Design and synthesis of inhibitors of plasmodium falciparum N -myristoyltransferase, a promising target for antimalarial drug discovery

Article abstract of DOI:10.1021/jm301160h

Design of inhibitors for N-myristoyltransferase (NMT), an enzyme responsible for protein trafficking in Plasmodium falciparum, the most lethal species of parasites that cause malaria, is described. Chemistry-driven optimization of compound 1 from a focuse

Full text of DOI:10.1021/jm301160h

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)
benzofuran-2-carboxylate (1)
NH
O
N
COOEt
O
This compound was synthesized and characterized according to literature procedures.
1, 2 1H-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,
13
J=7.6 Hz, 2H), 2.70 (s, 3H), 2.38-2.31 (m, 2H), 1.43 (t, J=7.2 Hz, 3H). 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
O
piperidine-1-carboxylate (I-1)
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-1-
NBoc
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
1
was collected as a while solid (1.51 g, 88% yield). H-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), 2-
bromo-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).
t-Butyl 4-[(2-formyl-3-methyl-1-benzofuran-4-yl)oxy]piperidine-1-
NBoc
O
carboxylate (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-4-
yl)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
1
vacuo, to afford the title compound as yellow oil (37 mg, 77% yield). H-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).
(3-Methyl-4-{[1-(triphenylmethyl)piperidin-4-yl]oxy}-1-
CPh3
N
O
benzofuran-2-yl) methanol (I-5)
CH2OH
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%
1
yield). H-NMR (CDCl3, 400 MHz): δ 7.68-7.41 (m, 5H), 7.38-7.30 (m, 6H), 7.25-
7.14 (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
Prepared from 2, 6-dihydroxy acetophone (2 g, 13.1 mmol), 1-
O
O
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
1
compound was obtained as an off-white solid (420 mg, 17% yield). H-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-4-
yl)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

Ethyl 3-methyl-4-(piperidin-4-yloxy)benzofuran-2-carboxylate (2)
NH
O
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
1
mg, 51% overall yield). H-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,
13
2H), 2.22-2.14 (m, 2H). 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
1
give the title compound as a white solid in 44% yield. H-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)-1-
benzo-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), 2.17-
2.10 (m, 2H). 13C-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)-1-
benzofuran-2-carboxylate (6)
The general procedure to make compound 3 was followed to
1
give the title compound as a white solid in 10% yield. H-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), 2.35-
2.26 (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)-1-
benzofuran-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
13
(m, 2H). C-NMR (CD3OD, 125 MHz): δ 161.79, 157.46, 154.57, 140.99, 139.22,
130.14, 130.00, 129.56, 127.64, 127.14, 120.27, 106.50, 106.16, 69.97, 66.71, 42.03,
36.09, 28.28, 11.87. Calculated exact mass for the protonated molecule (C23H26NO4):
380.1862; measured accurate mass (ESI): 380.1856. LC-MS purity: 100%, Rt = 12.78
min.
3-Phenylpropyl-3-methyl-4-(piperidin-4-yloxy)-1-
benzofuran-2-carboxylate (8)
The general procedure to make compound 3 was followed to
1
give the title compound as an off-white solid in 8% yield. H-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), 3.32-
3.28 (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
1
give the title compound as an off-white solid in 39% yield. H-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), 7.07-
7.05 (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,
13
2H), 2.20-2.13 (m, 2H). 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.
N-isopropyl-3-methyl-4-(piperidin-4-yloxy)benzofuran-2-
NH
O
carboxamide(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), 3.46-
3.40 (m, 2H), 3.32-3.29 (m, 2H), 2.80 (s, 3H), 2.32-2.25 (m, 2H), 2.21-2.13 (m, 2H),
13
1.29 (d, J=6.4 Hz, 6H). 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-2-
NH
O
carboxamide (10)
CONH
O
The general procedure to make compound 9 was followed to
1
give the title compound as a white solid in 22% yield. H-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). 13C-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.
3-Methyl-N-phenyl-4-(piperidin-4-yloxy)benzofuran-2-
NH
O
carboxamide (11)
CONH
O
The general procedure to make compound 9 was followed to
1
give the title compound as an off-white solid in 37% yield. H-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). 13C-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.
N-benzyl-3-methyl-4-(piperidin-4-yloxy)benzofuran-2-
NH
O
carboxamide(12)
CONH
O
The general procedure to make compound 9 was followed to
1
give the title compound as an a white solid in 33% yield. H-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.
3-Methyl-N-(naphthalen-1-ylmethyl)-4-(piperidin-4-
NH
O
yloxy)-benzofuran-2-carboxamide (13)
CONH
O
The general procedure to make compound 9 was followed to
1
give the title compound as a white solid in 49% yield. H-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
O
methanone (14)
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 off-
1
white solid (17 mg, 42% overall yield). H-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),
13
3.38-3.30 (m, 2H), 2.84 (s, 3H), 2.38-2.28 (m, 2H), 2.25-2.14 (m, 2H). 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
N
O
O
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 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. 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), 7.62-
7.53 (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,
13
2H), 2.26-2.13 (m, 2H). 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
N
O
O
The general procedure to make compound 17 was followed to
1
give the title compound as a white solid in 29% yield. H-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,
13
2H), 2.19-2.09 (m, 2H). 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

4-({3-Methyl-2-[(E)-2-phenylethenyl]-1-benzofuran-4-yl}oxy)
piperidine(16)
NH
O
O
To a mixture of compound I-4 (50 mg, 0.14 mmol) and
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
1
from preparative HPLC (white solid, 4.9 mg, 8% overall yield). H-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.
4-({3-methyl-2-[(Z)-2-phenylethenyl]-1-benzofuran-4-yl}oxy)
NH
O
piperidine(15)
O
The title compound was isolated as 2nd major fraction from the above
1
preparative HPLC (off-white solid, 14.6 mg, 23% overall yield). H-
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,
13
2H), 2.21-2.12 (m, 2H). 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.
4-{[3-Methyl-2-(2-phenylethyl)-1-benzofuran-4-yl]oxy}
NH
O
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
1
the title compound as colourless oil (6.6 mg, 38% yield). H-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,
13
4H), 2.25-2.19 (m, 2H), 2.15-2.09 (m, 2H), 2.11 (s, 3H). 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

4-{[3-Methyl-2-(phenoxymethyl)-1-benzofuran-4-yl]oxy}
piperidine (19)
NH
O
CH2O
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
1
oil (0.4 mg, 2% overall yield). H-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),
13
3.30-3.26 (m, 2H), 2.48 (s, 3H), 2.30-2.22 (m, 2H), 2.20-2.11 (m, 2H). 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.
4-({2-[(Benzyloxy)methyl]-3-methyl-1-benzofuran-4-yl}oxy)-
NH
O
piperidine (21)
CH2O
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
1
oil (2 mg, 10% overall yield). H-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). 13C-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-1-
benzofuran-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
1
solid (14.7 mg, 24% overall yield). H-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
13
(m, 2H). C-NMR (CD3OD, 100 MHz): δ 183.19, 157.14, 154.95, 148.89, 145.04,
136.18, 131.91, 130.81, 130.14, 129.73, 127.50, 123.26, 120.94, 106.64, 106.38,
70.04, 41.99, 28.26, 12.17. Calculated exact mass for the protonated molecule
(C23H24NO3): 362.1756; measured accurate mass (ESI): 362.1750. LC-MS purity:
97%, Rt = 13.17 min.
1-[3-Methyl-4-(piperidin-4-yloxy)-1-benzofuran-2-yl]-3-
phenylpropan-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
1
white solid (3.3 mg, 41% yield). H-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), t-
Butyl 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}
obtained as colourless oil (60 mg, 40% yield).
piperidine-1-carboxylate,
was
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),
13
3.47-3.37 (m, 2H), 3.29-3.19 (m, 2H), 2.26-2.16 (m, 2H), 2.12-2.04 (m, 2H). C-
S22

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-4-
yl]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
1
(8.0 mg, 36% overall yield). H-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).
13C-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
n
2
3
4
IC50 (µM)
220
n
2
3
4
IC50 (µM)
>1000
51
9.0
130
300
a
The IC50 values were averaged from two independent dose-response curves; the
variation was generally <30%.
Table S2. Investigation of basicity of the C-4 side chain against PfNMTa
X-R1
IC50 (µM)
8.5
X-R1
IC50 (µM)
110
150
220
a
The IC50 values were averaged from two independent dose-response curves; the
variation was generally <30%.
Table S3. Exploration of R1 groups in the C-4 side chain against PfNMTa
S24

R1
IC50 (µM)
9.0
R1
IC50 (µM)
84
108
51
102
500
140
8.5
51
54
13
370
32
>100
180
>100
a
The IC50 values were averaged from two independent dose-response curves; the
variation was generally <30%.
Table S4. Investigation of aryl R3 substituents against PfNMT and HsNMT1a,b
S25

R3
IC50 (PfNMT, µM)
S.I.c
H
0.27
5.6
>100
>100
>100
>50
4-Cl
4-Me
3-Cl
2.1
0.99
0.62
0.68
0.98
1.9
3-Me
3-OMe
2-Cl
90
>100
>100
>100
2-Me
a 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

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.
S28

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.
S29