Design, synthesis and systematic evaluation of cytotoxic 3-heteroarylisoquinolinamines as topoisomerases inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.05.047

A series of 3-heteroarylisoquinolinamines were designed, synthesized and evaluated for cytotoxicity, topoisomerases (topos) inhibitory activities and cell cycle inhibition. Several of the 3-heteroarylisoquinolines exhibited selective cytotoxicity against human ductal breast epithelial tumor (T47D) cells over non-cancerous human breast epithelial (MCF-10A) and human prostate cancer (DU145) cells. Most of the derivatives showed greater cytotoxicity in human colorectal adenocarcinoma (HCT-15) cells than camptothecin (CPT), etoposide and doxorubicin (DOX). Generally, 3-heteroarylisoquinolinamines displayed greater affinity for topo I than topo II. 3-Heteroarylisoquinolinamines with greater topo I inhibitory effect exhibited potent cytotoxicity. Piperazine-substituted derivative, 5b, with potent topo I and moderate topo II activities intercalated between DNA bases and interacted with topos through H-bonds at the DNA cleavage site of a docking model. Moreover, flow cytometry indicated that cytotoxic 3-heteroarylisoquinolinamines led to accumulation of human cervical (HeLa) cancer cells in the different phases of the cell cycle before apoptosis. Taken together, 3-heteroarylisoquinolinamines possessed potent cytotoxicity with topos and cell cycle inhibitory activities.

Full text of DOI:10.1016/j.ejmech.2014.05.047

European Journal of Medicinal Chemistry 82 (2014) 181e194
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and systematic evaluation of cytotoxic
3-heteroarylisoquinolinamines as topoisomerases inhibitors
,
1
,
,
Hue Thi My Van a 2, Hyunjung Woo b 2, Hyung Min Jeong a, Daulat Bikram Khadka a,
Su Hui Yang a, Chao Zhao a, Yifeng Jin a, Eung-Seok Lee c, Kwang Youl Lee a,
Youngjoo Kwon b **, Won-Jea Cho a
,
, *
a College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757, Republic of Korea
b College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top 5 Program, Ewha Womans University, Seoul 120-750,
Republic of Korea
c College of Pharmacy, Yeungnam University, Kyongsan 712-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-heteroarylisoquinolinamines were designed, synthesized and evaluated for cytotoxicity,
topoisomerases (topos) inhibitory activities and cell cycle inhibition. Several of the 3-
heteroarylisoquinolines exhibited selective cytotoxicity against human ductal breast epithelial tumor
(T47D) cells over non-cancerous human breast epithelial (MCF-10A) and human prostate cancer (DU145)
cells. Most of the derivatives showed greater cytotoxicity in human colorectal adenocarcinoma (HCT-15)
cellsthan camptothecin (CPT), etoposide and doxorubicin (DOX). Generally, 3-heteroarylisoquinolinamines
displayed greater affinity for topo I than topo II. 3-Heteroarylisoquinolinamines with greater topo I
inhibitory effect exhibited potent cytotoxicity. Piperazine-substituted derivative, 5b, with potent topo I and
moderate topo II activities intercalated between DNA bases and interacted with topos through H-bonds at
the DNA cleavage site of a docking model. Moreover, flow cytometry indicated that cytotoxic 3-
heteroarylisoquinolinamines led to accumulation of human cervical (HeLa) cancer cells in the different
phases of the cell cycle before apoptosis. Taken together, 3-heteroarylisoquinolinamines possessed potent
cytotoxicity with topos and cell cycle inhibitory activities.
Received 24 October 2013
Received in revised form
13 May 2014
Accepted 20 May 2014
Available online 21 May 2014
Keywords:
3-Heteroarylisoquinolinamine
Selective cytotoxicity
Topoisomerase I
Topoisomerase II
Molecular docking
Cell cycle arrest
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
The enzymatic function of topos involves DNA strand(s) cleav-
age, DNA swivel/strands passage and religation of broken strand(s).
DNA topological consequences such as supercoils, knots and
catenation arise during cellular processes like transcription, repli-
cation, recombination, and chromosome condensation and segre-
gation in fast-growing and dividing cells [1e3]. These problems
need to be addressed for the vitality of cells. In fact, nature has
reserved a specialized class of enzymes called topoisomerases
(topos) to handle DNA supercoiling and entanglements. The human
DNA scission and resealing take place through reversible trans-
esterifications between the tyrosyl unit of topos and the DNA
phosphate group. During this activity, a short-lived covalent topo-
DNA complex with scissile end(s) is formed and is termed ‘topo-
isomerase cleavage complex’ (topocc). The topocc can be trapped
by chemical entities to inhibit the DNA resealing activity of topos.
Stabilization of topocc in turn causes permanent DNA damage and
triggers apoptotic cell death. Thus, these topo inhibitors convert
functional topo into a fatal component of the cell. Apart from this,
topo inhibitors can also interfere with functions of topos at other
steps [5].
genome encodes six topos [4,5]. Among them, topo I and topo II (a,
b
) are established as important targets for cancer treatment.
Topos are highly expressed in tumors. Increased levels of topos
have been reported to be found in colorectal, prostate and breast
cancers, and leukemia [6e8]. The difference in expression of topos
between normal and tumor tissues makes topos inhibitors reliable
chemotherapeutic agents. Topos inhibitors hinder vital activities
necessary for rapidly growing and dividing cells. In addition, topos
* Corresponding author.
** Corresponding author.
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), wjcho@chonnam.ac.kr
(W.-J. Cho).
1
Present address: Organic Chemistry Department, Hanoi University of Pharmacy,
13-15 Le Thanh Tong, HoanKiem, Hanoi, Viet Nam.
2
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.05.047
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

182
H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
inhibitors that specifically freeze topocc cause DNA damage and
have deleterious effects in cancer cells versus normal cells. There-
fore, intensive work has been done in developing antitumor agents
that target topos. Among the large number of chemical classes
evaluated as topos inhibitors, 3-arylisoquinolinamine-based com-
pounds are the most promising (Fig. 1).
6-Amino-substituted benzo[c]phenathridines (1) represent the
constrained form of 3-arylisoquinolinamines. Benzo[c]phenan-
thridin-6-ylamines were basically developed as water-soluble de-
rivatives of the natural alkaloids, fagaronine and nitidine [9]. Unlike
the parent compounds, they lack the iminium charge responsible
for acute toxicity and low antitumor activity in few cases. Amino-
substituted nitidine (1) displays potent cytotoxicity and inhibitory
effects towards both topo I and II. Likewise, as part of our work to
develop water-soluble non-camptothecin (CPT) topo I inhibitors
with 3-arylisoquinolinamine scaffold, 5-amino-substituted indeno
[1,2-c]isoquinolines (2) were principally designed to preserve the
essential tetracyclic planar scaffold and C11 keto group necessary
for topo I inhibition and to incorporate amino alkyl tail or hetero-
cyclic amines to improve their aqueous solubility [10,11].
Fig. 2. 4-Amino-2-phenylquinazoline (4) and 3-heteroarylisoquinolinamine (5).
corresponding azidoisoquinoline 5g. Primary isoquinolinamine 5f
was obtained on heating isoquinolinyl chloride 9a with ammonia in
presence of NaHCO3 and DMF (Scheme 2). Alternatively, 5f was also
obtained by coupling of lithiated tolunitrile 7c and thiophenenitrile
7a assisted by hexamethylphosphoramide (HMPA).
In addition to the rigidified isoquinolinamines, flexible 3-
arylisoquinolinamines (3) have been developed by our research
groups as potent cytotoxic agents with topo I inhibitory activity and
the appropriate physiochemical properties and pharmacokinetic
profile necessary for oral drug use [12e15]. Apart from this, we
have also developed 4-amino-2-phenylquinazoline (4) with an
additional N in the isoquinoline ring (Fig. 2) [16]. Replacement of
the isoquinoline ring by quinazoline is advantageous as the tricyclic
compound attains planar conformation to intercalate between DNA
bases. Considering this fact, it can be expected that 3-pyrrole- and
3-thiophene-substituted isoquinolinamines (5) may attain a flat
structure and be potential topos inhibitors. Based on these ratio-
nales, we herein describe the synthesis and systematic evaluation
3. Results and discussion
3.1. Cytotoxicity
Cytotoxicity of 3-heteroarylisoquinolinamines (5) was assessed
using non-cancerous (human breast epithelial, MCF-10A) and tu-
mor (human ductal breast epithelial tumor, T47D; human prostate
cancer, DU145; and human colorectal adenocarcinoma, HCT-15)
cells [17]. Cytotoxicity results are summarized as IC50 values in
Table 2, and structureecytotoxicity relationships of heterocyclic
amine-substituted isoquinolines are summarized in Table 3.
Piperidine (5a, 5h and 5n), morpholine (5e, 5l and 5r), eNHBn (5m)
and eNH2 (5f) substituted 3-heteroarylisoquinolinamines and their
corresponding HCl salts exhibited selective cytotoxicity towards
tumorigenic T47D cells and non-toxic effects in normal MCF-10A
cells. In contrast, doxorubicin (DOX), a chemotherapeutic agent
approved as adjuvant therapy for breast cancer, was cytotoxic to
both malignant and non-malignant cell populations. Beside MCF-
10A, the aforementioned compounds had no effect on the
viability of prostate cancer DU145 cells. Among the set of 3-
heteroarylisoquinolinamines inactive towards MCF-10A and
DU145, piperidine substituted analogs (5a, 5h, 5n and their HCl
of 3-heteroarylisoquinolinamines as
inhibitors.
a novel class of topos
2. Chemistry
3-Heteroarylisoquinolinamines 5 were mainly synthesized ac-
cording to the synthetic pathway reported earlier [13]. Synthesis
was initiated by cycloaddition of lithiated toluamides 6 and het-
eroaryl nitriles 7 to obtain 3-heteroarylisoquinolones 8 (Scheme 1).
In the next step, isoquinolones 8 were aromatized into iso-
quinolinyl chlorides 9 by phosphorus oxychloride (POCl3). Finally,
1-amination of imine chlorides 9 with heterocyclic (piperidine,
piperazines, morpholine and homopiperazine) and aliphatic
(benzyl) amines produced the desired isoquinolinamines 5aee and
5het (Table 1 and Fig. 3). Piperidinyl, piperazinyl and morpholinyl
derivatives were further treated with concentrated HCl to afford
HCl salts. Treatment of 9b with sodium azide afforded the
salts) showed most potent activity towards T47D (IC50
2.56 0.12e8.03 0.35 M).
:
m
In contrast, piperazine- and homopiperazine-substituted ana-
logs lacked remarkable selectivity for MCF-10A, T47D and DU145
cells. N40-unsubstituted piperazinyl isoquinolines (5b, 5i and 5o)
and their corresponding salts were more toxic to MCF-10A, T47D
and DU145 cells than N40-alkylated piperazines. Among the
piperazine analogs, compounds 5p$HCl and 5q$HCl are worth
mentioning as they were less toxic towards MCF-10A and DU145
(IC50
heteroarylisoquinolinamines against T47D (IC50
2.84 0.1 M, respectively). Similarly, homopiperazine derivative
>
20
mM) and represent the most potent 3-
:
1.02 0.04,
m
5s was more toxic towards T47D (IC50: 1.32 0.02 mM) than MCF-
10A and DU145.
3-Heteroarylisoquinolinamines showed potent cytotoxic effects
against human colorectal adenocarcinoma (HCT-15) cells. Piperi-
dine (5a, 5h and 5n), N40-unsubstituted piperazine (5b, 5i and 5o),
N40-methyl piperazine (5c, 5j and 5p), N40-ethyl piperazine (5k and
5q), homopiperazine (5s) and eNH2 (5f) substituted 3-
heteroarylisoquinolinamines or their HCl salts exhibited greater
cytotoxicity than CPT, etoposide and DOX (Table 3). The low activity
of the positive control drugs against HCT-15 cells is due to multiple
Fig. 1. 3-Arylisoquinolinamine-based topos inhibitors: 6-amino-substituted benzo[c]
phenanthridine (1), 5-amino-substituted indeno[1,2-c]isoquinolines (2) and 3-
arylisoquinolinamines (3).

H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
183
Scheme 1. Synthesis of 3-heteroarylisoquinolinamines 5. Reagents and conditions: (i) n-BuLi, dry THF, ꢀ78 ꢁC; (ii) POCl3, 50 ꢁC; (iii) Amine, K2CO3, DMF, 110 ꢁC; (iv) NaN3, DMF,
100 ꢁC; (v) c-HCl, acetone.
drug resistance. The drug efflux membrane transporter, multidrug
resistance protein 1 (MDR1; also known as p-glycoprotein, P-gp or
as ATP-binding cassette sub-family B member 1, ABCB1), which is
overexpressed in HCT-15 cells, reduces intracellular accumulation
of CPT, etoposide and DOX by actively extruding them from the cell
[4,18e20]. In addition, the fraction of the drug (DOX) absorbed into
the cell is further metabolized by cytochrome P450 3A (CYP3A)
[19]. Despite these hurdles, the potent cytotoxicity profile of the
above-mentioned 3-heteroarylisoquinolinamines suggests that
they are unlikely to be substrates of MDR1 and CYP3A. N40-Ethyl
piperazine (5d), morpholine (5l and 5r) and benzyl (5m) de-
rivatives and their HCl salts were less toxic than DOX but showed
greater or similar activity to CPT and etoposide on HCT-15. Com-
pound 5e showed no significant cytotoxicity (IC50 ¼ 21.26
Table 1
Synthesis of 3-thiophenylisoquinolinamines 5a-s.
R1, R2, R3
R
-NHBn -N3 -NH2
N
N
N
N
N
O
N
N
Me
N
H
N
Et
N
Me
0.43 mM). The azide-substituted derivative 5g is the only compound
that had no effects in all of the cells used.
H, H, H
Me, H, H
H, Me, H 5h
H, H, Me 5n
5a
e
5b
5c
5d
5e
e
e
e
5s
e
e
5f
e
e
e
e
e
e
e
e
5g
e
5i
5o
5j
5p
5k
5q
5l
5r
5m
e
3.2. Topoisomerases inhibition
e
The topoisomerases inhibitory activities of the 3-
heteroarylisoquinolinamines were determined by ‘DNA relaxation
assay’ [17]. The topos inhibitory activities of HCl salts were gener-
ally greater than those of free amines, probably due to improved
affinity of the positively charged chemical species for negatively
charged DNA (Table 2, Figs. 4 and 5). Topos inhibition by 3-
thiophenylisoquinolinamines showed a distinct pattern in rela-
tion to chemical structures (Table 4). The activities were mainly
dependent upon the position of the methyl group in the iso-
quinoline ring and upon the type of heterocyclic amine at C1.
Piperidine-substituted analogs (5a, 5a$HCl) were inactive
against topo I while they showed moderate activity against topo II.
In contrast, their 7-Me derivatives (5n, 5n$HCl) showed superior
activity only against topo I. Piperidine derivative 5h and its HCl salt
displayed unique topos inhibition properties. 5h was selective
against topo II, while the selectivity of its HCl salt switched to topo I.
N40-unsubstituted piperazinyl isoquinolines (5b$HCl and 5i$HCl)
showed superior activity against topo I and moderate to low ac-
tivity against topo II. However, 7-Me derivative 5o lacked signifi-
cant activities towards both topos. N40-methyl-substituted
piperazine derivatives were inactive toward topo II. In addition to
Fig. 3. 3-Pyrrolylisoquinolinamines 5t.
Scheme 2. Synthesis of 3-heteroarylisoquinolinamine 5f. Reagents and conditions: (i) NH4OH, NaHCO3, DMF, 50 ꢁC; (ii) LiNMe2, HMPA, dry THF, ꢀ78 ꢁC; (iii) c-HCl, acetone.

184
H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
Table 2
Topos (I and II) inhibitory activities and cytotoxicity (IC50) of 3-heteroarylisoquinolinamines 5 and their corresponding HCl salts.
Compound
Topo I (%) inhibition
100 20
Topo II (%) inhibition
100 20
IC50 (mM)
m
M
mM
m
M
m
M
MCF-10Ad
T47De
DU145f
HCT-15g
Camptothecin
Etoposide
74.0/74.5a/
62.6b/72.3c
35.5/23.8a/
34.7b/41.7c
0.27 0.1
0.28 0.01
4.01 2.45
10.07 0.58
86.9/78.6a/
87.9b/86.2c
31.1/49.9a/
42.5b
12.23 1.47
2.57 0.08
13.85 1.75
9.45 0.28
Doxorubicin
5a
5a$HCl
5b
5b$HCl
5c
5c$HCl
5d
5d$HCl
5e
5e$HCl
5f
5f$HCl
5g
5h
5h$HCl
5i
5i$HCl
5j
5j$HCl
5k
5k$HCl
5l
5l$HCl
5m
5n
5n$HCl
5o
5p
0.93 0.03
>50
>50
44.12 0.69
18.1 0.46
18.1 0.84
38.78 0.22
49.01 0.32
23.77 0.15
>50
>50
>50
>50
>50
>50
>50
7.25 0.02
8.18 0.05
13.29 0.37
14.91 0.2
33.94 0.02
28.85 0.25
>50
48.7 0.38
>50
>50
0.84 0.04
3.22 0.05
7.26 0.25
9.53 0.44
4.51 0.17
15.44 0.43
16.08 0.5
17.41 0.42
8.08 0.39
24.41 0.05
14.77 0.31
7.93 0.18
17.48 0.57
>50
2.56 0.12
8.03 0.35
8.74 0.29
7.57 0.22
9.35 0.08
18.38 0.27
8.45 0.22
17.74 0.19
6.76 0.34
7.88 0.14
6.57 0.35
7.38 0.25
3.02 0.05
1.14 0.06
1.96 0.06
1.02 0.04
4.15 0.14
2.84 0.1
4.88 0.12
>50
>50
17.6 0.26
8.25 0.01
40.25 1.26
44.45 0.61
>50
46.61 0.08
>50
47.03 0.82
>50
>50
>50
3.76 0.12
3.61 0.1
1.04 0.06
1.25 0.04
5.45 0.29
2.44 0.09
7.9 0.39
7.42 0.23
9.29 0.44
21.26 0.43
14.74 0.3
1.2 0.06
24.39 1.18
>50
5.45 0.24
1.88 0.08
1.31 0.06
0.94 0.04
4.37 0.23
3.19 0.14
2.41 0.14
4.4 0.25
7.58 0.35
7.79 0.39
8.62 0.14
3.74 0.18
0.89 0.07
1.29 0.05
1.64 0.02
1.04 0.02
3.63 0.13
7.71 0.26
7.58 0.2
9.52 0.44
1.5 0.05
0.0
0.0
ND
ND
0.6
4.8
18.2
76.6
ND
ND
ND
ND
ND
ND
ND
ND
0.0
0.0
0.0
ND
ND
ND
ND
ND
ND
ND
0.0
0.0
0.0
ND
0.0
ND
6.5
1.1
3.7
ND
48.1
42.9
3.7
56.0
0.0
0.0
9.1
3.2
21.8
61.3
14.6
22.5
4.8
42.8
2.9
14.8
40.9
0.0
1.8
0.5
ND
0.0
ND
ND
ND
ND
0.0
0.0
ND
0.0
ND
1.1
ND
ND
0.0
ND
ND
ND
ND
ND
1.7
1.1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
73.0
100.0
97.5
80.8
2.6
15.0
0.0
0.0
0.0
0.0
2.1
0.0
45.9
59.0
59.6
11.2
5.1
0.0
0.0
0.0
0.0
>50
>50
8.31 0.17
11.7 0.27
32.97 0.1
39.98 0.36
17.21 0.03
22.11 0.27
>50
>50
>50
>50
>50
4.15 0.08
16.25 0.56
24.7 0.17
29.4 0.14
31.07 0.02
47.32 0.13
48.27 0.11
14.69 0.6
0.0
0.0
0.0
11.3
66.3
68.9
13.7
3.6
22.1
0.0
0.0
0.0
0.0
0.0
5.1
0.0
89.6
77.5
10.7
29.6
35.9
22.8
59.5
77.9
71.9
18.3
>50
5.54 0.17
11.58 0.21
20.87 0.02
43.14 0.13
42.9 0.06
35.62 0.47
49.06 1.38
20.59 0.36
5p$HCl
5q
5q$HCl
5r
5r$HCl
5s
6.38 0.32
7.63 0.26
1.32 0.02
8.0
Each data represents mean S.D. from three different experiments performed in triplicate.
ND: Not determined.
a
Values for compound 5g.
Values for compounds 5hem and their corresponding HCl salts.
Values for compounds 5nes and their corresponding HCl salts.
MCF-10A: non-cancerous human breast epithelial cells.
T47D: human ductal breast epithelial tumor cells.
DU145: human prostate cancer cells.
HCT-15: human colorectal adenocarcinoma cells.
b
c
d
e
f
g
Table 3
topo II, 6-Me and 7-Me derivatives of this subset were also inactive
Structureecytotoxicity relationships of piperidine-, piperazine- and morpholine-
or had minimal effect on topo I. However, 5c$HCl exhibited greater
substituted 3-thiophenylisoquinolinamines.
topo I inhibitory activity at both 100 and 20 m
M. Similar to N40-
methyl-substituted piperazines, N40-ethyl-substituted piperazines
were also inactive against topo II. Moreover, 5d, 5k and their HCl
salts had minimal or no effect on topo I. 7-Me analog 5q showed
minimal topo I activity whereas 5q$HCl showed similar activity to
CPT at 100 mM. Morpholine derivatives (5e$HCl and 5l$HCl) were
inactive against topo I but exhibited moderate activity against topo
II. 7-Me analogs 5r and 5r$HCl showed superior and similar activity
to CPT against topo I, respectively. Benzylamine-substituted de-
rivative (5m) showed moderate activity only against topo II.
Homopiperazine (5s), azide (5g) and eNH2 (5f) derivatives had no
significant activities against both topos.
3-Heteroarylisoquinolinamines like 5b, 5c, 5i, 5n, 5q, 5r and
their HCl salts with topo I inhibitory activity similar to or greater
than CPT showed potent cytotoxicity, indicating that topo I is major
target for such antitumor agents. Moderate topo I or II activities but
X
MCF-10A
T47D
DU145
HCT-15
a
b
CH2
NH, NMe, NEt
O
✗
✓
✗
<DOXc
<DOX
>DOXd
✓
✗
✓
✓
✓
✗
a
IC50 > 50
IC50 < 50
IC50 < 3.76 0.12
IC50 > 3.76 0.12
m
m
M.
M.
b
c
m
m
M (IC50 of doxorubicin).
M (IC50 of doxorubicin).
d

H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
185
Fig. 4. Topoisomerase I inhibitory activity of 3-heteroarylisoquinolinamines 5 and their corresponding HCl salts. Lane D: pBR322 only, lane T: pBR322 þ topo I, lane C:
pBR322 þ topo I þ CPT, remaining lanes: pBR322 þ topo I þ 3-heteroarylisoquinolinamines and their corresponding salts.
Fig. 5. Topoisomerase II inhibitory activity of 3-heteroarylisoquinolinamines 5 and their corresponding HCl salts. Lane D: pBR322 only, lane T: pBR322 þ topo II, lane E:
pBR322 þ topo II þ etoposide, remaining lanes: pBR322 þ topo II þ 3-heteroarylisoquinolinamines and their corresponding salts.
strong cytotoxic effects of compounds such as 5a, 5h$HCl, 5l$HCl
and 5m indicate that targets other than topos may also be
Table 4
Structure and topoisomerases inhibition relationships of piperidine, piperazine and
responsible for cell death. On the other hand, compounds such as
5d, 5f, 5j, 5k, 5o, 5p, 5s and their HCl salts are likely to exert toxic
effects on cells through different mechanisms. Moreover, low
cytotoxic effects of 5e$HCl despite moderate topo II inhibitory ac-
tivity may be due to the physiochemical properties of the com-
pound that prevent cell penetration or cause wide distribution
within cells, or may be due to cellular metabolic factors that
degrade the compound before reaching the designated target.
morpholine-substituted 3-thiophenylisoquinolinamines.
3.3. Molecular docking
R1, R2, R3
X
Topo Ia
Topo IIb
H, H, H
H, H, Me
CH2
CH2
NH
NH
NMe
NEt
O
0
þþ
Hypothetical binding models of DNA-topo I/II complexes and 3-
heteroarylisoquinolinamines (5b and 5b$HCl) exhibiting potent
topo I and moderate topo II inhibition were constructed by mo-
lecular docking tools to understand the possible interactions
responsible for topos inhibition. The Surflex-Dock program avail-
able in Sybyl-X 2.0 (winnt_os5x) was used to dock the selected 3-
heteroarylisoquinolinamines into the pockets formed after
removal of ligands from the 3D crystal structures of topotecan-
þþþþ
þþþ/þþþþ
0
0 - þþþþ
0 - þþþ
0
0
H, H/Me, H
H, H, Me
H, H/Me, H/Me
H, H/Me, H/Me
H, H/Me, H
H, H, Me
þ/þþ
0
0
0
þþ
O
þþþþ
0
a,
b
Semi quantitative representation of topos inhibition. The ratios of topos inhi-
M of the individual compounds to CPT and etoposide are assigned as
bition by 100
m
DNA-topo I (PDB: 1K4T) [21] and amsacrine-DNA-topo IIb (PDB:
4G0U) [22] ternary complexes. The 5b-DNA-topo I docking model
shows that the adenine base (A113) of the non-scissile DNA strand
follows: 0.0e0.25, 0 (inactive); 0.25e0.5, þ (low activity); 0.5e0.8, þþ (moderate
activity); 0.8e1.0, þþþ (similar activity as CPT or etoposide); >1.0, þþþþ (greater
activity than CPT or etoposide).

186
H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
Fig. 6. Hypothetical binding models of 3-heteroaylisoquinolinamine 5b with (A and C) DNA-topo I and (B and D) DNA-topo IIb complexes. Carbon units are light green in 5b, purple
in nucleotides and gray in amino acids. H-bonds are illustrated as yellow discontinuous lines. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
overlapped the thiophene ring of 5b and indicates possible
p
e
p
The docking model of 5b with DNA-topo II
the compound was bound to DNA and topo II
b
complex shows that
by both pep
stacking interactions between the aromatic rings (Fig. 6A, C). In
addition, the isoquinoline N of 5b was associated with Arg364 of
topo I by H-bond.
b
stacking and H-bond interactions. The aromatic rings of 5b attained
planar conformation and intercalated between DNA bases, parallel
to the plane of the bases, at the cleavage site (Fig. 6B, D). The
piperazine moiety protruded into the minor groove and was
associated with Glu522 by H-bond. Identical intercalative and H-
bond associations with DNA and topo IIb have been displayed by
the anticancer drugs amsacrine and mitoxantrone, and the anti-
cancer agent ametantrone [22]. The similarity in binding modes of
Intercalation of a chemical species between DNA base pairs at
the cleavage site is a major factor that inhibits topos function [21].
This topos inhibitor displaces the reactive eOH of the down-
stream (ꢀ1) base and prevents transesterification with a phos-
photyrosine unit of the upstream base (þ1) to join the cleaved
DNA stand and release topos. The stacking interactions between
the polycyclic topos inhibitors and DNA bases are governed by
multiple factors as dispersion forces, electrostatic attraction and
charge transfer interactions. The stacking interaction energies
calculated by an ab initio quantum mechanics (at the MP2/6-
31g(d) level of theory) has been reported to highly correlate
with actual binding orientation of CPT at the cleavage site [23].
Thus, the ab initio quantum mechanics calculation can be a
valuable tool for validation of hypothetical molecular models of
chemical species (e.g. 5b) generated by docking studies. Besides
intercalation, the H-bond interaction with Arg364 amino acid of
topo I may be essential for topo I inhibitory activity as seen in CPT,
indenoisoquinoline (MJ238) and indolocarbazole (SA315F) [24].
The importance of contact with Arg364 is further supported by
decreased affinity of CPT and indolocarbazole toward topo I with
Arg364His mutation. Considering these facts, the docking model
of 5b with DNA-topo I complex supports its potent topo I inhib-
itory activity.
5b with well-known topo IIb inhibitors proves the topo II inhibitory
activity of 5b$HCl.
3.4. Cell cycle inhibition
To gain better understanding of the preliminary mechanism of
cytotoxicity of 3-heteroarylisoquinolinamines, the interference of
cell cycle progression by the compounds was studied. The N40-
unsubstituted piperazinyl isoquinolines (5b, 5b$HCl, 5i, 5i$HCl and
5o), N40-ethyl-substituted piperazines (5d, 5d$HCl and 5k) and
primary isoquinolinamines (5f and its corresponding HCl salt) were
selected for cell cycle analysis on the basis of their toxic effects in
human breast adenocarcinoma (MCF-7), human gastric cancer
(MKN-28), human lung adenocarcinoma epithelial (A549), human
cervical cancer (HeLa) and mouse melanoma (B16) cells (Fig. 7). The
cell viability of at least 2 types of cancer cells due to the selected
analogs was below 50%.

H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
187
Fig. 7. Effect of 3-heteroarylisoquinolinamines on the viability of (A) human breast adenocarcinoma (MCF-7), (B) human gastric cancer (MKN-28), (C) human lung adenocarcinoma
epithelial (A549), human cervical cancer (HeLa) and (D) mouse melanoma (B16) cells. Cell viability was determined after 48 h by MTT assay. CPT was used as a positive control at
0.04, 0.4 and 4
mM. Similarly, DOX was also used as a positive control at 0.1, 1 and 10
m
M. Each column is the mean percent of viable cells from two independent experiments.
The effects of different concentrations of the selected derivatives
on the cell cycle of HeLa cells were investigated by determining the
cell population in each phase of the cell cycle by flow cytometry.
The number of cells in the G0/G1 phase increased by 2, 4 and 10%
phase. Accordingly, apoptotic cells also increased in a dose-
dependent manner. Similar observations have previously been re-
ported for structurally alike 6,7-dimethoxy-3-(3-methoxyphenyl)
isoquinolin-1-aime (10, CWJ-082), which cause
a-tubulin poly-
upon treatment with compound 5b at 5, 10 and 25
(Fig. 8). However, the cells underwent apoptosis upon increasing
the concentration to 50 M. Likewise, the HCl salt of 5b led to HeLa
m
M, respectively
merization, induce mitotic arrest and subsequent caspase-
dependent apoptosis (Fig. 9) [25]. These findings further suggest
that the cytotoxic effects of primary isoquinolinamine 5f are prin-
m
cell accumulation in G0/G1 phase. The HCl salt was more effective
than the free base 5b to inhibit cell cycle progression and to induce
apoptosis. 10% rise in the number of G0/G1 phase cells was attained
cipally related to targets other than topos. 5f$HCl (50 mM) also
inhibited cell cycle progression at G2/M phase with a 6% rise in cell
number compared to the control.
at 10
up at 25
m
M and 85% of sub-G0/G1 phase cells (apoptotic debris) piled
m
M. Similarly, N40-unsubstituted piperazines (5i, 5i$HCl
4. Conclusion
and 5o) slightly accumulated the cells in G0/G1 phase before
apoptosis (Fig. S1). N40-ethyl piperazine derivative 5d showed no
definite pattern of cell cycle inhibition, whereas its HCl salt and 5k
3-Heteroarylisoquinolinamines were designed and synthesized
under the presumption that heteroaromatic rings retain the planar
structure necessary for expressing topos inhibitory activities. The
cytotoxic effect of 3-heteroarylisoquinolinamines was highly
dependent upon the type of amine substituted at C1. Piperidine,
morpholine and benzyl amines-substituted derivatives were cyto-
toxic to T47D tumor cells, while they were ineffective towards non-
cancerous breast epithelial MCF-10A and cancerous prostate DU145
cells. Generally, 3-heteroarylisoquinolinamines showed superior
induced accumulation in
concentrations.
In contrast to piperazines, the primary isoquinolinamine 5f,
which lacked topos inhibitory activities, arrested the cells in G2/M
phase. The number of cells in G2/M phase increased significantly by
S phase at 25 mM and lower
43% at 50
mM. The amount of cell accumulation at G2/M phase is
concentration-dependent and is largely at the expense of G0/G1

Fig. 8. Effects of 3-heteroarylisoquinolinamines (5b, 5b$HCl, 5d, 5d$HCl 5f and 5f$HCl) on cell cycle progression and apoptosis in human cervical cancer (HeLa) cells. M1, apoptotic
cells (sub-G0/G1); M2, G0/G1 phase cells; M3, S phase cells; M4, G2/M phase cells.

H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
189
dry THF (5 mL) was added drop-wise to a mixture of n-butyllithium
(7.5 mL of 1.6 M in hexane, 12 mmol) in dry THF (20 mL) at ꢀ78 ꢁC.
The reaction mixture was then stirred at the same temperature for
6 h. The reaction was quenched with water and extracted with ethyl
acetate. The organic layer was washed with brine, dried over so-
dium sulfate and concentrated in vacuo. The crude product was
purified by column chromatography with n-hexane-ethyl acetate
(2:1) to afford compound 8a as yellow solid (400 mg, 35%). Mp:
198e200 ꢁC. IR (cmꢀ1): 3451 (NeH), 1647 (C]O). 1H NMR
(300 MHz, CDCl3)
d
(ppm): 11.16 (s, 1H, NH), 8.42 (d, J ¼ 8.1 Hz, 1H,
Fig. 9. 6,7-Dimethoxy-3-(3-methoxyphenyl)isoquinolin-1-aime (10, CWJ-082).
8-H), 7.86 (d, J ¼ 3.6 Hz, 1H, Ar), 7.66 (t, J ¼ 6.9 Hz, 1H, Ar), 7.56 (d,
J ¼ 7.5 Hz, 1H, Ar), 7.48 (t, J ¼ 6.9 Hz, 1H, Ar), 7.39 (d, J ¼ 5.1 Hz, 1H,
Ar), 7.19 (t, J ¼ 5.1 Hz, 1H, Ar), 6.82 (s, 1H, 4-H).
cytotoxicity against human colorectal adenocarcinoma HCT-15
cells compared with the established anticancer drugs used as
positive control. These observations suggest that 3-
heteroarylisoquinolinamines can be developed into oral drugs for
treatment of breast cancer. Topos inhibitory activities were also
largely dependent upon the position of the methyl group and
the type of amine substituted to the isoquinoline ring. HCl salts
showed greater topos inhibition than free bases. 3-
Heteroarylisoquinolinamines were more effective toward topo I
inhibition than topo II. Hypothetical binding mode of compound 5b
with topo I/II-DNA showed that the aromatic ring(s) intercalated
between DNA bases and associated with Arg364 and Glu522 amino
acids of topo I and II, respectively, through H-bonds. Cell cycle in-
hibition experiments showed that 3-heteroarylisoquinolinamines
arrested cell cycle and caused apoptotic cell death. All these find-
ings highlight that 3-heteroaylisoquinolinamines might be devel-
oped into new therapeutic agents to fight cancer.
5.1.2. 5-Methyl-3-thiophen-2-yl-2H-isoquinolin-1-one (8b)
The procedure described for 8a was used with N,N-diethyl-2,3-
dimethyl-benzamide 6b (2.05 g, 10 mmol), thiophene-2-
carbonitirle 7a (1.36 g, 12.5 mmol), and n-butyllithium (9.6 mL of
2.5 M in hexane, 24 mmol) in dry THF to afford compound 8b (1.3 g,
54%). 1H NMR (300 MHz, CDCl3)
d (ppm): 11.85 (s, 1H, NH), 8.28 (d,
J ¼ 7.8 Hz,1H, 8-H), 7.98 (d, J ¼ 2.7 Hz,1H, Ar), 7.48e7.46 (m,1H, Ar),
7.38e7.32 (m, 2H, Ar), 7.20e7.17 (m, 1H, Ar), 6.89 (s, 1H, 4-H), 2.56
(s, 1H, 5-CH3).
5.1.3. 6-Methyl-3-thiophen-2-yl-2H-isoquinolin-1-one (8c)
The procedure described for compound 8a was used with N,N-
diethyl-2,4-dimethyl-benzamide 1e (4.52 g, 22 mmol), thiophene-
2-carbonitrile 7a (3 g, 27.5 mmol) and n-butyllithium (33 mL of
1.6 M in hexane, 52.9 mmol) in dry THF (50 mL) to afford compound
8c as yellow solid (2.14 g, 40%). Mp: 224e225 ꢁC. IR (cmꢀ1): 3434
(NeH), 1652 (C]O). 1H NMR (300 MHz, CDCl3)
d (ppm): 10.8 (s, 1H,
5. Experimental section
NH), 8.30 (d, J ¼ 8.1 Hz, 1H, 8-H), 7.80e7.79 (m, 1H, Ar), 7.39e7.35
(m, 2H, Ar), 7.30 (d, J ¼ 8.1 Hz, 1H, Ar), 7.19e7.16 (m, 1H, Ar), 6.75 (s,
1H, 4-H), 2.49 (s, 3H, 6-CH3).
5.1. Chemistry
Melting points were determined by the capillary method with a
MEL-TEMP® capillary melting point apparatus and were uncor-
rected. IR spectra were obtained on a JASCO FT/IR 300E Fourier
5.1.4. 7-Methyl-3-thiophen-2-yl-2H-isoquinolin-1-one (8d)
The procedure described for compound 8a was used with N,N-
diethyl-2,5-dimethyl-benzamide 6d (5.27 g, 25.7 mmol), thio-
phene-2-carbonitrile 7a (3.5 g, 32.1 mmol) and n-butyllithium
(39 mL of 1.6 M in hexane, 61.7 mmol) in dry THF (50 mL) to afford
compound 8d as yellow solid (3.18 g, 50%). Mp: 219e222 ꢁC. IR
(cmꢀ1): 3439 (NeH), 1639 (C]O). 1H NMR (300 MHz, CDCl3)
transform infrared spectrometer using KBr pellets. 1H NMR and 13
C
NMR spectra were recorded with Varian Unity Plus 300 MHz and
Varian Unity Inova 500 MHz spectrometers at the Korea Basic
Science Institute. The chemical shifts are reported in parts per
million (ppm) downfield to TMS (
d
¼ 0). The coupling constants J
d
(ppm): 10.78 (s, 1H, NH), 8.21 (s, 1H, 8-H), 7.77e7.75 (m, 1H, Ar),
are given in Hertz. The data are reported in the following order:
chemical shift, multiplicity, coupling constant, and number of
protons. Multiplicity of proton signals are reported as s: singlet, d:
doublet, t: triplet, q: quartet, m: multiplet, dd: double doublet.
Mass spectra were obtained on Shimadzu LCMS-2010 EV liquid
chromatograph mass spectrometer using the electron spray ioni-
zation (ESI) method. High resolution mass spectrum was obtained
on Waters Synapt HDMS mass spectrometer. Elemental analyses
were performed using Thermo Fischer Flash 2000 elemental
analyzer and the values are within 0.4% of the theoretical values.
Column chromatography was performed with Merck silica gel 60
(70e230 mesh). TLC was performed using plates coated with silica
gel 60 F254 (Merck). Chemical reagents were purchased from Sig-
maeAldrich and Tokyo Chemical Industry Co., Ltd. and were used
without further purification. Solvents were distilled prior to use;
THF was distilled from sodium/benzophenone. All reactions were
conducted under an atmosphere of nitrogen in oven-dried glass-
ware with magnetic stirring.
7.51e7.45 (m, 2H, Ar), 7.37 (dd, J ¼ 5.1, 1.2 Hz, 1H, Ar), 7.19e7.16 (m,
1H, Ar), 6.79 (s, 1H, 4-H), 2.50 (s, 3H, 7-CH3).
5.1.5. 3-(1,5-Dimethyl-1H-pyrrol-2-yl)-5-methyl-2H-isoquinolin-1-
one (8e)
The procedure described for compound 8a was used with N,N-
diethyl-2,3-dimethyl-benzamide 6b (1.02 g, 4.99 mmol), 1,5-
dimethyl-1H-pyrrole-2-carbonitrile 7b (750 mg, 6.24 mmol) and
n-butyllithium (5 mL of 2.5 M in hexane, 12 mmol) in dry THF to
afford compound 8e (1 g, 79%). 1H NMR (300 MHz, CDCl3)
d (ppm):
10.12 (s, 1H, NH), 8.24 (d, J ¼ 8.1 Hz, 1H, 8-H), 7.47 (d, J ¼ 6.9 Hz, 1H,
6-H), 7.32 (t, J ¼ 7.8 Hz,1H, 7-H), 6.58 (s, 1H, 4-H), 6.45 (d, J ¼ 3.6 Hz,
1H, 300-H), 6.02 (dd, J ¼ 3.6, 0.6 Hz, 1H, 400-H), 3.66 (s, 3H, NCH3),
2.53 (s, 3H, 5-CH3), 2.32 (s, 3H, 500-CH3).
5.1.6. 1-Chloro-3-thiophen-2-yl-isoquinoline (9a)
Compound 8a (300 mg, 1.32 mmol) and POCl3 (10 mL) were
stirred at 50 ꢁC overnight. Excess POCl3 and volatile byproducts
were removed by vacuum distillation. The residue was then care-
fully treated with ice-water, neutralized with saturated NaHCO3
solution and extracted with ethyl acetate. The organic layer was
washed with brine, dried over Na2SO4 and concentrated in vacuo.
5.1.1. 3-Thiophen-2-yl-2H-isoquinolin-1-one (8a)
A solution of N,N-diethyl-2-methyl-benzamide 6a (955 mg,
5 mmol) and thiophene-2-carbonitrile 7a (681 mg g, 6.25 mmol) in

190
H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
The crude product was purified by column chromatography with n-
hexane-ethyl acetate to afford 9a as yellow solid (213 mg, 66%). Mp:
106e107 ꢁC. IR (cmꢀ1): 3030 (Are-H). 1H NMR (300 MHz, CDCl3)
Calcd. for C18H18N2S $ 0.1C4H8O2: C, 72.88; H, 6.25; N, 9.24; S, 10.57.
Found C, 73.29; H, 6.09; N, 9.40; S, 10.17.
d
(ppm): 8.27 (d, J ¼ 8.4 Hz, 1H, Ar), 7.83e7.78 (m, 2H, Ar), 7.71e7.66
5.1.12. 1-Piperazin-1-yl-3-thiophen-2-yl-isoquinoline (5b)
(m, 2H, Ar), 7.61e7.55 (m, 1H, Ar), 7.40e7.38 (m, 1H, Ar), 7.14e7.11
(m, 1H, Ar).
The procedure described for compound 5a was used with
compound 9a (270 mg, 1.1 mmol), piperazine (142 mg, 1.6 mmol)
and K2CO3 (761 mg, 5.5 mmol) in DMF to afford compound 5b as
ivory solid (236 mg, 72%). The free base was converted to 5b$HCl
salt (brown solid). Mp: 152e154 ꢁC. IR (cmꢀ1): 3403 (NeH),
5.1.7. 1-Chloro-5-methyl-3-thiophen-2-yl-isoquinoline (9b)
The procedure described for 9a was used with compound 8b
(1.1 g, 4.55 mmol) and POCl3 to afford compound 9b (800 mg, 67%).
3066(Are-H), 2947, 2832. 1H NMR (300 MHz, CDCl3)
d (ppm): 8.02
1H NMR (300 MHz, CDCl3)
d
(ppm): 7.99 (d, J ¼ 9.0 Hz, 1H, Ar), 7.78
(d, J ¼ 8.4 Hz, 1H, Ar), 7.74 (d, J ¼ 8.1 Hz, 1H, Ar), 7.65 (d, J ¼ 2.8 Hz,
1H, Ar), 7.59e7.54 (m, 2H, Ar), 7.46e7.43 (m, 1H, Ar), 7.35e7.33 (m,
1H, Ar), 7.12e7.09 (m, 1H, Ar), 3.61e3.48 (m, 4H, 10-N(CH2)2),
3.22e3.20 (m, 4H, 40-N(CH2)2), 2.96 (s, 1H, NH). 13C NMR (5b$HCl)
(s, 1H, 4-H), 7.62 (d, J ¼ 3.7 Hz, 1H, Ar), 7.40e7.32 (m, 3H, Ar), 7.07
(dd, J ¼ 4.9, 3.7 Hz, 1H, Ar), 2.57 (s, 3H, 5-CH3).
5.1.8. 1-Chloro-6-methyl-3-thiophen-2-yl-isoquinoline (9c)
(125 MHz, DMSO-d6) d
(ppm): 159.3 (1-C), 144.9 (3-C), 143.1 (200-C),
The procedure described for compound 9a was used with
compound 8c (2 g, 8.3 mmol) and POCl3 (20 mL) to afford com-
pound 9c as yellow solid (1.9 g, 88%). Mp: 88e89 ꢁC. IR (cmꢀ1):
138.6 (4a-C),130.7 (6-C),128.5,127.5,127.4,126.5,125.4,124.0,119.6
(8a-C), 109.7 (4-C), 47.6 (20-C, 60-C), 42.5 (30-C, 50-C). MS (ESI) m/z
296 (MþH)þ. Anal. Calcd. for C17H17N3S $ 0.45CH2Cl2: C, 62.82; H,
5.41; N, 12.6. Found C, 62.86; H, 5.45; N, 12.64.
3023 (Are-H). 1H NMR (300 MHz, CDCl3)
d (ppm): 8.14 (d,
J ¼ 8.7 Hz, 1H, Ar), 7.75 (s, 1H, Ar), 7.68 (dd, J ¼ 4.8, 1.2 Hz, 1H, Ar),
7.55 (s, 1H, Ar), 7.42e7.37 (m, 2H, Ar), 7.14e7.11 (m, 1H, Ar), 2.53 (s,
3H, 6-CH3).
5.1.13. 1-(4-Methyl-piperazin-1-yl)-3-thiophen-2-yl-isoquinoline
(5c)
The procedure described for compound 5a was used with
compound 9a (500 mg, 2.04 mmol), N-methylpiperazine (306 mg,
3.06 mmol) and K2CO3 (1.4 g, 10.2 mmol) in DMF to afford com-
pound 5c as brown solid (600 mg, 95%). The free base was con-
verted to 5c$HCl salt (brownish yellow solid). Mp: 64e66 ꢁC. IR
(cmꢀ1): 3050 (Are-H), 2969, 2885, 2931, 2832. 1H NMR (300 MHz,
5.1.9. 1-Chloro-7-methyl-3-thiophen-2-yl-isoquinoline (9d)
The procedure described for compound 9a was used with
compound 8d (2 g, 8.3 mmol) and POCl3 (20 mL) to afford com-
pound 9d as yellow solid (1.95 g, 85%). Mp: 90e92 ꢁC. IR (cmꢀ1):
3048 (Are-H). 1H NMR (300 MHz, CDCl3)
d
(ppm): 1H NMR
(300 MHz, CDCl3)
d
: 8.02e8.01 (m, 1H, Ar), 7.79 (s, 1H, Ar),
CDCl3)
d
(ppm): 8.02 (d, J ¼ 8.2 Hz, 1H, Ar), 7.73 (d, J ¼ 8.2 Hz, 1H,
7.70e7.65 (m, 2H, Ar), 7.51 (dd, J ¼ 9.4, 1.6 Hz, 1H, Ar), 7.37 (dd,
Ar), 7.64 (dd, J ¼ 3.6, 1.1 Hz, 1H, Ar), 7.55e7.52 (m, 2H, Ar), 7.44e7.39
(m, 1H, Ar), 7.33 (dd, J ¼ 5.0, 1.1 Hz, 1H, Ar), 7.10 (dd, J ¼ 5.1, 3.7 Hz,
1H, Ar), 3.58e3.55 (m, 4H, 10-N(CH2)2), 2.72e2.68 (m, 4H, 40-
N(CH2)2), 2.40 (s, 3H, NCH3). 13C NMR (5c$HCl) (125 MHz, DMSO-
J ¼ 5.0, 1.1 Hz, 1H, Ar), 7.13e7.10 (m, 1H, Ar), 2.54 (s, 3H, 7-CH3).
5.1.10. 1-Chloro-3-(1,5-dimethyl-1H-pyrrol-2-yl)-5-methyl-
isoquinoline (9e)
d6)
d
(ppm): 158.8 (1-C), 144.9 (3-C), 143.1 (200-C), 138.6 (4a-C),
The procedure described for compound 9a was used with
compound 8e (1 g, 3.96 mmol) and POCl3 to afford compound 9e
130.7 (6-C), 128.5, 127.6, 127.4, 126.5, 125.3, 124.1, 119.5 (8a-C), 109.7
(4-C), 52.1 (30-C, 50-C), 47.5 (20-C, 60-C), 42.2 (40-NCH3). MS (ESI) m/z
310 (MþH)þ. Anal. Calcd. for C18H19N3S $ 0.1C4H8O2 $ 0.1H2O: C,
69.05; H, 6.30; N, 13.13; S, 10.02. Found C, 69.01; H, 6.16; N, 13.13; S,
10.10.
(600 mg, 55%). 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.08 (d, J ¼ 8.0
Hz, 1H, Ar), 7.79 (s, 1H, 4-H), 7.45e7.36 (m, 2H, Ar), 6.51 (d,
J ¼ 3.6 Hz, 1H, 300-H), 5.98 (dd, J ¼ 3.5, 0.6 Hz, 1H, 400-H), 3.84 (s, 3H,
NCH3), 2.61 (s, 3H, 5-CH3), 2.29 (s, 3H, 500-CH3).
5.1.14. 1-(4-Ethyl-piperazin-1-yl)-3-thiophen-2-yl-isoquinoline
(5d)
5.1.11. 1-Piperidin-1-yl-3-thiophen-2-yl-isoquinoline (5a)
A mixture of compound 9a (100 mg, 0.4 mmol), piperidine
(52 mg, 0.6 mmol) and potassium carbonate (283 g, 2.05 mmol) in
DMF was refluxed overnight. The reaction mixture was cooled to
room temperature, diluted with water and extracted with ethyl
acetate. The organic layer was washed with brine, dried over
Na2SO4 and concentrated in vacuo. The crude product was purified
by column chromatography with CH2Cl2eMeOH (15:1) to afford
compound 5a as dark green solid (95 mg, 79%). The free base was
dissolved in acetone and then carefully treated with concentrated
HCl dropwise. The solid obtained was filtered off and dried to
obtain 5a$HCl salt (light brown solid). Mp: 84e85 ꢁC. IR (cmꢀ1):
The procedure described for compound 5a was used with
compound 9a (250 mg, 1.02 mmol), 1-ethylpiperazine (175 mg,
1.53 mmol) and K2CO3 (704 mg, 5.1 mmol) in DMF to afford com-
pound 5d as dark green liquid (260 mg, 79%). The free base was
converted to 5d$HCl salt (yellow solid). 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.01 (s, 1H, Ar), 7.74e7.72 (m, 1H, Ar), 7.64 (dd, J ¼ 3.6,
1.1 Hz,1H, Ar), 7.58e7.53 (m, 2H, Ar), 7.44e7.39 (m,1H, Ar), 7.33 (dd,
J ¼ 5.0, 1.1 Hz, 1H, Ar), 7.10 (dd, J ¼ 5.0, 3.6 Hz, 1H, Ar), 3.58 (t,
J ¼ 4.7 Hz, 4H, 10-N(CH2)2), 2.74 (t, J ¼ 4.7 Hz, 4H, 40-N(CH2)2), 2.54
(q, J ¼ 7.2 Hz, 2H, NCH2CH3), 1.17 (t, J ¼ 7.2 Hz, 3H, NCH2CH3). 13
C
NMR (5d$HCl) (125 MHz, DMSO-d6) d (ppm): 158.9 (1-C), 144.9 (3-
3071 (Are-H), 2927, 2846. 1H NMR (300 MHz, CDCl3)
d
(ppm): 7.98
C), 143.1 (200-C), 138.6 (4a-C), 130.7 (6-C), 128.5, 127.6, 127.4, 126.5,
125.3, 124.1, 119.5 (8a-C), 109.7 (4-C), 50.7 (30-C, 50-C), 50.0 (40-
NCH2CH3), 47.5 (20-C, 60-C), 8.8 (40-NCH2CH3). MS (ESI) m/z 324
(MþH)þ.
(d, J ¼ 8.2 Hz, 1H, Ar), 7.69e7.66 (m, 1H, Ar), 7.62 (dd, J ¼ 3.6, 1.1 Hz,
1H, Ar), 7.56e7.48 (m, 2H, Ar), 7.40e7.35 (m, 1H, Ar), 7.30 (dd,
J ¼ 1.1, 5.1 Hz, 1H, Ar), 7.09e7.06 (m, 1H, Ar), 3.46e3.42 (m, 4H,
N(CH2)2), 1.86e1.79 (m, 4H, 30-CH2, 50-CH2), 1.77e1.65 (m, 2H, 40-
CH2). 13C NMR (125 MHz, CDCl3)
d
(ppm): 161.5 (1-C), 146.3 (3-C),
5.1.15. 1-Morpholin-4-yl-3-thiophen-2-yl-isoquinoline (5e)
143.9 (200-C), 138.9 (4a-C), 129.6 (6-C), 127.8, 127.2, 126.2, 125.8,
The procedure described for compound 5a was used with
compound 9a (400 mg, 1.6 mmol), morpholine (428 mg, 4.9 mmol)
and K2CO3 (1.1 g, 8.2 mmol) in DMF to afford compound 5e as ash
colored solid (386 mg, 80%). The free base was converted to 5e$HCl
salt (light yellow solid). Mp: 125e126 ꢁC. IR (cmꢀ1): 3063 (Are-H),
125.2,123.2,120.8 (8a-C),108.6 (4-C), 52.4 (20-C, 60-C), 26.1 (30-C, 50-
C), 24.9 (40-C). 13C NMR (5a$HCl) (125 MHz, DMSO-d6)
d (ppm):
160.2 (1-C),145.0 (3-C),142.9 (200-C),138.6 (4a-C),130.5 (6-C),128.4,
127.5, 127.3, 126.1, 125.5, 124.0, 119.8 (8a-C), 108.9 (4-C), 52.þ2 (20-C,
60-C), 25.4 (30-C, 50-C), 24.2 (40-C). MS (ESI) m/z 295 (MþH) . Anal.
2955, 2844. 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.03 (d, J ¼ 8.3 Hz,

H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
191
1H, Ar), 7.75 (d, J ¼ 7.9 Hz, 1H, Ar), 7.65 (dd, J ¼ 3.7, 1.1 Hz, 1H, Ar),
7.60e7.53 (m, 2H, Ar), 7.44 (t, J ¼ 7.6 Hz, 1H, Ar), 7.34 (dd, J ¼ 5.0,
1.1 Hz, 1H, Ar), 7.12 (dd, J ¼ 5.0, 3.7 Hz, 1H, Ar), 4.00e3.97 (m, 4H,
(dd, J ¼ 5.0, 1.1 Hz, 1H, Ar), 7.25e7.22 (m, 1H, Ar), 7.11e7.08 (m, 1H,
Ar), 3.46e3.43 (m, 4H, N(CH2)2), 2.48 (s, 3H, 6-CH3), 1.87e1.80 (m,
4H, 30-CH2, 40-CH2), 1.74e1.67 (m, 2H, 40-CH2). 13C NMR (5h$HCl)
O(CH2)2), 3.53e3.50 (m, 4H, N(CH2)2). 13C NMR (125 MHz, CDCl3)
d:
(125 MHz, DMSO-d6) d
(ppm): 160.0 (1-C), 144.9 (3-C), 142.8 (200-C),
160.3 (1-C), 145.9 (3-C), 143.9 (200-C), 138.9 (4A-C), 129.9 (6-C),
127.9, 127.4, 126.4, 125.6, 125.4, 123.4, 120.4 (8a-C), 109.4 (4-C), 66.9
(30-C, 50-C), 51.6 (20-C, 60-C). 13C NMR (5e$HCl) (125 MHz, DMSO-d6)
140.4 (6-C), 138.9 (4a-C),128.3,128.1,127.2,126.3,125.4,123.9,118.0
(8a-C), 108.6 (4-C), 52.2 (20-C, 60-C), 25.4 (30-C, 50-C), 24.2 (40-C),
21.3 (6-CCH3). MS (ESI) m/z 309 (MþH)þ. Anal. Calcd. for C19H20N2S:
C, 73.99; H, 6.54; N, 9.08. Found C, 73.86; H, 6.44; N, 9.45.
d
(ppm): 159.9 (1-C),145.2 (3-C),143.2 (200-C),138.6 (4a-C),130.4 (6-
C), 128.4, 127.5, 127.3, 126.2, 125.4, 123.9, 119.6 (8a-C), 109.1 (4-C),
66.1 (30-C, 50-C), 51.4 (20-C, 60-C). MS (ESI) m/z 297 (MþH)þ. Anal.
Calcd. for C17H16N2OS: C, 68.89; H, 5.44; N, 9.45; S, 10.82. Found C,
68.72; H, 5.48; N, 9.36; S, 10.74.
5.1.19. 6-Methyl-1-piperazin-1-yl-3-thiophen-2-yl-isoquinoline
(5i)
The procedure described for compound 5a was used with
compound 9c (600 mg, 2.3 mmol), piperazine (594 mg, 6.9 mmol)
and K2CO3 in DMF to afford compound 5i as brownish green solid
(495 mg, 70%). The free base was then converted to 5i$HCl salt
(light yellow). Mp: 72e74 ꢁC. IR (cmꢀ1): 3418 (NeH), 3067(Are-H),
5.1.16. 3-Thiophen-2-yl-isoquinolin-1-ylamine (5f)
A mixture of compound 9a (100 mg, 0.4 mmol), ammonium
hydroxide (28% NH3 content, 77 mg), sodium bicarbonate (172 mg,
2.04 mmol) was heated at 50 ꢁC for 48 h. The reaction mixture was
diluted with water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over sodium sulfate and
concentrated in vacuo. The crude product was purified by column
chromatography to afford compound 5f as dark semi-solid (23 mg,
24%). The free base was then converted to 5f$HCl salt (ivory solid).
Alternatively, a solution of o-tolunitrile (351 mg, 3 mmol) and
thiophene-2-carbonitrile (654 mg, 6 mmol) in dry THF was added
dropwise to a mixture of LiNMe2 (5 mL, 3.3 mmol) and hexame-
thylphosphoramide (596 mg, 3.3 mmol) in dry THF at ꢀ78 ꢁC. The
reaction was maintained at the same temperature overnight. The
reaction was quenched with water and extracted with ethyl acetate.
The organic layer was washed with brine, dried over sodium sulfate
and concentrated in vacuo. The crude product was purified by
column chromatography to afford compound 5f as dark semi-solid
2933, 2836. 1H NMR (300 MHz, CDCl3)
d
(ppm): 7.91 (d, J ¼ 8.5 Hz,
1H, Ar), 7.63 (dd, J ¼ 3.6, 1.0 Hz, 1H, Ar), 7.51e7.49 (m, 2H, Ar), 7.32
(dd, J ¼ 5.0, 1.0 Hz, 1H, Ar), 7.25 (dd, J ¼ 8.5, 1.6 Hz, 1H, Ar), 7.10 (dd,
J ¼ 5.0, 3.7 Hz, 1H, Ar), 3.51e3.47 (m, 4H, 10-N(CH2)2), 3.19e3.16 (m,
4H, 40-N(CH2)2), 2.59 (s, 1H, NH), 2.49 (s, 3H, 6-CH3). 13C NMR
(5i$HCl) (125 MHz, DMSO-d6)
d (ppm): 159.1 (1-C), 145.0 (3-C),
143.2 (200-C), 140.5 (6-C), 138.9 (4a-C), 128.5, 128.4, 127.3, 126.4,
125.2, 123.9, 117.8 (8a-C), 109.4 (4-C), 47.5 (20-C, 60-C), 42.5 (30-C, 50-
C), 21.4 (6-CCH3). MS (ESI) m/z 310 (MþH)þ. Anal. Calcd. for
C18H19N3S $ 0.85H2O: C, 66.58; H, 6.42; N, 12.94; S, 9.87. Found C,
66.28; H, 6.02; N, 12.67; S, 9.47.
5.1.20. 6-Methyl-1-(4-methyl-piperazin-1-yl)-3-thiophen-2-yl-
isoquinoline (5j)
(140 mg, 20%). 1H NMR (300 MHz, CDCl3)
d (ppm): 7.64e7.58 (m,
The procedure described for compound 5a was used with
compound 9c (600 mg, 2.3 mmol), N-methylpiperazine (690 mg,
6.9 mmol) and K2CO3 (1.6 g, 11.5 mmol) in DMF to afford compound
5j as light brown solid (592 mg, 80%). The free base was then
converted to 5j$HCl salt (yellow solid). Mp: 85e87 ꢁC. IR (cmꢀ1):
3067 (Are-H), 2969, 2882, 2935, 2839. 1H NMR (300 MHz, CDCl3)
3H, Ar), 7.49 (m,1H, Ar), 7.35 (s,1H, 4-H), 7.30e7.28 (m, 2H, Ar), 7.06
(dd, J ¼ 5.0, 3.6 Hz, 1H, Ar), 5.35 (s, 2H, NH2). 13C NMR (5f$HCl)
(125 MHz, DMSO-d6) d
(ppm): 155.6 (1-C), 137.2 (3-C), 134.9 (200-C),
132.5 (4a-C), 129.3, 128.7, 128.2, 127.8, 127.7, 125.5, 116.6 (8a-C),
107.0 (4-C). MS (ESI) m/z 268 (MþCH3CNþH)þ, 227 (MþH)þ.
d
(ppm): 7.90 (d, J ¼ 8.6 Hz, 1H, Ar), 7.63 (d, J ¼ 3.7 Hz, 1H, Ar),
7.49e7.47 (m, 2H, Ar), 7.33e7.31 (m, 1H, Ar), 7.26e7.22 (m, 1H, Ar),
7.11e7.08 (m, 1H, Ar), 3.56 (m, 4H, 10-N(CH2)2), 2.70e2.67 (m, 4H,
40-N(CH2)2), 2.48 (s, 3H, 6-CH3), 2.40 (s, 3H, NCH3). 13C NMR
5.1.17. 1-Azido-5-methyl-3-thiophen-2-yl-isoquinoline (5g)
To a solution of compound 9b (500 mg, 1.92 mmol) in DMF,
sodium azide (187 mg, 2.88 mmol) was added and the reaction
mixture was heated at 100 ꢁC for overnight. The reaction mixture
was cooled, diluted with water and extracted with ethyl acetate.
The organic layer was washed with brine, dried over sodium sulfate
and concentrated in vacuo. The crude product was purified by
column chromatography to afford compound 5g as ivory solid
(450 mg, 87%). Mp: 172e174 ꢁC. IR (cmꢀ1): 3104 (Are-H), 2972,
(5j$HCl) (125 MHz, DMSO-d6)
d (ppm): 158.7 (1-C), 145.0 (3-C),
143.2 (200-C), 140.5 (6-C), 138.9 (4a-C), 128.5, 128.4, 127.3, 126.5,
125.1, 124.0, 117.8 (8a-C), 109.4 (4-C), 52.1 (30-C, 50-C), 47.5 (20-C, 60-
C), 42.2 (40-NCH3), 21.4 (6-CCH3). MS (ESI) m/z 324 (MþH)þ. Anal.
Calcd. for C19H21N3S $ 0.05C4H8O2 $ 0.05C3H7NO: C, 70.11; H, 6.61;
N, 12.89; S, 9.67. Found C, 69.94; H, 6.58; N, 12.79; S, 9.82.
2863. 1H NMR (300 MHz, CDCl3)
d (ppm): 8.62e8.59 (m, 1H, Ar),
8.31 (dd, J ¼ 3.8, 1.1 Hz, 1H, Ar), 7.77 (s, 1H, 4-H), 7.65e7.61 (m, 2H,
5.1.21. 1-(4-Ethyl-piperazin-1-yl)-6-methyl-3-thiophen-2-yl-
isoquinoline (5k)
Ar), 7.58 (dd, J ¼ 5.1, 1.1 Hz, 1H, Ar), 7.28e7.25 (m, 1H, Ar), 2.76 (s,
3H, 5-CH3). 13C NMR (125 MHz, CDCl3)
d
(ppm): 148.5 (1-C), 134.7
The procedure described for compound 5a was used with
compound 9c (600 mg, 2.3 mmol), 1-ethylpiperazine (787 mg,
6.9 mmol) and K2CO3 (1.6 g, 11.5 mmol) in DMF to afford compound
5k as dark green liquid (700 mg, 90%). The free base was then
converted to 5k$HCl salt (yellow solid). 1H NMR (300 MHz, CDCl3)
(3-C), 132.5 (200-C), 132.4 (4a-C), 130.8, 130.1, 128.8, 128.5, 128.4,
128.2, 122.8, 118.5, 110.5 (4-C), 19.5 (5-CCH3). Anal. Calcd. for
C
14H10N4S: C, 63.14; H, 3.78; N, 21.04; S, 12.04. Found C, 62.94; H,
3.87; N, 20.74; S, 12.28.
d
(ppm): 7.90 (d, J ¼ 8.5 Hz, 1H, Ar), 7.62 (dd, J ¼ 3.6, 1.1 Hz, 1H, Ar),
5.1.18. 6-Methyl-1-piperidin-1-yl-3-thiophen-2-yl-isoquinoline
(5h)
7.48e7.46 (m, 2H, Ar), 7.31 (dd, J ¼ 5.0, 1.1 Hz, 1H, Ar), 7.23 (dd,
J ¼ 8.5,1.6 Hz,1H, Ar), 7.09 (dd, J ¼ 5.0, 3.6 Hz,1H, Ar), 3.57e3.54 (m,
4H, 10-N(CH2)2), 2.73e2.70 (m, 4H, 40-N(CH2)2), 2.52 (q, J ¼ 7.2 Hz,
2H, NCH2CH3), 2.47 (s, 3H, 6-CH3), 1.15 (t, J ¼ 7.2 Hz, 3H, NCH2CH3).
The procedure described for compound 5a was used with
compound 9c (600 mg, 2.3 mmol), piperidine (587 mg, 6.9 mmol)
and K2CO3 in DMF to afford compound 5h as light yellow solid
(632 mg, 89%). The free base was then converted to 5h$HCl salt
(light yellow solid). Mp: 106e108 ꢁC. IR (cmꢀ1): 3062 (Are-H),
13C NMR (5k$HCl) (125 MHz, DMSO-d6)
d (ppm): 145.0 (1-C), 143.2
(3-C), 140.6 (200-C), 138.9 (6-C), 128.5 (4a-C), 128.4, 127.3, 126.5,
125.1, 124.0, 117.8 (8a-C), 109.4 (4-C), 50.7 (30-C, 50-C), 50.0 (40-
NCH2CH3), 47.5 (20-C, 60-C), 21.4 (6-CCH3), 8.8 (40-NCH2CH3). MS
(ESI) m/z 338 (MþH)þ.
2927, 2853. 1H NMR (300 MHz, CDCl3)
d
(ppm): 7.91 (d, J ¼ 8.5 Hz,
1H, Ar), 7.62 (dd, J ¼ 3.6, 1.1 Hz, 1H, Ar), 7.48e7.45 (m, 2H, Ar), 7.31

192
H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
5.1.22. 6-Methyl-1-morpholin-4-yl-3-thiophen-2-yl-isoquinoline
(5l)
145.6 (3-C),142.5 (200-C),136.6 (4a-C),135.5 (7-C),132.2 (6-C), 128.2,
127.3, 126.8, 124.3, 123.3, 119.9 (8a-C), 108.6 (4-C), 52.2 (20-þC, 60-C),
45.5 (30-C, 50-C), 21.6 (7-CCH3). MS (ESI) m/z 310 (MþH) . Anal.
Calcd. for C19H20N2S $ 0.05C4H8O2 $ 0.4H2O: C, 68.09; H, 6.34; N,
13.09; S, 9.99. Found C, 67.99; H, 6.14; N, 13.01; S, 10.15.
The procedure described for compound 5a was used with
compound 9c (600 mg, 2.3 mmol), morpholine (600 mg, 6.9 mmol)
and K2CO3 in DMF to afford compound 5l as off white solid (460 mg,
64%). The free base was then converted to 5l$HCl salt (yellow solid).
Mp: 117e119 ꢁC. IR (cmꢀ1): 3066 (Are-H), 2965, 2878, 2910, 2828.
5.1.26. 7-Methyl-1-(4-methyl-piperazin-1-yl)-3-thiophen-2-yl-
isoquinoline (5p)
1H NMR (300 MHz, CDCl3)
d
(ppm): 7.91 (d, J ¼ 8.5 Hz, 1H, Ar),
7.64e7.62 (m, 1H, Ar), 7.52e7.51 (m, 2H, Ar), 7.33 (dd, J ¼ 3.9, 1.1 Hz,
1H, Ar), 7.27e7.25 (m, 1H, Ar), 7.12e7.09 (m, 1H, Ar), 3.99e3.96 (m,
The procedure described for compound 5a was used with
compound 9d (600 mg, 2.3 mmol), N-methylpiperazine (690 mg,
6.9 mmol) and K2CO3 in DMF to afford compound 5p as dark solid
(700 mg, 94%). The free base was then converted to 5p$HCl salt
(yellow solid). Mp: 78e80 ꢁC. IR (cmꢀ1): 3047 (Are-H), 2972, 2839,
4H, O(CH2)2), 3.52e3.48 (m, 4H, N(CH2)2), 2.50 (s, 3H, 6-CH3). 13
C
NMR (5l$HCl) (125 MHz, DMSO-d6)
d (ppm): 159.8 (1-C), 145.2 (3-
C), 143.1 (200-C), 140.3 (6-C), 138.9 (4a-C), 128.3, 128.2, 127.2 (8a-C),
126.4 (4-C), 125.3, 123.9, 117.9, 108.8, 66.1 (30-C, 50-C), 51.4 (20-C, 60-
C), 21.3 (6-CCH3). MS (ESI) m/z 311 (MþH)þ. Anal. Calcd. for
2925, 2821. 1H NMR (300 MHz, CDCl3)
d (ppm): 7.78 (s, 1H, Ar),
7.65e7.61 (m, 2H, Ar), 7.53 (s, 1H, Ar), 7.39 (dd, J ¼ 8.4, 1.7 Hz, 1H,
Ar), 7.31 (dd, J ¼ 5.0, 1.1 Hz, 1H, Ar), 7.09 (dd, J ¼ 5.0, 3.6 Hz, 1H, Ar),
3.55 (m, 4H, 10-N(CH2)2), 2.72e2.69 (m, 4H, 40-N(CH2)2), 2.51 (s, 3H,
C
18H18N2OS: C, 69.65; H, 5.84; N, 9.02; S, 10.33. Found C, 70.22; H,
5.98; N, 9.02; S, 10.41.
7-CH3), 2.41 (s, 3H, NCH3). 13C NMR (125 MHz, CDCl3)
d(ppm): 159.9
5.1.23. Benzyl-(6-methyl-3-thiophen-2-yl-isoquinolin-1-yl)-amine
(5m)
(1-C), 146.2 (3-C), 143.1 (200-C), 137.0 (4a-C), 135.3 (7-C), 131.8 (6-C),
127.8, 127.2, 126.0, 124.5, 123.0, 120.6 (8a-C), 109.1 (4-C), 55.0 (30-C,
50-C), 50.9 (20-C, 60-C), 46.2 (40-NCH3), 22.0 (7-CCH3). MS (ESI) m/z
324 (MþH)þ. Anal. Calcd. for C19H21N3S $ 0.2H2O: C, 69.78; H, 6.6;
N, 12.85; S, 9.8. Found C, 69.70; H, 6.50; N, 12.75; S, 10.00.
The procedure described for compound 5a was used with
compound 9c (1.4 g, 5.4 mmol), benzylamine (4.62 g, 43.2 mmol)
and K2CO3 (3.72 g, 27 mmol) in DMF to afford compound 5m as
gray solid (1.4 g, 78%). Mp: 102e104 ꢁC. IR (cmꢀ1): 3462 (NeH),
3064, 2914. 1H NMR (300 MHz, CDCl3)
d
(ppm): 7.73 (s, 1H, Ar),
5.1.27. 1-(4-Ethyl-piperazin-1-yl)-7-methyl-3-thiophen-2-yl-
isoquinoline (5q)
7.61e7.59 (m, 2H, Ar), 7.51e7.40 (m, 6H, Ar), 7.36 (s, 1H, Ar),
7.22e7.16 (m, 2H, Ar), 5.49 (t, J ¼ 4.8 Hz, 1H, NH), 4.99 (d, J ¼ 5.1 Hz,
The procedure described for compound 5a was used with
compound 9d (600 mg, 2.3 mmol), 1-ethylpiperazine (787 mg,
6.9 mmol) and K2CO3 in DMF to afford compound 5q as brown solid
(760 mg, 97%). The free base was then converted to 5q$HCl salt
(yellow solid). Mp: 90e91 ꢁC. IR (cmꢀ1): 3042 (Are-H), 2972, 2832,
1H, NCH2), 2.50 (s, 3H, 6-CH3). 13C NMR (125 MHz, CDCl3)
d(ppm):
154.1 (1-C), 146.5 (3-C), 144.5 (200-C), 139.99, 139.94, 138.1, 128.5,
128.2, 127.7, 127.3, 127.2, 126.6, 125.9, 122.9, 121.3, 115.4 (8a-C),
104.8 (4-C), 45.7 (NCH2), 21.6 (6-CCH3). MS (ESI) m/z 331 (MþH)þ.
Anal. Calcd. for C21H18N2S: C, 76.33; H, 5.49; N, 8.48; S, 9.70. Found
C, 76.07; H, 5.53; N, 8.26; S, 9.85.
2914, 2815. 1H NMR (300 MHz, CDCl3)
d (ppm): 7.78 (s, 1H, Ar),
7.62e7.59 (m, 2H, Ar), 7.50 (s, 1H, Ar), 7.37 (dd, J ¼ 6.7, 1.7 1H, Ar),
7.30 (dd, J ¼ 3.9, 1.1 Hz, 1H, Ar), 7.09e7.06 (m, 1H, Ar), 3.56e3.53 (m,
4H, 10-N(CH2)2), 2.74e2.71 (m, 4H, 40-N(CH2)2), 2.56e2.49 (m, 5H,
NCH2CH3, 7-CH3), 1.16 (t, J ¼ 7.2 Hz, 3H, NCH2CH3). 13C NMR
5.1.24. 7-Methyl-1-piperidin-1-yl-3-thiophen-2-yl-isoquinoline
(5n)
The procedure described for compound 5a was used with
compound 9d (600 mg, 2.3 mmol), piperidine (587 mg, 6.9 mmol)
and K2CO3 in DMF to afford compound 5n as light yellowish green
solid (528 mg, 74%). The free base was then converted to 5n$HCl
salt (yellow solid). Mp: 116e117 ꢁC. IR (cmꢀ1): 3050 (Are-H), 2924,
(125 MHz, CDCl3) d
(ppm): 159.9 (1-C), 146.3 (3-C), 143.2 (200-C),
137.0 (4a-C), 135.3 (7-C), 131.8 (6-C), 127.8, 127.2, 126.0, 124.5, 122.9,
120.7 (8a-C), 109.0 (4-C), 52.8 (30-C, 50-C), 52.5 (40-NCH2CH3), 50.9
(20-C, 60-C), 22.0 (7-CCH3), 12.0 (40-NCH2CH3). MS (ESI) m/z 338
(MþH)þ. Anal. Calcd. for C20H23N3S: C, 71.18; H, 6.87; N, 12.45; S,
9.50. Found C, 70.84; H, 6.90; N, 12.34; S, 9.44.
2845. 1H NMR (300 MHz, CDCl3)
d (ppm): 7.79 (s, 1H, Ar), 7.64e7.60
(m, 2H, Ar), 7.50 (s, 1H, Ar), 7.38 (dd, J ¼ 8.4, 1.6 Hz, 1H, Ar), 7.30 (dd,
J ¼ 5.0,1.1 Hz,1H, Ar), 7.09 (dd, J ¼ 5.0, 3.6 Hz,1H, Ar), 3.45e3.42 (m,
4H, N(CH2)2), 2.51 (s, 3H, 7-CH3), 1.89e1.81 (m, 4H, 30-CH2, 50-CH2),
1.74e1.68 (m, 2H, 40-CH2). 13C NMR (5n$HCl) (125 MHz, DMSO-d6)
5.1.28. 7-Methyl-1-morpholin-4-yl-3-thiophen-2-yl-isoquinoline
(5r)
The procedure described for compound 5a was used with
compound 9d (600 mg, 2.3 mmol), morpholine (594 mg, 6.9 mmol)
and K2CO3 in DMF to afford compound 5r as gray solid (590 mg,
82%). The free base was then converted to 5r$HCl salt (cream
colored solid). Mp: 124e126 ꢁC. IR (cmꢀ1): 3052 (Are-H), 2965,
d
(ppm): 159.6 (1-C),145.0 (3-C), 142.1 (200-C), 136.7 (4a-C),135.7 (7-
C),132.5 (6-C),128.3,127.4,127.0,124.3,123.7,120.0 (8a-C),109.1 (4-
C), 52.2 (20-C, 60-C), 25.3 (30-C, 50-C), 24.1 (40-C), 21.6 (7-CCH3). MS
(ESI) m/z 309 (MþH)þ. Anal. Calcd. for C19H20N2S $ 0.1C4H8O2
$
0.1H2O: C, 73.03; H, 6.63; N, 8.78; S, 10.05. Found C, 72.73; H, 6.53;
N, 8.84; S, 10.32.
2862, 2911, 2833. 1H NMR (300 MHz, CDCl3)
d (ppm): 7.80e7.79 (m,
1H, Ar), 7.67 (d, J ¼ 8.3 Hz, 1H, Ar), 7.63 (dd, J ¼ 3.6, 1.1 Hz, 1H, Ar),
7.57 (s, 1H, Ar), 7.42 (dd, J ¼ 8.3, 1.5 Hz, 1H, Ar), 7.33 (dd, J ¼ 5.0,
1.1 Hz, 1H, Ar), 7.11 (dd, J ¼ 5.0, 3.6 Hz, 1H, Ar), 4.01e3.98 (m, 4H,
O(CH2)2), 3.52e3.48 (m, 4H, N(CH2)2), 2.52 (s, 3H, 7-CH3). 13C NMR
5.1.25. 7-Methyl-1-piperazin-1-yl-3-thiophen-2-yl-isoquinoline
(5o)
The procedure described for compound 5a was used with
compound 9d (600 mg, 2.3 mmol), piperazine (594 mg, 6.9 mmol)
and K2CO3 in DMF to afford compound 5o as brown solid (545 mg,
76%). The free base was then converted to 5o$HCl salt. Mp:
128e130 ꢁC. IR (cmꢀ1): 3418 (NeH), 3047 (Are-H), 2979, 2843,
(5r$HCl) (125 MHz, DMSO-d6) d (ppm): 159.4 (1-C), 145.3 (3-C),
142.4 (200-C), 136.7 (4a-C), 135.8 (7-C), 132.4 (6-C), 128.3, 127.4,
126.9, 124.2, 123.5, 119.8 (8a-C), 109.1 (4-C), 66.1 (30-C, 50-C), 51.3
(20-C, 60-C), 21.5 (7-CCH3). MS (ESI): m/z 311 (MþH)þ. HRMS (ESI):
m/z 311.1218 (MþH)þ (calcd for C18H19N2OS, 311.1218).
2910, 2817. 1H NMR (300 MHz, CDCl3)
d (ppm): 7.80 (s, 1H, Ar),
7.67e7.61 (m, 2H, Ar), 7.54 (s, 1H, Ar), 7.41 (dd, J ¼ 8.4, 1.6 Hz, 1H,
Ar), 7.32 (dd, J ¼ 5.0, 1.1 Hz, 1H, Ar), 7.10 (dd, J ¼ 5.0, 1.1 Hz, 1H, Ar),
3.48e3.45 (m, 4H, 10-N(CH2)2), 3.19e3.15 (m, 4H, 40-N(CH2)2), 2.52
5.1.29. 7-Methyl-1-(4-methyl-[1,4]diazepan-1-yl)-3-thiophen-2-yl-
isoquinoline (5s)
The procedure described for compound 5a was used with
compound 9d (550 mg, 2.1 mmol), 1-methylhomopiperazine
(s, 3H, 7-CH3). 13C NMR (125 MHz, DMSO-d6)
d (ppm): 160.1 (1-C),

H.T. My Van et al. / European Journal of Medicinal Chemistry 82 (2014) 181e194
193
(718 mg, 6.3 mmol) and K2CO3 (1.45 g, 10.5 mmol) in DMF to afford
was incubated with and without the compounds in assay buffer
[10 mM TriseHCl (pH 7.9) containing 50 mM NaCl, 50 mM KCl,
5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15 mg/mL bovine serum
albumin] for 30 min at 30 ꢁC. The reaction in a final volume of
20 mL was quenched by adding 3 mL of 7 mM EDTA. The DNA
samples were electrophoresed on 1% agarose gel at 25 V for 4 h
with TAE as the running buffer. The gels were stained for 30 min in
an aqueous solution of ethidium bromide (0.5 mg/mL). The DNA
bands were visualized by transillumination with UV light and the
supercoiled DNA was quantified using AlphaImager™ (Alpha
Innotech Corporation).
compound 5s as dark semi-solid (570 mg, 80%). 1H NMR (300 MHz,
CDCl3)
d (ppm): 7.80 (s, 1H, Ar), 7.60e7.57 (m, 2H, Ar), 7.41 (s, 1H,
Ar), 7.36 (dd, J ¼ 8.4, 1.6 Hz, 1H, Ar), 7.30e7.28 (m, 1H, Ar), 7.08 (dd,
J ¼ 5.0, 3.6 Hz, 1H, Ar), 3.92 (t, J ¼ 4.8 Hz, 2H, 10-NCH2), 3.86 (t,
J ¼ 6.0 Hz, 2H, 10-NCH2), 2.92e2.89 (m, 2H, 40-NCH2), 2.76e2.72 (m,
2H, 40-NCH2), 2.48 (s, 3H, 7-CH3), 2.44 (s, 3H, NCH3), 2.16e2.08 (m,
2H, 60-CH3). 13C NMR (125 MHz, CDCl3)
d(ppm): 159.4 (1-C), 146.3
(3-C), 142.4 (200-C), 137.4 (4a-C), 134.1 (7-C), 131.3 (6-C), 127.6, 126.9,
125.6, 124.9, 122.5 (8-C), 119.6 (8a-C), 107.1 (4-C), 57.8 (30-C), 57.3
(50-C), 51.8 (20-C), 51.2 (70-C), 46.2 (40-C), 27.7 (60-C), 21.8 (7-CCH3).
MS (ESI) m/z 338 (MþH)þ.
5.4. Molecular docking
5.1.30. Benzyl-[3-(1,5-dimethyl-1H-pyrrol-2-yl)-5-methyl-
isoquinolin-1-yl]-amine (5t)
The docking study was performed in Sybyl-X 2.0 (winnt_os5x)
using the Surflex Dock program. The structure of topotecan-DNA-
topo I was downloaded from Protein Data Bank (PDB: 1K4T). An
atom of Hg, a molecule of PEG and open carboxylate form of top-
otecan were deleted. The ligand (topotecan) was extracted. Hy-
drogens were added and minimization was performed using the
MMFF94s force field with MMFF94 charges, using a conjugate
gradient method, distance-dependent dielectric constant and
converging to 0.01 kcal/mol$Å. The eSH group of G11 nucleotide of
scissile DNA strand was changed to eOH. Protomol, an idealized
representation of a ligand that makes every potential interaction
with the binding site, was generated on the basis of ligand mode. 3-
Heteroarylisoquinolinamine 5b and topotecan were constructed in
Sybyl, energy minimized with MMFF94s force field and MMFF94
charges and stored in a Sybyl database. The compounds in the Sybyl
database were docked into the binding site by Surflex Dock on the
basis of protomol constructed earlier. The extracted topotecan was
considered a reference molecule. The docking protocol was able to
reproduce the position of topotecan (manually constructed and
stored in the Sybyl database) in the binding site with 0.61 Å root-
mean-square deviation (rmsd) of the heavy atoms of the extrac-
ted topotecan.
The procedure described for compound 5a was used with
compound 9e (500 mg, 1.84 mmol), benzylamine (990 mg,
9.23 mmol) and K2CO3 (1.27 g, 9.23 mmol) in DMF to afford com-
pound 5t as dark brown semi-solid (454 mg, 72%). 1H NMR
(300 MHz, CDCl3)
d
(ppm): 7.51 (d, J ¼ 8.2 Hz, 1H, Ar), 7.38e7.16 (m,
8H, Ar), 6.46 (d, J ¼ 3.5 Hz,1H, 300-H), 5.94 (dd, J ¼ 3.5, 0.6 Hz,1H, 400-
H), 5.50 (s,1H, NH), 4.83 (d, J ¼ 5.3 Hz, 2H, NCH2), 3.64 (s, 3H, NCH3),
2.57 (s, 3H, 5-CH3), 2.23 (s, 3H, 500-CH3). 13C NMR (150 MHz, CDCl3)
d(ppm): 154.2 (1-C), 145.0 (3-C), 139.9, 137.3, 133.9, 133.4, 131.7,
130.0, 128.5, 127.5, 127.0, 124.3, 119.2, 115.7, 108.5, 106.3, 104.9 (4-C),
45.8 (NCH2), 32.3 (100-NCH3), 19.4 (5-CCH3), 12.7 (500-CCH3).
5.2. Cytotoxicity
Four lines of cancer cells, MCF-10A, T47D, DU145 and HCT-15,
were cultured according to the supplier’s instructions. The cells
were seeded in 96-well plates at a density of 2e4 ꢂ 104 cells per
well and incubated overnight in 0.1 mL of media supplied with 10%
Fetal Bovine Serum (Hyclone, USA) in a 5% CO2 incubator at 37 ꢁC.
On day 2, the culture medium in each well was replaced with 0.1-
mL aliquots of medium containing graded concentration of the
compounds. On day 4, 5
mL of the cell counting kit-8 solution
Similarly, the structure of amsacrine-DNA-topo II
loaded from Protein Data Bank (PDB: 4G0U). A topo II
b
was down-
(Dojindo, Japan) was added to each well and incubated for an
additional 4 h under the same conditions. The absorbance of each
well was determined using an Automatic ELISA Reader System (Bio-
Rad3550) at a wavelength of 450 nm. To determine the IC50 values,
the absorbance readings at 450 nm were fitted to a four-parameter
logistic equation. The positive controls, CPT, etoposide and DOX,
were purchased from Sigma.
b
momomer, a
molecule of amsacrine and 3 Mg2þ associated with the monomer
were deleted. Rest of the procedures was performed in a similar
manner as explained for topo I. The docking protocol was able to
reproduce the position of manually constructed amsacrine in
binding site with 0.61 Å rmsd of the heavy atoms of the extracted
amsacrine.
5.3. Topoisomerases inhibition
5.5. Cell viability
Topo I inhibitory activity was determined by assessing the
relaxation of supercoiled DNA pBR322. A mixture of pBR322
(100 ng) and recombinant human DNA topo I (0.4 units; Topo-GEN
INC., USA) was incubated with and without the compounds at 37 ꢁC
for 30 min in relaxation buffer [10 mM TriseHCl (pH 7.9), 150 mM
NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5% glycerol].
Cell viability was determined by MTT assay. MCF-7, MKN-28,
A549, HeLa and B16 cancer cells were cultured in 96-well plates
and treated with compounds under test for 48 h. MTT (0.5
mg/mL)
was added 4 h before the end of culture. Finally, 100 L DMSO was
m
added to each well and the absorbance was measured after 30 min
at 570 nm using a microplate spectrophotometer.
The reaction in a final volume of 10
mL was quenched by adding
2.5 L of stop solution containing 5% sarcosyl, 0.0025% bromo-
m
phenol blue and 25% glycerol. The DNA samples were electro-
phoresed on 1% agarose gel at 15 V for 7 h with TAE (Tris-acetate-
EDTA) as the running buffer. The gels were stained for 30 min in an
aqueous solution of ethidium bromide (0.5 mg/mL). The DNA bands
were visualized by transillumination with UV light and the super-
coiled DNA was quantified using AlphaImager™ (Alpha Innotech
Corporation).
5.6. Cell cycle inhibition
Cell cycle phase distribution analysis of HeLa cells was per-
formed by flow cytometry after DNA staining. Cells were incubated
with various concentrations of compounds under test for 24 h
before they were harvested by centrifugation. Harvested cells were
washed twice with PBS, fixed in 70% ethanol (in PBS) on ice over-
The DNA topo IIa inhibitory activity of the compounds was
measured as follows. A mixture of supercoiled plasmid DNA
pBR322 (200 ng) and human DNA topo IIa (1 unit; Usb Corp., USA)
night and then resuspended in PBS containing 10 mg/mL propidium
iodide (PI), 0.5 mg/mL RNase, and 0.1% Triton X-100. The cells were
then incubated at 37 ꢁC in the dark for 30 min and analyzed using

194
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Acknowledgments
This work was supported by a grant from the Ministry of Health
& Welfare, Republic of Korea. (Grant: HI12C1640).
Appendix A. Supplementary data
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