B. Kafle, H. Cho et al.
FULL PAPERS
Synthesis
(Z)-4-[4-(4-Chlorobenzyloxy)benzylidene]-3-[4-
(trifluoromethyl)phenyl]isoxazol-5(4H)one (B3)
The syntheses of aldehydes, A to J, and isoxazolones, 1 to 7, are de-
scribed in the Supporting Information.
Rf =0.5 (EtOAc/n-hexane 1:4); m.p.: 1958C; IR (KBr): n˜ =1737, 1585,
1558, 1330, 1183, 1092 cmÀ1 1H NMR (400 MHz, CDCl3): d=5.15 (s,
;
2H), 7.06 (d, J=9.2 Hz, 2H), 7.36 (brs, 4H), 7.46 (brs, 1H) 7.73 (d, J=
8 Hz, 2H), 7.83 (d, J=7.6 Hz, 2H), 8.42 ppm (d, J=9.2 Hz, 2H);
13C NMR (100 MHz, CDCl3): d=69.5, 115.02, 115.3, 115.9 (d, J=3.8 Hz),
119.0, 120.5 (d, J=3.8 Hz), 121.1, 122.2, 124.1, 126.1, 128.7, 128.8, 129.5,
130.6, 130.9, 132.4 (q, J=30.2 Hz), 134.1, 134.2 (1C) 137.3, 151.9, 156.8,
157.1 (1C), 163.4, 163.7 (1C), 168.6 ppm; elemental analysis: calcd (%)
for C24H15ClF3NO3 (457.8): C 62.96, H 3.30, N 3.06; found: C 63.41, H
2.78, N 3.37.
Library Synthesis Procedure
The solubility of the aldehydes and isoxazolones in isopropanol at 658C
was determined by dissolving 0.05 mmol of aldehydes A–J and isoxazo-
lones 1–7 in isopropanol (Table 5). Seventy test tubes (10 mL capacity)
Table 5. Volume of isopropanol used to dissolve 0.05 mmol of aldehydes
and isoxazolnes at 658C.
(Z)-3-(3-Bromophenyl)-4-{4-[4-
(trifluoromethoxy)benzyloxy]benzylidene}isoxazol-5(4H)one (C2)
Aldehydes
iPrOH [mL]
Isoxazolones
iPrOH [mL]
A
B
C
D
E
F
G
H
I
0.2
0.2
0.2
0.2
1.6
0.2
0.4
0.2[a]
1.0
1.2
1
2
3
4
5
6
7
0.4
0.4
0.4
3.0
4.0
1.4
3.0
Rf =0.44 (EtOAc/n-hexane 1:4); m.p.: 1238C; IR (KBr): n˜ =1753, 1585,
1515, 1277, 1178, 1098 cmÀ1 1H NMR (400 MHz, CDCl3): d=5.17 (s,
;
2H), 7.07 (d, J=9.2 Hz, 2H), 7.24–7.70 (m, 3H), 7.41–7.51 (m, 4H), 7.52
(d, J=6.8 Hz, 1H), 7.72 (d, J=8 Hz, 1H), 7.75 (brs, 1H), 8.42 ppm (d,
J=9.2 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=69.4, 114.9, 115.3, 120.3
(q, J=256.2 Hz; OCF3), 121.1, 123.2, 126.1, 127.3, 128.9, 129.5, 130.7,
131.5, 133.8, 134.3, 137.3, 149.1, 151.9, 162.9, 163.7, 168.5 ppm; elemental
analysis: calcd (%) for C24H15BrF3NO4 (518.3): C 55.62, H 2.92, N 2.70;
found: C 55.43, H 2.68, N 2.91.
J
[a] H was a liquid and dissolved in 0.2 mL of iPrOH for convenience.
(Z)-4-{4-[4-(Trifluoromethoxy)benzyloxy]benzylidene}-3-[4-
(trifluoromethyl)phenyl]isoxazol-5(4H)one (C3)
Rf =0.4 (EtOAc/n-hexane 1:4); m.p.: 1698C; IR (KBr): n˜ =1737, 1588,
1558, 1332, 1282, 1179, 1092 cmÀ1 1H NMR (400 MHz, CDCl3): d=5.17
;
were set in a 10ꢀ7 array. A solution of a benzaldehyde derivative A
(0.05 mmol) in isopropanol was placed in the first column of test tubes
(10 tubes). A solution of B was then placed in the second column. Other
aldehyde derivatives were placed in a similar way in each column of test
(s, 2H), 7.07 (d, J=8.8 Hz, 2H), 7.25 (d, J=8.8 Hz, 2H), 7.45–7.47 (m,
3H), 7.43 (d, J=8 Hz, 2H), 7.83 (d, J=8.4 Hz, 2H), 7.62 ppm (d, J=
9.2 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=69.5, 115.2, 115.4, 115.9,
119.1, 120.6, 121.2, 122.3, 124.2, 126.2, 129.0, 129.6, 130.7, 130.4, 132.4 (q,
J=33.3 Hz), 134.4, 137.4, 149.1, 152.0, 156.9, 157.2 (1C), 163.5, 164.0
(1C), 168.7 ppm; elemental analysis: calcd (%) for C25H15F6NO4 (507.4):
C 59.18, H 2.98, N 2.76; found: C 59.01, H 2.89, N 2.91.
tubes. In the second step,
a solution of isoxazolone derivative 1
(0.05 mmol) in isopropanol was added into the first row of test tubes (7
tubes). Solutions of 2–7 were then added into the next rows of test tubes.
The total volume of isopropanol in each tube was 0.60 to 6.6 mL. The re-
sulting solutions were kept at 658C in a dry heating block for 4 h. In
most of the test tubes, precipitates formed during the reaction or on cool-
ing to room temperature. In some cases (F2, F3, F4, F6, F7, G2, G3, G4)
no precipitate formed, but TLC showed complete consumption of start-
ing materials. Addition of hexane (0.5 mL) and cooling to À508C result-
ed in precipitation of the reaction product. The supernatant liquid was
removed and the precipitate was washed with isopropanol (0.5 mL). The
solvent was evaporated by heating in a dry heating block and the product
analyzed by TLC, in which all the products exhibited essentially a single
spot. 1H NMR spectra of randomly selected crude samples of A7, E4,
and J7 also revealed the absence of noticeable impurities as shown in the
Supporting Information. The 70 compounds, thus obtained, were dis-
solved in DMSO to a 100 mm concentration and used for enzyme assay to
determine the inhibitory activity against PTP1B and other PTPs. Six of
the compounds that inhibited more than 90% of the PTP1B enzyme ac-
tivity were further purified by recrystallization in isopropanol for IC50 de-
termination and structural characterization including elemental and spec-
troscopic analysis.
(Z)-3-[4-(Pyridin-2-yl)phenyl]-4-{4-[4-
(trifluoromethoxy)benzyloxy]benzylidene}isoxazol-5(4H)one (C5)
Rf =0.06 (EtOAc/n-hexane 1:4); m.p.: 1898C; IR (KBr): n˜ =1737, 1585,
1
1558, 1288, 1266, 1178, 1092, 870, 776 cmÀ1; H NMR (400 MHz, CDCl3):
d=5.20 (s, 2H), 7.07 (d, J=8.8 Hz, 2H), 7.24–7.26 (m, 3H), 7.30–7.31
(m, 1H), 7.46 (d, J=8.8 Hz, 2H), 7.56 (brs, 1H), 7.70 (d, J=8.4 Hz, 2H),
7.80–7.82 (m, 2H), 8.18 (d, J=8.0 Hz, 2H), 8.42 (d, J=8.8 Hz, 2H),
8.74 ppm (d, J=5.2 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=69.4,
115.4, 115.7, 120.4 (q, J=253.3 Hz OCF3), 120.8, 121.2, 122.9, 126.3,
127.7, 128.0, 129.0, 129.2, 134.4, 137.0, 137.3, 141.8, 149.9, 152.0, 156.2,
163.6, 164.0, 168.9 ppm; elemental analysis: calcd (%) for C29H19F3N2O4
(516.1): C 67.44, H 3.71, N 5.42; found: C 67.29, H 3.50, N 5.58.
(Z)-Methyl 4-(5-oxo-4-{4-[4-(trifluoromethoxy)benzyloxy]benzylidene}-
4,5-dihydroisoxazol-3-yl)benzoate (C7)
Rf =0.2 (EtOAc/n-hexane 1:4); m.p.: 1728C; IR (KBr): n˜ =1768,1711,
1583, 1517, 1277, 1179, 1087, 771 cmÀ1 1H NMR (400 MHz, CDCl3): d=
;
4.00 (s, 3H), 5.17 (s, 2H), 7.07 (d, J=9.2 Hz, 2H), 7.24–7.26 (m, 3H),
7.45–7.48 (m, 4H), 7.68 (d, J=8 Hz, 2H), 8.22 (d, J=8.4 Hz, 2H),
8.41 ppm (d, J=9.2 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=52.5, 69.5,
115.2, 115.4, 120.3 (q, J=256.2 Hz; OCF3), 121.2, 126.2, 128.8, 128.9,
129.0, 130.3, 131.9, 132.3, 134.3, 137.4, 149.1, 151.9, 163.5, 163.8, 166.2,
168.6, 174.0 ppm; elemental analysis: calcd (%) for C26H18ClF3NO6 (457):
C 62.78, H 3.65, N 2.82; found: C 62.68, H 3.57, N 2.81.
(Z)-4-[4-(2-Chlorobenzyloxy)benzylidene]-3-[4-
(trifluoromethyl)phenyl]isoxazol-5(4H)one (A3)
Rf =0.5 (EtOAc/n-hexane 1:4); m.p.: 1718C; IR (KBr): n˜ =1746, 1588,
1
1563, 1327, 1277, 1174, 1114, 1092 cmÀ1; H NMR (400 MHz, CDCl3): d=
5.25 (s, 2H), 7.10 (d, J=8.4 Hz, 2H), 7.29–7.30 (m, 2H), 7.42–7.44 (m,
1H), 7.47 (brs, 1H), 7.50–7.52 (m, 1H), 7.74 (d, J=8.8 Hz, 2H), 7.84 (d,
J=8.4 Hz, 2H), 8.43 ppm (d, J=8.8 Hz, 2H); 13C NMR (100 MHz,
CDCl3): d=67.4, 115.1, 115.3, 115.9 (d, J=3.8 Hz), 119.0, 120.5 (d, J=
3.8, Hz), 121.1, 122.2, 124.1, 126.1, 127.0, 128.8, 129.5 (3C), 130.6, 130.9,
132.4 (q, J=32.6 Hz), 132.7, 133.3, 137.3, 151.9, 156.8, 157.10 (1C), 163.4,
163.6 (1C), 168.6 ppm; elemental analysis: calcd (%) for C24H15ClF3NO3
(457.8): C 62.96, H 3.30, N 3.06; found: C 62.93, H 2.84, N 3.27.
Enzyme Assay: IC50 Determination
The enzyme activity was measured at 378C by monitoring the hydrolysis
of pNPP in an enzyme reaction buffer. The absorbance at 405 nm was
measured to determine the amount of p-nitrophenol released. Enzymes
were diluted before the experiment to appropriate concentrations in
2078
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 2073 – 2079