Design and synthesis of 6,6-fused heterocyclic amides as raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.12.112

Compounds belonging to several scaffolds-quinazolines, quinolines and quinoxalines-were designed and synthesized as Raf kinase inhibitors. Scaffolds were assessed for in vitro BrafV600E inhibition, and overall kinase selectivity. Pharmacokinetic parameters for one of the scaffolds were also determined.

Full text of DOI:10.1016/j.bmcl.2011.12.112

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1678–1681
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 6,6-fused heterocyclic amides as raf kinase inhibitors
⇑
Savithri Ramurthy , Abran Costales, Johanna M. Jansen, Barry Levine, Paul A. Renhowe,
Cynthia M. Shafer, Sharadha Subramanian
Global Discovery Chemistry/Oncology & Exploratory Chemistry, Novartis Institutes for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds belonging to several scaffolds—quinazolines, quinolines and quinoxalines—were designed
and synthesized as Raf kinase inhibitors. Scaffolds were assessed for in vitro BrafV600E inhibition, and
overall kinase selectivity. Pharmacokinetic parameters for one of the scaffolds were also determined.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 5 October 2011
Revised 22 December 2011
Accepted 23 December 2011
Available online 30 December 2011
Keywords:
Raf kinase inhibitors
Potency
Selectivity
Quinazolines
The Ras-mitogen activated protein kinase (MAPK) signaling
pathway was the first signaling pathway elucidated from the cell
membrane to the nucleus.1 The MAPK signaling pathway consists
of the Ras/Raf/MEK/ERK signal transduction cascade which is a
vital mediator of a number of cellular activities including growth,
proliferation, survival and other aspects of cellular behavior that
can contribute to the transformed phenotype, making it an attrac-
tive pathway to target in several cancer types. The three Raf iso-
forms (Raf-1 or c-Raf, A-Raf and B-Raf) are all able to interact
with Ras and activate the MAP kinase pathway.2–5
efficacy in patients expressing the BrafV600E mutation suggesting
its mechanism of action is through inhibition of VEGFR rather than
RAF.15,16 Recently PLX-403217 has been launched as a Raf inhibitor.
However, several other RAF inhibitors (e.g. GSK2118436)18 are in
clinical trials and have shown evidence of clinical benefit. Our
group has previously disclosed the benzimidazole amide series
containing orally available and potent Raf inhibitors.19,20 In partic-
ular, the 3-t-butylphenylaminobenzimidazole amide 1 (Fig. 1)
potently inhibited BrafV600E and the phosphorylation of the down-
stream target ERK in the SKMEL-28 cell line with an EC50 of 0.3 lM.
Inhibition of the Raf/MEK/ERK pathway at the level of Raf
kinases is expected to be effective against tumors driven by this
pathway. It has been shown that the B-Raf mutation V600E in skin
nevi is a critical step in the initiation of melanocytic neoplasia.6
Furthermore, activating mutations in the kinase domain of B-Raf
occur in roughly 66% of malignant melanomas, 40–70% of papillary
thyroid carcinomas, 12% of colon carcinomas and 14% of liver can-
cers.5,7–9 The many effects of Raf kinases on cancer cell growth and
survival, together with the high prevalence of mutation in mela-
noma, for which there is no good treatment, make Raf an attractive
target for anticancer therapy.
To further explore the structure activity relationship of the benz-
imidazole core for inhibition of BrafV600E, the quinazolines, quino-
lines and quinoxaline amide series (Fig. 1) were synthesized21 and
compared to the benzimidazole amide series. In addition to the
O
O
N
N
N
H
HN
R
N
1 R = 3-tertbutylphenyl
The first reported RAF inhibitor, BAY43-9006 (Sorafenib),10–14
while effective in renal cell carcinoma (RCC), has shown a lack of
O
Z
X
O
Y
N
H
N
HN
R
Abbreviations: MAPK, Ras-mitogen activated protein kinase; VEGFR, vascular
endothelial growth factor receptor; CSFR1, colony stimulating factor-1 receptor;
RTK’s, receptor tyrosine kinases; STK’s, serine threonine kinases; TK, tyrosine
kinases; KDR, kinase domain receptor.
Quinazoline: X = N; Y = N; Z = CH
Quinoline: X = N; Y = Z = CH
Quinoxaline: X = N; Y = CH; Z = N
⇑
Corresponding author.
E-mail address: savithri.ramurthy@novartis.com (S. Ramurthy).
Figure 1. 6,6-Fused heterocyclic pyridyl amides designed as analogues of the
benzimidazole pyridyl amide.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.112

S. Ramurthy et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1678–1681
1679
in vitro BrafV600E SAR, pharmacokinetic profiles for a representative
example will be presented.
Z
X
OH
Y
Z
X
O
For all the scaffolds, acquiring the key intermediate 2-chloro-6-
methoxyheterocycles (2–4) was essential. A survey of literature
procedures for the preparation of appropriately substituted quina-
zolines rapidly led us to the conclusion that it would be necessary to
develop alternate synthetic routes for both the intermediates and
final products (Scheme 1). Methylation of 5-hydroxy-2-nitrobenz-
aldehyde 5 provided the corresponding methyl ether. Protection
of the aldehyde with glycol in the presence of tosic acid afforded
the dioxalane, which upon reduction yielded 6 in 60–70% yield.
Treatment with ethylchloroformate followed by deprotection affor-
ded the precursor 7. Ring closure was effected using condensed
ammonia under forcing conditions, followed by chlorination to
afford the key intermediate 2-chloro-6-methoxy quinazoline (2).
2-Chloro-6-methoxyquinoline (3) was obtained from commercially
available 8 by selective chlorination at the 2-position of the hetero-
cyclic ring followed by methylation. Synthesis of 2-chloro-6-meth-
oxy quinoxaline (4) required only chlorination of 9. Introduction of
anilines was effected under SNAr conditions for all the above men-
tioned scaffolds (Scheme 2). O-Arylation was then performed using
KHMDS and chloro-pyridylacetamide22 (13) in DMF as solvent.
Table 1 highlights the structure–activity relationship of the ani-
line and amide functionalities in the quinazoline series. As observed
in the benzimidazole series19 ortho substituents (17–19) on the
a,b
Y
N
H
Cl
2-4
10-12
O
Cl
N
H
O
N
Z
X
O
13
Y
N
H
c
N
N
H
14-16
Scheme 2. Reagents and conditions: (a) 3-t-Butylaniline, EtOH, 80 °C; (b) 48% HBr,
140 °C, microwave, 6 min; (c) KHMDS, K2CO3, DMF, microwave, 170 °C, 6 min.
Table 1
Structure–activity relationship of the quinazoline series
O
R'
O
R"
N
N
H
N
N
H
N
Compound R0
R00
BrafV600E IC50 M)23
(l
aniline led to significant loss of affinity >1.0
lM, offering no advan-
1
—
H
2-Br
2-Et
2-OPh
3-Cl
3-Br
3-t-Bu
3-CF3
3-OCF3
3-i-Pr
4-F
—
0.12
2.2
24.0
1.3
tage over the unsubstituted phenyl analog (1a) against BrafV600E
.
1a
17
18
19
20
21
14
22
23
24
25
26
27
28
29
30
31
32
33
34
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Meta-and para substituents (14, 19–24) were in general similarly
potent compared to meta- and para-substituents in the benzimid-
azole series (e.g., 1). In particular, the 3-OCF3 substituted phenyl
23 had the best affinity in the meta-substituted sub-series. For sub-
stituents in the para-position of the phenyl ring, affinity appeared
to improve as the hydrophobic nature of the substituents increased
(25–30). However, larger groups in the para-position of the phenyl
ring such as phenoxy (31) and 3-pyridyl (32) showed a decrease in
affinity indicating size/shape restrictions in this area of the binding
pocket. The similar binding affinity of phenoxy 31 and O–Me 29
analogs suggests an intricate balance between hydrophobicity
and size/shape. Interestingly, appendage of solubilizing groups on
the amide led to degradation of potency which was counter to the
SAR seen in the benzimidazole series.
5.3
0.66
0.18
0.045
0.64
0.07
0.15
0.60
0.31
0.067
0.001
0.39
0.08
0.60
25.0
8.6
4-Br
4-CF3
4-i-Pr
4-OMe
4-OCF3
4-OPh
4-(3)-Pyridyl Me
4-Br
4-Br
2-Hydroxy-ethylamino
Piperidinyl-ethyl
1.3
O
O
a, b, c
OH
O
O
O
H
Table 2
Comparison of the 6,6-heterocyclic series
NO2
H2N
5
7
6
2
O
O
O
d, e
f, g
N
Z
Y
O
N
N
H
Cl
N
COOEtHN
N
H
X
Compound
X
Y
Z
BrafV600E IC50
(lM)
1
16
15
14
—
N
N
N
—
—
N
CH
CH
0.12
OH
O
O
h, i
CH
CH
N
0.025
0.026
0.045
HO
HO
N
Cl
N
8
9
3
N
N
N
N
O
h
Table 2 compares the three scaffolds to the benzimidazole 1.
The biochemical assay data indicated quinazoline 14 to be equipo-
tent with 1 whilst quinoline 15 and quinoxazoline 16 are roughly
sixfold more potent than benzimidazole 1.
In order to understand the binding mode for the quinazoline
series, 14 was docked in the active site of the public domain crystal
Cl
4
Scheme 1. Synthesis of intermediates 2–4. Reagents and conditions: (a) K2CO3,
MeI, DMF; (b) Glycol, tosic acid; (c) PtO2, H2, EtOH; (d) EtOCOCl, THF; (e) concd HCl;
(f) NH3 at 0–130 °C; (g) POCl3, 95–100 °C, 4 h; (h) POCl3, THF, 65 °C; (i) K2CO3, MeI,
DMF.

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S. Ramurthy et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1678–1681
Figure 2. Binding site model for compound 14 (in purple), derived by docking into the crystal structure of B-Raf (PDB accession code 1UWH). The left picture shows a cartoon
representation of the kinase with selected residues in stick model (Glu501, Cys532, Phe593 and Asp594) and the co-crystallized Sorafenib in green. The right picture zooms in
and displays the extra radius binding surface around 14, colored by surface properties: red = hydrogen-bond donor, blue = hydrogen-bond acceptor, green = hydrophobic
surface, white = aromatic surface. Ortho positions in optimal van-der-Waals contact with the protein are indicated with arrows.
structure published for B-Raf (PDB accession code 1UWH).24 Figure
Supplementary data
2 (left) shows the overlap of the docking model with the co-crystal-
lized conformation of Sorafenib. The model suggests a very similar
binding mode when comparing 14 with Sorafenib.25 Specific inter-
actions between 14 and the B-Raf protein include hydrogen bonds
to (1) the backbone NH and C@O of Cys532 in the hinge region
through the pyridyl-amide moiety, (2) the backbone NH of
Asp594 through the quinazoline nitrogen, and (3) the side chain
COO– of Glu501 through the aniline NH. This model uses the
‘DFG-out’ conformation of the protein where the substituted ani-
line displaces Phe593, causing it to swing out and interact with
the aromatic systems in the hinge region and the selectivity pock-
et. The extra radius surface model in Figure 2 (right) shows that the
ortho carbons of the aniline are in optimal van-der-Waals contact
with the binding pocket, providing a rationale for the drop in affin-
ity with substitution in that position. The model indicates there is
space available for meta- and para-substitution, in accordance with
the observed SAR.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.112.
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While all three series yielded potent BrafV600E inhibitors in the
biochemical assay, none of these inhibited phosphorylation of
ERK in cells (SKMEL-28, RafV600E EC50 >10
lM). This observation
was consistent with the majority of the compounds from our origi-
nal benzimidazole series. This discrepancy between biochemical
and cellular potency could be due to permeability and solubility
limitations. Compounds from all series were tested in the Caco2
assay and shown to have poor permeability (Papp A–B <1 ꢀ 10ꢁ6
cm/s). Solubility at pH7 was measured and shown to be <1 lM.
In addition, it should be noted that in the biochemical assay, the
purified kinase domain of BrafV600E is not representative of the full
length protein in cells where BrafV600E exists as a complex with
chaperones, cytoskeleton, phosphatases and kinases.23
The kinase profiles of three representative compounds, 14–16
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AGC kinases. The compounds exhibited a fairly narrow kinase pro-
file, inhibiting four RTKs (CSFR1, Flt3, KDR and cABL) out of 50 ki-
11. Khire, U. R.; Bankston, D.; Barbosa, J.; Brittelli, D. R.; Caringal, Y.; Carlson, R.;
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N.; Kingery-Wood, J.; Lee, W.; Liu, X.-G.; Lowinger, T. B.; McAlexander, I.;
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A.; Wolanin, D. Bioorg. Med. Chem. Lett. 2004, 14, 783.
nases with an IC50 <1
l
M. The pharmacokinetic properties for one
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R.; Lee, W.; Smith, R. A.; Blum, C. L. PCT Int. Appl.: WO 9852559, 1998, p 53.
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of the prototypes in the quinazoline series, 24, were determined.
Following a single 20-mg/kg oral administration to female mice
in 15% captisol, 24 exhibited a clearance of 53.1 mL/min/kg, a vol-
ume of distribution of 4717 mL/kg, a half-life of 468 min, and an
oral bioavailability of 35%.
In conclusion, we developed three series of biochemically potent
BrafV600E inhibitors. Lack of cellular potency of these compounds
discouraged us from continuing further work on these series.
17. Flaherty, K. T.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthu, G. A.; Sosman, J.;
O’Dwyer, P. J.; Lee, R. J.; Grippo, J. F.; Nolop, K.; Chapman, P. B. N. Eng. J. Med.
2010, 363, 809.
18. Kefford, R.; Arkenau, H.; Brown, M. P.; Millward, M.; Infante, J. R.; Long, G. V.;
Ouellet, D.; Curtis, M.; Lebowitz, P. F.; Falchook, G. S. Annual ASCO meeting,
Chicago, IL, 2010, abstract 8503.
Acknowledgments
The authors wish to acknowledge Payman Amiri for enzyme
and cell based assays and Keshi Wang for formulation and bioanal-
ysis of these compounds.

S. Ramurthy et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1678–1681
1681
19. Ramurthy, S.; Subramanian, S.; Aikawa, M.; Payman, A.; Costales, A.; Dove, J.;
Fong, S.; Jansen, J. M.; Levine, B.; Ma, S.; McBride, C. M.; Michaelian, J.; Pick, T.;
Poon, D. J.; Girish, S.; Shafer, C. M.; Stuart, D.; Sung, L.; Renhowe, P. A. J. Med.
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20. Ramurthy, S.; Aikawa, M.; Amiri, P.; Costales, A.; Hashash, A.; Jansen, J. M.;
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24. Ligands were docked in the protein model from the 1UWH structure
downloaded from the PDB. The docking program Glide SP was used through
Maestro 8.0 (by Schrödinger, LLC). Selected poses for each of the ligands were
further minimized with the Embrace routine in Maestro, to allow optimization
of the ligands and selected residues. The forcefield OPLS_2005 was used with
the solvent model for0 water and the LBFGS minimizer in energy difference
mode. Residues in a 6 ÅA shell were allowed to move except for their C-
a atoms
21. Ramurthy, S.; Renhowe, P. A.; Subramanian, S. U.S. Pat. Appl. Publ. 2005, p 42.
US 20050085482.
22. Synthetic methodology and characterization data for 2 and 3 are included in
the Supplementary data.
which were restrained in a separate shell with a force constant of 200. That
0
0
same force constant was applied to an additional 4 ÅA shell outside the 6 ÅA shell.
25. Wan, P. T. C.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.;
Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R. Cell 2004, 116,
855.
23. Information on kinetic assays included in the Supplementary data.