Synthesis and structure activity relationships of a series of 4-amino-1H-pyrazoles as covalent inhibitors of CDK14

Article abstract of DOI:10.1016/j.bmcl.2019.05.024

The TAIRE family of kinases are an understudied branch of the CDK kinase family, that have been implicated in a number of cancers. This manuscript describes the design, synthesis and SAR of covalent CDK14 inhibitors, culminating in identification of FMF-0

Full text of DOI:10.1016/j.bmcl.2019.05.024

Accepted Manuscript
Synthesis and structure activity relationships of a series of 4-amino-1H-pyra-
zoles as covalent inhibitors of CDK14
Fleur M. Ferguson, Zainab M. Doctor, Scott B. Ficarro, Jarrod A. Marto, Nam
Doo Kim, Taebo Sim, Nathanael S. Gray
PII:
S0960-894X(19)30321-X
https://doi.org/10.1016/j.bmcl.2019.05.024
BMCL 26444
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
3 April 2019
14 May 2019
14 May 2019
Please cite this article as: Ferguson, F.M., Doctor, Z.M., Ficarro, S.B., Marto, J.A., Doo Kim, N., Sim, T., Gray,
N.S., Synthesis and structure activity relationships of a series of 4-amino-1H-pyrazoles as covalent inhibitors of
CDK14, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.024
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Synthesis and structure activity relationships of a series of 4-
amino-1H-pyrazoles as covalent inhibitors of CDK14
#
, a, b
#, a, b
, Scott B. Ficarroa, c, d, Jarrod A. Martoa, c, d, e
Fleur M. Ferguson
, Zainab M. Doctor
f
g,h
, a
Nam Doo Kim , Taebo Sim , Nathanael S. Gray*
a
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215,
b
USA
c
Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.
d
e
f
Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02215, USA.
Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
Daegu-Gyeongbuk Medical Innovation Foundation, Republic of Korea
g
h
#
Chemical Kinomics Research Center, Korea Institute of Science and Technology, Republic of Korea.
KU-KIST Graduate School of Converging Science and Technology, Korea University, Republic of Korea.
These authors contributed equally. * Corresponding author: nathanael_gray@dfci.harvard.edu
The TAIRE family of kinases are an understudied branch of the CDK kinase family, that have
been implicated in a number of cancers. This manuscript describes the design, synthesis and
SAR of covalent CDK14 inhibitors, culminating in identification of FMF-04-159-2, a potent,
covalent CDK14 inhibitor with a TAIRE kinase biased selectivity profile.
Keywords: Covalent kinase inhibitor; CDK14; TAIRE kinase; CDK15; CDK16; CDK17;
CDK18; CDK inhibitor; mitosis; cell cycle.
CDK14 is a member of the understudied
TAIRE subfamily of cyclin-dependent
kinases, named after the “TAIRE”
sequence motif in their cyclin binding site,
and comprising of CDKs 14-18.(
in numerous cancers including colorectal
(
4)
(5)
cancer , ovarian cancer
and gastric
(
6)
cancer.
compounds
However,
to
selective
interrogate
tool
the
1),(2),(3)
pharmacological consequences of CDK14
inhibition were, until recently, unavailable.
CDK14 overexpression has been reported
Scheme 1. Representative synthesis of 4-amino-1H-pyrazole analogs. See also supporting information.

1
Table 1. Single ring R analogs of AT7519. A) CDK14 IC50s were measured using a LanthaScreen
binding assay. IC50s were calculated as the average of three replicates, and are reported ± the standard
errorB) Antiproliferative activity against the HCT116 cell line was measured using a CellTiter-Glo assay.
IC50s were calculated as the average of three replicates, and are reported ± the standard error.
(
11)
We recently reported the identification and
characterization of FMF-04-159-2 (100),
the first covalent CDK14 inhibitor with
other CDK kinases.
A selective CDK14
inhibitor is consequently also expected to
have mild to insignificant effects on
HCT116 cell growth. Therefore we used
(
7)
TAIRE-kinase bias.
Off-target CDK2
activity was identified, and experimental
conditions to minimize CDK2 engagement
and maximize CDK14 engagement were
reported. Here we describe the structure
activity relationships of a series of 4-amino-
potency in
proliferation assay as
a
CellTiter-Glo (Promega)
first pass
a
approximation of compound selectivity for
CDK14 when prioritizing molecules for
progress through further rounds of
characterization (Supporting Figure 1). As
previously reported, AT7519 is a potent
inhibitor of HCT116 cell proliferation, with
an IC50 of 132 nM.
CDK14 contains
cysteine, C218, located at the beginning of
(
7)
1H-pyrazole
analogs
for
CDK14
biochemical and cellular potency, and
measure their effects on HCT116
proliferation. We show cellular engagement
of CDK14 by lead molecules, and
demonstrate their covalent nature by
MS/MS studies. This data is of interest, as
(
7)
a
uniquely placed
(
12)
the αD helix, proximal to the ATP pocket.
4
-amino-1H-pyrazole is
a
widely used
This SAR
In order to improve the potency and
selectivity of AT7519 towards CDK14, we
sought to design a covalent inhibitor.
Examination of the co-crystal structure of
CDK2 in complex with AT7519 (PDB:
2VU3) revealed that the 4-aminopiperidine
is oriented towards the αD helix in
(
8)
kinase inhibitor scaffold.
analysis aids development of further,
improved CDK14 inhibitors, and in addition,
provides insight into how CDK14 activity
can be removed from 4-amino-1H-pyrazole
analogs targeted towards other kinases.
In search of chemical leads for CDK14, we
performed a CDK14 cellular engagement
screen of a library of reported CDK
inhibitors using a biotin JNK-IN-7 pulldown
assay. This identified the multi-targeted
CMCG kinase inhibitor AT7519 as an
efficient CDK14 inhibitor at 1 µM.
result was confirmed using a LanthaScreen
CDK14 binding assay (Table 1).
(
10)
CDK2.
Assuming a conserved binding
1
mode, this substituent (R ) should provide
a suitable vector for targeting CDK14
C218.
(
9)
1
Therefore analogs containing varied R
substituents incorporating acrylamide and
(
10)
This
(E)-4-(dimethylamino)but-2-enamide
2
warheads (R ) were synthesized according
to Scheme 1.
SiRNA mediated CDK14 knockdown does
not cause significant proliferation defects in
the HCT116 cell line, unlike knockdown of
Initially, molecules containing a single
saturated or unsaturated ring were
synthesized (Table 1, compounds 11-24).

Table 2: Extended R1 analogs of AT7519. A) CDK14 IC50s were measured using a LanthaScreen
binding assay. IC50s were calculated as the average of three replicates, and are reported ± the standard
errorB) Antiproliferative activity against the HCT116 cell line was measured using a CellTiter-Glo assay.
IC50s were calculated as the average of three replicates, and are reported ± the standard error. –R is
used to denote reversible control compounds (without an alkene group).
These analogs lost significant potency
relative to AT7519, potentially due to the
loss of a hydrogen bonding interaction with
the piperidine NH seen in CDK2
regiochemistry not well tolerated at this
position (e.g. 27 vs 31). Preference for a
1,3 aminobenzamide regiochemistry of the
second ring was observed with the most
potent analogs, 27 and 28, combining
these two regiochemistries, in line with
computational docking results.
(PDB:2VU3), which was not compensated
for by covalent inhibition.
Replacing the aminobenzamide (28) with
an aminobenzylamine (36) dramatically
increased potency against CDK14. Again,
F210
E211
a
strong
preference
for
a
1,4
aminopiperidine regiochemistry and a 1,3
aminobenzylamine regiochemistry was
observed (Table 2, 36 vs 34). The most
potent analogs, 35 and 36, inhibited
CDK14 with IC50s below 1 nM in the
LanthaScreen biochemical assay. 36 was
chosen for follow-up studies due to its
lower toxicity in the cell titer glo assay,
relative to 35. Both compounds show
increased toxicity relative to AT7519,
indicating off-target activity. Compounds
containing an aminobenzylsulfonamide
Y212
V213
Q260
H214
C218
Figure 1: Docking of compound 10 into a
homology model of CDK14. View from above.
JNK3 contains a cysteine in an equivalent
region of the kinase to CDK14.
Examination of the structure of JNK3 in
complex with JNK-IN-7 (PDB: 3V6S)
indicated that a longer distance between
the hinge binding motif and the acrylamide
is required in order to successfully form a
covalent bond. Therefore analogs were
prepared containing two linked cyclic
aliphatic or aromatic rings decorated with
acrylamide or (E)-4-(dimethylamino)but-2-
enamide warheads (Table 2, 25-70).
Docking studies into a CDK14 homology
model built from the X-ray structure of
CDK12 (PDB: 4NST) predicted that a
range of linked two ring systems, with 1,4-
followed
similar
trends
to
the
aminobenzamide series. Compound 36 is
highly potent against CDK14, and also
displays increased toxicity against HCT116
cells.
IC50
IC50
(
9)
Compound CDK14 Compound CDK14
(nM)
(nM)
9
1000
500
> 1000
1000
46
10
57
68
1000
50
> 1000
> 1000
3
6
5
6
7
5
Table 3: Cellular target engagement of lead
compounds in biotin pull-down assay, estimated
based on testing of three compound
concentrations.
regiochemistry in ring
1 and a 1,3-
regiochemistry in ring 2 would best allow
for C218 covalent engagement (Figure
Inspired by the acrylamide bearing
substituent in JNK-IN-7, amide linked
(
7) (13)
1).
1
biphenyls were introduced to R (Table 1
Molecules containing a piperidine linked to
(9)
compounds 43-52).
Although these
1
an aminobenzamide at
reduced potency for CDK14 relative to
AT7519 (Table 2, 25-32). 1,4
R
displayed
compounds displayed reduced affinity, they
also exhibited the same regiochemical
preferences. Interestingly in this series, the
E)-4-(dimethylamino)but-2-enamide
1
aminopiperidine regiochemistry at R is
strongly preferred, with 1,3 aminopiperidine

Table 4: FMF-03-198 analogs with a modified heterocyclic core. A) CDK14 IC50s were measured using
a LanthaScreen binding assay. IC50s were calculated as the average of three replicates, and are
reported ± the standard error. B) Antiproliferative activity against the HCT116 cell line was measured
using a CellTiter-Glo assay. IC50s were calculated as the average of three replicates, and are reported ±
the
standard
error.
To verify that the 6 lead compounds and
two reversible control compounds identified
in Table 2 were able to engage CDK14 in
cells we performed a pull-down experiment.
Cells were treated for 4 h with compounds
at various doses, and then lysed and
treated with biotinylated JNK-IN-7 ,
followed by streptavidin coated beads.
CDK14 capture was assayed by western
blot. (Table 3, SI Figure 1). Of these
compounds, 10 was able to potently block
CDK14 pulldown at 50 nM, and 36, 46 and
27 also showed effects at 500 – 1000 nM
concentrations (Supporting Figure 2).
Compounds 56 and 68 were not active, or
weakly active in the cellular assay
(Supporting Figure 2). The reversible
control molecules also showed no activity
in this assay, indicating a dependency on
covalent bond formation for activity in cells.
Covalent bond formation by 10 and 36 was
verified by incubating compounds with
purified, recombinant CDK14 followed by
warhead was preferred to the acrylamide
warhead. 46 was the most potent of this
subseries against CDK14, but also had
increased toxicity relative to AT7519.
In an attempt to improve selectivity in a
manner
analogous
to
JNK-IN-8,
(
9)
compounds were synthesized with ortho-
methylation of the 1,4-diaminoaniline in ring
1
(47, 48). Unfortunately, this methylation
was not tolerated by CDK14.
Substitution of the ring 1 piperidine group
for a pyrrolidine resulted in compounds with
a less favorable CDK14 : HCT116 potency
profile (e.g. 72 vs 35) with the exception of
68, which was taken forward for validation.
Removal of the linking atoms between
rings 1 and 2 afforded 3(piperidine-1-
yl)anilines 53-56. This series maintained
acceptable CDK14 potency and the
dimethylamino-substituted analogs 54 and
56 had low HCT116 toxicity. 56 was
chosen for follow up studies.
Finally, the propyl amide analogs of the
most potent molecules were synthesized to
examine the effects of removal of the
covalent warhead. 57 was ~ 10-fold less
potent against CDK14 compared to 9 and
MS/MS
analysis.
Both
compounds
achieved complete labeling of CDK14
when incubated for 3 h at r.t., at a 10:1
molar ratio of compound to CDK14/Cyclin
Y protein. Digest experiments followed by
MS analysis showed that C218 was
exclusively labeled.
10, and displayed similar toxicity against
HCT116 cells to 10. Compound 75 was
significantly less potent against both
CDK14 and HCT116 cells than 35 and 36.
This indicated that the covalent binding
component improved binding towards
CDK14, but also towards off-targets that
alter cell proliferation.
To evaluate the cellular targets of 10 and
36 more broadly, we performed KiNativ
profiling at 1 μM compound concentration
(
14) (7)
(Figure 1).
This revealed that whilst
both compounds were potent against

3
Table 5: FMF-03-198 analogs with varied R substituents. A) CDK14 IC50s were measured using a
LanthaScreen binding assay. IC50s were calculated as the average of three replicates, and are reported
±
the standard error. B) Antiproliferative activity against the HCT116 cell line was measured using a
CellTiter-Glo assay. IC50s were calculated as the average of three replicates, and are reported ± the
standard error. –R is used to denote reversible control compounds (without an alkene group).
CDK14, they also inhibited a large number
of other kinases.
As hinge binding is a common feature of
Type I kinase inhibitors, analogs of FMF-
pocket.(10) Substitution of the 2,6-dichloro
phenyl ring of 9 and 10 for a more polar
2,6-difluoro phenyl ring (88, 89) yielded a
modest reduction in toxicity and maintained
potent CDK14 inhibition.
03-198-2 with altered hinge binding motifs
predicted to reduce hydrogen bonding
interactions were synthesized, in an
attempt to increase the selectivity for
CDK14 by reducing the reversible binding
affinity, but maintaining the ability to bind
covalently (Table 4, 76-87).
IC50
IC50 CDK14
Compound
Compound CDK14
nM)
(nM)
(
9
1
50
500
1000
96
104
101
> 1000
> 1000
> 1000
100
07
1
Table 6. Cellular target engagement of
additional lead compounds in biotin pull-down
assay, estimated based on testing of three
compound concentrations.
Unfortunately, none of these analogs
demonstrated potent inhibition in the
CDK14 binding assay (Table 4). Therefore,
3
the 2,6-dichlorobenzamide substituent (R )
The
bulkier
2-chloro,
6-methoxy
was varied in an effort to improve
selectivity (Table 5). In the co-crystal
structure of AT7519 in complex with CDK2,
the 2,6-benzamide group fills a small
hydrophobic pocket, and prior studies have
demonstrated that CDK2 binding activity
can be tuned by altering it’s substituents,
by introducing steric clashes with the back
substitution yielded a further reduction in
toxicity, whilst maintaining potency for
CDK14 (90, 91). The reversible propyl
amide 93 maintained comparable potency
towards CDK14 and comparable potency
against HCT116 cells as 90, indicating this
effect was primarily driven by changes in
the reversible binding profile of the

compounds. However, further increase in
the bulk of the 6-substituent to an ethoxy
group afforded a more toxic compound
with an IC50 of 39.6 nM for CDK14
compared to 256 nM for CDK2, as
measured by the NanoBRET assay. This
improved cellular potency for CDK14 over
CDK2, and other off-targets of compound
(
7)
KiNativ profiling of lead compounds
9
8
7
6
5
4
3
2
1
0
100 offered an improvement in the relative
cellular selectivity for CDK14 compared to
AT7519. Future work is required to remove
activity against these targets from CDK14 /
TAIRE kinase probes, therefore use of
washout conditions is recommended when
using 100 to probe the pharmacological
1
0
36
91
50 - 75 % I
100
>
90 % I
75 - 90 % I
35 - 50 % I
(
7)
consequences of CDK14 inhibition.
No. of kinases inhibited
90 % I 75 - 90 % I 50 - 75 % I 35 - 50 % I
CDK14
> 90 % I
Y
Compound
Total
>
Compound 100 represents a significant
advance towards developing chemical
probes for the TAIRE kinase family, and is
the first targeted covalent CDK14 inhibitor.
Acknowledgements
This work was funded by the following
grants; R21CA188881, R01CA219850,
DFCI Strategic Research Initiative
10
36
91
7
8
8
4
7
5
5
2
8
3
5
1
3
7
0
2
25
23
18
9
Y
N
Y
100
Figure 2: KiNativ profiling results of lead
compounds at 1 µM compound concentration. %
I, percent inhibition relative to DMSO only
control.
(
92), indicative of a narrow SAR window at
the 6-position. 2,6-dimethoxy substitution
96), 2,4,6-methoxy substitution (104) and
,4,6-chloro substitution (100) patterns
Disclosures
(
2
J.A.M. is a member of the SAB of Devices.
N.S.G. is a founder, SAB member, and
equity holder in Gatekeeper, Syros, Petra,
C4, B2S, and Soltego. The Gray lab
receives or has received research funding
from Novartis, Takeda, Astellas, Taiho,
Jansen, Kinogen, Her2llc, Deerfield, and
Sanofi. N.S.G., F.M.F., and Z.M.D. are
inventors on a patent application covering
chemical matter in this publication owned
by Dana-Farber Cancer Institute. S.B.F,
N.D.K. and T.S. have no conflicts to
declare
resulted in reduced CDK14 potency, but
also dramatically reduced HCT116 toxicity.
Addition of a methoxy group to the 3-
position of 10 to afford 97 didn’t
significantly affect CDK14 binding or
toxicity.
Finally,
the
un-substituted
compound 107 exhibited potent CDK14
inhibition and reduced HCT116 toxicity.
The active compounds from this series
were taken forward for cellular target
engagement studies (Table 6, Supporting
Figure 2). Of these, only 91 and 100 were
able to completely inhibit CDK14 pull-down
at concentrations below 1 μM.
Compound 91 and 100 were both
evaluated using the KiNativ platform for
cellular selectivity at concentrations of 1 μM
and compared to the profiles of 10 and 36.
Compound 91 demonstrated a comparable
number of targets compared to 10 and 36,
despite its reduced cytotoxicity. However,
compound 100 showed a favorable profile,
with a dramatically reduced number of
targets.
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