various pathogenic yeasts and fungi that are not easily amenable to traditional genetic analysis. Materials and Methods General 1H (400 MHz) and 13C (100 MHz) NMR spectra were obtained from a JEOL JNM-EX 400 spectrometer. Column chromatogra- phy was performed by manual flash chromatography (wet packed silica, 0.04–0.063 mm) or by automated column chromatography on a Biotage SP-4 system using pre-packed columns. Microwave reactions were performed in a Biotage Initiator reactor with a fixed hold time. X-ray structures with inhibitors were used as the starting point for all dockings. Docking was performed by using Glide (Schro¨ dinger) with extra precision (XP) settings and standard parameters for ligand docking [28,29]. Synthesis 4-(1-benzyl-4-(4-fluorophenyl)-1H-1,2,3-triazol-5-yl)-2-ch- loropyridine (3a). The triazole halide (575 mg, 1.5 mmol) was dissolved in dioxane/water 3:1 (13.5 mL/4.5 mL) in a micfrowave vial. 2-Chloropyridine-4-boronic acid (354 mg, 2.25 mmol) and K2CO3 (622 mg, 4.5 mmol) were added. The mixture was degassed with N2 for 5 minutes before Pd(PPh3)4 (34.7 mg, 30.0 mmol) was added. The reaction mixture was heated in the microwave for 30 minutes at 150uC. The solution was concenftrated under reduced pressure, and the residue was dissolved in dichloro- methane and filtered through Celite. The filtrate was concentrated under reduced pressure and the crude product was purified by flash column chromatography on silica gel (hexane: ethyl acetate = 4: 1) yielding 2 (76%, 418 mg) as a white solid. 1H NMR (CDCl3): d 8.42 (1H, d, J = 5.0 Hz), 7.46–7.42 (2H, m), 7.30 (3H, m), 7.03 (6H, m), 5.47 (2H, s). 13C NMR: d 162.9 (d, C-F J = 248 Hz), 159.9, 152.7, 150.7, 145.0, 139.3, 129.2, 128.9 (d, C-F J = 8.5 Hz), 128.8, 127.4, 126.0 (d, C-F J = 3.1 Hz), 125.1, 123.2, 116.0 (d, C-F J = 21.5 Hz), 52.9. HRMS [M+1]+ calculated for C20H15ClFN4+: 365.0964; found 365.0959. 4-(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-2-chloropyri- dine (3b). The triazole halide (315 mg, 0.87 mmol) was dissolved in dioxane/water 3:1 (9 mL/3 mL) in a microwave vial. 2-Chloropyridine-4-boronic acid (206 mg, 1.3 mmol) and K2CO3 (362 mg, 2.6 mmol) were added. The mixture was degassed with N2 for 5 minutes before Pd(PPh3)4 (20.2 mg, 17.4 mmol) was added. The reaction mixture was heated in the microwave for 30 minutes at 150uC. The solution was con- fcentrated under reduced pressure and the residue was dissolved in dichloromethane and filtered through Celite. The filtrate was concentrated under reduced pressure and the crude product was purified by flash column chromatography on silica gel (hexane: ethyl acetate = 4: 1) yielding 2 (89%, 268.9 mg) as a white solid. 1H NMR (CDCl3): d 8.41 (1H, dd, J = 5.1 Hz, 0.6 Hz), 7.48– 7.46 (2H, m), 7.29–7.32 (6H, m), 7.06 (1H, m), 7.02–7.04 (2H, m), 6.96 (1H, dd, J = 5.1 Hz, 1.5 Hz), 5.47 (2H, s). 13C NMR: d 152.3, 150.4, 145.6, 139.3, 134.5, 129.7, 129.6, 128.9, 128.7, 128.6, 128.4, 127.2, 126.9, 125.0, 123.1, 52.7. HRMS [M+1]+ calculated for C20H16ClN4+: 347.1058; found 347.1047. General procedure for Hartwig-Buchwald C-N bond coup- ling. An oven-dried microvial was charged with Pd(OAc)2 (1 mol%), 2-biphenylcyclohexylphosphine (2 mol%), NaOtBu (1.4 eq.), and compound 2 (1 eq). The vial was sealed with a cap, evacuated, and filled with N2. Dry toluene (0.5 ml) and amine (1.5 eq.) were added through the septum. The reaction mixture was heated at 100uC for the specified time. The reaction mixture was diluted with CH2Cl2 (2 ml) and filtered through Celite. The filtrate A Hog1 Inhibitor for Analyzing Kinase Signaling was concentrated under reduced pressure and the residue was purified on SP-4. 4-(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-N-isopropylp- yridin-2-amine (4a). Compound 4a was prepared according to the general procedure with a reaction time of 3 hours. The crude product was purified by flash column chromatography on silica gel (hexane:ethyl acetate = 2:1) yielding 4a (68%, 39.2 mg) as a white solid. 1H NMR (CDCl3): d 8.11 (d, 1H, J = 5.1 Hz), 7.60 (m, 2H), 7.28 (m, 6H), 6.34 (dd, 1H, J = 5.1 Hz, 1.2 Hz), 6.01 (s, 1H), 5.44 (2H, s), 4.55 (d, 1H, J = 7.7 Hz), 3.61 (m, 1H), 1.11 (d, 6H, J = 6.4 Hz). 13C NMR: d 158.5, 149.6, 144.8, 137.6, 135.5, 132.4, 130.5, 128.9, 128.7, 128.3, 128.1, 127.5, 126.9, 112.9, 107.3 2, 52.3, 43.2, 22.9. HRMS [M+1]+ calculated for C23H24N5+: 370.2026; found 370.2007. 4-(1-benzyl-4-(4-fluorophenyl)-1H-1,2,3-triazol-5-yl)-N-iso- propyl-pyridin-2-amine (4b). Compound 4b was prepared according to the general procedure with a reaction time of 3 hours. The crude product was purified by flash column chromatography on silica gel (hexane: ethyl acetate = 2: 1) yielding 4a (68%, 39.2 mg) as a white solid. 1H NMR (CDCl3): d 8.13 (dd, 1H, J = 5.1 Hz, 0.7 Hz), 7.57 (m, 2H), 7.27–6.95 (m, 7H, aromatic H), 6.33 (dd, 1H, J = 5.1 Hz, 1.4 Hz) 5.98 (s, 1H), 5.44 (2H, s), 4.57 (d, 1H, J = 7.9 Hz), 3.61 (m, 1H), 1.12 (d, 6H, J = 6.4 Hz). 13C NMR: d 162.5 (d, C-F J = 248 Hz), 158.6, 149.7, 144.0, 137.4, 135.4, 132.2, 128.9, 128.5 (d, C-F J = 8.5 Hz), 128.4, 127.5, 126.7 (d, C-F J = 3.1 Hz), 115.5 (d, C-F J = 21.5 Hz), 112.8, 107.2, 52.4, 43.2, 22.9. HRMS [M+1]+ calculated for C23H23FN5+: 388.1932; found 388.1850. 4-(1-benzyl-4-(4-fluorophenyl)-1H-1,2,3-triazol-5-yl)-N-iso- propyl-pyridin-2-amine (4c). Compound 4c was prepared according to the general procedure with a reaction time of 3 hours. The crude product was purified by flash column chro- matography on silica gel (hexane:ethyl acetate = 2:1) yielding 4c (74%, 41.6 mg) as a yellow oil. 1H NMR (MeOD3): d 8.17 (dd, J = 0.7, 5.2 Hz, 1H), 7.47 (m, 2H), 7.25 (m, 3H), 7.07-7.00 (m, 4H), 6.76 (dd, J = 1.4, 5.2 Hz, 1H), 6.68 (dd, J = 0.7, 1.3 Hz, 1H), 5.56 (s, 2H), 4.23 (dd, J = 10.5, 4.5 Hz, 1H), 4.07 (dd, J = 10.5, 7.4 Hz, 1H), 3.30 (m, 1H), 1.16 (d, 3H). 13C NMR (MeOD3): d 165.6, 164.2 (d, C-F J = 247 Hz), 149.3, 145.6, 140.1, 136.5, 133.0, 130.3 (d, C-F J = 8.5 Hz), 129.9, 129.4, 128.5, 127.6 (d, C-F J = 3.1 Hz), 119.1, 116.7 (d, C-F J = 22.3 Hz), 113.2, 72.7, 53.6, 47.0, 19.0. HRMS [M+1]+ calculated for C23H23FN5O+: 404.1881. 4-(1-benzyl-4-(4-fluorophenyl)-1H-1,2,3-triazol-5-yl)-N-(4- methyl-cyclohexyl)pyridin-2-amine (4d). Compound 4d was prepared according to the general procedure with a reaction time of 24 hours. The crude product was purified by flash column chromatography on silica gel (toluene:ethyl acetate = 97:3) yielding 4d (70%, 85.2 mg) as a white solid. 1H NMR (CDCl3): d 8.12 (app. t, 1H), 7.58 (m, 2H), 7.29–6.97 (m, 7H, aromatic H), 6.32 (m, 1H), 5.97 (d, 1H, J = 6.3 Hz), 5.44 (2H, s), 1.90 (m, 1H), 1.68–0.86 (m, 13H). 13C NMR: d 162.6 (d, C-F J = 247 Hz), 158.6, 149.7, 143.9, 137.4, 135.4, 132.2, 128.9, 128.7 (d, C-F J = 7.7 Hz), 128.4, 127.4, 126.7 (d, C-F J = 3.1 Hz), 115.7 (d, C-F J = 21.5 Hz), 112.7, 107.0, 52.4, 50.6, 46.9, 33.9, 33.2, 32.1, 30.8, 29.7 and 29.2, 22.3. HRMS [M+1]+ calculated for C27H29FN5+: 442.2402; found 442.2420. (S)-4-(1-benzyl-4-(4-fluorophenyl)-1H-1,2,3-triazol-5-yl)-N- (1-phenylethyl)pyridin-2-amine (4e). Compound 4e was pre- pared according to the general procedure with a reaction time of 70 hours. The crude product was purified by flash column chromatography on silica gel (heptane:ethyl acetate = 4:1) yielding 4e (64%, 79.2 mg) as yellow solid. 1H NMR (CDCl3): d 8.07 (app. d, 1H), 7.43 (m, 2H), 7.32–7.19 (m, 8H, aromatic H), 6.93–6.88 (m, 4H, aromatic H), 6.21 (dd, 1H, J = 5.1 Hz, 1.4 Hz), 5.96 (s, 1H), 5.45 (d, J = 5.6 Hz, 1H), 4.94–5.36 (m, 2H), 4.53 (m, 1H), 1.52 (d, PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e20012