Imidazo[1,2-a]pyridin-6-yl-benzamide analogs as potent RAF inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.10.047

A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAF^V600E. Medicinal chemistry techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.

Full text of DOI:10.1016/j.bmcl.2017.10.047

Accepted Manuscript
Imidazo[1,2-a]pyridin-6-yl-benzamide Analogs as Potent RAF Inhibitors
Aaron Smith, Zhi-Jie Ni, Daniel Poon, Zilin Huang, Zheng Chen, Qiong Zhang,
Laura Tandeske, Hanne Merritt, Kevin Shoemaker, John Chan, Susan Kaufman,
Kay Huh, Jeremy Murray, Brent A. Appleton, Sandra W. Cowan-Jacob,
Clemens Scheufler, Takanori Kanazawa, Johanna M. Jansen, Darrin Stuart,
Cynthia M. Shafer
PII:
S0960-894X(17)31042-9
https://doi.org/10.1016/j.bmcl.2017.10.047
BMCL 25376
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 August 2017
18 October 2017
19 October 2017
Please cite this article as: Smith, A., Ni, Z-J., Poon, D., Huang, Z., Chen, Z., Zhang, Q., Tandeske, L., Merritt, H.,
Shoemaker, K., Chan, J., Kaufman, S., Huh, K., Murray, J., Appleton, B.A., Cowan-Jacob, S.W., Scheufler, C.,
Kanazawa, T., Jansen, J.M., Stuart, D., Shafer, C.M., Imidazo[1,2-a]pyridin-6-yl-benzamide Analogs as Potent RAF
Inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.047
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Imidazo[1,2-a]pyridin-6-yl-benzamide Analogs as Potent RAF
Inhibitors
Aaron Smith, Zhi-Jie Ni, Daniel Poon, Zilin Huang, Zheng Chen, Qiong Zhang, Laura
Tandeske, Hanne Merritt, Kevin Shoemaker, John Chan, Susan Kaufman, Kay Huh,
Jeremy Murray, Brent A. Appleton, Sandra W. Cowan-Jacoba, Clemens Scheuflera,
Takanori Kanazawa, Johanna M. Jansen, Darrin Stuart and Cynthia M. Shafer*
Global Discovery Chemistry/Oncology & Exploratory Chemistry, Novartis Institutes for Biomedical Research, 5300 Chiron
Way, Emeryville CA 94608
aNovartis Institutes of Biomedical Research, Novartis Campus, CH-4056 Basel, Switzerland
Received Month XX, 2017; Accepted Month XX, 2017
Abstract— A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAFV600E. Medicinal chemistry
techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.
The Ras-mitogen activated protein kinase (MAPK)
signaling pathway transmits signals from receptors on
the cell membrane to cytoplasmic and nuclear factors
that are responsible for cell proliferation and survival.
This pathway (RAS/RAF/MEK/ERK) is frequently
disregulated in cancer, leading to uncontrolled
proliferation and tumor growth. Activating mutations
in B-RAF kinase (e.g. B-RAFV600E
occur in
approximately 60% of melanomas.1
These
observations have inspired a number of drug discovery
efforts which led to the FDA approval of vermurafenib
(Roche) and dabrafenib (GSK) for the treatment of
metastatic melanoma.2 Novartis has been active in the
development of B-RAF inhibitors including the
RAF2653 and encorafenib (LGX818) programs.4-5
Figure 1. B-RAFV600E imidazo[1,2-a]pyridine scaffold
)
The imidazo[1,2-a]pyridine series originated as a core
from
a lipid kinase program. We envisioned
incorporating an ortho-methyl phenyl and amide
moiety similar to that in imatinib (GleevecTM) to
transform the Type I scaffold into a Type II scaffold
(for a review of classifications by type, see references
8 and 9). It was hypothesized that a Type II version of
the imidazo[1,2-a]pyridine scaffold (Figure 1) would
lead to a novel series for B-RAFV600E inhibition with
new opportunities to tackle selectivity issues vs P38
and VEGFR2 that were observed with the Type I
imidazo[1,2-a]pyridine series. While we do not
expect Type II inhibitors to be inherently more
selective,10 sequence and structure variations exist
between B-RAFV600E and the anti-targets in the
hydrophobic pocket created by the DFG-out
conformation, which is induced by the Type II
inhibitors. An early prototype exhibited B-RAFV600E
biochemical potency and thus a medicinal chemistry
effort was initiated to explore the structure activity
Herein, we describe the discovery of a novel scaffold
that potently inhibits B-RAFV600E kinase—the
imidazo[1,2-a]pyridin-6-yl-benzamide amides (Figure
1). The structure-activity relationship between B-
RAFV600E inhibition, selectivity versus P38 mitogen-
activated protein kinase and vascular endothelial
growth factor receptor 2, VEGFR2, as well as the
pharmacokinetic profile for this scaffold will be
discussed. P38 and VEGFR2 were two kinase anti-
targets of interest due to potential adverse events such
as acneiform skin rash for inhibitors of P386 and
hypertension for inhibitors of VEGF receptors.7

relationship of this series.
other ortho position of the ring would retain the
selectivity improvement against P38 seen in 4 and
potency against B-RAFV600E as seen in 2. Synthesis of
the alternate pyridyl regioisomer (5) did retain similar
biochemical affinity and target modulation potency
compared to 2. However the improvement in P38
selectivity observed in 4 was not maintained.
Initial efforts retained the ortho-methyl phenyl moiety
of imatinib and probed the effect of the amide on the
B-RAFV600E structure activity relationship and
selectivity profile (R, Figure 1). Compounds which
had a formamide (R = H) or an acetamide (R = Me)
were devoid of B-RAFV600E inhibition at the
concentrations tested.11 However when R was a
phenyl (1, Table 1), potent biochemical inhibition (61
nM) as well as in vitro target modulation (A375 pERK
EC50) of 0.54 µM and a 20-fold selectivity over P38
was observed. Evaluation of the ortho position of the
phenyl group with a variety of moieties provided
compounds with inferior biochemical potencies and
consistently poor target modulation results.11
Substitution at the meta position of the phenyl ring,
however, yielded compounds with striking
biochemical affinities (2-6) with 2 and 3 giving the
best combination of target modulation and inhibition
of cellular proliferation. Compound 3, in particular,
provided a good balance between inhibition of
biochemical, cell proliferation and target modulation
assays, as well as exhibiting selectivity against P38
and VEGFR2. Incorporation of a nitrogen into the
ring of 2 to give 4 resulted in a slight loss in
biochemical affinity and a much larger decrease in
Compound 6 which incorporates a t-butyl group at the
meta position, gave the best target modulation and
inhibition of cellular proliferation observed for any
analogs synthesized in this series. However, this
compound was also very potent against the anti-targets
P38 and VEGFR2 as well as a number of other kinases
tested.10
Compound 2 had good biochemical, target modulation
and cellular potencies but suffered from low solubility
(<0.4 µM, PBS buffer, pH 7.0). In an attempt to
improve the solubility, basic amines such as those in 7
and 8 were incorporated on the phenyl ring.
Biochemical affinity suffered slightly but target
modulation and inhibition of cellular proliferation
remained similar to 2. The solubility improvement for
8 was a modest 11-fold to 4.6 µM. For 7, however,
the improvement in solubility was much more
dramatic (300-fold to 122 µM). The angular methyl is
thought to disrupt the planarity of the two rings
leading to the improved solubility.
target modulation potency.
however, improved P38 selectivity 100-fold, and we
wondered whether incorporating a nitrogen at the
This modification,
Table 1. Structure activity relationship of the amide substituent R
B-
A375
P38 IC50 (µM,
fold selectivity vs
VEGFR2 IC50 (µM,
fold selectivity vs B-
A375 EC50
(µM)
RAFV600E pERK EC50
Cmpd
1
R
B-RAFV600E
)
RAFV600E
)
IC50 (µM)
(µM)
0.061
0.54
0.10
1.6 (x3)
11 (x18)
0.0002
0.0003
0.006
0.23
0.48
>10
0.086
0.22 (x1100)
3.1 (x10,000)
>25 (x>4000)
0.32 (x1600)
2.7 (x9000)
-
2
3
4
0.070
0.13
0.28
0.0009
0.77
0.66 (x700)
2.0 (x2000)
5

0.0003
0.003
0.025
0.24
0.057
0.34
0.002 (x7)
0.044 (x146)
6
7
0.002 (x0.7)
-
0.005
0.13
0.43
<0.011 (x<2)
0.11 (x20)
8
meta-down position, resulting in similar potency
compared to compound 2.
A crystal structure of 2 in P38 (Figure 3) was
obtained, which showed an identical binding mode to
the one observed in B-RAF. The space available at the
meta-up position in the P38 structure is much more
limited due to the residue change in the αC-helix of
Val504 in B-RAF to Leu74 in P38 and the slight shift
in the αC-helix orientation between the two proteins
(Figure 3). We know from a public domain structure
(1KV2)12 that P38 can accommodate larger
substituents in the meta-up position. This is also
evident from our own work where 7, with two bulky
meta-substituents, exhibits potent P38 activity.
However, in order to fit a substituent in the meta-up
position in P38, the aryl group is required to twist
slightly out of plane with the amide because of the
more restricted space. In the case of 4, this twist
breaks the intramolecular hydrogen bond between the
pyridyl N and the amide NH, which will penalize the
binding affinity for P38 and thereby increase the B-
RAF selectivity of 4.
Figure 2. Crystal structure of 2 in wt-B-RAF (PDB code 6B8U).
To elucidate the binding mode of the imidazo[1,2-
a]pyridine series, the crystal structure of 2 bound to
wt-B-RAF was solved. Compound 2 was shown to
bind to the inactive DFG-out conformation of B-RAF
(Figure 2). The trifluoromethylphenyl ring occupies
the hydrophobic pocket while the central amide linker
is engaged in hydrogen bonds between the side chain
of Glu501 and the amide-NH of Asp594. A single
hydrogen bond is observed between the acetamide and
the backbone NH of hinge residue Cys532.
The crystallographic binding mode provides some
rationale for the observed SAR and selectivity trends
in Table 1. Hydrophobic meta-substituents, like the
CF3 in 2, fit nicely in a hydrophobic pocket that holds
Phe595 in the DFG-in conformation. The space that
gets created by the DFG-out flip for Type II inhibitors
can comfortably accommodate substituents as large as
a t-Bu. We hypothesize that the drop in potency for 4
is related to the fact that it is energetically more
favorable for the ortho-N to be in an intra-molecular
hydrogen bond with the amide –NH and therefore the
CF3 flips to the other meta position (“up” in the view
of Figure 2). While this conformation can be
accommodated in B-RAF, it does not satisfy the
hydrophobic hotspot in the alternative meta-down
position, occupied by the CF3 of 2. Regioisomer 5
allows the intra-molecular hydrogen bond while
projecting the CF3 into the hydrophobic hotspot at that
Figure 3. Overlay of crystal structure of 2 in wt-B-RAF (PDB code
6B8U, protein grey/ compound yellow) with crystal structure of 2 in
P38 (PDB code 5mz3, protein cyan/compound orange); overlay
performed using ligand superposition.
Because this series was identified from a lipid kinase
program, the selectivity of this scaffold versus lipid
kinases was of interest; however, when tested against
lipid kinases, 2 was found to have IC50’s > 9 µM. The

selectivity of 2 was also evaluated against a panel of
74 kinases and found to inhibit six kinases with IC50’s
below 1 µM (PDGFRα 0.008 µM, Kit 0.052 µM,
EphA4 0.074 µM, EphB4 0.15 µM, Lyn 0.17 µM and
VEGFR2 0.67 µM). Inhibition of these additional
kinases is potentially the reason that the inhibition of
the cellular growth is more potent than the target
modulation in a number of the analogs described in
this
paper.
Scheme 1. Synthetic procedure for imidazo[1,2-a]pyridine 2
O
O
a
CF3
O
O
CF3
NH2
N
B
B
+
Cl
H
O
O
9
10
11
F
F
O
O
I
b
c
I
I
F
N
N
N
H2N
HN
HN
N
N
N
12
13
14
O
O
d
CF3
N
N
H
HN
N
2
Reagents and conditions: (a) THF, 99%; (b) K2CO3, THF, MeOH, water, reflux, 51%; (c) Et3N,
DMAP, Ac2O, CH2Cl2, 83%; (d) 11, Pd(dppf)Cl2-DCM, Na2CO3, DME, 110 oC, 14%.
The pharmacokinetic properties of 2 were examined.
Following a single 20-mg/kg oral administration to
female mice in 10% EtOH/18% captisol, 2 exhibited
moderate clearance (18 mL/min/kg), a large volume of
distribution (2150 mL/kg), moderate half-life (147
min), and good oral bioavailability (73%).
imidazo[1,2-a]pyridine series to a Type II scaffold was
successful in creating potent B-RAFV600E inhibitors.
Improvements in selectivity against P38 and VEGFR2
were achieved with concomitant retention of
acceptable target modulation and cellular potency as
in 3 while solubility was significantly improved in 7.
During the course of our work, the combination of the
meta-chloro in 3 with the meta-substituted solubilizing
group in 7 was not synthesized although it could have
provided the best combination of selectivity and
physicochemical properties. This suggests exploration
further of this series could provide optimized analogs.
The synthesis of 2 in Scheme 1 shows the typical
method of synthesizing analogs from the imidazo[1,2-
a]pyridine series. The synthesis of the right hand
portion of the molecule was achieved through an
amidation of the boronic acid 9 with acid chloride 10
to yield intermediate 11. The left hand portion of the
molecule is synthesized by starting with the known
imidazopyridine 12.13 Deprotection of the amine
followed by amidation yields 14. Compounds 11 and
14 are then coupled through a Suzuki-Miyurama
reaction to yield 2.
Acknowledgments. The authors would like to thank
Dazhi Wang and Weiping Jia for their analytical
support, Elke Koch and Celine Be for their technical
support in protein purification, Aude Izaac for her
crystallization technical support and Yongjin Xu for
discussions on kinase selectivity.
In conclusion, the transformation of the Type I

Supporting Information available: Experimental
details for the synthesis and characterization of select
compounds and procedures for the biochemical and
cellular assays are included in the supporting
information. This material is available free of charge
via the internet at http://pubs.acs.org.
Smith, A.; Wan, Y.; Wang, X.; Xie, Y.; Zhang, Q.;
Appleton, B.; Dollinger, G.; Isbell, J.; Li, C.; Li, N.;
Sethuraman, V.; Stuart, D.; Sun, F.; Turner, N.; Xu, Y.;
Zang, R.; Poon, D.; Tellew, J., Discovery of LGX818, a
potent, selective RAF kinase inhibitor for treatment of
BRAFV600E-positive melanoma. 245th ACS National
Meeting, April 7-11 (2013), MEDI-277.
References and Notes
6. De Buck, S.; Heieber, W.; Vitaliti, A.; Straube, F.;
Emotte, C.; Bruin, G.; Woessner, R., Population PK-PD
Model for Tolerance Evaluation to the p38 MAP Kinase
Inhbitor BCT197. Pharmacometrics Syst. Pharmacol.
2015, 4, 691-700.
1. Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.;
Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett,
M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.;
Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.;
Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.;
Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake,
H.; Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave,
D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-Trench,
G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.;
Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.;
Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.;
Paterson, H.; Marais, R.; Marshall, C. J.; Wooster, R.;
Stratton, M. R.; Futreal, P. A., Mutations of the BRAF
gene in human cancer. Nature 2002, 417, 949-954.
2. Asati, V.; Bharti, S.K.; Mahapatra, D.K., Mutant B-
RAF Kinase Inhibitors as Anticancer Agents. Anti-
Cancer Agents Med Chem 2016, 16, 1558-1575.
7. Hayman, S.R.; Leung, N.; Grande, J.P.; Garovic,
V.D., VEGF Inhibition, Hypertension, and Renal
Toxicity. Curr. Oncol. Rep. 2012, 14, 285-294.
8. Mobitz, H., The ABC of Protein Kinase
conformations. Biochim. Biophys. Acta 2015, 1555-1566.
9. Roskoski Jr., R., Classification of small molecule
protein kinase inhibitors based upon the structures of
their drug-enzyme complexes. Pharmacol. Res. 2016,
103, 26-48.
10. Zhao, Z.; Wu, H.; Wang, L.; Liu, Y.; Knaff, S.; Liu,
Q.; Gray, N.S., Exploration of Type II Binding Mode: A
Privileded Approach for Kinase Inhibitor Focused Drug
Discovery? ACS Chem. Biol. 2014, 9, 1230-1241.
11. Unpublished results.
3. Williams, T.E.; Subramanian, S.; Verhagen, J.;
McBride, C.M.; Costales, A.; Sung, L.; Antonio-McCrea,
W.; McKenna, M.; Louie, A.K.; Ramurthy, S.; Levine,
B.; Shafer, C.M.; Machajewski, T.; Renhowe, P.A.;
Appleton, B.A.; Amiri, P.; Chou, J.; Stuart, D.; Aardalen,
K.; Poon, D., Discovery of RAF265: A Potent mut-B-
RAF inhibitor for the treatment of metastatic melanoma.
ACS Med. Chem. Lett. 2015, 6(9), 961-965.
12. Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P.;
Gilmore, T.; Graham, A.; Grob, P.; Hickey, E.; Moss, N.;
Pav, S.; Regan, J., Inhibition of p38 MAP kinase by
utilizing a novel allosteric binding site. Nature Struct.
Biol. 2002, 9, 268 - 272.
13. Hamdouchi, C.; Sanchez, C.; Ezquerra, J.,
Chemoselective
aminoimidazo[1,2-a]pyridines
bis(trifluoroacetate) (PIFA). Synthesis 1998, 6, 867-872.
arylsulfenylation
of
2-
4. http://www.arraybiopharma.com/product-
pipeline/encorafenib-lgx818/.
by phenyliodine(III)
5.
Chen, Z.; Costales, A.; Dillon, M.; Huang, S.;
Huang, Z.; Jin, J.; Liu, Z.; Machajewski, T.; Madera, A.;
Antonios-McCrea, W.; McKenna, M.; Pan, S.; Pecchi,
S.; Renhowe, P.; Sendzik, M.; Shafer, C.; Sharma, A.;

Tabel of Contents graphic
Imidazo[1,2-a]pyridin-6-yl-benzamide Analogs as Potent RAF Inhibitors
Aaron Smith, ZJ Ni, Daniel Poon, Zilin Huang, Zheng Chen, Qiong Zhang, Laura Tandeske, Hanne Merritt,
Kevin Shoemaker, John Chang, Sue Kaufman, Kay Huh, Jeremy Murray, Brent Appleton, Sandra W. Cowan-
Jacob^, Clemens Scheufler^, Takanori Kanazawa, Hanneke M. Jansen, Darrin Stuart and Cynthia M. Shafer*
Global Discovery Chemistry/Oncology & Exploratory Chemistry, Novartis Institutes for Biomedical Research, 5300 Chiron
Way, Emeryville CA 94608 USA
^Novartis Institutes of Biomedical Research, Novartis Campus, CH-4056 Basel, Switzerland
A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs were designed as inhibitors of B-RAFV600E. Medicinal chemistry techniques
were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.