Design and Discovery of N-(3-(2-(2-Hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, a Selective, Efficacious, and Well-Tolerated RAF Inhibitor Targeting RAS Mutant Cancers: The Path to the Clinic

Article abstract of DOI:10.1021/acs.jmedchem.9b00161

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.

Full text of DOI:10.1021/acs.jmedchem.9b00161

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Drug Annotation
Design and Discovery of N-(3-(2-(2-hydroxyethoxy)-6-morpholinopyridin-4-
yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide
(LXH254)- A selective, efficacious and well-tolerated RAF
inhibitor targeting RAS mutant cancers: The path to the clinic
Savithri Ramurthy, Benjamin Robert Taft, Robert J. Aversa, Paul Barsanti, Matthew T Burger, Yan
Lou, Gisele Nishiguchi, Alice Rico, Lina Setti, Aaron Smith, Sharadha Subramanian, Victoriano N. A.
Tamez, Huw R. Tanner, Lifeng Wan, Cheng Hu, Brent A Appleton, Mulugeta Mamo, Laura Tandeske,
John E. Tellew, Shenlin Huang, Qin Yue, Apurva Chaudhary, Hung Tian, Raman Iyer, A. Quamrul
Hassan, Lesley A. Mathews Griner, Laura R. LaBonte, Vesselina G. Cooke, Anne Van-Abbema, Hanne
Merritt, Kalyani Gampa, Fei Feng, Jing Yuan, Yuji Mishina, Yingyun Wang, jacob haling, Sepideh Vaziri,
Mohammad Hekmatnejad, Valery Polyakov, Xu(Richard) zang, Vijay Sethuraman, Payman Amiri,
Mallika Singh, William R Sellers, Emma Lees, Wenlin Shao, Michael P Dillon, and Darrin D. Stuart
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00161 • Publication Date (Web): 06 May 2019
Downloaded from http://pubs.acs.org on May 6, 2019
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Journal of Medicinal Chemistry
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Pharmacology
Griner, Lesley; Novartis
LaBonte, Laura; Novartis Institutes for BioMedical Research Inc,
Oncology Medicinal Chemisttry
Cooke, Vesselina; Novartis
Van-Abbema, Anne; Novartis Institute for BioMedical Research, Global
Discovery Chemistry
Merritt, Hanne; Novartis Institute for BioMedical Research, Global
Discovery Chemistry
Gampa, Kalyani; Novartis
Feng, Fei; Novartis
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Yuan, Jing; Novartis
Mishina, Yuji ; Novartis Institutes for BioMedical Research Inc
Wang, Yingyun; Novartis Institute of Biomedical research
haling, jacob; Genomics Institute of the Novartis Research Foundation
Vaziri, Sepideh; Genomics Institute of the Novartis Research Foundation,
Medicinal Chemistry
Hekmatnejad, Mohammad; Novartis Institutes for BioMedical Research
Emeryville, Biochemical Lead Discovery
Polyakov, Valery; Novartis, NIBR
zang, Xu(Richard); Novartis, NIBR
Sethuraman, Vijay; Novartis, NIBR
Amiri, Payman; Novartis Institute of Biomedical research
Singh, Mallika; Novartis Institute for BioMedical Research, Global
Discovery Chemistry
Sellers, William; Novartis Institutes for BioMedical Research Inc
Lees, Emma; Novartis Institutes for BioMedical Research Inc
Shao, Wenlin; Astra Zeneca,
Dillon, Michael; Ideaya Biosciences ,
Stuart, Darrin; Novartis
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Design and Discovery of N-(3-(2-(2-hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-
trifluoromethyl)isonicotinamide (LXH254)- A selective, efficacious and well-tolerated RAF inhibitor targeting
RAS mutant cancers: The path to the clinic
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Savithri Ramurthy* , Benjamin R. Taft , Robert J. Aversa , Paul A. Barsanti , Matthew T. Burger , Yan Lou ,
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Gisele A. Nishiguchi , Alice Rico , Lina Setti , Aaron Smith , Sharadha Subramanian , Victoriano Tamez , Huw
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Shenlin Huang , Qin Yue , Apurva Chaudhary , Hung Tian , Raman Iyer , A. Quamrul Hassan , Lesley A.
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Mathews Griner , Laura R. La Bonte , Vesselina G. Cooke , Anne Van Abbema , Hanne Merritt , Kalyani
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R. Sellers , Emma Lees , Wenlin Shao , Michael P. Dillon , Darrin D. Stuart
1 Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville,
California 94608, United States
2
Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue,
Cambridge, Massachusetts 02139, United States
3
. Genomics Institute of the Novartis Research Foundation, 10675 John Hopkins Dr, San Diego, California 92121
4. Oncology, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge,
Massachusetts 02139, United States
5
. Oncology, Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, California 94608,
United States
6
. Technical Research & Development, Global Drug Development, Novartis Pharmaceuticals Corp., One Health
Plaza, East Hanover , New Jersey, 07936, USA
7
. Process Research and Development, Chemical and Analytical Development, Novartis Institute for Biomedical
Research, One Health Plaza, East Hanover, New Jersey 07936, U.S.A.
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Journal of Medicinal Chemistry
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ABSTRACT
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Direct pharmacological inhibition of RAS has remained elusive and efforts to target CRAF have been challenging due to
the complex nature of RAF signaling, downstream of activated RAS and the poor overall kinase selectivity of putative RAF
1
inhibitors. Herein, we describe 15 (LXH254) , a selective B/C RAF inhibitor, which was developed by focusing on drug-
0
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like properties and selectivity. Our previous tool compound 3 (RAF709), a was potent, selective, efficacious, and well-
2
tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further
investigation of close analogs. A structure-based approach led to a pyridine series with an alcohol side-chain that could
interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and
TDI led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being
tested in phase 1 clinical trials.
INTRODUCTION
The RAS-RAF-MEK-ERK or MAPK pathway plays a prominent role in transmitting signals from the cell membrane to the
nucleus. Extracellular growth factors bind and activate cell surface receptor tyrosine kinases resulting in turnover of RAS-
GDP (H-, N-, KRAS) to RAS-GTP in turn resulting in the engagement and activation of RAF kinases (A-, B-, CRAF), thus
initiating the MAPK cascade. Just as this pathway is critical for growth, differentiation and homeostasis of normal tissues,
it also plays a central role in the uncontrolled growth and invasiveness of human cancer. The RAS genes and BRAF are
mutationally activated in approximately one-third of human tumors and genetic inactivation of CRAF prevents the
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development of KRAS mutant tumors in genetically engineered mice. This dependence on signaling
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downstream of mutant RAS has provided therapeutic opportunities for targeting the RAF, MEK and ERK kinases which
are eminently more druggable than RAS, with the possible exception of KRAS G12C for which covalent inhibitor
approaches have been undertaken. 3-5
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Clinical proof-of-concept for targeting RAF has been demonstrated with drugs like dabrafenib (1) which potently inhibit
BRAFV600E and provide significant tumor regression and increased survival in melanoma patients. However, the therapeutic
potential of these drugs is restricted to tumors with BRAFV600 mutations because in this context BRAF functions as a
constitutively active monomer while in the RAS mutant setting, BRAF and CRAF function as dimers which are resistant to
inhibition by these drugs.6-9 Therefore, testing the therapeutic potential of targeting RAF downstream of mutant RAS will
require compounds optimized to inhibit RAF dimers.10 Furthermore, robust evaluation of this therapeutic hypothesis will
require inhibitors with highly selective profiles so that the pharmacology is not compromised by off-target toxicity. For
example, LY3009120 (2), a pan-RAF inhibitor that effectively inhibits active RAF homo-and hetero-dimers has
demonstrated preclinical activity in tumors with RAS mutations. However, 2 inhibits several other kinases, including those
that have important roles in cancer such as Ephrin receptors, JNK and SRC family members which could also lead to off-
1
1
target toxicities. Such a profile could preclude dose-escalation to achieve potent RAF suppression in tumors and ultimately
compromise the therapeutic utility of such a compound. Therefore, a highly selective RAF inhibitor will be invaluable in
assessing the therapeutic utility in RAS mutant and atypical BRAF mutant tumors.
Chart 1
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Journal of Medicinal Chemistry
RESULTS AND DISCUSSION
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Our previous disclosure detailed the medicinal chemistry efforts to identify highly selective RAF inhibitors that potently
suppress the RAF-MEK-ERK pathway in RAS/RAF mutant tumor cells without causing paradoxical activation. This led
to the discovery of a key compound, 3, which enabled numerous early pharmacology studies in RAS/RAF mutant xenograft
models and validated the hypothesis that a wild-type B/CRAF inhibitor could prevent tumorigenesis in a RAS mutant setting
with minimal paradoxical activation.
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A key concern with 3 was the observed high clearance in human microsomes (Clint = 94 µL/min/mg), limiting the projected
human exposure and preventing the compound from progressing as a clinical candidate. For this reason, we set out to
understand the clearance mechanism and design analogs that could mitigate the high intrinsic human clearance. A cross
species metabolism study was performed for 3 in rat, dog and human hepatocytes, where a number of metabolites were
identified by LC/MS/MS (Figure 1). The major metabolites observed were an O-dealkylation product (M3) and tetrahydro-
pyranyl ring oxidation (M4-M5) in all species. In addition, the amide hydrolysis product (M1 and M2) was found to be
relatively abundant in rat hepatocytes compared to other species. No human unique metabolites were identified (Figure 1).
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Figure 1. Chemical structures of metabolites of 3 in rat(R), dog (D), and human (H) hepatocyte (quantification based
on UV response).
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With this knowledge, a focused set of THP analogs, in addition to the metabolite 4, were synthesized and human microsomal
clearance was determined (Table 1). Not surprisingly, metabolite 4 showed 10-fold lower human Clint (9.4 vs 94), although
when tested in a Calu-6 cellular proliferation (CP) assay, the compound was four-fold less potent (EC 3.9 μM vs 0.95
50
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μM). This might be attributed to the significant increase in polarity and negative impact on cellular permeability as
measured in the Caco-2 assay (AB/BA:0.3/43). The fluoro-tetrahydropyranyl analog 5 retained good cellular potency and
permeability, but this substitution had only a modest impact on the human Clint (65.5 vs 94). The methoxy-
tetrahydropyranyl and deutero analogs (6,7) both showed good cellular potency, but neither had improved human Clint, and
in fact, 6 had the highest measured Clint of all the analogs tested.
Table 1: Potency, ADME, and solubility data for tetrahydropyranyl analogues.
Cmpd
R
CRAF
pMEK Calu-6
EC50 (µM)
CP Calu-6
EC50 (µM)
Caco-2
Clint
human microsomal
Sol
cLogP
(
IC50 (µM)
Papp AB/BA (x10-6 cm/s)
(µM)
µL/min/mg)
3
0.0003
0.021
0.95
15/13
94
200
4.2
4
5
H
0.0009
0.064
0.005
3.91
0.34
0.3/43
16/20
9.4
55
25
2.3
4.5
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.0003
65.5
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Journal of Medicinal Chemistry
6
0
0
.0017
.0004
0.021
0.006
1.76
NA
302
88
43
4.3
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0.40
NA
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Table 2: Potency, ADME, and solubility data for CORE analogues.
Cmpd
R
CRAF
pMEK Calu-6
EC50 (µM)
CP Calu-6
EC50 (µM)
Caco-2
Clint
human microsomal
Sol
cLogP
(
IC50 (µM)
Papp AB/BA
(µM)
µL/min/mg)
(
x10-6 cm/s)
8
0.0004
0.0011
0.008
0.052
0.88
1.35
4.4/51
7.7
59.6
33.9
3.3
9
6.3/49
15.5
1.5
Given the limited success in lowering human microsomal clearance by fine-tuning 3, we hypothesized that
increasing the polarity of the molecules might help mitigate oxidative metabolism. Analogs were synthesized
and a representative set is shown in Table 2. Though polar analogs 8 and 9 had significantly lower human
microsomal Clint compared to 3 and acceptable in vitro potency profile, these compounds had poor in vivo efficacy and a
poor tolerability profile limiting their further progression.
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2
We next re-evaluated the original pyrimidine hit scaffold as this series had lower intrinsic clearance (43 µL/min/mg) while
maintaining potent inhibition of pMEK and proliferation in Calu-6 cells. During this time, we considered many strategic
options, one of which was to further optimize the cellular antiproliferative effect and pMEK EC . Although the protein-
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ligand interactions of our lead compounds were highly optimized, we re-examined early X-ray co-crystal structures to
identify opportunities for growing into new vectors and gaining positive interactions with the protein. Careful examination
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of crystal structure of 10 (IC : 0.04 uM) with BRAF (Figure 2a) revealed no hinge binding, unlike typical type 2 B/C RAF
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kinase inhibitors which rely on interaction with the hinge. We attributed the potency to a very weak H-bonding interaction
(N…O distance is slightly more than 3 Å) with the backbone carbonyl of F595 which could impact binding (Figure 2a),
and is unique to the best of our knowledge, for Type 2 B/CRAF inhibitors. This prompted the team to search for compounds
that are capable of interacting both with the hinge and with F595. Docking studies allowed us to hypothesize that compounds
from our lead series could interact with F595 by extending an H-bond donor from the second carbon atom of the pyrimidine
analog as with 11 (Table 3). This strategy led to the synthesis of many new analogs as represented in Table 3 and we found
that interaction with F595 led to compounds with significant improvement in cellular potency, although we do not have a
clear mechanistic understanding for this observation. The F595 H-bonding interaction was best exemplified by 12, which
included an amino-ethanol sidechain at the second carbon of the pyrimidine ring and is a ‘matched-pair’ with 11. As can
be seen in the docking model of 12 (Figure 2b), the sidechain extends directly toward the F595 carbonyl without disrupting
the conformation or other primary interactions. The oxygen atom of the sidechain is 2.9 angstroms away from the carbonyl
°
of F595 and has an approach angle of 118 , strongly suggesting an H-bond with the protein. Compound 12, showed a 33-
fold improvement in cell activity compared to 11 (CP Calu-6 EC = 0.16 vs 5.28 μM), despite having slightly lower
5
0
permeability (Table 3).
A
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Journal of Medicinal Chemistry
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Figure 2: A: X-ray structure of 10 in BRAF (PDB 6N0Q) B: Docking Models of 11 and 12 showing H-bond interaction
with F595
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3: Potency, ADME, and TDI for pyridine/pyrimidine analogues.
Cmpd
R
CRAF
pMEK Calu-
6 EC50 (µM)
CP Calu-6
EC50 (µM)
Clint
human
*Caco-2
**Sol ***cLogP ****TDI
(
IC50
Papp
(µM)
(kobs
microsomal
µL/min/mg)
(µM)
AB/BA (x10-
min-1)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
cm/s)
1
1
0
0
0
.0001
.0002
.0006
--
5.28
0.16
0.51
6.22
0.47
19/41
8/30
5/53
3/34
9/19
43.3
48.0
20.5
10.4
13.5
13
4.0
4.1
3.2
2.1
3.5
1
2
0.061
0.008
0.027
0.014
2.1
13
36.3
0.035
0.035
0.01
1
4
0.0007
730.
2
1
5
0
.0002
20.0
*
Forward and reverse flux were measured via transit across 21-day cultured Caco-2 monolayers at pH7.4 with quantification
via LC/MS/MS. **Solubility (shake–flask assay) was measured with Cl-free PBS at pH 6.8. ***cLogP was calculated using
Biobyte software.
1
3
****Time-dependent inhibition (TDI; k ) measured as described by Zimmerlin et. Al., Time dependent CYP3A4
obs
inactivation screening assay (k ):Test compound at 10 µM is incubated with 0.5 mg/mL human liver microsome in
obs
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Journal of Medicinal Chemistry
°
phosphate buffer containing 1 mM NADPH at 37 C for 0, 5, 15, and 30 minutes. The incubation mixture is then diluted
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
°
2
0 times and incubated with CYP3A4 substrate midazolam (20 µM) at 37 C for 6 minutes to determine residual CYP3A4
enzyme activity. The enzyme activity vs. incubation time is plotted to obtain the initial rate of decline which is defined as
kobs value.
Despite the improved cellular antiproliferative effect of 12, the compound had low solubility, high lipophilicity and sub-
optimal Clint (HLM). With this in mind, analogs with greater polarity in the aryl-rings were investigated, exemplified here
by 13 and 14 (Table 3). When comparing 12, 13 and 14, there was a clear trend that Clint (HLM) could be mitigated and
solubility could be improved by increasing polarity albeit at the cost of reduced permeability and decreased cellular potency.
In addition, during extended safety profiling of this chemical series, we observed a Cyp3A4 TDI signal which did not
correlate with lipophilicity. It appeared that all pyridine analogs 13 and 14 bearing an amino-ethanol sidechain had a high
risk for TDI (kobs > 0.03) and this led us to hypothesize that the issue was associated with the electron-rich bis-amino
pyridine functionality embedded in each compound. In an effort to make the rings electron deficient, the amino-ethanol
side chain was changed to an ethylene glycol side chain (replacing nitrogen with oxygen). This strategy led to the discovery
of 15 which maintained the critical F595 H-bond, but exhibited low risk for Cyp3A4 TDI (kobs = 0.01) with the best balance
of ADME properties and good cellular potency.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
X-ray structure of 15. The X-ray co-crystal structure of 15 in BRAF, as illustrated in Figure 3, showed a binding mode
2
similar to 3. As with 3, the central toluyl tightly occupies a narrow hydrophobic pocket formed by a number of the
sidechains including K483 and the gatekeeper residue T529. The CF pyridyl moiety occupies the hydrophobic pocket
3
formed by rotation of the DFG group, i.e. DFG-out, similar to the structure of compound 3 and other Type 2 kinase
14
inhibitors. The biggest difference with previously known structures was that the -hydroxyl of the glycolic moiety of 15
formed a strong hydrogen bond to the carbonyl of F595 manifested by 2.6 angstroms distance between the oxygen atoms
°
and an approach angle of 120 . All other hydrogen bonds seemed to be retained in the co-crystal, which might explain the
strong potency of the compound.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3: X-ray structure of 15 in BRAF (PDB 6N0P)12
1
5
In vitro profiling of 15 . The biochemical activity of 15 was further characterized against purified full-length BRAF
where the IC was 0.0004 µM, and consistent with the potency against CRAF. Off-rate can be an important attribute of
5
0
potency, affecting both the cellular activity in the face of feedback and the PK/PD relationship in vivo. The dissociation rate
constant for 15 was measured using the rapid dilution method and full-length CRAF (with activating mutations
Y340E/Y341E) kinase assay. The compound has a slow dissociation rate constant (T > 6.5 hrs), as there was little recovery
1
/2
of enzyme activity after 6 hours of incubation in the kinase assay.
In cellular assays, the dose-response of 15 was measured using a pMEK assay in Calu-6 cells with an EC = 0.05 µM,
5
0
minimal paradoxical activation (Figure 4) and inhibition of proliferation with EC = 0.28 µM. These data are clearly distinct
5
0
from Compound 1 which demonstrated clear paradoxical activation in the pMEK assay (Figure 4) with maximum activity
at 3 µM and no resultant inhibition up to the highest concentration tested (24 μM).
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1
2
3
4
5
6
7
8
9
1
00
1
5
0
15
0
-
-100
150
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
Log [M] (compound), 2 hours
Figure 4: pMEK activity in Calu-6 cells of 15 vs 1
The ability of 15 to stabilize RAF dimerization was measured using the CRAF:BRAF NanoBiT™ luciferase
complementation assay (Promega). Consistent with most other RAF inhibitors11,16 15 stabilized BRAF-CRAF
dimers with an EC = 0.16 µM (Figure 5), while 1 yielded no significant effect on RAF dimerization as reported
5
0
previously. Additional supporting data was revealed in the X-ray crystal structure of 15 (Figure 3), where each
BRAF protomer in the dimer structure is occupied with compound. This also demonstrated that (at least under
the conditions of crystallization), binding of 15 to one protomer did not preclude binding to the other protomer in
the dimer structure. Altogether, these data demonstrate that 15 stabilizes RAF dimers but is relatively equipotent
at inhibiting both protomers since it induces minimal paradoxical activation but effectively suppresses signaling.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Effect of 1 and 15 on the RAF dimerization in HCT116 cells.
2
Kinase Selectivity of 15. Consistent with our early in vivo tool compound , 15 was found to be highly selective when
evaluated using the KINOMEscanTm screening platform. Of the 456 kinases tested, 15 showed a high level of selectivity
(Figure 6) , demonstrating greater than 98% on-target binding to BRAF, BRAFV600E, and CRAF at 1 µM and very few off-
targets with DDR1 (>99%), DDR2 (84%), PDGFRb (>99%) the only kinases with binding >80% at 1 µM. The Kd values
for these kinases was determined and were consistent with 15 being a potent inhibitor of BRAF (Kd = 1.3 nM) and CRAF
(Kd = 3.6 nM) with similar potency against DDR1 (Kd = 1.8 nM) and less against DDR2 (Kd = 10 nM) and PDGFRb (Kd
=
14 nM). To the best of our knowledge, this compound along with our earlier series, exhibit an extremely high level of
kinase selectivity relative to other type 2 RAF inhibitors with a selectivity score of S(35) = 0.025. 17,18
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Journal of Medicinal Chemistry
Figure 6: KINOMEscan profile of 15
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Pharmacokinetics of 15. In pre-clinical pharmacokinetic experiments (Table 4), 15 had low to moderate clearance in
mouse (19 mL/min/kg), rat (31 mL/min/kg) and dog (3.5 mL/min/kg). Cmax in mouse (1.6 µM), rat (0.5 µM) and dog (0.4
µM) reached pharmacologically active concentrations and acceptable oral availability was observed in mouse (65%), rat
(38%) and dog (79%).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4: PK profile of 15 across species
Dose
AUCinf
(p.o.,
Cmax
Cl
Vss
F( po)
Species
(i.v./p.o,
(p.o., µM)
(mL/min/kg)
(L/kg)
(%)
mg/kg)
M*hr)
Mouse
Rat
2 / 4
2 / 4
1.6
0.5
0.4
4.3
1.8
3.1
19
31
2.1
5.4
1.7
65
38
79
Dog
0.2 / 0.4
3.5
*
: formulation (i.v./p.o): 25%PEG300 + 5%Solutol
PK/PD of 15. The PK/PD/efficacy relationship of 15 was examined in nude rats bearing Calu-6 (KRASQ61K) human NSCLC
xenograft tumors. The plasma exposure and pMEK levels in tumor tissue were determined following a single oral
administration of 15 across a dose range of 15, 35, 75, or 150 mg/kg. For each treatment group, tumor and blood samples
were collected at 1, 4, 7, 24, and 48 hours post-dose. The effect of 15 on pMEK levels in the tumor was determined using
an MSD immunoassay. The free plasma exposures of 15 were shown to be dose-proportional, corresponding with dose-
dependent reductions in pMEK levels in tumors (Figure 7). At the higher dose level of 150 mg/kg, free plasma
concentrations of 15 were maintained above the in vitro cellular pMEK IC value in Calu-6 for at least 24 hours, correlating
5
0
with a sustained in vivo pMEK target inhibition of ~70% for the same duration.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7 PK/PD analysis of 15 in the human Calu-6 NSCLC xenograft in rats. Percent pMEK levels (left axis), and free
exposure (right axis) following a single dose of 15, at time points indicated, in Calu-6 tumor bearing rats.
Efficacy and tolerability of 15 in the Calu-6 xenograft model. The antitumor efficacy and tolerability of 15 were
determined in the Calu-6 xenograft nude rat model. Rats were treated with vehicle or 15 at 15, 35, 75 or 150 mg/kg p.o.
daily beginning 13 days after tumor implantation and continued until day 31. The anti-tumor activity was determined by
assessing %T/C or % regression on day 31 post-implant (18 days of treatment). Treatment with 15 resulted in dose-
dependent anti-tumor activity with 15 mg/kg achieving 29% T/C. Treatment with 35 mg/kg resulted in tumor stasis
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Journal of Medicinal Chemistry
(
%T/C=9%) and both 75 mg/kg and 150 mg/kg resulted in mean tumor regression of 21% and 56 % respectively (Figure
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
a). All doses were well-tolerated with no significant body weight loss and no signs of toxicity or mortality (Figure 8b).
Following the last dose of 15 in the efficacy study, plasma was collected, the concentration of 15 was measured and area
under the curve (AUC) calculated for each dose (Table 5). 15 showed dose proportional plasma exposures and total plasma
concentrations of AUC0-24=104510 nM*h at steady state was associated with the lowest dose achieving tumor regression.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8 Efficacy of 15 in Calu-6 xenograft in rats. Tumor volumes (a) or percent body weight change from initial (b)
treatment groups vs. vehicle control.
Table 5: Mean plasma pharmacokinetic parameters of 15 on day 31
1
5mg/kg
35mg/kg
25210
2347
3
75mg/kg
104510
8680
2
150mg/kg
216680
12671
5.5
AUC (nM*h)
Cmax (nM)
Tmax (h)
7913
878
3
Tlast (h)
24
24
24
24
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With these encouraging rodent pharmacology activities and pharmacokinetic properties across species, 15 was
profiled further with respect to developability and drug like properties. The melting point of 15 as a free base is
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
192 C. Free form 15 has a log P>3, log D>4 (at pH 6.5) and pKa of 3.86. Its crystalline form solubility is 2
µg/mL. To increase the oral bioavailability, an amorphous solid dispersion formulation was developed. The solid
dispersion was formed by combining drug and a stabilizing polymer in a suitable ratio along with other adjuvants
under high thermomechanical stress followed by cooling and milling. The permeability (A-B) of 15 is medium
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-6
to high at 9*10 cm/s. The stability in human plasma is high, with >90% intact after a 3 h incubation and the
human plasma protein binding is 98%. In a manual patch clamp hERG assay, the compound exhibited a >10 uM
IC .
50
On the basis of favorable cellular potency, kinase selectivity, preclinical pharmacology and physical properties,
1
5 was advanced into human clinical trials.
Chemistry:
Scheme 1. Synthesis for analogs 3-9.
Reagents and Conditions. (a) O(CH CH Br) , NaH, DMF, 0 °C to RT. (b) morpholine, EtOH, RT. (c) N-(6-
2
2
2
methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide,
PdCl (dppf)∙CH Cl DME, 2M Na CO , 120 °C. (d) R-OH, NaH, dioxane or DMF, 90°C. (e) NaNO , H SO ,
2
2
2,
2
3
2
2
4
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Journal of Medicinal Chemistry
acetic acid, 0-25 °C, then water, 25 °C. (f) Cs CO , MeI, DMF. (g) morpholine, DMF, DIEA, 120 °C. (h) 4-
2
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, PdCl (dppf)∙CH Cl , DME, 2M Na CO , 120 °C.
2
2
2
2
3
(i) Aryl-CO H, HATU, DIEA, DMF.
2
Compounds 3-9 were synthesized according to Scheme 1. In order to enable last step diversification and obtain
final compounds exemplified in Table 1, intermediate 18 was synthesized from commercially available 16 via
bis-alkylation with O(CH CH Br) Subsequent Suzuki-Miyaura coupling reaction between 18 and N-(6-methyl-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2.
2
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide yielded a fluoro-
pyridine intermediate which was penultimate to the desired analogs. Treatment of this intermediate with the
desired pre-formed alkoxide in dioxane or dimethylformamide allowed for isolation of final products (3-7) upon
HPLC purification (Scheme 1). The pyridazine analog 8 was prepared in a similar fashion starting from
2
commercial tri-chloride 17. Compound 4 was prepared as reported in our last paper. Compound 9 was prepared
starting from amino-pyridazine 20 via hydrolysis of the corresponding diazonium species and subsequent N-
methylation to give 21. S 2 addition of morpholine, followed by Suzuki-Miyaura coupling of 4-methyl-3-
N
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline provided intermediate 22, which was transformed to final
compound 9 via typical amide coupling conditions. Scheme and synthesis of compound 10 is reported. 2
Synthesis and experimental procedure for Compound 10 is reported in supporting information.
Scheme 2. Synthesis of analogs 12-15.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and Conditions. (a) morpholine, DIPEA, EtOH, RT or 55°C. (b) N-(4-methyl-3-(4,4,5,5-tetramethyl-
,3,2-dioxaborolan-2-yl)phenyl)-3-(trifluoromethyl)benzamide, PdCl (dppf)∙CH Cl , DME, 2M Na CO 120 °C.
1
2
2
2
2
3,
(
c) mCPBA, DCM, RT. (d) R-OH, NaH, dioxane, NMP, 150 °C or R-NH K CO DMSO 55°C. (e) (2,6-
2
,
2
3,
,
difluoropyridin-4-yl)boronic acid, PdCl (dppf)∙CH Cl , DME, 2M Na CO 60 °C. (f) H 10% Pd/C, EtOH. (g)
2
2
2
2
3,
2,
Aryl-CO H, HATU, DIPEA, DMF.
2
The synthesis of analogs 12-15 is outlined in Scheme 2. Compound 12 was synthesized starting from
dichloropyrimidine 23 which was first subjected to S 2 reaction with morpholine, followed by Suzuki-Miyaura
N
coupling
with
N-(4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-(trifluoromethyl)
benzamide, and then oxidation via mCPBA to yield intermediate 24. Compound 24 was converted to analog 12
via S 2 reaction with 2-aminoethanol.
N
Compounds 13-15 were prepared from commercial aryl- and pyridyl-nitro compounds 25 and 26. Intermediates
2
9 and 30 were synthesized via Suzuki-Miyaura coupling reaction with (2,6-difluoropyridin-4-yl)boronic acid
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Journal of Medicinal Chemistry
followed by S 2 reaction with morpholine. Hydrogenation then yielded intermediates 31 and 32. Introduction
N
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of the side-chain groups at the 2-position of pyridine was performed through S Ar reaction, and subsequent
N
coupling of the anilines with carboxylic acids yielded 13-15 upon HPLC purification.
Scheme 3. Optimized synthesis of 15.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and Conditions. (a) (2,6-difluoropyridin-4-yl)boronic acid, Pd-XPhos precatalyst, 0.5 M K PO , THF
3
4
,
3
5-60 °C, 64% yield. (b) morpholine, K CO , DMSO, 40°C, quantitative yield. (c) 2-((tetrahydro-2H-pyran-2-
2 3
yl)oxy)ethanol, NaH, dioxane, 60-70 °C, 72% yield. (d) 2-(trifluoromethyl)-isonicotinic acid, EDC∙HCl, HOAT,
DIPEA, DMF, RT; then 2 M aqueous HCl, RT, 94% yield.
An optimized synthetic route for 15, used to supply larger quantities of material for in vivo studies, is oulined
in Scheme 3. A direct Suzuki-Miyaura coupling of 3-bromo-4-methylaniline 33 with the previously used
boronic ester was possible using the Pd-XPhos precatalyst. Installation of the morpholine and
ethylyene glycol moities were accomplished by successive S Ar reactions, providing aniline 35.
N
Coupling of the aniline with the 2-(trifluoromethyl)-isonicotinic acid was followed by in situ acidic
deprotection of the glycolic THP protecting group to afford 15 in high yield.
CONCLUSION:
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Despite the clinical efficacy of RAF and MEK inhibitors in BRAFV600 mutant melanoma19-21 they are ineffective
in RAS mutant tumors, leaving a significant unmet medical need. This class of RAF inhibitors approved for the
treatment of BRAFV600mut melanoma, including 2, induce paradoxical activation in BRAF wild-type cells and can
induce growth of RASmut tumors. However, given the central role of CRAF in driving mutant KRAS-driven
tumorigenesis, there remains a significant interest in developing RAF inhibitors that will be efficacious in RAS
mutant tumors. Our work demonstrates that type 2 RAF inhibitors have the potential to be effective in RAS mutant
tumors because they induce minimal paradoxical activation compared to RAF inhibitors with other binding modes
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
(
i.e. type 1 or type 1.5). A previously disclosed in vivo tool 3 gave the first evidence supporting this hypothesis,
but could not be progressed further due to high intrinsic clearance in human microsomes. Due to limitations with
the in vivo profile, we focused our efforts towards balancing physicochemical properties such as solubility and
Cl(int) HLM with cellular potency, by tweaking the electronics of the ring system. This approach led to the
identification of 15, a compound shown to be highly kinase-selective and cellularly potent in a RAS mutant cell
line (Calu-6) with minimal paradoxical activation. We believe this profile will enable the clinical development of
the compound as a single-agent or in combination therapy. With the combination of potent in vitro activity
and low to moderate CL, 15 demonstrates in vivo target modulation (pMEK), single agent antitumor
activity in the Calu-6 rat xenograft model, and drug-like properties suitable for development. 15 was
advanced into human studies and is currently being assessed in phase I trials.
EXPERIMENTAL SECTION:
General Methods. The compounds and/or intermediates were characterized by high performance liquid
chromatography (HPLC) using a Waters Millennium chromatography system with a 2695 separation module
(Milford, MA). The analytical columns were Alltima C-18 reversed phase, 4.6 mm x 50 mm, flow 2.5 mL/min,
from Alltech (Deerfield, IL). A gradient elution was used, typically starting with 5% acetonitrile/95% water and
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Journal of Medicinal Chemistry
progressing to 100% acetonitrile over a period of 10 min. All solvents contained 0.1% trifluoroacetic acid (TFA).
Compounds were detected by ultraviolet light (UV) absorption at either 220 or 254 nm. HPLC solvents were
from Burdick and Jackson (Muskegan, MI) or Fisher Scientific (Pittsburgh, PA). Mass spectrometric analysis
was performed on an LCMS instrument: a Waters system (Alliance HT HPLC and a Micromass ZQ mass
spectrometer, Eclipse XDB-C18, 2.1 mm x 50 mm; solvent system, 5-95% acetonitrile in water with 0.1% TFA;
flow rate 0.8 mL/min; molecular weight range 200-800; cone voltage 20 V; column temperature 40 °C). All
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
masses were reported as those of the protonated parent ions. H nuclear magnetic resonance (NMR) analyses
described herein were performed on some of the compounds with a Varian 400-MR MHz NMR (Palo Alto, CA)
1
spectrometer operating at a frequency of 399.89 MHz for H or Bruker DRX-500 NMR spectrometer operating
1
at a frequency of 500.13 MHz for H. The spectral reference was either TMS or the known chemical shift of the
solvent. The spectra were recorded at a temperature of 298 K. Preparative separations were carried out using a
Teledyne ISCO chromatography system, or by HPLC using a Waters 2767 sample manager, C-18 reversed phase
column, 30 x 50 mm, flow 75 mL/min. Typical solvents employed for the Teledyne ISCO chromatography system
and were dichloromethane, methanol, ethyl acetate, and heptane. Typical solvents employed for the reverse phase
HPLC were varying concentrations of acetonitrile and water with 0.1% trifluoroacetic acid. The purity of all
compounds screened in the biological assays was examined by LC-MS analysis and were found to be ≥95%.
Experimental details for the synthesis and characterization of compounds 3, 4 and 11 are reported earlier.2
General procedure for S Ar: To a mixture of sodium hydride, 60% in mineral oil (3-4 mmol) in dioxane or
N
DMF at ambient temperature was added the appropriate alcohol (1.6 mmol). To the mixture was added N-(6'-
2
fluoro-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide and the mixture was stirred
for 2 h at 90 °C. The cooled reaction mixture was poured into water and extracted twice with EtOAc). The
combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated. The mixture
was purified by reverse phase HPLC to give the desired product.
General procedure for the amide formation reaction: To the appropriate aniline (0.050 mmol) in DMF
(Volume: 0.75 mL) at RT were added HATU (0.050 mmol) and Huenig's Base (0.137 mmol) and the mixture
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was stirred overnight. The reaction was filtered, dissolved in DMSO and then purified via reverse phase HPLC
to give the desired product.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General Procedure for the Suzuki-Miyaura reaction: To the appropriate heteroaryl halide (0.10 mmol) and
the appropriate boronic ester (0.12 mmol) in DME (1 mL) were added PdCl (dppf)∙CH Cl adduct (0.010 mmol)
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and 2M aqueous sodium carbonate (0.50 mmol). The reaction mixture was irradiated at 120 °C in a Biotage
Initiator microwave for 10-12 min. The cooled reaction mixture was diluted with 2:1 DCM:MeOH (15 mL) and
filtered. The filtrate was concentrated and purified by reverse phase HPLC to give the desired product as its TFA
salt.
N-(6'-(((3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-
(trifluoromethyl)benzamide (5): To a stirred solution of 3-fluorodihydro-3-fluorodihydro-2H-pyran-4(3H)-one
o
(
100 mg, 0.847 mmol) in THF (5 mL) at -78 C was added L-selectride (1 M in THF, 0.847 mL, 0.847 mmol)
and the mixture was warmed to RT and stirred until no bubbling was observed. To the mixture was added 4-(5-
bromo-2-fluoropyridin-3-yl)morpholine (221 mg, 0.847 mmol) and the reaction mixture was refluxed for 2h. The
mixture was concentrated and purified by preparatory HPLC to give 4-(5-bromo-2-(((3R,4S)-3-fluorotetrahydro-
2
H-pyran-4-yl)oxy)pyridin-3-yl)morpholine (54 mg, 17.6% yield). LCMS m/z [M + H] =361/363, Rt = 0.88 min.
A mixture of 4-(5-bromo-2-(((3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)oxy)pyridin-3-yl)morpholine (54 mg,
.15 mmol), N-(6-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-3-
trifluoromethyl)benzamide(60.7 mg, 0.149 mmol), 2M sodium carbonate solution (0.224 mL, 0.448 mmol) and
PdCl (dppf).CH Cl adduct (6 mg, 0.0007 mmol) in DME (2mL) was stirred at RT overnight. The reaction
0
(
2
2
2
mixture was concentrated and purified by preparatory HPLC. The racemic mixture obtained was purified by
chiral HPLC to give N-(6'-(((3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)oxy)-2-methyl-5'-morpholino-[3,3'-
bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (9.9 mg, 11.6% yield). LCMS m/z [M + H] =561.2, Rt = 0.78min.
+
HRMS m/z (M +1) calcd 561.2119, obsd 561.2115.
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Journal of Medicinal Chemistry
The following compounds were prepared by the above method starting from the appropriate heteroaryl-halide
and the appropriate alcohol
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N-(6'-(((3S,4S)-3-methoxytetrahydro-2H-pyran-4-yl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-
+
3-(trifluoromethyl)benzamide (6): Obtained in 2% yield. LCMS m/z [M + H ] = 573.1, Rt = 0.76 min. HRMS
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
m/z (M +1) calcd 573.2319, obsd 573.2312.
N-(2-methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl-4-d)oxy)-[3,3'-bipyridin]-5-yl)-3-
+
1
(
trifluoromethyl)benzamide (7): Obtained in 6% yield. LCMS m/z [M + H ] = 544.1, Rt = 0.8 min. H NMR
(
500 MHz, d -DMSO) δ ppm 1.72 (ddd, J = 12.8, 8.3, 4.0 Hz, 2H), 2.05 (dt, J = 9.1, 4.0 Hz, 2H), 3.11 (t, J = 4.5
6
Hz, 3H), 3.58 (ddd, J = 11.6, 8.2, 3.2 Hz, 2H), 3.77 (t, J = 4.5 Hz, 4H), 3.85 (dt, J = 10.9, 4.9 Hz, 2H), 7.29 (d, J
=
2.1 Hz, 1H), 7.90 – 7.77 (m, 2H), 8.03 (d, J = 7.8 Hz, 1H), 8.26 (s, 1H), 8.31 (d, J = 7.9 Hz, 1H), 8.35 (s, 1H),
+
9.03 (d, J = 2.3 Hz, 1H), 10.93 (s, 1H). HRMS m/z (M+ + 1) calcd, obsd. HRMS m/z (M +1) calcd 544.2276,
obsd 544.2271.
N-(6-methyl-5-(5-morpholino-6-((tetrahydro-2H-pyran-4-yl)oxy)pyridazin-3-yl)pyridin-3-yl)-3-
(trifluoromethyl)benzamide (8): Obtained in 43% yield. LCMS (m/z) (M+H) = 544.2, Rt = 0.75 min. LCMS (m/z)
1
(
M+H) = 472.3, Rt = 0.88 min. H NMR (400 MHz, d -DMSO)  ppm 1.03 (t, J=7.04 Hz, 1 H) 1.76 (dtd, J=12.62,
6
8
.36, 8.36, 3.91 Hz, 2 H) 2.05 - 2.17 (m, 2 H) 3.32 - 3.38 (m, 4 H) 3.57 (ddd, J=11.44, 8.51, 3.13 Hz, 2 H) 3.69 -
.77 (m, 4 H) 3.79 - 3.88 (m, 2 H) 5.51 (tt, J=8.02, 3.91 Hz, 1 H) 7.06 (s, 1 H) 7.79 (t, J=7.83 Hz, 1 H) 7.98 (d,
3
J=7.83 Hz, 1 H) 8.20 (d, J=2.35 Hz, 1 H) 8.28 (d, J=7.83 Hz, 1 H) 8.32 (s, 1 H) 8.88 (d, J=2.74 Hz, 1 H) 10.68
+
(
s, 1 H). HRMS m/z (M + 1) calcd, obsd. HRMS m/z (M+ + 1) calcd 544.2166, obsd 544.216.
2
-(2-cyanopropan-2-yl)-N-(4-methyl-3-(1-methyl-5-morpholino-6-oxo-1,6-dihydropyridazin-3-
yl)phenyl)isonicotinamide (9): To a cooled solution (0-5 °C) of sodium nitrite (1.350 g, 19.57 mmol) in
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concentrated H SO (10.1 mL, 189 mmol) was added 4-bromo-6-chloropyridazin-3-amine (1.7 g, 8.16 mmol) in
2
4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acetic acid (33 mL). The reaction mixture was then stirred at 0 °C for 30 min before warming to RT. It was stirred
for 1 h followed by the addition of water (51 mL), and stirred at RT for a further 4 h. The reaction mixture was
then extracted with EtOAc, and the organic layer was dried over MgSO , filtered, and concentrated in vacuo to
4
yield a brown oil which was further purified by flash column chromatography over silica gel, eluting with 100%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
heptanes to 80% EtOAc:heptanes to yield 4-bromo-6-chloropyridazin-3(2H)-one (1.42 g, 6.78 mmol) in 83%
1
yield. LCMS m/z [M + H] =210.9/212.9, Rt = 0.42 min. H NMR (400 MHz, d -DMSO) δ ppm 8.08 - 8.32 (m,
6
1
H) 13.25 - 13.71 (m, 1 H)
To a solution of 4-bromo-6-chloropyridazin-3(2H)-one (500 mg, 2.387 mmol) and Cs CO (933 mg, 2.86 mmol)
2
3
in DMF (33 mL) was added MeI (0.224 mL, 3.58 mmol) dropwise over 20 min. The resulting mixture was stirred
for 3 h. The reaction mixture was then diluted with saturated aqueous NH Cl and then extracted twice with
4
EtOAc. The combined organics were dried over MgSO , filtered and concentrated in vaccuo to yield a brown
4
solid. The oil was further purified by flash column chromatography over silica gel, eluting with 100% heptanes
to 80% EtOAc:heptanes to give 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one as an off white solid (423 mg,
1
.9 mmol) in 79% yield.
To a solution of 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (300 mg, 1.343 mmol) in DMF (4.5 mL) was
added DIPEA (0.234 mL, 1.343 mmol) and morpholine (0.117 mL, 1.343 mmol) at RT. The resulting mixture
was heated to 120 °C for 5 h. The reaction mixture was then diluted with water and extracted twice with EtOAc.
The combined organics were dried over MgSO , filtered and concentrated in vacuo to give 6-chloro-2-methyl-4-
4
morpholinopyridazin-3(2H)-one (300mg, 1.3 mmol) in 97% yield. LCMS m/z [M + H] =230/232, Rt = 0.64 min.
The solid was utilized without further purification.
A solution of 6-chloro-2-methyl-4-morpholinopyridazin-3(2H)-one (100 mg, 0.435 mmol), Na CO (302 mg,
2
3
2
.85 mmol) and 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (112 mg, 0.479 mmol) and
PdCl (dppf)∙CH Cl adduct (178 mg, 0.5 mmol) in DME (1.45 mL) and water (0.726 mL) was heated under
2
2
2
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Journal of Medicinal Chemistry
microwave irradiation for 40 min at 120 °C. The reaction mixture was then diluted with EtOAc and water, and
the aqueous layer was then separated and extracted twice with EtOAc. The combined organics were dried over
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MgSO , filtered and concentrated in vacuo. The residue was purified by reverse phase HPLC to afford 9 as a
4
1
brown solid in 24% yield. LCMS (m/z) (M+H) = 473.4, Rt = 0.84 min. H NMR (500 MHz, d -DMSO) δ ppm
6
1
7
.75 (s, 6 H) 2.29 (s, 3 H) 3.36 - 3.51 (m, 4 H) 3.57 - 3.76 (m, 7 H) 6.59 (s, 1 H) 7.30 (d, J=8.22 Hz, 1 H) 7.65-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.78 (m, 2 H) 7.85 (d, J=3.91 Hz, 1 H) 7.94 - 8.06 (m, 1 H) 8.79 (d, J=5.09 Hz, 1 H) 10.56 (s, 1 H). HRMS m/z
+
(
M +1) calcd 473.2296, obsd 473.2291.
N-(4-methyl-3-(1-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)phenyl)-3-
trifluoromethyl)benzamide (10): NaH (60% in mineral oil, 1.64 g, 41.0 mmol) was added portionwise to a
(
stirred solution of 1H-benzo[d]imidazol-2(3H)-one (5 g, 37.3 mmol) in dry DMF (100 mL) at RT that was
maintained under an atmosphere of argon. After 75 min, a solution of di-tert-butyl dicarbonate (8.14 g, 37.3
mmol) in dry DMF (20 mL) was added dropwise, and the mixture was stirred at RT for 22 hours. The solvent
was removed in vacuo, and the residue diluted with sat. NH Cl solution and extracted twice with EtOAc. The
4
combined organics were dried over MgSO , filtered and concentrated in vacuo. The residue was purified by flash
4
column chromatography over silica gel using a mixture of hexane and EtOAc (7:3) as eluent to furnish tert-butyl
2
-oxo-2,3-dihydro-1H-benzo[d]imidazole-1-carboxylate (6.92 g, 29.5 mmol, 79 % yield) as a white solid. LCMS
1
(m/z) (M+H) = 235.1, Rt = 0.76 min. H NMR (400 MHz, CDCl ) δ 1.70 (s, 9 H) 7.05 - 7.21 (m, 3 H) 7.67 - 7.84
3
13
(
m, 1 H) 9.16 -9.30 (m, 1 H). C NMR (101 MHz, CDCl ) δ ppm 28.13 (s, 1 C) 76.70 (s, 1 C) 77.01 (s, 1 C)
3
7
7.33 (s, 1 C) 85.04 (s, 1 C) 109.90 (s, 1 C) 114.51 (s, 1 C) 122.12 (s, 1 C) 124.18 (s, 1 C) 126.91 (s, 1 C) 127.67
(s, 1 C) 148.61 (s, 1 C) 153.27 (s, 1 C).
Bromine (0.263 mL, 5.12 mmol) was added dropwise to a stirred solution of tert-butyl 2-oxo-2,3-dihydro-1H-
benzo[d]imidazole-1-carboxylate (1 g, 4.27 mmol) and sodium acetate (0.366 g, 5.55 mmol) in acetic acid (13
mL) at RT. After 10 min, a precipitate formed and the mixture was continually stirred at RT for 2 h. The mixture
was diluted with ice/water, and the yellow solid was filtered off and dried in air to give tert-butyl 6-bromo-2-oxo-
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2
,3-dihydro-1H-benzo[d]imidazole-1-carboxylate (1.2937 g, 4.13 mmol, 97 % yield) as a white solid. LCMS
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(m/z) (M+H) = 334.9/336.9, Rt = 0.93 min. H NMR (400 MHz, CDCl ) δ 1.69 (s, 10 H) 6.92 - 6.96 (m, 1 H)
3
7.28 - 7.33 (m, 1 H) 7.92 -8.00 (m, 1 H) 9.03 - 9.15 (m, 1 H). 13C NMR (101 MHz, CDCl ) δ ppm 28.13 (s, 1 C)
3
7
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6.70 (s, 1 C) 77.01 (s, 1 C) 77.33 (s, 1 C)85.04 (s, 1 C) 109.90 (s, 1 C) 114.51 (s, 1 C) 122.12 (s, 1 C) 124.18 (s,
C) 126.91 (s, 1 C) 127.67 (s, 1 C)148.61 (s, 1 C) 153.27 (s, 1 C).
0
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A mixture of tert-butyl 6-bromo-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-1-carboxylate (300 mg, 0.958 mmol),
MeI (0.090 mL, 1.437 mmol) and potassium carbonate (212 mg, 1.533 mmol) in acetonitrile (5 mL) was stirred
at RT under argon for 16 h. As the reaction was incomplete by LCMS after this time, another 212 mg of potassium
carbonate and 0.090 mL of methyl iodide were added into this mixture. It was stirred under argon at RT for
another 5 hours. Upon completion of the reaction, EtOAc was added and the organic layer was washed with water,
dried over sodium sulfate, filtered and concentrated. The crude residue was purified by flash column
chromatography over silica gel, eluting with 50-100% ethyl aceatate in hexanes, to give tert-butyl 6-bromo-3-
methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-1-carboxylate (120.6 mg, 0.369 mmol, 38.5 % yield). LCMS
(m/z) (M+H) = 228.9/230.9.
Tert-butyl 6-bromo-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-1-carboxylate (120.6 mg, 0.369 mmol)
was dissolved in 2 mL of 1:1 TFA/DCM. The reaction mixture was stirred at RT for 30 minutes under argon. The
solution was concentrated to remove most of solvent and TFA, basified to neutral with saturated aqueous sodium
carbonate solution. The solution was extracted with ethyl acetate and the organic layer was separated, dried over
sodium sulfate, filtered and concentrated. The crude residue was purified by flash column chromatography over
silica gel to yield 5-bromo-1-methyl-1H-benzo[d]imidazol-2(3H)-one in quantitative yield.
A mixture of 5-bromo-1-methyl-1H-benzo[d]imidazol-2(3H)-one (70 mg, 0.308 mmol), 4-methyl-3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (216 mg, 0.925 mmol), and cesium carbonate (100 mg, 0.308 mmol)
in dioxane (6 mL) and water (1.5 mL) was purged under argon for 3 minutes, Pd(PPh ) (35.6 mg, 0.031 mmol)
3 4
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Journal of Medicinal Chemistry
was added into the mixture and heated in microwave oven at 100 °C for 20 min. The reaction mixture was
partitioned between ethyl acetate and water and the organic layer was separated, washed with water and brine,
dried over sodium sulfate, filtered and concentrated. It was purified by flash column chromatography over silica
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gel eluting with 20% ethyl acetate in heptane to yield 5-(5-amino-2-methylphenyl)-1-methyl-1H-
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benzo[d]imidazol-2(3H)-one (60 mg, 0.237 mmol, 77%). LCMS (m/z) (M+H) = 254.1/230.9 Rt = 0.45 min.
To a mixture of 5-(5-amino-2-methylphenyl)-1-methyl-1H-benzo[d]imidazol-2(3H)-one (60 mg, 0.237 mmol) in
ethyl acetate (2 mL) at RT were added DIPEA (0.084 mL, 0.474 mmol) and 3-(trifluoromethyl)benzoyl chloride
(0.039 mL, 0.261 mmol) dropwise. The mixture was stirred at RT for 30 min. The reaction mixture was quenched
with water and the reaction mixture was partitioned between ethyl acetate and water. The organic layer was
separated, washed with water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was
purified by flash column chromatography over silica gel , eluting with 20% ethyl acetate in heptane, to yield N-
(4-methyl-3-(1-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)phenyl)-3-(trifluoromethyl)benzamide
1
(43.9 mg, 0.103 mmol, 43.6 % yield). LCMS (m/z) (M+H) = 425.6 Rt = 0.95 min. H NMR (400 MHz, CDCl )
3
δ ppm 2.19 (s, 3 H), 3.29 (s, 3 H), 6.89 (d, J=1.2 Hz, 1 H), 6.95 - 7.01 (m, 1 H), 7.12 (d, J=7.8 Hz, 1 H), 7.24 (d,
J=8.2 Hz, 1 H), 7.62 (s,2 H), 7.71 - 7.79 (m, 1 H), 7.88 - 7.99 (m, 1 H), 8.16 - 8.33 (m, 2 H), 10.39 (s, 1 H), 10.81
+
- 10.93 (m, 1 H). HRMS m/z (M +1) calcd 426.1424, obsd 426.1418.
N-(3-(2-((2-hydroxyethyl)amino)-6-morpholinopyrimidin-4-yl)-4-methylphenyl)-3-
(trifluoromethyl)benzamide (12): To a solution of triethylamine (0.057 mL, 0.410 mmol) and 4,6-dichloro-2-
(methylthio)pyrimidine (100 mg, 0.513 mmol) in ethanol (2.56 mL) at RT was added morpholine (0.046 mL,
0.513 mmol) in one portion. The resulting mixture was stirred at RT for 6 hrs. The precipitate formed was filtered
and washed with ethanol to give 4-(6-chloro-2-(methylthio)pyrimidin-4-yl)morpholine (83 mg, 66%). LCMS
(m/z) (M+H) = 246/247.9, Rt = 0.74 min.
The general procedure for the Suzuki-Miyaura reaction was followed for the next step utilizing the modified
work-up: the reaction mixture was partitioned between water and EtOAc. The aqueous was further washed with
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