Synthesis and in vitro evaluation of [¹⁸F](R)-FEPAQ: A potential PET ligand for VEGFR2

Abstract

Synthesis and in vitro evaluation of [¹⁸F](R)-N-(4-bromo-2-fluorophenyl)-7-((1-(2-fluoroethyl)piperidin-3-yl)methoxy)-6-methoxyquinazolin-4-amine ((R)-[¹⁸F]FEPAQ or [¹⁸F]1), a potential imaging agent for the VEGFR2, using phosphor image autoradiography are described. Synthesis of 2, the desfluoroethyl precursor for (R)-FEPAQ was achieved from t-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (3) in five steps and in 50% yield. [¹⁸F]1 was synthesized by reaction of sodium salt of compound 2 with [¹⁸F]fluoroethyl tosylate in DMSO. The yield of [¹⁸F]1 was 20% (EOS based on [¹⁸F]F⁻) with >99% radiochemical purity and specific activity of 1–2 Ci/μmol (n = 10). The total synthesis time was 75 min. The radiotracer selectively labeled VEGFR2 in slide-mounted sections of human brain and higher binding was found in surgically removed human glioblastoma sections as demonstrated by in vitro phosphor imager studies. These findings suggest [¹⁸F]1 may be a promising radiotracer for imaging VEGFR2 in brain using PET.

A key protein in the regulation of angiogenesis and vasculogenesis is vascular endothelial growth factor (VEGF), which is overexpressed in virtually all human tumors.^1–6 In addition to tumors, VEGF exerts neuroprotective actions directly through the inhibition of programmed cell death (PCD) or apoptosis, and the stimulation of neurogenesis.^7–9 Apart from angiogenesis, VEGF is a mediator of multiple processes enhancing blood brain barrier (BBB) permeability for glucose, and antioxidant activation, which indirectly results in neuroprotection.¹⁰ VEGF protects against neuronal death from hypoxia and glucose deprivation.^7,8,11,12 VEGF signaling through VEGFR2 was also shown to be required for antidepressants (fluoxetine, desipramine) to increase cell proliferation. Chronic antidepressant administration increases VEGF expression in both neurons and endothelial cells in the hippocampus. ^13–45 Thus, VEGF is a key component in the regulation of neuron and vessel growth in brain and other pathologies. There are excellent reviews that detail the therapeutic potential of VEGF and its receptors in CNS.^6,7,16,17 VEGF effects are mediated by a family of receptor tyrosine kinases (TKs), including VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1) and VEGFR-3 (Flt-4).^7,18 Of these, VEG-FR-2 appears to mediate almost all of the known cellular responses to VEGF. Antagonism of the VEGF pathway results in inhibition of angiogenesis and tumor growth in a number of tumor model systems. ^19–21 Although there has been significant advance in the imaging of tumors, there is a lack of agents for imaging and quantifying indices of angiogenesis. Inhibiting VEGF expression and interfering with angiogenesis may be a useful treatment target for tumors. It is reported that tumor perfusion changes dynamically during anti-VEGF treatment and that specific molecular markers in tumors and vasculature can be correlated with these perfusion changes.^22–25 Noninvasive imaging of VEGFR2 is of great potential clinical importance, as it may identify factors contributing to tumor resistance. Positron Emission Tomography (PET) imaging of VEGFR2 in tumor patients at baseline and in response to chemotherapy treatment may also provide novel information about the mechanisms that contribute to therapy resistance, and allow more effective use of VEGF inhibition. The multimodality molecular imaging of VEGF and VEGFR with radiotracers have recently been reviewed.^26–28 Efforts to image VEGF/VEGFR first focused VEGFR-specific uptake and the dynamic nature of the receptor expression in tumors as well as biological responses on tracing the distribution of the ligand by targeting with anti-VEGF antibodies labeled with 124-I and 89-Zr.^29–31 VEGFR-specific uptake and the dynamic nature of the receptor expression in tumors as well as biological responses to myocardial infarction and ischemia have been successfully demonstrated using these tracers.^31–33 However, imaging the expression of a ligand in circulation using long-lived antibodies as imaging probes requires waiting days until the unbound tracer has cleared sufficiently from the blood and the nonspecific uptake has declined sufficiently for good target/nontarget contrast. Mutated-VEGF121 (VEGFDEE) has also been developed to increase specificity for VEGFR2 and the results obtained were promising for potential translation to clinical studies. ³⁴ However, these peptide ligands do not cross BBB and hence the tracers are limited to imaging studies outside the brain. An understanding of the potential therapeutic value associated with binding to VEGFR2 in brain requires development of selective non-peptide PET ligands. Therapeutic drugs that act by binding to VEGFR2 can be evaluated and their therapeutic dose determined by an occupancy study using such a specific PET tracer. A specific PET tracer could also serve as a biological marker for angiogenesis and for the evaluation of diseases in which changes in VEGF receptor binding occurs. For example, VEGFR2 changes in tissue proliferation or angiogenesis can be quantified in vivo using PET. However, there is currently no good radiotracer for the measurement of VEG-FR2 binding expression in tumors and to monitor their role in angiogenesis. Racemic [¹¹C]PAQ was reported as the first VEGFR2 selective PET tracer which was proved specific for kidney tumors in rodents.³⁵ Radiolabeling of racemic [¹⁸F]sunitinib, [¹¹]vandetanib and [¹¹C]chloro-vandetanib has been recently reported, however, biological evaluation of these tracers are not reported yet.³⁶ All these radiotracers are 4- or 3-piperidinyl analogues of vandetanib (rINN, Caprelsa ZD6474), the first VEGFR2 drug approved by FDA.^37,38 Moreover, it has been reported that the (R)-enantiomer of vandetanib is 10-fold more active than (S)-enantiomer, also there is a better selectivity of (R)-enantiomer with respect to EGFR and VEGFR1.³⁹ Herein we report the synthesis and evaluation of (R)-enantiomer of a viable [F-18]analogue of PAQ [¹⁸F]fluoroethyl PAQ ([¹⁸F]FEPAQ, [¹⁸F]1) as a potential candidate for VEGFR2 imaging with PET.

We synthesized (R)-fluoroethyl-PAQ ((R)-FEPAQ) and the corresponding desfluoroethyl-PAQ with appropriate modifications of literature methods (Scheme 1).^35,39 In brief, reaction of 4-hydroxy-6-methoxyquinazolin-7-yl benzoate (5) with POCl₃ and N,N-diisopropylethyl amine and the coupling of the intermediate choroquinoline formed with 4-bromo-2-fluoroaniline (6) in one pot, afforded 4-((4-bromo-2-fluorophenyl)amino)-6-methoxy quinazolin-7-ol (7) in 70% yield.³⁹ Compound 7 upon reaction with (R)-tert-butyl 3-((tosyloxy)methyl)piperidine-1-carboxylate (4), which in turn was obtained by the tosylation of commercial (R)-tert-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (3), resulted in the isolation of BOC protected precursor using column chromatography (2–3% methanol in methylene chloride). The BOC protection was removed by treatment with trifluoro acetic acid in methylene chloride (1:1) to afford the radiolabeling precursor (R)-desfluoroethyl-PAQ (2) in 50% yield.³⁵ The nonradioactive standard (R)-FEPAQ (1) was obtained by reaction of compound 2 with fluoroethyl-bromide in 60% yield.⁴⁸ The VEGFR2 binding affinity (IC₅₀) of compound 1 was found to be 40 nM based on VEG-FR2 incubation mobility-shift KDR kinase assay using microfluidic chip to measure the conversion of a fluorescent peptide substrate to a phosphorylated product.⁴⁹ The radiolabeling of compound 1 was achieved in two steps (Scheme 1).⁴⁰ The radiolabeling was attempted under several conditions and the optimal method we adopted is present here. [¹⁸F]Fluoroethyl tosylate ([¹⁸F]FE-OTs) is prepared first by reacting [¹⁸F]F⁻ with ethylene glycol ditosylate using a modified procedure reported elsewhere.⁴¹ The crude product was purified using solid phase extraction using C-18 Sep-Pak^® to yield 70% of [¹⁸F]FE-OTs. The [¹⁸F]FE-OTs obtained is azeotropically dried and reacted with freshly prepared sodium salt of 2 in DMSO. The radiolabeled product was further purified using semi-preparative HPLC to afford [¹⁸F]1 in 20 ± 5% yield (n = 10). The chemical identity of [¹⁸F]1 was confirmed by co-injecting with nonradiolabeled (R)-1 by HPLC technique. The total time required for the radiosynthesis was 75 min. The radiochemical purity of [¹⁸F]1 was found to be >99% and the specific activity in the ranges of 1–2 Ci/μmol (EOB).

Scheme 1.

Synthesis and radiosynthesis of (R)-FEPAQ and (R)-[¹⁸F]FEPAQ. Reagents and conditions: (i) (Ts)₂O, THF, 6 h; (ii) POCl₃, CH₂Cl₂; (iii) K₂CO₃, THF; (iv) 4, K₂CO₃, THF; (v) TFA, CH₂Cl₂; (vi) fluoroethyl bromide/[¹⁸F]FEOTs, DMSO.

After synthesizing the radioligand in consistent yield and sufficient specific activity, [¹⁸F]1 was tested in surgically removed human glioblastoma using phosphor image autoradiography to determine its binding to VEGFR2 (Fig. 1).⁴² We choose glioblastoma model for the evaluation of [¹⁸F]1 because it is the most common brain malignancy, with high levels of VEGF and VEGFR2 expression when compared to other non-neural solid cancers.^43–45 Additionally, studies in animal models have shown that VEGFR2 inhibitors suppress the growth of gliomas in vivo and cause regression of blood vessels.^46,47 Frozen brain sections were used for the phosphor image study in quadruplicate. Slide-mounted sections were incubated in Tris buffer (pH 7.4) containing 0.18 nM of [¹⁸F]1 for 60 min at room temperature. Adjacent sections were incubated with ZM-323881 or (R)-PAQ, the two known selective VEGFR2 antagonists (1 μM) to determine nonspecific binding. After the incubation, sections were washed with ice-cold buffer and slides were quickly dried under a stream of cold air and exposed to a phosphor-imaging screen with high- and low-activity standards for 60 min. Screens are scanned with a Packard Cyclone phosphor-imaging system and analyzed with OptiQuant Acquisition and Analysis software (Packard). Displacement of total binding was observed with both VEGFR2 ligands as evident from Figure 1.

Figure 1.

Phosphor image of [¹⁸F]1 in glioma sections. T: total binding; NS1: nonspecific binding defined by blockade with ZM-323881; NS2: nonspecific defined by blockade with (R)-PAQ.

In summary, we successfully synthesized [¹⁸F]1, a potential imaging agent for VEGFR2. The total time required for the radiosynthesis was 75 min from EOS using [¹⁸F]FE-OTs in DMSO. [¹⁸F]1 was obtained in 20 ± 5% yield (EOS) with excellent radiochemical purities and specific activity. Phosphor image studies indicate that this newly developed [¹⁸F]1 ligand binds to VEGFR2 in surgically removed human glioma. The results obtained indicate the feasibility of using [¹⁸F]1 as a potential imaging agent to visualize VEGFR2 in brain using PET.