A useful EGFR-TK ligand for tumor diagnosis with SPECT: development of radioiodinated 6-(3-morpholinopropoxy)-7-ethoxy-4-(3′-iodophenoxy)quinazoline

Objective Epidermal growth factor receptor tyrosine kinase (EGFR-TK) represents an attractive target for tumor diagnosis agents. Previously, radioiodinated 4-(3-iodophenoxy)-6,7-diethoxyquinazoline (PHY) was reported to possess good characteristics as a tumor imaging agent. We have explored the feasibility of developing tumor diagnosis ligands superior to radioiodinated PHY. Methods New phenoxyquinazoline derivatives were designed with various side chains introduced to the 6th position of PHY. The IC50 values of the new derivatives to interrupt EGFR-TK phosphorylation were evaluated and compared to well-known EGFR-TK inhibitors. Tumor uptake studies of the new 125I-labeled derivatives were conducted with A431 tumor-bearing mice. Selectivity and binding characteristics were analyzed by in vitro blocking studies and a binding assay. Furthermore, SPECT/CT scans were performed using A431 tumor-bearing mice. Results Six quinazoline derivatives were designed and synthesized, and among these, 6a–d were found to have relatively high EGFR-TK inhibitory potency. In tumor uptake studies, [125I]6a ([125I]PYK) was found to have the highest tumor uptake and longest retention in tumors. In contrast, [125I]PYK was rapidly cleared from peripheral tissues, resulting in a high tumor-to-tissue ratio 24 h after injection. Moreover, the EGFR-TK selectivity of [125I]PYK was confirmed by pretreatment experiments with specific EGFR-TK inhibitors. Furthermore, [125I]PYK provided clear SPECT images of tumors. Conclusions Radioiodinated PYK, one of the newly synthesized quinazoline derivatives, was found to be a desirable ligand for EGFR-TK SPECT imaging. [125I]PYK showed high tumor accumulation and selective EGFR-TK binding and also succeeded in delivering high contrast imaging of tumors. These favorable characteristics of [125I]PYK suggest that the 123I-labeled counterpart, [123I]PYK, would have great potential for diagnostic SPECT tumor imaging.


Introduction
Epidermal growth factor receptor (EGFR) plays a key role in signal transduction pathways that regulate fundamentally important cellular functions. EGFR consists of an outer membrane EGF binding site and an inner membrane tyrosine kinase domain (EGFR-TK). EGFR-TK phosphorylation is stimulated by epidermal growth factor (EGF) or transforming growth factor a (TGFa) binding to the extracellular ligand-binding domain of EGFR and subsequent receptor dimerization. The intracellular tyrosine residues then undergo phosphorylation, which is followed by second messenger signaling and subsequent downstream events that lead to cellular proliferation, differentiation, motility, adhesion, and apoptosis [1,2]. EGFR is often overexpressed in cancers [3][4][5][6], thus inhibition of EGFR-TK as a target for cancer chemotherapy has proven to be effective in both preclinical and clinical settings.
EGFR-TK represents an attractive target for the development of new antitumor agents [7,8]. Among the EGFR-TK inhibitors used or tested clinically as antitumor agents are the quinazoline derivatives ZD1839 (Astra-Zeneca) [9,10] and OSI774 (Pfizer) [10,11]. One of the most potent compounds in the EGFR-TK inhibitor series is the quinazoline derivative PD153035 [12,13], which exhibits well-defined selectivity and tremendously high affinity for EGFR-TK (K i : 5 pM) coupled with remarkable cytotoxicity against several types of tumors in vitro such as the A431 epidermoid carcinoma cell line (IC 50 : 29 pM). In an effort to develop new cancer diagnostic agents, several PET and SPECT ligands have been synthesized and evaluated for imaging of EGFR-TK. The feasibility of targeting EGFR for tumor imaging has been experimentally demonstrated using 18 F or 11 C-labeled EGFR-TK inhibitors [14][15][16][17][18][19][20]; however, these labeled compounds were also observed to produce high radioactivity in the blood and peripheral tissues. Therefore, tumor images with these radioligands were affected by the radioactivity in peripheral tissues a short time after injection.
In this paper, several new quinazoline derivatives were designed on the basis of essential requirements for radiophamaceuticals and structure-activity relationships. New 125 I-labeled derivatives with various side chains introduced into the 6th position of PHY were synthesized, and biological studies with these compounds were carried out including analysis of SPECT/CT images. From these results, the new radioiodinated derivatives were evaluated for their use in SPECT tumor imaging.

Materials and methods
[ 125 I]NaI and [ 32 P]ATP were purchased from Tokyo Biomedical (Tokyo, Japan) and Muromachi Yakuhin (Tokyo, Japan), respectively. All other chemicals were of reagent grade and were purchased from Sigma-Aldrich Japan (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), Wako Pure Chemical Industry (Osaka, Japan) or Tokyo Chemical Industry (Tokyo, Japan). 4-(3-Iodophenoxy)-6,7-diethoxyquinazoline (1, PHY) was synthesized according to our previous paper [22]. 4-(3-chloropropyl) morpholine and 1-(3-chloropropyl)-4-methylpiperazine were synthesized by the method of Adams et al. [23]. The HPLC system employed included a M-600 pump, a Lambda-Max 481 ultraviolet detector (Waters, MA), a 170 NaI radioactivity detector (Beckman, CA), and a 5 C 18 -AR column (10 9 250 mm, Nacalai Tesque, Kyoto, Japan). Male ddY mice and balb/c nude mice were obtained from Japan SLC Inc. (Shizuoka, Japan). Animals were housed with ad libitum access to food and drinking water for at least one week before initiating experiments. Animals were housed and experiments were performed according to guidelines stipulated by the Osaka University of Pharmaceutical Sciences Animal Care and Use Committee.
Yield  A solution of 5 (0.60 g, 1.47 mmol) and potassium carbonate (1.0 g, 7.24 mmol) in DMF (15 mL) was stirred at 40°C for 20 min, and then 4-(3-chloropropyl)morpholine was added. This mixture was stirred and heated at 90°C for 3.5 h. The reaction mixture was poured onto a crushed ice/water mixture and extracted with ethyl acetate (3 9 50 mL). The organic layers were combined, extracted with brine (2 9 100 mL), dried over Na 2 SO 4 , and the solvent was removed. The crude material was recrystallized from methanol to afford the product.

Radiolabeling
New radioiodinated quinazoline derivatives were prepared by an iododestannylation reaction under non-carrier-added conditions from the corresponding tributyltin precursor (7a-d) as outlined in scheme 2. Aqueous hydrogen peroxide (10 lL, 30 %) was added to a mixture of [ 125 I]NaI (10 lL, 37.0 MBq, 74 TBq/mmol), 0.1 M HCl (25 lL) and the tributylstannyl precursor 7a-d (0.01 mg in 10 lL ethanol) in a sealed vial. After stirring for 10 min at room temperature, the reaction mixture was quenched with aqueous sodium bisulfite (0.1 mg in 10 lL). The mixture was isolated by HPLC using 0.1 M citrate buffer/MeOH or 0.1 M NaH 2 PO 4 buffer/MeOH as the eluent at a flow rate of 3.0 mL/min. The fraction corresponding to [ 125 I]6a-d was collected, and the solvent was removed in vacuo. Retention times, radiochemical yields, and radiochemical purities of the tracers were measured by HPLC.
Cell culture A431 human carcinoma cell line was cultured in serumsupplemented Dulbecco's modified Eagle's medium (DMEM) containing 10 % heat-inactivated fetal bovine serum and penicillin/streptomycin mixed solution at 37°C. The cells were split weekly in a 1:10 ratio using trypsin/ EDTA. They were then transferred to 100 mm cell culture dishes and allowed to grow until reaching confluence (about 0.5 million cells). Subsequently, the cells were detached using either trypsin/EDTA (0.25 %, 1.0 mL) or by scraping with a cell scraper into DMEM when they reached sub confluence. NALM6 leukemia cell line [24] as a nonadherent cell was cultured in DMEM containing 10 % heatinactivated fetal bovine serum and penicillin/streptomycin mixed solution at 37°C. The cells were split weekly into a 1:10 ratio. They were then transferred to culture flask and allowed to grow until about 0.5 million cells were obtained.

Measurement of inhibitory potency against EGFR-TK
The inhibitory potency of the new derivatives against EGFR-TK was measured using a Signa TECT Ò Protein Kinase Assay System (Promega, WI) according to the protein tyrosine kinase assay protocol of the attached document. The EGF receptor used was an immunopurified receptor from human A431 cells (Promega, WI). The substrate used was based on a portion of biotinylated protein tyrosine kinase selective peptide. EGF receptors (1 lg of protein, 5 lL), sodium vanadate (2.5 mM, 2.5 lL), biotinylated protein tyrosine kinase selective peptide (2.5 mM, 2.5 lL) and the dissolved test compound in water including 1 % DMSO (desired concentration, 7.5 lL) were added to the PTK assay 5X buffer (5 lL), and the mixture was preincubated at 30°C for 5 min. Next, [ 32 P]ATP (0.5 mM ATP, 18.5 kBq, 2.5 lL) was added to the reaction mixture. The reaction was allowed to proceed for 10 min at 30°C and then was stopped by the addition of guanidine hydrochloride solution (7.5 M, 13 lL). Each reaction mixture (12.5 lL) was spotted onto a biotin capture membrane. After all of the samples had been spotted, they were washed and rinsed as follows Tumor uptake studies of radioiodinated ligands using tumor-bearing mice A suspension of 1 9 10 7 A431 cells in 0.1 mL of medium was inoculated subcutaneously in male balb/c nude mice (4 weeks old, 20-25 g, n = 3). After approximately 2 weeks, when the solid tumor had grown to optimal size, the mice were used for tumor uptake studies. [ 125 I]6a-d (37 kBq, 74 TBq/mmol) in 0.1 mL of saline was injected intravenously into tumor-bearing mice via the lateral tail vein. At 1 or 24 h after administration, the animals were sacrificed. Samples of tumor were excised and weighed. Radioactivity was measured using a well-type NaI(Tl) scintillation gamma counter. The results were expressed in terms of percentage of injected dose per gram of tumor.

Biodistribution of the [ 125 I]PYK using normal and tumor-bearing mice
[ 125 I]PYK (37 kBq, 74 TBq/mmol) in 0.1 mL saline was injected intravenously into ddY male mice (5 weeks old, 25-30 g, n = 4) via the lateral tail vein. At the desired time (5 min-24 h) interval after administration, the animals were sacrificed by decapitation. Samples of blood and tissues of interest were excised and weighed. Radioactivity was measured using a well-type NaI(Tl) scintillation gamma counter. The results were expressed in terms of percentage of injected dose per gram of blood or tissue.

Effect of inhibitor pretreatment on in vivo tumor uptake of [ 125 I]PYK
For pretreatment experiments, tumor-bearing mice (6 weeks old, 20-25 g, n = 3) were given an intravenous injection of various inhibitors (1 mg/kg) 5 min before administration of [ 125 I]PYK (37 kBq, 74 TBq/mmol). The animals were sacrificed 24 h after injection of the radioligands, and the ligand distribution was studied as described above. The differences between non-treatment and pretreatment data were examined by Student's paired t tests.

Binding assay of [ 125 I]PYK
Assays were carried out as described in the previous section. Crude P 2 membrane fractions (A431or NALM6, 0.1 mg of protein, 10 lL) and [ 125 I]PYK (1-300 nM, 100 lL, 0.74 kBq) were used. Non-specific binding was determined in the presence of PD153035 (10 lM). Specific binding was defined as the difference between the total and non-specific binding. Scatchard plot analysis of the saturation binding data was used to determine the equilibrium dissociation constant (K d ) and the maximal number of binding sites (B max ).

SPECT/CT imaging
The mice underwent SPECT/CT studies when A431 tumors had grown to optimal size (approximately 2 weeks after inoculation). 125 I gamma camera imaging and image processing were performed using a small-animal imaging system with pinhole collimation (aperture diameter = 1 mm, focal length = 9 cm) and a 15-to 45 keV photo peak energy window (PET/SPECT/CT FX system, Gamma Medica Ideas). The A431 tumor-bearing mice were injected with approximately 14.8 MBq of [ 125 I]PYK via the tail vein under isoflurane anesthesia. 24 h after injection, SPECT scans were performed at 64 projections over 360°(ROR = 4.5 cm, 60 s/projection). Reconstructed data from SPECT and CT were visualized and coregistered using AMIRA 3.1.

Results
Chemical synthesis 4-(3-Iodophenoxy)-6,7-diethoxyquinazoline derivatives were designed and synthesized by the reaction scheme outlined in scheme 1. 4-Chloro-6,7-diethoxyquinazoline (1) was synthesized according to a modified procedure of Van Brocklin et al. [25]. Treatment with methanesulphonic acid in the presence of methionine dealkylated only the ethoxy group at the 6th position of 1. The resulting free phenolic function   administration and summarized in Table 3. [ 125 I]PYK was transported well into various organs and disappeared slowly from the blood. In the liver highest uptake and rapid clearance were observed. The lungs, kidney, heart, pancreas, spleen, and brain uptakes were similar pattern to that of the liver. Furthermore, the increase in [ 125 I]PYK uptake in the stomach and thyroid as deiodination parameters were not found. Therefore, it is suggested the good stability of [ 125 I]PYK in in vivo. Moreover, biodistribution studies of [ 125 I]PYK were performed in A431 tumor-bearing mice with time points 1, 6, 12, and 24 h after intravenous administration. The results are summarized in Table 4. The accumulation of [ 125 I]PYK in the tumor was high, and the tumor radioactivity level gradually decreased to 1.53 ± 0.15 % ID/g tissue from 1 to 24 h after imaging agent injection. In contrast with the high tumor uptake and retention of [ 125 I]PYK, accumulation in the blood and muscle was low, which resulted in good tumor/blood (57.0, Fig. 3a) and tumor/muscle (45.5, Fig. 3a) ratios 24 h after injection. [ 125 I]PYK also was found to have a relatively high uptake into the lungs, liver, and kidneys 1 h after injection; however, clearance of [ 125 I]PYK from these organs was more rapid than from tumor tissue. In particular, the radioactivity accumulated in the liver 24 h after injection was approximately 1/50 of the radioactivity 1 h after injection. Thus, tumor-to-organ ratios for [ 125 I]PYK 24 h after injection had desirable values (Fig. 3b: tumor to lung: 8.5, tumor to kidney: 4.3, tumor to liver: 3.0) for tumor diagnostic imaging.

In vitro EGFR-TK selectivity of [ 125 I]PYK
An in vitro pharmacological blocking study was performed to examine the degree of specific binding of [ 125 I] PYK using A431 tumor cell membranes. As shown in Fig. 4, a significant reduction of [ 125 I]PYK binding to EGFR-TK was found after pretreatment with EGFR-TK inhibitors such as m-IPQ, PD153035, and ZD1839. With genistein or RG13022 pretreatment, [ 125 I]PYK binding was slightly decreased. The reduced radioactivity measured after treatment with these inhibitors correlated with the avidity of EGFR-TK. On the other hand, non-EGFR-TK inhibitors, such as AG17 (PDGFR-TK), HNMPA (IGFR-TK) and VEGFR inhibitor I (vascular endothelial growth factor tyrosine kinase), failed to  As shown in Fig. 5, a significant decrease of [ 125 I]PYK uptake into tumors was found after pretreatment with EGFR-TK inhibitors. On the other hand, pretreatment with AG17 (PDGFR-TK) and HNMPA (IGFR-TK) had no effect on the tumor uptake of [ 125 I]PYK; however, VEGFR inhibitor I was found to decrease its tumor uptake.

Binding assay of [ 125 I]PYK
The crude P 2 membrane fraction from A431 or NAlM6 cells were used in further binding experiments for pharmacological characterization. The saturation binding of [ 125 I]PYK was analyzed using Scatchard analysis (Fig. 6)    Discussion EGFR-TK activation is involved in the proliferation, invasion, metastasis, angiogenesis, and suppression of apoptosis of cancer cells [1,2]. Therefore, EGFR-TK is an attractive molecular target for tumor diagnosis. Several radiolabeled quinazoline derivatives that have high inhibitory potency against EGFR-TK have been studied for diagnostic tumor imaging. Among these quinazoline EGFR-TK inhibitors, PD153035 has a very high inhibitory potency. The Parke-Davis Institute group and Tsou et al. have reported various structure-activity relationships of quinazoline derivatives [26,[28][29][30][31][32][33]. From their results, it was thought that various modifications of the quinazoline structure to provide a radiopharmaceutical would be possible. Thus, PD 153035 was selected as a lead compound for development of a new EGFR-TK diagnostic agent. We have previously reported radioiodinated 4-(3-iodophenoxy)-6,7-diethoxyquinazoline (PHY) as a phenoxyquinazoline derivative that has good characteristics for an EGFR-TK imaging ligand. In this study, our aim was to develop a radiopharmaceutical superior to radioiodinated PHY for tumor diagnoses that provided high tumor accumulation and selective interaction with the target molecule. On the basis of previous work in the development of various radiopharmaceuticals, several new phenoxyquinazoline derivatives with various side chains introduced on to the 6th position of the quinazoline were designed. Six phenoxyquinazoline derivatives were easily synthesized from the key compound 5 by reactions outlined in Scheme 1. Inhibitory potencies of these derivatives for EGFR-TK were measured ( Table 1). The IC 50 values were measured for the new inhibitors as well as for PD153035 and traditional EGFR-TK inhibitors as standards, and the new inhibitors were compared with these standard compounds to evaluate their inhibitory potency. The four quinazoline derivatives 6a-d were found to possess high inhibitory potency, while 6e and 6f displayed low inhibitory potency. In order to have high inhibitory potency against EGFR-TK, it is necessary that an inhibitor bind strongly to the ATP binding site. Thus, introduction of a hydrophilic side chain to the 6th position of the quinazoline providing a positive charge interaction was effective, particularly for hydrophilic side chains linked by a three carbon chain. These results were in agreement with previous structure-activity relationships [26,[28][29][30][31][32][33].
In initial screenings to assess the relative potential of the new radiolabeled quinazoline derivatives to act as diagnostic agents for tumor imaging by in vivo tumor uptake studies in A431 tumor-bearing mice (Fig. 2) In a biodistribution study (Tables 3, 4), accumulation of [ 125 I]PYK in tumors was high, with gradual decrease in the tumor radioactivity level. In contrast with the high tumor uptake and retention of [ 125 I]PYK, accumulation in peripheral organs was low, which resulted in good tumor to organ radioactivity ratios 24 h after injection. The clearance of [ 125 I]PYK from these peripheral organs was a desirable attribute for a radiopharmaceutical. The tumor uptake of various radiolabeled quinazoline derivatives as PET ligands has previously been reported. These ligands were observed to have relatively high tumor uptake 1 h post injection [14][15][16]; however, they were also found to induce high radioactivity in peripheral tissues 1 h post   (Fig. 3). Selective interaction of [ 125 I]PYK with EGFR-TK on A431 tumor cell membranes was confirmed by pretreatment experiments with various EGFR-TK and other tyrosine kinase inhibitors. As shown in Fig. 4, significant decreases in [ 125 I]PYK binding were found after pretreatment with selective EGFR-TK inhibitors. In addition, the reduced rate of [ 125 I]PYK binding correlated with the inhibitory potency of these competitive EGFR-TK inhibitors. Alternatively, non-EGFR-TK inhibitors failed to block [ 125 I]PYK binding. In in vitro pretreatment study, the selectivity of [ 125 I]PYK to EGFR-TK was very clear and there was no effect on [ 125 I]PYK binding by the pretreatment of non-EGFR-TK. On the other hand, in in vivo, [ 125 I]PYK tumor accumulation was found decreases by the EGFR-TK inhibitors pretreatment and a slight decrease by VEGFR inhibitor I pretreatment (Fig. 5). The reason about slight reduction of [ 125 I]PYK tumor accumulation by VEGFR inhibitor I was not clear, but it is known that the VEGFR inhibition causes various pharmacologic interactions such as decrease in tumor blood vessel permeability, vasoconstriction on tumor blood vessel [34,35]. Accordingly, it was considered that this phenomenon might be caused by pharmacologic interactions on tumor blood vessel with VEGFR inhibitor I pretreatment.
In the binding assay of [ 125 I]PYK using A431 tumor cell membranes (Fig. 6), linear Scatchard plots indicated that [ 125 I]PYK bound to a single population of binding sites with high affinity (K d value; 51.3 ± 11.1 nM; Hill's constant value; 1.02). On the other hand, in the NALM6 tumor cell as a negative control, the [ 125 I]PYK binding to EGFR-TK in tumor cell membranes was undetectable. Quinazoline derivatives have been reported to bind to the ATP binding domain of EGFR-TK [12,26,36,37]. Hence, [ 125 I]PYK presumably binds to the same ATP binding site analogous to other quinazoline derivatives.
These in vivo and in vitro data suggest that [ 125 I]PYK has desirable characteristics for EGFR-TK SPECT imaging. Accordingly, our design of new EGFR-TK ligands for tumor diagnosis was appropriate. In fact, the [ 125 I]PYK-SPECT imaging of tumors was very clear without radioactivity in the lungs as a ROI (Fig. 7). Thus, [ 125 I]PYK might be useful as a diagnostic SPECT imaging agent for lung cancer.
Radioiodinated PYK showed high contrast imaging of tumors with EGFR-TK selectivity. Therefore, radioiodinated PYK-SPECT is expected to allow visualization of primary and metastatic tumor lesions that express active mutant EGFR-TK. Currently, we are exploring the feasibility of using radioiodinated PYK as a predictor for NSCLC therapy with gefitinib, and results of this study will be reported soon.

Conclusion
The iodinated quinazoline derivatives (6a-d) were found to have relatively high inhibitory potency against EGFR-TK.