Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models

Article abstract of DOI:10.1021/acs.jmedchem.7b00708

The observed structure-activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

Full text of DOI:10.1021/acs.jmedchem.7b00708

Article
pubs.acs.org/jmc
Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors
and Efficacy in Rodent Hypertension Models
Ken Yamada,*,†,§,# Julian Levell,†,#,∥ Taeyong Yoon,†,#,⊥ Darcy Kohls,†,# David Yowe,† Dean F. Rigel,‡,□
Hidetomo Imase,† Jun Yuan,† Kayo Yasoshima,†,§ Keith DiPetrillo,‡,∇ Lauren Monovich,† Lingfei Xu,‡
Meicheng Zhu,†,○ Mitsunori Kato,†,§ Monish Jain,† Neeraja Idamakanti,†,◆ Paul Taslimi,†,⬢
Toshio Kawanami,†,§ Upendra A. Argikar,† Vidya Kunjathoor,† Xiaoling Xie,† Yukiko I. Yagi,§,¶
Yuki Iwaki,†,△ Zachary Robinson,†,▼ and Hyi-Man Park†,§,#
†Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts 02139-4133, United States
‡Novartis Institutes for BioMedical Research, Novartis Pharmaceuticals Corporation, East Hanover, New Jersey 07936-1080, United
States
§Novartis Institutes for BioMedical Research, Novartis Pharma K.K., Tsukuba, Ibaraki 300-2611, Japan
S
* Supporting Information
ABSTRACT: The observed structure−activity relationship of
three distinct ATP noncompetitive With-No-Lysine (WNK)
kinase inhibitor series, together with a crystal structure of a
previously disclosed allosteric inhibitor bound to WNK1, led to
an overlay hypothesis defining core and side-chain relationships
across the different series. This in turn enabled an efficient
optimization through scaffold morphing, resulting in com-
pounds with a good balance of selectivity, cellular potency, and
pharmacokinetic profile, which were suitable for in vivo proof-
of-concept studies. When dosed orally, the optimized
compound reduced blood pressure in mice overexpressing
human WNK1, and induced diuresis, natriuresis and kaliuresis
in spontaneously hypertensive rats (SHR), confirming that this
mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.
for chronic administration as an antihypertensive agent.9−11
Allosteric inhibition was also potentially beneficial for achieving
INTRODUCTION
■
In pseudohypoaldosteronism-II (PHAII) patients, mutations in
With-No-Lysine (WNK) kinases have been described as
causing hypertension with a paradoxical decrease in aldoster-
one, suggesting a mechanism of blood pressure regulation
potent efficacy in the cellular context, where ATP concen-
tration is very high.8 At the time, compound 1 was the only one
that cocrystallized with WNK1, revealing an allosteric binding
independent of the renin−angiotensin−aldosterone system.1,2
mode (Figure 1b, 5TF9). However, similar to compound 1,
both compounds 2 and 3 showed the best fit for ATP
We have previously shown that WNK476, a small molecule
ATP-competitive inhibitor of WNK kinase catalytic activity,
noncompetitive inhibition upon enzyme kinetic studies
(Figures S1a−c). This suggested the possibility of similar
allosteric inhibition across the three compounds.
induces diuresis and blood pressure reduction in various rodent
models of hypertension.3 The finding was consistent with
In order to rationalize this observation we developed an
previous reports of WNK-mediated phosphorylation of
overlay hypothesis based on docking models that we could test
oxidative-stress response 1 (OSR1) and STE20/SPS1-related
with structure−activity relationship (SAR) studies. Aligning the
proline/alanine-rich kinase (SPAK), that in turn regulate, via
phosphorylation, the functions of renal electrolyte transporters
hydrophobic chlorophenyl of 1 with the benzyl of compound 2
and cyclohexylmethyl of compound 3 positioned the amino-
thiazole of compound 1 congruent with the methoxyphenyl of
compound 2 and the terminal phenyl of the biphenyl from
compound 3. Furthermore, docking models suggested that the
protonated amine of both compounds 2 and 3 can form a
hydrogen bond with Glu268 (Figure 1c,d). The reverse binding
such as the sodium chloride cotransporter (NCC) and the
sodium potassium chloride cotransporter (NKCC1),4−6 as well
as the cellular phenotypes observed with a recently reported
WNK-SPAK disruptor.7
Prior to the discovery of the ATP-competitive WNK476, we
had identified compounds 1, 2, and 3 as allosteric inhibitors of
WNK kinases8 (Figure 1a). This mechanism of inhibition was
thought to be advantageous vs binding in the ATP site, in terms
of achieving the exquisite selectivity required to ensure safety
Received: May 22, 2017
© XXXX American Chemical Society
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Figure 1. Summary of SAR and overlay models of three scaffolds. (a) Structures of compounds 1−3 and their inhibition of WNK1 kinase activity.
Preliminary SAR suggested that the aminothiazole of compound 1, methoxyphenyl of compound 2, and phenyl of compound 3 occupy a tight
pocket, whereas the chlorophenyl of compound 1, phenyl of 2, and cyclohexyl of compound 3 occupy a looser hydrophobic pocket. (b) Crystal
structure of compound 1 (yellow) bound to WNK1 (purple) in an allosteric pocket adjacent to the ATP binding site occupied by AMP-PNP (light
blue). (c) Close-up view of the binding pocket from the same angle as panel b with an overlay model of compound 2 (orange) on top of compound
1 (gray). The methoxyphenyl motif occupies the pocket occupied by the aminothiazole of compound 1. The predicted hydrogen bonding contact of
protonated amine and Glu268 is indicated as green dots. (d) Overlay model of compound 3 (green) on top of compound 1 (gray). The terminal
phenyl of biphenyl motif occupies the aminothiazole pocket of compound 1. (e) Alternative overlay of compound 2 (orange) on top of compound 1
(gray). (f) Alternative overlay of compound 3 (green) on top of compound 1 (gray).
modes for compounds 2 and 3 (Figure 1e,f) would not make
the hydrogen bond contact with Glu268 and, hence, would
seem less likely. Our SAR studies around compounds 1−3 gave
a clear trend to favor the former overlay models (Figure 1c,d).
Thus, a tight SAR around the methoxyphenyl group of
compound 2 was observed, which was consistent with binding
to the small and rigid hydrophobic pocket around the
aminothiazole of compound 1 (Figure 1a). Contrary to the
tight SAR for the aminothiazole or methoxyphenyl, a broader
selection of hydrophobic motifs was tolerated as replacements
for the chlorophenyl, phenyl, or cyclohexyl of the three
compounds, suggesting that they all occupy a more flexible
hydrophobic pocket and disfavor the reverse binding option for
compounds 2 and 3. Although potent, the pharmacokinetic
profiles of compounds 1, 2, and 3 were inadequate for in vivo
efficacy validation of this mode of WNK inhibition. Therefore,
we exchanged substituents and core motifs across the three
scaffolds in parallel, in order to accelerate the identification of
an in vivo tool compound.
boost in potency, most likely arising from the increased
planarity. Compound 6, a close analogue of compound 5, with
a fluoro-substituent in the 5-position of the indole was
successfully cocrystallized with WNK1 (Figure 3a, PDB
5WDY). This structure confirmed the allosteric binding mode
for the scaffold as well as the proposed overlay orientation of
compound 2 with compound 1. It also showed the tight pocket
around the methoxyphenyl moiety as well as the proposed
hydrogen bond of the protonated amine linker with Glu268.
Although the poor lipophilic efficiency12 (LipE) of compound
3 was much improved in compound 6 (−2.9 vs −0.33), we
envisioned that further improvement in lipophilic efficiency
through truncation of the cyclohexyl moiety would be required
for a viable path forward to an in vivo tool compound. To this
end, installation of a methyl group adjacent to the indole
linkage in compound 5 introduced torsional strain to mimic the
binding conformation, and this change enabled retention of
potency when the cyclohexyl moiety was truncated to an
isobutyl group. Compound 7 represents one of the best
candidates from the series for in vivo proof of concept, although
it still suffers from low LipE (0.12).
RESULTS AND DISCUSSION
■
Many analogues of compound 2 were explored in cocrystal
studies, but it was the modification of the piperazine to the (R)-
aminopyrrolidine (Figure 2b, compound 8), which allowed the
generation of a cocrystal structure within this scaffold class,
confirming the allosteric binding mode and proposed overlay
A highly efficient optimization of compound 3 was carried out
by initially engrafting the methoxy group of compound 2 to
give a dramatic 60-fold improvement in potency (Figure 2a,
compound 4). Introduction of a pyridyl nitrogen to simulate
the quinoline nitrogen of compound 2 gave an additional 7-fold
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Figure 2. Morphing based optimization of the two series. (a) Introduction of methoxy to the phenyl group of compound 3 gave >100-fold
improvement in potency in compound 4, and introduction of a pyridyl nitrogen to allow for planarity gave further 4-fold improvement in potency to
give compound 5. The crystal structure with WNK1 was obtained with a fluorinated analogue compound 6. Replacement of cyclohexyl with a
simpler isobutyl group resulted in slightly less hydrophobic compound 7, which was equipotent to compound 5, likely owing to the introduced
torsional strain. (b) Replacement of piperazine and introduction of a para-chloro group gave compound 8, which was the only structure from this
scaffold that cocrystallized with WNK1. Removal of the extra phenyl ring of compound 2 and installment of a para-chloro group gave equipotent
compound 9, whose methoxyphenyl motif was replaced with aminothiazole to give compound 10 with nearly 10-fold improvement in potency.
Introduction of a chlorine substituent on the central pyridine ring gave compound 11 with a further 50-fold improvement in potency, most likely due
to the introduction of torsional strain.
Figure 3. Allosteric binding modes of the optimized WNK inhibitors. (a) Ternary complex of compound 6 and AMP-PNP bound to WNK1,
confirming the predicted orientation of binding and the hydrogen bond contact of the proton of the secondary amine with Glu268. (b) Ternary
complex of compound 8 and AMP-PNP bound to WNK1, confirming the predicted orientation of binding and the hydrogen bond contact of
protonated piperazine nitrogen with Glu268. (c) Docking model of compound 11 (yellow) overlaid on top of compound 8 (gray) bound to WNK1
and AMP-PNP. Confidence in the binding mode is enhanced by the crystal structures of three closely related scaffolds.
hypothesis for compound 3 vs compounds 1 and 2 (Figure 3b,
PDB 5WE8). This structure also highlighted several oppor-
tunities for compound optimization. Replacement of quinoline
with pyridine to simulate the biaryl of compound 3 was also
supported by the lack of obvious binding interactions in the X-
ray structure with this second ring system (Figure 2b).
Simultaneous grafting of the para-chloro group from
compound 1 onto the benzyl group gave compound 9 with
minimal loss of potency but overall improved LipE (1.23 vs
0.756). Replacement of methoxyphenyl of compound 9 with
aminothiazole from compound 1 gave compound 10 with a 10-
fold boost in potency, most likely due to forming a hydrogen
bonding interaction with Val281. This modification also had a
significant effect on improving the LipE (3.02) due to the lower
lipophilicity of the aminothiazole vs the methoxyphenyl. A
chlorine atom was introduced adjacent to the amide linkage in
compound 10 in order to create torsional strain and mimic the
expected binding conformation where the plane of the amide
and pyridine are almost perpendicular. This modification
further improved the potency by 25-fold and also LipE (3.7),
giving the optimized compound 11.
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Figure 4. Profiles of optimized compounds. (a) In vitro profiles of optimized compounds 7, 11, and 12 and pharmacokinetics in Sprague−Dawley
rats (0.5 mg kg−1 i.v., 1.5 mg kg−1 p.o.) for compounds 11 and 12. (b) Michaelis−Menten curves showing ATP noncompetitive binding for
compound 4. (c) Kinase selectivity data for compound 11 based on AMBIT KINOMEscan. Green dots indicate less than 35% inhibition, and red
dots indicate greater than 35% inhibition at 10 μM screening concentration. Blue dot indicates on target activity. *Images generated using TREEspot
Software Tool and reprinted with permission from KINOMEscan, a division of DiscoveRx Corporation; Copyright Discoverx Corporation 2010.
Figure 5. Pharmacokinetics and pharmacodynamics of compound 12. (a) When dosed at 30 mg kg−1 p.o. in FVB mice overexpressing human
WNK1, compound 12-treated mice showed significant reductions in systolic blood pressure (SBP) vs untreated mice (time-weighted average, vs 24
h before treatment). In a separate cohort of mice dosed at 30 mg kg−1 p.o., the plasma concentration reached nearly 10 μM at early time points and
declined to approximately 1 μM after 7 h. Data in panel a show mean SEM, n = 4 per group for blood pressure studies, n = 3 for pharmacokinetic
study; **p < 0.01, ***p < 0.001 compared to vehicle (two-way analysis of variance (ANOVA) with Bonferroni post-test). (b,c) Compound 12 dose-
dependently increased urinary volume (b) and urinary sodium and potassium excretion (c) in SHRs over a 7 h period. Data in panels b and c show
mean SEM, n = 4 per group; *p < 0.05 compared to vehicle (one-way analysis of variance (ANOVA) with Bonferroni post-test).
Compounds 7 and 11 showed IC50 < 2 μM in the cellular
OSR1 phosphorylation assay with reasonable aqueous
solubility, albeit with still rather high microsomal clearance
(Figure 4a). Based on the overall lipophilic efficiency of
compound 11 over 7, we decided to further profile compound
11 for suitability for in vivo proof of concept study for allosteric
WNK1 inhibition. Consistent with the allosteric mode of
inhibition, compound 11 showed ATP noncompetitive
inhibition (Figure 4b). When tested against a panel of 440
human kinases at 10 μM concentration, 2500-fold above
enzyme IC50 value, compound 11 showed excellent selectivity
with only a few significant off-target kinase inhibitions, most
notably Burton’s tyrosine kinase (BTK) and feline encephalitis
virus-related (FER) kinase, neither of which are implicated for
blood pressure regulation (Figure 4c, Table S1). This excellent
selectivity profile is consistent with the predicted allosteric
binding mode outside the highly conserved ATP-pocket
(Figure 3c).
also observed that some compounds demonstrated high
selectivity against WNK2 and WNK4 but never against
WNK3. Our homology model supported this general
observation that achieving selectivity within WNK family
members is challenging, particularly for WNK3 where the
residues surrounding the allosteric pocket were essentially
identical with WNK1 (Figure S2b). For WNK2 and WNK4,
there were some notable differences in the allosteric pocket,
such as Phe in place of Ser286 and Val in place of Ala269, for
WNK2 and WNK4, respectively (Figure S2a,c). However,
beyond the fact that the selectivity seems to be affected by
changes in the linker, which lies in proximity to the noted
amino acid differences that are situated between the glycine rich
loop and helix C, it remains to be determined how exactly
compound 11 achieves the apparent selectivity over WNK4 or
WNK2.
Based on the promising in vitro profiles, compound 11 was
used for rat PK and showed moderate clearance but relatively
low oral bioavailability and absolute exposure (Figure 4a, Table
S1). Metasite13,14 and StarDrop P450 analysis15 both suggested
N-demethylation as the major metabolic liability for compound
11, and we had also noticed chemical stability issues for
solutions of N-methylaminothiazole derivatives. We considered
It was a fortuitous finding that compound 11 showed nearly
1000-fold selectivity for WNK1 vs WNK4 and 57-fold
selectivity for WNK1 vs WNK2 (Figure S3a−d), providing
an alternative tool compound to previously disclosed pan-WNK
inhibitor WNK463.3 During the optimization process, it was
D
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a variety of replacements such as cyclopropyl for this methyl in
analogous series to block metabolism, but all suffered from
significant loss in potency. Deuteration has also been shown to
improve pharmacokinetic profiles, due to the kinetic isotope
effect,16,17 so we explored the perdeuteromethyl analogue of
compound 11 to assess whether this would provide sufficient
improvement to enable in vivo PD/efficacy studies (Figure 4a).
Compound 12 did show an improved rat PK profile, including
lower clearance, improvement in absolute oral exposure, and a
2-fold improvement in oral bioavailability (Figure 4a, Table
S1). Although we did not carry out full selectivity profiling with
compound 12, we had a high confidence that this would not
change significantly vs compound 11, and therefore, when
dosed in vivo, the molecule would exert its effect via specific
inhibition of WNK kinases and not be affected by off-target
kinase inhibition.
In FVB mice overexpressing human WNK1 with implanted
radiotelemetry devices,3 single oral doses at 10, 30, and 100 mg
kg−1 of compound 12 resulted in dose-dependent reductions in
systolic blood pressure both in terms of peak and time-
weighted average (TWA) vs baseline (Figure 5a, Table S2).
The observed blood pressure reduction at 30 mg kg−1
coincided with a plasma exposure at or above ∼2 μM (∼3-
fold above cellular IC50) in a separate cohort of animals (Figure
5a). In another preliminary study, a single oral dose of 30 mg
kg−1 of compound 7 resulted in a similar blood pressure
reduction (Table S2), providing further evidence that allosteric
inhibition of WNK kinase activities result in robust reduction in
blood pressure.
To further understand the effect of allosteric WNK inhibitors
on urinary output/electrolyte excretion and hemodynamic
parameters, single ascending oral doses of compound 12 were
given on successive days (vehicle, 10, 30, then 100 mg kg−1) to
spontaneously hypertensive rats (SHR) chronically instru-
mented with ascending aortic flow probes and aortic catheters/
radiotransmitters for continuous recording of cardiac output
and blood pressure.18 Consistent with our previous findings on
WNK1−4 kinase inhibition,3 compound 12 induced dose-
dependent diuresis, natriuresis, and kaliuresis, from 10 to 100
mg kg−1 (Figure 5b,c). While the observed diuresis and
natriuresis were similar to that observed from a single oral dose
of amiloride at 10 mg kg−1, kaliuresis was observed only with
the WNK inhibitor (Figure S5a,b), consistent with previously
reported WNK physiology.1,3 In terms of hemodynamic
parameters, compound 12 showed trends toward reduction of
blood pressure, stroke volume, and total peripheral resistance,
while increasing heart rate (Figure S6a,b).
WNK4. Topics of further investigation include the structural
basis as well as functional and pharmacological consequences of
such WNK family selectivity.
EXPERIMENTAL SECTION
■
The crystal structure of compound 1 bound to human WNK1 was
disclosed previously (PDB 5TF9, purple ribbons). The crystal
structures of compound 6 (PDB 5WDY, orange ribbons) and
compound 8 (PDB 5WE8, green ribbons) were resolved independ-
ently utilizing the same human WNK1 construct and conditions as
disclosed previously. Docking models in Figures 1c−f and 3c were
created using the X-ray structure of compounds 1 and 8, respectively,
using Glide-SP v5.0 and MacroModel v9.6 (Schrodinger, LLC, New
̈
York). In vitro determination of WNK kinase catalytic activity utilized
recombinant WNK kinase domains purified from E. coli. In vitro WNK
kinase activity was assessed by a mobility shift assay of peptide
substrate. Enzyme kinetics analyses were performed with GraphPad
Prism for Windows version 7. WNK-mediated phosphorylation of
exogenous OSR1 in HEK293 cells was shown by AlphaLISA. In vivo
cardiovascular studies utilized telemetry to monitor blood pressure and
heart rate in conscious WNK1 transgenic mice and aortic flow probes
and aortic catheters/radiotransmitters to continuously record cardiac
output and blood pressure in conscious SHRs. Metabolic cages were
used for urine collection, and venous blood samples were collected for
plasma electrolyte and pharmacokinetic analysis. LipE values were
calculated with the formulas proposed by Ryckmans et al:12 LipE =
pIC50 − cLogP. A detailed summary of experimental procedures for
the chemical synthesis, assays, and X-ray can be found in the
Supporting Information.
Chemical Synthesis. Unless otherwise specified, all solvents and
reagents were obtained from commercial sources and used without
further purification. All reactions were performed under nitrogen
atmosphere unless otherwise noted. Normal-phase flash chromatog-
1
raphy was performed using Merck silica gel 60 (230−400 mesh). H
and 13C NMR spectra were recorded on a Bruker DRX or a Bruker
1
AV400 (400 MHz for H and 100 MHz for 13C). Chemical shifts are
reported in parts per million (ppm) downfield from tetramethylsilane
(0.00 ppm) or residual peaks from the corresponding solvent as an
internal standard and coupling constants (J) in Hz. Multiplicity
abbreviation are as follows: s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, app = apparent, br = broad. LC−MS analyses
were performed on an HPLC system with a C18 column coupled to a
single quad mass spectrometer with electrospray ionization (ESI).
High-resolution mass spectra were obtained with Acquity Xevo G2
QTof system with UPLC (Acquity UPLC BEH C18 column, 2−98%
acetonitrile in water with 0.1% formic acid) coupled to time-of-flight
detection after electrospray ionization.
Chemical Stability. Methylaminothiazoles 10 and 11 showed
decomposition in 10 mM DMSO stock solution if kept at room
temperature over a prolonged (few months) period of time. Aliquots
of the DMSO stock solutions were thus kept frozen for assays and
periodically checked for purity by either WNK1 inhibition assay and/
or LC−MS.
CONCLUSION
■
Chemical Purity. All compounds were checked for purity before
assay by LC−MS and confirmed to have >95% purity.
A dynamic morphing of three independent allosteric WNK1
inhibitors based on an overlay hypothesis and a docking model
enabled an efficient optimization that led to the discovery of
potent and selective allosteric inhibitors with improved
physicochemical properties. Compound 12 showed efficacy in
rodent models of hypertension and volume overload.
Demonstration of both hemodynamic as well as renal effects
of WNK1 inhibition through a different mode of binding than
our previous report (allosteric vs ATP-site)3 provides another
line of evidence that the catalytic activity of WNK kinases play a
fundamental role in cardiovascular homeostasis. One of the
advantages of an allosteric binding site over the ATP-site for
WNK inhibition is the potential to achieve selectivity among
WNK family members such as for WNK1 vs WNK2 and
Synthesis of Compounds 3−7. 1-([1,1′-Biphenyl]-3-yl)-1H-
indole. A mixture of 1H-indole (1 g, 8.54 mmol), K3PO4 (3.81 g,
17.93 mmol), and copper(I) iodide (0.081 g, 0.427 mmol) in toluene
(10 mL) was purged with nitrogen for a few minutes, then 3-bromo-
1,1′-biphenyl (1.71 mL, 10.3 mmol) and cyclohexane-1,2-diamine
(0.209 mL, 1.71 mmol) were added. The reaction mixture was refluxed
for 2 days. After it was cooled, hexane was added to the mixture. The
resultant suspension was filtered through a short silica gel pad, and the
filtrate was concentrated in vacuo. The residue was purified by silica gel
flash chromatography to give 1-([1,1′-biphenyl]-3-yl)-1H-indole (420
mg, 18%). ESI-MS m/z: 270.3 [M + H]+.
1-([1,1′-Biphenyl]-3-yl)-1H-indole-3-carboxaldehyde. To a solu-
tion of 1-([1,1′-biphenyl]-3-yl)-1H-indole (420 mg, 1.56 mmol) in
DMF (10 mL) was added phosphorus oxychloride (0.174 mL, 1.87
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1
mmol) dropwise at 0 °C. The resultant solution was allowed to stir at
room temperature for 1 h, then poured onto crushed ice. The
suspension was adjusted to pH 6 with 2 N aqueous NaOH solution
with cooling. The crystalline product was collected by filtration and
washed with water to give 1-([1,1′-biphenyl]-3-yl)-1H-indole-3-
carbaldehyde (360 mg, 70%, 90% pure). ESI-MS m/z: 298.2 [M +
H]+.
(395 mg, 881 mmol, quant). H NMR (400 MHz, chloroform-d) δ
ppm 1.46 (s, 9 H) 3.14−3.40 (m, 4 H) 3.56−3.70 (m, 2 H) 3.83−3.91
(m, 1 H), 3.93 (s, 3 H), 3.97−4.05 (m, 1 H), 7.44 (t, J = 7.96 Hz, 1
H), 7.54−7.62 (m, 1 H), 7.67−7.72 (m, 1 H), 7.74−7.83 (m, 4 H),
8.17−8.27 (m, 1 H). ESI-MS m/z: 448 [M + H]+.
(2-(3-Methoxyphenyl)quinolin-4-yl)(piperazin-1-yl)methanone.
To a 50 mL flask containing tert-butyl 4-(2-(3-methoxyphenyl)-
quinoline-4-carbonyl)piperazine-1-carboxylate (365 mg, 0.815 mmol)
was added methylene chloride (2 mL) and trifluoroacetic acid (2 mL).
The mixture was stirred at room temperature for 18 h, concentrated
under reduced pressure, suspended in 1 N aqueous sodium hydroxide,
and extracted twice with methylene chloride. The combined organic
fractions were dried over sodium sulfate, filtered, and concentrated to
give (2-(3-methoxyphenyl)quinolin-4-yl)(piperazin-1-yl)methanone as
Compound 3: 1-(1-([1,1′-Biphenyl]-3-yl)-1H-indol-3-yl)-N-
(cyclohexylmethyl)methanamine. To a mixture of 1-([1,1′-biphen-
yl]-3-yl)-1H-indole-3-carbaldehyde (130 mg, 0.393 mmol) in MeOH
(2 mL) was added cyclohexylmethanamine (0.051 mL, 0.393 mmol).
The resultant suspension was warmed to 50 °C with stirring, and THF
(2 mL) was then added to give a solution. After the resultant solution
was stirred for 30 min, sodium cyanoborohydride (41.2 mg, 0.590
mmol) was added. The reaction mixture was stirred at room
temperature until the conversion was completed. One milliliter of 1
N HCl solution was added to the reaction mixture. The resultant
mixture was stirred for 30 min, then adjusted to pH 8 with 1 N
aqueous NaOH solution and diluted with water. The cloudy solution
was extracted with dicholoromethane once. The extract was dried over
Na2SO4 and concentrated. The residue was purified by silica gel flash
chromatography, eluting with hexane/ethyl acetate (1:1) to give 1-(1-
([1,1′-biphenyl]-3-yl)-1H-indol-3-yl)-N-(cyclohexylmethyl)-
1
a colorless foam (281 mg, 0.810 mmol, 99% yield). H NMR (400
MHz, chloroform-d) δ ppm 2.69−2.78 (m, 2 H), 3.05 (t, J = 5.04 Hz,
2 H), 3.17−3.24 (m, 2 H), 3.81−3.91 (m, 1 H), 3.93 (s, 3 H), 3.96−
4.07 (m, 1 H), 7.01−7.07 (m, 1 H), 7.44 (t, J = 7.81 Hz, 1 H), 7.58
(ddd, J = 8.31, 7.05, 1.26 Hz, 1 H), 7.70 (d, J = 8.56 Hz, 1 H), 7.74−
7.80 (m, 3 H), 7.83 (d, J = 7.55 Hz, 1 H), 8.21 (d, J = 8.56 Hz, 1 H).
ESI-MS m/z: 348 [M + H]+.
Compound 2: (4-Benzylpiperazin-1-yl)(2-(3-methoxyphenyl)-
quinolin-4-yl)methanone. To a solution of (2-(3-methoxyphenyl)-
quinolin-4-yl)(piperazin-1-yl)methanone (0.1 mmol, 37.4 mg) and
N,N-diisopropylethylamine (0.15 mmol; 19.4 mg) in dichloromethane
(1 mL) was added benzyl bromide (0.105 mmol, 18.0 mg). The
reaction mixture was stirred for 5 h and then quenched with H2O. The
organic layer was extracted with dichloromethane, passed through a
phase separator, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (eluent:
dichloromethane/ethanol) to give (4-benzylpiperazin-1-yl)(2-(3-
1
methanamine (91 mg, 59%). H NMR (300 MHz, DMSO-d6) δ ppm
7.80−7.33 (m, 12H), 7.25−7.05 (m, 2H), 3.88 (s, 2H), 2.45 (d, 2H),
1.83−1.52 (m, 5H), 1.42 (m, 1 H), 1.28−0.78 (m, 5H). ESI-MS m/z:
282.0 [M − NC7H15]+.
Compound 4: 1-Cyclohexyl-N-((1-(3′-methoxybiphenyl-3-yl)-1H-
indol-3-yl)methyl)methanamine (13 mg, 12%). 1H NMR (400 MHz,
chloroform-d) δ 7.89−7.40 (m, 8H), 7.39−7.25 (m, 4H), 7.13−6.93
(m, 1H), 4.14 (s, 2H), 3.98 (s, 3H), 2.71 (d, J = 6.7 Hz, 2H), 2.01−
1.54 (m, 6H), 1.47−0.93 (m, 5H). ESI-MS m/z: 312.2 [M − NC7H15
]+.
1
methoxyphenyl)quinolin-4-yl)methanone (34.5 mg, 79%). H NMR
(400 MHz, DMSO-d6) δ ppm 2.2 (br s, 1H), 2.39 (br s, 1H), ∼2.50
(br s, 1H, seen as shoulder on DMSO peak), 2.60 (br s, 1H), 3.13 (br
s, 1H), 3.20 (br s, 1H), 3.51 (br s, 2H), 3.69 (br s, 1H), 3.89 (s, 3H),
3.94 (br s, 1H), 7.10 (dd, J = 8.1, 2.2 Hz), 7.25 (m, 1H), 7.31 (m, 3H),
7.48 (t, J = 7.9 Hz), 7.67 (t, J = 7.7 Hz), 7.79−7.90 (m, 4H), 8.14−
8.15 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ ppm 41.1, 46.54,
52.74, 55.2, 61.64, 112.28, 115.48, 115.67, 119.62, 122.84, 124.51,
126.94, 127.38, 128.12, 128.75, 129.64, 129.88, 130.4, 137.69, 139.45,
143.35, 147.44, 155.52, 159.68, 165.57. HRMS m/z: 438.2168 (M +
H, C28H27N3O2, requires 438.2182).
Compound 5: 1-Cyclohexyl-N-((1-(2-(3-methoxyphenyl)pyridin-
1
4-yl)-1H-indol-3-yl)methyl)methanamine (19 mg, 13%). H NMR
(400 MHz, methanol-d4) δ 8.79−8.69 (m, 1H), 8.04 (s, 1H), 7.96 (d, J
= 1.8 Hz, 1H), 7.86−7.74 (m, 2H), 7.62−7.49 (m, 3H), 7.48−7.27 (m,
3H), 7.02 (ddd, J = 8.2, 2.5, 0.9 Hz, 1H), 4.44 (s, 2H), 3.89 (s, 3H),
2.88 (d, J = 6.7 Hz, 2H), 1.88−1.59 (m, 6H), 1.36−0.92 (m, 5H). ESI-
MS m/z: 426.4 [M + H]+.
Compound 6: 1-Cyclohexyl-N-((6-fluoro-1-(2-(3-methoxyphenyl)-
pyridin-4-yl)-1H-indol-3-yl)methyl)methanamine (40 mg, 62.5%).
1H NMR (400 MHz, chloroform-d) δ 8.68 (d, J = 6.06 Hz, 1 H),
Compound 8: (R)-N-(1-(4-Chlorobenzyl)pyrrolidin-3-yl)-2-(3-me-
thoxyphenyl)-N-methylquinoline-4-carboxamide (28 mg, 58%). H
1
7.85−7.95 (m, 2 H), 7.51−7.58 (m, 2 H), 7.43−7.47 (m, 3 H), 7.24−
7.30 (m, 1 H), 7.09−7.15 (m, 1 H), 6.97 (dt, 1 H), 4.16 (s, 2 H), 3.93
(s, 3 H), 2.93 (d, 2 H), 1.63−1.85 (m, 6H), 1.12−1.32 (m, 3 H),
0.92−1.07 (m, 2 H). ESI-MS m/z: 444.4 [M + H]+.
NMR (400 MHz, DMSO-d6, 413 K) δ ppm 1.98 (m, 1H), 2.34 (br s,
1H), 2.81 (m, 2H), 3.00 (br s, 3H), 3.55 (m, 2H), 3.90 (s, 3H), 7.07
(dd, J = 2.6, 0.9 Hz, 0.5H), 7.09 (m, 0.5H), 7.28−7.31 (m, 4H), 7.44−
7.46 (m, 1H), 7.59 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.71 (dd, J = 8.4, 0.8
Hz, 1H), 7.78−7.81 (m, 3H), 7.89 (s, 1H), 8.12 (dt, J = 8.45, 0.92
Hz). 13CNMR (100 MHz, DMSO-d6) δ ppm 27.44, 27.57, 28.53,
28.93, 31.49, 51.48, 52.76, 52.92, 53.14, 55.28, 55.29, 55.31, 56.42,
56.65, 57.19, 58.12, 58.17, 58.41, 112.36, 112.45, 115.34, 115.44,
115.48, 115.7, 115.77, 119.68, 119.7, 119.75, 122.64, 122.98, 123,
124.36, 124.61, 124.68, 127.3, 127.43, 127.55, 128.18, 128.23, 129.7,
129.77, 129.95, 129.98, 130.02, 130.06, 130.11, 130.42, 130.52, 131.32,
131.35, 137.92, 137.97, 138.14, 139.58, 139.6, 139.61, 144.23, 144.32,
144.39, 147.51, 147.52, 147.57, 155.71, 159.78, 159.79, 166.89, 166.94,
167.26. HRMS m/z: 486.1932 (M + H, C29H28ClN3O2, requires
486.1948).
Compound 9: (4-(4-Cchlorobenzyl)piperazin-1-yl)(2-(3-
methoxyphenyl)pyridin-4-yl)methanone (43 mg, 51%). ESI-MS
m/z: 422 [M + H]+. 1H NMR (400 MHz, chloroform-d) δ ppm
2.35−2.42 (m, 2 H), 2.50−2.57 (m, 2 H), 3.37−3.44 (m, 2 H), 3.51
(s, 2 H), 3.78−3.86 (m, 2 H), 3.90 (s, 3 H), 6.97−7.02 (m, 1 H), 7.20
(dd, J = 4.93, 1.39 Hz, 1 H), 7.23−7.26 (m, 2 H), 7.28−7.32 (m, 2 H),
7.39 (t, J = 7.96 Hz, 1 H), 7.52−7.56 (m, 1 H), 7.57−7.59 (m, 1 H),
7.67−7.72 (m, 1 H), 8.74 (d, J = 4.80 Hz, 1 H). ESI-MS m/z: 428 [M
+ H]+.
Compound 7: N-((1-(2-(3-Methoxyphenyl)-5-methylpyridin-4-yl)-
1
1H-indol-3-yl)methyl)-2-methylpropan-1-amine (610 mg, 80%). H
NMR (400 MHz, DMSO-d6) δ 0.89 (d, J = 6.57 Hz, 6H), 1.67−1.78
(m, 1H), 2.16 (s, 3H), 2.43 (d, J = 6.57 Hz, 2H), 3.83 (s, 3H), 3.92 (s,
2H), 6.98−7.03 (m, 1H), 7.11−7.22 (m, 3H), 7.39 (t, J = 7.58 Hz,
1H), 7.52 (s, 1H), 7.69 (s, 1H), 7.70 (d, J = 10.11 Hz, 1H), 7.77 (d, J
= 7.58 Hz, 1H), 7.95 (s, 1H), 8.74 (s, 1H). HRMS m/z: 400.2387 (M
+ H, C26H29N3O, requires 400.2383).
Synthesis of Compounds 2, 8, and 9. tert-Butyl 4-(2-(3-
Methoxyphenyl)quinoline-4-carbonyl)piperazine-1-carboxylate. A
50 mL flask was charged with 2-(3-methoxyphenyl)quinoline-4-
carboxylic acid (200 mg, 0.717 mmol), N-boc-piperazine (160 mg,
0.860 mmol), 1-hydroxy-7-azabenzotriazole (49 mg, 0.359 mmol),
N,N-dimethylformamide (2.1 mL), 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride (165 mg, 0.860 mmol), and N,N-
diisopropylethylamine (0.150 mL, 0.860 mmol). The mixture was
stirred at room temperature for 18 h, then diluted with water and
extracted with diethyl ether. The combined organic fractions were
washed with dilute aqueous hydrochloric acid followed by saturated
aqueous sodium bicarbonate and brine, dried over magnesium sulfate,
concentrated, and purified by silica gel chromatography (ethyl
acetate−hexanes) to give tert-butyl 4-(2-(3-methoxyphenyl)-
quinoline-4-carbonyl)piperazine-1-carboxylate as a colorless foam
Synthesis of Compound 10. Methyl 2-(2-Bromoacetyl)-
isonicotinate. To a solution of methyl 2-acetylisonicotinate (1 g,
F
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Journal of Medicinal Chemistry
Article
5.58 mmol) in acetic acid was added bromine (575 μL, 11.2 mmol)
and hydrogen bromide in acetic acid (33 wt %, 144 μL) at room
temperature. The mixture was stirred at room temperature overnight.
The reaction mixture was concentrated in vacuo. The residue was
basified with 1 N aqueous NaOH, and extracted with dichloro-
methane. The organic layer was concentrated in vacuo. The residue
was purified by silica gel column chromatography (heptane/ethyl
acetate) to give methyl 2-(2-bromoacetyl)isonicotinate (350 mg,
24%). ESI-MS m/z: 260.1 [M + H]+.
Methyl 2-(2-(Methylamino)thiazol-4-yl)isonicotinate. To a sol-
ution of methyl 2-(2-bromoacetyl)isonicotinate (329 mg, 1.275 mmol)
in methanol (10 mL) was added 1-methylthiourea (172 mg, 1.91
mmol) at room temperature. The mixture was stirred at 60 °C for 2 h.
The reaction mixture was quenched with 15 mL of 1 N aqueous
NaHCO3, extracted with dichloromethane, and passed through phase
separator. The organic solvent was removed in vacuo. The residue was
purified by silica gel column chromatography (heptane/ethyl acetate)
to give methyl 2-(2-(methylamino)thiazol-4-yl)isonicotinate (260 mg,
82%). ESI-MS m/z: 250.4 [M + H]+.
mmol) in one portion. This reaction mixture was stirred for 2 h at rt.
N-Methyl-thiourea (280 mg, 3.11 mmol) was added into the reaction
mixture, which was stirred for 2 h. Insoluble material was removed by
filtration, rinsing with dichloromethane. The organic extract was
passed through an SCX column, which was rinsed with methanol and
eluted with 20:2:1 EtOAc/MeOH/Et3N. The crude material was
purified by silica gel chromatography with ethyl acetate/methanol/
triethylamine as coeluents to give (5-chloro-2-(2-(methylamino)-
thiazol-4-yl)pyridin-4-yl)(4-(4-chlorobenzyl)piperazin-1-yl)methanone
1
(700 mg, 64%). HNMR (400 MHz, DMSO-d6) δ ppm 2.92 (d, J =
11.2 Hz, 3H), 3.04 (brs, 2H), 3.18−3.20 (m, 1H), 3.28−3.30 (m, 1H),
3.35−3.43 (m, 2H), 3.49−3.67 (m, 2H), 4.33−4.41 (m, 2H), 4.56−
4.61 (m, 1H), 7.41 (m, 1H), 7.54 (brs, 2H), 7.62−7.66 (m, 2H) 7.80
(brs, 1H), 7.88 (s, 1H), 8.69 (s, 1H), 11.56 (brs, 0.5H), 11.75 (brs,
0.5H). 13CNMR (100 MHz, DMSO-d6) δ ppm 169.50, 163.58,
163.40, 151.27, 151.10, 148.84, 148.01, 142.50, 142.31, 134.35, 133.31,
128.70, 128.35, 128.13, 124.63, 124.48, 118.45, 106.52, 57.71, 57.41,
50.56, 49.99, 49.86, 49.46, 42.88, 42.26, 39.91, 37.72, 30.91. HRMS
(TOF) calcd. for C21H22Cl2N5OS [M + H] +462.0922, found
462.0933.
2-(2-(Methylamino)thiazol-4-yl)isonicotinic Acid. To a solution of
methyl 2-(2-(methylamino)thiazol-4-yl)isonicotinate (260 mg, 1.043
mmol) in methanol (10 mL) was added 1 N sodium hydroxide
solution (8.34 mL) at room temperature. The mixture was stirred at
60 °C for 2 h. The organic solvent was removed in vacuo. The residue
was acidified by the addition of 1 N HCl. The precipitate formed was
collected and washed with water to give 2-(2-(methylamino)thiazol-4-
yl)isonicotinic acid (194 mg, 79%). ESI-MS m/z: 236.3 [M + H]+.
Compound 10: (4-(4-Chlorobenzyl)piperazin-1-yl)(2-(2-
(methylamino)thiazol-4-yl)pyridin-4-yl)methanone. A mixture of 2-
(2-(methylamino)thiazol-4-yl)isonicotinic acid (2.1 mmol, 500 mg), 1-
(4-chlorobenzyl)piperazine (3.2 mmol, 672 mg), (benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (3.19
mmol, 1.4 g) and triethylamine (8.5 mmol, 1.18 mL) in N-
methylpyrrolidinone (15 mL) was stirred overnight at room
temperature. The reaction mixture was quenched with H2O and
extracted three times with ethyl acetate. The combined organic layers
were washed with brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. Purification by reverse phase HPLC gave (4-
(4-chlorobenzyl)piperazin-1-yl)(2-(2-(methylamino)thiazol-4-yl)-
pyridin-4-yl)methanone (120 mg, 13%). 1HNMR (400 MHz, DMSO-
d6) δ ppm 2.37−2.53 (m, 4H), 2.89 (d, J = 3.2 Hz, 3H), 3.35 (brs,
2H), 3.47−3.64 (m, 4H), 7.25 (d, J = 3.2 Hz, 1H), 7.36−7.40 (m,
5H), 7.67 (q, J = 3.2 Hz, 1H), 7.81 (brs, 1H), 8.61 (d, J = 3.2 Hz, 1H).
13CNMR (100 MHz, DMSO-d6); δ ppm 169.42, 166.79, 152.86,
Synthesis of Compound 12. tert-Butyl (4-(5-Chloro-6-(4-(4-
chlorobenzyl)piperazine-1-carbonyl)pyridin-2-yl)thiazol-2-yl)-
carbamate. To a solution of (4-(4-chlorobenzyl)piperazin-1-yl)(2,5-
dichloropyridin-4-yl)methanone (550 mg, 1.43 mmol) and tributyl(1-
ethoxyvinyl)tin (0.5 mL, 1.487 mmol) in 1,4-dioxane (20 mL) was
added Pd(PPh3)4 (70 mg, 0.061 mmol), and the mixture was stirred at
100 °C for 7 h. After letting it cool to rt, the reaction mixture was
added into 2 M aqueous potassium fluoride solution (5 mL). To this
reaction mixture was added N-bromosuccucinimide (300 mg, 1.69
mmol) in one portion. This reaction mixture was stirred for 30 min at
rt. Insoluble material was removed via filtration, rinsing with methanol
(3 × 20 mL). The resulting filtrate was cooled to 0 °C into which a
solution of N-Boc-thiourea (400 mg, 2.27 mmol) was added. This
reaction mixture was stirred for 2 h, letting warm it to rt. After removal
of solvent, the resulting crude material was dissolved in dichloro-
methane and washed with 1 N aqueous NaOH solution and brine. The
organic layer was dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (heptane/ethyl acetate) to give tert-butyl (4-(5-
chloro-6-(4-(4-chlorobenzyl)piperazine-1-carbonyl)pyridin-2-yl)-
thiazol-2-yl)carbamate (550 mg, 70%). 1H NMR (400 MHz, CDCl3) δ
ppm 1.58 (s, 9H), 2.40−2.46 (m, 2H), 2.56 (br s, 2H), 3.26−3.31 (m,
2H), 3.52 (s, 2H), 3.82−3.88 (m, 2H), 7.26−7.32 (m, 4H), 7.66 (s,
1H), 7.85 (s, 1H), 7.99 (br s, 1H), 8.59 (s, 1H). ESI-MS m/z: 548.0
[M + H]+.
149.64, 149.56, 144.08, 138.24, 131.54, 130.67, 128.16, 119.28, 117.45,
105.55, 60.50, 51.74, 46.61, 41.16, 30.81. ESI-MS m/z: 428.3 [M +
H]+.
Compound 12: (5-Chloro-2-(2-((methyl-d3)amino)thiazol-4-yl)-
pyridin-4-yl)(4-(4-chlorobenzyl)piperazin-1-yl)methanone. To a sus-
pension of tert-butyl (4-(5-chloro-6-(4-(4-chlorobenzyl)piperazine-1-
carbonyl)pyridin-2-yl)thiazol-2-yl)carbamate (530 mg, 0.966 mmol)
and potassium carbonate (340 mg, 2.46 mmol) in DMF (15 mL) was
added CD3I (161 mg, 1.11 mmol) dropwise at 0 °C, and the mixture
was stirred for 2 h letting warm to rt. After removal of the solvent
under reduced pressure, the resulting material was suspended in water
(50 mL) and stirred for 30 min at rt to form a brown precipitate. The
resulting precipitate was collected via filtration and rinsed with cold
water and hexane to give tert-butyl (4-(5-chloro-4-(4-(4-
chlorobenzyl)piperazine-1-carbonyl)pyridin-2-yl)thiazol-2-yl)(methyl-
Synthesis of Compound 11. (4-(4-Chlorobenzyl)piperazin-1-
yl)(2,5-dichloropyridin-4-yl)methanone. A solution of 2,5-dichlor-
oisonicotinic acid (3.0 g, 15.7 mmol) in thionyl chloride (15 mL) was
refluxed for 2 h. After removal of solvents under reduced pressure, the
residue was dissolved in dichloromethane (15 mL). Triethylamine
(6.53 mL, 46.9 mmol) was added to the mixture followed by 1-(4-
chlorobenzyl)piperazine (3.95 g, 18.8 mmol). The mixture was stirred
for 2 h at room temperature before addition of water and
dichloromethane. The organic layer was separated. The aqueous
layer was extracted with dichloromethane. The combined organic layer
was dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (heptane/ethyl acetate) to give (4-(4-chlorobenzyl)piperazin-1-
yl)(2,5-dichloropyridin-4-yl)methanone (5.5 g, 92%).
Compound 11: (5-Chloro-2-(2-(methylamino)thiazol-4-yl)-
pyridin-4-yl)(4-(4-chlorobenzyl)piperazin-1-yl)methanone. To a sol-
ution of (4-(4-chlorobenzyl)piperazin-1-yl)(2,5-dichloropyridin-4-yl)-
methanone (790 mg, 2.05 mmol), and tributyl(1-ethoxyvinyl)tin (0.75
mL, 2.24 mmol) in 1,4-dioxane (20 mL) was added Pd(PPh3)4 (120
mg, 0.104 mmol), and the mixture was stirred at 100 °C for 20 h. After
letting it cool to rt, the reaction mixture was poured into 2 M aqueous
potassium fluoride solution (5 mL) and stirred for 2 h. To this
reaction mixture was added N-bromosuccucinimide (400 mg, 2.25
1
d3)carbamate (430 mg, 79%). H NMR (400 MHz, CDCl3) δ ppm
1.53 (s, 9H), 2.32−2.38 (m, 2H), 2.48 (br s, 2H), 3.18−3.27 (m, 2H),
3.43 (s, 2H), 3.70−3.72 (m, 1H), 3.82−3.86 (m, 1H), 7.17−7.23 (m,
4H), 7.64 (s, 1H), 7.92 (s, 1H), 8.49 (s, 1H). ESI-MS m/z: 565.4 [M
+ H]+.
To a suspension of tert-butyl (4-(5-chloro-4-(4-(4-chlorobenzyl)-
piperazine-1-carbonyl)pyridin-2-yl)thiazol-2-yl)(methyl-d3)carbamate
(50 mg, 0.088 mmol) in methanol (6 mL) was added 3 M aqueous
HCl solution (0.3 mL, 0.90 mmol), and the mixture was stirred for 2 h
at 60 °C. The mixture was cooled, and the solvent evaporated under
reduced pressure gave compound 12: (5-chloro-2-(2-((methyl-d3)-
amino)thiazol-4-yl)pyridin-4-yl)(4-(4-chlorobenzyl)piperazin-1-yl)-
methanone hydrochloride (41 mg, 86%). 1HNMR (400 MHz, DMSO-
G
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Journal of Medicinal Chemistry
Article
d6) δ ppm 3.04 (brs, 2H), 3.20 (brs, 1H), 3.29−3.42 (m, 3H), 3.52−
3.62 (m, 2H), 4.35−4.40 (m, 2H), 4.59 (brs, 1H), 7.44 (brs, 1H),
7.54−7.55 (m, 2H), 7.63 (brs, 2H), 7.92−7.97 (m, 1H), 8.70 (s, 1H),
11.45 (brs, 0.5H), 11.58 (brs, 0.5H). 13CNMR (100 MHz, DMSO-d6)
δ ppm 169.59, 163.73, 158.30, 158.05, 150.89, 148.86, 147.05, 142.48,
134.38, 133.29, 128.73, 124.91, 118.56, 116.16, 114.24, 106.65, 57.75,
50.58, 50.07, 49.93, 42.88, 42.34, 37.76, 30.71. HRMS (TOF) calcd.
for C21H25Cl2N5OS [M + H] +465.1156, found 465.1110.
ACKNOWLEDGMENTS
■
The authors thank Dr. Jason Elliott and Jovita Marcinkeviciene
for their guidance in preparation of this manuscript and Drs.
Genji Iwasaki, Lawrence Hamann, Jason Elliott, and Brian
Jones for their guidance on medicinal chemistry strategy. The
authors also thank the project team members Pramod Pandey,
Jongchan Lee, and Chunhua Wang for protein production for
biophysical and X-ray studies, Jessica Voltz for performing
WNK1−4 mobility shift assays, Gang Zheng, Jey Jayaseelan,
Jianning Ying, Sarah Mowbray, Waan-Jeng Huang, and Yang
Luo for designing/performing cellular OSR1 assays, Lin Deng
for performing PK studies, Tracy Herr for performing in vivo
studies with human WNK1 transgenic mice, Fumin Fu, Jing
Liu, Michael Beil, and Wei Chen for performing in vivo studies
with SHR, and Karl Gunderson for NMR characterization.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00708.
Detailed methods on protein production, purification,
assay, and crystallization conditions (PDF)
Molecular formula strings and some data (CSV)
ABBREVIATIONS USED
■
WNK, With-No-Lysine; ATP, adenosine triphosphate; AMP-
PNP, adenylyl-imidodiphosphate; OSR1, oxidative-stress re-
sponse 1; SPAK, STE20/SPS1-related proline/alanine-rich
kinase; LipE, lipophilic efficiency
AUTHOR INFORMATION
Corresponding Author
* ken.yamada@novartis.com.
ORCID
■
Ken Yamada: 0000-0001-7899-6192
Upendra A. Argikar: 0000-0002-0939-0813
REFERENCES
■
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∇K.D.: Insmed, Inc., Lake Hiawatha, New Jersey 07034, United
States.
○M.Z.: Transamerica, 6 Rumbrook Road, Leominster,
Massachusetts 01453, United States.
◆N.I.: Takeda Pharmaceuticals U.S.A., Inc., Cambridge,
Massachusetts 02139, United States.
⬢Merck Research Laboratories, 33 Louis Pasteur Avenue,
Boston, Massachusetts, 02115.
¶Y.I.Y.: FujiFilm Corporation, Kaiseimachi, Ashigarakami-gun,
Kanagawa 258-8577, Japan.
△Y.I.: Janssen Pharmaceutical K.K., 2-5 Yarai-cho, Shinjuku,
Tokyo 162-0805, Japan.
▼Z.R.: Short Path Distillery, Inc., 59 Magnolia Street,
Arlington, Massachusetts 02474, United States.
Author Contributions
#These authors contributed equally to the work. T.Y., J.L.,
K.Yas., T.K., J.Y., L.M., Y.M., and H.I. designed and prepared
the compounds, D.K. and X.X. performed X-ray crystallog-
raphy, M.K. performed modeling, D.F.R., K.D.P., and L.X.
designed and performed in vivo studies, Y.Y., P.T. and H.M.P.
designed and performed mobility shift assays, D.Y., N.I., Y.Y.,
and H.M.P. designed and/or performed cellular OSR1 assays,
U.A.A. and M.J. were responsible for pharmacokinetic studies,
V.K. performed statistical analysis, and H.M.P. and K.Yam.
directed the drug discovery program. All authors have given
approval to the final version of the manuscript.
Notes
The authors declare the following competing financial
interest(s): All authors were employed by Novartis Institutes
for BioMedical Research during the period of their research
described in this manuscript. All research was fully funded by
Novartis.
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