Ethyl-8-chloro-1-[4-(1H-pyrazol-1-yl)benzyl]-4-thioxo-1,4-dihydrocinnoline-3-carboxylate [(Example 60, Step 4) 188 mg, 0.442 mmol] was dissolved in 1,2-dimethoxyethane (1.5 mL) and treated with a mixture of (±)-trans-3-hydrazinotetrahydro-2H-pyran-4-ol and (±)-trans-4-hydrazinotetrahydro-2H-pyran-3-ol (117 mg, 0.885 mmol, 2 equiv) and potassium carbonate (183 mg, 1.327 mmol, 3 equiv). The mixture was placed into an oil bath preheated to 90° C. for 35 minutes, cooled to ambient temperature, diluted with water and extracted with ethyl acetate (3×50 mL). The combined organic extracts were washed once with brine, dried with sodium sulfate; filtered and concentrated in vacuo. The residue was purified by silica gel gradient chromatography (100:0 to 0:100; hexanes:ethyl acetate), providing an inseparable mixture of regioisomers, which was further purified by chiral HPLC (Chiralcel OJ Column, 100% methanol) to produce a first-, second-, and third-eluting peak. The first-eluting peak was determined to the titled compound (enantiopure, relative trans stereochemical configuration), of which the absolute stereochemistry is unknown: 1H-NMR (400 MHz, CDCl3) δ 8.22 (1H, d, J=8.1 Hz), 7.87 (1H, s), 7.71-7.59 (4H, m), 7.46 (1H, t, J=8.1 Hz), 7.29-7.20 (2H, m), 6.44 (1H, s), 6.33 (2H, s), 4.52-4.43 (1H, m), 4.26-4.17 (2H, m), 4.11-4.04 (1H, m), 3.55 (1H, t, J=12.0 Hz), 3.32 (1H, t, J=10.3 Hz), 2.32-2.19 (1H, m), 2.14-2.06 (1H, br d J=12.4 Hz). ppm; low resolution mass spectrometry (ES+) m/z 477.3 [(M+H)+; calculated for C24H22ClN6O3: 477.5].
