Discovery of (R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one (AMG 337), a Potent and Selective Inhibitor of MET with High Unbound Target Coverage and Robust in Vivo Antitumor Activity

Article abstract of DOI:10.1021/acs.jmedchem.5b01716

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Full text of DOI:10.1021/acs.jmedchem.5b01716

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Drug Annotation
Discovery of (R)-6-(1-(8-fluoro-6-(1-methyl-1H-pyrazol-4-yl)-
[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-
naphthyridin-5(6H)-one (AMG 337), a Potent and Selective Inhibitor of MET
with High Unbound Target Coverage and Robust In Vivo Antitumor Activity
Alessandro A Boezio, Katrina W Copeland, Karen Rex, Brian K Brian K. Albrecht, David
Bauer, Steve F Bellon, Christiane Bode, Martin A Broome, Deborah Choquette, Angela
Coxon, Isabelle Dussault, Satoko Hirai, Richard T. Lewis, Min-Hwa Jasmine Lin, Julia Lohman
Suchomel, Jingzhou Liu, Emily A Peterson, Michele Potashman, Roman Shimanovich,
Yohannes Teffera, Douglas A. Whittington, Karina R Vaida, and Jean-Christophe Harmange
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01716 • Publication Date (Web): 26 Jan 2016
Downloaded from http://pubs.acs.org on February 3, 2016
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Journal of Medicinal Chemistry
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Discovery of (R)ꢀ6ꢀ(1ꢀ(8ꢀfluoroꢀ6ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀ[1,2,4]triazolo[4,3ꢀa]pyridinꢀ
3ꢀyl)ethyl)ꢀ3ꢀ(2ꢀmethoxyethoxy)ꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (AMG 337), a Potent and
Selective Inhibitor of MET with High Unbound Target Coverage and Robust In Vivo
Antitumor Activity
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Alessandro A. Boezio,*,† Katrina W. Copeland,† Karen Rex,‡ Brian K. Albrecht,† David
Bauer,† Steven F. Bellon,† Christiane Boezio,† Martin A. Broome,‡ Deborah Choquette,† Angela
Coxon,‡ Isabelle Dussault,‡ Satoko Hirai,† Richard Lewis,† MinꢀHwa Jasmine Lin,† Julia Lohman,†
Jingzhou Liu,† Emily A. Peterson,† Michele Potashman,† Roman Shimanovich,† Yohannes Teffera,†
Douglas A. Whittington,† Karina R. Vaida† and JeanꢀChristophe Harmange†
†Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States
‡Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States
KEYWORDS: MET, oncology, naphthyridinone
ABSTRACT: Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET)
has been implicated in several human cancers and is an attractive target for small molecule drug discovery.
Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of
MET kinase activity, desirable preꢀclinical pharmacokinetics, significant inhibition of MET
phosphorylation in mice and robust tumor growth inhibition in a METꢀdependent mouse efficacy model.
MANUSCRIPT TEXT:
Introduction
The receptor tyrosine kinase MET and hepatocyte growth factor (HGF), its endogenous ligand, are
implicated in several cellular processes relevant to cancer, including cell proliferation, cell migration and
invasive growth. These also play an important role in embryonic development and wound healing.1
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However, deregulation of the MET/HGF pathway can lead to tumorigenesis and metastasis.2 Amplification
of the MET gene, overexpression of MET and/or HGF and constitutive activation conferred by sequence
mutations are some of the mechanisms of deregulation found in human cancers.3
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Several approaches to inhibiting the HGF/MET pathway are currently being tested in the clinic.4
The appeal of an ATPꢀcompetitive, small molecule inhibitor acting via the intracellular kinase domain is
based on its potential to block both ligandꢀdependent and ligandꢀindependent activities of MET.5 We
previously reported the discovery of a potent, orally active MET inhibitor (Class II) for the treatment of
cancer,6 and more recently disclosed four distinct series (represented by 1,7 2,8 39 and 410, Table 1) of
potent and uniquely selective MET inhibitors with a common uꢀshape binding mode (Class Ib). However,
each series suffered from its own unique liabilities. Efforts to improve upon these series led to the
discovery of a naphthyridinone series of MET inhibitors (represented by 5, Figure 1), leading to the
discovery of 2311 (Table 3). Herein, we describe the design, synthesis, pharmacokinetics and in vivo
efficacy of this class of compounds.
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Results and Discussion
The previously reported compounds (1-4)7ꢀ10 all show impressive enzymatic and cellular MET
potencies. Compound 1, in the Oꢀlinked series, was the first of the Uꢀshaped inhibitors disclosed. 1
exhibited singleꢀdigit nanomolar potency in our cellular assay, suitable PK, and showed >90% inhibition of
HGFꢀinduced phosphorylation of MET in a mouse liver pharmacodynamic (PD) model for up to 6 h (vide
infra). However, this series was later found to exhibit timeꢀdependent inhibition (TDI) of CYP3A4.12 To
overcome this liability, the Nꢀlinked series represented by compound 2 was developed. This series showed
similar potency and in vivo tumor response to compound 1, but no longer suffered from TDI. Despite the
advances made in the Oꢀ and Nꢀlinked series, both suffered from poor solubility and shorterꢀthanꢀdesired
duration of coverage in our PD model (>90% inhibition at 30 mg/kg up to 6 h was observed for both
series).
Table 1. Representative MET compounds (1-4) from four distinct series
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MeO
MeO
MeO
MeO
1
2
3
4
5
6
7
N
N
N
N
O
N
O
N
N
F
O
Me
S
O
Me
F
F
O
HN
N
Me
N
S
Me
N
N
N
Me
N
N
N
N
N
N
N
N
N
N
N
F
8
9
"O-Linked"
"N-Linked"
"Triazolopyridinone"
"Triazolopyridine"
10
11
Compound
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MET IC50
Biochemicala
Cellularb
4 nM
8 nM
6 nM
3 nM
0.6 nM
2 nM
1 nM
5 nM
LipEc (cLogP)
Solubility
4.2 (4.2)
5.6 (2.6)
6.4 (2.8)
5.4 (3.5)
85 / 4 / 17
29 / 2.0
>200 / 3 / 13
37 / 9 / 40
21 / 1.5
>200 / 52 / 126
>50 / >50
0.01 N HCl / PBS / SIF (ꢀg/mL)
CYP3A4 TDId
>50 / 26
IC50 / IC50 (w/Preinc.) (ꢀM)
Rat pharmacokineticse
Cl (L/h/kg)
0.06g
58
0.19i
26
0.56j
31
0.57j
26
Clu (L/h/kg)f
F (%)
22h
3.5
31h
1.8
43k
3.4
46k
4.2
T1/2 (h)
Liver PD responsel
Dose (mg/kg)
30
6 h
30
6 h
10
10
6 h
Last time pt >90% inhibition
Plasma Cu at time pt
12 h
34 nM
9 nM
20 nM
141 nM
aInhibition of MET kinase activity, n ≥ 2. bInhibition of HGFꢀmediated MET phosphorylation in PC3 cells,
n ≥ 2. cLipE = pIC50 – clog P. 13,14 dInhibition of CYP3A4 (midazolam 1'ꢀhydroxylation) IC50 shift with and
without preꢀincubation of the compound in the presence of NADPH. eIn vivo experiments were carried out
with male SpragueꢀDawley rats n = 3. Unbound fraction (fu) was measured using equilibrium dialysis;
Concentration = 5 g/mL. iv, 0.3 mg/kg (DMSO). po, 2 mg/kg (2% hydroxypropyl methylcellulose 1%
f
g
h
i
Tween 80 in water, pH 2.2 adjusted with hydrochloric acid). iv, 0.25 mg/kg (20% hydroxypropyl betaꢀ
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j
cyclodextrin in water, pH 3.5 adjusted with methanesulfonic acid). iv, 0.25 mg/kg (DMSO). kpo, 2 mg/kg,
(2% hydroxypropyl methylcellulose 1% Tween 80 in water, pH 2.2 adjusted with methanesulfonic acid).
lSee references 7, 8, 9 and 10 for PD measurements.
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In an attempt to improve solubility and increase the in vivo exposure of 1 and 2, compound 3 was
designed de novo while maintaining the same key binding interactions with MET. Despite little
improvement in solubility, this new series showed outstanding efficacy and extended coverage in a mouse
PD model. This compound displayed >90% inhibition of HGFꢀinduced phosphorylation of MET at 10
mg/kg up to 12 h, with an associated unbound plasma concentration of 20 nM providing 11ꢀfold coverage
of the cellular IC50. As antitumor activity in a xenograft model (vide infra) was strongly correlated with
high and extended inhibition in our PD model (>90% inhibition for >12 h) was preferable.15 Upon further
investigation, it was determined that this series of compounds unfortunately also suffered from TDI of
CYP3A4. In a parallel investigation, the use of a triazolopyridine ring instead of a triazolopyridinone ring
led to the identification of 4, which maintained potency and did not exhibit TDI liabilities while displaying
good solubility. However, 4 suffered from lesser sustained efficacy in the PD model relative to compound
3.
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N
MeO
N
MeO
N
O
N
N
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N
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O
Me
O
Me
O
Me
S
O
Me
Me
N
Me
N
N
N
N
N
N
N
N
N
F
F
3
4
5
Figure 1. Origin of the naphthyridinone series, represented by hybrid 5
In order to merge efficacy with mitigated TDI liability, two key design elements derived from 3 and
4 were hybridized to afford compound 5. First, our attention was drawn to the superior efficacy of
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compound 3 in the PD timeꢀcourse model. We hypothesized that the favorable Uꢀshaped bindingꢀmode of 3
is reinforced by a key intramolecular interaction between the carbonyl of the pyridone and the hydrogen of
the quinoline as shown in the Figure 1. Second, upon juxtaposition of 3 and 4, we attributed the CYP3A4
TDI activity to the triazolopyridinone ring of 3, since both series share a characteristic quinoline element.
Accordingly, the fluorotriazolopyridine portion of 4 (in green), was combined with a naphthyridinone ring
system to afford 5, in which direction of the internal Hꢀbond interaction, exhibited by 3, has been reversed
(in orange). This new scaffold was found to maintain singleꢀdigit nanomolar inhibition of MET
phosphorylation (MET Biochemical IC50 3 nM, Cellular IC50 9 nM).
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The synthesis of 5 required the individual construction of coupling partners 9a and 13a (Scheme 1). Stille
crossꢀcoupling of 5ꢀchloroꢀ2,3ꢀdifluoropyridine (6) and commercially available isoxazole stannane 7
yielded intermediate 8a, which was treated with hydrazine to form pyridyl hydrazine 9a. Coupling partner
13a was prepared as a racemic mixture in two steps from naphthyridinone 10a through alkylation with
ethyl 2ꢀbromopropanoate (11) followed by hydrolysis of the resultant ester (12a) under acidic conditions.
Acid 13a underwent HATUꢀmediated amide coupling with hydrazine 9a, after which cyclization under
dehydrative conditions provided the racemic fluorotriazolopyridine product in 52% yield over two steps.
Finally, separation of racemic 5 by chiral chromatography afforded the active (R)ꢀenantiomer of 5.16
Scheme 1. Synthesis of 5
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a
Reagents and conditions: (i) Pd(OAc)2, XꢀPhos, 1,4ꢀdioxane/water, 100 °C, 18 h, 60%; (ii) NH2NH2, iꢀ
PrOH, 65 °C, 3 h, 90%; (iii) Cs2CO3, THF, 60 °C, 3 h, quantitative; (iv) aq. HCl, 60 °C, 7 h; (v) 13a,
HATU, Hunig’s base, DMF, 23 °C, 3 h; (vi) PPh3, TMSN3, DEAD, THF, 23 °C, 3 h, 52% over two steps,
then chiral separation.
Alternatively, an enantioselective synthesis of compound 5 was achieved via preparation of a chiral acid
coupling partner 15 (Scheme 2).17 Mitsunobu reaction between naphthyridinone 10a and (S)ꢀ(ꢀ)ꢀmethyl
lactate (14), followed by ester hydrolysis afforded compound 15 in high yield and >95% ee.18 HATU
mediated amide coupling of 9a with 15, followed by cyclization under dehydrative conditions yielded 5 in
high yield and with complete retention of enantiomeric excess.
Scheme 2. Enantioselective synthesis of 5
a
Reagents and conditions: (i) PPh3, DEAD, THF, 0 to 23 °C, 10 h; (ii) aq. HCl, THF, 75 °C, 10 h, 67%
over two steps; (iii) 9a, HATU, Hunig’s base, MeCN, 0 °C, 30 min, 77%; (iv) PPh3, TMSN3, DEAD, THF,
20 °C, 1 h, 83%.
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Figure 2. Coꢀcrystal structure of MET and compound 5
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A coꢀcrystal of 5 within the catalytic domain of unphosphorylated MET was obtained19 (Figure 2)
indicating that the inhibitor adopts the desired Uꢀshaped binding mode around Met1211. Furthermore, Our
hypothesis that the naphthyridinone carbonyl and Hꢀ5' of the fluorotriazolopyridine ring (3.3Å) would
participate in an intramolecular Hꢀbond to reinforce the optimal binding mode was confirmed. Figure 3
provides a 2D representation20 of the key contacts made by 5 in the activeꢀsite of MET. The
naphthyridinone nitrogen atom Nꢀ1 participates in a singleꢀpoint hydrogen bond with linker residue
Met1160, while Nꢀ1' of the fluorotriazolopyridine interacts with the backbone –NH of Asp 1222.21 In
addition, a faceꢀtoꢀface πꢀstacking interaction is observed between Tyr1230 and the fluorotriazolopyridine
ring system. Furthermore, a twoꢀpoint binding contact between Cꢀ7' and Cꢀ4'' and the lone pairs of the
Arg1208 carbonyl group is apparent. Confirmation that the (R)ꢀenantiomer is preferred by the protein was
demonstrated by the methyl substituent filling a small, lipophilic pocket in MET, defined by the side chains
of Val1092, Leu1157, Ala1226, and Lys1110.22 Finally, the fluorine atom at the 8'ꢀposition fills a small
cleft within the active site, where it sits perpendicular to the backbone carbonyl of Asn1209.
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Figure 3. Ligand interaction diagram of MET and compound 5
Having identified a structurally unique scaffold with nanomolar inhibition of MET phosphorylation
we further characterized compound 5. The solubility of 5 was superior to compounds 1, 2 and 3 (0.01 N
HCl / PBS / SIF (ꢀg/mL): 172 / 34 / 55) and showed no timeꢀdependent inhibition of CYP3A4 (preꢀ
incubation IC50 >50 ꢀM, postꢀincubation IC50 >50 ꢀM). To supplement these promising data, the metabolic
stability and pharmacokinetic properties of 5 were evaluated (Table 2). Compound 5 demonstrated low
intrinsic clearance in rat, mouse and human liver microsomes. Additionally, the associated plasma free
fraction (fu) was high across species relative to compounds 1ꢀ4. For example, rat free fraction for 1: 0.001;
2: 0.007; 3: 0.018 and 4: 0.022 in comparison to 5: 0.196. Consistent with its low turnover in rat liver
microsomes compound 5 was cleared at a low to moderate rate after dosing in male SpragueꢀDawley rats.
While 5 showed similar total clearance (Cl) to compounds 1-4 in rat, i.e. 0.38(L/h/kg) vs 0.06, 0.19, 0.56
and 0.57(L/h/kg) respectively, the unbound clearance (Clu) was significantly improved for 5 compare to 1ꢀ4
(1.9 (L/h/kg) vs 58, 26, 31, and 26 (L/h/kg) respectively), which could be advantageous for lowering human
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dose requirements. Furthermore, compound 5 demonstrated both a reasonable bioavailability and halfꢀlife
for further in vivo testing.
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Table 2. in vitro ADME and in vivo PK parameters of 5
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Compound
5
8
RLM Cla (ꢀL/min/mg)
MLM Cla (ꢀL/min/mg)
HLM Cla (ꢀL/min/mg)
<5
14
b
Plasma fu rat
0.196
0.245
0.320
mouse
human
Rat pharmacokineticsc
Cl (L/h/kg)d
Clu (L/h/kg)
Vss (L/kg)
0.38
1.9
0.4
0.8
44
T1/2 (h)
F (%)e
aIn vitro (RLM = rat liver microsomes; MLM = mouse liver microsomes; HLM = human liver
microsomes). Plasma unbound fraction (fu) was measured using equilibrium dialysis; concentration = 5
b
c
d
ꢀg/mL. In vivo experiments were carried out using male SpragueꢀDawley rats n = 3. iv, 0.25 mg/kg
(DMSO). epo, 2 mg/kg (2% hydroxypropyl methylcellulose 1% Tween 80 in water, pH 2.2).
To demonstrate efficacy and target coverage over time of compound 5 in vivo, a PD assay
measuring changes in HGFꢀinduced phosphorylation of MET in mouse liver was used (Figure 4). Mice
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were administered a single dose of 5 at 10 mg/kg by oral gavage. Human recombinant HGF was injected
intravenously at 1, 3, 6, 9, 12 or 24 hours postꢀdose. Liver and blood were harvested 5 minutes after
administration of recombinant HGF. MET phosphorylation was inhibited at least 91% for 9 hours with an
associated unbound plasma concentration of 193 nM, covering both the cellular IC50 and IC90.
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Figure 4. Compound 5 inhibited HGFꢀinduced phosphorylation of MET over time in the mouse liver PD
assay after oral administration at 10 mg/kg (10 mL/kg dose volume, formulated in 2% hydroxypropyl
methylcellulose 1% Tween 80 in water, pH 2.2 adjusted with methanesulfonic acid). Data represent the
mean +/ꢀ standard deviation (n = 3 mice per group). Statistical significance was determined by ANOVA
followed by Bonferroni/Dunn post hoc test. * = p<0.0001 compared to HGF control. Red circles represent
the mean terminal unbound concentration of 5 in the plasma +/ꢀ standard deviation (n = 3 mice per group).
On the basis of the desirable solubility, lack of TDI and robust in vivo efficacy of compound 5 in our PD
models, we sought a general synthetic method to allow for rapid preparation of naphthyridinone analogs
substituted at the Cꢀ3 position; substitution at this position had proved pivotal for improving
physicochemical properties, metabolism and potency in previous series (e.g. compounds 3 and 4). In
addition, variation at the Cꢀ6' position was also important, since both the 3ꢀmethylꢀisoxazole and the Nꢀ
methyl pyrazole were good partners with the triazolopyridine core (e.g. compound 4). Finally, since
fluorine at the 8ꢀposition was not always necessary in previous series, variation of this substituent was also
included in the SAR.
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The synthesis of compound 5 analogs began with a Suzuki reaction between 5ꢀchloroꢀ2,3ꢀ
difluoropyridine (6a) or 5ꢀbromoꢀ2ꢀfluoropyridine (6b) and (3ꢀmethylisoxazolꢀ5ꢀyl)boronic acid or a Stille
reaction with 1ꢀmethylꢀ4ꢀ(tributylstannyl)ꢀ1Hꢀpyrazole, yielding fluoropyridine intermediates 8bꢀd
(Scheme 3). 8bꢀd were combined with hydrazine in iꢀPrOH and warmed to afford hydrazines 9bꢀd.
Naphthyridinones substituted at the Cꢀ3 position were derived through an annulation reaction between
commercially available ethyl 5ꢀbromoꢀ2ꢀmethylnicotinate (16) and 1,3,5ꢀtriazine under basic conditions
producing 3ꢀbromoꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (17) in 91% yield.23 Substitution of bromine was achieved
by heating with NaOMe/MeOH or MeOCH2CH2ONa/MeOCH2CH2OH in the presence of 3,4,7,8ꢀ
tetramethylꢀ1,10ꢀphenanthroline and CuI as catalysts to provide naphthyridinones intermediates 10b,c.
Exposure of 10b,c and (±)ꢀethyl 2ꢀiodopropanoate to Cs2CO3 in THF at elevated temperature generated
racemic ethyl esters 12b,c, which were converted to the corresponding carboxylic acids 13b,c through
treatment with an aqueous solution of HCl. HATU mediated coupling of the 13b-c with hydrazines 9a-d
yielded the hydrazide intermediates, which underwent dehydration with DEAD, TMSN3 and PPh3 to give
triazolopyridines 5, 18-25 after chiral separation.24
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Scheme 3. Synthesis of naphthyridinones (5, 18-25)
3
4
5
6
7
8
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aReagents and conditions: (i) for 8b: 3ꢀmethylꢀ5ꢀ(tributylstannyl)isoxazole, Pd2(dba)3, P(tꢀBu)3•BF4, CsF,
1,4ꢀdioxane, 90 °C, 12 h, 66%; for 8c: 1ꢀmethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀ
pyrazole, Pd(OAc)2, XꢀPhos, K3PO4, 1,4ꢀdioxane/water, 100 °C, 45 min, 81%; for 8d: 1ꢀmethylꢀ4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrazole, PdCl2(dppf), Cs2CO3, 1,4ꢀdioxane/water, 90 °C, 3 h,
87%; (ii) NH2NH2, iꢀPrOH, for 9b: 60 °C, 2 h, 96%; for 9c: 65 °C, 3 h, 97%; for 9b: 60 °C, 48 h, 98%;
(iii) tꢀBuOK, DMSO, 80 °C, 1 h, 91%; (iv) 3,4,7,8ꢀtetramethylꢀ1,10ꢀphenanthroline, CuI, for 10b: MeONa,
MeOH, 130 °C, 24 h, 89% (75% pure); for 10c: t-BuONa, MeOCH2CH2OH, 124 °C, 21 h, 85% (v)
Cs2CO3, THF, for 12b: DMF, 70 °C, 18 h, 40%; 12c: THF, 60 °C, 90 min, 53%; (vi) aq. HCl, for 13b: 80
°C, 10 h, 85%; for 13c: THF, 65 °C, 4 h, 78%; (vii) for HATU, Hunig’s base, MeCN or DMF, 23 °C, 1ꢀ3
h, 23ꢀ92%; (viii) PPh3, TMSN3, DEAD, THF, 23 °C, 1ꢀ3 h, 20ꢀ73%; then chiral separation.
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Table 3. Representative naphthyridinones
3
4
5
6
N
O
R3
N
Me
R1
7
N
N
8
N
9
R2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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52
53
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59
60
Biochemical
IC50 (nM)a
Cellular
IC50(nM)b
Compound
R1
R2
F
R3
LipEc
7.3
7.2
7.5
7.4
7.8
7.6
7.9
7.0
7.5
cLogP
1.2
H
3
2
1
2
2
1
1
9
3
9
5
5
F
MeO
1.6
18
19
20
21
22
23
24
25
F
MeOCH2CH2O
MeOCH2CH2O
H
7
1.5
H
F
7
1.3
8
0.9
F
MeO
6
1.3
F
MeOCH2CH2O
MeO
5
1.1
H
H
11
8
1.1
MeOCH2CH2O
1.0
aInhibition of MET kinase activity, n ≥ 2. bInhibition of HGFꢀmediated MET phosphorylation in PC3 cells,
n ≥ 2. cLipE = pIC50 ꢀ clog P.
Consistent with SAR completed in the series involving compounds 3 and 4, enzyme and cellular
potencies were maintained across multiple variations made within the naphthyridinone series with LipE
ranging from 7.0 to 7.9 (Table 3). Cꢀ6' substitution with methylisoxazoles (eg. 5, 18-20) and
methylpyrazoles (e.g. 21-25) appeared interchangeable with respect to potency. The addition of methoxy
(18, 22, 24) or methoxyethoxy (19, 20, 23, 25) groups at the Cꢀ3 position of the naphthyridinone, installed
for the purposes of improving solubility, were also well tolerated and maintained or slightly increased the
enzymatic potency (2ꢀ3x) when compared to the unsubstituted naphthyridinones 5 or 21. Finally,
replacement of fluorine with hydrogen only had an impact on potency in the pyrazole analogs (22 vs 24 and
23 vs 25), showing a slight decrease in enzyme potency (4ꢀ6x).
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Table 4. in vitro ADME and in vivo PK parameters of naphthyridinones 5, 18-25
5
6
7
8
9
Compound
5
8
18
ꢀꢀ
19
16
14
20
20
9
21
8
22
12
7
23
6
24
9
25
RLM Cla (ꢀL/min/mg)
MLM Cla (ꢀL/min/mg)
HLM Cla (ꢀL/min/mg)
<5
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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30
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<5
14
ꢀꢀ
23
7
<5
<5
8
7
ꢀꢀ
<5
9
<5
8
b
Plasma fu rat
0.196
0.245
0.320
ꢀꢀ
ꢀꢀ
ꢀꢀ
0.080 0.190 0.271 0.139 0.174 0.206 0.296
0.317 0.435 0.376 0.353 0.366 0.409 0.424
0.409 0.338 0.478 0.309 0.419 0.286 0.498
mouse
human
Rat pharmacokineticsc
Cl (L/h/kg)d
Clu (L/h/kg)
Vss (L/kg)
0.38
1.9
0.4
0.8
44
0.46
ꢀꢀ
0.20
2.4
0.7
2.9
54
0.29
1.5
1.0
2.4
59
0.28
1.0
0.7
4.0
31
0.12
0.9
0.5
3.6
43
0.27
1.6
0.9
3.0
30
0.65
3.2
1.2
1.7
36
0.70
2.4
1.3
1.8
25
0.5
1.1
31
T1/2 (h)
F (%)e
aIn vitro (RLM = rat liver microsomes; MLM = mouse liver microsomes; HLM = human liver
b
microsomes). Plasma unbound fraction (fu) were measured using equilibrium dialysis; Concentration = 5
ꢀg/mL. cIn vivo experiments with male SpragueꢀDawley rats (n = 3). div, 0.25 mg/kg (DMSO). epo, 2 mg/kg
(2% hydroxypropyl methylcellulose 1% Tween 80 in water, pH 2.2 adjusted with methanesulfonic acid)
The metabolic stability and pharmacokinetic properties of 18-25 were evaluated (Table 4). For all
compounds, low microsomal clearance was observed across species. The free fraction in rat, mouse and
human plasma were high, consistent with data collected for compound 5. No obvious differentiation was
observed based upon their respective unbound clearances (Clu) and oral bioavailabilities (F). Therefore, the
in vivo halfꢀlife was used as the determining factor to prioritize the candidates for additional profiling.
Analogs represented by compound 4, bearing a MeOCH2CH2Oꢀ solubilizing group demonstrated a
consistent crossꢀspecies metabolic profile in contrast to their Hꢀ or MeOꢀ counterparts.25 Consistent with
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previous observations, when inhibitors 20 and 23 were incubated with NADPH in the presence of liver
microsomes, their metabolic profiles were relatively consistent across species, showing demethylation of
the MeOCH2CH2Oꢀ side chain and subsequent oxidation to the acid. Consequently, compounds 19, 20 and
23 were selected for advancement into a PD timeꢀcourse study with the expectation that their longer halfꢀ
lives would translate into extended duration of coverage.
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Figure 5. The effect of 19, 20 and 23 on HGFꢀinduced phosphorylation of MET over time in the mouse
liver PD assay after oral administration at 10 mg/kg (10 mL/kg dose volume, formulated in 2%
hydroxypropyl methylcellulose 1% Tween 80 in water, pH 2.2 adjusted with methanesulfonic acid). Data
represent the mean +/ꢀ standard deviation (n = 3 mice per group). Statistical significance was determined by
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ANOVA followed by Bonferroni/Dunn post hoc test. * = p<0.0001 compared to HGF control. Red circles
represent the mean terminal unbound concentration of compound 19, 20 or 23 in the plasma +/ꢀ standard
deviation (n = 3 mice per group).
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When dosed at 10 mg/kg, unlike compounds 5 and 19, compounds 20 and 23 exhibited extended
inhibition of MET activity in mice, achieving >90% inhibition of HGFꢀinduced phosphorylation of MET
for at least 12 hours (Figure 5). Furthermore, compounds 20 and 23 maintained approximately 50%
inhibition over a 24 hour period. Based on these results, 20 and 23 were selected as structurally diverse
candidates for PK profiling in a second preclinical species.
Table 5. Dog PK parameters of 20 and 23
Compound
Dog pharmacokineticsa
Cl (L/h/kg)
20
23
0.30b
0.68
1.4
0.23c
0.46
1.1
Clu (L/h/kg)d
Vss (L/kg)
T1/2 (h)
4.3
5.9
19300
53e
21000
49f
10 mg/kg AUC0→∞ (ꢀg*h/L)
F (%)
94100
87e
82600
64f
30 mg/kg AUC0→∞ (ꢀg*h/L)
F (%)
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aIn vivo experiments with male beagle dogs (n = 3). biv 0.425 mg/kg (20% hydroxypropyl betaꢀcyclodextrin
1
2
3
4
5
6
7
c
in water). iv 0.713 mg/kg (20% hydroxypropyl betaꢀcyclodextrin in water, pH 3.5 adjusted with
methanesulfonic acid). dUnbound fraction (fu) were measured using equilibrium dialysis; Concentration = 5
ꢀg/mL. epo (2% hydroxypropyl methylcellulose 1% Tween 80 in water, pH 2.0 adjusted with
f
methanesulfonic acid). po (2% hydroxypropyl methylcellulose 1% Tween 80 in water, pH 2.2 adjusted
with methanesulfonic acid).
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23
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Compounds 20 and 23 showed low clearance (0.30 and 0.23 (L/h/kg)) and moderate volumes of
distribution (1.4 and 1.1 L/kg) respectively, with comparable halfꢀlives (4.3 and 5.9 h) following IV
administration to beagle dogs (Table 5). After oral dosing at 10 and 30 mg/kg, the AUCs increased
proportionally. The bioavailability for 20 was 53% at 10 mg/kg and 87% at 30 mg/kg in comparison to
49% and 64% for compound 23 at the same doses. Overall, both compounds had reasonable dog iv and po
PK properties.
Ultimately, 23 was advanced and evaluated in a METꢀdependent xenograft model (Figure 6).
Specifically, the NIHꢀ3T3/TPRꢀMet model derived from NIH3T3 cells transfected with human TPRꢀMet,
an oncogenic form of MET, was employed in this study.26 Following oral administration (q.d.) of 23 at 0.1,
0.3, 1, 3 and 10 mg/kg over 22 days, doseꢀdependent tumor growth inhibition was observed with an ED50 of
0.3 mg/kg and an ED90 of 0.8 mg/kg with no adverse effect on body weight.
Tumor Growth
Plasma Concentration
Figure 6. Effect of compound 23 on tumor growth in the NIHꢀ3T3/TPRꢀMet xenograft model. Compound
23 was administered by oral gavage once per day beginning 24 hours post tumor cell implantation (10
mL/kg dose volume, formulated in 20% hydroxypropyl betaꢀcyclodextrin in water, pH 3.5 adjusted with
methanesulfonic acid). Results are expressed as mean ± standard error (n = 12 per group). Asterisk denotes
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p <0.0005 compared to treatment with vehicle. Vehicle: 20% HPBCD, pH 3.5 adjusted with
methanesulfonic acid).
5
6
7
8
9
Illustrated by the graph of unbound plasma concentration of 23 over time, doses covering the
cellular IC90 for at least 12 hours (3 and 10 mg/kg) resulted in >90% tumor growth inhibition. Compound
23 did not suffer from TDI of CYP3A4 (IC50 >50 ꢀM, IC50 w/ preincubation >50 ꢀM), exhibited good
solubility in its hydrate form (0.01 N HCl / PBS / SIF (ꢀg/mL): 174 / 77 / >200) and displayed exquisite
selectivity against a panel of 402 tyrosine and serine/threonine kinases.27 These attributes when coupled
with its long halfꢀlife in rat and dog, its predictable metabolism and good human dose projection convinced
us to select compound 23 (AMG 337) as the clinical candidate.
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Conclusions
In summary, through rational design guided by our previous work on MET (eg. compounds 1-4), we have
generated a new naphthyridinone scaffold that demonstrated nanomolar inhibition of MET kinase activity,
improved solubility, a favorable TDI profile and robust in vivo efficacy in mouse models of METꢀdriven
tumorigenesis. Moreover, many of the compounds defined by the naphthyridinone scaffold, including 23,
exhibit high LipE and good pharmacokinetics in rat and dog with high unbound concentration in plasma.
Compound 23 showed potent and sustained inhibition of MET phosphorylation in a mouse liver
pharmacodynamic model and significantly inhibited tumor growth in a METꢀdependent mouse efficacy
model. 23 is currently in phase two clinical trials.
Experimental Section
Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used as
obtained. Anhydrous organic solvents were purchased from Aldrich packaged under N2 in Sure/Seal™
bottles and used directly. Reactions were monitored using Agilent 1100 Series LCMS with ultraviolet light
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(UV) detection at 215 nm and 254 nm and a low resolution electrospray mode (ESI). HRMS data was
recorded on Agilent LCꢀMS TOF (time of flight), Model G1969A instrument. Medium pressure liquid
chromatography (MPLC) was performed on a CombiFlash® Companion® (Teledyne Isco) with Redisep®
normalꢀphase silica gel (35ꢀ60 micron) columns and UV detection at 254 nm. Purity was measured using
Agilent 1100 Series high performance liquid chromatography (HPLC) with UV detection at 215, 254 and
280 nm (15 min; 1.5 mL/min flow rate; eluting with a binary solvent system A and B using a gradient
elution (A: water with 0.1% TFA; B: MeCN with 0.1% TFA). Unless otherwise noted, the purity of all
compounds was ≥ 95%. Compounds bearing a stereogenic center were purified by preparative supercritical
fluid chromatography (SFC) or preparative HPLC using a chiral column. Enantiomeric excess for
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1
compounds bearing a stereogenic center were determined using analytical HPLC. H NMR spectra were
recorded on a Bruker AVꢀ400 (400 MHz) spectrometer at ambient temperature. Chemical shifts are
reported in parts per million (δ), and are calibrated using residual undeuterated solvent as an internal
reference. Data for 1H NMR spectra are reported as follows: chemical shift (δ ppm) (multiplicity, coupling
constant (Hz), integration). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad, or combinations thereof. Differential scanning calorimetry (DSC) was
performed on a TA Instruments Q100 calorimeter at in an aluminum Tzero pan under dry N2, flowing at 50
mL/min.
Preparation of (
R
)-6-(1-(8-Fluoro-6-(3-methylisoxazol-5-yl)-[1,2,4]triazolo[4,3-
)-one (5) (racemic route):
a]pyridin-3-yl)ethyl)-
1,6-naphthyridin-5(6
H
5-(5,6-Difluoropyridin-3-yl)-3-methylisoxazole (8a): A pressure vessel was charged with 5ꢀchloroꢀ2,3ꢀ
difluoropyridine (6a) (5.00 g, 33.4 mmol), 3ꢀmethylꢀ5ꢀ(tributylstannyl)isoxazole (7) (18.7 g, 50.2 mmol),
XPhos (2.23 g, 4.68 mmol), PdOAc2 (0.53 g, 2.34 mmol) and 1,4ꢀdioxane (170 mL). The vessel was then
purged with Ar, sealed and heated at 100 °C for 16 h. After cooling to room temperature, the mixture was
concentrated under reduced pressure and purified by MPLC eluting with a gradient of 10ꢀ30% EtOAc in
hexanes. The resulting solid was triturated with hexanes and filtered to afford 5ꢀ(5,6ꢀdifluoropyridinꢀ3ꢀyl)ꢀ
3ꢀmethylisoxazole (8a) (3.91 g, 60% yield) as a yellow amorphous solid. 1H NMR (400 MHz, DMSOꢀd6) δ
ppm 8.52 ꢀ 8.59 (m, 2 H) 7.07 (s, 1H) 2.31 (m, 3H). LRMS (ESI): m/z (M + H) calcd 197.1; found 197.0.
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5-(5-Fluoro-6-hydrazinylpyridin-3-yl)-3-methylisoxazole (9a): A pressure vessel was charged with 5ꢀ
(5,6ꢀdifluoropyridinꢀ3ꢀyl)ꢀ3ꢀmethylisoxazole (8a) (3.68 g, 18.8 mmol), NH2NH2 (3.53 mL, 113 mmol) and
iꢀPrOH (95.0 mL). The vessel was sealed and warmed to 65 °C for 3 h. Upon cooling to room temperature,
the desired product precipitated out of solution. The solid was filtered and washed with iꢀPrOH (2 × 20
mL). The solid was triturated with a saturated solution of NaHCO3 (50 mL), filtered and washed with
water (50 mL) to afford 5ꢀ(5ꢀfluoroꢀ6ꢀhydrazinylpyridinꢀ3ꢀyl)ꢀ3ꢀmethylisoxazole (9a) (3.46 g, 90% yield)
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as a white amorphous solid. H NMR (400 MHz, DMSOꢀd6) δ ppm 8.42 ꢀ 8.44 (m, 1 H) 8.37 ꢀ 8.38 (m, 1
H) 7.76 (dd, J=12.23, 1.86 Hz, 1 H) 6.70 (s, 1 H) 4.33 (br. s., 2 H) 2.25 (s, 3 H). LRMS (ESI): m/z (M + H)
calcd 209.1; found 209.2.
(±)-Ethyl 2-(5-oxo-1,6-naphthyridin-6(5H)-yl)propanoate (12a): A pressure vessel was charged with
1,6ꢀnaphthyridinꢀ5(6H)ꢀone (10a) (3.00 g, 21.0 mmol), Cs2CO3 (13.0 g, 41.0 mmol), ethyl 2ꢀ
bromopropanoate (11) (5.30 mL, 41.0 mmol) and THF (70.0 mL). The vessel was sealed and heated at 60
°C for 3 h. Upon cooling to room temperature, the reaction was diluted with water (200 mL) and extracted
with ethyl acetate (2 × 200 mL). The organic phase was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by MPLC eluting with a gradient of 0ꢀ5%
DCM:MeOH (90:10) in DCM to afford (±)ꢀEthyl 2ꢀ(5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)propanoate (12a)
1
(5.29 g, quantitative) as a yellow oil. H NMR (400 MHz, DMSOꢀd6) δ ppm 8.94 (dd, J=4.55, 1.81 Hz, 1
H) 8.52 (ddd, J=8.09, 1.83, 0.73 Hz, 1H) 7.76 (d, J=7.73 Hz, 1H) 7.53 (dd, J=8.07, 4.55 Hz, 1H) 6.76 (dd,
J=7.63, 0.68 Hz, 1H) 5.32 (q, J=7.17 Hz, 1H) 4.13 (q, J=7.14 Hz, 2H) 1.62 (d, J=7.24 Hz, 3H) 1.15 (t,
J=7.09 Hz, 3H). LRMS (ESI): m/z (M + H) calcd 247.1; found 247.2.
(±)-2-(5-Oxo-1,6-naphthyridin-6(5H)-yl)propanoic acid hydrochloride (13a): A roundꢀbottom flask
equipped with a reflux condenser was charged with ethyl (±)ꢀethyl 2ꢀ(5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀ
yl)propanoate (12a) (5.29 g, 21.5 mmol) and a 6N HCl solution (10.0 mL, 330 mmol). The reaction was
heated at 60 °C for 7 h. Additional conc. HCl (~5 mL) was added over the course of the 7 h until complete
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hydrolysis was observed. The reaction mixture was cooled to room temperature and concentrated under
reduced pressure to afford (±)ꢀ2ꢀ(5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)propanoic acid hydrochloride (13a) as a
dark yellow amorphous solid which was used without further purification. 1H NMR (400 MHz, DMSOꢀd6)
δ 12.92 (br. s., 2H), 9.07 (dd, J=1.76, 5.09 Hz, 1H), 8.81 (ddd, J=0.60, 1.80, 7.80 Hz, 1H), 7.97 (d, J=7.73
Hz, 1H), 7.74 (dd, J=5.04, 8.07 Hz, 1H), 6.91 (dd, J=0.59, 7.73 Hz, 1H), 5.35 (q, J=7.20 Hz, 1H), 1.62 (d,
J=7.34 Hz, 3H) LRMS (ESI): m/z (M + H) calcd 219.1; found 219.2.
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(R
)-6-(1-(8-Fluoro-6-(3-methylisoxazol-5-yl)-[1,2,4]triazolo[4,3-
a]pyridin-3-yl)ethyl)-1,6-
naphthyridin-5(6 )-one (5): A roundꢀbottom flask was charged with (±)ꢀ2ꢀ(5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ
H
6(5H)ꢀyl)propanoic acid hydrochloride (13a) (151 mg, 593 µmol), 5ꢀ(5ꢀfluoroꢀ6ꢀhydrazinylpyridinꢀ3ꢀyl)ꢀ3ꢀ
methylisoxazole (9a) (136 mg, 652 µmol), HATU (293 mg, 771 µmol) in DMF (3 mL) under an
atmosphere of N2. The reaction mixture was treated with Hunig's base (311 µL, 1.78 mmol) and stirred at
room temperature for 3 h. The reaction mixture was concentrated under reduced pressure and the residue
was then quickly passed through a plug of silica gel eluting with a gradient of 0ꢀ100%
DCM:MeOH:NH4OH (90:10:10) in DCM, concentrated under reduced pressure and use without further
purification.
In a roundꢀbottom flask the product was then suspended in THF (6.00 mL) under an atmosphere of N2 and
treated with PPh3 (233 mg, 889 µmol) and TMSN3 (118 µL, 889 µmol). DEAD (141 µL, 889 µmol) was
introduced and the reaction was allowed to stirred for 3 h. The reaction mixture was passed through a
conditioned Isolute ® SPE column (SCXꢀ2) and washed with MeOH (3 × 25 mL). The compound was then
released from the column using a solution of ammonia in MeOH (2.0 M, 3 × 25 mL) and the solution was
concentrated under reduced pressure. The residue was purified by MPLC eluting with a gradient of 0ꢀ100%
DCM:MeOH:NH4OH (90:10:10) in DCM to afford (±)ꢀ6ꢀ(1ꢀ(8ꢀfluoroꢀ6ꢀ(3ꢀmethylisoxazolꢀ5ꢀyl)ꢀ
[1,2,4]triazolo[4,3ꢀa]pyridinꢀ3ꢀyl)ethyl)ꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (5) (120 mg, 52% yield over two
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steps) as a white amorphous solid. H NMR (400 MHz, DMSOꢀd6) δ 8.92 (dd, J=1.81, 4.55 Hz, 1H), 8.81
(d, J=1.08 Hz, 1H), 8.62 (ddd, J=0.73, 1.81, 8.12 Hz, 1H), 7.84 (dd, J=1.12, 11.49 Hz, 1H), 7.77 (d, J=7.82
Hz, 1H), 7.54 (dd, J=4.60, 8.12 Hz, 1H), 7.02 (q, J=7.10 Hz, 1H), 7.01 (s, 1H), 6.78 (dd, J=0.54, 7.78 Hz,
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1H), 2.27ꢀ2.34 (m, 3H), 2.00 (d, J=7.14 Hz, 3H). HRMS (ESI): m/z (M + H) calcd 391.1313; found
391.1328. The enantiomers of (±)ꢀ5 were seperated by high performance liquid chromatography (HPLC)
through repeated 1.5 mL injections of a 14 mg/mL solution onto a Chiralpak IAꢀH, 3 cm × 25 cm (i.d. ×
length) column. Isocratic elution with EtOH:Heptane (1:1) at a flowrate of 45 mL/min provided 52 mg of
peak 1 (ent-5) (ee > 99%) and 53 mg of peak 2 (5) (ee > 99%).
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Alternative preparation of (
3-yl)ethyl)-1,6-naphthyridin-5(6
2-(5-Oxo-1,6-naphthyridin-6(5H
R
)-6-(1-(8-Fluoro-6-(3-methylisoxazol-5-yl)-[1,2,4]triazolo[4,3-
)-one (5) (enantioselective route):
)-yl)propanoic acid hydrochloride (15): A roundꢀbottom flask
a]pyridin-
H
(R
)-
equipped with an addition funnel was charged with 1,6ꢀnaphthyridinꢀ5(6H)ꢀone (10a) (90.8 g, 620 mmol),
PPh3 (260 g, 994 mmol) and (S)ꢀ(ꢀ)ꢀlactic acid methyl ester (14) (74.0 mL, 780 mmol) in THF (1.20 L) and
the mixture was cooled in an ice bath. To this mixture was added DEAD (150 mL, 930 mmol) dropꢀwise,
while maintaining a reaction temperature below 15 °C Following addition, the reaction temperature was
stabilized at 10 °C, the ice bath was removed. An additional portion of THF (200 mL) was introduced and
the reaction mixture stirred for an additional 10 h. The reaction mixture was concentrated under reduced
pressure and azeotroped with EtOAc (2 × 250 mL). The reaction vessel was equipped with an overhead
stirrer and the reaction mixture was partitioned between a 6N HCl solution (1.0 L) and Et2O (1.5 L). The
layers were separated and the organic layer was extract with 6N HCl (2 × 300 mL). The combined aqueous
layers were filtered through a fritted filter and transferred to a 3ꢀneck roundꢀbottom flask equipped with a
condenser and overhead stirrer. The reaction mixture was heated to 75 °C for 10 h then allowed to cool to
room temperature. Additional 6N HCl solution (500 mL) was introduced and the reaction mixture was
washed with EtOAc (3 × 500 mL). The aqueous layer was concentrated under reduce pressure to produce a
thick slurry (~500 mL). The slurry was cooled 0 °C and the precipitate was filtered and washed with cold
MeCN to afford (R)ꢀ2ꢀ(5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)propanoic acid hydrochloride (15) (107 g, 67%
yield over two steps) as a light yellow amorphous solid. An analytical sample was confirmed to be >95%
ee. Analysis performed by HPLC, Chiralpak ADꢀH, 4.6 x 250 mm, 25% EtOH w/ 0.1% TFA and 75%
heptane, flowrate = 1 mL/min, detection = 215 nm.
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(R
)-6-(1-(8-Fluoro-6-(3-methylisoxazol-5-yl)-[1,2,4]triazolo[4,3-
a]pyridin-3-yl)ethyl)-1,6-
5
6
7
naphthyridin-5(6 )-one (5): A 250ꢀmL, twoꢀneck, roundꢀbottom flask equipped with a 25ꢀmL addition
H
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funnel was charged with a suspension of (R)ꢀ2ꢀ(5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)propanoic acid
hydrochloride (15) (4.00 g, 16.0 mmol), 1ꢀ(3ꢀfluoroꢀ5ꢀ(3ꢀmethylisoxazolꢀ5ꢀyl)pyridinꢀ2ꢀyl)hydrazine (9a)
(3.60 g, 17.0 mmol) and HATU (9.00 g, 24.0 mmol) in MeCN (50 mL) under an atmosphere of N2. The
reaction mixture was cooled to 0 ºC in an ice bath before Hunig's base (8.2 mL, 47 mmol) was added dropꢀ
wise to the rapidly stirred suspension over 30 minutes, while maintaining an internal temperature below 10
ºC in order to avoid racemization.28 Following addition, the reaction was warmed to room temperature over
30 minutes and concentrated under reduced pressure. The residue was then quickly passed through a plug
of silica gel and eluted with a gradient of 0ꢀ100% DCM:MeOH:NH4OH (90:10:10) in DCM to afford (2R)ꢀ
N'ꢀ(3ꢀfluoroꢀ5ꢀ(3ꢀmethylisoxazolꢀ5ꢀyl)pyridinꢀ2ꢀyl)ꢀ2ꢀ(5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)propanehydrazide
which was used without further purification.
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In a roundꢀbottom flask, crude (2R)ꢀN'ꢀ(3ꢀfluoroꢀ5ꢀ(3ꢀmethylisoxazolꢀ5ꢀyl)pyridinꢀ2ꢀyl)ꢀ2ꢀ(5ꢀoxoꢀ1,6ꢀ
naphthyridinꢀ6(5H)ꢀyl)propanehydrazide (1.90 g, 4.70 mmol) was resuspended in THF (50 mL). PPh3 (1.80
g, 7.00 mmol) was introduced and the reaction vessel was purged with N2. The reaction was cooled to 0 ºC
and treated with TMSN3 (930 ꢀL, 7.00 mmol) follow by slow addition of DEAD (1.10 mL, 7.00 mmol) via
syringe, while maintaining an internal the reaction temperature below 30 ºC. After 1 h, the reaction mixture
was concentrated under reduced pressure and partitioned between EtOAc (300 mL) and 2N HCl solution
(300 mL). The aqueous layer was collected and the organic layer was extracted with 2N HCl solution (2 ×
300 mL). The combined aqueous layers were cooled to 0 ºC and neutralized with an aqueous 6N NaOH
solution to pH 7. The precipitate was filtered and washed with cold H2O (2 × 50.0 mL) and cold EtOH
(50.0 mL). The solid was purified by slow recrystallization in EtOH to afford (R)ꢀ6ꢀ(1ꢀ(8ꢀfluoroꢀ6ꢀ(3ꢀ
methylisoxazolꢀ5ꢀyl)ꢀ[1,2,4]triazolo[4,3ꢀa]pyridinꢀ3ꢀyl)ethyl)ꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (5) (1.50 g, 83%
yield). An analytical sample was confirmed to be >99% ee. Analysis performed by HPLC, Chiralpak ADꢀ
H, 4.6 x 100 mm, EtOH:Heptane (1:1), flowrate = 1 mL/min, detection = 215 nm. The isolated solid exists
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as crystalline anhydrous free base form II displaying two melts: a melt at 200ꢀ239 ºC and final melt at 292ꢀ
299 ºC. 1H NMR and LRMS data are consistent with compound 5 from the racemic synthesis.
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Preparation of 18-25:
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5-(6-Fluoropyridin-3-yl)-3-methylisoxazole (8b): A 3ꢀneck roundꢀbottom flask equipped with an
overhead stirrer, temperature probe and reflux condenser was charged with 3ꢀmethylꢀ5ꢀ
(tributylstannyl)isoxazole (233 g, 625 mmol), 5ꢀbromoꢀ2ꢀfluoropyridine (6b) (100 g, 568 mmol), (tꢀ
Bu)3P•BF4 (16.5 g, 56.8 mmol), Pd2(dba)3 (26.0 g, 28.4 mmol) and 1,4ꢀdioxane (1.25 L). CsF (173 g, 1.14
mol) was slowly added to the stirred reaction mixture. Following addition, the reaction mixture was
warmed to 90 °C (internal temperature). After 12 h, the reaction mixture was cooled to room temperature
and concentrated under reduced pressure. The crude material was suspended in EtOAc (1.0 L) and filtered
through a silicaꢀgel plug. The plug was eluted with EtOAc (~3.0 L) and the filtrate was concentrated under
reduced pressure. The resulting solid was triturated with acetonitrile (~200 mL) and allowed to age at 0 °C
for 30 minutes. The solid was filtered and washed with cold acetonitrile (25 mL) and hexanes (25 mL). The
filtrate was concentrated under reduced pressure and the solid was triturated with Et2O (200 mL). The
resultant solid was filtered and washed with Et2O (25 mL) and hexanes (25 mL). The two batches of solid
were combined to afford 5ꢀ(6ꢀfluoropyridinꢀ3ꢀyl)ꢀ3ꢀmethylisoxazole (8b) (66.4 g, 66 % yield) as a pale
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yellow amorphous solid. H NMR (400 MHz, CHLOROFORMꢀd) δ 8.64 (d, J=2.45 Hz, 1H), 8.18 (ddd,
J=2.49, 7.43, 8.56 Hz, 1H), 7.07 (ddd, J=0.68, 3.01, 8.53 Hz, 1H), 6.44 (s, 1H), 2.40 (s, 3H) LRMS (ESI):
m/z (M + H) calcd 179.1; found 179.1.
5-(6-Hydrazinylpyridin-3-yl)-3-methylisoxazole (9b): A 3ꢀneck roundꢀbottom flask equipped with an
overhead stirrer, internal temperature probe and reflux condenser was charged with 5ꢀ(6ꢀfluoropyridinꢀ3ꢀ
yl)ꢀ3ꢀmethylisoxazole (8b) (66.4 g, 373 mmol) in iꢀPrOH (760 mL). To this suspension, hydrazine (70.2
mL, 2.24 mol) was added and the reaction mixture was heated at 60 °C (internal temperature) for 2 h. The
reaction mixture was cooled to 0 °C and allowed to age for 1 hour. The precipitate was filtered and washed
with cold iꢀPrOH to afford 5ꢀ(6ꢀhydrazinylpyridinꢀ3ꢀyl)ꢀ3ꢀmethylisoxazole dihydrofluoride (9b) (82.6 g, 96
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% yield). 1H NMR (400 MHz, DMSOꢀd6) δ 8.44 (dd, J=0.68, 2.35 Hz, 1H), 8.02 (s, 1H), 7.82 (dd, J=2.35,
8.80 Hz, 1H), 6.79 (d, J=8.80 Hz, 1H), 6.59 (s, 1H), 4.28 (br. s., 2H), 3.30 (br. s., 1H), 2.24 (s, 3H) LRMS
(ESI): m/z (M + H) calcd 191.1; found 191.1.
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2,3-Difluoro-5-(1-methyl-1H-pyrazol-4-yl)pyridine (8c): A 3ꢀneck flask equipped with an overhead
stirrer, internal temperature probe and reflux condenser under an atmosphere of N2 was charged with
Pd(OAc)2 (6.01 g, 26.8 mmol), XꢀPhos (25.5 g, 53.5 mmol), 1ꢀmethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrazole (134 g, 642 mmol), and a mixture of 1,4ꢀdioxane (1550 mL) and water
(150 mL). To this mixture was added 5ꢀchloroꢀ2,3ꢀdifluoropyridine (6a) (80.0 g, 535 mmol) and K3PO4
(341 g, 1.61 mol). The internal temperature was raised to 97 °C and the reaction mixture was stirred for 45
min. The reaction mixture was allowed to cool to ~80 °C and filtered through a pad of celite which was
subsequently washed with EtOAc (500 mL). The organic layer was washed with an aqueous solution of
NaCl sat. (250 mL). The organic layer was concentrated under reduced pressure, the residue was triturated
with warm Et2O (500 mL), cooled to room temperature and the solid was filtered and washed with Et2O
(100 mL) to afford 2,3ꢀdifluoroꢀ5ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)pyridine (8c) (84.9 g, 81% yield) as an offꢀ
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white amorphous solid. H NMR (400 MHz, DMSOꢀd6) δ 8.23ꢀ8.31 (m, 2H), 8.28 (s, 1H), 7.99 (d, J=0.68
Hz, 1H), 3.87 (s, 3H). LRMS (ESI): m/z (M + H) calcd 196.1; found 196.2.
3-Fluoro-2-hydrazinyl-5-(1-methyl-1H-pyrazol-4-yl)pyridine (9c): A 3ꢀneck roundꢀbottom flask
equipped with an overhead stirrer, internal temperature probe and reflux condenser was charged with 2,3ꢀ
difluoroꢀ5ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)pyridine (8c) (65.4 g, 335 mmol) and NH2NH2•H2O (114 mL, 2.35
mol) in iꢀPrOH (170 mL). The reaction mixture was heated at 65 °C for 3 h and then diluted with iꢀPrOH
(75 mL) and cooled in an ice bath. The solid was filtered and triturated with an aqueous solution of
saturated NaHCO3 (20 mL), filtered and washed with Et2O to afford 3ꢀfluoroꢀ2ꢀhydrazinylꢀ5ꢀ(1ꢀmethylꢀ1Hꢀ
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pyrazolꢀ4ꢀyl)pyridine (9c) (67.2 g, 97% yield) as a white amorphous solid. H NMR (400 MHz, DMSOꢀd6)
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δ 8.16 (t, J=1.50 Hz, 1H), 8.05 (d, J=0.50 Hz, 1H), 7.80 (d, J=0.78 Hz, 1H), 7.68 (s, 1H), 7.59 (dd, J=1.86,
3
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12.72 Hz, 1H), 4.12 (s, 2H), 3.84 (s, 3H). LRMS (ESI): m/z (M + H) calcd 208.1; found 208.3.
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2-Fluoro-5-(1-methyl-1
H
-pyrazol-4-yl)pyridine (8d):
A
pressure vessel was charged with
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PdCl2(dppf)•DCM adduct (3.25 g, 3.98 mmol), Cs2CO3 (38.9 g, 119 mmol), 1ꢀmethylꢀ4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrazole (9.93 g, 47.7 mmol), 5ꢀbromoꢀ2ꢀfluoropyridine (6b)
(7.00 g, 39.8 mmol) in 1,4ꢀdioxane (100 mL) and water (10 mL). The reaction was purged with an
atmosphere of Ar, sealed and stirred at 90 °C for 3 h. The reaction was cooled to room temperature,
concentrated under reduced pressure and purified by MPLC eluting with a gradient of 0ꢀ10% DCM:MeOH
(90:10) in DCM to afford 2ꢀfluoroꢀ5ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)pyridine (8d) (6.10 g, 87% yield) as a tan
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amorphous solid. H NMR (400 MHz, DMSOꢀd6) δ 8.46 (ddd, J=0.68, 1.25, 2.57 Hz, 1H), 8.22 (d, J=0.50
Hz, 1H), 8.14 (ddd, J=2.45, 7.82, 8.51 Hz, 1H), 7.94 (d, J=0.78 Hz, 1H), 7.18 (ddd, J=0.64, 2.98, 8.51 Hz,
1H), 3.87 (s, 3H). LRMS (ESI): m/z (M + H) calcd 178.1; found 178.2.
2-Hydrazinyl-5-(1-methyl-1H-pyrazol-4-yl)pyridine (9d): A pressure vessel was charged with 2ꢀfluoroꢀ
5ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)pyridine (8d) (6.13 g, 34.6 mmol), NH2NH2 (5.43 mL, 173 mmol) in iꢀPrOH
(200 mL) and stirred at 65 °C. Additional hydrazine (5.43 mL, 173 mmol) was added to the reaction
mixture every 12 h over 2 days in order to achieve full consumption of the starting material. The reaction
was partially concentrated (~50%) and cooled in an ice bath. The resultant solid was filtered and washed
with cold iꢀPrOH to afford 2ꢀhydrazinylꢀ5ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)pyridine (9d) (6.40 g, 98% yield) as
1
a gray amorphous solid. H NMR (400 MHz, DMSOꢀd6) δ 8.22 (dd, J=0.68, 2.35 Hz, 1H), 7.97 (s, 1H),
7.72 (d, J=0.68 Hz, 1H), 7.63 (dd, J=2.35, 8.61 Hz, 1H), 7.31 (s, 1H), 6.71 (dd, J=0.73, 8.66 Hz, 1H), 3.74ꢀ
3.96 (m, 3H), 3.54 (br. s., 2H). LRMS (ESI): m/z (M + H) calcd 190.1; found 190.2.
3-Bromo-1,6-naphthyridin-5(6H)-one (17): A 3ꢀneck roundꢀbottom flask equipped with an internal
temperature probe and reflux condenser under an atmosphere of N2 was charged with ethyl 5ꢀbromoꢀ2ꢀ
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methylnicotinate (16) (100 g, 410 mmol), 1,3,5ꢀtriazine (37.0 g, 456 mmol) and dry DMSO (100 mL). The
solution was stirred at room temperature for 30 min and tꢀBuOK (52.4 g, 467 mmol) was added, followed
by dry DMSO (100 mL). A significant exotherm was observed with an internal temperature rising to 84 °C.
The reaction stirred for 30 min and the reaction was heated to 80 °C for an additional 30 min. The reaction
mixture was then cooled to room temperature and diluted with water (600 mL) where a precipitate formed.
AcOH (27.0 mL, 470 mmol) was added and the solution was stirred at room temperature for 30 minutes.
The reaction was then degassed (Warning! HCN is generated during this reaction) by sparging the solution
with N2 for 18 h. The exhaust gas was scrubbed by passage through 6N NaOH (500 mL) to remove excess
HCN. Following the purge, the resultant solid was collected by filtration and washed with H2O and then
Et2O. The resultant orange brown solid was triturated in refluxing MeCN (150 mL) for 15 minutes, then
cooled to room temperature. The solid was filtered to afford 3ꢀbromoꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (17)
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(83.9 g, 91% yield) as a tan solid. H NMR (400 MHz, DMSOꢀd6) δ 11.75 (br. s, 1H), 9.00 (d, J=2.45 Hz,
1H), 8.59 (dd, J=0.78, 2.45 Hz, 1H), 7.49 (d, J=7.34 Hz, 1H), 6.62 (dd, J=0.73, 7.38 Hz, 1H). LRMS (ESI):
m/z (M + H) calcd 225.0/227.0; found 225.0/227.0.
3-Methoxy-1,6-naphthyridin-5(6H)-one (10b): A sealed Parr reactor was charged with NaOMe (120 g,
2.22 mol) and MeOH (400 mL) followed by 3ꢀbromoꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (17) (50.0 g, 220 mmol),
3,4,7,8ꢀtetramethylꢀ1,10ꢀphenanthroline (10.5 g, 44.0 mmol), and CuI (4.23 g, 22.0 mmol). The reactor
was sealed and heated at 130 °C and stirred for 24 h. The reaction was then cooled to room temperature and
the pressure was released. The mixture was transferred to a roundꢀbottom flask and cooled to 0 °C and the
reaction was neutralized with H2O (500 mL) and aqueous 6N HCl solution to pH 7. The solution was
diluted with H2O (500 mL) and extracted with EtOAc (3 × 1.0 L). The organic layer was concentrated
under pressure and the solid was dissolved in EtOAc:MeOH (5:1, 1.5 L) and the insoluble brown solid was
removed by filtration. The organic layer was washed with an aqueous solution nꢀ(2ꢀ
hydroxyethyl)ethylenediaminetriacetic acid trisodium salt hydrate (10 wt%, 500 mL). The aqueous phase
was separated and extracted with EtOAc (2 × 500 mL), the organic layers were combined and concentrated
under reduced pressure to afford 3ꢀmethoxyꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (10b) (35.0 g, 89% yield) as a
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yellow amorphous solid contaminated with 3,4,7,8ꢀtetramethylꢀ1,10ꢀphenanthroline (25 wt%). H NMR
(400 MHz, METHANOLꢀd4) δ 8.63 (d, J=3.03 Hz, 1H), 8.04 (d, J=3.03 Hz, 1H), 7.30 (d, J=7.43 Hz, 1H),
6.75 (d, J=7.43 Hz, 1H), 3.98 (s, 3H). LRMS (ESI): m/z (M + H) calcd 177.1; found 177.0.
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3-(2-Methoxyethoxy)-1,6-naphthyridin-5(6H)-one (10c): A 3ꢀneck roundꢀbottom flask equipped with
overhead stirrer, temperature probe and reflux condenser under an atmosphere of N was charged with 2ꢀ
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methoxyethanol (1.00 L, 12.7 mol) followed by portionꢀwise addition of tꢀBuONa (247 g, 2.57 mol)
(temperature increased to ~70°C). Following addition, the reaction mixture stirred for an additional 15
minutes. The condenser was removed and the vessel fitted with a shortꢀpath distillation apparatus. The
temperature was raised to 130 °C and ~140 g of distillate was collected (mostly tꢀBuOH). The reaction was
then refitted with a reflux condenser and cooled to 50 °C. 3ꢀBromoꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (17) (100
g, 444 mmol), 3,4,7,8ꢀtetramethylꢀ1,10ꢀphenanthroline (23.0 g, 97.0 mmol) and CuI (8.46 g, 44.4 mmol)
where introduced and the reaction temperature was raised to 125 °C (internal temperature) for 24 h. The
reaction was cooled to room temperature, neutralized with AcOH (153 mL), diluted with EtOAc (2.5 L),
stirred briefly. The solution was filtered through a plug of silica gel, which was eluted with 5% MeOH in
DCM (5 L). The filtrate was concentrated under reduced pressure, azeotroped with PhMe and the product
recrystalized from iꢀPrOH to afford 3ꢀ(2ꢀmethoxyethoxy)ꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (10c) (83.4 g, 85%
yield) as a yellow amorphous solid contaminated with a copper impurity (broad NMR). An analytical
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sample was purified by MPLC eluting with a gradient of 0ꢀ100% DCM:MeOH (90:10) in DCM. H NMR
(400 MHz, DMSOꢀd6) δ 11.46 (br. s, 1H), 8.67 (d, J=3.03 Hz, 1H), 7.90 (d, J=2.84 Hz, 1H), 7.29 (d,
J=7.63 Hz, 1H), 6.60 (d, J=7.34 Hz, 1H), 4.25ꢀ4.32 (m, 2H), 3.69ꢀ3.74 (m, 2H), 3.32 (s, 3H). LRMS (ESI):
m/z (M + H) calcd 121.1; found 221.2.
(±)ꢀEthyl 2-(3-methoxy-5-oxo-1,6-naphthyridin-6(5H)-yl)propanoate (12b): A roundꢀbottom flask
under an atmosphere of N2 was charged with 3ꢀmethoxyꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (10b) (7.61 g, 43.2
mmol), Cs2CO3 (6.9 mL, 86 mmol) and THF (100 mL) followed by addition of ethyl 2ꢀbromopropionate
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(9.0 mL, 69 mmol) in DMF (50 mL) and heated at 70 °C for 18 h. The reaction was cooled to room
temperature and concentrated under reduced pressure. The residue was diluted with water and extracted
with EtOAc (3X). The organic layer was dried over MgSO4, filtered and concentrated under reduced
pressure. The crude mixture was purified by MPLC eluting with 50% EtOAc in hexanes to afford (±)ꢀethyl
2ꢀ(3ꢀmethoxyꢀ5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)propanoate (12b) (3.0 g, 40% yield) as an offꢀwhite
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amorphous solid. H NMR (400 MHz, DMSOꢀd6) δ 8.70 (d, J=3.13 Hz, 1H), 7.90 (dd, J=0.59, 3.03 Hz,
1H), 7.61 (d, J=7.73 Hz, 1H), 6.74 (dd, J=0.64, 7.68 Hz, 1H), 5.30 (q, J=7.14 Hz, 1H), 4.12 (q, J=7.11 Hz,
2H), 3.93 (s, 3H), 1.61 (d, J=7.14 Hz, 3H), 1.15 (t, J=7.00 Hz, 3H). LRMS (ESI): m/z (M + H) calcd 277.1;
found 277.2.
(±)ꢀEthyl 2-(3-(2-methoxyethoxy)-5-oxo-1,6-naphthyridin-6(5H)-yl)propanoate (12c): A 3ꢀneck roundꢀ
bottom flask was equipped with a internal temperature probe, overhead stirrer, and reflux condenser under
an atmosphere of N2, then charged with 3ꢀ(2ꢀmethoxyethoxy)ꢀ1,6ꢀnaphthyridinꢀ5(6H)ꢀone (10c) (59.5 g,
270 mmol), Cs2CO3 (176 g, 540 mmol) and THF (600 mL). Ethyl 2ꢀbromopropanoate (56.3 mL, 432
mmol) was added to the suspension and the mixture was heated to 60 °C (internal temperature) for 90
minutes. The reaction was cooled to room temperature and filtered through a plug of silica gel eluting with
EtOAc and the filtrate was concentrated under reduced pressure. The crude material was purified by MPLC
eluting with a gradient of 50ꢀ100% EtOAc in DCM to afford (±)ꢀethyl 2ꢀ(3ꢀ(2ꢀmethoxyethoxy)ꢀ5ꢀoxoꢀ1,6ꢀ
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naphthyridinꢀ6(5H)ꢀyl)propanoate (12c) (45.5 g, 53% yield) as yellow oil. H NMR (400 MHz, DMSOꢀd6)
δ 8.70 (d, J=3.03 Hz, 1H), 7.92 (d, J=2.84 Hz, 1H), 7.61 (d, J=7.73 Hz, 1H), 6.73 (d, J=8.02 Hz, 1H), 5.31
(q, J=7.14 Hz, 1H), 4.25ꢀ4.33 (m, 2H), 4.12 (q, J=7.11 Hz, 2H), 3.65ꢀ3.78 (m, 2H), 3.32 (s, 3H), 1.61 (d,
J=7.24 Hz, 3H), 1.15 (t, J=7.09 Hz, 3H). LRMS (ESI): m/z (M + H) calcd 321.1; found 321.0.
(±)ꢀ2-(3-Methoxy-5-oxo-1,6-naphthyridin-6(5H)-yl)propanoic acid hydrochloride (13b): A pressure
vessel was charged with ethyl (±)ꢀ2ꢀ(3ꢀmethoxyꢀ5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)propanoate (12b) (3.30
g, 11.9 mmol) in aqueous 6N HCl solution (39.8 mL, 239 mmol) and stirred overnight at 80 °C. The
reaction was concentrated under reduced pressure to afford (±)-2ꢀ(3ꢀmethoxyꢀ5ꢀoxoꢀ1,6ꢀnaphthyridinꢀ
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