Supporting Information. Design and Synthesis of BACE1 Inhibitors with In

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1 Supporting Information Design and Synthesis of BACE1 Inhibitors with In Vivo Brain Reduction of β-amyloid Peptides Britt-Marie Swahn,*, Karin Kolmodin, Sofia Karlström, Stefan von Berg, Peter Söderman, Jörg Holenz, Stefan Berg, Johan Lindström, Marie Sundström, Dominika Turek, Jacob Kihlström, Can Slivo, Lars Andersson, David Pyring, Didier Rotticci, Liselotte Öhberg, Annika Kers, Krisztian Bogar, Margareta Bergh, Lise-Lotte lsson, Juliette Janson, Susanna Eketjäll, # Biljana Georgievska, # redrik Jeppsson # and Johanna älting # Department of Medicinal Chemistry; # Department of euroscience; Department of DMPK; AstraZeneca R&D Södertälje, SE , Södertälje, Sweden; Discovery Sciences; AstraZeneca R&D Mölndal, SE Mölndal, Sweden *Corresponding Author Phone: +46 (0) brittmarieswahn@gmail.com TABLE CTETS Synthetic procedures and analytical data for compounds 23, 24, 25, (S)-25, 26, 27, 28, 29, 30, 31, 32, (S)-32, 33, 34, 35, 36, 37, 38, 39, 40, (R)-42 S2-S24 Synthetic procedures for intermediates S25-S34 S1

2 Br 1-(3-Bromophenyl)-1-pyridin-4-yl-1H-isoindol-3-amine (23a). To a solution of tertbutyllithium (1.7 M in pentane, 3.5 ml, 6.0 mmol) in tetrahydrofuran (20 ml) at -105 C a solution of 4-iodopyridine (0.68 g, 3.3 mmol) in tetrahydrofuran (10 ml) was added drop wise. Then a solution of -[(3-bromophenyl)(2-cyanophenyl)methylene]-2-methylpropane-2- sulfinamide (1.2 g, 3.0 mmol) in tetrahydrofuran (10 ml) was added. After 1 h at -105 C the reaction was quenched by addition of water. The resulting mixture was partitioned between ethyl acetate and water, and the organic phase was dried over MgS 4 and concentrated in vacuo. The residue was dissolved in methanol (25 ml) and treated with hydrochloric acid (2 M in diethylether, 3.0 ml, 6.0 mmol) over night. The reaction mixture was concentrated and then partitioned between aq saturated sodium bicarbonate and chloroform. The organic phase was dried over MgS 4 and concentrated. Purification by flash chromatography afforded the title compound (0.66 g, 61% yield). 1 H MR (400MHz, CDCl 3 ) δ ppm (m, 2H), (m, 6H), (m, 3H), (m, 1H), 5.16 (br s, 2H). MS (ESI, positive ion) m/z: 364, 366 (M+1). 3-Pyridin-4-yl-3-(3-pyrimidin-5-yl-phenyl)-3H-isoindol-1-ylamine trifluoroacetate (23). Synthetic procedure as described for H MR (500 MHz, DMS-d 6 /TA-d) δ ppm 9.23 (s, 1 H), 9.14 (s, 2 H), 9.01 (d, J = 6.8 Hz, 2 H), 8.41 (d, J = 7.8 Hz, 1 H), 8.11 (d, J = 6.8 Hz, 2 H), S2

3 8.07 (d, J = 7.9 Hz, 1 H), 7.95 (q, J = 7.1 Hz, 2 H), 7.84 (t, J = 7.7 Hz, 1 H), 7.72 (s, 1 H), 7.64 (t, J = 7.9 Hz, 1 H), 7.38 (d, J = 7.9 Hz, 1 H). MS (ESI, positive ion) m/z: 364 (M+1). Br C 3 1-(3-Bromophenyl)-1-(2-(trifluoromethyl)pyridin-4-yl)-1H-isoindol-3-amine (24a). To tetrahydrofuran (30 ml) tert-butyllithium (6.65 ml, 11.3 mmol) was added during 2 min at -100 C. A solution of 4-iodo-2-(trifluoromethyl)pyridine (1.68 g, 6.16 mmol) in tetrahydrofuran (15 ml) was added during 15 min at -100 C followed by the addition of (E)--((3-bromophenyl)(2- cyanophenyl)methylene)-2-methylpropane-2-sulfinamide (2.0 g, 5.1 mmol) in tetrahydrofuran (15 ml) during 15 min at -100 C. The reaction mixture was kept at -90 C for 25 min and then the temperature was raised to rt during 25 min. 100 ml H 2 was added and the mixture was washed with 2x150 ml EtAc. The organic phase was dried with a 2 S 4 and concentrated. The residue was dissolved in MeH (30 ml) and 1 M HCl in Et 2 (10 ml), then the mixture was stirred for 1.5 h and concentrated. Saturated aq ahc 3 (100 ml) was added and the mixture was extracted with 3x100 ml DCM. The organic phase was dried with a 2 S 4 and concentrated. Purification by flash chromatography gave the title compound (1.85 g, 83% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm (m, 2 H), (m, 7 H), 7.64 (d, J=1.0 Hz, 1 H), 8.62 (d, J=5.0 Hz, 1 H). MS (ESI, positive ion) m/z: 432, 434 (M+1). C 3 S3

4 1-(3-(Pyrimidin-5-yl)phenyl)-1-(2-(trifluoromethyl)pyridin-4-yl)-1H-isoindol-3-amine (24). 1-(3-Bromophenyl)-1-(2-(trifluoromethyl)pyridin-4-yl)-1H-isoindol-3-amine (85 mg, 0.20 mmol), pyrimidin-5-ylboronic acid (36.5 mg, 0.29 mmol), CsC 3 (128 mg, 0.39 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) chloride (8.1 mg, 9.83 µmol) were added into a ml microwave vial and then DME (0.9 ml), water (0.15 ml) and ethanol (0.45 ml) were added. The atmosphere was changed to argon and the reaction mixture was heated in a microwave oven at 100 C for 20 min. H 2 (50 ml) was added to the reaction mixture, which was then extracted with 4x25 ml EtAc, dried (a 2 S 4 ) and concentrated. The residue was purified by flash chromatography to give the title compound (34 mg, 40% yield). 1 H MR (400MHz, CDCl 3 ) δ ppm (m, 1 H), (m, 7 H), (m, 1 H), 7.68 (d, J=1.0 Hz, 1 H), 8.64 (d, J=4.6 Hz, 1 H), 8.86 (s, 2 H), 9.19 (s, 1 H). MS (ESI, positive ion) m/z: 432 [M+H]. Br 1-(3-Bromophenyl)-4-fluoro-1-(pyridin-4-yl)-1H-isoindol-3-amine (25a). Using -((3- bromophenyl)(2-cyano-3-fluorophenyl)methylene)-2-methylpropane-2-sulfinamide (17) (4.1 g, 10 mmol) and 4-iodopyridine (2.26 g, 11.0 mmol) and following the procedure used to prepare 24a gave the title compound (3.4 g, 88% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm (m, 5 H), 7.43 (d, J=1.7 Hz, 1 H), (m, 1 H), (m, 1 H), 7.63 (d, J=7.6 Hz, 1 H), 8.48 (d, J=6.1 Hz, 2 H). MS (ESI, positive ion) m/z: 382, 384 (M + 1). S4

5 4-luoro-1-(pyridin-4-yl)-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine (25). Using 1- (3-bromophenyl)-4-fluoro-1-(pyridin-4-yl)-1H-isoindol-3-amine (0.76 g, 2 mmol) and pyrimidine-5-boronic acid (270 mg, 2.2 mmol) and following the procedure used to prepare 24 gave the title compound (330 mg, 43% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm 6.68 (br. s., 2 H), (m, 3 H), (m, 2 H), 7.54 (td, J=7.88, 4.73 Hz, 1 H), 7.64 (s, 1 H), 7.68 (dt, J=6.07, 2.17 Hz, 1 H), 7.77 (d, J=7.57 Hz, 1 H), (m, 2 H), 9.02 (s, 2 H), 9.18 (s, 1 H). MS (ESI, positive ion) m/z: 382 (M + 1). (S)-4-luoro-1-(pyridin-4-yl)-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine ((S)-25). 4-fluoro-1-(pyridin-4-yl)-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amin (1 g, 2.62 mmol) was dissolved in methanol (30 ml) and the resulting solution was injected (30 stacked injections) on a Chiralcel D column (20 x 250 mm), using methanol/c2 (30:70) as eluent at a flow rate of 50 ml/min. Detection was monitored at 220 nm and isomer 1, title compound was collected (414 mg, >99 % enantiomerically pure). 1 H MR (500MHz, DMS-d 6 ) δ ppm 6.69 (br. s., 2 H), (m, 3 H), (m, 2 H), (m, 1 H), (m, 1 H), (m, 1 H), (m, 1 H), (m, 2 H), 9.02 (s, 2 H), 9.18 (s, 1 H). MS (ESI, positive ion) m/z: 382 (M + 1). S5

6 4-luoro-1-(3-(5-fluoropyridin-3-yl)phenyl)-1-(pyridin-4-yl)-1H-isoindol-3-amine (26). Using 1-(3-bromophenyl)-4-fluoro-1-(pyridin-4-yl)-1H-isoindol-3-amine (25a) (96 mg, 0.25 mmol) and 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (61 mg, 0.28 mmol) and following the procedure used to prepare 24 gave title compound (25 mg, 25% yield). 1 H MR (400 MHz, DMS-d 6 ) δ ppm 6.66 (br. s., 2 H), (m, 3 H), (m, 2 H), 7.55 (td, J=7.96, 4.80 Hz, 1 H), (m, 1 H), (m, 1 H), 7.76 (d, J=7.58 Hz, 1 H), (m, 1 H), (m, 2 H), 8.57 (d, J=2.78 Hz, 1 H), 8.65 (t, J=1.77 Hz, 1 H). MS (ESI, positive ion) m/z: 399 (M + 1). C 3 4-luoro-1-(3-(pyrimidin-5-yl)phenyl)-1-(2-(trifluoromethyl)pyridin-4-yl)-1H-isoindol-3- amine (27). Using -((3-bromophenyl)(2-cyano-3-fluorophenyl)methylene)-2-methylpropane-2- sulfinamide 17 (1.18 g, 2.89 mmol) and 4-bromo-2-(trifluoromethyl)pyridine (0.72 mg, 3.18 mmol) and following the procedure used to prepare 24a gave 1-(3-bromophenyl)-4-fluoro-1-(2- (trifluoromethyl)pyridin-4-yl)-1h-isoindol-3-amine 27a (0.75 mg, 58% yield). MS (ESI, positive ion) m/z: 450, 452 (M + 1). Using 1-(3-bromophenyl)-4-fluoro-1-(2-(trifluoromethyl)pyridin-4- yl)-1h-isoindol-3-amine (56 mg, 0.12 mmol) and 5-pyrimidinylboronic acid (17 mg, 0.14 mmol) and following the procedure used to prepare 24 gave the title compound (26 mg, 46% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 5.93 (br. s., 2 H), (m, 1 H), (m, 2 H), (m, 5 H), 7.57 (s, 1 H), (m, 1 H), 8.78 (d, J=2.27 Hz, 2 H), 9.09 (d, J=2.27 Hz, 1 H). MS (ESI, positive ion) m/z: 450 (M + 1). S6

7 C C 3 5-(3-(3-amino-4-fluoro-1-(2-(trifluoromethyl)pyridin-4-yl)-1H-isoindol-1- yl)phenyl)nicotinonitrile (28) Using 1-(3-bromophenyl)-4-fluoro-1-(2-(trifluoromethyl)pyridin- 4-yl)-1H-isoindol-3-amine 27a (24 mg, 0.05 mmol) and 5-cyanopyridin-3-ylboronic acid (8 mg, 0.05 mmol) and following the procedure used to prepare 24 gave the title compound (10 mg, 38% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 5.56 (br. s., 2 H), (m, 1 H), (m, 2 H), (m, 5 H), (m, 1 H), 7.98 (t, J=1.89 Hz, 1 H), 8.56 (d, J=5.05 Hz, 1 H), 8.75 (d, J=1.77 Hz, 1 H), 8.86 (d, J=2.02 Hz, 1 H). MS (ESI, positive ion) m/z: 399 (M + 1). 1-(2,6-Dimethylpyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine (29). 1-(3-Bromophenyl)-1-(2,6-dimethylpyridin-4-yl)-4-fluoro-1H-isoindol-3-amine (70 mg, 0.17 mmol) and 5-pyrimidinylboronic acid (23 mg, 0.19 mmol) was reacted following the procedure used to prepare 31 to give the title compound (35 mg, 50% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 2.46 (s, 6 H), 5.52 (br. s, 2 H), 6.89 (s, 2 H), 7.11 (dd, J=9.35, 8.59 Hz, 1 H), (m, 2 H), (m, 4 H), 8.87 (s, 2 H), 9.17 (s, 1 H). MS (ESI, positive ion) m/z: 410 (M + 1). S7

8 Br 1-(3-Bromophenyl)-1-(2-cyclopropylpyridin-4-yl)-4-fluoro-1H-isoindol-3-amine (30a). tert-buli (0.73 ml, 1.24 mmol) was added drop wise over 3 minutes to dry TH (5 ml) in a flask cooled to -100 C under argon. 4-Bromo-2-cyclopropylpyridine (123 mg, 0.62 mmol) in dry TH (2 ml) was added drop wise. The mixture was stirred for 5 min then -((3-bromophenyl)(2- cyano-3-fluorophenyl)methylene)-2-methylpropane-2-sulfinamide 17 (253 mg, 0.62 mmol) in dry TH 2 ml was added dropwise. The mixture was stirred cold for 45 min. MeH (5.0 ml) was added followed by HCl in MeH (0.75 ml, 0.93 mmol). The mixture was allowed to reach rt and stirred over night. The solvents were evaporated and the residue was partitioned between EtAc and saturated aq ahc 3. The aq layer was extracted with EtAc, the organic phases were combined, dried over a 2 S 4 and concentrated. The crude product was purified by flash chromatographt to afford the title compound (100 mg, 38% yield). 1 H MR (600 MHz, DMSd 6 ) δ ppm (m, 4 H), (m, 1 H), 6.66 (br. s., 2 H), (m, 1 H), 7.18 (d, J=1.14 Hz, 1 H), (m, 3 H), 7.42 (t, J=1.71 Hz, 1 H), (m, 1 H), (m, 1 H), 7.64 (d, J=7.40 Hz, 1 H), 8.27 (d, J=5.12 Hz, 1 H). MS (ESI, positive ion) m/z: 422, 424 (M + 1). S8

9 1-(2-Cyclopropylpyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine (30). The compound was prepared in analogy with compound 24 in 62% yield, using 1-(3- bromophenyl)-1-(2-cyclopropylpyridin-4-yl)-4-fluoro-1h-isoindol-3-amine and pyrimidin-5- ylboronic acid. 1 H MR (600 MHz, DMS-d 6 ) δ ppm (m, 4 H), (m, 1 H), 6.63 (br. s., 2 H), (m, 1 H), (m, 1 H), (m, 1 H), (m, 2 H), (m, 1 H), 7.64 (s, 1 H), (m, 1 H), 7.79 (d, J=7.69 Hz, 1 H), 8.27 (d, J=5.12 Hz, 1 H), 9.02 (s, 2 H), 9.17 (s, 1 H). MS (ESI, positive ion) m/z: 422 (M + 1). C 2-luoro-6-(2-methoxy-pyridine-4-carbonyl)-benzonitrile (31c). Tetrakistriphenylphosphine palladium (0) (2.6 g, 2.2 mmol) was added in small portions to a solution of 3-fluoro-2- cyanozinc iodide (0.5 M in TH) (88 ml, 44 mmol) at 0 o C. A solution of 2-methoxyisonicotinoyl chloride (7.7 g, 45 mmol) in anhydrous TH (70 ml) was then added dropwise and the resulting reaction mixture was stirred at 0 o C for 1 h. The reaction was quenched by addition of water (150 ml) and the mixture was extracted with ethyl acetate (2 x 150 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to afford the title compound (8.2 g, 73% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 4.00 (s, 3 H), 6.99 (s, 1 H), 7.22 (d, J= 5.2 Hz, 1 S9

10 H), (m, 2 H), (m, 1 H), 8.39 (d, J= 5.1 Hz, 1 H). 19 MR (400 MHz, CDCl 3 ) δ ppm C S -((2-Cyano-3-fluoro-phenyl)-(2-methoxy-4-pyridyl)methylene)-2-methyl-propane-2- sulfinamide (31b). A solution of 2-fluoro-6-(2-methoxy-pyridine-4-carbonyl)-benzonitrile (8.2 g, 32.0 mmol) in dry TH (70 ml) was added to a solution of titanium (IV) ethoxide (16.6 ml, 80.0 mmol) in dry TH (35 ml) at rt. 2-Methyl-2-propanesulfinamide (4.46 g, 36.8 mmol) was then added and the resulting mixture was heated at reflux temperature for 22 h. The reaction mixture was cooled to rt and methanol (160 ml) was added, followed by addition of saturated aq a 2 C 3 (16 ml). The resulting mixture was filtered through a pad of sodium sulfate and the solids were washed thoroughly with ethyl acetate. The filtrate was concentrated in vacuo and the residue was purified by flash chromatography to afford the title compound (5.6 g, 49% yield). 1 H MR (400 MHz, CDCl 3 ) δ 1.39 (s, 9 H), 3.96 (s, 3 H), 6.99 (s, 1 H), 7.12 (m, 1 H), 7.20 (d, J = 7.0 Hz, 1 H), 7.32 (t, J = 8.6 Hz, 1 H), 7.68 (m, 1 H) 8.27 (d, J = 5.4 Hz, 1 H). 19 MR (400 MHz, CDCl 3 ) δ ppm , MS (ESI, positive ion) m/z: 360 (M+1). Br S10

11 1-(3-Bromophenyl)-4-fluoro-1-(2-methoxypyridin-4-yl)-1H-isoindol-3-amine (31a). n-butyllithium (1.7 ml, 4.17 mmol) (2.5 M in hexanes) was dropwise added to 1,3- dibromobenzene (0.50 ml, 4.17 mmol) in diethyl ether (25.0 ml) at -78 C under argon. The resulting mixture was stirred at -78 C for 1 h. Additional n-butyllithium (2.5 M in hexanes) (0.8 ml, 2.0 mmol) was added and the resulting reaction mixture was stirred at -78 C for 30 min. - ((2-cyano-3-fluorophenyl)(2-methoxypyridin-4-yl)methylene)-2-methylpropane-2-sulfinamide (1.5 g, 4.17 mmol) in diethyl ether (10 ml) was added dropwise and stirring was continued for 1 h at -78 C. Hydrochloric acid (25 ml, 12.5 mmol) (0.5 M in methanol) was added and the resulting reaction mixture was stirred at rt over night and concentrated in vacuo. The residue was partitioned between saturated aq a 2 C 3 and EtAc and extracted three times with EtAc. The combined organic layers were dried (a 2 S 4 ), filtered and concentrated in vacuo. Purification by flash chromatography gave the title compound (0.82 g, 47% yield). 1 H MR (500 MHz, DMSd 6 ) δ ppm 3.79 (s, 3 H), 6.61 (d, J=0.95 Hz, 1 H), 6.71 (br. s., 2 H), 6.87 (dd, J=5.44, 1.50 Hz, 1 H), (m, 3 H), 7.42 (t, J=1.81 Hz, 1 H), 7.46 (ddd, J=7.84, 1.62, 1.42 Hz, 1 H), 7.53 (td, J=7.84, 4.81 Hz, 1 H), 7.63 (d, J=7.57 Hz, 1 H), 8.05 (d, J=5.36 Hz, 1 H). MS (ESI, positive ion) m/z: 412, 414 (M+1). 4-luoro-1-(2-methoxypyridin-4-yl)-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine (31). 1-(3-Bromophenyl)-4-fluoro-1-(2-methoxypyridin-4-yl)-1H-isoindol-3-amine (100 mg, 0.24 mmol), 5-pyrimidinylboronic acid (32 mg, 0.25 mmol), Pd(II)Cl 2 dppf*ch 2 Cl 2 (9.9 mg, 0.01 mmol), cesium carbonate (0.06 ml, 0.73 mmol) and DME:EtH:water (6:3:1) (3 ml) were mixed in a microwave vial and heated in a microwave reactor at 150 C for 20 min. The resulting S11

12 product mixture was filtered and purified by preparative HPLC to give the title compound (58 mg, 54% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm (m, 1 H), 4.11 (d, J=6.31 Hz, 2 H), (m, 2 H), (m, 2 H), (m, 1 H), (m, 1 H), (m, 1 H), (m, 1 H), (s, 1 H). MS (ESI, positive ion) m/z: 412 (M+1). C 2-(2-Difluoromethyl-pyridine-4-carbonyl)-6-fluoro-benzonitrile (32c). To a degassed solution of 2-difluoromethyl-thioisonicotinic acid S-ethyl ester (100 mg, 0.46 mmol) and PdCl 2 (PPh 3 ) 2 (0.065 g, 0.09 mmol) in anhydrous toluene (5 ml) 2-cyano-3-fluorophenylzinc iodide (0.5 M in TH) (0.97 ml, 0.48 mmol) was added dropwise. The reaction mixture was stirred at rt for 1 h. Saturated aq H 4 Cl (10 ml) was added followed by saturated aq a 2 C 3 (20 ml) and the resulting mixture was extracted with ethyl acetate (2 x 50 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to afford the title compound (74 mg, 58% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 6.73 (t, J=55.1 Hz, 1 H), 7.46 (d, J=7.82 Hz, 1 H), 7.54 (t, J=8.01 Hz, 1 H), (m, 2 H), 7.92 (s, 1 H), 8.92 (d, J=5.08 Hz, 1 H). MS (ESI, positive ion) m/z: 277 (M+1). C S S12

13 -((2-Cyano-3-fluorophenyl)(2-(difluoromethyl)pyridin-4-yl)methylene)-2- methylpropane-2-sulfinamide (32b). To a solution of 2-(2-difluoromethyl-pyridine-4- carbonyl)-6-fluoro-benzonitrile (70 mg, 0.25 mmol) in dry TH (7 ml) was added titanium (IV) ethoxide (145 mg, 0.63 mmol) slowly at rt. 2-Methyl-2-propanesulfinamide (37 mg, 0.31 mmol) was then added in one portion and the reaction mixture was heated at reflux temperature for 24 h. The reaction mixture was cooled to rt and methanol (3 ml) was added, followed by saturated aq a 2 C 3 (0.3 ml). The mixture was stirred at rt for 1 h, filtered through a pad of diatomaceous earth and magnesium sulfate The solids were washed thoroughly with ethyl acetate. The filtrate was concentrated in vacuo and the residue was purified by flash chromatography to afford the title compound (63 mg, 66% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 1.41 (s, 9 H), 6.68 (t, J=55.5 Hz, 1 H), 7.21 (d, J=7.03 Hz, 1 H), 7.37 (t, J=8.60 Hz, 1 H), (m, 1 H), 7.73 (br. s., 2 H), 8.78 (d, J=5.08 Hz, 1 H). 19 MR (376 MHz, CDCl 3 ) δ ppm , MS (ESI, positive ion) m/z: 380 (M+1). Br 1-(3-bromophenyl)-1-(2-(difluoromethyl)pyridin-4-yl)-4-fluoro-1H-isoindol-3-amine (32a). n-butyllithium (2.5 M in hexane) (6.5 ml, 16.2 mmol) was added over 6 min to 1,3- dibromobenzene (1.9 ml, 16.2 mmol) in diethyl ether (40 ml) at -78 C under argon atmosphere. The reaction was stirred for 25 min, then -((2-cyano-3-fluorophenyl)(2- (difluoromethyl)pyridin-4-yl)methylene)-2-methylpropane-2-sulfinamide (5.6 g, 14.7 mmol) in TH (23 ml) was added over 12 min. The reaction was kept at -78 C for 2 h and subsequently methanol (20 ml) was added. The reaction was stirred for 30 min at rt and then concentrated in S13

14 vacuo. The residue was partitioned between saturated aq a 2 C 3 and DCM (x3). The combined organic layers were washed with water, dried (a 2 S 4 ), filtered and concentrated in vacuo. Purification by flash chromatography gave the title compound (4.28 g, 67% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm (m, 3 H), (m, 3 H), 7.44 (t, J=1.66 Hz, 1 H), (m, 2 H), 7.54 (d, J=1.26 Hz, 1 H), 7.57 (td, J=7.88, 4.89 Hz, 1 H), 7.68 (d, J=7.57 Hz, 1 H), 8.60 (d, J=5.20 Hz, 1 H). MS (ESI, positive ion) m/z: 432, 434 (M+1). 1-(2-(Difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3- amine (32). 1-(3-Bromophenyl)-1-(2-(difluoromethyl)pyridin-4-yl)-4-fluoro-1H-isoindol-3- amine (0.168 g, 0.39 mmol), 5-pyrimidinylboronic acid (0.063 g, 0.51 mmol), Pd(II)Cl 2 dppf*ch 2 Cl 2 (0.016 g, 0.02 mmol), cesium carbonate (0.093 ml, 1.17 mmol) and DME:EtH:water (6:3:1) (10.0 ml) were added to a microwave vial and heated at 150 C in a microwave reactor for 20 min. Additional 5-pyrimidinylboronic acid (0.048 g, 0.39 mmol) was added. The reaction mixture was heated at 150 C in a microwave reactor for an additional 20 min. The reaction mixture was filtered through a syringe filter, concentrated in vacuo, redissolved in methanol and purified by preparative HPLC to afford the title compound (0.12 g, 60% yield). 1H MR (400 MHz, DMS-d 6 ) δ ppm 6.92 (t, J=54.82 Hz, 1 H), 7.30 (dd, J=9.47, 8.21 Hz, 1 H), (m, 3 H), (m, 2 H), (m, 1 H), 7.70 (dt, J=6.82, 1.89 Hz, 1 H), 7.82 (d, J=7.58 Hz, 1 H), 8.59 (d, J=5.05 Hz, 1 H), 9.03 (s, 2 H), 9.18 (s, 1 H). MS (ESI, positive ion) m/z: 432 (M+1). S14

15 (S) 1-(2-(Difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol- 3-amine ((S)-32). 1-(2-(Difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1Hisoindol-3-amine was subjected to enatiomer separation using SC preparative chromatography to yield the title compound with an enantiomeric purity of 99%. 1 H MR (400 MHz, DMS-d 6 ) δ ppm (m, 3 H), 7.30 (dd, J=9.60, 8.08 Hz, 1 H), (m, 3 H), (m, 2 H), (m, 1 H), 7.70 (dt, J=6.82, 1.89 Hz, 1 H), 7.82 (d, J=7.58 Hz, 1 H), 8.59 (d, J=5.30 Hz, 1 H), 9.03 (s, 2 H), 9.18 (s, 1 H). MS (ESI, positive ion) m/z: 432 (M+1). 1-(2-(Difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(5-(prop-1-ynyl)pyridin-3-yl)phenyl)- 1H-isoindol-3-amine (33). A mixture of 1-(3-bromophenyl)-1-(2-(difluoromethyl)pyridin-4-yl)- 4-fluoro-1H-isoindol-3-amine (150 mg, 0.35 mmol), 5-(prop-1-ynyl)pyridin-3-ylboronic acid (112 mg, 0.69 mmol), Pd(II)Cl 2 dppf*ch 2 Cl 2 (14 mg, 0.02 mmol), aq 2 M K 2 C 3 (0.35 ml, 0.69 mmol) and dioxane (5 ml) was heated in a microwave reactor at 130 C for 15 min. The mixture was concentrated and the resulting residue was taken up in DCM (5 ml) and water (3 ml) and poured into a phase separator. The organic layer was collected, concentrated and purified by preparative HPLC to give the title compound (76 mg, 47% yield). 1 H MR (500 MHz, DMSd 6 ) δ ppm 2.10 (s, 3 H), (m, 1 H), 7.40 (d, J=7.88 Hz, 2 H), (m, 2 H), 7.61 S15

16 (d, J=5.04 Hz, 2 H), 7.71 (br. s., 2 H), 7.82 (d, J=7.57 Hz, 1 H), 8.01 (br. s., 1 H), 8.57 (s, 1 H), 8.65 (br. s., 1 H), 8.74 (br. s., 1 H). MS (ESI, positive ion) m/z: 469 (M+1). 4-luoro-1-(4-methoxyphenyl)-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine (34). 4- Methoxyphenylmagnesium bromide (6.0 ml, 3.0 mmol) was added to a solution of -((3- bromophenyl)(2-cyano-3-fluorophenyl)methylene)-2-methylpropane-2-sulfinamide 17 (0.41 g, 1.0 mmol) in TH (10 ml) at 0 ºC under an argon atmosphere. The resulting mixture was stirred and allowed to reach rt over night. The mixture was quenched with saturated aq. H 4 Cl, diluted with saturated aq. ahc 3 and extracted with DCM. The organic phase was dried and concentrated. The residue was dissolved in MeH (10 ml) and HCl (2 M in diethyl ether, 2 ml, 4 mmol) was added and the mixture was stirred at rt for 2.5 h. The mixture was concentrated and purified by flash chromatography to give 1-(3-bromophenyl)-4-fluoro-1-(4-methoxyphenyl)-1Hisoindol-3-amine 34a. A mixture of 1-(3-bromophenyl)-4-fluoro-1-(4-methoxyphenyl)-1Hisoindol-3-amine (103 mg, 0.25 mmol), 5-pyrimidinylboronic acid (37 mg, 0.3 mmol), 2M aq K 2 C 3 (0.38 ml, 0.75 mmol) and Pd(II)Cl 2 dppf*ch 2 Cl 2 (10 mg, 0.01 mol) in DM was heated in a microwave reactor at 150 ºC for 15 min. The mixture was diluted with water (5 ml) and extracted with DCM (15 ml). The organic layer was concentrated and the resulting residue was dissolved in MeH and purified by preparative HPLC to give the title compound (0.061 g, 59% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm 3.69 (s, 3 H), 6.50 (br. s., 2 H), (m, 2 H), (m, 3 H), (m, 2 H), 7.49 (td, J=7.80, 4.89 Hz, 1 H), (m, 1 H), (m, 2 H), 8.99 (s, 2 H), 9.17 (s, 1 H). MS (ESI, positive ion) m/z: 411 (M + 1). S16

17 4-luoro-1-(5-methoxy-4,6-dimethylpyridin-2-yl)-1-(3-(pyrimidin-5-yl)phenyl)-1Hisoindol-3-amine (35). tert-buli (1.04 ml, 1.77 mmol) was added dropwise to TH ( 5 ml) at ºC under an argon atmosphere. A solution of 6-bromo-3-methoxy-2,4-dimethylpyridine (191 mg, 0.88 mmol) in TH (3mL) was added dropwise followed by the addition of -((3- bromophenyl)(2-cyano-3-fluorophenyl)methylene)-2-methylpropane-2-sulfinamide 17 (300 mg, 0.74 mmol) in TH (7 ml). The resulting reaction mixture was left on the thawing cooling bath for 30 min then the mixture was stirred at rt for 1 h. HCl in MeH (3.54 ml, 4.42 mmol) was added and the resulting mixture was stirred at rt for 1 h. The mixture was concentrated and purified by flash chromatography to give 1-(3-bromophenyl)-4-fluoro-1-(5-methoxy-4,6- dimethylpyridin-2-yl)-1h-isoindol-3-amine 35a (272 mg, 84 %). MS (ESI, positive ion) m/z: 440, 442 (M+1). 1-(3-bromophenyl)-4-fluoro-1-(5-methoxy-4,6-dimethylpyridin-2-yl)-1Hisoindol-3-amine (272 mg, 0.62 mmol), pyrimidin-5-ylboronic acid (92 mg, 0.74 mmol), cesium carbonate (604 mg, 1.85 mmol) and Pd(II)Cl 2 dppf*ch 2 Cl 2 (50.4 mg, 0.06 mmol) were dissolved in DME:EtH:Water (6:3:1) (5 ml) and irradiated in a microwave oven for 20 min at 150 C. EtAc, water and brine were added and the organic phase was collected, dried, filtered and purified with preparative HPLC to give the title compound (45 mg, 17% yield). 1 H MR (600 MHz, DMS-d 6 ) δ ppm 2.20 (s, 3 H), 2.39 (s, 3 H), 3.64 (s, 3 H), 6.56 (br. s., 2 H), 7.21 (t, J=7.90 Hz, 1 H), (m, 2 H), 7.52 (d, J=7.83 Hz, 2 H), 7.60 (d, J=7.40 Hz, 1 H), 7.71 (s, 1 H), 7.96 (d, J=7.69 Hz, 1 H), 8.96 (s, 2 H), 9.17 (s, 1 H). MS (ESI, positive ion) m/z: 440 (M+1). S17

18 Br 5-(3-Amino-1-(3-bromophenyl)-4-fluoro-1H-isoindol-1-yl)-2-methoxy-3- methylbenzonitrile (36a). Magnesium turnings (37.3 mg, 1.53 mmol) were stirred under Ar(g) for 10 min and LiCl (0.5 M in TH) (1.84 ml, 0.92 mmol) was added followed by DIBAL-H (1 M in TH) (6.14 µl, 6.14 µmol). The mixture was stirred for 5 min and then 5-bromo-2- methoxy-3-methylbenzonitrile (194 mg, 0.86 mmol) in TH (1 ml) was added in one portion and stirred for 2 h. The mixture was transferred to -((3-bromophenyl)(2-cyano-3- fluorophenyl)methylene)-2-methylpropane-2-sulfinamide 17 (250 mg, 0.61 mmol) in TH (3 ml) at rt and stirred over night. MeH (4 ml) was added and the mixture was treated with HCl in MeH (1.47 ml, 1.84 mmol). ahc 3 (saturated) was added and the mixture was extracted with EtAc. The combined organic phases were washed with brine, dried over MgS 4 and concentrated. The crude product was purified by flash chromatography to give the title compound (88 mg, 32% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm 2.21 (s, 3 H), 3.87 (s, 3 H), 6.64 (br. s., 2 H), (m, 3 H), (m, 3 H), (m, 2 H), 7.70 (d, J=7.57 Hz, 1 H). MS (ESI, positive ion) m/z: 450, 452 (M + 1). 5-(3-Amino-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-1-yl)-2-methoxy-3- methylbenzonitrile (36). Pyrimidin-5-ylboronic acid (36 mg, 0.29 mmol), 5-(3-amino-1-(3- bromophenyl)-4-fluoro-1h-isoindol-1-yl)-2-methoxy-3-methylbenzonitrile (88 mg, 0.20 mmol), cesium carbonate (191 mg, 0.59 mmol) and Pd(II)Cl 2 dppf*ch 2 Cl 2 (7.9 mg, 9.8 µmol) were S18

19 dissolved in DME:EtH:water (6:3:1) (2 ml) and irradiated in a microwave oven for 20 min at 140 C. EtAc, water and brine were added and the organic phase was collected, filtered through celite, washed with brine, dried and concentrated. The title compound was isolated using preparative HPLC (55 mg, 63% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm 2.20 (s, 3 H), 3.86 (s, 3 H), (m, 1 H), (m, 3 H), 7.55 (m, 2 H), (m, 2 H), 7.84 (d, J=7.57 Hz, 1 H), 9.03 (s, 2 H), 9.17 (s, 1 H). MS (ESI, positive ion) m/z: 450 (M + 1). Br 5-(3-Amino-1-(3-bromophenyl)-4-fluoro-1H-isoindol-1-yl)-1,3-dimethylpyridin-2(1H)-one (37a). tert-buli (1.97 ml, 3.15 mmol) was added dropwise to a solution of 5-bromo-1,3- dimethylpyridin-2(1h)-one (333 mg, 1.65 mmol) in TH (1.5 ml) at -100 C under a nitrogen atmosphere. After 5 min a solution of -((3-bromophenyl)(2-cyano-3-fluorophenyl)methylene)- 2-methylpropane-2-sulfinamide 17 (611 mg, 1.5 mmol) in TH (2 ml) was added. After 30 min on the thawing cooling bath and 1 h at rt, HCl (1.25 M in MeH) (4.8 ml, 6.0 mmol) was added and the resulting mixture was stirred at rt for 1 h. The mixture was concentrated and the resulting residue was dissolved in DCM and saturated aq ahc 3. The organic phase was separated, concentrated and purified by flash chromatography to give the title compound (110 mg, 17% yield). 1 H MR (400 MHz, DMS-d 6 ) δ ppm 1.94 (s, 3 H), 3.35 (s, 3 H), 6.61 (br. s., 2 H), 7.24 (s, 2 H), (m, 2 H), (m, 1 H), (m, 2 H), (m, 1 H), (m, 1 H). MS (ESI, positive ion) m/z: 426, 428 (M + 1). S19

20 5-(3-Amino-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-1-yl)-1,3-dimethylpyridin- 2(1H)-one (37). Using 5-(3-amino-1-(3-bromophenyl)-4-fluoro-1H-isoindol-1-yl)-1,3- dimethylpyridin-2(1h)-one (110 mg, 0.26 mmol) and pyrimidine-5-boronic acid (38 mg, 0.31 mmol) and following the procedure used to prepare 41 gave title compound (38 mg, 35% yield). 1 H MR (400 MHz, DMS-d 6 ) δ ppm 1.92 (s, 3 H), 3.36 (s, 3 H), 6.57 (br. s., 2 H), (m, 3 H), (m, 2 H), (m, 1 H), (m, 2 H), 7.72 (d, J=7.58 Hz, 1 H), (m, 2 H), 9.18 (s, 1 H). MS (ESI, positive ion) m/z: 426 (M + 1). 5-(3-Amino-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-1-yl)-1-ethyl-3- methylpyridin-2(1h)-one (38). Using 5-(3-amino-1-(3-bromophenyl)-4-fluoro-1H-isoindol-1- yl)-1-ethyl-3-methylpyridin-2(1h)-one 20 (51 mg, 0.12 mmol) and pyrimidin-5-ylboronic acid (18.7 mg, 0.15 mmol) and following the procedure used to prepare 41 gave title compound (36 mg, 71% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm (m, 1 H), (m, 2 H), 7.74 (d, J=7.57 Hz, 1 H), (m, 2 H), 7.52 (td, J=7.88, 4.73 Hz, 1 H), (m, 2 H), (m, 3 H), 6.56 (br. s., 2 H), (m, 2 H), 1.93 (s, 3 H), 1.14 (t, J=7.09 Hz, 3 H). MS (ESI, positive ion) m/z: 496 (M + 1). S20

21 5-(3-Amino-4-fluoro-1-(3-(5-fluoropyridin-3-yl)phenyl)-1H-isoindol-1-yl)-1-ethyl-3- methylpyridin-2(1h)-one (39). Using 5-(3-amino-1-(3-bromophenyl)-4-fluoro-1H-isoindol-1- yl)-1-ethyl-3-methylpyridin-2(1h)-one 20 (51 mg, 0.12 mmol) and 5-fluoropyridin-3-ylboronic acid (16.3 mg, 0.12 mmol) and following the procedure used to prepare 41 gave title compound (37 mg, 70% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm 8.66 (t, J=1.73 Hz, 1 H), 8.57 (d, J=2.52 Hz, 1 H), (m, 1 H), 7.73 (d, J=7.57 Hz, 1 H), (m, 2 H), (m, 1 H), (m, 2 H), (m, 3 H), 6.56 (br. s., 2 H), (m, 2 H), 1.93 (s, 3 H), 1.14 (t, J=7.09 Hz, 3 H). MS (ESI, positive ion) m/z: 457 (M + 1). C 5-(3-(3-Amino-1-(1-ethyl-5-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-1H-isoindol- 1-yl)phenyl)nicotinonitrile (40). Using 5-(3-amino-1-(3-bromophenyl)-4-fluoro-1H-isoindol-1- yl)-1-ethyl-3-methylpyridin-2(1h)-one 20 (51 mg, 0.12 mmol) and 5-cyanopyridin-3-ylboronic acid (17.1 mg, 0.12 mmol) and following the procedure used to prepare 41 gave title compound (6 mg, 11% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm 9.06 (d, J=2.21 Hz, 1 H), 9.00 (d, J=2.21 Hz, 1 H), 8.55 (t, J=2.05 Hz, 1 H), 7.75 (d, J=7.57 Hz, 1 H), (m, 2 H), (m, 3 H), (m, 3 H), 6.56 (br. s., 2 H), (m, 2 H), 1.92 (s, 3 H), (m, 3 H). MS (ESI, positive ion) m/z: 464 (M + 1). S21

22 C Br 2-(3-Bromobenzoyl)-4,6-difluorobenzonitrile (42c). A solution of 2-bromo-4,6- difluorobenzonitrile (5.46 g, 25.1 mmol) in TH (12 ml) was added slowly over a period of 30 min to a heterogeneous mixture of Rieke zinc in TH (17.9 ml, 27.3 mmol) under argon atmosphere at 0 C. When the addition was completed the mixture was stirred at 0 C for an additional 20 min and then 4 h at rt. The stirring was stopped and the mixture was allowed to stand over night. The solution was separated from the sedimentation, containing excess of Rieke zinc, and was transferred into a dropping funnel under dry and inert conditions. 3-Bromobenzoyl chloride (5 g, 22.8 mmol), copper(i) cyanide (0.35 ml, 11.4 mmol) and anhydrous lithium chloride (0.57 ml, 27.3 mmol) were mixed in MeC (20 ml) stirred under inert conditions and warmed to 50 C. The previously prepared clear zincate solution was added quickly and the reaction mixture was stirred at 50 C for 1 h. Then water (40 ml) and saturated aq H 4 Cl (40 ml) were added. The mixture was extracted with DCM (3x50 ml). The separated organic phase was washed with saturated aq a 2 C 3, brine, dried over anhydrous sodium sulfate, filtered and concentrated. lash chromatographic purification afforded the title compound (3.76 g, 51% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm (m, 2 H), 7.43 (t, J=7.83 Hz, 1 H), 7.72 (dq, J=7.83, 0.84 Hz, 1 H), 7.83 (ddd, J=8.02, 1.96, 1.14 Hz, 1 H), 7.96 (t, J=1.77 Hz, 1 H). MS (ESI, positive ion) m/z: 324 (M+1). C S Br S22

23 -((3-Bromophenyl)(2-cyano-3,5-difluorophenyl)methylene)-2-methylpropane-2- sulfinamide (42b). 2-(3-Bromobenzoyl)-4,6-difluorobenzonitrile (0.98 g, 3.04 mmol) and 2- methyl-2-propanesulfinamide (0.52 g, 4.26 mmol) were added to DCM (12 ml) and stirred at room temperature for 10 min, followed by addition of titanium(iv) ethoxide (1.8 g, 7.9 mmol). The mixture was stirred under reflux conditions for 44 h, and was then cooled to rt and diluted with DCM (20 ml) and water (20 ml). The precipitation was filtered off and the filtercake was washed with DCM (20 ml) and methanol (20 ml). The filtrate was concentrated and the aq residue was extracted with DCM (3x20 ml). The combined organic layer was dried over anhydrous MgS 4, filtered and concentrated. lash chromatographic purification gave the title compound (0.43 g, 33% yield). 1 H MR (400 MHz, DMS-d 6 ) δ ppm 1.29 (s, 9 H), (m, 1 H), 7.58 (d, J=8.08 Hz, 1 H), 7.78 (t, J=1.77 Hz, 1 H), (m, 2 H). MS (ESI, positive ion) m/z: 369 (M+1). The observed mass corresponds to the fragment without the tertbutyl group. Br 5-(3-Amino-1-(3-bromophenyl)-4,6-difluoro-1H-isoindol-1-yl)-1-ethyl-3-methylpyridin- 2(1H)-one (42a). Isopropylmagnesium chloride lithium chloride complex, 1.3 M in TH solution (2.26 ml, 2.94 mmol) was added to a solution of 5-bromo-1-ethyl-3-methylpyridin-2(1H)-one in TH (4 ml) at rt under argon and stirred for 5 min. Then this mixture was added quickly to a solution of -((3-bromophenyl)(2-cyano-3,5-difluorophenyl)methylene)-2-methylpropane-2- sulfinamide (500 mg, 1.18 mmol) in TH (3 ml) at rt under argon. After stirring for 4 h HCl in MeH (2.8 ml, 3.5 mmol) was added and the mixture was stirred for an additional 20 min. The reaction mixture was concentrated and the residue was dissolved in DCM (10 ml) and washed S23

24 with saturated aq a 2 C 3 (5 ml). The organic phase was concentrated and the residue was purified by flash chromatography to afford the title compound (249 mg, 48% yield). 1 H MR (400 MHz, DMS-d 6 ) δ ppm 1.94 (s, 3 H), 3.37 (s, 3 H), 6.66 (br. s., 2 H), (m, 2 H), (m, 1 H), (m, 2 H), (m, 2 H), 7.61 (dd, J=8.21, 1.89 Hz, 1 H). MS (ESI, positive ion) m/z: 459 (M+1). (R)-5-(3-Amino-4,6-difluoro-1-(3-(5-(prop-1-ynyl)pyridin-3-yl)phenyl)-1H-isoindol-1-yl)- 1-ethyl-3-methylpyridin-2(1H)-one ((R)-42). 5-(3-Amino-1-(3-bromophenyl)-4,6-difluoro-1Hisoindol-1-yl)-1,3-dimethylpyridin-2(1H)-one (150 mg, 0.34 mmol), 5-(prop-1-ynyl)pyridin-3- ylboronic acid (71 mg, 0.44 mmol), Pd(II)Cl 2 dppf*ch 2 Cl 2 (13.9 mg, 0.02 mmol) and 2 M potassium carbonate aqueous solution (0.51 ml, 1.01 mmol) were mixed with degassed 2-methyl tetrahydrofuran (4 ml) under argon and heated to 80 C for 30 min. Then it was cooled to room temperature. Water (4 ml) was added and the mixture was extracted with 2-methyl tetrahydrofuran. The combined organic phase was washed with brine, dried over anhydrous MgS 4, filtered and concentrated. lash chromatographic purification of the residue afforded 5- (3-amino-4,6-difluoro-1-(3-(5-(prop-1-ynyl)pyridin-3-yl)phenyl)-1H-isoindol-1-yl)-1-ethyl-3- methylpyridin-2(1h)-one (112 mg, 69% yield). 1 H MR (500 MHz, DMS-d 6 ) δ ppm 1.93 (s, 3 H), (m, 3 H), 3.38 (s, 3 H), 6.60 (br. s., 2 H), (m, 3 H), (m, 2 H), (m, 2 H), 7.73 (dd, J=8.04, 1.73 Hz, 1 H), 7.99 (t, J=2.05 Hz, 1 H), 8.56 (d, J=1.89 Hz, 1 H), 8.73 (d, J=2.21 Hz, 1 H). MS (ESI, positive ion) m/z: 495 (M+1). This material, was subjected to enatiomer separation using SC preparative chromatography to yield the title S24

25 compound with an enantiomeric purity of >99.5 %. 1 H MR (400 MHz, DMS-d 6 ) δ ppm 1.16 (t, J=7.07 Hz, 3 H), 1.93 (s, 3 H), 2.10 (s, 3 H), (m, 2 H), 6.62 (br. s., 2 H), (m, 3 H), (m, 2 H), (m, 2 H), 7.77 (dd, J=8.21, 1.89 Hz, 1 H), 7.99 (t, J=2.15 Hz, 1 H), 8.56 (d, J=2.02 Hz, 1 H), 8.73 (d, J=2.27 Hz, 1 H). MS (ESI, positive ion) m/z: 495 (M+1). Synthetic procedures for intermediates: Cl 2-Methoxy-isonicotinoyl chloride. Thionyl chloride (30.0 ml, 0.39 mol) was added to a suspension of 2-methoxy-isonicotinic acid (15.0 g, 97.9 mmol) in anhydrous toluene (150 ml). The mixture was heated at 80 o C under nitrogen for 3 h, cooled to room temperature and filtered. The filtrate was concentrated in vacuo to afford the title compound (7.7 g, 46% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 4.00 (s, 3 H) 5.30 (d, J= 5.3 Hz, 1H) 7.38 (s, 1 H) 8.36 (d, J = 5.3 Hz, 1 H). Pyridine-2,4-dicarboxylic acid diethyl ester. Concentrated H 2 S 4 (20 ml) was added drop wise to a suspension of pyridine-2,4-dicarboxylic acid monohydrate (25.0 g, mmol) in ethanol (400 ml) and the resulting reaction mixture was heated at 70 o C for 14 hours. The S25

26 reaction mixture was cooled to room temperature, poured slowly into ice-cold saturated ahc 3 solution and the mixture was extracted with EtAc. The combined extracts were washed with saturated ahc 3 dried over MgS 4 and concentrated in vacuo. The crude material was cooled to 0 o C and triturated with Et 2 /hexane to afford the title compound (25.7 g, 85% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm (m, 6 H), (m, 4 H), 8.01 (d, J=4.76 Hz, 1 H), 8.61 (s, 1 H), 8.89 (d, J=4.76 Hz, 1 H). 2-ormyl-isonicotinic acid ethyl ester. DIBAL-H (1M in TH, 67.0 ml, 67.0 mmol) was added drop wise to a solution of pyridine-2,4-dicarboxylic acid diethyl ester (3.0 g, mmol) in dry TH (60 ml) at -78 o C. The reaction mixture was stirred for 2.5 h at -78 o C and then poured slowly into an ice-cold mixture of AcH (10 ml) and water (50 ml). The resulting mixture was allowed to warm to room temperature and stirred for 1 h. The mixture was then treated with saturated ahc 3 solution to adjust the ph to ~ 8 and extracted with EtAc. The combined extracts were washed with saturated ahc 3 solution, dried over MgS 4 and concentrated in vacuo. The residue was triturated with hexane to afford 2-formyl-isonicotinic acid ethyl ester (2.6 g, 86% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 1.44 (t, J=7.03 Hz, 3 H), 4.46 (q, J=7.16 Hz, 2 H), (m, 1 H), 8.49 (s, 1 H), 8.95 (d, J=4.69 Hz, 1 H), (s, 1 H). S26

27 2-Difluoromethyl-isonicotinic acid ethyl ester. To an ice-cold solution of crude 2-formylisonicotinic acid ethyl ester (4.79 g, 26.8 mmol) in anhydrous DCM (120 ml) was added Deoxo- luor (11.8 g, 53.5 mmol) slowly. The resulting mixture was allowed to warm to r.t. over 6 h. The mixture was then neutralized by the cautious addition of saturated ahc 3. The mixture was further diluted with DCM (200 ml) and the organic phase was separated. The aqueous layer was extracted with dichloromethane (150 ml). The combined extracts were dried over a 2 S 4 and concentrated in vacuo. The residue was purified by flash column chromatography using 20% ethyl acetate in hexane to afford 2-difluoromethyl-isonicotinic acid ethyl ester (3.77 g, 70% yield): 1 H MR (400 MHz, CDCl 3 ) δ ppm 1.44 (t, J=7.0 Hz, 3 H), 4.45 (q, J=7.0 Hz, 2 H), 6.70 (t, J=55.3 Hz, 1 H), 7.98 (d, J=5.08 Hz, 1 H), 8.19 (s, 1 H), 8.82 (d, J=5.08 Hz, 1 H). 19 MR (376 MHz, CDCl 3 ) δ ppm H 2-Difluoromethyl-isonicotinic acid. Lithium hydroxide (1.38 g, 57.4 mmol) was added in one portion to a solution of 2-difluoromethyl-isonicotinic acid ethyl ester (5.77 g, 28.7 mmol) in MeH (90 ml) and the mixture was stirred at rt over night. HCl in Et 2 (2.0 M, 28.7 ml, 57.4 mmol) was added slowly and the mixture was stirred for 5 min. The mixture was concentrated S27

28 and freeze dried to afford 2-difluoromethyl-isonicotinic acid (6.39 g, containing lithium chloride) which was used in the next step without further purification: 1 H MR (400 MHz, DMS-d 6 ) δ ppm 7.12 (t, J=54.7 Hz, 1 H), 8.03 (d, J=3.91 Hz, 1 H), 8.10 (s, 1 H), 8.90 (d, J=5.08 Hz, 1 H). 19 MR (376 MHz, DMS-d 6 ) δ ppm S 2-Difluoromethyl-thioisonicotinic acid S-ethyl ester. xalyl chloride (5.08 g, 40 mmol) was added slowly to a suspension of 2-difluoromethyl-isonicotinic acid (20 mmol, contaminated with lithium chloride) in anhydrous dichloromethane (100 ml) at 0 o C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was concentrated in vacuo to remove excess oxalyl chloride and the residue was redissolved in anhydrous dichloromethane (100 ml), and cooled to 0 o C. Triethylamine (4.05 g, 40 mmol) was added slowly, followed by ethanethiol (1.49 g, 24 mmol) and the resulting mixture was stirred for 2 hours. Water (100 ml) was then added; the organic layer was separated, dried over sodium sulphate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (gradient elution 5% to 10% ethyl acetate in hexanes) to yield 2-difluoromethylthioisonicotinic acid S-ethyl ester (3.8 g, 88% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm (m, 3 H) 3.15 (q, J=7.42 Hz, 2 H) 6.70 (t, J= Hz, 1 H) 7.86 (d, J=4.30 Hz, 1 H) 8.08 (s, 1 H) 8.82 (d, J=5.08 Hz, 1 H). 19 MR (376 MHz, CDCl 3 ) δ ppm MS (ESI, positive ion) m/z: 218 (M+1). S28

29 H Br 6-Bromo-2,4-dimethyl-3-hydroxypyridine. A solution of bromine (4.2 ml, 81.2 mmol) in anhydrous pyridine (80 ml) was added dropwise to a solution of 2,4-dimethyl-3-hydroxypyridine (10.0 g, 81.2 mmol) in anhydrous pyridine (160 ml). The mixture was stirred at rt for 1 h, concentrated under reduced pressure and then further dried under vacuum. The residue was taken up in water (100 ml) and the resulting mixture was stirred for 0.5 h at room temperature. The precipitated solid was collected by filtration, washed with water and air dried overnight to afford 6-bromo-2,4-dimethyl-3-hydroxypyridine (8.7g, 53% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 7.10 (s, 1 H), 4.73 (br, s, 1 H, H), 2.45 (s, 3 H), 2.23 (s, 3 H) Br 6-Bromo-3-methoxy-2,4-dimethyl-pyridine. A mixture of 6-bromo-2,4-dimethyl-3- hydroxypyridine (8.7 g, 43.1 mmol), iodomethane (4.0 ml, 64.6 mmol) and potassium carbonate (11.9 g, 86.1 mmol) in acetone (250 ml) was heated at reflux temperature for 3 hours. The reaction mixture was then cooled to room temperature and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography using 20% ethyl acetate in hexane to afford 6-bromo-3-methoxy-2,4-dimethylpyridine as a yellowish oil (7.9g, 85% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 7.14 (s, 1 H), 3.72 (s, 3 H), 2.48 (s, 3 H), 2.26 (s, 3 H) MS (ESI, positive ion) m/z: 216, 218 (M+1). S29

30 S 2,6-Dimethyl-thioisonicotinic acid S-ethyl ester. Triethylamine (18.8 ml, mmol) was added to a suspension of 2,6-dimethyl-isonicotinic acid (5.1 g, 33.7 mmol) in anhydrous dichloromethane (110 ml). The mixture was stirred at room temperature for 5 min, cooled to 0 o C and isobutyl chloroformate (6.62 ml, 50.6 mmol) was added dropwise. The reaction mixture was stirred at 0 o C for 30 min, ethanethiol (3.74 ml, 50.6 mmol) was added dropwise and stirring at 0 o C was continued for an additional 30 min. The reaction mixture was diluted with dichloromethane (100 ml), washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The crude product was purified by flash chromatography using 30% ethyl acetate in hexanes to afford 2,6-dimethyl-thioisonicotinic acid S-ethyl ester (5.1 g, 78% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm 7.40 (s, 2 H), 3.10 (q, J = 7.4 Hz, 2 H), 2.60 (s, 6 H), 1.36 (t, J = 7.4 Hz, 3 H) C 2-(2,6-Dimethyl-pyridine-4-carbonyl)-6-fluoro-benzonitrile. To a degassed solution of 2,6- dimethyl-thioisonicotinic acid S-ethyl ester (4.3 g, 22.0 mmol) in anhydrous toluene (80 ml) was added Pd(PPh 3 ) 2 Cl 2 (1.55 g, 2.2 mmol), followed by dropwise addition of 2-cyano-3- fluorophenylzinc iodide (0.5M in TH, 44.0 ml, 22.0 mmol). The reaction mixture was stirred at rt for 2 h with continued bubbling of nitrogen. Water (50 ml) and saturated aqueous H 4 Cl (100 ml) were added and the mixture was extracted with ethyl acetate (2 x 200 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated under S30

31 reduced pressure. The residue was purified by flash chromatography using a gradient of 25% to 100% ethyl acetate in hexanes to afford 2-(2,6-dimethyl-pyridine-4-carbonyl)-6-fluorobenzonitrile (3.4 g, 61% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm (m, 1 H), 7.49 (t, J = 8.4 Hz, 1 H), 7.45 (d, J = 7.7 Hz, 1 H), 7.24 (s, 2 H), 2.62 (s, 6 H). 19 MR (400 MHz, CDCl 3 ) δ ppm C S 2-Methyl-propane-2-sulfinic acid (2-cyano-3-fluoro-phenyl)-(2,6-dimethyl-pyridin-4-yl)- methyleneamide. A solution of 2-(2,6-dimethyl-pyridine-4-carbonyl)-6-fluoro-benzonitrile (4.9 g, 19.3 mmol) in dry TH (60 ml) was added to a solution of titanium (IV) ethoxide (10.0 ml, 48.2 mmol) in dry TH (20 ml) at rt. 2-Methyl-2-propanesulfinamide (2.68 g, 22.2 mmol) was then added in one portion and the reaction mixture was heated at reflux temperature for 20 h. The reaction mixture was cooled to rt and methanol (120 ml) was added, followed by saturated sodium bicarbonate solution (10 ml). The resulting suspension was filtered through a pad of sodium sulfate and the solids were washed thoroughly with ethyl acetate. The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography using a gradient of 50% to 100% ethyl acetate in hexanes to give 5.8 g of title compound. urther purification by preparative HPLC using a gradient of 39% to 100 % to acetonitrile in water afforded 2-methyl-propane-2-sulfinic acid (2-cyano-3-fluoro-phenyl)-(2,6-dimethyl-pyridin-4- yl)-methyleneamide (4.1 g, 60% yield). 1 H MR (400 MHz, CDCl 3 ) δ ppm (m, 1 H), S31

32 7.34 (t, J = 8.7 Hz, 1 H), 7.20 (d, J = 7.4 Hz, 1 H), 7.02 (s, 2 H), 2.56 (s, 6 H), 1.40 (s, 9 H). 19 MR (400 MHz, CDCl 3 ) δ ppm , MS (ESI, positive ion) m/z: 358 (M+1) CH Br 2-Bromo-4,6-difluorobenzaldehyde. Isopropylmagnesium chloride lithium chloride complex (127 ml, 164 mmol) was added slowly over a period of 25 min to a solution of commercially available bromo-3,5-difluoro-2-iodobenzene (50 g, 156 mmol) in 2-methyltetrahydrofuran (300 ml) at -10 C under nitrogen. When the addition was complete the mixture was stirred for 30 min. Then 4-formylmorpholine (17.34 ml, mmol) was added and the mixture was stirred for 4 h meanwhile it was allowed to warm up to room temperature. Then it was quenched with 1 M HCl aqueous solution (175 ml). To the mixture water (250 ml) was added and extracted with EtAc (2*100 ml). The combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and dried under reduced pressure at room temperature overnight. 2-bromo- 4,6-difluorobenzaldehyde was obtained as yellow solid (33.1 g, 96 % yield). 1 H MR (400 MHz, DMS-d 6 ) δ ppm (m, 1 H) (m, 1 H) (s, 1 H). MS (ESI, positive ion) m/z: 221 (M+1). Reference: J. D. Kehlbeck; E. J. Dimise; S. M. Sparks; S. errara; J. M. Tanski; C. M. Anderson, Synthesis 2007, C Br S32

33 2-Bromo-4,6-difluorobenzonitrile (11). 2-Bromo-4,6-difluorobenzaldehyde (291 g, 1318 mmol), lauryl sulfate sodium salt (76 g, 263 mmol), iodobenzene diacetate (637 g, 1977 mmol) and ammonium acetate (524 g, 6590 mmol) in water (1200 ml) was stirred at 70 C. After 30 min the mixture was cooled to rt and natrium thiosulfate (235 g, 1.48 mol) and water (250 ml) were added and stirred for additional 10 min. Then dichloromethane (500 ml) was added and extracted. The separated organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to yield 2-bromo-4,6-difluorobenzonitrile. 1 H MR (500 MHz, DMS-d 6 ) δ ppm (m, 1 H) (m, 1 H). MS (ESI, positive ion) m/z: 218 (M+1). Reference: C. Zhu; L. Ji; Y. Wei, Synthesis 2010, C ZnBr 2-Cyano-3,5-difluorophenylzinc bromide. To a vigorously stirred suspension of Rieke Zinc (5 g in 100 ml of TH), a solution of 2-bromo-4,6-difluoro-benzonitrile (2.6 g, 11.4 mmol) in dry TH (20 ml) was added dropwise over 1 h at 10 C. The mixture was allowed to reach room temperature, stirred for 30 minutes and then stored at -10 C. Br 5-Bromo-1-ethyl-3-methylpyridin-2(1H)-one. Commercially available 5-bromo-3- methylpyridin-2-ol (60 g, 319 mmol) and potassium carbonate (88 g, 638 mmol) were slurried in S33

34 dimethoxyethane (400 ml). Iodoethane (28 ml, 351 mmol) was added and the mixture was stirred at room temperature overnight. Then it was filtered and the filtercake was washed with EtAc (300 ml). The filtrate was extracted with water, washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated. The obtained orange solid was recrystallized from EtAc (50 ml) by addition of heptane (300 ml). The precipitate was filtered and washed with heptane (100 ml) and purified by flash chromatography to obtaine the title compound (39 g, 57 %). 1 H MR (400 MHz, DMS-d 6 ) δ ppm 1.19 (t, J=7.20 Hz, 3 H) (m, 3 H) 3.89 (q, J=7.07 Hz, 2 H) 7.44 (dd, J=2.78, 1.26 Hz, 1 H) 7.89 (dd, J=2.78, 0.76 Hz, 1 H). MS (ESI, positive ion) m/z: 218 (M+1). Reference: M. Ando et al. Bioorganic & Medicinal Chemistry 17 (2009) pp S34