Design, synthesis, structure-activity relationships and mechanism of action of new quinoline derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2018.11.048

A series of new quinoline derivatives was designed, synthesized and evaluated as potential antitumor agents. The results indicated that most compounds exhibited potent antiproliferative activity, and 7-(4-fluorobenzyloxy)–N-(2-(dimethylamino)- ethyl)quinolin-4-amine 10g was found to be the most potent antiproliferative agent against human tumor cell lines with an IC₅₀ value of less than 1.0 μM. Preliminary structure-activity relationships analysis suggested that (1) the large and bulky alkoxy substituent in position-7 might be a beneficial pharmacophoric group for antiproliferative activity; (2) the amino side chain substituents in position-4 facilitated the antiproliferative activity of this class of compounds; and (3) the length of the alkylamino side chain moiety affected the antiproliferative potency, with two CH₂ units being the most favorable. Further investigation of the mechanism of action of this class of compounds demonstrated that the representative compound 10g triggered p53/Bax-dependent colorectal cancer cell apoptosis by activating p53 transcriptional activity. Moreover, the results showed that compound 10g effectively inhibited tumor growth in a colorectal cancer xenograft model in nude mice. Thus, these quinoline derivatives might serve as candidates for the development of new antitumor drugs.

Full text of DOI:10.1016/j.ejmech.2018.11.048

Accepted Manuscript
Design, synthesis, structure-activity relationships and mechanism of action of new
quinoline derivatives as potential antitumor agents
Shangze Li, Lihua Hu, Jianru Li, Jiongchang Zhu, Feng Zeng, Qiuhua Huang, Liqin
Qiu, Runlei Du, Rihui Cao
PII:
S0223-5234(18)31007-9
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.048
EJMECH 10906
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 August 2018
Revised Date: 18 November 2018
Accepted Date: 20 November 2018
Please cite this article as: S. Li, L. Hu, J. Li, J. Zhu, F. Zeng, Q. Huang, L. Qiu, R. Du, R. Cao, Design,
synthesis, structure-activity relationships and mechanism of action of new quinoline derivatives as
potential antitumor agents, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.11.048.
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ACCEPTED MANUSCRIPT
Graphic abstract
A series of new quinoline derivatives was designed, synthesized and evaluated
as promising antitumor agents. The representative compound 10g could trigger
p53/Bax-dependent colorectal cancer cell apoptosis by activating p53 transcriptional
activity.

ACCEPTED MANUSCRIPT
Design, synthesis, structure-activity relationships and mechanism of
action of new quinoline derivatives as potential antitumor agents
Shangze Lic, #, Lihua Hub, #, Jianru Lia, Jiongchang Zhua, Feng Zengb, Qiuhua Huanga,
*
Liqin Qiua, Runlei Dub, Rihui Cao a
aSchool of Chemistry, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275,
P R China
bCollege of Life Sciences, Wuhan University, 299 Ba Yi Road, Wuchang, 430072, P R China
cMedical Science Research Center, Zhongnan hospital of Wuhan University, Hubei, Wuhan
430071, P R China
*Corresponding
author.
Tel:
86-20-84110918
Fax:
86-20-84112245,
E-mail:
caorihui@mail.sysu.edu.cn (R. Cao). # Shangze Li and Lihua Hu contributed equally to this work.
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Abstract
A series of new quinoline derivatives was designed, synthesized and evaluated as
potential antitumor agents. The results indicated that most compounds exhibited
potent antiproliferative activity, and 7-(4-fluorobenzyloxy)-N-(2-(dimethylamino)-
ethyl)quinolin-4-amine 10g was found to be the most potent antiproliferative agent
against human tumor cell lines with an IC50 value of less than 1.0 µM. Preliminary
structure-activity relationships analysis suggested that (1) the large and bulky alkoxy
substituent in position-7 might be a beneficial pharmacophoric group for
antiproliferative activity; (2) the amino side chain substituents in position-4 facilitated
the antiproliferative activity of this class of compounds; and (3) the length of the
alkylamino side chain moiety affected the antiproliferative potency, with two CH2
units being the most favorable. Further investigation of the mechanism of action of
this class of compounds demonstrated that the representative compound 10g triggered
p53/Bax-dependent colorectal cancer cell apoptosis by activating p53 transcriptional
activity. Moreover, the results showed that compound 10g effectively inhibited tumor
growth in a colorectal cancer xenograft model in nude mice. Thus, these quinoline
derivatives might serve as candidates for the development of new antitumor drugs.
Key Words: Synthesis; quinoline derivative; antiproliferative; structure-activity
relationships; mechanism of action.
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1. Introduction
Apoptosis is a programmed cellular suicide process that can remove damaged
cells, such as cancer cells, from multicellular organisms. In cancer cells, the apoptotic
pathway is often inactivated at the cellular level, which results in drug resistance in
cancer[1]. Therefore, developing a strategy to increase apoptosis activity is a potential
approach for cancer therapy.
The transcription factor p53 mediates multiple biological processes, such as cell
cycle arrest, apoptosis, metabolism and autophagy[2]. Over half of patients with tumors
have mutation of p53 protein, and thus, p53 is considered to be crucial for suppressing
tumorigenesis[3]. The post-translational modifications, including phosphorylation,
acetylation, ubiquitination and sumoylation, are the primary mechanism by which p53
is regulated. Phosphorylation of p53 at several serine/threonine residues has been
shown to promote nuclear localization, stabilization, DNA binding and subsequent
transcriptional activation. Induction of apoptotic death in cancer cells is considered the
major mechanism by which p53 prevents tumor development and progression.
Quinolines represent a large group of naturally occurring and synthetic products
associated with a wide spectrum of pharmacological functions, including
antibacterial[4-6], antitumor[7-9] and anti-HIV[10] activities. Since the discovery of
nalidixic acid by Lesher in 1962, intense research efforts have been spent in search of
more potent antibacterial agents, and the structure-activity relationships of quinolines
as antibacterial agents have also been extensively investigated[5-7, 9, 11].
Initially, quinolines were found to be specific inhibitors of bacterial type II
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topoisomerases, such as DNA gyrase[12]. Recently, topoisomerase IV[13], Pim-1
kinase[14], EGFR tyrosine kinase[15], tyrosine-protein kinase Met (c-Met)[16],
Penicillin-Binding Proteins[17] and histone deacetylase (HDAC)[18] were also
discovered to be pharmacological targets of this class of compounds. Due to structural
and functional similarities between bacterial DNA gyrase and human topoisomerase II,
it is speculated that this class of compounds also have potential antitumor activity.
Consequently, in the past decade, considerable attention has been focused on the
antitumor effects of quinolines, and a larger number of quinolines have been reported
as promising antitumor agents[7, 14-16, 19-26].
Antitume, 7-chloro-4(1H)-quinolone 11 (Fig. 1), was approved by the State
Food and Drug Administration of China (SFDA) as an antitumor drug for late
mammary cancer and non-small cell lung cancer treatment in 2003[27]. The
advantage of this compound as an antitumor drug is its low toxicity, easy
administration and cheap synthesis. However, its poor solubility and moderate
antitumor activity in vivo have limited its wide application in clinical cancer therapy.
Our group recently demonstrated that introducing a flexible amino side chain into
position-3 of the quinoline nucleus contributes to improving the water solubility and
in vitro antiproliferative activity of this class of compounds[28]. In a continuing effort
to develop novel quinoline derivatives endowed with better pharmacological profiles,
we designed and synthesized a series of new quinolines bearing an alkoxy group in
position-7 as well as a flexible alkylamino side chain at position-4. Herein, we report
the preparation of new quinoline derivatives, their antiproliferative activity,
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preliminary structure-activity relationships and mechanism of action as antitumor
agents.
2. Chemistry
The synthetic routes of quinolines 7, 9a-k and 10a-h are outlined in Scheme 1.
The reaction of m-anisidine 1 with ethoxymethylenemalonic ester 2 afforded the
crude Schiff base 3[29]. Cyclization of intermediate 3 in refluxing diphenyl ether
gave carboxylate 4[29], followed by hydrolysis with 10% sodium hydroxide solution
to provide carboxylic acid 5[29]. Decarboxylation of carboxylic acid 5 in refluxing
diphenyl ether afforded 7-methoxyquinolin-4(1H)-one 6. The conversion of 6 to
4-chloro-7-methoxy-quinoline 7 occurred readily by treatment with phosphorous
oxychloride. The demethylation of 4-chloro-7-methoxy-quinoline 7 was accomplished
readily using 48% hydrobromic acid as the reaction solvent to give
4-chloroquinolin-7-ol 8[30]. Compounds 9a-k bearing various alkoxy groups in
position-7 of the quinoline nucleus were synthesized from intermediate 8 by the
action of sodium hydride in dry DMF followed by addition of the appropriate
alkylating and arylating agents[30]. The amination of 9a, 9c, 9f, 9h and 9i with an
excess of N,N-diethylethylenediamine or 3-(diethylamino)propylamine by refluxing
provided the target compounds 10a-h in good yields[31]. The chemical structure of
1
all the synthesized new compounds was characterized by ESI-MS, HRMS, H NMR
and 13C NMR.
3. Results and Discussion
3.1 Antiproliferative activity in vitro
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The antiproliferative activity of quinolone derivatives 7, 9a-k and 10a-h against
a panel of human tumor cell lines was investigated and compared with the reference
drug cisplatin. To improve the solubility in aqueous solution, all compounds were
prepared in the form of hydrochloride by the usual methods before use. The
compounds 10a-h showed good water solubility (more than 10mg/mL). The tumor
cell line panel consisted of human colorectal carcinoma (HCT-116, RKO and DLD1),
liver carcinoma (HepG2), gastric carcinoma (BGC-823), non-small-cell lung
carcinoma (NCI-H1650) and ovarian carcinoma (SK-OV-3) cells. The results are
summarized in Table 1.
As shown in Table 1, the lead compound 7-chloro-4(1H)-quinolone 11 failed to
display antiproliferative activity against human tumor cell lines at a concentration of
10 µM. Compound 7 bearing a methoxy group in position-7 of the quinoline nucleus
only showed selective antiproliferative activity against the HCT-116, RKO, HepG2
and NCI-H1650 human tumor cell lines, with IC50 values of 8.82, 9.65, 6.07 and 8.16
µM, respectively. However, compounds 9a-k having relatively large and bulky alkoxy
substituents in position-7 displayed more potent antiproliferative activity against
several human tumor cell lines, with IC50 values of lower than 5.0 µM. Among all the
7-alkoxy substituted quinoline derivatives 9a-k, compound 9k exhibited the most
interesting antiproliferative activity against the HCT-116 and NCI-H1650 human
tumor cell lines, with IC50 values of 0.80 and 0.63 µM, respectively. These results
suggested that a large and bulky substituent at position-9 might be advisable
pharmacophoric group for antiproliferative activities.
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Our group previous investigations[28, 31-34] demonstrated that introducing a
flexible amino side chain into various skeleton molecules contributes to improving the
water solubility and in vitro antiproliferative activity. Logically, we speculated that
introducing an aminoethylamino side chain group into position-4 of quinolines 9
would impart improved water solubility and enhanced antiproliferative activity.
Consequently, a series of novel quinolone derivatives 10a-h bearing a flexible amino
side chain with a two or three methylene spacer in posititon-4 were prepared. As
predicted, most of the compounds exhibited more potent antiproliferative potency
against human tumor cell lines, with IC50 values lower than 2.0 µM. Although the
antiproliferative potency of compounds 10-b bearing an ethoxy group in position-7
showed no distinct difference with their parent compound 9a, compounds 10c, 10e
and 10g displayed more potent antiproliferative activity than their parent compounds
9b, 9f and 9i, with IC50 values ranging from 0.37 to 1.86 µM. Interestingly,
compounds 10c, 10e and 10g bearing a flexible amino side chain with a two
methylene spacer displayed more potent cytotoxic potency than compounds 10d, 10f
and 10h, which contained a three methylene spacer. Particularly, compound 10g,
bearing a 4-fluorobenzyl group in position-7 and a flexible amino side chain with a
two methylene spacer in position-4, was found to be the most potent antiproliferative
agent, with IC50 values lower than 1.0 µM against all the human tumor cell lines.
These results indicated that (1) the amino side chain substituents were beneficial
pharmacophoric groups for enhancing the antiproliferative activity and (2) the length
of the alkylamino side chain moiety affected their antiproliferative potency, with two
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CH2 units being more favorable.
3.2 Growth inhibition of human colorectal cancer cells by compound 10g
To investigate the inhibition effects of compound 10g on cell viability and cell
proliferation, we performed CCK8 and colony formation assays. The results of CCK8
assays showed that compound 10g decreased the viability of HCT116, RKO and
DLD1 cells in a time-dependent manner (Fig. 2A). The number of colony units
decreased significantly with an increase in compound 10g concentration (Fig. 2B). In
addition, phase-contrast micrographs revealed that compound 10g induced cell
morphology alterations, such as cell shrinkage and reduced cell number (Fig. 2C).
To assess whether compound 10g could kill cancer cells more effectively than
normal cells, we examined the susceptibility of normal human colorectal and hepatic
cell lines to compound 10g. Unexpectedly, compound 10g did not induce an obvious
difference in viability between cancer cells and normal cells (FHC) in colorectal
cancer cells, except at a high dose of compound 10g (Fig. 2D). These experiments
confirmed that compound 10g had significant inhibitory effects on human colorectal
cancer cells in vitro.
3.3 Compound 10g induced apoptosis in HCT116 cells
Drugs often elevate the activity of apoptosis pathways to exert anticancer effects.
We first examined whether compound 10g could induce apoptosis in HCT116
colorectal cancer cells. PI and Annexin V double staining was carried out to
quantitatively evaluate apoptosis, and obviously increased early and late phase
apoptosis were induced by compound 10g in a dose-dependent manner (Fig. 3A).
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Next, we analyzed markers of apoptosis at the protein level. Immunoblot
analysis showed increased cleaved Proline-rich acidic protein (PARP) and decreased
pro-caspase3 and pro-caspase9 after exposure to different doses of compound 10g
(Fig. 3B). Caspases play an important role in apoptosis progression as effectors. To
further verify whether compound 10g-induced apoptotic cell death was caspase
dependent, z-VAD-fmk, a general caspase inhibitor, was used. As shown in Fig. 3C,
the apoptosis induced by compound 10g was reversed by treatment with the
pan-caspase inhibitor z-VAD-fmk. The level of the apoptosis marker cleaved PRAP
was also reduced after addition of z-VAD-fmk (Fig. 3D). These results revealed that
compound 10g induces caspase-dependent apoptosis in colorectal cancer cells.
3.4 Compound 10g-induced cell death is independent on ROS and autophagy
In addition to apoptosis, inducing intracellular ROS accumulation, autophagy and
cell cycle arrest can also reduce cancer cell growth and viability[35-37]. To test
whether compound 10g-induced cell growth inhibition is exclusively dependent on
apoptosis, we performed experiments to test these three biological processes. We
constructed an HCT116 colorectal cancer cell line that stably expressed GFP-LC3 to
determine whether compound 10g could induce GFP-LC3 puncta. Moderate and
relatively high concentrations of compound 10g were chosen to treat the cells for 24 h.
Although we observed a few GFP-LC3 puncta when the cells were exposed to 10 µM
compound 10g, it was not sufficient to strongly induce cell death (Fig. 4A).
To test if the ROS level is changed after treatment with compound 10g, we used
DCFH-DA to stain cells treated with 5 or 10 µM compound 10g. Flow cytometry
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analysis showed that the fluorescence intensity in compound 10g treated cells was not
altered compared with untreated cells; however, the positive control cells displayed an
obvious elevation in ROS levels (Fig. 4B). This suggests that compound 10g has no
effect on ROS production at a concentration of 5 or 10 µM.
We use PI stain to analysis cell cycle progression, and the effect of 5 µM
compound 10g on cell cycle distribution was analyzed at 24 and 48 h via flow
cytometry. An obvious change in cell cycle arrest was not observed compared with the
vehicle group under these conditions (Fig. 4C and 4D).
3.5 Apoptosis was induced by compound 10g by targeting of p53 transcriptional
activity
p53 and Bax play crucial roles in the intrinsic apoptosis pathway[38, 39]. To
further investigate the possible mechanism by which compound 10g induces apoptosis,
p53- and Bax-deficient HCT116 cells were used to determine cell viability after
exposure to compound 10g. Interestingly, p53-deficient HCT116 cells displayed
tremendous resistance to compound 10g, and as the downstream gene of p53, Bax
deficiency also showed moderate resistance to compound 10g-induced apoptosis (Fig.
5A). Colony formation assays showed results consistent with the cell viability assays
(Fig. 5B). Furthermore, the increase in the number of Annexin V-positive cells
induced by 5 µM compound 10g was significantly blocked in p53- and Bax-deficient
HCT116 cells (Fig. 5C). Immunoblotting showed that the protein levels of the p53
target genes.
Bax, PUMA and p21 were significantly increased and that of PARP was
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decreased after treatment with increasing doses of compound 10g, but this was not
observe in the p53-deficient HCT116 cells (Fig. 5D). These results show that
compound 10g-induced cell apoptosis is dependent on p53. We noted that the protein
level of p53 was not changed after exposure to compound 10g (Fig. 5D), which
implies that the transcriptional activity of p53 may be regulated by this compound.
Because the phosphorylation and location of p53 is indicative of the transcriptional
activity, we next investigated whether the phosphorylation and location of p53 is
changed after treatment with compound 10g. Phosphorylation at serines 15 and 392 of
p53 regulates p53 activation and DNA binding ability[40, 41]. Therefore, we
determined whether phosphorylation of these two sites is induced by compound 10g.
As shown in Fig. 5E, the phosphorylation of p53 at serine 15 and 392 was increased
when cells were treated with compound 10g for the indicated times (Fig. 5E).
To study the subcellular localization of p53, fractionation analysis was employed,
and HCT116 cells were exposed to different doses of compound 10g. As shown in Fig.
5F, the protein level of p53 was increased in the nucleus after exposure to compound
10g for different time courses. Collectively, these results suggest that compound 10g
could exclusively induce p53-dependent cell apoptosis by targeting p53
transcriptional activity.
3.6 Compound 10g displays antitumor activity in vivo
To examine whether compound 10g induced apoptosis would result in anticancer
effects in vivo, we used a xenograft mouse model. The tumors grew quickly in control
group mice who were only treated with vehicle; however, more than half the tumor
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growth was suppressed in mice treated with compound 10g at an 80 mg/kg dose.
Tumor weight was significantly reduced after compound 10g treatment (Fig. 6A and
6B). These results suggested that compound 10g could exhibit antitumor activity in
vivo.
4. Conclusions
A series of quinolone derivatives were designed, synthesized and evaluated as
promising antiproliferative agents on the basis of the chemical structure of the lead
compound 7-chloro-4(1H)-quinolone 11. Preliminary structure-activity relationship
analysis indicated that (1) a large and bulky substituent in position-7 might be a
beneficial pharmacophoric group for antiproliferative activity; (2) the amino side
chain substituents facilitated antiproliferative activity; and (3) the length of the
alkylamino side chain moiety affected the antiproliferative potency, with two CH2
units being more favorable.
Numerous anti-cancer drugs induce cancer cell apoptosis via modulation of the
p53 pathway. However, p53 mutations are frequent not only in colorectal cancer but
also in the majority of other human tumors[42-44]. Bax also serves as an essential
effector of the mitochondrial apoptotic pathway and p53-mediated apoptosis.
Induction of p53 and/or Bax-dependent apoptosis by drugs has been used as a strategy
to kill cancer cells[45-47]. Our investigation demonstrated that compound 10g could
trigger p53/Bax-dependent colorectal cancer cell apoptosis by activating p53
transcriptional activity. In addition, compound 10g was found to significantly
abrogate tumorigenicity in a xenograft model.
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5. Experimental Section
5.1 Reagents and general methods
Z-VAD (OMe)-FMK (HY-16658) was purchased from MedChem Express.
Rabbit monoclonal antibodies against Bax (#5023) and Caspase 3 (#9665) and rabbit
polyclonal antibodies against Caspase 9 (#9502), cleaved PARP (#9541), p21, PUMA,
p-p53(ser15), and p-p53(ser392) were obtained from Cell Signaling Technology
(Beverly, MA). The antibody against total PARP and anti-mouse p53 monoclonal
antibody (sc-126) were purchased from Santa Cruz Biotechnology. Antibodies
targeting GAPDH and LaminA/C and the anti-mouse monoclonal β-Tubulin antibody
(AbM59005-37B-PU) were obtained from BPI.
ESI-MS spectra were obtained with a VG ZAB-HS spectrometer. 1H NMR and
13C NMR spectra were recorded on a Mercury-Plus 300 spectrometer at 300 MHz and
75 MHz, respectively, using TMS as the internal standard and CDCl3 or DMSO-d6 as
the solvent; chemical shifts (δ) are expressed in ppm. HRMS spectra were obtained
with an ESI-Q-TOF maxis 4G spectrometer. All reactions were monitored by TLC,
and spots were visualized with UV light. Silica gel F254 was used for analytical
thin-layer chromatography (TLC), and silica gels were used for column
chromatography.
5.2. Preparation of 7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5)
A mixture of ethyl 7-methoxyl-4-oxo-1,4-dihydroquinoline-3-carboxylate 4 (20
mmol), NaOH (80 mmol), ethanol (100 mL) and H2O (200 mL) was refluxed for 2 h,
and the ethanol was removed on a rotary evaporator. The mixture was acidified with
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dilute HCl. The precipitate was collected, washed well with H2O and dried in a
vacuum to provide compound 5. Compound 5 prepared in this manner was used
directly in the next step without further purification.
5.3 7-Methoxyquinolin-4(1H)-one (6)
A mixture of 7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 5 (10
mmol) and diphenyl ether (30 mL) was heated under reflux for 1 h. Then, the reaction
mixture was allowed to cool to room temperature, and petroleum ether (20 mL) was
added. The precipitate formed was collected by filtration and washed well with
petroleum ether. The pure 4-oxoquinoline 6 was obtained by recrystallization from a
large amount of hot water as white needle crystals (1.6 g, 86%). ESI-MS m/z 176
1
[M+H]+. H NMR (300 MHz, DMSO-d6): δ 11.63 (1H, s, NH), 7.93 (1H, d, J= 8.7
Hz, ArH), 7.76 (1H, d, J= 7.5 Hz, ArH), 6.91 (1H, d, J=2.4 Hz, ArH), 6.88 (1H, dd,
J= 8.7 Hz, J= 2.4 Hz, ArH ) 5.91 (1H, d, J= 7.5 Hz, ArH), 3.83(3H, s, CH3).
5.4 4-Chloro-7-methoxyquinoline (7)
A mixture of 7-methoxyquinolin-4(1H)-one 6 (10 mmol) and phosphorous
oxychloride (30 mL) was heated for 3 h. Most of phosphorous oxychloride was
removed by evaporation under reduced pressure, and the residue was poured into ice
water. Then, the mixture was made alkaline with ammonium hydroxide; the
precipitate formed was collected by filtration, washed well with water and dried in a
vacuum; and compound 7 was obtained as white solid (1.7 g, 88%). ESI-MS m/z 194
1
[M+H]+. H NMR (300 MHz, CDCl3): δ 8.67 (1H, d, J= 4.8 Hz, ArH), 8.09 (1H, d,
J= 9.0 Hz, ArH), 7.42 (1H, d, J=2.4 Hz, ArH), 7.33 (1H, d, J=4.8 Hz, ArH), 7.28 (1H,
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dd, J=9.0 Hz, J=2.4 Hz, ArH), 3.97 (3H, s, CH3). 13C NMR (75 MHz, CDCl3) δ 161.3,
151.0, 150.2, 142.4, 125.3, 121.7, 120.8, 119.3, 107.7, 55.9.
5.5 Preparation of 4-chloroquinolin-7-ol (8)
A mixture of 4-chloro-7-methoxyquinoline 7 (1.93 g, 10 mmol) and 48%
hydrobromic acid (50 mL) was refluxed. After completion of the reaction as indicated
by TLC, the mixture was cooled and poured onto ice. The aqueous mixture was
alkalized to pH 6 using 10% NaOH solution. The resulting precipitate was filtered,
washed with water and dried in vacuum to give 8 (1.76 g, 98%). The material was
used without further purification for the following step.
5.6 General procedure for compounds 9a-k
A mixture of 4-chloroquinolin-7-ol 8 (0.72 g, 4 mmol) and anhydrous DMF (10
mL) was stirred at room temperature until clear, and then, 60% NaH (0.4 g, 10 mmol)
and halogenated alkane (20-30 mmol) were added. The mixture was stirred at room
temperature. After completion of the reaction as indicated by TLC, the solution was
poured into H2O (100 mL) and extracted with ethyl acetate. The organic phase was
made acidic with concentrated hydrochloric acid. Upon removal of solvent, the
residue was crystallized from acetone to afford a yellow solid. The solid was
dissolved in water and made basic with sodium bicarbonate, and the aqueous mixture
was extracted with ethyl acetate. The organic phase was washed with water and brine
and then dried over anhydrous sodium sulfate, filtered and evaporated. The resulting
oil was purified by column chromatography using a mixture of dichloromethane and
methanol 100:1 as the eluent to successfully afford the target products 9a-k in good
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yield.
5.6.1 4-Chloro-7-ethoxyquinoline (9a)
Starting from ethyl bromide (15 mmol), compound 9a was isolated as a yellow
1
solid (0.56 g, 68%), mp 70-71 . ESI-MS m/z (M+H)+ 207.8. H NMR (300 MHz,
CDCl3): δ 8.66 (1H, d, J =4.8 Hz, ArH), 8.09 (1H, d, J =9.0 Hz, ArH), 7.39 (1H, d, J
=1.8 Hz, ArH), 7.32 (1H, d, J =4.8 Hz, ArH), 7.29-7.20 (1H, m, ArH), 4.19 (2H, q,
J =6.9 Hz, CH2), 1.50 (3H, t, J =6.9 Hz, CH3). 13C NMR (75 MHz, CDCl3): δ 160.3,
150.6, 149.8, 142.0, 124.9, 121.2, 120.7, 118.8, 107.8, 63.7, 14.6. HRMS (ESI)
calcd for C11H11ClNO [M+H]+ 208.0524, found 208.0515.
5.6.2 4-Chloro-7-isopropoxyquinoline (9b)
Starting from 2-bromopropane (30 mmol), compound 9b was isolated as a
1
yellow solid (0.48 g, 54%), mp 65-66 . ESI-MS m/z [M+H]+ 221.8. H NMR (300
MHz, CDCl3): δ 8.65 (1H, d, J =4.8 Hz, ArH), 8.08 (1H, d, J=9.3 Hz, ArH), 7.39 (1H,
s, ArH), 7.30 (1H, d, J = 4.8 Hz, ArH), 7.27-7.20 (m, 1H, ArH), 4.82-4.67 (1H, m,
CH), 1.43 (6H, d, J =6.0 Hz, CH3). 13C NMR (75 MHz, CDCl3): δ 159.3, 150.5,
149.7, 142.1, 125.0, 121.5, 121.0, 118.7, 108.8, 70.2, 21.7. HRMS (ESI) calcd for
C12H13ClNO [M+H]+ 222.0680, found 222.0676.
5.6.3 7-(Allyloxy)-4-chloroquinoline (9c)
Starting from allyl bromide (20 mmol), compound 9c was isolated as a yellow
1
solid (0.69 g, 79%), mp 36-37 . ESI-MS m/z (M+H)+ 219.8. H NMR (300 MHz,
CDCl3): δ 8.66 (1H, d, J = 4.5 Hz, ArH), 8.09 (1H, d, J =9.0 Hz, ArH), 7.41 (1H, s,
ArH), 7.29 (2H, dd, J =16.0 Hz, J = 6.3 Hz, ArH), 6.10-6.16 (1H, m, CH2=CH), 5.48
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(1H, d, J =17.2 Hz, CH2=CH), 5.34 (1H, d, J =10.5 Hz, CH2=CH), 4.69 (2H, d, J
13
=4.8 Hz, OCH2). C NMR (75 MHz, CDCl3): δ 159.9, 150.5, 149.9, 142.1, 132.2,
125.1, 121.4, 120.8, 119.0, 118.1, 108.4, 68.9. HRMS (ESI) calcd for C12H10ClNO
[M+H]+ 220.0524, found 220.0535.
5.6.4 4-Chloro-7-butoxyquinoline (9d)
Starting from 1-bromobutane (30 mmol), compound 9d was isolated as a yellow
1
solid (0.46 g, 49%), mp 37-38 . ESI-MS m/z [M+H]+ 236.1. H NMR (300 MHz,
CDCl3): δ 8.65 (1H, d, J =4.8 Hz, ArH), 8.08 (1H, d, J =9.3 Hz, ArH), 7.39 (1H, d, J
=1.8 Hz, ArH), 7.31 (1H, d, J =4.8 Hz, ArH), 7.29-7.23 (1H, m, ArH), 4.12 (2H, t, J
=6.6 Hz, CH2), 1.92-1.79 (2H, m, CH2), 1.62-1.47 (m, 2H, CH2), 1.00 (3H, t, J =7.2
13
Hz, CH3). C NMR (75 MHz, CDCl3): δ 160.5, 150.7, 149.8, 142.0, 124.9, 121.2,
120.8, 118.8, 108.0, 68.0, 31.0, 19.2, 13.8. HRMS (ESI) calcd for C13H14ClNO
[M+H]+ 236.0837, found 236.0844.
5.6.5 4-Chloro-7-isobutoxyquinoline (9e)
Starting from 1-bromo-2-methylpropane (30 mmol), compound 9e was isolated
1
as a white solid (0.84 g, 89%), mp 57-58 . ESI-MS m/z [M+H]+ 235.8. H NMR
(300 MHz, CDCl3): δ 8.65 (1H, d, J =4.8 Hz, ArH), 8.09 (1H, d, J =9.3 Hz, ArH),
7.38 (1H, d, J =2.4 Hz, ArH), 7.31 (1H, d, J =4.8 Hz, ArH), 7.28 (1H, dd, J =9.2 Hz,
J =2.4 Hz, ArH), 3.88 (2H, d, J =6.5 Hz, CH2), 2.18 (1H, dp, J =13.2 Hz, J =6.6 Hz,
CH), 1.07 (6H, d, J =6.7 Hz, CH3). 13C NMR (75 MHz, CDCl3): δ 160.7, 150.8,
149.9, 142.2, 125.0, 121.3, 121.0, 118.9, 108.1, 74.7, 28.1, 19.3. HRMS (ESI) calcd
for C13H14ClNO [M+H]+ 236.0837, found 236.0851.
17

ACCEPTED MANUSCRIPT
5.6.6 4-Chloro-7-(hexyloxy)quinoline (9f)
Starting from 1-bromohexane (30 mmol), compound 9f was isolated as a yellow
1
oil (0.72 g, 68%). ESI-MS m/z (M+H)+ 263.9. H NMR (300 MHz, CDCl3): δ 8.65
(1H, d, J =4.8 Hz, ArH), 8.07 (1H, d, J =9.0 Hz, ArH), 7.38 (1H, d, J =2.4 Hz, ArH),
7.30 (1H, d, J =4.8 Hz, ArH), 7.26 (1H, dd, J =9.0 Hz, J = 2.4 Hz, ArH), 4.11 (2H, t,
J =6.6 Hz, CH2(CH2)4CH3), 1.94-1.79 (2H, m, CH2CH2[CH2]3CH3), 1.49(2H, m,
[CH2]2CH2[CH2]2CH3), 1.37 (4H, m, [CH2]3CH2CH2CH3), 0.91 (3H, t, J =6.9 Hz,
CH3). 13C NMR (75 MHz, CDCl3): δ 160.7, 150.8, 149.9, 142.2, 125.0, 121.3, 121.0,
118.9, 108.1, 68.4, 31.6, 29.0, 25.8, 22.7, 14.1. HRMS (ESI) calcd for C15H18ClNO
[M+H]+ 264.1150, found 264.1159.
5.6.7 4-Chloro-7-(octyloxy)quinoline (9g)
Starting from 1-bromooctane (30 mmol), compound 9g was isolated as a yellow
oil (0.87 g, 75%). 1H NMR (400 MHz, CDCl3) δ 8.68 (d, J = 4.8 Hz, 1H, ArH), 8.11
(d, J = 9.2 Hz, 1H, ArH), 7.41 (d, J = 1.6 Hz, 1H, ArH), 7.33 (d, J = 4.8 Hz, 1H, ArH),
7.27 (d, J = 3.6 Hz, 1H, ArH), 4.12 (t, J = 6.4 Hz, 2H, CH2(CH2)6CH3), 1.92 – 1.81
(m, 2H, CH2CH2(CH2)5CH3), 1.55 – 1.45 (m, 2H, CH2CH2CH2(CH2)4CH3), 1.42 –
13
1.22 (m, 8H, CH2CH2CH2(CH2)4CH3), 0.89 (t, J = 6.4 Hz, 3H, CH2(CH2)6CH3). C
NMR (100 MHz, CDCl3) δ 160.7, 150.8, 149.9, 142.2, 125.0, 121.3, 121.0, 118.9,
107.9, 68.3, 31.8, 29.4, 29.2, 29.0, 26.0, 22.7, 14.1. HRMS (ESI) calcd for
C17H22ClNO [M+H]+ 292.1463, found 292.1465.
5.6.8 7-Benzyloxy-4-chloro-quinoline (9h)
18

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Starting from benzyl bromide (15 mmol), compound 9h was isolated as a white
1
solid (0.50 g, 47%), mp 87-88 . ESI-MS m/z [M+H]+ 269.8. H NMR (300 MHz,
CDCl3): δ 8.67 (1H, d, J =4.8 Hz, ArH), 8.12 (1H, d, J =9.3 Hz, ArH), 7.52-7.30 (8H,
13
m, ArH), 5.21 (2H, s, CH2Ph). C NMR (75 MHz, CDCl3): δ 160.2, 150.7, 150.0,
142.2, 135.9, 128.5, 128.1, 127.5, 125.2, 121.6, 120.9, 119.2, 108.7, 70.3. HRMS
(ESI) calcd for C16H13ClNO [M+H]+ 270.0680, found 270.0678.
5.6.9 7-(4-Fluorobenzyloxy)-4-chloro-quinoline (9i)
Starting from 4-fluorobenzyl bromide (20 mmol), compound 9i was isolated as a
1
yellow solid (1.02 g, 89%), mp 98-99 . ESI-MS m/z [M+H]+ 287.9. H NMR (300
MHz, CDCl3): δ 8.67 (1H, d, J =4.8 Hz, ArH), 8.12 (1H, d, J =9.3 Hz, ArH),
7.49-7.42 (3H, m, ArH), 7.36-7.30 (2H, m, ArH), 7.08 (2H, t, J =8.7 Hz, ArH), 5.17
(2H, s, CH2Ph). 13C NMR (75 MHz, CDCl3): δ 162.4 (d, J = 246.4 Hz), 159.9, 150.6,
150.0, 142.2, 131.7 (d, J = 2.2 Hz), 129.4 (d, J = 8.1 Hz), 125.2, 121.6, 120.8, 119.2,
115.44 (d, J = 21.5 Hz), 108.6, 69.5. HRMS (ESI) calcd for C16H12ClFNO [M+H]+
288.0586, found 288.0587.
5.6.10 4-Chloro-7-(phenethyloxy)quinoline (9j)
Starting from (2-bromoethyl)benzene (20 mmol), compound 9j was isolated as a
1
yellow solid (0.26 g, 23%), mp 84-85 . ESI-MS m/z [M+H]+ 283.8. H NMR (300
MHz, CDCl3): δ 8.64 (1H, d, J =3.9 Hz, ArH), 8.07 (1H, d, J =9.0 Hz, ArH), 7.40 (1H,
s, ArH), 7.36-7.19 (7H, m, ArH), 4.34 (2H, t, J =6.7 Hz, OCH2CH2), 3.18 (2H, t, J
=6.6 Hz, OCH2CH2). 13C NMR (75 MHz, CDCl3): δ 160.1 150.6, 149.8, 142.0, 137.7,
128.7, 128.3, 126.4, 124.9, 121.3, 120.7, 118.9, 108.1, 68.7, 35.4. HRMS (ESI) calcd
19

ACCEPTED MANUSCRIPT
for C17H14ClNO [M+H]+ 284.0837, found 284.0842.
5.6.11 7-(3-Phenylpropoxy)-4-chloroquinoline (9k)
Starting from (3-bromopropyl)benzene (30 mmol), compound 9k was isolated as
1
a white solid (0.64 g, 54%), mp 51-52 . ESI-MS m/z [M+H]+ 297.9. H NMR (300
MHz, CDCl3): δ 8.64 (1H, d, J =4.2 Hz, ArH), 8.08 (1H, d, J =9.0 Hz, ArH), 7.36 (1H,
s, ArH), 7.33-7.15 (7H, m, ArH), 4.10 (2H, t, J =6.0 Hz, OCH2CH2CH2), 2.84 (2H, t,
13
J =7.4 Hz, OCH2CH2CH2), 2.26-2.10 (2H, m, OCH2CH2CH2). C NMR (75 MHz,
CDCl3): δ 160.4 150.6, 149.8, 142.0, 140.9, 128.2(2C), 125.8, 124.9, 121.3, 120.7,
118.9, 108.0, 67.2, 32.1, 30.5. HRMS (ESI) calcd for C18H16ClNO [M+H]+ 298.0993,
found 299.1011.
5.7 General procedure for compounds 10a-h
A mixture of 4-chloro-7-alkoxyquiline 9 (2.0 mmol) and trimethyl orthoformate
(53 g, 500 mmol) was heated under reflux for 2 h. After completion of the reaction as
indicated by TLC, the solution was poured into H2O (100 mL) and extracted with
ethyl acetate. The organic phase was washed with water and brine and then dried over
anhydrous sodium sulfate, filtered and evaporated. The resulting oil was purified by
column chromatography using a mixture of dichloromethane and methanol 100:1 as
the eluent to successfully afford the target products 10a-h in good yield.
5.7.1 N-(2-(Dimethylamino)ethyl)-7-ethoxyquinolin-4-amine (10a)
Starting
from
4-chloro-7-ethoxyquinoline
9a
(2.0
mmol)
and
N,N-diethylethylenediamine (4.0 mmol), a yellow solid (0.38 g, 74%) was obtained,
mp 84-85 . ESI-MS m/z [M+H]+ 287.9. 1H NMR (300 MHz, CDCl3)：δ 8.44 (1H, d,
20

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J =5.1 Hz, ArH), 7.61 (1H, d, J = 9.0 Hz, ArH), 7.28 (1H, s, ArH)), 7.04 (1H, d, J
=9.0 Hz, ArH), 6.25 (1H, d, J =5.4 Hz, ArH), 5.98 (1H, s, NH), 4.13 (2H, q, J =6.9
Hz, OCH2CH3), 3.20 (2H, dd, J =10.2 Hz, J =5.1 Hz, HNCH2CH2), 2.75 (2H, t, J
=5.7 Hz, NHCH2CH2), 2.56(4H, q, J =6.9 Hz, N[CH2CH3]2), 1.46 (3H, t, J =6.9 Hz,
OCH2CH3), 1.04 (6H, t, J =7.1 Hz, N[CH2CH3]2). 13C NMR (75 MHz, CDCl3): δ
159.2, 151.1, 150.0, 149.7, 120.6, 116.9, 113.2, 108.5, 97.8, 63.3, 50.6, 46.4, 39.7,
14.7, 12.0. HRMS (ESI) calcd for C17H25N3O [M+H]+ 288.2070, found 288.2078.
5.7.2 N-(3-(dimethylamino)propyl)-7-ethoxyquinolin-4-amine (10b)
Starting
from
4-chloro-7-ethoxyquinoline
9a
(2.0
mmol)
and
3-(diethylamino)propylamine (4.0 mmol), a yellow oil (0.41 g, 75%) was obtained.
1
ESI-MS m/z [M+H]+ 302.1. H NMR (400 MHz, CDCl3): δ 8.41 (1H, d, J =5.4 Hz,
ArH), 7.83 (1H, s, NH), 7.64 (1H, d, J =9.0 Hz, ArH), 7.30 (1H, s, ArH), 7.02 (1H, d,
J =9.0 Hz, ArH), 6.23 (1H, d, J =5.4 Hz, ArH), 4.16 (2H, q, J = 6.9 Hz, CH2CH3),
3.38 (2H, d, J =4.5 Hz, NCH2CH2CH2), 2.65 (6H, dt, J =14.4 Hz, J =6.0 Hz,
CH2N[CH2CH3]2), 1.97-1.83 (2H, m, NHCH2CH2CH2), 1.47 (3H, t, J =6.8 Hz,
CH2CH3), 1.09 (6H, t, J =6.8 Hz, N[CH2CH3]2). 13C NMR (100 MHz, CDCl3): δ
159.0, 150.5, 150.3, 149.5, 121.4, 116.0, 113.1, 107.8, 96.5, 62.9, 52.5, 46.4, 43.5.
HRMS (ESI) calcd for C18H27N3O [M+H]+ 302.2227, found 302.2223.
5.7.3 7-(Allyloxy)-N-(2-(dimethylamino)ethyl)quinolin-4-amine (10c)
Starting from 7-allyloxy-4-chloro-quinoline 9c (2.0 mmol) and
N,N-diethylethylenediamine (4.0 mmol), a yellow oil (0.43 g, 80%) was obtained.
1
ESI-MS m/z [M+H]+ 300.1. H NMR (300 MHz, CDCl3): δ 8.43 (1H, d, J = 5.4 Hz,
21

ACCEPTED MANUSCRIPT
ArH), 7.62 (1H, d, J =9.0 Hz, ArH), 7.30 (1H, d, J =2.1 Hz, ArH), 7.08 (1H, dd, J
=9.3 Hz, J =1.8 Hz, ArH), 6.28 (1H, d, J =5.4 Hz, ArH), 6.09 (1H, dd, J =15.9 Hz, J =
10.5 Hz, J =5.1 Hz, CH2=CH), 5.97 (1H, s, NH), 5.46 (1H, d, J =17.2 Hz, CH2=CH),
5.31 (1H, d, J =10.5 Hz, CH2=CH), 4.65 (2H, d, J =5.1 Hz, OCH2CH=CH2), 3.25 (2H,
dd, J =10.5 Hz, J =5.4 Hz, NHCH2CH2), 2.80 (2H, t, J =5.7 Hz, NHCH2CH2), 2.59
13
(4H, q, J =7.2 Hz, [CH2CH3]2), 1.07 (6H, t, J =7.2 Hz, [CH2CH3]2). C NMR (75
MHz, CDCl3): δ 158.4, 150.4, 149.5, 149.3, 132.2, 120.8, 117.1, 116.3, 113.1, 108.2,
97.2, 68.1, 50.3, 46.0, 39.5, 11.4. HRMS (ESI) calcd for C18H25N3O [M+H]+
300.2070, found 300.2076.
5.7.4 7-(Allyloxy)-N-(3-(dimethylamino)propyl)quinolin-4-amine (10d)
Starting from 7-allyloxy-4-chloro-quinoline 9c (2.0 mmol) and
3-(diethylamino)propylamine (4.0 mmol), a yellow oil (0.18 g, 32%) was obtained.
1
ESI-MS m/z [M+H]+ 313.9. H NMR (300 MHz, CDCl3): δ 8.40 (1H, d, J = 5.4 Hz,
ArH), 7.83 (1H, s, NH), 7.64 (1H, d, J =9.0 Hz, ArH), 7.03 (1H, dd, J =9.0 Hz, J =1.5
Hz, ArH), 6.20 (1H, d, J =5.4 Hz, ArH), 6.08 (1H, ddd, J = 21.6 Hz, J =10.5 Hz, J =
5.1 Hz, CH=CH2), 5.45 (1H, d, J =17.2 Hz, CH=CH2), 5.29 (1H, d, J =10.5 Hz,
CH=CH2), 4.63 (2H, d, J = 5.1 Hz, OCH2CH=CH2), 3.34 (2H, d, J =3.9 Hz,
NCH2[CH2]2), 2.61 (6H, dd, J =13.8 Hz, J =6.6 Hz, CH2N[CH2CH3]2), 1.94-1.81 (m,
2H, NCH2CH2CH2), 1.07 (6H, t, J =7.2 Hz, N[CH2CH3]2). 13C NMR (75 MHz,
CDCl3): δ 158.8, 151.0, 150.5, 149.8, 132.8, 121.7, 117.7, 116.5, 113.6, 108.7, 97.0,
68.7, 53.4, 47.0, 44.4, 24.5, 11.6. HRMS (ESI) calcd for C19H27N3O [M+H]+
314.2227, found 314.2232.
22

ACCEPTED MANUSCRIPT
5.7.5 7-Hexyloxy-N-(3-(dimethylamino)ethyl)quinolin-4-amine (10e)
Starting from 4-chloro-7-(hexyloxy)quinoline 9f (2.0 mmol) and N,N-diethyl-
ethylenediamine (4.0 mmol), a yellow solid (0.54 g, 85%) was obtained, mp 92-93
.
1
ESI-MS m/z [M+H]+ 344.0. H NMR (300 MHz, CDCl3): δ 8.43 (1H, d, J = 5.4 Hz,
ArH), 7.61 (1H, d, J =9.0 Hz, ArH), 7.28 (1H, d, J =2.1 Hz, ArH), 7.04 (1H, dd, J
=9.0 Hz, J = 2.1 Hz, ArH), 6.26 (1H, d, J =5.4 Hz, ArH), 5.98 (1H, s, NH), 4.06 (2H,
t, J = 6.6 Hz, OCH2[CH2]4CH3), 3.22 (2H, dd, J =10.2 Hz, J =5.4 Hz, NHCH2CH2),
2.77 (2H, t, J =5.8 Hz, NHCH2CH2), 2.57 (4H, q, J =6.9 Hz, N[CH2CH3]2), 1.89-1.76
(2H, m, OCH2CH2[CH2]3CH3), 1.54-1.42 (m, 2H, O[CH2]2CH2[CH2]2CH3), 1.34 (4H,
d, J =3.6 Hz, O[CH2]3[CH2]2CH3, 1.05 (6H, t, J =7.2 Hz, N[CH2CH3]2), 0.90 (3H, t, J
13
=6.6 Hz, O[CH2]5CH3). C NMR (75 MHz, CDCl3): δ 159.5, 151.1, 145.0, 149.8,
120.6, 117.1, 113.2, 108.6, 97.8, 68.0, 50.7, 46.5, 39.8, 31.6, 29.1, 25.8, 22.6, 14.1,
12.1. HRMS (ESI) calcd for C21H33N3O [M+H]+ 344.2696, found 344.2707.
5.7.6 7-Benzyloxy-N-(3-(dimethylamino)ethyl)quinolin-4-amine (10f)
Starting from 7-benzyloxy-4-chloro-quinoline 9h (2.0 mmol) and N,N-diethyl-
ethylenediamine (4.0 mmol), a yellow solid (0.37 g, 58%) was obtained, mp 93-94 .
ESI-MS m/z [M+H]+ 349.9. 1H NMR (300 MHz, CDCl3): δ 8.44 (1H, d, J =4.8 Hz,
ArH), 7.63 (1H, d, J =9.0 Hz, ArH), 7.47 (2H, d, J =7.2 Hz, ArH), 7.34 (4H, dd, J
=13.5 Hz, J =7.8 Hz, ArH), 7.13 (1H, d, J =9.0 Hz, ArH), 6.28 (1H, d, J =5.1 Hz,
ArH), 5.99 (1H, s, NH), 5.17 (2H, s, OCH2Ph), 3.25 (2H, t, J =4.8 Hz, NHCH2CH2),
2.80 (t, J =5.4 Hz, 2H, NHCH2CH2), 2.59 (4H, q, J =6.9 Hz, N[CH2CH3]2), 1.07 (6H,
t, J =6.9 Hz, N[CH2CH3]2). 13C NMR (75 MHz, CDCl3): δ 159.1, 151.1, 149.9, 136.5,
23

ACCEPTED MANUSCRIPT
128.4, 127.9, 127.5, 120.9, 117.1, 113.6, 109.6, 109.2, 105.2, 98.0, 70.0, 50.8, 46.5,
39.8, 12.1. HRMS (ESI) calcd for C22H27N3O [M+H]+ 350.2227, found 350.2232.
5.7.7 7-(4-Fluorobenzyloxy)-N-(2-(dimethylamino)ethyl)quinolin-4-amine (10g)
Starting from 7-(4-fluorobenzyloxy)-4-chloro-quinoline 9i (2.0 mmol) and
N,N-diethylethylenediamine (4.0 mmol), a white solid (0.38 g, 56%) was obtained,
mp 67-68 . ESI-MS m/z [M+H]+ 367.7. 1H NMR (300 MHz, CDCl3): δ 8.44 (1H, d,
J =5.4 Hz, ArH), 7.63 (1H, d, J =9.0 Hz, ArH), 7.42 (2H, dd, J =8.4 Hz, J =5.4 Hz,
ArH), 7.36 (1H, d, J = 2.4 Hz, ArH), 7.10 (1H, dd, J =9.0 Hz, J =2.4 Hz, ArH), 7.04
(2H, t, J =8.7 Hz, ArH), 6.27 (1H, d, J =5.4 Hz, ArH), 5.99 (1H, d, J =2.4 Hz, NH),
5.11 (2H, s, OCH2Ph[p-F]), 3.23 (2H, dd, J =10.5 Hz, J =5.4 Hz, NHCH2CH2), 2.78
(2H, t, J =6.0 Hz, NHCH2CH2), 2.58 (4H, q, J =7.2 Hz, N[CH2CH3]2), 1.06 (6H, t, J
=7.2 Hz, N[CH2CH3]2). 13C NMR (75 MHz, CDCl3): δ 162.32 (d, J = 246.0 Hz),
159.0, 151.0, 149.9, 149.8, 132.3, 129.32 (d, J = 7.9 Hz), 121.0, 117.0, 115.33 (d, J =
21.5 Hz), 113.6, 109.1, 98.0, 69.3, 50.8, 46.6, 39.9, 12.1. HRMS (ESI) calcd for
C22H26FN3O [M+H]+ 368.2133, found 368.2139.
5.7.8 7-(4-Fluorobenzyloxy)-N-(3-(dimethylamino)propyl)quinolin-4-amine (10h)
Starting from 7-(4-fluorobenzyloxy)-4-chloro-quinoline 9i (2.0 mmol) and
3-(diethylamino)propylamine (4.0 mmol), a yellow oil (0.48 g, 68%) was obtained.
1
ESI-MS m/z [M+H]+ 382.4. H NMR (300 MHz, CDCl3): δ 8.41 (1H, d, J =5.1 Hz,
ArH), 7.83 (1H, s, NH), 7.66 (1H, d, J =9.0 Hz, ArH), 7.45-7.36 (2H, m, ArH), 7.34
(1H, s, ArH), 7.13-6.95 (3H, m, ArH), 6.20 (1H, d, J =5.1 Hz, ArH), 5.08 (2H, s,
OCH2Ph[p-F]), 3.32 (2H, s, NHCH2(CH2)2), 2.67-2.53 (6H, m, CH2N[CH2CH3]2),
24

ACCEPTED MANUSCRIPT
13
1.84 (2H, s, NHCH2CH2CH2), 1.06 (6H, t, J =6.9 Hz, N[CH2CH3]2). C NMR (75
MHz, CDCl3): δ 162.0 (d, J = 245.7 Hz), 158.6, 151.0, 150.3, 149.7, 132.11 (d, J =
2.0 Hz), 129.0 (d, J = 8.0 Hz), 121.7, 116.1, 115.0 (d, J = 21.4 Hz), 113.6, 108.8, 96.8,
68.9, 53.0, 46.7, 44.1, 24.3, 11.4. HRMS (ESI) calcd for C23H28FN3O [M+H]+
382.2289, found 382.2298.
5.8 Cell lines and cell culture
The human cancer cells (HCT116, RKO, DLD1, HepG2, BGC-823, NCI-H1650
and SK-OV-3) were purchase from American Type Culture Collection (ATCC,
Rockville, MD). HCT116 p53-/- and HCT116 Bax-/- cell lines were kindly provided by
Dr. Bert Vogelstein (The Sidney Kimmel Comprehensive Cancer Center, The Johns
Hopkins University Medical Institutions, Baltimore). All cells were cultured in
McCoy’s 5A medium (AppliChem) supplemented with 10% fetal bovine serum
(HyClone, Logan, UT, USA), 100 units·mL-1 streptomycin and 100 units·mL-1
penicillin at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
5.9 Cytotoxicity in vitro
Cell proliferative assay were carried out using 96 microtitre plate cultures and
MTT staining. Cells were grown in RPMI-1640 medium containing 10% (v/v) fetal
calf serum and 100 µM penicillin and 100 µM streptomycin. Cultures were
propagated at 37℃ in a humified atmosphere containing 5% CO2, Cells in logarithmic
phase were diluted to a density of 50,000 cells/mL in culture medium and treated with
various concentrations of compounds 9a-k and 10a-h in 96-well plates for 48 h in
final volumes of 200 µL. Then 20 µL MTT (5 mg/mL) was added to each well, and
25

ACCEPTED MANUSCRIPT
the cells were incubated for additional 4 h. After carefully removing the medium, the
precipitates were dissolved in 200 µL of DMSO, shaken mechanically for 2 min, and
then absorbance values at a wavelength of 570 nm were taken on a spectrophotometer
(Bio-Rad iMark microplate absorbance Reader, USA). IC50 values were calculated
using percentage of growth versus untreated control. Cisplatin and
7-chloro-4(1H)-quinolone 11 were used as positive controls and DMSO was used as
the solution for drugs. Final concentration of DMSO in the growth medium was 2%
(v/v) or lower, concentration without effect on cell replication. In all of these
experiments, three replicate wells were used to determine each point.
5.10 Cell viability assay
Cells were seeded into 96-well plates at a density of 2×104 per well, cultured for
24 h, and then treated with increasing doses of the tested compounds 7, 9a-k and
10a-h for 48 h. Cell viability was assayed using Cell Counting Kit-8 (CCK-8,
Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s protocol. The
concentration required to inhibit cell growth by 50% (IC50) was calculated from
viability curves as previously reported.
5.11 Colony formation assay
The indicated cells were counted and seeded in 12-well plates for colony
formation assays. Cells were cultured overnight, and then, the medium was replaced
with medium containing compound 10g at the indicated concentration. After 24 h of
exposure, the medium was changed back to standard culture medium, and the cells
were cultured for 9-10 days. The colonies were dyed with 1% crystal violet dye
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containing 4% paraformaldehyde for 30 min and washed with water. The colonies
were then photographed and counted using ImageJ software.
5.12 Apoptosis and cell cycle assay
Apoptotic cells were quantitated using an Annexin V-FITC apoptosis detection
kit after treatment with compound 10g at 0, 2.5 and 5 µM for 24 h. To investigate the
mechanisms by which compound 10g induces apoptosis, cells were pretreated with 20
µM z-VAD-FMK for 12 h and then treated with 2.5 µM or 5 µM compound 10g for a
an additional 24 h. Following treatment, the cells were trypsinized, washed twice with
cold PBS and resuspended in 100 µl of binding buffer. Then, Annexin V-FITC and PI
were added to the cells. After incubation for 15 min at 37 in the dark, an additional
400 µl of binding buffer was added to the cells, and they were then subjected to FACS
cytometry. The results were analyzed with Summit 4.3 software. For cell cycle
analysis, the cells were fixed in 70% ice-cold ethanol, incubated at 4°C for 1 h, and
then centrifuged at 1000 g for 5-10 min. The cell pellet was washed and suspended in
0.5 mL of PBS containing 50 µg/mL RNase A at 37°C for 30 min. Then, 50 µg/mL PI
solution was added and incubated with cells for another 30 min at room temperature
in the dark. The cells were analyzed via flow cytometry to assess cell cycle
distribution.
5.13 Autophagy and ROS assays
The GFP-LC3 cell line was used to examine autophagy. GFP puncta were
observed using a fluorescence microscope and counted after exposure to the indicated
concentration of compound 10g for 12 h. For the ROS assay, cells were exposed to
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the indicated concentration of compound 10g and then stained with DCFH-DA
followed by FACS analysis.
5.14 Immunoblotting analysis
Cells were treated with compound 10g at the indicated concentration for 0-24 h
and harvested at 80% confluence. Then, the cells were lysed with SDS sample buffer
and boiled for 5-10 min. Protein concentration was determined with a BCA protein
assay kit (Thermo), and 20 µg samples were loaded into SDS-PAGE gels. Proteins
were transferred to polyvinylidene difluoride (PVDF) membranes, which were then
blocked with TBST containing 5% dry fat milk and incubated with primary antibodies
overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody
for 1 h at room temperature. Then, the immunoblots were visualized with an
Immobilon Western Chemiluminescent HRP Substrate kit (Merck Millipore).
5.15 Xenograft tumor model
To further examine the anticancer role of compound 10g in vivo, we established a
human colorectal cancer xenograft model in 6-week-old male C57 mice by
subcutaneously injecting CT26-CL25 cells (3×106) into the flanks of the mice. The
mice were randomly divided into two groups, a vehicle control group and a compound
10g treatment group, when the tumor grew to 3-5 mm in diameter. Compound 10g (80
mg/kg body weight) was administered once every three days for 21 days. The tumors
were photographed, and tumor weights were obtained on day 21.
5.16 Statistical analysis
The data given in the text were expressed as means ± SD. The significance of
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