Design, Synthesis, and Biological Characterization of a Caspase 3/7 Selective Isatin Labeled with 2-[¹⁸F]fluoroethylazide

Imaging of programmed cell death (apoptosis) is important in the assessment of therapeutic response in oncology and for diagnosis in cardiac and neurodegenerative disorders. The executioner caspases 3 and 7 ultimately effect cellular death, thus providing selective molecular targets for in vivo quantification of apoptosis. To realize this potential, we aimed to develop 18F-labeled isatin sulfonamides with high metabolic stability and moderate lipophilicity while retaining selectivity and affinity for caspase 3/7. A small library of isatins modified with fluorinated aromatic groups and heterocycles was synthesized. A lead compound incorporating 2′-fluoroethyl-1,2,3-triazole was identified with subnanomolar affinity for caspase 3. “Click labeling” provided the 18F-labeled tracer in 65 ± 6% decay-corrected radiochemical yield from 2-[18F]fluoroethylazide. The compound showed high stability in vivo with rapid uptake and elimination in healthy tissues and tumor. The novel 18F-labeled isatin is a candidate radiotracer for further preclinical evaluation for imaging of apoptosis

Affinity of FED6 and uptake of [¹⁸F]FED6 in NSCLC cell lines.

A. IC50 values of FED6 obtained from measuring changes in p-EGFR Y1068 in NSCLC cell lines following incubation with FED6 for 3 h. Data are mean IC50 ± SE obtained from three separate experiments. B. Protein normalised uptake of [18F]FED6 in NSCLC cell lines following 1 h incubation with 0.22MBq [18F]FED6. C. Protein expression of total EGFR, and ABC transporters ABCG2 and ABCB1 in NSCLC cells. MCF7MX and 3T3-MDR1 were used as positive controls for ABCG2 and ABCB1 expression, respectively.</p

[¹⁸F]FED6 uptake in ABC transporter expressing cells.

A. Protein normalised uptake of [18F]FED6 in 3T3 and 3T3-MDR1 cells, following 1 h incubation with 0.22MBq of [18F]FED6, with or without pre-treatment with zosuquidar at 0.3 μM for 1 h. B. Protein normalised uptake of [18F]FED6 in MCF7 and MCF7MX cells, following 1 h incubation with 0.22MBq of [18F]FED6, with or without pre-treatment with 10 μM fumitremorgin C (FTC) for 1 h. Data are mean ± SE of triplicates, repeated twice. Statistical differences between data obtained following an unpaired t-test are indicated on graph; (** = P < 0.01).</p

Growth inhibitory effect associated with a series of cyanoquinoline molecules in 3T3, 3T3-MDR1, MCF7, and MCF7MX cells.

<p>A. Ratios of GI50 values in 3T3-MDR1 over GI50 values in 3T3 cells with and without pre-treatment with zosuquidar (zos). B. Ratios of GI50 values in MCF7MX over GI50 values in MCF7 cells with and without pre-treatment with fumitremorgin C (FTC). Data indicate mean ratios ± SE of ratios obtained from three separate experiments.</p

The effect of cyanoquinoline inhibitors on growth of ABC transporter-proficient or ABC transporter-deficient cell lines.

<p>The effect of cyanoquinoline inhibitors on growth of ABC transporter-proficient or ABC transporter-deficient cell lines.</p

Comparative affinities FED20, gefitinib and FED6 in TKI sensitive and resistant NSCLC cell lines and [¹⁸F]FED20 uptake in the cell lines.

<p>Typical of p-EGFR Y1068 and EGFR protein expression following 3 h incubation with increasing concentrations of A. FED20, B. gefitinib, or C. FED6 in PC9 and PC9ER cells; summary IC50 values for all cell lines determined from densitometry analysis of p-EGFR Y1068 in the western blots are shown. D. Protein normalised uptake of [¹⁸F]FED20 in TKI sensitive and resistant cells following 1 h incubation with 0.55 MBq of [¹⁸F]FED20 (low specific activity; 7.3 GBq/μmol). E. Protein normalised uptake of [¹⁸F]FED20 in TKI sensitive and resistant cells following 1 h incubation with 0.74 MBq of [¹⁸F]FED20 (higher specific activity; 52 GBq/μmol). (* = p < 0.05, ** = P < 0.01).</p

Efflux ratios of cyanoquinoline compounds in caco2 transwell assay.

<p>A. Efflux ratios of selected cyanoquinoline compounds. B. Efflux ratios of FED6 and vinblastine with or without pre-treatment with 10 mg/mL of verapamil or 10μM of fumitremorgin C (FTC) for 1 h. All data shows mean ± SE of triplicates, repeated twice. Statistically significant differences between data obtained following an unpaired t-test are indicated on graph; (* = p < 0.05, ** = P < 0.01).</p

Modulation of [¹⁸F]FED6 uptake following ABCG2 knockdown or drug inhibition.

<p>Protein normalised uptake of [¹⁸F]FED6 and corresponding protein expression by western blot in A. MCF7MX, B. MCF7, and C. A431 following 1 h incubation with 0.22MBq [¹⁸F]FED6. MCF7MX and A431 cells were transfected with 25 nM ABCG2- or scrambled-siRNA for 72 h prior to cell uptake. Non-transfected cells were pre-treated with or without 10 μM fumitremorgin C (FTC) or 10 μM gefitinib for 1 h. Data are mean ± SE from triplicates, repeated twice. β-actin was used as a loading control. (** = P < 0.01).</p

Physicochemical properties of cyanoquinoline compounds.

<p>Molecular weight (MW), the sum of oxygen and nitrogen atoms (N+O), calculated Log P (Marvin Sketch; <a href="https://www.chemaxon.com/products/marvin/marvinsketch" target="_blank">https://www.chemaxon.com/products/marvin/marvinsketch</a>), and polar surface area (PSA). LogarIthmic value of acid dissociation constant (pKA).</p

Summary of cell lines used.

<p>Summary of cell lines used.</p