Development of 3-aryl-1-isoquinolinamines as potent antitumor agents based on CoMFA

Article abstract of DOI:10.1016/j.ejmech.2010.08.042

Various substituted 3-aryl-1-isoquinolinamines were designed and synthesized based on the previously constructed CoMFA model. Most of the synthesized compounds showed excellent potency in eight different human tumor cell lines as expected. In order to fin

Full text of DOI:10.1016/j.ejmech.2010.08.042

European Journal of Medicinal Chemistry 45 (2010) 5493e5497
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Development of 3-aryl-1-isoquinolinamines as potent antitumor
agents based on CoMFA
Su Hui Yang a, Hue Thi My Van a, Thanh Nguyen Le a, Daulat Bikram Khadka a, Suk Hee Cho a,
,
Kyung-Tae Lee b, Eung-Seok Lee c, Young Bok Lee d, Chang-Ho Ahn d, Won-Jea Cho a
*
a College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Buk-gu, Gwangju 500-757, Korea
b College of Pharmacy, Kyung-Hee University, Seoul 130-701, Korea
c College of Pharmacy, Yeungnam University, Kyongsan 712-749, Korea
d Rexahn Pharmaceuticals, Inc., Rockville, MD 20850, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Various substituted 3-aryl-1-isoquinolinamines were designed and synthesized based on the previously
constructed CoMFA model. Most of the synthesized compounds showed excellent potency in eight
different human tumor cell lines as expected. In order to find the exact cytotoxic mechanism of these 3-
aryl-1-isoquinolinamines, we analyzed the cell cycle dynamics by flow cytometry and found that 3-aryl-
1-isoquinolinamine 6k-treated HeLa cells were arrested in G2/M phase, which is related to apoptosis.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 10 June 2010
Received in revised form
3 August 2010
Accepted 17 August 2010
Available online 24 August 2010
Keywords:
CoMFA
Synthesis
Antitumor agents
Cell cycle arrest
Apoptosis
1. Introduction
on developing potent water-soluble 3-arylisoquinolines by
applying a previously constructed CoMFA model; our former
The major purpose of a QSAR study is the prediction of the
activity of new compounds on the basis of previously synthesized
compounds. Among the modern 3D QSAR techniques, CoMFA has
generally been used in drug design because it affords fast genera-
tion of a QSAR equation with a contour map for analysis of newly
synthesized molecules [1]. Moreover, CoMFA models have also
been used for virtual screening to find lead compounds, although it
has limitations regarding reasonable activity prediction of novel
compounds that contain different 3D geometries [2].
In our continuous effort to develop potent antitumor agents, we
investigated the synthesis, biological evaluation and QSAR study of
3-arylisoquinoline compounds such as indeno[1,2-c]isoquinolines
1 [3], isoindolo[2,1-b]isoquinolinones 2 [4], 12-oxobenzo[c]phe-
nanthridinones 3 and benz[b]oxepines 4 [5] as the rigid forms of
the 3-aryl rings via 3-arylisoquinolinone as a key intermediate
(Fig. 1). However, we did not develop any compounds that sur-
passed the activity of the original hit compound 5, which was found
during the natural alkaloid synthesis [6]. Our research then focused
CoMFA model provided significantly high cross-validated r2 (q2)
value of 0.715 as well as a high conventional r2 value of 0.927 [7]. To
date, most of the CoMFA studies on diverse molecules have
provided reasonable statistical values that could explain the bio-
logical activities, but application of CoMFA for finding novel
bioactive compound has been rare [2].
In this work, we synthesized various 3-aryl-1-isoquinolin-
amines 6 based on the validation of their antitumor activities
through the CoMFA model. Moreover, cell cycle dynamics analyses
with apoptotic induction studies were performed because the
mode of action of 3-aryl-1-isoquinolinamines remained unclear;
some molecules were suggested to be partial inhibitors of top-
oisomerase I.
The CoMFA QSAR model was constructed from 58 diverse
substituted 3-arylisoquinolines with varied cytotoxicities as shown
in Table 1 [7]. The steric and electrostatic contributions to the
equation were 40.1 and 59.9%, respectively, and the reasonable
F value (96.804) with low standard deviation value (0.251) sug-
gested its potential for application.
Among the synthesized compounds, 3-aryl-1-isoquinolinamine
6a exhibited the most potent cytotoxicities against human tumor
* Corresponding author. Tel.: þ82 62 530 2933; fax: þ82 62 530 2911.
E-mail address: wjcho@jnu.ac.kr (W.-J. Cho).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.042

5494
S.H. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 5493e5497
O
R2
R2
N
R
R2
O
O
12-Oxobenzo[c]phenanthridinone (3)
R1
N
R2
N
O
R
O
Benz[b]oxepines(4)
Isoindolo[2,1-b]isoquinolines (2)
MOMO
O
O
R2
R2
MeO
MeO
R1
NH
R2
R1
N
NMe
O
R3
3-Arylisoquinoline (6)
O
R1
3-Arylisoquinolinone
NMe
5
O
Indeno[1,2-c]isoquinolines (1)
Me
OMOM
R1
R2
NEt2
CN
O
Fig. 1. Structure of constrained structures of 3-arylisoquinolines; indeno[1,2-b]isoquinoline 1, isoindolo[2,1-b]isoquinoline 2, 12-oxobenzo[c]phenanthridine 3 and benz[b]oxepine
4. Structural modification of 3-arylisoquinolinone to 3-arylisoquinolines 6.
cell lines. Therefore, we designed synthetically possible derivatives,
and their cytotoxic activities were virtually calculated using our
previously constructed CoMFA model as shown in Table 2. As
a result, most of the designed compounds exhibited sub-micro-
molar activities except dimethylamino-substituted compounds
6nep on the C-5 (R1) position.
position to afford the desired 3-aryl-1-isoquinolinamines 6 in
moderate yield as shown in Scheme 1. The free amines were then
treated with concentrated HCl in acetone to give the HCl salt forms
of the amines 6 [9].
3. Results and discussion
3.1. Chemistry
2. Chemistry
3.1.1. General procedure for synthesis of 6aem
In order to synthesize 3-aryl-1-isoquinoliamines 6, the previ-
ously reported lithiated toluamideebenzonitrile cycloaddition
method was used [8]. N-diethyl-o-toluamides 7 were reacted with
n-BuLi to give the anions, which were then treated with benzoni-
trile 8 to provide the 3-arylisoquinolines 9 in moderate yield.
Treatment of 9 with POCl3 afforded the corresponding imine
chloride 10, which was then treated with p-methoxybenzylamine
in the presence of K2CO3 in DMF to give the PMB-protected iso-
quinolinamine 11 in good yield. In our previous study, benzylamine
at the C-1 position was introduced and catalytic hydrogenation
reaction in the presence of PdeC was used to obtain the amine in
moderate yield. However, the above reaction condition could not be
applied to several substituted analogs. Finally, we found that tri-
fluoroacetic acid was useful to remove the PMB group on the C-1
A solution of p-methoxybenzylamine compound 11 in CH2Cl2 in
the presence of trifluoroacetic acid was stirred at room temperature
for overnight. To this solution sat. aq. NaHCO3 was added and
reaction mixture was extracted with CH2Cl2. The combined organic
extracts were washed with water, dried, and concentrated to give
the residue which was then purified by column chromatography on
silica gel to afford 3-aryl-1-isoquinolinamines 6 (n-hexane: ethyl
acetate ¼ 1:1 was used). To a solution of 3-aryl-1-isoquinolinamine
6 in acetone was added concentrated HCl to afford the precipitate
which was collected and washed with acetone to give the desired
hydrochloride salt.
3.1.2. 6-Methyl-3-o-tolylisoquinolin-1-amine hydrochloride salt
(6a)
1H NMR (DMSO-d6)
d: 13.84 (s, 1H), 9.40 (s, 1H), 8.79e7.57 (m,
7H), 7.38 (s, 1H), 2.75 (s, 3H), 2.61 (s, 3H). mp: 253e258 ꢀC (dec.).
LCMS: m/z (%) 248 (Mþ, 100).
Table 1
CoMFA correlation statistics for 3-arylisoquinolines.
Type
ma Nb Sc
F
r2
Cross-Validd RCe
SEP
q2 Stericf Elecg
58 0.251 96.804 0.924 0.485 0.715 40.1 59.9
3.1.3. 3-(3,4-Dimethoxyphenyl)-6-methylisoquinolin-1-amine (6b)
1H NMR (300 MHz, CDCl3)
d: 7.67e7.64 (m, 2H), 7.54 (m, 1H),
Conformer 1
Defined Region
6
7.45 (s, 1H), 7.27 (s, 1H), 7.18 (m, 1H), 6.91 (d, J ¼ 8.5 Hz, 1H), 5.76 (s,
2H), 3.96 (s, 3H), 3.89 (s, 3H), 2.50 (s, 3H). mp: 270e273 ꢀC (dec.).
LCMS: m/z (%) 294 (Mþ, 100).
a
Number of components.
b
c
d
e
f
Number of compounds.
Standard deviation.
Obtained from the leave-one-out cross-validation.
Relative contribution.
Steric field descriptors.
3.1.4. 3-(Benzo[d][1,3]dioxol-6-yl)-6-methylisoquinolin-1-amine
(6c)
1H NMR (300 MHz, CDCl3)
d
: 7.63 (d, J ¼ 8.5 Hz, 1H), 7.54 (m,
g
Electrostatic field descriptors.
2H), 7.45 (s, 1H), 7.22 (m, 2H), 6.88 (d, J ¼ 8.5 Hz, 1H), 5.98

S.H. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 5493e5497
5495
Table 2
Observed and calculated cytotoxicity (-log IC50 value) values against A-549 of the 3-aryl-1-isoquinolinamines based on the CoMFA model.
No.
Compound
R1
R2
R3
R4
Yield(%)
Obsda
Calcdb
Diffc
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
H
H
H
H
H
H
H
H
H
H
H
H
eCH3
eCH3
eCH3
eCH3
eCH3
eCH3
eCH3
H
H
H
H
H
H
H
H
2-methyl
67
79
71
67
68
89
75
92
91
71
60
78
89
73
60
40
6.79
6.25
6.85
6.77
6.42
6.97
6.74
6.35
7.27
6.89
6.12
6.55
6.36
<5.0
<5.0
<5.0
6.58
6.27
6.37
6.87
6.57
6.34
6.72
6.98
7.10
6.89
6.19
6.01
6.12
5.01
4.86
5.24
0.21
0.02
0.48
0.10
0.33
0.63
0.02
0.63
0.17
0
3,4-dimethoxy
3,4-methylenedioxide
4-methoxy-2-methyl
2,6-dimethyl
3-methoxy
3-methyl
3-methoxy
2-methyl
H
eCH3
eCH3
eOCH3
eOCH3
eOCH3
eOCH3
H
9
H
10
11
12
13
14
15
16
eOCH3
eOCH3
eOCH3
eOCH3
H
3-methoxy
3,5-dimethoxy
2-methyl
0.07
0.54
0.24
e
H
NMe2
NMe2
NMe2
H
H
H
H
H
2-methyl
3,4-dimethoxy
e
e
a
observed.
calculated.
differences.
b
c
(s, 2H), 5.40 (s, 2H), 2.46 (s, 3H). mp: 152e161 ꢀC. LCMS: m/z (%)
3.1.9. 3-(3-Methoxyphenyl)-7-methylisoquinolin-1-amine (6h)
1H NMR (300 MHz, CDCl3)
: 7.74 (s, 1H), 7.61 (d, 1H), 7.54e7.45
278 (Mþ, 100).
d
(m, 3H), 7.37 (t, 1H), 7.29 (s, 1H), 6.93 (m, 1H), 3.89 (s, 3H), 2.49
3.1.5. 3-(4-Methoxy-2-methylphenyl)-6-methylisoquinolin-1-
(s, 3H). mp: 95e113 ꢀC. LCMS: m/z (%) 264 (Mþ, 100).
amine (6d)
1H NMR (300 MHz, CDCl3)
7.37e7.28 (m, 2H), 7.15e6.91 (m, 2H), 6.85 (s, 1H), 6.76 (m, 2H), 3.78
(s, 3H), 2.47 (s, 3H), 2.37 (s, 3H). mp: 152e166 ꢀC. LCMS: m/z (%) 278
(Mþ, 100).
d
: 7.97 (d, J ¼ 8.5 Hz, 1H), 7.42 (s, 1H),
3.1.10. 7-Methyl-3-o-tolylisoquinolin-1-amine (6i)
1H NMR (300 MHz, CDCl3)
d: 7.63e7.58 (m, 2H), 7.47e7.40 (m,
2H), 7.27e7.20 (m, 3H), 7.05 (s, 1H), 5.41 (s, 2H), 2.51 (s, 3H) 2.38
(s, 3H). mp: 104e107 ꢀC. LCMS: m/z (%) 248 (Mþ, 100).
3.1.6. 6-Methyl-3-(2,6-dimethylphenyl)isoquinolin-1-amine (6e)
3.1.11. 6,7-Dimethoxy-3-phenylisoquinolin-1-amine (6j)
1H NMR (300 MHz, CDCl3)
d
: 7.72 (d, 1H), 7.48 (s, 1H), 7.32
1H NMR (300 MHz, CDCl3)
d: 7.97e7.95 (m, 2H), 7.45e7.30 (m,
(d, 1H), 7.20e7.08 (m, 3H), 6.89 (s, 1H), 5.26 (s, 2H), 2.52 (s, 3H), 2.12
4H), 7.05 (s, 1H), 6.94 (s, 1H), 5.29 (s, 2Hþ), 3.94 (s, 3H), 3.82 (s, 3H).
(s, 6H). mp: 198e205 ꢀC. LCMS: m/z (%) 262 (Mþ, 100).
mp: 128e141 ꢀC. LCMS: m/z (%) 280 (M , 100).
3.1.7. 6-Methyl-3-m-tolylisoquinolin-1-amine (6f)
3.1.12. 6,7-Dimethoxy-3-(3-methoxyphenyl)isoquinolin-1-amine
1H NMR (300 MHz, CDCl3)
d
: 9.36 (s, 2H), 8.44 (d, J ¼ 8.4 Hz, 1H),
(6k)
7.83e7.79 (m, 2H), 7.55 (s, 1H), 7.46e7.39 (m, 2H), 7.28 (d, J ¼ 8.1 Hz,
1H), 7.14 (s, 1H), 2.57 (s, 3H), 2.46 (s, 3H). mp: 263e271 ꢀC (dec.).
LCMS: m/z (%) 248 (Mþ, 100).
1H NMR (300 MHz, CDCl3)
d: 7.53e7.46 (m, 2H), 7.30 (m, 1H),
7.22 (s, 1H), 7.18 (s, 1H), 6.90 (s, 1H), 6.88 (m, 1H), 3.92 (s, 3H), 3.89
(s, 3H), 3.82 (s, 3H). mp: 254e259 ꢀC (dec.). LCMS: m/z (%) 310
(Mþ, 100).
3.1.8. 6-Methyl-3-p-tolylisoquinolin-1-amine (6g)
1H NMR (300 MHz, CDCl3)
J ¼ 8.4 Hz, 1H), 7.51 (s, 1H), 7.35 (s, 1H), 7.29e7.24 (m, 3H), 5.43
(s, 2H), 2.50 (s, 3H), 2.40 (s, 3H). mp: 259e262 ꢀC (dec.). LCMS: m/z
(%) 248 (Mþ, 100).
d
: 7.94 (d, J ¼ 8.1 Hz, 2H), 7.70 (d,
3.1.13. 3-(3,5-Dimethoxyphenyl)-6,7-dimethoxyisoquinolin-1-
ylamine (6l)
1H NMR (300 MHz, CDCl3)
d: 7.38 (s, 1H), 7.24 (s, 1H), 7.21 (s, 1H),
7.06 (s, 1H), 7.04 (s, 1H), 6.49 (s, 1H), 5.10 (s, 2H), 4.00 (s, 3H), 3.98
R1
R1
R2
R3
Me
NEt2
R4
R2
R3
POCl3
n-BuLi
R4
+
THF
NH
NC
O
O
7
8
9
R1
R1
R1
R4
R4
R4
PMBNH2
K2CO3
R2
R3
R2
R3
R2
R3
1) TFA/ CH2Cl2
N
N
2) c-HCl, acetone
N
DMF
Cl
.
NH2
NHPMB
HCl
10
6
11
Scheme 1. The synthesis of 3-aryl-1-isoquinolinamines 6aep. (a) R1 ¼ H, R2 ¼ Me, R3 ¼ H, R4 ¼ 2-Me; (b) R1 ¼ H, R2 ¼ Me, R3 ¼ H, R4 ¼ 3,4-dimethoxy; (c) R1 ¼ H, R2 ¼ Me, R3 ¼ H,
R4 ¼ 3,4-methylenedioxide; (d) R1 ¼ H, R2 ¼ Me, R3 ¼ H, R4 ¼ 4-methoxy-2-methyl; (e) R1 ¼ H, R2 ¼ Me, R3 ¼ H, R4 ¼ 2,6-dimethyl; (f) R1 ¼ H, R2 ¼ Me, R3 ¼ H, R4 ¼ 3-OMe; (g)
R1 ¼ H, R2 ¼ Me, R3 ¼ H, R4 ¼ 3-Me; (h) R1 ¼ H, R2 ¼ H, R3 ¼ Me, R4 ¼ 3-MeO; (i) R1 ¼ H, R2 ¼ H, R3 ¼ Me, R4 ¼ 2-Me; (j) R1 ¼ H, R2 ¼ OMe, R3 ¼ OMe, R4 ¼ H; (k) R1 ¼ H, R2 ¼ OMe,
R3 ¼ OMe, R4 ¼ 3-OMe; (l) R1 ¼ H, R2 ¼ OMe, R3 ¼ OMe, R4 ¼ 3,5-dimethoxy; (m) R1 ¼ H, R2 ¼ OMe, R3 ¼ OMe, R4 ¼ 2-Me; (n) R1 ¼ NMe2, R2 ¼ H, R3 ¼ H, R4 ¼ H; (o) R1 ¼ NMe2,
R2 ¼ H, R3 ¼ H, R4 ¼ 2-Me; (p) R1 ¼ NMe2, R2 ¼ H, R3 ¼ H, R4 ¼ 3,4-dimethoxy.

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S.H. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 5493e5497
Table 3
Inhibition of cell growth (IC50
,
m
M) by 3-aryl-1-isoquinolinamine compounds 6aep against human cancer cell lines.
No. Compound MDA-MB-231(breast) PANC-1(pancreas) HCT116(colon) PC3(prostate) OVCAR-3(ovary) SK-MEL-28(melanoma) Caki-1(kidney) SNB-19(glioblastoma)
1
2
3
4
5
6
7
8
9
6aa
6b
6c
6d
6e
6f
6g
6h
6i
0.15
0.66
0.13
0.15
0.14
0.57
0.15
0.17
0.075
0.44
0.14
0.40
0.54
0.24
1.75
0.22
0.24
0.21
1.28
0.20
0.47
0.20
0.67
0.19
0.70
0.83
0.19
0.85
0.16
0.17
0.17
0.59
0.18
0.21
0.12
0.52
0.16
0.77
0.78
0.24
2.69
0.19
0.25
0.24
1.19
0.22
0.42
0.26
0.58
0.18
0.72
1.25
0.15
0.57
0.16
0.17
0.16
0.47
0.15
0.24
0.10
0.27
0.074
0.19
0.38
0.35
1.41
0.22
0.29
0.23
0.72
0.25
0.41
0.18
0.75
0.23
0.69
0.91
0.12
0.51
0.11
0.12
0.12
0.69
0.16
0.14
0.14
0.59
0.17
0.54
0.67
0.26
2.15
0.23
0.28
0.24
0.75
0.21
0.48
0.16
0.71
0.22
0.40
0.56
10 6j
11 6k
12 6l
13 6m
14 6n
15 6o
16 6p
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
a
This compound was reported and used as the reference molecule for cytotoxicity comparison with other compounds.
(s, 3H), 3.86 (s, 6H). mp: 246e254 ꢀC (dec.). LCMS: m/z (%) 340
3.2. Biological evaluation
(Mþ, 100).
The in vitro cytotoxicity experiments were carried out with the
synthesized 3-aryl-1-isoquinolinamines 6 against several tumor
cell lines including Human MDA-MB-231 (breast), PC3 (prostate),
HCT116 (colon), OVCAR-3 (ovary), Caki-1 (kidney), PANC-1
(pancreas), SNB-19 (glioblastoma) and SK-MEL-28 (melanoma)
cells from the American Type Culture Collection (ATCC) (Manassas,
VA). A-549 (lung) cells for CoMFA study and HeLa (cervical) cell
lines for flow cytometry were used. The growth inhibition assays of
the 3-aryl-1-isoquinolinamine analogs against eight different
human tumor cell lines were carried out using the Sulforhodamine
B method [10].
3.1.14. 6,7-Dimethoxy-3-o-tolylisoquinolin-1-ylamine (6m)
1H NMR (300 MHz, CDCl3)
d: 7.45e7.03 (m, 7H), 5.01 (s, 2H), 4.02
(s, 3H), 4.02 (s, 3H), 2.40 (s, 3H). mp: 259e268 ꢀC (dec.). LCMS: m/z
(%) 294 (Mþ, 100).
3.1.15. N5,N5-Dimethyl-3-phenylisoquinolin-1,5-diamine (6n)
1H NMR (300 MHz, CDCl3)
d: 8.08 (m, 2H), 7.82 (s, 1H), 7.48e7.33
(m, 5H), 7.18 (m, 2H), 5.24 (s, 2H), 2.89 (s, 6H). mp: 235e250 ꢀC
(dec.). LCMS: m/z (%) 263 (Mþ, 95).
3.1.16. N5,N5-Dimethyl-3-o-tolylisoquinolin-1,5-diamine (6o)
As expected, most of the 3-aryl-1-isoquinolinamine derivatives
exhibited potent cytotoxicities, as shown in Table 3. The IC50 values
1H NMR (300 MHz, DMSO-d6)
d: 13.87 (s, 1H), 8.42 (d, 1H),
7.82e7.71 (m, 2H), 7.50e7.33 (m, 5H), 2.91 (s, 6H), 2.42 (s, 3H). mp:
of the compounds were less than 1 mM or even 0.1 mM against seven
212e219 ꢀC (dec.). LCMS: m/z (%) 277 (Mþ, 100).
tumor cell lines, suggesting that the potency of these compounds
was not greatly dependent on the substitution pattern of the
modified phenyl ring. Among these compounds, 6i showed equal or
better growth inhibitory activity against MDA-MB-231 breast
cancer cells compared to 6a. When we prepared 5-dimethylamino-
substituted 3-aryl-1-isoquinolinamines 6nep, no compounds
exceeded the activity of 6a against several tumor cell lines and this
3.1.17. 3-(3,4-Dimethoxyphenyl)-N5,N5-dimethylisoquinolin-1,5-
diamine (6p)
1H NMR (300 MHz, DMSO-d6)
d: 9.69 (s, 2H), 8.40 (d, 1H),
7.93e7.91 (m, 2H), 7.75e7.67 (m, 3H), 7.13 (d, 1H), 3.94 (s, 3H), 3.82
(s, 3H), 3.03 (s, 6H). 242e248 ꢀC (dec.). LCMS: m/z (%) 323 (Mþ,100).
Fig. 2. Effects of compound 6k on cell cycle progression in HeLa cells. Cells were treated with or without compound 6k (50e500 nM for 24 h), washed and then collected. The cells
were stained with propidium iodide and then DNA contents were analyzed by flow cytometry.

S.H. Yang et al. / European Journal of Medicinal Chemistry 45 (2010) 5493e5497
5497
Fig. 3. Effects of compound 6k on apoptotic induction in HeLa cells. Cells were treated with or without compound 6k (50e500 nM for 24 h) and then co-stained with PI and FITC-
conjugated annexin V. The externalization of phosphatidylserine (PS) was detected by flow cytometry after 6k treatment.
result was a good match with the CoMFA model prediction as
shown in Table 2. The average activity difference between the
observed values with calculated ones was only 0.26 and it means
our constructed CoMFA model is quite predictive.
determine their mode of action. Although some 3-arylisoquinoline
derivatives showed moderate topoisomerase I inhibitory activities,
no confirmed cytotoxic mechanism was clarified. In order to find
the exact cytotoxic mechanism of these 3-aryl-1-isoquinolin-
amines, we analyzed the cell cycle dynamics by flow cytometry and
found that 3-aryl-1-isoquinolinamine 6k-treated HeLa cells were
arrested in G2/M phase, which is related to apoptosis.
Finding water-soluble potent cytotoxic compounds will provide
valuable information to researchers developing antitumor chemo-
therapeutic agents.
To assess whether compound 6k-induced cytotoxicity of cells
was mediated via alterations in the cell cycle [11], the effects of 6k
on cell cycle progression were evaluated. As shown in Fig. 2,
compound 6k-induced a significant dose-dependent increase in the
cell population in the G2/M phase of the cell cycle. The cell cycle
progression of G2/M phase was 35.80, 48.59, 73.06, 83.14 and
86.98% with 50, 100, 200, 300 and 500 nM compound 6k, respec-
tively. This increase in the G2/M phase cell population was
accompanied by a concomitant decrease in the G1-phase cell
population.
Acknowledgement
This study was financially supported by Special Research
Apoptosis is an essential physiological process for the normal
development and maintenance of tissue homeostasis. Most anti-
cancer agents eradicate cancer cells by inducing apoptosis. To see
whether the cytotoxic effect of compound 6k derived from the
induction of apoptosis, HeLa cells were treated with 6k
(50e500 nM for 24 h), and then the externalization of phosphati-
dylserine (PS) was analyzed using Annexin V and PI double staining
[12]. As shown in Fig. 3, cell numbers increased up to 70.12% in the
lower-right quadrant, representing the increased early apoptotic
cells (Annexin V-positive). The other 12.52% of cells belonged to the
late apoptotic stage. This finding indicates that compound 6k-
induced cell death in HeLa cells is apoptotic rather than necrotic
cell death.
Program of Chonnam National University, 2009.
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