Synthesis and evaluation of novel 18F-labeled quinazoline derivatives with low lipophilicity for tumor PET imaging

Article abstract of DOI:10.1002/jlcr.3538

Four novel ¹⁸F-labeled quinazoline derivatives with low lipophilicity, [¹⁸F]4-(2-fluoroethoxy)-6,7-dimethoxyquinazoline ([¹⁸F]I), [¹⁸F]4-(3-((4-(2-fluoroethoxy)-7-methoxyquinazolin-6-yl)oxy)propyl)morpholine ([¹⁸F]II), [¹⁸F]4-(2-fluoroethoxy)-7-methoxy-6-(2-methoxyethoxy)quinazoline ([¹⁸F]III), and [¹⁸F]4-(2-fluoroethoxy)-6,7-bis(2-methoxyethoxy)quinazoline ([¹⁸F]IV), were synthesized via a 2-step radiosynthesis procedure with an overall radiochemical yield of 10% to 38% (without decay correction) and radiochemical purities of >98%. The lipophilicity and stability of labeled compounds were tested in vitro. The log P values of the 4 radiotracers ranged from 0.52 to 1.07. We then performed ELISA to measure their affinities to EGFR-TK; ELISA assay results indicated that each inhibitor was specifically bounded to EGFR-TK in a dose-dependent manner. The EGFR-TK autophosphorylation IC₅₀ values of [¹⁸F]I, [¹⁸F]II, [¹⁸F]III, and [¹⁸F]IV were 7.732, 0.4698, 0.1174, and 0.1176?μM, respectively. All labeled compounds were evaluated via cellular uptake and blocking studies in HepG2 cell lines in vitro. Cellular uptake and blocking experiment results indicated that [¹⁸F]I and [¹⁸F]III had excellent cellular uptake at 120-minute postinjection in HepG2 carcinoma cells (51.80?±?3.42%ID/mg protein and 27.31?±?1.94%ID/mg protein, respectively). Additionally, biodistribution experiments in S180 tumor-bearing mice in vivo indicated that [¹⁸F]I had a very fast clearance in blood and a relatively high uptake ratio of tumor to blood (4.76) and tumor to muscle (1.82) at 60-minute postinjection. [¹⁸F]III had a quick clearance in plasma, and its highest uptake ratio of tumor to muscle was 2.55 at 15-minute postinjection. These experimental results and experiences were valuable for the further exploration of novel radiotracers of quinazoline derivatives.

Full text of DOI:10.1002/jlcr.3538

Synthesis and evaluation of novel F-18 labeled
quinazoline derivatives with low lipophilicity for
tumor PET imaging
Yan Chong, Jin Chang, Wenwen Zhao, Yong He, Yuqiao Li, Huabei
Zhang, and Chuanmin Qi*
Key Laboratory of Radiopharmaceuticals, College of Chemistry, Beijing Normal University,
Beijing 100875, People’s Republic of China
Corresponding Author: Tel. +86-136 8138 0097; Fax. +86-10-58802075;
E-mail: qicmin@sohu.com
ABSTRACT
Four novel F-18 labeled quinazoline derivatives with low lipophilicity, [18F]4-(2-
fluoroethoxy)-6,7-dimethoxyquinazoline
([18F]I),
([18F]II),
[18F]4-(3-((4-(2-fluoroethoxy)-7-
methoxyquinazolin-6-yl)oxy)propyl)morpholine
[18F]4-(2-fluoroethoxy)-7-
methoxy-6-(2-methoxyethoxy)quinazoline ([18F]III) and [18F]4-(2-fluoroethoxy)-6,7-bis(2-
methoxyethoxy)quinazoline ([18F]IV), were synthesized via a two-step radiosynthesis
procedure with an overall radiochemical yield of 10-38% (without decay correction) and
radiochemical purities of > 98%. The lipophilicity and stability of labeled compounds were
tested in vitro. The log P values of the four radiotracers ranged from 0.52 to 1.07. We then
performed ELISA to measure their affinities to EGFR-TK. ELISA assay results indicated that
each inhibitor was specifically bound to EGFR-TK in a dose-dependent manner. The EGFR-
TK autophosphorylation IC50 values of [18F]I, [18F]II, [18F]III, and [18F]IV were 7.732 μM,
0.4698 μM, 0.1174 μM, and 0.1176 μM, respectively. All labeled compounds were evaluated
via cellular uptake and blocking studies in HepG2 cell lines in vitro. Cellular uptake and
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doi: 10.1002/jlcr.3538
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blocking experiment results indicated that [18F]I and [18F]III had excellent cellular uptake at
120 min post-injection in HepG2 carcinoma cells (51.80±3.42 %ID/mg protein and
27.31±1.94 %ID/mg protein, respectively). Additionally, biodistribution experiments in S180
tumor-bearing mice in vivo indicated that [18F]I had a very fast clearance in blood and a
relatively high uptake ratio of tumor to blood (4.76) and tumor to muscle (1.82) at 60 min
post-injection. [18F]III had a quick clearance in plasma, and its highest uptake ratio of tumor
to muscle was 2.55 at 15 min post-injection. These experimental results and experiences were
valuable for the further exploration of novel radiotracers of quinazoline derivatives.
Key Words: fluoride-18; quinazoline derivatives; EGFR; PET imaging probes
1. Introduction
The epidermal growth factor receptor (EGFR) has been reported to be closely related to
human tumor diseases1. Nearly 80 percent of cancer cell lines have an over-expression of
EGFR2. The activation of EGFR by specific ligands could influence the RAS-ERK and PI3K-
AKT signal transduction pathways1. Consequently, this will cause abnormalities in cell
division, proliferation, survival, apoptosis, migration and other biological reactions.
Quinazoline derivative is one of EGFR tyrosine kinase inhibitor found in the early stage3.
This type of small molecular inhibitor has outstanding specific binding affinities to EGFR
and has attracted the attention of many researchers. Therefore, many EGFR tyrosine kinase
inhibitors of quinazoline derivatives, such as Gefitinib4,5, Erlotinib6, Lapatinib7, Vandetanib8,
and Afatinib9, have been approved to be used for the treatment of cancer, especially non-
small cell lung cancer (NSCLC), in recent decades.
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With the rapid development of nuclear medical technology, positron emission tomography
(PET) has become a noninvasive and reliable method with which to diagnose cancer10.
Among positron nuclides, fluoride-18 has been favored as a medical PET nuclide for its
convenient half-life (109.8 min) and emission energy (635 keV)11,12. Moreover, researchers
have tried to find appropriate F-18 labeled radiotracers to realize the accurate diagnosis of
tumor diseases in an early stage. To date, the most successful and commonly used F-18
labeled tumor PET imaging probe is [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG)13, which
has been used to reflect glucose metabolism in tumor cells. Many researchers have developed
additional novel PET radiotracers for the diagnosis of specific tumor diseases, e.g., [18F]3’-
deoxy-3’-fluorothymidine ([18F]FLT)14,15, [18F]O-(2-fluoroethyl-L-tyrosine ([18F]FET)16, and
[18F] fluoro-misonidazole ([18F]FMISO)17.
Due to the high affinity of quinazoline derivatives to over-expressed EGFR in tumor cell
lines, researchers have attempted to design radioisotope labeled quinazoline derivatives as
radiotracers to target EGFR for the diagnosis of tumor diseases18. [11C]Gefitinib19,
[11C]Erlotinib20, [18F]Gefitinib21-23, [18F]Afatinib20 and other tumor PET imaging probes of
quinazoline derivatives have been developed and evaluated. However, these radiotracers
encountered some drawbacks in PET imaging: low tumor uptake, poor solubility and high
non-specific binding18. Therefore, we tried to develop novel F-18 labeled quinazoline
derivatives in order to overcome these shortcomings.
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Scheme 1 Structures of some radioisotope labeled quinazoline derivatives
Helen Su et al. revealed the intrinsic reason for the poor PET imaging effect of F-18
labeled quinazoline derivatives with their research on [18F]Gefitinib24. They concluded that 4-
anilinoquinazoline derivatives had rather high lipophilicity, which would result in
considerable non-specific binding on the lipophilic cell membrane and low clearance in
plasma. Consequently, the low uptake ratio of tumor to background would hinder the use of
this type of probe as a promising PET imaging radiotracer due to the high lipophilicity.
Enlightened by their thesis and conclusion, we attempted to improve the water solubility of
quinazoline derivatives to decrease the non-specific binding in vivo and increase the
clearance rate in plasma. Because the 4-anilino group severely influenced the lipophilicity of
quinazoline derivatives, we replaced this group with a 2-fluoroethoxy group to increase the
hydrophilicity. Although this substitution may weaken the specific binding ability of
quinazoline derivatives to EGFR, we anticipate that our novel radiotracers will have a rapid
clearance in plasma to increase the uptake ratio of tumor to background. Therefore, we finally
designed and synthesized four F-18 labeled quinazoline derivatives: [18F]4-(2-fluoroethoxy)-
6,7-dimethoxyquinazoline ([18F]I), [18F]4-(3-((4-(2-fluoroethoxy)-7-methoxyquinazolin-6-
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yl)oxy)propyl)morpholine
methoxyethoxy)quinazoline
([18F]II),
[18F]4-(2-fluoroethoxy)-7-methoxy-6-(2-
and
[18F]4-(2-fluoroethoxy)-6,7-bis(2-
([18F]III)
methoxyethoxy)quinazoline ([18F]IV). Then, we performed biological activity studies and
evaluated these radiotracers rationally.
2. Results and Discussion
2.1. Chemistry
We referred to our previous article (Shilei Li et al.)25 and simply optimized the synthesis
methods of I, II, III, and IV (Scheme 1-4). Compound I was prepared through a five-step
synthesis procedure as shown in Scheme 1. Compound 5 was obtained through a series of
reactions that included esterification, nitration, hydrogenation reduction and cyclization with
formamide. Compound 5 was the precursor of radiotracer [18F]I. It was substituted by 2-
fluoroethyl tosylate with K2CO3 as a base to produce compound I. The 2-fluoroethyl tosylate
(Compound 6) was prepared via the tosylation of 2-fluoroethanol with KOH as a strong base.
Similarly, compound II was also obtained via a 2-fluoroethyl tosylate substitution with
compound 7 (Scheme 2). Compounds III and IV were prepared in a similar fashion (Scheme
3-4). After the initial 1-bromo-2-methoxyethane substitution reaction, the remainder of the
synthesis section was similar to the five-step synthesis method of compound I. The overall
synthesis process could be finished efficiently with high yields and less purification.
Precursors for radiosynthesis (Compounds 5, 7, 13, 18) could also be easily prepared with
high yields.
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Scheme 2 Synthesis of compound I. Reagents and conditions: (a) EtOH, conc. H2SO4, Reflux; (b) 65% HNO3, CH3COOH,
0C; (c) H2, Pd/C, CH3OH, 40C; (d) HCONH2, 160-170C; (e) TsOCH2CH2F (Compound 6), DMF, K2CO3, 90-100C.
Scheme 3 Synthesis of compound II. Reagents and conditions: (a) TsOCH2CH2F (Compound 6), DMF, K2CO3, 90-100C.
Scheme 4 Synthesis of compound III. Reagents and conditions: (a) CH3CH2OH, conc. H2SO4, Reflux; (b)
CH3OCH2CH2OBr, Bu4NBr, K2CO3, CH3CN, Reflux; (c) 65% HNO3, CH3COOH, 0C; (d) H2, Pd/C, CH3OH, 40C; (e)
HCONH2, 160-170C; (f) TsOCH2CH2F, DMF, K2CO3, 90-100C.
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Scheme 5 Synthesis of compound IV. Reagents and conditions: (a) CH3OCH2CH2OBr, Bu4NBr, K2CO3, CH3CN, Reflux;
(b) 65% HNO3, CH3COOH, 0C; (c) H2, Pd/C, CH3OH, 40C; (d) HCONH2, 160-170C; (e) TsOCH2CH2F, DMF, K2CO3,
90-100C.
2.2. Radiochemistry
Scheme 6 Radiochemical synthesis of [18F]I, [18F]II, [18F]III, and [18F]IV. Reagents and conditions: (a) K18F/K2.2.2
anhydrous CH3CN, K2CO3, 100C, 15 min; (b) Anhydrous DMF, K2CO3, 100C, 15-30 min.
,
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All radiotracers were prepared with the [18F]KF-K2.2.2 complex via the same two-step
labeling procedure as shown in Scheme 5. The procedure included nucleophilic fluoro-
substitution of glycol-1,2-ditosylate and the substitution of 2-[18F]fluoroethyl tosylate with
precursors (Compounds 5, 7, 13, 18). The radiosynthesis procedure, including HPLC
separation, could be finished within 90 min. The overall radioactivity yield of these four
labeled products without decay correction was approximately 10%-38% and radiochemical
purities were all above 98%. The molar activity of four labeled compounds ranged from 61.0-
69.5 GBq/μmol. All related HPLC data and RCY of [18F]I, [18F]II, [18F]III, and [18F]IV are
summarized in Table 1.
Table 1. Retention time, HPLC conditions and overall radioactivity yield of I, II, III, and IV.
Retention
Time (min)
11.4
Radio-HPLC Conditions (solvent,
flow rate)
CH3OH:H2O=60:40[v:v], 2 mL/min
same as above
The Overall
Radioactivity Yield*
28.1%
Compounds
[18F]I
[18F]II
[18F]III
[18F]IV
13.6
11.8
12.8
10.9%
37.9%
36.7%
same as above
same as above
*Without decay correction
2.3. Stability in vitro
The radio-HPLC analysis results indicated that all labeled compounds were stable for at
least 2 hours in bovine serum at 37C without radioautolysis. Related radio-HPLC of labeled
compounds is shown in the supplemental materials.
2.4. Partition Coefficients
Partition coefficients (log P) of the four labeled compounds are shown in Table 2. The log
P values of the four radiotracers ranged from 0.52 to 1.07. Compared with the log P value of
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[18F]Gefitinib (3.46) reported in related article24, all labeled compounds had noticeably
decreased lipophilicity as we expected.
Table 2. Partition coefficient values of [18F]I, [18F]II, [18F]III, and [18F]IV (log P value, mean±SD,
n=3).
Compound
[18F]I
Partition coefficient (log P)
1.07±0.03
[18F]II
0.52±0.02
0.97±0.02
0.80±0.07
[18F]III
[18F]IV
2.5 ELISA
We performed ELISA to evaluate biological activities of target compounds. We also
selected Gefitinib as a comparative inhibitor of EGFR-TK in this experiment. The blocking
rate-dose curves and IC50 values of inhibitors of EGFR-TK are shown in Figure 1 and Table
3, respectively.
Figure 1. Blocking rate-inhibitor dose curves of ELISA (n=4): red line: I, green line: II, blue line:
III, purple line: IV, and black line: Gefitinib
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Table 3. The IC50 values of inhibitors of EGFR tyrosine kinase (95% confidence interval is shown in
parentheses, n=4).
Inhibitor
IC50 value of EGFR-TK autophosphorylation (mol/L)
7.732×10-6 (3.246×10-6-1.842×10-5)
4.698×10-7 (2.011×10-7-1.097×10-6)
1.174×10-7 (4.577×10-8-3.013×10-7)
1.176×10-7 (4.806×10-8-2.875×10-7)
1.347×10-10 (7.802×10-11-2.325×10-10)
I
II
III
IV
Gefitinib
Generally, the blocking rate of all inhibitors appeared to be dose-dependent. The results
also demonstrated that all inhibitors were specifically bound to EGFR-TK. The IC50 values of
EGFR-TK autophosphorylation for compounds I, II, III and IV were 7.732 μM, 0.4698 μM,
0.1174 μM, and 0.1176 μM, respectively. The four inhibitors without a 4-anilino group on the
quinazoline ring still retained specific binding abilities to the target receptor to some extent.
However, their affinities for EGFR-TK were lower than that of Gefitinib, which had an IC50
value of 0.1347 nM. This finding indicated that the 4-anilino group on quinazoline
derivatives made a significant contribution to the specific binding to EGFR-TK. Replacing
the anilino group with a 2-fluoroethoxy group would sacrifice the inhibitor’s affinity to
EGFR. However, we were searching for quinazoline derivatives with low lipophilicity, which
could make them to have a faster clearance rate in the blood and less non-specific binding.
We had to make this change though it would actually lead to a decreased affinity for the
receptor.
Compound III had the lowest IC50 value of EGFR-TK autophosphorylation among the four
inhibitors we synthesized. Compound IV had nearly the same biological activity as
compound III. This result indicated that replacing methoxy with 2-methoxyethoxy at the C-7
position of quinazoline hardly influenced the affinity of 4-(2-fluoroethoxy)-quinazoline
derivatives. However, the substituent group at the C-6 position of quinazoline could influence
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the IC50 value. Compounds I and II were less potent than compound III. We supposed that
the length of the substituent group at the C-6 position should not be too long or too short. The
lipophilicity of the substituent group at the C-6 position was irrelevant to the affinity of 4-(2-
fluoroethoxy)-quinazoline derivatives.
2.6. Cellular Uptake and Blocking Studies
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Figure 2. Cellular uptake and blocking studies of the radiotracers [18F]I (a), [18F]II (b), [18F]III (c)
and [18F]IV (d) in HepG2 human hepatocellular carcinoma cells (n=4).
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The cellular uptake and blocking studies were performed in HepG2 human hepatocellular
carcinoma cells, which had a medium level of EGFR over-expression. According to the
cellular uptake results of each labeled compound (Figure 2a-d), [18F]I and [18F]III
noticeably increased uptake in 2 hours. The highest cellular uptake values of [18F]I and
[18F]III were 51.80±3.42 %ID/mg and 27.31±1.94 %ID/mg protein, respectively, at 120 min.
[18F]I and [18F]III had excellent cellular association with HepG2 cell lines. In contrast,
[18F]IV showed a declining trend in cellular uptake as time elapsed. The highest cellular
uptake value was only 5.85±0.43 %ID/mg protein within the first 15 min. We supposed that
[18F]IV had better hydrophilicity and, therefore, did not penetrate the lipophilic cell
membrane well. Additionally, [18F]IV had poor stability in organisms according to the latter
result of the biodistribution experiment. These potential factors resulted in a decreased
cellular uptake of [18F]IV. Similarly, [18F]II had the weakest cellular uptake because it had
the lowest lipophilicity.
According to blocking experiments results, compounds [18F]I, [18F]III and [18F]IV
significantly reduced cellular uptake when they were incubated with 10 μM Gefitinib, an
EGFR tyrosine kinase inhibitor. The blocking percentages of Gefitinib to these three
radiotracers were often over 50%. This finding indicated that Gefitinib had a competitive
relationship with [18F]I, [18F]III and [18F]IV in terms of cellular association. To some extent,
it also demonstrated that [18F]I, [18F]III and [18F]IV bound specifically to EGFR in HepG2
carcinoma cells. However, we did not observe an obvious decline in the cellular uptake of
[18F]II when it was incubated with a 10 μM inhibitor. The cellular uptake value changed
slightly and was maintained at a very low level, which suggested that [18F]II had high non-
specific binding to EGFR in HepG2 cells. We concluded that [18F]II had the lowest
lipophilicity (log P=0.52), which hindered the ability of the molecule to reach the target
receptor inside of cell membrane and resulted in a high non-specific cellular association.
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2.7. Biodistribution in S180 Tumor-Bearing Mice
Table 3. Biodistribution data of [18F]I in S180 tumor-bearing mice (%ID/g, mean±SD, n=4).
Organ
Blood
Heart
Liver
Spleen
5 min
5.31±0.07
3.47±0.45
6.02±0.92
2.62±0.46
4.20±0.83
11.90±2.13
2.35±0.27
2.15±0.68
1.88±0.33
2.83±0.52
1.50±0.64
3.26±0.12
1.15
15 min
2.27±1.03
2.15±0.06
2.06±0.79
0.89±0.59
1.46±0.59
4.95±2.15
5.92±2.74
0.30±0.11
0.93±0.49
0.87±0.56
0.53±0.26
1.58±1.12
1.81
30 min
1.45±0.44
1.13±0.23
0.84±0.25
0.74±0.33
0.92±0.29
1.49±0.61
7.55±1.11
0.82±0.10
2.75±0.12
1.15±0.15
0.70±0.25
1.82±0.04
1.59
60 min
0.23±0.05
1.10±0.29
0.65±0.19
0.39±0.04
0.70±0.26
1.07±0.33
2.28±1.43
0.48±0.23
1.89±0.74
0.60±0.09
0.75±0.36
1.09±0.31
1.82
120 min
0.58±0.18
0.28±0.07
0.11±0.02
0.17±0.09
0.25±0.06
0.37±0.05
0.74±0.33
0.21±0.08
1.86±0.88
0.66±0.31
0.18±0.10
0.52±0.06
0.79
Lung
Kidney
Intestine
Stomach
Bone
Muscle
Brain
Tumor
Tumor/Muscle
Tumor/Blood
0.61
0.70
1.26
4.76
0.90
Table 4. Biodistribution data of [18F]II in S180 tumor-bearing mice (%ID/g, mean±SD, n=4).
Organ
Blood
Heart
Liver
Spleen
5 min
2.30±0.68
3.36±1.06
10.03±2.94
5.89±1.55
4.38±1.11
7.77±2.02
2.40±0.83
8.72±0.69
2.47±1.23
2.61±0.87
0.58±0.28
1.56±1.09
0.60
15 min
1.51±0.31
1.86±0.61
9.45±0.07
4.90±0.06
4.90±0.46
4.60±1.10
7.53±2.64
8.52±0.92
4.07±0.81
1.56±0.23
0.38±0.14
1.87±0.39
1.20
30 min
1.28±0.18
1.49±0.31
6.61±0.27
2.29±0.63
1.47±0.27
3.35±0.49
9.28±2.44
11.09±0.37
2.40±1.29
1.22±0.32
0.34±0.04
1.90±0.27
1.56
60 min
120 min
0.52±0.05
0.51±0.10
1.53±0.60
0.49±0.12
0.58±0.05
1.39±0.26
0.81±0.17
0.92±0.20
3.28±0.35
1.08±0.51
1.05±0.31
1.62±0.55
Lung
Kidney
Intestine
Stomach
Bone
Muscle
Brain
20.77±0.57 31.79±1.57
4.43±2.29
2.40±1.41
0.76±0.19
0.24±0.06
1.06±0.30
1.40
1.41±0.18
3.45±0.25
0.43±0.12
0.26±0.09
0.92±0.11
2.14
Tumor
Tumor/Muscle
Tumor/Blood
0.83
1.24
1.49
1.31
1.76
Table 5. Biodistribution data of [18F]III in S180 tumor-bearing mice (%ID/g, mean±SD, n=4).
Organ
Blood
Heart
Liver
Spleen
Lung
Kidney
Intestine
Stomach
Bone
5 min
15 min
30 min
60 min
120 min
0.68±0.13
0.73±0.24
0.44±0.36
0.46±0.13
1.74±0.09
0.49±0.10
1.10±0.64
0.46±0.14
1.74±0.67
0.45±0.09
2.93±0.43
2.41±0.06
2.95±0.72
1.61±0.18
3.12±0.35
3.13±1.36
1.83±0.07
1.47±0.27
2.21±0.50
1.87±0.15
1.77±0.27
1.58±0.30
1.24±0.61
1.03±0.25
1.68±0.20
1.82±0.59
3.30±0.53
1.67±0.52
1.52±0.80
0.87±0.27
1.18±0.11
1.24±0.11
1.00±0.12
0.74±0.11
5.32±0.16
1.22±0.15
4.95±0.10
0.68±0.14
1.98±0.43
0.84±0.13
1.12±0.05
1.01±0.23
0.46±0.02
0.51±0.06
1.76±0.02
0.68±0.16
2.12±0.22
1.80±1.14
1.93±1.33
0.86±0.14
Muscle
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Brain
Tumor
Tumor/Muscle
Tumor/Blood
1.03±0.18
2.05±0.20
1.10
0.76±0.14
2.23±0.49
2.55
0.63±0.08
1.52±0.06
1.81
0.44±0.13
1.22±0.32
1.43
0.45±0.11
0.94±0.17
2.08
0.70
1.25
1.29
1.09
1.31
Table 6. Biodistribution data of [18F]IV in S180 tumor-bearing mice (%ID/g, mean±SD, n=4).
Organ
Blood
Heart
Liver
Spleen
5 min
15 min
4.03±0.58
3.39±0.14
3.35±0.75
2.29±1.09
3.23±0.47
2.71±0.50
2.62±1.09
2.55±0.71
3.35±0.34
2.49±0.55
1.92±0.04
3.52±0.14
1.41
30 min
3.23±0.12
3.08±0.18
2.34±0.46
2.30±0.04
4.14±0.53
1.89±0.65
2.97±0.47
1.35±0.17
2.87±0.67
2.43±0.15
1.86±0.13
4.07±1.13
1.67
60 min
3.56±0.32
3.33±0.45
2.08±0.81
2.04±0.69
3.11±0.35
2.12±0.25
3.73±1.25
1.62±0.46
8.54±2.86
1.93±0.32
2.10±0.28
4.47±0.58
2.32
120 min
1.81±0.14
1.69±0.44
1.63±0.19
1.28±0.24
2.75±0.61
1.31±0.22
1.61±0.07
0.96±0.33
10.31±0.88
1.65±0.21
1.22±0.14
2.64±0.30
1.60
3.94±0.66
3.16±0.98
2.32±0.62
3.01±0.47
3.36±1.13
3.51±0.25
3.70±0.30
1.94±0.55
2.57±1.09
2.47±0.75
1.20±0.24
3.43±0.50
1.39
Lung
Kidney
Intestine
Stomach
Bone
Muscle
Brain
Tumor
Tumor/Muscle
Tumor/Blood
0.87
0.87
1.26
1.26
1.46
The biodistribution experiments of all radiotracers were executed in S180 tumor-bearing
mice. According to the biodistribution data summarized in Table 3-6, all radiotracers had
high initial uptake in the kidney and liver, which then quickly decreased as time elapsed. In
addition, we observed an obvious accumulation in the intestine. Nearly all other organs and
tissues had the highest uptake 5 min after the injection, which then consecutively declined as
time passed.
[18F]I had the highest tumor uptake (3.26±0.12 %ID/g) during the initial 5 min, and then,
[18F]I cleared quickly as time passed. Although this radiotracer did not have an accumulation
process in the tumor, it actually had a much faster clearance rate in blood than in the tumor so
that it could have a relatively high tumor to blood ratio (approximately 4.76) at 60 min post-
injection. The tumor to muscle uptake ratio value was highest (1.82) at the same time. In
conclusion, 60 min post-injection should be the best imaging time of [18F]I. This compound
has the potential to be an EGFR targeted tumor radiotracer.
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In contrast to [18F]I, we found that [18F]II accumulated in the tumor. The maximum uptake
value of [18F]II was 1.90±0.27 %ID/g at 30 min post-injection. Compared with [18F]I, the
lower lipophilicity of [18F]II did not contribute to an increase in clearance rate in the blood
but diminished tumor absorption. The uptake ratios of tumor to muscle and tumor to blood
were often less than 2, which suggested that [18F]II was not suitable as a PET tumor imaging
probe.
[18F]III had a maximum uptake value of 2.23±0.49 %ID/g at 15 min post-injection and the
tumor uptake remained above 1.50 %ID/g in the initial 30 min. However, the uptake in the
tumor was low due to the increased hydrophilicity. The clearance of [18F]III in blood was
quite fast so that the uptake ratio of tumor to blood remained steady 15 min post-injection.
The highest uptake ratio of tumor to muscle was 2.55 at 15 min post-injection. Therefore, the
15 min post-injection time point should be appropriate for tumor imaging.
For the radiotracer [18F]IV, we observed that the bone uptake of [18F]IV noticeably
increased as time elapsed. This result indicated that the [18F]IV radiotracer had severe
defluorination in vivo. The accumulation of free fluoride-18 in bones led to an increase in
radioactivity. However, we the reason why only [18F]IV had severe defluorination in vivo is
unknown. Propose that a substitution group at the C-7 position had an influence on the
molecular stability or metabolism in vivo. Although [18F]IV had the highest tumor uptake
value among all four labeled compounds, it could not serve as a tumor imaging probe due to
poor stability in vivo.
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3. Experimental Section
3.1 General
All chemical reagents and solvents were commercially available. Cell culture medium,
other related biological evaluation and experimental reagents and kits were purchased from
Yeasen Biological Inc in Shanghai. No-carrier-added fluoride-18 was provided by the PET
Center of Xuanwu Hospital and The General Hospital of the People’s Liberation Army via
the 18O(p, n)18F nuclear reaction. Semi-preparative HPLC reversed-phase columns (C18
column, 250×10 mm, particle size: 10 μm) were purchased from Agela™ Venusil. HPLC
separation was carried out on a Shimadzu™ LC-20A High-Performance Liquid
Chromatography system. C18 Sep-Pak Cartridges were purchased from Waters™ and
activated with 2 mL of methanol and 2×10 mL of water before use. Melting points were
measured in capillary tubes with an RY-1 melting point apparatus and were uncorrected.
Nuclear magnetic resonance spectra (1H and 13C-NMR) were performed on a Bruker™
1
13
spectrometer (400 MHz for H-NMR and 100 MHz for C-NMR), and chemical shifts (δ
values, ppm) were reported downfield from TMS. Mass spectra were measured with a
Bruker™ Apex IV FTM instrument using a low-resolution electrospray ionization (ESI)
source at the Mass Spectroscopy Center of Beijing Normal University. The radioactive
counts were measured with a Wizard™ 1470 Gamma Counter, and the radioactivities were
obtained via an RM-905a Radioactivity Meter. HepG2 hepatocellular carcinoma cells were
purchased from the Cancer Hospital of the Chinese Academy of Medical Science and
cultured in DMEM (including 10% FBS) in 5% CO2 atmosphere at 37C. The ICR mice were
supported by the Experimental Animal Department of Peking University Health Science
Center.
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3.2. Synthesis
The synthesis methods of the intermediate products (Compounds 1-18) are described in a
1
13
related reference25. The target compounds’ chemical synthesis method and H-NMR, C-
NMR and MS data of target compounds are shown as below. Related spectra are shown in
the supplemental data.
Preparation of 4-(2-fluoroethoxy)-6,7-dimethoxyquinazoline (I). Compound 5 (206 mg, 1.0
mmol) and compound 6 (432 mg, 2.0 mmol) were added to 5 mL of DMF. The reaction
solution was stirred at 90-100C for 2 hours. The solution was poured into 50 mL of ice
water under stirring and then extracted with 10 mL of ethyl acetate (10mL×8). The organic
layer was washed with saturated NaHCO3 solution (10 mL×1), saturated NaCl solution (10
mL×1) and distilled water (10 mL×2) in sequence and dried with Na2SO4. The solvent was
removed under vacuum conditions to obtain a light yellow solid. The crude product was
recrystallized from ethyl acetate and petroleum ether (v:v=3:1) to yield a light yellow target
1
product (45 mg, 18% yield). m.p. 168-169C. H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H,
ArH), 7.63 (s, 1H, ArH), 7.14 (s, 1H, ArH), 4.77 (dt, J= 47.2 Hz, 4.4 Hz, 2H, -CH2-), 4.33 (dt,
J=27.2 Hz, 4.4 Hz, 2H, -CH2-), 4.03 (s, 6H, -OCH3). 13C NMR (100 MHz, CDCl3) δ 160.94
(s), 155.57 (s), 149.98 (s), 146.01 (s), 145.06 (s), 115.86 (s), 108.51 (s), 105.98 (s), 81.75 (d,
J=168.8 Hz), 56.846 (s), 47.71 (d, J=19.5 Hz). MS (ESI+) m/z: 253.09 [M+H]+.
Preparation of 4-(3-((4-(2-fluoroethoxy)-7-methoxyquinazolin-6-yl)oxy)propyl)-morpholine
(II). The synthesis method of compound II was similar to the preparation procedure of
compound I. The initial compound 7 (0.319 g, 1.0 mmol) was commercially available.
Additionally, the product could be recrystallized from methanol to afford pure product as a
white solid (100 mg, 27% yield). m.p. 127-128C. 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H,
ArH), 7.55 (s, 1H, ArH), 7.03 (s, 1H, ArH), 4.67 (dt, J=47.1 Hz, 4.4 Hz, 2H, -CH2-), 4.22 (dt,
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J=27.2 Hz, 4.6 Hz, 2H, -CH2-), 4.13 (m, J=6.72 Hz, 2H, -CH2-), 3.90 (s, 3H, -OCH3), 3.65 (s,
4H, -CH2-), 2.47 (m, J=6.6 Hz, 2H, -CH2-), 2.38 (d, J=7.24 Hz, 4H, -CH2-), 2.00 (m, J=6.88
Hz, 4H, -CH2-). 13C NMR (100 MHz, CDCl3) δ 160.96 (s), 155.86 (s), 149.41 (s), 145.95 (s),
144.95 (s), 115.80 (s), 108.59 (s), 107.10 (s), 81.75 (d, J=168.8 Hz), 67.49 (s), 56.79 (s),
55.86 (s), 54.24 (s), 54.24 (s), 47.70 (d, J=19.5 Hz), 26.61 (s). MS (ESI+) m/z: 366.16
[M+H]+.
Preparation of 4-(2-fluoroethoxy)-7-methoxy-6-(2-methoxy-ethoxy)quinazoline (III). The
synthesis method of compound III was similar to the preparation procedure of compound I.
The product was recrystallized from methanol to yield a cotton-like yellow solid (112 mg, 33%
yield), m.p. 141-142C. 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H, ArH), 7.54 (s, 1H, ArH),
7.03 (s, 1H, ArH), 4,66 (dt, J= 47.1 Hz, 4.4 Hz, 2H, -CH2-), 4.26 (m, J=4.6 Hz, 1H, -CH2-),
4.20 (m, J=4.6 Hz, 3H, -CH2-), 3.90 (s, 1H, -OCH3), 3.77 (m, J=3.84 Hz, -CH2-), 3.39 (s, 3H,
-OCH3). 13C NMR (100 MHz, CDCl3) δ 160.92 (s), 155.97 (s), 146.04 (s), 145.95 (s), 145.19
(s), 115.70 (s), 108.64 (s), 107.28 (s), 81.75 (d, J=168.7 Hz), 71.14 (s), 68.98 (s), 59.76 (s),
56.76 (s), 47.70 (d, J=19.5 Hz). MS (ESI+) m/z: 297.1 [M+H]+.
Preparation of 4-(2-fluoroethoxy)-6,7-bis(2-methoxyethoxy)quinazoline (IV). The synthesis
method of compound IV was similar to the preparation procedure of compound I. The
product was recrystallized from methanol to yield a white solid (56 mg, 16% yield). m.p.
118-119C. 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H, ArH), 7.54 (s, 1H, ArH), 7.03 (s, 1H,
ArH), 4.66 (dt, J= 47.1 Hz, 4.4 Hz, 2H, -CH2-), 4.26 (s, 1H, -CH2-), 4.18 (m, J=4.72 Hz, 5H,
-CH2-), 3.76(m, J=4.8 Hz, 4H, -CH2-), 3.39(s, 6H, -OCH3). 13C NMR (100 MHz, CDCl3) δ
160.90 (s), 155.28 (s), 146.04 (s), 145.93 (s), 146.01 (s), 145.07 (s), 115.94 (s), 109.83 (s),
107.86 (s), 81.76 (d, J=168.7 Hz), 71.08 (s), 69.10 (s), 59.87 (s), 47.70 (d, J=19.5 Hz).
MS(ESI+) m/z: 341.13 [M+H]+.
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3.3. Radiosynthesis
All radiotracers were prepared via the following labeling procedure. [18F]fluoride, which
was generated from the 18O(p, n)18F nuclear reaction, was captured by a QMA anion
exchange cartridge. Then, 4-8 mg K2.2.2 cryptate and 6-8 mg potassium carbonate were
dissolved in a mixture of the solvent of 1 mL acetonitrile and 0.5 mL water. The fluoride-18
anion was recovered by eluting the QMA anion exchange cartridge with the mixture solution.
The solvent was azeotroped under a nitrogen stream at 110-120C to dry the mixture. This
drying process was repeated no fewer than three times. A total of 1 mL of anhydrous
acetonitrile supplement was added into the vial after drying each time. Then, 2-4 mg of the
initial compound, glycol-1,2-ditosylate, was reacted with the [18F]KF-K2.2.2 cryptate complex
and potassium carbonate at 100C for 15 min in anhydrous acetonitrile solvent to yield 2-
[18F]fluoroethyl tosylate. When the reaction was finished and the acetonitrile solvent was
evaporated, 1-2 mg of purified precursor (Compound 5, 7, 13, 18) dissolved in anhydrous
DMF was co-heated with newly prepared 2-[18F]fluoroethyl tosylate at 100C for 15-30 min.
The reaction mixture was diluted with water and then passed through a C18 Sep-Pak
cartridge. After eluting with 10 mL of water to wash out excess radioactive fluoride, the
cartridge was finally eluted with 2 mL of acetonitrile to gather the labeled product. The
product solution was finally purified with a reversed-phase HPLC C18 Column (Agela™
Venusil, 250×10 mm, particle size: 10 μm, CH3OH:H2O=60:40[v:v], 2 mL/min) to separate
the desired F-18 labeled product at the retention time. The retention time of the four labeled
compound could refer to Table 1.
3.4. Plasma Stability Studies
The stability of all labeled compounds was tested by HPLC analysis via the same method.
A solution of the labeled product in saline (approximately 100 μCi) was incubated in 1 mL of
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bovine serum for 2 h at 37C. Then, the plasma was centrifuged for 3 min at 1000 rpm. The
supernatant was passed through a 0.22-μm filter membrane and the stability of the product
was analyzed via radio-HPLC with a reversed-phase HPLC C18 Column (Agela™ Venusil,
250×10 mm, particle size: 10 μm, CH3OH:H2O=60:40[v:v], 2 mL/min).
3.5. Partition Coefficients
The partition coefficients (log P) were measured via the following procedure. The log P
value was determined by the radioactivity of 1-octanol and PBS (0.01 M, pH 7.2-7.4) at the
same volume. Initially, 0.1 mL of the labeled compound (approximately 30-50 μCi) saline
solvent was added to 1.9 mL PBS. Then, 2 mL of a water phase solution was mixed with 2
mL of 1-octanol. The mixture solution was centrifuged for 5 min at 1000 rpm. After the
partition equilibrium, 0.2 mL of solution in the water and oil phase was collected to measure
the radioactivity with a gamma counter. The log P value was calculated by the following
formula: log P = log[(CPM(n-octanol)-CPM(background)) /(CPM(PBS)-CPM(background))].
3.6. ELISA
The ELISA was carried out based on the following procedure. A Corning 96-well plate
was first coated with 0.01 M PBS diluted Poly(Glu:Tyr) 4:1 peptide solution at 37C for 24 h.
Then, the solution was removed and the plate was washed with 0.01 M PBST (containing
0.05‰ Tween 20) 3 times. After the plate was dry, 1 μL of different concentrations of
inhibitor solutions (ranging from 10-2 to 10-9 M) were added. Then 49 μL of 1 μM ATP’s
HEPES-MnCl2 buffer solution was added to each well. Ten minutes later, 50 μL of 10 ng/mL
recombinant human EGFR HEPES buffer solution was added to each well to activate the
phosphorylation reaction. The plate was shaken under 37C for 2 h. When the reaction was
finished, the reaction solutions were removed and the plate was washed. A total of 100 μL of
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0.01 M PBST (including 0.5% BSA and 0.05‰ Tween 20) with P-Tyr-100 mouse mAb
(1:1000) solution was added to the wells and reacted at 37C. Forty-five minutes later, all
solutions were removed and the plate was washed again. Then, 100 μL of 0.01 M PBST
(including 0.5% BSA and 0.05‰ Tween 20) with diluted goat-anti-mouse peroxidase
conjugated antibody (1:1000) solution was added. The reaction was stopped by washing as
the previous step. Finally, 100 μL of o-phenylenediamine working solution (diluted with pH
5.4 citrate buffer) was added to each well and reacted for 30 minutes in the darkness. 50 μL
of 2 M H2SO4 was added to each well to stop the chromogenic reaction, and the OD value of
each well was measured with a microplate reader at 490 nm. The blocking rate (%) of
specific concentrations of the inhibitor was calculated from [OD(without inhibitor)-OD(with
inhibitor)]/[OD(without inhibitor)-OD(background)]*100. The IC50 value of each inhibitor
was fit by GraphPad Prism 5.0 software.
3.7. Cellular Uptake and Blocking Studies
To prepare the cellular uptake experiment, 1×105 HepG2 cells were inoculated in each well
of a 24-well plate and incubated for 24 h in a 5% CO2 atmosphere at 37C. For the cellular
uptake assay, 1 μCi of the labeled compound was added to each well and incubated at 37C
for specific times (15, 30, 45, 60, 75, 90, and 120 min). All media was then removed and the
cells were washed with cold PBS (containing 0.2% BSA). The cells in each well were lysed
with 1 M NaOH, the solution was collected and the radioactive counts were measured with a
gamma counter. For the blocking study, 1 μCi of the labeled compound and 10-5 M Gefitinib
inhibitor were simultaneously added to each well and incubated under the same conditions.
Then, the cells were lysed and collected. The cellular uptake values of radiotracers are shown
in terms of the percentage of the injection dose per microgram protein that the cell contained
with a decay correction normalized as the mean±SD (%ID/mg, n=4). The blocking
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percentage (%) at the specific time was calculated as [CPM(without inhibitor)-CPM(with
inhibitor)]/CPM(without inhibitor)*100. The cellular association figures of each labeled
compound were drawn with GraphPad Prism 5.0 software.
3.8. Biodistribution in S180 Tumor-Bearing Mice
Female ICR mice with S180 tumors were used for biodistribution experiments. At first,
ICR mice were inoculated with S180 tumor cells (approximate 5×106 cells) in saline solution
in the left forelimb by means of a subcutaneous injection. The experiment was performed one
week after the inoculation. Each tumor-bearing mouse was injected with 0.1 mL of
radiotracer saline solution (approximately 5 μCi) by means of a tail vein injection and
euthanized at 5, 15, 30, 60, or 120 min post-injection. Required organs and tissue were
excised and collected quickly. Then, radioactive counts of all organs and tissue were
measured with a gamma counter, and masses of organs and tissue were weighed with an
electronic analysis balance. The standard uptake values (SUVs) of radiotracers in organs and
tissue were shown in the form of the percentage of the injection dose per gram with decay
correction normalized as the mean±SD (%ID/g, n=4). SUVs (%) were calculated via the
formula SUV(organ or tissue) = CPM(organ or tissue)/[CPM(1% injection dose)*Mass(organ
or tissue)]*100.
4. Conclusions
To summarize, we successfully synthesized four novel radiotracers with low lipophilicity.
All inhibitors had specific binding affinities to EGFR that appeared to be dose-dependent in
ELISA. Due to the replacement of the 4-anilino group, the affinities to EGFR-TK of all these
inhibitors were weaker than that of Gefitinib. In the cellular uptake study, the two
radiotracers [18F]I and [18F]III, which had relatively high lipophilicity, exhibited high
cellular uptake in HepG2 cell lines. The blocking studies in HepG2 cell lines demonstrated
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that they were specifically bound to EGFR as well. In the biodistribution study, [18F]I and
[18F]III had relatively higher tumor uptake and better clearance rates in plasma than the other
two radiotracers. Overall, [18F]I was the most potential radiotracer for tumor imaging. From
this research, we also concluded that we should control the lipophilicity of the quinazoline
radiotracer at a proper level. We should balance the relationship between the specific binding
ability to the receptor and the clearance rate in non-target organs and tissue in vivo as well.
These conclusions will guide our concept of radiotracer molecular design in future research.
Furthermore, experiments and PET studies will be carried out to evaluate the potency of
[18F]I and other radiotracers that appear to be promising tumor imaging radiotracers.
Acknowledgements
We acknowledge with thanks the financial support from the National Natural Science
Foundation of China (No.21571021). We also appreciate the cyclotron operator teams at the
PET center of Xuanwu Hospital and The General Hospital of the People’s Liberation Army
for their support and technical assistance with radionuclides.
Conflicts of Interest
The authors declare no conflicts of interest.
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