Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent

Article abstract of DOI:10.1016/j.bmcl.2010.05.036

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the 'click-to-chelate' protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12, [^99mTc(CO)₃]13, and [^99mTc(CO)₃]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)₃]12, [Re(CO)₃]13, and [Re(CO)₃]14, were shown to have high binding affinities (0.13 μM, 0.06 μM, and 0.16 μM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12-14) to c-Met receptor positive U87MG cells.

Full text of DOI:10.1016/j.bmcl.2010.05.036

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4240–4243
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a
potential c-met receptor kinase positive tumor imaging agent
Eun-Mi Kim, Min-Hee Joung, Chang-Moon Lee, Hwan-Jeong Jeong, Seok Tae Lim, Myung-Hee Sohn,
*
Dong Wook Kim
Department of Nuclear Medicine, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-712, Republic of Korea
Cyclotron Research Center, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-712, Republic of Korea
Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-712, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 December 2009
Revised 6 May 2010
Accepted 12 May 2010
Available online 15 May 2010
The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis
and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized techne-
tium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase
peptide synthesis and the ‘click-to-chelate’ protocol for the introduction of tricarbonyl technetium-99m,
as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met bind-
ing affinity, cellular uptake, and stability. The 99mTc labeled cMBP derivatives ([99mTc(CO)3]12,
Keywords:
c-Met
Click chemistry
c-Met binding peptide
Technetium-99m
[
99mTc(CO)3]13, and [99mTc(CO)3]14) were prepared in 85-90% radiochemical yields. The cold surrogate
cMBP derivatives, [Re(CO)3]12, [Re(CO)3]13, and [Re(CO)3]14, were shown to have high binding affinities
(0.13 lM, 0.06 lM, and 0.16 lM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addi-
tion, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these 99mTc
labeled cMBP derivatives ([99mTc(CO)3]12–14) to c-Met receptor positive U87MG cells.
Ó 2010 Elsevier Ltd. All rights reserved.
The hepatocyte growth factor (HGF)–mesenchymal-epithelial
transition factor (Met) molecular pathway affects cancer develop-
ment at different stages, from initiation to metastatic behavior, in
many tumor types.1–4 Since Met was discovered as an oncogene5,
the HGF/c-Met signaling pathway has come into the spotlight as a
promising therapeutic target for inhibiting tumor growth and vari-
ous strategies are currently in development to disrupt the HGF–
Met signal transduction pathway.6–9 Kim et al. previously reported
thatmonoclonalantibodySFN68inhibitstheHGF–c-Metinteraction
and blocks the biological function of c-Met mediated by HGF.10 To
define the epitope, they screened out the epitope-mimicking pep-
cations, such as drug discovery by the combinatorial approach and
molecular imaging by the tagging of biological systems, because
this reaction is able to proceed selectively and efficiently in aque-
ous media in the presence of various bioactive molecules such as
peptides, proteins, nucleotides, and whole organisms, affording
the 1,2,3-triazole in high yield as a stable linker for conjugation be-
tween two (bio)molecular components.13–15 In particular, Mindt
and co-workers reported that the 1,2,3-triazole derivatives ob-
tained from this click reaction of azido-biomolecules and propargyl
glycine could be regarded as a potent precursor for the preparation
of technetium-99m (Tc-99m) labeled radiopharmaceuticals for
single photon emission computed tomography (SPECT) because
these 1,2,3-triazole chelators can act as efficient ligand systems
for the formation of tricarbonyl Tc-99m complexes through the
‘click-to-chelate’ approach from [M(CO)3(H2O)3]+ (M = 99mTc,
Re).16–18
Technetium-99m labeled tracers are particularly useful: (a)
SPECT camera is widely used and easily available for noninvasive
imaging to detect disease in humans; (b) technetium-99m is read-
ily available at low cost from a 99Mo/99mTc generator system; (c)
finally, technetium-99m has ideal physical properties (t1/2 = 6 h,
141 keV).19,20 In particular, tricarbonyl technetium-99m is re-
garded as an attractive core for the introduction of technetium-
99m into biomolecules due to its small size and high chemical sta-
bility.21,22 In this work, we report the preparation of 99mTc labeled
tide,
NH2-Lys1-Ser2-Leu3-Ser4-Arg5-His6-Asp7-His8-Ile9-His10
-
His11-His12-Ac (c-Met binding peptide; cMBP), from a phage display
of a combinatorial peptide library. We recently reported the phar-
macokinetic evaluation of 125I-radiolabeled cMBPs and cMBP-X-
Cy5.5 (X: linker) for targeting c-Met receptor expression in U87MG
tumor models.11,12 In these previous studies, we found that cMBP
could specifically target the c-Met receptor and was a good candi-
date biomolecule to use a molecular imaging agent.
Nowadays, the copper(I)-catalyzed Huisgen 1,3-dipolar cyclo-
addition of azides and alkynes, which is commonly called click
chemistry, plays a crucial role in a wide range of biomedical appli-
* Corresponding author. Tel.: +82 63 250 2396; fax: +82 63 255 1172.
E-mail address: kimdw@chonbuk.ac.kr (D.W. Kim).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.036

E.-M. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4240–4243
4241
Figure 1. Structures of target molecules. The target molecules are divided into three parts: the ‘click-to-chelate’ prosthetic group, glycine containing linkage and cMBP
moiety.
cMBP derivatives as potential c-Met receptor kinase positive tumor
SPECT imaging agents using the ‘click-to-chelate’ protocol for the
incorporation of tricarbonyl technetium-99m. As shown in Figure
1, these radiolabeled cMBP analogues were designed with three
components: they consist of a cMBP moiety in order to have high
tected by the 9-fluorenylmethoxycarbonyl (Fmoc) group using
Cu(I) as a catalyst. After the removal of the Fmoc protecting group
under basic condition, the 1,2,3-triazole-cMBP derivatives (12, 13,
and 14) as precursors for 99mTc(CO)3 labeling were cleaved from
the solid phase by treatment with a mixture of triisopropylsilane,
thioanisol, water, ethanedithiol, and trifluoroacetic acid
(2.5:2.5:2.5:2.5:90), and the precursors were purified by reverse
phase HPLC.
target affinity toward the c-Met receptor tyrosine kinase,
a
(Gly)n-bAla-Lys linker to connect between cMBP and the radiola-
beled prosthetic group while taking into consideration their phar-
macokinetics (to improve their lipophilicity), and a ‘click-to-
chelate’ prosthetic group to introduce tricarbonyl technetium-
99m into the 1,2,3-triazole ligand with minimal steric interference,
thereby maintaining the favorable interaction with the target.
Scheme 1 illustrates the synthesis of the precursor for the tri-
carbonyl 99mTc labeled cMBP derivatives. The preparation of resins
containing cMBP and (Gly)n-bAla-Lys linker (2, 3, and 4) by solid
phase peptide synthesis were followed by the amide condensation
between the resins (2, 3, and 4) and azidoacetic acid (1) affording
the azido-cMBP resins (6, 7, and 8), in order to introduce the azide
group for the click reaction. The 1,2,3-triazole moiety of the resins
(9, 10, and 11) was prepared by 1,3-dipolar cycloaddition reaction
of the azide functionalized cMBP resins and propargyl glycine pro-
Metal coordination reaction of the precursors 12–14 with
[
99mTc(CO)3(OH2)3]+, which was readily prepared by a known
method,16–18 in aqueous media for 30 m at 80 °C provided the
radioactive cMBP complexes—[99mTc(CO)3]12, 99mTc(CO)3]13,
[
and [99mTc(CO)3]14—in 90%, 89%, and 85% radiochemical yield,
respectively (Scheme 2). The reaction was monitored by radio-
TLC scanner until the [99mTc(CO)3(OH2)3]+ peak disappeared. The
radiochemical purities of [99mTc(CO)3(OH2)3]+ was more than
98%. Nonradioactive cMBP complexes—[Re(CO)3]12, [Re(CO)3]13,
and [Re(CO)3]14 were also prepared using a similar procedure to
that employed for the above radioactive cMBP complexes except
for the use of [Re(CO)3(OH2)3]+ instead of [99mTc(CO)3(OH2)3]+ for
in vitro assays.
Scheme 1. Synthesis of 1,2,3-triazole precursors. Reagents and conditions: (i) NaN3, water, 4 °C, 16 h; (ii) DIC, HOBT, DMF, rt, 2 h; (iii) Cu(I)Br, DIPEA, DMF, Fmoc-L-
propargylglycine, rt, 4 h; (iv) 20% piperidine in DMF, rt, 10 m (Fmoc deprotection) (v) triisopropylsilane, thioanisol, water, ethanedithiol, trifluoroacetic acid (2.5/2.5/2.5/2.5/
90), rt, 3 h (cleavage).

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E.-M. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4240–4243
Scheme 2. Reagents and conditions: (i) [Re(OH2)3(CO)3]+, or [99mTc(OH2)3(CO)3]+, water, 80 °C, 30 m.
110
100
90
120
a
b
100
80
99mTc(CO)3]12
99mTc(CO)3]13
99mTc(CO)3]14
[
[
99mTc(CO)3]12
99mTc(CO)3]13
99mTc(CO)3]14
[
[
60
40
20
0
80
[
[
70
20
0
5 m
30 m
60 m 90 m
Time
2 h
3 h
10 m 30 m
1 h
2 h
3 h
4 h
Time
Figure 2. In vitro human serum stability (a) and transchelation challenge test (b) from [99mTc(CO)3]12, [99mTc(CO)3]13, and [99mTc(CO)3]14 as a function of the histidine
concentration.
Table 1
20000
Competitive binding affinity assay of cold surrogate cMBP derivatives ([Re(CO)3]12,
sample
sample + cold compound
[Re(CO)3]13, and [Re(CO)3]14) using recombinant chimeric c-Met/Fc protein,a and the
partition coefficient (P) of the 99mTc radiolabeled cMBP complexesb
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Entry
Compounds
IC50
(l
M)
Log Pc
1
2
3
4
cMBP
0.12
0.13
0.06
0.16
—
[Re(CO)3]12
[Re(CO)3]13
[Re(CO)3]14
À2.6 0.02
À2.0 0.01
À2.0 0.03
a
The results of the competitive binding experiment show that the binding of the
I-125 labeled cMBP to the HGF receptor was inhibited in a concentration dependent
manner by the non-radiolabeled Re complexes.
b
The log P values were determined with the three 99mTc radiolabeled complexes,
[
99mTc(CO)3]12, [99mTc(CO)3]13, and [99mTc(CO)3]14.
c
Data are expressed as means SD of duplicate samples.
99mTc(CO)3]12
12
[
99mTc(CO)3]13
13
[
99mTc(CO)3]14
14
[
To investigate their stability, the solutions of these radiolabeled
Figure 3. Cellular uptake and blocking study of 99mTc radiolabeled cMBP complexes
([99mTc(CO)3]12, [99mTc(CO)3]13, and [99mTc(CO)3]14) in U87MG cells. The cells
were incubated with 99mTc radiolabeled cMBP complexes (5.2 nmol) for 1 h. For the
blocking studies, 99mTc radiolabeled cMBP complexes were co-treated with the cold
cMBP peptide (86 nmol). The plate was then incubated for 1 h at 37 °C. All of the
tests were done in triplicate. The data are presented as the mean SD.
cMBP complexes were adjusted with physiological saline to a con-
centration of 37 MBq/mL, and aliquots of 100 L of these solutions
were added to 500 L human plasma and incubated at 37 °C. The
l
l
percentage of the complexes remaining after 2 h was over 80%
(Fig. 2). In the trans-chelation stability study using histidine (concn
0.1 mM) and cystein (concn 0.1 mM), the labeling efficiency of all
of the complexes was also over 90% after 3 h (Fig. 2). These results
showed that all of the 99mTc radiolabeled cMBP complexes have
sufficient stability for image acquisition.
Table 1 illustrated the competitive c-Met receptor binding assay
for the three cold surrogate Re complexes ([Re(CO)3]12–14) using a
cMet/Fc chimeric recombinant protein, and the partition coeffi-
cient (P) study with the three 99mTc radiolabeled complexes
([99mTc(CO)3]12–14). The IC50 values of [Re(CO)3]12–14 (0.13,
thetic group and (Gly)n-bAla-Lys linker in the 99mTc radiolabeled
cMBP complexes have no influence on the binding of the c-Met
receptor. The determination of log P shows that the insertion of
glycine at the linker allowed the lipophilicity to be improved; the
log P values of [99mTc(CO)3]12, [99mTc(CO)3]13, and [99mTc(CO)3]14
were À2.6 0.02, À2.0 0.01, and À2.0 0.03, respectively.
As shown in Figure 3, cellular uptake and blocking studies were
performed using a c-Met receptor positive tumor cell line such as
U87MG. The [99mTc(CO)3]13 complex showed higher cellular up-
take than both [99mTc(CO)3]12 and [99mTc(CO)3]14 in the U87MG
cells, with the results being similar to those of the c-Met receptor
binding study. Consequently, [99mTc(CO)3]13 containing the three
glycines (Gly-Gly-Gly) in the linker moiety had the highest c-Met
0.06, and 0.16 lM, respectively), and free cMBP (0.12 lM) were ob-
tained by competitive displacement studies using 125I-cMBP as a
radioligand. The binding affinities of these cold complexes were
in the nanomolar range and similar or higher to that of the free
cMBP. Thus, this result showed that the ‘click-to-chelate’ pros-

E.-M. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4240–4243
4243
protein binding affinity as well as cellular uptake, therefore could
References and notes
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This work was supported by research funds of Chonbuk Na-
tional University in 2010, Nuclear Research & Development Pro-
gram of the Korea Science and Engineering Foundation (KOSEF)
grant funded by the Korean government (MEST). (Grant code:
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gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.036.