Discovery of Novel c-Mesenchymal-Epithelia transition factor and histone deacetylase dual inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112651

Clinically, a single agent that simultaneously inhibits multiple targets has been widely used in cancer treatment to overcome complicated dose design and anti-cancer resistance. Inspired by the synergistic effects between c-Met and HDAC in tumor developme

Full text of DOI:10.1016/j.ejmech.2020.112651

Journal Pre-proof
Discovery of Novel c-Mesenchymal-Epithelia Transition Factor and Histone
Deacetylase Dual Inhibitors
Hao Hu, Fei Chen, Yuhong Dong, Ming Li, Sicong Xu, Mingze Qin, Ping Gong
PII:
S0223-5234(20)30623-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112651
EJMECH 112651
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 January 2020
Revised Date: 6 July 2020
Accepted Date: 7 July 2020
Please cite this article as: H. Hu, F. Chen, Y. Dong, M. Li, S. Xu, M. Qin, P. Gong, Discovery of Novel c-
Mesenchymal-Epithelia Transition Factor and Histone Deacetylase Dual Inhibitors, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112651.
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Graphical abstract

‹
‹
‹
A series of novel c-Met/HDAC Dual Inhibitors was designed and synthesized.
14x showed potent cytotoxicity against tested cell lines and better enzymatic inhibition.
Cell apoptosis study revealed 14x was very effective in the induction of apoptosis in a
dose-dependent manner in HCT-116 cells.
‹
‹
Cell cycle analysis showed that 14x significantly caused G2/M-phase arrest in HCT-116 cells.
Docking mode indicated 14x could form critical bonding interactions with c-Met and HDAC.

Discovery of Novel c-Mesenchymal-Epithelia Transition Factor and
Histone Deacetylase Dual Inhibitors
*
*
1
Hao Hu, Fei Chen, Yuhong Dong, Ming Li, Sicong Xu, Mingze Qin , Ping Gong
Key Laboratory of Structure-Based Drug Design and Discovery. Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, PR China
Abstract
Clinically, a single agent that simultaneously inhibits multiple targets has been widely used in cancer
treatment to overcome complicated dose design and anti-cancer resistance. Inspired by the synergistic effects
between c-Met and HDAC in tumor development, a novel series of c-Met/HDAC bifunctional inhibitors was
designed and synthesized by merging the pharmacophores of HDAC inhibitor into a c-Met inhibitor. All the target
compounds were evaluated for their biological activity, the most potent compound, 14x, exhibited strong
inhibition against HDAC1 with an IC50 of 18.49 nM and remarkable inhibitory activity against c-Met with an IC50
of 5.40 nM, respectively. In addition, 14x efficiently inhibited the proliferation of HCT-116, MCF-7 and A549 cell
lines with IC50 values of 0.22 μM, 1.59 μM and 0.22 μM, respectively, which were superior to the reference
compounds Cabozantinib and SAHA. Futhermore, 14x induced apoptosis and cause cell cycle arrest in G2/M
phase. Docking experiments on c-Met and HDAC enzymes revealed the key interactions between 14x with the
target protein. These results indicated that 14x was a potent dual c-Met/HDAC inhibitor and deserved for further
investigation.
Keywords: c-Met; HDAC; synthesis; antitumor activity
1. Introduction
c-Mesenchymal-epithelial transition factor (c-Met) is a prototype member of a subfamily of heterodimeric
receptor tyrosine kinases (RTKs) which is also known as hepatocyte growth factor receptor (HGFR) [1]. The
activation of c-Met by HGF induces several complex signal pathways that result in cell proliferation, survival,
motility, angiogenesis and invasion [2-3]. However, the overexpression of HGF has been linked to human cancers,
thus c-Met kinase has emerged as a promising target for cancer treatment. During the last few years, several small
1Correspondence: Prof. Ping Gong and Mingze Qin, Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China.
E-mail: gongpinggp@126.com (P.G.); qinmingze001@126.com (M.Q.)

molecule c-Met kinase inhibitors were under investigation which offered significant therapeutic opportunities for
the treatment of tumors. At present, two kinase inhibitors targeting c-Met have been apaproved by Food and Drug
Administration (FDA) (Fig. 1). Crizotinib, a dual inhibitor of c-Met/ALK kinase, was approved by FDA in August
2011 for the treatment of patients with ALK-positive advanced or metastatic non-small cell lung cancer (NSCLC).
Cabozantinib (XL-184), first launched in the U.S in 2013, is used for the treatment of prostate cancer, multiple
myeloma, small cell lung cancer and others carcinoma. In 2019, the FDA approved Cabozantinib for patients with
advanced hepatocellular carcinoma (HCC) who had been previously treated with Sorafenib [4-5]. However, the
current development of c-Met inhibitors is also not going smoothly. Savolitinib, an exquisitely selective c-Met
inhibitor, was found with all therapies in the setting for advanced NSCLC with which patients ultimately develop
therapeutic resistance [6-8]. Crizotinib case studies had reported on-target c-Met mutations including Y1230H/C,
D1228N/H and D1231Y single-point alterations in NSCLC [9]. Although Cabozantinib tend to have a broader
activity spectrum to overcome the acquired resistance, an expected increased tolerability burden comparing to
Crizotinib and Savolitinib had been indicated [10-11]
.
Epigenetic aberrations contribute to tumor generation and development. Among the numerous epigenetic
enzymes, human histone deacetylases (HDACs) are a family of 18 enzymes that have key effects on numerous
cellular functions [12-13]. The over-expression of HDACs is observed in different human cancers, therefore, they
are regarded as promising antitumor drug targets [14]. Hence, the developments of HDAC inhibitors are
proceeding intensively in academia and pharmaceutical companies. Five HDAC inhibitors have been launched,
namely, Vorinostat (SAHA), Romidepsin (FK228), Belinostat (PXD-101), Panobinostat (LBH-589), and
Chidamide (Fig. 1). In addition, multiple small molecule HDAC inhibitors are also being investigated in clinical
trials. Although HDAC inhibitors and other epigenetic agents showed efficacy in hematologic malignancies,
challenges still remain in treating against solid tumors [15-17].
Fig. 1. Structures of several representative c-Met and HDAC inhibitors.

Recently, molecular hybridization paradigm became an interesting and smart way to defeat the multifaceted
cancer disease by a single molecular entity that acts via several mechanisms just like a magic bullet. Considerable
studies have demonstrated that c-Met and its downstream signaling pathways can be influenced by HDACs both
directly and indirectly. In addition, HDACi, which inhibit HGF production [18], can also disrupt the deacetylation
of nonhistone proteins such as tubulin, Hsp90, p53, and Bcl-2. Hence, the HDACi can make the
pro-growth/pro-survival client proteins (e.g., Raf, AKT, c-Src, etc.) degradation [19-21]. As part of the ongoing
research, we decided to design and synthesize a serial of dual c-Met/HDAC inhibitors, with the aim of achieving
multitargeted therapy to overcome anti-cancer resistance.
2. Results and discussion
2.1. In silico analysis and design of compounds
In our previous study, we found a compound AC-386 [22] through an in-house high-throughput screening.
By using a molecular docking study, we found that the quinoline and pyridazinone moiety of the AC-386 occupy
the hinge region of the ATP binding site to form two hydrogen bonds with the residues of c-Met protein.
Furthermore, the NH of amide bond form one hydrogen bond with Asp1222 residues (Fig. 2). Therefore, the
modifications of both two moieties could not be well tolerated. Noting that the substituent at C7 of the quinoline
moiety extends into the solvent exposed region of the protein, which suggested that this site can be modified.
Besides, most of HDAC inhibitors share a general pharmacophore comprising three parts (Fig. 3), a zinc binding
group, a hydrophobic linker and a surface recognition cap [23]. For the classic pharmacophoric model of HDAC
inhibitors, the zinc binding group (ZBG) is the most important part of HDAC inhibitors, in which hydroxamic
acid and ortho-aminoanilide are the two privileged zinc binding groups [24]. In addition, compounds possessing a
hydrazide moiety as the zinc binding group have also been reported [25-26]. Based on our previous molecular
docking study, we incorporated hydroxamic acid functionality into C7 of the quinoline pharmacophore of c-Met
inhibitors with a proper spacer expect to obtain dual c-Met/HDAC inhibitors. Inspired by the aforementioned
evidence, using pharmacophore fusion strategy, novel hydroxamic dual c-Met/HDAC inhibitors were rationally
designed and synthesized (Fig. 3).

Fig. 2. (A) Proposed binding mode of AC-386 in the ATP pocket of c-Met (PDB code 3LQ8). (B) 2D molecular docking model of
AC-386 with protein crystal structure of c-Met.
Fig. 3. Design strategy of dual c-Met/HDAC inhibitors.
2.2. Chemistry
2.2.1. Synthesis of target compounds 14a-14y.
The synthetic route to obtain the desired target compounds (Scheme 1) is given in the following: the key
intermediates 8a-8g were treated with intermediates 12a-12m to give condensed amide products 13a-13y, which
were then ammoniated via NH2OH in dry methanol to give the target hydroxamic acid compounds 14a-14y [27].

Scheme 1. Preparation of derivatives of 14a-14y. Reagents and conditions: (a): HATU, Et3N, DCM, 40 , 3-5 h; (b): NH2OH.HCl,
NaOH, anhydrous, CH3OH, rt, 2-6 h;
The synthesis of key intermediates 8a-8g is summarized in Scheme 2. Commercially available
acetovanillone 1 reacted with different bromo esters to afford 2a-2d, which were treated with fuming nitric acid to
obtain 3a-3d, respectively. Treating 3a-3d with DMF-DMA provided 4a-4d, which were cyclized to give
intermediates 5a-5d, respectively. After chlorination of quinolin-4(1H)-one 5a-5d, the desired intermediates 6a-6d
were obtained. 7a-7g were obtained through nucleophilic substitution with p-nitrophenol or
2-fluoro-4-nitrophenol in chlorobenzene, which were reduced by H2/Pd to yield the desired intermediates 8a-8g.
Scheme 2. Preparation of intermediates 8a-8g. Reagents and conditions: (a) different bromo esters, K2CO3, DMF, 80 , 3-4 h; (b)
fuming HNO3, anhydrous DCM, -5 , 6-8 h; (c) DMF-DMA, toluene, 110 °C, 7-8 h; (d) Fe powder, AcOH, r.t, 30 min, 80 °C, 1-2 h;
(e) POCl3, 85 , 6-7 h; (f) 2-fluoro-4-nitrophenol/p-nitrophenol, PhCl, 140 , 20-24 h; (g) H2, Pd/C(10%),CH3OH, 40 , 3-5 h.
The substituted dihydropyridazine-4-carboxylic acid 12a-12m, which were used for the central amidation
step as described in Scheme 3 [28-30] were prepared from the corresponding substituted anilines 9a-9j. Azo ester
intermediates 10a-10m were obtained from benzenediazonium chloride by reaction with different ethyl acyl
acetate, which were reacted with DMF-DMA via cyclization to obtained 11a-11m. The cleavage of the ethyl esters
11a-11m with NaOH provided 12a-12m.

Scheme 3. Preparation of intermediates 12a-12m. Reagents and conditions: (a) i: conc HCl, NaNO2, H2O, 0 °C,0.5 h; ii: different
ethyl acyl acetate, AcONa, H2O/EtOH, rt, 5-8 h; (b) DMF-DMA, toluene, 110 °C, 5-8 h; (c) NaOH, H2O, 40°C, 2-5 h;
2.3. Biological evaluation
2.3.1. HDAC1 and c-Met enzymatic activity assay
Initially, the abilities of compounds 14a-14y to inhibit HDAC1 and c-Met were preliminarily screened at 100
nM concentration, respectively, with SAHA, Cabozantinib and AC-386 as the positive controls.
Table 1. HDAC1 and c-Met inhibitory activities of target compounds 14a-14y
HDAC1inhibition @
c-Met inhibition @
Compd.
n
R1
R2
R3
100nMa
100nMa
1
1
1
3
3
3
3
H
H
H
F
H
2-CF3
3-CF3,4-Cl
2-CH3
2-CH3
H
1.2%
5.2%
NIb
56.3%
50.2%
63.5%
95.5%
98.3%
93.5%
94.0%
14a
14b
14c
14d
14e
14f
CH3
CH3
CH3
CH3
H
8.0%
2.5%
6.0%
7.1%
F
F
2-CH3
2-F
F
CH3
14g

3
3
3
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
H
H
H
F
CH3
CH3
H
2-F
2-CH3
H
9.2%
10.7%
12.6%
61.1%
53.3%
62.1%
36.9%
86.6%
89.1%
40.0%
65.3%
35.3%
53.0%
59.5%
62.0%
47.6%
88.0%
86.4%
90.8%
98.5%
96.4%
89.1%
95.0%
90.4%
95.7%
88.6%
78.6%
61.1%
63.8%
46.3%
63.1%
76.7%
76.5%
72.8%
96.3%
78.2%
14h
14i
14j
CH3
CH3
H
2-F
14k
14l
F
H
H
H
H
H
H
H
H
F
2-CH3
2-CH3
H
14m
14n
14o
14p
14q
14r
14s
14t
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
2-CH3
3-Cl,4-F
2,4-(CH3)2
3-CF3
2-CH3
2-CH3
3-CH3
4-CH3
H
F
14u
14v
14w
14x
14y
H
H
H
H
CH3
CH3
H
CH2CH3
H
SAHAc
88.4%
2.80%
XL-184c
AC-386 c
93.1%
93.8%
―
—
a All compounds were assayed at least twice, and the inhibitory values were averaged.
b NI = no inhibition.
c Used as a positive control.
The biological data listed in Table 1 showed that all compounds showed similar or superior c-Met inhibitory

activities even compared with the positive controls, however only half of the compounds inhibited HDAC1 over
50% rate at 100 nM. The SARs on the length of the linker were firstly studied. Compounds 14a, 14b and 14c with
a one-carbon linker did not show any appreciable inhibition against HDAC1 (inhibition rates ranging from 0 % to
5.2%). As the length of the linker was increased to three carbons, a weak HDAC1 potency was still observed.
However, the c-Met activity was enhanced about 1.5-fold (14c: inhibition rate 63.5 % vs 14i: inhibition rate
98.5%). As the linker length increased further (e.g., 14k and 14l), HDAC1 potency was boosted by over 20-fold
(e.g., 14l vs 14e). Of particular note, compounds with six-carbon linker exhibited notable HDAC1 potency (e.g.,
14o, 14p and 14x), comparable to that of the launched HDAC inhibitors (e.g., SAHA). This study clearly showed
that the linker length is highly important for HDAC1 activity, probably because the short linker could not lead
their hydroxamate groups to the zinc ion to form an effective interaction. These results also suggested that six
carbon atoms chain of the linker region was the optimal group to maintain the inhibitory activities for HDAC1.
Interestingly, the linker length had no significant influence on c-Met inhibition of these dual inhibitors. (e.g., 14i:
inhibition rate 98.5%; 14n: inhibition rate 95.7%; 14p: inhibition rate 78.6%).
Further studies were performed to determine how R1 affects activity. Compound 14i with a H atom on the
phenyl ring exhibited better activity than 14d with a fluorine atom. The increase in activity can also be observed
by comparing compounds 14p (HDAC1: inhibition rate 89.1 %; c-Met: inhibition rate 78.6%) with 14t (HDAC1:
inhibition rate 53.0 %; c-Met: inhibition rate 63.1%).
Additional investigations were performed to study the effect of different substituents on the phenyl ring on
the enzymatic activity. Introduction of electron-withdrawing groups on the phenyl ring led to a slightly loss in
c-Met inhibitory activity as compared with compounds with electron-donating groups (14c vs 14b; 14i vs 14h).
Once we identified suitable substituents, we then investigate which position affects activity, the results showed
that the ortho-substitution is better than the meta and the para-substitution of the phenyl ring (14p vs 14v vs 14w).
The last investigation was performed by introducing different R2 to the dihydropyridazine ring to explore
whether the size of R2 group was closely related to antitumor activity. However, the introduction of methyl or
ethyl group can not lead to an obvious improvement in activity (e.g., 14x：HDAC1: inhibition rate 88.0 %; c-Met:
inhibition rate 96.3%; 14o: HDAC1: inhibition rate 86.6 %; c-Met: inhibition rate 88.6%; 14y: HDAC1: inhibition
rate 86.4 %; c-Met: inhibition rate 78.2%).
Considering the potent activity of compounds 14o, 14x and 14y in inhibition of c-Met and HDAC1, the IC50
values were determined. As showed in Table 2, the tested compounds exhibited excellent c-Met enzymatic
potency with IC50 values ranging from 5.40 to 19.86 nM and moderate HDAC1 enzymatic potency with IC50

values ranging from 18.49 to 34.84 nM. In particular, compound 14x showed the most potent activity against
HDAC1 with an IC50 of 18.49 nM, which was comparable to that of SAHA (IC50 = 14.15 nM). However, the
potency of 14x against c-Met (IC50 = 5.40 nM) was slightly superior to AC-386 (IC50 = 7.42 nM), and was
6.9-fold higher than that of XL184 (IC50 = 37.28 nM).
Table 2. Enzymatic assays for HDAC1/c-Met inhibition.
Compd.
14o
HDAC1 IC50 (nM) ± SDa
34.03 ± 0.22
18.49 ± 0.18
34.84 ± 0.36
14.15 ± 0.25
NT
c-Met IC50 (nM) ± SDa
19.86 ± 0.10
5.40 ± 0.05
14.23 ± 0.15
NT
14x
14y
SAHAb
XL-184b
AC-386b
37.28 ± 1.10
7.42 ± 0.20
NT
a SD: standard deviation.
b Positive control.
NT= not tested.
2.3.2. In vitro cytotoxic activities
After the synthesis and evaluation for enzymatic activity assays of these compounds, antiproliferative effects
of these compounds against HCT-116 (human colon cancer), A549 (human lung adenocarcinoma) and MCF-7
(human breast cancer) cell lines were tested using MTT assay.
Table 3. The antiproliferative activities of 14a-14y against HCT-116, MCF-7and A549 cell lines in vitro
.
IC50 a (μmol/L) ± SD
MCF-7
Compd.
n
R1
R2
R3
HCT-116
9.50 ± 0.40
0.35 ± 0.03
9.26 ± 1.01
2.04 ± 0.03
0.66 ± 0.15
A549
1
H
H
H
F
H
2-CF3
3-CF3,4-Cl
2-CH3
2-CH3
H
6.32 ± 0.41
3.48 ± 0.20
8.78 ± 2.13
2.60 ± 0.12
1.90 ± 0.23
>20
14a
14b
14c
14d
14e
1
1
3
3
CH3
CH3
CH3
CH3
1.59 ± 0.31
5.98 ± 0.10
2.09 ± 0.30
1.90 ± 0.11
F

3
3
3
3
3
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
F
F
H
CH3
CH3
CH3
H
2-CH3
2-F
3.98 ± 0.07
1.12 ± 0.05
0.95 ± 0.08
0.64 ± 0.03
0.94 ± 0.04
0.59 ± 0.02
0.69 ± 0.14
0.70 ± 0.02
0.29 ± 0.03
0.15 ± 0.02
0.66 ± 0.05
0.37 ± 0.06
0.44 ± 0.06
0.59 ± 0.02
2.30 ± 0.41
4.39 ± 0.89
0.37 ± 0.03
0.40 ± 0.02
0.22 ± 0.09
0.30 ± 0.01
0.55 ± 0.13
4.67 ± 0.05
0.37± 0.02
3.20 ± 0.14
2.10 ± 0.17
2.65 ± 0.21
1.60 ± 0.01
7.41 ± 1.10
2.32 ± 0.04
2.33 ± 0.10
4.36 ± 0.50
1.83 ± 0.06
1.61 ± 0.10
1.83 ± 0.22
2.20 ± 0.10
2.43 ± 0.21
0.75 ± 0.11
2.69 ± 0.11
3.65 ± 0.15
1.68 ± 0.10
2.56 ± 0.15
1.59 ± 0.06
1.21 ± 0.22
2.00 ± 0.20
5.98 ± 0.15
0.63 ± 0.05
5.74 ± 0.61
1.37 ± 0.02
3.92 ± 0.36
4.46 ± 0.51
9.26 ± 0.46
1.32 ± 0.50
4.17 ± 0.09
3.92 ± 0.17
1.82 ± 0.21
0.42 ± 0.04
3.13 ± 0.37
0.38 ± 0.04
1.84± 0.33
0.78 ± 0.05
4.28 ± 0.32
>20
14f
14g
H
H
H
F
2-F
14h
2-CH3
H
14i
14j
CH3
CH3
H
2-F
14k
F
H
14l
H
H
H
H
H
H
H
F
2-CH3
2-CH3
H
14m
14n
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
14o
2-CH3
3-Cl,4-F
2,4-(CH3)2
3-CF3
2-CH3
2-CH3
3-CH3
4-CH3
H
14p
14q
14r
14s
14t
F
14u
H
H
H
H
CH3
CH3
H
0.28 ± 0.09
0.16 ± 0.04
0.22 ± 0.04
0.23 ± 0.03
1.70 ± 0.12
7.80 ± 0.40
1.12 ± 0.10
14v
14w
14x
CH2CH3
H
14y
SAHAb
XL-184b
AC-386b
a
Inhibition values represent the average of at least three independent experiments.
b Used as a positive control.
Data in Table 3 illustrated that the target compounds 14a-14y exhibited moderate to potent antiproliferation

activities against one or more tested cancer cells with potencies in the single digit micromole range, which were
mostly in accordance with the trend in enzymatic activity. Similarly, a clear dependency of the potency on the
linker length is present, where the antiproliferative potency initially increased with the elongation of the linker.
Compounds 14o, 14s, 14v, 14x and 14y with six carbon linkers were the more potent compounds. Interestingly,
most of the compounds were more potent against HCT-116 cell line than the other two cancer cell lines (MCF-7
and A549), indicating that the target compounds might be used in treating human colon cancer. Among them,
compound 14x exhibited the best activity against all of the tested cell lines with IC50 values of 0.22 μM, 1.59 μM
and 0.22 μM against HCT-116, MCF-7 and A549 cell lines respectively.
We further examined the HCT-116, MCF-7 and A549 cell lines with a phase-contrast light microscope for
changes in cell morphology in response to 72 h treatment with 0.5 μM, 1.0 μM and 4.0 μM 14x (Fig.4). Through
electronic microscope, some cells appear to be rounded, formation of membrane bubbles and apoptotic bodies. To
our surprise, 14x did not only affect cell morphology but also strongly inhibits cell proliferation even at the
concentration of 0.5 μM. Because compound 14x showed the highest potency in inhibition of both cell
proliferation and c-Met/HDAC enzyme, it was advanced for further evaluation.
Fig.4. HCT-116, MCF-7 and A549 cell states after treatment with vehicle,0.5 μM, 1.0 μM L and 4.0 μM 14x for 72h. Figures are
representative of n ≥ 3 experiments.
2.3.3. Cell apoptosis study

In addition, we wanted to understand the mechanism of cell death, 14x was selected for apoptosis study in
HCT-116 cells using annexin V-FITC and PI staining and analyzed by flow cytometry (Fig. 5). After treating
HCT-116 cells with 0.2 μM, 1.0 μM and 5.0 μM of 14x for 48 h, the percentage of apoptotic cells was 4.19%,
11.53% and 21.48%, respectively, which displayed a higher apoptosis level than control (3.48%). It was evident
that 14x is effective in the induction of cancer cell apoptosis in a dose-dependent manner.
Fig. 5. Compound 14x induced apoptosis in HCT-116 cells. Cells were treated with various concentrations of 14x for 48 h and then
analyzed the Annexin V-FITC/PI staining test by flow cytometry analysis. Values represent the mean ± S.D, n = 3. The percentage of
cells in each part is indicated.
2.3.4. Cell cycle analysis
To probe the effect of compound 14x on various phases of cell cycle progression，flow cytometry experiment
was performed on HCT-116 cells. After treatment of HCT-116 cells with 14x for 48 h at indicated concentrations
(0.2 μM, 1.0 μM, 5.0 μM), the cells were fixed and stained with PI, the DNA content was evaluated by flow
cytometric method. The obtained results were compared with non-treated HCT-116 cells as control. As shown in
Fig. 6-7, it was clearly observed that the percentage of cells in G2/M-phase for compound 14x was increased from
11.74% (control group) to 14.59%, 80.03%, and 81.22%. These results confirmed that compound 14x
significantly caused G2/M-phase arrest in HCT-116 cells in a dose dependent manner.

Fig. 6. Effect of compound 14x on the cell cycle distribution of HCT-116 cells. The experiments were performed three times and a
representative experiment is shown.
120
100
4.87
15.1
5.5
13.28
80
60
40
20
0
55.37
30.04
57.63
30.63
G1
S
G2/M
81.22
80.03
14.59
B
11.74
A
C
D
Fig. 7. Quantitative analysis of cell cycle distributions; (A) Non-treated cells as control group; (B) treated with 14x at 0.2 μM; (C)

treated with 14x at 1.0 μM; (D) treated with 14x at 5.0 μM.
2.3.5. Binding Mode of the c-Met/HDAC Dual Inhibitors.
To investigate the binding mode of compound 14x with c-Met and HDAC1, molecular docking studies were
performed [23,31]. As shown in Fig. 8: A, B (PDB ID: 3LQ8), the quinoline scaffold fits well into the c-Met ATP
binding pocket and forms a hydrogen bond with the residue of Met1160. The H atom of the amide and the N atom
of pyridazinone scaffold of compound 14x formed two hydrogen bonds with Asp1222. In addition, the carbonyl of
pyridazinone formed hydrogen bond with Lys1110. These interactions were identified as the key driving force for
the c-Met binding. It was also revealed that the hydroxamic tail formed two hydrogen bonds with residues of
His1162 and Asn1171, which resulted in the improvement of c-Met activity comparing to the hit AC-386.
Compound 14x bound with HDAC1 (PDB ID:1C3S), mainly through ZBG (Fig. 8: C, D). The hydroxamic acid
of 14x coordinated to the catalytic Zn2+ of HDAC1 and formed hydrogen bond with His170.
A

B
C

D
Fig. 8. (A) Proposed 3D binding mode of compound 14x in the active site of c-Met (PDB: 3LQ8). (B) Proposed 2D binding mode of
compound 14x in the active site of c-Met (PDB: 3LQ8). (C) Proposed 3D binding mode of compound 14x in the active site of
HDAC1 (PDB: 1C3S). (D) Proposed 2D binding mode of compound 14x in the active site of HDAC1 (PDB: 1C3S). The figure was
generated using PyMol (http://www.pymol.org/).
3. Conclusions
In conclusion, on the basis of structural information for HDAC as well as the available structure-activity
relationship data of our previous c-Met inhibitor AC-386, we designed a novel chemical series of dual inhibitors
simutaneously targeting c-Met and HDAC. In biochemical assays, most of analogues showed potent inhibition of
c-Met and HDAC1 enzymatic activity. In addition, the SAR analyses around the linker and substituents led us to
find a lead molecule 14x. The results of antiproliferative activity indicated that 14x significantly exhibited
inhibitory activity against HCT-116, MCF-7 and A549 cell lines. Cell apoptosis study found 14x was highly
effective in the induction of apoptosis in a dose-dependent manner. Cell cycle analysis of 14x by flow cytometry
showed cell cycle arrest in G2/M phase. Compound 14x could bind well with active sites of c-Met and HDAC1 in
molecular docking study. Overall, 14x was a potential lead compound for cancer therapy. Extensive investigations
of the pharmacokinetics and in vivo activity of 14x are ongoing in our lab and the results will be reported in due
course.

4. Experimental
4.1 Chemistry
Unless otherwise specified, all materials were obtained from commercial suppliers and were used without
further purification. Reactions’ time and purity of the products were monitored by TLC on FLUKA silica gel
aluminum cards (0.2 mm thickness) with fluorescent indicator 254 nm. Column chromatography was run on silica
gel (200–300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). All melting points were
obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were
uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA).
The reverse phase HPLC was conducted on an Agilent 1260 Infinity chromatograph, which was equipped with
ZORBAX SB-C18 column (250 mm × 4.6 mm). The mobile phase A was methanol, and mobile phase B was 30
mM NaH2PO4 in water (pH 2.5). The gradient of 5−95% A was run at a flow rate of 1.0 mL/min over 30 min.
High-resolution mass spectra (HRMS) were measured with an Agilent Accurate-Mass Q-TOF 6530 in ESI mode
1
(Agilent, Santa Clara, CA, USA). HNMR and 13C NMR spectra were performed using Bruker spectrometers
(BrukerBioscience, respectively, Billerica, MA, USA) with TMS as an internal standard.
4.1.1 Preparation of ethyl 2-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)acetate (8a) [29]
4.1.1.1 Preparation of ethyl 2-(4-acetyl-2-methoxyphenoxy)acetate (2a)
To a solution of 4-hydroxy-3-methoxy-acetophenone (20.0 g, 120.5 mmol) in DMF (200 mL) was added
K2CO3 (17.4 g, 126.5 mmol) and ethyl 2-bromoacetate (20.9 g, 126.5 mmol). The reaction mixture was then
stirred at 80 °C for 3 h. The mixture was then poured into cold water (500 mL) with vigorously agitating, and the
resulting precipitate was filtered-off, washed with water, and dried under vacuum to afford the title compound 2a
(27.5 g, 90.5%) as a white solid. MS (ESI) m/z: 253.15 [M+H] +.
4.1.1.2 Preparation of ethyl 2-(4-acetyl-2-methoxy-5-nitrophenoxy)acetate (3a)
A stirred solution of 2a (25.0 g, 99.2 mmol) in CH2Cl2 (250 mL) was cooled to 10 °C, fuming HNO3 (46.8 g,
495.8 mmol) was added at an appropriate rate keeping the temperature below -5 °C. The reaction mixture was
allowed to stir at -5 °C for 6 h, then poured into cold water (200 mL), the organic layer was separated and washed
with water (200 mL), then concentrated under reduced pressure to afford crude 3a as a light yellow solid (24.3 g,
82.5%). MS (ESI) m/z: 298.13 [M+H] +.
4.1.1.3 Preparation of ethyl (E)-2-(4-(3-(dimethylamino)acryloyl)-2-methoxy-5-nitrophenoxy)acetate (4a)

20.0 g (67.3 mmol) of 3a was suspended in 200 mL of toluene at room temperature, then DMF-DMA (24.0 g,
202.0 mmol) was added to a solution. The reaction was heated to 110°C until TLC showed the completion of the
reaction. After cooling to rt, the resultant solid was collected by filtration, washed with toluene (30 mL), and then
dried under vacuum to yield 4a as a yellow solid (14.4 g, 60.8%). MS (ESI) m/z: 353.23 [M+H] +.
4.1.1.4 Preparation of ethyl 2-((6-methoxy-4-oxo-1,4-dihydroquinolin-7-yl)oxy)acetate (5a)
Fe powder (11.1 g, 199.0 mmol) was added to a solution of 4a (14.0 g, 39.8 mmol) in acetic acid (70 mL) at
60 °C in batches, then the mixture was stirred at 80 °C for 2 h. The hot solution was filtered through celite and
washed with hot acetic acid. The combined filtrate was cooled to rt, the resultant solid was collected by filtration
and washed with acetic acid (10 mL) to afford 5a as a pale solid (7.2 g, 65.0%). MS (ESI) m/z: 278.33 [M+H] +.
4..1.1.5 Preparation of ethyl 2-((4-chloro-6-methoxyquinolin-7-yl)oxy)acetate (6a)
A mixture consisting of 7.0 g (25.3 mmol) of 5a and 56.0 mL (8 v/m) of phosphorus oxychloride was
refluxed for 8 h whereby a clear solution was formed. Thereafter, the excess unreacted phosphorus oxychloride
was evaporated in vacuo and the residual oil was poured into ice water. The precipitate formed under vigorous
stirring conditions, thereby was collected by vacuum filtration, dried under reduced pressure to afford product 6a
as a white solid (6.3 g, 83.7%). MS (ESI) m/z: 296.10 [M+H] +.
4.1.1.6 Preparation of ethyl 2-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)acetate (7a)
4-nitrophenol (4.2 g 30.5 mmol) was added to a stirred solution of 6a (6.0 g, 20.3 mmol ) in 60 mL
chlorobenzene, then the reaction mixture was refluxed for 25 h whereby a clear solution was formed. Then
evaporated in vacuo, the resulting precipitate was added to 100 mL DCM, the organic portion was washed with 10%
w/w aqueous sodium bicarbonate solution (3 x 50 mL), the organic layer was evaporated in vacuo and dried under
reduced pressure to afford product 7a as a pale solid (4.8 g, 60.4%). MS (ESI) m/z: 399.18 [M+H] +.
4.1.1.7 Preparation of ethyl 2-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)acetate (8a)
10% Pd/C (0.45 g, 10%w/w) was added to a stirred solution of 7a (4.5 g, 11.3 mmol ) in 45 mL methanol
under H2, then the mixture was stirred at 40 °C for 4 h. After completion of reaction, it was allowed to cool to
room temperature. The solution was filtered through celite and washed with methanol (10 mL), then solvent was
evaporated in vacuo and dried under reduced pressure to afford product 8a as a rubricans solid. (4.0 g, 96.2%).
MS (ESI) m/z (%): 369.32 [M+H]+.
The preparation of the key intermediates 8b-8g are the same way as to preparation 8a, So the synthesis
method would not be listed here.
4.1.2 Preparation of ethyl 4-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)butanoate (8b)

Pale solid; total yield: 12.7%; MS (ESI) m/z (%): 397.22 [M+H]+.
4.1.3 Preparation of ethyl 4-((4-(4-amino-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)butanoate (8c)
Black solid; total yield: 10.5%; MS (ESI) m/z (%): 415.21 [M+H]+.
4.1.4 Preparation of ethyl 6-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)hexanoate (8d)
Black solid; total yield: 9.8%; MS (ESI) m/z (%): 425.33 [M+H]+.
4.1.5 Preparation of ethyl 6-((4-(4-amino-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)hexanoate (8e)
Black solid; total yield: 12.8%; MS (ESI) m/z (%): 443.42 [M+H]+.
4.1.6 Preparation of ethyl 7-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)heptanoate (8f)
Black solid; total yield: 14.1%; MS (ESI) m/z (%): 439.38 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.42 (d, J =
5.3 Hz, 1H), 7.50 (s, 1H), 7.34 (s, 1H), 6.93 (d, J = 8.7 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 6.36 (d, J = 5.2 Hz, 1H), 5.18
(s, 2H), 4.12 (t, J = 6.5 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 2.30 (t, J = 7.4 Hz, 2H), 1.80 (p, J = 6.7 Hz, 2H),
1.57 (p, J = 7.4 Hz, 2H), 1.47 (p, J = 7.5 Hz, 2H), 1.37 (q, J = 8.1 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H).
4.1.7 Preparation of ethyl 7-((4-(4-amino-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)heptanoate (8g)
Black solid; total yield: 15.1%; MS (ESI) m/z (%): 457.35 [M+H]+.
4.1.8 General procedure for preparation intermediates (10a-10m)
To a mixture of substituted phenyl amine (60.0 mmol) and 6 M/L HCl (60 mL), NaNO2 (5 g, 72.0 mmol) in
H2O (200 mL) was added dropwise at 0 ℃. After the completion of addition, the reaction mixture was stirred at 0 ℃
for 30 min, then added into a mixture of different ethyl acyl acetate (63.0 mmol), anhydrous sodium acetate (180.0
mmol) and EtOH (200 mL) at 0 ℃and stirred for another 2 h. Then the mixture was filtered, and the residue was
dried to afford light yellow solids 10a-10m in 70-95% yields.
4.1.8.1 Preparation of ethyl 3-oxo-2-(2-(2-(trifluoromethyl)phenyl)hydrazono)butanoate (10a)
Yellow solid; yield: 74.1%; MS (ESI) m/z (%): 303.12 [M+H]+.
4.1.8.2 Preparation of ethyl 2-(2-(4-chloro-3-(trifluoromethyl)phenyl)hydrazono)-3-oxopentanoate (10b)
Light yellow solid; yield: 79.8%; MS (ESI) m/z (%): 351. 25 [M+H]+.
4.1.8.3 Preparation of ethyl 3-oxo-2-(2-(o-tolyl)hydrazono)pentanoate (10c)
Yellow solid; yield: 84.2%; MS (ESI) m/z (%): 263.20 [M+H]+.
4.1.8.4 Preparation of ethyl 3-oxo-2-(2-(o-tolyl)hydrazono)butanoate (10d)
Yellow solid; yield: 88.1%; MS (ESI) m/z (%): 249.15 [M+H]+.
4.1.8.4 Preparation of ethyl 2-(2-(2-fluorophenyl)hydrazono)-3-oxopentanoate (10e)
Yellow solid; yield: 95.0%; MS (ESI) m/z (%): 267.11 [M+H]+.

4.1.8.5 Preparation of ethyl 3-oxo-2-(2-phenylhydrazono)pentanoate (10f)
Light yellow solid; yield: 70.1%; MS (ESI) m/z (%): 249.15 [M+H]+.
4.1.8.6 Preparation of ethyl 2-(2-(3-chloro-4-fluorophenyl)hydrazono)-3-oxopentanoate (10g)
Yellow solid; total yield: 82.7%; MS (ESI) m/z (%): 301.11 [M+H]+.
4.1.8.7 Preparation of ethyl 2-(2-(2,4-dimethylphenyl)hydrazono)-3-oxopentanoate (10h)
Light yellow solid; yield: 77.5%; MS (ESI) m/z (%): 277.18 [M+H]+.
4.1.8.8 Preparation of ethyl 3-oxo-2-(2-(m-tolyl)hydrazono)pentanoate (10i)
Yellow solid; yield: 92.3%; MS (ESI) m/z (%): 263.15 [M+H]+.
4.1.8.9 Preparation of ethyl ethyl 3-oxo-2-(2-(p-tolyl)hydrazono)pentanoate (10j)
Light yellow solid; yield: 90.6%; MS (ESI) m/z (%): 263.15 [M+H]+.
4.1.8.10 Preparation of ethyl 3-oxo-2-(2-phenylhydrazono)butanoate (10k)
Yellow solid; yield: 79.5%; MS (ESI) m/z (%): 235.10 [M+H]+.
4.1.8.11 Preparation of ethyl 3-oxo-2-(2-(3-(trifluoromethyl)phenyl)hydrazono)pentanoate (10l)
Light yellow solid; yield: 82.1%; MS (ESI) m/z (%): 317.19 [M+H]+.
4.1.8.12 Preparation of ethyl 3-oxo-2-(2-phenylhydrazono)hexanoate (10m)
Light yellow solid; yield: 94.4%; MS (ESI) m/z (%): 263.15 [M+H]+.
4.1.9 General procedure for preparation intermediates (11a-11m)
Appropriate intermediate 10a-10m (20.0 mmol) was suspended in 50 mL of toluene at room temperature,
then DMF-DMA (60.0 mmol) was added to a solution. The reaction was heated to 110 °C until TLC showed the
completion of the reaction. After cooling to rt, the resultant solid was collected by filtration, washed with toluene
(20 mL), and then dried under vacuum to yield 11a-11m as yellow solids in 66-82% yields.
4.1.9.1 Preparation of ethyl 4-oxo-1-(2-(trifluoromethyl)phenyl)-1,4-dihydropyridazine-3-carboxylate (11a)
Yellow solid; yield: 84.0%; MS (ESI) m/z (%): 313.10 [M+H]+.
4.1.9.2
Preparation
of
ethyl
1-(4-chloro-3-(trifluoromethyl)phenyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxylate (11b)
Yellow solid; yield: 79.7%; MS (ESI) m/z (%): 361.15 [M+H]+.
4.1.9.3 Preparation of ethyl 5-methyl-4-oxo-1-(o-tolyl)-1,4-dihydropyridazine-3-carboxylate (11c)
Yellow solid; yield: 74.9%; MS (ESI) m/z (%): 273.19 [M+H]+.
4.1.9.4 Preparation of ethyl 4-oxo-1-(o-tolyl)-1,4-dihydropyridazine-3-carboxylate (11d)
Yellow solid; yield: 78.4%; MS (ESI) m/z (%): 259.22 [M+H]+.

4.1.9.5 Preparation of ethyl 1-(2-fluorophenyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxylate (11e)
Yellow solid; yield: 65.9%; MS (ESI) m/z (%): 277.19 [M+H]+.
4.1.9.6 Preparation of ethyl 5-methyl-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxylate (11f)
Yellow solid; yield: 74.6%; MS (ESI) m/z (%): 259.22 [M+H]+.
4.1.9.7 Preparation of ethyl 1-(3-chloro-4-fluorophenyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxylate
(11g)
Yellow solid; total yield: 82.2%; MS (ESI) m/z (%): 311.10 [M+H]+.
4.1.9.8Preparation of ethyl 1-(2,4-dimethylphenyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxylate (11h)
Light yellow solid; yield: 70.1%; MS (ESI) m/z (%): 287.15 [M+H]+.
4.1.9.9 Preparation of ethyl 5-methyl-4-oxo-1-(m-tolyl)-1,4-dihydropyridazine-3-carboxylate (11i)
Yellow solid; yield: 66.3%; MS (ESI) m/z (%): 273.19 [M+H]+.
4.1.9.10 Preparation of ethyl 5-methyl-4-oxo-1-(p-tolyl)-1,4-dihydropyridazine-3-carboxylate (11j)
Yellow solid; yield:69.9%; MS (ESI) m/z (%): 273.19 [M+H]+.
4.1.9.11 Preparation of ethyl 4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxylate (11k)
Yellow solid; yield: 79.3%; MS (ESI) m/z (%): 245.15 [M+H]+.
4.1.9.12 Preparation of ethyl 4-oxo-1-(3-(trifluoromethyl)phenyl)-1,4-dihydropyridazine-3-carboxylate (11l)
Yellow solid; yield: 81.0%; MS (ESI) m/z (%): 327.32 [M+H]+.
4.1.9.13 Preparation of ethyl 5-ethyl-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxylate (11m)
Yellow solid; yield: 80.1%; MS (ESI) m/z (%): 273.19 [M+H]+.
4.1.10 General procedure for preparation intermediates (12a-12m)
NaOH (100.0 mmol) was added to a stirred solution of intermediate 11a-11m (20.0 mmol ) in 40 mL H2O,
then the reaction mixture was heated at 40 °C for 2-5 h whereby a clear solution was formed. Then the reaction
mixture was cool to room temperature, acidified (6 N HCl) to afford substituted acids (12a-12m) as white
precipitates in 90-96% yields.
4.1.10.1 Preparation of 4-oxo-1-(2-(trifluoromethyl)phenyl)-1,4-dihydropyridazine-3-carboxylic acid (12a)
Yellow solid; yield: 94.2%, MS (ESI) m/z (%): 285.09 [M-H]-;
4.1.10.2
Preparation
of
1-(4-chloro-3-(trifluoromethyl)phenyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxylic acid (12b)
White solid; yield: 91.7%, MS (ESI) m/z (%): 333.13 [M+H]+;
4.1.10.3 Preparation of 5-methyl-4-oxo-1-(o-tolyl)-1,4-dihydropyridazine-3-carboxylic acid (12c)

White solid; yield: 94.9%, MS (ESI) m/z (%): 245.17 [M+H]+;
4.1.10.4 Preparation of 4-oxo-1-(o-tolyl)-1,4-dihydropyridazine-3-carboxylic acid (12d)
White solid; yield: 90.2%, MS (ESI) m/z (%): 231.13 [M+H]+;
4.1.10.5 Preparation of 1-(2-fluorophenyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxylic acid (12e)
Yellow solid; yield: 92.4%, MS (ESI) m/z (%): 249.53 [M+H]+; 1H NMR (600 MHz, DMSO-d6) δ 15.16 (s, 1H),
8.98 (dd, J = 1.8, 1.0 Hz, 1H), 7.77 (td, J = 7.9, 1.7 Hz, 1H), 7.70–7.63 (m, 1H), 7.58 (ddd, J = 10.8, 8.4, 1.3 Hz, 1H),
7.47 (td, J = 7.8, 1.3 Hz, 1H), 2.11 (d, J = 1.0 Hz, 3H).
4.1.10.6 Preparation of 5-methyl-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxylic acid (12f)
Light yellow solid; yield: 96.5%, MS (ESI) m/z (%): 229.24 [M-H]-;
4.1.10.7 Preparation of 1-(3-chloro-4-fluorophenyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxylic acid
(12g)
Yellow solid; total yield: 94.9%, MS (ESI) m/z (%): 283.13 [M+H]+;
4.1.10.8 Preparation of 1-(2,4-dimethylphenyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxylic acid (12h)
Light yellow solid; yield: 90.3%, MS (ESI) m/z (%): 259.21 [M+H]+;
4.1.10.9 Preparation of 5-methyl-4-oxo-1-(m-tolyl)-1,4-dihydropyridazine-3-carboxylic acid (12i)
White solid; yield: 94.1%, MS (ESI) m/z (%): 245.17 [M+H]+;
4.1.10.10 Preparation of 5-methyl-4-oxo-1-(p-tolyl)-1,4-dihydropyridazine-3-carboxylic acid (12j)
White solid; yield: 90.9%, MS (ESI) m/z (%): 245.23 [M+H]+; 1H NMR (600 MHz, DMSO-d6) δ 15.57 (s, 1H), 9.23
(s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 2.40 (s, 3H), 2.15 (s, 3H).
4.1.10.11 Preparation of 4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxylic acid (12k)
White solid; yield: 95.3%, MS (ESI) m/z (%): 215.26 [M-H]-; 1H NMR (600 MHz, DMSO-d6) δ 15.08 (s, 1H), 9.11
(d, J = 7.8 Hz, 1H), 7.78 (d, J = 7.3 Hz, 2H), 7.63 (t, J = 7.9 Hz, 2H), 7.54 (t, J = 7.4 Hz, 1H), 7.07 (d, J = 7.7 Hz, 1H).
4.1.10.12 Preparation of 5-methyl-4-oxo-1-(3-(trifluoromethyl)phenyl)-1,4-dihydropyridazine-3-carboxylic acid
(12l)
White solid; yield: 91.0%, MS (ESI) m/z (%): 298.99 [M+H]+;
4.1.10.13 Preparation of 5-ethyl-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxylic acid (12m)
White solid; yield: 90.4%, MS (ESI) m/z (%): 245.56 [M+H]+; 1H NMR (600 MHz, DMSO-d6) δ 15.48 (s, 1H), 9.09
(s, 1H), 7.82 (d, J = 7.4 Hz, 2H), 7.64 (t, J = 7.9 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1H), 2.59 (q, J = 7.5 Hz, 2H), 1.20 (t, J =
7.5 Hz, 3H).
4.1.11 General procedure for preparation of intermediates (13a-13y)

A mixture of 8a-8g (1.0 mmol) and different intermediates 12a-12m (1.3 mmol), HATU (1.3 mmol) and TEA
(1.3 mmol) in DCM (15 mL) was stirred at reflux for 3-5 h. After being cooled to rt, the mixture was washed
successively with 10% aqueous potassium carbonate solution (20 mL×3) and brine (20 mL×2), the organic phase
was dried over anhydrous sodium sulfate. The solid was removed by filtration, and the filtrate was concentrated to
yield the 13a-13y respectively, yeilds 70.3-82.5%. Without any purification, the intermediates were used for next
procedure.
4.1.12 General procedure for preparation of target compounds (14a-14y)
A solution of sodium hydroxide (4.0 g, 100.0 mmol) in methanol (14 mL) was added to a stirred solution of
hydroxylamine hydrochloride (4.7 g, 67.2 mmol) in methanol (24 mL) at 0 °C. The reaction mixture was stirred at
0 °C for 1 h. The precipitate was removed by filtration and the filtrate was collected to provide fresh
hydroxylamine solution which was stored in a refrigerator before use. The appropriate esters 13a-13y (1.0 mmol)
was added to the above freshly prepared hydroxylamine solution (15 mL) at 0 °C, then NaOH (0.04 g,1.0 mmol)
was added to the solution. The reaction mixture was stirred at room temperature for 2-6 h. The reaction mixture
was neutralized with 6 N HCl. The formed precipitate was collected by filtration, washed with water, dried in
vacuo and purified by silica gel chromatography to afford target compounds 14a-14y in 41.2-87.5% yields.
4.1.12.1.
N-(4-((7-(2-(hydroxyamino)-2-oxoethoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-4-oxo-1-(2-(trifluoromethyl)phenyl
)-1,4-dihydropyridazine-3-carboxamide (14a)
White solid; Yield: 79.1%; M.p.: 188-190 °C; HRMS (ESI) (m/z): [M+H]+ calcd for C30H23F3N5O7,
622.1466;
found, 622.1472; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.89 (s, 1H), 10.91 (s, 1H), 9.08 (s, 1H), 8.78 (d, J =
7.8 Hz, 1H), 8.48 (d, J = 5.3 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.94–7.76 (m, 4H), 7.54
(s, 1H), 7.36 (s, 1H), 7.30 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 7.7 Hz, 1H), 6.51 (d, J = 5.2 Hz, 1H), 4.65 (s, 2H), 3.95
(s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 169.18 , 163.86 , 159.75 , 159.18 , 151.10 , 150.00 , 149.30 ,148.94 ,
147.44 , 146.08 ,144.93 , 140.57 , 135.52 ,134.38 , 131.21 , 129.05 , 127.69 , 123.74 , 121.92 ,121.64 , 121.57 ,
119.57 , 115.59 ,109.07 , 103.41 , 99.38 , 66.05 , 55.72 .
4.1.12.2
1-(3-chloro-4-(trifluoromethyl)phenyl)-N-(4-((7-(2-(hydroxyamino)-2-oxoethoxy)-6-methoxyquinolin-4-yl)oxy)phe
nyl)-5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide (14b)
Light yellow solid; Yield: 86.2%; M.p.: 182-184 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C31H24ClF3N5O7,

670.1245; found, 670.1250; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.99 (s, 1H), 10.90 (s, 1H), 9.21 (s, 1H),
9.07 (s, 1H), 8.49 (d, J = 5.3 Hz, 1H), 8.33 (d, J = 2.7 Hz, 1H), 8.19 (dd, J = 8.8, 2.7 Hz, 1H), 8.09–7.99 (m, 1H),
7.92–7.84 (m, 2H), 7.55 (s, 1H), 7.37 (s, 1H), 7.32 (d, J = 8.8 Hz, 2H), 6.53 (d, J = 5.3 Hz, 1H), 4.65 (s, 2H), 3.96
(s, 3H), 2.14 (s, 3H).
4.1.12.3
N-(4-((7-(2-(hydroxyamino)-2-oxoethoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-5-methyl-4-oxo-1-(o-tolyl)-1,4-dih
ydropyridazine-3-carboxamide (14c)
Light yellow solid; Yield: 87.5%; M.p.: 198-200 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C31H28N5O7,
582.1962; found, 582.1972; 1H NMR (400 MHz, DMSO-d6) δ 12.52 (s, 1H), 11.96 (s, 1H), 10.91 (s, 1H), 9.09 (d,
J = 1.6 Hz, 1H), 8.80 (d, J = 1.1 Hz, 1H), 8.49 (d, J = 5.3 Hz, 1H), 8.03 (dd, J = 12.8, 2.4 Hz, 1H), 7.61–7.41 (m,
7H), 7.37 (s, 1H), 6.52 (d, J = 5.2 Hz, 1H), 4.66 (s, 2H), 3.97 (s, 3H), 2.24 (s, 3H), 2.18 (s, 3H). 13C NMR (151
MHz, DMSO-d6) δ 170.00 , 164.25 , 160.49 , 159.59 , 154.71 , 153.08 , 151.60 , 149.85 , 149.35 , 146.39 ,
143.69 , 143.23 , 143.00 , 133.48 , 131.89 , 131.58 , 130.34 , 127.56 , 126.28 , 124.72 , 117.10 , 115.37 , 109.45 ,
102.76 , 99.65 , 66.47 , 56.18 , 17.48 , 13.21 .
4.1.12.4
N-(3-fluoro-4-((7-(4-(hydroxyamino)-4-oxobutoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-5-methyl-4-oxo-1-(o-tolyl
)-1,4-dihydropyridazine-3-carboxamide (14d)
Yellow solid; Yield: 70.0%; M.p.: 164-166 °C; HRMS (ESI) (m/z): [M+H]+ calcd for C33H31FN5O7,
628.2105;
found, 628.2081; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.52 (s, 1H), 10.48 (s, 1H), 8.81 (s, 1H), 8.75 (s, 1H),
8.48 (d, J = 5.2 Hz, 1H), 8.03 (dd, J = 12.7, 2.4 Hz, 1H), 7.61–7.42 (m, 7H), 7.40 (s, 1H), 6.50 (d, J = 5.2 Hz, 1H),
4.16 (t, J = 6.4 Hz, 2H), 3.96 (s, 3H), 2.24 (s, 3H), 2.20 (t, J = 7.4 Hz, 2H), 2.12 (s, 3H), 2.05 (q, J = 6.9 Hz, 2H).
13C NMR (151 MHz, DMSO-d6) δ 169.99 , 168.95 , 160.49 , 159.56 , 152.21 , 150.10 , 149.93 , 149.25 , 146.74 ,
143.72 , 143.23 , 143.00 , 137.50 , 136.64 , 133.48 , 131.89 , 131.56 , 130.34 , 127.56 , 126.28 , 124.72 , 117.09 ,
114.90 , 109.18 , 108.95 , 102.54 , 99.44 , 68.08 , 56.17 , 29.14 , 25.00 , 17.49 , 13.21 .
4.1.12.5
N-(3-fluoro-4-((7-(4-(hydroxyamino)-4-oxobutoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-5-methyl-4-oxo-1-phenyl-
1,4-dihydropyridazine-3-carboxamide (14e)
Yellow solid; Yield: 80.1%; M.p.: 182-184 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C32H29FN5O7,
614.1924;

found, 614.1901; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.30 (s, 1H), 10.46 (s, 1H), 9.11 (d, J = 1.1 Hz, 1H),
8.73 (d, J = 1.7 Hz, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.05 (dd, J = 12.8, 2.4 Hz, 1H), 7.88–7.81 (m, 2H), 7.64 (dd, J
= 8.6, 7.2 Hz, 2H), 7.61–7.46 (m, 4H), 7.40 (s, 1H), 6.51 (d, J = 5.1 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.97 (s, 3H),
2.20 (t, J = 7.4 Hz, 2H), 2.15 (s, 3H), 2.04 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 170.06 , 168.96 , 160.64 ,
159.58 , 154.74 , 153.11 , 152.21 , 149.94 , 149.25 , 146.73 , 144.47 , 143.41 , 140.02 , 137.51 , 131.84 , 130.11 ,
129.04 , 124.75 , 121.83 , 117.04 , 114.89 , 109.11 , 108.95 , 102.52 , 99.44 , 68.08 , 56.19 , 29.14 , 24.99 , 13.34 .
4.1.12.6
N-(3-fluoro-4-((7-(4-(hydroxyamino)-4-oxobutoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-4-oxo-1-(o-tolyl)-1,4-dihy
dropyridazine-3-carboxamide (14f)
Yellow solid; Yield: 73.0%; M.p.:149-151 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C32H29FN5O7,
614.2002;
found, 614.1985; 1H-NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H), 10.45 (s, 1H), 8.73 (s, 1H), 8.71 (d, J = 5.9 Hz,
1H), 8.48 (d, J = 5.2 Hz, 1H), 8.00 (dd, J = 12.7, 2.4 Hz, 1H), 7.58–7.42 (m, 7H), 7.40 (s, 1H), 6.93 (d, J = 7.7 Hz,
1H), 6.50 (d, J = 5.2 Hz, 1H), 4.16 (t, J = 6.5 Hz, 2H), 3.96 (s, 3H), 2.25 (s, 3H), 2.20 (t, J = 7.4 Hz, 2H), 2.04 (p,
J = 7.0 Hz, 2H).
4.1.12.7
N-(3-fluoro-4-((7-(4-(hydroxyamino)-4-oxobutoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-1-(2-fluorophenyl)-5-met
hyl-4-oxo-1,4-dihydropyridazine-3-carboxamide (14g)
Yellow solid; Yield: 74.9%; M.p.: 164-166 °C; HRMS (ESI) (m/z): [M+H]+ calcd for C32H28F2N5O7,
632.1892;
1
found, 632.1874; H-NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 10.47 (s, 1H), 8.88 (s, 1H), 8.74 (s, 1H), 8.49
(d, J = 5.3 Hz, 1H), 8.01 (dd, J = 12.8, 2.5 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.71–7.43 (m, 6H), 7.40 (s, 1H), 6.51
(d, J = 5.4 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.97 (s, 3H), 2.20 (t, J = 7.4 Hz, 2H), 2.11 (s, 3H), 2.04 (m, 2H).
4.1.12.8
1-(2-fluorophenyl)-N-(4-((7-(4-(hydroxyamino)-4-oxobutoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-5-methyl-4-oxo
-1,4-dihydropyridazine-3-carboxamide (14h)
White solid; Yield: 81.4%; M.p.:170-172 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C32H29FN5O7, 614.1989;
found, 614.1976; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.06 (s, 1H), 10.48 (s, 1H), 8.87 (s, 1H), 8.76 (s, 1H),
8.48 (d, J = 5.2 Hz, 1H), 7.86 (d, J = 8.6 Hz, 2H), 7.80 (s, 1H), 7.71–7.42 (m, 3H), 7.39 (s, 1H), 7.31 (d, J = 8.5
Hz, 2H), 6.50 (d, J = 5.2 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.95 (s, 3H), 2.21 (t, J = 7.4 Hz, 2H), 2.11 (s, 3H), 2.05

(m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 169.92 , 168.99 , 160.23 , 160.03 , 155.93 , 154.27 , 152.16 , 150.32 ,
149.81 , 149.14 , 146.67 , 145.48 , 142.78 , 136.09 , 132.06 , 131.58 , 130.92 , 127.68 , 125.90 , 122.03 , 121.87 ,
117.53 , 117.40 , 115.54 , 108.84 , 103.60 , 99.59 , 68.06 , 56.12 , 29.15 , 25.01 , 13.16 .
4.1.12.9
N-(4-((7-(4-(hydroxyamino)-4-oxobutoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-5-methyl-4-oxo-1-(o-tolyl)-1,4-dih
ydropyridazine-3-carboxamide (14i)
Light yellow solid; Yield: 81.3%; M.p.: 187-189 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C33H32N5O7,
610.2243; found, 610.2234; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): δ 12.41 (s, 1H), 10.48 (s, 1H), 8.77 (d, J =
15.6 Hz, 2H), 8.48 (d, J = 5.2 Hz, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.61–7.42 (m, 5H), 7.38 (s, 1H), 7.31 (d, J = 8.5
Hz, 2H), 6.50 (d, J = 5.1 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.95 (s, 3H), 2.24 (s, 3H), 2.20 (t, J = 7.3 Hz, 2H), 2.12
(s, 3H), 2.05 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 170.10 , 168.99 , 160.16 , 160.08 , 152.13 , 150.31 ,
149.79 , 149.20 , 146.77 , 143.77 , 143.26 , 142.89 , 136.10 , 133.49 , 131.86 , 131.46 , 130.30 , 127.53 , 126.28 ,
122.02 , 121.90 , 115.55 , 108.93 , 103.61 , 99.58 , 68.05 , 56.11 , 29.16 , 25.02 , 17.49 , 13.22 .
4.1.12.10
N-(4-((7-(4-(hydroxyamino)-4-oxobutoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-4-oxo-1-phenyl-1,4-dihydropyrida
zine-3-carboxamide (14j)
Light yellow solid; Yield: 81.0%; M.p.: 172-174 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C31H28N5O7,
582.1934; found, 582.1916; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.90 (s, 1H), 10.47 (s, 1H), 9.03 (d, J = 7.8
Hz, 1H), 8.75 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 8.02–7.73 (m, 4H), 7.63 (t, J = 7.8 Hz, 2H), 7.56–7.47 (m, 2H),
7.38 (s, 1H), 7.32 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 7.8 Hz, 1H), 6.50 (d, J = 5.2 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H),
3.95 (s, 3H), 2.20 (t, J = 7.4 Hz, 2H), 2.04 (t, J = 7.1 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 169.21 , 168.56 ,
159.78 , 159.71 , 151.72 , 149.94 , 149.39 , 148.83 , 147.98 , 146.38 , 142.88 , 141.74 , 135.69 , 129.71 , 128.58 ,
121.67 , 121.45 , 121.36 , 120.36 , 115.12 , 108.54 , 103.19 , 99.18 , 67.64 , 55.72 , 28.73 , 24.60 .
4.1.12.11
N-(3-fluoro-4-((7-((6-(hydroxyamino)-6-oxohexyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-1-(2-fluorophenyl)-5-
methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide (14k)
Yellow solid; Yield: 71.3%; M.p.: 167-169 °C; HRMS (ESI) (m/z): [M+H]+ calcd for C34H32F2N5O7,
660.2214;
found, 660.2202; 1H NMR (600 MHz, DMSO-d6) δ 12.17 (s, 1H), 10.38 (s, 1H), 8.88 (s, 1H), 8.71 (s, 1H), 8.48 (d,
J = 5.3 Hz, 1H), 8.01 (dd, J = 12.7, 2.5 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.65 (d, J = 6.7 Hz, 1H), 7.62–7.53 (m,

3H), 7.49 (dt, J = 11.2, 8.2 Hz, 2H), 7.40 (s, 1H), 6.50 (d, J = 5.2 Hz, 1H), 4.15 (t, J = 6.4 Hz, 2H), 3.96 (s, 3H),
2.11 (s, 3H), 2.01 (t, J = 7.3 Hz, 2H), 1.82 (q, J = 7.2 Hz, 2H), 1.60 (p, J = 7.7 Hz, 2H), 1.45 (m, 2H). 13C NMR
(151 MHz, DMSO-d6) δ 169.37 , 168.99 , 160.03 , 159.16 , 153.86 , 151.95 , 149.55 , 148.78 , 146.36 , 145.07 ,
142.46 , 137.01 , 136.26 , 131.70 , 131.21 , 130.62 , 127.27 , 125.53 , 124.34 , 117.15 , 117.03 , 116.64 , 114.40 ,
108.72 , 108.44 , 102.08 , 98.96 , 68.21 , 55.76 , 32.21 , 28.22 , 25.19 , 24.88 , 12.75 .
4.1.12.12
N-(3-fluoro-4-((7-((6-(hydroxyamino)-6-oxohexyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-5-methyl-4-oxo-1-phe
nyl-1,4-dihydropyridazine-3-carboxamide (14l)
White solid; Yield: 73.5%; M.p.: 134-136 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C34H33FN5O7, 642.2298;
found, 642.2279; 1H NMR (600 MHz, DMSO-d6) δ (ppm) 12.30 (s, 1H), 10.37 (s, 1H), 9.12 (s, 1H), 8.69 (s, 1H),
8.49 (d, J = 5.1 Hz, 1H), 8.05 (d, J = 12.8 Hz, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.67–7.48 (m, 6H), 7.41 (s, 1H), 6.51
(d, J = 5.3 Hz, 1H), 4.15 (t, J = 6.5 Hz, 2H), 3.97 (s, 3H), 2.15 (s, 3H), 2.01 (t, J = 7.3 Hz, 2H), 1.82 (t, J = 7.4 Hz,
2H), 1.60 (q, J = 7.4 Hz, 2H), 1.46 (q, J = 7.6 Hz, 2H) .
4.1.12.13
N-(4-((7-((6-(hydroxyamino)-6-oxohexyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-4-oxo-1-(o-tolyl)-1,4-dihydrop
yridazine-3-carboxamide (14m)
Yellow green solid; Yield: 81.0%; M.p.: 137-139 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C34H34N5O7,
624.2388; found, 624.2364; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.08 (s, 1H), 10.37 (s, 1H), 8.79 – 8.59 (m,
2H), 8.47 (d, J = 5.2 Hz, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.59–7.42 (m, 5H), 7.39 (s, 1H), 7.30 (d, J = 8.5 Hz, 2H),
6.92 (d, J = 7.6 Hz, 1H), 6.49 (d, J = 5.2 Hz, 1H), 4.14 (t, J = 6.6 Hz, 2H), 3.94 (s, 3H), 2.25 (s, 3H), 2.00 (t, J =
7.3 Hz, 2H), 1.81 (m, 2H), 1.67–1.55 (m, 2H), 1.45 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 169.22 , 169.01 ,
159.79 ,159.52 , 151.90 , 149.96 , 149.42 , 148.71 , 147.21 , 146.34 , 144.65 , 142.69 , 135.59 , 133.10 , 131.48 ,
129.92 , 127.18 , 125.90 , 121.65 , 121.52 , 120.03 , 115.05 ,108.36 , 103.15 , 99.12 , 68.18 , 55.70 , 32.21 , 28.23 ,
25.20 , 24.89 , 17.11 .
4.1.12.14
N-(4-((7-((6-(hydroxyamino)-6-oxohexyl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-5-methyl-4-oxo-1-(o-tolyl)-1,4-
dihydropyridazine-3-carboxamide (14n)
Yellow solid; Yield:78.5%; M.p.:175-177 ºC; HRMS (ESI) (m/z): [M+H]+ calcd for C35H36N5O7, 638.2554;
found, 638.2532; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 12.41 (s, 1H), 10.37 (s, 1H), 8.79 (s, 1H), 8.70 (s, 1H),
8.47 (d, J = 5.2 Hz, 1H), 7.86 (d, J = 8.9 Hz, 2H), 7.61–7.42 (m, 5H), 7.39 (s, 1H), 7.30 (d, J = 8.9 Hz, 2H), 6.50