Structure-guided development of covalent TAK1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.11.035

TAK1 (transforming growth factor-β-activated kinase 1) is an essential intracellular mediator of cytokine and growth factor signaling and a potential therapeutic target for the treatment of immune diseases and cancer. Herein we report development of a ser

Full text of DOI:10.1016/j.bmc.2016.11.035

Accepted Manuscript
Structure-guided development of covalent TAK1 inhibitors
Li Tan, Deepak Gurbani, Ellen L. Weisberg, John C. Hunter, Lianbo Li, Douglas
S. Jones, Scott B. Ficarro, Samar Mowafy, Chun-Pong Tam, Suman Rao,
Guangyan Du, James D. Griffin, Peter K. Sorger, Jarrod A. Marto, Kenneth D.
Westover, Nathanael S. Gray
PII:
S0968-0896(16)31236-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.035
BMC 13402
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 August 2016
14 November 2016
18 November 2016
Please cite this article as: Tan, L., Gurbani, D., Weisberg, E.L., Hunter, J.C., Li, L., Jones, D.S., Ficarro, S.B.,
Mowafy, S., Tam, C-P., Rao, S., Du, G., Griffin, J.D., Sorger, P.K., Marto, J.A., Westover, K.D., Gray, N.S.,
Structure-guided development of covalent TAK1 inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.11.035
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Structure-guided development of covalent TAK1 inhibitors
1
,2,#
3#
4,5
3
3
Li Tan , Deepak Gurbani , Ellen L. Weisberg , John C. Hunter , Lianbo Li , Douglas S.
6
,7
1,2
1,2,8
1,2
1,2,6
Jones , Scott B. Ficarro , Samar Mowafy , Chun-Pong Tam , Suman Rao , Guangyan
1
,2
4,5
6,
1,2
3
Du , James D. Griffin , Peter K. Sorger , Jarrod A. Marto , Kenneth D. Westover *,
1
,2
Nathanael S. Gray *
1
4
Department of Cancer Biology and Medical Oncology, Dana Farber Cancer Institute, Boston,
2
5
MA 02115; Department of Biological Chemistry and Molecular Pharmacology and Medicine,
6
HMS LINCS Center and Laboratory of Systems Pharmacology, Harvard Medical School,
3
Boston, MA 02215; Departments of Biochemistry and Radiation Oncology, The University of
7
Texas, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390; Department
of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;
8
Misr International University, Km 28 Cairo, Ismailia Rd., Ahmed Orabi Dist., Cairo, Egypt
#
*
These authors contributed equally.
Email addresses for correspondence:
Kenneth.Westover@UTSouthwestern.edu (KDW); Nathanael_Gray@dfci.harvard.edu (NSG)

Abstract:
TAK1 (transforming growth factor-β-activated kinase 1) is an essential intracellular mediator of
cytokine and growth factor signaling and a potential therapeutic target for the treatment of
immune diseases and cancer. Herein we report development of a series of 2,4-disubstituted
pyrimidine covalent TAK1 inhibitors that target Cys174 , a residue immediately adjacent to the
‘DFG-motif’ of the kinase activation loop. Co-crystal structures of TAK1 with candidate
compounds enabled iterative rounds of structure-based design and biological testing to arrive at
optimized compounds. Lead compounds such as 2 and 10 showed greater than 10-fold
biochemical selectivity for TAK1 over the closely related kinases MEK1 and ERK1 which
possess an equivalently positioned cysteine residue. These compounds are smaller, more easily
synthesized, and exhibit a different spectrum of kinase selectivity relative to previously reported
macrocyclic natural product TAK1 inhibitors such as 5Z-7-oxozeanol.

1
. Introduction
TAK1 (transforming growth factor-β-activated kinase 1) is a serine/threonine kinase
belonging to the MAPK kinase kinase (MAP3K) family initially identified because of its
responsiveness to TGF-β and bone morphogenetic protein (BMP) in preosteoblast cells [1].
Knockout of TAK1 in mice is embryonically lethal, causing severe neural tube deformities early
in gestation [2, 3]. TAK1 mediates responsiveness to environmental stress to control
transcription and apoptosis. TAK1 activity also appears to be involved in multiple inflammatory
conditions and cancers motivating interest in the development of TAK1 inhibitors for therapeutic
purposes.
TAK1 mediates activation of immune processes stimulated by pro-inflammatory
cytokines such as tumor necrosis factor alpha (TNFα), toll-like receptor (TLR) ligands and
interleukin-1 (IL-1) [4-7]. In B cells, conditional TAK1 knockout shows that TAK1 is essential
for mitogenic responses to receptor-mediated stimuli including TLR, anti-CD40 and anti-IgM
antibodies [8]. In T cells, conditional TAK1 knockout reduces the development of Treg cells
expressing Foxp3 [9-11]. In macrophages, TAK1 has been shown to function in innate immune
responses, whereby pattern recognition receptors utilize TAK1 to activate NFκB through MyD88
[
12]. TAK1 is associated with inflammatory disorders such as kidney fibrosis [13] and Crohn’s
disease [14] and depletion of TAK1 decreases levels of inflammatory infiltrates and damps
cytokine responses. TAK1 has also been studied in ischemic stroke models, where short-term
inhibition of TAK1 blocked activation of p38 and JNK following oxygen and glucose
deprivation [15].
Additionally, TAK1 is associated with multiple cancers including lymphoma [16],
ovarian cancer [17], colon cancer [18], neuroblastoma [19] and pancreatic cancer [20], possibly

related to modulation of inflammation in the cellular microenvironment [21]. Work by Singh and
colleagues has shown that TAK1 is required for survival of some KRAS-dependent colon cancer
cell lines and demonstrated that TAK1 inhibition induces apoptosis via modulation of WNT
signaling [18]. Recent work by Ansell and colleagues revealed that TAK1 is an essential
mediator of activated MyD88 signaling, a protein commonly mutated and constitutively active in
a subtype of non-Hodgkin lymphomas called Waldenstrom’s Macroglobulinemia (WM) [22]. In
addition, TAK1 activity has been associated with tumor aggressiveness in ovarian cancer [17].
A number of small molecule inhibitors of TAK1 kinase activity have been reported. 5Z-
7
- oxozeaenol (5Z7), a natural resorcylic lactone isolated from fungi, was identified as a TAK1
inhibitor in a screen searching for inhibitors of TAK1 catalytic activity. Subsequent studies
showed 5Z7 prevents IL-1 induced activation of TAK1, JNK, MAPK and NFκB in cell culture
by irreversible covalent binding to Cys174, located in the ATP-binding pocket of TAK1 [23].
Anti-TAK1 activity by 5Z7 has been demonstrated in multiple experimental systems [24, 25].
However, resorcylic acids lactones are known to inhibit multiple kinases [26], and broad-based
kinase profiling has demonstrated that 5Z7 is a potent inhibitor of MEK1/2, FLT3, KIT, PDGFR,
TGFRB and other kinases [27]. Improving the selectivity of 5Z7 and related molecules through
chemical modification is synthetically challenging, although reversible resorcylic acid lactones
were recently reported to have improved selectivity and pharmacokinetic properties [28]. AZ-
TAK1 is a thiophenecarboxamide reported to inhibit TAK1 signaling in mantle cell lymphoma
cancer cells and promote cell death [16]. ABC-FP, an aminofuropyridine, was reported as a
biochemically potent TAK1 inhibitor with good activity in a mouse ovarian tumor model [29].
Finally, LYTAK1, an orally available pyrrolopyrimidine, was reported to inhibit NF-κB activity
and potentiate the cytotoxicity of chemotherapeutic agents in pancreatic cancer [20]. Herein, we

report a new series of covalent TAK1 inhibitors based on a 2,4-disubstituted pyrimidine scaffold
that is well suited to further chemical modification.
2
2
. Results and Discussion
.1. Rationale
Previously we reported a series of reversible type-II kinase inhibitors including NG25,
which potently inhibit TAK1 [30]. These studies were motivated by the hypothesis that alternate
chemotypes might improve upon the selectivity and potency of existing TAK1 inhibitors such as
5
Z7. In addition to NG25, kinome profiling of our kinase inhibitor library identified compound 1
as a potent TAK1 inhibitor with an enzymatic IC50 of 34 nM in a fixed time-point LanthaScreen
binding assay (Life Technology, SelectScreen) [31]. 1 is similar to the 2,4-disubstituted
pyrimidine scaffold that we used to make WZ4002, a previously reported covalent inhibitor of
EGFR (Scheme 1A) [32].

Scheme 1. Structures of WZ4002, 1 and 2 (A) and synthesis of 2 (B). Reagents and conditions: i)
CO , DMSO, RT; ii) TFA, 2-BuOH, 100 °C; iii) Raney nickel, H , MeOH; iv) acryloyl
chloride, sat. NaHCO , THF, 0 °C ~RT.
K
2
3
2
3
To understand the mechanism of action of 1 as a TAK1 inhibitor and further evolve the
compound we solved a crystal structure of the TAK1-TAB1 protein in complex with 1 (PDB
5
J9L). The data collection and refinement statistics are given in Table S1. 1 exhibited a binding
mode to TAK1 similar to that of WZ4002 bound to EGFR (Fig. S1). The pyrrolopyrimidine base
of 1 is seen at the ATP binding site of TAK1 forming hydrogen bonds with hinge region residues
Glu105 and Ala107, and hydrophobic contacts with Val50, Val90 and Phe176. The aniline ring
with piperazinyl tail of 1 is bent 120º from this base and stabilized via hydrophobic contacts with
the α-carbon of Gly110 on one side and nonpolar aliphatic side chain of Val42 on the other. The
reactive acrylamide arm with its connecting phenyl ring is turned 114º extending towards solvent
channel with hydrophobic contacts to Pro160 and Leu163. There is no electron density
connecting Cys174 to the acrylamide moiety of 1 presumably due to an inappropriate trajectory
of the two groups (Fig. S2A). Nevertheless, we noted that the 2-position of the 4-
acrylamidophenyl group of 1 is located only 4.5 Å away from the thiol side chain of Cys174 of
TAK1 (Fig. 1A) suggesting that introducing an acrylamide at this position might provide a better
trajectory for approaching the cysteine residue. Based on this rationale we designed compound 2
with a structure similar to WZ4002 but changed the acrylamide from the meta-position as found
in WZ4002 to the ortho-position. With this change in substitution pattern we anticipated 2 would
react with Cys174, which is located immediately before the DFG-motif (DFG-1 position),
whereas WZ4002 was designed to target the gatekeeper-plus-7 (GK+7) cysteine [33]. The 5-

chlorine atom of the pyrimidine core in 2 was introduced to interact with the methionine GK of
TAK1 in a similar manner to WZ4002 interacting with EGFR T790M GK mutants [32]. We
chose not to include a 2-methoxy in the aniline tail, as in WZ4002, to avoid a potentially
disadvantageous collision with the bulky side chain of Tyr106 of TAK1 (Scheme 1A).
Fig. 1 X-ray co-crystal structure of TAK1-1 (yellow stick-cyan ribbon) (PDB 5J9L) overlaid
with TAK1-2 (purple stick-green ribbon) (PDB 5J8I). (A) TAK1-2 covalently binds to Cys174
and (B) hydrogen bonds (blue dashes) extend between the anilinopyrimidine moiety of 1 or 2
and the hinge region of TAK1.
Synthesis of compound 2 was readily achieved with a high overall yield in four steps
starting with 2,4,5-trichloropyrimidine. The 4-chloride group was substituted with a 2-
nitrophenol under basic conditions, followed by substitution of the 2-chloride with a 4-(4-
methylpiperazin-1-yl) aniline under acidic conditions to give 4. The nitro group of 4 was reduced
using hydrogenation and the resulting aniline was acrylated to create 2 (Scheme 1B). Compared
to 1, 2 showed potent TAK1 inhibition in a fixed time-point assay with an apparent IC50 of 5.1
nM. Mass spectrometry analysis of purified TAK1 incubated with 2 showed exclusive labeling

of Cys174 (Fig S3). To investigate the structural basis for covalent binding we solved the co-
crystal structure of the TAK1-TAB1 kinase domain in complex with 2 at a resolution of 2.4 Å
(
Fig.1B). In this structure (PDB 5J8I), the anilinopyrimidine moiety of 2 makes the expected
bidentate hinge hydrogen bonds with Ala107, and continuous electron density is observed
between the acrylamide warhead and Cys174, indicative of covalent bond formation (Fig. S2,
1
B). The orientation and interaction of the anilinopyrimidine portion of the compound closely
resemble the interactions observed in the TAK1-1 co-crystal structure, however, 1 with its
pyrrole hydrogen donor forms an additional hydrogen bond with Glu105. The chlorine of 2
interacts with the gatekeeper Met104, and also with the reacted covalent warhead. The phenyl
linker of 2 is orientated more co-planar to the pyrimidine core and closer to Cys174 compared
with 1. The reacted ortho-acrylamide interacts with the oxygen linkage and the chlorine on the
pyrimidine core intramolecularly, whereas the unreacted para-acrylamide of 1 protrudes toward
the solvent.
2
.2. Structure-activity relationship (SAR)
Given that MEK, ERK and TAK1 kinases possess analogous cysteine residues at the
DFG-1 position [33], and 5Z7 is also capable of inhibiting MAP kinases [23, 27], we also tested
the biochemical activity of 2 against MEK1 and ERK2. 5Z7 was used for comparison given that
it is widely used in the literature to investigate TAK1-mediated signaling and evaluate the
therapeutic potential of TAK1 inhibition as explained above. 5Z7 is also relevant to the currently
reported compounds because it also operates by a covalent mechanism. Relative to TAK1, 2
exhibited 15-fold lower potency against MEK1 (LanthaScreen, IC50 = 78 nM) whereas 5Z7
showed slightly greater potency against MEK1 (IC50 = 2.9 nM) than TAK1 (IC50 = 5.6 nM).
Against ERK2, 2 exhibited weak activity with an IC50 of 7.0 μM (Life Technologies,

SelectScreen) [34] while 5Z7 was about 10-fold more active (IC50 = 738 nM). To systematically
elucidate the structural requirements for potent inhibition of TAK1, 2 was divided into three
moieties: the covalent linker (R
these moieties was varied sequentially and over 20 analogs synthesized.
Table 1. SAR of R
1
), the pyrimidine core (R
2
), and the aniline tail (R ). Each of
3
1
a
Enzymatic IC50s (nM)
TAK1 MEK1 ERK2
R
1
ID
5
Z7
2
5
5
.6
.1
2.9
78
738
7890
5
6
7
8
9
5
8
0
3
142
2480
16
1090
3.3
277
>
10000 >10000
1630

1
1
0
1
2
5
2500
4810
>10000
1640
a
IC50s against TAK1 and MEK1 were obtained with LanthaScreen binding assays,
IC50s against ERK2 were obtained with Z’-Lyte activity assays; all assays were performed with a
compound incubation time of 60 min.
The first series of analogs focused on modifying the covalent linker (R ) (Table 1).
1
Replacing the linkage between the phenyl group and the pyrimidine core from ether to amine
decreased the potency against TAK1 and reduced the selectivity window between TAK1 and
MEK1 (5). During our SAR studies, AstraZeneca reported a series of selective and potent
ERK1/2 inhibitors, which are also based on the 2,4-disubstituted pyrimidine scaffold, and
possess the same covalent linker as 5 [35]. Interestingly, these ERK1/2 inhibitors were also
reported to inhibit MEK1, but not TAK1, whereas 5 exhibits only weak activity against ERK2.
The thioether linkage decreased the potency even more, but exhibited better selectivity for TAK1
relative to MEK1 (6). 7, utilizing an acrylate instead of an acrylamide as the covalent warhead,
exhibited good potency but less selectivity over MEK1 or ERK2 as compared to 2. Replacing the
acrylamide with a propionamide (8, 9) resulted in inactivity against either TAK1 or MEK1, as
expected. With a more reactive α-chloroacetamide warhead, 10 showed slightly decreased
potency but much better selectivity over MEK1 (100-fold). Addition of para-acetamide in the
phenyl ring results in a dramatic loss of potency (11).

Fig. 2 (A) X-ray co-crystal structures of TAK1-2 (green, PDB 5J8I) overlaid with TAK1-10
(
(
orange, PDB 5E7R) showing interactions between the oxygen linkage for warhead and Asn161
blue dashes). (B) X-ray co-crystal structures of TAK1-5 (cyan, PDB 5J7S) and TAK1-7 (gray,
PDB 5JH6). Nitrogen linkage results in loss of interactions with Asn161.
In addition to TAK1-2, we also determined the co-crystal structures of TAK1-5 (PDB
5
J7S), TAK1-7 (PDB 5JH6) and TAK1-10 (PDB 5E7R). Comparing these structures we
observed that positions of the carbonyl groups varied due to differences in the linkers. In 2 and
0, the carbonyl of the acrylamide interacts with the side chain of Asn-161 along with an
1
interaction with its oxygen linkage (Fig. 2A). In both 7 and 5, the carbonyl group can be seen
protruding away from Asn161 making interactions with the nitrogen linkage (Fig 2B). Despite
the conformational differences between these compounds, the covalent bonds with Cys174 of
TAK1 are still formed due to the flexibility of the warheads and the DFG-motif. Interestingly,
due to a shorter covalent warhead, 10 induces a shift in the position of the DFG-motif, which
causes a shift in the conformation of the TAK1 kinase upon covalent binding. The carbonyl of
the warhead in 10 specifically interacts with the side chain of Asn161 (Fig. 3). Cumulatively, the

biochemical and structural information suggest that the covalent bond is indispensable for
affinity, and that different linkages for the electrophiles result in conformational changes in the
inhibitors, consequently affecting their potency and selectivity.
DFG-motif
Asn161
Fig. 3 Changes in the overall conformation of DFG-motif between TAK1-7 (gray, PDB 5JH6)
and TAK1-10 (orange, PDB 5E7R). The carbonyl warhead group interaction is shown (black
lines).
Table 2. SAR of R
2
a
Enzymatic IC50s (nM)
TAK1 MEK1 ERK2
R
2
ID
2
5
.1
78
7890
9230
1
1
2
3
950
4470
1630
9
2

1
1
1
1
4
5
6
7
7
5
5
7
1300
763
1670
5600
2
3
750 >10000
010
6810
>10000
a
IC50s against TAK1 and MEK1 were obtained with LanthaScreen binding assays, IC50s against
ERK2 were obtained with Z’-Lyte activity assays; all assays were performed with a compound
incubation time of 60 min.
.
After optimization of the covalent linker moiety, SAR of the pyrimidine core (R ) was
2
investigated (Table 2). We found that removing the 5-chlorine resulted in a dramatic decrease in
potency (12), and substituents at 5-position such as methyl, methoxy or cyclopropyl all led to a
greater than 10-fold decrease in potency (13-15). Addition of a 6-amino group, which provides
an additional hydrogen bond donor, did not help the hinge binding as in 1 but instead abrogated
anti-TAK1 activity (16). Cyclization of the substitutions at 4- and 5-positions with a fused ring is
a common strategy to optimize 2,4-disubstituted pyrimidine scaffolds [36]. However, the
pyrrolopyrimidine core led to reduced activity in this case (17). In combination with the SAR of
1
R , these results suggest that interactions between the 5-chlorine and the acrylamide warhead or
the gatekeeper residue facilitate the warhead’s reaching Cys174, whereas the bicyclic core
probably disfavors the warhead from adapting the preferred conformation for a covalent reaction.

Table 3. SAR of R
3
a
Enzymatic IC50s (nM)
R
3
ID
TAK1 MEK1
ERK2
2
5.1
78
7890
b
1
1
8
9
342
b
b
4
3
.4
.2
8.9
11
361
736
2
0
2
1
1
7
7
4
2
.7
.3
.0
.6
.4
9.5
9.8
26
225
3140
242
2
2
2
2
2
3
4
5
8.2
8.5
4620
90.3
a
IC50s against TAK1 and MEK1 were obtained with LanthaScreen binding assays, all assays
b
were performed with a compound incubation time of 60 min; R
1
of 18 is same as R of 7.
1
In addition, we investigated the SAR of the aniline tail (R ) (Table 3). Addition of a
3
methoxy group to the 2-position of the aniline (18) led to loss of potency as expected, likely due
to steric clashes with Tyr106. Substitution with a 4-morpholinylaniline tail (19) maintained

potency against TAK1 but showed less selectivity over MEK1. Switching the substitutions from
- to 3-position slightly increased potency (20, 21). Next we exploited 1-substituted-pyrazol-3-
4
amine tails, which often maintain the potency for covalent inhibitors while reducing interaction
with non-covalent off-targets compared to the aniline tails [36]. 1-methyl-1-H-pyrazol-3-amine
(
20) showed similar potency but decreased selectivity compared to 19. Compounds with further
substitutions at the 1-methyl of the amine tails maintained potency (23-25) with slightly
improved selectivity over MEK1 (23, 25). As the reversible counterpart of 25, 26 showed
dramatically decreased potency, which is consistent with the aniline series (8, 9). The co-crystal
structure of TAK1-22 was determined (PDB 5JK3) and compared to the structures of 2, 5, 7 or
1
0 bound to TAK1. The major difference between binding modes of these analogs involved the
4
-methylpiperazin-1-yl group, which adopts the two lowest energy conformations (boat and
chair), demonstrating flexibility, and is approximately 4.5Å towards the side chain of Asn114,
whereas the methylpyrazole ring of 22 is planar, making hydrophobic contacts with Gly110 and
Val42 from either side. Interactions with Asn114 can be exploited for in future studies as a
means to optimize selectivity since the analogous residues differ in MEK1 (Gln153) or ERK2
(
Lys114) (Fig. 4).
Tyr106
Cys174
Ala107
Asn161
Asn114

Fig. 4 X-ray co-crystal structure of TAK1-22 (blue, PDB 5JK3), overlaid with TAK1-2 (green,
PDB 5J8I) and TAK1-7 (gray, PDB 5JH6) showing key interactions (blue lines). The flexibility
of the piperazinyl moiety adopting different conformations extending towards Asn114 is also
seen.
Although IC50 values are time-dependent for covalent compounds, for practical reasons
IC50 values were used to rank order compounds for SAR. We considered these values to be a
reasonable first estimate of potency and reactivity because reaction times were strictly
maintained at 60 min across assays. However, to further characterize key compounds and as an
additional demonstration of their covalent nature we performed additional time-dependent
experiments. We tested 2, 5 and 25 at a shorter incubation time of 10 min and observed
decreased potency with short incubation time, with IC50s of 360, 125 and 7.6 nM, respectively,
consistent with covalent binding. For one of our most promising lead compounds, 5, we
-
6
-1 -1
determined a Kinact/K
i
value of 6.87 x 10 nM s (Fig. S4) which is in the range of other
clinically used covalent inhibitors such as afatinib or neratinib [37].
.3. Kinome selectivity
2
Approximately 50 human kinases possess a cysteine immediately preceding the DFG-
motif [33]. We therefore surveyed the selectivity of inhibitor 5 across a panel of 468 kinases and
mutants using an in vitro ATP-site competition binding assay (KINOMEscan [38]) at a
concentration of 1 μM (Fig S5, Table S2). This analysis revealed that 5 binds to TAK1 and ten
other kinases with a cysteine at the DFG-1 position (Fig S5), and possesses a selectivity score,
defined as the percentage of kinases with scores less than 1 (S(1)) [39], of 0.10. In contrast, its
non-covalent counterpart, 9, possesses a same S(1) of 0.10 as 5 but hits no kinases with a

cysteine at the DFG-1 position. Instead 9 shows strong inhibition against new targets such as
MAP3K2/3, WEE1/2 and PYK2. We also profiled 7, 23 and 25 for their kinome selectivity at 1
μM. 7 was a more promiscuous inhibitor with an S(1) of 0.19. However, 23 and 25 showed
significantly improved selectivity with S(5) of 0.1 and 0.03 respectively, although, TAK1 was
not ranked among the strongest hits (Fig S5, Table S4-6).
In these data we also noted that 5Z7 did not display strong binding to TAK1 at a
concentration of 1 μM, and only exhibited a moderate inhibition score against TAK1 at 10 μM
(
Table S7). These effects are likely related to the nature of the various binding assays.
KINOMEscan utilizes a common “tracer” with variable binding affinity across kinases which
can influence relative inhibition scores [38]. On the other hand the SelectScreen assays is
optimized for each kinase. In this case SelectScreen assays were performed in a dose-response
format which provides more reliable measurements than single-point KINOMEscan results.
Taken together, the fact that 5 and 9 show distinct inhibitory profiles despite having nearly
identical chemical structures, except in the electrophile portion, strongly supports that covalent
binding is a determining factor for the kinome selectivity of these inhibitors. Along these lines 5,
2
3, 25 and 5Z7 preferentially bind to kinases that possess a cysteine at the DFG-1 position as
compared to 7 suggesting the 2,4-substituted pyrimidine template may provide a general scaffold
for further optimization of kinase selectivity (Table S2-7).
2
.4. Mouse liver microsomal stability
Table 4. Mouse liver microsomal stability
MLM MLM MLM
T
min)
1/2
ID
T
(min)
1/2
ID
T
(min)
1/2
ID
(
2
5
6.1
14 15.3 21
<1
8.1
10.8 15 12.8 22

7
5.7
3
8.8
17 10.5 23
4.2
4
6.9
1
0
3
19
20
3.8
23
24
25
1
The in vitro metabolic stability of some analogs was measured using mouse liver microsomes
(
MLM) to calculate half-lives (T1/2) [40]. Based on the data in Table 4, for the current 2,4-
disubstituted pyrimidine scaffold it can be concluded that amine linkage is more stable than the
ether linkage; the α-chloroacetamide and the acrylate warheads are more labile than the
acrylamide; electron-donor groups at the 5-position of the pyrimidine core make the inhibitors
more stable; and modifications of the aniline tail moiety dramatically decrease stability with
half-lives ranging from less than 1 min to 23 min.
3
. Conclusion
In summary, we report a structure-guided approach for development of TAK1 inhibitors
with single or double-digit nanomolar enzymatic IC50 values based on a common 2,4-
T790M
disubstituted pyrimidine scaffold as our previously described EGFR
covalent inhibitors [32].
Several of our inhibitors exhibited greater than 10-fold selectivity for TAK1 over MEK1 with
compound 10 exhibiting around 100-fold selectivity. Most of the inhibitors we described were
weakly active or inactive against ERK2, despite the fact that potent and selective ERK1/2
inhibitors have been developed and reported recently based on a very similar scaffold [35]. The
five newly determined co-crystal structures of TAK1 bound to newly synthesized compounds
guided design choices for covalent targeting of the DFG-1 cysteine in TAK1 and for improving
potency and selectivity. The inhibitors described here provide a synthetically straight-forward
pharmacophore and a solid structural basis for future optimization of selective covalent TAK1

inhibitors. In the accompanying communication we discuss the application of these inhibitors to
TAK1-centered polypharmacology.
4
4
. Experimental
.1. Chemistry
Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and
1
were used without further purification. H NMR spectra were recorded on 600 or 500 MHz
(
Varian AS600 or Bruker A500), and chemical shifts are reported in parts per million (ppm, )
downfield from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin
multiplicities are described as s (singlet), br (broad singlet), d (doublet), t (triplet), q (quartet),
and m (multiplet). Mass spectra were obtained on a Waters Micromass ZQ instrument.
Preparative HPLC was performed on a Waters Sunfire C18 column (19 x 50 mm, 5µM) using a
gradient of 15-95% methanol in water containing 0.05% trifluoroacetic acid (TFA) over 22 min
(
28 min run time) at a flow rate of 20 mL/min. Purities of assayed compounds were in all cases
greater than 95%, as determined by reverse-phase HPLC analysis.
,5-dichloro-4-(2-nitrophenoxy)pyrimidine (3). 2-Nitrophenol (840 mg, 6.0 mmol) and
2
potassium carbonate (800 mg, 6.0 mmol) were combined in dimethyl sulfoxide (DMSO) (10 mL)
and stirred for 15 min, then 2,4,5-trichloropyrimidine (560 µL, 5.0 mmol) was added and the
mixture was stirred overnight. The mixture was then diluted with ethyl acetate and washed with
water and brine, dried over Na
2
SO , filtered and concentrated. The crude product was purified by
4
column chromatography (hexane:ethyl acetate = 3:1) to yield 1.2 g (70%) of 3 as a white solid.
+
MS (ESI) m/z 286 (M+H) .
5
-chloro-N-(4-(4-methylpiperazin-1-yl)phenyl)-4-(2-nitrophenoxy)pyrimidin-2-amine (4).
To 3 (570 mg, 2.0 mmol) and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline (390 mg, 2.0 mmol)

in sec-butanol (4 mL) was added trifluoroacetic acid (154 µL, 2.0 mmol) and the mixture was
stirred overnight at 75 C. The mixture was then concentrated, neutralized with ammonia in
methanol and purified by column chromatography (dichloromethane:methanol = 10:1) to yield
+
2
65 mg (60%) of 4 as a pale-yellow solid. MS (ESI) m/z 441 (M+H) .
N-(2-((5-chloro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-
yl)oxy)phenyl)acrylamide (2). To 4 (88 mg, 0.2 mmol) in methanol (10 mL) was added 1 mL
Raney nickel suspension in methanol. The reaction mixture was stirred for 3 h under 1 atm of
hydrogen. The mixture was then filtered with celite, and the filtrate was concentrated and dried
under vacuum to give a crude product as a white solid. To the obtained white solid in
tetrahydrofuran (3 mL) was added saturated NaHCO solution (3 mL), the stirred mixture was
3
then cooled to 0 C, and acryloyl chloride was added (25 µL, 0.3 mmol) dropwise. The reaction
mixture was stirred at 0 C for 10 min, another batch of acryloyl chloride was added (8 µL, 0.1
mmol) dropwise. After another 5 min the mixture was allowed to recover to room temperature
(
RT), and diluted with ethyl acetate and washed with water and brine, dried over Na
2
SO , filtered
4
and concentrated. The crude product was then purified by reverse phase HPLC to give 58 mg (63%
1
for 2 steps) of 2 as a white solid. H NMR (600 MHz, DMSO-d
6
) δ 9.68 (br, 1H), 9.58 (s, 1H),
9
6
1
.52 (br, 1H), 8.40 (s, 1H), 8.05 (m, 1H), 7.32 (dd, J = 8.4, 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H),
.72 (m, 2H), 6.55 (dd, J = 16.8, 10.8 Hz, 1H), 6.20 (d, J = 16.8 Hz, 1H), 5.71 (d, J = 10.8 Hz,
H), 3.66 (m, 2H), 3.50 (m, 2H), 3.14 (m, 2H), 2.85 (s, 3H), 2.83 (m, 2H). MS (ESI) m/z 465
+
(
M+H) .
2
,5-dichloro-N-(2-nitrophenyl)pyrimidin-4-amine (28). To 2,4,5-trichloropyrimidine (560 µL
mg, 5.0 mmol) and 2-nitroaniline (700 mg, 5.0 mmol) in sec-butanol (25 mL) was added
trifluoroacetic acid (383 µL, 5.0 mmol) and the mixture was stirred overnight at 60 C. The

mixture was then concentrated, neutralized with ammonia in methanol and purified by column
chromatography (hexane:ethyl acetate = 2:1) to yield 640 mg (45%) of 28 as a yellow solid. MS
+
(
ESI) m/z 285 (M+H) .
2
4
5
-chloro-N -(4-(4-methylpiperazin-1-yl)phenyl)-N -(2-nitrophenyl)pyrimidine-2,4-diamine
(
29). To 28 (570 mg, 2.0 mmol) and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline (390 mg, 2.0
mmol) in sec-butanol (4 mL) was added trifluoroacetic acid (154 µL, 2.0 mmol) and the mixture
was stirred overnight at 85 C. The mixture was then concentrated, neutralized with ammonia in
methanol and purified by column chromatography (dichloromethane:methanol = 10:1) to yield
+
6
80 mg (76%) of 29 as a pale-yellow solid. MS (ESI) m/z 440 (M+H) .
N-(2-((5-chloro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-
yl)amino)phenyl)acrylamide (5). To 29 (88 mg, 0.2 mmol) in methanol (10 mL) was added 1
mL Raney nickel suspension in methanol. The reaction mixture was stirred for 3 h under 1 atm
of hydrogen. The mixture was then filtered with celite, and the filtrate was concentrated and
dried under vacuum to give a crude product as a white solid. To the obtained white solid in
tetrahydrofuran (3 mL) was added saturated NaHCO solution (3 mL), the stirred mixture was
3
then cooled to 0 C, and acryloyl chloride was added (25 µL, 0.3 mmol) dropwise. The reaction
mixture was stirred at 0 C for 10 min, another batch of acryloyl chloride was added (8 µL, 0.1
mmol) dropwise. After another 5 min the mixture was allowed to recover to room temperature
(
RT), and diluted with ethyl acetate and washed with water and brine, dried over Na
2
SO , filtered
4
and concentrated. The crude product was then purified by reverse phase HPLC to give 55 mg (60%
1
for 2 steps) of 5 as a white solid. H NMR (600 MHz, DMSO-d
6
) δ 10.16 (s, 1H), 9.67 (br, 1H),
9
.20 (br, 1H), 8.56 (s, 1H), 8.09 (s, 1H), 7.74 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 6.6 Hz, 1H), 7.40
(
d, J = 9.0 Hz, 2H), 7.32 (dd, J = 7.2, 7.2 Hz, 1H), 7.24 (dd, J = 7.8, 7.2 Hz, 1H), 6.78 (d, J = 9.6

Hz, 1H), 6.50 (dd, J = 16.8, 10.8 Hz, 1H), 6.30 (d, J = 17.4 Hz, 1H), 5.79 (d, J = 10.8 Hz, 1H),
+
3
.66 (m, 2H), 3.49 (m, 2H), 3.14 (m, 2H), 2.84 (s, 3H), 2.83 (m, 2H). MS (ESI) m/z 464 (M+H) .
-((2,5-dichloropyrimidin-4-yl)amino)phenol (30). To 2-aminophenol (550 mg, 5.0 mmol) in
2
dioxane (10 mL) was added 2,4,5-trichloropyrimidine (560 µL mg, 5.0 mmol), the mixture was
stirred overnight at RT. The mixture was then diluted with ethyl acetate and washed with water
and brine, dried over Na
2
SO
4
, filtered and concentrated to give 1.02 g (80%) of the crude product
+
3
2
0 as a white solid, which was directly used in the next step. MS (ESI) m/z 256 (M+H) .
-((5-chloro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenol (31).
To 30 (510 mg, 2.0 mmol) and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline (575 mg, 3.0 mmol)
in sec-butanol (5 mL) was added trifluoroacetic acid (230 µL, 3.0 mmol) and the mixture was
stirred overnight at 100 C. The mixture was then concentrated, neutralized with ammonia in
methanol and purified by column chromatography (dichloromethane:methanol = 10:1) to yield
+
6
80 mg (76%) of 31 as a white solid. MS (ESI) m/z 411 (M+H) .
2
-((5-chloro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl
acrylate (7). To 31 (82 mg, 0.2 mmol) in dimethylformamide (2 mL) was added
diisopropylethylamine (53 µL, 0.3 mmol), the stirred mixture was then cooled to -60 C, and
acryloyl chloride (17.8 µL, 0.22 mmol) was added dropwise. The reaction mixture was stirred at
-
60 C for 10 min, allowed to recover to RT (room temperature) gradually in 30 min, and
purified by reverse phase HPLC to give 64 mg (TFA salt, 56% for 2 steps) of 7 as a white solid.
1
H NMR (600 MHz, DMSO-d ) δ 8.96 (br, 1H), 8.36 (s, 1H), 8.04 (s, 1H), 7.72 (br, 1H), 7.34 (d,
6
J = 9.0 Hz, 2H), 7.30 (m, 3H), 6.70 (d, J = 9.0 Hz, 2H), 6.37 (d, J = 16.8, 1H), 6.26 (dd, J = 17.4,
1
0.2 Hz, 1H), 5.79 (d, J = 10.8 Hz, 1H), 3.02 (m, 4H), 2.54 (m, 4H), 2.28 (br, 3H), 2.84 (s, 3H).
+
MS (ESI) m/z 465 (M+H) .

4
.2. X-Ray crystallography procedures
TAK1–TAB1 fusion protein (TAK1 kinase domain residues 31–303 fused with C-terminal
TAB1 domain residues 468–497) was expressed in insect cells, purified and crystallized as
reported previously [30]. Crystals grew within 2 days by vapor diffusion in hanging drop at 20ºC
by mixing protein with equal volumes of reservoir solution consisting of 0.65–0.75 M sodium
citrate, 0.2 M NaCl, 0.1 M Tris, pH 7.0, and 5 mM adenosine. Crystals containing adenosine
were then back-soaked with excess of inhibitors at 250 µM - 500 µM dissolved in reservoir
solution for 6-8h. The crystals were then flash frozen with 20% ethyleneglycol as cryoprotectant
for data collection. Diffraction data were collected at Argonne Advanced Photon Source
(
beamline 19-D) and processed with HKL2000 and HKL3000 [41]. The structure was
determined by molecular replacement using Phaser [42] with reported TAK1 structure (PDB
code: 2YIY or 4O91) as search model. Coot was used for model building [43] and refinement
was carried out using both Phenix, version 1.10-2155 [44] and Refmac, version 5.8.0049. [45-
4
1
7], PyMol (The PyMOL Molecular Graphics System, version 1.6.0.0) and Meastro (version
.5.014) from Schrödinger, LLC. For demonstration of key interactions, LIGPLOT software was
used [48].
.3. Enzymatic assays
4
Enzymatic inhibition of MEK2 and ERK1 was tested in Z’-Lyte assays with ATP concentrations
near the Km for each kinases. Compound activity against TAK1 was tested in LanthaScreen
binding assays. All protocols are available from Life Technologies [49]. The MLM assays were
previously reported and are commercially available from Scripps Florida [40].
Acknowledgements

NSG and PKS were supported by LINCS Grant U54-HL127365. KDW was supported by CPRIT
R1207 and CPRIT RP140233. LT and NSG were supported by NIH CA 154303-05 and the
Pediatric Low Grade Astrocytoma (PLGA) foundation. Results shown in this article are derived
from work performed at Argonne National Laboratory, Structural Biology Center at the
Advanced Photon Source. Argonne is operated by U Chicago Argonne, LLC, for the U.S.
Department of Energy, Office of Biological and Environmental Research under contract
DEAC02-06CH11357.
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diagrams of protein-ligand interactions, Protein Eng, 8 (1995) 127-134.
49] Z'-Lyte assays: http://www.lifetechnologies.com/content/dam/LifeTech/migration/files/drug-
[
discovery/pdfs.par.60256.file.dat/20130430%20ssbk%20customer%20protocol%20and%20assay%20con
ditions.pdf ; TTK assay:
http://tools.lifetechnologies.com/content/sfs/manuals/TTK_LanthaScreen_Binding.pdf

Graphical abstract