Discovery of Potent Benzolactam IRAK4 Inhibitors with Robust in Vivo Activity

Article abstract of DOI:10.1021/acsmedchemlett.9b00380

IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.

Full text of DOI:10.1021/acsmedchemlett.9b00380

Subscriber access provided by - Access paid by the | UCSF Library
Letter
Discovery of Potent Benzolactam IRAK4 Inhibitors with Robust in vivo Activity
Naomi S. Rajapaksa, Alberto Gobbi, Joy Drobnick, Steven Do, Aleksandr Kolesnikov, Jun Liang,
Yongsheng Chen, Swathi Sujatha-Bhaskar, Zhiyu Huang, Hans Brightbill, Ross Francis, Christine
Yu, Edna F. Choo, Kevin DeMent, Yingqing Ran, Le An, Claire Emson, Jonathan Maher, John
Wai, Brent S. McKenzie, Patrick J. Lupardus, Ali A. Zarrin, James R. Kiefer, and Marian C. Bryan
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00380 • Publication Date (Web): 11 Nov 2019
Downloaded from pubs.acs.org on November 12, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Discovery of Potent Benzolactam IRAK4 Inhibitors with Robust in
vivo Activity
Naomi S. Rajapaksa,† Alberto Gobbi,† Joy Drobnick,† Steven Do,† Aleksandr Kolesnikov,†,¢ Jun Liang,†
Yongsheng Chen,‡ Swathi Sujatha-Bhaskar,† Zhiyu Huang,† Hans Brightbill,† Ross Francis,† Christine
Yu,† Edna F. Choo,† Kevin DeMent,†,∇ Yingqing Ran,†,^ Le An,† Claire Emson,†,○ Jonathan Maher,†
John Wai,‡ Brent S. McKenzie,† Patrick J. Lupardus,†, # Ali A. Zarrin,†,□ James R. Kiefer,† and Marian
C. Bryan†*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AUTHOR ADDRESS †Genentech, Inc., One DNA Way, South San Francisco, California 94080, United States. ‡ WuXi
Apptech, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, P. R. China
KEYWORDS. Benzolactam, IRAK4, Lupus, Pyrazolopyrimidine.
ABSTRACT: IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines
implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of
these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent
studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a
human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 which
achieves robust in vivo inhibition of cytokines relevant to human disease.
A)
Identification of selective interleukin-1 receptor associated
kinase 4 (IRAK4) inhibitors remains of significant interest due
to their potential to intervene in immunological diseases.1-4
Specifically, IRAK4 forms a critical node in multiple
proinflammatory signaling pathways via the myddosome
complex.5 This complex transmits and amplifies the response of
interleukin-1 receptor (IL-1R) family and toll-like receptors
(TLRs, except for TLR3) to inflammatory cues.6-7 Upon
activation, IRAK4 phosphorylates IRAK1, triggers multiple
signaling cascades leading to gene transcription and initiation
of the inflammatory response. As part of the response, multiple
disease-relevant cytokines are released including Interferon
alpha (IFNα), Tumor necrosis factor alpha (TNFα), and
Interleukin-6 (IL-6). Secretion of IFNα leads to upregulation of
several inflammatory proteins and patients with systemic lupus
erythematosus (SLE) often show enhanced IFNα serum levels
correlating with disease activity.8 IL-6 is also dysregulated in
SLE, causing both systemic effects and local inflammation.
Therefore, there is strong interest in IRAK4 inhibition to
suppress this pathway.1,2,5
optimization of a series of selective pyrazolopyrimidine
inhibitors .13
O
CONH2
O
H
N
N
N
N
F
N
H
HO
N
N
HN
N
CN
O
NC
B) HN
O
NH
N
O
F
1: PF-06650833
2: BMS-986126
3
N
N
O
N
NH
HO
O
N
O
4
Recent IRAK4 inhibitors indicate a diversity of chemical
Figure 1. A) Examples of recently disclosed IRAK4 inhibitors with
B) Co-crystal structure of 4 with IRAK4 (1.8 Å, PDB ID 6O95).
matter,
including
isoquinoline
PF-06650833
(1),9
aminopyrimidine BMS-986126 (2),10 and quinazoline (3)11
(Figure 1) with multiple small molecule inhibitors have entered
into clinical trials for immunological diseases such as PF-
06650833 (1) and BAY1834845 for rheumatoid arthritis and
psoriasis. Indeed, a recent evaluation of the literature highlights
that the discovery of IRAK4 inhibitors continues to be an active
area of research.12 We also recently reported the discovery and
These molecules afford potent and selective inhibitors but
lack adequate oral bioavailability.13 The subsequent semi-
saturated dihydrobenzofurans provide more favorable
physicochemical properties while maintaining potency and
improving
selectivity,
leading
to
hydroxymethyl
dihydrobenzofuran 4 (Figure 1), a highly potent, orally
bioavailable IRAK4 inhibitor with dose-dependent reduction of
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 8
IL-6 in a CpG (deoxycytidyl deoxyguanosine oligonucleotide)-
inhibitor with an LLE of 6.3. Encouraged by this result, we
evaluated 6-membered lactam 10 and pyridone 11 which would
present the carbonyl oxygen in the same vicinity.
induced mouse PK/PD model.13 Despite the initial success of 4,
further profiling revealed inconsistent inhibition of multiple
disease-relevant cytokines, suggesting suboptimal pathway
suppression. Concomitantly, we developed a human whole
blood (HWB) assay to directly assess the effect of molecules on
both IL-6 and IFNα. This assay in particular captures the effects
in plasmacytoid dendritic cells (pDCs), which are the most
potent producers of IFNα in the blood.14
1
2
3
4
5
6
7
8
Unfortunately, both compounds lost potency, resulting in
decreased LLE. Sulfur-based scaffolds 12-14 showed a similar
trend as the indanone and indanols. Sulfone 12 and one
sulfoxide enantiomer (13) both had improved LLE (6.0 and 6.1,
respectively) when compared with 5, while the other sulfoxide
(14) lost nearly 100-fold potency. Finally, sultam 15 showed an
encouraging level of potency with 19 nM biochemical potency
and an LLE of 6.4.
Profiling 4 in HWB showed only moderate and highly
variable potency for both cytokines (Figure 2A, IL-6 IC50 460
nM ± 160 nM, IFNα IC50 400 nM ± 170 nM). Most
disconcertingly, 4 demonstrated variable, suboptimal maximal
inhibition (MI) for either cytokine (Figure 2B, IL-6 72% ±
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Evaluation of pyrazolopyrimidine IRAK4
inhibitors with semi-saturated scaffolds
CMPD
5
STRUCTURE
IRAK4 Ki (nM)a LogDb
LLE
7.4%, IFNα 56%
±
12%). Investigation of other
dihydrobenzofurans revealed a trend for incomplete inhibition
with moderate-to-low activity against IL-6 (Table S1). When
we profiled other tool molecules and scaffolds, values for both
showed improved consistency relative to 4 (Table S1).
5.3 ± 2.1
2.7
5.6
4.7
(n = 4)
6
30 ± 6.5
2.8
(n = 4)
N
N
O
7c
N
6.1 ± 1.2
2.2
NH
6.0
4.8
(n = 4)
N
HO
O
N
N
O
8c
N
160 ± 22
2.1
NH
(n = 4)
N
HO
O
9
26 ± 3.6
1.3
6.3
5.0
5.2
6.0
(n = 6)
Figure 2. Comparison of 4 and 19 in HWB assays. A) IC50 and B)
MI for IL-6 and INFα. Each point reflects at least two donors.
10
11
12
80 ± 18
2.0
(n = 4)
As the inconsistent pathway suppression was scaffold-
dependent, we explored alternate cores, hypothesizing that
modification would identify more potent IRAK4 inhibitors with
improved universal cytokine suppression. In addition, we chose
to focus on semi-saturated scaffolds with low lipophilicity
(LogD < 3) to maintain desired drug-like properties. Noticing
the higher lipophilic ligand efficiency LLE (8.1 using LogD) of
isoquinoline 1 and other previously assessed scaffolds13 relative
to 4 (LLE = 6.6), we aimed to identify alternate scaffolds with
improved LLE versus parent 5 (Table 1).
210 ± 42
1.5
(n = 4)
14 ± 3
1.8
(n = 3)
N
Previous structure-activity relationships indicated the
necessity of a hydrogen bond acceptor in the scaffold to engage
in a water-mediated H-bond.13 We incorporated alternative
functionality capable of either participating in such an
interaction or displacing a conserved water molecule (Figure
1B). Indanone 6, which is roughly liponeutral to
dihydrobenzofuran 5 and extends the H-bond acceptor
functionality further from the core, resulted in a 5-fold loss in
biochemical potency. Reduction of the ketone to secondary
alcohols 7 and 8 revealed significant activity differences
between the enantiomers. Lactam 9 performed similarly to
ketone 6, and despite an approximately 4-fold loss in
biochemical potency relative to 5, it was a more efficient
N
O
13d
N
22 ± 2.9
1.5
NH
6.2
(n = 4)
S
N
O
O
N
N
O
14d
15
N
490 ± 150
1.6
NH
N
4.7
6.4
(n = 4)
S
O
O
19 ± 1
1.3
(n = 4)
ACS Paragon Plus Environment

Page 3 of 8
ACS Medicinal Chemistry Letters
aData are geometric mean of at least two independent
experiments ± standard deviation for the number (n) of
experiments conducted. bLogD measured at pH 7.4.
c,dEnantiomeric with undetermined absolute stereochemistry.
mouse and human in vitro assays. However, 18 was only
moderately permeable. Fluorinated tertiary alcohol 19 had a
very similar potency profile to 18 (Ki = 1.2 nM, IL-6 IC50 = 0.16
μM) with improved permeability. Sultam 20, containing the
same solvent front group was not nearly as potent as 19 and only
showed modest potency improvements over methyl sultam 15.
This data along with the moderate mouse in vitro metabolic
stability of 20 encouraged us to focus our further optimization
studies on the lactam scaffold (A) over the sultam (B).
1
2
3
4
5
6
7
8
From this survey, sultam 15, sulfoxide 13, sulfone 12, and
lactam 9 stood out as efficient scaffolds based on LLE.
Computational docking studies with 9 and 15 suggested that the
N-methyl substituents pointed toward a solvent-exposed region
of the binding pocket. From our previous work, we knew this
vector could be used to optimize potency and physicochemical
properties13 and evaluated several lactam N-substituents, as
shown in Table 2.
At this point, we further evaluated compound 19 to ensure
that the scaffold changes were having the desired effect on
reproducibility and MI in the HWB assay. Encouragingly, 19
showed superior potency to 4 when assessed by IL-6, as
described above, or IFNα, which repeated over multiple runs
(Figure 2A). Lactam 19 also gave improved MI with less
variability across the two cytokines (Figure 2B). Of critical
importance is that the kinase selectivity of the scaffold was
maintained relative to 6. 13 Lactam 19 inhibits only 11 kinases
at >70% of a total 221 kinases when assayed at 1 µM with >100-
fold selectivity over the most potent off-target (Table S3, S4).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Transition to a high-throughput IL-6 HWB assay and
installation in the screening cascade could then identify
compounds with improved and consistent potency and MI.
While the observed biochemical-to-HWB potency shifts
generally correlated with human plasma protein binding, they
could also reflect the effects of permeability, physicochemical
properties, and solubility. Encouragingly, both 9 and 15
provided comparable or improved inhibition of IL-6 relative to
5, albeit with significant variability and low-to-moderate MI
(Table 2). Replacing the lactam N-substituent with polar
functionality (Table 2, compounds 16-19) was well tolerated
and provided a path to improved and less variable biochemical
and HWB potency.
We obtained a 1.7Å resolution X-ray co-crystal structure of
19 with IRAK4 (Figure 3). Consistent with the binding mode of
the dihydrobenzofurans,13 the pyrazolopyrimidine amide binds
in close proximity to gatekeeper residue Tyr262 and forms two
hydrogen bonding interactions with the hinge region via the
amide oxygen with the backbone NH of Met265 and the
polarized CH of the pyrazole with the backbone carbonyl of
Val263.
Cyclic ether 16 and acyclic methyl ether 17 provided single-
digit nanomolar biochemical potency and sub-micromolar
HWB potency with MI’s of 71% and 76%, respectively, but
with poor metabolic stability. Tertiary alcohol 18 improved
HWB potency (IL-6 IC50 = 0.19 μM, MI =78%) and stability in
Table 2. Solvent front optimization
N
N
N
N
O
O
N
N
NH
N
NH
N
R
N
R
N
S
O
O
O
O
O
A
B
CMPD
CORE
R
IRAK4 Ki
(nM)a
HWB
IL-6 IC50 (nM)a
MI
(M)
LMb /Hepc
(H)
LMb /Hepc
MDCK
(AB)d
(%)
5.0 ± 2.1
(n = 4)
26 ± 3.6
(n = 6)
19 ± 1.0
(n = 4)
3.6 ± 9.4
(n = 3)
0.38 ± 3.6
(n = 4)
7.2 ± 4.8
(n = 3)
5
9
N/A
A
N/A
58 ± 12
38 ± 3.6
67 ± 10
81 / 75
27 / 21
31 / 21
5.3 / 8.6
<3.9 / <6.2
<3.9 / <6.2
14.4
11.2
17.3
15
B
3.7 ± 0.91
(n = 4)
0.47 ± 0.15
(n = 3)
16
A
71 ± 4.4
73 / ND
13 / ND
ND
1.9 ± 0.49
(n = 6)
0.32 ± 0.55
(n = 5)
17
18
19
20
A
A
A
B
76 ± 7.3
78 ± 9.4
82 ± 9.2
60 ± 13
14 / 71
14 / 21
14 / 21
59 / 32
<3.9 / 15.3
<3.9 / <6.2
<3.9 / <6.2
<3.9 / <6.2
11.2
4.8
9.1
5.5
2.2 ± 0.31
(n = 14)
0.16 ± 0.82
(n = 5)
1.4 ± 0.32
(n = 30)
0.19 ± 0.079
(n = 10)
7.8 ± 0.87
(n = 4)
1.2 ± 0.46
(n = 3)
aData are geometric mean of at least two independent experiments ± standard deviation for the number (n) of experiments conducted.
bH/M LM : predicted hepatic clearance in liver microsomes for human (H) and mouse (M). Reported in units of mL/min/kg. cH/M Hep :
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 8
d
predicted hepatic clearance in hepatocytes for human (H) and mouse (M). Reported in units of mL/min/kg. AB, apical-to-basolateral
permeability as measured in Madin–Darby canine kidney (MDCK) cells. Reported in units of ×10–6 cm/s.
1
2
3
4
5
6
7
8
including I185, V200, and L318, further buttress 19 in the
binding site. Having optimized the solvent front substituent, we
turned our attention to the ATP ribose pocket. Incorporating
extended ribose pocket groups further improved HWB potency.
Difluoroethyl-piperazine 21 afforded HWB potency = 81 nM
without significantly compromising in vitro stability or
permeability.
9
An even more pronounced potency increase was realized
with azetidinyl-piperidine 22 (IC50 = 26 nM), though with
greatly compromised permeability. We hypothesized this was
driven by the highly basic tertiary amine and evaluated analogs
with tempered basicity (23-25). As predicted, difluoroazetidine
23 substantially lowered the pKa and restored permeability.
This compound also lost 4-fold in IL-6 IC50 relative to 22 and
was only predicted to be moderately stable in mouse based on
in vitro assays, though. Oxetanyl-piperazine 24 similarly
modulated basicity, though still poorly permeable. The non-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
basic close analog difluorocyclobutyl-piperazine 25 balanced
Figure 3. X-ray co-crystal structure of lactam 19 (gold) bound to
permeability and in vitro stability with HWB IC50 of 100 nM.
IRAK4 (green) (PDB ID XXXX). Polar interactions between the
Based on these compound profiles, we evaluated the mouse
in vivo pharmacokinetic profiles of lactams 19, 21, and 25.
Compound 19 (1 mg/kg) had low plasma clearance, in good
agreement with the hepatocyte stability assay and was 34% oral
bioavailable. Despite stable-to-moderate stability in liver
microsomes and hepatocytes, 21 and 25 displayed high plasma
clearances in vivo (58 and 79 mL/min/kg). However, 19
showed consistent agreement between in vitro and in vivo
clearance across species.
ligand and protein are indicated by dashed lines.
The carbonyl of the lactam makes two hydrogen bond
contacts to solvent molecules, including a water-mediated
interaction with Asp272. As anticipated, the lactam N-
substituent extends into a solvent-exposed region. A polarized
CH engages the backbone carbonyl of Pro266 in a hydrogen-
bonding interaction, which may contribute to the slight
improvement in biochemical potency of 19 as compared to 18.
Numerous van der Waals contacts to aliphatic residues,
Table 3. Ribose pocket group optimization
N
N
O
N
NH
R
N
F
O
HO
d
Cmpd
Structure
IRAK4 Ki
(nM)a
HWB IL-6
IC50 (µM)a
MI (%)
(M)
(H)
MDCK
(AB)c
cpKaMB
LM / Hepb
LM / Hepb
1.4 ± 0.32
(n = 30)
0.19 ± 0.079
(n = 10)
N
O
82 ± 9.2
85 ± 4.5
14 / 21
29 / 21
<3.9 / <6.2
<3.9 / <6.2
9.2
5.1
<3.0
4.4
19
21
2.6 ± 0.64
(n = 20)
0.081 ± 0.039
(n = 6)
N
F
N
F
1.9 ± 0.45
0.026 ± 0.013
N
87 ± 5.7
75 ± 7.5
83 ± 2.9
45 / 33
47 / 40
17 / 21
<3.9 / <6.2
<3.9 / <6.2
<3.9 / <6.2
0.1
16.2
0.7
9.0
6.1
6.0
22
23
24
N
(n = 4)
(n = 6)
N
3.4 ± 0.36
0.11 ± 0.046
N
F
(n = 4)
(n = 8)
F
N
3.7 ± 0.76
0.078 ± 0.02
N
(n = 8)
(n = 9)
O
ACS Paragon Plus Environment

Page 5 of 8
ACS Medicinal Chemistry Letters
N
3.5 ± 0.50
0.1 ± 0.23
1
2
N
84 ± 2.6
24 / 27
<3.9 / <6.2
11.2
4.0
25
F
(n = 6)
(n = 6)
F
3
4
5
6
b
aData are geometric mean of at least two independent experiments ± SD for the number (n) conducted. H/M LM: predicted hepatic
clearance in liver microsomes for human (H) or mouse (M), H/M Hep: predicted hepatic clearance in hepatocytes for human (H) or mouse
c
(M). Both reported in units of mL/min/kg. AB, apical-to-basolateral permeability as measured in Madin–Darby canine kidney (MDCK)
cells. Reported in units of ×10–6 cm/s.d Calculated basic pKa (cpKaMB) computationally determined using Moka v.2.6.
7
8
9
Table 4. PK data for relevant examples
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
b
b
CMPD SPECIES
PPB
(%)
LM / Hepa
(mL/min/kg)
14 / 21
CLP
VSS
T1/2b (h)
AUC∞b
(µM*h)
F%b
(L/kg)
(mL/min/kg)
MOUSE
RAT
94.2
91.4
77.6
66.8
72.4
91.3
94.7
29
2.2
1.9
1.6
4.1
5.4
1.2
3.8
3.1
2.2
34
31
<6.1/<10
8.6 / <7.8
8.1 / <8.8
<3.9 /<6.2
29 / 21
9.2
0.95
3.7
19
DOG
11.3
15.8
109
47
CYNO
3.7
HUMAN
MOUSE
MOUSE
21
25
58
79
0.96
1.8
0.18
0.28
0.5
0.4
ND
ND
24 / 27
aLM: predicted hepatic clearance in liver microsomes for indicated species, Hep: predicted hepatic clearance in hepatocytes for indicated
species. bParameters were determined from i.v. (1 mg/kg) and p.o. (1 mg/kg) PK studies.
On the basis of robust and consistent HWB potency and
bioavailability of 19, this molecule was advanced into further in
vivo studies. Attenuation of disease-relevant cytokines was
assessed upon stimulation of the TLR pathway via treatment
with the TLR7/8 agonist resiquimod , also known as R848.
Stimulation of the TLR on or within the antigen-presenting cells
(e.g. dendritic cells) leads to cell activation via the myddasome
complex. 15 This is followed by induction of pro-inflammatory
cytokines, chemokines, and the release of large amounts of type
I interferons together with upregulation of costimulatory
molecules. This model has previously been used to evaluate
other IRAK4 small molecules inhibitors including previous tool
molecule 4.16,17
In this in vivo model, C57 black 6 mice were dosed with
either vehicle, a nanosuspension of 4 or a nanosuspension of 19
across a dose range 1h prior to stimulation with R848. At 1h
post R848 stimulation, blood samples were obtained from the
animals and cytokine levels were measured. Three prototypic
proinflammatory cytokines measured in this study were IFNα,
IL-6, and TNFα. 18 Stimulation of IFNα leads to differentiation
of monocytes into dendritic-like cells, induction of natural killer
and natural killer T cells, and promotion of Interferon gamma
(IFNγ) production, among other roles.18 IL-6 has numerous
functions, playing important roles in immune regulation and
inflammation as well as autoimmune diseases. Finally, TNFα is
another potent inflammatory mediator whose roles include
induction of cytokine production, activation or expression of
adhesion molecules, and growth stimulation.
Both 4 and 19 demonstrate dose-dependent increases in
plasma concentration upon treatment (Figure 4A). In addition,
all three cytokines increased upon treatment with R848 in
vehicle-treated animals (Figure 4B; Figure S2 in the Supporting
Information). Although mice showed
a dose-dependent
inhibition of IL-6 secretion compared with vehicle-treated
animals (Figure S2), a greater discrepancy between the two
compounds was shown for the other two disease-relevant
cytokines. The mouse TNFα WB IC50 are within 2-fold (IC50
=
234 nM for 19 vs 425 nM for 4), though compound 19 showed
pronounced and dose-dependent inhibition of TNFα at doses as
low as 3 mg/kg, whereas compound 4 only achieved TNFα
inhibition at 100 mg/kg (Figure S2 in the Supporting
Information).
Figure 4. Effect of compound 4 and 19 on the proinflammatory
IFNα in an R848-induced mouse PD model. A) Plasma
concentration upon dosing a nanosuspension of either 4 or 19 in
MCT, 2 h after compound treatment and 1 h after stimulation
with R848. B) IFNα levels as determined by ELISA, 2 h after
compound treatment and 1 h after stimulation with R848.
A strong dose-dependent inhibition IFNα was also seen for
lactam 19 at doses as low as 3 mg/kg (Figure 4B) while
benzofuran 4 failed to show inhibition of IFNα across doses.
The variable response of 4, despite compound exposure
recapitulates the findings of the HWB assay and confirms the
decision to pursue alternate cores in order to ensure more robust
and consistent inhibition. Together, these findings show that
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 8
∇K. D.: Takeda Bio Development Center Ltd., 9625 Towne
compound 19 is therefore a strong tool molecule for elucidation
of the role of IRAK4 inhibition across disease modalities that
would be affected for several key proinflammatory cytokines.
1
2
3
4
5
6
7
8
Center Drive, San Diego, California 92121, United States.
○C. E.: Medimmune, Inc., 1 Medimmune Way, Gaithersburg,
Maryland 20878, United States.
We previously identified a series of potent and selective
dihydrobenzofurans that inhibited IRAK4 in a biochemical and
cellular assay, were orally available, and showed inhibition of
the cytokine IL-6 in a PK/PD experiment in a dose-dependent
manner. However, this molecule was hampered by insufficient
and variable inhibition of other cytokines of interest as
demonstrated in our newly initiated HWB assay. As such,
optimization of the scaffold was undertaken which led to the
discovery of the lactams presented herein. These molecules not
only maintained excellent biochemical potency and selectivity
but also showed robust inhibition in the HWB assay.
Optimization of pharmacokinetic properties yielded 19, a
highly desirable tool molecule. Compound 19 shows robust
inhibition of multiple cytokines of interest in a TLR-challenge
mouse model and provides a valuable tool for further in vivo
experiments across disease models.
¢A. K.: 623 38th Ave. San Francisco, CA 94121, United States.
^Y. R.: 836 Polaris Ave., Foster City, CA 94404, United States.
#P. J. L.: Synthekine, Inc., 1700 Owens Street, Suite 500, San
Francisco, California 94157, United States.
□A.Z.: TRex Bio 863 Mitten Rd, Burlingame, CA 94010, United
States.
9
Author Contributions. The manuscript was written through
contributions of all authors. / All authors have given approval
to the final version of the manuscript.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Funding Sources. The authors declare no competing financial
interest.
ACKNOWLEDGMENT
Crystal diffraction data were collected at Southeast Regional
Collaborative Access Team (SER-CAT) 22-ID (or 22-BM)
beamline at the Advanced Photon Source, Argonne National
Laboratory. SER-CAT is supported by its member institutions (see
Experimental Procedures
A
modular synthetic route was devised from key
nitrobenzoate intermediate 26 to prepare a diverse array of
benzolactam IRAK4 inhibitors (Scheme 1). Upon treatment of
primary bromide 26 with a primary amine, the inhibitor core
www.ser-cat.org/members.html),
and
equipment
grants
(S10_RR25528 and S10_RR028976) from the National Institutes
of Health.
(27) was efficiently synthesized by
a one-step SN2
displacement/lactam formation. The ribose pocket group was
installed through a subsequent SNAr displacement of the aryl
chloride. Nitro-arene 28 was then reduced to the corresponding
aniline (29) under various conditions, and the
pyrazolopyrimidine hinge binding element was readily installed
under amide coupling conditions to furnish IRAK4 inhibitor 30.
ABBREVIATIONS
AB, apical-to-basolateral permeability; cpKaMB, calculated most
basic pKa; CpG, TLR agonist deoxycytidyl deoxyguanosine
oligodinucleotides; ELISA, Enzyme-linked immunosorbent assay;
Hep, predicted hepatic clearance in hepatocytes; HWB, human
whole blood; IFNα, interferon alpha; IFNγ, interferon gamma; IL-
1R, interleukin-1 receptor; IL-6, interleukin-6; IRAK4, interleukin-
1 receptor associated kinase 4; LLE, lipophilic ligand efficiency;
LM, predicted hepatic clearance in liver microsomes; MCT, 0.5%
methyl-cellulose/0.2% tween-80; MDCK, Madin-Darby canine
kidney cells; MI, maximal inhibition; PBS, phosphate-buffered
saline; PD, pharmacodynamic; PK, pharmacokinetic; R848, TLR
agonist resiquimod; SLE, systemic lupus erythematosus; TLR, toll-
like receptor; TNFα, tumor necrosis factor alpha
Scheme 1. Synthesis of benzolactam IRAK4 inhibitors
c
NO2
NO2
NO2
Cl
a
b
Br
R1
N
R1
N
O
R2
Cl
N
O
R3
O
O
26
27
28
N
N
O
N
N
N
N
NH2
d
N
NH
R1
N
R1
R2
N
N
N
N
R2
O
O
N
R3
Cl
31
OH
O
O
R3
29
30
32
Reagents and conditions: (a) NH2R1, triethylamine, methanol
(48%-quant.); (b) NHR2R3, potassium carbonate (63%-quant.); (c)
tin(II) chloride dihydrate, or Fe, ammonium chloride, or H2, Pd/C
(quant.); (d) 31, triethylamine, or 32, 7-Azabenzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate, 2,4,5-
trimethylpyridine (13%-quant.).
REFERENCES
1. Bryan, M. C.; Rajapaksa, N. S. Kinase inhibitors for the treatment
of immunological disorders: recent advances. J. Med. Chem.
2018, 61 (20), 9030–9058.
2. Patra, M. C.; Choi, S. Recent Progress in the Molecular
Recognition and therapeutic importance of interleukin-1 receptor-
associated kinase 4. Molecules 2016, 21, 1529-1544.
3. Chaudhary, D.; Robinson, S.; Romero, D. L. Recent advances in
the discovery of small molecule inhibitors of interleukin-1
receptor associated kinase 4 (IRAK4) as a therapeutic target for
inflammation and oncology disorders. J. Med. Chem. 2015, 58,
96–110.
4. De Nardo, D.; Balka, K. R.; Gloria, Y. C.; Rao, V. R.; Latz, E.;
Masters, S. L. J. Interleukin-1 receptor-associated kinase 4
(IRAK4) plays a dual role in myddosome formation and Toll-like
receptor signaling. J. Biol. Chem. 2018, 293, 15195–15207.
5. Lin, S.-C.; Lo, Y.-C.; Wu, H. Helical assembly in the MyD88-
IRAK4-IRAK2 complex in TLR/IL-1R signaling. Nature, 2010,
465, 885–890.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Assay conditions, experimental procedures, and crystallography
data (PDF)
AUTHOR INFORMATION
Corresponding Author
6. Ozato, K.; Tsujimura, H.; Tamura, T. Toll-like receptor signaling
and regulation of cytokine gene expression in the immune system.
BioTechniques, 2002, 33, S66–S75.
*E-mail: bryan.marian@gene.com.
Present Addresses
ACS Paragon Plus Environment

Page 7 of 8
ACS Medicinal Chemistry Letters
7. Takeuchi, O.; Akira, S. Pattern recognition receptors and
Yang, R.; Torres, L.; Zhang-Hoover, J.; Northrup, A.
Identification of quinazoline based inhibitors of IRAK4 for the
treatment of inflammation. Bioorg. Med. Chem. Lett. 2017, 27,
2721–2726.
inflammation. Cell, 2010, 140, 805–820.
1
2
3
4
5
6
7
8
8. Ohl, K.; Tenbrock, K. J. Inflammatory cytokines in systemic
lupus erythematosus. Biomed. Biotechnol. 2011, 2011, 432595:
1–14.
12. McElroy, W. T. Interleukin-1 receptor-associated kinase 4
(IRAK4) inhibitors: an updated patent review (2016-2018).
Expert Opin. Ther. Pat. 2019, 29, 243-259.
9. Lee, K. L.; Ambler, C. M.; Anderson, D. R.; Boscoe, B. P.; Bree,
A. G.; Brodfuehrer, J. I.; Chang, J. S.; Choi, C.; Chung, S.;
Curran, K. J.; Day, J. E.; Dehnhardt, C. M.; Dower, K.; Drozda,
S. E.; Frisbie, R. K.; Gavrin, L. K.; Goldberg, J. A.; Han, S.;
Hegen, M.; Hepworth, D.; Hope, H. R.; Kamtekar, S.; Kilty, I. C.;
Lee, A.; Lin, L.-L.; Lovering, F. E.; Lowe, M. D.; Mathias, J. P.;
Morgan, H. M.; Murphy, E. A.; Papaioannou, N.; Patny, A.;
Pierce, B. S.; Rao, V. R.; Saiah, E.; Samardjiev, I. J.; Samas, B.
M.; Shen, M. W. H.; Shin, J. H.; Soutter, H. H.; Strohbach, J. W.;
Symanowicz, P. T.; Thomason, J. R.; Trzupek, J. D.; Vargas, R.;
Vincent, F.; Yan, J.; Zapf, C. W.; Wright, S. W. Discovery of
13. Bryan, M. C.; Drobnick, J.; Gobbi, A.; Kolesnikov, A.; Chen, Y.;
Rajapaksa, N.; Ndubaku, C.; Feng, J.; Chang, W.; Francis, R.; Yu,
C.; Choo, E. F.; DeMent, K.; Ran, Y.; An, L.; Emson, C.; Huang,
Z.; Sujatha-Bhaskar, S.; Brightbill, H.; DiPasquale, A.; Maher, J.;
Wai, J.; McKenzie, B. S.; Lupardus, P. J.; Zarrin, A. A.; Kiefer,
J. R. Development of Potent and Selective Pyrazolopyrimidine
IRAK4 Inhibitors. J. Med. Chem. 2019, 62, 6223-6240.
14. Fitzgerald-Bocarsly, P.; Dai, J.; Singh, S. Plasmacytoid dendritic
cells and type I IFN: 50 years of convergent history. Cytokine
Growth Factor Rev. 2008, 19, 3–19.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
clinical
candidate
1-{[(2S,3S,4S)-3-ethyl-4-fluoro-5-
oxopyrrolidin2-yl]methoxy}-7-methoxyisoquinoline-6-
carboxamide (PF06650833), a potent, selective inhibitor of
interleukin-1 receptor associated kinase 4 (IRAK4), by fragment-
based drug design. J. Med. Chem. 2017, 60, 5521–5542.
15. Van, L. P.; Bardel, E.; Gregoire, S.; Vanoirbeek, J.; Schneider, E.;
Dy, M.; Thieblemont, N. Treatment with the TLR7 agonist R848
induces regulatory T-cell-mediated suppression of established
asthma symptoms. Eur. J. Immunol. 2011, 41, 1992–1999.
16. Genung, N. E.; Guckian, K. M. Small molecule inhibition of
interleukin-1 receptor-associated kinase 4 (IRAK4). Prog. Med.
Chem., 2017, 56, 117–163.
17. Lim, J.; Altman, M. D.; Baker, J.; Brubaker, J. D.; Chen, H.;
Chen, Y.; Fischmann, T.; Gibeau, C.; Kleinschek, M. A.; Leccese,
E.; Lesburg, C.; Maclean, J. K. F.; Moy, L. Y.; Mulrooney, E. F.;
Presland, J.; Rakhilina, L.; Smith, G. F.; Steinhuebel, D.; Yang,
R. Discovery of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide inhibitors of IRAK4. ACS Med.
Chem. Lett. 2015, 6, 683–688.
10. Dudhgaonkar, S.; Ranade, S.; Nagar, J.; Subramani, S.; Prasad, D.
S.; Karunanithi, P.; Srivastava, R.; Venkatesh, K.; Selvam, S.;
Krishnamurthy, P.; Mariappan, T. T.; Saxena, A.; Fan, L.;
Stetsko, D. K.; Holloway, D. A.; Li, X.; Zhu, J.; Yang, W.-P.;
Ruepp, S.; Nair, S.; Santella, J.; Duncia, J.; Hynes, J.; McIntyre,
K. W.; Carman, J. A. Selective IRAK4 inhibition attenuates
disease in murine lupus models and demonstrates steroid sparing
activity. J. Immunol. 2017, 198, 1308–1319.
11. Smith, G. F.; Altman, M. D.; Andresen, B.; Baker, J.; Brubaker,
J. D.; Chen, H.; Chen, Y.; Childers, M.; Donofrio, A.; Ferguson,
H.; Fischer, C.; Fischmann, T. O.; Gibeau, C.; Hicks, A.; Jin, S.;
Kattar, S.; Kleinschek, M. A.; Leccese, E.; Lesburg, C.; Li, C.;
Lim, J.; Liu, D.; Maclean, J. K. F.; Mansoor, F.; Moy, L. Y.;
Mulrooney, E. F.; Necheva, A. S.; Presland, J.; Rakhilina, L.;
18. (18) Turner, M. D.; Nedjai, B.; Hurst, T.; Pennington, D. J.
Cytokines and chemokines: At the crossroads of cell signalling
and inflammatory disease. Biochim. Biophys. Acta. 2014, 1843,
2563–2582.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 8
Insert Table of Contents artwork here
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
ACS Paragon Plus Environment