Discovery of novel 7-azaindole derivatives bearing dihydropyridazine moiety as c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.03.045

A series of 7-azaindole derivatives bearing the dihydropyridazine scaffold were synthesized and evaluated for their c-Met kinase inhibitory, and antiproliferative activity against 4 cancer cell lines (HT29, A549, H460, U87MG) were evaluated in?vitro. Most compounds showed moderate to excellent potency. Compared to foretinib, the most promising analog 34 (c-Met IC₅₀: 1.06?nM, a multitarget tyrosine kinase inhibitor) showed a 6.4-, 7.8-, and 3.2-fold increase in activity against HT29, A549, and H460?cell lines, respectively. Structure activity relationship studies indicated that mono-EWGs (such as R₂?=?F) at 4-position of moiety D was a key factor in improving the antitumor activity.

Full text of DOI:10.1016/j.ejmech.2017.03.045

Accepted Manuscript
Discovery of novel 7-azaindole derivatives bearing dihydropyridazine moiety as c-Met
kinase inhibitors
Qidong Tang, Linxiao Wang, Yongli Duan, Wenhui Wang, Shunmin Huang, Zhi Jia,
Shuang Jia, Wufu Zhu, Ping Wang, Rong Luo, Pengwu Zheng
PII:
S0223-5234(17)30202-7
10.1016/j.ejmech.2017.03.045
EJMECH 9305
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2017
Accepted Date: 22 March 2017
Please cite this article as: Q. Tang, L. Wang, Y. Duan, W. Wang, S. Huang, Z. Jia, S. Jia, W. Zhu,
P. Wang, R. Luo, P. Zheng, Discovery of novel 7-azaindole derivatives bearing dihydropyridazine
moiety as c-Met kinase inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.03.045.
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ACCEPTED MANUSCRIPT
Graphical abstract
A series of 7-azaindole derivatives bearing dihydropyridazine moiety (20–51) were designed,
synthesized and evaluated for their activity against c-Met kinase and cancer cell lines. The most
promising compound 34 showed excellent in vitro activity.

ACCEPTED MANUSCRIPT
Discovery of novel 7-azaindole derivatives bearing dihydropyridazine moiety as c-Met kinase
inhibitors
Qidong Tang a, ,†, Linxiao Wang a,†, Yongli Duan a, Wenhui Wang a, Shunmin Huang a, Jia Zhi a, Shuang Jia
a, Wufu Zhu a, Ping Wang a, Rong Luo b, Pengwu Zheng a,*
a School of Pharmacy, Jiangxi Science and Technology Normal University, Nanchang 330013, PR China
b Jiangxi Province Institute of Materia Medica, Nanchang 330000, PR China
Abstract
A series of 7-azaindole derivatives bearing the dihydropyridazine scaffold were
synthesized and evaluated for their c-Met kinase inhibitory, and antiproliferative activity
against 4 cancer cell lines (H460, A549, H460, U87MG) were evaluated in vitro. Most
compounds showed moderate to excellent potency, and compared to foretinib, the most
promising analog 34 (c-Met IC50: 1.06 nM, a multitarget tyrosine kinase inhibitor) showed a
6.4-, 7.8-, and 3.2-fold increase in activity against H460, A549, and H460 cell lines,
respectively. Structure activity relationship studies indicated that mono-EWGs (such as R2 =
F) at 4-position of moiety D was a key factor in improving the antitumor activity.
Keywords: c-Met; Receptor tyrosine kinase; Anti-tumor; 7-azaindole derivatives;
dihydropyridazine
1. Introduction
The HGF/MET signaling pathway is critical in mediating a wide range of normal
physiological functions including cell proliferation, invasion, motility, survival, and
morphogenesis [1–4]. c-Met has been shown to be frequently amplified or overexpressed in
various cancers, including brain, colorectal, gastric, lung, head, neck, and stomach
cancers[5–7]. Both amplifications/overexpression are associated with poor clinical outcomes
[8], underscoring the importance of increased c-Met signaling in these cancer types [9–12].
Corresponding authors. Tel./fax: +86 791 83802393.
E-mail addresses: tangqidongcn@126.com (Q. Tang), zhengpw@126.com (P. Zheng).
† These authors contribute equally to this work.
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Developing HGF/MET inhibitors is one of the research hotspots in molecular targeted therapy
for the treatment of human cancer.
Recently, the research of small-molecule c-Met inhibitors has obtained remarkable
achievements, resulting in the marketing of cabozantinib [13] (1, a small molecule that
inhibits the activity of multiple tyrosine kinases, including c-Met, RET, and VEGFR-2/KDR,
marketed in 2012) [14], and several candidates currently under clinical trials, such as foretinib
(2, GSK1363089), compounds 3 and 4 [15–17]. Based on the structure characteristics of these
inhibitor, we can obtain a general skeleton which contained four parts: moiety A, B, C, and D
can be summarized (Fig. 1). The main modification of these derivatives was focused on the
moiety C (a 5-atom linker) between moiety B and moiety D, which has two obvious structural
characteristics. One is the ‘5 atoms regulation’, which means six chemical bonds distance
existing between moiety B and moiety D; the other is the linker containing hydrogen, oxygen,
and nitrogen atoms which could form hydrogen-bond donor or acceptor [18–19].
(Figure 1. should listed here)
Dihydropyridazine fragment was widely used as a building block in the design of
anticancer agents. For example, compounds 6–8 (Fig. 2) displayed a multitude of biological
activities [20–22]. In this work, dihydropyridazine was introduced to moiety C to form the
5-atom linker, because the two nitrogen atoms in dihydropyridazine as the hydrogen-bond
acceptor have high ability to form hydrogen-bonding interactions with c-Met.
7-substituted-pyrrolo[2,3-b]pyridine was used as the moiety A. Substituted phenyl ring was
reserved as the moiety B and D. Small substituent X, R1, and R2 were introduced to
investigate their effects on activity of the target compounds. Accordingly, we designed a
novel series of 7-substituted-pyrrolo[2,3-b]pyridine derivatives bearing dihydropyridazine
moiety (Fig. 3).
(Figure 2. should listed here)
(Figure 3. should listed here)
2. Chemistry
2.1. Synthesis of 3-substituted-4-((1-substituted-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline
The
synthesis
of
the
key
intermediates
of
3-substituted-4-((1-substituted-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline was achieved from
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the
commercially
available
4-chloro-1H-pyrrolo[2,3-b]pyridine
or
4-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridine as shown in Scheme 1 [23–24].
(Scheme 1. should be listed here)
2.2. Synthesis of the target compounds of dihydropyridazine-based 7-azaindole
The target compounds 20–51 were prepared as illustrated in Scheme 2. Using substituted
aniline 14a–l as starting material, diazo-reaction was proceed to afford the intermediate 15a–l
in high yield, which was condensed with ethyl acetylacetate to get intermediates 16a–l. The
cyclization was proceed to convert 16a–l to the corresponding pyridazinone 17a–l in the
presence of (ethoxycarbonylmethylene)triphenylphosphorane under basic conditions [25].
Simple procedures such as hydrolysis and acyl chlorination were used to convert ethyl
4-methyl-6-oxo-1-substitutedphenyl-1,6-dihydropyridazine-3-carboxylate 17a–l to the
corresponding acyl chloride 19a–l; the reactions proceeded with 20% H2SO4 and thionyl
chloride, respectively. Reaction of anilines 13a–d with acyl chloride 19a–l promoted by
DIPEA in dichloromethane at room temperature yielded the target compounds 20–51 [26].
(Scheme 2. should be listed here)
3. Results and Discussion
3.1. In vitro enzymatic assays
The c-Met enzymatic assays of all synthesized 7-azaindole derivatives were evaluated
using homogeneous time-resolved fluorescence (HTRF) assay [27–28]. The results expressed
as the half-maximal inhibitory concentration (IC50) values presented in Table 1 using foretinib
as a positive control. The IC50 values are the average of three independent experiments.
As illustrated in Table 1, these novel 7-azaindole derivatives bearing dihydropyridazine
fragment were found to be active against c-Met kinase with IC50 values ranging from
1.06–56.25 nM; 5 of them (23, IC50 = 8.42 nM; 32, IC50 = 5.02 nM; 33, IC50 = 5.61 nM; 34,
IC50 = 1.06 nM; 34, IC50 = 9.21 nM) showed high potency with IC50 values in single-digit
nanomole range, which indicated that it’s a good design strategy to use 7-azaindole as moiety
A and introduce dihydropyridazine fragment to moiety C to form the 5-atom linker.
According to the data shown in Table 1, enzymatic assays data showed no preference for
activity when the methyl was introduced to X group, indicating that the X group contributed
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little to the c-Met inhibitory efficacy. For example, the activity of compound 20 (IC50 = 18.26
nM; X = H, R1 = H, R2 = H) was comparable to compound 40 (IC50 = 19.54 nM; X = CH3, R1
= H, R2 = H), and the same trend was observed in compounds 21/41, 22/42, 23/43, and so on.
The results suggested that the introduction of fluoro atoms (R1 = F) to the aminophenoxy
part of target compounds is benefit for the activity, which indicated that the
electron-withdrawing groups (EWGs, such as F) on moiety B benefited to the potency. That’s
why compounds 32–39 showed the best activity among these target compounds. For example,
the activity of compound 34 (IC50 = 1.06 nM; X = H, R1 = F, R2 = 4-F) was higher than
compound 23 (IC50 = 8.42 nM; X = H, R1 = H, R2 = 4-F), and the same trend was observed in
compounds 32/21, 33/22, 36/26, and so on.
Further investigations were performed to study the effect of different substituents on the
phenyl ring (moiety B) on potency. Introduction of mono-electron-withdrawing groups
(mono-EWGs, such as F and Br) on the phenyl ring showed good influences on the activity,
while incorporation of the mono-EWGs at 4-position of the phenyl showed a higher
preference. For example, compound 40 (R2 = H) showed a c-Met IC50 value 19.54 nM.
Introduction of mono-EDGs at 4-position of the phenyl (43, R2 = 4-F, IC50 = 9.21 nM,
increased 2.1-fold) increased the inhibitory efficacy to a greater extent than that of
mono-EDGs at 2-position and 3-position of the phenyl (41, R2 = 2-F, IC50 = 14.41 nM,
increased 1.4-fold; 42, R2 = 3-F, IC50 = 15.27 nM, increased 1.3-fold). However, the
introduction of strong EWGs to the phenyl ring (moiety B) led to an obvious decline in
efficiency, such as compound 24 (R2 = 2-CF3, IC50 = 39.52 nM) and 45 (R2 = 2-CF3, IC50 =
46.27 nM) caused the potency to be lowered by 2.2- and 2.4-fold, respectively. The
incorporation of double electron-withdrawing groups (double-EWGs) and mono-donating
groups (mono-EDGs) showed negative trend in potency of the compounds, such as 29 (R2 =
3,4-(Cl)2, IC50 = 56.25 nM), 30 (R2 = 3-Cl-4-F, IC50 = 42.15 nM), 27 (R2 = 4-OCH3, IC50 =
46.91 nM), and so on. Therefore, this could be demonstrated that mono-EWGs (the halogen
atoms) had a positive effect on c-Met kinase activity, but strong-EWGs, double-EWGs, and
EDGs showed negative results.
(Table 1. should be listed here)
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3.2. In vitro cytotoxic activities
Using foretinib as the positive control, the cytotoxic activities of all target compounds
20–51 have been evaluated against the HT-29 and A549 cell lines using the MTT assay [26,
29–30]. Some potent compounds were further evaluated against the H460 and U87MG cell
lines. The results expressed as IC50 values are shown in Table 2 as the mean values are the
average of three independent experiments.
The data listed in Table 2 revealed that compounds 20–51 possessed moderate to strong
cytotoxicity against the four tested cell lines in the single-digit µM range, and 12 of them
were more potent than foretinib against one or more cell lines. The IC50 values of the most
promising compound 34 were 0.039, 0.046, 0.091, and 0.76 µM against the HT29, A549,
H460, and U87MG cell lines, respectively, indicating that this compound was 6.4-, 7.8-, 3.2-,
and 1.3-times more active than foretinib (IC50 values: 0.25, 0.36, 0.29, and 0.96 µM,
respectively). The cytotoxic activities of these analogs had similar structure-activity
relationships (SARs) with summarized in the c-Met kinase level: (a) the X group (X = H/CH3)
contributed little to the cytotoxic activities; (b) the EWGs (such as R1 = F) on moiety B
benefited to the potency; (c) the number and electronic property of substituents (R2 group) on
moiety D were key factors in improving the cytotoxic activities; (d) mono-EWGs (such as R2
= F) at 4-position of moiety D showed a higher preference for the potency.
(Table 2. should be listed here)
3.3. Enzymatic selectivity assays
In order to examine whether compound 34 is a selective c-Met inhibitor, it was screened
against other 5 tyrosine kinases. As shown in Table 3, compound 34 also exhibited high
inhibitory effects against Flt-3 (IC50 = 1.32 nM) and PDGFR-β (IC50 = 3.26 nM). Moreover,
compound 34 exhibited inhibitory effects against c-Kit, VEGFR-2, and EGFR even though
the potency was 300.8-, 633.8-, and 684.2-fold lower than that of c-Met. These data indicated
that compound 34 is a promising multitarget inhibitor of tyrosine kinases.
(Table 3. should be listed here)
4. Binding model analysis
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To further elucidate the binding mode of compounds, docking analysis was performed.
The three-dimensional structure of the c-Met (PDB code: 3LQ8) were obtained from RCSB
Protein Data Bank. The docking simulation was conducted using SURFLEX-DOCK module
of SYBYL 8.1 package version. The binding model was exemplified by the interaction of
compound 34 with c-Met. As shown in Fig. 4, the pyrrolo[2,3-b]pyridine fragment formed
two hydrogen bonds with MET1160. In the 5-atom linker, the oxygen atom of the amide and
nitrogen atom of 1,6-dihydropyridazine in 2-position formed two hydrogen bonds with
LYS1110 and ASP1222, respectively. Therefore, compound 34 formed four
hydrogen-bonding interactions with c-Met.
(Figure 4. should listed here)
5. Conclusions
In summary, a series of 7-azaindole derivatives bearing the dihydropyridazine scaffold
were designed and synthesized. c-Met kinase and 4 human cancer cell lines (HT29, A549,
H460, and U87MG) were used to evaluate the potency of the synthesized compounds.
Compared with foretinib, 1 of the derivatives showed higher c-Met kinase inhibitory activity,
and 12 were more potent against one or more cell lines. Compound 34 (c-Met IC50 = 1.39 nM,
a multitarget tyrosine kinase inhibitor) showed the strongest cytotoxic activities against
HT-29, A549, and H460 cell lines; which was 6.4, 7.8, and 3.2 times more active than
foretinib against the 3 cell lines, respectively. Analysis of SARs indicated that X group (X =
H/CH3) contributed little to the c-Met and cytotoxic activities, and the EWGs (such as R1 = F)
on moiety B benefited to the potency. The number, position, and electrical properties of the
substituents on the phenyl ring (moiety D) have significantly influence on the potency, and
mono-EWGs (such as R2 = F) at 4-position of the phenyl ring showed a higher preference.
Further studies on SARs and the mechanism of action of these compounds are underway in
our laboratory.
6. Experimental
6.1. Chemistry
All the materials were obtained from commercial suppliers and used without purification,
unless otherwise specified. TLC analysis was carried out on silica gel plates GF254 (Qindao
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Haiyang Chemical, China). All melting points were obtained on a Büchi Melting Point B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. Yields were not
optimized. Elemental analysis was determined on a Carlo-Erba 1106 Elemental analysis
instrument (Carlo Erba, Milan, Italy). Mass spectra (MS) were taken in ESI mode on Agilent
1100 LC-MS (Agilent, Palo Alto, CA, USA). NMR spectra were performed using Bruker 400
MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal
standard.
6.2. General procedure for the key intermediates 13a–d
4-chloro-1H-pyrrolo[2,3-b]pyridine
9
(9.1
g,
60
mmol)
or
4-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridine 10 (10.0 g, 60 mmol) reacted with
4-nitrophenol 11a (12.5 g, 90 mmol) or 2-fluoro-4-nitrophenol 11b (14.1 g, 90 mmol) in
diphenyl ether at 190°C for 1 h respectively. The mixture was cooled to 60°C and poured into
ethyl acetate (350 mL) in an ice bath for 30–50 min. The mixture was filtered off and dried to
give light yellow solid 12a–d.
An appropriate nitro compound 12a–d (30 mmol) was refluxed with hydrazine hydrate
(400 mmol), Ferric chloride (15 mmol) in ethanol (150 mL) and monitored by TLC. The
reaction solution was cooled to room temperature. The mixture was filtered, and the filtrate
was concentrated in vacuum, after that water was added and stirred at room temperature for
30 min. And then, the yellow solid was filtered off and dried to yield corresponding anilines
13a–d.
6.2.1. 4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline (13a)
1
Light yellow solid; Yield: 55%; M.p.: 182–185°C; H NMR (400 MHz, DMSO-d6) δ
11.61 (s, 1H), 8.01 (d, J = 5.3 Hz, 1H), 7.30 (d, J = 2.3 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.63
(d, J = 8.5 Hz, 2H), 6.28 (d, J = 5.4 Hz, 1H), 6.21 (s, 1H), 5.08 (s, 2H); MS (ESI) m/z(%):
226.5 [M+H]+.
6.2.2. 3-fluoro-4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline (13b)
1
Light yellow solid; Yield: 49%; M.p.: 184–188°C; H NMR (400 MHz, DMSO-d6) δ
11.67 (s, 1H), 8.03 (d, J = 5.3 Hz, 1H), 7.33 (s, 1H), 7.02 (t, J = 9.0 Hz, 1H), 6.52 (d, J = 13.3
Hz, 1H), 6.44 (d, J = 8.6 Hz, 1H), 6.29 (d, J = 5.5 Hz, 1H), 6.24 (s, 1H), 5.42 (s, 2H); MS
(ESI) m/z(%): 244.2 [M+H]+.
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6.2.3. 4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline (13c)
1
Light yellow solid; Yield: 53%; M.p.: 188–191°C; H NMR (400 MHz, DMSO-d6) δ
8.01 (d, J = 5.3 Hz, 1H), 7.31 (d, J = 2.5 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 6.62 (d, J = 8.6 Hz,
2H), 6.26 (d, J = 5.3 Hz, 1H), 6.23 (m, 1H), 5.10 (s, 2H), 3.96 (s, 3H); MS (ESI) m/z(%):
240.8 [M+H]+.
6.2.4. 3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline (13d)
Light yellow solid; Yield: 56%; M.p.: 192–195°C; 1H NMR (DMSO-d6) 8.02 (m, 1H),
7.32 (s, 1H), 7.01 (t, J = 9.0, 1H), 6.51 (dd, J = 13.2, 1H), 6.43 (dd, J = 8.6, 1H), 6.28 (d, J =
5.4 Hz, 1H), 6.24 (m, 1H), 5.42 (s, 2H), 4.09 (s, 3H); MS (ESI) m/z(%): 258.7 [M+H]+.
6.3. General procedure for the preparation of compounds 22–51
NaNO2 (8 g, 0.115 mol) in H2O (260 mL) was added dropwise to a mixture of
substituted phenyl amine 14a–l (0.1 mol) and 15% HCl (90 mL) at 0°C. The reaction mixture
was stirred at 0°C for 40 min, and then added into a mixture of anhydrous sodium acetate
(22.5 g, 0.29 mol), ethyl acetylacetate (13 g, 0.1 mol), and EtOH (300 mL) at 0°C. The
mixture was stirred, filtered, and dried to afford light yellow solids (16a–l).
Carbethoxymethylene)triphenylphosphorane (8.40 g, 24 mmol) and appropriate
intermediate 16a–l (20.0 mmol) and were dissolved into 80 mL of dried toluene and refluxed
for 18 h. The solvent was concentrated in vacuum, and the solid was dissolved and
recrystallized using EtOH to afford 17a–l as white solids.
A mixture of 17a–l (15 mmol) and 20%H2SO4 (120 mL) was heated at 100°C for 5–10 h
and monitored by TLC. The reaction mixture was poured onto ice/water with vigorously
stirring and the precipitated was collected by filtration and dried to give 18a–l as light yellow
solids, which was added to thionyl chloride (50 mL) and refluxed for 6–8 h. The reaction
mixture was evaporated to yield the corresponding carbonyl chloride 19a–l in 80-85% yields,
and used for the next step immediately without further purification.
To a solution of an appropriate aniline 13a–d (2 mmol) and diisopropylethylamine (2.4
mmol) in dichloromethane (40 mL), an appropriate carbonyl chloride 19a–l (4 mmol)
dissolved in dried dichloromethane (20 mL) was added drop-wise in an ice bath. And then,
the reaction was removed to room temperature for 10–15 h and monitored by TLC. The
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mixture was washed with 10% K2CO3 (20 mL × 3) followed by brine (20 mL × 1), and the
organic phase was separated, dried, and concentrated in vacuum. The crude product was
purified by chromatography on silica gel using MeOH/CH2Cl2 to afford the target compounds
20–51 as white solids.
6.3.1
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-phenyl-1,6-dihy
dropyridazine-3-carboxamide (20)
1
Yield: 52%; M.p.: 145–146°C; H NMR (400 MHz, DMSO-d6) δ 12.22(s,1H), 11.56 (s,
1H), 9.04 (s, 1H), 8.26 (s, 1H), 7.79 (d, J = 6.8 Hz, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.56 (d, J =
7.0 Hz, 2H), 7.40 (s, 2H), 7.20 (d, J = 8.8 Hz, 2H), 6.94 (s, 1H), 6.66 (s, 1H), 6.61 (s, 1H),
2.68 (s, 3H); MS m/z (ESI): 438.2 [M+H]+; Anal. calcd. for C25H19N5O3 (%): C, 68.64; H,
4.38; N, 16.01; Found (%): C, 68.63; H, 4.36; N, 16.02.
6.3.2
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(2-fluorophenyl)-4-methyl-6-oxo
-1,6-dihydropyridazine-3-carboxamide (21)
Yield: 59%; M.p.: 148–149°C; 1H NMR (400 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.94 (s,
1H), 8.09 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.55–7.49 (m, 2H), 7.46 (d, J = 6.9 Hz,
1H), 7.35 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 6.93 (s, 1H), 6.69 (d, J = 8.5 Hz, 1H),
6.48 (s, 1H), 2.61 (s, 3H); MS m/z (ESI): 456.1 [M+H]+; Anal. calcd. for C25H18FN5O3 (%): C,
65.93; H, 3.98; N, 15.38; Found (%): C, 65.92; H, 3.99; N, 15.36.
6.3.3
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(3-fluorophenyl)-4-methyl-6-oxo
-1,6-dihydropyridazine-3-carboxamide (22)
Yield: 55%; M.p.: 146–147°C; 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 11.56 (s,
1H), 8.12 (s, 1H), 7.80 (d, J = 6.9 Hz, 2H), 7.48 (d, J = 6.6 Hz, 2H), 7.43 (d, J = 5.8 Hz, 2H),
7.37 (s, 1H), 7.19 (d, J = 5.0 Hz, 2H), 6.91 (s, 1H), 6.69 (d, J = 8.5 Hz, 1H), 6.51 (s, 1H), 2.58
13
(s, 3H); C NMR (100 MHz, DMSO-d6) δ 161.90, 158.92, 157.64, 151.57, 151.33, 144.61,
143.32, 142.34, 135.54, 130.66, 130.21, 125.07, 122.25 (2C), 122.12 , 121.16 (2C), 115.59,
115.38, 113.56, 113.30, 110.54, 102.61, 97.54, 18.59; MS m/z (ESI): 456.1 [M+H]+; Anal.
calcd. for C25H18FN5O3 (%): C, 65.93; H, 3.98; N, 15.38; Found (%): C, 65.91; H, 3.99; N,
15.37.
6.3.4
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-4-methyl-6-oxo
-1,6-dihydropyridazine-3-carboxamide (23)
Yield: 56%; M.p.: 144–145°C; 1H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H), 11.64 (s,
1H), 8.97 (s, 1H), 7.82 (d, J = 10.4 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 7.54 (d, J = 6.1 Hz, 2H),
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7.08 (t, J = 8.3 Hz, 2H), 6.94 (s, 1H), 6.87 (s, 1H), 6.67 (s, 1H), 6.62 (s, 1H), 2.51 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 162.22, 159.02, 157.47, 155.10, 151.55, 144.61, 143.27,
141.69, 130.13, 128.32 (2C), 128.23 (2C), 125.30, 124.12, 117.21, 115.94 (2C), 115.72 (2C),
109.84, 109.37, 101.30, 97.15, 18.61; MS m/z (ESI): 456.1 [M+H]+; Anal. calcd. for
C25H18FN5O3 (%): C, 65.93; H, 3.98; N, 15.38; Found (%): C, 65.92; H, 3.97; N, 15.39.
6.3.5
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromet
hyl)phenyl)-1,6-dihydropyridazine-3-carboxamide (24)
Yield: 57%; M.p.: 150–151°C; 1H NMR (400 MHz, DMSO-d6) δ 12.69 (s, 1H), 12.10 (s,
1H), 8.12 (d, J = 12.8 Hz, 1H), 7.97 (d, J = 7.9 Hz, 2H), 7.88 (d, J = 7.5 Hz, 2H), 7.80 (s, 1H),
7.74 (d, J = 5.6 Hz, 2H), 7.37 (s, 1H), 7.22 (d, J = 7.7 Hz, 2H), 6.72 (d, J = 5.8 Hz, 1H), 6.50
(s, 1H), 2.27 (s, 3H); MS m/z (ESI): 506.1 [M+H]+; Anal. calcd. for C26H18F3N5O3 (%): C,
61.78; H, 3.59; N, 13.86; Found (%): C, 61.76; H, 3.58; N, 13.88.
6.3.6
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(3-(trifluoromet
hyl)phenyl)-1,6-dihydropyridazine-3-carboxamide (25)
Yield: 58%; M.p.: 148–149°C; 1H NMR (400 MHz, DMSO-d6) δ 12.21 (s, 1H), 8.95 (s,
1H), 8.11 (d, J = 6.9 Hz, 1H), 7.91 (d, J = 12.6 Hz, 2H), 7.78 (d, J = 11.9 Hz, 2H), 7.73–7.65
(m, 2H), 7.39 (d, J = 7.3 Hz, 1H), 7.24 (d, J = 5.6 Hz, 2H), 6.97 (s, 1H), 6.70 (d, J = 9.5 Hz,
1H), 6.50 (s, 1H), 2.69 (s, 3H); MS m/z (ESI): 506.1 [M+H]+; Anal. calcd. for C26H18F3N5O3
(%): C, 61.78; H, 3.59; N, 13.86; Found (%): C, 61.77; H, 3.59; N, 13.87.
6.3.7
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(o-tolyl)-1,6-dih
ydropyridazine-3-carboxamide (26)
Yield: 52%; M.p.: 143–144°C; 1H NMR (400 MHz, DMSO-d6) δ 12.20 (s, 1H), 11.64 (s,
1H), 8.23 (d, J = 6.3 Hz, 1H), 7.71 (d, J = 6.7 Hz, 2H), 7.48–7.37 (m, 3H), 7.32 (d, J = 6.7 Hz,
2H), 7.14 (s, 1H), 6.99 (s, 1H), 6.95 (s, 1H), 6.71 (d, J = 4.6 Hz, 1H), 6.61 (s, 1H), 2.70 (s,
3H), 2.22 (s, 3H); MS m/z (ESI): 452.2[M+H]+; Anal. calcd. for C26H21N5O3 (%): C, 69.17; H,
4.69; N, 15.51; Found (%): C, 69.16; H, 4.68; N, 15.52.
6.3.8
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-methoxyphenyl)-4-methyl-6-o
xo-1,6-dihydropyridazine-3-carboxamide (27)
Yield: 53%; M.p.: 142–143°C; 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.64 (s,
1H), 8.13 (d, J = 5.4 Hz, 1H), 7.85 (d, J = 9.7 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.71–7.63 (m,
3H), 7.41 (d, J = 3.3 Hz, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.10 (s, 1H), 6.45 (d, J = 5.4 Hz, 1H),
6.18 (d, J = 3.4 Hz, 1H), 3.80 (s, 3H), 2.40 (s, 3H);. MS m/z (ESI): 468.2 [M+H]+; Anal. calcd.
10

ACCEPTED MANUSCRIPT
for C26H21N5O4 (%): C, 66.80; H, 4.53; N, 14.98; Found (%): C, 66.81; H, 4.54; N, 14.97.
6.3.9
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(2,5-dimethylphenyl)-4-methyl-6
-oxo-1,6-dihydropyridazine-3-carboxamide (28)
Yield: 55%; M.p.: 146–147°C;1H NMR (400 MHz, DMSO-d6) δ 12.65 (s, 1H), 10.55 (s,
1H), 8.13 (d, J = 5.4 Hz, 1H), 7.78 (d, J = 8.9 Hz, 2H), 7.41 (d, J = 3.4 Hz, 1H), 7.28 (d, J =
7.6 Hz, 1H), 7.23 (s, 1H), 7.21 (m, 2H), 7.19 (s, 1H), 7.06 (s, 1H), 6.46 (d, J = 5.4 Hz, 1H),
6.19 (d, J = 3.4 Hz, 1H), 2.41 (s, 3H), 2.34 (s, 3H), 2.07 (s, 3H); MS m/z (ESI): 466.2 [M+H]+;
Anal. calcd. for C27H23N5O3 (%): C, 69.66; H, 4.98; N, 15.04; Found (%): C, 69.65; H, 4.97;
N, 15.05.
6.3.10 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(2,4-dichlorophenyl)-4-methyl-6
-oxo-1,6-dihydropyridazine-3-carboxamide (29)
Yield: 57%; M.p.: 153–154°C; 1H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H), 11.64 (s,
1H), 8.25 (s, 1H), 7.78 (d, J = 7.5 Hz, 2H), 7.63 (s, 1H), 7.46–7.38 (m, 2H), 7.21 (d, J = 7.2
Hz, 2H), 7.17 (s, 1H), 6.95 (s, 1H), 6.65 (d, J = 6.7 Hz, 1H), 6.61 (s, 1H), 2.70 (s, 3H); MS
m/z (ESI): 507.1 [M+H]+; Anal. calcd. for C25H17Cl2N5O3 (%): C, 59.30; H, 3.38; N, 13.83;
Found (%): C, 59.28; H, 3.37; N, 13.84.
6.3.11 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(3-chloro-4-fluorophenyl)-4-met
hyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (30)
Yield: 58%; M.p.: 151–152°C; 1H NMR (400MHz, DMSO-d6) δ 12.57 (s, 1H), 11.64 (s,
1H), 8.24 (d, J = 5.8 Hz, 1H), 8.17 (dd, J = 6.3, 2.7 Hz, 1H), 7.90 (d, J = 5.2 Hz, 1H), 7.86 (d,
J = 8.9 Hz, 2H), 7.73 (t, J = 9.0 Hz, 2H), 7.51 (d, J = 3.3 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H),
6.60 (d, J = 5.7 Hz, 1H), 6.34 (d, J = 3.3 Hz, 1H), 2.13 (s, 3H); MS m/z (ESI): 491.1 [M+H]+;
Anal. calcd. for C25H17ClFN5O3 (%): C, 61.29; H, 3.50; N, 14.30; Found (%): C, 61.28; H,
3.49; N, 14.31.
6.3.12 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-bromo-2-fluorophenyl)-4-met
hyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (31)
Yield: 55%; M.p.: 153–154°C; 1H NMR (400 MHz, DMSO-d6) δ 12.20 (s, 1H), 11.64 (s,
1H), 8.89 (m, 1H), 8.11 (d, J = 13.4 Hz, 1H), 7.78 (d, J = 7.9 Hz, 2H), 7.50–7.43 (m, 3H),
7.36–7.33 (m, 2H), 7.22 (d, J = 5.9 Hz, 2H), 6.92 (s, 1H), 6.69 (m, 1H), 6.49 (s, 1H), 2.60 (s,
3H); MS m/z (ESI): 535.0 [M+H]+; Anal. calcd. for C26H19BrFN5O3 (%): C, 56.19; H, 3.21; N,
13.11; Found (%):C, 56.18; H, 3.21; N, 13.12.
6.3.13 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(2-fluorophenyl)-4-met
11

ACCEPTED MANUSCRIPT
hyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (32)
Yield: 56%; M.p.: 148–149°C; 1H NMR (400 MHz, DMSO-d6) δ 12.55 (s, 1H), 12.13 (s,
1H), 8.75 (s, 1H), 8.14 (s, 1H), 8.00 (d, J = 12.4 Hz, 1H), 7.55–7.52 (m, 4H), 7.50 (s, 1H),
7.45–7.39 (m, 2H), 6.55 (d, J = 8.5 Hz, 1H), 6.38 (s, 1H), 2.20 (s, 3H); MS m/z (ESI):474.1
[M+H]+; Anal. calcd. for C25H17F2N5O3 (%): C, 63.42; H, 3.62; N, 14.79; Found (%): C,
63.41; H, 3.61; N, 14.81.
6.3.14 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(3-fluorophenyl)-4-met
hyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (33)
Yield: 51%; M.p.: 150–151°C; 1H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 10.90 (s,
1H), 8.21 (d, J = 5.7 Hz, 1H), 8.11 (d, J = 3.6 Hz, 1H), 8.00 (d, J = 12.8 Hz, 1H), 7.75 (d, J =
11.7 Hz, 1H), 7.57–7.43 (m, 3H), 7.34 (d, J = 8.2 Hz, 2H), 7.15 (s, 1H), 6.91 (d, J = 4.2 Hz,
1H), 6.70 (d, J = 5.4 Hz, 1H), 2.47 (s, 3H); MS m/z (ESI): 474.1 [M+H]+; Anal. calcd. for
C25H17F2N5O3 (%): C, 63.42; H, 3.62; N, 14.79; Found (%): C, 63.41; H, 3.61; N, 14.80.
6.3.15 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl)-4-met
hyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (34)
Yield: 55%; M.p.: 147–148°C; 1H NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 10.80 (s,
1H), 8.07 (d, J = 5.2 Hz, 1H), 7.90 (d, J = 12.7 Hz, 1H), 7.59 (s, 1H), 7.44–7.31 (m, 5H), 7.07
(s, 1H), 6.92 (s, 1H), 6.39 (d, J = 5.2 Hz, 1H), 6.24 (s, 1H), 2.41 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 163.02, 162.22, 160.59, 159.11, 157.55, 151.59, 144.68, 143.36, 141.78, 137.17,
130.21, 128.42 (2C), 128.30 (2C), 125.38, 124.23, 117.27, 116.06, 115.82, 109.36, 109.17,
101.29, 97.21, 18.71; MS m/z (ESI): 474.1 [M+H]+; Anal. calcd. for C25H17F2N5O3 (%): C,
63.42; H, 3.62; N, 14.79; Found (%): C, 63.41; H, 3.60; N, 14.81.
6.3.16 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-4-methyl-6-oxo-1-(2-(trifl
uoromethyl)phenyl)-1,6-dihydropyridazine-3-carboxamide (35)
Yield: 53%; M.p.: 149–150°C; 1H NMR (400 MHz, DMSO-d6) δ 12.83 (s, 1H), 12.16 (s,
1H), 8.12–8.04 (m, 3H), 7.89 (d, J = 7.6 Hz, 1H), 7.80 (s, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.63
(d, J = 10.6 Hz, 1H), 7.54 (d, J = 9.4 Hz, 1H), 7.40 (s, 1H), 7.31 (d, J = 8.3 Hz, 1H), 6.72 (d, J
13
= 5.7 Hz, 1H), 6.57 (s, 1H), 2.26 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 170.11, 160.07,
157.45, 155.25, 152.79, 151.55, 144.63, 143.98, 143.09, 141.14, 137.63, 136.90, 134.73,
131.61, 131.06, 129.34, 128.11, 125.31, 124.29, 116.95, 109.85, 109.21, 108.99, 101.31,
97.16, 13.09; MS m/z (ESI): 524.1 [M+H]+; Anal. calcd. for C26H17F4N5O3 (%): C, 59.66; H,
12

ACCEPTED MANUSCRIPT
3.27; N, 13.38; Found (%): C, 59.64; H, 3.26; N, 13.39.
6.3.17 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-4-methyl-6-oxo-1-(o-tolyl
)-1,6-dihydropyridazine-3-carboxamide (36)
Yield: 59%; M.p.: 146–147°C; 1H NMR (400 MHz, DMSO-d6) δ 12.50 (s, 1H), 12.16 (s,
1H), 8.79 (s, 1H), 8.17 (s, 1H), 8.00 (d, J = 12.4 Hz, 1H), 7.59–7.52 (m, 4H), 7.50 (s, 1H),
7.47–7.42 (m, 2H), 6.53 (d, J = 8.5 Hz, 1H), 6.35 (s, 1H), 2.24 (s, 3H), 2.20 (s, 3H); MS m/z
(ESI): 470.2 [M+H]+; Anal. calcd. for C26H20FN5O3 (%): C, 66.52; H, 4.29; N, 14.92; Found
(%): C, 66.50; H, 4.29; N, 14.94.
6.3.18 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-methoxyphenyl)-4-m
ethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (37)
Yield: 54%; M.p.: 145–146°C; 1H NMR (400 MHz, DMSO-d6) δ 12.83 (s, 1H), 10.80 (s,
1H), 8.16 (d, J = 5.5 Hz, 1H), 8.04 (d, J = 4.1 Hz, 1H), 7.95 (d, J = 12.6 Hz, 1H), 7.63 (d, J =
8.2 Hz, 2H), 7.31 (d, J = 8.7 Hz, 1H), 7.12–7.06 (m, 3H), 6.97 (d, J = 8.8 Hz, 1H), 6.84 (d, J
= 4.0 Hz, 1H), 6.64 (d, J = 5.6 Hz, 1H), 3.76 (s, 3H), 2.41 (s, 3H); MS m/z (ESI): 486.2
[M+H]+; Anal. calcd. for C26H20FN5O4 (%): C, 64.33; H, 4.15; N, 14.43; Found (%): C, 64.32;
H, 4.14; N, 14.45.
6.3.19 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (38)
Yield: 58%; M.p.: 148–149°C; 1H NMR (400 MHz, DMSO-d6) δ 12.43 (s, 1H), 11.15 (s,
1H), 8.95 (s, 1H), 8.15 (s, 1H), 7.93 (d, J = 9.7 Hz, 1H), 7.54–7.48 (m, 3H), 7.40 (d, J = 3.5
Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 6.92 (s, 1H), 6.65 (s, 1H), 6.45 (s, 1H), 2.64 (s, 3H); MS
m/z (ESI): 524.1 [M+H]+; Anal. calcd. for C25H16Cl2FN5O3 (%): C, 57.27; H, 3.08; N, 13.36;
Found (%): C, 57.25; H, 3.07; N, 13.35.
6.3.20 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-bromo-2-fluorophen
yl)-4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (39)
Yield: 51%; M.p.: 147–148°C; 1H NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H), 11.10 (s,
1H), 8.98 (s, 1H), 8.15 (s, 1H), 7.90 (d, J = 9.7 Hz, 1H), 7.52–7.46 (m, 3H), 7.37 (d, J = 3.5
Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 6.95 (s, 1H), 6.68 (s, 1H), 6.55 (s, 1H), 2.68 (s, 3H); MS
m/z (ESI): 552.0 [M+H]+; Anal. calcd. for C25H16BrF2N5O3 (%): C, 54.36; H, 2.92; N, 12.68;
Found (%): C, 54.34; H, 2.91; N, 12.67.
6.3.21 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-phenyl
-1,6-dihydropyridazine-3-carboxamide (40)
13

ACCEPTED MANUSCRIPT
Yield: 54%; M.p.: 162–163°C; 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 9.04 (s,
1H), 8.26 (s, 1H), 7.79 (d, J = 6.8 Hz, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 7.0 Hz, 2H),
7.40 (s, 1H), 7.22 (br, 2H), 7.20 (s, 1H), 6.94 (s, 1H), 6.66 (s, 1H), 6.61 (s, 1H), 4.30 (s, 3H),
2.68 (s, 3H); MS m/z (ESI): 452.2 [M+H]+; Anal. calcd. for C26H21N5O3 (%): C, 69.17; H,
4.69; N, 15.51; Found (%): C, 69.16; H, 4.67; N, 15.50.
6.3.22 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(o-toly
l)-1,6-dihydropyridazine-3-carboxamide (41)
Yield: 57%; M.p.: 155–156°C; 1H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 8.80 (s,
1H), 8.14 (d, J = 5.3 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 7.9 Hz, 1H), 7.51 (d, J =
6.6 Hz, 2H), 7.47 (d, J = 7.3 Hz, 1H), 7.42 (d, J = 3.3 Hz, 1H), 7.23 (d, J = 8.7 Hz, 2H), 6.49
(d, J = 5.4 Hz, 1H), 6.22 (d, J = 3.3 Hz, 1H), 3.82 (s, 3H), 2.24 (s, 3H), 2.12 (s, 3H); MS m/z
(ESI): 466.2 [M+H]+; Anal. calcd. for C27H23N5O3 (%): C, 69.66; H, 4.98; N, 15.04; Found
(%): C, 69.65; H, 4.97; N, 15.05.
6.3.23 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(3-fluorophenyl)-4-met
hyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (42)
Yield: 54%; M.p.: 161–162°C; 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.05 (d,
J = 8.9 Hz, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 6.7 Hz, 2H),
7.32 (d, J = 2.6 Hz, 1H), 7.13 (d, J = 9.2 Hz, 2H), 7.00 (s, 1H), 6.38 (d, J = 5.0 Hz, 1H), 6.11
13
(d, J = 3.5 Hz, 1H), 3.73 (s, 3H), 2.32 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 161.89,
158.91, 157.74, 151.27, 150.50, 144.51, 143.31, 142.27, 135.58, 130.57, 130.23, 129.05,
122.25 (2C), 122.10, 121.11 (2C), 115.57, 115.37, 113.54, 113.28, 110.77, 102.79, 96.60,
31.55, 18.59; MS m/z (ESI): 470.2 [M+H]+; Anal. calcd. For C26H20FN5O3 (%): C, 66.52; H,
4.29; N, 14.92; Found (%): C, 66.51; H, 4.28; N, 14.93.
6.3.24 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-4-met
hyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (43)
Yield: 59%; M.p.: 159–160°C; 1H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H), 8.97 (s,
1H), 7.82 (d, J = 10.4 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 7.54 (d, J = 6.1 Hz, 2H), 7.08 (t, J =
8.3 Hz, 2H), 6.94 (s, 1H), 6.87 (s, 1H), 6.67 (s, 1H), 6.62 (s, 1H), 4.34 (s, 3H), 2.51 (s, 3H);
MS m/z (ESI): 470.2 [M+H]+; Anal. calcd. for C26H20FN5O3 (%): C, 66.52; H, 4.29; N, 14.92;
Found (%): C, 66.51; H, 4.29; N, 14.93.
6.3.25 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(3-bromophenyl)-4-me
14

ACCEPTED MANUSCRIPT
thyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (44)
Yield: 55%; M.p.: 154–155°C; 1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 8.13 (d,
J = 5.2 Hz, 1H), 8.01 (s, 1H), 7.80 (d, J = 8.7 Hz, 2H), 7.76 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H),
7.50 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 2.3 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 7.08 (s, 1H), 6.47
(d, J = 5.3 Hz, 1H), 6.20 (d, J = 2.1 Hz, 1H), 3.81 (s, 3H), 2.40 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 161.95, 159.00, 157.80, 151.34, 150.57, 144.57, 143.43, 142.42, 142.37, 135.66,
131.55, 130.98, 130.24, 129.12, 128.79, 125.25, 122.29 (2C), 121.49, 121.19 (2C), 110.80,
102.85, 96.67, 31.62, 18.67; MS m/z (ESI): 530.1 [M+H]+; Anal. calcd. for C26H20BrN5O3
(%): C, 58.88; H, 3.80; N, 13.20; Found (%): C, 58.87; H, 3.81; N, 13.19.
6.3.26 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(2-(trifluoromethyl)ph
enyl)-4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (45)
Yield: 53%; M.p.: 158–159°C; 1H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H), 8.78 (s,
1H), 8.10 (d, J = 5.6 Hz, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 7.3 Hz, 1H), 7.83 (d, J =
9.4 Hz, 2H), 7.73 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 3.3 Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 6.46
(d, J = 5.6 Hz, 1H), 6.18 (d, J = 3.4 Hz, 1H), 3.75 (s, 3H), 2.01 (s, 3H); MS m/z (ESI): 520.2
[M+H]+; Anal. calcd. for C27H20F3N5O3 (%): C, 62.43; H, 3.88; N, 13.48; Found (%): C,
62.42; H, 3.87; N, 13.49.
6.3.27 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(3-(trifluoromethyl)ph
enyl)-4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (46)
Yield: 52%; M.p.: 156–157°C; 1H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H) , 8.88 (s,
1H), 8.23 (s, 1H), 7.91 (d, J = 15.9 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 4.5 Hz, 2H),
7.16 (d, J = 8.5 Hz, 2H), 6.95 (s, 1H), 6.56 (s, 1H), 6.46 (s, 1H), 4.08 (s, 3H), 2.69 (s, 3H);
MS m/z (ESI): 520.2 [M+H]+; Anal. calcd. for C27H20F3N5O3 (%): C, 62.43; H, 3.88; N, 13.48;
Found (%): C, 62.42; H, 3.87; N, 13.49.
6.3.28 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(2,5-dimethylphenyl)-4
-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (47)
Yield: 56%; M.p.: 164–165°C; 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.13 (d,
J = 5.4 Hz, 1H), 7.78 (d, J = 8.9 Hz, 2H), 7.41 (d, J = 3.4 Hz, 1H), 7.28 (d, J = 7.6 Hz, 1H),
7.23 (s, 1H), 7.21 (d, J = 2.1 Hz, 2H), 7.19 (s, 1H), 7.06 (s, 1H), 6.46 (d, J = 5.4 Hz, 1H), 6.19
(d, J = 3.4 Hz, 1H), 3.81 (s, 3H), 2.41 (s, 3H), 2.34 (s, 3H), 2.07 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 162.14, 159.06, 157.78, 151.31, 150.57, 144.56, 143.38, 142.13, 140.51, 136.55,
15

ACCEPTED MANUSCRIPT
135.58, 131.90, 130.84, 130.13, 129.79, 129.12, 128.28, 122.41 (2C), 121.10 (2C), 110.82,
102.86, 96.66, 31.61, 20.77, 18.78, 17.26; MS m/z (ESI): 480.2 [M+H]+; Anal. calcd. for
C28H25N5O3 (%): C, 70.13; H, 5.26; N, 14.60; Found (%):C, 70.12; H, 5.25; N, 14.62.
6.3.29 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(2,4-dichlorophenyl)-4
-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (48)
Yield: 53%; M.p.: 167–168°C; 1H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H), 8.25 (s,
1H), 7.78 (d, J = 7.5 Hz, 2H), 7.63 (s, 1H), 7.46–7.38 (m, 2H), 7.21 (d, J = 7.2 Hz, 2H), 7.17
(s, 1H), 6.95 (s, 1H), 6.65 (d, J = 6.7 Hz, 1H), 6.61 (s, 1H), 4.30 (s, 3H), 2.70 (s, 3H); MS m/z
(ESI): 521.1 [M+H]+; Anal. calcd. for C26H19Cl2N5O3 (%): C, 60.01; H, 3.68; N, 13.46; Found
(%): C, 60.00; H, 3.67; N, 13.44.
6.3.30 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(3-chloro-4-fluorophe
nyl)-4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (49)
1
Yield: 57%; M.p.: 163–164°C; H NMR (400MHz, DMSO-d6) δ 12.07 (s, 1H), 9.12 (s,
1H), 8.24 (d, J = 5.8 Hz, 1H), 8.17 (dd, J = 6.3, 2.7 Hz, 1H), 7.90 (d, J = 5.2 Hz, 1H), 7.86 (d,
J = 8.9 Hz, 2H), 7.73 (t, J = 9.0 Hz, 1H), 7.51 (d, J = 3.3 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H),
6.60 (d, J = 5.7 Hz, 1H), 6.34 (d, J = 3.3 Hz, 1H), 3.88 (s, 3H), 2.13 (s, 3H); MS m/z (ESI):
504.1 [M+H]+; Anal. calcd. for C26H19ClFN5O3 (%): C, 61.97; H, 3.80; N, 13.90; Found (%):
C, 61.96; H, 3.78; N, 13.92.
6.3.31 N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-bromo-2-fluorophe
nyl)-4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (50)
Yield: 58%; M.p.: 158–159°C; 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.13 (d,
J = 5.4 Hz, 1H), 7.85 (d, J = 9.7 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.71-7.63 (m, 2H), 7.41 (d,
J = 3.3 Hz, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.10 (s, 1H), 6.45 (d, J = 5.4 Hz, 1H), 6.18 (d, J =
13
3.4 Hz, 1H), 3.80 (s, 3H), 2.40 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 161.68, 158.40,
157.69, 151.34, 150.53, 144.51, 143.95, 143.02, 135.49, 131.10, 129.48, 129.07, 128.79,
128.47, 123.03, 122.20 (2C), 121.06 (2C), 120.27, 120.05, 110.78, 102.84, 96.59, 31.55,
18.71; MS m/z (ESI): 549.1 [M+H]+; Anal. calcd. for C26H19BrFN5O3 (%): C, 56.95; H, 3.49;
N, 12.77; Found (%): C, 56.94; H, 3.48; N, 12.79.
6.3.32 N-(3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(3-(trifluorom
ethyl)phenyl)-4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (51)
Yield: 55%; M.p.: 155–156°C; 1H NMR (400 MHz, DMSO-d6) δ 12.82 (s, 1H), 8.29 (d,
J = 5.0 Hz, 1H), 8.08 (d, J = 12.1 Hz, 2H), 7.89 (d, J = 7.6 Hz, 1H), 7.84–7.72 (m, 2H), 7.63
16

ACCEPTED MANUSCRIPT
(d, J = 7.8 Hz, 1H), 7.54 (d, J = 7.0 Hz, 1H), 7.30 (s, 1H), 7.20 (d, J = 1.7 Hz, 1H), 6.71 (d, J
13
= 6.1 Hz, 1H), 6.65 (s, 1H), 4.31 (s, 3H), 2.26 (s, 3H); C NMR (100 MHz, DMSO-d6) δ
170.11, 160.06, 157.55, 155.19, 152.75, 150.45, 144.53, 143.94, 143.08, 141.12, 136.98,
134.72, 131.61, 131.07, 129.33, 129.27, 128.16, 128.09, 124.25, 116.96, 110.04, 109.21,
108.97, 101.49, 96.21, 31.57, 13.08; MS m/z (ESI): 538.1 [M+H]+; Anal. Calcd. for
C27H19F4N5O3 (%): C, 60.34; H, 3.56; N, 13.03; Found (%):C, 60.33; H, 3.54; N, 13.02.
6.4. Pharmacology
6.4.1. Tyrosine kinases assay
The tyrosine kinases activities were evaluated using homogeneous time-resolved
fluorescence (HTRF) assays, as previously reported protocol [26]. Briefly, 20 µg/mL poly
(Glu, Tyr) 4:1 (Sigma) was preloaded as a substrate in 384-well plates. Then 50 µL of 10 mM
ATP (Invitrogen) solution diluted in kinase reaction buffer (50 mM HEPES, pH 7.0, 1 M
DTT, 1 M MgCl2, 1 M MnCl2, and 0.1% NaN3) was added to each well. Various
concentrations of compounds diluted in 10 µL of 1% DMSO (v/v) were used as the negative
control. The kinase reaction was initiated by the addition of purified tyrosine kinase proteins
diluted in 39 µL of kinase reaction buffer solution. The incubation time for the reactions was
30 min at 25°C, and the reactions were stopped by the addition of 5 µL of Streptavidin-XL665
and 5 µL Tk Antibody Cryptate working solution to all of wells. The plates were read using
Envision (PerkinElmer) at 320 nm and 615 nm. The inhibition rate (%) was calculated using
the following equation: % inhibition = 100 - [(Activity of enzyme with tested compounds -
Min)/ (Max - Min)] × 100 (Max: the observed enzyme activity measured in the presence of
enzyme, substrates, and cofactors; Min: the observed enzyme activity in the presence of
substrates, cofactors and in the absence of enzyme). IC50 values were calculated from the
inhibition curves [26–28].
6.4.2. MTT assay in vitro
The anti-proliferative activities of compounds 20–51 were evaluated against HT-29,
H460, MKN-45, A549 and U87MG cell lines using the standard MTT assay in vitro, with
foretinib as the positive control, as previously reported protocol [26]. The cancer cell lines
were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine
17

ACCEPTED MANUSCRIPT
serum (FBS). Approximate 4 × 103 cells, suspended in MEM medium, were plated onto each
well of a 96-well plate and incubated in 5% CO2 at 37°C for 24 h. The compounds tested at
the indicated final concentrations were added to the culture medium and the cell cultures were
continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5 µg/mL,
and incubated with cells at 37°C for 4 h. The formazan crystals were dissolved in 100 mL of
DMSO each well, and the absorbency at 492 and 630 nm (for the reference wavelength) was
measured with an ELISA reader. All of the compounds were tested three times in each cell
line. The results expressed as IC50 (inhibitory concentration 50%) were the averages of three
determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner
(Bliss) software [29–30].
7. Acknowledgements
We gratefully acknowledge the generous support provided by National Natural Science
Foundation of China (NSFC No. 81660572), Science and Technology Project Founded by the
Education Department of Jiangxi Province (GJJ150797), Top-notch talent project of Jiangxi
Science & Technology Normal University (2016QNBJRC002), and Research Fund for the
Doctoral Program of Jiangxi Science and Technology Normal University (No. 3000990351).
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Legends
Fig. 1. The representative c-Met kinase inhibitors and the corresponding general structure.
Fig. 2. Anticancer agents bearing dihydropyridazine fragments.
Fig. 3. Design strategy for the 7-azaindole derivatives bearing dihydropyridazine moiety.
Fig. 4. Binding poses of compound 34 with c-Met. The proteins were displayed by grid ribbon. Compound
34 were displayed by multicolor sticks. H-bonding interactions between the 34 and c-Met were indicated
with dashed lines in black.
Scheme 1. Reagents and conditions: (i) Diphenyl Ether, 190°C, 50
Carbon, 80°C, 77 80%.
–55%; (ii) FeCl3, N2H4·H2O, Activated
–
Scheme 2. Reagents and conditions: (i) HCl, NaNO2, 0°C, 1 h, 90
CH3COONa, EtOH/H2O, 0°C, 0.5 h, r.t., 1.5 h, 86 94%; (iii) Ph3P=CHCOOC2H5, dioxane, 120°C, 10
h, 53 59%; (ⅳ) 20%H2SO4, 100°C, 5 10 h, 70 76%; (ⅴ) SOCl2, reflux, 6 h, 92 94%; (ⅵ) appropriate
aniline, carbonyl chloride, DIPEA, 0°C, 1 h, rt., 7
–
95%; (ii) Ethyl acetylacetate,
–
–15
–
–
–
–
–
10 h, 51–59%.
20

ACCEPTED MANUSCRIPT
Table 1
c-Met kinase activities of the target compounds 20–51
Table 2
Cytotoxic activities of compounds 20–51 against HT-29, A549, H460 and U87MG cancer cell lines in vitro
Table 3
Inhibition of tyrosine kinases by compound 34
21

Table 1
c-Met kinase activities of the target compounds 20–51
ACCEPTED MANUSCRIPT
Compd.
20
X
R1
H
H
H
H
H
H
H
H
H
H
H
H
F
R2
c-Met IC50 (nM)
18.26 ± 1.62
12.58 ± 0.93
11.63 ± 1.05
8.42 ± 0.58
H
H
H
2-F
21
H
3-F
22
H
4-F
23
H
2-CF3
3-CF3
2-CH3
4-OCH3
2,5-(CH3)2
2,4-(Cl)2
3-Cl-4-F
2-F-4-Br
2-F
39.52 ± 2.18
35.29 ± 1.72
19.14 ± 1.62
46.91 ± 2.29
33.51 ± 1.48
56.25 ± 2.06a
42.15 ± 1.39
41.23 ± 1.86
5.02 ± 0.53
24
H
25
H
26
H
27
H
28
H
29
H
30
H
31
H
32
H
F
3-F
33
5.61 ± 0.63
H
F
4-F
34
1.06 ± 0.12
H
F
2-CF3
2-CH3
4-OCH3
2,4-(Cl)2
2-F-4-Br
H
11.23 ± 1.58
10.25 ± 0.36
18.25 ± 1.35
22.39 ± 1.36
18.24 ± 1.03
19.54 ± 2.06
14.41 ± 1.29
15.27 ± 2.92
9.21 ± 2.36
35
H
F
36
H
F
37
H
F
38
H
F
39
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
F
40
2-F
41
3-F
42
4-F
43
3-Br
10.46 ± 1.20
46.27 ± 1.75
32.15 ± 1.80
41.25 ± 1.43
49.16 ± 2.39
45.31 ± 1.50
36.18 ± 2.28
29.35 ± 1.71
2.05 ± 0.16
44
2-CF3
3-CF3
2,5-(CH3)2
2,4-(Cl)2
3-Cl-4-F
2-F-4-Br
3-CF3
45
46
47
48
49
50
51
Foretinibb
a Bold values show the IC50 values of the target compounds lower than 10 nM.
b Used as the positive control.

Table 2
ACCEPTED MANUSCRIPT
Cytotoxic activities of compounds 20–51 against HT-29, A549, H460 and U87MG cancer cell lines in vitro.
IC50 (µM) ± SD
X
R1
R2
Compd.
HT29
A549
H460
0.32 ± 0.03
ND b
U87MG
1.02 ± 0.10
ND
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
0.35 ± 0.02
0.19 ± 0.02 a
0.21 ± 0.02
0.14 ± 0.01
0.89 ± 0.04
1.18 ± 0.12
1.36 ± 0.09
2.35 ± 0.03
5.36 ± 0.21
9.51 ± 0.27
6.25 ± 0.18
7.32 ± 0.22
0.10 ± 0.02
0.093 ± 0.006
0.039 ± 0.002
0.31 ± 0.03
0.13 ± 0.02
0.28 ± 0.03
0.24 ± 0.02
0.29 ± 0.03
0.39 ± 0.03
0.21 ± 0.01
0.32 ± 0.02
0.10 ± 0.02
0.24 ± 0.02
0.61 ± 0.04
1.53 ± 0.10
1.39 ± 0.23
4.15 ± 0.25
3.38 ± 0.17
5.37 ± 0.16
0.36 ± 0.03
0.25 ± 0.02
0.49 ± 0.03
0.30 ± 0.02
0.42 ± 0.02
0.23 ± 0.02
0.61 ± 0.04
0.92 ± 0.03
0.81 ± 0.02
2.42 ± 0.10
3.32 ± 0.11
2.47 ± 0.13
5.71 ± 0.19
1.84 ± 0.16
0.21 ± 0.04
0.16 ± 0.03
0.046 ± 0.003
0.32 ± 0.02
0.42 ± 0.03
0.39 ± 0.03
0.51 ± 0.04
0.62 ± 0.03
0.46 ± 0.04
0.38 ± 0.03
0.41 ± 0.04
0.25 ± 0.02
1.41 ± 0.14
2.06 ± 0.13
2.32 ± 0.10
4.61 ± 0.15
8.15 ± 0.11
5.73 ± 0.12
4.51 ± 0.16
0.62 ± 0.04
0.36 ± 0.02
20
H
2-F
21
H
3-F
3.42 ± 0.11
5.46 ± 0.14
1.92 ± 0.12
ND
22
0.19 ± 0.02
0.16 ± 0.02
0.76 ± 0.03
ND
H
4-F
23
H
2-CF3
3-CF3
2-CH3
4-OCH3
2,5-(CH3)2
2,4-(Cl)2
3-Cl-4-F
2-F-4-Br
2-F
24
H
25
H
0.35 ± 0.02
0.32 ± 0.04
ND
3.23 ± 0.12
8.39 ± 0.13
ND
26
H
27
H
28
H
0.96 ± 0.04
ND
9.25 ± 0.26
ND
29
H
30
H
ND
ND
31
H
6.32 ± 0.15
2.35 ± 0.14
0.76 ± 0.05
3.27 ± 0.11
2.63 ± 0.16
ND
32
0.20 ± 0.03
0.32 ± 0.04
0.091 ± 0.004
0.42 ± 002
0.38 ± 0.03
ND
H
F
3-F
33
H
F
4-F
34
H
F
2-CF3
2-CH3
4-OCH3
2,4-(Cl)2
2-F-4-Br
H
35
H
F
36
H
F
37
H
F
ND
ND
38
H
F
ND
ND
39
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
F
0.35 ± 0.03
0.34 ± 0.02
0.32 ± 0.03
0.18 ± 0.02
0.48 ± 0.03
ND
4.39 ± 0.17
4.91 ± 0.16
8.82 ± 0.19
4.36 ± 0.15
5.69 ± 0.18
ND
40
2-F
41
3-F
42
4-F
43
3-Br
44
2-CF3
3-CF3
2,5-(CH3)2
2,4-(Cl)2
3-Cl-4-F
2-F-4-Br
3-CF3
45
5.34 ± 0.06
ND
9.43 ± 0.14
ND
46
47
ND
ND
48
2.73 ± 0.04
ND
12.54 ± 0.19
ND
49
50
0.52 ± 0.04
0.29 ± 0.03
2.31 ± 0.12
0.96 ± 0.10
51
Foretinib c
a Bold values show the IC50 values of the target compounds lower than the values of the positive control.
b ND: Not determined.
c Used as the positive control.

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Table 3
Inhibition of tyrosine kinases by compound 34
Kinase
Flt-3
Enzyme IC50 (nM)
1.32
PDGFR-β
c-Kit
3.26
318.5
671.8
725.3
VEGFR-2
EGFR

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