Discovery of highly potent, selective, and brain-penetrable leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors

Article abstract of DOI:10.1021/jm301020q

There is a high demand for potent, selective, and brain-penetrant small molecule inhibitors of leucine-rich repeat kinase 2 (LRRK2) to test whether inhibition of LRRK2 kinase activity is a potentially viable treatment option for Parkinson's disease patients. Herein we disclose the use of property and structure-based drug design for the optimization of highly ligand efficient aminopyrimidine lead compounds. High throughput in vivo rodent cassette pharmacokinetic studies enabled rapid validation of in vitro-in vivo correlations. Guided by this data, optimal design parameters were established. Effective incorporation of these guidelines into our molecular design process resulted in the discovery of small molecule inhibitors such as GNE-7915 (18) and 19, which possess an ideal balance of LRRK2 cellular potency, broad kinase selectivity, metabolic stability, and brain penetration across multiple species. Advancement of GNE-7915 into rodent and higher species toxicity studies enabled risk assessment for early development.

Full text of DOI:10.1021/jm301020q

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pubs.acs.org/jmc
Discovery of Highly Potent, Selective, and Brain-Penetrable Leucine-
Rich Repeat Kinase 2 (LRRK2) Small Molecule Inhibitors
Anthony A. Estrada,*,† Xingrong Liu,∥ Charles Baker-Glenn,∞ Alan Beresford,○ Daniel J. Burdick,†
Mark Chambers,∞ Bryan K. Chan,† Huifen Chen,† Xiao Ding,∥ Antonio G. DiPasquale,●
Sara L. Dominguez,‡ Jennafer Dotson,† Jason Drummond,§ Michael Flagella,⊥ Sean Flynn,∞ Reina Fuji,⊥
Andrew Gill,× Janet Gunzner-Toste,† Seth F. Harris,# Timothy P. Heffron,† Tracy Kleinheinz,§
Donna W. Lee,⊥ Claire E. Le Pichon,‡ Joseph P. Lyssikatos,† Andrew D. Medhurst,× John G. Moffat,§
Susmith Mukund,# Kevin Nash,× Kimberly Scearce-Levie,‡ Zejuan Sheng,‡ Daniel G. Shore,† Thuy Tran,†
Naimisha Trivedi,∞ Shumei Wang,† Shuo Zhang,‡ Xiaolin Zhang,∥ Guiling Zhao,† Haitao Zhu,‡
and Zachary K. Sweeney†,▽
‡
§
†Department of Discovery Chemistry, Department of Neurosciences, Department of Biochemical and Cellular Pharmacology,
∥Department of Drug Metabolism and Pharmacokinetics, ⊥Department of Safety Assessment, and #Department of Structural Biology,
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
∞Department of Chemistry, Department of Biochemical and Cellular Pharmacology, and ○Department of Drug Metabolism and
×
Pharmacokinetics, BioFocus, Chesterford Research Park, Saffron Walden, CB10 1XL, United Kingdom
●X-ray Crystallographic Facility, University of CaliforniaBerkeley, 32 Lewis Hall, Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: There is a high demand for potent, selective, and brain-penetrant
small molecule inhibitors of leucine-rich repeat kinase 2 (LRRK2) to test whether
inhibition of LRRK2 kinase activity is a potentially viable treatment option for
Parkinson’s disease patients. Herein we disclose the use of property and
structure-based drug design for the optimization of highly ligand efficient
aminopyrimidine lead compounds. High throughput in vivo rodent cassette
pharmacokinetic studies enabled rapid validation of in vitro−in vivo correlations.
Guided by this data, optimal design parameters were established. Effective
incorporation of these guidelines into our molecular design process resulted in
the discovery of small molecule inhibitors such as GNE-7915 (18) and 19, which
possess an ideal balance of LRRK2 cellular potency, broad kinase selectivity,
metabolic stability, and brain penetration across multiple species. Advancement
of GNE-7915 into rodent and higher species toxicity studies enabled risk
assessment for early development.
monocytes, etc., and roles in immunology, inflammatory
bowel disease, cancer, and leprosy have been proposed.21,22
However, the utility of these inhibitors for studying the role of
LRRK2 in PD is limited, as they either do not sufficiently cross
the blood−brain barrier or do not demonstrate inhibition of
kinase activity in rodent brains.23
Our group has demonstrated the in vivo inhibition of LRRK2
autophosphorylation of serine 1292 (Ser1292) in the brains of
transgenic mice using a potent, selective, and brain-penetrable
tool compound (7); the discovery and characterization of this
autophosphorylation site are described elsewhere.24,26 The
moderate total plasma (∼150 nM) and brain (∼100 nM)
concentrations of 7 required to significantly inhibit LRRK2
INTRODUCTION
■
Parkinson’s disease (PD) is the second most common
neurodegenerative disease and affects approximately 1 in 100
people over the age of 60. A disease-modifying or neuro-
protective therapy is highly desired, as the current standard of
care is limited to symptomatic treatment.1
In the past decade, the LRRK2 gene, which encodes a large,
multidomain protein, has been linked to familial PD.2−6
Furthermore, the autosomal dominant G2019S kinase domain
variant has been associated with both familial and idiopathic
PD.7−9 Numerous reports have been published that suggest
that the kinase activity of LRRK2 is involved in Parkinson’s
disease pathophysiogenesis.10−17 Some of the small molecule
inhibitors disclosed in these reports are highly selective for
LRRK2 and could serve as useful tool compounds for
examination of LRRK2 function in the periphery.18−20 This is
critical as LRRK2 is expressed in kidney, lung, spleen,
Received: July 12, 2012
Published: September 17, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Featured Article
kinase activity in vivo provide further support for LRRK2 as a
potentially druggable target for PD.
based drug discovery to successfully optimize a series of
diaminopyrimidine inhibitors that led to the identification of 1
as a potent, selective, and brain penetrable LRRK2 small
molecule inhibitor.25 Compound 1, originally disclosed in a
2011 Genentech patent application,35 was subsequently used to
demonstrate in vivo inhibition of Ser910 and Ser935
phosphorylation in mouse brain.36 In this contribution we
report the expanded profiling of 1 and our subsequent lead
optimization efforts to identify a LRRK2 small molecule
inhibitor suitable for progression to higher species toxicity
studies and early development evaluation.
We recently reported the construction of a JAK2-based
LRRK2 homology model that was used to detect several
binding site residues that impart general kinase selectivity.25
Through a matched molecular pair activity cliff analysis we were
able to identify an amino acid near the hinge binding region
(Leu1949) as a selectivity hotspot.27−32 As illustrated in Figure
1, small ortho-substituents, such as the methoxy group in 2,4-
RESULTS AND DISCUSSION
■
As previously reported,25 1 has a LRRK2 biochemical potency
of 3 nM (Table 1), a JAK2 biochemical selectivity index of
>1067× and only inhibits wild-type and G2019S mutant
LRRK2 at greater than 50% inhibition at 1 μM (345-fold over
Ki) in a representative Invitrogen panel of 63 kinases. At this
stage of lead optimization, the JAK2 biochemical assay proved
to be a useful barometer for general kinase selectivity. In order
to determine the cellular activity of 1, we measured the
inhibition of LRRK2 autophosphorylation in HEK293 cells
using our LRRK2 cellular assay.24 As shown in Table 1, the
phospho-LRRK2 (pLRRK2) cellular IC50 of 1 was 29 nM,
translating into an enzyme-to-cell shift of approximately 10-
fold. With the cellular assay in place, we explored structure−
activity relationships (SARs) of the aminopyrimidine C-4 and
C-5 positions. The examples shown in Table 1 demonstrate
good biochemical (<10 nM) and cellular activities (<70 nM)
with the exception of aminopyrimidine C-5 fluoro (5) and C-5
cyano (6) analogues. The rationale for this observation was that
C-5 groups with increased lipophilicity and size are able to
interact more favorably with the gatekeeper Met1947 residue.
Furthermore, all of the examples in Table 1 possess desirable
CNS MPO scores37 and are highly efficient as measured by
lipophilic ligand efficiency (LLE)38 and ligand-efficiency-
dependent lipophilicity (LELP)39 parameters with scores that
are in line with CNS marketed drugs and clinical candidates.40
The optimal C-4/C-5 combination proved to be the C-4
aminomethyl/C-5 trifluoromethyl substituted aminopyrimidine
(i.e., 7, pLRRK2 IC50 of 9 nM).
Figure 1. Docking model of 1 (cyan) in LRRK2 (green with L1949 in
yellow). The side chain of Y931 of JAK2 is also shown (magenta).
Intermolecular hydrogen bond interactions are shown as yellow
dashed lines.
diaminopyrimidine 1, are well tolerated in LRRK2 but are
disfavored in JAK2 and approximately 290 other kinases that
contain a larger Phe or Tyr residue in this binding site region
near the hinge.25,33,34 This finding allowed us to use structure-
Table 1. Representative Aminopyrimidine SAR for C-4/C-5 Substitution Combinations
a
b
c
compd
R1
Cl
R2
CNS MPO score
LRRK2 Ki (nM)
LLE
LELP
JAK2 selectivity lndex
pLRRK2: IC50 (nM)
1
2
3
4
5
6
7
NHMe
OMe
5.4
5.6
5.1
5.2
5.5
4.7
5.1
3
9
6.6
5.7
6.8
6.0
5.8
6.9
6.6
4.2
5.3
4.3
5.4
3.6
2.4
5.0
>1067
>360
1546
>661
>55
29
69
Cl
Br
NHMe
OMe
2
13
Br
5
53
F
NHMe
NHMe
NHMe
58
11
2
418
105
9
CN
CF3
>288
>1928
a
b
c
Biochemical assay. (JAK2 Ki)/(LRRK2 Ki). Cellular assay.
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a
Table 2. Rat in Vitro and in Vivo PK Profiles of Aminopyrimidines
b
d
e
f
hepatocytes Clhep
% rat
PPB
Cl (Clu)
iv t1/2
(h)
MDR1 Papp, A−B
c
g
h
f
i
j
compd
(mL min−1 kg−1), h/r
(mL min−1 kg−1
)
F (%)
(×10−6cm/s)
MDR1 P-gp ER (B−A /A−B ) Bu/Pu CSF/Pu
1
2
4
6
7
4.9/24.3
1.8/4.4
12/16
91.8
94.6
96.6
83.5
86.3
51 (616)
9.8 (183)
5.7 (167)
4.4 (27)
24 (156)
0.46
0.83
0.67
1.1
64
106
58
4.4
8.2
2.8
0.8
1.4
6.8
1.2
0.17
0.23
0.10
0.08
0.50
0.29
0.84
0.18
0.19
0.48
5.6
2.1/5.4
1.8/7.6
117
80
3.1
1.2
18.2
a
Compound dosed po (1 mg/kg) as a suspension in MCT and iv (0.5 mg/kg) as a 60% PEG solution or 20−60% NMP solution for systemic PK
b
c
d
and iv as a 60% NMP solution for brain PK. In vitro stability in cryopreserved hepatocytes. h/r = human/rat. Clu = unbound clearance = total
clearance/[(100 − % rat PPB)/100]. MDCK-MDR1 human P-gp transfected cell line. A−B, apical-to-basolateral. Efflux ratio. B−A, basolateral-
to-apical. Unbound brain/unbound plasma AUC ratio. CSF/unbound plasma AUC ratio.
e
f
g
h
i
j
The rationale for installing the aminopyrimidine C-5
trifluoromethyl group in 7 was not limited to increasing the
activity for LRRK2 by providing optimal lipophilic contact with
the Met gatekeeper. We also hypothesized that the
trifluoromethyl group would impart a more favorable DMPK
profile in comparison to other C-5 substituents because of the
relative decreased oxidation potential of the pyrimidine ring
system. The increased lipophilicity of the trifluoromethyl group
should also favor blood−brain barrier penetration.
was achieved despite the presence of two HBDs and an amide
functionality, both of which may be considered undesirable for
crossing the blood−brain barrier. However, the small molecule
X-ray crystal structure of 7 (Figure 2) suggests that
In order to test this hypothesis, the DMPK profiles of
selected analogues from Table 1 were assessed (Table 2). Rat
was employed as the primary rodent species for exploratory in
vivo pharmacokinetic (PK) studies. In order to accurately assess
the brain penetration of multiple compounds, we utilized a
cassette dosing approach that provided brain, plasma, and CSF
AUC ratios at 0.25, 1, and 3 h time points.41 Using in vitro PPB
and BPB values, we then converted total brain/plasma (B/P)
and total CSF/plasma (CSF/P) to unbound brain/unbound
plasma (Bu/Pu) and CSF/unbound plasma (CSF/Pu) ratios.
This methodology proved to be an extremely critical resource
for our program and enabled the rapid generation of
meaningful in vitro MDR1 efflux ratio−in vivo unbound
brain concentration correlations for medicinal chemistry design
and inhibitor evaluation (vide infra).
In initial rat pharmacokinetic experiments, the previously
reported aminopyrimidine lead (1)25 was cleared rapidly from
plasma and demonstrated substantial brain penetration impair-
ment (Bu/Pu = 0.17, Table 2). The reduced CNS penetration
was consistent with a moderate MDR1 efflux ratio (ER (B−A)/
(A−B) = 2.8). While this stands in contrast to the previously
reported mouse Bu/Pu ratio of 0.61, studies with P-gp/Bcrp
knockout mice also suggested that 1 was an efflux substrate.25
Additionally, 1 exhibited potent time-dependent inhibition
(TDI) of CYP1A2. By converting the C-4 aminomethyl in 1 to
a C-4 methoxy (2), we were able to remove one hydrogen
bond donor (HBD) and achieve a 5-fold reduction in total
clearance and also eliminate TDI concerns. Additionally, this
modification improved the passive permeability (A−B of 4.4 to
8.2 × 10−6 cm/s, Table 2) and attenuated P-gp efflux (ER of
2.8 to 0.8, Table 2). Unfortunately, we quickly discovered that
most C-4 methoxy analogues suffered from high brain protein
binding and suboptimal Bu/Pu ratios. For inhibitor 6, despite
the extremely low Clu (27 mL min−1 kg−1), the lack of brain
exposure (Bu/Pu = 0.08) as predicted by in vitro MDR1 data
(ER = 6.8) and poor cellular activity (105 nM, Table 1)
resulted in deprioritization of C-5 cyano analogues.
Figure 2. Small molecule X-ray crystal structure of 7 with suggested
intramolecular hydrogen bonds in green.
intramolecular hydrogen bonds are formed between a fluorine
of the C-5 trifluoromethyl group and the C-4 aminomethyl N-
H,42,43 as well as the aniline N-H and the methoxy group.
These interactions likely increase passive permeability and
reduce the susceptibility to efflux by P-gp and/or other
transporters.44 The crystal structure also confirms the role of
the amide group in reducing planarity, which is important for
improving solubility and minimizing CYP inhibition.45,46
Inhibitor 7 is also stable in human hepatocytes (Clhep = 1.8
mL min−1 kg−1) and the rat CSF levels correlate well with the
measured unbound brain concentration.47,48 These data, along
with the PK/PD analysis for 7 illustrating robust in vivo
inhibition of LRRK2 autophosphorylation at Ser1292 in the
brains of G2019S transgenic mice,24 provided strong support
for additional profiling of 7 and structurally related compounds.
Initial Invitrogen kinase selectivity profiling of 7 at 0.1 μM
(178 kinases, 60-fold over LRRK2 Ki; 0 kinases at >50%
inhibition) and 1 μM (63 kinases, 600-fold over LRRK2 Ki; 0
kinases at >80% inhibition) demonstrates that 7 is likely to be a
highly selective inhibitor for LRRK2. In order to ensure that we
were including all structurally related kinases in our screening
panels, we re-evaluated the ATP binding site sequences of all
human kinases to identify those that have either a high ATP
binding site sequence identity to LRRK2 or a combination of a
Met gatekeeper and a small residue such as Leu, Val, Cys, or
Ala at the Leu1949 position. We found 41 kinases that met
these criteria. Of the 11 of these that were available at
Invitrogen and were not represented in our original panels, only
Finally, as we had hypothesized (vide supra), 7 provided a
desirable balance between in vivo stability, oral exposure, and
brain penetration. Good CNS penetration for this compound
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TTK (MPS1) was inhibited by several of our lead compounds.
Compound 7, for example, inhibited TTK activity by 55% at
0.1 μM and 98% at 1 μM. A TTK Lanthascreen assay provided
a Ki of 25 nM for 7, which translates to a biochemical TTK
selectivity index of 13×.49 Because of the structural homology
between LRRK2 and TTK, as well as potential toxicity
concerns (vide infra), it was decided that an increased TTK
selectivity index was desirable.
In an attempt to introduce additional TTK selectivity while
maintaining the desirable attributes of 7, we examined the
inhibitor binding site residue differences between a published
TTK crystal structure (magenta, PDB code 3GFW)50 and the
LRRK2 homology model (green) with 7 (cyan) docked in
TTK (Figure 3). The Cys604 TTK residue is smaller than
Leu1949, supporting the lack of selectivity imparted by the o-
methoxy group that serves as the key selectivity handle for
many kinases with larger residues at this position. Additionally,
both TTK and LRRK2 have Met gatekeepers. However, our
analysis suggested that we may be able to exploit the Ser1954
(LRRK2) to Asp608 (TTK) residue difference by adding
substituents at C-5′ of the front pocket phenyl ring (para to the
methoxy group) and introducing unfavorable steric and
electrostatic interactions with the bulkier, negatively charged
Asp608 side chain. The estimated distance from C-5′ of docked
7 (cyan) to the Asp608 side chain carboxyl oxygen is
approximately 3.9 Å. Thus, relatively small substituents were
anticipated to clash with the Asp608 side chain and provide an
increased TTK selectivity index.
In addition to the lack of TTK selectivity, we also discovered
that 7 induced micronuclei formation at low concentrations in a
human peripheral blood lymphocyte in vitro assay and in a
mouse in vivo micronucleus study at multiple doses. While
TTK has not been previously implicated in in vitro genotoxicity
findings,51 the role of TTK in cell-cycle checkpoint assembly
raised some concern that in vitro genotoxicity could be linked
to TTK inhibition and further supported our decision to
identify compounds with greater TTK selectivity.52 Guided by
the structural insight described above, we therefore designed
the phenyl C-5′ substituted small molecule inhibitors shown in
Table 3.
Phenyl C-5′ chloro (8, Table 3) and methoxy (9, Table 3)
substituted matched pairs of 7 provided clear support for the
TTK selectivity hypothesis as the biochemical TTK selectivity
index increased from 13× in 7 to 53× and 275×, respectively.
These compounds also maintained desirable LRRK2 cellular
activities (7 and 21 nM). Desirable selectivity indices over JAK2
and TTK were also achievable with chlorine as the C-2′ JAK
selectivity handle and methoxy at C-5′ (10 and 11). Expanded
kinase profiling (189 kinases) at 1 μM demonstrated excellent
selectivity for both 10 (250-fold over LRRK2 Ki; 0 kinases at
>75% inhibition) and 11 (345-fold over LRRK2 Ki; only two
kinases at >80% inhibition). Thus, TTK and JAK2 biochemical
assays were incorporated into our screening cascade as off-
target counterscreens and served as robust indicators of general
kinase selectivity for new compounds.
Figure 3. Key residue differences between LRRK2 homology model
(green) and TTK X-ray structure (magenta) with 7 (cyan) docked in
TTK. Hydrogen bond interactions between the TTK hinge residue
Gly605 and 7 are shown as yellow dashed lines. The red arrow shows
the measured distance between C-5′ of 7 and the side chain carboxyl
oxygen of Asp608.
After the TTK selectivity issues were addressed, the in vitro
and in vivo DMPK profiles of phenyl 2′,5′ substituted
analogues 8−12 were assessed (Table 4). The plasma protein
Table 3. Preliminary Investigation of Phenyl 2′,5′-Substitution for Increased TTK Selectivity
a
b
c
d
compd
R1
R2
R3
LRRK2 Ki (nM)
JAK2 selectivity index
TTK selectivity index
pLRRK2 IC50 (nM)
7
Me
Me
Me
Me
Et
OMe
OMe
OMe
Cl
H
2
2
1
4
3
>1928
>1368
>2540
>808
13
53
9
7
8
Cl
9
OMe
OMe
OMe
275
105
77
21
24
27
10
11
Cl
>1096
a
b
c
d
Biochemical assay. (JAK2 Ki)/(LRRK2 Ki). (TTK Ki)/(LRRK2 Ki). Cellular assay.
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a
Table 4. Rat in Vitro and in Vivo PK Profiles of Phenyl 2′,5′-Substituted Aminopyrimidines 8−12
b
d
e
f
hepatocytes Clhep
% rat
Cl (Clu)
iv t1/2
(h)
MDR1 Papp, A−B
c
g
h
f
i
j
compd
(mL min−1 kg−1), h/r
PPB (mL min−1 kg−1
)
F (%)
(×10−6 cm/s)
MDR1 P-gp ER (B−A /A−B ) Bu/Pu CSF/Pu
8
2.8/22
0.8/15.2
0.8/3.7
0.9/26
5.7/14
99.1
91.4
90.8
96.3
95.5
4.7 (551)
1.1
1.2
0.83
1.2
2.5
8.1
4.4
1.9
3.4
1.1
1.6
1.3
0.11
0.80
0.72
0.77
0.41
2.0
k
9
15 (170)
101
48
k
10
11
59 (640)
10.4
8.3
1.8
k
19 (514)
47
2.9
l
k
12
10 (227)
66
8.5
a
Compound dosed po (1 mg/kg) as a suspension in MCT and iv (0.5 mg/kg) as a 60% PEG solution or 20−60% NMP solution for systemic PK
b
c
d
and iv as a 60% NMP solution for brain PK. In vitro stability in cryopreserved hepatocytes. h/r = human/rat. Clu = unbound clearance = total
e
f
g
h
clearance/[(100 − % rat PPB)/100]. MDCK-MDR1 human P-gp transfected cell line. A−B, apical-to-basolateral. Efflux ratio. B−A, basolateral-
to-apical. Unbound brain/unbound plasma AUC ratio. CSF/unbound plasma AUC ratio. Blood clearance is reported. Morpholine-d8 analogue of
11.
i
j
k
l
binding of 8 was unexpectedly high, resulting in an unbound
clearance (551 mL min−1 kg−1) 3.5-fold higher than 7 and an
unacceptable Bu/Pu ratio of 0.11. Despite showing slight MDR1
efflux with the human P-gp cell line, dimethoxy analogue 9
demonstrated an in vivo profile comparable to that of 7. While
9 did not reveal any covalent adducts from in vitro trapping
studies with glutathione, we ultimately decided not to pursue
this compound because of concerns regarding electrophilic
metabolites that might be formed from oxidation of the
electron-rich phenyl ring. The excellent brain penetration of 10
was promising (Bu/Pu = 0.77); however, a large in vitro−in vivo
(iviv) disconnect resulted in a clearance higher than liver blood
flow. As a result of significant N-demethylation observed from
in vitro metabolite identification studies, the aminoethyl
analogue of 10 (11) was prepared and successfully reduced
the total clearance while maintaining comparable brain
penetration. Both 10 and 11 also demonstrated significant in
vitro oxidative metabolism of the morpholine ring. Thus, in an
attempt to further reduce the metabolic turnover, the
morpholine-d8 analogue (12) of 11 was synthesized and
reduced the clearance significantly, presumably because of
kinetic deuterium isotope effects.53
Figure 4. Vortex charts illustrating (A) binned cLogD (pH 7.4) values
for aminopyrimidines colored by in vitro human hepatocyte stability
measured from cryopreserved hepatocytes and (B) binned TPSA
values for aminopyrimidines colored by MDCK-MDR1 efflux ratios
(ER), Vortex, version 2012.02.13219.
As previously mentioned, 7 demonstrated genotoxicity at low
concentrations. This is a concern for an indication that
potentially requires chronic dosing from an early age.
Therefore, 10 and 11 were chosen for in vitro analyses in a
human peripheral blood lymphocyte genotoxicity assay. While
we were pleased to find that both parent molecules did not
induce micronuclei formation, incubation of 11 with S9 fraction
revealed the formation of potentially clastogenic metabolites.
Since the in vitro metabolite profile of deuterated morpholine
analogue 12 was similar to that of 11, these molecules were
deprioritized.
At this point in our program, we had established reliable
metabolic stability and permeability iviv correlations with a
wide range of aminopyrimidines, including those already
illustrated. These relationships were used to rapidly triage
newly synthesized molecules prior to in vivo evaluation, and
their associated physicochemical property ranges provided
useful guidelines at the design stage. For example, retrospective
analysis revealed that analogues with a cLogD between 1.8 and
3.3 had the highest probability (60%) of being metabolically
stable in human hepatocytes (Figure 4A). While inhibitors with
a cLogD < 1.8 also demonstrated good stability, most of these
compounds were P-gp efflux substrates. Most analogues with
TPSA < 90 showed a low propensity for P-gp efflux (85%
exhibited an efflux ratio of <3, Figure 4B). Thus, the application
of refined design parameters (1.8 < cLogD < 3.3 and TPSA <
90)54 for future analogues in this series was adopted to increase
our probability of success and ultimately reduce the number of
compounds synthesized prior to nomination for development.
With several molecules already demonstrating fairly opti-
mized activity, selectivity, and ADME profiles within the
aminopyrimidine scaffold, our goal was to make subtle
improvements while staying within the desirable property
ranges. This focus led to the alternative phenyl substitution
patterns shown in Table 5. C-5′ methyl analogues 13 and 14
displayed good TTK selectivity, with the latter also possessing
good LRRK2 cellular activity. In an attempt to maintain
acceptable selectivity indices while reducing both molecular
weight and lipophilicity, various methoxy/fluoro combinations
were targeted (15−19, Table 5). 15 demonstrated excellent
kinase selectivity when tested against 189 kinases at 1 μM (135-
fold over LRRK2 Ki; 0 kinases at >75% inhibition); however,
the LRRK2 cellular activity for 15 (63 nM) was suboptimal.
Inhibitor 16, which lacked a C-5′ group, was even less selective
for TTK than 7 and confirmed the necessity for a substituent at
C-5′. Simply shifting the fluorine from C-3′ to C-5′ (17)
restored the biochemical TTK selectivity index to greater than
50× while also increasing affinity for LRRK2. Maintaining the
methoxy/fluoro arrangement at C-2′/C-5′ and varying amino-
alkyl R1 substitution resulted in single-digit nanomolar LRRK2
cellular activities for GNE-7915 (18, 9 nM, R1 = aminoethyl)
and 19 (4 nM, R1 = aminocyclopropyl). Expanded Invitrogen
kinase profiling (187 kinases) at 0.1 μM for both 18 (100-fold
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Table 5. Aminopyrimidine Phenyl 2′,3′,5′-Substitution SAR
a
b
c
d
compd
R1
R2
Cl
R3
R4
LRRK2 Ki (nM)
JAK2 selectivity index
TTK selectivity index
pLRRK2 IC50 (nM)
13
14
15
16
17
18
19
Me
Me
Me
Me
Me
Et
Me
Me
OMe
H
H
H
H
F
7
2
>489
>1871
189
78
387
106
11
43
11
63
35
16
9
OMe
F
7
OMe
OMe
OMe
OMe
12
6
>262
>533
>3200
>3200
F
H
H
62
F
1
53
cPr
F
H
1
118
4
a
b
c
d
Biochemical assay. (JAK2 Ki)/(LRRK2 Ki). (TTK Ki)/(LRRK2 Ki). Cellular assay.
Figure 5. KinomeScan selectivity tree for 18 at 0.1 μM (100-fold over LRRK2 Ki). Of 451 profiled kinases, only LRRK2, TTK, and ALK
demonstrated <35% inhibition of % control (>65% probe displacement).
a
Table 6. Rat in Vitro and in Vivo PK Profiles of Phenyl 2′,5′-Substituted Aminopyrimidines 14 and 17−19
b
d
e
f
hepatocytes Clhep
% rat
PPB
Cl (Clu)
iv t1/2
(h)
MDR1 Papp, A−B
c
g
h
f
i
j
compd
(mL min−1 kg−1), h/r
(mL min−1 kg−1
)
F (%)
(×10−6 cm/s)
MDR1 P-gp ER (B−A /A−B ) Bu/Pu CSF/Pu
14
17
18
19
4.2/22
3.9/11
3.2/14
4.9/15
95.8
95.7
96.6
97.6
28 (667)
12 (279)
8.3 (244)
6.8 (296)
1.2
4.2
3.1
6.0
46
79
40
65
5.0
3.3
3.1
2.2
0.9
1.3
0.88
0.15
0.50
0.61
2.1
0.28
0.60
0.27
10.4
10.9
a
Compound dosed po (1 mg/kg) as a suspension in MCT and iv (0.5 mg/kg) as a 60% PEG solution or 20−60% NMP solution for systemic PK
b
c
d
and iv as a 60% NMP solution for brain PK. In vitro stability in cryopreserved hepatocytes. h/r = human/rat. Clu = unbound clearance = total
clearance/[(100 − % rat PPB)/100]. MDCK-MDR1 human P-gp transfected cell line. A−B, apical-to-basolateral. Efflux ratio. B−A, basolateral-
to-apical. Unbound brain/unbound plasma AUC ratio. CSF/unbound plasma AUC ratio.
e
f
g
h
i
j
over LRRK2 Ki) and 19 (250-fold over LRRK2 Ki) resulted in
only TTK showing greater than 50% inhibition. Selectivity
profiling using the DiscoverX KinomeScan55 competitive
binding assay panel, which included 392 unique kinases, was
also performed for 18 at 0.1 μM (Figure 5). Binding of >50%
probe displacement was detected for 10 kinases and of >65%
for only LRRK2, TTK, and ALK, further supporting the
excellent LRRK2 selectivity for 18. Cerep receptor profiling,
including expanded brain panels, suggested that 18 and 19 only
inhibited 5-HT2B with >70% inhibition at 10 μM. 18 and 19
were confirmed to be moderately potent 5-HT2B antagonists in
in vitro functional assays.56
The DMPK profiles of the most promising compounds from
Table 5 were then assessed (Table 6). Inhibitor 14 was
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a
Table 7. SAR and DMPK Profiles of Aminopyrimidines 20−25
a
Compound dosed po (1 mg/kg) as a suspension in MCT and iv (0.5 mg/kg) as a 60% PEG solution or 20−60% NMP solution for systemic PK
b
c
d
e
and iv as a 60% NMP solution for brain PK. Biochemical assay. (TTK Ki)/(LRRK2 Ki). Cellular assay. Clu = unbound clearance = total
clearance/[(100 − % rat PPB)/100]. MDCK-MDR1 human P-gp transfected cell line. efflux ratio. A:B, apical-to-basolateral; B:A, basolateral-to-
apical. Unbound brain/unbound plasma AUC ratio. Blood clearance is reported.
f
g
h
i
j
moderately cleared as predicted by in vitro measurements and
demonstrated good metabolic stability in human hepatocytes. It
also displayed good in vivo brain penetration. Compound 17
also showed a strong iviv correlation with respect to metabolic
clearance; however, the Bu/Pu ratio of 0.15 was suboptimal.
Both 18 and 19 demonstrated excellent in vitro DMPK and in
vivo rat PK profiles: (a) minimal turnover in human
hepatocytes; (b) low total and unbound clearance values as
predicted by rat hepatocytes; (c) long half-lives and good oral
exposure; (d) high passive permeability, no human P-gp efflux,
and good brain penetration in rats. Having established the lead
profiles of 14, 18, and 19 as benchmarks, additional SAR
exploration was carried out to address specific areas that we felt
could potentially be improved upon.
As amides are often substrates for P-gp, one approach to
improve brain penetration of our current leads was to introduce
amide isosteres such as tetrazole 20 (Table 7). Indeed, 20 did
exhibit good brain exposure (Bu/Pu = 0.65); however, poor
solubility, relatively modest LRRK2 cellular activity, and mild
CYP1A2 TDI precluded further advancement. Similar
tetrazoles, triazoles, pyrazoles, and other heterocyclic replace-
ments showed promising brain exposure, but most suffered
from a combination of poor solubility and time-dependent
inhibition of CYP1A2. Alkylsulfones were also investigated as
amide replacements. Representative of this series, 21 showed
good in vivo stability and brain PK (Bu/Pu = 0.70); however,
the alkylsulfones were compromised by time-dependent
inhibition of CYP1A2, poor solubility, and interestingly,
inadequate kinase selectivity. These observations are consistent
with the notion that the morpholinoamide functionality of the
primary series of inhibitors is optimal for decreasing inhibitor
planarity and improving solubility.57,58 Pyridine replacement of
the phenyl ring was also conducted to improve solubility and
gauge whether or not the C-5′ nitrogen lone pair could serve as
a suitable TTK selectivity handle. 22 provided support for this
hypothesis, as it showed a 63-fold selectivity TTK index.
Unfortunately, although 22 exhibited optimal cellular activity,
unbound clearance, and bioavailability, poor brain penetration
prevented further profiling. Scission of the amide bond in 22
and removal of the morpholine nitrogen to avoid bis-aniline
formation afforded 23. Unfortunately, while 23 and various
other saturated heterocycles enhanced brain penetration,
inadequate levels of TTK selectivity were obtained. Finally,
several morpholine derivatives were targeted, as the morpholine
ring had been identified as a major site of metabolism and an
altered metabolic profile for unpredictable toxicity/genotoxicity
could prove desirable. This strategy is exemplified by 24 and 25
where the intent was to minimize P-gp recognition of the
introduced polarity through steric congestion (tertiary alco-
hols) and intramolecular hydrogen bonding (secondary amide
N-H and arylfluorine). Good LRRK2 activity and in vivo
metabolic clearance was achieved, but a combination of poor
solubility and insufficient unbound brain levels again precluded
further progression.
Lead compounds 14, 18, and 19 were further evaluated in
PK/PD studies. Thus, BAC transgenic mice expressing human
LRRK2 protein with the G2019S Parkinson’s disease mutation
were given either a single oral dose (po) or intraperotineal (ip)
injection. Brain (hippocampus) and peripheral (spleen) tissues
were harvested 1−24 h postdose to assess pSer1292 levels and
compound concentration.24 Figure 6A shows robust, concen-
tration-dependent knockdown of pLRRK2 in the brain after
oral dosing with 18 at both 15 and 50 mg/kg and after ip
dosing at both 10 and 50 mg/kg (see Supporting Information
for spleen PK/PD graphs of 18, 19, and 14). By use of a
pharmacodynamic inhibition model to fit the data, 18 had a
calculated in vivo unbound brain IC50 of 7 nM, which is
consistent with the cellular IC50 of 9 nM.59 Taken together with
the recently published PK/PD data for 7,24 we adopted a
screening PK/PD protocol that involved ip dosing at both 10
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studies. As depicted in Table 8, both compounds exhibited low
total and unbound plasma clearance values and long half-lives.
The bioavailability for 18 at 1 mpk (17%) and 20 mpk (24%)
was slightly lower than expected, especially when one considers
the minimal contribution from first pass hepatic extraction. It is
known, however, that compounds predominantly metabolized
by CYP enzymes can demonstrate higher levels of first-pass
intestinal metabolism in cynomolgus monkeys than in
humans.60 The decline in oral exposure from 1 to 20 mpk
with 19 is suggestive of solubility-limited absorption.61
As the advancement of previous leads had been halted
because of genotoxicity concerns, 18 was chosen for evaluation
of in vivo genotoxic liabilities. Sprague−Dawley rats were
administered 18 daily for 2 days at 100 mg/kg via oral gavage
and assessed for the presence of micronucleated polychromatic
erythrocytes in their bone marrow. Inhibitor 18 did not induce
micronucleus formation, even with an approximate unbound
AUC(0−24h) plasma concentration of 5.95 μM·h. 18 was then
progressed to 7-day repeat-dose toxicity studies in rats (10, 50,
and 100 mg/kg) and cynomolgus monkeys (10, 25, and 65 mg/
kg). The compound was well tolerated in rats after oral
administration with day 7 mean unbound plasma AUC(0−24h)
concentrations of 0.8, 3.9, and 4.7 μM·h, respectively. High
drug exposures were also achieved in cynomolgus monkeys
with day 7 mean unbound plasma AUC(0−24h) concentrations of
0.8, 3.1, and 7.8 μM·h, respectively. Additionally, cyno terminal
brain and CSF levels measured 24 h after receiving the last dose
on day 7 confirmed good brain penetration (Bu/Pu = 0.6 and
CSF/Pu = 0.82). Taken together, these results indicate that 18
can be used to preclinically assess the consequences of
inhibiting LRRK2 kinase activity in the brain. Full disclosure
of toxicity studies performed with 18 and other amino-
pyrimidines will be disclosed in due course.
CHEMISTRY
■
The syntheses of aminoethyl (28) and aminocyclopropyl (29)
2,4-diaminopyrimidine rings are shown in Scheme 1. Scheme 2
Figure 6. In vivo G2019S LRRK2 transgenic mouse PK/PD results
measuring brain pSer1292 autophosphorylation. The closed 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) 18 [15 mg/kg, po, at
1, 3, and 6 h (n = 4/dose); 50 mg/kg, po, at 1, 3, 6, and 12 h (n = 4/
dose) and 24 h (n = 1); 10 mg/kg, ip, at 1 and 2.5 h (n = 3/dose); 50
mg/kg, ip, at 1 and 6 h (n = 3/dose)]; (B) 19 [10 and 50 mg/kg, ip, at
1 and 6 h (n = 3/dose)]; (C) 14 [10 and 50 mg/kg, ip, at 1 and 6 h (n
= 3/dose)].
a
Scheme 1. Synthesis of 2,4-Diaminopyrimidine Cores
a
Reagents and conditions: (a) CuI, KF, TMSCF3, DMF, Δ, 69%; (b)
and 50 mg/kg with tissue collection at 1 and 6 h postdose to
assess future compounds with promising profiles. In these
studies, 19 and 14 also demonstrated on-target reduction of
brain pLRRK2 (Figure 6B and Figure 6C) with calculated in
vivo unbound brain IC50 of 27 nM for 19 and 24 nM for 14.
As a result of their encouraging in vitro and in vivo profiles,
18 and 19 were both progressed to cynomolgus monkey PK
c-PrNH2, THF, 34%.
describes the two-step syntheses of 2 and 4 from known (30)25
or commercially available (31) starting materials. Tetrasub-
stituted precursors for 13 and 14 are derived from anilines 33
and 34 (Scheme 3) via a Sandmeyer reaction to provide
a
Table 8. In Vivo Cyno PK Profiles of 18 and 19
b
c
compd
hepatocytes Clhep (mL min−1 kg−1), cyno
% cyno PPB
Cl (Clu) (mL min−1 kg−1
)
iv t1/2 (h)
F (%), 1 mg/kg
F (%), 20 mg/kg
18
19
14
94.5
95.5
11 (200)
3.8 (84)
7.7
17
24
22
9.2
10.7
105
a
b
Compound dosed po (1 and 20 mg/kg) with crystalline material as a suspension in MCT and iv (0.5 mg/kg) as a 20−60% NMP solution. In vitro
c
stability in cryopreserved hepatocytes. Clu = unbound clearance = total clearance/[(100 − % cyno PPB)/100].
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provide 12, 18, and 19 involved a TFA catalyzed SNAr of
anilines 42 and 43 and then peptide coupling as the final step.
The synthesis of aminopyrimidine amide isosteres 20 and 21
is depicted in Scheme 5. 20 is derived from Buchwald−Hartwig
coupling of 4-amino-3-chlorobenzonitrile 64 and 4-amino-
methyl-2-chloro-5-trifluoromethylpyrimidine (60), followed by
tetrazole formation (66) and methylation. Nucleophilic
arylfluorine displacement with sodium thiomethoxide, m-
CPBA oxidation to the corresponding sulfone 69, nitro
reduction, and acid catalyzed coupling of the resulting aniline
70 with 4-aminomethyl-2-chloro-5-trifluoromethylpyrimidine
(60) yielded 21. Scheme 6 illustrates the independent
syntheses of pyridine analogues 22 and 23 as well as
morpholine amide derivatives 24 and 25. Hence, palladium
catalyzed aminocarbonylation of 6-chloro-4-methoxypyridin-3-
amine 71 with morpholine and Mo(CO)6 followed by
Buchwald coupling afforded 22. The synthesis of 23
commenced with palladium catalyzed cross-coupling of 2-
chloro-4-methoxy-5-nitropyridine (73) followed by concom-
itant dihydropyran and nitro reduction to give 75. Buchwald
coupling then provided the final target (23). Amide derivative
24 was synthesized in the same manner as 18 (Scheme 4),
using 1-amino-2-methylpropan-2-ol instead of morpholine.
Lastly, bromination of toluene derivative 76, amide formation,
and subsequent intramolecular lactam formation to give 78
followed by nitro reduction and standard SNAr coupling
afforded isoindolone 25.
Scheme 2. Synthesis of Aminopyrimidine-4-methoxy
Analogues 2 and 4
a
a
Reagents and conditions: (a) 2,5-dichloro-4-methoxypyrimidine,
Cs2CO3, Xantphos, Pd2(dba)3, dioxane, microwave, 15%; (b) 5-
bromo-2-chloro-4-methoxypyrimidine, TFA, n-BuOH, microwave,
35%; (c) morpholine, HBTU, DIPEA, DMF, 56%.
Scheme 3. Synthesis of Tetrasubstituted Phenyl
Intermediates for the Synthesis of 13, 14, and 15
a
CONCLUSION
■
Starting from highly ligand efficient leads 1 and 7, the
successful development of small molecule LRRK2 inhibitors
suitable for higher species toxicity studies and early develop-
ment evaluation is described. Optimization of LRRK2 cellular
activity through appropriate C-4/C-5 aminopyrimidine sub-
stitution was achieved while imparting broad kinase selectivity,
including structurally related kinases such as TTK, through
strategic decoration of the phenyl ring. We were also able to
effectively balance physicochemical properties, which enabled
the generation of inhibitors with excellent in vitro and in vivo
DMPK profiles including minimal turnover in human
hepatocytes and low unbound clearance and good oral
exposure in rodents and cynomologus monkeys. Of equal
importance was the demonstrated ability of multiple com-
pounds to penetrate the blood−brain barrier while maintaining
such a well-balanced profile. Taken together, these efforts
culminated in the discovery of GNE-7915 (18) and 19, highly
potent, selective, metabolically stable and brain-penetrant
LRRK2 inhibitors with potent in vivo unbound brain IC50
values measured from G2019S transgenic mice PK/PD studies.
GNE-7915 achieved high exposures in the periphery and brain
throughout a 7-day dosing regimen in both rat and cynomolgus
monkey toxicity studies. A detailed report of toxicity studies
and associated histopathology findings with GNE-7915 and
other aminopyrimidine LRRK2 inhibitors will be disclosed in a
subsequent publication.
a
Reagents and conditions: (a) acetone, conc HCl, H2O, NaNO2, then
CuCN, NaCN; (b) AcOH, conc H2SO4, H2O, Δ; (c) t-BuOK, MeOH,
THF, 80%; (d) KOH, EtOH, H2O, 68%.
arylnitriles 35 and 36 followed by hydrolysis to afford
carboxylic acids 37 and 38. The precursor for 15 is generated
from regioselective methoxide displacement of 39 to provide
40 (Scheme 3), followed by hydrolysis to yield benzoic acid 41.
Scheme 4 illustrates the representative syntheses of amino-
pyrimidine tetra- and pentasubstituted morpholinoamides.
Standard amide formation from the associated benzoic acid
(41−43) followed by acid catalyzed SNAr or Buchwald−
Hartwig coupling between anilines 44−46 and a 4-aminoalkyl-
2-chloro-5-trifluoromethylpyrimidine derivative furnished an
array of products. The second method used to synthesize
various aminopyrimidine targets commences from morpholine
amide formation of the respective p-nitrobenzoic acids (37, 38,
47−49) followed by nitro reduction and SNAr coupling
between anilines 55−59 and 60. The third protocol used to
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 Companion
chromatography system using gradient elution or with prepacked silica
gel cartridges from Biotage using a Biotage SP4 or an Isolera 4 MPLC
system using gradient elution. NMR spectra were recorded on Bruker
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a
Scheme 4. Synthesis of Aminopyrimidine Phenyl Tetra- and Pentasubstituted Morpholinoamides
a
Reagents and conditions: (a) morpholine, HATU or HBTU, DIPEA, CH2Cl2; (b) TFA, n-BuOH, Δ; (c) Cs2CO3, Xantphos, Pd(OAc)2, dioxane,
microwave; (d) PTSA, dioxane, Δ; (e) TFA, n-BuOH, microwave; (f) Pd/C, MeOH, H2 or SnCl2·2H2O, EtOH, H2O, Δ or Fe dust, EtOH, NH4Cl,
b
H2O, Δ; (g) n-BuOH, microwave; (h) 2-chloro-N-alkyl-5-(trifluoromethyl)pyrimidin-4-amine, PTSA, dioxane, Δ. Morpholine-d8 analogue.
Avance 400, Bruker DPX 400M, Bruker Avance III 400, and Bruker
AV 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, m = multiplet. Preparative HPLC was performed on a Polaris
C18 5 μm column (50 mm × 21 mm), a Waters Sunfire OBD
Phenomenex Luna phenylhexyl column (150 mm × 19 mm), or a
Waters Xbridge phenyl column (150 mm × 19 mm), eluting with
mixtures of water−acetonitrile or water−methanol, optionally
containing a modifier (0.1% v/v formic 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 LCMS 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 (a) HPLC (Waters
Acquity UPLC column, 21 mm × 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 or (b) HPLC (Phenomenex Luna C18 (2)
column, 4.6 mm × 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
(Waters Xterra MS C18 column, 4.6 mm × 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 mm × 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; (e) HPLC (Phenomenex, Gemini NX C18 column, 4.6 mm ×
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.
The syntheses of compounds 1, 3, 5, 6, and 7 have been described
previously.25
(4-(5-Chloro-4-methoxypyrimidin-2-ylamino)-3-
methoxyphenyl)(morpholino)methanone (2). To a solution of
(4-amino-3-methoxyphenyl)(morpholino)methanone (30, 0.490 g, 2.1
mmol) in 1,4-dioxane (7 mL) were added 2,5-dichloro-4-methoxypyr-
imidine (0.370 g, 2.1 mmol), Cs2CO3 (1.4 g, 4.1 mmol), Xantphos
(0.100 g, 0.21 mmol), and Pd2(dba)3 (0.095 g, 0.10 mmol), and the
reaction mixture was stirred at 140 °C for 25 min under microwave
irradiation. The reaction mixture was filtered, concentrated, purified by
flash chromatography, 0−100% EtOAc/heptane, and further purified
by preparative HPLC to afford the title compound (117 mg, 15%
1
yield). H NMR (400 MHz, DMSO-d6) δ 8.31 (m, 2H), 8.16 (d, J =
8.2 Hz, 1H), 7.08 (d, J = 1.3 Hz, 1H), 7.01 (dd, J = 8.2, 1.3 Hz, 1H),
4.00 (s, 3H), 3.89 (s, 3H), 3.61 (br s, 4H), 3.52 (br s, 4H). LCMS, m/
z = 379 [M + H]+.
(4-(5-Bromo-4-methoxypyrimidin-2-ylamino)-3-
methoxyphenyl)(morpholino)methanone (4). To a solution of 4-
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was then concentrated. The residue was purified by preparative HPLC
to afford 66 mg (37% yield, two steps) of the title compound as a
white solid. H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.24 (s,
Scheme 5. Synthesis of Aminopyrimidineamide Isosteres 20
and 21
a
1
1H), 8.12 (s, 1H), 7.32 (d, J = 4.0 Hz, 1H), 7.06 (s, 1H), 3.89 (s, 3H),
3.49−3.71 (m, 6H), 3.15−3.24 (m, 2H), 2.94 (d, J = 4.3 Hz, 3H).
LCMS, m/z = 446 [M + H]+.
(2,5-Dimethoxy-4-(4-(methylamino)-5-(trifluoromethyl)-
pyrimidin-2-ylamino)phenyl)(piperidin-1-yl)methanone (9,
Method e). To a solution of (4-amino-2,5-dimethoxyphenyl)-
(morpholino)methanone (59, 200 mg, 0.750 mmol) in n-BuOH (2
mL) were added 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-
amine (60, 159 mg, 0.750 mmol) and two drops of TFA. The mixture
was stirred at 100 °C under microwave irradiation for 1 h. After the
mixture was cooled and concentrated, the residue was purified by
preparative HPLC to afford the title compound (80 mg, 24% yield,
1
three steps). H NMR (500 MHz, CDCl3) δ 8.34 (s, 1H), 8.18 (s,
1H), 6.86 (s, 1H), 5.34 (s, 1H), 3.89 (s, 3H), 3.84 (s, 3H), 3.77−3.84
(m, 4H), 3.60−3.66 (m, 2H), 3.28−3.39 (m, 2H), 3.15 (d, J = 4.5 Hz,
3H). LCMS, m/z = 442 [M + H]+.
( 5 - C h l o r o - 2 - m e t h o x y - 4 - ( 4 - ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-ylamino)phenyl)(morpholino)-
methanone (10, Method b). The title compound was prepared in a
manner analogous to that for 8 (22% yield, two steps). 1H NMR (400
MHz, DMSO-d6) δ 8.59 (s, 1H), 8.20 (s, 1H), 7.88 (s, 1H), 7.32 (s,
1H), 7.25 (s, 1H), 3.81 (s, 3H), 3.60 (s, 4H), 3.53 (t, J = 4.3 Hz, 2H),
3.17 (s, 2H), 2.90 (d, J = 4.3 Hz, 3H). LCMS, m/z = 446 [M + H]+.
(5-Chloro-4-(4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)-2-methoxyphenyl)(morpholino)methanone (11,
Method c). A mixture of 2-chloro-N-ethyl-5-(trifluoromethyl)-
pyrimidin-4-amine (28; 0.25 g, 1.11 mmol), cesium carbonate (0.72
g, 2.22 mmol), 4-amino-5-chloro-2-methoxyphenyl)(morpholino)-
methanone (44; 0.6 g, 2.22 mmol), palladium(II) acetate (5 mg,
0.022 mmol), and Xantphos (19 mg, 0.033 mmol) in degassed dry
dioxane (4 mL) was sonicated in an ultrasonic bath for 2 min. The
mixture was degassed further with bubbling N2 for 2 min and heated at
100 °C for 2 h. The mixture was cooled, diluted with EtOAc (25 mL),
and washed with water (2 × 20 mL). The combined aqueous washes
were extracted with EtOAc (2 × 25 mL). The organic extracts were
combined and filtered through a phase separator, and the solvent was
removed. Purification of the residue by flash chromatography, 0−100%
EtOAc/isohexane, followed by trituration with Et2O/EtOAc gave the
a
Reagents and conditions: (a) 60, Cs2CO3, Xantphos, Pd2(dba)3,
dioxane, Δ, 45%; (b) NaN3, NH4Cl, DMF, Δ, 85%; (c) MeI, K2CO3,
acetone, Δ, 70%; (d) MeSNa, EtOH, Δ, 91%; (e) m-CPBA, CH2Cl2,
48%; (f) Pd/C, EtOH, H2, 99%; (g) 60, TFA, 2-methoxyethanol, Δ,
19%.
1
title compound (0.15 g, 30% yield) as a white solid. H NMR (400
MHz, CDCl3) δ 8.38 (s, 1H), 8.22 (s, 1H), 7.64 (s, 1H), 7.31 (s, 1H),
5.19 (br s, 1H), 3.87 (s, 3H), 3.79−3.77 (m, 4H), 3.65−3.58 (m, 4H),
3.32 (br d, J = 14.8 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H); LCMS, m/z =
460 [M + H]+.
amino-3-methoxybenzoic acid (31, 1.77 g, 10.6 mmol) in n-BuOH (62
mL) was added 5-bromo-2-chloro-4-methoxypyridine (2.25 g, 10.1
mmol) and TFA (0.194 mL, 2.52 mmol), and the reaction mixture was
stirred at 125 °C for 30 min under microwave irradiation. The formed
precipitate was collected by filtration, washing with n-BuOH and then
Et2O to provide 1.25 g of crude 32, which was used without further
purification.
To the crude carboxylic acid from above (32, 365 mg, 1.03 mmol)
dissolved in DMF (10 mL) were added morpholine (0.110 mL, 1.26
mmol), DIPEA (0.215 mL, 1.24 mmol), and HBTU (469 mg, 1.24
mmol), and the reaction mixture was stirred at room temperature for 1
h. The mixture was diluted with water and extracted with EtOAc (3 ×
20 mL), and the combined organic layers were dried (Na2SO4),
filtered, and concentrated. The residue was purified by preparative
HPLC to afford the title compound (246 mg, 56% yield, two steps).
1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 8.30 (s, 1H), 8.16 (d,
(5-Chloro-4-(4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)-2-methoxyphenyl)(D8-morpholino)methanone (12).
The title compound was prepared in a manner analogous to that for
1
18 (26% yield, two steps). H NMR (400 MHz, CDCl3) δ 8.38 (s,
1H), 8.22 (s, 1H), 7.64 (br s, 1H), 7.31 (s, 1H), 5.19 (br s, 1H), 3.87
(s, 3H), 3.65−3.58 (m, 2H), 1.30 (t, J = 7.2 Hz, 3H). LCMS, m/z =
468 [M + H]+.
(5-Chloro-2-methyl-4-(4-(methylamino)-5-(trifluoromethyl)-
pyrimidin-2-ylamino)phenyl)(morpholino)methanone (13,
Method d). A mixture of (4-amino-5-chloro-2-methylphenyl)-
(morpholino)methanone (56, 79 mg, 0.31 mmol), 2-chloro-N-
methyl-5-(trifluoromethyl)pyrimidin-4-amine (60, 66 mg, 0.31
mmol), and PTSA (59 mg, 0.31 mmol) in dioxane (4 mL) was
heated to 100 °C for 4 h. Saturated sodium bicarbonate solution (10
mL) and CH2Cl2 (10 mL) were added, and the organic phase was
passed through a hydrophobic frit. The solvent was removed in vacuo
and the residue was purified by preparative HPLC to give the title
J = 8.2 Hz, 1H), 7.08 (d, J = 1.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H),
3.98 (s, 3H), 3.89 (s, 3H), 3.61 (br s, 4H), 3.52 (br s, 4H). LCMS, m/
z = 423 [M + H]+.
1
compound (71 mg, 53% yield) as an off-white solid. H NMR (400
MHz, CDCl3) δ 8.55 (s, 1H), 8.21 (d, J = 1.1 Hz, 1H), 7.57 (s, 1H),
7.22 (s, 1H), 5.30 (br s, 1H), 3.79 (br d, J = 13.9 Hz, 4H), 3.60 (br s,
2H), 3.32 (br s, 2H), 3.11 (d, J = 4.7 Hz, 3H), 2.32 (s, 3H). LCMS,
m/z = 430 [M + H]+.
5 - M e t h o x y - 2 - m e t h y l - 4 - ( 4 - ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-ylamino)phenyl)(morpholino)-
methanone (14 Method d). The title compound was prepared in a
( 2 - C h l o r o - 5 - m e t h o x y - 4 - ( 4 - ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-ylamino)phenyl)(morpholino)-
methanone (8, Method b). To a solution of 2-chloro-N-methyl-5-
(trifluoromethyl)pyrimidin-4-amine (60, 0.10 g, 0.47 mmol) and (4-
amino-2-chloro-5-methoxyphenyl)(morpholino)methanone (55, 0.11
g, 0.40 mmol) in n-BuOH (2.0 mL) was added TFA (0.051 mL, 0.66
mmol). The mixture was stirred at 75 °C for 2 h. The reaction mixture
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a
Scheme 6. Synthesis of Pyridine Analogues and Morpholinoamide Derivatives
a
Reagents and conditions: (a) Mo(CO)6, Pd(PPh3)4, 4-DMAP, DIPEA, morpholine, dioxane, microwave, 50%; (b) 28, BrettPhos, BrettPhos
precatalyst, NaO-t-Bu, t-BuOH, Δ, 35%; (c) 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Na2CO3, KOAc, Pd(amphos)-
Cl2, CH3CN, degassed H2O, microwave, 79%; (d) Pd/C, EtOH, H2; (e) 60, BrettPhos, BrettPhos precatalyst, NaO-t-Bu, t-BuOH, Δ, 52%, two
steps; (f) 28, PTSA, dioxane, Δ; (g) 1-amino-2-methylpropan-2-ol, HATU, DIPEA, CH2Cl2, 95%, two steps; (h) SOCl2, MeOH, Δ, 82%; (i) NBS,
CH3CN, AIBN, Δ, 31%; (j) 1-amino-2-methylpropan-2-ol, Et3N, MeOH, Δ; (k) Fe dust, EtOH, NH4Cl, H2O, Δ, 77%, two steps; (l) PTSA,
dioxane, Δ, 33%.
1
manner analogous to that for 13 (42% yield). H NMR (400 MHz,
CDCl3) δ 8.47 (s, 1H), 8.18 (s, 1H), 7.74 (s, 1H), 6.71 (s, 1H), 5.25
(br s, 1H), 3.88 (s, 3H), 3.80 (br d, J = 18.0 Hz, 4H), 3.59 (br s, 2H),
3.31 (br s, 2H), 3.12 (d, J = 4.7 Hz, 3H), 2.28 (s, 3H). LCMS, m/z =
426 [M + H]+.
( 2 - F l u o r o - 5 - m e t h o x y - 4 - ( 4 - ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-ylamino)phenyl)(morpholino)-
methanone (17, Method e). The title compound was prepared in a
1
manner analogous to that for 9 (40% yield). H NMR (400 MHz,
DMSO-d6) δ 8.33 (d, J = 12.0 Hz, 1H), 8.25 (s, 1H), 8.11 (s, 1H),
7.35 (q, J = 4.3 Hz, 1H), 7.03 (d, J = 6.2 Hz, 1H), 3.89 (s, 3H), 3.64−
3.55 (m, 6H), 3.32−3.31 (m, 2H), 2.94 (d, J = 4.3 Hz, 3H). LCMS,
m/z = 430 [M + H]+.
( 5 - F l u o r o - 2 - m e t h o x y - 4 - ( 4 - ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-ylamino)phenyl)(morpholino)-
methanone (15, Method d). The title compound was prepared in a
1
manner analogous to that for 13 (20% yield). H NMR (400 MHz,
(4-(4-(Ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-
2-fluoro-5-methoxyphenyl)(morpholino)methanone (18). A
mixture of 2-chloro-N-ethyl-5-(trifluoromethyl)pyrimidin-4-amine
(28, 70 g, 310 mmol), 4-amino-2-fluoro-5-methoxybenzoic acid (43,
49 g, 265 mmol), and PTSA (30 g, 169 mmol) in 1,4-dioxane (400
mL) was stirred at 100 °C for 2 h (the reaction mixture remained as a
suspension despite heating). The reaction mixture was then cooled to
room temperature. The insoluble solids were collected by filtration,
washed with 1,4-dioxane (100 mL) and Et2O (50 mL), and air-dried
for 12 h to give crude 4-(4-(ethylamino)-5-(trifluoromethyl)-
pyrimidin-2-ylamino)-2-fluoro-5-methoxybenzoic acid 62. The materi-
al (108 g) was used in the next step without further purification.
To a cooled (0 °C) mixture of crude 4-(4-(ethylamino)-5-
(trifluoromethyl)pyrimidin-2-ylamino)-2-fluoro-5-methoxybenzoic
acid (62, 108 g, 289 mmol) in CH2Cl2 (500 mL) were added HBTU
(125 g, 330 mmol), morpholine (50 mL, 572 mmol), and DIPEA (150
mL, 863 mmol). The reaction mixture was stirred at 0 °C for 15 min
before warming to room temperature. The reaction mixture was stirred
at room temperature for an additional 45 min. The mixture was then
diluted with saturated sodium bicarbonate solution, and the aqueous
CDCl3) δ 8.35 (d, J = 6.7 Hz, 1H), 8.22 (s, 1H), 7.39 (br s, 1H), 7.07
(d, J = 10.6 Hz, 1H), 5.30 (t, J = 4.7 Hz, 1H), 3.86 (s, 3H), 3.85−3.73
(m, 4H), 3.63 (br d, J = 11.9 Hz, 2H), 3.32 (br d, J = 20.3 Hz, 2H),
3.12 (d, J = 4.7 Hz, 3H). LCMS, m/z = 430 [M + H]+.
( 2 - F l u o r o - 3 - m e t h o x y - 4 - ( 4 - ( m e t h y l a m i n o ) - 5 -
(trifluoromethyl)pyrimidin-2-ylamino)phenyl)(morpholino)-
methanone (16, Method g). To a solution of (4-amino-2-fluoro-3-
methoxyphenyl)(morpholino)methanone (58, 600 mg, 2.36 mmol) in
n-BuOH (8 mL) was added 2-chloro-N-methyl-5-(trifluoromethyl)-
pyrimidin-4-amine (60, 498 mg, 2.36 mmol). The reaction mixture
was stirred at 100 °C under microwave irradiation for 1 h. After the
mixture was cooled and concentrated, the residue was purified by flash
chromatography, 10−33% EtOAc/petroleum ether, to afford a white
solid, which was triturated with 10% EtOAc/petroleum ether to afford
the title compound as a white solid (700 mg, 69% yield, three steps).
1H NMR (500 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.21 (s, 1H), 8.11 (d,
J = 8.5 Hz, 1H), 7.28 (d, J = 4.5 Hz, 1H), 7.10−7.07 (m, 1H), 3.90 (d,
J = 1.5 Hz, 3H), 3.64 (s, 4H), 3.55 (s, 2H), 3.29 (s, 2H), 2.91 (d, J =
4.0 Hz, 3H). LCMS, m/z = 430 [M + H]+.
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layer was extracted with CH2Cl2 (2 × 100 mL). The combined extracts
were washed with saturated sodium bicarbonate solution, dried over
Na2SO4, filtered, and concentrated.
CH2Cl2, and filtered through Celite, eluting with CH2Cl2, and the
filtrate was concentrated. The residue was adsorbed onto silica and
purified by flash chromatography, 0−10% MeOH/CH2Cl2. The
product-containing fractions were combined, concentrated and the
residue was further purified by preparative HPLC to give the title
compound (26.5 mg, 35% yield) as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ 8.99 (s, 1H), 8.37 (s, 1H), 8.17 (s, 1H), 7.31 (s, 1H),
7.15 (t, J = 5.5 Hz, 1H), 3.93 (s, 3H), 3.70−3.49 (m, 8H), 3.45−3.35
(m, 2H), 1.10 (t, J = 7.1 Hz, 3H). LCMS, m/z = 427 [M + H]+.
N2-(4-Methoxy-6-(tetrahydro-2H-pyran-4-yl)pyridin-3-yl)-
N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine (23). To
a round-bottom flask was added 2-(3,6-dihydro-2H-pyran-4-yl)-4-
methoxy-5-nitropyridine (74, 197 mg, 0.8286 mmol). The flask was
purged with N2, and EtOH (10 mL) followed by Pd/C (20 wt %, 88
mg, 0.08 mmol) was added. H2 was bubbled through the solution for 5
min, and the reaction mixture was then stirred overnight at room
temperature under an atmosphere of H2. The reaction mixture was
filtered through Celite, eluting with CH2Cl2, and the filtrate was
concentrated to give crude 4-methoxy-6-(tetrahydro-2H-pyran-4-
yl)pyridin-3-amine (75, 170 mg) that was used without further
purification.
The crude product was first purified by SFC to give approximately
95 g of desired product. The material was then redissolved in 350 mL
of refluxing i-PrOH. The solution was allowed to cool slowly
(overnight) to room temperature with moderate stirring. The resulting
suspension was cooled in an ice bath for 20 min. The solid (83.5 g)
was collected by filtration. The isolated solid was redissolved in 400
mL of refluxing i-PrOH and allowed to cool to room temperature
overnight with stirring. Again, the resulting suspension was cooled in
an ice bath for 20 min. The white precipitate was collected by filtration
to give a white crystalline solid. The solid was air-dried for 5 h and
placed under vacuum for 3 h to give the desired product (74.5 g, 64%
1
yield, two steps). H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 12 Hz,
1H), 8.20 (s, 1H), 7.84 (s, 1H), 6.91 (d, J = 5.9 Hz, 1H), 5.21 (br s,
1H), 3.74−3.85 (m, 4H), 3.69−3.71 (m, 2H), 3.58−3.68 (m, 2H),
3.44 (br s, 2H), 1.33 (t, J = 7.2 Hz, 3H). LCMS, m/z = 444 [M + H]+.
(4-(4-(Cyclopropylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)-2-fluoro-5-methoxyphenyl)(morpholino)methanone
(19). The title compound was prepared in a manner analogous to that
for 18 (16% yield, two steps). 1H NMR (400 MHz, CDCl3) δ 8.77 (d,
J = 12.6 Hz, 1H), 8.20 (s, 1H), 7.94 (s, 1H), 6.91 (d, J = 6.0 Hz, 1H),
5.48 (s, 1H), 3.92 (s, 3H), 3.84−3.76 (m, 4H), 3.68 (t, J = 4.5 Hz,
2H), 3.44 (br s, 2H), 2.94−2.87 (m, 1H), 1.03−0.96 (m, 2H), 0.70−
0.65 (m, 2H). LCMS, m/z = 456 [M + H]+.
To a vial were added 2-chloro-N-methyl-5-(trifluoromethyl)-
pyrimidin-4-amine (60, 93 mg, 0.44 mmol), crude 4-methoxy-6-
(tetrahydro-2H-pyran-4-yl)pyridin-3-amine (75, 87 mg, 0.42 mmol),
BrettPhos (23 mg, 0.042 mmol), BrettPhos palladium(II) phenethyl-
amine chloride (33 mg, 0.042 mmol), and NaO-t-Bu (50 mg, 0.50
mmol). The vial was purged with N2, and t-BuOH (3.0 mL) was
added. N2 was bubbled through the reaction mixture for 3 min, and
the mixture was then heated to 110 °C in an oil bath for 3 h. The
reaction mixture was cooled to room temperature, diluted with
CH2Cl2, filtered through Celite, eluting with CH2Cl2, and the filtrate
was concentrated. The residue was adsorbed onto silica and purified by
flash chromatography, 0−100% EtOAc/heptane. The product-
containing fractions were combined, concentrated and the residue
was further purified by preparative HPLC to give the title compound
N2-(2-Chloro-4-(1-methyl-1H-tetrazol-5-yl)phenyl)-N4-meth-
yl-5-(trifluoromethyl)pyrimidine-2,4-diamine (20). A mixture of
N2 -(2-chloro-4-(2H-tetrazol-5-yl)phenyl)-N4 -methyl-5-
(trifluoromethyl)pyrimidine-2,4-diamine (66, 80 mg, 0.22 mmol),
K2CO3 (45 mg, 0.32 mmol), and methyl iodide (15 μL, 0.24 mmol) in
acetone (2 mL) was heated at 40 °C for 1 h. The mixture was cooled
and filtered. The filtrate was concentrated under reduced pressure.
Purification of the residue by flash chromatography, 0−100% EtOAc/
isohexane, gave the title compound (10 mg, 12% yield). 1H NMR (400
MHz, CDCl3) δ 8.90 (d, J = 8.7 Hz, 1H), 8.24 (s, 1H), 7.86 (d, J = 2.1
Hz, 1H), 7.79 (s, 1H), 7.68 (dd, J = 8.8, 2.1 Hz, 1H), 5.33 (s, 1H),
4.22 (s, 3H), 3.13 (d, J = 4.7 Hz, 3H). LCMS, m/z = 385 [M + H]+.
The isomeric N-Me product N2-(2-chloro-4-(2-methyl-2H-tetrazol-5-
yl)phenyl)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine (58
mg, 70% yield) was also isolated from the reaction mixture. 1H
NMR (400 MHz, CDCl3) δ 8.76 (d, J = 8.7 Hz, 1H), 8.23−8.19 (m,
2H), 8.05 (dd, J = 8.7, 2.0 Hz, 1H), 7.72 (s, 1H), 5.29 (s, 1H), 4.40 (s,
3H), 3.12 (d, J = 4.7 Hz, 3H). LCMS, m/z = 385 [M + H]+.
N2-(5-Fluoro-2-methoxy-4-(methylsulfonyl)phenyl)-N4-
methyl-5-(trifluoromethyl)pyrimidine-2,4-diamine (21). To 5-
fluoro-2-methoxy-4-(methylsulfonyl)aniline (70, 115.5 mg, 0.53
mmol) and 2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine
(60, 117 mg, 0.55 mmol) dissolved in 2-methoxyethanol (5.0 mL, 63
mmol) was added TFA (0.04 mL, 0.53 mmol), and the reaction
mixture was heated to 100 °C for 60 h. The mixture was diluted with
water and saturated sodium bicarbonate solution, extracted with
EtOAc (10 mL), and concentrated under reduced pressure. The
residue was purified by flash chromatography, 0−50% EtOAc/heptane,
followed by preparative HPLC to afford the title compound (38.9 mg,
1
(83 mg, 52% yield, two steps) as a white solid. H NMR (400 MHz,
DMSO-d6) δ 8.77 (s, 1H), 8.26 (s, 1H), 8.10 (s, 1H), 7.11−7.04 (m,
1H), 6.98 (s, 1H), 3.99−3.92 (m, 3H), 3.89−3.86 (m, 4H), 3.48−3.39
(m, 3H), 2.93−2.84 (m, 5H), 1.82−1.73 (m, 5H). LCMS, m/z = 384
[M + H]+.
4-(4-(Ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-
2-fluoro-N-(2-hydroxy-2-methylpropyl)-5-methoxybenzamide
(24). The title compound was prepared in a manner analogous to that
for 18 (95% yield, two steps). 1H NMR (400 MHz, CDCl3) δ 8.51 (d,
J = 15.6 Hz, 1H), 8.21 (s, 1H), 7.93 (s, 1H), 7.58 (d, J = 7.2 Hz, 1H),
7.26−7.15 (m, 1H), 5.23 (br s, 1H), 3.96 (s, 3H), 3.65−3.61 (m, 2H),
3.52−3.50 (m, 2H), 1.34 (t, J = 7.2 Hz, 3H), 1.21 (s, 6H). LCMS, m/z
= 446 [M + H]+.
5-(4-(Ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-
2-(2-hydroxy-2-methylpropyl)-6-methoxyisoindolin-1-one
(25). The synthesis of 25 was carried out using the same procedure as
1
that for 13, method d (38 mg, 33%). H NMR (400 MHz, CDCl3) δ
8.69 (s, 1H), 8.21 (s, 1H), 7.95 (s, 1H), 7.33 (s, 1H), 5.18 (br s, 1H),
4.53 (s, 2H), 3.98 (s, 3H), 3.63−3.56 (m, 4H), 3.39 (s, 1H), 1.34−
1.32 (m, 3H), 1.30 (s, 6H). LCMS, m/z = 439 [M + H]+.
1
2-Chloro-N-ethyl-5-(trifluoromethyl)pyrimidin-4-amine (28).
Into a 3000 mL four-necked round-bottom flask purged and
maintained with an inert atmosphere of N2 was placed copper(I)
iodide (224 g, 1.17 mmol) and potassium fluoride (68 g, 1.17 mmol).
The mixture was heated to 150 °C under reduced pressure for 2 h.
After the mixture was cooled to room temperature, a solution of
trimethyl(trifluoromethyl)silane (167 g, 1.17 mmol) in DMF (500
mL) and NMP (500 mL) was added by syringe. The mixture was
stirred for 30 min, followed by the addition of a solution of 2-chloro-5-
iodo-4-methoxypyrimidine (26, 278 g, 980.6 mmol) in DMF (500
mL) and NMP (500 mL) by syringe. The resulting solution was stirred
at 35 °C for 48 h and quenched by the addition of 10 L of water. The
solids were filtered out. The filtrate was extracted with EtOAc (3 ×
5000 mL). The combined organic layers were washed with brine (3 ×
2000 mL), dried over Na2SO4, and concentrated. The residue was
19% yield). H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 13.1 Hz,
1H), 8.3 (s, 1H), 8.30 (s, 1H), 7.50−7.40 (m, 1H), 7.30 (d, J = 6.4 Hz,
1H), 3.96 (s, 3H), 3.29 (s, 3H), 2.96 (d, J = 4.2 Hz, 3H). LCMS, m/z
= 395 [M + H]+.
(5-((4-(Ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)-
amino)-4-methoxypyridin-2-yl)(morpholino)methanone (22).
To a vial were added 2-chloro-N-ethyl-5-(trifluoromethyl)pyrimidin-
4-amine (28, 41.8 mg, 0.185 mmol), (5-amino-4-methoxypyridin-2-
yl)(morpholino)methanone (72, 41.9 mg, 0.177 mmol), BrettPhos
(9.7 mg, 0.018 mmol), BrettPhos palladium(II) phenethylamine
chloride (14.1 mg, 0.018 mmol), and NaO-t-Bu (21.0 mg, 0.212
mmol). The vial was purged with N2, and t-BuOH (2.0 mL) was
added. N2 was bubbled through the reaction mixture for 3 min. Then
the vial was capped and heated to 100 °C in an oil bath for 12 h. The
reaction mixture was cooled to room temperature, diluted with
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purified by flash chromatography, 0−5% EtOAc/petroleum ether, to
amount of water and dried at 50 °C under vacuum to afford the title
1
1
afford the title compound (153 g, 69% yield) as a white solid. H
compound (190 mg, 68% yield). H NMR (400 MHz, DMSO-d6) δ
NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 5.40 (s, 1H), 3.66−3.57 (m,
2H), 1.32−1.27 (t, J = 7.2 Hz, 3H). LCMS, m/z = 226 [M + H]+.
2-Chloro-N-cyclopropyl-5-(trifluoromethyl)pyrimidin-4-
amine (29). To 2,4-dichloro-5-(trifluoromethyl)pyrimidine (27, 1.25
g, 5.8 mmol) in MeOH (4 mL) at 0 °C was added cyclopropylamine
(0.69 mL, 10 mmol). The mixture was allowed to stir at room
temperature for 4 h, and then CH2Cl2 (20 mL) and water (20 mL)
were added. The mixture was passed through a hydrophobic frit, and
the solvent was removed. Purification of the residue by flash
chromatography, 0−100% CH2Cl2/isohexane, gave the title com-
pound (472 mg, 34% yield) as an off-white solid. 1H NMR (400 MHz,
CDCl3) δ 8.27 (d, J = 1.1 Hz, 1H), 5.56 (br s, 1H), 3.03−2.93 (m,
1H), 0.99−0.89 (m, 2H), 0.65−0.60 (m, 2H). LCMS, m/z = 238 [M +
H]+.
11.8 (br s, 1H), 7.40 (d, J = 12.0 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H),
6.00 (br s, 2H), 3.75 (s, 3H). LCMS, m/z = 186 [M + H]+.
(4-Amino-5-chloro-2-methoxyphenyl)(morpholino)-
methanone (44). The title compound was prepared in a manner
analogous to that for 46 (carried forward as crude yellow paste).
LCMS, m/z = 271 [M + H]+.
(4-Amino-5-fluoro-2-methoxyphenyl)(morpholino)-
methanone (45). The title compound was prepared in a manner
1
analogous to that for 46 (72% yield). H NMR (400 MHz, CDCl3) δ
8.02 (s, 2H), 6.95 (d, J = 10.5 Hz, 1H), 6.30 (d, J = 7.1 Hz, 1H), 3.76
(s, 3H), 3.72−3.64 (m, 4H), 3.58 (t, J = 4.9 Hz, 2H), 3.40 (t, J = 4.9
Hz, 2H). LCMS, m/z = 255 [M + H]+.
(4-Amino-2-fluoro-5-methoxyphenyl)(morpholino)-
methanone (46). A mixture of 4-amino-2-fluoro-5-methoxybenzoic
acid (43, 4.2 g, 22.7 mmol), HATU (10.4 g, 27.2 mmol), and
morpholine (5 mL, 56.8 mmol) in CH2Cl2 was stirred in a cold water
bath, and DIPEA (10 mL, 56.8 mmol) was added. After 10 min the
mixture was allowed to warm to room temperature and stirred for 1 h.
The mixture was diluted with CH2Cl2 (100 mL) and washed with
saturated sodium bicarbonate solution (3 × 75 mL) and water (100
mL). The separated organic phase was passed through a hydrophobic
frit, and the solvent was concentrated. The crude product was
triturated with CH2Cl2/Et2O and the filtered solid dried under vacuum
to give the title compound (4.10 g, 71% yield) as an off-white solid. 1H
NMR (400 MHz, CDCl3) δ 6.80 (d, J = 6.0 Hz, 1H), 6.36 (d, J = 10.7
Hz, 1H), 4.12 (s, 2H), 3.84 (s, 3H), 3.83−3.61 (m, 6H), 3.42 (s, 2H).
LCMS, m/z = 255 [M + H]+.
(4-Amino-2-chloro-5-methoxyphenyl)(morpholino)-
methanone (50). The title compound was prepared in a manner
analogous to that for 46 (63% yield). 1H NMR (400 MHz, DMSO-d6)
δ 8.14 (s, 1H), 7.47 (s, 1H), 3.95 (s, 3H), 3.51−3.72 (m, 6H), 3.15−
3.19 (m, 2H). LCMS, m/z = 301 [M + H]+.
(5-Chloro-2-methyl-4-nitrophenyl)(morpholino)methanone
(51). The title compound was prepared in a manner analogous to that
for 46 (95% yield). 1H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 7.36
(s, 1H), 3.83−3.76 (m, 4H), 3.66−3.58 (m, 2H), 3.28−3.20 (m, 2H),
2.37 (s, 3H). LCMS, m/z = 285 [M + H]+.
5-Chloro-2-methyl-4-nitrobenzonitrile (34). To 5-chloro-2-
methyl-4-nitroaniline (33, 5 g, 26.8 mmol) in a mixture of acetone
(17.5 mL) and water (19 mL) at 0 °C was added concentrated HCl
(5.6 mL). A solution of sodium nitrite (2.25 g, 32.6 mmol) in water
(7.5 mL) was added dropwise, and the mixture was allowed to stir at 0
°C for 30 min. The mixture was then added dropwise to a mixture of
copper cyanide (3.75 g, 42 mmol) and sodium cyanide (5.5 g, 112
mmol) in water (25 mL) and EtOAc (12.5 mL). The mixture was
allowed to stir at room temperature for 1 h, and then water (50 mL)
was added. The mixture was extracted with EtOAc (3 × 100 mL), and
the combined organic fractions were washed with 2 M NaOH(aq) (50
mL) and brine (50 mL). The organic fraction was passed through a
hydrophobic frit, and the solvent was removed in vacuo. The residue
was suspended in a 1:1 mixture of diethyl ether/isohexane. The solid
formed was removed by filtration and washed with isohexane and
dried. Further fractions were isolated from the filtrate and combined
with the initial solid fraction to give the title compound (3.22 g, 61%
1
yield) as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.80 (s,
1H), 7.80 (s, 1H), 2.63 (s, 3H). LCMS, m/z = 197 [M + H]+.
5-Methoxy-2-methyl-4-nitrobenzonitrile (36). The title com-
pound was prepared in a manner analogous to that for 34 (90% yield).
1H NMR (400 MHz, CDCl3) δ 7.72 (s, 1H), 7.31 (s, 1H), 3.98 (s,
3H), 2.55 (s, 3H). LCMS, m/z = 192 [M + H]+.
(5-Methoxy-2-methyl-4-nitrophenyl)(morpholino)-
5-Chloro-2-methyl-4-nitrobenzoic Acid (37). A mixture of 5-
chloro-2-methyl-4-nitrobenzonitrile (34, 1 g, 5 mmol) in AcOH (10
mL), water (10 mL), and concentrated H2SO4 (10 mL) was heated to
120 °C for 5 h. Water (100 mL) was added and the solid was removed
by filtration and dried to give the title compound (905 mg, 84% yield)
as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 7.76
(s, 1H), 2.70 (s, 3H). LCMS, m/z = 214 [M + H]+.
5-Methoxy-2-methyl-4-nitrobenzoic Acid (38). The title
compound was prepared in a manner analogous to that for 37 (58%
yield). 1H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.70 (s, 1H), 3.99
(s, 3H), 2.62 (s, 3H). LCMS, m/z = 210 [M + H]+.
methanone (52). The title compound was prepared in a manner
1
analogous to that for 46 (59% yield). H NMR (400 MHz, CDCl3) δ
7.72 (s, 1H), 6.90 (s, 1H), 3.94 (s, 3H), 3.85−3.76 (m, 4H), 3.61 (dd,
J = 5.7, 4.0 Hz, 2H), 3.23 (dd, J = 5.7, 4.0 Hz, 2H), 2.30 (s, 3H).
LCMS, m/z = 281 [M + H]+.
(2-Fluoro-3-methoxy-4-nitrophenyl)(morpholino)-
methanone (53). The title compound was prepared in a manner
analogous to that for 46 (carried forward as crude). LCMS, m/z = 285
[M + H]+.
(2,5-Dimethoxy-4-nitrophenyl)(morpholino)methanone
(54). The title compound was prepared in a manner analogous to that
for 46 (carried forward as crude). LCMS, m/z = 297 [M + H]+.
(4-Amino-2-chloro-5-methoxyphenyl)(morpholino)-
methanone (55). To a solution of (2-chloro-5-methoxy-4-
nitrophenyl)(morpholino)methanone (50, 825 mg, 2.74 mmol) in
EtOH (10 mL) were added iron dust (0.90 g, 16 mmol), NH4Cl (0.90
g, 17 mmol), and water (0.5 mL). The suspension was stirred at 90 °C
under a N2 atmosphere for 1 h. The reaction mixture was allowed to
cool to room temperature and filtered through a pad of Celite. The
filtrate was concentrated. The resulting solid residue was resuspended
in EtOAc, sonicated for 1 min, and filtered. The filtrate was
concentrated to give the crude title compound (743 mg), which was
used without further purification. LCMS, m/z = 271 [M + H]+.
(4-Amino-5-chloro-2-methylphenyl)(morpholino)-
methanone (56). To (5-chloro-2-methyl-4-nitrophenyl)-
(morpholino)methanone (51, 244 mg, 0.86 mmol) in EtOH (6
mL) and water (0.6 mL) was added SnCl2·2H2O (776 mg, 3.44
mmol). The mixture was heated to 65 °C for 5 h, and then 2 M
aqueous sodium hydroxide solution (10 mL) and CH2Cl2 (10 mL)
were added. The organic phase was passed through a hydrophobic frit
and the solvent was removed in vacuo to give the title compound (203
4-Amino-5-fluoro-2-methoxybenzonitrile (40). To a stirred
solution of MeOH (26 mL) in THF (15 mL) under N2 was added t-
BuOK (1.5 g, 13.0 mmol) in small portions over 10 min. The resulting
solution was stirred at room temperature for 5 min, and then 4-amino-
2,5-difluorobenzonitrile (39, 1.0 g, 6.5 mmol) was added. The mixture
was then warmed to 70 °C and stirred for 3.5 h. The mixture was
cooled and diluted with EtOAc (25 mL), washed with water and brine,
dried over MgSO4, and concentrated to dryness under reduced
pressure. The residue was triturated with diethyl ether/isohexane to
1
afford the title compound (0.86 g, 80% yield). H NMR (400 MHz,
CDCl3) δ 7.15 (d, J = 10.4 Hz, 1H), 6.26 (d, J = 7.2 Hz, 1H), 4.25 (br
s, 2H), 3.84 (s, 3H). LCMS, m/z = 167 [M + H]+.
4-Amino-5-fluoro-2-methoxybenzoic Acid (41). A solution of
4-amino-5-fluoro-2-methoxybenzonitrile (40, 250 mg, 1.51 mmol) and
KOH in a mixture of EtOH (5 mL) and water (5 mL) was heated at
reflux for 30 h. The mixture was cooled and treated with 30% aqueous
H2O2 (0.25 mL). The mixture was stirred for 10 min and then heated
to reflux for 18 h. After return to room temperature, water (3 mL) was
added and 6 N HCl solution was added until pH 4 was obtained. The
mixture was filtered and the collected solid was washed with a small
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Journal of Medicinal Chemistry
Featured Article
1
mg, 93% yield) as an off-white solid. H NMR (400 MHz, CDCl3) δ
at room temperature. The solvent was removed under reduced
7.07 (s, 1H), 6.60 (s, 1H), 4.10 (br s, 2H), 3.84−3.53 (br m, 6H),
3.36−3.24 (br m, 2H), 2.21 (s, 3H). LCMS, m/z = 255 [M + H]+.
(4-Amino-5-methoxy-2-methylphenyl)(morpholino)-
methanone (57). The title compound was prepared in a manner
analogous to that for 56 (92% yield). 1H NMR (400 MHz, CDCl3) δ
6.60 (s, 1H), 6.52 (s, 1H), 3.87−3.52 (br m, 6H), 3.84 (br s, 2H), 3.82
(s, 3H), 3.36−3.23 (br m, 2H), 2.17 (s, 3H). LCMS, m/z = 251 [M +
H]+.
(4-Amino-2-fluoro-3-methoxyphenyl)(morpholino)-
methanone (58). To a mixture of (2-fluoro-3-methoxy-4-
nitrophenyl)(morpholino)methanone (53, 1.2 g, 4.2 mmol) in
MeOH (50 mL) was added Pd/C (10 wt %, 100 mg). The reaction
mixture was stirred under H2 (1 atm) at room temperature for 1 h.
The insoluble material was filtered off and the filtrate was concentrated
under reduced pressure to afford the crude title compound as a white
solid, which was used without further purification. LCMS, m/z = 255
[M + H]+.
pressure and the residue was purified by flash chromatography, 0−50%
1
EtOAc/heptane, to afford the title compound (0.41 g, 48% yield). H
NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 5.2
Hz, 1H), 4.03 (s, 3H), 3.28 (s, 3H). LCMS, m/z = not detected.
5-Fluoro-2-methoxy-4-(methylsulfonyl)aniline (70). To 1-
fluoro-4-methoxy-2-(methylsulfonyl)-5-nitrobenzene (69, 0.41 g, 1.6
mmol) in EtOH (80 mL) was added Pd/C (10 wt %, 18 mg, 0.16
mmol), and the reaction mixture was stirred under H2 (1 atm) for 12
h. The reaction mixture was filtered through Celite, and the solvents
were removed under reduced pressure. The residue was purified by
flash chromatography, 0−70% EtOAc/heptane, to afford the title
1
compound (0.36 g, 99% yield). H NMR (400 MHz, CDCl3) δ 7.19
(d, J = 6.2 Hz, 1H), 7.46 (d, J = 11.0 Hz, 1H), 4.44 (s, 3H), 3.88 (s,
3H), 3.16 (s, 3H). LCMS, m/z = 220 [M + H]+.
(5-Amino-4-methoxypyridin-2-yl)(morpholino)methanone
(72). To a microwave vial were added 6-chloro-4-methoxypyridin-3-
amine (71, 100 mg, 0.63 mmol), molybdenum hexacarbonyl (183 mg,
0.69 mmol), Pd(PPh3)4 (36.5 mg, 0.03 mmol), and 4-DMAP (46.7
mg, 0.38 mmol). 1,4-Dioxane (10 mL) was then added followed by
DIPEA (0.22 mL, 1.26 mmol) and morpholine (0.66 mL, 7.57 mmol).
N2 was then bubbled through reaction mixture for 3 min. Then the vial
was capped and heated to 150 °C in a microwave for 40 min. The
reaction mixture was then filtered through Celite, eluting with CH2Cl2,
and the filtrate was concentrated. The crude residue was adsorbed
onto silica and purified by flash chromatography, 0−10% MeOH/
CH2Cl2, to give the title compound (75 mg, 50% yield) as a beige
(4-Amino-2,5-dimethoxyphenyl)(morpholino)methanone
(59). The title compound was prepared in a manner analogous to that
for 58 (carried forward as crude). LCMS, m/z = 267 [M + H]+.
3-Chloro-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-
2-ylamino)benzonitrile (65). A mixture of 2-chloro-N-methyl-5-
(trifluoromethyl)pyrimidin-4-amine (60, 211 mg, 1 mmol), 4-amino-
3-chlorobenzonitrile (64, 305 mg, 2 mmol), cesium carbonate (0.65 g,
2 mmol), Xantphos (17 mg, 0.03 mmol), and Pd2(dba)3 (5 mg, 0.02
mmol) in dioxane (3 mL) was sonicated in an ultrasonic bath for 1
min. The mixture was then degassed under a stream of N2 for 5 min.
The tube was sealed, and the mixture was heated at 100 °C for 18 h.
The reaction mixture was cooled and diluted with EtOAc (15 mL).
The organic layer was washed with water (2 × 20 mL), and the
combined aqueous extracts were further washed with EtOAc (2 × 15
mL). The combined organics were passed through a phase separation
cartridge, and the solvent was removed under reduced pressure.
Purification of the residue by flash chromatography, 0−100% EtOAc/
isohexane, gave the title compound as a white solid (140 mg, 45%
1
foam. H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 5.40 (s, 1H),
3.66−3.57 (m, 2H), 1.32−1.27 (t, J = 7.2 Hz, 3H). LCMS, m/z = 238
[M + H]+.
2-(3,6-Dihydro-2H-pyran-4-yl)-4-methoxy-5-nitropyridine
(74). To a microwave vial were added 2-chloro-4-methoxy-5-
nitropyridine (73, 200 mg, 1.06 mmol), 2-(3,6-dihydro-2H-pyran-4-
yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (312 mg, 1.48 mmol),
Na2 CO3 (158 mg 1.48 mmol), bis(di-tert-butyl(4-
dimethylaminophenyl)phosphine)dichloropalladium(II) (75 mg, 0.11
mmol), and KOAc (147 mg, 1.48 mmol). CH3CN (10 mL) and
degassed water (2 mL) were added, and N2 was bubbled through the
solution for 3 min. The vial was capped and submitted to microwave
heating at 140 °C for 40 min. The reaction mixture was diluted with
CH2Cl2, filtered through Celite, eluting with CH2Cl2, and the filtrate
was concentrated. The crude residue was adsorbed onto silica and
purified by flash chromatography, 0−100% EtOAc/heptane, to give
the title compound (197 mg, 79% yield). 1H NMR (300 MHz,
CDCl3) δ 8.25 (s, 1H), 5.40 (s, 1H), 3.66−3.57 (m, 2H), 1.32−1.27
(t, J = 7.2 Hz, 3H). LCMS, m/z = 237 [M + H]+.
Methyl 5-Methoxy-2-methyl-4-nitrobenzoate (76). To 5-
methoxy-2-methyl-4-nitrobenzoic acid (38, 1.27 g, 6 mmol) in
MeOH (15 mL) was added SOCl2 (0.44 mL, 6 mmol) dropwise.
The mixture was heated to reflux for 3 h and then allowed to cool. The
solvent was removed, and the residue was partitioned between CH2Cl2
(20 mL) and saturated sodium bicarbonate solution (20 mL). The
organic phase was passed through a hydrophobic frit, and the solvent
was removed. Purification of the residue by flash chromatography, 0−
100% EtOAc/isohexane, gave the title compound (1.11 g, 82% yield)
as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H), 7.60
(s, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 2.55 (s, 3H). LCMS, m/z = 226
[M + H]+.
1
yield). H NMR (400 MHz, CDCl3) δ 8.85 (d, J = 8.8 Hz, 1H); 8.23
(d, J = 1.1 Hz, 1H), 7.80 (s, 1H), 7.68 (d, J = 1.9 Hz, 1H), 7.58−7.53
(m, 1H); 5.32 (br s, 1H), 3.11 (d, J = 4.7 Hz, 3H). LCMS, m/z = 328
[M + H]+.
N2-(2-Chloro-4-(2H-tetrazol-5-yl)phenyl)-N4-methyl-5-
(trifluoromethyl)pyrimidine-2,4-diamine (66). To a solution of 3-
chloro-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-
benzonitrile (65, 135 mg, 6.41 mmol) in DMF (2 mL) were added
NaN3 (80 mg, 1.24 mmol) and NH4Cl (66 mg, 1.24 mmol). The
mixture was heated at 125 °C for 18 h. The mixture was cooled,
filtered and the solid washed with EtOAc. The filtrate was
concentrated under reduced pressure, and the crude residue was
diluted with water (5 mL) and Et2O (5 mL). A precipitate formed
which was filtered and washed with further portions of water and Et2O.
The solid was coevaporated with CH2Cl2 and MeOH and then dried
in a vacuum oven to yield the title compound (130 mg, 85% yield). 1H
NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.26−8.19 (m, 2H), 8.14
(d, J = 2.0 Hz, 1H), 8.03−7.96 (m, 1H), 7.30−7.22 (m, 1H), 2.89 (t, J
= 4.4 Hz, 3H). LCMS, m/z = 371 [M + H]+.
(2-Fluoro-5-methoxy-4-nitrophenyl)(methyl)sulfane (68). To
1,2-difluoro-4-methoxy-5-nitrobenzene (67, 0.72 g, 3.8 mmol) in
EtOH (100 mL) was added sodium thiomethoxide (0.30 g, 4.1 mmol)
in water (0.96 mL, 53 mmol), and the reaction mixture was heated to
reflux for 3 h. The solvent was removed under reduced pressure and
the residue was purified by flash chromatography, 0−40% EtOAc/
Methyl 2-(Bromomethyl)-5-methoxy-4-nitrobenzoate (77).
A mixture of methyl 5-methoxy-2-methyl-4-nitrobenzoate (76, 1.11 g,
5 mmol) and NBS (1.07 g, 6 mmol) in MeCN (20 mL) was degassed,
and then AIBN (18 mg, 0.11 mmol) was added. The mixture was
heated to 70 °C for 18 h. CH2Cl2 (50 mL) and water (50 mL) were
added, and the organic phase was passed through a hydrophobic frit.
The solvent was removed, and purification of the residue by flash
chromatography, 0−100% EtOAc/isohexane, gave the title compound
(477 mg, 31% yield) as an off-white solid. 1H NMR (400 MHz,
CDCl3) δ 7.92 (s, 1H), 7.66 (s, 1H), 4.88 (s, 2H), 4.02 (s, 3H), 4.00
(s, 3H). LCMS, m/z = 304 [M + H]+.
1
heptane, to afford the title compound (0.75 g, 91% yield). H NMR
(400 MHz, CDCl3) δ 7.68 (d, J = 9.3 Hz, 1H), 6.83 (d, J = 6.1 Hz,
1H), 3.98 (s, 3H), 2.55 (s, 3H). LCMS, m/z = not detected.
1-Fluoro-4-methoxy-2-(methylsulfonyl)-5-nitrobenzene
(69). (2-Fluoro-5-methoxy-4-nitrophenyl)(methyl)sulfane (68, 0.75 g,
3.5 mmol) was dissolved in CH2Cl2 (30 mL), and m-CPBA (77%
pure) (1.0 g, 4.5 mmol) was added portionwise. The mixture was
stirred for 16 h at room temperature. Additional m-CPBA (77% pure)
(1.5 g, 6.92 mmol) was added and the mixture stirred for another 24 h
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Journal of Medicinal Chemistry
Featured Article
2-(2-Hydroxy-2-methylpropyl)-6-methoxy-5-nitroisoindolin-
1-one (78). To methyl 2-(bromomethyl)-5-methoxy-4-nitrobenzoate
(77, 220 mg, 0.72 mmol) in MeOH (6 mL) were added Et3N (0.12
mL, 0.86 mmol) and 1-amino-2-methylpropan-2-ol (77 mg, 0.86
mmol). The mixture was heated to 70 °C for 5 h, and then EtOAc (30
mL) and 1 M HCl(aq) (30 mL) were added. The layers were separated,
and the aqueous layer was extracted with EtOAc (2 × 30 mL). The
combined organic fractions were passed through a hydrophobic frit,
and the solvent was removed. The residue was used directly in the next
reaction without any purification. LCMS, m/z = 281 [M + H]+.
5-Amino-2-(2-hydroxy-2-methylpropyl)-6-methoxyisoindo-
lin-1-one (79). To crude 2-(2-hydroxy-2-methylpropyl)-6-methoxy-5-
nitroisoindolin-1-one (78, 0.72 mmol) in a mixture of EtOH (15 mL)
and water (1.5 mL) were added iron powder (134 mg, 2.4 mmol) and
NH4Cl (160 mg, 3 mmol). The resulting mixture was heated at 80 °C
for 18 h, then cooled to room temperature, filtered through a pad of
Celite, and rinsed through with MeOH. The filtrate was evaporated,
and the residue was dissolved in CH2Cl2 (100 mL) and washed with
water (3 × 50 mL). The organic phase was dried over MgSO4, filtered,
and concentrated under reduced pressure to give the title compound
of Medicine for kindly providing the G2019S BAC transgenic
mice.
ABBREVIATIONS USED
■
LRRK2, leucine-rich repeat kinase 2; CNS, central nervous
system; PD, Parkinson’s disease; JAK2, Janus kinase 2; MMP,
matched molecular pair; MPO, multiparameter optimization;
LLE, lipophilic ligand efficiency; LELP, ligand-efficiency-
dependent lipophilicity; P-gp, P-glycoprotein; MDCK-MDR1,
Madin−Darby canine kidney cells-multidrug resistance protein
1; ER, efflux ratio; CSF, cerebrospinal fluid; PPB, plasma
protein binding; BPB, brain protein binding; AUC, area under
the curve; BAC, bacterial artificial chromosome; FVB, Friend
virus B-type; PEG, polyethylene glycol; NMP, N-methyl-2-
pyrrolidone; MCT, methylcellulose/Tween
REFERENCES
■
1
(138 mg, 77% yield, two steps). H NMR (400 MHz, CDCl3) δ 7.21
(1) Lees, A. J.; Hardy, J.; Revesz, T. Parkinson’s disease. Lancet 2009,
373, 2055−2066. Erratum in Lancet 2009, 374, 684.
(s, 1H), 6.68 (s, 1H), 4.40 (br s, 2H), 4.21 (s, 2H), 3.90 (s, 3H), 3.74
(s, 1H), 3.55 (s, 2H), 1.27 (s, 6H). LCMS, m/z = 251 [M + H]+.
Molecular Modeling. Homology models of LRRK2 were
constructed using the modeling program MOE, version 2009.10
(Chemical Computing Group, Montreal),62 and AMBER9963 force
field. The human LRRK2 sequence was 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
(2) Zimprich, A.; Biskup, S.; Leitner, P.; Lichtner, P.; Farrer, M.;
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,
T. M.; Wszolek, Z. K.; Gasser, T. Mutations in LRRK2 cause
autosomal-dominant parkinsonism with pleomorphic pathology.
Neuron 2004, 44, 601−607.
(3) Paisan-Ruiz, C.; Jain, S.; Evans, E. W.; Gilks, W. P.; Simon, J.; van
der Brug, M.; Lopez de Munain, A.; Aparicio, S.; Gil, A. M.; Khan, N.;
Johnson, J.; Martinez, J. R.; Nicholl, D.; Carrera, I. M.; Pena, A. S.; de
Silva, R.; Lees, A.; Marti-Masso, J. F.; Perez-Tur, J.; Wood, N. W.;
Singleton, A. B. Cloning of the gene containing mutations that cause
PARK8-linked Parkinson’s disease. Neuron 2004, 44, 595−600.
(4) Mata, I. F.; Wedemeyer, W. J.; Farrer, M. J.; Taylor, J. P.; Gallo,
K. A. LRRK2 in Parkinson’s disease: protein domains and functional
insights. Trends Neurosci. 2006, 29, 286−293.
(5) Greggio, E.; Cookson, M. R. Leucine-rich repeat kinase 2
mutations and Parkinson’s disease: three questions. ASN Neuro 2009,
1, e00002.
(6) Gandhi, P. N.; Chen, S. G.; Wilson-Delfosse, A. L. Leucine-rich
repeat kinase 2 (LRRK2): a key player in the pathogenesis of
Parkinson’s disease. J. Neurosci. Res. 2009, 87, 1283−1295.
(7) Dachsel, J. C.; Farrer, M. J. LRRK2 and Parkinson disease. Arch.
Neurol. 2010, 67, 542−547.
force field (Schrodinger, Inc., New York, NY).66 Inhibitor docking
̈
studies were carried out using docking program Glide SP with one
hydrogen bond constrained to the carbonyl oxygen of hinge residue
Ala1950. The docking poses were evaluated based on a combination of
criteria including the Glide docking score (cutoff of −6), favorable
intermolecular interactions with the hinge and other parts of the ATP-
binding pocket, and low strain energy of the bound ligand (Estrain < 2
kcal/mol).
ASSOCIATED CONTENT
■
S
* Supporting Information
Spleen PK/PD graphs for 14, 18, and 19 and additional
experimental procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
(8) 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.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 650-225-4934. Fax: 650-742-4943. E-mail: estrada.
anthony@gene.com.
Present Address
(9) Simon
́ ́
-Sanchez, J.; Schulte, C.; Bras, J. M.; Sharma, M.; Gibbs, J.
▽Novartis Institutes for Biomedical Research (NIBR), 4560
Horton Street, Emeryville, CA 94608-2916.
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.
(10) 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.
(11) Greggio, E.; Jain, S.; Kingsbury, A.; Bandopadhyay, R.; Lewis, P.;
Kaganovich, A.; van der Brug, M. P.; Beilina, A.; Blackinton, J.;
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Analytical, Bioanalytical, NMR, Pharmacokinetic,
Biopharmacology, and Safety Assessment colleagues for their
contributions. We thank Vivienne Allen, Linda Bao, Leo
Berezhkovskiy, Po-Chang Chiang, Kang-Jye Chou, Jason
Halladay, Quynh Ho, Claire Holt, Heather Kennedy, Emile
Plise, Veronica Rivera, Kimberley Smith, Hilda Solanoy, and
Herve
́
Van de Poel for their individual contributions. We also
̈
thank Dr. Zhenyu Yue and his group at the Mount Sinai School
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Featured Article
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.
(12) Cookson, M. R. The role of leucine-rich repeat kinase 2
(LRRK2) in Parkinson’s disease. Nat. Rev. Neurosci. 2010, 11, 791−
797.
(27) Hu, X.; Hu, Y.; Vogt, M.; Stumpfe, D.; Bajorath, J. MMP-Cliffs:
systematic identification of activity cliffs on the basis of matched
molecular pairs. J. Chem. Inf. Model. 2012, 52, 1138−1145.
(28) Muller, K. The power of MMPA and a teaching lesson in
medicinal chemistry. J. Med. Chem. 2012, 55, 1815−1816.
(29) Stumpfe, D.; Bajorath, J. Exploring activity cliffs in medicinal
chemistry. J. Med. Chem. 2012, 55, 2932−2942.
(13) Rudenko, I. N.; Chia, R.; Cookson, M. R. Is inhibition of kinase
activity the only therapeutic strategy for LRRK2-associated Parkinson’s
disease? BMC Med. 2012, 10, 20−27.
(30) Griffen, E.; Leach, A. G.; Robb, G. R.; Warner, D. J. Matched
molecular pairs as a medicinal chemistry tool. J. Med. Chem. 2011, 54,
7739−7750.
(14) 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
Parkinson’s disease models. Hum. Mol. Genet. 2011, 20, 3933−3942.
(15) Lee, B. D.; Shin, J.-H.; VanKampen, J.; Petrucelli, L.; West, A.
B.; Ko, H. S.; Lee, Y.-I.; Maguire-Zeiss, K. A.; Bowers, W. J.; Federoff,
H. J.; Dawson, V. L.; Dawson, T. M. Inhibitors of leucine-rich repeat
kinase-2 protect against models of Parkinson’s disease. Nat. Med. 2010,
16, 998−1000.
(31) Hu, Y.; Bajorath, J. Chemical transformations that yield
compounds with distinct activity profiles. ACS Med. Chem. Lett.
2011, 2, 523−527.
(32) Milletti, F.; Hermann, J. C. Targeted kinase selectivity from
kinase profiling data. ACS Med. Chem. Lett. 2012, 3, 383−386.
(33) Galkin, A. V.; Melnick, J. S.; Kim, S.; Hood, T. L.; Li, N.; Li, L.;
Xia, G.; Steensma, R.; Chopiuk, G.; Jiang, J.; Wan, Y.; Ding, P.; Liu, Y.;
Sun, F.; Schultz, P. G.; Gray, N. S.; Warmuth, M. Identification of
NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-
ALK. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 270−275;Erratum in
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2025.
(16) For a recent review on LRRK2 small molecule kinase inhibitors,
(34) Anderson, D. R.; Meyers, M. J.; Vernier, W. F.; Mahoney, M.
W.; Kurumbail, R. G.; Caspers, N.; Poda, G. I.; Schindler, J. F.; Reitz,
D. B.; Mourey, R. J. Pyrrolopyridine inhibitors of mitogen-activated
protein kinase activated protein kinase 2 (MK-2). J. Med. Chem. 2007,
50, 2647−2654.
(35) Baker-Glenn, C.; Burdick, D. J.; Chambers, M.; Chan, B. K.;
Chen, H.; Estrada, A.; Gunzner, J. L.; Shore, D.; Sweeney, Z. K.; Wang,
S.; Zhao, G. Aminopyrimidine derivatives as LRRK2 modulators and
their preparation and use for the treatment of Parkinson’s disease.
WO2011151360A1, 2011.
(36) Choi, H. G.; Zhang, J.; Deng, X.; Hatcher, J. M.; Patricelli, M. P.;
Zhao, Z.; Alessi, D. R.; Gray, N. S. Brain penetrant LRRK2 inhibitor.
ACS Med. Chem. Lett. 2012, 3, 658−662.
(37) Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. Moving
beyond the rules: the development of a central nervous system
multiparameter optimization (CNS MPO) approach to enable
alignment of druglike properties. ACS Chem. Neurosci. 2010, 1,
435−449.
see the following: Kramer, T.; Lo Monte, F.; Goring, S.; Amombo, G.
̈
M. O.; Schmidt, B. Small molecule kinase inhibitors for LRRK2 and
their application to Parkinson’s disease models. ACS Chem. Neurosci.
2012, 3, 151−160.
(17) Lee, B. D.; Dawson, V. L.; Dawson, T. M. Leucine-rich repeat
kinase 2 (LRRK2) as a potential therapeutic target in Parkinson’s
disease. Trends Pharmacol. Sci. 2012, 33, 365−373.
(18) Deng, X.; Dzamko, N.; Prescott, A.; Davies, P.; Liu, Q.; Yang,
Q.; Lee, J.-D.; Patricelli, M. P.; Nomanbhoy, T. K.; Alessi, D. R.; Gray,
N. S. Characterization of a selective inhibitor of the Parkinson’s disease
kinase LRRK2. Nat. Chem. Biol. 2011, 7, 203−205.
(19) Ramsden, N.; Perrin, J.; Ren., Z.; Lee, B. D.; Zinn, N.; Dawson,
V. L.; Tam, D.; Bova, M.; Lang, M.; Drewes, G.; Bantscheff, M.; Bard,
F.; Dawson, T. M.; Hopf, C. Chemoproteomics-based design of potent
LRRK2-selective lead compounds that attenuate Parkinson’s disease-
related toxicity in human neurons. ACS Chem. Biol. 2011, 6, 1021−
1028.
(20) Nichols, P. L.; Eatherton, A. J.; Bamborough, P.; Jandu, K. S.;
Philps, O. J.; Andreotti, D. Novel Compounds. WO 2011/038572 A1,
2011.
(21) Lewis, P. A.; Manzoni, C. LRRK2 and human disease: a
complicated question or a question of complexes? Sci. Signaling 2012,
5, pe2.
(22) Drolet, R. E.; Sanders, J. M.; Kern, J. T. Leucine-rich repeat
kinase 2 (LRRK2) cellular biology: a review of recent advances in
identifying physiological substrates and cellular functions. J. Neuro-
genet. 2011, 25, 140−151.
(23) Zhang, J.; Deng, X.; Choi, H. G.; Alessi, D. R.; Gray, N. S.
Characterization of TAE684 as a potent LRRK2 kinase inhibitor.
Bioorg. Med. Chem. Lett. 2012, 22, 1864−1869.
(24) Sheng, Z.; Zhang, S.; Bustos, D.; Kleinheinz, T.; Le Pichon, C.
E.; Dominguez, S. L.; Solanoy, H. O.; Drummond, J.; Zhang, X.; Ding,
X.; Cai, F.; Song, Q.; Li, X.; Yue, Z.; van der Brug, M. P.; Burdick, D.
J.; Gunzner-Toste, J.; Chen, H.; Liu, X.; Estrada, A. A.; Sweeney, Z. K.;
Scearce-Levie, K.; Moffat, J. G.; Kirkpatrick, D. S.; Zhu, H. Ser1292
autophosphorylation is a biomarker of LRRK2 kinase activity and
mediates cellular effects of PD mutations. Sci. Transl. Med. 2012, in
press.
(25) Chen, H.; Chan, B. K.; Drummond, J.; Estrada, A. A.; Gunzner-
Toste, J.; Liu, X.; Liu, Y.; Moffat, J. G.; Shore, D.; Sweeney, Z. K.;
Tran, T.; Wang, S.; Zhao, G.; Zhu, H.; Burdick, D. J. Discovery of
selective LRRK2 inhibitors guided by computational analysis and
molecular modeling. J. Med. Chem. 2012, 55, 5536−5545.
(26) Li, X.; Patel, J. C.; Wang, J.; Avshalumov, M. V.; Nicholson, C.;
Buxbaum, J. D.; Elder, G. A.; Rice, M. E.; Yue, Z. Enhanced striatal
dopamine transmission and motor performance with LRRK2 over-
expression in mice is eliminated by familial Parkinson’s disease
mutation G2019S. J. Neurosci. 2010, 30, 1788−1797.
(38) Leeson, P. D.; Springthorpe, B. The influence of drug-like
concepts on decision-making in medicinal chemistry. Nat. Rev. Drug
Discovery 2007, 6, 881−890.
(39) Keseru, G. M.; Makara, G. M. The influence of lead discovery
̋
strategies on the properties of drug candidates. Nat. Rev. Drug
Discovery 2009, 8, 203−212.
(40) For impact of lipophilic efficiency parameters on compound
quality, see the following: Tarcsay, A.; Nyíri, K.; Keseru, G. M. Impact
̋
of lipophilic efficiency on compound quality. J. Med. Chem. 2012, 55,
1252−1260. For impact on CNS drugs and candidates, see the
following: Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman,
M. D.; Verhoest, P. R.; Villalobos, A.; Will, Y. Defining desirable
central nervous system drug space through the alignment of molecular
properties, in vitro ADME, and safety attributes. ACS Chem. Neurosci.
2010, 1, 420−434.
(41) Liu, X.; Ding, X.; Deshmukh, G.; Liederer, B. M.; Hop, C. E. C.
A. Use of cassette dosing approach to assess brain penetration in drug
discovery. Drug Metab. Dispos. 2012, 40, 963−969.
(42) Hennig, L.; Ayala-Leon, K.; Angulo-Cornejo, J.; Richter, R.;
Beyer, L. Fluorine hydrogen short contacts and hydrogen bonds in
substituted benzamides. J. Fluorine Chem. 2009, 130, 453−460.
(43) Fritz, H.; Winkler, T. Weitreichende kopplung zwischen
protonen und fluor in 2-fluorbenzamiden. Helv. Chim. Acta 1974,
57, 836−839.
(44) Hitchcock, S. A. Structural modifications that alter the P-
glycoprotein efflux properties of a compound. J. Med. Chem. 2012, 55,
4877−4895.
(45) Lovering, F.; Bikker, J.; Humblet, C. Escape from Flatland:
increasing saturation as an approach to improving clinical success. J.
Med. Chem. 2009, 52, 6752−6756.
9432
dx.doi.org/10.1021/jm301020q | J. Med. Chem. 2012, 55, 9416−9433

Journal of Medicinal Chemistry
Featured Article
(46) Ishikawa, M.; Hashimoto, Y. Improvement in aqueous solubility
in small molecule drug discovery programs by disruption of molecular
planarity and symmetry. J. Med. Chem. 2011, 54, 1539−1554.
(47) Liu, X.; Smith, B. J.; Chen, C.; Callegari, E.; Becker, S. L.; Chen,
X.; Cianfronga, J.; Doran, A. C.; Doran, S. D.; Gibbs, J. P.; Hosea, N.;
Liu, J.; Nelson, F. R.; Szewc, M. A.; Van Deusen, J. Evaluation of
cerebrospinal fluid concentration and plasma free concentration as a
surrogate measurement for brain free concentration. Drug Metab.
Dispos. 2006, 34, 1443−1447.
(66) Maestro Suites, version 2009; Schrodinger, Inc.: New York, 2009.
̈
(48) Lin, J. H. CSF as a surrogate for assessing CNS exposure: an
industrial perspective. Curr. Drug Metab. 2008, 9, 46−59.
(49) Invitrogen Lanthascreen Eu kinase binding assay kit for TTK.
(50) Kwiatkowski, N.; Jelluma, N.; Filippakopoulos, P.;
Soundararajan, M.; Manak, M. S.; Kwon, M.; Choi, H. G.; Sim, T.;
Deveraux, Q. L.; Rottmann, S.; Pellman, D.; Shah, J. V.; Kops, G. J. P.;
Knapp, S.; Gray, N. S. Small-molecule kinase inhibitors provide insight
into Mps1 cell cycle function. Nat. Chem. Biol. 2010, 6, 359−368.
(51) Olaharski, A. J.; Gonzaludo, N.; Bitter, H.; Goldstein, D.;
Kirchner, S.; Uppal, H.; Kolaja, K. Identification of a kinase profile that
predicts chromosome damage induced by small molecule kinase
inhibitors. PLoS Comput. Biol. 2009, 5, e1000446.
(52) Lan, W.; Cleveland, D. W. A chemical tool box defines mitotic
and interphase roles for Mps1 kinase. J. Cell Biol. 2010, 190, 21−24.
(53) Wilberg, K. B. The deuterium isotope effect. Chem. Rev. 1955,
55, 713.
(54) Hitchcock, S. A.; Pennington, L. D. Structure−brain exposure
relationships. J. Med. Chem. 2006, 49, 7559−7583.
(55) Fabian, M. A.; Biggs, W. H.; Treiber, D. K.; Atteridge, C. E.;
Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P.
T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.;
́
Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J.-M.; Mehta,
S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.;
Zarrinkar, P. P.; Lockhart, D. J. A small molecule−kinase interaction
map for clinical kinase inhibitors. Nat. Biotechnol. 2005, 23, 329−336.
(56) Porter, R. H.; Benwell, K. R.; Lamb, H.; Malcolm, C. S.; Allen,
N. H.; Revell, D. F.; Adams, D. R.; Sheardown, M. J. Functional
characterization of agonists at recombinant human 5-HT2A, 5-HT2B
and 5-HT2C receptors in CHO-K1 cells. Br. J. Pharmacol. 1999, 128,
13−20.
(57) Zhou, S.-F.; Wang, B.; Yang, L.-P.; Liu, J.-P. Structure, function,
regulation and polymorphism and the clinical significance of human
cytochrome P450 1A2. Drug Metab. Rev. 2010, 42, 268−354.
(58) Zhou, S.-F.; Yang, L.-P.; Zhou, Z.-W.; Liu, Y.-H.; Chan, E.
Insights into the substrate specificity, inhibitors, regulation, and
polymorphisms and the clinical impact of human cytochrome P450
1A2. AAPS J. 2009, 11, 481−494.
(59) Cellular assay is triple mutant LRRK2 (R1441G/Y1699C/
G2019S), and BAC transgenic mice are G2019S mutant LRRK2.
(60) Akabane, T.; Tabata, K.; Kadono, K.; Sakuda, S.; Terashita, S.;
Teramura, T. A comparison of pharmacokinetics between humans and
monkeys. Drug Metab. Dispos. 2010, 38, 308−316.
(61) Roller, S.; Cui, D.; Laspina, C.; Miller-Stein, C.; Rowe, J.; Wong,
B; Prueksaritanont, T. Preclinical pharmacokinetics of MK-0974, an
orally active calcitonin-gene related peptide (CGRP)-receptor
antagonist, mechanism of dose dependency and species differences.
Xenobiotica 2009, 39, 33−45.
(62) MOE, version 2009.10; Chemical Computing Group Inc.:
Montreal, Quebec, Canada.
(63) Wang, J.; Cieplak, P.; Kollman, P. A. How well does a restrained
electrostatic potential (RESP) model perform in calculating conforma-
tional energies of organic and biological molecules? J. Comput. Chem.
2000, 21, 1049.
(64) Bairoch, A.; Boeckmann, B.; Ferro, S.; Gasteiger, E. Swiss-Prot:
juggling between evolution and stability. Briefings Bioinf. 2004, 5, 39−
55.
(65) Thompson, J. D.; Higgins, D. G.; Gibson, T. J. CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position specific gap penalties and weight
matrix choice. Nucleic Acids Res. 1994, 22, 4673−4680.
9433
dx.doi.org/10.1021/jm301020q | J. Med. Chem. 2012, 55, 9416−9433