M. L. Curtin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4505–4509
4509
15. The cellular IC50 values represent inhibition of KDR
phosphorylation in NIH3T3 cells stably transfected with
full length human KDR. Cells were stimulated with
human recombinant VEGF (50 ng/mL) for 10 min in the
presence of compound, after which the cells were lysed and
phosphorylated KDR determined by ELISA. KDR
ELISA consisted of an anti-KDR (R&D Systems) capture
antibody and anti-phosphotyrosine (4G10, UBI) horse-
radish peroxidase-conjugated detection antibody. Inhibi-
tion of VEGF induced phosphorylation was determined
relative to a nonphosphorylated control, and IC50 values
calculated by nonlinear regression analysis of the concen-
tration–response curve. Each IC50 determination was
preformed with five concentrations and each assay point
determined in duplicate.
In summary, a series of potent KDR isoindolinone
inhibitors has been identified. Optimal substitution
afforded analogs with significant enzymatic and cellular
potency as well as selectivity against several non-VEGF
tyrosine kinases. A 3D kinase overlay model suggested
that the predominant isoindolinone/KDR interactions
include hydrogen-bonds between the inhibitor lactam
moiety and the protein backbone as well as the urea
functionality and a glutamate residue.
References and notes
1. Hunter, T. Cell 2000, 100, 113–127.
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5. Dvorak, H. F. J. Clin. Oncol. 2002, 20, 4368–4380.
6. For a recent review see: Manley, P. W.; Bold, G.; Bruggen,
J.; Fendrich, G.; Furet, P.; Mestan, J.; Schnell, C.; Stolz,
B.; Meyer, T.; Meyhack, B.; Stark, W.; Strauss, A.; Wood,
J. Biochim. Biophys. Acta 2004, 1697, 17–27.
7. Burchat, A. F.; Calderwood, D. J.; Friedman, M. M.;
Hirst, G. C.; Li, B.; Rafferty, P.; Ritter, K.; Skinner, B. S.
Bioorg. Med. Chem. Lett. 2002, 12, 1687–1690.
8. KDR IC50 values were determined by an assay of
nonphosphorylated KDR active kinase domain (soluble
C-terminal domain with a N-terminal 6-His sequence to
facilitate purification, cloned, and expressed in baculovi-
rus) using an ATP concentration of 1 mM. A biotinylated
peptide substrate containing a single tyrosine was used in a
microtiter plate assay using HTRFâ methodology as
described in Kolb, A. J.; Kaplita, P. V.; Hayes, D. J.; Park,
Y.-W.; Pernell, C.; Major, J. S.; Mathias, G. Drug Discov.
Today 1998, 3, 333–342. Inhibition constants are the mean
of two determinations performed with seven
concentrations of the test compounds.
9. Dumas, J. Curr. Opin. Drug Discov. Dev. 2002, 5, 718–727.
10. Toledo, L. M.; Lydon, N. B.; Elbaum, D. Curr. Med.
Chem. 1999, 6, 775–805.
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Morimoto, M.; Tomita, F. Biochem. Biophys. Res. Com-
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12. Gingrich, D. E.; Reddy, D. R.; Iqbal, M. A.; Singh, J.;
Aimone, L. D.; Angeles, T. S.; Albom, M.; Yang, S.; Ator,
M. A.; Meyer, S. L.; Robinson, C.; Ruggeri, B. A.;
Dionne, C. A.; Vaught, J. L.; Mallamo, J. P.; Hudkins, R.
L. J. Med. Chem. 2003, 46, 5375–5388.
16. The model was constructed by using the published crystal
structure of unliganded KDR enzyme (PDBentry 1VR2)
in: McTigue, M. A.; Wickersham, J. A.; Pinko, C.;
Showalter, R. E.; Parast, C. V.; Tempczyk-Russell, A.;
Gehring, M. R.; Mroczkowski, B.; Kan, C.-C.; Villafr-
anca, J. E.; Appelt, K. Structure 1999, 7, 319–330. This
structure corresponds to KDR with a doubly-phosphor-
ylated activation loop in a closed conformation, but with
much of the loop disordered and presumed to be
conformationally mobile. The modeling studies reported
here focused on regions of the protein that were crystal-
lographically well defined. No modifications to the protein
structure were made to account for the flexible activation
loop.
17. The crystallographically observed orientation of 2 in
CDK2 kinase (PDBentry 1AQ1) was used as a source
of the staurosporine orientation.
18. A 3D overlay of kinase crystal structures of KDR, CDK2,
and p38 MAP kinases was created by selecting 12 spatially
equivalent residues from each of the three active sites. Six
of these residues were derived from the hinge region and
six were from the other regions of the active site. A least-
squares superposition of the alpha carbons from these 12
residues provided a reasonable active site overlay. The 12
residues for each enzyme are listed as follows: KDR (PDB
1VR2) Ala866, Lys868, Val914, Ile915, Val916, Glu917,
Phe918, Cys919, Asn1033, Leu1035, Cys1045, and
Asp1046; CDK2 (PDB1AQ1) Ala31, Lys33, Leu78,
Val79, Phe80, Glu81, Phe82, Leu83, Asn132, Leu134,
Ala144, and Asp145; p38 MAP (PDB1KV1) Ala51,
Lys53, Leu104, Val105, Thr106, His107, Leu108, Met109,
Asn155, Ala157, Leu167, Asp168.
19. For the crystal structure of urea 27 bound to p38 MAP
kinase (PDBentry 1KV1), see: Pargellis, C.; Tong, L.;
Churchill, L.; Cirillo, P. F.; Gilmore, T.; Graham, A. G.;
Grob, P. M.; Hickey, E. R.; Moss, N.; Pav, S.; Regan, J.
Nature Struct. Biol. 2002, 9, 268–272.
13. The given IC50 was taken from Ref. 12 and was run using
an ATP concentration of 16 lM.
14. For an earlier synthetic report of related isoindolinones,
see: Rupert, K. C.; Dodd, J. H.; Henry, J. R. Heterocycles
1997, 45, 2217–2221.
Cl
O
N
N
N
H
N
H
27