Supporting Information Zhang et al. 10.1073/pnas.1219457110 SI Experimental Procedures with brine (2.5 L), dried over anhydrous sodium sulfate, and filtered. Synthesis and Characterization of PLX647. PLX647, 5-(1H-pyrrolo The solvents were removed at reduced pressure to provide ∼560 g [2,3-b]pyridin-3-ylmethyl)-N-[[4-(trifluoromethyl)phenyl]methyl] of crude product. This material was triturated with methyl tert-butyl pyridin-2-amine, was synthesized from commercially available ether:hexanes (1:1.3) to yield compound 4 (360 g, 79.0%) as a yel- 1 5-bromopyridin-2-amine, 4-(trifluoromethyl)benzaldehyde and low solid. HNMRδ (CDCl3) 9.84 (s, 1H), 8.57 (s, 1H), 7.95 (dd, 1H-pyrrolo[2,3-b]pyridine following the synthetic route depicted 1H, J = 2.0, 8.8 Hz), 7.65 (d, 2H, J = 8.4Hz),7.50(d,2H,J= 8.4 Hz), in Scheme S1. 6.50 (d, 1H, J = 8.8 Hz), 5.72 (s, 1H), 4.77 (d, 2H, J = 5.2 Hz). Step 3 Step 1 Step 2 1 2 3 4 Step 4 Step 5 7a R = H 5 6 PLX647 7b R = CH3 Scheme S1. Step 1. Synthesis of 5-bromo-N-[[4-(trifluoromethyl)phenyl]methyl]pyridin- Step 3. Synthesis of tert-butyl N-(5-formyl-2-pyridyl)-N-[[4-(trifluoromethyl) 2-amine (compound 3). To a suspension of 5-bromopyridin-2-amine phenyl]methyl]carbamate (compound 5). To a solution of 6-[[4-(tri- (compound 1, 446.8 g, 2.58 mol) in acetonitrile (7.2 L) under an fluoromethyl)phenyl]methylamino]pyridine-3-carbaldehyde (com- atmosphere of nitrogen at room temperature, 4-(trifluoromethyl) pound 4, 485.5 g, 1.73 mol) in anhydrous THF (3.7 L), under benzaldehyde (compound 2,452.0g,2.60mol),triethylsilane nitrogen, di-tert-butyl dicarbonate (589.0 g, 2.70 mol), 4-dimethy- (1,250 mL, 7.82 mol), and trifluoroacetic acid (600 mL, 7.84 mol) laminopyridine (11.8 g, 0.097 mol), and N,N-diisopropylethyl- were added sequentially via addition funnel. The reaction was amine (550.0 mL, 3.16 mol) were added sequentially via additional refluxed (∼73 °C) for 16 h. The reaction mixture was cooled, funnel over 30 min. The reaction was stirred at room temperature concentrated under reduced pressure, and poured into a mixture for 1 h, poured into water (3 L), and extracted twice with ethyl of water (5 L) and hexane (3 L). The organic layer was separated acetate ( 3L and 1 L). The organic layers were combined, washed and discarded. Saturated potassium carbonate solution was added with brine (1.5 L), dried over anhydrous sodium sulfate, filtered, to the aqueous layer with stirring until a pH ∼9 and was extracted and concentrated to give crude product 5 (709 g, 107.6%), which twice with ethyl acetate (3 L each). The organic layers were com- was used in the next step without further purification. bined, washed with brine (2 L), dried over anhydrous sodium sul- Step 4. Synthesis of tert-butyl N-[5-[hydroxy(1H-pyrrolo[2,3-b]pyridin-3- fate, and filtered. Solvents were removed under reduced pressure yl)methyl]-2-pyridyl]-N-[[4-(trifluoromethyl)phenyl]methyl] carbamate to provide ∼995 g of crude product, which upon recrystallization (compound 7). To a solution 1H-pyrrolo[2,3-b]pyridine (compound from methyl tert-butyl ether/hexane (2:3) yielded 656.2 g of com- 6, 142.9 g, 1.21 mol) in methanol (4.5 L), under nitrogen, tert-butyl 1 fl pound 3 (76.6% yield) as white needles. HNMRδ (CDCl3) 8.16 N-(5-formyl-2-pyridyl)-N-[[4-(tri uoromethyl)phenyl]methyl] (d, 1H, J = 2.4 Hz), 7.62 (d, 2H, J = 8.0 Hz), 7.52 (dd, 1H, J = 2.4, carbamate (compound 5, 478.1 g, 1.26 mol) and sodium hydroxide 8.8Hz),7.48(d,2H,J= 8.0Hz),6.33(d,1H,J= 8.8 Hz), 5.12 (145 g, 3.63 mol) were added. The reaction was stirred at room (s, 1H), and 4.61 (d, 2H, J = 6.0 Hz). temperature for 15 h, poured into water (12 L), and the product Step 2. Synthesis of 6-[[4-(trifluoromethyl)phenyl]methylamino]pyridine-3- was extracted twice with ethyl acetate (9 L and 4 L). The com- carbaldehyde (compound 4). To a cooled (dry ice–acetone bath) bined organic layers were washed twice with brine (2.5 L each), solution of 5-bromo-N-[[4-(trifluoromethyl)phenyl]methyl]pyridin- dried over sodium sulfate, filtered, and concentrated under re- 2-amine (compound 3, 541.7 g, 1.63 mol) in anhydrous THF (6.5 L) duced pressure to provide a crude mixture of the hydroxy 7a and under nitrogen was added n-butyllithium (2.5 M in hexane, 1,970 mL, methoxy 7b (682 g, 117%) which was used in the next step without + + 4.93 mol) via additional funnel maintaining the internal temperature further purification. MS (electrospray ionization) [M+H ] = below –57 °C. The reaction mixture was stirred for 1.5 h at ≤–65 °C 499.4 (7a). before the addition of anhydrous dimethylformamide (265 mL, Step 5. Synthesis of 5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethyl)-N-[[4- 3.42 mol) over a period of 30 min. The reaction mixture was al- (trifluoromethyl)phenyl]methyl]pyridin-2-amine (PLX647). To a solution lowed to warm up to about 0 °C in 1 h. Saturated NH4Cl aqueous of tert-butyl N-[5-[hydroxy(1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]- solution (3 L) was added to quench the reaction and the products 2-pyridyl]-N-[[4-(trifluoromethyl)phenyl] methyl] carbamate and were extracted with ethyl acetate (8 L). The organic layer was washed tert-butyl N-[5-[methoxy(1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]- Zhang et al. www.pnas.org/cgi/content/short/1219457110 1of10 2-pyridyl]-N-[[4-(trifluoromethyl)phenyl]methyl]carbamate (7a were grown in ESF921 media (Expression Systems) at 2.0 × 106 and 7b, 682 g, 1.37 mol) in anhydrous acetonitrile (4.1 L), under cells/mL and were infected with a multiplicity of infection (MOI) = 5. nitrogen, triethylsilane (700 mL, 4.38 mol) and trifluoroacetic Cells were harvested 72 h postinfection and lysed using 10 mM acid (500 mL, 6.54 mol) were added via additional funnel over K-phosphate (pH 8.0), 25 mM NaCl, 0.5 mM PMSF, and pro- a period of 25 min. The reaction mixture was refluxed (∼66 °C) tease inhibitor mixture (EMD Millipore). Following clarification for 22 h, cooled, concentrated under reduced pressure, and by centrifugation the supernatant was adjusted to 40 mM K- poured into water (4 L) and hexane (4 L). The organic layer was phosphate (pH 8.0), 200 mM NaCl, and 30 mM imidazole. FMS separated and discarded. The pH of the aqueous layer was ad- proteins were purified through a combination of IMAC and further justed by the addition of a saturated aqueous solution of po- purified by ion-exchange and size-exclusion chromatography. The tassium carbonate with stirring until the pH solution was at ∼9 clarified cell extract was bound to an IMAC column (HiTrap, and the product was extracted twice with ethyl acetate (3 L each). nickel-charged chelating Sepharose Fast Flow; GE Healthcare) The organic layers were combined, washed with water (2 L) and then washed using 40 mM phosphate (pH 8.0), 200 mM NaCl, and brine (2 L), dried over anhydrous sodium sulfate, filtered, and 30 mM imidazole and eluted using 25 mM Hepes (pH 7.0), 150 mM concentrated to give crude product (546 g). The solid was tritu- NaCl, and 300 mM imidazole. Following elution, 10 mM DTT was rated with methanol and dried to give PLX647 (262.8 g, 58.9%) as added to the eluent. Pooled fractions were further diluted with + + a yellow solid. MS (electrospray ionization) [M+H ] = 383.1. 1H 20 mM Hepes (pH 7.0) before loading over a cation exchange NMR δ (DMSO-d6) 11.36 (s, 1H), 8.15 (s, 1H), 7.92 (s, 1H), 7.81 column (HiTrap SP Fast Flow; GE Healthcare), proteins separated/ (1H, d, J = 7.2 Hz), 7.62 (d, 2H, J = 7.6 Hz), 7.48 (d, 2H, J = eluted using a NaCl gradient. Final purity was achieved by further 7.2 Hz), 7.27 (d, 1H, J = 7.6 Hz), 7.23 (s, 1H), 7.00 (t, 1H,, J = chromatography using a Superdex S200 gel filtration column (26/ 4.0 Hz), 6.95 (d, 1H, J = 7.2 Hz), 6.44 (d, 1H, J = 8.0 Hz), 4.51 60; GE Healthcare) in 20 mM Hepes (pH 7.0), 150 mM NaCl, and 13 (d, 2H, J = 4.0 Hz), 3.81 (s, 2H). C NMR δ (DMSO-d6) 157.50, 10 mM DTT. 149.32, 147.23, 146.65, 142.97, 137.84, 128.24, 127.66 (q, JCF = Crystallization and structure determination. The KIT protein, at 31.7 Hz), 127.20, 125.56 (q, JCF = 5.6 Hz), 125.01, 124.98 (q, JCF = a concentration of ∼5 mg/mL, was incubated with 1 mM PLX647 217 Hz), 123.80, 119.60, 115.38, 113.76, 108.61, 44.40, 27.94. before setting up the 24-well crystallization trays using the sitting drop method at 20 °C. The protein was diluted 1:1 with mother Protein Purification, Crystallization, and Structure Determination. liquor consisting of 1.6 M ammonium sulfate, 2.0 M sodium Purification of KIT proteins.