Synthesis and preclinical evaluation of [11C]EAI045 as a PET tracer for imaging tumors expressing mutated epidermal growth factor receptor

Abstract

Background: Mutations in the epidermal growth factor receptor (EGFR) kinase domain are common in non-small cell lung cancer. Conventional tyrosine kinase inhibitors target the mutation site in the ATP binding pocket, thereby inhibiting the receptor's function. However, subsequent treatment resistance mutations in the ATP binding site are common. The EGFR allosteric inhibitor, EAI045, is proposed to have an alternative mechanism of action, disrupting receptor signaling independent of the ATP-binding site. The antibody cetuximab is hypothesized to increase the number of accessible allosteric pockets for EAI045, thus increasing the potency of the inhibitor. This work aimed to gain further knowledge on pharmacokinetics, the EGFR mutation-targeting potential, and the influence of cetuximab on the uptake by radiolabeling EAI045 with carbon-11 and tritium.

Results: 2-(5-fluoro-2-hydroxyphenyl)-2-((2-iodobenzyl)amino)-N-(thiazol-2-yl)acetamide and 2-(5-fluoro-2-hydroxyphenyl)-N-(5-iodothiazol-2-yl)-2-(1-oxoisoindolin-2-yl)acetamide were synthesized as precursors for the carbon-11 and tritium labeling of EAI045, respectively. [¹¹C]EAI045 was synthesized using [¹¹C]CO in a palladium-catalyzed ring closure in a 10 ± 1% radiochemical yield (decay corrected to end of [¹¹C]CO₂ production), > 97% radiochemical purity and 26 ± 1 GBq/µmol molar activity (determined at end of synthesis) in 51 min. [³H]EAI045 was synthesized by a tritium-halogen exchange in a 0.2% radiochemical yield, 98% radiochemical purity, and 763 kBq/nmol molar activity. The ability of [¹¹C]EAI045 to differentiate between L858R/T790M mutated EGFR expressing H1975 xenografts and wild-type EGFR expressing A549 xenografts was evaluated in female nu/nu mice. The uptake was statistically significantly higher in H1975 xenografts compared to A549 xenografts (0.45 ± 0.07%ID/g vs. 0.31 ± 0.10%ID/g, P = 0.0166). The synergy in inhibition between EAI045 and cetuximab was evaluated in vivo and in vitro. While there was some indication that cetuximab influenced the uptake of [³H]EAI045 in vitro, this could not be confirmed in vivo when tumor-bearing mice were administered cetuximab (0.5 mg), 24 h prior to injection of [¹¹C]EAI045.

Conclusions: EAI045 was successfully labeled with tritium and carbon-11, and the in vivo results indicated [¹¹C]EAI045 may be able to distinguish between mutated and non-mutated EGFR in non-small cell lung cancer mouse models. Cetuximab was hypothesized to increase EAI045 uptake; however, no significant effect was observed on the uptake of [¹¹C]EAI045 in vivo or [³H]EAI045 in vitro in H1975 xenografts and cells.

Keywords: EAI045; EGFR; Epidermal growth factor receptor; TKI; Tyrosine kinase inhibitor.

Scheme 1

Iodination of EAI045, followed by the tritiodehalogenation: (i) N-iodosuccinimide, trifluoroacetic acid, dichloromethane, room temperature, 2.5 h; (ii) tritium gas, palladium on calcium carbonate, ethanol, room temperature, 4 h

Scheme 2

Petasis approach for the synthesis of [¹¹C]EAI045 precursor, (i) methanol, room temperature, 22 h, 29%; (ii) dichloromethane, room temperature, 2.5 h, 56%; (iii) dichloromethane, room temperature, 19 h, 0%

Scheme 3

Synthesis of [¹¹C]EAI045 precursor (3a): (i) trimethylsilyl cyanide, ammonia in methanol, 45 °C, 30 min; (ii) hydrochloric acid in water, reflux/room temperature, overnight; (iii) di-tert-butyl dicarbonate, sodium hydroxide, water/tert-butanol, room temperature, overnight, 5% yield over three steps; (iv) N,N-diisopropylethylamine, propylphosphonic anhydride, ethyl acetate/dimethylformamide, room temperature, overnight, 15% yield; (v) trifluoroacetic acid, dichloromethane, room temperature, 15 min; (vi) 1,8-diazabicyclo(5.4.0)undec-7-ene, sodium sulfate, methanol, 55 °C, 1 h, Sodium cyanoborohydride, 55°C, 1h, 9% yield

Scheme 4

Radiolabeling of EAI045; (i) Tris(dibenzylideneacetone)dipalladium(0), 1,1′-bis(diphenylphosphino)ferrocene, tetrahydrofuran, dimethyl sulfoxide, 100 °C, 10 min

Fig. 1

Significant variation in uptake of [³H]EAI045 over time (n = 3/time point) was observed in control cell line A549 vs. H1975 and HCC827 cell lines in vitro (P < 0.006)

Fig. 2

[³H]EAI045 uptake as dependent on cetuximab concentration in A549, H1975 and HCC827 (n = 3/concentration) in vitro

Fig. 3

A [³H]EAI045 uptake in H1975 cells was shown to be concentration dependent. Specific binding of [³H]EAI045 uptake in H1975 cell was determined by co-incubating with 10 µM non-labeled EAI045 (n = 3/data point). B The specific binding was shown to be significantly higher at lower [³H]EAI045 concentrations

Fig. 4

Metabolism of [¹¹C]EAI045 in female nu/nu mice bearing A549 xenografts (n = 4 per time point)

Fig. 5

Biodistribution of [¹¹C]EAI045 in female nu/nu mice (n = 6 at 5 min, n = 7 at 30 min and n = 8 at 60 min p.i.)

Fig. 6

a Tumor uptake and retention comparison of [¹¹C]EAI045 in A549 (n = 8/time point) and H1975 (n = 4 at 5 min, n = 6 at 30 min, and n = 8 at 60 min p.i.) xenografted mice. b Tumor-to-blood ratio and c tumor-to-muscle ratio at 60 min p.i. comparison of [¹¹C]EAI045 in H1975 (N = 4, n = 8) and A549 (N = 4, n = 8) xenografted mice

Fig. 7

The influence of cetuximab pretreatment on [¹¹C]EAI045 a biodistribution in xenografted mice (N = 16, n = 8/treatment), b tumor uptake, c tumor-to-blood ratio, and d tumor-to-muscle ratio in A549 (N = 8) and H1975 (N = 8) xenografted (n = 8/treatment) mice at 60 min p.i