M. Andrzejewska et al. / Bioorg. Med. Chem. 11 (2003) 3997–4002
4001
H2O, 55%). TLC (CHCl3–MeOH, 9:1): Rf 0.56. UV
(MeOH/H2O, 1:9): (pH 2): 267 (8500), 302 (5300), (pH
Biological evaluation
6): 291 (6000), 302 (4800), (pH 12): 272 (10,400) 302
1
Materials. CK1 and CK2 were partially purified from
rat liver cytosol as previously described.37 G-CK was
purified from Golgi apparatus of rat lactating mam-
mary gland as described by Lasa et al.27 Synthetic pep-
tide substrates were kindly provided by Dr. O. Marin
(Padova, Italy).
(6500). H NMR(Me SO-d6) d (ppm): 14.2 (bs, H–N).
2
MS m/z: 466 (62, M+ꢀ2), 467 (5, M+ꢀ1), 468 (100,
M+), 469 (9, M++1), 470 (83, M++2), 471 (7,
M++3), 472 (31, M++4). Anal. calcd for C7HN2Br4Cl
(468.168): C, 17.96; H, 0.22; N, 5.98. Found: C, 17.80;
H, 0.34; N, 5.77.
Phosphorylation. Phosphorylation assays were per-
formed by incubating the three protein kinases CK1,
CK2 and G-CK in the absence and in the presence of
various inhibitors under their optimal conditions. In
detail, the activities of protein kinases CK1 and CK2
were tested in 50 mM Tris–HCl pH 7.5 containing
10 mM MgCl2, 100 mM NaCl, 20 mM g33P-ATP (spe-
cific activity of about 1000 cpm/pmol) and the specific
peptide substrate (100 m M RRRADDSDDDDD and
200 m M RRKHAAIGDDDDAYSITA for CK2 and
CK1, respectively). The activity of G-CK was assayed
on the specific peptide substrate KIEKFQSEEQQQ
(0.5 mM) under identical conditions except that MnCl2
was replacing MgCl2 and NaCl was omitted.
4,6,7-Tribromo-5-chloro-2-trifluoromethylbenzimidazole
(8e). Similar to 6 starting from 7e: (mp 261–263 ꢁC,
from EtOH/H2O, 82%). TLC (CHCl3–MeOH, 9:1): Rf
0.80. UV (MeOH/H2O, 1:9): (pH 2): 279 (9100), 303
(5200), (pH 6): 280 (8800), 301 (6400), (pH 12): 301
(10,600). 1H NMR(Me 2SO-d6) d (ppm): 13.6 (bs, H–N).
MS m/z: 456 (82, M+ꢀ2), 457 (8, M+ꢀ1), 458 (100,
M+), 459 (9, M++1), 460 (47, M++2), 461 (37,
M++3), 462 (8, M++4). Anal. calcd for
C8HN2Br3ClF3 (457.269): C, 21.01; H, 0.22; N, 6.13.
Found; C, 20.88; H, 0.34; N, 5.93.
4,6-Dibromo-5,7-dichloro-2-trifluoromethylbenzimidazole
(8f). Similar to 6 starting from 7f: (mp 264–266 ꢁC, from
EtOH/H2O, 87%). TLC (CHCl3–MeOH, 9:1): Rf 0.63.
UV (MeOH/H2O, 1:9): (pH 2): 276 (7200), 302 (3200),
(pH 6): 291 (6000), 302 (4800), (pH 12): 300 (10,000). 1H
Acknowledgements
NMR(Me SO-d6) d (ppm): 14.0 (bs, H–N). MS m/z:
The study was supported (M.A. and Z.K.) by the
Foundation for the Development of Diagnostic and
Therapy, Warsaw, Poland, and by grants from the Ita-
lian MIUR(F.M.). The authors are indebted to Prof. Dr.
Lorenzo A. Pinna for critical reading of the manuscript
and helpful discussion.
2
410 (39, M+ꢀ2), 411 (4, M+ꢀ1), 412 (100, M+), 413
(10, M++1), 414 (92, M++2), 415 (9, M++3), 416
(32, M++4). Anal. calcd for C8HN2Br2Cl2F3 (415.818):
C, 23.11; H, 0.24; N, 6.74. Found: C, 22.91; H, 0.40; N,
6.55.
4,7-Dibromo-5,6-dichloro-2-trifluoromethylbenzimidazole
(8g). Similar to 6 starting from 7g: (mp 252–254 ꢁC,
from EtOH/H2O, 87%). TLC (CHCl3–MeOH, 9:1): Rf
0.62. UV (MeOH/H2O, 1:9): (pH 2): 279 (10,200), (pH
References and Notes
1. Lonn, U.; Lonn, S. Eur. J. Biochem. 1987, 164, 541.
2. Dobrowolska, G.; Muszynska, G.; Shugar, D. Biochim.
Biophys. Acta 1991, 1080, 221.
3. Tamm, I.; Seghal, P. B. Advances Virus Res. 1978, 22, 187.
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Exp. Med. 1954, 99, 227.
5. Zou, R.; Drach, J. C.; Townsend, L. B. J. Med. Chem.
1997, 40, 811.
6. Zou, R.; Kawashima, E.; Freeman, G. A.; Koszalka, G. W.;
Drach, J. C.; Townsend, L. B. Nucleos. Nucleot. Nucl. 2000,
19, 125.
7. Koszalka, G. W.; Chamberlain, S. D.; Daluge, S. M.;
Boyd, F. L.; Tidwell, J. H.; Martin, M. T.; Harvey, R. J.;
Frick, L. W.; Perkins, D. G.; Wang, L. H.; Drach, J. C.;
Townsend, L. B.; Biron K. K. XII International Roundtable:
Nucleosides, Nucleotides and their Biological Application, La
Jolla, CA, Sept. 15–19, 1996; OP 41, 56.
8. Chamberlain, S.D.; Chan, J.H.; Tidwell, J.H.; Peckham,
G.E.; Harvey, R.J.; Dornsife, R.E.; Frick, L.W.; Townsend,
L.B.; Drach, J.C.; Koszalka, G.W. Abstract of Papers, 213th
National Meeting of the American Chemical Society, San
Francisco, CA, April 13–17, 1997; Carbohydrates 022.
9. Burton, E.; Lambie, A. J.; Ludgate, J. C.; Newbold, G. T.;
Percival, A.; Saggers, D. T. Nature 1965, 208, 1166.
10. Navarete-Vazquez, G.; Cedillo, R.; Hernandez-Campos,
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1
6): 291 (8400), 300 (8100), (pH 12): 300 (12,200). H
NMR(Me SO-d6) d (ppm): 14.0 (bs, H–N). MS m/z:
2
410 (38, M+ꢀ2), 411 (5, M+ꢀ1), 412 (100, M+), 413
(9, M++1), 414 (88, M++2), 415 (9, M++3), 416 (29,
M++4). Anal. calcd for C8HN2Br2Cl2F3 (415.818): C,
23.11; H, 0.24; N, 6.74. Found: C, 22.93; H, 0.41; N,
6.58.
4,5,6,7-Tetrabromo-2-mercaptobenzimidazole (9). To the
stirred solution of 6 (510 m, 1 mmol) in EtOH (25 mL)
thiourea (83 mg, 1.1 mmol) was added. The reaction
mixture was refluxed for 5 h. A precipitate formed was
filtered, dissolved in EtOH/aq ammonia (1:1) and pre-
cipitated by addition of acetic acid to pH 4–5. The
purification was repeated to give chromatographically
pure 9 (200 mg, 43%); mp >320 ꢁC (decomp.) (for ana-
lysis cryst. from EtOH). TLC (CHCl3–MeOH, 9:1):
0.81. UV (MeOH/H2O, 1:9): (pH 2): 256 (16,800, 293
(4600), 327 (12,900), (pH 6): 255 (17,600), 292 (4600),
327 (14,900), (pH 12): 293 (9100). 1H NMR(Me 2SO-d6)
d (ppm): 13.3 (bs, H–N). MS m/z: 464 (70, M+ꢀ2), 465
(7, M+ꢀ1), 466 (100, M+), 467 (10, M++1), 468 (67,
M++2), 469 (6, M++3), 470 (19, M++4). Anal. calcd
for C7H2 N2Br4S (465.783): C, 18.05; H, 0.43; N, 6.01.
Found: C, 18.23; H, 0.55; N, 5.85.