◦
After cooling to 25 C, the mixture was acidified with 10% HCl
References
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were dried (Na2SO4) and evaporated. Purification of the residue by
recrystallization (hexane–CHCl3) afforded 0.10 g (92%) of acid 3a
as a white solid (mp 100 ◦C). 1H NMR (400 MHz, CD3COCD3)
d 2.05 (s, 3H, Ar-CH3), 2.08 (s, 3H, Ar-CH3), 4.10 (s, 2H, SCH2),
4.58 (s, 2H, 2H2), 6.59 (s, 1H, H3ꢀꢀ), 6.66 (d, J = 8.5, 1H, H6ꢀ),
7.06 (d, J = 8.5, 1H, H5ꢀ), 7.10 (s, 1H, H3ꢀ), 7.48 (d, J = 8.0 Hz,
2H, H2ꢀꢀꢀ + H6ꢀꢀꢀ), 7.58 (d, J = 8.0 Hz, 2H, H3ꢀꢀꢀ + H5ꢀꢀꢀ) ppm.
13C NMR (100 MHz, CD3COCD3) d 15.2 (q, Ar-CH3), 16.2 (q,
Ar-CH3), 35.2 (t, CH2S), 65.5 (t, C2), 112.6 (d, C6ꢀ), 125.3 (s, CF3,
1JC–F = 270.7 Hz), 126.4 (d, 2C, C3ꢀꢀꢀ + C5ꢀꢀꢀ, 3JC–F = 3.7 Hz), 127.0
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2
(s), 128.4 (s), 129.0 (s, C4ꢀꢀꢀ, JC–F = 32.2 Hz), 129.7 (d, 2C, C2ꢀꢀꢀ
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135.6 (s), 139.5 (s), 141.9 (s), 156.8 (s), 170.1 (s, C1) ppm. IR
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=
(NaCl) m 3400 − 2700 (br, O–H), 2925 (w, C–H), 1734 (s, C O),
1614 (m), 1490 (s), 1325 (s), 1228 (m), 1124 (s), 1069 (s), 1016 (w),
840 (m) cm−1. MS m/z (%) 453 ([M + 1]+, 4), 452 (M+, 17), 257
(18), 256 (47), 255 ([M–C9H9O3S]+, 100), 139 (10). HRMS (EI+)
calcd for C22H19F3O3S2 452.0728, found 452.0732. Anal. Calcd for
C22H19F3O3S2: C, 58.39; H, 4.23; S, 14.17. Found: C, 58.06; H,
4.16; S, 14.15%.
Cell culture
3 Several other dibromothiophene derivatives (2,5-dibromothiophene,
ethyl
4,5-dibromothiophene-2-carboxylate
or
ethyl
2,5-
HeLa cells were routinely maintained in DMEM supplemented
with 5% FCS and ligand assays were performed in the same
medium.
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Stable reporter cell lines
A stable Gal4-hPPARb/d cell line was generated as described
previously to facilitate assessment of agonistic activity at PPARb/d
with minor modifications.37 Briefly, HeLa cells were cotransfected
with Gal4-hPPARb/dand (17m)5-bglobin-Luc-Neo reporter gene.
At 48 h post-transfection, the cell line was selected with Geneticin
at 0.8 mg mL−1 and puromycin at 0.3 lg mL−1. Clones expressing
PPARb/d agonist-inducible luciferase activity were selected as
described.
5 R. Pereira, C. Gaudon, B. Iglesias, P. Germain, H. Gronemeyer and
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Ramsden, European Patent No. WO 0292590, 2002; P. J. Beswick,
C. C. F. Hamlett, V. Patel, M. L. Sierra and N. G. Ramsden, Chem.
Abstr., 2002, 137, 384743.
7 H.-L. Elbe, L. Assmann, R. Tiemann, U. Schulz, H. Haenssler and
H.-W. Dehne, European Patent No. EP 0755185, 1995; H.-L. Elbe, L.
Assmann, R. Tiemann, U. Schulz, H. Haenssler and H.-W. Dehne,
Chem. Abstr., 1995, 124, 55787.
Assessment of agonistic activity
To test the different compounds, identical aliquots of the Gal-
hPPARb/d reporter cell line were seeded and exposed in parallel
to the known agonist GW501516 1a, the test ligand, and vehicle
for 16 h. Cells were lysed in 100 lL of lysis buffer (25 mM Tris
phosphate [pH 7.8], 2 mM EDTA, 1 mM DTT, 10% glycerol, and
1% Triton X-100). A 100 lL portion of luciferin buffer (20 mM
Tris phosphate [pH 7.8], 1.07 mM MgCl2, 2.67 mM MgSO4, 1 mM
EDTA, 33.3 mM DTT, 0.53 mM ATP, 0.47 mM luciferin, and
0.27 mM coenzyme A) was added prior to direct photon counting.
8 S. Kano, Y. Yuasa, T. Yokomatsu and S. Shibuya, Heterocycles, 1983,
20, 2035–2037.
9 O. Meth-Cohn and G. van Vuuren, J. Chem. Soc., Perkin Trans. 1, 1986,
233–243.
10 The boronates (6, M = B(OMe)2 or B(OiPr)2) were prepared by
bromine–lithium exchange (n-BuLi, THF, −78 ◦C) followed by trap-
ping with B(OMe)3 or B(OiPr)3 and were used without isolation.
The organostannane (6, M = SnBu3) was prepared by trapping
with Bu3SnCl the organolithium generated as indicated above, and
purified by reverse-phase column chromatography (V. Farina, J. Org.
Chem., 1991, 56, 4985–4987). The organozinc halide (6, M = ZnCl)
was prepared by treating with ZnCl2 the organolithium generated as
indicated above and was used without isolation.
Acknowledgements
We are grateful to the Spanish MEC (SAF2004-07131-FEDER),
the European Community (ANTICANCER RETINOIDS
QLK3-2002-02029 and EPITRON Contract No. LSHC-CT2005-
518417) and Xunta de Galicia (PGIDIT05PXIC31403PN).
4524 | Org. Biomol. Chem., 2006, 4, 4514–4525
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The Royal Society of Chemistry 2006
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