B. S. Park et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4720–4723
4723
5-methylphenyl group to exhibit NOE effect as shown in Figure
References and notes
2d. The final hydroxyl product 1 was obtained by demethylation
of the methoxy group of compound 10 using 10 equiv of boron-
trifluoride–dimethylsulfide complex in dichloromethane.
The screening results of the target compound 1 over 45 differ-
ent kinases have revealed that the inhibitory activity of the com-
pound was not exhibited over almost all of the tested kinases,
while high potency and activity was selectively shown at ROS1
kinase only (Table 1).22
1. Alaoui-Jamali, M. A. Biomed. Pharmacother. 2006, 60, 629.
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13. Charest, A.; Lane, K.; McMahon, K.; Park, J.; Preisinger, E.; Conroy, H.; Housman,
D. Gene. Chromosome Canc. 2003, 37, 58.
The compound was tested initially at a single dose concentra-
tion of 10 lM. At this concentration, 94% inhibition of the enzy-
matic activity of ROS1 kinase was observed, while the inhibition
in activity was below 30% in all of the other kinases, and in the
range of 20–30% in 5 kinases only (ABL1, Aurora A, EPHA1, RAF1
and RON/MST1R).
Compound 1 was further tested over ROS1 kinase in order to
determine its IC50, where a 10-dose IC50 mode with threefold
14. Jun, H. J.; Woolfenden, S.; Coven, S.; Lane, K.; Bronson, R.; Housman, D.;
Charest, A. Cancer Res. 2009, 69, 2180.
15. Holland, E. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6242.
16. Hess, S. M.; Anderson, J. G.; Bierbach, U. Bioorg. Med. Chem. Lett. 2005, 15, 443.
17. Van de Kelft, E. Acta Neurochir. 1997, 139, 589.
serial dilutions starting at 20 lM concentration was applied
against Staurosporine23–25 as a reference standard. The com-
pound has showed an IC50 value of 199 nM, while the IC50 va-
lue for the non-selective kinase inhibitor Staurosporine was
0.07 nM.
18. Ohgaki, H.; Kleihues, P. Am. J. Pathol. 2007, 170, 1445.
19. Wipf, P.; Mahler, S. G.; Okumura, K. Org. Lett. 2005, 7, 4483.
20. Turner, F. A.; Gearien, J. E. J. Org. Chem. 1959, 24, 1952.
21. De Frutos, O.; Atienza, C.; Echavarren, A. M. Eur. J. Org. Chem. 2001, 1, 163.
22. Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.;
Campbell, B. T.; Chan, K. W.; Ciceri1, P.; Davis, M. I.; Edeen, P. T.; Faraoni1, R.;
Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.;
Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. Nat. Biotechnol. 2008,
26, 127.
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24. Gescher, A. Crit. Rev. Oncol. Hematol. 2000, 34, 127.
25. Yang, S.; Malaviya, R.; Wilson, L. J.; Argentieri, R.; Chen, X.; Yang, C.; Wang, B.;
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26. Preparation of compound 1: To a solution of compound 10 (75 mg, 0.16 mmol)
in dichloromethane (3 mL) was added borontrifluoride–methyl sulfide
The high selectivity of compound 1 to ROS1 kinase and the
diminished activity over the other kinases could be attributed
to the increased bulkiness exerted by the substituents at pyrim-
idine nucleus. This increase in bulkiness seems to hinder the fit-
ting of the compound to the binding sites of most of these
kinases and to exclude it from their binding pockets. However,
the selective inhibition of ROS1 kinase might be owed to a un-
ique difference in the geometry of the binding pocket of this en-
zyme that enables the fitting and interaction of compound 1. An
indirect inhibitory effect of compound 1 at ROS1 kinase, proba-
bly through binding at an allosteric binding site, is another pos-
sible assumption for the reason behind this selective inhibition.
The absolute reason is still unclear and we believe that it worth
further exploration for the mechanism of its unique inhibitory
effect.
In conclusion, a highly potent and selective inhibitor for ROS1
kinase has been synthesized and can be used as a promising lead
for new selective inhibitors for ROS1 kinase. It worth also to men-
tion that until now, no selective inhibitor is available for ROS1 ki-
nase, and the development of new selective inhibitors for such
kinase might open the way for new selective therapeutics for
astrocytomas. Screening of compound 1 against glioblastoma-de-
rived tumors is currently undergoing.
complex (172 lL, 1.65 mmol) in a dropwise manner at room temperature
and under N2 atmosphere. The resulting suspension was stirred for 12 h, and
then the mixture was concentrated under vacuum. The residue was partitioned
between ethylacetate (100 mL) and brine (50 mL). The organic layer was
separated and dried over anhydrous MgSO4, then evaporated under vacuum.
The residue was purified by column chromatography (silica gel, methanol-
dichloromethane 1:30, v/v) to yield the pure hydroxyl product 1 as a white
powder (32.5 mg, 45%); mp 236–237 °C; 1H NMR (CD3OD) d 1.09 (d, J = 5.7 Hz,
3H), 2.21 (s, 3H), 3.25 (s, 1H), 3.38 (br s, 1H), 3.87 (br s, 1H), 5.32 (s, 2H), 6.65 (s,
1H), 6.69 (s, 1H), 6.76 (s, 1H), 6.86 (s, 1H), 7.38–7.41 (m, 1H), 8.14 (d, J = 8.1 Hz,
1H), 8.28 (s, 1H), 8.49 (d, J = 3.5 Hz, 1H), 8.81 (s, 1H); IR (KBr) 3425, 2926, 2360,
1586, 1454, 1349, 1162 cmÀ1
.
27. Kinase assays were performed at Reaction Biology Corporation using the
‘HotSpot’ assay platform. Kinase assay protocol; reaction buffer: base reaction
buffer; 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35,
0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. Required cofactors
were added individually (if needed) to each kinase reaction. Reaction
Procedure: To a freshly prepared buffer solution was added any required
cofactor for the enzymatic reaction, followed by the addition of the selected
kinase at a concentration of 20 lM. The contents were mixed gently, then the
compound under test (compound 1) dissolved in DMSO was added to the
reaction mixture in the appropriate concentration. 339-ATP (specific activity
Acknowledgments
500
mixture was incubated at room temperature for 2 h. Initial screening over 45
kinases: Compound was tested by single dose duplicate made at
concentration of 10 M. Staurosporine was used as a control compound in a
5-dose IC50 mode with 10-fold serial dilutions starting at 20 M. Reaction was
carried out at 10 ATP concentration. Testing against ROS1 kinase:
compound was tested in 10-dose IC50 mode with threefold serial
dilutions starting at 20 M. Staurosporine was used as a control compound
in a 10-dose IC50 mode with fivefold serial dilutions starting at 20 M. Reaction
was carried out at 10 M ATP concentration.
lCi/lL) was added to the mixture in order to initiate the reaction, and the
This research was supported by Korea Institute of Science and
Technology and Pioneer Research Program for converging
technology through the Korea Science and Engineering Foundation
funded by the Ministry of Education, Science and Technology
(M10711060001-08M1106-00110). We also appreciate to Dr. Sean
W. Deacon and Dr. Haiching Ma from Reaction Biology Corporation
for kinase screening.
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