Huntington's disease JNK inhibitor optimization

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Article
Lead Optimization Towards Proof of Concept Tools for Huntington?s
Disease Within a 4-(1H-Pyrazol-4-yl)pyrimidine Class of pan-JNK Inhibitors
John Wityak, Kevin F McGee, Michael P Conlon, Ren Hua Song, Bryan C Duffy, Brent Clayton, Michael
Lynch, Gwen Wang, Emily Freeman, James Haber, Douglas B. Kitchen, David D Manning, Jiffry Ismail, Yuri
Khmelnitsky, Peter C Michels, Jeff Webster, Macarena Irigoyen, Michele Luche, Monica Hultman, Mei Bai,
IokTeng D Kuok, Ryan Newell, Marieke Lamers, Philip Leonard, Dawn Yates, Kim Matthews, Lynette Ongeri,
Steve Clifton, Tania Mead, Susan Deupree, Pat Wheelan, Kathyrn A Lyons, Claire Wilson, Alex Kiselyov,
Leticia Toledo-Sherman, Maria Beconi, Ignacio Muñoz-Sanjuan, Jonathan Bard, and Celia Dominguez
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5013598 • Publication Date (Web): 11 Mar 2015
Downloaded from http://pubs.acs.org on March 16, 2015
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Muñoz-Sanjuan, Ignacio; CHDI Foundation Inc.,
Bard, Jonathan; CHDI Foundation Inc.,
Dominguez, Celia; CHDI Management Inc., Advisors to CHDI Foundation
Inc.
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Lead Optimization Towards Proof of Concept Tools for Huntington’s Disease Within a 4-
(1H-Pyrazol-4-yl)pyrimidine Class of pan-JNK Inhibitors
John Wityak,f
Kevin F. McGee,a
Michael P. Conlon,a
Ren Hua Song,a
Bryan C. Duffy,a
Brent
Clayton,a
Michael Lynch,a
Gwen Wang,a
Emily Freeman,a
James Haber,a
Douglas B. Kitchen,a
David D. Manning,a
Jiffry Ismail,a
Yuri Khmelnitsky,a
Peter Michels,a
Jeff Webster,a
Macarena
Irigoyen,a
Michele Luche,a
Monica Hultman,a
Mei Bai,a
IokTeng D. Kuok,a
Ryan Newell,a
Marieke Lamers,b
Philip Leonard,b
Dawn Yates,b
Kim Matthews,b
Lynette Ongeri,b
Steve
Clifton,b
Tania Mead,b
Susan Deupree,c
Pat Wheelan,c
Kathy Lyons,d
Claire Wilson,e
Alex
Kiselyov,f
Leticia Toledo-Sherman,f
Maria Beconi,f
Ignacio Muñoz-Sanjuan,f
Jonathan Bard,f
and Celia Dominguezf
a
Albany Molecular Research Inc. (AMRI), 26 Corporate Circle, Albany, NY 12212-5098,
b
BioFocus Discovery Services, Charles River Laboratories, Chesterford Research Park, CB10
1XL, UK, c
Tandem Labs, 2202 Ellis Road, Durham, NC 27703, d
Kathryn A. Lyons,
Pharmacokinetics Consultant to CHDI, P.O. Box 64, Holland, NY 14080, e
Evotec, 114 Milton
Park, Abingdon, OX14 4SA, UK, f
CHDI Foundation, Inc., 6080 Center Drive, Suite 100, Los
Angeles, CA 90045
Abstract
Through medicinal chemistry lead optimization studies focused on calculated properties and
guided by x-ray crystallography and computational modeling, potent pan-JNK inhibitors were
identified that showed sub-micromolar activity in a cellular assay. Using in vitro ADME
profiling data, 9t was identified as possessing favorable permeability and a low potential for
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efflux, but was rapidly cleared in liver microsomal incubations. In a mouse pharmacokinetics
study, compound 9t was brain penetrant after oral dosing, but exposure was limited by high
plasma clearance. Brain exposure at a level expected to support modulation of a
pharmacodynamic marker in mouse was achieved when the compound was co-administered with
the pan-cytochrome P450 inhibitor 1-aminobenzotriazole.
Introduction
The c-Jun N-terminal serine/threonine protein kinases (JNKs) are a mitogen-activated protein
kinase family that regulates signal transduction events in response to environmental stress. To
date, three distinct jnk genes have been identified (jnk1, jnk2, and jnk3), expressing 10 isoforms
and splice variants of JNK proteins. Whereas JNK1 and JNK2 are ubiquitously expressed, JNK3
is present primarily in brain, with lower expression found in testis, heart, and pancreatic β cells.1
Activation of JNK has been implicated in chronic neurodegenerative disorders such as
Parkinson’s and Alzheimer’s diseases.2,3
Genetic ablation of the murine jnk3 gene resulted in
mice that were resistant to the excitotoxic glutamate-receptor agonist kainic acid, leading to a
reduction in seizure activity.4
Recent reports describe orally bioavailable, ATP-competitive,
JNK inhibitors that have shown beneficial effects in vitro and in vivo.5,6
In addition, JNK
substrate-competitive peptides have shown beneficial effects in models of ischemia and
Alzheimer’s disease.7,8
Of particular interest were reports implicating increased JNK expression and activity in cellular9,
10, 11, 12. 13, 14
and in vivo models15,16
of Huntington’s disease (HD), an autosomal dominant,
progressive neurodegenerative disease that is characterized clinically by motor, cognitive, and
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behavioral deficits. In addition, a role for JNK3 in fast axonal transport (FAT) was demonstrated
in squid axoplasm perfused with mutant huntingtin protein, where JNK3 was shown to
phosphorylate kinesin-1 heavy chain and decrease FAT.17
Due to its reported role in neuronal
cell death, apoptosis, cargo transport, and its restricted tissue distribution, JNK3 is an attractive
target for potential therapeutic intervention in HD. We therefore desired a potent, selective, and
brain penetrant JNK3 inhibitor for proof of concept (POC) studies.
There have been many reports of JNK inhibitors from a wide variety of ATP competitive and
non-competitive chemotypes.18,19
Review of this literature revealed several compounds for
benchmarking efforts, the results of which are summarized in the Supplementary Information
section. Our minimum requirements for a POC compound is a pan-JNK inhibitor having > 100-
fold selectivity against p38 MAPK (itself a partially validated HD target of interest) and
adequate cellular potency and brain exposure to affect a pharmacodynamic (PD) marker.
Potential choices considered for a cellular assay were inhibition of phosphorylation of c-Jun or
ATF-2 substrates with reduction of phosphorylated c-Jun (p-c-Jun), or ATF-2 as the PD marker.
In addition, a POC compound would need to have adequate ADME properties, as determined in
solubility, permeability/efflux, and microsomal stability assays. Attesting to the challenge, none
of the benchmark compounds were judged suitable for advancement into mouse HD models.
Concurrent with these benchmarking studies, and to further facilitate identification of novel
starting points, a docking-based virtual screen was conducted based on the x-ray crystal structure
of JNK3 in complex with an imidazole-pyrimidine inhibitor (1pmq) from the Protein Data Bank
(PDB, www.rcsb.org). A compound library of approximately two hundred thousand compounds
from Asinex’s “privileged” collections was docked against a protein grid generated from the
1pmq structure. Of the 1100 virtual hits selected for wet screening a set of approximately 90
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were confirmed as actual hits. Among these, pyrazole 1 was identified as an attractive starting
point for medicinal chemistry and was advanced into hit to lead studies based upon favorable
JNK3 potency, chemical tractability, and a small base of published SAR including potent activity
in a cellular p-c-Jun assay.20
Profiling studies revealed 1 to be a pan-JNK inhibitor with
favorable permeability, but suffering a high rate of metabolism in mouse liver microsomes
(mLM) and poor selectivity against p38α.
Table 1. Activity Profile of Pyrazole 1.
JNK3
IC50 ± SD
(nM)
JNK1
IC50 ± SD
(nM)
JNK2
IC50 ± SD
(nM)
p38αααα IC50
± SD
(nM)
Caco-2
Papp A-B
(nm·sec-1
)
Caco-2
Papp B-A
(nm·sec-1
)
mLM Clint
(µµµµL/min/mg)
hLM Clint
(µµµµL/min/mg)
433 ± 208 737 ± 281 161 ± 77 24 ± 18 467 242 81.8 < 23.1
The x-ray crystal structure of human JNK3 in complex with 1 at 2.3 Å resolution confirmed the
mode of binding indicated by the docking studies, showing a single hydrogen bond between the
pyridine ring to the backbone NH of Met149 in the linker region of the protein (Figure 1). As
has been observed in several JNK3 structures, including 1pmq, a water molecule mediates a
hydrogen bond interaction between the pyrazole ring and the charged terminal amino group of
Lys93. The chlorophenyl substituent occupies the hydrophobic region I and appeared to cause
an induced-fit movement of the hydrophobic side chain of Met146 towards the back of the
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binding pocket, as compared to its position in the structure of JNK3 in complex with adenosine.
This movement further causes a perturbation in the side chain position of Ile124. We noted that
in the structures of JNK3 complexes in the PDB that the hydrophobic side chain of Met146 is
observed to occupy two predominant rotameric states depending of the hydrophobic nature of the
bound inhibitor.21,22
We postulated that inhibitors bearing a hydrophobic group such as in
pyrazole 1 may induce the side chain of Met146 to adopt the rotameric state mimicking the
corresponding pocket of p38, which could explain the lack of selectivity observed against p38.
An analysis and superposition of crystal structures of p38 and JNK3 with compounds of similar
chemotypes containing groups occupying the hydrophobic pocket reveals such similarity23,24
.
This knowledge base informed our SAR exploration around this chemotype and resulted in the
design of compounds that would induce the engagement of the Met146 side chain as observed in
the adenosine structure, thus blocking the hydrophobic pocket of hydrophobic region 1. Docking
grids were prepared for JNK1, JNK2, JNK3, and p38; docking and scoring was used as a tool to
guide the medicinal chemistry efforts of the group.
A B
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Figure 1. 2Fo-Fc electron density map contoured at 1σ showing A) pyrazole 1 binding in the
JNK3 active site, B) a three dimensional schematic depicting the binding of pyrazole 1 (green) to
JNK3 (yellow) overlapped with the binding of adenosine in the JNK3 active site (aqua)
highlighting the alternate positions of Met146 and its effect of Ile124.
Synthesis of inhibitors
We chose to focus an SAR study around the 4-(pyrazol-4-yl)pyrimidine scaffold. Synthesis of
these pyrazoles was readily accomplished using a variation on the Knorr pyrazole synthesis
illustrated in Scheme 1. Alkylation of 4-methylpyrimidine 2 with ester 3 in the presence of
sodium hexamethyldisilazide gave a mixture of keto and enol tautomers 4 and 4’. Reaction with
dimethylformamide dimethylacetal (DMF-DMA) then afforded the intermediate enaminone,
which was followed by cyclization in the presence of hydrazine or a mono-substituted hydrazine
to give pyrazole 5. Oxidation using mCPBA then provided sulfone 6. For compounds in which
R2
is a hydrogen atom, the introduction of a tetrahydropyran (THP) protecting group at this
stage, giving 7, avoids the large excess of amine necessary in the subsequent amination step.
Protection in this way suppresses a competing dimerization process which was prominent with
poorly nucleophilic amines (NH2R1
). Microwave assisted amination to provide 8 followed by
THP removal under acidic conditions affords 9. It should be noted that in every instance, the
regiochemistry of cyclization with a mono-substituted hydrazine was confirmed as the 2,3-
regioisomer (as depicted in 7-9) through Heteronuclear Multiple Bond Correlation (HMBC) and
NOE NMR experiments (see Supporting Information).
Scheme 1. General synthesis of pyrazolesa
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a
Reagents and conditions: (a) NaHMDS, THF, 60 °C, 4 d; (b) DMF-DMA, tol, 110 °C, 6 h, then
NH2NHR2
, RT, 16 h; (c) mCPBA, CH2Cl2, 0 °C, 16 h; (d) 3,4-dihydro-2H-pyran, p-TsOH, RT, 10 min;
(e) NH2R1
, dioxane, MW, 160 °C, 1 h; (f) HCl-dioxane, MeOH, RT, 2 h.
The synthesis of 3-trifluoromethyl pyrazole 9o required an alternative synthetic route due to the
poor reactivity of enol ether 10 (Scheme 2). Enol-ether 11a, prepared by standard methods, was
treated with triethyl orthoformate to provide enol ether 12. Cyclization with methyl hydrazine
provided pyrazole 13, which was converted to 2-thiomethylpyrimidine 14 following a two-step
cyclization procedure. Oxidation to the activated sulfone 15, followed by displacement with
excess trans-4-aminocyclohexanol gave 9o.
Scheme 2. Synthesis of 3-trifluoromethylpyrazole 9oa
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a
Reagents and conditions: (a) HC(OEt)3, Ac2O, reflux, 19 h; (b) NH2NHCH3, THF, -10 °C, 1.25 h; (c)
DMF-DMA, tol, 110 °C, 20 h; (d) methyl carbamimidothioate, NaOMe, i-PrOH, 12 h; (e) mCPBA,
CH2Cl2, 0 °C, 16 h; (f) NH2R1
, dioxane, 160 °C, 1 h.
The synthesis of 5-chloropyrimidine 9mm is shown in Scheme 3. Cyclization of ethyl
acetoacetate 11b with S-methylisothiouronium sulfate gave pyrimidone 16 in good yield.
Chlorination using sulfuryl chloride provided chloride 17, which was further chlorinated using
phosphorus oxychloride to give dichloride 18. Regioselective hydrogenolysis of the 4-chloro-
substituent led to 5-chloropyrimidine 19. It should be noted that chloride 19 was quite volatile,
and caution must be exercised during solvent removal. Reaction of 19 with N-methoxy-N-
methyl-1-(trifluoromethyl)cyclopropanecarboxamide then gave ketone 20. Pyrazole formation
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using DMF•DMA followed by hydrazine provided pyrazole 21 in poor isolated yield. It was
likely that the presence of the chloride on the pyrimidine modulated reactivity. Several
subsequent steps required more forcing conditions, resulting in lower yields. Oxidization to
methylsulfone 22 using mCPBA, THP protection to give pyrazole 23, and methylsulfone
displacement using 4,4-difluorocyclohexylamine under microwave assisted heating afforded 24.
Deprotection under standard conditions completed the synthesis of 9mm in low overall yield.
Scheme 3. Synthesis of 5-chloropyrimidine 9mma
a
Reagents and conditions: (a) S-methyl isothiouronium sulfate, Na2CO3, H2O, RT, 16 h, 43%; (b) SO2Cl2,
FeCl3, AcOH, Ac2O, 100 °C, 36 h, 69%; (c) POCl3, DMA, 115 °C, 15 h, 75%; (d) H2, Pd-C, NaOH, H2O,
RT, 24 h, 75%; (e) N-methoxy-N-methyl-1-(trifluoromethyl)cyclopropanecarboxamide, NaHMDS, THF,
0 °C-RT, 16 h, 79%; (f) DMF-DMA, MeOH, 110 °C, 6 h; then NH2NH2, THF, RT, 16 h, 5%; (g)
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mCPBA, CH2Cl2, RT, 16 h, 49%; (h) 3,4-dihydro-2H-pyran, p-TsOH, 0 °C, 5 min, 99%; (i) 4,4-
difluorocyclohexylamine, DMSO, Et3N, MW, 130 °C, 3 h, 17%; (j) HCl-dioxane, MeOH, RT, 2 d, 85%.
Preparation of pyrimidine 25 is shown in Scheme 4. Reaction of cyclopropanecarboxylic acid 26
with N-methoxy methylamine afforded Wienreb amide 27, which was subsequently used to
acylate 2-chloro-4-methylpyridine to give 28 in good yield. Pyrazole formation to provide 29 and
protection as the THP to give 30 was followed by Buchwald–Hartwig reaction with 3,3-
difluorocyclobutylamine (31), giving aminopyridine 32. Removal of the THP under standard
conditions then afforded 25.
Scheme 4. Synthesis of 2-aminopyridine 25a
a
Reagents and conditions: (a) NH(OCH3)CH3 • HCl, EDC, HOBt, Et3N, CH2Cl2, RT, 90%; (b) LHMDS,
2-chloro-4-methylpyridine, THF, 55%; (c) DMF-DMA, MeOH, 110 °C, 6 h; then NH2NH2, THF, RT, 16
h, 68%; (d) 3,4-dihydro-2H-pyran, p-TsOH, 0 °C, 5 min, 95%; (e) 31, (tBu3P)2Pd(0), NaOtBu, dioxane,
130 °C, 16 h; then HCl, i-PrOH, RT, 2 d, 16%.
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Results and Discussion
Based upon the binding mode of 1, we expected to gain potency through the addition of an amine
containing substituent at the pyrimidine 2-position. This would complete the double hinge
hydrogen bond interaction with Met149 and would introduce the possibly of gaining favorable
interactions with residues in the sugar region. Our SAR investigation began with preparation of
a series of analogs in which the R3
group was fixed as 4-chlorophenyl with the incorporation of a
small set of amines at the 2-position (Table 2). The incorporation of a 2-amino moiety on the
pyrimidine resulted in a one to two order of magnitude improvement in JNK3 activity relative to
1, with 4-hydroxycyclohexylamine 9c showing potent activity but little selectivity against JNK1,
JNK2, or p38α. The lack of selectivity against JNK1 and JNK2 was expected, as the ATP
binding site of the JNK isoforms are highly conserved (98% homology), with the only residue
differences being Met115 in JNK1/3 versus Leu77 in JNK2, and Leu144 in JNK2/3 versus
Ile106 in JNK1. Several groups have used these differences to gain some degree of selectivity
among the JNK isoforms.25,26,27,28,29
The lack of selectivity against p38 was also predicted since
the chlorophenyl substituent had been preserved and was expected to occupy the hydrophobic
region I, as depicted in Figure 1, in both the JNKs and p38.
Table 2. JNK and p38α Potency of (4-Chlorophenyl)pyrazol-3-yl Derivativesa
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Cmpd R1
JNK3
IC50 ± SD
(nM)
JNK1
IC50 ± SD
(nM)
JNK2
IC50 ± SD
(nM)
p38αααα IC50
± SD
(nM)
9a 36 ± 9.3 119 ± 15.5 26 ± 2.1 6.2 ± 1.6
9b 81 ± 18 269 ± 50.2 62 ± 15 113 ± 74.1
9c 5.4 ± 1.7 26 ± 13 15 ± 2.8 3.2 ± 1.5
9d 14 ± 6.2 32 ± 9.3 19 ± 2.6 14 ± 5.5
a
Values accompanied by standard deviation were averaged from at least two independent experiments.
The next set of analogs kept the trans-4-hydroxycyclohexylamine at R1
constant and probed the
R3
position (Table 3). Most significantly, selectivity against p38α was achieved by replacing the
3-aryl moiety with saturated groups. tert-Butyl derivative 9e was the most potent compound of
this set, showing an IC50 value of 13 nM. Compounds 9f-h were also potent JNK3 inhibitors,
affording inhibition constants of approximately 20 nM. Cyclopropyl 9i gave up 2-fold potency
to this group, with tetrahydropyran 9j another 2-fold less potent. The importance of a lipophilic
substituent at R3
to potency was further established by the order of magnitude loss seen with
pyrazole 9k. As expected, little selectivity was observed against the JNK1 or JNK2 isoforms.
Table 3. JNK and p38α Potency of trans-4-Hydroxycyclohexylamine Derivativesa
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Cmpd R3
JNK3
IC50 ± SD
(nM)
JNK1
IC50 ± SD
(nM)
JNK2
IC50 ± SD
(nM)
p38αααα IC50
± SD
(nM)
9e 13 ± 1.0 16 ± 3.5 17 ± 0.0 > 10000
9f 16 ± 3.5 22 ± 3.5 24 ± 7.8 5560
9g 22 ± 4.6 24 ± 0.71 28 ± 4.9 > 10000
9h 26 ± 1.1 22 ± 2.8 28 ± 1.4
5940 ±
1420
9i 42 ± 4.9 26 ± 2.8 43 ± 5.6 > 10000
9j 73 ± 28 33 ± 8.1 51 ± 10
7450 ±
2295
9k H 487 ± 57.8 266 ± 99.7 550 ± 106 NTb
a
b
NT indicates not tested.
Noting the generally favorable selectivity against p38α for this set, we obtained a co-
structure of JNK3 with pyrazole 9e at 2.3 Å resolution (Figure 2). Inspection of the structure
revealed the expected double hinge interaction of the N1 pyrimidine and NH-linker moiety with
the carbonyl and NH of Met149, and a water-mediated hydrogen bonding interaction with the
side chain of Lys93. The cyclohexyl ring pointed towards the solvent interphase and occupied
the hydrophobic region II, with its hydroxyl substituent forming a hydrogen bond to Gln155.
Unexpectedly, the pyrazole had rotated about the pyrazole pyrimidine bond, bringing the tert-
butyl group and the cyclohexanol in close proximity due to an apparent favourable
intramolecular hydrophobic contact. In this “horseshoe” conformation the tert-butyl group
occupied the sugar pocket and made hydrophobic interactions with Val78, Ala91, and Leu206.
Interestingly, since no portion of compound 9e occupied the hydrophobic region I, this allowed
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the side chain of Met146 to populate its “natural” adenosine-bound rotamer, and thus occupy the
hydrophobic region I, leading to favorable selectivity against p38. The relatively weak JNK3
activity of 9k, which is devoid of a hydrophobic moiety at R3
, highlights the importance of the
placement of a hydrophobic moiety in the ribose pocket, aiding intramolecular stabilization of
the horseshoe conformation.
A B
Figure 2. 2Fo-Fc electron density map contoured at 1σ showing A) 9e binding in the JNK3
active site, B) a three dimensional schematic showing the binding of 9e (pink) to JNK3 (aqua)
overlapped with the binding of 1 (green) in the JNK3 active site (yellow).
Compounds 9e and 9j were taken into in vitro ADME assays (Table 4). Both compounds
showed low solubility, were rapidly metabolized in mLM, and had good stability in human liver
microsomal incubations (hLM). Permeability and P-gp mediated efflux were determined in an
MDCK-MDR1 transfected cell line. Tetrahydropyran 9e displayed good permeability but had
moderate to high P-gp efflux, whereas 9j showed low permeability accompanied by moderate to
high efflux.
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Table 4. Results of In Vitro ADME Profiling
Cmpd
No.
JNK3
IC50
(nM)
Aq. Sol.
(mg/mL)
mLM Clint
(µµµµL/min/
mg)
hLM Clint
(µµµµL/min/
mg)
MDCK
Papp A-B
(nm-s-1
)
MDCK
-MDR1
EER
9e 13 0.028 498.8 < 23.1 253 9
9j 73 0.037 39.8 < 23.1 38 14
Our strategy to improve efflux to an acceptable level was to reduce the number of hydrogen bond
donors. Tactics included alkylation of the pyrazole ring and replacement of the cyclohexanol
moiety, resulting in the compounds of Table 5. Alkylation of the pyrazole as in compounds 9l-o
in all cases resulted in high permeability and a low effective efflux ratio (EER), but other than
for 9m, loss of JNK3 potency was observed. This may be due to the loss of a productive
interaction of the pyrazole moiety with Lys93, or the introduction of a possibly unfavorable
interaction as in the case of trifluoroethyl 9n. Replacement of the cyclohexanol moiety with
groups that did not bear a hydrogen bond donor as in 9p-u also had acceptable permeability, low
EER, and retained acceptable JNK3 potency, with the one notable exception being 3-
methoxypropyl 9s. The permeability and efflux data indicated that efflux was not the result of
specific recognition by the transporter of the pyrazole’s free NH and suggest that P-gp efflux
may be avoided in this series by simply keeping the hydrogen bond donor count to 2 or less.
With regard to JNK potency these results appeared to point to the need for a cyclic or α-
branched alkyl substituent such as isopropyl to maximize hydrophobic interactions with residues
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in the sugar pocket. Of particular interest was pyrimidine 33, which was not a JNK3 inhibitor, a
surprising result, given the potency of pyridine 1. Docking studies support these results, as
docking of compound 33 with grids derived from the X-ray structure of JNK3 in complex with
either 9e or 1 did not generate acceptable binding poses. We propose that in the absence of an
alkyl amino group at the pyrimidine 2-position, internal energy stabilization of the isopropyl
group is no longer present, and thus no rotamer of the pyrazole-pyrimidine group is favored.
Table 5. Permeability and Efflux Profiling of Selected JNK Inhibitorsa
9
N N
R3
N
N
N
H
R1
R2
33
HN N
N
N
Cmpd R1
R2
R3
JNK3
IC50 ±
SD (nM)
Aq. Sol.
(mg/mL)
MDCK
Papp A-B
(nm-s-1
)
MDCK-
MDR1
EER
9l 113 ± 16 0.040 428 0.5
9m 24 ± 6.2 0.043 466 1.5
9n 339 ± 98 0.060 291 0.7
9o CH3 CF3 219 ± 14 0.058 494 1.6
9p H 110 ± 16 0.031 203 1.9
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9q H 24 ± 4.0 0.0026 107 2.2
9r H 13 ± 0.0 0.034 321 1.2
9s H 493 ± 45 0.036 NTb
NT
9t H 94 ± 22 0.0039 265 1.8
9u H 16 ± 2.5 0.018 166 5.3
33 --- --- --- > 100000 0.018 NT NT
a
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NT indicates not tested.
Another round of SAR was carried out to further investigate the R1
position while keeping the R3
position as either 1-methylcyclopropyl or 1-trifluoromethylcyclopropyl. As all compounds thus
far showed poor metabolic stability, we hoped to identify compounds having improved stability
in microsomal incubations. Bearing in mind our desire to attain compounds with blood-brain
barrier permeability, we attempted to keep the hydrogen bond donor count to two or less,
maintain the polar surface area (PSA) to less than 90, and hold the cLogP in the range of 2-4.
Keeping R3
as 1-trifluoromethylcyclopropyl, compounds 9v-y ranged in activity, with IC50
values of 66 nM (cyclopentyl 9w) to 237 nM (cyclopropylmethyl 9y). None of these analogs
was as potent as cyclohexyl 9q (IC50 = 24 nM), whose potency was surprising when compared to
9f or 9g, both of which can make an additional hydrogen bonding interaction of the hydroxyl
moiety with the side chain of Gln155. These findings suggest that these additional interactions
come at some cost, presumably from desolvation. The potencies of cyclic ethers 9z-bb were also
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unexpected when compared to 9q and 9w; it was postulated that these ether moieties might also
interact with Gln155, which can flip to present either a hydrogen bond donor or acceptor to the
ligand. The complete lack of potency of 9cc was especially surprising. Docking studies of 9cc
with the grid generated from the crystal structure of JNK3 with structurally similar 9e generated
several favorable ‘horseshoe-shaped” ligand binding poses showing good overlay with the x-ray
position and binding interactions of 9e. The data in table 6 shows a correlation of JNK3 potency
with the lipophilicity of R1
; we speculate that the oxetanyl moiety was not sufficiently lipophilic
to provide the necessary hydrophobic shielding to drive a productive double hinge hydrogen
bonding interaction. Next investigated were the 4- and 3-piperidines 9dd-hh, however, none of
these showed potent JNK3 activity, with the most potent compound, trifluoroethyl 9ff, having
attenuated basicity, suggesting that basicity in this region is not well-tolerated. Replacement of
the monocyclic R1
with a much larger multi-ring substituted aniline moiety as in 9ii showed
potency consistent with literature reports;5
however, ligand efficiency, PSA, and solubility
suffered relative to the other potent compounds from this series. The isomeric tertiary alcohols
9jj and 9kk were approximately 4-fold less potent than 9g. The final three compounds from
Table 6 examined the effect of fluorination of the cyclobutyl moiety (9ll), comparison with its
pyridinyl analog (25), and the effect of chlorination at the 5-position of the pyrimidine ring
(9mm). Fluorination of the cyclobutyl resulted in a 10-fold loss of potency when compared to
9v. Comparison of pyrimidine 9ll with pyridine 25 demonstrated that this change results in no
significant difference with respect to JNK3 potency. In addition, both compounds showed
similar selectivity (10-fold) against p38α (data not shown). Literature precedent indicated that
incorporation of a 5’-chloro group on a related pyrimidine scaffold helps to improve JNK3
potency by ca. 2 fold.30
A 5-chlorinated analog of 9t (9mm) was prepared in an attempt to
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encourage the “horseshoe” conformation and improve activity, but this instead resulted in a 4-
fold potency loss, which may be explained by the chloro-substituent obstructing co-planarity of
the pyrimidine and pyrazole rings. Metabolic stability of this series continued to be poor, with
typical half-life values of < 20 min in mouse liver microsomes.
Table 6. JNK3 Potency of 3-Cyclopropylpyrazolesa
Cmpd R1
R4
R5
JNK3
IC50 ±
SD (nM)
Aq. Sol.
(mg/mL)
PSA cLogP
9v H CF3 90 ± 29 0.0058 66 2.6
9w H CF3 66 ± 5.8 0.0032 66 3.2
9x H CF3 111 ± 23 0.0028 66 4.3
9y H CH3 237 ± 35 0.029 66 2.8
9z H CF3 120 ± 30 76 1.3
9aa H CF3 151 ± 35 0.018 76 2.0
9bb H CF3
742 ±
123
0.046 76 1.5
9cc H CF3 > 100000 0.034 63 1.9
9dd H CF3
2632 ±
486
0.054 70 2.3
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9ee H CH3
1890 ±
416
0.038 78 1.5
9ff H CF3 298 ± 69 0.016 70 2.6
9gg H CF3
19118 ±
1427
0.045 78 2.0
9hh H CF3
2398 ±
350
0.048 78 2.0
9ii H CF3 109 ± 12 0.0019 110 3.8
9jj H CF3 77 ± 27 0.059 87 2.2
9kk H CF3 81 ± 28 0.0010 87 2.2
9ll H CF3
793 ±
100
0.007 66 2.0
25a --- ---
964 ±
157
0.015 54 2.4
25b S
N
--- --- 78 ± 38 0.0002 95 4.6
9mm Cl CF3 441 ± 92 66 3.6
a
Activity in cellular assays
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In a rat model of HD, increased phosphorylation of c-Jun, accompanied by an increase in protein
aggregates and a loss of DARPP-32 immunoreactivity was observed after lentiviral-mediated
expression of htt171-82Q.31
Presumably, a reduction in the activation of c-Jun would result in
neuroprotection and phenotypic improvements. To support compound prioritization for
subsequent in vivo testing and to aid in estimating the dose required to modulate a p-c-Jun
pharmacodynamic endpoint, compounds were tested in a cellular assay to assess JNK-mediated
phosphorylation of c-Jun. The cell-based assay was conducted at Life Technologies (Carlsbad,
CA) using their LanthaScreen technology. In this assay TNF-α was used to stimulate JNK
activation in HeLa cells stably expressing GFP-c-Jun 1–79. Phosphorylation was determined by
measuring the TR-FRET signal between a terbium-labeled anti-p-c-Jun antibody and GFP after
lysis of the cells. Given the challenge of achieving compound exposure in the brain necessary to
modulate p-c-Jun levels in vivo, we held the assumption that a sub-500 nanomolar IC50 against
cellular c-Jun activation would be desirable. As shown in Table 7, despite nanomolar
biochemical potency of 9c, and double-digit nanomolar potency for many of the other
compounds, the cellular potency of these inhibitors was disappointing. Notable was the potency
of 25b (IC50 = 0.5 µM), which was the only aminopyridine in this set. This finding prompted
testing of several other aminopyridines from our collection; however, none showed sub-
micromolar potency in the c-Jun assay (data not shown). The activity of SP600125 (34)32
is
included in Table 7 as an assay standard; its potency was consistent with prior literature values.
Difficulty in efficiently translating the biochemical potency of JNK inhibitors to potency against
c-Jun phosphorylation in a cellular context is well-documented. Upon exposure to activating
stimuli, c-Jun is rapidly phosphorylated. In addition, the c-jun transcription response element is
constitutively occupied and this phosphorylation occurs while the proteins are bound to the c-jun
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promoter, activating transcription, and leading to c-Jun induction.33
Conversely, there have been
several JNK inhibitors with reported sub-micromolar cell-based activity; however, when we
attempted to recapitulate these results in either the Life Technologies assay, or in an internally
developed NIH3T3-cell-based c-Jun MSD assay (data not shown), we were unable to reasonably
match the published potencies. It is perhaps of interest to note that the potency of a recently
reported covalent inhibitor JNK-IN-11 (35)34
was an exception. In the Life Technologies assay,
35 showed an IC50 of 0.1 uM and had similar translation of biochemical to cellular potency as
some of the other inhibitors studied. Unfortunately, this compound potently inhibited a number
of other kinases, including p38, when tested at 1 µM concentration in selectivity profiling
conducted at Cerep (see Supporting Information). As stated earlier, this activity would be a
potential confound in interpretation of efficacy results. In addition, it cannot be ruled out that the
potent inhibition of p-c-Jun observed may be the result of off-target activity against a variety of
kinase families.
Due in part to the cellular c-Jun results, compounds were also tested in a second cellular assay,
the LPS-induced TNF-α secretion assay in PBMC conducted at Cerep (Celle l'Evescaul, France).
It is known that LPS activates the JNKs to induce TNF-α production,35
and that this can be
suppressed in macrophages by 34.36
The compounds in Table 7 showed markedly better potency
and translation against this readout. Dexamethasone is included as the assay positive standard,
and its potency was as expected. Interestingly, the dual JNK/p38 inhibitor 9c was approximately
equipotent with JNK inhibitor 9r. The expectation was that significantly improved activity
might have resulted from potent inhibition of these two MAPK families. Aminopyridine 25b
was also one of the more potent compounds in this assay. These results, while encouraging, do
little to advance JNK inhibition as an HD therapeutic strategy, but may become relevant if a
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connection between elevated TNF-α and HD disease progression can be established. At present
they demonstrate that the compounds can cross cell membranes and modulate functional activity
within a cell at a concentration that might reasonably be achievable in brain.
Table 7. Cellular Activity of Selected JNK Inhibitorsa
Cmpd
JNK3 IC50
(nM)
p38αααα
IC50
(nM)
p-c-Jun
IC50 (µµµµM)
Ratio p-c-
Jun/JNK3
LPS-
TNF-αααα
IC50 (µµµµM)
Ratio
TNF-
αααα/JNK3
9c 5 16 8.0 1600 0.081 16
9r 13 4574 2.5 192 0.093 7
9f 16 5559 2.2 138 0.45 28
9m 24 3052 1.6 67 5.8 242
9q 24 >10000 2.0 83 0.89 37
25b 78 NT 0.51 7 0.15 2
9t 94 6084 > 10 NA 0.18 2
9ii 109 >10000 3.0 28 0.18 2
9x 111 >10000 2.7 24 0.35 3
9z 121 >10000 > 10 NA 1.5 12
34 59 >10000 2.8 48 0.89 15
35 0.5b
NT 0.1 200 NT NA
dexamethasone NT NT NT --- 0.0051 ---
a
NT indicates not tested. NA indicates not calculated.
b
Value from Ref 33.
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ADME-PK
In preparation for planned in vivo pharmacodynamic evaluation in mice of a p-c-Jun biomarker,
several of the compounds from Tables 3, 5, and 6 were profiled for microsomal stability;
however, despite the structural diversity queried, all showed rapid rates of metabolism in both
mouse and rat liver microsomes (data not shown). From these studies 9t emerged as an early
example of a compound having met most of our criteria for progression into further studies. The
JNK3 potency (IC50 = 94 nM), cellular potency (IC50 = 180 nM, LPS-TNF-α assay), cellular
permeability (MDCK-WT Papp A-B = 265 nm-s-1
), and P-gp-mediated efflux (EER = 1.8) of 9t
were acceptable. While its stability in mLM was poor/moderate (Clint = 142 mL/min.mg), its
rate of disappearance was slower than most compounds tested from this series. When assayed at
10 µM in receptor panels of 144 diverse and 75 kinase targets, 9t showed > 50% of control
specific binding in 9 out of the 219 assays: adenosine A3 (51%), Na+
channel (83%),
norepinephrine transporter (92%), CDC2/CDK1 (79%), CDK2 (77%), CDK5 (72%), GSK3β
(82%), HGK (54%), and JNK1 (97%). The full report can be found in the Supporting
Information section.
As a follow-up to the receptor profiling study, IC50 values were determined against CDK5 and
GSK3β, since these are targets implicated in HD (Table 8). While activity against these would
not affect a p-c-Jun readout, they might impact an efficacy readout in an HD model. The data
suggests that selectivity against these kinases, which have relatively close homology to JNK3,
may be difficult to achieve with this chemotype, although the selectivity observed for 9m shows
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that > 50-fold is possible. This level of selectivity was possibly driven by favorable hydrophobic
interactions of the R2
isopropyl moiety with Leu206 in JNK3 and, assuming a similar binding
conformation with GSK3β and CDK5, unfavorable, or less favorable, interactions with Cys199
(GSK3β) and Ala143 (CDK5).
Table 8. CDK5 and GSK3β Inhibition of Selected JNK3 Inhibitors
Cmpd
JNK3
IC50 (nM)
CDK5
IC50 (nM)
GSK3ββββ
IC50 (nM)
9j 73 1100 260
9u 16 87 280
9m 24 1400 1800
9t 88 430 380
In a mouse BBB penetration study, 9t was dosed as an i.v. bolus in mouse at 5 mpk. It showed a
brain to plasma ratio of 1.8 : 1, reaching a Cmax in brain of 1967 ng-eq/mg at a Tmax of 0.25 h.
Clearance of the compound was rapid; the half-life was approximately 15 min. By 2 hours the
brain concentration had fallen to 100 ng-eq/mg. Considering the high rate of microsomal
metabolism noted for the series, attempts were made to identify specific site(s) of metabolism. A
metabolite identification study of 9t in mLM revealed extensive metabolism involving both the
R1
cyclohexyl and R3
cyclopropyl groups. While it was not possible to quantify the relative
abundance of metabolites due to unknown ionization efficiencies of the various species, clear
evidence was obtained demonstrating R1
and R3
hydroxylation(s), dehydration(s), and
dealkylation to the 2-aminopyrimidine (structures of these putative metabolites and their
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respective extracted single ion mass chromatograms can be found in the Supporting Information
section).
Thus far, the incorporation of hydrophobic groups at both R1
and R3
necessary to achieve a level
of biochemical potency that might translate into sub-micromolar cellular potency was
incompatible with metabolic stability in mouse. Anticipating that metabolism issues might
dictate the need to proceed into an in vivo pharmacodynamic proof of concept study with a high
clearance compound, we undertook an in vitro cytochrome P450-mediated metabolism
suppression study of 9t in mLM. Using the cytochrome P450 (CYP450) inhibitors listed in Table
9, the objective was to identify which CYP450 isoforms were principally responsible for
metabolism as evidenced by reduction in the rate of clearance. Table 9 lists the specific
inhibitors of human CYP450 isoforms studied and the results are summarized in Figure 3. The
results from co-administration of each inhibitor with 9t suggest that human CYP450-2B6-like,
human CYP450-2C19-like, and human CYP450-3A4-like activities are mainly responsible for
its metabolism in mLM. It is important to caution that the CYP450 isoforms in mouse are not
well-characterized, and that the precise CYP450 isoforms being inhibited in mLM by this panel
of human CYP450 inhibitors is unknown. The CYP450-3A4/5 inhibitor ketoconazole was
particularly effective at slowing the rate of clearance when dosed at both 3x and 30x its Ki. In
addition, pan-CYP450 inhibition using SKF-525a was more effective than inhibition of any
individual isoform; it strongly suppressed the rate of metabolism when co-administered at 3x Ki.
Table 9. Human CYP450 Inhibitors for Metabolism Suppression Study
Human CYP450
Isoform
Selective
Inhibitor
Ki (µM)
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1A2 α-Naphthoflavone 0.01
2B6 Clopidogrel 0.5
2D6 Quinidine 0.2
2C8 Montelukast 0.07
2C9 Sulfaphenazole 0.3
2C19 Ticlopidine 1.2
3A4/5 Ketoconazole 0.09
Figure 3. Suppression of metabolism of 9t in mLM incubations by co-administration with
known inhibitors of human CYP450 isoforms.
These in vitro findings were then extended demonstrating that in vivo suppression of CYP450-
mediated metabolism of 9t could result in enhanced brain exposure after oral dosing in mice.
Thus, 9t was dosed orally at 30 mpk to one cohort, a second cohort was co-administered 30 mpk
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9t with 50 mpk orally of the CYP450-3A4 inhibitor ketoconazole, and a third group was co-
administered 30 mpk 9t and 30 mpk orally of 1-aminobenzotriazole (1-ABT), a potent pan-
CYP450 inhibitor (Figure 4). The brain concentration at 8 h was approximately 100-fold
improved with 1-ABT co-administration relative to administration of 9t alone. The
concentration of 9t in brain was 7.3 µM by 0.5 h post-dose and remained above this level
through 4 h, reaching a Cmax of 11.4 µM at 1 h, and then falling to 4.8 µM at 8 h. Working back
from this exposure level and assuming that a brain concentration would likely need to be at some
multiple of the cellular IC50 value, we felt confident that if elevated levels of TNF-α could be
correlated with HD disease progression, 9t would have the potency necessary to go forward as a
PD tool.
It is important to note that co-administration with the CYP450 inhibitor did not increase BBB
permeability or alter the brain to plasma ratio; it only served to decrease clearance, thus
improving exposure to all tissues. The plasma exposure of 9t was also similarly improved (not
shown). For a highly metabolized compound such as 9t, suppression of metabolism 1) improved
exposure to levels that might allow modulation of a PD marker in brain; 2) ensures that PD
activity can be attributed to the parent compound and directly linked to JNK inhibition; 3) since
1-ABT has been shown to be relatively non-toxic in a rat toxicology study,37
it may permit a
highly metabolized compound to enter into a mouse HD efficacy study requiring chronic dosing.
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Figure 4. Brain concentrations of 9t over time in mice dosed orally with A) 30 mpk 9t (▲), B)
30 mpk 9t co-administered with 50 mpk ketoconazole (◊), C) 30 mpk 9t co-administered with 30
mpk 1-ABT (♦).
Conclusions
Despite numerous reports of JNK inhibitors, there remains a need for sharp tools having
sufficient cellular potency and pharmacokinetic profile to support in vivo proof of concept
studies in models of Huntington’s disease. With a focus on identification of compounds having
calculated properties aligned with BBB penetration for HD, we have expanded the scope of the
SAR of the 1H-pyrazol-4-yl)pyrimidine chemotype through incorporation of new substituents
that bind in the sugar pocket, while significantly expanding the SAR with respect to binding in
0.01
0.1
1
10
100
0 2 4 6 8 10 12 14 16 18 20 22 24
BrainConc(µµµµM)
Time (hr) post 9t dose
9t 30 mg/kg PO
9t 30 mg/kg PO + keto 50 mg/kg PO
9t 30 mg/kg PO + 1-ABT 30 mg/kg PO
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hydrophobic region II. Through our studies, a binding model to rationalize the observed
selectivity for JNK3 versus p38 was established, one which favors small groups at R3
that allow
Met146 of JNK3 to occupy hydrophobic region I. Three different cellular assays were used to
stratify compounds for further studies. In two different assays used to assess the activity of
compounds against a p-c-Jun readout, only the irreversible inhibitor 34 showed a level of activity
consistent with a pharmacodynamic tool. While 34 has the necessary level of cellular potency, its
permeability in an MDCK-MDR1 assay, and its kinase selectivity were insufficient (see
Supporting Information). In an LPS-TNF-α secretion assay, all compounds showed a much
improved translation of biochemical potency, with most showing sub-micromolar IC50 values.
Our focus on calculated properties resulted in compounds showing low efflux, leading to the
identification of 9t as a brain penetrant pan-JNK inhibitor in mouse. All compounds from this
chemotype were highly and rapidly metabolized, showing high in vivo clearance in mouse;
however, this liability could be overcome through in vivo pan-CYP450 inhibition. Additional
studies will be needed to establish a correlation between TNF-α levels and HD disease
progression to demonstrate whether 9t is suitable for oral dosing in pharmacodynamic and
efficacy models.
Experimental Section
Unless otherwise noted, reagents and solvents were used as received from commercial suppliers.
All non-aqueous reactions were carried out under an atmosphere of dry nitrogen (unless
otherwise noted). Proton nuclear magnetic resonance spectra were obtained on a Bruker
AVANCE 300 spectrometer at 300 MHz or Bruker AVANCE 500 spectrometer at 500 MHz.
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Spectra are given in ppm (δ) and coupling constants, J values, are reported in hertz (Hz).
Tetramethylsilane was used as an internal standard for 13
C and 1
H nuclear magnetic resonance.
Mass spectra were obtained on either a Perkin Elmer Sciex 100 mass spectrometer (APCI),
Varian 1200L single quadrapole mass spectrometer (ESI) or a Waters Acquity SQD (ESI and
APCI). LC–MS analyses were obtained using a Varian 1200L single quadrapole mass
spectrometer (ESI, HP-LCMS) or a Waters Acquity SQD (ESI and APCI, UP-LCMS). HPLC
analyses were obtained using a Grace Alltima C18 column, 3µ, (7 × 53 mm) with UV detection
at 254 nm (unless otherwise noted) using standard solvent gradient program (Methods 1–5). All
final compounds were of ≥95% purity as assessed by 1
H NMR and using one of the analytical
HPLC methods noted above.
Method 1
Time
(min)
Flow
(mL/min)
%A %B
0.0 3.0 90.0 10.0
5.0 3.0 0.0 100.0
6.0 3.0 0.0 100.0
A = 95% Water/Acetonitrile with 0.05% v/v Trifluoroacetic Acid
B = 95% Acetonitrile/Water with 0.05% v/v Trifluoroacetic Acid
Method 2
Time
(min)
Flow
(mL/min)
%A %B
0.0 3.0 70.0 30.0
5.0 3.0 0.0 100.0
6.0 3.0 0.0 100.0
Method 3
Time
(min)
Flow
(mL/min)
%A %B
0.0 3.0 100.0 0.0
10.0 3.0 0.0 100.0
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11.0 3.0 0.0 100.0
Method 4
Time
(min)
Flow
(mL/min)
%A %B
0.0 3.0 100.0 0.0
5.0 3.0 0.0 100.0
6.0 3.0 0.0 100.0
Method 5
Time
(min)
Flow
(mL/min)
%A %B
0.0 0.75 90.0 10.0
20.0 0.75 0.0 100.0
25.0 0.75 0.0 100.0
A = Water with 0.01% v/v Trifluoroacetic Acid
B = Acetonitrile with 0.01% v/v Trifluoroacetic Acid
hJNK3αααα1, p38αααα and ββββ In Vitro Kinase Assays for Compound IC50 Determinations
Compounds were prepared as 10 mM stocks in 100% DMSO from fresh powder. The compound
stock solution was serially diluted 1:3 in DMSO for a 10-point concentration dose response in
duplicate and transferred to assay plates with a final DMSO assay concentration of one percent.
Control compounds such as JNK inhibitor JNK-40138
(36) and p38 inhibitor SB 23906339
(37)
were also included in each test plate to monitor assay performance.
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Figure 5. Structures of assay standards 36 and 37.
The Kinase Glo® assay platform from Promega (Cat. # V6714) was used to determine
compound IC50 for hJNK3α1. In this format, ATP in the reaction is measured after the addition
of the Kinase-Glo® Reagent. The assay was performed at room temperature in 384-well plates
(Corning Cat. # 3572). Each well received 8 nM JNK3α1 (Invitrogen, Cat. # PR6983A), 2 µM
ATF-2 (BPS Biosciences, Cat. # 40520), and 1 µM ATP (Cell Signaling Technology, Cat. #
9804) in 50 mM Tris-HCl pH 7.5, 2 mM EGTA, 2 mM DTT, and 10 mM MgCl2 and test
compounds in a 20 µL final reaction volume. The kinase reaction was initiated with the addition
of JNK3α1 kinase and incubated 30 minutes prior to the addition of the Kinase-Glo® Reagent as
per manufacture’s recommendation. The plate was incubated for an additional 15 minutes at
room temperature and luminescence was measured in an Analyst GT reader (Molecular Devices,
using the default luminescence settings). The luminescence produced is inversely related to
kinase activity. Data were analyzed by calculating the percent of inhibition and each IC50 was
determined using the 4-parameter logistic equation (model 205, Excel fit -IDBS curve-fitting
software).
The γ32
P-ATP radioactive assay platform was used to determine compound IC50 for human p38α
and β isoforms. The assay was performed at room temperature in 96-well plates (Corning, Cat.
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#3363). Each well received 10 nM hp38α or 40 nM hp38β (Millipore), 2 or 3 µM ATF-2
respectively (BPS Biosciences), 50 µM ATP (Cell Signaling Technology, Cat. # 9804) and 1.125
µCi γ-32
P-ATP (PerkinElmer, Cat. # BLU502A250UC) in 50 mM Tris-HCl pH 7.5, 2 mM
EGTA, 2 mM DTT, and 10 mM MgCl2 and test compounds in a 20 µL final reaction volume.
The kinase reaction was initiated with the addition of the p38α or β kinase and incubated 30
minutes for the hp38α assay and 40 minutes for the hp38β. Reactions were terminated with the
addition of 150 μL of 150 mM phosphoric acid to each well. The reaction mixture was
transferred to a pretreated- Immobilon filter plate based on manufacture’s recommendation
(Millipore, Cat. # MAIPNOB50). After vacuum filtration, the filter plate was washed four times
with 300 μL of 150 mM phosphoric acid. After the final wash, the membrane was allowed to air
dry at room temperature, 50 µL of EcoScint scintillation cocktail (National Diagnostics Cat. #
LS-271) was added, and radioactivity measured using a TriLux reader (Perkin Elmer). Data were
analyzed by calculating the percent of inhibition and each IC50 was determined using the 4-
parameter logistic equation (model 205, Excel fit-IDBS software).
Compound Synthesis
4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-N-(cyclopropylmethyl)pyrimidin-2-amine (9a)
Preparation of 1-(4-Chlorophenyl)-2-(2-(methylthio)pyrimidin-4-yl)ethenol/ethanone (4
and 4’). To a stirred solution at 0 °C of 2 (2.10 g, 15.0 mmol) and methyl 4-chlorobenzonate
(3b, 2.64 g, 15.0 mmol) in THF (30.0 mL) was added lithium hexamethyldisilazide (30 mL of a
1.0 M solution in THF, 30.0 mmol) and after the addition was complete the reaction mixture was
warmed to room temperature. After 16 h the reaction mixture was quenched with saturated
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ammonium chloride solution and the layers were separated. The aqueous layer was extracted
using ethyl acetate (2 × 25 mL). The combined organic phase was washed with 1.0 N
hydrochloric acid, water, saturated sodium chloride, dried (MgSO4), filtered, and concentrated
under reduced pressure. The residue was triturated using heptane/methylene chloride to provide a
mixture of (Z)-1-(4- chlorophenyl)-2-(2-(methylthio)pyrimidin-4-yl)ethanol (4) and 1-(4-
chlorophenyl)-2-(2- (methylthio)pyrimidin-4-yl)ethanone (4’), 2.56:1 ratio, 4.09 g, 98%
combined yield, as a bright yellow crystalline solid: 1
H NMR (300 MHz, CDCl3) δ 2.51 (s,
1.17H), 2.62 (s, 3H), 4.34 (s, 0.78H), 5.96 (s, 1H), 6.66 (d, J = 5.4 Hz, 1H), 6.97 (d, J = 5.1 Hz,
0.39H), 7.38–7.42 (m, 2H), 7.43–7.47 (m, 0.78H), 7.75–7.79 (m, 2H), 7.98 (d, J = 8.7 Hz,
0.78H), 8.32 (d, J = 5.4 Hz, 1H), 8.46 (d, J = 5.1 Hz, 0.39H), 14.60 (br s, 1H); MS (APCI) m/z
279 [M + H]+
.
Preparation of 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-2-(methylthio)pyrimidine (5). A
stirred solution of 4 and 4’ (1.00 g, 3.59 mmol) and DMF•DMA (0.640 g, 5.38 mmol) in toluene
(10 mL) was heated at reflux for 18 h. After this time the reaction mixture was cooled to room
temperature and concentrated under reduced pressure. To the resulting residue was added
anhydrous hydrazine (0.235 g, 7.18 mmol) and ethanol (9.0 mL) and the mixture was stirred at
room temperature for 16 h. After this time the mixture was concentrated under reduced pressure
and the residue was recrystallized using ethyl acetate/heptane to provide 2-methylthiopyrimidine
5 (0.822 g, 85%) as an off-white solid: 1
H NMR (300 MHz, CDCl3) δ 2.43 (s, 3H), 2.99 (br s,
1H), 6.84 (d, J = 5.1 Hz, 1H), 7.42–7.50 (m, 4H), 8.21 (s, 1H), 8.35 (d, J = 5.1 Hz, 1H); MS
(ESI) m/z 303 [M + H]+
.
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Preparation of 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-2-(methylsulfonyl)pyrimidine (6).
To a stirred suspension at 0 °C of 5 (0.773 g, 2.56 mmol) in methylene chloride (12.0 mL) was
added m-CPBA (1.18 g, 5.11 mmol) portion-wise and then the reaction mixture was warmed to
room temperature. After 16 h a saturated sodium bicarbonate solution (20 mL) was added to the
reaction mixture and then stirred for 5 min. The organic layer was separated. The aqueous layer
was extracted with ethyl acetate (2 × 15 mL). The combined organic phase was washed with
sodium thiosulfate, saturated sodium chloride and dried (MgSO4), filtered, and concentrated
under reduced pressure to provide 2-(methylsulfonyl)pyrimidine 6 (0.847 g, 99%) as an off-
white solid: mp 194–195 °C; 1
H NMR (500 MHz, CDCl3) δ 3.23 (s, 3H), 4.95 (br s, 1H), 7.33
(d, J = 5.0 Hz, 1H), 7.45–7.50 (m, 4H), 8.34 (s, 1H), 8.69 (d, J = 5.0 Hz, 1H); MS (APCI) m/z
335 [M + H]+
.
Preparation of 4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-N-(cyclopropylmethyl)pyrimidine-
2-amine (9a). A microwave tube charged with a stir bar and suspension of 6 (0.300 g, 0.90
mmol), cyclopropanemethylamine (0.128 g, 1.80 mmol) in dioxane (3.0 mL) was irradiated (400
W, 130 °C) for 30 min. After this time the reaction mixture was concentrated under reduced
pressure. The residue was purified using flash column chromatography (silica gel; 40-80% ethyl
acetate/heptanes, gradient elution) to provide 9a (0.118 g, 41%) as an off-white solid: mp 221–
222 °C; 1
H NMR (500 MHz, DMSO-d6) d 0.12 (br s, 2H), 0.94–0.83 (m, 2H), 0.94–0.83 (m,
1H), 2.86 (br s, 2H), 6.58 (s, 1H), 6.95 (s, 1H), 7.56–7.41 (m, 4H), 8.04–8.32 (m, 2H), 13.30–
13.38 (m, 1H); 13
C NMR (125 MHz, DMSO-d6) δ 162.1, 157.8, 140.2, 132.1 (low intensity),
131.1, 130.9, 130.6, 128.2, 127.8, 117.9, 117.4, 44.8, 10.8, 3.1; HRMS: Calcd. for C17H16ClN5:
326.1172, Found: 326.1180; HPLC: Method 1, tR = 3.24 min, (> 99% AUC).
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4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-N-(cyclohexylmethyl)pyrimidine-2-amine (9b).
The compound was prepared using a method similar to that described for the preparation of 9a,
0.164 g, 50% as a white solid: mp 222–223 °C; 1
H NMR (500 MHz, DMSO-d6) δ 0.73 (br s,
2H), 1.11 (br s, 3H), 1.33–1.60 (m, 6H), 2.80 (br s, 2H), 6.59–6.65 (br s, 1H), 6.87 (s, 1H), 7.41–
7.56 (m, 4H), 8.03–8.31 (m, 2H), 13.29–13.37 (m, 1H); 13
C NMR (125 MHz, DMSO-d6) δ
162.4, 157.8, 140.2, 133.3 (low intensity), 131.1, 130.8, 130.5, 128.2, 127.7, 117.9, 117.4, 46.6,
37.0, 30.3, 26.1, 25.3; HRMS: Calcd. for C20H22ClN5: 368.1642, Found: 368.1646; HPLC:
Method 1, tR = 3.85 min, (> 99% AUC).
trans-4-(4-(3-Chlorophenyl)-1H-pyrazol-4-yl)-pyrimidin-2-ylamino)cyclohexanol (9c). The
compound was prepared using a method similar to that described for the preparation of 9a, with
the addition of an extra preparative HPLC purification step after flash column chromatography
(Phenomenex Luna 10µ C18(2) 100Å column (21.2 × 250 mm); 10–60% then 60–100% [95:5
CH3CN:H2O]/[95:5 H2O:CH3CN, 0.05% TFA], gradient elution, integration at 254 nm) and then
washed using saturated sodium bicarbonate, 0.114 g, 17% as a white solid: mp 248–250 °C; 1
H
NMR (500 MHz, DMSO-d6) δ 1.10–1.16 (m, 4H), 1.62–1.73 (m, 4H), 3.25–3.40 (br s, 2H), 4.48
(br s, 1H), 6.66 (br s, 1H), 6.85 (s, 1H), 7.46–7.55 (m, 4H), 8.13 (d, J = 5.5 Hz, 1H), 8.27 (br s,
1H), 13.34 (br s, 1H); 13
C NMR (125 MHz, DMSO-d6) δ 161.2, 160.1, 157.4, 148.3, 140.5,
133.2, 132.4, 131.2, 130.6, 128.0, 117.8, 106.6, 68.4, 48.4, 34.0, 30.3; HRMS: Calcd. for
C19H20ClN5O: 370.1435, Found: 370.1425; HPLC: Method 1, tR = 2.60 min, (> 99% AUC).
4-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-N-(pyridine-2-yl)pyrimidin-2-amine (9d). The
compound was prepared using a method similar to that described for the preparation of 9a, 0.032
g, 5% as an off-white solid: mp 246–247 °C; 1
H NMR (500 MHz, DMSO-d6) δ 6.96–7.01 (m,
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2H), 7.47–7.57 (m, 5H), 7.65 (d, J = 8.0 Hz, 1H), 8.18–8.47 (m, 3H), 9.85 (br s, 1H), 13.47–
13.60 (m, 1H); 13
C NMR (125 MHz, DMSO-d6) δ 160.3, 158.6, 158.5, 158.0, 157.9, 157.7,
152.1, 152.0, 151.9, 145.6, 145.5, 145.2, 139.0, 138.6, 133.0, 130.7, 128.5, 117.1, 112.8, 110.9;
HRMS: Calcd. for C18H13ClN6: 349.0968, Found: 349.0976; HPLC: Method 1, tR = 2.99 min, (>
99% AUC).
trans-4-(4-(3-tert-Butyl-1H-pyrazol-4-yl)-pyrimidin-2-ylamino)cyclohexanol (9e). The
compound was prepared using a method similar to that described for the preparation of 9a,
0.189g, 38% as a white solid: mp 232–234 °C; 1
H NMR (500 MHz, CD3OD) δ 1.35–1.39 (m,
4H), 1.46–1.52 (m, 9H), 1.96–1.98 (m, 2H), 2.02–2.05 (m, 2H), 3.30–3.32 (m, 2H), 3.57–3.58
(m, 1H), 3.89 (br s, 1H), 6.71–6.76 (m, 1H), 7.78–7.94 (m, 1H), 8.12 (d, J = 5.0 Hz, 1H); 13
C
NMR (125 MHz, CD3OD) δ 29.93, 30.56, 32.05, 33.76, 35.17, 50.30, 70.70, 109.9, 118.3, 142.9,
151.9, 158.3, 162.8, 164.4; HRMS: Calcd. for C17H25N5O: 316.2137, Found: 316.2149; HPLC:
Method 1, tR = 2.30 min, (98.6% AUC).
trans-4-(4-(3-(1-Methylcyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol
(9f). The compound was prepared using a method similar to that described for the preparation of
9a, 0.125 g, 40% as an off-white solid: mp; no clear melt observed; 1
H NMR (500 MHz, DMSO-
d6) d 0.73–0.83 (m, 2H), 0.92–0.94 (m, 2H), 1.23–1.30 (m, 2H), 1.38–1.44 (m, 5H), 1.86–1.93
(m, 4H), 3.46 (s, 2H), 3.71 (br s, 1H), 4.11 (br s, 1H), 7.25 (d, J = 6.5 Hz, 1H), 8.29–8.38 (m,
2H), 8.50–8.69 (m, 1H); 13
C NMR (125 MHz, DMSO-d6) δ 166.5, 153.4, 151.7, 145.6, 139.9,
116.2, 105.7, 67.5, 48.8, 33.5, 29.8, 24.0, 13.6, 13.4; HRMS: Calcd. for C17H23N5O: 314.1981,
Found: 314.1983; HPLC: Method 1, tR = 2.18 min, (97.5% AUC).
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trans-4-(4-(3-(1-(Trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-
ylamino)cyclohexanol (9g). The compound was prepared using a method similar to that
described for the preparation of 9a, 0.064 g, 19% as a white solid: mp 239–240 °C; 1
H NMR
(500 MHz, DMSOd6) δ 1.21–1.33 (m, 6H), 1.44–1.53 (m, 2H), 1.80–1.88 (m, 4H), 3.38–3.41
(m, 1H), 3.81 (br s, 1H), 4.52 (d, J = 5.4 Hz, 1H), 6.74 (br s, 1H), 6.81–6.83 (m, 1H), 8.02–8.28
(m, 2H), 13.19–13.56 (m, 1H); 19F {1H} (282 MHz, CDCl3) δ –67.27; 13
C NMR (125 MHz,
DMSO-d6) δ 161.5, 159.4, 157.6, 143.8, 139.9, 130.9, 120.3, 106.3, 68.3, 48.2, 34.2, 30.5, 11.3;
HRMS: Calcd. for C17H20F3N5O: 368.1698, Found: 368.1691; HPLC: Method 3, tR = 3.57 min,
(> 99% AUC).
trans-4-(4-(3-Cyclobutyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol (9h). The
compound was prepared using a method similar to that described for the preparation of 9a, 0.167
g, 53% as a light yellow solid: mp; no clear melt observed; 1
H NMR (500 MHz, DMSO-d6) d
1.28–1.46 (m, 4H), 1.92–2.03 (m, 6H), 2.31–2.36 (m, 4H), 3.48 (br s, 1H), 3.69–3.94 (m, 1H),
4.32 (m, 1H), 5.50 (br s, 1H), 7.20 (br s, 1H), 8.24 (br s, 1H), 8.50 (br s, 2H), 13.18 (br s, 1H);
13
C NMR (125 MHz, DMSO-d6) δ 167.1, 152.9, 152.1, 145.0, 138.7, 114.4, 105.4, 67.5, 49.8,
33.6, 32.4, 29.7, 27.8, 18.1; HRMS: Calcd. for C17H23N5O: 314.1981, Found: 314.1981; HPLC:
Method 1, tR = 2.30 min, (98.4% AUC).
4-(3-Cyclopropyl-1H-pyrazol-4-yl)-N-isopropylpyrimidin-2-amine (9i). The compound was
prepared using a method similar to that described for the preparation of 9a, 0.116 g, 33% as a
H NMR (500 MHz, DMSO-d6) δ 0.85–1.00 (m, 4H), 1.16–1.30
(m, 4H), 1.81–1.84 (m, 2H), 1.89–1.92 (m, 2H), 2.89 (br s, 1H), 3.35–3.40 (m, 1H), 3.63 (br s,
1H), 4.51 (d, J = 4.5 Hz, 1H), 6.74 (br s, 1H), 6.82 (d, J = 5.5 Hz, 1H), 7.93–8.24 (m, 1H), 8.15
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(s, 1H), 12.56–12.68 (m, 1H); 13
C NMR (125 MHz, DMSO-d6) δ 161.5, 157.6, 151.8, 144.7,
139.5, 130.0, 117.6, 117.2, 105.4, 68.4, 49.1, 34.2, 30.2, 8.5, 8.0, 7.1; HRMS: Calcd. for
C16H21N5O: 300.1824, Found: 300.1833; HPLC: Method 1, tR = 2.09 min, (> 99% AUC).
trans-4-(4-(3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)pyrimidin-2-
ylamino)cyclohexanol (9j). The compound was prepared using a method similar to that
described for the preparation of 9a, 0.077 g, 23% as a white powder: mp 229 °C; 1
H NMR (300
MHz, DMSO-d6) δ 1.05–1.40 (m, 4H), 1.60–2.00 (m, 8H), 3.55–4.05 (m, 7H), 4.55–4.61 (m,
1H), 6.78 (d, J = 4.5 Hz, 2H), 7.95–8.30 (m, 2H), 12.84–12.96 (m, 1H); 13
C NMR (125 MHz,
DMSO-d6) δ 161.7, 160.6, 157.7, 153.9, 146.2, 139.4, 130.3, 115.3, 105.8, 68.3, 67.3, 48.5, 34.3,
33.3, 31.9, 31.3, 30.3; HRMS: Calcd. for C18H25N5O2: 344.2087, Found: 344.2090; HPLC:
Method 1, tR = 1.98 min, (> 99% AUC, integration at 230 nm).
trans-4-(4-(1-cyclopentyl-3-cyclopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanol
(9l). The compound was prepared using a method similar to that described for the preparation of
9a, 0.155g, 59% as a white foam: mp No clear melt observed; 1
H NMR (500 MHz, CDCl3) δ
0.60–0.63 (m, 2H), 1.08–1.12 (m, 2H), 1.25–1.33 (m, 2H), 1.40 (br s, 1H), 1.41–1.49 (m, 2H),
1.67–1.71 (m, 2H), 1.81–1.86 (m, 1H), 1.96–2.03 (m, 4H), 2.06–2.10 (m, 4H), 2.17–2.19 (m,
2H), 3.66–3.70 (m, 1H), 3.48–3.91 (m, 1H), 4.88 (d, J = 3.9 Hz, 1H), 5.03–5.09 (m, 1H), 6.79 (d,
J = 5.0 Hz, 1H), 7.89 (s, 1H), 8.21 (d, J = 5.0 Hz, 1H); 13
C NMR (125 MHz, DMSO-d6) δ 161.6,
159.4, 157.3, 141.9, 137.9, 119.1, 107.1 (low intensity), 68.4, 58.4, 48.7, 34.2, 32.4, 30.3, 24.3,
7.7, 5.4; HRMS: Calcd. for C21H29N5O: 368.2450, Found: 368.2455; HPLC: Method 1, tR = 3.09
min, (98.3% AUC).
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trans-4-(4-(1-Isopropyl-3-(1-methylcyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-
ylamino)cyclohexanol (9m). The compound was prepared using a method similar to that
described for the preparation of 9a, 0.085 g, 33% as a white solid: mp; no clear melt observed;
1
H NMR (500 MHz, DMSO-d6) δ 0.67 (br s, 2H), 0.92 (br s, 2H), 1.17–1.33 (m, 4H), 1.42 (d, J
= 6.5 Hz, 6H), 1.45 (s, 3H), 1.82–1.89 (m, 4H), 3.36–3.41 (m, 1H), 3.80 (br s, 1H), 4.51 (d, J =
5.5 Hz, 1H), 4.91– 4.96 (m, 1H), 6.73 (br s, 1H), 7.84 (s, 1H), 8.17 (br s, 1H); 13
C NMR (125
MHz, DMSO-d6) δ 161.7, 159.6, 157.5, 144.1, 138.3, 117.9, 106.8, 68.4, 49.1, 48.5, 34.3, 30.5,
24.2, 22.5, 15.0, 11.2; HRMS: Calcd. for C20H29N5O: 356.2450, Found: 356.2463; HPLC:
Method 1, tR = 3.63 min, (> 99% AUC).
trans-4-(4-(3-Cyclopropyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)pyrimidin-2-
ylamino)cyclohexanol (9n). The compound was prepared using a method similar to that
described for the preparation of 9a, 0.251 g, 57% as a white foam: mp No clear melt observed;
1
H NMR (500 MHz, DMSO-d6) δ 0.58–0.64 (m, 2H), 1.13–1.15 (m, 2H), 1.18–1.33 (m, 4H),
1.82–1.95 (m, 5H), 3.36–3.41 (m, 1H), 3.71 (br s, 1H), 4.50 (d, J = 2.5 Hz, 1H), 5.15 (q, J = 9.0
Hz, 2H), 6.84 (d, J = 5.0 Hz, 1H), 7.93 (s, 1H), 8.23 (s, 1H); 19
F {1
H} NMR (282 MHz, CDCl3)
δ –68.75; 13
C NMR (125 MHz, DMSO-d6) δ 161.6, 158.7, 157.6, 143.9, 139.7, 123.7 (q, J =
280.4 Hz), 120.4, 107.5 (low intensity), 68.4, 49.5 (q, J = 34.0 Hz), 48.7, 34.2, 30.3, 7.7, 5.2;
HRMS: Calcd. for C18H22F3N5O: 382.1855, Found: 382.1848; HPLC: Method 1, tR = 2.75 min,
(98.7% AUC).
(4-(3-Isopropyl-1H-pyrazol-4-yl)-N-(trans-4-methoxycyclohexyl)-pyrimidin-2-amine (9r).
H NMR (500 MHz, DMSO-d6) δ 1.17–1.31 (m,
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10H), 1.94 (d, J = 11.0 Hz, 2H), 2.03 (d, J = 11.0 Hz, 2H), 3.08–3.14 (m, 1H), 3.30 (s, 3H),
3.66–3.72 (m, 1H), 3.87–4.09 (m, 1H), 6.77–6.79 (m, 2H), 7.93–8.31 (m, 1H), 8.13 (d, J = 3.0
Hz, 1H), 12.73–12.89 (m, 1H); 13
C NMR (125 MHz, DMSO-d6) δ 161.6, 160.7, 157.7, 148.7,
139.3, 130.1, 115.9, 114.7, 105.5, 77.9, 55.0, 49.0, 30.3, 30.0, 26.4, 24.8, 22.4, 21.7; HRMS:
Calcd. for C17H25N5O: 316.2137, Found: 316.2132; HPLC: Method 1, tR = 2.62 min, (98.5%
AUC).
trans-4-(4-(3-Isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexanecarbonitrile (9u).
H NMR (500 MHz, DMSO-d6) δ 1.29–1.35 (m,
8H), 1.51–1.58 (m, 2H), 1.94–1.97 (m, 2H), 2.07–2.09 (m, 2H), 2.66–2.71 (m, 1H), 3.71 (br s,
1H), 3.84–4.06 (m, 1H), 6.79 (d, J = 5.0 Hz, 1H), 6.90 (br s, 1H), 7.93–8.27 (m, 1H), 8.14 (d, J =
5.0 Hz, 1H), 12.74–12.89 (m, 1H); 13
C NMR (125 MHz, DMSO-d6) δ 161.5, 160.8, 157.6,
156.1, 148.8, 139.4, 130.2, 123.0, 116.0, 114.8, 105.8, 48.0, 30.6, 28.2, 26.6, 24.8, 22.5, 21.8;
HRMS: Calcd. for C17H22N6: 311.1984, Found: 311.1984; HPLC: Method 1, tR = 2.74 min,
(>99% AUC).
N-(Cyclopropylmethyl)-4-(3-(1-methylcyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine
(9y). The compound was prepared using a method similar to that described for the preparation of
9a, 0.064 g, 25% as a white solid: mp 158–160 °C; 1
H NMR (500 MHz, DMSO-d6) δ 0.20–0.22
(m, 2H), 0.38–0.44 (m, 2H), 0.70–0.82 (m, 2H), 0.87–0.89 (m, 2H), 1.06–1.11 (m, 1H), 1.34–
1.38 (m, 3H), 3.22 (br s, 2H), 6.88–6.94 (m, 2H), 7.92–8.20 (m, 2H), 12.72–12.95 (m, 1H); 13
C
NMR (125 MHz, DMSO-d6) δ 162.2, 159.6, 157.6, 153.7, 146.3, 139.7, 130.4, 118.1, 117.2,
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106.1, 44.8, 24.4, 24.2, 14.6, 13.8, 12.3, 11.1, 3.1; HRMS: Calcd. for C15H19N5: 270.1719,
Found: 270.1718; HPLC: Method 1, tR = 3.47 min, (> 99% AUC).
N-(1-Ethylpiperidin-4-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1Hpyrazol-4-yl)pyrimidin-
2-amine (9dd). The compound was prepared using a method similar to that described for the
preparation of 9a, 0.058 g, 19% as a crystalline white solid: mp 220–222 °C (dec); 1
H NMR (500
MHz, DMSO-d6) d 0.90–1.10 (m, 3H), 1.23–1.40 (m, 2H), 1.40–1.60 (m, 4H), 1.74–1.95 (m,
4H), 2.25–2.39 (m, 2H), 2.80–2.92 (m, 2H), 3.85 (br s, 1H), 6.70–6.85 (m, 2H), 8.10–8.30 (m,
2H), 13.20–13.60 (m, 1H); 19
F{1
H} (282 MHz, DMSO-d6) δ –67.27 (s); 13
C NMR (125 MHz,
DMSO-d6) δ 161.7, 159.2, 157.8, 126.2 (q, J = 274.2 Hz), 120.4, 106.5, 52.1, 51.7, 47.4, 31.8,
20.4, 12.3, 11.4; HRMS: Calcd. for C18H23F3N6: 381.2015, Found: 381.2029; HPLC: Method 1,
tR = 1.94 min, (97.3% AUC).
trans-4-(4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrimidin-2-
ylamino)cyclohexanol (9o)
Preparation of 3-(Ethoxymethylene)-1,1,1-trifluoropentane-2,4-dione 13. A stirred solution
of 1,1,1-trifluoropentane-2,4-dione 12 (12.7 g, 82.4 mmol), acetic anhydride (8.41 g, 82.4
mmol), and triethyl orthoformate (24.4 g, 164.8 mmol) were heated at reflux for 19 h. After this
time the reaction mixture was cooled to room temperature and concentrated under reduced
pressure. The residue was vacuum distilled under reduced pressure to provide 13 (6.01 g, 35%)
(55:45 mixture of E,Z isomers by NMR integration) as a red oil: bp 70–85 °C at 2 mm Hg; 1
H
NMR (300 MHz, CDCl3) δ 1.40–1.49 (m, 3H), 2.32 (s, 1.35H), 2.42 (s, 1.65H), 4.30–4.40 (m,
2H), 7.69 (s, 0.55H), 7.96 (s, 0.45H); 19
F{1
H} NMR (282 MHz, CDCl3) δ –76.43, –71.92.
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Preparation of 1-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)ethanone 14. To a stirred
solution at –10 °C of methyl hydrazine (0.657 g, 14.3 mmol) in THF (500 mL) was added a
solution of E- and Z- 3-(ethoxymethylene)-1,1,1-trifluoropentane-2,4-dione 20 (3.00 g, 14.3
mmol) in THF (250 mL) dropwise over 1 h. After the addition was complete the resulting
reaction mixture was stirred at –10 °C for 1.25 h, then allowed to warm to room temperature
over 1 h. After this time the reaction mixture was concentrated under reduced pressure, and the
residue was purified using flash column chromatography (silica gel; 20–85% ethyl
acetate/heptanes, gradient elution) to provide 14 (1.92 g, 70%) as a light-yellow solid: mp 79–80;
1
H NMR (300 MHz, CDCl3) δ 2.48 (s, 3H), 3.99 (s, 3H), 7.93 (s, 1H); MS (ESI) m/z 253 [M +
H]+
.
Preparation of 4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-2-(methylthio)pyrimidine
15. A stirred solution of 1-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)ethanone 14 (0.850 g,
4.42 mmol) and DMF•DMA (1.05 g, 8.85 mmol) in toluene (10 mL) was heated at reflux for 18
h. After this time additional DMF•DMA (1.05 g, 8.85 mmol) was added and heating at reflux
was continued for an additional 2 h. After this time the reaction mixture was concentrated under
reduced pressure, the residue was treated with methyl carbamimidothioate•½H2SO4 (0.923 g,
6.64 mmol) and sodium methoxide (30 wt.%, 1.17 mL, 6.64 mmol) in 2-propanol (20 mL), and
the resulting reaction mixture was heated at reflux for 12 h. After this time the reaction mixture
was cooled to room temperature, acidified to pH 3 with 1 N hydrochloric acid and extracted with
ethyl acetate (2 × 50 mL). The combined organic layers were washed with aqueous saturated
sodium chloride solution (30 mL), dried (Na2SO4), filtered, and concentrated under reduced
pressure. The residue was purified using flash column chromatography (silica gel; 25–85% ethyl
acetate/heptanes, gradient elution) to provide 15 (0.097 g, 8%) as a golden oil: 1
H NMR (300
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MHz, CDCl3) δ 2.59 (s, 3H), 4.00 (s, 3H), 7.17 (d, J = 5.1 Hz, 1H), 8.11 (s, 1H), 8.51 (d, J = 5.1
Hz, 1H); 19
F{1
H} NMR (282 MHz, CDCl3) δ -60.42; LC-MS (ESI) m/z 275 [M + H]+
.
Preparation of 4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-2-(methylsulfonyl)-
pyrimidine 16. The title compound was prepared by a similar method described for the
preparation of 6. 0.098 g, 92% as a white solid: 1
H NMR (300 MHz, CDCl3) δ 3.39 (s, 3H), 4.05
(s, 3H), 7.70 (d, J = 5.1 Hz, 1H), 8.31 (s, 1H), 8.87 (d, J = 5.1 Hz, 1H); 19
F{1
H} NMR (282
MHz, CDCl3) δ –60.69; MS (ESI) m/z 307 [M + H]+
.
Preparation of Trans-4-(4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)pyrimidin-2-
ylamino)cyclohexanol (9o). The compound was prepared using a method similar to that
described for the preparation of 9a, 0.061 g, 56% as an off-white powder: mp 191–192 °C; 1
H
NMR (300 MHz, DMSO-d6) δ 1.10–1.35 (m, 4H), 1.80–1.92 (m, 4H), 3.38 (br s, 1H), 3.73 (br s,
1H), 3.96 (s, 3H), 4.51 (d, J = 4.5 Hz, 1H), 6.74 (br d, J = 4.8 Hz, 1H), 6.92 (br s, 1H), 8.24 (br
d, J = 4.8 Hz, 1H), 8.55 (br s, 1H); 19
F {1
H} NMR (282 MHz, DMSO-d6) δ –59.26; 13
C NMR
(125 MHz, DMSO-d6) δ 161.6, 158.5, 156.9, 134.4, 122.4, 120.3, 119.5, 105.7, 68.4, 48.5, 34.2,
30.2; HRMS: Calcd. for C15H18F3N5O: 342.1542, Found: 342.1536; HPLC: Method 1, tR = 2.47
min, (98.4% AUC).
N-(4,4-Difluorocyclohexyl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-
yl)pyrimidin-2-amine (9t)
Preparation of 2-Methylsulfonyl)-4-(1-(tetrahydro-2H-pyran-2-yl)-3-(1-
(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidine (6t). To a stirred solution of 2-
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(methylsulfonyl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1Hpyrazol-4-yl)pyrimidine (6t) (1.06 g,
3.19 mmol) in 3,4-dihydropyrane (5.0 mL) was added p-toluenesulfonic acid (0.061 g, 0.32
mmol) at room temperature. After 10 min the reaction mixture was concentrated under reduced
pressure. The residue was partitioned between saturated sodium bicarbonate and chloroform, and
separated. The combined organics were dried (MgSO4), filtered and concentrated under reduced
pressure. The residue was purified using flash column chromatography (silica gel; 5-20% ethyl
acetate/methylene chloride, gradient elution) to provide 7t (1.03 g, 78%) as an off-white solid:
1
H NMR (500 MHz, CDCl3) δ 1.34–1.41 (m, 2H), 1.28–1.73 (m, 5H), 2.00–2.05 (m, 2H), 2.13–
2.16 (m, 1H), 3.36 (s, 3H), 3.69–3.74 (m, 1H), 4.09–4.12 (m, 1H), 5.41 (dd, J = 9.5, 2.5 Hz, 1H),
7.82 (d, J = 5.0 Hz, 1H), 8.44 (s, 1H), 8.82 (d, J = 5.0 Hz, 1H); 19
F {1
H} (282 MHz, CDCl3) δ –
68.45 (s); MS (ESI) m/z 417 [M + H]+
.
Preparation of N-(4,4-Difluorocyclohexyl)-4-(1-tetrahydro-2H-pyran-2-yl)-3-(1-
(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine (8t). The compound was
prepared from 7t using a method similar to that described for the preparation of 9a to provide 8t
(0.178 g, 47%) as an off-white solid: 1
H NMR (300 MHz, CDCl3) δ 1.20–1.25 (m, 2H), 1.46–
1.47 (m, 2H), 1.61–1.71 (m, 6H), 2.01–2.14 (m, 8H), 3.67–3.76 (m, 1H), 4.04–4.11 (m, 2H),
5.12 (br s, 1H), 5.41 (dd, J = 9.3, 3.0 Hz, 1H), 6.93 (d, J = 5.4 Hz, 1H), 8.16 (s, 1H), 8.25 (d, J =
5.4 Hz, 1H); 19
F {1
H} (282 MHz, CDCl3) δ –68.42 (s), –68.39 (s); MS (ESI) m/z 472 [M + H]+
.
Preparation of N-(4,4-Difluorocyclohexyl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-
1Hpyrazol-4-yl)pyrimidin-2-amine (9t, CHDI-00372893). To a stirred solution of 8t (0.178 g,
0.38 mmol) in methanol (3.0 mL) was added hydrochloric acid (0.283 mL of a 4.0 M solution in
dioxane, 1.13 mmol,) at room temperature. After 1.5 h the reaction mixture was concentrated
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under reduced pressure. The residue was basified with sodium bicarbonate solution and the
precipitate was collected by filtration. This solid was purified by trituration with methylene
chloride followed by methanol to provide 9t (0.052 g, 13%) as a white solid: 1
H NMR (300
MHz, DMSO-d6) δ 1.21–1.26 (m, 2H), 1.43–1.66 (m, 4H), 1.84–2.09 (m, 6H), 3.95–4.14 (m,
1H), 6.87 (d, J = 5.1 Hz, 1H), 6.99 (br s, 1H), 8.05–8.31 (m, 2H), 13.23–13.59 (m, 1H); 19
F{1
H}
(282 MHz, CDCl3) δ –67.22 (s), –67.56 (s); 13
C NMR (125 MHz, DMSO-d6) δ 161.5, 159.3,
158.9, 157.8, 143.7, 140.0, 130.9, 125.6, 123.7, 121.8, 120.3, 106.7, 106.5, 46.3, 31.6 (t, J = 23.9
Hz), 28.0, 27.9, 21.3, 11.5, 11.2; HRMS: Calcd. for C17H18F5N5: 388.1561, Found: 388.1554;
HPLC: Method 1, tR = 3.22 min, (97.4% AUC).
trans-4-((4-(1H-Pyrazol-4-yl)pyrimidin-2-yl)amino)cyclohexanol (9k). The compound was
prepared using a method similar to that described for the preparation of 9t, 0.0658 g, 58% as a
H NMR (500 MHz, DMSO-d6) 1.24–1.33 (m, 4H), 1.81–1.89 (m,
4H), 3.33– 3.40 (m, 1H), 3.67–3.75 (m, 1H), 4.51 (d, J = 4.5 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H),
6.81 (d, J = 5.5 Hz, 1H), 8.02 (br s, 1H), 8.17 (d, J = 4.5 Hz, 1H), 8.32 (br s, 1H), 13.42 (br s,
1H); 13
C NMR (125 MHz, DMSO-d6) δ 161.4, 158.9, 157.5, 137.4, 128.0, 120.5, 104.8, 68.1,
48.5, 33.8, 30.0; HRMS: Calcd. for C13H17N5O: 260.1511, Found: 260.1510; HPLC: Method 4,
tR = 2.30 min., (98.2% AUC).
N-Cyclohexyl-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine
Dihydrochloride (9q). The compound was prepared using a method similar to that described for
the preparation of 9t, 0.158 g, 94% as a crystalline yellow-brown solid: 1
H NMR (500 MHz,
DMSO-d6) δ 1.19–1.89 (m, 15H), 4.10 (s, 1H), 7.19 (s, 1H), 8.25–8.35 (m, 2H), 8.62 (s, 1H); 19
F
{1
H} (282 MHz, DMSO-d6) δ –67.36 (s); 13
C NMR (125 MHz, DMSO-d6) δ 166.0, 153.5,
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146.3, 143.5, 136.6, 126.0 (q, J = 274.2 Hz), 118.4, 106.1, 48.9, 32.0, 24.8, 24.1, 20.6, 11.4;
HRMS: Calcd. for C17H20F3N5: 352.1749, Found: 352.1757; HPLC: Method 1, tR = 3.36 min,
(98.7% AUC).
N-Cyclobutyl-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine
(9v). The compound was prepared using a method similar to that described for the preparation of
9t, 0.096 g, 87% as a white solid: mp; no clear melt observed; 1
H NMR (300 MHz, DMSO-d6) δ
1.27 (br s, 2H), 1.53 (br s, 2H), 1.60–1.74 (m, 2H), 2.00–2.10 (m, 2H), 2.27–2.30 (m, 2H), 4.64
(br s, 1H), 7.15 (d, J = 6.3 Hz, 1H), 8.28 (d, J = 6.3 Hz, 1H), 8.52 (br s, 2H), 13.74 (br s, 1H); 19
F
{1
H} (282 MHz, DMSO-d6) δ –67.37 (s); 13
C NMR (125 MHz, DMSO-d6) δ 165.6, 153.2,
146.8, 143.5, 136.3, 126.0 (q, J = 273.8 Hz), 118.5, 106.3, 45.4, 30.0, 20.8, 14.5, 11.4; HRMS:
Calcd. for C15H16F3N5: 324.1436, Found: 324.1446; HPLC: Method 1, tR = 3.00 min, (98.5%
AUC).
N-Cyclopentyl-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine
(9w). The compound was prepared using a method similar to that described for the preparation of
9t, 0.143 g, 90% as a white solid: mp; no clear melt observed; 1
H NMR (300 MHz, DMSO-d6)
δ 1.28 (br s, 2H), 1.50–1.61 (m, 6H), 1.71–1.73 (m, 2H), 1.94–1.96 (m, 2H), 4.48 (br s, 1H), 7.19
(d, J = 6.3 Hz, 1H), 8.31 (d, J = 6.6 Hz, 1H), 8.45–8.59 (m, 2H), 13.92 (br s, 1H); 19
F {1
H} (282
MHz, DMSO-d6) δ –67.39 (s); 13
C NMR (125 MHz, DMSO-d6) δ 165.8, 153.7, 146.4, 143.2,
136.5, 126.0 (q, J = 272.9 Hz), 118.5, 106.0, 52.3, 32.0, 23.0, 20.7, 11.4; HRMS: Calcd. for
C16H18F3N5: 338.1593, Found: 338.1595; HPLC: Method 1, tR = 3.19 min, (> 99% AUC).
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N-Cycloheptyl-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine
(9x). The compound was prepared using a method similar to that described for the preparation of
9t, 0.079 g, 50% as an off-white solid: mp; no clear melt; 1
H NMR (300 MHz, DMSO-d6) δ 1.27
(br s, 2H), 1.42–1.70 (m, 12H), 1.88–1.95 (m, 2H), 4.27 (br s, 1H), 7.16 (d, J = 6.0 Hz, 1H), 8.30
(d, J = 6.3 Hz, 1H), 8.35 (br s, 1H), 8.52–8.57 (m, 1H), 13.49 (br s, 1H); 19
F {1
H} (282 MHz,
DMSO-d6) δ –67.35 (s); 13
C NMR (125 MHz, DMSO-d6) δ 165.9, 153.3, 146.5, 136.4, 126.0 (q,
J = 274.2 Hz), 118.6, 106.0, 51.2, 34.1, 27.5, 23.0, 20.6, 11.4; S: Calcd. for C18H22F3N5:
N-(Tetrahydro-2H-pyran-4-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-
yl)pyrimidin-2-amine Hydrochloride (9z). The compound was prepared using a method
similar to that described for the preparation of 9t, 0.057 g, 28% as a white solid: mp; no clear
melt observed; 1
H NMR (500 MHz, DMSO-d6) δ 1.26 (br s, 2H), 1.50 (br s, 2H), 1.54–1.62 (m,
2H), 1.83 (d, J = 11.0 Hz, 2H), 3.35–3.39 (m, 2H), 3.90 (d, J = 11.0 Hz, 2H), 4.23 (br s, 3H),
7.11 (br s, 1H), 8.00 (br s, 1H), 8.29 (d, J = 6.0 Hz, 1H), 8.50 (br s, 1H); 19
F {1
H} (282 MHz,
CDCl3) δ –67.55 (s); 13
C NMR (125 MHz, DMSO-d6) δ 165.7, 153.7, 146.9, 143.5, 136.6, 126.1
(q, J = 274.2 Hz), 118.5, 106.4, 65.6, 46.5, 32.0, 20.6, 11.5; HRMS: Calcd. for C16H18F3N5O:
354.1542, Found: 354.1544; HPLC: Method 1, tR = 2.48 min, (97.6% AUC).
N-(Tetrahydro-2H-pyran-3-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-
yl)pyrimidin-2-amine (9aa). The compound was prepared using a method similar to that
described for the preparation of 9t, 0.089 g, 80% as a white solid: mp 228–230 °C; 1
H NMR
(300 MHz, DMSOd6) δ 1.29 (br s, 2H), 1.50 (br s, 2H), 1.54–1.74 (m, 3H), 1.93–1.95 (m, 1H),
3.30 (br s, 1H), 3.30 (br s, 1H), 3.34 (br s, 1H), 3.70–3.73 (m, 1H), 3.81–3.86 (m, 1H), 4.22 (br s,
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1H), 7.24 (br s, 1H), 8.32–8.64 (m, 3H); 19
F {1
H} (282 MHz, CDCl3) δ –67.46 (s); 13
C NMR
(125 MHz, DMSO-d6) δ 165.8, 154.0, 146.7, 143.3, 136.7, 126.0 (q, J = 272.9 Hz), 118.3, 106.4,
69.7, 66.9, 46.4, 28.3, 23.6, 20.9, 11.3, 11.2; HRMS: Calcd. for C16H18F3N5O: 354.1542, Found:
354.1536; HPLC: Method 1, tR = 2.69 min, (98.7% AUC).
Preparation of N-(tetrahydrofuran-3-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-
4-yl)pyrimidin-2-amine Hydrochloride (9bb). The compound was prepared using a method
similar to that described for the preparation of 9t, 129.4 mg, 52% as a white solid: 1
H NMR (300
MHz, DMSO-d6): δ 1.29 (m, 2H), 1.52 (m, 2H), 1.88–1.98 (m, 1H), 2.19–2.31 (m, 1H), 3.65–
3.68 (m, 1H), 3.69–3.72 (m, 1H), 3.80–3.94 (m, 2H), 4.68 (m, 1H), 7.23 (d, J = 6.3 Hz, 1H),
8.35 (d, J = 6.3 Hz, 1H), 8.60 (bs, 2H); 19
F {1
H} (282 MHz, DMSO-d6) δ –67.33 (s); 13
C NMR
(125 MHz, DMSO-d6) δ 164.8, 153.9, 147.0, 142.6, 136.1, 125.6 (q, J = 274.2 Hz), 118.2, 106.1,
71.7, 65.8, 51.1, 31.6, 20.1, 11.0; HRMS: Calcd. for C15H16F3N5O: 340.1385, Found: 340.1385;
HPLC: Method 1, tR = 2.52 min, (> 99% AUC).
N-(Oxetan-3-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyridin-2-amine
(9cc). The compound was prepared using a method similar to that described for the preparation
of 9t, 0.024 g, 46% as a yellow solid: mp; no clear melt; 1
H NMR (500 MHz, DMSO-d6) δ 1.23
(s, 2H), 1.51–1.53 (m, 2H), 3.51 (br s, 2H), 4.16–4.20 (m, 1H), 4.22–4.27 (m, 1H), 4.27–4.47 (m,
1H), 5.08 (br s, 1H), 6.77 (br s, 1H), 6.92 (s, 1H), 7.89 (br s, 1H), 8.29 (br s, 1H); 19
F {1
H} (282
MHz, CDCl3) δ –67.16; MS (ESI) m/z 325 [M + H]+.
; HRMS: Calcd. for C15H15F3N4O:
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4-(3-(1-Methylcyclopropyl)-1H-pyrazol-4-yl)-N-(piperidin-4-yl)pyrimidin-2-amine
Dihydrochloride (9ee). The compound was prepared using a method similar to that described
for the preparation of 9t, 0.033 g, 39% as an off-white solid: mp; no clear melt; 1
H NMR (500
MHz, DMSO-d6) δ 0.85–0.95 (m, 4H), 1.38 (s, 3H), 1.75–1.83 (m, 2H), 2.05–2.10 (m, 2H),
3.00–3.10 (m, 2H), 3.30–3.36 (m, 2H), 7.20 (d, J = 5.5 Hz, 1H), 8.20 (br s, 1H), 8.33 (d, J = 5.5
Hz, 1H), 8.81 (br s, 1H), 8.97 (br s, 1H); HRMS: Calcd. for C16H22N6: 299.1984, Found:
299.1992; HPLC: Method 1, tR = 2.34 min, (> 99% AUC).
N-(1-(2,2,2-Trifluoroethyl)piperidin-4-yl)-4-(3-(1-(trifluoromethyl)cyclopropyl)-1H-
pyrazol-4-yl)pyrimidin-2-amine Dihydrochloride (9ff)
Preparation of 2,2,2-Trifluoro-1-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethanone (37). To a
stirred solution at 0 °C of 1,4-dioxa-8-azaspiro[4.5]decane (38) (20.0 g, 0.140 mol),
triethylamine (28.3 g, 0.279 mol), and DMAP (0.86 g, 0.007 mol) in dichloromethane (490 mL)
was added trifluoroacetic anhydride (32.3 g, 0.154 mol) dropwise over 20 min. After the addition
was complete the reaction mixture was stirred at 0 °C for 1 h, then warmed to room temperature
over 4 h. After this time the reaction was quenched with saturated aqueous sodium bicarbonate
(100 mL) and separated. The organic layer was washed with water (100 mL), saturated sodium
chloride (100 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The
yellow oil was dissolved in hot ethyl acetate (200 mL) and precipitate was filtered. The solution
was cooled to room temperature, passed through a plug of silica gel and the filtrate was
concentrated under reduced pressure to afford 37 (32.4 g, 97%) as a yellow oil, which
crystallized to a waxy solid upon standing: 1
H NMR (300 MHz, CDCl3) δ 1.76 (t, J = 6.0 Hz,
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4H), 3.67 (t, J = 5.7 Hz, 2H), 3.77 (t, J = 6.0 Hz, 2H), 4.00 (s, 4H); 19
F {1
H} (282 MHz, CDCl3)
δ –68.91 (s); MS (ESI) m/z 240 [M + H]+
.
Preparation of 8-(2,2,2-Trifluoroethyl)-1,4-dioxa-8-azaspiro[4.5]decane (39). To a stirred
solution of BH3-THF complex in THF (1.0 M; 69.0 mL, 69.0 mmol) was added a solution of 37
(15.0 g, 62.7 mmol) in THF (69.0 mL) dropwise over 10 min. The resulting reaction solution
was heated at reflux for 30 min, cooled to room temperature, an additional portion of BH3-THF
complex in THF (1.0 M; 69.0 mL, 69.0 mmol) was added, and the reaction mixture was heated at
reflux for 24 h. After this time the reaction mixture was cooled to room temperature, carefully
quenched with methanol (100 mL), and the resulting reaction mixture was heated to reflux for 30
min. After this time the reaction mixture was cooled to room temperature and concentrated under
reduced pressure to provide 39 (7.50 g, 53%) as a light yellow oil: 1
H NMR (300 MHz, CDCl3) δ
1.73–1.77 (m, 4H), 2.74–2.79 (m, 4H), 3.01 (q, J = 9.6 Hz, 2H), 3.96 (s, 4H); 19
F {1
H} (282
MHz, CDCl3) δ –69.27 (s); MS (ESI) m/z 226 [M + H]+
.
Preparation of 1-(2,2,2-Trifluoroethyl)piperidin-4-one oxime (40). A stirred solution of 39
(7.43 g, 32.9 mmol) in a 1.0 M hydrochloric acid (125 mL) was heated at reflux for 6 h. After
this time the reaction mixture was cooled in an ice/water bath, the solution was made basic (pH
9) with saturated sodium carbonate, and extracted with MTBE (3 × 150 mL). The combined
organics were washed with saturated sodium chloride, dried (Na2SO4), filtered, and concentrated
under reduced pressure to prove the crude ketone (4.71 g, 79%). The crude ketone was taken up
in absolute ethanol (130 mL) and treated with sodium acetate (3.20 g, 39.0 mmol) and
hydroxylamine hydrochloride (2.17 g. 31.2 mmol). The resulting reaction mixture was stirred at
room temperature for 18 h. After this time the reaction mixture was concentrated under reduced
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pressure, the residue was partitioned between ethyl acetate (200 mL) and water (200 mL), and
separated. The organic layer was washed with saturated sodium chloride, dried (Na2SO4),
filtered, and concentrated under reduced pressure to provide 40 (4.59 g, 90%) as a clear,
colorless oil: 1
H NMR (300 MHz, CDCl3) δ 2.37 (t, J = 6.3 Hz, 2H), 2.67 (t, J = 6.3 Hz, 2H),
2.70–2.84 (m, 4H), 3.05 (q, J = 9.6 Hz, 2H), 7.86 (s, 1H); 19
F {1
H} (282 MHz, CDCl3) δ –69.26
(s); Note: Could not obtain adequate ionization for a mass spectrum.
Preparation of 1-(2,2,2-Trifluoroethyl)piperidin-4-amine (36). A Parr bottle charged with a
solution of 40 (3.51 g, 17.9 mmol) in absolute ethanol (45.0 mL) was purged with nitrogen and
Raney nickel catalyst (Grade 2800 50 wt. % slurry in water) (1.8 mL) was added to the solution.
The reactor bottle was purged with hydrogen (3×), the pressure was maintained at 30–40 psi, and
shaken for 72 h at room temperature. After this time the reaction bottle was purged with
nitrogen, the catalyst was carefully removed over a pad of Celite, and the filtrate was
concentrated under reduced pressure to afford 36 (2.28 g, 70%) as a light-yellow oil: 1
H NMR
(300 MHz, CDCl3) δ 1.42–1.50 (m, 2H), 1.70–1.86 (m, 2H), 1.90 (br s, 2H), 2.30–2.47 (m, 2H),
2.59–2.72 (m, 1H), 2.80-3.05 (m, 4H); 19
F {1
H} (282 MHz, CDCl3) δ –69.05 (s); Note: Could
not obtain adequate ionization for a mass spectrum.
Preparation of N-(1-(2,2,2-Trifluoroethyl)piperidin-4-yl)-4-(3-(1-
(trifluoromethyl)cyclopropyl)-1H-pyrazol-4-yl)pyrimidin-2-amine Dihydrochloride (9ff).
The compound was prepared using a method similar to that described for the preparation of 9t
using 36 as the amine, 0.113 g, 30% as an off-white powder: mp 84–87 °C; 1
H NMR (500 MHz,
DMSO-d6) δ 1.25 (s, 2H), 1.51 (s, 2H), 1.65–2.10 (m, 6H), 2.80–3.00 (m, 2H), 3.10–4.90 (m,
6H), 7.26 (d, J = 5.5 Hz, 1H), 8.38 (d, J = 5.5 Hz, 1H), 8.66 (br s, 1H), 8.88 (br s, 1H); 19
F {1
H}
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Huntington's disease JNK inhibitor optimization

Huntington's disease JNK inhibitor optimization

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