTc]Tc-10 (10
MBq/100 µL to KB tumor-bearing BALB/c nude mice via the tail vein. Mice were anesthetized with
isoflurane and scanned by nanoScan-SPECT/CT. The scanning parameters for each imaging modality
employed a γ-ray energy window of 140 keV ± 10%, matrix size of 256 × 256, acquisition time of 5–15 s
per angular step of 18°, and reconstruction algorithm of ordered-subset expectation maximization with
nine iterations. For integrated CT, tube voltage of 45 kVp, exposure time of 1.5 s per projection, and
reconstruction algorithm of cone-beam filtered back-projection were used. To test the specific uptake of
99mTc-10, blocking study was performed by co-injection of free FA (100 µg/100 µL).
6. Acknowledgement
This research was partially supported by National R&D Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Science NRF-
2017M2A2A7A01071134 and NRF-2015M2C2A1047687. This research was also supported by
the Ministry of Health and Welfare (Grant Number: HI15C3093).
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Graphic abstract
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