Synthesis, 11C labeling and biological properties of derivatives of the tyrphostin AG957

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Abstract

Four analogues of AG957, a known inhibitor of the tyrosine kinase p210bcr-abl, have been synthesized and tested for their growth inhibitory effect against the BCR/ABL-positive FDrv210C cells as well as the epidermal growth factor (EGF) receptor-positive Baf/ERX cells. All compounds that can undergo oxidation to the corresponding quinone demonstrated inhibition of FDrv210C cells and Baf/ERX cells. Compounds that cannot become oxidized showed significantly less inhibition of BCR/ABL- or EGF receptor-mediated cell proliferation. The 11C-labeled compounds were prepared by labeling 4-aminobenzoic acid using [11C]CH3I, which afforded the corresponding 11C-labeled methyl ester in excellent yields. Subsequent condensation of the amino group with an appropriately substituted hydroxy benzaldehyde formed the respective Schiff base. Reduction of this compound with NaBH3CN gave the 11C-labeled inhibitors in an overall radiochemical yield of 17.3±2.1% (n=3; not decay corrected) and an average specific radioactivity of 40 GBq/µmol (1.1 Ci/µmol) at the end of synthesis. The total synthesis time from EOB including HPLC purification and formulation was 45 min.

Introduction

AG957, a derivative of the natural compound lavendustin A, is an inhibitor of the tyrosine kinase p210bcr-abl, which is found almost exclusively in the leukocytes of patients suffering from chronic myeloid leukemia (CML) [1], [2], [3]. Due to its selective expression in CML, the BCR/ABL oncogene is an attractive target not only for drug development but also as a diagnostic indicator using nuclear medicine imaging tools. The feasibility of external imaging of myeloid leukemia has been demonstrated using a humanized monoclonal antibody labeled with I-131 [4]. However, no suitable imaging agents for CML using PET or SPECT are currently available. Until recently, AG957 has only been labeled with the β-emitters tritium and C-14 [5], and, more recently, we have developed a procedure for the radiolabeling of AG957 at the methyl ester position using the PET radionuclide C-11 [6]. During the biological evaluation of this radiotracer, we have discovered that AG957 (1) is readily oxidized in solution and forms the corresponding quinone, NSC 676537 (2) (Fig. 1). The rapid appearance of such radioactive metabolites may limit the use of [11C] 1 as a molecular imaging agent in PET. This has prompted our search for biologically active analogues of AG957 that do not undergo oxidation as readily as the parent compound but retain its pharmacological properties.

In this study, we have synthesized 4-(N-2,4-dihydroxybenzyl) amino methyl benzoate (3) and 4-(N-2-hydroxybenzyl) amino methyl benzoate (4), which cannot undergo oxidation to a quinone, as well as 4-(N-2,3-dihydroxybenzyl) amino methyl benzoate (5) and 4-(N-3,4-dihydroxybenzyl) amino methyl benzoate (6), where oxidation to the corresponding quinone is more difficult and leads to the formation of an unstable o-quinone (Fig. 2).

The biological activity of these compounds was then compared with the parental AG957 and its oxidized form in a cellular proliferation assay against Baf/ERX [7] and FDrv210C [8] cells. The aim of our study was to identify the most promising compound for PET imaging studies in order to continue further investigations regarding the pharmacokinetic properties of this compound.

Section snippets

General

[11C]CO2 was produced by the 14N(p,α)11C nuclear reaction using a target gas that consisted of 98.03% nitrogen and 1.97% oxygen. A 10 MeV proton beam was generated using the IBA Cyclone 10/5 cyclotron at Austin Health. Typical irradiation parameters were 40 µA/h for 30 min, which produced 22.2–25.9 GBq (600–700 mCi) of [11C]CO2. Except for LiAlH4, all chemicals and solvents were purchased from Sigma-Aldrich. LiAlH4 (1 M in THF) was obtained in 1 ml vials from ABX Advanced Biochemical Compounds.

Growth inhibition assays

The factor-independent cell line FD.rv210 clone C (FDrv210C) transfected with MPZEN (bcr-abl) under the control of a retroviral promoter [10] was maintained in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS) (JRH Bisociences, CSL Ltd) at 37°C, 5% CO2 [8]. Baf/ERX cells were derived from the pre-B mouse cell line Baf/3 [7] that had been transfected with the epidermal growth factor (EGF) receptor. Baf/ERX cells were maintained at 37°C, 5% CO2 in RPMI 1640 medium containing

Chemistry

The synthesis of all four novel inhibitors was achieved by reacting methyl 4-amino benzoate with the appropriate hydroxy benzaldehyde to form the corresponding Schiff base, which was then reduced using NaBH3CN to form the putative novel inhibitor [6], [12]. This strategy is outlined in Scheme 1. The compounds were then purified by column chromatography on silica gel and recrystallized from ethyl acetate/cyclohexane.

Radiochemistry

The 11C labeling of the novel inhibitors 3, 4, 5 and 6 was achieved by following

Discussion

We have demonstrated that compounds 1, 2, 5 and 6 inhibit the growth of EGF receptor expressing Baf/ERX cells as well as FDrv210C cells. This finding is consistent with reports from Levitzki et al [12], who determined an IC50 value of 0.25 µM for AG957 in a growth inhibition assay on EGF receptor-positive A431 cells. However, the mechanism of action of tyrosine kinase inhibitors is still not fully understood.

In CML, the BCR/ABL fusion protein induces permanent activation of Ras, which is

Conclusion

In conclusion, we have demonstrated that the ability to undergo oxidation is a crucial factor to the inhibitory effect of AG957 and its analogues. These findings strongly support a mechanism of action by which an oxidized species of the inhibitor interacts with the tyrosine kinase, potentially by forming a covalent bond. However, more sophisticated experiments need to be performed in order to elucidate the mechanism of action of AG957 and its analogues.

Our assays also show that AG957 and its

Acknowledgments

This work was supported in part by the National Health and Medical Research Council Project Grant No. 234709 and the Austin Hospital Medical Research Foundation Grant No. 2-1551. E.C.N. was supported, in part, by National Health and Medical Research Council project Grant No. 164809.

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