A radiobrominated tyrosine kinase inhibitor for egfr with l858r/t790m mutations in lung carcinoma

Article abstract of DOI:10.3390/ph14030256

Activating double mutations L858R/T790M in the epidermal growth factor receptor (EGFR) region are often observed as the cause of resistance to tyrosine kinase inhibitors (TKIs). Third‐generation EGFR‐TKIs, such as osimertinib and rociletinib (CO‐1686), wa

Full text of DOI:10.3390/ph14030256

pharmaceuticals
Article
A Radiobrominated Tyrosine Kinase Inhibitor for EGFR with
L858R/T790M Mutations in Lung Carcinoma
1
,2
, Kenji Mishiro 3 , Ryuichi Nishii , Akira Makino , Yasushi Kiyono , Kazuhiro Shiba ,
4
5
5
6
Muammar Fawwaz
Seigo Kinuya 7 and Kazuma Ogawa
1,3,
*
1
Graduate School of Medical Sciences, Kanazawa University, Kakuma-machi, Kanazawa,
Ishikawa 920-1192, Japan; muammar.fawwaz@umi.ac.id
Faculty of Pharmacy, Universitas Muslim Indonesia, Urip Sumoharjo KM. 10, Makassar 90-231, Indonesia
Institute for Frontier Science Initiative, Kanazawa University, Kakuma-machi, Kanazawa,
Ishikawa 920-1192, Japan; mishiro@p.kanazawa-u.ac.jp
2
3
4
5
6
7
National Institute of Radiological Sciences (NIRS), QST, Inage-ku, Chiba 263-8555, Japan;
nishii.ryuichi@qst.go.jp
Biomedical Imaging Research Center (BIRC), University of Fukui, Eiheiji-cho, Yoshida-gun,
Fukui 910-1193, Japan; amakino@u-fukui.ac.jp (A.M.); ykiyono@u-fukui.ac.jp (Y.K.)
Advanced Science Research Center, Kanazawa University, Takara-machi, Kanazawa,
Ishikawa 920-8640, Japan; shiba@med.kanazawa-u.ac.jp
Department of Nuclear Medicine, Institute of Medical, Pharmaceutical and Health Sciences,
Kanazawa University, Takara-machi, Kanazawa, Ishikawa 920-8641, Japan; kinuya@med.kanazawa-u.ac.jp
Correspondence: kogawa@p.kanazawa-u.ac.jp; Tel./Fax: +81-76-234-4460
*
Abstract: Activating double mutations L858R/T790M in the epidermal growth factor receptor
EGFR) region are often observed as the cause of resistance to tyrosine kinase inhibitors (TKIs).
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
(
ꢀ
ꢁꢂꢃꢄꢅꢆ
Third-generation EGFR-TKIs, such as osimertinib and rociletinib (CO-1686), was developed to target
such resistance mutations. The detection of activating L858R/T790M mutations is necessary to select
sensitive patients for therapy. Hence, we aimed to develop novel radiobromine-labeled CO-1686 as a
positron emission tomography (PET) imaging probe for detecting EGFR L858R/T790M mutations.
Nonradioactive brominated-CO1686 (BrCO1686) was synthesized by the condensation of N-(3-[{2-
chloro-5-(trifluoromethyl)pyrimidin-4-yl}amino]-5-bromophenyl) acrylamide with the correspond-
ing substituted 1-(4-[4-amino-3-methoxyphenyl]piperazine-1-yl)ethan-1-one. The radiobrominated
Citation: Fawwaz, M.; Mishiro, K.;
Nishii, R.; Makino, A.; Kiyono, Y.;
Shiba, K.; Kinuya, S.; Ogawa, K.
A Radiobrominated Tyrosine Kinase
Inhibitor for EGFR with L858R/T790M
Mutations in Lung Carcinoma.
Pharmaceuticals 2021, 14, 256.
77
https://doi.org/10.3390/ph14030256
[ Br]BrCO1686 was prepared through bromodestannylation of the corresponding tributylstanny-
7
7
lated precursor with [ Br]bromide and N-chlorosuccinimide. Although we aimed to provide a
7
7
76
Academic Editor: Irina Velikyan
novel PET imaging probe, Br was used as an alternative radionuclide for Br. We fundamentally
7
7
evaluated the potency of [ Br]BrCO1686 as a molecular probe for detecting EGFR L858R/T790M
using human non-small-cell lung cancer (NSCLC) cell lines: H1975 (EGFR L858R/T790M), H3255
Received: 7 February 2021
Accepted: 10 March 2021
Published: 12 March 2021
(
(
EGFR L858R), and H441 (wild-type EGFR). The BrCO1686 showed high cytotoxicity toward H1975
IC50 0.18 0.06 M) comparable to that of CO-1686 (IC 0.14 0.05 M). In cell uptake experiments,
±
µ
±
µ
50
7
7
the level of accumulation of [ Br]BrCO1686 in H1975 was significantly higher than those in H3255
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
7
7
and H441 upon 4 h of incubation. The radioactivity of [ Br]BrCO1686 (136.3% dose/mg protein)
was significantly reduced to 56.9% dose/mg protein by the pretreatment with an excess CO-1686.
These results indicate that the binding site of the radiotracers should be identical to that of CO-1686.
The in vivo accumulation of radioactivity of [77Br]BrCO1686 in H1975 tumor (4.51
than that in H441 tumor (3.71 0.13) 1 h postinjection. Our results suggested that [ Br]BrCO1686
±
0.17) was higher
7
7
±
has specificity toward NSCLC cells with double mutations EGFR L858R/T790M compared to those in
EGFR L858R and wild-type EGFR. However, the in vivo accumulation of radioactivity in the targeted
tumor needs to be optimized by structural modification.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
Keywords: tyrosine kinase inhibitors; L858R/T790M; imaging probes; bromine-labeled; PET
4
.0/).
Pharmaceuticals 2021, 14, 256. https://doi.org/10.3390/ph14030256
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 256
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1
. Introduction
Overexpression of epidermal growth factor receptor (EGFR) and mutation in the
tyrosine kinase (TK) domain have been shown to be highly associated with a variety
of tumors, including non-small-cell lung cancer (NSCLC) [ ]. Activation of EGFR-TK
is involved in the proliferation, metastasis, angiogenesis, invasion, and suppression of
apoptosis [ ]. Therefore, EGFR is an attractive target for tumor imaging and therapy [ ].
As the therapeutic effect of EGFR-TK inhibitors (TKIs) depends on EGFR mutation
status, it is important to determine this status before administrating them [ ]. Imaging
1
2
3
3
agents for determining EGFR status could serve as an alternative strategy to predict the
drug efficacy instead of gene diagnosis with repeated invasive biopsy [4,5].
EGFR-TKIs are effective molecular targeted drugs for NSCLC patients with L858R
mutation [
6,
7
]. However, almost all patients acquired resistance to the first-generation
]. A secondary EGFR mutation in codon 790
threonine-790 to methionine) of exon 20 of this gene has been reported in approximately
EGFR-TKIs, such as gefitinib and erlotinib [
(
8
5
0% of all cases that develop resistance to EGFR-TKI therapy [
6
]. Crystal structure analysis
].
This mutation is believed to be induced by the treatment with first-generation TKIs because
it is rarely detected in untreated patients [ ]. Thus, to overcome the resistance, the third-
showed that T790M hinders the interaction of EGFR with the first-generation EGFR-TKIs [
7
8
generation EGFR-TKIs possessing a high-affinity against EGFR with L858R/T790M double
mutations were developed. At the same time, companion diagnosis is also important for
the third-generation EGFR-TKIs. We recently synthesized and evaluated a radiotracer,
1
25
[
I]ICO1686, which is a derivative of one of the third-generation EGFR-TKIs, CO-1686,
125
with a high affinity for EGFR with L858R/T790M double mutations. [ I]ICO1686 could
detect the EGFR with L858R/T790M double mutations in vitro; however, it showed insuffi-
cient tumor uptake and low tumor/blood ratio of radioactivity [9].
Radiobromine has some advantages over other radiohalogens. For example, 76Br is
a promising radionuclide for positron emission tomography (PET) with a relatively long
half-life (16.2 h). It makes it possible to deliver the radiopharmaceuticals to distant sites
and to take delayed PET imaging, such as at one day postinjection, which is impossible
18
for F-labeled probes. Meanwhile, bromine has chemical properties similar to those of
iodine. Therefore, for the synthesis of a radiobromine-labeled probe, a synthetic scheme
for a radioiodine-labeled probe can be applied without substantial modification. More-
over, the smaller size of bromine than for iodine may not hinder the binding site [10].
Hence, we aimed to provide a novel radiobromine-labeled CO-1686 to determine the
EGFR L858R/T790M mutation for the selection of patients sensitive to third-generation
EGFR-TKIs.
In this study, a non-radioactive brominated N-(3-{[2-({4-[4-acetylpiperazin-1-yl]-
2
-methoxyphenyl}amino)-5- (trifluoromethyl)pyrimidin-4-yl]amino}-5-bromophenyl)
7
7
acrylamide (BrCO1686, Scheme 1, 9) and a radioactive brominated 9 ([ Br]BrCO1686,
Scheme 2, [ Br]9) were synthesized and evaluated in vitro and in vivo using three
7
7
different human NSCLC cell lines; H1975 (EGFR with L858R/T790M dual mutations),
H3255 (EGFR with L858R mutation), and H441 (wild-type EGFR). Although we are
interested in developing 76Br-labeled probes for PET, in these initial studies, Br was
used because of its longer half-life (57.0 h).
77

Pharmaceuticals 2021, 14, 256
3 of 14
O
N
O
O
OH
NO2
HN
(b)
NO2
O
N
N
(
a)
(c)
O
N
2
O
N
F
O N
2
H N
2
F
1
3
H
N
O N
2
NO2
O N
2
NO2
H
N
2
NH
2
H N
2
O
(d)
(e)
(f)
O
Br
4
Br
Br
6
5
O
Cl
Br
7
Cl
Br
8
Br
N
3
(i)
F3C
O
N
(
g)
N
N
O
(h)
N
N
O
O
N
N
N
H
O
N
N
H
N
N
H
N
N
H
H
H
H
CF3
CF3
9
◦
◦
Scheme 1. Synthesis of non-radioactive brominated CO-1686. (a) K CO , CH I, acetone, 0 C, 5 h; (b) DIPEA, DMA, 90 C,
2
3
3
◦
overnight; (c) Pd/C 10%, H , MeOH, rt, 5 h; (d) H SO , NBS, 80 C, 1.5 h; (e) SnCl 2H O, ethanol, rt, 3 h; (f) Boc O, MeCN,
·
2
2
4
2 2 2
◦
◦
5
0 C, overnight; (g) 2,4-dichloro-5(trifluoromethyl) pyrimidine, DIPEA, n-butanol, 0 C, 2 h, rt, 4 h; (h-1) HCl/ethyl acetate,
rt, 1 h; (h-2) acryloyl chloride, DIPEA, dichloromethane, −30 C to rt, 2 h; (i) TFA, 1,4-dioxane, 60 C, 3 h.
◦
◦
O
O
77Br
N
Sn(n-Bu)3
F3C
N
O
N
F3C
O
N
(a)
O
N
1
O
N
N
N
H
N
N
H
N
N
H
N
N
H
H
H
77
0
[ Br]9
7
7
77
–
◦
Scheme 2. Radiosynthesis of compound [ Br]9. (a) [ Br]Br , acetic acid, NCS, ethanol, 80 C, 60 min.
2. Results
2.1. Probe Design and Synthesis
A derivative of CO-1686, brominated compound 9, was prepared by multistep synthe-
sis starting from commercially available 1,3-dinitrobenzene (Scheme 1). A bromine atom
was introduced in the diaminophenyl group of CO-1686 using N-bromosuccinimide (NBS).
The bromine was attached in this substituent because this would not substantially influ-
ence the affinity of CO-1686 toward EGFR L858R/T790M, according to the complex crystal
structure of CO-1686 and T790M EGFR [11]. The most important substituents playing a
pivotal role in the binding of CO-1686 to EGFR L858R/T790M are the anilinopyrimidine
core, methoxy, and trifluoromethyl groups [11,12].
2.2. Cytotoxicity Assays
The half-maximal inhibitory concentration (IC ) of
9
was determined by 2-(2-methoxy-
50
4
-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt
(
WST-8) assay. Our previous study showed that iodinated CO-1686 (ICO1686) has a similar
cytotoxicity effect with parent compound CO-1686 toward mutated NSCLC. In this study,
the cytotoxicity of toward H1975 and H3255 was similar to those of ICO1686 and CO-1686,
as shown in Table 1. This suggests that the bromine substituent on did not significantly
9
9

Pharmaceuticals 2021, 14, 256
4 of 14
influence the activity toward mutated EGFR, in terms of both active mutant L858R EGFR
and double mutations L858R/T790M EGFR. In contrast, the IC50 of
9 toward H441 was
significantly lower than that of ICO1686 (p = 0.0178). The data were statistically analyzed
and presented as mean ± standard deviation (SD).
Table 1. The IC50 of 9, ICO1686, CO-1686, and gefitinib on NSCLC cell lines by WST-8 assay.
Mutation
Status
IC50 (µM)
Cells Lines
9
ICO1686 *
CO-1686 *
Gefitinib *
H1975
H3255
H441
L858R/T790M
L858R
Wild type
0.18 ± 0.06
0.20 ± 0.01
0.64 ± 0.04
0.20 ± 0.05
0.50 ± 0.21
1.84 ± 0.44
0.14 ± 0.05
0.15 ± 0.02
0.26 ± 0.04
>10
0.02 ± 0.02
>10
Data represent the mean ± SD of three separate experiments. * Data from reference [9].
7
7
77
2
.3. Radiosynthesis of [ Br]BrCO1686 ([ Br]9)
77
The [ Br]
9
was synthesized by a bromodestannylation reaction with the correspond-
ing tributyltin precursor 10, as shown in Scheme 2. This radiotracer was synthesized using
◦
N-chlorosuccinimide (NCS) as an oxidizing agent in an acidic solution at 80 C in moderate
radiochemical yield (45%). After purification using reversed phase-high-performance
liquid chromatography (RP-HPLC), the radiochemical purity was over 99%. The identity
77
of [ Br]9 was confirmed by comparing its retention time with that of nonradioactive 9 in
7
7
the HPLC analyses (Figure S1). These analyses showed that the retention time of [ Br]
(
9
9.13 min) was slightly shorter than that of [1 I]ICO1686 (11.76 min).
25
2
.4. Determination of Partition Coefficient
7
7
77
The log p value for [ Br]
9
was 1.72
±
0.21, which indicates that [ Br]
9
has appro-
7
7
priate lipophilicity for passive membrane penetration. The log p value of [ Br]
9
was
significantly lower than that of [1 I]ICO1686, which was 1.84
25
±
0.01 (p = 0.0097) [9]. This
result is consistent with the reverse-phase HPLC analysis, in which the retention time of
7
7
125
[
Br]9 was shorter than that of [ I]ICO1686.
2
.5. Stability Assessment
77
The stability profile of [ Br]
9
in phosphate-buffered saline (PBS) and murine plasma
77
is presented in Supporting Information (Figure S2). [ Br]
murine plasma after incubation for 1 h at 37 C. Its purity decreased to 57% after 24 h
of incubation.
9
was >80% intact in PBS and
◦
2.6. Cellular Uptake Studies
Data on the radioactivity uptake are expressed as percent dose per milligram pro-
77
125
tein (% dose/mg protein), as shown in Figure 1. [ Br]9 and [ I]ICO1686 exhibited
preferential accumulation in H1975 (L858R/T790M double mutations EGFR) upon 4 h of
incubation, which was significantly higher than that observed with H3255 (L858R active
mutant EGFR) and H441 (wild-type EGFR). In the in vitro blocking experiment toward
7
7
H1975 cells, the accumulation of radioactivity of [ Br]
[
9
(136.3% dose/mg protein) and
125
I]ICO1686 (151.3% dose/mg protein) was reduced to 56.9 and 67.7% dose/mg protein,
respectively, by the pretreatment with an excess CO-1686 (p < 0.0001 and p < 0.0001, respec-
tively). Meanwhile, pretreatment with excess gefitinib toward H1975 was meaningless but
significantly reduced the accumulation of both radiotracers in H3255 cells.

Pharmaceuticals 2021, 14, 256
5 of 14
) [125I]ICO1686 and (
B
) [ Br]
77
9
in H1975, H3255, and H441 cell lines
Figure 1. The cell uptake of (
A
after 4 h incubation. Significance was determined using Dunnett’s multiple-comparison test or
unpaired Student’s t-test (* p < 0.05) ns: not significant.
2
.7. Biodistribution Studies
The biodistributions of [ Br]
summarized in Table 2. These data are expressed as percent injected dose per gram
% ID/g). At 4 h after injection, the distribution pattern in normal organs was similar
among the groups, with the highest uptake in the intestine and liver. The accumulation
7
7
and [125I]ICO1686 were studied in normal mice, as
9
(
7
7
125
of [ Br]
9 in blood was much higher than that of [ I]ICO1686. The accumulation of
both radiotracers in mice bearing H1975 and H441 tumors is summarized in Table 3.
7
7
The accumulation of [ Br]
[
9
in H1975 tumor at 1 h postinjection was higher than that of
1
25
77
I]ICO1686. As shown in Table 4, regarding the biodistribution of [ Br]bromide ion
in normal mice, it was retained for a long time in the blood and some organs.

Pharmaceuticals 2021, 14, 256
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Table 2. Biodistribution of [ Br]9 and [125I]ICO1686 at 10 min, 1, 4, and 24 h after i.v. injection in ddY mice.
77
Time after Injection
Tissues
[77
Br]9
1
0 min
1 h
4 h
24 h
1.99
Blood
Liver
Kidney
3.57
11.04
11.68
25.38
2.83
5.11
3.78
8.72
4.57
6.17
2.76
3.18
0.45
(0.21)
(0.55)
(0.94)
(5.45)
(0.57)
(3.46)
(2.53)
(1.25)
(0.89)
(1.45)
(0.83)
(0.55)
(0.30)
3.24
3.83
4.21
26.49
3.18
4.83
3.58
4.97
2.93
7.00
1.66
2.02
0.95
(0.30)
(0.38)
(0.67)
(2.77)
(0.19)
(1.82)
(0.10)
(1.97)
(0.24)
(0.58)
(1.18)
(0.55)
(0.14)
3.19
3.72
2.81
5.23
47.73
2.92
3.04
5.08
1.87
6.68
1.54
1.13
0.74
(0.18)
(0.30)
(1.17)
(1.06)
(7.88)
(2.02
(0.28)
(0.28)
(1.25)
(1.02)
(0.62)
(0.78)
(0.50)
(0.28)
1.04
1.33
1.00
0.98
1.14
1.13
1.62
0.80
2.21
0.89
0.47
0.42
13.48
35.01
(0.21)
(0.25)
(0.15)
(0.11)
(0.46)
(0.09)
(0.22)
(0.21)
(0.33)
(0.93)
(0.20)
(0.09)
(1.28)
(6.85)
Small intestine
Large intestine
Spleen
Pancreas
Lung
Heart
‡
Stomach
Bone
Muscle
Brain
Urine
Feces
I]ICO1686
Blood
Liver
-
-
-
-
-
-
[125
1.41
19.21
9.66
36.32
0.94
2.73
2.03
3.61
2.24
2.60
0.74
1.20
0.08
(0.38)
(2.04)
(1.19)
(12.00)
(0.18)
(0.56)
(0.20)
(0.40)
(0.49)
(1.23)
(0.19)
(0.14)
(0.01)
0.17
7.40
2.40
40.99
1.65
0.55
0.86
0.59
0.30
1.64
0.16
0.23
0.01
(0.02)
(1.01)
(0.56)
(4.13)
(0.34)
(0.09)
(0.15)
(0.25)
(0.08)
(0.71)
(0.03)
(0.09)
(0.01)
0.06
6.45
0.75
6.08
81.54
0.20
0.12
0.16
0.08
0.76
0.05
0.04
0.01
(0.01)
(1.21)
(0.18)
(1.99)
(10.19)
(0.07)
(0.04)
(0.03)
(0.04)
(0.61)
(0.08)
(0.01)
(0.00)
0.13
1.40
0.54
0.30
0.52
0.13
0.05
0.14
0.20
0.05
0.09
0.04
0.00
(0.06)
(0.17)
(0.10)
(0.11)
(0.55)
(0.04)
(0.01)
(0.02)
(0.25)
(0.02)
(0.05)
(0.02)
(0.00)
(0.24)
(6.53)
Kidney
Small intestine
Large intestine
Spleen
Pancreas
Lung
Heart
‡
Stomach
Bone
Muscle
Brain
‡
Urine
Feces
-
-
-
-
-
-
1.63
71.54
‡
Data presented as % ID/g tissue. Each value represents mean ± SD for four mice. ‡ Presented as % ID/organ.
Table 3. Biodistribution of double tracers [ Br]9 and [125I]ICO1686 at 1 and 6 h after i.v. injection in tumor-bearing mice.
77
Time after Injection
Tissues
1
h
6 h
1 h
6 h
[77
Br]9
[125I]ICO1686
(0.20)
Blood
Liver
Kidney
5.64
15.89
7.75
64.52
24.33
9.19
12.95
11.42
5.36
1.31
1.93
3.42
1.49
(0.19)
(0.72)
(0.85)
(3.35)
(17.81)
(2.12)
(5.14)
(1.52)
(1.07)
(0.39)
(1.51)
(1.19)
(0.16)
(0.17)
(0.13)
5.35
6.76
4.91
12.46
59.40
2.04
4.28
6.29
2.99
1.27
1.95
1.79
1.39
3.48
3.30
(0.99)
(1.78)
(0.80)
(5.93)
(18.22)
(2.36)
(0.91)
(0.52)
(1.05)
(0.89)
(2.50)
(0.30)
(0.17)
(0.50)
(0.54)
0.50
30.32
2.64
86.29
27.65
1.80
2.71
1.62
0.61
1.49
0.42
0.36
0.04
0.68
0.44
0.17
10.16
1.05
11.72
95.22
0.25
0.36
0.37
0.53
1.17
0.22
0.10
0.02
0.10
0.19
(0.03)
(3.50)
(0.41)
(8.01)
(32.67)
(0.07)
(0.22)
(0.15)
(0.86)
(1.68)
(0.15)
(0.05)
(0.03)
(0.02)
(0.06)
(2.78)
(1.45)
(2.99)
(21.75)
(0.75)
(0.87)
(0.67)
(0.20)
(0.52)
(0.16)
(0.18)
(0.01)
(0.11)
(0.06)
Small intestine
Large intestine
Spleen
Pancreas
Lung
Heart
‡
Stomach
Bone
Muscle
Brain
H1975
H441
4.51
3.71
Data presented as % ID/g tissue. Each value represents mean ± SD for four mice. ‡ Presented as % ID/organ.

Pharmaceuticals 2021, 14, 256
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77
Table 4. Biodistribution of [ Br]bromide at 15 min, 1, 4, and 24 h after i.v. injection in ddY mice.
Time after Injection
Tissues
1
5 min
1 h
4 h
24 h
Blood
Liver
Kidney
Small
intestine
Large
intestine
Spleen
Pancreas
Lung
6.96
2.45
5.10
(0.58)
(0.29)
(0.83)
6.98
2.61
4.58
(0.45)
(0.17)
(0.21)
6.42
2.35
4.51
(0.64)
(0.30)
(0.58)
3.48 (0.36)
1.18 (0.12)
2.62 (0.34)
3
.94
(0.24)
3.92
(0.16)
3.18
(0.29)
1.99 (0.22)
3
.12
(0.44)
2.78
(0.22)
2.37
(0.30)
1.55 (0.16)
4.74
5.09
8.02
3.72
3.94
3.72
2.28
1.08
2.45
(0.31)
(0.78)
(0.95)
(0.47)
(0.54)
(0.44)
(0.18)
(0.13)
(0.50)
4.30
4.38
7.12
3.23
4.59
3.25
1.92
1.49
3.02
(0.26)
(0.27)
(0.48)
(0.09)
(1.18)
(0.24)
(0.11)
(0.07)
(0.35)
3.75
3.80
5.81
2.41
5.44
3.00
1.63
1.45
2.46
(0.73)
(0.37)
(0.59)
(0.20)
(1.23)
(0.38)
(0.14)
(0.14)
(0.42)
2.25 (0.36)
2.22 (0.32)
3.62 (0.32)
1.50 (0.19)
4.18 (1.00)
1.78 (0.30)
0.98 (0.04)
0.84 (0.09)
1.27 (0.28)
20.74 (4.69)
1.33 (0.49)
Heart
Stomach
Bone
‡
Muscle
Brain
‡
Neck
Urine
Feces
‡
-
-
-
-
-
-
‡
Data presented as % ID/g tissue. Each value represents mean ± SD for four mice. ‡ Presented as % ID/organ.
3. Discussion
The modulation of EGFR expression has been used as a target for therapy and diag-
nosis due to its highly specific targeting capability. The therapeutic effects of EGFR-TKIs
in NSCLC can be assessed by PET imaging of EGFR, which can provide more accurate
information to aid in the selection of patients for individualized therapy [13]. In recent
years, several PET probes based on EGFR-TKI derivatives for detecting EGFR mutation
and/or predicting treatment response in NSCLC have been reported [13].
In our previous study, an in vitro kinase inhibition assay with ICO1686 and CO-1686
showed their high selectivity toward double mutations EGFR L858R/T790M compared
with that toward wild-type EGFR [9]. Therefore, we conducted a further study by designing
77
76
and synthesizing radiobrominated [ Br]9 as a surrogate of [ Br]9, which can be used as a
molecular imaging agent for EGFR L858R/T790M because bromine could be a bioisostere
of iodine. A bromine atom was introduced in the diaminophenyl group of CO-1686, as
was done with the iodine derivative in the previous study. As expected, introducing
bromine at this position did not significantly affect the CO-1686 affinity toward EGFR
L858R/T790M because it does not play a key role in the binding activity of CO-1686 to
EGFR L858R/T790M based on the complex crystal structure analysis [11]. As a result, the
IC50 of 9 was similar to that of the iodinated compounds ICO1686 and CO-1686.
Concerning 77Br labeling, radiobromination was achieved in the presence of an oxidiz-
ing agent NCS with a moderate radiochemical yield of approximately 45%. Radiobromine
was directly introduced to the aromatic group by a destannylation reaction with a positively
charged radiobromine generated in situ [14].
Cellular uptake studies of both the radiotracers showed a significant difference among
77
125
the three cells. Preferential accumulation of [ Br]9 and [ I]ICO1686 was seen in H1975,
which has EGFR double mutations. In the in vitro blocking studies, both the radiotracers
showed specificity in H1975 cells. The methoxyl group contributes to the specificity of CO-
1686 toward EGFR, and the trifluoromethyl substituent of CO-1686 is beneficial for forming
hydrophobic interactions with Met790, which is not afforded by a wild-type gatekeeper
residue with Thr790 [11]. The specificity of both the radiotracers toward H1975 cells
was evaluated by two independent experiments using two blocking agents, CO-1686 and
77
125
gefitinib. As expected, the uptake of [ Br]
9 and [ I]ICO1686 in H1975 was significantly
reduced by almost 60% upon pretreatment with CO-1686, which is known to bind to

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7
7
EGFR-TK with L858R/T790M double mutations. In contrast, the uptake of [ Br]
[
9
and
125
I]ICO1686 in H1975 was not significantly reduced with gefitinib, which is known to
be ineffective to EGFR-TK with L858R/T790M double mutations. These results indicate
that the radiotracers bound to EGFR-TK with L858R/T790M double mutations compete
with CO-1686. Moreover, gefitinib only reduced the radioactivity uptake of radiotracers in
H3255. Although H1975 cells carry the EGFR L858R mutation, the presence of secondary
mutation T790M interferes with the affinity of first-generation TKIs, such as gefitinib, to
the ATP binding site [15,16].
In the biodistribution study, [ Br]9 and [125I]ICO1686 were co-injected into the mice
to minimize the number and increase the reliability of the data in the biodistribution study.
At 4 h postinjection, the distribution patterns in normal organs were similar among the
groups, with the highest uptake in the intestine and liver. High uptake in the liver and
small intestine indicated that radiotracers were mainly eliminated through hepatobiliary
clearance because both radiotracers have relatively hydrophobic structures. However, the
77
7
7
125
clearance rate of [ Br]
was eliminated at a rate of up to 71% in feces, while only 35% of [ Br]
at 24 h postinjection. The high radioactivity of radiobromine-labeled tracers in the blood
is strongly related to the free bromide ions [17 19]. Therefore, the radioactivity in the
blood can be used as an index of the debromination of radiobromine-labeled compounds
9 was slow from the blood and other organs. The [ I]ICO1686
77
9
was eliminated
–
77
in vivo
[20]. In this study, the accumulation of [ Br]9 in the blood was much higher
than that of [ I]ICO1686, which suggested that debromination occurred in vivo. Our
experiment on the biodistribution of [ Br]bromide showed long-term retention in the blood
and some organs (Table 4). In this context, we confirmed that radioactivity in the blood
after the injection of [ Br]
125
77
77
9
should be caused by free bromide ions from debrominated
7
7
76
[
Br]
9
. Debromination has also been observed with some radiotracers, such as 4-[ Br]-
76
76
bromo-
α
-methyl-L-phenylalanine (4-[ Br]BAMP) and 3-[ Br]-bromo-
[ Br]BAMT) [14,21].
In biodistribution studies using tumor-bearing mice, the accumulation of [ Br]
higher than that of [ I]ICO1686 in H1975 tumors (Table 3). According to a biodistribution
study of [ Br]bromide, the blood clearance of [ Br]bromide was slow, and free bromide
was distributed throughout the whole body (Table 4). The accumulation of free radiobro-
α-methyl-L-tyrosine
7
6
(
7
7
9
was
1
25
77
77
77
mide generated by the debromination of [ Br]
the tumor and all organs. Thus, we assume that after injection of [ Br]
9 would contribute to its high uptake in
77
9
, the specificity to
tumors with EGFR L858R/T790M mutations should be low. Therefore, it is desirable to
develop more stable and selective analogs that highly accumulate in target tumors.
Previous studies have suggested that the high and rapid hepatobiliary excretion of
radiotracers must have been derived from the high lipophilicity, which may hinder the
accumulation of these radiotracers in the targeted tumor [22,23]. Some studies have shown
that the introduction of a short-chain PEG in radiotracers improved pharmacokinetics
and accumulation in the tumor, presumably because the hepatobiliary excretion rate was
reduced due to the increased hydrophilicity [12
, ,24]. Therefore, we suppose that the
23
77
structural modification of [ Br]
accumulation.
9, such as PEG introduction, might improve its tumor
4. Material and Methods
4.1. General Chemistry
Solvents and reagents were purchased from Nacalai Tesque, Inc. (Kyoto, Japan), Tokyo
Chemical Industry, Co., Ltd. (Tokyo, Japan), Fujifilm Wako Pure Chemical Corporation
Osaka, Japan), Kanto Chemical, Co., Inc. (Tokyo, Japan), Merck (Darmstadt, Germany),
(
and Chemescene (Monmouth Junction, NJ, USA), and used as provided. CO-1686 was
purchased from AdooQ BioScience (Irvine, Canada). Gefitinib was purchased from Fujifilm
Wako Pure Chemical Corporation (Osaka, Japan). Thin-layer chromatography (TLC) was
performed on silica plates 60 F254 (Merck, Darmstadt, Germany).

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Nuclear magnetic resonance (NMR) spectra were recorded on a JNM-ECS400 (400 MHz)
or JNM-ECA600 (600 MHz) spectrometer (JEOL Ltd., Tokyo, Japan). The mass spectrometry
was performed on a JMS-T700 (JEOL Ltd., Tokyo, Japan) on fast atom bombardment mass
spectrometry (FAB-MS). HPLC analyses and purification were carried out on a Shimadzu
Prominence HPLC system (Shimadzu Corp., Kyoto, Japan). The optical density in WST-8
®
assays were measured on an Infinite F200 Pro microplate reader (TECAN, Männedorf,
Switzerland). Radioactivity was determined using an auto gamma counter ARC-7010B
(Hitachi, Ltd., Tokyo, Japan).
4.2. Probe Design and Synthesis
Design and synthesis procedures were shown in Schemes 1 and 2. Intermediate
compounds were synthesized according to the previous studies, with a slight modifi-
cation [9,25–28]. For compounds and tin precursor 10, materials synthesized in the
previous study [ ] were used. NMR spectra are provided in the Supporting Information
1–3
9
(
Figures S3–S8).
4
.2.1. Synthesis of 1-bromo-3,5-dinitrobenzene (4)
Compound (7.0 g, 70%) was synthesized according to the previous report [28]. H
NMR (400 MHz, CDCl3):
1
4
1
3
δ
8.72 (2H, d, J = 1.6 Hz), 9.01 (1H, t, J = 1.6 Hz). C NMR
(
[
100 MHz, CDCl ):
M + H] : m/z = 245.9298, found 245.9276.
δ
117.7, 123.8, 132.2, 149.1. HRMS (FAB+) calculated for C H BrN O
3
6
3
2
4
+
4
.2.2. Synthesis of 5-bromobenzene-1,3-diamine (5)
Compound (5.26 g, 99%) was synthesized according to the previous report [29]. H
NMR (400 MHz, CDCl3):
1
5
1
3
δ
3.60 (4H, br s), 5.91 (1H, s), 6.25 (2H, d, J = 2.0 Hz). C NMR
(
[
100 MHz, CDCl ):
M + H] : m/z = 185.9777, found 185.9793.
δ
100.1, 108.8, 123.7, 148.5. HRMS (FAB+) calculated for C H BrN
3
6
7
2
+
4.2.3. Synthesis of tert-butyl (3-amino-5-bromophenyl)carbamate (6)
To a stirred mixture of 5 (5.26 g, 28.13 mmol, 1.0 eq.) and acetonitrile (50 mL), di-tert-
◦
butyl dicarbonate (Boc O) (6.14 g, 28.13 mmol, 1 eq.) was added at 50 C and the mixture
2
was stirred overnight under nitrogen atmosphere. After the reaction was completed,
the solvent was removed by evaporation. The crude product was purified by column
chromatography on silica gel (hexane/ethyl acetate = 7/3) and concentrated under reduced
1
pressure to afford
(
6
(4.45 g, 55%) as a crystal orange. H NMR (400 MHz, CDCl ):
δ
1.51
3
13
9H, s), 3.73 (2H, s), 6.42 (1H, s), 6.50 (1H, s), 6.79 (2H, s). C NMR (100 MHz, CDCl3):
δ
28.2, 80.8, 103.3, 111.3, 112.6, 123.1, 140.5, 148.3, 152.5. HRMS (FAB+) calculated for
+
C H BrN O [M + H] : m/z = 286.0300, found 286.0317.
11
15
2
2
4
.2.4. Synthesis of tert-butyl(3-{[2-chloro-5-(trifluoromethyl)pyrimidin-4-yl]amino}-5-
bromophenyl)carbamate (7)
To a stirred mixture of
6 (4.45 g, 15.50 mmol, 1.0 eq.) and n-butanol (31 mL), 2,4-
dichloro-5(trifluoromethyl) pyrimidine (2 mL, 1 eq., purchased from Chemscene), N,N-
◦
diisopropylethylamine (DIPEA) (5 mL, 2 eq.) were added at 0 C and stirred for 2 h. The
stirring was continued at room temperature for 4 h. After the reaction was completed,
the solvent was removed by evaporation. The crude product was purified using column
chromatography on silica gel (hexane/ethyl acetate = 7/3) and concentrated under reduced
1
pressure to afford
7
(4.1 g, 85%) as pale-yellow solid
.
H NMR (400 MHz, CDCl ):
δ
1.54
3
9H, s), 6.59 (1H, s), 7.00 (1H, s), 7.39 (1H, s), 7.48 (1H, s), 7.66 (1H, s), 8.46 (1H, s). 3C NMR
δ 28.2, 81.7, 106.8 (q, JCF = 30.5 Hz), 110.6, 118.6, 119.6, 122.8, 123.4
q, JCF = 271.8 Hz), 137.8, 140.2, 152.3, 156.1 (q, J = 4.8 Hz), 157.1, 163.6. HRMS (FAB+)
1
(
(
(
100 MHz, CDCl3):
CF
+
calculated for C H BrClF N O [M + H] : m/z = 465.9854, found 465.9842.
16
15
3
4
2

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4
.2.5. Synthesis of N-(3-{[2-chloro-5-(trifluoromethyl)pyrimidin-4-yl]amino}-5-
bromophenyl)acrylamide (8)
A mixture of (4.1 g, 8.77 mmol, 1.0 eq.) and 4.0 M hydrochloric acid (HCl)/ethyl
7
acetate (1.0 mL) was stirred at room temperature for 1 h. After completion of the reaction,
the mixture was concentrated under reduced pressure to give a corresponding de-Boc
product as a colorless solid. The crude product was used for the next reaction without
further purification. To a stirred mixture of the crude product (3.0 g) and DIPEA (4.3 mL,
2
4.48 mmol, 3 eq.) in dichloromethane (81 mL), acryloyl chloride (1 mL, 12.24 mmol, 1.5 eq.)
◦
was added at
−
30 C and warmed to room temperature. After stirring for 2 h, the crude
product was diluted with ethyl acetate, washed with 1 M HCl, saturated aqueous NaHCO ,
3
and brine successively. The crude product was purified using column chromatography
on silica gel (dichloromethane/methanol = 200/1), crystalized from chloroform/hexane
1
to afford
8
(200 mg, 10%) as a white solid. H NMR (400 MHz, CDCl3):
δ
5.84 (2H,
d, J = 7.2 Hz), 6.24 (1H, dd, J = 11.2, 6.8 Hz), 6.47 (1H, d, J = 11.2 Hz), 7.43 (1H, s), 7.50
13
(
(
1
[
1H, s), 7.66 (1H, s), 7.98 (1H, s), 8.63 (1H, s). C NMR (100 MHz, (CD ) SO):
δ
101.3
q, JCF = 70.5 Hz), 109.8, 116.2, 117.6, 121.6, 123.9 (q, JCF = 270.8 Hz), 127.8, 131.8, 140.8, 141.5,
57.0 (q, JCF = 4.8 Hz), 161.3, 163.7, 166.2. HRMS (FAB+) calculated for C H BrClF N O
3
2
14
9
3
4
+
M + H] : m/z = 419.9702, found 419.9600.
4
.2.6. Synthesis of N-(3-{[2-({4-[4-acetylpiperazin-1-yl]-2-methoxyphenyl}amino)-5-
(
trifluoromethyl)pyrimidin-4-yl]amino}-5-bromophenyl)acrylamide (9)
To a stirred mixture of compound
8 (120 mg, 0.28 mmol, 1.0 eq.) and 2 M trifluoroacetic
acid (TFA) in dioxane (0.6 mL), compound
temperature. The mixture was warmed to 60 C and stirred for 3 h. After completion of
the reaction, the pH of the reaction mixture was adjusted to neutral (7–8) by the addition of
3
(70 mg, 0.28 mmol, 1 eq.) was added at room
◦
saturated aqueous NaHCO , and the mixture was extracted with ethyl acetate. The organic
3
phase was separated, dried over anhydrous Na SO , filtered, and concentrated under
2
4
reduced pressure. The crude product was purified by an isocratic HPLC system equipped
®
with a Cosmosil 5SL-II (20 ID
×
250 mm) column (Nacalai Tesque) with a mobile phase
of ethyl acetate/methanol (97/3) at a flow rate of 9.5 mL/min. The column temperature
was maintained at 40 C. The product was concentrated under reduced pressure to afford
80 mg, 45%) as a white solid. H NMR (400 MHz, CDCl ):
◦
9
1
(
δ
2.16 (3H, s), 3.12–3.20 (4H, m),
3
3
.64 (2H, t, J = 4.4 Hz), 3.79 (2H, t, J = 4.8 Hz), 3.91 (3H, s), 5.80 (1H, d, J = 10.8 Hz), 6.21
(
1H, dd, J = 15.2, 10.4 Hz), 6.44 (1H, d, J = 15.6 Hz), 6.54–6.60 (2H, m), 7.11 (1H, s), 7.25
1H, s), 7.43 (1H, s), 7.56 (1H, s), 7.66 (1H, s), 7.74 (1H, s), 8.07 (1H, d, J = 8.8 Hz), 8.30 (1H, s).
(
1
3
C NMR (100 MHz, (CD ) SO): δ 16.9, 41.2, 47.3, 51.0, 51.5, 60.2, 113.0 (q, JCF = 40.5 Hz),
3
2
1
1
16.2, 124.5, 127.4, 135.3, 136.9, 138.9, 142.1, 146.4 (q, JCF = 334.9 Hz), 147.1, 149.6, 154.8,
65.5, 167.1, 177.1, 180.9, 184.2 (q, JCF = 6.0 Hz), 186.8, 191.2, 194.4, 200.7. HRMS (FAB+)
+
calculated for C H BrF N O [M + H] : m/z = 633.1324, found 633.1311.
27
27
3
7
3
4
.3. Cytotoxicity Assays
WST-8 assay was used to evaluate IC50 of
and H3255) as described previously [ ]. The reference compounds; CO-1686, ICO-1686,
9
toward NSCLC cell lines (H1975, H441
9
and gefitinib, have been evaluated in our previous study using the same source of cell
lines [9].
4
.4. Production of Bromine-77
77Br was produced by 77
Se(p,n) Br reaction with and separated according to a previ-
77
ous study at the University of Fukui [20,30].
7
7
77
4
.5. Radiosynthesis of [ Br]BrCO1686 ([ Br]9)
7
7
Radiotracers, [ Br]
responding precursor 10 (Scheme 1). A solution of [ Br]bromide (2.5 MBq, 1
charged into a sealed vial containing 10 (1 mg/mL, 10 L) in ethanol, acetic acid (5%, 30
9 was synthesized by a bromodestannylation reaction of the cor-
77
µ
L) was
µ
µL),

Pharmaceuticals 2021, 14, 256
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◦
and NCS (20 mg/mL, 10
µ
L). After vortexed for 5 min, the mixture was shaken at 80 C
for 60 min. The reaction was quenched by aqueous sodium hydrogen sulfite (5 mg/mL,
1
0
µ
L), and the reaction solution was purified by a gradient HPLC system equipped with a
®
Cosmosil 5C18 MS-II (4.6 ID
phase (water/methanol = 30/70–0/100 in 20 min) at a flow rate of 1 mL/min. The col-
umn temperature was maintained at 40 C. The [ Br]9 was isolated after confirmation by
×
150 mm) column (Nacalai Tesque) with a varying mobile
◦
77
comparing the retention times of the radiobrominated and nonradioactive
analysis. Radioactivity was determined by an auto well gamma counter.
9 in the HPLC
4
.6. Determination of Partition Coefficient
77
To determine the lipophilicity, the partition coefficient of [ Br]
9
was measured as
described previously [31]. The partition coefficient (log P) was expressed as the ratio of
radioactivity in n-octanol and radioactivity in 0.1 M phosphate buffer (pH 7.4).
4.7. Stability Assessment
To confirm the stability, we measured the purity of radiotracer after incubation in PBS
and mouse plasma, respectively, as described previously [9].
4
.8. Cellular Uptake Studies
5
5
5
H1975 (1
×
10 cells/well), H441 (1
×
10 cells/well), and H3255 (2
×
10 cells/well)
NSCLC cells were cultured in 6 well plates. Cells were incubated in medium with 10%
FBS and 100 IU/mL penicillin–streptomycin at 37 C in a humidified atmosphere with
◦
5
% CO for 24 h. The cell culture was then incubated in an FBS-free medium containing
2
125
77
[
I]ICO1686, which was prepared by the method reported previously [
3.7 kBq/well, respectively) for 4 h, and then washed with 1 mL of ice-cold PBS. The cells
were dissolved by a 1.0 M NaOH aqueous solution (0.5 mL 2). The cell lysate was
transferred into a test tube. The radioactivity of Br was measured by an auto well gamma
9] and [ Br]9
(
×
77
counter at a 100–600 keV energy range. The crossover of 1 I activity into the Br channel
25
77
125
was negligible. After one month of experiments, the radioactivity of I was measured
at a 16–71 keV energy range because the radioactivity of Br was negligible at the time.
In the blocking assays, CO-1686 or gefitinib (100 M, respectively) was co-incubated with
77
µ
the radiotracer, and the cellular uptake was assessed using the same method as mentioned
above. The BCA protein assay kit protocol was used to determine the protein concentration.
4.9. Tumor Model
Six weeks old male ddY mice and four weeks old female BALB/c nu/nu mice were
purchased from Japan SLC Inc. (Hamamatsu, Japan). Tumor-bearing mice were prepared
by subcutaneous injection of cells suspended in 100
µ
L medium to the female BALB/c
6
nu/nu mice. H1975 cells (5
×
10 cells) were implanted in the right shoulder, and H441
6
cells (3
×
10 cells) were implanted in the left shoulder. The tumor reached palpable size
after post-inoculation 12 and 9 days for H1975 and H441, respectively.
4
.10. Biodistribution Studies
Mice were injected intravenously with double tracers [ Br]
25 kBq) in 100 L of saline solution containing 1% tween-80 and 10% ethanol via the lateral
77
(10 kBq) and [125I]ICO1686
9
(
µ
tail vein. The ddY mice were sacrificed by decapitation at 10 min, 1, 4, and 24 h. Meanwhile,
the tumor-bearing mice were sacrificed at 1 h and 6 h postinjection (4 mice/group). The
77
selected tissues and organs were collected, weighed, and counted the radioactivity of Br
using an auto well gamma counter. One month later, 1 I activity was determined. The
biodistribution data were expressed as% ID/g along with the SD. The same procedures
25
77
were applied to perform the biodistribution study of [ Br]bromide ion (111 kBq).

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4.11. Statistical Analysis
Statistical methods, one-way ANOVA followed by a Dunnett’s multiple-comparison
test for cellular uptake studies in H1975 cells. Unpaired Student’s t-test was used to confirm
the significances in the cytotoxicity assays, determination of partition coefficient, and the
cellular uptake studies toward H3255 and H441 cells. All values are given as mean
and p < 0.05 was considered statistically significant.
±
SD,
5
. Conclusions
77
Radiobrominated [ Br]
9
was successfully synthesized with high radiochemical purity.
This radiotracer showed in vitro specificity toward NSCLC cells with EGFR L858R/T790M
double mutations compared with that in EGFR L858R and wild-type EGFR. However, the
in vivo accumulation in the target tumor was insufficient to visualize the tumor. Therefore,
it is desirable to develop more stable and selective analogs that are rapidly cleared from the
body and highly accumulate in the target tumor. The structural modification by introducing
77
PEG could be an alternative to improve the tumor uptake of [ Br]9.
Supplementary Materials: The followings are available online at https://www.mdpi.com/1424
-
8247/14/3/256/s1, Figure S1: The chromatograms of (a) nonradioactive brominated compound
7
7
9
(BrCO1686) and (
radiolabeled compound [77Br]
dinitrobenzene ( ), Figure S4: The NMR spectra of 5-bromobenzene-1,3-diamine (
NMR spectra of tert-butyl (3-amino-5-bromophenyl)carbamate ( ), Figure S6: The NMR spectra
of tert-butyl(3-{[2-chloro-5-(trifluoromethyl)pyrimidin-4-yl]amino}-5-bromophenyl)carbamate ( ),
Figure S7: The NMR spectra of N-(3-{[2-chloro-5-(trifluoromethyl)pyrimidin-4-yl]amino}-5- bro-
mophenyl)acrylamide ( ), Figure S8: The NMR spectra of N-(3-{[2-({4-[4-acetylpiperazin-1-yl]-2-
b
) radioactive compound [77Br]
in PBS and plasma, Figure S3: The NMR spectra of 1-bromo-3,5-
), Figure S5: The
9 ([ Br]BrCO1686), Figure S2: The stability of
9
4
5
6
7
8
methoxyphenyl}amino)-5-(trifluoromethyl)pyrimidin-4-yl]amino}-5-bromophenyl)acrylamide (9).
Author Contributions: Conceptualization, K.O. and R.N.; methodology, K.O. and K.M.; validation,
K.O., K.M., M.F., R.N., K.S. and S.K.; formal analysis, M.F. and K.M.; investigation, M.F.; resources,
R.N., A.M., Y.K.; writing—original draft preparation, M.F.; writing—review and editing, K.M. and
K.O.; supervision, K.O. and K.M.; project administration, K.O.; funding acquisition, K.O. and R.N.
All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported in part by the Terumo Life Science Foundation and Grants-in-
Aid for Scientific Research (16H05397) from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Animal experiments were conducted in accordance with the guidelines of our institution. The animal
handling procedure was approved by the Kanazawa University Animal Care Committee. (Approval
number: AP-163723).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors greatly acknowledge the Indonesia Endowment Fund for Education
(
LPDP) and the Ministry of Research, Technology and Higher Education of the Republic of Indonesia
for providing a BUDI-LN scholarship.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Gupta, R.; Dastane, A.M.; Forozan, F.; Riley-Portuguez, A.; Chung, F.; Lopategui, J.; Marchevsky, A.M. Evaluation of EGFR
abnormalities in patients with pulmonary adenocarcinoma: The need to test neoplasms with more than one method. Mod. Pathol.
009, 22, 128–133. [CrossRef] [PubMed]
Yarden, Y.; Sliwkowski, M.X. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2001, 2, 127–137. [CrossRef] [PubMed]
2
2
.

Pharmaceuticals 2021, 14, 256
13 of 14
3
4
5
.
.
.
Altine, B.; Gai, Y.; Han, N.; Jiang, Y.; Ji, H.; Fang, H.; Niyonkuru, A.; Bakari, K.H.; Rajab Arnous, M.M.; Liu, Q.; et al. Preclinical
Evaluation of a Fluorine-18 18F-Labeled Phosphatidylinositol 3-Kinase Inhibitor for Breast Cancer Imaging. Mol. Pharm. 2019, 16,
563–4571. [CrossRef] [PubMed]
Yeh, H.H.; Ogawa, K.; Balatoni, J.; Mukhapadhyay, U.; Pal, A.; Gonzalez-Lepera, C.; Shavrin, A.; Soghomonyan, S.; Flores, L.,
nd; Young, D.; et al. Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung
4
2
carcinomas using PET/CT. Proc. Natl. Acad. Sci. USA 2011, 108, 1603–1608. [CrossRef] [PubMed]
Pal, A.; Balatoni, J.A.; Mukhopadhyay, U.; Ogawa, K.; Gonzalez-Lepera, C.; Shavrin, A.; Volgin, A.; Tong, W.; Alauddin, M.M.;
1
8
Gelovani, J.G. Radiosynthesis and initial in vitro evaluation of [ F]F-PEG6-IPQA–a novel PET radiotracer for imaging EGFR
expression-activity in lung carcinomas. Mol. Imaging Biol. 2011, 13, 853–861. [CrossRef] [PubMed]
Sullivan, I.; Planchard, D. Next-Generation EGFR Tyrosine Kinase Inhibitors for Treating EGFR-Mutant Lung Cancer beyond
First Line. Front. Med. (Lausanne) 2016, 3, 76. [CrossRef] [PubMed]
6.
7.
8.
9.
Singh, M.; Jadhav, H.R. Targeting non-small cell lung cancer with small-molecule EGFR tyrosine kinase inhibitors. Drug Discov.
Today 2018, 23, 745–753. [CrossRef]
Pao, W.; Wang, T.Y.; Riely, G.J.; Miller, V.A.; Pan, Q.; Ladanyi, M.; Zakowski, M.F.; Heelan, R.T.; Kris, M.G.; Varmus, H.E. KRAS
mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med. 2005, 2, e17. [CrossRef] [PubMed]
Fawwaz, M.; Mishiro, K.; Nishii, R.; Sawazaki, I.; Shiba, K.; Kinuya, S.; Ogawa, K. Synthesis and Fundamental Evaluation of
Radioiodinated Rociletinib (CO-1686) as a Probe to Lung Cancer with L858R/T790M Mutations of Epidermal Growth Factor
Receptor (EGFR). Molecules 2020, 25, 2914. [CrossRef]
1
1
1
1
1
0. Zhou, D.; Kim, S.H.; Chu, W.; Voller, T.; Katzenellenbogen, J.A. Evaluation of aromatic radiobromination by nucleophilic
substitution using diaryliodonium salt precursors. J. Label. Comp. Radiopharm. 2017, 60, 450–456. [CrossRef]
1. Yan, X.E.; Zhu, S.J.; Liang, L.; Zhao, P.; Choi, H.G.; Yun, C.H. Structural basis of mutant-selectivity and drug-resistance related to
CO-1686. Oncotarget 2017, 8, 53508–53517. [CrossRef] [PubMed]
2. Song, Y.; Xiao, Z.; Wang, K.; Wang, X.; Zhang, C.; Fang, F.; Sun, X.; Shen, B. Development and Evaluation of 18F-IRS for Molecular
Imaging Mutant EGF Receptors in NSCLC. Sci. Rep. 2017, 7, 3121. [CrossRef]
3. Chen, W.; Shen, B.; Sun, X. Analysis of Progress and Challenges of EGFR-Targeted Molecular Imaging in Cancer With a Focus on
Affibody Molecules. Mol. Imaging 2019, 18, 1536012118823473. [CrossRef] [PubMed]
4. Hanaoka, H.; Ohshima, Y.; Suzuki, Y.; Yamaguchi, A.; Watanabe, S.; Uehara, T.; Nagamori, S.; Kanai, Y.; Ishioka, N.S.;
7
6
Tsushima, Y.; et al. Development of a Widely Usable Amino Acid Tracer: Br-alpha-Methyl-Phenylalanine for Tumor PET
Imaging. J. Nucl. Med. 2015, 56, 791–797. [CrossRef]
1
5. Su, H.; Seimbille, Y.; Ferl, G.Z.; Bodenstein, C.; Fueger, B.; Kim, K.J.; Hsu, Y.T.; Dubinett, S.M.; Phelps, M.E.; Czernin, J.; et al.
1
8
Evaluation of [ F]gefitinib as a molecular imaging probe for the assessment of the epidermal growth factor receptor status in
malignant tumors. Eur. J. Nucl. Med. Mol. Imaging 2008, 35, 1089–1099. [CrossRef]
1
1
1
1
2
2
6. Guo, A.; Villen, J.; Kornhauser, J.; Lee, K.A.; Stokes, M.P.; Rikova, K.; Possemato, A.; Nardone, J.; Innocenti, G.; Wetzel, R.; et al.
Signaling networks assembled by oncogenic EGFR and c-Met. Proc. Natl. Acad. Sci. USA 2008, 105, 692–697. [CrossRef]
7. Bruehlmeier, M.; Roelcke, U.; Bläuenstein, P.; Missimer, J.; Schubiger, P.A.; Locher, J.T.; Pellikka, R.; Ametamey, S.M. Measurement
of the extracellular space in brain tumors using 76Br-bromide and PET. J. Nucl. Med. 2003, 44, 1210–1218. [PubMed]
8. Rossin, R.; Berndorff, D.; Friebe, M.; Dinkelborg, L.M.; Welch, M.J. Small-animal PET of tumor angiogenesis using a (76)Br-labeled
human recombinant antibody fragment to the ED-B domain of fibronectin. J. Nucl. Med. 2007, 48, 1172–1179. [CrossRef] [PubMed]
9. Lee, H.; Finck, B.N.; Jones, L.A.; Welch, M.J.; Mach, R.H. Synthesis and evaluation of a bromine-76-labeled PPARgamma
antagonist 2-bromo-5-nitro-N-phenylbenzamide. Nucl. Med. Biol. 2006, 33, 847–854. [CrossRef]
0. Ogawa, K.; Masuda, R.; Mizuno, Y.; Makino, A.; Kozaka, T.; Kitamura, Y.; Kiyono, Y.; Shiba, K.; Odani, A. Development of a novel
radiobromine-labeled sigma-1 receptor imaging probe. Nucl. Med. Biol. 2018, 61, 28–35. [CrossRef]
1. Ohshima, Y.; Hanaoka, H.; Watanabe, S.; Sugo, Y.; Watanabe, S.; Tominaga, H.; Oriuchi, N.; Endo, K.; Ishioka, N.S. Preparation
76
and biological evaluation of 3-[ Br]bromo-α-methyl-L-tyrosine, a novel tyrosine analog for positron emission tomography
imaging of tumors. Nucl. Med. Biol. 2011, 38, 857–865. [CrossRef] [PubMed]
2
2. Slobbe, P.; Windhorst, A.D.; Walsum, M.S.-v.; Schuit, R.C.; Smit, E.F.; Niessen, H.G.; Solca, F.; Stehle, G.; van Dongen, G.A.M.S.;
18
Poot, A.J. Development of [ F]afatinib as new TKI-PET tracer for EGFR positive tumors. Nucl. Med. Biol. 2014, 41, 749–757.
CrossRef] [PubMed]
[
2
3. Ogawa, K.; Takeda, T.; Yokokawa, M.; Yu, J.; Makino, A.; Kiyono, Y.; Shiba, K.; Kinuya, S.; Odani, A. Comparison of Radioiodine-
or Radiobromine-Labeled RGD Peptides between Direct and Indirect Labeling Methods. Chem. Pharm. Bull. (Tokyo) 2018, 66,
6
51–659. [CrossRef] [PubMed]
2
2
2
4. Xu, X.; Yuan, L.; Yin, L.; Jiang, Y.; Gai, Y.; Liu, Q.; Wang, Y.; Zhang, Y.; Lan, X. Synthesis and Preclinical Evaluation of 18F-PEG3-FPN
for the Detection of Metastatic Pigmented Melanoma. Mol. Pharm. 2017, 14, 3896–3905. [CrossRef]
5. Lux, J.; Chan, M.; Elst, L.V.; Schopf, E.; Mahmoud, E.; Laurent, S.; Almutairi, A. Metal Chelating Crosslinkers Form Nanogels
with High Chelation Stability. J. Mater. Chem. B 2013, 1, 6359–6364. [CrossRef] [PubMed]
6. Zhou, W.; Ercan, D.; Chen, L.; Yun, C.H.; Li, D.; Capelletti, M.; Cortot, A.B.; Chirieac, L.; Iacob, R.E.; Padera, R.; et al. Novel
mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature 2009, 462, 1070–1074. [CrossRef] [PubMed]

Pharmaceuticals 2021, 14, 256
14 of 14
2
7. Koura, M.; Yamaguchi, Y.; Kurobuchi, S.; Sumida, H.; Watanabe, Y.; Enomoto, T.; Matsuda, T.; Okuda, A.; Koshizawa, T.;
Matsumoto, Y.; et al. Discovery of a 2-hydroxyacetophenone derivative as an outstanding linker to enhance potency and
β-selectivity of liver X receptor agonist. Bioorganic Med. Chem. 2016, 24, 3436–3446. [CrossRef]
2
2
3
8. Fischer, C.; Sparr, C. Synthesis of 1,5-bifunctional organolithium reagents by a double directed ortho-metalation: Direct transfor-
mation of esters into 1,8-dimethoxy-acridinium salts. Tetrahedron 2018, 74, 5486–5493. [CrossRef]
9. Michael, D.A.; Brian, M.A.; Kenneth, L.A.; Sathesh, B.; Jason, B.; Kaleen, K.C.; Bernard, C.; Maria, E. Aminopyrimidines as syk
inhibitors. U.S. Patent 2011/0245205 A1, 6 October 2011.
0. Ogawa, K.; Kanbara, H.; Kiyono, Y.; Kitamura, Y.; Kiwada, T.; Kozaka, T.; Kitamura, M.; Mori, T.; Shiba, K.; Odani, A. Development
and evaluation of a radiobromine-labeled sigma ligand for tumor imaging. Nucl. Med. Biol. 2013, 40, 445–450. [CrossRef]
[PubMed]
3
1. Ogawa, K.; Shiba, K.; Akhter, N.; Yoshimoto, M.; Washiyama, K.; Kinuya, S.; Kawai, K.; Mori, H. Evaluation of radioiodinated
vesamicol analogs for sigma receptor imaging in tumor and radionuclide receptor therapy. Cancer Sci. 2009, 100, 2188–2192.
[CrossRef] [PubMed]