Synthesis, β-haematin inhibition, and in vitro antimalarial testing of isocryptolepine analogues: SAR study of indolo[3,2-c]quinolines with various substituents at C2, C6, and N11

Article abstract of DOI:10.1016/j.bmc.2014.03.030

A series of indolo[3,2-c]quinolines were synthesized by modifying the side chains of the ω-aminoalkylamines at the C6 position and introducing substituents at the C2 position, such as F, Cl, Br, Me, MeO and NO₂, and a methyl group at the N11 po

Full text of DOI:10.1016/j.bmc.2014.03.030

Bioorganic & Medicinal Chemistry 22 (2014) 2629–2642
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, b-haematin inhibition, and in vitro antimalarial testing of
isocryptolepine analogues: SAR study of indolo[3,2-c]quinolines with
various substituents at C2, C6, and N11
Ning Wang a, Kathryn J. Wicht b, Kento Imai a, Ming-qi Wang a, Tran Anh Ngoc a, Ryo Kiguchi a,
Marcel Kaiser c,d, Timothy J. Egan b, , Tsutomu Inokuchi a,
⇑
⇑
a Division of Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku,
Okayama 700-8530, Japan
b Department of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa
c Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
d University Basel, Petersplatz 1, CH-4003 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of indolo[3,2-c]quinolines were synthesized by modifying the side chains of the
x-aminoalkyl-
Received 31 January 2014
Accepted 17 March 2014
Available online 26 March 2014
amines at the C6 position and introducing substituents at the C2 position, such as F, Cl, Br, Me, MeO and
NO2, and a methyl group at the N11 position for an SAR study. The in vitro antiplasmodial activities of the
derivative agents against two different strains (CQS: NF54 and CQR: K1) and the cytotoxic activity against
normal L6 cells were evaluated. The test results showed that compounds 6k and 6l containing the
branched methyl groups of 3-aminopropylamino at C6 with a Cl atom at C2 exhibited a very low cyto-
toxicity with IC50 values above 4000 nM, high antimalarial activities with IC50 values of about 11 nM
for CQS (NF54), IC50 values of about 17 nM for CQR (K1), and RI resistance indices of 1.6. Furthermore,
the compounds were tested for b-haematic inhibition, and QSAR revealed an interesting linear correlation
between the biological activity of CQS (NF54) and three contributing factors, namely solubility, hydro-
philic surface area, and b-haematin inhibition for this series. In vivo testing of 6l showed a reduction
in parasitaemia on day 4 with an activity of 38%.
Keywords:
Indolo[3,2-c]quinoline
Antiplasmodial activity
Chloroquine sensitive and resistant
C9-alkylamino-substituted isocryptolepine
In vitro test
b-Haematin inhibition
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
structurally unique artemisinin with an endoperoxide function
was introduced in the 1970s as a result of a search for a new drug
Malaria is a mortal disease caused by Plasmodium parasites,
which are spread between humans by infected mosquitoes.1 In
2012, malaria caused about 219 million clinical cases and approx-
imately 0.66 million deaths.2 Currently, a number of effective anti-
malarial drugs and treatments are available, but drug resistance
remains a problem because of the rapid evolution and adaptation
of the malaria parasite.3 Moreover, current antimalarial drugs are
still unaffordable for underdeveloped countries where people are
the most vulnerable.4 Therefore, novel and economical alternatives
are needed.
from Chinese herbal medicines.6 However, the Plasmodium falcipa-
rum strains resistant to CQ are now widespread in endemic areas7,8
and evidence of in vitro and in vivo resistance even against the
most recently introduced artemisinin-based combination therapy
(ACT) has also now been demonstrated for the therapy of uncom-
plicated P. falciparum infections.9,10
The mechanism of action of chloroquines against the malaria
parasites was explored by one of our groups.11 It was argued that
the intra-erythrocytically active antimalarial agents act by binding
to haematin,12–14 blocking b-haematin formation and leaving toxic
haematin in the parasite.15 This suggests that the mode of action of
such intra-erythrocytically active antimalarials is to simply com-
plex haem and prevent the conversion to b-haematin. Based on
these results, the design of antimalarial drugs has been envisaged
based on the 4-aminoquinoline cores and a search for new antima-
larial agents with the chloroquine-related core has continued to be
actively pursued.16–18
Chloroquine (CQ, I), historically, the most effective and cheapest
therapeutic agent, found long-lasting use for more than half a cen-
tury as a specific drug for the treatment of malaria patients.5 The
⇑
Corresponding authors. Tel.: +27 21 650 2528; fax: +27 21 650 5195 (T.J. Egan),
tel.: +81 86 251 8210; fax: +81 86 251 8021 (T.I.).
E-mail addresses: Timothy.Egan@uct.ac.za (T.J. Egan), inokuchi@cc.okayama-u.ac.jp
(T. Inokuchi).
http://dx.doi.org/10.1016/j.bmc.2014.03.030
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2630
N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
Due to the appearance of resistance to chloroquine, other chlo-
submitted for in vivo drug screening against the Plasmodium berg-
hei malaria model in Swiss mice.
roquine-related antimalarial drugs with quinoline motifs have
been proposed.19,20 Examples include amodiaquine (II) (trade
names Camoquin, Flavoquine)21 and amopyroquine (III)22, both
of which contain a 4-aminoquinoline core. Amodiaquine has been
shown to be more effective than chloroquine in treating chloro-
quine-resistant Plasmodium falciparum malaria infections.
The other leading antimalarial drug with a quinoline motif is
mefloquine (IV).23 Mefloquine remains effective against human
malaria parasites. These close analogues of chloroquine as well as
certain chloroquine analogues, such as lumefantrine, maintain
activity against chloroquine-resistant parasite strains, Indeed,
resistance to chloroquine is not believed to arise from any change
in the drug target, but rather primarily from mutations in a mem-
brane protein, PfCRT, found in the parasite digestive vacuole that is
thought to transport chloroquine away from its site of action.24,25
This effect is compound specific. As a result, it may be possible to
design new antimalarials based on the same mechanism of action
as that of chloroquine.
Finding new lead compounds from plant products is one of the
pivotal strategies in the search for new drugs against infectious
diseases.26,27 In this regard, the constituents of the roots of the
climbing shrub Cryptolepis sanguinolenta, growing in some African
countries, is promising since an aqueous macerate or decoction of
this root is used for the treatment of endemic disease such as ma-
laria fever.28,29 Among the known constituents of this plant, cry-
ptolepine (V),30,31 neocryptolepine (VI),32 and isocryptolepine
(VII)33are attractive as the lead candidates for developing more ac-
tive derivatives (see Fig. 2), since these compounds show antima-
larial activities though with a weak potency.34,35
2. Chemistry
Since Timari’s first accomplishment of the total synthesis of iso-
cryptolepine in 1997,46 a variety of synthetic schemes was pro-
posed for this structural motif and its analogues comprising
either a pyridine or an indole ring closure in the final stage.47–51
For example, a one-step procedure for the construction of 5,11-
dehydroindolo[3,2-c]quinoline-6-one using isatin and 2-amino-
benzylamine was described by Bergman in 2003,52 in which isatin
consist of a 2(1H)-quinolone unit and 2-aminobenzylamine formed
the indole part. Later, Tzeng and co-workers53 applied this method
to the synthesis of a series of 11H-indolo[3,2-c]quinoline deriva-
tives modified with an amino group at the C6 position. Mohan ap-
plied the Fischer indole synthesis for construction of the
isocryptolepines in 2005,54 which highlighted the one-step synthe-
sis with a good yield, but only one example was demonstrated. A
similar approach was later reported by Kumar in 200955 using 1-
methyl-1,2,3,4-tetrahydroquinolin-4-one and phenylhydrazine.
Furthermore, a multistep method was described by Choshi and
co-workers56 for isocryptolepine using the methyl 2-(indol-2-
yl)benzoate, in which the microwave-assisted tandem Curtius
rearrangement and electrocyclic ring closure of an aza 6
system was employed as the key operation.
p-electron
In our approach to construct a series of indolo[3,2-c]quinoline
derivatives, the 5,11-dihydro-indolo[3,2-c]quinolin-6-ones (3)
were chosen as the key intermediates. As shown in Scheme 1,
compound 3, obtained from 1 and 2by heating in acetic acid,
was then converted to 6-chloro-11H-indolo[3,2-c]quinoline (4)
by dehydrative chlorination with POCl3, and then amino groups
were introduced at the C6 of 4 by the ArSN reaction with various
amines. According to this protocol, various R1 substituents at the
C2 position, such as F, Cl, Br, Me, MeO and NO2, can be introduced
using isatin bearing the respective substituents. Appropriate
In particular, the structure of isocryptolepine (VII) has attracted
our attention, since this compound can be envisaged to possess the
4-aminoquinoline subunit, which can be recognized by dissecting
out the tetracyclic indolo[4,3-c]quinoline core of VIII at the bond
indicated by the wavy line.36
Isocryptolepine (VII) and its C2-, C3-, C8-, or C9-substituted
derivatives were evaluated for their in vitro antiplasmodial activity
against the CQS and CQR strains of P. falciparum.37 All the modified
compounds showed a higher activity than the parent isocryptole-
pine (VII), and 8-bromo-2-chloroisocryptolepine was the most po-
amines, comprising
no)alkylamines, -hydroxyalkylamines, cyclic secondary amines,
x-aminoalkylamines, x-(N,N-dialkylami-
x
and arylamines, reacted with 4 by conventional heating or by
applying MW irradiation in a short time, yielding the 6-amino-
11H-indolo[3,2-c]quinolines 5. Some terminal amino groups of 5
were further transformed into urea 6 (X = O, S), sulfonamides 7
and carbonyl-amides 8 as shown in Scheme 2.
We then obtained the N-methylated analogues 10. The methyl-
ation of 4 by heating with NaH followed by MeI produced the 6-
chloro-11-methyl-indolo[3,2-c]quinoline (9), which was heated
with the appropriate amines under MW irradiation to yield 10.
The terminal amino group was coupled by the treatment of 10 with
the isocyanate to give 11 as shown in Scheme 3.
tent derivative with
a selectivity index >100 versus the
mammalian cell. On the other hand, the derivatives of the indo-
lo[3,2-c]quinoline structures VIII were examined with their 5N-
oxide for antimalarial activities by varying the substituents at C3,
C6, C8, and N11.38,39
Among the tested derivatives, 3-chloro-11-(dimethylamino-3-
propyl)-8-methoxyindolo[3,2-c]quinoline 5N-oxide had the stron-
gest blood schizontocidal antimalarial activity against the P. berghei
strain in vitro.38 Later, Go et al. developed indolo[3,2-c]quinoline
with a 4-methylpiperazino group, which was about 104 times
more active in vitro than chloroquine,40,41 but its cytotoxicity
was not determined.
3. Results and discussion
These examples suggested that the indolo[3,2-c]quinoline core
has an inherent potential as a lead antimalarial agent and the
introduction of substituents to the appropriate positions in this
motif could improve its biological activities.
3.1. Antiplasmodial activity and cytotoxicity
As a result of the initial screening of the C6-substituted indo-
lo[3,2-c]quinolines, prepared from 4a using various amines, trends
arising from the effect of the C6-substituents on the in vitro anti-
plasmodial activity against the CQS (NF54) cells were elucidated.
As part of our ongoing program aimed at finding antimalarial
agents based on the plant-derived lead compounds from Cryptol-
epis sanguinolenta,42–44 we have prepared a series of indolo[3,2-
c]quinoline derivatives by varying the substituents at C6 and
N11. The antimalarial activities and the cytotoxicity were im-
proved by modification of the substituents at C6 and the effect of
the N-methyl group at N11 was also investigated. Furthermore,
the compounds were tested for b-haematin inhibition45 for a better
understanding of the mechanism of action of the indolo[3,2-
c]quinoline derivatives. The most favorable compound was
Thus, the
x-aminoalkylamino groups of 5a–5g are more effective
than the alkylamino group of 5h–5j, and the benzeneamino group
of 5l is less effective (Table 1).
In the next step, the cooperative effect of the substituent at the
C2 position was screened using halogens and a methyl group as a
substituent on the 6-(3-aminopropylamino)indolo[3,2-c]quino-
lines. 2-Chloro-substituted 5p had the highest activity relative to

N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
2631
O
HN
H2N
H2N
i
R1
ii
+
O
R1
N
H
N
O
1
2
3
10
9
11
HN
8
HN
N
1
2
iii
R1
R1
3
7
6
N
5
Cl
R3
4
5
4
R1=H/1-Br/2-F/2-Cl/2-Br/2-Me/2-MeO/2-NO2
Scheme 1. Synthesis of indolo[3,2-c]quinolines with amino group at the C-6 position. Reagents and conditions: (i) AcOH, reflux, 8–20 h; (ii) POCl3, 130 °C, 8 h; (iii)
appropriate amines, 90–140 °C, 20 min–4 h.
i
HN
PhNCX
H
X=O or S
R1
N
Ph
X
N
NH(CH2)nNH
6
ii
R3SO2Cl
R3=Ph or Me
HN
HN
N
R1
N
5
NH(CH2)nNH2
NH(CH2)3NHSO2 R3
7
R1=H/1-Br/2-F/2-Cl/2-Br/2-Me/2-MeO/2-NO2
n=3, n=6
ii
R4COCl
HN
N
R4=2-thenoyl or diethylcarbamyl
O
R4
NH(CH2)3NH
8
Scheme 2. Synthesis of indolo[3,2-c]quinolines by further modifications of the terminal amino group. Reagents and conditions: (i) THF, rt; (ii) THF, Et3N, rt.
10
9
11
Me
Me
9
Me
10
HN
8
N
N
N
N
1
N
N
2
R1
3
i
7
ii
iii
R1
R1
O
6
Cl
N
5
Cl
R2
N
H
N
H
N
H
4
4
11
R1=H/2-F/2-Cl/2-Br/2-Me/2-MeO
Scheme 3. Synthesis of indolo[3,2-c]quinolines by N-11 methylation and amination at the C6 position. Reagents and conditions: (i) MeI, NaH, 0 °C–rt, 4 h; (ii) appropriate
amines, 140 °C, 24 h; (iii) THF, rt.
the 2-bromo-substituted 5n and 2-fluoro-substituted 5m, while
the 2-methyl-substituted 5o was the least effective among the
tested compounds.
activity against CQS (NF54), while the toxicity against the L6 cells
decreased to half that of the non-branched (3-aminopropylamino)-
substituted 5p.
We also examined the effect of branched 3-aminopropylamino-
substituents at the C6 position against the CQS (NF54) cell. Thus,
the (3-amino-2-methylpropylamino) and (3-amino-2,2-dimethyl-
propylamino)-substituted 5q and 5r showed a slightly decreased
We next examined the
oalkylamino)-substituted indolo[3,2-c]quinolines using phenyliso-
cyanate and phenylthioisocyanate (Table 2). remarkable
improvement in the activity against CQS (NF54) was attained with
x-modification of the 6-(x-amin-
A

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N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
Table 1
Antiplasmodial activity against P. falciparum (CQS, NF54; CQR, K1) and cytotoxicity toward L6 Cells of indolo[3,2-c]quinoline derivatives 4 and 5
R3
N
R1
N
R2
No.
R1
R2
Cl
R3
L6 cells
IC50 nM
NF54
IC50 nM
SIa
L6/NF54
K1
IC50 nM
SIa
L6/K1
RIb
K1/NF54
b-Haematin inhibition
lM
c
c
c
Isocryptolepine
4a
1190
192956.1
780
NTd
1.5
7.6
H
H
H
10209.7
13.7
18.9
45.8
>1000
116.3
N
H
NH2
NH2
5a
H
626.8
739.1
816.5
1182.1
82.7
NTd
6.0
N
H
5b
5c
5d
H
H
H
H
H
H
36.1
12.6
15.0
20.5
64.8
78.8
62.0
81.0
49.6
N
H
NH2
NH2
25.1
90.2
32.5
13.1
2.0
6.0
N
H
NTd
NTd
62.4
N
H
N
N
NH2
5e
5f
H
H
H
H
1776.4
4108.7
26.4
63.2
67.3
65.0
NH
N
171.0
N
H
N
5g
5h
5i
H
H
H
H
H
H
H
H
606.1
890.6
717.4
2051.1
9.4
64.3
6.3
25.1
NTd
NTd
NTd
24.1
2.66
83.6
OH
N
H
140.6
226.5
113.4
200
N
H
OH
3.2
286.7
147.8
O
N
H
5j
18.1
N
H
5k
5l
H
H
H
H
2934.5
18.6
157.8
48.0
NTd
NTd
30.6
44.1
OMe
15213.0
316.7
N
H
OMe
N
H
NH2
NH2
NH2
NH2
NH2
NH2
5m
5n
5o
5p
5q
5r
2-F
H
H
H
H
H
H
638.9
891.0
322.0
1120.7
1918.4
13.0
8.1
49.1
110.0
8.9
NTd
NTd
NTd
67.7
59.0
51.0
14.3
19.4
60.9
12.4
12.7
20.7
N
H
2-Br
2-Me
2-Cl
2-Cl
2-Cl
N
H
36.1
6.2
N
H
180.8
65.0
72.1
16.6
32.5
36.1
10.9
2
N
H
29.5
25.5
N
H
1839.3
14.5
2
Podophylotoxin
Chloroquine
Artemisinin
9.4
4.3
209.5
2.8
22.3
0.7
30–33
11–14
Amodiaquine
a
b
c
Selectivity Index is the ratio of IC50 for cytotoxicity versus antiplasmodial activity (L6/P.f.).
Resistance index is the ratio of IC50 for the resistant versus the sensitive strain (K1/NF54).
The IC50 values are the means of two independent assays; the individual values vary by less than a factor of 2.
Not tested.
d
the 6-(3-phenylureido)propylamino)-substituted 6c having an IC50
2.4 nM, compared to 13.7 nM of the unmodified 5a. In contrast, the
activity of the 6-(3-phenylthioureido)propylamino)-substituted 6b
slightly decreased to 23.5 nM compared to 13.7 nM of 5a. Subse-
quently, the effect of substituents, such as 2-F, 1-Br, 2-Br, 2-Me,
2-MeO, 2-NO2, at the C1 or C2 position of the 6-(3-phenylurei-
do)propylamino)-substituted indolo[3,2-c]quinolines was exam-
ined. However, distinct synergistic effects of the substituents at
the C1 or C2 were not found. Indeed, the activity was weaker for
all these derivatives.
Modification of the 3-aminopropylamino group at C6 with
other groups, such as methanesulfonyl 7a, benzenesulfonyl 7b,
N,N-diethylamidoyl 8a, or 2-thiophenoyl 8b, were tested. However,
no significant improvements in the in vitro antiplasmodial activity
were achieved, though the cytotoxicity was improved by the
x
-modifications.
As a result of the valuation of the substituent effects at the
C2 and C6 positions, and modification and branching of their
pendant groups at C6, we have succeeded in improving the SI
values to about 377 with IC50 values of about 11 nM for

N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
2633
Table 2
Antiplasmodial activity against P. falciparum (CQS, NF54; CQR, K1) and cytotoxicity toward L6 Cells of indolo[3,2-c]quinoline derivatives 6, 7 and 8
No.
R1
R2
R3
L6 cells
ICc50 nM
NF54
SIa
L6/NF54
K1
SIa
L6/K1
RIb
K1/NF54
b-Haematin inhibition
lM
ICc50 nM
ICc50 nM
Isocryptolepine
1190
780
1.5
O
H
N
6a
6b
H
H
H
H
861.5
22.1
23.5
39.0
10.6
24.4
35.3
1.1
2.1
16.2
18.5
N
H
N
H
S
O
O
O
O
249.1
1152.6
1504.2
3624.2
1281.1
965.7
49.3
53.7
49.2
NTd
5.1
21.5
30.6
N
H
N
H
N
H
6c
6d
6e
6f
H
H
H
H
H
H
H
H
H
H
2.4
25.7
288.7
22.5
23.6
17.1
24.2
27.0
10.9
480.3
58.5
12.6
56.9
40.9
22.4
1.9
23.9
22.7
22.0
13.9
17.2
14.8
15.2
14.4
14.6
N
H
N
H
N
H
2-F
N
H
N
H
N
H
1-Br
2-Br
2-Me
2-MeO
2-NO2
2-Cl
2-Cl
N
H
N
H
N
H
36.9
42.5
19.6
NTd
34.7
22.7
77.4
1.6
1.8
1.1
N
H
N
H
N
H
O
6g
6h
6i
N
H
N
N
H
H
O
1517.8
4642.7
2568.0
4105.3
88.8
191.8
95.1
376.6
N
H
N
H
N
H
O
N
H
N
N
H
H
O
O
O
6j
50.0
17.5
51.4
234.6
235.9
1.9
1.6
1.6
N
H
N
H
N
H
6k
N
H
N
H
N
H
6l
2-Cl
H
H
H
H
4004.4
1063.9
810.6
10.6
54.3
72.0
377.8
19.6
11.3
16.9
NTd
92.9
11.7
118.5
24.2
N
H
N
H
N
H
O
S
O
7a
7b
N
H
N
H
O
S
O
H
8.7
1.3
1.2
N
H
N
H
O
8a
8b
H
H
H
H
3568.8
3495.6
41.1
72.4
86.8
48.3
NTd
39.2
24.1
N
H
N
H
N
O
S
84.9
41.2
N
H
N
H
a
b
c
Selectivity Index is the ratio of IC50 for cytotoxicity versus antiplasmodial activity (L6/P.f.).
Resistance index is the ratio of IC50 for the resistant versus the sensitive strain (K1/NF54).
The IC50 values are the means of two independent assays; the individual values vary by less than a factor of 2.
Not tested.
d
compounds 6k and 6l, whose C6-(3-aminopropylamino) group
was modified with phenylthiourea and branched with methyl
groups.
In thisstudy, we alsoinvestigatedtheeffect oftheintroduction ofa
methyl group at the C11 position. A series of 11-methyl-indolo
[3,2-c]quinoline derivatives (10 and 11) was investigated (Table 3).
Their antimalarial activity was not improved relative to the
compound 5 series, and they exhibited a higher cytotoxicity against
the L6 cells.
We then selected some compounds for testing against the CQR
(K1) strain of P. falciparum. All the tested compounds showed a po-
tent antimalarial activity against the CQS strains (IC50 value rang-
ing from 16 to 91 nM), and low RI values between 1 and 6. The
RI provides a quantitative measurement of the antiplasmodial

2634
N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
Table 3
Antiplasmodial activity against P. falciparum (CQS, NF54; CQR, K1) and cytotoxicity toward L6 Cells of indolo[3,2-c]quinoline derivatives 9, 10 and 11
No.
R1
R2
R3
L6 cells
ICc50 nM
NF54
SIa
L6/NF54
K1
SIa
L6/K1
RIb
K1/NF54
b-Haematin inhibition
lM
ICc50 nM
ICc50 nM
Isocryptolepine
1190
17284.0
780
NTd
1.5
3.3
9
H
Cl
Me
Me
7386.0
72.3
2.3
3.9
>1000
24.2
N
H
NH2
NH2
N
10a
H
279.2
258.7
700.8
824.5
576.2
457.0
619.8
425.2
313.4
291.6
191.6
137.6
85.4
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
1.2
N
H
10b
10c
10d
10e
10f
10g
10h
10i
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
273.7
93.2
31.3
58.3
48.6
35.4
95.4
40.9
52.7
31.4
32.9
0.9
7.5
ꢀ1000
>1000
26.6
41.2
39.9
28.0
38.4
25.9
25.8
29.5
56.9
N
H
H
N
H
NH2
NH2
N
2-Br
2-Br
2-Br
2-Cl
2-Cl
2-Cl
2-F
26.3
9.9
N
H
N
H
9.4
N
H
NH2
NH2
N
17.5
4.5
N
H
N
H
7.7
N
H
NH2
NH2
NH2
O
10j
5.5
N
H
10k
10l
2-Me
2-MeO
6.1
N
H
4.2
11
H
Me
107.7
7.3
14.8
NTd
22.7
N
H
N
H
N
H
a
b
c
Selectivity Index is the ratio of IC50 for cytotoxicity versus antiplasmodial activity (L6/P.f.).
Resistance index is the ratio of IC50 for the resistant versus the sensitive strain (K1/NF54).
The IC50 values are the means of two independent assays; the individual values vary by less than a factor of 2.
Not tested.
d
improving the antiplasmodial activity against the CQR (K1) strains
than the other groups with an IC50 value of 19.6 nM. It is very
interesting that 6k and 6l containing branched methyl groups at
the C6-side chain exhibited significantly increased cytotoxicity val-
ues ((IC50 = 4100 or 4000 nM) relative to compound 6j, and anti-
malarial activity increased especially against the CQR (K1) strains.
3.2. b-Haematin inhibition and QSAR
Inspection of the activity data revealed the importance of the R2
amine side chain for high b-haematin and parasite activity in this
series. This is evident from the weak parasite activity determined
for 4a and 9, both of which contain only a chloro substituent at
the R2 position (Fig. 3-green). Interestingly, these compounds also
have very a weak b-haematin activity, which suggests that the
amine side chain is vital for the molecular interaction with haem
at pH 4.8. This may well be due to the hydrogen bonding capabil-
ities of the amine group which could assist the interaction of the
fused-ring system with haem in order to prevent the formation
of b-haematin. A scattered but statistically significant correlation
(r2 = 0.41, P < 0.0001) was observed between the log of the IC50
for b-haematin inhibition, log(bHi), and the log(IC50) of the biolog-
ical activity against the NF54 strain (Fig. 4a). This direct linear
trend showed that while haemozoin inhibition is likely a contribut-
ing factor to the parasite activity and hence a probable mechanism
of action, there are, as expected, other factors which likely play a
significant role.
Figure 1. Chloroquine and its related antimalarial agents.
Figure 2. Structures of indoloquinolines from Cryptolepis sanguinolenta.
In order to define these factors, a linear regression with combi-
nations of the predicted physical properties for each compound
was carried out. Figure 4b shows the best statistical fit that
contains physically relevant coefficients. The result of the analysis
activity against the CQR strains relative to that against the CQS
strains and reveals promising drug discovery leads.57 The introduc-
tion of the MeO group at the C2 position (6h) was more effective in

N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
2635
Figure 3. All compounds arranged by increasing biological activity. The least active compounds are those with Cl at the C6 position (4a, 9), the 2,4-dimethoxy phenyl ring on
the side chain (5l), the compound with a R1 = bromo at the C1 position (6e) (as opposed to the C2 position 6f) and an alcohol (5i and 5h) or tetrahydrofuran (THF) group on the
amine side chain (5j). On the other hand, the compounds containing phenylurea on the amine side chain show the greatest activity (6a–6d, 6f–6l and 11).
5
a
4
3
2
1
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
β-haematin inhibition log(IC50) (μM)
5
b
4
3
2
1
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Predicted NF54 Log(IC50) (nM)
Figure 4. Linear correlation analysis revealed (a) a relationship between activities such that log(NF54IC50) = 0.772⁄log(bHiIC50) + 0.32, r2 = 0.41, P <0.0001. (b) QSAR analysis
between biological activity and predicted physical properties improved the statistics; log(NF54IC50) = 0.193⁄(Sol.) À 0.027 ⁄(%Hydrophil.) À 10.069⁄(1/bHi) À 2.208, r2 = 0.61,
P <0.0001, statistically significant both for individual parameters (t = 4.13, 4.99, 3.87 > tcrit = 2.69) and overall correlation (F = 23.01 > Fcrit = 4.25) at 99% confidence level.
predicted that the biological IC50 correlates with the sum of terms
related to the solubility, hydrophilicity and 1/bHiIC50. By incorpo-
rating these extra parameters, the statistics were improved so that
less scatter was observed (r2 = 0.61). The equation for the predicted
NF54 log(IC50) indicates that an increase in the solubility parame-
ter (Sol.) decreased the activity, a result that has previously been
observed in other series,42 while increasing the percentage of the
hydrophilic surface area (%Hydrophil.) on the molecule increases
the activity. These parameters represent the hydrophobic/hydro-
philic balance required for optimal biological activity. Further-
more, a lower bHi IC50 results in a lower logNF54 IC50 as
expected. The compounds with the highest solubility are the
hydroxylated 5h and 5i (Fig. 1-blue), which, together with their
slightly higher bHi IC50s, could account for their weak activity.
The compounds with R2 = Cl (Fig. 3-green) also have a high solubil-
ity and low% hydrophilic surface area, however, their lack of activ-
ity is primarily due to their lack of b-haematin inhibition. It is
important to note that when these two compounds are excluded,
the trend in Figure 4b remains statistically significant. The other
compound with a lower activity is 5l which possesses a relatively
small% hydrophilic surface area, indicating that the compound is
slightly too hydrophobic for optimal activity. Figure 3 also demon-
strates that generally, the phenylurea moiety on the amine side
chain results in the highest biological activity. Further investiga-
tion of the phenylurea-containing compounds reveals that they
have a moderate solubility (average phenylurea Sol. = average
non-phenylurea Sol.), however, the average% hydrophilic area is
8% higher for the phenylurea compounds which could explain their
greater NF54 activity.
3.3. In vivo antimalarial activity
Compound 6l with the lowest cytotoxicity (IC50 above
4004 nM), low resistant index of 1.6 and strong antiplasmodial

2636
N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
activity in vitro (11 nM) was selected for an in vivo drug testing
model against Plasmodium berghei in mice. The in vivo study was
carried out according to the standard protocol following the ‘4
Day Test’ by the FACS analysis. The activity was calculated as the
difference between the mean percent parasitaemia for the control
(n = 5 mice) and treated groups expressed as a percentage relative
to the control group. After daily intraperitoneal dosing at 50 mg/kg
for four consecutive days, this compound showed a significant
reduction in parasitaemia on day 4 with an activity of 38%. While
not strongly active in vivo, this result suggests that modifications
to improve the water solubility might produce an analogue with
an improved in vivo activity.
formed solids were collected by filtration. The crude product was
purified by flash chromatography using AcOEt-2N ammonia in
MeOH (10:1 V/V) as the eluent to yield pure 5 or 10 as solids.
5.1.2.1.
mine (5a).
N1-(11H-Indolo[3,2-c]quinolin-6-yl)propane-1,3-dia-
Yield: 93%, mp: >230 °C decompose; 1H NMR d
8.42 (d, J = 7.9 Hz, 1H), 8.25 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 8.1 Hz,
2H), 7.48 (dd, J = 8.2, 7.0 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.27 (dt,
J = 14.6, 7.4 Hz, 2H), 7.07 (s, 1H), 3.80 (t, J = 5.9 Hz, 2H), 2.75 (t,
J = 5.7 Hz, 2H), 1.83 (m, 2H); 13C NMR d 153.4, 146.6, 141.1,
138.5, 128.4, 126.6, 124.3, 122.0, 121.9, 121.0 (2C), 120.5, 114.5,
111.8, 103.1, 40.5 39.6, 32.0. HPLC purity 99.1%. HRMS (ESI) calcd
for C18H18N4 [MÀH]À Exact Mass: 289.1453, found 289.1456.
5.1.2.2. N1-(11H-Indolo[3,2-c]quinolin-6-yl)butane-1,4-diamine
(5b).
4. Conclusion
Yield: 78%, mp: 166–168 °C; 1H NMR
d 8.40 (d,
We have prepared a series of indolo[3,2-c]quinolines by varying
the substituents at the C2 position, and modifying the terminal
amino group of the C6-aminoalkylamino side chain with phenyl-
isocyanate. A methyl group was then introduced at N11 by meth-
J = 7.6 Hz, 1H), 8.25 (d, J = 7.6 Hz, 1H), 7.66 (d, J = 6.9 Hz, 2H),
7.48 (t, J = 7.3 Hz, 1H), 7.41 (t, J = 7.3 Hz, 1H), 7.27 (dt, J = 17.1,
7.2 Hz, 2H), 6.62 (s, 1H), 3.72 (s, 2H), 2.64 (t, J = 6.6 Hz, 2H), 1.78
(m, 2H), 1.52 (dd, J = 13.5, 6.7 Hz, 2H); 13C NMR d 153.4, 146.6,
141.2, 138.5, 128.4, 126.7, 124.3, 122.0, 121.9, 121.2, 121.1,
120.5, 114.5, 111.8, 103.2, 42.0, 40.8, 31.4, 27.3. HPLC purity
ylation. All the synthesized derivatives showed
a potent
antiplasmodial activity against CQS (NF54), and CQR (K1) strains
in vitro. Compared to 5a, the 2-chloro-substituted derivative 5p
was the most effective, and the urea derivatives 6c had an in-
creased activity against the CQS strain (NF54). The sulfonyl deriv-
atives 7 and carbonyl derivatives 8 were less effective. Compounds
6k and 6l containing branched methyl groups exhibited signifi-
cantly higher cytotoxicity values, and antimalarial activity espe-
cially increased against the CQR strain (K1). Compounds 10 and
11 bearing an N11-methyl group did not show an improved anti-
malarial activity and they exhibited a higher cytotoxicity against
L6 cells. Most of the compounds have a very effective b-haematin
inhibition activity. The linear correlation analysis revealed there
were three contributing factors, namely, water solubility, hydro-
philic surface area, and b-haematin inhibition that influence the
biological activity of this series against CQS (NF54) parasites.
99.9%. HRMS (ESI) calcd for
303.1610, found 303.1614.
C
19H20N4 [MÀH]À Exact Mass:
5.1.2.3.
mine (5c).
N1-(11H-Indolo[3,2-c]quinolin-6-yl)pentane-1,5-dia-
Yield: 80%, mp: 168–169 °C; 1H NMR d 8.41 (d,
J = 7.6 Hz, 1H), 8.26 (m, 1H), 7.66 (d, J = 7.2 Hz, 2H), 7.49 (t,
J = 7.1 Hz, 1H), 7.41 (t, J = 7.4 Hz, 1H), 7.28 (dt, J = 14.9, 7.2 Hz,
2H), 6.56 (s, 1H), 3.72 (s, 2H), 2.56 (s, 2H), 1.77 (d, J = 5.4 Hz, 2H),
1.43 (s, 4H); 13C NMR d 153.4, 146.6, 141.2, 138.5, 128.4, 126.7,
124.3, 122.0, 121.9, 121.2, 121.1, 120.5, 114.5, 111.8, 103.2, 42.2,
41.0, 33.8, 29.8, 24.6. HPLC purity 99.3%. HRMS (ESI) calcd for
C
20H22N4 [MÀH]À Exact Mass: 317.1766, found 317.1761.
5.1.2.4. N1-(11H-Indolo[3,2-c]quinolin-6-yl)hexane-1,6-diamine
(5d).
Yield: 78%; 1H NMR d 8.38 (d, J = 7.9 Hz, 1H), 8.23 (d,
5. Experimental
5.1. Chemistry
J = 7.7 Hz, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.47 (dd, J = 11.2, 4.0 Hz,
1H), 7.40 (t, J = 7.5 Hz, 1H), 7.27 (dt, J = 18.5, 7.4 Hz, 2H), 6.54 (d,
J = 5.5 Hz, 1H), 3.69 (dd, J = 13.1, 6.6 Hz, 2H), 2.51 (d, J = 5.9 Hz,
2H), 1.75 (t, J = 6.9 Hz, 2H), 1.40 (m, 6H); 13C NMR d 153.4, 146.6,
141.2, 138.5, 128.4, 126.7, 124.3, 121.9, 121.8, 121.2, 121.1,
120.5, 114.5, 111.8, 103.2, 42.1, 40.9, 33.8, 29.9, 27.1, 26.8. HPLC
purity 97.8%. HRMS (ESI) calcd for C21H24N4 [MÀH]À Exact Mass:
331.1923, found 331.1916.
5.1.1. General
The commercially obtained reagents were used without further
purification. Column chromatographies were achieved on a silica
gel column (230–400 mesh) using a gradient solvent system
(n-hexane/ ethyl acetate as the eluent unless otherwise specified).
The 1H NMR and 13C NMR spectra were measured on the Varian
INOVA-600 spectrometer with DMSO-d6 as the solvent unless
otherwise indicated. Chemical shifts (d ppm) were determined
using tetramethylsilane (TMS) as the internal reference. Melting
points were determined on a J-Science RFS-10 hot stage micro-
scope. High resolution mass spectra were obtained on a Bruker
micrOTOF II-SKA spectrometer. Purity was verified using HPLC sys-
tems. HPLC was performed on Waters e2695 Separations Module
using Waters 2998 Photodiode Array (PDA) detector equipped with
5.1.2.5.
N-(3-(4-(3-Aminopropyl)piperazin-1-yl)propyl)-11H-
Yield: 47%. 1H NMR d
indolo[3,2-c]quinolin-6-amin (5e).
12.47 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.23 (dd, J = 7.9, 1.1 Hz, 1H),
7.63 (dd, J = 10.8, 8.2 Hz, 2H), 7.48 (d, J = 1.4 Hz, 1H), 7.41 (s, 1H),
7.33–7.22 (m, 2H), 6.64 (s, 1H), 3.72 (d, J = 6.1 Hz, 2H), 2.53 (t,
J = 6.7 Hz, 4H), 2.44 (t, J = 6.7 Hz, 6H), 2.34– 2.23 (m, 4H), 1.95–
1.85 (m, 2H), 1.54–1.42 (m, 2H); 13C NMR d 153.4, 146.5, 141.1,
138.5, 128.4, 126.6, 124.3, 121.93, 121.8, 121.2 (2C), 120.9, 114.5,
111.8, 103.2, 57.1 (2C), 56.3, 53.6 (2C), 53.2 (2C), 30.7, 26.5 (2C).
HPLC purity 98.1%. HRMS (ESI) calcd for C25H32N6 [MÀH]À Exact
Mass: 415.2610, found 415.2609.
a Symmetry C18 column (4.6 Â 150 mm, 5
lm). Water (A) and ACN
(B) were used as eluents. A 100–10%, B, 35 min gradient was used
with a flow rate of 1 ml/min. 0.1%TFA was added to solvent A and
B. 254 nm was used as wavelength.
5.1.2.6.
(5f).
6-(1,4-Diazepan-1-yl)-11H-indolo[3,2-c]quinoline
Yield: 83%, mp: 216–218 °C; 1H NMR d 12.61 (s, 1H),
5.1.2. General procedure for the synthesis of 6-
aminoindolo[3,2-c]quinolines 5 and 10
6-Chlorooindolo[3,2-c]quinoline 4 or 9 (0.4 mmol) and an ex-
cess of the appropriate amine (2.0 mmol) heated together at 90–
100 °C for 20 min–24 h. Completion of the reaction was monitored
by TLC. The reaction mixture then was added to water, and the
8.35 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 8.3 Hz,
1H), 7.68 (d, J = 8.0 Hz, 1H), 7.56 (dd, J = 8.1, 7.1 Hz, 1H), 7.42 (m,
2H), 7.31 (t, J = 7.5 Hz, 1H), 3.86 (s, 2H), 3.80 (d, J = 2.3 Hz, 2H),
2.98 (d, J = 2.3 Hz, 2H), 2.88 (t, J = 5.6 Hz, 2H), 1.88 (s, 2H); 13C
NMR d 157.6, 145.1, 142.7, 138.9, 128.6, 127.7, 124.6, 123.0,
122.5, 122.1, 122.0, 120.7, 115.5, 112.0, 106.0, 55.4, 50.7, 49.1,

N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
2637
48.6, 31.1. HPLC purity 100%. HRMS (ESI) calcd for C20H20N4
115.1, 112.6, 104.6, 103.6, 99.2, 56.8, 55.8. HPLC purity 99.8%.
HRMS (ESI) calcd for C23H19N3O [MÀH]À Exact Mass: 368.1399,
found 368.1398.
[MÀH]À Exact Mass: 315.1610, found 315.1608.
5.1.2.7. N1-(11H-Indolo[3,2-c]quinolin-6-yl)-N3,N3-dimethylpro-
pane-1,3-diamine (5g).
Yield: 90%; 1H NMR d 12.45 (s, 1H),
5.1.2.13. N1-(2-Fluoro-11H-indolo[3,2-c]quinolin-6-yl)propane-
8.25 (t, J = 8.1 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.48 (t, J = 7.6 Hz,
1H), 7.40 (t, J = 7.6 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.26 (t,
J = 7.4 Hz, 1H), 7.14 (s, 1H), 3.75 (dd, J = 11.4, 5.9 Hz, 2H), 2.46 (t,
J = 6.2 Hz, 2H), 2.24 (s, 6H), 1.89 (dd, J = 12.7, 6.3 Hz, 2H); 13C
NMR d 153.4, 146.7, 141.2, 138.6, 128.5, 126.7, 124.3, 122.0,
122.0, 121.2, 120.6, 120.5, 114.6, 112.0, 103.2, 58.9, 45.8 (2C),
41.0, 26.7. HPLC purity 99.9%. HRMS (ESI) calcd for C20H22N4
[MÀH]À Exact Mass: 317.1766, found 317.1759.
1,3-diamine (5m).
Yield: 87%, mp: 222–224 °C; 1H NMR d
8.42 (d, J = 8.0 Hz, 1H), 8.01 (dd, J = 9.6, 3.0 Hz, 1H), 7.65 (dd,
J = 9.0, 5.3 Hz, 2H), 7.442 (m, 1H), 7.36 (m, 1H), 7.33 (m, 1H),
7.07 (s, 1H), 3.76 (d, J = 5.2 Hz, 2H), 2.73 (t, J = 6.2 Hz, 2H), 1.81
(m, 2H); 13C NMR d 156.2 (J = 237.1 Hz), 153.0, 143.4, 140.6
(J = 3.6 Hz), 138.5, 128.5 (J = 8.4 Hz,), 124.6, 121.7, 121.2, 120.7,
116.8 (J = 24.1 Hz), 114.4 (J = 9.6 Hz), 111.9, 106.2 (J = 23.1 Hz),
103.6, 40.5, 39.7, 32.9. HPLC purity 99.6%. HRMS (ESI) calcd for
C
18H17FN4 [MÀH]À Exact Mass: 307.1359, found 307.1364.
5.1.2.8.
(5h).
2-(11H-Indolo[3,2-c]quinolin-6-ylamino)ethanol
Yield: 35%. 1H NMR d 12.50 (s, 1H), 8.35 (d, J = 7.9 Hz,
5.1.2.14. N1-(2-Bromo-11H-indolo[3,2-c]quinolin-6-yl)propane-
1,3-diamine (5n).
Yield: 82%, mp: 181–183 °C; 1H NMR d
1H), 8.26 (d, J = 7.8 Hz, 1H), 7.66 (t, J = 8.8 Hz, 2H), 7.51 (t,
J = 7.6 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.30 (dd, J = 17.0, 7.8 Hz,
2H), 6.64 (d, J = 5.1 Hz, 1H), 5.41 (s, 1H), 3.80 (m, 2H), 3.75 (t,
J = 5.2 Hz, 2H); 13C NMR d 153.7, 146.2, 141.3, 138.6, 128.7,
126.4, 124.5, 122.1, 121.9, 121.5, 121.1, 120.7, 114.6, 112.0,
103.2, 61.5, 44.2. HPLC purity 100%. HRMS (ESI) calcd for
8.50 (s, 1H), 8.44 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.58
(m, 2H), 7.42 (t, J = 7.5 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.25 (s,
1H), 3.79 (s, 2H), 2.75 (t, J = 6.1 Hz, 2H), 1.81 (m, 2H); 13C NMR d
153.7, 145.3, 140.0, 138.6, 1301.0, 128.640, 124.7, 124.2, 121.6,
121.2, 120.78, 116.1, 112.8, 112.0, 103.6, 40.5, 39.7, 32.8. HPLC pur-
ity 96.3%.
C
17H15N3O [MÀH]À Exact Mass: 276.1137, found 276.1141.
5.1.2.9.
(5i).
3-(11H-Indolo[3,2-c]quinolin-6-ylamino)propan-1-ol
Yield: 96%, mp: 199–201 °C; 1H NMR d 12.51 (s, 1H),
5.1.2.15. N1-(2-Methyl-11H-indolo[3,2-c]quinolin-6-yl)propane-
1,3-diamine (5o).
Yield: 61%, mp: 229–231 °C; 1H NMR d 8.38
8.36 (d, J = 7.9 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.65 (dd, J = 13.0,
8.3 Hz, 2H), 7.50 (t, J = 7.4 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.29
(dd, J = 14.6, 7.3 Hz, 2H), 6.80 (s, 1H), 3.81 (m, 2H), 3.60 (t,
J = 5.7 Hz, 2H), 1.89 (m, 2H); 13C NMR d 153.5, 146.0, 141.1,
138.5, 128.5, 126.1, 124.3, 121.9, 121.8, 121.2, 120.8, 120.5,
114.4, 111.8, 102.9, 59.4, 38.6, 32.9. HPLC purity 100%. HRMS
(ESI) calcd for C18H17N3O [MÀH]À Exact Mass: 290.1293, found
290.1296.
(d, J = 7.8 Hz, 1H), 8.03 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.55 (d,
J = 8.4 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.26
(t, J = 7.4 Hz, 1H), 3.76 (d, J = 5.8 Hz, 2H), 2.73 (t, J = 5.6 Hz, 2H),
2.46 (s, 3H), 1.84–1.76 (m, 2H); 13C NMR d 153.1, 144.8, 141.0,
138.6, 130.1, 129.9, 126.5, 124.2, 122.0, 121.4, 121.1, 120.5,
114.4, 111.9, 103.24, 40.5, 39.7, 33.0, 21.5. HPLC purity 96.2%.
HRMS (ESI) calcd for C19H21N4 [M+H]+ Exact Mass: 305.1766, found
305.1745.
5.1.2.10.
c]quinolin-6-amine (5j).
N-((Tetrahydrofuran-2-yl)methyl)-11H-indolo[3,2-
Yield: 42%, mp: 185–187 °C; 1H
5.1.2.16. N1-(2-Chloro-11H-indolo[3,2-c]quinolin-6-yl)propane-
1,3-diamine (5p).
Yield: 95%, mp: >230 °C decompose; 1H
NMR d 12.48 (s, 1H), 8.30 (m, 1H), 8.25 (m, 1H), 7.67 (dd,
J = 10.4, 5.0 Hz, 2H), 7.50 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H),
7.29 (dt, J = 17.5, 7.5 Hz, 2H), 6.49 (t, J = 5.3 Hz, 1H), 4.30 (m, 1H),
3.86 (m, 1H), 3.77 (dd, J = 7.8, 3.5 Hz, 2H), 3.66 (d, J = 7.3 Hz, 1H),
1.93 (m, 2H), 1.79 (m, 2H); 13C NMR d 153.3, 146.4, 141.2, 138.6,
129.0, 126.64, 124.7, 122.0, 121.8, 121.3, 120.9, 120.6, 114.6,
111.9, 103.0, 77.6, 67.6, 44.9, 29.1, 25.6. HPLC purity 100%. HRMS
(ESI) calcd for C20H19N3O [MÀH]À Exact Mass: 316.1450, found
316.1444.
NMR d 8.45 (d, J = 8.0 Hz, 1H), 8.35 (d, J = 2.2 Hz, 1H), 7.65 (dd,
J = 15.5, 8.5 Hz, 2H), 7.48–7.40 (m, 2H), 7.30 (t, J = 7.5 Hz, 1H),
7.24 (s, 1H), 3.79 (d, J = 4.2 Hz, 2H), 2.75 (t, J = 6.1 Hz, 2H), 1.81
(m, J = 6.2 Hz, 2H); 13C NMR d 153.6, 145.1, 140.1, 138.6, 128.4,
128.3, 124.8, 124.7, 121.7, 121.2, 121.1, 120.8, 115.4, 112.0,
103.6, 40.5, 39.8, 32.9. HPLC purity 98.5%. HRMS (ESI) calcd for
C
18H18ClN4 [M+H]+ Exact Mass: 325.1220, found 325.1192.
5.1.2.17. N1-(2-Chloro-11H-indolo[3,2-c]quinolin-6-yl)-2-meth-
ylpropane-1,3-diamine (5q).
Yield: 97%, mp: 214–216 °C; 1H
5.1.2.11.
(5k).
N-Benzyl-11H-indolo[3,2-c]quinolin-6-amine
NMR d 8.42 (d, J = 8.0 Hz, 1H), 8.32 (d, J = 2.5 Hz, 1H), 7.65 (d,
J = 8.1 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.55 (s, 1H), 7.45 (dd,
J = 8.8, 2.5 Hz, 1H), 7.44–7.40 (m, 1H), 7.32–7.25 (m, 1H), 3.77
(dd, J = 7.7, 5.3 Hz, 1H), 3.54 (dd, J = 8.5, 4.1 Hz, 1H), 2.71 (dd,
J = 12.4, 4.6 Hz, 1H), 2.59 (dd, J = 12.4, 7.0 Hz, 1H), 1.98 (t,
J = 3.9 Hz, 1H), 0.97 (d, J = 6.9 Hz, 3H); 13C NMR d 153.6, 145.1,
140.1, 138.6, 128.4, 128.3, 124.8, 124.7, 121.67, 121.1, 121.1,
120.8, 115.3, 112.0, 103.6, 47.4, 46.5, 35.6, 17.0. HPLC purity
99.5%. HRMS (ESI) calcd for C19H20ClN4 [M+H]+ Exact Mass:
339.1376, found 339.1352.
Yield: 48%, mp: 205–207 °C; 1H NMR d 12.51 (s, 1H),
8.53 (d, J = 7.9 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.1 Hz,
1H), 7.66 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.44 (t,
J = 7.6 Hz, 1H), 7.33–7.26 (m, 5H), 7.18 (t, J = 7.0 Hz, 1H), 5.01 (d,
J = 5.8 Hz, 2H); 13C NMR d 153.0, 146.4, 142.0, 141.4, 138.6, 128.5
(3C), 127.9 (2C), 126.8, 126.7, 124.4, 122.0, 121.9, 121.3, 121.2,
120.6, 114.7, 111.9, 103.1, 44.0. HPLC purity 100%. HRMS (ESI)
calcd for C22H17N3 [MÀH]À Exact Mass: 322.1344, found 322.1336.
5.1.2.12. N-(2,4-Dimethoxyphenyl)-11H-indolo[3,2-c]quinolin-
6-amine (5l).
Yield: 96%, mp: 220–221 °C; 1H NMR d 12.70
5.1.2.18.
dimethylpropane-1,3-diamine (5r).
N1-(2-Chloro-11H-indolo[3,2-c]quinolin-6-yl)-2,2-
Yield: 71%, mp: 226–
(s, 1H), 8.94 (d, J = 8.8 Hz, 1H), 8.34 (d, J = 7.6 Hz, 1H), 8.16 (d,
J = 7.9 Hz, 1H), 7.97 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.75 (d,
J = 8.0 Hz, 1H), 7.59 (dd, J = 11.1, 4.0 Hz, 1H), 7.50 (t, J = 7.5 Hz,
1H), 7.44 (t, J = 7.4 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 6.76 (d,
J = 2.6 Hz, 1H), 6.65 (dd, J = 8.8, 2.6 Hz, 1H), 4.03 (s, 3H), 3.79 (s,
3H); 13C NMR d 155.1, 150.5, 149.7, 145.8, 141.4, 138.8, 128.9,
127.3, 124.9, 124.1, 122.6, 122.1, 121.4, 121.2, 120.5, 119.6,
228 °C; 1H NMR d 8.38 (d, J = 7.9 Hz, 1H), 8.38 (m, 1H), 7.81 (s,
1H), 7.66 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 8.9 Hz, 1H), 7.47–7.40 (m,
2H), 7.30 (t, J = 7.5 Hz, 1H), 3.63 (s, 2H), 2.55 (s, 2H), 0.99 (s, 6H);
13C NMR d 153.9, 145.1, 140.1, 138.7, 128.5, 128.2, 124.9, 124.8,
121.7, 1212, 121.1, 121.0, 115.4, 112.1, 103.7, 51.9, 50.6, 24.7,

2638
N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
14.6 (2C). HPLC purity 98.6%. HRMS (ESI) calcd for C20H22ClN4
[M+H]+ Exact Mass: 353.1533, found 353.1518.
7.20 (s, 1H), 4.31 (s, 3H), 3.78 (d, J = 5.3 Hz, 2H), 2.74 (t,
J = 6.1 Hz, 2H), 1.81 (m, 2H); 13C NMR d 153.4, 146.0, 140.0,
139.5, 128.9, 128.2, 124.9, 124.8, 121.7, 121.2, 121.1, 120.4,
115.8, 110.5, 103.8, 40.6, 39.7, 33.6, 32.8. HPLC purity 98.0%.
5.1.2.19.
pane-1,3-diamin (10a).
N1-(11-Methyl-11H-indolo[3,2-c]quinolin-6-yl)pro-
Yield: 62%; 1H NMR
8.52 (d,
d
J = 8.2 Hz, 1H), 8.45 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 8.2 Hz, 1H),
7.71 (d, J = 8.2 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.7 Hz,
1H), 7.33 (t, J = 7.5 Hz, 1H), 7.27 (t, J = 7.5 Hz, 1H), 7.05 (s, 1H),
4.33 (d, J = 9.1 Hz, 3H), 3.79 (d, J = 5.6 Hz, 2H), 2.74 (t, J = 6.0 Hz,
2H), 1.82 (dd, J = 12.2, 6.0 Hz, 2H); 13C NMR d 153.2, 147.5, 140.6,
140.0, 128.2, 127.2, 124.4, 122.8, 121.1, 121.0, 120.9, 120.7,
115.1, 110.3, 103.4, 40.4, 39.7, 33.9, 32.9. HPLC purity 88.2%.
5.1.2.26. N1-(2-Chloro-11-methyl-11H-indolo[3,2-c]quinolin-6-
yl)-2,2-dimethylpropane-1,3-diamine (10h).
Yield: 67%,
mp: 180–182 °C; 1H NMR d 8.46 (d, J = 2.3 Hz, 1H), 8.44 (d,
J = 7.9 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H),
7.51 (dd, J = 6.5, 2.4 Hz, 2H), 7.35 (s, 1H), 4.35 (s, 3H), 3.62 (d,
J = 4.4 Hz, 2H), 2.54 (s, 2H), 0.99 (s, 6H); 13C NMR d 153.6, 146.0,
140.1, 139.4, 128.7, 128.2, 124.8, 124.8, 121.7, 121.3, 120.9,
120.5, 115.8, 110.5, 103.9, 52.1, 50.7, 35.5, 33.6, 24.7 (2C). HPLC
purity 96.5%.
5.1.2.20. 2,2-Dimethyl-N1-(11-methyl-11H-indolo[3,2-c]quino-
lin-6-yl)propane-1,3-diamine (10b).
Yield: 52%, mp: 127–
129 °C; 1H NMR d 8.52 (d, J = 8.3 Hz, 1H), 8.42 (d, J = 7.9 Hz, 1H),
7.82 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.61 (s, 1H), 7.54–
7.44 (m, 2H), 7.34 (t, J = 7.5 Hz, 1H), 7.27 (dd, J = 8.1, 7.0 Hz, 1H),
4.34 (s, 3H), 3.64 (s, 2H), 2.53 (s, 2H), 0.99 (s, 6H); 13C NMR d
153.4, 147.4, 140.5, 140.0, 128.1, 127.0, 124.4, 122.8, 121.0,
121.0, 120.8, 120.7, 115.1, 110.3, 103.4, 51.9, 50.4, 35.9, 33.9,
24.7 (2C). HPLC purity 99.2%.
5.1.2.27. N1-(2-Chloro-11-methyl-11H-indolo[3,2-c]quinolin-6-
yl)-N3,N3-dimethylpropane-1,3-diamine (10i).
Yield: 61%,
mp: 119–121 °C; 1H NMR d 8.42 (d, J = 2.3 Hz, 1H), 8.29 (d,
J = 8.0 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H),
7.49 (dd, J = 8.9, 2.4 Hz, 2H), 7.37 (s, 1H), 7.26 (s, 1H), 4.31 (s,
3H), 3.73 (d, J = 5.2 Hz, 2H), 2.45 (t, J = 6.3 Hz, 2H), 2.24 (s, 6H),
1.90–1.83 (m, 2H); 13C NMR d 153.3, 146.0, 140.0, 139.4, 128.9,
128.2, 124.9, 124.8, 121.7, 121.1, 120.5, 120.4, 115.7, 110.5,
103.8, 58.8, 45.8(2C), 41.1, 33.6, 26.5. HPLC purity 100%.
5.1.2.21. N1,N1-Dimethyl-N3-(11-methyl-11H-indolo[3,2-c]quin-
olin-6-yl)propane-1,3-diamin (10c).
Yield 82%, mp: 158–
160 °C; 1H NMR d 8.53 (d, J = 8.3 Hz, 1H), 8.30 (d, J = 7.9 Hz, 1H),
7.83 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 0.9 Hz,
1H), 7.47 (d, J = 7.3 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.28 (s, 1H),
7.15 (s, 1H), 4.35 (s, 3H), 3.75 (d, J = 5.6 Hz, 2H), 2.46 (t,
J = 6.3 Hz, 2H), 2.25 (s, 6H), 1.88 (m, 2H); 13C NMR d 153.1, 147.5,
140.5, 139.9, 128.1, 127.3, 124.4, 122.8, 121.1, 120.9, 120.7,
120.4, 115.1, 110.4, 103.4, 58.9, 45.8 (2C), 41.1, 33.9, 26.6. HPLC
purity 99.8%.
5.1.2.28. N1-(2-Fluoro-11-methyl-11H-indolo[3,2-c]quinolin-6-
yl)propane-1,3-diamine (10j).
Yield: 36%, mp: 116–118 °C;
1H NMR d 8.44 (d, J = 7.9 Hz, 1H), 8.18 (dd, J = 10.8, 2.4 Hz, 1H),
7.77 (t, J = 10.0 Hz, 1H), 7.70 (dd, J = 9.1, 5.8 Hz, 1H), 7.45 (dd,
J = 16.9, 9.1 Hz, 1H), 7.36 (ddd, J = 10.7, 8.5, 2.6 Hz, 1H), 7.31 (t,
J = 7.5 Hz, 1H), 4.29 (d, J = 9.8 Hz, 3H), 3.75 (t, J = 5.9 Hz, 2H), 2.74
(t, J = 6.1 Hz, 2H), 1.86–1.78 (m, 2H); 13C NMR
d
156.1(J = 237.1 Hz), 152.8, 144.2, 140.0, 139.9(J = 3.0 Hz), 128.8
(J = 9.1 Hz), 124.7, 121.2, 121.0, 120.4, 116.6 (J = 24.1 Hz), 114.6
(J = 10.6 Hz) 110.3, 107.3 (J = 24.2 Hz), 103.8, 40.0, 39.5, 33.4,
32.3. HPLC purity 99.2%.
5.1.2.22. N1-(2-Bromo-11-methyl-11H-indolo[3,2-c]quinolin-6-
yl)propane-1,3-diamine (10d).
Yield: 58%, mp: 153–155 °C;
1H NMR d 8.55 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.2 Hz,
1H), 7.61 (t, J = 8.1 Hz, 2H), 7.48 (d, J = 7.7 Hz, 1H), 7.33 (t,
J = 7.5 Hz, 1H), 7.21 (s, 1H), 4.30 (s, 3H), 3.77 (d, J = 3.9 Hz, 2H),
2.74 (t, J = 6.2 Hz, 2H), 1.81 (m, 2H); 13C NMR d 153.4, 146.2,
140.0, 139.3, 130.8, 129.1, 124.8, 124.6, 121.1, 121.0, 120.3,
116.4, 112.9, 110.4, 103.7, 40.4, 39.8, 33.6, 32.6. HPLC purity 90.5%.
5.1.2.29.
yl)propane-1,3-diamin (10k).
N1-(2,11-Dimethyl-11H-indolo[3,2-c]quinolin-6-
Yield: 46%, mp: 112–114 °C;
1H NMR d 8.45 (d, J = 7.8 Hz, 1H), 8.32 (s, 1H), 7.82 (d, J = 8.3 Hz,
1H), 7.63 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.39–7.32 (m,
2H), 4.37 (s, 3H), 3.78 (t, J = 6.1 Hz, 2H), 2.75 (t, J = 5.8 Hz, 2H),
2.51 (s, 3H), 1.86–1.79 (m, 2H); 13C NMR d 152.8, 145.5, 140.3,
139.9, 129.9, 129.7, 126.9, 124.3, 121.9, 121.0, 120.8, 120.7,
114.9, 110.1, 103.4, 39.1, 33.8, 31.9, 22.9, 21.5. HPLC purity 99.1%.
5.1.2.23. N1-(2-Bromo-11-methyl-11H-indolo[3,2-c]quinolin-6-
yl)-2,2-dimethylpropane-1,3-diamine (10e).
Yield: 61%,
mp: 217–219 °C; 1H NMR d 8.54 (s, 1H), 8.42 (d, J = 7.9 Hz, 1H),
7.81 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 6.1 Hz, 2H), 7.49 (t, J = 7.7 Hz,
1H), 7.34 (t, J = 7.5 Hz, 1H), 4.30 (s, 3H), 3.62 (s, 2H), 2.55 (s, 2H),
0.99 (s, 6H); 13C NMR d 153.6, 146.1, 140.0, 139.2, 130.8, 129.0,
124.7, 124.6, 121.2, 120.9, 120.4, 116.5, 112.8, 110.4, 103.8, 52.0,
50.6, 35.4, 33.6, 24.7 (2C). HPLC purity 93.1%.
5.1.2.30. N1-(2-Methoxy-11-methyl-11H-indolo[3,2-c]quinolin-
6-yl)propane-1,3-diamine (10l).
Yield: 53%, mp: 79–81 °C;
1H NMR d 8.43 (d, J = 7.9 Hz, 1H), 7.90 (s, 1H), 7.80 (m, 1H), 7.65
(d, J = 9.0 Hz, 1H), 7.47 (s, 1H), 7.31 (s, 1H), 7.20 (m, 1H), 6.87
(m, 1H), 4.36 (d, J = 4.3 Hz, 3H), 3.91 (s, 3H), 3.74 (s, 2H), 2.73
(t, J = 6.2 Hz, 2H), 1.881 (m, 2H); 13C NMR
d 153.8, 152.1,
5.1.2.24. N1-(2-Bromo-11-methyl-11H-indolo[3,2-c]quinolin-6-
142.5, 140.2, 140.0, 128.5, 124.4, 121.1, 120.8, 120.7, 118.0,
115.0, 110.2, 103.9, 103.8, 55.8, 40.5, 39.7, 33.6, 32.9. HPLC purity
98.4%.
yl)-N3,N3-dimethylpropane-1,3-diamine (10f).
Yield: 53%,
d 8.56 (d, J = 1.7 Hz, 1H), 8.30 (d,
mp: 132–134 °C; NMR
J = 8.0 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.65 (m, 2H), 7.49 (d,
J = 7.3 Hz, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.28 (s, 1H), 4.32 (s, 3H),
3.74 (d, J = 5.3 Hz, 2H), 2.46 (t, J = 6.3 Hz, 2H), 2.24 (s, 6H), 1.81
(m, 2H); 13C NMR d 153.4, 146.3, 140.0, 139.3, 130.9, 129.3,
124.9, 124.7, 121.2, 120.6, 120.4, 116.5, 113.0, 110.6, 103.8, 58.9,
45.9 (2C), 41.15, 33.7, 26.5. HPLC purity 93.0%.
5.1.3. General procedure for the synthesis of compounds 6 and
11
11H-Indolo[3,2-c]quinolin-6-amine (5, 30 mg) and was com-
pletely dissolved in THF (1 mL), and then a solution of isocyanate
(1.2 equiv) and THF (1 mL) were added drop by drop under stirring
at room temperature for 2–6 h. TLC monitoring was used to ensure
complete of the reaction. After reaction was finished, the reaction
mixture was evaporated to dryness. The crude product was puri-
fied by flash chromatography using n-hexane-AcOEt (1:1) as rhe
eluent to yield pure products.
5.1.2.25. N1-(2-Chloro-11-methyl-11H-indolo[3,2-c]quinolin-6-
yl)propane-1,3-diamine (10g).
Yiel: 67%, mp: 171–173 °C;
1H NMR d 8.46 (d, J = 7.9 Hz, 1H), 8.43 (s, 1H), 7.81 (d, J = 8.3 Hz,
1H), 7.68 (d, J = 8.8 Hz, 1H), 7.49 (m, 2H), 7.34 (t, J = 7.4 Hz, 1H),

N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
2639
5.1.3.1.
phenylurea (6a).
1-(6-(11H-Indolo[3,2-c]quinolin-6-ylamino)hexyl)-3-
Yield: 61%; 1H NMR d 12.40 (s, 1H), 8.37
NMR d 12.52 (s, 1H), 8.57 (s, 1H), 8.48 (m, 2H), 7.66 (d, J = 8.1 Hz,
1H), 7.61 (d, J = 8.8 Hz, 1H), 7.56 (dd, J = 8.8, 2.3 Hz, 1H), 7.43 (t,
J = 8.0 Hz, 3H), 7.31 (t, J = 7.5 Hz, 1H), 7.22 (t, J = 7.9 Hz, 2H), 6.88
(t, J = 7.3 Hz, 2H), 6.33 (s, 1H), 3.76 (d, J = 6.1 Hz, 2H), 3.26 (d,
J = 6.2 Hz, 2H), 1.88 (m, 2H); 13C NMR d 156.1, 153.7, 145.2,
141.0, 140.0, 138.6, 131.0, 129.1 (2C), 128.8, 124.8, 124.2, 121.6,
121.5, 121.3, 120.8, 118.2 (2C), 116.1, 112.9, 112.0, 103.6, 38.1,
37.2, 30.7. HPLC purity 91.3%. HRMS (ESI) calcd for C25H23BrN5O
[M+H]+ Exact Mass: 488.1086, found 488.1057.
(m, 2H), 8.22 (m, 1H), 7.63(m, 2H), 7.45 (m, 1H), 7.41–7.30 (m,
3H), 7.25 (dt, J = 19.4, 7.4 Hz, 2H), 7.18 (dd, J = 10.3, 5.1 Hz, 2H),
6.84 (d, J = 6.9 Hz, 1H), 6.56 (s, 1H), 6.10 (s, 1H), 3.69 (d,
J = 5.5 Hz, 2H), 3.08 (d, J = 5.7 Hz, 2H), 1.76 (d, J = 5.8 Hz, 2H),
1.48–1.35 (m, 6H); 13C NMR d 155.6, 153.4, 146.5, 141.1, 141.0,
138.5, 129.1 (3C), 128.4, 126.7, 124.3, 121.9, 121.3, 121.2, 121.1,
120.5, 118.0 (2C), 114.5, 111.8, 103.2, 40.9, 39.5, 30.3, 29.8, 26.9,
26.8. HPLC purity 94.5%. HRMS (ESI) calcd for C28H30N5O [M+H]+
Exact Mass: 452.2450, found 452.2437.
5.1.3.7. 1-(3-(2-Methyl-11H-indolo[3,2-c]quinolin-6-ylamino)
propyl)-3-phenylurea (6g).
Yield: 76%, mp: 140–142 °C; 1H
5.1.3.2. 1-(3-(11H-Indolo[3,2-c]quinolin-6-ylamino)propyl)-3-
phenylthiourea (6b).
NMR d 12.37 (s, 1H), 8.57 (s, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.04 (s,
1H), 7.62 (dd, J = 17.8, 8.2 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.39 (t,
J = 7.4 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.22
(t, J = 7.8 Hz, 2H), 6.88 (t, J = 7.3 Hz, 1H), 6.64 (d, J = 5.0 Hz, 1H),
6.35 (d, J = 5.1 Hz, 1H), 3.76 (d, J = 5.9 Hz, 2H), 3.27 (d, J = 6.0 Hz,
2H), 2.47 (s, 3H), 1.91–1.84 (m, 2H); 13C NMR d 156.1, 153.0,
144.7, 141.0, 140.9, 138.5, 130.1, 123.0, 129.1(2C), 126.6, 124.2,
121.9, 121.4, 121.3, 121.1, 120.4, 118.2(2C), 114.3, 111.8, 103.3,
38.1, 37.3, 30.8, 215. HPLC purity 96.3%. HRMS (ESI) calcd for
Yield: 84%; 1H NMR d 12.50 (s, 1H),
9.64 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 7.9 Hz, 1H), 8.00
(m, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.50 (m, 1H), 7.48–7.38 (m, 4H),
7.29 (ddd, J = 22.0, 15.2, 7.5 Hz, 4H), 7.10 (t, J = 7.4 Hz, 1H), 6.79
(m, 1H), 3.79 (dd, J = 12.2, 6.1 Hz, 2H), 3.69 (s, 2H), 2.01 (m, 2H);
13C NMR d 180.7, 153.3, 141.2, 139.5, 138.5 (2C), 129.2, 128.5,
126.4, 124.8, 124.4, 123.7, 122.0, 121.8 (2C), 121.4, 121.2, 120.6,
114.5, 111.9, 103.0, 42.2, 38.33, 29.9. HPLC purity 99.4%. HRMS
(ESI) calcd for C25H24N5S [M+H]+ Exact Mass: 426.1752, found
426.1731.
C
26H26N5O [M+H]+ Exact Mass: 424.2137, found 424.2115.
5.1.3.8. 1-(3-(2-Methoxy-11H-indolo[3,2-c]quinolin-6-ylamino)
propyl)-3-phenylurea (6h).
Yield: 85%, mp: 129–131 °C; 1H
5.1.3.3. 1-(3-(11H-Indolo[3,2-c]quinolin-6-ylamino)propyl)-3-
phenylurea (6c).
Yield: 98%; 1H NMR d 12.47 (s, 1H), 8.61
NMR d 12.38 (s, 1H), 8.58 (d, J = 3.7 Hz, 1H), 8.44 (d, J = 7.6 Hz,
1H), 7.78 (s, 1H), 7.65 (d, J = 7.9 Hz, 2H), 7.46–7.38 (m, 3H), 7.28
(s, 1H), 7.22 (t, J = 7.8 Hz, 2H), 7.14 (d, J = 9.0 Hz, 1H), 6.89 (d,
J = 7.4 Hz, 1H), 6.59 (m, 1H), 6.36 (d, J = 5.0 Hz, 1H), 3.89 (s, 3H),
3.74 (d, J = 5.0 Hz, 2H), 3.27 (m, 2H), 1.88 (dd, J = 5.9, 3.9 Hz, 2H);
13C NMR d 156.2, 154.3, 152.1, 141.1, 141.0, 138.6, 129.1 (2C),
128.0, 124.5, 122.0, 121.5, 121.3, 120.5, 118.9, 118.2 (2C), 118.1,
114.6, 111.8, 103.5, 102.6, 55.9, 38.2, 37.3, 30.6. HPLC purity
95.7%. HRMS (ESI) calcd for C26H26N5O2 [M+H]+ Exact Mass:
440.2087, found 440.2047.
(s, 1H), 8.49 (d, J = 7.9 Hz, 1H), 8.28 (d, J = 7.9 Hz, 1H), 7.73 (d,
J = 8.3 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.48 (m, 3H), 7.43 (t,
J = 7.5 Hz, 1H), 7.29 (dt, J = 19.3, 7.5 Hz, 2H), 7.24 (t, J = 7.1 Hz,
2H), 6.90 (dd, J = 10.7, 4.0 Hz, 1H), 6.79 (t, J = 5.0 Hz, 1H), 6.39 (t,
J = 5.4 Hz, 1H), 3.82 (dd, J = 11.8, 5.7 Hz, 2H), 3.32 (dd, J = 11.9,
5.8 Hz, 2H), 1.93 (dd, J = 12.5, 6.2 Hz, 2H); 13C NMR d 156.2,
153.4, 146.5, 141.2, 141.1, 138.6, 129.1 (2C), 128.4, 126.7, 124.4,
122.0, 121.9, 121.5, 121.2, 121.1, 120.6, 118.2 (2C), 114.6, 111.9,
103.2, 38.1, 37.3, 30.9. HPLC purity 98.1%. HRMS (ESI) calcd for
C
25H24N5O [M+H]+ Exact Mass: 410.1981, found 410.1963.
5.1.3.9. 1-(3-(2-Nitro-11H-indolo[3,2-c]quinolin-6-ylamino)pro-
5.1.3.4.
propyl)-3-phenylurea (6d).
1-(3-(2-Fluoro-11H-indolo[3,2-c]quinolin-6-ylamino)
Yield: 83%, mp: 207–209 °C; 1H
pyl)-3-phenylurea (6i).
Yield: 60%, mp: >300 °C decompose;
1H NMR d 12.92 (s, 1H), 9.33 (d, J = 2.6 Hz, 1H), 8.58 (s, 1H), 8.54
(d, J = 8.0 Hz, 1H), 8.23 (dd, J = 9.2, 2.6 Hz, 1H), 7.71 (dd, J = 17.8,
8.6 Hz, 2H), 7.47 (t, J = 7.6 Hz, 1H), 7.41 (dd, J = 13.1, 6.8 Hz, 3H),
7.35 (t, J = 7.5 Hz, 1H), 7.22 (t, J = 7.9 Hz, 2H), 6.88 (t, J = 7.3 Hz,
1H), 6.32 (t, J = 5.8 Hz, 1H), 3.83 (q, J = 6.4 Hz, 2H), 3.26 (q,
J = 6.4 Hz, 2H), 1.92–1.85 (m, 2H); 13C NMR d 156.1, 155.5, 150.6,
141.2, 141.0, 140.4, 138.6, 129.1 (2C), 1271, 125.2, 122.6, 121.5,
121.4, 121.3, 121.2, 119.5, 118.2 (2C), 113.4, 112.3, 103.6, 38.3,
37.2, 30.7. HPLC purity 95.6%. HRMS (ESI) calcd for C25H23N6O2
[M+H]+ Exact Mass: 455.1832, found 455.1799.
NMR d 12.46 (s, 1H), 8.57 (s, 1H), 8.47 (d, J = 8.0 Hz, 1H), 8.02
(dd, J = 9.6, 2.9 Hz, 1H), 7.72 (dd, J = 9.1, 5.4 Hz, 1H), 7.67 (d,
J = 8.1 Hz, 1H), 7.43 (d, J = 7.6 Hz, 3H), 7.34 (dd, J = 10.3, 7.3 Hz,
1H), 7.32–7.29 (m, 1H), 7.22 (q, J = 5.8 Hz, 2H), 6.88 (dd, J = 11.6,
4.2 Hz, 1H), 6.76 (s, 1H), 6.34 (s, 1H), 3.76 (q, J = 6.4 Hz, 2H), 3.27
(q, J = 6.4 Hz, 2H), 1.90–1.86 (m, 2H); 13C NMR
d 156.2
(J = 237.1 Hz), 156.11, 153.0, 143.3, 141.0, 140.6 (J = 3.6 Hz),
138.6, 129.1(2C), 128.5 (J = 8.4 Hz), 124.6, 121.7, 121.5, 121.3,
120.7, 118.2(2C), 116.8 (J = 24.1 Hz), 114.4 (J = 9.6 Hz), 111.9,
106.2 (J = 23.1 Hz), 103.6, 38.1, 37.3, 30.7. HPLC purity 98.4%.
HRMS (ESI) calcd for C25H23FN5O [M+H]+ Exact Mass: 428.1887,
found 428.1873.
5.1.3.10. 1-(3-(2-Chloro-11H-indolo[3,2-c]quinolin-6-ylamino)
propyl)-3-phenylurea (6j).
Yield: 93%; 1H NMR d 12.51 (s,
1H), 8.58 (s, 1H), 8.49 (d, J = 7.9 Hz, 1H), 8.34 (d, J = 2.3 Hz, 1H),
7.66 (m, 2H), 7.44 (dd, J = 14.9, 8.1 Hz, 4H), 7.31 (t, J = 7.5 Hz,
1H), 7.22 (t, J = 7.7 Hz, 2H), 6.88 (t, J = 7.0 Hz, 2H), 6.34 (t,
J = 5.4 Hz, 1H), 3.78 (m, 2H), 3.328 (m, 2H), 1.88 (m, 2H); 13C
NMR d 156.1, 153.6, 145.0, 141.0, 140.0, 138.6, 129.0 (2C), 128.5,
128.4, 125.0, 124.8, 121.6, 121.4, 121.3, 121.1, 120.8, 118.2 (2C),
115.4, 112.0, 103.7, 38.1, 37.2, 30.7. HPLC purity 99.8%. HRMS
(ESI) calcd for C25H23ClN5O [M+H]+ Exact Mass: 444.1591, found
444.1573.
5.1.3.5.
propyl)-3-phenylurea (6e).
1-(3-(1-Bromo-11H-indolo[3,2-c]quinolin-6-ylamino)
Yield: 86%, mp: >230 °C decom-
pose; 1H NMR d 11.77 (s, 1H), 8.57 (s, 1H), 8.51 (d, J = 8.0 Hz,
1H), 7.99 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.50 (d,
J = 7.5 Hz, 1H), 7.48–7.41 (m, 3H), 7.37 (m, 2H), 7.22 (t, J = 7.5 Hz,
2H), 6.96 (s, 1H), 6.88 (m, 1H), 6.32 (s, 1H), 3.78 (d, J = 6.2 Hz,
2H), 3.27 (d, J = 6.2 Hz, 2H), 1.89 (m, 2H); 13C NMR d 156.2,
153.5, 148.5, 141.1, 138.7, 138.5, 129.1 (2C), 128.7, 127.0, 126.3,
124.9, 121.5, 121.2, 1209, 120.7, 118.2 (2C), 115.9, 114.1, 113.5,
104.8, 38.2, 37.3, 30.8. HPLC purity 99.8%. HRMS (ESI) calcd for
5.1.3.11. 1-(3-(2-Chloro-11H-indolo[3,2-c]quinolin-6-ylamino)-
C
25H23BrN5O [M+H]+ Exact Mass: 488.1086, found 488.1064.
2-methylpropyl)-3-phenylurea (6k).
Yield: 78%, mp: 143–
145 °C; 1H NMR d 12.50 (s, 1H), 8.58 (s, 1H), 8.52 (d, J = 8.0 Hz,
1H), 8.32 (d, J = 2.5 Hz, 1H), 7.67–7.63 (m, 2H), 7.45–7.40 (m,
4H), 7.33–7.27 (m, 1H), 7.24–7.17 (m, 2H), 6.93 (t, J = 6.1 Hz, 1H),
5.1.3.6.
propyl)-3-phenylurea (6f).
1-(3-(2-Bromo-11H-indolo[3,2-c]quinolin-6-ylamino)
Yield: 79%, mp: 126–128 °C; 1H

2640
N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
6.89–6.85 (m, 1H), 6.42 (t, J = 6.1 Hz, 1H), 3.69 (dd, J = 12.6, 6.6 Hz,
1H), 3.62–3.55 (m, 1H), 3.18 (qd, J = 13.7, 7.8 Hz, 2H), 2.10 (dd,
J = 12.7, 6.8 Hz, 1H), 0.96 (d, J = 6.8 Hz, 3H); 13C NMR d 156.4,
153.7, 145.0, 141.0, 140.2, 138.6, 129.2(2C), 128.5, 128.4, 124.9,
124.8, 121.6, 121.5, 121.2, 121.1, 120.8, 118.2(2C), 115.4, 112.0,
103.6, 43.6, 42.8, 34.5, 16.3. HPLC purity 100%. HRMS (ESI) calcd
for C26H25ClN5O [M+H]+ Exact Mass: 458.1748, found 458.1726.
121.4, 121.0, 120.6, 114.5, 111.9, 103.0, 40.9, 38.0, 30.0. HPLC pur-
ity 100%. HRMS (ESI) calcd for C24H23N4O2S [M+H]+ Exact Mass:
431.1542, found 431.1519.
5.1.5. General procedure for the synthesis of compound 8
11H-Indolo[3,2-c]quinolin-6-amine (5a, 30 mg) and was
completely dissolved in THF (1 mL), and then a solution of acyl
chloride (1.2 equiv) and THF (1 mL) were added drop by drop
with stirring, and finally 2.0 equiv of triethylamine was added,
the reaction was carried out at room temperature for 2–6 h. TLC
monitoring was used to ensure complete of the reaction. After
reaction was finished, the crude product was purified by flash
chromatography using hexane-AcOEt (1:1) as the eluent to yield
pure products.
5.1.3.12. 1-(3-(2-Chloro-11H-indolo[3,2-c]quinolin-6-ylamino)-
2,2-dimethylpropyl)-3-phenylurea (6l).
Yield: 84%, mp:
138–140 °C; 1H NMR d 12.52 (s, 1H), 8.71 (s, 1H), 8.65 (d,
J = 8.0 Hz, 1H), 8.34 (d, J = 2.0 Hz, 1H), 7.70–7.64 (m, 2H), 7.46
(dd, J = 17.4, 8.2 Hz, 4H), 7.34 (t, J = 7.5 Hz, 1H), 7.24 (t, J = 7.9 Hz,
2H), 7.06 (s, 1H), 6.90 (t, J = 7.3 Hz, 1H), 6.68 (t, J = 6.0 Hz, 1H),
3.64 (d, J = 6.3 Hz, 2H), 3.10 (d, J = 6.4 Hz, 2H), 0.95 (s, 6H); 13C
NMR d 156.8, 154.0, 144.9, 140.9, 140.2, 138.6, 129.1(2C), 128.4,
128.3, 124.9, 124.8, 121.7, 121.6, 121.0(2C), 120.9, 118.3(2C),
115.4, 112.0, 103.5, 46.8, 46.1, 37.2, 24.0(2C). HPLC purity 98.0%.
HRMS (ESI) calcd for C27H27ClN5O [M+H]+ Exact Mass: 472.1904,
found 472.1863.
5.1.5.1. 3-(3-(11H-Indolo[3,2-c]quinolin-6-ylamino)propyl)-1,1-
diethylurea (8a).
Yield: 78%, mp: 83–85 °C; 1H NMR d 12.44
(s, 1H), 8.49 (d, J = 7.9 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.65 (d,
J = 8.1 Hz, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H),
7.32–7.16 (m, 2H), 6.88 (s, 1H), 6.32 (t, J = 4.9 Hz, 1H), 3.73 (dd,
J = 11.2, 5.5 Hz, 2H), 3.28–3.17 (m, 6H), 1.82 (dd, J = 11.9, 5.8 Hz,
2H), 1.08–0.94 (m, 6H); 13C NMR d 157.7, 153.3, 146.5, 141.2,
138.5, 128.4, 126.5, 124.3, 121.9(2C), 121.1(2C), 120.6, 114.5,
111.8, 103.2, 40.6(2C), 37.9, 37.8, 31.0, 14.3(2C). HPLC purity 97.1%.
5.1.3.13. 1-(3-(11-Methyl-11H-indolo[3,2-c]quinolin-6-ylamino)
propyl)-3-phenylurea (11).
Yield: 72%, mp: 218–220 °C; 1H
NMR d 8.56 (s, 1H), 8.54 (d, J = 8.3 Hz, 1H), 8.49 (d, J = 7.9 Hz,
1H), 7.83 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.50 (dd,
J = 16.2, 8.2 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 7.5 Hz,
1H), 7.28 (t, J = 7.6 Hz, 1H), 7.22 (t, J = 7.7 Hz, 2H), 6.88 (t,
J = 7.3 Hz, 1H), 6.74 (d, J = 5.6 Hz, 1H), 6.33 (d, J = 5.7 Hz, 1H),
4.36 (s, 3H), 3.77 (q, J = 6.3 Hz, 2H), 3.27 (dd, J = 12.5, 6.3 Hz, 2H),
1.88 (m, 2H); 13C NMR d 1561, 153.2, 147.4, 141.1, 140.6, 134.0,
129.1(2C), 128.1, 127.3, 124.4, 122.8, 121.4, 121.2, 121.0, 120.9,
120.7, 118.2(2C), 115.1, 110.3, 103.4, 38.1, 37.3, 33.9, 30.8. HPLC
purity 100%.
5.1.5.2. N-(3-(11H-Indolo[3,2-c]quinolin-6-ylamino)propyl)thi-
ophene-2-carboxamide (8b).
Yield: 90%, mp: 200–202 °C;
1H NMR d 12.47 (s, 1H), 8.64 (t, J = 5.4 Hz, 1H), 8.45 (d, J = 7.9 Hz,
1H), 8.26 (d, J = 7.9 Hz, 1H), 7.80–7.70 (m, 2H), 7.66 (t, J = 7.8 Hz,
2H), 7.48 (dd, J = 8.2, 7.0 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.29 (dt,
J = 17.9, 7.5 Hz, 2H), 7.14 (ddd, J = 8.8, 5.7, 3.2 Hz, 1H), 6.75 (s,
1H), 3.79 (dd, J = 12.1, 6.1 Hz, 2H), 3.46–3.43 (m, 2H), 1.99 (dt,
J = 6.1, 3.9 Hz, 2H); 13C NMR d 161.8, 153.3, 146.4, 141.2, 140.6,
138.5, 131.0, 128.5, 128.3(2C), 126.6, 124.4, 122.0, 121.9, 121.2,
121.4, 120.6, 114.5, 111.9, 103.2, 38.3, 37.5, 30.2. HPLC purity
96.5%.
5.1.4. General procedure for the synthesis of compound 7
N1-(11H-indolo[3,2-c]quinolin-6-yl)propane-1,3-diamine (5a,
30 mg) and was completely dissolved in THF (1 mL), and then a
solution of arenesulfonyl chloride (1.2 equiv) and THF (1 mL) were
added drop by drop with stirring, and finally 2.0 equiv of triethyl-
amine was added, the reaction was carried out at room tempera-
ture for 2–6 h. TLC monitoring was used to ensure complete of
the reaction. After reaction was finished, the crude product was
purified by flash chromatography using hexane/AcOEt (1:1) as
the eluent to yield pure products.
5.2. Biological testing assay
5.2.1. Activity against Plasmodium falciparum
In vitro activity against erythrocytic stages of P. falciparum was
determined using a 3H-hypoxanthine incorporation assay,58,59
using the chloroquine and pyrimethamine resistant P. falciparum
K1 strain that originate from Thailand (Thaitong et al. 1983)60
and strain susceptible to known antimalarial drugs (P. falciparum
NF54) (Ponnudurai et al. 1981),61 and all the test compounds
were compared for activity with the standard drug chloroquine
(Sigma C6628). Compounds were dissolved in DMSO at 10 mg/
mL and added to parasite cultures incubated in RPMI 1640 med-
ium without hypoxanthine, supplemented with HEPES (5.94 g/l),
NaHCO3 (2.1 g/l), neomycin (100 U/mL), AlbumaxR (5 g/l) and
washed human red cells A+ at 2.5% haematocrit (0.3% parasita-
emia). Serial drug dilutions of eleven 3-fold dilution steps cover-
5.1.4.1.
methanesulfonamide (7a).
N-(3-(11H-Indolo[3,2-c]quinolin-6-ylamino)propyl)
Yield: 66%, mp: 193–2195 °C;
1H NMR d 12.51 (s, 1H), 8.41 (d, J = 7.9 Hz, 1H), 8.26 (d, J = 7.9 Hz,
1H), 7.70 (d, J = 8.2 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.51 (t,
J = 7.6 Hz, 1H), 7.44 (dt, J = 15.2, 6.8 Hz, 2H), 7.30 (dd, J = 15.4,
7.8 Hz, 2H), 6.68 (m, 1H), 3.80 (dd, J = 12.0, 5.9 Hz, 2H), 3.10 (q,
J = 6.2 Hz, 2H), 2.85 (d, J = 1.2 Hz, 3H), 1.97 (m, 2H); 13C NMR d
153.5, 146.2, 141.3, 138.6, 128.7, 126.3, 124.5, 122.1, 121.9,
121.5, 121.2, 120.7, 114.6, 112.0, 103.1, 40.7, 39.8, 38.2, 30.4. HPLC
purity 98.8%. HRMS (ESI) calcd for C19H21N4O2S [M+H]+ Exact
Mass: 369.1385, found 369.1359.
ing a range from 100 to 0.002
plates were incubated in a humidified atmosphere at 37 °C; 4%
CO2, 3% O2, 93% N2. After 48 h 50
of 3H-hypoxanthine
(=0.5 Ci) was added to each well of the plate. The plates were
lg/mL were prepared. The 96-well
l
L
l
5.1.4.2. N-(3-(11H-Indolo[3,2-c]quinolin-6-ylamino)propyl)ben-
zenesulfonamide (7b).
incubated for a further 24 h under the same conditions. The
plates were then harvested with a Betaplate™ cell harvester
(Wallac, Zurich, Switzerland), and the red blood cells transferred
onto a glass fibre filter then washed with distilled water. The
dried filters were inserted into a plastic foil with 10 mL of scintil-
lation fluid, and counted in a Betaplate™ liquid scintillation coun-
ter (Wallac, Zurich, Switzerland). IC50 values were calculated from
sigmoidal inhibition curves by linear regression (Huber 1993)62
using Microsoft Excel.
Yield: 91%, mp: 88–90 °C; 1H NMR d
12.46 (s, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.24 (dd, J = 8.0, 1.2 Hz, 1H),
7.96 (t, J = 6.1 Hz, 1H), 7.71 (dd, J = 5.2, 3.3 Hz, 2H), 7.65 (dd,
J = 16.0, 8.1 Hz, 2H), 7.54–7.49 (m, 2H), 7.44 (dd, J = 10.7, 4.8 Hz,
2H), 7.42–7.38 (m, 1H), 7.28 (dd, J = 7.9, 7.1 Hz, 2H), 6.59 (d,
J = 5.9 Hz, 1H), 3.69 (q, J = 6.3 Hz, 2H), 2.89 (q, J = 6.7 Hz, 2H),
1.87–1.80 (m, 2H); 13C NMR d 153.4, 146.2, 141.2, 140.9, 138.5,
132.7, 129.5(2C), 128.6, 126.8(2C), 126.5, 124.4, 122.0, 121.8,

N. Wang et al. / Bioorg. Med. Chem. 22 (2014) 2629–2642
2641
5.2.2. In vitro cytotoxicity against L6 cells
Assays were performed in 96-well microtiter plates, each well
containing 100 L of RPMI 1640 medium supplemented with 1%
-glutamine (200 mM) and 10% fetal bovine serum, and 4000 L6
cells (a primary cell line derived from rat skeletal myoblasts)
(Page et al., 1993 and Ahmed et al., 1994).63,64 Serial drug
dilutions of eleven 3-fold dilution steps covering a range from
References and notes
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100 to 0.002
plates were inspected under an inverted microscope to assure
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l
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Blue was then added to each well and the plates incubated for
another 2 h. Then the plates were read with a Spectramax Gemini
XS microplate fluorometer (Molecular Devices Cooperation,
Sunnyvale, CA, USA) using an excitation wave length of 536 nm
and an emission wave length of 588 nm. The IC50 values were
calculated by linear regression (Huber 1993)62 from the sigmoidal
dose inhibition curves using SoftmaxPro software (Molecular
Devices Cooperation, Sunnyvale, CA, USA).
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50.
5.2.3. Detergent mediated assay for b-haematin inhibition
The b-haematin formation inhibition assay method described
by Carter et al.60,64 was modified for manual liquid delivery. Three
stock solutions of the samples were prepared by dissolving the pre-
weighed compound in DMSO and after sonication, diluting with
DMSO to give 20 mM, 2 mM and 0.4 mM solutions of each sample.
These were delivered to a 96-well plate in duplicate to give con-
18. Radl, S. Archiv der Pharmazie (Weinheim, Germany) 1996, 329, 115.
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We are grateful to Okayama University for its support and to
the Advanced Science Research Center for the NMR experiments
and EA analyses. We are thankful to Prof. S. Nakashima, Okayama
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for the scholarship supports to NW. T.J.E. acknowledges the
National Research Foundation and Medical Research Council of
South Africa and University of Cape Town for the financial
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the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.030.

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