Design and Synthesis of Inhibitors of Plasmodium falciparumN

Supporting Information

Design and Synthesis of Inhibitors of Plasmodium falciparum N-Myristoyltransferase, a Promising Target for Anti-Malarial Drug Discovery Zhiyong Yu†, James A. Brannigan‡, David K. Moss§, A. Marek Brzozowski‡, Anthony J. Wilkinson‡, Anthony A. Holder§, Edward W. Tate†* and Robin J. Leatherbarrow†* †

Department of Chemistry, Imperial College London, London, SW7 2AZ, U.K.

‡

York Structural Biology Laboratory, Department of Chemistry, University of York,

York, YO10 5DD, U.K. §

Division of Parasitology, MRC National Institute for Medical Research, The

Ridgeway, Mill Hill, London, NW7 1AA, U.K.

Contents SYNTHETIC PROCEDURES ........................................................................................................... 2 SUPPLEMENTARY TABLES ......................................................................................................... 24 SUPPLEMENTARY FIGURES ....................................................................................................... 28 REFERENCES ............................................................................................................................. 31

S1

SYNTHETIC PROCEDURES Ethyl 3-methyl-4-(3-(pyridin-3-ylmethylamino)propoxy)

NH

O

N COOEt

benzofuran-2-carboxylate (1)

O

This compound was synthesized and characterized according to literature procedures. 1, 2 1

H-NMR (CD3OD, 400 MHz): δ 8.82 (s, 1H), 8.74 (s, 1H), 8.20 (d, J=8.0 Hz, 1H),

7.70 (dd, J=8.0, 5.2 Hz, 1H), 7.39 (t, J=8.0 Hz, 1H), 7.13 (d, J=8.4 Hz, 1H), 6.79 (d, J=8.0 Hz, 1H), 4.44 (s, 2H), 4.41 (q, J=7.2 Hz, 2H), 4.28 (t, J=5.6 Hz, 2H), 3.41 (t, J=7.6 Hz, 2H), 2.70 (s, 3H), 2.38-2.31 (m, 2H), 1.43 (t, J=7.2 Hz, 3H).

13

C-NMR

(CD3OD, 100 MHz): δ 160.48, 155.71, 155.04, 148.96, 148.69, 140.07, 139.52, 128.70, 125.74, 124.88, 118.21, 104.63, 104.00, 64.65, 60.69, 47.96, 45.02, 25.78, 13.23, 10.38. Calculated exact mass for the protonated molecule (C21H25N2O4): 369.1814; measured accurate mass (ESI): 369.1806. LC-MS purity: 100%, Rt = 11.74 min.

t-Butyl 4-{[2-(ethoxycarbonyl)-3-methyl-1-benzofuran-4-yl]oxy} NBoc

piperidine-1-carboxylate (I-1)

O COOEt O

To a stirred solution of ethyl 4-hydroxy-3-methylbenzofuran-2-

carboxylate (2 g, 9.1 mmol), t-Butyl 4-hydrxypiperidine-1-carboxylate (4.56 g, 22.7 mmol) and triphenylphosphine (5.95 g, 22.7 mmol) in anhydrous THF (20 mL) was added DIAD (4.34 mL, 22.7 mmol) dropwise at room temperature. The resulting mixture was stirred at the same temperature for 4 hours, and then concentrated in vacuo. The residue was purified by column chromatography over silica gel, eluting with hexane: ethyl acetate, 5:1 to afford the title compound as colourless oil (3.52 g, 96% yield). 1H-NMR (CDCl3, 400 MHz): δ 7.32 (t, J=8.0 Hz, 1H), 7.13 (d, J=8.0 Hz, S2

1H), 6.65 (d, J=8.0 Hz, 1H), 4.74-4.69 (m, 1H), 4.46 (q, J=7.2 Hz, 2H), 3.68-3.61 (m, 2H), 3.58-3.52 (m, 2H), 2.78 (s, 3H), 2.04-1.97 (m, 2H), 1.96-1.86 (m, 2H), 1.50 (s, 9H), 1.46 (t, J=7.2 Hz, 3H).

4-({1-[(tert-butoxy)carbonyl]piperidin-4-yl}oxy)-3-methyl-1NBoc O

benzofuran-2-carboxylic acid (I-2) COOH O

LiOH·H2O (1.16 g, 27.5 mmol) was added to a solution of compound

I-1 (1.85 g, 4.6 mmol) in MeOH/H2O (40 mL, 1:1). The mixture was stirred for 4 hours at 50°C. The volatile components were then evaporated in vacuo. The residue was dissolved in 30 mL H2O, followed by the addition of 3 mL of concentrated HCl while stirring. Precipitation occurred in the resulting solution and the title compound was collected as a while solid (1.51 g, 88% yield). 1H-NMR (CD3OD, 400 MHz): δ 7.40 (t, J=8.0 Hz, 1H), 7.12 (d, J=8.0 Hz, 1H), 6.65 (d, J=8.0 Hz, 1H), 4.74-4.69 (m, 1H), 3.69-3.62 (m, 2H), 3.56-3.50 (m, 2H), 2.76 (s, 3H), 2.06-1.99 (m, 2H), 1.94-1.86 (m, 2H), 1.50 (s, 3H).

2-Benzoyl-3-methyl-1-benzofuran-4-ol (I-3) A mixture of 2, 6-dihydroxy acetophenone (500 mg, 3.3 mmol), 2bromo-1-phenylethan-1-one (690 mg, 3.4 mmol) and potassium carbonate (1.36 g, 10 mmol) in acetonitrile (20 mL) was stirred at refluxing temperature for 24 hours. The reaction mixture was filtered through a celite pad and the filtrate was concentrated in vacuo. The residue was purified by column chromatography over silica gel, to afford the title compound as a light yellow solid (34 mg, 41% yield). 1H-NMR (CD3COCD3, 400 MHz): δ 9.31 (brs, 1H), 8.07 (d, J=7.2 Hz, 2H), 7.68 (t, J=7.2 Hz, 1H), 7.61 (t, S3

J=7.2 Hz, 2H), 7.33 (t, J=8.0 Hz, 1H), 7.06 (d, J=8.0 Hz, 1H), 6.75 (d, J=8.0 Hz, 1H), 2.81 (s, 3H).

NBoc O

t-Butyl 4-[(2-formyl-3-methyl-1-benzofuran-4-yl)oxy]piperidine-1carboxylate (I-4)

CHO O

LiAlH4 (0.26 g, 6.8 mmol) was added slowly to a solution of I-1 (1.81

g, 4.5 mmol) in anhydrous THF (20 mL) at 0˚C. The resulting mixture was gradually warmed up to room temperature for 2 hours. 100 µL of water was then added to the reaction mixture, followed by the filtration through a celite pad. The filtrate was concentrated in vacuo, to give t-butyl 4-((2-(hydroxymethyl)-3-methylbenzofuran-4yl)oxy)piperidine-1-carboxylate as a light-yellow solid (0.55g, 68% yield) without further purification. To a solution of the above compound (50 mg, 0.13 mmol) in anhydrous DCM, activated manganese dioxide (560 mg, 6.5 mmol) was added at room temperature, and the resulting mixture was stirred at the same temperature for 24 hours. After that, the reaction mixture was filtered over a celite pad and the filtrate was concentrated in vacuo, to afford the title compound as yellow oil (37 mg, 77% yield). 1H-NMR (CDCl3, 400 MHz): δ 9.96 (s, 1H), 7.38 (t, J=8.0 Hz, 1H), 7.11 (d, J=8.0 Hz, 1H), 6.66 (d, J=8.0 Hz, 1H), 4.74-4.69 (m, 1H), 3.69-3.62 (m, 2H), 3.56-3.50 (m, 2H), 2.76 (s, 3H), 2.06-1.99 (m, 2H), 1.94-1.86 (m, 2H), 1.50 (s, 3H).

N

CPh3

(3-Methyl-4-{[1-(triphenylmethyl)piperidin-4-yl]oxy}-1benzofuran-2-yl) methanol (I-5)

O CH2 OH O

S4

Prepared from ethyl 4-hydroxy-3-methylbenzofuran-2-carboxylate (500 mg, 2.25 mmol) and 1-(triphenylmethyl)piperidin-4-ol (1.93 g, 5.5 mmol) in anhydrous THF (8 mL) according to the preparation of compound I-1, Ethyl 3-methyl-4-{[1(triphenylmethyl)-piperidin-4-yl]oxy}-1-benzofuran-2-carboxylate was obtained as a white solid (800 mg, 65% yield). LiAlH4 (85 mg, 2.2 mmol) was added slowly to a solution of the above compound (800 mg, 1.45 mmol) in anhydrous THF (10 mL) at 0˚C. The resulting mixture was gradually warmed up to room temperature for 2 hours. 100 µL of water was then added to the reaction mixture, followed by filtration through a celite pad. The filtrate was concentrated in vacuo, to give the title compound as colourless oil (750 mg, 90% yield). 1H-NMR (CDCl3, 400 MHz): δ 7.68-7.41 (m, 5H), 7.38-7.30 (m, 6H), 7.257.14 (m, 4H), 7.10 (t, J=8.0 Hz, 1H), 6.98 (d, J=8.0 Hz, 1H), 6.54 (d, J=8.0 Hz, 1H), 4.70 (s, 2H), 4.06-3.94 (m, 1H), 2.24-2.11 (m, 2H), 2.12-1.97 (m, 4H), 1.91-1.83 (m, 2H).

1-(4-Hydroxy-3-methyl-1-benzofuran-2-yl)ethan-1-one (I-6) OH

O

O

Prepared from 2, 6-dihydroxy acetophone (2 g, 13.1 mmol), 1-

chloropropan-2-one (1.05 mL, 13.1 mmol) and potassium carbonate (5.4 g, 39.4 mmol) in acetonitrile (30 mL) according to the preparation of compound I-5, the title compound was obtained as an off-white solid (420 mg, 17% yield). 1H-NMR (CD3COCD3, 400 MHz): δ 9.27 (brs, 1H), 7.32 (t, J=8.0 Hz, 1H), 7.05 (d, J=8.0 Hz, 1H), 6.72 (d, J=8.0 Hz, 1H), 2.76 (s, 3H), 2.53 (s, 3H).

S5

2-(Benzylsulfanyl)-1-benzofuran-4-ol (I-7)3 To a solution of 2, 6-dihydroxyacetophenone (2 g, 13.2 mmol) in ethanol/water (20 mL, 1:1), ethyl carbazate (1.36 g, 13.2 mmol) was added. The resulting mixture was refluxed for 24 hours and kept overnight at 4 °C. The precipitate was filtered off, washed with diethyl ether and dried under vacuum, to afford N'-1-(2,6-dihydroxyphenyl) ethylidene-ethoxycarbohydrazide as a yellow solid (720 mg, 24% yield). To a solution of the above compound (600 mg, 2.52 mmol) in DMF (3 mL), thionyl chloride (3.6 mL) was added at the temperature of -20 ˚C. After the liberation of gas ceased, the reaction mixture was warmed up to room temperature and maintained for a further 3 hours. The excess of thionyl chloride and DMF were removed under pressure. The residue was re-dissolved by EtOAc (50 mL) and partitioned with 5% NaHCO3 (50 mL). The aqueous layer was further extracted by EtOAc (2 x 30 mL). The combined organic layers were sequentially washed by water and brine, dried over anhydrous sodium sulphate, concentrated in vacuo, to afford 2-(1, 2, 3-Thiadiazol-4yl)benzene-1,3-diol without further purification as a yellow solid (260 mg, 54% yield). A mixture of 2-(1, 2, 3-Thiadiazol-4-yl)benzene-1,3-diol (250 mg, 1.3 mmol), benzyl chloride (165 µL, 1.4 mmol) and potassium carbonate (270 mg, 1.9 mmol) in dry acetone (10 mL) was stirred at 60 ˚C for 6 hours. After that, the reaction mixture was evaporated under pressure to dryness and the residue was purified by column chromatography over silica gel, to afford the title compound as a white solid (75 mg, 24% yield). 1H-NMR (CD3COCD3, 400 MHz): δ 7.34-7.23 (m, 5H), 7.14 (t, J=8.0 Hz, 1H), 7.01 (d, J=8.0 Hz, 1H), 6.86 (s, 1H), 6.68 (d, J=8.0 Hz, 1H), 4.23 (s, 2H). m/z (ESI) 257.1 (M+H+).

S6

NH O

Ethyl 3-methyl-4-(piperidin-4-yloxy)benzofuran-2-carboxylate (2) A mixture of compound I-1 (10 mg, 0.025 mmol) and TFA (50 µL)

COOEt O

in DCM (1 mL) was stirred at room temperature for 2 hours. The

reaction mixture was evaporated under pressure to dryness, which was further purified by semi-preparative reverse phase HPLC to give the title compound as a white solid (9.8 mg, 94% yield). 1H-NMR (CD3OD, 400 MHz): δ 7.41 (t, J=8.4 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 6.88 (d, J=8.0 Hz, 1H), 4.98-4.93 (m, 1H), 4.42 (q, J=7.2 Hz, 2H), 3.50-3.40 (m, 2H), 3.33-3.28 (m, 2H), 2.80 (s, 3H), 2.34-2.24 (m, 2H), 2.22-2.12 (m, 2H), 1.43 (t, J=7.2 Hz, 3H). 13C-NMR (CD3OD, 100 MHz): δ 161.87, 157.38, 154.55, 141.09, 130.09, 127.02, 120.30, 106.50, 106.16, 69.95, 62.13, 42.01, 28.28, 14.63, 11.82. Calculated exact mass for the protonated molecule (C17H22NO4): 304.1549; measured accurate mass (ESI): 304.1561. LC-MS purity: 100%, Rt = 11.39 min.

Phenyl

3-methyl-4-(piperidin-4-yloxy)-1-benzofuran-2-

carboxylate(3) A mixture of compound I-2 (44 mg, 0.12 mmol), EDCI (25 mg, 0.13 mmol), hydroxybenzotriazole (22 mg, 0.15 mmol) in anhydrous acetonitrile (3 mL) was stirred at room temperature for 30 minutes, and then treated with phenol (12.1 mg, 0.13 mmol) and DIPEA (41 µL, 0.24 mmol). The resulting mixture was further stirred at room temperature for another 12 hours. After that, the solution was evaporated to dryness in vacuo. The residue was re-dissolved in ethyl acetate (20 mL) and washed with 0.5 M NaOH and brine sequentially. The organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo, to produce the N-Boc precursor without further purification. S7

The Boc-deprotection in DCM (2 mL) containing 5% TFA was carried out according to the preparation of compound 2, to afford the title compound as a white solid (28.1 mg, 51% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 7.50-7.43 (m, 3H), 7.32 (t, J=7.6 Hz, 1H), 7.26 (d, J=7.6 Hz, 2H), 7.18 (d, J=8.0 Hz, 1H), 6.90 (d, J=8.0 Hz, 1H), 4.94-4.90 (m, 1H), 3.48-3.41 (m, 2H), 3.35-3.29 (m, 2H), 2.85 (s, 3H), 2.35-2.27 (m, 2H), 2.22-2.14 (m, 2H).

13

C-NMR (CD3OD, 100 MHz): δ 160.02, 157.66, 154.69,

151.71, 140.26, 130.67, 130.62, 129.16, 127.21, 122.79, 120.29, 106.62, 106.19, 70.09, 41.99, 28.26, 12.05. Calculated exact mass for the protonated molecule (C21H22NO4): 352.1549; measured accurate mass (ESI): 352.1545. LC-MS purity: 100%, Rt = 12.17 min.

Benzyl

3-methyl-4-(piperidin-4-yloxy)-1-benzofuran-2-

carboxylate(4) The general procedure to make compound 3 was followed to give the title compound as a white solid in 44% yield. 1H-NMR (CD3OD, 400 MHz): δ 7.50 (d, J=8.0 Hz, 2H), 7.44-7.35 (m, 4H), 7.14 (d, J=8.0 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 5.40 (s, 2H), 4.95-4.91 (m, 1H), 3.45-3.39 (m, 2H), 3.31-3.27 (m, 2H), 2.78 (s, 3H), 2.33-2.23 (m, 2H), 2.15-2.10 (m, 2H). 13C-NMR (CD3OD, 100 MHz): δ 161.59, 157.47, 154.57, 140.88, 137.29, 130.21, 129.67, 129.46, 127.48, 120.29, 106.53, 106.20, 69.95, 67.70, 41.98, 28.25, 24.21, 11.94. Calculated exact mass for the protonated molecule (C22H24NO4): 366.1705; measured accurate mass (ESI): 366.1708. LC-MS purity: 100%, Rt = 12.38 min.

S8

Naphthalen-1-ylmethyl 3-methyl-4-(piperidin-4-yloxy)-1benzo-furan-2-carboxylate (5) The general procedure to make compound 3 was followed to give the title compound as a white solid in 20% yield. 1H-NMR (CD3OD, 400 MHz): δ 8.19 (d, J=8.0 Hz, 1H), 7.96 (t, J=7.2 Hz, 2H), 7.69 (d, J=7.2 Hz, 1H), 7.63-7.51 (m, 3H), 7.38 (t, J=8.0 Hz, 1H), 7.12 (d, J=8.0 Hz, 1H), 6.83 (d, J=8.0 Hz, 1H), 4.92-4.86 (m, 1H), 3.42-3.35 (m, 2H), 3.31-3.25 (m, 2H), 2.71 (s, 3H), 2.30-2.21 (m, 2H), 2.172.10 (m, 2H).

13

C-NMR (CD3OD, 100 MHz): δ 161.62, 157.50, 154.53, 140.90,

135.34, 132.61, 130.18, 128.96, 127.42, 126.36, 124.70, 120.25, 106.47, 106.20, 69.84, 66.01, 41.92, 28.19, 11.92. Calculated exact mass for the protonated molecule (C26H26NO4): 416.1862; measured accurate mass (ESI): 416.1854. LC-MS purity: 99%, Rt = 13.10 min.

Cyclohexylmethyl 3-methyl-4-(piperidin-4-yloxy)-1benzofuran-2-carboxylate (6) The general procedure to make compound 3 was followed to give the title compound as a white solid in 10% yield. 1H-NMR (CD3OD, 400 MHz): δ 7.42 (t, J=8.0 Hz, 1H), 7.16 (d, J=8.0 Hz, 1H), 6.88 (d, J=8.0 Hz, 1H), 5.00-4.94 (m, 1H), 4.19 (d, J=6.4 Hz, 2H), 3.50-3.41 (m, 2H), 3.32-3.27 (m, 2H), 2.81 (s, 3H), 2.352.26 (m, 2H), 2.24-2.12 (m, 2H), 1.93-1.69 (m, 6H), 1.42-1.25 (m, 3H), 1.21-1.07 (m, 2H). 13C-NMR (CD3OD, 125 MHz): δ 161.99, 157.46, 154.56, 141.14, 130.10, 126.94, 120.31, 106.52, 106.20, 71.15, 69.94, 41.99, 38.63, 30.83, 28.27, 27.45, 26.83, 11.96. Calculated exact mass for the protonated molecule (C22H30NO4): 372.2175; measured accurate mass (ESI): 372.2167. LC-MS purity: 100%, Rt = 12.46 min.

S9

2-Phenylethyl-3-methyl-4-(piperidin-4-yloxy)-1benzofuran-2-carboxylate (7) The general procedure to make compound 3 was followed to give the title compound as a white solid in 9% yield. 1H-NMR (CD3OD, 400 MHz): δ 7.41 (t, J=8.0 Hz, 1H), 7.34-7.30 (m, 4H), 7.27-7.22 (m, 1H), 7.14 (d, J=8.0 Hz, 1H), 6.87 (d, J=8.0 Hz, 1H), 5.02-4.92 (m, 1H), 4.59 (t, J=6.8 Hz, 2H), 3.49-3.40 (m, 2H), 3.32-3.25 (m, 2H), 3.11 (t, J=6.8 Hz, 2H), 2.71 (s, 3H), 2.35-2.26 (m, 2H), 2.23-2.10 (m, 2H).

13

C-NMR (CD3OD, 125 MHz): δ 161.79, 157.46, 154.57, 140.99, 139.22,


3-Phenylpropyl-3-methyl-4-(piperidin-4-yloxy)-1benzofuran-2-carboxylate (8) The general procedure to make compound 3 was followed to give the title compound as an off-white solid in 8% yield. 1H-NMR (CD3OD, 400 MHz): δ 7.43 (t, J=8.0 Hz, 1H), 7.32-7.19 (m, 5H), 7.17 (d, J=8.0 Hz, 1H), 6.89 (d, J=8.0 Hz, 1H), 5.01-4.92 (m, 1H), 4.37 (t, J=6.4 Hz, 2H), 3.50-3.39 (m, 2H), 3.323.28 (m, 2H), 2.82 (t, J=7.6 Hz, 2H), 2.82 (s, 3H), 2.36-2.25 (m, 2H), 2.25-2.07 (m, 4H). 13C-NMR (CD3OD, 125 MHz): δ 161.89, 157.45, 154.56, 142.48, 141.07, 130.14, 129.49, 127.07, 120.31, 106.53, 106.21, 69.95, 65.42, 42.01, 33.21, 31.55, 28.28,

S10

11.94. Calculated exact mass for the protonated molecule (C24H28NO4): 394.2018; measured accurate mass (ESI): 394.2018. LC-MS purity: 100%, Rt = 13.14 min.

(3-Methoxyphenyl) methyl 3-methyl-4-(piperidin-4-yloxy)1-benzofuran-2-carboxylate (26) The general procedure to make compound 3 was followed to give the title compound as an off-white solid in 39% yield. 1H-NMR (CD3OD, 400 MHz): δ 7.40 (t, J=8.0 Hz, 1H), 7.33 (t, J=8.0 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H), 7.077.05 (m, 2H), 6.95-6.92 (m, 1H), 6.85 (d, J=8.0 Hz, 1H), 5.37 (s, 2H), 4.94-4.91 (m, 1H), 3.83 (s, 3H), 3.46-3.40 (m, 2H), 3.33-3.28 (m, 2H), 2.78 (s, 3H), 2.32-2.25 (m, 2H), 2.20-2.13 (m, 2H).

13

C-NMR (CD3OD, 100 MHz): δ 160.17, 159.98, 156.06,

153.17, 139.44, 137.33, 129.34, 128.81, 126.10, 120.10, 118.87, 113.54, 113.42, 105.11, 104.77, 68.54, 66.16, 54.32, 40.55, 26.83, 10.56. Calculated exact mass for the protonated molecule (C23H26NO5): 396.1811; measured accurate mass (ESI): 396.1798. LC-MS purity: 100%, Rt = 12.65 min.

NH O

N-isopropyl-3-methyl-4-(piperidin-4-yloxy)benzofuran-2carboxamide(9)

CONH O

To a solution of compound I-2 (30 mg, 0.08 mmol) in DMF/DCM

(2 mL, 1:1) was sequentially added DIPEA (15 µL, 0.088 mmol), PyBOP (46 mg, 0.088 mmol). The above mixture was stirred at room temperature for 30 minutes, followed by the addition of isopropylamine (7.6 µL, 0.088 mmol). The resulting mixture was further stirred at room temperature for 3 hours. After that, the reaction mixture was diluted with DCM (15 mL) and partitioned with 0.5M NaOH solution (15

S11

mL), and then the aqueous layer was further extracted by DCM (10 mL). The combined organic layers were washed brine, dried over anhydrous sodium sulphate and concentrated in vacuo. The residue was purified by column chromatography over silica gel, to afford the N-Boc precursor. The Boc-deprotection in DCM (2 mL) containing 5% TFA was carried out according to the preparation of compound 2, to afford the title compound as a white solid (11.5 mg, 34% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 7.37 (t, J=8.4 Hz, 1H), 7.16 (d, J=8.4 Hz, 1H), 6.87 (d, J=8.4 Hz, 1H), 4.98-4.93 (m, 1H), 4.30-4.96 (m, 1H), 3.463.40 (m, 2H), 3.32-3.29 (m, 2H), 2.80 (s, 3H), 2.32-2.25 (m, 2H), 2.21-2.13 (m, 2H), 1.29 (d, J=6.4 Hz, 6H).

13

C-NMR (CD3OD, 100 MHz): δ 161.52, 156.45, 154.30,

143.11, 129.19, 123.31, 120.78, 106.62, 106.05, 69.78, 42.32, 41.92, 28.23, 22.58, 11.52. Calculated exact mass for the protonated molecule (C18H25N2O3): 317.1865; measured accurate mass (ESI): 317.1867. LC-MS purity: 100%, Rt = 10.75 min.

N-cyclohexyl-3-methyl-4-(piperidin-4-yloxy)benzofuran-2NH

carboxamide (10)

O CONH O

The general procedure to make compound 9 was followed to

give the title compound as a white solid in 22% yield. 1H-NMR (CD3OD, 400 MHz): δ 7.37 (t, J=8.4 Hz, 1H), 7.16 (d, J=8.4 Hz, 1H), 6.87 (d, J=8.4 Hz, 1H), 4.97-4.92 (m, 1H), 3.94-3.86 (m, 1H), 3.49-3.38 (m, 2H), 3.32-3.29 (m, 2H), 2.80 (s, 3H), 2.35-2.23 (m, 2H), 2.23-2.12 (m, 2H), 2.00-1.91 (m, 2H), 1.91-1.76 (m, 2H), 1.76-1.66 (m, 1H), 1.50-1.35 (m, 4H), 1.35-1.19 (m, 1H).

13

C-NMR (CD3OD, 100 MHz): δ 161.46,

156.45, 154.30, 143.11, 129.18, 123.36, 120.80, 106.64, 106.05, 69.79, 49.72, 41.93, 33.76, 28.25, 26.57, 26.44, 11.54. Calculated exact mass for the protonated molecule

S12

(C21H29N2O3): 357.2178; measured accurate mass (ESI): 357.2162. LC-MS purity: 100%, Rt = 12.20 min.

NH O

3-Methyl-N-phenyl-4-(piperidin-4-yloxy)benzofuran-2carboxamide (11)

CONH O


give the title compound as an off-white solid in 37% yield. 1H-NMR (CDCl3, 400 MHz): δ 8.32 (s, 1H, CONH), 7.72 (d, J=7.2 Hz, 2H), 7.40 (t, J=7.2 Hz, 2H), 7.33 (t, J=8.0 Hz, 1H), 7.17 (t, J=7.2 Hz, 1H), 7.09 (d, J=8.0 Hz, 1H), 6.69 (d, J=8.0 Hz, 1H), 4.67-4.58 (m, 1H), 3.23-3.17 (m, 2H), 2.89 (s, 3H), 2.87-2.81 (m, 2H), 2.15-2.08 (m, 2H), 1.89-1.81 (m, 2H).

13

C-NMR (CDCl3, 100 MHz): δ 158.18, 154.82, 154.17,

141.00, 137.59, 129.08, 128.13, 124.89, 124.39, 119.91, 105.44, 104.11, 73.08, 43.53, 31.89, 11.20. Calculated exact mass for the protonated molecule (C21H23N2O3): 351.1709; measured accurate mass (ESI): 351.1696. LC-MS purity: 92%, Rt = 11.74 min.

NH O

N-benzyl-3-methyl-4-(piperidin-4-yloxy)benzofuran-2carboxamide(12)

CONH O


give the title compound as an a white solid in 33% yield. 1H-NMR (CD3OD, 400 MHz): δ 7.40-7.32 (m, 5H), 7.26 (t, J=7.2 Hz, 1H), 7.14 (d, J=8.4 Hz, 1H), 6.85 (d, J=8.4 Hz, 1H), 4.92-4.87 (m, 1H), 4.58 (s, 2H), 3.45-3.39 (m, 2H), 3.32-3.28 (m, 2H), 2.80 (s, 3H), 2.34-2.24 (m, 2H), 2.20-2.12 (m, 2H). 13C-NMR (CD3OD, 100 MHz): δ 162.32, 156.54, 154.34, 142.97, 140.18, 129.55, 129.33, 128.51, 128.20, 123.78,

S13

120.76, 106.64, 106.06, 69.79, 43.54, 41.90, 28.22, 11.56. Calculated exact mass for the the protonated molecule (C22H25N2O3): 365.1865; measured accurate mass (ESI): 365.1875. LC-MS purity: 100%, Rt = 11.55 min.

NH O

3-Methyl-N-(naphthalen-1-ylmethyl)-4-(piperidin-4yloxy)-benzofuran-2-carboxamide (13)

CONH O


give the title compound as a white solid in 49% yield. 1H-NMR (CD3OD, 400 MHz): δ 8.18 (d, J=8.4 Hz, 1H), 7.90 (d, J=8.4 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.58-7.44 (m, 4H), 7.33 (t, J=8.4 Hz, 1H), 7.10 (d, J=8.4 Hz, 1H), 6.80 (d, J=8.4 Hz, 1H), 5.05 (s, 2H), 4.89-4.84 (m, 1H), 3.43-3.37 (m, 2H), 3.32-3.26 (m, 2H), 2.80 (s, 3H), 2.28-2.21 (m, 2H), 2.18-2.10 (m, 2H). 13C-NMR (CD3OD, 100 MHz): δ 162.23, 156.51, 154.29, 142.92, 135.33, 134.98, 132.62, 129.79, 129.31, 129.13, 127.36 126.86, 126.61, 126.44, 124.31, 123.85, 120.72, 106.61, 106.06, 69.75, 41.87, 41.59, 28.19, 11.59. Calculated exact mass for the protonated molecule (C26H27N2O3): 415.2022; measured accurate mass (ESI): 415.2020. LC-MS purity: 100%, Rt = 12.50 min.

(3-Methyl-4-(piperidin-4-yloxy)benzofuran-2-yl)(phenyl) NH

methanone (14)

O COPh O

Prepared from a solution of compound I-3 (34 mg, 0.14 mmol), t-

Butyl 4-hydrxypiperidine-1-carboxylate (84 mg, 0.42 mmol), triphenylphosphine (109 mg, 0.42 mmol) and DIAD (79 µL, 0.42 mmol) in anhydrous THF (2 mL) according to the procedure of making compound I-1, the N-Boc precursor was obtained.

S14

The N-Boc deprotection was carried out in DCM containing 5% TFA (2 mL) according to the preparation of compound 2, to afford the title compound as an offwhite solid (17 mg, 42% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 8.01 (d, J=7.2 Hz, 2H), 7.66 (t, J=7.6 Hz, 1H), 7.56 (t, J=7.6 Hz, 2H), 7.46 (t, J=8.4 Hz, 1H), 7.18 (d, J=8.4 Hz, 1H), 6.91 (d, J=8.4 Hz, 1H), 5.02-4.95 (m, 1H), 3.51-3.42 (m, 2H), 3.38-3.30 (m, 2H), 2.84 (s, 3H), 2.38-2.28 (m, 2H), 2.25-2.14 (m, 2H).

13

C-NMR

(CD3OD, 100 MHz): δ 186.00, 155.91, 153.53, 147.07, 137.86, 132.42, 129.35, 129.19, 128.05, 127.20, 119.12, 105.23, 104.91, 68.60, 40.57, 26.84, 11.08. Calculated exact mass for the protonated molecule (C21H22NO3): 336.1600; measured accurate mass (ESI): 336.1603. LC-MS purity: 100%, Rt = 12.37 min.

4-{[3-Methyl-2-(3-phenyl-1,2,4-oxadiazol-5-yl)-1-benzofuran-

NH O

4-yl]oxy}piperidine (17) N O

O N

A mixture of compound I-2 (25 mg, 0.066 mmol), EDCI (14 mg,

0.073 mmol), hydroxybenzotriazole (12 mg, 0.086 mmol) in anhydrous acetonitrile (2 mL) was stirred at room temperature for 30 minutes, and then treated with N'hydroxy-benzene-carboximidamide (10 mg, 0.073 mmol) and DIPEA (24 µL, 0.13 mmol). The resulting mixture was further stirred at room temperature for 12 hours. After that, the solution was evaporated to dryness in vacuo. The residue was redissolved in ethyl acetate (20 mL) and washed with 0.5 M NaOH and brine sequentially. The organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo. The residue was then dissolved in dry toluene over 4Å molecular sieve and the mixture was stirred at 110 ˚C for 12 hours. After that, the reaction mixture was filtered off and the filtration was concentrated in vacuo, to give the N-Boc precursor without further purification. S15

The Boc-deprotection in DCM (2 mL) containing 5% TFA was carried out according to the preparation of compound 2, to afford the title compound as a white solid (6.3 mg, 20% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 8.14 (d, J=7.6 Hz, 2H), 7.627.53 (m, 3H), 7.43 (t, J=8.0 Hz, 1H), 7.22 (d, J=8.0 Hz, 1H), 6.90 (d, J=8.0 Hz, 1H), 4.99-4.95 (m, 1H), 3.50-3.44 (m, 2H), 3.37-3.31 (m, 2H), 2.94 (s, 3H), 2.38-2.26 (m, 2H), 2.26-2.13 (m, 2H).

13

C-NMR (CD3OD, 100 MHz): δ 170.00, 169.76, 158.24,

154.51, 137.38, 132.64, 130.37, 130.10, 128.46, 127.83, 126.01, 120.24, 106.94, 106.12, 70.09, 42.02, 28.30, 11.78. Calculated exact mass for the protonated molecule (C22H22N3O3): 376.1661; measured accurate mass (ESI): 376.1659. LC-MS purity: 100%, Rt = 13.33 min.

4-{[2-(3-Benzyl-1,2,4-oxadiazol-5-yl)-3-methyl-1-

NH O

benzofuran-4-yl]oxy}piperidine (25) N O

O N


give the title compound as a white solid in 29% yield. 1H-NMR (CD3OD, 400 MHz): δ 7.46-7.24 (m, 6H), 7.18 (d, J=8.0 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 4.99-4.90 (m, 1H), 4.17 (s, 2H), 3.49-3.38 (m, 2H), 3.33-3.26 (m, 2H), 2.83 (s, 3H), 2.36-2.26 (m, 2H), 2.19-2.09 (m, 2H).

13

C-NMR (CD3OD, 100 MHz): δ 171.17, 169.92, 158.18,

154.46, 137.34, 137.04, 130.30, 130.06, 129.72, 128.16, 125.85, 120.21, 106.91, 106.09, 70.04, 41.97, 32.86, 28.25, 11.67. Calculated exact mass for the protonated molecule (C23H24N3O3): 390.1818; measured accurate mass (ESI): 390.1819. LC-MS purity: 100%, Rt = 12.86 min.

S16

NH O

4-({3-Methyl-2-[(E)-2-phenylethenyl]-1-benzofuran-4-yl}oxy) piperidine(16) To a mixture of compound I-4 (50 mg, 0.14 mmol) and

O

benzyltriphenyl phosphonium bromide (65 mg, 0.15 mmol) in DCM/water (2 mL, 1:1), 10M of NaOH solution (50 µL) was added. The resulting mixture was stirred at room temperature for 1 hour. After that, the reaction mixture was diluted with DCM (20 mL) and washed sequentially with water and brine (15 mL each). The organic layer was concentrated to give the mixture of the cis/trans N-Boc precursors. The Boc-deprotection in DCM (2 mL) containing 5% TFA was carried out according to the preparation of compound 2, to isolate the title compound as 1st major fraction from preparative HPLC (white solid, 4.9 mg, 8% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 7.41 (d, J=8.0 Hz, 2H), 7.34-7.26 (m, 3H), 7.17 (t, J=8.0 Hz, 1H), 6.89 (d, J=8.0 Hz, 1H), 6.78 (d, J=8.0 Hz, 1H), 6.72 (d, J=12.4 Hz, 1H), 6.51 (d, J=12.4 Hz, 1H), 4.89-4.85 (m, 1H), 3.44-3.37 (m, 2H), 3.31-3.26 (m, 2H), 2.54 (s, 3H), 2.32-2.22 (m, 2H), 2.19-2.11 (m, 2H). 13C-NMR (CD3OD, 125 MHz): δ 156.93, 152.96, 150.05, 138.72, 132.03, 130.13, 128.88, 128.52, 127.58, 126.68, 120.99, 116.34, 106.39, 105.56, 69.57, 41.95, 28.32, 11.06. Calculated exact mass for the protonated molecule (C22H24NO2): 334.1807; measured accurate mass (ESI): 334.1813. LC-MS purity: 96% (trans:cis = 3:1), Rt = 13.22 min.

NH

4-({3-methyl-2-[(Z)-2-phenylethenyl]-1-benzofuran-4-yl}oxy)

O

piperidine(15) O

The title compound was isolated as 2nd major fraction from the above preparative HPLC (off-white solid, 14.6 mg, 23% overall yield). 1H-

NMR (CD3OD, 400 MHz): δ 7.58 (d, J=7.2 Hz, 2H), 7.38 (t, J=8.0 Hz, 2H), 7.27 (t, S17

J=7.2 Hz, 1H), 7.23-7.18 (m, 3H), 7.07 (d, J=8.0 Hz, 1H), 6.78 (d, J=8.0 Hz, 1H), 4.90-4.82 (m, 1H), 3.46-3.40 (m, 2H), 3.32-3.27 (m, 2H), 2.53 (s, 3H), 2.35-2.22 (m, 2H), 2.21-2.12 (m, 2H).

13

C-NMR (CD3OD, 100 MHz): δ 157.20, 153.02, 150.95,

138.43, 129.81, 129.62, 128.88, 127.58, 126.80, 121.67, 115.10, 114.66, 106.55, 105.43, 69.62, 41.94, 28.34, 10.52. Calculated exact mass for the protonated molecule (C22H24NO2): 334.1807; measured accurate mass (ESI): 334.1799. LC-MS purity: 97%, Rt = 13.64 min.

NH O

4-{[3-Methyl-2-(2-phenylethyl)-1-benzofuran-4-yl]oxy} piperidine(18)

O

A mixture of compound 15 (13 mg, 0.039 mmol), palladium on

carbon (10% w/w, 5 mg, 0.008 mmol) and 1,4-cyclohexadiene (70 µL) in ethanol (1 mL) was stirred at 80 ˚C for 4 hours. The reaction mixture was then concentrated in vacuo and the residue was purified by semi-preparative reverse phase HPLC to give the title compound as colourless oil (6.6 mg, 38% yield). 1H-NMR (CD3OD, 500 MHz): δ 7.23 (t, J=8.0 Hz, 2H), 7.18-7.11 (m, 4H), 7.03 (d, J=8.0 Hz, 1H), 6.74 (d, J=8.0 Hz, 1H), 4.85-4.81 (m, 1H), 3.40-3.35 (m, 2H), 3.30-3.25 (m, 2H), 3.00 (brs, 4H), 2.25-2.19 (m, 2H), 2.15-2.09 (m, 2H), 2.11 (s, 3H).

13

C-NMR (CD3OD, 125

MHz): δ 157.06, 153.11, 152.38, 142.37, 129.55, 129.31, 127.08, 125.06, 121.20, 111.15, 106.34, 105.55, 69.41, 41.90, 35.44, 29.07, 28.31, 10.17. Calculated exact mass for the protonated molecule (C22H26NO2): 336.1964; measured accurate mass (ESI): 336.1964. LC-MS purity: 100%, Rt = 13.30 min.

S18

NH O

4-{[3-Methyl-2-(phenoxymethyl)-1-benzofuran-4-yl]oxy} piperidine (19)

CH 2O O

To a stirred solution of compound I-5 (30 mg, 0.06 mmol),

phenol (14.1 mg, 0.15 mmol) and triphenylphosphine (39 mg, 0.15 mmol) in anhydrous THF (2 mL) was added DIAD (29 µL, 0.15 mmol) at room temperature. The resulting mixture was stirred at the same temperature for 4 hours, and then concentrated in vacuo. The residue was purified by column chromatography over silica gel, to afford the N-trityl precursor. The trityl-deprotection was carried out in DCM (1 mL), containing 0.1% of TFA and 0.2% of water, at room temperature for 12 hours. After that, the reaction mixture was concentrated in vacuo and the residue was purified by semi-preparative reverse phase HPLC, with no TFA in the mobile phases, to afford the title compound as colourless oil (0.4 mg, 2% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 7.32-7.28 (m, 2H), 7.24 (t, J=8.0 Hz, 1H), 7.09 (d, J=8.0 Hz, 1H), 7.04 (d, J=8.0 Hz, 2H), 6.98 (t, J=8.0 Hz, 1H), 6.81 (d, J=8.0 Hz, 1H), 5.15 (s, 2H), 4.96-4.89 (m, 1H), 3.43-3.38 (m, 2H), 3.30-3.26 (m, 2H), 2.48 (s, 3H), 2.30-2.22 (m, 2H), 2.20-2.11 (m, 2H).

13

C-NMR

(CD3OD, 125 MHz): δ 159.96, 153.18, 148.70, 138.03, 130.54, 126.75, 122.40, 120.63, 116.18, 115.81, 106.33, 105.89, 69.55, 61.90, 41.98, 28.34, 10.50. Calculated exact mass for the protonated molecule (C21H24NO3): 338.1756; measured accurate mass (ESI): 338.1772. LC-MS purity: 100%, Rt = 12.44 min.

NH O

4-({2-[(Benzyloxy)methyl]-3-methyl-1-benzofuran-4-yl}oxy)piperidine (21)

CH 2O O

A solution of I-5 (30 mg, 0.06 mmol) and sodium hydride (60% S19

w/w, 7.2 mg, 0.18 mmol) in anhydrous DMF was stirred at room temperature for 30 minutes, and then treated with benzyl bromide (10.7 µL, 0.09 mmol). The resulting mixture was further stirred at room temperature for 12 hours. After that, the reaction mixture was quenched with 100 µL of cold water, diluted with EtOAc (20 mL) and sequentially washed with water and brine (each for 20 mL). The organic layer was concentrated in vacuo and the residue was purified by column chromatography over silica gel, to give the N-trityl precursor. The trityl-deprotection was carried out in DCM (1 mL), containing 0.1% of TFA and 0.2% of water, at room temperature for 12 hours. After that, the reaction mixture was concentrated in vacuo and the residue was purified by semi-preparative reverse phase HPLC, with no TFA in the mobile phases, to afford the title compound as colourless oil (2 mg, 10% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 7.40-7.28 (m, 5H), 7.23 (t, J=8.0 Hz, 1H), 7.09 (d, J=8.0 Hz, 1H), 6.81 (d, J=8.0 Hz, 1H), 4.91-4.85 (m, 1H), 4.64 (s, 2H), 4.60 (s, 2H), 3.46-3.39 (m, 2H), 3.30-3.27 (m, 2H), 2.43 (s, 3H), 2.30-2.22 (m, 2H), 2.20-2.12 (m, 2H).

13

C-NMR (CD3OD, 125 MHz): δ 156.06,

151.72, 148.30, 137.87, 128.02, 127.62, 127.45, 125.19, 119.22, 114.14, 104.84, 104.45, 71.65, 68.06, 61.44, 40.53, 26.90, 9.14. Calculated exact mass for the protonated molecule (C22H26NO3): 352.1913; measured accurate mass (ESI): 352.1909. LC-MS purity: 100%, Rt = 12.34 min.

(2E)-1-[3-Methyl-4-(piperidin-4-yloxy)-1-benzofuran-2-yl]3-phenylprop-2-en-1-one (23) Prepared from a solution of compound I-6 (190 mg, 1.0 mmol), t-Butyl 4-hydrxypiperidine-1-carboxylate (500 mg, 2.5 mmol), triphenylphosphine

S20

(657 mg, 2.5 mmol) and DIAD (480 µL, 2.5 mmol) in anhydrous THF (4 mL) according to the procedure of making compound I-1, t-Butyl 4-[(2-acetyl-3-methyl-1benzofuran-4-yl)oxy]piperidine-1-carboxylate was obtained as a light yellow solid (318 mg). Above solid (50 mg, 0.13 mmol) and benzaldehyde (15 µL, 0.15 mmol) in ethanol (1 mL) was added 0.25 M NaOH solution (1 mL). The mixture was stirred at room temperature for 3 hours, and then diluted with 20 mL EtOAc, followed by the wash with water and brine (20 mL each). The organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo. The residue was purified by column chromatography over silica gel, to give the N-Boc precursor. The N-Boc deprotection was carried out in DCM containing 5% TFA (2 mL) according to the preparation of compound 2, to afford the title compound as a yellow solid (14.7 mg, 24% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 7.86-7.75 (m, 4H), 7.51-7.45 (m, 4H), 7.24 (d, J=8.0 Hz, 1H), 6.89 (d, J=8.0 Hz, 1H), 5.02-4.93 (m, 1H), 3.50-3.39 (m, 2H), 3.33-3.27 (m, 2H), 2.88 (s, 3H), 2.36-2.24 (m, 2H), 2.24-2.12 (m, 2H).

13

C-NMR (CD3OD, 100 MHz): δ 183.19, 157.14, 154.95, 148.89, 145.04,


1-[3-Methyl-4-(piperidin-4-yloxy)-1-benzofuran-2-yl]-3phenylpropan-1-one (22)

S21

Prepared from a solution of compound 23 (8 mg, 0.017 mmol), palladium on carbon (10% w/w, 7 mg, 0.004 mmol) and 1,4-cyclohexadiene (60 µL) in ethanol (1 mL), according to the preparation of compound 18, the title compound was obtained as a white solid (3.3 mg, 41% yield). 1H-NMR (CD3OD, 400 MHz): δ 7.44 (t, J=8.0 Hz, 1H), 7.29-7.28 (m, 4H), 7.22-7.18 (m, 1H), 7.17 (d, J=8.0 Hz, 1H), 6.88 (d, J=8.0 Hz, 1H), 4.98-4.95 (m, 1H), 3.46-3.38 (m, 2H), 3.33-3.27 (m, 2H), 3.30 (t, J=7.6 Hz, 2H), 3.04 (t, J=7.6 Hz, 2H), 2.82 (s, 3H), 2.34-2.25 (m, 2H), 2.23-2.12 (m, 2H). 13C-NMR (CD3OD, 100 MHz): δ 194.25, 157.00, 154.95, 148.16, 142.45, 130.56, 129.42, 127.10, 126.01, 125.94, 120.65, 106.52, 106.33, 69.94, 42.50, 42.00, 30.75, 28.27, 11.90. Calculated exact mass for the protonated molecule (C23H26NO3): 364.1913; measured accurate mass (ESI): 364.1904. LC-MS purity: 100%, Rt = 13.10 min.

4-{[2-(Benzylsulfanyl)-1-benzofuran-4-yl]oxy}piperidine (20) Prepared from a solution of compound I-7 (90 mg, 0.35 mmol), tButyl 4-hydrxypiperidine-1-carboxylate (180 mg, 0.88 mmol), triphenylphosphine (240 mg, 0.88 mmol) and DIAD (170 µL, 0.88 mmol) in anhydrous THF (2 mL) according to the procedure of making compound I-1, t-Butyl 4-{[2-(benzylsulfanyl)-1-benzofuran-4-yl]oxy}

piperidine-1-carboxylate,

was

obtained as colourless oil (60 mg, 40% yield). The N-Boc deprotection was carried out in DCM containing 5% TFA (2 mL) according to the preparation of compound 2, to afford the title compound as colourless oil (9.7 mg, 93% yield). 1H-NMR (CD3OD, 400 MHz): δ 7.27-7.18 (m, 6H), 7.14 (d, J=8.0 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 6.79 (s, 1H), 4.87-4.79 (m, 1H), 4.17 (s, 2H), 3.47-3.37 (m, 2H), 3.29-3.19 (m, 2H), 2.26-2.16 (m, 2H), 2.12-2.04 (m, 2H).

S22

13

C-

NMR (CD3OD, 100 MHz): δ 157.90, 149.48, 148.69, 137.54, 128.54, 128.04, 126.91, 125.25, 119.76, 108.89, 106.51, 104.41, 68.78, 40.37, 38.59, 26.95. Calculated exact mass for the protonated molecule (C20H22NO2S): 340.1371; measured accurate mass (ESI): 340.1369. LC-MS purity: 100%, Rt = 12.80 min.

4-{[2-(Phenylmethane)sulfonyl-1-benzofuran-4-yl]oxy} piperidine(24) A mixture of t-Butyl 4-{[2-(benzylsulfanyl)-1-benzofuran-4yl]oxy} piperidine-1-carboxylate (20 mg, 0.046 mmol) and 3-chloroperbenzoic acid (77% w/w, 16 mg, 0.07 mmol) in DCM (1 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo, to give the N-Boc precursor without further purification. The N-Boc deprotection was carried out in DCM containing 5% TFA (2 mL) according to the preparation of compound 2, to afford the title compound as pink oil (8.0 mg, 36% overall yield). 1H-NMR (CD3OD, 400 MHz): δ 7.52 (t, J=8.0 Hz, 1H), 7.46 (s, 1H), 7.35-7.21 (m, 6H), 6.99 (d, J=8.0 Hz, 1H), 4.98-4.92 (m, 1H), 4.72 (s, 2H), 3.48-3.39 (m, 2H), 3.29-3.19 (m, 2H), 2.28-2.18 (m, 2H), 2.14-2.07 (m, 2H). 13

C-NMR (CD3OD, 100 MHz): δ 157.59, 151.61, 148.00, 130.60, 129.39, 128.55,

128.22, 127.77, 117.35, 112.47, 106.75, 105.11, 68.95, 60.60, 40.31, 26.78. Calculated exact mass for the protonated molecule (C20H22NO4S): 372.1270; measured accurate mass (ESI): 372.1263. LC-MS purity: 100%, Rt = 10.91 min.

S23

SUPPLEMENTARY TABLES

Table S1. Investigation of optimal length of the C-4 side chain against PfNMTa

a

n

IC50 (µM)

n

IC50 (µM)

2

220

2

>1000

3

9.0

3

51

4

130

4

300

The IC50 values were averaged from two independent dose-response curves; the

variation was generally 100

180

The IC50 values were averaged from two independent dose-response curves; the

variation was generally 100

4-Cl

5.6

>100

4-Me

2.1

>100

3-Cl

0.99

>50

3-Me

0.62

90

3-OMe

0.68

>100

2-Cl

0.98

>100

2-Me

1.9

>100

The IC50 values were averaged from two independent dose-response curve; the

standard deviation was within 30% of the resulting values; b 2-OMe and 4-OMe analogues were not synthesized due to their acid liabilities; c S.I. = selectivity index, calculated as IC50(HsNMT1) / IC50(PfNMT).

S26

DLS i02 / 0.9795 ADSC Q315 CCD 720 x 0.25 71–1.59 (1.63–1.59) 10.3 (65.9) 12.2 (2.9) 99.7 (99.9) 7.2 (7.0) 161262 17.1 / 21.9 11552 9827 93 192 1416 16.6 15.2 24.5 11.5 25.8 0.024 2.307

DLS i03 / 0.9763 ADSC Q315 CCD 900 x 0.2 42–1.56 (1.60–1.56) a 7.8 (37.1) 15.2 (2.2) 98.0 (82.8) 5.9 (2.7)

161442 18.8 / 23.8 11496 9897 N/A 192 1384

13.8 12.5 N/A 9.2 22.8

0.021 2.123

0.019 2.634

16.3 14.8 32.7 12.0 23.7

163266 23.8 / 29.1 11104 9661 87 192 1153

DLS i041 / 0.9173 CMOS Pilatus 2M 2220 x 0.1 37–1.58 (1.62–1.58) 10.0 (63.4) 14.2 (2.9) 98.4 (97.6) 7.9 (7.7)

PvNMT-NHM-25 4B13 57.32, 119.13, 178.46 P212121

0.020 2.281

13.0 12.2 12.6 9.6 20.0

194832 25.2 / 30.2 11054 9615 87 192 1137

DLS i041 / 0.9173 CMOS Pilatus 2M 2220 x 0.1 33–1.50 (1.54–1.50) 9.8 (62.2) 14.6 (3.4) 99.6 (99.6) 7.9 (7.9)

PvNMT-NHM-26 4B14 57.38, 118.96, 179.13 P212121

Rsym = ΣhΣl |Ihl- |/ ΣhΣl, where Il is the lth observation of reflection h and is the weighted average intensity for all observations l of reflection h.

0.022 2.112

17.2 16.1 32.5 12.3 24.8

118372 18.5 / 23.9 11137 9693 81 192 1250

DLS i02 / 0.9795 ADSC Q315 CCD 360 x 0.5 55–1.79 (1.84–1.79) 11.4 (66.1) 14.5 (3.0) 100 (100) 7.2 (7.2)

PvNMT-NHM-22 4B12 57.47, 121.92, 178.25 P212121

d

Root-mean-square deviation of bond lengths or bond angles from ideal geometry.

S27

Rcryst = ∑||Fo| − |Fc||/∑|Fo| where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.Rfree is the Rcryst calculated with 5% of the reflections omitted from refinement.

c

PvNMT-NHM-13 4B11 57.34, 119.03, 175.47 P212121

PvNMT-NHM 4B10 57.37, 119.06, 176.40 P212121

Highest resolution shell is shown in parentheses.

b

a

PDB accession code Cell dimensions a, b, c (Å) Space Group Data collection Beamline / Wavelength Detector type Images x oscillation (°) Resolution (Å) Rsym (%) b I/σI Completeness (%) Redundancy Refinement No. unique reflections Rwork / Rfreec No. atoms Protein Ligand Co-factor Water B-factors (Å2) All atoms Protein Ligand Co-factor Water R.m.s.deviations d Bond lengths (Å) Bond angles (°)

Table S5. X-ray data collection and refinement statistics

SUPPLEMENTARY FIGURES

Figure S1. Alignment of PvNMT and PfNMT The sequences of the PvNMT and PfNMT have been aligned with the program MultAlign (Version 5.4.1); PvNMT shares 81% residue identity with PfNMT in primary sequence; out of 23 residues forming the ligand binding pocket, only 2 residues are different between these two enzymes (Y212 and Y334 in PvNMT are replaced by F212 and F334 in PfNMT).The colour codes are as follows: red, exact match; blue underline, residues forming the ligand binding site; green star, different residues between these two enzymes out of the residues forming the ligand binding pocket.

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Figure S2. Overlay of a benzofuran inhibitor and a peptide substrate in PvNMT 26 (PDB code: 4B14, C green), the peptide substrate (PDB: 1IID, C cyan); N blue, O red.

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S30

adopt a chair conformation but refine with slightly different ring puckers in the three independent molecules in each asymmetric unit.

(Top Panel). The final, refined electron density map (2Fo – Fc contoured at a level of 1 σ) is shown in blue (Bottom Panel). The piperidine rings of the ligands

An unbiased “omit” difference density map from early stages of refinement (Fo – Fc contoured at a level of 2.5 σ) associated with each ligand is shown in green

The ligands are shown in approximately the same orientation and in cylinder representation, colored by atom; carbon (gray), oxygen (red) and nitrogen (blue).

Figure S3. Electron density maps relating to bound ligands

REFERENCES

1.

Belanger, P. C.; Dufresne, C.; Lau, C. K.; Scheigetz, J. An improved synthesis

of ethyl 4-hydroxy-3-methylbenzofuran-2-carboxylate. Org. Prep. Proced. Int. 1988, 20, 299-302. 2.

Masubuchi, M.; Ebiike, H.; Kawasaki, K.; Sogabe, S.; Morikami, K.; Shiratori,

Y.; Tsujii, S.; Fujii, T.; Sakata, K.; Hayase, M.; Shindoh, H.; Aoki, Y.; Ohtsuka, T.; Shimma, N. Synthesis and biological activities of benzofuran antifungal agents targeting fungal N-myristoyltransferase. Bioorg. Med. Chem. 2003, 11, 4463-4478. 3. S.;

Abramov, M. A.; Dehaen, W.; D'Hooge, B.; Petrov, M. L.; Smeets, S.; Toppet, Voets,

M.

Nucleophilic

Intramolecular

Cyclization

Alkynechalcogenolates. Tetrahedron 2000, 56, 3933-3940.

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Reactions

of

Design and Synthesis of Inhibitors of Plasmodium falciparumN

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