Synthesis of new 3-arylisoquinolinamines: Effect on topoisomerase I inhibition and cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2003.09.001

To investigate the structure-activity relationships of 3-arylisoquinolines, diverse substituted 3-aryisoquinolinamines were synthesized and tested in vitro antitumor activity against four tumor cell lines. Some of the compounds showed potent topoisomerase I inhibitory activity. Docking study of 7d with topoisomerase I-DNA complex was also performed.

Full text of DOI:10.1016/j.bmcl.2003.09.001

Bioorganic & Medicinal Chemistry Letters 13(2003) 4451–4454
Synthesis of New 3-Arylisoquinolinamines: Effect on
Topoisomerase I Inhibition and Cytotoxicity
Won-Jea Cho,* Sun Young Min, Thanh Nguyen Le and Tae Sung Kim
College of Pharmacy, Chonnam National University, Yong-Bong dong, Buk-gu, Kwangju 500-757, South Korea
Received 24 July 2003; revised 30 August 2003; accepted 4 September 2003
Abstract—To investigate the structure–activity relationships of 3-arylisoquinolines, diverse substituted 3-aryisoquinolinamines were
synthesized and tested in vitro antitumor activity against four tumor cell lines. Some of the compounds showed potent topo-
isomerase I inhibitory activity. Docking study of 7d with topoisomerase I–DNA complex was also performed.
# 2003Elsevier Ltd. All rights reserved.
Introduction
this investigation we found the amide carbonyl group of
isoquinolin-1-one played an important role for exhibit-
ing the cytotoxicities. When the carbonyl group was
transformed to amines, water solubility was increased
with retaining the activities. Among the synthesized
compounds, CWJ-a-5 was tested in vivo assay using
BDF1 mice (P388 leukemia) and resulted in 160 T/C%
with low toxicity.13 The high oral bioavailability as well
as promising pharmacokinetic data of CWJ-a-5 served
as useful information in future clinical studies of related
compounds14 (Fig. 1).
Eukaryotic DNA topoisomerase I (top I) is an essential
enzyme that act to relax supercoiled DNA during the
transcription, replication and mitosis.1 Intracellular
levels of top I are elevated in a number of human solid
tumors, relative to the respective normal tissues, sug-
gesting that controlling the top I level is important to
treat cancer.2 Top I poisons show their antitumor
activities by stabilizing the cleavable ternary complex
consisting of top I enzyme, DNA, and drug.3 Thus, top
I is a promising target for the development of new can-
cer chemotherapeutics against a number of solid
tumors. Camptothecin is a representative top I inhibitor
and its derivatives, topotecan and irinotecan, have been
launched as clinically used drugs.4 Several non-camp-
tothecin top I inhibitors have been reported over the
last decade. These include benzo[c]phenanthridines,5
indenoisoquinolines,6 indolocarbazones,7 saintopin,8
and benzophenazines.9 The X-ray crystal structure of
human top I covalently joined to double-stranded DNA
base pair and bound to the topotecan was also
revealed.10,11
Our research was next focused on introducing the var-
ious amines on C-1 position replacing N-methylpiper-
azine in 3-arylisoquinolines. For further investigation of
isoquinolinamines on the antitumor cytotoxicities with
top I inhibition activities, a systematic synthesis was
performed to get interesting results. Based on the X-ray
structure of ternary top I–DNA complex with topotecan,
a docking study was also carried out.
We recently reported the synthesis and biological eval-
uation of 3-arylisoquinolines related to the lead com-
pound (CWJ-a-5) with the 3-D-QSAR study.12 Most of
these compounds were shown to be highly cytotoxic
against several kinds of human tumor cell lines. From
Figure 1. The structure of 1-(4-methylpiperazinyl)-3-phenylisoquino-
line hydrochloride (CWJ-a-5).
*Corresponding author. Tel.: +82-625-302-933; fax: +82-625-302-
911; e-mail: wjcho@chonnam.ac.kr
0960-894X/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.001

4452
W.-J. Cho et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4451–4454
Synthesis
ian), SK-MEL-2 (melanoma), and HCT 15 (colon)
using sulforhodamine B (SRB) assay.15 The top I inhi-
bitory activity was carried out by the supercoiled DNA
unwinding assay method.16 The IC50 cytotoxicity values
obtained with cell lines are summarized in Table 1 and
the top I inhibitory results are shown in Figure 2.
The coupling reaction of N-methyl-o-tuluamide (1) with
benzonitrile (2) was accomplished via dilithio species
using n-BuLi in moderate yield. The imine chloride (4)
derived from the corresponding amide by treating
POCl3 was reacted with various amines in refluxing
DMF to provide the 1-substituted isoquinolines in good
yield. For the preparation of primary amines (9), the
benzylamines (8) was performed to a catalytic hydro-
genation reaction with 5% Pd/C on 80 psi hydrogen
atmosphere to afford the corresponding amines in
moderate yield. 3-Naphthylisoquinoline (14) was also
obtained efficiently using the above method as shown in
Scheme 1.
N-Ethylpiperazine compound 5 proved to be less or
slightly more active than the lead compound (CWJ-a-5),
depending on the cell lines tested. However, the N-
methylhomopiperazine compound 6f (0.44 mM, A 549),
primary amine 9b (0.14 mM, SK-MEL-2) and naphtha-
lene compound 14 (0.52 mM, A 549) displayed sub-
micromolar cytotoxicity concentrations. Both of these
compounds possess 6-methyl and 20-methyl substituents
on aromatic rings. Previous work on our laboratory has
found that alkyl groups on aromatic rings of 3-aryliso-
quinolines enhanced the cytotoxic activities and molec-
ular modeling results also supported the hydrophobic
favoring effect.12 Weak cytotoxicity was observed for
ethylenediamine analogues 7 (>300 mM) although they
are potent top I inhibitors as shown in Figure 2. In this
Biological Results and Discussion
The cytotoxicity experiment of the synthesized com-
pounds was performed in vitro against four human
tumor cell lines such as A 549 (lung), SKOV-3(ovar-
Scheme 1. The synthesis of 3-arylisoquinolinamines.

W.-J. Cho et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4451–4454
4453
Table 1. Synthetic yield of 3-arylisoquinolines, IC50 cytotoxicity (m mole) and topoisomerase I activity
b
No.
Compd
R1
R2
Yielda
A549
SK-OV-3SK-MEL-2
HCT15
Top activity
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
8a
8b
8c
8d
8e
9a
9b
9c
10a
H
6-Me
6-Me
6-Me
6-Me
H
H
H
H
H
6-Me
6-Me
6-Me
6-Me
6-Me
H
H
6-Me
H
6-Me
H
6-Me
H
H
H
H
H
20-Me
40-Me
20-Cl
H
30-Me
40-Me
40-Cl
40-Br
20-Me
H
20-Me
20-Cl
40-Me
H
87
78
88
77
79
90
86
89
91
78
79
57
67
82
62
67
89
88
78
72
46
67
57
46
48
90
87
—
—
—
55.35
18.16
20.98
26.94
13.36
10.84
8.57
68.91
41.80
61.96
63.37
194.91
17.01
8.22
95.73
7.22
2.42
8.65
8.07
+
11.92
12.85
14.40
5.58
8.38
8.85
6.18
8.55
4.12
0.99
>300
>300
>300
>300
82.97
++
++
+
+++
+++
+
++
++
++
++
+++
+
+
+++
+
5.58
11.09
9.20
7.84
9.78
5.26
1.22
9.34
9
11.77
6.04
0.44
19.96
3.85
0.70
>300
>300
>300
>300
101.84
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1.20
>300
>300
>300
>300
176.75
102.93167.74
170.94
168.12
122.11
2.732.03 1.48
0.17
3.14
7.95
10.24
>300
>300
>300
>300
87.36
30-Me
30-Me
40-Me
H
85.05
107.65
+
145.82
192.86
156.05
878.73111.3
133.28
94.37
+
171.43
64.22
ntc
nt
H
1.87
nt
20-Me
20-Me
H
0.21
1.81
4.36
9.17
186.50
3.64
13.71
1.17
—
0.14
2.11
7.70
6.45
183.44
1.98
14.53
4.78
—
0.17
1.92
7.24
7.65
252.57
1.93
13.94
1.67
—
++
nt
++
+
+
+
nt
10b
11
14
20-Me
H
—
H
—
—
—
145.30
0.52
13.56
0.97
—
CWJ-a-5
Doxorubicin
Camptothecin
—
—
—
nt
++
—
aChemical yield represents the final step for the preparation of each compounds.
bActivity of the compounds is expressed semi-quantitatively as follows: +; weak activity, ++; similar activity as the camptothecin; +++; greater
activity than the camptothecin.
cnt, not tested.
In general compounds 5, 6 and 7 that contain ethylene-
diamine units show relatively potent top I inhibitory
activity.
Docking study was carried out using Sybyl 6.7 (Tripos,
Inc.)17 on a Silicon Graphics Indigo 2 workstation.
Construction of protein–ligand was based on X-ray
structure of ternary top I-DNA-topotecan complex
(PDB entry 1K4T).12 Ligand was manually docked into
Figure 2. Topoisomerase I inhibitory activities of the compounds:
Lane 1,16: pBR322 DNA; lane 2, 17: pBR322 DNA+ top I; lane 3,
18: pBR322 DNA+ top I+camptothecin (0.1 mg/mL); lane 19:
pBR322+top I+camptothecin (0.01 mg/mL); the others follows the
lane number (prepared compound number, 0.1 mg/mL), 4 (6a), 5 (14),
6 (6b), 7 (6c), 8 (6d), 9 (6e), 10 (6f), 11 (10a), 12 (11), 13( 10b), 14 (9b),
20 (5a), 21 (5b), 22 (5c), 23( 5d), 24 (5e), 25 (7a), 26 (7b), 27 (7c), 28
(7d), 29 (8a), 30 (8b), 31 (8c). Thicker band of the supercoiled form
implies more potent inhibitory activity of the compound. The semi-
quantitative comparison of the inhibitory activities was exhibited in
Table 1. Detailed assay procedure is also described in ref 19.
the active site of topotecan binding region. The ethyl-
enediamine group of compound 7d was considered to
have strong hydrogen bonding with carboxyl group of
Asp 533 (2.221 A), phosphoester group of Guanine 12
(1.490 A) and amide carbonyl moiety of Gln 633 (2.739 A)
as shown in Figure 3. On the other hand, primary
amines 9b did not have good interaction to this binding
series of isoquinolinamines, top I inhibitory activity did
not correlate particularly well with cytotoxicity, at least
not in any quantitative sense, since the most potent
cytotoxic compound 9b was not the most top I inhi-
bitor. This is not the unusual result because cell mem-
brane penetration ability to reach the target affects the
cytotoxicity. Benzylamines 8a–e were all less potent
than the CWJ-a-5. The acetylated amine 11 showed
dramatically decreased cytotoxicity compared to 10a.
Interestingly, amine 9b exhibited very potent cytotoxi-
city against daunorubicin-resistant AML-2 subline
(0.0027 mg/mL) and doxorubicin-resistant subline DX-
100 (0.0056 mg/mL) as shown in Table 2.
Table 2. Cytotoxicity (mg/mL) of 9b against daunorubin and
doxorubicin resistant cells18
Compd
AML-2a
D100c
WTb
DX-100d
0.0056
9b
Doxorubicin
Daunorubicin
0.0078
0.01
0.001
0.0027
1.933
0.52
.20
0.43
aAML-2 means acute myelogenous leukaemia.
bWT: wild type AML-2 leukemia.
cD100: daunorubicin (100 nM) resistant AML-2.
dDX-100: doxorubicin (100 ng/mL) resistant AML-2.

4454
W.-J. Cho et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4451–4454
Figure 3. Hypothetical docking model of 7d with topoisomerase I–DNA complex.
Pommier, Y.; Staker, L. S.; Cushman, M. J. Med. Chem. 2003,
site due to the lacks of the length of chain to reach the
protein functional group.
46, 3275.
7. Yamashita, Y.; Fujii, N.; Murakaya, C.; Ashizawa, T.;
Okabe, M.; Nakano, H. Biochemistry 1992, 31, 12069.
8. Yamashita, Y.; Kawada, S.; Fujii, N.; Nakano, H. Bio-
chemistry 1991, 30, 5838.
9. Vicker, N.; Burgess, L.; Chuckowree, I. S.; Dodd, R.;
Fokes, A. J.; Hardick, D.; Hancox, T. C.; Miller, W.; Milton,
J.; Sohal, S.; Wang, S.; Wren, S. P.; Charlton, P. A.; Danger-
field, W.; Liddle, C.; Mistry, P.; Stewart, A. J.; Denny, W. A.
J. Med. Chem. 2002, 45, 721.
In summary, amine substituents at the C-1 position of
3-arylisoquinolines provide novel, potent and water
soluble top I inhibitors with significant antitumor cyto-
toxicity. The selective cytotoxicity of 9b against doxo-
rubicin- and daunorubicin-resistant tumor cells is highly
significant. Finally, these compounds represent a pro-
mising new class of non-camptothecin top I inhibitors
worthy of a further study.
10. Redinbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.;
Hol, W. G. J. Science 1998, 279, 1504.
11. Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.;
Burgin, A. B., Jr.; Stewart, L. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 15387.
Acknowledgements
12. Cho, W.-J.; Kim, E.-K.; Park, I. Y.; Jeong, E. Y.; Kim,
T. S.; Le, T. N.; Kim, D.-D.; Lee, E.-S. Bioorg. Med. Chem.
2002, 10, 2953.
13. Cho, W.-J.; Park, M.-J.; Chung, B.-H.; Lee, C.-O. Bioorg.
Med. Chem. Lett. 1998, 8, 41.
The financial support from Korea Research Foundation
(KRF-2002-015-EP0142) of Korea is appreciated.
14. Kim, K. E.; Cho, W.-J.; Chang, S. J.; Yong, C. S.; Lee,
C. H.; Kim, D. D. Int. J. Pharm. 2001, 217, 101.
15. (a) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.;
Simon, R. M.; Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks,
A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113. (b) Ske-
han, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd,
M. R. J. Natl. Cancer Inst. 1990, 82, 1107.
References and Notes
1. (a) Wang, J. C. Annu. Rev. Biochem. 1996, 65, 635. (b) Liu,
L. F. Annu. Rev. Biochem. 1989, 58, 351.
2. Chen, A. Y.; Liu, L. F. Annu. Rev. Pharmacol. Toxicol.
1994, 34, 191.
3. Pommier, Y.; Pourquier, P.; Fan, Y.; Strumberg, D. Bio-
chim. Biophys. Acta 1998, 1400, 83 .
16. Fukuda, M.; Nishio, K.; Kanzawa, F.; Ogasawara, H.;
Ishida, T.; Arioka, H.; Bojanowski, K.; Oka, M.; Saijo, N.
Cancer Res. 1996, 576, 789.
17. The Sybyl program (Version 6.7) was supplied by Tripos
Associates, 1699 South Hanley Road, Suite 303, St. Louis,
MO 63144, USA.
4. Rajendra, R.; Gounder, M. K.; Saleem, A.; Schellens,
J. H. M.; Ross, D. D.; Bates, S. E.; Sinko, P.; Rubin, E. H.
Cancer Res. 2003, 63, 3228, and references cited therein.
5. (a) Makhey, D.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.;
LaVoie, E. J. Med. Chem. Res. 1995, 5, 1. (b) Janin, Y. L.;
Croisy, A.; Rious, J.-L.; Bisagni, E. J. Med. Chem. 1993, 36,
3686. (c) Singh, S. K.; Ruchelman, A. L.; Li, T.-K.; Liu, A.;
Liu, L. F.; LaVoie, E. J. J. Med. Chem. 2003, 46, 2254.
6. (a) Jayaraman, M.; Fox, B. M.; Hollingshead, M.; Kohl-
hagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2002,
45, 242. (b) Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.;
18. Kim, H.-S.; Lee, T.-B.; Choi, C.-H. Biochem. Biophys.
Res. Commun. 2001, 281, 109.
19. 500 ng supercoiled pBR 322 DNA was incubated with 1
unit topoisomerase I in the absence or presence of camp-
tothecin or the compounds for 30 min at 37 ꢀC. The reaction
mixtures were analyzed on 1% agarose gel followed by ethi-
dium bromide staining.