Design and synthesis of Imidazo[1,2-b]pyridazine IRAK4 inhibitors for the treatment of mutant MYD88 L265P diffuse large B-cell lymphoma

Article abstract of DOI:10.1016/j.ejmech.2020.112092

Harboring MYD88 L265P mutation triggers tumors growth through the activation of NF-κB by interleukin-1 receptor associated kinase 4 (IRAK4) in diffuse large B-cell lymphoma (DLBCL), highlighting IRAK4 as a therapeutic target for tumors driven by aberrant MYD88 signaling. Herein, we report the design, synthesis, and structure?activity relationships of imidazo[1,2-b]pyridazines as potent IRAK4 inhibitors. The representative compound 5 exhibited excellent IRAK4 potency (IRAK4 IC₅₀ = 1.3 nM) and favorable kinase selectivity profile. It demonstrated cellular selectivity for activated B cell–like (ABC) subtype DLBCL with MYD88 L265P mutation in cytotoxicity assay. The kinase inhibitory efficiency of compound 5 was further validated by Western blot analysis of phosphorylation of IRAK4 and downstream signaling in OCI-LY10 and TMD8 cells. Besides, combination of compound 5 and BTK inhibitor ibrutinib synergistically reduced the viability of TMD8 cells. These results indicated that compound 5 could be a promising IRAK4 inhibitor for the treatment of mutant MYD88 DLBCL.

Full text of DOI:10.1016/j.ejmech.2020.112092

Journal Pre-proof
Design and synthesis of Imidazo[1,2-b]pyridazine IRAK4 inhibitors for the treatment of
mutant MYD88 L265P diffuse large B-cell lymphoma
Yun Chen, Gang Bai, Yi Ning, Shi Cai, Tao Zhang, Peiran Song, Jinpei Zhou, Wenhu
Duan, Jian Ding, Hua Xie, Huibin Zhang
PII:
S0223-5234(20)30059-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112092
EJMECH 112092
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 November 2019
Revised Date: 21 January 2020
Accepted Date: 21 January 2020
Please cite this article as: Y. Chen, G. Bai, Y. Ning, S. Cai, T. Zhang, P. Song, J. Zhou, W. Duan, J.
Ding, H. Xie, H. Zhang, Design and synthesis of Imidazo[1,2-b]pyridazine IRAK4 inhibitors for the
treatment of mutant MYD88 L265P diffuse large B-cell lymphoma, European Journal of Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112092.
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Graphical abstract

Design and Synthesis of Imidazo[1,2-b]pyridazine IRAK4
Inhibitors for the Treatment of Mutant MYD88 L265P Diffuse
Large B-Cell Lymphoma
a,f,#
b,c,d,#
b,c
a
b,c
b,c
Yun Chen , Gang Bai
, Yi Ning , Shi Cai , Tao Zhang , Peiran Song , Jinpei
e
c, f
b,c,d
b,c,*
a,*
Zhou , Wenhu Duan , Jian Ding
, Hua Xie
, Huibin Zhang
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, PR China
c
University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
d
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, PR China
e
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR
China
f
Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu
Chong Zhi Road, Shanghai 201203, PR China
#
Equal contribution: Both authors contributed equally to this work.
Correspondence to: Huibin Zhang, E-mail : zhanghb80@163.com, Tel: 86-25-83271302;
Hua xie, E-mail : hxie@simm.ac.cn, Tel: 86-21-50805897.
*
Abstract
Harboring MYD88 L265P mutation triggers tumors growth through the activation of NF-κB by
interleukin-1 receptor associated kinase 4 (IRAK4) in diffuse large B-cell lymphoma (DLBCL),
highlighting IRAK4 as a therapeutic target for tumors driven by aberrant MYD88 signaling. Herein,
we report the design, synthesis, and structure−acꢀvity relaꢀonships of imidazo[1,2-b]pyridazines
as potent IRAK4 inhibitors. The representative compound 5 exhibited excellent IRAK4 potency
(
IRAK4 IC = 1.3 nM) and favorable kinase selectivity profile. It demonstrated cellular selectivity
50
for activated B cell–like (ABC) subtype DLBCL with MYD88 L265P mutation in cytotoxicity assay.
The kinase inhibitory efficiency of compound 5 was further validated by Western blot analysis of
phosphorylation of IRAK4 and downstream signaling in OCI-LY10 and TMD8 cells. Besides,
combination of compound 5 and BTK inhibitor ibrutinib synergistically reduced the viability of
1

TMD8 cells. These results indicated that compound 5 could be a promising IRAK4 inhibitor for the
treatment of mutant MYD88 DLBCL.
Key words: Interleukin-1 receptor associated kinase 4; Imidazo[1,2-b]pyridazine; Diffuse large
B-cell lymphoma; Drug design; Antitumor agents
Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype representing
roughly 30-35% of all non-Hodgkin lymphomas case.[1] Based on the gene expression profiling
DLBCL could be divided into two major molecular subtypes termed germinal center B-cell-like
(
GCB) and activated B-cell-like (ABC) which has a poor prognosis.[2] ABC-DLBCL is addicted to
constitutive B-cell receptor (BCR) signaling and NF-κB activation rendering them sensitive to
Bruton's tyrosine kinase (BTK) inhibitors ibrutinib.[3, 4] However, harboring MYD88 L265P variant
which provides an alternative pathway to activate NF-κB was found to convey resistance to
ibrutinib in BCR-independent patients,[5] and this recurrent gain of function mutation occurred
in 29% of ABC-DLBCL patients.[6] Therefore, novel therapeutic drugs need to be developed to
tackle this urgent problem.
IRAK4 is an essential effector to promote tumor progression in MYD88 L265P mutated DLBCL.[6]
Mutated MYD88 protein induces dimerization of IRAK4 via death domain and initiates
trans-autophosphorylation of IRAK4 without external ligands stimulation.[7, 8] Subsequently, it
triggers the allosteric activation of IRAK1,[7] phosphorylation of the IKKβ which resulting in the
degeneration of IκB and constitutive NF-κB activation.[8-10] Recently, Zhang et al. reported that
downregulation of phosphorylated IRAK4 and NF-κB could disrupt tumor–stroma IL-1β-IRAK4
feedforward circuitry and increase chemotherapeutic efficacy in PDAC.[11, 12] Remarkably,
individuals who lack IRAK4 show only increased susceptibility to certain pyogenic infections
without extensive viral or fungal infection risk prior to adolescence.[13, 14] These suggest that
IRAK4 is a promising anti-tumor target and selective IRAK4 inhibitors may have anti-neoplastic
activity while avoiding severe side effect.
In the past decade, small molecule IRAK4 inhibitors of various chemotypes have been described
and reviewed,[15, 16] with notable contributions from groups including Pfizer,[17] Curis,[18]
2

BMS,[19, 20] Nimbus,[21] AstraZeneca,[22, 23] and Genentech[24] (Figure 1). It has been
reported that, in pyrazolo[1,5-a]pyrimidine IRAK4 inhibitors, an intramolecular hydrogen bond
between the amide N−H and pyrimidine nitrogen stabilizes the structure into a planar
conformation that allows for a favorable slanted face-to-edge π−π interaction with the
gatekeeper Tyr262 and moreover pyrazolo[1,5-a]pyrimidine moiety binds to the hinge region
effectively through the backbone of Met265 and Val263 via the amide carbonyl and the unusual
C2 hydrogen. [24-26] Given that potential selectivity issues of the pyrazolo[1,5-a]pyrimidine
scaffold,[27] a series of new IRAK4 Inhibitors were designed through scaffold-hopping strategy
(
Figure 2). We retained all of the intra- and intermolecular interactions between
pyrazolo[1,5-a]pyrimidine moiety and IRAK4 to afford imidazo[1,2-b]pyridazine scaffold.
Meanwhile, in consideration of the atypical hydrogen bond provided by C2 hydrogen of pyrazole
ring, we designed an imidazo[1,5-a]pyrimidine scaffold to determine whether integrated “hinge
binding element” was essential to IRAK4 potency (Figure 2). Most of IRAK4 inhibitors described
in the literature were focused on anti-inflammatory and autoimmune disease, but few of IRAK4
inhibitors involved in the treatment of DLBCL were reported. Herein, we reported a series of
imidazo[1,2-b]pyridazines as IRAK4 inhibitors focusing on the application of the treatment of
DLBCL.
O
F
O
H
HN
N
N
N
O
N
F
N
H
N
HO
N
O
O
N
NH
N
N
O
O
O
N
O
HN
N
N
OH
H N
2
BMS
BMS-986126
Pfizer
PF-06650833
Curis
CA-4948
O
N
H N
O
2
O
N
N
N
O
O
HO
N
HN
N
HO
NH
O
N
O
N
N
O
S
N
N
H
Nimbus
AstraZeneca
Genentech
ND-2158
Figure 1. Representative small molecule inhibitors of IRAK4.
3

Figure 2. Rational design of target compounds by scaffold hopping strategy.
Results and Discussion
Enzymatic activity and cytotoxicity assay of alternative scaffold representations
All alternative scaffold representations listed in Table 1 were evaluated for IRAK4 inhibitory
potency using Z-lyte kinase assay. After optimizing the Ser/Thr protein kinase detection platform,
IC values of these compounds were determined by inhibition rate curve of various dosages. As
50
is shown in Table 1, the imidazo[1,2-b]pyridazine derivatives (compounds 3 and 5) showed equal
potency (IC = 8.7 nM and 1.3 nM, respectively) to their counterparts pyrazolo[1,5-a]pyrimidines
50
(
compounds 1 and 4) against IRAK4. The imidazo[1,5-a]pyrimidine derivative 2 characterized by
nitrogen atom at the 2-position, completely lost activity compared to compounds 1 and 3,
indicating that C2 proton from pyrazole or imidazole ring was vital to retain IRAK4 potency.
Corresponding methylated derivatives of 4 and 5 at C2 position (compounds 4-Me and 5-Me)
exhibited significant loss of IRAK4 potency compared to compounds 4 and 5. It further confirmed
that the importance of the C2 proton from pyrazole or imidazole ring.
These compounds were also tested cytotoxicity against DLBCL cell lines in vitro (Table 1).
Compound 5 selectively suppressed OCI-LY10 and TMD8 cells with MYD88 L265P mutation (IC =
50
0
.7 μM and 1.2 μM, respectively), but not Ramos and HT cell lines with WT MYD88 (IC = 11.4
50
μM and 40.1 μM, respectively). Whereas compound 4 inhibited all cells mentioned above
without selectivity presumably due to the potential off-target toxicity. These results indicated
that compound 5 possessed higher target selectivity and less toxicity than 4.
Based on the excellent IRAK4 potency and selective cytotoxicity of 5, imidazo[1,2-b]pyridazine
scaffold represented an exciting start point for the development of a novel IRAK4 inhibitor against
ABC DLBCL.
4

Table 1
Enzymatic activity and cytotoxicity assay of alternative scaffold representations.
IRAK4
OCI-LY10
TMD8
Ramos
HT
Compd
[
a]
[a]
[a]
[a]
[a]
IC (nM)
IC (μM)
50
IC (μM)
50
IC (μM)
50
IC (μM)
50
50
9
.3 ± 2.5
5.5 ± 0.4
7.3 ± 2.4
19.0 ± 8.5
29.2 ± 6.9
>
1000
.7 ± 1.0
.5 ± 0.8
> 50
> 50
> 50
> 50
8
2
10.7 ± 2.1
0.2 ± 0.0
17.8 ± 6.1
0.2 ± 0.0
44.3 ± 6.8
0.6 ± 0.0
41.7 ± 1.8
2.7 ± 0.7
1
.3 ± 0.1
0.7 ± 0.1
1.2 ± 0.4
11.4 ± 3.8
40.1 ± 7.1
[
[
b]
b]
[b]
[b]
[b]
>
1000
1000
NT
NT
NT
NT
NT
[b]
[b]
[b]
>
NT
NT
NT
[
[
a] Data are the mean ± SD of at least three independent experiments.
b] NT represents for no test.
SARs of imidazo[1,2-b]pyridazines and cellular activity evaluation
The modification of compound 5 was first focused on replacement of (3R)-3-aminopiperidine
moiety with various amines, and compounds 6-13 was obtained (Table 2). Biological results
suggested that R-configuration in 5 was important for maintaining IRAK4 inhibitory potency, and
the S-isomer 6 displayed a 10-fold less potency against IRAK4. The 5-membered ring (7, IRAK4
IC = 7.7 nM) and 7-membered ring (9, IRAK4 IC = 9.0 nM) exhibited minimal decrease in
50
50
5

potency, which indicated the size of the ring influences slightly the activity. Nevertheless,
compound 8 lost potency completely, suggesting that the 5-membered ring compared to
6
-membered ring may have different SARs. The position of amino in aminopiperidine group is
vital to IRAK4 inhibitory potency. The 4-substituted analogue 10 resulted in almost 10-fold loss of
potency. In addition, replacement of (3R)-3-aminopiperidine group (5) with a piperazine group
(
12) displayed 20-fold loss of potency. Although a tertiary amine attached to the scaffold (3) could
be tolerated, the (3R)-3-methylaminopiperidine analogue (13) showed a complete loss of potency.
Throughout the SAR studies, compound 5 still showed highest potency.
Then SARs of pyrazole moiety were investigated. Compared with 5, substituents with
trifluoromethyl (14), cyan (15), and formamide (16) were 7-, 12-, and 4-fold less potent,
respectively. Replacing the difluoromethyl group with a methyl (17) resulted in dramatic
reduction in potency, suggesting that the electron-withdrawing groups in pyrazole moiety could
be beneficial for IRAK4 potency. Modifications of the trifluoromethyl-pyrazole 14, selected for
synthetic feasibility over difluoromethyl-pyrazole 5, were then undertaken to investigate the
vectors to the solvent front region. All of the compounds demonstrated disappointing results.
The potency decreased with larger substituents toward solvent (18 and 19). When hydrophilic
groups 4-piperidyl (20) and 4-tetrahydropyranyl (21) were introduced to pyrazole moiety, IRAK4
potency was eroded presumably due to steric clashed with the IRAK4. Through the thorough
investigation of SARs, we found that 5 remained the most effective IRAK4 inhibitor.
We subsequently selected potent compounds in enzymatic assays to access their cell killing
ability against DLBCL cell lines in vitro (Table 4). All of these compounds displayed selective
cytotoxicity against MYD88 L265P mutation cell lines, OCI-LY10 and TMD8, without obvious
cytotoxicity against Ramos and HT cell lines. Among these compounds, the IC50 values of 9
(
OCI-LY10 IC = 0.8 μM and TMD8 IC = 1.2 μM) showed a similar level to 5.
50 50
The binding model of 5 with IRAK4 was predicted by Glide docking in Schrodinger (Figure 3). The
results suggested that compound 5 could insert ATP pocket of IRAK4 deeply and form a single
hydrogen bond interaction provided by the amide oxygen with Met265 of the hinge region. The
(
3R)-3-aminopiperidine group, occupying the ATP ribose pocket, donates extensive
hydrogen-bonding interaction to Asp329, Asn316, and Ala315. The computational modeling
results might explain the excellent enzyme potency of compound 5.
6

Table 2
SAR of imidazo[1,2-b]pyridazines 3 and 5-13.
1
[a]
Compd
R
IRAK4 IC (nM)
50
3
8.7 ± 1.0
1.3 ± 0.1
36.2 ± 7.2
7.7 ± 1.3
> 1000
5
6
7
8
9
9.0 ± 1.3
80.3 ± 42.3
17.5 ± 4.5
65.5 ± 26.7
> 1000
1
1
1
1
0
1
2
3
[
a] Data are the mean ± SD of at least three independent experiments.
7

Table 3
SAR of imidazo[1,2-b]pyridazines 5 and 14-21.
3
4
[a]
Compd
R
R
IRAK4 IC (nM)
50
5
CHF2
CF3
Me
Me
1.3 ± 0.1
23.5 ± 13.0
40.8 ± 24.5
12.5 ± 4.8
> 1000
1
1
1
1
1
1
2
2
4
5
6
7
8
9
0
1
CN
Me
CONH2
Me
Me
Me
CF3
Et
32.3 ± 18.4
77.0 ± 41.3
52.2 ± 34.1
49.7 ± 24.5
CF3
i-Pr
CF3
4-piperidyl
4-THP
CF3
[
a] Data are the mean ± SD of at least three independent experiments.
Table 4
Cytotoxicity assay of potent compounds.
OCI-LY10
TMD8
Ramos
HT
Compd
[
a]
[a]
[a]
[a]
IC (μM)
IC (μM)
50
IC (μM)
50
IC (μM)
5
0
50
6
7
9
22.3 ± 3.8
3.6 ± 0.5
0.8 ± 0.1
8.8 ± 1.0
2.8 ± 0.3
5.8 ± 0.1
3.6 ± 0.5
10.5 ± 1.2
22.3 ± 8.0
8.3 ± 2.9
1.2 ± 0.4
17.7 ± 5.1
5.9 ± 2.4
14.8 ± 6.0
6.1 ± 2.8
11.1 ± 4.5
> 50
45.0 ± 4.6
7.2 ± 1.6
> 50
> 50
> 50
35.9 ± 3.1
> 50
1
1
1
1
2
1
4
5
8
1
> 50
> 50
28.8 ± 11.1
> 50
> 50
> 50
> 50
> 50
[
a] Data are the mean ± SD of at least three independent experiments.
8

Figure 3. Binding-mode analysis of 5 with IRAK4 (PDB code: 4Y73). (A) The (3R)-3-aminopiperidine moiety
donated extensive hydrogen-bonding interaction to ATP pocket. (B) The difluoromethyl-pyrazole moiety extended
to solvent and the (3R)-3-aminopiperidine moiety occupied ribose binding region of the ATP pocket.
Selectivity profiling of compounds 5 and 4
We further investigated the kinase selectivity profile of compounds 5 and 4 against a panel of
kinases (Figure 4). At the concentration of 0.1 μM, 5 showed less than 50% inhibition against
most of tested kinases, and displayed greater than 50% inhibition in only three kinases, including
FLT3, FLT3-ITD and c-Kit. These three kinases have been reported to be crucial in the
development of acute myeloid leukemia (AML), indicating a potential indication of these
structures.[9] By comparison, compound 4 showed more than 50% inhibition against eight
kinases, including FLT3, FLT3-ITD, c-Kit, RET, SRC, VEGFR3, VEGFR2, and ABL. These data
demonstrated that compound 5 possessed much higher selectivity than 4, which was consistent
with the more selective anti-proliferation function of 5 against tumor cells than 4.
9

Figure 4. Selectivity profiling of compounds 5 and 4 against a panel of kinases. Inhibitory activities of the
compounds (at the concentration of 0.1 μM) against these kinases were determined by using ELISA assay.
Compound 5 inhibits the viability of DLBCL cells by targeting IRAK4 signaling
To test whether the anti-tumor function of these IRAK4 inhibitors through inhibiting IRAK4 and
downstream signaling activation, we conducted Western blot analysis of OCI-LY10 and TMD8 cell
lines by incubating with or without compound 5 at indicated concentrations for 2h. The results
showed that compound 5 dose-dependently decreased the autophosphorylation of IRAK4
(
Thr-345/Ser-346) compared to vehicle control in OCI-LY10 cells (Figure 5A, B). Moreover, the
phosphorylation of downstream molecules, IKKβ and NF-κB transcription factor (p65), could also
be effectively blocked upon compound 5 treatment. The inhibitory effect on IRAK4 and its
downstream signaling in TMD8 cells was also examined, and similar results were observed
(
Figure 5C, D). These results indicated that compound 5 inhibited DLBCL cells by targeting IRAK4
signaling.
1
0

Figure 5. Compound 5 effectively inhibited the phosphorylation of IRAK4 and the activation of downstream
effectors IKKβ and p65 in OCI-LY10 (A, B) and TMD8 (C, D) cells. All protein samples treated with compound 5 or
DMSO were collected after two hours incubation and analyzed by Western blot. Protein quantitative data of
p-IRAK4, p-IKKβ and p-p65 were analyzed with Image J. Significance threshold was fixed at *p < 0.05, **p < 0.01,
***p < 0.001.
Combination of compound 5 with ibrutinib synergistically exhibits anti-tumor activity to DLBCL
cells
Previous study proved that combination of IRAK4 inhibitor and BTK inhibitor synergistically
decreased the cell viability in cells harboring gain-of-function MYD88 L265P mutation.[23] Hence,
we treated the BCR-dependent TMD8 cells with compound 5 and ibrutinib at different
concentrations for 72 h. The result showed that this combination strategy exhibited synergistic
anti-tumor effects against TMD8 cells compared to each treatment alone (Figure 6). According to
the combination index (CI) value obtained by the CalcuSyn software,[28] we found that
combining compound 5 at 0.3 μM with ibrutinib at 6.3 nM exerted maximum synergistic
response against TMD8 cells growth with CI of 0.13 (Figure 6). These results revealed that IRAK4
is a promising drug target for DLBCL treatment and the combined use of ibrutinib and IRAK4
inhibitors will obtain more benefits in future clinical utilization.
1
1

+
Figure 6. Synergistic anti-tumor efficacy of compound 5 and BTK inhibitor ibrutinib in MYD88 L265P TMD8 cells.
Cells were incubated with various doses of ibrutinib and compound 5 for 72 h. Experiment was conducted by at
least three times and all date were represented as mean ± SD. Statistical significance was analyzed by Student's
t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
Chemistry
These key intermediates 25, 27, 29, and 33a-33e were prepared as shown in Scheme 1. Both of
intermediates 25, 27, and 29 were synthesized from methyl 1-methyl-4-nitro-1H-pyrazole-3-
carboxylate (22), which have been previously disclosed.[25] Nitrification of 3-(trifluoromethyl)-
1
H-pyrazole (30) with fuming nitric acid provided 4-nitro-3-(trifluoromethyl)-1H-pyrazole 31.
Then nucleophilic substitution between 31 and iodoalkanes resulted in 32a-32c. It could not get
corresponding 32d-32e directly through nucleophilic substitution with iodide of 4-Boc-piperidyl
and 4-THP. Then p-toluenesulfonate of 4-Boc-piperidyl and 4-THP was used to react with 31, and
3
3
2d-32e was afforded. Hydrogenation of 32a-32e nitro group with Pd/C delivered intermediates
3a-33e.
The general synthetic route toward 3 and 5-21 was depicted in Scheme 2. An Aldol condensation
between 6-chloropyridazin-3-amine (34) and N,N-dimethylformamide dimethyl acetal yielded
compound 35. Nucleophilic aromatic substitution of 35 with ethyl bromoacetate followed by ring
closure
provided
ethyl
6-chloroimidazo[1,2-b]pyridazine-3-carboxylate
(36).
The
6
-methoxyimidazo[1,2-b]pyridazine-3-carboxylic acid instead of intermediate 37 was obtained
when 36 was hydrolyzed with LiOH in methanol/tetrahydrofuran/water, and intermediate 37 was
obtained in the mixed solvent without methanol. Subsequent condensation of 37 with
1
2

intermediate 25 provided key intermediate 38, which was converted to corresponding target
compounds 3, 5, and 6-13 through electrophilic aromatic substitution and subsequent
deprotection. Compound 39 was synthesized by the S Ar and hydrolysis reaction from 36.
N
Likewise, condensation of 39 with corresponding intermediates 33a-33e and subsequent
deprotection afforded target compounds 14-21.
Electrophilic aromatic substitution of 40 with tert-butyl (3R)-3-aminopiperidine-1-carboxylate
and hydrolysis provided carboxylic acid 41. Then condensation of intermediate 41 with 25 and
subsequent deprotection delivered target compound 2. Target compounds 1 and 4 could be
afforded through similar processes (Scheme 3).
Through similar synthetic process of 4 and 5, compounds 4-Me and 5-Me could be synthesized
from ethyl 5-chloro-2-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate 44 and ethyl 6-chloro-2-
methylimidazo[1,2-b]pyridazine-3-carboxylate 46 respectively (Scheme 4).
Scheme 1. Synthesis of Intermediates 25, 27, 29, and 33a-33e: a) DIBALH, DCM, 0 °C to rt; b) IBX, DMSO, rt; (c) DAST, DCM,
-
2 3 3 3 2 4
40 °C to rt; d) H , Pd/C, MeOH, rt; e) NH /MeOH, 50 °C, sealed tube; f) POCl , DIPEA, 0 °C to rt; g) Fuming HNO /H SO , 0 °C to rt;
2 3
h) tetrahydro-2H-pyran-4-yl 4-methylbenzenesulfonate or tert-butyl 4-(tosyloxy)piperidine-1-carboxylate, Cs CO , DMF, 70 °C; i)
2 3
iodoalkane, K CO , DMF, rt.
1
3

Scheme 2. Synthesis of target compounds 3 and 5-21: a) N,N-dimethylformamide dimethyl acetal, 100 °C; b) i. ethyl
2 2
bromoacetate, MeCN, 85 °C; ii. DIPEA, rt; c) LiOH·H O, THF/H O, 35 °C; d) intermediate 25, EDCI, HOBT, DIPEA, DMF, rt; e)
2
amines, KF, DIPEA, DMSO, 100 °C; f) tert-butyl (3R)-3-aminopiperidine-1-carboxylate, KF, DMSO, 100 °C; g) LiOH·H O,
2
MeOH/THF/H O, 30 °C; h) intermediates 27, 29 and 33a-e, EDCI, HOBT, DIPEA, DMF, rt; i) TFA, DCM, rt.
Scheme 3. Synthesis of target compounds 1, 2, and 4: a) tert-butyl (3R)-3-aminopiperidine-1-carboxylate or tert-butyl
2 2
(R)-3-aminopiperidine-1-carboxylate, KF, DMSO, 100 °C; b) LiOH·H O, MeOH/THF/H O, 50 °C; c) intermediate 25, HATU, DIPEA,
MeCN, rt; d) TFA, DCM, rt.
1
4

Scheme 4. Synthesis of target compounds 4-Me and 5-Me: a) tert-butyl (R)-3-aminopiperidine-1-carboxylate, KF, DMSO, 100 °C;
2 2 2 2
b) LiOH·H O, MeOH/THF/H O, 50 °C; c) intermediate 25, HATU, DIPEA, MeCN, rt; d) TFA, DCM, rt; e) LiOH·H O, THF/H O, 35 °C.
Conclusion
In summary, we designed and synthesized a series of imidazo[1,2-b]pyridazine IRAK4 inhibitors
and analyzed SARs. Among these compounds, 3, 5, 7, and 9 showed superior IRAK4 inhibitory
activity and cytotoxicity against MYD88 L265P mutation cell lines. In particular, compound 5
displayed excellent IRAK4 potency (IC = 1.3 nM) and 10-fold greater selectivity on MYD88 L265P
50
mutation cell lines (OCI-LY10 IC = 0.7 μM and TMD8 IC = 1.2 μM) than WT MYD88 cell lines.
50
50
Besides, compound 5 demonstrated a favorable selectivity profile and only three kinases (FLT3,
FLT3-ITD, and c-Kit) were observed for > 50% inhibition rate. Considering selectivity profile of 5, it
displayed cellular selectivity for ABC-DLBCL with MYD88 mutation in cytotoxicity assay
consequently. Western blot assay showed that compound 5 could inhibit the phosphorylation of
IKKβ and activation of NF-κB in OCI-LY10 and TMD8 cell lines. Finally, inhibition of NF-κB activity
translated to synergistic effect on the loss of cell viability in TMD8 cells treated with a
combination of compound 5 and ibrutinib. These results demonstrated that compound 5 could
be a promising IRAK4 inhibitor and worthy of further investigation for the treatment of mutant
MYD88 DLBCL.
Experimental Section
Chemistry
All purchased solvents and chemicals were used without further purification unless otherwise
noted. Flash chromatography was performed using silica gel (300-400 mesh). All reactions were
1
5

1
monitored by thin layer chromatography (TLC) or high performance liquid chromatography. H
1
3
NMR and C NMR were generated in CDCl , DMSO-d , or CD OD on Varian Mercury 300, 400, or
3
6
3
5
00 NMR spectrometers. Mass spectrometry was conducted using an Agilent 6110 Quadrupole
LC/MS (ESI-MS mode). High resolution mass spectrometry was conducted using an Agilent G6520
Q-TOF spectrometer (ESI-MS mode). The purity of all tested compounds was determined by the
Agilent infinity 1260 HPLC system coupled with a diode array detector (DAD) and the Eclipse Plus
C18 column (4.6 × 150 mm, 5 μm).
(R)-5-(3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]
pyrimidine-3-carboxamide trifluoroacetate (1)：A solution of ethyl 5-chloropyrazolo[1,5-a]
pyrimidine-3-carboxylate 42 (569 mg, 2.52 mmol), tert-butyl (R)-piperidin-3-ylcarbamate (909 mg,
4
.54 mmol) and potassium fluoride (1.47 g, 25.2 mmol) was stirred at 100 °C for 2 h. The mixture
was allowed to cool to room temperature and H O was added upon which precipitate formed.
2
The precipitate was filtered off and washed with water and dried to afford the white solid. Then
the solid dissolved in methanol/tetrahydrofuran/water (6 mL/6 mL/2 mL), followed by addition of
lithium hydroxide monohydrate (527 mg, 12.6 mmol) was stirred overnight at 50 °C for 2 h. The
solvent was removed in vacuo. The residue was added water (4 mL) and adjusted pH to 3-4 with
1
mol/L hydrochloric acid upon which precipitate formed. The precipitate was filtered off, washed
1
with water and dried to afford compound 43a as a pale yellow solid (800 mg, 88%). H NMR (400
MHz, Chloroform-d) δ 8.44–8.18 (m, 2H), 6.56 (d, J = 8.0 Hz, 1H), 4.64 (s, 1H), 3.97 (s, 2H), 3.66 (s,
2
1
H), 3.48 (d, J = 12.9 Hz, 1H), 2.04 (d, J = 11.0 Hz, 1H), 1.86 (s, 1H), 1.67 (dd, J = 27.6, 11.4 Hz, 2H),
-
.44 (s, 9H). MS (ESI): 360.3 [M-H] .
A solution of 43a (75 mg, 0.217 mmol), intermediate 25 (35 mg, 0.228 mmol), EDCI (48 mg, 0.249
mmol), HOBT (34 mg, 0.249 mmol) and N,N-diisopropylethylamine (80 μL, 0.484 mmol) in DMF (2
mL) was stirred overnight at room temperature. The reaction was taken up in ethyl acetate,
washed with water and brine. The organic phase was dried over anhydrous sodium sulfate,
filtered, and concentrated. The residue was purified by prep-TLC (5% MeOH in DCM) to afford
tert-butyl (R)-(1-(3-((3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)carbamoyl)pyrazolo[1,5-a]
1
6

pyrimidin-5-yl)piperidin-3-yl)carbamate (42 mg, 41%). Then it dissolved in dichloromethane (4 mL)
which was added trifluoroacetic acid (1 mL). The mixture was stirred at room temperature for 2 h.
The solvent was removed under reduced pressure and the residue was purified by triturating
1
with methanol/diethyl ether (1 mL/5 mL) to afford compound 1 as a white solid (28 mg, 65%). H
NMR (400 MHz, DMSO-d ) δ 9.36 (s, 1H), 8.88 (d, J = 7.9 Hz, 1H), 8.33 (d, J = 15.8 Hz, 2H), 8.06 (s,
6
3
H), 7.10 (t, J = 53.9 Hz, 1H), 6.82 (d, J = 7.9 Hz, 1H), 4.15 (d, J = 14.0 Hz, 2H), 3.88 (s, 3H), 3.56 (t,
13
J = 10.8 Hz, 2H), 2.11–1.98 (m, 1H), 1.91–1.77 (m, 1H), 1.77–1.52 (m, 2H). C NMR (126 MHz,
DMSO-d ) δ 158.97, 156.60, 145.49, 137.13, 133.43 (t, J = 27.3 Hz), 124.47, 118.50, 112.82 (t, J =
6
+
2
29.4 Hz), 100.23, 98.10, 47.67, 46.42, 43.98, 27.62, 21.48. ESI-HRMS m/z [M+H] calcd for
C H F N O: 391.1801, found: 391.1792.
17 21 2 8
(R)-2-(3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)imidazo[1,5-a]
pyrimidine-8-carboxamide trifluoroacetate (2): A solution of ethyl 2-chloroimidazo[1,5-a]
pyrimidine-8-carboxylate 40 (366 mg, 1.62 mmol), tert-butyl (R)-piperidin-3-ylcarbamate (585 mg,
2
.92 mmol) and potassium fluoride (942 mg, 16.2 mmol) was stirred at 100 °C for 2 h. The
mixture was allowed to cool to room temperature and H O was added upon which precipitate
2
formed. The precipitate was filtered off and washed with water and dried to afford the white
solid. Then the solid dissolved in methanol/tetrahydrofuran/water (8 mL/8 mL/3.5 mL), followed
by addition of lithium hydroxide monohydrate (680 mg, 16.2 mmol) was stirred at 50 °C overnight.
The solvent was removed in vacuo. The residue was added water (5 mL) and adjusted pH to 3-4
with 1 mol/L hydrochloric acid upon which precipitate formed. The precipitate was filtered off,
1
washed with water and dried to afford compound 41 as a pale yellow solid (467 mg, 80%). H
NMR (300 MHz, DMSO-d ) δ 11.20 (s, 1H), 8.57–8.42 (m, 1H), 7.91 (dd, J = 3.8, 2.2 Hz, 1H), 7.01
6
(
d, J = 6.8 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H), 4.12 (s, 2H), 3.48–3.21 (m, 2H overlapped with
H O)3.13 (s, 1H), 1.82 (d, J = 18.8 Hz, 2H), 1.58–1.45 (m, 2H), 1.39 (t, J = 3.0 Hz, 9H). MS (ESI):
2
-
3
60.3 [M-H] .
A solution of 41 (100 mg, 0.277 mmol), intermediate 25 (37 mg, 0.251 mmol), HATU (192 mg,
.503 mmol) and N,N-diisopropylethylamine (105 μL, 0.629 mmol) in acetonitrile (5 mL) was
0
stirred for 2 h at room temperature. The reaction was taken up in ethyl acetate, washed with
water and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and
1
7

concentrated. The residue was purified by flash column chromatography eluting with 1% to 3%
MeOH in DCM to afford tert-butyl(R)-(1-(8-((3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)
carbamoyl)imidazo[1,5-a]pyrimidin-2-yl)piperidin-3-yl)carbamate (45 mg, 36%). Then it dissolved
in dichloromethane (4 mL) which was added trifluoroacetic acid (1 mL). The mixture was stirred
at room temperature for 2 h. The solvent was removed under reduced pressure and the residue
was purified by triturating with diethyl ether (1 mL/5 mL) to afford compound 2 as a white solid
1
(
31 mg, 67%). H NMR (300 MHz, DMSO-d ) δ 9.39 (s, 1H), 8.61 (d, J = 7.9 Hz, 1H), 8.33 (s, 1H),
6
8
.24–8.06 (m, 3H), 8.05 (s, 1H), 7.10 (t, J = 54.0 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 4.22 (d, J = 13.6
Hz, 1H), 4.05 (s, 2H), 3.87 (s, 3H),3.87 (s, 3H), 3.49 (dd, J = 13.3, 8.2 Hz, 2H), 2.02 (d, J = 7.0 Hz,
1
3
1
1
4
3
H), 1.82 (s, 1H), 1.76–1.52 (m, 2H). C NMR (126 MHz, DMSO-d ) δ 159.00, 155.18, 139.24,
6
33.32 (t, overlapped), 133.15, 125.62, 124.09, 118.92, 115.17, 112.80 (t, J = 229.7 Hz), 47.39,
+
6.42, 44.09, 27.70, 21.59. ESI-HRMS m/z [M+H] calcd for C H F N O: 391.1801, found:
17 21 2 8
91.1807.
(R)-6-(3-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)imidazo[1,2-b]
pyridazine-3-carboxamide (3): This compound was prepared from tert-butyl (R)-piperidin-3-
1
ylcarbamate as described in the synthesis of 8 as an off-white solid (78%). H NMR (300 MHz,
DMSO-d ) δ 9.99 (s, 1H), 8.38 (s, 1H), 8.29–7.94 (m, 5H), 7.37 (d, J = 10.1 Hz, 1H), 7.31 (s, 1H),
6
3
1
2
2
.98 (s, 1H), 3.91 (s, 3H), 3.77 (s, 1H), 3.51–3.27 (m, 3H), 2.10–1.95 (m, 1H), 1.94–1.78 (m, 1H),
1
3
.78–1.55 (m, 2H). C NMR (126 MHz, DMSO-d ) δ 155.46, 155.26, 138.11, 137.64, 134.50 (t, J =
6
6.8 Hz), 127.06, 125.83, 121.46, 117.25, 113.02, 112.29 (t, J = 230.9 Hz), 49.78, 46.46, 45.32,
+
8.30, 21.32. ESI-HRMS m/z [M+H] calcd for C H F N O: 391.1801, found: 391.1790.
1
7 21 2 8
(R)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-5-](piperidin-3-ylamino)pyrazolo[1,5-a]
pyrimidine-3-carboxamide trifluoroacetate (4): This compound was prepared from tert-butyl (R)-
1
3
-aminopiperidine-1-carboxylate as described in the synthesis of 1 as an off-white solid (72%). H
NMR (300 MHz, DMSO-d ) δ 9.27 (s, 1H), 8.86–8.53 (m, 3H), 8.26 (d, J = 7.0 Hz, 2H), 8.18 (d, J =
6
7
3
1
.1 Hz, 1H), 7.11 (t, J = 54.2 Hz, 1H), 6.48 (d, J = 7.7 Hz, 1H), 4.41 (s, 1H), 3.88 (s, 3H), 3.20 (s, 2H),
1
3
.10 (s, 2H), 1.99 (d, J = 9.9 Hz, 2H), 1.88–1.58 (m, 2H). C NMR (126 MHz, DMSO-d ) δ 159.21,
6
56.45, 145.86, 144.80, 136.30, 134.02 (t, J = 28.7 Hz), 125.01, 118.36, 115.74, 112.73 (t, J =
1
8

+
2
30.7 Hz), 101.65, 100.66, 45.36, 43.22, 42.97, 26.91, 18.89. ESI-HRMS m/z [M+H] calcd for
C H F N O: 391.1801, found: 391.1793.
17 21 2 8
(R)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-6-(piperidin-3-ylamino)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (5): This compound was prepared from tert-butyl
(
(
R)-3-aminopiperidine-1-carboxylate as described in the synthesis of 8 as a light-yellow solid
1
95%). H NMR (300 MHz, DMSO-d ) δ 9.94 (s, 1H), 8.66 (s, 2H), 8.27 (s, 1H), 8.09 (s, 1H), 7.97 (d,
6
J = 9.8 Hz, 1H), 7.57 (d, J = 6.8 Hz, 1H), 7.36–6.82 (m, 2H), 4.15 (s, 1H), 4.01–3.77 (m, 3H),
1
3
3
1
.31–2.87 (m, 4H), 2.13–1.85 (m, 2H), 1.84–1.57 (m, 2H). C NMR (126 MHz, DMSO-d ) δ 155.86,
6
53.28, 138.11, 136.28, 135.40 (t, J = 27.4 Hz), 126.64, 126.37, 121.63, 117.09, 115.20, 112.20 (t,
+
J = 231.6 Hz), 44.99, 43.88, 43.02, 26.91, 18.99. ESI-HRMS m/z [M+H] calcd for C H F N O:
1
7 21 2 8
3
91.1801, found: 391.1797.
(
R)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-2-methyl-5-(piperidin-3-ylamino)pyrazolo
1,5-a]pyrimidine-3-carboxamide trifluoroacetate (4-Me): solution of ethyl
-chloro-2-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate 44 (CAS NO. 1431654-56-6, 50 mg,
.209 mmol), tert-butyl (R)-3-aminopiperidine-1-carboxylate (84 mg, 0.417 mmol) and potassium
[
A
5
0
fluoride (121 mg, 2.09 mmol) was stirred at 100 °C for 2 h. The mixture was allowed to cool to
room temperature and the reaction was taken up in ethyl acetate (15 mL), and the organics were
washed with water and brine. The organic phase was dried over anhydrous sodium sulfate,
filtered, and concentrated. The residue was purified by prep-TLC (5% MeOH in DCM) to afford
ethyl (R)-5-((1-(tert-butoxycarbonyl)piperidin-3-yl)amino)-2-methylpyrazolo[1,5-a]pyrimidine-
+
3
0
-carboxylate as pale-yellow solid (83 mg, 99%). MS (ESI): 404.3 [M+H] . Then the solid (50 mg,
.124 mmol) dissolved in methanol/tetrahydrofuran/water (1 mL/1 mL/0.3 mL), followed by
addition of lithium hydroxide monohydrate (31 mg, 0.744 mmol) was stirred at 50 °C overnight.
The solvent was removed in vacuo. The residue was adjusted pH to 3-4 with 1 mol/L hydrochloric
acid upon which precipitate formed. The precipitate was filtered off, washed with water and
1
dried to afford intermediate 45 as a white solid (36 mg, 75%). H NMR (400 MHz, Chloroform-d) δ
1
3
0.59 (brs, 1H), 8.17 (s, 1H), 6.16 (s, 1H), 5.76 (brs, 1H), 4.13 (s, 1H) 3.88–3.13 (m, 4H), 2.62 (s,
-
H), 2.10–1.82 (m, 2H), 1.81–1.67 (m, 2H). MS (ESI): 374.1 [M-H] .
1
9

Compound 4-Me was prepared from intermediates 45 and 25 by the similar procedure of
1
synthesis of 2 as a light-yellow solid (64%). H NMR (400 MHz, DMSO-d ) δ 9.40 (s, 1H), 8.57 (d, J
6
=
7.3 Hz, 3H), 8.30 (s, 1H), 8.04 (s, 1H), 7.10 (t, J = 54.2 Hz, 1H), 6.41 (d, J = 7.6 Hz, 1H), 4.48–4.29
1
3
(
m, 1H), 3.87 (s, 3H), 3.32–2.94 (m, 4H), 2.13–1.89 (m, 2H), 1.82–1.55 (m, 2H). C NMR (151 MHz,
DMSO-d ) δ 160.23, 156.27, 154.95, 146.74, 135.75, 134.04 (t, J = 29.1 Hz), 125.19, 118.44,
6
1
18.03, 116.04, 112.79 (t, J = 229.7 Hz), 100.91, 97.46, 45.43, 43.11, 42.97, 26.98, 18.91, 14.64.
+
ESI-HRMS m/z [M+H] calcd for C H F N O : 405.1957, found: 415.1966.
18
23
2
8
2
(R)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-2-methyl-6-(piperidin-3-ylamino)imidazo
[
1,2-b]pyridazine-3-carboxamide trifluoroacetate (5-Me): Intermediate 47 was was prepared
from intermediate 25 and ethyl 6-chloro-2-methylimidazo[1,2-b]pyridazine-3-carboxylate 46 (CAS
1
NO.14714-18-2) by the similar procedure of synthesis of 37 as white solid (56%). H NMR (400
MHz, Chloroform-d) δ 10.79 (s, 1H), 8.36 (s, 1H), 7.98 (d, J = 9.4 Hz, 1H), 6.83 (t, J = 54.5 Hz, 1H),
+
3
.92 (t, J = 1.2 Hz, 3H), 2.89 (s, 3H). MS (ESI): 341.1 [M+H] .
Compound 5-Me was prepared from intermediate 47 and tert-butyl (R)-3-aminopiperidine-1-
1
carboxylate by the similar procedure of synthesis of 8 as a white solid (71%). H NMR (400 MHz,
DMSO-d ) δ 10.08 (s, 1H), 8.55 (s, 2H), 8.30 (s, 1H), 7.88 (d, J = 9.7 Hz, 1H), 7.45 (d, J = 6.7 Hz,
6
1
4
1
H), 7.12 (t, J = 54.3 Hz, 1H), 6.90 (d, J = 9.7 Hz, 1H), 4.20–4.05 (m, 1H), 3.90 (s, 3H), 3.24–2.92 (m,
1
3
H), 2.58 (s, 3H), 2.07–1.86 (m, 2H), 1.76–1.56 (m, 2H). C NMR (151 MHz, DMSO-d ) δ 156.84,
6
52.94, 146.59, 135.76, 135.37 (t, J = 27.2 Hz), 126.44, 125.68, 117.18, 116.99, 115.11, 112.29 (t,
+
J = 231.3 Hz), 45.00, 43.94, 43.02, 26.99, 19.04, 15.79. ESI-HRMS m/z [M+H] calcd for
C H F N O : 405.1957, found: 415.1961.
18
23
2
8
2
(
S)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-6-(piperidin-3-ylamino)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (6): This compound was prepared from tert-butyl
3S)-3-aminocyclohexane-1-carboxylate as described in the synthesis of 8 as a white solid (56%).
(
1
H NMR (400 MHz, DMSO-d ) δ 9.94 (s, 1H), 8.62 (s, 2H), 8.27 (s, 1H), 8.08 (s, 1H), 7.97 (d, J = 9.7
6
Hz, 1H), 7.56 (d, J = 6.8 Hz, 1H), 7.13 (t, J = 54.2 Hz, 1H), 6.93 (d, J = 9.8 Hz, 1H), 4.15 (s, 1H), 3.91
1
3
(
s, 3H), 3.20 (s, 2H), 3.07 (m, 2H), 1.99 (d, J = 9.9 Hz, 2H), 1.69 (s, 2H). C NMR (126 MHz,
DMSO-d ) δ 155.84, 153.32, 138.05, 136.13, 135.41 (t, J = 27.5 Hz), 126.57, 126.37, 121.65,
6
2
0

1
17.67, 117.09, 115.30, 112.21 (t, J = 231.2 Hz), 45.02, 43.89, 43.02, 26.91, 18.99. ESI-HRMS m/z
+
[
M+H] calcd for C H F N O: 391.1801, found: 391.1791.
1
7 21 2 8
(R)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-6-(pyrrolidin-3-ylamino)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (7): This compound was prepared from tert-butyl
1
(
R)-3-aminopyrrolidine-1-carboxylate as described in the synthesis of 8 as a white solid (61%). H
NMR (400 MHz, DMSO-d ) δ 9.94 (s, 1H), 8.62 (s, 2H), 8.27 (s, 1H), 8.08 (s, 1H), 7.97 (d, J = 9.7 Hz,
6
1
3
H), 7.56 (d, J = 6.8 Hz, 1H), 7.13 (t, J = 54.2 Hz, 1H), 6.93 (d, J = 9.8 Hz, 1H), 4.15 (s, 1H), 3.91 (s,
13
H), 3.20 (s, 2H), 3.14–2.95 (m, 2H), 1.99 (d, J = 9.9 Hz, 2H), 1.69 (s, 2H). C NMR (126 MHz,
DMSO-d ) δ 156.49, 153.72, 138.72, 137.06, 135.14 (t, J = 27.8 Hz), 127.36, 126.77, 121.98,
6
+
1
18.84, 117.60, 115.36, 112.73 (t, J = 230.9 Hz), 50.20, 49.66, 44.49, 30.24. ESI-HRMS m/z [M+H]
calcd for C H F N O: 377.1644, found: 377.1651.
16 19 2 8
(R)-6-(3-aminopyrrolidin-1-yl)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (8): A mixture of N,N-dimethylformamide dimethyl
acetal (8.40 g, 70.5 mmol) and 6-chloropyridazin-3-amine 34 (8.70 g, 67.2 mmol) was stirred at
1
10°C for 2 h. After cooling, The off-white solid precipitated was collected by filtration and
washed with ethyl acetate (20 mL) and dried to afford (E)-N'-(6-chloropyridazin-3-yl)-N,N-
1
dimethylformimidamide 35 (8.90 g, 72%). H NMR (300 MHz, DMSO-d ) δ 8.47 (s, 1H), 7.57 (d, J =
6
9
.1 Hz, 1H), 7.14 (d, J = 9.1 Hz, 1H), 3.12 (s, 3H), 3.01 (s, 3H).
A mixture of compound 35 (2.35 g, 12.7 mmol) and ethyl bromoacetate (3.88 mL, 38.2 mmol) in
acetonitrile (20 mL) was stirred at 80 °C for 3 h. After cooling to room temperature, a large
amount of solids precipitated, which was filtered off with suction, washed with diethyl ether (20
mL), recrystallized with ethyl acetate/acetonitrile (2:5, 35 mL) to afford yellow crystalline solid
(
1.8 g). The solid was dissolved in a solution of N,N-diisopropylethylamine (1.69 mL, 10.2 mmol)
in acetonitrile (25 mL). The reaction mixture was stirred for 3 h at room temperature. The solvent
was removed in vacuo and the residue was triturated with water to afford ethyl
1
6
-chloroimidazo[1,2-b]pyridazine-3-carboxylate 36 as a light brown solid (960 mg, 83%). H NMR
2
1

(
300 MHz, DMSO-d ) δ 8.46–8.31 (m, 2H), 7.63 (d, J = 9.5 Hz, 1H), 4.36 (q, J = 7.1 Hz, 2H), 1.34 (t, J
6
=
7.1 Hz, 3H).
A mixture of compound 36 (600 mg, 2.66 mmol) and Lithium hydroxide monohydrate (335 mg,
.98 mmol) in tetrahydrofuran/water (10 mL/2.5 mL) was stirred at 35 °C for 4 h. The solvent was
7
removed in vacuo. The residue was added water (4mL) and adjusted pH to 3-4 with 1 mol/L
hydrochloric acid upon which precipitate formed. The precipitate was filtered off, washed with
water and dried to afford 6-chloroimidazo[1,2-b]pyridazine-3-carboxylic acid 37 as an off-white
1
solid (519 mg, 98%). H NMR (400 MHz, DMSO-d ) δ 13.30 (s, 1H), 8.63–8.20 (m, 2H), 7.61 (d, J =
6
9
.5 Hz, 1H).
A solution of compound 37 (300 mg, 1.52 mmol), 3-(difluoromethyl)-1-methyl-1H-pyrazol-4-
amine 25 (203 mg, 1.38 mmol), EDCI (317 mg, 1.66 mmol), HOBT (223 mg, 1.66 mmol) and
N,N-diisopropylethylamine (570 μL, 3.45 mmol) in DMF (10 mL) was stirred overnight at room
temperature. The reaction was taken up in ethyl acetate (50 mL), and the organics were washed
with water and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and
concentrated. The residue was purified by flash column chromatography eluting with 1% MeOH
1
in DCM to afford compound 38 as a white solid (303 mg, 67%). H NMR (300 MHz, DMSO-d ) δ
6
8
3
.47 (s, 1H), 7.57 (d, J = 9.1 Hz, 1H), 7.14 (d, J = 9.1 Hz, 1H), 3.12 (s, 3H), 3.01 (s, 3H). MS (ESI):
+
49.2 [M+Na] .
A mixture of intermediate 38 (40 mg, 0.122 mol), tert-butyl (R)-pyrrolidin-3-ylcarbamate (46 mg,
0
0
.244 mmol), potassium fluoride (71 mg, 1.22 mmol), and N,N-diisopropylethylamine (61 μL,
.367 mmol) in DMSO (2 mL) was stirred at 100°C for 45 minutes. The reaction was taken up in
ethyl acetate (15 mL), and the organics were washed with water and brine. The organic phase
was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by
prep-TLC (6% MeOH in DCM) to afford tert-butyl (R)-(1-(3-((3-(difluoromethyl)-1-methyl-1H-
pyrazol-4-yl)carbamoyl)imidazo[1,2-b]pyridazin-6-yl)pyrrolidin-3-yl)carbamate as
a
colorless
1
liquid (76 mg, 90%). H NMR (400 MHz, Chloroform-d) δ 10.23 (s, 1H), 8.39 (s, 1H), 8.30 (s, 1H),
7
.80 (d, J = 9.8 Hz, 1H), 6.92 – 6.57 (m, 2H), 4.85 (s, 1H), 4.42 (s, 1H), 3.92 (d, J = 1.3 Hz, 3H), 3.85
2
2

(
dd, J = 10.8, 6.1 Hz, 1H), 3.69 (dq, J = 14.4, 7.2 Hz, 2H), 3.44 (dd, J = 10.9, 4.5 Hz, 1H), 2.36 (dq, J
13.5, 7.0 Hz, 1H), 2.12 – 2.00 (m, 1H), 1.46 (s, 9H).
=
A solution of tert-butyl (R)-(1-(3-((3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)carbamoyl)
imidazo[1,2-b] pyridazin-6-yl)pyrrolidin-3-yl)carbamate (76 mg, 0.159 mmol) in dichloromethane
(
4 mL) was added trifluoroacetic acid (1 mL). The mixture was stirred at room temperature for 2 h.
The solvent was removed under reduced pressure and the residue was purified by triturating
1
with methanol/diethyl ether (1 mL/5 mL) to afford compound 8 as a white solid (77 mg, 98%). H
NMR (400 MHz, DMSO-d ) δ 10.15 (s, 1H), 8.39 (s, 1H), 8.19 (s, 3H), 8.17 (s, 1H), 8.09 (d, J = 9.9
6
Hz, 1H), 7.30–6.96 (m, 2H), 4.04 (s, 1H), 3.91 (s, 3H), 3.81 (dd, J = 11.9, 5.9 Hz, 1H), 3.77–3.56 (m,
1
3
3
1
1
H), 2.38 (dq, J = 14.7, 8.0 Hz, 1H), 2.14 (dd, J = 13.1, 5.8 Hz, 1H). C NMR (126 MHz, DMSO-d ) δ
6
55.50 , 152.72 , 137.74 , 136.61 , 134.60 (t, J = 28.2 Hz), 126.69 , 125.95 , 121.22 , 117.15 ,
+
14.95 , 112.84 , 112.30 (t, J = 230.3 Hz), 50.80 , 49.51 , 44.86 , 28.96 . ESI-HRMS m/z [M+H]
calcd for C H F N O: 377.1644, found: 377.1645.
16 19 2 8
(
R)-6-(azepan-3-ylamino)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (9): This compound was prepared from tert-butyl
R)-3-aminoazepane-1-carboxylate as described in the synthesis of 8 as a light-yellow solid (65%).
(
1
H NMR (400 MHz, DMSO-d ) δ 9.97 (s, 1H), 8.79 (d, J = 58.3 Hz, 2H), 8.27 (s, 1H), 8.11 (s, 1H),
6
7
4
1
1
2
4
.97 (d, J = 9.8 Hz, 1H), 7.57 (d, J = 6.9 Hz, 1H), 7.14 (t, J = 54.1 Hz, 1H), 6.95 (d, J = 9.7 Hz, 1H),
.30 (s, 1H), 3.91 (s, 3H), 3.48–3.39 (m, 1H), 3.34–3.18 (m, 2H), 3.07 (s, 1H), 2.12–1.96 (m, 1H),
1
3
.96–1.68 (m, 4H), 1.69–1.50 (m, 1H). C NMR (126 MHz, DMSO-d ) δ 155.99, 153.10, 138.15,
6
36.21, 135.32 (t, J = 26.9 Hz), 126.61, 126.25, 121.47, 117.75, 117.16, 115.34, 112.10 (t, J =
+
31.3 Hz), 47.99, 47.52, 46.98, 31.29, 25.39, 21.77. ESI-HRMS m/z [M+H] calcd for C H F N O:
1
8 23 2 8
05.1957, found: 405.1949.
6
-(4-aminopiperidin-1-yl)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (10): This compound was prepared from tert-butyl
1
piperidin-4-ylcarbamate as described in the synthesis of 8 as a white solid (88%). H NMR (400
MHz, DMSO-d ) δ 9.96 (s, 1H), 8.41 (s, 1H), 8.16 (s, 1H), 7.99 (d, J = 4.7 Hz, 3H), 7.47 (d, J = 10.1
6
Hz, 1H), 7.17 (t, J = 53.8 Hz, 1H), 4.29 (d, J = 13.3 Hz, 2H), 3.91 (s, 3H), 3.39–3.30 (m, 1H),
2
3

1
3
3
.17–2.98 (m, 2H), 2.02 (d, J = 12.5 Hz, 2H), 1.60 (qd, J = 12.8, 4.4 Hz, 2H). C NMR (126 MHz,
DMSO-d ) δ 155.42, 154.97, 138.05, 137.72, 134.19 (t, J = 28.5 Hz), 127.12, 125.78, 121.47,
6
+
1
17.25, 112.76, 112.43 (t, J = 230.1 Hz), 47.27, 43.94, 28.92. ESI-HRMS m/z [M+H] calcd for
C H F N O: 391.1801, found: 391.1798.
17 21 2 8
6
-((2-aminoethyl)amino)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (11): This compound was prepared from tert-butyl
1
(
2-aminoethyl)carbamate as described in the synthesis of 8 as a white solid (95%). H NMR (400
MHz, DMSO-d ) δ 10.09 (s, 1H), 8.35 (s, 1H), 8.12 (s, 1H), 7.97 (d, J = 9.8 Hz, 1H), 7.74 (s, 1H), 7.14
6
(
t, J = 53.9 Hz, 1H), 6.91 (d, J = 9.8 Hz, 1H), 3.90 (s, 3H), 3.58 (q, J = 5.6 Hz, 2H), 3.09 (q, J = 5.7 Hz,
1
3
2
1
H). C NMR (126 MHz, DMSO-d ) δ 155.59, 154.13, 137.98, 135.94, 126.27, 125.86, 121.52,
6
+
17.27, 115.59, 115.00, 112.33 (t, J = 230.5 Hz), 38.59, 37.52. ESI-HRMS m/z [M+H] calcd for
C H F N O: 351.1492, found: 351.1488.
14 17 2 8
N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-6-(piperazin-1-yl)imidazo[1,2-b]pyridazine-3-
carboxamide trifluoroacetate (12): This compound was prepared from tert-butyl piperazine-1-
1
carboxylate as described in the synthesis of 8 as a white solid (89%). H NMR (400 MHz, DMSO-d )
6
δ 9.89 (s, 1H), 9.06 (s, 2H), 8.40 (s, 1H), 8.19 (s, 1H), 8.17 (d, J = 10.0 Hz, 1H), 7.47 (d, J = 10.1 Hz,
1
3
1
H), 7.17 (t, J = 53.8 Hz, 1H), 3.91 (s, 3H), 3.81 (t, J = 5.2 Hz, 4H), 3.28 (t, J = 5.2 Hz, 4H). C NMR
(
126 MHz, DMSO-d ) δ 155.30, 154.80, 138.28, 137.93, 134.18 (t, J = 28.2 Hz), 127.35, 125.71,
6
+
1
21.56, 117.28, 112.61, 112.52 (t, J = 230.1 Hz), 42.63, 42.19. ESI-HRMS m/z [M+H] calcd for
C H F N O: 377.1644, found: 377.1638.
16 19 2 8
(R)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-6-(methyl(piperidin-3-yl)amino)imidazo
[
1,2-b]pyridazine-3-carboxamide trifluoroacetate (13): This compound was prepared from tert-
butyl (R)-3-(methylamino)piperidine-1-carboxylate as described in the synthesis of 8 as a white
1
solid (35%). H NMR (400 MHz, Methanol-d ) δ 8.28 (s, 1H), 8.18 (s, 1H), 7.93 (s, 1H), 7.37 (s, 1H),
4
6
1
1
.88 (t, J = 54.2 Hz, 1H), 4.36 (d, J = 12.0 Hz, 1H), 3.93 (s, 3H), 3.42 (s, 2H), 3.13 (s, 3H), 3.00 (s,
1
3
H), 2.00 (d, J = 65.1 Hz, 4H). C NMR (126 MHz, Methanol-d ) δ 158.18, 156.47, 139.76, 138.3 ,
4
37.08 (t, J = 28.1 Hz), 127.77, 127.50, 123.16, 118.40, 113.76 (t, J = 230.9 Hz), 113.60, 53.55,
2
4

+
4
6.17, 44.56, 39.76, 31.29, 27.44, 23.05. ESI-HRMS m/z [M+H] calcd for C H F N O: 405.1957,
18 23 2 8
found: 405.1961.
(R)-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-6-(piperidin-3-ylamino)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (14): A mixture of ethyl 6-chloroimidazo[1,2-b]
pyridazine-3-carboxylate 36 (600 mg, 2.66 mmol), tert-butyl (R)-3-aminopiperidine-1-carboxylate
(
960 mg, 4.79 mmol) and potassium fluoride (1.54 g, 26.6 mmol) in DMSO (8 mL) was stirred
overnight at 100°C. The reaction was taken up in ethyl acetate (50 mL), and the organics were
washed with water and brine. The organic phase was dried over anhydrous sodium sulfate,
filtered, and concentrated. The residue was purified by flash column chromatography eluting
with
1%
to
2%
MeOH
in
DCM
to
afford
intermediate
ethyl
(
R)-6-((1-(tert-butoxycarbonyl)piperidin-3-yl)amino)imidazo[1,2-b]pyridazine-3-carboxylate (620
1
mg, 60%) as a light yellow oil. H NMR (300 MHz, DMSO-d ) δ 8.00 (s, 1H), 7.84 (d, J = 9.7 Hz, 1H),
6
7
1
.08 (d, J = 6.1 Hz, 1H), 6.90 (d, J = 9.7 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 4.02–3.65 (m, 2H), 1.97 (s,
H), 1.79 (s, 1H), 1.61 (s, 1H), 1.38 (s, 3H), 1.35–1.25 (m, 4H), 1.12 (s, 6H).
A mixture of ethyl (R)-6-((1-(tert-butoxycarbonyl)piperidin-3-yl)amino)imidazo[1,2-b]pyridazine-3
carboxylate (620 mg, 1.59 mmol) and lithium hydroxide monohydrate (200 mg, 4.78 mmol) in
-
methanol/tetrahydrofuran/water (9 mL/9 mL/3 mL) was stirred at 35 °C for 4 h. The solvent was
removed in vacuo. The residue was added water (4 mL) and adjusted pH to 3-4 with 1 mol/L
hydrochloric acid upon which precipitate formed. The precipitate was filtered off, washed with
water and dried to afford (R)-6-((1-(tert-butoxycarbonyl)piperidin-3-yl)amino)imidazo[1,2-b]
1
pyridazine-3-carboxylic acid 39 as an off-white solid(320 mg, 56%). H NMR (300 MHz, DMSO-d )
6
δ 12.48 (s, 1H), 7.97 (s, 1H), 7.85 (d, J = 9.7 Hz, 1H), 7.14 (d, J = 6.1 Hz, 1H), 6.90 (d, J = 9.7 Hz, 1H),
3
1
.72 (s, 2H), 3.38 (s, 2H), 3.29–3.10 (m, 1H), 2.06–1.88 (m, 1H), 1.79 (s, 1H), 1.61 (s, 1H),
-
.50–1.32 (m, 3H), 1.13 (s, 7H). MS (ESI): 360.3 [M-H] .
A solution of intermediate 39 (70 mg, 0.193 mmol), 1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-
amine 33a (35 mg, 0.213mmol), EDCI (45 mg, 0.232 mmol), HOBT (32 mg, 0.232 mmol) and
N,N-diisopropylethylamine (80 μL, 0.484 mmol) in DMF (2 mL) was stirred overnight at room
temperature. The reaction was taken up in ethyl acetate, washed with water and brine. The
2
5

organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue
was purified by prep-TLC (6% MeOH in DCM) to afford tert-butyl (R)-3-((3-((1-methyl-3-
(
trifluoromethyl)-1H-pyrazol-4-yl)carbamoyl)imidazo[1,2-b]pyridazin-6-yl)amino)piperidine-1-
carboxylate (60 mg, 61%). Then it dissolved in dichloromethane (4 mL) which was added
trifluoroacetic acid (1 mL). The mixture was stirred at room temperature for 2 h. The solvent was
removed under reduced pressure and the residue was purified by triturating with
1
methanol/diethyl ether (1 mL/5 mL) to afford compound 14 as a white solid (40 mg, 78%). H
NMR (400 MHz, DMSO-d ) δ 9.87 (s, 1H), 8.67 (s, 2H), 8.32 (s, 1H), 8.09 (s, 1H), 7.98 (d, J = 9.8 Hz,
6
1
1
H), 7.59 (d, J = 7.1 Hz, 1H), 6.92 (d, J = 9.8 Hz, 1H), 4.07 (s, 1H), 3.96 (s, 3H), 3.23 (d, J = 11.8 Hz,
13
H), 3.11 (d, J = 19.2 Hz, 2H), 2.99 (s, 1H), 2.09–1.88 (m, 2H), 1.66 (d, J = 7.0 Hz, 2H). C NMR
(
126 MHz, DMSO-d ) δ 156.40, 153.30, 138.19, 136.33, 131.79 (q, J = 35.8 Hz), 128.17, 126.72,
6
1
21.52 (q, J = 268.2 Hz), 121.47, 117.02, 115.21, 45.23, 44.17, 42.89, 27.14, 19.29. ESI-HRMS m/z
+
[
M+H] calcd for C H F N O: 409.1707, found: 407.1719.
1
7 20 3 8
(R)-N-(3-cyano-1-methyl-1H-pyrazol-4-yl)-6-(piperidin-3-ylamino)imidazo[1,2-b]pyridazine-3-
carboxamide trifluoroacetate (15): This compound was prepared from intermediate 29 as
1
described in the synthesis of 14 as an off-white solid (69%). H NMR (300 MHz, DMSO-d ) δ 10.30
6
(
(
s, 1H), 8.66 (s, 2H), 8.41 (s, 1H), 8.12 (s, 1H), 7.99 (d, J = 9.8 Hz, 1H), 7.65 (d, J = 6.8 Hz, 1H), 6.95
d, J = 9.8 Hz, 1H), 4.27 (s, 1H), 3.98 (s, 3H), 3.36–3.24 (m, 1H), 3.24–2.97 (m, 3H), 1.99 (d, J = 11.8
1
3
Hz, 2H), 1.74 (d, J = 9.2 Hz, 2H). C NMR (126 MHz, DMSO-d ) δ 155.74, 153.17, 138.31, 136.66,
6
1
26.77, 125.24, 124.58, 121.20, 115.18, 113.56, 45.06, 44.25, 43.07, 26.81, 19.15. ESI-HRMS m/z
+
[
M+H] calcd for C H N O: 366.1785, found: 366.1795.
1
7 20 9
(R)-N-(3-carbamoyl-1-methyl-1H-pyrazol-4-yl)-6-(piperidin-3-ylamino)imidazo[1,2-b]pyridazine-
3
-carboxamide trifluoroacetate (16): This compound was prepared from intermediate 27 as
1
described in the synthesis of 14 as an off-white solid (quant). H NMR (300 MHz, DMSO-d ) δ
6
1
2
0.68 (s, 1H), 8.88 (s, 1H), 8.53 (s, 1H), 8.36 (s, 1H), 8.17 (d, J = 10.6 Hz, 2H), 7.94 (d, J = 10.2 Hz,
H), 7.56 (s, 1H), 6.91 (d, J = 9.7 Hz, 1H), 4.63 (s, 1H), 3.95 (s, 3H), 3.46 (d, J = 11.8 Hz, 2H), 2.82
1
3
(
dd, J = 47.4, 10.9 Hz, 2H), 1.97 (d, J = 22.2 Hz, 2H), 1.80–1.52 (m, 2H). C NMR (126 MHz,
DMSO-d ) δ 165.79, 155.22, 153.64, 138.03, 136.83, 131.97, 126.28, 124.92, 122.06, 122.00,
6
2
6

+
1
15.31, 46.43, 43.63, 43.06, 28.10, 20.97. ESI-HRMS m/z [M+H] calcd for C H N O : 384.1891,
17 22 9 2
found: 384.1901.
(R)-N-(1,3-dimethyl-1H-pyrazol-4-yl)-6-(piperidin-3-ylamino)imidazo[1,2-b]pyridazine-3-
carboxamide trifluoroacetate (17): This compound was prepared from 1,3-dimethyl-1H-pyrazol-
1
4
-amine as described in the synthesis of 14 as a white solid (95%). H NMR (400 MHz,
Methanol-d ) δ 8.18 (s, 1H), 7.90 (d, J = 10.1 Hz, 2H), 7.04 (d, J = 9.8 Hz, 1H), 4.23 (d, J = 7.0 Hz,
4
1
2
H), 3.85 (s, 3H), 3.49 (dd, J = 12.0, 3.2 Hz, 1H), 3.25 (d, J = 7.4 Hz, 1H), 3.15 (d, J = 12.2 Hz, 1H),
13
.27 (s, 3H), 2.24–2.18 (m, 1H), 2.18–2.06 (m, 1H), 1.90–1.76 (m, 2H). C NMR (126 MHz,
DMSO-d ) δ 155.77, 153.14, 139.33, 137.63, 135.21, 126.43, 124.98, 122.19, 116.98, 114.92,
6
+
4
3
5.26, 44.52, 42.95, 38.47, 27.08, 19.43, 11.23. ESI-HRMS m/z [M+H] calcd for C H N O:
17 23 8
55.1989, found: 355.1998.
(R)-N-(1-ethyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-6-(piperidin-3-ylamino)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (18): This compound was prepared from
1
intermediate 33b as described in the synthesis of 14 as a light-yellow solid (66%). H NMR (400
MHz, DMSO-d ) δ 9.87 (s, 1H), 8.67 (s, 2H), 8.38 (s, 1H), 8.08 (s, 1H), 7.98 (d, J = 9.7 Hz, 1H), 7.58
6
(
d, J = 7.2 Hz, 1H), 6.92 (d, J = 9.8 Hz, 1H), 4.25 (q, J = 7.3 Hz, 2H), 4.08 (s, 1H), 3.22 (s, 1H), 3.14 (s,
1
3
1
H), 3.08 (s, 1H), 2.99 (s, 1H), 2.10–1.86 (m, 2H), 1.66 (s, 2H), 1.42 (t, J = 7.3 Hz, 3H). C NMR
(
126 MHz, DMSO-d ) δ 156.37, 153.30, 138.22, 136.42, 131.63 (q, J = 35.5 Hz), 126.76, 121.5 (q, J
6
=
1
267.8 Hz), 121.45, 117.81, 116.92, 115.45, 115.14, 47.50, 45.27, 44.15, 42.88, 27.20, 19.32,
+
5.07. ESI-HRMS m/z [M+H] calcd for C H F N O: 423.1863, found: 423.1869.
18 22 3 8
(R)-N-(1-isopropyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-6-(piperidin-3-ylamino)imidazo[1,2-b]
pyridazine-3-carboxamide trifluoroacetate (19): This compound was prepared from
1
intermediate 33c as described in the synthesis of 14 as a light-yellow solid (39%). H NMR (400
MHz, DMSO-d ) δ 9.87 (s, 1H), 8.68 (s, 2H), 8.39 (s, 1H), 8.08 (s, 1H), 7.98 (d, J = 9.8 Hz, 1H), 7.58
6
(
d, J = 7.2 Hz, 1H), 6.92 (d, J = 9.8 Hz, 1H), 4.64 (h, J = 6.7 Hz, 1H), 4.09 (s, 1H), 3.24 (d, J = 11.8 Hz,
1
6
H), 3.15 (s, 1H), 3.06 (s, 1H), 2.99 (s, 1H), 2.09–1.87 (m, 2H), 1.64 (d, J = 9.7 Hz, 2H), 1.46 (d, J =
13
.6 Hz, 6H). C NMR (126 MHz, DMSO-d ) δ 156.36, 153.31, 138.25, 136.45, 131.33 (q, J = 35.7
6
2
7

Hz), 126.78, 125.19, 121.60 (q, J = 268.9 Hz), 121.46, 116.81, 115.53, 115.11, 54.51, 45.32, 44.15,
+
4
4
2.87, 27.23, 22.33, 19.35. ESI-HRMS m/z [M+H] calcd for C H F N O: 437.2020, found:
19 24 3 8
37.2032.
(R)-6-(piperidin-3-ylamino)-N-(1-(piperidin-4-yl)-3-(trifluoromethyl)-1H-pyrazol-4-yl)imidazo
[
1,2-b]pyridazine-3-carboxamide trifluoroacetate (20): This compound was prepared from
1
intermediate 33d as described in the synthesis of 14 as a light-yellow solid (87%). H NMR (400
MHz, DMSO-d ) δ 9.91 (s, 1H), 8.92 (d, J = 11.3 Hz, 1H), 8.79 (s, 2H), 8.59 (d, J = 11.1 Hz, 1H), 8.43
6
(
(
s, 1H), 8.11 (s, 1H), 7.99 (d, J = 9.7 Hz, 1H), 7.63 (d, J = 7.3 Hz, 1H), 6.94 (d, J = 9.6 Hz, 1H), 4.67
td, J = 10.8, 10.4, 5.5 Hz, 1H), 4.11 (s, 1H), 3.45 (d, J = 12.8 Hz, 2H), 3.23 (d, J = 12.1 Hz, 1H),
3
.19–2.89 (m, 5H), 2.24 (d, J = 13.2 Hz, 2H), 2.15 (t, J = 12.4 Hz, 2H), 1.99 (d, J = 11.8 Hz, 2H), 1.66
1
3
(
s, 2H). C NMR (126 MHz, DMSO-d ) δ 156.37, 153.37, 136.43, 131.82 (q, J = 35.9 Hz), 126.75,
6
1
2
25.87, 121.49, 121.44 (q, J = 268.7 Hz), 117.14, 115.28, 56.27, 45.32, 44.08, 42.86, 42.23, 28.39,
+
7.21, 19.31. ESI-HRMS m/z [M+H] calcd for C H F N O: 478.2285, found: 478.2282.
21 29 3 9
(R)-6-(piperidin-3-ylamino)-N-(1-(tetrahydro-2H-pyran-4-yl)-3-(trifluoromethyl)-1H-pyrazol-4-yl)
imidazo[1,2-b]pyridazine-3-carboxamide trifluoroacetate (21): This compound was prepared
1
from intermediate 33e as described in the synthesis of 14 as a light-yellow solid (68%). H NMR
(
400 MHz, DMSO-d ) δ 9.88 (s, 1H), 8.71 (s, 2H), 8.42 (s, 1H), 8.09 (s, 1H), 7.98 (d, J = 9.7 Hz, 1H),
6
7
.60 (s, 1H), 6.93 (d, J = 9.8 Hz, 1H), 4.56 (d, J = 11.5 Hz, 1H), 4.09 (s, 1H), 4.04–3.88 (m, 2H), 3.47
(
t, J = 11.4 Hz, 2H), 3.24 (s, 1H), 3.15 (s, 1H), 3.02 (d, J = 31.5 Hz, 2H), 2.08–1.88 (m, 6H), 1.66 (s,
1
3
2
1
5
4
H). C NMR (126 MHz, DMSO-d ) δ 156.38, 153.34, 138.17, 136.29, 131.58 (q, J = 36.3Hz),
6
26.70, 125.59, 121.53 (q, J = 268.2 Hz), 121.46, 117.58 (d, J = 4.3 Hz), 116.92, 115.22, 65.75,
+
8.38, 45.33, 44.17, 42.87, 32.53, 27.25, 19.38. ESI-HRMS m/z [M+H] calcd for C H F N O :
21 26 3 8 2
79.2125, found: 479.2128.
Biological studies
Kinase activity inhibition assay: IRAK4 recombinant human protein (PV3362) was obtained from
Thermo Fisher scientific. Kinase activity was evaluated using Z'-LYTE™ kinase assay kit (PV3180,
2
8