U. R. Khire et al. / Bioorg. Med. Chem. Lett. 14 (2004) 783–786
Table 3. Carboxamides in the diphenyl urea class
785
References and notes
1. Dumas, J. Exp. Opin. Ther. Pat. 2001, 11, 405, and refer-
ences cited therein. Chandra Kumar, S.; Madison, V.
Exp. Opin. Emerg. Drugs 2001, 6, 303.
2. Bridges, A. Curr. Med. Chem. 1999, 6, 825.
3. Lyons, J. F.; Wilhelm, S. M.; Hibner, B.; Bollag, G.
Endocr.-Relat. Cancer 2001, 8, 219.
Compd
X
Y
R1
R2
IC50 (nM)9
4. Smith, R. A.; Barbosa, J.; Blum, C. L.; Bobko, M. A.;
Caringal, Y. V.; Dally, R.; Johnson, J. S.; Katz, M. E.;
Kennure, N.; Kingery-Wood, J.; Lee, W.; Lowinger, T. B.;
Lyons, J.; Marsh, V.; Rogers, D. H.; Swartz, S.; Walling,
T.; Wild, H. Bioorg. Med. Chem. Lett. 2001, 11, 2775.
Due to slight modifications of the biochemical assay, IC50
values obtained during the initial phase of the program
were slightly lower than those determined during the later
phase, such as described in this report. For example,
compound 1 was initially found to have IC50=0.54 mM
(n=6).
28
29
30
31
32
33
34
35
36
37
38
39
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
N
N
N
N
H
H
H
H
H
H
H
Me
H
H
H
H
Me
3-Pyridyl
4-(Me2N)-Phenyl
6-MeO-3-Pyridyl
(4-Morpholinyl)phenyl
Me
Et
130
100
410
150
210
53
460
330
500
63
Me
i-Bu
CH
CH
CH
4-Morpholinyl-(CH2)2
Me
Me2N-(CH2)2
N
N
61
100
5. Lowinger, T. B.; Riedl, B.; Dumas, J.; Smith, R. A. Curr.
Pharm. Des. 2002, 8, 99.
6. Riedl, B.; Lowinger, T. B.; Bankston, D.; Barbosa, J.;
Brittelli, D. R.; Carlson, R.; Dumas, J.; Hibner, B.;
Kadono, H.; Katz, M.: Kennure, N.; Khire, U.; Lee, W.;
Monahan, M.-K.; Natero, R.; Renick, J.; Rong, H.;
Scott, W. J.; Sibley, R. N.; Smith, R. A., Wood, J. 92nd
Meeting of the American Association for Cancer Research,
New Orleans, March 24–28, 2001; Abstract 4956.
Table 4. Carboxamides in the diphenyl urea class
7. Miller, S.; Osterhout, M.; Dumas, J.; Khire, U.; Low-
inger, T.; Riedl, B.; Scott, W.; Smith, R.; Wood, J.;
Rodriguez, M.; Wang, M. PCTInt. Appl., WO 99/32436.
Chem. Abstr. 1999, 131, 58658.
8. Riedl, B.; Dumas, J.; Khire, U.; Lowinger, T. B.; Scott,
W. J.; Smith, R. A.; Wood, J. E.; Monahan, M.-K.;
Natero, R.; Renick, J.; Sibley, R. N. PCTInt. Appl., WO
00/42012. Chem. Abstr. 2000, 133, 120157.
Compd
R1
R2
IC50 (nM)9
40
41
42
43
H
H
Me
H
Me
Et
Me
6
27
170
60
4-Morpholinyl-(CH2)2
Table 5. Aqueous solubility of selected ureas
9. In vitro Raf Kinase activity screen: Human Raf-1 (c-Raf)
and Mek were cloned, expressed and purified at Onyx
Pharmaceuticals. Briefly, Sf-9 cells were infected with
baculovirus encoding epitope-tagged c-Raf or Mek
(EYMPME; EE-tag on the C-terminus). Epitope-tagged
Raf and Mek were then purified from infected Sf-9 cell
lysates by immuno-affinity chromatography, and were stored
at ꢀ80 ꢁC in the following storage buffers. Raf kinase: 20
mM Tris pH 8.0, 1mM EDTA, 1mM DTT, 20 mM Leu-
peptin, 1% v/v NP40, 50% glycerol. Mek kinase: 25 mM Tris
pH 7.8, 10 mM NaCl, 1 mM EDTA, 1 mM DTT, 4 mM
Leupeptin, 50% glycerol. In order to assay for Raf-1 activity,
Raf and Mek were diluted together with reaction buffer (200
mM Tris pH 8.2, 100 mM NaCl and 20 mM 2-mercapto-
ethanol) to 4 and 20 mg/mL, respectively, and 20 mL of this
enzyme-substrate mixture was added to each well of a 96-well
plate. The kinase reaction was initiated by adding 25 mL of 10
mM g-[33P]ATP (sp. Act. 400 Ci/mol) for incubation at 32 ꢁC
for 25 min. Filtration onto a phosphocellulose mat was used
to harvest protein, and 1% phosphoric acid was used to wash
away unbound radio-nucleotide. Following drying by
microwave heating, the filter was enclosed in a plastic sample
bag, scintillation fluid was added, and a b-plate counter was
used to measure filter-bound radioactivity. To screen for
inhibitors, test compounds were serially diluted from 10 mM
stock solutions in DMSO, using a liquid handling robot, into
10% DMSO in water to 10 times the final desired concentra-
tions (1% final DMSO concentration). Five microliters of
these serially diluted stocks or matching DMSO containing
vehicle was added to the enzyme-substrate mixture, prior to
the addition of radiolabelled ATP. IC50 values were
calculated using a four-parameter non-linear curve-fitting
program. At least two independent IC50 determinations
Compd
Solubility, pH 2.7
(mg/mL)
Solubility, pH 7.2
(mg/mL)
38
39
25
43
26
ꢂ4
129
141
39
ꢂ4
41
ꢂ5
ꢂ5
ꢂ5
36
Conditions: (a) pH 7.2, 0.5ꢃPBS; or pH 2.7, 3 mM citrate, 1.4 mM
KCl, 68 mM NaCl; (b) agitate at rt for 3 h; (c) centrifuge and analyze
by NMR.11
In summary, novel Raf-1 kinase inhibitors from the
urea class have been prepared. The carboxamide group
of BAY 43-9006 and its analogues was shown to be a
suitable position for the introduction of water-solubi-
lizing groups. Improvements of aqueous solubilities by
up to 10-fold were realized without significant impact
on Raf-1 kinase potency.
Acknowledgements
We would like to thank our colleagues at Onyx Phar-
maceuticals for their support in this collaborative effort,
particularly Dr. John Lyons and Dr. Vivienne Marsh,
who developed the Raf-1 kinase assay. We also would
like to thank Mr. Anthony Paiva and Mr. Stuart Cole-
man for mass spectrometry support.