Omega-carboxypyridyl substituted ureas as Raf kinase inhibitors: SAR of the amide substituent

Article abstract of DOI:10.1016/j.bmcl.2003.11.041

Bis-aryl ureas have been disclosed previously as a potent class of Raf kinase inhibitors. Modifications in the amide portion led to an improvement in aqueous solubility, an important characteristic for an oral drug. Based on this finding, we hypothesize t

Full text of DOI:10.1016/j.bmcl.2003.11.041

Bioorganic & Medicinal Chemistry Letters 14 (2004) 783–786
Omega-carboxypyridyl substituted ureas as Raf kinase inhibitors:
SAR of the amide substituent
Uday R. Khire,a,* Donald Bankston,a James Barbosa,b David R. Brittelli,a
Yolanda Caringal,a Robert Carlson,b Jacques Dumas,a Todd Gane,a Sarah L. Heald,a
Barbara Hibner,b Jeffrey S. Johnson,a Michael E. Katz,b Nancy Kennure,b
Jill Kingery-Wood,a Wendy Lee,a Xiao-Gao Liu,a Timothy B. Lowinger,a
Ian McAlexander,a Mary-Katherine Monahan,a Reina Natero,a Joel Renick,a
Bernd Riedl,a Hong Rong,b Robert N. Sibley,a Roger A. Smitha and Donald Wolanina
aDepartment of Chemistry Research, Bayer Research Center, 400 Morgan Lane, West Haven, CT 06516, USA
bDepartment of Cancer Research, Bayer Research Center, 400 Morgan Lane, West Haven, CT 06516, USA
Received 15 September 2003; accepted 6 November 2003
Abstract—Bis-aryl ureas have been disclosed previously as a potent class of Raf kinase inhibitors. Modifications in the amide
portion led to an improvement in aqueous solubility, an important characteristic for an oral drug. Based on this finding, we
hypothesize that this portion of the molecule is directed towards the solvent in Raf-1.
# 2003 Elsevier Ltd. All rights reserved.
Most of the known small molecule protein kinase inhi-
bitors bind to a highly conserved ATP-binding pocket
and in general are flat, aromatic molecules that mimic
the adenine portion of ATP.1 Therefore, relatively
poor drug-like properties such as high logP and low
aqueous solubility are major challenges. For example,
the introduction of water-solubilizing groups to the 6-
and 7-positions of the quinazoline EGFR inhibitors
was an important step towards the optimization of their
physico-chemical properties.2
Introduction of an N-methyl carboxamide at the meta
position of the distal phenyl ring (2) increased activity
by almost 10-fold.5 Replacement of the distal phenyl
ring with a 4-pyridyl ring (3) also significantly improved
the potency. Combination of these two structural fea-
tures leads to highly potent ureas, such as 4. An addi-
tional optimization effort where the isoxazole ring of 4
is replaced by other five-membered heterocycles as well
as appropriately substituted phenyl groups led to a
clinical candidate BAY 43-9006 5.3,5
We have previously reported our focus on Raf-1 kinase
as a validated target for the treatment of cancer.3 The
lead compound 1 [N-(5-tert-butyl-3-isoxazolyl)-N0-(4-
phenoxyphenyl)urea, Fig. 1] was identified by screening
of a combinatorial chemistry library, and exhibits an
IC50 value of 1100 nM against recombinant human Raf-
1 kinase.4 Optimization of 1 led to a series of potent,
orally active Raf-1 kinase inhibitors,5ꢀ8 and culminated
in the identification of a clinical candidate BAY 43-9006
(5).3,5
We sought to identify a site for the introduction of
water solubilizing groups without affecting the Raf-1
inhibitory potency. In this article, we wish to report our
study of the structure–activity relationships of the
amide portion of the molecule, directed towards opti-
mizing aqueous solubility.10
Our group has previously disclosed synthetic routes to
ureas similar to those described in this report, such as
1–5.7,8 Furthermore, the general preparation of ureas
26–27 and 37–39 is depicted in Figure 2. Reaction of
5-hydroxynicotinic acid methyl ester with 1-fluoro-4-
nitrobenzene, followed by reduction of the nitro group,
urea formation, hydrolysis of the ester and subsequent
amide formation provides access to amide analogues.
* Corresponding author. Tel.: +1-203-812-5203; fax: +1-203-812-
6182; e-mail: uday.khire.b@bayer.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.041

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U. R. Khire et al. / Bioorg. Med. Chem. Lett. 14 (2004) 783–786
Table 1. Substitution of the carboxamide group
Compd
R1
R2
Raf-1 kinase IC50 (nM)9
2
6
7
8
9
10
11
H
H
H
Me
H
H
Me
Et
n-Pr
Me
CH2Ph
Ph
3-Pyridyl
120
130
140
5800
460
370
68
H
Table 2. Carboxamides related to diphenyl urea BAY 43-9006 (4)
Figure 1. Urea-based Raf-1 kinase inhibitors.
Compd
X
Y
R1
R2
IC50 (nM)9
12
13
14
15
16
17
18
19
20
21
5
22
23
24
25
26
27
CH CH
CH CH
CH CH
CH CH
CH CH
CH CH
CH CH
CH CH
CH CH
CH CH
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
Me
4-Morpholinyl-(CH2)2
1-Piperidinyl-(CH2)2
2-Et-pyrrolidin-1-yl-(CH2)
3-Pyridyl
4-(Me2N)-phenyl
PhNH-(CH2)2
MeO-(CH2)2
4-MeO-3-pyridyl
(4-Morpholinyl)phenyl
Me
130
70
82
270
44
230
160
110
130
160
12
CH
CH
CH
CH
CH
N
N
N
N
N
N
Et
Me
i-Pr
26
300
2300
73
140
50
Figure 2. Preparation of ureas 26–27 and 37–39.
4-Morpholinyl-(CH2)2
4-Morpholinyl-(CH2)2
Me
CH
CH
Initial evaluation of the effect of amide substituents larger
than methyl revealed no improvement, but demonstrated
the existence of an extra space in this binding region
of Raf-1 kinase (Table 1). For example, replacement of the
N-methyl group of 2 with larger alkyl groups such as ethyl
or n-propyl led to similar Raf-1 potencies9 (entries 6 and
7). In contrast, modification of the primary amide group to
a secondary amide as in urea 8 was detrimental to the
potency, suggesting the importance of a hydrogen-bond
donor in this part of the molecule. Increasing the size of the
N-substituent to benzyl or phenyl led to a less dramatic
loss in potency (Table 1, entries 9 and 10). Similar trends
were observed with urea derivatives where the isoxazole
heterocycle was replaced by a substituted phenyl group
(Tables 2–4). Interestingly, replacement of the N-phenyl
group in 10 with an N-3-pyridyl group 11 improved
potency, indicating that polar amides could be beneficial
to the Raf-1 potency.
N
Introduction of a pyridine in place of the distal phenyl
ring in such analogues provided compounds with
retained high potency (e.g., 25 and 26 vs 13, Table 2);
however, additive effects seen previously in the case of 3
versus 1, 4 versus 2, and 33 versus 28 were not realized.
Interestingly, N-alkylnicotinamide analogues such as
26–27 and 37–39 (Tables 2 and 3) showed Raf-1 poten-
cies similar to those of the isomeric N-alkyl 2-pyr-
idinecarboxamides.
Selected examples were evaluated in an equilibrium-
based aqueous solubility assay, according to a high-
throughput Nuclear Magnetic Resonance (NMR) flow
technology assay protocol developed in-house.11 The
effect of N-methyl amide substitution with polar amides
on aqueous solubility is shown in Table 5. Compound
39 is more than 10-fold more soluble than the corre-
sponding N-methyl amide analogue 38 at physiological
pH. As expected from these basic analogues, very good
aqueous solubility was observed at lower pH (Table 5),
which could potentially provide improved absorption
through the gastrointestinal membrane.11
Based on this observation, various polar carboxamides
were synthesized; the results are shown in Tables 2–4. In
most of these examples, introduction of water-solubiliz-
ing groups provided compounds with retained Raf-1
inhibitor potency, and large groups were well tolerated.
We hypothesize that this portion of the molecule is
directed towards the solvent when the compound is
bound to Raf-1 kinase.

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.

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U. R. Khire et al. / Bioorg. Med. Chem. Lett. 14 (2004) 783–786
were performed on each compound, and the mean value is
reported. In all cases, standard deviations were less than
50% of the mean IC50 value.
cussion on importance of the aqueous solubility as a drug-
like property: Lipinski, C. A.; Lombardo, F.; Dominy, B.
W.; Feeney, P. J. Adv. Drug Deliv. Rev. 2001, 46, 3, and
references cited therein. Huuskonen, J. Combin. Chem.,
High Throughput Screen. 2001, 4, 311.
10. For water solubilizing groups we specifically focused on
substituents with heteroatoms that could add hydro-
philicity or provide a site for salt formation. For a dis-
11. Heald, S. H.; Blake, P. C. Am. Pharm. Rev. In press.