IRAK-4 inhibitors. Part II: A structure-based assessment of imidazo[1,2-a]pyridine binding

Article abstract of DOI:10.1016/j.bmcl.2008.04.039

A potent IRAK-4 inhibitor was identified through routine project cross screening. The binding mode was inferred using a combination of in silico docking into an IRAK-4 homology model, surrogate crystal structure analysis and chemical analogue SAR.

Full text of DOI:10.1016/j.bmcl.2008.04.039

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3291–3295
IRAK-4 inhibitors. Part II: A structure-based assessment
of imidazo[1,2-a]pyridine binding
George M. Buckley,a Thomas A. Ceska,b Joanne L. Fraser,a Lewis Gowers,a
a,
a
Colin R. Groom,a Alicia Perez Higueruelo,a,* Kerry Jenkins, Stephen R. Mack,
*
Trevor Morgan,a David M. Parry,a William R. Pitt,a Oliver Rausch,a
Marianna D. Richarda and Verity Sabina
aUCB, Granta Park, Great Abington, Cambridge CB21 6GS, UK
bUCB, 216 Bath Road, Slough, Berkshire SL1 4EN, UK
Received 12 March 2008; revised 15 April 2008; accepted 16 April 2008
Available online 22 April 2008
Dedicated to the memory of our friend and colleague David Rainey.
Abstract—A potent IRAK-4 inhibitor was identified through routine project cross screening. The binding mode was inferred using a
combination of in silico docking into an IRAK-4 homology model, surrogate crystal structure analysis and chemical analogue SAR.
ꢀ 2008 Elsevier Ltd. All rights reserved.
At UCB it is a routine practice to cross screen final com-
pounds and advanced intermediates across kinase pro-
jects in early Discovery phase (Hit-to-Lead through to
Lead Optimisation). Through this approach we identi-
fied compound 1, from our JNK kinase programme,1
which possessed significant potency in an IRAK-4 en-
zyme assay2 but was poorly active against JNK-1 and
JNK-2. There are many precedents for 2-aminopyrimi-
dine-based kinase inhibitors, with this motif typically
binding to the hinge backbone within the ATP-binding
site.3 This initially raised concerns over novelty and
the potential for promiscuity associated with this scaf-
fold. A close analogue example, 2, from our JNK
program exhibited this binding mode in a JNK-3 co-
crystal structure (Fig. 1).4 Attempts to crystallise IRAK
constructs in-house were unsuccessful and there were no
reported IRAK crystal structures in the public domain
at this time.5
N
N
1
N
NH
N
N
H
IRAK-4, IC50 216nM
JNK-1, IC50 3801nM
JNK-2, 59% inhib. @ 10μM
Some key analogues were prepared to explore the nature
of the binding in IRAK-4 (Scheme 1). The ‘methyl
capped’ compound, 4, retained similar levels of potency
to 1 suggesting that the bidentate HBA/HBD-binding
mode was not responsible for activity. This was further
supported by the O-linked pyrimidine, 5, which despite
lacking the aminopyrimidine-binding motif, still exhib-
ited low-micromolar activity. The regioisomeric
pyridines 6 and 7 showed contrasting SAR, with the 2,6-
pyridine isomer 6 having low-nanomolar potency whilst
the 2,4-pyridine isomer 7 showed little activity, despite
having a more accessible bidentate-binding motif.
Keywords: IRAK; IRAK-4; JNK; Kinase; Kinase inhibitor; IRAK-4
inhibitor; Homology model; Imidazopyridine; Imidazo[1,2-a]pyridine;
Binding mode; JNK crystal structure; GASP; Docking; Hinge binder;
Hinge region; Bidentate hydrogen bond; b-Turn; Protein modelling;
IRAK-4 homology model; IRAK-4 crystal structure; Aminopyrimi-
dine; Immunity; Anti-inflammatory; Hydrogen bond; P·G motif.
In the absence of an IRAK-4 crystal structure, a set of
homology models were built using structural informa-
tion from crystallographically elucidated kinases
*
Corresponding authors. Tel.: +44 1223 896300; e-mail addresses:
alicia@cryst.bioc.cam.ac.uk; drkerryjenkins@yahoo.co.uk
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.039

3292
G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3291–3295
N
N
N
N
N
NH
N
NH
N
N
N
O
CH3
4, IRAK-4, IC50 200nM
5, IRAK-4, IC50 1189nM
JNK-1, 33% inhib. @ 10μM
JNK-2, 35% inhib. @ 10μM
JNK-1, 24% inhib. @ 10μM
JNK-2, 30% inhib @ 10μM
N
N
N
N
H
N
NH
NH
N
N
N
H
H
H
6, IRAK-4, IC50 35nM
7, IRAK-4, 31% inhib. @ 10μM
JNK-1, 0% inhib. @ 10μM
JNK-2, 0% inhib. @ 10μM
JNK-1, 39% inhib. @ 10μM
JNK-2, 15% inhib. @ 10μM
Figure 1. Structurally related aminopyrimidine 2 in JNK-3 crystal
structure showing typical aminopyrimidine interaction with the hinge
region (PDB code 3CGF).4 IRAK-4 IC50 3 lM, JNK-3 IC50 270 nM.
N
H2N
HN
NH
N
NH
NH
NH
N
N
boc
possessing similarity to IRAK-4 with respect to either
the overall kinase domain or the ATP-binding site (for
a more detailed discussion see the Supplementary
information).
N
e
N
N
N
N
H
N
N
N
1
N
boc
N
N
c
N
N
O
d, e
N
N
N
N
H
Docking the cross-screening hit 1 (see Fig. 4 in supple-
mentary section) into the IRAK-4 homology models
suggested that the imidazopyridine nitrogen atom (N1)
bound to the hinge backbone (Met265) and the amino-
pyrimidine moiety merely served as a linking scaffold
rather than as the key binding feature. This binding
mode was observed with a closely related 2-methylated
aminopyrimidine analogue 8 (crystallised in JNK-3) that
is incapable of forming the usual aminopyrimidine
bidentate interaction (Fig. 2).6
f, g, h
N
a, b
CH3
4
5
HO
N
N
N
N
N
boc
N
i, j
N
N
SO2CH3
O
N
k
B(OH)2
N
N
H2N
N
N
N
N
boc
N
Cl
N
Pyridine 6 also docked favourably in this mode, adopt-
ing a ‘U-shaped’ conformation with the protonated
piperidine ring nitrogen sitting in the anionic pocket
formed by Asp329 and the carbonyl backbones of
Asn316 and Ala315. Conformational analysis of the li-
gand showed that the docked binding conformation clo-
sely resembled its global minima conformation (Fig. 3).7
NH
l
m, j
Br
N
Cl
N
N
H
7
n
H2N
N
N
N
e
N
boc
N
N
N
boc
o
NH
N
N
N
N
Docking experiments suggested that the poorly active
compound 7 preferentially docked to the hinge region
through the pyridine nitrogen (Fig. 4). Our homology
model and subsequent crystal structure data5 showed
that the IRAK-4 hinge region does not have the 2nd car-
bonyl (Pro266) available to interact with the 2-amino
group as it is engaged a type 1. b-Turn. Thus a bidentate
HBD/HBA pairing was not a viable binding mode. In
the ‘best docked’ mode the piperidine did not access
the anionic pocket and the imidazopyridine experienced
some notable close contacts.
N
H
N
Br
H
6
Scheme 1. Synthesis of hit 1 and analogues 4–7. Reagents and
conditions: (a) AlCl3, 0 ꢁC; Ac2O, 40 ꢁC; (b) DMF–DMA, reflux (35%,
two steps); (c) 1-Boc-4-guanidinopiperidine, NaH, DMF, reflux (60%);
(d) NaH, DMF, rt, MeI, 60 ꢁC (43%); (e) HCl, Et2O, DCM, MeOH
(63–100%); (f) Thiourea, NaOMe, nBuOH, 150 ꢁC, microwave; (g)
MeI, nBuOH, (44%, two steps); (h) mCPBA, DCM, (77%); (i) 1-Boc-4-
hydroxypiperidine, NaH, DMF, 80 ꢁC (49%); (j) TFA, DCM (6–43%);
(k) NBS, CHCl3 (86%); (l) 2-Chloropyridyl-4-boronic acid, Pd(PPh3)4,
Na2CO3, DME, H2O, 150 ꢁC, microwave (13%); (m) 4-Amino-1-Boc
piperidine, NaOtBu, Pd(OAc)2, 1,3-bisdiphenylphosphinopropane,
DME, 130 ꢁC, microwave (28%); (n) iPrMgCl, THF, À78 ꢁC;
nBu3SnCl, À78 ꢁC–rt, 2,6-dibromopyridine, Pd(PPh3)4, rt–66 ꢁC
(75%); (o) 4-Amino-1-Boc piperidine, NaOtBu, Pd(OAc)2,
tBu3PHBF4, DME, 85 ꢁC (76%).
Conformational analysis revealed that compound 7 is
more skewed than 6 in its low-energy conformations.
This is most likely due to the steric differences arising
from the regioisomeric pyridine methines interacting

G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3291–3295
3293
Figure 2. Structurally related aminopyrimidine 8 demonstrating alter-
native binding mode in JNK-3 crystal structure (PDB code: 3CGO).6
IRAK-4 IC50 400 nM, JNK-3, IC50 3 lM.
Figure 4. Suggested ‘best docked’ binding mode for 7. Single hydrogen
bond acceptor interaction through pyridine (yellow hashed line). Close
contacts with imidazopyridine evident (magenta hashed lines).8
Figure 3. Docking mode of 6 (white) overlayed with the lowest energy
conformation (orange). (Image generated with the program PyMol.)8
differently with both the imidazopyridine and aminopi-
peridine rings. This distorts the conformation of 7 from
the optimal ‘U-shape’ adopted by 6 and prevents the
favourable positioning of the piperidine nitrogen into
the anionic pocket. In this conformation the piperidine
of 7 is likely to significantly clash with the protein active
site surface. This can be seen from the overlay of 6 with
7 in its nearest local minima conformation (Fig. 5) and is
a plausible explanation for the difference in potency seen
between the two pyridine regioisomers.
Figure 5. Docking mode of 6 (orange) overlayed with 7 (yellow) in its
closest local minima to the ‘U-shape’ conformation.8
inhibited this enzyme. This further corroborated the sig-
nificance of the hydrogen bond accepting nitrogen in
this position in the bicyclic appendage. Interestingly,
indazole 11 showed moderately good IRAK-4 potency.
It is plausible that either nitrogen could act as a hydro-
gen bond acceptor through tautomerism, or alterna-
tively this heterocycle might bind through a different
mode altogether. Indeed it is noteworthy that com-
pounds 9 and 10 exhibit little or no JNK potency whilst
indazole 11 is modestly potent against JNK. This per-
haps implicates the indazole in some alternative mode
(or mixed modes) of binding. Isoquinoline 12 showed
a significant but modest potency, possibly reflecting
These data encouraged us to pursue 2,6-pyridines as a
discrete chemical series from the original pryimidine
hit. We then sought to further explore the role of the
imidazo[1,2-a]pyridine motif (Table 1 and Scheme 2).
Replacement of this bicycle with isoelectronic benzimid-
azole, 9, gave rise to a potent IRAK-4 inhibitor whilst
the approximately isosteric indole, 10, only very weakly

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G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3291–3295
Table 1. Alternative bicyclic heterocycles
There are many approaches to hit finding, such as
HTS/MTS of large screening decks against isolated
targets, high content cell-based screening, ligand-based
approaches (modification of natural ligands or known
pharmacologically active compounds) and structure-
based approaches (in silico screening, fragment screen-
ing by NMR or X-ray crystallography, de novo de-
sign). Here we have demonstrated the potential of
project cross-fertilisation through routine cross screen-
ing of medicinal chemistry target compounds and ad-
vanced intermediates. This simple and pragmatic
approach generated a new lead for our IRAK pro-
gram. This fortuitous hit may have been overlooked
from its perceived similarity to existing kinase inhibi-
tors. However, the binding mode of the hit molecule
was analysed in the absence of target crystallographic
structural information using a combination of in silico
modelling, synthetic analogues and surrogate kinase
(JNK-3) crystallography. This gave us confidence that
a novel binding mode was in operation and that the
aminopyrimidine motif could be changed to a new
and potentially more selective scaffold. The homology
modelling helped to guide the lead optimisation and
led to the generation of a highly potent series of
IRAK-4 inhibitors with good drug-like properties
and is the subject of another communication.9 The
validity of the IRAK homology models was subse-
quently corroborated by close overlay with published
crystallographic data.5
R
NH
9 - 12
N
N
H
Compound
R
IC50 nM (or % inhibition
at 10 lM)
IRAK-4, 70 nM
JNK-1 (0%)
JNK-2 (0%)
N
9
N
N
IRAK-4 (30%)
JNK-1 (0%)
JNK-2 (0%)
10
11
12
H
IRAK-4, 456 nM
JNK-1, 2080 nM
JNK-2, 1760 nM
N
N
IRAK-4, 1934 nM
JNK-1 (5%)
JNK-2 (0%)
N
N
N
N
b, c
b, c
N
benzimidazole
NH
NH
N
N
a
Br
N
H
Br
Br
N
9
a
Acknowledgements
Br
N
N
N
indole
N
The authors extend their appreciation to the Analytical
Sciences Department (for NMR, LC–MS and prepara-
tive HPLC), the enzyme assay and protein expression
biologists and to the members of the JNK kinase project
team.
N
H
10
H
N
N
boc
N
g, c
N
d, e, f
N
NH
N
N
N
H
N
H
Supplementary data
Br
11
12
N
H2N
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.04.039.
B(OH)2
N
Br
N
Br
boc
boc
N
N
N
NH
N
h
i, c
N
F
H
N
H
References and notes
Scheme 2. Synthesis of bicyclic replacement analogues, 9–12. Reagents
and conditions: (a) NaH, indole or benzimidazole, NMP (33–75%); (b)
4-Amino-1-Boc piperidine, NaOtBu, Pd(OAc)2,tBu3PHBF4, DME,
120 ꢁC, microwave (13–52%); (c) HCl, Et2O, DCM (27–70%); (d) I2,
1. (a) Alam, M.; Beevers, R. E.; Ceska, T.; Davenport, R. J.;
Dickson, K. M.; Fortunato, M.; Gowers, L.; Haughan, A.
F.; James, L. A.; Jones, M. A.; Kinsella, N.; Lowe, C.;
Meissner, J. W. G.; Nicolas, A.-L.; Perry, B. G.; Phillips, D.
J.; Pitt, W. R.; Platt, A.; Ratcliffe, A. J.; Sharpe, A.; Tait, L.
Bioorg. Med. Chem. Lett. 2007, 17, 3463; (b) Ratcliffe, A. J.;
World Patent WO038001, 2006.
i
KOH, DMF, (90%); (e) PrMgCl, THF, 0 ꢁC; nBu3SnCl, 0 ꢁC–rt, 2,6-
dibromopyridine, Pd(PPh3)4, rt–66 ꢁC (64%); (f) Boc2O, Et3N, DMAP,
MeCN (82%); (g) 4-Amino-1-Boc piperidine, Cs2CO3, Pd(OAc)2,
tBu3PHBF4, DME, 85 ꢁC (47%); (h) 4-Amino-1-Boc piperidine, THF,
140 ꢁC, microwave (50%); (i) Isoquinoline-4-boronic acid, Na2CO3,
nBu4NBr, Pd(PPh3)4, DME, H2O, reflux (40%).
2. Buckley, G. M. et al. Bioorg. Med. Chem. Lett. 2008, 18,
3211.
3. Some illustrative examples of aminopyrimidine kinase
hinge binders include: (a) Bingham, A. H.; Davenport, R.
J.; Gowers, L.; Knight, R. L.; Lowe, C.; Owen, D. A.;
Parry, D. M.; Pitt, W. R. Bioorg. Med. Chem. Lett. 2004,
19, 409; (b) Sayle, K. L.; Bentley, J.; Boyle, F. T.; Calvert,
A. H.; Cheng, Y.; Curtin, N. J.; Endicott, J. A.; Golding, B.
the somewhat off-linear hydrogen bond interaction to
Met265 seen in the best docked molecules (see Fig. 5
in the Supplementary section).

G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3291–3295
3295
T.; Hardcastle, I. R.; Jewsbury, P.; Mesguiche, V.; Newell,
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6. Coordinates submitted to the PDB: code 3CGO.
7. Conformational Searches were carried out with the pro-
gram Sybyl (Tripos International, 1699 South Hanley
Road, St. Louis, Missouri 63144, USA) using the grid
search with 30ꢁ increment for both rotatable bonds (linker
between Imidazopyridine–pyridine and between pyridine–
amino group) with further minimisation for each
conformer.
8. DeLano, W. L.; http://www.pymol.org.
9. Buckley, G. M. et al. Bioorg. Med. Chem. Lett. 2008,
doi:10.1016/j.bmcl.2008.04.042.
4. Coordinates submitted to the PDB: code 3CGF.
5. Since this work was carried out, researchers at Amgen
(formerly Tularik) have published the first X-ray crystal
structures of IRAK-4. Our homology model is in very close
agreement to this structural data (see Supplementary data).
Wang, Z.; Liu, J.; Sudom, A.; Ayres, M.; Li, S.; Wesche,
H.; Powers, J. P.; Walker, N. P. C. Structure 2006, 14, 1835.