Share:

PATENT

WO 2014066834

https://www.google.com/patents/WO2014066834A1?cl=en

EXAMPLE 1

4-({2-[(Aminosulfonyl)amino]ethyl}amino)- V-(3-bromo-4-fluorophenyl)- V -hydroxy- l,2,5-oxadiazole-3-carboximidamide

Step 1: 4-Amino-N’-hydroxy-l,2,5-oxadiazole-3-carboximidamide

[00184] Malononitrile (320.5 g, 5 mol) was added to water (7 L) preheated to 45 °C and stirred for 5 min. The resulting solution was cooled in an ice bath and sodium nitrite (380 g, 5.5 mol) was added. When the temperature reached 10 °C, 6 N hydrochloric acid (55 mL) was added. A mild exothermic reaction ensued with the temperature reaching 16 °C. After 15 min the cold bath was removed and the reaction mixture was stirred for 1.5 hrs at 16-18 °C. The reaction mixture was cooled to 13 °C and 50% aqueous hydroxylamine (990 g, 15 mol) was added all at once. The temperature rose to 26 °C. When the exothermic reaction subsided the cold bath was removed and stirring was continued for 1 hr at 26-27 °C, then it was slowly brought to reflux. Reflux was maintained for 2 hrs and then the reaction mixture was allowed to cool overnight. The reaction mixture was stirred in an ice bath and 6 N hydrochloric acid (800 mL) was added in portions over 40 min to pH 7.0. Stirring was continued in the ice bath at 5 °C. The precipitate was collected by filtration, washed well with water and dried in a vacuum oven (50 °C) to give the desired product (644 g, 90%). LCMS for C3H6N5O2

(M+H)+: m/z = 144.0. ¹³C MR (75 MHz, CD3OD): δ 156.0, 145.9, 141.3. Step 2: 4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride [00185] 4-Amino-N^,-hydroxy-l ,2,5-oxadiazole-3-carboximidamide (422 g, 2.95 mol) was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 Ν hydrochloric acid (1.475 L, 3 eq.) and this suspension was stirred at 42 – 45 °C until complete solution was achieved. Sodium chloride (518 g, 3 eq.) was added and this solution was stirred in an ice/water/methanol bath. A solution of sodium nitrite (199.5 g, 0.98 eq.) in water (700 mL) was added over 3.5 hrs while maintaining the temperature below 0 °C. After complete addition stirring was continued in the ice bath for 1.5 hrs and then the reaction mixture was allowed to warm to 15 °C. The precipitate was collected by filtration, washed well with water, taken in ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g) and stirred for 1 hr. This suspension was filtered through sodium sulfate (200 g) and the filtrate was concentrated on a rotary evaporator. The residue was dissolved in methyl i-butyl ether (5.5 L), treated with charcoal (40 g), stirred for 40 min and filtered through Celite. The solvent was removed in a rotary evaporator and the resulting product was dried in a vacuum oven (45 °C) to give the desired product (256 g, 53.4%). LCMS for C3H4CIN4O2 (M+H)+: m/z = 162.9. ¹³C NMR (100 MHz, CD3OD): 5 155.8, 143.4, 129.7.

Step 3: 4-Amino-N’-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3-carboximidamide [00186] 4-Amino-N-hydroxy-l ,2,5-oxadiazole-3-carboximidoyl chloride (200.0 g, 1.23 mol) was mixed with ethyl acetate (1.2 L). At 0-5 °C 2-methoxyethylamine [Aldrich, product # 143693] (119.0 mL, 1.35 mol) was added in one portion while stirring. The reaction temperature rose to 41 °C. The reaction was cooled to 0 – 5 °C. Triethylamine (258 mL, 1.84 mol) was added. After stirring 5 min, LCMS indicated reaction completion. The reaction solution was washed with water (500 mL) and brine (500 mL), dried over sodium sulfate, and concentrated to give the desired product (294 g, 1 19%) as a crude dark oil.

LCMS for C₆Hi_{2 5}0₃ (M+H)⁺: m/z = 202.3. 1H NMR (400 MHz, DMSO- ): δ 10.65 (s, 1 H), 6.27 (s, 2 H), 6.10 (t, J = 6.5 Hz, 1 H), 3.50 (m, 2 H), 3.35 (d, J = 5.8 Hz, 2 H), 3.08 (s, 3 H).

Step 4: N’-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidamide

[00187] 4-Amino-N-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3- carboximidamide (248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210 g, 3.7 mol) was added. The reaction was refluxed at 100 °C overnight (15 hours). TLC with 50% ethyl acetate (containing 1% ammonium hydroxide) in hexane indicated reaction completed (product Rf = 0.6, starting material Rf = 0.5). LCMS also indicated reaction completion. The reaction was cooled to room temperature and extracted with ethyl acetate (3 x 1 L). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (201 g, 81%) as a crude off-white solid. LCMS for C6H12N5O3 (M+H)⁺: m/z = 202.3 ^LH NMR (400 MHz, OMSO-d₆): δ 10.54 (s, 1 H), 6.22 (s, 2 H), 6.15 (t, J = 5.8 Hz, 1 H), 3.45 (t, J= 5.3 Hz, 2 H), 3.35 (m, 2 H), 3.22 (s, 3 H). Step 5: N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride

[00188] At room temperature N’-hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5- oxadiazole-3-carboximidamide (50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueous solution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) was added followed by water (250 mL) and ethyl acetate (250 mL). At 3-5 °C a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0 g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at 3 – 8 °C for 2 hours and then room temperature over the weekend. LCMS indicated reaction completed. The reaction solution was extracted with ethyl acetate (2 x 200 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (49.9 g, 126%) as a crude white solid. LCMS for

C₆HioClN₄03 (M+H)⁺: m/z = 221.0. ^!H NMR (400 MHz, DMSO-d₆): δ 13.43 (s, 1 H), 5.85 (t, J= 5.6 Hz, 1 H), 3.50 (t, J= 5.6 Hz, 2 H), 3.37(dd, J= 10.8, 5.6 Hz, 2 H), 3.25 (s, 3 H).

Step 6 : N-(3-Bromo-4-fluorophenyl)-N’-hydroxy-4- [(2-methoxyethyl)amino] – 1 ,2,5- oxadiazole-3-carboximidamide [00189] N-Hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5-oxadiazole-3-carboximidoyl chloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixture was heated to 60 °C. 3-Bromo-4-fluoroaniline [Oakwood products, product # 013091] (43.6 g, 0.229 mol) was added and stirred for 10 min. A warm sodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was added over 15 min. The reaction was stirred at 60 °C for 20 min. LCMS indicated reaction completion. The reaction solution was cooled to room temperature and extracted with ethyl acetate (2 x 300 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (76.7 g, 98%) as a crude brown solid. LCMS for Ci₂Hi₄BrF₅0₃ (M+H)⁺: m/z = 374.0, 376.0. 1H NMR (400 MHz, DMSO- t_f): δ 11.55 (s, 1 H), 8.85 (s, 1 H), 7.16 (t, J= 8.8 Hz, 1 H), 7.08 (dd, J= 6.1, 2.7 Hz, 1 H), 6.75 (m, 1 H), 6.14 (t, J= 5.8 Hz, 1 H), 3.48 (t, J = 5.2 Hz, 2 H), 3.35 (dd, J= 10.8, 5.6 Hz, 2 H), 3.22 (s, 3 H).

Step 7: 4-(3-Bromo-4-fluorophenyl)-3-{4- [(2-methoxyethyl)amino]-l,2,5-oxadiazol-3- yl}-l,2,4-oxadiazol-5(4H)-one

[00190] A mixture of N-(3-bromo-4-fluorophenyl)-N’-hydroxy-4-[(2- methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidamide (76.5 g, 0.204 mol), 1,1 ‘- carbonyldiimidazole (49.7 g, 0.307 mol), and ethyl acetate (720 mL) was heated to 60 °C and stirred for 20 min. LCMS indicated reaction completed. The reaction was cooled to room temperature, washed with 1 N HC1 (2 x 750 mL), dried over sodium sulfate, and concentrated to give the desired product (80.4 g, 98%) as a crude brown solid. LCMS for

(M+H)⁺: m/z = 400.0, 402.0. 1H NMR (400 MHz, DMSO-c½): δ 7.94 (t, J = 8.2 Hz, 1 H), 7.72 (dd, J = 9.1, 2.3 Hz, 1 H), 7.42 (m, 1 H), 6.42 (t, J= 5.7 Hz, 1 H), 3.46 (t, J = 5.4 Hz, 2 H), 3.36 (t, J= 5.8 Hz, 2 H), 3.26 (s, 3 H).

Step 8: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-l,2,5-oxadiazol-3- yl}-l,2,4-oxadiazol-5(4H)-one

[00191] 4-(3-Bromo-4-fluoroplienyl)-3-{4-[(2-metlioxyethyl)amino]-l,2,5-oxadiazol- 3-yl}-l,2,4-oxadiazol-5(4H)-one (78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At -67 °C boron tribromide (37 mL, 0.392 mol) was added over 15 min. The reaction was warmed up to -10 °C in 30 min. LCMS indicated reaction completed. The reaction was stirred at room temperature for 1 hour. At 0 – 5 °C the reaction was slowly quenched with saturated sodium bicarbonate solution (1.5 L) over 30 min. The reaction temperature rose to 25 °C. The reaction was extracted with ethyl acetate (2 x 500 mL, first extraction organic layer is on the bottom and second extraction organic lager is on the top). The combined organic layers were dried over sodium sulfate and concentrated to give the desired product (75 g, 99%) as a crude brown solid. LCMS for Ci₂HioBrFN₅0₄ (M+H)⁺: m/z = 386.0, 388.0.

1H NMR (400 MHz, DMSO-^): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.70 (m, 1 H), 7.68 (t, J = 8.7 Hz, 1 H), 6.33 (t, J = 5.6 Hz, 1 H), 4.85 (t, J= 5.0 Hz, 1 H), 3.56 (dd, J= 10.6, 5.6 Hz, 2 H), 3.29 (dd, J= 11.5, 5.9 Hz, 2 H).

Step 9 : 2-({4- [4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro- 1 ,2,4-oxadiazol-3-yl] – l,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate

[00192] To a solution of 4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]- l,2,5-oxadiazol-3-yl}-l,2,4-oxadiazol-5(4H)-one (1.5 kg, 3.9 mol, containing also some of the corresponding bromo-compound) in ethyl acetate (12 L) was added methanesulfonyl chloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature. Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, during which time the reaction temperature increased to 35 °C. After 2 h, the reaction mixture was washed with water (5 L), brine (1 L), dried over sodium sulfate, combined with 3 more reactions of the same size, and the solvents removed in vacuo to afford the desired product (7600 g, quantitative yield) as a tan solid. LCMS for C HnBrFNsOeS a (M+Na)⁺: m/z = 485.9, 487.9. ^!H NMR (400 MHz, DMSO- d₆): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J = 8.7 Hz, 1 H), 6.75 (t, J = 5.9 Hz, 1 H), 4.36 (t, J = 5.3 Hz, 2 H), 3.58 (dd, J = 11.2, 5.6 Hz, 2 H), 3.18 (s, 3 H).

Step 10: 3-{4-[(2-Azidoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- l,2,4-oxadiazol-5(4H)-one

To a solution of 2-({4-[4-(3-bromo-4-f uorophenyl)-5-oxo-4,5-dihydro-l ,2,4- oxadiazol-3-yl]-l ,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate (2.13 kg, 4.6 mol, containing also some of the corresponding bromo-compound) in dimethylformamide (4 L) stirring in a 22 L flask was added sodium azide (380 g, 5.84 mol). The reaction was heated at 50 °C for 6 h, poured into ice/water (8 L), and extracted with 1 : 1 ethyl acetate:heptane (20 L). The organic layer was washed with water (5 L) and brine (5 L), and the solvents removed in vacuo to afford the desired product (1464 g, 77%) as a tan solid. LCMS for CnHgBrFNsOs a

(M+Na)⁺: m/z = 433.0, 435.0. ^!H NMR (400 MHz, DMSO-J₆): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J = 5.7 Hz, 1 H), 3.54 (t, J = 5.3 Hz, 2 H), 3.45 (dd, J= 1 1.1 , 5.2 Hz, 2 H).

Step 11: 3-{4-[(2-Aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-

1.2.4- oxadiazol-5(4H)-one hydrochloride

[00194] Sodium iodide (1080 g, 7.2 mol) was added to 3-{4-[(2-azidoethyl)amino]-

1.2.5- oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-l ,2,4-oxadiazol-5(4H)-one (500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for 30 min during which time a mild exotherm was observed. Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution in methanol (1 L) dropwise at a rate so that the temperature did not exceed 35 °C, and the reaction was allowed to stir for 3.5 h at ambient temperature. The reaction was neutralized with 33 wt% solution of sodium thiosulfate pentahydrate in water (-1.5 L), diluted with water (4 L), and the pH adjusted to 9 carefully with solid potassium carbonate (250 g – added in small portions: watch foaming). Di-ieri-butyl dicarbonate (318 g, 1.45 mol) was added and the reaction was allowed to stir at room temperature. Additional potassium carbonate (200 g) was added in 50 g portions over 4 h to ensure that the pH was still at or above 9. After stirring at room temperature overnight, the solid was filtered, triturated with water (2 L), and then MTBE (1.5 L). A total of 11 runs were performed (5.5 kg, 13.38 mol). The combined solids were triturated with 1 : 1 THF:dichloromethane (24 L, 4 runs in a 20 L rotary evaporator flask, 50 °C, 1 h), filtered, and washed with dichloromethane (3 L each run) to afford an off- white solid. The crude material was dissolved at 55 °C tetrahydrofuran (5 mL/g), treated with decolorizing carbon (2 wt%) and silica gel (2 wt%), and filtered hot through celite to afford the product as an off-white solid (5122 g). The combined MTBE, THF, and dichloromethane filtrates were concentrated in vacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethyl acetate gradient, 30 L) to afford more product (262 g). The combined solids were dried to a constant weight in a convection oven (5385 g, 83%).

In a 22 L flask was charged hydrogen chloride (4 N solution in 1 ,4-dioxane, 4 L, 16 mol). tert-Butyl [2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l ,2,4- oxadiazol-3-yl]-l ,2,5-oxadiazol-3-yl}amino)ethyl]carbamate (2315 g, 4.77 mol) was added as a solid in portions over 10 min. The slurry was stirred at room temperature and gradually became a thick paste that could not be stirred. After sitting overnight at room temperature, the paste was slurried in ethyl acetate (10 L), filtered, re-slurried in ethyl acetate (5 L), filtered, and dried to a constant weight to afford the desired product as a white solid (combined with other runs, 5 kg starting material charged, 41 13 g, 95%). LCMS for

Ci₂H_nBrFN₆0₃ (M+H)⁺: m/z = 384.9, 386.9. 1H NMR (400 MHz, DMSO-^): δ 8.12 (m, 4 H), 7.76 (m, 1 H), 7.58 (t, J = 8.7 Hz, 1 H), 6.78 (t, J = 6.1 Hz, 1 H), 3.51 (dd, J = 1 1.8, 6.1 Hz, 2 H), 3.02 (m, 2 H).

Step 12: tert-Butyl ({[2-({4-[4-(3-bromo-4-nuorophenyl)-5-oxo-4,5-dihydro-l,2,4- oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate [Aldrich, product # 142662] (149 mL, 1.72 mol) and dichloromethane (1.5 L) and cooled using an ice bath to 2 °C. teri-Butanol (162 mL, 1.73 mol) in dichloromethane (200 mL) was added dropwise at a rate so that the temperature did not exceed 10 °C. The resulting solution was stirred at room temperature for 30-60 min to provide tert-bvAy\ [chlorosulfonyl]carbamate.

A 22 L flask was charged with 3- {4-[(2-aminoethyl)amino]- 1 ,2,5-oxadiazol-3- yl}-4-(3-bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one hydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After cooling to -15 °C with an ice/salt bath, the solution oi tert- Vmtvl i Vi 1 r>rosulfonyl]carbamate (prepared as above) was added at a rate so that the temperature did not exceed -10 °C (addition time 7 min). After stirring for 10 min, triethylamine (1085 mL, 7.78 mol) was added at a rate so that the temperature did not exceed -5 °C (addition time 10 min). The cold bath was removed, the reaction was allowed to warm to 10 °C, split into two portions, and neutralized with 10% cone HC1 (4.5 L each portion). Each portion was transferred to a 50 L separatory funnel and diluted with ethyl acetate to completely dissolve the white solid (-25 L). The layers were separated, and the organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford an off- white solid. The solid was triturated with MTBE (2 x 1.5 L) and dried to a constant weight to afford a white solid. A total of 4113 g starting material was processed in this manner (5409 g, 98%). 1H NMR (400 MHz, DMSO-^): δ 10.90 (s, 1 H), 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J = 8.6 Hz, 1 H), 6.58 (t, J = 5.7 Hz, 1 H), 3.38 (dd, J= 12.7, 6.2 Hz, 2 H), 3.10 (dd, J= 12.1 , 5.9 Hz, 2 H), 1.41 (s, 9 H).

Step 13: N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

[00198] To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) was added tert-bvXyl ({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate (1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixture was stirred at room temperature for 1.5 h, the solvents removed in vacuo, and chased with dichloromethane (2 L). The resulting solid was treated a second time with fresh 98:2 trifluoroacetic acid:water (8.9 L), heated for 1 h at 40- 50 °C, the solvents removed in vacuo, and chased with dichloromethane (3 x 2 L). The resulting white solid was dried in a vacuum drying oven at 50 °C overnight. A total of 5409 g was processed in this manner (4990 g, quant, yield). LCMS for C₁₂H₁₂BrFN₇0₅S (M+H)⁺: m/z = 463.9, 465.9. 1H NMR (400 MHz, DMSO- ): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.7 Hz, 1 H), 6.67 (t, J = 5.9 Hz, 1H), 6.52 (t, J= 6.0 Hz, 1 H), 3.38 (dd, J = 12.7, 6.3 Hz, 2 H), 3.11 (dd, J = 12.3, 6.3 Hz). Step 14: 4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N’- hydroxy-l,2,5-oxadiazole-3-carboximidamide

[00199] To a crude mixture of N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5- dihydro-l,2,4-oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide (2.4 mol) containing residual amounts of trifluoroacetic acid stirring in a 22 L flask was added THF (5 L). The resulting solution was cooled to 0 °C using an ice bath and 2 N NaOH (4 L) was added at a rate so that the temperature did not exceed 10 °C. After stirring at ambient temperature for 3 h (LCMS indicated no starting material remained), the pH was adjusted to 3-4 with concentrated HC1 (-500 mL). The THF was removed in vacuo, and the resulting mixture was extracted with ethyl acetate (15 L). The organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford a solid. The solid was triturated with MTBE (2 x 2 L), combined with three other reactions of the same size, and dried overnight in a convection oven to afford a white solid (3535 g). The solid was recrystallized (3 x 22 L flasks, 2:1 watenethanol, 14.1 L each flask) and dried in a 50 °C convection oven to a constant weight to furnish the title compound as an off-white solid (3290 g, 78%). LCMS for CnHnBrF yC S (M+H)⁺: m/z = 437.9, 439.9. i NMR (400 MHz, DMSO-J^): δ 11.51 (s, 1 H), 8.90 (s, 1 H), 7.17 (t, J= 8.8 Hz, 1 H), 7.11 (dd, J= 6.1, 2.7 Hz, 1 H), 6.76 (m, 1 H), 6.71 (t, J = 6.0 Hz, 1 H), 6.59 (s, 2 H), 6.23 (t, J= 6.1 Hz, 1 H), 3.35 (dd, J= 10.9, 7.0 Hz, 2 H), 3.10 (dd, J= 12.1, 6.2 Hz, 2 H).

PATENT

WO 2010005958

https://www.google.com/patents/WO2010005958A2?cl=en

EXAMPLES Example 1

4-({2-[(Aminosulfonyl)amino]ethyl}amino)-7V-(3-bromo-4-fluorophenyl)-iV’-hydroxy- l,2,5-oxadiazole-3-carboximidamide

Step A: 4-Amino-N’-hydroxy-l,2,5-oxadiazole-3-carboximidamide

Malononitrile [Aldrich, product # M1407] (320.5 g, 5 mol) was added to water (7 L) preheated to 45 ⁰C and stirred for 5 min. The resulting solution was cooled in an ice bath and sodium nitrite (380 g, 5.5 mol) was added. When the temperature reached 10 ⁰C, 6 N hydrochloric acid (55 mL) was added. A mild exothermic reaction ensued with the temperature reaching 16 ⁰C. After 15 min the cold bath was removed and the reaction mixture was stirred for 1.5 hrs at 16-18 ⁰C. The reaction mixture was cooled to 13 ⁰C and 50% aqueous hydroxylamine (990 g, 15 mol) was added all at once. The temperature rose to 26 ⁰C. When the exothermic reaction subsided the cold bath was removed and stirring was continued for 1 hr at 26-27⁰C, then it was slowly brought to reflux. Reflux was maintained for 2 hrs and then the reaction mixture was allowed to cool overnight. The reaction mixture was stirred in an ice bath and 6 N hydrochloric acid (800 mL) was added in portions over 40 min to pH 7.0. Stirring was continued in the ice bath at 5 ⁰C. The precipitate was collected by filtration, washed well with water and dried in a vacuum oven (50 ⁰C) to give the desired product (644 g, 90%). LCMS for C₃H₆N₅O₂ (M+H)+: m/z = 144.0. ¹³C NMR (75 MHz, CD₃OD): δ 156.0, 145.9, 141.3. Step B: 4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride

4-Amino-N’-hydroxy-l,2,5-oxadiazole-3-carboximidamide (422 g, 2.95 mol) was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 Ν hydrochloric acid (1.475 L, 3 eq.) and this suspension was stirred at 42 – 45 ⁰C until complete solution was achieved. Sodium chloride (518 g, 3 eq.) was added and this solution was stirred in an ice/water/methanol bath. A solution of sodium nitrite (199.5 g, 0.98 eq.) in water (700 mL) was added over 3.5 hrs while maintaining the temperature below 0 ⁰C. After complete addition stirring was continued in the ice bath for 1.5 hrs and then the reaction mixture was allowed to warm to 15 ⁰C. The precipitate was collected by filtration, washed well with water, taken in ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g) and stirred for 1 hr. This suspension was filtered through sodium sulfate (200 g) and the filtrate was concentrated on a rotary evaporator. The residue was dissolved in methyl f-butyl ether (5.5 L), treated with charcoal (40 g), stirred for 40 min and filtered through Celite. The solvent was removed in a rotary evaporator and the resulting product was dried in a vacuum oven (45 ⁰C) to give the desired product (256 g, 53.4%). LCMS for C₃H₄ClN₄O₂(M+H)+: m/z = 162.9. 13c NMR (100 MHz, CD₃OD): δ 155.8, 143.4, 129.7.

Step C: 4-Amino-N’-hydroxy-N-(2-methoxyethyl)- 1 ,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride (200.0 g, 1.23 mol) was mixed with ethyl acetate (1.2 L). At 0-5⁰C 2-methoxyethylamine [Aldrich, product # 143693] (119.0 mL, 1.35 mol) was added in one portion while stirring. The reaction temperature rose to 41 ⁰C. The reaction was cooled to 0 – 5 °C. Triethylamine (258 mL, 1.84 mol) was added. After stirring 5 min, LCMS indicated reaction completion. The reaction solution was washed with water (500 mL) and brine (500 mL), dried over sodium sulfate, and concentrated to give the desired product (294 g, 119%) as a crude dark oil. LCMS for C₆Hi₂N₅O₃ (M+H)⁺: m/z = 202.3. ¹H NMR (400 MHz, DMSO-J₆): δ 10.65 (s, 1 H), 6.27 (s, 2 H), 6.10 (t, J= 6.5 Hz, 1 H), 3.50 (m, 2 H), 3.35 (d, J= 5.8 Hz, 2 H), 3.08 (s, 3 H).

Step D: N’-Hydroxy-4-[(2-methoxyethyl)amino]-l ,2,5-oxadiazole-3-carboximidamide

4-Amino-N’-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3-carboximidaniide (248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210 g, 3.7 mol) was added. The reaction was refluxed at 100 ⁰C overnight (15 hours). TLC with 50% ethyl acetate (containing 1% ammonium hydroxide) in hexane indicated reaction completed (product Rf= 0.6, starting material Rf = 0.5). LCMS also indicated reaction completion. The reaction was cooled to room temperature and extracted with ethyl acetate (3 x 1 L). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (201 g, 81%) as a crude off-white solid. LCMS for C₆H₁₂N₅O₃ (M+H)⁺: m/z = 202.3 ¹H NMR (400 MHz, DMSO-Gk): δ 10.54 (s, 1 H), 6.22 (s, 2 H), 6.15 (t, J= 5.8 Hz, 1 H), 3.45 (t, J= 5.3 Hz, 2 H), 3.35 (m, 2 H), 3.22 (s, 3 H).

Step E: N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride

Ν. ,Ν O

At room temperature N’-hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3- carboximidamide (50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueous solution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) was added followed by water (250 mL) and ethyl acetate (250 mL). At 3-5 ⁰C a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0 g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at 3 – 8 ⁰C for 2 hours and then room temperature over the weekend. LCMS indicated reaction completed. The reaction solution was extracted with ethyl acetate (2 x 200 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (49.9 g, 126%) as a crude white solid. LCMS for C₆Hi₀ClN₄O₃ (M+H)⁺: m/z = 221.0. ¹H NMR (400 MHz, DMSO-J₆): δ 13.43 (s, 1 H), 5.85 (t, J= 5.6 Hz, 1 H), 3.50 (t, J= 5.6 Hz, 2 H), 3.37(dd, J= 10.8, 5.6 Hz, 2 H), 3.25 (s, 3 H).

Step F: N-(3-Bromo-4-fluorophenyl)-N’-hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5- oxadiazole-3 -carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixture was heated to 60 °C. 3-Bromo-4- fluoroaniline [Oakwood products, product # 013091] (43.6 g, 0.229 mol) was added and stirred for 10 nrn^‘n. A warm sodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was added over 15 min. The reaction was stirred at 60 ⁰C for 20 min. LCMS indicated reaction completion. The reaction solution was cooled to room temperature and extracted with ethyl acetate (2 x 300 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (76.7 g, 98%) as a crude brown solid. LCMS for Ci₂Hi₄BrFN₅O₃ (M+H)⁺: m/z = 374.0, 376.0. ¹H NMR (400 MHz, DMSO-J₆): δ 11.55 (s, 1 H), 8.85 (s, 1 H), 7.16 (t, J= 8.8 Hz, 1 H), 7.08 (dd, J= 6.1, 2.7 Hz, 1 H), 6.75 (m, 1 H), 6.14 (t, J= 5.8 Hz, 1 H), 3.48 (t, J= 5.2 Hz, 2 H), 3.35 (dd, J= 10.8, 5.6 Hz, 2 H), 3.22 (s, 3 H).

Step G: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-l,2,5-oxadiazol-3-yl}- 1 ,2,4-oxadiazol-5(4H)-one

A mixture of N-(3-bromo-4-fluorophenyl)-N’-hydroxy-4-[(2-methoxyethyl)amino]-l,2,5- oxadiazole-3-carboximidamide (76.5 g, 0.204 mol), l,r-carbonyldiimidazole (49.7 g, 0.307 mol), and ethyl acetate (720 mL) was heated to 60 ⁰C and stirred for 20 min. LCMS indicated reaction completed. The reaction was cooled to room temperature, washed with 1 Ν HCl (2 x 750 mL), dried over sodium sulfate, and concentrated to give the desired product (80.4 g, 98%) as a crude brown solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z = 400.0, 402.0. ¹H NMR (400 MHz, OMSO-d₆): δ 7.94 (t, J= 8.2 Hz, 1 H), 7.72 (dd, J= 9.1, 2.3 Hz, 1 H), 7.42 (m, 1 H), 6.42 (t, J= 5.7 Hz, 1 H), 3.46 (t, J= 5.4 Hz, 2 H), 3.36 (t, J= 5.8 Hz, 2 H), 3.26 (s, 3 H).

Step H: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-liydroxyethyl)amino]-l,2,5-oxadiazol-3-yl}- 1 ,2,4-oxadiazol-5(4H)-one

4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyetliyl)amino]-l,2,5-oxadiazol-3-yl}-l,2,4- oxadiazol-5(4H)-one (78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At -67 ⁰C boron tribromide (37 mL, 0.392 mol) was added over 15 min. The reaction was warmed up to -10 ⁰C in 30 min. LCMS indicated reaction completed. The reaction was stirred at room temperature for 1 hour. At 0 – 5 ⁰C the reaction was slowly quenched with saturated sodium bicarbonate solution (1.5 L) over 30 min. The reaction temperature rose to 25 ⁰C. The reaction was extracted with ethyl acetate (2 x 500 mL, first extraction organic layer is on the bottom and second extraction organic lager is on the top). The combined organic layers were dried over sodium sulfate and concentrated to give the desired product (75 g, 99%) as a crude brown solid. LCMS for C₁₂H₁₀BrFN₅O₄ (M+H)⁺: m/z = 386.0, 388.0. ¹H NMR (400 MHz, DMSO-^₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.70 (m, 1 H), 7.68 (t, J= 8.7 Hz, 1 H), 6.33 (t, J= 5.6 Hz, 1 H), 4.85 (t, J= 5.0 Hz, 1 H), 3.56 (dd, J= 10.6, 5.6 Hz, 2 H), 3.29 (dd, J= 11.5, 5.9 Hz, 2 H).

Step I: 2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]-l,2,5- oxadiazol-3-yl}amino)ethyl methanesulfonate

To a solution of 4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-l,2,5-oxadiazol- 3-yl}-l,2,4-oxadiazol-5(4H)-one (1.5 kg, 3.9 mol, containing also some of the corresponding bromo-compound) in ethyl acetate (12 L) was added methanesulfonyl chloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature. Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, during which time the reaction temperature increased to 35 ⁰C. After 2 h, the reaction mixture was washed with water (5 L), brine (I L), dried over sodium sulfate, combined with 3 more reactions of the same size, and the solvents removed in vacuo to afford the desired product (7600 g, quantitative yield) as a tan solid. LCMS for

Ci₃HnBrFN₅O₆SNa (M+Na)⁺: m/z = 485.9, 487.9. ¹H NMR (400 MHz, DMSCW₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J- 5.9 Hz, 1 H), 4.36 (t, J= 5.3 Hz, 2 H), 3.58 (dd, J= 11.2, 5.6 Hz, 2 H), 3.18 (s, 3 H).

Step J: 3-{4-[(2-Azidoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- 1 ,2,4-oxadiazol-5(4H)-one

To a solution of 2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate (2.13 kg, 4.6 mol, containing also some of the corresponding bromo-compound) in dimethylformamide (4 L) stirring in a 22 L flask was added sodium azide (380 g, 5.84 mol). The reaction was heated at 50⁰C for 6 h, poured into ice/water (8 L), and extracted with 1 : 1 ethyl acetate:heptane (20 L). The organic layer was washed with water (5 L) and brine (5 L), and the solvents removed in vacuo to afford the desired product (1464 g, 77%) as a tan solid. LCMS for C₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z =

433.0, 435.0. ¹H NMR (400 MHz, DMSO-*/*): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J= 5.7 Hz, 1 H), 3.54 (t, J= 5.3 Hz, 2 H), 3.45 (dd, J= 11.1, 5.2 Hz, 2 H).

Step K: 3-{4-[(2-Aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- 1 ,2,4-oxadiazol-5(4H)-one hydrochloride

Sodium iodide (1080 g, 7.2 mol) was added to 3-{4-[(2-azidoethyl)amino]-l,2,5-oxadiazol-3- yl}-4-(3-bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one (500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for 30 min during which time a mild exotherm was observed. Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution in methanol (1 L) dropwise at a rate so that the temperature did not exceed 35 ⁰C, and the reaction was allowed to stir for 3.5 h at ambient temperature. The reaction was neutralized with 33 wt% solution of sodium thiosulfate pentahydrate in water (~1.5 L), diluted with water (4 L), and the pΗ adjusted to 9 carefully with solid potassium carbonate (250 g – added in small portions: watch foaming). Di-fe/t-butyl dicarbonate (318 g, 1.45 mol) was added and the reaction was allowed to stir at room temperature. Additional potassium carbonate (200 g) was added in 50 g portions over 4 h to ensure that the pΗ was still at or above 9. After stirring at room temperature overnight, the solid was filtered, triturated with water (2 L), and then MTBE (1.5 L). A total of 11 runs were performed (5.5 kg, 13.38 mol). The combined solids were triturated with 1 : 1 TΗF:dichloromethane (24 L, 4 runs in a 20 L rotary evaporator flask, 50 ⁰C, 1 h), filtered, and washed with dichloromethane (3 L each run) to afford an off- white solid. The crude material was dissolved at 55 ⁰C tetrahydrofuran (5 mL/g), treated with decolorizing carbon (2 wt%) and silica gel (2 wt%), and filtered hot through celite to afford the product as an off-white solid (5122 g). The combined MTBE, THF, and dichloromethane filtrates were concentrated in vacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethyl acetate gradient, 30 L) to afford more product (262 g). The combined solids were dried to a constant weight in a convection oven (5385 g, 83%).

In a 22 L flask was charged hydrogen chloride (4 N solution in 1,4-dioxane, 4 L, 16 mol). fert-Butyl [2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]carbamate (2315 g, 4.77 mol) was added as a solid in portions over 10 min. The slurry was stirred at room temperature and gradually became a thick paste that could not be stirred. After sitting overnight at room temperature, the paste was slurried in ethyl acetate (10 L), filtered, re-slurried in ethyl acetate (5 L), filtered, and dried to a constant weight to afford the desired product as a white solid (combined with other runs, 5 kg starting material charged, 4113 g, 95%). LCMS for C₁₂H_nBrFN₆O₃ (M+H)⁺: m/z

= 384.9, 386.9. ¹H NMR (400 MHz, DMSO-J₆): δ 8.12 (m, 4 H), 7.76 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.78 (t, J= 6.1 Hz, 1 H), 3.51 (dd, J= 11.8, 6.1 Hz, 2 H), 3.02 (m, 2 H).

Step L: tert-Butyl ({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-diliydro-l,2,4-oxadiazol- 3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate [Aldrich, product #

142662] (149 mL, 1.72 mol) and dichloromethane (1.5 L) and cooled using an ice bath to 2 ⁰C. tert-Butanol (162 mL, 1.73 mol) in dichloromethane (200 mL) was added dropwise at a rate so that the temperature did not exceed 10 ⁰C. The resulting solution was stirred at room temperature for 30-60 min to provide tert-butyl [chlorosulfonyljcarbamate.

A 22 L flask was charged with 3-{4-[(2-aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3- bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one hydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After cooling to -15 ⁰C with an ice/salt bath, the solution of tert-butyl [chlorosulfonyl]carbamate (prepared as above) was added at a rate so that the temperature did not exceed -10 ⁰C (addition time 7 min). After stirring for 10 min, triethylamine (1085 mL, 7.78 mol) was added at a rate so that the temperature did not exceed -5 ⁰C (addition time 10 min). The cold bath was removed, the reaction was allowed to warm to 10 ⁰C, split into two portions, and neutralized with 10% cone HCl (4.5 L each portion). Each portion was transferred to a 50 L separatory funnel and diluted with ethyl acetate to completely dissolve the white solid (~25 L). The layers were separated, and the organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford an off-white solid. The solid was triturated with MTBE (2 x 1.5 L) and dried to a constant weight to afford a white solid. A total of 4113 g starting material was processed in this manner (5409 g, 98%). *Η NMR (400 MHz, OMSO-d₆): δ 10.90 (s, 1 H), 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.6 Hz, 1 H), 6.58 (t, J= 5.7 Hz, 1 H), 3.38 (dd, J= 12.7, 6.2 Hz, 2 H), 3.10 (dd, J = 12.1, 5.9 Hz, 2 H), 1.41 (s, 9 H). Step M: N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dmydro-l ,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) was added tert-butyl ({[2- ({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-diliydro-l,2,4-oxadiazol-3-yl]-l,2,5-oxadiazol-3- yl}amino)ethyl]amino}sulfonyl)carbamate (1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixture was stirred at room temperature for 1.5 h, the solvents removed in vacuo, and chased with dichloromethane (2 L). The resulting solid was treated a second time with fresh 98:2 trifluoroacetic acid:water (8.9 L), heated for 1 h at 40-50 ⁰C, the solvents removed in vacuo, and chased with dichloromethane (3 x 2 L). The resulting white solid was dried in a vacuum drying oven at 50 ⁰C overnight. A total of 5409 g was processed in this manner (4990 g, quant, yield). LCMS for C]₂H₁₂BrFN₇O₅S (M+H)⁺: m/z = 463.9, 465.9.

¹H NMR (400 MHz, OM$>O-d₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.7 Hz, 1 H), 6.67 (t, J= 5.9 Hz, IH), 6.52 (t, J= 6.0 Hz, 1 H), 3.38 (dd, J= 12.7, 6.3 Hz, 2 H), 3.11 (dd, J= 12.3, 6.3 Hz).

Step N: 4-( {2-[(Aminosulfonyl)amino]ethyl} amino)-N-(3-bromo-4-fluorophenyl)-N- hydroxy-l,2,5-oxadiazole-3-carboximidamide

To a crude mixture of N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4- oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide (2.4 mol) containing residual amounts of trifluoroacetic acid stirring in a 22 L flask was added THF (5 L). The resulting solution was cooled to 0 °C using an ice bath and 2 Ν NaOH (4 L) was added at a rate so that the temperature did not exceed 10 ⁰C. After stirring at ambient temperature for 3 h (LCMS indicated no starting material remained), the pH was adjusted to 3-4 with concentrated HCl (-500 mL). The THF was removed in vacuo, and the resulting mixture was extracted with ethyl acetate (15 L). The organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford a solid. The solid was triturated with MTBE (2 x 2 L), combined with three other reactions of the same size, and dried overnight in a convection oven to afford a white solid (3535 g). The solid was recrystallized (3 x 22 L flasks, 2: 1 water: ethanol, 14.1 L each flask) and dried in a 50 ⁰C convection oven to a constant weight to furnish the title compound as an off-white solid (3290 g, 78%). LCMS for C_nH₁₄BrFN₇O₄S (M+H)⁺: m/z = 437.9, 439.9. ¹H NMR (400 MHz, DMSO-J₆): δ 11.51 (s, 1 H), 8.90 (s, 1 H), 7.17 (t, J= 8.8 Hz, 1 H), 7.11 (dd, J= 6.1, 2.7 Hz, 1 H), 6.76 (m, 1 H), 6.71 (t, J= 6.0 Hz, 1 H), 6.59 (s, 2 H), 6.23 (t, J= 6.1 Hz, 1 H), 3.35 (dd, J= 10.9, 7.0 Hz, 2 H), 3.10 (dd, J= 12.1, 6.2 Hz, 2 H).

The final product was an anhydrous crystalline solid. The water content was determined to be less than 0.1% by Karl Fischer titration.

CLIP

INCB24360

Company:Incyte Corp.
Target: IDO1
Disease: Cancer

 

TEAMWORK

Incyte’s team (from left): Andrew Combs, Dilip Modi, Joe Glenn, Brent Douty, Padmaja Polam, Brian Wayland, Rick Sparks, Wenyu Zhu, and Eddy Yue.

Credit: Incyte

Tweet