1584 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Johnson et al.
the reaction was diluted with H2O (15 mL) and the precipitate
filtered, washed with H2O, collected, and dried in vacuo. Refer
to the Supporting Information for specific synthetic details and
characterization data for inhibitors 3b-l analogous to that
reported for 3a below.
Benzaldehyde O-(3-Carboxyphenyl)oxime (3a). Benz-
aldehyde (21.0 µL, 0.207 mmol) was subjected to the repre-
sentative coupling procedure with 3 (31.8 mg, 0.208 mmol) as
outlined above (method 2), yielding 3a as a white solid (29.0
mg, 58%): 1H NMR (500 MHz, 1:1 CD3OD:DMSO-d6) δ 7.39-
7.45 (m, 5H), 7.59-7.64 (m, 1H), 7.71-7.78 (m, 3H), 8.58 (s,
1H); 13C NMR (125 MHz, DMSO-d6) δ 114.5, 118.8, 123.3,
127.7, 129.1, 129.3, 130.8, 131.1, 132.3, 153.3, 158.9, 166.9;
MALDI-FTMS (DHB) 242.0812 m/z [MH]+, C14H12NO3 re-
quires 242.0812; RP-HPLC 98% pure.
the reaction was stirred at room temperature. After 4 h the
reaction was diluted with H2O (500 mL) and extracted with
EtOAc (4 × 100 mL), and the combined organics were washed
with H2O (2 × 100 mL), dried over Na2SO4, filtered, and
concentrated. Flash chromatographic purification over silica
(5-10% gradient EtOAc in hexanes) afforded 19 as a clear
syrup (6.02 g, 58%): 1H NMR (500 MHz, CDCl3) δ 1.36 (t, J )
7.0 Hz, 3H), 2.12 (s, 3H), 4.20 (q, J ) 7.0 Hz, 2H), 5.34 (s,
2H), 7.15-7.19 (m, 2H), 7.31-7.35 (m, 2H), 7.36-7.41 (m, 2H),
7.42-7.36 (m, 2H), 8.00-8.14 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 14.3, 14.4, 63.1, 66.4, 113.4, 122.9, 128.1, 128.1, 128.6,
131.5, 136.3, 163.5, 166.3, 166.4; GC-MS 228 m/z [M - 85]+,
benzyl 4-hydroxybenzoate C14H12O3 requires 228.
Ethyl N-(2-Carboxyphenoxy)acetimidate (20). LiOH‚
H2O (431 mg, 10.3 mmol) was added to a solution of ethyl-N-
(2-benzyloxycarbonylphenoxy)acetimidate 18 (802 mg, 2.56
mmol) in a 9/3/3 mL mixture of THF/MeOH/H2O and the
reaction was stirred at room temperature. After 6 h the
reaction was diluted with H2O (100 mL), washed with CH2Cl2
(4 × 30 mL), acidified to pH ∼5.0-5.5 with 0.5 N HCl, and
extracted with EtOAc (4 × 30 mL). The combined organics
were washed with H2O (30 mL) and brine (30 mL), dried over
Na2SO4, filtered, and concentrated to afford 20 as a white solid
(459 mg, 80%): 1H NMR (500 MHz, acetone-d6) δ 1.34 (t, J )
6.9 Hz, 3H), 2.18 (s, 3H), 4.20 (q, J ) 6.9 Hz, 2H), 7.02 (ddd,
J ) 0.9, 7.3, 7.8 Hz, 1H), 7.51 (ddd, J ) 1.8, 7.3, 8.7 Hz, 1H),
7.60 (dd, J ) 0.9, 8.7 Hz, 1H), 7.86 (dd, J ) 1.8, 7.8 Hz, 1H);
13C NMR (125 MHz, acetone-d6) δ 14.6, 14.7, 63.8, 115.2, 118.5,
121.3, 132.4, 134.5, 160.2, 166.8, 167.4; ESI-MS 224 m/z [MH]+,
C11H14NO4 requires 224.
Ethyl N-(4-Carboxyphenoxy)acetimidate (21). LiOH‚
H2O (550 mg, 13.1 mmol) was added to a solution of ethyl N-(4-
benzyloxycarbonylphenoxy)acetimidate 19 (1.04 g, 3.32 mmol)
in a 9/3/3 mL mixture of THF/MeOH/H2O and the reaction
was stirred at room temperature. After 24 h the reaction was
diluted with H2O (100 mL), washed with CH2Cl2 (4 × 30 mL),
acidified to pH ∼5.0-5.5 with 0.5 N HCl, and extracted with
EtOAc (3 × 30 mL). The combined organics were washed with
H2O (30 mL) and brine (30 mL), dried over Na2SO4, filtered,
and concentrated to afford 21 as a white crystalline solid (698
mg, 94%): 1H NMR (500 MHz, CDCl3) δ 1.37 (t, J ) 6.9 Hz,
3H), 2.14 (s, 3H), 4.22 (q, J ) 6.9 Hz, 2H), 7.18-7.22 (m, 2H),
8.04-8.08 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 14.5, 14.6,
63.3, 113.7, 122.1, 132.3, 164.2, 166.7, 172.0; ESI-MS 224 m/z
[MH]+, C11H14NO4 requires 224.
Representative Procedure for the Coupling of Ethyl
N-(2-Carboxyphenoxy)acetimidate 20 or Ethyl N-(4-
Carboxyphenoxy)acetimidate 21 to Arylaldehydes a-l
Yielding Oxime Ethers 2a-l and 4a-l, Respectively
(Method 3). To a solution of acetimidate 20 or 21 (∼0.1-0.3
mmol, 1 equiv) and aldehyde (∼0.1-0.3 mmol, 1 equiv) in 1,4-
dioxane (2.0 mL) was added 70% HClO4 (0.9 equiv) and the
reaction was stirred at room temperature. Upon completion
as determined by reverse phase HPLC (2-6 h), the reaction
was diluted with H2O (20 mL) and the precipitate filtered,
washed with H2O, collected, and dried in vacuo. Refer to the
Supporting Information for specific synthetic details and
characterization data of inhibitors 2c-l and 4b-l analogous
to 2a as shown below.
Benzyl 2-Fluorobenzoate (16). Benzyl alcohol (2.60 mL,
25.1 mmol) was added slowly to a stirring solution of 2-fluo-
robenzoic acid (2.92 g, 20.8 mmol), 4-(dimethylamino)pyridine
(251 mg, 2.05 mmol), and 1,3-dicyclohexylcarbodiimide (5.16
g, 25.0 mmol) in anhydrous CH2Cl2 at room temperature under
an Ar atmosphere. After 18 h the precipitate was filtered off
and washed with CH2Cl2, and the filtrate was concentrated
with silica to a powder. Flash chromatographic purification
over silica (10% to 20% gradient EtOAc in hexanes) afforded
16 as a clear, colorless liquid (4.20 g, 88%): 1H NMR (500 MHz,
CDCl3) δ 5.39 (s, 2H), 7.14 (ddd, J ) 1.1, 8.4, 9.5 Hz, 1H), 7.20
(dt, J ) 1.1, 7.7 Hz, 1H), 7.31-7.36 (m, 1H), 7.37-7.41 (m,
2H), 7.44-7.48 (m, 2H), 7.49-7.55 (m, 1H), 7.96 (dt, J ) 1.8,
7.3 Hz); 13C NMR (125 MHz, CDCl3) δ 66.93, 117.0 (d, JC-F
)
22.1 Hz), 118.7 (d, JC-F ) 9.6 Hz), 124.0 (d, JC-F ) 3.8 Hz),
128.1, 128.2, 128.6, 132.2, 134.6 (d, JC-F ) 9.6 Hz), 135.7, 162.1
(d, JC-F ) 260 Hz), 164.2 (d, JC-F ) 3.8 Hz); GC-MS230 m/z
[M]+, C14H11FO2 requires 230.
Benzyl 4-Fluorobenzoate (17). Benzyl alcohol (4.05 mL,
39.1 mmol) was added slowly to a stirring solution of 4-fluo-
robenzoic acid (5.00 g, 35.7 mmol), 4-(dimethylamino)pyridine
(432 mg, 3.53 mmol), and 1,3-dicyclohexylcarbodiimide (8.15
g, 39.5 mmol) in anhydrous CH2Cl2 at room temperature under
an argon atmosphere. After 18 h the reaction was worked up
according to procedures as outlined for the synthesis of 16.
Flash chromatographic purification over silica (10% EtOAc in
hexanes) afforded 17 as a clear, colorless liquid (7.75 g, 94%):
1H NMR (500 MHz, CDCl3) δ 5.35 (s, 2H), 7.06-14 (m, 2H),
7.32-7.41 (m, 3H), 7.41-7.47 (m, 2H), 8.06-8.12 (m, 2H); 13
NMR (125 MHz, CDCl3) δ 66.84, 115.5 (d, JC-F ) 22.0 Hz),
C
126.4 (d, JC-F ) 2.9 Hz), 128.2, 128.3, 128.6, 132.3 (d, JC-F
)
9.6 Hz), 135.9, 165.5, 165.8 (d, JC-F ) 253 Hz); GC-MS 230
m/z [M]+, C14H11FO2 requires 230.
Ethyl N-(2-Benzyloxycarbonylphenoxy)acetimidate
(18). To a stirring solution of ethyl N-hydroxyacetimidate (2.06
g, 20.0 mmol) in anhydrous DMF (60 mL) under Ar was added
tBuOK (2.21 g, 19.7 mmol) all at once. After 30 min benzyl
2-fluorobenzoate 16 (4.13 g, 17.9 mmol) was added as a
solution in DMF (20 mL) and the reaction was stirred at room
temperature. After 3 h the reaction was diluted with H2O (400
mL) and extracted with EtOAc (3 × 100 mL), and the combined
organics were washed with H2O (3 × 50 mL) and brine (50
mL), dried over Na2SO4, filtered, and concentrated. Flash
chromatographic purification over silica (5-10% gradient
EtOAc in hexanes) afforded 18 as a clear syrup (3.61 g, 64%):
1H NMR (500 MHz, CDCl3) δ 1.34 (t, J ) 7.0 Hz, 3H), 2.00 (s,
3H), 4.18 (q, J ) 7.0 Hz, 2H), 5.34 (s, 2H), 6.96 (dt, J ) 1.1,
7.7 Hz, 1H), 7.30-7.35 (m, 1H), 7.35-7.39 (m, 2H), 7.42-7.48
(m, 3H), 7.55 (dd, J ) 1.1, 8.4 Hz, 1H), 7.90 (dd, J ) 1.8, 7.7
Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 14.4, 14.5, 63.1, 66.6,
114.5, 117.0, 120.4, 128.2, 128.4, 128.5, 131.7, 133.8, 136.1,
159.5, 165.8, 166.7; GC-MS 313 m/z [M]+, C18H19NO4 requires
313, 228 m/z [M - 85]+, benzyl 2-hydroxybenzoate C14H12O3
requires 228.
Benzaldehyde-O-(2-carboxyphenyl)oxime (2a). HClO4
(70%, 14.0 µL, 0.163 mmol), benzaldehyde (18.0 mL, 0.177
mmol), and 20 (39.0 mg, 0.175 mmol) were subjected to the
representative coupling procedure as outlined above (method
3), yielding 2a as a white solid (26.1 mg, 62%): 1H NMR (500
MHz, acetone-d6) δ 7.15 (dt, J ) 0.9, 7.8 Hz, 1H), 7.48-7.55
(m, 3H), 7.59 (dt, J ) 1.8, 7.8 Hz, 1H), 7.71 (d, J ) 8.2 Hz,
1H), 7.83-7.89 (m, 3H), 8.66 (s, 1H), 10.8-11.5 (broad s, 1H);
13C NMR (125 MHz, DMSO-d6) δ 115.7, 119.7, 122.1, 127.8,
129.0, 130.8, 130.9, 131.1, 133.1, 153.4, 157.7, 166.8; MALDI-
FTMS (DHB) 242.0811 m/z [MH]+, C14H12NO3 requires
242.0812; RP-HPLC >99% pure.
Ethyl N-(4-Benzyloxycarbonylphenoxy)acetimidate
(19). To a stirring solution of ethyl N-hydroxyacetimidate (3.79
g, 36.8 mmol) in anhydrous DMF (150 mL) under Ar was
Fibril Formation Assay. Wild type TTR was purified from
an Escherichia coli expression system as described previ-
ously.70 Disposable cuvettes (Fisher #14 385 938) were charged
t
added BuOK (4.09 g, 36.4 mmol) all at once. After 40 min
benzyl 4-fluorobenzoate 17 (7.63 g, 17.9 mmol) was added and