570 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Vang et al.
=8.7 Hz), 4.58 (t, 2H, J=6.6 Hz), 3.77 (s, 3H), 2.60 (t, 2H, J=6.9
Hz), 2.39 (m, 2H); 13C NMR (CDCl3/CD3OD) δ 172.1, 160.3,
160.1, 157.6, 152.3, 140.2, 134.3, 132.6, 128.7, 128.4, 126.5, 126.3,
126.1, 125.5, 123.1, 122.9, 122.8, 122.6, 122.0, 121.5, 118.1, 114.2,
105.5, 98.2, 55.5, 33.0, 26.4, 19.1. HRMS (FAB) calcd for
C32H27N4O6 (M þ H)þ m/z 563.1931, found 563.1955.
6-Hydroxy-3-(1-(4-(6-methoxybenzo[d]thiazol-2-ylamino)-4-
oxobutyl)-1H-1,2,3-triazol-4-yl)-2-m-tolylbenzofuran-5-carboxylic
Acid (630). 630 was synthesized similarly to 522. 1HNMR(CDCl3/
CD3OD) δ 8.25 (s, 1H), 7.80 (s, 1H), 7.53 (m, 2H), 7.46 (d, 1H, J =
7.8 Hz), 7.18 (m, 2H), 7.11 (d, 1H, J=7.8 Hz), 6.94 (s, 1H), 6.91 (m,
1H), 4.52 (m, 2H), 3.80 (s, 3H), 2.54 (m, 2H), 2.35 (m, 2H), 2.31(s,
3H); 13C NMR (CDCl3/CD3OD) δ 172.5, 170.9, 169.4, 156.8,
142.3, 138.5, 133.1, 130.2, 129.9, 128.6, 127.8, 124.4, 123.0, 121.3,
115.4, 106.7, 104.3, 98.1, 88.4, 60.9, 56.0, 32.3, 25.5, 21.6. HRMS
(FAB) cacld for C30H26N5O6S (M þ H)þ m/z 584.1604, found
584.1619.
Microplate Luminometer, Promega). The level of NFAT/AP-1
activation for a sample was calculated as the ratio between firefly
and Renilla luciferase activity for the same sample. Typically,
each sample was run in triplicate. Compounds giving firefly/
Renilla luciferase ratios >2-fold higher than the corresponding
ratios for the control samples (DMSO) were selected for retest-
ing in a dose-response format, using the same assay.
TCR Time Courses, Cell Lysis, and Immunoblotting. Jurkat
TAg T cells were treated with small-molecule compound or DMSO
as described above. Thereafter, cells were TCR stimulated (OKT3,
500 ng/mL) for the indicated time periods and then lysed in lysis
buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 1% Triton X-100,
50 mM NaF, 5 mM Na3VO4, 1 mM PMSF, 5 mM EDTA).
Proteins were later resolved by SDS-PAGE, transferred onto
PVDF membranes, and blotted with the indicated antibodies.
These procedures were as before.9
IC50 Measurements. The PTP-catalyzed hydrolysis of DiFM-
UP in the presence of compound was assayed at 30 ꢀC in a 60 μL
96-well format reaction system in 50 mM Bis-Tris, pH 6.0 assay
buffer having an ionic strength of 50 mM (adjusted with NaCl)
and containing 1 mM dithiotreitol, 5% DMSO, and 1% PEG8000.
At various concentrations of the compound, the initial rate at
fixed DiFMUP concentration (equal to the corresponding Km
value for each PTP) was determined using a FLx800 microplate
reader (Bio-Tek Instruments, Inc.), an excitation wavelength of
360 nm and measuring the emission of the fluorescent reaction
product 6,8-difluoro-7-hydroxy-4-methylcoumarin at 460 nm.
The nonenzymatic hydrolysis of the substrate was corrected by
measuring the control without addition of enzyme. The IC50
values were calculated as described before.27
Acknowledgment. This work was supported by Grants
1R21CA132121 (to L.T.) and MLSCN U54 HG003914 (to
T.M.) from the National Institutes of Health. Support came
also from the Norwegian Cancer Society (to T.V.) and from
the Oxnard Foundation. We acknowledge resources of the
University of Miami Center for Computational Science
(CCS). We also thank Gladys M. Tjørhom and Jorun Solheim
for technical assistance and Kjetil Tasken for support.
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