Synthesis and evaluation of novel HCV replication inhibitors

David C. McGowana,*, Mourad D. Khamlichi,b Alex De Groot,a Frederik Pauwels, Frédéric Delouvrouy, Kristof Van Emelen,a Kenneth Simmen,a and Pierre Raboissona

aJanssen Infectious Diseases BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium

bVillapharma Research, Parque Tecnológico de Fuente Álamo. Ctra. El Estrecho-Lobosillo, Km. 2,5- Av. Azul 30320 Fuente Álamo de Murcia, Murcia, Spain

Supporting Information

Reagents and solvents were purchased from commercial sources and used without purification. Anhydrous solvents were obtained from Aldrich and used directly. Silica gel chromatography was generally performed with prepacked silica gel cartridges (from Biotage, Interchim, or Teledyne-Isco). The purity of all compounds were determined by UPLC, conducted on Waters: Acquity® UPLC® -DAD and SQD systems, Waters HSS T3 column (1.8 µm, 2.1 x 100 mm, at 40°C). The mobile phase consisted of 10 mM CH3COONH4 in 95% H2O + 5% CH3CN (A) and CH3CN (B). A gradient program was used as follows: From 100% A to 5% A in 2.10 min, to 0% A in 0.90 min, to 5% A in 0.5min, at a flow rate of 0.8 mL/min. All compounds exhibited greater than 95% purity. 1H and 13C (APT) NMR experiments were performed at 27°C on a Bruker Avance I - 600 MHz. TMS or residual solvent shift of deuterated dimethysulfoxide (1H : 2.5 ppm, 13C : 39.51 ppm) was used as internal standard for calibration of spectra. The following standard abbreviations were used for signal multiplicities: singlet (s), doublet (d), triplet (t), multiplet (m), and broad signal (br. s.). NMR abbreviations: s = singlet, br. s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet.

Preparation of Intermediates

(S)-t-butyl 2-(6-nitro-1H-benzo[d]imidazol-2-yl)pyrrolidine-1-carboxylate (1). Into a 500 mL round bottom flask equipped with a magnetic stir bar was placed boc-L-proline (14g, 65.3 mmol), pyridine/DMF (100 mL, 1/1), then CDI (11.65 g, 71.8 mmol). The mixture is allowed to stir for 2 hours at 45°C. 4-nitro-1, 2-phenylenediamine (10 g, 65.3 mmol) is added. The reaction is allowed to cool to room temperature and stirred 15 hours. The solvents were removed under reduced pressure and the mixture was reconstituted in acetic acid (100 mL) and heated to 100°C for 1 hour. The solvents were removed under reduced pressure. Ethyl acetate (100 mL) was added and was washed with a NaHCO3 (sat. aq. 4 x 50 mL). The organic layer was dried over sodium sulfate, the solids were removed via filtration, and the solvents of the filtrate were

removed under reduced pressure to afford the titled compound as an orange solid (17.4 g, 80%). LC-MS [M+H]+ m/z = 333 (M+H), and a purity above 95%.

(S)-tert-butyl 2-(6-amino-1H-benzo[d]imidazol-2-yl)pyrrolidine-1-carboxylate (2). Into a 500 mL round bottom flask equipped with a magnetic stir bar was placed 1 (17 g, 51.15 mmol), ethyl acetate (200 mL), and ethanol (200 mL). The flask was sparged with nitrogen and 10% Pd/C (1g) is introduced. The flask is sealed, the atmosphere is removed and replaced with hydrogen. The mixture was allowed to stir vigorously for 15 hours. LC-MS shows complete conversion to the desired product. The flask was opened, sparged with nitrogen, and filtered through packed celite under a blanket of nitrogen gas. The solvents of the filtrate were removed under reduced pressure to afford a viscous oil. 1,4-Dioxane is added (100 mL) followed by HCl in dioxane (0.1M, 50 mL) to form the HCl salt. The solvents were removed under reduced pressure to afford the titled compound as a tan solid (16.1 g, 93% yield). LC-MS [M+H]+ m/z = 303.

(S)-6-azido-2-(pyrrolidin-2-yl)-1H-benzo[d]imidazole (3). A solution of NaNO2 in water (14.8 mL) was added drop-wise to a solution of 2 in water (24 mL) and conc. HCl (14 mL) at 0ºC. The mixture was stirred for 1 h. A solution of NaN3 in water (7.4 mL) was added drop-wise and stirred for 30 min. The product was partitioned with EtOAc and NaHCO3 (sat. aq.), dried over MgSO4, the solids were removed by filtration, and the filtrate was concentrated to dryness. The crude was purified by silica gel chromatography using a CH2Cl2 / CH3OH gradient (9:1 to 5:1), to afford the titled compound as a tan solid (1.89 g, 47%). LC-MS [M+H]+ m/z = 229.1. Retention time: 1.28min.

(S)-1-(2-(6-azido-1H-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)-2-phenylethanone (4a). Phenylacetic acid (0.752 g, 5.52 mmol), DIPEA (1.9 mL, 11.04 mmol) and HATU (2.099 g, 5.52 mmol) were dissolved in DMA (11 mL) and stirred for 5 minutes at rt. Then 3 (0.63 g, 2.76 mmol) was added and the stirring continued overnight. Crude material was diluted with ethyl acetate and washed two times with NaHCO3

(1M), brine and water, dried over MgSO4, filtered and concentrated under vacuum. The crude material was purified by chromatography column using a dichloromethane / methanol (100/0 to 90/10) gradient to afford the titled compound (0.9 g, 94%). LC-MS [M+H]+ m/z = 347.1. Retention time: 2.28min.

(R)-1-((S)-2-(6-azido-1H-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)-2-hydroxy-2-phenylethanone (4b). The titled compound was prepared using a procedure analogous to the one used to prepare 4a, with the exception that (R)-(-)-mandelic acid was used. LC-MS [M+H]+ m/z = 363.1. Retention time: 1.95min.

Methyl ((S)-1-((S)-2-(6-azido-1H-benzo[d]imidazol-2-yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (4c). The titled compound was prepared using a procedure analogous to the one used to prepare 4a, with the exception that (S)-2-((methoxycarbonyl)amino)-3-methylbutanoic acid [74761-42-5] was used. LC-MS [M+H]+ m/z = 386.2. Retention time: 2.17min.

(S)-5-(4-bromophenyl)-2-(pyrrolidin-2-yl)-1H-imidazole (6). Synthesis of the titled compound, via the boc-protected precursor (5), were carried out according to the procedure described in Bioorganic and Medicinal Chemistry Letters 22 (2012) 3488-3491.

(S)-1-(2-(5-(4-bromophenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-2-phenylethanone (7a). DIPEA (1.9 mL, 11.3 mmol) and HATU (2.1 g, 5.6 mmol) were added to a solution of 2-phenylacetic acid (0.773g, 5.6 mmol) in DMA (11 mL), the mixture stirred for 15 minutes at rt. Then 6 (1.03 g, 2.8 mmol) was added and stirred at r.t. overnight. The mixture was washed with EtOAc, NaHCO3 (sat. aq.) and brine. The organic layers were dried over magnesium sulfate, the solids were removed by filtration and the filtrate was concentrated under reduced pressure. The product was purified by flash chromatography on silica gel, using DCM/MeOH (99:1- 20:1) gradient to afford the titled compound (1.23 g, 85%).

(R)-1-((S)-2-(5-(4-bromophenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-2-hydroxy-2-phenylethanone (7b). The titled compound was prepared using a procedure analogous to the one used to prepare 7a, with the exception that (R)-(-)-mandelic acid was used. LC-MS [M+]+ m/z = 426.1. [M+2]+ m/z = 428.1. Retention time: 2.01min.

Methyl ((S)-1-((S)-2-(5-(4-bromophenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (7c). The titled compound was prepared using a procedure analogous to the one used to prepare 7a, see J. Med. Chem., 2014, 57 (5), pp 2058–2073, with the exception that (S)-2-((methoxycarbonyl)amino)-3-methylbutanoic acid [74761-42-5] was used. 1H NMR (300 MHz, CHLOROFORM-d) δ 7.66 (br d, J=8.52 Hz, 1H), 7.48 (d, J=8.25 Hz, 3H), 7.28 (s, 1H), 5.44 (br d, J=9.21 Hz, 1H), 4.24-4.37 (m, 1H), 3.59-3.89 (m, 4H), 2.82 (s, 3H), 2.28-2.40 (m, 1H), 1.91-2.26 (m, 4H), 0.88 (d, J=6.74 Hz, 6H). LC-MS [M+]+ m/z = 449.0. [M+2]+ m/z = 451.0. Retention time: 0.84min.

(S)-2-phenyl-1-(2-(5-(4-((trimethylsilyl)ethynyl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)ethanone (8a). In a sealed tube was placed 7a (1.91 g, 4.6 mmol), Pd(PPh3)2Cl2(0.313 g, 0.4 mmol), CuI (0.17 g, 0.8 mmol) and PPh3 (0.234 g, 0.8 mmol) and were dissolved in Et3N (37.2 mL) and stirred at r.t. for 15 min. Then TMS-acetylene (3.8 mL, 27.9 mmol) in Et3N (18.6 mL) was added. The mixture was heated to 110 ºC and stirred overnight. The solvent was removed under reduced pressure and the crude was purified via silica column chromatography (DCM/MeOH, 99:1- 20:1) to afford the titled compound as a brown oil (1.8 g, 90%) brown oil.

(R)-2-hydroxy-2-phenyl-1-((S)-2-(5-(4-((trimethylsilyl)ethynyl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)ethanone (8b). The titled compound was prepared using the same procedure as the one used to prepare 8a. LC-MS [M+H]+ m/z = 444.2. Retention time: 2.91min.

Methyl ((S)-3-methyl-1-oxo-1-((S)-2-(5-(4-((trimethylsilyl)ethynyl)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)butan-2-yl)carbamate (8c). The titled compound was prepared using the same procedure as the one used to prepare 8a. LC-MS [M+H]+ m/z = 467.2. Retention time: 3.05min.

(S)-1-(2-(5-(4-ethynylphenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-2-phenylethanone (9a). K2CO3 (1.1 g, 7.9 mmol) was added to a stirred solution of 8a in methanol (16.4 mL) and allowed to stir at rt for 2h. The solids were removed by filtration and the solvent of the filtrate was removed under reduced pressure. The crude was washed with EtOAc, H2O and brine. The organic layer was dried over magnesium sulfate, the solids were removed by filtration and the solvent of the filtrate was concentrated under reduced pressure to afford the titled compound as a brown solid (1.27 g, 91 %).

(R)-1-((S)-2-(5-(4-ethynylphenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-2-hydroxy-2-phenylethanone (9b). The titled compound was deprotected using the same procedure as the one used to prepare 9a.

Methyl ((S)-1-((S)-2-(5-(4-ethynylphenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (9c). The titled compound was deprotected using the same procedure as the one used to prepare 9a. LC-MS [M+H]+ m/z = 395.2. Retention time: 1.99min.

Spectra of final compounds

101H NMR (DMSO-d6, 600 MHz)

1013C NMR (DMSO-d6, 101 MHz)

111H NMR (DMSO-d6, 600 MHz)

1113C NMR (DMSO-d6, 101 MHz)

121H NMR (DMSO-d6, 600 MHz)

1213C NMR (DMSO-d6, 101 MHz)

131H NMR (DMSO-d6, 600 MHz)

1313C NMR (DMSO-d6, 101 MHz)

141H NMR (DMSO-d6, 600 MHz)

1413C NMR (DMSO-d6, 101 MHz)

151H NMR (DMSO-d6, 600 MHz)

1513C NMR (DMSO-d6, 101 MHz)

161H NMR (DMSO-d6, 600 MHz)

1613C NMR (DMSO-d6, 101 MHz)

171H NMR (DMSO-d6, 600 MHz)

1713C NMR (DMSO-d6, 101 MHz)

181H NMR (DMSO-d6, 600 MHz)

1813C NMR (DMSO-d6, 101 MHz)