Synthesis, structure-activity relationships and preliminary mechanism of action of novel water-soluble 4-quinolone-3-carboxamides as antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2017.09.017

A series of novel water-soluble 4-quinolone-3-carboxamides was prepared and evaluated as antiproliferative agents. Preliminary results indicated that most compounds tested in this study showed potent antiproliferative potencies against human tumor cell li

Full text of DOI:10.1016/j.ejmech.2017.09.017

Accepted Manuscript
Synthesis, structure-activity relationships and preliminary mechanism of action of
novel water-soluble 4-quinolone-3-carboxamides as antiproliferative agents
Zeyan Zhang, Xingpeng Xiao, Tong Su, Jinyi Wu, Jianwei Ren, Jiongchang Zhu,
Xiaodong Zhang, Rihui Cao, Runlei Du
PII:
S0223-5234(17)30699-2
10.1016/j.ejmech.2017.09.017
EJMECH 9732
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 March 2017
Revised Date: 8 September 2017
Accepted Date: 11 September 2017
Please cite this article as: Z. Zhang, X. Xiao, T. Su, J. Wu, J. Ren, J. Zhu, X. Zhang, R. Cao, R. Du,
Synthesis, structure-activity relationships and preliminary mechanism of action of novel water-soluble 4-
quinolone-3-carboxamides as antiproliferative agents, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.09.017.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphic abstract
A series of novel water-soluble 4-quinolone-3-carboxamides was prepared and
evaluated as antitumor agents. The representative compound 8k could trigger
p53/Bax- independent colorectal cancer cell apoptosis via inducing ROS
accumulation.

ACCEPTED MANUSCRIPT
Synthesis, structure-activity relationships and preliminary
mechanism of action of novel water-soluble
4
-quinolone-3-carboxamides as antiproliferative agents
a
c
a
b
a
Zeyan Zhang , Xingpeng Xiao , Tong Su , Jinyi Wu , Jianwei Ren ,
b
a
*, b
*, a
Jiongchang Zhu ,Xiaodong Zhang , Rihui Cao , Runlei Du
a
College of Life Sciences, Wuhan University, 299 Ba Yi Road, Wuchang, 430072, P R China
b
School of Chemistry, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275,
P R China
c
Department of Anesthesiology，Renmin Hospital of Wuhan University，Wuhan 430060，P R
China
*
Corresponding authors Tel: 86-20-84110918 fax: 86-20-84112245, E-mail addressed:
runleidu@163.com (Runlei Du); caorihui@mail.sysu.edu.cn (Rihui Cao)
Zeyan Zhang and Xingpeng Xiao contributed equally to this work.
1

ACCEPTED MANUSCRIPT
Abstract
A series of novel water-soluble 4-quinolone-3-carboxamides was prepared and
evaluated as antiproliferative agents. Preliminary results indicated that most
compounds tested in this study showed potent antiproliferative potencies against
human tumor cell lines, and compound 8k was found to be the most potent
antiproliferative agents with IC50 value of lower than 10 µM against nine human
tumor cell lines. These results suggested that (1) the alkylamino side chain substituent
was the advisable pharmacophoric group for the enhanced antiproliferative activities;
(2) the length of the alkylamino side chain moiety also affected their antiproliferative
potencies, and three methylene units were more favorable; (3) introducing arylated
alkyl substituent into N1-position of quinolone facilitated antiproliferative activities
of this class of compounds. Further investigations on mechanism of action of this
class of compound demonstrated that the representative compound 8k could trigger
p53/Bax- independent colorectal cancer cell apoptosis via inducing ROS
accumulation.
Keywords: Synthesis; 4-quinolone; antiproliferative; structure-activity relationships;
mechanism of action.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Cancer is a major public health problem worldwide. About 25% deaths in the
United States is due to cancer[1]. Colorectal cancer (CRC) is the third most common
cancer in the world and the fourth most frequent cause of cancer-related deaths[2].
Evasion of apoptosis is a key hallmark of cancer[3, 4]. Apoptosis is a genetically
programmed cellular suicide process that can remove unwanted, redundant, or
damaged cells, including cancer cells, from multicellular organisms. Induction of cell
apoptosis has been a popular strategy for cancer therapy[5].
Quinolone nucleus is common to many natural and synthetic alkaloids with a
broad spectrum of pharmacological properties including antibacterial, antitumor and
anti-HIV activities[6-8]. Since the discovery of nalidixic acid by Lesher in 1962,
many efforts have spent in searching for more potent antibacterial agents, and several
generations of quinolones which exhibit excellent antibacterial activities have been
discovered and used clinically in the past 40 years.
Quinolones are initially discovered to exert their antibacterial effects by
inhibiting essential type II bacterial topoisomerases such as DNA gyrase[9].
Subsequently, topoisomerase IV[10] and penicillin-binding proteins (PBPs) were also
found to be the pharmacological target of this class of compounds. Due to structural
and functional similarities between bacterial DNA gyrase and mammalian
topoisomerase II, some quinolones were found to inhibit both bacterial and
mammalian topoisomerase II. Further investigation demonstrated that mammalian
topoisomerase II inhibitory potency was correlated well with mammalian in vitro
3

ACCEPTED MANUSCRIPT
cytotoxicity and in vivo antitumor activity. Consequently, numerous interests in
quinolones have been stimulated by their potential antitumor activities, and a large
number of quinolone derivatives acting as antitumor agents were reported [11-15].
7-Chloro-4(1H)-quinolone 1 (Fig. 1), a typical quinolone, was discovered as
potent antitumor agent by inhibiting DNA synthesis and damaging DNA template of
tumor cell. Its capsule was approved by the State Food and Drug Administration of
China (SFDA) as antitumor drug for late mammary cancer and non-small cell lung
cancer treatment in 2003[16]. Compound 1 is characteristic of low toxicity, ease of
administration and cheap synthesis, however, the poor solubility of compound 1
restricted its utility for cancer therapy.
Our previous investigations[17-19] demonstrated that introducing a flexible
amino side chain into many lead compounds contributed to the development of
potential bioactive agents with good water solubility. As a part of our efforts to
develop new quinolones as antitumor agents, we described the synthesis and
antiproliferative evaluation of new quinolones bearing an alkyl substituent at
N1-position and a flexible alkylamino side chain at position-3. Our primary objectives
are (1) to improve the water solubility of quinolones; (2) to enhance the antitumor
potency of this class of compounds; (3) to elucidate the structure-activity relationships
of these compounds; (4) to disclose mechanism of action of this class of compounds.
To the best of our knowledge, all quinolones bearing a flexible alkylamino side chain
in position-3 are novel.
2
. Chemistry
4

ACCEPTED MANUSCRIPT
The synthetic routes of novel 4(1H)-quinolones 6a-d are outlined Scheme 1. The
reaction of anilines 2a-q with ethoxymethylenemalonic ester 3 afforded Schiff bases
4
a-d. Cyclization of intermediate 4a-d in refluxing diphenyl ether gave ethyl
4
(1H)-quinolone-3-carboxylates 5a-d[20]. Amination of carboxylates 5a-d with an
excess of diamines by subjecting to microwave irradiation (150℃, 30 min) provided
the amidated derivatives 6a-d, bearing a flexible alkylamine side chain in
position-3[17-19].
The reaction of ethyl 7-chloro-4(1H)-quinolone-3-carboxylate 5b with
appropriate alkyl halogenide by the action of sodium hydride in anhydrous DMF
furnished ethyl 7-chloro-1-alkyl-4-quinolone-3-carboxylate 7a-f. Amination of
carboxylates 7a-f with an excess of diamines without solvent by subjecting to
microwave irradiation (150℃, 30 min) gave the amidated derivatives 8a-k in 53-68%
yield (Scheme 1). The chemical structure of all the newly synthesized compounds
1
13
was characterized by MS, HRMS, H NMR and C NMR spectra.
3
. Results and Discussion
3
.1 Antiproliferative activity and structure-activity relationships analysis
The antiproliferative potencies of quinolone derivatives 6a-d, 7a-f, 8a-k against
a panel of human tumor cell lines were investigated and compared with the reference
drugs 7-chloro-4(1H)-quinolone 1 and cisplatin. In order to improve the solubility in
aqueous solution, all compounds were prepared in the form of hydrochloride by the
usual methods before use. As predicted, the hydrochloride salt of novel quinolones
6
a-d and 8a-k bearing an alkylamino side chain showed excellent water solubility
5

ACCEPTED MANUSCRIPT
(more than 10mg/ml). The tumor cell line panel was consisted of human laryngeal
carcinoma (Hep-2), breast carcinoma (MCF-7), gastric carcinoma (BGC-823), liver
carcinoma (HepG2), cervical carcinoma (Hela), prostate carcinoma (PC-3) and
colorectal carcinoma (HCT-8, HCT-116 and RKO). The results are summarized in
Table 1.
As shown in Table 1, the lead compound 7-chloro-4(1H)-quinolone 1 failed to
display antiproliferative activities against nine human tumor cell lines at the
concentration of 50.0 µM, and the poor water-soluble property might be contributed
to its weak activity. While compounds 6a-d, the condensation products of compounds
5
a-d with 3-(diethylamino) propylamine, showed potent antiproliferative potencies
against most of human tumor cell lines with IC values lower than 50.0 µM. The
5
0
influence of substituent in position-7 of quinolone on antiproliferative activities
indicated that their antiproliferative potencies followed the tendency of 7-Cl > 7-Br >
7
F >7-OCH3.
Compounds 7a-d bearing a short alkyl group at N1-position exhibited moderate
or no antiproliferative activity at the concentration of 50.0 µM. Interestingly,
introducing a benzyl or (3-phenyl) propyl substituent into N1-position of ethyl
7
-chloro-4(1H)-quinolones-3-carboxylate 5b led to compounds 7e and 7f, respectively,
which showed remarkable antiproliferative activity against most of human tumor cell
lines with IC50 values lower than 20.0 µM. These results suggested that the
N1-arylated alkyl substituent of quinolones played a very important role in the
modulation of the antiproliferative potencies.
6

ACCEPTED MANUSCRIPT
The antiproliferative potency increased greatly after converting the
quinolone-3-carboxylates 7a-f to its 3-carboxamide derivatives 8a-k. Of all
3
-carboxamide derivatives, compounds 8i-k bearing an arylated alkyl substituent at
N1 of quinolone exhibited more potent antiproliferative effects than compounds 8a-i
having a short alkyl group at N1 of quinolone. Especially, compounds, bearing a
(3-phenyl) propyl group in N1 of quinolone, were found to be the most potent
antiproliferative agents with IC values lower than 10.0 µM against seven human
5
0
tumor cell lines investigated. The results further confirmed that the N1-arylated alkyl
substituent of quinolones facilitated the antiproliferative potencies. In addition,
compounds 8b, 8d, 8f, 8h, 8j and 8k bearing a flexible amino side chain with three
methylene spacer displayed more potent antiproliferative potencies than compounds
8
a, 8c, 8e, 8g and 8i having two methylene spacer. It is worth mentioning that
compounds 8j and 8k exhibited more potent antiproliferative activity than the positive
control drug cisplatin. These results indicated that (1) the alkylamino side chain
substituent was the advisable pharmacophoric group for the enhanced antiproliferative
activities; (2) the length of the alkylamino side chain moiety also affected their
antiproliferative potencies, and three methylene units might be more favorable.
3
.2 Growth inhibition of human cancer cells by compound 8k
To test the potential application of compound 8k in the treatment of cancers, the
inhibitory effect of compound 8k on growth of human cancer cell lines, HCT116 and
RKO, was investigated using the Cell Counting Kit-8(CCK-8) assay. Compared with
the vehicle treated cells, HCT116 and RKO exhibited markedly inhibited growth in a
7

ACCEPTED MANUSCRIPT
dose-dependent manner after treatment with compound 8k for 24h (Fig. 2A). The IC50
values of compound 8k (the concentrations for decreasing the growth rate of cells by
5
0%) were 5.79±0.5 µM for HCT116, 4.87±0.5 µM for RKO. To test whether
compound 8k affected tumorigenicity-correlated responses in vitro，plate colony
formation assay was performed (Fig. 2B). The colony-forming ability of HCT116 and
RKO was significantly (p<0.05) impaired when treated with compound 8k for 24h
dose-dependently (Fig. 2C).
3
.3 Compound 8k induced apoptosis in HCT116 and RKO
In normal viable cells, phosphatidylserine (PS) is located on the cytoplasmic
surface of the cell membrane. However, in the intermediate stages of apoptosis, PS is
translocated from the inner to the outer leaflet of the membrane. The translocation of
phosphatidylserine (PS) from the inner to the outer leaflet of the cell membrane is an
early event in apoptosis [21]. To determine whether the compound 8k induced growth
inhibition of cancer cells was associated with apoptosis, Annexin V-FITC/PI binding
assay was performed to examine the integrity of cell membrane and the
externalization of PS. As control, the binding of Annexin V to vehicle-treated
HCT116 was 1.3%, which increased to 18.2% and 38.2% when treated with 2 µM and
4
µM compound 8k for 24 h respectively (Fig. 3A).
At the late stage of apoptosis, some of the fragmentized DNA leaches out when
cells are fixed in ethanol and subsequently rehydrated. So these cells contain less
DNA and can be observed as a hypo-diploid or 'sub-G1' peak in a DNA histogram[22].
To further confirmed 8k killed cancer cells via inducing apoptosis, we performed PI
8

ACCEPTED MANUSCRIPT
staining assay, and the results revealed that the proportion of‘sub-G1’cells increased
when HCT116 was treated with increasing dosage of compound 8k (Fig. 3B). In
addition, the cleaved PARP(c-PARP), a marker for apoptosis, was increased along
with the increasing concentrations of compound 8k treatment (Fig. 3C). Together,
compound 8k killed cancer cells, at least in part, by triggering apoptosis.
3
.4 Compound 8k induce apoptosis via caspase-dependent pathway
As caspases are effector molecules of the extrinsic and intrinsic apoptotic
pathways[23], we next examined whether compound 8k induce cells apoptosis via a
caspase-dependent pathway. As shown in Fig. 4A, the increasing of Annexin V
-/-
positive cells in compound 8k treated HCT116 WT or HCT116 Bax could be
reversed by pan-caspase inhibitor Z-VAD-FMK (CASPi) pretrement. In addition, the
-/-
increasing c-PARP in compound 8k treated HCT116 WT or HCT116 Bax also were
blunted by CASPi pretrement (Fig. 4B). These results reveal that compound 8k
induce cancer cell apoptosis at least partially depend on caspase.
3
.5 Compound 8k-induced cell apoptosis was through p53- and Bax-independent
mechanisms
p53 and Bax are two of the most famous pro-apoptotic genes[24]. To verified
-/-
whether they were required for compound 8k induced apoptosis, HCT116 p53 and
-/-
HCT116 Bax cells were employed. First, the knock-out of these two genes was
confirmed by western blotting as in our previously paper[25]. Then compound 8k
inhibited the viability of both these cell strains was revealed by the CCK-8 assay and
colony formation (Fig. 2B and Fig. 4C). Furthermore, the proportion of Annexin V
positive cells were increased when treated these two cells with dosage increasing
9

ACCEPTED MANUSCRIPT
compound 8k (Fig. 4D). At last, the c-PARP also increased in both cells treated with
compound 8k (Fig. 4E). Collectively, these results suggest that the compound 8k
inducing cell apoptosis is independent on both p53 and Bax.
3
.6 Intracellular ROS are involved in compound 8k-induced apoptosis
Some chemotherapeutic agents can induce apoptosis through ROS generation in
cancer cells[26-28]. Through H2DCFDA-based flow cytometry, ROS was detected
remarkably accumulation in HCT116 treated with 8 µM compound 8k for 24h, which
could be reversed by the free radical scavengers L-NAC or GSH (Fig. 5A). To
determine whether intracellular ROS accumulation were involved in compound 8k
induced cancer cells death, colony formation was performed. Compound 8k potently
inhibited colony formation of HCT116 in a dose-dependent manner, and ROS
scavengers (both GSH and L-NAC) largely alleviated this inhibition (Fig. 5B and C).
Furthermore, CCK-8 assay indicated that when treated with 4 µM, 16 µM and 32 µM
for 24h, the viability of HCT116 were significantly higher in the GSH and L-NAC
protecting groups in compared to the control group (Fig. 5D). As expected, the
c-PARP increased when HCT116 was treated with increasing of compound 8k dosage,
and there were weaker increases of the c-PARP when cells were pretreated with ROS
scavenger L-NAC (Fig. 5E). Together, these results suggest that compound 8k
induces intracellular ROS accumulation in HCT116 which is involved in compound
8
k induced apoptosis.
4
. Conclusion
With the aim of improving the pharmacokinetic properties and enhancing the
1
0

ACCEPTED MANUSCRIPT
antitumor potencies of quinolone derivatives, in the present investigation, a series of
water-soluble quinolone derivatives bearing an alkyl substituent at N1 and a flexible
alkyl amino side chain at position-3 was synthesized and their antiproliferative
potential against seven human tumor cell line in culture were investigated.
Preliminary results evidenced the importance of the alkylamino side chain group in
position-3 and the nature of alkyl substituent at N1. The 3-phenylpropyl substituent in
position N1-position was the suitable pharmacophore giving rise to the most potent
antiproliferative agents and the optimal length of the alkylamino side chain moiety
was three methylene units.
In addition, we presented evidence to show that compound 8k was effective in
killing colorectal colon (CRC) cancer cells. Cell death by compound 8k was due to
apoptosis as characterized by the increased Annexin V binding, ‘sub-G1’ fraction and
cleavage of PARP-1. Moreover, our results suggested that compound 8k induced
ROS-triggering apoptosis depended on caspase, but not p53 and Bax. Many
anti-cancer agents induce cancer cell apoptosis via the p53 modulated pathway[29,
3
0]. However, p53 mutations are frequent not only in colorectal cancer but also in
majority of other human tumors[31]. Bax belongs to the bcl-2 family, which serves as
an essential effector of the mitochondrial apoptotic pathway and p53-mediated
apoptosis [24, 32]. Induction p53 and/or Bax-dependent apoptosis by drugs has been a
strategy to kill cancer cell[33, 34]. Similarly, Bax is another most frequently
inactivated genes in CRC, which resulted from MSI caused frameshift mutations[35,
3
6]. Hence, it’s necessary to figure out whether the compound 8k induced apoptosis is
11

ACCEPTED MANUSCRIPT
p53- and/or Bax-dependent or not. In our study, we have demonstrated that compound
8
k could trigger apoptosis independent on p53 and Bax. So compound 8k may serve
as a potential therapeutic drug for treating cancer with p53 and/or Bax mutation.
The modulation of intracellular ROS levels has been a selective anticancer
strategy for that cancer cells have a higher basal level of ROS[28, 37, 38].
Mechanistic insights showed that compound 8k triggered CRC apoptosis via inducing
ROS accumulation. And lowering the ROS level to mimic normal cells with low basal
levels of ROS by free radical scavengers protected cells from compound 8k inducing
apoptosis. Hence, compound 8k may be a potentially selective killer of CRC. Future
studies are needed to test this possibility in primary cells and to clarify the more
detailed molecular mechanisms of compound 8k in inducing apoptosis in cancer cells.
5
. Experimental protocols
5
.1 Reagents and general methods
All reagents were purchased from commercial suppliers and were dried and
purified when necessary. Melting points were determined with a Kofler micromelting
point apparatus without correction. ESI-MS spectra were obtained from VG ZAB-HS
1
13
spectrometer. H NMR and C NMR spectra were recorded on a Mercury-Plus 300
spectrometer at 300 MHz and 75 MHz respectively, using TMS as internal standard
and CDCl or DMSO-d as solvent and chemical shifts (δ) were expressed in ppm.
3
6
HRMS were obtained from ESI-Q-TOF maxis 4G spectrometer. All reactions were
monitored by TLC and spots were visualized with UV light or iodine. Silica gel F254
were used in analytical thin-layer chromatography (TLC) and silica gel were used in
1
2

ACCEPTED MANUSCRIPT
column chromatography respectively.
.2 General procedure for compounds 5a-d
mixture of the respective aniline 2a-d (100 mmol) and
5
A
ethoxymethylenemalonic ester 3 (25.3 g, 110 mmol) was stirred at RT for 10 min, and
then the mixture was stirred and heated at 110 ℃ for 1.5 h. The reaction mixture was
evaporated in vacuum to remove the residual ethanol and provide the condensation
products 4a-d. Compounds 4a-d prepared in these ways can be used directly in the
next step without further purification.
The molten condensation products 4a-d above (100 mmol) was poured slowly
into boiling diphenyl ether (150 mL). The mixture was heated under reflux for 2 h.
The solution was allowed to cool to room temperature and petroleum ether (75 mL)
was added. The precipitate formed was collected by filtration, washed well with
petroleum ether. It was found that the diphenyl ether could be satisfactorily removed
from the product by resuspending the crude product in petroleum ether or ethanol
(500 mL), stirring well, and refiltering. Compounds 5a-d had very poor solubility
1
even in DMSO-d , so it was too difficult to characterize their chemical structure by H
6
1
3
NMR and C NMR spectra.
5
.2.1 Ethyl 7-fluoro-4(1H)-quinolone-3-carboxylate (5a)
Starting from m-fluoroaniline 2a (100 mmol), compound 5a was obtained as
white solid (16.9 g, 72%), Mp >250 ℃.
Ethyl 7-chloro-4(1H)-quinolone-3-carboxylate (5b)
Starting from m-chloroaniline 2b (100 mmol), compound 5b was obtained as
1
3

ACCEPTED MANUSCRIPT
white solid (19.6 g, 78%), Mp >250 ℃.
5
.2.2 Ethyl 7-bromo-4(1H)-quinolone-3-carboxylate (5c)
Starting from m-bromoaniline 2c (100 mmol), compound 5c was obtained as
white solid (22.2 g, 75%), Mp >250 ℃.
5
.2.3 Ethyl 7-methoxy-4(1H)-quinolone-3-carboxylate (5d)
Starting from m-methoxyaniline 2d (100 mmol), compound 5d was obtained as
white solid (16.3 g, 66%), Mp >250 ℃.
5
.3 General procedure for compounds 6a-d
The mixture of
-substituted-4(1H)-quinolone-3-carboxylates 5a-d (5.0 mmol) and 3-(diethylamino)
the
corresponding
ethyl
7
propylamine (5.0 ml) was stirred under microwave irradiation at 150 ℃ for 30 min.
The resulting solution was partitioned between water and methylene chloride. The
aqueous phase was extracted with methylene chloride and the combined organic
layers were washed with brine, dried over Na SO , filtered, evaporated. The residue
2
4
oil was purified by a column chromatography, using a mixture of chloroform and
methanol 50:1 as an eluent, to successfully afford the desirable products.
5
.3.1 N-[3-(diethylamino)propyl]-7-fluoro-4(1H)-quinolone-3-carboxamide (6a)
Starting from compound 5a (5.0 mmol), compound 6a was isolated as white
+
1
solid (0.72 g, 45%). Mp 188.4-189.6 ℃. ESI-MS m/z 320 [M+H] . H NMR (300
MHz, CDCl ): δ 10.45 (s, 1H, CONH), 8.82 (s, 1H, ArH), 8.35-8.40 (m, 1H, ArH),
3
7
.23-7.27 (m, 1H, ArH), 7.08-7.14 (m, 1H, ArH), 3.56 (q, J =5.1Hz, 2H,
NHCH CH CH N(CH CH ) ), 2.53-2.60 (m, 6H, NHCH CH CH N(CH CH ) ),
2
2
2
2
3 2
2
2
2
2
3 2
1
4

ACCEPTED MANUSCRIPT
1
.78-1.88 (m, 2H, NHCH CH CH N(CH CH ) ), 1.02 (t, J = 6.9 Hz, 6H,
2
2
2
2
3 2
1
3
N(CH CH ) ). C NMR (75 MHz, CDCl ) δ 176.6, 166.4, 163.4, 144.6, 141.8, 129.1,
2
3 2
3
1
23.8, 114.2, 111.3, 104.8, 50.5, 47.0, 38.0, 27.4, 11.6. HRMS (ESI) calcd for
+
C H FN O [M+H] 320.1769, found 320.1769.
17
22
3
2
5
.3.2 7-Chloro-N-[3-(diethylamino)propyl]-4(1H)-quinolone-3-carboxamide (6b)
Starting from compound 5b (5.0 mmol), compound 6b was isolated as white
+
1
solid (0.87 g, 52%). Mp 197.2-198.0 ℃. ESI-MS m/z 336 [M+H] . H NMR (300
MHz, CDCl ): δ 10.48 (s, 1H, CONH), 8.84 (s, 1H, ArH), 8.34 (d, J = 8.7 Hz, 1H,
3
ArH), 7.63 (d, J = 1.5 Hz, 1H, ArH), 7.36 (dd, J = 8.7, 1.5 Hz, 1H, ArH), 3.55 (q, J
=5.1
Hz,
2H,
NHCH CH CH N(CH CH ) ),
2.58-2.65
(m,
6H,
2
2
2
2
3 2
NHCH CH CH N(CH CH ) ), 1.78-1.94 (m, 2H, NHCH CH CH N(CH CH ) ), 1.07
2
2
2
2
3 2
2
2
2
2
3 2
1
3
(
t, J = 7.2 Hz, 6H, N(CH CH ) ). C NMR (75 MHz, CDCl ) δ 176.7, 166.4, 144.6,
2 3 2 3
1
40.9, 138.8, 127.9, 125.8, 125.4, 118.9, 111.5, 50.5, 47.1, 38.1, 27.4, 11.6. HRMS
+
(
ESI) calcd for C H ClN O [M+H] 336.1473, found 336.1477.
1
7
22
3
2
5
.3.3 7-Bromo-N-[3-(diethylamino)propyl]-4(1H)-quinolone-3-carboxamide (6c)
Starting from compound 5c (5.0 mmol), compound 6c was isolated as white solid
+
1
(
0.93 g, 49%). Mp 187.2-187.9 ℃. ESI-MS m/z 380 [M+H] . H NMR (300 MHz,
CDCl ): δ 10.52 (s, 1H, CONH), 8.84 (s, 1H, ArH), 8.26 (d, J = 8.7 Hz, 1H, ArH),
3
7
2
1
.81 (d, J = 1.5 Hz, 1H, ArH), 7.51 (dd, J = 8.7, 1.5 Hz, 1H, ArH), 3.56 (q, J =5.1 Hz,
H, NHCH CH CH N(CH CH ) ), 2.58-2.65 (m, 6H, NHCH CH CH N(CH CH ) ),
2
2
2
2
3 2
2
2
2
2
3 2
.82-1.91 (m, 2H, NHCH CH CH N(CH CH ) ), 1.06 (t, J = 7.2 Hz, 6H,
2
2
2
2
3 2
13
N(CH CH ) ). C NMR (75 MHz, CDCl ) δ 176.8, 166.5, 144.5, 141.1, 128.6, 127.9,
2
3 2
3
1
5

ACCEPTED MANUSCRIPT
1
27.2, 125.8, 122.1, 111.5, 50.6, 47.1, 38.1, 27.4, 11.7. HRMS (ESI) calcd for
+
C H BrN O [M+H] 380.0968, found 380.0973.
17
22
3
2
5
.3.4 N-[3-(diethylamino)propyl]-7-methoxy-4-(1H)-quinolone-3-carboxamide (6d)
Starting from compound 5d (5.0 mmol), compound 6d was isolated as white
+
1
solid (0.96 g, 58%). Mp 141.6-142.3 ℃. ESI-MS m/z 332 [M+H] . H NMR (300
MHz, CDCl ): δ 10.50 (s, 1H, CONH), 8.78 (s, 1H, ArH), 8.30 (d, J = 9.0 Hz, 1H,
3
ArH), 7.00 (dd, J = 9.0, 1.8 Hz, 1H, ArH), 6.91 (d, J = 1.8 Hz, 1H, ArH), 3.89 (s, 3H,
CH O), 3.54 (q, J =5.4Hz, 2H, NHCH CH CH N(CH CH ) ), 2.51-2.59 (m, 6H,
3
2
2
2
2
3 2
NHCH CH CH N(CH CH ) ), 1.78-1.87 (m, 2H, NHCH CH CH N(CH CH ) ), 1.02
2
2
2
2
3 2
2
2
2
2
3 2
1
3
(
t, J = 7.2 Hz, 6H, N(CH CH ) ). C NMR (75 MHz, CDCl ) δ 176.6, 166.3, 162.9,
2 3 2 3
1
43.1, 141.1, 127.6, 120.7, 115.8, 110.8, 99.7, 55.6, 50.3, 46.7, 37.8, 27.2, 11.4.
+
HRMS (ESI) calcd for C H N O [M+H] 332.1969, found 332.1977.
1
8
25
3
3
5
.4 General procedure for compounds 7a-f
A mixture of ethyl 7-chloro-4(1H)-quinolone-3-carboxylate 5b (10 mmol) and
anhydrous DMF (60 mL) was stirred at RT for 10 min, and then 60% NaH (1.2 g, 30
mmol) and alkyl halogenide (15-30 mmol) were added. The mixture was continued to
stir at RT. After completion of the reaction as indicated by TLC, the solution was
poured into H O (150 mL), and extracted with ethyl acetate. The organic phase was
2
made acidic with concentrated hydrochloric acid. Upon removal of solvent, the
residue was crystallized from acetone to afford yellow solid. The solid was dissolved
in water and made basic with sodium bicarbonate, and the aqueous mixture extracted
with ethyl acetate. The organic phase was washed with water and brine, then dried
1
6

ACCEPTED MANUSCRIPT
over anhydrous sodium sulfate, filtered and evaporated. The crude product was
purified by a column chromatography, using a mixture of ethyl acetate and petroleum
ether (3:1) as an eluent, to successfully afford the desirable products in good yield.
5
.4.1 Ethyl 7-chloro-1-methyl-4-quinolone-3-carboxylate (7a)
Starting from compound 5b (10 mmol) and iodomethane (15 mmol), compound
a was isolated as white solid (1.7 g, 65%). Mp 178.8-180.2 ℃. ESI-MS m/z 288
7
+
1
[
M+Na] . H NMR (300 MHz, CDCl ): δ 8.42-8.45 (m, 2H, ArH)), 7.38-7.41 (m, 2H,
3
ArH), 4.39 (q, J = 7.2 Hz, 2H, OCH CH ), 3.86 (s, 3H, NCH ), 1.42 (t, J = 7.2 Hz,
2
3
3
1
3
3
1
H, OCH CH ). C NMR (75MHz, CDCl ): δ 172.9, 164.9, 151.2, 141.3, 138.2,
2 3 3
28.8, 127.1, 125.8, 117.7, 110.8, 60.6, 41.8, 15.1. HRMS (ESI) calcd for
+
C H ClNO [M+Na] 288.0398, found 288.0397.
13
12
3
5
.4.2 Ethyl 7-chloro-1-ethyl-4-quinolone-3-carboxylate (7b)
Starting from compound 5b (10 mmol) and iodoethane (15 mmol), compound 7c
was isolated as white solid (1.9 g, 69%). Mp 163.8-164.4 ℃. ESI-MS m/z 302
+
1
[
M+Na] . H NMR (300 MHz, CDCl ): δ 8.44-8.47 (m, 2H, ArH), 7.25-7.43 (m, 2H,
3
ArH), 4.39 (q, J =7.2Hz, NCH CH ), 4.21(q, J =7.2Hz, OCH CH ), 1.56 (t, J =7.2Hz,
2
3
2
3
1
3
NCH CH ), 1.41(t, J =7.2Hz, OCH CH ). C NMR (75 MHz, CDCl ): δ 173.5,
2
3
2
3
3
1
65.2, 148.9, 139.4, 139.1, 129.5, 127.5, 125.5, 115.8, 111.5, 61.1, 49.2, 14.7. HRMS
+
(
ESI) calcd for C H ClNO [M+Na] 302.0554, found 302.0564.
1
4
14
3
5
.4.3 Ethyl 1-allyl-7-chloro-4-quinolone-3-carboxylate (7c)
Starting from compound 5b (10 mmol) and allyl bromide (15 mmol), compound
c was isolated as white solid (2.2 g, 75%). Mp 188.0-189.6 ℃. ESI-MS m/z 314
7
1
7

ACCEPTED MANUSCRIPT
+
1
[
M+Na] . H NMR (300 MHz, CDCl ): δ 8.48 (s, 1H, ArH), 8.42 (d, J =8.7Hz, 1H,
3
ArH), 7.34-7.39 (m, 2H, ArH), 5.94-5.07(m, 1H, CH CH=CH ), 5.40 (d, J = 10.5 Hz,
2
2
1
H, CH CH=CH ), 5.20 (d, J = 17.1 Hz, 1H, CH CH=CH ), 4.79 (m, 2H,
2 2 2 2
CH CH=CH ), 4.38 (q, J = 6.9Hz, 2H, OCH CH ), 1.41 (t, J = 6.9 Hz, 3H,
2
2
2
3
13
OCH CH ). C NMR (75 MHz, CDCl ): δ 173.8, 165.2, 149.7, 140.0, 139.2, 130.5,
2
3
3
1
29.4, 127.3, 125.8, 119.8, 116.5, 111.6, 61.2, 56.2, 14.8. HRMS (ESI) calcd for
+
C H ClNO [M+Na] 314.0565, found 314.0565.
15
14
3
5
.4.4 Ethyl 1-butyl-7-chloro-4-quinolone-3-carboxylate (7d)
Starting from compound 5b (10 mmol) and n-iodobutane (30 mmol), compound
d was isolated as white solid (1.9 g, 62%). Mp 105.8-106.7 ℃. ESI-MS m/z 330
7
+
1
[
M+Na] . H NMR (300MHz, DMSO-d ): δ 8.62 (s, 1H, ArH), 8.17 (d, J =8.7Hz, 1H,
6
ArH), 7.87 (d, J =1.8 Hz, 1H, ArH), 7.47 (dd, J =8.7Hz, 1.8Hz, 1H, ArH), 4.33 (t,
J=7.2 Hz, 2H, NCH CH CH CH ), 4.20 (q, J=7.2Hz, 2H, OCH CH ), 1.64-1.73 (m,
2
2
2
3
2
3
2
H, NCH CH CH CH ), 1.24-1.34 (m, 5H, CH CH CH CH OCH CH ), 0.88 (t,
2 2 2 3 2 2 2 3, 2 3
13
J=7.2 Hz, 3H, NCH CH CH CH ). C NMR (75MHz, CDCl ): δ 172.8, 165.0, 150.5,
2
2
2
3
3
1
40.3, 138.4, 129.2, 127.5, 125.8, 117.4, 111.1, 60.7, 53.3, 31.3, 19.9, 15.1, 14.3.
+
HRMS (ESI) calcd for C H ClNO [M+Na] 330.0867, found 330.0870.
1
6
16
3
5
.4.5 Ethyl 1-benzyl-7-chloro-4-quinolone-3-carboxylate (7e)
Starting from compound 5b (5mmol) and benzyl bromide (15mmol), compound
e was isolated as white solid (2.9 g, 85%). Mp 147.9-148.8 ℃. ESI-MS m/z 364
7
+
1
[
M+Na] . H NMR (300MHz, DMSO-d ): δ 8.54 (s, 1H, ArH), 8.43 (d, J=8.1Hz, 1H,
6
ArH), 7.30-7.38 (m, 5H, ArH), 7.14-7.18 (m, 2H, ArH), 5.34 (s, 2H, CH Ph), 4.38 (q,
2
1
8

ACCEPTED MANUSCRIPT
1
3
J=7.2 Hz, 2H, OCH CH ), 1.41 (t, J=7.2Hz, 3H, OCH CH ). C NMR (75MHz,
2
3
2
3
CDCl ) δ 173.6, 165.0, 150.0, 140.1, 139.1, 134.0, 129.5, 129.4, 128.8, 127.5, 126.4,
3
+
1
3
5
25.7, 116.7, 111.7, 61.1, 57.5, 14.8. HRMS (ESI) calcd for C H ClNO [M+Na]
19 16 3
64.0711, found 364.0714.
.4.6 Ethyl 7-chloro-1-(3-phenylpropyl)-4-quinolone-3-carboxylate (7f)
Starting from compound 5b (10 mmol) and 1-(3-bromopropyl)benzene (50
mmol), compound 7f was isolated as white solid (2.4 g, 65%). Mp 136.6-137.8 ℃.
+
1
ESI-MS m/z 392 [M+Na] . H NMR (300 MHz, CDCl ): δ 8.44 (d, J = 8.7 Hz, 1H,
3
ArH), 8.37 (s, 1H, ArH), 7.43-7.15 (m, 7H, ArH), 4.40 (q, J = 7.2 Hz, 2H,
OCH CH ), 4.10 (t, J = 7.2 Hz, 2H, NCH CH CH Ph), 2.77 (t, J = 7.2 Hz, 2H,
2
3
2
2
2
NCH CH CH Ph), 2.32–2.20 (m, 2H, NCH CH CH Ph), 1.42 (t, J = 7.2 Hz, 3H,
2
2
2
2
2
2
13
OCH CH ). C NMR (75 MHz, CDCl ): δ 173.2, 164.9, 149.1, 139.2, 139.1, 138.8,
2
3
3
1
1
5
29.4, 128.6, 128.1, 127.9, 127.3, 126.5, 125.3, 115.3, 111.2, 60.8, 52.9, 32.3, 29.6,
+
4.4. HRMS (ESI) calcd for C H ClNO [M+Na] 392.1024, found 392.1032.
2
1
20
3
.5 General procedure for compounds 8a-k
The mixture of the corresponding
-chloro-4-quinolone-3-carboxylate (5.0 mmol) and alkylamines (5.0 mL) was stirred
1-substituted
ethyl
7
under microwave irradiation at 150℃ for 15 min. The resulting solution was
partitioned between water and methylene chloride. The aqueous phase was extracted
with methylene chloride and the combined organic layers were washed with brine,
dried over Na SO , filtered, evaporated. The crude oil was purified by a column
2
4
chromatography, using a mixture of chloroform and methanol 50:1 as an eluent, to
1
9

ACCEPTED MANUSCRIPT
successfully afford the desirable products.
5
.5.1 7-Chloro-N-[2-(diethylamino)ethyl]-1-methyl-4-quinolone-3-carboxamide (8a)
Starting from compound 7a (5.0 mmol) and N,N-diethylethylenediamine (5.0
mL), compound 8a was isolated as white solid (1.1 g, 68%). Mp 163.6-163.7 ℃.
+
1
ESI-MS m/z 336 [M+H] . H NMR (300 MHz, CDCl ): δ 9.92 (s, 1H, CONH), 8.73
3
(s, 1H, ArH), 8.45 (d, J = 8.4 Hz, 1H, ArH), 7.43-7.50 (m, 2H, ArH), 3.91 (s, 3H,
NCH ), 3.55 (q, J =6.3 Hz, 2H, NHCH CH N(CH CH ) ), 2.61-2.67 (m, 6H,
3
2
2
2
3 2
NCH CH CH N(CH CH ) ), 1.79-1.91(m, 2H, NCH CH CH N(CH CH ) ), 1.08 (t,
2
2
2
2
3 2
2
2
2
2
3 2
13
J = 7.2 Hz, 6H, N(CH CH ) ). C NMR (75 MHz, CDCl ): δ 176.0, 164.6, 148.8,
2
3 2
3
1
40.7, 139.5, 129.1, 126.3, 125.9, 116.0, 112.7, 52.1, 47.5, 41.8, 37.7, 12.2. HRMS
+
(
ESI) calcd for C H ClN O [M+H] 336.1473, found 336.1476.
1
7
22
3
2
5
.5.2
7-Chloro-N-[3-(diethylamino)propyl]-1-methyl-4-quinolone-3-carboxamide
(8b)
Starting from compound 7a (5.0 mmol) and 3-(diethylamino)propylamine (5.0
mL), compound 8b was isolated as white solid (0.9 g, 54%). Mp 142.7-143.1 ℃.
+
1
ESI-MS m/z 350 [M+H] . H NMR (300 MHz, CDCl ): δ .92 (s, 1H, CONH), 8.73 (s,
3
1
H, ArH), 8.45 (d, J = 8.7 Hz, 1H, ArH), 7.43-7.50 (m, 2H, ArH), 3.91 (s, 3H,
NCH ), 3.55 (q, J =6.3 Hz, 2H, NHCH CH N(CH CH ) ), 2.61-2.67 (m, 6H,
3
2
2
2
3 2
NCH CH CH N(CH CH ) ), 1.79-1.91(m, 2H, NCH CH CH N(CH CH ) ), 1.08 (t,
2
2
2
2
3 2
2
2
2
2
3 2
13
J = 7.2 Hz, 6H, N(CH CH ) ). C NMR (75 MHz, CDCl ) δ 176.1, 164.6, 148.9,
2
3 2
3
1
40.7, 139.6, 129.0, 125.9, 116.0, 112.7, 50.6, 47.1, 41.8, 38.0, 27.4, 11.9. HRMS
+
(
ESI) calcd for C H ClN O [M+H] 350.1630, found 350.1638.
1
8
24
3
2
2
0

ACCEPTED MANUSCRIPT
5
.5.3 7-Chloro-N-[2-(diethylamino)ethyl]-1-ethyl-4-quinolone-3-carboxamide (8c)
Starting from compound 7b (5.0 mmol) and N,N-diethylethylenediamine (5.0
mL), compound 8c was isolated as white solid (1.0 g, 58%). Mp 154.0-154.8 ℃.
+
1
ESI-MS m/z 350 [M+H] . H NMR (300 MHz, CDCl ): δ 9.95 (s, 1H, CONH),
3
8
.77 (s, 1H, ArH), 8.44 (d, J=8.4Hz, 1H, ArH), 7.50 (d, J =1.5Hz, ArH), 7.43(d, J
8.4Hz, 1.5 Hz, ArH), 4.29 (q, J =7.2Hz, 2H, NCH CH ), 3.55 (q, J =6.6 Hz, 2H,
=
2
3
NHCH CH N(CH CH ) ), 2.82 (t, J =6.9Hz, 2H, NCH CH N(CH CH ) ), 2.64 (q, J
2
2
2
3 2
2
2
2
3 2
=
7.2Hz, 4H, N(CH CH ) ), 1.57 (t, J =7.2Hz, 3H, NCH CH ), 1.10 (t, J =7.2Hz, 6H,
2 3 2 2 3
1
3
N(CH CH ) ). C NMR (75 MHz, CDCl ): δ 175.9, 164.7, 147.6, 139.6, 139.4, 129.3,
2
3 2
3
1
26.6, 125.6, 115.7, 112.9, 52.1, 49.3, 47.5, 37.7, 14.8, 12.3. HRMS (ESI) calcd for
+
C H ClN O [M+H] 350.1630, found 350.1636.
18
24
3
2
5
.5.4
7-Chloro-N-[3-(diethylamino)propyl]-1-ethyl-4-quinolone-3-carboxamide
(8d)
Starting from compound 7b (5.0 mmol) and 3-(diethylamino)propylamine (5.0
mL), compound 8d was isolated as white solid (1.06 g, 57%). Mp 127.4-128.5 ℃.
+
1
ESI-MS m/z 364 [M+H] . H NMR (300 MHz, CDCl ): δ 9.92 (s, 1H, CONH), 8.77
3
(s, 1H, ArH), 8.45 (d, J = 8.7 Hz, 1H, ArH), 7.51 (d, J = 1.8 Hz, 1H, ArH), 7.43 (dd,
J = 8.7, 1.8 Hz, 1H, ArH), 4.28 (q, J = 7.2 Hz, 2H, NCH CH ), 3.49 (q, J = 6.9 Hz,
2
3
2
1
1
H, NCH CH CH N(CH CH ) ), 2.54 (m, 6H, NCH CH CH N(CH CH ) ),
2 2 2 2 3 2 2 2 2 2 3 2
.86-1.74 (m, 2H, NCH CH CH N(CH CH ) ), 1.57 (t, J = 7.2 Hz, 3H, NCH CH ),
2
2
2
2
3 2
2
3
13
.04 (t, J = 7.2 Hz, 6H, N(CH CH ) ). C NMR (75 MHz, CDCl ): δ 176.0, 164.6,
2
3 2
3
2
1

ACCEPTED MANUSCRIPT
1
47.7, 139.5, 129.3, 126.6, 125.7, 115.7, 112.9, 50.7, 49.4, 47.2, 38.0, 27.6, 14.9, 12.1.
+
HRMS (ESI) calcd for C H ClN O [M+H] 364.1786, found 364.1791.
1
9
26
3
2
5
.5.5 1-Allyl-7-chloro-N-[2-(diethylamino)ethyl]-4-quinolone-3-carboxamide (8e)
Starting from compound 7c (5.0 mmol) and N,N-diethylethylenediamine (5.0
mL), compound 8e was isolated as white solid (0.96 g, 53%). Mp 149.8-150.8 ℃.
+
1
ESI-MS m/z 362 [M+H] . H NMR (300 MHz, CDCl ): δ 10.00 (s, 1H, CONH), 8.75
3
(s, 1H, ArH), 8.44 (d, J = 8.7 Hz, 1H, ArH), 7.46 (d, J = 1.5 Hz, 1H, ArH), 7.42 (dd,
J = 8.7, 1.5 Hz, 1H, ArH), 5.94-6.07 (m, 1H, CH CH=CH ), 5.40 (d, J = 10.2 Hz, 1H,
2
2
CH CH=CH ), 5.20 (d, J = 17.1 Hz, 1H, CH CH=CH ), 4.81-4.84 (m, 2H,
2
2
2
2
CH CH=CH ), 3.64 (q, J = 6.6 Hz, 2H, NHCH CH N(CH CH ) ), 2.82 (t, J = 6.6 Hz,
2
2
2
2
2
3 2
2
H, NHCH CH N(CH CH ) ), 2.74 (q, J = 6.9 Hz, 4H, N(CH CH ) ), 1.17 (t, J = 7.2
2 2 2 3 2 2 3 2
1
3
Hz, 6H, N(CH CH ) ). C NMR (75 MHz, CDCl ): δ 176.0, 164.6, 148.3, 140.1,
2
3 2
3
1
39.4, 130.3, 129.1, 126.4, 125.8, 119.9, 116.4, 112.9, 56.2, 52.1, 47.5, 37.8. 12.2.
+
HRMS (ESI) calcd for C H ClN O [M+H] 362.1630, found 362.1639.
1
9
24
3
2
5
.5.6 1-Allyl-7-chloro-N-[3-(diethylamino)propyl]-4-quinolone-3-carboxamide (8f)
Starting from compound 7c (5.0 mmol) and 3-(diethylamino)propylamine (5.0
mL), compound 8f was isolated as white solid (1.24 g, 66%). Mp 121.9-122.3 ℃.
+
1
ESI-MS m/z 376 [M+H] . H NMR (300 MHz, CDCl ) δ 9.91 (s, 1H, CONH), 8.76 (s,
3
1
1
5
=
H, ArH), 8.45 (d, J = 8.7 Hz, 1H, ArH), 7.46 (d, J = 1.8 Hz, 1H, ArH), 7.40-7.44 (m,
H, ArH), 5.94-6.07 (m, 1H, CH CH=CH), 5.40 (d, J = 10.5 Hz, 1H, CH CH=CH ),
2
2
2
.20 (d, J = 17.1 Hz, 1H, CH CH=CH ), 4.81-4.83 (m, 2H, CH CH=CH ), 3.50 (q, J
2
2
2
2
6.6
Hz,
2H,
NHCH CH CH N(CH CH ) ),
2.56-2.63
(m,
6H,
2
2
2
2
3 2
2
2

ACCEPTED MANUSCRIPT
NHCH CH CH N(CH CH ) ), 1.79-1.89 (m, 2H, NHCH CH CH N(CH CH ) ), 1.08
2
2
2
2
3 2
2
2
2
2
3 2
1
3
(
t, J = 7.2 Hz, 6H, N(CH CH ) ). C NMR (75 MHz, CDCl ) δ 171.4, 159.8, 143.4,
2 3 2 3
1
2
5
35.4, 134.7, 125.5, 124.4, 121.7, 121.1, 115.3, 111.7, 108.3, 51.5, 46.0, 42,5, 33.3,
+
2.8, 7.4. HRMS (ESI) calcd for C H ClN O [M+H] 376.1786, found 376.1793.
2
0
26
3
2
.5.7 1-Butyl-7-chloro-N-[2-(diethylamino)ethyl]-4-quinolone-3-carboxamide (8g)
Starting from compound 7d (5.0 mmol) and N,N-diethylethylenediamine (5.0
mL), compound 8g was isolated as white solid (1.23g, 65%). Mp 168.3-168.5 ℃.
+
1
ESI-MS m/z 378 [M+H] . H NMR (300 MHz, CDCl ): δ 9.96 (s, 1H, CONH), 8.72
3
(s, 1H, ArH), 8.46 (d, J = 8.7 Hz, 1H, ArH), 7.49 (d, J = 1.5 Hz, 1H, ArH), 7.43 (dd,
J = 8.7, 1.5 Hz, 1H, ArH), 4.20 (t, J = 7.2 Hz, 2H, NCH CH CH CH ), 3.58 (q, J =
2
2
2
3
6
.6 Hz, 2H, NHCH CH N(CH CH ) ), 2.73 (t,
J
=
6.6 Hz, 2H,
2
2
2
3 2
NHCH CH N(CH CH ) ), 2.66 (q, J =6.9Hz, 4H, N(CH CH ) ), 1.84-1.94 (m, 2H,
2
2
2
3 2
2
3 2
NCH CH CH CH ), 1.40-1.52 (m, 2H, NCH CH CH CH ), 1.10 (t, J =7.2Hz, 6H,
2
2
2
3
2
2
2
3
1
3
N(CH CH ) ), 1.01 (t, J = 7.2 Hz, 3H, NCH CH CH CH ). C NMR (75 MHz,
2
3 2
2
2
2
3
CDCl ): δ 175.9, 164.7, 148.2, 139.9, 139.4, 129.4, 126.7, 125.7, 115.8, 112.7, 54.3,
3
+
5
3
5
2.2, 47.6, 37.8, 31.2, 20.3, 13.9, 12.3. HRMS (ESI) calcd for C H ClN O [M+H]
20 28 3 2
78.1943, found 378.1951.
.5.8 1-Butyl-7-chloro-N-[3-(diethylamino)propyl]-4-quinolone-3-carboxamide (8h)
Starting from compound 7d (5.0 mmol) and 3-(diethylamino)propylamine (5.0
mL), compound 8h was isolated as white solid (1.22 g, 63%). Mp 132.0-132.4 ℃.
+
1
ESI-MS m/z 392 [M+H] . H NMR (300 MHz, CDCl ): δ 9.96 (s, 1H, CONH), 8.72
3
(s, 1H, ArH), 8.45 (d, J = 8.7 Hz, 1H, ArH), 7.49 (d, J = 1.5 Hz, 1H, ArH), 7.43 (dd,
2
3

ACCEPTED MANUSCRIPT
J = 8.7, 1.5 Hz, 1H, ArH), 4.21 (t, J = 7.5 Hz, 2H, NCH CH CH CH ), 3.50 (q,
2
2
2
3
J=6.6Hz,
2H,
NHCH CH CH N(CH CH ) ),
2.65-2.71
4H, NCH CH CH CH
2 2 2 3,
(m,
6H,
2
2
2
2
3 2
NHCH CH CH N(CH CH ) ),
1.84-1.94
(m,
2
2
2
2
3 2
NHCH CH CH N(CH CH ) ), 1.40-1.52 (m, 2H, NCH CH CH CH ), 1.13 (t, J =
2
2
2
2
3 2,
2
2
2
3
13
7
.2 Hz, 6H, N(CH CH ) ), 1.02 (t, J = 7.2 Hz, 3H, NCH CH CH CH ). C NMR (75
2 3 2 2 2 2 3
MHz, CDCl ): δ 176.0, 164.6, 148.2, 139.9. 139.4, 129.3, 126.6, 125.7, 115.9, 112.7,
3
5
4.3, 50.8, 47.2, 38.1, 31.2, 27.6, 20.3, 14.0, 12.1. HRMS (ESI) calcd for
+
C H ClN O [M+H] 392.2099, found 392.2109.
21
30
3
2
5
.5.9
1-Benzyl-7-chloro-N-[2-(diethylamino)ethyl]-4-quinolone-3-carboxamide
(8i)
Starting from compound 7e (5mmol) and N,N-diethylethylenediamine (5.0 mL),
compound 8i was isolated as white solid (1.4 g, 68%). Mp 204.2-205.2 ℃. ESI-MS
+
1
m/z 412 [M+H] . H NMR (300 MHz, CDCl ): δ 9.94 (s, 1H, CONH), 8.88 (s, 1H,
3
ArH), 8.45 (d, J = 9.0 Hz, 1H, ArH), 7.32-7.39 (m, 5H, ArH), 7.15-7.17 (m, 2H,
ArH), 5.40 (s, 2H, CH Ph), 3.57 (q, J = 6.6 Hz, 2H, CONHCH CH N(CH CH ) )),
2
2
2
2
3 2
2
.72 (t, J = 6.6 Hz, 2H, CONHCH CH N(CH CH ) ), 2.63 (q, J = 7.2 Hz, 4H,
2 2 2 3 2
1
3
N(CH CH ) ), 1.09 (t, J = 7.2 Hz, 6H, N(CH CH ) ). C NMR (75MHz, CDCl ): δ
2
3 2
2
3 2
3
1
1
76.1, 164.6, 148.9, 140.2, 139.4, 134.0, 129.6, 129.2, 128.9, 126.7, 126.4, 125.6,
16.7, 113.0, 57.8 52.1, 47.6, 37.8, 12.3. HRMS (ESI) calcd for C H ClN O
2
2
3
26
3
+
[
M+H] 412.1798, found 412.1798.
5
.5.10 1-Benzyl-7-chloro-N-[2-(diethylamino)propyl]-4-quinolone-3-carboxamide
(8j)
2
4

ACCEPTED MANUSCRIPT
Starting from compound 7e (5.0 mmol) and 3-(diethylamino)propylamine (5.0
mL), compound 8j was isolated as white solid (1.2 g, 56%). Mp 182.3-183.2 ℃.
+
1
ESI-MS m/z 426 [M+H] . H NMR (300MHz, DMSO-d ): δ 10.04 (s, 1H, CONH ),
6
8
2
2
.85(1H, s, ArH), 8.41 (d, J=9.0Hz, 1H, ArH), 7.34-7.40(m, 5H, ArH), 7.14-7.16(m,
H, ArH), 5.42 (s, 2H, CH Ph), 3.55 (q, J =6.3Hz, 2H, NHCH CH CH N(CH CH ) ),
2
2
2
2
2
3 2
.90-3.01 (m, 6H, NHCH CH CH N(CH CH )
3 2
), 1.82-1.87(m, 2H,
2
2
2
2
1
3
NHCH CH CH N(CH CH ) ), 1.29 (t, J =7.2Hz, 6H, N(CH CH ) ). C NMR
2
2
2
2
3 2
2
3 2
(
75MHz, CDCl ): δ 176.2, 164.5, 149.0, 140.2, 139.5, 133.9, 129.6, 129.2, 129.0,
3
1
26.6, 126.4, 125.9, 116.7, 113.0, 57.9, 50.7, 47.2, 38.1, 27.5, 12.0. HRMS (ESI)
+
calcd for C H ClN O [M+H] 426.1943, found 426.1945.
2
4
28
3
2
5
.5.11 7-Chloro-N-[3-(diethylamino)propyl]-1-(3-phenylpropyl)-4-quinolone-3-
carboxamide (8k)
Starting from compound 7f (5.0 mmol) and 3-(diethylamino)propylamine (5.0
mL), compound 8k was isolated as white solid (1.4 g, 63%). Mp 136.2-136.7 ℃.
+
1
ESI-MS m/z 454 [M+H] . H NMR (300 MHz, CDCl ): δ 9.91 (s, 1H, CONH), 8.71
3
(s, 1H, ArH), 8.43 (d, J = 6.6 Hz, 1H, ArH), 7.41-7.19 (m, 7H, ArH), 4.16 (t, J =6.0
Hz, 2H, NCH CH CH Ph), 3.48 (q, J = 4.8 Hz, 2H, NHCH CH CH ), 2.76 (t, J =5.4
2
2
2
2
2
2
Hz, 2H, NCH CH CH Ph), 2.51-2.56 (m, 6H, NHCH CH CH N(CH CH ) ),
2
2
2
2
2
2
2
3 2
2
.19-2.27
(m,
2H,
NCH CH CH Ph),
1.75-1.82
(m,
2H,
2
2
2
13
NHCH CH CH N(CH CH ) ), 1.02 (t, J = 7.2 Hz, 6H, N(CH CH ) ). C NMR (75
2
2
2
2
3 2
2
3 2
MHz, CDCl ): δ 175.8, 164.5, 148.0, 139.6, 139.4, 129.1, 128.9, 128.3, 126.7, 126.4,
3
1
25.6, 115.6, 112.6, 53.4, 50.5, 47.0, 37.9, 32.7, 30.2, 27.3, 11.8. HRMS (ESI) calcd
2
5

ACCEPTED MANUSCRIPT
+
for C H ClN O [M+H] 454.2256, found 454.2259.
2
6
32
3
2
5
.6 General procedure for the hydrochloride salt of 6a-d, 7a-f and 8a-k.
The target compounds 6a-d, 7a-f and 8a-k 5b (2 mmol) was dissolved in CH Cl
2
2
(20 mL) and stirred at RT for 5 min, respectively. Then 4M HCl/ethanol (10 mL) was
added, and the mixture was stirred at RT for 30 min, then removed the solvent
under reduced pressure to obtain the target hydrochloride salt as yellow solid.
5
.7 Cell lines and cell culture
The Human colon cancer cells (Hep-2, MCF-7, BGC-823, HepG2, Hela, PC-3,
HCT-8, HCT116 and RKO ) were purchase from American type culture collection
-/-
-/-
(
ATCC, Rockville, MD). HCT116 p53 and HCT116 Bax 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
-1
-1
(
Hyclone, Logan, UT, USA), 100 mg·mL streptomycin and 100 units·mL penicillin
o
at 37 C in a humidified atmosphere of 5% CO and 95% air.
2
5
.8 Cell viability assay
Cells were seeded into 96-well flat-bottom plates at a density of 3×10 per well
4
and cultured for 24 h, then treated with increasing dosage of compound 8k for the 24
h. The cells viability was assayed using Cell Counting Kit-8 (CCK-8, Dojindo
Laboratories, Kumamoto, Japan) cells were incubated with 10 µl of CCK-8 solution
for 1 h at 37, according to the manufacturer’s instruction. Cell viability was then
determined by a spectrophotometer set (ELx800, BioTek, USA) at a wavelength of
2
6

ACCEPTED MANUSCRIPT
4
50 nm.
5
.9 Colony formation assay
After being seeded into 12-well plates at a density of 300 per well for 48 h,
cells were treated with indicated does of compound 8k. After 24 h, the medium with
drug were replaced with fresh medium. About 10 days later, cell colonies were stained
with crystal violet for 15 min and photographed. Finally, the numbers of colonies
were scored.
5
.10 Annexin V-FITC/PI binding assay
Cells were plated in 6-well plates at a density of 5×10 , and the cells were
5
treated with compound 8k after 80% confluence. After 24 h, cells were collected with
trypsin and washed with ice-cold PBS buffer (137mM NaCl, 2.7 mM KCl, 10 mM
Na HPO , 2 mM KH PO , pH 7.4) for twice and resuspended in 100 µL binding
2
4
2
4
buffer. Then, 5 µL Annexin V-FITC and 5 µL PI (FITC Annexin V Apoptosis
Detection Kit I (BD, CAT#556547) were added to the cells. After incubated for 15
min at room temperature in the dark, the cells were added an additional 400 µL
binding buffer and subjected to FACS cytometry. The results were analyzed with
Summit version 4.3 software.
5
.11 PI staining
5
5
×10 Cells were seeded in 6-well plates to reach 80% confluence and treat
with compound 8k. After exposure to 8k for 24 h, cells were collected with trypsin
o
and washed with PBS buffer, then fixed with 75% ethanol at -20 C overnight. The
fixed cells were re-suspended in 500 µL PBS buffer containing 0.2 mg/mL RNase A
2
7

ACCEPTED MANUSCRIPT
o
for 30min at 37 C to digest RNA, and then add PI to 50 µg/mL to stain the cells for
0min in the dark. Finally, the cells were analyzed with flow cytometer. The results
were analyzed with Summit version 4.3 software.
3
5
.12 Intracellular ROS generation examination
Treated cells were collected and washed twice with PBS. Then the cells were
re-suspended in serum-free McCoy’s 5A medium, followed by the addition of 10 nM
H2DCFDA (carboxy-20,70-dichlorodihydrofluorescin diacetate; Invitrogen) and
o
incubation at 37 C in the dark for 20 min. The cells were then washed twice with
PBS and the relative ROS levels of the cells were measured through flow cytometry
analysis. The experimental data were analyzed using FCS Express version 3 software
(DeNovo, Los Angeles, CA, USA).
5
.13 Western Blot Analysis
Cells seeded in 60 mm plates were applied with indicated experimental
treatments. Treated cells were collected and washed with PBS. Then the cell lysates
were extracted using SDS sample buffer(2% SDS; 50 mM Tris-HCl, pH 6.8; 10%
glycerol; 12.5 mM EDTA) and the protein concentration was detected using
bicinchoninic acid protein assay kit (Pierce). Identical quantities of proteins were
separated by 10% SDS-PAGE and transferred to PVDF membranes (PVDF, Millipore,
cat # IPVH00010, Merck KGaA, Darmstadt, Germany). The membranes were
blocked with 5% non-fat milk and the probed with indicated primary antibodies
o
overnight at 4 C. After incubated with specific horseradish peroxidase conjugated
secondary antibodies, the bands were revealed by ECL detection system (Millipore).
2
8