Design and synthesis of orally bioavailable benzimidazole reverse amides as pan RAF kinase inhibitors

Article abstract of DOI:10.1021/ml5002272

Benzimidazole reverse amides were designed and synthesized as Pan RAF kinase inhibitors. Investigation of the structure-activity relationship of the compounds revealed that they were potent in vitro and exhibited desirable in vivo properties.

Full text of DOI:10.1021/ml5002272

Letter
pubs.acs.org/acsmedchemlett
Design and Synthesis of Orally Bioavailable Benzimidazole Reverse
Amides as Pan RAF Kinase Inhibitors
Sharadha Subramanian, Abran Costales, Teresa E. Williams, Barry Levine, Christopher M. McBride,
Daniel Poon, Payman Amiri, Paul A. Renhowe, Cynthia M. Shafer, Darrin Stuart, Joelle Verhagen,
and Savithri Ramurthy*
Global Discovery Chemistry/Oncology & Exploratory Chemistry, Novartis Institutes for Biomedical Research, 4560 Horton Street,
Emeryville, California 94608, United States
S
* Supporting Information
ABSTRACT: Benzimidazole reverse amides were designed and synthesized as Pan
RAF kinase inhibitors. Investigation of the structure−activity relationship of the
compounds revealed that they were potent in vitro and exhibited desirable in vivo
properties.
KEYWORDS: MAP, ras-mitogen activated protein kinase, VEGF, vascular endothelial growth factor receptor, CSF1R,
colony stimulating factor-1 receptor, RTKs, receptor tyrosine kinases, STKs, serine threonine kinases, TKs, tyrosine kinases
he RAS-RAF-MEK-ERK pathway is a key driver of
oncogenic signals, and mutations in RAS and BRAF occur
T
in approximately 30% of all human cancers.1,2 In normal tissues
the RAS family of GTPases transmit signals from growth factor
receptors on the cell membrane to trigger activation of the
RAF-MEK-ERK kinase cascade leading to the promotion of
proliferation and survival signals in the cytoplasm and nucleus.
Disregulation of the pathway through mutation occurs most
frequently at the level of RAS, where oncogenic mutations lead
the stabilization of RAS in the active GTP-bound form resulting
in constitutive activation of downstream effectors. Activating
mutations in BRAF occur in approximately 8% of human
tumors, most notably in 50% of human melanomas with the
most frequent mutation resulting in a valine to glutamic acid
mutation at codon 600 (BRAFV600E).2 BRAFV600E is con-
stitutively active and functions completely independent of RAS.
Given the central role this pathway plays in cancer it has
been the center of intense drug discovery efforts for over 20
years. While RAS has remained an elusive target for drug
discovery, numerous inhibitors have entered human clinical
trials targeting RAF, MEK, and, more recently, ERK. Most
notably, the RAF inhibitors vemurafenib and dabrafenib as well
as the MEK inhibitor trametinib have been approved for the
Figure 1. Design strategy of reverse amide series.
prompted us to explore modifications on the pyridyl amide
portion of the molecule, an area that had not been extensively
investigated. The amide functionality was transposed to form
the “reverse” amide compound 2, which showed better
correlation between biochemical potency (BRAFV600E IC50
=
0.003 μM) and the cell based activity (EC50 = 0.044 μM). In
this letter, the structure−activity relationship (SAR) of the
reverse amide series and the in vitro and in vivo profiles of
representative examples will be discussed.
Synthesis of the reverse amide series was attained by the
route outlined in Scheme 1. O-Arylation of chloropyridyl-t-
butyl ester 4 with the commercially available nitroaminophenol
3 followed by N-methylation and hydrolysis yielded the
corresponding acid 5. Curtius rearrangement of 5 yielded
pyridyl t-butylcarbamate 6. Hydrolysis of the t-butyl ester in 6
afforded the key intermediate 7, which was acetylated to give 8.
Hydrogenation of the nitro group in 8 gave the phenelene
diamine, which was then converted to the thiourea and cyclized
to afford analogues (9−18).
treatment of human melanoma tumors expressing BRAFV600E
.
The significant therapeutic benefit provided by vemurafenib
(PLX-4032) and dabrafenib confirms that RAF is an attractive
target for drug development.3
Our group has previously disclosed a series of benzimidazole
amides as potent RAF inhibitors.4,5 Out of several compounds
synthesized, only compound 1 potently inhibited BRAFV600E
(IC50 = 0.044 μM) and exhibited the best downstream target
modulation pERK (EC50 = 0.3 μM, Figure 1) in the amide
series.4,5 The poor correlation between the biochemical
potency and cell-based activity in the benzimidazole amides
Received: April 23, 2014
Accepted: July 11, 2014
Published: July 17, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Incorporating basic amines onto the amide was found to
significantlly impact the solubility of this series. After
considerable work, the piperidinyl and methylpiperazinyl
amide 20−25 (Table 2) were found to be superior in terms
Scheme 1
Table 2. SAR of the Reverse Amides
a
Reagents and conditions: (a) K2CO3, t-BuOK, DMAc; (b) CH3I,
10% aqueous NaOH, TBAB, CH2Cl2; (c) TFA, CH2Cl2; (d) Et3N,
isobutylchloroformate; (e) NaN3, water, 0°C; (f) t-BuOH, toluene
100°C; (g) TFA, CH2Cl2; (h) acetic anhydride, pyridine, dioxane,
85°C; (i) 10%Pd/C, H2, MeOH; (j) R′ArNCS, MeOH; (k) FeCl3,
MeOH.
The SAR from our previous series was utilized to begin
exploration of the SAR of the reverse amides.4 Lipophilic
groups at the meta and para positions of ring A exhibited good
biochemical potency (9−18). The tert-butyl (2 and 13) and the
isopropyl groups (10 and 15) at the meta and para positions
exhibited good biochemical and cellular potency. The same
trend was observed in 18 with 2-F and 5-CF3 substitution on
ring A. A trifluoromethoxy group at the meta (11) and para
positions (16) on ring A were biochemically potent as 2, but
the cell potencies were 2-fold less. On the basis of the p-ERK
EC50 values, the trifluoromethyl group at the para position (17)
on ring A proved superior than at the meta position (12) with
an 8-fold improvement over 12. In general, the reverse amide
series showed improved and consistent cellular activity
compared to the amide series (Table 1).4
While the reverse amide series provided compounds that
were highly potent in the both enzymatic and cellular assays, it
was observed that 2 exhibited solubility of 0.28 μg/mL at pH 7
and inhibited CYP3A4 at 0.5 μM. Thus, in exploring the SAR,
the focus was broadened to include improving physicochemical
properties while retaining the biochemical and cellular potency.
of potency and solubility as expected. However, because basic
amines can cause an inhibition of hERG, we monitored this
parameter during our optimization. The piperidinyl amides
could be incorporated by coupling 7 with the corresponding
carboxylic acids (Scheme 2).6 The N-methyl piperazinyl reverse
a
Scheme 2
Table 1. SAR of the Benzimidazole Reverse Amide Series
compd
R′
BRAFV600E IC50 (μM)
p-ERK EC50 (μM)
2
3-t-Bu
3-Et
0.003
0.04
0.1
a
Reagents and conditions: (a) BOP, DIEA, DMF, 60°C; (b) 10% Pd/
9
<0.0004
<0.0004
0.0004
0.003
C, H2, MeOH; (c) R′NCS, MeOH; (d) FeCl3, MeOH.
10
11
12
13
14
15
16
17
18
3-iPr
0.05
0.1
3-OCHF3
3-CF3
4-t-Bu
4-Et
0.4
amides were derived by the acylation of 7 with chloroacetyl
chloride followed by nucleophilic substitution with the
corresponding amine.6 The remainder of the syntheses were
similar to Scheme 1 to give the desired products.
The piperidinyl analogue of 2, compound 20, had a similar
potency profile to 2 but greatly improved solubility (27 μg/mL
at pH 7). It was also found that bulky groups on the basic
0.009
0.05
0.08
0.09
0.1
0.001
4-iPr
0.004
4-OCHF3
4-CF3
2-F, 5-CF3
0.001
0.004
0.05
0.09
0.001
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ACS Medicinal Chemistry Letters
Letter
nitrogen of the piperidine ring improved the CYP profile. For
example, 22 was 6-fold less potent against CYP3A4 (3 μM)
than 2, while also having improved solubility (581 μg/mL at
pH 7). Since 22 met our criteria for potency, solubility, and
CYP profile, a preliminary rat PK study of 22 was performed.
Following a single dose 10 mg/kg oral administration 22
exhibited high clearance (90 mL/min/kg), volume of
distribution (17784 mL/kg), adequate half-life (251 min),
and poor oral bioavailability (15%). We hypothesized that the
poor bioavailability could be due to the presence of an oxidative
metabolism prone t-butyl group on ring A. Replacement of the
t-butyl group with 2-fluoro-5-trifluoromethyl (23) maintained
cell potency and solubility (561 μg/mL at pH 7). To confirm
our hypothesis concerning oral bioavailability, we carried out a
rat PK study with 23. Following a single dose 10 mg/kg oral
administration, 23 proved superior to 22 with an oral
bioavailability of 54%, improved CL (49 mL/min/kg), and
similar half-life and volume of distribution. Upon further
investigation, it was found that replacing the piperidinyl group
in 23 with an isopropyl methylpiperazinyl (24) also gave a
potent compound that exhibited solubility at 28 μg/mL at pH 7
and CYP3A4 inhibition at 14 μM. Further, replacing the
isopropyl in 24 with an ethyl produced 25, which exhibited the
best combination of cellular potency and clearance along with
CYP3A4 inhibition >25 μM. Because of this superior profile,
compound 25 was further evaluated.
Figure 2. Efficacy and pathway inhibition of 25 in the HT29
(BRAFV600E) human colorectal carcinoma xenograft model: (A)
vehicle (diamonds), 10 mg/kg/day (squares), 30 mg/kg/day
(triangles), and 100 mg/kg (circles). (B) HT29 tumors from mice
dosed with 25 at 30 and 100 mg/kg and tumors were harvested at 4
and 24 h after final dose, and lysates were analyzed by Western blot for
pERK and total ERK.
The kinase profile of 25 was examined against 50 kinases
including receptor tyrosine kinases (RTKs), serine/threonine
kinases (STKs), tyrosine kinases (TKs), and AGC kinases.
Compound 25, in addition to being a pan-Raf inhibitor,
inhibited four RTKs: CSF1R kinase, VEGFR kinase, c-Kit, and
PDGFR with IC50 values < 10 nM, respectively. Additionally,
25 inhibited one of the Src family of kinases, Lck with IC50
value of 240 nM.
The PK profile of 25 was examined in rat. Compound 25
exhibited acceptable clearance (32 mL/min/kg), volume of
distribution (12737 mL/kg), half-life (340 min), and oral
bioavailability (40%) after oral administartion of 10 mg/kg.
Because of its favorable PK profile, 25 was evaluated in a
mouse tumor xenograft model. The SK-MEL-28 cell line could
being cleaved to the pyridyl amine. The pyridyl amine of 25 did
not exhibit cell potency (pERK >10 μM) and hence might not
have contributed to the tumor growth inhition. The increased
plasma stability of the piperidinyl amides over the methyl
piperazinyl amides could be attributed to the steric bulk on the
carbon α to the amide carbonyl group. Further the hERG signal
for 25 was 2.3 μM, which we considered suboptimal.
In conclusion, a novel series of potent Raf inhibitors was
developed.8 This series had significantly improved physico-
chemical properties compared to the earlier benzimidazole
amide series.4 In addition, 25 demonstrated target inhibition in
vivo with good antitumor activity. Further work on this scaffold
to improve the inhibition of cellular proliferation, ERK
phosphorylation, hERG, and plasma stability led us to identify
compound RAF265, which has since entered human clinical
trials.
not be grown in vivo in nude mice so the HT29 (B-RAFV600E
)
human colorectal tumor xenograft model was used to test the
effects of 25 on tumor growth in vivo.7 It demonstrated
significant antitumor activity (Figure 2) at doses 10 mg/kg/day
(ΔT/ΔC = 52%), 30 mg/kg/day (ΔT/ΔC = 72%), and 100
mg/kg/day (ΔT/ΔC = 75%) over the course of 16 days of
treatment. It was very well tolerated at the 10 and 30 mg/kg
doses; however, in the 100 mg/kg/day group there was an
average body weight loss of 13%. The compound demonstrated
time dependent inhibition of pERK consistent with inhibition
of BRAFV600E in tumors (Figure 2B). These data demonstrate
that the novel reverse amide 25 is efficacious in human
colorectal xenografts.
Mouse plasma stability studies in vitro were conducted on the
reverse acetamides, piperidinyl, and the methyl piperazinyla-
mides. The stability of 17, 23, 24, and 25 were examined at 37
°C at an initial concentration of 1 μM. We observed that 100%
of 17 and 52% of 23 remained after 45 min of incubation
showing that the reverse acetamides and piperidinyl amides
exhibited good to moderate plasma stability. However, only 1%
of 24 and 25 remained after 45 min showing that
methylpiperazinylamides possessed poor plasma stability. For
other compounds in this series, it was found that the amide was
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental data and procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(S.R.) E-mail: savithri.ramurthy@novartis.com.
Notes
The authors declare no competing financial interest.
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ACS Medicinal Chemistry Letters
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ACKNOWLEDGMENTS
■
The authors wish to acknowledge Paul Barsanti for mouse
plasma stability study data and Dazhi Tang for HRMS analysis
of these compounds.
ABBREVIATIONS
■
MAPK, ras-mitogen activated protein kinase; VEGFR, vascular
endothelial growth factor receptor; CSFR1, colony stimulating
factor-1 receptor; RTKs, receptor tyrosine kinases; STKs, serine
threonine kinases; TKs, tyrosine kinases; KDR, kinase domain
receptor; SAR, structure−activity relationship; DMAc, dime-
thylacetamide
REFERENCES
■
(1) Bos, J. L. Ras oncogenes in human cancer: A review. Cancer Res.
1989, 49, 4682−4689.
(2) 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.; Gustereson, 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.
(3) Flaherty, K. T.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G.
A.; Sosman, J. A.; O’Dwyer, P. J.; Lee, R. J.; Grippo, J. F.; Nolop, K.;
Chapman, P. B. Inhibition of mutated, activated BRAF in metastatic
melanoma. N. Engl. J. Med. 2010, 363 (9), 809−819.
(4) 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. Design and synthesis of orally
bioavailable benzimidazoles as Raf kinase inhibitors. J. Med. Chem.
2008, 51 (22), 7049−52.
(5) Ramurthy, S.; Aikawa, M.; Amiri, P.; Costales, A.; Hashash, A.;
Jansen, J. M.; Lin, S.; Ma, S.; Renhowe, P. A.; Shafer, C. M.;
Subramanian, S..; Sung, L. Design and synthesis of 5,6-fused
heterocyclic amides as Raf Kinase inhibitors. Bioorg. Med. Chem. Lett.
2011, 21 (11), 3286−3289.
(6) Synthetic methodology and characterization data for 25 are
included in the Supporting Information.
(7) Tumors were established 10 days following subcutaneous
implantation of 3 × 106 cells/mouse. Mice were randomized into 4
groups, and oral dosing commenced with vehicle (5 mM citrate) or 25
at 10, 30, and 100 mg/kg daily for 17 days.
(8) Ramurthy, S.; Subramanian, S.; Verhagen, J.; Poon, D. J.; Hansen,
T.; Shafer, C.; McBride, C.; Levine, B. H.; Costales, A.; Renhowe, P.
Substituted benzazoles and use thereof as inhibitors of Raf kinase.
Patent WO037273, 2005.
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dx.doi.org/10.1021/ml5002272 | ACS Med. Chem. Lett. 2014, 5, 989−992