Synthesis and preliminary evaluation of 18F-icotinib for EGFR-targeted PET imaging of lung cancer

Article abstract of DOI:10.1016/j.bmc.2018.12.034

Epidermal growth factor receptor (EGFR) has emerged as an attracting target in the field of imaging and treatment for non-small cell lung cancer (NSCLC). Radiolabeled EGFR-tyrosine kinase inhibitors (EGFR-TKIs) specifically targeting EGFR are deemed as pr

Full text of DOI:10.1016/j.bmc.2018.12.034

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1
8
Synthesis and preliminary evaluation of F-icotinib for EGFR-targeted PET
imaging of lung cancer
a,b
c,d
e
a,b
a,⁎
a,⁎
Xinmiao Lu , Cheng Wang , Xiao Li , Peilin Gu , Lina Jia , Lan Zhang
a
b
c
Shanghai Institute of Applied Physics (SINAP), Chinese Academy of Sciences, Shanghai 201800, PR China
University of Chinese Academy of Sciences, Beijing 100049, PR China
Department of Nuclear Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, PR China
Institute of Nuclear Medicine, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, PR China
Department of Nuclear Medicine, Changhai Hospital, Shanghai 200433, PR China
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
EGFR
Epidermal growth factor receptor (EGFR) has emerged as an attracting target in the field of imaging and
treatment for non-small cell lung cancer (NSCLC). Radiolabeled EGFR-tyrosine kinase inhibitors (EGFR-TKIs)
specifically targeting EGFR are deemed as promising probes for the imaging of NSCLC. This study aimed to label
NSCLC
PET imaging
18
18
icotinib (one kind of EGFR-TKI) with F through click reaction to develop a new EGFR-targeting PET probe- F-
1
8
F-icotinib
18
icotinib. F-icotinib was obtained in 44.81% decay-corrected yield in 100 min synthesis time with 34 GBq/μmol
Click reaction
specific activity and > 99% radiochemical purity at the end of synthesis. The identity of the product was con-
1
8
19
firmed by co-injection with F-icotinib and F-icotinib. The Log P was 1.28 ± 0.04 (n = 6). The tracer dis-
1
8
played excellent stability after incubation for 4 h in vitro. F-icotinib showed satisfying binding ability to A549
NSCLC cells, which could be inhibited by icotinib. PET imaging studies demonstrated a specific uptake of the
radiotracer (0.90 ± 0.24% ID/g) in A549 tumor-bearing mice, while lower uptake was observed in heart, lung
and spleen at 1.5 h post injection. Inmunohistochemical staining confirmed that the A549 tumor was EGFR-
1
8
positive. Therefore, we considered that F-icotinib was a highly promising compound for EGFR-based tumor
PET imaging.
1
. Introduction
gefitinib has high non-specific absorption and strong background signal
1
0 11
in in vivo evaluation.
C-gefitinib has obvious accumulation in the
11
Lung cancer has the highest morbidity and mortality all over the
intestine, and the application is limited by nuclide C, which only has a
11 18
world. Non-small cell lung cancer (NSCLC) accounting for > 80% of
lung cancer is one of the most fatal diseases with a low five-year sur-
half-life of 20 min.
F-FEA-erlotinib reported in 2017 displayed ex-
tremely high uptake in liver and intestine, and the accumulation in the
heart and kidneys was also obvious, according to the biodistribution
1
vival rate.
1
2
11
13,14
Epidermal growth factor receptor (EGFR) was overexpressed on the
studies.
C-erlotinib is the most promising PET probe,
it can
2
,3
18
surface of NSCLC cells and closely related to the neovascularization,
detect the lymph node metastases which can’t be distinguished by F-
FDG. But it still has high accumulation in the intestine and was also
4
,5
proliferation, metastasis and invasion of tumor cells.
Targeted
1
1
therapy against EGFR using EGFR-tyrosine kinase inhibitor (EGFR-
limited by the usage of C.
6
15
TKI) is effective in clinical treatments due to their great specificity and
Icotinib is one kind of EGFR-TKI molecule with quinoline struc-
7
–9
targeting ability to EGFR.
ture, which has great specificity and targeting ability to EGFR. Phar-
macokinetic analysis shows that the in vivo half-life of icotinib is 6 h
Positron emission tomography (PET) imaging technology especially
combined with computed tomography (CT) is the most advanced ima-
ging technology with great specificity and sensitivity. The specific
sensitivity of radiolabeling EGFR-TKI to EGFR has been already
exploited to design PET probes for diagnosis and imaging for NSCLC
1
6
(half-life of gefitinib ≈40 h and half-life of erlotinib ≈36 h ), and the
1
7
concentration in blood reaches the peak in 2 h post injection (p.i.),
which demonstrates that icotinib has higher potential to be the pre-
cursor to design a PET probe. Since the in vivo effective time of a probe
only needs to meet the time required for specific accumulation and
(
Fig. 1), yet with some inadequate more or less. For example, 18F-
⁎
Corresponding authors at: P. O. Box 800-204, Shanghai 201800, PR China.
E-mail addresses: jialina@sinap.ac.cn (L. Jia), zhanglan@sinap.ac.cn (L. Zhang).
https://doi.org/10.1016/j.bmc.2018.12.034
Received 7 November 2018; Received in revised form 24 December 2018; Accepted 27 December 2018
0968-0896/ © 2018 Published by Elsevier Ltd.
Please cite this article as: Lu, X., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2018.12.034

X. Lu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
O
O
F
18F
F
N
N
N
HN
N
HN
N
Cl
N
HN
N
Cl
HN
N
Cl
O
O
H311C O
O
O
N
O
O
N
O
O
N
N
N
H311C O
H C
3
O
O
18F
11C-gefitinib
18F-gefitinib
18F-FEA-erlotinib
11C-erlotinib
Fig. 1. EGFR-TKI PET imaging probes.
diagnostic observation. Longer in vivo retention time makes no sense
but produces excess radiation damage. Meanwhile, icotinib has a crown
ether structure, which could provide higher hydrophilicity. Therefore,
reacted at room temperature until 2-fluoroethyl-4-toluenesulfonate (1)
disappeared. The reaction mixture was filtered, and the filtrate con-
taining 1-azido-2- fluoroethane (2) was used without isolation for
subsequent reactions.
1
8
icotinib labeled with F may show great potential to improve the
quality of NSCLC PET imaging. So it is valuable to use icotinib as the
precursor to develop a PET probe targeting EGFR.
2.2.3. Synthesis of 19F-icotinib (4)
The copper(I) catalyzed cycloaddition between azides and alkynes
To a stirred solution of copper sulfate (0.15 mmol) and sodium as-
corbate (0.30 mmol) in 12 mL PBS (pH 6.0) was added a solution of
icotinib (3) (26.50 mg, 0.068 mmol) in DMF (1 mL). After addition of 2-
fluoroethylazide (2) (0.70 mmol) in 2 mL DMF, stirring was continued
(
CuAAC) to form 1,2,3-triazoles is the most famous ‘click reaction’.
Because of the advantage of mild reaction conditions, tolerance of
1
8,19
solvents and pH, high chemoselectivity and perfect regioselectivity,
this reaction was introduced into the field of radiopharmaceutical and
at 50 °C for 1 h. The reaction mixture was quenched with H O (30 mL),
2
1
8
has achieved great success, especially for F labeled PET probes. Sev-
eral innovated PET probes have been developed based on CuAAC re-
and extracted with ethyl acetate (3 × 20 mL). After drying over sodium
sulfate, the solvent was removed with reduced pressure to yield
2
0–22
18
19
action.
Notably, F-HX
4
prepared by CuAAC reaction is in clinical
31.21 mg click product F-icotinib (4) in 95.71% ‘click reaction’ yield.
+
+
studies for non-invasive detection of hypoxia in patients with head and
TOF-ESI-MS (ESI) [M+H]
481.15, [M+Na]
503.15,
2
2
neck, or lung cancer. Therefore, the aim of this study is to synthesize
the EGFR targeting PET probe based on icotinib via CuAAC reaction and
evaluate its practicality as a PET tracer in NSCLC.
(C24
H
25
O N F, calculated molecular weight [MW] = 480.49).
4 6
1
H NMR (400 MHz, CD OD): δ8.65(s, 1H), δ8.54(s, 1H), δ8.34(s,
3
1H), δ8.22(s, 1H), δ7.93–7.97(t, 1H), δ7.57–7.58(d, 1H), δ7.46–7.50(t,
1
H), δ7.33(s, 1H), δ4.75–4.98(t, 2H), δ4.31–4.34(m, 4H),
13
2
. Experimental section
δ3.77–3.82(m, 4H), δ3.66(m, 4H), δ2.90(s, 1H), δ2.74(s, 1H). C NMR
(
100 MHz, CD OD): δ162.79, δ157.11, δ156.47, δ153.82, δ150.25,
3
2.1. General information
δ146.98, δ140.50, δ131.39, δ130.69, δ130.11, δ129.54, δ122.35,
δ122.07, δ120.81, δ119.08,δ110.74, δ83.20, δ81.52, δ73.42, δ70.94,
δ70.85, δ70.43, δ69.26, δ68.87
All reagents were purchased commercially from Sinopharm
Chemical Reagent Co, Ltd. (Shanghai, China) except for special in-
structions. Icotinib was purchased from Adooq Bioscience. All reagents
and solvents were used without further purification unless noted
otherwise. Agilent 1100 High Performance Liquid Chromatography
2.3. Synthesis of 18F-icotinib (7)
2.3.1. Synthesis of 2-azidoethyl-4-toluenesulfonate (5)
(
HPLC) equipped with UV-Vis absorbance detector and radioactive
1,2-Bis(tosyloxy)ethane (1.24 g, 3.35 mmol) was added to 100 mL
DMF, then sodium azide (total 0.22 g, 3.38 mmol) was added every
0.5 h, (0.073 g × 3). The mixture reacted at room temperature for 24 h.
2-azidoethyl-4-toluenesulfonate (5) (0.46 g, 1.91 mmol) was obtained
after purifying by column chromatography in 57.01% yield.
1
8
detector was used for purification and quality control of F-icotinib
with absorbance at 254 nm. A semi-preparative column (Agilent XDB
C18, 9.4 × 300 mm, with a flow rate of 2 mL/min, UV at 254 nm) was
used in the experiments. The mobile phase of HPLC was H
acetonitrile (B).
2
O (A) and
1
H NMR (400 MHz, CD OD): δ7.83–7.85(t, 2H), δ7.48–7.50(t, 2H),
3
A549 xenograft mice were obtained from National Rodent
δ4.15–4.18(t, 2H), δ3.51–3.53(t, 2H), δ2.48(s, 3H).
6
Experimental Animal Seed Center (Shanghai, China). 5 × 10 A549
cells in 100 μL phosphate-buffered saline (PBS) were inoculated sub-
cutaneously into the shoulder of 6–8 week-old male athymic nu/nu
nude mice. Tumor was taken out and crushed into 1 mm pieces after
achieving 5 mm, then were surgically implanted subcutaneously under
isoflurane anaesthesia to the athymic nu/nu nude mice. Tumors were
allowed to grow to 5–10 mm before imaging studies were commenced.
18
2
.3.2. Synthesis of F-1-azido-2-fluoroethane (6)
18
F-fluoride (n.c.a) (5–20 mCi) was captured on QMA Cartridge and
was eluted with 2 mL mixture containing 28.8 mg K2.2.2 and 6.8 mg
CO dissolved in 1.92 mL acetonitrile and 0.08 mL H O into reaction
vial. The F-fluoride/K /K2.2.2 mixture was dried three times by
azeotropic distillation with dried acetonitrile under nitrogen purge at
15 °C in metal bath. After cooling down, 5.0 mg 2-azidoethyl-4-to-
K
2
3
2
18
2
CO
3
1
2
2
3
.2. Synthesis of reference compound 19F-icotinib (4)
.2.1. Synthesis of 2-fluoroethyl-4-toluenesulfonate (1)
luenesulfonate (5) in 0.4 mL of acetonitrile was added into dried
1
8
−
F–F . The reaction vessel was sealed and heated to 115 °C for 10 min.
After cooling down, the mixture of product was distilled at 85 °C for
1
8
2
-Fluoroethanol (20 mL) and p-toluenesulfonyl chloride (7.15 g,
15 min. Distilled product was collected to obtain F-1-azido-2- fluor-
7.53 mmol) was added to 80 mL dichloromethane and reacted under
oethane (6) solution (3.0–12 mCi).
the catalysis of 17.40 mL triethylamine and 0.20 g 4-dimethylamino-
pyridine (DMAP) at room temperature for 24 h. 2-Fluoroethyl-4-tolue-
nesulfonate (1) (4.23 g, 19.40 mmol) was collected in 51.70% yield
after purifying by column chromatography.
1
8
2
.3.3. Synthesis of F-icotinib (7)
18
F-1-azido-2-fluoroethane (6) (200 μL) and icotinib 3 (diluted in
3
00 μL DMF) were added to 200 μL PBS (pH = 6) and reacted under the
catalysis of 0.5 M copper sulfate and 1.5 M sodium ascorbate for
15 min. The mixture was then filtered by a 0.22 μm filter membrane
and the radiolabeling yield of click reaction was determined by radio-
HPLC. The relative percentage content of the peak of 18F-icotinib (7)
2
.2.2. Synthesis of 1-azido-2- fluoroethane (2)
-Fluoroethyl-4-toluenesulfonate (1) (4.23 g, 19.40 mmol) and so-
dium azide (3.78 g, 58.15 mmol) was added into 55 mL DMF and
2
2

X. Lu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
was determined by radio-HPLC with the normalization method of peak
and the wells were washed twice with PBS. The solution was transferred
1
8
area. The filtered mixture was diluted with 6 mL water, and the F-
icotinib was trapped on a Sep-Pak light C18 cartridge, which was then
into a counting tube and the radioactivity was measured with a γ
1
8
counter. The uptake fractions of F-icotinib were calculated as per-
centage of applied dose per million cells. Sextuplet measurements were
applied in this experiment.
1
8
rinsed with water (6 mL). The F-icotinib was then eluted with 400 μL
acetonitrile and diluted with 500 μL water. The product was isolated via
semi-preparative
radio-HPLC.
The
gradient
was
(A),
0
3
–15 min–16 min–23 min-24 min,
65%–65%–30%–30%–65%
2.7. PET-CT imaging
5%–35%–70%–70%–35% (B) and the flow rate was 2 mL/min. The
1
8
18
F-icotinib fraction was collected and diluted with water (8 mL). F-
MicroPET-CT imaging was performed on A549 xenograft mice using
icotinib was trapped on a Sep-Pak light C18 cartridge and rinsed with
a microPET/CT scanner (SuoerNova). All of the animal experiments
followed the specifications for laboratory animal studies provided by
Renji Hospital, Shanghai Jiao Tong University. Mice bearing A549
1
8
water (6 mL), then F-icotinib was eluted by 300 μL ethanol. After
removing part of ethanol, the product was formulated with 0.9% NaCl
(
containing 10% ethanol) for further use.
18
tumor were injected with approximately 1.3–1.5 MBq, 150 μL of F-
Since the use of copper was deemed as the default of click reaction,
18
icotinib via the tail vein. To validate the specificity of F-icotinib, mice
the concentration of copper ions in the preparation was tested using
ICP-MS.
with A549 tumor were injected with excess fold of icotinib (0.1 mg,
18
2
00 μL) via the tail vein before the administration of F-icotinib. Then
We also optimized the condition of click reaction, such as reaction
temperature, precursor concentration and catalyst dosage, to get the
highest radiolabeling yield.
mice were anesthetized with 5% isoflurane delivered in 66%/33% ni-
trogen/oxygen prior to performing PET imaging experiments and the
images at 1.5 h p.i. were reconstructed with the three dimensional or-
dered-subset expectation maximization (OSEM) algorithm.
2.4. Octanol/water partition coefficient
Small-animal CT imaging was performed for anatomical reference
and a 3 min acquisition was performed after PET. Mice were anesthe-
tized with isoflurane (1–2%) throughout the period of imaging.
1
8
18
To determine the lipophilicity of F-icotinib, 5 μL formulated F-
icotinib was added into the mixture of 595 μL PBS (pH 7.4) and 600 μL
octanol. The mixture was vigorously vortexed for 2 min followed by
centrifugation (12000 rpm, 5 min). From each layer, an aliquot of
2.8. Immunohistochemical analysis
1
00 μL was removed and counted in a γ-counter. The partition coeffi-
Mice bearing A549 tumor were sacrificed. The tumor tissues were
cient (log P) was then calculated as a ratio of counts in the octanol
fraction to the counts in the water fraction. The experiment was re-
peated 6 times.
extracted for analyzing the expression status of EGFR. Formalin-fixed,
paraffin-embedded tumor tissue sections (thickness, 4–7 μm) were de-
paraffinized in xylene and rehydrated in gradient ethanol. Heat-induced
antigen retrieval was conducted in 10 mmol/L citrate buffer.
Endogenous peroxidase was blocked by incubation with 3% hydrogen
peroxidase for 10 min. The slides were then incubated with primary
antibodies rabbit anti-EGFR (1:100, Abcam, ab52894), followed by
peroxidase conjugated secondary antibodies for 0.5 h at 37 °C. Color
development was treated with DAB (3, 3′-diaminobenzidine tetra-
hydrochloride) and counterstaining was performed with hematoxylin.
The optical density (OD) value was measured by image analysis.
2.5. In vitro stability
1
8
The formulated
F-icotinib was incubated in 0.5 mL PBS
(
pH = 7.4) and 0.5 mL mouse serum for 4 h at 37 °C. The in vitro sta-
bility of the probe at different time points (including 0.5 h, 1 h, 2 h, 3 h
and 4 h) was analyzed by radio-thin-layer chromatography (radio-TLC)
(
silica gel; methanol/ethyl acetate, 1:1 [v/v]; Rf = 0.5). Each test had a
parallel sample. The in vitro stability of the probe in mouse serum and
PBS after incubating for 4 h was also analyzed by radio-HPLC.
3
. Results
2.6. In vitro cell uptake
3.1. Chemistry and radiochemistry
A549 Cells were grown in DMEM containing 10% fetal calf serum
The reference compound 19F-icotinib (4) was synthesized in a three-
1
9
and antibiotics (penicillin) under 37 °C, 5% CO
2
. A549 cells were
step procedure as shown in Fig. 2. F-icotinib (4) was obtained
with > 99% purity (HPLC) without further purification. The structure
was characterized by MS and NMR spectroscopy (see more in
Supporting information).
seeded 24 h prior to the experiment in DMEM medium in 24-well plates
5
at densities of 1 × 10 cells per well. On the day of the experiment,
1
8
1
5 μCi (50 μL) of F-icotinib was added to the cells for the study group,
18
18
while 15 μCi (50 μL) of F-icotinib and 10 μg icotinib (50 μL) were
added for the control group. After being incubated at 37 °C for 60 min,
culture was removed and cells were washed twice with cold PBS to
remove the free probes. Cells were dissolved with 1 mL NaOH (1.0 M)
2-Azidoethyl-4-toluenesulfonate (5), F-1-azido-2-fluoroethane (6)
1
8
and F-icotinib (7) were synthesized as shown in Fig. 3. 2-Azidoethyl-
1
8
4-toluenesulfonate (5) was obtained in a yield of 57.01%. F-1-azide-2-
fluoroethane (6) was prepared via nucleophilic fluorination of 2-
Fig. 2. The synthesis process of reference compound 19F-icotinib (4).
3

X. Lu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 3. Synthesis of 2-azidoethyl-4-toluenesulfonate (5), 18F-1-azido-2-fluoroethane (6) and 18F-icotinib (7).
Table 1
Yield of ‘click’ radiolabeling reaction of 18F-icotinib under different reaction conditions.
Entry
Icotinib/mg
CuSO
4
/μL
Sodium ascorbate/μL
PBS/μL
Temperature/°C
Yield of ‘click’ radiolabeling*
1
2
3
4
5
6
1
1
2
2
4
4
20
60
20
60
60
60
20
60
20
60
60
60
67
50
50
50
50
50
80
18.4 ± 1.3% (n = 3)
33.3 ± 2.8% (n = 3)
19.9 ± 3.2% (n = 3)
43.6 ± 5.3% (n = 3)
71.1 ± 4.8% (n = 3)
> 99%
200
67
200
200
200
*
Yields were determined by radio-HPLC.
azidoethyl 4-benzenesulfonate (5). The radiolabeling yield of (6)
injection.
was > 99% and the distillation efficiency was > 72%. The radio-
1
8
chemical purity of
F-1-azide-2-fluoroethane (6) after distillation
3.2. Octanol/water partition coefficient
was > 99% and chemical impurities such as K2.2.2 and labeling pre-
cursor could be removed from the labeling product and had no influ-
ence to the subsequent reaction.
The radioactivity counts distributing in the water layer and oil layer
1
8
was recorded. The measured log
P
value of
F-icotinib was
The conditions of click reaction were optimized and the results were
1
.28 ± 0.04 (n = 6), according to our experiments.
1
8
showed in Table 1. Under the optimized condition, F-icotinib was
prepared in > 99% radiolabeling yield with the reaction condition of
3.3. In vitro stability
icotinib (0.01 mmol), CuSO (3.0 equiv) and sodium ascorbate (9.0
4
1
8
equiv) reacting with F-1-azide-2-fluoroethane (6) in a solution of PBS
18
The in vitro stability of F-icotinib was determined by radio-TLC
(
pH 6.0), DMF and acetonitrile (200 μL:300 μL:200 μL) for 15 min at
and radio-HPLC. The radio-TLC (see more in Supporting Information)
1
8
8
0 °C. After purification, F-icotinib was obtained in 44.81% decay-
18
and radio-HPLC results showed that F-icotinib kept completely intact
corrected yield in 100 min synthesis time with 34 GBq/μmol specific
activity at the end of synthesis (EOS) and > 99% radiochemical purity.
after 4 h incubation in 0.01 M PBS (pH 7.4) and mouse serum. Stability
18
analysis chromatograms of F-icotinib by HPLC after 4 h incubation
1
8
19
F-icotinib was identified by co-elution with reference compound F-
were shown in Fig. 5.
icotinib (t = 12.65 min) by HPLC (Fig. 4).
R
The copper ion concentration of the formulated product determined
by ICP-MS was 20 ppb, which fulfilled the requirements for intravenous
3.4. In vitro cell uptake
The cellular uptake results showed the uptake of 18F-icotinib in the
A549 cells harboring EGFR expression was high (19.50 ± 4.85% ap-
1
8
plied radioactivity per million cells (n = 6)). The uptake of F-icotinib
in the A549 cells was reduced when cells were incubated with icotinib
at the same time (10.59 ± 0.91% applied radioactivity per million
1
8
cells (n = 6)). The result suggested that F-icotinib exhibited specifi-
city for EGFR.
3.5. PET-CT imaging
The microPET-CT images of A549 xenograft mice at 1.5 h p.i. were
1
8
shown in Fig. 6. Obvious accumulation of F-icotinib was seen in
tumor, and the maximum SUV (Standardized Uptake Value) and
average SUV in tumor were 0.65 and 0.35, respectively. The tumor
1
8
uptake of F-icotinib at 1.5 h p.i. based on PET quantification was
0
.90 ± 0.24 %ID/g. Tracer uptake was predominantly observed in the
intestine (SUVmax = 25.87). The tracer was eliminated from the
kidney rapidly (SUVmax = 0.91). There was no obvious uptake in the
lung and muscle which corresponded to high tumor-to-lung and
-
muscle radios (3.5, and 7.0 respectively). Negligible bone uptake
Fig. 4. HPLC analysis of 18F-icotinib (7) co-injected with 19F-icotinib (4).
(
SUVmax = 0.05 at 1.5 h p.i.) was observed.
4

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Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 5. The in vitro stability of 18F-icotinib (7) in PBS (pH = 7.4) and mouse serum.
The excess icotinib will block the tumor EGFR, which would lead to
4. Discussion
1
8
the decreased accumulation of F-icotinib in the tumor. So when ico-
tinib was used as a blocker in PET imaging, radioactivity uptake in the
tumor was dramatically reduced. The uptake in mice was 0.3 ± 0.11 %
ID/g, and the maximum SUV of tumor was 0.05 and the average SUV
was 0.01 (35-fold decreased), respectively.
Targeted therapy with EGFR-TKI is effective for the clinical treat-
ment of NSCLC. However, only patients with high expression and/or
specific mutations of EGFR demonstrate high sensitivity to the EGFR-
targeted therapy, an indiscriminate use of EGFR targeting drugs would
make the overall efficiency poor. To achieve reasonable and effective
therapy for NSCLC patients, reliable diagnosis and accurate images are
of great values. In this study, we synthesized and biologically evaluated
3
.6. Immunohistochemical analysis
1
8
the EGFR targeting PET probe- F-icotinib based on icotinib, which is a
reversible EGFR-TKI and has been approved to be used in the treatment
of advanced NSCLC in 2011 in China.
To validate the EGFR expression in A549 tumors harvested from the
mice we used in PET imaging experiments, tumor specimens were
analyzed for EGFR. Typical results of immunohistochemical analysis of
A549 tumors were showed in Fig. 7, which indicated that in both
blocked and unblocked imaging experiments, A549 tumors were EGFR-
positive and there was no obvious difference of EGFR expression level.
OD values of blocked and unlocked xenograft sections were
According to the structure activity relationship between icotinib
2
3,24
and EGFR protein,
the amino substituent and nitrogen atoms on the
quinoline ring play a pivotal role in the drug efficacy. It is beneficial for
the inhibitory activity of quinazoline EGFR-TKI that the radiolabeling
of icotinib with the ‘click reaction’ between azides and alkynes to in-
troduce of 1,2,3-triazoles to the 3′-position of icotinib molecules. So,
0
.24 ± 0.02 (n = 6) and 0.27 ± 0.04 (n = 6), respectively.
1
8
F-icotinib was synthesized using the efficient click reaction.
Fig. 6. MicroPET/CT images acquired at 1.5 h post-injection with 1.3–1.8 MBq 18F-icotinib (7) in A549 xenograft mice anesthetized with 5% isoflurane.
5

X. Lu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 7. Immunohistochemical staining of A549 tumors with anti-EGFR antibody. (a) Tumors in mice injected with 18F-icotinib (7); (b) tumors in mice co-injected
with 18F-icotinib (7) and icotinib, (original magnification, ×200).
According to the results given by the condition experiments in
Table 1, it was evident that higher radiolabeling yield can be obtained
by increasing reaction temperature, dosage of catalyst and concentra-
tion of icotinib. In short, thanks to the great virtue of click reaction, the
results in both chemistry and radiochemistry process were extremely
satisfying and the radiochemical yield (44.81%) and specific activity
has great potential to be translated into application in clinic as a reli-
able tool for tumor imaging.
Acknowledgement and declarations
None.
(
34 GBq/μmol) were also acceptable. From all aspects, it did help a lot
to improve the nuclide utilization and lower the initial radioactivity of
Funding
1
8 −
F
.
1
8
The Log P value of F-icotinib was 1.28 ± 0.04 (n = 6), which
This study was financially supported by grants from the National
Nature Science Foundation of China (NSFC) under Contract NO.
11505269 and the Strategic Priority and Research Program of Chinese
Academy of Science under Contract NO. XDA02000000. The authors
also acknowledge Shanghai Atom KeXing pharmaceutical Co., Ltd. for
meant that the probe is a little lipophilic. And by all means, EGFR-TKIs
drugs or probes generally reach their targets by passive diffusion
through the cell membrane. Therefore the lipophilicity is generally
1
8
necessary. Compared with F-FEA-erlotinib reported recently, which
1
8
12 18
18 −
was also labeled with F via CuAAC reaction,
F-icotinib demon-
providing
F .
strated reduced radioactivity accumulation in the liver at their own
observation time point (0.96 ± 0.21%ID/g at 90 min p.i. to
Appendix A. Supplementary data
1
5.2 ± 3.7 %ID/g at 60 min p.i.), likely due to the lower lipophilicity
18 18 18
of F-icotinib (Log P value of F-FEA-erlotinib was 2.36 ± 0.01). F-
icotinib kept totally intact after 4 h incubation. No defluorination or
radioactive metabolic degradation was observed, which reflected the
great in vitro stability of our probe. The cell assays demonstrated that
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2018.12.034.
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