Design and synthesis of orally bioavailable benzimidazoles as raf kinase inhibitors

Article abstract of DOI:10.1021/jm801050k

A series of arylaminobenzimidazoles was designed and synthesized as Raf kinase inhibitors. Exploration of the structure-activity relationship resulted in compounds that are potent in vitro and show desirable in vivo properties.

Full text of DOI:10.1021/jm801050k

J. Med. Chem. 2008, 51, 7049–7052
7049
Design and Synthesis of Orally Bioavailable
Benzimidazoles as Raf Kinase Inhibitors
Savithri Ramurthy,* Sharadha Subramanian, Mina Aikawa,
Payman Amiri, Abran Costales, Jeff Dove, Susan Fong,
Johanna M. Jansen, Barry Levine, Sylvia Ma,
Christopher M. McBride, Jonah Michaelian, Teresa Pick,
Daniel J. Poon, Sandhya Girish, Cynthia M. Shafer,
Darrin Stuart, Leonard Sung, and Paul A. Renhowe
NoVartis Institutes for Biomedical Research,
4560 Horton Street, EmeryVille, California 94608
Figure 1. Evolution of the 2-arylaminobenzimidazole scaffold.
ReceiVed August 20, 2008
physicochemical properties and the lack of downstream modula-
tion on the MAP kinase axis on cells persuaded us to pursue a
program to identify Raf kinase inhibitor(s) with an improved
profile. Our initial effort involved trying to improve the
physicochemical properties of 1 by eliminating the urea while
retaining Raf kinase activity. Tying the urea back onto ring A
(Figure 1, path a) led to 2 with lower affinity for Raf kinase,17
while closing the urea onto the B ring gave 3 which, while less
potent than 1, still potently inhibited C and mutant B isoforms
of Raf. Further SAR exploration centered on this series.
Removing the substituents on the aryl ring of 3 (ring A) to
give the unadorned aryl ring (5, Table 1) led to a dramatic loss
of affinity, indicating substitution on the aryl ring is needed for
potent Raf inhibition. Systematic evaluation of various substit-
uents at the 2-, 3- and 4-positions on ring A indicated that
substitution on the 3- and 4-positions of the ring led to higher
affinity for Raf than substitution on the 2-position (e.g., 7 and
8 vs 6; 10 and 11 vs 9). In particular, a bromine at the 4-position
(8) improved affinity nearly 200-fold in B-RafV600E compared
to 5 while a trifluoromethyl group (11) further improved potency
770-fold over 5. Incorporating a tert-butyl group at C-4 of ring
A (13), while still quite potent, began to show some erosion of
activity compared to 11. In this case, moving the substituent to
the meta position gave improved affinity (12).
Overall, it was concluded that incorporation of liphophilic
substituents was needed for potent Raf inhibition. Further
investigation of ring A arenes indicated that neither heteroatoms
in the ring nor polar substituents on the ring were tolerated.17
At this point, introduction of a methyl on the benzimidazole
NH was explored giving 18 and the regioisomer 425 where the
methyl is on the nitrogen meta to the oxygen linker of the
pyridine. Compound 18 exhibited 45-fold better affinity in c-Raf
over the other regioisomer 4. Compared to the unmethylated
analogues, the majority of the methylated analogues (14-23)
exhibited significantly improved enzymatic potency (5- to 60-
fold) against both isoforms of Raf. Compound 18 with a
4-bromo substituent exhibited an affinity for B-RafV600E that
was 5-fold better than 1. However, further extending the methyl
on the benzimidazole nitrogen to an ethyl (28) significantly
decreased potency against both Raf isoforms.
Abstract: A series of arylaminobenzimidazoles was designed and
synthesized as Raf kinase inhibitors. Exploration of the structure-activity
relationship resulted in compounds that are potent in vitro and show
desirable in vivo properties.
The Ras-mitogen activated protein kinase (MAPKa) signaling
pathway was the first signaling pathway elucidated from the
cell membrane to the nucleus.1 The MAPK signaling path
consists of the Ras/Raf/MEK/ERK signal transduction cascade
which is a vital mediator of a number of cellular fates including
growth, proliferation, survival, and other aspects of cellular
behavior that can contribute to the transformed phenotype,
making it an attractive pathway to target in several cancer types.
The three Raf isoforms (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
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 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 and 12% of colon
carcinomas.5,7-9 The many effects of Raf kinases on cancer
cell growth and survival, together with the high prevalence of
mutation in melanoma, for which there is no good treatment,
make Raf a very attractive target for anticancer therapy.
Raf kinase inhibitors have been reported previously, and the
most advanced small molecule inhibitor, 1 (BAY43-9006,
Sorafenib), has been developed and launched for the treatment
of renal cell carcinoma.10-14 However, its lack of activity in
tumors expressing mutant B-Raf, for example, melanoma, may
be because the mechanism of action is through inhibition of
VEGFR rather than Raf.15,16 In this communication, we describe
the discovery of a series of the arylaminobenzimidazole amides
that potently inhibit Raf kinase. The structure-activity relation-
ship for c-Raf and B-RafV600E, PK, and efficacy for this scaffold
will also be discussed.
At the time our program began, 1, a potent biochemical
inhibitor of Raf was in clinical trials. However, the poor
* To whom correspondence should be addressed. Phone: 510-923-7767.
Fax: 510-923-3360. E-mail: savithri.ramurthy@novartis.com.
a Abbreviations: MAPK, Ras-mitogen activated protein kinase; VEGFR,
Vvascular endothelial growth factor receptor; PDGFR platelet-derived
growth factor receptor; CSF1R, colony stimulating factor-1 receptor; RTKs,
receptor tyrosine kinases; STKs, serine threonine kinases; Lck, leukocyte
specific protein tyrosine kinase.
In addition to evaluating the SAR of aromatic A rings,
aliphatic groups were also assessed. Replacing the aryl ring with
10.1021/jm801050k CCC: $40.75
2008 American Chemical Society
Published on Web 10/23/2008

7050 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Table 1. SAR Evaluating R1 and R2
Letters
Table 2. SAR of Amide
IC50 (µM)19
EC50 (µM)
SK- p-
MEL-28 Erk
IC50 (µM)
B-RafV600E c-Raf SK-MEL-28 p-Erk
EC50 (µM)
B-
c-
Raf
compd R1
R2
RafV600E
compd R3
R4
1
5
3
6
7
8
9
0.011
7.7
0.28
2.4
1.3
0.039
9.0
0.30
0.011
0.026
0.063
0.13
0.028
0.039
0.025
0.002
1.9
0.001
3.5
0.076
0.49
0.21
0.019
0.95
0.056
0.020
0.008
0.015
0.048
0.015
0.010
0.004
0.001
0.15
0.006
0.009
0.15
0.004
0.009
1.1
1.3
4.3
29
18
30
31
32
33
34
H
H
H
H
H
H
H
Me
Et
0.001
0.001
0.014
0.017
0.004
0.041
0.78
2.8
1.9
>10
>10
H
H
H
H
H
H
H
H
H
H
phenyl
4-Cl-3-CF3
2-Br
3-Br
4-Br
2-CF3
3-CF3
4-CF3
3-t-Bu
4-t-Bu
0.002
0.013
0.031
0.011
0.007
2.7
4.6
>10
2-morpholinoethyl
2-hydroxyethyl
2-propylpiperidine
>10
0.61
7.4
Me Me
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
7.1
5.9
replacement of the methyl group with the polar primary
carboxamide (29) retained Raf potency.
Me phenyl
Me 4-Cl-3 CF3
Me 2-Br
Me 3-Br
Me 4-Br
Me 2-CF3
Me 3-CF3
Me 4-CF3
Me 2-t-Bu
Me 3-t-Bu
Me 4-t-Bu
Me cyclohexyl
Me cyclohexylmethyl
Me 3-(4-pyridyl)
>10
7.3
9.8
>5.0
1.9
One of the goals of our program was to remove the urea in
1 while retaining potent Raf kinase activity and improving the
physicochemical properties, in particular solubility. The solubil-
ity of our compounds was measured and compared to 1, which
had a solubility of <0.005 µg/mL at pH 7. The solubility of 18
at pH 7 was 4.9 µg/mL, significantly better than 1. Incorporation
of a solubilizing group on the amide like the 2-morpholinoethyl
group of 31 resulted in further improved aqueous solubility of
7.3 µg/mL at pH 7 and 29 µg/mL at pH 5.5.
The benzimidazole amides in general have a narrow kinase
profile. Example 23 was tested against 50 kinases including
receptor tyrosine kinases (RTKs), serine/threonine kinases
(STKs), and tyrosine kinases (TKs). Compound 23, in addition
to being a pan-Raf inhibitor, inhibits four RTKs, CSF1R kinase,
VEGFR kinase, c-Kit, and PDGFRꢀ with IC50 values of 19,
18, 10, and 10 nM, respectively. Additionally, 23 inhibits two
members of Src family of kinases, cAbl and Lck, with IC50
values of 80 and 60 nM, respectively. However, the compound
was not tested in the respective cell based assays.
To rationalize the observed SAR from a 3D structural
perspective, 18 was docked in the active site of the public
domain crystal structure published for B-Raf (PDB accession
code 1UWH).20 Figure 2 (top) shows the overlay of that docking
model with the cocrystallized conformation of 1. The model
suggests a very similar binding mode for both compounds, with
matching occupancy of hydrophobic pockets and matching
hydrogen bonds to the hinge domain (the backbone NH and
CdO of Cys532) and to the Asp594/Glu501 pair in the catalytic
region; selected residues interacting with 18 are shown in a
cartoon representation in Figure 2 (bottom).
The N-methyl group of 18 occupies a hydrophobic subpocket
of the selectivity pocket, rationalizing the increased affinity for
this substitution. The observed preference for phenyl with
lipophilic substituents as ring A is explained by the fact that
the model places that ring in the pocket that is normally occupied
by the phenylalanine from the DFG motif (Phe593) in the active,
“DFG-in”, conformation. Binding of 1 has been shown to induce
the “DFG-out” conformation where the substituted phenyl takes
the place of Phe593 in this hydrophobic environment.20 The
modeling work on 18 suggests that it too binds to the protein
in the “DFG-out” conformation. The fact that solubilizing groups
are tolerated on the pyridyl end of the scaffold is explained by
the model, which places the solubilizing groups in the solvent-
exposed part of the binding site.
>10
>10
4.6
1.3
0.008
0.088
2.7
0.045
0.14
4.4
0.14
0.014
0.068
0.89
3.0
0.28
0.45
0.028
0.010
0.018
>10
2.3
>10
5.9
Et
4-Br
a cyclohexyl ring (25) led to an analogue with similar affinity
as the unsubstituted aryl ring (5). Interestingly, extending the
cyclohexyl ring by a methylene unit significantly increased
B-RafV600E affinity (26).
The inhibition of cellular proliferation in SK-MEL-28 cells
(B-RafV600E, melanoma cell line) was evaluated for a number
of compounds and compared to 1. In addition, inhibition of the
p-Erk signal was measured in SK-MEL-28 cells to determine
whether the compounds are acting via inhibition of Raf. 1
inhibited the proliferation of cellular growth and the p-Erk signal
but with much lower potency compared to its biochemical
activity. Compound 20 with a 3-trifluoromethyl moiety, how-
ever, exhibited cell inhibition and p-Erk modulation with similar
activity as 1. The discrepancy between biochemical and cell
potency could be explained, that in the biochemical assay
purified kinase domain of Raf is being tested compared to the
cellular assay in which Raf exists as a complex with chaperons,
cytoskeleton, phosphatases, and kinases.18 Compound 23 with
the 3-tert-butyl exhibited the most impressive inhibition of cell
proliferation (0.89 µM) and inhibition of Erk phosphorylation
(0.28 µM) in this series.
As mentioned earlier, only lipophilic groups on the arene gave
good enzymatic potency, but they had limited solubility. The
pyridyl end of the molecule was explored as an opportunity to
modify the scaffold’s physicochemical properties. The initial
evaluation of the effect of amide substituents larger than methyl
revealed no improvement (Table 2). For example, ethyl (30)
gave similar potency as methyl (18). Modification of the
secondary amide (18) to a tertiary amide (34) was detrimental,
suggesting the importance of a hydrogen bond donor in this
portion of the molecule. Incorporating a solubilizing group
connected by a linker led to a slight loss in potency (31-33)
and reduced inhibition of Erk phosphorylation. Interestingly,
The benzimidazole core was easily constructed beginning with
the commercially available aminonitrophenol (35) (Scheme 1).

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7051
Scheme 2a
a Reagents and conditions: (a) K2CO3, t-BuOK, DMAC; (b) CH3I, 10%
aqueous NaOH, TBAB, CH2Cl2; (c) H2, 10% Pd/C, MeOH; (d) 4-bro-
mophenyl isothiocyanate, MeOH, room temp, 16 h; (e) EDCI, HF,16 h,
room temp; (f) TFA, CH2Cl2; (g) HNR3R4, HBTU, DIEA, THF, room temp.
Figure 2. Docking model of 18 in B-Raf active site. Top: a surface
representation of the active site colored by atom-type (oxygen ) red,
nitrogen ) blue, sulfur ) yellow, carbon and hydrogen ) white),
showing the overlay of 18 (cyan) with the cocrystallized conformation
of 1 in green.20 Bottom: a cartoon representation of the kinase with
selected residues in stick model (Glu501, Cys532, Phe593, Asp594)
and 18 in cyan.
Figure 3. Efficacy and pathway inhibition of 27 in the HT29
(B-RafV600E) human colorectal carcinoma xenograft model: (A) 10
(mg/kg)/day (open squares), 30 (mg/kg)/day (gray squares), or 100 (mg/
kg)/day (black squares). Circles represent vehicle treatment. / indicates
p < 0.05 (one-way ANOVA). (B) HT29 tumors from mice dosed with
27 at 100 mg/kg for 5 days were harvested 1, 4, and 24 h after the
final dose, and tumor lysates were analyzed by Western blot for
phospho-ERK (pERK) and total ERK (ERK).
Scheme 1a
and the carboxylic acid was coupled with amines to give the
desired products (29-33).
The PK parameters of 18 and 27 were examined. Following
a single 20 mg/kg oral administration to female mice in 5%
captisol, 18 exhibited a low clearance (0.4 (mL/min)/kg), low
volume of distribution (183 mL/kg), long half-life (325 min),
and almost complete oral bioavailability. Compound 27 exhib-
ited a higher clearance (7.1 (mL/min)/kg), slightly higher volume
of distribution (406 mL/kg), and shorter half-life (105 min) than
18 but also had almost complete oral bioavailability, suggesting
the compounds of this class were suitable for further in vivo
evaluation.
Compound 27 was selected for evaluation in a mouse tumor
xenograft model because of better solubility over other com-
pounds in this series. The SK-MEL-28 cell line could not be
grown in vivo in nude mice, and therefore, the HT29 (B-
RafV600E) human colorectal tumor xenograft model was used
to test the effect of the compound on tumor growth in vivo.
Subcutaneous tumors were established to a size of approximately
100 mm3 before oral dosing commenced. HT29 human colon
carcinoma tumor cells were harvested from in vitro cultures
and implanted subcutaneously into female nu/nu mice (2 × 106
cells/mouse). Ten days after implant, mice were randomized
by tumor volume (∼100 mm3) into four groups (10/group) and
then treated by oral gavage with vehicle (10% Captisol) or 27
at 10, 30, or 100 mg/kg daily for 28 days. Tumor volume and
body weight were measured twice weekly (n ) 10/group).
Compound 27 demonstrated significant antitumor activity
(Figure 3A) at doses of 30 (mg/kg)/day (∆T/∆C ) 19%) and
100 (mg/kg)/day (15% tumor regression) over the course of 28
days of treatment. The compound was very well-tolerated at
a Reagents and conditions: (a) KHMDS, K2CO3, DMF, 90°C, 16 h; (b)
TFAA, (CH3)2SO4, NaOH, CH3CN, 16 h, room temp; (c) H2, 10% Pd/C,
MeOH; (d) R2NCS, MeOH, room temp, 16 h; (e) MeI, MeOH, 16 h, room
temp; (f) EDCI, THF, 16 h, room temp.
Our key intermediate (37) was obtained by O-arylation of
chloropyridylacetamide (36) with 35.21 The nitro group was then
reduced to give 38. Alternatively, the amino of 37 could first
be methylated and then reduced to give 39.22,23 The phenylene-
diamine (38 or 39) was then treated with an isothiocyante to
afford thioureas 39a and 39b. Ring closure of thioureas was
affected by sulfur activation with methyl iodide24 or 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) to give the
2-arylaminobenzimidazoles 3-13 and 14-27.25,26
To incorporate a variety of amides on the pyridyl ring, the
aminonitrophenol (35) was coupled with tert-butyl 4-chloro-
pyridine-2-carboxylate (40) (Scheme 2). The remainder of the
synthesis followed that of the methylamide series shown in
Scheme 1 until the end when the ester (42) was deprotected

7052 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Letters
RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer
Res. 2003, 63, 1454–1457.
30 (mg/kg)/day; however, in the 100 mg/kg/day group there
was an average 10% body weight loss and one mortality. The
compound showed time dependent inhibition of pERK consistent
with inhibition of B-RafV600E in tumors (Figure 3B). On the
other hand, as this compound has similar profile as 23, we cannot
discount the contribution of VEGFR/other kinase inhibition to
efficacy. These data simply demonstrate the efficacy of the
novel, orally active 2-aminobenzimidazole 27 in human col-
orectal xenografts.
In conclusion, we developed a novel series of potent Raf
inhibitors that significantly improved the solubility compared
to the urea 1. In addition, favorable pharmacokinetics was
demonstrated for 18 and 27, which was indicative of the series.
Further work on this scaffold to improve the inhibition of
cellular proliferation and Erk phosphorylation was done to
identify compound Raf-265, which is in phase I clinical trials
with results to be communicated in due course.
(9) Brose, M. S.; Volpe, P.; Feldman, M.; Kumar, M.; Rishi, I.; Gerrero,
R.; Einhorn, E.; Herlyn, M.; Minna, J.; Nicholson, A.; Roth, J. A.;
Albelda, S. M.; Davies, H.; Cox, C.; Brignell, G.; Stephens, P.; Futreal,
P. A.; Wooster, R.; Stratton, M. R.; Weber, B. L. BRAF and RAS
mutations in human lung cancer and melanoma. Cancer Res. 2002,
62, 6997–7000.
(10) 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. Discovery of heterocyclic ureas as
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(11) Khire, U. R.; Bankston, D.; Barbosa, J.; Brittelli, D. R.; Caringal, Y.;
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Acknowledgment. The authors acknowledge Ahmad Hashash
and Keshi Wang for formulation and bioanalytics done on these
compounds.
(13) Lyons, J. F.; Wilhelm, S.; Hibner, B.; Bollag, G. Discovery of a novel
Raf kinase inhibitor. Endocr.-Relat. Cancer 2001, 8, 219–225.
(14) Kumar, C. C.; Madison, V. Drugs targeted against protein kinases.
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(15) Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong,
H.; Chen, C.; Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.; Shujath,
J.; Gawlak, S.; Eveleigh, D.; Rowley, B.; Liu, L.; Adnane, L.; Lynch,
M.; Auclair, D.; Taylor, I.; Gedrich, R.; Voznesensky, A.; Riedl, B.;
Post, L. E.; Bollag, G.; Trail, P. BAY 43-9006 exhibits broad spectrum
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receptor tyrosine kinases involved in tumor progression and angio-
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Supporting Information Available: Experimental details for
the synthesis and characterization of all compounds; procedure on
biochemical and cellular assay. This material is available free of
charge via the Internet at http://pubs.acs.org.
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(8) Kimura, E. T.; Nikiforova, M. N.; Zhu, Z.; Knauf, J. A.; Nikiforov,
Y. E.; Fagin, J. A. High prevalence of BRAF mutations in thyroid
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JM801050K