The effect of the [${}^{18}$F]-PEG group on tracer qualification of [4-(phenylamino)-quinazoline-6-YL]-amide moiety—An EGFR putative irreversible inhibitor

Abstract

Previous reports have designated the labeled derivatives of [4-(phenylamino)-quinazoline-6-yl]-amide group as the most promising EGFR-PET imaging agent candidates. To further improve tracer qualifications and increase stability and solubility, additional derivatives of this group substituted at the 7-position with various lengths of fluoro-polyethyleneglycol (F-PEG) chains were synthesized. These novel derivatives inhibited EGFR autophosphorylation with ${\mathrm{IC}}_{50}$ values of 5–40 nM. The compounds were successfully labeled with fluorine-18 at the PEG chain via a three-step radiosynthesis route. The labeled final products were obtained with a 13–32% decay corrected radiochemical yield, 99% radiochemical purity, and high specific activity.

Introduction

Overexpression of the EGFR has been demonstrated in numerous human epithelial tumors. Furthermore, correlation between EGFR overexpression and metastasis formation, therapy resistance, poor prognosis and short survival has been recently described (Tokunaga et al., 1995; Shimada and Imamura, 1996; Rae and Lippman, 2004, Levitzki, 2003). Thus, the EGFR-TK has become a major target for the development of specific anticancer drugs (Levitzki and Mishani, 2006). Examples of such therapies include FDA-approved reversible EGFR-TK inhibitors such as Gefitinib (Iressa${}^{\mathrm{TM}}$, ZD1839; AstraZeneca, Wilmington, PA) and Erlotinib (Tarceva${}^{\mathrm{TM}}$; Genentech, San Francisco, CA) (http://www.fda.gov/cder/drug/advisory/iressa.htm; http://www.fda.gov/bbs/topics/news/2004/NEW01139.html), and compounds currently under clinical trials such as Lapatinib (GW572016, GlaxoSmithkline), PKI-166, and the irreversible inhibitor CI-1033 (Fig. 1). Erlotinib and Gefitinib yield similar results in that they are both effective only in a small percentage of patients in whom EGFR possesses activating mutations in the kinase domain (Paez et al., 2004, Tsao et al., 2005, Pao et al., 2004, Takano et al., 2005, Lynch et al., 2004). Non-small cell lung cancer patients who initially respond to Gefitinib and Erlotinib become resistant due to secondary mutations in the EGFR, in addition to the primary mutations that made them responsive to these inhibitors (Pao et al., 2005). Concurrent with the multitude of efforts aimed at targeting and inhibiting the EGFR in cancerous cells, the role that EGFR overexpression plays in cancer development is gradually unraveling. Since accurate measurements of EGFR phosphorylation in human tumor are lacking, it is actually not possible to assess whether the poor response to Gefitinib or Erlotinib is indeed due to a lack of the specific activating mutations, the absence of a survival function of EGFR, or to an insufficient long-term occupancy of the receptor by reversible inhibitors. Consequently, there has been a growing interest in the use of EGFR-TK inhibitors as radiotracers for molecular imaging of EGFR overexpressing tumors via nuclear medicine modalities such as Positron Emission Tomography (PET) (Wang et al., 2006, Pal et al., 2006, Mishani et al., 2005, Mishani et al., 1999).

Current pre-clinical data show that cell lines expressing the Gefitinib-resistant mutants are inhibited by irreversible EGFR inhibitors, suggesting that these drugs may have future clinical utility (Gazdar and Minna, 2005, Kobayashi et al., 2005, Kwak et al., 2005, Yoshimura et al., 2006, Tsou et al., 2001). Therefore, in recent reports (Ortu et al., 2002; Mishani et al., 2005; Vasdev et al., 2005, Dissoki et al., 2006, Abourbeh et al., 2007, Pal et al., 2006), we and others have focused on the design and development of novel irreversible inhibitors as PET imaging agent candidates. One group of compounds that was tested, the 4-dimethylamino-but-2-enoic acid [4-(phenylamino)-quinazoline-6-yl]-amide group held a favorable profile, characterized by a remarkable inhibitory potency toward the EGFR, sufficient selectivity with respect to other tested tyrosine kinase receptors (Abourbeh et al., 2007), and elevated chemical and biological stabilities compared with previously studied irreversible inhibitors (Ortu et al., 2002). The lead compound in this group, ML04, was labeled with C-11 (Mishani et al., 2004) and F-18 (Dissoki et al., 2006) (Fig. 2), and its potential as an EGFR-PET imaging agent was evaluated (Abourbeh et al., 2007). Although the biological stability of ML04 was improved relative to previously described irreversible labeled inhibitors, it did not yield adequate PET images in tumor-bearing animal models, probably due to its low bioavailability, solubility under physiological conditions and fast clearance of the labeled inhibitor from blood. We report here the development of novel Fluorine-18 labeled inhibitors 1a–d (Scheme 1, Scheme 2, Scheme 3) based on the 4-anilinoquinazoline structure which contains the chloroacetamide and dimethylamino-but-2-enoic acid as in ML05 and ML04, respectively (Fig. 2) attached at the 6-position of the quinazoline ring (Mishani et al., 2005). Presently, the chemical modifications that were introduced to the molecule include varied lengths $(n=2,4,6)$ of fluorinated polyethyleneglycol (PEG) (Zhang et al., 2005) chains attached at the 7-position of the quinazoline ring of ML05. Since PEG chains of 2 did not improve the biological characteristics of ML05, and PEG chain of 6 resulted in lower affinity of ML05 to the EGFR, only PEG chain of 4 was utilized for the ML04 derivative.