Design, synthesis and biological evaluation of novel N-sulfonylamidine-based derivatives as c-Met inhibitors via Cu-catalyzed three-component reaction

Article abstract of DOI:10.1016/j.ejmech.2020.112470

In our continuing efforts to develop novel c-Met inhibitors as potential anticancer candidates, a series of new N-sulfonylamidine derivatives were designed, synthesized via Cu-catalyzed multicomponent reaction (MCR) as the key step, and evaluated for their in vitro biological activities against c-Met kinase and four cancer cell lines (A549, HT-29, MKN-45 and MDA-MB-231). Most of the target compounds showed moderate to significant potency at both the enzyme-based and cell-based assay and possessed selectivity for A549 and HT-29 cancer cell lines. The preliminary SAR studies demonstrated that compound 26af (c-Met IC₅₀ = 2.89 nM) was the most promising compound compared with the positive foretinib, which exhibited the remarkable antiproliferative activities, with IC₅₀ values ranging from 0.28 to 0.72 μM. Mechanistic studies of 26af showed the anticancer activity was closely related to the blocking phosphorylation of c-Met, leading to cell cycle arresting at G2/M phase and apoptosis of A549 cells by a concentration-dependent manner. The promising compound 26af was further identified as a relatively selective inhibitor of c-Met kinase, which also possessed an acceptable safety profile and favorable pharmacokinetic properties in BALB/c mouse. The favorable drug-likeness of 26af suggested that N-sulfonylamidines may be used as a promising scaffold for antitumor drug development. Additionally, the docking study and molecular dynamics simulations of 26af revealed a common mode of interaction with the binding site of c-Met. These positive results indicated that compound 26af is a potential anti-cancer candidate for clinical trials, and deserves further development as a selective c-Met inhibitor.

Full text of DOI:10.1016/j.ejmech.2020.112470

Journal Pre-proof
Design, synthesis and biological evaluation of novel N-sulfonylamidine-based
derivatives as c-Met inhibitors via Cu-catalyzed three-component reaction
Xiang Nan, Jing Zhang, Hui-Jing Li, Rui Wu, Sen-Biao Fang, Zhi-Zhou Zhang, Yan-
Chao Wu
PII:
S0223-5234(20)30441-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112470
EJMECH 112470
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 April 2020
Revised Date: 9 May 2020
Accepted Date: 13 May 2020
Please cite this article as: X. Nan, J. Zhang, H.-J. Li, R. Wu, S.-B. Fang, Z.-Z. Zhang, Y.-C. Wu, Design,
synthesis and biological evaluation of novel N-sulfonylamidine-based derivatives as c-Met inhibitors via
Cu-catalyzed three-component reaction, European Journal of Medicinal Chemistry (2020), doi: https://
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Graphical Abstract
Design, synthesis and biological evaluation of novel N-sulfonylamidine-based derivatives as c-Met
inhibitors via Cu-catalyzed three-component reaction
Xiang Nan, Jing Zhang, Hui-Jing Li, Rui Wu, Sen-Biao Fang, Zhi-Zhou Zhang, Yan-Chao Wu

Design, synthesis and biological evaluation of novel N-sulfonylamidine-based derivatives as c-Met
inhibitors via Cu-catalyzed three-component reaction
Xiang Nan a, Jing Zhang b, Hui-Jing Li a, d, *, Rui Wu b, *, Sen-Biao Fang c, *, Zhi-Zhou Zhang a, d, Yan-Chao Wu a, d, *
a School of Marine Science and Technology, Harbin Institute of Technology, Weihai 264209, PR China
b Department of Pharmacy, the 3201 Affiliated Hospital of Xi'an Jiaotong University, Hanzhong 723000, PR China
c School of Computer Science and Engineering, Central South University, Changsha 410082, PR China
d Weihai Institute of Marine Biomedical Industrial Technology, Wendeng District, Weihai 264400, PR China
Corresponding authors
*Hui-Jing Li: lihuijing@iccas.ac.cn; *Rui Wu: yjk_3201@163.com; *Sen-Biao Fang: fangsb@csu.edu.cn; *Yan-Chao Wu:
ycwu@iccas.ac.cn

Abstract
In our continuing efforts to develop novel c-Met inhibitors as potential anticancer candidates,
a
series of new
N-sulfonylamidine derivatives were designed, synthesized via Cu-catalyzed multicomponent reaction (MCR) as the key step, and
evaluated for their in vitro biological activities against c-Met kinase and four cancer cell lines (A549, HT-29, MKN-45 and
MDA-MB-231). Most of the target compounds showed moderate to significant potency at both the enzyme-based and cell-based
assay and possessed selectivity for A549 and HT-29 cancer cell lines. The preliminary SAR studies demonstrated that compound
26af (c-Met IC50 = 2.89 nM) was the most promising compound compared with the positive foretinib, which exhibited the
remarkable antiproliferative activities, with IC50 values ranging from 0.28 to 0.72 μM. Mechanistic studies of 26af showed the
anticancer activity was closely related to the blocking phosphorylation of c-Met, leading to cell cycle arresting at G2/M phase
and apoptosis of A549 cells by a concentration-dependent manner. The promising compound 26af was further identified as a
relatively selective inhibitor of c-Met kinase, which also possessed an acceptable safety profile and favorable pharmacokinetic
properties in BALB/c mouse. The favorable drug-likeness of 26af suggested that N-sulfonylamidines may be used as a promising
scaffold for antitumor drug development. Additionally, the docking study and molecular dynamics simulations of 26af revealed a
common mode of interaction with the binding site of c-Met. These positive results indicated that compound 26af is a potential
anti-cancer candidate for clinical trials, and deserves further development as a selective c-Met inhibitor.
Keywords: N-Sulfonylamidine; c-Met inhibitors; Cu-catalyzed three-component reaction; biological evaluation; docking study.

1. Introduction
Mesenchymal-epithelial transition factor (c-Met), also termed as hepatocyte growth factor receptor (HGFR), is a structurally
distinct member of heterodimeric transmemeric receptor tyrosine kinase composed of an extracellular α chain and a membrane
spanning β chain that are connected by a disulfide bond [1,2]. Phosphorylated c-Met further triggers the activation of
downstream signaling pathways such as the Ras/MAPK, c-Src and PI3K/Akt pathways that contributes to the regulation of many
physiological processes including cell proliferation, survival, motility, migration, morphogenic differentiation and angiogenesis
[3–7]. Aberrant c-Met signaling is implicated in the development and progression in a variety of human solid cancers such as
thyroid cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancers, renal cancer, etc [8-12]. More
importantly, convincing evidences disclosed that both overexpression of c-Met and MET amplification have been correlated with
advanced disease stage and poor prognosis [13-15]. Furthermore, crosstalk between c-Met and other receptor tyrosine kinases
promotes aggressive tumor behavior and resistance to conventional cancer therapy [16,17]. Therefore, inhibition of the abnormal
activation of c-Met/HGF signaling pathway represents one of the most promising therapeutic targets in the discovery of
anticancer drugs.
Up to now, many strategies have applied to regulate the abnormal activation of c-Met/HGF signaling pathway and small
molecule c-Met kinase inhibitors as one of the most promising approaches has been attracted more and more attention [18-24].
The advantage of small molecule inhibitors via the intracellular kinase domain is that they can effectively block both
ligand-dependent and independent activation of c-Met [25-31]. Although the development of small-molecule c-Met tyrosine
kinase inhibitors (TKIs) started much later than other strategies, significant progress has been made recently with a considerable
number of compounds entering clinical trials or being approved as anticancer drugs (Fig. 1). The reported small-molecule
inhibitors can be roughly divided into two types according to their structures and binding modes with the kinase catalytic domain
of c-Met, namely type I and type II [32-34]. As reported recently, type II inhibitors may be more effective than type I inhibitors
against the mutations near the active site of c-Met because type II inhibitors are assumed to be able to walk away from the
gatekeeper and bind beyond the entrance of the c-Met active site [35-39]. Various type II c-Met inhibitors have been developed
to narrow the gap between bioactive compounds and promising anticancer candidates, leading to some type II c-Met inhibitors
entering clinical trials [40-43]. However, a poor selectivity profile may make the related type II Met inhibitors display a certain
degree of toxicity in various organs, or lead to suboptimal dosing for the c-Met inhibition in clinical applications [44,45].
Accordingly, discovery of novel type II c-Met inhibitors with improved selectivity profiles, particularly to VEGFR-2, and
minimal side effects becomes a high priority [46].

Fig. 1. The representative c-Met kinase inhibitors of different types.
As shown in Fig. 1, the structures of type II c-Met inhibitors can be disconnected into four units (block A–D) according to
their structures and interaction characteristics with protein molecular. Usually, the modification of moiety A and moiety B is
constrained since the binding modes of c-Met kinase with type II inhibitors. The modification of moiety C, including linear and
cyclic fragments between moieties A and B, is the most successful strategy to develop new c-Met inhibitors, which is known as
‘5 atoms regulation/hydrogen-bond donors or acceptors’ [47-50]. These structural characteristics indicated that exploring a
suitable linker might be a feasible way to discover novel type II c-Met inhibitors. Although many efforts have been paid to the
construction of the 5-atom linker (Fig. 2), one-pot synthesis of the 5-atom linker is still a significant challenge. Multicomponent
reactions (MCRs) deemed to satisfy this criterion in view of their ability to generate structurally diverse molecules in a simple
one-pot reaction [51,52]. It is worth to note that the marriage of MCRs and combinatorial synthesis has innovated medicinal
chemistry by boosting diversification of organic molecules to discover new drug candidates. Amidines [53-55] and sulfonates
[56,57] have attracted continuous attention due to their fundamental roles in many selective drugs with low toxicity,
sulfonylamidine (Fig. 2) could be a potential linker candidate for c-Met inhibitors. To our delight, the sulfonylamidine scaffold
also conformed to the characteristics of the ‘5 atoms regulation’ and contained both hydrogen-bond donor and acceptor. In
this context, we hope that by using sulfonylamidine as the 5-atom linker could facilitate the discovery of highly selective c-Met
inhibitors. Accordingly, we reported for the first time the one-pot synthesis of the sulfonylamidine linker via Cu-catalyzed
modified Click chemistry [58-61], which provided a rapid approach towards the synthesis of new-designed c-Met inhibitors.
Meanwhile, various substituents were introduced into the moiety A, moiety D as well as the moiety C (5-atom linker) to
investigate the effects on activity. All target compounds were evaluated for their c-Met kinase activity and antiproliferative
activities against four cancer cell lines, including H460, HT-29, MKN-45 and MDA-MB-231. Moreover, the cell apoptosis and

cycle arrest, western blotting, acute toxicity, selectivity index, kinase selectivity and pharmacokinetic profiles and docking study
of the representative compound 26af were further explored. Inspiringly, 26af showed remarkable antiproliferative effects, high
selectivity to c-Met vs VEGFR-2, favorable pharmacokinetic properties and an acceptable safety profile, which suggested 26af is
a potential anti-cancer candidate for clinical trials, and deserves further development as a selective c-Met inhibitor.
Fig. 2. Various moiety C structures with linear or cyclic 5-atom linkers.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of 3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)aniline (5) and 3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)aniline
(9)
As shown in Scheme 1, 3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)aniline 5 was synthesized from commercially available
3-amino-2-thiophenecarboxylic acid methyl ester, which underwent saponification reaction followed by decarboxylation reaction
under basic conditions to afford 3-aminothiophene 1 with good overall yield [62]. Condensation of 1 with Mander’s reagent and
subsequent intramolecular cyclization in hot diphenyl ether provided 4H-thieno[3,2-b]pyridin-7-one 3 in 65% overall yield [63].
7-Chloro-thieno[3,2-b]pyridine 4 was obtained in 83% yield by treating 3 on exposure of phosphorus oxychloride. Etherification
of 4 with 4-amino-2-fluorophenol generated amide 5 in 68% yield, and 20% of 4 was recovered. The intermediate 9 was also
synthesized starting from 3-amino-2-thiophenecarboxylic acid methyl ester as illustrated in Scheme 1. This commercially
available material underwent a sequence of formylation, cyclization, and chlorination to afford 8 in 47% overall yield [64].
Coupling of 4-chloro-thieno[3,2-d]pyrimidine 8 with 4-amino-2-fluorophenol in the presence of NaH to yield the intermediate 9

in 76% yield [65].
Scheme 1. Reagents and conditions: (a) i) 2 M NaOH, reflux, 30 min; ii) oxalic acid, 1-propanol, 38 °C, 45 min; (b) triethyl orthoformate,
2,2-dimethyl-1,3-dioxane-4,6-dione, 85 °C, overnight; (c) Ph2O, 240 °C, 30 min; (d) POCl3, DMF (cat.),
0 °C → reflux, 2 h; (e)
4-amino-2-fluorophenol, NaH, DMSO, 0 °C → 60 °C, overnight; (f) HCOOH, Ac2O, 0 °C → r.t., 12 h; (g) ammonium formate, formamide, 150 °C,
8 h; (h) oxalyl chloride, DMF (cat.), CH2Cl2, 0 °C → reflux, 3 h.
2.1.2. Synthesis of 4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline (14)
The synthesis of intermediate 14 was depicted in Scheme 2. Regioselective nitration of 3,4-dimethoxybenzoic acid followed
hydrogenation with H2 in the presence of Raney nickel afforded 2-amino-4,5-dimethoxybenzoic acid 11 in 57% overall yield,
which underwent an intramolecular cyclization in formamidine to give quinazolinone 12 with good efficiency [66]. Subsequently,
chlorination of quinazolinone 12 using phosphorous oxychloride followed by etherification with 4-amino-2-fluorophenol
provided amide 14 in 48% overall yield [65].
Scheme 2. Reagents and conditions: (a) HNO3 (20%), 60 °C, 5 h; (b) H2, Raney nickel, 2-propanol, 40 °C, 5 h; (c) formamide, 150 °C, 8 h; (d)
POCl3, 0 °C → reflux, 9 h; (e) 4-amino-2-fluorophenol, NaH, DMSO, 0 °C → 60 °C, overnight.
2.1.3. Synthesis of 6,7-disubstituted-4-phenoxyquinolines 22a–f
As shown in Scheme 3, the key intermediates of 6,7-disubstituted-4-phenoxyquinolines 22a–f were synthesized from
commercially available 4-hydroxy-3-methoxyacetophenone, which was alkylated with iodomethane, 1-bromobutane or
1-bromo-3-chloropropane under basic reaction conditions to provide 15a–c in excellent yields. Regioselective nitration and
subsequent aminomethylenation with N,N-dimethylformamide dimethyl acetal (DMF-DMA) to afford intermediates 17a–c,
which underwent an intramolecular cyclization in the presence of iron powder and acetic acid to give 18a–c with good overall
efficiency. Compounds 18a–c were converted into compounds 19a–c on exposure of phosphorus oxychloride in high yields.
Treatment of 19c with different secondary amines under basic conditions to generate the intermediates 20a–d. Compounds
19a–b and 20a–d were subjected to a two-step sequence of etherification and hydrogenation for the elegant synthesis of amides
22a–f.

Scheme 3. Reagents and conditions: (a) CH3I, K2CO3, acetone, r.t.,
4 h; (b) 1-bromobutane, K2CO3, DMF, 80 °C, 10 h; (c)
1-bromo-3-chloropropane, K2CO3, DMF, r.t., overnight; (d) concentrated nitric acid (20%), 0 °C, overnight; (e) DMF-DMA, toluene, reflux, 10 h;
(f) Fe (powder), AcOH, 80 °C, 2 h; (g) POCl3, DMF (cat.), reflux, 1 h; (h) secondary amines, DMF, NaI, 85 °C, 10 h; (i) 2-fluoro-4-nirophenol,
chlorobenzene, 140 °C, 20 h; (j) SnCl2.2H2O, EtOH, 70 °C, 6 h.
2.1.4. Synthesis of benzylsulfonyl azides 25a–k
The condensation of thiourea with different substituted alkyl chlorides 23a–k followed by chlorosulfonation with
N-chlorosuccinimide afforded sulfonyl chlorides 24a–k [67]. Further substitution of 24a–k with sodium azide provided sulfonyl
azides 25a–k smoothly in moderate overall yields.
Scheme 4. (a) reflux, 1 h; (b) NCS, 2 M HCl, CH3CN, 0 °C → r.t., 0.5 h; (c) NaN3, 0 °C→ r.t., 2 h.
2.1.5. Synthesis of target compounds bearing sulfonylamidine scaffold
A variety of amines 5, 9, 14 and 22a–f were successfully employed as an efficient reacting partner in the Cu-catalyzed
three-component reaction in the presence of pyridine catalyst to afford the desired N-sulfonylamidine-containing target
compounds 26a–am in moderate to excellent yields [68,69]. The coupling reaction has extremely broad substrate scope with all
three components of alkyne, sulfonyl azide and amine. The reaction took place under very mild reaction conditions and displayed
excellent functional group compatibility. The reaction possibly proceeds through a ketenimine intermediate, which is generated
in situ from the Cu-triazole cycloadduct followed by Dimorph rearrangement via α-diazoimine species readily react with an
amine along with release of N2 gas. Altogether, the Cu-catalyzed three-component coupling reaction could be an efficient and
convenient method to combine several active moieties into one molecule, and could be convenient to implement structural
modification via alternation of diverse starting materials. All newly synthesized target compounds were purified by column
chromatography and their structures were characterized by NMR, MS and elemental analysis.

Scheme 5. General synthetic route of target compounds 26a–am.
2.2. Biological evaluation
2.2.1. In vitro enzymatic assays and structure-activity relationships
The c-Met enzymatic assays of all newly synthesized target compounds were evaluated using homogeneous time-resolved
fluorescence (HTRF) assay. Foretinib was used as a positive control, and the results expressed as the half-maximal inhibitory
concentration (IC50) values were presented in Table 1, as mean values of experiments performed in triplicate.
As illustrated in Table 1, these novel derivatives bearing N-sulfonylamidine moiety were found to be active against c-Met
kinase with IC50 values ranging from 2.89 to 76.55 nM; three of them (26af, IC50= 2.89 nM; 26ah, IC50= 4.02 nM; 26aj, IC50=
3.83 nM) showed comparable potency with foretinib (IC50= 2.15 nM), indicating that the introduction of the new moiety
N-sulfonylamidine as ‘five-atom linker’ maintained the potent c-Met kinase inhibitory efficacy. The initial SARs study of moiety
A was performed. Compounds 26a (IC50 =37.43 nM, R = H, R1 = n-butyl) and 26b (IC50 = 28.62 nM, R = 4-F, R1 = n-butyl)
bearing the block thieno[3,2-b]pyridine-7-yl A1 only displayed moderate c-Met kinase inhibitory activity compared with the
positive foretinib. The replacement of the thieno[3,2-b]pyridine A1 with thieno[3,2-d]pyrimidine A2 (26c, IC50 = 60.25 nM, R =
H, R1 = n-butyl; 26d, IC50 = 42.48 nM, R = 4-F, R1 = n-butyl) and 6,7-dimethoxyquinazoline A3 (26e, IC50 = 74.58 nM, R = H,
R1 = n-butyl; 26f, IC50 = 50.87 nM, R = 4-F, R1 = n-butyl) all resulted in a significant loss of potency. Inspiringly, compounds
26g–h (26g, IC50 = 25.74 nM, R = H, R1 = n-butyl; 26h, IC50 = 20.12 nM; R = 4-F, R1 = n-butyl) bearing the block
6,7-dimethoxyquinoline A4 displayed greater potency than compounds 26a–f, suggesting that the quinoline pharmacophore is
more suitable for vander Waals interactions with the backbone of c-Met kinase. Accordingly, quinoline derivatives were further
studied in the following work.
Considering the vital role of 5-atom linker on activity, comprehensive quinoline analogs with diverse R1 substituents,
including phenyl, methoxymethyl, hydrogen atom, 1-cyclohexenyl, 3-pyridyl and 3-thienyl were investigated to further discover
the structure-activity relationships. The SARs based on IC50 values indicated that the R1 group played an important role for the
c-Met kinase inhibitory efficacy. The introduction of phenyl (26i, IC50 = 76.55 nM, R = H, R1 = Phenyl) led to 2.9-fold decrease
compared with 26g (IC50 = 25.74 nM, R = H, R1 = n-butyl). Simultaneously, five-membered heterocyclic groups were also

explored, such as 3-pyridyl 26q (IC50 =60.58 nM, R = H, R1 = 3-pyridyl) and 3-thienyl 26s (IC50 = 55.58 nM, R = H, R1 =
3-thienyl), which led to 2.4- and 2.2-fold loss activity in against c-Met kinase compared with 26g, indicating that the
electron-rich five membered ring made an adverse effect on c-Met inhibitory potency. Moreover, the replacement n-butyl (26g,
IC50 = 25.74 nM, R = H) with 1-cyclohexenyl 26o (IC50 = 50.54 nM, R = H, R1 = 1-cyclohexenyl) resulted in 1.9-fold loss
activity. However, with the introduction of H atom on R1, compound 26m (IC50 = 20.36 nM, R = H, R1 = H, increased 1.3-fold)
displayed a slight increase on c-Met inhibitory efficacy in comparison with 26g. To our delight, introduction of methoxymethyl
26k (IC50 = 15.64 nM, R = H, R1 = methoxymethyl) showed a significant increase on c-Met inhibitory activity. The above results
suggested that electronic effect and steric hindrance of R1 group were sensitive to the c-Met kinase inhibitory activities. Further
studies about the effect of R1 group are in progress in our laboratory and will be reported upon in the future.
With the suitable R1 group (methoxymethyl) in hand, the influence of substituents on the phenyl ring R was further studied.
The SARs based on IC50 values also revealed that the R group also played an important role for the c-Met kinase inhibitory
efficacy due to its essential interactions with the hydrophobic pocket of c-Met. In comparison with 26k (IC50 = 15.64 nM, R = H)
bearing no substituent on phenyl ring, the incorporation of mono-donating groups (mono-EDGs) showed negative tendency in
potency, such as 26u (IC50= 18.72 nM, R = 2-CH3), 26v (IC50 = 22.45 nM, R = 3-CH3) and 26w (IC50 = 27.86 nM, R = 4-CH3). In
view of these results, the utilization of EDGs was not further pursed. Inspiringly, introduction of mono-electron-withdrawing
groups (mono-EWGs, such as F and Cl) on the phenyl ring showed positive influences on activity. It was worth to note that
incorporation of the mono-EWGs at 4-position of the phenyl (26l, IC50 = 9.12 nM, R = 4-F, increased 1.7-fold) showed a higher
preference than that of mono-EWGs at 3-position (26y, IC50 = 11.36 nM, R = 3-F, increased 1.4-fold) and 2-position of the
phenyl (26x, IC50 = 13.28 nM, R = 2-F, increased 1.2-fold). However, the introduction of double electron-withdrawing groups
(26ab, IC50 = 8.38 nM, R = 3,4-di-Cl) showed a slight decrease on c-Met inhibitory efficacy in comparison with
mono-electron-withdrawing derivative (26z, IC50 = 5.35 nM, R = 4-Cl), which suggested a mono-substituted phenyl ring is more
desirable than a disubstituted phenyl ring. The introduction of strong EWGs to phenyl ring derivative 26ac (IC50 = 46.32 nM, R =
4-CF3, decreased 2.9-fold) resulted in a significantly decrease in activities compared with 26k, suggesting that the phenyl
probably need a properly electron density. The hindrance effect of the bulky substituents likely damped the potency because of
the more steric analog 26aa (IC50 = 24.65 nM, R = 4-Br, decreased 1.6-fold) showed obvious decreased activity compared with
26k, indicating that the hydrophobic pocket of c-Met was fairly sensitive to the size of substituents.
Based on the understanding of the cocrystal structural between c-Met and foretinib, the quinoline portion (moiety A) on the
left side not only forms hydrogen bonds with the Pro1158 and Met1160 residues and also maintains vander Waals interactions
with the c-Met backbone. In addition, this quinoline system is directed toward a solvent-exposed area, which provides an
opportunity for the introduction of polar and hydrophilic groups. Encouraged by these results, analogues contained polar groups

on 7-position of quinoline were prepared for SAR exploration. The replacement the methoxy (26z, IC50 = 5.35 nM, A4, R = 4-Cl,
R1 = methoxymethyl) at the 7-position of quinoline with n-butoxy (26ad, IC50 =12.12 nM, A5, R = 4-Cl, R1 = methoxymethyl)
led to a 2.3-fold decrease in c-Met inhibitory activity, which revealed the length of carbon tether was significant to activity.
According to the previous research, a three-carbon tether between these amines and the quinoline has been determined to be the
optimal length for improving the potency. Thus, different amines at the end of this tether were investigated, and all of them
improved the inhibitory potency of c-Met compared with 26z and 26ab, which can be explained by the vander Waals interaction
between the carbon tether and the c-Met backbone. Based on pharmacological data in Table 1, it was found that the introduction
of water-soluble group at 7-position of quinoline nucleus had a marked influence on c-Met inhibitory efficacy in the preferential
order of morpholinyl > N-methyl piperazinyl > piperidinyl > 4-methyl piperidinyl.
The pharmacological data above indicated that the favorable moiety A (quinoline pharmacophore), and the appropriate
electronic density and steric hindrance on the 5-atom linker are the main reasons for the inhibitory activity of c-Met kinase.
Moreover, the pharmacological data demonstrated that the hydrophobic pocket of c-Met kinase can accommodate the
mono-EWGs of the phenyl ring (moiety D), especially the Cl atom at para-position of phenyl rings. Additionally, the
introduction of different hydrophilic groups at 7-position of quinoline nucleus made a positive influence on c-Met inhibitory
efficacy.
Table 1. In vitro c-Met kinase activities of target compounds 26a-am.
Compd.
26a
A
R1
R
H
c-Met IC50 (nM)
37.43 ± 2.56
28.62 ± 2.74
60.25 ± 4.36
42.48 ± 3.25
74.58 ± 4.93
50.87 ± 3.45
25.74 ± 1.98
20.12 ± 1.64
76.55 ± 5.82
A1
A1
A2
A2
A3
A3
A4
A4
A4
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
Phenyl
26b
26c
4-F
H
26d
26e
4-F
H
26f
4-F
H
26g
26h
26i
4-F
H

26j
26k
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A4
A5
A5
A6
A6
A7
A7
A8
A8
A9
A9
Phenyl
4-F
H
63.26 ± 4.85
15.64 ± 1.64
9.12 ± 1.26
Methoxymethyl
Methoxymethyl
26l
4-F
H
26m
26n
H
20.36 ± 2.12
16.55 ± 1.84
50.54 ± 4.66
44.37 ± 3.35
60.58 ± 4.68
52.72 ± 6.29
55.58 ± 4.47
4-F
H
H
26o
1-Cyclohexenyl
1-Cyclohexenyl
3-Pyridyl
3-Pyridyl
3-Thienyl
4-F
26p
H
26q
4-F
26r
26s
H
4-F
26t
3-Thienyl
44.25 ± 3.46
18.72 ± 1.54
22.45 ± 2.78
27.86 ± 3.23
13.28 ± 1.76
11.36 ± 0.98
5.35 ± 0.63
24.65 ± 1.86
8.38 ± 0.85
46.32 ± 3.24
12.12 ± 0.95
16.53 ± 1.69
2.89 ± 0.35
6.26 ± 0.84
4.02 ± 0.51
8.14 ± 0.83
3.83 ± 0.46
7.18 ± 0.68
5.12 ± 0.65
8.22 ± 0.84
2.15 ± 0.18
26u
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
Methoxymethyl
2-Methyl
3-Methyl
4-Methyl
2-F
26v
26w
26x
26y
3-F
26z
4-Cl
26aa
26ab
26ac
26ad
26ae
26af
26ag
26ah
26ai
26aj
26ak
26al
26am
Foretinib
4-Br
3,4-di-Cl
4-CF3
4-Cl
3,4-di-Cl
4-Cl
3,4-di-Cl
4-Cl
3,4-di-Cl
4-Cl
3,4-di-Cl
4-Cl
3,4-di-Cl
2.2.2. In vitro antiproliferative assays of target compounds against four human tumor cell lines
The MTT assay was used to evaluate the cytotoxicity of target compounds to four human tumor cell lines, including three
c-Met-addicted cancer cell lines A549 (human lung carcinoma), HT-29 (human colon adenocarcinoma), MKN-45 (human
gastric cancer) and a c-Met less-sensitive MDA-MB-231 (triple-negative human breast cancer), taking foretinib as positive
control. The results were expressed as half-maximal inhibitory concentration (IC50) values and listed in Table 2, as mean values
of independent experiments performed in triplicate.
As shown in Table 2, most of the target compounds showed moderate to excellent cytotoxic activities to the four tumor cell
lines tested in vitro, with IC50 values ranging from 0.28 to 32.49 μM. The activity of four target compounds to certain cancer
lines was similar or higher than that of foretinib, indicating that the introduction of N-sulfonylamidines as the 5-atom linker

maintained significant cytotoxicity. Higher potency was observed that quinoline nucleus as the moiety A in comparison with
other pharmacophores such as thieno[3,2-b]pyridine A1, thieno[3,2-d]pyrimidine A2 and 6,7-dimethoxyquinazoline A3. What's
more, most of the target compounds displayed good antiproliferative potency against A549, while the antiproliferative potency
against the other three cell lines was relatively poor, indicating that this series of target compounds may possess selectivity for
A549 cell. To our delight, the most promising compound 26af exhibited remarkable cytotoxicity against A549, HT-29 and
MDA-MB-231 with IC50 values of 0.28 ± 0.04, 0.32 ± 0.03, 0.72 ± 0.06 µM, respectively, which were 1.2–1.5 folds more active
than foretinib. The study on structure-activity relationships (SARs) revealed that these analogs showed similar properties as
summarized in the c-Met kinase level mentioned above: (I) quinoline nucleus as the moiety A generally exhibited higher potency
than other pharmacophores such as thieno[3,2-b]pyridine A1, thieno[3,2-d]pyrimidine A2 and quinazoline A3; (II) the target
compounds showed excellent selectivity toward A549 cell; (III) R1 were substituted with methoxymethyl afforded favorable
cytotoxicity against four human tumor cell lines; (IV) the EWGs (such as F and Cl) on the moiety D benefited to the potency,
and the potency of para-substituted phenyl ring (moiety D) was higher than that of ortho- and meta-substituted phenyl rings; (V)
bulky electron-withdrawing groups on the phenyl ring (such as Br and CF3) led to an obvious decrease in cytotoxicity; (VI)
different cyclic tertiary amino groups at the 7-position of quinoline were advantageous for the potent cytotoxicity.
Table 2. Cytotoxic activities for target compounds 26a-am against A549, HT-29, MKN-45 and MDA-MB-231 cell lines in vitro.
IC50 (µmol/L) ± SD
Compd.
A549
HT-29
MKN-45
11.46 ± 1.32
9.17 ± 1.28
21.85 ± 1.12
12.46 ± 1.57
ND
MDA-MB-231
15.28 ± 0.97
14.55 ± 1.75
25.55 ± 3.34
16.82 ± 2.65
ND
26a
26b
26c
26d
26e
26f
8.68 ± 0.95
6.23 ± 0.76
17.28 ± 2.03
10.34 ± 1.33
27.12 ± 1.84
18.63 ± 2.12
7.55 ± 0.82
4.60 ± 0.24
28.45 ± 2.65
20.36 ± 1.82
3.14 ± 0.42
1.42 ± 0.16
5.16 ± 0.52
3.76 ± 0.25
12.15 ± 0.85
9.43 ± 0.94
18.12 ± 1.35
13.16 ± 0.74
14.26 ± 0.74
10.06 ± 1.22
9.63 ± 0.86
7.06 ± 1.14
18.64 ± 1.54
11.15 ± 0.75
30.52 ± 2.43
22.14 ± 2.68
8.26 ± 0.69
5.18 ± 0.52
32.63 ± 2.24
23.66 ± 2.18
3.52 ± 0.28
1.68 ± 0.22
5.85 ± 0.35
4.12 ± 0.38
14.46 ± 1.44
10.32 ± 1.45
20.56 ± 1.85
15.45 ± 1.66
16.46 ± 0.85
11.24 ± 0.96
23.36 ± 1.58
9.28 ± 1.08
5.54 ± 0.63
26.33 ± 1.95
ND
31.76 ± 3.46
12.83 ± 1.44
9.04 ± 0.65
ND
26g
26h
26i
26j
32.49 ± 2.74
6.64 ± 0.57
4.24 ± 0.33
9.29 ± 0.63
7.65 ± 0.52
20.45 ± 1.88
19.65 ± 1.69
28.28 ± 3.16
22.30 ± 2.54
23.83 ± 3.15
19.52 ± 1.45
26k
26l
4.05 ± 0.36
2.15 ± 0.25
5.99 ± 0.63
4.55 ± 0.42
16.26 ± 1.14
15.24 ± 1.26
22.86 ± 1.75
18.63 ± 1.45
ND
26m
26n
26o
26p
26q
26r
26s
26t
11.86 ± 1.34

26u
26v
4.15 ± 0.36
7.54 ± 0.56
8.02 ± 0.68
2.85 ± 0.35
2.14 ± 0.18
0.62 ± 0.09
7.14 ± 0.65
0.71 ± 0.08
11.35 ± 0.86
2.56 ± 0.32
3.53 ± 0.48
0.28 ± 0.04
0.47 ± 0.08
0.35 ± 0.05
0.67 ± 0.07
0.32 ± 0.03
0.61 ± 0.09
0.38 ± 0.05
0.72 ± 0.12
0.41 ± 0.05
4.85 ± 0.61
8.02 ± 0.75
8.74 ± 0.94
3.16 ± 0.39
2.47 ± 0.41
0.91 ± 0.07
8.03 ± 0.92
0.88 ± 0.12
13.24 ± 1.12
2.85 ± 0.28
4.05 ± 0.51
0.32 ± 0.03
0.59 ± 0.07
0.41 ± 0.06
0.78 ± 0.05
0.36 ± 0.04
0.74 ± 0.06
0.55 ± 0.04
0.88 ± 0.09
0.46 ± 0.03
5.16 ± 0.64
9.15 ± 1.16
9.75 ± 1.22
3.26 ± 0.43
3.52. ± 0.45
1.05 ± 0.13
11.28 ± 1.46
1.16 ± 0.15
15.26 ± 1.46
3.05 ± 0.61
4.36 ± 0.72
0.48 ± 0.06
0.72 ± 0.05
0.65 ± 0.06
0.88 ± 0.09
0.61 ± 0.07
0.82 ± 0.11
0.76 ± 0.08
1.15 ± 0.15
0.064 ± 0.007
8.19 ± 0.91
13.06 ± 1.52
13.66 ± 1.56
5.67 ± 0.63
5.34 ± 0.63
2.32 ± 0.27
12.12 ± 1.38
3.32 ± 0.27
20.37 ± 2.04
5.46 ± 0.63
7.19 ± 0.64
0.72 ± 0.06
1.04 ± 0.12
1.12 ± 0.23
0.93 ± 0.13
1.24 ± 0.15
1.35 ± 0.16
1.56 ± 0.14
1.98 ± 0.18
0.95 ± 0.09
26w
26x
26y
26z
26aa
26ab
26ac
26ad
26ae
26af
26ag
26ah
26ai
26aj
26ak
26al
26am
Foretinib
Bold values show the IC50 values of the prepared compounds lower than the values of the positive control foretinib. ND: Not determined.
a Used as a positive control.
2.2.3. cell apoptosis and cycle arrest
In view of A549 cell line displayed higher sensitivity than the other three tested cancer cell lines to 26af in the preliminary
cytotoxicity profile, A549 cells were used in our mechanistic study. To investigate the specific mechanisms of antiproliferative
activity associated with 26af on human tumor cells, the effect of 26af on A-549 cell apoptosis and cells cycle analysis was
confirmed indirectly using Flow Cytometer by double staining with FITC-annexin V and propidium iodide. Initially, we
investigated the effect of 26af on apoptosis induction. As shown in Fig. 3, the flow cytometric analysis showed significant
increase in the percentage of Annexin-FITC/PI-positive apoptotic cells after A549 cells were treated with 26af with a series of
concentrations (0.25, 0.5, and 1.0 µM). Specifically, compared with the control group, compound 26af (1.0 µM) could
significantly induce the late apoptotic (30.99%) and early apoptotic (15.69%), the similar trend was observed in other
concentrations (0.25, 0.5 µM). The results indicated that compound 26af can induce apoptosis in a concentration-dependent
manner in the early and late stages of apoptosis. Moreover, the percentage of Annexin-FITC/PI-positive apoptotic cells of 26af
was higher than (annexin V positive cell population: 26af versus foretinib, early apoptosis: 15.69% versus 11.25%; late apoptosis:
30.99% versus 22.90%) that of foretinib when treated with the same concentration (1.0 µM), which could be concluded that
compound 26af can induce apoptosis of A549 more efficient than foretinib. Subsequently, we examined the effect of 26af on cell
cycle distribution and induction of apoptosis in different phases in A549 cells. As shown in Fig. 4, after the cells were treated

with vehicle or various concentrations of 26af (0.25, 0.5, and 1.0 µM), DNA content of the control group displayed a typical
histogram in exponentially growing cells. The increasing concentration of 26af caused the percentage of A549 cells in G2/M
phase was significantly increased by a concentration dependent manner, similar with the effect of foretinib on A549 cells. Taken
together, these synergistic results demonstrated that 26af displayed a significant inhibitory effect on the proliferation of A549
cells, which may be achieved by cell cycle arrest at G2/M phase of cell mitosis and subsequently apoptosis induction in cellular.
Fig. 3. The effect of 26af on A549 cells apoptosis by Annexin-FITC/PI double staining.
Fig. 4. The effect of 26af on A549 cells cycle distribution cycle distribution by propidium iodide staining with RNase.
2.2.4. Western blotting analysis of c-Met
Biochemical and cellular cytotoxic data demonstrated that compound 26af was an excellent c-Met inhibitor. In order to further
investigate whether the c-Met kinase inhibition of 26af in a cell-free system can be recapitulated in vitro, western blotting assay
as part of the mechanistic validation was carried out to determine the effect of 26af on c-Met activation in A549 cells. A549 cells
were treated with different concentrations of 26af for 1 h or DMSO as negative control. Afterwards, the cells were harvested,
lysed and subjected to western blotting analysis with the antibodies of phospho-Met (Tyr1234/1235), in which the level of
GAPDH served as loading control. As shown in Fig. 5, compound 26af showed excellent inhibition against c-Met
phosphorylation in a concentration-dependent manner. These data are consistent with the biochemical and cellular cytotoxic
potency (Table 1, 2), which suggested that compound 26af inhibited the activity of c-Met contributes mainly to the suppression
of A549 cell proliferation regardless of the mechanistic complexity in c-Met signaling pathways and crosstalk with other receptor
tyrosine kinases.

Fig. 5. Effect of compound 26af on c-Met kinase phosphorylation in A549 cells.
2.2.5. Acute toxicity test
To explore the safety profile of these N-sulfonylamidine derivatives, the acute toxicity of 26af was evaluated in mice. Sixty
8-week-old male BALB/c mice were randomized into six groups (n = 10) to receive 0 (vehicle only), 100, 200, 300, or 400
mg/kg of 26af intraperitoneal injection on day zero. One group was kept without treatment as a normal control. Animals were
observed for 15 days. Treatment with 26af at 400, 300 mg/kg killed 50%, 30% of the mice, respectively. At the lower dose (0,
100, and 200 mg/kg), all treated animals showed no abnormalities, anaphylactic responses, allergic reactions, significant body
weight loss and even animal deaths and were as healthy as the normal control animals throughout the experiment. These results
(Table 3) suggested that administration of 26af at or below 200 mg/kg (i.p.) may be safe for mice.
Table 3. Acute toxicity profile of compound 26af.
Does (mg/kg)
400
Mice (N)
Deaths (N)
Survival on day 15 (%)
10
10
10
10
10
10
5
3
0
0
0
0
50
70
300
200
100
100
100
100
100
Vehicle
Normal
2.2.6. Selectivity index
In order to find out whether these N-sulfonylamidine derivatives are selective to normal cells, the cytotoxicity of compound
26af against Human umbilical vein endothelial cells (HUVEC) and human normal colorectal mucosa epithelial cell (FHC) were
evaluated. As shown in Table 4, the results demonstrated that compound 26af was found to be more selective to normal cells
than to cancer cells in comparison with foretinib. The selectivity index of compound 26af to HUVEC, FHC cells were 4.2 and
19.1, respectively, which was superior to that of the positive foretinib (selectivity index to HUVEC, 0.9; FHC, 8.3). Based on the
results, we could infer that compound 26af has moderate selectivity towards cancer cells over normal cells.
Table 4. The cytotoxicity of compound 26af against HUVEC, FHC, and HT-29 cell lines.
IC50 (µmol/L) ± SD
Compd.
HUVEC
1.35 ± 0.21
0.42 ± 0.05
FHC
HT-29
26af
6.12 ± 0.56
3.83 ± 0.42
0.32 ± 0.03
0.46 ± 0.06
Foretinib

2.2.7. Enzymatic selectivity assays
Considering its remarkable potency against c-Met kinase in vitro and in vivo, compound 26af was selected to further
investigate the kinase selectivity profile. The inhibitory activity of 26af against a panel of kinases including c-Met family
member Ron, highly homologous kinase ALK and other seven tyrosine kinases were assayed using the homogeneous time
resolved fluorescence (HTRF) method (Table 5). In contrast to its high potency against c-Met (IC50 = 2.89 nM), 26af also
exhibited high inhibitory effects against c-Kit (IC50 = 4.26 nM) and Flt-3 (IC50 = 7.28 nM). Moreover, 26af demonstrated
moderate selectivity against PDGFRα, PDGFRβ, Ron, VEGFR-2 and Flt-4 kinase (133-fold, 196-fold, 143-fold, 233-fold,
124-fold, respectively). Additionally, 26af exhibited a slight or no tyrosine kinase inhibitory activity against EGFR and ALK
(IC50 > 10 µM). Compared with foretinib, 26af significantly increased the selectivity for VEGFR-2, which provide a novel
scaffold to investigate the selectivity and reduce VEGFR-2 related side effects. In summary, all the evidences mentioned above
suggested that 26af was a relatively selective inhibitor of c-Met kinase.
Table 5. Kinase selectivity profile of compound 26af and foretinib.
Enzyme IC50 (nM)
Enzyme
26af
Foretinib
c-kit
PDGFRα
PDGFRβ
Ron
4.26
6.52
384
5.52
565
10.63
3.92
412
VEGFR-2
EGFR
Flt-3
673
4.26
>10,000
7.28
3180
5.24
ALK
>10,000
358
>5,000
4.95
Flt-4
c-Met
2.89
2.15
2.2.8. Pharmacokinnetic (PK) profile of the selected compound 26af
With its remarkable in vitro enzyme and cell potencies, the pharmacokinetic properties of oral and intravenous administrated
26af were further evaluated in BALB/c mouse, and the results were summarized in Table 6. After oral administration, compound
26af (8 mg/kg) presented favorable PK profiles, in briefly, rapid absorption (Tmax= 2.5 h), high maximum concentration (Cmax
=
1228.4 ng/mL), high plasma exposure (AUC0-∞ = 6.8 µg.h.mL-1), accepted elimination half-life (T1/2 = 3.5 h), and well clearance
(1.18 L.h-1.kg-1). After intravenous injecting 26af (2 mg/kg), the maximum concentration was 675.6 ng/mL, plasma exposure was
2.3 µg h mL-1, and elimination half-life was 1.8 h. To our delight, compound 26af had a moderate oral bioavailability (74%) in
mouse. Given the promising overall PK profiles and the novelty of N-sulfonylamidine moiety, which suggested compound 26af
could be a potential candidate for cancer therapy deserving further study.

Table 6. Pharmacokinetic profiles of compound 26af in BALB/c mouse.
Route
i.v.
Does (mg/kg)
T1/2 (h)
1.8
Cmax (ng.mL-1)
675.6
Tmax (h)
AUC0-∞ (µg.h.mL-1)
CL (L.h-1.kg-1)
F (%)
2
8
－
2.3
6.8
－
p.o.
3.5
1228.4
2.5
1.18
74
2.3. Molecular docking studies and molecular dynamics (MD) simulations
To further explore the binding modes of target compounds with the active site of c-Met, molecular docking simulation studies
were carried out by using Autodock 4.2 package. Based on the in vitro inhibition results, we selected compound 26af, the best
c-Met inhibitor in this study, as ligand example, and the structures of c-Met/VEGFR-2 were selected as the docking model (PDB
ID code: 3LQ8, 3U6J, respectively). In blind docking process, the one conformation with the lowest binding energy was used to
predict the protein-aptamer binding spots. The binding modes of compound 26af with c-Met or VEGFR-2 were shown in Fig. 6A,
6B, respectively. As shown in Fig. 6A, the nitrogen atom of quinoline, the oxygen atom of sulfonyl group in compound 26af
formed two H-bond interactions with residue Met1160 and Lys1110 of c-Met, respectively. One π-π interaction between the
2-fluorophenyl ring of compound 26af and Phe1223 has been formed simultaneously. Inspiringly, the nitrogen atom of
morpholine formed one H-bond interaction with residue Asn1171. In contrast, the nitrogen atom of quinoline, the nitrogen atom
of NH in 26af only formed two H-bond interactions with VEGFR-2 residue Cys919 and Thr916 as shown in Fig. 6B. The
differences between the two binding modes (c-Met vs VEGFR-2) might be the main reasons that the c-Met selective preferred
than VEGFR-2 in terms of compound 26af. More importantly, the complex structure of 26af/c-Met (Fig. 6A, -20.57 kcal/mol)
with lower binding energy from precise docking process compared with 26af/VEGFR-2 (Fig. 6B, -8.53 kcal/mol).
The results obtained from the above docking analysis provoked us to explore the dynamic behavior of c-Met-26af/foretinib
complexes. A total of 10 ns simulation time was conducted and MDs trajectory was employed for extracting the refined binding
model (Fig. 6C). The binding interface were mainly located at three regions, part A, part B and part C, one schematic diagram
for each region were shown in Fig. 6D, Fig. 6E and Fig. 6F. In part A, the terminal 4-chlorophenyl ring of 26af fitted into the
hydrophobic pocket which was formed hydrophobic amino residues Val1155, Leu1157, Thr1126, Met1131, Ile1130, Phe1134,
Phe1200, Leu1195, Val1139 and Leu1140, etc. Compared with foretinib, the terminal phenyl ring moiety of 26af was not fully
exposed to the hydrophobic pocket, which made the hydrophobic effects of 26af seemed to be relatively weaker than that of
foretinib. In part B, the oxygen atom of sulfonyl group in compound 26af formed a H-bond with residue Lys1110. Additionly, a
salt-bridge effects was observed between the oxygen atom of sulfonyl group and residue Lys1110 because of Lys belongs to
polar amino acid. Moreover, the branch chain of -CH2-CH2-O-CH3 formed hydrophobic interactions with another hydrophobic
pocket that consisted of Asp1222, Asn1209, Phe1223, Met1211, Leu1140 and Leu1157, etc. The results suggested that the
interactions of N-sulfonylamidine moiety might be the main reasons that the antiproliferative activities of 26af was superior to
that of foretinib. For part C, the nitrogen atom of quinoline in compound 26af formed one H-bond with residue Met1160, which

was similar with the complex of foretinib/c-Met. To our surprise, the nitrogen atom of morpholine formed another H-bond
interaction with the residue Asn1171. In summary, these results of the docking studies and MD simulations showed that
4-phenoxyquinoline derivatives containing N-sulfonylamidine moiety could act synergistically to interact with the binding sites
of c-Met, suggesting that the N-sulfonylamidine moiety could serve as an excellent platform from which to develop potential
antitumor candidates.
6A
6C
6B
6D
6E
6F
Part B
Fig. 6. (A) Binding pose of compound 26af with the active site of c-Met. Compound 26af was showed in colored sticks, green: carbon atom, blue:
nitrogen atom, pink: oxygen atom, yellow: sulfur atom. (B) Binding pose of compound 26af with the active site of VEGFR-2. (C) Overlay of 26af
(green color) in the same cavity along with foretinib (pink color) as the reference molecule (A, B and C). (D) The interaction details of part A. (E)
The interaction details of part B. (F) The interaction details of part C.
3. Conclusion
In summary, different N-sulfonylamidine derivatives as potent c-Met inhibitors were designed, synthesized and evaluated
employing a well-planned optimization strategy to investigate the influence of linkers as well as different moieties A and D on
the expected antitumor activity. Most of the target compounds demonstrated potent antiproliferative activities in A549, HT-29,
MKN-45 and MDA-MB-231 cell lines as well as c-Met kinase, which suggested the introduction of N-sulfonylamidineas as the
5-atom linker maintained or even increased the potent activity. The preliminary SAR studies was carried out, and 26af was
identified as the most potent and relatively selective c-Met inhibitor at both the enzyme-based and cell-based assay compared
with that of foretinib. The cell cycle and apoptosis results proved that compound 26af presented a significant inhibitory effect on
the proliferation of A549 cells, which may be achieved by periodic blocking and apoptosis induction in the G2/M phase.
Furthermore, 26af inhibited phosphorylation levels of c-Met protein in A549 cells by a dose-dependent manner. In addition, the

safety profiles of 26af were further studied and no obvious toxicity was observed in acute toxicity tests. Molecular docking of
26af into ATP binding site of c-Met and VEGFR-2 was performed, and the results suggested that 26af could bind with the active
site of c-Met better than that of VEGFR-2. Meanwhile, the preliminary results in vivo reflected that compound 26af possessed
promising overall PK profiles, and the favorable drug-likeness of 26af indicated that N-sulfonylamidine could be used a novel
scaffold for the development of c-Met inhibitors. Collectively, these positive results highlight that c-Met inhibitor 26af is a
potential antitumor candidate, warranting further investigation and development.
4. Experimental
4.1. Chemistry
Common reagents and materials were purchased from commercial sources and were used without further purification unless
otherwise noted. Organic solvents were routinely dried and/or distilled prior to use and stored over molecular sieves under argon.
Analytical thin layer chromatography (TLC) was performed on precoated silica gel 60 GF254 plates. Flash column
chromatography was run on silica gel (200-300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China).
1
Visualization on TLC was achieved by use of UV light (254 nm) or iodine. H and 13C NMR spectra were recorded on Bruker
Avance III 400 MHz NMR using tetramethylsilane (TMS) as internal standard. The chemical shifts are expressed in ppm and
coupling constants are given in Hz. Data for 1H NMR are recorded as follows: chemical shift (δ, ppm), multiplicity (s = singlet; d
= doublet; t = triplet; q = quarter; p = pentet; m = multiplet; br = broad), coupling constant (Hz), integration. Data for 13C NMR
are reported in terms of chemical shift (δ, ppm). Mass spectra were recorded on a Bruker Daltonics APEXII49e spectrometer
with ESI source as ionization. Melting points were measured by using a Gongyi X-5 microscopy digital melting point apparatus
and are uncorrected. Elemental analysis was performed on an Elemental Analyzer vario EL Cube instrument. All yields are
unoptimized and generally represent the result of a single experiment.
4.1.1.
General
procedure
for
preparation
of
3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)aniline
(5)
,
3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)aniline (9) and 4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline (14)
According to the reported methods as shown in Scheme 1 and Scheme 2, the preparation of intermediates 5, 9 and 14 were
obtained in a multistep process start with commercially available 3-amino-2-thiophenecarboxylic acid methyl ester and
3,4-dimethoxybenzoic acid, respectively. And thus the details of synthesis were not be listed here.
4.1.1.1. 3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)aniline (5)
Yellow solid, m.p.: 112–114 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.49 (d, J = 5.2 Hz, 1 H), 8.13 (d, J = 5.6 Hz, 1 H), 7.57 (d,
J = 5.6 Hz, 1 H), 7.10 (t, J = 9.2 Hz, 1 H), 6.57 (d, J = 5.2 Hz, 1 H), 6.53 (dd, J = 2.4, 13.2 Hz, 1 H), 6.45 (dd, J = 2.0, 8.4 Hz, 1
H), 5.55 (br s, 2 H). 13C NMR (100 MHz, DMSO-d6) δ 160.1, 158.7, 154.4 (d, J = 242.1 Hz), 149.5, 148.9 (d, J = 10.3 Hz), 132.0,

128.8 (d, J = 12.5 Hz), 124.9, 124.1 (d, J = 1.6 Hz), 120.5, 110.0 (d, J = 2.1 Hz), 102.8, 101.3 (d, J = 20.7 Hz). ESI-MS: m/z
261.1 [M+H]+.
4.1.1.2. 3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)aniline (9)
Brown solid, m.p.: 159–161 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.70 (s, 1 H), 8.46 (d, J = 5.2 Hz, 1 H), 7.66 (d, J = 5.6 Hz,
1 H), 7.08 (t, J = 9.2 Hz, 1 H), 6.50 (dd, J = 2.0, 12.8 Hz, 1 H), 6.42 (dd, J = 1.6, 8.4 Hz, 1 H), 5.47 (br s, 2 H). 13C NMR (100
MHz, DMSO-d6) δ 163.5, 163.1, 154.3 (d, J = 241.4 Hz), 154.2, 148.7 (d, J = 10.3 Hz), 137.3, 127.9 (d, J = 13.1 Hz), 124.2,
116.2, 109.5 (d, J = 1.8 Hz), 101.0 (d, J = 20.9 Hz). ESI-MS: m/z 262.1 [M+H]+.
4.1.1.3. 4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline (14)
Brown solid, m.p.: 225–227 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1 H), 7.52 (s, 1 H), 7.40 (s, 1 H), 7.02 (t, J = 8.4 Hz,
1 H), 6.52 (dd, J = 2.4 Hz, J = 12.8 Hz, 1 H), 6.42 (dd, J = 2.0 Hz, J = 8.4 Hz, 1 H), 5.39 (br s, 2 H), 3.96 (s, 3 H) , 3.94 (s, 3 H).
13C NMR (100 MHz, DMSO-d6) δ 164.5, 155.7, 154.3 (d, J = 241.2 Hz), 152.2, 150.0, 148.7, 148.3 (d, J = 10.3 Hz), 128.5 (d, J
= 13.0 Hz), 124.1, 109.5, 109.2, 106.7, 101.1 (d, J = 21.0 Hz), 100.5, 56.1, 55.9. ESI-MS: m/z 316.1 [M+H]+.
4.1.2. General procedure for preparation of 3-fluoro-4-(6,7-disubstituted quinolin-4-yloxy)anilines (22a-f)
The preparation of intermediates 22a–f has been illustrated in detail in our previous work [70] as shown in Scheme 3, so the
synthesis method would not be listed here.
4.1.2.1. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluoroaniline (22a)
White solid, m.p.: 193–195 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 5.6 Hz, 1 H), 7.51 (s, 1 H), 7.38 (s, 1 H), 7.07 (t,
J = 8.4 Hz, 1 H), 6.56 (dd, J = 2.4, 13.2 Hz, 1 H), 6.47 (dd, J = 2.0, 8.8 Hz, 1 H), 6.39 (d, J = 5.2 Hz, 1 H), 5.50 (s, 2 H), 3.93 (s,
6 H). 13C NMR (100 MHz, DMSO-d6) δ 160.0, 154.3 (d, J = 242.0 Hz), 152.4, 149.1, 148.8, 148.5 (d, J = 10.4 Hz), 146.1, 129.2
(d, J = 12.5 Hz), 124.0, 114.4, 110.0, 107.7, 101.4, 101.2, 98.9, 55.6. ESI-MS: m/z 315.2 [M+H]+.
4.1.2.2. 4-((7-Butoxy-6-methoxyquinolin-4-yl)oxy)-3-fluoroaniline (22b)
Light yellow solid, m.p.: 196–198 °C. 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1 H), 7.57 (s, 1 H), 7.40 (s, 1 H), 7.02
(t, J = 8.8 Hz, 1 H), 6.55 (dd, J = 2.8, 12.0 Hz, 1 H), 6.50–6.47 (m, 1 H), 6.39 (d, J = 5.2 Hz, 1 H), 4.19 (t, J = 6.8 Hz, 2 H), 4.03
(s, 3 H), 1.96-1.88 (m, 2 H), 1.58–1.49 (m, 2 H), 0.99 (t, J = 7.2 Hz, 3 H). 13C NMR (100 MHz, CDCl3) δ 160.8, 155.0 (d, J =
246.8 Hz), 152.3, 149.7, 148.5, 146.5, 145.6 (d, J = 9.5 Hz), 132.5 (d, J = 12.6 Hz), 124.3, 115.3, 110.0 (d, J = 2.9 Hz), 108.3,
103.7 (d, J = 21.2 Hz), 101.7, 99.6, 68.6, 56.1, 30.7, 19.2, 13.8. ESI-MS: m/z 357.2 [M+H]+.
4.1.2.3. 3-Fluoro-4-((6-methoxy-7-(3-(morpholin-4-yl)propoxy)quinolin-4-yl)oxy)aniline (22c)
1
Light yellow solid, m.p.: 217–219 °C. H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1 H), 7.56 (s, 1 H), 7.41 (s, 1 H),
7.02 (t, J = 8.8 Hz, 1 H), 6.57–6.53 (m, 1 H), 6.49 (dd, J = 1.6, 8.4 Hz, 1 H), 6.39 (d, J = 5.2 Hz, 1 H), 4.26 (t, J = 6.4 Hz, 2 H),
4.02 (s, 3 H), 3.71 (t, J = 4.4 Hz, 4 H), 2.57 (t, J = 7.2 Hz, 2 H), 2.48–2.44 (m, 4 H), 2.14–2.10 (m, 2 H). 13C NMR (100 MHz,

CDCl3) δ 160.8, 155.1 (d, J = 247.6 Hz), 152.1, 149.7, 148.9, 146.7, 145.7 (d, J = 9.6 Hz), 132.5 (d, J = 12.7 Hz), 124.4, 115.5,
111.0 (d, J = 2.9 Hz), 108.6, 103.7 (d, J = 21.2 Hz), 101.9, 99.7, 67.2, 67.0, 56.2, 55.4, 53.7, 26.0. ESI-MS: m/z 450.2 [M+Na]+.
4.1.2.4. 3-Fluoro-4-((6-methoxy-7-(3-(piperdine-1-yl)propoxy)quinolin-4-yl)oxy)aniline (22d)
1
Light yellow solid, m.p.: 194–196 °C. H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1 H), 7.57 (s, 1 H), 7.38 (s, 1 H),
7.02 (t, J = 8.8 Hz, 1 H), 6.56 (dd, J = 2.4, 12.0 Hz, 1 H), 6.50 (d, J = 8.4 Hz, 1 H), 6.40 (d, J = 5.2 Hz, 1 H), 4.24 (t, J = 6.0 Hz,
2 H), 4.01 (s, 3 H), 2.86–2.82 (m, 4 H), 2.40–2.36 (m, 2 H), 1.88–1.85 (m, 4 H), 1.61–1.57 (m, 4 H). 13C NMR (100 MHz,
DMSO-d6) δ 160.6, 154.8 (d, J = 241.1 Hz), 152.3, 149.8, 149.3, 149.0 (d, J = 10.8 Hz), 146.7, 129.7 (d, J = 12.7 Hz), 124.4,
114.9, 110.6, 108.9, 102.0 (d, J = 5.5 Hz), 101.8, 99.6, 67.2, 56.2, 55.6, 54.6, 26.6, 26.1, 24.6. ESI-MS: m/z 426.3 [M+H]+.
4.1.2.5. 3-Fluoro-4-((6-methoxy-7-(3-(4-methylpiperazine-1-yl)propoxy)quinolin-4-yl)oxy)aniline (22e)
1
Light yellow solid, m.p.: 202–204 °C. H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1 H), 7.57 (s, 1 H), 7.40 (s, 1 H),
7.02 (t, J = 8.8 Hz, 1 H), 6.58 (dd, J = 2.4, 12.0 Hz, 1 H), 6.54 (dd, J = 2.4, 9.2 Hz, 1 H), 6.41 (d, J = 5.2 Hz, 1 H), 4.26 (t, J = 6.4
Hz, 2 H), 4.03 (s, 3 H), 2.64–2.50 (m, 8 H), 2.16 (s, 3 H), 2.11 (t, J = 10.8 Hz, 2 H), 1.91–1.87 (m, 2 H). 13C NMR (100 MHz,
CDCl3) δ 160.7, 155.1 (d, J = 246.5 Hz), 152.2, 149.7, 148.8, 146.7, 145.8 (d, J = 9.5 Hz), 132.4 (d, J = 12.5 Hz), 124.3 (d, J =
1.9 Hz), 115.4, 111.0 (d, J = 2.7 Hz), 108.6, 103.6 (d, J = 21.2 Hz), 101.8, 99.7, 67.3, 56.1, 55.1, 54.9, 53.1, 46.0, 26.3. ESI-MS:
m/z 441.3 [M+H]+.
4.1.2.6. 3-Fluoro-4-((6-methoxy-7-(3-(4-methylpiperdine-1-yl)propoxy)quinolin-4-yl)oxy)aniline (22f)
1
Light yellow solid, m.p.: 190–192 °C. H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1 H), 7.59 (s, 1 H), 7.40 (s, 1 H),
7.05 (t, J = 8.8 Hz, 1 H), 6.54 (dd, J = 2.4, 12.0 Hz, 1 H), 6.50 (dd, J = 2.8, 8.8 Hz, 1 H), 6.41 (d, J = 5.2 Hz, 1 H), 4.26 (t, J = 6.4
Hz, 2 H), 4.02 (s, 3 H), 2.96 (d, J = 11.6 Hz, 2 H), 2.61–2.57 (m, 2 H), 2.16–2.14 (m, 2 H), 1.96 (t, J = 10.8 Hz, 2 H), 1.65 (d, J =
10.4 Hz, 2 H), 1.32–1.29 (m, 3 H), 0.96 (d, J = 6.0 Hz, 3 H). 13C NMR (100 MHz, CDCl3) δ 160.7, 155.1 (d, J = 246.5 Hz), 152.2,
149.7, 148.8, 146.7, 145.8 (d, J = 9.5 Hz), 132.5 (d, J = 12.7 Hz), 124.4, 115.4, 111.0, 108.7, 103.7 (d, J = 21.1 Hz), 101.8, 99.7,
67.5, 56.1, 55.4, 54.0, 34.2, 30.8, 26.4, 21.9. ESI-MS: m/z 440.2 [M+H]+.
4.1.3. General procedure for preparation of sulfonyl azides 24a–k
Various alkyl halides 23a–k (2.5 mmol) and thiourea (0.19 g, 2.5 mmol) were refluxed together in EtOH (2.5 mL) for 1 h.
After removal of EtOH at reduced pressure, the obtained solid was slowly added to a mixture of NCS (1.33 g, 10 mmol), 2 M
HCl (0.68 mL) and MeCN (4 mL) over 15 min. Upon completion of the reaction (TLC monitoring), the solvent was removed
under vacuum. Then H2O (5 mL) was added to the residue and stirred for 15 min, the resulting solid was filtered and dried under
an infrared lamp to afford the corresponding sulfonyl chlorides 24a-k. Without further purification, 24a-k was added in portions
to a solution of NaN3 (195 mg, 3 mmol) in acetone/H2O (10 mL, acetone/H2O = 1:1) at 0 °C. After being stirred at room
temperaturefor 4 h, the reaction mixture was concentrated in vacuo and H2O (10 mL) was added to the residue. The aqueous

solution was extracted with CHCl3 (3 × 10 mL). The combined organic layers were washed with brine, dried over Na2SO4,
filtered and concentrated under reduced pressure to afford sulfonyl azides 25a–k.
4.1.3.1. Phenylmethanesulfonyl azide (25a)
1
White solid, yield: 65%, m.p.: 50–52 °C. H NMR (400 MHz, CDCl3) δ 7.48–7.44 (m, 5 H), 4.54 (s, 2 H). 13C NMR (100
MHz, CDCl3) δ 131.0, 129.9, 129.3, 126.7, 61.6. ESI-MS: m/z 198.0 [M+H]+.
4.1.3.2. (2-Methylphenyl)methanesulfonyl azide (25b)
1
White solid, yield: 57%, m.p.: 38–40 °C. H NMR (400 MHz, CDCl3) δ 7.42 (d, J = 7.6 Hz, 1 H), 7.34 (d, J = 6.4 Hz, 1 H),
7.29–7.27 (m, 2 H), 4.61 (s, 2 H), 2.48 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 138.7, 131.9, 131.3, 130.1, 126.8, 125.2, 59.4,
19.7. ESI-MS: m/z 212.1 [M+H]+.
4.1.3.3. (3-Methylphenyl)methanesulfonyl azide (25c)
White solid, yield: 66%, m.p.: 39–41 °C. 1H NMR (400 MHz, CDCl3) δ 7.29–7.27 (m, 1 H), 7.24–7.22 (m, 2 H), 7.20 (s, 1 H),
4.44 (s, 2 H), 2.34 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 139.1, 131.6, 130.6, 129.1, 128.0, 126.5, 61.9, 21.3. ESI-MS: m/z
212.1 [M+H]+.
4.1.3.4. (4-Methylphenyl)methanesulfonyl azide (25d)
1
White solid, yield: 78%, m.p.: 56–58 °C. H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 8.0 Hz, 2 H), 7.24 (d, J = 7.6 Hz, 2 H),
4.49 (s, 2 H), 2.39 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 140.1, 130.8, 129.9, 123.6, 61.7, 21.3. ESI-MS: m/z 212.1 [M+H]+.
4.1.3.5. (2-Fluorophenyl)methanesulfonyl azide (25e)
White solid, yield: 69%, m.p.: 40–42 °C. 1H NMR (400 MHz, CDCl3) δ 7.51–7.43 (m, 2 H), 7.25–7.16 (m, 2 H), 4.63 (s, 2 H).
13C NMR (100 MHz, CDCl3) δ 161.4 (d, J = 249.0 Hz), 132.7 (d, J = 2.2 Hz), 132.1 (d, J = 8.2 Hz), 125.0 (d, J = 3.7 Hz), 116.1
(d, J = 21.2 Hz), 114.4 (d, J = 14.2 Hz), 54.9. ESI-MS: m/z 216.0 [M+H]+.
4.1.3.6. (3-Fluorophenyl)methanesulfonyl azide (25f)
White solid, yield: 74%, m.p.: 48–50 °C. 1H NMR (400 MHz, CDCl3) δ 7.48–7.43 (m, 1 H), 7.27–7.17 (m, 3 H), 4.55 (s, 2 H).
13C NMR (100 MHz, CDCl3) δ 162.8 (d, J = 246.7 Hz), 130.9 (d, J = 8.3 Hz), 128.8 (d, J = 7.8 Hz), 126.8 (d, J = 3.0 Hz), 118.0
(d, J = 22.3 Hz), 117.1 (d, J = 21.0 Hz), 61.3. ESI-MS: m/z 216.1 [M+H]+.
4.1.3.7. (4-Fluorophenyl)methanesulfonyl azide (25g)
White solid, yield: 82%, m.p.: 43–45 °C. 1H NMR (400 MHz, CDCl3) δ 7.47–7.44 (m, 2 H), 7.14 (t, J = 8.4 Hz, 2 H), 4.51 (s,
2 H). 13C NMR (100 MHz, CDCl3) δ 163.7 (d, J = 249.1 Hz), 132.9 (d, J = 8.7 Hz), 122.6, 116.5 (d, J = 21.8 Hz), 61.0. ESI-MS:
m/z 216.1 [M+H]+.
4.1.3.8. (4-Chlorophenyl)methanesulfonyl azide (25h)
1
White solid, yield: 85%, m.p.: 65–67 °C. H NMR (400 MHz, CDCl3) δ 7.44–7.39 (m, 4 H), 4.50 (s, 2 H). 13C NMR (100

MHz, CDCl3) δ 136.3, 132.3, 129.6, 125.2, 61.1. ESI-MS: m/z 232.0 [M+H]+.
4.1.3.9. (4-Bromophenyl)methanesulfonyl azide (25i)
1
White solid, yield: 85%, m.p.: 71–73 °C. H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.4 Hz, 2 H), 7.34 (d, J = 8.4 Hz, 2 H),
4.48 (s, 2 H). 13C NMR (100 MHz, CDCl3) δ 132.6, 132.5, 125.7, 124.5, 61.2. ESI-MS: m/z 276.1 [M+H]+.
4.1.3.10. (3,4-Dichlorophenyl)methanesulfonyl azide (25j)
1
White solid, yield: 65%, m.p.: 72–74 °C. H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 2.0 Hz, 1 H), 7.53 (d, J = 8.0 Hz, 1 H),
7.31 (dd, J = 2.4, 8.4 Hz, 1 H), 4.47 (s, 2 H). 13C NMR (100 MHz, CDCl3) δ 134.7, 133.6, 132.8, 131.3, 130.1, 126.7, 60.5.
ESI-MS: m/z 288.0 [M+Na]+.
4.1.3.11. (4-Trifluoromethylphenyl)methanesulfonyl azide (25k)
1
White solid, yield: 48%, m.p.: 81–83 °C. H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.0 Hz, 2 H), 7.61 (d, J = 8.0 Hz, 2 H),
4.59 (s, 2 H). 13C NMR (100 MHz, CDCl3) δ 132.1 (q, J = 32.8 Hz), 131.5, 130.7, 126.3 (q, J = 3.7 Hz), 123.7 (q, J = 270.8 Hz),
61.2. ESI-MS: m/z 266.0 [M+H]+.
4.1.4. General synthetic procedure for target compounds 26a-am
To a stirred mixture of CuI (0.042 mmol), sulfonyl azide (0.48 mmol) and alkyne (0.52 mmol) in CHCl3 (10 mL), amine
nucleophile (0.4 mmol) was added slowly at room temperature under an N2 atmosphere. Pyridine (0.52 mmol) was added prior to
the addition of nucleophiles. After the reaction was completed, as monitored with TLC, the reaction mixture was diluted by
adding CHCl3 (10 mL) and saturated aqueous NH4Cl solution (20 mL). The mixture was stirred for an additional 30 min and two
layers were separated. The aqueous layer was extracted with CHCl3 (3 × 20 mL). The combined organic layers were dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using a
mixture of hexane and ethyl acetate as eluent to afford compounds 26a–am.
4.1.4.1. N'-(Benzylsulfonyl)-N-(3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)phenyl)hexanamidine (26a)
White solid, yield: 74%, m.p.: 99–101 °C. 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1 H), 7.78 (d, J = 5.2 Hz, 1 H), 7.58 (d, J =
5.2 Hz, 1 H), 7.46–7.33 (m, 5 H), 7.24–7.15 (m, 2 H), 6.68 (s, 1 H), 6.50 (d, J = 4.8 Hz, 1 H), 4.35 (s, 2 H), 2.10–1.97 (m, 2 H),
1.74–1.53 (m, 2 H), 1.30–1.21 (m, 4 H), 0.86 (d, J = 7.2 Hz, 3 H). 13C NMR (100 MHz, CDCl3) δ 168.0, 160.0, 158.6, 153.9 (d, J
= 248.5 Hz), 148.8, 137.1 (d, J = 11.3 Hz), 136.8 (d, J = 9.1 Hz), 131.5, 131.2, 130.8, 129.7, 128.6, 128.5, 124.7, 123.3, 118.2,
111.5 (d, J = 22.0 Hz), 103.2, 61.1, 35.6, 31.6, 27.9, 22.2, 13.9. Anal. Calcd. For C26H26FN3O3S2: C, 61.04; H, 5.12; N, 8.21.
Found: C, 61.05; H, 5.10; N, 8.23. ESI-MS: m/z 512.2 [M+H]+.
4.1.4.2. N-(3-Fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)phenyl)-N'-((4-fluorobenzyl)sulfonyl)hexanamidine (26b)
1
White solid, yield: 78%, m.p.: 92–94 °C. H NMR (400 MHz, CDCl3) δ 8.53 (d, J =4.8 Hz, 1 H), 7.79 (d, J = 5.2 Hz, 1 H),
7.59 (d, J = 5.2 Hz, 1 H), 7.42–7.39 (m, 2 H), 7.20–7.02 (m, 4 H), 6.84–6.78 (m, 1 H), 6.51 (d, J = 5.2 Hz, 1 H), 4.31 (s, 2 H),

2.04–1.94 (m, 2 H), 1.76–1.56 (m, 2 H), 1.32–1.27 (m, 4 H), 0.86 (t, J = 6.8 Hz, 3 H). 13C NMR (100 MHz, CDCl3) δ 168.1,
162.9 (d, J = 246.3 Hz), 160.1, 158.4, 153.9 (d, J = 247.9 Hz), 148.6, 137.1 (d, J = 13.3 Hz), 136.8 (d, J = 7.0 Hz), 132.5 (d, J =
7.8 Hz), 131.7, 125.7, 124.6, 123.3, 122.4, 118.2, 115.6 (d, J = 21.5 Hz), 111.6 (d, J = 22.9 Hz), 103.2, 60.2, 35.7, 31.6, 27.9,
22.2, 13.8. Anal. Calcd. For C26H25F2N3O3S2: C, 58.96; H, 4.76; N, 7.93. Found: C, 58.98; H, 4.77; N, 7.95. ESI-MS: m/z 530.1
[M+H]+.
4.1.4.3. N'-(Benzylsulfonyl)-N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)hexanamidine (26c)
White solid, yield: 82%, m.p.: 123–125 °C. 1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1 H), 8.02 (d, J = 5.2 Hz, 1 H), 7.60 (d, J =
5.6 Hz, 1 H), 7.56–7.32 (m, 6 H), 7.18–7.10 (m, 1 H), 6.70–6.64 (m, 1 H), 4.35 (s, 2 H), 1.82–1.53 (m, 4 H), 1.34–1.21 (m, 4 H),
0.88 (t, J = 6.8 Hz, 3 H). 13C NMR (100 MHz, CDCl3) δ 167.9, 163.4, 163.1, 154.0, 153.9 (d, J = 247.5 Hz), 136.4 (d, J = 9.0
Hz), 136.1 (d, J = 13.9 Hz), 135.9, 131.2, 130.9, 129.7, 128.6, 128.4, 124.4, 123.7, 118.1, 111.4 (d, J = 22.7 Hz), 61.1, 35.6, 31.5,
27.7, 22.2, 13.9. Anal. Calcd. For C25H25FN4O3S2: C, 58.58; H, 4.92; N, 10.93. Found: C, 58.60; H, 4.90; N, 10.94. ESI-MS: m/z
513.2 [M+H]+.
4.1.4.4. N-(3-Fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-N'-((4-fluorobenzyl)sulfonyl)hexanamidine (26d)
White solid, yield: 84%, m.p.: 118–120 °C. 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1 H), 8.01 (d, J = 4.8 Hz, 1 H), 7.56 (d, J =
5.2 Hz, 2 H), 7.42–7.33 (m, 2 H), 7.20–7.07 (m, 2 H), 6.99 (t, J = 8.0 Hz, 1 H), 6.79–6.74 (m, 1 H), 4.27 (s, 2 H), 1.72–1.53 (m,
2 H), 1.27–1.23 (m, 6 H), 0.83 (t, J = 6.4 Hz, 3 H). 13C NMR (100 MHz, CDCl3) δ 168.0, 163.4, 162.9, 162.8 (d, J = 245.7 Hz),
153.9, 153.8 (d, J = 248.2 Hz),136.4 (d, J = 9.4 Hz), 132.2, 136.1, 132.6 (d, J = 7.7 Hz), 125.7, 124.3, 123.7, 118.2, 117.4, 115.6
(d, J = 21.5 Hz), 111.6 (d, J = 22.3 Hz), 60.2, 35.6, 31.5, 27.7, 22.2, 13.9. Anal. Calcd. For C25H24F2N4O3S2: C, 56.59; H, 4.56; N,
10.56. Found: C, 56.61; H, 4.59; N, 10.55. ESI-MS: m/z 531.1 [M+H]+.
4.1.4.5. N'-(Benzylsulfonyl)-N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)hexanamidine (26e)
White solid, yield: 68%, m.p.: 132–134 °C. 1H NMR (400 MHz, CDCl3) δ 8.56 (s, 1 H), 7.56–7.30 (m, 8 H), 7.21–7.13 (m, 1
H), 6.69–6.62 (m, 1 H), 4.31 (s, 2 H), 4.04 (s, 3 H), 4.03 (s, 3 H), 2.14–2.11 (m, 2 H), 1.70–1.53 (m, 2 H), 1.27–1.20 (m, 4 H),
0.83 (t, J = 6.8 Hz, 3 H). 13C NMR (100 MHz, CDCl3) δ 167.8, 164.6, 156.2, 153.9 (d, J = 246.7 Hz), 152.5, 150.5, 149.5, 136.7
(d, J = 13.5 Hz), 136.1 (d, J = 9.2 Hz), 131.2, 130.9, 129.8, 128.6, 128.4, 123.8, 118.1, 111.5 (d, J = 24.0 Hz), 106.7, 100.9, 61.1,
56.5, 56.4, 35.6, 31.6, 27.7, 22.2, 13.9. Anal. Calcd. For C29H31FN4O5S: C, 61.47; H, 5.51; N, 9.89. Found: C, 61.45; H, 5.54; N,
9.90. ESI-MS: m/z 589.2 [M+Na]+.
4.1.4.6. N-(4-((6,7-Dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-N'-((4-fluorobenzyl)sulfonyl)hexanamidine (26f)
White solid, yield: 75%, m.p.: 124–126 °C. 1H NMR (400 MHz, CDCl3) δ 8.56 (s, 1 H), 7.55–7.53 (m, 2 H), 7.42–7.34 (m, 2
H), 7.28 (s, 1 H), 7.20 (t, J = 8.0 Hz, 1 H), 7.12–7.05 (m, 1 H), 7.00 (t, J = 8.0 Hz, 1 H), 6.79–6.74 (m, 1 H), 4.26 (s, 2 H), 4.04 (s,
3 H), 4.02 (s, 3 H), 2.19–2.14 (m, 2 H), 1.71–1.53 (m, 2 H), 1.26–1.23 (m, 4 H), 0.83 (t, J = 6.4 Hz, 3 H). 13C NMR (100 MHz,

CDCl3) δ 167.9, 164.6, 162.9 (d, J = 246.3 Hz), 156.2, 153.9 (d, J = 247.5 Hz), 152.4, 150.5, 149.5, 136.9 (d, J = 12.0 Hz), 135.0
(d, J = 8.5 Hz), 132.6 (d, J = 8.2 Hz), 125.7, 123.9, 118.2, 115.5 (d, J = 21.6 Hz), 111.7 (d, J = 23.4 Hz), 110.1, 106.7, 100.9,
60.2, 56.4, 35.6, 31.5, 27.7, 22.2, 13.9. Anal. Calcd. For C29H30F2N4O5S: C, 59.58; H, 5.17; N, 9.58. Found: C, 59.59; H, 5.15; N,
9.61. ESI-MS: m/z 607.2 [M+Na]+.
4.1.4.7. N'-(Benzylsulfonyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)hexanamidine (26g)
White solid, yield: 76%, m.p.: 141–143 °C. 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 5.2 Hz, 1 H), 7.65 (d, J = 11.6 Hz, 1 H),
7.56 (s, 1 H), 7.50–7.41 (m, 3 H), 7.37 (s, 1 H), 7.30–7.29 (m, 2 H), 7.22–7.20 (m, 1 H), 7.12 (t, J = 8.4 Hz, 1 H), 6.36 (d, J = 4.4
Hz, 1 H), 4.33 (s, 2 H), 4.02 (s, 3 H), 3.98 (s, 3 H), 2.78–2.63 (m, 2 H), 1.75–1.72 (m, 2 H), 1.22–1.20 (m, 4 H), 0.80 (t, J = 6.0
Hz, 3 H). 13C NMR (100 MHz, CDCl3) δ 167.9, 160.1, 154.0 (d, J = 246.5 Hz), 153.1, 149.8, 148.5, 146.7, 137.8 (d, J = 12.7 Hz),
136.3 (d, J = 9.5 Hz), 130.8, 129.8, 128.6, 128.4, 123.4, 118.2, 115.6, 111.7 (d, J = 23.3 Hz), 107.4, 102.2, 99.5, 61.1, 56.2, 56.1,
35.6, 31.6, 27.9, 22.2, 13.8. Anal. Calcd. For C30H32FN3O5S: C, 63.70; H, 5.70; N, 7.43. Found: C, 63.71; H, 5.72; N, 7.46.
ESI-MS: m/z 566.2 [M+H]+.
4.1.4.8. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N'-((4-fluorobenzyl)sulfonyl)hexanamidine (26h)
White solid, yield: 68%, m.p.: 152–154 °C. 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 4.4 Hz, 1 H), 7.65 (d, J = 11.6 Hz, 1 H),
7.57 (s, 1 H), 7.51–7.38 (m, 3 H), 7.16 (s, 1 H), 7.01 (t, J = 8.0 Hz, 2 H), 6.83–6.76 (m, 1 H), 6.37 (d, J = 5.2 Hz, 1 H), 4.31 (s, 2
H), 4.04 (s, 3 H), 4.01 (s, 3 H), 2.26–2.17 (m, 2 H), 1.74–1.54 (m, 2 H), 1.25–1.24 (m, 4 H), 0.82 (t, J = 5.6 Hz, 3 H). 13C NMR
(100 MHz, CDCl3) δ 168.0, 162.9 (d, J = 245.2 Hz), 160.1, 154.0 (d, J = 249.3 Hz), 153.2, 149.8, 148.4, 146.7, 137.9 (d, J = 13.9
Hz), 136.3 (d, J = 10.0 Hz), 132.5 (d, J = 7.6 Hz), 125.7, 123.4, 118.2, 115.6, 115.5 (d, J = 21.5 Hz), 111.9 (d, J = 23.2 Hz),
107.4, 102.2, 99.5, 60.2, 56.2, 56.1, 35.7, 31.6, 27.9, 22.2, 13.8. Anal. Calcd. For C30H31F2N3O5S: C, 61.74; H, 5.35; N, 7.20.
Found: C, 61.76; H, 5.35; N, 7.21. ESI-MS: m/z 606.2 [M+Na]+.
4.1.4.9. N'-(Benzylsulfonyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-2-phenylacetamidine (26i)
1
Light yellow solid, yield: 54%, m.p.: 104–106 °C. H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 4.8 Hz, 1 H), 7.55 (s, 1 H),
7.48–7.47 (m, 3 H), 7.41 (s, 2 H), 7.40–7.27 (m, 6 H), 7.12 (t, J = 8.4 Hz, 1 H), 6.86–6.82 (m, 2 H), 6.33 (d, J = 5.2 Hz, 1 H),
4.40 (s, 2 H), 4.36 (s, 2 H), 4.05 (s, 3 H), 4.03 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 164.8, 159.9, 154.0 (d, J = 249.3 Hz),
153.1, 149.8, 148.5, 146.8, 138.4 (d, J = 13.4 Hz), 135.3 (d, J = 8.6 Hz), 132.8, 130.9, 130.0, 129.8, 129.6, 128.6, 128.5, 128.4,
123.5, 118.3, 115.5, 111.9 (d, J = 22.7 Hz), 107.7, 102.2, 99.3, 61.1, 56.2, 56.1, 40.6. Anal. Calcd. For C32H28FN3O5S: C, 65.63;
H, 4.82; N, 7.18. Found: C, 65.64; H, 4.81; N, 7.20. ESI-MS: m/z 586.2 [M+H]+.
4.1.4.10. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N'-((4-fluorobenzyl)sulfonyl)-2-phenylacetamidine (26j)
Light yellow solid, yield: 62%, m.p.: 93–95 °C. 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 5.2 Hz, 1 H), 7.55 (s, 1 H), 7.51 (d,
J = 11.6 Hz, 1 H), 7.45–7.42 (m, 5 H), 7.28 (d, J = 6.4 Hz, 2 H), 7.14 (t, J = 8.4 Hz, 1 H), 7.05 (t, J = 8.4 Hz, 2 H), 6.93–6.80 (m,

2 H), 6.33 (d, J = 4.8 Hz, 1 H), 4.39 (s, 2 H), 4.36 (s, 2 H), 4.05 (s, 3 H), 4.04 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 164.8,
164.1, 161.7, 161.1 (d, J = 247.0 Hz), 154.0 (d, J = 248.7 Hz), 153.1, 149.8, 148.5, 146.9, 138.5 (d, J = 12.8 Hz), 135.2 (d, J =
9.6 Hz), 132.8, 132. 6 (d, J = 8.2 Hz), 129.9, 129.6, 128.4, 125.7, 123.5, 118.3, 115.6 (d, J = 21.5 Hz), 112.0 (d, J = 22.4 Hz),
107.6, 102.2, 99.4, 60.1, 56.2, 56.1, 40.6. Anal. Calcd. For C32H27F2N3O5S: C, 63.67; H, 4.51; N, 6.96. Found: C, 63.69; H, 4.52;
N, 6.97. ESI-MS: m/z 603.2 [M]+.
4.1.4.11. N'-(Benzylsulfonyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-(3-methoxy)propanamidine (26k)
Yellow solid, yield: 73%, m.p.: 148–150 °C. 1H NMR (400 MHz, CDCl3) δ 8.85 (br s, 1 H), 8.51 (d, J = 5.2 Hz, 1 H), 7.61 (dd,
J = 2.4, 12.0 Hz, 1 H), 7.58 (s, 1 H), 7.46–7.44 (m, 3 H), 7.37–7.32 (m, 3 H), 7.19 (t, J = 8.4 Hz, 1 H), 7.05–7.03 (m, 1 H), 6.38
(d, J = 5.2 Hz, 1 H), 4.35 (s, 2 H), 4.07 (s, 3 H), 4.05 (s, 3 H), 3.70 (t, J = 5.2 Hz, 2 H), 3.44 (s, 3 H), 3.21 (t, J = 5.2 Hz, 2 H). 13C
NMR (100 MHz, CDCl3) δ 165.4, 160.0, 154.1 (d, J = 248.8 Hz), 153.0, 149.7, 148.6, 146.8, 138.0 (d, J = 12.3 Hz), 135.8 (d, J =
9.3 Hz), 130.8, 129.8, 128.6, 128.4, 123.5, 118.1 (d, J = 3.2 Hz), 115.5, 111.7 (d, J = 22.6 Hz), 107.7, 102.2, 99.4, 68.8, 60.9,
58.9, 56.2, 56.1, 33.9. Anal. Calcd. For C28H28FN3O6S: C, 60.75; H, 5.10; N, 7.59. Found: C, 60.73; H, 5.12; N, 7.60. ESI-MS:
m/z 554.2 [M+H]+.
4.1.4.12. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N'-((4-fluorobenzyl)sulfonyl)-(3-methoxy)propanamidine (26l)
Yellow solid, yield: 76%, m.p.: 145–147 °C. 1H NMR (400 MHz, CDCl3) δ 8.89 (br s, 1 H), 8.51 (d, J = 5.2 Hz, 1 H), 7.63 (dd,
J = 1.6, 12.0 Hz, 1 H), 7.58 (s, 1 H), 7.44 (s, 2 H), 7.42–7.40 (m, 1 H), 7.20 (t, J = 8.4 Hz, 1 H), 7.06–7.02 (m, 3 H), 6.38 (d, J =
5.2 Hz, 1 H), 4.32 (s, 2 H), 4.07 (s, 3 H), 4.05 (s, 3 H), 3.73 (t, J = 5.2 Hz, 2 H), 3.45 (s, 3 H), 3.24 (t, J = 4.8 Hz, 2 H). 13C NMR
(100 MHz, CDCl3) δ 165.4, 162.9 (d, J = 246.2 Hz), 159.9, 154.1 (d, J = 248.4 Hz), 153.0, 149.7, 148.6, 146.9, 138.1 (d, J = 12.4
Hz), 135.7 (d, J = 9.3 Hz), 132.5 (d, J = 8.2 Hz), 125.7 (d, J = 3.0 Hz), 123.5, 118.1 (d , J = 2.9 Hz), 115.6 (d, J = 21.5 Hz),
111.8 (d, J = 22.8 Hz), 107.7, 102.2, 99.3, 68.8, 60.0, 58.9, 56.2, 56.1, 34.0. Anal. Calcd. For C28H27F2N3O6S: C, 58.84; H, 4.76;
N, 7.35. Found: C, 58.87; H, 4.75; N, 7.37. ESI-MS: m/z 572.3 [M+H]+.
4.1.4.13. N'-(Benzylsulfonyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)acetamidine (26m)
1
Light yellow solid, yield: 46%, m.p.: 126–128 °C. H NMR (400 MHz, CDCl3) δ 8.75 (br s, 1 H), 8.46 (d, J = 5.2 Hz, 1 H),
7.64 (d, J = 11.6 Hz, 1 H), 7.57 (s, 1 H), 7.50–7.43 (m, 3 H), 7.38 (s, 1 H), 7.35–7.32 (m, 2 H), 7.20–7.11 (m, 2 H), 6.38 (d, J =
4.8 Hz, 1 H), 4.35 (s, 2 H), 4.04 (s, 3 H), 4.01 (s, 3 H), 2.46 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 164.3, 160.2, 154.0 (d, J =
248.4 Hz), 153.2, 149.8, 148.4, 146.6, 138.0 (d, J = 12.3 Hz), 136.1 (d, J = 9.2 Hz), 130.9, 129.6, 128.7, 128.6, 123.5, 118.2 (d, J
= 3.1 Hz), 115.6, 111.7 (d, J = 22.6 Hz), 107.3, 102.3, 99.5, 61.0, 56.2, 21.9. Anal. Calcd. For C26H24FN3O5S: C, 61.29; H, 4.75;
N, 8.25. Found: C, 61.30; H, 4.77; N, 8.24. ESI-MS: m/z 532.2 [M+Na]+.
4.1.4.14. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N'-((4-fluorobenzyl)sulfonyl)acetamidine (26n)

1
Light yellow solid, yield: 49%, m.p.: 119–121 °C. H NMR (400 MHz, CDCl3) δ 8.68 (br s, 1 H), 8.48 (d, J = 5.2 Hz, 1 H),
7.65 (d, J = 12.0 Hz, 1 H), 7.57 (s, 1 H), 7.43–7.40 (m, 2 H), 7.38 (s, 1 H), 7.18–7.11 (m, 2 H), 7.03 (t, J = 8.4 Hz, 2 H), 6.38 (d,
J = 4.8 Hz, 1 H), 4.32 (s, 2 H), 4.05 (s, 3 H), 4.02 (s, 3 H), 2.53 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 164.3, 163.0 (d, J =
247.1 Hz), 160.1, 154.0 (d, J = 248.0 Hz), 153.3, 149.9, 148.5, 146.7, 138.2 (d, J = 12.7 Hz), 136.0 (d, J = 9.5 Hz), 132.6 (d, J =
8.0 Hz), 125.6 (d, J = 3.3 Hz), 123.5, 118.2, 115.7 (d, J = 21.5 Hz), 115.6, 111.8 (d, J = 22.7 Hz), 107.4, 102.3, 99.5, 60.1, 56.2,
22.0. Anal. Calcd. For C26H23F2N3O5S: C, 59.20; H, 4.39; N, 7.97. Found: C, 59.21; H, 4.37; N, 7.94. ESI-MS: m/z 528.1
[M+H]+.
4.1.4.15. N'-(Benzylsulfonyl)-2-(cyclohexen-1-yl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)acetamidine (26o)
White solid, yield: 68%, m.p.: 141–143 °C. 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 4.4 Hz, 1 H), 7.60–7.58 (m, 2 H), 7.47
(d, J = 11.6 Hz, 2 H), 7.44–7.43 (m, 2 H), 7.37–7.33 (m, 3 H), 7.19 (t, J = 8.4 Hz, 1 H), 7.02–7.00 (m, 1 H), 6.38 (d, J = 4.8 Hz, 1
H), 4.36 (s, 2 H), 4.06 (s, 3 H), 4.04 (s, 3 H), 3.63 (s, 2 H), 2.13–2.10 (m, 2 H), 1.95–1.94 (m, 2 H), 1.65–1.60 (m, 4 H). 13C
NMR (100 MHz, CDCl3) δ 164.6, 159.9, 154.2 (d, J = 249.0 Hz), 153.1, 149.8, 148.7, 147.0, 138.4 (d, J = 12.5 Hz), 135.4 (d, J =
9.2 Hz), 132.1, 130.9, 130.7, 129.9, 128.6, 128.5, 123.6, 117.9 (d, J = 2.8 Hz), 115.6, 111.7 (d, J = 22.6 Hz), 107.8, 102.3, 99.4,
61.0, 56.2, 43.0, 28.5, 25.5, 22.7, 21.8. Anal. Calcd. For C32H32FN3O5S: C, 65.18; H, 5.47; N, 7.13. Found: C, 65.17; H, 5.49; N,
7.13. ESI-MS: m/z 590.2 [M+H]+.
4.1.4.16. 2-(Cyclohexen-1-yl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N'-((4-fluorobenzyl)sulfonyl)acetamidine
(26p)
White solid, yield: 71%, m.p.: 132–134 °C. 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 4.8 Hz, 1 H), 7.61 (dd, J = 1.6, 12.0 Hz,
1 H), 7.57 (s, 1 H), 7.51 (s, 1 H), 7.43–7.40 (m, 3 H), 7.21 (t, J = 8.4 Hz, 1 H), 7.06–6.98 (m, 3 H), 6.37 (d, J = 4.8 Hz, 1 H), 4.32
(s, 2 H), 4.07 (s, 3 H), 4.04 (s, 3 H), 3.66 (s, 2 H), 2.14–2.10 (m, 2 H), 1.97–1.95 (m, 2 H), 1.66–1.60 (m, 4 H). 13C NMR (100
MHz, CDCl3) δ 164.7, 163.0 (d, J = 246.4 Hz), 159.9, 154.2 (d, J = 249.4 Hz), 153.1, 149.8, 148.7, 147.0, 138.5 (d, J = 12.2 Hz),
135.3 (d, J = 8.6 Hz), 132.6 (d, J = 8.2 Hz), 132.0, 130.8, 125.8 (d, J = 2.5 Hz), 123.7, 118.0, 115.6 (d, J = 21.4 Hz), 111.8 (d, J
= 23.0 Hz), 107.9, 102.2, 99.4, 60.2, 56.2, 43.1, 28.5, 25.5, 22.7, 21.8. Anal. Calcd. For C32H31F2N3O5S: C, 63.25; H, 5.14; N,
6.91. Found: C, 63.26; H, 5.12; N, 6.92. ESI-MS: m/z 630.2 [M+Na]+.
4.1.4.17. N'-(Benzylsulfonyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-2-(pyridin-3-yl)acetamidine (26q)
Yellow solid, yield: 42%, m.p.: 121–123 °C. 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1 H), 8.46 (d, J = 4.8 Hz, 1 H), 8.17 (s, 1
H), 7.70 (d, J = 7.2 Hz, 1 H), 7.58 (d, J = 12.0 Hz, 1 H), 7.55 (s, 1 H), 7.46–7.45 (m, 2 H), 7.39 (s, 1 H), 7.36–7.29 (m, 4 H), 7.14
(t, J = 8.4 Hz, 1 H), 7.06–7.04 (m, 1 H), 6.66–6.62 (m, 1 H), 6.36 (d, J = 4.8 Hz, 1 H), 4.40 (s, 2 H), 4.24 (s, 2 H), 4.04 (s, 3 H),
4.02 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 163.5, 159.9, 154.1 (d, J = 248.1 Hz), 153.1, 150.1, 149.8, 148.6, 148.5, 146.9,
138.4 (d, J = 11.9 Hz), 137.5, 135.8 (d, J = 7.6 Hz), 131.0, 130.6, 129.6, 128.7, 124.4, 124.1, 123.6, 118.5, 115.6, 112.0 (d, J =

22.9 Hz), 107.7, 102.3, 99.4, 61.1, 56.2, 37.8. Anal. Calcd. For C31H27FN4O5S: C, 63.47; H, 4.64; N, 9.55. Found: C, 63.44; H,
4.66; N, 9.56. ESI-MS: m/z 587.2 [M+H]+.
4.1.4.18. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N'-((4-fluorobenzyl)sulfonyl)-2-(pyridin-3-yl)acetamidine
(26r)
Yellow solid, yield: 44%, m.p.: 112–114 °C. 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1 H), 8.46 (d, J = 4.8 Hz, 1 H), 8.15 (s, 1
H), 7.74 (d, J = 7.2 Hz, 1 H), 7.61 (d, J = 12.0 Hz, 1 H), 7.55 (s, 1 H), 7.44–7.41 (m, 2 H), 7.38 (s, 1 H), 7.34 –7.31 (m, 1 H),
7.24–7.22 (m, 1 H), 7.15 (t, J = 8.4 Hz, 1 H), 7.06–7.03 (t, J = 8.0 Hz, 3 H), 6.35 (d, J = 4.8 Hz, 1 H), 4.37 (s, 2 H), 4.27 (s, 2 H),
4.04 (s, 3 H), 4.02 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 164.2, 163.6, 161.8, 159.9, 154.0 (d, J = 248.9 Hz), 153.1, 151.2 (d, J
= 245.9 Hz), 149.8, 148.6, 146.9, 138.5 (d, J = 11.8 Hz), 137.4, 135.8 (d, J = 8.5 Hz), 132.7 (d, J = 8.1 Hz), 130.6, 125.5, 124.5,
123.6, 118.4, 115.7 (d, J = 21.5 Hz), 115.6, 112.1 (d, J = 21.8 Hz), 107.6, 102.3, 99.4, 60.2, 56.2, 37.9. Anal. Calcd. For
C31H26F2N4O5S: C, 61.58; H, 4.33; N, 9.27. Found: C, 61.60; H, 4.31; N, 9.30. ESI-MS: m/z 605.2 [M+H]+.
4.1.4.19. N'-(Benzylsulfonyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-2-(thien-3-yl)acetamidine (26s)
Light yellow solid, yield: 62%, m.p.: 116–118 °C. 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 5.2 Hz, 1 H), 7.55 (s, 1 H), 7.51
(d, J = 12.0 Hz, 1 H), 7.47–7.35 (m, 7 H), 7.16–7.12 (m, 2 H), 7.00 (d, J = 4.4 Hz, 1 H), 6.89–6.87 (m, 1 H), 6.34 (d, J = 5.2 Hz,
1 H), 4.39 (s, 2 H), 4.37 (s, 2 H), 4.05 (s, 3 H), 4.03 (s, 3 H). 13C NMR (100 MHz, DMSO-d6) δ 163.7, 159.1, 153.1 (d, J = 245.1
Hz), 152.6, 149.5, 148.8, 146.4, 136.9 (d, J = 12.1 Hz), 136.6 (d, J = 9.7 Hz), 134.7, 130.9, 130.5, 128.5, 128.1, 127.9, 126.1,
123.8, 123.3, 118.5 (d, J = 2.4 Hz), 114.5, 110.7 (d, J = 22.6 Hz), 107.8, 102.2, 98.9, 59.9, 55.7, 34.4. Anal. Calcd. For
C30H26FN3O5S2: C, 60.90; H, 4.43; N, 7.10. Found: C, 60.91; H, 4.45; N, 7.08. ESI-MS: m/z 614.2 [M+Na]+.
4.1.4.20. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-N'-((4-fluorobenzyl)sulfonyl)-2-(thien-3-yl)acetamidine (26t)
1
Light yellow solid, yield: 56%, m.p.: 107–109 °C. H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 4.0 Hz, 1 H), 7.55 (s, 1 H),
7.42–7.42 (m, 3 H), 7.40 (s, 1 H), 7.29–7.26 (m, 2 H), 7.15 (t, J = 8.4 Hz, 1 H), 7.04 (t, J = 8.4 Hz, 3 H), 6.88–6.86 (m, 1 H),
6.33 (d, J= 5.2 Hz, 1 H), 4.40 (s, 2 H), 4.34 (s, 2 H), 4.05 (s, 3 H), 4.02 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 164.4, 164.2,
161.7, 158.7 (d, J = 246.4 Hz), 154.0 (d, J = 248.7 Hz), 153.1, 149.8, 148.6, 146.8, 138.5 (d, J = 11.9 Hz), 135.2 (d, J = 8.8 Hz),
132.6 (d, J = 8.4 Hz), 132.5, 128.5, 127.9, 125.6 (d, J = 2.3 Hz), 125.5, 123.5, 118.3, 115.6 (d, J = 21.5 Hz), 111.9 (d, J = 22.5
Hz), 107.6, 102.2, 99.4, 60.1, 56.2, 35.2. Anal. Calcd. For C30H25F2N3O5S2: C, 59.10; H, 4.13; N, 6.89. Found: C, 59.09; H, 4.15;
N, 6.90. ESI-MS: m/z 610.2 [M+H]+.
4.1.4.21. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-3-methoxy-N'-((2-methylbenzyl)sulfonyl)propanamidine (26u)
Yellow solid, yield: 78%, m.p.: 115–117 °C. 1H NMR (400 MHz, CDCl3) δ 8.84 (br s, 1 H), 8.51 (d, J = 5.2 Hz, 1 H), 7.58 (s,
1 H), 7.56 (dd, J = 2.0, 12.0 Hz, 1 H), 7.43 (s, 1 H), 7.41 (d, J = 7.2 Hz, 1 H), 7.22–7.16 (m, 4 H), 7.10–7.08 (m, 1 H), 6.39 (d, J
= 5.2 Hz, 1 H), 4.42 (s, 2 H), 4.07 (s, 3 H), 4.05 (s, 3 H), 3.66 (t, J = 5.2 Hz, 2 H), 3.43 (s, 3 H), 3.17 (t, J = 5.2 Hz, 2 H), 2.41 (s,