（合 成 実 験 ）
Ge neral Met hods. All reactions involving air- or moisture-sensitive reagents or inter mediates were perfor med under an inert atmosphere of nitrogen or argon in dry glassware.
Unless other wise noted, solvents and reagents were reagent grade a nd used without purification. Microwave reactions were perfor med using a Biota ge Initiator microwave reactor (Biotage Japan Ltd.). Analytical TLC was carried on Merck silica gel 60 GF254 plates or Fuji Silysia Chemical Chr omatorex NH -TLC plates. Silica gel column chr omatography was perfor med using 0.045–0.075 mm Wakogel C-300, Merck 0.063–0.200 mm silica gel 60, Fuji Silysia Chemical 100 –200 mesh Chr omatorex NH silica, or Purif -Pack (Si or NH, Moritex Corp.) using the indicated eluents. Yields are unoptimized.
Preparative high perfor ma nce liquid chr omatography (Prep-HPLC) was perfor med at conditions: Column: Welchrom C18 (150 mm x 20 mm); Wavelength 220 nm; Mobile phase:
A CH3CN (0.1% TFA); B water (0.1% TFA); Flow rate: 25 mL /min; Injection volume: 2 mL;
Run time: 30 min; Equilibrat ion: 5 min. Proton nuclear ma gnetic resona nce (1H NMR) spectra were recorded on 200 MHz, 300 MHz, 400 MHz, and/or 500 Mz spectr ometers (Bruker DPX-300, Bruker Avance II-300, Bruker Avance III-400 plus, Bruker Avance III 400 MHz Ultrashield 400 PlusTM Digital, or Br uker Ava nce NMRS -400 NMR spectr ometers).
Chemical shifts are given as δ values (ppm), using tetramethylsilane as the internal standard.
Coupling constants are reported in hertz (Hz). Spectral splitting patter ns are designated as follow: s, singlet; br, broad; d, doublet; t, triplet; m, multiplet. Liq uid chromatography-mass spectra (LC−MS) spectra were recorded on a Shimazu LC -20AD or Agilent LC/MSD 1200 Series spectrometer, outfitted with a n L -column2 ODS (3.0 mm i. d. × 50 mm, 3 μ m, CERI), Xbridge C18 (4.6 mm i.d. × 50 mm, 3.5 μm), SunFire C18 (4.6 mm i.d. × 50 mm, 3.5 μm), Zorbax Ext C18 (4.6 mm i.d. × 50 mm, 5 μm), or Restek Ultra AQ C18 (2.1 mm i.d. × 30 mm, 3.0 μm) column. LC-MS samples were eluted under basic or acidic conditio ns.
Basic conditions: Flow rate = 1.5 mL/min; Mobile phase: A = ultrapure water/ CH3CN = 90/10 (5 mM NH4OAc), B = ultrapure water/ CH3CN = 10/90 (5 mM NH4OAc); gradient: 5–
90% B over 0.9 min followed by 90% B isocratic over 1.1 min. Or flow rate = 2.0 mL/min;
mobile phase: A = water (10 mM NH4HCO3), B = CH3CN; gradient: 5–95% B in 1.2 minutes.
Or flow rate = 1.5 mL/min; mobile phase: A = water/ CH3CN = 90/10 (10 mM NH4OAc), B
= water/ CH3CN = 70/30 (10 mM NH4OAc); C = water/ CH3CN = 10/90 (10 mM NH4OAc) ; gradient: 0–100% B in 1.5 minutes, followed by 0 –100% over 3.0 min. Acidic conditions:
Flow rate = 1.5 mL/min; mobile phase: A = ultrapure water (0.05% TFA), B = CH3CN (0.05%
TFA); gradient: 5–90% B over 0.9 minutes followed by 90% B isocratic over 1.1 m in. Or flow rate = 1.5 mL/min; mobile phase: A = water (0.01% TFA), B = CH3CN (0.01% TFA);
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gradient: 5–95% B in 1.2 minutes. Or flow rate = 1.5 mL/min; mobile phase: A = water/
CH3CN = 98/2 (0.05% HCO2H), B = water/ CH3CN = 90/10 (0.05% HCO2H); C = water / CH3CN = 2/98 (0.05% HCO2H); gradient: 0–100% B in 0.75 minutes, followed by 0 –100%
over 1.0 min. MS spectra were recor ded using a Shima dzu LCMS -2020 or an Agilent 6130 spectrometer with electrospray ionization. High perfor ma nce liquid chromatograph y (HPLC) spectra were obtained on a Shimazu LC - 20AD outfitted with a L-column2 ODS (2.1 mm i.d. x 30 mm, 2 μm, CERI) or Capcell Pak C18AQ (Shiseido). HPLC samples were eluted under basic or acidic conditions. Basic condition: Flow rate = 0.5 mL/min; mobile phase: A = 10 mM aqueous solution of NH4HCO3 (0.2% H2CO3), B = CH3CN (0.2% H2CO3);
gradient: 5–95% B in 5.2 minutes; Injection volume of sa mple: 10 μL. MS spectra wer e recor ded using Corona Ultra RS with electr ospray ionization. HPLC spectra were used to confirm ≥95% purity for each compound shown in the SAR tables. Elemental analyses (Anal.) was conducted at Sumika Chemical Analysis Service, Ltd.
2,4-Difluoro-3-(1H-pyrazolo[3,4-b]pyridin-5-ylethynyl)aniline (98). 3-ethynyl-2,4-difluor oaniline (94, 1.0 g, 6.5 mmol), 5 -bromo- 1H-pyrazolo[3,4-b]pyridine (99, 1.3 g, 6.5 mmol), triethyla mine (2.7 ml, 19.6 mmol), P dCl2(Amphos) (0.088 g, 0.13 mmol), copper(I) iodide (0.044 g, 0.23 mmol) were dissolved in DMSO (15 ml), and the mixture was stirred at 120 °C for 1 h under microwave irradiation. The reaction with sa me scale was repeated one time in a separate reaction tube. Both mixtur es were combined, and saturated aqueous NaHCO3 (50 mL) and EtOAc (50 mL) were added. The insoluble materials were filtered off and washing with the mixture of EtOAc - THF (3:1). From the filtrate, the combined or ganic layers were collected. From the aqueous layer, extraction wi th EtOAc-THF (5:1, 30 mL) was carried out 3 times. The combined organic layers were washed with brine, dried over MgSO4, a nd concentrated under vacuum. The residue was purified by column chr omatography (NH silica gel, eluted with 0 –100% EtOAc in hexane) and treated with EtOAc/IPE/hexane (20ml/10ml/10ml). The resulting precipitate was collected by filtration to give 98 (2.2 g, 61%) as pale yellow solid. 1H NMR (300 MHz, DMSO -d6) δ 5.22 (2H, s), 6.77–7.00 (2H, m), 8.21 (1H, s), 8.51 (1H, d, J = 2.1 Hz), 8.67 (1H, d, J = 2.1 Hz), 13.97 (1H, br s). MS (API) m/z calculated for C14H8F2N4 + H+ (M + H)+ : 271.1. Found: 271.0 (M + H)+.
5-Bromo-N-[2,4-difluoro(1H-pyrazolo[3,4-b]pyridin-5-ylet hy nyl)phe nyl]pyridine -3-sulfo namide (93a). Compound 98 (149 mg, 0.552 mmol) was dissolved in pyridine (5 ml) , and to this solution was added 5-bromopyridine -3-sulfonyl chloride (95a, 157 mg, 0.612 mmol) at -15 ºC. After stirring at -15 ºC for 1 h, compound 95a (143 mg, 0.552 mmol) was added to the mix ture, which was stirred at -15 ºC for 30 min. MeOH (2 dr ops) was adde d
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into the reaction mixture, and the whole was stirred at room temperature for 10 min. The solvent was evaporated, and the residue was purified by column chromatography (silica gel, eluted with 25–60% EtOAc in hexane) to give 93a (158 mg, 58%) as a white solid. 1H NMR (300 MHz, DMSO -d6) δ 7.01–7.52 (2H, m), 8.22 (1H, d, J = 1.3 Hz), 8.32 (1H, s), 8.53 (1H, d, J = 2.0 Hz), 8.67 (1H, d, J = 2.0 Hz), 8.83 (1H, d, J = 1.9 Hz), 9.04 (1H, d, J = 2.2 Hz), 10.24–11.01 (1H, m), 13.52–14.28 (1H, m). 1 3C NMR (75 MHz, DMSO-d6): 162.10, 158.91 (d, J = 4.4 Hz), 155.12, 151.38, 151.14, 145.88, 137.82, 137.12, 134.51, 134.22, 129.84, 121.01, 120.74, 114.43, 112.67, 111.06, 102.23, 98.14, 76.64. MS (API) m/z calculated for C1 9H1 0BrF2N5O2S + H+ (M + H)+ : 490.0. Found: 489.9 (M + H)+. HPLC purity 99.8% . mp: 251–253 ºC.
2,5-Dic
hloro-N-[2,4-difluoro-3-(1H-pyrazolo[3,4-b]pyridin-5-ylethy nyl)phe nyl]be nze nesulfo namide ( 93b). Compound 98 (553 mg, 2.05 mmol) was dissolved in pyridine (10 ml) , and to this solution was added 2,5-dichlorobenzene-1-sulfonyl chloride (95b, 528 mg, 2.15 mmol) at 0 °C. After stirring at 0 °C for 1 h and at room temperature for 1 h, the mixture was treated with MeOH at room temperature and concentrate d under vacuum. The residue was purified by column chr omatography (silica gel, eluted wit h 10–100% EtOAc in hexane then 0 –40% MeOH in EtOAc) and recrystallized from EtOH (40 ml) and water (36 ml) at 50 °C. The collected solid (including di -sulfona mide b yproduct) was dissolved with EtOH (40 mL) and 4 N aqueous LiOH (3 ml) was added to the mixture.
After stirring at 50 °C for 1 h, the mixture was neutralized with 1 N aqueous HCl at room temperature and concentrated under vacuum to half volume. To the mixture (yellow solution) was added water (30 mL) at 50 °C and stirred for 2 days at room temperature. The resulting solid was collected by filtration and washed with water to give 93b (653 mg, 67%) as a pale-yellow solid. 1H NMR (300 MHz, DMSO-d6) 7.16–7.29 (1H, m), 7.35 (1H, td, J
= 8.9, 5.9 Hz), 7.71 –7.82 (2H, m), 7.88 (1H, dd, J = 1.9, 0.9 Hz), 8.22 (1H, d, J = 1.3 Hz), 8.54 (1H, d, J = 2.0 Hz), 8.67 (1H, d, J = 2.0 Hz), 10.79 (1H, s), 13.98 (1H, br s). 13C NMR (75 MHz, DMSO-d6): 162.10 (d, J = 3.9 Hz), 158.76 (d, J = 4.4 Hz), 155.28 (d, J = 5.5 Hz), 151.38, 151.13, 139.22, 134.92, 134.49, 134.27, 134.19, 132.59, 130.33 (d, J = 15.4 Hz), 129.66 (d, J = 10.4 Hz), 120.88 (dd, J = 12.7, 3.9 Hz), 114.42, 112.55 (dd, J = 22.0, 3.9 Hz), 111.07, 102.09 (dd, J = 20.9, 18.7 Hz), 98.12, 76.66. Anal. Calcd for C2 0H10Cl2F2N4O2S: C, 50.12; H, 2.10; N, 11.69. Found: C, 50.05; H, 1.90; N, 11.59. MS (API) m/z calculated for C2 0H10Cl2F2N4O2S + H+ (M + H)+ : 479.0. Found: 479.0 (M + H)+. HPLC purit y
>99.9%. mp: 219–221 ºC.
Ethyl 2,5-dichloro-3-{[2,4-difluoro-3-(1H-pyrazolo[3,4-b]pyridin-5-ylethynyl)phe nyl]sulfamoyl}benzoate ( 101). Thionyl chloride (1.9 ml, 26.0 mmol) was
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added dropwise to water (2 ml, 2.0 mmol) at 0 °C. After stirring at room temperature for 16 h, copper(I) chloride (20 mg, 0.20 mmol) was added to the mixture and the mixture was cooled to 0 °C ( mixture called as "A "). Sodium nitrite (152 mg, 2.2 mmol) in water (2 ml, 2.0 mmol) was added dropwise to the mixture of ethyl 3-a mino-2,5-dichlorobenzoic acid (100, 520 mg, 1.6 mmol) in concentrated aqueous HCl (2 ml, 24 mmol) at 0 °C. After stirring at 0 °C for 10 min, the mixture was added dropwise to the mixture "A" fr om a) at 0 °C, and the mixture was stirred at 0 °C for 1 h. The mixture was treated with water at r oom temperature and extracted with EtOAc. The combined orga nic layers were washed with brine, dried over MgSO4 and concentrated under vacuum. This product was dissolved in pyridine (2 ml), an d to this solution was added compound 98 (161 mg, 0.60 mmol) at 0 °C. After stirring at 0 °C for 2 h, the mixture was treated with MeOH and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0 –50% EtOAc in hexane) and recrystallized from EtOH/water to give 101 (87 mg, 27%) as a n off-white solid. 1H NMR (300 MHz, DMSO -d6) 1.33 (3H, t, J = 7.1 Hz), 4.37 (2H, q, J = 7.2 Hz), 7.14–7.30 (1H, m), 7.36 (1H, td, J = 8.9, 5.8 Hz), 8.04 (1H, d, J = 2.6 Hz), 8.14 (1H, d, J = 2.6 Hz), 8.22 (1H, d, J = 1.3 Hz), 8.54 (1H, d, J = 2.0 Hz), 8.67 (1H, d, J = 2.0 Hz), 10.93 (1H, s), 13.98 (1H, s).
MS (API) m/z calculated for C2 3H14Cl2F2N4O4S + H+ (M + H)+ : 551.0. Found: 550.9 (M + H)+.
2,5-Dic hloro-N-[2,4-difluoro(1H-pyrazolo[3,4-b]pyridin-5-ylet hy nyl)phe nyl]
-3-(hydroxymet hyl) benzenesulfonamide ( 93c). To a solution of 101 (77 mg, 0.14 mmol) in THF (2 ml) was added LAH (1 5.9 mg, 0.418 mmol) at 0 °C. After stirring at roo m temperature for 16 h, the mix ture was neutralized with saturated aqueous NH4Cl at 0 °C and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4 and concentrated under vacuum. The residue was purified by column chr omatography (silica gel, eluted with 5 –100% EtOAc in hexane) and preparative HPLC (L -Column 2 ODS, eluted with H2O in acetonitrile containing 0.1% TFA ). The desired fraction was neutralized with saturated aqueous NaHCO3 and extracted with EtOAc. The comb ined orga nic la yers were separated, dried over MgSO4 and concentrated under vacuum to give 93c (34 mg, 48%) as a white solid. 1H NMR (300 MHz, DMSO -d6) 4.63 (2H, d, J = 5.7 Hz), 5.74 (1H, t, J = 5.8 Hz), 7.02–7.24 (1H, m), 7.24–7.47 (1H, m), 7.80 (2H, s), 8.22 (1H, d, J
= 1.3 Hz), 8.53 (1H, d, J = 1.3 Hz), 8.67 (1H, d, J = 2.0 Hz), 10.78 (1H, br s), 13.98 (1H, br s). 13C NMR (75 MHz, DMSO -d6): 157.96, 155.02, 151.40, 151.13, 145.55, 140.37, 134.49, 134.16, 132.12, 131.45, 128.57, 128.43, 127.65, 122.79, 114.42, 112.30 (d, J = 21.5 Hz), 111.19, 101.90, 97.81, 76.98, 60. 49. MS (API) m/z calculated for C21H1 2Cl2F2N4O3S - H+ (M - H)- : 507.0. Found: 506.9 (M - H)-. HPLC purity 96.2%. mp: 236–238 ºC.
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N-{3-[(2-aminopyrimidin-5-yl)et hy nyl]-2,4-difluorophe
nyl}-2,5-dichloro-3-(hydroxymet hyl) benzenesulfo namide ( 93d). Thionyl chloride (6.0 ml, 82.2 mmol) was added dr opwise to water (6 ml) at 0 °C. After stirring at room temperature for 16 h , copper(I) chloride (0.06 3 g, 0.64 mmol) was added to the mixture a nd the mixture was coole d to 0 °C ( mixture called as "A"). Sodium nitrite (486 mg, 7. 05 mmol) in water (6 ml) was added dr opwise to the mixture of 100 (1.50 g, 6.41 mmol) in concentrated aqu eous HCl (6 ml, 72 mmol) at 0 °C. After stirring at 0 °C for 30 min, the mixture was added dropwise t o the mixture "A" at 0 °C, and the mixture was further stirred at 0 °C for 15 min. The mixture was treated with water at room temperature and extracted w ith EtOAc. The combine d orga nic layers were washed with brine, dried over MgSO4 and concentrated under vacuum.
This pr oduct was dissolved in pyridine (15 ml), and 94 (982 mg, 6.41 mmol) was added to the mixture at 0 °C. After stirring at 0 °C for 2 h, the mixture was treated with MeOH and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0–50% EtOAc in hexane) to give cr ude ester product (1.7 3 g). The product was dissolved in THF (15 ml), and LiBH4 (0.140 g, 6.41 mmol) was added to the mixture at room temperature. After stirring at 65 °C for 5 h, the reaction was q uenched with saturated aqueous NH4Cl at room temperature a nd extracted with EtOAc. The combined orga nic layers were washed with brine, dr ied over MgSO4 and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0 –100% EtOAc i n hexane) to give 102 (1.15 g) as a crude product. The crude 102 (1.15 g) was dissolved in DMSO (20 ml) , and to this solution were added 5- iodopyrimidin-2-a mine (103, 0.713 g, 3.23 mmol), Cs2CO3 (3.82 g, 11.7 mmol) and Pd(PCy3)2Cl2 (0.144 g, 0.210 mmol) at room temperature. After stirring at 120 °C for 2 h under microwave irra diation, the mixture was filtered through a pad of Celite, and the filtrate was treated with water and extracted wit h EtOAc. The combined organic la yers were washed with brine, dried over MgSO4 a nd concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 0–100% EtOAc in hexane) and recrystallized from EtOH (30 ml)/water (45 ml) at 60 °C. The obtained solid was purified by column chroma tography (silica gel, eluted wit h 0–100% EtOAc in hexane) and purified by preparative HPLC (L -Column 2 ODS, eluted with H2O in acetonitrile containing 0.1% TFA). The desired fraction was neutralized with saturated aqueous NaHCO3 and extracted with EtOAc. The co mbined organic layers wer e dried over MgSO4 and concentrated under vacuum. The residue was recr ystallized fr om EtOH (15 mL)/water (30 mL) at 60 °C to give 93d (0.240 g, 7.7% from 100) as a white solid.
1H NMR (300 MHz, DMSO -d6) 4.63 (2H, d, J = 5.6 Hz), 5.75 (1H, t, J = 5.7 Hz), 7.05–7.47 (4H, m), 7.66–7.92 (2H, m), 8.43 (2H, s), 10.48 –10.85 (1H, m). 13C NMR (75 MHz, DMSO-d6): 167.72, 161.72 (d, J = 4.4 Hz), 161.02, 158.34 (t, J = 6.3 Hz), 154.90 (d, J = 5.5 Hz), 145.69, 139.47, 132.25, 131 ,90, 128.93 (d, J = 9.4 Hz), 128.52, 127.65, 120.93 (dd, J = 12.7,
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3.9 Hz), 112.40 (dd, J = 22.0, 3.9 Hz), 105.03, 102.42 (dd, J = 20.9, 18.2 Hz), 96.16, 78.32, 60.46. Anal. Calcd for C19H12Cl2F2N4O3S: C, 47.03; H, 2.49; N, 11.55. Found: C, 47.15;
H, 2.78; N, 11.42. MS (API) m/z calculated for C19H1 2Cl2F2N4O3S + H+ (M + H)+ : 485.0.
Found: 485.2 (M + H)+. mp: 219–221 ºC.
5Chloro2metho xypyridine 3sulfonyl c hloride (1 06). To a solution of 1,2 -dibenzyldisulfane (8.87 g, 36.0 mmol) and 5-chloro-2- methoxypyridin-3-a mine (104, 4.76 g, 30.0 mmol) in acetonitrile (63.1 ml) was added dr opwise p entyl nitrite (8.79 ml, 66.0 mmol) over 20 min at 80 °C. After stirring at 80 °C for 30 min, the mixture concentrated under vacuum. The residue was purified by silica gel column chr omatography ( silica gel, elute d with 0–10% EtOAc in hexane) to obtain cr ude product 105 (5.40 g) as orange oil. This ma terial was used in the next reaction without further purification. NCS (16.3 g, 123 mmol) was added dropwise for over 10 min to a solution of cr ude 105 (5.40 g) in AcOH (34.9 ml) and water (11.0 ml) at 25 °C. After stirring at room temperature over night , the mixture was diluted with water, and extracted with EtOAc. The combined or ganic layers were washed with brine, dried over MgSO4, and concentrated under vacuum. The mixture was concentrated under vacuum. The residue was purified by silica gel column chr omatography silica gel, eluted with 0 –20% EtOAc in hexane) to yield 106 (4.40 g, 53% in 2 steps) as orange oil. 1H NMR (300 MHz, DMSO -d6) 3.86 (3H, s), 7.91 (1H, d, J = 2.6 Hz), 8.17 (1H, d, J
= 2.6 Hz).
N-(3-((2-amino pyrimidin-5-yl)ethynyl)-2,4-difluorophe nyl)
-5-chloro-2-met hoxy pyridine -3-sulfon amide acetic acid salt ( 93e). Compound 94 (2.78 g, 18.2 mmol), 106 (4.40 g, 18.2 mmol) were dissolved pyridine (43.1 ml) , and the mixture was stirred at room temperature over night. Then, MeOH (10 ml) was added to the mixture, and the mixture was stirred at room temperature for 10 min. The mixture was concentrated under vacuum.
The residue was purified by silica gel column chromatography ( silica gel, eluted with 0 –50%
EtOAc in hexane) to give cr ude 107 (5.02 g) as a beige solid. This material was used in the next reaction without further purification. The crude 107 (1.24 g, 3.46 mmol), 103 (1.15 g, 5.18 mmol), Pd(PCy3)2Cl2 (170 mg, 0.240 mmol), Cs2CO3 (4.51 g, 13.8 mmol) were dissolved in DMSO (16.5 ml) , and the mixture was stirred at 120 °C for 3 h under N2. The mixtur e was diluted with water and brine, a nd extracted with EtOAc. The combined orga nic la yers were washed with brine, dried over MgSO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography ( silica gel, eluted with 0 –20% EtOAc in hexane) t o yield crude pr oduct. The cr ude pr oduct was subjected to a mino silica gel column chr omatography ( silica gel, eluted with 0–50% MeOH in EtOAc) to remove bypr oducts. The amino silica gel, including the desired product, was subjected to water/EtOAc/AcOH (100
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ml/100 ml/18 ml). After stirring at room tempe rature for 10 min, the insoluble materials were filtered off, and further elution with EtOAc/AcOH (30 ml/6 ml) was carried out 4 times.
From the combined filtrate, the comb ined or ganic layers were collected, washed with water and brine, dried over MgSO4, and concentrated under vacuum. The residue was dissolve d with EtOAc/THF/saturated aqueous NaHCO3 (120 ml/30 ml/30 ml). The combined organi c layers were washed with saturated aqueous NaHCO3, brine, dried over MgSO4, and concentrated under vacuum. This solid was triturated with EtOAc, a nd the precipitate was collected b y filtration to yield cr ude pr o duct as a white solid (872 mg). The obtained soli d was purified b y silica gel column chr omatography (( silica gel, eluted with 0 –100% EtOAc i n hexane), and triturated with EtOAc. The precipitate was collected b y filtration to yield fre e for m of the desire d product (635 mg). Additional batches with 4.05 times scale and 4.12 times scale were carried out in a similar manner to obtain free for m of the desired product (4.83 g). All obtained free for m of the desired product were combined, and dissolved i n AcOH (24.8 ml) and DMSO (66 ml) at 50 °C. The solution was filtered to remove small insoluble materials with washing with AcOH (24.8 ml). Water (50 ml) was added dropwise to the filtrate at 50 °C. The mixture was allowed to cool to 25 °C for 30 min. The precipitate was collected by filtration, washed with EtOH/water (1/10, 33 mL) 3 times a nd dried under vacuum at 50 °C to yield 93e (5.53 g, 20% in 2 steps) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.94 (3H, s), 7.14–7.37 (4H, m), 8.07 (1H, d, J = 2.6 Hz), 8.43 (2H, s), 8.52 (1H, d, J = 2.6 Hz), 10.46 (1H, s). Anal. Calcd for C1 8H1 2ClF2N5O3S·1.0AcOH: C, 46.93; H, 3.15; N, 13.68. Found: C, 46.85; H, 3.17; N, 13.64. MS (API) m/z calculated for C18H1 2ClF2N5O3S + H+ (M + H)+ : 452.0. Found: 452.0 (M + H)+.
5-Chloro-N-[2,4-difluoro-3-({2-[(trans-4-hydroxycyclo
hexyl)amino]pyrimidin-5-yl}ethy nyl)phe nyl] -2-met hoxypyridine -3-sulfonamide (93f). Compound 107 (1.25 g, 3.48 mmol), 2-chloro-5-iodopyrimidine (1.1 0 g, 4.53 mmol), DIEA (6.75 g, 52.3 mmol), copper(I) iodide (0.133 g, 0.698 mmol), Pd(PCy3)Cl2 (0.257 g, 0.348 mmol) were dissolved in DMSO (11 ml), and the mixture was stirred at 60 °C for 1 h. The residue was diluted with EtOAc, washed with water, saturated aqueous NaHCO3 and brine, dried over MgSO4 and concentrated under vacuum. The residue was purified by column chr omatography (silica gel, eluted wit h 0–100% EtOAc in hexane) to give cr ude 108 (1.52 g) as a pale pink solid. This crude product was used for the next step without further purification and dissolved in acetonitrile (13 ml).
To the mixture were a dded trans-4-a minocyclohexanol (109, 0.743 g, 6.45 mmol), DIE A (0.834 g, 6.45 mmol). After stirring at 60 °C over night, the mixture was neutralized wit h sat. aqueous NaHCO3 and extracted with EtOAc. The combined organic layers were washe d with water and brine, dried over MgSO4 and concentrated under vacuum. The residue wa s purified b y column chromatography (silica gel, eluted with 0 –100% EtOAc in hexane) and
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then subjected to NH silica gel, and the silica gel was washed well with EtOAc/MeOH (0 – 30% MeOH in EtOAc) to remove impur ity. The silica gel including the object was suspended with EtOAc/water/AcOH (20ml/20ml/4ml). After stirring at room temperatur e for 10 min, the mixture was filtrated. The silica gel was tr eated with EtOAc/water/AcOH (20 ml/20 ml/4 ml), and filtrated twice. All filtrate was collected, and the or ganic layers were separated. The combined or ganic layers were wa shed with excess 2 N aqueous NaOH, brine, dried over MgSO4 and concentrated under vacuum. The residue (1. 87 g) was purified b y column chromatography(silica gel, eluted with 0 – 100% EtOAc in hexane) a nd recr ystallized from EtOH (20 ml) and water (20 ml) at 80 °C to give 93f (598 mg, 31% in 2 steps) as a whit e solid. 1H NMR (300 MHz, DMSO-d6) δ 1.12–1.38 (4H, m), 1.78–1.90 (4H, m), 3.35–3.48 (1H, m), 3.61–3.75 (1H, m), 3.93 (3H, s), 4.56 (1H, d, J = 4.3 Hz), 7.14–7.23 (1H, m), 7.31 (1H, td, J = 8.9, 6.0 Hz), 7.74 (1H, d, J = 7.9 Hz), 8.06 (1H, d, J = 2.6 Hz), 8.40–8.50 (2H, m), 8.52 (1H, d, J = 2.5 Hz), 10.45 (1H, s). Anal. Calcd for C24H2 2ClF2N5O4S·0.5H2O·1.0EtOH: C, 51.61; H, 4.83; N, 11.57. Found: C, 51.61; H, 4.91;
N, 11.74. MS (API) m/z calculated for C2 4H22ClF2N5O4S + H+ (M + H)+ : 550.1. Found : 550.1 (M + H)+.
3-(Benzylsulfa nyl) -2,5-dichlorobenzyl acetate ( 112). Amyl nitrite (32 ml, 240.8 mmol) was added dropwise to a mixture of 100 (19.4 g, 82.9 mmol) and dibenzyl disulfide (24.5 g, 99.5 mmol) in acetonitrile (240 ml) at 70 °C. After stirring at 70 °C under a dry atmospher e (CaCl2 tube) for 1 h, the mixture was concentrated under vacuum. The residue was purified by column chromatography (NH silica gel, eluted with 0 –15% EtOAc in hexane), and the n washed with hexane to give the crude pr oduct 110 (17.6 g) as orange oil. This pr oduct wa s subjected t o the next reaction without further purification. At the next step, NaBH4 (7.8 g, 206.3 mmol) was added to a solution of calcium chloride (11.5 g, 103.62 mmol) in EtOH (130 ml) at 0 °C. After being stirred at 0 °C for 20 min, the cr ude 100 (17.6 g) in dried THF (130 ml) was added to the reaction mixture. After stirring at 0 °C for 10 min and at roo m temperature for 2 h under N2 gas, the reaction was quenched with saturated aqueous NH4Cl at 0 °C and extracted with EtOAc. The combined or ganic layers were w ashed with brine, dried over MgSO4 and concentrated under vacuum to give the crude pr oduct 111 (14.0 g) as a pale yellow solid. This product was subjected to the next reaction without purification.
Acetic a nhydride (5.3 ml, 56.2 mmol) was added dropwise to a solution of the cr ude 111 (14.0 g), DMAP (1.1 g, 9.4 mmol) and triethyl a mine (13 ml, 93.3 mmol) in dried THF (160 ml) at room temperature. After stirring at 50 °C under a dry atmosphere (CaCl2 tube) for 2 h, the mixture was diluted with water at r oom temperature and extracted with EtOAc. The combined orga nic layers were washed with brine, dried over MgSO4 and concentrated under vacuum. The residue was purified by column chromatography (silica gel, eluted with 15–
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30% EtOAc in hexane), a nd washed with hexane to give 112 (11.7g, 66% in 3 steps) as a colorless powder. 1H NMR (300 MHz, DMSO-d6) δ 2.10 (3H, s), 4.39 (2H, s), 5.11 (2H, s), 7.24–7.39 (4H, m), 7.41-7.48 (3H, m).
2,5-Dic hloro-3-(chlorosulfonyl)benzyl acetate (113). Compound 112 (8.6 g, 25.2 mmol) was dissolved in AcOH (50 ml)/THF(10 ml)/water (10 ml), and to this solution was adde d NCS (13.4 g, 100.4 mmol) at 0 °C. After stirring at room temperature for 1 h, the mixture was concentrated under vacuum, diluted with water and extracted with EtOAc. The combined orga nic layers were washed with brine, dried over MgSO4 and concentrated under vacuum. The residue was purified by column chromatography (silica gel, elut ed with 5–
30% EtOAc in hexane) to give 113 (7.7 g, 97 %) as a colorless solid. 1H NMR (300 MHz, DMSO-d6) δ 2.12 (3H, s), 5.14 (2H, s), 7.52 (1H, d, J = 2.6 Hz), 7.85 (1H, d, J = 2.6 Hz).
2,5-Dic hloro-3-[(3-et hy nyl-2,4-difluorophe nyl)sulfamoyl]be nzyl acetate (114).
Compound 113 (7.7 g, 24.4 mmol) was dissolved in dried THF (10 ml) , and to this solution was added a solution of 94 (3.5 g, 22.9 mmol) in pyridine (30 ml) at 70 °C. After stirring at 70 °C under a dry atmosphere (CaCl2 tube) for 1 h, the mixture was concentrated under vacuum, and the residue was diluted with water at room temperature and extracted with EtOAc.
The combined orga nic la yers were washed with brine, dried over MgSO4 and concentrated under vacuum. The residue was purified by column chr omatography (silica gel, eluted wit h 20–50% EtOAc in hexane), and washed with IPE to give 114 (7.9 g, 79 %) as a colorless powder. 1H NMR (300 MHz, DMSO-d6) δ 2.13 (3H, s), 4.87 (1H, s), 5.22 (2H, s), 7.12 – 7.22 (1H, m), 7.27 –7.38 (1H, m), 7.86 (1H, d, J = 2.6 Hz), 7.90 (1H, d, J = 2.5 Hz), 10.80 (1H, s). MS (API) m/z calculated for C17H11Cl2F2NO4S - H+ (M - H)- : 432.0. Found: 432.1 (M - H)-.
2,5-Dic
hloro-3-({3-[(2-chloropyrimidin-5-yl)ethynyl]-2,4-difluorophe nyl}sulfamoyl) be nzyl acetate ( 115). Compound 114 (7.6 g, 17.4 mmol) , Pd(PCy3)2Cl2 (1.3 g, 1.7 mmol), DIEA (30 ml, 171.8 mmol), copper (I) iodide (0.66 g, 3.5 mmol), 2-chlor o-5-iodopyrimidine (4.4 g, 18.3 mmol) were dissolved in DMSO (60 ml), and the mixture was stirred at 60 °C under N2 for 1 h. The mixture was diluted with water and EtOAc at room temperature a nd the insoluble material was removed b y filtration, and th e filtrate was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4 and concentrated under vacuum. The residue was purified b y col umn chr omatography (silica gel, eluted with 30 –100% EtOAc in hexane), and washed with EtOAc/IPE to give 115 (5.3 g, 56%) as a pale yellow powder. 1H NMR (300 MHz, DMSO-d6) δ 2.12 (3H, s), 5.22 (2H, s), 7.22 –7.31 (1H, m), 7.35–7.46 (1H, m), 7.84–7.94 (2H, m),
173
9.03 (2H, s), 10.90 (1H, s). MS (API) m/z calculated for C21H12Cl3F2N3O4S - H+ (M - H)- : 544.0. Found: 544.0 (M - H)-.
2,5-Dic hloro-N-{2,4-difluoro-3-[(2-{[(2R)-1-hydroxypropa n-2-yl]amino}pyrimidin-5-yl)ethy nyl]phe nyl}-3-(hydroxymet hyl)benze nesulfonamide ( 93g). Compound 115 (1.00 g, 1.83 mmol), (R)-2-a minopropan-1-ol (0.275 g, 3.66 mmol) and DIEA (0.650 ml, 3.72 mmol) were dissolved in dried THF (10 ml) , and the mix ture was stirred at 60 °C under a dr y atmosphere (CaCl2 tube) overnight. The mixture was diluted with water at room temperature and extracted with EtOAc. The combined orga nic layers were washed with brine, dried over MgSO4 and concentrated under vacuum. The residue was dissolved in MeOH (10 ml), and 2 N aqueous NaOH (5.00 ml, 10.0 mmol) was added to the solution at room temperature.
After stirring at room temperature for 1 h, t he mixture was neutralized with 1 N aqueous HCl at room temperature an d extracted with EtOAc. The combined orga nic layers were washe d with brine, dried over MgSO4 and concentrated under vacuum. The residue was washed with EtOAc to give crude product as a pale yellow powder. The solid was crystallized fr om acetone/water and dried under vacuo to give 93g (0.822 g, 83%) as a colorless cr ystal. 1H NMR (300 MHz, DMSO-d6) 1.13 (3H, d, J = 6.6 Hz), 3.25–3.39 (1H, m), 3.41 –3.52 (1H, m), 3.93–4.08 (1H, m), 4.63 (2H, d, J = 5.1 Hz), 4.72 (1H, br s), 5.77 (1H, t, J = 5.6 Hz), 7.14–7.33 (2H, m), 7.58 (1H, d, J = 8.1 Hz), 7.77–7.80 (1H, m), 7.80 –7.83 (1H, m), 8.46 (2H, br s), 10.74 (1H, s). Anal. Calcd for C22H18Cl2F2N4O4S: C, 48.63; H, 3.34; N, 10.31.
Found: C, 48.58; H, 3.52; N, 10.13. MS (API) m/z calculated for C2 2H18Cl2F2N4O4S + H+ (M + H)+ : 543.0. Found: 543.0 (M + H)+.
<生 物 そ の 他 の 実 験>
Materials. GCN2 (1-1649 amino acids of accession number Q9P2K8.3) and GST-PERK (536-1116 amino acids of accession number NP_004827.3) were purchased from Carna Biosciences (Kobe, Japan). DMEM high glucose, GFP-eIF2α protein, LanthaScreen® Tb-peIF2a (pSer52) Antibody Kit, TR-FRET Dilution Buffer 1 M HEPES solution, penicillin-streptomycin solution, and goat serum were obtained from Thermo Fischer Scientific (Waltham, MA). DMEM without amino acids were custom-made by Katayama Kagaku (Osaka, Japan). ATP, DTT, Triton-X100 and Proclin300 were from Sigma Aldrich (St. Louis, MO). Tween-20 and Brij-35 detergent was obtained from Merck Millipore (Billerica, MA).
EGTA was from Dojindo (Kumamoto, Japan). ATF-4 (D4B8) Rabbit mAb, phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb and DRAQ were purchased from Cell Signaling (Danvers, MA). IRDye® 800CW Goat anti-Rabbit Secondary Antibody was obtained from LI-COR Biosciences (Lincoln, NE). Dialyzed FBS for incell western assay was from JRScientific
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(Woodland, CA). Tb-labeled anti GST antibody (Tb-GST) was purchased from Cisbio (Codolet, France). The other reagents were obta ined from Wako (Osaka, Japan).
GCN2 and PERK enzyme assay. The enzyme reaction was run in the enzyme assay buffer, which consisted of 25 mM HEPES, 10 mM MgCl2, 2 mM DTT, 0.01% Tween-20, and 1 mM EGTA. In the reaction mixture, the final concentrations of GFP-eIF2α and ATP were as follows; 130 nM and 190 μM for GCN2, 130 nM and 4 μM for PERK, respectively. To evaluate the potency of inhibitors after 60 min incubation, 3 -fold concentration of compounds (2 μl) and 3-fold concentration of enzymes (2 μl) were mixed in each well of a white flat-bottomed 384-well plate (Greiner Bio-One, Frickenhausen, Germany). After 60 min incubation at room temperature, the enzymatic reaction was initiated by addition of 2 μl of 3-fold concentration of substrate premix (GFP -eIF2 and ATP). The reaction was proceeded for 60 min at room temperature. The final concentration of GST-GCN2 and GST-PERK in the reaction mixture were 1 nM and 0.05 nM, respectively. Throughout this study, the enzymatic reaction was terminated by additi on of 2 μl of 4×detection buffer (4 nM Tb-peIF2a (pSer52) Antibody, 44 mM EDTA in TR-FRET Dilution Buffer) and the plate was further incubated for 60 min. Time-resolved fluorescence was monitored by using an EnVision plate reader (PerkinElmer, Waltham, MA). The solution in each well was excited by using a laser (λ = 337 nm) reflected by a dichroic mirror (D400/D505 (PerkinElmer)), and fluorescence signals from terbium (Tb) and GFP were detected by using two emission filters (CFP 486 (Perkin Elmer) for Tb and Emission 520 (PerkinElmer) for GFP). Amount of phosphorylated GFP-eIF2α was monitored by TR-FRET ratio (GFP fluorescence/Tb fluorescence). The values of 0% and 100% inhibition control were set to the TR -FRET ratio in the presence and absence of enzymes, respectively. The percentage of inhibition was calculated by using equation (1),
(1)
, where T is the value of the wells containing test compounds, and μH and μL are the mean values of the 0% and 100% inhibition control wells, respectively. IC50 values were calculated by fitting a sigmoidal dose-response curve to a plot of assay read out over inhibition concentration. All fits were performed using XLfit (IDBS, Guildford, UK).
GCN2 dilution assay. Dilution assay was carried out using binding assay buffer (50 mM Hepes, 10 mM MgCl2, 1 mM EGTA, 0.1 mM DTT, 0.01% Brij-35). The 200 × IC60 -8 0
(measured in the presence of the Kd (70 nM) of GCN2 probe, which was prepared in-house, after 2 h incubation) of 7.5 nl test inhibitors were dispensed in a white flat -bottomed 384-well plate using Access Echo555 (Labcyte, Sunnyvale, CA). Subsequently, 1.5 μl of a
10-
−
−
=
L H
100 H
inhibition of
Percentage
T
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fold concentration of Tb/GCN2 premix (2.1 nM Tb-GST and 10 nM GST-GCN2) was added to each well, and the plate was sealed with a plate sealer (Eidia, Japan) in a moist chamber.
After 2 h incubation at room temperature, the preincubated sample was diluted with 13.5 μl of TR-FRET assay buffer containing a concentration 20 times the Kd (1400 nM) of BODIPY-FL labeled GCN2 probe. Subsequently, time-course change in the TR-FRET signal was measured using an EnVision plate reader. The solution in each well was excited by using a laser (λ = 337 nm) reflected by a dichroic mirror (D400/D505, PerkinElmer), and fluorescence signals from terbium (Tb) and BODIPY-FL were detected by using two emission filters (CFP 486 (Perkin Elmer) for Tb and Emission 515 (PerkinElmer) for BODIPY-FL). TR-FRET ratio (BODIPY-FL fluorescence/Tb fluorescence) was utilized as binding signal. The values of the 0% and 100% controls were the signals obtained in the presence and absence of GCN2, respectively. The half-time of the reaction was obtained by fitting the time -course data to single exponential curve using Prism 5 software (GraphPad, La Jolla, CA). The chemical structure of the GCN2 probe used in this assay is below.
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In vitro PD marker (ATF4 and p-eIF2α) detection using in cell western. U2OS cells were cultured in the maintenance medium (DMEM high glucose containing 10% FBS, 100 U ml− 1 penicillin, and 100 μg ml−1 streptomycin) and were seeded at 2×104 cells/well on a 96-well polystyrene plate (Corning, Corning, NY). After overnight incubation at 37 °C/5% CO2, the maintenance medium was removed from the plate and the cells were washed wi th 150 l of PBS. Subsequently, test compounds in 50 l of the assay medium (DMEM without amino acid containing 10% dialyzed FBS, 100 U ml−1 penicillin, and 100 μg ml− 1 streptomycin) were added to the plate and incubated for 4 hr at 37 °C/5% CO2. The cells were fixed by adding 50 μl/well of MildForm® 20NM (for ATF4 assay) or 4% paraformaldehyde (for p -eIF2 assay) and the plate was incubated for 10 min at room temperatu re. Subsequently, cells were washed with 100 μl PBS per well. For cell permeabilization and prevention of nonspecific antibody binding, 100 μl of GSTX blocking buffer (0.1% Triton-X100, 10 % goat serum, and 0.05% Proclin300 in PBS) was added to each well and the plate was incubated for 2 h at room temperature. After removing the solution and washing the cell with100 μl/well of PBST (PBS containing 0.1% TritonX -100) one time, the cells were incubated with 40 μl of 1000-fold diluted anti ATF-4 (D4B8) Rabbit mAb (for ATF4 assay) or anti phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb (for p-eIF2α assay) in 5-fold diluted GSTX blocking buffer (diluted with PBS), followed by overnight incubation at 4 °C. After removing the solution, cells were washed with 100 μL of PBST three times and 40 μl of 2000-fold diluted IRDye®
800CW Goat anti-Rabbit Secondary Antibody solution containing 10000 -fold diluted DRAQ in 5-fold diluted GSTX blocking buffer was added to each well followed by 1 -h incubation at room temperature. After washing the cells with 200 μl of PBST 5 times, 100 μl of PBS was added to each well and fluorescence intensity of washed cells was measured using Odyssey (LI-COR Biosciences). In order to normalize the number of cells in each well, fluorescence signal of IRDye® 800 CW corresponding to ATF4 expression level was corrected with remaining cells staining with DRAQ for nuclear visualization. Signals for 100%
inhibition were obtained from cells cultured in the control medium (DMEM high glucose containing 10% dialyzed FBS, 100 U ml− 1 penicillin, and 100 μg ml− 1 streptomycin). The percentage of inhibition in the presence of each concentration of compounds was calculated by using equation (1). IC50 values were calculated by fitting a sigmoidal dose -response curve to a plot of assay read out over inhibition concentration. All fits were performed using XLfit.
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Cellular PERK inhibition assay by quantitative PCR. U2OS cells were cultured in DMEM medium (containing 10% FBS, 100 U ml− 1 penicillin, and 100 μg ml− 1 streptomycin) and were seeded at 4×103 cells/well on a 96-well polystyrene plate (Corning). After overnight incubation at 37 °C/5% CO2, cells were treated with test compounds for 2 h followed by the addition of 100 nM thapsigargin (Sigma Aldrich) for 2 h to induce CHOP using D300e digital dispenser (TECAN). After washing with 150 μl of PBS, RNA extraction and cDNA synthesis was performed using FastLane Cell Probe kit (Qiagen). Real-time quantitative PCR was performed on ABI StepOne real-time PCR system (Applied Biosystems) using TaqMan gene expression assays for CHOP and GAPDH. Relative gene expression was calculated using the ΔΔCT method following the manufacturer ’s instructions. CHOP mRNA was normalized to GAPDH mRNA. IC5 0 values were calculated usi ng GraphPad Prism version 6 (GraphPad Software).
In vitro cell viability assay in combination with asparaginase. CCRF-CEM cells were cultured in RPMI medium (containing 10% FBS, 100 U ml−1 penicillin, and 100 μg ml− 1 streptomycin) and were seeded at 5×103 cells/well on a 96-well clear bottom white polystyrene plate (Corning). After overnight incubation at 37 °C/5% CO2, cells were treated with test compounds together with asparaginase (Sigma Aldrich) for 72 h using D300e digital dispenser (Tecan). Cell viability was assessed using CellTiter-Glo luminescent cell viability assay (Promega).
In vitro PD marker (ATF4 and p-eIF2α) detection using western blotting. CCRF-CEM cells were cultured in RPMI medium (containing 10% FBS, 100 U ml− 1 penicillin, and 100 μg ml− 1 streptomycin) and were seeded at 5×105 cells/well on a 12-well plate (Corning).
After overnight incubation at 37 °C/5% CO2, cells were treated with test compounds together with asparaginase (Sigma) for 4 h. Whole-cell lysates were prepared using a lysis buffer (10% glycerol, 1% sodium dodecyl sulfate, and 62.5 mmol/l Tris -HCl [pH 7.5] with protease and phosphatase inhibitors, cOmplete Mini and PhosSTOP, respectively; Sigma Aldrich).
Cell lysates were electrophoresed using a gradient SDS–PAGE gel containing 7.5%–15%
acrylamide (Perfect NT gel, DRC, Japan) and transferred to a nitrocellulose membrane using the iBlot Gel Transfer System (Thermo Fisher Scientific). After incubation for 1 h with blocking buffer [StartingBlock T20 (PBS), Thermo Fisher Scientific] at room temperature, membranes were labeled with primary antibodies by overnight incubation at 4 °C, followed by 1 h of incubation with HRP-conjugated secondary antibodies (Cell Signaling Technology, San Antonio, TX) at room temperature. Blots were incubated with ECL western blotting detection reagents (GE Healthcare, Chicago, IL), scanned using ImageQuant LAS -4000 (GE Healthcare), and quantified using ImageQuant TL (GE Healthcare). The following
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antibodies were used for the western blot analysis: anti-ATF4 (#11815, 1:1000), -eIF2α (#5324, 1:2000), -phospho-eIF2α (#5324, 1:2000), and -GCN2 (#3302, 1:1000) (Cell Signaling Technology); and phospho-Thr899-GCN2 (#ab75836, 1:1000, Abcam, Cambridge, UK). An eIF2α protein was used as loading control.
In vivo PD assay in combination with asparaginase. A suspension of CCRF-CEM cells (1
× 107 cells/site) was subcutaneously injected into the right flanks of 6 -week-old female SCID mice (C.B17/Icr-scid/scid Jcl; CLEA, Japan). Tumor volume was calculated as volume = L
× l2 × 1/2, where L represents the longest diameter across the tumor and l represents the corresponding perpendicular distance. GCN2 inhibitors were dissolved in 0.5%
methylcellulose and asparaginase (Kyowa Hakko Kiri n, Japan) was dissolved in 5% glucose.
When tumor volume reached 200-500 mm3, the mice were pre-treated with 1,000 U/kg asparaginase (intraperitoneally) for 24 h and then treated with GCN2 inhibitors (orally) and asparaginase again for 8 h. The mice were housed and maintained in accordance with the institutional guidelines established by the Institutional Animal Care and Use Committee, in a facility accredited by the American Association for Accreditation of Laboratory Animal Care.
All animal experimental protocols were approved by the Institutional Animal Care and Use Committee.
For western blot analysis, tumors were homogenized with Lysing Matrix I (MP Biomedicals, Santa Ana, CA) in the lysis buffer (10% glycerol, 1% sodium dodecyl sulfate, and 62.5 mmol/l Tris-HCl [pH 7.5] with protease and phosphatase inhibitors, cOmplete Mini and PhosSTOP, respectively; Sigma -Aldrich). Western blotting was performed as shown above.
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Crystallography experiments. Human GCN2 (eukaryotic translation initiation factor 2 alpha kinase 4), residues 577-1013 (residues 658-780 deleted) were cloned into the pSxB27 vector, and expressed using strain Sf9 of the baculoviral expression system with two sets of mutations. SBVC-2222, crystallized with compound 92, containing the foll owing four mutations: D848N, T899A, T904A, K807A. SBVC -2307, crystallized with compound 93d and compound 93e, containing the following mutations: D848N, T889A, and T904A. The cell pellet from 10 L of cell culture was suspended into 800 ml lysis buffer (25 mM Tris pH 8.0, 250 mM NaCl, 20 mM Imidazole, 10% Glycerol, 0.5 mM TCEP) and 3 Roche cOmplete Protease Inhibitor tablets. The lysate was run through a microfluidizer at 18,000 psi prior to being centrifuged for 1 hour at 14,000 RPM (Beckman, JA -14 rotor). The supernatant was affinity purified rolling overnight at 4 oC with 10 ml Probond Nickel resin (Invitrogen).
The resin was washed with 1 L lysis buffer followed by 1 L wash buffer (25 mM Tris pH 8.0, 500 mM NaCl, 20 mM Imidazole, 10% Glycerol, 0.5 mM TCEP), and eluted with 25 mM Tris pH 8.0, 250 mM NaCl, 250 mM Imidazole, 10% Glycerol, 0.5 mM TCEP. TEV protease digestion was performed overnight at 4 oC. The digested GCN2-TEV solution was further purified by size exclusion chromatography (20 mM Tris pH 7.6, 150 mM NaCl, 0.5 mM TCEP) and GCN2 eluted as a dimer. The GCN2 containing fractions from the SEC were pooled and passed over a Probond Nickel resin column using a shallow imidazole gradient. Fractions containing GCN2 were pooled and buff er exchanged back into SEC buffer (20 mM Tris pH 7.6, 150 mM NaCl, 0.5 mM TCEP). After the final buffer exchange the protein was concentrated to approximately 33 mg/ml in 2.15 ml for crystallization. Crystals were grown with 1 mM compound mixed 1:1 with the mother liquor containing 10% PEG 5000 MME, 0.7 M LiCl, 0.1 M NaCitrate pH 5.6, and 3% Ethylene Glycol. The resulting crystals were briefly passed through a drop containing mother liquor plus 30% Glycerol before being flash frozen in liquid nitrogen and exposed to X-rays for data collection at beamline 5.0.2 and 5.0.3 at the ALS. GCN2 + compound 92 crystals belong to the orthorhombic space group C2221, and contain two protein molecules in the asymmetric unit. GCN2 + compound 93d and GCN2 + compound 93e crystals belong to the hexagonal space group P6122, and contain one protein molecule in the asymmetric unit. The data was reduced by the HKL2000 software package.94 The structures were determined by the molecular replacement method using MOLREP95 of the CCP4 program suite. Multiple cycles of model building with XtalView9 6 or COOT97 and refinement with REFMAC9 8 were performed to improve model quality. The coordinates and structure factors have been deposited in the RCSB Protein Data Bank with the accession codes 6N3L, 6N3N and 6N3O.
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D a t a r e d u c t i o n a n d r e f i n e me n t s t a t i s t i c s f o r t h e X - r a y s t r u c t u r e s o f G C N 2 / c o mp o u n d s c o mp l e x .
Data Collection PDB ID 6N3L
Compound Compound 92
Wavelength (Å) 0.9764848
Space group C2221
Unit cell dimensions (Å)
a=83.9,b=122.2, c=120.7
α=β=γ=90°
Resolution (Å) 2.61
Unique reflections 18894
Redundancy 6.8
Completeness (%) 99.6 (97.8)
I/σ(I) 9.2 (1.4)
Rs yma 0.179 (>1.0)*
CC ½ (highest resolution
shell) 0.653
Refinement
Molecules in asymmetric unit 2
Reflections used 17799
RMS Bonds (Å) 0.010
RMS Angles (°) 1.392
Average B value (Å2) 60.7
R-valueb 0.224
R freeb 0.291
Data Collection PDB ID 6N3N
Compound Compound 93d
Wavelength (Å) 1.0
Space group P6122
Unit cell dimensions (Å) a=82.1,b=82.1,
c=192.6
181
α=90°,β=90°, γ=120°
Resolution (Å) 3.01
Unique reflections 8253
Redundancy 7.7
Completeness (%) 99.1 (99.8)
I/σ(I) 22.5 (1.6)
Rs yma 0.074 (1.298)
CC ½ (highest resolution shell) 0.504 Refinement
Molecules in asymmetric unit 1
Reflections used 7751
RMS Bonds (Å) 0.008
RMS Angles (°) 1.171
Average B value (Å2) 132.7
R-valueb 0.214
R freeb 0.276
Data Collection PDB ID 6N3O
Compound Compound 93e
Wavelength (Å) 1.0
Space group P6122
Unit cell dimensions (Å)
a=82.9,b=82.9, c=193.7
α=90°,β=90°, γ=120°
Resolution (Å) 2.40
Unique reflections 16229
Redundancy 18.6
Completeness (%) 100.0 (100.0)
I/σ(I) 23.1 (1.1)
Rs yma 0.099 (3.621)
CC ½ (highest resolution shell) 0.460
182 Refinement
Molecules in asymmetric unit 1
Reflections used 15338
RMS Bonds (Å) 0.007
RMS Angles (°) 1.196
Average B value (Å2) 87.1
R-valueb 0.199
R freeb 0.252
aRsym = ΣhΣj |<I(h)> I(h)j | / ΣhΣj <I(h)>, where <I(h)> is the mean intensity of symmetry -related reflections. bR-value = Σ | |Fobs| - |Fcalc| | / Σ |Fobs|. Rfree for 5% of reflections excluded from refinement.
Values in parentheses are for the highest resolution shell. * Limitations in older versions of HKL2000 did not provide Rsym values over 1.0.
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Docking study utilizing GCN2 homology model. Compound 91 was docked into the GCN2 model with vemurafenib using Glide (ver. 6.4). In a grid generation process, the standard default settings were used without "Scaling factor" which is modified to 0.5 becau se the template structure is not a crystal structure but a model. In a docking calculation, two H -bond constraints for NH moieties of main chain of Cys805 and Asp866 were set. SP mode was used for docking precision and common moiety between 91 and vemurafenib was set for core structure of pose constraint docking with setting 1.5 A as "Tolerance". As output options,
"Write out at most" option was changed to "10 poses per ligand" and "Number of poses per ligand to include" option was changed to "30". The st andard default settings were used for other settings.
The GCN2 model with vemurafenib was constructed by homology modeling using the crystal structure of B-Raf bound to vemurafenib (PDB ID, 3OG7) as a template structure. Before the homology modeling, the template structure was modified by the followings. The structure of T529-Y538 parts of the B-Raf structure was replaced by the corresponding parts of the crystal structure of GCN2 with compound 92. The structure of H539-E549 parts of the B-Raf structure was replaced by the corresponding parts of the crystal structure of GCN2 with 92. The structure of I625-P632 parts of the B-Raf structure was replaced by the corresponding parts of the crystal structure of B -Raf with BR2(PDB ID, 3SKC). In the homology modeling procedure, amino acid sequence alignment between GCN2 and B -Raf was prepared by using ClustalW. Homology modeling was conducted by Modeller with the standard default settings and 10 models were generated as an output. After the preparation of GCN2 model structure, binding model of GCN2 with vemurafenib was constructed.
Vemurafenib was positioned by superposing the crystal structure of B -Raf with vemurafenib to GCN2 model and complexed structure between GCN2 and vemurafenib was energy -minimized using the MMFF94s force field in MOE (version 2014, Chemical Computing Group, Montreal, Canada) to obtain the final docking models. During the minimization procedure, the following conditions were adopted. The dielectric constant was set to 4r, where r is t he distance between two interacting atoms. The residues, which are 8 Å away from each compound, were fixed. A harmonic force constraint against the initial atomic positions of the backbone was added, using 0.3 Å as a force constant. And atomic charges for the protein and the compounds were set according to the AMBER99 force field and the AM1 -BCC method, respectively. After minimization in MOE, the most plausible binding model was selected based on the intE value (interaction energy) in MOE.
184
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