Discovery and optimization of a potent and selective indazolamine series of IRAK4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127686

IRAK4 is a key mediator of innate immunity. There is a high interest in identifying novel IRAK4 inhibitors for the treatment of inflammatory autoimmune diseases. We describe here a highly potent and selective IRAK4 inhibitor (HS271) that exhibited superio

Full text of DOI:10.1016/j.bmcl.2020.127686

Journal Pre-proofs
Discovery and Optimization of A Potent and Selective Indazolamine Series of
IRAK4 Inhibitors
Wenqiang Zhai, Yongping Lu, Yabo Zhu, Mengguang Zhou, Cheng Ye,
Zheng-Zheng Shi, Wenjian Qian, Taishan Hu, Lei Chen
PII:
S0960-894X(20)30797-6
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127686
BMCL 127686
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
21 August 2020
5 November 2020
10 November 2020
Please cite this article as: Zhai, W., Lu, Y., Zhu, Y., Zhou, M., Ye, C., Shi, Z-Z., Qian, W., Hu, T., Chen, L.,
Discovery and Optimization of A Potent and Selective Indazolamine Series of IRAK4 Inhibitors, Bioorganic &
Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127686
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Bioorganic & Medicinal Chemistry Letters
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Discovery and Optimization of A Potent and Selective Indazolamine Series of
IRAK4 Inhibitors
a
a
a
a
a
a
Wenqiang Zhai , Yongping Lu , Yabo Zhu , Mengguang Zhou , Cheng Ye , Zheng-Zheng Shi , Wenjian
,
a
a
a
Qian* , Taishan Hu , Lei Chen
aZhejiang Hisun Pharmaceutical Co. Ltd., China, 46 Waisha Rd., Taizhou 318099, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
IRAK4 is a key mediator of innate immunity. There is a high interest in identifying novel IRAK4
inhibitors for the treatment of inflammatory autoimmune diseases. We describe here a highly
potent and selective IRAK4 inhibitor (HS271) that exhibited superior enzymatic and cellular
activities, as well as excellent pharmacokinetic properties. HS271 displayed robust in vivo anti-
inflammatory efficacy as evaluated in rat models of LPS induced TNFα production collagen-
induced arthritis.
Keywords:
IRAK4
2
009 Elsevier Ltd. All rights reserved.
Inflammation
Indazole
TNFα
Collagen-induced arthritis
Interleukin-1 receptor associated kinase 4 (IRAK4) is an
In the exploration of various scaffolds, the indazole compound
intracellular serine-threonine kinase that belongs to the IRAK 120 was chosen as a starting point for medicinal chemistry efforts
1
-2
family. IRAK4 is a key signaling mediator downstream of IL-1 (Figure 1).
family receptors (IL-1Rs) and Toll like receptors (TLRs), and plays
an essential role in the initiation and control of innate immune
O
F3C
N
3
responses.
4
3
HN
OH
5
N
Multiple genetic evidences have demonstrated the associations
of IRAK4 with inflammation. IRAK4-deficient or kinase dead
knock-in mice showed severe defects in cytokine responses in
2
N
1
6
7
OH
1
IRAK4 IC50 (nM): 8.6
THP-1 IC50 (nM): 128
IRAK1 IC50 (uM): 3.6
Solubility (PBS 7.4, uM): 6.7
Microsomal Clearance CLint (mL/min/Kg, Human /Mouse /Rat): 1.28/ 3.91/ 3.11.
Rat PK: Rat PK @ 5mpk,po (Cmax/AUC/t1/2/F) :2313/18400/3.6/84%.
4
arthritic models .Additionally, cells derived from IRAK4-deficient
individuals have impaired responses to ILR/TLR receptor
5
-6
stimulation.
Therefore, modulation of IRAK4 activity has
emerged as a promising strategy to control inflammatory
responses. IRAK4 has been recognized as an attractive target for
the treatment of inflammatory autoimmune diseases such as
Figure 1. Key properties of compound 1
7
-8
9,10
rheumatoid arthritis (RA) and other related diseases
.
Firstly, we screened various compounds with substitutes on the
N2 position of compound 1. To our delight, the replacement of
tertiary alcohol with amines delivered improved biochemical and
cellular activities. Thus, a series of indazolamine derivatives were
further synthesized (Table 1). The dimethylamino compound 2 was
first examined, and found that 15-fold enhancement in the
biochemical assay, and 2-fold enhancement in the cell-based assay
as compared with the compound 1. As compared with compound
There has been considerable interest in developing orally
available IRAK4 inhibitors. Although diverse molecules have been
1
1-19
reported , there is still a need for potent and selective IRAK4
inhibitors with improved potency and pharmacokinetic (PK)
profiles. Herein, we report a series of indazolamine IRAK4
inhibitors which showed potent anti-inflammatory activities in
cell-based assays, excellent PK properties in preclinical models，
and more importantly, robust in vivo efficacy, as reflected by
suppressed LPS-induced TNFα production and alleviated collagen-
induced arthritis (CIA) in rat models.
2
, the diethylamino analog 3 delivered loss in kinase potency and
cellular potency. The azetidino derivatives 4 and 5 were explored
with poor cellular potency. Additional SAR studies on the N2
position, including amide, sulfonamide and pyrazole derivatives

(compound 6-10), were unable to identify any better compounds the C7 position could only tolerate a small group. Therefore,
than compound 2.
2 2
additional small substituents such as F, CN, NH and NMe were
employed. However, none of these (compound 18-21) could retain
the potency well. Given the key dimethylamino group of
compound 2, compound 22 that combined the C7-methoxy and
dimethylamino group was rationally designed. However,
compound 22 was proved to be less potent than compound 2 and
Table 1. SAR of Indazolamines at the N2 Position
O
F3C
N
HN
R1
N
N
1
6. Furthermore, replacement of the dimethylamino fragment of
OH
the compound 22 with a piperidine ring, the analog 23, showed an
acceptable THP-1 activity. The demethylation of compound 23
was unable to retain the potency (compound 24 vs compound 23).
Cmpd
R1
IRAK4 IC50 (nM)
.6
0.55
THP-1 IC50 (nM)
OH
1
2
8
128
55
Table 3. SAR for C-7 migration
N
O
3
F3C
N
1
1
6.7
0.6
514
N
HN
R3
NH
R3
4
5
Cmpd
1
IRAK4 IC50 (nM)
THP-1 IC50 (nM)
128
1400
N
OH
N
1
76
28
NDa
N
8
.6
N
HO
NHSO2Et
6
7
NDa
1
2
O
N
0.55
55
CN
N
N
N
36.7
>2000
256
HO
1
3
OH
OH
OH
SO2Et
8
9
N
N
N
NDa
5
4
7
7
>2000
N
H
OH
N
730
O
14
NDa
1
0
170
N
NDa
N
N
1
13
N
aND: Not Detect
1
1
5
6
Secondly, the R2 moiety was evaluated in the scaffold of
>
2000
NDa
110
compound 2. As shown in Table 2, two derivatives 11 and 12 were
designed and synthesized. The bi-aryl compound 12 was found
with comparable activity as compound 2.
N
O
OH
OH
2
Table 2. SAR of R substituents
N
N
8
.3
N
N
R2
O
O
O
O
F3C
N
HN
N
HN
OH
17
N
N
NDa
N
N
2
02
09
OH
1
OH
Cmpd
R2
IRAK4 IC50 (nM) THP-1 IC50 (nM)
1
8
OH
OH
OH
N
N
N
NDa
3
1
2
8.6
128
55
N
N
N
F
0
.55
19
F3C
N
S
NDa
1
600
1
1
1
2
34.8
198
CN
NH2
N
F3C
N
2
2
2
0
1
2
7.7
58.7
O
N
NDa
>
>
2000
2000
96
OH
N
NDa
N
Thirdly, the migration of C6 tertiary alcohol of compound 1 was
explored (Table 3). Nevertheless, the IRAK4 potency of compound
3 was completely lost. While the cyclopropyl substituent
compound 14) showed moderate potency on IRAK4 kinase and
was subjected to further optimization. A large morpholine
substituent (compound 15) and a small methoxy one (compound
N
1
(
N
N
N
N
223
125
375
N
N
N
O
O
O
2
2
3
4
1
6) were chosen to be synthesized. Gratifyingly, the introduction
N
5
.7
of a methoxy group (compound 16) improved the IRAK4 -
inhibitory activity as well as THP-1 potency significantly. Inspired
by the discovery, further modification at the C7 position was
undertaken. The introduction of an ethoxy group (compound 17)
led to a substantial loss in IRAK4 potency, which suggested that
NH
4
1

aND: Not Detect
(Figure 3A). Compound 16 could also effectively block TNFα
production with a maximum inhibition at 53% (Figure 3B).
Therefore, compound 2 (hereafter named HS271) was chosen for
further investigations.
As we know, the co-crystal structure of lead compound 1 and
IRAK4 protein was not available. Three strategies including bio-
isosteres replacement, C7-migration and aryl modification were
conducted for the lead compound 1 (Figure 2). It has been found
out that 1) N-7 position exploration revealed that the
dimethylamine was the best one, the bulky group in N-7 was
unfavorable; 2) bi-aryl derivative retained the potency well; 3)
C7-migration developed a new series, the methoxy group were
the best choice. Several functional groups were not tolerated well
in the C-7 position, including the bulky alkyl group, basic amino
derivatives, and electron-withdrawing group.
400
A
3
2
1
00
00
00
0
4
6.01%
*
62.11%
**
9
1.00%
***
Vehicle
15mpk
50mpk 150mpk
O
N
O
Compound 2
N
HN
OH
N
N
HO
200
B
12
bi-aryl
1
1
50
00
50
0
3
1.76%
O
O
F3C
N
F3C
N
bioisosteres
51.75%
52.65%
HN
N
HN
OH
*
*
N
**
N
N
N
HO
HO
1
2
C7-migration
O
Vehicle 15mpk 50mpk 150mpk
Compound 16
F3C
N
HN
OH
N
N
O
Figure 3. Evaluation of compound 2 and 16 in a rat model of LPS-induced
16
TNF response. *P<0.05, **P<0.01，***P<0.001, vs vehicle
Figure 2. Three Strategies for lead optimization of compound 1
With these promising biochemical and cellular potency data,
compound 2, 12, 16 and 23 were selected for further studies. As
shown in Table 4, compound 2, 16 and 23 exhibited excellent
selectivity against IRAK1. In a rat PK study, the bi-aryl compound
HS271 was evaluated using a rat model of collagen induced
arthritis (CIA) (Figure 4). It led to a significant reduction in paw
swelling as compared to vehicle control, with a minimum effective
dose at 15 mg/kg QD. Notably, at 150 mg/kg QD, HS271
eliminated the paw swelling.
1
2 exhibited inferior exposure as compared with others. Moreover,
compound 23 displayed a hERG inhibition risk (hERG IC50 = 6.9
μM). Taken together, compound 2 and 16 were advanced into in
vivo efficacy studies.
The HS271 was stable in liver microsome assays across other
species, including rat, mouse, monkey, and human (Table 5) and a
good correlation between metabolic stability and the in vivo PK
profile (Table 6).
Table 4. Enzymatic selectivity, PK exposure (rat), and hERG
inhibition of compound 2, 12, 16 and 23
Cmpd
2a,e
12b,f
NDd
21
16a,e
14.4
3323
23a,f
10.4
344
IRAK1 IC50 (μM)
7.2
C
max (ng/mL)
2107
AUC0-24h
11920
104
34533
5080
(ng/mL·h)
T
1/2 (h)
3.3
8.3
3.1
NDd
NDd
3.5
5.5
6.5
19.7
6.9
Cl (mL/min/kg)c
e
hERG IC50 (μM)
16.5
>30
a
PO 10 mg/kg; PO: peroral administration; b PO 2 mg/kg; Cl data was tested
c
d
e
@
1 mg/kg IV; IV: intravenous injection; ND: Not Detect; Formulation:
DMA:30%Solutol HS-15:Saline = 5:5:90 (v/v/v); Formulation:
DMSO:PEG200 = 5:95 (v/v); hERG: Human ether-a-go-go related gene.
f
e
Figure 4. Evaluation of HS271 (compound 2) in a rat model of collagen
induced arthritis (CIA). PO: peroral administration; QD: quaque die.
Table 5. In Vitro ADME Properties of HS271
The rat systemic LPS induced TNFα response model was
conducted to assess compound 2 and 16 for their in vivo efficacy
in blocking cytokine production (Figure 3). Male Sprague-Dawley
rats were orally administered (PO) with either vehicle or compound
at different dosages. At 1 h post-dosing, the animals were
stimulated with LPS at 1 mg/kg intravenously, and blood was
collected 3 h post-stimulation for TNFα assay. Compound 2
achieved up to 91% TNFα inhibition in a dose-dependent manner
Micorsomal
8.82
(mouse
)
5.25
15
116
1.96
Clearance Clint
(human)
(rat)
(dog)
(monkey)
(mL/min/kg)

6
-
P
1)
P
app × 10 (cm·s
2
7.27 (A-B), 22.70 (B-A)
In conclusion, we have identified a novel indazolamine series of
IRAK4 Inhibitors. The pre-clinical candidate HS271 exhibited
superior biochemical and cellular activities, as well as excellent
ADME properties. HS271 displayed robust anti-inflammatory
efficacy in rat models of acute inflammation and CIA. To date, the
IRAK4 inhibitor HS271 has advanced into pre-clinical
development.
app (B-A)/ Papp
0.83 (efflux ratio)
(A-B)
Human
hepatocyte
stability t1/2
Human
583 min
PPBa
98.6 (human)
(%)
Acknowledgment
Solubility (μM)
PPB: Plasma Protein Binding
602 (PBS 7.4), 92.3 (FassIF 6.5)
This work was funded in total by Zhejiang Hisun Pharmaceutical Co. Ltd.
a
Table 6. In Vivo Pharmacokinetic Profiles of HS271 in Mouse,
Appendix A. Supplementary data
Rat, Dog, and Monkey
Supplementary data to this article can be found online at
species
mouse
rat
dog
monkey
IVd POe
IVd
1
POe IVd
10 0.5
POe
2
IVd
POe
1
.
References and Notes Li, S.; Strelow, A.; Fontana, E. J. et al.
Proc. Nat. Acad. Sci. U.S.A. 2002, 99. 5567.
Janssens, S.; Beyaert, R. Mol. Cell 2003, 11. 293.
dose
mg/kg)
1
2
0.5
2
(
2.
3
.
Suzuki, N.; Suzuki, M.; Yeh, W.-C. Trends Immunol. 2002, 23.
t
1/2 (h)
1.62 NAc
mL/min/kg 17.1 NAc
2.43 NAc
2.01
8.3
3.31 0.72 6.40
NAc 55.6 NAc
NAc 2.63 NAc
1.96 3.50
4.86 NAc
5
03.
CL
4.
5
6
Suzuki, N.; Suzuki, S.; Duncan, G. S. et al. Nature 2002, 416, 750.
Picard, C.; Puel, A.; Bonnet, M. et al. Science 2003, 299, 2076.
Ku, C.-L.; von Bernuth, H.; Picard, C. et al. J. Exp. Med. 2007,
.
.
(
)
2
04, 2407.
Huang, Q.; Ma, Y.; Adebayo, A. et al. Arthritis Rheum. 2007, 56,
192.
7
8
.
.
V
ss(L/kg)
1.30
0.67 NAc
1870 3670
2
Joosten, L. A.; Netea, M. G. Arthritis Rheum. 2009, 60, 1571.
Chaudhary, D.; Robinson, S.; Romero, D. L. J. Med. Chem. 2015,
5
AUC0-24h
1192
9
66 1300 2023
148 85.3
0
9.
(ng/mL▪h)
8, 96.
Scott, J. S.; Degorce, S. L.; Anjum, R. et al. J. Med. Chem. 2017,
0, 10071.
1. Lee, K. L.; Ambler, C. M.; Anderson, D. R. et al. J. Med. Chem.
017, 60, 5521.
2. Bryan, M. C.; Drobnick, J.; Gobbi, A. et al. J. Med. Chem. 2019,
2, 6223.
3. Smith, G. F.; Altman, M. D.; Andresen, B. et al. Bioorg. Med.
Chem. Lett. 2017, 27, 2721.
4. Hanisak, J.; Seganish, W. M.; McElroy, W. T. et al. Bioorg. Med.
Chem. Lett. 2016, 26, 4250.
NAc 67.3
NAc
58.2 NAc
NAc
10.
F (%)
14.4
49
6
a
b
Formulation: DMA:30%Solutol HS-15:Saline = 5:5:90 (v/v/v); Formulation:
DMA:30%Solutol HS-15:Saline = 10:10:80 (v/v/v); NA: Not Applicable;
IV: intravenous injection; PO: peroral administration.
1
1
1
1
c
2
d
e
6
Furthermore, the cytochrome P450 inhibitions of HS271 were
investigated to assess possible drug-drug interactions. No
inhibition was observed (IC50 values > 30μM) in major cytochrome
P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6,
CYP3A4t, CYP3A4m, CYP2B6 and CYP2C8).
15. Seganish, W. M.; Fischmann, T. O.; Sherborne, B. et al. ACS Med.
Chem. Lett. 2015, 6, 942.
The synthesis of HS271 was straightforward, as shown in
Scheme 1. The indazolamine core 26 from commercially available
ortho-nitrobenzaldehyde (compound 25) via a mild, one-pot
16. Rajapaksa, N. S.; Gobbi, A.; Drobnick, J. et al. ACS Med. Chem.
Lett. 2020, 11, 327.
1
7. McElroy, W. T.; Tan, Z.; Ho, G. et al. ACS Med. Chem. Lett. 2015,
, 677.
8. Lim, J.; Altman, M. D.; Baker, J. et al. ACS Med. Chem. Lett.
015, 6, 683.
6
2
1
condensation−Cadogan reductive cyclization
synthesized. The intermediate 27 was prepared under KNO
con H SO conditions, and then reduced under 1 atm H to furnish
the amino intermediate 28. Subsequently, the intermediate 28 was
was efficient
1
3
and
2
2
4
2
19. McElroy, W. T. Expert Opin. Ther. Pat. 2019, 29, 243.
20. Tobias, T.; Johannes, P.; Nicolas, G. WO Patent 2017186700,
2007.
2
2
coupled with 6-(trifluoromethyl)picolinoyl chloride to afford the
amide product 29. Finally, the Grignard reaction was conducted
under MgMeCl and LiCl conditions to successfully deliver HS271.
The overall yield was approximately 43%.
2
1. Genung, N. E.; Wei, L.; Aspnes, G. E. et al. Org. Lett. 2014, 16,
114.
3
2
2. The 6-(trifluoromethyl)picolinoyl chloride was synthesized from
the commercial available material 6-(trifluoromethyl)picolinic acid
(CAS No.: 131747-42-7). The detail was found in support
Scheme 1. Synthetic Route of HS271
information.
1
. H2N
N
CHO
MeOH
N
Con. H SO
2
KNO3
69%
4
N
N
MeOOC
NO2
2
8
.
Bu3P
N
N
MeOOC
4%
25
26
O
F3C
N
O2N
N
H2N
N
Cl
Pd/C, H2
HOAc, MeOH
N
N
DIPEA, DCM
MeOOC
MeOOC
83% over two steps
28
27
O
O
F3C
N
F3C
N
HN
N
HN
MgMeCl, LiCl
N
N
N
89%
HO
MeOOC
N
N
29
HS271

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Discovery and Optimization of A
Potent and Selective Indazolamine
Series of IRAK4 Inhibitors
Leave this area blank for abstract info.
Wenqiang Zhai, Yongping Lu, Yabo Zhu, Mengguang Zhou, Cheng Ye, Zheng-Zheng Shi, Wenjian
Qian*, Taishan Hu, Lei Chen
O
F3C
N
HN
N
N
N
OH
2
IRAK4 IC50 (nM): 0.55
THP-1 IC50 (nM): 55
IRAK1 IC50 (uM): 7.2