Radiosynthesis and biological evaluation of novel receptor tyrosine kinase inhibitors for in vivo targeting of EGFR

Objectives: The overexpression of the epidermal growth factor receptor (EGFR) in tumours underlines the recent interest in EGFR as an attractive target for the development of new cancer imaging agents. EGFR-tyrosine kinase inhibitors (EGFR-TKI) based on the 4-anilinoquinazoline scaffold have been explored as potential probes for EGFR imaging [1, 2]. However up to now, no optimal radiotracer is still available. Herein, we report the synthesis, radiosynthesis (¹²⁵I- and ¹⁸F) and biological evaluation of three novel halogenated 6-substituted 4-anilinoquinazoline based EGFR-TKI as potential biomarkers.
Methods: The halogenated 6-substituted-4-anilinoquinazolines (2a, 2b and 2c) were obtained by reaction of 6-amino-4-anilinoquinazoline (1) with 3-/4-iodobenzoyl and 4-fluorobenzoyl chlorides as depicted in Scheme 1 [3].
Studies to inhibit EGFR autophosphorylation and A431 cellular proliferation were performed by Western blot and MTT colorimetric assays, respectively. ¹²⁵I-radiolabelled anilinoquinazolines, [¹²⁵I]-2a/b were prepared via destannylation of the corresponding tributylstannyl precursors with [¹²⁵I]NaI. Cellular uptake studies were conducted in intact A431 cells. Optimization of the radiosynthesis of the ¹⁸F-radiolabeld anilinoquinazoline [¹⁸F]-2c was attempted by nucleophilic substitution of the trimethylammonium- and nitro-6-substituted 4-anilinoquinazoline precursors using different reaction temperatures and solvents.
Results: The 6-substituted-4-anilinoquinazolines (2a, 2b and 2c) were synthesised in high chemical yield (>90%) and fully characterized. The fluorinated quinazoline 2c with IC₅₀ < 0.1 μM was shown to be the most potent inhibitor of EGFR autophosphorylation. Furthermore the three compounds are potent inhibitors of A431 cell proliferation as demonstrated by IC₅₀ values (0.78µM (2a), 3.37 (2b), 2.43 µM (2c)). The ¹²⁵I-radioiodinated analogues, obtained in high radiochemical purity and specific activity, displayed a relative high celular uptake (>35%) in intact A431 cells. Despite of all attempts the radiofluorinated compound [¹⁸F]-2c was only formed in a modest labeling yield (4%) what, up to now has hampered further radiopharmacological investigation.
Conclusions: Three novel halogenated 4-anilinoquinazoline-based EGFR-TK inhibitors, 2a, 2b and 2c, have been successful prepared and radiolabelled. The in vivo inhibition study of EGFR autophosphorylation and A431 cell proliferation proved that all three compounds show inhibitory properties at the micromolar level. In summary, data from this study suggest that this class of quinazolines derivatives encompasses promising derivatives with the potential to act as EGFR-TKI and should be further explored as biomarkers for SPECT and PET.
Research Support: The authors acknowledge FCT and DAAD for financial support. C. Neto thanks FCT for a Ph.D. grant (SFRH/BD/31319/2006).
References:
[1] Noble MEM. et al., (2004), Science, 303, 5665, 1800-5.
[2] Levitzki A. et al., (2003), Acc Chem Res, 36, 6, 462-9. [3] Hicks JW. et al., Molecules (2010), 15, 11, 8260-78.
[3] Fernandes C. et al., Bioorg Med Chem, (2007), 15, 12, 3974-80.