G. Noronha et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5546–5550
5549
Table 4. In vitro evaluation of 42 in different kinases
3. Wiener, J. R.; Windham, T. C.; Estrella, V. C.; Parikh, N.
U.; Thall, P. F.; Deavers, M. T.; Bast, R. C.; Mills, G. B.;
Gallick, G. E. Gynecol. Oncol. 2003, 88, 73.
4. Ito, H.; Gardner-Thorpe, J.; Zinner, M. J.; Ashley, S. W.;
Whang, E. E. Surgery 2003, 134, 221.
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7. Yeatman, T. J. Nat. Rev. 2004, 4, 470.
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M.; Cravens, A.; McSharry, H.; Iwakura, A.; Yoon, Y.;
Himes, N.; Burstein, D.; Doukas, J.; Soll, R.; Losordo, D.;
Cheresh, D. J. Clin. Invest. 2004, 113, 885.
Kinase
IC50 lM
Kinase
IC50 lM
Src
Yes
0.006
0.001
0.013
0.021
0.023
1.32
VEGFr2
Raf1-MEK1
Abl
0.555
0.069
0.020
5.99
Lck
Lyn
Fyn
Blk
Abl (T315I)
EphB4
Raf1
0.064
0.068
0.006
0.659
2.71
EGFr
FGFr1
Tie2
0.965
0.96
0.678
PDGFrb
CSK
p70S6K
9. Susva, M.; Missbach, M.; Green, J. Trends Pharmacol.
Sci. 2000, 21, 489.
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D.; Zhang, R. L.; Chopp, M.; Cheresh, D. A. Nat. Med.
2001, 7, 222.
11. Boschelli, D. H.; Wu, B.; Sosa, A. C. B.; Durutlic, H.; Ye,
F.; Raifeld, Y.; Golas, J. M.; Boschelli, F. J. Med. Chem.
2004, 47, 6666.
12. Boschelli, D. H.; Wu, B.; Sosa, A. C. B.; Durutlic, H.;
Chen, J. J.; Wang, Y.; Golas, J. M.; Lucas, J.; Boschelli, F.
J. Med. Chem. 2005, 48, 3891.
13. Boschelli, D. H.; Wu, B.; Sosa, A. C. B.; Chen, J. J.;
Golas, J. M.; Boschelli, F. Bioorg. Med. Chem. Lett. 2005,
15, 4681.
14. Manetti, F.; Locatelli, G. A.; Maga, G.; Schenone, S.;
Modugno, M.; Forli, S.; Corelli, F.; Botta, M. J. Med.
Chem. 2006, 49, 3278.
3-aminophenyl ring. Similarly, the 2-(1-pyrrolidinyl)eth-
oxy group at the 3-position (43) and at the 4-position
(42) results in compounds with 60-fold improvement in
potency compared to the corresponding phenyl ana-
logue (15). Conformationally, restricted analogues
(48–51) showed weaker potency over the more flexible
analogues (44, 45, and 54–56). Earlier studies in the pyri-
dopyrimidinone series have shown that the water-solu-
ble group on the core does not play a significant role
in improving the potency.27 However, we have shown
in this benzotriazine series that a water-soluble group
plays a significant role in improving the potency of the
molecules.
The binding of 42 at the ATP pocket of Src kinase and
the key amino acid interactions with 42 are shown in
Figure 1. The 2,6-dimethyl phenyl group is positioned
deep inside the hydrophobic pocket. The ring nitrogen
at the 2-position is involved as a hydrogen bond accep-
tor with the NH of Met-341, while the 3-amino group is
involved as a hydrogen bond donor with the carbonyl of
Met-341 at the hinge region of the ATP binding pocket.
The pyrrolidine nitrogen interacts with Asp-348. We
have examined compound 42 for kinase selectivity
(Table 4). Compound 42 is equipotent against other
Src family members (Yes, Lck, Lyn, Fyn, and Blk),
PDGFrb and EphB4 receptor tyrosine kinases but has
greatly reduced activity against VEGFr2, EGFr, and
FGFr1. This benzotriazine series has significantly differ-
ent selectivity than the structurally similar Src focused
pyridopyrimidinone series,27 which has pronounced
activity against FGFr1 and reduced activity versus
PDGFrb.
15. Carraro, F.; Naldini, A.; Pucci, A.; Locatelli, G. A.;
Maga, G.; Schenone, S.; Bruno, O.; Ranise, A.; Bondav-
alli, F.; Brullo, C.; Fossa, P.; Menozzi, G.; Mosti, L.;
Modugno, M.; Tintori, C.; Manetti, F.; Botta, M. J. Med.
Chem. 2006, 49, 1549.
16. Dalgarno, D.; Stehle, T.; Narula, S.; Schelling, P.; van
Schravendijk, M.; Adams, S.; Andrade, L.; Keats, J.;
Ram, M.; Jin, L.; Grossman, T.; MacNeil, I.; Metcalf, C.;
Shakespeare, W.; Wang, Y.; Keenan, T.; Sundaramoorthi,
R.; Bohacek, R.; Weigele, M.; Sawyer, T. Chem. Biol.
Drug Des. 2006, 67, 46.
17. Wang, Y.; Metcalf, C.; Shakespeare, W.; Sundaramoorthi,
R.; Keenan, T.; Bohacek, R.; Van Schravendijk, M.;
Violette, S.; Narula, S.; Dalgarno, D.; Haraldson, C.;
Keats, J.; Liou, S.; Mani, U.; Pradeepan, S.; Ram, M.;
Adams, S.; Weigele, M.; Sawyer, T. Bioorg. Med. Chem.
Lett. 2003, 13, 3067.
18. The IC50 values for compounds were determined using a
luminescence-based kinase assay with recombinant Src
obtained from Invitrogen. In white, flat-bottomed, 96-well
plates (Nunc) parallel assays were run at room tempera-
ture at a final volume of 50 lL. Each well contained 40 lL
of buffer consisting of 40 mM Tris buffer, pH 7.4,
containing 50 mM MgCl2, 800 lM EGTA, 350 lM Triton
X-100, 2 mM b-mercaptoethanol, 250 lM peptide sub-
strate (PTK2; Promega), and an appropriate amount of
Src (75–25 ng/well) such that the assay was linear over
60 min. The final concentrations of TargeGen compounds
for IC50 value determinations ranged from 1000 to
0.01 lM by adding the appropriate amount of compound
in 2.5 lL DMSO; the DMSO present in each assay was
constant at 5%. The reaction was initiated by the addition
of 10 lL ATP to a final assay concentration of 3 lM.
After the reaction had proceeded for 60 min, 50 lL of
Kinase-Glo reagent (Promega) was added to terminate the
reaction. This solution was then allowed to proceed for an
additional 10 min to maximize the luminescence reaction.
Values were then measured using an Ultra 384 instrument
(Tecan) set for luminosity measurements. Two control
In summary, we have developed a novel benzotriazine
series as potent Src inhibitors. We have identified
the key structural requirements for improving the
activity in this series, whose SAR is distinct from that
of other structurally analogous Src-inhibitors. Future
studies will focus on further lead optimization of
this series, pharmacokinetic properties, and in vivo
pharmacology.
References and notes
1. Trevino, J. G.; Summy, J. M.; Gallick, G. E. Mini. Rev.
Med. Chem. 2006, 6, 109.
2. Talamonti, M. S.; Roh, M. S.; Curley, S. A.; Gallick, G.
E. J. Clin. Invest. 1993, 91, 53.