18F-Labeled Pyrido[3,4-d]pyrimidine as an Effective Probe for Imaging of L858R Mutant Epidermal Growth Factor Receptor

Article abstract of DOI:10.1021/acsmedchemlett.6b00520

In nonsmall-cell lung carcinoma patients, L858R mutation of epidermal growth factor receptor (EGFR) is often found, and molecular target therapy using EGFR tyrosine kinase inhibitors is effective for the patients. However, the treatment frequently develops drug resistance by secondary mutation, of which approximately 50% is T790M mutation. Therefore, the ability to predict whether EGFR will undergo secondary mutation is extremely important. We synthesized a novel radiofluorinated 4-(anilino)pyrido[3,4-d]pyrimidine derivative ([¹⁸F]APP-1) and evaluated its potential as a positron emission tomography (PET) imaging probe to discriminate the difference in mutations of tumors. EGFR inhibition assay, cell uptake, and biodistribution study showed that [¹⁸F]APP-1 binds specifically to the L858R mutant EGFR but not to the L858R/T790M mutant. Finally, on PET imaging study using [¹⁸F]APP-1 with tumor-bearing mice, the H3255 tumor (L858R mutant) was more clearly visualized than the H1975 tumor (L858R/T790M mutant).

Full text of DOI:10.1021/acsmedchemlett.6b00520

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Letter
18F-Labeled pyrido[3,4-d]pyrimidine as an effective probe for
imaging of L858R-mutant epidermal growth factor receptor
Hiroyuki Kimura, Haruka Okuda, Masumi Ishiguro, Kenji Arimitsu, Akira Makino, Ryuichi Nishii,
Anna Miyazaki, Yusuke Yagi, Hiroyuki Watanabe, Ikuo Kawasaki, Masahiro Ono, and Hideo Saji
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00520 • Publication Date (Web): 20 Mar 2017
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18F-Labeled pyrido[3,4-d]pyrimidine as an effective probe for imag-
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Hiroyuki Kimura,1,2‡* Haruka Okuda,1‡ Masumi Ishiguro,3 Kenji Arimitsu,2,3 Akira Makino,4 Ryuichi
Nishii,5 Anna Miyazaki,1 Yusuke Yagi,1 Hiroyuki Watanabe,1 Ikuo Kawasaki,3 Masahiro Ono,1 and
Hideo Saji1*
1Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
2Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto, Japan
3School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, Hyogo, Japan
4Biomedical Imaging Research Center (BIRC), University of Fukui, Fukui, Japan
5National Institute of Radiological Sciences, Chiba, Japan
KEYWORDS: Epidermal growth factor receptor tyrosine kinase (EGFR-TK), L858R-mutant EGFR, positron emission tomogra-
phy, fluorin-18, 4-(anilino)pyrido[3,4-d]pyrimidine
ABSTRACT: In non-small-cell lung carcinoma patients, L858R mutation of epidermal growth factor receptor (EGFR) is often
found, and molecular target therapy using EGFR tyrosine kinase inhibitors is effective for the patients. However, the treatment fre-
quently develops drug resistance by secondary mutation, of which approximately 50% is T790M mutation. Therefore, the ability to
predict whether EGFR will undergo secondary mutation is extremely important. We synthesized a novel radiofluorinated 4-
(anilino)pyrido[3,4-d]pyrimidine derivative ([18F]APP-1) and evaluated its potential as a positron emission tomography (PET) im-
aging probe to discriminate the difference in mutations of tumors. EGFR inhibition assay, cell-uptake, and biodistribution study
showed that [18F]APP-1 binds specifically to the L858R mutant EGFR but not to the L858R/T790M mutant. Finally, on PET imag-
ing study using [18F]APP-1 with tumor-bearing mice, the H3255 tumor (L858R mutant) was more clearly visualized than the
H1975 tumor (L858R/T790M mutant).
Non-small-cell lung carcinoma (NSCLC) is a class of lung
cancer that accounts for 80% of lung cancer cases and is a
major cause of cancer-related deaths. In approximately 10%–
30% of patients with NSCLC, a somatic activating mutation
occurs in the tyrosine kinase (TK) domain of the epidermal
growth factor receptor (EGFR) and causes the exon 21 muta-
tion, resulting in leucine being replaced by arginine at position
858 (L858R mutant) or exon 19 deletions in the EGFR gene.1
The increased kinase activity of mutant EGFR plays a key role
in cell proliferation and angiogenesis in cancer cells. There-
fore, EGFR-TK inhibitors (EGFR-TKIs), such as gefitinib
(Iressa®), erlotinib (Tarceva®), and afatinib (Giotriff®), are
used in NSCLC therapy to bind to the ATP domain in mutant
EGFR-TK.2
However, patients who initially respond to EGFR-TKIs typ-
ically develop drug resistance within 6–12 months after the
start of therapy, thereby inevitably disrupting the therapy.3 The
most common factor is the occurrence of secondary mutation
of the mutant EGFR gene, which leads to methionine replac-
ing threonine at position 790 of EGFR (T790M mutation). The
T790M mutation occurs in approximately 50% of patients
with resistance to EGFR-TKIs.3,4 Therefore, predicting wheth-
er EGFR undergoes this secondary mutation is extremely im-
portant for planning the treatment of EGFR-TKIs. For clinical
settings, genetic testing is conducted via biopsy to find muta-
tions and provide a definitive cancer diagnosis before starting
the therapy.5 However, because of the invasiveness of this
procedure, such testing is done only when the effect of molec-
ular targeting drugs decreases over the course of treatment.
Therefore, developing noninvasive diagnostics techniques that
can determine the therapeutic efficiency of EFGR-TKIs has
become necessary.
Positron emission tomography (PET) is a molecular imag-
ing technique that enables noninvasive detection of specific
molecules in living systems, and is expected, in particular, to
detect the presence of mutant EGFR. Furthermore, some ra-
diolabeled EGFR-TKIs, including [18F]gefitinib, [18F]afatinib
and [11C]erlotinib, have been investigated using PET probes
for EGFR-TK-positive tumor imaging.6–12 However, the use of
these compounds does not permit visualization of an EGFR-
TK-positive tumor. In addition, the use of [11C]erlotinib re-
portedly allows primary and secondary mutant tumors to be
differentiated by visualizing the former but not the latter,12 and
it leads to unclear images of L858R mutant tumors, similar to
the results for other radiolabeled EGFR-TKIs.
Previous investigations of a series of 4-(anilino)pyrido[3,4-
d]pyrimidine derivatives as EGFR-TKIs claim that these com-
pounds show lower half-maximal inhibitory concentration
(IC50) to EGFR-TK compared with 4-(anilino)quinazoline.13-15
Therefore, the radiolabeled 4-(anilino)pyrido[3,4-d]pyrimidine
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derivative is also expected to become an imaging-probe candi- The inhibition assay of APP-1 involved using erlotinib and
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date for tumors targeted by EGFR-TKIs. In addition, we
aimed drug design on the grounds that incorporating a Michael
acceptor unit at position 6 of pyrido[3,4-d]pyrimidine and
quinazoline scaffolds leads to irreversible inhibition of EGFR-
TK through a covalent modification of cysteine 797 in the
ATP binding domain16-18 and that the piperazinyl group is use-
ful to afford suitable water solubility and induce the radio-
labeling site. Moreover, if the accumulation of the derivative
differs between the primary and secondary mutant tumor, we
hope to use 4-(anilino)pyrido[3,4-d]pyrimidine as an imaging
probe to identify whether EGFR mutations are present. In the
present study, we synthesize a novel radiofluorinated 4-
(anilino)pyrido[3,4-d]pyrimidine derivative APP-1 (Figure 1)
and evaluate its ability to discriminate between primary and
secondary mutations in in vitro and in vivo experiments.
AZD9291 to inhibit the L858R mutant and L858R/T790M
mutant EGFR-TK; the results for IC50 appear in Table 1. Erlo-
tinib inhibits the L858R mutant EGFR kinase but not the
L858R/T790M mutant,12 and AZD9291 (osimertinib, Tagris-
so®), developed by AstraZeneca to inhibit the L858R/T790M
mutant EGFR-TK and recently approved by the US Food and
Drug Administration, inhibits both the L858R and
L858R/T790M mutants of EGFR-TK.19 As a result, both APP-
1 and erlotinib inhibit L858R mutant EGFR-TK somewhat
more than L858R/T790M mutant of EGFR-TK (15.6 0.8 nM
vs 326 64 nM and 12.5 6.0 nM vs 4040 1270 nM, re-
spectively), and AZD9291 potently inhibits mutant EGFR-TK
(12.3 3.1 nM vs 14.5 5.3 nM). Thus, radioactive APP-1 is
also expected to become a probe candidate for imaging
EGER-TK-positive tumors. In addition, APP-1 is found to be
selective, although the range of selectivity is smaller than that
of erlotinib. However, whether its selectivity suffices to visu-
ally discriminate between L858R- and L858R/T790M-mutant
tumors remains to be determined. Therefore, we continue
evaluating the potential of APP-1 for use in PET imaging.
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Table 1. IC50 for APP-1, erlotinib, and AZD9291.
IC50 (nM)
Compound
L858R
L858R/T790M
APP-1
Erlotinib
AZD9291
15.6 0.8
12.5 6.0
12.3 3.1
326 64
4040 1270
14.5 5.3
The values represent the mean SE (n = 3).
Figure 1. Chemical structure of EGFR-targeting PET imaging
probes.
We synthesized 18F-labeled [18F]APP-1 from precursor 3 in a
two-step reaction. First, we prepared radioactive 2-
[18F]fluoroethyl tosylate by a nucleophilic displacement reac-
tion of ethyleneglycol-1,2-ditosylate with the [18F]fluoride
anion. After purification by preparative high-performance
liquid chromatography (HPLC) and solid-phase extraction, 2-
[18F]fluoroethyl tosylate was reacted with compound 3 for 20
min at 110°C to yield [18F]APP-1 in a radiochemical yield of
3.2 0.94% [n = 5, end of synthesis (EOS), from potassium
[18F]fluoride] after preparative HPLC. The total operation took
less than 110 min. The isolated [18F]APP-1 was identified by a
HPLC analysis with co-injection of APP-1 (Figure 2). The
radiochemical purity and the specific activity exceeded 95%
and 40.4 GBq/µmol, respectively.
The nonradioactive APP-1 was synthesized as indicated in
Scheme 1. First, 6-aminopyrido[3,4-d]pyrimidine 1 was pre-
pared from 2-fluoro-5-aminopyridine via the synthetic route
reported by Rewcastle et al.14 Compound 1 was coupled with
4-bromocrotonic acid by using 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride as the condensing agent fol-
lowed by the addition of 1-(tert-butyloxycarbonyl)piperazine
to obtain amide 2 at an 91% yield. The deprotection of the
tert-butyloxycarbonyl group of compound 2 gave amine 3.
Finally, compound 3 was reacted with 2-fluoroethyl tosylate to
obtain APP-1.
Scheme 1. Synthesis of nonradioactive APP-1 and its precursor 3.
Reagents and conditions: (a) 4-bromocrotonic acid, 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride, DIPEA, DMF, rt, 1 h
followed by 1-(tert-butyloxycarbonyl)piperazine, rt, 30 min, 91%; (b) TFA, dichloromethane, rt, 85%; (c) 2-fluoroethyl tosylate, Et3N,
DMF, rt, 45%.
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Scheme 2. Radiosynthesis of [18F]APP-1.
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d
OTs
OTs
18F
TsO
3
4
2-[18F]fluoroethyl tosylate
ethylene-1,2-ditosylate
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e
HN
N
Br
HN
N
Br
H
N
H
N
N
N
N
N
HN
O
N
N
O
N
18F
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[
18F]APP-1
3
Reagent and conditions: (d) potassium [18F]fluoride, Kryptofix 2.2.2, MeCN, 90°C, 5 min; and (e) Et3N, DMF, 110°C, 20 min, 3.2
0.94% radiochemical yield (n = 5, EOS from potassium [18F]fluoride).
H1975 cells. Furthermore, upon adding AZD9291 as inhibitor,
the H3255-cell uptake decreases (104% 8.6% dose/mg pro-
tein to 46.8% 7.6% dose/mg protein, P < 0.01), whereas the
uptake of H1975 cells remains stable. This result suggests that
the different uptake is caused by the specific binding of
[18F]APP-1 to L858R mutant EGFR-TK. Because the inhibi-
tion rate of H3255 did not change even when 5 µM or more of
AZD9291 was added (data not shown), incomplete blockade
of H3255 cells may represent nonspecific binding.
We studied the biodistribution of [18F]APP-1 in H3255-
tumor-bearing mice (Table 2). The highest accumulation of
[18F]APP-1 occurred in the intestines [small intestine: 44.94%
injected dose per gram (ID/g) at 1 h post-injection, colon:
59.65% ID/g at 3 h post-injection] and was excreted over time.
The accumulation of [18F]APP-1 in bone was low. Therefore,
[18F]APP-1 was stable in vivo. The accumulation of [18F]APP-
1 in tumors was retained for at least 3 h (3.62% ID/g at 1 h
Figure 2. HPLC analysis of [18F]APP-1 co-injected with nonradi-
oactive APP-1. HPLC conditions: the column was a Cosmosil
Table 2. Biodistribution of [18F]APP-1 in H3255-tumor-
bearing mice.
5C18-AR-II 10 mm × 250 mm, flow rate was 5.0 mL/min, UV
excitation at 280 nm, and mobile phase systems were MeCN
(0.1% TFA) : H2O (0.1% TFA) = 20:80 (0 min) to 40:60 (20
min).
Injected dose/g (%)
Tissue
1 h after injection 3 h after injection
We next investigated the cellular uptake of [18F]APP-1 by
two human NSCLC cells: H3255 cells expressing the L858R
mutant EGFR and H1975 cells expressing the L858R/T790M
mutant. The results appear in Figure 3. The uptake of
[18F]APP-1 in H3255 cells was twice as much as that in the
Blood
1.60 0.33
1.61 0.38
3.48 0.51
17.93 7.12
44.94 5.51
6.96 0.61
7.86 1.23
5.80 0.78
4.37 0.50
8.58 1.25
0.81 0.46
0.63 0.21
3.62 0.87
1.12 0.08
0.72 0.12
1.18 0.14
3.64 1.68
14.70 4.31
59.65 11.68
2.78 0.38
1.12 0.12
1.08 0.17
2.05 0.22
1.32 0.40
0.29 0.06
3.80 0.88
Heart
Lung
Stomach
Small Intestine
Colon
Liver
Pancreas
Spleen
Kidney
Bone
Muscle
Tumor (H3255)
Ratios
Tumor/Blood
Tumor/Muscle
Tumor/Lung
2.25 0.18
6.15 1.88
1.04 0.20
3.35 0.66
13.37 4.02
3.21 0.54
Figure 3. Accumulation of [18F]APP-1 in H3255 and H1975 cells
after incubation with and without AZD9291. The values represent
the mean ± standard deviation (n = 3; *P < 0.01; “n.s.” means
“not significant”).
The values represent the mean SD (n = 5).
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post-injection, 3.80% ID/g at 3 h post-injection). Because the Table 3. Ratios of accumulated radioactivity in H3255 and
accumulation of [11C]erlotinib for 1 h in H3255 tumors is re-
ported to be decreasing over time,9 we assume that [18F]APP-1
strongly binds to EGFR-TK in H3255 tumors by irreversible
binding associated with a covalent bond via the Michael ac-
ceptor group. However, the slow clearance from normal tissue
must be improved. At each time point, Table 2 also gives the
tumor-to-blood, tumor-to-muscle, and tumor-to-lung ratios.
All ratios increase over time and exceed three at 3 h post-
injection. Therefore, we expect the use of [18F]APP-1 for visu-
alization of H3255 tumors in mice.
Furthermore, we studied in vivo blocking to determine the
ability of [18F] to discriminate between L858R and
L858R/T790M mutant EGFRs (Figure 4). Co-administration
of excess AZD9291 significantly reduced the accumulation of
[18F]APP-1 in H3255 tumors (54% inhibition) at 3 h post-
injection. However, accumulation in H1975 tumors was not
blocked by excess AZD9291. These results correspond to
H1975 tumor-bearing mice after PET-CT imaging.
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2
3
4
5
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Ratio
Tumor
Tumor/Blood
Tumor/Muscle
Tumor/Lung
H3255
H1975
3.12
0.74
6.80
2.95
3.25
0.74
those of the cell-uptake study (Figure 3). In other words, these
results confirm that, in mice with mutant-EGFR-TK tumors,
[18F]APP-1 binds specifically to L858R mutant EGFR-TK but
not to L858R/T790M mutant EGFR-TK.
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Furthermore, we performed PET imaging of [18F]APP-1 in
H3255- or H1975-tumor-bearing mice (Figure 5). The images
at 3 h post-injection of [18F]APP-1 show that H3255 tumors
are more clearly visualized than H1975 tumors. In addition,
we measured the radioactivity in each organ and tissue after
image acquisition and calculated the tumor-to-blood, tumor-
to-muscle, and tumor-to-lung ratios (Table 3). The contrast
between H3255 tumors and surrounding tissue is higher than
that when using [11C]erlotinib.13 These results suggest that
[18F]APP-1 is effective as an imaging probe that targets L858R
mutant EGFR.
We designed and synthesized
a radiofluorinated 4-
(anilino)pyrido[3,4-d]pyrimidine derivative, APP-1, as a PET
imaging agent to discriminate between L858R and
L858R/T790M mutant EGFRs. When used in an EGFR-TK
inhibition assay, APP-1 was the strongest inhibitor of the
L858R mutant EGFR-TK, and a weak inhibitor of the
L858R/T790M mutant EGFR-TK. The cell-uptake study
shows that [18F]APP-1 binds specifically to the L858R mutant
EGFR-TK but not to the L858R/T790M mutant. Further as-
sessment of the biodistribution revealed that [18F]APP-1 re-
sults in a high tumor-to-tissue ratio in H3255-tumor-bearing
mice, while co-injection of AZD9291 suggests that the accu-
mulation of [18F]APP-1 in tumors is specific to EGFR-TK. In
addition, for PET imaging, the H3255 tumor is more clearly
visualized than the H1975 tumor. Based on these results, we
conclude that [18F]APP-1 has a potential to be used as a PET
imaging probe to discriminate between L858R and
L858R/T790M mutant EGFRs in NSCLC.
Figure 4. Effect of co-administration of AZD9291 on biodistribu-
tion of [18F]APP-1 (3 h post-injection). The graphs show the mean
%ID/g of four mice with the error bars giving the standard devia-
tion (*P < 0.01).
ASSOCIATED CONTENT
Supporting Information
Full experimental procedures and characterization data for all new
compounds described in this study. The Supporting Information is
available free of charge on the ACS Publications website at
DOI:XXX. (PDF)
AUTHOR INFORMATION
Corresponding Author
* E-mail: hsaji@pharm.kyoto-u.ac.jp.
* E-mail: hkimura@mb.kyoto-phu.ac.jp.
Author Contributions
Figure 5. PET-CT image of [18F]APP-1 in H3255- [(A), (B)] or in
H1975- [(C), (D)] tumor-bearing mice at 3 h post-injection. Pan-
els (A) and (C) show coronal images, while panels (B) and (D)
show transverse images.
The manuscript was written through contributions of all authors,
and all the authors approve the final version of the manuscript.
‡These authors have contributed equally.
Funding Sources
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Hendrikse, N. H. Development of [11C]erlotinib positron
This work was supported by the Practical Research for Innovative
emission tomography for in vivo evaluation of EGF receptor
mutational status. Clin. Cancer. Res. 2013, 19, 183–193.
(12) Abourbeh, G.; Itamar, B.; Salnikov, O.; Beltsov, S.; Mishani,
E. Identifying erlotinib-sensitive non-small cell lung carci-
noma tumors in mice using [11C]erlotinib PET. EJNMMI Res.
2015, 5, 4.
1
2
3
4
Cancer Control from the Japan Agency for Medical Research and
Development (AMED) and Grants-in-Aid for Scientific Research
from the Japan Society for the Promotion of Science.
Notes
The authors declare no competing financial interest.
5
(13) Bridges, A. J.; Zhou, H.; Cody, D. R.; Rewcastle, G. W.;
McMichael, A.; Showalter, H. D. H.; Fry, D. W.; Kraker, A.
J.; Denny, W. A. Tyrosine kinase inhibitors. 8. An unusually
steep structure-activity relationship for analogues of 4-(3-
bromoanilino)-6,7-dimethoxyquinazoline (PD 153035), a po-
tent inhibitor of the epidermal growth factor receptor. J. Med.
Chem. 1996, 39, 267–276.
(14) Rewcastle, G. W.; Palmer, B. D.; Thompson, A. M.; Bridges,
A. J.; Cody, D. R.; Zhou, H.; Fry, D. W.; McMichael, A.;
Denny, W. A. Tyrosine kinase inhibitors. 10. isomeric 4-[(3-
bromophenyl)amino]pyrido[d]pyrimidines are potent ATP
binding site inhibitors of the tyrosine kinase function of the
epidermal growth factor receptor. J. Med. Chem. 1996, 39,
1823–1835.
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ABBREVIATIONS
ATP, adenosine triphosphate; DIPEA, N,N- diisopropylethyla-
mine;
Boc,
tert-butyloxycarbonyl;
DMF,
N,N-
dimethylformamide; EGFR, epidermal growth factor receptor;
EGFR-TK, epidermal growth factor receptor tyrosine kinase;
EGFR-TKIs, epidermal growth factor receptor tyrosine kinase
inhibitors; ID, injected dose; MeCN, acetonitrile; NSCLC, non-
small-cell lung carcinoma; PET, positron emission tomography;
HPLC, high-performance liquid chromatography; rt, room tem-
perature; TFA, trifluoroacetic acid.
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18F-Labeled pyrido[3,4-d]pyrimidine as an effective probe for imaging of L858R-mutant epidermal growth factor re-
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Hiroyuki Kimura, Haruka Okuda, Masumi Ishiguro, Kenji Arimitsu, Akira Makino, Ryuichi Nishii, Anna Miyazaki, Yusuke
Yagi, Hiroyuki Watanabe, Ikuo Kawasaki, Masahiro Ono, and Hideo Saji
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