Discovery of highly potent, selective, and brain-penetrant aminopyrazole Leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors

Article abstract of DOI:10.1021/jm401654j

Leucine-rich repeat kinase 2 (LRRK2) has drawn significant interest in the neuroscience research community because it is one of the most compelling targets for a potential disease-modifying Parkinson's disease therapy. Herein, we disclose structurally div

Full text of DOI:10.1021/jm401654j

Article
pubs.acs.org/jmc
Discovery of Highly Potent, Selective, and Brain-Penetrant
Aminopyrazole Leucine-Rich Repeat Kinase 2 (LRRK2) Small Molecule
Inhibitors
Anthony A. Estrada,*,† Bryan K. Chan,*,† Charles Baker-Glenn,∇ Alan Beresford,◆ Daniel J. Burdick,†
Mark Chambers,∇ Huifen Chen,† Sara L. Dominguez,‡ Jennafer Dotson,† Jason Drummond,§
Michael Flagella,⊥ Reina Fuji,⊥ Andrew Gill,○ Jason Halladay,∥ Seth F. Harris,# Timothy P. Heffron,†
Tracy Kleinheinz,§ Donna W. Lee,⊥ Claire E. Le Pichon,‡ Xingrong Liu,∥ Joseph P. Lyssikatos,†
Andrew D. Medhurst,○ John G. Moffat,§ Kevin Nash,○ Kimberly Scearce-Levie,‡ Zejuan Sheng,‡
Daniel G. Shore,† Susan Wong,∥ Shuo Zhang,‡ Xiaolin Zhang,∥ Haitao Zhu,‡ and Zachary K. Sweeney†
†
‡
§
∥
Departments of Discovery Chemistry, Neurosciences, Biochemical and Cellular Pharmacology, Drug Metabolism and
Pharmacokinetics, ⊥Safety Assessment, and #Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080,
United States
Departments of ∇Chemistry, ○Biochemical and Cellular Pharmacology, and ◆Drug Metabolism and Pharmacokinetics, BioFocus,
Chesterford Research Park, Saffron Walden, CB10 1XL, United Kingdom
S
* Supporting Information
ABSTRACT: Leucine-rich repeat kinase 2 (LRRK2) has drawn significant
interest in the neuroscience research community because it is one of the most
compelling targets for a potential disease-modifying Parkinson’s disease therapy.
Herein, we disclose structurally diverse small molecule inhibitors suitable for
assessing the implications of sustained in vivo LRRK2 inhibition. Using
previously reported aminopyrazole 2 as a lead molecule, we were able to
engineer structural modifications in the solvent-exposed region of the ATP-
binding site that significantly improve human hepatocyte stability, rat free brain
exposure, and CYP inhibition and induction liabilities. Disciplined application
of established optimal CNS design parameters culminated in the rapid
identification of GNE-0877 (11) and GNE-9605 (20) as highly potent and
selective LRRK2 inhibitors. The demonstrated metabolic stability, brain
penetration across multiple species, and selectivity of these inhibitors support
their use in preclinical efficacy and safety studies.
therapy could benefit both sporadic and familial cases of
PD.9−11 Multiple LRRK2 mutations have been reported to
demonstrate increased kinase activity in vitro and in vivo, with
the most common being the autosomal dominant G2019S
mutation that resides in the activation loop of the kinase
domain.12−18 As a result, several groups have recently reported
structurally diverse small molecule LRRK2 kinase inhibitors.
These molecules should facilitate efforts to unlock the normal
functions of LRRK2, the role of LRRK2 in PD and other
diseases,19,20 and the consequences of prolonged inhibition in
preclinical species.21−35
Our group was the first to report on a series of potent,
selective, and brain-penetrable aminopyrimidine LRRK2
inhibitors.24,36 The demonstrated kinase selectivity was
achieved through the discovery and exploitation of a selectivity
hotspot that was identified through kinase counterscreening
INTRODUCTION
■
Parkinson’s disease (PD) is a degenerative disorder of the
central nervous system (CNS) that affects approximately 5% of
the population greater than 80 years of age.1 It is characterized
pathologically by gradual loss of dopaminergic neurons and
dopamine secretion in the substantia nigra, and a disease-
modifying or neuroprotective therapy remains a major unmet
medical need. Currently available PD treatments only address
the dopamine deficiencies characteristic of the disorder, and the
most widely used symptomatic treatment, levodopa (L-dopa),
often causes L-dopa-induced dyskinesias (LID) after prolonged
use.2,3 The LRRK2 gene is a multidomain protein comprising
2527 amino acids and is expressed throughout the brain and
other peripheral organs. The association of this protein with PD
was established in 2004, and it has since been determined that
mutations in the LRRK2 gene are the most common cause of
familial PD.4−8 Furthermore, LRRK2 genetic polymorphisms
have been linked to idiopathic PD in terms of age of onset,
symptoms, and pathology, suggesting that a LRRK2-derived
Received: October 25, 2013
© XXXX American Chemical Society
A
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and a matched molecular pair activity cliff analysis.24,37−42 Lead
optimization using property- and structure-based drug design
provided highly efficient LRRK2 inhibitors such as GNE-7915
(1, Figure 1)25 with excellent selectivity against the broader
1 μM). A docking overlay of inhibitors 1 and 2 using our
LRRK2 homology model can be visualized in Figure 2.24
Figure 1. Previously reported LRRK2 diaminopyrimidine inhib-
itors.25,26
kinome and good free brain-to-plasma ratios across preclinical
species. Experiments employing 1 and additional lead
compounds demonstrated potent cellular activity and robust
PK/PD responses in G2019S bacterial artificial chromosome
(BAC) transgenic mice. These studies were enabled by the
discovery of Ser1292 as a direct, specific autophosphorylation
site suitable for use as an in vitro and in vivo biomarker of
LRRK2 activity.43 In an effort to reduce size, improve aqueous
solubility (thermodynamic solubility of crystalline 1 at pH 7.4 <
2 μg mL−1), and eliminate the potential for ortho-quinoneimine
reactive metabolite formation, we successfully designed a series
of aminopyrazoles such as 2 (LELP44,45 = 5.7; thermodynamic
solubility of crystalline 2 at pH 7.4 = 20 μg mL−1) as aniline
bioisosteres (Figure 1).26 In this contribution, we report our
successful efforts to identify improved aminopyrazole LRRK2
inhibitors that possess profiles suitable for early development
evaluation.
Figure 2. Docking model of 1 (cyan) and 2 (magenta) in LRRK2
(green). The side-chain Y931 of JAK2 is also shown (yellow).
Intermolecular hydrogen-bond interactions are shown as yellow
dashed lines.
Gratifyingly, JAK2 continued to serve as a reliable indicator of
general kinase selectivity for aminopyrazoles with a chloro or
methyl substituent ortho to the aminopyrimidine hinge binder
(an unfavorable proximity of the chloro substituent of inhibitor
2 to Y931 of JAK2 can be seen in Figure 2).24,25 All of the
inhibitors disclosed herein have a JAK2 biochemical selectivity
index of >150-fold unless otherwise specified. Compound 2
also possesses good human liver microsomal (LM) stability and
shows a favorable rat in vitro−in vivo stability correlation with
excellent free-drug clearance (Table 1). Unfortunately, upon
RESULTS AND DISCUSSION
■
As previously reported,26 aminopyrazole 2 has LRRK2
biochemical and cellular potencies of 9 and 28 nM, respectively
(Table 1), and demonstrates very good kinase selectivity (185
kinases, 116-fold over LRRK2 Ki, 1 kinase at >75% inhibition at
a
Table 1. Summarized Profile of Aminopyrazole 2 and Representative SAR for Fused Morpholinopyrazoles
h
LM Clhep
hep Clhep
MDR1
i
d
f
LRRK2 Ki
pLRRK2 IC50
(mL min−1 kg−1
)
(mL min−1 kg−1
)
rat CI (Clu)
P-gp ER
(B:A/A:B)
rat
b
c
e
e
g
j
k
compd
R1
R2
Me
R3
(nM)
(nM)
h/r
h/r
(mL min−1 kg−1
)
Bu/Pu
2
3
4
5
9
28
20
18
49
42
3/13
10/18
21(84)
0.9
0.7
1.0
1.0
1.3
0.37
H
Me
H
0.9
3
11/47
14/17
8/32
12/−
H
H
l
Me
Me
(S)-Me
H
6
8/−
3/19
l
6
H
H
3
4/11
43 (105)
0.2
a
Compounds were dosed po (1 mg kg−1) as an aqueous suspension with 1% methylcellulose, iv (0.5 mg kg−1) as either a 60% PEG solution or a
b
c
20−60% NMP solution for systemic PK, and iv (0.5 mg kg−1) as a 60% NMP solution for brain PK. Biochemical assay. Cellular assay; all
biochemical and cellular assay results represent the arithmetic mean of a minimum of two determinations, and these assays generally produced
results within 3-fold of the reported mean. Liver microsome-predicted hepatic clearance. h/r = human/rat. In vitro stability in cryopreserved
hepatocytes. Clu = unbound clearance = total clearance/fup, where fup is the unbound plasma free fraction. MDCK-MDR1 human P-gp transfected
d
e
f
g
h
i
j
k
l
cell line. Efflux ratio. Basolateral-to-apical/apical-to-basolateral. Unbound brain/unbound plasma AUC ratio. Absolute stereochemistry unknown.
B
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expanded profiling, we observed higher turnover when inhibitor
2 was incubated with human hepatocytes (10 mL min−1 kg−1,
Table 1), and in vitro metabolite identification studies revealed
high levels of glucuronidated parent (>60%) after 3 h.
Additionally, the unbound brain/unbound plasma concen-
tration (Bu/Pu) ratio for 2 was suboptimal (Bu/Pu = 0.37, Table
1), and bioavailability in cynomolgus monkey pharmacokinetic
(PK) studies was poor (oral bioavailability 5% at 1 and 20 mg
kg−1), potentially because of high intestinal metabolism via
glucuronidation. As a consequence, there was significant
uncertainty in the human PK prediction for compound 2 and
thus further progression was halted.
Table 2. Investigation of Pyrazole Amide Substitutions
d
LM Clhep
MDR1
e
b
pLRRK2 IC50 (mL min−1 kg−1
)
P-gp ER
f
a
c
compd
R1
(nM)
h/r
(B:A/A:B)
Our initial strategy to address the issues with aminopyrazole
2 led to the fused morpholinopyrazoles shown in Table 1. We
hypothesized that cyclization of the tertiary alcohol would
potentially improve the Bu/Pu concentration ratio by lowering
topological polar surface area (TPSA) and removing one
hydrogen-bond donor (HBD). Additionally, we hoped to
improve metabolic stability by reducing the number of rotatable
bonds, increasing rigidity, and attenuating the observed
glucuronidation with tertiary alcohol 2. Thus, morpholinopyr-
azoles 3, 4, and 5 (Table 1) were prepared. Although all of
these fused bicycles showed similar cellular potencies as 2 and
lacked P-gp-mediated efflux in vitro, desirable microsomal
stability improvements were not achieved. Pleasingly, mono-
methyl-substituted morpholine 6 (Table 1) demonstrated low
turnover in both human LM and hepatocytes and retained
good cellular activity. Assessment of the rat in vivo PK profile of
6 revealed low free-drug clearance (105 mL min−1 kg−1), but a
slight in vitro−in vivo disconnection reduced our confidence in
the improved in vitro human stability relative to our previous
lead (2). Additionally, brain cassette rat PK studies of 6
revealed an unacceptable Bu/Pu concentration ratio (0.2, Table
1).25,46 As a result, further evaluation of 6 was halted in favor of
pursuing inhibitors with superior profiles.
In addition to pursuing bicyclic pyrazoles, a separate parallel
strategy to identify polar, metabolically stable pyrazole N-
substituents was developed. We have previously shown that
simple, linear N-alkyl-substituted pyrazoles exhibited potent
reversible and time-dependent inhibition (TDI) of CYP1A2
(IC50 values typically <1 μM) and that branching, as in
inhibitor 2, reproducibly attenuates CYP1A2 inhibition. A
similar strategy was adopted for the pyrazole amides shown in
Table 2. A gem-dimethyl linker was designed to reduce the risk
of CYP1A2 inhibition, to improve metabolic stability versus the
methylene counterpart, and to facilitate potential intra-
molecular hydrogen bonding between the amide N−H and
pyrazole nitrogen via the Thorpe−Ingold effect.47,48 We were
hopeful that the latter would reduce the efflux potential of the
amide inhibitors because amides are known substrates of P-gp
and the TPSA of 7−10 (>95 Å2) are above suggested values for
brain-penetrant small molecules.49,50
7
Me
Et
36
6/8
5.5
4.0
1.3
1.5
8
99
8/27
7/36
9/27
9
iPr
140
53
10
−CH2CF3
a
Cellular assay; all cellular assay results represent the arithmetic mean
of a minimum of two determinations, and these assays generally
produced results within 3-fold of the reported mean. Liver
microsome-predicted hepatic clearance. h/r = human/rat. MDCK-
MDR1 human P-gp transfected cell line. Efflux ratio. Basolateral-to-
apical/apical-to-basolateral.
b
c
d
e
f
successful in reducing the P-gp efflux for 9 and 10, modest
cellular potencies of compounds in this series discouraged
further progression.51
A series of gem-disubstituted cyano pyrazole LRRK2
inhibitors were then prepared via dehydration of the precursor
amide. We were hopeful that removing the amide functionality
and maintaining a low MW would enhance brain penetration.
Validation of this strategy was provided with GNE-0877 (11,
Table 3), which showed significantly enhanced LRRK2 cellular
potency (3 nM) and low turnover in human liver microsomes
and hepatocytes with no evidence of glucuronidation. We
hypothesize that the significant enhancement in cellular
potency is primarily due to a better fit and tighter binding of
the less bulky gem-dimethyl cyano group. The flexibility
afforded by the lack of intramolecular hydrogen bonding should
allow the methyl groups to form better van der Waals contacts
with the hydrophobic residues in the protein, whereas the
cyano group can form favorable electrostatic interactions with
the side chain of Arg1957. Invitrogen kinase-selectivity profiling
(188 kinases) of aminopyrazole 11 at 0.1 μM (145-fold over
LRRK2 Ki) resulted in only four kinases showing greater than
50% inhibition (Aurora B = 51%, RSK2 = 52%, RSK4 = 62%,
and RSK3 = 68%) and suggested that 11 is a highly selective
LRRK2 inhibitor. Furthermore, 11 possessed a 212-fold
biochemical-selectivity index over TTK (Ki = 150 nM),
which was previously highlighted as an off-target kinase of
concern because of the suggested role of TTK in the
maintenance of chromosomal stability.25
The in vivo rat clearance for inhibitor 11 was within 2-fold of
measured in vitro stability, and good oral bioavailability (88%)
was achieved at 50 mg kg−1 following administration of a
methylcellulose/tween (MCT) suspension. Crystalline 11 also
demonstrated good thermodynamic solubility (pH 7.4
solubility = 100 μg mL−1). Importantly, the absence of in
vitro P-gp efflux translated to a good in vivo rat free brain-to-
plasma ratio (0.6) and corresponding CSF-to-free plasma ratio
(0.9).52,53 Despite previous success in eliminating CYP1A2
inhibition with alkyl branching of the pyrazole N-substituents,26
11 was found to be a reversible CYP1A2 inhibitor (IC50 = 0.7
μM), but this compound did not exhibit TDI of CYP1A2.
The first amide synthesized (7, Table 2) possessed good
LRRK2 cellular activity and LM stability and was devoid of
CYP inhibition. Unlike morpholinopyrazole 6, compound 7
also showed a promising in vitro−in vivo correlation (rat iv Cl
= 12 mL min−1 kg−1; Clu = 27 mL min−1 kg−1). Unfortunately,
as predicted by the high TPSA (96 Å2), 7 was effluxed in the
MDCK-MDR1 cell line and exhibited poor in vivo brain
penetration (rat Bu/Pu = 0.08). Attempts to mask further or
block P-gp recognition of the amide motif led to the
preparation of ethyl- (8), isopropyl- (9), and trifluoroethyl-
substituted (10) secondary amides. Although we were
C
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a
Table 3. gem-Disubstituted Aminopyrazole SAR and DMPK Profiles
a
Compounds were dosed po (1 mg kg−1) as an aqueous suspension with 1% methylcellulose, iv (0.5 mg kg−1) as either a 60% PEG solution or a
b
c
20−60% NMP solution for systemic PK, and iv (0.5 mg kg−1) as a 60% NMP solution for brain PK. Biochemical assay. Cellular assay; all
biochemical and cellular assay results represent the arithmetic mean of a minimum of two determinations, and these assays generally produced
results within 3-fold of the reported mean. Liver microsome-predicted hepatic clearance. h/r = human/rat. In vitro stability in cryopreserved
hepatocytes. Clu = unbound clearance = total clearance/fup, where fup is the unbound plasma free fraction. MDCK-MDR1 human P-gp transfected
d
e
f
g
h
i
j
k
l
cell line. Efflux ratio. Basolateral-to-apical/apical-to-basolateral. Unbound brain/unbound plasma AUC ratio. NT = not tested.
Additional profiling suggested that 11 was a pan-inducer of
CYP enzymes in human hepatocytes regulated through nuclear
receptors PXR (3A4), AhR (1A2), and CAR (2B6). There are
several literature examples where minor structural modifications
and/or stereoisomers can alleviate the induction potential of
small molecule inhibitors.54 We therefore targeted close-in
analogues of 11 such as pyrazole regioisomer 12 (Table 3),
spiro-cyclopropyl matched pair 13, spiro-cyclopropyl chlor-
opyrazole 14, and carbon-linked gem-dimethyl nitrile 15.
Although these compounds exhibited similar in vitro and in
vivo profiles as 11 and several showed reduced CYP1A2
potential liabilities, all of these inhibitors suffered from variable
levels of CYP induction.
It was then hypothesized that incorporating polarity more
distal to the pyrazole ring while maintaining branched pyrazole
N-alkyl substitutions for CYP1A2 inhibition relief55,56 may be
necessary to attenuate the observed human CYP induc-
tion.57−59 To minimize the number of rotatable bonds, the
compact N-oxetyl pyrazole analogue (16, Table 4) was
synthesized. Because of the structural similarity of the oxetane
to the N-capping group of compound 2, it was hoped that the
N-oxetyl pyrazole would retain the favorable properties of 2
without suffering from glucuronidation of the related alcohol.
Indeed, 16 demonstrated good LRRK2 cellular potency
(pLRRK2 IC50 = 29 nM) and excellent stability upon
incubation in hepatocytes (human hep Clhep = 2 mL min−1
kg−1). Unfortunately, although 16 did not show significant
reversible CYP inhibition, time-dependent inhibition of
CYP1A2 was observed (IC50 = 2 μM). This result suggested
that more sterically encumbered substitutions, such as a
tetrahydropyranyl (THP) group, may be required for
attenuation of CYP1A2 inhibition. As predicted, THP analogue
17 showed excellent LRRK2 potency and in vitro metabolic
stability similar to that of 16, whereas it showed no evidence of
time-dependent inhibition of CYP1A2.
Because of its favorable cellular potency and in vitro
metabolic stability, 17 was progressed into in vivo PK
evaluation. Intravenous dosing of 17 at 0.5 mpk in rats
returned a total plasma clearance of 74 mL min−1 kg−1 and a
volume of distribution of 1.1 L kg−1. The resultant short half-
life (0.17 h), potential for extrahepatic clearance, undesired in
vitro−in vivo disconnection, and suboptimal brain penetration
(Bu/Pu = 0.3) halted the progression of 17. Other minor
modifications of 17 involving substitution of the THP ring and
incorporation of asymmetry (data not shown) did not reduce in
vivo clearance.
With the uncertainties surrounding the efficacy drivers for
disease modification, it was a program goal to ensure that
adequate half-lives were achieved by our LRRK2 inhibitors. We
envisioned that the incorporation of a weakly basic group into
the solvent-exposed region of our inhibitor would potentially
increase the volume of distribution and thereby extend the in
D
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a
Table 4. Oxetyl, Pyranyl, and Piperidinyl Aminopyrazole SAR and DMPK Profiles
a
Compounds were dosed po (1 mg kg−1) as an aqueous suspension with 1% methylcellulose, iv (0.5 mg kg−1) as either a 60% PEG solution or a
b
c
20−60% NMP solution for systemic PK, and iv (0.5 mg kg−1) as a 60% NMP solution for brain PK. Biochemical assay. Cellular assay; all
biochemical and cellular assay results represent the arithmetic mean of a minimum of two determinations, and these assays generally produced
results within 3-fold of the reported mean. Liver microsome-predicted hepatic clearance. h/r = human/rat. In vitro stability in cryopreserved
hepatocytes. Clu = unbound clearance = total clearance/fup, where fup is the unbound plasma free fraction. MDCK-MDR1 human P-gp transfected
d
e
f
g
h
i
j
k
l
cell line. Efflux ratio. Basolateral-to-apical/apical-to-basolateral. Unbound brain/unbound plasma AUC ratio. Absolute stereochemistry unknown.
vivo half-life without compromising potency. Replacement of
the THP group in 17 with a piperidine-N-oxetyl moiety
afforded 18 (Table 4). As predicted, 18 showed good LRRK2
biochemical (LRRK2 Ki = 4 nM) and cellular potency
(pLRRK2 IC50 = 20 nM) with no evidence of reversible or
time-dependent CYP inhibition. The in vivo rat clearance (Clp
= 22 mL min−1 kg−1) correlated well with the in vitro metabolic
stability results (rLM = 16.3 mL min−1 kg−1). In addition, the
volume of distribution (1.7 L kg−1) was slightly higher than 17,
leading to a significantly improved half-life (2.1 h). Despite
attempts to modulate the basicity of the piperidine nitrogen
with the electron-withdrawing oxetyl group, inhibitor 18 (cpKa
= 7.6) showed significant in vivo brain-penetration impairment
(Bu/Pu = 0.06). MDCK-MDR1 results confirmed 18 to be a
substrate of P-gp (ER = 5.1). Nonetheless, the low unbound
clearance suggested that the inhibitor possessed excellent
systemic metabolic stability, which supported further opti-
mization of 18.
fluoro-piperidinyl system to give 19. Although MDCK-MDR1
results suggested an improved efflux ratio (ER = 2.2),
fluoropiperidine 19 showed minimal improvement in in vivo
unbound brain penetration (Bu/Pu = 0.08). One potential
explanation for the impaired brain penetration could be
attributed to efflux by the BCRP transporter (MDCK-BCRP
ER = 4.2). During the optimization leading to tertiary alcohol 2,
it was recognized that the replacement of the methylpyrazole
with a chloropyrazole within the current series of LRRK2
aminopyrazole inhibitors could translate to improved brain
penetration. In addition to the obvious structural difference, it
was also envisioned that such a methyl-to-chloro substitution
would increase the lipophilicity of the inhibitor (ΔcLogD7.4
=
+0.4), thereby potentially leading to improved intrinsic
permeability. Indeed, notable improvement in the MDCK-
MDR1 and MDCK-BCRP efflux ratios (ER = 0.80 and 0.90,
respectively) was observed for chloropyrazole analogue GNE-
9605 (20). Despite the minor increase in lipophilicity, 20
retained excellent predicted human metabolic stability when
assayed in human liver microsomes and hepatocytes. In
addition, no reversible or time-dependent inhibition of any of
To reduce further the basicity of the inhibitor without
significantly altering the structure and overall physicochemical
properties, the piperidinyl group in 18 was exchanged for a 3-
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the major CYP isoforms was observed. Similar to chloropyr-
azole 2, less than 0.2% of trapping adducts were observed when
20 was incubated in human liver microsomes in the presence of
potassium cyanide, and no adducts were detected after
incubation with glutathione and methoxyamine. Importantly,
our strategy to attenuate the CYP induction observed with
other aminopyrazole inhibitors by installing polarity distal to
the pyrazole ring proved successful, as 20 was not an inducer of
any CYP isoform tested (3A4, 1A2, and 2B6).
Inhibitor 20 was highly potent against LRRK2 in both
biochemical (Ki = 2.0 nM) and cellular (IC50 = 18.7 nM)
assays. When assayed against a 178-membered Invitrogen
kinase panel (0.1 μM; 50-fold over LRRK2 Ki), 20 only
inhibited 1 kinase greater than 50% (TAK1-TAB1 = 54%). In
addition, 20 did not exhibit any inhibitory activity against a
representative panel of receptors and ion channels. In vivo rat
PK studies with 20 demonstrated a total plasma clearance of 26
mL min−1 kg−1 (Table 4) with excellent oral bioavailability
(90%). As predicted by the in vitro permeability data, unbound
brain-to-plasma and CSF-to-unbound plasma AUC ratios were
between 0.5 and 1.0.
Promising aminopyrazole inhibitors 11 and 20 were
evaluated for their ability to inhibit in vivo LRRK2 Ser1292
autophosphorylation using BAC transgenic mice expressing
human LRRK2 protein with the G2019S Parkinson’s disease
mutation (Figure 3). Tissues from the hippocampus (brain)
were harvested at 1, 3, and 6 h post intraperotineal (ip)
injection at 10 and 50 mg kg−1 to evaluate pharmacodynamic
knockdown.43 Using free-drug concentrations, robust concen-
tration-dependent inhibition of Ser1292 autophosphorylation
was observed for both inhibitors. In vivo unbound brain IC50
values of 3 and 20 nM were calculated for 11 and 20,
respectively, using a pharmacodynamic inhibition model.
Cynomolgus monkey PK studies were carried out with 11
and 20 because these inhibitors possessed well-balanced
profiles and satisfied our target candidate profile criteria. Both
11 and 20 demonstrated excellent in vitro−in vivo correlations
and free-drug clearance values ≤100 mL min−1 kg−1. As
anticipated from in vitro human MDR1 permeability data and
in vivo rat brain-penetration studies, 11 and 20 exhibited
excellent brain penetration in higher species (cyno CSF/Pu =
1.2 and 1.1, respectively, Table 5). For several reasons, inhibitor
11 was chosen as a representative of the pyrazole series for
further evaluation in preclinical toxicity and genotoxicity
studies: (a) approximately 10-fold more potent in vivo
unbound brain IC50 for 11 compared to 20, (b) in vivo
multiday PK studies in rat and cyno with 11 demonstrated
excellent and sustained oral exposure without evidence of
autoinduction, which may suggest species-dependent nuclear
receptor binding,60,61 and (c) human induction potential of 11
could be derisked in early clinical trials. In assessing the
genotoxicity risk of pyrazole 11, it was determined that the
inhibitor did not induce increases in micronucleated poly-
chromatic erythrocytes after administration of 11 daily for 2
days at 200 mg kg−1 (mean unbound plasma concentration =
4.8 μM). The results of preclinical multiday toxicity studies in
both rats and monkeys employing 11 and previously disclosed
aminopyrimidine 1 will be disclosed shortly.
Figure 3. In vivo G2019S LRRK2 transgenic mouse PK/PD results
measuring brain pSer1292 autophosphorylation. The circles represent
the observed data, and the lines represent the predicted data from a
direct inhibition model. Percent inhibition is normalized to pSer1292
levels observed in mice dosed with vehicle alone (n = 3). Plotted data
are shown for mice treated with (A) 11 [10 and 50 mg kg−1, ip, at 1, 3,
and 6 h (n = 3/dose)] and (B) 20 [10 and 50 mg kg−1, ip, at 1 and 6 h
(n = 3/dose)].
trimethylsilyldiazomethane followed by debenzylation afforded
pyrazoles 23. Installation of the nitro group and activation of
the hydroxyl group were achieved via NBS bromination and
nitration. Treatment of crude 24 with sodium hydride triggered
the ring-closing alkylation to give the [5,6]-fused nitro-
pyrazoles. Reduction of the nitropyrazole to the corresponding
aminopyrazole followed by a standard SNAr reaction with 2-
chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (26)
provided 3−6.
The syntheses of 7−11 were carried out as outlined in
Scheme 2. Alkylation of 5-methyl-4-nitropyrazole with methyl
2-bromo-2-methylpropanoate followed by saponification of the
methyl ester yielded common intermediate 28. Amide coupling,
nitro-group reduction, and acid- or base-catalyzed addition to
26 yielded inhibitors 7−10. Dehydration of primary amide 34
using phosphorus oxychloride yielded 11. Close-in analogues of
11, inhibitors 12 and 13, were prepared similarly according to
Scheme 3. The preparation of 5-chloropyrazole 14 began with
the cyclopropanation of acetate 39 via a bis-alkylation protocol
(Scheme 4). Regioselective lithiation of the resulting nitro-
pyrazole followed by trapping with hexachloroethane installed
the C5-chloro substitution. The synthesis was then completed
CHEMISTRY
■
Fused bicyclic inhibitors 3−6 were prepared according to
Scheme 1. Propargylation of alcohols 21 yielded the cyclo-
addition precursors 22. Formal cycloadditions between 22 and
F
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a
Table 5. In Vivo Cyno PK Profiles of 11 and 20
hepatocytes Clhep
b
(mL min−1 kg−1
cyno
)
CI (Clu)
iv t1/2
(h)
F(%)
c
d
compd
% cyno PPB
(mL min−1 kg−1)
1 mg kg−1
CSF/Pu
11
20
19
13
80 3
82.1
19.7 (100)
7.8 (43)
2.2
4.0
35
74
1.2
1.1
a
Compounds were dosed po (1 mg kg−1) with crystalline material as an aqueous suspension with 1% methylcellulose and iv (0.5 mg kg−1) as a 20−
b
c
60% NMP solution. In vitro stability in cryopreserved hepatocytes. Clu = unbound clearance = total clearance/fup, where fup is the unbound plasma
free fraction. CSF/unbound plasma AUC ratio.
d
give aminopyrazoles 45 and 46 (4-amino-3-methylpyrazole
analogues not shown). Finally, palladium-catalyzed coupling
with 26 afforded a mixture of the isomeric products, which
were separated by preparative HPLC.
Scheme 1. Synthesis of Fused Morpholinopyrazole
Analogues
a
Piperidine-based inhibitors 18−20 were synthesized as
outlined in Schemes 7 and 8. Installation of the N-piperidinyl
moiety was achieved via alkylation or Mitsunobu reactions. N-
Boc deprotection by treatment with HCl in dioxane and
reductive amination using oxetan-3-one provided the N-oxetyl-
piperidine intermediates. Installation of the 5-chloro sub-
stitution to give 53 (Scheme 8) was accomplished via lithiation
and hexachloroethane trapping as described above. Standard
reduction of the nitropyrazole using either iron or palladium-
catalyzed hydrogenation set the stage for the acid-catalyzed
SNAr reactions to give the final inhibitors as enantiomeric
mixtures. The individual enantiomers were separated by SFC
purification.
a
Reagents and conditions: (a) NaH, 3-bromoprop-1-yne, THF, 0 °C;
(b) Me3SiCHN2, 135 °C, microwave; (c) H2, Pd(OH)2, EtOH, 100
°C; (d) NBS, CH3CN; (e) HNO3, 0 °C; (f) NaH, DMF, 0 °C; (g)
hydrazine hydrate, Raney nickel, THF, MeOH, 0 °C; (h) 26, t-BuOH,
100 °C.
a
CONCLUSIONS
Scheme 2. Synthesis of N-Alkyl Pyrazole Analogues
■
Expanding upon the identification of aminopyrazoles as aniline
bioisosteres, we identified 11 and 20 as highly efficient, LRRK2-
specific small molecule inhibitors with ADME profiles suitable
for multiday efficacy and toxicity studies. Both inhibitors
demonstrated robust, concentration-dependent knockdown of
pLRRK2 in the brain of G2019S transgenic mice expressing
human LRRK2 protein. Advantages of the aminopyrazole series
include elimination of potential anilino-derived ortho-quinonei-
mine reactive metabolite formation, good overall aqueous
solubility, and low free-drug clearance values demonstrated by
multiple compounds. The aminopyrazole scaffold is also
structurally differentiated from the majority of LRRK2
inhibitors disclosed in the literature, which should prove
beneficial in assessing the risk of prolonged inhibition of
LRRK2 in vivo. A detailed report of toxicity studies and
associated histopathology findings with 11 and previously
disclosed anilino-aminopyrimidine LRRK2 inhibitor 1 will be
reported shortly.
a
Reagents and conditions: (a) NaH, methyl 2-bromo-2-methylpropa-
noate, DMF, 70%; (b) LiOH, THF-H2O, 90%; (c) (i) (COCl)2,
CH2Cl2, (ii) R-NH2, THF; (d) Pd/C, H2, MeOH; (e) 26, Et3N, n-
BuOH, 120 °C; (f) 26, TFA, 2-methoxyethanol, 70 °C; (g) POCl3, 90
°C, 42%.
EXPERIMENTAL SECTION
■
All chemicals were purchased from commercial suppliers and used as
received. Flash chromatography was carried out with prepacked SiO2
cartridges from either ISCO or SiliCycle on an ISCO CombiFlash
chromatography system using gradient elution or with prepacked silica
gel cartridges from Biotage using a Biotage SP4 or an Isolara 4 MPLC
system using gradient elution. NMR spectra were recorded on a
Bruker Avance 400, Bruker DPX 400M, or Bruker Avance III 400 or
500 NMR spectrometers and referenced to tetramethylsilane. The
following abbreviations are used: br = broad signal, s = singlet, d =
doublet, dd = doublet of doublets, t = triplet, q = quartet, and m =
multiplet. Preparative HPLC was performed on a Polaris C18 5 μM
column (50 × 21 mm), a Waters Sunfire OBD Phenomenex Luna
Phenyl Hexyl column (150 × 19 mm), or a Waters Xbridge Phenyl
column (150 × 19 mm), eluting with mixtures of water−acetonitrile or
water−methanol, optionally containing a modifier (0.1% v/v formic
in a manner analogous to that which yielded compound 11. C-
linked analogue 15 was prepared via a straightforward two-step
sequence (Scheme 5). Cyclization between methylhydrazine
and 2,2-dimethyl-3-oxopentanedinitrile (42) provided amino-
pyrazole 43. Acid-catalyzed coupling with 2-chloro-N-ethyl-5-
(trifluoromethyl)pyrimidin-4-amine (44) yielded 15.
Compounds 16 and 17 were synthesized according to
Scheme 6. Alkylation of 5-methyl-4-nitropyrazole yielded an
isomeric mixture of the corresponding N-alkyl-5-methyl-4-
nitropyrazole and N-alkyl-3-methyl-4-nitropyrazole. The mix-
tures of regioisomers were then reduced via hydrogenation to
G
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a
Scheme 3. Synthesis of Analogues 12 and 13
a
Reagents and conditions: (a) Cs2CO3, methyl 2-bromoacetate, DMF, 89%; (b) NaH, CH3I, DMF, 39%; (c) (i) LiOH, THF, H2O, (ii) (COCl)2,
cat. DMF, DCM, (iii) NH4OH, THF, 86%; (d) Pd/C, H2, EtOH; (e) 26, TFA, 2-methoxyethanol, 90 °C; (f) POCl3, 90 °C; (g) NaH, ethylene
dibromide, DMF, 0 °C, 40%.
a
Scheme 4. Synthesis of Inhibitor 14
a
Reagents and conditions: (a) NaH, ethylene dibromide, DMF, 0 °C, 20%; (b) LiHMDS, Cl3CCCl3, −78 °C, THF, 54%; (c) (i) LiOH, water, THF,
(ii) (COCl)2, cat. DMF, CH2Cl2, (iii) NH4OH, THF, 69%, three steps; (d) iron dust, NH4Cl, EtOH, quant.; (e) 26, TFA, 2-methoxyethanol, 70 °C;
(f) POCl3, 90 °C, 30%, two steps.
a
a
Scheme 5. Synthesis of Inihibitor 15
Scheme 7. Synthesis of N-Piperidinyl Inhibitors
a
Reagents and conditions: (a) methylhydrazine sulfate, HCl, EtOH,
reflux, 28%; (b) 44, TFA, n-BuOH, 100 °C, microwave, 11%.
a
a
Scheme 6. Synthesis of Inhibitors 16 and 17
Reagents and conditions: (a) Cs2CO3, N-Boc-4-bromopiperidine,
DMF, 120 °C, 52%; (b) ( )-(cis)-tert-butyl 3-fluoro-4-hydroxypiper-
idine-1-carboxylate, PPh3, diisopropyl azodicarboxylate, THF, quant.;
(c) HCl, 1,4-dioxane, 55 °C; (d) oxetan-3-one, DIPEA, NaBH(OAc)3,
acetic acid, DCE; (e) Pd/C, H2, EtOH; (f) 26, TFA, 2-
methoxyethanol, 95 °C.
acid or 10 mM ammonium bicarbonate). Low-resolution mass spectra
were recorded on a Sciex 15 mass spectrometer in ES+ mode, a
Micromass ZQ single quadrapole LC−MS in ES+, ES− mode, or a
Quattro Micro LC-MS-MS in ES+, ES− mode. All final compounds
were purified to >95% chemical and optical purity, as assayed by either
(a) HPLC (Waters Acquity UPLC column 21 × 50 mm, 1.7 μM) with
gradient of 0−90% acetonitrile (containing 0.038% TFA) in 0.1%
aqueous TFA, with UV detection at λ = 254 and 210 as well as CAD
detection with an ESA Corona detector, (b) HPLC (Phenomenex
Luna C18 (2) column 4.6 × 100 mm, 5 μM) with gradient of 5−95%
acetonitrile in water (with 0.1% formic acid in each mobile phase),
with UV DAD detection between λ = 210 and 400 nm, (c) HPLC
a
Reagents and conditions: (a) Cs2CO3, 3-iodo-oxetane or 4-bromo-
tetrahydropyran, 100 °C, DMF; (b) Pd/C, H2, EtOH; (c) 26,
Cs2CO3, XPhos, Pd2(dba)3, 1,4-dioxane, 135 °C, microwave.
H
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a
propanamide (7). To the solution of N,2-dimethyl-2-(3-methyl-4-
nitro-1H-pyrazol-1-yl)propanamide (29, 0.4 g, 1.77 mmol) in MeOH
(25 mL) was added Pd/C (80 mg, 10 wt %). The reaction was stirred
under a hydrogen atmosphere (1 atm) for 1 h. The mixture was
filtered through Celite and concentrated under reduced pressure to
give crude 2-(4-amino-3-methyl-1H-pyrazol-1-yl)-N,2-dimethylpropa-
namide (0.41 g), which was used in the next step without further
purification.
Scheme 8. Synthesis of Inhibitor 20
To a solution of 2-(4-amino-3-methyl-1H-pyrazol-1-yl)-N,2-dime-
thylpropanamide (0.41 g, 1.83 mmol) in n-BuOH (15 mL) were
added 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (26,
0.43 g, 2.01 mmol) and triethylamine (0.77 mL, 5.49 mmol), and the
reaction mixture was stirred at 120 °C for 1 h before it was cooled to
room temperature. Water (20 mL) was added, the aqueous phase was
extracted with ethyl acetate (3×), and the combined organic phase was
washed with brine, dried over anhydrous sodium sulfate, filtered, and
concentrated to give a residue that was purified by silica gel
chromatography (CH2Cl2/MeOH, 20:1) to give 7 (0.18 g, 23%
a
Reagents and conditions: (a) ( )-(cis)-tert-butyl 3-fluoro-4-hydrox-
ypiperidine-1-carboxylate, PPh3, diisopropyl azodicarboxylate, THF;
(b) TFA, DCM, 58% over two steps; (c) oxetan-3-one, DIPEA,
NaBH(OAc)3, acetic acid, DCE, 85%; (d) LiHMDS then C2Cl6, THF,
−78 °C, 65%; (e) iron dust, NH4Cl, EtOH, 90 °C; (f) 26, TFA, 2-
methoxyethanol, 90 °C, 40%, two steps.
1
yield) as a white solid. H NMR (400 MHz, DMSO) δ 9.09 (s, 1H),
(Waters Xterra MS C18 column 4.6 × 100 mm, 5 μM) with gradient
of 5−95% acetonitrile in water (with 10 mM ammonium bicarbonate
in the aqueous mobile phase), with UV DAD detection between λ =
210 and 400 nm, (d) HPLC (Supelco, Ascentis Express C18 or
Hichrom Halo C18 column 4.6 × 150 mm, 2.7 μM) with gradient of
4−100% acetonitrile in water (with 0.1% formic acid in each mobile
phase), with UV DAD detection between λ = 210 and 400 nm, or (e)
HPLC (Phenomenex, Gemini NX C18 column 4.6 × 150 mm, 3 μM)
with gradient of 4.5−100% acetonitrile in water (with 10 mM
ammonium bicarbonate in the aqueous mobile phase), with UV DAD
detection between λ = 210 and 400 nm.
8.14 (s, 1H), 8.10 (s, 1H), 7.20 (s, 1H), 2.90 (s, 3H), 2.55 (s, 3H),
2.18 (s, 3H), 1.63 (s, 6H). LCMS, m/z = 372 [M + H]+.
N-Ethyl-2-methyl-2-(3-methyl-4-((4-(methylamino)-5-
(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-
propanamide (8). The title compound was prepared in a manner
1
analogous to 7 (37% yield). H NMR (500 MHz, CDCl3) δ 8.08 (s,
1H), 7.91 (s, 1H), 6.55 (brs, 1H), 5.36 (brs, 1H), 5.19 (brs, 1H),
3.23−3.28 (m, 2H), 3.03 (d, J = 4.5 Hz, 3H), 2.19 (s, 3H), 1.82 (s,
6H), 1.06 (t, 3H). LCMS, m/z = 386 [M + H]+.
N-Isopropyl-2-methyl-2-(3-methyl-4-((4-(methylamino)-5-
(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-
propanamide (9). The title compound was prepared in a manner
analogous to 7. 1H NMR (500 MHz, CD3OD) δ 8.03 (s, 1H), 7.85 (s,
1H), 4.00−3.97 (m, 1H), 2.99 (s, 3H), 2.23 (s, 3H), 1.76 (s, 6H), 1.11
(d, J = 7.0 Hz, 6H). LCMS, m/z = 400 [M + H]+.
2 - M e t h y l - 2- ( 3 - m e t h y l - 4 - ( ( 4- ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-N-
(2,2,2-trifluoroethyl)propanamide (10). The title compound was
prepared in a manner analogous to 7. 1H NMR (500 MHz, CDCl3) δ
8.13 (s, 2H), 7.02 (s, 1H), 6.79 (s, 1H), 5.24 (s, 1H), 3.87−3.80 (m,
2H), 3.05 (d, J = 4.5 Hz, 3H), 2.30 (s, 3H), 1.84 (s, 6H). LCMS, m/z
= 440 [M + H]+.
The synthesis of compound 2 was described previously.26
N2-(6,6-Dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-
3-yl)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine (3).
To a solution of 6,6-dimethyl-3-nitro-6,7-dihydro-4H-pyrazolo[5,1-
c][1,4]oxazine (25, 35.0 mg, 0.177 mmol) in THF/MeOH (3 mL/3
mL) were added Raney Ni (100 mg) and hydrazine hydrate (1 mL) at
0 °C. After being stirred for 16 h, the insoluble material was filtered
off. The filtrate was concentrated in vacuo to give 6,6-dimethyl-6,7-
dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-amine (26 mg, 89% yield).
LCMS, m/z = 168 [M + H]+.
A microwave vial equipped with a magnetic stirrer was charged with
6,6-dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-amine (13
mg, 0.078 mmol), 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-
amine (26, 20 mg, 0.095 mmol), and t-BuOH (0.5 mL). The mixture
was heated at 100 °C under microwave irradiation for 15 min. After
concentration, the residue was purified by preparative HPLC to give 3
(9.5 mg, 36% yield). 1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 7.67
(s, 1H), 6.79 (s, 1H), 5.18 (s, 1H), 4.83 (s, 2H), 3.93 (s, 2H), 3.00 (d,
J = 4.5 Hz, 3H), 1.63 (s, 6H). LCMS, m/z = 343 [M + H]+.
N2-(6,7-Dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-3-yl)-N4-
methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine (4). The title
2 - M e t h y l - 2- ( 3 - m e t h y l - 4 - ( ( 4- ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-
propanenitrile (11). A solution of 2-methyl-2-(3-methyl-4-((4-
(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-
1-yl)propanamide (34, 250 mg, 0.7 mmol) in POCl3 (5 mL) was
stirred at 90 °C for 1 h. The POCl3 was removed by evaporation. The
mixture was then slowly poured onto ice (10 mL). The pH of the
solution was adjusted to 8 with saturated sodium carbonate. The
aqueous phase was extracted with EtOAc (3×). The combined organic
phase was washed with brine, dried over anhydrous sodium sulfate,
filtered, and concentrated to give a residue that was purified by
1
compound was prepared in a manner analogous to 3 (34% yield). H
1
recrystallization to give 11 (100 mg, 42% yield) as a white solid. H
NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.60 (s, 1H), 6.37 (s, 1H),
5.13 (s, 1H), 4.81 (s, 2H), 4.19 (t, J = 5.0 Hz, 2H), 4.11 (t, J = 5.0 Hz,
2H), 3.01 (d, J = 4.8 Hz, 3H). LCMS, m/z = 315 [M + H]+.
N2-((6S)-4,6-Dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]-
oxazin-3-yl)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-dia-
mine (5). The title compound was prepared in a manner analogous to
NMR (300 MHz, DMSO) δ 9.18 (s, 1H), 8.29 (s, 1H), 8.14 (s, 1H),
7.10 (s, 1H), 2.91 (d, 3H), 2.22 (s, 3H), 1.94 (s, 6H). HRMS (ES) m/
z: [M + H]+ calcd for C14H16F3N7H+, 340.1492; found, 340.1484.
2 - M e t h y l - 2- ( 5 - m e t h y l - 4 - ( ( 4- ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-
propanenitrile (12). The title compound was synthesized from 2-(4-
amino-5-methyl-1H-pyrazol-1-yl)-2-methylpropanamide (36) in a
1
3 (22% yield). H NMR (400 MHz, DMSO) δ 9.10 8.57 (m, 1H),
8.06 (s, 1H), 7.81 7.34 (m, 1H), 6.95 (s, 1H), 5.31 (s, 1H), 4.29 4.18
(m, 1H), 4.09 (dd, J = 12.4, 3.1 Hz, 1H), 3.64 (dd, J = 12.3, 9.5 Hz,
1H), 2.85 (s, 3H), 1.31 (d, J = 5.6 Hz, 3H), 1.25 (d, J = 6.2 Hz, 3H).
LCMS, m/z = 343 [M + H]+.
1
manner analogous to 11 (17% yield). H NMR (400 MHz, DMSO)
δ 8.93 (s, 1H), 8.07 (s, 1H), 7.79 (s, 1H), 6.95 (s, 1H), 2.83 (s, 3H),
2.41 (s, 3H), 1.95 (s, 6H). LCMS, m/z = 340 [M + H]+.
N4-Methyl-N2-(4-methyl-6,7-dihydro-4H-pyrazolo[5,1-c]-
[1,4]oxazin-3-yl)-5-(trifluoromethyl)pyrimidine-2,4-diamine
(6). The title compound was prepared in a manner analogous to 3
1-(3-Methyl-4-((4-(methylamino)-5-(trifluoromethyl)-
p y r i m i d i n - 2 - y l ) a m i n o ) - 1 H - p y r a z o l - 1 - y l ) -
cyclopropanecarbonitrile (13). A mixture of 1-(4-amino-3-methyl-
1H-pyrazol-1-yl)cyclopropanecarboxamide (38, 0.30 g, 1.7 mmol), 2-
chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (26, 0.40 g,
1.9 mmol), and trifluoroacetic acid (0.1 mL) in 2-methoxyethanol
(5 mL) was stirred at 90 °C for 45 min. The reaction was cooled to
room temperature and diluted with saturated sodium bicarbonate. The
1
(14% yield). H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.55 (s,
1H), 5.15 (s, 1H), 4.96 (m, 1H), 4.32−3.93 (m, 4H), 2.99 (d, J = 4.7
Hz, 3H), 1.47 (d, J = 6.5 Hz, 3H). LCMS, m/z = 329 [M + H]+.
N,2-Dimethyl-2-(3-methyl-4-((4-(methylamino)-5-
(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-
I
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1
product was then extracted with EtOAc (3×). The combined extracts
were washed with brine, dried over sodium sulfate, filtered, and
concentrated. The crude product was purified by silica gel
chromatography (0−100% EtOAc in heptane) to give 1-[3-methyl-4-
[[4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl]amino]pyrazol-
1-yl]cyclopropanecarboxamide (0.31 g, 50%). LCMS, m/z = 256 [M +
H]+.
A mixture of 1-[3-methyl-4-[[4-(methylamino)-5-(trifluoromethyl)-
pyrimidin-2-yl]amino]pyrazol-1-yl]cyclopropanecarboxamide (0.30 g,
0.84 mmol) in POCl3 (3 mL) was stirred at 90 °C for 1 h. The
reaction was slowly poured onto an ice-cold 1 M sodium carbonate
solution. The product was extracted with EtOAc (3×). The combined
extracts were washed with saturated sodium bicarbonate and then
brine, dried over sodium sulfate, filtered, and concentrated. The crude
product was purified by preparative HPLC to give 13 (176 mg, 62%
yield). 1H NMR (400 MHz, DMSO) δ 9.09 (s, 1H), 8.12 (s, 2H), 7.03
(s, 1H), 2.91 (d, J = 4.4 Hz, 3H), 2.16 (s, 3H), 1.87 1.78 (m, 2H), 1.78
1.70 (m, 2H). LCMS, m/z = 338. [M + H]+.
to give the title compound (68 mg, 26% yield). H NMR (400 MHz,
DMSO) δ 8.84 (s, 1H), 8.04 (s, 1H), 7.71 (s, 1H), 6.89 (s, 1H), 4.54
(t, J = 6.5 Hz, 2H), 4.43 (t, J = 6.1 Hz, 2H), 4.07 (s, 1H), 3.45− 3.43
(m, 1H), 2.89−2.79 (m, 5H), 2.12 (s, 3H), 2.04−1.72 (m, 6H).
LCMS, m/z = 412 [M + H]+.
N2-(1-((cis)-3-Fluoro-1-(oxetan-3-yl)piperidin-4-yl)-3-methyl-
1H-pyrazol-4-yl)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-
diamine (19). The title compound was prepared in a manner
analogous to 18 (11% yield). The enantiomers were separated by
chiral SFC purification. 1H NMR (400 MHz, DMSO) δ 8.89 (s, 1H),
8.05 (s, 1H), 7.78 (s, 1H), 6.92 (s, 1H), 4.86 (m, 1H), 4.56 (m, 2H),
4.46 (m, 2H), 4.27 (m, 1H), 3.58 (m, 1H), 3.22−3.11 (m, 1H), 2.96−
2.71 (m, 4H), 2.29−1.98 (m, 6H), 1.90 (m, 1H). LCMS, m/z = 430
[M + H]+.
N2-(5-Chloro-1-((trans)-3-fluoro-1-(oxetan-3-yl)piperidin-4-
yl)-1H-pyrazol-4-yl)-N4-methyl-5-(trifluoromethyl)pyrimidine-
2,4-diamine (20). A mixture of ( )-(trans)-4-(5-chloro-4-nitro-1H-
pyrazol-1-yl)-3-fluoro-1-(oxetan-3-yl)piperidine (53, 2.2 g, 3.9 mmol),
iron dust (1.6 g, 29 mmol), and ammonium chloride (1.5 g, 29 mmol)
in ethanol (20 mL) was stirred at 90 °C for 30 min. The reaction was
filtered and concentrated. The residue was sonicated with 100 mL of
EtOAc for 5 min. The mixture was filtered to remove all insoluble
solids. The filtrate was then concentrated to give crude ( )- (trans)-4-
(5-chloro-4-amino-pyrazol-1-yl)-3-fluoro-1-(oxetan-3-yl)piperidine
(1.9 g).
1-(5-Chloro-4-((4-(methylamino)-5-(trifluoromethyl)-
p y r i m i d i n - 2 - y l ) a m i n o ) - 1 H - p y r a z o l - 1 - y l ) -
cyclopropanecarbonitrile (14). The title compound was prepared
1
in a manner analogous to 11 (yield =30%). H NMR (400 MHz,
DMSO) δ 9.12 (s, 1H), 8.11 (s, 1H), 7.94 (s, 1H), 7.06 (m, 1H), 2.84
(d, J = 2.7 Hz, 3H), 2.10−1.98 (m, 2H), 1.89 1.76 (m, 2H). LCMS,
m/z = 358 [M + H]+.
2-(5-((4-(Ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)-
amino)-1-methyl-1H-pyrazol-3-yl)-2-methylpropanenitrile
(15). To a solution of 2-(5-amino-1-methyl-1H-pyrazol-3-yl)-2-
methylpropanenitrile (43, 100 mg, 0.61 mmol) in n-BuOH (1 mL)
were added 2-chloro-N-ethyl-5-(trifluoromethyl) pyrimidin-4-amine
(44, 137 mg, 0.61 mmol) and a drop of trifluoroacetic acid. After
heating in microwave oven at 100 °C for 1 h, the resulting solution was
concentrated in vacuo, and the residue was purified by preparative
HPLC to afford the title compound as a white solid (23 mg, 11%). 1H
NMR (500 MHz, DMSO) δ 9.54 (s, 1H), 8.17 (s, 1H), 7.24 (s, 1H),
6.36 (s, 1H), 3.67 (s, 3H), 3.44−3.38 (m, 2H), 1.62 (s, 6H), 1.10 (t, J
= 7.0 Hz, 3H). LCMS, m/z = 354 [M + H]+.
To a mixture of the crude ( )-(trans)-4-(5-chloro-4-amino-pyrazol-
1-yl)-3-fluoro-1-(oxetan-3-yl)piperidine (1.9 g) and 2-chloro-N-
methyl-5-(trifluoromethyl)pyrimidin-4-amine (26, 1.5 g, 6.9 mmol)
in 2-methoxyethanol (25 mL) was added TFA (0.60 mL, 7.7 mmol).
The reaction was stirred at 90 °C for 15 min. The mixture was then
diluted with saturated sodium bicarbonate and extracted with EtOAc
(3×). The combined extracts were washed with brine, dried over
sodium sulfate, filtered, and concentrated. The crude product was
purified by preparative HPLC, chiral SFC, and recrystallized in
1
isopropanol to give 20 (0.70 g, 40% yield). H NMR (400 MHz,
DMSO) δ 8.91 (s, 1H), 8.08 (s, 1H), 7.87 (s, 1H), 7.00 (s, 1H), 5.03
4.79 (m, 1H), 4.56 (m, 1H), 4.46 (m, 2H), 3.68−3.51 (m, 1H), 3.26−
3.12 (m, 1H), 2.92−2.73 (m, 3H), 2.54 (s, 2H), 2.20−1.88 (m, 3H).
HRMS (ES) m/z: [M + H]+ calcd for C17H20ClF4N7OH+, 450.1427;
found, 450.1418.
N4-Methyl-N2-(5-methyl-1-(oxetan-3-yl)-1H-pyrazol-4-yl)-5-
(trifluoromethyl)pyrimidine-2,4-diamine (16). A microwave tube
was charged a mixture of 3-methyl-1-(oxetan-3-yl)-1H-pyrazol-4-
amine and 5-methyl-1-(oxetan-3-yl)-1H-pyrazol-4-amine (45, 0.26 g,
1.7 mmol), 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine
(26, 0.28 g, 1.3 mmol), cesium carbonate (0.87 g, 2.7 mmol), XPhos
(64 mg, 0.13 mmol), tris(dibenzylideneacetone)dipalladium(0) (61
mg, 0.067 mmol), and 1,4-dioxane (16 mL). The reaction was sealed
and heated in a microwave reactor at 135 °C for 30 min. The reaction
mixture was then filtered and concentrated. The crude product was
((2-Methyl-2-(prop-2-ynyloxy)propoxy)methyl)benzene
(22). To a mixture of NaH (5.0 g) in THF (50 mL) at 0 °C was added
dropwise 1-(benzyloxy)-2-methylpropan-2-ol (21, 9.36 g, 51.9 mmol).
After being stirred at room temperature for 30 min, 3-bromoprop-1-
yne (12.4 g, 104 mmol) was added dropwise at 0 °C. The mixture was
then stirred overnight at reflux. The reaction was quenched by the
addition of saturated ammonium chloride. The resulting mixture was
extracted with ethyl acetate (3×). The organic layer was combined,
dried over sodium sulfate, and concentrated. The residue was purified
by silica gel chromatography (petroleum ether/EtOAc, 20:1) to afford
1
purified by preparative HPLC to give 16 (22 mg, 4.9% yield). H
NMR (400 MHz, DMSO) δ 8.91 (s, 1H), 8.05 (s, 1H), 7.86 (s, 1H),
6.94 (s, 1H), 5.53 (m, 1H), 4.95−4.91 (m, 2H), 4.90 4.83 (m, 2H),
2.85 (s, 3H), 2.14 (s, 3H). LCMS, m/z = 329 [M + H]+.
1
the title compound (9.7 g, 86% yield) as pale-yellow oil. H NMR
N4-Methyl-N2-(5-methyl-1-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazol-4-yl)-5-(trifluoromethyl)pyrimidine-2,4-diamine (17).
The title compound was prepared in a manner analogous to 16
(500 MHz, CDCl3) δ 7.34−7.35 (m, 5H), 4.56 (s, 2H), 4.18 (d, J =
2.0 Hz, 2H), 3.38 (s, 2H), 2.34 (t, J = 2.0 Hz, 1H), 1.25 (s, 6H).
2-((1H-Pyrazol-5-yl)methoxy)-2-methylpropan-1-ol (23). A
mixture of ((2-methyl-2-(prop-2-ynyloxy)propoxy)methyl)benzene
(1.00 g, 4.58 mmol) and (diazomethyl)trimethylsilane (2.29 mL)
was stirred at 135 °C under microwave irradiation for 1 h. Removal of
the solvent under vacuum afforded 3-((1-(benzyloxy)-2-methylpro-
pan-2-yloxy)methyl)-1H-pyrazole (1.19 g, 80% yield). The crude
residue was carried to the next step without further purification.
LCMS, m/z = 261 [M + H]+.
A mixture of 3-((1-(benzyloxy)-2-methylpropan-2-yloxy)methyl)-
1H-pyrazole (5.5 g, 21 mmol) and 10% Pd(OH)2 on carbon (2.2 g) in
EtOH (100 mL) was stirred at 100 °C under H2 (4 atm) for 12 h. The
insoluble material was filtered off, and the filtrate was concentrated to
afford 2-((1H-pyrazol-5-yl)methoxy)-2-methylpropan-1-ol (3.0 g, 83%
yield). LCMS, m/z = 171 [M + H]+.
1
(yield =8.2%). H NMR (400 MHz, DMSO) δ 8.83 (s, 1H), 8.04 (s,
1H), 7.65 (s, 1H), 6.89 (s, 1H), 4.33 (m, 1H), 3.95 (m, 2H), 3.47 (m,
2H), 2.84 (s, 3H), 2.21 (s, 3H), 2.02 (m, 2H), 1.82 1.67 (m, 2H).
LCMS, m/z = 357 [M + H]+.
N4-Methyl-N2-(3-methyl-1-(1-(oxetan-3-yl)piperidin-4-yl)-
1H-pyrazol-4-yl)-5-(trifluoromethyl)pyrimidine-2,4-diamine
(18). A suspension of 4-(3-methyl-4-nitro-1H-pyrazol-1-yl)-1-(oxetan-
3-yl)piperidine (49) and minor amount of 4-(5-methyl-4-nitro-
pyrazol-1-yl)-1-(oxetan-3-yl)piperidine (0.158 g, 0.593 mmol) and
palladium on carbon (10 wt %, 0.1 g) in ethanol (15 mL) was stirred
under a hydrogen atmosphere for 18 h. The reaction mixture was then
filtered through Celite and concentrated. The residue was redissolved
in 2-methoxythanol (3 mL) and mixed with 2-chloro-N-methyl-5-
(trifluoromethyl)pyrimidin-4-amine (26, 0.13 g, 0.63 mmol) and TFA
(0.1 mL, 1 mmol). The resulting mixture was stirred at 95 °C for 1 h.
The reaction was allowed to cool to room temperature and
concentrated. The crude product was purified by preparative HPLC
2-Methyl-2-((4-nitro-1H-pyrazol-5-yl)methoxy)propyl Ni-
trate (24). To a solution of 2-((1H-pyrazol-3-yl)methoxy)-2-
methylpropan-1-ol (3.00 g, 17.6 mmol) in acetonitrile (30 mL) was
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Journal of Medicinal Chemistry
Article
added NBS (3.45g, 19.4 mmol) in one portion. After overnight
stirring, the mixture was concentrated. The residue was purified by
preparative HPLC to give 2-((4-bromo-1H-pyrazol-5-yl)methoxy)-2-
methylpropan-1-ol (1.8 g, 41%). LCMS, m/z = 249 [M + H]+.
Fuming nitric acid (3.0 mL) was added to 2-((4-bromo-1H-pyrazol-
3-yl)methoxy)-2-methylpropan-1-ol (300 mg, 1.2 mmol) at 0 °C. After
being stirred for 1 h at 0 °C, the reaction was quenched with ice. The
mixture was extracted with ethyl acetate (3×). The organic layers were
washed with water and brine, dried over sodium sulfate, and
concentrated to give crude 2-methyl-2-((4-nitro-1H-pyrazol-5-yl)-
methoxy)propyl nitrate (260 mg, 52% yield). LCMS, m/z = 261 [M
+ H]+.
6,6-Dimethyl-3-nitro-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]-
oxazine (25). To a cooled (0 °C) solution of 2-methyl-2-((4-nitro-
1H-pyrazol-5-yl)methoxy)-propyl nitrate (580 mg, 1.12 mmol) in
DMF (15 mL) was added NaH (89.0 mg, 2.23 mmol, 60%
dispersion). The mixture was stirred overnight at room temperature.
This reaction was quenched by ice, and the mixture was extracted with
ethyl acetate (30 mL × 3). The organic layers were washed with water
and brine, dried over sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by silica gel chromatography, eluting
with (5−20% EtOAc in petroleum ether) to give 6,6-dimethyl-3-nitro-
6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine (43.5 mg, 20% yield).
LCMS, m/z = 198 [M + H]+.
N-Ethyl-2-methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)-
propanamide (30). The title compound was prepared in a manner
analogous to 29. LCMS, m/z = 241 [M + H]+.
N-Isopropyl-2-methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)-
propanamide (31). The title compound was prepared in a manner
analogous to 29. LCMS, m/z = 255 [M + H]+.
2-Methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)-N-(2,2,2-
trifluoroethyl)propanamide (32). The title compound was
prepared in a manner analogous to 29. LCMS, m/z = 295 [M + H]+.
2-Methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)propanamide
(33). To the solution of 2-methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-
yl)propanoic acid (2.5 g, 11.7 mmol) in CH2Cl2 (50 mL) was added
dropwise oxalyl chloride (2.97 g, 23.4 mmol). The reaction was stirred
at room temperature for 2 h and then concentrated under reduced
pressure to remove the solvent. The residual solid was dissolved in
THF (30 mL), which was then added dropwise into a stirred solution
of ammonium hydroxide (50 mL). The reaction was stirred at room
temperature for 1 h. The solution was then concentrated under
reduced pressure and partitioned between EtOAc (50 mL) and water
(100 mL), and the aqueous phase was extracted with EtOAc (3×).
The combined organic was washed with saturated ammonium chloride
(50 mL), dried over anhydrous sodium sulfate, filtered, and
concentrated to give the crude product 2-methyl-2-(3-methyl-4-
nitro-1H-pyrazol-1-yl)propanamide (2.5 g, quant.) as white solid
that was used in the next step without further purification.
2-Methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)propanoic
Acid (28). To a solution of 5-methyl-4-nitropyrazole (27, 10 g, 78.6
mmol) in DMF (50 mL) was added NaH (4.72 g, 118.0 mmol, 60%
dispersion) in several portions. The reaction mixture was stirred at
room temperature for 2 h followed by the addition of methyl 2-bromo-
2-methylpropanoate (21.3 g, 118.0 mmol). The resulting mixture was
stirred for an additional 12 h before water (250 mL) was added to
quench the reaction. The aqueous phase was extracted with EtOAc
(3×), and the combined organic phase was washed with brine (100
mL), dried over anhydrous sodium sulfate, filtered, and concentrated
to dryness to give a residue that was purified by silica gel
chromatography to give methyl 2-methyl-2-(3-methyl-4-nitro-1H-
pyrazol-1-yl)propanoate (11.5 g, 80% yield) as a white solid. 1H
NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 3.78 (s, 3H), 2.57 (s, 3H),
1.89 (s, 6H). LCMS, m/z = 228 [M + H]+.
2 - M e t h y l - 2- ( 3 - m e t h y l - 4 - ( ( 4- ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-
propanamide (34). To the solution of 2-methyl-2-(3-methyl-4-nitro-
1H-pyrazol-1-yl)propanamide (2.5g, 11.7 mmol) in MeOH (50 mL)
was added Pd/C (1 g, 10 wt %). The reaction was stirred under a
hydrogen atmosphere (1 atm) for 1 h at room temperature. The
solution was filtered and concentrated under reduced pressure to give
the crude 2-(4-amino-3-methyl-1H-pyrazol-1-yl)-2-methylpropana-
mide (2.0 g, 93% yield), which was used in the next step without
further purification.
To a solution of 2-(4-amino-3-methyl-1H-pyrazol-1-yl)-2-methyl-
propanamide (250 mg, 1.37 mmol) in 2-methoxyethanol (5 mL) were
added 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (290
mg, 1.37 mmol) and trifluoroacetic acid (156 mg, 1.37 mmol). The
reaction was stirred at 70 °C for 30 min. The reaction mixture was
cooled to room temperature followed by the addition of water (10
mL). The pH of the aqueous solution was adjusted to 8 by adding
saturated sodium carbonate. The aqueous phase was extracted with
EtOAc (3×). The combined organic phase was dried over anhydrous
sodium sulfate, filtered, and concentrated to give a residue that was
purified by silica gel chromatography (CH2Cl2/MeOH, 20:1) to give
2-methyl-2-(3-methyl-4-(4-(methylamino)-5-(trifluoromethyl)-
pyrimidin-2-ylamino)-1H- pyrazol-1-yl)propanamide (250 mg, 51%
yield) as white solid.). LCMS, m/z = 358 [M + H]+.
Methyl 2-(5-Methyl-4-nitro-1H-pyrazol-1-yl)acetate and
methyl 2-(3-methyl-4-nitro-1H-pyrazol-1-yl)acetate (35 and
37). A microwave vial equipped with a magnetic stirrer was charged
with methyl 2-bromoacetate (7.22 g, 47.2 mmol), 5-methyl-4-
nitropyrazole (5 g, 39.4 mmol), cesium carbonate (19.2 g, 59.1
mmol), and DMF (40 mL). The reaction mixture was stirred at room
temperature for 1 h. It was then filtered to remove all insoluble solids.
The filtrate was concentrated and purified by silica gel chromatography
(petroleum ether/EtOAc, 3:1) to afford the mixture of methyl 2-(5-
methyl-4-nitro-1H-pyrazol-1-yl)acetate and methyl 2-(3-methyl-4-
nitro-1H-pyrazol-1-yl)acetate as a yellow oil (7.0 g, 89% yield).
LCMS, m/z = 200 [M + H]+.
2-(4-Amino-5-methyl-1H-pyrazol-1-yl)-2-methylpropana-
mide (36). To a mixture of methyl 2-(5-methyl-4-nitro-1H-pyrazol-1-
yl)acetate and methyl 2-(3-methyl-4-nitro-1H-pyrazol-1-yl)acetate (4.5
g, 23 mmol) in DMF (50 mL) were added NaH (60% dispersion, 2.3
g, 56 mmol) and iodomethane (3.5 mL, 56 mmol). The reaction was
stirred at room temperature for 4 h. The mixture was diluted with
saturated ammonium chloride and extracted with EtOAc (3×). The
combined extracts were washed with brine, dried over sodium sulfate,
filtered, and concentrated. The crude product was purified by silica gel
chromatography (0−100% EtOAc in heptane) to give a mixture of
To a solution of methyl 2-methyl-2-(3-methyl-4-nitro-1H-pyrazol-1-
yl)propanoate (2 g, 8.81 mmol) in THF (40 mL) and water (10 mL)
was added LiOH (250 mg, 10.6 mmol), and the resulting mixture was
stirred at room temperature for 5 h. THF was removed under reduced
pressure, the aqueous phase was extracted with EtOAc (2×), and the
organic phases were discarded. The pH value of the aqueous phase was
adjusted to 1 to 2 by adding a 3 N HCl solution, and the mixture was
extracted with EtOAc (3×). The combined organic phase was washed
with brine (20 mL), dried over anhydrous sodium sulfate, filtered, and
1
concentrated in vacuo to give crude 28 (1.4 g, 74% yield). H NMR
(300 MHz, CD3OD) δ 8.71 (s, 1H), 2.53 (d, J = 1.1 Hz, 3H), 1.88 (s,
6H).
N,2-Dimethyl-2-(3-methyl-4-nitro-1H-pyrazol-1-yl)-
propanamide (29). To the solution of 2-methyl-2-(3-methyl-4-nitro-
1H-pyrazol-1-yl)propanoic acid (1.4 g, 6.47 mmol) in CH2Cl2 (30
mL) was added dropwise oxalyl chloride (1.64 g, 12.9 mmol), and the
reaction was stirred at room temperature for 2 h. The reaction mixture
was then concentrated under reduced pressure to remove the solvent.
The residual solid was redissolved in THF (30 mL), and methylamine
(6.5 mL, 12.94 mmol, 2 M in THF) was added dropwise. The reaction
was stirred at room temperature for 12 h. The reaction solution was
concentrated under reduced pressure and partitioned between EtOAc
(15 mL) and water (10 mL), and the aqueous phase was extracted
with EtOAc (2×). The combined organic phase was washed with
saturated ammonium chloride (10 mL), dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo to give a residue that was
purified by silica gel chromatography (petroleum ether/EtOAc, 2:1) to
1
give 29 (920 mg, 63% yield) as white solid. H NMR (300 MHz,
CDCl3) δ 8.33 (s, 1H), 6.29 (s, 1H), 2.79 (d, J = 4.8 Hz, 3H), 2.58 (s,
3H), 1.84 (s, 6H). LCMS, m/z = 358 [M + H]+.
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Journal of Medicinal Chemistry
Article
methyl 2-methyl-2-(5-methyl-4-nitro-pyrazol-1-yl)propanoate and
methyl 2-methyl-2-(3-methyl-4-nitro-pyrazol-1-yl)propanoate (2.0 g,
39% yield). LCMS, m/z = 228 [M + H]+.
brine, dried over sodium sulfate, filtered, and concentrated. The crude
product was purified by silica gel chromatography (0−5% CH3OH in
CH2 Cl2 ) to give 1-(3-methyl-4-nitro-pyrazol-1-yl)-
cyclopropanecarboxamide (0.53 g, 86% over two steps). LCMS, m/z
= 211 [M + H]+.
The mixture of methyl 2-methyl-2-(5-methyl-4-nitro-pyrazol-1-
yl)propanoate and methyl 2-methyl-2-(3-methyl-4-nitro-pyrazol-1-
yl)propanoate (1.56 g, 6.87 mmol) was then dissolved in THF (20
mL). To the solution were then added water (5 mL) and LiOH (0.17
g, 6.87 mmol). The resulting mixture was stirred overnight at room
temperature. The reaction was then diluted with brine and washed
with diethyl ether. The aqueous layer was acidified to pH 2 by adding a
2 M HCl solution. The mixture was then extracted with EtOAc (4×).
The combined extracts were washed with brine, dried over sodium
sulfate, filtered, and concentrated to give a crude mixture of 2-methyl-
2-(5-methyl-4-nitro-pyrazol-1-yl)propanoic acid and 2-methyl-2-(3-
methyl-4-nitro-pyrazol-1-yl)propanoic acid (1.3 g, 88% yield). LCMS,
m/z = 214 [M + H]+.
To a crude mixture of 2-methyl-2-(5-methyl-4-nitro-pyrazol-1-
yl)propanoic acid and 2-methyl-2-(3-methyl-4-nitro-pyrazol-1-yl)-
propanoic acid (0.63 g, 2.9 mmol) were added CH2Cl2 (10 mL),
oxalyl chloride (0.6 mL, 7 mmol), and 1 drop of DMF. The reaction
was stirred at room temperature for 2 h prior to concentrating to a
solid. The solid was redissolved in THF (5 mL), which was then added
dropwise to a stirred solution of ammonium hydroxide (10 mL). After
stirring at room temperature for 35 min, the reaction was diluted with
brine and extracted with EtOAc (4×). The combined extracts were
washed with brine, dried over sodium sulfate, filtered, and
concentrated to give a crude mixture of 2-methyl-2-(5-methyl-4-
nitro-pyrazol-1-yl)propanamide and 2-methyl-2-(3-methyl-4-nitro-pyr-
azol-1-yl)propanamide (0.60 g, 96% yield). LCMS, m/z = 213 [M +
H]+.
A suspension of the mixture of 2-methyl-2-(5-methyl-4-nitro-
pyrazol-1-yl)propanamide and 2-methyl-2-(3-methyl-4-nitro-pyrazol-
1-yl)propanamide (0.65 g, 3.1 mmol) and palladium on carbon (10 wt
%, 0.2 g) in EtOH (10 mL) was stirred under a hydrogen atmosphere
(1 atm) for 1 h. The mixture was filtered through Celite and
concentrated to give a mixture of 2-(4-amino-5-methyl-pyrazol-1-yl)-2-
methyl-propanamide and 2-(4-amino-3-methyl-pyrazol-1-yl)-2-methyl-
propanamide (0.58 g, quant.). The crude product was carried to the
next step without purification. LCMS, m/z = 183 [M + H]+.
1 - ( 4 - A m i n o - 3 - m e t h y l - 1 H - p y r a z o l - 1 - y l ) -
cyclopropanecarboxamide (38). To a solution of a mixture of
methyl 2-(5-methyl-4-nitro-pyrazol-1-yl)acetate and methyl 2-(3-
methyl-4-nitro-pyrazol-1-yl)acetate (0.70 g, 3.5 mmol) in DMF (10
mL) were added NaH (0.30 g, 7.5 mmol, 60% dispersion) and
ethylene dibromide (0.80 mL, 9.2 mmol) at 0 °C. The reaction was
stirred at room temperature for 2 h. The reaction was then diluted
with saturated ammonium chloride and extracted with EtOAc. The
organic extracts were washed with brine, dried over sodium sulfate,
filtered, and concentrated. The crude product was purified by silica gel
chromatography (0−100% EtOAc in heptane) to give methyl 1-(5-
methyl-4-nitro-pyrazol-1-yl)cyclopropanecarboxylate (0.30 g, 38%
yield). LCMS, m/z = 226 [M + H]+.
A
suspension of 1-(3-methyl-4-nitro-pyrazol-1-yl)-
cyclopropanecarboxamide (0.50 g, 2.4 mmol) and palladium on
carbon (10 wt %, 0.2 g) in EtOH (5 mL) was stirred under a hydrogen
atmosphere (1 atm) for 3 h. The reaction was then filtered through
Celite and concentrated. The crude material (0.30 g, 71% yield) was
used in the next step without purification. LCMS, m/z = 181 [M +
H]+.
Methyl 1-(4-Nitro-1H-pyrazol-1-yl)cyclopropanecarboxylate
(40). To a solution of methyl 2-(4-nitropyrazol-1-yl)acetate (39, 1.5 g,
8.1 mmol) in DMF (25 mL) were added a 60% dispersion of NaH
(0.81 g, 20 mmol) and ethylene dibromide (0.85 mL, 9.7 mmol) at 0
°C. The reaction was stirred at 0 °C for 10 min prior to stirring at
room temperature for 2 h. The reaction was then diluted with
saturated ammonium chloride and extracted with EtOAc (3×). The
organic extracts were washed with brine, dried over sodium sulfate,
filtered, and concentrated. The crude product was purified by silica gel
chromatography (0−100% EtOAc in heptane) to give methyl 1-(4-
nitropyrazol-1-yl)cyclopropanecarboxylate (0.35 g, 20%). LCMS, m/z
= 212 [M + H]+.
1 - ( 4 - A m i n o - 5 - c h l o r o - 1 H - p y r a z o l - 1 - y l ) -
cyclopropanecarboxamide (41). To as a solution of methyl 1-(4-
nitropyrazol-1-yl)cyclopropanecarboxylate (40, 0.35 g, 1.7 mmol) in
THF (15 mL) was added a 1 M solution of LiHMDS in THF (3.3 mL,
3.3 mmol) at −78 °C. The reaction was stirred at −78 °C for 30 min
before the addition of a solution of hexachloroethane (0.47 g, 2.0
mmol) in THF (5 mL). The reaction was then stirred at −78 °C for
30 min before warming to room temperature. The reaction was diluted
with saturated ammonium chloride and extracted with EtOAc (3×).
The combined extracts were washed with brine, dried over sodium
sulfate, filtered, and concentrated. The crude product was purified by
silica gel chromatography (0−100% EtOAc in heptane) to give methyl
1-(5-chloro-4-nitro-pyrazol-1-yl)cyclopropanecarboxylate (0.22 g, 54%
yield). LCMS, m/z = 246 [M + H]+.
To a solution of methyl 1-(5-chloro-4-nitro-pyrazol-1-yl)-
cyclopropanecarboxylate (0.15 g, 0.61 mmol) in THF (8 mL) were
added water (2 mL) and lithium hydroxide (22 mg, 0.92 mmol). The
reaction was stirred at room temperature for 7 h. The mixture was
then diluted with water and washed with diethyl ether. The aqueous
layer was then acidified to pH 1 by adding concentrated hydrochloric
acid followed by extraction by EtOAc (3×). The combined extracts
were washed with brine, dried over sodium sulfate, filtered, and
concentrated to give 1-(5-chloro-4-nitro-pyrazol-1-yl)-
cyclopropanecarboxylic acid (97 mg, 69%). The crude product was
carried to the next step without purification.
To a solution of the crude 1-(5-chloro-4-nitro-pyrazol-1-yl)-
cyclopropanecarboxylic acid (97 mg, 0.42 mmol) in CH2Cl2 (2 mL)
were added oxalyl chloride (0.5 mL) and 1 drop of DMF. The reaction
was stirred at room temperature for 45 min prior to concentration in
vacuo. The residue was the redissolved in THF (10 mL), which was
then added dropwise to a stirring mixture of ammonium hydroxide (10
mL) and THF (10 mL). The reaction was stirred at room temperature
for 1 h. The mixture was then diluted with brine and extracted with
EtOAc (3×). The combined extracts were washed with brine, dried
over sodium sulfate, filtered, and concentrated to give crude 1-(5-
chloro-4-nitro-pyrazol-1-yl)cyclopropanecarboxamide (99 mg, quant.
yield).
A
mixture of methyl 1-(5-methyl-4-nitro-pyrazol-1-yl)-
cyclopropanecarboxylate (0.50 g, 2.2 mmol), lithium hydroxide (0.05
g, 2 mmol) in THF (10 mL), and water (5 mL) was stirred at room
temperature for 24 h. The reaction was diluted with brine and washed
with diethyl ether. The aqueous layer was then acidified to pH 2 by the
addition of a 2 M HCl solution. The mixture was then extracted with
EtOAc (4×). The combined extracts were washed with brine, dried
over sodium sulfate, and concentrated to give crude 1-(3-methyl-4-
nitro-1H-pyrazol-1-yl)cyclopropanecarboxylic acid (0.62 g, quant.).
The material was carried to the next step without purification.
A mixture of the crude 1-(5-chloro-4-nitro-pyrazol-1-yl)-
cyclopropanecarboxamide (90 mg, 0.39 mmol), ammonium chloride
(62 mg, 1.1 mmol), and iron dust (66 mg, 1.17 mmol) in ethanol (10
mL) was stirred at 90 °C for 30 min. The reaction was the filtered
through Celite and concentrated. To the residue was added EtOAc (50
mL), and the mixture was sonicated for 2 min. The mixture was then
filtered to remove all insoluble solids. The filtrate was concentrated to
give 1-(4-amino-5-chloro-pyrazol-1-yl)cyclopropanecarboxamide (0.09
To
a solution of 1-(3-methyl-4-nitro-1H-pyrazol-1-yl)-
cyclopropanecarboxylic acid (0.62 g, 2.9 mmol) in CH2Cl2 (5 mL)
were added oxalyl chloride (0.78 mL, 8.8 mmol) and 1 drop of DMF.
The reaction was stirred for 30 min prior to being concentrated under
vacuum. The residue was dissolved in THF (2 mL) and added to a
stirred mixture of ammonium hydroxide (10 mL) in THF (10 mL).
After 30 min of stirring, the mixture was diluted with water and
extracted with EtOAc (3×). The combined extracts were washed with
L
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g, quant.). The crude material was carried onto the next step without
further purification. LCMS, m/z = 201 [M + H]+.
To a suspension of 4-(3-methyl-4-nitro-pyrazol-1-yl)piperidine
hydrochloride (0.25 g, 1.2 mmol) in 1,2-dichloroethane (5 mL)
were added DIPEA (0.3 mL, 1.8 mmol) and oxetan-3-one (0.21 g, 3.0
mmol). The suspension was stirred at room temperature for 15 min
prior to the addition of sodium triacetoxyborohydride (0.53 g, 2.4
mmol) and glacial acetic acid (0.082 mL, 1.4 mmol). The reaction was
stirred at room temperature for 2 h. The reaction was diluted with
saturated sodium bicarbonate and extracted with CH2Cl2 (3×). The
combined extracts were washed with water, dried over sodium sulfate,
filtered, and concentrated. The crude product was purified by silica gel
chromatography (0−100% EtOAc in heptane) to give a mixture of 4-
(3-methyl-4-nitro-pyrazol-1-yl)-1-(oxetan-3-yl)piperidine and 4-(5-
methyl-4-nitro-pyrazol-1-yl)-1-(oxetan-3-yl)piperidine. (0.15 g, 48%
yield). LCMS, m/z = 267 [M + H]+.
2-(5-Amino-1-methyl-1H-pyrazol-3-yl)-2-methylpropaneni-
trile (43). To a solution of 2,2-dimethyl-3-oxo-pentanedinitrile (42,
900 mg, 6.62 mmol) and methylhydrazine sulfate (953 mg, 20 mmol)
in EtOH (10 mL) was added concentrated HCl (1.7 mL, 198 mmol).
After heating at reflux overnight, the solvent was removed in vacuo,
and the residue was purified by silica gel chromatography (CH2Cl2/
MeOH, 20:1) to give the 43 as a white solid (300 mg, 28% yield).
LCMS, m/z = 165 [M + H]+.
5-Methyl-1-(oxetan-3-yl)-1H-pyrazol-4-amine (45). To a
mixture of 3-methyl-4-nitro-pyrazole (0.80 g, 6.3 mmol), cesium
carbonate (4.1 g, 12 mmol) in DMF (10 mL) was added 3-iodo-
oxetane (3.47 g, 19 mmol). The mixture was stirred at 100 °C for 3 h.
The reaction was diluted with water and extracted with EtOAc (3×).
The combined extracts were washed with brine, dried over sodium
sulfate, filtered, and concentrated. The crude product was purified by
silica gel chromatography (20−100% EtOAc in heptane) to give a
mixture of 5-methyl-4-nitro-1-(oxetan-3-yl)-1H-pyrazole and 3-meth-
yl-4-nitro-1-(oxetan-3-yl)-1H-pyrazole (0.85 g, 74% yield). LCMS, m/
z = 184 [M + H]+.
To a solution of a mixture of 5-methyl-4-nitro-1-(oxetan-3-yl)-1H-
pyrazole and 3-methyl-4-nitro-1-(oxetan-3-yl)-1H-pyrazole (0.137 g,
0.75 mmol) in ethanol (2 mL) was added Pd/C (10 wt %, 0.10 g).
The mixture was stirred under a hydrogen atmosphere for 24 h. The
reaction was filtered through Celite and concentrated to give a mixture
of 5-methyl-1-(oxetan-3-yl)-1H-pyrazol-4-amine and 3-methyl-1-(ox-
etan-3-yl)-1H-pyrazol-4-amine (83 mg, 73% yield). LCMS, m/z = 154
[M + H]+.
( )-(trans)-3-Fluoro-4-(3-methyl-4-nitro-1H-pyrazol-1-yl)-1-
(oxetan-3-yl)piperidine (50). The title compound was prepared in a
manner analogous to 49 (81% yield). LCMS, m/z = 285 [M + H]+.
(
)-trans-3-Fluoro-4-(4-nitro-1H-pyrazol-1-yl)piperidine
(52). To a mixture of ( )-cis-butyl 3-fluoro-4-hydroxypiperidine-1-
carboxylate (25 g, 114 mmol), 4-nitropyrazole (51, 15.48 g, 137
mmol), and PPh3 (44.8 g, 171 mmol) in THF (800 mL) was added
diisopropyl azodicarboxylate (34.54 g, 171 mmol) dropwise at 0 °C
under a nitrogen atmosphere. The reaction was stirred at 30 °C for 20
h and then concentrated in vacuo, and the residue was diluted with
EtOAc (100 mL) and washed with water (50 mL) and brine (50 mL).
The organic layer was dried over magnesium sulfate, filtered, and
concentrated, and the residue was purified by silica gel chromatog-
raphy (petroleum ether/EtOAc, 5:1) to give the crude product as a
yellow solid. To a solution of the yellow solid in CH2Cl2 (200 mL)
was added TFA (60 g, 0.53 mol), and the mixture was stirred at room
temperature for 2 h. The reaction mixture was then concentrated in
vacuo, the residue was diluted with DCM (100 mL) and water (50
mL), and the pH value was adjusted to 8 with a saturated K2CO3
solution. The organic layer was dried over magnesium sulfate, filtered,
and concentrated. The residue was purified by silica gel chromatog-
raphy (10% MeOH in CH2Cl2) to give the title compound as a yellow
solid (14.3 g, 58% yield over two steps). LCMS, m/z = 215 [M + H]+.
( )-(trans)-4-(5-Chloro-4-nitro-1H-pyrazol-1-yl)-3-fluoro-1-
(oxetan-3-yl)piperidine (53). To a solution of ( )-(trans)-3-fluoro-
4-(4-nitro-1H-pyrazol-1-yl)piperidine (12.8 g, 60 mmol), oxetan-3-one
(4.75 g, 66 mmol), and ZnCl2 (82 mg, 0.6 mmol) in MeOH (200 mL)
was added NaBH3CN (5.67 g, 90 mmol) at 0 °C. The reaction was
stirred at 30 °C for 20 h and then quenched with ice water, and the
solvent was removed in vacuo. The residue was diluted with EtOAc
(100 mL) and washed with water and brine. The organic layer was
then dried over magnesium sulfate, filtered, and concentrated. The
residue was purified by silica gel chromatography (CH2Cl2/MeOH,
15:1) to give ( )-(trans)-3-fluoro-4-(4-nitro-1H-pyrazol-1-yl)-1-(ox-
etan-3-yl)piperidine as a yellow solid (13.5 g, 85%). LCMS, m/z = 271
[M + H]+.
5-Methyl-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-amine
(46). The title compound was prepared in a manner analogous to 45
(72% yield, two steps). LCMS, m/z = 182 [M + H]+.
tert-Butyl 4-(3-Methyl-4-nitro-1H-pyrazol-1-yl)piperidine-1-
carboxylate (47). A mixture of 3-methyl-4-nitropyrazole (1.47 g,
11.6 mmoL), N-Boc-4-bromopiperidine (3.7 g, 14 mmol), and cesium
carbonate (7.5 g, 23 mmol) in DMF (15 mL) was stirred in a sealed
reaction tube at 120 °C for 2 days. The reaction was then diluted with
water and extracted with EtOAc (3×). The combined extracts were
washed with brine, dried over sodium sulfate, filtered, and
concentrated. The crude product was then purified by silica gel
chromatography (0−100% EtOAc in heptane) to give a 2:1 mixture of
tert-butyl 4-(3-methyl-4-nitro-1H-pyrazol-1-yl)piperidine-1-carboxylate
and tert-butyl 4-(5-methyl-4-nitro-1H-pyrazol-1-yl)piperidine-1-car-
boxylate as an off-white solid (1.9 g, 52% combined yield). The
crude product was carried to the next step without purification. LCMS,
m/z = 311 [M + H]+.
( )-(trans)-tert-Butyl 3-Fluoro-4-(3-methyl-4-nitro-1H-pyra-
zol-1-yl)piperidine-1-carboxylate (48). To a solution of 5-
methyl-4-nitro-1H-pyrazole (1.2 g, 9.6 mmol), tert-butyl (cis)-3-
fluoro-4-hydroxypiperidine-1-carboxylate (2.1 g, 9.6 mmol), and
triphenylphosphine (2.8 g, 11 mmol) in THF (8 mL) was added
diisopropyl azodicarboxylate (2.4 mL, 11 mmol). The reaction was
stirred at room temperature for 18 h. The mixture was then diluted
with brine and extracted with EtOAc (3×). The combined extracts
were washed with brine, dried over sodium sulfate, filtered, and
concentrated. The crude product was purified by silica gel
chromatography (0−100% EtOAc in heptane) to give a mixture of
the title compound (3.24 g, quant.) containing a trace amount of
(trans)-tert-butyl 3-fluoro-4-(5-methyl-4-nitro-1H-pyrazol-1-yl)-
piperidine-1- carboxylate. LCMS, m/z = 329 [M + H]+.
To a solution of ( )-(trans)-3-fluoro-4-(4-nitro-1H-pyrazol-1-yl)-1-
(oxetan-3-yl)piperidine (13.5 g, 50 mmol) in THF (150 mL) was
added LiHMDS (60 mL, 1 M in THF, 60 mmol) dropwise at −70 °C
under a nitrogen atmosphere. The reaction was stirred at −70 °C for 2
h, and C2Cl6 (14.2 g, 60 mmol) in THF (30 mL) was then added. The
mixture was allowed to warm to room temperature and stirred for 2 h.
The mixture was quenched with ice water and concentrated in vacuo.
The residue was diluted with EtOAc (100 mL) and washed with water
and brine. The organic layer was dried over magnesium sulfate,
filtered, and concentrated. The residue was purified by silica gel
chromatography (50% EtOAc in petroleum ether) to give the
( )-(trans)-4-(5-chloro-4-nitro-1H-pyrazol-1-yl)-3-fluoro-1-(oxetan-3-
yl)piperidine as a white solid (11.9 g, 65% yield). 1H NMR (500 MHz,
CDCl3) δ 8.26 (s, 1H), 5.15−5.01 (m, 1H), 4.74−4.71 (m, 2H),
4.66−4.63 (m, 2H), 4.51−4.43 (m, 1H), 3.73−3.67 (m, 1H), 3.28−
3.26 (m, 1H), 2.93−2.89 (m, 1H), 2.44−2.35 (m, 1H), 2.19−2.05 (m,
3H). LCMS, m/z = 305 [M + H]+.
4-(3-Methyl-4-nitro-1H-pyrazol-1-yl)-1-(oxetan-3-yl)-
piperidine (49). To a solution of tert-butyl 4-(3-methyl-4-nitro-1H-
pyrazol-1-yl)piperidine-1-carboxylate (0.65 g, 2.1 mmol) in 1,4-
dioxane (5 mL) was added a 4 M HCl in 1,4-dioxane solution (5
mL, 20 mmol). The reaction was stirred at 55 °C for 3 h. The resulting
precipitate was collected by filtration to give a 2:1 mixture of crude
hydrochloride salts of 4-(3-methyl-4-nitro-1H-pyrazol-1-yl)piperidine
and 4-(5-methyl-4-nitro-1H-pyrazol-1-yl)piperidine (0.51 g, quant.).
The crude product was carried to the next step without purification.
Molecular Modeling. Homology models of LRRK2 were
constructed using the modeling program MOE version 2009.1062
and AMBER9963 force field. The human LRRK2 sequence was
M
dx.doi.org/10.1021/jm401654j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
retrieved from Swiss-Prot64 and aligned to template structure
sequences using ClustalW65 followed by manual fine-tuning of
residues adjacent to loop regions, insertions, and deletions. The
models were further refined with bound ligand using the Macromodel
utility implemented in Maestro and OPLS2005 force field.66 Inhibitor
docking studies were carried out using docking program Glide SP with
one hydrogen-bond constraint to the carbonyl oxygen of hinge residue
Ala1950. The docking poses were evaluated on the basis of a
combination of criteria including the Glide docking score (cutoff =
−6), favorable intermolecular interactions with the hinge and other
parts of the ATP-binding pocket, low strain energy of the bound ligand
(Estrain < 2 kcal/mol), and so forth.
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in Parkinson’s disease. Expert Opin. Pharmacother. 2006, 7, 1715−
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Lincoln, S.; Kachergus, J.; Hulihan, M.; Uitti, R. J.; Calne, D. B.;
Stoessl, A. J.; Pfeiffer, R. F.; Patenge, N.; Carbajal, I. C.; Vieregge, P.;
Asmus, F.; Muller-Myhsok, B.; Dickson, D. W.; Meitinger, T.; Strom,
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PARK8-linked Parkinson’s disease. Neuron 2004, 44, 595−600.
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K. A. LRRK2 in Parkinson’s disease: Protein domains and functional
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mutations and Parkinson’s disease: Three questions. ASN Neuro 2009,
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ASSOCIATED CONTENT
* Supporting Information
■
S
Additional experimental procedures and references. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*(A.A.E.) Phone: 650-225-4934. Fax: 650-742-4943. E-mail:
estrada.anthony@gene.com.
*(B.K.C.) Phone: 650-467-3195. Fax: 650-742-4943. E-mail:
chan.bryan@gene.com.
(10) Satake, W.; Nakabayashi, Y.; Mizuta, I.; Hirota, Y.; Ito, C.; Kubo,
M.; Kawaguchi, T.; Tsunoda, T.; Watanabe, M.; Takeda, A.;
Tomiyama, H.; Nakashima, K.; Hasegawa, K.; Obata, F.; Yoshikawa,
T.; Kawakami, H.; Sakoda, S.; Yamamoto, M.; Hattori, N.; Murata, M.;
Nakamura, Y.; Toda, T. Genome-wide association study identifies
common variants at four loci as genetic risk factors for Parkinson’s
disease. Nat. Genet. 2009, 41, 1303−1307.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank our Analytical, Bioanalytical, NMR, Pharmacokinetic,
Biopharmacology, and Safety Assessment colleagues for their
contributions. We also thank John Atherall, Linda Bao, Leo
Berezhkovskiy, Diane Carrera, Jonathan Cheong, Po-Chang
Chiang, Kang-Jye Chou, Xiao Ding, Quynh Ho, Claire Holt,
Heather Kennedy, Emile Plise, Veronica Rivera, Kimberley
(11) Simon
́ ́
-Sanchez, J.; Schulte, C.; Bras, J. M.; Sharma, M.; Gibbs, J.
R.; Berg, D.; Paisan-Ruiz, C.; Lichtner, P.; Scholz, S. W.; Hernandez,
D. G.; Kruger, R.; Federoff, M.; Klein, C.; Goate, A.; Perlmutter, J.;
̈
Bonin, M.; Nalls, M. A.; Illig, T.; Gieger, C.; Houlden, H.; Steffens, M.;
Okun, M. S.; Racette, B. A.; Cookson, M. R.; Foote, K. D.; Fernandez,
H. H.; Traynor, B. J.; Schreiber, S.; Arepalli, S.; Zonozi, R.; Gwinn, K.;
van der Brug, M.; Lopez, G.; Chanock, S. J.; Schatzkin, A.; Park, Y.;
Hollenbeck, A.; Gao, J.; Huang, X.; Wood, N. W.; Lorenz, D.; Deuschl,
G.; Chen, H.; Riess, O.; Hardy, J. A.; Singleton, A. B.; Gasser, T.
Genome-wide association study reveals genetic risk underlying
Parkinson’s disease. Nat. Genet. 2009, 41, 1308−1312.
Smith, Hilda Solanoy, and Herve
individual contributions.
́
Van de Poel for their
̈
ABBREVIATIONS USED
■
AUC, area under the curve; BAC, bacterial artificial
chromosome; BCRP, breast cancer resistance protein; Bu,
unbound brain concentration; CNS, central nervous system;
CSF, cerebrospinal fluid; CYP, cytochrome P450; DIPEA, N,N-
diisopropylethylamine; ER, efflux ratio; h, human; ip, intra-
perotineal; iv, intravenous; JAK2, janus kinase 2; LELP, ligand-
efficiency-dependent lipophilicity; LM, liver microsome;
LRRK2, leucine-rich repeat kinase 2; MCT, methylcellulose/
tween; MDCK-MDR1, Madin-Darby canine kidney cells-
multidrug resistance protein 1; NMP, N-methyl-2-pyrrolidone;
PD, Parkinson’s disease; PE, petroleum ether; PEG, poly-
(ethylene glycol); P-gp, P-glycoprotein; PK/PD, pharmacoki-
netics/pharmacodynamics; pLRRK2, phospho-LRRK2; PPB,
plasma protein binding; Pu, unbound plasma concentration; r,
rat; SFC, supercritical fluid chromatography; TDI, time-
dependent inhibition; TPSA, topological surface area; Vd,
volume of distribution; XPhos, 2-dicyclohexylphosphino-
2′,4′,6′-triisopropylbiphenyl
(12) West, A. B.; Moore, D. J.; Biskup, S.; Bugayenko, A.; Smith, W.
W.; Ross, C. A.; Dawson, V. L.; Dawson, T. M. Parkinson’s disease-
associated mutations in leucine-rich repeat kinase 2 augment kinase
activity. Proc. Natl. Acad. Sci.U.S.A. 2005, 102, 16842−16847.
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Kaganovich, A.; van der Brug, M. P.; Beilina, A.; Blackinton, J.;
Thomas, K. J.; Ahmad, R.; Miller, D. W.; Kesavapany, S.; Singleton, A.;
Lees, A.; Harvey, R. J.; Harvey, K.; Cookson, M. R. Kinase activity is
required for the toxic effects of mutant LRRK2/dardarin. Neurobiol.
Dis. 2006, 23, 329−341.
(14) Cookson, M. R. The role of leucine-rich repeat kinase 2
(LRRK2) in Parkinson’s disease. Nat. Rev. Neurosci. 2010, 11, 791−
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(15) Rudenko, I. N.; Chia, R.; Cookson, M. R. Is inhibition of kinase
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(16) Liu, Z.; Hamamichi, S.; Lee, B. D.; Yang, D.; Ray, A.; Caldwell,
G. A.; Caldwell, K. A.; Dawson, T. M.; Smith, W. W.; Dawson, V. L.
Inhibitors of LRRK2 kinase attenuate neurodegeneration and
Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila
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