Discovery of 6-aryl-azabenzimidaoles that inhibit the TBK1/IKK-ε kinases

Article abstract of DOI:10.1016/j.bmcl.2013.12.123

The discovery and optimization of a series of 6-aryl-azabenzimidazole inhibitors of TBK1 and IKK-ε is described. Various internal azabenzimidazole leads and reported TBK1/IKK-ε inhibitors were docked into a TBK1 homology model. The resulting overlays insp

Full text of DOI:10.1016/j.bmcl.2013.12.123

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1138–1143
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 6-aryl-azabenzimidaoles that inhibit the TBK1/IKK-
kinases
e
a
b
c
a
Jeffrey W. Johannes a, , Claudio Chuaqui , Scott Cowen , Erik Devereaux , Lakshmaiah Gingipalli ,
⇑
Audrey Molina d, Tao Wang e, David Whitston c, Xiaoyun Wu f, Hai-Jun Zhang g, Michael Zinda c
a Department of Cancer Chemistry, Oncology IMED, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, United States
b Chemistry Innovation Center, Discovery Sciences, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, United States
c Department of Cancer Bioscience, Oncology IMED, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, United States
d Acetylon Pharmaceuticals, Inc., Seaport Center, 70 Fargo Street, Suite 205, Boston, MA 02210, United States
e Biogen Idec, 12 Cambridge Center, Cambridge, MA 02142, United States
f Department of Infection Chemistry, Infection IMED, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, United States
g Biortus Biosciences, Building A5, 6 Dongsheng West Road, Jiangyin, Jiangsu Province 214437, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and optimization of a series of 6-aryl-azabenzimidazole inhibitors of TBK1 and IKK-e is
Received 28 October 2013
Revised 29 December 2013
Accepted 31 December 2013
Available online 13 January 2014
described. Various internal azabenzimidazole leads and reported TBK1/IKK-
a TBK1 homology model. The resulting overlays inspired a focused screen of 6-substituted azabenzimi-
dazoles against TBK1/IKK- . This screen resulted in initial hit compound 3. The TBK1/IKK- enzyme
e inhibitors were docked into
e
e
and cell potency of this compound was further improved using structure guided drug design. Systematic
exploration of the C6 aryl group led to compound 19, a potent inhibitor of TBK1 with selectivity against
cell cycle kinases CDK2 and Aurora B. Further elaboration and optimization gave compound 25, a single
Keywords:
TBK1
digit nM inhibitor of TBK1. These compounds may serve as in vitro probes to evaluate TBK1/IKK-
oncology target.
e as an
IKK-
e
Azabenzimidazole
Kinase inhibitor
Ó 2014 Elsevier Ltd. All rights reserved.
TANK-binding kinase 1 (TBK1)1 and inhibitor of nuclear factor-
HEK293-TLR3 NF-
HEK293 cells containing an ISRE-firefly luciferase reporter con-
struct are stimulated with poly I:C (a synthetic analog of double
j
B ISRE-firefly luciferase cell line. In this assay,
j
B kinase subunit epsilon (IKK-
serine/threonine kinases which are involved in a number of cell
signaling pathways.3 TBK1 and IKK-
, together referred to as the
IKK-related kinases, are biologically distinct4 from the cannonical
B kinases IKK- , IKK-b, and IKK- that are involved in the regu-
lation of NF-
B;5,6 however, some crosstalk has been demonstrated
between the members of the IKK family.7 The biology of TBK1 and
IKK- is complex and multifaceted. They have been shown to
e
)2 are a pair of highly homologous
e
stranded RNA). Treatment with a TBK1/IKK-e inhibitor inhibits
the TLR3 dependent signaling cascade by reducing levels of the
phospho-IRF3 homodimer bound to the ISRE promoter.12 In order
to have increased confidence in the phenotypic outcomes of cell-
based target validation work, additional enzyme selectivity (at
KM ATP) against the cell-cycle disrupting kinases CDK2 and Aurora
B (AurB) was sought.13 AurB and CDK2 were chosen because they
were readily available as part of a small AZ internal panel of ki-
nases that were routinely run and would give us an initial read
on kinase selectivity. We focused on these two kinases in particular
since small molecule inhibition of these kinases has been linked to
effects on cell proliferation and apoptosis in certain cellular
contexts.14,15 In addition, a few select compounds (compounds
18–20, vide infra) were submitted to a diverse panel of 125 kinases
Ij
a
c
j
e
interact with numerous molecules and phosphorylate a number
of targets,8 leading to a role in innate immunity, inflammation,
autophagy, proliferation, and insulin signaling.9 In recent years,
several publications have emerged linking TBK1 and IKK-e to onco-
genesis, sparking interest in the IKK-related kinases as a cancer
target.10
Following the initial publications linking TBK1/IKK-e to oncoge-
nicity, we initiated a drug discovery program to develop appropri-
ate biological tools for internal target validation work. The goal
was to identify in vitro probe compounds that were dual TBK1/
to measure % kinase activity remaining at 1
compound 20 showed less than 50% activity remaining for only
15 kinases (not including TBK1 or IKK-
Supplementary data for details).
lM. As an example,
IKK-
e
enzyme inhibitors.11 In addition, we sought compounds that
e) out of 125 (see the
could also inhibit a luciferase reporter signal in a stably transfected
Recently, a few research groups have reported TBK1 inhibi-
tors,16 and we reported the discovery of the azabenzimidazole
class of TBK1 inhibitors.17 This paper describes further work to
⇑
Corresponding author. Tel.: +1 781 839 4241.
E-mail address: Jeffrey.Johannes@astrazeneca.com (J.W. Johannes).
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.123

J. W. Johannes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1138–1143
1139
O
S
O
R
O
S
O
HN
N
O
N
O
N
HN
N
N
Br
O
1
O
HO
N
H
7
N
2
2
R = cyclobutyl
6
O
4
R = cyclopentyl
N
H
N
N
3
Figure 1. TBK1/IKK-e inhibitors from internal work and literature references.
Figure 3. Overlay of reported GSK IKK-
e
inhibitor 1 (pink) and 6-aryl-azabenzim-
idazole hit 3 (green) docked into a TBK1 homology model.
differences between the inherent conformational and interaction
preferences of the differing gatekeeper residues. To follow up on
this idea, we selected compounds from our collection of azabenz-
imidazoles which featured substituents at the 6-position of the
azabenzimidazole ring. An initial focused screen of 6-substituted
azabenzimidazoles uncovered compound 3 (Fig. 1). The enzyme
and cell data for these four leads are presented in Table 1.
In our hands, GSK compound 1 had a TBK1 enzyme IC50 of
0.093
lM, which also translated into good cell potency
(0.072
lM). Moreover, this compound exhibited excellent selectiv-
ity against cell-cycle kinases CDK2 and AurB. In contrast, enzyme
and cell potent 6-Br-azabenzimidazoles 2 and 4 had poor selectiv-
ity against AurB. The newly identified hit compound 3 was only a
0.859 lM inhibitor of TBK1 but demonstrated proof-of-concept
that a large aromatic group could be tolerated at the 6-position
Figure 2. Overlay of reported GSK IKK-e inhibitor 1 (pink) and azabenzimidazole 2
of the azabenzimidazole scaffold.
(green) docked into a TBK1 homology model.
Docking of hit compound 3 into a homology model of TBK1
along with 1 indicated that the phenyl group at the 6-position of
the azabenzimidazole occupied roughly the same position as the
thiophene of 1 (Fig. 3). Additionally, comparison back to azabenz-
imidazoles 4 and 2 suggested that 3 could be further elaborated at
the 7-position, possibly building in the potent interactions that the
propylamino carboxamide group provides. However, we were con-
scious of the possibility that there might be a steric clash if substit-
uents existed at the 6 and 7 positions simultaneously.
Initially, compounds 5 and 6, bearing aryl groups at the 6-posi-
tion and N-Me at the 7-position of the azabenzimidazole were
made to quickly evaluate the significance of this concern (Table 2).
A slight drop in potency was observed; however, the thiophene
compound inspired by the GSK lead 1 was more potent than the
phenyl. These observations focused our efforts on placing a thio-
phene at the 6-postion and leaving the 7-position unsubstituted.
Three thiophene isomers were prepared which sought to maintain
optimize the azabenzimidazole series for improved kinase selectiv-
ity and cell potency in the TBK1/IKK-
At the time our work initiated, an initial survey of the literature
revealed an interesting series of IKK- inhibitors reported by
GSK.18 Compound 1 (Fig. 1) was reported as a potent IKK-
inhib-
itor with moderate selectivity against a small panel of 10 kinases,
including other I B kinases. Docking one of our previously re-
e reporter assay.
e
e
j
ported azabenzimidazoles 2 (Fig. 1) along with GSK compound 1
into a homology model of TBK119 suggested that the bromine atom
at the 6-position of 2 was positioned in the same pocket as the thi-
ophene of 1 (Fig. 2). This led to the hypothesis that aryl groups at
the 6-position of the azabenzimidazole series may be tolerated by
the methionine gatekeeper residue of TBK1 and IKK-e. Since Aurora
B and CDK2 have leucine and phenylalanine gatekeepers respec-
tively, we hoped to gain selectivity over these kinases by exploiting
Table 1
TBK1, IKK-e, CDK2, AurB, and TBK1 cell reporter data for the initial leads
Compound
TBK1 enzyme IC50
(lM)
IKK-
e
enzyme IC50
(lM)
CDK2 enzyme IC50
(l
M)
Aurora B enzyme IC50
(l
M)
HEK293 TBK1 reporter IC50 (lM)
1
2
3
4
0.093
0.004
0.859
0.010
0.469
0.021
>30
>4.91
>5.32
0.038
0.072
0.006
0.076
2.79
0.093
0.008

1140
J. W. Johannes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1138–1143
Table 2
Discovery of a 6-thiophenyl azabenzimidazole inhibitor of TBK1/IKK-
e
R2
N
R1
N
O
N
H
Compound
R1
R2
TBK1 enzyme IC50
1.80
(l
M)
IKK-
e
enzyme IC50
(l
M)
HEK293 TBK1 reporter IC50 (lM)
O
5
NHCH3
HO
HO
O
O
6
7
NHCH3
1.04
3.30
S
S
HO
HO
H
0.256
0.867
0.395
>30
>30
O
S
8
H
0.129
O
HO
HO
9
H
H
1.05
11.6
>30
>30
O
O
10
0.049
0.114
S
the position of the carboxylic acid while moving the sulfur atom to
different positions around the ring. Two of these isomers, 7 and 8,
showed an improvement over hit 3 in terms of TBK1 enzyme po-
tency. Compound 10 showed a dramatic improvement, bringing
TBK1 enzyme potency, but started to show single digit micromolar
activity in our cell assay. Carboxamide 14 maintained a similar
TBK1 enzyme potency to the acid, but impressively showed a huge
improvement in cell potency, resulting in a compound that was
268 nM in our HEK293 TBK1 reporter assay. A spot check of the
CDK2 and AurB enzyme IC50’s for this compound showed an
improvement in selectivity for AurB but similar selectivity for
CDK2 when compared to 6-Br azabenzimidazole 4 (vide supra).
Driven by this result, we explored substituents on the carbox-
amide nitrogen in an effort to probe interactions with the P-loop
and further enhance the kinase selectivity of the current lead. This
idea lent itself well to a parallel synthesis approach via amide bond
coupling between a thiophene carboxylic acid building block and a
diverse set of amines. A library was made and screened against
the TBK1 IC50 down to 0.049 lM. Despite strong enzyme potency,
this compound was inactive in the HEK293 TBK1 reporter assay.
In fact, all three thiophene carboxylic acids were inactive in the
cell. Interestingly, the furan analog 9 was ꢀ10-fold less active at
the enzyme level than its thiophene counterpart.
Based on these data, we chose to focus on optimizing the cell
potency of 10. Our hypothesis was that the lack of any cell activity
was due to the carboxylic acid group on the thiophene. Docking re-
sults (not shown) indicated that the carboxylic acid may interact
with the conserved lysine residue in the kinase. Our design goal
was to replace this carboxylic acid with a non-ionizable group that
possessed lone pairs which could still interact with the conserved
lysine. Table 3 shows the results when the acid of 10 is replaced
with methyl ketone, cyano, methyl carboxamide, or carboxamide.
Methyl ketone analog 12 and methyl carboxamide 13 had reduced
TBK1, IKK-e, CDK2, and AurB, and the resulting TBK1 active
compounds were profiled in the HEK293 TBK1 cell reporter assay.
A selected set of compounds with potent TBK1 enzyme IC50’s, good
kinase selectivity versus CDK2 and AurB, and cell activity are
presented in Table 4. These compounds are all secondary amides,
Table 3
SAR for a putative lysine interaction and cell activity with improved AurB selectivity
R1
S
N
O
N
H
N
Compound R1
TBK1 enzyme IC50
M)
IKK-
e
enzyme IC50
CDK2 enzyme IC50
M)
Aurora B enzyme IC50
M)
HEK293 TBK1 reporter IC50
M)
(
l
(lM)
(
l
(l
(l
11
12
13
14
CN
C(O)CH3
C(O)NHCH3 0.203
C(O)NH2 0.030
>30
0.650
>30
6.75
2.56
>30
12.1
2.30
1.78
0.851
0.268
0.205
0.984

J. W. Johannes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1138–1143
1141
Table 4
Selected thiophene amides with improved cell potency and AurB selectivity
O
R1HN
S
N
O
N
H
N
Compound R1
TBK1 enzyme IC50
M)
IKK-
e
enzyme IC50
CDK2 enzyme IC50
M)
Aurora B enzyme IC50
M)
HEK293 TBK1 reporter IC50
M)
(
l
(l
M)
(
l
(
l
(l
O
15
16
0.098
0.100
0.412
0.330
>30
>30
1.38
1.68
0.170
0.136
HO
17
18
19
20
0.219
0.143
0.077
0.084
1.09
>30
>30
>30
>30
2.82
0.089
0.078
0.037
0.030
1.75
18.8
5.92
1.44
0.273
0.497
Table 5
Incorporating substitution at the 6 position of the azabenzimidazole and impact on kinase selectivity while maintaining cell potency
HN
O
A
N
R1
N
O
N
H
Number R1
A
TBK1 enzyme IC50
M)
IKK-
e
enzyme IC50
CDK2 enzyme IC50
Aurora B enzyme IC50
M)
HEK293 TBK1 reporter IC50
(
l
(l
M)
(
lM)
(l
(
lM)
4
21
Br
Br
NH 0.010
0.076
>30
2.79
0.093
0.156
0.008
O
0.759
0.460
>30
>30
O
HO
22
23
O
1.10
1.3
>3
S
S
NH 0.046
NH 0.036
0.448
0.158
>30
0.889
0.165
H2N
HO
O
S
24
25
13.6
0.299
0.284
2.17
N
O
H2N
NH 0.009
NH 0.388
0.042
3.23
1.45
0.007
N
S
NH
O
26
>30
2.47
0.128
N
S
and most of them feature a small branched alkyl group. In general,
the compounds have TBK1 enzyme IC50’s of 100 nM and all are
sponding 7-position of the 6-aryl-azabenzimidazole series. Based
on the drop in TBK1 enzyme potency between hit compound 3
and 6-7-disubstituted compound 5 (vide supra), we surmised that
there was a steric interaction between the phenyl group ortho-CH
and the NH at the 7-position causing the phenyl and/or the N-alkyl
groups to rotate out of the plane of the azabenzimidazole ring
which would preclude a favorable binding conformation (Fig. 4).
We posited two solutions to this issue. One idea involved replacing
the C7 NH with oxygen, which presents a lone pair towards the thi-
>30
and 19
l
M against CDK2, with AurB selectivity varying between 1
M. Compound 18 is a selective inhibitor of TBK1 and is
l
78 nM in our cell assay. Analogs 19 and 20 are even more potent
in the cell assay while maintaining some selectivity against AurB.
Having exhaustively explored the 6-position, we set out to build
in additional cell potency by re-introducing the propylamino-car-
boxamide group from the 7-position of 4 back onto the corre-

1142
J. W. Johannes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1138–1143
Br
NH2
NH2
ophene CH, and the other changed the C6 heterocycle from a thio-
phene to a thiazole, which switches an aryl CH for an aryl nitrogen
that can accept a hydrogen bond from the neighboring C7 NH
group.
Br
N
HO
O
a
c
b
+
O
O
N
H
N
N
I-1
I-2
The key feature of compound 4 was its exquisite TBK1 enzyme
potency and single-digit nM activity in the TBK1 reporter assay.
However, the AurB selectivity of this compound was poor, less than
10-fold at the enzyme level versus TBK1. Armed with new selectiv-
ity enhancing 6-aryl groups, we set out to synthesize compounds
bearing functionality at both the 6 and 7-position of the azabenz-
imidazole system (Table 5). To test the first hypothesis that intro-
duction of an ether linked propyl-carboxamide group would pair
well with larger groups, we made the matched pair 4 and 21,
which differed only by the atom linker at C7 of the azabenzimidaz-
ole. Unfortunately, this ether linked analog showed a dramatic loss
in TBK1 enzyme potency relative to the nitrogen linker. As a final
test of this hypothesis, we made 22, which placed the thiophene
carboxamide at the C6 position alongside the C7 ether linked pro-
pyl-carboxamide. Compound 22 was slightly more potent than the
6-Br analog 21, but when compared to the C7-unsubstituted com-
pound 10 (vide supra), 22 was 10ꢁ less active upon introduction of
the C7 ether linked appendage.
We next focused our efforts on our second hypothesis, pairing a
C6 aryl group with the C7-nitrogen linkage via the introduction of a
thiazole group. This specific thiazole isomer was chosen because it
provided an internal H-bond acceptor to the C7 NH, maintained a
heterocyclic sulfur atom, and was synthetically accessible. Inter-
mediate thiocarboxamide 23 showed initial promise; it had a
TBK1 enzyme IC50 of 46 nM and improved selectivity versus AurB
compared to C6-bromo analog 4, but reduced cell potency. Further
elaboration of this thiocarboxamide resulted in thiazole 24, a 34
nM TBK1 enzyme inhibitor with poor cell potency, presumably
again due to the acid moiety. As expected, the carboxamide analog
25 gave a compound that was 7 nM in the TBK1 cell reporter assay.
NH
O
S
O
HO
S
N
N
O
O
N
H
N
H
N
N
10
19
Scheme 1. Synthesis of compounds 10 and 19. Reagents and conditions: (a) POCl3
(2 equiv), CH3CN, 150 °C, mw, 1 h, 59%; (b) 4-boronothiophene-2-carboxylic acid
(1.1 equiv), Cs2CO3 (2.5 equiv), sodium 3,30,300-phosphinetriyltribenzenesulfonate
tetrahydrate (0.2 equiv), Pd(OAc)2 (0.05 equiv), H2O:CH3CN (2:1), 150 °C, mw,
5 min; (c) HATU (1 equiv), DIEA (2 equiv), DMA, 2-methylpropan-1-amine
(1 equiv), 55% (2 steps).
The synthesis of a series of 6,7-substituted azabenzimidazoles is
outlined in Scheme 2. Again, POCl3 was used to condense diamino
pyridine I-3 and p-anisic acid to give the bicyclic heterocycle 6-Br-
7-Cl-azabenzimidazole I-4. Heating an excess of 1,3-diaminopro-
pane with the 7-chloro-azabenzimidazole I-4 in butanol gave pri-
mary amine I-5 in good yield. Amide bond formation to give 4
was achieved using cyclopentanecarbonyl chloride under Schot-
ten–Baumann conditions. Treatment of the bromide 4 with several
equivalents of copper cyanide gave nitrile I-6 after workup with
ammonia. Exposure of the nitrile to successive amounts of ammo-
nium sulfide over 48 h resulted in thioamide 23. The thiazole ring
was constructed via a Hantzsch thiazole synthesis to give carbox-
ylic acid 24. Finally, HATU coupling with either ammonium
hydroxide or isobutylamine gave 25 and 26 respectively.
The compounds described herein may be useful in vitro probes
25 was potent inhibitor of TBK1 and IKK-
e
enzymes with a slight
for further validation work on TBK1 and IKK-
We have used structure based design to morph the thiophene
group from a reported IKK- inhibitor onto the 6-position of an
azabenzimidazole scaffold. This led to the discovery of 6-aryl-aza-
benzimidazoles 19 and 20, which have an improved kinase selec-
tivity profile versus CDK2 and AurB over C6-bromo compound 4
e as oncology targets.
improvement in selectivity versus the C6 Br compound 4. Interest-
ingly, further elaboration of the carboxamide with the isobutyl
group present in compound 19 (Table 4, above) caused a loss in
TBK1 enzyme potency and cell reporter potency while also reduc-
ing AurB enzyme potency. It is plausible that the terminal C7 cyclo-
pentyl group and isobutyl groups attempt to occupy similar
binding pockets in the enzyme.
The synthesis of advanced 6-aryl-7-H-azabeznimidazole 19 is
outline in Scheme 1. The synthesis began via POCl3-mediated het-
erocycle formation between diamino pyridine I-1 and p-anisic acid
to give the 6-Br-azabenzimidazole I-2.20 Suzuki coupling between
commercially available 4-boronothiophene-2-carboxylic acid and
unprotected azabenzimidazole I-2 was possible utilizing a water-
soluble phosphine ligand to give the 6-aryl-azabenzimidazole
10.21 The synthesis of 19 was completed by HATU coupling be-
tween 2-methylpropan-1-amine and acid 10.
e
while still demonstrating TBK1/IKK-e enzyme potency and low
double digit nM potency against a cell based reporter assay. We
have also uncovered compound 25, which has single digit nM po-
tency in the cell reporter assay and very good TBK1 enzyme po-
tency. Compound 25, along with compounds 1, 4, 17, 18, 19, 20,
and 26, have slightly lower IC50’s in the cell assay versus the
TBK1 enzyme assay, and compounds 2, 15, 16, and 23 show only
a very small drop from enzyme to cell. In either case, the cell and
enzyme IC50’s are within the error of the assays. This is a surprising
and interesting result. Since the TBK1 and IKK-e enzyme assays are
run at KM ATP concentrations and the cell contains mM levels of
ATP, one may expect a drop in potency from enzyme to cell. One
explanation for why this is not observed here relies on the fact that
TBK1 contains several interacting domains and loops that are in-
volved in the homodimerization, trans-autophosphorylation, and
subsequent activation of the kinase.9 If these compounds bind to
an inactive conformation of the kinase, the affinity of that form
for ATP could be significantly reduced, in which case ATP would
not compete as strongly with the inhibitor as expected.22 In this
situation, one might observe very little drop-off from enzyme to
cell with highly permeable compounds.
R
R
S
O
R
N
R
S
R
R
HN
N
N
H
N
N
R
R
R
N
H
N
HN
N
S
Acknowledgments
N
H
N
The authors would like to thank Judith Stanway for insect cell
culture, Anna Valentine for protein purification, Claire Brassington
Figure 4. Two possible solutions to relieve the steric interaction between the C6
and C7 substituents.

J. W. Johannes et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1138–1143
1143
Cl
N
NH2
Br
NH2
NH2
Cl
N
HN
N
a
c
b
Br
Br
N
N
I-3
O
O
N
H
N
H
O
O
I-4
I-5
HO
O
HN
O
HN
O
R
O
NH
N
H
O
HO
g
f
HN
N
HN
N
HN
N
N
N
R
N
N
N
S
S
O
O
O
N
H
N
H
N
H
24
25 R = H
4
R = Br
I-6 R = CN
23 R = C(S)NH2
d
e
26 R = isobutyl
Scheme 2. Synthesis of a series of 6,7-substituted azabenzimidazoles. Reagents and conditions: (a) CH3CN, POCl3 (3 equiv), 150 °C, mw, 1 h, 80%; (b) 1,3-diaminopropane
(10 equiv), butanol, 160 °C, mw, 3 h, 80%; (c) cyclopentanecarbonyl chloride (1 eq), satd aq NaHCO3:DCM (1:1), 70%; (d) copper(I) cyanide (20 equiv), DMF, 160 °C, mw, 4 h
(NH4OH/BuOH workup); (e) ammonium sulfide (8 equiv), MeOH:DMF (2:1), 100 °C, mw, 1 h, then additional ammonium sulfide (8 equiv), 23 °C, 48 h, 62% (2 steps); (f)
bromopyruvic acid (1 equiv), EtOH, 80 °C, mw, 1 h, 60%; (g) HATU (1 equiv), DIEA (2 equiv), DMA, 2-methylpropan-1-amine (1 equiv), 5%.
HEPES buffer with a kinase and its substrates (for TBK1, 3 nM recombinant full
for protein crystallization, Nicholas Larsen for structural biology
experiments, and Ray Chen for helpful discussions.
length TBK1 (Life Technologies, Madison. WI), 1.8
KRRRAL(pS)VASLPGL, Primm Biotech, Cambridge, MA), 30
MgCl2; for IKK- 4 nM IKK- 1.5 peptide substrate (5FAMA
KELDQGSLCTpSFVGTLQ-NH2, 21st Century Biochemicals, Marlborough, MA),
ATP, and 10 mM MgCl2). For the 10-point IC50 test, inhibitor
concentrations were usually started from 10 M, followed by nine half-log
l
M CK1tide (5FAM-Ahx-
lM ATP, and 10 mM
e,
e,
lM
Supplementary data
5 lM
l
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
12.123.
dilutions. Reactions were kept at room temperature for a pre-defined duration
of time, quenched using HEPES buffer containing EDTA, and then analyzed
using microfluidic mobility shift assay on a Caliper LC3000 system. The ratio of
product (phosphorylated peptide) to total peptide was used to calculate
percentage inhibition for each inhibitor concentration. IC50’s were obtained by
fitting the percentage inhibition data to
equation using Abase (IDBS).
a four-parameter sigmoidal IC50
References and notes
12. TBK1 cell assay: On day 1, plate 40,000 HEK293-TLR3 NF-
jB-ISRE-Luciferase
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IKKe was evaluated by IC50’s. In a typical experiment, a series of kinase
catalyzed reactions with different concentrations of an inhibitor were set up in