Design, synthesis and biological evaluation of novel 4-phenoxy-6,7-disubstituted quinolines possessing (thio)semicarbazones as c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.02.003

In continuing our efforts to identify small molecules able to inhibit c-Met kinase, three series of novel 6,7-disubstituted-4-phenoxyquinoline derivatives (23a-w, 26a-d and 30a-d) bearing (thio)semicarbazone scaffold were designed, synthesized and evaluated for their cytotoxicity. The biological data revealed that most compounds exhibited moderate-to-excellent activity against HT-29, MKN-45, A549 cancer cell lines and relative poor potency toward MDA-MB-231 cell as well as hardly any cytotoxicity in normal PBL cell. Eleven compounds were further examined for their inhibitory activity against c-Met kinase and three compounds (23h, 23n and 26a) demonstrated good inhibitory activity. This work resulted in the discovery of a potent c-Met inhibitor 23n, bearing 2-hydroxy-3-allylphenyl group at R² moiety, as a valuable lead molecule, which possessed remarkable cytotoxicity and high selectivity against A549 and HT-29 cell lines with IC₅₀ values of 11 nM and 27 nM. Besides, it displayed excellent c-Met kinase inhibition on a single-digital nanomolar level (IC₅₀ = 1.54 nM). Meanwhile, the results from preliminarily in vivo study reflected that compound 23n showed promising overall PK profiles, consistent with the efficacy in both MKN-45 and HT-29 tumor xenograft mice model. These results clearly indicated that compound 23n is a potent and highly selective c-Met inhibitor and its favorable in vitro and in vivo profiles warrant further investigation.

Full text of DOI:10.1016/j.bmc.2016.02.003

Accepted Manuscript
Design, synthesis and biological evaluation of novel 4-phenoxy-6,7-disubstitu-
ted quinolines possessing (thio)semicarbazones as c-Met kinase inhibitors
Xin Zhai, Guanglong Bao, Limei Wang, Mingke Cheng, Meng Zhao, Sijia Zhao,
Hongyang Zhou, Ping Gong
PII:
S0968-0896(16)30075-X
http://dx.doi.org/10.1016/j.bmc.2016.02.003
BMC 12805
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
4 December 2015
2 February 2016
3 February 2016
Please cite this article as: Zhai, X., Bao, G., Wang, L., Cheng, M., Zhao, M., Zhao, S., Zhou, H., Gong, P., Design,
synthesis and biological evaluation of novel 4-phenoxy-6,7-disubstituted quinolines possessing
(thio)semicarbazones as c-Met kinase inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.02.003
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Design, synthesis and biological evaluation of novel 4-phenoxy-6,7-disubstituted quinolines
possessing (thio)semicarbazones as c-Met kinase inhibitors
Xin Zhaia,*, Guanglong Baoa, Limei Wanga, Mingke Chengb, Meng Zhaoa, Sijia Zhaoa, Hongyang Zhoua, Ping
Gonga,*
a Key Laboratory of Structure-based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, P.R. China; and b Chong Qing Yaopharma CO., LTD, Chongqing 401121, P.R. China.
Abstract
In continuing our efforts to identify small molecules able to inhibit c-Met kinase, three series of novel
6,7-disubstituted-4-phenoxyquinoline derivatives (23a-w, 26a-d and 30a-d) bearing (thio)semicarbazone
scaffold were designed, synthesized and evaluated for their cytotoxicity. The biological data revealed that most
compounds exhibited moderate-to-excellent activity against HT-29, MKN-45, A549 cancer cell lines and
relative poor potency toward MDA-MB-231 cell as well as hardly any cytotoxicity in normal PBL cell. Eleven
compounds were further examined for their inhibitory activity against c-Met kinase and three compounds (23h,
23n and 26a) demonstrated good inhibitory activity. This work resulted in the discovery of a potent c-Met
inhibitor 23n, bearing 2-hydroxy-3-allylphenyl group at R2 moiety, as a valuable lead molecule, which
possessed remarkable cytotoxicity and high selectivity against A549 and HT-29 cell lines with IC50 values of 11
nM and 27 nM. Besides, it displayed excellent c-Met kinase inhibition on a single-digital nanomolar level (IC50
= 1.54 nM). Meanwhile, the results from preliminarily in vivo study reflected that compound 23n showed
promising overall PK profiles, consistent with the efficacy in both MKN-45 and HT-29 tumor xenograft mice
model. These results clearly indicated that compound 23n is a potent and highly selective c-Met inhibitor and its
favorable in vitro and in vivo profiles warrant further investigation.
Keywords: Quinoline; (Thio)semicarbazone; Anti-tumor activity; c-Met kinase inhibitor; Pharmacokinetic
profile; Xenograft mice model.
1. Introduction
c-Met, also referred to as the surface receptor of hepatocyte growth factor receptor (HGFR), is a
heterodimeric transmembrane receptor tyrosine kinase, that mediates activation of several signaling pathways
implicated in aggressive cancer phenotypes.1-3 Aberrant c-Met/HGF signaling, resulting from MET genomic
amplification, c-Met/HGF overexpression or c-Met mutations, has been identified in a variety of human
malignances, and in many instances has been correlated with advanced disease stage and poor prognosis.4-6 This
emphasizes c-Met as an attractive therapeutic target, triggering a number of approaches to disrupt aberrant c-Met
signaling.
Cabozantinib (1) (Fig. 1), a novel oral multi-kinase inhibitor mainly targeting on c-Met and vascular
endothelial growth factor receptor 2 (VEGFR-2), was approved by U.S. FDA in November 2012 for the
treatment of patients with progressive metastatic medullary thyroid cancer (MTC).7 Recently, significant
* Corresponding authors. Tel./fax: +86 24 2398 6429.
E-mail address: zhaixin_syphu@126.com (X. Zhai); gongpinggp@126.com (P. Gong).
1

progress has been made on the development of c-Met inhibitors with several compounds reaching clinical trials,
including Foretinib (2) (Fig. 1),8 Kirin (3),9 AM7 (4)10 and AMG458 (6)11 (Fig. 1) as well as several newly
developed compounds bearing 6,7-disubstituted-4-phenoxyquinoline frameworks as the primary
pharmacophoric scaffolds. The structure-activity relationships (SARs) of quinoline-based inhibitors indicated
that quinoline pharmacophores and linkers between the phenyl ring A and part B were responsible for the
interaction with the backbone of the corresponding kinase.12,13 Furthermore, the part B of phenyl ring probably
extended into the hydrophobic pocket formed by tyrosine residues to enhance the inhibitory activity.14 By means
of Discovery Studio 3.0,15 the spatial distances (SD) of linkers in the representative inhibitors 2, 4, and 5 [1]
were obtained as 6.327 Å (PDB: 3LQ8), 6.549 Å (PDB: 2RFN), 6.434 Å (PDB: 3CE3), respectively, which
would be attribute to the fitting into the hydrophobic pocket of c-Met. What′s more, whether the structures of
linkages were constrained (4, 5 and 6) or not (1, 2, and 3), it was found that six chemical bonds space, which
was characterized by the illustrated “5 atoms regulation” in our previous work,16 were retained by summarizing
the structures of a large number of quinoline based inhibitors.
As a continuation of our study towarding identification of potent c-Met inhibitors, we decide to take
advantage of the widely-used 4-phenoxyquinoline skeleton as a privileged scaffold of protein tyrosine kinases
(PTK) inhibitors and modify it with three key linkages, thereby designing three series of quinoline analogs. In
light of our early study,17,18 we revealed that semicarbazone moiety, a hybrid linkage derived from two fused
pharmacophoric domains of diaryl urea from Sorafenib19 and N-acylhydrazone from PAC-120 (Fig. 1), were also
widely used as building blocks in several antitumor agents on account of the presence of hydrogen bond donors
and acceptors as well as its flexible skeleton (Fig. 1). To our delight, examination from corresponding software
indicated that whose SD value is approximate 6.496 Å, which is similar to that of compounds 2, 4, 5 and meet
the “5 atoms regulation”. Accordingly, incorporation of semicarbazone scaffold into
4-phenoxy-6,7-disubstituted quinoline artwork led to a series of hybrids 23a-w. Furthermore, enlightened by the
structural characteristics of 3 in Fig.1, compounds 26a-d bearing thiosemicarbazones instead of semicarbazones
were synthesized based on bioisosterism. Moreover, in order to identify the linkage between phenyl ring A and
part B, compounds 30a-d bearing reversed semicarbazone were prepared. In view of our previous works,17,18 the
potential 3-carbon tether at the 7-position of quinoline moiety was reserved, while the morpholino group was
replaced by three other water-soluble substituents, including piperidin-1-yl, 4-methylpiperidin-1-yl and
pyrrolidin-1-yl groups, to extend the SARs of the new designed compounds. Furthermore, various substituents
(R2) were introduced with the purpose of exploring the influence of substituents on antitumor activity by
regulating the electronic or steric effect at the phenyl ring (moiety B). Herein, in this report we disclosed the
synthesis and c-Met inhibition study of these three series of new analogues.
2

Fig. 1. The structures of small-molecular c-Met inhibitors bearing 6,7-disubstituted-4-phenoxyquinoline scaffold, Sorafenib, PAC-1
and the designed compounds.
2. Chemistry
Compounds 23a-w were synthesized according to the procedures outlined in Scheme 1. Commercially
available 4-hydroxy-3-methoxyacetophenone 9 was alkylated with 1-bromo-3-chloropropane in the presence of
potassium carbonate to provide 10.21 Nearly regioselective nitration with fuming nitric acid, subsequent
aminomethylenation by means of modified Vilsmeier–Haack reagent N,N-dimethylformamide dimethylacetal
(DMF-DMA) gave rise to 12.22 The intramolecular cyclization by iron powder and acetic acid proceeded
smoothly to afford the quinolinol core 13, which was subjected to a nucleophilic substitution with different
amines (morpholine, piperidine, 4-methylpiperidine and pyrrolidine) to generate the desired intermediates 14a–d.
The resultant hydroxyl moieties 14a–d were converted into the corresponding 15a–d on exposure to phosphorus
oxychloride. SN2 reactions of chloride in the 15a–d with 2-fluoro-4-nitrophenol furnished 16a–d, which were
reduced by iron powder and concentrated hydrochloric acid in ethanol/water (9: 1 v/v) to provide anilines 17a–d.
Amide derivatives 18a–d were introduced by further treatment with phenyl chloroformate 17a–d. Subsequently,
the key intermediate semicarbazides 19a–d were available via hydrazinolysis of 18a–d with 80% hydrazine
monohydrate in refluxing 1,4-dioxane. The final products 23a-w were obtained via the condensation of 19a–d
with appropriate aldehydes 22a-n in isopropanol at 80 °C with catalytic amounts of acetic acid.23
In order to synthesis of aldehyde 22k, a three-step reaction sequence was utilized. An initial alkylation of
commercially available salicylaldehyde 20j with 3-bromoprop-1-ene and potassium carbonate in
N,N-dimethylformamide acquired 21j. Subsequently, Claisen rearrangement delivered one desired aldehyde 22j
at 200 °C. Finally, 22j was methylated with iodomethane in the darkness under basic condition using anhydrous
potassium carbonate in dry acetone to bring out another target aldehyde 22k.24
3

Scheme 1. Reagents and conditions: (i) 1-bromo-3-chloropropane, potassium carbonate, N,N-dimethylformamide, r.t.; (ii) fuming
nitric acid, dichloromethane, 10 °C; (iii) N,N-dimethylformamide dimethyl acetal, toluene, reflux; (iv) iron powder, acetic acid,
80 °C; (v) amines, acetonitrile, reflux; (vi) phosphorus oxychloride, acetonitrile, reflux; (vii) 2-fluoro-4-nitrophenol, chlorobenzene,
reflux; (viii) Fe (powder), conc. hydrochloric acid, ethanol/water (9: 1 v/v), reflux; (ix) phenylchloroformate, anhydrous potassium
carbonate, dry acetone, 0 °C to r.t.; (x) 80% hydrazine monohydrate, 1,4-dioxane, reflux; (xi) aldehyde, acetic acid, 2-propanol,
reflux; (xii) 3-bromoprop-1-ene, potassium carbonate, N,N-dimethylformamide, r.t.; (xiii) 200 °C; (xiv) iodomethane, potassium
carbonate, acetone, reflux.
Scheme 2 depicts the sequence of reactions that lead to the construction of compounds 26a-d utilizing 17c as
the starting material, which was obtained according to the reactions in Scheme 1. In the beginning, treatment of
17c with thiophosgen in saturated sodium bicarbonate aqueous solution obtained isothiocyanate 24,25 which was
then reacted with 80% hydrazine monohydrate in trichloromethane at room temperature to receive the key
intermediate thiosemicarbazide 25. The requisite benzylidenethiosemicarbazide derivatives 26a-d were
progressed as previously described reaction in Scheme 1.
Scheme 2. Reagents and conditions: (i) thiophosgen, salt. aq. sodium bicarbonate, trichloromethane, 0 °C to r.t.; (ii) 80% hydrazine
hydrate, trichloromethane, r.t.; (iii) acetic acid (cat. amount), 2-propanol, reflux.
The preparation of analogues 30a-d was illustrated in Scheme 3. Nucleophilic displacement of chloride group
in the intermediate 15c with 3-fluoro-4-hydroxybenzaldehyde afforded benzaldehyde 27, which were condensed
with semicarbazides 29a-d in isopropanol to yield desired compounds 30a-d. Herein, semicarbazides 29a-d
were installed from appropriate anilines 22p-s, which were readily acylated by phenyl chloroformate in dry
acetone with potassium carbonate to furnish 28a-d. Subsequently, hydrazinolysis of 28a-d with an excess of
80% hydrazine hydrate generated the intermediates 29a-d.
4

Scheme 3. Reagents and conditions: (i) 3-fluoro-4-hydroxybenzaldehyde, chlorobenzene, reflux; (ii) acetic acid (cat. amount),
2-propanol, reflux. (iii) phenyl chloroformate, anhydrous potassium carbonate, dry acetone, 0 °C to r.t.; (iv) 80% hydrazine hydrate,
1,4-dioxane, reflux.
1
The chemical structures of obtained compounds were confirmed by H NMR, NOESY NMR, 13C NMR, and
MS spectrum. All the target compounds might exist in the E or Z isomeric form due to the imine bonds formed
in condensation procedures. Despite the possibility of the formation of two diastereoisomers, only the
(E)-isomer was obtained from the experimental procedure adopted here. As for compounds 23a-w, 26a-d and
1
30a-d, a detailed analysis of the H NMR spectra revealed that only one –CH–N– signal existed for each
compound. In addition, for the representative compound 23f, the NOESY effects which resulted from the E
isomer was observed between the proton of –NH–N– (H1, δ 11.04 ppm, singlet) and the proton of –CH–N– (H2,
δ 8.49 ppm, singlet). This effect should not be observed in the (Z)-isomer due to the larger intramolecular
distance of H1 and H2.16, 17, 26, 27
Fig. 2. The NOESY effects of representative compound 23f.
3. Biology
3.1. HTRF kinase assay
The c-Met kinase activity was performed by homogeneous time-resolved fluorescence (HTRF) assays
previously reported protocol.28 In addition, the most promising compounds 23n was evaluated against other six
tyrosine kinases (Ron, KDR, PDGFR-α, c-kit, Flt-3 and EGFR) using the same screening method. Briefly, 20
μg/mL poly (Glu, Tyr) 4:1 (Sigma) was preloaded as a substrate in 384-well plates. Then 50 μL of 10 mM ATP
(Invitrogen) solution diluted in kinase reaction buffer (50 mM HEPES, pH 7.0, 1 mM DTT, 1 mM MgCl2, 1 mM
MnCl2, 0.1% NaN3) was added to each well. Various concentrations of compounds were diluted in 10 μL of 1%
DMSO (v/v), with blank DMSO solution as the negative control. The kinase reaction was initiated by the
addition of purified tyrosine kinase proteins diluted in 39 μL of kinase reaction buffer solution. Reactions were
5

incubated for 30 min at 25 °C and stopped by the addition of 5 μL Streptavidin-XL665 and 5 μL Tk Antibody
Cryptate working solution to all of wells. The plate was read by Envision (Perkinelmer) at 320 nm and 615 nm.
The inhibition rate (%) was calculated using the following equation: % inhibition = 100-[(Activity of enzyme
with tested compounds-Min)/(Max-Min)]-100 (Max: the observed enzyme activity measured in the presence of
enzyme, substrates, and co-factors; Min: the observed enzyme activity in the presence of substrates, cofactors
and in the absence of enzyme). Half-maximal inhibitory concentration (IC50) values were calculated from the
inhibition curves.
3.2. Cell proliferation assay
The anti-proliferativity of compounds 23a-w, 26a-d and 30a-d were evaluated against human nonsmall-cell
lung cancer cell (A549), human colorectal cancer cell (HT-29) and human gastric cancer cell (MKN-45), a c-Met
less sensitive human breast cancer cell (MDA-MB-231) as well as a normal human peripheral blood lymphocyte
cell (PBL) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, taking
Foretinib and PAC-1 as positive controls.29 The cancer cells were cultured in minimum essential medium (MEM)
supplemented with 10% fetal bovine serum (FBS). Approximately 4×103 cells, suspended in MEM medium,
were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37 °C for 24 h. The test compounds
were added to the culture medium at the indicated final concentrations and the cell cultures were continued for
72 h. Fresh MTT was added to each well at a final concentration of 5 μg/mL and incubated with cells at 37 °C
for 4 h. The formazan crystals were dissolved in 100 μL DMSO per each well, and the absorbency at 492 nm
(for the absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with the
ELISA reader. All of the compounds were tested three times in each of the cell lines. The results expressed as
IC50 values were the mean ± SD and were calculated by using the Bacus Laboratories Incorporated Slide
Scanner (Bliss) software.
3.3. Pharmacokinetic (PK) parameters obtained in mice
To evaluate the bioavailability of compound 23n in male mice, a single dose was administered as a solution in
PEG 400/water (70:30) by either tail vein injection (iv, 20 mg/kg) or oral gavage (po, 35 mg/kg).30 The mice
were fasted overnight prior to dosing and fed 4 h postdose. A total of 18 mice were used in the study (n = 9 each
for iv and po group). Three serum samples were collected from each mouse, the first two samples by
retro-orbital bleed (~100 µL/20-25 g mouse) and the third sample by cardiac puncture. Blood samples were
collected at 0.17, 0.5, 1, 3, 6, 8, 10, 12, and 24 h time points following iv dosing and at 0.25, 0.5, 1, 3, 6, 8, 10,
12, and 24 h following oral dosing. Blood samples were allowed to clot on ice and were centrifuged, and serum
was harvested. Serum samples were stored at -20 °C until analysis. Concentrations of parent compound were
later determined by LC/MS/MS. Composite serum concentration-time profiles were constructed for PK analysis.
3.4. In vivo antitumor efficacy studies
Initial antitumor efficacy studies were carried out in vivo to investigate the efficacy of desired compounds
in HT-29 and MKN-45 xenograft nude mice models using Foretinib as positive control.31 Two cell lines, HT-29
cell (1×107/100 μL PBS per mice) and MKN-45 cell (1×107/100 μL PBS per mice), were subcutaneously (s.c.)
injected into the right flank of 7- to 8-week old male SCID mice or Balb/c nude mice. Cell numbers were
confirmed by try pan blue staining prior to injection. When the average s.c. tumor volume reached 100 mm3,
6

mice were randomly divided into various treatment and control groups (six mice per group). Tumor size was
measured once every two days with a caliper (calculated volume = shortest diameter 2×longest diameter/2).
Body weight, diet consumption and tumor size were recorded once every two days. After two or four weeks,
mice were sacrificed and tumors were excised and stored at 80 °C until further analysis.
4. Results and discussion
4.1. In vitro cytotoxicity and structure activity relationships
The cytotoxicity of the target compounds 23a-w, 26a-d and 30a-d were evaluated in vitro against three
c-Met-addicted cancer cell lines including A549, HT-29, MKN-45, MDA-MB-231 and PBL cell lines by
standard MTT assay taking Foretinib and PAC-1 as positive controls. The results were expressed as IC50 values
and presented in Tables 1 and 2, as mean values of experiments performed in triplicate.
As illustrated in Tables 1 and 2, most compounds exhibited moderate-to-excellent cytotoxicity, with IC50
values ranging from 0.011 to 1.6 μM against c-Met-addicted A549, HT-29 and MKN-45 cell lines. Compared
with Foretinib, three of them (23h, 30a, 30d) exhibited equivalent or even higher potency against A549 and
HT-29 cell lines. Particularly compound 23n displayed promising cytotoxicity with IC50 values of nanomolar
level on A549, HT-29 and MKN-45 cells. As a general trend, the title compounds were more potent in A549 and
HT-29 than they were in MKN-45 cells. By contrast, the compounds exhibited poor activity against
MDA-MB-231 cell line and hardly any antitumor potency on PBL cells.
The SARs commenced by the introduction of a carbon tether, which contains different tertiary amines, off of
the quinoline ring at 7-position was examined first. A three-carbon tether between-amines and the quinoline had
previously been determined to be the optimal length for improving the potency; therefore, four kinds of amines
at the end of this tether were examined. Of these amines, piperidinyl and 4-methylpiperidinyl derivatives 23f-g
and 23s-u was proved to be superior to that of 23a-b and 23c-e bearing morpholino and pyrrolidinyl groups,
respectively. Accordingly, piperidinyl and 4-methylpiperidinyl derivatives were further studied in the following
work.
In terms of phenyl ring, the influence of substituents were explored either. The pharmacological data from
Table 1 referred to substituted-benzene analogs disclosed that variations of R2 made a moderate impact on the
cytotoxicity. For compounds 23a-g and 23s-u, the presence of the electron-withdrawing groups such as nitro
(23b, 23d and 23t) imparted enhanced activity. By contrast, the electron-donating groups such as methoxyl (23a,
23c, 23f and 23s) weakened the biological activity slightly. While, compounds bearing bulky groups at
4-position, such as 23j with 4-trifluoromethoxy group, 23l possessing 4-dimethylamino group and 23e, 23g, 23u
bearing trifluoromethyl group, decreased or even vanished the antitumor potency against A549 and HT-29 cell
lines, which suggested that the substitution at this position is fairly sensitive to a substituent’s size.
Based on earlier study, we rationalized modifications of the phenyl group could modulate the π-stacking
interactions with corresponding receptors allowing for increasing potency. However, compared with piperidinyl
analog 21b bearing phenyl group (IC50 = 0.16 μM (A549), 0.18 μM (HT-29) and 0.58 μM (MKN-45)) [17],
incorporation of heterocyclic groups, such as the thiophen-2-yl, pyridine-4-yl and 1H-indol-2-yl groups in
compounds 23p, 23q and 23r, led to 1.10- to 5.25-folds decrease in antitumor tendency against A459, HT-29
and MKN-45 cells. Fortunately, compound 23n bearing 2-hydroxy-3-allylphenyl group, the optimal
pharmacophore of PAC-1,20,32 was screening out displaying nanomolar level inhibition in cell-based assay with
IC50 values of 11, 27 and 39 nM against A549, HT-29 and MKN-45 cancer cell lines, which were 15.4, 9.6 and
1.7 folds more active than that of Foretinib (0.17, 0.26 and 0.023 μM), respectively. The dramatic boost in
7

tendency might be ascribe to the introduction of 2-hydroxy-3-allyl group conduced to conjugation on
corresponding receptors necessary for activity. This hypothesis could be confirmed by comparing 23n with its
methylation compound 23o, as 23o caused significant loss of potency on all three c-Met-addicted cancer cell
lines.
Attention was turned to focus on linkage between A and B moiety in the subsequent efforts. Initially, the
replacement of semicarbazone with thiosemicarbazone was carried out to investigate the effects of the oxygen
atom. Although thiosemicarbazone derivatives 26a-d displayed a slightly boost in the subsequent
kinase-biochemical assays (Table 3), their activity against tested cancer cells decreased somewhat, which
corroborated the results from date 23h (0.13, 0.26, 0.27 and 0.79 μM toward tested cell lines) vs. 26d (0.23, 0.42,
0.82 and 1.4 μM against tested lines). In the following work, a new series of derivatives 30a-d conjugated with
reversed semicarbazone were synthesized to identify the linkage. The pharmacological data revealed that 30a-d
exhibited elevated tendency than 26a-d against all the tested cell lines in comparison 26a-b with 30a-b,
especially for 30d, which showed excellent activity 0.098, 0.11 and 0.14 μM against A549, HT-29 and MKN-45
cancer cell lines, respectively.
Table 1 Structures and cytotoxicity of the target compounds 23a-w against A549, HT-29, MKN-45 and MDA-MB-231 cancer cell
lines in vitro.
IC50 a (μM)
Compd
R1
R2
A549
ND b
ND
HT-29
MKN-45
MDA-MB-231
PBL
N-morpholino
N-morpholino
pyrrolidin-1-yl
4-methoxylphenyl
4-nitrophenyl
23a
23b
23c
0.90±0.13
0.71±0.02
0.69±0.05
0.77±0.05
0.75±0.03
0.75±0.01
1.9±0.25
1.7±0.14
1.3±0.11
17.25±2.62
ND
4-methoxylphenyl
0.68±0.06
0.46±0.10
0.79±0.10
0.54±0.12
0.66±0.08
0.13±0.02
ND
26.48±2.51
pyrrolidin-1-yl
pyrrolidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
4-methylpiperidin-1-
4-methylpiperidin-1-
4-methylpiperidin-1-
4-methylpiperidin-1-
4-methylpiperidin-1-
-
4-nitrophenyl
4-trifluoromethylphenyl
4-methoxylphenyl
4-trifluoromethylphenyl
2-chloro-4-fluorophenyl
2,4-dichlorophenyl
4-trifluoromethoxyphen
2,4-dimethoxylphenyl
4-N,N-dimethylphenyl
4-hydroxylphenyl
2-hydroxyl
ND
23d
23e
0.31±0.05
0. 81±0.12
0.59±0.09
0.66±0.10
0.26±0.01
0.37±0.05
0.51±0.08
1.6±0.32
0.47±0.07
1.3±0.21
0.97±0.12
1.8±0.25
0.84±0.11
1.1±0.15
0.79±0.17
0.75±0.06
0.82±0.12
ND
ND
23f
0.66±0.05
0.63±0.08
0.27±0.07
0.46±0.07
0.63±0.08
1.99±0.35
0.87±0.12
0.85±0.09
0.039±0.004
0.67±0.11
0.98±0.17
0.83±0.05
0.74±0.07
0.47±0.08
0.42±0.09
1.0±0.23
125.56±8.35
ND
23g
ND
23h
23i
ND
ND
23j
0.59±0.05
1.4±0.10
ND
ND
23k
23l
ND
1.0±0.31
1.4±0.17
0.79±0.08
0.44±0.10
0.79±0.11
1.4±0.11
0.98±0.12
1.26±0.15
1.0±0.21
0.87±0.14
1.3±0.13
0.91±0.12
1.5±0.17
ND
ND
23m
23n
23o
0.78±0.10
0.011±0.001
0.25±0.04
0.84±0.10
0.78±0.08
0.79±0.06
0.43±0.09
0.38±0.03
0.65±0.07
ND
0.47±0.05
0.027±0.002
0.46±0.07
0.94±0.12
0.82±0.09
0.77±0.05
0.61±0.12
0.23±0.02
0.74±0.13
0.47±0.10
0.44±0.04
0.26±0.03 d
0.83±0.07
106.91±7.28
ND
2-methoxyl-4-allylpheny
pyridine-4-yl
ND
23p
23q
23r
1H-indole-3-yl
ND
thiophene-2-yl
68.68±4.12
23.02±2.64
ND
4-methoxylphenyl
4-nitrophenyl
23s
23t
4-trifluoromethylphenyl
2,4-dichlorophenyl
2-chloro-4-fluorophenyl
-
ND
23u
23v
ND
0.35±0.07
0.53±0.03
0.023±0.004 e
0.82±0.13
23w
Foretini
PAC-1
0.19±0.01
0.17±0.02 c
ND
79.49±6.65
ND
-
-
4.6±0.33
123.81±9.64
a The average of three independent experiments;
b ND: not determined;
8

c Reported value is 0.029 mM;24
d Reported value is 0.165 mM;24
e Reported value is 0.008 mM.25
Table 2 Structures and cytotoxicity of compounds 26a-d and 30a-d against A549, HT-29, MKN-45 and MDA-MB-231 cancer cell
lines in vitro.
IC50 a (μM)
Compd.
R2
A549
HT-29
MKN-45
MDA-MB-231
PBL
phenyl
26a
26b
26c
0.33±0.02
0.21±0.02
0.61±0.10
0.19±0.05
0.71±0.08
0.74±0.15
0.50±0.10
1.2±0.17
1.4±0.15
1.3±0.12
78.36±6.54
3,4-difluorophenyl
4-chlorophenyl
ND
ND
ND
0.23±0.02
0.28±0.02
ND
ND
34.12±2.72
25.47±2.32
ND
2-chloro-4-fluorophenyl
26d
30a
0.42±0.09
0.19±0.03
0.49±0.08
0.27±0.07
0.11±0.01
0.26±0.03
0.83±0.07
0.82±0.21
0.18±0.02
0.32±0.05
0.060±0.004
0.14±0.01
0.023±0.004
0.82±0.13
1.4±0.15
1.2±0.13
0.96±0.12
0.80±0.11
1.0±0.13
ND
phenyl
3,4-difluorophenyl
30b
4-bromophenyl
ND
ND
30c
2-bromo-4-fluorophenyl
30d
0.098±0.01
0.17±0.02
ND
36.43±2.86
ND
-
-
Foretinib
PAC-1
4.6±0.33
123.81±9.64
a The average of three independent experiments.
4.2. In vitro enzymatic assays and selectivity
The c-Met kinase assays of eleven selected analogues (23a, 23c, 23f, 23n, 23r, 23s, 23w, 26a, 26d, 30a, 30d)
were performed by HTRF assay. Served as the positive control, Foretinib was determined simultaneously. As
shown in Table 3, all the eleven tested compounds exhibited c-Met enzymatic potency, reflecting that the
inhibition of c-Met is a candidate underlying mechanism for the antitumor effect. In particular, compound 23n
was distinguished with an IC50 value of 1.54 nM, which was comparable to that of Foretinib (IC50 = 1.16 nM).
Table 3 c-Met kinase activity of the selected compounds and foretinib in vitro.
Compd.
23a
IC50 a (nM)
56.42±4.36
72.33±7.63
13.51±1.15
1.54±0.12
54.25±5.26
15.67±1.28
Compd.
23w
IC50 a (nM)
26.26±2.43
8.92±1.13
16.83±1.62
62.82±5.84
121.42±9.26
1.16±0.17
23c
26a
23f
26d
23n
23r
30a
30d
23s
Foretinibb
a Data presented is the mean±SD value of three independent determinations;
b Used as positive control, reported value is 4 nM.24
Then we were prompted to investigate their specifical inhibition against c-Met kinase, the optimal compound
23n was screened against six selected kinases inhibited by most of reported quinoline-based multi-targeted
kinase inhibitors further, which are potential biological targets for the designed compounds. As shown in Table 4,
compound 23n inhibited c-Met family member Ron kinase either, with IC50 value of 14.5 nM. In contrast to its
high potency against c-Met, compound 23n barely inhibited the kinase activity of other tested tyrosine kinases.
9

It exhibited preferable selectivity versus KDR (IC50 = 210.2 nM), PDGFR-α (IC50 = 212.3 nM), c-kit (IC50
162.5 nM), Flt-3 (IC50 = 366.7 nM), and no inhibition activity against EGFR (IC50 > 100 μM).
=
Furthermore, although 23n exhibited similar c-Met kinase inhibition to the Foretinib, the results of c-Met
kinase assay in Table 3 showed that most of compounds were less active than Foretinib. It suggested that the
compounds might act through some other mechanisms rather than only by inhibiting c-Met and Ron. Further
studies on the mechanisms of these compounds are in progress.
Table 4 Inhibition of tyrosine kinases by compound 23n.
Kinases IC50 a in nM
Compd.
Ron
14.5
KDR
210.2
PDGFR-α
c-kit
Flt-3
EGFR
212.3
162.5
366.7
23n
＞100 000
a Data presented is the mean value of three independent determinations.
4.3. PK profiles of the selected compounds
Before assessing the in vivo pharmacological activity, the metabolic stability and PK properties were
evaluated first. The PK parameters of selected analogues in mice by either iv (20 mg/kg) or po (35 mg/kg) are
compiled in Table 5. 23h and 30d were metabolically unstable and rapidly cleared in vivo. For 26a, its Tmax value
is relatively longer than others. Delightedly, differing from their similar enzymatic and cellular potency in vitro,
compound 23n demonstrated remarkable potency in its PK properties, which was confirmed by single oral dose
administration. It displayed a longer half-lives (t1/2, 13.75 ± 1.74 h) and mean residence times (MRT0→∞, 23.41 ±
2.33 h), favorable clearance (CLz/F, 2.23 ± 0.55 L/h/kg), and so on. Given the promising overall PK profiles and
structural novelty, compound 23n was selected as the lead for the subsequent evaluation in vivo.
Table 5 The pharmacokinetic profiles of Selected Compounds
AUC0→∞
(mg·h /L)b
0.91 ± 0.19
16.50 ± 4.54
7.34 ± 2.69
1.21 ± 0.19
Clz/F
Vz/F
T1/2z
Cmax
Tmax
Compd.
MRT0→∞ (h)a
(L/h/kg)c
(L/kg)d
(h)e
(μg/L)f
(h)g
23h
23n
26a
30d
13.18 ± 2.26
23.41 ± 2.33
24.28 ± 6.23
14.77 ± 1.78
80.02 ± 17.81
2.23 ± 0.55
3.92 ± 1.55
30.21 ± 4.46
370.98 ± 194.58
43.88 ± 10.04
107.09 ± 30.62
346.22 ± 10.59
3.30 ± 0.26
13.75 ± 1.74
13.48 ± 4.35
5.07 ± 1.31
0.078 ± 0.011
0.68 ± 0.19
0.087 ± 0.022
0.073 ± 0.012
0.75 ± 0.29
6.00 ± 1.63
12.00 ± 1.68
7.33 ± 1.16
Values are presented as the mean ± SD in three independent experiments.
Dosed in DMSO: Cremophor EL: 0.9% saline (20: 20: 60 (v/v/v));
a Mean residence time;
b Area under the plasma concentration-time curve for 0-∞ h after dosing;
c Clearance (statistical moment parameter);
d Apparent volume of distribution (statistical moment parameter);
e Half-lives;
f Maximum plasma concentration after oral dosing;
g Time to reach Cmax
.
4.4. In vivo antitumor activity
10

On the basis of its encouraging PK profiles, 23n was advanced for in vivo efficacy studies in HT-29 and
MKN-45 xenograft nude mice models taking Foretinib as reference drug. It was found that when administered
orally twice daily (BID) for 24 days at a dose of 50 mg/kg, 23n significantly inhibited tumor growth of 70.18%
(p < 0.001) vs. control group in MKN-45 xenograft mice model (Fig. 3A). In order to further confirm its tumor
growth arrest in vivo, the test to another well-accepted c-Met-dependent HT-29 xenograft mice model was
extended. At the same dosing regimens, 23n shrank tumor growth significantly, and the inhibition ratio was
65.86% (p < 0.01) in a 27-day study, compared with the vehicle control (Fig. 3B). Meanwhile, Foretinib
exhibited equivalent or better suppression than 23n with tumor growth inhibitory rate of 78.23% and 73.68% on
MKN-45 and HT-29 xenograft mice model at the same dosage without any significant differences, respectively.
Furthermore, it was well tolerated with no overt signs of toxicity or changes in body weight in two xenograft
models (Fig. 3C and 3D).
Fig. 3. Effects of compound 23n and Foretinib in the MKN-45 (A) and HT-29 (B) xenograft models (mean tumor volume in mm3 ±
SEM, **p < 0.01, ***p < 0.001, vs. vehicle control group). Tumor-bearing Balb/c nude mice were randomly divided into groups in
which 6 mice were contained per group. While the tumor volume reached 100 mm3, 23n and Foretinib were administrated po (BID)
at a dose of 50 mg/kg or vehicle alone for 24 or 27 days. (C) and (D) shown are the average body weights for 23n-treated,
Foretinib-treated mice groups and vehicle groups in the MKN-45 and HT-29 xenograft models, respectively.
5. Conclusions
In summary, thirty-one novel quinoline-based selective c-Met kinase inhibitors (23a-w, 26a-d and 30a-d)
were designed and synthesized. Most compounds displayed moderate-to-excellent activity against HT-29,
MKN-45, A549 cancer cell lines and relative poor potent toward MDA-MB-231 cell as well as hardly any
cytotoxicity to normal PBL cell. The exploration of preliminary SARs indicated that the hybridization of
quinoline pharmacophore and semicarbazone as a privileged structure is of great importance for the good
antiproliferative potency and 4-methylpiperidin-1-yl was favorable for the better tendency. Besides, the
identification of inhibitor 23n, bearing 2-hydroxy-3-allylphenyl group at R2 moiety, as a valuable lead molecule,
11

which possessed excellent c-Met kinase inhibition on a single-digital nanomolar level (IC50 = 1.54 nM).
Moreover, it displayed remarkable cytotoxicity and high selectivity against A549 and HT-29 cell lines with IC50
values of 11 nM and 27 nM. Meanwhile, the results from preliminarily in vivo study reflected that compound
23n showed promising overall PK profiles and shrank tumor growth significantly in MKN-45 and HT-29
xenograft mice model. In conclusion, compound 23n is worth further studying as a new potential antitumor
agent owing to its pronounced in vitro and in vivo antitumor activity. Further studies on structural optimization
and the mechanisms are currently underway and will be reported in the future.
6. Experimental
6.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used without further
purification. All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik,
1
Flawil, Switzerland) and were uncorrected. H NMR and 13C NMR spectra were generated on a Bruker
ARX-300 or ARX-600 spectrometers (Bruker Bioscience, Billerica, MA, USA). Column chromatography was
carried out on silica gel (200-300 mesh). Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS
(Agilent, Palo Alto, CA, USA). Elemental analysis was determined on a Carlo-Erba 1106 Elemental analysis
instrument (Carlo Erba, Milan, Italy).
6.2. 4-(3-Chloropropoxy)-3-methoxyacetophenone (10)
To a solution of 4-hydroxy-3-methoxy-acetophenone (6.00 g, 36.1 mmol) in DMF (25 mL) was added K2CO3
and 1-bromo-3-chloropropane (6.98 g, 50.6 mmol). The reaction mixture was then stirred at r.t. for 10 h. The
mixture was then poured into cold water (100 mL) with vigorously agitating, and the resulting precipitate was
filtered off, washed with water, and dried under vacuum to afford the title compound 10 (8.27 g) as a white solid,
yield: 93.8%. m.p.: 61-63 °C. MS (ESI) m/z: 242.2, 244.1 [M+H]+.
6.3. 4-(3-Chloropropoxy)-5-methoxy-2-nitroacetophenone (11)
A stirred solution of 10 (2.00 g, 8.22 mmol) in CH2Cl2 (10 mL) was cooled to 10 °C, and fuming nitric acid
(95%, 1.30 g, 20.6 mmol) was added at a rate such that the temperature did not exceed 10 °C. The reaction
mixture was allowed to stir at 10 °C for 2 h, then transferred with stirring to cold water (20 mL). After 15 min,
the organic layer was separated and the aqueous layer washed with additional CH2Cl2 (10 mL). The organic
portions were combined and washed with 10% w/w aqueous sodium bicarbonate solution (2×10 mL), then water
(10 mL), then concentrated under reduced pressure to afford crude 11, which was recrystallized from 90%
1
ethanol to afford 11 (1.91 g) as a light yellow solid, yield: 80.1%. H NMR (400 MHz, CDCl3) δ 7.64 (s, 1H),
6.76 (s, 1H), 4.26 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H), 3.77 (t, J = 6.6 Hz, 2H), 2.50 (s, 3H), 2.29-2.37 (m, 2H). m.p.:
60-61 °C. MS (ESI) m/z: 287.0, 289.0 [M+H]+.
6.4. 1-(4-(3-Chloropropoxy)-5-methoxy-2-nitrophenyl)-3-(dimethylamino)prop-2-en-1-one (12)
DMF-DMA (4.15 g, 34.8 mmol) was added to a solution of 11 (2.00 g, 6.95 mmol) in toluene (10 mL). The
reaction was heated to 110 °C until TLC showed the completion of the reaction. After cooling to 10 °C, the
resultant solid was collected by filtration, washed with toluene (5 mL) and then dried under vacuum to yield 12
(1.84 g) as a yellow solid, yield: 75.8%. m.p.: 117-119 °C. MS (ESI) m/z: 342.2, 344.1 [M+H]+.
12

6.5. 6-Methyloxy-7-(4-(3-chloropropoxy))-4-quiolin-ol (13)
Fe (powder, 1.23 g, 22.0 mmol) was added to a solution of 12 (1.50 g, 4.40 mmol) in acetic acid (12 mL) at
40 °C, then the mixture was stirred at 80 °C with vigorous agitation for 2 h. The hot solution was filtered
through celite and washed with hot acetic acid. The combined filtrate was cooled to 20 °C, and the resultant
solid was collected by filtration which was recrystallized from acetic acid (6 mL) and washed with water to
afford 13 (0.820 g) as a light yellow solid , yield: 65.0%. m.p.: 232-234 °C. MS (ESI) m/z: 267.0, 269.0 [M+H]+.
6.6. General procedure for preparation of 4-hydroxyquinolines (14a–d)
To a stirring solution of 13 (10.0 g, 37.4 mmol) in acetonitrile (100 mL) at 25 °C was added appropriate
secondary amines (0.374 mol), and the resulting reaction mixture was heated to 80 °C for 8 h, then cooled to
room temperature. Approximately 60 mL acetonitrile was removed under reduced pressure. The residue was
cooled to 10 °C, and the resultant precipitate was filtered, washed with ethyl acetate (35 mL) to give the
corresponding 4-hydroxyquinolines 14a-d.
6.6.1. 6-Methyloxy-7-(4-(3-morpholino-propoxy))-4-quiolinol (14a)
Yellow solid, yield: 93.9%. m.p.: 187-189 °C. MS (ESI) m/z: 319.3 [M+H]+.
6.6.2. 6-Methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-ol (14b)
Yellow solid, yield: 84.5%. m.p.: 167-169 °C. MS (ESI) m/z: 303.4 [M+H]+.
6.6.3. 6-Methyloxy-7-(4-(3-(piperidin-1-yl)propoxy))-4-quiolin-ol (14c)
White solid, yield: 88.7%. m.p.: 176-77 °C. MS (ESI) m/z: 317.3 [M+H]+.
6.6.4. 6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-ol (14d)
Sallow solid, yield: 87.9%. m.p.: 182-184 °C. MS (ESI) m/z: 331.4 [M+H]+.
6.7. General procedure for preparation of 4-chloroquinolines (15a–d)
A solution of an appropriate dry 4-hydroxyquinoline (20.0 mmol) and POCl3 (40 mL) in dry acetonitrile (40
mL) was refluxed on an oil-bath for approximate 6 h. After cooling to ambient temperature, the contents were
concentrated under reduced pressure and the pale residue was poured into ice-water (100 mL) with vigorous
agitation. The solution was treated with concentrated ammonium hydroxide to achieve pH to 8 while
maintaining the temperature below 20 °C, and the mixture was extracted with CH2Cl2 (320 mL). The combined
organic layer was washed with brine, then water, and dried over anhydrous Na2SO4, concentrated under reduced
pressure to give corresponding 4-chloroquinolines 15a–d.
6.7.1. 4-Chloro-6-methyloxy-7-(4-(3-morpholino-propoxy))quinoline (15a)
White solid, yield: 82.4%. m.p.: 173-176 °C. MS (ESI) m/z: 336.2, 338.2 [M+H]+.
6.7.2. 4-Chloro-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinoline (15b)
White solid, yield: 83.1%. m.p.: 172-174 °C. MS (ESI) m/z: 321.4 [M+H]+.
6.7.3. 4-Chloro-6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinoline (15c)
White solid, yield: 85.9%. m.p.: 187-188 °C. MS (ESI) m/z: 335.5 [M+H]+.
13

6.7.4. 4-Chloro-6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinoline (15d)
White solid, yield: 81.4%. m.p.: 179-181 °C. MS (ESI) m/z: 349.3 [M+H]+.
6.8. General procedure for preparation of nitro compounds (16a-d)
A stirring mixture of an appropriate 15a-d (20.0 mmol) and 2-fluoro-4-nitrophenol (24.1 mmol) in
chlorobenzene (40 mL) was heated to 140 °C for about 20 h. The reaction was considered complete when less
than 5% starting material remained. After cooling to room temperature, the reaction mixture was concentrated
under reduced pressure to yield a pale solid. The solid was dissolved in CH2Cl2 (80 mL), and washed with
saturated K2CO3 aqueous solution (2×20 mL), then water (20 mL), and dried over anhydrous Na2SO4,
concentrated under reduced pressure to afford a wheat solid, which was recrystallized from anhydrous ethanol
(30 mL) to yield corresponding 16a–d.
6.8.1. 4-(2-Fluoro-4-nitrophenoxy)-6-methoxy-7-(3-morpholino-propoxy) quinoline (16a)
Light yellow solid, yield: 62.0%. m.p.: 135-136 °C; MS (ESI) m/z (%): 458.1 [M+H]+.
6.8.2. 4-(2-Fluoro-4-nitrophenoxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy) quinoline (16b)
Yellow solid, yield: 76.3%. m.p.: 140-141 °C; MS (ESI) m/z (%): 442.2 [M+H]+.
6.8.3. 4-(2-Fluoro-4-nitrophenoxy)-6-methoxy-7-(3-(piperidin-1-yl)propoxy) quinoline (16c)
Yellowish-white solid, yield: 80.4%. m.p.: 137-138 °C; MS (ESI) m/z (%): 456.4 [M+H]+.
6.8.4. 4-(2-Fluoro-4-nitrophenoxy)-6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinoline (16d)
Yellow solid; yield: 78.7%. m.p.: 133-134 °C; MS (ESI) m/z (%): 470.6 [M+H]+.
6.9. General procedure for preparation of anilines (17a-d)
A mixture of 16a-d (20.0 mmol), Fe (powder, 0.200 mol) and concentrated hydrochloric acid (36.5%, 1.00
mL) in ethanol/water (100 mL, 9:1 v/v) was refluxed with vigorous agitation for 2 h. The hot solution was
filtered through celite and the filter cake was washed with hot ethanol. The combined filtrate was concentrated
under reduced pressure to afford a yellow solid, which was recrystallized from anhydrous ethanol (60 mL) to
yield corresponding anilines 17a-d.
6.9.1. 3-Fluoro-4-(6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yloxy) aniline (17a)
White solid, yield: 92.0%. 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 5.1 Hz, 1H), 7.58 (s, 1H), 7.44 (s, 1H),
7.04 (t, J = 8.4 Hz, 1H), 6.54-6.59 (dd, J1 = 12.0 Hz, J2 = 2.4 Hz, 1H), 6.49-6.52 (m, 1H), 6.42 (d, J = 5.1 Hz,
1H), 4.27 (t, J = 6.6 Hz, 2H), 4.04 (s, 3H), 3.77-3.88 (br, 2H), 3.74 (t, J = 4.5 Hz, 4H), 2.60 (t, J = 6.9 Hz, 2H),
2.49-2.52 (m, 4H), 2.10-2.19 (m, 2H). m.p.: 217-218 °C. MS (ESI) m/z: 427.1 [M+H]+.
6.9.2. 3-Fluoro-4-(6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yloxy) aniline (17b)
Light yellow solid, yield: 72.3%. m.p.: 208-209 °C. MS (ESI) m/z: 412.5 [M+H]+.
6.9.3. 3-Fluoro-4-(6-methoxy-7-(3-(piperdine-1-yl)propoxy)quinolin-4-yloxy) aniline (17c)
Gray solid, yield: 85.5%. m.p.: 196-197 °C. MS (ESI) m/z: 426.3 [M+H]+.
6.9.4. 3-Fluoro-4-(6-methoxy-7-(3-(4-methylpiperdine-1-yl)propoxy)quinolin-4-yloxy)aniline (17d)
14

White solid, yield: 77.4%. 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 5.3 Hz, 1H), 7.58 (s, 1H), 7.41 (s, 1H),
7.03 (t, J = 8.7 Hz, 1H), 6.56 (dd, J1 = 11.8, J2 = 2.6 Hz, 1H), 6.50 (dd, J1 = 9.0, J2 = 2.9 Hz, 1H), 6.39 (dd, J =
5.3, 0.8 Hz, 1H), 4.25 (t, J = 6.7 Hz, 2H), 4.03 (s, 3H), 3.82 (s, 2H), 2.94 (m, 2H), 2.57 (m, 2H), 2.15 (m, 2H),
1.98 (t, J = 10.9 Hz, 2H), 1.63 (d, J = 10.4 Hz, 2H), 1.28 (m, 3H), 0.93 (d, J = 6.0 Hz, 3H). m.p.: 193-194 °C.
MS (ESI) m/z: 440.2 [M+H]+.
6.10. General procedure for preparation of phenylcarbamates (18a-d)
To the mixture of 17a-d (10.0 mmol) and anhydrous K2CO3 (2.76 g, 20.0 mmol) in dry acetone (60 mL),
phenyl chloroformate (1.88 mL, 15.0 mmol) was added dropwise while maintaining the temperature between
0 °C and 5 °C. After the addition was completed, the mixture was warmed to room temperature for another 3 h,
and the solvent was evaporated under reduced pressure. The residue was dissolved in CH2Cl2 (20 mL), and
washed with water (3×10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure to afford
corresponding phenylcarbamates 18a–d, which were immediately used in the next step without further
purification.
6.10.1. Phenyl 3-fluoro-4-(6-methoxy-7-(3-morpholino-propoxy)-quinolin-4-yloxy)phenylcarbamate (18a)
Yellow oil, yield: 90.2%. m.p.: 219–221 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 8.44 (d, J = 5.1
Hz, 1H), 7.64-7.68 (m, 1H), 7.49 (s, 1H), 7.39-7.44 (m, 4H), 7.36 (s, 1H), 7.20-7.27 (m, 3H), 6.41 (d, J = 5.1 Hz,
1H), 4.17 (t, J = 6.3 Hz, 2H), 3.91 (s, 3H), 3.52–3.65 (br, 4H), 2.52-2.62 (br, 4H), 2.31-2.43 (br, 2H), 1.89-2.06
(m, 2H). MS (ESI) m/z: 548.3, 549.3 [M+H]+.
6.10.2. Phenyl 3-fluoro-4-(6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy))-quinolin-4-yloxy) phenylcarbamate
(18b)
Yellow oil, yield: 85.0%. m.p.: 204-206 °C. MS (ESI) m/z: 532.2 [M+H]+.
6.10.3. Phenyl 3-fluoro-4-(6-methoxy-7-(3-(piperdine-1-yl)propoxy))-quinolin-4-yloxy) phenylcarbamate
(18c)
Light yellow oil, yield: 83.1%. m.p.: 210-212 °C. MS (ESI) m/z: 546.4 [M+H]+.
6.10.4.
Phenyl
3-fluoro-4-(6-methoxy-7-(3-(4-methylpiperdine-1-yl)propoxy))-quinolin-4-yloxy)
phenylcarbamate (18d)
Yellow oil, yield: 82.3%. m.p.: 226-228 °C. MS (ESI) m/z: 560.3 [M+H]+.
6.11.1. N1-(3-Fluoro-4-(6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yloxy)phenyl)semicarbazide (19a)
A mixture of 18a (4.40 g, 8.01 mmol) and 80% hydrazine monohydrate (10 mL) in 1,4-dioxane (20 mL) was
refluxed overnight with vigorous agitation. Most of the solvent was evaporated under reduced pressure when
white solid appeared. After cooling to 10 °C, the resulting precipitate was filtered off, washed with water, and
dried under vacuum to afford the title compound 19a (1.80 g) as a white solid, yield: 46.7%. 1H NMR (400 MHz,
DMSO-d6) δ 9.03 (s, 1H), 8.47 (d, J = 5.4 Hz, 1H), 7.83-7.88 (m, 1H), 7.63 (s, 1H), 7.53 (s, 1H), 7.44-7.48 (m,
1H), 7.40 (s, 1H), 7.33 (t, J = 9.0 Hz, 1H), 6.43 (d, J = 5.4 Hz, 1H), 4.32-4.59 (br, 2H), 4.20 (t, J = 6.6 Hz, 2H),
3.95 (s, 3H), 3.58-3.61 (m, 4H), 2.45-2.48 (m, 2H), 2.33-2.44 (br, 4H), 1.94-2.03 (m, 2H). m.p.: 188-190 °C. MS
(ESI) m/z: 485.3 [M+H]+.
6.11.2. N1-(3-Fluoro-4-(6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yloxy)phenyl)semicarbazide
15

(19b)
White solid, yield: 43.9%. 1H NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.47 (d, J = 5.1 Hz, 1H), 7.83-7.87
(m, 1H), 7.63 (s, 1H), 7.53 (s, 1H), 7.44-7.47 (m, 1H), 7.38 (s, 1H), 7.33 (t, J = 9.0 Hz, 1H), 6.42 (d, J = 5.1 Hz,
1H), 4.29-4.57 (br, 2H), 4.20 (t, J = 6.3 Hz, 2H), 3.95 (s, 3H), 2.58 (t, J = 6.3 Hz, 2H), 2.46 (s, 4H), 1.94-2.03
(m, 2H), 1.70 (s, 4H). m.p.: 177-178 °C. MS (ESI) m/z: 470.3 [M+H]+.
6.11.3.
(19c)
N1-(3-Fluoro-4-(6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yloxy)phenyl)semicarbazide
The intermediate 19c, which was purified by silica gel column chromatography (eluent, CH2Cl2/ MeOH/ Et3N
= 100:1:1 to 100:10:1), was obtained from 18c by the general procedure as a white solid, yield: 39.6%. 1H NMR
(400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.47 (d, J = 5.1 Hz, 1H), 7.82-7.87 (m, 1H), 7.60 (s, 1H), 7.53 (s, 1H),
7.41–7.49 (m, 1H), 7.38 (s, 1H), 7.32 (t, J = 9.0 Hz, 1H), 6.43 (d, J = 5.1 Hz, 1H), 4.30-4.52 (br, 2H), 4.18 (t, J =
6.3 Hz, 2H), 3.95 (s, 3H), 2.39-2.47 (m, 6H), 1.91-2.01 (m, 2H), 1.45–1.58 (m, 4H), 1.33-1.44 (m, 2H). m.p.:
181-182 °C. MS (ESI) m/z: 484.3 [M+H]+.
6.11.4.
N1-(3-Fluoro-4-(6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yloxy)phenyl)semicarbazide
(19d)
The intermediate 19d, which was purified by silica gel column chromatography (eluent, CH2Cl2/ MeOH/ Et3N
= 100:1:1 to 100:10:1), was obtained from 18d by the general procedure as a white solid, yield: 38.9%. 1H NMR
(400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.47 (d, J = 5.1 Hz, 1H), 7.82-7.86 (m, 1H), 7.58 (s, 1H), 7.41 (s, 1H),
7.03 (t, J = 8.7 Hz, 1H), 6.54-6.59 (dd, J1 = 12.0 Hz, J2 = 2.7 Hz, 1H), 6.48-6.52 (m, 1H), 6.41 (d, J = 5.1 Hz,
1H), 4.32-4.49 (br, 2H), 4.25 (t, J = 6.6 Hz, 2H), 4.03 (s, 3H), 2.89-2.98 (m 2H), 2.57 (m, 2H), 2.11-2.20 (m,
2H), 1.94-2.02 (m, 2H), 1.58-1.68 (m, 2H), 1.21-1.36 (m, 3H), 0.94 (d, J = 6.0 Hz, 3H). m.p.: 191-193 °C. MS
(ESI) m/z: 498.2 [M+H]+.
6.12.General procedure for the synthesis of semicarbazones (23a-w)
To a solution of 19a-d (0.200 g, 0.414 mmol) in isopropanol (2 mL), 1.1 equiv. of aldehydes or ketones and
acetic acid (1 drop) were added, and the mixture was refluxed for 5-6 h until TLC showed the completion of the
reaction. After cooling to room temperature, the resultant precipitate was filtered and dried under vacuum.
6.12.1.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-methoxybenzyli
dene)semicarbazide (23a)
White solid (88.5%); m.p.: 221-223 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 9.19 (s, 1H), 8.51 (d,
J = 5.1 Hz, 1H), 7.90-7.96 (m, 2H), 7.83 (d, J = 9.0 Hz, 2H), 7.64-7.68 (m, 1H), 7.58 (s, 1H), 7.46 (s, 1H), 7.41
(t, J = 9.0 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 6.50 (d, J = 5.1 Hz, 1H), 4.27 (t, J = 6.0 Hz, 2H), 3.97 (s, 3H), 3.81
(s, 3H), 3.71-3.79 (br, 4H), 2.94-3.19 (br, 6H), 2.15-2.24 (m, 2H); MS (ESI) m/z: 604.2 [M+H]+. Anal. calcd. for
C32H34FN5O6 (%): C, 63.67; H, 5.68; N, 11.60. Found (%): C, 63.62; H, 5.70; N, 11.58.
6.12.2.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-
N4-(4-nitrobenzylidene)semicarbazide (23b)
Yellow solid (92.4%); m.p.: 208-209 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 9.27 (s, 1H), 8.50 (d,
J = 5.4 Hz, 1H), 8.01-8.04 (m, 3H), 7.89-7.94 (dd, J1 = 13.2 Hz, J2 = 2.4 Hz, 1H), 7.63-7.66 (m, 1H), 7.55 (s,
16

1H), 7.39-7.45 (m, 4H), 6.47 (d, J = 5.1 Hz, 1H), 4.21 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 3.60 (s, 4H), 2.29-2.50
(br, 6H), 1.95-2.04 (m, 2H); MS (ESI) m/z: 619.4 [M+H]+. Anal. calcd. for C31H31FN6O7 (%): C, 60.19; H, 5.05;
N, 13.59. Found (%): C, 60.17; H, 5.07; N, 13.61.
6.12.3.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-methoxybe
nzylidene)semicarbazide (23c)
White solid (79.6%); m.p.: 217-196 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 9.18 (s, 1H), 8.49 (d,
J = 5.1 Hz, 1H), 7.94-7.96 (m, 1H), 7.86-7.91 (m, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.64-7.67 (m, 1H), 7.55 (s, 1H),
7.37-7.43 (m, 2H), 7.01 (d, J = 9.0 Hz, 2H), 6.47 (d, J = 5.1 Hz, 1H), 4.21 (t, J = 6.6 Hz, 2H), 3.96 (s, 3H), 3.81
(s, 3H), 2.61 (t, J = 7.2 Hz, 2H), 2.41-2.48 (br, 4H), 1.95-2.04 (m, 2H), 1.66-1.75 (m, 4H). MS (ESI) m/z: 588.2
[M+H]+. Anal. calcd. for C32H34FN5O5 (%): C, 65.40; H, 5.83; N, 11.92. Found (%): C, 65.42; H, 5.85; N,
11.90.
6.12.4.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-nitrobenzy
lidene)semicarbazide (23d)
1
Yellow solid (84.8%); m.p.: 201-203 °C; H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.40 (s, 1H), 8.50
(d, J = 5.4 Hz, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.01 (s, 1H), 7.98 (d, J = 8.7 Hz, 1H), 7.89-7.94 (dd, J1 = 13.5 Hz,
J2 = 2.4 Hz, 1H), 7.63-7.68 (m, 1H), 7.55 (s, 1H), 7.40-7.46 (m, 2H), 6.47 (d, J = 5.4 Hz, 1H), 4.21 (t, J = 6.6 Hz,
2H), 3.96 (s, 3H), 2.62 (t, J = 6.3 Hz, 2H), 2.51-2.58 (m, 4H), 1.96-2.05 (m, 2H), 1.71 (s, 4H); MS (ESI) m/z:
603.3 [M+H]+. Anal. calcd. for C31H31FN6O6 (%): C, 61.79; H, 5.19; N, 13.95. Found (%): C, 61.82; H, 5.21; N,
13.93.
6.12.5.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-(trifluoro
methyl)benzylidene)semicarbazide (23e)
1
Yellow solid (77.9%); m.p.: 228-230 °C; H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.31 (s, 1H), 8.49
(d, J = 5.1 Hz, 1H), 8.01-8.03 (m, 3H), 7.89-7.94 (dd, J1 = 13.2 Hz, J2 = 2.1 Hz, 1H), 7.63-7.66 (m, 1H), 7.55 (s,
1H), 7.39-7.45 (m, 4H), 6.47 (d, J = 5.1 Hz, 1H), 4.22 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 2.69 (t, J = 6.9 Hz, 2H),
2.59 (s, 4H), 1.98-2.07 (m, 2H), 1.74 (s, 4H); MS (ESI) m/z: 626.4 [M+H]+. Anal. calcd. for C32H31F4N5O4 (%):
C, 61.43; H, 4.99; N, 11.19. Found (%): C, 61.45; H, 4.95; N, 11.21.
6.12.6.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-methoxyben
zylidene)semicarbazide (23f)
1
White solid (82.1%); m.p.: 219-221 °C; H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 9.17-9.44 (br, 1H),
8.49 (d, J = 5.4 Hz, 1H), 7.89-7.95 (m, 2H), 7.79-7.83 (m, 2H), 7.62-7.66 (m, 1H), 7.55 (s, 1H), 7.37-7.43 (m,
2H), 7.01 (d, J = 8.7 Hz, 2H), 6.46 (d, J = 5.4 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 3.81 (s, 3H), 2.43 (t,
J = 7.2 Hz, 2H), 2.30-2.39 (br, 4H), 1.92-2.01 (m, 2H), 1.48-1.55 (m, 4H), 1.36-1.43 (m, 2H); MS (ESI) m/z:
602.5 [M+H]+. Anal. calcd. for C33H36FN5O5 (%): C, 65.88; H, 6.03; N, 11.64. Found (%): C, 65.90; H, 6.05; N,
11.66.
6.12.7.
17

(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-(trifluorom
ethyl)benzylidene)semicarbazide (23g)
White solid (79.5%); m.p.: 235-237 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 9.27 (s, 1H), 8.50 (d,
J = 5.4 Hz, 1H), 8.04 (s, 1H), 8.01 (d, J = 1.5 Hz, 2H), 7.94 (dd, J1 = 13.2 Hz, J2 = 2.4 Hz, 1H), 7.63-7.66 (m,
1H), 7.55 (s, 1H), 7.39-7.45 (m, 4H), 6.47 (d, J = 5.1 Hz), 4.19 (t, J = 6.6 Hz, 2H), 3.96 (s, 3H), 2.44 (t, J = 7.2
Hz, 2H), 2.36 (s, 4H), 1.91-2.01 (m, 2H), 1.48-1.55 (m, 4H), 1.34-1.42 (m, 2H). MS (ESI) m/z: 656.5 [M+H]+.
Anal. calcd. for C33H33F4N5O4 (%): C, 61.97; H, 5.20; N, 10.95. Found (%): C, 61.93; H, 5.23; N, 10.91.
6.12.8.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(2-chloro-4-flu
orobenzylidene)-semicarbazide (23h)
1
White solid (84.7%); m.p.: 213-215 °C; H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 9.33 (s, 1H),
8.45-8.49 (m, 2H), 8.34 (s, 1H), 7.88-7.92 (dd, J1 = 13.5 Hz, J2 = 2.1 Hz, 1H), 7.63-7.66 (m, 1H), 7.52-7.55 (m,
2H), 7.40-7.46 (m, 2H), 7.32-7.38 (m, 1H), 6.47 (d, J = 5.1 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 2.43
(t, J = 6.9 Hz, 2H), 2.27-2.40 (br, 4H), 1.91-2.01 (m, 2H), 1.44-1.53 (m, 4H), 1.31-1.43 (m, 2H); MS (ESI) m/z:
624.2 [M+H]+. Anal. calcd. for C32H32ClF2N5O4 (%): C, 61.59; H, 5.17; N, 11.22. Found (%): C, 61.63; H, 5.20;
N, 11.20.
6.12.9.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(2,4-dichlorob
enzylidene)- semicarbazide (23i)
White solid (89.5%); m.p.: 204-206 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 9.33 (s, 1H), 8.49 (d,
J = 5.4 Hz, 1H), 8.44 (d, J = 8.7 Hz, 1H), 8.34 (s, 1H), 7.87-7.92 (dd, J1 = 13.5 Hz, J2 = 2.4 Hz, 1H), 7.70 (d, J =
2.1 Hz, 1H), 7.62-7.65 (m, 1H), 7.54 (s, 1H), 7.50-7.54 (dd, J1 = 8.7 Hz, J2 = 2.1 Hz, 1H), 7.40-7.45 (m, 2H),
6.47 (d, J = 5.4 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 2.44 (t, J = 6.9 Hz, 2H), 2.36 (s, 4H), 1.92-2.01
(m, 2H), 1.48-1.55 (m, 4H), 1.37-1.42 (m, 2H); MS (ESI) m/z: 640.3 [M+H]+. Anal. calcd. for C32H32Cl2FN5O4
(%): C, 60.00; H, 5.04; N, 10.93. Found (%): C, 60.03; H, 5.01; N, 10.95.
6.12.10.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-(trifluorom
ethoxy)benzylidene)semicarbazide (23j)
White solid (75.4%); m.p.: 239-241 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.31 (s, 1H), 8.49 (d,
J = 5.1 Hz, 1H), 8.00-8.05 (m, 3H), 7.89-7.94 (d, J = 13.5 Hz, 1H), 7.63-7.66 (m, 1H), 7.55 (s, 1H), 7.40-7.44
(m, 4H), 6.47 (d, J = 5.1 Hz, 1H), 4.19 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H), 2.43-2.48 (m, 2H), 2.40 (s, 4H),
1.91-2.03 (m, 2H), 1,46-1.56 (m, 4H), 1.34-1.45 (m, 2H); MS (ESI) m/z: 656.2 [M+H]+. Anal. calcd. for
C33H33F4N5O5 (%): C, 60.45; H, 5.07; N, 10.68. Found (%): C, 60.47; H, 5.03; N, 10.71.
6.12.11.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(2,4-dimethox
ybenzylidene)semicarbazide (23k)
White solid (82.0%); m.p.: 227-228 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 9.74 (s, 1H), 8.49 (d,
J = 5.1 Hz, 1H), 8.24 (s, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.89-7.95 (dd, J1 = 13.5 Hz, J2 = 2.4 Hz, 1H), 7.63-7.66
(m, 1H), 7.54 (s, 1H), 7.36-7.42 (m, 2H), 6.59-6.62 (m, 2H), 6.46 (d, J = 5.1 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H),
3.96 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 2.44 (t, J = 6.9 Hz, 2H), 2.37 (s, 4H), 1.92-2.01 (m, 2H), 1.48-1.55 (m,
18

4H), 1.35-1.45 (m, 2H); MS (ESI) m/z: 632.3 [M+H]+. Anal. calcd. for C34H38FN5O6 (%): C, 64.65; H, 6.06; N,
11.09. Found (%): C, 64.65; H, 6.04; N, 11.11.
6.12.12.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-(dimethyla
mino)benzylidene)-semicarbazide (23l)
White solid (68.9%); m.p.: 226-229 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.11 (s, 1H), 8.49 (d,
J = 5.1 Hz, 1H), 7.91-7.96 (m, 1H), 7.86 (s, 1H), 7.61-7.68 (m, 3H), 7.54 (s, 1H), 7.36-7.42 (m, 2H), 6.75 (d, J =
8.7 Hz, 2H), 6.46 (d, J = 5.1 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 2.97 (s, 6H), 2.43 (t, J = 6.9 Hz, 2H),
2.35 (s, 4H), 1.91-2.01 (m, 2H), 1.47-1.55 (m, 4H), 1.33-1.44 (m, 2H); MS (ESI) m/z: 615.2 [M+H]+. Anal.
calcd. for C34H39FN6O4 (%): C, 66.43; H, 6.40; N, 13.67.. Found (%): C, 66.45; H, 6.40; N, 13.63.
6.12.13.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-hydroxyben
zylidene)semicarbazide (23m)
White solid (60.1%); m.p.: 201-203 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 9.21 (s, 1H), 8.49 (d,
J = 5.4 Hz, 1H), 7.90-7.95 (m, 2H), 7.62-7.69 (m, 3H), 7.55 (s, 1H), 7.36-7.42 (m, 2H), 6.86 (d, J = 8.7 Hz, 2H),
6.47 (d, J = 5.1 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H), 2.53-2.62 (br, 2H), 2.38-2.48 (br, 4H), 1.96-2.06
(m, 2H), 1.48-1.62(m, 4H), 1.34-1.45 (m, 2H); MS (ESI) m/z: 588.4 [M+H]+. Anal. calcd. for C32H34FN5O5 (%):
C, 65.40; H, 5.83; N, 11.92. Found (%): C, 65.36; H, 5.86; N, 11.93.
6.12.14.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(3-allyl-2-hydr
oxybenzylidene)semicarbazide (23n)
White solid (64.1%); m.p.: 197-198 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 10.44-10.79 (br, 1H),
9.63 (s, 1H), 8.49 (d, J = 5.1 Hz, 1H), 8.26 (s, 1H), 7.80-7.85 (dd, J1 = 13.5 Hz, J2 = 2.1 Hz, 1H), 7.54 (s, 1H),
7.47-7.51 (m, 1H), 7.37-7.43 (m, 3H), 7.16 (d, J = 7.2 Hz, 1H), 6.88 (t, J = 7.5 Hz, 1H), 6.47 (d, J = 5.4 Hz, 1H),
5.93-6.06 (m, 1H), 5.02-5.10 (m, 2H), 4.19 (t, J = 5.7 Hz, 2H), 3.96 (s, 3H), 3.41 (d, J = 6.6 Hz, 2H), 2.45-2.49
(m, 2H), 2.41 (s, 4H), 1.94-2.03 (m, 2H), 1.49-1.56 (m, 4H), 1.34-1.45 (m, 2H); MS (ESI) m/z: 628.4 [M+H]+.
Anal. calcd. for C35H38FN5O5 (%): C, 66.97; H, 6.10; N, 11.16. Found (%): C, 66.95; H, 6.12; N, 11.14.
6.12.15.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(3-allyl-2-meth
oxybenzylidene)-semicarbazide (23o)
White solid (74.4%); m.p.: 213-216 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H), 9.21 (s, 1H), 8.49 (d,
J = 5.4 Hz, 1H), 8.28 (s, 1H), 8.15 (d, J = 7.5 Hz, 1H), 7.90-7.95 (dd, J1 = 13.5 Hz, J2 = 2.1 Hz, 1H), 7.67 (d, J =
8.4 Hz, 1H), 7.55 (s, 1H), 7.38-7.44 (m, 2H), 7.24-7.27 (m, 1H), 7.15-7.20 (m, 1H), 6.47 (d, J = 5.4 Hz, 1H),
5.93-6.06 (m, 1H), 5.07-5.12 (m, 2H), 4.19 (t, J = 6.6 Hz, 2H), 3.96 (s, 3H), 3.73 (s, 3H), 3.42 (d, J = 5.7 Hz,
2H), 2.44 (t, J = 7.2 Hz, 2H), 2.36 (s, 4H), 1.92-2.01 (m, 2H), 1.45-1.55 (m, 4H), 1.30-1.45 (m, 2H); MS (ESI)
m/z: 642.7 [M+H]+. Anal. calcd. for C36H40FN5O5 (%): C, 67.38; H, 6.28; N, 10.91. Found (%): C, 67.41; H,
6.30; N, 10.88.
6.12.16.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(pyridin-4-ylm
19

ethylene)semicarbazide (23p)
White solid (72.1%); m.p.: 211-212 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J = 6.0 Hz, 2H), 8.48 (d, J
= 5.1 Hz, 1H), 8.03 (s, 1H), 7.84-7.90 (dd, J1 = 13.5 Hz, J2 = 2.1 Hz, 1H), 7.67 (d, J = 5.1 Hz, 2H), 7.54 (s, 1H),
7.47-7.52 (m, 1H), 7.34-7.40 (m, 2H), 6.46 (d, J = 5.1 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 2.43 (t, J =
7.2 Hz, 2H), 2.35 (s, 4H), 1.92-2.01 (m, 2H), 1.48-1.55 (m, 4H), 1.33-1.43 (m, 2H); MS (ESI) m/z: 573.2
[M+H]+. Anal. calcd. for C31H33FN6O4 (%): C, 65.02; H, 5.81; N, 14.68. Found (%): C, 65.00; H, 5.84; N, 14.65.
6.12.17.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-((1H-indol-2-yl
)methylene)semicarbazide (23q)
1
White solid (62.6%); m.p.: 239-242 °C; H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 10.52 (s, 1H), 8.92
(s, 1H), 8.49 (d, J = 5.4 Hz, 1H), 8.24-8.27 (m, 1H), 8.22 (s, 1H), 7.91-7.97 (dd, J1 = 13.2 Hz, J2 = 2.1 Hz, 1H),
7.84 (d, J = 2.7 Hz, 1H), 7.60 (m, 1H), 7.55 (s, 1H), 7.39-7.46 (m, 3H), 7.15-7.24 (m, 2H), 6.48 (d, J = 5.4 Hz,
1H), 4.19 (t, J = 6.3 Hz, 2H), 3.97 (s, 3H), 2.44 (t, J = 7.2 Hz, 2H), 2.36 (s, 4H), 1.92-2.01 (m, 2H), 1.48-1.55
(m, 4H), 1.35-1.44 (m, 2H); MS (ESI) m/z: 611.6 [M+H]+. Anal. calcd. for C34H35FN6O4 (%): C, 66.87; H, 5.78;
N, 13.76. Found (%): C, 66.85; H, 5.81; N, 13.74.
6.12.18.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(thiophen-2-yl
methylene)semicarbazide (23r)
White solid (61.2%); m.p.: 189-191 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 9.10 (s, 1H), 8.49 (d,
J = 5.1 Hz, 1H), 8.20 (s, 1H), 7.85-7.92 (dd, J1 = 13.2 Hz, J2 = 2.4 Hz, 1H), 7.66 (d, J = 5.1 Hz, 1H), 7.55-7.58
(m, 2H), 7.46 (m, 1H), 7.36-7.49 (m, 2H), 7.12-7.14 (m, 1H), 6.47 (d, J = 5.4 Hz, 1H), 4.21 (t, J = 6.3 Hz, 1H),
3.96 (s, 3H), 2.53-2.74 (br , 6H), 1.96-2.11 (m, 2H), 1.51-1.65 (m, 4H), 1.36-1.49 (m, 2H); MS (ESI) m/z: 578.2
[M+H]+. Anal. calcd. for C30H32FN5O4S (%): C, 62.38; H, 5.58; N, 12.12. Found (%): C, 62.36; H, 5.61; N,
12.10.
6.12.19.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-me
thoxybenzylidene)semicarbazide (23s)
White solid (82.1%); m.p.: 220-222 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 9.13 (s, 1H), 8.49 (d,
J = 5.1 Hz, 1H), 7.91-7.96 (dd, J1 = 13.5 Hz, J2 = 2.1 Hz, 1H), 7.86 (s, 1H), 7.68 (d, J = 8.7 Hz, 3H), 7.56 (s,
1H), 7.36-7.42 (m, 2H), 6.75 (d, J = 8.7 Hz, 2H), 6.47 (d, J = 5.1 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H),
3.81 (s, 3H), 2.87 (m, 2H), 2.45 (t, J = 6.6 Hz, 2H), 1.85-2.01 (m, 4H), 1.56-1.60 (m, 2H), 1.24-1.41 (m, 1H),
1.08-1.21 (m, 2H), 0.90 (d, J = 6.6 Hz, 3H); (ESI) m/z: 616.7 [M+H]+. Anal. calcd. for C34H38FN5O5 (%): C,
66.33; H, 6.22; N, 11.37. Found (%): C, 66.35; H, 6.20; N, 11.35.
6.12.20.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-nit
robenzylidene)semicarbazide (23t)
1
Yellow solid (87.9%); m.p.: 204-207 °C; H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 9.39 (s, 1H), 8.50
(d, J = 5.1 Hz, 1H), 8.29 (d, J = 8.7 Hz, 2H), 8.17 (d, J = 8.7 Hz, 2H), 8.09 (s, 1H), 7.89-7.94 (dd, J1 = 13.2 Hz,
J2 = 2.1 Hz, 1H), 7.64-7.67 (m, 1H), 7.55 (s, 1H), 7.40-7.47 (m, 2H), 6.47 (d, J = 5.4 Hz, 1H), 4.19 (t, J = 6.0 Hz,
2H), 3.96 (s, 3H), 2.78-3.02 (m, 2H), 2.53-2.61 (m, 2H), 1.97-2.01 (m, 4H), 1.57-1.61 (m, 2H), 1.28-1.45 (br,
20

1H), 1.05-1.22 (m, 2H), 0.90 (d, J = 6.3 Hz, 3H); MS (ESI) m/z: 631.8 [M+H]+. Anal. calcd. for C33H35FN6O6
(%): C, 62.85; H, 5.59; N, 13.33. Found (%): C, 62.83; H, 5.59; N, 13.31.
6.12.21.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-(tri
fluoromethyl)benzylidene)semicarbazide (23u)
White solid (79.1%); m.p.: 241-243 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.40-11.73 (br, 1H), 9.61-9.91 (br,
1H), 8.48 (d, J = 5.4 Hz, 1H), 8.08-8.14 (m, 3H), 7.91-7.96 (m, 1H), 7.73-7.81 (m, 2H), 7.63-7.67 (m, 1H), 7.59
(s, 1H), 7.38-7.47 (m, 2H), 6.48 (d, J = 5.1 Hz, 1H), 4.21 (t, J = 4.8 Hz, 2H), 3.97 (s, 3H), 2.98-3.05 (m, 2H),
2.61-2.65 (m, 2H), 1.92-2.09 (m, 4H), 1.60-1.65 (m, 2H), 1.32-1.49 (br, 1H), 1.20-1.31 (m, 2H), 0.91 (d, J = 6.3
Hz, 3H); (ESI) m/z: 654.5 [M+H]+. Anal. calcd. for C34H35F4N5O4 (%): C, 62.47; H, 5.40; N, 10.71. Found (%):
C, 62.45; H, 5.42; N, 10.68.
6.12.22.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(2,4-d
ichlorobenzylidene)-semicarbazide (23v)
White solid (82.1%); m.p.: 213-215 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.33 (s, 1H), 8.49 (d,
J = 5.4 Hz, 1H), 8.44 (d, J = 8.7 Hz, 1H), 8.34 (s, 1H), 7.87-7.92 (dd, J1 = 13.5 Hz, J2 = 2.4 Hz, 1H), 7.70 (d, J =
2.1 Hz, 1H), 7.62-7.65 (m, 1H), 7.54 (s, 1H), 7.50-7.53 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 7.39-7.45 (m, 2H),
6.47 (d, J = 5.1 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 2.87 (m, 2H), 2.45 (t, J = 6.6 Hz, 2H), 1.85-2.01
(m, 4H), 1.56-1.60 (m, 2H), 1.24-1.41 (m, 1H), 1.08-1.21 (m, 2H), 0.90 (d, J = 6.6 Hz, 3H); 13C NMR (100
MHz, DMSO-d6) δ 159.9, 154.7, 153.2, 152.4, 150.0, 149.3, 146.8, 138.7, 136.6, 135.5, 135.0, 133.8, 131.0,
129.6, 129.2, 128.1, 124.2, 117.1, 114.9, 109.0, 108.9, 102.4, 99.5, 67.2, 56.2, 55.2, 53.9 (2C), 34.4 (2C), 30.8 ,
26.6, 22.3; MS (ESI) m/z: 654.4 [M+H]+. Anal. calcd. for C33H34Cl2FN5O4 (%): C, 60.55; H, 5.24; N, 10.70.
Found (%): C, 60.53; H, 5.20; N, 10.72.
6.12.23.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(2-chl
oro-4-fluorobenzylidene)-semicarbazide (23w)
1
White solid (92.0%); m.p.: 218-219 °C; H NMR (400 MHz, DMSO-d6) δ 11.12(s, 1H), 9.30 (s, 1H),
8.44-8.49 (m, 2H), 8.34 (s, 1H), 7.87-7.93 (dd, J1 = 13.5 Hz, J2 = 2.4 Hz, 1H), 7.62-7.65 (m, 1H), 7.54 (s, 1H),
7.50-7.54 (m, 1H), 7.37-7.45 (m, 2H), 7.31-7.36 (m, 1H), 6.47 (d, J = 5.4 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96
(s, 3H), 2.84-2.88 (m, 2H), 2.46 (t, J = 6.6 Hz, 2H), 1.86-2.00 (m, 4H), 1.55-1.60 (m, 2H), 1.24-1.41 (m, 1H),
1.08-1.21 (m, 2H), 0.90 (d, J = 6.6 Hz, 3H); MS (ESI) m/z: 638.4 [M+H]+. Anal. calcd. for C33H34ClF2N5O4 (%):
C, 62.11; H, 5.37; N, 10.98. Found (%): C, 62.13; H, 5.33; N, 11.02.
6.13.
4-(4-(7-(3-(Piperidin-1-yl)propoxy)-6-methoxyquinolin-4-yloxy)-3-fluorophenyl)thiosemicarbazide (25)
Intermediate 17c (5.00 g, 11.7 mmol) was dissolved in CHCl3 (50 mL) and sat. aq. NaHCO3 (50 mL) was
added. The resulting biphasic solution was cooled to 0 °C and thiophosgene (2.03 g, 17.6 mmol) was then
carefully added via syringe. After the addition was completed, the reaction was allowed to warm to room
temperature and stirred for 6 h. The organic layer was separated and the aqueous layer was extracted with CHCl3
20 mL for twice. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced
pressure to afford intermediate 24 (4.82 g, 87.2%) as a yellow oil which was used in the next step as soon as
21

possible without further purification.
To a solution of 24 (4.80 g, 10.2 mmol) in CHCl3 (40 mL) was added 80% hydrazine monohydrate (40 mL),
and the biphasic solution was vigorously stirred for 3 h at room temperature. The organic layer was separated
and the aqueous layer was extracted with CHCl3 10 mL for twice. The combined organic layers were dried over
Na2SO4, filtered, and concentrated under reduced pressure to get a brown oil which was purified by silica gel
column chromatography (eluent, CH2Cl2/ MeOH/ Et3N = 100: 2: 1 to 100: 10: 1) to afford 25 (2.84 g) as a
yellow solid, yield: 53.2%. m.p.: 105-109 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.47 (d, J = 5.1 Hz,
1H), 7.80-7.86 (m, 1H), 7.58 (s, 1H), 7.52 (s, 1H), 7.40-7.49 (m, 1H), 7.40 (s, 1H), 7.31 (t, J = 9.0 Hz, 1H), 6.46
(d, J = 5.1 Hz, 1H), 4.32-4.48 (br, 2H), 4.19 (t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 2.39-2.47 (m, 6H), 1.91-2.03 (m,
2H), 1.43-1.57 (m, 4H), 1.31-1.43 (m, 2H). MS (ESI) m/z: 500.4 [M+H]+.
6.14.General procedure for the synthesis of thiosemicarbazide (26a-d)
To a solution of 44 (0.200 g, 0.404 mmol) in isopropanol (2 mL), 1.1 equiv. of appropriate aldehyde and
acetic acid (1 drop) were added, and the mixture was refluxed for 6-7 h until TLC showed the completion of the
reaction. After cooling to 0 °C, the resultant precipitate was filtered and dried under vacuum.
6.14.1.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-benzylidene-th
iosemicarbazide (26a)
Yellow solid (78.4%); m.p.: 121-123 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 10.27 (s, 1H), 8.53
(d, J = 5.4 Hz, 1H), 8.21 (s, 1H), 7.92-7.97 (m, 3H), 7.62-7.65 (m, 1H), 7.56 (s, 1H), 7.44-7.50 (m, 4H), 7.42 (s,
1H), 6.48 (d, J = 5.4 Hz, 1H), 4.21 (t, J = 6.3 Hz, 2H), 3.97 (s, 3H), 2.53-2.74 (m, 6H), 1.96-2.11 (m, 2H),
1.51-1.64 (m, 4H), 1.37-1.48 (m, 2H); MS (ESI) m/z: 588.6 [M+H]+. Anal. calcd. for C32H34FN5O3S (%): C,
65.40; H, 5.83; N, 11.92. Found (%): C, 65.38; H, 5.85; N, 11.88.
6.14.2.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(3,4-difluorobe
nzylidene)-thiosemicarbazide (26b)
1
Yellow solid (79.4%); m.p.: 137-138 °C; H NMR (400 MHz, DMSO-d6) δ 12.02-12.29 (br, 1H), 10.37 (s,
1H), 8.53 (d, J = 5.1 Hz, 1H), 8.16 (s, 1H), 7.91 (d, J = 12.6 Hz, 1H), 7.47-7.71 (m, 6H), 7.42 (s, 1H), 6.47 (d, J
= 5.1 Hz, 1H), 4.21 (t, J = 6.0 Hz, 2H), 3.97 (s, 3H), 2.55-2.69 (m, 6H), 2.01-2.07 (m, 2H), 1.48-1.65 (m, 4H),
1.31-1.47 (m, 2H); MS (ESI) m/z: 624.2 [M+H]+. Anal. calcd. for C32H32F3N5O3S (%): C, 61.62; H, 5.17; N,
11.23. Found (%): C, 61.64; H, 5.18; N, 11.20.
6.14.3.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(4-chlorobenzy
lidene)-thiosemicarbazide (26c)
Yellow solid (82.7%); m.p.: 128-130 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.96-12.17 (br, 1H), 10.23-10.43
(br, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.18 (s, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.89-7.94 (dd, J1 = 12.6 Hz, J2 = 2.4 Hz,
1H), 7.58-7.62 (m, 1H), 7.46-7.55 (m, 4H), 7.41 (s, 1H), 6.47 (d, J = 5.4 Hz, 1H), 4.19 (t, J = 6.3 Hz, 2H), 3.96
(s, 3H), 2.43 (t, J = 7.2 Hz, 2H), 2.27-2.39 (br, 4H), 1.92-2.01 (m, 2H), 1.45-1.58 (m, 2H), 1.34-1.45 (m, 2H);
MS (ESI) m/z: 622.3 [M+H]+. Anal. calcd. for C32H33ClFN5O3S (%): C, 61.78; H, 5.35; N, 11.26. Found (%): C,
61.80; H, 5.32; N, 11.24.
22

6.14.4.
(E)-N1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-N4-(2-chloro-4-flu
orobenzylidene)-thiosemicarbazide (26d)
1
Yellow solid (77.3%); m.p.: 136-138 °C; H NMR (400 MHz, DMSO-d6) δ 12.12-12.29 (br, 1H), 10.39 (s,
1H), 8.51-8.59 (m, 3H), 7.88-7.92 (dd, J1 = 12.6 Hz, J2 = 1.8 Hz, 1H), 7.59-7.62 (m, 3H), 7.50 (t, J = 9.6 Hz,
1H), 7.41 (s, 1H), 7.33-7.39 (m, 1H), 6.47 (d, J = 5.1 Hz, 1H), 4.20 (t, J = 6.3 Hz, 2H), 3.97 (s, 3H), 2.45 (t, J =
7.2 Hz, 2H), 2.23-2.43 (br, 4H), 1.93-2.02 (m, 2H), 1.45-1.59 (m, 4H), 1.32-1.45 (m, 2H); MS (ESI) m/z: 640.2
[M+H]+. Anal. calcd. for C32H32ClF2N5O3S (%): C, 60.04; H, 5.04; N, 10.94. Found (%): C, 60.07; H, 5.02; N,
10.98.
6.15.General procedure for the synthesis of semicarbazides (29a-d)
To the mixture of variant amines (50.0 mmol) and K2CO3 (13.8 g, 0.100 mol) in dry acetone (50 mL), phenyl
chloroformate (75.0 mmol) was added dropwisely while maintaining the temperature between 0 and 5 °C. After
the addition was completed, the mixture was warmed to room temperature for another 3 h, and the solvent was
evaporated under reduced pressure. The residue was washed with water 50 mL for thrice, filtered, and dried
under vacuum to afford corresponding carbamates 29a-d.
A mixture of an appropriate carbamate 29a-d (20.0 mmol) and 80% hydrazine hydrate (10 mL) in 1,4-dioxane
(10 mL) was refluxed overnight with vigorous agitation. After cooling to room temperature, the resulting
precipitate was filtered-off, washed with water, and dried under vacuum to afford corresponding semicarbazides
30a-d.
6.15.1. N-Phenylhydrazinecarboxamide (29a)
1
White solid (77.6%); H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H), 7.52 (d, J = 7.5 Hz, 2H), 7.36 (s, 1H),
7.22 (t, J = 7.5 Hz, 2H), 6.91 (t, J = 7.5 Hz, 1H), 4.33 (s, 2H). MS (ESI) m/z: 152.2 [M+H]+.
6.15.2. N-(3,4-Difluorophenyl)hydrazinecarboxamide (29b)
White solid (87.1%); MS (ESI) m/z: 188.0 [M+H]+.
6.15.3. N-(4-Bromophenyl)hydrazinecarboxamide (29c)
White solid (86.9%); MS (ESI) m/z: 230.2, 232.1 [M+H]+.
6.15.4. N-(2-Bromo-4-fluorophenyl)hydrazinecarboxamide (29d)
White solid (81.9%); MS (ESI) m/z: 248.1, 250.3 [M+H]+.
6.16. 3-Fluoro-4-(6-methoxy-7-(3-(piperidin-1-yl)propoxy)-quinolin-4-yloxy)benzaldehyde (27)
A solution of 4-chloroquinoline (20.0 mmol) and 3-fluoro-4-hydroxybenzaldehyde (3.40 g, 24.0 mmol) in
chlorobenzene (35 mL) was refluxed for 17 h. The reaction was considered complete when < 5% starting
material remained. After cooling to room temperature, the reaction mixture was concentrated under reduced
pressure to yield a pale solid. The solid was dissolved in CH2Cl2 (60 mL), and washed with saturated K2CO3
aqueous solution 20 mL for twice, then water (20 mL), and dried over anhydrous Na2SO4, concentrated under
reduced pressure to afford brown solid which was purified by silica gel column chromatography (eluent,
CH2Cl2/MeOH/Et3N = 100:2:1 to 100:5:1) to afford corresponding benzaldehydes 27. White solid (41.9%); m.p.:
159-161 °C; MS (ESI) m/z: 439.3, 440.3 [M+H]+.
23

6.17. General procedure for the synthesis of compounds (30a-d)
To a solution of 30a-d (0.500 mmol) in isopropanol (3 mL), 1.1 equiv. of appropriate 15a–r and one drop of
acetic acid were added, and the mixture was refluxed for 4-6 h until TLC showed the completion of the reaction.
After cooling to room temperature, the resultant precipitate was filtered and dried under vacuum to give the
corresponding compounds.
6.17.1.
(E)-2-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)benzylidene)-N-phenylsemi
carbazide (30a)
White solid (84.3%); m.p.: 202-204 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 9.00 (s, 1H), 8.51 (d,
J = 5.4 Hz, 1H), 8.20-8.24 (d, J = 11.7 Hz, 1H), 8.00 (s, 1H), 7.70-7.73 (m, 1H), 7.67 (d, J = 8.1 Hz, 2H),
7.47-7.53 (m, 2H), 7.42 (s, 1H), 7.31 (t, J = 8.1 Hz, 2H), 7.03 (t, J = 7.2 Hz, 1H), 6.53 (d, J = 5.4 Hz, 1H), 4.22
(t, J = 6.3 Hz, 2H), 3.96 (s, 3H), 3.50-3.66 (br, 4H), 2.27-2.48 (m, 6H), 1.91-2.04 (m, 2H); MS (ESI) m/z: 572.4
[M+H]+. Anal. calcd. for C32H34FN5O4 (%): C, 67.24; H, 6.00; N, 12.25. Found (%): C, 67.26; H, 6.03; N, 12.22.
6.17.2.
(E)-N1-(3,4-Difluorophenyl)-N4-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)be
nzylidene)semicarbazide (30b)
White solid (82.6%); m.p.: 219-221 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 8.94 (s, 1H), 8.52 (d,
J = 5.1 Hz, 1H), 8.05 (s, 1H), 7.92-8.02 (m, 2H), 7.69-7.73 (m, 1H), 7.64-7.68 (dd, J1 = 8.4, J2 = 3.0 Hz, 1H),
7.49-7.54 (m, 2H), 7.45 (s, 1H), 7.26-7.33 (m, 1H), 6.57 (d, J = 5.4 Hz, 1H), 4.26 (t, J = 5.7 Hz, 2H), 3.96 (s,
3H), 2.75-3.22 (m, 6H), 2.15-2.31 (m, 2H), 1.67-1.83 (m, 4H), 1.42-1.60 (br, 2H); MS (ESI) m/z: 608.2 [M+H]+.
Anal. calcd. for C32H32F3N5O4 (%): C, 63.25; H, 5.31; N, 11.53. Found (%): C, 63.22; H, 5.33; N, 11.51.
6.17.3.
(E)-N1-(4-Bromophenyl)-N4-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)benzy
lidene)semicarbazide (30c)
White solid (86.9%); m.p.: 193-196 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 9.15 (s, 1H), 8.52 (d,
J = 5.4 Hz, 1H), 8.19-8.23 (dd, J1 = 12.3 Hz, J2 = 1.5 Hz, 1H), 8.01 (s, 1H), 7.73 (m, 1H), 7.68 (d, J = 9.0 Hz,
1H), 7.45-7.55 (m, 5H), 6.54 (d, J = 5.1 Hz, 1H), 4.25 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H), 2.75-3.06 (br, 6H),
2.13-2.23 (m, 2H), 1.64-1.79 (m, 4H), 1.44-1.55 (br, 2H); MS (ESI) m/z: 650.3 [M+H]+. Anal. calcd. for
C32H33BrFN5O4 (%): C, 59.08; H, 5.11; N, 10.77. Found (%): C, 59.06; H, 5.13; N, 10.73.
6.17.4.
(E)-N1-(2-Bromo-4-fluorophenyl)-N4-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)o
xy)benzylidene)semicarbazide (30d)
White solid (86.6%); m.p.: 206-208 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 9.31 (s, 1H), 8.50 (d,
J = 5.4 Hz, 1H), 8.45 (d, J = 6.3 Hz, 1H), 8.01 (s, 1H), 7.88-7.93 (dd, J1 = 13.0 Hz, J2 = 1.8 Hz, 1H), 7.63-7.66
(m, 1H), 7.55 (s, 1H), 7.51-7.54 (m, 1H), 7.39-7.49 (m, 2H), 7.32-7.38 (m, 1H), 6.47 (d, J = 5.4 Hz, 1H), 4.21 (t,
J = 6.3 Hz, 2H), 3.96 (s, 3H), 3.58-3.61 (m, 4H), 2.44-2.48 (m, 2H), 2.33-2.44 (br, 4H), 1.95-2.03 (m, 2H); MS
(ESI) m/z: 668.3 [M+H]+. Anal. calcd. for C32H32BrF2N5O4 (%): C, 57.49; H, 4.82; N, 10.48. Found (%): C,
57.51; H, 4.80; N, 10.51.
24

Acknowledgments
This work was supported by Liaoning Baiqianwan Talents Program (No. 2013921042), Program for Liaoning
Excellent Talents in University (No. LR2014030) and the Special Scientific Research Fund of Shenyang
Pharmaceutical University (No. ZCJJ2014401).
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26

Graphical Abstract
IC50 (nM)
MKN-45
39
Compd.
23n
A549
11
HT-29
27
c-Met
1.54
Three series of novel 6, 7-disubstituted-4-phenoxyquinoline derivatives bearing semicarbazone scaffold were
synthesized and evaluated for their cytotoxicity. Compound 23n was further examined for its c-Met kinase
activity in vitro as well as pharmacokinetic profiles and antitumor efficacy in vivo.
27