The discovery of 2-amino-3,5-diarylbenzamide inhibitors of IKK-α and IKK-β kinases

Article abstract of DOI:10.1016/j.bmcl.2007.04.088

A potent and selective series of 2-amino-3,5-diarylbenzamide inhibitors of IKK-α and IKK-β is described. The most potent compounds are 8h, 8r and 8v, with IKK-β inhibitory potencies of pIC₅₀ 7.0, 6.8 and 6.8, respectively. The series has excell

Full text of DOI:10.1016/j.bmcl.2007.04.088

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3972–3977
The discovery of 2-amino-3,5-diarylbenzamide inhibitors of
IKK-a and IKK-b kinases
John A. Christopher,a,* Barbara G. Avitabile,b, Paul Bamborough,a
Aurelie C. Champigny,a Geoffrey J. Cutler,b Susan L. Dyos,a Ken G. Grace,a
Jeffrey K. Kerns,c Jeremy D. Kitson,a Geoffrey W. Mellor,b James V. Morey,a,à
Mary A. Morse,a Carolyn F. O’Malley,a Champa B. Patel,a Nicholas Probst,a
William Rumsey,c Clive A. Smithb and Michael J. Wilsona
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., 709 Swedeland Road, King of Prussia, PA 19406, USA
Received 5 April 2007; revised 20 April 2007; accepted 25 April 2007
Available online 29 April 2007
Abstract—A potent and selective series of 2-amino-3,5-diarylbenzamide inhibitors of IKK-a and IKK-b is described. The most
potent compounds are 8h, 8r and 8v, with IKK-b inhibitory potencies of pIC50 7.0, 6.8 and 6.8, respectively. The series has excellent
selectivity, both within the IKK family over IKK-e, and across a wide variety of kinase assays. The potency of 8h in the IKK-b
enzyme assay translates to significant cellular activity (pIC50 5.7–6.1) in assays of functional and mechanistic relevance.
Ó 2007 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
way is well documented in chronic inflammatory
disease and consequently identification of selective
IKK-b inhibitors has been a particular goal for
anti-inflammatory drug discovery.2,3 More recently a
non-redundant role for IKK-a has been elucidated, in
mediating signal transduction from TNFR family
members, such as co-stimulation receptors CD40 and
BAFFR on B lymphocytes.4 The identification of iso-
form selective IKK inhibitors will provide pharmacolo-
gical reagents to further address the differential role of
these kinases in health and disease.
The IjB (IKK) family of kinases represent an area of in-
tense research since they are central regulators of NF-jB
transcription factors, controlling gene expression in in-
nate and adaptive immune responses.2 Four kinases in
this family have been identified based on sequence
homology (IKK-a, -b, -e and TBK1). The most widely
studied family member to date is IKK-b which is ubiq-
uitously expressed and mediates activation of NF-jB
p50/RelA in response to pro-inflammatory stimuli such
as tumour necrosis factor-a (TNF-a) and lipopolysac-
charide (LPS). The role of this canonical NF-jB path-
The discovery and development of small-molecule inhib-
itors of IKK-family kinases represents a significant area
of research, as described in a recent review of the patent
literature by Lowinger and colleagues.5 We observed
that small-molecule IKK-family inhibitors of differing
chemotypes contain a common hydrogen-bonding phar-
macophore, Figure 1. For example, thiophene 1, thieno-
pyridine 2 and fused pyrazole 3 exhibit a common motif,
where the orientation of a primary amide is restricted by
an adjacent hydrogen-bonding functionality. In agree-
ment with a pharmacophore hypothesis proposed by
Morwick for thiophene inhibitors similar to 1, we as-
sumed that in each case the constrained, unsubstituted,
Keywords: Kinase; IKK; Inhibitor.
*
Corresponding author. E-mail: John.A.Christopher@gsk.com
Present address: Argenta Discovery Ltd, 8/9 Spire Green Centre,
Flex Meadow, Harlow, Essex CM19 5TR, UK.
à
Present address: University Chemical Laboratory, Lensfield Road,
Cambridge CB2 1EW, UK.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.088

J. A. Christopher et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3972–3977
3973
additional substitution at the 3-position of the phenyl
ring would allow a second point of diversity and could
provide additional favourable interactions within the ki-
nase active site.
NH2
NH
O
N
O
NH
2 O
NH2
S
NH2
NC
1
2
S
MeO
H2N
N
Scheme 1 summarises the synthetic route we used to tar-
get compounds 8, 11 and 12. 2-Amino-5-iodobenzoic
acid 5 provided the most versatile building block in
the 2-amino series; cyclisation with triphosgene to the
benzoxazinedione 6 allowed ring opening with ammonia
or methyl amine to provide carboxamides 7 after Suzu-
ki–Miyaura coupling. Optionally, this was followed by
bromination and Suzuki–Miyaura coupling to introduce
a 3-aryl substitutent, yielding 8.
H2NO2S
H2N
N
N
NH2
O
3
Figure 1. Literature IKK inhibitors 1–3.5
The analogous 2-hydroxy series was accessed in a simi-
lar fashion from 5-bromosalicylamide 9, a Suzuki–
Miyaura coupling and bromination sequence yielding
phenol 10. Suzuki–Miyaura coupling reactions on this
material were inefficient and unreliable, and temporary
protection by methylation, followed by microwave-med-
iated nucleophilic de-methylation after the coupling al-
lowed smooth access to the target compounds. This
route also provided access to methoxy compounds 11,
which we believed could be of interest, as the action of
the methoxy group as a hydrogen-bond acceptor from
the carboxamide could introduce the desired constraint
into the hydrogen-bond network.
amide group was essential for successful IKK
inhibition.6
Here, we report how we combined this hypothesis with
the synthetic tractability of an IKK-e selective series to
create potent inhibitors of IKK-a and IKK-b. As previ-
ously reported in this journal, the 5-(1H-benzimidazol-
1-yl)-3-alkoxy-2-thiophenecarbonitriles, such as 4,
Figure 2, are potent inhibitors of IKK-e, and are selec-
tive over IKK-a and IKK-b.7 With robust and exploit-
able chemistry in hand from the synthesis of 4 and
analogues, we sought to explore this template further
by adapting it to target other kinases of therapeutic
interest.
We investigated amino-substituted compounds 8a–s,
Table 1, assessing their IKK-a, -b and -e inhibitions in
enzyme activity assays.8,9 Amide substitution, where
R3 = Me, was not tolerated (8a, 8b; compare 8b with
8f); a finding in accordance with the structures of litera-
ture inhibitors 1–3 all lacking such substitution.
We rationalised that removing the hydrogen bond
acceptor capability of the benzimidazole group in 4,
converting the nitrile to a primary amide and combining
this with appropriate proximal alkoxy or amino func-
tionality on a phenyl ring to incorporate an extended
hydrogen-bonding network into a novel framework
would be profitable for IKK-b. We rationalised that
The most striking SAR is apparent in the R1 position.
First, we observed that compounds 8c and 8d which lack
R1 substitution are at best weakly active.10 A distinct
preference for para-substituted phenyl groups at R1 is
apparent, as without exception, all compounds lacking
this arrangement (e.g., 8e, k, m, o, p) fail to achieve
inhibitory potencies above pIC50 6.0 for any of the
IKK isoforms. Finer SAR emerged within compounds
which have para-substituted phenyl groups at R1.
S-linked sulfonamides (8f–h , l, q, r) yielded the greatest
MeO
MeO
N
N
4
IKK-α pIC50 < 4.8
IKK-β pIC50 < 4.8
IKK-ε pIC50 7.4
S
CN
O
MeO2S
Figure 2. IKK-e selective inhibitor 4.
R2
R2
I
I
(a)
(b), (c)
(d), (c)
OH
NHR3
NHR3
O
R1
HN
O
H2N
O
O
H2N
O
H2N
O
5
7
8
6
O
R2
Br
R2
R2
(c), (d)
(e), (c)
(f)
R1
NH2
R1
NH2
NH2
NH2
Br
HO
12
O
HO
9
HO
10
O
O
11
O
Scheme 1. Reagents and conditions: (a) triphosgene, THF, rt; (b) MeNH2 or 0.5 M NH3/dioxane, THF, rt; (c) aryl boronic acid, PdCl2(dppf), 2 M
(aq) Na2CO3, dioxane, water, 80 °C or microwave heating, 150 °C, 20 min; (d) NBS, acetic acid, rt; (e) MeI, K2CO3, acetone, rt; (f) LiI, pyridine,
microwave heating, 175 °C.

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J. A. Christopher et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3972–3977
Table 1. IKK-a, IKK-b and IKK-e inhibition by compounds 8a–s
Compound
IKK-a pIC50
IKK-b pIC50
IKK-e pIC50
R1
R2
R3
8a
8b
8c
8d
8e
8f
<4.8
<4.8
<4.8
<4.8
5.3
<4.8
<4.8
4.8
NDa
ND
(4-SO2Me)Ph
(4-SO2(1-pyrrolidine))Ph
H
4-FPh
4-FPh
Ph
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
<4.8
<4.8
<4.8
<4.8
<4.8
<4.8
<4.8
<4.8
ND
<4.8
5.8
H
4-Pyridyl
4-FPh
4-FPh
4-FPh
4-FPh
4-ClPh
4-ClPh
Ph
5.6
6.3
6.2
6.7
(4-SO2(1-pyrrolidine))Ph
(4-SO2NH2)Ph
(4-SO2NH2)Ph
(4-NHSO2Me)Ph
(4-NHSO2Me)Ph
(3-NHSO2Me)Ph
(4-SO2NH2)Ph
(3-SO2NH2)Ph
(4-CH2SO2NH2)Ph
(3-NMe2)Ph
8g
8h
8i
6.4
5.2
7.0
5.8
8j
4.9
5.0
5.8
5.1
8k
8l
Ph
Ph
6.0
6.6
5.1
<4.8
<4.8
<4.8
<4.8
5.4
8m
8n
8o
8p
8q
8r
8s
<4.8
5.1
Ph
Ph
5.4
<4.8
5.5
<4.8
5.4
Ph
Ph
4-Pyridyl
4-Pyridyl
4-SO2MePh
4-SO2MePh
7.1
5.7
6.6
6.8
5.8
ND
(4-SO2NH2)Ph
(4-SO2NH2)Ph
(4-SO2(1-pyrrolidine))Ph
5.3
6.4
ND
The error within each assay is estimated as 0.3 log units.
a ND, not determined.
potencies against IKK-a and -b, while homologated of
the sulfonamides (compare 8l with 8n) or N-linked sul-
fonamides (8i and 8j) were substantially less potent.
4-pyridyl R2 group, exhibit any inhibition of IKK-e,
with 8q also having significant selectivity for IKK-a over
IKK-b. A detailed discussion of the R2 SAR is outside
the scope of this manuscript, but from the data above
it is clear that this position provides an opportunity
for modulation of IKK isoform selectivity. Figure 3
illustrates inhibition data for key compounds.
It is clear that although the series generally has a degree
of selectivity for IKK-b over IKK-a this is typically
no greater than 10-fold, for example 8r and 8s, with a
methyl sulfone at the para-position of the R2
phenyl substituent. Only compounds 8p and 8q, with a
We then investigated O-substituted compounds 11a–d
and 12a–d, assessing the inhibition of IKK-a, IKK-b
and IKK-e. Key data are summarised in Table 2. Meth-
oxy substitution yielded inactive IKK inhibitors, both
without (11a) and with (11b–d) substitution at R1. Hy-
droxy substitution was more profitable; compounds
with substitution at both R1 and R2 (12b–12d) exhibited
encouraging inhibitory activities against IKK-a and
IKK-b.
pIC50
7.5
7
6.5
6
5.5
Encouraged by the potency of compounds in the amino-
series, representative molecule 8l was docked into the
ATP-site of a homology model of IKK-b, Figure 4a.11
The results agree with our hypothesis that the primary
carboxamide makes a pair of hydrogen bonds to the
hinge region, Glu97 and Cys99. This is tethered by an
internal hydrogen bond to the aniline NH2 group,
helping to maintain planarity.
*
*
*
*
*
*
5
4.5
4
8c
8h
8l
8m
8q
Figure 3. IKK-a (cyan), IKK-b (blue) and IKK-e (green) inhibition by
compounds 8c, h, l, m, q. *pIC50 < 4.8.
Table 2. IKK-a, IKK-b and IKK-e inhibition by compounds 11a–d and 12a–d
Compound
IKK-a pIC50
IKK-b pIC50
IKK-e pIC50
R1
R2
11a
11b
11c
11d
12a
12b
12c
12d
<4.8
<4.8
<4.8
<4.8
4.8
<4.8
<4.8
<4.8
<4.8
<4.8
5.8
NDa
ND
ND
ND
ND
<4.8
<4.8
<4.8
H
4-Pyridyl
Ph
4-Pyridyl
(4-SO2NH2)Ph
Ph
4-ClPh
4-Pyridyl
4-Pyridyl
4-ClPh
Ph
H
5.7
5.7
(4-SO2NH2)Ph
(4-NHSO2Me)Ph
(4-SO2NMe2)Ph
6.0
5.1
5.3
4-Pyridyl
The error within each assay is estimated as 0.3 log units.
a ND, not determined.

J. A. Christopher et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3972–3977
3975
helix. This amino acid pair is also found in CDK-2
(Asp86 and Lys89), where their side-chains interact
favourably with sulfonamides attached to several inhib-
itor chemotypes.12–14 Figure 4b shows the structure of a
sulfonamide-containing oxindole bound into CDK-2,
illustrating this interaction. By analogy, it is likely that
similar interactions contribute to the enhanced IKK-a
and IKK-b potency of sulfonamides in Table 1. Com-
paring the activity of substituted and unsubstituted sul-
fonamides (8r and 8s) it seems that the sulfonamide NH
does not contribute to activity, so the main interaction is
most likely hydrogen bonding between the sulfonamide
oxygens and either the Lys106 side-chain or the Asp103
backbone.
Our observations of the distinct preference for para-sub-
stitution at R1 in the amino series led us to explore this
region further. The synthetic route above (Scheme 1)
necessitates the introduction of R1 via a Suzuki–Miya-
ura coupling step, and the limited availability of appro-
priately functionalized boronic acids or esters led us to
adapt pentafluorophenylsulfonate ester chemistry to al-
low increased diversity.
As recently described by Avitabile and co-workers, the
pentafluorophenyl (PFP) group can be used as a
sulfonic acid protecting group, which is stable to
Suzuki–Miyaura conditions with the employment of a
suitable base.15 The PFP group can then be displaced
by amine nucleophiles to yield substituted sulfona-
mides, Scheme 2.16,17 We utilised this chemistry to pre-
pare compounds 8t–8y, which were assessed for their
IKK-a, IKK-b and IKK-e kinase inhibition, Table 3.
Substitution of the sulfonamide did not yield substan-
tial improvements in IKK-a or IKK-b enzyme inhibi-
tion (compare 8t–y with 8l in Table 2). However, the
tolerance of a variety of N-substituents offers the
opportunity to moderate the physicochemical proper-
ties of the molecule, a detailed description of which is
outside the scope of this manuscript. As with the
majority of compounds in Table 2, no significant inhi-
bition of IKK-e was observed.
Figure 4. (a) Model of IKK-b with docked compound 8l (Top). (b)
X-ray structure of CDK-2 with bound oxindole, PDB code 1fvv.12 H-
bonds are shown as orange dashed lines.
The R1 aromatic group points towards the solvent side
of the ATP site. The 4-position of R1 points towards
Asp103 and Lys106, located on the surface of a small
R2
R2
R2
I
(a)-(c)
(d), (e)
(f)
OH
NH2
NH2
NH2
H2N
13
O
H2N
14
O
H2N
15
O
H2N
O
8t-y
4HNO2S
R
(F5C6)O3S
Scheme 2. Reagents and conditions: (a) triphosgene, THF, rt; (b) 0.5 M NH3/dioxane, THF, rt; (c) aryl boronic acid, PdCl2(dppf), 2 M (aq) Na2CO3,
dioxane, water, 80 °C; (d) NBS, acetic acid, rt; (e) R2-boronic acid, PdCl2(dppf), Na2B4O7, dioxane, EtOH, reflux; (f) R1NH2, THF, reflux.
Table 3. IKK-a, IKK-b and IKK-e inhibition by compounds 8t–y
Compound
IKK-a pIC50
IKK-b pIC50
IKK-e pIC50
R4
R2
8t
6.0
6.0
6.2
6.1
6.0
6.1
6.5
6.5
6.8
6.6
6.5
6.6
<4.8
<4.8
<4.8
<4.8
<4.8
<4.8
–NH(CH2)3NMe2
Ph
Ph
Ph
Ph
Ph
Ph
8u
8v
8w
8x
8y
–NH(CH2)3(1-morpholine)
–NH(CH2)2NH2
–NH(CH2)3OMe
–NHcyclopropyl
–NH(CH2)2(1-pyrrolidine)
The error within each assay is estimated as 0.3 log units.

3976
J. A. Christopher et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3972–3977
Table 4. Inhibition of pro-inflammatory cytokine release in PBMCs by
8h
4. Bonizzi, G.; Karin, M. Trends Immunol. 2004, 25, 280.
5. Coish, P. D. G.; Wickens, P. L.; Lowinger, T. B. Expert
Opin. Ther. Patents 2006, 16, 1.
Cytokine
pIC50
6. Morwick, T.; Berry, A.; Brickwood, J.; Cardozo, M.;
Catron, K.; DeTuri, M.; Emeigh, J.; Homon, C.; Hrap-
chak, M.; Jacober, S.; Jakes, S.; Kaplita, P.; Kelly, T. A.;
Ksiazek, J.; Liuzzi, M.; Magolda, R.; Mao, C.; Marshall,
D.; McNeil, D.; Prokopowicz, A., III; Sarko, C.; Scouten,
E.; Sledziona, C.; Sun, S.; Watrous, J.; Wu, J. P.; Cywin,
C. L. J. Med. Chem. 2006, 49, 2898.
7. Bamborough, P.; Christopher, J. A.; Cutler, G. J.;
Dickson, M. C.; Mellor, G. W.; Morey, J. V.; Patel, C.
B.; Shewchuck, L. M. Bioorg. Med. Chem. Lett. 2006, 16,
6236.
TNF-a
IL-1b
IL-6
6.1
6.4
5.7
The cellular activity of 8h was assessed via the secretion
of the pro-inflammatory cytokines TNF-a, IL-1b and
IL-6 from peripheral blood mononuclear cells (PBMC)
stimulated by lipopolysaccharide (LPS).18 The IKK-b
enzyme potency of 8h translated to significant inhibition
of the secretion of these cytokines in the cellular assay
(Table 4), as expected for inhibition of the NF-jB sig-
nalling pathway. In addition, 8h was observed to inhibit
(pIC50 = 5.7) the nuclear translocation of NF-jB in a
TNF-a induced cellular assay, confirming the mecha-
nism of action of the compound.19
8. pIC50 = Àlog10 IC50; where the IC50 is the molar concen-
tration of compound required to inhibit the kinase activity
by 50%. IKK-b kinase inhibitory activity was determined
using
a time-resolved fluorescence resonance energy
transfer (TR-FRET) assay. Recombinant human IKK-b
(residues 1–737) was expressed in baculovirus as a C-
terminal GST-tagged fusion protein. IKK-b (typically 2–
5 nM final) diluted in assay buffer (50 mM HEPES,
10 mM MgCl2, 1 mM CHAPS, pH 7.4, with 1 mM DTT
and 0.01% w/v BSA) was added to wells containing
various concentrations of compound or DMSO vehicle
(less than 5% final). The reaction mixture was initiated by
the addition of GST-IjBa substrate (25 nM final)/ATP
(1 lM final), in a total volume of 6 ll. The reaction was
incubated for 30 min at room temperature, then termi-
nated by the addition of stop reagent (3 ll) containing
50 mM EDTA and detection reagents in buffer (100 mM
HEPES, pH 7.4, 150 mM NaCl and 0.1% w/v BSA).
Detection reagents comprise antiphosphoserine-IjBa-32/
36 monoclonal antibody 12C2 (Cell Signalling Technol-
ogy, Beverly Massachusetts, USA) labelled with W-1024
europium chelate (Wallac OY, Turku, Finland) and an
allophycocyanin-labelled anti-GST antibody (Prozyme,
San Leandro, California, USA). The reaction mixture
(9 ll total volume) was further incubated for at least
60 min at room temperature. The degree of phosphoryla-
tion of GST-IjBa was measured using a suitable time-
resolved fluorimeter as a ratio of specific 665 nm energy
transfer signal to reference europium 620 nm signal. IKK-
a kinase inhibitory activity was determined in an analo-
gous fashion, using 6-his-tagged full length IKK-a. The
error within both assays 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.
9. IKK-e inhibitory activity data were generated using
conditions previously described, see Ref. 7.
In addition to exhibiting selectivity within the IKK fam-
ily for IKK-a and IKK-b over IKK-e, the series pos-
sesses an outstanding overall kinase selectivity profile.
Compound 8h was screened in a panel of more than
50 kinase assays, including Alk5, CDK-2, EGFR,
ErbB2, GSK3b, PLK1, Src, VegFr2 and displayed
greater than 50-fold selectivity against all of them. In
addition, 8h was submitted to the Kinomescan assay
panel of 150 kinases (Ambit Biosciences), which utilise
kinases or kinase domains fused to T7 bacteriophage.20
The compound did not show a significant ability to dis-
place an immobilised ATP-site ligand from any of the
kinases in the panel.
In conclusion, we have applied knowledge from litera-
ture IKK inhibitors to a tractable IKK-e series to create
a novel series of potent and selective IKK-a and IKK-b
2-amino-3,5-diarylbenzamide inhibitors. The most po-
tent compounds in the series are 8h, 8r and 8v, with
IKK-b inhibitory potencies of pIC50 7.0, 6.8 and 6.8,
respectively. The series has excellent selectivity, both
within the IKK family over IKK-e, and across a wide
variety of kinase enzyme and binding assays. The po-
tency in the IKK-b enzyme assay translates to signifi-
cant cellular activity (pIC50 5.7–6.1) in assays of
functional and mechanistic relevance.
10. Similar IKK-b activity (IC50 = 14.5lM) has recently been
reported for compound 8c, see Ref. 6.
11. The sequence of IKK-b was aligned to a large panel of
protein kinase structures. CDK-2 (27% identical in the
kinase domain to IKK-b) was chosen as the template
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 active-like ATP site conformation. MODELLER
Acknowledgements
Duncan B. Judd, Karen E. Lackey, David D. Miller,
Katherine L. Widdowson, Rick Williamson and Rick
Cousins are thanked for their support and useful
discussions.
ˇ
(Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779) was
used to generate IKK-b 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). Docking was carried out
using Gold version 2.1 (Cambridge Crystallographic Data
Centre, www.ccdc.cam.ac.uk).
References and notes
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