Discovery of substituted pyrazol-4-yl pyridazinone derivatives as novel c-Met kinase inhibitors

Article abstract of DOI:10.1007/s12272-015-0703-7

A series of pyridazin-3-one substituted with morpholino-pyrimidine derivatives was synthesized and evaluated as tyrosine kinase inhibitors against c-Met enzyme, and anti-proliferative activities of Hs746T human gastric cancer cell line. Most of compounds exhibited good biological activity, while compound 10, 12a, 14a displayed excellent c-Met enzyme inhibitory activities and Hs746T cell-based activities.

Full text of DOI:10.1007/s12272-015-0703-7

Arch. Pharm. Res.
DOI 10.1007/s12272-015-0703-7
RESEARCH ARTICLE
Discovery of substituted pyrazol-4-yl pyridazinone derivatives
as novel c-Met kinase inhibitors
Eun-Young Kim2 Seung-Tae Kang2 Heejung Jung1,3 Chi Hoon Park1,3
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Chang-Soo Yun1,3 Jong Yeon Hwang1,3 Byung Jin Byun1 Chong Ock Lee1
Hyoung Rae Kim1 Jae Du Ha1 Do Hyun Ryu2 Sung Yun Cho1,3
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Received: 11 August 2015 / Accepted: 16 December 2015
Ó The Pharmaceutical Society of Korea 2016
Abstract A series of pyridazin-3-one substituted with
morpholino-pyrimidine derivatives was synthesized and
evaluated as tyrosine kinase inhibitors against c-Met
enzyme, and anti-proliferative activities of Hs746T human
gastric cancer cell line. Most of compounds exhibited good
biological activity, while compound 10, 12a, 14a displayed
excellent c-Met enzyme inhibitory activities and Hs746T
cell-based activities.
and Vande Woude 2008; Christensen et al. 2005; Ma et al.
2008). c-Met is accountable for proliferation, scattering,
invasion, and metastasis of tumor cells (Straussman et al.
2012; Turke et al. 2010). Hyperactivated signaling due to
dysregulation of the c-Met kinase is observed in many
cancers and correlates with tumorigenic invasive growth,
migration, proliferation, survival, and metastasis by
downstream signal transducers, such as MAPK, PIP3K,
and STAT pathways (Lai et al. 2009). Moreover, c-Met
amplification derives acquired resistance to gefitinib or
erlotinib treatment in 20 % of NSCLC patient along with
EGFR mutations (Engelman et al. 2007). Recently, it was
reported that 7 % of c-Met mutation were found in 412
lung adenocarcinoma patients samples by whole-exome
sequencing (WES) on tumor and germline DNA (Collis-
son et al. 2014). Despite the promising possibilities of
c-Met in drug development, clinical developments are
limited by low degree of benefits in clinical phases.
Recent studies revealed that selecting the right patient is
highly critical to c-Met inhibitor development (Garber
et al. 2014). There are at least 20 compounds in active
clinical development, and some of them reached the
clinical trials phase (Cui et al. 2014). Moreover, c-Met
has been recognized as an active component in the
development of EGFR and b-Raf inhibitor resistance by
the formation of heterodimers with HER3 and b-Raf
receptors, respectively (Straussman et al. 2012). Indeed,
development of c-Met inhibitors for regulation of c-Met
signaling pathway are an attractive strategy for the clin-
ical management of various tumors, such as liver, breast,
pancreas, lung, kidney, bladder, ovary, brain, prostate,
and many other cancers (Ma et al. 2008). Recently, we
have reported c-Met inhibitors with biologically active
scaffolds, and presented their biological activity (Kang
et al. 2014; Lee et al. 2012).
Keywords Mesenchymal epithelial transition factor Á c-
MET inhibitor Á Pyridazine derivatives Á Kinase inhibitor
Introduction
c-Met (mesenchymal epithelial transition factor) is a
receptor tyrosine kinase (RTK) family and an attractive
target for cancer therapy with its ligand, hepatocyte
growth factor (HGF) or scatter factor (SF) which is crucial
for normal development (Boccaccio and Comoglio 2006;
Gherardi et al. 2012). c-Met is known to be overexpressed
and mutated in a variety of human cancer types (Knudsen
& Sung Yun Cho
sycho@krict.re.kr
Jae Du Ha
jdha@krict.re.kr
1
Korea Research Institute of Chemical Technology,
PO Box 107, Daejeon 305-600, Korea
2
Department of Chemistry, Sungkyunkwan University,
Suwon 440-746, Korea
3
Department of Medicinal and Pharmaceutical Chemistry,
University of Science and Technology, Daejeon 305-550,
Korea
123

E.-Y. Kim et al.
J = 3.9 Hz, 1H), 8.46 (m, 1H), 7.89 (d, J = 9.0 Hz, 1H),
7.64 (d, J = 8.8 Hz, 1H), 7.49 (m, 1H).
Described herein is a series of hydrazinone-based inhi-
bitors of c-Met kinase, which were substituted with
pyrimidine substituents. In an effort to search c-Met kinase
inhibitors, substituted pyrazol-4-yl pyridazinone deriva-
tives were synthesized and evaluated toward c-Met kinase
inhibitory activity.
3-Chloro-6-(pyridin-4-yl)pyridazine
(1c) 1H
NMR
(300 MHz, CDCl3) d 8.81 (d, J = 5.5 Hz, 2H), 7.95 (d,
J = 5.5 Hz, 2H), 7.92 (d, J = 9.0 Hz, 1H), 7.67 (d,
J = 9.0 Hz, 1H).
Materials and methods
General methods
Synthesis of 2a–2c
A mixture of 3-chloro-6-(1-methyl-1H-pyrazol-4-yl)pyri-
dazine (494 mg, 2.5 mmol) in AcOH (20 mL) was stirred
at 120 °C for 4 h. The reaction mixture was concentrated
under reduced pressure, and water was added to the con-
centrates. The generated solids was filtered and dried to
afford 6-(1-methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-one
(431 mg, 98 %).
All chemicals were obtained from commercial suppliers
and used without further purification unless otherwise
specified. Thin layer chromatography (TLC) analyses were
performed with precoated silica gel 60 F254. 1H NMR
spectra were recorded with a Bruker 300 MHz spectrom-
eter at ambient temperature with the residual solvent peaks
as internal standards; chemical shifts are given in ppm
relative to Me4Si or the corresponding solvent signal (1H:
CDCl3 = 7.26 ppm). Flash chromatography was per-
formed on columns of Merck silica gel (230–400 mesh,
40–63 micro meter) with ethyl acetate/hexane or methanol/
dichloromethane as eluents. The Liquid Chromatography
high-resolution mass spectra (LC-HRMS, electron spray
ionization) experiments were performed with Synapt G2
(Waters MS Technology, Manchester, Q-TOF MS, UK)
mass analyzer. Data were acquired in the positive ion
mode. Calibration was performed with an external cali-
bration mixture immediately prior to analysis.
6-(1-Methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-one (2a) 1H
NMR (300 MHz, CDCl3) d 8.70 (d, J = 11.3 Hz, 1H), 7.96
(s, 1H), 6.94 (s, 1H), 6.62 (d, J = 11.3 Hz, 1H), 3.95 (s, 3H).
6-(Pyridin-3-yl)pyridazin-3(2H)-one
(2b) 1H
NMR
(300 MHz, D2O) d 8.98 (s, 1H), 8.58 (m, 2H), 7.94 (d,
J = 9.9 Hz, 1H), 7.78 (m, 1H), 7.07 (d, J = 9.9 Hz, 1H).
6-(Pyridin-4-yl)pyridazin-3(2H)-one
(2c) 1H
NMR
(300 MHz, D2O) d 8.64 (d, J = 4.5 Hz, 2H), 8.22 (d,
J = 4.5 Hz, 2H), 8.05 (dd, J = 1.9, 9.9 Hz, 1H), 7.07 (dd,
J = 1.9, 9.9 Hz, 1H).
Synthesis of (S)-(4-benzylmorpholin-2-yl)methanol (3) To
a stirred solution of 2-(benzylamino)ethanol (6.9 mL,
54 mmol) in water/2-propanol (2:5, 7 mL) was added (R)-
2-chloromethyloxirane (5 g, 54 mmol). The solution was
stirred for 13 h at room temperature. To the resulting
solution was added NH4OH (35 %wt, 70 mmol), and stir-
red for 4 h at the same temperature. The reaction mixture
was quenched with HCl (1 N), and the pH of the solution
was adjusted with HCl (1 N) solution to pH = 9. The
solution was extracted with CH2Cl2 (40 mL 9 2), and the
organic extracts were dried (Na2SO4), filtered, and con-
centrated under reduced pressure. Purification by column
chromatography (SiO2) afforded (S)-(4-benzylmorpholin-
2yl)methanol (5.7 g, 50 %). 1H NMR (300 MHz, CDCl3) d
7.33-7.26 (m, 5H), 3.89 (dt, J = 2.4, 11.2 Hz, 1H), 3.70
(dd, J = 2.4, 11.2 Hz, 1H), 3.72-3.48 (m, 5H), 2.70-2.64
(m, 2H), 2.18 (dt, J = 3.3, 11.2 Hz, 1H), 2.17 (brS, 1H),
2.04-1.97 (m, 1H).
Synthesis
Synthesis of 1a–1c
To
a
solution of 3,6-dichloropyridazine (500 mg,
3.4 mmol), 1-methylpyrazole-4-boronic acid pinacol ester
(560 mg, 2.7 mmol), K2CO3 (1.1 g, 8.1 mmol) in 1,4-
dioxane/H2O (2.5:1, 5 mL) was added PdCl2(dppf)2. After
stirred at 90 °C for 5 h, the reaction mixture was was
extracted with CH2Cl2 (40 mL), and the organic extracts
were dried (Na2SO4), filtered, and concentrated under
reduced pressure. Purification by column chromatography
(SiO2)
afforded
3-chloro-6-(1-methyl-1H-pyrazol-4-
yl)pyridazine (494 mg, 75 %).
3-Chloro-6-(1-methyl-1H-pyrazol-4-yl)pyridazine (1a) 1H
NMR (300 MHz, CDCl3) d 8.20 (d, J = 7.4 Hz, 1H), 7.98
(s, 1H), 7.95 (s, 1H), 7.50 (d, J = 7.4 Hz, 1H), 3.95 (s, 3H).
Synthesis of (S)-morpholin-2-ylmethanol (4) A mixture of
(S)-(4-benzylmorpholin-2-yl)methanol (5.5 g, 26 mmol)
and Pd/C (10 % Pd, 0.55 g) in MeOH (50 mL) was reacted
with hydrogen balloon. After stirring for 12 h, the reaction
3-Chloro-6-(pyridin-3-yl)pyridazine
(1b) 1H
NMR
(300 MHz, CDCl3) d 9.20 (d, J = 1.8 Hz, 1H), 8.76 (d,
123

Discovery of substituted pyrazol-4-yl pyridazinone derivatives as novel c-Met kinase inhibitors
mixture was filtered with Celite, and concentrated under
reduced pressure. The crude mixture of (S)-morpholin-2-yl
methanol was used for next step without purification (3.1 g,
yl)methyl)-6-(1-methyl-1H-pyrazol-4-yl)pyridazine-3(2H)-
one. (1.51 g, 60 %).
(S)-2-((4-(5-Bromopyrimidin-2-yl)morpholin-2-yl)methyl)-
6-(1-methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-one
99 %). 1H-NMR (300 MHz, CDCl3)
d 3.88 (d,
J = 11.2 Hz, 1H), 3.68-3.45 (m, 4H), 2.92-2.74 (m, 3H),
(7a) 1H NMR (300 MHz, CDCl3) d 8.29 (s, 2H), 7.81 (s,
1H), 7.77 (s, 1H), 7.41 (d, J = 9.6 Hz, 1H), 6.98 (d,
J = 9.6 Hz, 1H), 4.55 (d, J = 14.3 Hz, 1H), 4.49 (dd,
J = 13.4 Hz, J = 7.6 Hz, 1H), 4.34 (d, J = 13.4 Hz, 1H),
4.21-3.96 (m, 3H), 3.95 (s, 3H), 3.57 (dt, J = 2.4, 11.2 Hz,
1H), 3.25-3.10 (m, 1H), 3.01 (dd, J = 11.2, 13.4 Hz, 1H.
2.71 -2.58 (m, 1H).
Synthesis of (S)-(4-(5-bromopyrimidin-2-yl)morpholin-2-
yl)methanol (5) To a stirred solution of (S)-morphlin-2-yl
methanol (2.5 g, 21 mmol) in EtOH (40 mL) was added
5-bromo-2-chloropyrimidine (4.5 g, 23 mmol) and DIPEA
(7.4 ml, 42 mmol), and the resulting solution was stirred
for 13 h at room temperature. After the completion of
reaction, the reaction mixture was concentrated, extracted
with CH2Cl2. The organic extracts were dried (Na2SO4),
filtered, and concentrated under reduced pressure. Purifi-
cation by column chromatography (SiO2) afforded (S)-(4-
(5-bromopyrimidine-2-yl)morpholine-2-yl)methanol (5.09
g, 87 %). 1H NMR (300 MHz, CDCl3) d 8.31 (s, 2H), 4.46
(d, J = 11.2 Hz, 2H), 4.02 (dd, J = 2.5, 11.2 Hz, 1H),
3.80-3.52 (m, 4H), 3.12-3.00 (m, 1H), 2.93-2.83 (m, 1H).
(S)-2-((4-(5-Bromopyrimidin-2-yl)morpholin-2-yl)methyl)-
(7b) 1H
6-(pyridin-3-yl)pyridazin-3(2H)-one
NMR
(300 MHz, CDCl3) d 9.06 (s, 1H), 8.56 (m, 1H), 8.29 (s,
2H), 7.78 (dd, J = 2.1, 5.1 Hz, 1H), 7.66 (d, J = 9.6 Hz,
1H), 7.45 (m, 1H), 7.04 (d, J = 9.6 Hz, 1H), 4.55 (m, 2H),
4.34 (d, J = 13.2 Hz, 1H), 4.24 (dd, J = 4.5, 13.2 Hz,
1H), 4.10 (m, 1H), 4.03 (d, J = 11.4 Hz, 1H), 3.57 (dt,
J = 2.7, 11.4 Hz, 1H), 3.19 (m, 1H), 3.04 (dd, J = 3.0,
10.2 Hz, 1H).
(S)-2-((4-(5-Bromopyrimidin-2-yl)morpholin-2-yl)methyl)-
(7c) 1H
Synthesis of (S)-(4-(5-bromopyrimidin-2-yl)morpholin-2-
yl)methyl 4-methylbenzenesulfonate (6) To a solution of
(S)-(4-(5-bromopyrimidin-2-yl)morpholin-2-yl)methanol
(1.6 g, 5.8 mmol) in CH2Cl2 (20 mL) was added TsCl
(1.7 g, 8.7 mmol), Et3N (1.6 mL, 11 mmol), DMAP
(72 mg, 0.58 mmol). The resulting mixture was stirred for
2 h at room temperature. After the completion of reaction,
the reaction mixture was extracted with CH2Cl2 and the
organic extracts was dried (Na2SO4), filtered and concen-
trated under reduced pressure. Purification by column
chromatography (SiO2) afforded (S)-(4-(5-bromopyrim-
6-(pyridin-4-yl)pyridazin-3(2H)-one
NMR
(300 MHz, CDCl3) d 8.74 (m, 2H), 8.29 (s, 2H), 8.05 (m,
2H), 7.77 (s, 1H), 7.80 (d, J = 9.6 Hz, 1H), 7.15 (d,
J = 9.6 Hz, 1H), 4,55 (m, 2H), 4.34 (d, J = 13.2 Hz, 1H),
4.24 (dd, J = 4.5, 13.2 Hz, 1H), 4.10 (m, 1H), 4.03 (d,
J = 11.4 Hz, 1H), 3.57 (dt, J = 2.7, 11.4 Hz, 1H), 3.19
(m, 1H), 3.04 (dd, J = 3.0, 10.2 Hz, 1H).
Synthesis of (S)-6-(1-methyl-1H-pyrazol-4-yl)-2-((4-(5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)
morpholin-2-yl)methyl)pyridazin-3(2H)-one (9) To
a
idin-2-yl)
morpholin-2-yl)methyl
4-methylbenzen-
solution of (S)-2-((4-(5-bromopyrimidin-2-yl)morpholine-
2-yl)methyl)-6-(1-methyl-1H-pyrzol-4-yl)pyridazin-3(2H)-
one (200 mg, 0.46 mmol), 4,4,40,40,5,5,50,50-octamethyl-
2,20-bi(1,3,2-dioxaborolane) (177 mg, 0.69 mmol), KOAc
(182 mg, 1.9 mmol), dppf (26 mg, 0.046 mmol) in 1,4-
dioxane (2 mL) was added Pd(dppf)Cl2 (38 mg,
0.046 mmol). After stirring for 13 h at 80 °C, the reaction
mixture was extracted with CH2Cl2. The organic extracts
was dried (Na2SO4), filtered and concentrated under
reduced pressure. To the resulting crude mixture was added
H2O (1.6 mL), THF (1.6 mL) and NaBO3 (69 mg,
0.69 mmol), and the resulting mixture was stirred for 4 h at
room temperature. After concentrated, purification by col-
umn chromatography (SiO2) afforded (S)-2-((4-(5-hydrox-
ypyrimidin-2-yl)morpholin-2-yl)methyl)-6-(1-methyl-1H-
pyrzaeol-4-yl)pyridazine-3(2H)-one (50 mg, 30 %). 1H
NMR(300 MHz, CDCl3) d 8.11 (s, 2H), 7.83 (s, 1H), 7.81
(s, 1H), 7.44 (d, J = 9.3 Hz, 1H), 7.49 (d, J = 9.3 Hz,
1H), 4.48-3.90 (m, 6H), 3.94 (s, 3H), 3.58 (t, J = 10.3 Hz,
1
sulponate. (2.49 g, 99 %). H NMR (300 MHz, CDCl3) d
8.29 (s, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz,
2H), 4.49 (d, J = 13.0 Hz, 1H), 4.37 (d, J = 13.0 Hz, 1H),
4.14-4.05 (m, 2H), 3.93 (dd, J = 7.6, 13.4 Hz, 1H), 3.74-
3.65 (m, 1H), 3.54 (dt, J = 2.5, 11.2 Hz, 1H), 3.08-2.96
(m, 1H), 2.80 (dd, J = 11.2, 13.4 Hz, 1H), 2.45 (s, 3H).
Synthesis of 7a–7c
To a solution of (S)-(4-(5-bromopyridin-2-yl) morpholin -2-
yl)methyl 4-methylbenzensulponate (2.5 g, 5.8 mmol) in
DMF (20 mL) was added (6-(1-methyl-1H-pyrazol-4-
yl)pyridazin-3(2H)-one (1.2 g, 6.9 mmol), Cs2CO3 (2.8 g,
8.7 mmol). After stirring for 13 h at 50 °C, the reaction
mixture was extracted with CH2Cl2. The organic extracts
was dried (Na2SO4), filtered and concentrated under reduced
pressure. Purification by column chromatography (SiO2)
afforded
(S)-2-((4-(5-bromopyrimidin-2-yl)morpholin-2-
123

E.-Y. Kim et al.
1H), 3.07 (t, J = 10.3 Hz, 1H), 2.91 (t, J = 10.3 Hz, 1H);
LC/MS (M?1) calculated for C24H33N9O3 495.6, found
496.2.
methyl-1H-pyrazol-4-yl)-2-((4-(5-(1,2,3,6-tetrahydropy-
ridin-4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)pyridazin-3
(2H)-one (20 mg, 0.037 mmol) in formic acid (3 mL) was
added formaldehyde (37 %, 0.1 mL). After stirring at
95 °C for 13 h, the reaction mixture was adjusted to
pH = 10 by NaOH (1 N) solution. The resulting mixture
was extracted with ethyl acetate (20 mL). The organic
extracts was dried (Na2SO4), filtered and concentrated
under reduced pressure. Purification by column chro-
matography (SiO2) afforded (S)-2-((4-(5-(1-methyl-1,2,
3,6-tetrahydropyridin-4-yl)pyrimidin-2-yl)morpholin-2-yl)
methyl)-6-(1-methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-one.
Synthesis of (S)-2-((4-(5-(1-methyl-1,2,3,6-tetrahydropyridin-
4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)-6-(1-methyl-1H-
pyrazol-4-yl)pyridazin-3(2H)-one (10) tert-Butyl (S)-4-(2-(2-
((3-(1-methyl-1H-pyrazol-4-yl)-6-oxopyridazin-1(6H)-yl)
methyl)morpholino)pyrimidin-5-yl)-3,6-dihydropyridine-
1(2H)-carboxylate To a solution of (S)-2-((4-(5-bro-
mopyrimidin-2-yl)morpholin-2-yl)methyl)-6-(1-methyl-1H-
pyrazol-4-yl)pyridazin-3(2H)-one (100 mg, 0.23 mmol),
tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
3,6-dihydropyridine-1(2H)-carboxylate (106 mg, 0.35 mmol),
Cs2CO3 (224 mg. 0.69 mmol) in dimethoxyethane/H2O
(3:1, 1 mL) was added PdCl2(dppf)2 (9.4 mg, 11.5 mol),
and the reaction mixture was stirred at 80 °C for 2 h. The
resulting mixture was extracted with ethyl acetate (20 mL).
The organic extracts was dried (Na2SO4), filtered and
concentrated under reduced pressure. Purification by col-
umn chromatography (SiO2) afforded tert-butyl (S)-4-(2-
(2-((3-(1-methyl-1H-pyrazol-4-yl)-6-oxopyridazin-1(6H)-yl)
methyl)morpholino)pyrimidin-5-yl)-3,6-dihydropyridine-1
(2H)-carboxylate. (87 mg, 71 %): 1H NMR (300 MHz,
CDCl3) d 8.34 (s, 2H), 7.81 (s, 1H), 7.76 (s, 1H), 7.40 (d,
J = 9.4 Hz, 1H), 6.97 (d, J = 9.4 Hz, 1H), 5.90 (brs, 1H),
4.61 (d, J = 12.5 Hz, 1H), 4.50 (q, J = 6.6 Hz, 1H), 4.39
(d, J = 12.5 Hz, 1H), 4.28 (dd, J = 4.1, 13.1 Hz, 1H),
4.12-4.05 (m, 4H), 3.95 (s, 3H), 3.71-3.58 (m, 3H), 3.20
(dt, J = 1.8, 11 Hz, 1H), 3.03 (q, J = 6.6 Hz, 1H), 2.42
(brs, 2H), 1.48 (s, 9H).
1
(12 mg, 71 %). H NMR (300 MHz, CDCl3) d 8.35 (s,
2H), 7.81 (s, 1H), 7.77 (s, 1H), 7.40 (d, J = 9.4 Hz, 1H),
6.97 (d, J = 9.4 Hz, 1H), 5.94 (s, 1H), 4.61 (d, J =
12.5 Hz, 1H), 4.51 (q, J = 6.6 Hz, 1H), 4.39 (d, J =
12.5 Hz, 1H), 4.17 (dd, J = 4.1, 13.1 Hz, 1H), 4.11 (t, J =
5.5 Hz, 1H), 4.01 (d, J = 12.5 Hz, 1H), 3.95 (s, 3H), 3.82
(m, 2H), 3.59 (dt, J = 1.8, 11.0 Hz, 1H), 3.19 (dt, J = 1.8,
11.0 Hz, 2H), 3.10 (s, 2H), 3.02 (q, J = 6.6 Hz, 1H), 2.67
(t, J = 5.5 Hz, 1H), 2.50 (s, 2H), 2.41 (s, 3H); LC/MS
(M?1) calculated for C23H28N8O2 448.5, found 449.2.
(S)-2-((4-(5-(1-(2-Hydroxyethyl)-1,2,3,6-tetrahydropyridin-
4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)-6-(1-methyl-1H-
pyrazol-4-yl)pyridazin-3(2H)-one (10b) To a solution of
(S)-6-(1-methyl-1H-pyrazol-4-yl)-2-((4-(5-(1,2,3,6-tetrahy-
dropyridin-4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)pyri-
dazin-3(2H)-one (25 mg, 0.057 mmol) in DMF (1 mL)
was added 2-bromoethanol (0.16 mL, 2.01 mmol), Et3N
(0.61 mL, 4.32 mmol), K2CO3 (0.28 g, 1.82 mmol), and
the resulting mixture was heated at 60 °C for 3 h. After
completion of the reaction, the reaction mixture was
extracted with CH2Cl2. Organic extracts were dried dried
(Na2SO4), filtered and concentrated under reduced pres-
sure. Purification by column chromatography (SiO2)
afforded (S)-2-((4-(5-(1-(2-hydroxyethyl)-1,2,3,6-tetrahy-
dropyridin-4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)-6-
(1-methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-one (20 mg,
75 %). 1H NMR (300 MHz, CDCl3) d 8.35 (s, 2H), 7.81 (s,
1H), 7.79 (s, 1H), 7.77 (s, 1H), 7.40 (d, J = 9.5 Hz, 1H),
6.97 (d, J = 9.5 Hz, 1H), 5.94 (m, 1H), 4.60 (d,
J = 13.0 Hz, 1H), 4.50 (dd, J = 8.0, 10.0 Hz, 1H), 4.38
(d, J = 13.0 Hz, 1H), 4.17 (dd, J = 4.3, 13.0 Hz, 1H),
4.11 (m, 2H), 3.94 (s, 3H), 3.69 (t, J = 5.3 Hz, 2H), 3.58
(dt, J = 2.6, 11.3 Hz, 1H), 3.20 (m, 3H), 3.03 (dd,
J = 10.0, 13.0 Hz, 1H), 2.77 (t, J = 5.3 Hz, 1H), 2.67 (t,
J = 5.3 Hz, 2H), 2.50 (s, 2H); LC/MS (M?1) calculated
for C24H30N8O3 478.6, found 479.2.
(S)-6-(1-Methyl-1H-pyrazol-4-yl)-2-((4-(5-(1,2,3,6-tetrahy-
dropyridin-4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)pyri-
dazin-3(2H)-one (10) To a solution of (S)-tert-butyl-4-(2-
(2-((3-(1-methyl-1H-pyrzzol-4-yl)-6-oxopyridazin-1(6H)-yl)
methyl)morpholino)pyrimidin-5-yl)-5,6-dihydropyridine
(84 mg, 0.16 mmol) in CH2Cl2 (5 mL) was added HCl
(4 M, 3 mL). After stirring for 4 h, the reaction mixture
was concentrated. Trituration and filtration by ether affor-
ded (S)-6-(1-methyl-1H-pyrazol-4-yl)-2-((4-(5-(1,2,3,6-te-
trahydropyridin-4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)
1
pyridazin-3(2H)-one (62 mg, 83 %). H NMR (300 MHz,
MeOH-d4) d 8.52 (s, 2H), 8.13 (s, 1H), 7.95 (s, 1H), 7.78 (d,
J = 9.4 Hz, 1H), 7.03 (d, J = 9.4 Hz, 1H), 6.14 (s, 1H), 4.62
(d, J = 12.5 Hz, 1H), 4.48 (q, J = 6.6 Hz, 1H), 4.25 (dd,
J = 4.1, 13.1 Hz, 1H), 4.08 - 4.00 (m, 3H), 3.94 (s, 3H), 3.84
(s, 2H), 3.57 (d, J = 12.5 Hz, 1H), 3.46 (dt, J = 1.8, 11.0 Hz,
2H), 3.10 (q, J = 6.6 Hz, 2H), 2.78 - 2.72 (m, 2H); LC/MS
(M?1) calculated for C24H30N8O3 478.6, found 479.2.
Synthesis of (S)-6-(1-methyl-1H-pyrazol-4-yl)-2-((4-(5-(4-
((4-methylpiperazin-1-yl)methyl)phenyl)pyrimidin-2-yl)
morpholin-2-yl)methyl)pyridazin-3(2H)-one (11) (S)-4-(2-
(2-((3-(1-Methyl-1H-pyrazol-4-yl)-6-oxopyridazin-1(6H) yl)
(S)-2-((4-(5-(1-Methyl-1,2,3,6-tetrahydropyridin-4-yl)pyrim-
idin-2-yl)morpholin-2-yl)methyl)-6-(1-methyl-1H-pyrazol-4-
yl)pyridazin-3(2H)-one (10a) To a solution of (S)-6-(1-
123

Discovery of substituted pyrazol-4-yl pyridazinone derivatives as novel c-Met kinase inhibitors
methyl)morpholino)pyrimidin-5-yl)benzaldehyde To a solu-
tion of (S)-2-((4-(5-bromopyrimidin-2-yl)morpholin-2-yl)
methyl)-6-(1-methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-one
(30 mg, 0.069 mmol), (4-formylphenyl)boronic acid (12
mg, 0.076 mmol), K2CO3 (20 mg, 0.13 mmol) in 1,4-
dioxane/H2O(2:1, 3 mL) was added Pd(PPh3)4 (4 mg,
0.0035 mmol) under nitrogen atmosphere. After stirring for
4 h at 100 °C, the resulting mixture was extracted with
ethylacetate (20 mL). The organic extracts was dried
(Na2SO4), filtered and concentrated under reduced pres-
sure. Purification by column chromatography (SiO2)
afforded (S)-4-(2-(2-((3-(1-methyl-1H-pyrazol-4-yl)-6-ox-
pyridazin-1(6H)yl)methyl)morpholino)pyrimidin-5-yl)ben-
(3 mL, 1:1) was added tert-butyl 4-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)piperidine-1-car-
boxylate (76 mg, 0.20 mmol), Na2CO3 (50 mg, 0.41
mmol), Pd(PPh3)4 (4 mg, 0.003 mmol), and the reaction
mixture was stirred at 80 °C for 2 h. After completion of
the reaction, the reaction mixture was extracted with
EtOAc (20 mL) and water (10 mL). The organic extracts
were dried (Na2SO4), filtered and concentrated under
reduced pressure. Purification by column chromatography
(SiO2) afforded (S)-tert-butyl-4-(4-(2-(2-((6-oxo-3-(pyr-
idin-3-yl)pyridazin-1(6H)-yl)methyl) morpholino)pyrim-
idin-5-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (62 mg,
1
77 %). H NMR (300 MHz, CDCl3) d 9.06 (s,1H), 8.67
1
zaldehyde. (25 mg, 80 %). H NMR 300 MHz, CDCl3) d
(s,1H), 8.43 (s, 2H), 8.18 (d, J = 7.9 Hz, 1H), 7.68 (s, 2H),
7.58 (s, 1H), 7.45 (m, 1H), 7.09 (d, J = 9.0 Hz, 1H), 4,65
(m, 1H), 4.58 (dd, J = 7.5, 13.0 Hz, 1H), 4.42 (d,
J = 14.0 Hz, 1H), 4.31 (m, 3H), 4.29 (m, 1H), 4.05 (m,
1H), 3.63 (dt, J = 2.5, 11.0 Hz, 1H),), 3.26 (m, 1H), 3.10
(dd, J = 10.0, 13.5 Hz, 1H), 2.92 (s, 2H), 2.18 (d,
J = 13.0 Hz, 2H), 1.97 (m, 3H), 1.50 (s, 9H).
10.0 (s, 1H), 8.60 (s, 2H), 7.80 (s, 1H), 7.77 (s, 1H), 7.40
(d, J = 9.7 Hz, 1H), 6.98 (d, J = 9.7 Hz, 1H), 4.48 (d,
J = 14.3 Hz, 1H), 4.21 (dd, J = 7.6, 13.4 Hz, 1H), 3.94 (s,
3H), 3.60 (dt, J = 2.4, 11.2 Hz, 1H), 3.32 - 3.18 (m, 1H),
3.10 (dd, J = 11.2, 13.4 Hz, 1H).
(S)-6-(1-Methyl-1H-pyrazol-4-yl)-2-((4-(5-(4-((4-methylpiper-
azin-1-yl)methyl)phenyl)pyrimidin-2-yl)morpholin-2-yl)methyl)
pyridazin-3(2H)-one (11) To a solution of (S)-4-(2-(2-((3-
(1-methyl-1H-pyrazol-4-yl)-6-oxpyridazin-1(6H)yl)methyl)
morpholino)pyrimidin-5-yl)benzaldehyde (32 mg, 0.069
mmol) in CH2Cl2 (2 mL) was added 1-methylpiperazine
(16 mg, 0.13 mmol), NaBH(OAc)3 (23 mg, 0.10 mmol),
AcOH (5 lL, 0.083 mmol). After stirring for 3 h at room
temperature, the pH of reaction mixture was adjusted to
pH = 8 by K2CO3 (5 %), the resulting solution was
extracted with ethyl acetate (20 mL). The organic extracts
was dried (Na2SO4), filtered and concentrated under
reduced pressure. Purification by column chromatography
(SiO2) afforded (S)-6-(1-methyl-1H-pyrazol-4-yl)-2-((4-(5-
((4-methylpiperazin-1-yl)methyl)pyrimidin-2-yl)morpholin-2-yl)
methyl)pyridazin-3(2H)-one. (8 mg, 21 %). 1H NMR
(300 MHz, CDCl3) d 8.55 (s, 2H), 7.82 (s, 1H), 7.78 (s,
1H), 7.45 - 7.36 (m, 5H), 6.99 (d, J = 6.9 Hz, 1H), 4.69 (d,
J = 13.0 Hz, 1H), 4.53 (dd, J = 8.2, 13.0 Hz, 1H), 4.44
(d, J = 13.2 Hz, 1H), 4.24 - 4.00 (m, 3H), 3.95 (s, 3H),
3.62 (dt, J = 1.9, 11.2 Hz, 1H), 3.56 (s, 2H), 3.21 (m, 1H),
3.08 (dd, J = 10.1, 13.2 Hz, 1H), 2.69 - 2.45 (m, 6H), 2.36
(s, 3H); LC/MS (M?1) calculated for C29H35N9O2 541.7,
found 542.6.
(S)-2-((4-(5-(1-(Piperidin-4-yl)-1H-pyrazol-4-yl)pyrimidin-
2-yl)morpholin-2-yl)methyl)-6-(pyridin-3-yl)pyridazin-3(2H)-
one HCl salt (12a) To a solution of (S)-tert-butyl 4-(4-(2-
(2-((6-oxo-3-(pyridin-3-yl)pyridazin-1(6H)-yl)methyl) mor-
pholino)pyrimidin-5-yl)-1H-pyrazol-1-yl)piperidine-1-car-
boxylate (45 mg, 0.075 mmol) in CH2Cl2 (3 mL) was
added HCl (4 M in 1,4-dioxane, 3 mL). After stirring at
room temperature for 18 h, the resulting mixture was
extracted with EtOAc (20 mL). The organic extracts were
dried (Na2SO4), filtered and concentrated under reduced
pressure. Purification by column chromatography (SiO2)
afforded (S)-2-((4-(5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)
pyrimidin-2-yl)morpholin-2-yl)methyl)-6-(pyridin-3-yl)
pyridazin-3(2H)-one HCl salt (38 mg, 95 %). 1H NMR
(300 MHz, MeOH-d4) d 8.91 (s,1H), 8.67 (s,1H), 8.41 (s,
2H), 8.11 (d, J = 9.0 Hz, 1H), 7.69 (s, 2H), 7.58 (s, 1H), 7.44
(m, 1H), 7.09 (d, J = 9.0 Hz, 2H), 4,65 (m, 1H), 4.56 (dd,
J = 7.5, 13.0 Hz, 1H), 4.40 (d, J = 14.0 Hz, 1H), 4.30 (m,
3H), 4.27 (m, 1H), 4.07 (m, 1H), 3.64 (dd, J = 5.5, 11.0 Hz,
1H), 3.28 (m, 1H), 3.09 (dd, J = 9.0, 13.5 Hz, 1H), 2.91 (s,
2H), 2.19 (d, J = 13.0 Hz, 2H), 1.97 (m, 3H).
(S)-2-((4-(5-(1-(Piperidin-4-yl)-1H-pyrazol-4-yl)pyrimidin-
2-yl)morpholin-2-yl)methyl)-6-(pyridin-4-yl)pyridazin-3(2H)-
one HCl salt (12b)) 1H NMR (300 MHz, CDCl3) d 8.76
(s, 2H), 8.41 (s, 2H), 8.08 (s, 1H), 7.78 (d, J = 9.9 Hz,
2H), 7.58 (s, 1H), 7.54 (d, J = 6.0 Hz, 1H), 7.15 (d,
J = 9.9 Hz, 2H), 4,65 (m, 1H), 4.56 (dd, J = 7.5, 13.0 Hz,
1H), 4.40 (d, J = 13.0 Hz, 1H), 4.31 (m, 3H), 4.28 (m,
1H), 4.06 (m, 1H), 3.62 (dd, J = 5.5, 11.0 Hz, 1H), 3.26
(m, 1H), 3.10 (dd, J = 9.0, 13.5 Hz, 1H), 2.93 (s, 2H), 2.19
(d, J = 13.0 Hz, 2H), 1.97 (m, 3H).
Synthesis of 12a–12d
(S)-tert-Butyl-4-(4-(2-(2-((6-oxo-3-(pyridin-3-yl)pyridazin-
1(6H)-yl)methyl)morpholino) pyrimidin-5-yl)-1H-pyrazol-
1-yl)piperidine-1-carboxylate To as solution of (S)-2-((4-
(5-bromopyrimidin-2-yl)morpholin-2-yl)methyl)-6-(pyridin-
3-yl)pyridazin-3(2H)-one (58 mg, 0.14 mmol) in THF/water
123

E.-Y. Kim et al.
(S)-2-((4-(5-(1-(1-(2-Hydroxyethyl)piperidin-4-yl)-1H-pyra-
zol-4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)-6-(pyridin-3-
yl)pyridazin-3(2H)-one (12c) To a solution of (S)-2-((4-
(5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyrimidin-2-yl)mor-
pholin-2-yl)methyl)-6-(pyridin-3-yl)pyridazin-3(2H)-one
(190 mg, 0.38 mmol) in DMF (3 mL) was added 2-bro-
moethanol (0.14 mL, 1.93 mmol), Et3N (0.59 mL,
4.24 mmol), K2CO3 (0.24 g, 1.73 mmol), and the resulting
mixture was heated at 60 °C for 3 h. After completion of
the reaction, the reaction mixture was extracted with
CH2Cl2. Organic extracts were dried dried (Na2SO4), fil-
tered and concentrated under reduced pressure. Purification
by column chromatography (SiO2) afforded (S)-2-((4-(5-
(1-(1-(2-hydroxyethyl)piperidin-4-yl)-1H-pyrazol-4-yl)pyrim-
idin-2-yl)morpholin-2-yl)methyl)-6-(pyridin-3-yl)pyridazin-3
6.98 (d, J = 9.4 Hz, 1H), 4.54-4.44 (m, 2H), 4.27-4.14 (m,
3H), 4.02 (t, J = 5.4 Hz, 2H), 3.95 (s, 3H), 3.67-3.64 (m,
1H) 3.60 (dt, J = 2.5, 11.2 Hz, 1H), 3.17-3.08 (m, 2H),
2.96 (dd, J = 10.1, 13.0 Hz, 1H), 2.70 (t, J = 5.4 Hz, 2H),
2.33 (s, 6H); LC/MS (M?1) calculated for C21H28N8O3
440.5, found 441.2.
Synthesis of 14
(S)-6-(1-Methyl-1H-pyrazol-4-yl)-2-((4-(5-((1-methylpi-
peridin-4-yl)methoxy)pyrimidin-2-yl)morpholin-2-yl)methyl)
pyridazin-3(2H)-one (14) tert-Butyl (S)-4-(((2-(2-((3-(1-
methyl-1H-pyrazol-4-yl)-6-oxopyridazin-1(6H)-yl)methyl)
morphol -ino)pyrimidin-5-yl)oxy)methyl)piperidine-1-car-
boxylate To a solution of (S)-2-((4-(5-hydroxypyrimidin-
2-yl)morpholin-2-yl)methyl)-6-(1-methyl-1H-pyrazol-4-yl)
pyridazin-3(2H)-one (15 mg, 39 mmol) in DMF (0.5 mL)
was added tert-butyl 4-((tosyloxy)methyl)piperidine-1-
carboxylate (22 mg, 58 mmol), (KI (3 mg, 18 mmol), and
CS2CO3 (19 mg, 58 mmol), and the resulting mixture was
stirred at 90 °C for 15 h. The resulting mixture was
extracted with ethyl acetate (10 mL) and water (5 mL).
Organic extracts were dried (Na2SO4), filtered and con-
centrated under reduced pressure. Purification by column
chromatography (SiO2) afforded tert-butyl (S)-4-(((2-(2-
((3-(1-methyl-1H-pyrazol-4-yl)-6-oxopyridazin-1(6H)-yl)
1
(2H)-one (148 mg, 77 %). H NMR (300 MHz, CDCl3) d
8.40 (s, 2H), 7.62 (d, J = 9.4 Hz, 2H), 7.58 (m, 3H), 7.40
(m, 1H), 7.11 (m, 1H), 7.08 (d, J = 9.4 Hz, 1H), 4.62 (d,
J = 13.6 Hz, 1H), 4.51 (d, J = 7.6 Hz, 1H), 4.41 (d,
J = 13.6 Hz, 1H), 4.28 (d, J = 4.8 Hz, 1H), 4.24 (d,
J = 4.8 Hz, 1H), 4.15 (m, 2H), 4.02 (d, J = 11.5 Hz, 1H),
3.75 (m, 1H), 3.64 (t, J = 5.1 Hz, 2H), 3.48 (s, 2H), 3.22
(m, 1H), 3.05 (m, 3H), 2.64 (t, J = 5.1 Hz, 2H), 2.32 (m,
3H).
(S)-2-((4-(5-(1-(1-(2-Hydroxyethyl)piperidin-4-yl)-1H-pyrazol-
4-yl)pyrimidin-2-yl)morpholin-2-yl)methyl)-6-(pyridin-4-yl)pyri-
dazin-3(2H)-one (12d) 1H NMR (300 MHz, CDCl3) d
8.40 (s, 2H), 7.64 (d, J = 9.2 Hz, 2H), 7.58 (m, 3H), 7.42
(m, 1H), 7.11 (m, 1H), 7.09 (d, J = 9.2 Hz, 1H), 4.62 (d,
J = 13.4 Hz, 1H), 4.50 (d, J = 7.4 Hz, 1H), 4.40 (d,
J = 13.4 Hz, 1H), 4.27 (d, J = 4.8 Hz, 1H), 4.25 (d,
J = 4.8 Hz, 1H), 4.15 (m, 2H), 4.02 (d, J = 11.5 Hz, 1H),
3.74 (m, 1H), 3.65 (t, J = 5.1 Hz, 2H), 3.49 (s, 2H), 3.20
(m, 1H), 3.07 (m, 3H), 2.64 (t, J = 5.4 Hz, 2H), 2.31 (m,
3H).
methyl)
morphol-ino)pyrimidin-5-yl)oxy)methyl)piper-
1
idine-1-carboxylate (17 mg, 78 %). H NMR (300 MHz,
CDCl3) d 8.09 (s, 2H), 7.81 (s, 1H), 7.77 (s, 1H), 7.40 (d,
J = 9.4 Hz, 1H), 6.98 (d, J = 9.4 Hz, 1H), 4.51 - 4.47 (m,
2H), 4.21-4.19 (m, 1H), 4.01 (t, J = 5.4 Hz, 2H), 3.95 (s,
3H), 3.88-3.81 (m, 3H), 3.59 (dt, J = 2.8, 10.1, 11.2 Hz,
2H), 3.29-3.23 (m, 2H), 3.02 (ddd, J = 2.8, 10.1, 13.0 Hz,
4H), 1.88-1.83 (m, 4H), 1.46 (s, 9H).
(S)-6-(1-Methyl-1H-pyrazol-4-yl)-2-((4-(5-(piperidin-4-yl-
methoxy)pyrimidin-2-yl)morpholin-2-yl)methyl)pyridazin-
3(2H)-one (14a) To a solution of tert-butyl (S)-4-(((2-(2-
((3-(1-methyl-1H-pyrazol-4-yl)-6-oxopyridazin-1(6H)-yl)
methyl)morpholino)pyrimidin-5-yl)oxy)methyl)piperidine-
1-carboxylate (5.7 mg, 0.01 mmol) in CH2Cl2 (2 mL) was
added TFA (0.5 mL, 0.04 mmol). After stirring for 13 h at
room temperature, the reaction mixture was concentrated.
Trituration and filtration by ether afforded (S)-6-(1-methyl-
1H-pyrazol-4-yl)-2-((4-(5-(piperidin-4-ylmethoxy)pyrim-
idin-2-yl)morpholin-2-yl)methyl)pyridazin-3(2H)-one (3 mg,
52 %). 1H NMR (300 MHz, CDCl3) d 8.10 (s, 2H), 7.82 (s,
1H), 7.77 (s, 1H), 7.41 (d, J = 9.4 Hz, 1H), 7.00 (d,
J = 9.4 Hz, 1H), 4.52-4.40 (m, 2H), 4.30-4.16 (m, 1H),
4.03 (t, J = 5.4 Hz, 2H), 3.95 (s, 3H), 3.63-3.5 (m, 2H),
3.28-3.18 (m, 4H), 3.00-2.95 (m, 2H), 2.14-2.03 (m, 8H);
LC/MS (M?1) calculated for C23H30N8O3 466.5, found
453.2 (M?1-Me).
(S)-2-((4-(5-(2-(Dimethylamino)ethoxy)pyrimidin-2-yl)-
morpholin-2-yl)methyl)-6-(1-methyl-1H-pyrazol-4-yl)pyri-
dazin-3(2H)-one (13) To a solution of (S)-2-((4-(5-hy-
droxypyrimidin-2-yl)morpholin-2-yl)methyl)-6-(1-methyl-
1H-pyrazol-4-yl)pyridazin-3(2H)-one (20 mg, 0.054 mmol)
in DMF (1 mL) was added 2-(dimethylamino)ethyl
hydrochloride (23 mg, 0.16 mmol), Cs2CO3 (22 mg,
0.16 mmol), KI (9 mg, 0.054 mmol). After stirring for 2 h
at 90 °C, the resulting mixture was extracted with ethyl
acetate (20 mL). Organic extracts was dried (Na2SO4),
filtered and concentrated under reduced pressure. Purifi-
cation by column chromatography (SiO2) afforded (S)-2-
((4-(5-(2-(dimethylamino)ethoxy)pyrimidin-2-yl)morpholin-
2-yl)methyl)-6-(1-methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-
one (16 mg, 70 %). 1H NMR (300 MHz, CDCl3) d 8.12 (s,
2H), 7.81 (s, 1H), 7.77 (s, 1H), 7.40 (d, J = 9.4 Hz, 1H),
123

Discovery of substituted pyrazol-4-yl pyridazinone derivatives as novel c-Met kinase inhibitors
(S)-6-(1-Methyl-1H-pyrazol-4-yl)-2-((4-(5-((1-methylpi-
peridin-4-yl)methoxy)pyrimidin-2-yl)morpholin-2-yl)methyl)
pyridazin-3(2H)-one (14) To a solution of (S)-6-(1-
methyl-1H-pyrazol-4-yl)-2-((4-(5-(piperidin-4-ylmethoxy)
pyrimidin-2-yl)morpholin-2-yl)methyl)pyridazin-3(2H)-one
(8.6 mg, 0.015 mmol) in formic acid (1 mL) was added
formaldehyde (37 %, 17 lL). After stirring at 95 °C for
13 h, the pH of the reaction mixture was adjusted to pH 10
by NaOH (1 N) solution. The resulting mixture was
extracted with ethyl acetate (20 mL). The organic extracts
were dried (Na2SO4), filtered and concentrated under
reduced pressure. Purification by column chromatography
(SiO2) afforded (S)-6-(1-methyl-1H-pyrazol-4-yl)-2-((4-(5-
((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)morpholin-
acetate (20 mL). The organic extracts was dried (Na2SO4),
filtered and concentrated under reduced pressure. Purifi-
cation by column chromatography (SiO2) afforded (S)-6-
(1-methyl-1H-pyrazol-4-yl)-2-((4-(5-(2-(4-methylpiperazin-
1-yl)ethoxy)pyrimidin-2-yl)morpholin-2-yl)methyl) pyri-
dazin-3(2H)-one (13 mg, 56 %). 1H NMR (300 MHz,
CDCl3) d 8.10 (s, 2H), 7.81 (s, 1H), 7.76 (s, 1H), 7.40 (d,
J = 9.4 Hz, 1H), 6.97 (d, J = 9.4 Hz, 1H), 4.54-4.44 (m,
2H), 4.22-4.19 (m, 1H), 4.06 (t, J = 5.4 Hz, 2H), 3.95 (s,
3H), 3.60 (dt, J = 2.5, 11.2 Hz, 1H), 3.18-3.08 (m, 1H),
2.97 (dd, J = 10.1, 13.0 Hz,1H), 2.78 (t, J = 5.4 Hz, 2H),
2.62-2.52 (m, 7H), 2.32 (s, 3H), 2.03-1.96 (m, 4H); LC/MS
(M?1) calculated for C24H33N9O3 495.6, found 496.2.
(S)-6-(1-Methyl-1H-pyrazol-4-yl)-2-((4-(5-(2-(pyrrolidin-
1-yl)ethoxy)pyrimidin-2-yl)morpholin-2-yl)methyl)pyridazin-
3(2H)-one (15b) 1H NMR (300 MHz, CDCl3) d 8.11 (s,
2H), 7.81 (s, 1H), 7.77 (s, 1H), 7.40 (d, J = 9.4 Hz, 1H),
6.98 (d, J = 9.4 Hz, 1H), 4.55-4.45 (m, 2H), 4.27-4.22 (m,
1H), 4.10 (t, J = 5.4 Hz, 2H), 3.95 (s, 3H), 3.61 (dt,
J = 2.5, 11.2 Hz, 1H), 3.18-3.09 (m, 1H), 2.95 (dd,
J = 10.1, 13.0 Hz, 1H), 2.67 (t, J = 5.4 Hz, 2H), 2.28-
2.07 (m, 7H), 1.94-1.78 (m, 3H); LC/MS (M?1) calculated
for C23H30N8O3 466.5, found 467.2.
1
2-yl)methyl) pyridazin-3(2H)-one (5 mg, 61 %). H NMR
(300 MHz, CDCl3) d 8.09 (s, 2H), 7.81 (s, 1H), 7.76 (s,
1H), 7.39 (d, J = 9.4 Hz, 1H), 6.97 (d, J = 9.4 Hz, 1H),
4.54-4.47 (m, 2H), 4.28-4.23 (m, 1H), 4.06 (t, J = 5.4 Hz,
2H), 3.95 (s, 3H), 3.66-3.59 (m, 2H), 3.18-3.09 (m, 1H),
2.97 (q, J = 7.6 Hz, 1H), 2.72-2.65 (m, 2H), 2.29 (s, 3H),
2.28-2.18 (m, 2H), 2.04-1.93 (m, 5H), 1.82-1.73 (m, 2H);
LC/MS (M?1) calculated for C24H32N8O3 480.6, found
467.2 (M?1-Me).
Synthesis of 15a–15c
(S)-6-(1-Methyl-1H-pyrazol-4-yl)-2-((4-(5-(2-morpholi-
noethoxy)pyrimidin-2-yl)morpholin-2-yl)methyl)pyridazin-
3(2H)-one (15c) 1H NMR (300 MHz, CDCl3) d 8.09 (s,
2H), 7.79 (s, 1H), 7.75 (s, 1H), 7.39 (d, J = 9.4 Hz, 1H),
6.96 (d, J = 9.4 Hz, 1H), 4.53-4.40 (m, 2H), 4.27-3.94 (m,
5H), 3.93 (s, 3H), 3.72 (t, J = 4.4 Hz, 4H), 3.58 (dt,
J = 1.9, 11.2 Hz, 1H), 3.18-3.05 (m, 1H), 2.94 (dd,
J = 10.1, 13.0 Hz, 1H), 2.74 (t, J = 5.4 Hz, 2H), 2.54 (t,
(S)-2-((4-(5-(2-Chloroethoxy)pyrimidin-2-yl)morpholin-2-yl)
methyl)-6-(1-methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-one
To a solution of (S)-2-((4-(5-hydroxypyrimidin-2-yl)mor-
pholin-2-yl)methyl)-6-(1-methyl-1H-pyrazol-4-yl)pyridazin-
3(2H)-one (100 mg, 0.27 mmol) in DMF (1 mL) was
added 1,2-dichloroethane (28 lL, 0.54 mmol) Cs2CO3
(218 mg, 0.67 mmol), KI (5 mg, 0.027 mmol). After stir-
ring at 90 °C for 13 h, the resulting mixture was extracted
with dichloromethane (10 mL). The organic extracts was
dried (Na2SO4), filtered and concentrated under reduced
pressure. Purification by column chromatography (SiO2)
afforded (S)-2-((4-(5-(2-chloroethoxy)pyrimidin-2-yl)mor-
pholin-2-yl)methyl)-6-(1-methyl-1H-pyrazol-4-yl)pyridazin-
J = 4.4 Hz, 4H); LC/MS (M?1
)
calculated for
C223H30N8O4 482.5, found 483.2.
c-Met kinase assay
Inhibition of kinase activity against recombinant c-Met
protein was measured using homogeneous time-resolved
fluorescence (HTRF) assays. Recombinant proteins con-
taining c-Met kinase domain were purchased from Milli-
pore. Optimal enzyme, ATP, and substrate concentrations
were established using HTRF KinEASE kit (Cisbio)
according to the manufacturer’s instructions. Assays are
composed of the c-Met enzyme mixed with serially diluted
compounds and peptide substrates in a kinase reaction
buffer (250 mM HEPES (pH 7.0), 0.5 mM orthovanadate,
0.05 % BSA, 0.1 % NaN3, 5 mM MgCl2, 1 mM DTT).
Following the addition of reagents for detection, the Time
Resolved-Fluorescence Resonance Energy Transfer
(TRFRET) signal was measured using an EnVision multi-
label reader (Perkin Elmer). Dose–response curves were
1
3(2H)-one (81 mg, 70 %). H NMR (300 MHz, CDCl3) d
8.05 (s, 2H), 7.74 (s, 1H), 7.70 (s, 1H), 7.34 (d, J = 9.4 Hz,
1H), 6.91 (d, J = 9.4 Hz, 1H), 4.44-4.40 (m, 2H), 4.21-
3.91 (m, 6H), 3.88 (s, 3H), 3.71 (s, 1H), 3.55-3.49 (m, 2H),
3.11-3.02 (m, 1H), 2.94-2.87 (m 1H).
(S)-6-(1-Methyl-1H-pyrazol-4-yl)-2-((4-(5-(2-(4-methylpiper-
azin-1-yl)ethoxy)pyrimidin-2-yl)morpholin-2-yl)methyl)pyri-
dazin-3(2H)-one (15a) To a solution of (S)-2-((4-(5-(2-
chloroethoxy)pyrimidin-2-yl)morpholin-2-yl)methyl)-6-(1-
methyl-1H-pyrazol-4-yl)pyridazin-3(2H)-one (20 mg, 0.046
mmol) was added 1-methylpiperazine (19 mg, 0.18 mmol)
and KI (8.5 mg, 0.051 mmol). After stirring for 13 h at
70 °C, the resulting mixture was extracted with ethyl
123

E.-Y. Kim et al.
generated to determine IC50 using Prism version 5.01
(GraphPad). Reference compounds were included in each
assay for plate uniformity (crizotinib) and select com-
pounds were subjected to repeat experiments.
benzyl group by use of Pd catalyzed hydrogenation
(Scheme 2).
The synthesis of hydrazinones 9 was accomplished by
starting from hydroxymethylmorpholine 4 (Scheme 3). The
production of 5 was readily available by treatment of 2,5-
dibromopyrimidine 5 with hydroxymethylmorpholine 4 in
the presence of diisopropylamine. Simple treatment of 5 with
TsCl afforded 6, and the resulting compound 6 was used for
the synthesis of 7 in alkylation condition of 2 by use of
Cs2CO3. Synthesis of pinacolborane derivative 8 was affor-
ded by palladium-catalyzed cross coupling of bis(pinaco-
lato)diboron with 7. Introduction of OH was achieved by
NaBO3 oxidation of compound 8. Tetahydropyridine
attached hydrazinone 10 was prepared from bromopyrim-
idine derivative 7a by Pd-catalyzed cross coupling reaction
with the N-Boc protected tetrahydropyridine dioxoborolane,
and successive treatment of the resulting intermediate fol-
lowed by the reductive amination of the tetrahydropyridine
with formaldehyde (Scheme 4). Substituted pyrazoles were
introduced by the corresponding dioxoboroloane pyrazoles
under Pd-catalyzed cross-coupling condition followed by
deprotection of Boc or tetrahydropyranyl group for CH2-
CH2OTHP. A series of alkylated pyrimidine derivatives was
prepared from 9a to afford 13, 14, 15 (Scheme 5).
Proliferation assay
Cells were plated in 96-well plates (10,000 cells per well)
and serial dilutions of compounds were added. At the end
of the incubation period (72 h), cell viability was mea-
sured by a tetrazolium dye assay using WST-8 [2-(2-
methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disul-
fophenyl)-2H-tetrazolium, monosodium salt] (Dojindo,
Japan). IC50 was calculated by a nonlinear regression using
Prism version 5.01.
Results and discussion
Chemistry
Commercially available 3,6-dichloropyridazine was con-
verted into the corresponding substituted pyridazine
derivatives 1 by palladium catalyzed cross coupling of the
aromatic compounds (Scheme 1).
The resulting pyridazines 1 were readily hydrolyzed to
afford the pyridazinones 2 in the presence of AcOH and H2O.
Reaction of 2-(benzylamino)ethan-1-ol and epichlorohydrin
gave (S)-(4-benzylmorpholin-2-yl)methanol 3. 2-Hydrox-
ymethylmorpholine 4 was easily obtained by deprotection of
In vitro c-Met enzyme inhibitory activity and cell
cytotoxicity assay
The kinase inhibitory activities of the compounds were
evaluated by the well-established homogeneous time-re-
solved fluorescence (HTRF) method against recombinant
c-Met enzyme purchased from Millipore. A detailed SAR
was evolved and the basic structure of the scaffold
underlying biological data depend on substituents were
being well identified. Proper selection of substituents, site
selection for substitution, averting any steric congestion in
binding compartments in ATP binding hinge region would
effectively deliver a potent c-Met kinase inhibitors. The
compounds potently inhibited the kinase functions with
IC50 values between 19 and 96 nM. As an initial assess-
ment of the SAR focusing on the pyrimidine, functional
groups such as H, OH, tetrahydropyridine, phenyl, and
pyrazole, were introduced into the pyrimidine ring; the
results are summarized in Table 1. We first examined the
effect of hydroxyl (9) on pyrimidine, the inhibitory activity
was increased compared to that of hydrogen (7a). Intro-
duction of tetrahydropyridine functionalities (10, 10a, 10b)
significantly increase the inhibitory activity 10. Substitu-
tion of phenyl substituted piperazine exhibited almost
equivalent inhibitory activity to 10. Introduction of 3 (or
4)-pyridine instead of N-methyl pyrazole to pyridazinone
exhibited almost same inhibitory activities (12a, 12b).
Scheme 1 Reagents and conditions: (i) 1-methyl-4-(4,4,5,5-tetram-
ethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole or ArB(OH)2, Pd(dppf)2-
Cl2, K2CO3, 90 °C, 3 h, 81 %; (ii) AcOH, H2O, 120 °C, 4 h, 83 %
Scheme 2 Reagents and conditions: (i) (R)-epichlorohydrin, H2O/i-
PrOH, rt, 13 h; (ii) NH4OH, rt, 3 h, 50 % (2 steps); (iii) Pd/C, H2,
MeOH, 12 h, 98 %
123

Discovery of substituted pyrazol-4-yl pyridazinone derivatives as novel c-Met kinase inhibitors
i
Scheme 3 Reagents and conditions: (i) Pr2NH, EtOH, rt, 13 h, 87 %; (ii) TsCl, DMAP, Et3N, CH2Cl2, rt, 2 h, 99 %; (iii) 2, Cs2CO3, 50 °C,
13 h, 60 %; (iv) 4,4,40,40,5,5,50,50-octamethyl-2,20-bis(1,3,2-dioxaborolane), KOAc,, Pd(dppf) Cl2, dppf, dioxane, 80 °C, 13 h, then NaBO3,
H2O, THF, 30 % (2 steps)
Scheme 4 Reagents and conditions: (i) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate,
Pd(PPh3)2Cl2, DME/H2O (3:1), 80 °C, 3 h, 71 %; (ii) 4 M HCl in 1,4-dioxane, CH2Cl2, rt, 1 h, 83 %; (iii) HCHO (35 %), HCO2H, 95 °C,
13 h, 71 % or ClCH2CH2OH, Et3N, K2CO3, DMF, 60 °C, 3 h, 75 %; (iv) (4-formylphenyl)boronic acid, Pd(PPh3)4, K2CO3, 1,4-dioxane,
100 °C, 4 h, 80 %; (v) 1-methylpiperazine, NaBH(OAc)3, AcOH, rt, 3 h, 21 %; (vi) tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate, Pd(PPh3)2Cl2, Cs2CO3, THF/water (3:1), 80 °C, 2 h, 77 %; (vii) HCl (in 1,4-dioxane, 4 N),
CH2Cl2, 18 h, 95 % or ClCH2CH2OH, Et3N, K2CO3, DMF, 60 °C, 3 h, 77 %
Parent compound 9 exhibiting potent inhibitory activities
either c-Met enzyme or Hs746T cell cytotoxicity assay
which has structurally defective compartment derived from
hydroxy functionality. To complement the drawback of
hydroxyl, structural modification of 9 was performed by
introduction of alkyl substituents. Systematic study of the
123

E.-Y. Kim et al.
Scheme 5 Reagents and
conditions: i 2-
(dimethylamino)ethyl
hydrochloride, Cs2CO3, KI,
DMF, 90 °C, 2 h, 70 %; ii tert-
butyl 4-((tosyloxy)methyl)
piperidine-1-carboxylate,
Cs2CO3, DMF, 90 °C, 15 h,
78 %; iii TFA, rt, 13 h, 52 %; iv
HCHO (35 %), HCO2H, 95 °C,
13 h, 60 %; v 1,2-
dichloroethane, Cs2CO3, KI,
DMF, 90 °C, 13 h, 70 %; vi
amines, KI, DMF, 70, 13 h,
56 %
substitution of pyridine on pyridazinone and alkylation of
pyrimidin-5-ol revealed that inhibitory activity was slightly
improved when a piperidin-4-methanol (14a) and mor-
pholino-1-ethanol (15c) in enzyme activities. The other
substituents, such as N,N-dimethylaminoethan-1-ol (13), N-
methylmorpholinoethan-1-ol (14), and phenyl derivative
(15b) did not significantly contribute to increase the inhi-
bitory activity toward c-Met enzyme. All of the compounds
exhibited micro-molar range of Hs746T cell cytotoxicity
assay. Of the compounds tested 12a and 12b exhibited
moderate inhibitory activities toward Hs746T cell line.
And, none of the tested compounds have activities in
H1993 cell line as a negative control.
c-Met in complex with crizotinib (PDB ID: 2WGJ) were fixed
by the Protein Preparation Wizard and the low-energy 3D
structures of compounds were generated by the LigPrep (Cui
et al. 2011). The Glide module inStandardPrecision(SP) mode
was used to dock compounds into ATP binding site of c-Met.
To check the validity of the Glide docking protocols, the
crizotinib was also docked into c-Met and the docking results
were compared to its native conformation in the co-crystallized
form. The root mean square deviation (RMSD) of the docked
crizotinib was 0.20 as it seems exactly superimposed on the
native bound one, indicating that the Glide docking method
samples the correct ligand orientation. The key interactions of
compound 12a with excellent c-Met inhibitory activities in
both enzyme- and cell-based in vitro assays and the co-crystal
structure of crizotinib bound to the c-Met kinase domain are
presented in Fig. 1.
Pyrimidine ring has an important role in directing both
phenyl and the terminal pyridazinone ring, which is con-
sidered to have an interaction of p–p stacking with
Tyr1230 in molecular docking study (vide infra). More-
over, nitrogen of pyrimidine is an essential compartment to
induce hinge binding with Met1160 and morpholine was
assumed to be interacted with Pro1158. In current study,
we have considered all of the aforementioned data of the
SAR of our scaffold, and compound 12a and 12b exhibited
nano-molar range of inhibitory activities and also micro-
molar range of cell cytotoxicity assay in Hs746T cell lines.
As shown in Fig. 1, both classes of inhibitors bind to
ATP binding site of c-Met with a similar conformation and
in particular, crizotinib held in the active site through two
H-bond interactions with Pro1158 and Met1160, in addi-
tion to a relatively weak p–p stacking interactions formed
with Tyr1230 phenyl moiety. Upon analysis of the inter-
action of compound 12a in ATP binding site of c-Met, it is
observed that the pyrimidine nitrogen and pyridazinone
oxygen participate in the H-bond interactions with
Met1160 in the hinge region, similar to the pyridine
nitrogen of crizotinib, and Asp1222. In addition to H-bond
interaction, the 3-pyridyl substituted pyridazinone forms
strong p–p stacking interactions with aromatic ring of
Tyr1230 under the A-loop as did by the trihalogenated
benzyl moiety of crizotinib, which is critical for selective
c-Met inhibition (Timofeevski et al. 2009). Although there
is a favorable interaction between C2 atom of morpholine
Molecular modeling
Molecular docking studies of the synthesized compounds were
performed against human c-Met to understand the binding
interactions and identify if these compounds have analogous
binding mode to the c-Met inhibitor crizotinib. All the calcu-
lations was carried out using the Maestro v9.5 (Schrodinger,
Inc.) software (Maestro, version 9.5). Structural defects of
123

Discovery of substituted pyrazol-4-yl pyridazinone derivatives as novel c-Met kinase inhibitors
Table 1 SAR of substituent of R1 and R2 on pyridazinones
R2
N
N
R1
N
N
N
O
O
No
R1
R2
c-Met enzyme (IC50, lM
)
Cell-based assay
Hs746T IC50 (lM)
H1993 IC50 (lM)
9
a
a
OH
0.060
0.025
0.24
2.69
[10
4.21
10
HN
10a
10b
a
a
0.057
0.130
3.38
3.43
3.86
3.82
MeN
HO
N
11
a
0.039
0.035
1.16
1.9
[10
N
NMe
NH
12a
3-pyridyl (b)
3.6
N
N
N
N
d
12b
12c
4-pyridyl (c)
b
d
0.047
0.23
1.2
7.1
3.6
[10
NCH 2CH2OH
e
12d
13
c
e
0.41
10
[10
[10
a
0.082
3.90
O
NMe2
14
a
a
0.059
0.019
3.22
3.17
[10
[10
O
MeN
14a
O
HN
15a
15b
a
a
0.096
0.046
7.51
2.91
[10
[10
MeN
N
O
N
O
15c
a
0.040
3.79
[10
N
O
O
ring and Pro1158, the H-bond with Pro1158 is not found in
compound 12a. These results provide a rationale for
explaining the activity difference between compound 12a
and crizotinib. Met1211 has closer interactions with the
phenyl group and 2-aminopyridine of crizotinib and the
pyridazinone and morpholine of compound 12a via
hydrophobic interactions.
In conclusion, series of pyrazol-4-yl pyridazin-3(2H)-
ones were synthesized and evaluated against c-Met kinase
inhibitory activities. Pyrazol-4-yl pyridazin-3(2H)-one 14a
displayed most potent c-Met inhibitory activity with an
IC50 of 19 nM. This compound exhibited moderate inhi-
bitory activity of cell proliferation in Hs746T cell line with
an IC50 of 3.2 lM. Molecular docking studies performed
123

E.-Y. Kim et al.
´
Cui JJ, Tran-Dube M, Shen H, Nambu M, Kung P-P, Pairish M, Jia L,
Meng J, Funk L, Botrous I, McTigue M, Grodsky N, Ryan K,
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Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park
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¨
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Garber K (2014) MET inhibitors start on road to recovery. Nat Rev
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Gherardi E, Birchmeier W, Birchmeier C, Woude GV (2012)
Targeting MET in cancer: rationale and progress. Nat Rev
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Kang S-T, Kim E-Y, Archary R, Jung H, Park CH, Yun C-S, Hwang
JY, Choi SU, Chae C, Lee CO, Kim HR, Ha JD, Ryu D, Cho SY
(2014) Discovery of substituted 6-pheny-3H-pyridazin-3-one
derivatives as novel c-Met kinase inhibitors. Bioorg Med Chem
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Knudsen BS, Vande Woude G (2008) Showering c-MET-dependent
cancers with drugs. Curr Opin Genet Dev 18:87–96
Lai AZ, Abella JV, Park M (2009) Crosstalk in Met receptor
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Fig. 1 Proposed binding modes of crizotinib (salmon) and compound
12a (green) in ATP binding site of c-Met. H-bond interactions are
drawn as dashed red lines
against human c-Met revealed that this series of com-
pounds has highly analogous binding mode of crizotinib
and confirm the significance of SAR profile in designing
compounds of this series. All of the final potent compounds
possess the essential interaction with ATP binding site of
c-Met with H-bond interaction with Pro1158 and Met
1150, in addition to a relatively weak p–p stacking inter-
actions formed with Tyr1230 phenyl moiety. Further
investigation into the effect of substituents to improve
pharmacokinetic properties, oral bioavailability, and
in vivo Xenograft antitumor activity are in progress.
Ma PC, Tretiakova MS, MacKinnon AC, Ramnath N, Johnson C,
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T, Vokes EE, Husain AN, Salgia R (2008) Expression and
mutational analysis of MET in human solid cancers. Genes
Chromosom Cancer 47:1025–1037
Acknowledgments This work was supported by a grant from the
Korea Research Institute of Chemical Technology (KRICT) and
the National Research Foundation of Korea (H.R.K, NRF-2012
M3A9A9054902).
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