Identification of Pyrimidine-Based Lead Compounds for Understudied Kinases Implicated in Driving Neurodegeneration

Article abstract of DOI:10.1021/acs.jmedchem.1c00440

The pyrimidine core has been utilized extensively to construct kinase inhibitors, including eight FDA-approved drugs. Because the pyrimidine hinge-binding motif is accommodated by many human kinases, kinome-wide selectivity of resultant molecules can be p

Full text of DOI:10.1021/acs.jmedchem.1c00440

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Article
Identification of Pyrimidine-Based Lead Compounds for
Understudied Kinases Implicated in Driving Neurodegeneration
David H. Drewry, Joel K. Annor-Gyamfi, Carrow I. Wells, Julie E. Pickett, Verena Dederer,
Franziska Preuss, Sebastian Mathea, and Alison D. Axtman*
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ABSTRACT: The pyrimidine core has been utilized extensively to construct
kinase inhibitors, including eight FDA-approved drugs. Because the pyrimidine
hinge-binding motif is accommodated by many human kinases, kinome-wide
selectivity of resultant molecules can be poor. This liability was seen as an
advantage since it is well tolerated by many understudied kinases. We hypothesized
that nonexemplified aminopyrimidines bearing side chains from well-annotated
pyrimidine-based inhibitors with off-target activity on understudied kinases would
provide us with useful inhibitors of these lesser studied kinases. Our strategy paired
mixing and matching the side chains from the 2- and 4-positions of the parent
compounds with modifications at the 5-position of the pyrimidine core, which is
situated near the gatekeeper residue of the binding pocket. Utilizing this approach,
we imparted improved kinome-wide selectivity to most members of the resultant
library. Importantly, we also identified potent biochemical and cell-active lead
compounds for understudied kinases like DRAK1, BMP2K, and MARK3/4.
The IDG program aims to catalyze the characterization of all
proteins through stimulating research around those that are
most poorly studied.13 One arm of the IDG program supports
illumination of the dark kinome, which includes development
of high-quality chemical tools for these understudied kinases.
As we examined the data available for literature pyrimidine-
based TBK1 inhibitors, we noticed a range of understudied
kinases that were also inhibited by these compounds.
Several of the understudied kinases inhibited by pyrimidines
also have been genetically implicated in driving neuro-
degenerative diseases. The MARK family of kinases, for
example, phosphorylates tau protein in its repeat domain and
thereby regulates its affinity for microtubules, affecting the
aggregation of tau into neurofibrillary tangles. Observations of
AD brains show a strong correlation between cognitive
dysfunction and cortical neurofibrillary tangle density.14−16
Mutations in tau have also been shown to cause a form of
FTD.16 Furthermore, with a characterized role in dendrite
branching and spine development, understudied kinase AAK1
is suggested to play a role in several neurodegenerative
disorders, including AD and ALS.14,17,18
INTRODUCTION
■
Pyrimidines represent an important building block in the
medicinal chemistry arsenal. Compounds bearing a pyrimidine
core have proven to be bioactive and exhibit diverse
pharmacology, including anticonvulsant, analgesic, sedative,
antidepressive, antipyretic, anti-inflammatory, antiviral, anti-
HIV, antimicrobial, and antitumor activities.1 Pyrimidines are
very useful as kinase scaffolds that employ a nitrogen to make
key hydrogen bonds with the conserved hinge region found in
nearly all human kinases. Aminopyrimidines substituted with
an NH in the 2-position can make an additional hydrogen
bond with the kinase hinge. To date, eight FDA-approved
kinase inhibitors employ a pyrimidine as the key kinase hinge-
binding motif.2
The work we describe here stems from the synergistic
convergence of two separate interests that were satisfied
through diversification of the pyrimidine scaffold. The first of
these interests centers around the generation and use of kinase
inhibitors as tools to build deeper understanding of signaling in
neurodegenerative disease. TBK1, a kinase with links to
amyotrophic lateral sclerosis (ALS), frontotemporal dementia
(FTD), Huntington’s disease, and Alzheimer’s disease (AD), is
potently inhibited by several pyrimidine-bearing com-
pounds.3−12 A second interest of ours is identification of
high-quality tool molecules for understudied kinase targets.
Our pursuit of this interest relies on parallel chemical tool and
kinase assay development, efforts that are supported in part by
the NIH Illuminating the Druggable Genome (IDG) program.
Special Issue: New Horizons in Drug Discovery -
Understanding and Advancing Kinase Inhibitors
Received: March 10, 2021
© XXXX The Authors. Published by
American Chemical Society
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A

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Figure 1. Structures and broad selectivity profiling of aminopyrimidines that served as the basis for library design. All WT kinases inhibited > 90%
at 10 μM by MRT67307 and BX-912 and all WT kinases inhibited > 65% at 1 μM by GSK8612 are shown.
Figure 1 shows the structures and kinome-wide profiling
data generated at DiscoverX (scanMAX or KINOMEscan) for
three TBK1-targeting aminopyrimidines.19 The data for TBK1
inhibitors MRT67307 and BX-912 (designed for PDK1 but
potent inhibitor of TBK1) are already in the literature (LINCS
database).20−24 These two compounds were screened at 10
μM (Figure 1). GSK8612 was recently disclosed by
GlaxoSmithKline as a potent and selective TBK1 inhibitor.25
We opted to survey the kinome-wide selectivity of GSK8612 at
DiscoverX at 1 μM (Figure 1). While the screening
concentrations are different, it is apparent that these scaffolds
differentially and potently inhibit many kinases across the
kinome and that selectivity can be augmented through
structural changes.
Several kinases potently inhibited by aminopyrimidines like
BX-912, MRT67307, and/or GSK8612 are members of the
IDG nominated list of dark kinases. Thus, this scaffold was
considered an excellent starting point from which to design
high-quality chemical tools. Development of these tools will
enable elucidation of the function of those kinases that have
suffered from a dearth of characterization, including those on
the IDG list. High-quality chemical tools will also enable
B
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a
Scheme 1. Library Design and Preparation Strategy
a
Step A: Pyrimidine 1, amine 1, DIPEA, ethanol, −10 °C to 50 °C. Step B: Aminopyrimidine 2, amine 2, dioxane × HCl, butanol, 80 °C. Step C:
Aminopyrimidine X, 5% Pd/C, H2, TEA, methanol, r.t.
Figure 2. Library of aminopyrimidine analogs prepared.
further characterization of kinase-mediated signaling in neuro-
degenerative disease and facilitate the validation of therapeutic
hypotheses.
1. Seven final compounds were prepared with X = H, seven
with X = Br, and seven with X = cyclopropyl (Figure 2).
Analogs of BX-912 and MRT67307 (and the parent
compounds themselves) that varied only at the 5-position
(box in Scheme 1) were not prepared. A great deal of effort has
been dedicated to making close structural analogs of these two
compounds, and we did not want our work to be redundant.
Since it was not commercially available at the time and the
most selective of the parent scaffolds, GSK8612 and variants
with X = H and cyclopropyl were all synthesized. The method
used to prepare these analogs is outlined in Scheme 1. Briefly,
taking advantage of the inherent reactivity of 2,4-dichloropyr-
RESULTS AND DISCUSSION
■
Compound Design and Synthesis. Our library design
involved mixing and matching the side chains and cores from
published aminopyrimidine inhibitors MRT67307, BX-912,
and GSK8612 to furnish 21 total analogs. Specifically, we
paired mixing and matching the side chains from the 2- and 4-
positions of the parent compounds with modifications at the 5-
position of the pyrimidine core as shown in the box in Scheme
C
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imidines, iterative nucleophilic aromatic displacements were
executed, and specific brominated compounds (GSK8612, 6, 9,
10, and 13) were subsequently dehalogenated to furnish all
final compounds.
kinase panel at a single concentration (1 μM) in radiometric
enzymatic assays at Eurofins at the Km = ATP for each kinase.
Table S1 shows the results of this study, where % control is
reported for each compound for each kinase and lower values
indicate greater inhibition. The column labeled “no. kinases ≤
10 PoC in enzyme panel” in Table 1 captures the number of
kinases in this 16-member panel inhibited ≥ 90% by each
compound.
We had several expected outcomes from our strategy of
mixing and matching the side chains and cores from well
exemplified pyrimidine-based inhibitors. First, we intended to
develop more narrowly selective compounds by incorporating
these distinct side chains and 5-position modifications into
new compounds. Next, we wanted to generate preliminary
structure−activity relationships (SAR) for several understudied
kinases. In doing so, we aimed to leverage these scaffolds with
known inhibitory activity of dark kinases to identify more
optimal chemical starting points for development of high-
quality chemical tools. Finally, we wanted to develop focused
SAR around the 5-position (box in Scheme 1), which has not
been systematically investigated in the literature yet and is
proposed to play a key role in dictating both potency and
selectivity due to its proximity to the kinase gatekeeper residue
(Figure 3).
Table 1. Kinase Panel Profiling of Aminopyrimidine Library
no. kinases ≤ 10 PoC in
S10
(1 μM)
no. kinases with
b
c
compound
enzyme panel
PoC < 10
3
4
5
6
7
8
9
10
11
12
13
14
0
1
1
4
7
0
13
0
1
0
0.005
0.027
0.005
0.047
0.154
0.002
0.233
0.069
0.007
0.002
0.03
2
11
2
19
62
1
94
28
3
1
4
6
0
12
27
0
0.067
0
15
16
17
18
19
20
21
22
0
0
16
0
1
1
9
12
13
16
14
0.002
0.007
0.223
0.007
0.022
0.002
0.074
1
3
90
3
9
1
30
N.T.
N.T.
a
a
MRT67307
MRT68921
BX-795
BX-912
GSK8612
N.T.
a
a
N.T.
a
a
N.T.
N.T.
N.T.
8
a
a
N.T.
1
0.02
a
b
N.T.: not tested. S10 (1 μM): Percentage of screened kinases with
c
PoC values < 10 at 1 μM. Number of kinases with PoC values < 10 at
1 μM.
We found that, with the exception of two analogs
(compounds 9 and 18), all compounds in our library inhibited
fewer kinases in this custom kinase panel than published
compounds BX-912, BX-795 (analog of BX-912), MRT67307,
and MRT68921 (analog of MRT67307).20−23 In most cases,
we demonstrated a significant decrease in the number of
kinases potently inhibited by our aminopyrimidine analogs.
This becomes more meaningful when we consider that the
kinases selected in our custom panel represent some of the
most common off-target kinases inhibited by the parent
compounds. Gratifyingly, unlike the published compounds
from the BX and MRT series that elicited potent inhibition of
nearly all kinases in the panel, our compounds have cleaned-up
profiles, and we were able to dial out inhibition of certain
kinases through structural modification. We were pleased to
observe potent inhibition of several understudied kinases by
analogs that inhibited fewer kinases in this panel, including
BMP2K by 6; AAK1, DRAK1, DRAK2, and MARK1 by 7;
AAK1 and BMP2K by 13; and MARK1, MARK3, MARK4,
MLK1, and NUAK1 by 14.
Figure 3. Structural studies support optimization for understudied
MARK kinases. X-ray crystallography structure of human MARK3 in
complex with aminopyrimidine 9. Compound 9 (PDB code: 7P1L) is
shown in teal stick representation. The kinase structural elements
mediating the binding are labeled in orange. Parts of the G-rich loop,
including I62, were made transparent to provide a better view of the
interactions. Key interactions are indicated as dashed lines.
Targeted Kinase Inhibition Profiling. We selected a
small panel of representative kinases against which to profile
our library of 21 aminopyrimidine analogs. These kinases
include some of the original targets for which pyrimidine-based
inhibitors were prepared (JAK2, IKKε, TBK1, and ULK1), a
more well-studied kinase that is potently inhibited by many
analogs within this structural class (AURKB), and several
understudied kinases, many of which are both on the IDG list
(AAK1, BMP2K, DRAK1−2, MARK1−4, MLK1, MLK3, and
NUAK1) and of interest in the neuroscience space.14,15,17,26−29
We profiled our aminopyrimidine series and the parent
aminopyrimidines that influenced our design against this
D
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Table 2. NanoBRET Profiling of Entire Aminopyrimidine Library
IC50 values (nM)
b
c
c
b
compound
DRAK1
MARK3
MARK4
TBK1
3
4
5
6
7
8
9
10
11
12
13
14
>10000
874 103
>10000
>10000
126 27.6
>10000
638 91.1
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
872
>10000
>10000
>10000
>10000
4015
>10000
>10000
>10000
>10000
3537
>10000
214
8445
>10000
>10000
>10000
808
>10000
2680 230
7456
>10000
321 2.49
>10000
>10000
>10000
2050 196
>10000
6170 2190
>10000
3622
15
16
17
18
19
20
21
22
>10000
>10000
2311
4.47 0.406
>10000
>10000
>10000
58.2 3.21
43.2 2.69
>10000
>10000
>10000
137
>10000
>10000
>10000
2001
328 38.5
95
515
3333
>10000
>10000
>10000
>10000
72
>10000
>10000
>10000
509
>10000
>10000
>10000
338 22.5
>10000
>10000
>10000
2580 215
a
MRT67307
MRT68921
BX-795
BX-912
319
N.T.
a
a
a
N.T.
N.T.
N.T.
N.T.
a
a
a
N.T.
N.T.
a
N.T.
338 4.5
>10000
2327
339 39.5
GSK8612
>10000
a
b
N.T.: not tested. Compounds tested in singlicate (n = 1) in dose−response where error not shown, and compounds with SEM tested in triplicate
c
(n = 3) in dose−response. Compounds tested in singlicate (n = 1) in dose−response where error not shown, and compounds with SEM tested in
duplicate (n = 2) in dose−response.
When we examined the data generated via screening in the
Eurofins kinase panel versus compound structures for our
synthetic library, we noticed some predominating trends.
When varying the 5-position (X in Figure 2), more kinases
were tolerant of the bromo substituent, and thus those analogs
bearing X = Br inhibited the most kinases in this panel. When
X = Br, an average of 6.4 kinases (range of 0−16 kinases) were
inhibited ≥ 90%. The average number of kinases inhibited was
calculated by adding the number of kinases inhibited ≤ 10
percent of control (PoC) in the enzyme panel (Table 1,
column 2) and dividing by the total number of compounds.
For X = Br, this works out to be a sum of 45, divided by 7 total
compounds for an average of 6.4 kinases. We will use this
equation to calculate and report average kinase inhibition
throughout this section. Switching to X = H resulted in
compounds that inhibited the fewest kinases in the series, with
an average of only 0.1 kinases (range of 0−1 kinase),
demonstrating ≥ 90% inhibition in the Eurofins panel at 1
μM. Finally, cyclopropyl at position X was somewhere between
H and Br in terms of number of kinases inhibited, with an
average of 2.7 kinases (range of 0−9 kinases) inhibited ≥ 90%.
When considering the amine side chains in the 2- and 4-
positions, some general inhibition trends were also observed.
Incorporation of the sulfonamide-bearing side chain in the
pyrimidine 4-position (R1 position, Scheme 1) resulted in
compounds that inhibited the fewest number of kinases: an
average of 1.3 kinases (range of 0−4 kinases) ≥ 90% at 1 μM.
In the pyrimidine 2-position, the substituted pyrazole is the R2
substituent that resulted in aminopyrimidines with the
narrowest inhibition profiles: an average of 1.1 kinases
(range of 0−7 kinases) ≥ 90%. The ortho-methyl group on
the pyrazole ring likely contributes to this enhanced inhibition
profile when compared to other side chains at the R2 position.
While some of the analogs in our library, including those with a
5-position H and/or 4-position sulfonamide, did not inhibit
any kinases in our smaller panel, it is difficult to extrapolate
whether this translates to loss of all kinase inhibitory activity or
a narrower selectivity profile unless broader kinome-wide
screening is executed.
Library-Wide Cellular Target Engagement Studies.
The data generated by screening our aminopyrimidine analogs
in the panel of kinases at Eurofins motivated follow-up cell-
based studies. We used cellular target engagement assays to
determine whether potent enzyme inhibition corresponded
with potent binding in cells. The NanoBRET assay offers a
method through which cellular penetrance and binding of a
compound to its kinase target in cells can be simultaneously
assessed.30 Given the consistently potent inhibition of TBK1
by many compounds in our library (Table S1) and our interest
in this kinase, we profiled the entire library using the TBK1
NanoBRET assay in dose−response format (Table 2). We
found that potent enzymatic inhibition of TBK1 did not always
translate to potent engagement of TBK1 in cells. We note that
orthogonal assay formats, such an enzymatic and cellular target
engagement assay, can sometimes yield different results and,
for this reason, that its best to assess kinase inhibition via
multiple methods. All compounds that demonstrated at least
50% inhibition at 10 μM when tested using the TBK1
NanoBRET assay in the first dose−response experiment were
followed up with two additional replicates. Two members of
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our library, 7 and 18, and GSK8612 demonstrated
submicromolar IC50 values in the TBK1 NanoBRET assay.
These three compounds demonstrated PoC values ≤ 6 in the
TBK1 enzymatic assay (Table S1), making them among the
most potent inhibitors tested in this assay of those that we
synthesized. The validation of GSK8612 as a potent, cell-active
compound targeting TBK1 aligns well with the recent
publication that described its development and character-
ization, including validation of its activity in multiple cell-based
assays.25 Further, submicromolar activity in a NanoBRET assay
has translated to phenotypic results for multiple chemical
probes and is one part of the chemical probe criteria defined
for SGC-nominated chemical probes.17,31,32
This more comprehensive analysis of selectivity surfaced
many findings. We learned that trends within our smaller
curated enzymatic kinase panel (16 kinases) were generally
maintained in this larger profiling effort. Compounds that
inhibited the fewest kinases in the Eurofins enzymatic panel
were largely those that demonstrated the most favorable
selectivity profile in the DiscoverX scanMAX screening. We
identified several pyrimidine-based kinase inhibitors with
useful selectivity. To provide some context on a useful
selectivity threshold for tool compounds, we include kinase
inhibitors with an S10 (1 μM) < 0.04 in our kinase
chemogenomic set (KCGS) since they are the compounds
that when screened can be more easily used to correlate
phenotype with kinase target.33,34 Based on their selectivity
scores, 14 of 21, or 67%, of our novel inhibitors were found to
be KCGS eligible. We confirmed that kinases potently
inhibited in the smaller, biased enzymatic panel were also
inhibited in the DiscoverX profiling and identified additional
kinases that were differentially inhibited by certain analogs,
providing fodder for future projects.
When comparing the results from our smaller enzymatic
screening with kinome-wide screening, it is apparent that
results do not correlate perfectly. It is not unexpected that
these orthogonal assay types, enzymatic and binding, would
yield slightly different results. General structural trends
observed when analyzing the smaller kinase panel, however,
were generally conserved in broader profiling. The most
selective compounds, inhibiting an average of 2.6 of 403 WT
kinases, bear a 5-position H (8, 12, 15, 16, 17, 19, 20). As
described for the smaller enzyme panel, average kinase
inhibition here is being calculated by summing the number
of kinases with PoC < 10 in the kinome-wide profiling for each
compound (Table 1, column 4) and then dividing by the total
number of compounds. For 5-position H, this works out to be
18 divided by 7, which is equal to an average of 2.6 kinases
inhibited. The 5-position cyclopropyl aminopyrimidines (3, 4,
5, 11, 14, 21, and 22) inhibited an average of 10.9 of 403 WT
kinases, making them the next most selective when considering
just the 5-position substituent. Finally, the remaining
compounds (6, 7, 9, 10, 13, 18, and GSK8612) with a 5-
position bromo are among the least selective compounds when
screened broadly, inhibiting an average of 44.7 of 403 WT
kinases. Moving to the 4-position amine side chain, the
aminopyrimidine analogs bearing a sulfonamide side chain (5,
6, 11, 12, 13, 16, 19, 21, and GSK8612) were among the most
selective compounds profiled and only inhibited an average of
5.6 of 403 WT kinases. Similarly, the 2-position pyrazole side
chain imparted some selectivity to analogs 3, 4, 5, 7, 8, 10, 12,
15, and GSK8612, resulting in inhibition of an average of 12.8
of 403 WT kinases when broadly screened.
Shifting our attention to understudied kinases, we also
observed that some of our compounds potently inhibited
DRAK1, MARK3, and MARK4 (Table S1). As part of the IDG
program, we have interest in developing high-quality chemical
tools to help elucidate the function of these poorly
characterized kinases. For our aminopyrimidine series, dose−
response NanoBRET analysis yielded five analogs (4, 7, 9, 18,
and 22) and MRT67307 with submicromolar activity in the
DRAK1 NanoBRET assay. We repeated the NanoBRET assay
in dose−response for these six compounds and found all to
maintain activity over three replicates. Gratifyingly, 7, 9 18, 22,
and MRT67307 were the most potent compounds in the
DRAK1 enzymatic assay (Table S1), and all demonstrated
PoC ≤ 9. Finally, the entire series was tested in the MARK3
and MARK4 NanoBRET assays. We found four compounds
(9, 14, 18, and 22) with submicromolar IC50 values in the
MARK4 NanoBRET assay, two of which (9 and 18) also
demonstrated submicromolar IC50 values in the MARK3
NanoBRET assay. Several of the parent compounds also had
submicromolar IC50 values in the MARK3/4 NanoBRET
assays. For MARK3, the four most potent compounds in the
MARK3 enzymatic assay, all with PoC ≤ −1 (Table S1), were
the most efficacious in the MARK3 NanoBRET assay (Table
2). For MARK4, compounds 9, 18, MRT67307, and BX-912
were among the most potent compounds in the MARK4
enzymatic assay (Table S1, PoC ≤ 5), and all demonstrated
MARK4 NanoBRET IC50 values < 400 nM. In general, we
noted very good correlation between the orthogonal enzymatic
(Table S1) and cellular target engagement (Table 2) assay
formats for these understudied kinases. Based on our 16-kinase
enzyme inhibition panel, we felt confident that some of these
understudied kinase chemical leads inhibited fewer kinases
than their parent compounds, and we chose to assess their
kinome-wide selectivities to determine whether they require
further optimization in our pursuit of high-quality chemical
tools.
Assessment of Kinome-Wide Selectivity. The promis-
ing inhibition profiles of our aminopyrimidine series in the
custom enzymatic assay panel motivated a broader survey to
ascertain the kinome-wide selectivity of our library. All 21
novel analogs were screened at 1 μM via the DiscoverX
scanMAX platform, which includes 403 wild-type (WT)
human kinases. A selectivity score (S10) for each compound
is included in Table 1, representative of the percentage of
kinases that exhibit binding with a percent of control (PoC) <
10 at 1 μM. A final column is included in Table 1 that converts
this S10 (1 μM) value into the number of kinases bound with a
PoC < 10 in the DiscoverX panel. Figure S1 details the specific
WT kinases that bound with a PoC < 10 at 1 μM in the
DiscoverX scanMAX panel for each aminopyrimidine analog.
Taken together, our NanoBRET profiling (Table 2) and
kinome-wide screening (Table 1) enabled us to confirm that
GSK8612 is the most potent in cells, selective (S10 (1 μM) =
0.02), and the most useful TBK1 inhibitor from all that we
tested. We did not improve upon the activity of this published
compound through these synthetic efforts. Next, of the
DRAK1 active compounds in the NanoBRET assay, only 4
was selective enough to be considered a valuable tool molecule.
Given its S10 (1 μM) = 0.027 and submicromolar IC50 value in
the DRAK1 NanoBRET assay, compound 4 was nominated as
a dark kinase tool that can be used to help illuminate the
function of DRAK1. Information for compound 4 has been
posted on the Dark Kinase Knowledgebase.35 Finally,
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Table 3. Combined Enzymatic Data
a
b
c
compound
potently active
moderately active
MYLK2 = 479 nM
weakly active
15
8
DRAK2: 91%
MKNK2 = 2866 nM, DRAK2: 60%, YANK2 >
10000 nM
12
16
21
3
CSF1R = 233 nM
CSF1R = 305 nM
TYK2 = 33 nM
LRRK2 = 89 nM
JAK2: 62%
TBK1 = 128 nM, TRKA = 477 nM
BMPR2 = 2918 nM, AURKB: 61%, JAK2: 107%
DRAK2: 59%, NIM1 = 4398 nM, MYLK2 = 1119
nM
5
TBK1 = 149 nM
IKKε = 1199 nM
11
17
TBK1 = 187 nM, TRKA = 153 nM
JAK2: 87%, IKKε = 1314 nM, AURKB: 51%
DRAK1 = 325 nM, DRAK2 = 161 nM, AAK1 =
390 nM, SIK2 = 481 nM
MKNK2 = 769 nM, TYK2 = 720 nM, JAK2 >
10000 nM, BMP2K: 56%
19
CSF1R = 91 nM
TYK2 = 276 nM
JAK2: 54%, ERBB2 > 10000 nM, TBK1 = 2737 nM
GSK8612 TBK1 = 37 nM
LRRK2 = 159 nM, MAP2K5: 11%, CSF1R = 264 IKKε = 552 nM, DAPK3 > 10000 nM, NUAK2 =
nM
1151 nM, ULK3 = 946 nM, MKNK2 = 1369 nM
20
4
AURKB: 5%
NUAK1 = 176 nM, SIK2 = 185 nM
BMP2K: 58%, DRAK1: 78%, DRAK2: 65%, JAK2:
81%, ACVR1 = 537 nM, BMPR1B = 9028 nM
LRRK2 = 19 nM, DRAK2 = 62 nM
IKKε = 216 nM, DRAK1 = 202 nM, ULK3 = 343 ULK2 = 1742 nM, ULK1: 58%
nM, MKNK2 = 380 nM, TBK1 = 192 nM,
MAP2K5: 12%
13
6
AURKB: 6%, BMP2K = 40 nM, AAK1 = 74 nM,
TYK2 = 48 nM, STK16 = 82 nM
TBK1 = 192 nM, JAK2: 12%, BMPR2 = 488 nM, NUAK2 = 900 nM, CSNK2A2 = 2269 nM,
TRKA = 283 nM, NUAK1 = 317 nM
PIP5K1A > 10000 nM, MKNK2 = 1009 nM
TBK1 = 66 nM, JAK2: 3%, BMP2K = 38 nM,
STK16 = 88 nM, TYK2 = 67 nM, AURKB: 7%,
AAK1 = 80 nM
CSF1R = 192 nM, TRKA = 158 nM, NUAK1 =
171 nM
PRP4 > 10000 nM, PIP5K1A > 10000 nM,
CSNK2A2 = 2304 nM, CSNK2A1 > 10000 nM,
ULK3 = 1371 nM
a
b
Potently active: IC50 value < 100 nM or < 10% control at 1 μM (Table S1). All IC50 values determined in duplicate. Moderately active: IC50 value
c
200−500 nM or 10−49% control at 1 μM (Table S1). All IC50 values determined in duplicate. Weakly active: IC50 value > 500 nM or > 49%
control at 1 μM (Table S1). All IC50 values determined in duplicate.
selectivity profiling shows that all compounds with sub-
micromolar IC50 values in the MARK3/4 NanoBRET assays
require further optimization to reduce the number of off-target
kinases that are potently inhibited in addition to MARK3/4
(S10 (1 μM) = 0.067−0.233). Efforts are ongoing to improve
the selectivity of analogs we identified as cell-active in the
MARK3/4 NanoBRET assays. The weak cellular potency of 4
for DRAK1 and 14/22 for MARK4 make it difficult to judge
the selectivity window offered by these compounds. They
should be considered chemical starting points in need of
further optimization to improve upon this potency while
maintaining or improving kinome-wide selectivity.
Orthogonal Validation of scanMAX Results. The
scanMAX assay identifies potential targets for our compounds
across the kinome. We chose to follow up and validate the
scanMAX kinase binding results by further testing of selected
actives in enzyme inhibition assays. Our choice of kinases for
follow-up varied depending on the selectivity of the compound
in question, as measured by S10 (1 μM). Highly selective
compounds that are also potent on their kinase target have the
potential to be chemical probe candidates, and, as such, we
chose to execute thorough enzyme profiling to validate or
invalidate potential off-targets as well as further quantify
potency. Thus, for the five compounds in our library with an
leads, a few additional understudied kinases with PoC < 35% at
1 μM in the scanMAX platform for these six compounds were
also selected for follow-up. Lastly, for a final four compounds
(20, 4, 13, and 6) with S10 (1 μM) = 0.022−0.047, we selected
only certain kinases with PoC < 20% at 1 μM in the scanMAX
platform and/or PoC < 50% in our initial profiling at Eurofins
(Table S1) for targeted follow-up, with a bias toward kinases
that were frequently inhibited by other analogs in the series as
well as understudied kinases of interest. All follow-up
enzymatic assays were executed at the Km = ATP for each
respective kinase. Results from these studies combined with
the single-concentration enzymatic results from Table S1 are
displayed in Table 3. Compounds in Table 3 are listed in order
of their kinome-wide selectivity scores from most (15) to least
(6) selective.
In examining the data generated and collected in Table 3,
some interesting trends emerge. The most selective com-
pounds (S10 (1 μM) < 0.02: 15, 8, 12, 16, and 21) that were
comprehensively profiled via enzymatic assays potently
inhibited 0−1 kinase (potently active, Table 3). We classify
the kinases potently inhibited by these compounds as more
well-studied. Except for 17 and GSK8612, the same group of
most selective compounds (S10 (1 μM) < 0.02) inhibited 0−2
kinases with moderate potency (Table 3). Some less selective
compounds (13 and 6) for which we did selective enzymatic
follow-up were found to be potent inhibitors of 5−7 kinases,
including members of the understudied NAK family (AAK1,
BMP2K, and STK16).17 Several of the aminopyrimidines
tested demonstrated IC50 values of 200−500 nM or inhibition
equal to 10−49% control at 1 μM for understudied kinases.
Given their kinome-wide selectivity scores and modest
potency, these compounds represent potential chemical leads
for the development of chemical tools to study these poorly
characterized kinases. Many kinases were assigned the weakly
S10 (1 μM) < 0.002 (15, 8, 12, 16, and 21), we followed-up on
all kinases with PoC < 35% at 1 μM in the scanMAX platform
and/or PoC < 50% in our initial custom enzymatic profiling
panel at Eurofins (Table S1). One exception to this was
exclusion of AURKA follow-up for 21. For the six compounds
in our library with an S10 (1 μM) = 0.005−0.02 (3, 5, 11, 17,
19, and GSK8612), we carried out enzymatic assays on all
kinases with PoC < 20% at 1 μM in the scanMAX platform
and/or PoC < 50% in our initial enzymatic profiling at
Eurofins (Table S1). Given our interest in identifying chemical
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Table 4. Selected NanoBRET Follow-Up
a
IC50 values (nM)
compound
BMP2K
CSF1R
>10000
LRRK2
MYLK2
NUAK1
TYK2
3
6
12
13
>10000
1020
3000
7010
>10000
15
3670
16
19
>10000
5060
>10000
>10000
GSK8612
>10000
>10000
a
Compounds tested in singlicate (n = 1) in dose−response.
active designation based on weak potency in the respective
enzymatic assays. In some cases, such as with compounds 5
and 13, two kinases that share high structural homology were
inhibited with differential potencies. For compound 5, there is
an 8-fold difference in potency for TBK1 and IKKε, and for
compound 13, there is a nearly 3-fold difference between
NUAK1 and NUAK2.
Importantly, we see that the compounds with submicromo-
lar IC50 values in the respective NanoBRET assays
corresponded with enzymatic inhibition IC50 values < 250
nM for GSK8612 (TBK1 = 37 nM) and compound 4 (DRAK1
= 202 nM). TBK1, LRRK2, DRAK1/2, CSF1R, TYK2, and
TRKA were identified as frequently inhibited kinases by our
aminopyrimidine series. This list is comprised of some kinases
that pyrimidines are known to potently inhibit (TBK1 and
TYK2)36,37 as well as kinases that represent new targets for
optimization. Analysis of the compounds that inhibit these
kinases could inform next steps in new chemistry to develop
specific SAR for these kinases. Before embarking on next
synthetic steps, the narrow selectivity profiles of exemplars
within our series coupled with potent enzymatic data
motivated interrogation of the cell-based activity of some of
these compounds in the respective NanoBRET assays.
between our enzymatic potencies and NanoBRET IC50 values
prompted us to further investigate compound properties that
might impact cell permeability.
Assessment of Compound Properties. With exceptions,
we observed > 30−300-fold losses in potency when
considering enzymatic versus NanoBRET activities. This was
true for NanoBRET values reported both in Tables 2 and 4. A
< 10-fold loss in potency was only observed in four cases:
GSK8612 for TBK1, 15 for MYLK2, and 4 and 17 for DRAK1.
The overall trend of biochemical activity not translating to
cellular potency made us curious about the physicochemical
properties of our aminopyrimidines. To address this, we
evaluated the kinetic solubility and permeability (PAMPA) of
our library of compounds and included the parent compounds
as well (Table 5).
Results in Table 5 demonstrated that our compounds were
generally very soluble. With the exception of 5, 6, 7, 11, 13, 16,
18, and 21, the measured solubility was estimated to be > 75%
of the dose concentration, and thus the actual solubility may be
higher than Table 5 reflects. As nearly all compounds
Table 5. Kinetic Solubility and PAMPA Assay Results
Selective NanoBRET Assay Follow-Up. For compounds
in Table 3 that inhibited kinases with potency < 500 nM, we
elected to determine their cellular target engagement via the
NanoBRET assay. No more than two kinases were evaluated
per compound. Several single digit micromolar inhibitors of
specific kinases were identified among these selective amino-
pyrimidine compounds. Table 4 shows that these compounds
were most cell-active in the BMP2K NanoBRET assay. We
were excited to identify that weaker inhibition of MYLK2 in
the enzymatic assay (IC50 value = 479 nM) translated to single
digit micromolar activity in the MYLK2 NanoBRET assay.
Compound 15 potently inhibits the fewest kinases of the
compounds that we synthesized (Tables 1 and 3), but
demonstrates modest MYLK2 inhibitory potency. Given the
paucity of literature around MYLK2 and the distinct lack of
compound optimization efforts directed at MYLK2, 15
represents one of the most promising chemical starting points
for MYLK2 chemical probe development. The single digit
micromolar activity in cells will need to be improved upon via
medicinal chemistry optimization to yield analogs with a better
selectivity window versus off-target kinases. We also observed
that compounds that exhibited potent activity in the LRRK2
enzyme assay (3 and GSK8612, IC50 values < 160 nM) and in
the TYK2 enzyme assay (16 and 19, IC50 values < 280 nM)
did not have any activity in the respective NanoBRET assays
when tested at concentrations up to 10 μM. The disparity
compound
kinetic solubility (μM)
Pe (cm/s)
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
171.0
190.1
33.2
4.7
40.3
3.89 × 10−7
5.08 × 10−6
a
<LOQ
<LOQ
a
3.73 × 10−6
2.39 × 10−6
1.30 × 10−6
1.51 × 10−6
174.6
159.0
195.2
118.7
167.7
20.0
173.6
176.9
147.0
169.3
135.6
176.5
199.7
85.6
7.09 × 10−7
a
<LOQ
<LOQ
a
5.61 × 10−7
6.23 × 10−8
a
<LOQ
8.24 × 10−8
8.85 × 10−7
4.56 × 10−7
1.66 × 10−7
2.60 × 10−6
5.97 × 10−7
2.60 × 10−6
19
20
21
22
164.0
164.6
155.5
173.1
MRT67307
BX-912
GSK8612
1.39 × 10−7
a
<LOQ
a
<LOQ: Below limit of quantitation.
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demonstrated solubility > 10 μM, they were not considered
poorly soluble, and this was eliminated from consideration as
driving their poor cellular potency. The permeability data,
however, were a bit more varied for this aminopyrimidine
library. Six compounds (5, 6, 12, 13, 16, and GSK8612) were
below the limit of quantitation (LOQ) and/or precipitated in
the assay media, making it impossible to determine their
permeability. Several of these compounds were among the
analogs with the lowest kinetic solubility concentrations as
well. For reference, Pe (permeability coefficient) values < 1.50
× 10−6 cm/s correlate with the human fraction absorbed <
80% and is a generally accepted cutoff for low permeability. In
addition to the compounds already mentioned that were below
the LOQ, this cutoff adds the majority of our library as well as
some parent compounds to a low permeability category: 3, 9,
11, 14, 15, 17−20, 22, and BX-912.
If we start to consider which structural elements could be
compromising the solubility and/or permeability of our
compounds, some trends emerge. The sulfonamide side
chain is present in most of the least soluble compounds (5,
6, 11, 13, 16, and 21) and all poorly permeable compounds (5,
6, 12, 13, 16, and GSK8612). As compounds with suboptimal
solubility (7) or poor permeability (GSK8612) still proved
active in our NanoBRET assays, just looking at half of the data
is not sufficient. None of the compounds with both
problematic solubility and permeability (5, 6, 13, and 16)
were active in our NanoBRET assays, leading us to conclude
that these two factors together point to compounds that are
poorly cell permeable. Since the majority of compounds did
not fall into this final category, consideration of physicochem-
ical properties did not explain our cell-based results.
It is worth noting for compounds/kinases where enzymatic
data has been published, the control compounds used in the
NanoBRET assays can be considered as a benchmark. For
CSF1R, dasatinib has a published enzymatic IC50 = 0.57 nM,
and we determined its NanoBRET IC50 to be 18.3 nM. For
NUAK1, BX-795 has a published IC50 = 5 nM, and we
determined its NanoBRET IC50 to be 187 nM.19 In both cases,
single digit or subnanomolar enzymatic IC50 values translated
to submicromolar NanoBRET IC50 values, and a ∼34-fold loss
in potency was observed when moving to the cell-based assay.
While not universal, it appears that for this set of compounds
and kinase targets, exceptional enzymatic potency is key to
achieving cellular potency to overcome the more than 30-fold
drop-off in cellular potency.
Structural Studies and Lead Optimization Plan for
MARK Subfamily. The MARK3 cocrystal structure with 9
corroborates the high affinity that this compound demonstrates
in the MARK3 NanoBRET assay (Table 2). Figure 3 shows
that the activation segment of MARK3 folds back and packs
against compound 9 via residue V205, while maintaining a
DFG-in conformation. Several key hydrogen bonds are made
between 9 and the binding pocket. In addition, several
hydrogen bonds are present between the binding pocket and
the 2- and 4-position side chains of 9. The imidazole
nitrogen(s) can interact with the main chain carbonyl of I62
in the G-rich loop as well as E182 in the catalytic loop and
E139 in the αD helix. It has also been suggested that, although
weak, C−H−O hydrogen bonds can exist.38 While inhibitor
conformations have been synchronized, the free rotation of the
4-position side chain allows the imidazole to rotate, which
perturbs some of these interactions. These hydrogen bonds,
therefore, do not simultaneously coexist, but rather different
hydrogen-bond patterns emerge due to free rotation of the
imidazole ring. With respect to the 2-position side chain, K60
(not shown) is at a distance of 3.4 Å and Y134 is at a distance
of 3.2 Å from the morpholine ring. Since the morpholine ring
is highly flexible, these distances represent the proximity of the
mentioned residues to the ring rather than to a particular atom
within it. Unsupported geometries as well as the flexibilities of
both residues and the morpholine ring, however, suggest that
K60 and Y134 do not form strong hydrogen bonds with the
morpholine ring. A weak and likely transient interaction
between Y134 and the morpholine ring on 9 is shown in the
Figure 3 pose. Essential interactions are made between the 3-
position nitrogen of the pyrimidine core and 2-position NH
with A135 in the MARK3 hinge. Importantly, the 5-position
bromo is oriented such that it makes an interaction with the
sulfur within gatekeeper residue M132.
It is interesting to consider this cocrystal structure versus
those solved for MRT67307 with TBK1 (PDB codes: 4IM0
and 4IWQ) and ULK2 (PDB code: 6QAU), and BX-320, a
close structural analog of BX-912, with PDK1 (PDB code:
1Z5M).23,39,40 The gatekeeper in TBK1 and ULK2, like in
MARK3, is a methionine (M86 in TBK1 and M85 in ULK2),
while in PDK1 the gatekeeper is a leucine (L86).23,39,40 The
proximity of the cyclopropyl group in MRT67307 or bromine
in BX-320 to the gatekeeper residue varies, supporting this
position influences binding preference and excludes molecules
that bear a group that is too large at the 5-position of the
pyrimidine ring. Furthermore, the discussion around M85 in
ULK2 noted some flexibility of this residue, suggesting a
certain degree of plasticity of the back pocket when
accommodating bulky hydrophobic residues such as the
cyclopropyl group in MRT67307.40 Hinge-binding interactions
are maintained in all cases between the pyrimidine nitrogen at
position 1 and the amino side chain NH at position 2,
supporting that this essential part of the molecule cannot be
modified without substantial losses in binding affinity. When
MRT67307 binds to ULK2, the pendant morpholine on the 2-
position side chain makes hydrogen-bonding interactions via a
water molecule with D95 of ULK2.40 The morpholine is not
shown to hydrogen bond in TBK1 structures with MRT67307,
which could partially explain the loss of affinity of 9 for TBK1
(Table 2) and potent binding of 9 to ULK2 (Figure S1).39
Although the interactions between the morpholine ring and
K60/Y134 of MARK3 are weak, these residues may be key in
the development of more specific MARK3 binders. If the
flexibilities of this 2-position side chain and/or morpholine
ring are reduced, it may allow for formation of stronger
hydrogen bonds and increase MARK3 binding affinity.
Also, in comparing the structure of 9 bound to MARK3
versus both the ULK2 and TBK1 structures with MRT67307,
we observe that the binding mode of 9 induced a “folded”
conformation of the activation segment that partially protrudes
into the ATP binding pocket. Comparison of the various
cocrystal structures suggests that this could be due to a more
compact structure of 9, in which the 2-position imidazole
stacks onto the 4-position phenyl ring. MRT67307, in
comparison, adopts a more elongated binding conformation
that likely prevents this “folded” conformation. Six compounds
from our library have the 4-position imidazole side chain: 3, 9,
10, 14, 15, and 20. Of these, only 9 and 14 demonstrate
submicromolar affinity for MARK3 and/or MARK4 in the
corresponding NanoBRET assays. In addition to the 2-position
imidazole, both 9 and 14 share the 4-position phenyl ring in
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their structures. It is possible that the 2-position imidazole
cannot efficiently stack with the 4-position pyrazole shared by
3, 10, and 15 to allow for binding in the MARK3 pocket
created when the activation segment folds. This unique
compact binding mode that seems to be tied to inhibitor
side chain substituents should be considered in future design
efforts.
If we examine and compare the residues that line or
surround the active sites of these kinases (TBK1, ULK2, and
PDK1) versus some of our understudied kinases of interest
(DRAK1, MARK3/4, and BMP2K; Figure 4), we observe that
pyrimidine scaffold through design. Several cell-active com-
pounds were discovered that exhibit submicromolar Nano-
BRET cellular target engagement IC50 values against kinases
including TBK1, DRAK1, MARK3, and MARK4. Three of
these kinases (DRAK1, MARK3, and MARK4) are under-
studied, IDG kinases that need high-quality chemical tools to
be able to characterize their function. Examples of compounds
with sub- or nearly submicromolar activities for understudied
kinases and modest kinome-wide selectivity are included in
Figure 4. These aminopyrimidines represent promising
chemical starting points in our campaign to identify chemical
probes to enable elucidation of the biological function(s) of
lesser studied kinases. The cellular potency of these
compounds will need to be improved via chemical
optimization to furnish compounds more suitable for inter-
rogating the biology of these understudied kinases. The
aminopyrimidine represents an alternative chemotype to our
recently disclosed AAK1/BMP2K chemical probe and, as such,
inhibits a different panel of off-target kinases.17,18 Development
of this chemotype is expected to yield a chemical probe with a
different kinome-wide inhibition profile and thus slightly
different biological activities. Results here reiterate the need to
employ orthogonal biochemical and cell-based assays to
understand the actual selectivity and potency of kinase
inhibitors more fully. The methods described herein offer a
path for others to identify and develop high-quality inhibitors
for understudied kinases to facilitate illumination of the entire
druggable kinome.
EXPERIMENTAL SECTION
■
Chemistry. General Information. Reagents were purchased
from commercial suppliers and used without further characterization
or purification. Temperatures are given in degrees Celsius (°C), and
unless otherwise stated, operations were carried out at room or
ambient temperature (r.t.), typically around 25 °C. Evaporation of
solvent was carried out using a rotary evaporator under reduced
pressure with a bath temperature not exceeding 60 °C. Thin-layer
chromatography (TLC) was used to follow the course of reactions.
Figure 4. Promising chemical leads for understudied kinases.
the gatekeeper is either a methionine or leucine in all cases
(gatekeeper, Figure S2). The key hinge-binding residue in all
these kinases is maintained as either a cysteine or alanine
(hinge donor and outer hydrogen-bond acceptor, Figure S2).
Finally, the morpholine-anchoring residue is coming from the
αD helix in ULK2 rather than the conserved hinge tyrosine
(hinge, Figure S2) utilized to anchor the morpholine in our
MARK3 structure, supporting that this residue may be key to
the design of MARK-family specific molecules.
Using the MARK3 cocrystal structure as our guide, we will
build on the subfamily selectivity observed for compounds 14
and 22 versus 9 and 18. Given that the 5-position of these
compounds seems to dictate both subfamily and kinome-wide
selectivity, we hypothesize that further exploration of this
position is warranted. Y134 within the hinge of MARK family
members is not maintained by all kinases, and thus adding
interactions with this residue may impart additional selectivity
through precluding binding with other kinases where it is
another residue (phenylalanine in TBK1, for example). We aim
to use these learnings to develop chemical tools that
demonstrate specificity within the MARK family in addition
to improved kinome-wide selectivity.
1
Intermediates and products exhibited satisfactory H NMR and/or
microanalytical data. The following conventional abbreviations are
also used: equivalents (equiv), mmol (millimoles), mg (milligrams),
and h (hours). Reactions were carried out under a blanket of nitrogen
unless otherwise stated. Compounds were visualized using a UV
(ultraviolet) lamp (254 nM). 1H and 13C NMR spectra were collected
in DMSO-d6, acetonitrile-d4, chloroform-d, or methanol-d4 and
recorded on Varian Inova 400 Megahertz (MHz), Bruker DRX 500
MHz, Bruker Avance III 400 MHz, Varian VNMRS 500 MHz, or
Agilent ProPulse 600 MHz spectrometers, noting the magnet strength
in all cases. Peak positions are given in parts per million (ppm) and
calibrated based upon the shift of the indicated solvent. Coupling
constants (J values) are expressed in hertz (Hz), and multiplicities are
reported as follows: singlet (s), doublet (d), doublet(s) of doublets
(dd/ddd) or triplets (dt) or quartets (dq), triplet (t), triplet(s) of
doublets (td/ttd), quartet (q), pentet (p), pentet of doublets (pd),
heptet (h), and multiplet (m). An asterisk (*) was added after the line
listing of spectral data for compounds where a single peak could not
be unambiguously assigned in the 13C spectrum. Double asterisks
(**) were added after the line listing of spectral data for compounds
where a single peak as part of a multiplet could not be unambiguously
assigned in the 13C spectrum. Purity was assessed via LC−MS using
an Agilent mass spectrometer (column: Agilent Poroshell 120 SB-
C18, 4.6 mm × 30 mm, 2.7 μm with UHPLC Guard Infinity Lab
Poroshell 120 SB-C18, 4.6 mm × 5 mm, 2.7 μm). Syntheses of key
intermediates and several final products were performed by
ChemSpace LLC. Identity and purity of all final products were
confirmed upon arrival.
CONCLUSIONS
■
In summary, we provide details related to the synthesis and
extensive biological evaluation of a library of aminopyrimi-
dines. We have shown that selectivity can be built into the
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General Procedure for the Synthesis of Compound B:
Procedure A. To a solution of compound 1 (1 equiv) in ethanol
(10−20 mL) were added dropwise DIPEA (2 mL) and amine 1 (1
equiv) at −10 °C. The resulting mixture was heated to r.t. then stirred
at 50 °C for 16 h. Solvent was next evaporated from the reaction
mixture and to the resulting material was added water (30 mL). The
obtained precipitate was filtered, washed with water, isopropanol, and
hexane, and dried under vacuum at 50 °C to give compounds 2a−2g
(amount, yield, purity), which were used in the next step without
further purification.
(q, J = 279 Hz), 120.72, 119.89, 107.57, 49.90 (q, J = 34.0 Hz), 41.03,
8.12, 5.08, 1.98. HPLC purity: 100%. HRMS (ESI) ([M + H]+) Calcd
for C20H23F3N7O2S: 482.1588, found: 482.1582.
4-(((5-Bromo-2-((3-(morpholinomethyl)phenyl)amino)-
pyrimidin-4-yl)amino)methyl)benzenesulfonamide (6). 128 mg,
1
24% yield. H NMR (400 MHz, DMSO-d6)δ 9.19 (s, 1H), 8.06 (s,
1H), 7.73 (dd, J = 15.3, 7.0 Hz, 4H), 7.60 (s, 1H), 7.50 (d, J = 8.0 Hz,
2H), 7.43 (d, J = 8.2 Hz, 1H), 7.26 (s, 2H), 7.10 (t, J = 7.8 Hz, 1H),
6.81 (d, J = 7.4 Hz, 1H), 4.70 (d, J = 6.1 Hz, 2H), 3.52 (d, J = 4.8 Hz,
4H), 3.17 (d, J = 5.2 Hz, 2H), 2.28 (s, 4H). 13C NMR (126 MHz,
acetonitrile-d3) δ 158.62, 156.44, 145.33*, 144.50, 141.96*, 140.19,
133.48, 128.40, 127.58, 126.11, 122.70, 121.44, 119.72, 93.03*, 66.46,
62.67, 60.58, 53.36, 43.67, 24.26*. HPLC purity: 98.4%. HRMS (ESI)
([M + H]+) Calcd for C22H26BrN6O3S: 533.0970, found: 533.0955.
N-(3-((5-Bromo-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-
4-yl)amino)pyrimidin-4-yl)amino)propyl)cyclobutanecarboxamide
(7). 83.0 mg, 17% yield. 1H NMR (400 MHz, chloroform-d) δ 7.95 (s,
2H), 6.34 (s, 1H), 6.04−5.99 (m, 1H), 5.58−5.53 (m, 1H), 4.64 (q, J
= 8.5 Hz, 2H), 3.49 (q, J = 6.2 Hz, 2H), 3.35 (q, J = 6.4 Hz, 2H), 2.98
(p, J = 8.5 Hz, 1H), 2.33−2.20 (m, 5H), 2.19−2.08 (m, 2H), 2.04−
1.80 (m, 2H), 1.76 (p, J = 6.3 Hz, 2H). 13C NMR (151 MHz,
acetonitrile-d3) δ 173.06, 156.65, 156.48, 153.76, 138.90, 121.50**
(q, J = 279.8), 120.99, 119.51, 119.43, 49.88 (q, J = 34.2 Hz), 37.32,
35.47, 33.62, 26.93, 22.78, 15.55, 8.19.HPLC purity: 97.8%. HRMS
(ESI) ([M + H]+) Calcd for C18H24BrF3N7O: 490.1178, found:
490.1176.
N - ( 3 - ( ( 2 - C h l o r o p y r i m i d i n - 4 - y l ) a m i n o ) p r o p y l ) -
cyclobutanecarboxamide (2a). 330 mg, 50% yield, 95% purity by
LC−MS.
N-(3-((5-Bromo-2-chloropyrimidin-4-yl)amino)propyl)-
cyclobutanecarboxamide (2b). 2.20 g, 50% yield, 95% purity by
LC−MS.
N-(3-((2-Chloro-5-cyclopropylpyrimidin-4-yl)amino)propyl) cy-
clobutanecarboxamide (2c). 570 mg, 67% yield, 95% purity by
LC−MS.
4-(((5-Bromo-2-chloropyrimidin-4-yl)amino)methyl)-
benzenesulfonamide (2d). 6.40 g, 55% yield, 95% purity by LC−MS.
4-(((2-Chloro-5-cyclopropylpyrimidin-4-yl)amino)methyl)-
benzenesulfonamide (2e). 850 mg, 50% yield, 95% purity by LC−
MS.
N-(2-(1H-Imidazol-4-yl)ethyl)-5-bromo-2-chloropyrimidin-4-
amine (2f). 2.50 g, 25% yield, 95% purity by LC−MS.
N-(2-(1H-Imidazol-4-yl)ethyl)-2-chloro-5-cyclopropylpyrimidin-
4-amine (2g). 420 mg, 50% yield, 95% purity by LC−MS.
General Procedure for the Synthesis of Compounds 3−11,
13, 14, 17, 18, 21, 22, and GSK8612: Procedure B. To a solution
of compound 2 (1 equiv) and amine 2 (1 equiv) in butanol (2−5
mL) was added dioxane × HCl (10%w/w) (0.5−2 mL), and the
resulting mixture was stirred at 80 °C for 16 h. The reaction mixture
was next neutralized with aqueous ammonia and concentrated under
vacuum. The resulting material was purified by preparative HPLC
(2−7 min, 35−70% methanol (0.1% ammonium hydroxide), 30 mL/
min; column: YMC-ACTUS TRIART C18, 20 mm × 100 mm, 5 μm)
to give final compounds 3−11, 13, 14, 17, 18, 21, 22, and GSK8612
(amount, yield).
N-(3-((2-((3-Methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)-
amino)pyrimidin-4-yl)amino)-propyl)cyclobutanecarboxamide (8).
1
75.0 mg, 15% yield. H NMR (400 MHz, chloroform-d) δ 7.98 (s,
1H), 7.83 (d, J = 5.9 Hz, 1H), 6.33 (s, 1H), 5.82 (d, J = 5.6 Hz, 1H),
5.56 (s, 2H), 4.59 (q, J = 8.5 Hz, 2H), 3.41−3.25 (m, 4H), 2.95 (p, J
= 8.6 Hz, 1H), 2.31−2.18 (m, 5H), 2.13 (q, J = 9.1, 8.3 Hz, 2H),
2.01−1.79 (m, 3H), 1.72 (p, J = 6.3 Hz, 2H). 13C NMR (151 MHz,
acetonitrile-d3) δ 172.67, 161.10, 157.78, 153.04*, 138.64*, 125.45*,
121.63** (q, J = 280), 120.78, 119.87, 118.98, 49.87 (q, J = 34.0 Hz),
37.31, 33.92, 26.98, 22.74, 15.56, 8.19. HPLC purity: 100%. HRMS
(ESI) ([M + H]+) Calcd for C18H25F3N7O: 412.2073, found:
412.2069.
N4-(2-(1H-Imidazol-4-yl)ethyl)-5-cyclopropyl-N2-(3-methyl-1-
(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)pyrimidine-2,4-diamine (3).
N 4 - ( 2 - ( 1 H - I m i d a z o l - 4 - y l ) e t h y l ) - 5 - b r o m o - N 2 - ( 3 -
(morpholinomethyl)phenyl)pyrimidine-2,4-diamine (9). 190 mg,
1
1
71.0 mg, 36% yield. H NMR (400 MHz, DMSO-d6) δ 11.86 (s,
11% yield. H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 9.21
1H), 8.11 (s, 2H), 7.56 (s, 2H), 6.84 (d, J = 16.5 Hz, 2H), 5.02−4.91
(m, 2H), 3.63 (q, J = 6.8 Hz, 2H), 2.81 (t, J = 7.4 Hz, 2H), 2.16 (s,
3H), 1.41 (td, J = 10.7, 8.6, 5.9 Hz, 1H), 0.78 (d, J = 8.0 Hz, 2H),
0.40 (t, J = 5.1 Hz, 2H). 13C NMR (151 MHz, acetonitrile-d3) δ
160.51, 156.33, 150.93, 138.16, 134.55*, 132.59, 121.60** (q, J =
280.0 Hz), 120.39, 120.11, 113.02*, 107.40, 49.73 (q, J = 33.9 Hz),
38.56, 24.68, 8.17, 5.05, 1.84. HPLC purity: 95.0%. HRMS (ESI) ([M
+ H]+) Calcd for C18H22F3N8: 407.1920, found: 407.1918.
(s, 1H), 8.01 (s, 1H), 7.75 (s, 1H), 7.63−7.54 (m, 2H), 7.14 (t, J =
7.8 Hz, 2H), 6.83 (d, J = 7.5 Hz, 2H), 3.68 (q, J = 6.9 Hz, 2H), 3.54
(t, J = 4.6 Hz, 4H), 3.31 (s, 2H), 2.87−2.78 (m, 2H), 2.29 (s, 4H).
13C NMR (101 MHz, methanol-d4) δ 163.42*, 158.65, 158.56,
155.08, 140.37, 137.30, 134.65, 128.08, 122.67, 120.21, 118.15,
116.24*, 92.60, 66.28, 63.13, 53.22, 40.58, 26.34. HPLC purity:
95.9%. HRMS (ESI) ([M + H]+) Calcd for C20H25BrN7O:458.1304,
found: 458.1300.
N-(3-((5-Cyclopropyl-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-
N4-(2-(1H-Imidazol-5-yl)ethyl)-5-bromo-N2-(3-methyl-1-(2,2,2-
trifluoroethyl)-1H-pyrazol-4-yl)pyrimidine-2,4-diamine (10). 325
pyrazol-4-yl)amino)pyrimidin-4-yl)amino)propyl)-
1
cyclobutanecarboxamide (4). 57.0 mg, 11% yield. H NMR (400
1
mg, 17% yield. H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H),
MHz, chloroform-d) δ 8.01 (s, 1H), 7.70 (s, 1H), 6.21 (s, 1H), 5.94
(s, 1H), 5.59 (s, 1H), 4.62 (q, J = 8.5 Hz, 2H), 3.53 (q, J = 5.9 Hz,
2H), 3.36 (q, J = 6.3 Hz, 2H), 2.96 (p, J = 8.2 Hz, 1H), 2.31−2.19
(m, 5H), 2.18−2.10 (m, 2H), 2.03−1.82 (m, 2H), 1.77 (p, J = 6.7 Hz,
2H), 1.51−1.40 (m, 1H), 1.25 (s, 1H), 0.91 (dq, J = 5.7, 3.9 Hz, 2H),
0.53−0.45 (m, 2H). 13C NMR (101 MHz, chloroform-d) δ 175.72,
162.75, 157.86, 152.71, 140.54, 123.13 (q, J = 280 Hz), 122.38,
121.92, 110.04, 52.97 (q, J = 34.7 Hz), 39.91, 37.24, 36.04, 29.84,
25.43, 18.09, 10.99, 7.41, 4.56. HPLC purity: 100%. HRMS (ESI)
([M + H]+) Calcd for C21H29F3N7O: 452.2386, found: 452.2385.
4-(((5-Cyclopropyl-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyra-
zol-4-yl)amino)pyrimidin-4-yl)amino)methyl)benzenesulfonamide
8.55 (s, 1H), 8.09 (s, 1H), 7.95 (s, 1H), 7.55 (s, 1H), 7.08 (s, 1H),
6.87 (s, 1H), 4.98 (s, 2H), 3.60 (q, J = 6.9, 6.5 Hz, 2H), 2.78 (t, J =
7.1 Hz, 2H), 2.16 (s, 3H). 13C NMR (151 MHz, acetonitrile-d3) δ
156.58, 156.46, 153.77, 144.88*, 143.51*, 138.62, 132.63, 121.54 (q, J
= 279.9 Hz), 120.99, 119.43, 117.93, 49.74 (q, J = 34.1 Hz), 38.91,
24.47, 8.21. HPLC purity: 96.6%. HRMS (ESI) ([M + H]+) Calcd for
C15H17BrF3N8:445.0712, found: 445.0709.
4-(((5-Cyclopropyl-2-((3-(morpholinomethyl)phenyl)amino)-
pyrimidin-4-yl)amino)methyl)-benzenesulfonamide (11). 18.0 mg,
4% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.83 (s, 1H), 7.75 (d, J =
8.1 Hz, 2H), 7.66 (s, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.50−7.42 (m,
3H), 7.25 (s, 2H), 7.06 (t, J = 7.9 Hz, 1H), 6.75 (d, J = 7.7 Hz, 1H),
4.75 (d, J = 5.9 Hz, 2H), 3.57−3.50 (m, 5H), 3.17 (d, J = 5.5 Hz,
2H), 2.32−2.25 (m, 4H), 1.61−1.50 (m, 1H), 0.88−0.83 (m, 2H),
0.52 (d, J = 4.7 Hz, 2H). 13C NMR (101 MHz, methanol-d4) δ
162.60, 158.31, 152.19, 144.97, 142.02, 140.52, 137.00, 128.03,
127.12, 125.89, 122.35, 120.11, 118.04, 110.30, 66.24, 63.12, 53.13,
1
(5). 74.0 mg, 15% yield. H NMR (400 MHz, DMSO-d6) δ 8.01 (s,
1H), 7.79−7.70 (m, 3H), 7.59 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H),
7.38−7.33 (m, 1H), 7.26 (s, 2H), 4.87 (q, J = 9.2 Hz, 2H), 4.68 (d, J
= 6.1 Hz, 2H), 2.08 (s, 3H), 1.52 (s, 1H), 0.83 (d, J = 8.0 Hz, 2H),
0.48 (d, J = 5.0 Hz, 2H). 13C NMR (151 MHz, acetonitrile-d3) δ
160.37, 156.47, 151.51, 143.05, 139.49, 138.98, 125.27, 123.81, 123.0
K
https://doi.org/10.1021/acs.jmedchem.1c00440
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
43.32, 39.00, 7.14, 3.92. HPLC purity: 100%. HRMS (ESI) ([M +
H]+) Calcd for C25H31N6O3S:495.2178, found: 495.2162.
N-(3-((5-Bromo-4-((4-sulfamoylbenzyl)amino)pyrimidin-2-yl)-
amino)phenyl)pyrrolidine-1-carboxamide (13). 383 mg, 5% yield.
1H NMR (500 MHz, methanol-d4) δ 7.95 (d, J = 2.2 Hz, 1H), 7.80
MHz, methanol-d4) δ 176.49, 162.67, 158.29, 155.62, 151.52, 140.97,
139.70, 128.11, 114.28, 113.88, 112.08, 110.40, 45.61, 39.47, 39.00,
37.37, 36.13, 29.14, 25.06, 24.83, 17.57, 7.07, 3.81. HPLC purity:
100%. HRMS (ESI) ([M + H]+) Calcd for C26H36N7O2:478.2930,
found: 478.2914.
4-(((5-Bromo-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-
(dd, J = 8.4, 2.2 Hz, 2H), 7.71 (d, J = 2.4 Hz, 1H), 7.49−7.43 (m,
2H), 7.08 (dt, J = 4.6, 1.8 Hz, 2H), 6.95 (td, J = 4.3, 1.8 Hz, 1H), 4.77
(d, J = 2.0 Hz, 2H), 3.42 (dt, J = 6.8, 3.5 Hz, 4H), 1.94 (h, J = 2.5 Hz,
4H). 13C NMR (101 MHz, methanol-d4) δ 158.61, 156.19, 155.36,
153.60, 144.23, 141.95, 140.23, 139.55, 128.02, 127.27, 125.75,
114.94, 114.62, 112.77, 92.26, 45.57, 43.56, 25.01. HPLC purity:
92.6%. HRMS (ESI) ([M + H]+) Calcd forC22H25BrN7O3S:546.0923,
found: 546.0914.
yl)amino)pyrimidin-4-yl)-amino)methyl)benzenesulfonamide
1
(GSK8612). 379 mg, 73% yield. H NMR (400 MHz, DMSO-d6) δ
8.43 (s, 1H), 7.98 (s, 1H), 7.73 (d, J = 8.0 Hz, 3H), 7.62−7.53 (m,
1H), 7.41 (s, 2H), 7.27 (s, 2H), 4.90 (q, J = 9.1 Hz, 2H), 4.61 (s,
2H), 2.08 (s, 3H). 13C NMR (151 MHz, acetonitrile-d3) δ 156.82,
156.44, 154.25, 142.15, 139.72, 139.67, 125.39, 123.87, 123.25 (q, J =
281 Hz), 121.58, 119.20, 49.92 (q, J = 34.0 Hz), 41.30, 8.17. HPLC
purity: 100%. HRMS (ESI) ([M + H]+) Calcd for C17H18BrF3N7O2S:
520.0378, found: 520.0378.
N4 -(2-(1H-Imidazol-4-yl)ethyl)-5-cyclopropyl-N2 -(3-
(morpholinomethyl)phenyl)pyrimidine-2,4-diamine (14). 73.0 mg,
24% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 8.85 (s,
1H), 7.81 (s, 1H), 7.64−7.54 (m, 3H), 7.11 (t, J = 7.8 Hz, 1H),
6.90−6.81 (m, 2H), 6.77 (d, J = 7.5 Hz, 1H), 3.69 (q, J = 6.7 Hz,
2H), 3.54 (t, J = 4.6 Hz, 4H), 3.30−3.20 (m, 1H), 3.17 (d, J = 3.9 Hz,
1H), 2.85 (t, J = 7.2 Hz, 2H), 2.29 (d, J = 4.8 Hz, 4H), 1.44 (q, J =
4.8, 2.9 Hz, 1H), 0.79 (dt, J = 8.3, 3.1 Hz, 2H), 0.51−0.38 (m, 2H).
13C NMR (151 MHz, acetonitrile-d3) δ 160.24, 156.35, 150.71,
139.02, 136.45, 132.53, 126.05, 119.42, 116.81, 115.42, 114.90,
108.19, 64.35, 60.97, 51.28, 38.46, 24.40, 5.12, 1.85. HPLC purity:
100%. HRMS (ESI) ([M + H]+) Calcd for C23H30N7O:420.2512,
found: 420.2506.
General Procedure for the Synthesis of Compounds 12, 15,
16, 19, and 20: Procedure C. To a solution of 6, 9, 10, 13, or
GSK8612 (1 equiv) in methanol (20 mL) were added 5% Pd/C (0.5
equiv) and triethylamine (TEA, 3 equiv). The resulting mixture was
stirred under H2 (hydrogen) atmosphere at r.t. for 16 h. Next the
reaction mixture was filtered, and the filtrate was concentrated in
vacuo. The crude product was purified via preparative HPLC (2−7
min, 30−55% acetonitrile, 30 mL/min; column: SunFire C18, 19 mm
× 100 mm, 5 μm) to give 12, 15, 16, 19, and 20 (amount, yield).
4-(((2-((3-Methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)-
pyrimidin-4-yl)amino)methyl)benzenesulfonamide (12). 108 mg,
1
36% yield. H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.87 (s,
N-(3-((4-((3-(Cyclobutanecarboxamido)propyl)amino)pyrimidin-
1H), 7.77 (dd, J = 8.9, 6.9 Hz, 3H), 7.66 (s, 1H), 7.46 (d, J = 8.0 Hz,
2H), 7.30 (s, 2H), 5.92 (s, 1H), 4.90 (q, J = 9.1 Hz, 2H), 4.57 (s,
2H), 2.11 (s, 3H). 13C NMR (151 MHz, acetonitrile-d3) δ 161.02,
157.98, 153.76, 142.49, 139.69, 139.23, 125.44, 125.24, 123.88,
123.79, 121.45 (q, J = 279 Hz), 121.16, 119.67, 94.31*, 49.96 (q, J =
34.4 Hz), 41.30, 8.11. HPLC purity: 96.6%. HRMS (ESI) ([M + H]+)
Calcd for C17H19F3N7O2S:442.1273, found: 442.1270.
2-yl)amino)phenyl)pyrrolidine-1-carboxamide (17). 19.0 mg, 10%
1
yield. H NMR (400 MHz, chloroform-d) δ 7.94 (s, 1H), 7.82 (s,
1H), 7.24 (s, 1H), 7.22−7.03 (m, 3H), 6.95 (d, J = 8.2 Hz, 1H), 6.13
(s, 2H), 5.80 (d, J = 6.5 Hz, 1H), 5.56 (s, 1H), 3.41 (m, 6H), 3.26
(m, 2H), 2.95−2.78 (m, 1H), 2.19 (d, J = 9.8 Hz, 2H), 2.07−1.89 (m,
9H), 1.68 (d, J = 6.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
174.29, 162.95, 160.25, 154.80, 154.49, 141.70, 140.83, 128.16,
113.36, 113.15, 111.41, 98.08, 55.31, 46.08, 39.17, 36.78, 29.55, 25.47,
25.11, 18.22. HPLC purity: 100%. HRMS (ESI) ([M + H]+) Calcd
for C23H32N7O2: 438.2617, found: 438.2617.
N4-(2-(1H-Imidazol-5-yl)ethyl)-N2-(3-methyl-1-(2,2,2-trifluor-
oethyl)-1H-pyrazol-4-yl)pyrimidine-2,4-diamine (15). 86.0 mg, 10%
1
yield. H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.15 (d, J =
35.4 Hz, 2H), 7.73 (s, 1H), 7.53 (s, 1H), 7.18 (s, 1H), 6.83 (s, 1H),
5.85 (d, J = 5.8 Hz, 1H), 4.95 (q, J = 9.2 Hz, 2H), 3.51 (s, 2H), 2.75
(t, J = 7.5 Hz, 2H), 2.16 (s, 3H). 13C NMR (151 MHz, acetonitrile-
d3) δ 161.05, 157.72, 153.16*, 138.57*, 132.48, 122.48, 121.55 (q, J =
280 Hz), 120.74, 119.80, 116.93*, 114.19, 49.77 (q, J = 32.4 Hz),
38.55, 24.75, 8.19. HPLC purity: 100%. HRMS (ESI) ([M + H]+)
Calcd for C15H18F3N8:367.1607, found: 367.1596.
N-(3-((5-Bromo-4-((3-(cyclobutanecarboxamido)propyl)amino)-
pyrimidin-2-yl)amino)phenyl)pyrrolidine-1-carboxamide (18). 335
mg, 13% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 7.97
(s, 1H), 7.87 (t, J = 2.1 Hz, 1H), 7.62 (t, J = 5.4 Hz, 1H), 7.25 (d, J =
8.1 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 7.00−6.91 (m, 2H), 3.42 (q, J =
6.6 Hz, 2H), 3.33 (d, J = 6.4 Hz, 4H), 3.07 (q, J = 6.5 Hz, 2H), 2.93
(p, J = 8.4 Hz, 1H), 2.14−2.02 (m, 4H), 1.96 (q, J = 9.8 Hz, 2H),
1.90−1.79 (m, 4H), 1.78−1.60 (m, 3H). 13C NMR (101 MHz,
methanol-d4) δ 176.54, 158.67, 158.61, 155.60, 155.03, 140.53,
139.71, 128.13, 114.73, 114.19, 112.40, 92.61, 45.63, 39.47, 37.91,
36.26, 28.86, 25.06, 24.84, 17.58. HPLC purity: 98.1%. HRMS (ESI)
([M + H]+) Calcd for C23H31BrN7O2: 516.1723, found: 516.1707.
N-(3-((5-Cyclopropyl-4-((4-sulfamoylbenzyl)amino)pyrimidin-2-
yl)amino)phenyl)pyrrolidine-1-carboxamide (21). 16.0 mg, 8%
yield. 1H NMR (400 MHz, methanol-d4) δ 7.80 (d, J = 8.3 Hz,
2H), 7.70 (t, J = 2.1 Hz, 1H), 7.47−7.41 (m, 3H), 7.22 (t, J = 8.0 Hz,
1H), 7.15 (dt, J = 8.0, 1.5 Hz, 1H), 6.97 (dt, J = 8.0, 1.6 Hz, 1H), 4.82
(s, 2H), 3.46−3.41 (m, 4H), 1.98−1.92 (m, 4H), 1.57 (td, J = 7.9, 4.0
Hz, 1H), 1.02−0.97 (m, 2H), 0.61−0.56 (m, 2H). 13C NMR (101
MHz, methanol-d4) δ 163.32, 155.39, 152.73, 142.84, 142.38, 140.50,
139.89, 136.96, 128.85, 127.60, 125.88, 117.51, 117.11, 115.24,
112.53, 45.66, 43.99, 25.01, 23.61, 6.92, 4.27. HPLC purity: 100%.
HRMS (ESI) ([M + H]+) Calcd for C25H30N7O3S:508.2131, found:
508.2116.
N-(3-((4-((4-Sulfamoylbenzyl)amino)pyrimidin-2-yl)amino)-
phenyl)pyrrolidine-1-carboxamide (16). 23.0 mg, 8% yield. 1H
NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 7.93 (s, 1H), 7.88 (s,
1H), 7.80 (d, J = 5.8 Hz, 1H), 7.74 (m, 3H), 7.48 (d, J = 8.3 Hz, 2H),
7.26 (s, 2H), 7.18 (s, 1H), 6.99 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 8.7
Hz, 1H), 5.97 (s, 1H), 4.63 (s, 2H), 3.31 (d, J = 7.5 Hz, 4H), 1.82−
1.78 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 162.86, 160.14,
155.30*, 154.46, 144.67, 142.92, 141.44, 140.84, 128.18, 127.94,
126.07, 113.46, 113.27, 111.51, 97.85*, 46.09, 40.86, 25.46. HPLC
purity: 95.0%. HRMS (ESI) ([M
C22H26N7O3S:468.1818, found: 468.1814.
+
H]+) Calcd for
4-(((2-((3-(Morpholinomethyl)phenyl)amino)pyrimidin-4-yl)-
1
amino)methyl)benzenesulfonamide (19). 18.8 mg, 38% yield. H
NMR (400 MHz, methanol-d4) δ 7.84 (d, J = 8.4 Hz, 2H), 7.77 (d, J
= 6.0 Hz, 1H), 7.59 (t, J = 2.0 Hz, 1H), 7.52−7.47 (m, 2H), 7.36 (d, J
= 8.2 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 6.90 (dt, J = 7.5, 1.4 Hz, 1H),
6.01 (d, J = 6.0 Hz, 1H), 4.70 (s, 2H), 3.61 (t, J = 4.7 Hz, 4H), 3.35
(s, 2H), 2.36 (t, J = 4.5 Hz, 4H). 13C NMR (101 MHz, methanol-d4)
δ 163.16, 159.74, 154.25, 144.48, 142.16, 140.34, 137.04, 128.07,
127.30, 125.95, 122.68, 120.45, 118.38, 97.18, 66.23, 63.11, 53.14,
43.26. HPLC purity: 100%. HRMS (ESI) ([M + H]+) Calcd for
C22H27N6O3S: 455.1865, found: 455.1851.
N-(3-((4-((3-(Cyclobutanecarboxamido)propyl)amino)-5-cyclo-
propylpyrimidin-2-yl)amino)-phenyl)pyrrolidine-1-carboxamide
(22). 16.0 mg, 5% yield. 1H NMR (400 MHz, methanol-d4) δ 7.81 (t,
J = 2.1 Hz, 1H), 7.55 (d, J = 1.1 Hz, 1H), 7.25 (ddd, J = 8.1, 2.2, 1.1
Hz, 1H), 7.15 (t, J = 8.1 Hz, 1H), 6.97 (ddd, J = 8.0, 2.1, 1.0 Hz, 1H),
3.58 (t, J = 6.6 Hz, 2H), 3.47−3.42 (m, 5H), 3.24 (t, J = 6.7 Hz, 2H),
2.96 (pd, J = 8.6, 1.0 Hz, 1H), 2.24−2.14 (m, 2H), 2.10−2.01 (m,
2H), 1.98−1.93 (m, 5H), 1.84−1.75 (m, 3H), 1.46 (ttd, J = 8.1, 5.2,
1.1 Hz, 1H), 0.92−0.87 (m, 2H), 0.49−0.44 (m, 2H). 13C NMR (101
N4-(2-(1H-Imidazol-5-yl)ethyl)-N2-(3-(morpholinomethyl)-
1
phenyl)pyrimidine-2,4-diamine (20). 21.0 mg, 19% yield. H NMR
(400 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.92 (s, 1H), 7.80 (d, J = 16.9
Hz, 2H), 7.61 (s, 1H), 7.54 (s, 1H), 7.24 (s, 1H), 7.12 (t, J = 7.8 Hz,
1H), 6.79 (d, J = 10.4 Hz, 2H), 5.92 (d, J = 5.8 Hz, 1H), 3.54 (m,
L
https://doi.org/10.1021/acs.jmedchem.1c00440
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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6H), 3.31 (s, 2H), 2.79 (m, 2H), 2.29 (s, 4H). 13C NMR (101 MHz,
Methanol-d4) δ 163.14, 159.75, 153.70, 140.60, 137.24, 134.91,
134.58, 128.07, 122.50, 120.29, 118.18, 116.44, 97.18, 66.28, 63.20,
53.24, 40.31, 26.50. HPLC purity: 100%. HRMS (ESI) ([M + H]+)
Calcd for C20H26N7O: 380.2199, found: 380.2186.
Biological Evaluation: Enzymatic Assays. Eurofins kinase
enzymatic radiometric assays were carried out at the Km = ATP at
a single concentration (1 μM) in duplicate for each kinase in Table
S1. Eurofins kinase enzymatic radiometric assays were carried out at
the Km = ATP in dose−response (9-pt curve in duplicate) for each
kinase with an IC50 value listed in Table 3. Details about the substrate
used, protein constructs, controls, and assay protocol for each kinase
assay can be found at the Eurofins Web site: https://www.
eurofinsdiscoveryservices.com.
Library-Wide NanoBRET Assays. Human embryonic kidney
(HEK293) cells (hypotriploid, female, fetal) were purchased from
ATCC and grown in Dulbecco’s modified Eagle’s medium (DMEM,
Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS,
Corning). Cells were incubated in 5% CO2 at 37 °C and passaged
every 72 h with trypsin. They were not allowed to reach confluency.
Constructs for NanoBRET measurements of DRAK1 (DRAK1-
NLuc), MARK3 (NLuc-MARK3), MARK4 (NLuc-MARK4), and
TBK1 (NLuc-TBK1) included in Table 2 were kindly provided by
Promega. NanoBRET assays were executed as described previously.17
Preferred NLuc orientations are indicated in parentheses after each
construct. Assays were carried out in dose−response as described by
the manufacturer using 0.5 μM of tracer K-9 for DRAK1 and MARK3
and 0.5 μM of tracer K-5 for MARK4 and TBK1. Respective tracer
titration curves that we generated for DRAK1, MARK3, and MARK4
can be found at https://darkkinome.org/data.35 Tracer titration
curves for MARK3, MARK4, and TBK1 can also be found on the
Promega Web site.
Kinome Screening. The scanMAX assay platform was used to
assess the selectivity of each aminopyrimidine analog at 1 μM at
Eurofins DiscoverX Corporation. As described previously, this
commercial assay platform screens against 403 WT human kinases
and provides percent of control values.19 These percent of control
values are captured in Table 1.
Specific NanoBRET Assay Follow-Up. NanoBRET assays for
the six kinases in Table 4 were carried out in dose−response in
singlicate by Carna Biosciences. Assays were carried out according to
the manufacturer’s protocol.
Kinetic Solubility and Permeability (PAMPA). Kinetic
solubility analysis was carried out from 10 mM DMSO stocks of
compounds in phosphate buffered saline solution (PBS) at pH 7.4 by
Analiza, Inc. Following 24 h incubation in a Millipore solubility filter
plate, samples were vacuum filtered, and the filtrates collected for
analysis. Filtrates were injected into the nitrogen detector for
quantification via total chemiluminescent nitrogen determination
(CLND). Filtrates were quantified with respect to a calibration curve
generated using standards that span the dynamic range of the
instrument. Calculated solubility values are corrected for background
nitrogen present in the DMSO and the media.
PAMPA analysis was carried out by Analiza, Inc. using a Corning
Gentest Precoated PAMPA plate. DMSO stocks of compound diluted
in PBS at pH 7.4 were added to the donor compartment of the plate,
PBS at pH 7.4 was added to the acceptor compartment, and the plate
was left to incubate for 5h. Both the donor and acceptor
compartments were collected and analyzed by CLND. Donor and
acceptor samples were quantified using the calibration curve
generated using standards that span the dynamic range of the
instrument. Measured concentrations are corrected for background
nitrogen present in the DMSO and the media. Concentration values
from the donor and acceptor compartment are used in the calculation
of the effective permeability of the compound. Solubility of the
compound is determined experimentally rather than assuming full
solubility.
Crystallization and Structure Determination. The coding
sequence for the MARK3 residues 48−366 was cloned into the vector
pNIC-CT10HF. Accordingly, the expressed construct comprised a
TEV-cleavable His6 tag in its C-terminus. Expression in E. coli Rosetta
(DE3) was performed as previously described.41 For purification, the
pellet was resuspended in lysis buffer (50 mM HEPES/NaOH pH 7.4,
500 mM NaCl, 0.5 mM TCEP, 5% glycerol), and the cells lysed by
sonication. After clearance by centrifugation, the lysate was loaded
onto a Ni-NTA column. The bound His6-tagged protein was eluted in
lysis buffer containing 300 mM imidazole. TEV cleavage was
performed while dialyzing the sample overnight at 4 °C. The cleaved
tag, TEV, and contaminating proteins were removed by another Ni-
NTA step. Finally, MARK3 was subjected to gel filtration using an
AKTA Xpress system combined with an S200 column in KGF150
buffer (20 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 0.5 mM
TCEP, 5% glycerol).
100 nL of a solution containing the protein−ligand complex (14
mg/mL MARK348−366, 500 μM 9) was transferred to a 3-well
crystallization plate (Swissci), mixed with 50 nL of precipitant
solution (0.1 M sodium formate pH 7.0, 24% PEG3350), and
incubated at 4 °C. Crystals were spotted after 3 days and did not
change appearance after 6 days. They were mounted in precipitant
solution cryoprotected with additional 25% ethylene glycol. Data were
collected at Swiss Light Source (SLS) X06SA and analyzed, scaled,
and merged with the SLS automated data processing (adp) pipeline.42
The structure was solved by molecular replacement with Phaser43
using a MARK3 model as a template (PDB code: 2QNJ)44 and
refined with Phenix.45 The model and structure factors have been
deposited to the PDB with the code 7P1L (crystallographic
parameters are included in Table S2).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00440.
■
sı
Eurofins 16-kinase panel enzymatic data, an additional
figure that details kinome-wide selectivities of all
compounds, sequence alignment of kinases potently
inhibited by aminopyrimidines, X-ray crystallographic
data, LC chromatograms, and NMR spectra are included
(PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
■
Alison D. Axtman − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy and Division of Chemical
Biology and Medicinal Chemistry, UNC Eshelman School of
Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599, United States;
orcid.org/0000-0003-4779-9932;
Email: alison.axtman@unc.edu
Authors
David H. Drewry − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy, Division of Chemical Biology
and Medicinal Chemistry, UNC Eshelman School of
Pharmacy, and UNC Lineberger Comprehensive Cancer
Center, School of Medicine, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599, United
States; orcid.org/0000-0001-5973-5798
Joel K. Annor-Gyamfi − Structural Genomics Consortium,
UNC Eshelman School of Pharmacy and Division of
Chemical Biology and Medicinal Chemistry, UNC Eshelman
School of Pharmacy, University of North Carolina at Chapel
Statistics. Standard error of the mean (SEM) was calculated for
NanoBRET assays executed more than once. Calculated SEM is
included alongside IC50 values in Table 2.
M
https://doi.org/10.1021/acs.jmedchem.1c00440
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hill, Chapel Hill, North Carolina 27599, United States;
orcid.org/0000-0001-8510-2951
supported in part by the NC Biotechnology Center Institu-
tional Support grant 2018-IDG-1030, NIH 1U24DK116204
and U54AG065187, DoD ALSRP award AL190107, and ALS
Association grant ID wa1127.
Carrow I. Wells − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy and Division of Chemical
Biology and Medicinal Chemistry, UNC Eshelman School of
Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599, United States;
orcid.org/0000-0003-4799-6792
Julie E. Pickett − Structural Genomics Consortium, UNC
Eshelman School of Pharmacy and Division of Chemical
Biology and Medicinal Chemistry, UNC Eshelman School of
Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599, United States;
orcid.org/0000-0002-9535-8528
Verena Dederer − Institute for Pharmaceutical Chemistry,
Johann Wolfgang Goethe-University, 60438 Frankfurt am
Main, Germany; Structural Genomics Consortium,
Buchmann Institute for Molecular Life Sciences, Johann
Wolfgang Goethe-University, 60438 Frankfurt am Main,
Germany
Franziska Preuss − Institute for Pharmaceutical Chemistry,
Johann Wolfgang Goethe-University, 60438 Frankfurt am
Main, Germany; Structural Genomics Consortium,
Buchmann Institute for Molecular Life Sciences, Johann
Wolfgang Goethe-University, 60438 Frankfurt am Main,
Germany
ABBREVIATIONS USED
■
DIPEA, N,N-diisopropylethylamine; DMSO, dimethyl sulf-
oxide; HCl, hydrochloric acid; HPLC, high-performance liquid
chromatography; IC50, half maximal inhibitory concentration;
LC−MS, liquid chromatography−mass spectrometry; Km,
Michaelis constant; LINCS, Library of Integrated Network-
Based Cellular Signatures; NanoBRET, bioluminescence
resonance energy transfer using nanoluciferase; Nluc, nano-
luciferase; NMR, nuclear magnetic resonance; PAMPA,
parallel artificial membrane permeability assay; Pd/C, palla-
dium on carbon; v/v, volume for volume; w/w, weight for
weight
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Constructs for NanoBRET measurements of DRAK1,
MARK3, MARK4, and TBK1 were kindly provided by
Promega. Coral was used to make the kinome tree depicted
in our Table of Contents graphic. Coral was developed in the
Phanstiel Lab at UNC: http://phanstiel-lab.med.unc.edu/
CORAL.46 We used the TREEspot kinase interaction mapping
1,
software to prepare the kinome trees in Figure
and
Supplemental
Information: http://treespot.discoverx.com. We
thank ChemSpace LLC for synthetic support and Stefan
Knapp for guidance on x-ray crystallographic studies. We thank
the Department of Chemistry Mass Spectrometry Core
Laboratory at the University of North Carolina for their
assistance with mass spectrometry analysis. The Structural
Genomics Consortium is a registered charity (number
1097737) that receives funds from AbbVie, Bayer Pharma
AG, Boehringer Ingelheim, Canada Foundation for Innovation,
Eshelman Institute for Innovation, Genome Canada, Genen-
tech, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-
DD grant no. 115766], Janssen, Merck KGaA Darmstadt
Germany, MSD, Novartis Pharma AG, Ontario Ministry of
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