6356
D. J. Kopecky et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6352–6356
OH
O
OH
R1
N
R1
N
a
c, d
N
N
N
N
N
H
N
H
N
H
R1
N
R1
N
N
N
N
H
N
N
H
N
N
H
37 R1=Cl
32 R1=Cl
39
38 R1=Me
33 R1=Me
b
e
R2
NMe2
OH
R1
N
N
N
N
H
N
R1
N
N
H
N
N
N
H
N
N
H
28 R1=Cl, R2=NMe2
29 R1=Me, R2=NMe2
34
30 R1=Me, R2=NE t
2
31 R1=Me, R2=N(CH2)4
Scheme 4. Reagents and conditions: (a) OsO4, NMO, acetone/H2O, 87%; (b) i—NaIO4, acetone/H2O, ii—R2HÁHCl, Et3N, NaBH(OAc)3, 1,2-dichloroethane, 51–52% (compounds 28
and 29) or R2H, AcOH, NaBH(OAc)3, 1,2-dichloroethane, 21–25% (compounds 30 and 31); (c) p-TsCl, pyridine, CH2Cl2, 64%; (d) NaOMe, CHCl3/MeOH, 0 °C, 88%; (e) MeNH2,
EtOH, 40 °C, 27%.
5. Unpublished results.
6. The Ki assay for ACK1 has been described in detail in: Xiao, S.-H.; Farrelly, F.;
Table 4
Kinase selectivity for compounds 1a, 13, and 14 (Ki, l
M).a
Anzola, J.; Crawford, D.; Jiao, J.X; Liu J.; Ayres, M.; Li, S.; Huang, L.; Sharma R.;
Kayser F.; Wesche, H.; Young, S.W. Anal. Biochem. 2007, 267, 179. Briefly,
histidine(6) tagged truncated ACK1 (aa 117–489) were over expressed and
purified to apparent homogeneity from SF9 cells using baculovirus expression
Kinase
1a
13
14
ACK1
LCK
BTK
KDR
Tie-2
Jak3
0.020
0.013
0.078
>25
>8.3
0.074
0.003
0.00004
0.00025
1.59
1.16
0.17
0.003
0.011
0.125
>12.5
>2.5
system.
A biotinylated peptide (AIRLKEEEI-YFFFFAKKK-amide) substrate is
phosphorylated by ACK1 in the presence of various concentrations of ATP. The
phosphorylated peptide is detected with electrochemiluminescence using
phospho-tyrosine-specific mAb-labeled with ruthenium (II) tribipyridine (BV-
TagTM), and streptavidin coated paramagnetic beads. Kis were generated by
fitting the dose response data to the Morrison equation.
>12.5
a
7. ACK1 cell-based assay IC50 determination: The assay relies upon the fact that
ACK1 is heavily autophosphorylated in proliferating, adherent cells, and upon
detachment ACK1 becomes de-phosphorylated very rapidly. When attached to
plastic, ACK1 auto-phosphorylation is detectable within minutes, and reaches a
maximum within 2 h. For this experiment, 293 cells stably expressing Flag-
tagged ACK1 are detached from their culture dish with PBS/EDTA. After a PBS
wash, cells are pre-incubated in suspension with DMSO or compound. Next, the
samples are either lysed directly (control) or allowed to re-attach in medium
containing 10% FCS on tissue culture dishes for 2 h prior to lysis. The inhibition
of ACK1 auto-phosphorylation is determined by anti p-Tyr WB after Flag IP.
8. (a) Snow, R. J.; Cardozo, M. G.; Morwick, T. M.; Busacca, C. A.; Dong, Y.; Eckner,
R. J.; Jacober, S.; Jakes, S.; Kapadia, S.; Lukas, S.; Panzenbeck, M.; Peet, G. W.;
Peterson, J. D.; Prokopowicz, A. S.; Sellati, R.; Tolbert, R. M.; Tschantz, M. A.;
Moss, N. J. Med. Chem. 2002, 45, 3394; (b) Goldberg, D. R.; Butz, T.; Cardozo, M.
G.; Eckner, R. J.; Hammach, A.; Huang, J.; Jakes, S.; Kapadia, S.; Kashem, M.;
Lukas, S.; Morwick, T. M.; Penzenbeck, M.; Patel, U.; Pav, S.; Peet, G. W.;
Peterson, J. D.; Prokopowicz, A. S.; Snow, R. J.; Sellati, R.; Takahashi, H.; Tan, J.;
Tschantz, M. A.; Wang, X.-J.; Wang, Y.; Wolak, J.; Xiong, P.; Moss, N. J. Med.
Chem. 2003, 46, 1337.
Values are means of at least two experiments.
Acknowledgments
We are grateful to our colleagues Merrill Ayres for ACK1 protein
expression, and George Tonn and Monica Sweany for generating PK
data.
References and notes
1. (a) Manser, E.; Leung, T.; Salihuddin, H.; Tan, L.; Lim, L. Nature 1993, 363, 364;
(b) Manser, E.; Leung, T.; Salhihuddin, H.; Zhao, Z. S.; Lim, L. Nature 1994, 367,
40; (c) Manser, E.; Chong, C.; Zhao, Z. S.; Leung, T.; Michael, G.; Hall, C.; Lim, L. J.
Biol. Chem 1995, 270, 25070; (d) Nur, E. K. A.; Zhang, A.; Keenan, S. M.; Wang, X.
I.; Seraj, J.; Satoh, T.; Meiners, S.; Welsh, W. J. Mol. Cancer Res. 2005, 3, 297.
2. (a) Yang, W.; Lo, C. G.; Dispenza, T.; Cerione, R. A. J. Biol. Chem. 2001, 276,
17468; (b) Teo, M.; Tan, L.; Lim, L.; Manser, E. J. Biol. Chem. 2001, 276, 18392.
3. van der Horst, E. H.; Degenhardt, Y. Y.; Strelow, A.; Slavin, A.; Chinn, L.; Orf, J.;
Rong, M.; Li, S.; See, L.-H.; Nguyen, K. Q. C.; Hoey, T.; Wesche, H.; Powers, S.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15901.
9. All new compounds described herein were characterized by mass spectroscopy
and 1H NMR, and were determined to be of >95% purity by reverse-phase HPLC.
10. Monosubstituted hydrazines were synthesized by reacting hydrazine hydrate
with the appropriate alkyl halide in refluxing ethanol overnight followed by
distillation.
4. (a) Mahajan, N. P.; Whang, Y. E.; Mohler, J. L.; Earp, H. E. Cancer Res. 2005, 65,
10514; (b) Mahajan, N. P.; Liu, Y.; Majumder, S.; Warren, M. R.; Parker, C. E.;
Mohler, J. L.; Earp, H. S.; Whang, Y. E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8438.
11. For
a discussion of the relationship between physical properties with
successful drug development, see e.g.: Lipinski, C. A. J. Pharmacol. Toxicol.
Methods 2000, 44, 235.