Novel [99mTcN]2+ Labeled EGFR Inhibitors as Potential Radiotracers for Single Photon Emission Computed Tomography (SPECT) Tumor Imaging

The epidermal growth factor receptor (EGFR) is overexpressed in many cancers, including breast, ovarian, endometrial and non-small cell lung cancer. An EGFR-specific imaging agent could facilitate clinical evaluation of primary tumors or metastases. To achieve this goal, 4-(2-aminoethylamino)-6,7-dimethoxyquinazoline (ADMQ) was synthesized based on a 4-aminoquinazoline core and then conjugated with N-mercapto- acetylglycine (MAG) and N-mercaptoacetyltriglycine (MAG3), respectively, to give compounds 1 and 2. The final complexes [99mTcN]-1 and [99mTcN]-2 were successfully obtained with radiochemical purities of >99% and >98% as measured by radio-HPLC. No decomposition of the two complexes at room temperature was observed over a period of 2 h. Their partition coefficients indicated they were hydrophilic and the electrophoresis results showed they were negatively charged. Biodistribution in tumor-bearing mice demonstrated that the two new complexes showed tumor accumulation, high tumor-tomuscle (T/M) ratios and fast clearance from blood and muscle. Between the two compounds, the 99mTcN-MAG3-ADMQ ([99mTcN]-2) showed the better characteristics, with the tumor/muscle and tumor/blood ratios reached 2.11 and 1.90 at 60 min post-injection, 4.20 and 1.10 at 120 min post-injection, suggesting it could be a promising radiotracer for SPECT tumor imaging.

under stirring at 0-5 °C for 30 min and then at room temperature for 24 h to obtain 4. Compound 4 was hydrogenated with Pd/C in methanol at room temperature to obtain 5. Compound 5 was reacted with formamide at 165-170 °C under N 2 to obtain 6, which was treated with thionyl chloride and DMF to get chloro derivative 7 and then 7 coupled with ethylenediamine in i-PrOH at 80 °C to obtain 8. The synthesis of compounds 9-12 is shown in Scheme 2. Compound 9 in which the thiol group was protected by trityl chloride in advance was reacted with N-hydroxysuccinimide (NHS) using dicyclohexylcarbodiimide (DCC) as condensation reagent to obtain the active ester 10. The active ester 10 was reacted with the amine group of glycine to provide the Tr-MAG (11).  Tr-MAG 3 (12) was synthesized by the same procedure as 11, except using triglycine instead of glycine. Tr-MAG (11) was conjugated with compound 8 with DCC as condensation agent and 4-dimethylaminopyridine (DMAP) as nucleophilic agent to provide the compound 13. Tr-MAG 3 (12) was conjugated with compound 8 under the same conditions to give compound 14. Next, the thiol groups were deprotected in trifluoroacetic acid (TFA) to obtain 1. Compound 2 was prepared using the same method as product 1.  [27,28]. It is reasonable to suppose that the structure of the two complexes in this paper might be similar to those that have been reported in the previous work for the same N 3 S ligand system was employed to coordinate to 99m Tc. The radiochemical purity of the two complexes was routinely checked by radio-HPLC. The HPLC patterns of [ 99m TcN]-1 and [ 99m TcN]-2 are shown in Figure 1. It was observed that the retention time of [ 99m TcN] 2+ int was 1.8 min, while those of [ 99m TcN]-1 and [ 99m TcN]-2 were found to be 4.4 min and 3.0 min, respectively. The mean radiochemical purity of the two products was over 90% immediately after the preparation.

In Vitro Stability
The HPLC analysis results for the two complexes indicated that they were all stable in PBS after incubation for 2 h.

Paper Electrophoresis
Paper electrophoresis showed that about 90% of the initial activity remained in the positive electrode, indicating that they are negatively charged complexes.

Partition coefficients
As far as passive diffusion into tissues and cells is concerned, the lipophilicity of the molecule (generally denoted by log P) should be sufficiently high to allow penetration through the cell membrane. However, high log P values usually lead to slow clearance from blood, accumulation in metabolic tissue, and non-specific binding in tumors. The partition coefficients (

Biological Evaluation
Mice were sacrificed and major organs such as brain, liver, lungs, kidneys, spleen, stomach and heart, as well as the tumors and blood, were isolated to evaluate the tissue distribution of the two complexes. The biodistribution results are summarized in Tables 2 and 3. There were apparent similarities in the biodistribution patterns of the two compounds that demonstrated tumor accumulation, high tumor-to-muscle (T/M) ratio, tumor-to-blood (T/B) ratio and rapid washout from blood. Early hepatic and renal activity reflected the two compounds were excreted through the hepatobiliary as well as the renal system. The clearance rate in the tumor was slower than in other tissues or organs with time, so the activity in the tumor exceeded than that in most other tissue or organs. For [ 99m TcN]-1, the tumor-to-muscle (T/M) and tumor-to-blood (T/B) ratios reached 1.82 and 1.94 at 60 min, 1.17 and 1.75 at 120 min post-injection, respectively. For [ 99m TcN]-2, the tumor-to-muscle (T/M) and tumor-to-blood (T/B) ratios reached 2.11 and 1.90 at 60 min, 4.20 and 1.10 at 120 min, respectively. Between them, [ 99m TcN]-2 showed the better characteristics with the higher tumor/muscle ratio that reached to 4.2 at 120 min. The two compounds were hydrophilic so that they were unable to cross the blood brain barrier, thus making their brain uptake much lower. It is deemed worthwhile to modify the structure of ADMQ suitably to render it less hydrophilic to enhance the tumor uptake of its 99m Tc labeled complex.

General Information
Succinic dihydrazide (SDH) kit was obtained from BeijingShihong Pharmaceutical Center, Beijing Normal University (Beijing, China). 99 Mo/ 99m Tc generator was obtained from the China Institute of Atomic Energy (CIAE) (Beijing, China). THF was refluxed over sodium/benzophenone and CH 2 Cl 2 was refluxed over phosphorous CaH 2 . Other solvents were purchased as anhydrous. 1 H-and 13 C-NMR spectra were recorded on a Bruker model (Bruker, Karlsruhe, Germany) spectrometer operating in DMSO-d 6 or CDCl 3 at 400 MHz and 100 MHz, respectively. 1 H signals were reported in ppm. The IR spectra were recorded using KBr pellets in the 4,000-400 cm −1 region on a Nicolet-AVATAR 360 FT-IR spectrometer (Nicolet-AVATAR, Belmont, MA, USA). ESI-MS spectra were obtained on Waters LCT Premier XE (Waters, Milford, MA, USA). HPLC analyses were performed on a Shimadzu SCL-10 AVP (Shimadzu, Kyoto, Japan) equipped with a Packard 500 TR series flow scintillation analyzer (Shimadzu) A C-18 reversed-phase Alltima column (5 μm, 150 mm × 4.6 mm) was used for radiochemical purity analysis.

Synthesis
The preparation of compounds 3-8 was carried out by the similar procedure described in [30,31] with some modifications. The synthesis of compounds 9-12 was carried out by the same procedure described in [32]. The preparation and the analysis data of the compounds are shown below.
Ethyl 4,5-dimethoxy-2-nitrobenzoate (4). A solution of ethyl 3,4-dimethoxybenoate (6.0 g, 0.026 mol) in AcOH (23 mL) was added dropwise to nitric acid (6 mL, 65%) at 0-5 °C, and stirred at the same temperature for 30 min and then for 24 h at room temperature. Reaction progress was monitored by TLC and it was found to be complete after this time. The reaction mixture was poured onto ice/water to afford the yellow precipitate which was filtered and washed with ice water. The precipitate dried over P 2 O 5 to afford the product 4 (5.  (5). A solution of 4 (5.0 g, 0.020 mol) in methanol (30 mL) was hydrogenated with Pd/C (1.15 g) at room temperature. Reaction development was monitored by TLC and the reduction continued until no more hydrogen was consumed. The catalyst was filtered off and methanol removed to afford a brown precipitate 5 which was filtered through a glass funnel and dried under reduced pressure   (ADMQ, 8). To a solution of 7 (1.0 g, 4.5 mmol) in i-PrOH (30 mL) ethylenediamine (0.45 mL, 6.75 mmol) was added. The reaction mixture was heated to 80 °C and stirred for 4 h and then was quenched in ice water and filtered to afford the solid product 8 (0.9 g, yield 80.6%). IR (cm −1 ): ν 3504, 3419, 3340, 2986, 2071, 1625, 1598, 1541, 1511, 1421, 1251 Conjugation of Tr-MAG and ADMQ (Tr-MAG-ADMQ, 13). Triethylamine (0.13 mL, 0.9 mmol ) was added dropwise into a solution of 8 (148 mg, 0.6 mmol) in anhydrous dichloromethane (5 mL) and the mixture was stirred at room temperature for 5 min. After 11 and HOBt (90 mg, 0.66 mmol) were added to the mixture, DCC (138 mg, 0.66 mmol) in anhydrous dichloromethane (2 mL) was added dropwise to the solution at 0 °C. The mixture was stirred for 30 min at 0 °C and then overnight at room temperature to give a white precipitate which was removed by filtration. The organic phase was washed with saturated aqueous sodium bicarbonate, brine and dried over anhydrous sodium sulfate. After filtered and concentrated, the crude product was purified by column chromatography

Synthesis of 99m TcN-MAG-ADMQ ([ 99m TcN]-1)
Compound 13 (5 mg) was treated with trifluoroacetic acid under cation trapping conditions (5% triethylsilane) at room temperature for 5 min and then the solvent was removed under a stream of nitrogen to give a residue which was neutralized with 0.1 M NaOH. The solution was extracted with dichloromethane for three times, and then the aqueous phase was placed under nitrogen protection. Saline (1 mL) containing [ 99m TcO 4 ] − (15 MBq) was added to a kit containing stannous chloride dihydrate (0.05 mg), succinic dihydrazide (SDH, 5.0 mg) and propylenediamine tetraacetic acid (PDTA, 5.0 mg). The mixture was kept at room temperature for 15 min and then the MAG-ADMQ solution (1 mL) was added. The reaction mixture was allowed to stand for 15 min at 100 °C to give the final complex 99m TcN-MAG-ADMQ ([ 99m TcN]-1).

In Vitro Stability Study
Stability of the two complexes was studied using radio-HPLC analysis at different time intervals as soon as they were prepared. After the complexes were added to test tubes containing PBS solution (0.025 M, pH 7.4), the mixtures were incubated at 37 °C by shaking in a thermomixer. The radiochemical purity was measured at 30 min, 60 min and 120 min.

Radio-HPLC Analysis
The formation of the two complexes was routinely determined by Radio-HPLC. Water (solvent A) and acetonitrile (solvent B) were used for elution. For analysis of the product, the HPLC gradient system started with 100% A/0% B with a linear gradient to 0% A/100% B from 0 to 30 min. The flow rate was 1.0 mL/min. Sample (5 μL) was used for analysis. Recovery was determined by summing the total counts in all fractions and comparing them to the total injected activity.

Paper Electrophoresis
A 1 µL sample was spotted on a piece of Whatman 1 chromatography paper (length 15 cm) which was saturated with 0.05 M pH 7.4 phosphate buffer in an electrophoresis bath in advance. 150 V was applied for 2 h. The strip was then dried and divided into three segments. The distribution of radioactivity on each strip was determined.

Measurement of Partition Coefficients
The two complexes were each mixed with an equal volume of 1-octanol and phosphate buffer (0.025 M, pH 7.4) in a centrifuge tube. After vortexing at room temperature for 1 min, the mixture was centrifuged at 5,000 r/min for 5 min. The aliquot of each phase (0.1 mL) was pipetted and counted in a well-type NaI(Tl) detector and the process was repeated three times. The partition coefficient value was usually expressed as log P, where the partition coefficient, P, was calculated using the following equation: P = (cps in octanol-cps in background)/(cps in buffer-cps in background)

Biodistribution Study
Biodistribution studies of the two complexes were performed in Kunming female mice (18-22 g) bearing S 180 tumors, which grew to a leg diameter of 10-15 mm. 99m TcN-MAG-ADMQ, and 99m TcN-MAG 3 -ADMQ (0.1 mL, 7.4 × 105 Bq) were injected via the tail vein and the injected radioactivity was measured with a well-type NaI(Tl) detector, respectively. The mice were sacrificed at 30 min, 60 min and 120 min post-injection and the tumor, other organs of interest and blood were collected, weighed and measured for radioactivity. The results were expressed as the percent uptake of injected dose per gram of tissue (%ID/g). All biodistribution studies were carried out in compliance with the national laws related to the conduct of animal experimentation.

Conclusions
In summary, 4-(2-aminoethylamino)-6,7-dimethoxyquinazoline (ADMQ) was successfully synthesized and conjugated with MAG and MAG 3 , respectively. The two new compounds were labeled with technetium-99m in high yields through a ligand exchange reaction, which was easily used for the preparation of a radiopharmaceutical through a freeze-dried kit formulation. Moreover, [ 99m TcN]-2 demonstrates tumor accumulation, high tumor-to-muscle (T/M) ratios and rapid washout from blood. Thus, it appears of high interest to explore the [ 99m TcN]-2 as a potential single photon emission computed tomography (SPECT) tumor imaging agent.