Design and Discovery of N-(2-Methyl-5′-morpholino-6′-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3′-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers

Article abstract of DOI:10.1021/acs.jmedchem.6b01862

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date

Full text of DOI:10.1021/acs.jmedchem.6b01862

Article
Design and Discovery of N-(2-methyl-5'-morpholino-6'-((tetrahydro-2H-
pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A
potent, selective and efficacious RAF inhibitor targeting RAS mutant cancers
Gisele A. Nishiguchi, Alice Rico, Huw R. Tanner, Robert J. Aversa, Benjamin Robert Taft, Sharadha
Subramanian, Lina Setti, Matthew T Burger, Lifeng Wan, Victoriano N. A. Tamez, Aaron Smith, Yan Lou,
Paul Andrew Barsanti, Brent A. Appleton, Mulugeta Mamo, Laura Tandeske, Ina Dix, John E. Tellew,
Shenlin Huang, Lesley A. Mathews Griner, Vesselina G. Cooke, Anne Van Abbema, Hanne Merritt, Sylvia
Ma, Kalyani Gampa, Fei Feng, Jing Yuan, Yingyun Wang, Jacob R Haling, Sepideh Vaziri, Mohammad
Hekmat-Nejad, Johanna M. Jansen, Valery R Polyakov, Richard Zang, Vijay Sethuraman, Payman Amiri,
Mallika Singh, Emma Lees, Wenlin Shao, Darrin D. Stuart, Michael P Dillon, and Savithri Ramurthy
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2017
Downloaded from http://pubs.acs.org on May 30, 2017
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Zang, Richard; Novartis Institute of Biomedical research
Sethuraman, Vijay; Novartis,
Amiri, Payman; Novartis Institute of Biomedical research
Singh, Mallika; Novartis Institute of Biomedical research
Lees, Emma; Jounce therapeutics, Inc.
Shao, Wenlin; Astra Zeneca,
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Stuart, Darrin; Novartis
Dillon, Michael; Ideaya Biosciences ,
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Ramurthy, Savithri; Novartis Institute of Biomedical research
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Design and Discovery of Nꢀ(2ꢀmethylꢀ5'ꢀmorpholinoꢀ6'ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)oxy)ꢀ[3,3'ꢀ
bipyridin]ꢀ5ꢀyl)ꢀ3ꢀ(trifluoromethyl)benzamide (RAF709): A potent, selective and efficacious RAF
inhibitor targeting RAS mutant cancers
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Gisele A. Nishiguchi2, Alice Rico1, Huw Tanner1, Robert J. Aversa2, Benjamin R. Taft1, Sharadha
Subramanian1, Lina Setti1, Matthew T. Burger2, Lifeng Wan1, Victoriano Tamez2, Aaron Smith1, Yan
Lou1, Paul A. Barsanti1, Brent A. Appleton1, Mulugeta Mamo1, Laura Tandeske6, Ina Dix3, John E.
Tellew4, Shenlin Huang4, Lesley A. Mathews Griner5, Vesselina G. Cooke5, Anne Van Abbema6, Hanne
Merritt6, Sylvia Ma6, Kalyani Gampa5, Fei Feng5, Jing Yuan5, Yingyun Wang6, Jacob R. Haling4, Sepideh
Vaziri4, Mohammad HekmatꢀNejad6, Johanna M. Jansen1, Valery Polyakov1, Richard Zang1, Vijay
Sethuraman6, Payman Amiri6, Mallika Singh6, Emma Lees5, Wenlin Shao5, Darrin D. Stuart5 , Michael P.
Dillon2, Savithri Ramurthy*1
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 Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Werk
Klybeck, Postfach, CHꢀ4002 Basel, Switzerland
4 Genomics Institute of the Novartis Research Foundation, 10675 John Hopkins Dr, San Diego, C
California 92121
5 Oncology, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge,
Massachusetts 02139, United States
6 Oncology, Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, California
94608, United States
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ABSTRACT:
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RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical
need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in
genetically engineered mouse models and human tumor cells. To date, many small molecule approaches
are under investigation to target CRAF, yet kinaseꢀselective and cellular potent inhibitors remain
challenging to identify. Herein, we describe 14 (RAF709)1, a selective B/C RAF inhibitor, which was
developed through a hypothesisꢀdriven approach focusing on drugꢀlike properties. A key challenge
encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and
potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We
investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of
the crystal packing would improve solubility, which led to a change from Nꢀmethylpyridone to a
tetrahydropyranyl oxy pyridine derivative. 14 proved to be soluble, kinase selective and efficacious in a
KRAS mutant xenograft model.
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INTRODUCTION
The mitogenꢀactivated protein kinase (MAPK) signaling pathway plays a major role in the regulation of
multiple cellular functions. The pathway is activated through extracellular signals that in turn induce the
RAS proteins (Kꢀ, Nꢀ, HRAS) to exchange GDP for GTP, resulting in activation of the RAF/MEK/ERK
cascade. The MAPK pathway is often constitutively activated in human cancers through activating
mutations in the RAS proteins or BRAF. In fact, the RAS genes are the most frequently mutated
oncogenes in human tumors, however, targeting RAS directly as a cancer therapeutic has been
challenging. Recent studies have described approaches to selectively inhibit KRASG12C through covalent
2ꢀ4
targeting, resulting in a cellular active inhibitor in the low micromolar range.
While such covalent
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inhibitors demonstrate proofꢀofꢀconcept for targeting KRASG12Cꢀdriven tumors, other RAS mutant alleles
remain undruggable.
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In considering targets downstream of mutant RAS, emerging biology from genetically engineered mouse
models and tumor cell lines has begun to uncover which effectors are critical for tumorigenesis, providing
new opportunities for therapeutic development. For example, CRAF was shown to be the critical mediator
of mutant KRASꢀdriven cell proliferation and tumor development in both cellular and genetically
engineered mouse tumor models.5,6 CRAF was also shown to play a critical role in feedbackꢀmediated
pathway reactivation following MEK inhibitor7 treatment in KRAS mutant cancers. In addition, CRAF
plays an essential role in mediating paradoxical activation following BRAF inhibitor treatment.8ꢀ10 Thus
selective inhibitors that potently inhibit the activity of B/CRAF could be both effective in blocking
mutant RASꢀdriven tumorigenesis and preventing feedback mediated reactivation While selective RAF
inhibitors such as vemurafenib (1) have been developed to treat BRAFV600E mutant tumors, these
inhibitors are ineffective against tumors with wildꢀtype BRAF and in fact can accelerate the growth of
RAS mutant tumors through paradoxical activation of RAF dimers.11,12 LY3009120 (2) has recently been
described as an effective inhibitor of RAF dimers and demonstrates efficacy against some preclinical
models of RAS mutant tumors; however, it appears to inhibit several other kinases, including those that
have important roles in cancer such as Ephrin receptors, JNK and SRC family members.13 Thus, a potent
inhibitor of RAF dimers with an improved selectivity profile will be invaluable in assessing the
therapeutic utility and therapeutic index in RAS mutant tumors.
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Chart 1
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RESULTS AND DISCUSSION
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The major medicinal chemistry goal of this project was to identify highly selective RAF inhibitors that
would potently suppress the RAFꢀMEKꢀERK pathway in KRAS mutant tumor cells without causing
paradoxical activation. For this purpose, we developed a full length CRAF biochemical assay as well as
Caluꢀ6 (KRASQ61K) cell proliferation and pMEK target modulation assays in which inhibition and
activation of the pathway could be measured. Herein we describe our hit finding approach, the design and
the synthesis of analogues that led to the discovery of 14 (RAF709), a RAF inhibitor that is wellꢀtolerated
and efficacious in KRAS mutant xenograft models.
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In order to assess whether any existing RAF kinase inhibitor chemotypes were able to suppress pMEK in
KRAS mutant tumor cells without inducing paradoxical activation, we pursued an accelerated hit finding
strategy that centered on leveraging our existing RAF inhibitor knowledgebase. A dataset from 28k
compounds that had shown evidence of RAF inhibition in historical biochemical or cellular assays was
assembled. Subsets of this collection were selected for the CRAF biochemical assay and the pMEK target
modulation assay in Caluꢀ6 (KRASQ61K) cells. The overarching selection criterion was to ensure sampling
of all chemotypes for which there were analogues with good cellular potency in order to ensure we could
assess the potential for paradoxical activation. A set of 116 compounds with evidence of pMEK
suppression in Caluꢀ6 cells was selected for assessment of paradoxical activation.
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Figure 1. Boxꢀplot of the distribution of foldꢀactivation of pMEK in Caluꢀ6 cells for each of the
annotated types (mechanisms) of kinase inhibition 14 based on medicinal chemistry scaffold matches. The
116 compounds in this analysis had a CRAF IC50 varying from 42 nM to 30 pM and an EC50 in the pMEK
target modulation assay in Caluꢀ6 cells < 0.3 µM. The solid line at 2ꢀfoldꢀactivation indicates the team’s
assessment of a relevant cutꢀoff.
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From the analysis of this data set, there was a consistent trend towards type 2 inhibitors (DFG out, αCꢀ
helix in) exhibiting less paradoxical activation compared to type 1 (DFG in, αCꢀhelix in) and 1.5 (DFG in,
αCꢀhelix out)15,16 modes of inhibition (Figure 1). Compound 1 did not meet the criteria (pMEK >30 µM )
to be considered in the data set. On the basis of this observation, the team focused on type 2 inhibitors to
further progress towards our medicinal chemistry goal. In depth data mining and further profiling of the
hits uncovered several issues associated with type 2 inhibitors, including poor kinase selectivity, low
aqueous solubility and high lipophilicity. With these liabilities in mind, we hypothesized that increased
levels of saturation could be an approach to improve solubility and enhance kinase selectivity. It has been
documented that an increase in the fraction of sp3 atoms (Fsp3), defined as the ratio of sp3 atoms to the
total number of nonꢀhydrogen atoms in the molecule, is associated with decrease in melting point and
improved solubility for small molecules.17 At the same time, higher sp3 character within a given molecule
can also offer the opportunity for higher specificity in receptor/ligand complementarity and potentially
mitigate offꢀtargets binding.18 This hypothesis led the team to further probe our type 2 hits leading to the
prioritization of compound 3 (Figure 2), a starting point that has measurable solubility and an attractive
kinase selectivity profile relative to other hits. In a KINOMEscan binding displacement assay,19, 20 3 had
a selectivity score S(35) of 0.076 when screened at 10 µM, indicative of high kinase selectivity. The
morpholine moiety as a potential hinge binder was also viewed as a unique feature within RAF kinase
inhibitors.
In order to elucidate the binding mode, we obtained an Xꢀray coꢀcrystal structure of 3 complexed with the
kinase domain BRAF. As illustrated in Figure 2, the oxygen of the morpholine makes a Hꢀbond
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interaction to the backꢀbone NH of residue C532 in the hinge region (O…N distance is approximately 2.7
Å). The pyrimidine core is situated in a hydrophobic region, approximately 3.6 Å away from F595, which
could be consistent with an edge to face or Tꢀshaped piꢀinteraction. The exact type of this interaction is
difficult to ascertain due to the 2.4 Å resolution of the crystal structure. Because the two nitrogen atoms of
the pyrimidine core are facing the solvent, they are unlikely to adversely affect the potency of the
compound, but are beneficial for lowering lipophilicity and probably, enhancing its solubility. The central
phenyl is twisted out of the plane of the molecule due to the presence of the ‘flag’ methyl allowing the
amide moiety to engage the protein in two hydrogen bond interactions typical for type 2 inhibitors. The
carbonyl makes a Hꢀbond to D594 (O…N distance is approximately 2.7 Å) and the NH is 2.9 Å away
from E501 without destroying the salt bridge between K483 and E501. The tꢀbutyl phenyl occupies a
hydrophobic region referred to here as the induced fit pocket. The protein adopted an inactive
conformation characteristic for type 2 inhibitors with the DFG out and the αCꢀhelix in.21
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Figure 2. Xꢀray structure of 3 in the BRAF construct (PDB 5VAL)22 illustrating key features of protein
ligand interactions depiction of the crystalized ligand illustrating most important features of protein ligand
interactions, labeled key residues, and a ligand pocket surface. All distances are in Å.
We initiated hit optimization with 3 (Figure 2) aiming to improve the solubility and cellular potency.
Through hit expansion from our compound collection we identified analogue 4 (Table 1) with a lower
cLogP (4.1) and similar cellular potency as 3. From this compound, we synthesized the corresponding
reverseꢀamide match 5 (Table 1) in order to assess if there is a favorable amide orientation for the series.
Compound 5 had comparable biochemical potency and antiꢀproliferative effect relative to 4 with
improved target modulation, measurable solubility and reasonable clearance (rat and human liver
microsomes Clint: 79 and 43 µL/min/mg) making this a preferred amide isomer for subsequent analogs.
Table 1. Biochemical and cellular activity, permeability and solubility data for compounds 3, 4, 5,
and 6
CRAF IC50
(µM)
pMEK Caluꢀ6
EC50 (µM)
CP Caluꢀ6
EC50 (µM)
Cacoꢀ2 Papp AB/BA*
(x10ꢀ6 cm/s)
Sol**
(µM)
Cmpd
cLogP***
0.0005
0.002
0.2
0.7
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15/15
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<2
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0.0002
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0.7
^/1.8
<2
4.5
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*Forward and reverse flux were measured via transit across 21ꢀday cultured Cacoꢀ2 monolayers at pH7.4
with quantification via LC/MS/MS. **Solubility (shake–flask assay) was measured with Clꢀfree PBS at
pH 6.8. ***cLogP was calculated using Biobyte software. ^Poor recovery.
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Based on the BRAF coꢀcrystal structure with 3, we postulated that removal of the pyrimidine nitrogen
adjacent to the central phenyl should not affect biochemical potency since both nitrogens in the
pyrimidine ring face the solvent. The pyridine analogue 6 (Table 1) was found to have similar potency as
5 in the biochemical assay. The resulted torsional angle increase between the pyridine ring and the central
phenyl ring relative to the pyrimidineꢀcentral phenyl ring, led to the hypothesis that there is some
flexibility (around the hydrophobic pocket and the gate keeper residue T529) would be tolerated. . While
the poor solubility of compound 6 still remained a concern, the potent cellular activity provided
confidence in the series and led to the hypothesis that core modifications could be utilized to modulate
cell potency as well as the properties of the molecules. This turned our attention to the lipophilicity of the
cores and we focused on designing analogues with lower cLogP.
In an effort to decrease the cLogP, we investigated core modifications with more polar heterocycles in
conjunction with central and induced fit pyridyl modifications. Four representative examples of our SAR
set are illustrated in Table 2. We quickly learned from compounds 7, 8, 9 and 10 (Table 2) that intrinsic
clearance and solubility were consistently improved but the increased polarity eroded cellular potency in
many cases. We hypothesized that optimal polarity might be essential to balance potency and drugꢀlike
properties and strategized that making different core modifications would allow us to find the right
balance. The balanced profile of solubility, target modulation and antiꢀproliferative effect of pyridone 7
led us to select this compound over the other analogues for further profiling. Since kinase selectivity is
one of the liabilities with type 2 inhibitors, we screened this compound using the KINOMEscan binding
displacement assay against 456 human kinases and their relevant mutants at 1 µM (Figure 3). Of the 456
kinases tested, it showed a high level of selectivity, demonstrating greater than 99% binding to BRAF,
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BRAFV600E and 90% binding to CRAF with binding to only one offꢀtarget at >80% (PDGFRβ,89%) and a
selectivity score (Sꢀscore) of S(35) = 0.015. In cellular target modulation, it was potent and showed
minimal paradoxical activation in KRAS mutant cell lines.
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Figure 3. KINOMEscan profile of 7
Table 2. Potency, ADME and solubility data for analogues with a lower cLogP
Y
O
N
O
X
N
NH
O
Z
F
F
F
Cmpd
X
Y
Z
CRAF
pMEK Caluꢀ
6 EC50 (µM)
CP Caluꢀ
Cacoꢀ2
Clint
Sol
cLogP
IC50 (µM)
6 EC50
(µM)
Papp AB/BA
(x10ꢀ6 cm/s)
(human microsomal
µL/min/mg)
(µM)
CH CH CH 0.0004
0.076
0.5
20/22
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70
3.5
7
N
CH CH 0.0003
CH 0.0008
0.2
4.1
1.8
21/16
7/43
110
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3.2
2.8
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CH
N
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<7.7
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CH
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>30
3/39
<7.7
1040 1.5
10
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Compound 7 also demonstrated good permeability, minimum efflux based on Cacoꢀ2 assays and
moderate in-vitro clearance measured in rat (Clint: 46 µL/min/mg) and human liver microsomes (Clint: 51
µL/min/mg). Given its favorable in vitro ADME profile, the pharmacokinetics of 7 was evaluated in rats
(Sprague Dawley) at 1 mg/kg IV and 3 mg/kg PO. The results indicated that 7 had an oral total exposure
(AUC) of 0.438 µM*hr, high in-vivo clearance (53 mL/min/kg), Cmax of 0.15 µM and lowꢀmoderate
bioavailability of 22%. Formulating 7 proved challenging, potentially due to its solubility and this
hindered further in-vivo studies, especially at higher doses where a lack of doseꢀproportional exposure
was observed. More in depth physicochemical characterization of 7 revealed a melting point >230 °C
(decomposed) which clearly suggested that tight crystal packing in the solid phase might also have
contributed to the poor solubility and this was further supported by measuring the solubility from
crystalline material which gave a value of 4 µM. Therefore, lead optimization centered on further
addressing the solubility of the series without compromising the cellular potency.
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In order to further understand the chemical modifications required to disrupt crystal packing or lowering
the melting point we investigated the small molecule Xꢀray structure of 7.22 The molecules are arranged in
pairs oriented headꢀtoꢀtail so that the amide nitrogen of each molecule (N22) forms a moderately strong
Hꢀbond to the carbonyl oxygen (O12) of the pyridone ring of the second molecule (Figure 4). The
distance between N and O atoms is 2.99 Å. Additionally, the same pyridone rings are oriented in the
parallel planes with the distance of 4.21 Å between them which is consistent with the parallel displaced
piꢀinteraction.
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Figure 4. Small molecule Xꢀray structure of compound 7.
Based on this analysis we hypothesized that removal of the carbonyl would disrupt the intermolecular
hydrogen bond network and furthermore the introduction of sp3 character in close proximity to the core
could potentially disrupt the parallel displaced piꢀstacking interaction. To test our hypothesis, the 3ꢀ
morpholinoꢀ2ꢀoxypyridine core emerged as a valuable scaffold (Table 3). In this series, the pyridone
carbonyl was replaced by the ether oxygen and the directionality of the methyl provided a solvent
exposed vector. This core was initially derived from the pyridone alkylation reaction (Scheme 1) to
provide the minor Oꢀalkylated product, exemplified by 11 (Table 3). It is noteworthy to highlight the
serendipitous nature of the discovery of the Oꢀalkylated core ‘as a reaction by-product’.
While compound 11 was moderately potent in cellular assays and remained poorly soluble, we
hypothesized that the methoxy group was satisfying the disruption of the interꢀmolecular hydrogen
bonding network but was not large enough to disrupt the parallel piꢀinteraction. Given the Fsp3 of
compound 11 is 0.29, we envisioned that an increase in Fsp3 around the methoxy group might address the
solubility liability. As shown in Table 3, by increasing the size of the R1 substituent from methyl to
isopropyl, compound 12 was obtained. Although its solubility was still low, the fact that cell potency was
maintained was encouraging and larger substituents were investigated. A glycol moiety 13 (Table 3) was
tolerated and it provided a lower cLogP and higher Fsp3 with solubility trending in a favorable direction
at 24 µM, however an efflux liability was introduced. By exploring larger R1 substituents such as the
tetrahydropyran in 14 high throughput solubility was improved to 200 µM and the solubility of the highly
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stable crystalline material was measured to be 120 µM (melting point: 196 °C). Moreover, pMEK cellular
potency was preserved and low efflux was maintained. Overall, 14 provided the most favorable balance
of physicochemical properties and cellular potency and hence it was selected for further profiling.
9
10
11
12
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14
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Table 3. Effect of Increased Fsp3 on solubility, permeability and activity in the oxyꢀpyridine core
Cacoꢀ2
pMEK Caluꢀ6
EC50 (µM)
CP Caluꢀ6
EC50(µM)
Sol
Cmpd
R1
CRAF IC50(µM)
Papp AB/BA
(x10ꢀ6 cm/s)
cLogP
Fsp3
(µM)
Me
0.0004
0.0006
0.0005
0.25
0.3
1.6
2.9
2.5
16/14
ꢀ
<2
<2
24
4.6
5.4
3.7
0.29
0.35
0.32
11
12
13
14
ꢀCH2(CH3)2
ꢀCH2CH2OH
0.08
5/45
0.0005
0.02
0.95
15/13
200
4.2
0.39
Xꢀray structure of 14. The Xꢀray coꢀcrystal structure of 14 in BRAF illustrated in Figure 5 shows a
binding mode similar to 3 with respect to the morpholine and induced fit moiety. The central pyridine
occupies the space of the corresponding central toluyl tightly filling up a narrow hydrophobic pocket
formed by the sidechains of K483, I527, T529, and A481. The CF3 phenyl occupies the hydrophobic
inducedꢀfit pocket similar to the structure of compound 3. The tetrahydropyran faces a solvent opening in
the ATP binding pocket which can accommodate such a large group. The protein maintains the DFGꢀout
αCꢀHelixꢀin inactive conformation consistent with a type 2 mode of inhibition.
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Figure 5: Xꢀray structure of 14 in BRAF (PDB 5VAM)22
In vitro profiling of 1423. The biochemical activity of 14 was further characterized against purified fullꢀ
length BRAF where the IC50 measured 0.0004 µM which is inꢀline with the potency against CRAF. Offꢀ
rate is an important attribute of potency, impacting both the cellular activity in the face of feedback and
the PK/PD relationship in vivo. Therefore we attempted to measure the dissociation rate constant for 14
using the rapid dilution method and it appeared to have very slow dissociation kinetics (T1/2 > 6.5 hrs). In
cellular assays the doseꢀresponse of pMEK and pERK were measured in Caluꢀ6 cells with EC50 = 0.02
and 0.1 µM with minimal paradoxical activation (Figure 6A) and inhibition of proliferation with EC50 =
0.95 µM. Compound 1 clearly demonstrated paradoxical activation in the pMEK assay (Figure 6A) with
maximum activity at 3 µM and no resultant inhibition up to the highest concentration tested (30 ꢁM). The
ability of 14 to stabilize RAF dimerization was measured using the CRAF:BRAF NanoBiT™ luciferase
complementation assay (Promega). Consistent with most other RAF inhibitors, 14 stabilized BRAFꢀ
CRAF dimers with an EC50 = 0.8 µM (Figure 6B), while 1 demonstrated modest stabilization of dimers in
this assay. Additional supporting data was revealed in the Xꢀray crystal structure of 14 (Figure 5), where
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each BRAF protomer in the dimer structure was loaded with compound. This also demonstrated that (at
least under the conditions of crystallization), binding of 14 to one protomer did not preclude binding to
the other protomer in the dimer structure. Altogether, these data demonstrate that 14 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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Figure 6. A: pMEK inhibition and activation of 1 and 14 in Caluꢀ6 RAS mutant cells; B: Effect of 1 and
14 on the stimulation of dimerization in HCT116 cells.
Kinase Selectivity of 14. Consistent with this chemical series, 14 was found to be highly selective when
evaluated using the KINOMEscanTm screening platform. Of the 456 kinases tested19, 20, 14 showed a high
level of selectivity, demonstrating greater than 99% onꢀtarget binding to BRAF, BRAFV600E, and CRAF at
1 ꢀM and very few offꢀtargets with DDR1 (>99%), DDR2 (86%), FRK (92%), PDGFRb (96%) the only
kinases with binding >80% at 1 µM. To the best of our knowledge, this compound exhibits the highest
level of kinase selectivity amongst known type 2 RAF inhibitors with a selectivity score of S(35) =
0.018.20
Compounds such as 14 and 7 with a morpholine as the hinge binding moiety appeared to be remarkably
selective inhibitors of RAF kinases often exhibiting more than 10,000 fold difference in the binding
affinities even toward other structurally similar proteins like p38 and EGFR. The combination of a
morpholine, a central core attached to the perpendicular central phenyl and an appropriate group to fill the
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inducedꢀfit hydrophobic pocket (Figure 5) allowed us to fineꢀtune inhibitors to accommodate the unique
geometries of RAF kinases ensuring subꢀoptimal binding to other kinases.
7
8
9
Pharmacokinetics of 14. 14 was reasonably stable in plasma after a 3 hour incubation at 37 °C across
species [plasma stability (%remaining): rat 85%, mouse 82%, dog 95%, human 101%] and plasma protein
binding was measured to be 98% across species. In pharmacokinetic experiments (Table 4), 14 had
moderate clearance in mouse (35 ml/min/kg) and dog (14 ml/min/kg) and high clearance in rat (50
ml/min/kg). Cmax in mouse (1 µM), dog (0.5 µM) and rat (0.5 µM) reached pharmacologically active
concentrations and, acceptable oral availability was observed in mouse (68%), rat (24%) and dog (48%).
Compared to 7, 14 was less challenging to formulate due to higher intrinsic solubility which enabled
progression into PK/PD and efficacy studies at higher doses.
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Table 4: PK profile of 14 across species
AUCinf
(p.o.,
Dose
Cmax
Cl
Vss
F( po)
Species
(i.v./p.o,
mg/kg)
(p.o., µM)
(ml/min/kg)
(L/kg)
(%)
ꢀM*hr)
1 / 3
3 / 10
0.3 / 1
1.0
0.8
0.5
1.7
1.5
1.0
35
50
14
1.2
3.5
3.1
68^
24*
48*
Mouse
Rat
Dog
*: formulation (i.v./p.o): 20%PEG300 + 5%Solutol ^: formulation (i.v./p.o): 25% PEG300 + 5% Solutol
PK/PD of 14. The PK/PD relationship of 14 was examined in the Caluꢀ6 xenograft model in nude mice.
The plasma exposure of 14, and levels of pERK in tumor tissue were determined following a single oral
administration across a dose range of 10, 30 and 200 mg/kg. 14 showed doseꢀproportional increases in
plasma exposure and a corresponding doseꢀdependent inhibition of pERK in Caluꢀ6 tumors (Figure 7).
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At 24 hrs postꢀdose of 10 mg/kg 14, plasma levels were below the lower limit of quantitation and
there was a complete lack of pERK inhibition. At the highest dose level of 200 mg/kg free plasma
concentrations were maintained above the cellular in vitro pERK IC50 value for the entire 24 hours, which
was consistent with sustained inhibition of pERK ≥ 70%. It is interesting to note that at the 10 mg/kg and
30 mg/kg doses, the pathway seemed to be hyperactivated compared to baseline at the 24 hour time point.
The most likely explanation for this is that reactivation of the pathway occurred following the loss of
negative feedback when pERK was briefly suppressed by 14 at earlier time points. However, it may also
be possible that either lower concentrations of 14 or its slow dissociation rate could be contributing to the
pathway hyperactivation. Doseꢀdependent modulation of pMEK, DUSP6, and SPRY4 were similar to
those described for pERK (data not shown).23
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Figure 7. PK/PD analysis of 14 in the human Caluꢀ6 NSCLC xenograft in mice. Percent pERK
inhibition (left axis), and free exposure (right axis) following a single dose of 14, at time points indicated,
in Caluꢀ6 tumor bearing mice
Efficacy and tolerability of 14 in the Caluꢀ6 xenograft model. The antitumor efficacy and tolerability
of 14 were determined in the Caluꢀ6 xenograft nude mouse model. Mice were treated with vehicle or 14
at 10, 30, or 200 mg/kg p.o. daily beginning 12 days after tumor implantation and continued until day 27.
The antiꢀtumor activity was determined by assessing %T/C or % regression on day 27 postꢀimplant (15
days of treatment). Treatment with 14 resulted in doseꢀdependent antiꢀtumor activity with 10 mg/kg being
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subꢀefficacious (%T/C = 92%), 30 mg/kg resulted in measurable antiꢀtumor activity (%T/C=46%) and
200 mg/kg resulted in mean tumor regression of 92%, while the same high dose was not efficacious in the
PC3, KRAS WT model (Supporting Information Figure 1A). All doses were well tolerated with no
significant body weight loss and no signs of toxicity or mortalities were observed (Figure 8 and
Supplementary Figure 1B).
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Figure 8. Efficacy of 14 in Caluꢀ6 xenograft in mice. Tumor volumes (A) or percent body weight change
from initial (B) treatment groups vs. vehicle control.
Chemistry: Compounds from Tables 2 and 3 were synthesized according to Schemes 1, 2 and 3. The
synthesis of morpholineꢀcore bromide moieties are illustrated in Scheme 1. Starting with commercially
available 15, formation of the morpholine was obtained by reaction with 1ꢀbromoꢀ2ꢀ(2ꢀ
bromoethoxy)ethane in the presence of DIEA at 120 °C to provide product 16 in 70% yield (Scheme 1).
By reacting 16 with HCl in dioxane at reflux the corresponding pyridone was obtained which upon
treatment with K2CO3 and iodomethane an approximately 5:1 ratio of the Nꢀalkylated and Oꢀalkylated
products (17 and 16) was obtained and easily separable by column chromatography.
The pyrazinone bromide 19 was synthesized by starting with commercially available bisꢀbromide 18 and
treating with morpholine at 100 °C. Upon isolation of the product (19) Nꢀmethylation was accomplished
as described above to give a major isomer as the desired product 20.
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Bromide 22 was synthesized by starting with 21 and utilizing the procedure for the ring closure of the
morpholine as described above for the synthesis of 16 to give intermediate 22 in 76% yield.
7
8
9
Scheme 1. Synthesis of core/morpholine bromides
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Reagents and Conditions. (a) 1ꢀbromoꢀ2ꢀ(2ꢀbromoethoxy)ethane, DMF, DIEA, 120 °C, 70ꢀ76%; (b)
HCl, dioxane, 110 °C, 93%; (c) K2CO3, DMF, MeI, 76ꢀ91%; (d) Morpholine, 100 °C, 61%.
Intermediates containing the central phenyl or central pyridine and induced fit motifs were synthesized
according to Scheme 2. Starting with 23, reaction with 3ꢀ(trifluoromethyl)benzoyl chloride in DCM and
DIEA afforded intermediate 24. Upon borylation of 24, boronic ester intermediate 25 was isolated in
71% yield. Compound 28 was obtained by treating the commercially available bromide 26 with
bis(pinacolato)diboron, KOAc and Pd(dppf)Cl2ꢀDCM in dioxane to give boronate ester 27. Reduction of
the nitro functionality to the corresponding aniline 28 was accomplished with hydrogen and Pd/C in 99%
yield. Boronate ester 30 was easily prepared by reacting 29 with 3ꢀ(trifluoromethyl)benzoyl chloride in
DCM and DIEA.
Scheme 2. Synthesis of boronic ester intermediates
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Reagents and Conditions. (a) ArCOCl, DCM, DIEA, 96ꢀ100%; (b) Pd(dppf)Cl2ꢀDCM, dioxane, bisꢀ
(pinacolato)diboron, KOAc, 70ꢀ80%; (c) H2, Pd/C, 99%.
Final compounds (7, 8, 9 and 10) were obtained by the SuzukiꢀMiyaura reaction between the core
bromides and the appropriate boronic esters as illustrated in Scheme 3. Alternatively, final compounds
were also obtained via a SuzukiꢀMiyaura reaction with the core bromides and the boronic ester 28,
followed by amide bond formation with the appropriate carboxylic acid in the presence of EDC and
HOAt.
In order to enable last step diversification and obtain final compounds exemplified in Table 3,
intermediate 31 was synthesized via SuzukiꢀMiyaura coupling reaction between 22 and 25. Treatment of
this intermediate with the desired preꢀformed alkoxide in THF allowed for isolation of final products (12,
13, and 14) upon HPLC purification (Scheme 3).
Scheme 3. Final compounds synthesis
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Reagents and Conditions. (a) Pd(dppf)Cl2ꢀDCM, DME, 2M Na2CO3, 120 °C; (b) Pd(dppf)Cl2ꢀDCM,
DME, 2M Na2CO3, 120 °C, boronic ester, then DMF, EDC, HOAt, carboxylic acid; (c) R1ꢀOH, NaH,
Dioxane or DMF, 80ꢀ105 °C.
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CONCLUSION: Despite the clinical activity of RAF and MEK inhibitors in BRAFV600 mutant
melanoma, they are relatively ineffective in RAS mutant tumors, leaving a significant unmet medical
need. The class of RAF inhibitors approved for the treatment of BRAFV600mut melanoma, including
vemurafenib and dabrafenib, induce paradoxical activation in BRAF wildꢀtype cells and can induce
growth of RASmut tumors. However, given the central role of CRAF in driving mutant KRASꢀdriven
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 (i.e. type 1 or type 1.5). However, previous type 2 RAF inhibitors have suffered
from a lack of selectivity, potency and solubility. We set out to address these key issues in the design of a
nextꢀgeneration type 2 RAF inhibitor and in this manuscript we describe the discovery of 14. By
considering saturation as a characteristic to modulate kinase selectivity during hit selection we identified
our starting point, a morpholine analog 3 which demonstrated good selectivity. A strategy focused on
lowering lipophilicity to address solubility enabled the identification of 7, a potent type 2 RAF inhibitor
in KRAS mutant tumor cells with minimal paradoxical activation. Limitations associated with solubility
and formulation of 7 and challenges in balancing physicochemical properties with cellular potency
directed the effort towards increasing the Fsp3 character as a strategy to improve solubility by disrupting
crystal packing. This approach led to the identification of 14, a compound shown to be highly kinase
selective and cellularly potent in a KRAS mutant cell line (Caluꢀ6) with minimal paradoxical activation
which we believe would enable the compound to be combined with other known linear and/or lateral
pathway inhibitors. In-vivo evaluation of 14 indicated, it has a reasonable rodent PK profile suitable for
PK/PD/efficacy and tolerability studies. 14 was shown to modulate pERK/pMEK24 in-vivo and it
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demonstrated doseꢀdependent tumor regressions in the KRAS mutant Caluꢀ6 xenograft model with no
significant body weight loss.
7
8
9
EXPERIMENTAL SECTION
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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 Cꢀ18 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 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 XDBꢀC18, 2.1 mm
x 50 mm; solvent system, 5ꢀ95% acetonitrile in water with 0.1% TFA; flow rate 0.8 mL/min; molecular
weight range 200ꢀ800; cone voltage 20 V; column temperature 40 °C). All masses were reported as those
1
of the protonated parent ions. H nuclear magnetic resonance (NMR) analyses described herein were
performed on some of the compounds with a Varian 400ꢀMR MHz NMR (Palo Alto, CA) spectrometer
1
operating at a frequency of 399.89 MHz for H or Bruker DRXꢀ500 NMR spectrometer operating at a
1
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, Cꢀ18
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
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LCꢀMS analysis and were found to be ≥95%. Experimental details for the synthesis and characterization
of compounds 3 and 4 are described in the supporting information.
7
8
9
General Procedure for the SuzukiꢀMiyaura reaction. To the appropriate heteroaryl halide (0.10 mmol)
and the appropriate boronic ester (0.12 mmol) in DME (1 mL) was added PdCl2 (dppf).CH2Cl2 adduct
(0.010 mmol) and 2M aqueous sodium carbonate (0.50 mmol). The reaction mixture was irradiated at
120 °C in a Biotage Initiator microwave for 10ꢀ12 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.
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The following compounds were prepared by the above method starting from the appropriate halide and
boronic ester.
Nꢀ(4ꢀmethylꢀ3ꢀ(6ꢀmorpholinopyrimidinꢀ4ꢀyl)phenyl)ꢀ3ꢀ(trifluoromethyl)benzamide (5). Obtained in
51% yield. LCMS (m/z) (M+H) = 443.3, Rt = 0.75 min. 1H NMR (500 MHz, DMSOꢀd
) δ ppm 10.70 (s,
6
1H) 8.86 (s, 1H) 8.26 – 8.38 (m, 2H) 7.93 – 8.06 (m, 2H) 7.74 – 7.90 (m, 2H) 7.43 (d, J = 8.5 Hz, 1H)
7.22 (s, 1H) 3.89 (s, 4H) 3.64 – 3.79 (m, 4H) 2.33 (s, 3H) 13C NMR (126 MHz, DMSOꢀd
6
) δ 164.52,
161.50, 153.48, 137.38, 135.90, 132.32, 131.93, 131.66, 130.32, 129.83, 129.57, 128.82, 125.51, 124.66,
124.63, 123.35, 122.87, 121.72, 103.55, 66.15, 45.40,19.42. HRMS m/z (M++ 1) calcd 443.1695, obsd
443.1694.
Nꢀ(4ꢀmethylꢀ3ꢀ(2ꢀmorpholinopyridinꢀ4ꢀyl)phenyl)ꢀ3ꢀ(trifluoromethyl)benzamide (6). Obtained in
28% yield as a white solid. LCMS (m/z) (M+H) = 442.3, Rt = 0.76 min. 1H NMR (400 MHz, DMSOꢀd6)
δ ppm 10.40 ꢀ 10.61 (m, 1 H) 8.30 (s, 1 H) 8.26 (d, J=7.83 Hz, 1 H) 8.17 (d, J=5.48 Hz, 1 H) 7.90 ꢀ 8.03
(m, 1 H) 7.67 ꢀ 7.82 (m, 3 H) 7.27 ꢀ 7.41 (m, 1H) 7.00 (s, 1 H) 6.82 (d, J=5.48 Hz, 1 H) 3.44 ꢀ 3.59 (m, 8
H) 2.25 (s, 3 H) . 13C NMR (126 MHz, DMSOꢀd
) δ 164.39, 139.29, 137.33, 136.06, 132.28, 131.37,
6
130.74, 130.25, 130.22, 129.78, 129.53, 128.69, 125.52, 124.64, 123.35, 121.36, 121.14, 114.59, 109.70,
66.08, 45.95, 19.83. HRMS m/z (M+ + 1) calcd 442.1742, obsd 442.1742.
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Nꢀ(4ꢀmethylꢀ3ꢀ(1ꢀmethylꢀ5ꢀmorpholinoꢀ6ꢀoxoꢀ1,6ꢀdihydropyridinꢀ3ꢀyl)phenyl)ꢀ3ꢀ
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(trifluoromethyl)benzamide (7). Obtained in 15% yield as an offꢀwhite solid. LCMS (m/z) (M+H) =
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472.3, Rt = 0.88 min. 1H NMR (400 MHz, CD3OD) δ ppm 8.27 (s, 1H), 8.22 (d, J=8.2 Hz, 1H), 7.91 (d, J
=7.8 Hz, 1H), 7.71 ꢀ 7.79 (m, 1H), 7.56 ꢀ 7.66 (m, 2H), 7.36 (d, J =1.6 Hz, 1H), 7.31 (d, J =8.2 Hz, 1H),
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6.93 (d, J =2.0 Hz, 1H), 3.82 ꢀ 3.92 (m, 4H), 3.65 (s, 3H), 3.09 ꢀ 3.19 (m, 4H), 2.32 (s, 3H). C NMR
(126 MHz, DMSOꢀd ) δ 164.28, 157.93, 140.77, 138.05, 137.20, 136.17, 132.27, 131.49, 131.10, 130.64,
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130.23, 129.77, 129.52, 128.60, 125.54, 124.60, 123.38, 122.06, 121.93, 119.95, 118.61, 66.60,
49.38,37.66, 20.16. HRMS m/z (M+ + 1) calcd 472.1848, obsd 472.1844.
Nꢀ(4ꢀmethylꢀ3ꢀ(4ꢀmethylꢀ6ꢀmorpholinoꢀ5ꢀoxoꢀ4,5ꢀdihydropyrazinꢀ2ꢀyl)phenyl)ꢀ3ꢀ
(trifluoromethyl)benzamide (8). Obtained in 13% yield as a solid. LCMS m/z (M+H) = 473.1, Rt =
0.92 min. 1H NMR (400 MHz, CD3OD ) δ ppm 8.25 (s, 1 H) 8.20 (d, J=7.83 Hz, 1 H) 7.89 (d, J=7.83 Hz,
1 H) 7.67 ꢀ 7.80 (m, 2 H) 7.56 (dd, J=8.22, 2.35 Hz, 1 H) 7.26 (d, J=8.22 Hz, 1 H) 7.18 (s, 1 H) 3.80 (s, 8
H) 3.56 (s, 3 H) 2.39 (s, 3 H) . HRMS m/z (M+ + 1) calcd 473.1801, obsd 473.1801.
Nꢀ(1',2ꢀdimethylꢀ5'ꢀmorpholinoꢀ6'ꢀoxoꢀ1',6'ꢀdihydroꢀ[3,3'ꢀbipyridin]ꢀ5ꢀyl)ꢀ3ꢀ
(trifluoromethyl)benzamide (9). Obtained in 22% yield as a solid. LCMS m/z (M+H) = 473.0, Rt = 0.56
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min. H NMR (400 MHz, CD3OD) δ ppm 8.77 (d, J=2.35 Hz, 1 H), 8.29 (s, 1 H), 8.23 (d, J=7.83 Hz, 1
H), 8.12 (d, J=2.35 Hz, 1 H), 7.92 (d, J=8.22 Hz, 1 H), 7.68 ꢀ 7.80 (m, 1 H), 7.43 (d, J=2.35 Hz, 1 H),
6.93 (d, J=2.35 Hz, 1 H), 3.77 ꢀ 3.94 (m, 4 H), 3.64 (s, 3 H), 3.07 ꢀ 3.20 (m, 4 H), 2.52 (s, 3 H). 13C NMR
(126 MHz, DMSOꢀd ) δ 164.70, 157.97, 151.42, 141.01, 140.49, 135.61, 133.84, 132.72, 132.35, 131.10,
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130.34, 129.06, 124.70, 121.43, 116.76, 66.56, 49.34, 37.71, 23.21. HRMS m/z (M+ + 1) calcd 473.1801,
obsd 473.1804. m.p: 242 °C
Nꢀ(1',2ꢀdimethylꢀ5'ꢀmorpholinoꢀ6'ꢀoxoꢀ1',6'ꢀdihydroꢀ[3,3'ꢀbipyridin]ꢀ5ꢀyl)ꢀ2ꢀ
(trifluoromethyl)isonicotinamide (10). To 5ꢀbromoꢀ1ꢀmethylꢀ3ꢀmorpholinopyridinꢀ2(1H)ꢀone (546 mg,
2.0 mmol) and 6ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyridinꢀ3ꢀamine (492 mg, 2.1
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mmol) in DME (Volume: 11 mL) was added PdCl2 (dppf).CH2Cl2 adduct (163 mg, 0.2 mmol) and 2M
aqueous sodium carbonate (3.00 mL, 6 mmol). The reaction mixture was irradiated at 125 °C for 20 min.
LCꢀMS showed primarily conversion to product. The cooled reaction mixture was diluted with 2:1
DCM:MeOH (15 mL) and filtered. The filtrate was concentrated with silica gel and purified by flash
chromatography over silica gel (ethyl acetate with 0 to 15% methanol gradient) to give 5'ꢀaminoꢀ1,2'ꢀ
dimethylꢀ5ꢀmorpholinoꢀ[3,3'ꢀbipyridin]ꢀ6(1H)ꢀone (450 mg, 1.498 mmol, 74.9 % yield) as a brown solid.
LCMS m/z [M + H+] = 301.0. To a solution of 2ꢀ(trifluoromethyl)pyridineꢀ4ꢀcarboxylic acid (0.021 g,
0.110 mmol), HOAt (0.015 g, 0.110 mmol), and EDC.HCl (0.021 g, 0.110 mmol) in DMF (0.5 mL) was
added 5'ꢀaminoꢀ1,2'ꢀdimethylꢀ5ꢀmorpholinoꢀ[3,3'ꢀbipyridin]ꢀ6(1H)ꢀone (0.030 g, 0.100 mmol). The
mixture was stirred overnight at ambient temperature. The reaction mixture was diluted with DMSO,
filtered, and purified by HPLC. The clean fractions were lyophilized to give Nꢀ(1',2ꢀdimethylꢀ5'ꢀ
morpholinoꢀ6'ꢀoxoꢀ1',6'ꢀdihydroꢀ[3,3'ꢀbipyridin]ꢀ5ꢀyl)ꢀ2ꢀ(trifluoromethyl)isonicotinamide (13 mg, 0.022
mmol, 21.93 % yield) as a light yellow solid. LCMS (m/z) (M+H) = 474.0, Rt = 0.55 min. 1H NMR (400
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MHz, CD3OD) δ ppm 9.23 (d, J=2.35 Hz, 1 H) 8.99 (d, J=5.14 Hz, 1 H) 8.43 (d, J=2.35 Hz, 1 H) 8.37 (s,
1 H) 8.19 (dd, J=5.01, 1.54 Hz, 1 H) 7.54 (d, J=2.25 Hz, 1 H) 6.97 (d, J=2.30 Hz, 1 H) 3.83 ꢀ 3.91 (m, 4
H) 3.67 (s, 3 H) 3.14 ꢀ 3.22 (m, 4 H) 2.72 (s, 3 H) HRMS m/z (M+ + 1) calcd 474.1753, obsd 474.1755.
Nꢀ(6'ꢀmethoxyꢀ2ꢀmethylꢀ5'ꢀmorpholinoꢀ[3,3'ꢀbipyridin]ꢀ5ꢀyl)ꢀ3ꢀ(trifluoromethyl)benzamide
(11).
Obtained in 28% yield as a light yellow solid by following the general procedure for the SuzukiꢀMiyaura
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reaction. LCMS m/z [M + H+] = 473.3, Rt = 0.73 min. H NMR (400 MHz, CD3OD) δ ppm 9.32 (d,
J=2.4 Hz, 1H), 8.43 (d, J=2.4 Hz, 1H), 8.34 (s, 1H), 8.28 (d, J=8.2 Hz, 1H), 7.96 (d, J=7.8 Hz, 1H), 7.88
(d, J=2.0 Hz, 1H), 7.78 (t, J=7.8 Hz, 1H), 7.31 (d, J=2.0 Hz, 1H), 4.05 (s, 3H), 3.80 ꢀ 3.91 (m, 4H), 3.09 ꢀ
3.18 (m, 4H), 2.67 (s, 3H). HRMS m/z (M+ + 1) calcd 473.1801, obsd 473.1801.
General procedure for SNAr: To a mixture of sodium hydride, 60% in mineral oil (3.0ꢀ5.2 mmol) in 1,4ꢀ
dioxane or DMF (7 mL) at ambient temperature was added the appropriate alcohol (3.0ꢀ5.0 mmol). The
mixture was stirred for 15ꢀ30 min. Nꢀ(6'ꢀfluoroꢀ2ꢀmethylꢀ5'ꢀmorpholinoꢀ[3,3'ꢀbipyridin]ꢀ5ꢀyl)ꢀ3ꢀ
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(trifluoromethyl)benzamide (1.0 mmol) was added, and the reaction was stirred at 80ꢀ105 °C for 1ꢀ4 hr.
The cooled reaction mixture was poured into water (50 mL) and extracted with EtOAc (2x50 mL). The
combined organics were washed with brine (25 mL), dried over sodium sulfate, filtered, and concentrated
onto silica gel. The mixture was purified by flash chromatography (24 g silica gel, DCM with a 0ꢀ10%
MeOH gradient over 20 min). Pure product fractions were concentrated to give the desired product. The
products could be purified further by reverse phase HPLC or recrystallization from acetonitrile.
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The following compounds were prepared by the above method starting from the appropriate alcohol.
Nꢀ(6'ꢀisopropoxyꢀ2ꢀmethylꢀ5'ꢀmorpholinoꢀ[3,3'ꢀbipyridin]ꢀ5ꢀyl)ꢀ3ꢀ(trifluoromethyl)benzamide
trifluoroacetic acid salt (12). Obtained in 56% yield as an offꢀwhite solid. LCMS m/z [M + H+] = 501.1,
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Rt = 0.87 min. H NMR (400 MHz, DMSOꢀd6) δ ppm 10.92 (s, 1H), 9.03 (s, 1H), 8.33 (s, 1H), 8.21 ꢀ
8.31 (m, 2H), 8.02 (d, J=7.8 Hz, 1H), 7.78 ꢀ 7.86 (m, 2H), 7.24 (d, J=2.0 Hz, 1H), 5.35 (dt, J=12.2, 6.2
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Hz, 1H), 3.67 ꢀ 3.78 (m, 4H), 3.06 (m, 4H), 2.52 ꢀ 2.56 (s, 3H), 1.34 (d, J=6.3 Hz, 6H). C NMR (126
MHz, DMSOꢀd ) δ 164.99, 159.01, 158.72, 155.56, 149.30, 138.29, 135.95, 135.72, 135.31, 135.03,
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133.37, 132.48, 130.51, 129.96, 129.70, 129.33, 126.64, 125.56, 125.45, 124.82, 123.28, 68.51,
66.54,50.27, 22.41, 20.85. HRMS m/z (M+ + 1) calcd 501.2114, obsd 501.212. m.p: 204 °C
Nꢀ(6'ꢀ(2ꢀhydroxyethoxy)ꢀ2ꢀmethylꢀ5'ꢀmorpholinoꢀ[3,3'ꢀbipyridin]ꢀ5ꢀyl)ꢀ3ꢀ
(trifluoromethyl)benzamide (13). Obtained in 60% yield as a white solid. LCMS m/z [M + H+] = 503.2,
Rt = 0.66 min. 1H NMR (400 MHz, CDCl3) δ ppm 8.63 (d, J=2.4 Hz, 1H), 8.13 ꢀ 8.19 (m, 2H), 8.09 (d,
J=7.8 Hz, 1H), 7.95 (br. s., 1H), 7.85 (d, J=7.8 Hz, 1H), 7.76 (d, J=1.6 Hz, 1H), 7.67 (t, J=7.8 Hz, 1H),
7.11 (d, J=2.0 Hz, 1H), 4.59 ꢀ 4.67 (m, 2H), 3.97 ꢀ 4.06 (m, 2H), 3.85 ꢀ 3.95 (m, 4H), 3.11 ꢀ 3.20 (m, 4H),
2.52 (s, 3H). HRMS m/z (M+ + 1) calcd 503.1906, obsd 503.1907.
Nꢀ(2ꢀmethylꢀ5'ꢀmorpholinoꢀ6'ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)oxy)ꢀ[3,3'ꢀbipyridin]ꢀ5ꢀyl)ꢀ3ꢀ
(trifluoromethyl)benzamide (14). Obtained in 66% yield as an offꢀwhite solid. LCMS m/z [M + H+] =
1
543.1, Rt = 0.77 min. H NMR (400 MHz, CDCl3) δ ppm 8.62 (s, 1H), 8.16 (br. s., 2H), 8.10 (d, J=7.8
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Hz, 1H), 7.86 (d, J=7.8 Hz, 2H), 7.77 (d, J=2.0 Hz, 1H), 7.63 ꢀ 7.73 (m, 1H), 7.07 (d, J=2.4 Hz, 1H), 5.43
(dt, J=8.1, 4.0 Hz, 1H), 3.95 ꢀ 4.06 (m, 2H), 3.83 ꢀ 3.94 (m, 4H), 3.70 (ddd, J=11.5, 8.4, 3.1 Hz, 2H), 3.10
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ꢀ 3.25 (m, 4H), 2.54 (s, 3H), 2.17 (dt, J=8.7, 4.5 Hz, 2H), 1.89 (dtd, J=12.8, 8.49, 8.5, 3.9 Hz, 2H). C
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NMR (126 MHz, DMSOꢀd ) δ 164.73, 154.68, 151.28, 140.72, 138.01, 135.84, 133.88, 133.20, 132.37,
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130.33, 129.84, 129.59, 129.07, 128.89, 125.89, 125.51, 124.74, 123.34, 69.80, 66.64, 65.03,
50.40,32.19,23.22. HRMS m/z (M+ + 1) calcd 543.2219, obsd 543.2218. m.p: 196 °C
4ꢀ(5ꢀBromoꢀ2ꢀmethoxypyridinꢀ3ꢀyl)morpholine (16). To a solution of 5ꢀbromoꢀ2ꢀmethoxypyridinꢀ3ꢀ
amine (3.00 g, 14.8 mmol, 1.00 equiv) in DMF (40 mL) was added 1ꢀbromoꢀ2ꢀ(2ꢀbromoethoxy)ethane
(4.11 g, 17.7 mmol, 1.20 equiv.) and DIEA (7.74 mL, 44.3 mmol, 3.00 equiv.). The reaction mixture was
stirred at 120 °C for 24 h. Upon cooling to room temperature, the reaction was partitioned between water
and EtOAc, the aqueous phase was extracted three times with EtOAc, the organics were combined, dried
over Na2SO4, filtered and concentrated. The crude material was purified via silica gel flash
chromatography eluting with heptanes and ethyl acetate (0ꢀ20%) to give 4ꢀ(5ꢀbromoꢀ2ꢀmethoxypyridinꢀ3ꢀ
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yl)morpholine (2.80 g, 70% yield). LCMS m/z [M + H+] = 273.0/275.0. H NMR (400 MHz, CDCl3) δ
ppm 7.84 (d, J=2.0 Hz, 1H), 7.14 (d, J=2.0 Hz, 1H), 3.97 (s, 3H), 3.76 ꢀ 3.91 (m, 4H), 2.90 ꢀ 3.18 (m,
4H).
5ꢀbromoꢀ1ꢀmethylꢀ3ꢀmorpholinopyridinꢀ2(1H)ꢀone (17). A 0.3 M solution of 4ꢀ(5ꢀbromoꢀ2ꢀ
methoxypyridinꢀ3ꢀyl)morpholine (2.5 g, 9.15 mmol, 1.00 equiv.) in 4 M HCl in dioxane (3.8 mL, 45.8
mmol, 5 equiv.) was heated to 100 °C for 1 hr. Upon cooling to room temperature, the reaction was
concentrated to dryness under low pressure. The residue was suspended in water (20 mL) and neutralized
with solid sodium bicarbonate. The solid was filtered, rinsed with water (2x10 mL) and dried to give 5ꢀ
bromoꢀ3ꢀmorpholinopyridinꢀ2ꢀol in 93% yield (2.2 g). LCMS m/z [M + H+] = 258.9/260.9. To a 0.3 M
solution of 5ꢀbromoꢀ3ꢀmorpholinopyridinꢀ2ꢀol (500 mg, 1.9 mmol, 1.00 equiv.) in DMF (7 mL) was
added K2CO3 (560 mg, 4.1 mmol, 2.10 equiv.) and iodomethane (0.139 mL, 2.2 mmol, 1.1 equiv.). The
reaction mixture was stirred at ambient temperature overnight. LCꢀMS showed conversion to pyridone
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product and about 15ꢀ20% of Oꢀalkylated byꢀproduct. The reaction mixture was diluted with ethyl acetate
and filtered. The filtrate was concentrated under reduced pressure with silica gel. The crude mixture was
purified by flash chromatography eluting with heptanes and ethyl acetate (50ꢀ100%, then 10:1 ethyl
acetate:methanol gradient) to give 5ꢀbromoꢀ1ꢀmethylꢀ3ꢀmorpholinopyridinꢀ2(1H)ꢀone (400 mg, 76%
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yield). LCMS m/z [M + H+] = 272.8/274.9. H NMR (400 MHz, CDCl3) δ ppm 7.11 (d, J=2.4 Hz, 1H),
6.64 (d, J=2.4 Hz, 1H), 3.76 ꢀ 3.97 (m, 4H), 3.52 (s, 3H), 3.05 ꢀ 3.27 (m, 4H).
5ꢀbromoꢀ3ꢀmorpholinopyrazinꢀ2(1H)ꢀone (19). A solution of 3,5ꢀdibromopyrazinꢀ2ꢀol (500 mg, 1.97
mmol, 1.0 equiv.) was heated in morpholine (0.9 mL, 9.85 mmol, 5 equiv.) at 100 °C for 18 hours. Upon
overnight stirring, cooled to room temperature and filtered the precipitate. The filtrate was partitioned
between water and ethyl acetate, the organic phase was dried with sodium sulfate, filtered and
concentrated. The yellow solid was used without further purification (310 mg). LCMS m/z [M + H+] =
259.9/261.9.
5ꢀbromoꢀ1ꢀmethylꢀ3ꢀmorpholinopyrazinꢀ2(1H)ꢀone (20). To
a
solution of 5ꢀbromoꢀ3ꢀ
morpholinopyrazinꢀ2(1H)ꢀone (22 mg, 0.085 mmol, 1.0 equiv.) in DMF (1 mL, 0.085 M) at 0 °C was
added potassium carbonate (23 mg, 0.169 mmol, 2.0 equiv.), followed by iodomethane (0.005 mL, 0.085
mmol, 1.0 equiv.). The reaction was allowed to warm to room temperature and stirred for 2 hours.
Partitioned between ethyl acetate and water, the organic phase was dried with sodium sulfate, filtered and
concentrated to dryness to give 5ꢀbromoꢀ1ꢀmethylꢀ3ꢀmorpholinopyrazinꢀ2(1H)ꢀone in 91% yield as a
major isomer. LCMS m/z [M + H+] = 274.0/276.0. 1H NMR (400 MHz, CDCl3) δ ppm 6.77 (s, 1 H) 3.85
ꢀ 4.00 (m, 4 H) 3.66 ꢀ 3.83 (m, 4 H) 3.42 (s, 3 H).
4ꢀ(5ꢀBromoꢀ2ꢀfluoropyridinꢀ3ꢀyl)morpholine (22). To a stirred solution of 3ꢀaminoꢀ5ꢀbromoꢀ2ꢀ
fluoropyridine (10.0 g, 52.5 mmol, 1.00 equiv) in DMF (100 mL) at 0 °C was slowly added NaH (3.78 g,
157 mmol, 3.00 equiv). The mixture was allowed to warm to room temperature over 15 min and treated
with bis(2ꢀbromoethyl) ether (9.89 mL, 79.0 mmol, 1.50 equiv). The mixture was heated to 80 °C and
stirred for 3 h. The cooled reaction mixture was poured into water (500 mL) and stirred for 20 min. The
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mixture was filtered. The filter cake was washed with water (2 x 50 mL), allowed to air dry for a few
hours, and then washed with heptane (2 x 50 mL). The resulting solid was dried to give 20 (10.5 g, 76.4
% yield) as a light brown solid which was used without further purification. LCMS m/z [M + H+] =
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261.0/263.0. H NMR (400 MHz, CDCl3) δ ppm 7.81 (t, J=2.0 Hz, 1H), 7.32 (dd, J=9.0, 2.0 Hz, 1H),
3.84 ꢀ 3.91 (m, 4H) , 3.11 ꢀ 3.16 (m, 4H).
Nꢀ(5ꢀbromoꢀ6ꢀmethylpyridinꢀ3ꢀyl)ꢀ3ꢀ(trifluoromethyl)benzamide (24). To a solution of 3ꢀbromoꢀ2ꢀ
methylpyridinꢀ5ꢀamine (510 mg, 2.73 mmol, 1.00 equiv) in DCM (10 mL) was added DIEA (0.476 mL,
2.73 mmol, 1.00 equiv) and 3ꢀ(trifluoromethyl)benzoyl chloride (569 mg, 2.73 mmol, 1.00 equiv.). The
mixture was stirred at ambient temperature for 2 h. The reaction mixture was washed sequentially with
water (10 mL) and brine (10 mL), dried over sodium sulfate, filtered, and concentrated to give Nꢀ(5ꢀ
bromoꢀ6ꢀmethylpyridinꢀ3ꢀyl)ꢀ3ꢀ(trifluoromethyl)benzamide (980 mg, 100 % yield) as an offꢀwhite solid
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which was used without further purification. LCMS m/z [M + H+] = 359.0/361.0. H NMR (400 MHz,
DMSOꢀd6) δ ppm 10.70 (m, 1H), 8.78 (m, 1H), 8.46 (m, 1H), 8.28 (m, 2H), 7.98 (m, 1H), 7.78 (m, 1H),
2.55 (m, 3H).
Nꢀ(6ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ3ꢀ
(trifluoromethyl)benzamide (25). To
a
solution of Nꢀ(5ꢀbromoꢀ6ꢀmethylpyridinꢀ3ꢀyl)ꢀ3ꢀ
(trifluoromethyl)benzamide (10.5 g, 29.2 mmol, 1.00 equiv) in 1,4ꢀdioxane (9 mL) was added
bis(pinacolato)diboron (11.1 g, 43.9 mmol, 1.50 equiv), potassium acetate (5.74 g, 58.5 mmol, 2.00
equiv) and PdCl2(dppf).CH2Cl2 adduct (2.39 g, 2.92 mmol, 0.100 equiv). The reaction was stirred at
reflux for 2 h. The cooled reaction mixture was diluted with ethyl acetate (20 mL) and filtered through
Celite. The filtrate was concentrated and purified by flash chromatography over silica gel (220 g,
heptanes with 0ꢀ100% ethyl acetate gradient) to give Nꢀ(6ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaborolanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ3ꢀ(trifluoromethyl)benzamide (6.69 g, 70.7% yield) as an offꢀwhite solid.
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