Structure-based design, synthesis, and biological evaluation of imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.12.031

We identified novel potent inhibitors of p38 MAP kinase using structure-based design strategy. X-ray crystallography showed that when p38 MAP kinase is complexed with TAK-715 (1) in a co-crystal structure, Phe169 adopts two conformations, where one intera

Full text of DOI:10.1016/j.bmc.2017.12.031

Accepted Manuscript
Structure-based Design, Synthesis, and Biological Evaluation of Imidazo[1,2-
b]pyridazine-based p38 MAP Kinase Inhibitors
Akira Kaieda, Masashi Takahashi, Takafumi Takai, Masayuki Goto, Takahiro
Miyazaki, Yuri Hori, Satoko Unno, Tomohiro Kawamoto, Toshimasa Tanaka,
Sachiko Itono, Terufumi Takagi, Teruki Hamada, Mikio Shirasaki, Kengo
Okada, Gyorgy Snell, Ken Bragstad, Bi-Ching Sang, Osamu Uchikawa, Seiji
Miwatashi
PII:
S0968-0896(17)31781-9
https://doi.org/10.1016/j.bmc.2017.12.031
BMC 14136
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 September 2017
20 December 2017
22 December 2017
Please cite this article as: Kaieda, A., Takahashi, M., Takai, T., Goto, M., Miyazaki, T., Hori, Y., Unno, S.,
Kawamoto, T., Tanaka, T., Itono, S., Takagi, T., Hamada, T., Shirasaki, M., Okada, K., Snell, G., Bragstad, K.,
Sang, B-C., Uchikawa, O., Miwatashi, S., Structure-based Design, Synthesis, and Biological Evaluation of
Imidazo[1,2-b]pyridazine-based p38 MAP Kinase Inhibitors, Bioorganic & Medicinal Chemistry (2017), doi:
https://doi.org/10.1016/j.bmc.2017.12.031
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Structure-based Design, Synthesis, and Biological
Evaluation of Imidazo[1,2-b]pyridazine-based p38 MAP
Kinase Inhibitors
Akira Kaieda,a,* Masashi Takahashi,a Takafumi Takai,a Masayuki Goto,a Takahiro Miyazaki,a Yuri
Hori,a Satoko Unno,a Tomohiro Kawamoto,a Toshimasa Tanaka,a Sachiko Itono,a Terufumi Takagi,a
Teruki Hamada,a Mikio Shirasaki,a Kengo Okada,a Gyorgy Snell,b Ken Bragstad,b Bi-Ching Sang,b
Osamu Uchikawa,a and Seiji Miwatashia
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited., 26-1, Muraoka-higashi
2-chome, Fujisawa, Kanagawa 251-8555, Japan
b Takeda California, 10410 Science Center Drive, San Diego, California 92121, United States
KEYWORDS: p38 mitogen-activated protein kinase inhibitors, rheumatoid arthritis, structure-based
design, imidazo[1,2-b]pyridazine derivatives, pyridine N-oxide
ABSTRACT
We identified novel potent inhibitors of p38 MAP kinase using structure-based design strategy. X-ray
crystallography showed that when p38 MAP kinase is complexed with TAK-715 (1) in a co-crystal
structure, Phe169 adopts two conformations, where one interacts with 1 and the other shows no
interaction with 1. Our structure-based design strategy shows that these two conformations converge
into one via enhanced protein-ligand hydrophobic interactions. According to the strategy, we focused
1

on scaffold transformation to identify imidazo[1,2-b]pyridazine derivatives as potent inhibitors of p38
MAP kinase. Among the herein described and evaluated compounds, N-oxide 16 exhibited potent
inhibition of p38 MAP kinase and LPS-induced TNF-αproduction in human monocytic THP-1 cells,
and significant in vivo efficacy in rat collagen-induced arthritis models. In this article, we report the
discovery of potent, selective and orally bioavailable imidazo[1,2-b]pyridazine-based p38 MAP kinase
inhibitors with pyridine N-oxide group.
1. Introduction
The p38 mitogen-activated protein (MAP) kinase, a serine/threonine kinase, is widely expressed in
endothelial, immune, and inflammatory cells; it is a key component in the cascade leading to the
activation of pro-inflammatory cytokines, such as TNF-αand IL-1β, in immune cells.1-4 The p38 MAP
kinase is activated by inflammatory cytokines or extracellular stressors such as heat, mechanical wear,
osmotic shock, and ultraviolet light.5 Recent analysis of synovial tissue, extracted from patients with
rheumatoid arthritis (RA), suggests that the p38 MAP kinase is overproduced within the inflamed
tissue.6-7 Inhibition of p38 MAP kinase is, therefore, a promising therapeutic strategy for the treatment
of cytokine-driven disorders such as RA, inflammatory bowel disease (IBD), and chronic obstructive
pulmonary disease (COPD).8-9, 5
Whereas many p38 MAP kinase inhibitors have proceeded to clinical studies, there has been limited
progress in the clinical development of these compounds.10-11 However, this remains an area of intense
interest,12-18 and many compounds are in clinical development.
The p38 MAP kinase inhibitors reported to date can be divided into three types based on the chemical
structure and interaction with p38 MAP kinase (Figure 1).19 All three types occupy the ATP-binding site.
Both teardrop binder such as SB203580 and linear binder such as Scio-469 and VX-745 shows ATP
competitiveness.20-24 Another type, extended binder such as BIRB-79625, shows ATP non-
2

competitiveness due to induction of DFG-out conformation by binding to allosteric site of the ATP
pocket.25
Fig. 1. Classification of representative p38 MAP kinse inhibitors.
We previously reported that the pyridinylthiazole derivatives, such as
(1, TAK-715), are potent inhibitors
of p38 MAP kinase, which was classified in teardrop binder (Figure 2).26 In our further effort toward
discovering alternative classes of p38 MAP kinase inhibitors that could be developed as backups to 1,
we investigated novel core structures instead of the thiazole ring of 1. In this report, we describe
rational design using structure-based design, synthesis, SAR, and biological assessment of the
derivatives of imidazo[1,2-b]pyridazine.
3

Fig. 2. Chemical Structure of 1
2. Chemistry
The synthesis of imidazo[1,2-b]pyridazine analogs 8a-o and 10a-b followed a general route as
outlined in scheme 1. To the lithium salt of 2a-b in THF were added the appropriate propylenimines
3a-d to provide the corresponding ketones 4a-d and 4f.26-27 The Weinreb amide 3e, which was used
instead of the propyleneimines 3a-d, also led to the formation of ketone 4e. Bromination of 4a-f using
bromine in acetic acid afforded α–bromoketones 5a-f, which were treated with 3-aminopyridazine in
EtOH to give no desired product. It is likely that α–bromoketone alkylated the N1 of 3-aminopyridazine
because of the relatively high nucleophilicity of the N1 of 3-aminopyridazine. To decrease the
nucleophilicity of the N1, we used 3-amino-6-chloropyridazine instead of 3-aminopyridazine,
successfully achieving the cyclization to imidazo[1,2-b]pyridazines 6a-f. De-chlorination of 6a-f under
hydrogenesis condition led to the key intermediates 7a-f. Importantly, Et3N was necessary for HCl
scavenging, even though its presence diminished the catalytic ability of Pd/C. The subsequent amidation
with the appropriate acyl chloride produced 8a-o and 9a-b. Compounds 10a-b were prepared by de-
chlorination of 9a-b under the hydrogenesis condition.
4

N-Oxides 15a-b were synthesized as shown in scheme 2. Reductive de-chlorination of 11 generated
12a. Oxidation of 12a-b led to 13a-b, followed by hydrolysis to give carboxylic acids 14a-b.
Condensation between 14a-b and 7d afforded N-oxides 15a-b.
As shown in scheme 3, sulfate 16 was prepared using treatment with one equivalent of sulfonic acid
in acetic acid and ethanol. Acetic acid as a solvent was the most effective at dissolving 15a in a small
volume of solvent.
Scheme 1. Synthesis of compounds 8a-o and 10a-ba
5

6

a Reagents and conditions: (a) i) nBuLi, THF, −78 ºC then at 0 ºC; ii) 3a-e, −78 ºC; (b) Br2, AcOH, 80
ºC for 5a-e; (c) Br2, AcONa, AcOH, rt for 5f; (d) 3-aminopyridazine, EtOH, 80 ºC; (e) 3-amino-6-
chloropyridazine, EtOH, 80 ºC; (f) H2 (0.5 MPa), Pd/C, AcOH, 50 ºC for 7a; (g) H2 (0.1 MPa), Pd/C,
Et3N, DMF, rt for 7b-f; (h) i) acyl chloride derivative, Et3N, DCM, rt; ii) NH3/EtOH, rt for 8a-j, 8m,
9b; (i) i) acyl chloride derivative, pyridine, DMA, rt; ii) NH3/EtOH, THF, rt for 8k-l; (j) i) acyl chloride
derivative, Et3N, THF, rt; ii) NH3/EtOH, THF, rt for 8n; (k) i) 6-methylpyridine-3-carboxylic acid,
(COCl)2, Et3N, DMF, THF, rt; ii) Et3N, THF, rt; iii) NH3/EtOH, THF, rt for 8m; (l) i) 2-chloro-6-
methylpyridine-4-carboxylic acid, POCl3, 50 ºC; ii) Et3N, THF, rt; iii) NH3/EtOH, THF, rt for 9a; (m)
H2 (0.5 MPa), Pd/C, Et3N, DMF, 50 ºC for 10a-b.
Scheme 2. Synthesis of compounds 15a-ba
a
Reagents and conditions: (a) H2 (0.1 MPa), Pd/C, Et3N, DMF, rt for 12a; (b) H2O2 urea complex,
AcOH, rt; (c) 8 N NaOH (aq.), THF, MeOH, 70 °C; (d) 7d, HATU, iPr2EtN, pyridine, 70 ºC.
7

Scheme 3. Synthesis of sulfate 16a
a Reagents and conditions: (a) H2SO4, EtOH, AcOH, 100 °C.
3. Results and discussion
Based on a 2.00-Å resolution of the co-crystal structure of 1 bound to p38 MAP kinase, the nitrogen
atom of the pyridine ring, and the hydrogen atom of the amide NH group of 1, form typical hydrogen-
bond interactions with the NH and carbonyl oxygen on the main chain of Met109, respectively (Figure
3).28 Additionally, the nitrogen atom of the thiazole ring forms a hydrogen bond with the NH2 group on
the side chain of Lys53, and the oxygen atom of the amide carbonyl group interacts with the NH on the
main chain of Ala172. Interestingly, the side chain of Phe169 adopts two conformations, where one has
hydrophobic interactions with ethyl thiazole of 1, and the other shows no interaction with 1. We,
therefore, hypothesized that enhancing the hydrophobic interactions of the inhibitor with the side chain
of Phe169 would improve the inhibition of p38. To enhance the hydrophobic interactions, we initially
transformed ethyl thiazole of 1 into several fused rings and discovered that imidazo[1,2-b]pyridazine
derivative 8a strongly inhibited p38 (Figure 4, Table 1).
8

(A)
(B)
Fig. 3. (A) Structure of inhibitor 1. The atoms involved in the hydrogen-bond interactions with p38
MAP kinase are shown in blue. (B) X-ray co-crystal structure of 1 bound with p38 MAP kinase (PDB
code 6ANL). The blue dashed lines show hydrogen-bond interactions.
Fig. 4. Design concept of p38 MAP kinase inhibitors. The atoms involving hydrogen-bond interactions
with p38 MAP kinase are shown in blue.
The inhibitory potencies (IC50) of the synthesized compounds were estimated using a p38 MAP kinase
assay and an assay measuring LPS-induced production of TNF-α in human monocytic THP-1 cells
(Table 1). The pyridine 8a and pyrimidine 8f analogs showed stronger inhibition of p38 MAP kinase
than did 1. Pyridine 8a inhibited the LPS-induced production of TNF-α in THP-1 cells with an IC50
value of 58 nM, while the inhibitory activity of pyrimidine 8f was nearly equal to that of 1. We,
9

therefore, aimed to synthesize pyridine analogs that would show fewer differences between enzyme and
cellular functions. Hence, we focused on optimizing the side chains. Based on previous studies26-27, we
examined the effects of the substituents on phenyl groups. Replacing the methyl group in 8a with a
chloro group at the meta-position of 8b led to similar activity against p38 MAP kinase. Compound 8c,
possessing a chloro group at the para-position, showed reduced inhibitory activity. Introduction of the
smaller fluoro group at the para-position produced 8d, which was a more potent inhibitor than 8a. This
result is consistent with the co-crystal structure of 1, in which the space near the para-position on the
phenyl group is narrow. Additionally compound 8e was found to be more potent than 8g.
Table 1. Inhibitory activities of imidazo[1,2-b]pyridazine derivatives against p38 MAP kinase and
production of TNF-αin human THP-1 cells.
p38 MAPK
Production of TNF-α
Cmpd.
X
R1
R2
IC50a (nM)
IC50a (nM)
240 (170 - 350)
73 (36 - 150)
56 (50 - 63)
240 (110 - 540)
58 (35 - 94)
1
CH
N
Ph
3-Me
3-Me
8a
8f
Ph
260 (140 - 510)
10

CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
Ph
3-Cl
34 (28 - 41)
290 (200 - 440)
9.4 (8.4 - 10)
11 (10-13)
88 (40 - 190)
130 (69 - 250)
52 (34 - 81)
8b
8c
8d
8e
8g
8h
8i
Ph
4-Cl
Ph
3-Me, 4-F
3-Me, 4-F
3-Cl, 4-F
3-Me, 4-F
3-Me, 4-F
3-Me, 4-F
3-Me, 4-F
3-Me, 4-F
3-Me, 4-F
3-Me, 4-F
3-Me, 4-F
3-CF3-Ph
3-CF3-Ph
2-CH3-Ph
3-CH3-Ph
4-CH3-Ph
3-F-Ph
3-Cl-Ph
2-Py
13 (8.5-19)
210 (160-280)
84 (74 - 95)
11 (9.0 - 13)
23 (20 - 27)
12 (10 - 13)
14 (8.5 - 22)
23 (19 - 28)
2.2 (0.58 – 8.0)
5.4 (3.1 – 9.3)
>10000
1300 (610 - 2800)
19 (13 - 29)
1000 (110 - 9900)
18 (14 - 24)
8j
8k
8l
14 (7.8 - 24)
360 (220 - 610)
3.5 (2.2 – 5.5)
16 (9.0 - 29)
8m
10b
3-Py
4-Py
8n
a
IC50 values shown are the mean values of quadruple measurements. Numbers in parentheses
represent 95% confidence intervals.
We further investigated the effects of the amide side chain linked at the 2-position on the pyridine
ring. A methyl group, as an electron-donating substituent, was introduced to the ortho-, meta-, or para-
position (8h-j) on the phenyl group of the benzoyl moiety. Compared with 8d, the ortho-methyl 8h
11

showed decreased potency; the meta- and para-methyl analogs 8i and 8j exhibited tolerable inhibitory
activity, inhibiting the p38 MAP kinase with IC50 values of 11 and 23 nM, respectively. Introduction of
an electron-withdrawing substituent, such as a fluoro or a chloro group (8k, 8l), at a meta-position on
the phenyl group of the benzoyl moiety, produced such potent inhibitors as 8i. To control the
lipophilicity of inhibitors, we replaced the phenyl ring with the pyridine ring. The 2-pyridyl 8m showed
decreased inhibitory activity against p38, while the 3-pyridyl 10b or 4-pyridyl group 8n displayed more
potent inhibitory activity against p38 than did 8d. Additionally, in the human THP-1 cells, 10b and 8n
were more effective than 8d at inhibiting the production of TNF-α.
Non-substituted pyridine derivatives can inhibit CYPs by chelating the heme iron of CYP enzymes.26,
29, 30
As shown in Table 2, compounds 10b and 8n showed potent inhibition of CYP3A4. We, therefore,
introduced the methyl group into the adjacent position on the nitrogen of the pyridine ring.
Surprisingly, compound 8o showed increased inhibition of CYP3A4; compound 10a showed reduced
inhibition of CYP3A4 while maintaining potent inhibitory activity against p38 and production of TNF-
α. Although compound 10a showed tolerable oxidative metabolic stability in rat microsomes, there was
a distinct discrepancy in rat and human oxidative metabolic stability. Interestingly, compound 10a had
greater oxidative metabolic stability compared with that of 8n. These results suggest that the methyl
group plays a role in preventing oxidative metabolism to give the pyridine N-oxide derivative. In order
to confirm that the main metabolism of 10a was oxidation of pyridine, we examined the in vitro
metabolism of 10a in human liver microsomes and detected MS3 as a main metabolite at the rate of
17.8% (Table 3). On the basis of the LC/UV/MS analysis for 10a and the metabolite, MS3 was
predicted N-oxide analog (see experimental section, Figure 5). The pyridine N-oxide 15a, which was
indeed synthesized, exhibited a dramatically improved stability in human liver microsomes and reduced
the discrepancy between rat and human metabolic stabilities compared with 10a, with inhibitory activity
as potent as that of compound 10a. The structure of 15a was confirmed to be identical to that of MS3.
Although the oxygen atom of the N-oxide 15a can coordinate with the heme ion, 15a showed tolerable
12

inhibition of CYP3A4. Therefore, the N-oxide analog 15a was selected for further investigation as a
drug candidate.
Table 2. DMPK profiles and inhibitory activities of imidazo[1,2-b]pyridazine derivatives against p38
MAP kinase and production of TNF-αin human THP-1 cells.
p38
TNF-α
Solubility CYP3A4
inhibition b
Stability in liver
microsomes
Cmpd. R1
IC50a (nM)
IC50a (nM)
pH = 6.8
(%
Rat / human
(μg/mL)
inhibition) (μL/min/mg)
3-Py
2.2 (0.58 –
3.5 (2.2 – 5.5) 0.92
61
71/105
10b
8.0)
4-Py
5.4 (3.1 – 9.3) 16 (9.0 - 29)
0.75
85
86
75/177
53/104
8n
8o
6-Me-3-Py 1.8 (0.63 –
1.2 (0.93 – 1.6) 0.38
5.1)
2-Me-4-Py 4.4 (1.8 - 11)
15 (9.0 – 26)
0.15
2.4
42
55
65/132
23/89
10a
15b
6-Me-3-Py 2.7 (2.4 – 3.0) 16 (8.9 – 28)
13

N-oxide
2-Me-4-Py 5.4 (4.6 – 6.4) 18 (10 – 33)
N-oxide
0.91
38
16/32
15a
a
IC50 values shown are the mean values of quadruple measurements. Numbers in parentheses
represent 95% confidence intervals. b Compound concentration is 10 μM.
Table 3. HPLC and LC/MSn data of 10a and its metabolites after incubation with human liver
microsomes.
Metabolite Retention time
(min)
Ratio a
(%)
[M+H]+
(m/z)
439
Major fragment ions
33.4
23.0
72.5
17.8
10a
303, 321, 411, 421
303, 321, 409, 438
455
MS3
a Residual and production ratios after 30 min incubation with human liver microsomes.
14

Fig. 5. Predicted structure of the main metabolite MS3 after 30 min incubation with human liver
microsomes.
To understand the binding mode of the novel class of p38 MAP kinase inhibitors, 15a was co-
crystallized with p38 MAP kinase. The determined 1.6-Å-resolution X-ray structure of the complex
revealed that 15a binds to the hinge region of p38 MAP kinase, which adopts the single Phe169
conformation (Figure 6). In the complex, the interactions observed in the co-crystal structure of 1,
bound with p38 MAP kinase, were expectedly conserved. Additionally, the solvent-exposed oxygen
atom of the pyridine-N-oxide interacts with the side chain of His174. The 4-fluoro-3-methyl phenyl ring
fits well into the shallow inner hydrophobic pocket defined by the residues Thr106 and Leu104. Finally,
the imidazo[1,2-b]pyridazine moiety interacts with the side chains of Tyr35, Val38, and Phe169. This
interaction, strengthened by the scaffold transformation, contributes to increased inhibition of p38, as
was shown in the SAR study. In a kinase panel study to probe potential off-target liabilities, 15a
exhibited more than 100 fold selectivity over 40 kinases among 42 diverse kinases and showed several
fold selectivity against Her4 and CK1δ(data not shown).
Fig. 6. X-ray crystal structure of the complex of 15a bound with p38 MAP kinase (PDB code 5WJJ).
15

The pharmacokinetic properties of 15a and the sulfate 16, in rats, are shown in Table 4. Since the
solubility of 15a was low with the value of 0.91 μg/mL, the sulfate 16 was prepared. However 16
showed no improvement in solubility (data not shown). After exploration of administration medium,
usage of corn oil as media of 16 fortunately led to significant increases in rat Cmax and AUC compared
with those of 15a in 0.5% methyl cellulose suspension. Absorption and exposure of the sulfate 16 were
favorable in rats at oral doses of 3 mg/kg and were sufficient for further study.
Table 4. Pharmacokinetic properties of 15a and 16a in ratsb.
Cmax, po
(ng/mL)
75
Tmax, po AUCpo
MRT,
po
Vd
CL
F
animal
cmpd
(h)
(ng·h/mL)
(h)
(mL/kg (mL/h/kg (%)
)
)
Ratc
2.00
1.67
703
5.32
5.63
1380
1380e
600
600e
13.5
42.6
15a
16
Ratd
190
2130
a
b
c
H2SO4 salt of 15a. Mean values of measurements conducted in three animals. i.v. 0.2 mg/kg, p.o.
d
e
3.0 mg/kg in 0.5% methyl cellulose suspension. p.o. 3.0 mg/kg in corn oil suspension. Because the
main body of 16 and 15a is the same, Vd and CL of 15a are applied for 16.
Compound 16 was evaluated in a pseudoestablished model of collagen-induced arthritis (CIA) in rats
(Figure 7). The rats were administered compound 16 orally, with b.i.d dosing of 0.1, 0.3, 1, 3, and 10
mg/kg for 11 days; swelling of the paw was measured on day 14. Compound 16 showed dose-
16

dependent efficacy in achieving a marked reduction in paw swelling at a dose of 10 mg/kg. Statistically
significant reduction in paw swelling was also observed at doses of 1, 3, and 10 mg/kg.
Fig. 7. Anti-inflammatory effects of 16 in the model of collagen-induced arthritis in the rat (po, 11 days
treatment). **; p≤0.01 vs. control by Dunnett's test.
4. Conclusion
On the basis of X-ray crystallography of the complex of 1 with p38 MAP kinase, we focused on the
scaffold transformation of 2-ethylthiazole of 1. The designed imidazo[1,2-b]pyridazine derivatives were
identified as potent inhibitors of p38 MAP kinase. The extensive SAR analysis of imidazo[1,2-
b]pyridazine derivatives led to compound 10a, which strongly inhibited p38 and the production of
TNF-αin human THP-1 cells. During the course of optimizing the DMPK profile, the in vitro structural
analysis of the metabolites of compound 10a resulted in the discovery of pyridine N-oxide 15a which
exhibited a dramatically improved metabolic stability in human liver microsomes. Sulfate 16 also
17

potently inhibited p38 and production of TNF-α and showed an excellent DMPK profile and in vivo
efficacy.
5. Experimental Section
5.1. Chemistry
Melting points were determined using a Yanagimoto or a Büchi melting point apparatus B-545; the
values are presented uncorrected. 1H NMR and 13C NMR spectra were recorded on a Varian Gemini-200
1
(200 MHz), Varian Ultra-300 (300 MHz), or Bruker DPX-300 (300 MHz for H NMR and 75 MHz for
13C) spectrometer and are reported in parts per million (δ) relative to tetramethylsilane (TMS, δ 0.0
ppm). Data are reported as follows: chemical shifts, integration, multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; dd, doublet of doublets; br, broad signal; brs, broad singlet; m, multiplet (denotes
complex pattern)), and coupling constants (J, Hz). Unless otherwise specified, all solvents and reagents
were obtained from commercial suppliers and used without further purification. The yields were
unoptimized. Column chromatography was performed using Merck silica gel 60 (70–230 mesh). Thin-
layer chromatography (TLC) was performed on Merck silica gel plates 60F254. LC-MS analysis was
performed on a Shiseido CAPCELL PACK C-18 UG120 S-3 column (1.5 mmϕ× 35 mm) in a Waters
Alliance 2795 or an Agilent 1100 LC system equipped with a Waters 2487 absorbance detector and a
Micromass ZQ2000 mass spectrometer. Analytes were eluted using a linear gradient of water (0.05%
TFA)/acetonitrile (0.04% TFA) from 90:10 to 0:100 over 4 min at a flow rate of 0.5 mL/min. UV
detection was at 220 nm. Preparative HPLC was performed on a Shiseido CAPCELL PACK C-18
UG120 S-5 column (20 mmϕ × 50 mm), eluting at 25 mL/min with a gradient of water (0.1%
TFA)/acetonitrile (0.1% TFA). UV detection was at 220 nm. The purity of all compounds used in
biological studies was determined to be ≥ 95% by elemental analysis. Elemental analyses were
performed by Takeda Analytical Research Laboratories, Ltd. Experimentally determined hydrogen,
18

carbon, and nitrogen composition by elemental analysis was within ±0.4% of the expected value,
implying a purity of ≥95%. All experiments using animals were reviewed and approved by the Internal
Animal Care and Use Committee of Takeda Pharmaceutical Research Division.
The 2-tert-butoxycarbonylamino-4-methylpyridine (2a), N-(3-methylbenzoyl)propyleneimine (3a),
and N-(3-chlorobenzoyl)propyleneimine (3b) were prepared according to a previous report.11, 12
2-tert-Butoxycarbonylamino-4-methylpyrimidine (2b). Di(tert-butyl) dicarbamate (12 mL, 50
mmol) was added dropwise to a solution of 4-methylpyrimidine-2-amine (5.0 g, 46 mmol) in tert-
butanol over 1 h, and the solution was stirred at room temperature for 14 h. The solvent was evaporated
and the residue was purified by silica gel column chromatography (hexane/ethyl acetate= 1/1) to give a
solid precipitate. The solid precipitate was crystallized from isopropyl ether and hexane to give 2b as a
white solid (6.7 g, 70%), and this compound was used in the next reaction. Mp: 87-88 °C.
N-(4-Chlorobenzoyl)propyleneimine (3c). A solution of propyleneimine (12 mL, 0.15 mol) in
tetrahydrofuran (160 mL) was added to a 1 N aqueous solution of sodium hydroxide. To this mixture
was added 4-chlorobenzoyl chloride (25 g, 0.143 mol) dropwise at 0 °C. The mixture was stirred for 30
min then extracted with ethyl acetate. The extract was dried, and the solvent was distilled to give 3c as
1
an oil (25 g, 89%). H NMR (CDCl3) δ: 1.39 (3H, d, J = 5.5 Hz), 2.15 (1H, d, J = 2.9 Hz), 2.51-2.66
(2H, m), 7.39-7.47 (2H, m), 7.93-8.01 (2H, m).
N-(4-Fluoro-3-methylbenzoyl)propyleneimine (3d). This compound was prepared from 4-fluoro-3-
methylbenzoyl chloride as described in the synthesis of 3c, as a colorless oil (91%). 1H NMR (CDCl3) δ:
1.39 (3H, d, J = 5.4 Hz), 2.14 (1H, d, J = 3.4 Hz), 2.33 (3H, s), 2.51-2.61 (2H, m), 7.06 (1H, t, J = 8.8
Hz), 7.81-7.90 (2H, m).
3-Chloro-4-fluoro-N-methoxy-N-methylbenzamide (3e). To a solution of 3-chloro-4-fluorobenzoyl
chloride (25.0 g, 0.130 mol) in diethylether (40 mL) was dropwise added a solution of N, O-
dimethylhydroxylamine (13.3 g, 0.136 mol) and a 2N aqueous solution of sodium hydroxide (136 mL,
19

0.272 mol) in diethylether (80 mL) at 0 °C. The mixture was stirred at room temperature for 15 h. The
organic layer was separated and the aqueous layer was extracted with diethylether. The combined
organic layer was washed with brine, dried over sodium sulfate, and evaporated to give 3e as a pale
1
yellow oil (27.6 g, 98%). This compound was used in the next reaction. H NMR (CDCl3) δ: 3.37 (3H,
s), 3.55 (3H, s), 7.17 (1H, t, J = 8.7 Hz), 7.61-7.68 (1H, m), 7.82 (1H, dd, J = 2.2, 7.1 Hz).
2-(2-tert-Butoxycarbonylaminopyridin-4-yl)-1-(3-methylphenyl)ethanone (4a). To a solution of
2a (146 g, 0.700 mol) in anhydrous tetrahydrofuran (1300 mL) was dropwise added a solution of 1.6 M
n-butyl lithium in hexane (875 mL, 1.40 mol) at -78 °C. Then, the mixture was stirred at 0 °C for 30
min and cooled to −78 °C. To the mixture was dropwise added a solution of 3a (123 g, 0.700 mol) in
anhydrous tetrahydrofuran (130 mL). The mixture was stirred at −78 °C for 1 h and at room
temperature for 1 h. To the mixture was added brine (1300 mL), and the organic layer was separated.
The aqueous layer was extracted with ethyl acetate and combined organic layer was dried and
evaporated. The residue was crystallized from ethyl acetate to give 4a (185 g, 81%) as a yellow solid.
Mp: 144–146 °C. 1H NMR (CDCl3) δ: 1.53 (9H, s), 2.42 (3H, s), 4.28 (2H, s), 6.87 (1H, d, J = 5.1 Hz),
7.32-7.43 (2H, m), 7.75-7.83 (2H, m), 7.92 (1H, s), 8.06 (1H, brs), 8.21 (1H, d, J = 5.1 Hz).
2-(2-tert-Butoxycarbonylaminopyridin-4-yl)-1-(3-chlorophenyl)ethanone (4b). This compound
was prepared from 3b as described in the synthesis of 4a as a solid, and used in the next reaction. Mp:
152-153 °C (ethyl acetate/isopropyl ether). 1H NMR (CDCl3) δ: 1.53 (9H, s), 4.26 (2H, s), 6.85 (1H, dd,
J = 5.2, 1.8 Hz), 7.43 (1H, dd, J = 8.0, 7.7 Hz), 7.56 (1H, ddd, J = 8.0, 2.2, 1.1 Hz), 7.86 (1H, ddd, J =
7.7, 1.7, 1.1 Hz), 7.91 (1H, s), 7.95 (1H, brs), 7.97 (1H, dd, J = 2.2, 1.7 Hz), 8.22 (1H, dd, J = 5.2, 0.6
Hz).
2-(2-tert-Butoxycarbonylaminopyridin-4-yl)-1-(4-chlorophenyl)ethanone (4c). This compound
was prepared from 3c as described in the synthesis of 4a as a solid (81%), and used in the next reaction.
1
Mp: 155–156 °C. H NMR (CDCl3) δ: 1.53 (9H, s), 4.25 (2H, s), 6.84 (1H, dd, J = 1.5, 5.1 Hz), 7.44
(2H, d, J = 8.9 Hz), 7.89-7.90 (3H, m), 8.12 (1H, brs), 8.20 (1H, dd, J = 0.7, 5.1 Hz).
20

2-(2-tert-Butoxycarbonylaminopyridin-4-yl)-1-(4-fluoro-3-methylphenyl)ethanone (4d). This
compound was prepared from 3d as described in the synthesis of 4a as a white solid (85%), and used in
the next reaction. Mp: 143–144 °C. 1H NMR (CDCl3) δ: 1.53 (9H, s), 2.33 (3H, d, J = 1.7 Hz), 4.24 (2H,
s), 6.86 (1H, dd, J = 1.3, 5.1 Hz), 7.08 (1H, t, J = 8.9 Hz), 7.79-7.89 (2H, m), 7.92 (1H, s), 8.11 (1H, s),
8.21 (1H, d, J = 5.1 Hz).
2-(2-tert-Butoxycarbonylaminopyridin-4-yl)-1-(3-chloro-4-fluorophenyl)ethanone (4e). To
a
solution of 2a (1.91 g, 9.19 mmol) in tetrahydrofuran (20 mL) was dropwise added a solution of 1.6 M
n-butyl lithium in hexane (12.1 mL) at −78 °C over 10 min. Then, the mixture was stirred at 0 °C for 30
min and cooled to −78 °C. To the mixture was added a solution of 3e (2.00 g, 9.19 mmol) in
tetrahydrofuran (2 mL) dropwise over 10 min. The mixture was stirred at room temperature for 1 h. To
the mixture was added a saturated aqueous solution of sodium hydrogen carbonate, and the organic
layer was separated. The aqueous layer was neutralized with 1N hydrogen chloride and extracted with
ethyl acetate. The combined organic layer was washed with brine, dried over sodium sulfate, and
evaporated. The residue was washed with diisopropylether and dried to give 4a (2.67 g, 80%) as a white
solid. This compound was used in the next reaction. Mp: 141–143 °C. 1H NMR (CDCl3) δ: 1.53 (9H, s),
4.24 (2H, s), 6.84 (1H, dd, J = 1.5, 5.1 Hz), 7.20-7.28 (1H, m), 7.82-7.93 (3H, m), 8.07 (1H, dd, J = 2.3,
7.2 Hz), 8.21 (1H, d, J = 5.1 Hz).
2-(2-tert-Butoxycarbonylaminopyrimidin-4-yl)-1-(3-methylphenyl)ethanone (4f). This compound
was prepared from 2b as described in the synthesis of 4a as a solid, and used in the next reaction. Mp:
194–195 °C.
2-(2-Aminopyridin-4-yl)-2-bromo-1-(3-methylphenyl)ethanone hydrobromide (5a). Bromine
(29.2 mL, 0.566 mol) was added to a solution of 4a (185 g, 0.566 mol) in acetic acid (400 mL), and the
mixture was stirred at 80 °C for 2 h. The mixture was concentrated and the residue was crystallized
1
from acetonitrile-ethyl acetate to give 5a (171 g, 78%) as a solid. H NMR (DMSO-d6) δ: 2.41 (3H, s),
21

3.41 (1H, brs), 6.98 (1H, d, J = 6.8 Hz), 7.12 (1H, s), 7.20 (1H, s), 7.43-7.58 (2H, m), 7.87-8.00 (3H,
m), 8.17 (2H, brs).
2-(2-Aminopyridin-4-yl)-2-bromo-1-(3-chlorophenyl)ethanone
hydrobromide
(5b).
This
compound was prepared from 4b as described in the synthesis of 5a as a solid, and used in the next
reaction. 1H NMR (DMSO-d6) δ: 3.42 (3H, s), 6.98 (1H, dd, J = 6.9, 1.7 Hz), 7.12 (1H, s), 7.20 (1H, s),
7.64 (1H, t, J = 8.0 Hz), 7.80 (1H, d, J = 8.0 Hz), 7.96 (1H, d, J = 6.9 Hz), 8.05 (1H, d, J = 8.0 Hz),
8.15 (1H, s).
2-(2-Aminopyridin-4-yl)-2-bromo-1-(4-chlorophenyl)ethanone
hydrobromide
(5c).
This
compound was prepared from 4c as described in the synthesis of 5a as a white solid (87%), and used in
1
the next reaction. H NMR (DMSO-d6) δ: 6.97 (1H, d, J =6.6 Hz), 7.12 (1H, dd, J =2.4, 3.4 Hz), 7.21
(1H, d, J =3.4 Hz), 7.69 (2H, d, J = 8.6 Hz), 7.97 (1H, dd, J = 2.4, 6.6 Hz), 8.12 (2H, d, J =8.6 Hz),
8.19 (2H, brs), 13.34 (1H, brs).
2-(2-Aminopyridin-4-yl)-2-bromo-1-(4-fluoro-3-methylphenyl)ethanone hydrobromide (5d).
This compound was prepared from 4d as described in the synthesis of 5a as a solid (90%), and used in
the next reaction. 1H NMR (DMSO-d6) δ: 2.33 (3H, s), 6.98 (1H, dd, J =1.6, 6.7 Hz), 7.11 (1H, s), 7.21
(1H, s), 7.36 (1H, t, J = 9.0 Hz), 7.97 (1H, d, J = 6.7 Hz), 8.00-8.07 (1H, m), 8.10 (1H, dd, J =1.1, 7.9
Hz), 8.19 (2H, brs), 13.35 (1H, brs).
2-(2-Aminopyridin-4-yl)-2-bromo-1-(3-chloro-4-fluorophenyl)ethanone hydrobromide (5e).
Bromine (0.367 mL, 7.16 mmol) was added to a solution of 4e (2.61 g, 7.16 mmol) in acetic acid (15
mL), and the mixture was stirred at 70 °C for 3 h. The mixture was cooled to room temperature and the
precipitate was collected on a filter, washed with ethyl acetate, and dried to give 5a (2.66 g, 88%) as a
1
white solid. This compound was used in the next reaction. Mp: 231–233 °C. H NMR (DMSO-d6) δ:
6.96 (1H, dd, J =1.6, 6.6 Hz), 7.10 (1H, s), 7.16 (1H, d, J = 1.1 Hz), 7.68 (1H, t, J = 8.9 Hz), 7.96 (1H,
d, J = 6.6 Hz), 8.00-8.17 (3H, m), 8.36 (1H, dd, J = 2.3, 7.0 Hz).
22

2-(2-Aminopyrimidin-4-yl)-2-bromo-1-(3-methylphenyl)ethanone hydrobromide (5f). Bromine
(0.16 mL, 3.1 mmol) was added to a solution of 4f (1.00 g, 3.1 mmol) and sodium acetate (0.50 g, 6.11
mmol) in acetic acid (10 mL) at room temperature. The mixture was stirred at room temperature for 2 h
and concentrated. A saturated aqueous solution of sodium hydrogen carbonate was added to the residue
and the mixture was extracted with ethyl acetate. The organic layer was dried and evaporated to give 5e
as an oil. This compound was used in the next reaction.
4-[6-Chloro-2-(3-methylphenyl)imidazo[1, 2-b]pyridazin-3-yl]pyridin-2-amine (6a). 3-Amino-6-
chloropyridazine (6.71 g, 51.8 mmol) and 5a (20.0 g, 51.8 mmol) were stirred at 80 °C for 14 h in
ethanol (200 mL). The mixture was cooled to room temperature, and the solvent was removed by
evaporation under reduced pressure. A saturated aqueous sodium hydrogen carbonate was added to the
residue. The precipitated solid was collected by filtration, washed with water, and dried. The obtained
solid was dissolved in tetrahydrofuran, filtered, and the solvent was removed by evaporation under
reduced pressure. The obtained solid was recrystallized from ethyl acetate to give 6a (10.5 g, 60%) as a
solid. Mp: 247–248 °C. MS (ESI+): 336 (M+H). 1H-NMR (CDCl3) δ: 2.36 (3H, s), 4.51 (2H, brs), 6.80
(1H, dd, J = 1.4, 0.8 Hz), 6.83 (1H, ddd, J = 5.2, 4.1, 0.8 Hz), 7.11 (1H, d, J = 9.3 Hz), 7.14-7.24 (2H,
m), 7.36 (1H, d, J = 7.4 Hz), 7.58 (1H, s), 7.94 (1H, d, J = 9.3 Hz), 8.14 (1H, d, J = 5.2 Hz). Anal.
Calcd for C18H14ClN5+0.75H2O: C, 61.89; H, 4.47; N, 20.05. Found: C, 61.75; H, 4.39; N, 20.34.
4-[6-Chloro-2-(3-chlorophenyl)imidazo[1,
2-b]pyridazin-3-yl]pyridin-2-amine
(6b).
This
compound was prepared from 5b as described in the synthesis of 6a as a solid (43%). Mp: 268–269 °C
(tetrahydrofuran). MS (ESI+): 356 (M+H). 1H NMR (DMSO-d6) δ: 6.19 (2H, brs), 6.57 (1H, dd, J = 5.2,
1.4 Hz), 6.60 (1H, dd, J = 1.4, 0.8 Hz), 7.40-7.44 (2H, m), 7.47 (1H, d, J = 9.3 Hz), 7.52-7.59 (1H, m),
7.68-7.72 (1H, m), 8.06 (1H, dd, J = 5.2, 0.8 Hz), 8.31 (1H, d, J = 9.3 Hz). Anal. Calcd for C17H11Cl2N5:
C, 57.13; H, 3.11; N, 19.66. Found: C, 57.13; H, 3.08; N, 19.46.
4-[6-Chloro-2-(4-chlorophenyl)imidazo[1,
2-b]pyridazin-3-yl]pyridin-2-amine
(6c).
This
compound was prepared from 5c as described in the synthesis of 6a as a solid (63%). Mp: 183–184 °C.
23

1
MS (ESI+): 356 (M+H). H NMR (DMSO-d6) δ: 6.17 (2H, brs), 6.57 (1H, dd, J = 5.2, 1.2 Hz), 6.59
(1H, s), 7.46 (1H, d, J = 9.5 Hz), 7.47 (2H, d, J = 8.7 Hz), 7.65 (2H, d, J = 8.7 Hz), 8.04 (1H, d, J = 5.4
Hz), 8.29 (1H, dd, J = 9.5 Hz).
4-[6-Chloro-2-(4-fluoro-3-methylphenyl)imidazo[1, 2-b]pyridazin-3-yl]pyridin-2-amine (6d).
This compound was prepared from 5d as described in the synthesis of 6a as a solid (71%). Mp: 217–
1
218 °C. MS (ESI+): 354 (M+H). H NMR (DMSO-d6) δ: 2.24 (3H, d, J = 1.5 Hz), 6.21 (2H, brs), 6.58
(1H, dd, J = 5.3, 1.3 Hz), 6.63 (1H, s), 7.12-7.20 (1H, m), 7.34-7.42 (1H, m), 7.46 (1H, d, J = 9.5 Hz),
7.67 (1H, dd, J = 7.5, 1.5 Hz), 8.04 (1H, dd, J = 5.3, 0.4 Hz), 8.29 (1H, d, J = 9.5 Hz). Anal. Calcd for
C18H13ClFN5+3.5H2O: C, 51.87; H, 4.84; N, 16.80. Found: C, 51.85; H, 3.41; N, 18.42.
4-[6-Chloro-2-(3-chloro-4-fluorophenyl)imidazo[1, 2-b]pyridazin-3-yl]pyridin-2-amine (6e). This
compound was prepared from 5e as described in the synthesis of 6a as a pale yellow solid (50%). Mp:
1
237–239 °C. MS (ESI+): 374 (M+H). H NMR (DMSO-d6) δ: 6.19 (2H, s), 6.54-6.63 (2H, m), 7.42-
7.52 (2H, m), 7.54-7.63 (1H, m), 7.82 (1H, dd, J = 2.2, 7.3 Hz), 8.07 (1H, d, J = 5.1 Hz), 8.31 (1H, d, J
= 9.4 Hz).
4-[6-Chloro-2-(3-methylphenyl)imidazo[1, 2-b]pyridazin-3-yl]pyrimidin-2-amine (6f). This
compound was prepared from 5f as described in the synthesis of 6a as a solid (22%). Mp: 237–238 °C
1
(ethyl acetate/hexane). MS (ESI+): 337 (M+H). H-NMR (CDCl3) δ: 2.38 (3H, s), 5.10 (2H, s), 7.08
(1H, d, J = 5.1 Hz), 7.14 (1H, d, J = 9.5 Hz), 7.16-7.20 (1H, m), 7.24 (1H, t, J = 7.5 Hz), 7.40-7.50 (1H,
m), 7.61-7.65 (1H, m), 7.96 (1H, d, J = 9.5 Hz), 8.42 (1H, d, J = 5.1 Hz). Anal. Calcd for C17H13ClN6:
C, 60.63; H, 3.89; N, 24.95. Found: C, 60.28; H, 3.97; N, 24.32.
4-[2-(3-Methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-amine (7a).
4-[6-Chloro-2-(3-
methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyridine-2-amine (5.00 g, 14.9 mmol) and 10% palladium
carbon powder (50% water containing product) (1.00 g) were stirred in acetic acid under hydrogen
atmosphere at 50 °C for 3 h under the hydrogen pressure of 0.5 MPa. The reaction mixture was cooled
24

to room temperature and filtered, and the solvent was removed by evaporation under reduced pressure.
A saturated aqueous solution of sodium hydrogen carbonate was added to the residue, and the
precipitated solid was collected by filtration. The solid was washed with water and dried. The obtained
solid was recrystallized from ethanol to give 7a (2.43 g, 54%) as a yellow solid. Mp: 188–189 °C. MS
1
(ESI+): 302 (M+H). H NMR (CDCl3) δ: 2.37 (3H, s), 4.50 (2H, brs), 6.83 (1H, dd, J = 1.4, 0.8 Hz),
6.87 (1H, dd, J = 5.2, 1.4 Hz), 7.10 (1H, dd, J = 9.3, 4.4 Hz), 7.13-7.19 (1H, m), 7.22 (1H, t, J = 7.1
Hz), 7.37-7.42 (1H, m), 7.60-7.63 (1H, m), 8.01 (1H, dd, J = 9.3, 1.6 Hz), 8.14 (1H, dd, J = 5.2, 0.8
Hz), 8.33 (1H, dd, J = 4.4, 1.6 Hz). Anal. Calcd for C18H15N5+0.5H2O: C, 69.66; H, 5.20; N, 22.57.
Found: C, 69.72; H, 5.18; N, 22.40.
4-[2-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-amine
(7b).
4-[6-Chloro-2-(3-
chlorophenyl)imidazo[1,2-b]pyridazin-3-yl]pyridine-2-amine (5.62 g, 15.8 mmol), 10% palladium
carbon powder (50% water containing product) (0.56 g), and triethylamine (4.4 mL, 31.6 mmol) were
stirred in N, N-dimethylformamide (200 mL) under hydrogen atmosphere, at room temperature, for 1 h.
The mixture was filtered, and the solvent was removed by evaporation under reduced pressure. A
saturated aqueous solution of sodium hydrogen carbonate was added to the residue, and the precipitated
solid was collected by filtration. The solid was washed with water and dried to give 7b (4.83 g, 95%) as
1
an off-white solid. MS (ESI+): 322 (M+H). H NMR (DMSO-d6) δ: 6.13 (2H, brs), 6.59 (1H, dd, J =
5.3, 1.3 Hz), 6.65 (1H, s), 7.35 (1H, dd, J = 9.2, 4.3 Hz), 7.39-7.45 (2H, m), 7.56-7.62 (1H, m), 7.71-
7.76 (1H, m), 8.04 (1H, dd, J = 5.3, 0.6 Hz), 8.23 (1H, dd, J = 9.2, 1.7 Hz), 8.55 (1H, dd, J = 4.3, 1.7
Hz).
4-[2-(4-Chlorophenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-amine (7c). This compound was
prepared from 6c as described in the synthesis of 7b as a solid (61%). Mp: 252–253 °C (ethyl acetate).
1
MS (ESI+): 322 (M+H). H NMR (DMSO-d6) δ: 6.10 (2H, brs), 6.57 (1H, dd, J = 5.2, 1.7 Hz), 6.63
(1H, s), 7.32 (1H, dd, J = 9.1, 4.4 Hz), 7.46 (2H, d, J = 8.8 Hz), 7.67 (2H, d, J = 8.8 Hz), 8.01 (1H, d, J
25

= 5.2 Hz), 8.21 (1H, dd, J = 9.1, 1.7 Hz), 8.53 (1H, dd, J = 4.4, 1.7 Hz). Anal. Calcd for
C17H12ClN5+0.4H2O: C, 62.07; H, 3.92; N, 21.29. Found: C, 62.16; H, 4.20; N, 21.41.
4-[2-(4-Fluoro-3-methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-amine
(7d).
This
compound was prepared from 6d as described in the synthesis of 7b as a pale yellow solid (44%). Mp:
179–180 °C. MS (ESI+): 320 (M+H). 1H NMR (CDCl3) δ: 2.29 (3H, d, J = 1.9 Hz), 4.52 (2H, brs), 6.82
(1H, dd, J = 1.6, 0.6 Hz), 6.85 (1H, dd, J = 5.4, 1.6 Hz), 6.96 (1H, dd, J = 9.3, 8.5 Hz), 7.11 (1H, dd, J
= 9.2, 4.4 Hz), 7.35-7.41 (1H, m), 7.63 (1H, dd, J = 8.0, 2.1 Hz), 8.00 (1H, dd, J = 9.2, 1.7 Hz), 8.15
(1H, dd, J = 5.4, 0.6 Hz), 8.34 (1H, dd, J = 4.4, 1.7 Hz). Anal. Calcd for C18H14FN5: C, 67.70; H, 4.42;
N, 21.93. Found: C, 67.67; H, 4.43; N, 21.85.
4-[2-(3-Chloro-4-fluorophenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-amine
(7e).
This
compound was prepared from 6e as described in the synthesis of 7b as a pale yellow solid (50%). Mp:
201–203 °C. MS (ESI+): 340 (M+H). 1H NMR (DMSO-d6) δ: 6.59 (2H, brs), 6.68 (1H, dd, J = 1.5, 5.7
Hz), 6.81 (1H, s), 7.38 (1H, dd, J = 4.5, 9.1 Hz), 7.48 (1H, t, J = 8.9 Hz), 7.57-7.64 (1H, m), 7.85 (1H,
dd, J = 1.9, 7.2 Hz), 8.03 (1H, d, J = 5.3 Hz), 8.26 (1H, dd, J = 1.7, 9.3 Hz), 8.59 (1H, dd, J = 4.5, 1.5
Hz).
4-[2-(3-Methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyrimidin-2-amine (7f). This compound was
prepared from 6f as described in the synthesis of 7b as a pale yellow solid (90%). Mp: 250–251 °C. MS
(ESI+): 303 (M+H). 1H NMR (DMSO-d6) δ: 2.31 (3H, s), 6.09 (2H, brs), 6.56 (1H, dd, J = 5.2, 1.7 Hz),
6.66 (1H, s), 7.16 (1H, d, J = 7.4 Hz), 7.26 (1H, dd, J = 7.7, 7.4 Hz), 7.32 (1H, dd, J = 9.4, 4.4 Hz),
7.38 (1H, d, J = 7.7 Hz), 7.59 (1H, s), 8.01 (1H, d, J = 5.2 Hz), 8.21 (1H, dd, J = 9.1, 1.7 Hz), 8.53
(1H, dd, J = 4.4, 1.7 Hz).
N-{4-[2-(3-Methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-yl}benzamide (8a). To
a
solution of 7a (150 mg, 0.498 mmol) in dichloromethane (8 mL) were added triethylamine (208 μL,
1.49 mmol) and benzoyl chloride (173 μL, 1.49 mmol). The mixture was stirred at room temperature
26

for 30 min, and a saturated aqueous solution of sodium hydrogen carbonate was added to the reaction
mixture. An ammonia-ethanol solution (2.0 M, 10 mL) was added to the resultant mixture, and the
mixture was stirred at room temperature for 30 min. The solvent was removed by evaporation under
reduced pressure, and the obtained residue was purified by silica gel chromatography (ethyl acetate:
hexane = 3: 7– 8: 2) and recrystallized from ethyl acetate to give 8a (145 mg, 72%) as a solid. Mp:
178–180 °C. MS (ESI+): 406 (M+H). 1H NMR (CDCl3) δ: 2.37 (3H, s), 7.12-7.19 (3H, m), 7.23 (1H, t,
J = 7.5 Hz), 7.40 (1H, d, J = 7.5 Hz), 7.47-7.60 (3H, m), 7.63 (1H, s), 7.93-7.96 (2H, m), 8.05 (1H, dd,
J = 9.1, 1.6 Hz), 8.26 (1H, dd, J = 5.3, 0.8 Hz), 8.41 (1H, dd, J = 4.4, 1.6 Hz), 8.88 (1H, s), 8.92 (1H, d,
13
J = 0.8 Hz). C NMR (CDCl3) δ: 21.4, 77.2, 114.6, 117.7, 121.1, 122.3, 125.5, 126.0, 127.3, 128.4,
128.9, 129.4, 132.3, 133.3, 134.3, 138.4, 139.4, 139.5, 143.1, 145.2, 148.1, 152.1, 165.8 (s). Anal.
Calcd for C25H19N5O: C, 74.06; H, 4.72; N, 17.27. Found: C, 73.81; H, 4.71; N, 17.24.
N-{4-[2-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-yl}benzamide
(8b).
This
compound was prepared from 7b as described in the synthesis of 8a as a pale yellow solid (53%). Mp:
1
213–214 °C (ethanol). MS (ESI+): 426 (M+H). H NMR (CDCl3) δ: 7.13-7.20 (2H, m), 7.24-7.36 (2H,
m), 7.47-7.62 (4H, m), 7.78 (1H, dd, J = 1.9, 1.7 Hz), 7.90-7.96 (2H, m), 8.04 (1H, dd, J = 9.1, 1.7
Hz), 8.35 (1H, dd, J = 5.2, 0.6 Hz), 8.41 (1H, dd, J = 4.4, 1.7 Hz), 8.71 (1H, brs), 8.85 (1H, dd, J = 1.4,
0.8 Hz). 13C NMR (CDCl3) δ: 77.2, 114.7, 118.1, 120.9, 122.8, 125.7, 126.8, 127.3, 128.6, 128.7, 128.9,
129.8, 132.3, 134.2, 134.7, 135.3, 138.9, 139.6, 143.3, 143.4, 148.3, 152.2, 165.8 (s). Anal. Calcd for
C24H16ClN5O+0.2H2O: C, 67.12; H, 3.85; N, 16.31. Found: C, 67.38; H, 3.80; N, 16.19.
N-{4-[2-(4-Chlorophenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-yl}benzamide
(8c).
This
compound was prepared from 7c as described in the synthesis of 8a as an off-white solid (74%). Mp:
218–219 °C (ethanol). MS (ESI+): 426 (M+H). 1H NMR (CDCl3) δ: 7.13-7.20 (2H, m), 7.36 (2H, d, J =
8.7 Hz), 7.48-7.60 (3H, m), 7.65 (2H, d, J = 8.7 Hz), 7.91-7.98 (2H, m), 8.05 (1H, dd, J = 9.2, 1.7 Hz),
8.36 (1H, dd, J = 5.1, 0.8 Hz), 8.41 (1H, dd, J = 4.4, 1.7 Hz), 8.72 (1H, s), 8.85 (1H, dd, J = 1.4, 0.8
13
Hz). C NMR (CDCl3) δ: 77.3, 114.7, 118.0, 121.0, 122.5, 125.6, 127.3, 128.9, 128.9, 130.0, 132.0,
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132.4, 134.2, 134.6, 139.1, 139.6, 143.3, 143.7, 148.3, 152.2, 165.8 (s). Anal. Calcd for C24H16ClN5O:
C, 67.69; H, 3.79; N, 16.44. Found: C, 67.74; H, 3.81; N, 16.51.
N-{4-[2-(4-Fluoro-3-methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-yl}benzamide
(8d).
This compound was prepared from 7d as described in the synthesis of 8a as a pale yellow solid (83%).
1
Mp: 189–190 °C (ethanol). MS (ESI+): 424 (M+H). H NMR (CDCl3) δ: 2.29 (3H, d, J = 1.9 Hz), 6.96
(1H, dd, J = 9.3, 8.8 Hz), 7.14 (1H, dd, J = 9.1, 4.4 Hz), 7.17 (1H, dd, J = 5.2, 1.4 Hz), 7.35-7.41 (1H,
m), 7.47-7.66 (4H, m), 7.90-7.95 (2H, m), 8.03 (1H, dd, J = 9.1, 1.7 Hz), 8.33 (1H, dd, J = 5.2, 0.8
13
Hz), 8.40 (1H, dd, J = 4.4, 1.7 Hz), 8.69 (1H, brs), 8.86 (1H, dd, J = 1.4, 0.8 Hz). C NMR (CDCl3) δ:
14.6 (d, J = 3.30 Hz), 77.3, 114.7 (s), 115.2 (d, J = 23.1 Hz), 117.9, 120.9, 122.2, 125.3, 125.5, 127.3
(s), 127.9 (d, J = 8.3 Hz), 128.9 (s), 129.2 (d, J = 3.9 Hz), 131.9 (d, J = 5.5 Hz), 132.3, 134.2 (s), 139.4
(d, J = 17.6 Hz), 143.2, 144.3, 148.2, 152.2 (s), 161.6 (d, J = 247.0 Hz), 165.8 (s). Anal. Calcd for
C25H18FN5O: C, 70.91; H, 4.28; N, 16.54. Found: C, 70.77; H, 4.33; N, 16.53.
N-{4-[2-(4-Fluoro-3-methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-yl}-3-
trifluoromethylbenzamide (8e). This compound was prepared from 7d as described in the synthesis of
1
8a as a white solid (82%). Mp: 228–229 °C (hexane/ethyl acetate). MS (ESI+): 492 (M+H). H NMR
(CDCl3) δ: 2.29 (3H, d, J = 1.9 Hz), 6.94-7.02 (1H, m), 7.16 (1H, dd, J = 4.5, 9.2 Hz), 7.22-7.28 (1H,
m), 7.35-7.43 (1H, m), 7.61-7.71 (2H, m), 7.85 (1H, d, J = 8.0 Hz), 8.05 (1H, dd, J = 1.6, 9.2 Hz), 8.13
(1H, d, J = 7.6 Hz), 8.22 (1H, s), 8.36 (1H, d, J = 5.3 Hz), 8.41 (1H, dd, J = 1.6, 4.5 Hz), 8.75 (1H, s),
13
8.86 (1H, s). C NMR (CDCl3) δ: 14.6 (d, J = 3.8 Hz), 77.2, 114.7 (s), 115.2 (d, J = 23.1 Hz), 117.9,
121.2, 121.8, 122.0 (s), 124.4 (q, J = 3.7 Hz), 125.3, 125.4, 125.6 (s), 127.9 (d, J = 8.2 Hz), 128.9 (q, J
= 3.1 Hz), 129.2 (d, J = 3.8 Hz), 129.5, 130.4 (s), 131.5 (q, J = 33.3 Hz), 131.9 (d, J = 5.5 Hz), 135.0
(s), 139.5 (d, J = 11.0 Hz), 143.2, 144.4, 148.2, 151.8 (s), 161.7 (d, J = 247.0 Hz), 164.3 (s). Anal.
Calcd for C26H17F4N5O: C, 63.54; H, 3.49; N, 14.25. Found: C, 63.42; H, 3.50; N, 14.23.
N-{4-[2-(3-Methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyrimidin-2-yl}benzamide (8f).
This
compound was prepared from 7f as described in the synthesis of 8a as a pale yellow solid (45%). Mp:
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1
200–201 °C (ethanol). MS (ESI+): 407 (M+H). H NMR (CDCl3) δ: 2.39 (3H, s), 7.19-7.31 (3H, m),
7.44-7.53 (3H, m), 7.54-7.61 (1H, m), 7.64 (1H, d, J = 5.2 Hz), 7.66 (1H, s), 7.87-7.93 (2H, m), 8.08
13
(1H, dd, J = 9.3, 1.6 Hz), 8.46 (1H, dd, J = 4.4, 1.7 Hz), 8.61 (1H, brs), 8.81 (1H, d, J = 5.2 Hz). C
NMR (CDCl3) δ: 21.5, 77.2, 116.9, 118.5, 121.6, 125.8, 126.6, 127.5, 128.1, 128.8, 129.6, 129.8, 132.4,
133.5, 134.3, 138.1, 140.0, 143.3, 148.0, 157.3, 157.8, 159.0, 164.8 (s). Anal. Calcd for
C24H18N6O+0.2H2O: C, 70.30; H, 4.52; N, 20.50. Found: C, 70.34; H, 4.61; N, 20.20.
N-{4-[2-(3-Chloro-4-fluorophenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-yl}-3-trifluoromethyl
benzamide (8g). This compound was prepared from 7e as described in the synthesis of 8a as a white
1
solid (57%). Mp: 235–237 °C (hexane/ethyl acetate). MS (ESI+): 512 (M+H). H NMR (DMSO-d6) δ:
7.34-7.52 (3H, m), 7.55-7.64 (1H, m), 7.72-7.80 (1H, m), 7.87 (1H, dd, J = 2.2, 7.3 Hz), 7.94-8.01 (1H,
m), 8.29 (2H, dd, J = 1.5, 9.2 Hz), 8.37 (1H, s), 8.50 (1H, s), 8.56 (1H, dd, J = 0.6, 5.1 Hz), 8.61 (1H,
dd, J = 1.5, 4.5 Hz), 11.34 (1H, s). 13C NMR (DMSO-d6) δ: 115.9 (s), 117.8 (d, J = 21.5 Hz), 119.8 (s),
120.4 (d, J = 17.6 Hz), 121.4, 122.7 (s), 125.3 (d, J = 3.9 Hz), 126.3, 129.0 (s), 129.1 (d, J = 7.7 Hz),
129.6 (d, J = 32.5 Hz), 130.2 (d, J = 15.4 Hz), 131.8 (d, J = 3.9 Hz), 132.7, 135.4, 138.5, 139.6, 141.1,
144.9, 149.1, 153.1, 156.0, 159.3, 165.3 (s). Anal. Calcd for C25H14ClF4N5O: C, 58.66; H, 2.76; N,
13.68. Found: C, 58.55; H, 2.87; N, 13.59.
N-{4-[2-(4-Fluoro-3-methylphenyl)imidazo[1,2-b]pyridazin-3-yl]pyridin-2-yl}-2-
methylbenzamide (8h). This compound was prepared from 2-methylbenzoyl chloride as described in
the synthesis of 8d as a solid (65%). Mp: 265–266 °C (ethanol-tetrahydrofuran). MS (ESI+): 438
(M+H). 1H NMR (CDCl3) δ: 2.30 (3H, d, J = 1.9 Hz), 2.53 (3H, s), 6.95-7.02 (1H, m), 7.15 (1H, dd, J =
9.1, 4.4 Hz), 7.19 (1H, dd, J = 5.2, 1.7 Hz), 7.25-7.30 (2H, m), 7.36-7.42 (2H, m), 7.55-7.59 (1H, m),
7.63-7.66 (1H, m), 8.04 (1H, dd, J = 9.1, 1.7 Hz), 8.27 (1H, dd, J = 5.2, 0.8 Hz), 8.41 (1H, dd, J = 4.4,
1.7 Hz), 8.43 (1H, s), 8.82-8.86 (1H, m). Anal. Calcd for C26H20FN5O: C, 71.38; H, 4.61; N, 16.01.
Found: C, 71.23; H, 4.67; N, 15.89.
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