R. K. Kunz et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5115–5117
5117
formed between compound 19 and the KDR mutant is a 2.9-Å
hydrogen bond between the benzisoxazole nitrogen and the back-
bone amide of Asp810. Although the potential exists for a hydro-
gen bond between Glu-640 and the arylamino-benzisoxazole
hydrogen, it is not observed in the KDR mutant co-crystal struc-
ture. The equivalent interaction is present in the c-Kit/Gleevec
structure (with the amide hydrogen of Gleevec) and this discrep-
ancy could be a result of subtle structural differences between
the KDR mutant and wild-type c-Kit.
IC50 (μM)
Kinase
N
cKit
p38
KDR
Tie-2
Src
Lck
cFMS
PDGF-α
0.003
0.339
0.353
> 5.00
0.117
0.075
0.071
0.004
O
O
Cl
N
N
H
19
N
N
H
CF3
In summary, a new class of c-Kit inhibitors has been described,
which exhibits superior in vitro potencies compared to Gleevec.
Compound 19, exhibiting the greatest selectivity against p38,
was nearly 20-fold more potent against c-Kit as compared to Glee-
vec. Improvement in off-target selectivity against p38 was
achieved by modification of the arylamino group.
Figure 2. Kinase selectivity of benzisoxazole 19.
References and notes
1. Nocka, K.; Majumder, S.; Chabot, B.; Ray, P.; Cervone, M.; Bernstein, A.; Besmer,
P. Gene Dev.. 1989, 3, 816.
2. Heinrich, M. C.; Blanke, C. D.; Druker, B. J.; Corless, C. L. J. Clin. Oncol. 2002, 20,
1692.
3. Lennartsson, J.; Jelacic, T.; Linnekin, D.; Shivakrupa, R. Stem Cells 2005, 23, 16.
4. Eklund, K. K. Immunol. Rev. 2007, 217, 38.
5. Marshall, J. Nat. Rev. 2004, 4, 787.
6. Capdeville, R.; Buchdunger, E.; Zimmerman, J.; Matter, A. Nat. Rev. Drug Discov.
2002, 1, 493.
7. c-Kit intrinsic activity was measured by c-Kit-homogeneous time resolved
fluorescent assay.
8. Reported activity of Gleevec against c-Kit and p38 was measured internally.
9. Compound 1 was dosed intravenously at 5 mg/kg in rats. After a period of 1, 4,
and 8 h, the plasma, bile, and urine were sampled and analyzed for presence of
metabolites by LC–MS/MS.
10. Zhu, Z.; Espenson, J. H. J. Org. Chem. 1995, 60, 1326.
11. For in vivo validation of mast-cell activation using structurally distinct
chemotypes to that described here, see: (a) Hu, E.; Tasker, A.; White, R. D.;
Kunz, R. K.; Human, J.; Chen, N.; Bürli, R.; Hungate, R.; Novak, P.; Itano, A.;
Zhang, X.; Yu, V.; Nguyen, Y.; Tudor, Y.; Plant, M.; Flynn, S.; Xu, Y.; Meagher, K.
L.; Whittington, D. A.; Ng, G. Y. J. Med. Chem. 2008, 51, 3065; (b) Chen, N.; Bürli,
R. W.; Neira, S.; Hungate, R.; Zhang, D.; Yu, V.; Nguyen, Y.; Tudor, Y.; Plant, M.;
Flynn, S.; Meagher, K. L.; Lee, M. R.; Zhang, X.; Itano, A.; Schrag, M.; Xu, Y.; Ng,
G. Y.; Hu, E. Bioorg. Med. Chem. Lett. 2008, 18, 4137.
Figure 3. Co-crystal structure of compound 19 and V916T KDR mutant enzyme.
Oxygen atoms, red; nitrogen atoms, blue; chlorine atoms, orange; fluorine atoms,
magenta; sulfur, yellow. The KDR mutant residues are labeled in black, and the
corresponding wild-type c-Kit residues are shown in blue.
To gain a better understanding of the binding mode of com-
pound 19, a co-crystal structure was obtained with a KDR mutant
protein where the gatekeeper residue was mutated from valine to
threonine to mimic the active site of c-Kit. The 2.9-Å resolution
structure is shown below in Fig. 3.17 Consistent with the Gleevec/
c-Kit co-crystal structure,18,19 benzisoxazole 19 binds to the DFG-
out form of the enzyme. The gatekeeper threonine is engaged in
a 3.2 Å hydrogen bonding interaction with the amide hydrogen,
similar to the Gleevec aminopyrimidine–Thr-670 hydrogen bond
in c-Kit. The pyrimidine ring forms a hydrogen bond with the back-
bone amide adjacent to the Cys-673 residue in the linker region,
which mirrors the hydrogen bond formed between this residue
in c-Kit and the pyridine nitrogen of Gleevec. The third contact
12. An in vitro metabolite identification study of compound 23 was conducted in
rat hepatocytes at 20
lM at 37 °C, which produced no evidence of
benzisoxazole ring-opening.
13. Angell, R. M.; Baldwin, I. R.; Bamborough, P.; Deboeck, N. M.; Longstaff, T.;
Swanson, S. WO2004010995.
14. Unoptimized yields.
15. Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science
2002, 298, 1912.
16. Bunchdunger, E.; Cioffi, C. L.; Law, N.; Stover, D.; Ohno-Jones, S.; Druker, B. J.;
Lydon, N. B. J. Pharm. Exp. Ther. 2000, 295, 139.
17. PDB deposition code number for the co-crystal structure is 3DTW.
18. Mol, C. D.; Lim, K. B.; Vandana Sridhar, H.-Z.; Chien, E. Y. T.; Sang, B.-C.;
Nowakowski, J.; Kassel, D. B.; Cronin, C. N.; McRee, D. E. J. Biol. Chem. 2003, 278,
31461.
19. Mol, C. D.; Dougan, D. R.; Schneider, T. R.; Skene, R. J.; Kraus, M. L.; Schiebe, D.
N.; Snell, G. P.; Zou, H.; Sang, B.-C.; Wilson, K. P. J. Biol. Chem. 2004, 279, 31655.