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C. Leung et al. / Bioorg. Med. Chem. 10 (2002) 2309–2323
Neopentyl (3-bromophenyl)difluoromethanesulfonate (81).
Prepared in 87% yield from 80 using the same proce-
dure as that used for preparing 79 from 78 (colorless
7.84 (1H, s, Ar–H), 7.72 (1H, d, J=8.0 Hz, Ar–H), 7.64
(1H, d, J=7.3 Hz, Ar-H), 7.39 (1H, t, J=8.1 Hz, Ar–
H), 7.11 (2H, d, J=8.0 Hz, Ar–H), 6.82 (2H, d,
J=8.8 Hz, Ar–H), 4.19 (2H, s, CH2O), 3.93 (2H, t,
J=6.2 Hz, CH2), 2.59 (2H, q, J=7.6 Hz, CH2), 1.73–
1.85 (2H, m, CH2), 1.44–1.54 (2H, m, CH2), 1.21 (3H, t,
J=7.7 Hz, CH3), 1.03 (6H, s, CH3); 19F NMR (CDCl3)
d ꢀ24.19; 13C NMR (CDCl3) d 157.18, 136.53, 135.62
(t, JCF=1.8 Hz), 130.28, 130.21 (t, JCF=6.4 Hz), 128.70,
125.86 (t, JCF=6.0 Hz), 122.81, 120.15, 114.65, 82.99,
63.38, 34.85, 34.52, 28.00, 23.95, 23.64, 15.72. MS m/z
(relative intensity) 504 (28), 205 (59), 122 (100), 107 (65),
97 (29), 83 (25), 55 (52). HRMS calcd for
C22H27O4F2S1Br1 504.0782, found 504.0774.
1
oil). H NMR (CDCl3) d 1.00 (s, 9H), 4.11 (s, 2H), 7.36
(t, J=7.8 Hz, 1H), 7.62 (d, J=7.8 Hz, 1H), 7.71 (d,
J=7.8 Hz, 1H), 7.81 (s, 1H); 13C NMR (CDCl3) d
25.91, 32.23, 84.81, 122.83 (t, J=283.5 Hz), 123.90,
125.93 (t, J=6.1 Hz), 130.05 (t, J=22.7 Hz), 130.22 (t,
J=6.7 Hz), 130.39, 135.70; 19F NMR (CDCl3) d
ꢀ24.74. LR-EIMS m/z (relative intensity) 357 (1), 356
(3), 358 (18), 207 (93), 205 (100). HR-EIMS calcd for
C12H15SO3BrF2, 355.9893, found 355.9895.
Neopentyl-3-(5-acetyl-2-thienyl)methanesulfonate (82).
To a solution of 81 (0.476 g, 1.33 mmol) in deox-
ygenated DMF (20 mL), was added 5-acetyl-2-thie-
nylboronic acid (0.339 g, 1.99 mmol), K2CO3 (0.55 g,
4 mmol), Pd(OAc)2 (30 mg, 0.13 mmol) and H2O
(0.27 mL, 15 mmol). The reaction was stirred at room
temperature under nitrogen for 2 h. The reaction was
diluted with ether, washed with saturated NH4Cl, water
and brine, dried (MgSO4) and concentrated to give a
light-yellow solid. Flash chromatography of the crude
product (5:1 hexane/EtOAc) gave pure 82 in 87% yield
(white solid): mp 88–89 ꢁC. 1H NMR (CDCl3) d 1.00 (s,
9H), 2.56 (s, 3H), 4.13 (s, 2H), 7.36 (d, J=3.9 Hz, 1H),
7.54 (t, J=7.9 Hz, 1H), 7.65 (m, 2H), 7.82 (d, J=7.9 Hz,
1H), 7.91 (s, 1H); 13C NMR (CDCl3) d 25.911, 26.67,
32.22, 84.77, 120.73 (t, J=283.9 Hz), 124.72 (t,
J=6.0 Hz), 125.11, 127.37 (t, J=5.5 Hz), 129.16 (t,
J=22.2 Hz), 129.76, 130.14, 133.46, 134.22, 144.23,
150.32, 190.59; 19F NMR (CDCl3) d ꢀ24.74. EIMS m/z
402 (30), 332 (29), 251 (100). HREIMS calcd for
C18H20SO4F2, 402.0771, found 402.0785.
Kinetic studies with PTP1B
Rates of PTP1B-catalyzed dephosphorylation in the
presence or absence of inhibitors were determined using
fluorescein diphosphate (FDP at Km concentration
ꢀ20 mM) as substrate in assay buffer containing 50 mM
Bis-Tris (pH 6.5), 2 mM EDTA, 5 mM N,N0-dimethyl-
N,N0-bis(mercaptoacetyl)hydrazine (DMH) or dithio-
threitol (DTT), 0.01% triton X-100 and 5% DMSO.
For peptides 7–9 and compounds 50–73, assays were
carried out at 25 ꢁC in 96-well plates with total volume
of 200 mL per well. Reactions were initiated by the
addition of PTP1B (final concentration 0.2 mg/mL). The
phosphatase activity was followed by monitoring the
production of the fluorescent product fluorescein
monophosphate (FMP) continuously for 10 min using
the Cytofluor II plate reader (PerSeptive Biosystems),
with excitation at 440 nm (slit width 20 nm) and emis-
sion 530 nm (slit width 25 nm), or the SPECTRAmax
GEMINI XS (Molecular Devices) dual-scanning
microplate spectrofluorometer, with excitation at
485 nm and emission at 538 nm. For compounds 28–40,
assays were carried out in 1 mL cuvettes with total
volumes of 700 mL. The phosphatase activity was fol-
lowed by monitoring the production of FMP at 450 nm
using a Varian Cary 1 spectrophotometer. IC50’s were
determined using at least nine different inhibitor con-
centrations. For Ki determinations (peptides 7–9),
substrate concentrations of 15, 20, 25, 35, and 50 mM
were used. 1/v versus 1/[S] plots were obtained which
exhibited competitive inhibition patterns for all three
peptides. The slopes of these plots were determined
using Microsoft Excel. These slopes were replotted
against the concentration of the inhibitor using
Microsoft Excel and the Ki was obtained from the x-
intercept of this replot.
3 - (5 - Acetyl - 2 - thienyl)difluoromethanesulfonic acid,
ammonium salt (71). Prepared in 98% yield from 82
using the same procedure as thatꢁused for preparing 65
1
from 79 (white solid): dec. at 122 C. H NMR (D2O) d
2.21 (s, 3H), 6.97 (d, J=4.0 Hz, 1H), 7.18 (t, J=7.8 Hz,
1H), 7.32 (m, 2H), 7.40 (d, J=7.8 Hz, 1H), 7.52 (s, 1H);
13C NMR (D2O) d 25.54, 119.73 (t, J=277.2 Hz),
123.66 (t, J=6.5 Hz), 125.36, 127.06 (t, J=5.9 Hz),
128.55, 129.23, 131.00 (t, J=23.3 Hz), 132.47, 136.54,
141.53, 151.86, 195.19; 19F NMR (D2O) d ꢀ28.27. LR-
ESMS m/z (relative intensity) 331 (100). HR-ESMS
calcd for C13H9F2S2O4 330.9910, found 330.9916.
HPLC retention time=11.8 min.
[{5 - (4 - Ethylphenoxy) - 2,2 - dimethylpentyl}(3-bromo-
phenyl)]difluoromethane-sulfonate (83). To a solution of
4-ethylphenol (0.101 g, 0.83 mmol, 1 equiv) in 1:1 anhyd
THF/CH2Cl2 (45 mL) was added TMAD (0.287 g,
1.66 mmol, 2 equiv). Once the TMAD was completely
dissolved, a solution of 42 (0.665 g, 1.66 mmol, 2 equiv)
in 1:1 anhyd THF/CH2Cl2 (25 mL) was added. Tribu-
tylphosphine (0.427 mL, 0.35 g, 1.66 mmol, 2 equiv) was
then added dropwise to the reaction mixture over a
period of 5 min. The reaction was stirred at room tem-
perature for 3 h and then concentrated by rotary eva-
poration. Column chromatography (1:1, CH2Cl2/
hexane) of the crude residue yielded pure 83 as a white
Acknowledgements
We thank Sunia Afzaal, Gary Kruppa and Paul Speir at
BrukerDaltonics for obtaining the high-resolution
FTICR-ES mass spectra.
References and Notes
solid in 96% yield: mp 36–37 ꢁC. H NMR (CDCl3) d
1. Al-Obeidi, F. A.; Lam, K. S. Oncogene 2000, 19, 5690.
1