Ligand-protein interactions of selective casein kinase 1δ inhibitors

Article abstract of DOI:10.1021/jm4006324

Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.

Full text of DOI:10.1021/jm4006324

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Article
Ligand-Protein interactions of Selective Casein Kinase 1# inhibitors.
Scot Richard Mente, Eric P Arnold, Todd W Butler, Subramanyam Chakrapani, Ramalakshmi
Chandrasekaran, Kevin Cherry, Ken DiRico, Angela Doran, Katherine Fisher, Paul Galatsis, Michael E.
Green, Matthew Hayward, John M. Humphrey, John Knafels, Jianke Li, Shenping Liu, Michael Marconi,
Scott McDonald, Jeffrey F. Ohren, Vanessa Paradis, Blossom Sneed, Kevin Walton, and Travis T Wager
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4006324 • Publication Date (Web): 06 Aug 2013
Downloaded from http://pubs.acs.org on August 22, 2013
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Neuroscience
Walton, Kevin; Pfizer Worldwide Research and Development, Neuroscience
Wager, Travis; Pfizer, CNS
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Ligand-Protein Interactions of Selective
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Casein Kinase 1δ inhibitors.
Scot Mente*, Eric Arnold, Todd Butler, Subramanyam Chakrapani, Ramalakshmi
Chandrasekaran, Kevin Cherry, Ken DiRico, Angela Doran, Katherine Fisher, Paul Galatsis,
Michael Green, Matthew Hayward, John Humphrey, John Knafels, Jianke Li, Shenping Liu,
Michael Marconi, Scott McDonald, Jeff Ohren, Vanessa Paradis, Blossom Sneed, Kevin Walton
and Travis Wager
Pfizer Worldwide Research and Development, 700 Main Street, Cambridge, MA 02139, USA; 1
Eastern Point Road, Groton, CT 06340, USA.
*Address correspondence to this author at Pfizer Worldwide Research and Development, 700
Main Street, Cambridge, MA 02139, USA; Tel: 617-395-0711; E-mail: scot.mente@pfizer.com
ABSTRACT
Casein Kinase 1 (CK1) δ and ε are believed necessary enzymes for the regulation of circadian
rhythms in all mammals. Based on our previously published work demonstrating that a CK1ε
preferring compound to be ineffective circadian clock modulator, we have synthesized a series of
pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using
structure-based drug design, we have been able to exploit differences in the hinge region
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between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR,
brain exposure and the effect of these inhibitors on mouse circadian rhythms are described. The
in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient
central exposure levels is capable of modulating of circadian rhythms.
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INTRODUCTION
Casein kinase 1 protein kinases (CK1s) function as regulators of signal transduction pathways
and are involved in Wnt signaling and circadian rhythms.1-4 We have previously demonstrated
that dual CK1δ/ε inhibitors, such as PF-670462 (compound 1, chart 1), have robust phase delay
in animal models of circadian rhythm,5 while selective CK1ε compounds have been shown to be
ineffective clock modulators.6 As such, CK1δ potency appears to be a key factor in the design of
a circadian rhythm modulating agent.
A number of aspects can potentially confound the design of a successful CNS-active CK1δ
inhibitor. These include poor whole-cell potency, a lack of selectivity and incorrectly aligned
ADMET properties. Targeting a kinase for CNS indications may be particularly challenging,
due to the balancing of free drug levels necessary to achieve central exposure with potential
peripheral safety considerations. In fact, due to the polar nature of many kinase scaffolds,
peripheral free drug exposure may be as much as 2-3-fold higher than the relevant free central
exposure. Since many approved kinase drugs are known to have cardiotoxicity,7,8 imbalance
between peripheral and central drug levels is a clear area of concern. Leveraging the CNS drug
analysis and CNS MPO algorithm, molecules were designed in high desirability space (CNS
MPO > 4) to increase the odds of identifying compounds with good CNS exposure and aligned
ADME attributes.9,10 Although many have highlighted the potential benefits of drug
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promiscuity,11,12 we share in the opinion that overly promiscuous compounds are “forbiddingly
dangerous…without a rational strategy to control specificity…”13 and would be a higher risk
during clinical assessment.
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While the previously described compound 1 has proven to be a useful tool for demonstrating
the role of CK1δ inhibitors for modulation of circadian rhythms in vivo, it suffers from poor off-
target pharmacology. Recent structural examinations of 1 bound to CK1δ and CK1ε, suggest a
very similar binding to both isoforms14,15, and help explain why 1 is only slightly CK1δ
preferring. In addition to both CK1δ and CK1ε, 1 was active (>50% Inh at 1uM) against 6
kinases: PKAα, p38, MAP4K4, CK1α, LCK and EGFR. This non-optimal selectivity profile
necessitated the need for modification to build in greater selectivity and a more favorable CNS
profile with the ultimate goal of advancing a selective CK1δ inhibitor to the clinic to test the
circadian rhythm shift mechanism of action (MOA). We began our pursuit to identify a selective
CK1δ inhibitor by screening our internal compound collection for viable hits. Compounds 1 and
2 were identified from this HTS.
Chart 1.
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This paper describes the use of structure-based drug design to develop ATP-competitive CK1δ
preferring inhibitors. We report the x-ray structures of 4 pyrazole-substituted pyridine
compounds bound to CK1δ and compare their binding characteristics to those of a similar
inhibitor bound to p38, as this enzyme proved to be the most challenging of the off-target kinase
activities. We find that the binding mode of the more selective inhibitors is characterized by an
interaction with a glycine residue (Gly110) of the kinase. Although this residue is conserved in
p38 and CK1δ, the adjacent hinge residue, Pro87, results in a framework that is both rigid and
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unique. These inhibitors represent the first such CK1δ preferring ligands designed with a clear
effort toward gaining both enzyme activity and central exposure.
Chemistry
Scheme 1 illustrates the synthesis of CK1δ preferring inhibitors, 2-4. Commercially available
pyridine 8 and ethyl 4-fluorobenzoate 9 were condensed in presence of LHMDS to give ketone
10 which was directly treated with dimethylformamide dimethyl acetal (DMF-DMA) to afford
enamine 11 as a mixture of E,Z isomers in ~1:8 ratio. Reacting 11 with methyl hydrazine at 0o C
resulted in a mixture of N-methylpyrazole regio-isomers which, after trituration with 1:1
ether/heptane, gave compound 2 in 31% isolated yield over 3 steps.
Oxidation of 2 with mCPBA afforded N-oxide 13 in 95% yield which upon cyanation, with
trimethylsilylcyanide (TMSCN), resulted in an 86% yield of pyridylnitrile 14. Catalytic
hydrogenation using sponge nickel and 7N ammonia in MeOH gave amine 15 in 99% yield,
which upon heating with ethyl formate produced formamide 16. N-Methylation, using MeI,
resulted in 17 in 90% yield. Subsequent hydrolysis using methanolic hydrochloric acid provided
the target compound 4 in reasonable yield (56%).
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The desired target compound 3 could be prepared in 2 steps via standard reaction conditions.
Namely, pyridyl nitrile 14 could be hydrolyzed to methyl ester 18 using acidic methanol in 55%
yield. Alcohol 3 could then be obtained by LAH reduction of 18 in 20% yield.
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Scheme 1.a Synthesis of 2-substituted pyridyl anlogs 2-4.
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Reagents and conditions: i) 2 Eq. LHMDS, THF, 0 ◦C to rt, 3.75 h (crude); ii) 1.1 eq DMF-
DMA, reflux, 72 h (crude); iii) 1.2 eq methylhydrazine, EtOH, 0 ºC, 3h (31% over 3 steps); iv)
1.2 eq mCPBA, CH2Cl2, 0 ºC to rt, 18h (99%); v) 1.3 eq TMS-CN, 1.2 eq dimethylcarbamoyl
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chloride, CH2Cl2, rt, 16h (86%); vi) H2 (50 psi), catalytic sponge Ni, 7N ammonia in MeOH,
THF, rt, 2h (99%); vii) ethyl formate, 70 ºC, 3h (80%); viii) 1.5 eq MeI, 1.2 eq NaH, THF, rt,
18h (90%); ix) MeOH/12N HCl (10:1 v/v), reflux, 90 min, followed by ISOPROPYL
ALCOHOL/MeOH recryst 56%); x) 1.25M HCl/MeOH, reflux 20h, (55%); xi) 1M LAH/THF,
rt, 30 min (25%)
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Scheme 2.a Synthesis of fused oxazepine 5.
b Reagents and conditions: i) 2 eq n-BuLi, THF, -70 ºC then 1.5 eq 20, 1.5h (44%): ii) 0.9 eq
21, 2 mol% Pd(OAc)2, 4 mol% Xphos, 1.2 eq Cs2CO3, ACN/H2O (5:1), reflux, 3h (80%); iii) 1.4
eq 24, 1.7 eq NaH, THF, -20 to -5 ºC, 4h, (70%); iv) H2SO4 conc, 110o C, 10 min v) 5N NaOH,
pH 7.0 phosphate buffer, 110 oC, 3 h, 80% from 25; vi) 2.25 eq LAH/THF, 50 ºC, 2h; vii) 2 eq p-
toluenesulfonic acid, MeOH, 42% from 26.
Scheme 2 illustrates the synthesis of CK1δ/ε inhibitor 5. Pinacol borane 21 was prepared by
lithiation of bromopyrazole 19 followed by borolation with 20 in 44% yield. Subsequent Suzuki
coupling of iodide 22 with 21 using cesium carbonate, Xphos, and Pd(OAc)2 yielded
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diarylpyrazole 23 in 80% yield. SNAr displacement using deprotonated N-Boc ethanolamine 24
generated pyridylether 25 in 81% yield. Removal of the Boc protecting group afforded amine 26
and subsequent one-pot cyano-hydrolysis and ring closure yielded oxazepinone 27 in 80% over 2
steps. The desired oxazepine 5 was then prepared by reduction of 27 using lithium aluminum
hydride followed by conversion to the tosylate salt in 42% yield
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Scheme 3.a Synthesis of morpholine 6.
N
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tBuO
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o
Scheme 3c
Reagents and conditions: i) 5 mol% Pd(dppf)2Cl2, 2 eq CsF, DMF, 140 C,
2h(80%); ii) 1 eq. n-BuLi, toluene, 1eq 30, -78 ºC, 1.5h (22%iii) 10 eq. 1.0 M HCl/EtOH, EtOH,
rt, 12h (assumed 100%); iv) 4 eq. NaBH4, MeOH, -40 ºC to rt, 30 min (26%)
Scheme 3 illustrates the synthesis of CK1δ/ε inhibitor 6. The synthesis started with Suzuki
coupling of a commercially available pyridyl iodide 28 and boronate intermediate 21 that
resulted in 80% yield of intermediate 29 as a white solid. Temperature controlled halogen-metal
exchange of 29 followed by addition of morpholinone 30 at -78o C yielded keto-ether
intermediate 31 as an off-white foam which was subjected to acidic hydrolysis conditions and
subsequent ring closure to yield imine 32. Reduction of the imine with sodium borohydride
resulted in the isolation of racemic morpholine as white foam in 26% yield. Preparative chiral
HPLC was then used to separate the (-) antipode of 6 from its (+) antipode.
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Table 1. Structure, pharmacology and exposure data.
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a
IC50 values reported as geometric means of n > 2 independent experiments, unless otherwise
noted.
b Exposures are maximum concentrations at a 56 mg/kg dose, unless otherwise noted.
c32 mg/kg dose.
RESULTS AND DISCUSSION
Structure Based Drug Design. Compound 2 was a high throughput screening hit that was
previously co-crystallized with the p38 enzyme.16,17 The X-ray structure of 2 bound to p38
(Figure 1) shows the pyridine core interacting as a single point binder with the hinge (Met109),
the pyrazole nitrogen interacting through a water molecule with the catalytic Lys38, and the
para-fluoro aryl ring occupying the hydrophobic selectivity pocket of p38. In our hands, 2 was
found to also be a potent CK1δ inhibitor (IC50 = 48 nM), with moderate whole-cell activity (EC50
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Figure 1. Binding of inhibitors to p38a (red box; compound 2) and CK1δ (ribbon diagram and
green boxes; compounds 3-6). All structures share a common interaction of the pyridine core
interacting as a single point binder with the hinge (Met109/Leu85) of p38/CK1δ.
Also
conserved in all structures is the pyrazole nitrogen interacts through a water molecule with the
catalytic Lys38, as well as the para-fluoro aryl ring occupied the hydrophobic selectivity pocket
of p38. Selective CK1δ inhibitors (3-6) make additional hinge interaction with the proline
carbonyl of CK1δ. Compound 3-6 also hydrogen bond to the carbonyl oxygens of both residues
Leu85 and Gly86.
= 545 nM). Evaluation of 2 at a 1 μM concentration against a larger selectivity panel,
consisting of 103 kinases, found that 2 inhibited 9 kinases at greater than 50%: ABL (61); LYN
(70.5); MAPK14 (55); p38 (85); JNK2/3 (48); ROCK2 (52); EGFR (80); MAP4K4 (66);
SAPK2A (96).
The relative selectivity of compound 2 over this subset of the kinase pharmacology can be
attributed largely to the para-fluoro aryl group which interacts with the so-called ‘hydrophobic’
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selectivity pocket. This feature exists in both the CK1δ and p38 enzymes despite differences in
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primary sequence; in p38 this is comprised of Thr-Val-Leu residues, while in CK1δ/ε it is
comprised of Met-Val-Met residues.
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An obvious and important difference between these kinases was the proline residue (Pro87) of
the backbone of CK1δ that is not present in p38. This difference was targeted in the design of
novel and selective CK1δ inhibitors as exemplified by compound 3 (Figures 1 and 2). The
primary hydroxyl group of compound 3 makes a selective interaction with the proline (Pro87)
carbonyl of CK1δ that was in an altered conformation compared to p38 (Ala111). Although the
same carbonyl exists in both enzymes, the flexible hinge of p38 positioned this functionality in a
different orientation relative to CK1δ; shown in Figure 2. Previous studies have shown that p38
ligands containing the pyridine hinge binding motif can induce a peptide flip in the hinge region
by introduction of an amino group ortho to the pyridine nitrogen.18 With these ligands, the
amino group H-bonds to the backbone carbonyl of the hinge residue (Met109 in p38), yielding
improved potency and p38 selectivity. In contrast, the proline of CK1δ, holds that same
carbonyl at a different angle and renders the ligand-induced ‘peptide flipping’ all but impossible.
Compound 3 adds an additional methyl spacer between the pyridine acceptor and the putative
donor in an attempt to gain selectivity. Indeed, 3 proved to increase the p38 selectivity to
roughly 50x; whereas 2 was only 3x. However, the hydroxyl group was a concern due to its
potential for rapid metabolism.
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Figure 2. View of backbone carbonyls of CK1δ (white wire with compound 3) and p38 (green
wire). Comparing the angle between the carbonyl groups (C=O vector) gives values of 41 for
Leu85 (Met109 in p38), 117 for the Gly86 (Gly110 in p38) and 80 for Pro87 (Ala111 in p38).
Compound 4 was similarly designed to retain the new-found selectivity handle and maintain or
improve potency at reasonable levels of central exposure.19 This was accomplished through
inclusion of a methyl-amine group which also displaced nearby water molecules and shielded the
H-bond to the carbonyl.20 The potency of 4 improved roughly 2x over compound 3 and the
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selectivity over p38 widened to 230x. Importantly, compound 4 was completely selective in our
kinase panel (0/43, table 1) and showed large improvements in cellular activity. Additionally, 4,
when dosed subcutaneously (SC), possessed good central exposure (7.5 μM free brain exposure;
Cb,u/Cp,u = 0.78). However, the addition of a basic center was found to increase hERG-liability.
The next round of designs produced compounds 5-6 which have in common the inclusion of a
group designed to lower the basicity of the amine; a strategy which has been used to reduce the
affinity of basic compounds to the hERG channel.21-23 With the inclusion of the oxazepine ring,
compound 5 had improved CK1δ potency to 6.5 nM and also proved to be the most potent
compound in the whole cell assay at 39 nM. hERG activity of compound 5 was reduced ~6x
relative to compounds 4. Published hERG models have suggested the importance of ligand
properties on channel block such as basicity, hydrophobicity and ligand flexibility.25
Conceptually, compounds possessing a basic center are thought to interact with aromatic
residues (Phe656), leaving polar heterocycles, such as the pyrazole group of our ligands, to
interact with the channel via specific H-bonding interactions to a serine residue (Ser624) at the
top of the pore.25,26 The oxazepene, 5, may benefit from both the reduction of the pKa (5.93
versus 7-8), as well as conformational restriction which may hinder the spacing between
pharmacophoric elements.26 Although 5 maintained 100x selectivity over p38, at 654 nM, it
was markedly more potent versus p38 than 4. In addition, two additional kinase activities were
detected in the kinase panel (CK1α and MAP4K4). While the free plasma concentration (Cp,u
following SC dosing) of 5 was comparable to 4, free brain levels were now disproportionally
lower by roughly 2.5x (Cb,u/Cp,u = 0.33), narrowing the gains made from the reduction of hERG
activity.
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Morpholines are also well known to reduce basicity via a negative inductive effect. This
concept was introduced into the scaffold of compound 2 to produce compound 6. Docking27 of 6
into the CK1δ X-ray structure also suggested the ability to increase the potency of the ligand on
the receptor for this series through the creation of additional interactions with Ile15. This
compound was the most potent compound measured in the enzyme assay (5.3 nM) and very
potent in the whole cell assay (89.9 nM). Compound 6 also maintained good p38 and overall
kinase selectivity versus our panel.
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Figure 3. Mouse circadian rhythm shift (y-axis, in hours) versus compound (x-axis). Top and
lower bounds of box refer to 75th and 25th percentile values. Mean shifts are -1.5, -2.9 and -1.2
hours (p < 0.001 for all three) for compounds 4, 5 and 6 respectively.
Selected compounds from table 1 were screened in an in vivo mouse circadian rhythm
model.6 Figure 3 illustrates the magnitude of circadian rhythm shift observed in mice following
3 days of drug treatment (56 mg/kg subcutaneous dose). For the mice treated in these studies,
the shifts refer to the difference between the start of the active period after dosing versus before
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dosing. All compounds demonstrate significant (p < 0.001) phase shift delay, with 5 showing
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the largest mean shift of 2 hours and 55 minutes.
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CONCLUSIONS
We have illustrated that compounds 3-6 are potent CK1δ inhibitors that gain a large
degree of selectivity over p38 via a unique H-bonding interaction with the hinge region. In
particular, placement of a CH2-spaced heteroatom capable of donating a H-bond to the carbonyl
of the hinge Pro in CK1δ. Larger cyclic amines, oxazepine and morpholine, were designed to
maintain this motif and have been shown to shift mouse circadian rhythms in vivo. These
analogues demonstrate the utility of structure-based drug design to address issues of potency and
selectivity. However, these analogues also suffer mild hERG pharmacology. For instance, 5,
though active in vivo, is present at 8.8 μM maximum free plasma concentration at this dose;
which translates to an approximate 3-fold hERG TI (hERG EC50/Cp,u). Future publications will
highlight compounds designed to address this issue, while maintaining in vivo efficacy.
EXPERIMENTAL SECTION
General Information. All solvents and reagents were obtained from commercial sources and
were used as received. Compound 1 is commercially available (Tocris), 2 was prepared using
published methodology28, and 3-6 were prepared as discussed below. All reactions were
followed by TLC (TLC plates F254, Merck) or LCMS (liquid chromatography-mass
spectrometry) analysis. Varian 300, 400 or 500 MHz instruments were used to obtain 1H and 13C
NMR spectra in deuterated solvents. All proton shifts are reported in δ units (ppm) and were
13
measured relative to signals for chloroform (7.26 ppm) or methanol (3.30 ppm). All C shifts
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are reported in δ units (ppm) relative to the signals for chloroform (77 ppm) or methanol (49
ppm) with 1H decoupling. All coupling constants (J values) are reported in Hertz (Hz). GCMS
were recorded on an HP 6890 GC system equipped with a 5973 mass selective detector
(Stationary phase: HP-1, fused silica, 12 m x 0.202 mm x 0.33 µm, temperature limits: -60 ºC to
325 ºC, ramp rate = 30 ºC/ min, solvent delay = 0.4 min). Analytical analyses by UPLC were
performed on a Waters Acquity system with PDA detection (UV 210 nm) at 45º C, flow rate 0.5
mL/min, with a gradient of 95/5 buffer/acetonitrile (0 to 7.55 min), 10/90 buffer/acetonitrile
(7.55 to 7.85 min), 95/5 buffer/acetonitrile (8.10 to 10.30 min) using the following columns and
buffers: Waters BEH C8 column (2.1 x 100 mm, 1.7 um) with 50 mM sodium perchlorate/0.1%
phosphoric acid or 10 mM ammonium bicarbonate as buffer; Waters BEH RP C18 column (2.1 x
100 mm, 1.7 um) or Waters HSS T3 (2.1 x 100 mm, 1.8 um) column with 0.1% methanesulfonic
acid buffer. Mass spectra were recorded on a Micromass ADM atmospheric pressure chemical
ionization instrument (MS, APCI). High resolution mass spectra were obtained on an Agilent
6620 LC-MS TOF equipped with a Zorbax Eclipse column (50 mm x 4.6 mm, 1.8 micron XDB-
C18) using 0.1% formic acid aqueous as mobile phase A1 and acetonitrile containing 0.1%
formic acid as mobile phase B1. Column chromatography was carried out on silica gel 60 (32-60
mesh, 60 Ǻ) or on pre-packed Biotage™ columns. The purities of final compounds 3-6 as
measured by UPLC were found to be above 95%.
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1-(4-Fluorophenyl)-2-(pyridine-4-yl)ethanone (10). To a solution of 4-methylpyridine (48g,
500 mmol) in THF (100 mL) under N2 at 0 oC was slowly added LHMDS (1.0M in THF, 1,030
mL,1030 mmol). Once addition was complete, a dark brown solution resulted and this was
o
stirred at 0 C for 1h. Ethyl 4-fluorobenzoate (85g, 74 mL, 500 mmol) in THF (100 mL) was
added slowly via an addition funnel and stirring was continued at 0 oC for another 45 min before
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the reaction was slowly warmed to rt. After stirring for 2 h at rt, the reaction was quenched with
50 mL of water. This reaction mixture was cooled to 0 oC, the pH adjusted to 7.0 by the addition
of ~50 mL of 6N HCl and extracted with EtOAc (2x400 mL). The combined organic phases
were dried over Na2SO4, filtered and concentrated. The crude solid was triturated with 50 mL
heptanes to afford the title compound (108g) as a light yellow solid. This material was used
without purification in the subsequent step. A portion of this solid was subjected to silica gel
chromatography using 100% EtOAc to afford ~95% pure title compound as a light yellow
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colored solid. H NMR (CDCl3, 400 MHz) δ 8.45 (dd, J = 1.6, 4.3, 2H), 7.90-7.95 (m, 2H), 7.09
(dd, J = 1.6, 4.3, 2H), 7.01-7.07 (m, 2H), 4.13 (s, 2H). 13C (CDCl3, 100 MHz) δ 194.3, 166.6 (d,
J
= 255.5), 149.9, 143.3, 132.6 (d, J
= 3.7), 130.6 (d, J
= 9.6), 124.9, 113.6 (d, J
=
F-C
F-C
F-C
F-C
22.1), 44.4. HRMS calcd for C13H10FNO 216.0819 (M+H)+ , found 216.0816.
(Z)-(Dimethylamino)-1-(4-fluorophenyl)-2-(pyridin-4-yl)prop-2-en-1-one (11). To a 500
solution of ketone 10 (106g, 500 mmol) in THF (500 mL) was added dimethylformamide
dimethyl acetal (76 mL, 572 mmol) and the mixture was refluxed at 75 oC for 72 h. The reaction
was cooled to rt and concentrated down under reduced pressure to afford the title compound as a
reddish gum which was taken crude to the next step. A portion of this material was purified by
silica gel chromatography using 5% MeOH/EtOAc to afford 95% pure title compound as light
1
1
yellow solid. H NMR showed a ~1:8 mixture of E, Z isomers. H NMR (CDCl3, 400 MHz),
major Z-isomer : δ 8.44 (dd, J = 1.6, 4.5, 2H), 7.36-7.41 (m, 2H), 7.31 (s, 1H), 7.01 (dd, J = 1.6,
4.5, 2H), 6.88-6.95 (m, 2H), 2.72 (s, 6H). 13C (CDCl3, 100 MHz) δ 192.4, 161.2 (d, J
=
=
F-C
F-C
250.3), 154.5, 148.8, 145.7, 137.0 (d, J
= 3.7), 130.7 (d, J
= 8.8), 126.7, 114.7 (d, J
F-C
F-C
22.1), 108.5, 44.4. HRMS calcd for C16H15FN2O 271.1241(M+H)+ , found 271.1240.
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4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine (2). To a solution of enamine 11
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(140g, 520 mmol) in EtOH (200 mL) at 0 oC was added dropwise a solution of methylhydrazine
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(33 mL, 624 mmol) in EtOH (70 mL). After 3 h stirring at 0 C, the reaction was concentrated
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under reduced pressure, re-dissolved in 500 mL EtOAc, and washed with 75 mL water and 50
mL satd. brine. The organics were dried over Na2SO4, filtered and the concentrate was passed
through a silica plug and rinsed with 2L each EtOAc and 10% MeOH/EtOAc. The filtrate was
concentrated under reduced pressure and triturated with 1:1 heptanes/Et2O (200 mL). The title
compound (42 g, 31%) was recovered as a white solid after filtration and drying. 1H NMR
(CDCl3, 400 MHz) δ 8.49-8.52 (m, 2H), 7.60 (s, 1H), 7.42-7.47 (m, 2H), 7.14-7.17 (m, 2H),
7.02-7.09 (m, 2H), 4.00 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 162.9 (d, J
= 247.2), 150.3,
F-C
148.9, 141.2, 131.9 (d, J
= 8.0), 130.9, 129.0 (d, J
= 3.2), 122.9, 118.4, 114.7 (d, J
=
F-C
F-C
F-C
21.8), 39.5. HRMS calcd for C15H12FN3 254.1088 (M+H)+ , found 254.1080.
4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine 1-oxide (13). To a 0o C solution
of of diarylpyrazole 2 (42.0 g, 163.9 mmol) in 300 mL of DCM was added mCPBA (42 g, 197
mmol). The reaction was warmed to rt and stirred 18h then purified directly by silica gel
chromatography using 5-50% MeOH/EtOAc to afford 44 g (99%) of the title compound as white
1
solid. H NMR (CDCl3, 400 MHz) δ 8.04-8.08 (m, 2H), 7.57 (s, 1H), 7.36-7.42 (m, 2H), 7.07-
7.11 (m, 2H), 7.00-7.06 (m, 2H), 3.95 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 162.7 (d, J
=
F-C
248.7), 148.3, 139.0, 131.5, 130.2, 130.1(d, J F-C = 8.1), 128.5 (d, J F-C = 3.7), 124.6, 116.5, 115.7
(d, J F-C = 22.1), 39.2. HRMS calcd for C15H12FN3O 270.1037 (M+H)+ , found 270.1032.
4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)picolinonitrile (14). To a 0o C suspension of
N-oxide 13 (57 g, 212 mmol) and trimethylsilyl cyanide (28 g, 275 mmol) in 250 mL DCM was
added dropwise dimethyl carbamoyl chloride (24 mL, 254 mmol). Once addition was complete,
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the resulting clear solution was stirred at rt for 16 h. The reaction was treated with 150 mL sat.
K2CO3 and diluted with 250 mL of DCM. After one more extraction of the aqueous phase with
200 mL DCM, the combined organics were dried over Na2SO4, filtered and concentrated under
reduced pressure. The concentrate was slurried with 200 mL Et2O, and filtered to recover 51g
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(86%) of the title compound as a light yellow solid. H NMR (CDCl3, 400 MHz) δ 8.54 (dd, J =
5.3, 0.6, 1H), 7.68 (s, 1H), 7.56 (dd, J = 1.68, 0.8, 1H), 7.38-7.43 (m, 2H), 7.32 (dd, J = 5.1, 1.8,
1H), 7.05-7.12 (m, 2H), 4.01 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 162.6 (d, J
= 248.1),
F-C
151.2, 148.9, 142.5, 134.2, 130.9, 130.1 (d, J
= 8.1), 128.3 (d, J
= 3.7), 126.8, 125.0,
F-C
F-C
117.2, 116.1, 115.8 (d, J F-C = 21.4), 39.4. HRMS calcd for C16H11FN4 279.1041 (M+H)+, found
279.1036.
(4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)methanamine (15). To a
nitrogen sparged 2L pressure autoclave was added sponge nickel (Johnson Matthey type A-4000,
23g, 387 mmol), nitrile 14 (35 g, 125.8 mmol), and 7N ammonia in methanol (1225 mL, 8.58
mol). The vessel was purged with nitrogen (pressurized to 50 psi and then vented) 3x, then
purged with hydrogen (pressurized to 50 psi and vented) 3x. The mixture was pressurized to 50
psi with hydrogen and shaken for 2 h. After purging with nitrogen 3x and the reaction was
filtered (celite) and washed methanol (350 mL). The filtrate was concentrated under reduced
1
pressure to recover 36 g (99%) of the title compound as a pink gum. H NMR (MeOH-d4, 400
MHz) δ 8.47 (d, J = 5.1, 1H), 8.02 (s, 1H), 7.40-7.46 (m, 2H), 7.31 (br s, 1H), 7.21 (dd, J = 5.3,
1.8, 1H), 7.10-7.16 (m, 2H), 4.17 (s, 2H), 3.98 (s, 3H). 13C NMR (MeOH-d4, 100 MHz) δ 165.7
(d, J
= 245.9), 154.1, 150.7, 150.0, 144.0, 133.5, 131.9 (d, J
= 8.1), 130.6 (d, J
= 3.0),
F-C
F-C
F-C
123.5, 122.1, 118.7, 116.7 (d, J
(M+H)+ , found 283.1349.
= 22.1), 44.2, 39.4. HRMS calcd for C16H15FN4 283.1354
F-C
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N-((4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)methyl)formamide (16).
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6
o
Amine 15 (35 g, 124 mmol) in ethyl formate (500 mL) was heated to 70 C. After 3 h heating,
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the reaction was cooled to RT, concentrated under reduced pressure, passed through a 3.5”x5”
silica gel plug flushing first with 100% EtOAc to remove the less polar impurities and then
eluting with 25% MeOH in EtOAc to yield 31 g (80%) of the title compound as light yellow
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solid. H NMR (CDCl3, 400 MHz) showed a ~9:1 mixture of rotamers; major isomer - δ 8.36 (d,
J = 5.3, 1H), 8.27 (s, 1H), 7.61 (s, 1H), 7.37-7.43 (m, 2H), 7.14 (s, 1H), 7.00-7.06 (m, 4H), 4.52
(d, J = 5.3, 2H), 3.97 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 167.0 (d, J
= 247.4), 161.1,
F-C
156.1, 149.0, 148.6, 141.9, 130.7, 129.2 (d, J
= 8.1), 126.6 (d, J
= 3.0), 121.4, 120.5,
F-C
F-C
117.6, 113.9 (d, J
found 311.1301.
= 21.4), 42.9, 39.1. HRMS calcd for C17H15FN4O 311.1303 (M+H)+ ,
F-C
N-((4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine-2-yl)methyl)-N-methyl-
formamide (17). Sodium hydride (4.8 g,120 mmol) was added to formamide 16 (31 g, 99
mmol) in THF (992 mL). After 30 min stirring, methyl iodide (21 g, 149 mmol) was added.
The resulting suspension was stirred at rt for 18 h, quenched with 150 mL of saturated NaHCO3
and extracted with DCM (3x400 mL). The combined organic extracts were dried over Na2SO4
and then filtered through a 1”x3” silica plug layered with 1” MgSO4. The filtrate was
concentrated under reduced pressure to give 29 g (90%) of the title compound as light yellow
1
colored solid. H NMR (CDCl3, 400 MHz) δ ~1:1 mixture of rotamers, 8.40/8.35 (two d’s, 2H
total, J = 5.3), 8.16 (s, 1H), 8.04 (s, 1H), 7.59 (d, 2H, J = 2.3), 7.34-7.39 (m, 4H total), 6.96-7.06
(m, 8H total), 4.54 (s, 2H), 4.39 (s, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 2.87 (s, 3H), 2.73 (s, 3H). 13C
NMR (CDCl3, 100 MHz) δ ~1:1 mixture of rotamers, 163.8,163.7, 162.9, 162.5, 161.3, 156.1,
156.2, 149.8, 149.3, 148.5, 148.4, 142.0, 130.7, 130.6, 130.2, 130.1, 130.0, 128.9, 128.7, 121.5,
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121.1, 120.3, 119.9, 117.6, 117.5, 115.5, 115.4, 115.3, 115.2, 54.9, 49.5, 39.7, 34.7, 29.8.
HRMS calcd for C18H17FN4O 325.1459 (M+H) +, found 325.1455.
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1-(4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine-2-yl)-N-methylmethanamine
(4). N-Methylformamide 17 (29g, 89 mmol) in 580 mL of 1:10 v/v of 12 N HCl : MeOH was
refluxed for 30 min. The reaction was cooled to rt, concentrated under reduced pressure and
triturated with MeOH (6x50 mL) to afford 26 g of light cream colored solid. Crystallization
from boiling ISOPROPYL ALCOHOL/MeOH (4:1 ratio) afforded 18.5 g (56%) of the title
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compound as white cottony solid. H NMR (MeOH-d4, 400 MHz) δ 8.63 (d, J = 6.3, 1H), 8.53
(s, 1H), 8.26 (d, J = 1.8, 1H), 7.69 (dd, J = 6.3, 2.0, 1H), 7.51-7.56 (m, 2H), 7.19-7.26 (m, 2H),
4.60 (s, 2H), 4.05 (s, 3H), 2.88 (s, 3H). 13C NMR (MeOH-d4, 100 MHz) δ 162.2 (d, J
=
F-C
247.6), 153.2, 152.1, 147.3, 144.5, 136.4, 130.1 (d, J
= 8.7), 129.3 (d, J
= 3.6), 126.7,
F-C
F-C
125.1, 115.0 (d, J
= 22.3), 117.2, 50.3, 40.3, 34.6. HRMS calcd for C17H17FN4 297.151
F-C
(M+H)+, found 297.1504.
Methyl 4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)picolinate (18) Nitrile 14 (330 mg,
1.18 mmol) andHCl/MeOH (1.25 M, 16 mL, 20.4 mmol) were refluxed for 20 h, then cooled to
rt and concentrated under reduced pressure. The residue was dissolved in 100 mL DCM and
basified using 20 mL of satd. NaHCO3. The organics were dried over Na2SO4, filtered and
concentrated under reduced pressure to recover 370 mg of crude product as semisolid. This was
subjected to silica gel chromatography using 75-100% EtOAc/heptanes to afford 208 mg (55%)
1
of the title compound as a white solid. H NMR (CDCl3, 400 MHz) δ 8.51 (d, J = 5.1, 1H), 7.99-
8.01 (m, 1H), 7.64 (s, 1H), 7.34-7.39 (m, 2H), 7.21 (dd, J = 5.1, 1.8, 1H), 6.96-7.03 (m, 2H),
3.93 (s, 3H), 3.92 (s, 3H). 13C NMR (CDCl3, 100 MHz), δ 165.2, 162.3 (d, J F-C = 248.1), 149.4,
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148.2, 147.7, 141.9, 130.4, 129.7 (d, J F-C = 8.1), 128.2 (d, J F-C = 3.7), 124.8, 123.2, 116.7, 115.1
(d, J F-C = 21.4), 52.4, 38.8. HRMS calcd for C17H14FN3O2 312.1143 (M+H)+ , found 312.1148
(4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)methanol (3). To a solution
of ester 18 (180 mg, 0.57 mmol) in THF (6 mL) at RT was added dropwise a THF solution
ofLAH (1.0 M, 0.86 mL, 0.86 mmol). After 30 min stirring, the reaction was quenched with
dropwise addition of water and diluted with THF (50 mL). After the addition of celite, the slurry
was stirred for 30 min and then filtered through a pad of MgSO4. The filtrate was concentrated
under reduced pressure to recover 165 mg of desired alcohol as a pink colored gum. This was
subjected to silica gel chromatography using 5:95:0.1 MeOH:EtOAc:NH4OH (conc) to afford
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41mg (25%) of the title compound as a colorless gum which solidified upon standing at RT. H
NMR (CDCl3, 400 MHz) δ 8.39 (d, J=5.1, 1H), 7.59 (s, 1H), 7.39-7.44 (m, 2H), 7.16 (s, 1H),
6.99-7.06 (m, 3H), 4.68 (s, 2H), 3.97 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 162.4 (d, J F-C
247.4), 159.6, 148.6, 148.5, 141.8, 130.7, 128.3 (d, J = 8.1), 126.2 (d, J = 2.9), 121.3,
=
F-C
F-C
119.2, 117.9, 114.3 (d, J F-C = 21.4), 64.2, 39.1. HRMS calcd for C16H14FN3O 284.1194 (M+H)+,
found 284.1188.
3-(4-Fluorophenyl)-1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-
21. To a solution of n-BuLi (2.5M in hexanes, 500 mL, 1.25 mol) at -70o C was added slowly a
solution of commercially available pyrazole 19 (160 g, 0.63 mol) inTHF (1.2L). The resulting
solution was stirred at this temp for 1h. 2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
20 (175 g, 0.94 mol) was then added slowly maintaining the temp below -60o C. After addition,
the reaction was stirred at -70o C for 30 min and then quenched slowly by the addition of H2O.
The resulting slurry was filtered off and the filtrate was concentrated under reduced pressure.
This was taken up in EtOAc (2L) and washed with (250 mL)and brine (100 mL), dried over
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Na2SO4 and concentrated under reduced pressure to yield crude product which solidified upon
cooling to 0o C. After triturating with 500 mL hexanes, 84 g (44 %) of the title compound was
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obtained as white solid. H NMR (CDCl3, 400 MHz) δ 7.91-7.97 (m, 2H), 7.72 (s, 1H), 7.03-
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7.09 (m, 2H), 3.91 (s, 3H), 1.31 (s, 12H). 13C NMR (CDCl3, 100 MHz) δ 158.8 (d, J
=
F-C
245.9), 156.0, 139.8, 129.7 (d, J F-C = 3.7), 127.7 (d, J F-C = 8.1), 113.0 (d, J F-C = 21.4), 99.7,
83.3, 38.6, 24.7. HRMS calcd for C16H20BFN2O2 303.1678 (M+H)+, found 303.1672
3-Fluoro-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)picolinonitrile (23). Commercially
available 3-fluoro-4-iodopicolinonitrile 22 (50g, 0.20 mol), 3-(4-boronate 21 (67 g, 0.22 mol),
Pd(OAc)2 (0.91 g, 4.0 mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (Xphos)
(3.8 g, 8.0 mmol) and Cs2CO3 (79 g, 0.24 mol) in ACN (350 mL) and H2O (70 mL) were
refluxed for 3 h under N2. The reaction was cooled to RT, filtered and the filtrate was diluted
with 300 mL EtOAc. The organic layer was washed with water (200 mL) and brine (150 mL),
dried over Na2SO4 and concentrated under reduced pressure to afford crude product. This was
washed with 1:1 EtOAc/hexanes (200 mL) to yield 48 g (80 %) of the title compound as white
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solid. H NMR (CDCl3, 400 MHz) δ 8.30 (d, J=4.9, 1H), 7.87 (d, J=2.9, 1H), 7.40-7.45 (m, 2H),
7.26-7.30 (m, 1H), 7.08-7.15 (m, 2H), 4.05 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 163.8 (d, J F-
C = 248.9), 158.5 (d, J F-C = 268.7), 149.9, 146.6 (d, J F-C = 5.9), 132.7 (d, J F-C = 7.4), 130.7 (d, J
F-C = 7.4), 130.2 (d, J F-C = 7.4), 128.4 (d, J F-C = 3.7), 127.4 (d, J F-C = 2.2), 123.3 (d, J F-C = 16.9),
115.9 (d, J F-C = 22.1), 113.1 (d, J F-C = 4.4), 108.7, 39.4. HRMS calcd for C16H10F2N4 297.0946
(M+H)+, found 297.0941
tert-Butyl(2-((2-cyano-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-3-
yl)oxy)ethyl)carbamate (25).
To 23 (1.00 g, 3.38 mmol) and tert-butyl (2-
hydroxyethyl)carbamate 24 (777 mg, 4.82 mmol) in THF (17 mL) at -20 °C was added sodium
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hydride (228 mg, 5.70 mmol). The mixture was stirred for 3.5 h keeping the bath temperature
between -20 and -10 º C. The mixture was warmed to -5 °C for another 20 min, and was then
quenched with satd. ammonium chloride (5 mL). Water (10 mL) was added and the mixture was
extracted with 2 x 15 mL EtOAc. Extracts were washed with brine, dried with MgSO4, filtered
and concentrated to give a pale orange oil. The oil was diluted with 2 mL of ethyl acetate, and
with stirring an equal volume of isooctane was added resulting in a thick slurry. This was stirred
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overnight and the title compound (1.03 g, 70%) was isolated by filtration as a white powder. H
NMR ( CDCl3, 400 MHz) δ 8.22 (d, J=4.9, 1H), 7.80 (s, 1H), 7.32-7.37 (m, 2H), 7.19 (d, J=4.9,
1H), 6.96-7.03 (m, 2H), 5.02 (br s, 1H), 3.99 (s, 3H), 3.96 (t, J=5.1, 2H), 3.35 (dd, J=5.3, 5.3,
2H), 1.38 (s, 9H). 13C NMR (CDCl3, 100 MHz) δ 162.2 (d, J F-C = 248.1), 155.5, 155.4, 148.6,
145.7, 135.6, 131.9, 129.2 (d, J F-C = 8.1), 128.4, 128.2 (d, J F-C = 2.9), 128.0, 115.3 (d, J
=
F-C
22.1), 115.1, 110.9, 79.1, 73.4, 40.1, 38.9, 27.9. HRMS calcd for C23H24FN5O3 438.1941
(M+H)+, found 438.1936.
9-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-3,4-dihydropyrido[2,3-f][1,4]oxazepin-
5(2H)-one (27). To 7 mL of neat sulfuric acid was added with stirring carbamate 25 (2.00 g,
4.58 mmol). The mixture was heated to 110° C for 10 min, and then cooled to RT. The solution
was then added slowly to ~30 mL of crushed ice and water, and cooled to -10° C. To the cold
mixture was added slowly 5 N NaOH (45 mL) to bring the pH near neutral, followed by 1M pH
7 phosphate buffer to achieve a final pH of 7. The mixture was heated back to 110° C for 3 h,
and then was cooled to RT and stirred for 12 h. The tan solids were collected via filtration and
recrystallized from ethyl acetate/methanol (95:5) to yield 1.3 g (80%) of the title compound as a
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white solid. H NMR (DMSO-d6, 400 MHz) δ 8.52 (t, J=6.0, 1H), 8.31 (d, J=4.7, 1H), 8.01 (s,
1H), 7.34-7.40 (m, 2H), 7.14-7.23 (m, 3H), 3.94 (s, 3H), 3.89 (t, J=5.3, 2H), 3.20 (dd, J=5.3,
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10.9, 2H). 13C NMR (DMSO-d6, 100 MHz) δ 167.86, 161.8 (d, J
= 244.4), 148.0, 147.6,
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F-C
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146.8, 144.9, 135.5, 133.1, 130.0 (d, J F-C = 3.7), 129.5 (d, J F-C = 8.1), 127.3, 115.6 (d, J
=
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22.1), 112.4, 74.6, 39.0, 38.5. HRMS calcd for C18H15FN4O2 339.1179 (M+H)+, found 339.1251
9-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-2,3,4,5-tetrahydropyrido[2,3-
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f][1,4]oxazepine (5). Oxazepinone 27 (300 mg, 0.89 mmol) was added in 50 mg increments
over 10 min to a 50 ºC soln of LAH (1M in THF, 1.5 mL, 1.5 mmol) in THF (50 mL). The
mixture was stirred at 50° C for 1 h. An additional 0.5 mL of the lithium aluminum hydride
solution was added and the mixture stirred at 50° C for an additional hour. The mixture was
cooled to RT and quenched by the sequential addition of 0.1 mL water, 0.1 mL of 15% NaOH,
and 0.3 mL water. The mixture was stirred at RT for 4 days, and then was filtered through a bed
of celite. The filter cake was rinsed with THF (20 mL) and EtOAc (20 mL). The combined
filtrate was treated with decolorizing carbon, filtered, and concentrated to yield 234 mg of a pale
yellow solid. This was dissolved in EtOAc (20 mL) and treated with p-toluenesulfonic acid (285
mg, 1.50 mmol) as a solution in 1 mL methanol. An additional 0.5 mL of methanol was added
with gentle warming while a precipitate began to appear. The mixture was stirred at RT for 12 h
at which point the solids were collected via filtration to provide 280 mg (42%) of the title
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compound as a pale yellow solid. H NMR (MeOH-d4, 400 MHz) δ 8.38 (d, J = 5.9, 1H), 8.35
(s, 1H), 7.63 (d, J = 6.3, 1H), 7.46-7.51 (m, 2H), 7.15-7.22 (m, 2H), 4.87 (s, 2H), 4.44-4.48 (m,
2H), 4.04 (s, 3H), 3.78-3.82 (m, 2H). 13C NMR (MeOH-d4, 100 MHz) δ 164.8(d, J F-C = 247.4),
156.2, 152.5, 145.5, 141.7, 138.9, 137.2, 135.5 (d, J F-C = 8.1), 129.3 (d, J F-C = 3.0), 128.4, 115.9
(d, J F-C = 22.1), 112.4, 70.5, 50.3, 48.0, 40.1. HRMS calcd for C18H17FN4O 325.1459 (M+H)+,
found 325.1454.
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2-Bromo-4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridine (29). To a DMF (5 mL)
solution of commercially available 28 (0.095 g, 0.34 mmol), boronate 21 (0.100 g, 0.335 mmol),
and cesium fluoride (0.106 g, 0.67 mmol) under N2 was added [1,1’-
bis(diphenylphosphino)ferrocene]dichloro-palladium(II) (13 mg, 0.017 mmol). The mixture was
heated to 140o C for 2 h and then cooled to RT. The reaction was diluted with EtOAc (100 mL)
and filtered through a pad of celite and rinsed with another 10 mL of EtOAc. The filtrate was
washed with H2O (20 mL), brine (15 mL), dried over MgSO4 and filtered. The filtrate was
concentrated under reduced pressure to recover a residual oil. This was subjected to silica gel
chromatography using 60% EtOAc/heptanes to recover 100 mg of solid. Crystallization from
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hot EtOAc/heptanes yielded 89 mg of the title compound as a white solid (80% yield). H NMR
(CDCl3, 400 MHz) δ 8.22 (d, J = 5.3, 1H), 7.62 (s, 1H), 7.41-7.45 (m, 2H), 7.39 (m, 1H), 7.06 -
7.10 (m, 3H), 3.99 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ 162.8 (JF-C = 247.4), 150.0, 148.7,
143.8, 142.6, 130.8, 130.2 (JF-C = 8.1), 128.5 (JF-C = 2.9), 126.2, 121.5, 116.6, 115.6 (JF-C=21.4),
39.2. HRMS calcd for C15H11BrFN3 332.0193 (M+H)+, found 332.0197
tert-Butyl
(2-(2-(4-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-2-
oxoethoxy)ethyl)carbamate (31). A suspension of 29 (10.0 g, 30.0 mmol) in toluene (300 mL)
in a three neck flask fitted with a nitrogen inlet and a mechanical stirrer, was cooled to -78o C. n-
BuLi (2.5 M in hexanes,12.0 mL, 30.0 mmol) was added at such a rate to maintain the internal
temperature below -72o C. The reaction was then stirred at -78o C for 30 min and a solution of
tert-butyl 3-oxomorpholine-4-carboxylate (6.1 g, 30.1 mmol) in toluene (50 mL) was added
dropwise keeping the temperature below - 70o C. The reaction was stirred at -78o C for 1 h,
warmed to RT and stirred for 2 h, quenched with a mixture of saturated NH4Cl/EtOAc (1:5, 600
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mL). The organic phase was dried (MgSO4), evaporated to dryness in vacuo and the residue
purified by flash chromatography using a 0 - 100% gradient of ethyl acetate in heptane to yield
3.0 g (22%) of title compound as an off-white foam 1H NMR (CDCl3, 400 MHz ) δ 8.40 (d, J =
5.1, 1H), 7.93 (d, J = 1.6, 1H), 7.66 (s, 1H), 7.34-7.39 (m, 2H), 7.22 (dd, J = 1.9, 5.1, 1H), 6.98-
7.03 (m, 2H), 5.26 (br s, 1H), 5.05 (s, 2H), 3.95 (s, 3H), 3.64 (dd, J = 4.7, 10.2, 2H), 3.36 (dd, J =
5.1, 10.2, 2H), 1.41 (s, 9H). 13C NMR (CDCl3,100 MHz,) δ 197.5, 171.4, 163.2 (JF-C = 248.1),
156.3, 152.6, 149.4, 149.1, 142.6, 131.2, 130.5 (JF-C = 8.1), 129.0 (JF-C = 2.9), 126.4, 120.4,
117.6, 116.0 (JF-C = 22.1), 74.0, 71.1, 40.7, 39.6, 28.7. LCMS m/z 399 (M+H-tBu)
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5-(4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)-3,6-dihydro-2H-1,4-
oxazine (32). A solution of carbamate 31 (3.0 g, 6.6 mmol) in ethanol was treated with 1.0 M
HCl in ethanol (60.0 mL, 60.0 mmol) and the mixture was stirred at RT for 12 h and
concentrated under high pressure to give title compound (3.0 g, >100%). This crude imine was
used without further purification in next step. LCMS m/z 337 (M+H)
(-)-3-(4-(3-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)morpholine (6).
A
solution of 32 (2.7 g, 8.0 mmol) in methanol (20 mL) was cooled to - 40o C, NaBH4 (1.2 g, 32.0
mmol) was added in batches and the mixture stirred at RT for 30 min. The mixture was diluted
with DCM (150 mL) and washed with water, brine, dried (MgSO4) and concentrated in vacuo.
The residue was purified by flash chromatography eluting with EtOAc, 10% EtOAc/EtOAc,
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10% MeOH/DCM to yield 0.5 g (26%) of racemic title compound as colorless foam. H NMR (
CDCl3,400 MHz) δ 8.37 (d, J = 5.1, 1H), 7.58 (s, 1H), 7.37-7.41 (m, 2H), 7.20-7.25 (m, 1H), ),
6.98-7.04 (m, 3H), 3.96 (s, 3H), 3.93-4.00 (m, 2H), 3.86-3.89 (m, 1H), 3.51-3.58 (m, 1H), 3.39-
3.45 (m, 1H), 3.04-3.11 (m, 1H), 2.95-3.0 (m, 1H). LCMS m/z 339 (M+H)
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Chiral HPLC (Chiralcel OJ-H, 10/90 MeOH/CO2 with 0.2% isopropylamine) was used to
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separate the enantiomers. Enantiomer 1 - title compound 6 [α]D25: -29.5 (c = 1.0, MeOH) H
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NMR (CDCl3, 500 MHz) δ 8.42 (d, J = 5.1, 1H), 7.60 (s, 1H), 7.37-7.45 (m , 2H), 7.22 (s ,1H),
6.99-7.07 (m, 3H), 3.95-4.02 (m, 2 H), 3.97 (s, 3H), 3.85 (dd, J = 1.5, 11.2, 1H), 3.53-3.61 (m, 1
H), 3.41-3.46 (m, 1H), 3.07-3.14 (m, 1H), 2.97-3.02 (m, 1 H), 2.2 (br s, 1 H). 13C NMR (CDCl3,
100 MHz) δ 162.4 (d, JF-C = 247.4), 159.5, 149.5, 148.5, 141.5, 130.6, 130.1(JF-C = 8.1), 128.9
(JF-C = 3.7), 121.4, 120.3, 118.0, 115.5 (JF-C = 21.4), 72.2, 67.4, 60.3, 45.7, 39.1. HRMS:
Calculated for C19 H19 F N4 O: 339.1616 (M+H): Found: 339.1618 (M+H). Enantiomer 2:
[α]D24: 34.7 (c = 1.0, MeOH)
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Biology
Cell Free (Biochemical Assay): The CK1δ and CK1ε kinase assays were performed in a 20 µl
final volume in buffer containing 50 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM DTT, 100 µg/mL
BSA, 1 mM TCEP, 1.25% Glycerol, 0.0125% P104, 2.5 mM KCl, 10 µM ATP, and 42 µM
peptide substrate PLSRTLpSVASLPGL (Flotow et al.,1990). The final enzyme concentraionts
were 2 nM for CK1δ and 2.5 nM for Ck1ε. Assays were run in a panel format in the presence of
1 µl of compounds or 4% DMSO. The reactions were incubated for 60 min at 25°C for CK1δ
and 85 min at 25°C for CK1ε, followed by detection with use of 20 µl of Kinase-Glo Plus Assay
reagent according to the manufacturer’s instructions. Luminescent output was measured on the
Perkin-Elmer Envision plate reader.
Whole Cell (Translocation Assay): COS-7 cells were maintained at 37°C in 5% CO2 in
Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. Log-
phase cells were dislodged with 5 minute treatment of TrypLE Express and viable cell count was
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determined with Cedex cell counter. Cells were diluted in DMEM medium to a density of 1.5E5
viable cells/mL in 2/3 of the final volume of final transfected cell mix. Cells were cotransfected
with two plasmid DNAs, mPER3-GFP in pd2EGFP-N1 vector and human CK1 in pcDNA4/hisA
vector (for CK1ε at a ratio of 1:5 respectively for CK1δ at a ratio of 1:11 respectively), using
Lipofectamine 2000 reagent according to the manufacturer's recommendations. The transfection
mix contained approximately 0.83 ug/mL of DNA and 6 uL/mL of Lipofectamine 2000, in a
total of 1/3 of the final transfection volume in Opti-MEM medium. After 20 min at room
temperature, the cell mix was combined with the DNA transfection mix, per manufacturer
instructions. Transfected cell suspension (50 mL) was dispensed per well by multidrop dispenser
into Greiner 384-well Cellcoat (PDL) plates. Compounds were diluted with Opti-MEM I to a 4X
concentration before addition to plated cells. Inhibitors of CKIδ/ε were tested at a 11
concentrations to evaluate their ability to inhibit mPER3-GFP translocation to the nucleus. After
overnight exposure at 37°C in a CO2 incubator, cells were fixed by the addition of 12%
paraformaldehyde in phosphate-buffered saline (PBS) with 20% sucrose to a final concentration
of 4%, and then the cells were incubated for 30 min at room temperature. Fixative was removed,
and cells were washed with PBS and then stained with 0.4 µg/ml Hoechst dye in blocking buffer
containing 4% goat serum and 0.1% TritonX for 1 hour. In some experiments, GFP enhancing
antibodies were used when needed for signal amplification. Cells were washed again with PBS
and stored at 4°C in PBS or scanned immediately with the Cellomics ArrayScan VTI.
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Circadian Timing methods follow those procedures published previously7. All procedures
performed on these animals were in accordance with regulations and established guidelines and
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