Note: Descriptions are shown in the official language in which they were submitted.
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ASSAYS AND BIOMARKERS FOR LRRK2
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Serial No.
61/418,208 filed on 30 November 2010, which is incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] This invention pertains to assays for discovering compounds that
modulate
the function of LRRK2 and biomarkers for in vivo evaluation of LRRK2 activity.
BACKGROUND OF THE INVENTION
[0003] Neurodegenerative diseases such as Parkinson's disease, Lewy body
dementia
and Huntington's disease affect millions of individuals. Parkinson's disease
is a chronic,
progressive motor system disorder that afflicts approximately one out of every
1000 people,
with hereditary Parkinson's disease accounting for 5-10% of all of patients.
Parkinson's
disease is caused by progressive loss of mid-brain dopamine neurons, leaving
patients with
impaired ability to direct and control their movements. The primary
Parkinson's disease
symptoms are trembling, rigidity, slowness of movement, and impaired balance.
Many
Parkinson's disease patients also experience other symptoms such as emotional
changes,
memory loss, speech problems, and sleeping disorders.
[0004] The gene encoding the leucine-rich repeat kinase 2 protein (LRRK2)
has been
identified in association with hereditary Parkinson's disease (Paisan-Ruiz et
al., Neuron, Vol.
44(4), 2004, pp 595-600; Zimprich et al., Neuron, Vol. 44(4), 2004, 601-607).
In-vitro
studies show that LRRK2 proteins containing Parkinson's disease -associated
mutations
generally have an increased LRRK2 kinase activity and a decreased rate of GTP
hydrolysis
compared to the wild-type protein (Guo et al., Experimental Cell Research,
Vol. 313(16),
2007, pp. 3658-3670. Anti-LRRK2 antibodies have been used to label brainstem
Lewy
bodies associated with Parkinson's disease and cortical antibodies associated
with Lewis body
dementia, suggesting that LRRK2 may play an important role in Lewy body
formation and
pathogenesis associated with these diseases (Zhou et al., Molecular
Degeneration, 2006, 1:17
doi:10.1186/1750-1326-1-17). LRRK2 has also been identified as a gene
potentially
associated with increased susceptibility to Crohn's disease and susceptibility
to leprosy
(Zhang et al., New England J. Med. Vol. 361 (2009) pp.2609-2618.
[0005] Accordingly, compounds and compositions effective at modulating
LRRK2
activity may provide a treatment for neurodegenerative diseases such as
Parkinson's disease
and Lewy body dementia and for diseases such as leprosy and Crohn's disease.
Particularly,
there is a need for assays that enable the identification and evaluation of
compounds capable
of inhibiting LRRK2 kinase activity in cells. There is also a critical
requirement for
biomarkers that will enable the efficacy of inhibitors of LRRK2 kinase
activity to be
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evaluated in human patients and in animal models of Parkinson's disease.
Finally, there is a
continuing demand for techniques and biomarkers which might permit the
identification of
subjects at risk for Parkinson's Disease (PD) or with Parkinson's disease that
may be
responsive to treatment with LRRK2 inhibitors. The effectors of LRRK2 activity
are
unknown, which has impaired the ability of researchers to assess the ability
of compounds to
inhibit LRRK2 kinase activity in cells or in vivo.(N. Dzamko, 2010,
Biochemical J.)
Autophosphorylation is known to play an important role in the biological
activity of many
kinases, and it has been previously established that LRRK2 is capable of
autophosphorylation
at many sites in vitro.(Gloekner, 2010, J. Proteosome Res.; Kamakikawaji,
2009,
Biochemistry). These studies have found that LRRK2 functions primarily as a
serine/threonine kinase, and it has been suggested that phosphorylation at
threonine sites is
preferred. Importantly however, while several dozen autophosphorylation sites
have been
reported following in vitro studies, the vast majority of these sites are
appear not to be
phosphorylated in cells (Reference). Furthermore, there has been no report
describing a site
of autophosphorylation that can be identified in animals. An authentic
autophosphorylation
site that can be easily monitored in cells and in animals, as well as assays
that allow the
degree of autophosphorylation to be monitored under various conditions, will
be a critical tool
in the development of therapeutics targeting LRRK2.
SUMMARY OF THE INVENTION
[0006] Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) gene are the
most
common cause of familial Parkinson's disease (PD). Mutations in the LRRK2 gene
associated with Parkinson's disease modulate autophosphorylation of LRRK2.
Monitoring
changes in autophosphorylation of LRRK2 thus provides a tool for identifying
or evaluating
inhibitors of LRRK2 in vitro, and provides a potential biomarker for
monitoring LRRK2
activity in vivo.
[0007] The invention provides an assay/biomarkercapable of assessing
LRRK2
activity in the brain. Quantitative studies by the inventors demonstrated that
LRRK2
autophosphorylation on Ser1292 is among the most abundant autophosphorylation
events in
vitro and is detectable in transfected cells. Phosphospecific antibodies
directed against
pSer1292 were generated and used to show that LRRK2 familial mutations,
including
R1441G, Y1699C, G20195 and 12020T, all show increased p5er1292.
Autophosphorylation
of Ser1292 depends on LRRK2 kinase activity and GTP binding, and most likely
occurs in
cis. Mutation of Ser1292 to alanine (51292A) abolished the neuronal toxicity
caused by
LRRK2 PD mutation in cell-based models. In addition, expression of LRRK2 PD
mutant
proteins in neurons altered basal autophagy activity. S1292A mutation also
partially
ameliorates this phenotype. Use of LRRK2 kinase inhibitors show that LRRK2
kinase
inhibition effectively blocks S1292 autophosphorylation in cultured cells and
in LRRK2 BAC
transgenic animals. Accordingly, S1292 autophosphorylation represents a
biomarker of
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LRRK2 kinase activity in vivo and may contribute to the mechanism through
which LRRK2
causes Parkinson's disease. The terms "phosphorylation" and
"autophosphorylation" are used
interchangeably herein.
[0008] In one aspect of the invention, there is provided a method for
identifying
patients or subjects having Parkinson's disease or that are at risk of
developing Parkinson's
disease, the method comprising determining the level of S1292 phosphorylation
or
autophosphorylation in said patient, wherein an elevated level correlates with
having
Parkinson's disease or that are at risk of developing Parkinson's disease.
[0009] The method may further comprise obtaining or acquiring a LRRK2-
containing sample from the subject;
[0010] The method may further comprise measuring, monitoring or otherwise
detecting the amount or level of S1292 autophosphorylation in the subject
sample.
The method may further comprise comparing the amount or level of S1292
autophosphorylation in the subject sample with the amount or level of S1292
autophosphorylation in one or more control samples.
[0011] In one aspect of the invention, there is provided a method for
predicting the
sensitivity of a subject to a LRRK2 modulator, or efficacy of a LRRK2
modulator for treating
Parkinson's disease, the method comprising determining the level of S1292
phosphorylation
or autophosphorylation in said patient, whereby elevated levels of S1292
phosphorylation or
autophosphorylation correlate with efficacy of said inhibitor for treating
said disease.
[0012] The method may further comprise obtaining or acquiring a first
LRRK2-
containing sample from a subject.
[0013] The method may further comprise administering a LRRK2 modulator to
the
subject.
[0014] The method may further comprise obtaining or acquiring a second
LRRK2-
containing sample from the subject after administering the LRRK2 modulator to
the subject.
[0015] The method may further comprise measuring, monitoring or otherwise
detecting the amount or level of S1292 autophosphorylation in the first
subject sample.
[0016] The method may further comprise measuring, monitoring or otherwise
detecting the amount or level of S1292 autophosphorylation in the second
subject sample.
[0017] The method may further comprise comparing the amount or level of
51292
autophosphorylation in the first subject sample with the amount or level of
S1292
autophosphorylation in the second subject sample.
[0018] The invention also provides a method of selecting a patient to
receive a
LRRK2 inhibitor comprising measuring a patient's S1292 phosphorylation or
autophosphorylation profile, whereby a patient having an elevated S1292
autophosphorylation
profile is selected. The term "51292 profile" refers to the amount of 51292
autophosphorylation in a patient's sample compared to the amount in a control
sample(s). The
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S1292 profile can be expressed in terms of a ratio (e.g. amount of S1292
autophosphorylation
in a patient sample or cell divided by amount of 51292 autophosphorylation in
a control
sample or cell) or a subtraction (e.g. amount of 51292 in a patient sample or
cell minus the
amount of 51292 autophosphorylation in a control sample or cell). An
"increased 51292
profile" refers to a 51292 autophosphorylation profile that is determined to
have 51292
autophosphorylation levels that are substantially higher in the than control
samples. In one
example, a 51292 autophosphorylation profile is about 5% to 100% greater than
than in the
control sample.
[0019] In the subject methods, the LRRK2 modulator may be a compound of
formula I or formula II, or one of the LRRK2 modulators disclosed herein.
[0020] In one aspect of the invention there is provided a method for
identifying
subjects having Parkinson's disease or that are at risk of developing
Parkinson's disease, the
method comprising:
obtaining or acquiring a LRRK2-containing sample from a subject;
measuring, monitoring or otherwise detecting the amount or level of S1292
autophosphorylation in the subject sample; and
comparing the amount or level of S1292 autophosphorylation in the subject
sample with the amount or level of S1292 autophosphorylation in one or more
control
samples.
[0021] The method may further comprise determining if the amount or level
of
S1292 autophosphorylation in the subject sample is elevated with respect to or
otherwise
greater than that of the control sample(s). If the amount or level of 51292
autophosphorylation in the subject sample is elevated with respect to or
greater than that of
the control sample(s), the subject may be identified as having Parkinson's
disease or as being
at risk of developing Parkinson's disease.
[0022] By way of example and not of limitation, the LRRK2-containing
subject
sample may comprise cells, cell lysate, or solutions or suspensions thereof,
obtained from
tissue or serum from the subject. In one embodiment the subject sample may
comprise
monocytes (white blood cells) or lysate thereof The sample may be obtained
from the
subject via syringe or tissue extraction device. The sample thus obtained may
be subject to
cell separation, buffering, solvent extraction and/or exposure to lysing
enzyme(s) to expose or
otherwise make accessible the LRRK2 protein contained therein.
[0023] In certain embodiments the measuring of the level of 51292
autophosphorylation in the subject sample may be carried out using an antibody
that
associates with 51292 (i.e., an anti-phospho-51292-LRRK2 antibody), and
detecting the
antibody. The measuring may thus comprise:
transferring, affixing or immobilizing cells or cell lysate from the subject
sample onto an assay plate;
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applying an anti-phospho-S1292-LRRK2 antibody to the assay plate;
removing or washing away excess anti-phospho-S1292-LRRK2 antibody;
and
detecting anti-phospho-S1292-LRRK2 antibody bound to the assay plate.
100241 In certain embodiments the anti-phospho-S1292-LRRK2 antibody may
be a
monoclonal or polyclonal antibody that binds to an antigen peptide or protein
comprising the
sequence PNEMGKLSKIWDLPL, or CPNEMGKLSKIWDLPL, or a fragment thereof In
the above sequence the serine residue "S" is phosphorylated or
phosphorolatable. The antigen
peptide or protein thus can be represented as PNEMGKL-pS-KIWDLPL or CPNEMGKL-
pS-
KIWDLPL wherein "pS" is phosphorylated serine.
[0025] In another embodiment the antigen peptide or protein is between 12
and 20
residues in length, or between 14 and 18 residues in length, includes a
phosphorylated serine,
and has 90% homology with PNEMGKL-pS-KIWDLPL. In still another embodiment the
antigen peptide or protein is between 12 and 20 residues in length, or between
14 and 18
residues in length, includes a phosphorylated serine, and contains at least 10
consecutive
residues from within PNEMGKL-pS-KIWDLPL. The anti-phospho-S1292-LRRK2 antibody
may be applied to the assay plate as a solution.
[0026] In certain embodiments, detection of the anti-phospho-S1292-LRRK2
antibody may utilize ELISA detection, DELFIA detection, fluorescence
detection,
electrochemiluminescense detection, or other electrical, optical or
radioisotope techniques
capable of quantitative measurement or detection of antibodies on an assay
plate.
[0027] In certain embodiments, the comparing of the amount or level of
S1292
autophosphorylation in the subject sample with the amount or level of S1292
autophosphorylation in one or more control samples may be embodied in logical
elements of
computer software stored in a computer-readable medium. The comparing may thus
comprise, for example: inputting, via computer user interface, data
representing the amount or
level of S1292 autophosphorylation in the subject sample; inputting, via
computer user
interface, data representing the amount or level of S1292 autophosphorylation
in the control
sample(s); executing programming stored in the computer memory to generate
comparison
data; and displaying, by computer visual interface, the comparison data, in
alphanumeric
and/or graphical format, with the comparison data displayed in a manner that
shows or
determines whether or not the level of S1292 autophosphorylation in the
subject sample is
elevated with respect to or greater than that of the control sample(s).
[0028] Autophosphorylation level of LRRK2 S1292 may also be used as a
clinical
pharmacodynamic marker or companion diagnostic tool for determining if patient
subjects
will react favorably to administration of a particular LRRK2 modulator
therapy, and/or what
dosing level of a LRRK2 modulator or inhibitor may be optimal or desirable for
a subject. In
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this regard the invention provides a method for determining subject response
to therapy
involving a LRRK2 modulator, the method comprising:
obtaining or acquiring a first LRRK2-containing sample from a subject;
administering a LRRK2 modulator to the subject;
obtaining or acquiring a second LRRK2-containing sample from the subject
after administering the LRRK2 modulator to the subject;
measuring, monitoring or otherwise detecting the amount or level of S1292
autophosphorylation in the first subject sample;
measuring, monitoring or otherwise detecting the amount or level of S1292
autophosphorylation in the second subject sample; and
comparing the amount or level of S1292 autophosphorylation in the first
subject sample with the amount or level of S1292 autophosphorylation in the
second subject
sample.
100291 The method may further comprise determining if the amount or level
of
51292 autophosphorylation in the second subject sample is less than the amount
or level of
51292 autophosphorylation in the first subject sample. A reduction in 51292
autophosphorylation in the second subject sample with respect to the first
subject sample
indicates that the LRRK2 modulator is effective. The amount or level of
reduction of S1292
autophosphorylation further is indicative of how effective the LRRK2 modulator
is for the
subject, and thus what dosing levels may be appropriate for the subject.
[0030] By way of example and not of limitation, the first and second
LRRK2-
containing subject samples may comprise cells, cell lysate, or solutions or
suspensions
thereof, obtained from tissue or serum from the subject. In one embodiment the
subject
samples may comprise monocytes (white blood cells) or lysate thereof The
samples may be
obtained from the subject via syringe or tissue extraction device. The samples
thus obtained
may be subject to cell separation, buffering, solvent extraction and/or
exposure to lysing
enzyme(s) to expose or otherwise make accessible the LRRK2 protein contained
therein.
[0031] In certain embodiments the measuring of the level of 51292
autophosphorylation in the first and second subject samples may be carried out
using an
antibody that associates with 51292 (i.e., an anti-phospho-51292-LRRK2
antibody), and
detecting the antibody. The measuring may thus comprise
transferring, affixing or immobilizing cells or cell lysate from the first and
second samples onto an assay plate or plates;
applying an anti-phospho-S1292-LRRK2 antibody to the assay plate or
plates;
removing or washing away excess anti-phospho-S1292-LRRK2 antibody;
and
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detecting anti-phospho-S1292-LRRK2 antibody bound to the assay plate or
plates.
[0032] In certain embodiments the anti-phospho-S1292-LRRK2 antibody may
be a
monoclonal or polyclonal antibody that binds to an antigen peptide or protein
comprising the
sequence PNEMGKL-pS-KIWDLPL (PNEMGKLSKIWDLPL), CPNEMGKL-pS-
KIWDLPL (CPNEMGKLSKIWDLPL), or a fragment thereof, wherein "pS" is
phosphorylated serine as noted above. In another embodiment the antigen
peptide or protein
is between 12 and 20 residues in length, or between 14 and 18 residues in
length, includes a
phosphorylated serine, and has 90% homology with PNEMGKL-pS-KIWDLPL. In still
another embodiment the antigen peptide or protein is between 12 and 20
residues in length, or
between 14 and 18 residues in length, includes a phosphorylated serine, and
contains at least
consecutive residues from within PNEMGKL-pS-KIWDLPL. The anti-phospho-S1292-
LRRK2 antibody may be applied to the assay plate(s) as a solution.
[0033] In certain embodiments, detection of the anti-phospho-S1292-LRRK2
antibody from the first and second subject samples may utilize ELISA
detection, DELFIA
detection, fluorescence detection, electrochemiluminescense detection, or
other electrical,
optical or radioisotope techniques capable of quantitative measurement or
detection of
antibodies on an assay plate or plates.
[0034] In certain embodiments, the comparing of the amount or level of
S1292
autophosphorylation in the first subject sample with the amount or level of
S1292
autophosphorylation in the second subject sample may be embodied in logical
elements of
computer software stored in a computer-readable medium. The comparing may thus
comprise, for example: inputting, via computer user interface, data
representing the amount or
level of S1292 autophosphorylation in the first subject sample; inputting, via
computer user
interface, data representing the amount or level of S1292 autophosphorylation
in the second
subject sample; executing programming stored in the computer memory to
generate
comparison data; and displaying, by computer visual interface, the comparison
data, in
alphanumeric and/or graphical format, with the comparison data displayed in a
manner that
shows or determines whether or not the level of S1292 autophosphorylation in
the second
subject sample is less than or otherwise reduced with respect to that in the
first subject
sample.
[0035] The subject invention may be embodied in a kit that comprises:
one or more syringes or tools for acquiring a tissue sample or samples from a
subject;
an assay plate(s); and
a solution of anti-phospho-S1292-LRRK2 antibody.
[0036] The kit may further comprise buffer solution(s) for stabilizing
sample(s) from
the subject.
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[0037] The kit may further comprise a solution(s) of cell lysing reagent.
[0038] The kit may further comprise solution(s) or reagent(s) capable of
transferring,
affixing or immobilizing cells or cell lysate from the subject sample onto the
assay plate(s).
[0039] The kit may further comprise a solution for rinsing or removing
excess anti-
phospho-S1292-LRRK2 antibody from the assay plate(s).
[0040] The kit may further comprise a LRRK2 modulator solution and a
syringe or
other device for administration of the LRRK2 modulator solution.
[0041] The invention also provides an anti-phospho-S1292-LRRK2 antibody
characterized in that it is capable of binding to an antigen peptide or
protein comprising the
sequence CPNEMGKL-pS-KIWDLPL (CPNEMGKLSKIWDLPL), or a fragment thereof,
wherein "pS" is phosphorylated serine. In another embodiment, the anti-phospho-
S1292-
LRRK2 antibody is characterized in that it is capable of binding to an antigen
peptide or
protein comprising the sequence PNEMGKL-pS-KIWDLPL (PNEMGKLSKIWDLPL), or a
fragment thereof, wherein "pS" is phosphorylated serine. In another embodiment
the antigen
peptide or protein is between 12 and 20 residues in length, or between 14 and
18 residues in
length, includes a phosphorylated serine, and has 90% homology with PNEMGKL-pS-
KIWDLPL. In still another embodiment the antigen peptide or protein is between
12 and 20
residues in length, or between 14 and 18 residues in length, includes a
phosphorylated serine,
and contains at least 10 consecutive residues from within PNEMGKL-pS-KIWDLPL.
[0042] One aspect of the invention is an assay for screening compounds
and
compositions for inhibition of LRRK2 activity, the method comprising: exposing
LRRK2 or a
fragment thereof to a potential inhibitor compound; and detecting the level or
amount of
phosphorylation associated with a phosphorylation site on the LRRK2 protein.
[0043] In certain embodiments, the LRRK2 phoshorylation site may be
Serine 1292
(Ser1292 or S1292).
[0044] In certain embodiments, monitoring phosphorylation at the LRRK2
phosphorylation site may be carried out using an antibody associated with the
LRRK2
autophosphorylation site (anti-phospho-LRRK2 antibody). Detection of the anti-
phospho
antibody may utilize ELISA, DELFIA, fluorescence, electrochemiluminescense, or
other
electrical, optical or radioisotope techniques. In one embodiment the antibody
used is anti-
phospho-Serine 1292-LRRK2 antibody.
[0045] In certain embodiments, the LRRK2 used in the assay may be wild
type or a
mutated form associated with Parkinson's disease, such as the R1441G, R1441C,
Y1699C,
G2019S, 12020T mutations. In other embodiments, the LRRK2 protein used in the
assay may
feature combinations of Parkinson's Disease associated mutations (i.e.
R1441G/G2019S or
R1441G/Y1699C/G2019S). In further embodiments, the assay may feature mutations
known
to impact the kinase or GTPase activity of LRRK2 (i.e. D1994A). The LRRK2 may
be of
human, rabbit, mouse or other mammalian origin. The LRRK2 protein used in the
assay may
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be prepared by recombinant techniques using HEK293, CHO or other suitable cell
line
transfected or virally transduced with cDNA encoding all or part of LRRK2. The
LRRK2
may include an epitope tag to facilitate affinity purification, antibody
detection, or
immunoprecipitation.
[0046] In one embodiment, the subject method comprises: recombinantly
producing
LRRK2 in cells; exposing the cells to one or more potential LRRK2 inhibitor
compounds;
lysing the cells; exposing the cell lysate(s) to an anti-phospho-LRRK2
antibody; and
detecting anti-phospho-LRRK2 antibody bound to LRRK2 in the cell lysate.
[0047] In another embodiment, the method comprises: recombinantly
producing
LRRK2 in cells; exposing the cells to one or more potential LRRK2 inhibitor
compounds;
lysing the cells; immobilizing LRRK2 from the lysed cells; exposing the cell
lysate(s) to an
anti-phospho-LRRK2 antibody; and detecting anti-phospho-LRRK2 antibody bound
to
LRRK2 in the cell lysate.
[0048] In another embodiment, the method comprises: recombinantly
producing
LRRK2 in cells; immobilizing the cells in a plurality of wells of a first
plate; exposing the
cells in each well to a potential LRRK2 inhibitor compound; lysing the cells
in each well;
immobilizing LRRK2 from the lysed cells from each well of the first plate in a
corresponding
well of a second plate; exposing the immobilized LRRK2 to an anti-phospho-
LRRK2
antibody; and detecting anti-phospho-LRRK2 antibody bound to LRRK2.
Immobilizing the
LRRK2 from the lysed cells may comprise, for example, seeding or immobilizing
an antibody
for LRRK2 or an epitope tag thereon in the wells of the second plate, and
transferring the cell
lysate from the wells of the first plate to corresponding wells of the second
plate.
[0049] Another aspect of the invention comprises the utilization of a
phosphorylation
site of LRRK2 as a biomarker method for the in vivo detection of Parkinson's
disease
mutations in LRRK2 and for evaluation of the in vivo efficacy of LRRK2
inhibitors. The
method may comprise: administering a LRRK2 inhibitor to a subject; acquiring a
tissue or
serum sample from the subject; and detecting the level or amount of
phosphorylation
associated with a phosphorylation site on LRRK2 from the sample.
[0050] In one embodiment, the biomarker phosphorylation site may be
Serine 1292
(Ser1292 or S1292) of the LRRK2 protein.
[0051] Monitoring phosphorylation at the LRRK2 phosphorylation site may
be
carried out by detection of antibody associated with the LRRK2
autophosphorylation site
(anti-phospho-LRRK2 antibody) such as anti-phospho-Serine 1292-LRRK2 antibody.
anti-
phospho-Serine 1292-LRRK2 may be detected using standard procedures such as
ELISA,
DELFIA, fluorescence, electrochemiluminescense, or other electrical, optical
or radioisotope
techniques in combination with an anti-phospho-Serine 1292-LRRK2 antibody.
[0052] Another aspect of the invention is an antibody for an antigen
comprising the
sequence CPNEMGKL-pS-KIWDLPL, or a fragment thereof, wherein "pS" is
phosphorylated
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serine. In another embodiment the antigen is between 14 and 18 residues in
length, includes a
phosphorylated serine, and has 90% homology with CPNEMGKL-pS-KIWDLPL.
[0053] Another aspect of the invention is a kit or kits for in vitro
evaluation of
LRRK2 inhibitors. Such kits may comprise, for example: a solution containing
LRRK2
protein; and a solution containing an anti-phospho-LRRK2 antibody such as anti-
phospho-
Serl 292-LRRK2 antibody. The solutions may be stored in suitable tubes or
containers, and
the kit may further comprise containers, multiwell plates, buffer solutions
and tools
configured for carrying out the assay and biomarker methods of the invention
described
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] The invention will be more fully understood by reference to the
following
drawings, which are for illustrative purposes.
[0055] Figure. 1. Identification of an in vivo LRRK2 autophosphorylation
site. (A)
Schematic diagram depicting the domain architecture of LRRK2. (B, C) Annotated
MS/MS
spectra collected which unambiguously identify pSerl 292 and pThr1343.
Comparison of
these spectra to MS/MS derived from synthetic internal standards provides
additional
confirmation. (D, E) Extracted ion chromatograms for p5er1292 SFPNEMGKLS#K and
pThr1343 LMIVGNT#GSGK peptides from reactions of G20195-LRRK2 at times ranging
from 0 to 60 min. The isotopically labeled internal standards of pSerl 292
(13C615N1 Leu;
+7.017 Da) and pThr1343 (13C515N1 Val; +6.014 Da) are shown at the back of
each 3D plot,
with the signal in the front representing the light analyte peptide derived
from the digested
sample. (F, G) Extracted ion chromatograms for light and heavy pSerl 292
peptides from
FLAG-LRRK2 immunoprecipiated from transfected 293 cells. Light pSerl 292 can
be
detected from G20195-LRRK2, but not kinase dead D1994A LRRK2.
[0056] Figure 2. Kinase activity of LRRK2 is required for S1292
phosphorylation
(A) Western blots detecting LRRK2 autophosphorylation in lysates of HEK 293
cells
transiently expressing FLAG tagged LRRK2 WT, 51292A and D1994A. (B) Western
blots
detecting LRRK2 autophosphorylation in the brain lysates of BAC transgenic
mice over
expressing LRRK2 WT or kinase-dead mutant.
[0057] Figure 3. Familial Parkinson's disease mutations increase S1292
autophosphorylation (A) Representative Western blot image detecting p51292 and
total
LRRK2 signal from lysate of HEK293 cells transiently transfected with WT,
R1441G,
Y1699C, G20195, 12020T, G2385R and R1441G/G20195 (RG/GS) LRRK2. (B)
Quantitation of average fold of LRRK2 p51292 signal in mutants compared to
wild type,
signals were average of 4 independent experiments. (C) Representative Western
blot image
detecting p51292 and total LRRK2 from tissue lysate from different brain
regions of BAC
transgenic mice expressing Flag-tagged wild type (WT) and G2019S LRRK2. (D)
Quantitation of fold of pS1292 change in total brain lysate from BAC
transgenic mice
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expressing Flag-tagged wild type (WT) and G2019S LRRK2. N = 5 animals in each
genotype.
[0058] Figure 4. Biochemical mechanisms of LRRK2 autophosphorylation on
S1292 (A) GTP binding is required for S1292 autophosphorylation.
Representative western
blot image detecting p51292 and total LRRK2 from lysates transiently
expressing Flag-
tagged wild type, T1348N, G20195 and T1348N/G20195 LRRK2. (B) LRRK2 S1292A
protein cannot phosphorylate 51292 residue on LRRK2 kinase-dead mutant
(D1994A).
Schematic of two constructs co-transfected in HEK293 cells (left).
Representative image of
Western blot detecting p51292 from lysates of HEK293 cells transiently
expressing Flag-
tagged LRRK2 R1441G/Y1699C/G20195 (3M), 3M/S1292A, D1994A, and D1994A with
3M/S1292A (right). (C) Full-length LRRK2 cannot phosphorylate 51292 residue on
a
truncated kinase-dead LRRK2 (Di 994A). Schematic of two co-transfected
constructs (left).
Representative image of Western blot detecting p51292 and total LRRK2 from
HEK293 cells
expressing full-length and truncated LRRK2 carrying D1994A mutation.
[0059] Figure 5. 51292 is required for LRRK2 PD mutation induced neurite
outgrowth defects. (A) Neurolucida tracing of a representative GFP transfected
hippocampal
neuron. GFP signal was used for neurite tracing with Neurolucida software. An
overlay of
concentric circles, spaced at 20[tm intervals, centered at the cell body was
used for Sholl
analysis. (B) Representative Neurolucida tracing of GFP and LRRK2 co-
transfected
hippocampal neurons. GFP were co-transfected with empty vector, LRRK2 Wild-
type,
R1441G G20195, S1292A R1441G G20195 and R1441G G20195 D1994A alleles into
hippocampal cultures at day 6 in vitro (DIV 6). At DIV 17, neurons were fixed
and stained
with GFP and MAP2. Images of transfected neurons were taken with confocal
microscopy
and dendrites were traced with Neurolucida software by an observer blind to
the identity of
the allele. Scale bar: 100[tm. (C) Sholl analysis. Mean ( SEM) number of
intersections with
each circle in LRRK2 transfected cells were counted. Student t-test p values
between R1441G
G20195 and wild type, R1441G G20195 D1994A, S1292A R1441G G20195 were less
than
0.05 (indicated by *). N = 22-30 cells for each constructs. (D).
Quantification of total
dendritic length as in (B). n = 40-85 cells in three experiments. Mean
diamonds were shown
for each group. Student t-test p values between R1441G G20195 (red circle) and
wild type,
R1441G G20195 D1994A, S1292A R1441G G20195 were less than 0.001.
[0060] Figure 6A-D. Si 292A mutation partially ameliorates autophagy
impairment
induced by LRRK2 PD mutations.
[0061] Figure 7. LRRK2 kinase inhibitors block p51292
autophorsphorylation in a
cell-based assay. (A) Utilizing the pLRRK2 electrochemiluminescent 96 well
plate based
assay, an increased pLRRK(5er1292) signal was detected in LRRK2 cell lysates
induced with
lug/mL doxycline for 16 hours compared to non-induced LRRK cell lysates. (B)
Dose
dependent reduction of pS1292 signal in doxycline induced LRRK2 cells
incubated with
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Sutent (IC50 0.10uM) and G1023 (IC50 0.0016uM) for 16 hours. Inhibition was
normalized to
non-induced cell lysates as 100% inhibition. (C) 600 compound data set showing
robust
correlation of cellular pLRRK(Ser1292) inhibitor IC50 values and biochemical
LRRK2 Ki
values. Slope = 0.95, R2= 0.77. Open symbols are out of range IC50 values and
were
excluded from the regression fit.
[0062] Figure 8. LRRK2 kinase inhibitor G1023 reduces p51292
autophorsphorylation in vivo. Image of Western blot detecting p51292 and total
LRRK2 from
tissue lysates of spleen (A) and brain (B) from Flag-tagged G2019S LRRK2BAC
transgenic
animals. Animals were dosed with vehicle alone (2 animals), 10, 30 or 100
mg/kg of
G01581023 (3 animals each for the compoud dosed groups). (C) Quantitation of
pS1292/total
LRRK2 signal in (A) and (B). (D), (E).
[0063] Figure 9A, 9B. LRRK2 kinase inhibitor G1023 ameliorates neurite
outgrowth defect of G2019S neurons cultured from LRRK2 BAC transgenic mice.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0064] Unless otherwise stated, the following terms used in this
Application,
including the specification and claims, have the definitions given below. It
must be noted
that, as used in the specification and the appended claims, the singular forms
"a", "an," and
"the" include plural referents unless the context clearly dictates otherwise.
[0065] The definitions of certain terms as used in this specification are
provided
below. Definitions of other terms may be found in the glossary provided by the
U.S.
Department of Energy, Office of Science, Human Genome Project (http:
www.ornl.gov/sci/techresources/Human_Genome/glossary/). In practicing the
present
invention, many conventional techniques in molecular biology, microbiology and
recombinant DNA are used. These techniques are well-known and are explained
in, e.g.,
Current Protocols in Molecular Biology, Vols. I-III, Ausubel, ed. (1997);
Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A Practical Approach,
Vols. I and II,
Glover D, ed. (1985); Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic
Acid
Hybridization, Hames & Higgins, eds. (1985); Transcription and Translation,
Hames &
Higgins, eds. (1984); Animal Cell Culture, Freshney, ed. (1986); Immobilized
Cells and
Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the
series,
Methods in Enzymol. (Academic Press, Inc., 1984); Gene Transfer Vectors for
Mammalian
Cells, Miller & Cabs, eds. (Cold Spring Harbor Laboratory, N.Y., 1987); and
Methods in
Enzymology, Vols. 154 and 155, Wu & Grossman, and Wu, eds., respectively.
[0066] As used herein, the term "allele" means a particular form of a
gene or DNA
sequence at a specific chromosomal location (locus).
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[0067] As used herein, the term "antibody" includes, but is not limited
to, polyclonal
antibodies, monoclonal antibodies, humanized or chimeric antibodies and
biologically
functional antibody fragments sufficient for binding of the antibody fragment
to the protein.
Antibodies can be used in assays to determine the presence of variant proteins
and peptides
where the genetic polymorphisms of the invention are in the coding region of
the gene.
[0068] As used herein, the term "clinical response" means any or all of
the
following: a quantitative measure of the response, no response, and adverse
response (i.e.,
side effects).
[0069] As used herein, the term "clinical trial" means any research study
designed to
collect clinical data on responses to a particular treatment, and includes but
is not limited to
phase I, phase II and phase III clinical trials. Standard methods are used to
define the patient
population and to enrol subjects.
[0070] As used herein, the term "effective amount" of a compound is a
quantity
sufficient to achieve a desired therapeutic and/or prophylactic effect, for
example, an amount
which results in the prevention of or a decrease in the symptoms associated
with a disease that
is being treated, e.g., the diseases associated with LRRK2 mutant
polynucleotides and mutant
polypeptides identified herein (particularly Alzheimer's disease and
Parkinson's disease). The
amount of compound administered to the subject will depend on the type and
severity of the
disease and on the characteristics of the individual, such as general health,
age, sex, body
weight and tolerance to drugs. It will also depend on the degree, severity and
type of disease.
The skilled artisan will be able to determine appropriate dosages depending on
these and other
factors. Typically, an effective amount of the compounds of the present
invention, sufficient
for achieving a therapeutic or prophylactic effect, range from about 0.000001
mg per
kilogram body weight per day to about 10,000 mg per kilogram body weight per
day.
Preferably, the dosage ranges are from about 0.0001 mg per kilogram body
weight per day to
about 100 mg per kilogram body weight per day. The compounds of the present
invention can
also be administered in combination with each other, or with one or more
additional
therapeutic compounds.
[0071] As used herein, "expression" includes but is not limited to one or
more of the
following: transcription of the gene into precursor mRNA; splicing and other
processing of
the precursor mRNA to produce mature mRNA; mRNA stability; translation of the
mature
mRNA into protein (including codon usage and tRNA availability); and
glycosylation and/or
other modifications of the translation product, if required for proper
expression and function.
[0072] As used herein, the term "gene" means a segment of DNA that
contains all
the information for the regulated biosynthesis of an RNA product, including
promoters,
exons, introns, and other untranslated regions that control expression.
[0073] As used herein, the term "genotype" means an unphased 5' to 3'
sequence of
nucleotide pairs found at one or more polymorphic sites in a locus on a pair
of homologous
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chromosomes in an individual. As used herein, genotype includes a full-
genotype and/or a
sub-genotype.
100741 As used herein, the term "locus" means a location on a chromosome
or DNA
molecule corresponding to a gene or a physical or phenotypic feature, in
particular the
LRRK2 gene.
[0075] As used herein, the term "LRRK2 modulating agent" is any compound
that
alters (e.g., increases or decreases) the expression level or biological
activity level of LRRK2
polypeptide compared to the expression level or biological activity level of
LRRK2
polypeptide in the absence of the LRRK2 modulating agent. LRRK2 modulating
agent can be
a small molecule, polypeptide, carbohydrate, lipid, nucleotide, or combination
thereof The
LRRK2 modulating agent may be an organic compound or an inorganic compound.
[0076] As used herein, the term "mutant" means any heritable variation
from the
wild-type that is the result of a mutation, e.g., single nucleotide
polymorphism. The term
"mutant" is used interchangeably with the terms "marker", "biomarker", and
"target"
throughout the specification.
[0077] As used herein, the term "medical condition" includes, but is not
limited to,
any condition or disease manifested as one or more physical and/or
psychological symptoms
for which treatment is desirable, and includes previously and newly identified
diseases and
other disorders.
[0078] As used herein, the term "nucleotide pair" means the nucleotides
found at a
polymorphic site on the two copies of a chromosome from an individual.
[0079] As used herein, the term "polymorphic site" means a position
within a locus
at which at least two alternative sequences are found in a population, the
most frequent of
which has a frequency of no more than 99%.
[0080] As used herein, the term "phased" means, when applied to a
sequence of
nucleotide pairs for two or more polymorphic sites in a locus, the combination
of nucleotides
present at those polymorphic sites on a single copy of the locus is known.
[0081] As used herein, the term "polymorphism" means any sequence variant
present
at a frequency of >1% in a population. The sequence variant may be present at
a frequency
significantly greater than 1% such as 5% or 10% or more. Also, the term may be
used to refer
to the sequence variation observed in an individual at a polymorphic site.
Polymorphisms
include nucleotide substitutions, insertions, deletions and microsatellites
and may, but need
not, result in detectable differences in gene expression or protein function.
[0082] As used herein, the term "polynucleotide" means any RNA or DNA,
which
may be unmodified or modified RNA or DNA. Polynucleotides include, without
limitation,
single- and double-stranded DNA, DNA that is a mixture of single- and double-
stranded
regions, single- and double-stranded RNA, RNA that is mixture of single- and
double-
stranded regions, and hybrid molecules comprising DNA and RNA that may be
single-
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stranded or, more typically, double-stranded or a mixture of single- and
double-stranded
regions. In addition, polynucleotide refers to triple-stranded regions
comprising RNA or DNA
or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs
containing
one or more modified bases and DNAs or RNAs with backbones modified for
stability or for
other reasons.
[0083] As used herein, the term "polypeptide" means any polypeptide
comprising
two or more amino acids joined to each other by peptide bonds or modified
peptide bonds,
i.e., peptide isosteres. Polypeptide refers to both short chains, commonly
referred to as
peptides, glycopeptides or oligomers, and to longer chains, generally referred
to as proteins.
Polypeptides may contain amino acids other than the 20 gene-encoded amino
acids.
Polypeptides include amino acid sequences modified either by natural
processes, such as post-
translational processing, or by chemical modification techniques that are well-
known in the
art. Such modifications are well described in basic texts and in more detailed
monographs, as
well as in a voluminous research literature.
[0084] As used herein, the terms "phosphorylation" and
"autophosphorylation",
which are used interchangeably herein refer generally to the adding of a
phosphate group to
LRRK2 at a phosphorylation site thereon, such as Serine 1292 (Ser 1292 or
S1292).
[0085] As used herein, the term "SNP nucleic acid" means a nucleic acid
sequence,
which comprises a nucleotide that is variable within an otherwise identical
nucleotide
sequence between individuals or groups of individuals, thus existing as
alleles. Such SNP
nucleic acids are preferably from about 15 to about 500 nucleotides in length.
The SNP
nucleic acids may be part of a chromosome, or they may be an exact copy of a
part of a
chromosome, e.g., by amplification of such a part of a chromosome through PCR
or through
cloning. The SNP nucleic acids are referred to hereafter simply as "SNPs". A
SNP is the
occurrence of nucleotide variability at a single position in the genome, in
which two
alternative bases occur at appreciable frequency (i.e., >1%) in the human
population. A SNP
may occur within a gene or within intergenic regions of the genome. SNP probes
according to
the invention are oligonucleotides that are complementary to a SNP nucleic
acid.
[0086] As used herein, the administration of an agent or drug to a
subject or patient
includes self-administration and the administration by another. It is also to
be appreciated that
the various modes of treatment or prevention of medical conditions as
described are intended
to mean "substantial", which includes total but also less than total treatment
or prevention,
and wherein some biologically or medically relevant result is achieved.
[0087] As used herein, "Parkinson's disease" ("PD") means a degenerative
disorder
of the central nervous system that impairs motor skills, speech, and/or
cognitive function.
Symptoms of Parkinson's disease may include, for example, muscle rigidity,
tremor, slowing
of physical movement (bradykinesia) and loss of physical movement (akinesia).
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[0088] As used herein, "Lewy body disease" also called "Lewy body
demntia",
diffuse Lewy body disease", cortical Lewy body disease", means a
neurogenerative disorder
characterized anatomically by the presence of Lewy bodies in the brain.
[0089] As used herein, "Subject" means mammals and non-mammals. Mammals
means any member of the mammalia class including, but not limited to, humans;
non-human
primates such as chimpanzees and other apes and monkey species; farm animals
such as
cattle, horses, sheep, goats, and swine; domestic animals such as rabbits,
dogs, and cats;
laboratory animals including rodents, such as rats, mice, and guinea pigs; and
the like.
Examples of non-mammals include, but are not limited to, birds, and the like.
The term
"subject" does not denote a particular age or sex.
[0090] As used herein, "Therapeutically effective amount" means an amount
of a
compound that, when administered to a subject for treating a disease state, is
sufficient to
effect such treatment for the disease state. The "therapeutically effective
amount" will vary
depending on the compound, disease state being treated, the severity or the
disease treated,
the age and relative health of the subject, the route and form of
administration, the judgment
of the attending medical or veterinary practitioner, and other factors.
[0091] The terms "those defined above" and "those defined herein" when
referring
to a variable incorporates by reference the broad definition of the variable
as well as particular
definitions, if any.
[0092] "Treating" or "treatment" of a disease state includes, inter alia,
inhibiting the
disease state, i.e., arresting the development of the disease state or its
clinical symptoms,
and/or relieving the disease state, i.e., causing temporary or permanent
regression of the
disease state or its clinical symptoms.
[0093] All patents and publications identified herein are incorporated
herein by
reference in their entirety.
LRRK2
[0094] Referring to FIG. 1, there is shown a schematic representation of
the LRRK2
protein with N-terminus at the left and C-Terminus at the right. The LRRK2
protein is 2527
amino acids in length and belongs to the ROCO-protein superfamily. LRRK2
includes an
ANK (Ankyrin repeat) domain, a LRR (Leucine rich repeat) domain, a ROC (Ras in
complex
proteins) domain, a COR (C-terminal of Roc) linker domain, a kinase domain,
and a WD40
domain. The S1292 autophosphorylation site is located generally between the
LRR and ROC
domains as shown in FIG. 1. The R1441G mutation occurs in the ROC domain,
while the
Y1699C mutation is in the COR domain, and the G20195 and 12020T are found in
the Kinase
domain. The human LRRK2 protein and corresponding nucleic acid sequences are
well
known and are disclosed, for example, as SEQ. ID NO: 2 and SEQ ID NO: 1
respectively in
published application US 2010/0035251.
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Identification of S1292 as an in vivo autophosphorylation site
[0095] In vitro kinase assays were carried out using purified G20195 and
kinase
dead (D1994A) LRRK2. Reactions were performed in the presence or absence of
ATP (1
mM) and GTPgS (10 mM), then separated by SDS-PAGE. Coomassie stained LRRK2
bands
were subjected to in gel trypsin digestion followed by liquid-chromatography
tandem mass
spectrometry (LC-MS/MS, Aqua peptides (Sigma-Aldrich)). Database searching
revealed a
series of spectra which confidently matching to phosphopeptides of LRRKs. Of
the identified
sites, none were observed from the reactions with kinase dead Di 994A LRRK2.
Noteworthy
were a cluster of phosphopeptides beginning with pSer1292 at the C-terminal
portion of the
LRR domain and extending through the ROC domain (Figs. 1B, 1C).
Phosphorylation was
also observed on at least one of the residues within the kinase activation
loop (pSer2031; data
not shown). These results support a previous indicating that LRRK2
preferentially modifies
Thr residues over Ser, at least within the context of autophosphorylation
activity.
[0096] Among the identified LRRK2 autophosphorylation sites, an unusual
frequency of Met residues was noted in close proximity to LRRK2
phosphorylation sites, with
9/14 peptides displaying a Met between one and five positions upstream of the
modified
residue. While none of the phosphopeptides were observed in the reactions
lacking ATP and
GTPgS, the single exception was a spectra matching to the sequence
SFPNEMGKLS#K,
corresponding to pSer1292 (Fig. 1B).
[0097] To assess the relative abundance of phosphorylation in vitro and
assist with
validating these modifications in vivo, a series of internal standard peptides
were synthesized.
These peptides comprised the same sequences as identified LRRK2
phosphopeptides, but
included a single amino acid which was enriched in stable isotopes Included
among these
were isotopically labeled peptides directed towards pSer1292 were SFPNEMGKLS#K
(13C615N1 Leu; +7.017 Da), pThr1343 LMIVGNT#GSGK (13C515N1 Val; +6.014 Da),
pThr1357 TTLLQQLMKT#K (13C615N1 Leu +7.017 Da), and pThr1368
SDLGMQSAT#VGID VK (13C515N1 Val +6.014 Da). To comprehensively assess
phosphorylation status of pSer1357 and pSer1368, additional peptides were
required due
address ragged-end termini flanking these sequences.
[0098] In vitro kinase reactions were then performed using purified WT,
G20195,
and R1441C LRRK2 protein. Timepoints were collected at 0, 5, 10, 30 and 60
min, separated
by SDS-PAGE and Coomassie stained. Extracted ion chromatograms for pSer1292
and
pThr1343 peptides from the G2019S reactions are shown relative to their
corresponding
internal standards (Fig. 1D, 1E). As noted in the phosphomapping studies, a
small fraction of
5er1292 was phosphorylated in the purified G20195 LRRK2 at the onset of the
kinase
reaction. The raw MS data indicate that each phosphopeptide increases in
abundance, with
pSer1292 displaying a steady increase from 5 to 60 min compared to pThr1343
which
increases from 0 to 10 min before rapidly reaching a plateau. At each position
monitored,
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G2019S appears to undergo more rapid autophosphorylation than either WT or
R1441C
LRRK2 (Fig. 1E, 1F, 1G, 1H). Interestingly, individual sites display unique
kinetics, with
autophosphorylation of pThr1343 and pThr1357 rapidly reaching plateaus, while
phosphorylation at Ser1292 and Thr1368 continue robustly even out to the 60
min timepoint
(Fig. 1E, 1F, 1G, 1H). A modest but consistent difference is also observed
between the
activity of WT and R1441C LRRK2 (Fig. 1E, 1F, 1G, 1H).
[0099] In order to validate LRRK2 autophosphorylation activity in living
cells, an
experiment on FLAG-LRRK2 overexpressed in HEK293 cells using our quantitative
LC-
MS/MS assay was performed. The majority of autophosphorylation events
identified from in
vitro kinase assays were undetectable in immunoprecipitated LRRK2, even with
the use of
LRRK2 isotopic standards as sentries. The one exception was pSer1292.
Extracted ion
chromatograms of the heavy pSer1292 signal revealed a low level of pSer1292 on
FLAG-
G2019SLRRK2 (Fig. 1F), but not FLAG-D1994ALRRK2. An MS/MS acquired on the
internal
standard confirmed the identity of the heavy peptide, with perfect
coresolution of the native
peptide essential to confirming the presence of p5er1292 from 293 cells.
Neatly, an
analogous MS/MS spectrum was collected from the light form of this peptide,
but had been
coisolated with another ion, confounding automated identification efforts.
Manual inspection
and comparison of these two spectra permitted deconvolution and additional
validation of
p5er1292 within this sample.
[00100] Since pSer1292 was the most robust autophosphorylation site in
vitro and in
cultured cells, a rabbit polyclonal antibody was generated that specifically
recognizes this
phospho-epitope (anti-pLRRK2 antibody). 293 Cells were transfected with wild
type,
S1292A (phospho-site dead) and Di 994A (kinase dead) LRRK2 constructs. It was
found that
anti-pLRRK2 antibody detect a specific signal from lysates of cells
transfected with wild type
LRRK2, but not those with either S1292A or Di 994A mutations (Fig 2A),
demonstrating that
the anti-pLRRK2 antibody is specific to the phosphorylated S1292 epitope.
Furthermore, the
anti-pLRRK2 antibody detected a specific signal from brain lysates of BAC
transgenic mice
expressing wild type LRRK2, but not those expressing kinase-dead LRRK2 (Fig.
2B). These
results demonstrate that LRRK2 kinase activity is required for phosphorylation
of S1292
residue in vivo.
Parkinson's mutations increase LRRK2 autophosphorylation on Ser1292
[0100] The most common familial mutation of LRRK2 G2019S has been shown
to
increase its kinase activity in a variety of in vitro kinase assays, including
autophosphorylation, phosphorylation of MBP and LRRKtide. Beside G2019S, most
published results show that other PD mutations, including R1441G/C, Y1699C and
12020T,
do not increase LRRK2 kinase activity in vitro kinase. How PD mutations affect
LRRK2
kinase activity in vivo remains unknown because the lack of in vivo
substrates.
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[0101] Since LRRK2 autophosphorylation on Ser 1292 is dependent on its
kinase
activity, the affect of different LRRK2 PD mutations on LRRK2
autophosphorylation levels
was tested by transfecting 293 cells with Flag-tagged LRRK2 constructs
containing different
Parkinson's mutations. The levels of total LRRK2 and p51292 in whole cell
lysates were
assessed by Western blotting. We found that all of the four confirmed LRRK2
familial
Parkinson's mutations, including R1441G, Y1699C, G20195 and 12020T, showed
increased
LRRK2 autophosphorylation at S1292 site comparing to wild type LRRK2.
Furthermore,
combining two PD mutations R1441G and G20195 in the same construct increased
LRRK2
autophosphorylation level even higher (Figure 3A, B). Notably, G23 85R, a PD
risk factor
mutation found in Asian population, did not increase autophosphorylation on
Ser1292.
[0102] We also tested whether PD mutations increase LRRK2
autophosphorylation
in transgenic animals. We compared LRRK2 autophosphorylation levels in the
brain tissues
from transgenic animals expressing wild type or G2019S LRRK2 proteins. We
found that
total LRRK2 protein levels were comparable in the two lines of transgenic
mice, while the
phospho-LRRK2 level is much higher in tissues from LRRK2 G2019S BAC transgenic
mice
in all brain regions examined (Figure 3C). Quantitatively, LRRK2
autophosphorylation on
S1292 is more than 10 times higher in total brain lysates of transgenic
animals expressing
G2019S mutant LRRK2 than the ones expressing wild type LRRK2 (Figure 3D).
Together,
these data demonstrate that Parkinson's mutations of LRRK2 increase LRRK2
autophosphorylation on S1292 both in cultured cells and in animals.
Biochemical mechanisms of LRRK2 Ser1292 autophosphorylation
[0103] GTP binding is recognized as being required for LRRK2 kinase
activation in
vitro. LRRK2 T1348N mutation has been shown to abolish GTP binding in vitro.
However,
it is unclear how GTP binding affects LRRK2 kinase activity in vivo. To test
whether GTP
binding is required for LRRK2 autophosphorylation in vivo, LRRK2
autophosphorylation
levels in 293 cells transfected with LRRK2 T1348N and LRRK2 G20195/T1348N
mutant
constructs were examined. LRRK2 S1292 autophosphorylation was not detectable
in lysates
form cells expressing T1348N mutation. S1292 phosphorylation is also greatly
reduced in
LRRK2 G20195/T1348N transfected cells compared to those expressing LRRK2
G2019S
(Figure 4A). These results demonstrate that in vivo LRRK2 autophosphorylation
on S1292
requires GTP binding.
[0104] The mechanism of LRRK2 autophosphorylation may occur by two
possible
routes. One possibility is that one LRRK2 molecule may phosphorylate S1292 on
another
LRRK2 molecule, i.e. intermolecular phosphorylation. The other possibility is
that the kinase
domain of a LRRK2 molecule may phosphorylate its own S1292, i.e.
intramolecular
phosphorylation. To distinguish these two possibilities, LRRK2 51292A mutant
was
transfected with LRRK2 D1994A mutant. 51292A mutation abolishes the
autophosphorylation site, while Di 994A mutation abolishes LRRK2 kinase
activity. If
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LRRK2 autophosphorylation is inter-molecular, LRRK2 Si 292A can phosphorylate
LRRK2
Di 994A when these two constructs are co-transfected, and phospho-LRRK2 signal
would be
observed. However, LRRK2 autophosphorylation was not observed in lysates of
293 cells
co-transfected with S1292A and D1994A mutation (Figure 4B). This data suggest
that
LRRK2 autophosphorylation is likely to be intra-molecular.
[0105] To further validate intra-molecular autophosphorylation,
phosphorylation of a
N-terminal-truncated LRRK2 by full-length wild type LRRK2 was tested. Wild
type LRRK2
and N-terminal truncated LRRK2 (907-2527) were co-transfected with Di 994A
mutation.
Little 51292 phosphorylation was detected in truncated LRRK2 D1994A by Western
blot (Fig
4C). Therefore, the full-length wild type LRRK2 could not phosphorylate
truncated LRRK2.
Taken together, these data demonstrated that LRRK2 autophosphorylation is most
likely
intra-molecular.
5er1292 is required for neuronal toxicity induced by LRRK2 Parkinson's
mutations.
[0106] Ser1292 residue is located at the junction of LRR domain and
GTPase
domain of LRRK2. A protein homology analysis showed that 51292 is conserved
from worm
to human, while the overall amino acid identities are limited. The importance
of Ser1292
autophosphorylation for the biological function of LRRK2 was thus evaluated.
[0107] Expression of LRRK2 protein carrying PD mutations causes neuronal
toxicity in cultured primary neurons. Furthermore, the observed neuronal
toxicity requires the
kinase activity of LRRK2. Transfection of cultured primary hippocampal neurons
from El8
rat embryos with LRRK2 cDNA carrying different PD mutations and examined
neurite
morphology was carried out (Figure 5A) to show that hippocampal neurons
expressing
LRRK2 R1441G/G2019S double mutation exhibit robust neurite outgrowth defect
compared
with those expressing wild type LRRK2 or GFP only. Consistent with published
reports,
introducing a kinase-dead mutation D1994A to LRRK2 R1441G/G20195 double
mutation
abolished this phenotype. Furthermore, mutating Ser 1292 to Alanine in LRRK2
R1441G/G20195 double mutation also abolished the neurite outgrowth defects,
suggesting
5er1292 phosphorylation is important for LRRK2 R1441G/G20195 induced neurite
outgrowth phenotypes (Figure 5 B, D).
[0108] In addition to causing shortening of total neurite length,
expressing LRRK2
R1441G/G20195 double mutant protein also reduced the complexities of the
dendritic trees in
cultured primary hippocampal neurons, as shown by Sholl analysis. Furthermore,
LRRK2
G2019S/R1441G /D1994A mutant protein restored dendritic complexity of the
neurons,
suggesting that LRRK2 kinase activity is required. Importantly, mutating LRRK2
autophosphorylation site serine 1292 to alanine also ameliorated this
phenotype (Figure 5C).
Taken together, these data strongly suggest that autophosphorylation of
Ser1292 on LRRK2
plays important role in mediating LRRK2 PD mutation induced neuronal toxicity.
Ser1292 autophosphorylation is important for LRRK2 PD mutation induced
autophagy impairment
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[0109] The effect of Ser1292 autophosphorylation on neuronal toxicity
induced by
LRRK2 PD mutations was examined. We first considered whether Ser1292
autophosphorylation affects LRRK2 kinase activity. Flag-tagged wt and Si 292A
LRRK2
protein was purified from transiently transfected 293 cells. Kinase activities
were compared
in an in vitro kinase assay with LRRKtide as the substrate. No significant
difference between
LRRK2 WT and S1292A protein were observed.
[0110] Subcellular localization of LRRK2 PD mutant proteins was next
examined.
Flag-tagged LRRK2 R1441G/G2019S double mutant construct was transfected into
cultured
primary cortical neurons. Immunofluorescent cytochemistry was used to detect
Flag-tagged
LRRK2 proteins in transfected neurons. WT LRRK2 protein was found to show
diffused
distribution in neurons. However, in more than 80% of neurons transfected with
LRRK2
R1441G/G2019S double mutant, LRRK2 protein showed filamentous distribution of
LRRK
that co-localized with microtubule markers (Fig 6A). Furthermore, LRRK2 mutant
protein
also formed large puncta in some of the neurons (Fig 6B). To identify the
nature of the
filaments and the puncta, these neurons were co-stained with different celluar
markers.
LRRK2 filaments was found to be co-localized with microtubule, as shown by co-
staining
with tubulin. Furthermore, both LRRK2 filaments and puncta co-localized with
autophagy
adaptor protein p62 (Fig 6A, 6B). Additional cellular marker for autophagy
were examined.
LRRK2 puncta were also positive for LC3 and K63 poly-ubiquitin, which are
markers for
autophagosomes. These data suggest that LRRK2 PD mutation impaired basal
autophagy
activity in neurons.
[0111] To test whether LRRK2 kinase activity and/or LRRK2 51292
autophosphorylation modulate the effect of LRRK2 PD mutations on autophagy,
LRRK2
R1441G/G20195/D1994A and LRRK2 R1441G/G20195/S1292A mutant proteins were
expressed in cultured cortical neurons. Loss of kinase activity (by D1994A)
abolished
LRRK2 localization to microtubules and to autophagosomes (Fig 6C, 6D),
indicating that
kinase activity is important for impaired autophagy activity caused by LRRK2
PD mutations.
Interestingly, Si 292A mutation substantially reduced LRRK2 localization to
microtubules,
but had little effect on the formation of LRRK2 positive autophagosomes (Fig
6D). These
data suggest that LRRK2 autophosphorylation on S1292 contributes to impaired
autophagy
activity induced by LRRK2 PD mutations.
LRRK2 kinase inhibitors block S1292 autophosphorylation in cells
[0112] To test whether LRRK2 kinase inhibitors can block LRRK2
autophosphorylation in cells, an inducible HEK293 cell line expressing FLAG
epitope-tagged
LRRK2 R1441G/Y1699C/ G20195 triple mutant protein (referred here as LRRK2
cells) was
generated. This cell lines was then used to develop a 96-well plate-based
assay to quantify
phospho-51292 in cell lysates. The assay used electrochemiluminescent
detection technology
(Meso-Scale Discovery, MSD) in which LRRK2 was captured with solid phase-
immobilized
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anti-FLAG monoclonal antibody, followed by detection with anti-pSer1292
antibody and a
Ruthenium-conjugated anti-rabbit secondary antibody. We were thus able detect
a significant
increase in pS1292 signal in lysates of cells induced with doxycycline (Fig.
7A). Further,
when doxycycline-induced LRRK2 cells were incubated with Sutent, a known LRRK2
kinase
inhibitor (Ki ref), we observed a dose-dependent reduction of pS1292 signal,
with maximal
inhibition equivalent to the signal detected in lysate from non-induced cells
(Fig. 7B).
Subsequently, we tested over 600 compounds with varying potencies in the LRRK2
kinase
assay on these cells. The IC5Os for reduction of pS1292 signal correlated
robustly with the
biochemical Ki of LRRK2 for the compounds (Fig. 7C). These data demonstrate
that LRRK2
kinase inhibitors block S1292 autophosphorylation in cells, indicating that
p51292 can
therefore be used as a marker for LRRK2 kinase inhibition in vivo.
A potent, selective and brain-penetrable LRRK2 kinase inhibitor blocks phospho-
S1292 in
LRRK2 G2019S BAC transgenic mouse brain
[0113] Through a high-throughput chemical screen and lead optimization,
we
identified a highly potent and selective LRRK2 small molecule inhibitor, [3-
methoxy-4-(4-
methylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-pheny1]-morpholin-4-yl-
methanone
(G1023). The biochemical Ki of G1023 is about 4nm. In LRRK2 cell-based assay,
G1023
efficiently reduced pLRRK2 levels in a dose-dependent manner, with observed
cellular IC50
at 1 OnM (Fig. 7B).
[0114] To test whether G1023 can inhibit LRRK2 kinase activity in vivo,
BAC
transgenic mice expressing LRRK2 G2019S protein were injected with a single
dose of this
compound at 10, 30 and 100mg/kg. Periphery and brain tissues were harvested 6
hrs after
injection to measure compound concentration as well as phospho-LRRK2 levels.
[0115] To examine LRRK2 kinase inhibition in G1023 injected animals,
spleen,
lymph node and brain tissue were harvested 6 hrs after compound injection to
determine
phospho-LRRK2 levels. In periphery tissues, including spleen and lymph node,
G1023
reduced phospho-S1292 in a dose-dependent manner (Fig 8A, 8C). In brain
tissue, G1023
also reduced phospho-S1292 level effectively, although the dosage required for
reduction is
higher (Fig 8B, 8C). The calculated unbound concentration of G1023 in the
brain required
for phospho-S1292 reduction is consistent with the cellular IC50 for this
compound. These
data demonstrate that G1023 has high brain exposure and blocks LRRK2 kinase
activity in
vivo. Furthermore, these data also demonstrate that LRRK2 autophosphorylation
on 5er1292
can be used as a pharmacodynamic marker to measure the efficacy of LRRK2
kinase
inhibitors in vivo.
LRRK2 kinase inhibitor attenuates Parkinson's mutation induced neurite
outgrowth defect.
[0116] To test whether inhibition of LRRK2 kinase activity ameliorates PD
mutations induced neuronal toxicity, we used cultured embryonic hippocampal
neurons
derived from LRRK2 BAC transgenic mice. These neurons showed neurite outgrowth
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defects as demonstrated by shorter total neurite length when compared with
hippocampal
neurons derived from non-transgenic littermates (Fig 9). We treated the
neuronal cultures
with LRRK2 kinase inhibitor G1023. We found that G1023 reversed neurite
outgrowth
defect of neurons derived from LRRK2 BAC transgenic mice in a dose-dependent
manner.
These data show that chemical inhibition of LRRK2 kinase activity can
functionally block
LRRK2 PD mutation induced neuronal toxicity.
S1292 is an in vivo LRRK2 autophosphorylation site
[0117] From the results disclosed herein, it can be seen that S1292 is an
in vivo
LRRK2 autophosphorylation site. Several lines of evidence support that LRRK2
kinase
activity is required for phosphorylation of S1292. First, the kinase- dead
mutations abolished
S1292 phosphorylation in in vitro autophosphrylation assay (Figure 1),
transfected
heterologous cells (Figure 2A), and tissues from BAC transgenic mice
overexpressing
LRRK2 (Figure 2B) Second, a highly specific LRRK2 inhibitor blocks S1292
phosphorylation in both a LRRK2 cell line (Figure 7B) and a LRRK2 transgenic
mice (Figure
8). Finally, many structurally diversified small molecule LRRK2 kinase
inhibitors blocks
S1292 phosphorylation in cell-based assay, providing chemical validation that
LRRK2 kinase
activity is required for S1292 phosphorylation in cells. Importantly, the
concentrations
required for pLRRK2 reduction are closely related to LRRK2 biochemical Ki of
the
compounds. Taken together, all the evidence suggests that S1292 is an in vivo
LRRK2
autophosphoryaltion site.
[0118] While many other LRRK2 autophosphorylation sites have been
reported
previously, none were detectable in cells or animal models, even in
overexpression situations.
S1292 autophosphorylation is readily detectable in lysates from transfected
cells and
transgenic animals. S1292 is the only LRRK2 autophosphorylation site yet
identified that
exists in significant amount in vivo. Identification of the S1292
autophosphorylation site
provides a powerful biomarker tool for monitoring LRRK2 kinase activity in
mammalian
subjects.
Familial Parkinson's mutations increase LRRK2 autophosphorylation on S1292
[0119] All the confirmed familial Parkinson's disease mutations of LRRK2,
including R1441G, Y1699C, G20195 and 12020T, increase S1292
autophosphorylation when
expressed in 293T cells. Of these familial mutations, only G2019S has been
concretely
shown to increase LRRK2 kinase activity in vitro. G2019S is located in kinase
activation
loop. It has been suggested that G2019S mutation may change the conformation
of the kinase
activation loop hence activate LRRK2 kinase. However, 12020T, which is also
near kinase
activation loop, was not consistently found to increase LRRK2 kinase activity
in vitro. One
possible explanation is that the substrates used in the biochemical assays
were not
endogenous substrates. Therefore phosphorylation of these proteins may not
accurately
represent in vivo LRRK2 kinase activity. Herein it is shown that both
mutations can increase
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S1292 phosphorylation in cell-based systems, suggesting that both PD mutations
in LRRK2
kinase domain increase its kinase activity. R1441G and Y1 699C mutations are
located in the
ROC-COR domain. Both mutations have been shown to reduce LRRK2 GTPase
activity.
GTP binding has been shown to be required for LRRK2 kinase activity in vitro.
This
disclosure demonstrates that GTP binding is required for S1292
autophosphorylation in cell-
based system (Figure. 5A). Therefore, reduction of GTPase activity caused by
R1441G and
Y1 699C mutation would result in more LRRK2 protein in GTP-bound state, hence
increase
S1292 autophosphorylation.
[0120] Notably, the confirmed PD risk factor mutation G2385R located in
WD40
domain, was not found to increase S1292 autophosphorylation. One simple
explanation could
be that G23 85R mutation may not affect LRRK2 kinase activity or
autophosphorylation.
Alternatively, heterologous expression of LRRK2 in 293 cells may not provide
the suitable
cellular environment for LRRK2 autophosphorylation. When comparing S1292
autophosphorylation level of G20195 mutation in vitro, in 293 cells and in
animal models, the
levels of increase were 3, 5 and 10, respectively. This suggests that in vivo
51292
autophosphorylation is higher than in vitro.
The role of S1292 autophosphorylation in LRRK2 PD mutation induced neuronal
toxicity
[0121] Given that increased 51292 autophosphorylation is a unifying theme
for all
the familial Parkinson's mutations, it is reasonable to hypothesize that 51292
autophosphorylation may be important for LRRK2 function and its role in PD
pathogenesis.
In cell-based LRRK2 models, Parkinson's mutations have been shown to cause
cellular
toxicity in both cell lines and primary neurons. Importantly, kinase activity
has been shown
to be required for the toxicity. The fact that expression of LRRK2
R1441G/G20195
mutations in embryonic hippocampal neurons reduces neurite length and
complexity confirms
the published results. Also consistent with the published data, kinase-dead
mutation was
found to ameliorate the neurite outgrowth phenotypes. Importantly, Si 292A
mutation also
reduces neurite outgrowth phenotypes. S1292 autophosphorylation may be part of
the
mechanisms downstream of LRRK2 kinase activity to induce cellular toxicity.
Notably,
51292 is located at the junction of LRR domain and ROC domain. Phosphorylation
of this
site may induce protein conformation change and result in change of protein-
protein
interaction.
[0122] The mechanisms through which LRRK2 PD mutations induce neuronal
toxicity remain unknown. LRRK2 proteins carrying PD mutations were found to
accumulate
on microtubules when expressed in neurons. This is consistent with results in
cell lines and
primary neurons. These aggregates are positive for selective autophagy
substrate p62
(SQSTM1). Accumulation of p62 positive aggregates is a characteristic of
impaired
autophagy function. Since autophagy cargos in neurons are thought to traffick
along the
microtubule network en route to lysosomes, accumulation of p62 on microtubules
may
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suggest impaired autophagy trafficking in neurons expressing LRRK2 PD
mutations. Since
LRRK2 was observed accumulating in large puncta that stain positive for p62,
LC3 and Lys-
63 poly-ubiquitin, autolysosomal degradation pathway may be impaired.
Autophagy is an
important "house keeping" function in differentiated neurons, while loss of
autophagy leads to
neurodegeneration and results in neurologic defects. This suggests that LRRK2
PD mutations
may cause cellular toxicity through autophagy impairment. Interestingly, since
the 51292A
mutation does not ameliorate appearance of LRRK2-positive autolysomes, 5er1292
phosphorylation may function upstream of autolysomal degradation. Importantly,
kinase-
dead mutation (Dl 994A) abolished both microtubule and autolysosome
accumulation of
LRRK2, suggesting that kinase activity is required for both. It remains likely
that substrates
of the LRRK2 remain to be identified.
LRRK2 autophosphorylation as a marker for in vivo LRRK2 kinase activity
[0123] S1292 autophosphorylation serves as a robust marker for in vivo
LRRK2
kinase activity. Furthermore, S1292 autophosphorylation can be easily detected
in tissue
lysates from LRRK2 transgenic animals. This provides a pharmacodynamic assay
to monitor
the knockdown of LRRK2 kinase activity in vivo. Taken together, S1292
autophosphorylation can be used to develop compound screening tools for LRRK2
kinase
inhibitors.
LRRK2 Modulators
[0124] LRRK2 modulators or inhibitors usable with the invention may
comprise
compounds of formula I or compounds of formula II.
[0125] In one embodiment, the LRRK2 modulators are of formula I:
R5
I
R1
X R6 NO
R2L
N )n
)L I
N N \
H (R7)m
R3 I
or pharmaceutically acceptable salts thereof,
wherein:
m is from 0 to 3;
X is: -N1r-; -0-; or -S(0),- wherein r is from 0 to 2 and Ra is hydrogen or
Ci_6alkyl;
R1 is: Ci_6alkyl; halo-Ci_6alkyl; C2_6alkenyl; C2_6alkynyl; halo-Ci_6alkyl;
Ci_6alkoxy-C1_
6alkyl; hydroxy-Ci_6alkyl; amino-Ci_6alkyl; Ci_6alkylsulfonyl-Ci_6alkyl;
C3_6cycloalkyl
optionally substituted with Ci_6alkyl or halo; C3_6cycloalkyl-Ci_6alkyl
wherein the C3_
6cycloalkyl portion is optionally substituted with Ci_6alkyl;
tetrahydropyranyl;
tetrahydrofuranyl; tetrahydrofuranyl-Ci_6alkyl; oxetanyl; or oxetan-Ci_6alkyl;
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or R1 and Ra together with the atoms to which they are attached may form a
three to
six membered ring that may optionally include an additional heteroatom
selected from 0, N
and S, and which is substituted with oxo, halo or Ci_6alkyl;
R2 is: halo; Ci_6alkoxy; cyano; C2_6alkynyl; C2_6alkenyl; halo-Ci_6alkyl; halo-
C1-
6alkoxy; C3_6cycloalkyl wherein the C3_6cycloalkyl portion is optionally
substituted with C1_
6alkYl; C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is
optionally substituted
with Ci_6alkyl; tetrahydrofuranyl; tetrahydrofuranyl-Ci_6alkyl; acetyl;
oxetanyl; or oxetan-C1_
6alkyl;
R3 is: -OW; halo; cyano; Ci_6alkyl; halo-Ci_6alkyl; C3_6cycloalkyl optionally
substituted with Ci_6alkyl; C3_6cycloalkyl-Ci_6alkyl wherein the
C3_6cycloalkyl portion is
optionally substituted with Ci_6alkyl; tetrahydrofuranyl; tetrahydrofuranyl-
Ci_6alkyl; oxetanyl;
or oxetan-Ci_6alkyl;
R4 is: hydrogen; Ci_6alkyl; halo-Ci_6alkyl; Ci_6alkoxy-Ci_6alkyl;
C3_6cycloalkyl
optionally substituted with Ci_6alkyl or halo; C3_6cycloalkyl-Ci_6alkyl
wherein the C3_
6cYcloalkyl portion is optionally substituted with Ci_6alkyl or halo;
tetrahydrofuranyl;
tetrahydrofuranyl-Ci_6alkyl; oxetanyl; or oxetan-Ci_6alkyl;
R5 is: hydrogen; or Ci_6alkyl;
n is 0 or 1;
R6 is: hydrogen; Ci_6alkyl; Ci_6alkoxy-Ci_6alkyl; hydroxy-Ci_6alkyl; amino-
Ci_6alkyl;
C3_6cycloalkyl; C3_6cycloalkyl-Ci_6alkyl; heterocyclyl; or heterocyclyl-
Ci_6alkyl; wherein the
C3_6cycloalkyl, C3_6cycloalkyl-Ci_6alkyl, heterocyclyl and heterocyclyl-
Ci_6alkyl each may be
optionally substituted with one, two, three or four groups groups
independently selected from:
Ci_6alkyl; halo-Ci_6alkyl; Ci_6alkoxy; halo-Ci_6alkoxy; hydroxy; hydroxy-
Ci_6alkyl; halo;
nitrite; Ci_6alkyl-carbonyl; Ci_6alkyl-sulfonyl; C3_6cycloalkyl;
C3_6cycloalkyl-Ci_6alkyl; C3_
6cycloalkyl-carbonyl; amino; or heterocyclyl; or two of the groups together
with the atoms to
which they are attached may form a five or six-membered ring;
or R5 and R6 together with the nitrogen atom to which they are attached form a
three-
to seven-membered ring that optionally includes an additional heteroatom
selected from 0, N
and S(0)õ, and which is optionally substituted with one, two, three or four
groups
independently selected from: Ci_6alkyl; halo-Ci_6alkyl; Ci_6alkoxy; halo-
Ci_6alkoxy; hydroxy;
Ci_6alkoxy-Ci_6alkyl; hydroxy-Ci_6alkyl; halo, nitrite; Ci_6alkyl-carbonyl;
Ci_6alkyl-sulfonyl;
C3_6cycloalkyl; C3_6cycloalkyl-Ci_6alkyl; C3_6cycloalkyl-carbonyl; amino; or
heterocyclyl; or
two of the groups together with the atoms to which they are attached may form
a five or six-
membered ring; and
R7 is: halo; Ci_6alkyl; Ci_6alkoxy; halo-Ci_6alkyl; or halo-Ci_6alkoxy.
[0126] In certain embodiments of formula I, n is 0.
[0127] In certain embodiments of formula I, n is 1.
101281 In certain embodiments of formula I, m is from 0 to 2.
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[0129] In certain embodiments of formula I, m is 0 or 1.
[0130] In certain embodiments of formula I, m is 0.
[0131] In certain embodiments of formula I, m is 1.
[0132] In certain embodiments of formula I, r is 0.
[0133] In certain embodiments of formula I, r is 2.
[0134] In certain embodiments of formula I, X is -N1r-or -0-.
[0135] In certain embodiments of formula I, X is -NRa.
[0136] In certain embodiments of formula I, X is -0-.
[0137] In certain embodiments of formula I, X is -S(0)õ-.
[0138] In certain embodiments of formula I, X is -NH-or -0-.
[0139] In certain embodiments of formula I, Ra is hydrogen.
[0140] In certain embodiments of formula I, Ra is Ci_6alkyl.
[0141] In certain embodiments of formula I, R1 is: Ci_6alkyl; halo-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; amino-Ci_6alkyl; Ci_6alkylsulfonyl-Ci_6alkyl;
C3_6cycloalkyl; or C3_
6CYCloalkyl-Ci_6alkyl.
[0142] In certain embodiments of formula I, R1 is: Ci_6alkyl;
C3_6cycloalkyl
optionally substituted with Ci_6alkyl; or C3_6cycloalkyl-Ci_6alkyl wherein the
C3_6cycloalkyl
portion is optionally substituted with Ci_6alkyl.
[0143] In certain embodiments of formula I, R1 is: Ci_6alkyl; halo-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; amino-Ci_6alkyl; Ci_6alkylsulfonyl-Ci_6alkyl;
tetrahydrofuranyl;
tetrahydrofuranyl-Ci_6alkyl; oxetanyl; or oxetan-Ci_6alkyl.
[0144] In certain embodiments of formula I, R1 is: Ci_6alkyl; halo-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; amino-Ci_6alkyl; or Ci_6alkylsulfonyl-Ci_6alkyl.
[0145] In certain embodiments of formula I, R1 is Ci_6alkyl.
[0146] In certain embodiments of formula I, R1 is halo-Ci_6alkyl.
[0147] In certain embodiments of formula I, R1 is Ci_6alkoxy-Ci_6alkyl.
[0148] In certain embodiments of formula I, R1 is amino-Ci_6alkyl.
[0149] In certain embodiments of formula I, R1 is Ci_6alkylsulfonyl-
Ci_6alkyl
optionally substituted with Ci_6alkyl.
[0150] In certain embodiments of formula I, R1 is C3_6cycloalkyl
optionally
substituted with Ci_6alkyl.
[0151] In certain embodiments of formula I, R1 is C3_6cycloalkyl-
Ci_6alkyl wherein
the C3_6cycloalkyl portion is optionally substituted with Ci_6alkyl.
[0152] In certain embodiments of formula I, R1 is tetrahydropyranyl.
[0153] In certain embodiments of formula I, R1 is tetrahydrofuranyl.
[0154] In certain embodiments of formula I, R1 is tetrahydrofuranyl-
Ci_6alkyl;
oxetanyl.
101551 In certain embodiments of formula I, R1 is or oxetan-Ci_6alkyl.
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[0156] In certain embodiments of formula I, R1 is: methyl; ethyl; n-
propyl;
isopropyl; isobutyl; 3,3-dimethylpropyl; cyclopropyl; cyclobutyl; cyclopentyl;
cyclohexyl;
cyclopropylmethyl; cyclobutylmethyl; cyclopentylmethyl; cyclopropylethyl;
methoxyethyl;
oxetanyl; or tetrahydrofuranylmethyl.
[0157] In certain embodiments of formula I, R1 is: methyl; ethyl; n-
propyl;
isopropyl; isobutyl; 3,3-dimethylpropyl; cyclopentyl; cyclohexyl;
cyclopropylmethyl;
cyclobutylmethyl; cyclopentylmethyl; cyclopropylethyl; methoxyethyl; oxetanyl;
or
tetrahydrofuranylmethyl.
[0158] In certain embodiments of formula I, R1 is: methyl; ethyl; n-
propyl;
isopropyl; isobutyl; 3,3-dimethylpropyl; cyclopentyl; cyclohexyl;
cyclopentylmethyl;
methoxyethyl; oxetanyl; or tetrahydrofuranylmethyl.
[0159] In certain embodiments of formula I, R1 is: methyl; ethyl; n-
propyl;
isopropyl; or isobutyl.
[0160] In certain embodiments of formula I, R1 is methyl or ethyl.
[0161] In certain embodiments of formula I, R1 is methyl.
[0162] In certain embodiments of formula I, R1 is ethyl.
[0163] In certain embodiments of formula I, R1 is: cyclopropyl;
cyclobutyl;
cyclopentyl; cyclohexyl; cyclopropylmethyl; cyclobutylmethyl;
cyclopentylmethyl; or
cyclopropylethyl.
[0164] In certain embodiments of formula I, R1 is: cyclopentyl;
cyclohexyl; or
cyclopentylmethyl.
[0165] In certain embodiments of formula I, R2 is: halo; Ci_6alkoxy; halo-
Ci_6alkyl;
halo-Ci_6alkoxy; C3_6cycloalkyl wherein the C3_6cycloalkyl portion is
optionally substituted
with Ci_6alkyl; C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is
optionally
substituted with Ci_6alkyl; tetrahydrofuranyl; tetrahydrofuranyl-Ci_6alkyl;
oxetanyl; or oxetan-
Ci_6alkyl.
[0166] In certain embodiments of formula I, R2 is: halo; Ci_6alkoxy; halo-
Ci_6alkyl;
cyano; C2_6alkynyl; C2_6alkenyl; C3_6cycloalkyl; or C3_6cycloalkyl-Ci_6alkyl.
[0167] In certain embodiments of formula I, R2 is: halo; Ci_6alkoxy; halo-
Ci_6alkyl;
cyano; C3_6cycloalkyl; or C3_6cycloalkyl-Ci_6alkyl.
[0168] In certain embodiments of formula I, R2 is: halo; Ci_6alkoxy; halo-
Ci_6alkyl;
C3_6cycloalkyl; or C3_6cycloalkyl-Ci_6alkyl.
[0169] In certain embodiments of formula I, R2 is: halo; halo-Ci_6alkyl;
or cyano.
[0170] In certain embodiments of formula I, R2 is: halo; or halo-
Ci_6alkyl.
[0171] In certain embodiments of formula I, R2 is halo.
[0172] In certain embodiments of formula I, R2 is Ci_6alkoxy.
[0173] In certain embodiments of formula I, R2 is halo-Ci_6alkoxy.
101741 In certain embodiments of formula I, R2 is halo-Ci_6alkyl.
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[0175] In certain embodiments of formula I, R2 is C3_6cycloalkyl.
[0176] In certain embodiments of formula I, R2 is C3_6cycloalkyl-
Ci_6alkyl.
[0177] In certain embodiments of formula I, R2 is tetrahydrofuranyl.
[0178] In certain embodiments of formula I, R2 is tetrahydrofuranyl-
Ci_6alkyl.
[0179] In certain embodiments of formula I, R2 is oxetanyl.
[0180] In certain embodiments of formula I, R2 is oxetan-Ci_6alkyl.
[0181] In certain embodiments of formula I, R2 is halo, trifluoromethyl
or cyano.
[0182] In certain embodiments of formula I, R2 is chloro, trifluoromethyl
or cyano.
[0183] In certain embodiments of formula I, R2 is fluoro, chloro or
bromo.
[0184] In certain embodiments of formula I, R2 is chloro.
[0185] In certain embodiments of formula I, R2 is fluoro.
[0186] In certain embodiments of formula I, R2 is bromo.
[0187] In certain embodiments of formula I, R2 is trifluoromethyl.
[0188] In certain embodiments of formula I, R2 is methoxy.
[0189] In certain embodiments of formula I, R2 is cyano.
[0190] In certain embodiments of formula I, R2 is C2_6alkynyl.
[0191] In certain embodiments of formula I, R2 is C2_6alkenyl.
[0192] In certain embodiments of formula I, R3 is -OW.
[0193] In certain embodiments of formula I, R3 is: Ci_6alkyl; or halo-
Ci_6alkyl.
[0194] In certain embodiments of formula I, R3 is: halo; or -OW.
[0195] In certain embodiments of formula I, R3 is: halo; Ci_6alkoxy; halo-
Ci_6alkoxy;
C3_6cycloalkyloxy; or C3_6cycloalkyl-Ci_6alkyloxy.
[0196] In certain embodiments of formula I, R3 is: Ci_6alkoxy; halo-
Ci_6alkoxy; C3_
6CYClOalkylOXY; or C3_6cycloalkyl-Ci_6alkyloxy.
[0197] In certain embodiments of formula I, R3 is: halo; Ci_6alkoxy;
cyano; or halo-
Ci_6alkoxy.
[0198] In certain embodiments of formula I, R3 is: halo; Ci_6alkoxy; or
halo-C1-
6alkoxy.
[0199] In certain embodiments of formula I, R3 is: methoxy; halo;
trifluoromethoxy;
difluoromethoxy; 2-halo-ethoxy or 2,2,2-trihaloethoxy.
[0200] In certain embodiments of formula I, R3 is: methoxy; or halo.
[0201] In certain embodiments of formula I, R3 is: methoxy; chloro; or
fluoro.
[0202] In certain embodiments of formula I, R3 is methoxy.
[0203] In certain embodiments of formula I, R3 is chloro.
[0204] In certain embodiments of formula I, R3 is fluoro.
[0205] In certain embodiments of formula I, R3 is: Ci_6alkoxy; cyano; or
halo-C1_
6alkoxy.
102061 In certain embodiments of formula I, R3 is: Ci_6alkoxy; or halo-
Ci_6alkoxy.
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[0207] In certain embodiments of formula I, R3 is Ci_6alkoxy.
[0208] In certain embodiments of formula I, R3 is methoxy.
[0209] In certain embodiments of formula I, R3 is cyano.
[0210] In certain embodiments of formula I, R3 is C3_6cycloalkyl.
[0211] In certain embodiments of formula I, R3 is C3_6cycloalkyl-
Ci_6alkyl.
[0212] In certain embodiments of formula I, R3 is tetrahydrofuranyl. In
certain
embodiments of formula I, R3 is tetrahydrofuranyl-Ci_6alkyl.
[0213] In certain embodiments of formula I, R3 is oxetanyl.
[0214] In certain embodiments of formula I, R3 is oxetan-Ci_6alkyl.
[0215] In certain embodiments of formula I, R4 is: Ci_6alkyl; halo-
Ci_6alkyl; CI_
6alkoxy-Ci_6alkyl; C3_6cycloalkyl optionally substituted with Ci_6alkyl;
C3_6cycloalkyl-C1_
6alkyl wherein the C3_6cycloalkyl portion is optionally substituted with
Ci_6alkyl;
tetrahydrofuranyl; tetrahydrofuranyl-Ci_6alkyl; oxetanyl; or oxetan-Ci_6alkyl;
[0216] In certain embodiments of formula I, R4 is: Ci_6alkyl; halo-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; C3_6cycloalkyl; or C3_6cycloalkyl-Ci_6alkyl.
[0217] In certain embodiments of formula I, R4 is: Ci_6alkyl; halo-
Ci_6alkyl; or C3_
6CYC loalkyl.
[0218] In certain embodiments of formula I, R4 is Ci_6alkyl.
[0219] In certain embodiments of formula I, R4 is halo-Ci_6alkyl.
[0220] In certain embodiments of formula I, R4 is Ci_6alkoxy-Ci_6alkyl.
[0221] In certain embodiments of formula I, R4 is C3_6cycloalkyl.
[0222] In certain embodiments of formula I, R4 is C3_6cycloalkyl-
Ci_6alkyl.
[0223] In certain embodiments of formula I, R4 is tetrahydrofuranyl.
[0224] In certain embodiments of formula I, R4 is tetrahydrofuranyl-
Ci_6alkyl.
[0225] In certain embodiments of formula I, R4 is oxetanyl.
[0226] In certain embodiments of formula I, R4 is or oxetan-Ci_6alkyl.
[0227] In certain embodiments of formula I, R4 is: methyl; ethyl;
isopropyl;
cyclopropyl; cyclobutyl; cyclopropylmethyl; cyclobutylmethyl; 2-haloethyl; or
2,2,2-
trihaloethyl.
[0228]
[0229] In certain embodiments of formula I, R4 is methyl.
[0230] In certain embodiments of formula I, R5 is hydrogen.
[0231] In certain embodiments of formula I, R5 is Ci_6alkyl.
[0232] In certain embodiments of formula I, R5 is methyl.
[0233] In certain embodiments of formula I, R5 is ethyl.
[0234] In certain embodiments of formula I, R6 is hydrogen.
[0235] In certain embodiments of formula I, R6 is Ci_6alkyl.
102361 In certain embodiments of formula I, R6 is Ci_6alkoxy-Ci_6alkyl.
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[0237] In certain embodiments of formula I, R6 is hydroxy-Ci_6alkyl.
[0238] In certain embodiments of formula I, R6 is amino-Ci_6alkyl.
[0239] In certain embodiments of formula I, R6 is C3_6cycloalkyl
optionally
substituted with one, two or three groups independently selected from:
Ci_6alkyl; halo-C1_
6alkyl; Ci_6alkoxy; halo-Ci_6alkoxy; hydroxy; hydroxy-Ci_6alkyl; halo;
nitrite; Ci_6alkyl-
carbonyl; Ci_6alkyl-sulfonyl; C3_6cycloalkyl; C3_6cycloalkyl-Ci_6alkyl;
C3_6cycloalkyl-carbonyl;
amino; or heterocyclyl; or two of the groups together with the atoms to which
they are
attached may form a five or six-membered ring.
[0240] In certain embodiments of formula I, R6 is C3_6cycloalkyl-
Ci_6alkyl wherein
the C3_6cycloalkyl portion thereof is optionally substituted with one, two or
three groups
independently selected from: Ci_6alkyl; halo-Ci_6alkyl; Ci_6alkoxy; halo-
Ci_6alkoxy; hydroxy;
hydroxy-Ci_6alkyl; halo; nitrite; Ci_6alkyl-carbonyl; Ci_6alkyl-sulfonyl;
C3_6cycloalkyl; C3_
6cYcloalkyl-Ci_6alkyl; C3_6cycloalkyl-carbonyl; amino; or heterocyclyl; or two
of the groups
together with the atoms to which they are attached may form a five or six-
membered ring.
[0241] In embodiments of formula I wherein R6 is heterocyclyl, such
heterocycle
may be: azetidinyl; pyrrolidinyl; piperidinyl; piperazinyl; morpholinyl;
thiomorpholinyl; 3-
oxa-8-aza-bicyclo[3.2.1]oct-8-y1; 2-oxa-5-aza-bicyclo[2.2.1]hept-5-y1; or 8-
oxa-3-aza-
bicyclo[3.2.1]oct-3-y1; each optionally substituted as defined herein.
[0242] In embodiments of formula I wherein R6 is heterocyclyl, such
heterocycle
may be: azetidinyl; pyrrolidinyl; piperidinyl; piperazinyl; or morpholinyl;
each optionally
substituted as defined herein, i.e., such heterocycyl is optionally
substituted with one, two or
three groups independently selected from: Ci_6alkyl; halo-Ci_6alkyl;
Ci_6alkoxy; halo-C1_
6alkoxy; hydroxy; hydroxy-Ci_6alkyl; halo; nitrite; Ci_6alkyl-carbonyl;
Ci_6alkyl-sulfonyl; C3_
6cycloalkyl; C3_6cycloalkyl-Ci_6alkyl; C3_6cycloalkyl-carbonyl; amino; or
heterocyclyl; or two
of the groups together with the atoms to which they are attached may form a
five or six-
membered ring.
[0243] In certain embodiments of formula I, R6 is heterocyclyl optionally
substituted
with one, two or three groups independently selected from: Ci_6alkyl; halo-
Ci_6alkyl; C1_
6alkoxy; halo-Ci_6alkoxy; hydroxy; hydroxy-Ci_6alkyl; halo; nitrite; Ci_6alkyl-
carbonyl; C1-
6alkyl-sulfonyl; C3_6cycloalkyl; C3_6cycloalkyl-Ci_6alkyl; C3_6cycloalkyl-
carbonyl; amino; or
heterocyclyl; or two of the groups together with the atoms to which they are
attached may
form a five or six-membered ring.
[0244] In embodiments of formula I wherein R6 is heterocyclyl-Ci_6alkyl,
the
heterocyclyl portion thereof may be: azetidinyl; pyrrolidinyl; piperidinyl;
piperazinyl;
morpholinyl; thiomorpholinyl; 3-oxa-8-aza-bicyclo[3.2.1]oct-8-y1; 2-oxa-5-aza-
bicyclo[2.2.1]hept-5-y1; or 8-oxa-3-aza-bicyclo[3.2.1]oct-3-y1; each
optionally substituted as
defined herein, i.e., such heterocycyl portion is optionally substituted with
one, two or three
groups independently selected from: Ci_6alkyl; halo-Ci_6alkyl; Ci_6alkoxy;
halo-Ci_6alkoxy;
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hydroxy; hydroxy-Ci_6alkyl; halo; nitrite; Ci_6alkyl-carbonyl; Ci_6alkyl-
sulfonyl; C3_
6cycloalkyl; C3_6cycloalkyl-Ci_6alkyl; C3_6cycloalkyl-carbonyl; amino; or
heterocyclyl; or two
of the groups together with the atoms to which they are attached may form a
five or six-
membered ring..
[0245] In embodiments of formula I wherein R6 is heterocyclyl-Ci_6alkyl,
the
heterocyclyl portion thereof may be: azetidinyl; pyrrolidinyl; piperidinyl;
piperazinyl; or
morpholinyl; each optionally substituted as defined herein.
[0246] In certain embodiments of formula I, R6 is heterocyclyl-Ci_6alkyl
wherein the
heterocyclyl portion thereof is optionally substituted with one, two or three
groups
independently selected from: Ci_6alkyl; halo-Ci_6alkyl; Ci_6alkoxy; halo-
Ci_6alkoxy; hydroxy;
hydroxy-Ci_6alkyl; halo; nitrite; Ci_6alkyl-carbonyl; Ci_6alkyl-sulfonyl;
C3_6cycloalkyl; C3_
6cycloalkyl-Ci_6alkyl; C3_6cycloalkyl-carbonyl; amino; or heterocyclyl; or two
of the groups
together with the atoms to which they are attached may form a five or six-
membered ring.
[0247] In certain embodiments of formula I, R6 is: hydrogen; methyl;
ethyl;
isopropyl; or cyclopropyl.
[0248] In certain embodiments of formula I, R6 is: hydrogen; methyl;
ethyl;
isopropy1;2-amino-propyl; oxetan-3-y1; 2-methoxy-ethyl; 2-hydroxy-ethyl;
cyclopropyl;
piperidin-4-y1; 1-methyl-piperidin-4-y1; tert-butl; 2-hydroxy-2-methyl-propyl;
cyclobutyl; 1-
methyl-cyclobutyl; 2-hydroxy-propyl; 1-cyano-cyclopropyl; 3,3-difluoro-
cyclobutyl;
cyclopropylmethyl; 3-fluoro-cyclobutyl; or 2,2-difluoroethyl;
[0249] In certain embodiments of formula I, R6 is hydrogen.
[0250] In certain embodiments of formula I, R6 is methyl.
[0251] In certain embodiments of formula I, R6 is ethyl.
[0252] In certain embodiments of formula I, R6 is isopropyl.
[0253] In certain embodiments of formula I, R6 is 2-amino-propyl.
[0254] In certain embodiments of formula I, R6 is oxetan-3-yl.
[0255] In certain embodiments of formula I, R6 is 2-methoxy-ethyl.
[0256] In certain embodiments of formula I, R6 is 2-hydroxy-ethyl.
[0257] In certain embodiments of formula I, R6 is cyclopropyl.
[0258] In certain embodiments of formula I, R6 is piperidin-4-yl.
[0259] In certain embodiments of formula I, R6 is 1-methyl-piperidin-4-
yl.
[0260] In certain embodiments of formula I, R6 is tert-butl.
[0261] In certain embodiments of formula I, R6 is 2-hydroxy-2-methyl-
propyl.
[0262] In certain embodiments of formula I, R6 is cyclobutyl.
[0263] In certain embodiments of formula I, R6 is 1-methyl-cyclobutyl.
[0264] In certain embodiments of formula I, R6 is 2-hydroxy-propyl.
[0265] In certain embodiments of formula I, R6 is 1-cyano-cyclopropyl.
102661 In certain embodiments of formula I, R6 is 3,3-difluoro-
cyclobutyl.
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[0267] In certain embodiments of formula I, R6 is cyclopropylmethyl.
[0268] In certain embodiments of formula I, R6 is 3 -fluoro-cyclobutyl.
[0269] In certain embodiments of formula I, R6 is 2,2-difluoroethyl.
[0270] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form a three- to seven-membered ring that is
optionally
includes an additional heteroatom selected from 0, N and S(0)õ, and which is
optionally
substituted with one, two or three groups independently selected from
Ci_6alkyl, halo-C1_
6alkyl, Ci_6alkoxy, halo-C 1_6 alkoxy, hydroxy, hydroxy-Ci_6alkyl, halo,
nitrite, Ci_6alkyl-
carbonyl, Ci_6alkyl-sulfonyl, C3_6cycloalkyl, C3_6cycloalkyl-Ci_6alkyl,
C3_6cycloalkyl-carbonyl,
or heterocyclyl, or two of the groups together with the atoms to which they
are attached may
form a five or six-membered ring.
[0271] In embodiments of formula I wherein R5 and R6 together with the
nitrogen
atom to which they are attached form a three- to seven-membered ring that is
optionally
includes an additional heteroatom selected from 0, N and S(0)õ, such ring may
be: azetidinyl;
pyrrolidinyl; piperidinyl; piperazinyl; morpholinyl; thiomorpholinyl;
azepinyl; 3-oxa-8-aza-
bicyclo[3.2.1]oct-8-y1; 2-oxa-5-aza-bicyclo[2.2.1]hept-5-y1; or 8-oxa-3-aza-
bicyclo[3.2.1]oct-
3-y1; each optionally substituted as defined herein.
[0272] In embodiments of formula I wherein R5 and R6 together with the
nitrogen
atom to which they are attached form a three- to seven-membered ring that is
optionally
includes an additional heteroatom selected from 0, N and S(0)õ, such ring may
be: azetidinyl;
pyrrolidinyl; piperidinyl; piperazinyl; or morpholinyl; each optionally
substituted as defined
herein.
[0273] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form a morpholinyl group that is optionally
substituted once
or twice with groups independently selected from Ci_6alkyl, halo-Ci_6alkyl,
Ci_6alkoxy, halo-
Ci_6alkoxy, hydroxy, hydroxy-Ci_6alkyl, halo, nitrite, Ci_6alkyl-carbonyl,
Ci_6alkyl-sulfonyl,
C3_6cycloalkyl, C3_6cycloalkyl-Ci_6alkyl, C3_6cycloalkyl-carbonyl, amino, or
heterocyclyl, or
the two groups together with the atoms to which they are attached may form a
five or six-
membered ring.
[0274] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form a piperidinyl group that is optionally
substituted once or
twice with groups independently selected from Ci_6alkyl, halo-Ci_6alkyl,
Ci_6alkoxy, halo-C1_
6alkoxy, hydroxy, hydroxy-Ci_6alkyl, halo, nitrite, Ci_6alkyl-carbonyl,
Ci_6alkyl-sulfonyl, C3_
6cycloalkyl, C3_6cycloalkyl-Ci_6alkyl, C3_6cycloalkyl-carbonyl, amino, or
heterocyclyl, or the
two groups together with the atoms to which they are attached may form a five
or six-
membered ring.
[0275] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form a piperazinyl group that is optionally
substituted once or
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twice with groups independently selected from Ci_6alkyl, halo-Ci_6alkyl,
Ci_6alkoxy, halo-C1_
6alkoxy, hydroxy, hydroxy-Ci_6alkyl, halo, nitrite, Ci_6alkyl-carbonyl,
Ci_6alkyl-sulfonyl, C3_
6cycloalkyl, C3_6cycloalkyl-Ci_6alkyl, C3_6cycloalkyl-carbonyl, amino, or
heterocyclyl, or the
two groups together with the atoms to which they are attached may form a five
or six-
membered ring.
[0276] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form a pyrrolidinyl group that is optionally
substituted once
or twice with groups independently selected from Ci_6alkyl, halo-Ci_6alkyl,
Ci_6alkoxy, halo-
Ci_6alkoxy, hydroxy, hydroxy-Ci_6alkyl, halo, nitrite, Ci_6alkyl-carbonyl,
Ci_6alkyl-sulfonyl,
C3_6cycloalkyl, C3_6cycloalkyl-Ci_6alkyl, C3_6cycloalkyl-carbonyl, amino, or
heterocyclyl, or
the two groups together with the atoms to which they are attached may form a
five or six-
membered ring.
[0277] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form a group selected from: morpholin-4-y1; 4-
hydroxy-
pip eridin- 1 -yl; octahydro-pyrido [ 1 ,2-a] pyrazin-2-y1; 2-hydroxy-pip
eridin- 1 -yl; 4,4-dimethyl-
pip eridin- 1 -yl; 3 ,5 -dimethyl-pip eridin- 1 -yl; 1 -hydroxy-1 -methyl-
ethyl)-p ip eridin- 1 -yl; 3 -
hydroxy-pyrro lidin- 1 -yl; 4-methyl-pip eridin- 1 -yl; pip eridin- 1 -yl;
azetidin-1 -yl; 4,4-difluoro-
pip eridin- 1 -yl; 3 -methyl-pip eridin- 1 -yl; 4 -methoxy-pip eridin- 1 -yl;
3,3 -difluoro-pip eridin- 1 -
y1; 4-cyano -pip eridin-1 -yl; 4-fluoro-piperidin-1 -yl; 3 -methoxy-pip eridin-
1 -yl; 4- ethyl-
p ip erazin- 1 -yl; 4-acetyl-pip erazin- 1 -yl; 3 -trifluoromethyl-pip eridin-
1 -yl; 4-tert-butyl-
pip eridin-1 -yl; 2-hydroxy-ethyl)-pip erazin- 1 -yl; 2-methyl-pyrro lidin- 1 -
yl; 4-hydroxymethyl-
pip eridin-1 -yl; 2-methyl-pip eridin-1 -yl; pyrro lidin- 1 -yl; 4-
methanesulfonyl-pip erazin- 1 -yl; 3 -
trifluoromethyl-pyrro lidin- 1 -yl; 4-(2,2,2-trifluoro-ethyl)-pip erazin- 1 -
yl; 2 -methyl-morpho lin-
4-y1; (2,6-dimethyl-morpholin-4-y1; 2,2-diethyl-morpholin-4-y1; 3-
hydroxymethyl-morpholin-
4-y1; 2-isobutyl-morpholin-4-y1; 2-hydroxymethyl-morpholin-4-y1; 3,3-dimethyl-
morpholin-
4-y1; 4-methyl-pip erazin-1 -yl; 4-is opropyl-pip erazin-1 -yl; pip erazin- 1 -
yl; 3 -oxa-8 -aza-
bicyclo[3.2.1]oct-8-y1; (S)-3-methyl-morpholin-4-y1; 2-oxa-5-aza-
bicyclo[2.2.1]hept-5-y1; 8-
oxa-3 -aza-bicyc to [3 .2.1 ] oct-3 -yl; (R)-3 -methyl-morpho lin-4 -yl; 4 -
cyc loprop anecarb onyl-
pip erazin-1 -yl; 4-( 1 -hydroxy- 1 -methyl- ethyl)-pip eridin-1 -yl; 4-cyc
lobutyl-pip erazin-1 -yl;
(R)-3 -hydroxy-pyrro lidin- 1 -yl; 4 -oxetan-3 -yl-pip erazin- 1 -yl; 3 -
morpho lin-4-yl-azetidin- 1 -yl;
4-( 1 -methyl-pip eridin-4 -y1)-pip erazin- 1 -yl; 3,3 -difluoro-azetidin- 1 -
yl; 4-dimethylamino-
pip eridin-1 -yl; 4-pip eridin-4-yl-pip erazin-1 -yl; (4,4-difluoro-pip eridin-
1 -yl; (3 -morpho lin-4-
yl-azetidin-l-y1; 2-oxa-6-aza-spiro[3.3]hept-6-y1; 2-oxa-5-aza-
bicyclo[2.2.1]hept-5-y1); 4-
methoxy-piperidin-1-y1); [1,4]oxazepan-4-y1; 2R,6S)-2,6-dimethyl-morpholin-4-
y1; 3-
hydroxy-azetidin- 1 -yl; 3 -cyano-pyrro lidin- 1 -yl; 3 ,5 -dimethyl-pip
erazin- 1 -yl; (3 R,5 S)-
dimethyl-pip erazin-1 -yl; 3 -Fluoro-pyrro lidin- 1 -yl; (S)-3 -F luoro-pyrro
lidin- 1 -yl; pip erazin-1 -
y1; 3,3-Difluoro-pyrrolidin-1-y1; 3,3-Difluoro-azetidin-1-y1; 2,2,6,6-
tetrafluoro-morpholin-4-
yl; 2-methoxymethyl-pyrro lidin- 1 -yl; (S)-2-methoxymethyl-pyrro lidin- 1 -
yl; (1 S ,4 S)-2- oxa-5 -
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azabicyclo [2.2.1 ]heptan-5 -yl; (3 S,4 S)-3 ,4-difluoropyrro lidin- 1 -yl; 3
,4-di fluoropyrro li din- 1-yl;
and 3 -methoxypyrro lidin- 1-yl.
[0278] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form a group selected from: morpholin-4-y1; 4-
hydroxy-
pip eridin- 1 -yl; octahydro-pyrido [1 ,2- a] pyrazin-2-y1; 2-hydroxy-pip
eridin- 1 -yl; 4,4- dimethyl-
p ip eridin- 1 -yl; 3 ,5 - dimethyl-pip eridin- 1 -yl; 1 -hydroxy- 1 -methyl-
ethyl)-pip eridin- 1 -yl; 3 -
hydroxy-pyrro lidin-1 -yl; 4 -methyl-p ip eridin- 1 -yl; pip eridin- 1 -yl;
azetidin-1 -yl; 4,4-difluoro-
pip eridin- 1 -yl; 3 -methyl-pip eridin- 1 -yl; 4 -methoxy-p ip eridin- 1 -yl;
3,3 - difluoro-pip eridin- 1 -
y1; 4-cyano -pip eridin-1 -yl; 4- fluoro-pip eridin-1 -yl; 3 -methoxy-pip
eridin- 1 -yl; 4- ethyl-
p ip erazin- 1 -yl; 4-acetyl-pip erazin- 1 -yl; 3 -trifluoromethyl-pip eridin-
1 -yl; 4-tert-butyl-
pip eridin-1 -yl; 2-hydroxy-ethyl)-pip erazin- 1 -yl; 2-methyl-pyrro lidin- 1 -
yl; 4-hydroxymethyl-
pip eridin-1 -yl; 2-methyl-pip eridin-1 -yl; pyrro lidin- 1 -yl; 4-
methanesulfonyl-pip erazin- 1 -yl; 3 -
trifluoromethyl-pyrro lidin- 1 -yl; 4-(2,2,2-trifluoro- ethyl)-pip erazin- 1 -
yl; 2 -methyl-morpho lin-
4-y1; (2,6-dimethyl-morpholin-4-y1; 2,2-diethyl-morpholin-4-y1; 3 -
hydroxymethyl-morpholin-
4-y1; 2-isobutyl-morpholin-4-y1; 2-hydroxymethyl-morpholin-4-y1; 3,3-dimethyl-
morpholin-
4-y1; 4-methyl-piperazin-1-y1; 4-isopropyl-piperazin-1-y1; piperazin-l-y1; 3-
oxa-8-aza-
bicyclo[3.2.1]oct-8-y1; (S)-3-methyl-morpholin-4-y1; 2-oxa-5-aza-
bicyclo[2.2.1]hept-5-y1; 8-
oxa-3 -aza-bicyclo [3 .2.1 ] oct-3 -yl; (R)-3 -methyl-morpho lin-4 -yl; 4 -cyc
loprop anecarb onyl-
pip erazin-1 -yl; 4-( 1 -hydroxy- 1 -methyl- ethyl)-pip eridin-1 -yl; 4-cyc
lobutyl-pip erazin-1 -yl;
(R)-3 -hydroxy-pyrro lidin- 1 -yl; 4 -oxetan-3 -yl-pip erazin- 1 -yl; 3 -
morpho lin-4-yl- azetidin- 1 -yl;
4-( 1 -methyl-pip eridin-4 -y1)-pip erazin- 1 -yl; 3,3 - difluoro- azetidin- 1
-yl; 4- dimethylamino-
p ip eridin-l-y1; and 4-piperidin-4-yl-piperazin-1-yl.
[0279] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form morpholin-4-yl.
[0280] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-hydroxy-piperidin-1-yl.
[0281] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form octahydro-pyrido[1,2-a]pyrazin-2-yl.
[0282] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-hydroxy-piperidin-1-yl.
[0283] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4,4-dimethyl-piperidin-1-yl.
[0284] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3,5-dimethyl-piperidin-1-yl.
[0285] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 1-hydroxy-1-methyl-ethyl)-piperidin-1-yl.
[0286] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-hydroxy-pyrrolidin-1 -yl.
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[0287] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-methyl-piperidin-1-yl.
[0288] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form piperidin-l-yl.
[0289] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form azetidin-l-yl.
[0290] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4,4-difluoro-piperidin-1-yl.
[0291] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-methyl-piperidin-1-yl.
[0292] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-methoxy-piperidin-1-yl.
[0293] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3,3-difluoro-piperidin-1-yl.
[0294] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-cyano-piperidin-1-yl.
[0295] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-fluoro-piperidin-1-yl.
[0296] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-methoxy-piperidin-1-yl.
[0297] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-ethyl-piperazin-1-yl.
[0298] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-acetyl-piperazin-1-yl.
[0299] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-trifluoromethyl-piperidin-1-yl.
[0300] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-tert-butyl-piperidin-1-yl.
[0301] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form.
[0302] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-hydroxy-ethyl)-piperazin-1-yl.
[0303] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-methyl-pyrrolidin-1-yl.
[0304] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-hydroxymethyl-piperidin-1-yl.
[0305] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-methyl-piperidin-1-yl.
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[0306] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form pyrrolidin-l-yl.
[0307] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-methanesulfonyl-piperazin-1-yl.
[0308] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-trifluoromethyl-pyrrolidin-1-yl.
[0309] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-(2,2,2-trifluoro-ethyl)-piperazin-1-yl.
[0310] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-methyl-morpholin-4-yl.
[0311] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (2,6-dimethyl-morpholin-4-yl.
[0312] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2,2-diethyl-morpholin-4-yl.
[0313] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-hydroxymethyl-morpholin-4-yl.
[0314] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-isobutyl-morpholin-4-yl.
[0315] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-hydroxymethyl-morpholin-4-yl.
[0316] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3,3-dimethyl-morpholin-4-yl.
[0317] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-methyl-piperazin-1-yl.
[0318] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-isopropyl-piperazin-1-yl.
[0319] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form piperazin-l-yl.
[0320] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-oxa-8-aza-bicyclo[3.2.1]oct-8-yl.
[0321] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (S)-3-methyl-morpholin-4-yl.
[0322] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl.
[0323] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl.
[0324] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (R)-3-methyl-morpholin-4-yl.
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[0325] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-cyclopropanecarbonyl-piperazin-1-yl.
[0326] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-(1-hydroxy-1-methyl-ethyl)-piperidin-1-
yl.
[0327] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-cyclobutyl-piperazin-1-yl.
[0328] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (R)-3-hydroxy-pyrrolidin-1-yl.
[0329] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-oxetan-3-yl-piperazin-1-yl.
[0330] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-morpholin-4-yl-azetidin-1 -yl.
[0331] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-(1-methyl-piperidin-4-y1)-piperazin-1-
yl.
[0332] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3,3-difluoro-azetidin-1-yl.
[0333] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-dimethylamino-piperidin-1-yl.
[0334] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 4-piperidin-4-yl-piperazin-1-yl.
[0335] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (4,4-difluoro-piperidin-1 -yl.
[0336] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (3-morpholin-4-yl-azetidin-1-yl.
[0337] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-oxa-6-aza-spiro[3.3]hept-6-yl.
[0338] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-oxa-5-aza-bicyclo[2.2.1]hept-5-y1),In
certain
embodiments of formula I, R5 and R6 together with the nitrogen atom to which
they are
attached form 4-methoxy-piperidin-1-y1).
[0339] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form [1,4]oxazepan-4-yl.
[0340] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2R,6S)-2,6-dimethyl-morpholin-4-yl.
[0341] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-hydroxy-azetidin-1 -yl.
[0342] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-cyano-pyrrolidin-1-yl.
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[0343] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3,5-dimethyl-piperazin-1-yl.
[0344] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (3R,5S)-dimethyl-piperazin-1-yl.
[0345] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3-Fluoro-pyrrolidin-1-yl.
[0346] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (S)-3-Fluoro-pyrrolidin-1-yl.
[0347] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form piperazin-l-yl.
[0348] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3,3-difluoro-pyrrolidin-1-yl.
[0349] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 3,3-difluoro-azetidin-1-yl.
[0350] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2,2,6,6-tetrafluoro-morpholin-4-yl.
[0351] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form 2-methoxymethyl-pyrrolidin-1-ylln certain
embodiments of formula I, R5 and R6 together with the nitrogen atom to which
they are
attached form (S)-2-methoxymethyl-pyrrolidin-1-yl.
[0352] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-
yl.
[0353] In certain embodiments of formula I, R5 and R6 together with the
nitrogen
atom to which they are attached form (3S,4S)-3,4-difluoropyrrolidin-1-y1; 3,4-
difluoropyrrolidin-1-y1; and 3-methoxypyrrolidin-1-yl.
[0354] In certain embodiments of formula I, R7 is halo.
[0355] In certain embodiments of formula I, R7 is Ci_6alkyl.
[0356] In certain embodiments of formula I, R7 is Ci_6alkoxy.
[0357] In certain embodiments of formula I, R7 is halo-Ci_6alkyl.
[0358] In certain embodiments of formula I, R7 is halo-Ci_6alkoxy.
[0359] In certain embodiments of formula I, R7 is halo or methoxy.
[0360] In certain embodiments of formula I, R7 is fluoro, chloro or
methoxy.
[0361] In certain embodiments of formula I, R7 is fluoro or chloro.
[0362] In certain embodiments of formula I, R7 is methoxy.
[0363] In certain embodiments of formula I, R7 is chloro.
[0364] In certain embodiments of formula I, R7 is fluoro.
103651 In certain embodiments of formula I, the LRRK2 modulator compound
is:
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[4-(5-Fluoro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -morpho lin-
4 -yl-
methanone
[4-(5-Fluoro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4 -
hydroxy-pip eridin-
1 -y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-pheny1]-morpholin-
4-yl-
methanone
4-(5 -Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-N,N-dimethyl-
benzamide
445 -Chloro-4-methylamino-pyrimidin-2-ylamino)-N-cyclopropy1-3 -methoxy-
benzamide
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4 -
hydroxy-pip eridin-
1 -y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -difluoromethoxy-pheny1]-
morpholin-4-
yl-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-pheny1]-
(octahydro-
pyrido [ 1 ,2-a]pyrazin-2 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(2 -
hydroxy-pip eridin-
1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4,4-
dimethyl-
pip eridin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(3 ,5 -
dimethyl-
pip eridin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] - [4-( 1 -
hydroxy- 1 -
methyl-ethyl)-pip eridin- 1 -yl] -methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(3 -
hydroxy-
pyrro lidin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4 -methyl-
pip eridin-
1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -pip eridin-
1 -yl-
methanone
Azetidin- 1 -y144-(5 -chloro-4-methyl amino -pyrimidin-2-ylamino)-3 -methoxy-
phenyl] -
methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4,4-
difluoro-
pip eridin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(3 -methyl-
pip eridin-
1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4 -
methoxy-
pip eridin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(3,3 -
difluoro-
pip eridin- 1 -y1)-methanone
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1 - [4 -(5 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-benzoyl] -p
ip eridine-4 -
c arb onitrile
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4 -
fluoro-pip eri din- 1 -
y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(3 -
methoxy-
pip eridin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4 -ethyl-
pip erazin- 1 -
y1)-methanone
1- {4- [4 -(5 -Chloro-4-methylamino -pyrimi din-2 -ylamino)-3 -methoxy-b
enzoyl] -pip erazin- 1 -
y1} -ethanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(3 -
trifluoromethyl-
pip eridin- 1 -y1)-methanone
(4-tert-Butyl-pip eridin- 1 -y1)- [445 -chloro-4-methylamino-pyrimidin-2-
ylamino)-3 -methoxy-
phenyl] -methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] - [4 -(2-
hydroxy- ethyl)-
pip erazin- 1 -yl] -methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(2 -methyl-
pyrro lidin-
1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4 -
hydroxymethyl-
pip eridin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(2 -methyl-
pip eridin-
1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -pyrro
lidin- 1 -yl-
methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4 -
methane sulfonyl-
pip erazin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(3 -
trifluoromethyl-
pyrro lidin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] - [4 -
(2,2,2-trifluoro-
ethyl)-pip erazin- 1 -yl] -methanone
[4-(5 -Bromo -4 -methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morpholin-4-yl-
methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(2 -methyl-
morpho lin-
4-y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(2, 6-
dimethyl-
morph lin-4 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(2,2-
diethyl-
morph lin-4 -y1)-methanone
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[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(3 -
hydroxymethyl-
morpholin-4-y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(2 -is
butyl-
morpholin-4-y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(2 -
hydroxymethyl-
morpholin-4-y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(3,3 -
dimethyl-
morpholin-4-y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4 -
methyl-pip erazin-
1 -y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4 -is
opropyl-
pip erazin- 1 -y1)-methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -y1 amino)-3 -methoxy-phenyl] -morpholin-
4-yl-
methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -pip erazin-
1 -yl-
methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 - difluoromethoxy-phenyl] -
(4-hydroxy-
pip eridin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -ethoxy-phenyl] -morpholin-4-
yl-
methanone
[3 -Methoxy-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-phenyl] -
morpho lin-4 -
yl-methanone
4-(5 -Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-benzamide
445 -Chloro-4-methylamino-pyrimidin-2-ylamino)-N-ethyl-3 -methoxy-benzamide
445 -Chloro-4-methylamino-pyrimidin-2-ylamino)-N-isopropyl-3 -methoxy-
benzamide
4-(5 -Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-N-methyl-benzamide
[4-(5 -Chloro-4 -prop oxy-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -morpholin-4-
yl-
methanone
[4-(5 -Chloro-4 -ethyl amino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -
morpholin-4-yl-
methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(3 -oxa-8 -
aza-
bicyclo [3 .2.1 ] oct- 8 -y1)-methanone
[4-(5-Chloro-4-ethoxy-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -morpho lin-4 -
yl-methanone
[4-(5 -Chloro-4 -is oprop oxy-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morpholin-4-yl-
methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -((S)-3 -
methyl-
morpholin-4-y1)-methanone
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[4-(5-Chloro-4-methoxy-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -morpholin-4-yl-
methanone
[445 -Chloro-4 -methyl amino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(2 -oxa-
5 -aza-
bicyclo [2.2.1 ]hept-5-y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(8 -oxa-
3 -aza-
bicyclo [3 .2.1 ] oct-3 -y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -((R)-3 -
methyl-
morph lin-4-y1)-methanone
N-(3 -Amino -propy1)-4 -(5 -chloro-4-methylamino-pyrimidin-2 -ylamino)-3 -
methoxy-
b enzamide
[445 -Cyc lopropy1-4 -methyl amino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morph lin-4 -yl-
methanone
[445 -Chloro-4 -is obutylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -morph
lin-4 -yl-
methanone
[445 -Chloro-4 -propylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -morpholin-
4-yl-
methanone
[4-(5 -Chloro-4 -is opropylamino -pyrimidin-2 -y1 amino)-3 -methoxy-phenyl] -
morpholin-4-yl-
methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4 -
cyc loprop anecarb onyl-pip erazin- 1 -y1)-methanone
[445 -Bromo -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] - [4-( 1 -
hydroxy- 1 -
methyl- ethyl)-pip eridin- 1 -yl] -methanone
[445 -Bromo -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(3 -
trifluoromethyl-
pyrro lidin- 1 -y1)-methanone
1 - [4 -(5 -Bromo -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-benzoyl] -
pip eridine-4 -
c arb nitrite
[445 -Bromo -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4 -cyc
lobutyl-
pip erazin- 1 -y1)-methanone
[445 -Bromo -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] - [4 -
(2,2,2-trifluoro-
ethyl)-p ip erazin- 1 -yl] -methanone
[445 -Bromo -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4 -
methoxy-
pip eridin- 1 -y1)-methanone
[445 -Bromo -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -((R)-3 -
hydroxy-
pyrro lidin- 1 -y1)-methanone
[4-(5 -Bromo -4 -methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4 -
oxetan-3 -yl-
pip erazin- 1 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -is oprop oxy-phenyl] -
morpholin-4-yl-
methanone
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{4- [5 -Chloro-4-(cyclopropylmethyl-amino)-pyrimidin-2 -ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
[445 -Chloro-4 -cycl obutylamino -pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -
morpholin-4-yl-
methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -hydroxy-phenyl] -morpho
lin-4 -yl-
methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4-ethyl-
pip erazin- 1 -y1)-
methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-2-fluoro-5 -methoxy-phenyl] -
morpho lin-
4-yl-methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-3 -cyclobutoxy-phenyl] -
morpholin-4-yl-
methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -cyclobutoxy-pheny1]-
morpholin-4-yl-
methanone
[445 -Chloro-4 -ethyl amino-pyrimidin-2 -ylamino)-2 -fluoro-5 -methoxy-phenyl]
-morpholin-4-
yl-methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(3 -morpho
lin-4-yl-
azetidin- 1 -y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(3 -
morpho lin-4 -yl-
azetidin- 1 -y1)-methanone
[445 -Chloro-4 -cycl op entylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morpholin-4 -yl-
methanone
{4- [5 -Chloro-4-( 1 -methyl-cyclobutylamino)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
[445 -Chloro-4 -cycl ohexylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morpholin-4-yl-
methanone
(4- { 5 -Chloro-4- [(tetrahydro-furan-3 -ylmethyl)-amino]-pyrimidin-2-ylamino
} -3 -methoxy-
pheny1)-morpholin-4-yl-methanone
[445 -Io do-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -morpholin-4-
yl-
methanone
[445 -Bromo -4 -is oprop oxy-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morpholin-4-yl-
methanone
[445 -Bromo -4 -ethoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -morpholin-4 -
yl-methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4,4-
difluoro-pip eridin- 1 -
y1)-methanone
(4- { 5 -Chloro-4- [(tetrahydro-furan-2-ylmethyl)-amino]-pyrimidin-2-ylamino }
-3 -methoxy-
pheny1)-morpholin-4-yl-methanone
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{4- [5 -Chloro-4-(cyclobutylmethyl-amino)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
[4-(5-Iodo-4-methoxy-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -morpholin-4-yl-
methanone
[445 -Cyc lobuty1-4 -methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morph lin-4 -yl-
methanone
[445 -Cyc lopropy1-4 -methyl amino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morph lin-4 -yl-
methanone
{4- [5 -Chloro-4-(2-cyclopropyl-ethylamino)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
{4- [5 -Chloro-4-(2-methoxy-ethylamino)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
{4- [5 -Chloro-4-(cyclopentylmethyl-amino)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -((R)-2,2-di-
deutero-3 -
methyl-morpho lin-4 -y1)-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -(2,2,2-trifluoro-ethoxy)-
phenyl] -
morpholin-4-yl-methanone
[4- [5 -Chloro-4 -(2-methoxy-ethylamino)-pyrimidin-2-ylamino] -3 -(2,2,2-
trifluoro-ethoxy)-
phenyl] -morpholin-4-yl-methanone
[445 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-2 -fluoro-5 -methoxy-phenyl] -
morph lin-4 -yl-
methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -((S)-2,2-di-
deutero-3 -
methyl-morpho lin-4 -y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] - [441 -
methyl-
pip eridin-4 -y1)-pip erazin- 1 -yl] -methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -trifluoromethoxy-phenyl] -
morpho lin-4 -
yl-methanone
[445 -Cyc lobuty1-4 -methoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -morpho
lin-4 -yl-
methanone
[3 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-phenyl] -morpholin-
4-yl-
methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -y1 amino)-3 -ethoxy-phenyl] -morpholin-4-
yl-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(3,3 -
difluoro-
azetidin- 1 -y1)-methanone
{4- [5 -Bromo-4-(2-methoxy-ethylamino)-pyrimidin-2-ylamino] -3 -ethoxy-phenyl}
-morpho lin-
4-yl-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(4 -
dimethylamino-
pip eridin- 1 -y1)-methanone
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445 -Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-N-oxetan-3 -yl-
benzamide
445 -Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-N-(tetrahydro-pyran-
3 -y1)-
b enzamide
{4- [5 -Chloro-4-(tetrahydro-pyran-3 -ylamino)-pyrimidin-2-ylamino] -3 -
methoxy-phenyl} -
morpholin-4-yl-methanone
{4- [5 -Chloro-4-(2-methoxy-ethylamino)-pyrimidin-2-ylamino] -3 -cyclobutoxy-
phenyl} -
morpholin-4-yl-methanone
{4- [5 -Bromo-4-(2-methoxy-ethylamino)-pyrimidin-2-ylamino] -3 -cyclobutoxy-
phenyl} -
morpholin-4-yl-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -(4 -pip
eridin-4-yl-
pip erazin- 1 -y1)-methanone
{4- [5 -Chloro-4-(2,2,2-trifluoro-ethylamino)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
{4- [5 -Chloro-4-(2,2-difluoro-ethylamino)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -y1 amino)-3 -is oprop oxy-phenyl] -morph
lin-4-yl-
methanone
[3 -Bromo-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-phenyl] -morpholin-4-
yl-
methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -y1 amino)-3 -methoxy-phenyl] -[4-( 1 -
hydroxy- 1 -methyl-
ethyl)-p ip eridin- 1 -yl] -methanone
(4-(5 -bromo -4 -methoxypyrimidin-2-ylamino)-3 -methoxyphenyl) (3 -
(trifluoromethyl)pyrro lidin- 1 -yl)methanone
1 -(4 -(5 -bromo-4-methoxypyrimidin-2-ylamino)-3 -methoxyb enzoyl)pip eridine-
4 -c arb nitrite
(3 -methoxy-4-(5 -methoxy-4-(methylamino)pyrimidin-2-ylamino)phenyl)
(morpholino)methanone
N-(3 -aminopropy1)-4-(5-chloro-4-(methylamino)pyrimidin-2-ylamino)-3 -
methoxybenzamide
[3 -Methoxy-4-(4 -methylamino-5 -prop -1 -ynyl-pyrimidin-2-ylamino)-phenyl] -
morpholin-4-yl-
methanone
[3 -Methoxy-4-(4 -methoxy-5 -prop- 1 -ynyl-pyrimidin-2-ylamino)-phenyl] -
morpho lin-4 -yl-
methanone
[5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
morpho lin-
4-yl-methanone
{4- [5 -Bromo-4-(2-methoxy-ethoxy)-pyrimidin-2-ylamino] -3 -methoxy-phenyl} -
morpho lin-4-
yl-methanone
-Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-N,N-dimethyl-
benzamide
5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-b enzami
de
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-Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-N-(2-hydroxy-ethyl)-2-
methoxy-
benzamide
5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-N-(2-
methoxy-ethyl)-
benzamide
5- Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-N-methyl-b
enzamide
5- Chloro-4-(5 -chloro-4-methyl amino -pyrimidin-2 -ylamino)-N-cyc lopropy1-2 -
methoxy-
b enzamide
[5 -Chloro-4-(5 -chloro-4-methyl amino-pyrimidin-2-ylamino)-2 -methoxy-phenyl]
-(4,4-
difluoro -pip eridin- 1 -y1)-methanone
[5 -Chloro-4-(4 -ethylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-2-methoxy-
phenyl] -
morpholin-4-yl-methanone
5- Chloro-N-cyc lopropy1-4 -(4- ethylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-2-
methoxy-b enzamide
5- Chloro-4-(4 -ethyl amino -5 -trifluoromethyl-pyrimidin-2 -y1 amino)-2-
methoxy-N-(2-
methoxy- ethyl)-b enzamide
[5 -Chloro-4-(5 -chloro-4-methyl amino-pyrimidin-2-ylamino)-2 -methoxy-phenyl]
-pip erazin- 1 -
yl-methanone
[5 -Chloro-4-(5 -chloro-4-methyl amino-pyrimidin-2-ylamino)-2 -methoxy-phenyl]
-(4-
dimethylamino -pip eridin- 1 -y1)-methanone
[5 -Chloro-4-(5 -chloro-4-methyl amino-pyrimidin-2-ylamino)-2 -methoxy-phenyl]
-(3 -hydroxy-
pyrro lidin- 1 -y1)-methanone
5 - Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-N-oxetan-
3 -yl-
b enzamide
5- Chloro-N-cyc lopropy1-2 -methoxy-4-(4 -methylamino-5 -trifluoromethyl-
pyrimidin-2 -
ylamino)-b enzamide
5- Chloro-4-(5 -chloro-4-methyl amino -pyrimidin-2 -ylamino)-2-methoxy-N-( 1 -
methyl-
p ip eridin-4-y1)-benzamide
[5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2 -methoxy-phenyl] -
pyrro lidin-
1 -yl-methanone
N-tert-Butyl-5 -chloro-4-(5-chloro-4-methylamino-pyrimidin-2-ylamino)-2-
methoxy-
benzamide
5- Chloro-2-methoxy-N,N-dimethy1-4-(4 -methylamino-5 -trifluoromethyl-
pyrimidin-2 -
ylamino)-benzamide
5- Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-N-(2-
methoxy-ethyl)-
N-methyl-benzamide
5- Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-N-(2-hydroxy-2-
methyl-propy1)-
2-methoxy-benzamide
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[5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
(4-hydroxy-
pip eridin- 1 -y1)-methanone
[5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
(2-
hydroxymethyl-morpho lin-4-y1)-methanone
-Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-N-(2-hydroxy-ethyl)-2-
methoxy-
N-methyl-benzamide
5 -Chloro-4-(5 -chloro-4-methylamino -pyrimidin-2 -ylamino)-2-methoxy-N-( 1 -
methyl-
cyc lobuty1)-b enzamide
[5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
[1,4] oxazepan-4-yl-methanone
[5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
((2R,6S)-
2,6-dimethyl-morpholin-4-y1)-methanone
1- [5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-b
enzoyl] -
pip eridine-4-carb onitrile
5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-N-(2-hydroxy-propy1)-
2-
methoxy-b enzamide
[5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
(3 -hydroxy-
azetidin- 1 -y1)-methanone
5 -Chloro-4-(5 -chloro-4-methylamino -pyrimidin-2 -ylamino)-N-( 1 -cyano-
cyclopropy1)-2-
methoxy-b enzamide
1- [5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-b
enzoyl] -
pyrro lidine-3 -carb onitrile
[5 -Chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
((3R,5 S)-
dimethyl-pip erazin- 1 -y1)-methanone
5 -Chloro-2-methoxy-4-(4-methylamino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-
N-( 1 -
methyl-pip eridin-4-y1)-b enzamide
5 -Chloro-2-methoxy-4-(4-methylamino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-
N-oxetan-3 -
yl-b enzamide
5 -Chloro-N-(2 -hydroxy-2 -methyl-propy1)-2-methoxy-4-(4-methylamino-5 -
trifluoromethyl-
pyrimidin-2-ylamino)-benzamide
[5 -Chloro-2-methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
pip erazin- 1 -yl-methanone
1- [5 -Chloro-2-methoxy-4 -(4-methyl amino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-benzoyl] -
pyrro lidine-3 -carb onitrile
1- [2 -F luoro-5 -methoxy-4-(4-methylamino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-benzoyl] -
pyrro lidine-3 -carbonitrile
[5 -Chloro-2- ethoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morph lin-4-yl-methanone
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2-Fluoro-5 -methoxy-N-methyl-4 -(4-methyl amino-5 -trifluoromethyl-pyrimidin-2-
ylamino)-
benzamide
-Chloro-N-(2 -hydroxy-2 -methyl-propy1)-2-methoxy-N-methy1-4-(4 -methylamino-5
-
trifluoromethyl-pyrimidin-2-ylamino)-benzamide
(445 -cyc lopropy1-4-methoxypyrimidin-2 -ylamino)-3 -
methoxyphenyl)(morpholino)methanone
1- { 2 42 -Methoxy-4 -(morpho line-4 -c arb ony1)-phenylamino] -4 -methylamino-
pyrimidin-5 -y1} -
ethanone
{4- [5 -Chloro-4-(2 -methoxy-ethoxy)-pyrimidin-2-ylamino] -3 -methoxy-phenyl} -
morpho lin-4-
yl-methanone
{4- [5 -Bromo-4-(2-methoxy-ethylamino)-pyrimidin-2-ylamino]-2-fluoro-5-methoxy-
phenyl} -
morpholin-4-yl-methanone
{4- [5 -Bromo-4-(2 -methoxy-ethylamino)-pyrimidin-2-ylamino] -3 -is oprop oxy-
phenyl } -
morpholin-4-yl-methanone
{4- [5 -Chloro-4-(2 -methoxy-ethoxy)-pyrimidin-2-ylamino] -3 -cyclobutoxy-
phenyl} -
morpholin-4-yl-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2-ylamino)-3 -cyc lop entyloxy-phenyl] -
(2-o xa-6-aza-
spiro [3.3 ] hept-6-y1)-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2-ylamino)-3 -cyc lop entyloxy-phenyl] -
morpho lin-4 -
yl-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -(2 -oxa-6
-aza-
spiro [3.3 ] hept-6-y1)-methanone
2-(2 -methoxy-4 -(2,2,6, 6-tetrafluoromorpho line-4-carb onyl)phenylamino)-4-
(methylamino)pyrimidine-5 -carb onitrile
2-(4 -(4,4 -difluoropip eridine- 1 -carb ony1)-2-methoxyphenylamino)-4-
(methylamino)pyrimidine-5 -carb nitrite
[445 -Chloro-4 -methylamino-pyrimidin-2-ylamino)-3 -cyclopropyl-phenyl] -
morpholin-4-yl-
methanone
4-(5 -Cyano -4 -methyl amino-pyrimidin-2 -ylamino)-N-(3 ,3 -difluoro-
cyclobuty1)-3 -methoxy-
benzamide
445 -Cyan -4 -methyl amino-pyrimidin-2 -ylamino)-N-cyclopropylmethy1-3 -
methoxy-
benzamide
2- [4-((S)-3 -F luoro-pyrro lidine- 1 -carbonyl)-2-methoxy-phenylamino] -4-
methylamino-
pyrimidine-5 -carb nitrite
4-(5 -Cyano -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-N-methyl-
benzamide
4-(5 -Cyano -4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-N,N-dimethyl-
benzamide
445 -Cyan -4 -methylamino-pyrimidin-2 -ylamino)-N,N-diethy1-3 -methoxy-
benzamide
445 -Cyan -4 -methylamino-pyrimidin-2 -ylamino)-N-is opropy1-3 -methoxy-
benzamide
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2- [2 -Methoxy-4 -(pyrro lidine- 1 -carbonyl)-phenylamino] -4 -methylamino -
pyrimidine-5 -
c arb onitrile
2- [4 -(3 -Fluoro-azetidine- 1 -carbonyl)-2-methoxy-phenylamino] -4-
methylamino-pyrimidine-5 -
c arb onitrile
445 -Cyan -4 -methyl amino-pyrimidin-2 -ylamino)-N-ethy1-3 -methoxy-benzamide
2- [2 -Methoxy-4 -(pip eridine- 1 -carbonyl)-phenylamino] -4 -methylamino -
pyrimidine-5 -
c arb nitrite
445 -Cyan -4 -methyl amino-pyrimidin-2 -ylamino)-N-(2,2 -difluoro-ethyl)-3 -
methoxy-
benzamide
N-tert-Butyl-4 -(5 -cyano-4-methylamino-pyrimidin-2-ylamino)-3 -methoxy-b
enzamide
445 -Cyan -4 -methyl amino-pyrimidin-2 -ylamino)-N-ethyl-N-is opropy1-3 -
methoxy-
benzamide
2- [4-(3 ,3 -Difluoro-pyrro lidine- 1 -c arb ony1)-2 -methoxy-phenylamino] -4 -
methylamino-
pyrimidine-5 -carb onitrile
2- [4-(3 ,3 -Difluoro-azetidine- 1 -carbonyl)-2 -methoxy-phenyl amino] -4 -
methylamino-
pyrimidine-5 -carb onitrile
2- [4-((R)-3 -F luoro-pyrro lidine- 1 -carbonyl)-2-methoxy-phenylamino] -4-
methylamino-
pyrimidine-5 -carb nitrite
445 -Cyan -4 -methyl amino-pyrimidin-2 -ylamino)-N-ethy1-3 -methoxy-N-methyl-
b enzamide
2- [4 -(Azetidine- 1 -carbonyl)-2-methoxy-phenylamino] -4-methylamino-
pyrimidine-5-
carbonitrile
445 -Cyan -4 -methyl amino-pyrimidin-2 -ylamino)-N-is opropy1-3 -methoxy-N-
methyl-
b enzamide
{4- [5 -Chloro-4-(tetrahydro-furan-3 -ylamino)-pyrimidin-2 -ylamino] -3 -
methoxy-phenyl} -
morpholin-4-yl-methanone
[3 -Is oprop oxy-4 -(4-methyl amino-5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morph lin-4-yl-methanone
[3 -Ethoxy-4 -(4-methyl amino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-phenyl]
-morph lin-4 -
yl-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2-ylamino)-3 -cyclopropoxy-phenyl] -
morph lin-4-yl-
methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-3 -cycloprop oxy-phenyl] -
morpholin-4-yl-
methanone
{4- [5 -Chloro-4-(2 -methane sul fonyl-ethyl amino)-pyrimidin-2-ylamino] -3 -
methoxy-phenyl} -
morpholin-4-yl-methanone
2- [4 -(5 -Bromo -4 -methylamino-pyrimidin-2 -ylamino)-2,5-dimethoxy-phenyl] -
1 -morpholin-4-
yl-ethanone
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2- [4 -(5 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-2,5-dimethoxy-phenyl] -
1 -morpholin-4-
yl-ethanone
2- [4 -(5 -Chloro-4 -methylamino-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -1 -
morpholin-4-yl-
ethanone
2- [3 -Methoxy-4 -(4-methylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -1 -
morpholin-4-yl-ethanone
2- [4 -(5 -Chloro-4 -methoxy-pyrimidin-2 -ylamino)-2,5 -dimethoxy-phenyl] -1 -
morpholin-4-yl-
ethanone
2- [4 -(5 -Chloro-4 -methoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -1 -
morpho lin-4 -yl-
ethanone
2- [4 -(5 -Bromo -4 -methoxy-pyrimidin-2 -ylamino)-3 -methoxy-phenyl] -1 -
morpho lin-4 -yl-
ethanone
2- [2,5 -Dimethoxy-4 -(4-methylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-
phenyl] -1 -
morpholin-4-yl-ethanone
N-(3 -Amino -propy1)-5 -chloro-4-(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-
2-methoxy-
benzamide
{4- [5 -Bromo-4-(2-methoxy-ethylamino)-pyrimidin-2-ylamino] -3 -methoxy-phenyl
} -
morpholin-4-yl-methanone
{4- [5 -Chloro-4-(2-methoxy-propylamino)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
4-(5 -chloro-4-(methylamino)pyrimidin-2-ylamino)-N,N,3 -trimethylbenzamide
(4-(5-chloro-4-(methylamino)pyrimidin-2-ylamino)-3 -
methylphenyl)(morpholino)methanone
(4-(5-chloro-4-(methylamino)pyrimidin-2-ylamino)-3 -methylphenyl)(4-hydroxypip
eridin- 1 -
yl)methanone
(4-(4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-3 -
(trifluoromethoxy)phenyl) (morph lino)methanone
(4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-3 -
(trifluoromethoxy)phenyl) (morph lino)methanone
(5 -chloro-2-methoxy-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)phenyl)(p erdeuteromorpholino)methanone
(5 -fluoro-2-methoxy-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)phenyl)(morpholino)methanone
(4-(4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-5 -fluoro-2-
methoxyphenyl)(morpholino)methanone
[2-F luoro-5 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
morph lin-4-yl-methanone
445 -Cyan -4 -ethylamino-pyrimidin-2 -ylamino)-2 -fluoro-5 -methoxy-N,N-
dimethyl-
benzamide
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(4-(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-2-fluoro-3 -
methoxyphenyl)(morpholino)methanone
(1 S,4 S)-2 -oxa-5 -azabicyclo [2.2.1 ] heptan-5 -y1(4-(4 -(ethyl amino)-5 -
(trifluoromethyl)pyrimidin-2-ylamino)-2-fluoro-3 -methoxyphenyl)methanone
(1 S,4 S)-2 -oxa-5 -azabicyclo [2.2.1 ] heptan-5 -y1(2-fluoro-3 -methoxy-4-(4-
(methylamino)-5-
(trifluoromethyl)pyrimidin-2-ylamino)phenyl)methanone
-chl oro-4 -(5 -chl oro-4 -methoxypyrimidin-2 -ylamino)-2-methoxy-N-methylb
enzamide
2-(4-((3 S ,4 S)-3 ,4 -difluoropyrro lidine- 1 -carb ony1)-2-
methoxyphenylamino)-4 -
(methylamino)pyrimidine-5 -carb nitrite
(5 -chloro-2-methoxy-4-(4 -(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)phenyl)(3 -
methoxypyrro lidin- 1 -yl)methanone
(2-fluoro-5-methoxy-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)phenyl)(3 -
methoxypyrro lidin- 1 -yl)methanone
2-fluoro-N-(2-hydroxy-2-methylpropy1)-5-methoxy-N-methy1-4-(4-(methylamino)-5-
(trifluoromethyl)pyrimidin-2-ylamino)benzamide
((2S,6R)-2,6-dimethylmorpholino)(2-fluoro-5-methoxy-4-(4-(methylamino)-5-
(trifluoromethyl)pyrimidin-2-ylamino)phenyl)methanone
(2-fluoro-5-methoxy-4-(4-(2-methoxyethoxy)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)phenyl)(morpholino)methanone
(2- ethoxy-5 -fluoro-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)phenyl)(morpholino)methanone
(4-(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-3 -
isopropoxyphenyl)(morpholino)methanone
(4-(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-2-fluoro-3 -
isopropoxyphenyl)(morpholino)methanone
(4-(5 -chloro-4 -(pip eridin- 1 -yl)pyrimidin-2-ylamino)-3 -
methoxyphenyl)(morpholino)methanone
(4-(5 -chl oro-4 -(pyrro lidin- 1 -yl)pyrimidin-2-ylamino)-3 -
methoxyphenyl)(morpholino)methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -methoxy-
phenyl] -
morph lin-4-yl-methanone
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-3 -methoxy-phenyl] -
morph lin-4-
yl-methanone
[5 -Chloro-4-(5 -chloro-4-ethylamino-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
morpho lin-4-
yl-methanone
[445 -Chloro-4 -methoxy-pyrimidin-2 -ylamino)-2 -fluoro-5 -methoxy-phenyl] -
morpho lin-4 -yl-
methanone
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[5 -Chloro-4-(5 -chloro-4-methoxy-pyrimidin-2-ylamino)-2-methoxy-phenyl] -
morpholin-4-yl-
methanone
[3 -Chloro-4-(5 -chloro-4-methoxy-pyrimidin-2-ylamino)-phenyl] -morph lin-4-
yl-methanone
[2-Chloro-5-methoxy-4-(4-methoxy-5-trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morph lin-4-yl-methanone
[3 -Methoxy-4-(4-methoxy-5-trifluoromethyl-pyrimidin-2-ylamino)-phenyl] -
morpholin-4-yl-
methanone
{4- [5 -Chloro-4-(tetrahydro-pyran-4-yloxy)-pyrimidin-2-ylamino] -3 -methoxy-
phenyl} -
morpholin-4-yl-methanone
-Chloro-N-( 1 -cyano -cyclopropy1)-2-methoxy-4-(4-methylamino-5 -
trifluoromethyl-
pyrimidin-2-ylamino)-benzamide
5 -Chloro-N-( 1 -cyano -cyclopropy1)-2-methoxy-4-(4-methylamino-5 -
trifluoromethyl-
pyrimidin-2-ylamino)-benzamide
[5 -Chloro-2-methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
(2,6-dimethyl-morpholin-4-y1)-methanone
[3 -Methoxy-4-(4 -pyrro lidin- 1 -y1-5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -morpho lin-
4-yl-methanone
[3 -Cyclobutoxy-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morpholin-4-yl-methanone
N-(3,3 -Difluoro-cyclobuty1)-3 -methoxy-4-(4-methylamino -5 -trifluoromethyl-
pyrimidin-2-
ylamino)-benzamide
3 -Methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-N-oxetan-
3 -yl-
b enzamide
[3 -Methoxy-4-(4 -methyl amino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-
phenyl] -(8 - oxa-3 -
aza-bicyclo [3 .2.1 ] oct-3 -y1)-methanone
[3 -Methoxy-4-(4 -methyl amino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-
phenyl] -(2- oxa-5 -
aza-bicyclo [2.2.1 ] hept-5 -y1)-methanone
[3 -Methoxy-4-(4 -methyl amino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-
phenyl] -pyrro lidin- 1 -
yl-methanone
N,N-Diethyl-3 -methoxy-4 -(4-methyl amino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-
b enzamide
(4-Dimethylamino-pip eridin- 1-y1)- [3 -methoxy-4-(4-methylamino-5-
trifluoromethyl-
pyrimidin-2-ylamino)-phenyl] -methanone
(2,6-Dimethyl-morpholin-4-y1)[3 -methoxy-4-(4 -methyl amino -5 -
trifluoromethyl-pyrimidin-
2-ylamino)-phenyl] -methanone
1- [3 -Methoxy-4-(4-methylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
benzoyl] -
pip eridine-4-carb nitrite
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[3 -Methoxy-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-phenyl] -
[4 -(2,2,2-
trifluoro- ethyl)-p ip erazin- 1 -yl] -methanone
[3 -Methoxy-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-phenyl] -
(4-methoxy-
pip eridin- 1 -y1)-methanone
3 -Methoxy-N-(2-methoxy-ethyl)-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-
ylamino)-
benzamide
3 -Methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-N-( 1 -
methyl-
p ip eridin-4-y1)-benzamide
N-(4,4-Difluoro-cyclohexyl)-3 -methoxy-4-(4-methylamino-5-trifluoromethyl-
pyrimidin-2-
ylamino)-benzamide
[445 -Chloro-4-methylamino-pyrimidin-2-ylamino)-3 -(oxetan-3 -yloxy)-phenyl] -
morpholin-4-
yl-methanone
[3 -Cyc lopropylmethoxy-4-(4-methylamino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-phenyl] -
morph lin-4-yl-methanone
[3 -Cyclobutylmethoxy-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morpholin-4-yl-methanone
N-Ethyl-3 -methoxy-N-methy1-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-
ylamino)-
b enzamide
[445 -Bromo -4 -methylamino-pyrimidin-2-ylamino)-5 -chloro-2 -methoxy-phenyl] -
morpho lin-
4-yl-methanone
[3 -Cyc lopropy1-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-
phenyl] -
morph lin-4-yl-methanone
[3 -Bromo -4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-phenyl] -
morph lin-4 -
yl-methanone
[3 -Chloro-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-phenyl] -
morph lin-4 -
yl-methanone
[2-Chloro-5 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
morph lin-4-yl-methanone
[2-Chloro-4 -(5 -chloro-4-methylamino-pyrimidin-2-ylamino)-5-methoxy-phenyl] -
morpho lin-
4-yl-methanone
[4-(5 -Bromo -4 -methoxy-pyrimidin-2-ylamino)-2 -chloro-5 -methoxy-phenyl] -
morpho lin-4 -yl-
methanone
(5 -chloro-4-(5 -chloro-4-(methylamino)pyrimidin-2-ylamino)-2-
methoxyphenyl)(morpholino)methanone
[5 -Chloro-2 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
morph lin-4-yl-methanone
[2-Fluoro-3 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
morph lin-4-yl-methanone
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[3 -(2-Fluoro-ethoxy)-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morph lin-4-yl-methanone
(4-(5-chloro-4-methoxypyrimidin-2-ylamino)-3 -methoxyphenyl)(pyrrolidin- 1 -
yl)methanone
[5 -Ethoxy-2 -fluoro-4 -(4-methyl amino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
morpholin-4-yl-methanone
3 -Methoxy-N-(2-methoxy-ethyl)-N-methy1-4-(4-methylamino-5-trifluoromethyl-
pyrimidin-2-
ylamino)-benzamide
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-3 -methoxy-N-(2-
methoxy-ethyl)-N-
methyl-b enzamide
2-Fluoro-5 -methoxy-N-(2-methoxy- ethyl)-N-methy1-4-(4 -methylamino -5 -
trifluoromethyl-
pyrimidin-2-ylamino)-benzamide
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-5 -methoxy-
N-(2-methoxy-
ethyl)-N-methyl-benzamide
[2-Fluoro-5 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
((S)-2-methoxymethyl-pyrro lidin- 1 -y1)-methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -methoxy-
phenyl] -(( S)-
2-methoxymethyl-pyrro lidin- 1 -y1)-methanone
N-Ethy1-4-(4 -ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-5-
methoxy-N-(2-
methoxy-ethyl)-benzamide
N-Ethy1-2-fluoro-5 -methoxy-N-(2-methoxy-ethyl)-4-(4-methylamino -5 -
trifluoromethyl-
pyrimidin-2-ylamino)-benzamide
[5 -Ethoxy-4 -(4-ethylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-
phenyl] -
morpholin-4-yl-methanone
(4-(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-yl-amino)-3 -
isopropoxyphenyl)(morpholino)methanone
(2-fluoro-3 -is oprop oxy-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)phenyl) (morpholino)methanone
(4-(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-3 -
(trifluoromethoxy)phenyl) (morph lino)methanone
(2-fluoro-3 -is oprop oxy-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)phenyl) (morpholino)methanone
2-(2 -methoxy-4 -(morpho line-4 -c arb onyl)phenylamino)-4-
(methylamino)pyrimidine-5-
carbonitrile
4-Methoxy-2- [2 -methoxy-4-(morpho line-4 -c arb ony1)-phenylamino] -
pyrimidine-5 -c arb onitrile
4-Ethylamino-2- [5 -fluoro-2-methoxy-4 -(morpho line-4 -c arb ony1)-
phenylamino] -pyrimdine-5 -
carb nitrite
2- [5 -F luoro-2-methoxy-4-(morpho line-4 -c arb ony1)-phenyl amino] -4 -
methylamino-
pyrimidine-5 -carb onitrile
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2- [4 -((2R,6 S)-2,6 -Dimethyl-morpho line-4 -c arb ony1)-5 -fluoro-2-methoxy-
phenyl amino] -4-
methylamino-pyrimidine-5 -c arb onitrile
2- [4 -((2R,6 S)-2,6 -Dimethyl-morpho line-4 -c arb ony1)-5 -fluoro-2-methoxy-
phenyl amino] -4-
ethylamino-pyrimidine-5 -carbonitrile
4-(5 -Cyano -4 -methyl amino-pyrimidin-2 -ylamino)-2 -fluoro-5 -methoxy-N,N-
dimethyl-
benzamide
[5 -Chloro-2-methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] - [4-
(2-methoxy- ethyl)-p ip erazin- 1 -yl] -methanone
[5 -Chloro-2-methy1-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-
phenyl] -
morpholin-4-yl-methanone
[5 -Methoxy-2-methy1-4-(4 -methylamino-5 -trifluoromethyl-pyrimi din-2 -
ylamino)-phenyl] -
morph lin-4-yl-methanone
[2-F luoro-5 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
(hexahydro-pyrro lo [ 1 ,2-a] pyrazin-2 -y1)-methanone
[2-F luoro-5 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
( 1 S,5R)-8 -oxa-3 -aza-bicyclo [3 .2.1 ] oct-3 -yl-methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -methoxy-
phenyl] -
( 1 S,5R)-8 -oxa-3 -aza-bicyclo [3 .2.1 ] oct-3 -yl-methanone
2-Fluoro-5 -methoxy-N-methy1-4 -(4-methyl amino-5 -trifluoromethyl-pyrimidin-2-
ylamino)-N-
( 1 -methyl-pip eridin-4 -y1)-b enzamide
[2-F luoro-5 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -(4-
morph lin-4-yl-p ip eridin- 1 -y1)-methanone
[4-(5 -Chloro-4 -methoxy-pyrimidin-2 -y1 amino)-3 -hydroxy-phenyl] -morpholin-
4-yl-
methanone
[5 -Chloro-2-hydroxy-4-(4 -methylamino-5 -tri fluoromethyl-pyrimidin-2-
ylamino)-phenyl] -
morpholin-4-yl-methanone
[2-F luoro-5 -methoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
( 1 S,4 S)-2 -oxa-5 -aza-bicyclo [2.2.1 ] hept-5 -yl-methanone
[2,3 -Difluoro-4-(4-methylamino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-
phenyl] -morpho lin-
4-yl-methanone
[4-(5-Chloro-4-methylamino-pyrimidin-2-ylamino)-2,3 -difluoro-phenyl] -
morpholin-4-yl-
methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -methoxy-
phenyl] -
( 1 S,4 S)-2 -oxa-5 -aza-bicyclo [2.2.1 ] hept-5 -yl-methanone
[5 -Chloro-2-methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
( 1 S,4 S)-2 -oxa-5 -aza-bicyclo [2.2.1 ] hept-5 -yl-methanone
[2,5 -Difluoro-4-(4 -methylamino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-
phenyl] -morpho lin-
4-yl-methanone
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[445 -Chloro-4 -methylamino-pyrimidin-2-ylamino)-2,5 -difluoro-phenyl] -morph
lin-4-yl-
methanone
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-N-(2-
hydroxy-2-methyl-
propy1)-5 -methoxy-benzamide
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-N-(2-
hydroxy-2-methyl-
propy1)-5 -methoxy-N-methyl-benzamide
[5 -Chloro-2-methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -(3 -
morph lin-4-yl-azetidin- 1 -y1)-methanone
N-tert-Butyl-4-(4 -ethyl amino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-3 -
methoxy-benzamide
[3 -Fluoro-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-phenyl] -
morph lin-4 -
yl-methanone
[5 -Chloro-2-methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -(4-
oxetan-3 -yl-pip erazin- 1 -y1)-methanone
[5 -Chloro-2-methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
(3,3 -difluoro-pyrro lidin- 1 -y1)-methanone
[4-(4-Cyc lopropylamino-5 -tri fluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -
methoxy-
phenyl] -morph lin-4-yl-methanone
[4-(4-Azetidin- 1 -y1-5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -
methoxy-phenyl] -
morph lin-4-yl-methanone
-Fluoro-N-(2 -hydroxy-2 -methyl-propy1)-2 -methoxy-4-(4 -methylamino -5 -
trifluoromethyl-
pyrimidin-2-ylamino)-benzamide
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-5-fluoro-N-(2-
hydroxy-2-methyl-
propy1)-2-methoxy-benzamide
5 -Fluoro-N-(2 -hydroxy-2 -methyl-propy1)-2 -methoxy-N-methy1-4 -(4-
methylamino-5 -
trifluoromethyl-pyrimidin-2-ylamino)-benzamide
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-5-fluoro-N-(2-
hydroxy-2-methyl-
propy1)-2-methoxy-N-methyl-benzamide
[4-(4-Ethyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2,5 -difluoro-
phenyl] -morpholin-4-
yl-methanone
[2-F luoro-4-(4 -is opropylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-5 -
methoxy-phenyl] -
morpholin-4-yl-methanone
[3 -Methoxy-2-methy1-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morph lin-4-yl-methanone
[4-(4-Ethyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -
methoxy-phenyl] -(1 -
oxa-6-aza-spiro [3.3 ] hept-6-y1)-methanone
2-Fluoro-N-(2-hydroxy-2-methyl-propy1)-5 -methoxy-4-(4 -methylamino -5 -
trifluoromethyl-
pyrimi din-2 -ylamino)-b enzamide
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((3 S,4 S)-3 ,4-Difluoro-pyrro lidin- 1-y1)- [2 -fluoro-5 -methoxy-4-(4-
methylamino-5 -
trifluoromethyl-pyrimidin-2-ylamino)-phenyl] -methanone
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-5 -methoxy-
N-methyl-
b enzamide
2-Fluoro-N-(2-hydroxy-2-methyl-propy1)-3 -methoxy-4-(4 -methylamino -5 -
trifluoromethyl-
pyrimidin-2-ylamino)-benzamide
(3,3 -Difluoro-pyrro lidin- 1 -y1)45 -(2 -fluoro-ethoxy)-2-methoxy-4-(4 -
methylamino-5 -
tri fluoromethyl-pyrimidin-2-ylamino)-phenyl] -methanone
[5 -Chloro-2-methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -( 1 -
oxa-6-aza-spiro [3.3 ]hept-6-y1)-methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2,3 -difluoro-
phenyl] -morpholin-4-
yl-methanone
[4-(4-Cyc lopropylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-5 -methoxy-2 -
methyl-
phenyl] -morpholin-4-yl-methanone
[5 -Chloro-4-(4 -ethyl amino-5-trifluoromethyl-pyrimidin-2-ylamino)-2-methoxy-
phenyl] -(2-
oxa-6-aza-spiro [3.3 ]hept-6-y1)-methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-5 -methoxy-2 -methyl-
phenyl] -
morpholin-4-yl-methanone
4-(4-Cyclopropylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-N-(2-
hydroxy-2-
methyl-propy1)-5 -methoxy-benzamide
N-tert-Butyl-4 -(5 -cyano-4- ethylamino-pyrimidin-2 -ylamino)-3 -methoxy-b
enzami de
[3 -Difluoromethoxy-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
morpholin-4-yl-methanone
[2-Fluoro-5 -(2 -fluoro-ethoxy)-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-
2 -ylamino)-
phenyl] -morpholin-4-yl-methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -(2 -
fluoro-ethoxy)-
phenyl] -morpholin-4-yl-methanone
[2-Fluoro-5 -methoxy-4 -(4 -methoxy-5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morpholin-4-yl-methanone
[4-(4-Cyclopropylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-2,3 -difluoro-
phenyl] -
morpholin-4-yl-methanone
[4-(4-Cyclopropylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-2,5 -difluoro-
phenyl] -
morpholin-4-yl-methanone
[2-Chloro-5-fluoro-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-
pheny1]-
morpholin-4-yl-methanone
4-Ethylamino-2- [4-((S)-3 -fluoro -pyrro lidine- 1 -carbonyl)-2-methoxy-
phenylamino] -
pyrimidine-5 -carb nitrite
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-Chloro-2-methoxy-N-methy1-4-(4-methyl amino-5 -trifluoromethyl-pyrimidin-2-
ylamino)-N-
( 1 -methyl- 1 H-pyrazol-4 -y1)-b enzamide
(3,3 -Difluoro-pyrro lidin- 1 -y1)- [444 -ethylamino-5 -trifluoromethyl-
pyrimidin-2-ylamino)-2,5 -
dimethoxy-phenyl] -methanone
[4-(5-Chloro-4-methoxy-pyrimidin-2-ylamino)-3 -(2-fluoro-ethoxy)-phenyl] -
morpho lin-4 -yl-
methanone
[2-Methoxy-5 -methy1-4 -(4 -methylamino-5 -trifluoromethyl-pyrimi din-2 -
ylamino)-phenyl] -
morpholin-4-yl-methanone
[2-Chloro-4-(4 -ethyl amino-5-trifluoromethyl-pyrimidin-2-ylamino)-5 -fluoro-
phenyl] -
morpholin-4-yl-methanone
[3 -Chloro-2-fluoro-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-
pheny1]-
morpholin-4-yl-methanone
(3,3 -Difluoro-pyrro lidin- 1 -y1)- [4-(4 -ethylamino-5 -trifluoromethyl-
pyrimidin-2-ylamino)-5 -
(2-fluoro-ethoxy)-2-methoxy-phenyl] -methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2,6-difluoro-3 -
methoxy-phenyl] -
morpholin-4-yl-methanone
[2,6-Difluoro-3 -methoxy-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-
ylamino)-phenyl] -
morpholin-4-yl-methanone
[4-(4-Ethoxy-5 -trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-5 -methoxy-
phenyl] -
morpholin-4-yl-methanone
[2,5 -Dimethoxy-4 -(4-methylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
pheny1]-
morpholin-4-yl-methanone
(3,3 -Difluoro-pyrro lidin- 1 -y1)- [4-(4 -ethylamino-5 -trifluoromethyl-
pyrimidin-2-ylamino)-5 -
fluoromethoxy-2-methoxy-phenyl] -methanone
[5 -Methoxy-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-2 -
trifluoromethyl-
phenyl] -morpholin-4-yl-methanone
[445 -Chloro-4 -methylamino-pyrimidin-2-ylamino)-5 -methoxy-2 -trifluoromethyl-
phenyl] -
morpholin-4-yl-methanone
[5 -Chloro-2-fluoro-4 -(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-
pheny1]-
morpholin-4-yl-methanone
[4-(4-Cyclopropylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-2-fluoro-5 -(2 -
fluoro-
ethoxy)-phenyl] -morpholin-4-yl-methanone
[5 -Chloro-4-(4 -ethyl amino-5-trifluoromethyl-pyrimidin-2-ylamino)-2-methyl-
phenyl] -
morpholin-4-yl-methanone
[5 -Chloro-4-(4 -ethyl amino-5-trifluoromethyl-pyrimidin-2-ylamino)-2-
fluoromethoxy-
phenyl] -morpholin-4-yl-methanone
[5 -Fluoro-2-methy1-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-
phenyl] -
morpholin-4-yl-methanone
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[5 -Chloro-4-(4 -ethyl amino-5-tri fluoromethyl-pyrimidin-2-ylamino)-2-fluoro-
phenyl] -
morpholin-4-yl-methanone
{ 2-Fluoro-4 - [4-(( 1 R,2 S)-2 -fluoro-cyc lopropylamino)-5 -trifluoromethyl-
pyrimidin-2 -
ylamino] -5 -methoxy-phenyl} -morpho lin-4 -yl-methanone
(2,2-Dimethyl-morpho lin-4-y1)- [2-fluoro -5 -methoxy-4 -(4-methylamino-5 -
trifluoromethyl-
pyrimidin-2 -ylamino)-phenyl] -methanone
[3 -Cyc loprop oxy-4-(4 -methyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morph lin-4-yl-methanone
[5 -Chloro-2-difluoromethoxy-4 -(4- ethylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-
phenyl] -morph lin-4-yl-methanone
[5 -Chloro-2-difluoromethoxy-4 -(4-methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-
phenyl] -morph lin-4-yl-methanone
4-Ethylamino-2- [5 -fluoro-4-((S)-3 -fluoro-pyrro lidine- 1 -carbonyl)-2-
methoxy-phenylamino] -
pyrimidine-5 -carb onitrile
4-Ethylamino-2- [2 -fluoro-4-(( S)-3 -fluoro-pyrro lidine- 1 -carbonyl)-5 -
methoxy-phenylamino] -
pyrimidine-5 -carb onitrile
[3 -Chloro-2,6 -dimethoxy-4 -(4 -methyl amino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-
phenyl] -morph lin-4-yl-methanone
(2,2-Dimethyl-morpho lin-4 -y1)- [3 -methoxy-4-(4 -methylamino -5 -
trifluoromethyl-pyrimidin-
2-ylamino)-phenyl] -methanone
[5 -(2-Fluoro-ethoxy)-2 -methy1-4 -(4-methyl amino-5 -tri fluoromethyl-
pyrimidin-2-ylamino)-
phenyl] -morph lin-4-yl-methanone
[5 -(2-Fluoro-ethoxy)-2 -methoxy-4 -(4-methylamino-5 -trifluoromethyl-
pyrimidin-2-ylamino)-
phenyl] -pyrro lidin- 1 -yl-methanone
(5 -chloro-4-(4 -(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-y1 amino)-2-
methoxyphenyl)(p erdeuteromorpholino)methanone
[445 -Chloro-4 -methoxy-pyrimidin-2 -ylamino)-2,5 -dimethoxy-phenyl] -
morpholin-4-yl-
methanone
[5 -Fluoromethoxy-2-methy1-4 -(4 -methylamino -5 -trifluoromethyl-pyrimidin-2 -
ylamino)-
phenyl] -morph lin-4-yl-methanone
{ 4 - [Ethyl-(4-methylamino-5 -trifluoromethyl-pyrimidin-2 -y1)-amino] -5 -
fluoromethoxy-2 -
methyl-phenyl } -morpholin-4-yl-methanone
[5 -Fluoromethoxy-2-methoxy-4 -(4 -methylamino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-
phenyl] -pyrro lidin- 1 -yl-methanone
[4-(4-Cyc lobutylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -
methoxy-phenyl] -
morpholin-4-yl-methanone
[2-Fluoro-5-methy1-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morpholin-4-yl-methanone
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[445 -Chloro-4 -methoxy-pyrimidin-2 -ylamino)-5 -methoxy-2-methyl-phenyl] -
morpholin-4-yl-
methanone
[445 -Chloro-4 -ethoxy-pyrimidin-2 -ylamino)-5 -methoxy-2 -methyl-phenyl] -
morph lin-4 -yl-
methanone
[2-Fluoro-5-fluoromethoxy-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-
ylamino)-
phenyl] -pyrro lidin- 1 -yl-methanone
4-Cyc lopropylamino-2 - [4-(( S)-3 -fluoro-pyrro lidine- 1 -c arb ony1)-2 -
methoxy-phenylamino] -
pyrimi dine-5 -carb onitrile
4-Ethylamino-2- [4-((S)-3 -fluoro-pyrro lidine- 1 -carbonyl)-2-methoxy-5 -
methyl-phenylamino] -
pyrimi dine-5 -carb onitrile
4-Ethylamino-2- [2 -fluoro-4-(( S)-3 -fluoro-pyrro lidine- 1 -carbonyl)-
phenylamino] -pyrimidine-
-carb onitrile
[5 -Is oprop oxy-2 -methy1-4 -(4-methylamino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-phenyl] -
morph lin-4-yl-methanone
[5 -Ethoxy-2-methy1-4-(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-
phenyl] -
morpholin-4-yl-methanone
[5 -F luoromethoxy-2 -methy1-4-(4 -methylamino -5 -trifluoromethyl-pyrimidin-2
-ylamino)-
phenyl] -pyrro lidin- 1 -yl-methanone
[5 -Chloro-4-(4 -cyclopropylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-2-
fluoromethoxy-
phenyl] -morpholin-4-yl-methanone
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-5 -fluoro-2-methyl-
phenyl] -
morpholin-4-yl-methanone
(5 -methoxy-2-methy1-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)phenyl)(p erdeuteromorpholino)methanone
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-N-(2-hydroxy-2-
methyl-propy1)-5-
methoxy-2-methyl-b enzamide
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2 -ylamino)-2-fluoro-N-(2-
hydroxy- 1 , 1 -
dimethyl-ethyl)-5-methoxy-benzamide
2- [2 -Chloro-4-(( S)-3 -fluoro-pyrro lidine- 1 -carbonyl)-5 -methyl-
phenylamino] -4 -ethylamino-
pyrimi dine-5 -carb onitrile
2- [2 -Chloro-4-(( S)-3 -fluoro-pyrro lidine- 1 -carbonyl)-phenylamino] -4 -
ethylamino-pyrimidine-
5 -carb onitrile
[5 -Cycl opropylmethoxy-2-methy1-4 -(4-methylamino -5 -trifluoromethyl-
pyrimidin-2-
ylamino)-phenyl] -morpho lin-4 -yl-methanone
[5 -F luoromethoxy-2 -methoxy-4-(4 -methylamino -5 -trifluoromethyl-pyrimidin-
2-ylamino)-
phenyl] -pip eridin- 1 -yl-methanone
[5 -Chloro-4-(4 -ethylamino-5 -tri fluoromethyl-pyrimidin-2 -ylamino)-2 -
fluoromethoxy-
phenyl] -pyrro lidin- 1 -yl-methanone
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[4-(4-Cyclobutylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-5 -fluoro-2 -
methoxy-phenyl] -
morpholin-4-yl-methanone
[5 -Chloro-4-(4 -cycl obutylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2-
methoxy-phenyl] -
morpholin-4-yl-methanone
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2 -ylamino)-2-fluoro-N-( 1 -
hydroxymethyl-
cyclopropy1)-5-methoxy-benzamide
4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2 -ylamino)-2-fluoro-N-( 1 -
hydroxy-
cyclopropylmethyl)-5 -methoxy-benzamide
N-(2-Hydroxy-2-methyl-propy1)-5-methoxy-2-methy1-4-(4-methylamino-5-
trifluoromethyl-
pyrimidin-2-ylamino)-benzamide
2- [2 -Chloro-4-(( S)-3 -fluoro-pyrro lidine- 1 -carbonyl)-5 -methoxy-
phenylamino] -4 -ethylamino-
pyrimidine-5 -carb nitrite
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2 -fluoro-5 -methyl-
phenyl] -
morpholin-4-yl-methanone
[445 -Chloro-4 -methoxy-pyrimidin-2 -y1 amino)-5 -fluoromethoxy-2 -methyl-
phenyl] -
pyrro lidin- 1 -yl-methanone
-Fluoromethoxy-2-methoxy-N,N-dimethy1-4-(4-methylamino-5 -trifluoromethyl-
pyrimidin-
2-ylamino)-benzamide
[4-(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-5 -fluoro-2-
fluoromethoxy-phenyl] -
pyrro lidin- 1 -yl-methanone
[3 -Fluoro-2,6 -dimethoxy-4-(4 -methyl amino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-phenyl] -
morph lin-4-yl-methanone
(44442,3 -tetradeutero-cyclopropylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)-2-fluoro-
5 -methoxyphenyl)(morpho lino)methanone
[5 -Methoxy-4-(4 -methyl amino-5 -trifluoromethyl-pyrimi din-2 -ylamino)-2 -
trifluoromethoxy-
phenyl] -morph lin-4-yl-methanone
[4-(4 -Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-2,5 -dimethoxy-
phenyl] -morph lin-
4-yl-methanone
[2-Chloro-5 -methy1-4 -(4 -methylamino-5 -trifluoromethyl-pyrimidin-2 -
ylamino)-phenyl] -
morpholin-4-yl-methanone
(4-(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-2-fluoro-5-
methoxyphenyl)(p erdeuteromorpholino)methanone
(5 -bromo-2 -methoxy-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimidin-2 -
ylamino)phenyl) (morpholino)methanone
[4-(4-Cyclobutylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-5 -methoxy-2-
methyl-
phenyl] -morpholin-4-yl-methanone or
[2,5 -Dimethy1-4 -(4-methyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
phenyl] -
morpholin-4-yl-methanone.
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[0366] In another embodiment, the LRRK2 modulators are of formula II:
R1
X
R2j R4
N '/N
)L
N%...\--N
N
H R5 I
or pharmaceutically acceptable salts thereof,
wherein:
X is: -N1r-; or -0- wherein Ra is hydrogen or Ci_6alkyl;
R1 is: Ci_6alkyl; C2_6alkenyl; C2_6alkynyl; halo-Ci_6alkyl; Ci_6alkoxy-
Ci_6alkyl;
hydroxy-Ci_6alkyl; amino-Ci_6alkyl; Ci_6alkylsulfonyl-Ci_6alkyl;
C3_6cycloalkyl optionally
substituted one or more times with Ci_6alkyl; C3_6cycloalkyl-Ci_6alkyl wherein
the C3_
6cycloalkyl portion is optionally substituted one or more times with
Ci_6alkyl; heterocyclyl
optionally substituted one or more times with R7; or heterocyclyl-Ci_6alkyl
optionally
substituted one or more times with R7;
or X and R1 together form Ci_6alkyl; Ci_6alkoxy-Ci_6alkyl; C3_6cycloalkyl
optionally
substituted one or more times with R6; or C3_6cycloalkyl-Ci_6alkyl wherein the
C3_6cycloalkyl
portion is optionally substituted one or more times with R6;
or R1 and Ra together with the atoms to which they are attached may form a
three- to
six-membered heterocyclic ring optionally substituted one or more times with
R7;
R2 is: Ci_6alkyl; halo; Ci_6alkoxy; cyano; C2_6alkynyl; C2_6alkenyl; halo-
Ci_6alkyl;
halo-Ci_6alkoxy; C3_6cycloalkyl optionally substituted one or more times with
R6; C3_
6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is optionally
substituted one or more
times with R6; -ORb wherein Rb is Ci_6alkyl, C3_6cycloalkyl optionally
substituted one or more
times with R6, or C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion
is optionally
substituted one or more times with R6; or -C(0)-Re wherein Re is Ci_6alkyl,
Ci_6alkoxy, amino,
or heterocyclyl optionally substituted one or more times with R7;
R3 is: hydrogen; Ci_6alkyl; halo-Ci_6alkyl; C2_6alkenyl; C2_6alkynyl; hydroxy-
Ci_6alkyl;
Ci_6alkoxy-Ci_6alkyl; cyano-Ci_6alkyl; Ci_6alkylsulfonyl;
Ci_6alkylsulfonylCi_6alkyl; amino-C1_
6alkyl; C3_6cycloalkyl optionally substituted one or more times with R6;
C3_6cycloalkyl-C1_
6alkyl wherein the C3_6cycloalkyl portion is optionally substituted one or
more times with R6;
C3_6cycloalkyl-sulfonyl wherein the C3_6cycloalkyl portion is optionally
substituted one or
more times with R6; heterocyclyl optionally substituted one or more times with
R7;
heterocyclyl-Ci_6alkyl wherein the heterocyclyl portion is optionally
substituted one or more
times with R7; aryl optionally substituted one or more times with Rs; aryl-
Ci_6alkyl wherein
the aryl portion is optionally substituted one or more times with Rs;
heteroaryl optionally
substituted one or more times with Rs; heteroaryl-Ci_6alkyl wherein the
heteroaryl portion is
optionally substituted one or more times with Rs;or -Y-C(0)-Rd;
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Y is C2_6alkylene or a bond;
Rd is Ci_6alkyl, Ci_6alkoxy, amino, Ci_6alkyl-amino, di-Ci_6alkyl-amino, halo-
C1_
6alkyl-amino, di-halo-Ci_6alkyl-amino, halo-Ci_6alkyl, hydroxy-Ci_6alkyl,
hydroxy, CI_
6alkoxy-Ci_6alkyl, cyano-Ci_6alkyl, Ci_6alkylsulfonylCi_6alkyl, amino-
Ci_6alkyl, C3_6cycloalkyl
optionally substituted one or more times with R6, C3_6cycloalkyl-Ci_6alkyl
wherein the C3_
6cycloalkyl portion is optionally substituted one or more times with
R6rheterocycly1
optionally substituted one or more times with R7, or heterocyclyl-Ci_6alkyl
wherein the
heterocyclyl portion is optionally substituted one or more times with R7;
R4 is: hydrogen; Ci_6alkyl; halo; cyano; halo-Ci_6alkyl; C2_6alkenyl;
C2_6alkynyl; CI_
6alkoxy; Ci_6alkoxy-Ci_6alkyl; hydroxy-Ci_6alkyl; C3_6cycloalkyl optionally
substituted one or
more times with R6; C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl
portion is optionally
substituted one or more times with R6; or
R5 is: hydrogen; or Ci_6alkyl;
each R6 is independently: Ci_6alkyl; halo-Ci_6alkyl; Ci_6alkoxy; oxo; cyano;
halo; or
Y-C(0)-Rd;
each R7 is independently: Ci_6alkyl; halo-Ci_6alkyl; halo; oxo; Ci_6alkoxy;
C1_
6alkylsulfonyl; Ci_6alkoxy-Ci_6alkyl; cyano; -Y-C(0)-Rd; heterocyclyl;
heterocyclyl-Ci_6alkyl;
C3_6cycloalkyl; C3_6cycloalkyl-Ci_6alkyl; or C3_6cycloalkylsulfonyl; and
each R8 is independently: oxo; Ci_6alkyl; halo-Ci_6alkyl; halo; Ci_6alkyl-
sulfonyl; C1_
6alkoxy; Ci_6alkoxy-Ci_6alkyl; cyano; hetoeryclyl; heterocyclyl-Ci_6alkyl; -Y-
C(0)-Rd; C3_
6cYcloalkyl, C3_6cycloalkyl-Ci_6alkyl, or C3_6cycloalkyl-sulfonyl.
[0367] In certain embodiments of formula II, X is -NRa-or -0-.
[0368] In certain embodiments of formula II, X is -NRa.
[0369] In certain embodiments of formula II, X is -0-.
[0370] In certain embodiments of formula II, X is -NH-or -0-.
[0371] In certain embodiments of formula II, X is -NH-.
[0372] In certain embodiments of formula II, X is -0-.
[0373] In certain embodiments of formula II, Ra is hydrogen.
[0374] In certain embodiments of formula II, Ra is Ci_6alkyl.
[0375] In certain embodiments of formula II, R1 is: Ci_6alkyl;
C2_6alkenyl; C2_
6alkynyl; halo-Ci_6alkyl; Ci_6alkoxy-Ci_6alkyl; hydroxy-Ci_6alkyl; amino-
Ci_6alkyl; C1_
6alkylsulfonyl-Ci_6alkyl; C3_6cycloalkyl optionally substituted with
Ci_6alkyl; C3_6cycloalkyl-
Ci_6alkyl wherein the C3_6cycloalkyl portion is optionally substituted with
Ci_6alkyl;
heterocyclyl; or heterocyclyl-Ci_6alkyl.
[0376] In certain embodiments of formula II, R1 is: Ci_6alkyl; halo-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; amino-Ci_6alkyl; Ci_6alkylsulfonyl-Ci_6alkyl;
C3_6cycloalkyl; or C3_
6CYCloalkyl-Ci_6alkyl.
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[0377] In certain embodiments of formula II, Ri is: Ci_6allcyl;
C3_6cycloalkyl
optionally substituted with Ci_6alkyl; or C3_6cycloalkyl-Ci_6alkyl wherein the
C3_6cycloalkyl
portion is optionally substituted with Ci_6alkyl.
[0378] In certain embodiments of formula II, R1 is: Ci_6alkyl; halo-
Ci_6alkyl; CI_
6alkoxy-Ci_6alkyl; amino-Ci_6alkyl; Ci_6alkylsulfonyl-Ci_6alkyl;
tetrahydrofuranyl;
tetrahydrofuranyl-Ci_6alkyl; oxetanyl; or oxetan-Ci_6alkyl.
[0379] In certain embodiments of formula II, R1 is: Ci_6alkyl; halo-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; amino-Ci_6alkyl; or Ci_6alkylsulfonyl-Ci_6alkyl.
[0380] In certain embodiments of formula II, R1 is Ci_6alkyl or halo-
Ci_6alkyl.
[0381] In certain embodiments of formula II, R1 is Ci_6alkyl.
[0382] In certain embodiments of formula II, R1 is halo-Ci_6alkyl.
[0383] In certain embodiments of formula II, R1 is Ci_6alkoxy-Ci_6alkyl.
[0384] In certain embodiments of formula II, R1 is amino-Ci_6alkyl.
[0385] In certain embodiments of formula II, R1 is Ci_6alkylsulfonyl-
Ci_6alkyl
optionally substituted with C44a1k54.
[0386] In certain embodiments of formula II, R1 is C3_6cycloalkyl
optionally
substituted with Ci_6alkyl.
[0387] In certain embodiments of formula II, R1 is C3_6cycloalkyl-Ci_6alkyl
wherein
the C3_6cycloalkyl portion is optionally substituted with Ci_6alkyl.
[0388] In certain embodiments of formula II, R1 is heterocyclyl or
heterocyclyl-C1_
6alkyl.
[0389] In embodiments of formula II wherein R1 is heterocyclyl or
heterocyclyl-C1_
6alkyl, such heterocyclyl may be piperidinyl, piperazinyl, morpholinyl,
tetrahydropyranyl,
pyrrolidinyl, tetrahydrofuranyl or oxetanyl, each optionally substituted as
defined herein.
[0390] In embodiments of formula II wherein R1 is heterocyclyl or
heterocyclyl-C1_
6alkyl, such heterocyclyl may be tetrahydropyranyl, piperidinyl,
tetrahydrofuranyl or
oxetanyl, each optionally substituted as defined herein..
[0391] In certain embodiments of formula II, R1 is tetrahydrofuranyl.
[0392] In certain embodiments of formula II, R1 is tetrahydropyranyl.
[0393] In certain embodiments of formula II, R1 is tetrahydrofuranyl-
Ci_6alkyl or
oxetanyl.
[0394] In certain embodiments of formula II, R1 is tetrahydrofuranyl-
Ci_6alkyl.
[0395] In certain embodiments of formula II, R1 is oxetanyl.
[0396] In certain embodiments of formula II, R1 is or oxetan-Ci_6alkyl.
[0397] In certain embodiments of formula II, R1 is: methyl; ethyl; n-
propyl;
isopropyl; isobutyl; 3,3-dimethylpropyl; cyclopropyl; cyclobutyl; cyclopentyl;
cyclohexyl;
cyclopropylmethyl; cyclobutylmethyl; cyclopentylmethyl; cyclopropylethyl;
methoxyethyl;
oxetanyl; tetrahydropyranyl; 2,2-difluoroethyl; or tetrahydrofuranylmethyl.
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[0398] In certain embodiments of formula II, R1 is: methyl; ethyl; n-
propyl;
isopropyl; isobutyl; 3,3-dimethylpropyl; cyclopentyl; cyclohexyl;
cyclopropylmethyl;
cyclobutylmethyl; cyclopentylmethyl; cyclopropylethyl; methoxyethyl; oxetanyl;
tetrahydropyranyl; 2,2-difluoroethyl ; or tetrahydrofuranylmethyl.
[0399] In certain embodiments of formula II, R1 is: methyl; ethyl; n-
propyl;
isopropyl; isobutyl; 3,3-dimethylpropyl; cyclopentyl; cyclohexyl;
cyclopentylmethyl;
methoxyethyl; oxetanyl; tetrahydropyranyl; or tetrahydrofuranylmethyl.
[0400] In certain embodiments of formula II, R1 is 2,2-difluoroethyl.
[0401] In certain embodiments of formula II, R1 is: methyl; ethyl; n-
propyl;
isopropyl; or isobutyl.
[0402] In certain embodiments of formula II, R1 is methyl or ethyl.
[0403] In certain embodiments of formula II, R1 is methyl.
[0404] In certain embodiments of formula II, R1 is ethyl.
[0405] In certain embodiments of formula II, R1 is: cyclopropyl;
cyclobutyl;
cyclopentyl; cyclohexyl; cyclopropylmethyl; cyclobutylmethyl;
cyclopentylmethyl; or
cyclopropylethyl.
[0406] In certain embodiments of formula II, R1 is: cyclopentyl;
cyclohexyl; or
cyclopentylmethyl.
[0407] In certain embodiments of formula II, R1 is: cyclopropyl.
[0408] In certain embodiments of formula II, R1 and Ra together with the
atoms to
which they are attached may form a three- to six-membered heterocyclic ring.
[0409] In certain embodiments of formula II, R1 and Ra together with the
atoms to
which they are attached may form a three-membered heterocyclic ring.
[0410] In certain embodiments of formula II, R1 and Ra together with the
atoms to
which they are attached may form a four-membered heterocyclic ring.
[0411] In certain embodiments of formula II, R1 and Ra together with the
atoms to
which they are attached may form a five-membered heterocyclic ring.
[0412] In certain embodiments of formula II, R1 and Ra together with the
atoms to
which they are attached may form a six-membered heterocyclic ring.
[0413] In certain embodiments of formula II, X and R1 together form
Ci_6alkyl; C3_
6cycloalkyl optionally substituted with Ci_6alkyl; or C3_6cycloalkyl-Ci_6alkyl
wherein the C3_
6cYcloalkyl portion is optionally substituted with Ci_6alkyl.
[0414] In certain embodiments of formula II, X and R1 together form
Ci_6alkyl.
[0415] In certain embodiments of formula II, X and R1 together form
C3_6cycloalkyl
optionally substituted with Ci_6alkyl.
[0416] In certain embodiments of formula II, X and R1 together form
C3_6cycloalkyl-
Ci_6alkyl wherein the C3_6cycloalkyl portion is optionally substituted with
Ci_6alkyl.
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[0417] In certain embodiments of formula II, R2 is: Ci_6alkyl; halo;
Ci_6alkoxy;
cyano; C2_6alkynyl; C2_6alkenyl; halo-Ci_6alkyl; halo-Ci_6alkoxy;
C3_6cycloalkyl optionally
substituted with Ci_6alkyl; C3_6cycloalkyl-Ci_6alkyl wherein the
C3_6cycloalkyl portion is
optionally substituted with Ci_6alkyl; -ORb wherein Rb is Ci_6alkyl,
C3_6cycloalkyl optionally
substituted with Ci_6alkyl, or C3_6cycloalkyl-Ci_6alkyl wherein the
C3_6cycloalkyl portion is
optionally substituted with Ci_6alkyl; or -C(0)-Re.
[0418] In certain embodiments of formula II, R2 is: halo; Ci_6alkoxy; halo-
Ci_6alkyl;
halo-Ci_6alkoxy; C3_6cycloalkyl wherein the C3_6cycloalkyl portion is
optionally substituted
with Ci_6alkyl; C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is
optionally
substituted with Ci_6alkyl; tetrahydrofuranyl; tetrahydrofuranyl-Ci_6alkyl;
oxetanyl; or oxetan-
Ci_6alkyl.
[0419] In certain embodiments of formula II, R2 is: halo; Ci_6alkoxy; halo-
Ci_6alkyl;
cyano; C2_6alkynyl; C2_6alkenyl; C3_6cycloalkyl; or C3_6cycloalkyl-Ci_6alkyl.
[0420] In certain embodiments of formula II, R2 is: halo; Ci_6alkoxy; halo-
Ci_6alkyl;
cyano; C3_6cycloalkyl; or C3_6cycloalkyl-Ci_6alkyl.
[0421] In certain embodiments of formula II, R2 is: halo; Ci_6alkoxy; halo-
Ci_6alkyl;
C3_6cycloalkyl; or C3_6cycloalkyl-Ci_6alkyl.
[0422] In certain embodiments of formula II, R2 is: halo; halo-Ci_6alkyl or
cyano.
[0423] In certain embodiments of formula II, R2 is: fluoro; bromo; chloro;
iodo;
trifluoromethyl; or cyano.
[0424] In certain embodiments of formula II, R2 is: chloro;
trifluoromethyl; or cyano.
[0425] In certain embodiments of formula II, R2 is: halo; or halo-
Ci_6alkyl.
[0426] In certain embodiments of formula II, R2 is Ci_6alkyl.
[0427] In certain embodiments of formula II, R2 is halo.
[0428] In certain embodiments of formula II, R2 is Ci_6alkoxy.
[0429] In certain embodiments of formula II, R2 is halo-Ci_6alkoxy.
[0430] In certain embodiments of formula II, R2 is halo-Ci_6alkyl.
[0431] In certain embodiments of formula II, R2 is C3_6cycloalkyl.
[0432] In certain embodiments of formula II, R2 is C3_6cycloalkyl-
Ci_6alkyl.
[0433] In certain embodiments of formula II, R2 is tetrahydrofuranyl.
[0434] In certain embodiments of formula II, R2 is tetrahydrofuranyl-
Ci_6alkyl.
[0435] In certain embodiments of formula II, R2 is oxetanyl.
[0436] In certain embodiments of formula II, R2 is oxetan-Ci_6alkyl.
[0437] In certain embodiments of formula II, R2 is fluoro, chloro or bromo.
[0438] In certain embodiments of formula II, R2 is chloro.
[0439] In certain embodiments of formula II, R2 is fluoro.
[0440] In certain embodiments of formula II, R2 is bromo.
104411 In certain embodiments of formula II, R2 is bromo.
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[0442] In certain embodiments of formula II, R2 is iodo.
[0443] In certain embodiments of formula II, R2 is trifluoromethyl.
[0444] In certain embodiments of formula II, R2 is methoxy.
[0445] In certain embodiments of formula II, R2 is cyano.
[0446] In certain embodiments of formula II, R2 is C2_6alkynyl.
[0447] In certain embodiments of formula II, R2 is C2_6alkenyl.
[0448] In certain embodiments of formula II, R2 is -ORb wherein Rb is
Ci_6alkyl, C3_
6cycloalkyl optionally substituted with Ci_6alkyl, or C3_6cycloalkyl-Ci_6alkyl
wherein the C3_
6cycloalkyl portion is optionally substituted with Ci_6alkyl.
[0449] In certain embodiments of formula I, R2 is -C(0)-Re wherein Re is
Ci_6alkyl,
Ci_6alkoxy, amino, or heterocyclyl.
[0450] In certain embodiments of formula II, R3 is: Ci_6alkyl; halo-
Ci_6alkyl; C2-
6alkenYl; C2_6alkynyl; hydroxy-Ci_6alkyl; Ci_6alkoxy-Ci_6alkyl; cyano-
Ci_6alkyl; C1_
6alkYlsulfonyl; Ci_6alkylsulfonylCi_6alkyl; amino-Ci_6alkyl; C3_6cycloalkyl
optionally
substituted one or more times with R6; C3_6cycloalkyl-Ci_6alkyl wherein the
C3_6cycloalkyl
portion is optionally substituted one or more times with R6; heterocyclyl
optionally
substituted one or more times with R7; heterocyclyl-Ci_6alkyl wherein the
heterocyclyl portion
is optionally substituted one or more times with R7; aryl optionally
substituted one or more
times with Rs; heteroaryl optionally substituted one or more times with Rs; or
-Y-C(0)-Rd.
[0451] In certain embodiments of formula II, R3 is: hydrogen; Ci_6alkyl;
halo-C1-
6alkYl; C2-6alkenyl; C2_6alkynyl; hydroxy-Ci_6alkyl; C 1_6 alkoxy-Ci_6alkyl;
cyano-Ci_6alkyl; C1_
6alkylsulfonyl; Ci_6alkylsulfonylalkyl; amino-Ci_6alkyl; C3_6cycloalkyl
optionally substituted
with Ci_6alkyl; C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is
optionally
substituted with Ci_6alkyl; heterocyclyl; heterocyclyl-Ci_6alkyl; aryl;
heteroaryl; or -C(0)-Re.
[0452] In certain embodiments of formula II, R3 is: Ci_6alkyl; halo-
Ci_6alkyl;
hydroxy-Ci_6alkyl; Ci_6alkoxy-Ci_6alkyl; C3_6cycloalkyl optionally substituted
with Ci_6alkyl;
C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is optionally
substituted with C1_
6alkyl; heterocyclyl; heterocyclyl-Ci_6alkyl; or -C(0)-Rb wherein Rb is
Ci_6alkyl, Ci_6alkoxy,
amino, or heterocyclyl.
[0453] In certain embodiments of formula II, R3 is: Ci_6alkyl; hydroxy-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; C3_6cycloalkyl optionally substituted with Ci_6alkyl;
C3_6cycloalkyl-C1_
6alkyl wherein the C3_6cycloalkyl portion is optionally substituted with
Ci_6alkyl; heterocyclyl;
heterocyclyl-Ci_6alkyl; or -C(0)-Re wherein Re is Ci_6alkyl, Ci_6alkoxy,
amino, or
heterocyclyl.
[0454] In certain embodiments of formula II, R3 is: Ci_6alkyl; hydroxy-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; C3_6cycloalkyl optionally substituted one or more times
with R6; C3_
6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is optionally
substituted one or more
times with R6; heterocyclyl optionally substituted one or more times with R7;
heterocyclyl-C1_
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6alkyl wherein the heterocyclyl portion is optionally substituted one or more
times with R7; or
-C(0)-Rd.
[0455] In certain embodiments of formula II, R3 is: Ci_6alkyl; hydroxy-
Ci_6alkyl; C1_
6alkoxy-Ci_6alkyl; heterocyclyl; heterocyclyl-Ci_6alkyl; or -C(0)-Re wherein
Re is Ci_6alkyl,
Ci_6alkoxy, amino, or heterocyclyl.
[0456] In certain embodiments of formula II, R3 is: Ci_6alkyl; hydroxy-
Ci_6alkyl; CI_
6alkoxy-Ci_6alkyl; heterocyclyl; or heterocyclyl-Ci_6alkyl.
[0457] In certain embodiments of formula II, R3 is: Ci_6alkyl; hydroxy-
Ci_6alkyl; or
Ci_6alkoxy-Ci_6alkyl.
[0458] In embodiments of formula II wherein R3 is heterocyclyl or
heterocyclyl-C1_
6alkyl, such heterocyclyl may be piperidinyl, piperazinyl, morpholinyl,
tetrahydropyranyl,
pyrrolidinyl, tetrahydrofuranyl or oxetanyl.
[0459] In embodiments of formula II wherein R3 is heterocyclyl or
heterocyclyl-C1_
6alkyl, such heterocyclyl may be piperidinyl, piperazinyl, morpholinyl,
tetrahydropyranyl,
pyrrolidinyl, azetidinyl, tetrahydrofuranyl or oxetanyl, each optionally
substituted one or
more times, or one or two times, with R7 as defined herein.
[0460] In embodiments of formula II wherein R3 is heterocyclyl or
heterocyclyl-C1_
6alkyl, such heterocyclyl may be piperidinyl, morpholinyl, tetrahydropyranyl,
tetrahydrofuranyl or oxetanyl.
[0461] In embodiments of formula II wherein R3 is heterocyclyl or
heterocyclyl-C1_
6alkyl, such heterocyclyl may be piperidinyl, pyrrolidinyl, azetidinyl,
morpholinyl,
tetrahydropyranyl, tetrahydrofuranyl or oxetanyl, each optionally substituted
one or more
times, or one or two times, with R7 as defined herein.
[0462] In certain embodiments of formula II, R3 is: methyl; ethyl; n-
propyl;
isopropyl; 2-methoxy-ethyl; oxetan-3 -yl; 2 -(morpho lin-4 -y1)- ethyl ; 2-
hydroxy-2 -methyl-
propan-l-y1; tetrahydropyran-4-y1; or morpholin-4-yl-carbonyl.
[0463] In certain embodiments of formula I, R3 is: methyl; ethyl; n-propyl;
isopropyl; 2 -methoxy-ethyl ; oxetan-3 -yl; 2 -(morpho lin-4 -y1)- ethyl ; 2-
hydroxy-2 -methyl-
propan-l-y1; or tetrahydropyran-4-yl.
[0464] In certain embodiments of formula II, R3 is hydrogen.
[0465] In certain embodiments of formula II, R3 is Ci_6alkyl.
[0466] In certain embodiments of formula II, R3 is halo-Ci_6alkyl.
[0467] In certain embodiments of formula II, R3 is C2_6alkenyl.
[0468] In certain embodiments of formula II, R3 is C2_6alkynyl.
[0469] In certain embodiments of formula II, R3 is hydroxy-Ci_6alkyl.
[0470] In certain embodiments of formula II, R3 is Ci_6alkoxy-Ci_6alkyl.
[0471] In certain embodiments of formula II, R3 is C3_6cycloalkyl
optionally
substituted one or more times with R6.
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[0472] In certain embodiments of formula II, R3 is C3_6cycloalkyl
optionally
substituted with Ci_6alkyl.
[0473] In certain embodiments of formula II, R3 is C3_6cycloalkyl-Ci_6alkyl
wherein
the C3_6cycloalkyl portion is optionally substituted one or more times with
R6.
[0474] In certain embodiments of formula II, R3 isC3_6cycloalkyl-Ci_6alkyl
wherein
the C3_6cycloalkyl portion is optionally substituted with Ci_6alkyl.
[0475] In certain embodiments of formula II, R3 is heterocyclyl optionally
substituted one or more times with R7.
[0476] In certain embodiments of formula II, R3 is heterocyclyl.
[0477] In certain embodiments of formula II, R3 is heterocyclyl-Ci_6alkyl
wherein the
heterocyclyl portion is optionally substituted one or more times with R7.
[0478] In certain embodiments of formula II, R3 is heterocyclyl-Ci_6alkyl.
[0479] In certain embodiments of formula II, R3 is -C(0)-Re wherein Re is
C4_6a4417
G, . -- = = = - - .
[0480] In certain embodiments of formula II, R3 is cyano-Ci_6alkyl.
[0481] In certain embodiments of formula II, R3 is Ci_6alkylsulfonyl.
[0482] In certain embodiments of formula II, R3 is Ci_6alkylsulfonyl-
Ci_6alkyl.
[0483] In certain embodiments of formula II, R3 is amino-Ci_6alkyl.
[0484] In certain embodiments of formula II, R3 is aryl optionally
substituted one or
more times with Rs.
[0485] In certain embodiments of formula II, R3 is aryl.
[0486] In certain embodiments of formula II, R3 is phenyl optionally
substituted one
or more times, or one or two times, with Rs.
[0487] In certain embodiments of formula II, R3 is heteroaryl optionally
substituted
one or more times, or one or two times, with Rs.
[0488] In certain embodiments of formula II, R3 is heteroaryl.
[0489] In certain embodiments of formula II, R3 is C3_6cycloalkyl-sulfonyl
wherein
the C3_6cycloalkyl portion is optionally substituted one or more times with R6
[0490] In certain embodiments of formula II, R3 is: hydrogen; methyl;
ethyl; propyl;
isopropyl; butyl; cyclopropyl; cyclopropylmethyl; cyclobutyl; methanesulfonyl;
ethylsulfonyl;
cyclopropylsulfonyl; sec-butylsulfonyl; morpholin-4-yl-ethyl; oxetan-3-y1; 2-
methoxyethyl;
2-hydroxy-2-methyl-propyl; 3-hydroxy-2-methyl-propan-2-y1; 2-methoxy-propyl;
tetrahydro-
2H-pyran-4-y1; tetrahydrofuran-3-y1; 2,6-dimethyltetrahydro-2H-pyran-4-y1;
tetrahydro-2H-
pyran-3-y1); phenyl; 4-(methylsulfonyl)phenyl); 4-cyano-phenyl; 4-fluoro-
phenyl; 4-chloro-
phenyl; 3,5-difluorophenyl; 4-(dimethylamino-carbonyl)-phenyl); 4-
(cyclopropylsulfonyl)phenyl; 2,2,2-trifluoroethyl; 2-fluoroethyl;
difluoromethyl; 2-dimethyl-
1,3 -di oxan-5 -yl; 1 -methyl-cyclopropyl-carbonyl; 3 -methylpyridin-4-y1; 2-
methylpyridin-4-y1;
pyridin-2-y1; pyrimidin-2-y1; pyrimidin-5-y1; pyridin-2-ylmethyl; 1-(pyridin-2-
yl)ethyl;
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cyclopropylsulfonyl; 1-cyano-1 -methyl-ethyl (also called 2-cyano -prop an-2-
y1); 2-cyano-
ethyl; 1-cyano-ethyl; 2-cyano-2-methyl-propyl; 1-(2,2,2-
trifluoroethyl)piperidin-4-y1; 1-
(methylsulfonyl)azetidin-3 -yl; (3 -methyloxetan-3 -yl)methyl; (1 S,5 S)-8 -
oxabicyclo [3.2.1] octan-3 -yl; 1-(oxetan-3 -yl)pip eridin-4-y1; 1-acetyl-pip
eridin-4-y1; 1-
(cyc lopropyl-carb ony1)-p ip eridin-4-y1; 1-methyl-pip eridin-4-y1; 1-methy1-
2-oxo-p ip eridin-5-
yl; 2-oxo -pip eridin-5 -yl; 1-(is opropyl-c arb ony1)-pip eridin-4-y1; 1-
(oxetan-3-yl)azetidin-3 -yl;
1-(cyclopropyl-carbony1)-piperidin-4-y1; 2-methoxycyclopentyl; 3 -
methoxycyclopentyl; 1-
methoxy-2-methylprop an-2-y1; tetrahydro-2H-1,1 -dioxo-thiopyran-4-y1; 3 -
fluoro-1-(oxetan-
3 -yl)pip eridin-4-y1; 1-methoxyprop an-2-y1; 1-(2,2,2-trifluoroethyl)azetidin-
3 -y1); 1-(oxetan-3 -
yl)pyrro lidin-3 -yl ; 1 -is opropylazetidin-3 -yl; 3 -fluoro-l-methylpip
eridin-4-y1; 1-ethy1-3 -
fluoropip eridin-4-y1; 1-methylpyrro lidin-3-y1; 2-methoxyethyl)p ip eridin-4-
y1); 1-methy1-1-
(methylamino-carbony1)-ethyl; 2-methyl-2-morpholino-propyl; 4,4-
difluorocyclohexyl;
morpholin-4-yl-carbonyl; dimethylamino -carbonyl-methyl; methylamino-carbonyl-
methyl; 1-
methy1-1-(dimethylamino -c arb ony1)-ethyl; pyrrolidin-' -yl-carbonyl; 1-cyamo-
cyclopropyl; 1-
(pyrro lidin-' -yl-carbonyl)-ethyl; 1-(dimethylamino-carbony1)-ethyl; 1-
(methoxy-c arb ony1)-
ethyl; 1-(tert-butylamino-carb ony1)-1 -methyl-ethyl; 1-(2,2,2-trifluoro
ethyllamino-c arb ony1)-
1-methyl-ethyl; 1-(ethylamino-carbony1)-1-methyl-ethyl; 1-
(cyclopropylmethylamino-
carbony1)-1-methyl-ethyl; 1-(ethylamino-carbony1)-cyclobutyl; 1-(is opropyl
amino-c arb ony1)-
1-methyl-ethyl; 1-cyano-cyclobutyl; 2-methoxy-l-methyl-ethyl; 1-methy1-1-
(methoxy-
carbony1)-ethyl; 2-methoxy-2-methyl-prop an-1 -yl; 1-(oxetan-3 -y1)-pyrro
lidin-3 -yl;
isopropylsulfonyl; butane-2-sulfonyl; 1-(2-fluoro ethyl)-pip eri din-4-y1; 3 -
fluoro-l-methyl-
pip eridin-4-y1; 1-ethy1-3 -fluoro-pip eridin-4-y1; pyridin-3 -ylmethyl; 6-
methyl-pyridin-2-
ylmethyl; 2-(morpho lin-1 -y1)-1,1, dimethyl-ethyl; pyrimdin-2-yl-methyl; 3 -
fluoro-1-(oxetan-
3-y1)-p ip eridin-4-y1; 1 -(oxetan-3 -y1)-piperidin-3 -yl; 1-([1,3]Dioxolan-2-
ylmethyl)-pip eridin-
4-y1; pyridazin-3-ylmethyl; pip eridin-3-y1; pyrazin-2-ylmethyl; 2-hydroxy-3-
methyl-butan-1-
yl; 1-( [1,3 ]Dioxo lan-2-ylmethyl)-pyrro lidin-3 -yl; pyrimidin-4-ylmethyl; 1-
methy1-1H-
pyrazol-3-ylmethyl; I-methyl-144H- [1,2,4] triazol-3 -y1)-ethyl; 1-methyl-1 -
(5-methy1-4H-
[1,2,4] triazol-3 -y1)-ethyl; 3 -fluoro-p ip eridin-4-y1; 2-hydroxy-cyc lop
entyl; dimethyl-
[1,3 ] dioxan-5 -yl, 2-(5-methyl-1,3,4-oxadiazo 1-2-yl)prop an-2-y1; 2-(4-
methy1-4H-1,2,4-
triazol-3 -yl)prop an-2-y1; 2-(1-methy1-1H-1,2,4-triazol-3 -yl)prop an-2-y1; 2-
(1-methy1-1H-
pyrazo 1-4-yl)prop an-2-y1; 2-(1-methy1-1H-pyrazol-3-y1)propan-2-y1; 2-(1-
methy1-1H-pyrazol-
5-y1; 2-(4H-1,2,4-triazol-3 -yl)prop an-2-y1; or 1-methyl-1H-pyrazole-4-yl.
104911 In certain embodiments of formula II, R3 is: methyl; ethyl;
propyl; isopropyl;
butyl; cyclopropyl; cyclopropylmethyl; cyclobutyl; methanesulfonyl;
ethylsulfonyl;
cyclopropylsulfonyl; sec-butylsulfonyl; morpholin-4-yl-ethyl; oxetan-3-y1; 2-
methoxyethyl;
2-hydroxy-2-methyl-propyl; 3 -hydroxy-2-methyl-propan-2-y1; 2-methoxy-propyl;
tetrahydro-
2H-pyran-4-y1; tetrahydrofuran-3-y1; 2,6-dimethyltetrahydro-2H-pyran-4-y1;
tetrahydro-2H-
pyran-3 -y1); phenyl; 4-(methylsulfonyl)phenyl); 4-cyano -phenyl; 4-fluoro-
phenyl; 4-chloro-
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phenyl; 3 ,5-difluorophenyl; 4-(dimethylamino-carbonyl)-phenyl); 4-
(cyclopropylsulfonyl)phenyl; 2,2,2-trifluoroethyl; 2-fluoroethyl;
difluoromethyl; 2-dimethyl-
1 ,3 -dioxan-5-y1; 1 -methyl-cyclopropyl-carbonyl; 3 -methylpyridin-4-y1; 2-
methylpyridin-4-y1;
pyridin-2-y1; pyrimidin-2-y1; pyrimidin-5-y1; pyridin-2-ylmethyl; 1 -(pyridin-
2-yl)ethyl;
cyclopropylsulfonyl; 1 -cyano-1 -methyl-ethyl (also called 2-cyano -prop an-2-
y1); 2-cyano-
ethyl; 1 -cyano-ethyl; 2-cyano-2-methyl-propyl; 1 -(2,2,2-
trifluoroethyl)piperidin-4-y1; 1 -
(methylsulfonyl)azetidin-3 -yl; (3 -methyloxetan-3 -yl)methyl; (1 S,5 S)-8 -
oxabicyclo [3.2.1 ] octan-3 -yl; 1 -(oxetan-3 -yl)pip eridin-4-y1; 1 -acetyl-
piperidin-4-y1; 1 -
(cyc lopropyl-carb ony1)-p ip eridin-4-y1; 1 -methyl-pip eridin-4 -yl; 1 -
methy1-2-oxo-piperidin-5 -
y1; 2-oxo -pip eridin-5 -yl; 1 -(isopropyl-carbonyl)-pip eridin-4-y1; 1 -
(oxetan-3 -yl)azetidin-3 -yl;
1 -(cyclopropyl-carbonyl)-piperidin-4-y1; 2-methoxycyclopentyl; 3 -
methoxycyclopentyl; 1 -
methoxy-2-methylpropan-2-y1; tetrahydro-2H- 1,1 -dioxo-thiopyran-4-y1; 3 -
fluoro-1 -(oxetan-
3 -yl)pip eridin-4 -yl; 1 -methoxypropan-2-y1; 1 -(2,2,2-
trifluoroethyl)azetidin-3 -y1); 1 -(oxetan-3 -
yl)pyrro li din-3 -yl; 1 -is opropylazetidin-3 -yl; 3 -fluoro- 1 -
methylpiperidin-4-y1; 1 -ethy1-3 -
fluoropiperidin-4-y1; 1 -methylpyrrolidin-3 -yl; 2-methoxyethyl)p ip eridin-4-
y1); 1-methyl-1 -
(methylamino-carbonyl)-ethyl; 2-methyl-2-morpholino-propyl; 4,4-
difluorocyclohexyl;
morpholin-4-yl-carbonyl; dimethylamino -carbonyl-methyl; methylamino-carbonyl-
methyl; 1 -
methyl-1 -(dimethylamino-carbonyl)-ethyl; pyrrolidin-' -yl-carbonyl; 1 -cyamo-
cyclopropyl; 1 -
(pyrrolidin-' -yl-carbonyl)-ethyl; 1 -(dimethylamino-carbonyl)-ethyl; 1 -
(methoxy-c arb ony1)-
ethyl; 1 -(tert-butylamino-carbonyl)-1 -methyl-ethyl; 1 -(2,2,2-trifluoro
ethyllamino-c arb ony1)-
1 -methyl-ethyl; 1 -(ethylamino-carbonyl)- 1-methyl-ethyl; 1 -
(cyclopropylmethylamino-
carbony1)-1 -methyl-ethyl; 1 -(ethylamino-carbonyl)-cyclobutyl; 1 -(is
opropylamino-c arb ony1)-
1 -methyl-ethyl; 1 -cyano-cyclobutyl; 2-methoxy- 1-methyl-ethyl; 1-methyl-1 -
(methoxy-
carbony1)-ethyl; 2-methoxy-2-methyl-prop an-1 -yl; 1 -(oxetan-3 -y1)-
pyrrolidin-3 -yl;
isopropylsulfonyl; butane-2-sulfonyl; 1 -(2-fluoro ethyl)-pip eridin-4-y1; 3 -
fluoro-1 -methyl-
pip eridin-4 -yl; 1 -ethyl-3 -fluoro-piperidin-4-y1; pyri din-3 -ylmethyl; 6-
methyl-pyridin-2-
ylmethyl; 2-(morpho lin-1 -y1)- 1,1 , dimethyl-ethyl; pyrimdin-2-yl-methyl; 3 -
fluoro- 1 -(oxetan-
3-y1)-pip eridin-4-y1; 1 -(oxetan-3 -y1)-piperidin-3 -yl; 1 -( [1,3 ] Dioxo
lan-2-ylmethyl)-pip eridin-
4-y1; pyridazin-3 -ylmethyl; pip eridin-3 -yl; pyrazin-2-ylmethyl; 2-hydroxy-3
-methyl-butan- 1 -
y1; 1 -( [1,3 ]Dioxolan-2-ylmethyl)-pyrrolidin-3 -yl; pyrimidin-4-ylmethyl; 1-
methyl-1 H-
pyrazol-3 -ylmethyl; 1-methyl-1 -(4H- [1 ,2,4] triazol-3 -y1)-ethyl; 1-methyl-
1 -(5-methyl-4H-
[1 ,2,4] triazol-3 -y1)-ethyl; 3 -fluoro-piperidin-4-y1; 2-hydroxy-cyc lop
entyl; dimethyl-
[ 1 ,3 ] dioxan-5 -yl; 245-methyl-I ,3 ,4-oxadi azo 1-2-yl)prop an-2-y1; 2-(4-
methyl-4H- 1 ,2,4-
triazol-3 -yl)prop an-2-y1; 2-(1 -methyl-1 H-1 ,2,4 -triazol-3 -yl)prop an-2-
y1; 2-(1 -methyl- 1 H-
pyrazo 1-4-yl)prop an-2-y1; 2-(1 -methyl-1 H-pyrazol-3 -yl)propan-2-y1; 2-(1 -
methyl-1 H-
pyrazol-5-y1; 2-(4H-1 ,2,4 -triazol-3 -yl)prop an-2-y1; or 1-methyl-1 H-pyrazo
le-4 -yl..
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[0492] In certain embodiments of formula II, R3 is: hydrogen; methyl;
ethyl; n-
propyl; isopropyl; 2-methoxy-ethyl; oxetan-3-y1; 2-hydroxy-2-methyl-propan-1-
y1;
tetrahydropyran-4-y1; or morpholin-4-yl-carbonyl.
[0493] In certain embodiments of formula II, R3 is: methyl; ethyl; n-
propyl;
isopropyl; 2-methoxy-ethyl; oxetan-3 -yl; 2 -hydroxy-2 -methyl-prop an-1 -yl;
or
tetrahydropyran-4-yl.
[0494] In certain embodiments of formula II, R3 is: methyl; ethyl;
isopropyl; 2-
methoxy-ethyl; oxetan-3-y1; or 2-hydroxy-2-methyl-prop an-1 -yl.
[0495] In certain embodiments of formula II, R3 is: methyl; ethyl;
isopropyl; 2-
methoxy-ethyl; oxetan-3-y1; or 2-hydroxy-2-methyl-prop an-1 -yl.
[0496] In certain embodiments of formula II, R3 is: methyl; ethyl; or
isopropyl.
[0497] In certain embodiments of formula II, R3 is hydrogen.
[0498] In certain embodiments of formula II, R3 is methyl.
[0499] In certain embodiments of formula II, R3 is ethyl.
[0500] In certain embodiments of formula II, R3 is n-propyl.
[0501] In certain embodiments of formula II, R3 is isopropyl.
[0502] In certain embodiments of formula II, R3 is 2-methoxy-ethyl.
[0503] In certain embodiments of formula II, R3 is oxetan-3-yl.
[0504] In certain embodiments of formula II, R3 is 2-hydroxy-2-methyl-
propan-1-yl.
[0505] In certain embodiments of formula II, R3 is tetrahydropyran-4-yl.
[0506] In certain embodiments of formula II, R3 is morpholin-4-yl-carbonyl.
[0507] In certain embodiments of formula II, R3 is butyl.
[0508] In certain embodiments of formula II, R3 is cyclopropyl.
[0509] In certain embodiments of formula II, R3 is cyclopropylmethyl.
[0510] In certain embodiments of formula II, R3 is cyclobutyl.
[0511] In certain embodiments of formula II, R3 is methanesulfonyl.
[0512] In certain embodiments of formula II, R3 is ethylsulfonyl.
[0513] In certain embodiments of formula II, R3 is cyclopropylsulfonyl.
[0514] In certain embodiments of formula II, R3 is sec-butylsulfonyl.
[0515] In certain embodiments of formula II, R3 is morpholin-4-yl-ethyl.
[0516] In certain embodiments of formula II, R3 is 2-hydroxy-2-methyl-
propyl.
[0517] In certain embodiments of formula II, R3 is 3-hydroxy-2-methyl-
propan-2-yl.
[0518] In certain embodiments of formula II, R3 is 2-methoxy-propyl.
[0519] In certain embodiments of formula II, R3 is tetrahydro-2H-pyran-4-
yl.
[0520] In certain embodiments of formula II, R3 is tetrahydrofuran-3-yl.
[0521] In certain embodiments of formula II, R3 is 2,6-dimethyltetrahydro-
2H-
pyran-4-yl.
105221 In certain embodiments of formula II, R3 is tetrahydro-2H-pyran-3-
y1).
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[0523] In certain embodiments of formula II, R3 is phenyl.
[0524] In certain embodiments of formula II, R3 is 4-
(methylsulfonyl)pheny1).
[0525] In certain embodiments of formula II, R3 is 4-cyano-phenyl.
[0526] In certain embodiments of formula II, R3 is 4-fluoro-phenyl.
[0527] In certain embodiments of formula Iv, R3 is 4-chloro-phenyl.
[0528] In certain embodiments of formula II, R3 is 3,5-difluorophenyl.
[0529] In certain embodiments of formula II, R3 is 4-(dimethylamino-
carbony1)-
phenyl).
[0530] In certain embodiments of formula II, R3 is 4-
(cyclopropylsulfonyl)phenyl.
[0531] In certain embodiments of formula II, R3 is 2,2,2-trifluoroethyl.
[0532] In certain embodiments of formula II, R3 is 2-fluoroethyl.
[0533] In certain embodiments of formula II, R3 is difluoromethyl.
[0534] In certain embodiments of formula II, R3 is 2-dimethy1-1,3-dioxan-5-
yl.
[0535] In certain embodiments of formula II, R3 is 1-methyl-cyclopropyl-
carbonyl.
[0536] In certain embodiments of formula II, R3 is 3-methylpyridin-4-yl.
[0537] In certain embodiments of formula II, R3 is 2-methylpyridin-4-yl.
[0538] In certain embodiments of formula II, R3 is pyridin-2-yl.
[0539] In certain embodiments of formula II, R3 is pyrimidin-2-yl.
[0540] In certain embodiments of formula II, R3 is pyrimidin-5-yl.
[0541] In certain embodiments of formula II, R3 is pyridin-2-ylmethyl.
[0542] In certain embodiments of formula II, R3 is 1-(pyridin-2-yl)ethyl.
[0543] In certain embodiments of formula II, R3 is cyclopropylsulfonyl.
[0544] In certain embodiments of formula II, R3 is 1-cyano-1-methyl-ethyl
(also
called 2-cyano-propan-2-y1).
[0545] In certain embodiments of formula II, R3 is 2-cyano-ethyl.
[0546] In certain embodiments of formula II, R3 is 1-cyano-ethyl.
[0547] In certain embodiments of formula II, R3 is 2-cyano-2-methyl-propyl.
[0548] In certain embodiments of formula II, R3 is 1-(2,2,2-
trifluoroethyl)piperidin-
4-yl.
[0549] In certain embodiments of formula II, R3 is 1-
(methylsulfonyl)azetidin-3-yl.
[0550] In certain embodiments of formula II, R3 is (3-methyloxetan-3-
yl)methyl.
[0551] In certain embodiments of formula II, R3 is (1S,5S)-8-
oxabicyclo[3.2.1]octan-3-yl.
[0552] In certain embodiments of formula II, R3 is 1-(oxetan-3-yl)piperidin-
4-yl.
[0553] In certain embodiments of formula II, R3 is 1-acetyl-piperidin-4-yl.
[0554] In certain embodiments of formula II, R3 is 1-(cyclopropyl-carbony1)-
piperidin-4-yl.
105551 In certain embodiments of formula II, R3 is 1-methyl-piperidin-4-yl.
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[0556] In certain embodiments of formula II, R3 is 1-methy1-2-oxo-piperidin-
5-yl.
[0557] In certain embodiments of formula II, R3 is 2-oxo-piperidin-5-yl.
[0558] In certain embodiments of formula II, R3 is 1-(isopropyl-carbony1)-
piperidin-
4-yl.
[0559] In certain embodiments of formula II, R3 is 1-(oxetan-3-yl)azetidin-
3-yl.
[0560] In certain embodiments of formula II, R3 is 1-(cyclopropyl-carbony1)-
piperidin-4-yl.
[0561] In certain embodiments of formula II, R3 is 2-methoxycyclopentyl.
[0562] In certain embodiments of formula II, R3 is 3-methoxycyclopentyl.
[0563] In certain embodiments of formula II, R3 is 1-methoxy-2-methylpropan-
2-yl.
[0564] In certain embodiments of formula II, R3 is tetrahydro-2H-1,1-dioxo-
thiopyran-4-yl.
[0565] In certain embodiments of formula II, R3 is 3-fluoro-1-(oxetan-3-
yl)piperidin-
4-yl.
[0566] In certain embodiments of formula II, R3 is 1-methoxypropan-2-yl.
[0567] In certain embodiments of formula II, R3 is 1-(2,2,2-
trifluoroethyl)azetidin-3-
yl).
[0568] In certain embodiments of formula II, R3 is 1-(oxetan-3-
yl)pyrrolidin-3-yl.
[0569] In certain embodiments of formula II, R3 is 1-isopropylazetidin-3-
yl.
[0570] In certain embodiments of formula II, R3 is 3-fluoro-1-
methylpiperidin-4-yl.
[0571] In certain embodiments of formula II, R3 is 1-ethyl-3-
fluoropiperidin-4-yl.
[0572] In certain embodiments of formula II, R3 is 1-methylpyrrolidin-3-yl.
[0573] In certain embodiments of formula II, R3 is 2-methoxyethyl)piperidin-
4-y1).
[0574] In certain embodiments of formula II, R3 is 1-methy1-1-(methylamino-
carbony1)-ethyl.
[0575] In certain embodiments of formula II, R3 is 2-methyl-2-morpholino-
propyl.
[0576] In certain embodiments of formula II, R3 is 4,4-difluorocyclohexyl.
[0577] In certain embodiments of formula II, R3 is dimethylamino-carbonyl-
methyl.
[0578] In certain embodiments of formula II, R3 is methylamino-carbonyl-
methyl.
[0579] In certain embodiments of formula II, R3 is 1-methy1-1-
(dimethylamino-
carbony1)-ethyl.
[0580] In certain embodiments of formula II, R3 is pyrrolidin-'-yl-
carbonyl.
[0581] In certain embodiments of formula II, R3 is 1-cyano-cyclopropyl.
[0582] In certain embodiments of formula II, R3 is 1-(pyrrolidin-'-yl-
carbony1)-ethyl.
[0583] In certain embodiments of formula II, R3 is 1-(dimethylamino-
carbony1)-
ethyl.
105841 In certain embodiments of formula II, R3 is 1-(methoxy-carbony1)-
ethyl.
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[0585] In certain embodiments of formula II, R3 is 1-(tert-butylamino-
carbony1)-1-
methyl-ethyl.
[0586] In certain embodiments of formula II, R3 is 1-(2,2,2-
trifluoroethyllamino-
carb ony1)- 1-methyl-ethyl.
[0587] In certain embodiments of formula II, R3 is 1-(ethylamino-carbony1)-
1-
methyl-ethyl.
[0588] In certain embodiments of formula II, R3 is 1-
(cyclopropylmethylamino-
carb ony1)- 1-methyl-ethyl.
[0589] In certain embodiments of formula II, R3 is 1-(ethylamino-carbony1)-
cyclobutyl.
[0590] In certain embodiments of formula II, R3 is 1-(isopropylamino-
carbony1)-1-
methyl-ethyl.
[0591] In certain embodiments of formula II, R3 is 1-cyano-cyclobutyl.
[0592] In certain embodiments of formula II, R3 is dimethyl-[1,3]dioxan-5-
yl.
[0593] In certain embodiments of formula II, R3 is 2-methoxy-2-methyl-
propan-1-yl.
[0594] In certain embodiments of formula II, R3 is 2-methoxy-1 -methyl-
ethyl.
[0595] In certain embodiments of formula II, R3 is 1-methy1-1-(methoxy-
carbony1)-
ethyl.
[0596] In certain embodiments of formula II, R3 is 1-oxetan-3-yl-pyrrolidin-
3-yl.
[0597] In certain embodiments of formula II, R3 is isopropylsulfonyl.
[0598] In certain embodiments of formula II, R3 is butane-2-sulfonyl.
[0599] In certain embodiments of formula II, R3 is 1-(2-fluoroethyl)-
piperidin-4-yl.
[0600] In certain embodiments of formula II, R3 is 3-fluoro-1-methyl-
piperidin-4-yl.
[0601] In certain embodiments of formula II, R3 is 1-ethyl-3-fluoro-
piperidin-4-ylln
certain embodiments of formula II, R3 is pyridin-3-ylmethyl.
[0602] In certain embodiments of formula II, R3 is 6-methyl-pyridin-2-
ylmethyl.
[0603] In certain embodiments of formula II, R3 is 2-(morpholin-1-y1)-
1,1,dimethyl-
ethyl.
[0604] In certain embodiments of formula II, R3 is pyrimdin-2-yl-methyl.
[0605] In certain embodiments of formula II, R3 is 3-fluoro-1-(oxetan-3-y1)-
piperidin-4-yl.
[0606] In certain embodiments of formula II, R3 is 1-(oxetan-3-y1)-
piperidin-3-yl.
[0607] In certain embodiments of formula II, R3 is 1-([1,3]Dioxolan-2-
ylmethyl)-
piperidin-4-yl.
[0608] In certain embodiments of formula II, R3 is pyridazin-3-ylmethyl.
[0609] In certain embodiments of formula II, R3 is piperidin-3-yl.
[0610] In certain embodiments of formula II, R3 is pyrazin-2-ylmethyl.
106111 In certain embodiments of formula II, R3 is 2-hydroxy-3-methyl-butan-
1-yl.
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[0612] In certain embodiments of formula II, R3 is 1-([1,3]dioxolan-2-
ylmethyl)-
pyrrolidin-3-yl.
[0613] In certain embodiments of formula II, R3 is pyrimidin-4-ylmethyl.
[0614] In certain embodiments of formula II, R3 is 1-methyl-1H-pyrazol-3-
ylmethyl.
[0615] In certain embodiments of formula II, R3 is 1-methy1-1-(5-methy1-4H-
[1,2,4]triazol-3-y1)-ethyl.
[0616] In certain embodiments of formula II, R3 is 1-methy1-1-(4H-
[1,2,4]triazol-3-
y1)-ethyl.
[0617] In certain embodiments of formula II, R3 is 3-fluoro-piperidin-4-y1;
2-
hydroxy-cyclopentyl.
[0618] In certain embodiments of formula II, R3 is 2-(5-methy1-1,3,4-
oxadiazol-2-
yl)propan-2-yl.
[0619] In certain embodiments of formula II, R3 is 2-(4-methy1-4H-1,2,4-
triazol-3-
yl)propan-2-yl.
[0620] In certain embodiments of formula II, R3 is 2-(1-methy1-1H-1,2,4-
triazol-3-
yl)propan-2-yl.
[0621] In certain embodiments of formula II, R3 is 2-(1-methy1-1H-pyrazol-4-
y1)propan-2-y1; 2-( 1 -methyl- 1 H-pyrazol-3 -yl)propan-2-yl.
[0622] In certain embodiments of formula II, R3 is 2-(1-methyl-1H-pyrazol-5-
yl.
[0623] In certain embodiments of formula II, R3 is 2-(4H-1,2,4-triazol-3-
yl)propan-
2-yl.
[0624] In certain embodiments of formula II, R3 is 1-methyl-1H-pyrazole-4-
yl.
[0625] In embodiments of formula II wherein R3 is aryl, such aryl may be
unsubstituted phenyl or phenyl substituted one or more times with R8, or in
certain
embodiments, once, twice or three times with a group or groups independently
selected from
Ci_6alkyl, halo, halo-Ci_6alkyl, Ci_6alkoxy, hydroxy or cyano.
[0626] In embodiments of formula II wherein R3 is heteroaryl or heteroaryl-
C1_
6alkyl, such heteroaryl moiety may be pyrrolyl, pyrazolyl, imidazolyl,
thiazolyl, oxazolyl,
pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, isoxazolyl, isothiazolyl,
triazolyl, oxadiaolyl,
thiadiazolyl or tetrazolyl, each being unsubstituted or substituted once or
twice with R8, or in
certain embodiments, substituted once or twice with Ci_6alkyl.
[0627] In embodiments of formula II wherein R3 is heteroaryl or heteroaryl-
C1_
6alkyl, such heteroaryl moiety may be pyrazolyl, pyridinyl, pyrimidinyl,
pyrazinyl,
pyridazinyl, triazolyl or oxadiaolyl each being unsubstituted or substituted
once or twice with
R8, or in certain embodiments, substituted once or twice with Ci_6allcyl.
[0628] In embodiments of formula II wherein R3 is heteroaryl or heteroaryl-
C1_
6alkyl, such heteroaryl moiety may be pyrrolyl, pyrazolyl, imidazolyl,
thiazolyl, oxazolyl,
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pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl, each being unsubstituted or
substituted one
or more times with Rs.
[0629] In embodiments of formula II wherein R3 is heterocyclyl, such
heterocyclyl
moiety may be piperidinyl, pyrrolidinyl, oxetanyl, tetrahydropyranyl,
tetrahydrofuranyl,
azetidinyl, [1,3]dioxolanyl or tetrahydrothiopyranyl, each being unsubstituted
or substituted
one or more times with R7.
[0630] In embodiments of formula II wherein R3 is heterocyclyl-Ci_6alkyl,
such
heterocyclyl moiety may be piperidinyl, piperazinyl, morpholinyl,
thiomorpholinyl,
pyrrolidinyl, oxetanyl, tetrahydropyranyl, tetrahydrofuranyl, azetidinyl,
[1,3]dioxolanyl or
tetrahydrothiopyranyl, each being unsubstituted or substituted one or more
times with R7.
[0631] In certain embodiments of formula II, R3 is -Y-C(0)-Rd.
[0632] In certain embodiments of formula II, Y is a bond.
[0633] In certain embodiments of formula II, Y is C2_6alkylene.
[0634] In certain embodiments of formula II, Y is isopropylidine.
[0635] In certain embodiments of formula II, Y is methylene.
[0636] In certain embodiments of formula II, Y is ethylene.
[0637] In certain embodiments of formula II, Y is -C(CH3)2-.
[0638] In certain embodiments of formula II, Y is -CH2-.
[0639] In certain embodiments of formula II, Y is -CH(CH3)-.
[0640] In certain embodiments of formula II, Y is -CH2-C(CH3)2-.
[0641] In certain embodiments of formula II, Y is -C(CH3)2-CH2-.
[0642] In certain embodiments of formula II, Rd is Ci_6alkyl, Ci_6alkoxy,
amino, or
heterocyclyl.
[0643] In certain embodiments of formula II, Rd is Ci_6alkyl.
[0644] In certain embodiments of formula II, Rd is Ci_6alkoxy.
[0645] In certain embodiments of formula II, Rd is amino.
[0646] In certain embodiments of formula II, Rd is halo-Ci_6alkyl.
[0647] In certain embodiments of formula II, Rd is hydroxy-Ci_6alkyl.
[0648] In certain embodiments of formula II, Rd is Ci_6alkoxy-Ci_6alkyl.
[0649] In certain embodiments of formula II, Rd is cyano-Ci_6alkyl.
[0650] In certain embodiments of formula II, Rd is
Ci_6alkylsulfonylCi_6alkyl.
[0651] In certain embodiments of formula II, Rd is amino-Ci_6alkyl.
[0652] In certain embodiments of formula II, Rd is C3_6cycloalkyl
optionally
substituted one or more times with R6.
[0653] In certain embodiments of formula II, Rd is C3_6cycloalkyl-Ci_6alkyl
wherein
the C3_6cycloalkyl portion is optionally substituted one or more times with
R6.
[0654] In certain embodiments of formula II, Rd is heterocyclyl optionally
substituted one or more times with R7.
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[0655] In certain embodiments of formula II, Rd is heterocyclyl-Ci_6alkyl
wherein the
heterocyclyl portion is optionally substituted one or more times with R7.
[0656] In certain embodiments of formula II, Rd is 1-methyl-cyclopropyl;
methylamino; dimethylamino; pyrrolidin-1 -yl; methoxy; cyclopropyl-methyl;
ethyl; 2,2,2-
trifluoro-ethyl; tert-butyl; or isopropyl.
[0657] In certain embodiments of formula II, Rd is 1-methyl-cyclopropyl.
[0658] In certain embodiments of formula II, Rd is methylamino.
[0659] In certain embodiments of formula II, Rd is dimethylamino.
[0660] In certain embodiments of formula II, Rd is pyrrolidin-l-yl.
[0661] In certain embodiments of formula II, Rd is methoxy.
[0662] In certain embodiments of formula II, Rd is cyclopropyl-methyl.
[0663] In certain embodiments of formula II, Rd is ethyl.
[0664] In certain embodiments of formula II, Rd is 2,2,2-trifluoro-ethyl.
[0665] In certain embodiments of formula II, Rd is tert-butyl.
[0666] In certain embodiments of formula II, Rd is isopropyl.
[0667] In embodiments of formula II wherein Rd is heterocyclyl or
heterocyclyl-C1_
6alkyl, such heterocyclyl may be piperidinyl, piperazinyl, morpholinyl,
tetrahydropyranyl,
pyrrolidinyl, azetidinyl, tetrahydrofuranyl or oxetanyl, each optionally
substituted one or
more times, or one or two times, with R7 as defined herein.
[0668] In embodiments of formula II wherein Rd is heterocyclyl, such
heterocyclyl
moiety may be piperidinyl, pyrrolidinyl, oxetanyl, tetrahydropyranyl,
tetrahydrofuranyl,
azetidinyl, [1,3]dioxolanyl or tetrahydrothiopyranyl, each being unsubstituted
or substituted
one or more times with R7.
[0669] In embodiments of formula II wherein Rd is heterocyclyl-Ci_6alkyl,
such
heterocyclyl moiety may be piperidinyl, piperazinyl, morpholinyl,
thiomorpholinyl,
pyrrolidinyl, oxetanyl, tetrahydropyranyl, tetrahydrofuranyl, azetidinyl,
[1,3]dioxolanyl or
tetrahydrothiopyranyl, each being unsubstituted or substituted one or more
times with R7.
[0670] In certain embodiments of formula II, R4 is: hydrogen; Ci_6alkyl;
halo; halo-
Ci_6alkyl; Ci_6alkoxy-Ci_6alkyl; hydroxy-Ci_6alkyl; C3_6cycloalkyl optionally
substituted with
Ci_6alkyl; C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is
optionally substituted
with Ci_6alkyl; or -C(0)-Re wherein Re is Ci_6alkyl, Ci_6alkoxy, amino, or
heterocyclyl.
[0671] In certain embodiments of formula II, R4 is: Ci_6alkyl; halo; halo-
Ci_6alkyl;
Ci_6alkoxy-Ci_6alkyl; hydroxy-Ci_6alkyl; C3_6cycloalkyl optionally substituted
with Ci_6alkyl;
C3_6cycloalkyl-Ci_6alkyl wherein the C3_6cycloalkyl portion is optionally
substituted with C1_
6alkyl; or -C(0)-Re wherein Re is Ci_6alkyl, Ci_6alkoxy, amino, or
heterocyclyl.
[0672] In certain embodiments of formula II, R4 is: hydrogen; Ci_6alkyl;
halo; C3_
6cycloalkyl optionally substituted with Ci_6alkyl; C3_6cycloalkyl-Ci_6alkyl
wherein the C3_
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6cycloalkyl portion is optionally substituted with Ci_6alkyl; or -C(0)-Re
wherein Re is C1_
6alkyl, Ci_6alkoxy, amino, or heterocyclyl.
[0673] In certain embodiments of formula II, R4 is: hydrogen; Ci_6alkyl;
halo; or C3_
6cycloalkyl optionally substituted with Ci_6alkyl.
[0674] In certain embodiments of formula II, R4 is hydrogen or Ci_6alkyl.
[0675] In certain embodiments of formula II, R4 is hydrogen.
[0676] In certain embodiments of formula II, R4 is Ci_6alkyl.
[0677] In certain embodiments of formula II, R4 is halo.
[0678] In certain embodiments of formula II, R4 is cyano.
[0679] In certain embodiments of formula II, R4 is halo-Ci_6alkyl.
[0680] In certain embodiments of formula II, R4 is Ci_6alkoxy-Ci_6alkyl.
[0681] In certain embodiments of formula II, R4 is hydroxy-Ci_6alkyl.
[0682] In certain embodiments of formula II, R4 is C3_6cycloalkyl
optionally
substituted with Ci_6alkyl.
[0683] In certain embodiments of formula II, R4 is hydrogen or methyl.
[0684] In certain embodiments of formula II, R4 is C3_6cycloalkyl-Ci_6alkyl
wherein
the C3_6cycloalkyl portion is optionally substituted with Ci_6alkyl.
[0685] In certain embodiments of formula II, R4 is -C(0)-Re wherein Re is
Ci_6alkyl,
Ci_6alkoxy, amino, or heterocyclyl.
[0686] In certain embodiments of formula II, R4 is -C(0)-Re wherein Re is
heterocyclyl.
[0687] In embodiments of formula II wherein Re is heterocyclyl, such
heterocyclyl
may be pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl.
[0688] In embodiments of formula II wherein Re is heterocyclyl, such
heterocyclyl
may be piperidinyl, piperazinyl or morpholinyl.
[0689] In certain embodiments of formula II, R4 is: hydrogen; methyl;
isopropyl;
cyclopropyl; chloro; or morpholin-4-yl-carbonyl.
[0690] In certain embodiments of formula II, R4 is: hydrogen; methyl;
isopropyl;
cyclopropyl; or chloro.
[0691] In certain embodiments of formula II, R4 is hydrogen.
[0692] In certain embodiments of formula II, R4 is methyl.
[0693] In certain embodiments of formula II, R4 is isopropyl.
[0694] In certain embodiments of formula II, R4 is cyclopropyl.
[0695] In certain embodiments of formula II, R4 is chloro.
[0696] In certain embodiments of formula II, R4 is morpholin-4-yl-carbonyl.
[0697] In certain embodiments of formula II, R4 is 2-fluoro-ethyl.
[0698] In certain embodiments of formula II, R4 is C3_6cycloalkyl
optionally
substituted one or more times, or one or two times, with R6.
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[0699] In certain embodiments of formula II, R4 is C3_6cycloalkyl-Ci_6alkyl
wherein
the C3_6cycloalkyl portion is optionally substituted one or more times, or one
or two times,
with R6.
[0700] In certain embodiments of formula II, R4 is-Y-C(0)-Rd.
[0701] In certain embodiments of formula II, or R3 and R4 together with the
atoms to
which they are attached may form a 5- or 6-membered ring that optionally
includes a
heteroatom selected from 0, N and S.
[0702] In certain embodiments of formula II, R5 is hydrogen.
[0703] In certain embodiments of formula II, R5 is Ci_6alkyl.
[0704] In certain embodiments of formula II, R5 is methyl.
[0705] In certain embodiments of formula II, each R6 is independently
Ci_6alkyl;
halo-Ci_6alkyl; Ci_6alkoxy; cyano; or halo.
[0706] In certain embodiments of formula II, R6 is Ci_6alkyl; halo-
Ci_6alkyl; C1_
6alkoxy; or halo.
[0707] In certain embodiments of formula II, R6 is Ci_6alkyl; halo-
Ci_6alkyl; or halo.
[0708] In certain embodiments of formula II, R6 is Ci_6alkyl.
[0709] In certain embodiments of formula II, R6 is halo-Ci_6alkyl.In
certain
embodiments of formula II, R6 is Ci_6alkoxy.
[0710] In certain embodiments of formula II, R6 is cyano.
[0711] In certain embodiments of formula II, R6 is halo.
[0712] In certain embodiments of formula II, R6 is Y-C(0)-Rd.
[0713] In certain embodiments of formula II, R6 is oxo.
[0714] In certain embodiments of formula II, each R7 is independently
Ci_6alkyl;
halo-Ci_6alkyl; halo; Ci_6alkylsulfonyl; Ci_6alkoxy-Ci_6alkyl; cyano;
heterocyclyl; or C3_
6cycloalkylsulfonyl wherein the C3_6cycloalkyl portion is optionally
substituted one or more
times with R6.
[0715] In certain embodiments of formula II, R7 is Ci_6alkyl.
[0716] In certain embodiments of formula II, R7 is halo-Ci_6alkyl.
[0717] In certain embodiments of formula II, R7 is halo.
[0718] In certain embodiments of formula II, R7 is Ci_6alkylsulfonyl.
[0719] In certain embodiments of formula II, R7 is Ci_6alkoxy-Ci_6alkyl.
[0720] In certain embodiments of formula II, R7 is cyano.
[0721] In certain embodiments of formula II, R7 is -Y-C(0)-Rd.
[0722] In certain embodiments of formula II, R7 is heterocyclyl.
[0723] In certain embodiments of formula II, R7 is C3_6cycloalkylsulfonyl
wherein
the C3_6cycloalkyl portion is optionally substituted one or more times with
R6.
[0724] In certain embodiments of formula II, R7 is oxo.
107251 In certain embodiments of formula II, R7 is Ci_6alkoxy.
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[0726] In certain embodiments of formula II, R7 is heterocyclyl-Ci_6alkyl.
[0727] In certain embodiments of formula II, R7 is C3_6cycloalkyl.
[0728] In certain embodiments of formula II, R7 is C3_6cycloalkyl-
Ci_6alkyl.
[0729] In embodiments of formula II wherein R7 is heterocyclyl, such
heterocyclyl
moiety may be piperidinyl, pyrrolidinyl, oxetanyl, tetrahydropyranyl,
tetrahydrofuranyl,
azetidinyl, [1,3]dioxolanyl or tetrahydrothiopyranyl.
[0730] In embodiments of formula II wherein R7 is heterocyclyl-Ci_6alkyl,
such
heterocyclyl moiety may be piperidinyl, piperazinyl, morpholinyl,
thiomorpholinyl,
pyrrolidinyl, oxetanyl, tetrahydropyranyl, tetrahydrofuranyl, azetidinyl,
[1,3]dioxolanyl or
tetrahydrothiopyranyl.
[0731] In certain embodiments of formula II, each R8 is independently oxo;
CI_
6alkYl; halo-Ci_6alkyl; halo; Ci_6alkoxy; Ci_6alkoxy-Ci_6alkyl; cyano;
C3_6cycloalkyl optionally
substituted one or more times with R6, C3_6cycloalkyl-Ci_6alkyl wherein the
C3_6cycloalkyl
portion is optionally substituted one or more times with R6, or C3_6cycloalkyl-
sulfonyl
wherein the C3_6cycloalkyl portion is optionally substituted one or more times
with R6.
[0732] In certain embodiments of formula II, R8 is oxo.In certain
embodiments of
formula II, R7 is Ci_6alkyl.
[0733] In certain embodiments of formula II, R7 is halo-Ci_6alkylIn certain
embodiments of formula II, R7 is halo.In certain embodiments of formula II, R7
is Ci_6alkoxy.
[0734] In certain embodiments of formula II, R7 is Ci_6alkoxy-Ci_6alkyl.
[0735] In certain embodiments of formula II, R7 is cyano.
[0736] In certain embodiments of formula II, R7 is hetoeryclyl.
[0737] In certain embodiments of formula II, R7 is -Y-C(0)-Rd.
[0738] In certain embodiments of formula II, R7 is C3_6cycloalkyl
optionally
substituted one or more times with R6.
[0739] In certain embodiments of formula II, R7 is C3_6cycloalkyl-Ci_6alkyl
wherein
the C3_6cycloalkyl portion is optionally substituted one or more times with
R6.
[0740] In certain embodiments of formula II, R7 is C3_6cycloalkyl-sulfonyl
wherein
the C3_6cycloalkyl portion is optionally substituted one or more times with
R6.
[0741] In certain embodiments of formula II, R8 is oxo.
[0742] In certain embodiments of formula II, R8 is Ci_6alkyl.
[0743] In certain embodiments of formula II, R8 is halo-Ci_6alkyl.
[0744] In certain embodiments of formula II, R8 is halo.
[0745] In certain embodiments of formula II, R8 is Ci_6alkyl-sulfonyl.
[0746] In certain embodiments of formula II, R8 is Ci_6alkoxy.
[0747] In certain embodiments of formula II, R8 is Ci_6alkoxy-Ci_6alkyl.
[0748] In certain embodiments of formula II, R8 is cyano; hetoeryclyl.
107491 In certain embodiments of formula II, R8 is heterocyclyl-Ci_6alkyl.
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[0750] In certain embodiments of formula II, R8 is -Y-C(0)-Rd.
[0751] In certain embodiments of formula II, R8 is C3_6cycloalkyl.
[0752] In certain embodiments of formula II, R8 is C3_6cycloalkyl-Ci_6alkyl-
C3_
6CYC loalkyl-sulfonyl.
[0753] In embodiments of formula II wherein R8 is heterocyclyl, such
heterocyclyl
moiety may be piperidinyl, pyrrolidinyl, oxetanyl, tetrahydropyranyl,
tetrahydrofuranyl,
azetidinyl, [1,3] dioxolanyl or tetrahydrothiopyranyl.
[0754] In embodiments of formula II wherein R8 is heterocyclyl-Ci_6alkyl,
such
heterocyclyl moiety may be piperidinyl, piperazinyl, morpholinyl,
thiomorpholinyl,
pyrrolidinyl, oxetanyl, tetrahydropyranyl, tetrahydrofuranyl, azetidinyl,
[1,3]dioxolanyl or
tetrahydrothiopyranyl.
[0755] In certain embodiments of formula II, the compound may be:
N2-(1,5-dimethy1-1H-pyrazol-4-y1)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-
diamine
N4-methyl-N2-( 1 -(2-morpho lino ethyl)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N4-methyl-N2-( 1 -methyl- 1 H-pyrazol-4-y1) -5 -(trifluoromethyl)pyrimidine-
2,4-diamine
5-chloro-N2-(1-isopropy1-1H-pyrazol-4-y1)-N4-methylpyrimidine-2,4-diamine
N4-methyl-N2-(1 -methyl- 1 H-pyrazol-5-y1) -5-(trifluoromethyl)pyrimidine-2,4-
diamine
N4-methyl-N2-(1 -methyl- 1 H-pyrazol-3 -y1) -5-(trifluoromethyl)pyrimidine-2,4-
di amine
N2-(1,3-dimethy1-1H-pyrazol-4-y1)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-
diamine
5-Chloro-N-(1,5-dimethy1-1H-pyrazol-4-y1)-4-(tetrahydro-2H-pyran-4-
yloxy)pyrimidin-2-
amine
N4-methy1-5-(trifluoromethyl)-N2-(1,3,5-trimethyl-1H-pyrazol-4-y1)pyrimidine-
2,4-diamine
5- Chloro-N-( 1 -isopropyl- 1 H-pyrazol-4-y1)-4 -(tetrahydro-2H-pyran-4 -
yloxy)pyrimidin-2 -
amine
5-Chloro-N-(1,5-dimethy1-1H-pyrazol-4-y1)-4-methoxypyrimidin-2-amine
N-( 1 ,5 -dimethyl- 1 H-pyrazol-4 -y1)-4-(pyrro lidin- 1 -y1)-5 -
(trifluoromethyl)pyrimidin-2-amine
N2-( 1 -ethyl-5 -methyl-1 H-pyrazol-4-y1)-N4-methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
5-Chloro-N-(1,3-dimethy1-1H-pyrazol-4-y1)-4-methoxypyrimidin-2-amine
N4-methyl-N2-(3 -methyl-1 -(oxetan-3 -y1)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N2-(5 -chloro- 1 -methyl- 1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4 -
diamine
5- Chloro-4-methoxy-N-(3 -methyl-1 -(oxetan-3 -y1)- 1 H-pyrazol-4 -
yl)pyrimidin-2-amine
5- Chloro-4-methoxy-N-( 1 -(2-methoxyethyl)-3 -methyl- 1H-pyrazol-4-
yl)pyrimidin-2-amine
5-chloro-4-methoxy-N-(1-(2-methoxyethyl)-5-methy1-1H-pyrazol-4-y1)pyrimidin-2-
amine
5-Chloro-N-(5 -chloro- 1 -methyl- 1 H-pyrazol-4 -y1)-4-methoxypyrimidin-2-
amine
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2-Methyl-I -(3 -methyl-4 -(4 -(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)-1 H-
pyrazol- 1 -yl)prop an-2-ol
2-Methyl-I -(3 -methyl-4-(4-(methylamino)-5 -chloro-pyrimidin-2-ylamino)- 1 H-
pyrazol- 1 -
yl)prop an-2-ol
N2-(1 -(2-methoxyethyl)-3 -methyl-1 H-pyrazol-4 -y1)-N4-methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-(1 -(2-methoxyethyl)-5-methy1-1H-pyrazol-4-y1)-N4-methyl-5-
(trifluoromethyl)pyrimidine-2,4-diamine
5-Chloro-N-(1 -ethyl-3 -methyl-1 H-pyrazol-4 -y1)-4 -methoxypyrimidin-2-amine
5-Chloro-N4-methyl-N2-(3 -methyl-1 -(oxetan-3 -y1)- 1 H-pyrazol-4 -
yl)pyrimidine-2,4-diamine
N2-(1 -isopropyl-3 -methyl-1 H-pyrazol-4 -y1)-N4-methyl-5-
(trifluoromethyl)pyrimidine-2,4 -
diamine
5-Chloro-N-(1 -isopropyl-3 -methyl-1 H-pyrazol-4 -y1)-4 -methoxypyrimidin-2-
amine
5-Chloro-N2-( 1,3 -dimethyl- 1 H-pyrazol-4-y1)-N4-methylpyrimidine-2,4-diamine
5-Chloro-N2-( 1 -isopropyl-3 -methyl-1 H-pyrazol-4 -y1)-N4-methylpyrimidine-
2,4-diamine
N4-methyl-N2-(3 -methyl-1 -(tetrahydro-2H-pyran-4-y1)-1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N4-methyl-N2-(5 -methyl-1 -(tetrahydro-2H-pyran-4-y1)-1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N2-(2-Ethyl-2H-pyrazol-3 -y1)-5-fluoro-N4-methyl-pyrimidine-2,4-diamine
5-Fluoro-N4-methyl-N2-(2-methyl-2H-pyrazol-3 -y1)-pyrimidine-2,4-diamine
5-Fluoro-N4-methyl-N4-(2-propyl-2H-pyrazol-3 -y1)-pyrimidine-2,4-diamine
N2-(2,5-Dimethy1-2H-pyrazol-3 -y1)-5 -fluoro-N4-methyl-pyrimidine-2,4-diamine
N2-(3 -isopropyl- 1 -methyl- 1 H-pyrazol-5 -y1)-N4-methyl-5-
(trifluoromethyl)pyrimidine-2,4-
diamine
5-Chloro-N-(3 -cyclopropyl-1 -methyl- 1H-pyrazol-5-y1)-4 -methoxypyrimidin-2-
amine
N2-(3 -Cyclopropyl-1 -methyl- 1H-pyrazol-5-y1)-N4-methyl-5-
(trifluoromethyl)pyrimidine-2,4 -
diamine
5-Chloro-N-(3 -isopropyl- 1-methyl-1 H-pyrazol-5 -y1)-4 -methoxypyrimidin-2-
amine
5-Chloro-N2-(5-isopropyl-2-methyl-2H-pyrazol-3 -y1)-N4-methyl-pyrimidine-2,4-
diamine
5-Chloro-4-methoxy-N-(1,3 ,5-trimethy1-1 H-pyrazol-4 -yl)pyrimidin-2-amine
5-Chloro-N4-methyl-N2-(1,3 ,5-trimethy1-1 H-pyrazol-4-y1)-pyrimidine-2,4-di
amine
5-Chloro-N2-(5-cyclopropy1-2-methyl-2H-pyrazol-3 -y1)-N4-methyl-pyrimidine-2,4-
diamine
N4-Methyl-N2-(5-methyl- 1 - oxetan-3 -yl- 1 H-pyrazol-4-y1)-5-trifluoromethyl-
pyrimidine-2,4-
diamine
N2-(1 -isopropyl- 1H-pyrazol-5 -y1)-N4-methyl-5 -(trifluoromethyl)pyrimidine-
2,4-diamine
5-Chloro-N-(1 -ethyl-5 -methyl-1 H-pyrazol-4 -y1)-4 -methoxypyrimidin-2- amine
5-Chloro-N2-( 1 -ethyl-5 -methyl-1 H-pyrazol-4 -y1)-N4-methylpyrimidine-2,4-
diamine
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N2-(1 -ethyl-3 -methyl-1 H-pyrazol-4-y1)-N4-methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
5-Chloro-N2-( 1 -ethyl-3 -methyl-1 H-pyrazol-4 -y1)-N4-methylpyrimidine-2,4-
diamine
5-chloro-N2-(1 -is opropyl-1 H-pyrazol-5 -y1)-N4-methylpyrimidine-2,4-diamine
5-chloro-N-( 1 -isopropyl- 1 H-pyrazol-5 -y1)-4-methoxypyrimidin-2-amine
5-chloro-4-methoxy-N-(3 -methyl-1 -(methylsulfony1)- 1 H-pyrazol-4 -yl)pyrimi
din-2-amine
N2-(1 -ethyl-1H-pyrazol-3 -y1)-N4-methyl-5-(trifluoromethyl)pyrimidine-2,4-
diamine
5-chloro-4-methoxy-N-(5-methyl- 1 -phenyl-1 H-pyrazol-4-yl)pyrimidin-2-amine
N2-(1 -isopropyl- 1 H-pyrazol-3 -y1)-N4-methyl-5-(trifluoromethyl)pyrimidine-
2,4-diamine
N4-methyl-N2-(5-methy1-1 -(2,2,2-trifluoro ethyl)- 1 H-pyrazol-4 -y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-(1 -(2,2-dimethyl- 1,3 -dioxan-5-y1)-3 -methyl-1 H-pyrazol-4 -y1)-N4 -
methy1-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
5-chl oro-4 -methoxy-N-(5-methyl- 1 -(4-(methylsulfonyl)pheny1)-1 H-pyrazol-4 -
yl)pyrimi din-2-
amine
N4-ethyl-N2-( 1-methyl-1 H-pyrazol-3 -y1)-5-(trifluoromethyl)pyrimidine-2,4-
diamine
5-chl oro-N-( 1 ,5 -dimethyl- 1 H-pyrazol-4-y1)-4 -(oxetan-3 -yloxy)pyrimidin-
2-amine
5-chl oro-4 -(2,2-difluoro ethoxy)-N-( 1 ,5 -dimethyl-1 H-pyrazol-4 -
yl)pyrimidin-2-amine
5-chl oro-N-( 1 ,5 -dimethyl- 1 H-pyrazol-4-y1)-4 -(2,2,2-trifluoro
ethoxy)pyrimidin-2-amine
5-chloro-4-methoxy-N-(3 -methyl-1 -(tetrahydro-2H-pyran-4-y1)- 1 H-pyrazol-4-
yl)pyrimidin-2-
amine
(4-(4-(5 -chloro-4-methoxypyrimidin-2-ylamino)-3 -methyl- 1 H-pyrazol- 1 -
yl)pip eridin- 1 -y1)(1 -
methylcyclopropyl)methanone
(4-(4-(5 -chloro-4-methoxypyrimidin-2-y1 amino)-5-methyl- 1 H-pyrazol- 1 -
yl)pip eridin- 1 -y1)(1 -
methylcyclopropyl)methanone
4-(4-(5-chloro-4-methoxypyrimidin-2-ylamino)-3 -methyl-1 H-pyrazol- 1 -
yl)benzonitrile
5-chloro-4-methoxy-N-(3 -methyl-1 -(3 -methylpyridin-4-y1)-1 H-pyrazol-4 -
yl)pyrimidin-2-
amine
5-chl oro-N-( 1 -(cyclopropylsulfony1)-5 -methyl-1 H-pyrazol-4 -y1)-4-
methoxypyrimidin-2-
amine
5-chl oro-N-( 1 -(cyclopropylsulfony1)-3 -methyl-1 H-pyrazol-4 -y1)-4-
methoxypyrimidin-2-
amine
2-(4-(5-chloro-4-methoxypyrimidin-2-ylamino)-5 -methyl-1 H-pyrazol- 1 -y1)-2-
methylprop anenitrile
2-(4-(5-chloro-4-methoxypyrimidin-2-ylamino)-3 -methyl-1 H-pyrazol- 1 -y1)-2-
methylprop anenitrile
5-chloro-4-ethoxy-N-(5 -methyl-1 -(tetrahydro-2H-pyran-4-y1)-1 H-pyrazol-4 -
yl)pyrimidin-2-
amine
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(5-Chloro-4-methoxy-pyrimidin-2-y1)-[ 1 -(4-methanesulfonyl-phenyl)-3 -methyl-
1 H-pyrazol-
4-yl] -amine
(5-Chloro-4-methoxy-pyrimidin-2-y1)-(3 -methyl-1 -phenyl-1 H-pyrazol-4-y1)-
amine
(4-Methoxy-5-trifluoromethyl-pyrimidin-2-y1)-(3 -methyl-1 -phenyl- 1H-pyrazol-
4 -y1)-amine
(4-Methoxy-5-trifluoromethyl-pyrimidin-2-y1)-(5-methyl-1 -phenyl- 1H-pyrazol-4
-y1)-amine
(5-Chloro-4-methoxy-pyrimidin-2-y1)-( 1 -methane sulfony1-3 -methyl-1 H-
pyrazol-4 -y1)-amine
(5-Chloro-4-methoxy-pyrimidin-2-y1)[5-methy1-1 -(tetrahydro-pyran-4 -y1)- 1 H-
pyrazol-4-yl] -
amine
4- [4 -(5-Chloro-4 -methoxy-pyrimidin-2-ylamino)-3 -methyl-pyrazol-1 -y1]-N,N-
dimethyl-
benzamide
4- [4 -(5-Chloro-4 -methoxy-pyrimidin-2-ylamino)-5-methyl-pyrazol-1 -y1]-N,N-
dimethyl-
benzamide
4- [4 -(5-Chloro-4 -methoxy-pyrimidin-2-ylamino)-5-methyl-pyrazol-1 -y1]-
benzonitrile
N2-(5 -Methoxy- 1-methyl-1 H-pyrazol-4-y1)-N4 -methy1-5-trifluoromethyl-
pyrimidine-2,4-
diamine
(5-Chloro-4-methoxy-pyrimidin-2-y1)-[5-chloro-1 -(tetrahydro-pyran-4 -y1) -1 H-
pyrazol-4-yl] -
amine
(5-Chloro-4-methoxy-pyrimidin-2-y1)- {1 - [1 -(2-fluoro-ethyl)-p ip eridin-4-
yl] -3 -methyl- 1 H-
pyrazol-4-y1} -amine
N2- [1 -(1 - [1,3 ]Dioxolan-2-ylmethyl-pip eri din-4-y1)-5-methyl- 1 H-pyrazol-
4-yl] -N4-ethy1-5 -
trifluoromethyl-pyrimidine-2,4-diamine
N2- [1 -(1 - [1,3 ]Dioxolan-2-ylmethyl-pip eri din-4-y1)-3 -methyl- 1 H-
pyrazol-4-yl] -N4-ethy1-5 -
trifluoromethyl-pyrimidine-2,4-diamine
N2-(1,3 -Dimethyl-1 H-pyrazol-4 -y1)-5 -io do -N4-methyl-pyrimidine-2,4-
diamine
N4-methyl-N2-(5 -methyl-1 -(1 -(2,2,2-trifluoro ethyl)pip eridin-4-y1)- 1 H-
pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N4-methyl-N2-(3 -methyl-1 -(1 -(2,2,2-trifluoro ethyl)piperidin-4-y1)- 1 H-
pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
5-bromo-N4-methyl-N2-(5 -methyl-1 -(1 -(2,2,2-trifluoro ethyl)pip eridin-4-y1)-
1 H-pyrazol-4-
yl)pyrimidine-2,4 -diamine
5-bromo-N4-methyl-N2-(3 -methyl-1 -(1 -(2,2,2-trifluoro ethyl)pip eridin-4-y1)-
1 H-pyrazol-4-
yl)pyrimidine-2,4 -diamine
5-bromo-N4-methyl-N2-(3 -methyl-1 -(2,2,2-trifluoro ethyl)-1 H-pyrazol-4-
yl)pyrimidine-2,4-
diamine
5-bromo-N4-methyl-N2-(5 -methyl-1 -(2,2,2-trifluoro ethyl)-1 H-pyrazol-4-
yl)pyrimidine-2,4-
diamine
N4-ethyl-N2-(3 -methyl-1 -(oxetan-3 -y1)-1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
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5-chloro-N4-ethyl-N2-(3 -methyl-1 -(oxetan-3 -y1)-1 H-pyrazol-4-yl)pyrimidine-
2,4-diamine
5-bromo-N4-methyl-N2-(1 -methyl- 1 H-pyrazol-5-yl)pyrimidine-2,4-diamine
2-methyl-I -(5-methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)-1 H-
pyrazol- 1 -yl)prop an-2-ol
5-chloro-N4-methyl-N2-(3 -methyl-1 -(methylsulfony1)-1 H-pyrazol-4-
yl)pyrimidine-2,4-
diamine
N4-methyl-N2-(3 -methyl-1 -(methylsulfony1)-1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N4-methyl-N2-(3 -methyl-1 H-pyrazol-4-y1)-5 -(trifluoromethyl)pyrimidine-2,4-
diamine
5-bromo-N4-ethyl-N2-(3 -methyl-1 -(oxetan-3 -y1)-1 H-pyrazol-4 -yl)pyrimidine-
2,4 -diamine
N2-( 1 -(difluoromethyl)-3 -methyl-1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(difluoromethyl)-5 -methyl-1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
5-bromo -N4-ethyl-N2-( 1-ethyl-5-methyl-1 H-pyrazol-4 -yl)pyrimidine-2,4-
diamine
5-bromo-N2-(1 -(4-fluoropheny1)-3 -methyl-1 H-pyrazol-4 -y1)-N4 -methylpyrimi
dine-2,4 -
di amine
5-bromo-N4-methyl-N2-(3 -methyl-1 -phenyl-1 H-pyrazol-4 -yl)pyrimidine-2,4-
diamine
5-bromo-N4-methyl-N2-(5 -methyl-1 -phenyl-1 H-pyrazol-4 -yl)pyrimidine-2,4-
diamine
5-bromo-N4-methyl-N2-(1 -methyl- 1 H-pyrazol-4-yl)pyrimidine-2,4-diamine
N4-methyl-N2-(3 -methyl-1 -(1 -(methylsulfonyl)azetidin-3 -y1)-1 H-pyrazol-4 -
y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
5-bromo-N4-methyl-N2-(3 -methyl-1 -propyl- 1 H-pyrazol-4-yl)pyrimidine-2,4-
diamine
5-chloro-N4-methyl-N2-(3 -methyl-1 -((3 -methyloxetan-3 -yl)methyl)- 1 H-
pyrazol-4-
yl)pyrimidine-2,4 -diamine
5-bromo-N2-(1 -(3,5 -difluoropheny1)-5-methyl-1 H-pyrazol-4 -y1)-N4 -
methylpyrimidine-2,4-
diamine
5-bromo-N2-(1 -(3,5 -difluoropheny1)-3 -methyl-1 H-pyrazol-4 -y1)-N4 -
methylpyrimidine-2,4-
diamine
N4-methyl-N2-(3 -methyl-1 -(pyridin-2-y1)-1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-
2,4-diamine
N4-methyl-N2-(3 -methyl-1 -((3 -methyl oxetan-3 -yl)methyl)-1 H-pyrazol-4 -y1)-
5-
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(5 -methyl-1 -propyl- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N4-methyl-N2-(3 -methyl-1 -propyl- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
5-bromo-N2-(1 -isopropyl-3 -methyl-1 H-pyrazol-4-y1)-N4-methylpyrimidine-2,4-
diamine
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5-bromo-N2-(1 -(4-chloropheny1)-5-methyl- 1 H-pyrazol-4-y1)-N4-
methylpyrimidine-2,4 -
di amine
N2-( 1 -(4-chloropheny1)-5-methyl- 1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N4-methyl-N2-(3 -methyl-1 -(4 -(methyl sulfonyl)pheny1)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(5 -methyl-1 -(4 -(methylsul fonyl)pheny1)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -((1 S,5S)-8-oxabicyclo [3 .2. 1 ] octan-3 -y1)-3 -methyl-1 H-pyrazol-4
-y1)-N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -butyl-5-methyl- 1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4-
diamine
N4-methyl-N2-(3 -methyl-1 -(pyrimidin-2 -y1)- 1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-
2,4-diamine
N2 -(1 -(4-chloropheny1)-3 -methyl-1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N2-( 1 -(2-fluoroethyl)-3 -methyl-1 H-pyrazol-4-y1)-N4 -methyl-5 -
(trifluoromethyl)pyrimidine-
2,4-diamine
N4-methyl-N2-(3 -methyl-1 -(1 -(oxetan-3 -yl)pip eridin-4-y1)- 1 H-pyrazol-4-
y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N4-methyl-N2-(5 -methyl-1 -(1 -(oxetan-3 -yl)pip eridin-4-y1)- 1 H-pyrazol-4-
y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N2-( 1 -(2-fluoroethyl)-5 -methyl-1 H-pyrazol-4-y1)-N4 -methyl-5 -
(trifluoromethyl)pyrimidine-
2,4-diamine
1 -(4 -(444 -(ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-3 -methyl-1
H-pyrazol- 1 -
yl)p ip eridin- 1 -yl)ethanone
cyclopropy1(4-(5 -methy1-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)pip eridin- 1 -yl)methanone
cyclopropy1(4-(3 -methy1-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)pip eridin- 1 -yl)methanone
1 -(4 -(444 -(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-5 -methyl-1
H-pyrazol- 1 -
yl)p ip eridin- 1 -yl)ethanone
N2 -(5-chloro- 1 -isopropyl- 1 H-pyrazol-4 -y1)-N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N2 -(5-chloro- 1 -ethyl- 1 H-pyrazol-4 -y1)-N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(3 -methyl-1 -(pyrimidin-5-y1)- 1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-
2,4-diamine
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N4 -methyl-N2-(4 -methyl- 1 -(1 -methylp ip eridin-4 -y1)- 1 H-pyrazol-3 -y1)-
5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(5 -methyl-1 -(2 -methylpyridin-4 -y1)- 1 H-pyrazol-4 -y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(3 -methyl-1 -(2 -methylpyridin-4 -y1)- 1 H-pyrazol-4 -y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N4 -ethyl-N2 -(3 -methyl-1 -((3 -methyloxetan-3 -yl)methyl)- 1 H-pyrazol-4-y1)-
5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2 -(5 -chloro- 1 -cyc lopropyl- 1 H-pyrazol-4 -y1)-N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N2 -(5 -chloro- 1 -(cyclopropylmethyl)- 1 H-pyrazol-4 -y1)-N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
445 -methy1-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimidin-2 -ylamino)- 1
H-pyrazol- 1 -
yl)b enzonitrile
4-(3 -methy1-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimidin-2 -ylamino)- 1
H-pyrazol- 1 -
yl)b enzonitrile
N4-methyl-N2-(3 -methyl-1 -(tetrahydrofuran-3 -y1)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(5 -methyl-1 -(tetrahydrofuran-3 -y1)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
-(4 -(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-5 -methyl- 1 H-
pyrazol- 1 -y1)-1 -
methylp ip eridin-2-one
5 -(4 -(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-3 -methyl- 1 H-
pyrazol- 1 -y1)-1 -
methylp ip eridin-2-one
5-(3 -methy1-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimi din-2 -ylamino)-
1 H-pyrazol- 1 -
yl)piperidin-2-one
545 -methy1-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimi din-2 -ylamino)- 1
H-pyrazol- 1 -
yl)piperidin-2-one
N2-( 1 -is opropy1-5 -methyl- 1 H-pyrazol-4-y1)-N4 -methyl-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N,N-dimethy1-4-(5 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -
ylamino)- 1 H-
pyrazol- 1 -yl)benzamide
4-(4 -(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-5 -methyl- 1 H-
pyrazol- 1 -y1)-
N,N-dimethylb enzami de
N4 -ethyl-N2 -(5 -methyl- 1 -(tetrahydro-2H-pyran-4 -y1)- 1 H-pyrazol-4 -y1)-5
-
(trifluoromethyl)pyrimidine-2,4 -diamine
N4 -ethyl-N2 -(3 -methyl-1 -(tetrahydro-2H-pyran-4 -y1)- 1 H-pyrazol-4 -y1)-5 -
(trifluoromethyl)pyrimidine-2,4 -diamine
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N4 -ethyl-N2 -(3 -methyl-1 -(methylsul fony1)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)pyrimidine-
2,4-diamine
N2-( 1 -(4-(cyclopropylsulfonyl)pheny1)-3 -methyl-1 H-pyrazol-4 -y1)-N4 -
methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
4-(4-(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-5-methyl- 1 H-
pyrazol- 1 -
yl)b enzonitrile
N4 -ethyl-N2 -(5-methyl- 1 -(4-(methylsulfonyl)pheny1)- 1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
N,N-dimethy1-4-(3 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -
ylamino)- 1 H-
pyrazol- 1 -yl)benzamide
N2-( 1 -(cyclopropylmethyl)-5 -methyl-1 H-pyrazol-4-y1)-N4-methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(cyclopropylmethyl)-3 -methyl-1 H-pyrazol-4-y1)-N4-methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(4-(cyclopropylsulfonyl)pheny1)-5 -methyl-1 H-pyrazol-4 -y1)-N4-methy1-
5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2 -(5-chloro- 1 -(oxetan-3 -y1)- 1 H-pyrazol-4 -y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-
2,4-diamine
N4-ethyl-N2-(5-methyl- 1 -((3 -methyloxetan-3 -yl)methyl)- 1 H-pyrazol-4-y1)-5
-
(trifluoromethyl)pyrimidine-2,4-diamine
N2 -(1 -(cyclopropylsulfony1)-3 -methyl- 1H-pyrazol-4 -y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2 -(1 -(cyclopropylsulfony1)-3 -methyl-1 H-pyrazol-4-y1)-N4 -ethy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
5-chloro-N4-(2,2 -difluoro ethyl)-N2 -( 1 ,5 -dimethyl- 1 H-pyrazol-4-
yl)pyrimidine-2,4-diamine
5-chloro-4-methyl-N-(3 -methyl-1 -(4 -(methylsulfonyl)pheny1)- 1 H-pyrazol-4-
yl)pyrimidin-2-
amine
N2 -(1 -(4-(cyclopropylsulfonyl)pheny1)-5 -methyl-1 H-pyrazol-4 -y1)-N4-ethy1-
5-
(trifluoromethyl)pyrimidine-2,4-diamine
2-methyl-I -(445 -methy1-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)pip eridin- 1 -yl)prop an- 1-one
N4 -ethyl-N2-( 1 -methyl- 1 H-pyrazol-5-y1)-5-(trifluoromethyl)pyrimidine-2,4-
diamine
N2-(3 -cyc lopropyl- 1 -methyl- 1 H-pyrazol-4-y1)-N4-methyl-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N2-(5-cyc lopropyl- 1 -methyl- 1 H-pyrazol-4-y1)-N4-methyl-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N4-methyl-N2-(5-methyl- 1 -((3 -methyloxetan-3 -yl)methyl)- 1 H-pyrazol-4-y1)-
5 -
(trifluoromethyl)pyrimidine-2,4-diamine
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N2-(5-chloro- 1 -((3 -methyl oxetan-3 -yl)methyl)-1 H-pyrazol-4 -y1)-N4 -
methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
1 -(4 -(444 -(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-5 -methyl-1
H-pyrazol- 1 -
yl)piperidin-1 -y1)-2-methylprop an- 1-one
N4-ethyl-N2-(3 -methyl-1 -(1 -(oxetan-3 -yl)azetidin-3 -y1)- 1 H-pyrazol-4-y1)-
5-
(trifluoromethyl)pyrimidine-2,4-diamine
cyclopropy1(4-(4-(4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-3 -
methyl-1 H-
pyrazol- 1 -yl)piperidin-1 -yl)methanone
cyclopropy1(4-(4-(4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-5 -
methyl-1 H-
pyrazol-1 -yl)piperidin-1 -yl)methanone
1 -(5 -chloro-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-1H-
pyrazol-1 -y1)-
2-methylprop an-2-ol
N4 -ethyl-N2-( 1 -ethyl- 1 H-pyrazol-3 -y1)-5-(trifluoromethyl)pyrimidine-2,4-
diamine
(S)-N2-( 1 -(2-methoxypropy1)-5-methyl- 1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(2-methoxycyclopenty1)-3 -methyl-1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
(S)-N2-( 1 -(2-methoxypropy1)-3 -methyl-1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(1 -methoxy-2-methylpropan-2-y1)-3 -methyl-1 H-pyrazol-4 -y1)-N4-
methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(2,6 -dimethyltetrahydro-2H-pyran-4 -y1)-3 -methyl-1 H-pyrazol-4 -y1)-
N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
(R)-N2-( 1 -(2-methoxypropy1)-5-methyl- 1 H-pyrazol -4 -y1)-N4-methy1-5-
(trifluoromethyl)
pyrimidine-2,4 -diamine
N2-( 1 -(3 -methoxycyclopenty1)-3 -methyl-1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N4 -methyl-N2-(1 -methyl-5-(methylamino)-1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)pyrimidine-
2,4-diamine
N4-methyl-N2-(5 -methyl-1 -(methylsulfony1)- 1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)
pyrimidine-2,4 -diamine
N4-methyl-N2-(5 -methyl-1 -(tetrahydro-2H-1 , 1 -dioxo-thiopyran-4-y1)- 1 H-
pyrazol-4-y1)-5 -
(trifluoromethyl) pyrimidine-2,4-diamine
2-methyl-I -(4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-5-
(trifluoromethyl)-1 H-pyrazol-1 -yl)propan-2-ol
2-methyl-I -(4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-3 -
(trifluoromethyl)-1 H-pyrazol-1 -yl)propan-2-ol
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N2-( 1 -(3 -fluoro- 1 -(oxetan-3 -yl)pip eridin-4 -y1)-3 -methyl- 1 H-pyrazol-
4 -y1)-N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
(R)-N2 -( 1 -(1 -methoxyprop an-2 -y1)-3 -methyl-1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
1 -(3 -tert-buty1-4-(4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2 -ylamino)-
1 H-pyrazol- 1 -y1)-
2-methylprop an-2-ol
N4-methyl-N2-(3 -methyl-1 -(1 -(2,2,2-trifluoroethyl)azetidin-3 -y1)- 1 H-
pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(1 -methoxy-2-methylprop an-2-y1)-5-methyl- 1 H-pyrazol-4-y1)-N4-
methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
(R)-N4 -methyl-N2 -(3 -methyl-1 -(1 -(oxetan-3 -yl)pyrrolidin-3 -y1)- 1 H-
pyrazol-4 -y1)-5-
(trifluoromethyl) pyrimidine-2,4 -diamine
(R)-N2 -( 1 -(1 -methoxyprop an-2 -y1)-5-methyl- 1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N4 -methyl-N2-(4 -methyl- 1 H-pyrazol-5-y1)-5 -(trifluoromethyl)pyrimidine-2,4-
diamine
N4 -ethyl-N2 -(5-methyl- 1 -(1 -methylpip eridin-4-y1)- 1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N4 -ethyl-N2 -(3 -methyl-1 -(1 -methylpip eridin-4-y1)- 1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)
pyrimi dine-2,4 -diamine
N4-methyl-N2-(3 -methyl-1 -(1 -methylp ip eridin-4 -y1)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)
pyrimi dine-2,4 -diamine
N4-methyl-N2-(5 -methyl-1 -(1 -methylp ip eridin-4 -y1)- 1 H-pyrazol-4-y1)-5 -
(trifluoromethyl)
pyrimi dine-2,4 -diamine
(R)-N4 -methyl-N2 -(5-methyl- 1 -(1 -(oxetan-3 -yl)pyrrolidin-3 -y1)- 1 H-
pyrazol-4 -y1)-5-
(trifluoromethyl) pyrimidine-2,4 -diamine
N4-methyl-N2-(5 -methyl-1 -(pyridin-2 -ylmethyl)- 1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(3 -methyl-1 -(pyridin-2 -ylmethyl)- 1 H-pyrazol-4 -y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(1 -is opropylazetidin-3 -y1)-3 -methyl-1 H-pyrazol-4-y1)-N4-methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
1 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-y1 amino)- 1 H-
pyrazo le-5 -
c arb onitrile
N4 -ethyl-N2 -( 1 -(isopropylsulfony1)-3 -methyl-1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(isopropylsulfony1)-3 -methyl-1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
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N2-( 1 -(is opropylsulfony1)-5-methyl- 1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(s ec-butylsulfony1)-5-methyl- 1 H-pyrazol-4 -y1)-N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(sec-butylsulfony1)-3 -methyl-1 H-pyrazol-4 -y1)-N4-methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
1 -(4 -(4-(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-3 -isopropyl-
1 H-pyrazol- 1 -y1)-
2-methylprop an-2-ol
N2-( 1 -(3 -fluoro- 1 -methylpiperidin-4-y1)-3 -methyl-1 H-pyrazol-4-y1)-N4-
methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2-(5-isopropyl- 1 -methyl- 1 H-pyrazol-4-y1)-N4 -methyl-5 -
(trifluoromethyl)pyrimidine-2,4-
diamine
N4-methyl-N2-(3 -methyl-1 -(1 -(pyridin-2-yl)ethyl)- 1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(5 -methyl-1 -(1 -(pyridin-2-yl)ethyl)- 1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-(5-chloro- 1 -(tetrahydro-2H-pyran-4-y1)- 1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-(3 -isopropyl-1 -methyl- 1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-2,4 -
diamine
N2-(3 -cyc lobutyl- 1 -methyl- 1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4 -
diamine
N2-(5-cyc lobutyl- 1 -methyl- 1 H-pyrazol-4-y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4 -
diamine
N4-methyl-N2-(3 -methyl-1 -(tetrahydro-2H-pyran-3 -y1)- 1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N4-methyl-N2-(5 -methyl-1 -(tetrahydro-2H-pyran-3 -y1)- 1 H-pyrazol-4 -y1)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-(1 ,5-dimethyl- 1 H-pyrazol-4 -y1)-N4-((tetrahydro-2H-pyran-4-y1) methyl)-5-
(trifluoromethyl)pyrimidine-2,4-diamine
(R)-N4-methyl-N2-(3 -methyl-1 -(1 -methylpyrrolidin-3 -y1)- 1 H-pyrazol-4-y1)-
5 -
(trifluoromethyl)pyrimidine-2,4-diamine
1 -(5 -chloro-4 -(4 -(ethylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)- 1
H-pyrazol- 1 -y1)-2-
methylprop an-2-ol
1 -(3 -cyclopropy1-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-
1 H-pyrazol- 1 -
y1)-2-methylpropan-2-ol
1 -(3 -cyc lopropy1-4 -(4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-
1 H-pyrazol- 1 -
y1)-2-methylpropan-2-ol
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2-(5 -chloro-4-(4 -(methyl amino)-5-(trifluoromethyl)pyrimi din-2 -ylamino)- 1
H-pyrazol- 1 -y1)-
N,2-dimethylprop anamide
N2-( 1 -(1 -(2 -methoxyethyl)pip eridin-4 -y1)-3 -methyl-1 H-pyrazol-4-y1)-N4 -
methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2-( 1 -(1 -(2 -methoxyethyl)pip eridin-4 -y1)-5-methyl- 1 H-pyrazol-4-y1)-N4 -
methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
(R)-N4 -methyl-N2 -(5-methyl- 1 -(1 -methylpyrrolidin-3 -y1)- 1 H-pyrazol-4-
y1)-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2 -(5-chloro- 1 -(3 -fluoro- 1 -methylpip eridin-4 -y1)- 1 H-pyrazol-4-y1)-N4-
methyl-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N2 -(5-chloro- 1 -(1 -ethyl-3 -fluoropip eridin-4-y1)- 1 H-pyrazol-4 -y1)-N4-
methy1-5 -
(trifluoromethyl)pyrimidine-2,4-diamine
N4 -ethyl-N2-( 1 -(ethylsulfony1)-3 -methyl-1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-
2,4-diamine
N4 -ethyl-N2 -( 1 -(ethylsulfony1)-5 -methyl-1 H-pyrazol-4-y1)-5-
(trifluoromethyl)pyrimidine-
2,4-di amine
N4-methyl-N2-(3 -methyl-1 -(2 -methy1-2-morpho linopropy1)- 1 H-pyrazol-4 -y1)-
5-
(trifluoromethyl) pyrimidine-2,4 -diamine
N2-( 1 -(1 -ethyl-3 -fluoropiperidin-4 -y1)-3 -methyl-1 H-pyrazol-4-y1)-N4-
methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
N2 -(5-(dimethyl amino)- 1 -methyl- 1 H-pyrazol-4 -y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4-diamine
2-(5-chloro-4-(4 -(methyl amino)-5-(trifluoromethyl)pyrimi din-2 -ylamino)- 1
H-pyrazol- 1 -y1)-
2-methylprop an- 1 -ol
N2-( 1 -(ethylsulfony1)-3 -methyl-1 H-pyrazol-4-y1)-N4 -methy1-5-
(trifluoromethyl)pyrimidine-
2,4-diamine
2-Methyl-I -[3 -methy1-4 -(4 -methylamino-5-trifluoromethyl-pyrimidin-2 -
ylamino)-pyrazol- 1 -
yl] -prop an-2 -ol
N2-[ 1 -(2-Methoxy-ethyl)-3 -methyl-1 H-pyrazol-4-yl] -N4-methyl-5 -
trifluoromethyl-
pyrimidine-2,4-diamine
N2-[ 1 -(2-Methoxy-ethyl)-5-methyl- 1 H-pyrazol-4-yl] -N4-methyl-5 -
trifluoromethyl-
pyrimidine-2,4-diamine
5-Bromo -N2-( 1 -ethyl-3 -methyl-1 H-pyrazol-4-y1)-N4 -methyl-pyrimidine-2,4 -
diamine
N4-Methyl-N2- [3 -methyl-1 -(2,2,2 -trifluoro-ethyl)- 1 H-pyrazol-4-yl] -5-
trifluoromethyl-
pyrimidine-2,4 -diamine
5-Bromo -N2-( 1 -difluoromethy1-5 -methyl-1 H-pyrazol-4-y1)-N4-methyl-
pyrimidine-2,4-
di amine
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5-Bromo-N2-(1 -difluoromethy1-3 -methyl-1 H-pyrazol-4-y1)-N4-methyl-pyrimidine-
2,4-
di amine
5-Bromo -N2-(1,5-dimethyl- 1 H-pyrazol-4 -y1)-N4 -ethyl-pyrimidine-2,4-diamine
5-Bromo -N2- [1 -(4-fluoro-phenyl)-5 -methyl-1 H-pyrazol-4-yl] -N4 -methyl-
pyrimidine-2,4 -
di amine
5-Bromo -N4-methyl-N2-(5-methyl- 1 -propyl- 1 H-pyrazol-4-y1)-pyrimidine-2,4-
diamine
5-Bromo -N2- [1 -(4-chloro-phenyl)-3 -methyl-1 H-pyrazol-4 -yl] -N4-methyl-
pyrimi dine-2,4-
di amine
N2-( 1,5-Dimethy1-1 H-pyrazol-4 -y1)-N4 -ethy1-5-trifluoromethyl-pyrimidine-
2,4 -di amine
5- [4 -(4-Ethylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-3 -methyl-pyrazol-
1 -y1]-
piperidin-2-one
4- [4 -(4-Ethylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-3 -methyl-pyrazol-
1 -y1]-N,N-
dimethyl-benzamide
N2-[ 1 -(4-Cyclopropanesulfonyl-phenyl)-3 -methyl-1 H-pyrazol-4-yl] -N4-ethyl-
5 -
trifluoromethyl-pyrimidine-2,4-diamine
4- [4 -(4-Ethylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-3 -methyl-pyrazol-
1 -y1]-
benzonitrile
N4-Ethyl-N2- [1 -(4-methane sulfonyl-pheny1)-3 -methyl-1 H-pyrazol-4-yl] -5-
trifluoromethyl-
pyrimidine-2,4 -diamine
1- { 4 -[4 -(4-Ethylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-5 -methyl-
pyrazol-1 -yl] -
p ip eridin-1 -yl} -2-methyl-prop an- 1-one
1- { 4 -[4-(4-Ethylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-3 -methyl-
pyrazol-1 -yl] -
p ip eridin-1 -yl} -2-methyl-prop an- 1-one
N4-Methyl-N2- [3 -methyl-1 -(3 -methyl-pyridin-4-y1)-1 H-pyrazol-4 -yl] -5 -
trifluoromethyl-
pyrimidine-2,4 -diamine
N2- [1 -((R)-2-Methoxy-propy1)-3 -methyl-1 H-pyrazol-4-yl] -N4-methy1-5-
trifluoromethyl-
pyrimidine-2,4 -diamine
N2- [1 -(2,6-Dimethyl-tetrahydro -pyran-4 -y1)-5-methyl- 1 H-pyrazol-4 -yl] -
N4-methy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N2- [1 -(1,1 -Dioxo -hexahydro- 1 $1%6 &-thiopyran-4 -y1)-3 -methyl-1 H-
pyrazol-4-yl] -N4-
methy1-5-trifluoromethyl-pyrimidine-2,4 -diamine
N2- [1 -((R)-2-Methoxy-1 -methyl-ethyl)-5-methyl-1H-pyrazol-4-yl] -N4-methy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N2- [1 -((S)-2-Methoxy- 1 -methyl-ethyl)-3 -methyl-1 H-pyrazol-4 -yl] -N4-
methy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N4 -Methyl-N2- [3 -methyl-1 -((S)- 1 -oxetan-3 -yl-pyrrolidin-3 -y1)- 1 H-
pyrazol-4 -yl] -5-
trifluoromethyl-pyrimidine-2,4-diamine
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N4 -Methyl-N2- [5 -methyl-1 -((S)- 1 -oxetan-3 -yl-pyrrolidin-3 -y1)-1 H-
pyrazol-4 -yl] -5-
trifluoromethyl-pyrimidine-2,4-diamine
N2-[ 1 -(1 -Is opropyl-azetidin-3 -y1)-5-methyl- 1 H-pyrazol-4 -yl] -N4 -
methy1-5-trifluoromethyl-
pyrimidine-2,4 -diamine
N4-Ethyl-N2- [5 -methyl-1 -(prop ane-2-sulfony1)-1 H-pyrazol-4-yl] -5 -
trifluoromethyl-
pyrimidine-2,4-diamine
N2-(5-Cyclobuty1-1 -methyl-1 H-pyrazol-4 -y1)-N4 -ethy1-5 -trifluoromethyl-
pyrimidine-2,4 -
diamine
N2-(3-Cyclobuty1-1 -methyl- 1 H-pyrazol-4 -y1)-N4 -ethy1-5 -trifluoromethyl-
pyrimidine-2,4 -
diamine
N4-Ethyl-N2- { 1 - [1 -(2-methoxy-ethyl)-pip eridin-4-yl] -3 -methyl-1 H-
pyrazol-4 -yl} -5 -
trifluoromethyl-pyrimidine-2,4-diamine
N4-Ethyl-N2- { 1 - [1 -(2-methoxy-ethyl)-piperidin-4-yl] -5-methyl-I H-pyrazol-
4 -yl} -5 -
trifluoromethyl-pyrimidine-2,4-diamine
N2- {1 -[ 1 -(2-F luoro-ethyl)-pip eridin-4-yl] -5-methyl- 1 H-pyrazol-4-y1} -
N4 -methy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N2- {1 -[ 1 -(2-F luoro-ethyl)-pip eridin-4-yl] -3 -methyl- 1 H-pyrazol-4-y1} -
N4 -methy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N2-[5-Chloro-1 -(3 -fluoro-1 -methyl-pip eridin-4-y1)- 1 H-pyrazol-4-yl] -N4 -
methy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N2-( 1 -Ethanesulfony1-5 -methyl-1 H-pyrazol-4-y1)-N4 -methyl-5 -
trifluoromethyl-pyrimidine-
2,4-diamine
N4-Methyl-N2- [5 -methyl-1 -(2-methyl-2-morpholin-4-yl-propy1)- 1 H-pyrazol-4-
yl] -5 -
trifluoromethyl-pyrimidine-2,4-diamine
N4-Methyl-N2-(3 -methyl-1 -pyridin-3 -ylmethyl-1 H-pyrazol-4 -y1)-5 -
trifluoromethyl-
pyrimi dine-2,4 -diamine
N2-( 1 -Cyclopropanesulfony1-3 -cyclopropyl- 1 H-pyrazol-4-y1)-N4 -methy1-5-
trifluoromethyl-
pyrimi dine-2,4 -diamine
N4-Methyl-N2-(5 -methyl-1 -pyridin-3 -ylmethyl-1 H-pyrazol-4 -y1)-5 -
trifluoromethyl-
pyrimi dine-2,4 -diamine
(5-Chloro-4-methoxy-pyrimidin-2-y1)- { 1 - [1 -(2-fluoro-ethyl)-p ip eridin-4-
yl] -5-methyl- 1 H-
pyrazol-4-y1} -amine
N4-Methyl-N2- [3 -methyl-1 -(6-methyl-pyridin-2-ylmethyl)-1H-pyrazol-4-yl] -5-
trifluoromethyl-pyrimidine-2,4-diamine
N4-Ethyl-N2- [1 -(2-methoxy-ethyl)-3 -methyl-1 H-pyrazol-4-yl] -5-
trifluoromethyl-pyrimidine-
2,4-diamine
N4-Ethyl-N2- [1 -(2-methoxy-ethyl)-5-methy1-1H-pyrazol-4-y1]-5-trifluoromethyl-
pyrimidine-
2,4-diamine
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1 - [4 -(4-Ethylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-5 -methyl-
pyrazol- 1 -yl] -2-
methyl-prop an-2 -ol
1 - [4 -(4-Ethylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-3 -methyl-
pyrazol- 1 -yl] -2-
methyl-prop an-2 -ol
N2-[ 1 -(1,1 -Dimethy1-2-morpho lin-4 -yl-ethyl)-3 -methyl-1 H-pyrazol-4-yl] -
N4-ethyl-5 -
trifluoromethyl-pyrimidine-2,4-diamine
N2-[ 1 -(1,1 -Dimethy1-2-morpho lin-4 -yl-ethyl)-3 -methyl-1 H-pyrazol-4-yl] -
N4-methyl-5 -
trifluoromethyl-pyrimidine-2,4-diamine
N2-[ 1 -(3 -Fluoro- 1 -methyl-pip eridin-4 -y1)-3 -methyl-1 H-pyrazol-4 -yl] -
N4-methy1-5 -
trifluoromethyl-pyrimidine-2,4-diamine
N4 -Cyclopropyl-N2-(1 -methanesulfony1-3 -methyl-1 H-pyrazol-4-y1)-5-tri
fluoromethyl-
pyrimi dine-2,4 -diamine
N4 -Cyclopropyl-N2-(1 -methanesulfony1-5-methyl- 1 H-pyrazol-4-y1)-5-tri
fluoromethyl-
pyrimi dine-2,4 -diamine
1 -[3 -Chloro-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-
pyrazol- 1 -yl] -2-
methyl-prop an-2 -ol
2- [4 -(4-Ethylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-3 -methyl-pyrazol-
1 -yl] -N-
methyl-is obutyramide
N4-Methyl-N2-(3 -methyl-1 -pyrimidin-2 -ylmethyl- 1 H-pyrazol-4-y1)-5-tri
fluoromethyl-
pyrimi dine-2,4 -diamine
N2 -[5-Chloro- 1 -(3 -fluoro- 1 -oxetan-3 -yl-pip eridin-4-y1)- 1 H-pyrazol-4-
yl] -N4-methy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N2 -[5-Chloro- 1 -(3 -fluoro- 1 -oxetan-3 -yl-pip eridin-4-y1)- 1 H-pyrazol-4-
yl] -N4-ethy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N4 -Ethyl-N2- [5 -methyl-1 -((S)- 1 -oxetan-3 -yl-piperidin-3 -y1)- 1 H-
pyrazol-4 -yl] -5-
trifluoromethyl-pyrimidine-2,4-diamine
N4 -Ethyl-N2- [3 -methyl-1 -((S)- 1 -oxetan-3 -yl-piperidin-3 -y1)- 1 H-
pyrazol-4 -yl] -5-
trifluoromethyl-pyrimidine-2,4-diamine
N4-Methyl-N2-(5 -methyl-1 -pyridazin-3 -ylmethyl- 1 H-pyrazol-4-y1)-5 -
trifluoromethyl-
pyrimidine-2,4-diamine
N4-Methyl-N2-(3 -methyl-1 -pyridazin-3 -ylmethyl- 1 H-pyrazol-4-y1)-5 -
trifluoromethyl-
pyrimidine-2,4-diamine
N4 -Ethyl-N2- [5 -methyl-1 -((S)- 1 -methyl-pip eridin-3 -y1)- 1 H-pyrazol-4 -
yl] -5-trifluoromethyl-
pyrimidine-2,4-diamine
N4 -Ethyl-N2- [3 -methyl-1 -((S)- 1 -methyl-pip eridin-3 -y1)- 1 H-pyrazol-4 -
yl] -5-trifluoromethyl-
pyrimidine-2,4-diamine
3 - [5 -Chloro-4-(4 -methylamino-5-tri fluoromethyl-pyrimidin-2 -ylamino)-
pyrazol- 1 -yl] -2,2 -
dimethyl-prop ionitrile
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N4-Methyl-N2- [5 -methyl-1 -(6-methyl-pyridin-2-ylmethyl)-1H-pyrazol-4-yl] -5-
trifluoromethyl-pyrimidine-2,4-diamine
N4-Methyl-N2-(5 -methyl-1 -pyrimidin-2-ylmethyl- 1 H-pyrazol-4-y1)-5-
trifluoromethyl-
pyrimidine-2,4 -diamine
N4-Methyl-N2-(5 -methyl-1 -pyrazin-2-ylmethy1-1 H-pyrazol-4-y1)-5-
trifluoromethyl-
pyrimidine-2,4 -diamine
N4-Methyl-N2-(3 -methyl-1 -pyrazin-2-ylmethy1-1 H-pyrazol-4-y1)-5-
trifluoromethyl-
pyrimidine-2,4 -diamine
3 - [5 -Chloro-4-(4 -ethylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-pyrazol-
1 -yl] -2,2-
dimethyl-prop ionitrile
N4 -Ethyl-N2- [1 -(3 -fluoro- 1 -oxetan-3 -yl-piperidin-4-y1)-3 -methyl-1 H-
pyrazol-4 -yl] -5 -
trifluoromethyl-pyrimidine-2,4-diamine
3-Methyl-I - [5 -methy1-4 -(4-methylamino-5-trifluoromethyl-pyrimidin-2-
ylamino)-pyrazol- 1 -
yl] -butan-2-ol
3-Methyl-I -[3 -methy1-4 -(4-methylamino-5-trifluoromethyl-pyrimidin-2-
ylamino)-pyrazol- 1 -
yl] -butan-2-ol
N2-[ 1 -(1 - [1,3 ]Dioxolan-2-ylmethyl-pyrrolidin-3 -yl)-3 -methyl-1 H-pyrazol-
4 -yl] -N4 -ethy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N4-Methyl-N2-(5 -methyl-1 -pyrimidin-4 -ylmethyl- 1 H-pyrazol-4-y1)-5 -
trifluoromethyl-
pyrimidine-2,4-diamine
N4-Methyl-N2- [5 -methyl-1 -(1 -methyl-1 H-pyrazol-3 -ylmethyl)-1 H-pyrazol-4-
yl] -5-
trifluoromethyl-pyrimidine-2,4-diamine
N4-Methyl-N2- [3 -methyl-1 -(1 -methyl-1 H-pyrazol-3 -ylmethyl)-1 H-pyrazol-4-
yl] -5-
trifluoromethyl-pyrimidine-2,4-diamine
3 - [3 -Chloro-4-(4-methylamino-5 -trifluoromethyl-pyrimi din-2-ylamino)-
pyrazol- 1 -yl] -2,2-
dimethyl-prop ionitrile
N4-Ethyl-N2- {3 -methyl-1 - [1 -methyl-1 -(4H-[1,2,4]triazol-3 -y1)-ethyl] -1
H-pyrazol-4 -yl} -5-
trifluoromethyl-pyrimidine-2,4-diamine
N2-[ 1 -(1 - [1,3 ]Dioxolan-2-ylmethyl-pyrrolidin-3 -yl)-5-methy1-1 H-pyrazol-
4 -yl] -N4 -ethy1-5-
trifluoromethyl-pyrimidine-2,4-diamine
N4-Methyl-N2-(3 -methyl-1 -pyrimidin-4 -ylmethyl- 1 H-pyrazol-4-y1)-5-
trifluoromethyl-
pyrimi dine-2,4 -diamine
N2-(5-Fluoromethy1-1 -methyl-1 H-pyrazol-4 -y1)-N4-methy1-5-trifluoromethyl-
pyrimidine-
2,4-diamine
N4-Ethyl-N2- {3 -methyl-1 - [1 -methyl-1 -(5 -methyl-4H- [1,2,4]triazol-3-y1)-
ethyl] -1 H-pyrazol-
4-y1} -5-trifluoromethyl-pyrimidine-2,4-diamine
N4-Methyl-N2- {3 -methyl-1 - [1 -methyl-1 -(4H-[1,2,4]triazol-3 -y1)-ethyl] -1
H-pyrazol-4-y1} -5-
trifluoromethyl-pyrimidine-2,4-diamine
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N4 -Ethyl-N2- [1 -(3 -fluoro-pip eridin-4 -y1)-3 -methyl-1 H-pyrazol-4 -yl] -5-
trifluoromethyl-
pyrimidine-2,4-diamine
2- [5 -Methyl-4 -(4-methyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol- 1 -yl] -
cyclop entanol
2- [3 -Methyl-4 -(4-methyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol- 1 -yl] -
cyclop entanol
N4-ethyl-N2-(3 -methyl-1 -(2-(5 -methyl- 1,3 ,4-oxadiazol-2-yl)prop an-2-y1)-1
H-pyrazol-4 -y1)-
5-(trifluoromethyl) pyrimidine-2,4-diamine
N4 -ethyl-N2-(3 -methyl-1 -(2-(4-methyl-4H- 1,2,4 -triazol-3 -yl)prop an-2-y1)-
1 H-pyrazol-4-y1)-
5-(trifluoromethyl) pyrimidine-2,4-diamine
N4 -ethyl-N2-(3 -methyl-1 -(2-(1 -methyl-1 H- 1,2,4 -triazol-3 -yl)prop an-2-
y1)- 1 H-pyrazol-4-y1)-
5-(trifluoromethyl) pyrimidine-2,4-diamine
N4-methyl-N2-(3 -methyl-1 -(2-(5-methyl- 1,3 ,4 -oxadiazol-2-yl)prop an-2-y1)-
1 H-pyrazol-4 -
y1)-5-(trifluoromethyl) pyrimidine-2,4 -diamine
N4-methyl-N2-(3 -methyl-1 -(2-( 1-methyl-1 H-pyrazol-4 -yl)prop an-2-y1)-1 H-
pyrazol-4 -y1)-5-
(trifluoromethyl) pyrimidine-2,4 -diamine
N4-methyl-N2-(5 -methyl-1 -(2-( 1-methyl-1 H-pyrazol-4 -yl)prop an-2-y1)-1 H-
pyrazol-4 -y1)-5-
(trifluoromethyl) pyrimidine-2,4 -diamine
N4-ethyl-N2-(3 -methyl-1 -(2-(1 -methyl- 1 H-pyrazol-3 -yl)prop an-2-y1)-1 H-
pyrazol-4-y1)-5 -
(trifluoromethyl) pyrimidine-2,4 -diamine
N4-ethyl-N2-(3 -methyl-1 -(2-(1 -methyl- 1 H-pyrazol-5-yl)prop an-2-y1)-1 H-
pyrazol-4-y1)-5 -
(trifluoromethyl) pyrimidine-2,4 -diamine
N4-methyl-N2-(3 -methyl-1 -(2-( 1-methyl-1 H-pyrazol-5 -yl)prop an-2-y1)-1 H-
pyrazol-4 -y1)-5-
(trifluoromethyl) pyrimidine-2,4 -diamine
N4-methyl-N2-(3 -methyl-1 -(2-( 1-methyl-1 H-pyrazol-3 -yl)prop an-2-y1)-1 H-
pyrazol-4 -y1)-5-
(trifluoromethyl) pyrimidine-2,4 -diamine
N2-( 1 ',5-dimethy1-1 'H- 1,4'-bipyrazol-4 -y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4 -
di amine
N2-( 1 ',3 -dimethyl-1 'H- 1,4'-bipyrazol-4 -y1)-N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4 -
diamine
N2-( 1 -(2-(4H-1,2,4 -triazol-3 -yl)prop an-2-y1)-3 -methyl-1 H-pyrazol-4-y1)-
N4-methy1-5-
(trifluoromethyl)pyrimidine-2,4 -diamine
241,3 -Dimethyl-1 H-pyrazol-4 -ylamino)-4 -methylamino-pyrimidine-5 -c arb
nitrite
2-(1 -ethyl-5 -methyl- 1 H-pyrazol-4 -ylamino)-4 -(methylamino)pyrimidine-5 -c
arb nitrite
2-(1 -isopropyl-3 -methyl-1 H-pyrazol-4-ylamino)-4-(methyl amino)pyrimidine-5 -
c arb nitrite
2-(1 -ethyl-3 -methyl-1 H-pyrazol-4 -ylamino)-4 -(methylamino)pyrimidine-5 -c
arb nitrite
2-(3 -methyl-1 -phenyl- 1 H-pyrazol-4 -ylamino)-4 -(methylamino)pyrimidine-5-
carb nitrite
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2-(3 -methyl-1 -(1 (2,2,2-trifluoroethyl)pip eridin-4-y1) -1 H-pyrazol-4-
ylamino)-4-
(methylamino)pyrimidine-5-c arb onitrile
245-methyl-I -(1 (2,2,2-trifluoroethyl)p ip eridin-4-y1) -1 H-pyrazol-4-
ylamino)-4-
(methylamino)pyrimidine-5-c arb onitrile
2-(1 -methyl-1 H-pyrazol-4 -ylamino)-44methylamino)pyrimidine-5-carbonitrile
245-methyl-I -(2,2,2-trifluoro ethyl)- 1 H-pyrazol-4 -ylamino)-
44methylamino)pyrimidine-5 -
carbonitrile
2-(3 -methyl-1 -(2,2,2-trifluoro ethyl)- 1 H-pyrazol-4 -ylamino)-
44methylamino)pyrimidine-5 -
carbonitrile
245-methyl-I -phenyl- 1 H-pyrazol-4 -ylamino)-44methylamino)pyrimidine-5-
carbonitrile
245-methyl-I 4tetrahydro-2H-pyran-4 -y1)- 1 H-pyrazol-4-ylamino)-4-
(methylamino)pyrimidine-5-c arb onitrile
2-(3 -methyl-1 4tetrahydro-2H-pyran-4 -y1)- 1 H-pyrazol-4-ylamino)-4-
(methylamino)pyrimidine-5-c arb onitrile
2-( 1 -ethyl-5 -methyl-1 H-pyrazol-4 -ylamino)-4 4ethylamino)pyrimidine-5-
carbonitrile
2-(1 (4-fluoropheny1)-3 -methyl-1 H-pyrazol-4-ylamino)-4-
(methylamino)pyrimidine-5 -
carbonitrile
2-(1 4difluoromethyl)-3 -methyl-1 H-pyrazol-4 -ylamino)-4-
(methylamino)pyrimidine-5 -
carbonitrile
245-methyl-I -propyl-1 H-pyrazol-4 -ylamino)-4 4methylamino)pyrimidine-5-
carbonitrile
2-(1 -(3,5 -difluoropheny1)-3 -methyl-1 H-pyrazol-4 -ylamino)-
44methylamino)pyrimidine-5 -
carbonitrile
2-(1 (4-chloropheny1)-3 -methyl-1 H-pyrazol-4-ylamino)-4-
(methylamino)pyrimidine-5 -
carbonitrile
2-(1 (4-chloropheny1)-5 -methyl-1 H-pyrazol-4-ylamino)-4-
(methylamino)pyrimidine-5 -
carbonitrile
2-(3 -methyl-1 4pyridin-2-y1)-1H-pyrazol-4-ylamino)-44methylamino)pyrimidine-5-
carbonitrile
44ethylamino)-245-methy1-1 4tetrahydro-2H-pyran-4-y1)-1H-pyrazol-4-
ylamino)pyrimidine-
5-carbonitrile
44ethylamino)-2 -(3 -methyl-1 4tetrahydro-2H-pyran-4-y1)-1H-pyrazol-4-
ylamino)pyrimidine-
5-carbonitrile
44ethylamino)-2 -(3 -methyl-1 -(oxetan-3 -y1)- 1 H-pyrazol-4 -
ylamino)pyrimidine-5 -carbonitrile
2-(1 -is opropy1-5-methy1-1 H-pyrazol-4-ylamino)-4-(methylamino)pyrimidine-5 -
carbonitrile
2-(1,5-dimethyl- 1 H-pyrazol-4-ylamino)-4-methoxypyrimi dine-5-carbonitrile
2-(1,5-dimethyl- 1 H-pyrazol-4-ylamino)-442,2,2-trifluoroethylamino)pyrimidine-
5-
c arbonitrile
2-(1 -ethyl-5 -methyl- 1 H-pyrazol-4 -ylamino)-4 -methoxypyrimidine-5 -
carbonitrile
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4-(2,2-difluoroethylamino)-2-(1,5 -dimethyl-1 H-pyrazol-4 -ylamino)pyrimidine-
5-carb onitrile
2-(1,5-dimethyl- 1 H-pyrazol-4-ylamino)-4-(2,2,2-tri fluoroethoxy)pyrimidine-5-
c arb nitrite
2-(1 -(cyclopropylmethyl)-3 -methyl-1 H-pyrazol-4-ylamino)-4 -(methylamino)
pyrimidine-5-
c arb onitrile
2-(1 -(4,4 -difluorocyclohexyl)-5-methyl- 1 H-pyrazol-4-ylamino)-4-
(methylamino) pyrimidine-
5-carbonitrile
2-(3 -methyl-1 -(oxetan-3 -y1)- 1 H-pyrazol-4-ylamino)-4-(2,2,2-trifluoro
ethylamino)pyrimidine-
5-carb nitrite
2-(5 -chloro- 1 -(tetrahydro-2H-pyran-4-y1)-1H-pyrazol-4-ylamino)-4-
(methylamino)
pyrimidine-5-carbonitrile
2-(1 -Difluoromethy1-5-methy1-1H-pyrazol-4-ylamino)-4-methylamino-pyrimidine-5-
carbonitrile
2-(1,5-Dimethy1-1 H-pyrazol-4 -ylamino)-4 -ethylamino-pyrimidine-5-carb
nitrite
2-[i -(4-Fluoro-pheny1)-5-methy1-1 H-pyrazo 1-4 -ylamino] -4 -methylamino -
pyrimidine-5 -
c arb onitrile
4-Methylamino-2-(3 -methyl-1 -propyl- 1 H-pyrazol-4-ylamino)-pyrimidine-5 -c
arb nitrite
4-Methylamino-2-(5 -methyl-1 -oxetan-3 -y1-1 H-pyrazol-4-ylamino)-pyrimidine-5-
c arb onitrile
4-Methylamino-2-(3 -methyl-1 -oxetan-3 -y1-1 H-pyrazol-4-ylamino)-pyrimidine-5-
c arb onitrile
2-[i -(3,5 -Difluoro-pheny1)-5-methyl- 1 H-pyrazo 1-4 -ylamino] -4-methylamino-
pyrimidine-5-
carbonitrile
4-(2,2-Difluoro-ethoxy)-2-( 1,5-dimethyl- 1 H-pyrazol-4-ylamino)-pyrimidine-5 -
carbonitrile
2-[i -(4,4 -Difluoro-cyclohexyl)-3 -methyl-1 H-pyrazo 1-4 -ylamino] -4 -
methylamino-pyrimidine-
5-carb onitrile
N,N-dimethy1-2-(5 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)acetamide
N,N-dimethy1-2-(3 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)acetamide
N-methyl-2-(3 -methy1-4 -(4 -(methylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)-1 H-
pyrazol- 1 -yl)acetamide
N-methyl-2-(5 -methy1-4 -(4 -(methylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)-1 H-
pyrazol- 1 -yl)acetamide
N,N,2-trimethy1-2-(5-methy1-4-(4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-
ylamino)-
1 H-pyrazol- 1 -yl)propanamide
2-methyl-2-(5-methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)-1 H-
pyrazol- 1 -y1)-1 -(pyrrolidin-1 -yl)prop an-1 -one
2-methyl-2-(5-methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)-1 H-
pyrazol- 1 -yl)propanenitrile
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1 -(3 -methyl-4 -(4 -(methylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)- 1
H-pyrazol- 1 -
yl)cyclopropanecarbonitrile
(R)-2-(3 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -ylamino)-
1 H-pyrazol- 1 -
y1)-1 -(pyrro lidin- 1 -yl)prop an- 1 -one
(R)-N,N-dimethy1-2 -(3 -methy1-4-(4 -(methylamino)-5 -
(trifluoromethyl)pyrimidin-2-ylamino)-
1 H-pyrazol- 1 -yl)propanamide
( S)-2 -(3 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -
ylamino)- 1 H-pyrazol- 1 -
y1)-1 -(pyrro lidin- 1 -yl)prop an- 1 -one
3 -(5 -methyl-4 -(4 -(methylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)- 1
H-pyrazol- 1 -
yl)propanenitrile
3 -(3 -methyl-4 -(4 -(methylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)- 1
H-pyrazol- 1 -
yl)propanenitrile
methyl 2-methyl-2-(3 -methy1-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-
2 -ylamino)-
1 H-pyrazol- 1 -yl)prop ano ate
methyl 2-methyl-2-(5 -methy1-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-
2 -ylamino)-
1 H-pyrazol- 1 -yl)prop ano ate
2-(3 -ethy1-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-y1 amino)- 1 H-
pyrazol- 1 -y1)-2 -
methylprop anenitrile
(R)-2 -(5 -methy1-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -ylamino)-
1 H-pyrazol- 1 -
y1)-1 -(pyrro lidin- 1 -yl)prop an- 1 -one
(R)-N,N-dimethy1-2 -(5 -methy1-4-(4 -(methylamino)-5 -
(trifluoromethyl)pyrimidin-2-ylamino)-
1 H-pyrazol- 1 -yl)propanamide
( S)-2 -(5 -methy1-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -
ylamino)- 1 H-pyrazol- 1 -
y1)-1 -(pyrro lidin- 1 -yl)prop an- 1 -one
( S)-N,N-dimethy1-2 -(5 -methy1-4-(4 -(methylamino)-5 -
(trifluoromethyl)pyrimidin-2-ylamino)-
1 H-pyrazol- 1 -yl)propanamide
( S)-2 -(5 -methy1-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -
ylamino)- 1 H-pyrazol- 1 -
yl)propanenitrile
( S)-2 -(3 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -
ylamino)- 1 H-pyrazol- 1 -
yl)propanenitrile
2-(4 -(5 -chloro-4 -(methyl amino)pyrimidin-2 -ylamino)-3 -methyl-1 H-pyrazol-
1 -y1)-2 -
methylprop anenitrile
2-(5 -chloro-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimi din-2 -ylamino)-
1 H-pyrazol- 1 -y1)-
2-methylprop anenitrile
2-(3 -cyclopropy1-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimi din-2 -
ylamino)- 1 H-pyrazol- 1 -
y1)-2-methylpropanenitrile
2,2-dimethy1-3 -(3 -methy1-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)propanenitrile
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2,2-dimethy1-3 -(5 -methy1-4 -(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)propanenitrile
1 -(5 -chloro-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimi din-2 -ylamino)-
1 H-pyrazol- 1 -
yl)cyclopropanecarbonitrile
N-tert-butyl-2-methyl-2 -(3 -methy1-4 -(4-(methylamino)-5 -
(trifluoromethyl)pyrimidin-2 -
ylamino)- 1 H-pyrazol- 1 -yl)propanamide
2-methyl-2-(3 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2 -
ylamino)- 1 H-
pyrazol- 1 -y1)-N-(2,2,2-trifluoroethyl)propanamide
2-(5 -chloro-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimi din-2 -ylamino)-
1 H-pyrazol- 1 -y1)-
N-ethy1-2 -methyl propanamide
N-(cyclopropylmethyl)-2-methy1-2 -(5 -methy1-4-(4 -(methyl amino)-5 -
(trifluoromethyl)pyrimidin-2-ylamino)- 1 H-pyrazol- 1 -yl)propanamide
N-(cyclopropylmethyl)-2-methyl-2 -(3 -methy1-4-(4 -(methyl amino)-5 -
(trifluoromethyl)pyrimidin-2-ylamino)- 1 H-pyrazol- 1 -yl)propanamide
N-ethyl- 1 -(3 -methyl-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)cyclobutanecarboxamide
N-is opropy1-2-methy1-2 -(5 -methy1-4-(4-(methylamino)-5 -
(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-pyrazol- 1 -yl)propanamide
1 -(3 -methyl-4 -(4 -(methylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)- 1
H-pyrazol- 1 -
yl)cyclobutanecarbonitrile
2-(4-(4-(cyclopropylamino)-5 -(trifluoromethyl)pyrimidin-2-ylamino)-3 -methyl-
1 H-pyrazol- 1 -
y1)-2-methylpropanenitrile
N,2-dimethy1-2 -(3 -methy1-4-(4-(methylamino)-5 -(trifluoromethyl)pyrimidin-2-
ylamino)- 1 H-
pyrazol- 1 -yl)propanamide
1 -(5 -chloro-4-(4 -(methyl amino)-5 -(trifluoromethyl)pyrimidin-2 -ylamino)-
1 H-pyrazol- 1 -
yl)cyclopropanecarbonitrile
2- [4 -(5 -Chloro-4 -methoxy-pyrimidin-2 -ylamino)-5 -methyl-pyrazol- 1 -y1]-2-
methyl-propionic
acid methyl ester
2- [4 -(5 -Chloro-4 -methoxy-pyrimidin-2 -ylamino)-3 -methyl-pyrazol- 1 -y1]-2-
methyl-propionic
acid methyl ester
(S)-N,N-Dimethy1-2 43 -methy1-4 -(4-methylamino-5 -trifluoromethyl-pyrimidin-2
-ylamino)-
pyrazol- 1 -yl] -prop ionamide
R)-2-[3 -Methy1-4-(4-methylamino-5 -trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol- 1 -yl] -
propionitrile
2- [4 -(5 -Chloro-4 -methoxy-pyrimidin-2-ylamino)-3 -cyclopropyl-pyrazol- 1 -
yl] -2-methyl-
propionitrile
(R)-245 -Methy1-4 -(4-methylamino-5 -trifluoromethyl-pyrimidin-2 -ylamino)-
pyrazol- 1 -yl] -
propionitrile
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N-Ethyl-2- [3 -methy1-4 -(4-methylamino -5 -trifluoromethyl-pyrimidin-2-
ylamino)-pyrazol-1 -
y1]-isobutyramide
N-Ethy1-2-[5-methy1-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol-1-
y1]-isobutyramide
1- [5 -Methyl-4-(4-methyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol-1-yl] -
cyclobutanecarboxylic acid ethylamide
2- [5 -Methy1-4-(4-methyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol-1-yl] -N-
(2,2,2-trifluoro-ethyl)-isobutyramide
N-Isopropy1-243-methy1-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol-
1-yl] -isobutyramide
N-Methy1-2-[5-methy1-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol-1-
y1]-isobutyramide
1- [5 -Methyl-4-(4-methyl amino -5 -trifluoromethyl-pyrimidin-2-ylamino)-
pyrazol-1-yl] -
cyclobutanecarbonitrile
N-tert-Buty1-245-methy1-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-
ylamino)-pyrazol-
1-yl] -isobutyramide
2- [4 -(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-5 -methyl-
pyrazol-1 -yl] -N-
methyl-isobutyramide
2- [4-(4-Cyc lopropylamino-5-tri fluoromethyl-pyrimidin-2-ylamino)-3 -methyl-
pyrazol-1 -yl] -
N-methyl-isobutyramide
2- [4-(4-Cyclopropylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-5 -methyl-
pyrazol-l-yl] -
N-methyl-isobutyramide
2- [4 -(4-Ethylamino -5 -trifluoromethyl-pyrimidin-2-ylamino)-3 -methyl-
pyrazol-1 -yl] -2-
methyl-propionitrile or
2-(3 -chloro-4-(4-(methyl amino)-5-(trifluoromethyl)pyrimi din-2-ylamino)-1H-
pyrazol-1 -y1)-
2-methylpropanenitrile.
Experimental Examples
Mass spectrometry analysis
[0756] Excised gel pieces were reduced, alkylated, and subjected to
overnight
trypsin digestion. For phosphomapping analyses, digested peptides were
extracted, dried, and
resuspended in 10% acetonitrile /5% formic acid. For quantitative analysis, a
mixture of
isotopically labeled internal standard peptides (Cell Signaling Technologies,
Danvers, MA)
was introduced to the digested gel pieces prior to extracting the peptides,
and then following
extraction, dried peptides were resuspended in 3% acetonitrile/5% formic
acid/10% H202 at
least 30 min prior to analysis to allow for complete oxidation of methionine
containing
peptides. Peptides were separated using a standard gradient comprised of
H20/acetonitrile/formic acid delivered by a nanoAcquity UPLC (Waters, Milford
MA) and
analyzed on an LTQ-Orbitrap XL (Thermo, San Jose, CA) mass spectrometer using
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conditions previously described (Phu and Kirkpatrick, Mol cell Proteomics
2011) In the
present study, the mass spectrometer was operated in data-dependent mode with
a 60,000
resolution full MS scan collected in the Orbitrap and MS/MS for the eight most
intense ions
collected in the ion trap. Spectra data were converted to mzxml and searched
using the target-
decoy strategy against the human Uniprot database and rough filtered to 5% FDR
using a
linear discriminant analysis (Huttlin and Gygi, Cell 2011). The localizations
of putative
phosphorylation sites were optimized and confidence scores assigned using the
AScore
algorithm (Beausoliel and Gygi, Nat Biotech 2006), with scores of 13 and 19
representing the
95% and 99% confidence levels respectively. Confidently matching
phosphopeptides with
uncertain localizations (Ascore < 13) have not been reported. For the
remaining peptide-
spectral matches, manual validation of at least one representative spectra for
each unique
phosphopeptide sequence was required for inclusion herein. For quantitative
analysis, full
MS scans were integrated using Qualbrowser software (Thermo, San Jose, CA) to
determine
the peak areas for heavy and light m/z ions of the most abundant charge states
of each
LRRK2 phosphopeptide.
Western Blotting
[0757] Cultured cells and mouse brains were homogenized in RIPA buffer
(25mM
Tris-HC1 (pH 7.6), 150mM NaC1, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS)
containing Complete protease and phosphotase inhibitor cocktail (Roche).
Cell/tissue lysate
was cleared via centrifugation at 20,000g for 30 minutes at 4C. Protein
concentration of the
supernatant was measured using BCA assay (Pierce). 2Oug of lysate was loaded
onto 3-8%
Tris-Acetate gels or 4-12% Bis-Tris gels (Invitrogen) and immunoblotted with
the following
antibodies: phospho-LRRK2 (Custom generated), LRRK2 (Epitomics), FLAG, Tubulin
(Sigma). The Li-Cor Odyssey system was used for Western blot detection.
Generation of stable LRRK2 cell line
[0758] FLAG-tagged full length human LRRK2 was inserted into pTre2Hygro
vector (Clontech). QuickChange Site-directed mutagenesis kit was used to
introduce 3
Parkinson's mutations, R1441G, Y1699C and G20195 (Stratagene). The
tetracycline
regulated Tet-On system was used to generate a LRRK2 stable cell line (Gossen
et al).
HEK293 Tet-On cells (Clontech) was transfected with the LRRK2 plasmid
mentioned above
together with GFP. Hygromycin was used for stable cell selection and 48 single
clones were
selected using clone-disk. LRRK2 expression levels were compared using Western
blots to
identify the highest expresser line. The concentration of hygromycin used for
selection was
determined by a killing curve assay. Gossen, M., el al. (1995) Science 268:
1766-1769.
Inhibitor Treatment of LRRK2 Cells
[0759] HEK293 Tet-On LRRK2 G20195/R144G/Y1699C inducible stable cell line
was grown in complete media (High Glucose DMEM, 10% Tet System FBS, 1%
Glutamax ,
1%NEAA, 10Oug/mL G418, 3Oug/mL Hygromycin B). Cells were induced with lug/mL
of
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doxycycline for 16 hours at 37 degrees Celsius. Cells were treated with
inhibitors for one
hour, and then lysed with cold MSD Tris Lysis Buffer.
Immunoassay
[0760] Anti-FLAG M2 antibody (Sigma, Saint Louis, MO, USA) was
immobilized
onto a standard Binding 96 Well Small Spot Plate (MSD, Gaithersburg, MD, USA).
Antibody was diluted in low salt TBS buffer (16.6mM Tris, 50mM NaC1, 16.6mM
HEPES,
0.015% Triton X-100) to 333ug/mL and Sul was spotted and allowed to dry
overnight. Plates
were blocked for 1 hour with Sea Block (Thermo Scientific, Rockford, IL, USA),
then
washed with MSD Tris Wash Buffer.
[0761] Cell lysate was transferred to the assay plate and incubated for 2
hours at
room temperature. After washing with MSD Wash Buffer, Anti-phospho-Ser 1292
pLRRK
antibody was diluted to 0.370ug/mL in Complete Assay Buffer (20mM Tris, 150mM
NaC1,
0.05% Tween 20,10% Sea Block, 1% normal mouse serum), added and incubated at
room
temperature for one hour. After washing, SULFOTAG Goat Anti-Rabbit antibody
was
diluted to lug/mL in Complete Assay Buffer, added and incubated at room
temperature for
one hour. After washing plates with MSD Wash Buffer, lx MSD Read Buffer T was
added
and the plate was measured on the MSD Sector Imager 6000 according to
manufactures
instructions.
Hippocampal Culture and Transfection
[0762] Dissociated hippocampal neuron cultures were prepared as described
(Brewer
et al.) Briefly, 200 hippocampal neurons mm-2 were plated 24-well clear plates
coated or 8-
well culture chamber (BD) with 30 [tg/ml poly-D-lysine and 2 [tg/ml laminin.
For IHC, cells
were fixed in 4% formaldehyde, 4% sucrose in PBS for 10 min at room
temperature.
Antibodies were applied in GDB buffer (0.1% gelatin, 0.3% TX-100, 450 mM NaC1,
32%
0.1M Phosphate buffer [pH 7.4]). Images were acquired using an LSM710 confocal
system or
GE InCell automated imager. Image analysis was done using InCell Developer.-
G.J. Brewer
et al., "Optimizing survival of hippocampal neurons in B27-supplemented
Neurobasal, a new
serum-free medium combination". J. Neuro. Meth. 170:181-187. Neurons were co-
transfected with GFP and LRRK2 (1:20 ratio) at 6 days in vitro using
Lipofectamine 2000.
Immunofluorescence
[0763] Cells were fixed with 4% paraformaldehyde/4% sucrose in phosphate-
buffered saline (PBS, pH 7.4). Following permeabilization (0.1% Triton-X in
PBS), blocking
(5% NGS, 0.1% Triton X-100 in PBS) and primary antibody incubation, antibodies
were
visualized using secondary antibodies conjugated to Alexa 405, Alexa 488 or
Alexa
568(Invitrogen). The following antibodies were used for primary
immunostaining: mouse
anti-FLAG (Sigma), rabbit anti-Flag (Cell signaling), chicken anti-GFP
(Invitrogen), rabbit
anti-MAP2 (Millipore), rabbit beta-tubulin (Abcam), rabbit anti-P62 (Santa
Cruz), rabbit anti-
LC3 (Cell signaling), rabbit Lys63-specific anti-Ubiquitin (Millipore).
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Image Acquisition and Quantitative Analysis
[0764] For neurite outgrowth quantification, neurons were fixed 11 days
post-
transfection. Cells were stained with GFP and MAP2 for dendrite observation.
Confocal
images of transfected neurons were obtained using Zeiss LSM 710 20X (NA?)
objective.
Dendrites from neurons were traced using Neurolucida software
(MicroBrightField). Sholl
analysis and total dendritic length were analyzed with Neurolucida Explorer
(MicroBrightField). Statistical analysis was performed using JMP 9 software. P
values were
obtained using (non-paired Student's t test, one way ANOVA test ?). All image
acquisition
and analysis were done in a blind manner.
[0765] For LRRK2 cellular localization, neurons were fixed 3-4 days post-
transfection. Confocal images of transfected neurons were obtained using Zeiss
LSM 710
100X (NA?) or Leica 5P5 confocal microscope 63X (NA) with Z-section size of?.
Quantitative analysis of cellular localization of various LRRK2 constructs was
performed
blind to the experimental conditions.
Animal Study
[0766] G1023 was dissolved in 1% RC591 in water. Mice in treatment groups
(n=3
per group) received intraperitoneal injection at 10, 30, or 100 mg/kg. Vehicle
group (n=2)
received 1% RC591. Dosing volume was calculated based on animal's weight and
did not
exceed 500u1. Animals were taken down 6 hours post-dose. The brain, spleen,
and lymph
nodes were rapidly harvested and dissected. All animals were handled according
to IACUC
guidelines.
Cell-based Assay Using MSD Platform
[0767] Based on the foregoing, a cell-based assay was made using anti-FLAG
antibody capture of LRRK2 and anti-phospho-Serine 1292-LRRK2 antibody on Meso
Scale
Discovery (MSD, Gaithersburg MD) plates, with detection of captured anti-
phospho-Serine
1292-LRRK2 antibody with MSD sulfotag goat anti-rabbit antibody using the MSD
sector
reader according to the procedure in the Example below. FIG. 5A shows the
Western blot for
a tetracycline-On inducible LRRK2 R1441G/Y1699C/G20195 triple mutant cell line
expressing LRRK2 upon treatment with 1 ug/mL of doxycycline, with anti-phospho-
Serine
1292-LRRK2 antibody shown in the first (upper) row, and anti-FLAG epitope
(control) in the
second (lower) row. FIG. 5B is a graphical representation of the Western blot
illustration of
FIG. 5A, with relative luminescence shown on the vertical axis.
[0768] Using the assay with the LRRK2 inhibitor Sutent (SU-11248 or
sunitinib,
American Custom Chemicals Inc.) LRRK2 activity was measured by detection of
anti-
phospho-Serine 1292-LRRK2 antibody.
Inhibition of LRRK2 Autophosphorylation In vivo
[0769] The in vivo effect of LRRK2 inhibitors on S1292 autophosphorylation,
and
thus LRRK2 inhibition, can be determined with the invention. Three month old
BAC
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transgenic mice overexpressing LRRK2 G2019S were administered the LRRK2
inhibitor [3-
methoxy-4-(4-methylamino-5-trifluoromethyl-pyrimidin-2-ylamino)-pheny1]-
morpholin-4-yl-
methanone at 10 mg/kg, 30 mg/kg and 100 mg/kg, orally. After six hours,
hippocampus,
lymph node and spleen tissue were harvested and levels of autophosphorylation
at S1292 was
determined by detecting anti-phospho-Serine 1292-LRRK2 antibody as described
herein.
HEK293 R1441G/Y1699C/G20195 LRRK2 HEK 293 Tet-On cell line
[0770] Using standard mutagenesis technique, the R1441G, Y1699C, G20195
mutations and a N-terminal FLAG tag were introduced into the full length human
LRRK2
sequence (hererinafter "LRRK2-3M"). LRRK2-3M was cloned into pTre2Hyg vector
(Clontech, Cat. No. 631014) using NotI and EcoRV restriction sites. pTre2Hyg-
LRRK2-3M
in combination with pCaggs_GFP was then introduced into Clontech's Tet-On
HEK293 cells
following their standard protocol (http://www.clontech.com/images/pt/PT3898-
1.pdf).
LRRK2-3M Cell line handling and induction procedures
Materials:
DMEM with high glucose (Site Service)*
Glutamax 100X (Invitrogen 35050)*
NEAA 100X (Invitrogen 11140)*
Tet system FBS (Clontech 631105)*
G418 100mg/m1 stock dissolved in H20 (Clontech 631308)*
Pen/Strep (Invitrogen)*
Doxycycline lmg/m1 dissolved in H20 (Clontech)*
Hygromycin B 100mg/m1 stock (Invivogen)
* available online
DMEM medium for HEK293 Tet_On LRRK2 Triple Mutant GFP cells:
DMEM with glutamine (1000m1)
Add 100 ml Tet system FBS, f.c. 10%
Add 10 ml of 100X pen/strep solution
Add 10 ml of 100X Glutamax
Add 10 ml of 100X NEAA
Add 1 ml of 100mg/m1 G418, f.c. 10Oug/m1
Add 300u1 of 100mg/m1Hygromycin B, f.c. 3Oug/m1
Day 1:
Plate cells at 20% confluence (Varies based on difference in surface areas of
different plates)
Day 2:
16 ¨ 20 hours after plating, directly add Doxycycline (1mg/m1 stock) solution
to culture wells
yielding lug/m1 f.c. of Dox. (if the handling volume is too little, dilute
stock Dox to 10X or
100X in culture media first)
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Day 3:
24 hours after Dox induction, cells are ready for compound treatment
pLRRK2 antibody
[0771] Rabbits were immunized with peptide CPNEMGKL-pS-KIWDLPL-amide
according to standard procedures (Yenzyme). Serum from immunized rabbits were
first
purified by passing through an agarose column conjugated with peptide CPNEMGKL-
pS-
KIWDLPL-amide. Antibodies bound to the column were eluted. The purified
material then
was passed through an agarose column conjugated with the peptide
CPNEMGKLSKIWDLPL-amide to absorb the antibodies cross-reacting to un-
phosphorylated
peptide.
BAC Mice
[0772] BAC mice overexpressing G2019S and/or other Parkinson's disease
mutations may be prepared generally by the procedures of Marshall et al. in
"Generation of
BAC Transgenic Mice", Methods in MoL Biol. Vol. 256 (2004) pp. 159-182.
Assay using Meso Scale Discovery platform
[0773] HEK293 cells were transiently transfected with a plasmid containing
FLAG
epitope-tagged full-length LRRK2 cDNA under the control of the CMV promotor.
Transfections were carried out in 10-cm dishes using Fugene 6 reagent (Roche
Applied
Science, Mannheim, Germany) according to the manufacturer's instructions. 24
hours post-
transfection cells were harvested, counted, seeded into 96-well plates and
allowed to attach
for 1 hour prior to compound addition.
[0774] Cells were treated with compounds for 1 hour, then medium was
removed
and cells lysed in MSD (Meso Scale Discovery, Gaithersburg, MD) lysis buffer
(Meso Scale
Discovery, Gaithersburg, MD) supplemented with protease and phosphatase
inhibitors.
[0775] Assay detection plates were prepared by spotting 2ug of anti-FLAG
monoclonal antibody (Sigma Aldrich, St Louis, MO) in each well of 96-well MSD
plates,
followed by blocking with Sea-Block (Thermo Fisher, Rockford, IL). Cell
Lysates were
transferred to MSD plates, incubated for 2 hours, then washed. Captured
phospho-LRRK2
was detected by addition of anti-phospho-Ser1292-LRRK2, incubating for 2
hours, followed
by washing. Detection of captured antibody with MSD Sulfotag goat anti-rabbit
antibody
using the MSD Sector reader was carried out according to the manufacturer's
instructions.
Other Assay Formats
[0776] In a variation of the above assay format, determination of phospho-
Ser(1292)
in cell lysates is achieved using cells transfected (stably or transiently) or
virally transduced
with cDNA encoding all or part of LRRK2 protein, with or without fusion to
epitope tag
protein. Capture of overexpressed LRRK2 or fragment of LRRK2, with or without
fused
epitope tag or fusion protein partner, from cell lysate is done with
immobilized antibody,
directed against LRRK2 sequence, other epitope tag or fusion protein. Solid-
phase matrix for
CA 02818428 2013-05-16
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PCT/US2011/062468
110
capture can include ELISA plates, Meso Scale Discovery plates (Meso Scale
Discovery,
Gaithersberg, MD), Membranes (e.g. nitrocellulose, PVDF). Detection of
captured phospho-
Ser1292-LRRK2 with antibody against phospho-Ser1292 by methods including:
ELISA,
DELFIA, Electrochemiluminescence, fluorescence.
[0777] Another variation of the above assay format utilizes capturing
phospho-
5er1292-LRRK2 from cell lysates with immobilized anti-phospho-5er1292-LRRK2
antibody,
followed by detection of captured LRRK2 with antibody against LRRK2 sequence,
epitope
tag or fusion protein partner.
[0778] Plate-based detection of endogenous phospho-Ser(1292) from cell
lysates in
any of the formats described above may use anti-LRRK2 and anti-phospho-
Ser(1292)
antibodies.
[0779] Plate-based detection of phospho-Ser(1292) in intact cells may be
done by
Immunofluorescent detection of phospho-Ser(1292) in fixed and permeabilized
cells by
microscopic detection and quantitation of fluorescence intensity, or by bulk
fluorescence (e.g.
LiCor "In-Cell Western")
[0780] Plate-based detection of phospho-Ser(1292) in intact cells may
utilize:
Colorimetric/immunohistochemical detection and quantitation of anti-phospho-
Ser(1292)
binding to fixed and permeabilized cells; Western Blotting of total cell
lysates or
immunoprecipitated endogenous or transfected LRRK2 with anti-phospho-
Ser(1292); or
Western blotting of protein immunoprecipited with anti-phosphoSer(1292)
antibody followed
by detection with anti-LRRK or antibody against fusion protein/epitope tag.
[0781] While the present invention has been described with reference to
the specific
embodiments thereof, it should be understood by those skilled in the art that
various changes
may be made and equivalents may be substituted without departing from the true
spirit and
scope of the invention. In addition, many modifications may be made to adapt a
particular
situation, material, composition of matter, process, process step or steps, to
the objective spirit
and scope of the present invention. All such modifications are intended to be
within the scope
of the claims appended hereto.
