A carbon-11 labeled imidazo[1,2-a]pyridine derivative as a new potential PET probe targeting PI3K/mTOR in cancer

Abstract

The PI3K/Akt/mTOR pathway is frequently dysregulated in cancer due to its central role in cell growth, survival, and proliferation. Overactivation of the PI3K/Akt/mTOR pathway may occur through varying mechanisms including mutations, gene amplification, and upstream signaling events, ultimately resulting in cancer. Therefore, PI3K/Akt/mTOR pathway has emerged as an attractive target for cancer therapy and imaging. A promising approach to inhibit this pathway involves a simultaneous inhibition of both PI3K and mTOR using a dual inhibitor. Recently, a potent dual PI3K/mTOR inhibitor, 2,4-difluoro-N-(2-methoxy-5-(3-(5-(2-(4-methylpiperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (7), was discovered and demonstrated excellent kinase selectivity IC₅₀ (PI3K/mTOR) = 0.20/21 nM; good cellular growth inhibition IC₅₀ (HCT-116 cell) = 10 nM, modest plasma clearance, and acceptable oral bioavailability. Expanding on this discovery, here we present the synthesis of the carbon-11 labeled imidazo[1,2-a]pyridine derivative 2,4-difluoro-N-(2-methoxy-5-(3-(5-(2-(4-[¹¹C]methylpiperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (N-[¹¹C]7) as a new potential radiotracer for the biomedical imaging technique positron emission tomography (PET) imaging of PI3K/mTOR in cancer. The reference standard 7 and its N-demethylated precursor, 2,4-difluoro-N-(2-methoxy-5-(3-(5-(2-(piperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (11), were synthesized in 7 and 8 steps with 10% and 7% overall chemical yield, respectively. N-[¹¹C]7 was prepared from 11 using [¹¹C]methyl triflate ([¹¹C]CH₃OTf) through N-¹¹C-methylation and isolated by high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) formulation in 40-50% radiochemical yield decay corrected to end of bombardment (EOB) based on [¹¹C]CO₂. The radiochemical purity was > 99% and the molar activity (A_m) at EOB was in the range of 296-555 GBq/µmol (n = 5).

Introduction

Phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), and protein kinase B (PKB), also known as (AKA) Akt, are critical signaling molecules involved in the regulation of cellular growth, proliferation, and survival [1-3]. Overactivation of the PI3K/Akt/mTOR signaling pathway is one of the most commonly involved pathways in human cancers through promoting proliferation and simultaneously downregulating apoptosis [4]. For this reason, numerous drugs targeting these signaling molecules have been developed and are currently being evaluated in preclinical and clinical trials including GSK2126458 (GlaxoSmithKline) and Pictilisib, AKA GDC-0941 (Genentech) [5].

As the development of diagnostic imaging agents is akin to that of therapeutic drugs, the PI3K/Akt/mTOR pathway is an appealing target for the innovation of imaging probes [6]. The advanced biomedical imaging technique positron emission tomography (PET) is a promising molecular imaging modality for PI3K/Akt/mTOR pathway-related diseases, fueling the growing interest to design and evaluate new PET radiotracers for in vivo imaging of PI3K/Akt/mTOR pathway [7,8]. Imaging the PI3K/Akt/mTOR pathway will enable researchers and clinicians to identify patients with potential benefit from pathway inhibitors, monitor treatment progression, and elucidate mechanisms of drug resistance.

However, in vivo PET imaging probes currently available for PI3K/Akt/mTOR are limited and non-specific. Only ¹⁸F-fluoro-2-deoxy-glucose (¹⁸F-FDG) and ¹⁸F-fluorothymidine (¹⁸F-FLT) have been reported to evaluate early-response biomarkers for PI3K/Akt/mTOR inhibition in a breast cancer model [9,10]. Hence, there is an urgent need to develop PET probes for imaging the PI3K/Akt/mTOR pathway.

A potential strategy to inhibit and image the PI3K/Akt/mTOR pathway involves utilizing dual PI3K/mTOR inhibitors to simultaneously target multiple connections in the PI3K/Akt/mTOR pathway. In our previous work, we pioneered the synthesis of dual PI3K/mTOR inhibitor radiotracers, namely [¹¹C]GSK2126458 and [¹⁸F]GSK2126458 [11], as shown in Figure 1. Lan group subsequently synthesized carbon-11 and fluorine-18 labeled Pictilisib (GDC-0941) analogs [12,13] (Figure 1) and evaluated these PET tracers in breast cancer animal models. To date, no clinical evaluation of a PI3K/mTOR PET tracer has been reported. Nevertheless, there remains opportunity for pharmacodynamic and pharmacokinetic improvement. Novel radiolabeled PI3K/mTOR dual inhibitors will exhibit differences in metabolism as well as provide different molecular imaging applications in the diagnosis and treatment of diseases. Therefore, a new radiolabeled PI3K/mTOR dual inhibitor is required in the current study.

Figure 1.

PET PI3K/mTOR probes.

In this ongoing study, we synthesized a carbon-11 labeled potent and selective PI3K/mTOR dual inhibitor 2,4-difluoro-N-(2-methoxy-5-(3-(5-(2-(4-[¹¹C]methylpiperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (N-[¹¹C]7, Figure 1) as a new potential PET cancer imaging agent. 2,4-Difluoro-N-(2-methoxy-5-(3-(5-(2-(4-methylpiperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (7) is an imidazo[1,2-a]pyridine derivative recently developed for use as a cancer therapeutic [14]. This lead compound exhibited excellent kinase selectivity IC₅₀ (PI3K/mTOR) = 0.20/21 nM; good cellular growth inhibition IC₅₀ (HCT-116 cell) = 10 nM, modest plasma clearance, acceptable oral bioavailability, and has O- and N-methyl positions amenable to labeling with carbon-11. Therefore, its carbon-11 labeled radiotracer is expected to have high specific binding to PI3K/mTOR. Here we report the design, synthesis, and labeling of N-[¹¹C]7.

Materials and methods

General

All commercial reagents and solvents were purchased from Sigma-Aldrich and Fisher Scientific and used without further purification. [¹¹C]Methyl triflate ([¹¹C]CH₃OTf) was prepared according to a literature procedure [15]. Melting points were determined on WRR apparatus and were uncorrected. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance II 500 MHz NMR Fourier transform spectrometer at 500 and 125 MHz, respectively. Chemical shifts (δ) are reported in parts per million (ppm) relative to an internal standard tetramethylsilane (TMS, δ 0.0) (¹H NMR) and to the solvent signal (¹³C NMR), and coupling constants (J) are reported in hertz (Hz). Liquid chromatography-mass spectra (LC-MS) analysis was performed on Q Exactive Mass Spectrometer from Thermo Scientific, USA, consisting of an 1100 series HPLC connected to a diode array detector and a 1946D mass spectrometer configured for positive-ion/negative-ion electrospray ionization (ESI). The high-resolution mass spectra (HRMS) were obtained using a Waters/Micromass LCT Classic spectrometer. Chromatographic solvent proportions are indicated as volume: volume ratio. Thin-layer chromatography (TLC) was run using HS silica gel GF254 uniplates (5 × 10 cm²). Plates were visualized under UV light. Normal phase flash column chromatography was carried out on Combiflash Rf 150 silica gel 60 (300-400 mesh) with a forced flow of the indicated solvent system in the proportions described below. All moisture- and air-sensitive reactions were performed under a positive pressure of nitrogen maintained by a direct line from a nitrogen source. The purity of the synthesized compounds was confirmed by reversed-phase high-performance liquid chromatography (RP-HPLC) using an Agilent Extend 5 TC-C18 column (4.6 × 250 mm, 5 μm) on a Thermo UltiMate 3000 HPLC system, mobile phase 30% CH₃CN/70% 0.1% trifluoroacetic acid (TFA) in water, flow rate 1.0 mL/min and UV (254 nm) flow detector. Analytical RP-HPLC was performed using Waters ACQUITY Arc system, a Prodigy (Phenomenex) 5 µm C-18 column, 4.6 × 250 mm; mobile phase 25% CH₃CN: 75% 20 mM H₃PO₄; flow rate 1.2 mL/min; UV (254 nm) and γ-ray (PIN diode) flow detectors. Semi-preparative RP-HPLC was performed using a Prodigy (Phenomenex) 5 µm C-18 column, 10 × 250 mm; 20% CH₃CN/80% 20 mM H₃PO₄ mobile phase; 5 mL/min flow rate; UV (254 nm) and γ-ray (PIN diode) flow detectors. C18 Plus Sep-Pak cartridges were obtained from Waters Corporation (Milford, MA). Sterile Millex-FG 0.2 µm filter units were obtained from Millipore Corporation (Bedford, MA).

Ethyl 6-bromoimidazo[1,2-a]pyridine-3-carboxylate (1)

To a stirred solution of 2-amino-5-bromopyridine (340 mg, 1.95 mmol) in 95% EtOH (10 mL) was added 2-chloro-3-oxopropionic acid ethyl ester (750 mg, 4.98 mmol). The reaction solution was stirred at room temperature (RT) for 1 h, and then stirred for 24 h under reflux. The white solid was removed by filtration, and the filtrate was concentrated under reduced pressure. To the resulting residue, 20 mL of dichloromethane (DCM) was added, and the solution was washed with H₂O (30 mL × 2). The organic layer was dried over anhydrous Na₂SO₄ and filtered. The solvent was removed under vacuum, and the crude product was purified by silica gel column chromatography with petroleum ether/ethyl acetate (10:1) as eluent to afford 1 as a white solid (470 mg, 84%), mp 116.6-117.4°C. ¹H NMR (500 MHz, DMSO-d₆ ): δ 9.34 (d, J = 1.7 Hz, 1H), 8.31 (s, 1H), 7.82 (d, J = 9.5 Hz, 1H), 7.73 (dd, J = 9.5, 1.9 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H). LC-MS (ESI, m/z): Calcd for C₁₀H₉BrN₂O₂ (M+H⁺) 269.0, found: 269.1.

6-bromoimidazo[1,2-a]pyridine-3-carbohydrazide (2)

To a solution of compound 1 (120 mg, 0.45 mmol) in 95% ethanol (10 mL), hydrazine hydrate (450 mg, 8.92 mmol) was added, the reaction mixture was stirred at reflux for 13 h. The reaction mixture was cooled to RT. The precipitate was collected by filtration and washed with 95% ethanol, and the resulting solid was dried to afford compound 2 as white needle crystals (100 mg, 88%), mp 221.4-222.0°C. ¹H NMR (500 MHz, DMSO-d₆ ): δ 9.87 (s, 1H), 9.61 (d, J = 1.2 Hz, 1H), 8.29 (s, 1H), 7.72 (dd, J = 9.5, 0.7 Hz, 1H), 7.60 (dd, J = 9.5, 2.0 Hz, 1H), 4.52 (s, 1H). LC-MS (ESI, m/z): Calcd for C₈H₇BrN₄O ([M+H]⁺) 256.1, found: 256.9.

2-(6-bromoimidazo[1,2-a]pyridin-3-yl)-5-vinyl-1,3,4-oxadiazole (3)

To a solution of compound 2 (100 mg, 0.39 mmol) in 10 mL of DCM, two drops of acetic acid and acrolein (66 mg, 1.17 mmol) were added, and the reaction mixture was stirred at RT for 10 h. The reaction mixture was cooled down to 0°C, and then iodosobenzenediacetate (IBD) (118 mg, 0.59 mmol) was added. The reaction mixture was stirred at 0°C for 12 h. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography with petroleum ether/ethyl acetate (10:1) as eluent to afford 3 as a white solid white solid (64 mg, 56%), mp 176.4-177.3°C. ¹H NMR (500 MHz, DMSO-d₆ ): δ 9.42 (s, 1H), 8.49 (s, 1H), 7.88 (d, J = 9.5 Hz, 1H), 7.75 (dd, J = 9.5, 1.7 Hz, 1H), 6.95 (dd, J = 17.6, 11.3 Hz, 1H), 6.42 (d, J = 17.6 Hz, 1H), 6.03 (d, J = 11.3 Hz, 1H). LC-MS (ESI, m/z): Calcd for C₁₁H₇BrN₄O ([M+H]⁺) 292.1, found: 292.9.

N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (4)

To a solution of 5-bromo-2-hydroxy-3-nitropyridine (4.0 g, 19.8 mmol) in 40 mL of pyridine, 2,4-difluorobenzenesulfonyl chloride (3.2 mL, 23.8 mmol) was added dropwise at 0°C, the reaction mixture was then warmed up to RT and stirred for 10 h. The reaction was quenched by adding 30 mL of cold water. The precipitate was collected by filtration, washed with water, and dried to afford 4 as a pale pink solid (6.6 g, 88%), mp 161.1-162.0°C. ¹H NMR (500 MHz, DMSO-d₆ ): δ 10.46 (s, 1H), 8.13 (d, J = 2.3 Hz, 1H), 7.81-7.73 (m, 2H), 7.57 (s, 1H), 7.23 (d, J = 2.3 Hz, 1H), 3.61 (s, 3H). LC-MS (ESI, m/z): Calcd for C₁₂H₉BrF₂N₂O₃S ([M+H]⁺) 380.2, found: 380.9.

2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide (5)

To a solution of compound 4 (200 mg, 0.53 mmol) in 20 mL of anhydrous 1,4-dioxane, bis(pinacolato)diboron (147 mg, 0.58 mmol), PdCl₂(dppf)-CH₂Cl₂ (22 mg, 0.026 mmol) and KOAc (155 mg, 3.0 mmol) were add, the reaction flask was purged with nitrogen, and stirred under reflux for 3 h. After the reaction mixture was cooled down to RT, the solid was removed by filtration through celite and washed with DCM and methanol, and the resulting filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with petroleum ether/ethyl acetate (10:1) as eluent to afford 5 as a yellow solid (225 mg, 71%), mp 172.5-173.6°C. ¹H NMR (500 MHz, CDCl₃): δ 8.24 (d, J = 1.6 Hz, 1H), 8.02 (d, J = 1.6 Hz, 1H), 7.82 (m, 1H), 7.11 (s, 1H), 6.94-6.88 (m, 2H), 3.89 (s, 3H), 1.32 (s, 12H). LC-MS (ESI, m/z): Calcd for C₁₈H₂₁BF₂N₂O₅S ([M+Na]⁺) 449.1, found: 449.1.

2-(6-bromoimidazo[1,2-a]pyridin-3-yl)-5-(2-(4-methylpiperazin-1-yl)ethyl)-1,3,4-oxadiazole (6)

To a solution of compound 3 (210 mg, 0.72 mmol) in 10 mL of DCM, N-methylpiperazine (79 mg, 0.79 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (120 mg, 0.79 mmol) were added, and the reaction mixture was stirred at RT for 13 h. The reaction mixture was concentrated under reduced pressure, and the resulting product was purified by silica gel column chromatography with DCM/methanol (50:1) as eluent to afford 6 as a white solid (189 mg, 67%), mp 146.2-147.2°C. ¹H NMR (500 MHz, CDCl₃): δ 9.62 (s, 1H), 8.23 (s, 1H), 7.68 (d, J = 9.5 Hz, 1H), 7.51 (dd, J = 9.5, 1.5 Hz, 1H), 3.16 (t, J = 7.4 Hz, 2H), 2.92 (t, J = 7.4 Hz, 2H), 2.60 (s, 4H), 2.45 (s, 4H), 2.29 (s, 3H). LC-MS (ESI, m/z): Calcd for C₁₆H₁₉BrN₆O ([M+H]⁺) 392.3, found: 392.8.

2,4-difluoro-N-(2-methoxy-5-(3-(5-(2-(4-methylpiperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (reference standard, 7)

To a solution of compound 6 (200 mg, 0.51 mmol) and compound 5 (240 mg, 0.56 mmol) in 10 mL of 1,4-dioxane and 2 ml of water, potassium carbonate (110 mg, 0.77 mmol) and PdCl₂(dppf)-CH₂Cl₂ (37 mg, 0.046 mmol) were added at RT. The reaction mixture was heated and stirred at reflux for 2.5 h with nitrogen protected. After the reaction mixture cooled down to RT, the solid was removed by filtration through celite, and the resulting filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with DCM/methanol (50:1) as eluent to afford 7 as a white solid (182 mg, 58%), mp 127.1-128.2°C. ¹H NMR (500 MHz, CDCl₃): δ 9.49 (s, 1H), 8.29 (s, 1H), 8.17-8.13 (m, 1H), 8.12 (d, J = 1.8 Hz, 1H), 7.99 (d, J = 1.8 Hz, 1H), 7.85 (d, J = 9.3 Hz, 1H), 7.59 (d, J = 9.2 Hz, 1H), 7.27 (dd, J = 17.4, 9.5 Hz, 1H), 6.96 (t, J = 8.3 Hz, 1H), 3.99 (s, 3H), 3.20 (t, J = 7.3 Hz, 2H), 2.95 (t, J = 7.4 Hz, 2H), 2.57 (d, J = 68.0 Hz, 8H), 2.31 (s, 3H). HRMS (ESI, m/z): Calcd for C₂₈H₂₈F₂N₈O₄S ([M-H]^-) 609.1850, found: 609.1851.

2,4-difluoro-N-(2-hydroxy-5-(3-(5-(2-(4-methylpiperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (O-demethylated precursor, 8)

To a solution of compound 7 (120 mg, 0.20 mmol) in 5 mL of anhydrous acetonitrile, iodotrimethylsilane (79 μL, 0.40 mmol) was added at RT, the reaction mixture was heated and stirred under reflux for 4 h. After the reaction mixture cooled down to RT, the solvent was removed under reduced pressure, and the resulting residue was purified with preparative silica gel TLC to afford 8 as a white solid (96 mg, 82%), mp 158.2-159.3°C. ¹H NMR (500 MHz, DMSO-d₆ ): δ 12.34 (s, 1H), 9.27 (s, 1H), 8.36 (s, 1H), 8.04 (t, J = 8.5 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.78 (dd, J = 9.4, 1.8 Hz, 1H), 7.69 (s, 2H), 7.56-7.50 (m, 1H), 7.39-7.32 (m, 4H), 7.24-7.21 (m, 2H), 3.07 (dd, J = 14.1, 6.9 Hz, 4H), 2.74 (d, J = 3.6 Hz, 5H), 2.27 (s, 3H), 1.14 (d, J = 7.3 Hz, 3H). HRMS (ESI, m/z): Calcd for C₂₈H₂₈F₂N₈O₄S ([M-H]^-) 595.1693, found: 595.1698.

Tert-butyl 4-(2-(5-(6-bromoimidazo[1,2-a]pyridin-3-yl)-1,3,4-oxadiazol-2-yl)ethyl) piperazine-1-carboxylate (9)

To a solution of compound 3 (210 mg, 0.72 mmol) in 10 mL of dichloromethane, N-Boc piperazine (79 mg, 0.79 mmol) and DBU (120 mg, 0.79 mmol) were added at RT, the reaction mixture was stirred at RT for 13 h. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography with DCM/methanol (50:1) as eluent to afford 9 as a white solid (192 mg, 68%), mp 144.6-145.6°C. ¹H NMR (500 MHz, CDCl₃): δ 9.62 (s, 1H), 8.24 (s, 1H), 7.68 (d, J = 9.5 Hz, 1H), 7.52 (dd, J = 9.5, 1.6 Hz, 1H), 3.44 (d, J = 4.3 Hz, 4H), 3.18 (t, J = 7.2 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H), 2.51 (s, 4H), 1.47 (s, 9H). LC-MS (ESI, m/z): Calcd for C₂₀H₂₅BrN₆O₃ ([M+H]⁺) 477.1, found: 477.1.

Tert-butyl 4-(2-(5-(6-(5-(2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl) imidazo[1,2-a]pyridin-3-yl)-1,3,4-oxadiazol-2-yl)ethyl)piperazine-1-carboxylate (10)

To a solution of compound 9 (500 mg, 1.05 mmol) and compound 5 (493 mg, 1.16 mmol), in 10 mL of 1,4-dioxane and 2 mL of H₂O, potassium carbonate (435 mg, 3.15 mmol) and PdCl₂(dppf)-CH₂Cl₂ (75 mg, 0.092 mmol) were added. The reaction mixture was stirred at reflex for 3 h under nitrogen protected. After the reaction mixture cooled down to RT, the solid was removed by filtration through celite, and the resulting filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with DCM/methanol (50:1) as eluent to afford 10 as a white solid (125 mg, 53%), mp 99.8-100.6°C, ¹H NMR (500 MHz, CDCl₃): δ 9.37 (s, 1H), 8.21 (s, 1H), 8.07 (d, J = 6.5 Hz, 1H), 8.02 (s, 1H), 7.90 (s, 1H), 7.76 (d, J = 9.2 Hz, 1H), 7.52 (d, J = 9.2 Hz, 1H), 7.19 (t, J = 7.9 Hz, 1H), 6.91 (t, J = 8.8 Hz, 1H), 5.24 (s, 1H), 3.89 (s, 3H), 3.39 (s, 4H), 3.16 (t, J = 7.1 Hz, 2H), 2.91 (t, J = 7.1 Hz, 2H), 2.48 (s, 4H), 1.40 (s, 9H). LC-MS (ESI, m/z): Calcd for C₃₂H₃₄F₂N₈O₆S ([M+H]⁺) 697.2, found: 697.3.

2,4-difluoro-N-(2-methoxy-5-(3-(5-(2-(piperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (N-demethylated precursor, 11)

To a solution of compound 10 (500 mg, 0.72 mmol) in 6 mL of dichloromethane, 2 mL of TFA was added dropwise at RT, and the reaction mixture was stirred at RT for 12 h. After removed the solvent under reduced pressure, 20 mL of DI water was added. The pH of the resulting solution was adjusted to 7-8 with 3 N NaOH and extracted with dichloromethane (30 mL × 3). The combined organic layer was washed with 20 mL of saturated brine and 20 mL of water and then dried with anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure, and the resulting product was purified by silica gel column chromatography with DCM/methanol (100:1 to 9:1) as eluent to afford 11 as a white solid (339 mg, 79%), mp 137.0-138.1°C. ¹H NMR (500 MHz, DMSO-d₆ ): δ 9.37 (s, 1H), 8.38 (d, J = 3.7 Hz, 2H), 7.97-7.92 (m, 2H), 7.92-7.84 (m, 2H), 7.64-7.54 (m, 1H), 7.28 (dd, J = 8.5, 2.1 Hz, 1H), 3.73 (s, 3H), 3.22 (t, J = 7.0 Hz, 2H), 3.14-3.05 (m, 4H), 2.92 (t, J = 7.0 Hz, 2H), 2.73 (s, 4H). ¹³C NMR (125 MHz, DMSO-d₆ ): δ 164.5, 157.9, 157.5, 146.9, 142.4, 137.8, 132.9, 132.6, 132.5, 127.5, 126.3, 124.7, 124.4, 120.8, 118.3, 112.6, 112.4, 111.4, 106.6, 106.4, 106.2, 54.1, 54.1, 49.5, 43.3, 23.1. HRMS (ESI, m/z): Calcd for C₂₇H₂₆F₂N₈O₄S ([M+H]⁺) 597.1844, found: 597.1844.

2,4-difluoro-N-(2-methoxy-5-(3-(5-(2-(4-[¹¹C]methylpiperazin-1-yl)ethyl)-1,3,4-oxadiazol-2-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (target tracer, N-[¹¹C]7)

[¹¹C]CO₂ was produced by the ¹⁴N(p,α)¹¹C nuclear reaction in the small volume (9.5 cm³) aluminum gas target provided with the Siemens RDS-111 Eclipse cyclotron. The target gas consisted of 1% oxygen in nitrogen purchased as a specialty gas from Praxair, Indianapolis, IN. Typical irradiations used for the development were 58 μA beam current and 15 min on target. The production run produced approximately 25.9 GBq of [¹¹C]CO₂ at end of bombardment (EOB). N-demethylated precursor (11, 0.1-0.3 mg) was dissolved in CH₃CN (300 μL). To this solution was added aqueous NaOH (2 N, 2 μL). The mixture was transferred to a small reaction vial. No-carrier-added (high molar activity) [¹¹C]CH₃OTf that was produced by the gas-phase production method [15] within 12 min from [¹¹C]CO₂ through [¹¹C]CH₄ and [¹¹C]CH₃Br with silver triflate (AgOTf) column was passed into the reaction vial at RT until radioactivity reached a maximum (2 min), and then the reaction vial was isolated and heated at 80°C for 3 min. The contents of the reaction vial were diluted with aqueous NaHCO₃ (0.1 M, 1 mL). The reaction vial was connected to a 3 mL HPLC injection loop. The labeled product mixture solution was injected onto the semi-preparative HPLC column for purification. The product fraction was collected in a recovery vial containing 30 mL water. The diluted tracer solution was then passed through a C-18 Plus Sep-Pak cartridge and washed with water (3 × 10 mL). The cartridge was eluted with EtOH (3 × 0.4 mL) to release the labeled product, followed by saline (10-11 mL). The eluted product was then sterile-filtered through a Millex-FG 0.2 µm membrane into a sterile vial.

Results and discussion

Chemistry

Synthesis of the reference standard 7 and its O-demethylated precursor 8 and N-demethylated precursor 11 is depicted in Figure 2 according to the reported and modified procedures [11,14,16-19]. The synthesis of compound 1 involved a cyclization reaction between 2-amino-5-bromopyridine and 2-chloro-3-oxopropionic acid ethyl ester following a modified procedure and achieved a yield of 84%. This yield was 24% greater than the reported yield. The hydrazinolysis reaction between compound 1 and hydrazine hydrate in ethanol yielded the intermediate 2 in 88% yield. Hydrazide 2 was treated with acrolein to afford an acyl imine intermediate and then transformed to 3 in 56% yield. Coupling 2,4-difluorobenzenesulfonyl chloride with 5-bromo-2-methoxypyridin-3-amine in pyridine provided compound 4 in 88% yield. Compound 5 was generated via a palladium catalyzed cross-coupling reaction of bis(pinacolato)diboron with compound 4 in 71% yield. Compound 6 was prepared from compound 3 via an electrophilic addition with N-methylpiperazine in 67% yield. 6 was subsequently engaged in a Suzuki cross-couplings reaction with compound 5 to afford compound 7 as the reference standard in 58% yield. The O-demethylated precursor 8 was prepared from compound 7 by reacting with iodotrimethylsilane in 82% yield. Compound 3 was subjected to an electrophilic addition with N-Boc piperazine to provide intermediate 9 in 68% yield. Compound 10 was prepared from 9 via a Suzuki cross-couplings reaction with compound 5 in 53% yield. The N-demethylated precursor 11 was prepared from compound 10 by reacting with TFA in dichloromethane in 79% yield.

Figure 2.

Synthesis of the reference standard 7 and its O-demethylated precursor (8) and N-demethylated precursor (11). Reagents and conditions: (i) 2-chloro-3-oxopropionic acid ethyl ester, EtOH, reflux; (ii) hydrazine hydrate, EtOH, reflux; (iii) acrolein, acetic acid, DCM, RT, IBD; (iv) pyridine, RT; (v) bis(pinacolato)diboron, PdCl₂(dppf)-CH₂Cl₂, KOAc, 1,4-dioxane, reflux; (vi) N-methylpiperazine, DBU, RT; (vii) 5, K₂CO₃, PdCl₂(dppf)-CH₂Cl₂, 1,4-dioxane, reflux; (viii) iodotrimethylsilane, CH₃CN, reflux; (ix) N-Boc piperazine, DBU, DCM, RT; (x) 5, K₂CO₃, PdCl₂(dppf)-CH₂Cl₂, 1,4-dioxane, reflux; (xi) TFA, DCM, RT.

Radiochemistry

Synthesis of the carbon-11 labeled imidazo[1,2-a]pyridine derivative [¹¹C]7 labeled at both O- and N-positions is shown in Figure 3. O-Demethylated precursor 8 underwent O-¹¹C-methylation [20,21] using the reactive ¹¹C-methylating agent [¹¹C]CH₃OTf [15,22] in acetonitrile at 80°C under basic condition (2 N NaOH) and failed to yield the expected tracer, O-[¹¹C]7. The different bases including strong, mild, and weak bases were tested in the labeling reaction, but it was still unable to obtain O-[¹¹C]7. There are many N-positions in compound 8, and the ¹¹C-methylation on N-position is a potential competing reaction compared to O-position to form ¹¹C-labeled quaternary ammines, rather than O-[¹¹C]7. A representative semi-preparative RP-HPLC chromatographic profile for purification of O-[¹¹C]7 is shown in Figure 4, which shows several polar labeled products, and no relatively lipophilic O-[¹¹C]7 was identified. Similarly, N-Demethylated precursor 11 was labeled with [¹¹C]CH₃OTf through N-¹¹C-methylation [19,23] under the same condition to successfully produce the target tracer, N-[¹¹C]7. The labeled product was isolated by semi-preparative RP-HPLC with a C-18 column and subsequently concentrated by solid-phase extraction (SPE) [11,24] with a disposable C-18 Plus Sep-Pak cartridge to produce the corresponding pure radiolabeled compound N-[¹¹C]7 in 40-50% radiochemical yield, decay corrected to EOB, based on [¹¹C]CO₂. [¹¹C]Methyl iodide ([¹¹C]CH₃I) is a most commonly used ¹¹C-methylating agent, but [¹¹C]CH₃OTf is more reactive than [¹¹C]CH₃I [15,21,22], and thus, the radiochemical yield of N-[¹¹C]7 was relatively high.

Figure 3.

Synthesis of the target radiotracer N-[¹¹C]7. Reagents and conditions: (i) [¹¹C]CH₃OTf, CH₃CN, 2 N NaOH, 80°C, 3 min.

Figure 4.

A representative semi-preparative RP-HPLC chromatographic profile for the purification of O-[¹¹C]7: Red, radioactive HPLC trace; and Green, UV HPLC trace.

Automated radiosynthesis was performed in a self-designed, multi-purpose ¹¹C-radiosynthesis module [25-27]. Our radiosynthesis module facilitated the overall design of the reaction, purification, and reformulation processing, making it suitable for adaptation for human doses. Radiosynthesis included three stages: a labeling reaction, purification, and formulation. In order to prepare the device for subsequent syntheses and minimize radiation exposure to production personnel, automated pre-cleaning routines were employed. Prior to isolation in the hot cell, solvents and/or reagents can be loaded as prompted by the operating software. The overall synthesis time was 35-40 min from EOB. Our module enabled in-process measurement of ¹¹C-tracer molar activity (A_m, GBq/µmol at EOB) using semi-preparative RP-HPLC [25]. At the end of synthesis (EOS), the A_m of ¹¹C-tracer was determined again by analytical RP-HPLC, calculated, and decay-corrected to EOB of [¹¹C]CO₂. Both semi-preparative and analytical RP-HPLC methods gave similar A_m values. The A_m of N-[¹¹C]7 at EOB was in a range of 296-555 GBq/µmol (n = 5). The general method to increase the A_m of ¹¹C-tracer produced in our radiochemistry facility has been studied in our previous work [21]. A representative semi-preparative RP-HPLC chromatographic profile for purification of N-[¹¹C]7 is shown in Figure 5.

Figure 5.

A representative semi-preparative RP-HPLC chromatographic profile for the purification of N-[¹¹C]7: Red, radioactive HPLC trace; and Green, UV HPLC trace.

The radiochemical identity as well as chemical and radiochemical purities were determined by analytical RP-HPLC [28]. Figure 6 shows a representative chromatographic profile for analysis of N-[¹¹C]7. The identity of N-[¹¹C]7 was confirmed by comparing the retention time (t_R) to reference standard 7 at 8.919 min (Figure 6C). The radioactive HPLC chromatogram (Figure 6A) showed the main peak for N-[¹¹C]7 at 9.082 min with > 99% purity. Radiochemical purity was determined through γ-ray (PIN diode) flow detector to be > 99% in all batches. UV HPLC chromatogram (Figure 6B) showed a major cold peak at 8.776 min and a few minor cold peaks around 2-3 min, which were identified from the ethanol/saline solution (Figure 6D). The peak identification in UV chromatogram was further confirmed by HPLC co-injection [29] of N-[¹¹C]7 with 7 in ethanol/saline solution. Likewise, chemical purity was determined through UV flow detector to be > 99% in all batches. A few identifiable UV peaks from the EtOH/saline used in tracer formulation after HPLC-SPE purification and formulation contributed to minor impurities. However, there is no chemical purity limit for radiotracer release in PET tracer production as radiosynthesis is a micro-scale synthesis resulting in trace amounts of the radiotracer. EtOH, acetonitrile, and acetone were used as cleaning solvents in our ¹¹C-radiosynthesis module. These volatile organic impurities were analyzed and determined by a gas chromatography (GC) equipped with a capillary column and flame ionization detector (FID) to meet all established quality control (QC) criteria. The 1 h stability test of the radiotracer after time of terminal sterilizing filtration was performed by analytical HPLC and demonstrated no changes in chemical and radiochemical purity [29], suggesting N-[¹¹C]7 was stable for at least 1 h after terminal sterilizing filtration.

Figure 6.

A representative analytical RP-HPLC chromatographic profile for the target tracer’s N-[¹¹C]7 radiochemical identity, chemical and radiochemical purity. A: Radioactive chromatogram of N-[¹¹C]7; B: UV chromatogram of N-[¹¹C]7; C: UV chromatogram of reference standard 7; D: UV chromatogram of ethanol in saline.

Conclusion

In summary, a multiple step synthetic route with high yields has been developed to produce O-demethylated precursor 8, N-demethylated precursor 11, reference standard 7, and the target PET radiotracer, N-[¹¹C]7. Radiosynthesis utilized [¹¹C]CH₃OTf to carry out N-¹¹C-methylation at the piperazinyl nitrogen position of the precursor. The resulting labeled product was subsequently purified by RP-HPLC with a C18 column, followed by SPE with a C18 Plus Sep-Pak cartridge trap/release formulation. N-[¹¹C]7 was obtained in high radiochemical yield, chemical and radiochemical purities, molar activity, and with a reasonably short overall synthesis time. In conclusion, a potent and selective dual PI3K/mTOR inhibitor, labelled with carbon-11, has been successfully radiosynthesized. These results will facilitate future studies to evaluate N-[¹¹C]7 as a new PET agent for the imaging of PI3K/mTOR in cancer and potentially yield novel understandings into this important pathway. The in vitro and in vivo biological evaluations of N-[¹¹C]7 are currently underway, and the results will be reported in due course.

Disclosure of conflict of interest

None.