Synthesis and antiproliferative evaluation of certain indolo[3,2-c]quinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2010.01.033

The present report describes the synthesis and antiproliferative evaluation of certain indolo[3,2-c]quinoline derivatives. For the C₆ anilino-substituted derivatives, (11H-indolo[3,2-c]quinolin-6-yl)phenylamine (6a) was inactive. Structural optimization of 6a by the introduction of a hydroxyl group at the anilino-moiety resulted in the enhancement of antiproliferative activity in which the activity decreased in an order of para-OH, 7a > meta-OH, 8a > ortho-OH, 9a. For the C₆ alkylamino-substituted derivatives, 11a, 12a, 13a, 14a, and 15a exhibited comparable antiproliferative activities against all cancer cells tested and the skin Detroit 551 normal fibroblast cells. Three cancer cells, HeLa, A549, and SKHep, are very susceptible with IC₅₀ of less than 2.17 μM while PC-3 is relatively resistant to this group of indolo[3,2-c]quinolines. For the 2-phenylethylamino derivatives, compound 20a is active against the growth of HeLa with an IC₅₀ of 0.52 μM, but is less effective against the growth of Detroit 551 with an IC₅₀ of 19.32 μM. For the bis-indolo[3,2-c]quinolines, N,N-bis-[3-(11H-indolo[3,2-c]quinolin-6-yl)aminopropyl]amine hydrochloride (25) is more active than its N-methyl derivative 26 and the positive Doxorubicin. Mechanism studies indicated 25 can induce caspase-3 activation, γ-H2AX phosphorylation, cleavage of poly(ADP-ribose)polymerase and DNA fragmentation. These results provide evidence that DNA, topo I, and topo II are the primary targets of indolo[3,2-c]quinoline derivatives and that consequently inhibits proliferation and causes apoptosis in cancer cells.

Full text of DOI:10.1016/j.bmc.2010.01.033

Bioorganic & Medicinal Chemistry 18 (2010) 1948–1957
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiproliferative evaluation of certain
indolo[3,2-c]quinoline derivatives
d
Chih-Ming Lu a,b, Yeh-Long Chen a, Hui-Ling Chen c, Chyi-An Chen a, Pei-Jung Lu c, , Chia-Ning Yang ,
*
Cherng-Chyi Tzeng a,
*
a Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
b Graduate Institute of Pharmaceutical Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
c Institute of Clinical Medicine, National Cheng-Kung University, School of Medicine, 138 Sheng-Li Road, Tainan 704, Taiwan
d Institute of Biotechnology, National University of Kaohsiung, 700 Kaohsiung University Rd, Kaohsiung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present report describes the synthesis and antiproliferative evaluation of certain indolo[3,2-c]quin-
oline derivatives. For the C6 anilino-substituted derivatives, (11H-indolo[3,2-c]quinolin-6-yl)phenyla-
mine (6a) was inactive. Structural optimization of 6a by the introduction of a hydroxyl group at the
anilino-moiety resulted in the enhancement of antiproliferative activity in which the activity decreased
in an order of para-OH, 7a > meta-OH, 8a > ortho-OH, 9a. For the C6 alkylamino-substituted derivatives,
11a, 12a, 13a, 14a, and 15a exhibited comparable antiproliferative activities against all cancer cells tested
and the skin Detroit 551 normal fibroblast cells. Three cancer cells, HeLa, A549, and SKHep, are very sus-
Received 27 November 2009
Revised 13 January 2010
Accepted 14 January 2010
Available online 28 January 2010
Keywords:
Indolo[3,2-c]quinoline derivatives
Antiproliferative activity
Anticancer agents
ceptible with IC50 of less than 2.17
c]quinolines. For the 2-phenylethylamino derivatives, compound 20a is active against the growth of HeLa
with an IC50 of 0.52 M, but is less effective against the growth of Detroit 551 with an IC50 of 19.32 M.
lM while PC-3 is relatively resistant to this group of indolo[3,2-
l
l
For the bis-indolo[3,2-c]quinolines, N,N-bis-[3-(11H-indolo[3,2-c]quinolin-6-yl)aminopropyl]amine
hydrochloride (25) is more active than its N-methyl derivative 26 and the positive Doxorubicin. Mecha-
nism studies indicated 25 can induce caspase-3 activation,
c-H2AX phosphorylation, cleavage of poly
(ADP-ribose)polymerase and DNA fragmentation. These results provide evidence that DNA, topo I, and
topo II are the primary targets of indolo[3,2-c]quinoline derivatives and that consequently inhibits pro-
liferation and causes apoptosis in cancer cells.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
cryptolepine binds tightly to DNA and behave as a typical inter-
calating agent. It stabilizes the topoisomerase II-DNA covalent
The tetracyclic indoloquinoline ring system constitutes an
important structural moiety in natural products exhibiting
numerous biological activities. For example, cryptolepine (an
indolo[3,2-b]quinoline derivative) and isocryptolepine (an indo-
lo[3,2-c]quinoline derivative) are two indoloquinolines isolated
from the roots of the african plant Cryptolepis sanguinolenta
which had been used in traditional medicine against malaria.1–5
These two alkaloids, which only differ by the respective orienta-
tion of their indole and quinoline rings, display potent antipara-
sitic properties. Extensive investigations on the in vitro and
in vivo biological activities of cryptolepine revealed its wide
applications as anti-malarial,6 anti-microbial,7,8 anti-imflammato-
ry,9 and anticancer agents.10–13 Mechanism studies indicated that
complex and stimulates the cutting of DNA by topoisomerase II
(topo II). The isomeric isocryptolepine (also referred to as
cryptosanguinolentine), however, attracted only limited atten-
tion.14–22 For the past few years, we have synthesized certain
indolo[3,2-c]quinoline derivatives for anticancer evaluation on
the ground that these tetracyclic heterocycles may function in
a similar way with cryptolepine to intercalate into the DNA dou-
ble helix resulting in the inhibition of DNA replication and
transcription.23–26 Among them, (E)-1-[3-(11H-indolo[3,2-c]quin-
olin-6-ylamino)phenyl]ethanone oxime (1) demonstrated a mean
GI50 value of 1.70 lM in the NCI’s full panel of 60 human cancer
cells.23 In continuation of our study to explore more potent anti-
cancer drug candidates, we described herein the preparation and
antiproliferative evaluation of indolo[3,2-c]quinoline and the bis-
indolo[3,2-c]quinolines derivatives. To elucidate their mechanism
of action, we have also studied their interactions with DNA and
their effects on topo I and topo II.
* Corresponding authors. Tel.: +886
7 3121101x2684 (C.-C.T.); +886 6 235
3535x4415 (P.-J.L.).
E-mail addresses: pjlu2190@mail.ncku.edu.tw (P.-J. Lu), tzengch@kmu.edu.tw
(C.-C. Tzeng).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.033

C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
1949
HN
N
OH
Me
N
N
N
N
N
Me
Me
Cryptolepine
Isocryptolepine
Indolo[3,2-c]quinoline derivative, 1
Treatment of 5a with ethanolamine afforded 2-(11H-indolo[3,2-
c]quinolin-6-ylamino)ethanol (11) as depicted in Scheme 2. Prepa-
ration of its C6 alkylamino-substituted analogs 12–17 were achieved
by the reaction of 5 and the respective alkylamines. 6-[2-(4-
Hydroxyphenyl)ethylamino]-11H-indolo[3,2-c]quinoline hydro-
chloride (18a) was obtained by the treatment of 5a with 4-(2-ami-
noethyl)phenol. Accordingly, compounds 19a and 20a were
prepared by the treatment of 5a with 2-(3,4-dihydroxyphenyl)eth-
ylamine and 2-(3,4-dimethoxyphenyl)ethylamine, respectively.
Compound 18b was prepared from 5b by the same reaction
conditions.
2. Chemistry
Reaction of 2-fluoroisatin (2b) and 2-aminobenzylamine (3)
gave 5,11-dihydro-2-fluoroindolo[3,2-c]quinolin-6-one (4b) as
described in Scheme 1. Treatment of 4b with POCl3 afforded
6-chloro-2-fluoro-11H-indolo[3,2-c]quinoline (5b) in
a
good
overall yield. Preparation of 6-chloro-11H-indolo[3,2-c]quinoline
(5a) had been previously reported.23 Treatment of 5a with
substituted anilines gave (11H-indolo[3,2-c]quinolin-6-yl)phenyl-
amine (6a) and its derivatives 7a–10a respectively in a fairly
good yield.
HN
O
R
R
H2N
H2N
(ii)
(i)
+
O
N
H
O
N
H
3
4
a: R = H
b: R = F
2
HN
HN
6: R1 = H
(iii)
R
R
7: R1 = 4-OH
8: R1 = 3-OH
9: R1 = 2-OH
10: R1 = 4-OMe
R1
N
Cl
N
N
H
5
Scheme 1. Reagents and conditions: (i) AcOH, reflux for 8 h; (ii) POCl3, 150 °C, 48 h; (iii) aniline or substituted-aniline, s-BuOH, reflux for 18 h.
11 R1 = OH
12 R1 = N(Me)2
13 R1 = CH2N(Me)2
14 R1 = NHCH2CH2OH
15 R1 = NHCH2CH2NH2
HN
N
(i)
R
R1
N
H
HN
N
R
(ii)
X
16 X = NH
17 X = O
5
N
N
H
HN
N
18 R1 = OH, R2 = H
R1
R
(iii)
19 R1 = OH, R2 = OH
20 R1 = OMe, R2 = OMe
N
H
R2
Scheme 2. Reagents and conditions: (i) aliphatic-amine, ethoxyethanol, 140 °C, 24 h; (ii) 2-(piperazin-1-yl)ethanamine or 2-morpholinoethanamine, ethoxyethanol, 140 °C,
24 h; (iii) substituted-phenylethylamine, ethoxyethanol, 140 °C, 24 h.

1950
C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
Preparationof bis-indolo[3,2-c]quinolinesderivativesis depicted
OMe (an H-bonding acceptor). Compounds 7a and 8a exhibited
in Scheme 3. Reaction of 5a and 1,6-diaminohexane in 2-ethoxyeth-
anol gave a mixture of N-[6-(11H-indolo[3,2-c]quinolin-6-ylamino)
hexyl]-11H-indolo[3,2-c]quinolin-6-amine (21) and N-(6-amin-
selective cytotoxicity in which both compounds are inactive
against Detroit 551 with IC50 of 25.43 and 42.54 lM, respectively.
For the C6 alkylamino-substituted indolo[3,2-c]quinoline deriv-
atives, 11a, 12a, 13a, 14a, and 15a exhibited comparable antiprolif-
erative activities against all cancer cells tested and Detroit 551 as
shown in Table 1. Three cancer cells, HeLa, A549, and SKHep, are
ohexyl)-11H-indolo[3,2-c]quinolin-6-amine (23). Accordingly,
a
mixture of the bis-indolo[3,2-c]quinoline 22 and the aminoalkyl-
amine derivative 24 was obtained by the treatment of 5a with
1,7-diaminoheptane. N,N-Bis-[3-(11H-indolo[3,2-c]quinolin-6-
yl)aminopropyl]amine hydrochloride (25) was synthesized by the
reaction of 5a and dipropylenetriamine. Under similar reaction
conditions, the N-methyl derivative 26 was prepared from 5a and
N,N-bis(3-aminopropyl)methylamine.
very susceptible with IC50 of less than 2.17 lM while PC-3 is rela-
tively resistant to this group of indolo[3,2-c]quinolines. It is inter-
esting that these compounds are more active than the positive
Doxorubicin against the growth of A549. Compound 14a was more
active than its C2 methoxy derivative 14b implied the substitution
of an electron-donating group at C2 position is unfavorable. The
same trends were observed in which 16a > 16b and 17a > 17b.
Comparison of 15a and 16a indicated the cyclic 2-piperazinyleth-
ylamino substituent 16a is more cytotoxic than acyclic poly-
aminoalkylamino derivative 15a. The piperazinyl derivative 16a
is more active than its morpholino isomer 17a. For the 2-phenyl-
ethylamino derivatives, para-hydroxy 18a and meta, para-dime-
thoxy 20a are equally active against the growth of cancer cells
while meta, para-dihydroxy 19a was much less active. It is worth
to mention that compound 20a is active against the growth of HeLa
3. Pharmacological results and discussion
All the synthesized indolo[3,2-c]quinoline derivatives were
evaluated in vitro against a panel of four cancer cell lines including
human cervical epithelioid carcinoma (HeLa), non-small cell lung
cancer (A549), hepatocelluar carcinoma (SKHep), and prostate can-
cer (PC-3) using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide) assay. The skin Detroit 551 normal
fibroblast cells were also evaluated since a potential anticancer
drug candidate should selectively affect only tumor cells and not
somatic cells. The concentration that inhibited the growth of 50%
of cells (IC50) was determined from the linear portion of the curve
by calculating the concentration of agent that reduced absorbance
in treated cells, compared to control cells, by 50%. The IC50 results
of indolo[3,2-c]quinoline derivatives are summarized in Table 1.
For the C6 anilino-substituted indolo[3,2-c]quinoline derivatives,
(11H-Indolo[3,2-c]quinolin-6-yl)phenylamine (6a) was inactive.
Structural optimization of 6a by the introduction of a hydroxyl
group at the anilino-moiety resulted in the enhancement of anti-
proliferative activity in which the cytotoxicity decreased in an or-
der of para-OH, 7a > meta-OH, 8a > ortho-OH, 9a. The only
exception was HeLa cell in which 9a was more active than 8a.
Compound 7a was more active than 10a indicated a substituent
of para-OH (an H-bonding donor) is more favorable than a para-
with an IC50 of 0.52
lM, but is inactive against the growth of De-
troit 551 with an IC50 of 19.32
l
M. For the bis-indolo[3,2-c]quino-
lines 21–26, compound 23 is more active than Doxorubicin against
the growth of HeLa and A549 with IC50 values of 0.072 and
0.084 lM, respectively. N,N-Bis-[3-(11H-indolo[3,2-c]quinolin-6-
yl)aminopropyl]amine hydrochloride (25) is more active than its
N-methyl derivative 26 and the positive Doxorubicin against the
growth of all cancer cells tested.
To elucidate their mechanism of actions, compound 25 was se-
lected for evaluation of its effect on the topoisomerase I (topo I)
inhibitory activities and the unwinding of supercoiled plasmid
DNA as shown in Figure 1A. Compound 25 at 10 lM can almost
completely inhibit topo I activity in vitro. However, Figure 1B
shows that compound 25 did not inhibit topo I activity at the
low concentration (form 0.1 to 1.0 lM). Our results also show that
HN
NH
HN
(i)
+
N
N
H
(CH2)n
N
N
N
N
(CH2)nNH2
H
H
21 n = 6
22 n = 7
23 n = 6
24 n = 7
NH
N
HN
N
(ii)
5a
N
H
N
H
N
H
25
NH
N
HN
N
(iii)
N
H
N
N
H
Me
26
Scheme 3. Reagents and conditions: (i) Polymethylenediamine (n = 6 and n = 7), ethoxyethanol in a sealed tube, 130–140 °C, 4 days; (ii) dipropylenetriamine, pyridine, and
ethoxyethanol in a sealed tube, 100–120 °C, 4 days; (iii) N,N-bis(3-aminopropyl)methylamine, pyridine, and ethoxyethanol in a sealed tube, 100–120 °C, 4 days.

C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
1951
Table 1
Antiproliferative activity (IC50, l
M)a of indolo[3,2-c]quinoline derivatives
Compd
HeLa
A549
SKHep
PC-3
Detroit 551
DOX
6a
0.33 0.05
>30
2.23 0.30
>30
0.09 0.03
>30
1.76 0.71
>30
ND
ND
7a
8a
9a
1.54 0.27
10.34 2.83
4.69 1.63
11.40 0.59
0.62 0.14
0.61 0.16
0.85 0.28
0.74 0.06
1.49 0.12
0.65 0.15
0.68 0.27
5.35 0.48
0.87 0.10
4.32 0.77
0.59 0.09
1.33 0.43
3.77 0.49
0.52 0.34
0.31 0.05
1.19 0.17
0.072 0.01
2.67 0.33
0.068 0.02
0.89 0.18
3.87 1.57
10.35 1.51
17.69 6.40
10.94 1.80
0.99 0.07
0.83 0.04
0.50 0.03
0.70 0.08
0.93 0.05
1.03 0.09
0.53 0.02
6.20 0.12
1.13 0.08
6.73 0.17
5.74 0.21
9.19 0.76
10.28 0.89
5.39 0.60
0.50 0.07
1.19 0.27
0.084 0.01
2.23 0.39
0.11 0.02
0.43 0.09
5.70 1.17
7.23 1.62
21.89 7.78
7.78 0.84
1.02 0.18
1.60 0.62
0.79 0.08
0.72 0.11
1.31 0.27
2.17 0.37
0.38 0.04
2.67 0.20
1.29 0.02
5.29 0.38
5.24 0.56
7.78 0.07
6.66 0.54
5.83 1.42
1.47 0.13
1.61 0.41
2.51 0.68
2.54 0.50
0.09 0.02
0.28 0.03
5.43 0.90
18.40 3.95
>30
25.43 1.99
42.54 4.37
ND
10a
11a
12a
13a
14a
14b
15a
16a
16b
17a
17b
18a
18b
19a
20a
21
20.98 7.78
5.65 0.24
5.79 1.21
3.65 0.38
5.81 0.87
6.51 0.28
7.98 0.11
1.77 0.12
12.26 1.00
2.84 0.60
6.84 1.12
8.94 0.95
6.32 0.38
15.20 6.68
8.98 1.05
0.59 0.02
6.56 0.75
2.55 1.17
6.25 0.82
0.08 0.05
2.01 0.09
ND
3.15 0.46
2.21 0.14
2.18 0.35
1.74 0.14
ND
3.88 0.22
2.60 0.18
ND
ND
ND
4.30 0.65
ND
ND
19.32 3.13
ND
ND
ND
ND
22
23
24
25
1.98 0.04
2.14 0.18
26
a
Data are presented as the mean sem (standard error of the mean) from four to six separated experiments. Statistical analyses were performed using Bonferroni t-test
method after ANOVA for multigroup comparison and Student’s t-test method for two-group comparison. P = 0.05 was considered significant. Analysis of linear regression (at
least three data within 20–80% inhibition) was used to calculate IC50 values.
compound 25 can bind tightly to DNA and may behave as a typical
intercalating agent in a concentration of 10 M. In Figure 1C, our
results indicated that at a concentration of 10 M, compound 25
which was consistent with the results of DNA fragmentation. The
double-strand breaks of DNA were measured by the following his-
tone H2AX phosphorylation. Western blot analysis confirmed that
l
l
can inhibit topoisomerase II activity effectively when compared
with the positive Doxorubicin at the same concentration. An inter-
esting feature was that compound 25 inhibits topo II in a dose
dependent manner (1.0, 0.3, and 0.1 lM; lanes 3–5 in Fig. 1D).
These results suggested that compound 25 has preferentially
at a concentration of 0.1 lM, compound 25 induced significant
H2AX phosphorylation as shown in Figure 3B. These results indi-
cate that compound 25 exerts its cytotoxicity through the inhibi-
tion of cell cycle progression and caused apoptosis of cancer cells
followed by DNA fragmentation, and consequentially cell death.
inhibited DNA topo II relaxation activity.
4. Conclusion
To investigate the potential cell proliferative inhibition activity
of 25 in cancer cells, we first examined the effect of 25 on cell
proliferation and clonogenic survival in HeLa cells. As shown in
Figure 2A, compound 25 inhibited in HeLa cell proliferation in both
concentration- and time-dependent manners. The maximal prolif-
Certain indolo[3,2-c]quinoline derivatives have been synthe-
sized and evaluated for their antiproliferative activities.
Among them, N,N-bis-[3-(11H-indolo[3,2-c]quinolin-6-yl)-amino-
propyl]amine hydrochloride (25) is more active than its N-methyl
derivative 26 and the positive Doxorubicin against the growth of
all cancer cells tested. Mechanism studies indicated 25 inhibit topo
eration inhibition of 25 at 0.3
was 95–100%. Accordingly, the IC50 value for HeLa cells was
0.068 M. Moreover, we performed in vitro clonogenic assays to
lM (or 1.0 lM) after 48 h treatment
l
I activity only at a high concentration of 10 lM. For the topo II inhib-
itory activity, compound 25 inhibits topo II in a dose dependent
manner and is more active than Doxorubicin at the same concentra-
determine the antitumor activities of 25. As shown in Figure 2B,
our results show that clonogenicity (colony size and number) of
HeLa cells was reduced in a dose-dependent manner after expo-
tion of 10 lM. The clonogenicity (colony size and number) of HeLa
sure to 25 at 0.025, 0.05, 0.1, and 0.3 lM, the number of colonies
cells was reduced in a dose-dependent manner after exposure to
compound 25. Compound 25 caused inhibition of cell cycle progres-
sion in PC-3 cells at 48 h and resulted in a significant increase of the
sub-G1 phase population. Western blot analysis confirmed that
compound 25 induced significant H2AX phosphorylation. These re-
sults show that compound 25 exerts its cytotoxicity through the
inhibition of cell cycle progression and caused apoptosis of cancer
cells followed by DNA fragmentation, and consequentially cell
death.
was 48.2 5.6 (S.D.), 10.5 1.2, 6.0 1.0, and 4.2 0.6, respec-
tively, while in control the number of colonies was 235.0 13.8
(S.D.). This represents significantly decrease in the number of col-
onies formed from cells treated with 25.
To examine the mechanism responsible for compound 25-med-
iated cell proliferation inhibition, cell cycle distribution was
evaluated using flow cytometric analysis. Results from Figure 3A
indicated that treating cells with 25 caused a significant inhibition
of cell cycle progression in PC-3 cells at 48 h, resulting in a signif-
icant increase of the sub-G1 phase population compared with that
of the DMSO control. The observation of cells in sub-G1 phase indi-
cates DNA fragmentation and programmed cell death. Apoptosis
was also evident upon examination of common molecular markers
of apoptosis, including the cleavage of the caspase substrate PARP
from 116 kDa intact form into 85 kDa fragment product and cas-
pase-3 activation. Treatment of 25 induced both of these markers,
5. Experimental
5.1. General
Melting points were determined on an Electrothermal IA9100
melting point apparatus and are uncorrected. Nuclear magnetic

1952
C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
Figure 1. Effect of indolo[3,2-c]quinolines on DNA topoisomerases I and II. (A) Topo I inhibitory activities and the unwinding assay of supercoiled plasmid DNA by
camptothecin (CPT in 10 M) and 25 (10 M). (B) Similar reactions were carried out with 25 (1.0, 0.3, and 0.1 M in lines 3–5). (C) Topo II inhibitory activity of compound 25.
Lines 1 and 2 are both control experiments with supercoiled DNA, untreated or treated with topo II, respectively; lines 3–4 correspond to supercoiled pBR322 reacted with
topo II and 10 M of doxorubicin and 25. (D) Similar reactions were carried out with 25 (1.0, 0.3, and 0.1 M in lines 3–5).
l
l
l
l
l
resonance (1H and 13C) spectra were recorded on a Varian-Unity-
400 spectrometer. Chemical shifts were expressed in parts per mil-
lion (d) with tetramethylsilane (TMS) as an internal standard. Thin-
layer chromatography was performed on silica gel 60 F-254 plates
purchased from E. Merck and Co., The elemental analyses were
performed in the Instrument Center of National Science Council
at National Cheng-Kung University and National Taiwan University
using Heraeus CHN-O Rapid EA, and all values are within 0.4% of
the theoretical compositions.
107.08, 107.45 (J = 24.2 Hz), 111.84, 112.60 (J = 9.1 Hz), 117.04
(J = 24.3 Hz), 117.94 (J = 8.4 Hz), 120.97, 121.27, 124.22, 124.44,
134.64, 137.75, 139.95 (J = 3.1 Hz), 156.98 (J = 235.7 Hz), 159.65.
Anal. Calcd for C15H9FN2O: C, 71.42; H, 3.60; N, 11.11. Found: C,
71.11; H, 3.80; N 10.92.
A mixture of 4b (1.51 g, 6.0 mmol) and POCl3 (30 mL) was re-
fluxed for 8 h (TLC monitoring). After cooling, the reaction mixture
was poured into ice-H2O (150 mL) and the concentrated NaOH
solution was added until a pH of 10 was reached. The precipitate
thus formed was collected and crystallized from MeOH/DMF to
give 5b (1.48 g, 91% yield) as a yellow solid. Mp: 250–251 °C. IR
(KBr): 3265, 1630, 1591, 1513, 1458, 1357, 1241, 1191. 1H NMR
(DMSO-d6): 7.41–7.45 (m, 1H, Ar-H), 7.57–7.62 (m, 1H, Ar-H),
7.68 (ddd, 1H, J = 8.8, 8.8, 2.4 Hz, 3-H), 7.79 (d, 1H, J = 8.0 Hz, Ar-
H), 8.11 (dd, 1H, J = 9.2, 5.6 Hz, 4-H), 8.30 (dd, 1H, J = 9.2, 2.4 Hz,
1-H), 8.43 (d, 1H, J = 8.0 Hz, Ar-H), 13.11 (br s, 1H, NH). 13C NMR
(DMSO-d6): 106.46 (J = 24.2 Hz), 111.59, 112.30, 117.30
(J = 10.6 Hz), 118.50 (J = 25.3 Hz), 120.58, 121.42 (J = 2.3 Hz),
126.43, 131.18 (J = 10.1 Hz), 138.80, 141.31, 141.45, 141.50,
143.92, 159.53 (J = 243.3 Hz). Anal. Calcd for C15H8FClN2Á0.3H2O:
C, 65.23; H, 3.14; N, 10.14. Found: C, 65.21; H, 3.52; N, 10.20.
5.1.1. 6-Chloro-2-fluoro-11H-indolo[3,2-c]quinoline (5b)
A mixture of 5-fluoroisatin (0.83 g, 5 mmol), 2-aminobenzyl-
amine (1.22 g, 10 mmol), and acetic acid (30 mL) was refluxed for
8 h (TLC monitoring), the reaction mixture was poured into water.
The solid thus formed was collected and crystallized from MeOH to
give 0.85 g (67%) of 2-fluoro-11H-indolo[3,2-c]quinolin-6-one (4b).
Mp: >350 °C. IR (KBr): 3240, 1623, 1556, 1452, 1202. 1H NMR
(DMSO-d6): 7.28–7.32 (m, 1H, Ar-H), 7.39–7.45 (m, 2H, Ar-H),
7.51 (dd, 1H, J = 8.8, 4.8 Hz, 4-H), 7.66 (d, 1H, J = 8.4 Hz, Ar-H),
8.02 (dd, 1H, J = 9.6, 2.8 Hz, 1-H), 8.23 (d, 1H, J = 7.6 Hz, Ar-H),
11.54 (br s, 1H, NH), 12.60 (br s, 1H, NH). 13C NMR (DMSO-d6):

C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
1953
Figure 2. The effects of 25 on cell proliferative inhibition and colony formation in HeLa cells. (A) Cell proliferative inhibition effect of 25 in HeLa. Cell growth inhibition
activity of 25 was assessed by MTT. Adherent cells proliferated in 96-well plates (3000 cells/well) were incubated with different concentrations of (0.025, 0.05, 0.1, 0.3 and
1.0 lM) at various time intervals. (B) Representative dishes by colony forming assay.
5.1.2. (11H-Indolo[3,2-c]quinolin-6-yl)phenylamine (6a)
A mixture of 6-chloro-2-methoxy-11H-indolo[3,2-c]quinoline
(5a, 0.51 g, 2 mmol),12 aniline (0.28 g, 3 mmol), and 2-butanol
(20 mL) was refluxed for 18 h (TLC monitoring). The solvent was
removed in vacuo and the residue was suspended in H2O (50 mL).
The resulting precipitate that separated was collected, washed with
H2O, and dried to give a crude solid, which was recrystallized from
MeOH to give 6a (0.40 g, 65%). Mp: 339–340 °C. IR (KBr): 3059,
1643, 1587, 1539, 1496, 1434, 1388, 1355, 1216, 1155. 1H NMR
(DMSO-d6): 7.36–7.41 (m, 2H, Ar-H), 7.54–7.67 (m, 6H, Ar-H),
7.76–7.85 (m, 2H, Ar-H), 8.03 (d, 1H, J = 8.0 Hz, Ar-H), 8.32 (d, 1H,
J = 8.4 Hz, Ar-H), 8.69 (d, 1H, J = 7.2 Hz, Ar-H), 10.20 (br s, 1H, NH),
12.53 (br s, 1H, NH). 13C NMR (DMSO-d6): 102.16, 112.60, 113.52,
120.00, 121.19, 121.87 (2C), 123.01, 124.18, 125.14, 125.94,
126.32, 129.20, 129.82 (2C), 130.80, 136.03, 137.45, 138.94,
143.06, 148.34. Anal. Calcd for C21H15N3Á0.8H2O: C, 71.06; H, 4.83;
N, 11.84. Found: C, 71.41; H, 4.75; N, 11.88.
Compounds 7a–10a were prepared from 5a in analogy to that
described for compound 6a, using the appropriate substituted-
aniline.
5.1.3. 4-(11H-Indolo[3,2-c]quinolin-6-ylamino)phenol
hydrochloride (7a)
Yield (60%). Mp: 310–311 °C (MeOH). IR (KBr): 3083, 1637,
1608, 1510, 1455, 1398, 1266, 1228, 1163. 1H NMR (DMSO-d6):
7.98–7.01 (m, 2H, Ar-H), 7.39–7.45 (m, 3H, Ar-H), 7.54–7.75 (m,
3H, Ar-H), 7.82 (d, 1H, J = 8.0 Hz, Ar-H), 8.04 (d, 1H, J = 8.4 Hz, Ar-
H), 8.54 (d, 1H, J = 8.4 Hz, Ar-H), 8.63 (d, 1H, J = 8.0 Hz, Ar-H),
9.95 (br s, 2H, NH, OH), 12.21 (br s, 1H, NH), 14.02 (br s, 1H,
HCl). 13C NMR (DMSO-d6): 101.00, 112.57, 113.16, 116.45 (2C),
119.55, 121.25, 121.51, 121.91, 122.88, 124.84, 125.75, 126.98,
127.70, 130.63, 135.62, 138.76(2C), 142.45, 149.15, 156.99. Anal.
Calcd for C21H15N3OÁ1.0H2OÁ1.0HCl: C, 66.38; H, 4.43; N, 11.06.
Found: C, 66.01; H, 4.77; N, 11.00.

1954
C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
Figure 3. Effect of 25-mediated apoptosis and cell death in PC-3 cells associated with the apoptotic pathways. (A) Flow cytometric analysis of PC-3 cells. After treatment with
DMSO, 0.1, or 0.3 M of 25 for 48 h, cells were harvested, fixed, and stained with propidium iodide as described in the Experimental Section prior to analysis by flow
cytometry. (B) Western blot analysis of the effect of 25 on DNA damage (phophorylation of -H2AX), induce caspase-3 activation and PARP cleavage in PC-3 cells. Cells were
treated with 0.1 M of 25 and 10 M of CPT (camptothecin) for 48 h, respectively. The protein contents were normalized by probing the same membrane with b-actin
antibody. M: mock, D: DMSO.
l
c
l
l
5.1.4. 3-(11H-Indolo[3,2-c]quinolin-6-ylamino)phenol
hydrochloride (8a)
H), 8.09 (d, 1H, J = 8.0 Hz, Ar-H), 8.57 (d, 1H, J = 8.0 Hz, Ar-H),
8.66 (d, 1H, J = 7.2 Hz, Ar-H), 9.85 (br s, 1H, OH), 10.28 (br s, 1H,
NH), 12.20 (br s, 1H, NH), 14.07 (br s, 1H, HCl). 13C NMR (DMSO-
d6): 100.98, 112.68, 113.06, 117.09, 119.17, 119.77, 121.28,
121.48, 122.04, 122.56, 123.03, 125.05, 125.89, 128.42, 129.15,
130.82, 135.23, 138.82, 142.34, 148.74, 152.87. Anal. Calcd for
C21H15N3OÁ1.9H2OÁ1.0HCl: C, 63.66; H, 5.04; N, 10.61. Found: C,
63.50; H, 5.07; N, 10.58.
Yield (62%). Mp: 319–320 °C (MeOH). IR (KBr): 3426, 3178,
1639, 1594, 1457, 1408, 1250, 1215, 1186. 1H NMR (DMSO-d6):
6.84–6.87 (m, 1H, Ar-H), 7.01–7.04 (m, 2H, Ar-H), 7.33–7.85 (m,
6H, Ar-H), 8.07 (d, 1H, J = 8.0 Hz, Ar-H), 8.32 (d, 1H, J = 8.0 Hz, Ar-
H), 8.69 (dd, 1H, J = 8.0, 0.8 Hz, Ar-H), 9.98 (br s, 1H, OH), 10.27
(br s, 1H, NH), 12.95 (br s, 1H, NH), 14.18 (br s. 1H, HCl). 13C
NMR (DMSO-d6): 101.99, 111.53, 112.69, 113.34, 114.07, 114.89,
119.29, 121.26, 121.95, 122.07, 123.11, 125.41, 126.10. 130.72,
131.05, 135.13, 137.81, 138.97, 143.16, 148.12, 158.77. Anal. Calcd
for C21H15N3OÁ1.5H2OÁ1.0HCl: C, 64.54; H, 4.96; N, 10.75. Found: C,
64.27; H, 5.00; N, 10.65.
5.1.6. (11H-Indolo[3,2-c]quinolin-6-yl)-(4-
methoxyphenyl)amine (10a)
Yield (60%). Mp: 317–318 °C (MeOH). IR (KBr): 3064, 1639,
1605, 1509, 1455, 1397, 1247, 1175. 1H NMR (DMSO-d6): 3.85 (s,
1H, OCH3), 7.11–7.15 (m, 2H, Ar-H), 7.38–7.75 (m, 5H, Ar-H),
7.82 (d, 1H, J = 8.0 Hz, Ar-H), 8.00 (d, 1H, J = 8.0 Hz, Ar-H), 8.52
(d, 1H, J = 8.4 Hz, Ar-H), 8.62 (d, 1H, J = 7.2 Hz, Ar-H), 9.83 (br s,
1H, NH), 12.38 (br s, 1H, NH). 13C NMR (DMSO-d6): 55.44,
101.44, 112.48, 113.39, 114.93 (2C), 120.28, 121.21, 121.54,
5.1.5. 2-(11H-Indolo[3,2-c]quinolin-6-ylamino)phenol (9a)
Yield (61%). Mp: 339–340 °C (MeOH). IR (KBr): 3176, 1641,
1604, 1508, 1456, 1402, 1296, 1243, 1213, 1107. 1H NMR
(DMSO-d6): 7.00–7.77 (m, 8H, Ar-H), 7.84 (d, 1H, J = 8.0 Hz, Ar-

C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
1955
121.73, 122.77, 124.62, 125.66, 126.93, 129.54, 130.43, 135.21,
138.74 (2C), 142.43, 149.13, 157.95. Anal. Calcd for
C22H17N3OÁ1.1H2OÁ1.0HCl: C, 66.76; H, 5.11; N, 10.62. Found: C,
66.91; H, 5.15; N, 10.56.
Anal. Calcd for C19H20N4Á1.5HClÁ1.5H2O: C, 56.75; H, 6.15; N,
13.93. Found: C, 56.59; H, 5.76; N, 14.21.
5.1.11. 2-((2-Fluoro-11H-indolo[3,2-c]quinolin-6-
ylamino)methylamino)ethanol hydrochloride (14b)
5.1.7. 2-(11H-Indolo[3,2-c]quinolin-6-ylamino)ethanol
hydrochloride (11a)
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/10) and crystallized from EtOH in 76% yield.
Mp: 260 °C (dec). IR (KBr): 3386, 3179, 1585, 1530, 1501, 1458,
1416, 1371, 1220, 1196. 1H NMR (DMSO-d6): 3.13–3.17 (m, 2H,
CH2), 3.365–3.38 (m, 2H, CH2), 3.72 (br s, 2H, NHCH2), 3.97–4.00
(m, 2H, CH2O), 5.34 (br s, 1H, OH), 7.05 (br s, 1H, NH), 7.31–7.48
(m, 3H, Ar-H), 7.70 (d, 1H, J = 8.0 Hz, Ar-H), 7.81 (dd, 1H, J = 8.8,
5.2 Hz, 4-H), 8.18 (dd, 1H, J = 10.0, 2.8 Hz, 1-H), 8.50 (d, 1H,
J = 8.4 Hz, Ar-H), 9.28 (br s, 1H, NH), 12.87 (br s, 1H, HCl). 13C
NMR (DMSO-d6): 37.76, 47.69, 48.99, 56.35, 103.08, 106.15
(J = 22.8 Hz), 111.56, 114.30 (J = 9.9 Hz), 116.69 (J = 23.5 Hz),
120.22, 120.95, 121.09, 124.43, 127.96 (J = 8.3 Hz), 138.11, 140.39
(J = 3.8 Hz), 141.87, 152.44, 156.93 (J = 236.5 Hz). Anal. Calcd for
C19H19FN4OÁ1.0HCl: C, 60.88; H, 5.38; N, 14.95. Found: C, 61.00;
H, 5.59; N, 14.57.
A
mixture of 5a (0.51 g, 2 mmol), ethanolamine (0.24 g,
4 mmol), and 2-ethoxyethanol (20 mL) was heated at 140–150 °C
for 48 h (TLC monitoring). The solvent was removed in vacuo and
the residue was suspended in MeOH (50 mL). The resulting precip-
itate that separated was collected, washed with H2O, and dried to
give a crude solid, which was recrystallized from MeOH to give 11a
(0.38 g, 69%). Mp: 339–340 °C. IR (KBr): 3383, 3080, 1647, 1616,
1457, 1419, 1348, 1224, 1180. 1H NMR (DMSO-d6 + TFA-d): 3.86–
3.89 (m, 2H, CH2O), 4.00–4.03 (m, 2H, CH2N), 7.44–7.81 (m, 5H,
Ar-H), 8.18 (d, 1H, J = 8.4 Hz, Ar-H), 8.38 (br s, 1H, NH), 8.47–8.50
(m, 1H, Ar-H), 8.58 (d, 1H, J = 8.0 Hz, Ar-H), 12.40 (br s, 1H, NH),
13.59 (s, 1H, HCl). 13C NMR (DMSO-d6): 43.91, 60.63, 101.83,
111.88, 113.71, 120.62 (2C), 120.69, 121.29, 122.06, 123.66,
124.47, 128.91, 138.27, 140.95, 142.44, 152.16. Anal. Calcd for
C17H15N3OÁ1.0HCl: C, 65.07; H, 5.14; N, 13.39. Found: C, 65.02; H,
5.22; N, 13.36.
5.1.12. N-(2-(2-Aminoethylamino)ethyl)-(11H-indolo[3,2-
c]quinolin-6-yl)amine hydrochloride (15a)
Compounds 12a–14a, 14b, 15a–20a, and 16b–18b were pre-
pared from 5a or 5b in analogy to that described for compound
11a, using the appropriate substituted-alkylamines.
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/3) and crystallized from EtOH in 57% yield. Mp:
202–203 °C. IR (KBr): 3351, 1647, 1615, 1526, 1453, 1343, 1216.
1H NMR (DMSO-d6): 2.93–2.97 (m, 4H, 2 Â CH2), 3.06 (t, 2H,
J = 6.0 Hz, CH2NH2), 3.85 (t, 2H, J = 6.0 Hz, CH2NH), 7.27–7.53 (m,
4H, Ar-H), 7.66–7.70 (m, 2H, Ar-H), 8.31–8.42 (m, 2H, Ar-H),
12.71 (br s, 1H, NH). 13C NMR (DMSO-d6): 37.97, 39.66, 45.94,
48.06, 102.69, 111.49, 114.21, 120.10, 120.80, 121.03, 121.33,
121.76, 123.96, 126.02, 128.10, 138.13, 140.81, 145.64, 152.94.
Anal. Calcd for C19H20N4Á2.0HClÁ0.4H2O: C, 57.11; H, 6.00; N,
17.53. Found: C, 57.47; H, 6.30; N, 17.21.
5.1.8. N-(2-(Dimethylamino)ethyl)-(11H-indolo[3,2-c]quinolin-
6-yl)amine (12a)
The title compound was crystallized from EtOH in 69% yield.
Mp: 85–87 °C. 1H NMR (DMSO-d6): 2.32 (s, 6H, N(CH3)2), 2.68 (t,
2H, J = 6.4 Hz, CH2N), 3.80 (q, 2H, J = 6.4 Hz, NHCH2), 6.55 (br s,
1H, NH), 7.27–7.53 (m, 4H, Ar-H), 7.66–7.68 (m, 2H, Ar-H), 8.24–
8.27 (m, 2H, Ar-H), 12.53 (br s, 1H, NH). 13C NMR (DMSO-d6):
38.29, 45.21 (2C), 58.31, 102.68, 111.58, 114.15, 120.26 (2C),
120.92, 121.39, 121.60, 123.98, 126.23, 128.11, 138.15, 140.75,
145.98, 152.90. Anal. Calcd for C19H20N4Á0.7H2O: C, 71.79; H,
6.80; N, 17.67. Found: C, 71.94; H, 6.77; N, 17.80.
5.1.13. N-(11H-Indolo[3,2-c]quinolin-6-yl)-2-(piperazin-1-
yl)ethanamine (16a)
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/10 to 1/3) and crystallized from MeOH in 29%
yield. Mp: 210–211 °C. IR (KBr): 3402, 1570, 1526, 1451, 1344,
1267, 1120. 1H NMR (DMSO-d6): 2.47 (br s, 4H, piperazinyl-H),
2.68 (t, 2H, J = 6.4 Hz, CH2N), 2.77 (br s, 4H, piperazinyl-H), 3.75–
3.80 (m, 2H, NHCH2), 6.56 (t, 1H, J = 4.8 Hz, NH), 7.26–7.52 (m,
4H, Ar-H), 7.65–7.68 (m, 2H, Ar-H), 8.23–8.26 (m, 2H, Ar-H),
12.50 (br s, 1H, NH). 13C NMR (DMSO-d6): 37.37, 45.85 (2C),
54.01 (2C), 57.27, 102.70, 111.68, 114.17, 120.02, 120.26, 120.88,
121.38, 121.58, 123.98, 126.28, 128.08, 138.19, 140.69, 146.14,
152.91. Anal. Calcd for C21H23N5Á1.0H2O: C, 69.40; H, 6.93; N,
19.27. Found: C, 69.68; H, 6.92; N, 19.00.
5.1.9. N-(3-(Dimethylamino)propyl)-(11H-indolo[3,2-
c]quinolin-6-yl)amine (13a)
The title compound was crystallized from EtOH in 66% yield.
Mp: 73–75 °C. 1H NMR (DMSO-d6): 1.90 (quin, 2H, J = 6.4 Hz,
CH2), 2.27 (s, 6H, N(CH3)2), 2.34–2.48 (m, 2H, CH2N), 3.73–3.78
(m, 2H, NHCH2), 7.14 (br s, 1H, NH), 7.25–7.52 (m, 4H, Ar-H),
7.65–7.68 (m, 2H, Ar-H), 8.24–8.28 (m, 2H, Ar-H), 12.48 (br s, 1H,
NH). 13C NMR (DMSO-d6): 26.18, 40.46, 45.33 (2C), 58.31, 102.71,
111.48, 114.10, 120.10, 120.14, 120.69, 121.48, 121.54, 123.86,
126.18, 128.00, 138.09, 140.67, 146.16, 152.87. Anal. Calcd for
C20H22N4Á0.2H2O: C, 74.60; H, 7.01; N, 17.40. Found: C, 74.57; H,
7.15; N, 17.45.
5.1.14. 2-Fluoro-N-(piperazin-1-ylethyl)-11H-indolo[3,2-
c]quinolin-6-amine hydrochloride (16b)
5.1.10. 2-(2-(11H-Indolo[3,2-c]quinolin-6-
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/10 to 1/3) and crystallized from MeOH in 31%
yield. Mp: 278–279 °C. IR (KBr): 3383, 1645, 1614, 1458, 1418,
1115. 1H NMR (DMSO-d6): 2.77–2.87 (m, 8H, piperazinyl-H),
3.05 (br s, 2H, CH2N), 3.52 (m, 2H, NHCH2), 7.36–7.54 (m, 3H,
Ar-H), 7.74 (d, 1H, J = 8.4 Hz, Ar-H), 7.91 (dd, 1H, J = 9.2, 5.2 Hz,
4-H), 7.98 (d, 1H, J = 8.0 Hz, Ar-H), 8.28 (dd, 1H, J = 9.6, 2.8 Hz,
1-H), 12.45 (br s, 1H, NH). 13C NMR (DMSO-d6): 35.58, 48.59,
48.93, 52.50 (2C), 54.30, 106.25 (J = 24.2 Hz), 106.37, 111.94,
115.95 (J = 9.8 Hz), 117.17 (J = 25.1 Hz), 120.76, 120.94, 121.67,
124.93, 130.09 (J = 8.4 Hz), 138.57, 141.46, 141.66 (J =
ylamino)ethylamino)ethanol hydrochloride (14a)
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/10) and crystallized from EtOH in 61% yield.
Mp: 273–274 °C (dec). IR (KBr): 3337, 3077, 1643, 1612, 1456,
1419, 1343, 1219, 1086. 1H NMR (DMSO-d6): 3.14 (br s, 2H, CH2),
3.41–3.42 (m, 2H, CH2), 3.73 (t, 2H, J = 5.6 Hz, NHCH2), 4.43–4.45
(m, 2H, CH2O), 5.34 (br s, 1H, OH), 7.42–7.86 (m, 5H, Ar-H),
8.57–8.74 (m, 4H, Ar-H) 9.18 (br s, 2H, NH), 13.01 (br s, 1H, NH),
13.90 (br s, 1H, HCl). 13C NMR (DMSO-d6): 30.69, 45.88, 49.28,
56.44, 100.55, 112.63, 112.84, 118.93, 121.25 (2C), 121.93,
122.78, 125.12, 125.76, 130.60, 135.30, 138.53, 141.48, 149.33.

1956
C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
4.5 Hz), 156.83, 158.15 (J = 238.7 Hz). Anal. Calcd for
C21H22FN5Á0.5HClÁ2.0H2O: C, 60.39; H, 6.39; N, 16.77. Found: C,
60.52; H, 6.40; N, 16.81.
(J = 3.0 Hz), 142.90, 152.43, 155.57, 156.74 (J = 235.0 Hz). Anal.
Calcd for C23H18FN3OÁ1.5H2O: C, 69.33; H, 5.31; N, 10.55. Found:
C, 69.15; H, 5.31; N, 10.22.
5.1.15. N-(Morpholinoethyl)-11H-indolo[3,2-c]quinolin-6-
amine (17a)
5.1.19. 6-[2-(3,4-Dihydroxyphenyl)ethylamino]-11H-
indolo[3,2-c]quinoline hydrochloride (19a)
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/5) and crystallized from EtOH in 26% yield. Mp:
132–133 °C. IR (KBr): 3432, 3154, 1592, 1518, 1449, 1402, 1366,
1224, 1197, 1126. 1H NMR (DMSO-d6): 2.49–2.54 (m, 4H, mor-
pholinyl-H), 2.72 (t, 2H, J = 6.4 Hz, CH2N), 3.62–3.66 (m, 4H, mor-
pholinyl-H), 3.76–3.86 (m, 2H, NHCH2), 6.56 (t, 1H, J = 5.2 Hz,
NH), 7.25–7.70 (m, 6H, Ar-H), 8.23–8.30 (m, 2H, Ar-H) 12.48 (br
s, 1 h, NH). 13C NMR (DMSO-d6): 37.22, 53.22 (2C), 57.10, 66.36
(2C), 102.59, 111.51, 114.04, 120.05, 120.14, 120.76, 121.28,
121.44, 123.86, 126.17, 127.95, 138.04, 140.59, 145.98, 152.76.
Anal. Calcd for C21H22N5OÁ1.0H2O: C, 69.21; H, 6.64; N, 15.37.
Found: C, 69.07; H, 6.65; N, 15.38.
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/10) and crystallized from MeOH in 15% yield.
Mp: 202–203 °C. IR (KBr): 3418, 3180, 1648, 1613, 1526, 1459,
1346, 1251, 1116. 1H NMR (DMSO-d6): 2.96 (t, 2H, J = 7.4 Hz,
CH2), 4.05–4.10 (m, 2H, NCH2), 6.62–6.68 (m, 2H, Ar-H), 6.80–
6.81 (d, 1H, J = 1.6 Hz, Ar-H), 7.42–7.81 (m, 5H, Ar-H), 8.27–8.36
(m, 2H, Ar-H and NH), 8.50 (d, 1H, J = 8.0 Hz, Ar-H), 8.54 (d, 1H,
J = 8.0 Hz, Ar-H), 8.80 (br s, 1H, OH), 8.84 (br s, 1H, OH), 12.44
(br s, 1H, NH), 13.80 (br s, 1H, HCl). 13C NMR (DMSO-d6): 33.88,
43.93, 100.22, 112.66, 112.80, 115.51, 116.45, 118.86, 119.62,
120.74, 121.19, 121.95, 122.78, 124.81, 125.64, 129.14, 130.64,
138.53, 141.37, 143.81, 145.18, 149.24, 155.96. Anal. Calcd for
C23H19N3O2Á1.0HClÁ2.0H2O: C, 62.52; H, 5.47; N, 9.51. Found: C,
62.58; H, 5.45; N, 9.63.
5.1.16. 2-Fluoro-N-(morpholinoethyl)-11H-indolo[3,2-
c]quinolin-6-amine (17b)
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/5) and crystallized from EtOH in 38% yield. Mp:
116–117 °C. IR (KBr): 3355, 3282, 1595, 1527, 1454, 1413, 1338,
1195, 1112. 1H NMR (DMSO-d6): 2.60 (br s, 4H, morpholinyl-H),
2.77 (m, 2H, CH2N), 3.64–3.67 (m, 4H, morpholinyl-H), 3.80–3.84
(m, 2H, NHCH2), 6.62 (br s, 1H, NH), 7.33–7.48 (m, 3H, Ar-H),
7.68–7.73 (m, 2H, Ar-H), 8.06 (dd, 1H, J = 9.6, 3.2 Hz, 1-H), 8.31
(d, 1H, J = 8.0 Hz, Ar-H), 12.54 (br s, 1H, NH). 13C NMR (DMSO-
d6): 37.26, 53.21 (2C), 57.12 (2C), 66.24, 103.15, 105.96
(J = 22.7 Hz), 111.73, 114.11 (J = 9.1 Hz), 116.65 (J = 23.5 Hz),
102.43 (2C), 121.13, 124.40, 128.15 (J = 7.6 Hz), 138.17, 140.22
(J = 3.8 Hz), 142.68, 152.45, 156.77 (J = 235.7 Hz). Anal. Calcd for
C21H21FN4OÁ2.0H2O: C, 62.99; H, 6.29; N, 13.99. Found: C, 62.63;
H, 6.50; N, 13.79.
5.1.20. 6-[2-(3,4-Dimethoxyphenyl)ethylamino]-11H-
indolo[3,2-c]quinoline hydrochloride (20a)
The title compound was crystallized from MeOH in 66% yield.
Mp: 231–232 °C. IR (KBr): 3411, 3055, 1648, 1612, 1514, 1458,
1421, 1347, 1261, 1157. 1H NMR (DMSO-d6): 3.07 (t, 2H,
J = 7.4 Hz), 3.01 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 4.21–4.26 (m,
2H, NCH2), 6.78 (d, 1H, J = 8.0 Hz, Ar-H), 6.86 (dd, 1H, J = 1.6,
8.0 Hz, Ar-H), 7.05 (d, 1H, J = 1.6 Hz, Ar-H), 7.42–7.81 (m, 5H, Ar-
H), 8.34 (br s, 1H, NH), 8.38 (d, 1H, J = 8.0 Hz, Ar-H), 8.51–8.55
(m, 2H, Ar-H), 12.60 (br s, 1H, NH), 13.80 (br s, 1H, HCl). 13C
NMR (DMSO-d6): 34.32, 43.89, 55.28, 55.49, 100.14, 111.88,
112.66, 112.70, 113.06, 118.74, 120.68, 120.98, 121.18, 121.97,
122.72, 124.84, 125.71, 130.61, 130.86, 135.24, 138.53, 141.38,
147.37, 148.58, 149.21. Anal. Calcd for C25H23N3O2Á1.0HClÁ1.2H2O:
C, 65.92; H, 5.84; N, 9.22. Found: C, 65.89; H, 5.93; N, 9.29.
5.1.17. 6-[2-(4-Hydroxyphenyl)ethylamino]-11H-indolo[3,2-
c]quinoline hydrochloride (18a)
5.1.21. N-[6-(11H-indolo[3,2-c]quinolin-6-ylamino)hexyl]-11H-
indolo[3,2-c]quinolin-6-amine (21) and N-(6-aminohexyl)-11H-
indolo[3,2-c]quinolin-6-amine (23)
The title compound was crystallized from MeOH in 30% yield.
Mp: 320–321 °C. IR (KBr): 3415, 3088, 1645, 1611, 1513,
1458, 1426, 1345, 1218, 1170. 1H NMR (DMSO-d6): 3.03 (t, 2H,
J = 7.4 Hz, CH2), 4.10–4.16 (m, 2H, NCH2), 6.67–6.71 (m, 2H, Ar-
H), 7.18–7.22 (m, 2H, Ar-H), 7.42–7.47 (m, 1H, Ar-H), 7.54–7.63
(m, 2H, Ar-H), 7.76–7.82 (m, 2H, Ar-H), 8.32 (d, 1H, J = 8.4 Hz, Ar-
H), 8.38 (br s, 1H, NH), 8.50–8.54 (m, 2H, Ar-H), 9.29 (br s, 1H,
OH), 12.47 (br s, 1H, NH), 13.80 (br s, 1H, HCl). 13C NMR (DMSO-
d6): 33.69, 43.96, 100.16, 112.74, 113.08, 115.14 (2C), 118.74,
120.73, 121.20, 122.04, 122.79, 124.94, 125.74, 128.39, 129.96
(2C), 130.72, 135.21, 138.56, 141.42, 149.16, 155.93. Anal. Calcd
for C23H19N3OÁ1.0HClÁ1.2H2O: C, 67.14; H, 5.49; N, 10.21. Found:
C, 67.14; H, 5.47; N, 10.20.
A mixture of 5a (0.51 g, 2 mmol), 1,6-diaminohexane (0.47 g,
4 mmol), and 2-ethoxyethanol (20 mL) was heated in a sealed steel
bomb at 130–140 °C for 4 days (TLC monitoring). The reaction mix-
ture was partitioned between H2O (50 mL) and CH2Cl2 (50 mL). The
organic layer was separated, dried over MgSO4, and evaporated.
The resulting residue was purified by column chromatography
(MeOH/CH2Cl2 1/5 to 1/3) to give two fractions. The first fraction
was crystallized from EtOH to give bis-substituted product 21
(0.14 g, 13%) as
a yellow solid. Mp: 249–250 °C. IR (KBr):
3338,3173, 1643, 1614, 1528, 1455, 1417, 1217. 1H NMR (DMSO-
d6): 1.57–1.84 (m, 8H, 4 Â CH2), 3.91 (q, 4H, J = 6.0 Hz, 2 Â NHCH2),
7.34–7.38 (m, 2H, Ar-H), 7.47–7.51 (m, 4H, Ar-H), 7.63–7.67 (m,
2H, Ar-H), 8.23 (br s, 2H, 2 Â NH), 8.47 (d, 2H, J = 8.0, 1.6 Hz, Ar-
H), 8.55 (d, 2H, J = 8.0 Hz, Ar-H), 12.60 (br s, 1H, HCl), 13.45 (br s,
2H, 2 Â NH). 13C NMR (DMSO-d6): 26.05, 28.71, 41.84, 100.86,
112.30, 113.18, 120.78, 121.28, 121.39, 122.50, 123.60, 125.10,
5.1.18. 4-[2-(2-Fluoro-11H-indolo[3,2-c]quinolin-6-
ylamino)ethyl]phenol (18b)
The title compound was purified by column chromatography
(MeOH/CH2Cl2 1/10) and crystallized from EtOH in 13% yield.
Mp: 252–253 °C. IR (KBr): 3458, 3328, 1585, 1535, 1513, 1455,
1415, 1239, 1192. 1H NMR (DMSO-d6): 2.97 (t, 2H, J = 7.2 Hz,
CH2), 4.81–4.87 (m, 2H, NCH2), 6.63 (br s, 1H, NH), 6.71–6.75 (m,
2H, Ar-H), 7.15–7.18 (m, 2H, Ar-H), 7.29–7.47 (m, 3H, Ar-H), 7.67
(d, 1H, J = 8.0 Hz, Ar-H), 7.74 (dd, 1H, J = 9.2, 5.6 Hz, 4-H), 8.05
(dd, 1H, J = 9.6, 2.8 Hz, 1r-H), 8.32 (d, 1H, J = 8.0 Hz, Ar-H), 9.20
(s, 1H, OH), 12.48 (br s, 1H, NH). 13C NMR (DMSO-d6): 34.51,
42.88, 103.26, 105.88 (J = 22.7 Hz), 111.56, 114.06 (J = 9.1 Hz),
115.18 (2C), 116.61 (J = 24.3 Hz), 102.26, 120.87, 121.17, 124.32,
128.33 (J = 7.6 Hz), 129.63 (2C), 130.33, 138.12, 140.26
129.81,
138.40,
141.17,
150.29.
Anal.
Calcd
for
C36H32N6Á2.0HClÁ4.0H2O: C, 62.33; H, 6.10; N, 12.12. Found: C,
62.51; H, 6.11; N, 12.01.
The second fraction was crystallized from EtOH to give mono-
substituted product 23 (0.08 g, 12%) as a yellow solid. Mp: 84–
85 °C. IR (KBr): 3430, 3165, 1618, 1575, 1529, 1453, 1215. 1H
NMR (DMSO-d6): 1.41–1.78 (m, 8H, 4 Â CH2), 2.76 (t, 2H,
J = 7.2 Hz, CH2NH2), 3.70 (m, 2H, NHCH2), 5.59 (br s, 1H, NH),
7.24–7.30 (m, 2H, Ar-H), 7.41 (m, 1H, Ar-H), 7.48 (m, 1H, Ar-H),
7.63–7.67 (m, 2H, Ar-H), 8.28 (m, 1H, Ar-H), 8.40 (m, 1H, Ar-H).

C.-M. Lu et al. / Bioorg. Med. Chem. 18 (2010) 1948–1957
1957
13C NMR (DMSO-d6): 25.84, 26.32, 27.29, 29.24, 38.87, 40.36,
102.69, 111.45, 114.13, 120.05, 120.69, 120.77, 121.43, 121.69,
123.87, 126.22, 128.02, 138.12, 140.75, 146.10, 152.98. Anal. Calcd
for C21H24N4Á1.0HClÁ2.0H2O: C, 62.29; H, 7.229; N, 13.84. Found: C,
62.52; H, 7.26; N, 13.62.
EtOH to give 25 as a white powder. Yield: 21%; Mp: 76–77 °C. IR
(KBr): 3398, 1650, 1614, 1576, 1528, 1452, 1215. 1H NMR
(DMSO-d6): 2.31 (m, 4H), 2.89 (s, 3H), 3.35–3.49 (m, 4H), 4.11
(m, 4H), 7.39 (m, 2H, Ar-H), 7.51 (m, 2H, Ar-H), 7.61 (m, 2H, Ar-
H), 7.71–7.78 (m, 4H, Ar-H),), 8.45–8.50 (m, 4H, Ar-H), 8.55 (br s,
2H, NH), 8.66 (m, 2H, Ar-H), 10.60 (br s, 1H, NH), 12.71 (br s, 1H,
NH), 13.64 (br s, 1H, HCl). 13C NMR (DMSO-d6): 23.42, 40.21,
42.06, 52.19, 100.24, 112.49, 112.82, 118.88, 120.86, 121.15,
121.81, 122.65, 124.72, 125.48, 130.32, 135.40, 138.44, 141.36,
149.13. Anal. Calcd for C37H35N7Á1.2HClÁ0.9H2O: C, 70.30; H, 6.11;
N, 15.51. Found: C, 70.34; H, 6.04; N, 15.42.
5.1.22. N-[7-(11H-Indolo[3,2-c]quinolin-6-ylamino)heptyl]-
11H-indolo[3,2-c]quinolin-6-amine (22) and N-(7-
aminoheptyl)-11H-indolo[3,2-c]quinolin-6-amine (24)
From 5a and 1,7-diaminoheptane as described for 21 and 23.
Compound 22 was obtained in 8% yield (0.09 g) as a pale-yellow
solid. Mp: 330 °C (dec). IR (KBr): 3368, 3066, 1643, 1613, 1458,
1417, 1218. Mp: 330 °C (dec). IR (KBr): 1614, 1643, 3338. UV
(MeOH): 258 (4.89), 295 (4.31), 323 (4.31, 338 (4.39). 1H NMR
(DMSO-d6): 1.48 (br s, 6H, 3 Â CH2), 1.81 (br s, 4H, 2 Â CH2), 3.96
(q, 4H, J = 6.4 Hz, 2 Â NHCH2), 7.36–7.58 (m, 6H, Ar-H), 7.71–7.79
(m, 4H, Ar-H), 8.33 (br s, 2H, 2 Â NH), 8.43 (d, 2H, J = 8.0, 1.6 Hz,
Ar-H), 8.53–8.62 (m, 4H, Ar-H), 12.62 (br s, 2H, HCl), 13.82 (br s,
2H, 2 X NH). 13C NMR (DMSO-d6): 26.07, 28.57, 42.27, 100.27,
112.59, 112.87, 119.28, 120.78, 121.24, 121.82, 122.79, 124.57,
125.50, 130.41, 138.51, 141.31, 149.38. Anal. Calcd for
C37H34N6Á2.0HClÁ2.0H2O: C, 66.16; H, 6.00; N, 12.51. Found: C,
65.95; H, 6.16; N, 12.41.
Acknowledgments
Financial support of this work by the National Science Council
of the Republic of China is gratefully acknowledged. We also thank
National Cancer Institute (NCI) of the United States for the antican-
cer screenings and the National Center for High-Performance Com-
puting for providing computer resources and chemical database
services.
References and notes
Compound 24 was obtained in 32% yield (0.22 g) as a pale-yel-
low solid. Mp: 141–142 °C. IR (KBr): 3412, 3146, 1625, 1573, 1528,
1453, 1214. 1H NMR (DMSO-d6): 1.32–1.43 (m, 8H, 4 Â CH2), 1.76
(quin, 2H, J = 7.2 Hz, CH2), 2.52 (m, 2H, CH2), 3.69 (q, 2H, J = 6.8 Hz,
NHCH2), 6.56 (br s, 1H, NH), 7.24–7.31 (m, 2H, Ar-H), 7.38–7.50 (m,
2H, Ar-H), 7.63–7.66 (m, 2H, Ar-H), 8.23 (dd, 1H, J = 8.0, 1.6 Hz, Ar-
H), 8.38 (d, 1H, J = 8.0 Hz, Ar-H), 12.20–12.80 (br s, 2H, NH2). 13C
NMR (DMSO-d6): 26.47, 26.77, 28.95, 29.35, 32.92, 40.48, 41.50,
102.74, 111.41, 114.09, 120.07, 120.68, 120.76, 121.46, 121.54,
123.88, 126.24, 128.02, 138.09, 140.74, 146.13, 153.00. Anal. Calcd
for C22H26N4Á0.3HClÁ0.4H2O: C, 72.47; H, 7.49; N, 15.37. Found: C,
72.47; H, 7.49; N, 15.36.
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5.1.23. N,N-Bis-[3-(11H-indolo[3,2-c]quinolin-6-
yl)aminopropyl]amine hydrochloride (25)
A mixture of 5a (1.26 g, 5 mmol), dipropylenetriamine (1.31 g,
10 mmol), pyridine (0.8 mL), and ethoxyethanol (20 mL) was
heated in a sealed steel bomb at 100–120 °C for 4 days (TLC mon-
itoring). The reaction mixture was then cooled and evaporated in
vacuo to give a residue which was treated with 1 N HCl (30 mL)
and stirred at room temperature overnight. The resulting residue
was filtered off and the filtrate was treated with NaHCO3 to neu-
tralize HCl. The precipitate thus obtained was collected, purified
by FC (MeOH/CH2Cl2 = 1/5), and crystallized from EtOH to give
25 (0.45 g, 16%) as a white powder. Mp: 107–108 °C. IR (KBr):
3385, 3179, 1645, 1612, 1458, 1416, 1217. 1H NMR (DMSO-d6):
2.21 (m, 4H), 3.16 (m, 4H), 4.16 (m, 4H), 7.38 (m, 2H), 7.51 (m,
2H), 7.57 (m, 2H), 7.70–7.78 (m, 4H), 8.52 (m, 4H), 8.75 (m, 2H),
9.41 (br s, 2H), 12.71 (br s, 2H), 13.84 (br s, 2H, HCl). 13C NMR
(DMSO-d6): 25.37, 42.46, 44.18, 100.39, 112.60, 112.88, 118.94,
121.02, 121.24, 121.96, 122.72, 124.87, 125.63, 130.52, 135.48,
138.52, 141.44, 149.22. Anal. Calcd for C36H33N7Á2.4HClÁ3.0H2O:
C, 61.31; H, 5.92; N, 13.90. Found: C, 61.33; H, 5.72; N, 13.83.
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5.1.24. N,N-Bis-[3-(11H-indolo[3,2-c]quinolin-6-
yl)aminopropyl]-N-methylamine hydrochloride (26)
This compound was obtained from 5a and N,N-bis(3-aminopro-
pyl)methylamine as described for 25, which was crystallized from