IRAK-4 inhibitors. Part 1: A series of amides

Article abstract of DOI:10.1016/j.bmcl.2008.04.058

The synthesis and profile of a series of amides are described. Some of these compounds were potent IRAK-4 inhibitors and two examples were evaluated in vivo.

Full text of DOI:10.1016/j.bmcl.2008.04.058

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3211–3214
IRAK-4 inhibitors. Part 1: A series of amides
George M. Buckley, Lewis Gowers, Alicia Perez Higueruelo, Kerry Jenkins,
Stephen R. Mack, Trevor Morgan, David M. Parry, William R. Pitt, Oliver Rausch,
Marianna D. Richard, Verity Sabin* and Joanne L. Fraser
UCB, Department of Medicinal Chemistry, Granta Park, Great Abington, Cambridge, CB21 6GS, UK
Received 13 March 2008; revised 23 April 2008; accepted 23 April 2008
Available online 26 April 2008
Dedicated to the memory of our friend and colleague David Rainey.
Abstract—The synthesis and profile of a series of amides are described. Some of these compounds were potent IRAK-4 inhibitors
and two examples were evaluated in vivo.
Ó 2008 Elsevier Ltd. All rights reserved.
The interleukin-1 receptor associated kinases (IRAKs)
are a family of serine/threonine kinases involved in
mediating cellular signalling downstream of IL-1, IL-
18 and a number of Toll-like receptors.1 IRAK-4 is crit-
ical for the activation of intracellular signalling cas-
cades, such as the NFjB and MAPK pathways, which
are essential for the production of inflammatory cyto-
kines.2 Mice lacking IRAK-4 are viable and show com-
plete abrogation of inflammatory cytokine production
in response to IL-1, IL-18 or LPS.3 Similarly, human pa-
tients lacking IRAK-4 are severely immunocompro-
mised and are not responsive to these cytokines.4,5 The
role of IRAK-4 in innate immunity makes it an interest-
ing target for inhibition by small molecules.6 The SAR
of some N-acyl 2-aminobenzimidazole IRAK-4 inhibi-
tors7 1 and the crystal structure of one of these com-
plexed with the kinase domain of human IRAK-48
have recently been reported in the literature.
Through screening a library of small molecules against
IRAK-4, we identified the thiazole amide 2a as a prom-
ising starting point (IRAK-4 IC50 = 2.8 lM) for a
medicinal chemistry programme.
O
S
N
2a
H
N
Cl
N
Compound 2a was docked, using Gold,9 into a set of
homology models generated in-house with Modeller
software,10 using known structures of kinases possessing
similarities to IRAK-4 (Fig. 1).11 This study suggested
that the amide linker formed a key interaction with
the hinge region of IRAK-4, the terminal pyridine had
the potential for a p-stacking interaction with the tyro-
sine gatekeeper, and that space was available for substi-
tution around the aniline.
O
N
X
N
To investigate SAR around the aniline ring, compounds
with the general structure 2 were prepared (Scheme 1)
from 2-(3-pyridyl)-1,3-thiazole-4-carboxylic acid and a
diverse set of commercially available anilines selected
on the basis of electronics, lipophilicity, weight and
shape. Mono-substituted and multi-substituted anilines
were included, selecting such that the molecular weight
of the final compounds would not exceed 420. Single
point determinations of IRAK-4 by each compound at
H
Y
N
R
1
Keywords: IRAK; IRAK-4 inhibitor; Inflammation; Kinase; SAR;
Thiazole amide; Pyridine amide; Suzuki reaction; Docking; Micro-
somes; Solubility; In vivo exposure.
*
Corresponding author. Tel.: +44 01223 896300; fax: +44 1223
896400; e-mail: verity.sabin@UCB-group.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.058

3212
G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3211–3214
Table 1. IRAK-4 inhibition and rate of human microsomal clearance
by compounds 2a–2r
Compound
R
IRAK-4
Human
IC50 (lM)13 Clint
(lL/min/mg
protein)14
2a
2b
2c
2d
2e
2f
2-Cl
H
2-Me
2.8
7.2
7.3
>10
0.8
1.0
4.8
8.9
3.7
6.4
2.4
0.7
185
209
205
Not done
300
103
197
149
62
2-iPr
2-OMe
2-OCHF2
3-Me
3-OMe
2g
2h
2i
4-OMe
2j
4-Oxazole
4-Piperidine
4-(N-Methyl)
piperazine
4-Morpholine
2,6-Di-Cl
2,6-Di-Me
2,4-Di-OMe
59
40
2k
2l
24
Figure 1. Compound 2a docked into IRAK-4 homology model.
(Figure generated with the program PyMOL).12
2m
2n
2o
2p
2q
2r
2.8
36
>10
>10
1.2
Not done
Not done
111
R
O
O
2-OMe, 4-Morpholine 0.16
2-OMe, 4-(N-Methyl) 0.35
piperazine
20
14
a
HO
S
N
H
S
N
N
Values are means of at least two experiments.
N
N
2
to in vivo oxidation. 6-Bromo-2-pyridinecarboxylic acid
was coupled with 2-chloroaniline to give a versatile
bromoaryl intermediate, which was reacted with boro-
nate esters under Suzuki conditions to give 3a–3g
(Scheme 2). The IRAK-4 IC50 values for these com-
pounds were determined (Table 2). The similar potency
of the pyridine 3a to its thiazole analogue 2a gave us
confidence that SAR would track between the two
sub-series. The inactivity of the thiazole, pyrimidine
and amino pyridine compounds 3b, 3c and 3d suggested
that this part of the molecule was involved in something
more than a simple p-stacking interaction and that the
orientation and basicity of the nitrogen atom was key
for modulating activity against IRAK-4. The pyrazoles
3f and 3g were more successful, having improved
potency against IRAK-4 compared with 3a.
Scheme 1. Reagents: (a) (COCl)2, DMF, DCM; then appropriate
aniline, base, NMP, 18–60%.
10 lM were obtained on the whole library, giving a
range of activities from 0% to 98%. IRAK-4 IC50 values
and rates of in vitro clearance by human microsomes
were then determined for selected compounds 2b–2r
(Table 1).
Compared with the unsubstituted phenyl amide 2b,
ortho-substitution with chloro, methoxy and difluoro-
methoxy (2a, 2e and 2f, respectively) significantly im-
proved potency against IRAK-4, as did para-
substitution with electron-donating substituents such
as methoxy and nitrogen-linked heterocycles (2i, 2k, 2l
and 2m). These potency effects were additive, with the
most active examples in the set being the disubstituted
compounds 2q and 2r. The presence of nitrogen-linked
substituents at the para position also had a beneficial ef-
fect on the rate of turnover by human microsomes.
O
O
a
HO
N
H
N
N
Cl
Br
Br
Turning our attention to the other end of the molecule,
we next sought an alternative to the exposed terminal
pyridine ring to avoid potential complications associ-
ated with pyridine/pyridine-N-oxide cycling in vivo.
The pyridine N-oxide of 2a (readily formed by reacting
2a with urea hydroperoxide and trifluoroacetic anhy-
dride in DCM) was significantly less active (6.9 lM)
against IRAK-4 than the parent compound.
b
c
O
O
N
H
N
N
Cl
H
N
Cl
3a
Ar
3b-g
N
To enable us to quickly investigate SAR around the ter-
minal aromatic, we next changed the thiazole core to
pyridine with the pyridine nitrogen between the two
points of substitution. We found no evidence that a
2,6-disubstituted pyridine of this type would be prone
Scheme 2. Reagents and conditions: (a) 2-chloroaniline, HBTU,
DIPEA, DMF, 60%; (b) pyridyl boronate ester, Pd(PPh3)4, Na2CO3,
DME, H2O, lW, 150 °C, 5 min, 10%; (c) appropriate aryl boronate
ester, Pd(PPh3)4, Na2CO3, DME, H2O, lW, 150 °C, 5 min, 24–80%.

G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3211–3214
Table 2. Inhibition of IRAK-4 by compounds 3a–3g Table 4. In vivo blood exposure of 4a and 4b
3213
Compound
Ar
IRAK-4 IC50 (lM)13
Compound
Cmax (ng/mL)
Tmax (h)
AUC0–7 (ng h/mL)
4a
4b
652
266
7
1
7176
402
N
3a
3.4
Compounds 4a and 4b were progressed into an in vivo
PK study15 in Lewis rats dosed orally at 10 mpk (Table
4). Compound 4a was found to achieve higher levels of
exposure than 4b, as expected from the in vitro clear-
ance. 4a had the additional advantage of excellent aque-
ous solubility (3009 lM at pH 7.4).
3b
3c
3d
3e
3f
>10
>10
>10
1.6
N
S
N
N
N
In conclusion, a set of novel potent IRAK-4 inhibitors
has been prepared, and the rapid rate of microsomal
clearance associated with the original screening hit sig-
nificantly reduced. A selected example (4a) had suitable
properties, excellent solubility and good levels of in vivo
exposure.
NH2
H
N
N
0.66
0.71
NH
Acknowledgment
N
We thank the DMPK, Pharmaceutical Sciences and
Protein Expression departments for their contributions
to this work.
3g
NMe
N
Values are means of at least two experiments.
References and notes
Table 3. IRAK-4 inhibition and rate of rat and human microsomal
clearance by compounds 4a and 4b
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O
MeN
N
N
N
H
N
OMe
4a
N
O
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Science 2003, 299, 2076.
O
N
N
N
H
H
N
N
OMe
4b
Compound IRAK-4
14
Rat Clint
Human
14
IC50 (lM)13 (lL/min/mg Clint
protein)
(lL/min/mg protein)
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4a
4b
0.42
0.02
9
51
5
14
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Values are means of at least two experiments.
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R. J. Mol. Biol. 1997, 267, 727.
Following the synthetic route outlined in Scheme 2, but
using 2-methoxy-4-piperidin-1-yl aniline and 2-meth-
oxy-4-morpholin-4-yl aniline in place of 2-chloroaniline
in the first step, we combined some of the most promising
anilines from the thiazole series with some of our best
terminal aromatics from the pyridine series to give com-
pounds such as 4a and 4b. Both of these were sub-
micromolar inhibitors of IRAK-4 with acceptable rates
of clearance in both rat and human microsomes
(Table 3).
10. Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779.
11. Buckley, G. M.; Ceska, T. A.; Fraser, J. L.; Gowers, L.;
Groom, C. R.; Higueruelo, A. P.; Jenkins, K.; Mack, S.
R.; Morgan, T.; Parry, D. M.; Pitt, W. R.; Rausch, O.;
Richard, M. D.; Sabin, V. Bioorg. Med. Chem. Lett., in
press, doi:10.1016/j.bmcl.2008.04.039.
12. DeLano, W. L. http://www.pymol.org.
13. The IRAK-4 in vitro enzyme assay determined the effect
of test compounds on the phosphorylation of a fluorescein

3214
G. M. Buckley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3211–3214
labelled peptide detected using IMAP. It was performed in
14. To investigate rates of clearance by human and rat liver
microsomes, test compound (0.5 lM) and microsomal
protein (final concentration 1 mg/mL) were pre-incubated
at 37 °C for 10 min. Reactions were started by the addition
of NADPH and subsequent time points were stopped by
the addition of acetonitrile containing an internal standard
(ketoconazole). Samples were centrifuged 10,000 rpm for
10 min before analysis by LCMS. Disappearance of
compound was measured over 40 min.
15. Test compound was administered orally in a vehicle of 5%
DMSO/95% methylcellulose (0.5% w/v, 400 Cps) to a male
Lewis rat (Charles River). Serial blood samples were
collected by tail vein sampling and frozen before analysis.
Samples were analysed and concentrations quantified by
LCMS/MS. PK Parameters were calculated using Win-
NonLin (Pharsight).
black 384-well plates in a volume of 20 lL, comprising of
reaction buffer (10 mM Tris–HCl, 10 mM MgCl2, 0.1%
BSA, 1 mM DTT, 0.05% NaN3, pH 7.2), 30 lM ATP,
100 nM F-peptide substrate (5FAM-ERMRPRKRQ
GSVRRRV-NH2 Molecular Devices #RP7048), 2 nM
Baculo His-IRAK-4 (in-house) and test compound in 2%
DMSO. The kinase reaction was incubated for 1 h at room
temperature then terminated by addition of 60 lL Pro-
gressive Buffer A (Molecular Devices #R8124) containing
1/400 IMAP binding reagent (Molecular Devices
#R8124). The binding of IMAP to the phosphorylated
F-peptide substrate required 1 h of incubation at room
temperature. The fluorescence polarisation was
subsequently measured using
reader.
a Tecan Ultra plate