Synthesis and anticancer activity of aminodihydroquinoline analogs: Identification of novel proapoptotic agents

Article abstract of DOI:10.1016/j.bmcl.2013.04.038

A series of 2-aminodihydroquinoline analogs were synthesized and their in vitro cytotoxicities against metastatic breast adenocarcinoma cell line MDA-MB-231 were tested. Five out of 16 compounds exhibited promising activity and structure-activity relationship revealed major role of dialkylaminoethyl substituents on dihydroquinoline ring for the activity. Two compounds, 5f and 5h, presented cytotoxicity with IC₅₀ values of about 2 μM when the compounds were treated to the cells without serum. The cell proliferation was inhibited mildly when the cells cultured with serum. Flow cytometry analyses showed that those compounds arrested the cells at G2/M checkpoint when the cell cycle is active while they induce apoptosis when the cell growth is restricted due to the absence of growth factors. These results suggest the two novel compounds may have anticancer activity through cell cycle arrest and pro apoptosis mechanism.

Full text of DOI:10.1016/j.bmcl.2013.04.038

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3976–3978
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anticancer activity of aminodihydroquinoline analogs:
Identification of novel proapoptotic agents q
⇑
Eun Lee , SeulAa Han , Guo Hua Jin, Hwa Jin Lee, Woo-Young Kim, Jae-Ha Ryu, Raok Jeon
Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Ku, Seoul 140-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 February 2013
Revised 6 April 2013
Accepted 16 April 2013
Available online 25 April 2013
A series of 2-aminodihydroquinoline analogs were synthesized and their in vitro cytotoxicities against
metastatic breast adenocarcinoma cell line MDA-MB-231 were tested. Five out of 16 compounds exhib-
ited promising activity and structure–activity relationship revealed major role of dialkylaminoethyl sub-
stituents on dihydroquinoline ring for the activity. Two compounds, 5f and 5h, presented cytotoxicity
with IC50 values of about 2 lM when the compounds were treated to the cells without serum. The cell
proliferation was inhibited mildly when the cells cultured with serum. Flow cytometry analyses showed
that those compounds arrested the cells at G2/M checkpoint when the cell cycle is active while they
induce apoptosis when the cell growth is restricted due to the absence of growth factors. These results
suggest the two novel compounds may have anticancer activity through cell cycle arrest and pro apop-
tosis mechanism.
Keywords:
Aminodihydroquinoline
Cytotoxicity
Anticancer activity
Ó 2013 Elsevier Ltd. All rights reserved.
Cancer has become a major leading cause of death worldwide.1
Most conventional anti-cancer drugs have substantial side effects
as a result of the lack of selectivity between cancer cells and nor-
mal cells.2 Although significant progress has been made in the
development of novel chemotherapeutic agents for the treatment
of cancer, there is still an urgent need to develop new therapeutic
agents.3
R = H, alkyl,
(CH2) OH
n
X
R
(CH2) NR'2
n
N
N
H
X = H, Cl
Figure 1. Representative structure of aminodihydroquinoline analogs.
All these reasons and our interest in the identification of novel
anticancer agents prompted us to investigate dihydroquinoline
analogs, which were initially designed as an iNOS inhibitor in our
group regarding the previous reports.4 However, these class of
compound only revealed cytotoxic activity without inhibitory
activity of NO production.
Quinoline and dihydroquinoline scaffold attracts many atten-
tions in drug discovery due to their variable range of biological
activities, especially anticancer, antibacterial and anti-inflamma-
tory activities.5 In this context, dihydroquinoline analogs have
been the subject of investigations as a new class of antitumor
agents (Fig. 1). A novel class of dihydroquinoline analogs was ob-
tained by introducing alkyl amino substituents at C2 position of
the hydroquinoline backbone and their in vitro cytotoxicities were
evaluated against breast cancer cell line MDA-MB-231.
aniline analogs gave compounds 2 which were followed Friedel–
Crafts alkylation to yield dihydroquinolinones 3. The compounds
were treated with Lawensson’s reagent to convert oxygen atom
at C2 position to sulfur. Addition of various alkylamino groups to
dihydroquinoline-2-thiones 4 and following elimination under
the condition of ammonium hydroxide and mercury(II) chloride
gave 2-alkylaminodihydroquinolines 5.
The prepared 2-alkylaminodihydroquinoline analogs 5 were
tested for their cytotoxicity against MDA-MB-231 cell lines. MTT
assay was carried out to determine the cytotoxic activities. The
inhibitory properties of cell proliferation or cell survival of the
compounds are shown in Table 1. When the cells were treated with
the drugs without serum, five of the tested compounds (5f, 5g, 5h,
5k and 5l) revealed cytotoxicity against MDA-MB-231 cells up to
or more than 50% at concentration of 5
five compounds were obtained. Two compounds 5f and 5h which
have the highest toxicity with 2 M of IC50 value were subjected
lM. The IC50 values of these
Synthetic route for aminodihydroquinoline analogs 5a–5n were
depicted in Scheme 1. Acyl substitution of the starting molecule
l
to study on the mechanism of cytotoxicity. When the cells grew
well, the compounds showed moderate effect on cytotoxicity,
while the cells rarely grew due to the absence of serum, the growth
inhibitory effect were more potent (Fig. 2). Both of those com-
pounds showed similar cytotoxicty.
q
This paper is dedicated to Professor Young-Ger Suh on the occasion of his 60th
birthday.
⇑
Corresponding author. Tel.: +82 2 710 9571; fax: +82 2 715 9571.
E-mail address: rjeon@sookmyung.ac.kr (R. Jeon).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.038

E. Lee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3976–3978
3977
R1
R1
R2
5
R2
6
a
b
c
X
X
X
O
7
NH2
N
H
O
N
H
1a-c
3a-d
2a-c
1a: X = p-Cl
1b: X = m-Cl
1c: X = H
2a: X = p-Cl, R1 = R2 = Me
2b: X = m-Cl, R1 = R2 = Me
2c: X = H, R1 = H, R2 = Ph
3a: X = 6-Cl, R1 = R2 = Me
3b: X = 5-Cl, R1 = R2 = Me
3c: X = 7-Cl, R1 = R2 = Me
3d: X = H, R1 = H, R2 = Ph
R1
R2
R1
R2
d
X
X
R3
N
H
S
N
N
H
5a-n
4a-d
4a: X = 6-Cl, R1 = R2 = Me
4b: X = 5-Cl, R1 = R2 = Me
4c: X = 7-Cl, R1 = R2 = Me
4d: X = H, R1 = H, R2 = Ph
5a: X = 6-Cl, R1 = R2 = Me, R3 = H
5b: X = 6-Cl, R1 = R2 = Me, R3 = methyl
5c: X = 6-Cl, R1 = R2 = Me, R3 = ethyl
5d: X = 6-Cl, R1 = R2 = Me, R3 = propyl
5e: X = 6-Cl, R1 = R2 = Me, R3 = hydroxybutyl
5f: X = 6-Cl, R1 = R2 = Me, R3 = dimethyl aminopropyl
5g: X = 6-Cl, R1 = R2 = Me, R3 = dimethyl aminoethyl
5h: X = 6-Cl, R1 = R2 = Me, R3 = diethyl aminopropyl
5i: X = 6-Cl, R1 = R2 = Me, R3 = benzyl
5j: X = 6-Cl, R1 = R2 = Me, R3 = morpholinyl
5k: X = 5-Cl, R1 = R2 = Me, R3 = dimethyl aminopropyl
5l: X = 7-Cl, R1 = R2 = Me, R3 = dimethyl aminopropyl
5m: X = 6-Cl, R1 = R2 = Me, R3 = hydroxypropyl
5n: X = H, R1 = H, R2 = Ph, R3 = H
Scheme 1. Synthesis of 2-aminodihydroquinoline analogs. Reagents and conditions: (a) alkylacryloral chloride, TEA, THF; (b) aluminum chloride, dichlorobenzene; (c)
Lawensson’s reagent, toluene; (d) R3NH2, HgCl2, NH4OH, THF.
Table 1
Cytotoxicity of the synthesized compounds against MDA-MB-231
Compound
% To controla
IC50 (lM)
1
lM
5 lM
3a
4a
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
82.29**
85.74**
80.48***
91.9
89.7
90.7
( 0.08)
( 0.05)
( 0.03)
( 0.12)
( 0.06)
( 0.05)
( 0.03)
( 0.06)
( 0.16)
( 0.03)
( 0.29)
( 0.21)
( 0.02)
( 0.03)
( 0.13)
( 0.16)
81.71**
94.3
( 0.04)
( 0.07)
( 0.02)
( 0.07)
( 0.07)
( 0.02)
( 0.02)
( 0.04)
( 0.10)
( 0.02)
( 0.09)
( 0.13)
( 0.02)
( 0.04)
( 0.16)
( 0.22)
80.95***
92.3
85.1
82.44*
84.85*
37.99***
50.83***
36.1***
68.12**
70.26**
43.94***
46.3***
84.2
87.6
54.06***
66.94***
53.32***
88.8
2.1
7.0
2.0
91.4
60.45***
68.43***
84.48*
86.6
4.8
4.0
5m
5n
86.3
a
The cells were treated with DMSO (control) or the compounds (1 lM and 5 lM) for 72 h without the serum supplement were
subjected to MTT assay. Relative amount of viable cells (% to control and standard errors) were shown.
*
p <0.05.
p <0.01.
**
***
p <0.001, versus control.
To investigate the mechanism of the cytotoxicity, we performed
flow cytometry experiments for the compounds 5f and 5h (Table 2).
In the presence of serum, the cells were in the active cell cycle and
were found more in G2/M stages and less in G1/G0 in the presence
of the compounds than those of control cells suggesting that the
compounds might arrest the cells at the G2/M checkpoints. When
the cells were not in the active cell cycle due to the absence of ser-
um, the apoptotic cells increased significantly by the treatment of
compounds 5f and 5h. Since majority of cells in solid tumors are
shortage of oxygen and growth factors due to the angiogenic prob-
lems, proapoptotic effect of the compounds 5f and 5h in the low
serum condition may suggest the potential use of this compounds
as anticancer agents.
This biological result clearly showed that substitution of dial-
kylaminoethylamino group at C2 position of dihydroquinoline
was essential for the activity. The replacement of the terminal

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E. Lee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3976–3978
tertiary amine (5f) with alcohol (5e, 5m or 5j) was generally dele-
terious to the activity. Also, three methylene units (5f) in the
dimethylamino alkyl substituent seemed to be better for activity
than two methylene units (5g). The para-chloro substituted com-
pound 5f revealed better activity than meta- (5j) or ortho-substi-
tuted compounds (5k).
In summary, a series of aminodihydroquinoline analogs were
synthesized as novel proapoptotic agents. The biological studies
demonstrated that dialkylaminoethyl substituted dihydroquino-
lines are effective inhibitors of cell survival in the growth factor de-
prived condition. Flow cytometry analysis also suggested that the
representative compounds 5f and 5h arrested the cells at the G2/
M stages. Preliminary studies on structure–activity relationship re-
vealed that dialkylaminoethyl group at C2 position of dihydroquin-
oline ring is the key structural element for the activity. Thus, we
suggest dialkylaminoethyl substituted dihydroquinolines as a no-
vel lead which can be further developed into potential chemother-
apeutic agents in cancer therapy.
Acknowledgments
This work was supported by the National Research Foundation
of Korea grant funded by the Korea government (Project No. 2011-
0030699) and the SRC Research Center for Women’s Diseases of
Sookmyung Women’s University (2011).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
04.038.
References and notes
Figure 2. Cytotoxicity of compounds 5f (A) and 5h (B) against MDA-MB-231 cells in
the presence (grey box) or absence (opened box) of serum. Cell viability was
assessed by the MTT assay8 after treating with different concentrations of the
compounds for 72 h. The data represent mean SD (n = 6) for one experiment
performed in triplicate. ⁄p <0.05, and ⁄⁄⁄p <0.001 compared to the control (DMSO
treated) value, respectively.
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McDonald, F. M. Bioorg. Med. Chem. Lett. 2003, 13, 1981.
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Table 2
Cell cycle deregulation and cell death analyses of MDA-MB-231a by aminodihydro-
quinoline analogsb
6. Breast cancer cell, MDA-MB-231 was cultured in RPMI media with the
supplement of fetal bovine serum.
With serum
Control
Without serum
5f
5h
Control
5f
5h
4.77**
86.24
4.48*
4.27*
7. The cell cycle and dying cells were analyzed as described previously (Tate, C. R.;
Rhodes, L. V.; Segar, H. C.; Driver, J. L.; Pounder, F. N.; Burow, M. E.; Collins-
Burow, B. M. Breast Cancer Res. 2012, 14, R79). Briefly, the cells with or without
the drug treatment were fixed with EtOH and the nuclear DNA were labeled by
propidium iodide. The cells with different DNA amount were analyzed by FACS
Calibur (BD Biosciences, San Jose, CA).
SubG1
G0/G1
S
1.99
67.16
14.49
15.82
1.53
1.64
2.15
84.87
6.19
7.63**
81.6*
5.61
64.06**
14.76
61.75**
15.44
G2/M
19.22**
20.48**
6.53
4.73
8. General procedure for MTT assay: A total of 5000 MDA-MB-231 cells were
plated at 96-well plates with serum containing medium. The compounds were
treated the day after seeding at the indicated concentrations with or without
serum. Three days later, the MTT solution was added and the developed
formazon was dissolved with DMSO. The absorbance at 595 nm was determined
using a Biokinetics plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).
Sixiplicate wells were assayed for each condition.
a
The MDA-MB 231 cells treated with DMSO (control), 5f (5 lM), and 5h (5 lM)
for 72 h were subjected to the cell cycle analyses using propidium iodide and flow
cytometry. See Ref. 6.
b
See Ref. 7 for protocol.
*
p <0.05.
**
p <0.01, versus control.