Rational Design, Synthesis and Evaluation of Novel C6-Bicycloalkaneimidazole Containing Imidazo[1,2-b]pyridazines for ASK1 Inhibition

Article abstract of DOI:10.1002/bkcs.12275

Apoptosis signal-regulating kinase 1 (ASK1) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family that involves downstream phosphorylation of MAP kinases, c-Jun N-terminal kinases, and p38 MAP kinases. ASK1 inhibitors could possibly be beneficial for ameliorating the development and progression of diseases. Especially, ASK1 has been of interest as one of therapeutic targets for nonalcoholic fatty liver disease as the most common chronic liver diseases including simple steatosis and nonalcoholic steatohepatitis. In this manuscript, novel ASK1 inhibitor lead KTA-29 which has an imidazo[1,2-b]pyridazine core with novel C6-bicycloheptaneimidazole is disclosed. With the novel imidazo[1,2-b]pyridazine core, structure-activity-relationship study for ASK1 potency is described and KTA-29 affinity toward ASK1 with molecular modeling study is explained.

Full text of DOI:10.1002/bkcs.12275

Article
DOI: 10.1002/bkcs.12275
BULLETIN OF THE
Y. Lee et al.
KOREAN CHEMICAL SOCIETY
Rational Design, Synthesis and Evaluation of Novel C6-
Bicycloalkaneimidazole Containing Imidazo[1,2-b]pyridazines for
ASK1 Inhibition
Yujin Lee,†,‡ Jiyoon Jang,†,§ Maimoona Bibi,†,‡ Krishna Babu Duggirala,†,‡ Sang Hee Ji,†,¶ Ji
†,
†,¶,
Hun Lee,† Sunjoo Ahn,†,‡ Jin Sook Song,†, Chong Hak Chae, Seong Hwan Kim,
*
*
*
and
Kwangho Lee†,‡,
*
†Bio and Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 34114,
South Korea. *E-mail: jssong@krict.re.kr; chchae@krict.re.kr
‡Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113,
South Korea. *E-mail: kwangho@krict.re.kr
§Department of Chemistry, Sungkyunkwan University, Suwon 16419, South Korea
¶Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon
34134, South Korea. *E-mail: hwan@krict.re.kr
Received February 10, 2021, Accepted March 27, 2021
Apoptosis signal-regulating kinase 1 (ASK1) is a member of mitogen-activated protein kinase kinase
kinase (MAP3K) family that involves downstream phosphorylation of MAP kinases, c-Jun N-terminal
kinases, and p38 MAP kinases. ASK1 inhibitors could possibly be beneficial for ameliorating the develop-
ment and progression of diseases. Especially, ASK1 has been of interest as one of therapeutic targets for
nonalcoholic fatty liver disease as the most common chronic liver diseases including simple steatosis and
nonalcoholic steatohepatitis. In this manuscript, novel ASK1 inhibitor lead KTA-29 which has an imidazo
[1,2-b]pyridazine core with novel C6-bicycloheptaneimidazole is disclosed. With the novel imidazo[1,2-b]
pyridazine core, structure-activity-relationship study for ASK1 potency is described and KTA-29 affinity
toward ASK1 with molecular modeling study is explained.
Keywords: ASK1, NASH, Kinase, Imidazo[1,2-b]pyridazine, KTA-29
Introduction
18 mg selonsertib and 30% of those receiving 6 mg
selonsertib experienced a reduction of at least one stage in
hepatic fibrosis.8 Nonetheless, it is currently under clinical
study for treating diabetic kidney disease, pulmonary arte-
rial hypertension.9
Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) is
a member of mitogen-activated protein kinase kinase kinase
(MAP3K) family that involves downstream phosphoryla-
tion of MAP kinases (MAPKs), c-Jun N-terminal kinases
(JNKs), and p38 MAPKs.1 ASK1 is activated by various
stresses, so its activation is closely relevant to the pathogen-
esis of several diseases.2 Therefore, ASK1 inhibitors could
possibly be beneficial for ameliorating the development and
progression of diseases.3 Especially, ASK1 has been of
interest as one of therapeutic targets for nonalcoholic fatty
liver disease (NAFLD) as the most common chronic liver
diseases including simple steatosis and nonalcoholic
steatohepatitis (NASH).4,5
Besides selonsertib, Takeda also reported a novel
imidazo[1,2-a]pyridine-based selective ASK1 inhibitor
(Takeda 1) through structure-based design and optimiza-
tion.10 Although selonsertib and Takeda 1 have different
structure cores, they are potent and selective to the ASK1
inhibition. As a fast-follower approach, we decide to design
new ASK1 inhibitor combining structural features of
selonsertib and Takeda 1 to find novel structure (Figure 2).
First, the selonsertib core has been carefully investigated
and its pyridine and 4-isopropyl-1,2,4-triazole structural
conformation has been well described (shown blue). Sec-
ond, Takeda 1 has the key hydrogen bonding interactions
to ASK1 in common along as selonsertib does except the
selonsertib pyridine and 4-isopropyl-1,2,4-triazole interac-
tion. Therefore it is worth to hybridize the selonsertib pyri-
dine and 4-isopropyl-1,2,4-triazole into the Takeda 1 core.
To this end, with all the structural information together,
medicinal chemistry campaign searching for new ASK1
inhibitor with new core has initiated: to adapt selonsertib
Several ASK1 inhibitors have been reported (Figure 1).6
Selonsertib (GS-4997) is the only ASK1 inhibitor advanced
to clinical evaluation for NASH indication but recently
failed to achieve the desired endpoint. Although the
STELLAR-3 and STELLAR-4 phase III have failed to
show that selonsertib could cause fibrosis regression and
reduce clinical disease progression in patients with bridging
fibrosis or compensated cirrhosis due to NASH,7 43% of
patients with NASH and stage 2 or 3 liver fibrosis receiving
Bull. Korean Chem. Soc. 2021
© 2021 Korean Chemical Society, Seoul & Wiley-VCH GmbH.
Wiley Online Library
1

Article
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
pyridine and 4-isopropyl-1,2,4-triazole structural conforma-
tion, the Takeda 1 imidazo[1,2-a]pyridine core was
converted into imidazo[1,2-b]pyridazine. And then as for
the 4-isopropyl-1,2,4-triazole mimic, we designed
4,5-bicycloalkaneimidazoles for structural novelty and rigid
conformation as illustrated in Figure 2.
then quenched with tert-butyl alcohol to provide tert-butyl
(2-chloroacetyl)carbamate (1) as a white crystalline solid.11
The 1 was added to 3-amino-6-chloropyridazine in N,N-
dimethylacetamide in the presence of disodium hydrogen
phosphate to give tert-butyl (6-chloroimidazo[1,2-b]pyridazin-
2-yl)carbamate (2) as a bright brown crystalline solid. The
Boc of 2 was removed by treating 4 N hydrochloric acid in
1,4-dioxane to give 3, and then coupled with 4-isopropyl
benzoic acid using hexafluorophosphate azabenzotriazole tet-
ramethyl uronium (HATU) to give N-(6-chloroimidazo[1,2-b]
pyridazin-2-yl)-4-isopropylbenzamide (4) as a white solid.
Results and Discussion
Representative synthetic chemistry is described in Scheme 1.
2-Chloroacetamide and oxalyl chloride were condensed and
N
N
N
O
N
N
O
O
HN
N
N
N
N
N
N
N
N
N
N
N
H
N
H
O
N
N
F
Takeda 1
Selonsertib
Biogen
Figure 1. Various ASK1 inhibitors.
Figure 2. ASK1 medicinal chemistry strategy.
Scheme 1. Reagents and conditions: (a) DCE, reflux, 3 h; t-BuOH (67%); (b) 3-amino-6-chloropyridazine, Na2HPO4, DMA, 120 ꢀC, 2 h
(86%); (c) HCl, dioxane, rt, 4 h (quantitative); (d) 4-isopropylbenzoic acid, HATU, DIPEA, DMF, rt, o/n (34%); (e) formamidine acetate,
K2CO3, toluene, 80 ꢀC, o/n (17%); (f) K2CO3, DMSO, 180 ꢀC, o/n (15%).
Bull. Korean Chem. Soc. 2021
© 2021 Korean Chemical Society, Seoul & Wiley-VCH GmbH.
www.bkcs.wiley-vch.de
2

BULLETIN OF THE
Article
Imidazo[1,2-b]pyridazines for ASK1 Inhibition
KOREAN CHEMICAL SOCIETY
Table 1. Biochemical activities of KTA-29 and its analogs to
ASK1.
ASK1% remaining
activity @ 0.5 μM
Compound
R1
R2
Selonsertib
0
6
7
8
9
Me
Et
1,2-benzene
1,2-benzene
1,2-benzene
1,2-benzene
1,2-benzene
1,2-dimethyl
1,2-dimethyl
1,2-dimethyl
88
79
55
66
62
100
79
52
59
57
92
82
59
64
69
100
80
35
77
92
i-Pr
c-Pr
t-Bu
Me
Et
i-Pr
c-Pr
t-Bu
Me
Et
i-Pr
c-Pr
t-Bu
Me
Et
i-Pr
c-Pr
t-Bu
10
11
12
13
14
15
16
17
18
19
20
21
22
KTA-29
23
24
1,2-dimethyl
1,2-dimethyl
1,2-cyclohexane
1,2-cyclohexane
1,2-cyclohexane
1,2-cyclohexane
1,2-cyclohexane
1,2-cycloheptane
1,2-cycloheptane
1,2-cycloheptane
1,2-cycloheptane
1,2-cycloheptane
Final condensation of 4 with 1,4,5,6,7,8-hexahydrocyclohepta
[d]imidazole (5) provided the desired 4-isopropyl-N-(6-(5,-
6,7,8-tetrahydrocyclohepta[d]imidazol-1(4H)-yl)imidazo
[1,2-b]pyridazin-2-yl)benzamide (KTA-29) as a yellow
solid. All the structural analogs (6–24) were prepared
throughout the same chemistry.
Figure 3. (a) Predicted docking pose of KTA-29 (green ball and
stick model) superimposed with the selonsertib (magenta line, pdb
code: 6OYT) in the ASK1. Surface and ribbon models of flap
region are omitted for clarity, only the interacting residues are
labeled. Hydrogen bonds are shown as green dashed lines;
(b) RMSD profile of the ASK1 C-α atoms (gray line, left axis)
and KTA-29 (red line, right axis) during the MD simulations;
(c) interaction fraction plot of protein-ligand contacts during the
MD simulations. H-bonds, hydrophobic interactions and water
bridges are shown in green, purple and blue, respectively.
Newly prepared compounds were evaluated on % ASK1
remaining activity assay at 0.5 μM (Table 1).12 First, we
surveyed para-substitution effect (R1) on the C2-benzamide
side chain. Although the Takeda 1 has tert-butyl at that site,
other ASK1 inhibitors possess either cyclopropyl or isopro-
pyl for optimal potency. On our imidazo[1,2-a]pyridine
backbone, isopropyl is proven consistently optimal at the
para-substitution (R1) of the C2-benzamide. Second, for
structural novelty purpose, we designed symmetric or
cyclized R2 on the C6-imidazoles. Benzimidazole,
4,5-dimethylimidazole, 4,5-cyclohexaneimidazole and
4,5-cycloheptaneimidazole were employed as symmetric or
cyclized imidazoles. Among them, KTA-29, having isopro-
pyl at R1 and 1,2-cycloheptane at R2, was most potent in
this series.
To further elucidate the interactions between the ligands
and ASK1, molecular docking and molecular dynamics
simulations for each complex were conducted with the
Schrödinger Suite 2020-4.13
Dockings were performed using the Glide program with
default settings unless specified. The crystal structure of
Bull. Korean Chem. Soc. 2021
© 2021 Korean Chemical Society, Seoul & Wiley-VCH GmbH.
www.bkcs.wiley-vch.de
3

BULLETIN OF THE
Article
Imidazo[1,2-b]pyridazines for ASK1 Inhibition
KOREAN CHEMICAL SOCIETY
ASK1 (pdb code: 5VIL) was downloaded from Protein
Data Bank, and structural defects were fixed. Standard pre-
cision (SP) flexible ligand docking was done with cubit
grid size of 22 Å centered on the crystal ligand. Robustness
of docking protocol was verified via reproduction of crystal
ligand poses with good root mean square displacement of
0.2 Å. The best docked pose with the lowest Glide score
was selected for the subsequent binding pose analysis and
molecular dynamics simulation.
The Desmond program was used to carry out classical
molecular dynamics (MD) simulations for the complexes.
Each protein–ligand complex was solvated in simple point-
charge model using orthorhombic periodic boundary condi-
tions, with neutralizing Na+ or Cl− ions. Systems were
relaxed with NPT ensembles (isothermal–isobaric ensemble
(constant temperature, pressure and the number of parti-
cles)) with Desmond default settings. Simulations were run
for a total of 250 ns with constant temperature and pressure
of 300 K and 1.01325 bar. Analysis of simulation results
were performed using the SID and SEA program.
Ala707, and Leu810, giving additional stability to the com-
plex. The main chain NH of Gly759 has additional NHÁ Á Áπ
interaction with the terminal phenyl group. Taken over,
these data show that KTA compounds and selonsertib have
similar interaction profiles with the ASK1.
KTA-29 was next examined in a cell-based assay.
HepG2 cells are one of widely used cell-based models for
NAFLD and the accumulation of triglyceride (TG) is one
of the characteristics of NAFLD.14 Here, the cellular activ-
ity of KTA-29 was evaluated in HepG2 cells by measuring
TG content.15 As shown in Figure 4, TG accumulation was
significantly induced by 1 mM free fatty acid (FFA) mix-
ture containing palmitate/oleate (1:2 ratio), and the FFA-
induced TG accumulation was significantly inhibited by
0.3 μM selonsertib. In this study, KTA-29 fails to show
noticeable cellular activity at 3 μM, while selonsertib
inhibits TG accumulation at 0.3 μM.
Conclusion
Figure 3(a) shows the superimposed structure of KTA-29
and selonsertib at the ATP binding site of ASK1. Low-pro-
tein and ligand root-mean-square deviation (RMSD) pro-
files of less than 2.1 and 1.3 Å show the stability of our
docking and simulation systems. The binding affinity of the
compounds are mainly due to the bidentate hydrogen bond-
ing of the amide NH and imidazole nitrogen atoms to the
main chain C O and NH atoms of Val757 in the hinge
region. The cycloheptaneimidazole nitrogen also shows
strong hydrogen bonding propensity with Lys709 during
MD simulations, contributing to the overall ligand stability
shown in Figure 3(b).
In summary, as an exploratory ASK1 inhibitor program, we
designed new ASK1 inhibitor hybridizing the selonsertib’s
pyridine and 4-isopropyl-1,2,4-triazole with the Takeda’s
imidazo[1,2-a]pyridine: we introduced 4,5-bicyclo-
alkaneimidazoles at C6 position of imidazo[1,2-b]
pyridazine. During the course of this investigation, we iden-
tified KTA-29 having isopropyl at para-substitution of the
C2-benzamide and 1,2-cycloheptaneimidazole at the C6
position of imidazo[1,2-b]pyridazine, and attempted to
measure cellular activity for NAFLD indication model.
Synthetic chemistry to KTA-29 was described and its puta-
tive binding mode was also described using a molecular
docking model. Although KTA-29 was not potent enough
to show cellular activity, further investigation will be car-
ried out searching for novel and more potent ASK1
inhibitors.
The compounds are located in the hydrophobic pocket
composed of Leu686, Val694, Ala707 and Leu810 in
Figure 3(c). Especially, the imidazo[1,2-b]pyridazine is
clamped between hydrophobic residues, Leu686, Val694,
Experimental
Instrumentation and Analysis. Reagents used in this
study were purchased from Sigma-Aldrich (St. Louis, MO,
USA), TCI (Tokyo, Japan), Alfa (Auburn Hills, MI, USA),
Ark Pharm (Arlington Heights, IL, USA), and so forth, and
used without further purification. The purity of the synthe-
sized compounds and the progress of the reaction were con-
firmed by thin layer chromatography (TLC) using TLC
Silica gel 60 F254, 0.25 mm (Merck, St. Louis, MO, USA).
A ultraviolet (UV) lamp (254 and 365 nm) was used for
identification of the disadvantageous substances on the
TLC. Separation was done using either medium-pressure
liquid chromatography (CombiFlash NEXTGEN 300+
[Teledyne ISCO, Lincoln, NE, USA] using a column car-
tridge [4–120 g, RediSep®Rf]) or high-performance liquid
chromatography (HPLC) (YL9100 Semi-prep HPLC sys-
tem [YL9101S Vacuum Degasser, YL9111S Binary Pump,
YL9120S UV/Vis Detector] using Agilent 5 Prep-C18
Figure 4. Effect of KTA-29 on FFA-induced TG accumulation.
Effect of KTA-29 on FFA-induced TG accumulation was evalu-
ated in HepG2 cells by measuring TG content. Selonsertib was
used as the positive control. ##p < 0.01 (the control group
vs. FFA-treated group), *p < 0.05 (FFA-treated group vs. FFA
+ selonsertib-treated group).
Bull. Korean Chem. Soc. 2021
© 2021 Korean Chemical Society, Seoul & Wiley-VCH GmbH.
www.bkcs.wiley-vch.de
4

BULLETIN OF THE
Article
Imidazo[1,2-b]pyridazines for ASK1 Inhibition
KOREAN CHEMICAL SOCIETY
100 × 21.2 mm column cartridge). Mass spectroscopy of
the products was identified by Agilent 6130 Quandrupole
Liquid chromatography–mass spectrometry (LC/MS). High
resolution mass spectroscopy of the products was obtained
by EI mode, 70 eV, resolution 5000 using JEOL JMS-700
High Resolution Mass Spectrometer (HRMS). Nuclear
magnetic resonance (NMR) spectra for the structural analy-
sis of the products were measured by Bruker ultrashield
300-MHz, 400-MHz and 500-MHz NMR spectrometers.
Most of NMR solvents were purchased from Cambridge
Isotope Laboratories such as chloroform‑d, methanol‑d4,
and dimethylsulfoxide-d6. Molecular modeling studies were
performed using the Glide software in the Maestro11.5
software with SP options.
tert-Butyl (2-Chloroacetyl)carbamate (1). To the suspen-
sion of 2-chloroacetamide (2.50 g, 26.7 mmol) in
1,2-chloroethane (15.0 mL) was added dropwise oxalyl
chloride (2.80 mL, 32.1 mmol) at 0 ꢀC. After heating under
reflux for 3 h, the mixture was cooled to 0 ꢀC. tert-Butyl
alcohol/1,2-dichloroethane (8.00 mL, 1/1) were added to
the mixture and the mixture was stirred for 20 min. Satu-
rated aqueous sodium hydrogen carbonate solution was
added to the reaction mixture, and the mixture was
extracted with 1,2-dichloroethane. After washing with satu-
rated aqueous sodium hydrogen carbonate solution and
water, the extract was dried over anhydrous magnesium
sulfate and filtered. The organic filtrate was evaporated
organic filtrate was evaporated under reduced pressure to
give 6-chloroimidazo[1,2-b]pyridazin-2-amine (3) as a yel-
low solid (1.70 g, quantitative yield): H NMR (500 MHz,
DMSO‑d6) δ 7.68 (dd, J = 9.2, 0.7 Hz, 1H), 7.36 (s, 1H),
7.04 (d, J = 9.2 Hz, 1H), 5.66 (s, 2H); LC–MS: calcd for
C6H5ClN4: 168.0, found: 169.4 [M + H+].
1
N-(6-Chloroimidazo[1,2-b]pyridazin-2-Yl)-4-isopropyl-
benzamide (4). To a solution of 6-chloroimidazo[1,2-b]
pyridazin-2-amine (3, 1.00 g, 5.93 mmol) in N,N-
dimethylformamide (20.0 mL) were added 4-iso-
propylbenzoic acid (1.07 g, 6.52 mmol) and HATU
(2.71 g, 7.12 mmol) and diisopropylamine (2.07 mL,
11.9 mmol). After stirring at room temperature for over-
night, the reaction mixture was diluted with ethyl acetate
and washed with water several times. The organic layer
was washed with brine, dried over anhydrous magnesium
sulfate and filtered. The organic filtrate was evaporated
under reduced pressure and the residue was purified by
flash column chromatography on silica gel (hexane/ethyl
acetate = 100/0 à 50/50) to give N-(6-chloroimidazo[1,2-
b]pyridazin-2-yl)-4-isopropylbenzamide (4) as a white solid
1
(630 mg, yield 34%): H NMR (500 MHz, DMSO‑d6) δ
11.40 (s, 1H), 8.49 (s, 1H), 8.12 (d, J = 9.4 Hz, 1H), 8.05–
7.99 (m, 2H), 7.38 (dd, J = 13.1, 8.8 Hz, 3H), 3.03–2.91
(m, 1H), 1.24 (d, J = 6.9 Hz, 6H); LC–MS: calcd for
C16H15ClN4O: 314.1, found: 315.0 [M + H+].
1,4,5,6,7,8-Hexahydrocyclohepta[d]imidazole (5). To a
solution of 2-chlorocycloheptanone (5.00 g, 34.1 mmol) in
toluene (150 mL) was added formamidine acetate (17.8 g,
171 mmol) in the presence of potassium carbonate (23.6 g,
171 mmol). The reaction mixture was stirred at 80 ꢀC for
overnight. After cooling to room temperature, solvent was
removed under reduced pressure. The residue was par-
titioned with ethyl acetate and water three times. The com-
bined organic layer was washed with brine, dried over
anhydrous magnesium sulfate and filtered. The organic fil-
trate was evaporated under reduced pressure and the residue
was purified by flash column chromatography on silica gel
(dichloromethane/methanol = 100/0 à 70/30) to give
1,4,5,6,7,8-hexahydrocyclohepta[d]imidazole (5) as brown
solid (770 mg, yield 17%): 1H NMR (400 MHz,
chloroform‑d) δ 7.32 (s, 1H), 4.51 (s, 2H), 2.74–2.66
(m, 4H), 1.84–1.75 (m, 2H), 1.75–1.67 (m, 4H).
under
reduced
pressure
to
provide
tert-butyl
(2-chloroacetyl)carbamate (1) as a white crystal (3.45 g,
yield 67%): 1H NMR (300 MHz, DMSO‑d6) δ 10.63
(s, 1H), 4.42 (s, 2H), 1.44 (s, 9H).
tert-Butyl (6-Chloroimidazo[1,2-b]pyridazin-2-yl)carba-
mate (2). To a solution of 3-amino-6-chloropyridazine
(5.26 g, 40.6 mmol) in N,N-dimethylacetamide (150 mL)
were added tert-butyl (2-chloroacetyl)carbamate (1, 12.6 g,
65.0 mmol) and disodium hydrogen phosphate (14.4 g,
101 mmol), and the resulting mixture was stirred at 120 ꢀC
for 3 h. After cooling the mixture to room temperature,
water (40 mL) was added to the mixture, and the precipi-
tated crystals were filtered to give tert-butyl
(6-chloroimidazo[1,2-b]pyridazin-2-yl)carbamate (2) as a
bright brown solid (9.40 g, yield 86%): 1H NMR
(500 MHz, DMSO‑d6) δ 10.26 (s, 1H), 8.08–7.97 (m, 2H),
7.29 (d, J = 9.4 Hz, 1H), 1.49 (s, 9H); LC–MS: calcd for
C7H12ClNO3: 268.1, found: 269.3 [M + H+].
4-Isopropyl-N-(6-(5,6,7,8-tetrahydrocyclohepta[d]imida-
zol-1(4H)-yl)imidazo[1,2-b]pyridazin-2-yl)benzamide
6-Chloroimidazo[1,2-b]pyridazin-2-amine (3). To a sus-
pension of tert-butyl (6-chloroimidazo[1,2-b]pyridazin-
2-yl)carbamate (2, 2.65 g, 9.86 mmol) in ethyl acetate
(30.0 mL) was added 4 N hydrochloric acid in 1,4-dioxane
(20.0 mL), and the mixture was stirred at room temperature
for overnight. The solvent was removed under reduced
pressure. Water was added and the mixture was basified by
1 N aqueous sodium hydroxide solution to pH 7. Ethyl ace-
tate was added to the mixture and the mixture was extracted
twice. The combined organic layer was washed with brine,
dried over anhydrous magnesium sulfate and filtered. The
(KTA-29).
4-isopropylbenzamide
N-(6-chloroimidazo[1,2-b]pyridazin-2-yl)-
(4, 300 mg, 0.95 mmol),
1,4,5,6,7,8-hexahydrocyclohepta[d]imidazole (5, 130 mg,
0.95 mmol), and potassium carbonate (400 mg, 2.85 mmol)
were stirred in dimethyl sulfoxide (3.00 mL) at 160 ꢀC for
overnight. After cooling to room temperature, the reaction
mixture was diluted with ethyl acetate and washed with
water several times. The organic layer was washed with
brine, dried over anhydrous magnesium sulfate and filtered.
The organic filtrate was evaporated under reduced pressure
and the residue was purified by flash column
Bull. Korean Chem. Soc. 2021
© 2021 Korean Chemical Society, Seoul & Wiley-VCH GmbH.
www.bkcs.wiley-vch.de
5

BULLETIN OF THE
Article
Imidazo[1,2-b]pyridazines for ASK1 Inhibition
KOREAN CHEMICAL SOCIETY
Nunez, M. Sabat, J. Tyhonas, H. Wang, X. Wang, S. Swann,
ACS Med. Chem. Lett. 2017, 8, 316. (d) S. Hou, X. Yang, Y.
Tong, Y. Yang, Q. Chen, B. Wan, R. Wei, Y. Wang, Y.
Zhang, B. Kong, J. Huang, Y. Chen, T. Lu, Q. Hu, D. Du,
Eur. J. Med. Chem. 2021, 211, 113114.
chromatography on silica gel (ethyl acetate/methanol = 100/
0
à
90/10)
to
give
4-isopropyl-N-(6-(5,-
6,7,8-tetrahydrocyclohepta[d]imidazol-1(4H)-yl)imidazo
[1,2-b]pyridazin-2-yl)benzamide (KTA-29) as a yellow
solid (60.0 mg, yield 15%): 1H NMR (500 MHz,
DMSO‑d6) δ 11.40 (s, 1H), 8.50 (s, 1H), 8.24–8.20 (m,
1H), 8.06–8.02 (m, 2H), 7.85 (s, 1H), 7.43 (d, J = 9.4 Hz,
1H), 7.42–7.39 (m, 2H), 3.01–2.95 (m, 1H), 2.79–2.73 (m,
2H), 2.73–2.68 (m, 2H), 1.82–1.76 (m, 2H), 1.72–1.62 (m,
4H), 1.24 (d, J = 6.9 Hz, 6H); 13C NMR (100 MHz,
DMSO‑d6) δ 165.15, 153.21, 145.51, 143.87, 141.63,
134.79 (2C), 131.34, 128.66, 128.61, 126.87, 125.87,
115.52, 106.64, 33.90, 30.63, 29.75, 27.45, 27.41, 24.76,
24.07; HRMS (EI) calcd for C24H26N6O: 414.2168, found:
414.2160.
7. S. A. Harrison, V. W. Wong, T. Okanoue, N. Bzowej, R.
Vuppalanchi, Z. Younes, A. Kohli, S. Sarin, S. H. Caldwell,
N. Alkhouri, M. L. Shiffman, M. Camargo, G. Li, K. Kersey,
C. Jia, Y. Zhu, C. S. Djedjos, G. M. Subramanian, R. P.
Myers, N. Gunn, A. Sheikh, Q. M. Anstee, M. Romero-
Gomez, M. Trauner, Z. Goodman, E. J. Lawitz, Z. Younossi,
J. Hepatol. 2020, 73, 26.
8. R. Loomba, E. Lawitz, P. S. Mantry, S. Jayakumar, S. H.
Caldwell, H. Arnold, A. M. Diehl, C. S. Djedjos, L. Han,
R. P. Myers, G. M. Subramanian, J. G. McHutchison, Z. D.
Goodman, N. H. Afdhal, M. R. Charlton, Hepatology 2018,
67, 549 Erratum in: Hepatology 2018, 67(5), 2063.
Acknowledgments. The authors greatly acknowledge the
generous financial support from the Korea Research Insti-
tute of Chemical Technology (KK2131-30, SI2131-10 and
SI2131-40).
9. Clinical trials searched drug name “selonsertib”. URL https://
clinicaltrials.gov/ (accessed January 7, 2021).
10. Y. Terao, H. Suzuki, M. Yoshikawa, H. Yashiro, S.
Takekawa, Y. Fujitani, K. Okada, Y. Inoue, Y. Yamamoto,
H. Nakagawa, S. Yao, T. Kawamoto, O. Uchikawa, Bioorg.
Med. Chem. Lett. 2012, 22, 7326.
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
11. N. Sakai et al. (Takeda Pharmaceutical Company Limited),
WO 2008/016192.
12. The biochemical activities against ASK1 were tested using
the Eurofins Kinase Profiler screening platform (Eurofins
Pharma Discovery Services UK Limited, Dundee, UK). In the
assay, ASK1 (h) is incubated with 8 mM MOPS pH 7.0,
0.2 mM EDTA, 0.33 mg/mL myelin basic protein, 10 mM
MgAcetate and [gamma-33P]-ATP. The reaction is initiated
by the addition of the Mg/ATP mix. After incubation for
40 minutes at room temperature, the reaction is stopped by
the addition of phosphoric acid to a concentration of 0.5%.
10 μL of the reaction is then spotted onto a P30 filtermat and
washed four times for 4 min in 0.425% phosphoric acid and
once in methanol prior to drying and scintillation counting.
13. Schrödinger Release 2020-4: Maestro, Schrödinger, LLC,
New York, NY, 2020.
References
1. (a) A. Matsuzawa, K. Saegusa, T. Noguchi, C. Sadamitsu, H.
Nishitoh, S. Nagai, S. Koyasu, K. Matsumoto, K. Takeda, H.
Ichijo, Nat. Immunol. 2005, 6, 587e592. (b) H. Ichijo, E.
Nishida, K. Irie, P. tenDijke, M. Saitoh, T. Moriguchi, M.
Takagi, K. Matsumoto, K. Miyazono, Y. Gotoh, Science
1997, 275, 90e94. (c) A. Matsuzawa, H. Ichijo, J. Biol. Chem.
2001, 130, 1e8.
2. G. Kefala, K. Tziomalos, Expert Rev. Gastroenterol. Hepatol.
2019, 13, 189.
3. R. Brys, K. Gibson, T. Poljak, S. Van Der Plas, D. Amantini,
Prog. Med. Chem. 2020, 59, 101.
4. N. Chalasani, Z. Younossi, J. E. Lavine, A. M. Diehl, E. M.
Brunt, K. Cusi, M. Charlton, A. J. Sanyal, Hepatology 2012,
55, 2005.
5. G. Vernon, A. Baranova, Z. M. Younossi, Aliment
Pharmacol. Ther. 2011, 34, 274.
6. (a) S. Zhang, C. Huang, X. Lyu, P. Wang, Y. Zang, Z. Wang,
H. Wang, J. Li, Y. Zhao, Eur. J. Med. Chem. 2020, 195,
112277. (b) Z. Xin, M. K. Himmelbauer, J. H. Jones, I.
Enyedy, R. Gilfillan, T. Hesson, K. King, D. J. Marcotte, P.
Murugan, J. C. Santoro, F. Gonzalez-Lopez de Turiso, ACS
Med. Chem. Lett. 2020, 11, 485. (c) M. Lanier, J. Pickens,
S. V. Bigi, E. L. Bradshaw-Pierce, A. Chambers, Z. S.
Cheruvallath, D. Cole, D. R. Dougan, J. Ermolieff, T.
Gibson, P. Halkowycz, A. Hirokawa, A. Ivetac, J. Miura, E.
ꢀ
ꢀ
14. M. J. Gomez-Lechon, M. T. Donato, A. Martínez-Romero, N.
Jiménez, J. V. Castell, J. E. O’Connor, Chem. Biol. Interact
2007, 165, 106.
15. Cellular TG content measurement. Human hepatoma cell line
HepG2 (Korean Cell Line Bank, Seoul, Korea) were cultured
in Minimal Essential Medium (MEM; HyClone, Logan, UT,
USA) with 10% fetal bovine serum (HyClone, Logan, UT,
USA), 1% antibiotic-antimycotic solution (HyClone, Logan,
UT, USA) at 37ꢀC and 5% CO2. Cells were incubated in a
96-well plate at a density of 2 × 104 cells/well for 24 h. Then,
cells were treated with compounds and/or the mixture of
palmitic acid and oleic acid at a ratio of 1:2 that was incu-
bated in water bath at 70ꢀC for 30 min. After 2-day incuba-
tion, the cellular TG content was measured using Triglyceride
Quantification Kit (Sigma-Aldrich, St. Louis, MO, USA).
Bull. Korean Chem. Soc. 2021
© 2021 Korean Chemical Society, Seoul & Wiley-VCH GmbH.
www.bkcs.wiley-vch.de
6