5-(1H-Benzimidazol-1-yl)-3-alkoxy-2-thiophenecarbonitriles as potent, selective, inhibitors of IKK-ε kinase

Article abstract of DOI:10.1016/j.bmcl.2006.09.018

The identification and hit-to-lead exploration of a novel, potent and selective series of substituted benzimidazole-thiophene carbonitrile inhibitors of IKK-ε kinase is described. Compound 12e was identified with an IKK-ε enzyme potency of pIC₅₀

Full text of DOI:10.1016/j.bmcl.2006.09.018

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6236–6240
5-(1H-Benzimidazol-1-yl)-3-alkoxy-2-thiophenecarbonitriles as
potent, selective, inhibitors of IKK-e kinase
Paul Bamborough,a John A. Christopher,a,* Geoffrey J. Cutler,b Marion C. Dickson,a
Geoffrey W. Mellor,b James V. Morey,a, Champa B. Patelb and Lisa M. Shewchukc
aGlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
bGlaxoSmithKline R&D, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
cGlaxoSmithKline Inc., 5 Moore Drive, Research Triangle Park, NC 27709, USA
Received 21 August 2006; revised 4 September 2006; accepted 7 September 2006
Available online 25 September 2006
Abstract—The identification and hit-to-lead exploration of a novel, potent and selective series of substituted benzimidazole–thio-
phene carbonitrile inhibitors of IKK-e kinase is described. Compound 12e was identified with an IKK-e enzyme potency of
pIC50 7.4, and has a highly encouraging wider selectivity profile, including selectivity within the IKK kinase family.
Ó 2006 Elsevier Ltd. All rights reserved.
Protein kinases catalyse the transfer of the c-phosphate
from ATP to the side-chain hydroxyl group of tyrosine,
serine or threonine residues of proteins involved in the
regulation of diverse cellular functions. Aberrant kinase
activity is implicated in many diseases and makes this
target class attractive for the pharmaceutical industry.1
which are known to be involved in the regulation of
pro-inflammatory cytokines.3,6–11 Inhibition of IKK-e
has been less widely explored than inhibition of IKK-b
or the classical IKK-a/b/NEMO complex. Potent inhib-
itors of IKK-e with selectivity within the IKK family
will therefore be valuable in further clarifying the roles,
and defining the therapeutic value, of this kinase target.
The IjB (IKK) family of kinases represents an area of
intense research, most of which has centred on inhibi-
tion of IKK-b or the complex formed between the ki-
nases IKK-a and IKK-b and the regulatory sub-unit
NEMO (also known as IKK-1, IKK-2 and IKK-c,
respectively).2 IKK-e (also known as IKK-3, inducible
IKK or IKK-i) was initially identified as an LPS-in-
duced transcript from a macrophage cell-clone and later
as part of a PMA-inducible IjB kinase complex, differ-
ent from the classical IKK-a/b/NEMO complex.3,4 It
was shown to be predominantly expressed in cells and
tissues of the immune system (peripheral blood leuko-
cytes, thymus and spleen) and constitutively expressed
in human rheumatoid arthritis, osteoarthritis and nor-
mal synovium.5 Several observations suggest that
IKK-e might be involved in the regulation of transcrip-
tion factors, such as NF-jB, IRF3 and C/EBP, all of
As part of a hit identification exercise, benzimidazole 1
was identified as a moderately potent inhibitor of
IKK-e, pIC50 5.4 (Fig. 1).12 Extensive in-house kinase
cross-screening is routine within GSK, and substantial
selectivity data were readily available for 1. Analysis
of these data revealed a moderate selectivity profile, with
inhibition of Polo-like kinase 1 (PLK1, pIC50 6.9) being
one of the most significant off-target activities. The
PLK1 activity was not surprising, as 1 and analogues
originated from in-house medicinal chemistry efforts tar-
geted at this kinase. PLK1 has the potential to interfere
with several stages of mitosis and therefore presents an
intervention opportunity in the oncology arena.13
N
1
N
Keywords: IKK-e kinase inhibitors; IKK.
Corresponding author. Tel.: +44 0 1438 763578; e-mail: John.A.
Christopher@gsk.com
S
*
O
O
H2N
Present address: University Chemical Laboratory, Lensfield Road,
Cambridge CB2 1EW, UK.
Figure 1. Initial hit compound 1.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.018

P. Bamborough et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6236–6240
Table 1. IKK-e and PLK1 inhibition of compounds 2–5
6237
MeO
MeO
MeO
N
N
N
N
2,3
4,5
MeO
MeO2S
S
S
O
R1
R1
O
O
Compound
R1
IKK-b pIC50
IKK-e pIC50
PLK1 pIC50
2
3
4
5
CONH2
CN
<4.8
<4.8
<4.8
<4.8
<4.8
5.3
6.9
6.2
CONH2
CN
<4.8
6.1
6.6
<5.0
Identification of 1 and the availability of analogues from
the PLK1 drug development effort prompted further
screening of members of the series to gain a clearer pic-
ture of the IKK-e structure–activity relationships and
the wider kinase selectivity profile. From this exercise
it became apparent, as illustrated by the two pairs of
molecules in Table 1, Compounds 2–5, that replacement
of the amide moiety by a nitrile group was detrimental
to the PLK1 activity, but beneficial to the inhibitory ef-
fect upon IKK-e.14 Furthermore, compounds 2–5 were
observed to be inactive against IKK-b.15
R4
R3
R4
R3
N
N
N
(a)
N
S
S
O
CN
O
R2
R2
O
NH2
Scheme 1. Reagents and condition: (a) (F3CCO)2O, pyridine, CH2Cl2,
À10 °C.
accepted by N3 of the benzimidazole ring. The dime-
thoxy subsituents at the 5 and 6 positions of the benz-
imidazole ring point towards solvent at the edge of the
ATP site. The benzimidazole also makes hydrophobic
contacts with Ala31 and Leu134. The thiophene ring lies
alongside the gatekeeper Phe80. An internal hydrogen-
bond between the carboxamide NH2 and the ether oxy-
gen orientates the ether-linked R2 benzyl group (see
Scheme 1) so that its plane lies perpendicular to the
plane of the benzimidazole ring. The ortho-Cl atom
makes hydrophobic contacts with Val18 and Gly11 on
the N-terminal lobe. The opposite edge of the phenyl
ring is in the region of the backbone carbonyl of
Gln131 in the C-terminal lobe, although it makes no
specific interactions. The primary carboxamide substitu-
ent on the thiophene ring points towards the back pock-
et of the active site, where it makes a direct hydrogen-
bond with Glu51 via the NH2. However, the carbonyl
of the carboxamide lacks any hydrogen-bonding partner
protein atom or visible water.
Encouraged by these early indications of selectivity,
especially in the case of 5, we sought to obtain a li-
gand-bound crystal structure to clarify the binding
mode and aid our understanding of the origins of the
selectivity. Although IKK-e crystallography was not
available, amide 6 showed modest CDK-2 inhibitory
potency (pIC50 5.8). It was possible to soak this com-
pound into crystals of CDK-2/cyclin A to obtain a struc-
ture of the complex.16 The CDK-2 structure acted as a
surrogate for IKK-e and provided valuable information
which could be translated, with knowledge of the amino
acid differences in the ATP-binding sites, to aid our
understanding of the binding mode in the target kinase.
As illustrated in Figure 2, the structure shows that the
characteristic hydrogen-bond from the Leu83 residue
in the hinge region of the CDK-2 ATP-binding site is
MeO
MeO
N
N
N
N
7
6
S
S
O
O
O
O
H2N
HO
Cl
CDK-2 pIC50 5.8
IKK- pIC50 4.7
IKK- pIC50 < 4.8
PLK1 pIC50 6.2
It is believed that the binding mode of this series in
IKK-e is similar to that seen in CDK-2, and that N3
of the benzimidazole also forms a hydrogen-bond to
the hinge region (Cys89 in IKK-e). Comparison of the
sequences of IKK-e and PLK1 in the context of the
CDK-2 structure provides an explanation for the selec-
tivity differences between amide and nitrile analogues
in these kinases. A model of compound 12a docked into
a homology model of IKK-e is illustrated in Figure 3.17
Figure 2. X-ray structure of compound 6 complexed with CDK-2/
cyclin A.

6238
P. Bamborough et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6236–6240
pounds such as 5, where the amide is replaced by nitrile,
lose much of their potential to hydrogen-bond to any
buried water, and so are less potent against PLK1. It
is believed that in the ATP-binding site of IKK-e they
displace any buried water and the cyano group fills the
lipophilic cavity left behind.
A chemistry effort was initiated, with the aim of expand-
ing the SAR, enhancing the IKK-e potency and improv-
ing the selectivity profile. Two approaches were pursued
in parallel; first, selections of available molecules with
amide substitution on the thiophene were dehydrated
to the corresponding nitrile, using trifluoroacetic anhy-
dride in dichloromethane (Scheme 1). The precursor
amides were prepared by routes previously reported.19
Alternatively, the nitrile moiety was installed at an early
stage in the synthesis via the dehydration of 3-hydroxy-
2-thiophenecarboxamide 8, itself formed by the conju-
gate addition/cyclisation of 2-mercaptoacetamide onto
methyl 2-propiolate (Scheme 2). With nitrile 9 in hand,
installation of the benzimidazole portion was achieved
via reaction with sulfuryl chloride and displacement of
the intermediate chloride under conditions reported by
Corral.20 O-Alkylation under standard conditions then
furnished target compounds 12.
Figure 3. Compound 12a docked into the homology model of IKK-e.
Table 2 shows some of the aligned residues in the ATP-
binding sites of CDK-2, IKK-e, PLK1 and MK2. Espe-
cially relevant is the unusual histidine (His105) buried
deep in the back of the ATP site of PLK1. It is much
more common in protein kinases to find aliphatic resi-
dues at the equivalent position, for example Leu55 in
CDK2 and Leu59 in IKK-e. MK2, whose X-ray struc-
ture has been solved, has an analogous histidine
(His108) at the same position as His105 in PLK1. The
crystal structure of staurosporine bound to MK2 shows
a water molecule buried at the back of the ATP site that
appears to be hydrogen-bonded to both His108 and
Glu104.18 Since both residues are conserved in PLK1,
we believe that a similar situation exists, and that in cer-
tain ligand-bound situations a water molecule can
bridge between His105 and Glu101 at the back of the
ATP site. It is striking that even carboxylic acids can
be tolerated in the position of the amide in PLK1
(e.g., compound 7), presumably because the ionisable
histidine provides a counter to the charge.
Key data are summarized below in Table 3. In most
cases, R2 O-substitution was found to be beneficial to
the IKK-e activity, when compared to the unsubstituted
hydroxy compound 12g. O-Benzyl substitution appears
to be generally more favourable than O-alkyl (compare
12a to 5, 12f, 12h and 12j). Within the benzyl series, a
significant preference for ortho-substitution on the phen-
yl ring is apparent, as illustrated by comparison of sulf-
ones 12e and 3. a-Branching of the benzyl chain in 12d
and 12l is observed to be unfavourable (the (S)-enantio-
mers of 12d and 12l were not prepared), with a 10-fold
drop in potency observed between 12a and 12d. This is
interpreted to mean that there is a precise steric require-
ment which requires both ortho substitution on the ring
and the absence of an a-branch to position the benzylic
phenyl ring in a suitable position.
Replacement of the PLK1 His105 with leucine in both
CDK-2 and IKK-e means that the hydrogen-bonding
potential of a water at the same position cannot be sat-
isfied. Burial of water in this location in IKK-e would
therefore be expected to be less favourable than in
PLK1, and so compounds that bind with a hydrogen-
bond to this water are less potent. In contrast, com-
O
O
O
S
(a)
+
NH2
HS
NH2
OMe
OH
8
CN
Cl
S
S
(c)
(b)
CN
Table 2. ATP site comparison between human CDK-2, IKK-e, PLK1
and MK2
O
OH
10
9
CDK-2
IKK-e
PLK1
MK2
R4
R3
R4
R3
N
N
N
N
ILE10
LEU15
VAL23
ALA36
LEU59
VAL68
MET86
CYS89
LEU59
CYS67
LEU70
VAL78
VAL18
ALA31
LEU55
VAL64
PHE80
LEU83
GLN131
LEU134
ALA144
ALA80
HIS105
VAL114
LEU130
CYS133
GLY180
PHE183
GLY193
ALA91
HIS108
VAL118
MET138
LEU141
GLU190
LEU193
THR206
(d)
(e)
S
S
CN
CN
HO
O
R2
11
12
Scheme 2. Reagents and conditions: (a) NaOMe, MeOH, 0 °C; (b)
(F3CCO)2O, pyridine, CH2Cl2, rt; (c) Cl2SO2, CHCl3, rt; (d) substi-
tuted benzimidazole, triethylamine, CHCl3, rt; (e) alkyl bromide,
K2CO3, DMF, 100 °C, microwave heating.
GLY139
MET142
THR156

P. Bamborough et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6236–6240
6239
Table 3. IKK-e inhibition values
Compound
R2
R3
R4
IKK-e pIC50
3
–CH2(4-SO2MePh)
( )–CH2(2-Tetrahydrofuranyl)
–CH2(2-CF3Ph)
OMe
H
OMe
5.3
6.1
6.6
7.3
7.6
5.4
7.4
5.8
5.6
5.5
6.0
5.3
4.9
5.8
5
H
OMe
O(CH2)2-4-Morpholine
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
–CH2(2-CF3Ph)
–CH2(2-CF3Ph)
O(CH2)3OH
OMe
OMe
OMe
OMe
OMe
H
(R)-CHMe(2-CF3Ph)
–CH2(2-SO2MePh)
–(CH2)2-4-Morpholine
H
–CH2-Cyclopropyl
–CH2Ph
–(CH2)2-1-Piperidine
–CH2(4-CONH2Ph)
(R)-CHMe(2-ClPh)
OMe
OMe
OMe
OMe
OMe
OMe
Table 4. Kinase inhibitory potencies of 12e (values in pIC50
)
Alk-5
<5.3
EGFR
<5.0
ErbB2
<5.0
GSK-3
<4.8
IKK-a
IKK-b
IKK-e
Jnk-3
<4.8
p38a
<4.8
p38b
PLK1
6.1
<4.8
<4.8
7.4
<4.8
In most cases, the R3 and R4 substituents are identical in
this initial exploration of the SAR. From the CDK-2
crystal structure, which shows R3 and R4 pointing to-
wards solvent, it was anticipated that modifications
incorporating solubilising groups to alter the physico-
chemical properties of the series would be well tolerated
in these positions. Comparison of 12b and 12c with 12a
shows that this is the case, and in fact longer chains give
an increase in IKK-e activity, perhaps because of lipo-
philic interactions with Leu15 and other residues around
the edge of the ATP site.
also thank Karen E. Lackey and David D. Miller for
their guidance and support, and synthetic contributions
from James A. Linn, Kristen E. Nailor, Kevin W. Kuntz
and James M. Salovich are gratefully acknowledged.
Duncan B. Judd and Bethany Brown are thanked for
their assistance in the large-scale synthesis of 9.
References and notes
1. Parang, K.; Sun, G. Curr. Opin. Drug Disc. Dev. 2004, 7,
617.
2. Coish, P. D. G.; Wickens, P. L.; Lowinger, T. B. Expert
Opin. Ther. Patents 2006, 16, 1.
3. Shimada, T.; Kawai, T.; Takeda, K.; Matsumoto, M.;
Inoue, J-i.; Tatsumi, Y.; Kanamaru, A.; Akira, S. Int.
Immunol. 1999, 11, 1357.
4. Peters, R. T.; Liao, S.-M.; Maniatis, T. Mol. Cell 2000, 5,
513.
5. Aupperle, K. R.; Yamanishi, Y.; Bennett, B. L.; Mercurio,
F.; Boyle, D. L.; Firestein, G. S. Cell Immunol. 2001, 214,
54.
In addition to the highly encouraging IKK-e inhibitory
characteristics of the compounds, in-house cross-screen-
ing indicated that an excellent overall kinase selectivity
profile was maintained, a key finding being that all com-
pounds in Table 3 were essentially inactive (pIC50 < 4.8)
at IKK-a and IKK-b. Kinase data are summarized
above in Table 4 for 12e, which also has encouraging
developability characteristics; specifically good aqueous
solubility (60 lM) and inactivity (pIC50 < 4.3) against
the cytochrome P450 isozymes 1A2, 2D6, 2C9 and 3A4.
6. Nomura, F.; Kawai, T.; Nakanishi, K.; Akira, S. Genes
Cells 2000, 5, 191.
7. Sankar, S.; Chan, H.; Romanow, W. J.; Li, J.; Bates, R. J.
Cell. Signal. 2006, 18, 982.
In summary, the 5-(1H-Benzimidazol-1-yl)-3-alkoxy-2-
thiophenecarbonitrile series represents a novel, potent
and selective class of IKK-e inhibitors. The most potent
compounds obtained are 12b,12c and 12e, with enzyme
inhibitory potencies of pIC50 7.3, 7.6 and 7.4, respective-
ly. The series has excellent selectivity against IKK-a and
IKK-b, and provides an opportunity to examine the
therapeutic role of IKK-e and further aid the under-
standing of the IKK family of kinases.
8. Fitzgerald, K. A.; McWhirter, S. M.; Faia, K. L.; Rowe,
D. C.; Latz, E.; Golenbock, D. T.; Coyle, A. J.; Liao, S.-
M.; Maniatis, T. Nat. Immunol. 2003, 4, 491.
9. Sharma, S.; tenOever, B. R.; Grandvaux, N.; Zhou, G.-P.;
Lin, R.; Hiscott, J. Science 2003, 300, 1148.
10. McWhirter, S. M.; Fitzgerald, K. A.; Rosains, J.; Rowe,
D. C.; Golenbock, D. T.; Maniatis, T. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 233.
11. Kravchenko, V. V.; Mathison, J. C.; Schwamborn, K.;
Mercurio, F.; Ulevitch, R. J. J. Biol. Chem. 2003, 278,
26612.
12. pIC50 = Àlog10 IC50; where the IC50 is the concentration of
compound required to inhibit the kinase activity by 50%.
IKK-e inhibition data were generated as follows: recombi-
nant human IKK-e was expressed in baculovirus as a
FLAG-tagged fusion protein, and its activity assessed using
a time-resolved fluorescence resonance energy transfer
Acknowledgments
The GSK Screening and Compound Profiling and Com-
putational, Analytical and Structural Sciences groups
are thanked for the generation of kinase inhibition data,
and for solubility and P450 data for 12e. The authors

6240
P. Bamborough et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6236–6240
(TR-FRET) assay. Briefly, IKK-e (15 nM total protein)
16. The PDB deposition code for this structure is 2I40. The
expression, purification and crystallization of CDK-2/
Cyclin A was carried out as previously described; see:
Jeffrey, P. D.; Russo, A. A.; Polyak, K.; Gibbs, E.;
diluted in assay buffer (50 mM HEPES, 10 mM MgCl2,
1 mM Chaps, 1 DTT and 0.01% w/v BSA, pH7.4) was
added to wells containing various concentrations of com-
pound or DMSO vehicle (3% final). The reaction was
initiated by the addition of 15 ll of GST-IjBa substrate
(25 nM)/ATP (2 lM) in a total volume of 30 ll. The
reaction was incubated for 15 min at ambient temperature
and then stopped by the addition of 15 ll of 50 mM EDTA.
Detection reagent (15 ll) in buffer (100 mM Hepes, 150 mM
NaCl, 0.1% w/v BSA, pH 7.4) containing antiphosphoser-
ine-IjBa-32/36 monoclonal antibody, Clone 12C2 (Cell
Signalling Technology, Beverley, Massachusetts, USA)
labelled with W-1024 europium chelate (Perkin-Elmer,
Turku, Finland) and an allophycocyanin labelled anti-
GST antibody (Prozyme, San Leandro, California, USA)
was added and the reaction further incubated for at least
45 min at ambient temperature. The degree of phosphory-
lation of GST-IjBa was measured using a Packard
Discovery plate reader (Perkin-Elmer Life Sciences, Bea-
consfield, UK) as a ratio of specific 665 nm energy transfer
signal to reference europium 620 nm signal. The error of the
assay is estimated as 0.3 log units, based on the standard
deviation around the mean value of an inhibitor used as a
standard compound in every assay.
´
Hurwitz, J.; Massague, J.; Pavletich, N. P. Nature 1995,
376, 313. Crystals were soaked with 50 lM compound for
2 days prior to data collections. The structure was refined
˚
to an Rfactor of 20% at 2.75 A.
17. The sequence of IKK-e was aligned to a large panel of
protein kinase structures. CDK-2 was chosen as the
template despite its relatively low sequence similarity
(24% identity over the kinase domain) because of the
availability of CDK-2 crystal structures complexed with
molecules of interest. The kinase chain was extracted
from a complex of CDK-2 with Cyclin A to give an
ˇ
active-like ATP site conformation. MODELLER (Sali,
A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779) was used
to generate IKK-e coordinates. Limited refinement, in
the presence of a manually bound ligand from a different
chemical series to the one described here, was performed
using Discover running through InsightII (Accelrys,
www.accelrys.com).
18. Underwood, K. W.; Parris, K. D.; Federico, E.; Mosyak,
L.; Czerwinski, R. M.; Shane, T.; Taylor, M.; Svenson,
K.; Liu, Y.; Hsiao, C.-L.; Wolfrom, S.; Maguire, M.;
Malakian, K.; Telliez, J.-B.; Lin, L.-L.; Kriz, R. W.;
Seehra, J.; Somers, W. S.; Stahl, M. L. Structure 2003,
11, 627.
19. (a) Andrews, C. W. III.; Cheung, M.; Davis-Ward, R. G.;
Drewry, D. H.; Emmitte, K. A.; Hubbard, R. D.; Kuntz,
K. Int. Patent Appl. WO 04/014899; (b) Cheung, M.;
Emmitte, K. A. Int. Patent Appl. WO 05/037827.
20. Corral, C.; Lissavetzky, J. Synthesis 1984, 847.
´
13. Barr, F. A.; Sillje, H. H. W.; Nigg, E. A. Nat. Rev. Mol.
Cell Biol. 2004, 5, 429.
14. PLK1 kinase activities were determined using methods
described below.19a
15. IKK-b kinase activities were determined using methods
previously described; see: Baldwin, I. R.; Bamborough, P.;
Christopher, J. A.; Kerns, J. K.; Longstaff, T.; Miller, D.
D. Int. Patent Appl. WO 05/067923.