Synthesis and antimalarial testing of neocryptolepine analogues: Addition of ester function in SAR study of 2,11-disubstituted indolo[2,3-b]quinolines

Article abstract of DOI:10.1016/j.ejmech.2013.03.072

This report describes the synthesis, and in vitro and in vivo antimalarial evaluations of certain ester-modified neocryptolepine (5-methyl-5H-indolo[2,3-b] quinoline) derivatives. The modifications were carried out by introducing ester groups at the C2 and/or C9 position on the neocryptolepine core and the terminal amino group of the 3-aminopropylamine substituents at the C11 position with a urea/thiourea unit. The antiplasmodial activities of our 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 the ester modified neocryptolepine derivatives have higher antiplasmodial activities against both strains and a low cytotoxic activity against normal cells. The best results were achieved by compounds 9c and 12b against the NF54 strain with the IC₅₀/SI value as 2.27 nM/361 and 1.81 nM/321, respectively. While against K1 strain, all the tested compounds showed higher activity than the well-known antimalarial drug chloroquine. Furthermore, the compounds were tested for β-haematin inhibition and 12 were found to be more active than chloroquine (IC₅₀ = 18 μM). Structure activity relationship studies exposed an interesting linear correlation between polar surface area of the molecule and β-haematin inhibition for this series. In vivo testing of compounds 7 and 8a against NF54 strain on Plasmodium berghei female mice showed that the introduction of the ester group increased the antiplasmodial activity of the neocryptolepine core substantially.

Full text of DOI:10.1016/j.ejmech.2013.03.072

European Journal of Medicinal Chemistry 64 (2013) 498e511
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimalarial testing of neocryptolepine analogues:
Addition of ester function in SAR study of 2,11-disubstituted indolo
[2,3-b]quinolines
,
d
Wen-Jie Lu a, Kathryn J. Wicht b, Li Wang a, Kento Imai a, Zhen-Wu Mei a, Marcel Kaiser c
**
,
,
,
a,
*
Ibrahim El Tantawy El Sayed a e, Timothy J. Egan b , Tsutomu Inokuchi
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
e Chemistry Department, Faculty of Science, El Menoufeia University, Shebin El Koom, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
This report describes the synthesis, and in vitro and in vivo antimalarial evaluations of certain ester-
modified neocryptolepine (5-methyl-5H-indolo[2,3-b]quinoline) derivatives. The modifications were
carried out by introducing ester groups at the C2 and/or C9 position on the neocryptolepine core and the
terminal amino group of the 3-aminopropylamine substituents at the C11 position with a urea/thiourea
unit. The antiplasmodial activities of our 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
the ester modified neocryptolepine derivatives have higher antiplasmodial activities against both strains
and a low cytotoxic activity against normal cells. The best results were achieved by compounds 9c and
12b against the NF54 strain with the IC50/SI value as 2.27 nM/361 and 1.81 nM/321, respectively. While
against K1 strain, all the tested compounds showed higher activity than the well-known antimalarial
Received 20 December 2012
Received in revised form
30 March 2013
Accepted 31 March 2013
Available online 15 April 2013
Keywords:
5H-Indolo[2,3-b]quinoline
Antiplasmodial activity
CQS/CQR
drug chloroquine. Furthermore, the compounds were tested for
found to be more active than chloroquine (IC50 ¼ 18 M). Structure activity relationship studies exposed
an interesting linear correlation between polar surface area of the molecule and -haematin inhibition
b-haematin inhibition and 12 were
C2/C9-ester substituted neocryptolepine
In vivo test
m
b
for this series. In vivo testing of compounds 7 and 8a against NF54 strain on Plasmodium berghei female
mice showed that the introduction of the ester group increased the antiplasmodial activity of the neo-
cryptolepine core substantially.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
more than 90% of the cases. In spite of the intensive efforts to
combat malaria, the incidence of malaria has not decreased, espe-
Malaria is a potentially deadly tropical disease caused by para-
sites of the genus Plasmodium, which is transmitted via the bites of
infected mosquitoes [1]. According to the World Health Organiza-
tion (WHO), this disease led to about 216 million malarial infected
cases in 2010, and approximately 0.7 million died due to the non-
availability of proper treatment, mostly involving children under
5 years old [2]. The Plasmodium falciparum species, which is the
most virulent and deadly of the malaria parasites, is responsible in
cially in the tropical and subtropical areas [3].
As one of the most effective and cheapest therapeutic agents,
chloroquine (CQ) has been used for more than half a century as a
specific drug for the treatment of malaria patients [4]. The unique
endoperoxide structure of artemisinin emerged in the 1970s as the
results of efforts to find a new drug from plants [5]. These drugs are
still important regarding medical treatment and making efforts to
find more active derivatives from the leads are still continued [6,7].
However, the P. falciparum strains resistant to CQ are still spreading
in the endemic areas [8] and in vitro and in vivo resistances even
against the most recently introduced artemisinin-based combina-
tion therapy (ACT) have also been demonstrated as therapy for un-
complicated P. falciparum infections [9]. Therefore, the development
* Corresponding author. Tel.: þ81 86 251 8210; fax: þ81 86 251 8021.
** Corresponding author. Tel.: þ27 21 650 2528; fax: þ27 21 650 5195.
E-mail addresses: Timothy.Egan@uct.ac.za, inokuchi@cc.okayama-u.ac.jp
(T. Inokuchi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.072

W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
499
of new chemotherapeutic treatments for this disease is urgently
needed.
showed only some reduction in parasitaemia on day 4, with ac-
tivities of 15.4% and 22.1%, respectively. Until now, the SAR studies
of neocryptolepines (indolo[2,3-b]quinoline) have been conducted
by changing the halogen, alkyl, nitro, alkoxy or alkylthio, amino,
cyano groups as the substitution at the C2, C8, C9, and C11 positions
[15]. Therefore, a search for a more effective group amenable to
inducing a higher activity and less toxicity are urgently needed to
improve the in vivo potency.
The isolation of new lead compounds from plants is one of the
strategies in the search for new drugs against infectious diseases
[10e12]. The present work has been carried out as part of our
ongoing program to develop novel antimalarial drugs based on the
natural product neocryptolepine isolated from the roots of the
climbing shrub Cryptolepis sanguinolenta [13] that is used in Central
and West Africa in traditional medicine for the treatment of ma-
laria [14].
The indoloquinoline alkaloids [15,16] such as cryptolepine (5-
methyl-5H-indolo[2,3-c]quinoline) (I), neocryptolepine (5-methyl-
5H-indolo[2,3-b]quinoline, cryptotackieine) (II), isocryptolepine
(5-methyl-5H-indolo[3,2-c]quinoline, cryptosanguinolentine) (III),
and the non-natural isoneocryptolepine (5-methyl-5H-indolo[2,3-
c]quinoline) (IV) were reported to show potent antiplasmodial ac-
tivity against chloroquine-resistant P. falciparum [17], as shown in
Fig. 1. Due to the lower affinity of neocryptolepine for DNA and
topoisomerase II inhibition compared to cryptolepine and iso-
cryptolepine, the neocryptolepine core has been further used as the
lead for developing an antimalarial agent [18].
In our previous paper concerning the antiproliferative activity of
neocryptolepine derivatives [22], we demonstrated that the intro-
duction of an ester function on the indolo[2,3-b]quinoline core
improved both the antiproliferative activity against several cell
lines and the cytotoxicity index against the usual cell line [23]. A
similar effect was also demonstrated on the activity of other plant-
derived anticancer agents such as taxol [24], camptothecin [25],
bryostatin [26], and 5-fluorouracil [27]. In addition, similar
enhancement of the activities is demonstrated in the anti-
plasmodial and antitrypanosomal activities with bicyclic amides
and esters of dialkylamino acids [28]. These improvements in the
anticancer and antimalarial activity are believed to be due to
increasing both the lipophilicity [29] and bioavailability of the drug
by introducing an ester group into the core structure. Encouraged
by these significant examples, we examined the introduction of an
ester group to the neocryptolepine core and studied its influence on
the antiplasmodial activity. In this study, we report the further
investigation of the antimalarial potential of the neocryptolepine
core. An ester group, which may act as a potential lipophilic site,
was introduced at the C-2 position of the A-ring and/or the C-9
position of the D-ring in order to establish or extend the structure
activity relationship (SAR) study for these regions.
In 2002, Pieters and the co-authors reported that the 2-halo-
substituted neocryptolepines (2-bromo-, 2-chloro-, 2-fluoro-, and
2-iodo) are more active against P. falciparum than neocryptolepine
and less cytotoxic, the most active and selective compound being 2-
bromoneocryptolepine with an IC50 against the chloroquine-
resistant P. falciparum of 4.0
mM while the IC50 on the MRC-5 cells
is >32 M. Furthermore, the 2,9-disubstituted derivatives with a
m
cyano group at C9 and various substituents at C2 exhibited an
antitrypanosomal activity in the micromole range in the absence of
an obvious cytotoxicity [19]. Recently, El-Sayed et al. introduced
aminoalkylamino groups at C8 or C11 of neocryptolepine skeleton
by varying the substituents at C2, which showed an increased
antiplasmodial activity of the neocryptolepine core with the in vitro
selectivity index >500. The most active compounds bearing 5-
methyl and 8-(N1,N1-diethylpentane-1,4-diamine) side chains
showed an IC50 of 10 nM, which has the same potency as chloro-
quine and a selectivity index of 1800 [20].
2. Results and discussion
2.1. Chemistry
The C2- and C9-substituted 11-chloro-5-methyl-5H-indolo[2,3-
b]quinolines 6 were prepared using indole-3,5-dicaboxylate 2b or
5-bromo-indole-3-carboxylate 2c as the starting material. Accord-
ing to the method previously described by us [30], N-methylani-
lines were used for the three-step synthesis of 6. The dimethyl 1H-
indole-3,5-dicarboxylate (2b), a key starting compound, was first
prepared by installation of ester group at the C-3 position of methyl
1H-indole-5-carboxylate (1b) using the reaction with tri-
chloroacetyl chloride in the presence of pyridine, followed by
alkaline treatment with KOH at reflux in MeOH.
The obtained 5-substituted methyl indole-3-carboxylate 2 was
oxidatively combined with N-methylanilines 3 via chlorination with
N-chlorosuccinimide (NCS) in the presence of 1,4-dimethylpiperazine,
giving 2-(methyl(aryl)amino)-1H-indole-3,5-dicarboxylate 4. The
heating of 4 at 250 ꢀC in diphenyl ether induced intramolecular
acylation at the C-2 position of the aniline core, forming the tetracyclic
indolo[2,3-b]quinolinone 5. The treatment of 5 with POCl3 afforded
the desired chlorides 6 as a result of dehydrative chlorination.
Addition-substitution at the C11 position smoothly proceeded by
In our previous paper describing the SAR study of the 5-methyl-
5H-indolo[2,3-b]quinoline core, we modified and changed the
u-
aminoalkylamino side chains at C11 by varying the electron-
withdrawing or electron-donating nature of the C2 substituent.
The compound bearing (3-(3-phenylureido)propyl)amino at C11
and Cl at C2 showed an antimalarial activity 4 times more potent
than chloroquine (CQ) for CQS (NF54) with an IC50 of 2.2 nM and a
selectivity index of 1400, and the compound substituted with ((3-
phenylthioureido)pentyl)amino at C11 and MeO at C2 showed
a much greater potency than CQ for CQR (K1) with an IC50 of 2.2 nM,
a selectivity index of 1243 and a resistance index of 0.5 by K1/NF54
[21].
However, in spite of the significantly improved antimalarial
activities of some compounds against the NF54 strain, testing of the
in vivo drug screening model against Plasmodium berghei in Swiss
mice were not satisfactory, as the neocryptolepine derivatives
Fig. 1. Structures of indoloquinolines from Cryptolepis sanguinolenta.

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W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
heating with amines in THF to give various 11-aminoindolo[2,3-b]
quinolines 7e9 (Scheme 1).
was regarded as the best motif at the C2 position. Therefore, we can
confirm that the substitution of the ester group at either C2 or C9
position on the neocryptolepine core can both have a positive effect
on the antimalarial activity. Due to this discovery, we have further
synthesized several 2,9-disubstituted derivatives 9 by varying the
halogen (eCl, eBr) and ester group, and as expected, their anti-
malarial activity was improved and found higher than that of the
well-known antimalarial agent, chloroquine, with a range of IC50
value from 2.16 to 9.52 nM and also an improved SI value that
ranged from 56.4 to 361.
On the other hand, the solubility of an agent in an aqueous
medium can significantly influence its further application, despite
its high biological activity. We further tried to improve the solu-
bility of the agents by transforming the ester group into a carbox-
ylic acid substituent. The C9-ester substituted neocryptolepines
were hydrolysed into the C9-carboxylic acid analogues by treating
the methyl 11-amino-5H-indolo[2,3-b]quinoline-9-carboxylate 8
with NaOH(aq) in MeOH. The mixtures were refluxed overnight to
afford the C9-carboxylic acid substituted indolo[2,3-b]quinolines
11 in good yields, as shown in Scheme 2. The evaluation results of
their antimalarial activity are summarized in Table 2.
Unfortunately, it turned out that though the solubility of the
hydrolysed derivatives 11 in water was improved, their anti-
plasmodial activity against P. falciparum dramatically decreased
compared to their precursors 8a, 8c and 8d. Similar effect was also
mentioned on the comparable activity of the natural alkaloids
cryptolepinoic acid and methyl cryptolepinoate [31].
Further modifications of the 11-amino neocryptolepine de-
rivatives 7e9 at the terminal amino group of the lateral attachment
were carried out as outlined in Scheme 3. The amino group was
3. Pharmacology
3.1. Antiplasmodial activity and cytotoxicity
The introduction of an amino group at the C11 position and a
halo group at the C2 position on the 5-methylindole[2,3-b]quino-
line (neocryptolepine) core can significantly increase the anti-
plasmodial activities compared to the natural product against the
chloroquine-sensitive P. falciparum Ghana strain based on a previ-
ous study [19]. Based on these facts, we introduced the amino
group at the C11 position with the varying kinds of substituents at
the C2 position and the C9 position for the SAR study of the neo-
cryptolepine core.
The results of the studies on the antiplasmodial activity against
P. falciparum (CQS: NF54) and the cytotoxicity towards L6 cells
based on the synthesized derivatives 7e10 are summarized in
Table 1 along with the results of the antimalarial drug, chloroquine.
All the tested compounds, except the compound 8g, have signifi-
cant cytotoxic effect against the NF54 strains of P. falciparum. From
the data of series 8, we found that compound 8a, with a 3-
aminopropylamino group at the C11 position, contributed to the
antimalarial activity most with an IC50 value of 24.8 nM. In addition,
compared to the non-substituted compound 10, which was syn-
thesized in our previous study, the remarkable increase happened
after introducing the ester group at the C2 or C9 position (com-
pounds 7 and 8a). Moreover, the ester group showed a higher
contribution to the antiplasmodial activity than the Cl group that
Scheme 1. Preparation of ester substituted 11-aminoneocryptolepines 7e9. Reagents and conditions: (i) (a) pyridine, trichloroacetyl chloride, THF, (b) KOH, MeOH, reflux.
(ii) (a) N-Chlorosuccinimide, 1,4-dimethylpiperazine, (b) trichloroacetic acid. (iii) Diphenyl ether, reflux. (iv) POCl3, toluene, reflux. (v) Appropriate amines.

W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
501
Table 1
The antiplasmodial activity against P. falciparum (CQS: NF54) and cytotoxicity towards L6 cells of the neocryptolepine derivatives 7e9.
Compound
R1
R2
R3
Yield
L6 cells
IC50 nM
3194
NF54
SIa
b
-Haematin inhibition
IC50 nM
1580
L6/NF54
2.0
IC50
mM
Neocryptolepine
10 [20]
H
H
H
H
H
91%
92%
95%
86%
279
218
436
315
78.8
24.8
55.8
42.5
3.50
8.79
7.81
7.41
8a
8b
8c
CO2Me
CO2Me
CO2Me
48.9
51.5
62.5
8d
8e
H
H
CO2Me
CO2Me
87%
89%
268
639
33.3
278
8.05
9.49
122.8
8f
8g
7
H
CO2Me
CO2Me
H
91%
90%
93%
95%
88%
6190
3810
337
750
1770
8.28
9.52
7.57
8.25
2.15
40.7
146
105.5
55.5
H
CO2Me
CO2Me
Cl
104.4
13.0
9a
9b
CO2Me
CO2Me
1390
427
56.4
25.4
9c
9d
9e
Br
CO2Me
Br
83%
96%
91%
819
2.27
4.54
2.16
9.40
361
232
186
11.5
10.2
30.1
18.4
CO2Me
Cl
1050
Br
401
Podophylotoxin
Chloroquine
14.5
a
Selectivity Index is the ratio of IC50 for cytotoxicity versus antiplasmodial activity (L6/P.f.).

502
W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
vacuole membrane, haemozoin inhibition remains
a suitable
parasite-specific drug target. Recently, neutral lipids have been
shown to mediate haemozoin formation in the digestive vacuole
[34,35]. Furthermore, synthetic haemozoin (b-haematin) can form
under biomimetic conditions with detergent mediators, which
imitate the neutral lipids. As a result, an assay has been developed
which quantifies the ability of a compound to inhibit b-haematin
formation by measuring the UVevis absorbance of the haeme
pyridine complex at 405 nm, formed when pyridine is added to free
haematin [36,37].
Scheme 2. Hydrolysis of 8. Reagents and conditions: MeOH/NaOH(aq).
The compounds were tested for
(Tables 1 and 3) and many show high activity, with 12 samples
having lower IC50 values than chloroquine (18 M). Structure
activity relationship studies exposed an interesting linear corre-
lation (Fig. 2a) between polar surface area of the molecule and
b-haematin inhibition
transformed into urea/thiourea group by mixing with isocyanate or
isothiocyanate in dried THF with stirring at 0 ꢀC to room temper-
ature. Their antiproliferative activities against P. falciparum and L6
cells are summarized in Table 3.
m
b
-
As shown in Table 3, after the transforming of the 3-
aminopropylamine to the urea group, the cytotoxicity of all the
synthesized urea derivatives 12 against the L6 cells decreased,
especially the 2,9-diester substituted compound 12e with a sig-
nificant IC50 value of 26,500 nM. In addition, the mono-substituted
derivatives 12aed at the C2 or C9 position had improved IC50 values
ranging from 1.81 to 14.5 nM against P. falciparum (CQS: NF54). In
addition, the thiourea group showed a higher contribution to the
antiplasmodial activity against P. falciparum (CQS: NF54) than the
urea group, when compared between agents 12c/12d and agents
12e/12f. On the other hand, the antiplasmodial activity against
P. falciparum (CQS: NF54) of the C2 and C9 disubstituted ones 12eei
slightly decreased. All these thiourea/urea derivatives showed
significant activity against CQS (NF54), and some of them are
higher than the well-known antimalarial drug chloroquine
(IC50 ¼ 9.1 nM). Compound 12b gave the best result with an IC50
value of 1.81 nM and a high SI value of 321 among the compound 12
series.
haematin inhibition for this series. Intriguingly, the direct rela-
tionship may suggest that the site at which these molecules block
b
-haematin growth is hydrophilic in nature. The neocryptolepine
molecular structures bear similarities to the 4-aminoquinoline
based antimalarials. In vitro studies with these drugs have
revealed an increase in the amount of free haem in the parasite
digestive vacuole, suggesting haemozoin inhibition as the mech-
anism of action [38]. Whilst the neocryptolepine series shows
similar
b
-haematin inhibition activity, correlation analysis be-
-haematin IC50s revealed
tween whole cell NF54 IC50 values and
b
a very weak correlation (Fig. 2b). This is not entirely unexpected
owing to the large number of factors that might influence a
compound’s ability to cross four membranes to reach the site of
haemozoin formation (the red blood cell membrane, the para-
sitophorous vacuole membrane, the P. falciparum plasma mem-
brane and the food vacuole membrane).
In order to explore factors that might influence activity in
NF54 parasites, multiple regression analysis was carried out be-
tween the logged IC50 (log(NF54IC50)) and a range of predicted
physical properties for each molecule in the series as well as
3.2.
b-Haematin inhibition and QSAR
measured b-haematin inhibition IC50 values (Fig. 2c). The statis-
tically strongest correlation was observed for the 3-variable
model with polar surface area (PSA), mean water of hydration
(H2O$hyd) and the natural log of the hydrogen bond donors
(ln(HBD)). Fitted coefficients show a large dependence on the
hydration term, such that molecules which are well hydrated
have the greatest activity. Opposing this, with the second highest
weighting, is the PSA term which, when decreased, increases the
activity. Lastly, an increase in ln(HBD) results in greater activity.
Upon entering the parasite membrane, a highly hydrated mole-
cule would liberate many molecules of water, causing a favour-
able increase in entropy. A molecule with more hydrogen bond
The formation of haemozoin in the acidic food vacuole of
P. falciparum is vital for malaria parasite survival. Inhibiting this
crystallisation process causes an accumulation of cytotoxic hae-
matin, Fe(III)PPIX, an oxidation product of the haem molecule
which is released upon the parasite’s ingestion of haemoglobin
[32]. Several well-known antimalarial drugs (e.g. chloroquine) are
thought to interact with haematin at pH 4.8 through
p-stacking,
coordination and/or hydrogen bonding, blocking the growth face of
haemozoin and slowing down the crystal formation rate [33].
Despite the development of resistance, now widely accepted to be
the result of altered parasite transporter proteins in the food
Table 2
The antiplasmodial activity against P. falciparum (CQS: NF54) and cytotoxicity towards L6 cells of compounds 11.
Compound
R1
R2
R3
Yield
82%
79%
85%
L6 cells
IC50 nM
NF54
SIa
Solubility in H2O
Freely
IC50 nM
L6/NF54
11a
H
CO2H
CO2H
CO2H
113,000
20,000
66,200
1690
2550
2160
66.8
7.84
30.6
11b
H
Freely
11c
H
Freely
a
Selectivity Index is the ratio of IC50 for cytotoxicity versus antiplasmodial activity (L6/P.f.).

W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
503
hydration and polarity must be established for good activity. This
correlation therefore appears to be physically feasible and could
be used as a strategy for synthesizing derivatives with higher
potency. For example, compounds with the best activity (9ce12b)
all contain three HBDs, resulting in large H2O$hyd and not more
than one polar ester group. Furthermore the most active com-
pound (12b) contains a thiourea which contributes significantly
less to the PSA than the urea group without detrimentally
affecting the number of waters of hydration and with no decrease
in HBDs. In contrast, the least active compounds (8eeg) have a
moderate PSA, less than one HBD and notably lower H2O$hyd.
The series includes nine compounds containing the basic
amino group, which is considered vital for pH trapping in
the parasite acidic digestive vacuole. Previous work on 4-
aminoquinolines suggests that these nine compounds should
Scheme 3. Further modifications of the lateral amino group. Reagents and conditions:
PhNCX, THF, 0e25 ꢀC, 30 min.
donors would logically be better hydrated, and indeed there is a
significant correlation between these two variables. However,
should the molecule be too polar, it will not be able to pass
through the lipophilic membrane. Thus
a balance between
Table 3
Antiplasmodial activity against P. falciparum (CQS: NF54) and cytotoxicity towards L6 cells of compounds 12.
Compound
R1
R2
R3
Yield
93%
L6 cells
IC50 nM
NF54
SIa
b-Haematin inhibition
IC50 nM
L6/NF54
IC50 mM
12a
CO2Me
H
754
4.16
181
23.0
12b
12c
CO2Me
H
84%
92%
581
1.81
14.5
321
178
15.7
12.8
H
CO2Me
2580
12d
12e
H
CO2Me
CO2Me
96%
90%
2820
6.03
16.7
467
10.8
4.6
CO2Me
26,500
1590
12f
CO2Me
CO2Me
CO2Me
84%
89%
627
14.4
13.6
43.5
246
7.5
12g
Cl
3340
14.9
12h
12i
12j
Br
CO2Me
90%
83%
89%
6740
1120
8.05
7.15
837
157
73
16.0
10.1
CO2Me
Br
Cl
Br
408
5.59
9.40
17.8
18.4
Podophylotoxin
Chloroquine
14.5
a
Selectivity Index is the ratio of IC50 for cytotoxicity versus antiplasmodial activity (L6/P.f.).

504
W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
Fig. 2. Quantitative structureeactivity relationships were found for the neocryptolepine series. (a) An increase in polar surface area (PSA) was found to improve b-haematin
inhibition activity according to 1/
activity with the
b
HIC50 ¼ 0.001788(PSA) e 0.09485, r2 ¼ 0.58, P < 0.0001. (b) A weak correlation (r2 ¼ 0.43 and P ¼ 0.001) was observed for the log of NF54
b
-haematin activity, showing that the biological activity of a compound depends on more factors than its ability to inhibit haemozoin, if this is indeed the
target. (c) Predicted vs experimental NF54 activity based on multiple linear regression where log(NF54IC50) ¼ ꢁ0.299(H2O.hyd) þ 0.028(PSA) ꢁ 0.652(ln(HBD)) þ 3.108
(r2 ¼ 0.904, P < 0.0001); statistically significant for both individual parameters (t ¼ 5.40, 5.78, 6.85, 4.34 > tcrit ¼ 2.88) and overall correlation (F ¼ 56.811 > Fcrit ¼ 5.09) at 99%
confidence level.
Table 4
Antiplasmodial activity against P. falciparum (CQS: NF54, CQR: K1) and cytotoxicity towards L6 cells of neocryptolepine derivatives.
NO
L6 cells IC50 nMc
NF54 IC50 nMc
SIa L6/NF54
K1 IC50 nMc
SIa L6/K1
RIb K1/NF54
Neocryptolepine
7
8a
9a
9b
9d
9e
12a
12b
12c
12d
12e
12f
12g
12i
12j
3194
337
218
1390
427
1050
401
1580
8.28
24.8
9.52
7.57
4.54
2.16
4.16
1.81
14.5
6.03
16.7
14.4
13.6
7.15
5.59
2.02
40.7
8.79
146
56.4
232
186
181
321
178
1696
22.1
30.3
11.9
20.2
15.9
19.2
56.1
16.1
68.4
64.4
40.8
50.4
23.3
19.7
16.8
1.88
15.2
7.19
117
21.1
66.0
20.9
13.4
36.1
37.7
43.8
650
1.07
2.67
1.22
1.25
2.67
3.5
8.89
13.5
8.89
4.71
10.7
2.44
3.50
1.71
2.75
3
754
581
2580
2820
26500
627
3340
1120
408
467
1590
43.5
246
157
73
12.4
143
56.9
24.3
Podophylotoxin
Chloroquine
14.5
9.40
210
22.3
a
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 less than a factor 2.
b
c

W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
505
accumulate at the target site much more readily than those with
5. Experimental
no basic amino side chains and consequently display better ac-
tivities [29,39]. Upon inspection, it is evident that the basic
groups do not influence the NF54 activity and as a result, it
appears unlikely that the target lies within the digestive vacuole.
While it appears that haemozoin may not be the target of these
compounds, the result is not completely definitive, because of
the observation of a weak correlation as shown in Fig. 2b.
Combrinck et al. [38] have shown the ability of antimalarials to
increase free haem in cultured P. falciparum by using cell frac-
tionation. Such a study could provide further evidence for the
mechanism of action of this series.
5.1. Chemistry: general and methods
The commercially obtained reagents were directly used without
further purification. The 1H NMR and 13C NMR spectra were
measured on the Varian INOVA-600 or Varian INOVA-400 spec-
trometer. High resolution mass spectra were obtained on a Bruker
micrOTOF II-SKA spectrometer.
5.1.1. General procedure for the preparation of dimethyl 1H-indole-
3,5-dicarboxylate (2b)
We then selected some compounds with different sub-
stituents at the terminal amino group and the C2, C9 positions
for further measurement of another strain e CQR (K1). As shown
in Table 4, the RI provides a quantitative measurement of the
antiplasmodial activity against the CQR strains relative to that
against the CQS strains and reveals promising drug discovery
leads [40]. Most of the tested compounds showed promising
antimalarial activities against both strains and a low resistance
index (RI). Gratifyingly, we found that all the compounds were
significantly more active against the CQR (K1) than CQ, having
IC50 values that ranged from 11.9 nM to 68.4 nM for K1 (CQ,
IC50 ¼ 210 nM) and RI values ranging from 1.22 to 13.5 (CQ,
RI ¼ 22.3).
Pyridine (3.54 mmol) was added to a suspension of methyl 1H-
indole-5-carboxylate (1b, 2.72 mmol) in anhydrous THF (6 mL) at
0
ꢀC. A solution of trichloroacetyl chloride (3.54 mmol) in THF
(6 mL) was added dropwise via an addition funnel over 1 h. The
reaction mixture was then allowed to warm to room temperature
to stir over night. The reaction mixture was quenched with 1 M HCl,
dried over Na2SO4, and concentrated under vacuum. The resulting
solids were then dissolved in MeOH (54 mL), and KOH(s) was
added. The reaction mixture was heated to reflux for 5 h, stirred at
ambient temperature for 1 h, followed by concentration under
vacuum. The solids were purified by chromatography (SiO2, 25%
EtOAc/Hexanes). 1H NMR (400 MHz, DMSO-d6)
d: 12.28 (s, 1H), 8.69
(d, J ¼ 1.5 Hz, 1H), 8.23 (s, 1H), 7.83 (dd, J ¼ 8.6, 1.7 Hz, 1H), 7.57 (dd,
J ¼ 8.6, 0.5 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.34 (s, 2H); 13C NMR
3.3. In vivo antimalarial activity
(150 MHz, DMSO-d6)
d: 167.0, 164.4, 139.0, 134.3, 125.2, 123.3, 122.7,
112.5, 107.4, 51.9, 50.9.
The monoester substituted compounds 7 and 8a with high
antimalarial activities against the NF54 strain were subjected to
an in vivo drug-screening model against Plasmodium berghei fe-
male mice in Switzerland. The in vivo study was carried out ac-
cording to the standard protocol following the “4 Day Test” [41]
by FACS analysis. Activity was calculated as the difference be-
tween the mean percent parasitaemia for the control (n ¼ 5
mice) and treated groups expressed as a percent relative to the
control group. After daily intraperitoneal dosing 20 mg/kg for
four consecutive days, the neocryptolepine derivatives 7 and 8a
showed some reduction in parasitaemia on day 4 and day 7, with
activities of 33.4% and 72.5%, which were improved remarkably
after the introduction of the ester group with respect to the
maximum activity of 22.1% at the dose of 50 mg/kg for the C2/C9
non-substituted compound 1-(3-(5-methyl-5H-indolo[2,3-b]qui-
nolin-11-ylamino)propyl)-3-phenylurea [20] in our previous
study.
5.1.2. Methyl 5-bromo-1H-indole-3-carboxylate (2c)
White solids; 1H NMR (400 MHz, CDCl3)
: 8.68 (s, 1H), 8.33
d
(d, J ¼ 1.9 Hz, 1H), 7.92 (d, J ¼ 3.0 Hz, 1H), 7.37 (dd, J ¼ 8.6, 1.9 Hz,
1H), 7.29 (dd, J ¼ 8.6, 0.4 Hz, 1H), 3.93 (s, 3H); 13C NMR
(100 MHz, CDCl3)
112.9, 108.6, 51.3.
d: 165.1, 134.6, 131.8, 127.4, 126.3, 124.2, 115.7,
5.1.3. General procedure for the preparation of methyl 2-((4-
(methoxycarbonyl)phenyl)(methyl)amino)-1H-indole-3-
carboxylate (4a)
To the argon flushed solution of methyl indole-3-carboxylate (2a,
2.5 mmol) in CH2Cl2 (5 mL), a mixture of N-chlorosuccinimide
(2.75 mmol) and N,N0-dimethylpiperazine (1.25 mmol) in CH2Cl2
(1 mL) was added dropwise at 0 ꢀC. The mixture was stirred at 0 ꢀC
for 2 h, and a solution of trichloroacetic acid (0.63 mmol) and methyl
4-(methylamino)benzoate (3a, 5 mmol) in CH2Cl2 (2 mL) was then
added. The reaction was allowed to warm to room temperature and
further stirred for 3.5 h. The mixture was consecutively washed with
saturated aqueous NaHCO3, 1 N HCl, brine, and water. The organic
phase was dried over anhydrous MgSO4 and concentrated under
reduced pressure. The crude product was purified by column
chromatography (SiO2, Hexanes/EtOAc ¼ 7/1) to give the white
4. Conclusion
We have described the synthesis, in vitro and in vivo anti-
malaria evaluations of the C2 and/or C9 position ester-modified
neocryptolepine derivatives against NF54 and K1 strains as well
as the
b
-haematin inhibition. The cytotoxicity of these series of
solids, mp: 217e219 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d: 12.25 (br s,
compounds against normal cell lines was also evaluated. Struc-
ture activity relationship studies were discussed by changing the
kind of substituents at C2 and/or C9 and by introducing and
modifying of side chains at C11 of the neocryptolepine core.
1H), 8.00 (d, J ¼ 7.92 Hz, 1H), 7.81 (d, J ¼ 8.80 Hz, 2H), 7.39 (d,
J ¼ 7.63 Hz, 1H), 7.29e7.15 (m, 2H), 6.73 (d, J ¼ 8.80 Hz, 2H), 3.78 (s,
3H), 3.66 (s, 3H), 3.37 (s, 3H); 13C NMR (150 MHz, DMSO-d6)
d: 166.1,
163.5, 151.7, 145.3, 132.6, 130.7, 125.8, 122.7, 121.5, 121.0, 119.0, 112.9,
111.8, 98.4, 51.6, 50.6, 39.1.
Correlation between antiplasmodial activity and b-haematin ac-
tivity was weak. Instead, factors likely to be involved in uptake of
these compounds into the parasite appear to be most predictive
of biological activity. The introduction of an ester group indeed
significantly contributed to their high activities against
P. falciparum and low cytotoxicity against the normal cell line,
especially, to improving their in vivo activity greatly. The thiourea
group at the terminal amino group at C11 also showed a
contribution to the antiplasmodial activity.
5.1.4. Dimethyl 2-(methyl(phenyl)amino)-1H-indole-3,5-
dicarboxylate (4b)
White solids, mp: 168e169 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 9.09
(s,1H), 8.80e8.75 (m,1H), 7.85 (dd, J ¼ 8.5,1.7 Hz,1H), 7.25e7.19 (m,
3H), 6.96e6.85 (m, 3H), 3.89 (s, 3H), 3.79 (s, 3H), 3.44 (s, 3H); 13C
NMR (100 MHz, CDCl3)
d: 168.2, 164.3, 149.3, 146.5, 134.9, 129.3,
126.3, 124.0, 123.5(2C), 121.3, 117.1, 110.3, 97.6, 51.9, 51.0, 40.2.

506
W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
ESI-HRMS: m/z calcd. for C19H17N2O4 [M ꢁ H]e 337.1188. Found
5.1.11. Methyl 5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]
quinoline-9-carboxylate (5b)
337.1166.
Pale grey solids, mp: >300 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d:
5.1.5. Dimethyl 2-((4-(methoxycarbonyl)phenyl)(methyl)amino)-1H-
12.45 (s, 1H), 8.86e8.81 (m, 1H), 8.40 (d, J ¼ 7.8 Hz, 1H), 7.94e7.86
(m, 1H), 7.78 (q, J ¼ 9.1 Hz, 2H), 7.55 (d, J ¼ 8.4 Hz, 1H), 7.42 (t,
J ¼ 7.1 Hz, 1H), 3.98 (s, 3H), 3.90 (s, 3H); 13C NMR (100 MHz, DMSO-
indole-3,5-dicarboxylate (4c)
White solids, mp: 234e236 ꢀC; 1H NMR (400 MHz, CDCl3)
d:
9.58 (s, 1H), 8.84 (d, J ¼ 1.3 Hz, 1H), 7.96 (dd, J ¼ 8.5, 1.6 Hz, 1H),
7.80e7.73 (m, 2H), 7.38 (d, J ¼ 8.5 Hz, 1H), 6.70e6.63 (m, 2H), 3.93
(s, 3H), 3.82 (d, J ¼ 3.5 Hz, 6H), 3.42 (s, 3H); 13C NMR (100 MHz,
d6) d: 171.6, 166.9, 147.8, 139.2, 137.9, 131.5, 125.8, 124.7, 124.2, 123.8,
122.5, 122.0, 121.6, 115.5, 110.7, 102.1, 51.9, 33.4. ESI-HRMS: m/z
calcd. for C18H13N2O3 [M ꢁ H]e 305.0932. Found 305.0867.
CDCl3) d: 168.0, 167.3, 163.9, 150.9, 146.7, 135.0, 131.1, 125.7, 124.7,
124.4, 124.0, 120.6, 113.5, 110.9, 100.8, 52.0, 51.8, 51.2, 39.7. ESI-
HRMS: m/z calcd. for C21H20N2O6 [M þ Na]þ 419.1219. Found
419.1217.
5.1.12. Dimethyl 5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]
quinoline-2,9-dicarboxylate (5c)
Pale grey solids, mp: >300 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d:
12.53 (s, 0H), 8.91 (d, J ¼ 2.1 Hz,1H), 8.78 (d, J ¼ 1.3 Hz,1H), 8.17 (dd,
5.1.6. Dimethyl 2-((4-chlorophenyl)(methyl)amino)-1H-indole-3,5-
J ¼ 8.9, 2.2 Hz, 1H), 7.89 (dd, J ¼ 8.4, 1.7 Hz, 1H), 7.82 (d, J ¼ 9.0 Hz,
dicarboxylate (4d)
1H), 7.51 (d, J ¼ 8.4 Hz, 1H), 3.95 (s, 3H), 3.91 (d, J ¼ 4.7 Hz, 6H); 13
C
White solids, mp: 188e189 ꢀC; 1H NMR (600 MHz, CDCl3)
d: 8.79
NMR (150 MHz, DMSO-d6) d: 171.1, 166.9, 165.9, 147.8, 142.0, 137.8,
(s, 1H), 8.70 (s, 1H), 7.92 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.26 (d, J ¼ 8.4 Hz,
131.2, 127.7, 124.6, 124.1, 123.4, 122.8(2C), 121.8, 116.1, 111.0, 102.7,
52.2, 52.0, 33.8. ESI-HRMS: m/z calcd. for C20H17N2O5 [M þ H]þ
365.1137. Found 365.1140.
2H), 7.21e7.16 (m, 2H), 6.82e6.77 (m, 2H), 3.93 (s, 3H), 3.84 (s, 3H),
3.43 (s, 3H); 13C NMR (100 MHz, CDCl3)
d: 168.01, 164.1, 148.4, 145.4,
134.8, 129.1, 126.1, 125.9, 124.3, 123.8(2C), 117.5, 110.4, 98.8, 52.1,
51.1, 40.1. ESI-HRMS: m/z calcd. for C19H18ClN2O4 [M þ H]þ
373.0960. Found 373.0965.
5.1.13. Methyl 2-chloro-5-methyl-11-oxo-6,11-dihydro-5H-indolo
[2,3-b]quinoline-9-carboxylate (5d)
Pale grey solids, mp: >300 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d:
5.1.7. Dimethyl 2-((4-bromophenyl)(methyl)amino)-1H-indole-3,5-
12.54 (s,1H), 8.81 (s, 1H), 8.29 (s,1H), 7.92 (d, J ¼ 8.3 Hz,1H), 7.86 (d,
dicarboxylate (4e)
J ¼ 8.5 Hz,1H), 7.80 (d, J ¼ 9.0 Hz,1H), 7.56 (d, J ¼ 8.2 Hz,1H), 3.98 (s,
White solids, mp: 193e194 ꢀC; 1H NMR (400 MHz, CDCl3)
d
:
3H), 3.90 (s, 3H); 13C NMR (150 MHz, DMSO-d6)
d: 170.3, 166.8,
8.85 (s, 1H), 8.79 (s, 1H), 7.91 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.34e7.26 (m,
147.9, 138.0(2C), 131.2, 126.9, 125.9, 124.6(2C), 123.6, 122.7, 121.8,
118.1, 111.0, 102.5, 51.9, 33.8. ESI-HRMS: m/z calcd. for C18H14ClN2O3
[M þ H]þ 341.0693. Found 341.0679.
3H), 6.75e6.67 (m, 2H), 3.92 (s, 3H), 3.83 (s, 3H), 3.41 (s, 3H); 13C
NMR (100 MHz, CDCl3) d: 168.0, 164.0, 148.1, 145.9, 134.7, 132.1,
126.1, 124.4, 123.9(2C), 117.7, 113.2, 110.4, 99.1, 52.0, 51.1, 40.0. ESI-
HRMS: m/z calcd. for C19H18BrN2O4 [M þ H]þ 417.0450. Found
417.0452.
5.1.14. Methyl 2-bromo-5-methyl-11-oxo-6,11-dihydro-5H-indolo
[2,3-b]quinoline-9-carboxylate (5e)
Pale grey solids, mp: >300 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d:
5.1.8. Methyl 5-bromo-2-((4-(methoxycarbonyl)phenyl)(methyl)
12.57 (s, 1H), 8.82 (d, J ¼ 1.3 Hz, 1H), 8.44 (d, J ¼ 2.4 Hz, 1H), 7.96e
amino)-1H-indole-3-carboxylate (4f)
7.87 (m, 2H), 7.81 (d, J ¼ 9.1 Hz, 1H), 7.57 (d, J ¼ 8.4 Hz, 1H), 3.99 (s,
White solids, mp: 217e218 ꢀC; 1H NMR (600 MHz, CDCl3)
d
: 9.08
3H), 3.90 (s, 3H); 13C NMR (150 MHz, DMSO-d6)
d: 170.2, 166.9,
(s, 1H), 8.29 (d, J ¼ 1.9 Hz, 1H), 7.81e7.76 (m, 2H), 7.37 (dd, J ¼ 8.5,
147.9, 138.3,138.1, 133.9,127.8, 126.3,124.6,123.6,122.7,121.8,118.3,
114.8, 111.0, 102.5, 51.9, 33.7. ESI-HRMS: m/z calcd. for C18H13BrN2O3
[M þ Na]þ 407.0007. Found 407.0009.
2.0 Hz, 1H), 7.22 (d, J ¼ 8.6 Hz, 1H), 6.69e6.64 (m, 2H), 3.84 (s, 3H),
3.81 (s, 3H), 3.42 (s, 3H); 13C NMR (150 MHz, CDCl3)
d: 167.3, 163.8,
151.0, 146.1, 131.1, 130.7, 127.8, 126.4, 124.4, 120.6, 115.7, 113.5, 112.5,
99.7, 51.9, 51.2, 39.7. ESI-HRMS: m/z calcd. for C19H18BrN2O4
[M þ H]þ 417.0450. Found 417.0457.
5.1.15. Methyl 9-bromo-5-methyl-11-oxo-6,11-dihydro-5H-indolo
[2,3-b]quinoline-2-carboxylate (5f)
Pale grey solids, mp: >300 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d:
5.1.9. Methyl 5-bromo-2-((4-chlorophenyl)(methyl)amino)-1H-
12.34 (s, 1H), 8.88 (d, J ¼ 2.1 Hz, 1H), 8.22 (s, 1H), 8.15 (dd, J ¼ 8.8,
indole-3-carboxylate (4g)
2.1 Hz, 1H), 7.79 (d, J ¼ 9.0 Hz, 1H), 7.38 (d, J ¼ 1.7 Hz, 2H), 3.92 (s,
White solids, mp: 193e194 ꢀC; 1H NMR (600 MHz, CDCl3)
d: 8.36
3H), 3.90 (s, 3H); 13C NMR (150 MHz, DMSO-d6)
d: 171.1,165.8,147.2,
(s, 1H), 8.24 (d, J ¼ 1.9 Hz,1H), 7.31 (dd, J ¼ 8.5, 1.9 Hz,1H), 7.23e7.16
142.0, 133.6, 131.2, 127.7, 125.5(2C), 123.9, 122.7, 122.2, 115.9, 113.7,
113.0, 102.0, 52.2, 33.7. ESI-HRMS: m/z calcd. for C18H13BrN2O3
[M þ Na]þ 407.0007. Found 407.0007.
(m, 2H), 7.11 (d, J ¼ 8.5 Hz, 1H), 6.79e6.75 (m, 2H), 3.82 (s, 3H), 3.41
(s, 3H); 13C NMR (150 MHz, CDCl3)
d: 164.0, 147.9, 145.5, 130.4,
129.2, 128.2, 125.9(2C), 124.1, 117.5, 115.5, 112.0, 97.9, 51.1, 40.1. ESI-
HRMS: m/z calcd. for C17H15BrClN2O2 [M þ H]þ 393.0005. Found
393.0006.
5.1.16. 9-Bromo-2-chloro-5-methyl-5H-indolo[2,3-b]quinolin-11(6H)-
one (5g)
Pale grey solids, mp: >300 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d:
5.1.10. General procedure for the preparation of methyl 5-methyl-11-
oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-2-carboxylate (5a)
Methyl 2-((4-(methoxycarbonyl)phenyl)(methyl)amino)-1H-indole-
3-carboxylate (4a, 1.5 mmol) was heated at reflux in diphenyl ether
(8 mL) at 250 ꢀC for 3 h. The solids were filtered and washed with
diethyl etherto give palegreysolids, mp:>300ꢀC;1H NMR (600 MHz,
12.27 (s, 1H), 8.22 (dd, J ¼ 5.2, 1.8 Hz, 2H), 7.77 (d, J ¼ 9.1 Hz, 1H),
7.73 (dd, J ¼ 9.0, 2.6 Hz, 1H), 7.44e7.30 (m, 2H), 3.91 (s, 3H); 13C
NMR (150 MHz, DMSO-d6) d: 170.2, 147.3, 137.8, 133.7, 131.0, 126.7,
125.6(2C), 125.4, 124.6, 122.1, 117.8, 113.5, 112.9, 101.7, 33.6. ESI-
HRMS: m/z calcd. for C16H10BrClN2O [M þ Na]þ 382.9563. Found
382.9565.
DMSO-d6)
7.84(d, J¼ 8.80Hz,1H), 7.48(d, J¼ 7.63Hz,1H), 7.36e7.17 (m, 2H), 3.98
(s, 3H), 3.91 (s, 3H); 13C NMR (150 MHz, DMSO-d6)
: 171.0, 165.9,
d
: 12.21 (s,1H), 8.96 (d, J ¼ 2.05 Hz,1H), 8.28e8.12 (m, 2H),
5.1.17. General procedure for the preparation of methyl 11-chloro-
5-methyl-5H-indolo[2,3-b]quinoline-2-carboxylate (6a)
d
146.9, 142.0, 134.7, 130.9, 127.8, 124.2, 123.7, 123.2, 122.5, 121.5, 120.3,
115.8, 111.0, 102.9, 52.1, 33.7.
A
suspension of methyl 5-methyl-11-oxo-6,11-dihydro-5H-
indolo[2,3-b]quinoline-2-carboxylate (5a, 1 mmol) in phosphoryl

W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
507
chloride was heated at reflux for 3 h and then poured into an ice-
bath with stirring. Removal of the liquid was by filtration, and the
solids were sequentially washed with aqueous saturated NaHCO3,
and water. The orange product was dried under vacuum over P2O5,
2.4 Hz, 1H), 7.66 (d, J ¼ 9.1 Hz, 1H), 7.62 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.54
(d, J ¼ 8.4 Hz, 1H), 4.30 (s, 3H); 13C NMR (150 MHz, CDCl3)
d: 155.0,
153.6, 135.6, 135.5, 132.8, 131.8, 128.6, 126.5, 125.4, 125.0, 124.8,
120.08, 119.1, 115.9, 113.1, 33.5. ESI-HRMS: m/z calcd. for
mp: 281e283 ꢀC; 1H NMR (600 MHz, CDCl3)
d
: 8.95 (s, 1H), 8.29 (d,
C
16H10BrCl2N2 [M þ H]þ 378.9404. Found 378.9406.
J ¼ 8.22 Hz, 2H), 7.74e7.57 (m, 2H), 7.52 (t, J ¼ 7.48 Hz, 1H), 7.22 (t,
J ¼ 7.48 Hz,1H), 4.24 (s, 3H), 4.00 (s, 3H); 13C NMR (150 MHz, CDCl3)
5.2. General procedure for the preparation of 2,9-disubstituted 5-
methyl-5H-indolo[2,3-b]quinolin-11-amine (7e9)
d: 166.0, 155.1, 154.5, 139.2, 135.7, 131.3, 130.1, 128.2, 125.4, 123.9,
123.8, 123.5, 120.9, 118.5, 117.8, 114.3, 52.4, 33.4.
11-Chloro-5-methyl-5H-indolo[2,3-b]quinoline
derivatives
5.1.18. Methyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-9-
carboxylate (6b)
(6aeg, 1 mmol) was heated at reflux with a large excess of an
appropriate amine in THF for 3e4 h. The reaction was monitored by
TLC. Then the mixture was washed with water and extracted with
CH2Cl2. The organic phase was dried over MgSO4 and concentrated
under vacuum. The crude product was purified by column chro-
matography using eluent changed from AcOEt to AcOEt-2 N
ammonia in MeOH (10:1) to give the final product.
Orange solids, mp: 248e249 ꢀC; 1H NMR (400 MHz, CDCl3)
d:
9.01 (s, 1H), 8.45 (d, J ¼ 8.2 Hz, 1H), 8.20 (d, J ¼ 8.4 Hz, 1H), 7.88 (t,
J ¼ 7.7 Hz, 1H), 7.81 (d, J ¼ 8.6 Hz, 1H), 7.70 (d, J ¼ 8.4 Hz, 1H), 7.60 (t,
J ¼ 7.6 Hz, 1H), 4.43 (d, J ¼ 7.3 Hz, 3H), 3.98 (s, 3H); 13C NMR
(150 MHz, CDCl3) d: 167.4, 157.6, 156.1, 137.1, 136.5, 131.5, 130.9,
126.0, 125.4, 123.4, 122.9(2C), 121.5, 119.2, 116.7, 114.4, 51.9, 33.4.
ESI-HRMS: m/z calcd. for C18H14ClN2O2 [M þ H]þ 325.0744. Found
325.0797.
5.2.1. Methyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]
quinoline-2-carboxylate (7)
Yellow solids, mp: 149e150 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d:
5.1.19. Dimethyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-2,9-
9.12 (d, J ¼ 1.8 Hz, 1H), 8.25 (dd, J ¼ 9.0, 1.8 Hz, 1H), 7.99 (d,
J ¼ 7.7 Hz, 1H), 7.90 (d, J ¼ 9.1 Hz, 1H), 7.52 (d, J ¼ 7.5 Hz, 1H), 7.29 (t,
J ¼ 7.6 Hz, 1H), 7.09 (t, J ¼ 7.0 Hz, 1H), 4.17 (s, 3H), 3.99 (t, J ¼ 6.5 Hz,
2H), 3.93 (s, 3H), 2.67 (t, J ¼ 6.3 Hz, 2H), 1.78e1.74 (m, 2H); 13C NMR
dicarboxylate (6c)
Orange solids, mp: 278e280 ꢀC; 1H NMR (600 MHz, CDCl3)
d:
9.14 (d, J ¼ 1.8 Hz, 1H), 9.03 (d, J ¼ 1.5 Hz, 1H), 8.51 (dd, J ¼ 8.9,
1.8 Hz, 1H), 8.24 (dd, J ¼ 8.4, 1.6 Hz, 1H), 7.91 (d, J ¼ 9.0 Hz, 1H), 7.79
(d, J ¼ 8.4 Hz, 1H), 4.55 (s, 3H), 4.06 (s, 3H), 3.99 (s, 3H); 13C NMR
(150 MHz, DMSO-d6) d: 165.9, 156.2, 151.9, 148.1, 140.2, 130.2, 126.5,
124.5, 124.3, 122.1, 121.3, 118.5, 116.9, 115.3, 115.1, 103.7, 52.2, 47.4,
39.9, 33.1, 32.5. ESI-HRMS: m/z calcd. for C21H21N4O2 [M ꢁ H]e
361.1670. Found 375.1666.
(150 MHz, CDCl3) d: 167.2, 165.7, 156.4, 155.7, 139.1, 138.0, 132.2,
131.7, 128.6, 125.8, 125.1, 124.4, 123.0, 122.8, 119.2, 117.1, 115.0, 52.7,
52.1, 34.4. ESI-HRMS: m/z calcd. for C20H16ClN2O4 [M þ H]þ
383.0799. Found 383.0799.
5.2.2. Methyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-
b]quinoline-9-carboxylate (8a)
5.1.20. Methyl 2,11-dichloro-5-methyl-5H-indolo[2,3-b]quinoline-9-
Yellow solids, mp: 170e171 ꢀC; 1H NMR (600 MHz, DMSO-d6)
carboxylate (6d)
d
: 8.63 (d, J ¼ 1.4 Hz, 1H), 8.52 (d, J ¼ 7.7 Hz, 1H), 7.93e7.87 (m,
Orange solids, mp: 283e284 ꢀC; 1H NMR (600 MHz, CDCl3)
d:
2H), 7.83 (t, J ¼ 7.7 Hz, 1H), 7.52 (d, J ¼ 8.4 Hz, 1H), 7.46 (t,
J ¼ 7.4 Hz, 1H), 4.19 (s, 3H), 4.00 (t, J ¼ 6.6 Hz, 2H), 3.86 (s, 3H),
2.71 (t, J ¼ 6.3 Hz, 2H), 1.84e1.78 (m, 2H); 13C NMR (150 MHz,
8.90 (d, J ¼ 1.2 Hz, 1H), 8.33 (d, J ¼ 2.3 Hz, 1H), 8.17 (dd, J ¼ 8.4,
1.7 Hz, 1H), 7.73 (dd, J ¼ 9.1, 2.3 Hz, 1H), 7.63 (dd, J ¼ 21.2, 8.7 Hz,
2H), 4.33 (s, 3H), 3.97 (s, 3H); 13C NMR (150 MHz, CDCl3)
d: 167.3,
DMSO-d6) d: 167.4, 158.3, 155.7, 149.1, 137.5, 131.1, 125.6,
157.6, 155.9,135.9,135.1, 131.8, 131.6,129.2,125.7,125.3,124.7, 122.8,
124.1(2C), 123.6, 121.2, 118.3, 116.0, 115.5(2C), 102.6, 51.6, 47.2,
33.1, 32.5. ESI-HRMS: m/z calcd. for C21H21N4O2 [M ꢁ H]e
361.1670. Found 361.1639.
122.3, 120.4, 117.0, 116.2, 52.1, 33.9. ESI-HRMS: m/z calcd. for
C
18H13Cl2N2O2 [M þ H]þ 359.0354. Found 359.0357.
5.1.21. Methyl 2-bromo-11-chloro-5-methyl-5H-indolo[2,3-b]
quinoline-9-carboxylate (6e)
5.2.3. Methyl 11-(4-aminobutylamino)-5-methyl-5H-indolo[2,3-b]
quinoline-9-carboxylate (8b)
Orange solids, mp: 289e290 ꢀC; 1H NMR (600 MHz, CDCl3)
d:
Yellow oil; 1H NMR (600 MHz, DMSO-d6)
d
: 8.62 (t, J ¼ 6.1 Hz,
9.06 (d, J ¼ 1.5 Hz, 1H), 8.60 (d, J ¼ 2.2 Hz, 1H), 8.27 (dd, J ¼ 8.4,
1H), 8.53 (s, 1H), 7.92 (dd, J ¼ 8.3, 7.0, 2H), 7.85 (t, J ¼ 7.7 Hz, 1H),
7.53 (d, J ¼ 8.4 Hz, 1H), 7.49 (t, J ¼ 7.5 Hz, 1H), 4.20 (s, 3H), 3.90 (t,
J ¼ 7.1 Hz, 2H), 3.87 (s, 3H), 2.67 (t, J ¼ 7.4 Hz, 2H), 1.85e1.76 (m,
1.7 Hz, 1H), 7.96 (dd, J ¼ 9.1, 2.2 Hz, 1H), 7.74 (dd, J ¼ 20.0, 8.7 Hz,
2H), 4.46 (s, 3H), 3.99 (s, 3H); 13C NMR (150 MHz, CDCl3)
d: 167.5,
158.5, 156.5, 135.7, 134.4, 131.7, 130.9, 128.8, 128.5, 125.9, 125.2,
2H), 1.48 (d, J ¼ 6.4 Hz, 2H); 13C NMR (150 MHz, DMSO-d6)
d:
123.3, 122.2, 120.8, 117.3, 116.3, 52.1, 33.6. ESI-HRMS: m/z calcd. for
167.4, 158.4, 155.9, 149.0, 137.4, 131.2, 125.8, 124.0(2C), 123.7, 121.4,
118.4, 116.1, 115.7, 115.5, 103.0, 51.7, 47.3, 38.6, 32.5, 27.6, 24.9. ESI-
HRMS: m/z calcd. for C22H25N4O2 [M þ H]þ 377.1978. Found
377.1978.
C
18H13BrClN2O2 [M þ H]þ 402.9849. Found 402.9848.
5.1.22. Methyl 9-bromo-11-chloro-5-methyl-5H-indolo[2,3-b]
quinoline-2-carboxylate (6f)
Orange solid, mp: >300 ꢀC; 1H NMR (600 MHz, CDCl3)
d
: 9.14 (d,
5.2.4. Methyl 11-(2-(dimethylamino)ethylamino)-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylate (8c)
J ¼ 1.8 Hz, 1H), 8.53 (d, J ¼ 1.9 Hz, 1H), 8.48 (dd, J ¼ 8.9, 1.9 Hz, 1H),
7.84 (d, J ¼ 9.0 Hz, 1H), 7.70e7.60 (m, 2H), 4.43 (s, 3H), 4.04 (d,
Yellow solids, mp: 172e173 ꢀC; 1H NMR (400 MHz, DMSO-d6)
J ¼ 8.0 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d
: 166.0, 155.4, 153.4,
d
: 8.70 (s, 1H), 8.54 (d, J ¼ 8.3 Hz, 1H), 7.97e7.91 (m, 2H), 7.86 (t,
139.6,137.9, 137.2, 132.8,132.0,128.7, 126.5, 124.7, 124.3,119.4, 118.7,
114.7, 113.6, 52.6, 33.6. ESI-HRMS: m/z calcd. for C18H13BrClN2O2
[M þ H]þ 402.9849. Found 402.9850.
J ¼ 7.8 Hz, 1H), 7.55 (d, J ¼ 8.4 Hz, 1H), 7.48 (t, J ¼ 7.6 Hz, 1H), 7.26
(t, J ¼ 4.5 Hz, 1H), 4.21 (s, 3H), 4.00 (dd, J ¼ 10.8, 5.5 Hz, 2H), 3.87
(s, 3H), 2.65 (t, J ¼ 6.1 Hz, 2H), 2.27 (s, 6H); 13C NMR (150 MHz,
CDCl3) d: 168.2, 157.4, 155.6, 149.3, 138.3, 130.5, 127.0, 125.3, 123.8,
5.1.23. 9-Bromo-2,11-dichloro-5-methyl-5H-indolo[2,3-b]quinoline (6g)
122.5, 120.6, 119.7, 116.1, 115.6, 114.8, 106.0, 58.2, 51.5, 45.2, 44.7,
32.6. ESI-HRMS: m/z calcd. for C22H23N4O2 [M ꢁ H]e 375.1826.
Found 375.1806.
Orange solids, mp: 288e290 ꢀC; 1H NMR (600 MHz, CDCl3)
d:
8.45 (d, J ¼ 1.9 Hz, 1H), 8.37 (d, J ¼ 2.3 Hz, 1H), 7.76 (dd, J ¼ 9.1,

508
W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
5.2.5. Methyl 11-(3-(dimethylamino)propylamino)-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylate (8d)
(t, J ¼ 6.4 Hz, 2H), 1.86e1.77 (m, 2H); 13C NMR (150 MHz, DMSO-d6)
d
: 167.4,158.1,155.8,147.9, 136.1,130.7,125.8(2C),123.9,123.2,118.6,
Yellow solids, mp: 105e106 ꢀC; 1H NMR (600 MHz, CDCl3)
d
:
117.5, 116.7, 116.2, 104.6, 103.1, 51.8, 46.8, 39.9 (overlap with DMSO-
d6 peaks), 33.3, 32.6. ESI-HRMS: m/z calcd. for C21H22ClN4O2
[M þ H]þ 397.1431. Found 397.1432.
8.65 (d, J ¼ 1.4 Hz, 2H), 8.07 (dd, J ¼ 8.4, 1.5 Hz, 1H), 8.00 (d,
J ¼ 8.3 Hz, 1H), 7.70 (dd, J ¼ 11.9, 4.4 Hz, 2H), 7.65 (d, J ¼ 8.6 Hz, 1H),
7.35 (t, J ¼ 7.6 Hz, 1H), 4.23 (d, J ¼ 1.2 Hz, 3H), 4.20e4.10 (m, 2H),
3.94 (s, 3H), 2.72e2.64 (m, 2H), 2.39 (s, 6H), 1.95e1.73 (m, 2H); 13
C
5.2.11. Methyl 11-(3-aminopropylamino)-2-bromo-5-methyl-5H-
NMR (150 MHz, CDCl3)
126.2, 124.1, 123.6, 123.4, 120.7, 119.1, 115.9, 115.8, 114.5, 103.0, 59.5,
d
: 168.4, 158.3, 155.5, 149.0, 137.5, 130.3,
indolo[2,3-b]quinoline-9-carboxylate (9c)
Yellow solids, mp: 109e112 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d:
51.5, 50.1, 45.4, 32.5, 26.3. ESI-HRMS: m/z calcd. for
C
8.76 (s, 1H), 8.57 (s, 1H), 7.97e7.88 (m, 2H), 7.84 (d, J ¼ 9.2 Hz, 1H),
7.52 (d, J ¼ 8.4 Hz, 1H), 4.16 (s, 3H), 3.95 (t, J ¼ 6.7 Hz, 2H), 3.86 (s,
3H), 2.67 (t, J ¼ 6.4 Hz, 2H), 1.82 (t, J ¼ 6.5 Hz, 2H); 13C NMR
23H25N4O2[M ꢁ H]e 389.1983. Found 389.1949.
5.2.6. Methyl 11-(3-hydroxypropylamino)-5-methyl-5H-indolo
[2,3-b]quinoline-9-carboxylate (8e)
(150 MHz, DMSO-d6) d: 167.4, 158.1, 155.8, 147.9, 136.4, 133.4, 126.1,
125.9,123.9(2C),118.6,117.7,117.3, 116.2,113.5,103.1, 51.7, 46.6, 39.3,
33.1, 32.6, 32.3. ESI-HRMS: m/z calcd. for C21H22BrN4O2 [M þ H]þ
441.0926. Found 441.0927.
Orange solids, mp: 223e224 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d: 8.60e8.50 (m, 2H), 7.94e7.88 (m, 2H), 7.86e7.81 (m, 1H), 7.52 (d,
J ¼ 8.4 Hz, 1H), 7.50e7.43 (m, 2H), 4.57 (t, J ¼ 4.7 Hz, 1H), 4.19 (s,
3H), 3.97 (q, J ¼ 6.5 Hz, 2H), 3.87 (s, 3H), 3.47 (dd, J ¼ 10.8, 5.9 Hz,
5.2.12. Methyl 11-(3-aminopropylamino)-9-bromo-5-methyl-5H-
indolo[2,3-b]quinoline-2-carboxylate (9d)
2H), 1.90 (p, J ¼ 6.4 Hz, 2H); 13C NMR (150 MHz, DMSO-d6)
d: 167.4,
158.3, 155.8, 149.2, 137.4, 131.2, 125.7, 124.0(2C), 123.6, 121.3, 118.4,
116.0, 115.6(d), 103.1, 58.4, 51.6, 45.8, 40.0(overlap with DMSO-d6
peaks), 33.6, 32.5. ESI-HRMS: m/z calcd. for C21H20N3O3[M ꢁ H]e
362.1492. Found 362.1483.
Yellow solids, mp: 162e163 ꢀC; 1H NMR (600 MHz, DMSO-
d6)
d
: 9.10 (d, J ¼ 1.6 Hz, 1H), 8.24 (dd, J ¼ 8.9, 1.7 Hz, 1H), 8.10
(d, J ¼ 1.9 Hz, 1H), 7.88 (d, J ¼ 9.0 Hz, 1H), 7.46 (d, J ¼ 8.4 Hz, 1H),
7.39 (dd, J ¼ 8.4, 1.9 Hz, 1H), 4.14 (s, 3H), 3.97 (t, J ¼ 6.6 Hz, 2H),
3.92 (s, 3H), 2.71 (t, J ¼ 6.2 Hz, 2H), 1.83e1.77 (m, 2H); 13C NMR
5.2.7. Methyl 5-methyl-11-(4-methyl-1,4-diazepan-1-yl)-5H-indolo
(150 MHz, DMSO-d6) d: 165.8, 156.5, 150.5, 149.0, 140.4, 130.7,
[2,3-b]quinoline-9-carboxylate (8f)
126.7(2C), 126.2, 124.0, 121.5, 118.5, 115.6, 114.9, 110.4, 102.3,
52.2, 47.5, 39.9 (overlap with DMSO-d6 peaks), 32.9, 32.6. ESI-
HRMS: m/z calcd. for C21H22BrN4O2 [M þ H]þ 441.0926. Found
441.0927.
Orange solids, mp: 179e180 ꢀC; 1H NMR (400 MHz, CDCl3)
d:
8.93 (s, 1H), 8.47 (d, J ¼ 8.2 Hz, 1H), 8.26e8.18 (m, 1H), 7.80 (dd,
J ¼ 3.7, 1.7 Hz, 2H), 7.71 (d, J ¼ 8.4 Hz, 1H), 7.50 (ddd, J ¼ 8.1, 4.2,
1.7 Hz, 1H), 4.37 (d, J ¼ 0.8 Hz, 3H), 3.97 (d, J ¼ 0.9 Hz, 3H), 3.93e
3.87 (m, 2H), 3.78 (t, J ¼ 6.1 Hz, 2H), 3.14 (d, J ¼ 4.5 Hz, 2H), 3.05e
2.90 (m, 2H), 2.63 (s, 3H), 2.39e2.23 (m, 2H); 13C NMR (100 MHz,
5.2.13. N-(3-Aminopropyl)-9-bromo-2-chloro-5-methyl-5H-indolo
[2,3-b]quinolin-11-amine (9e)
CDCl3)
d: 168.1, 159.5, 157.8, 152.3, 138.5, 130.7, 129.7, 126.8, 125.7,
Yellow solids, mp: 152e154 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d:
122.8, 122.0, 121.5, 121.0, 120.8, 116.8, 115.0, 61.3, 57.9, 52.9, 52.2,
51.9, 47.2, 33.3, 29.2. ESI-HRMS: m/z calcd. for C24H27N4O2 [M þ H]þ
403.2134. Found 403.2135.
8.59 (d, J ¼ 1.8 Hz, 1H), 8.03 (d, J ¼ 1.7 Hz, 1H), 7.86 (d, J ¼ 9.2 Hz,
1H), 7.82 (dd, J ¼ 9.1, 2.0 Hz, 1H), 7.44 (d, J ¼ 8.4 Hz, 1H), 7.39 (dd,
J ¼ 8.4, 1.9 Hz, 1H), 4.12 (s, 3H), 3.91 (t, J ¼ 6.6 Hz, 2H), 2.63 (t,
J ¼ 6.3 Hz, 2H), 1.77 (t, J ¼ 6.5 Hz, 2H); 13C NMR (150 MHz, DMSO-
5.2.8. Methyl 5-methyl-11-morpholino-5H-indolo[2,3-b]quinoline-
d6) d: 156.6,150.9,148.1,136.3,130.7,126.9,126.1,125.2,124.2,123.3,
9-carboxylate (8g)
118.3, 117.3, 116.5, 109.9, 102.9, 46.9, 39.5 (overlap with DMSO-d6
peaks), 33.3, 32.5. ESI-HRMS: m/z calcd. for C19H19BrClN4 [M þ H]þ
417.0482. Found 417.0480.
Orange solids, mp: 213e215 ꢀC; 1H NMR (400 MHz, CDCl3)
d:
9.01 (d, J ¼ 1.6 Hz, 1H), 8.50 (d, J ¼ 8.2 Hz, 1H), 8.20 (dd, J ¼ 8.4,
1.7 Hz, 1H), 7.83 (d, J ¼ 4.1 Hz, 2H), 7.74 (d, J ¼ 8.4 Hz, 1H), 7.57e7.47
(m, 1H), 4.41 (s, 3H), 4.18e4.09 (m, 4H), 3.98 (s, 3H), 3.75e3.66 (m,
5.3. General procedure for the preparation of 9-ester substituted
11-amino-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylic acid
(11aec)
4H); 13C NMR (100 MHz, CDCl3)
d: 167.8, 158.4, 156.7, 151.3, 138.1,
131.0, 129.7, 126.5, 126.0, 122.4, 122.0, 121.0(2C), 120.7, 116.5, 115.1,
67.6, 51.9, 51.0, 33.7. ESI-HRMS: m/z calcd. for C22H22N3O3 [M þ H]þ
376.1661. Found 376.1662.
Methyl
11-amino-5-methyl-5H-indolo[2,3-b]quinoline-9-
carboxylate 8a, 8c and 8d was dissolved in MeOH and heated
at reflux with 20% NaOH (aq.) overnight. The reaction was
monitored by TLC. Then the mixture was neutralized with 1 N
HCl (aq.) and concentrated to remove the MeOH under vacuum.
The crude product was purified by reverse-phase chromatog-
raphy using eluent changed from H2O to H2O/CH3CN (50:1) to
give carboxylic acid.
5.2.9. Dimethyl 11-(3-aminopropylamino)-5-methyl-5H-indolo
[2,3-b]quinoline-2,9-dicarboxylate (9a)
Yellow solids, mp: 75e76 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d:
9.17 (s, 1H), 8.63 (s, 1H), 8.28 (d, J ¼ 8.1 Hz, 1H), 7.97e7.89 (m, 2H),
7.55 (d, J ¼ 8.4 Hz, 1H), 4.20 (s, 3H), 4.02 (t, J ¼ 6.6 Hz, 2H), 3.94 (s,
3H), 3.87 (s, 3H), 2.75 (t, J ¼ 6.3 Hz, 2H), 1.87e1.82 (m, 2H); 13C NMR
(150 MHz, DMSO-d6) d: 167.4, 165.9, 158.2, 155.4, 148.9, 140.2, 130.7,
126.6, 125.7, 124.1, 123.7, 122.0, 119.0, 116.4, 115.8, 115.2, 102.5, 52.3,
51.6(2C), 47.1, 32.8, 32.5. ESI-HRMS: m/z calcd. for C23H25N4O4
[M þ H]þ 421.1876. Found 421.1877.
5.3.1. 11-(3-Aminopropylamino)-5-methyl-5H-indolo[2,3-b]
quinoline-9-carboxylic acid (11a)
White solids, mp: >300 ꢀC; 1H NMR (600 MHz, D2O)
d: 8.10 (d,
J ¼ 8.2 Hz, 1H), 7.90 (t, J ¼ 7.5 Hz, 1H), 7.71 (d, J ¼ 8.4 Hz, 1H), 7.58
(dd, J ¼ 9.9, 4.9 Hz, 2H), 7.53 (s, 1H), 7.17 (dd, J ¼ 7.9, 4.8 Hz, 1H),
3.69e3.57 (m, 5H), 2.93e2.83 (m, 2H), 2.06e1.91 (m, 2H); 13C NMR
5.2.10. Methyl 11-(3-aminopropylamino)-2-chloro-5-methyl-5H-
indolo[2,3-b]quinoline-9-carboxylate (9b)
Yellow solids, mp: 185e186 ꢀC; 1H NMR (600 MHz, DMSO-d6)
d:
(150 MHz, D2O) d: 168.9, 151.6, 146.9, 139.0, 135.8, 133.7, 127.4, 125.1,
8.59 (d, J ¼ 2.0 Hz, 1H), 8.54 (s, 1H), 7.89 (dd, J ¼ 8.4, 1.4 Hz, 1H), 7.84
(d, J ¼ 9.2 Hz, 1H), 7.79 (dd, J ¼ 9.1, 2.1 Hz, 1H), 7.50 (d, J ¼ 8.3 Hz,
1H), 4.12 (s, 3H), 3.93 (t, J ¼ 6.7 Hz, 2H), 3.86 (s, 3H), 2.67
123.7, 123.3, 122.4, 118.8, 116.2, 115.4, 111.0, 97.9, 44.8, 36.4, 34.3,
27.9. ESI-HRMS: m/z calcd. for C20H19N4O2 [M ꢁ H]e 347.1513.
Found 347.1505.

W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
509
5.3.2. 11-(2-(Dimethylamino)ethylamino)-5-methyl-5H-indolo[2,3-
5.4.3. Methyl 5-methyl-11-(3-(3-phenylureido)propylamino)-5H-
b]quinoline-9-carboxylic acid (11b)
indolo[2,3-b]quinoline-9-carboxylate (12c)
White solids, mp: 276e280 ꢀC; 1H NMR (400 MHz, DMSO-d6)
Pale yellow solids, mp: 209e211 ꢀC; 1H NMR (400 MHz, DMSO-
d
: 9.03 (d, J ¼ 8.0 Hz, 2H), 8.57 (s, 1H), 8.15 (dd, J ¼ 19.0, 8.6 Hz,
d6)
d
: 8.61 (d, J ¼ 7.5 Hz, 1H), 8.57 (d, J ¼ 1.5 Hz, 1H), 8.42 (s, 1H),
2H), 8.04 (t, J ¼ 7.8 Hz, 1H), 7.82 (d, J ¼ 8.4 Hz, 1H), 7.71 (t,
J ¼ 7.7 Hz, 1H), 4.37 (dd, J ¼ 11.4, 5.7 Hz, 2H), 4.30 (s, 3H), 3.55 (t,
7.97e7.87 (m, 2H), 7.87e7.81 (m, 1H), 7.54 (t, J ¼ 6.4 Hz, 2H), 7.48
(ddd, J ¼ 8.1, 6.9, 1.1 Hz, 1H), 7.33 (dd, J ¼ 8.6, 1.1 Hz, 2H), 7.23e7.15
(m, 2H), 6.90e6.84 (m, 1H), 6.18 (t, J ¼ 5.8 Hz, 1H), 4.20 (s, 3H), 3.91
(q, J ¼ 6.6 Hz, 2H), 3.85 (s, 3H), 3.19 (q, J ¼ 6.5 Hz, 2H), 1.97e1.90 (m,
J ¼ 5.5 Hz, 2H), 2.78 (s, 6H); 13C NMR (150 MHz, DMSO-d6)
d:
167.6, 152.5, 149.3, 140.71, 136.7, 133.3, 127.7, 125.2, 124.9, 124.5
(2C), 120.6, 116.9, 112.2, 99.5, 56.0, 43.1, 42.8, 35.7, 34.4. ESI-
HRMS: m/z calcd. for C21H21N4O2 [M ꢁ H]e 361.1670. Found
361.1653.
2H); 13C NMR (150 MHz, DMSO-d6)
d: 167.4, 158.4, 155.9, 155.6,
149.2, 140.4, 137.4, 131.2, 128.6, 125.9, 123.9(2C), 123.7, 121.4, 121.1,
118.5, 117.8, 116.1, 115.8, 115.6, 103.5, 51.7, 45.3, 36.5, 32.6, 31.6. ESI-
HRMS: m/z calcd. for C28H26N5O3 [M ꢁ H]e 480.2036. Found 480.
2002.
5.3.3. 11-(3-(Dimethylamino)propylamino)-5-methyl-5H-indolo
[2,3-b]quinoline-9-carboxylic acid (11c)
Pale Yellow solids, mp: 280e282 ꢀC; 1H NMR (400 MHz, DMSO-
5.4.4. Methyl 5-methyl-11-(3-(3-phenylthioureido)propylamino)-
5H-indolo[2,3-b]quinoline-9-carboxylate (12d)
d6)
d
: 8.97 (s, 1H), 8.89 (d, J ¼ 8.5 Hz, 1H), 8.52 (s, 1H), 8.12 (dd,
J ¼ 16.6, 8.6 Hz, 2H), 8.05e7.98 (m, 1H), 7.79 (d, J ¼ 8.4 Hz, 1H), 7.70
(t, J ¼ 7.7 Hz, 1H), 4.26 (s, 3H), 4.01 (dd, J ¼ 12.9, 6.5 Hz, 2H), 3.15e
3.04 (m, 2H), 2.70 (s, 6H), 2.31 (dt, J ¼ 14.0, 7.2 Hz, 2H); 13C NMR
Pale yellow solids, mp: 172e175 ꢀC; 1H NMR (400 MHz, DMSO-
d6)
d: 9.45 (s, 1H), 8.60 (d, J ¼ 7.5 Hz, 1H), 8.57 (d, J ¼ 1.4 Hz, 1H),
7.96e7.89 (m, 2H), 7.87e7.80 (m, 1H), 7.71 (s, 1H), 7.54 (d, J ¼ 8.4 Hz,
1H), 7.52e7.45 (m, 2H), 7.23 (d, J ¼ 4.3 Hz, 4H), 7.06 (dt, J ¼ 8.6,
4.5 Hz, 1H), 4.20 (s, 3H), 3.93 (dd, J ¼ 13.0, 6.5 Hz, 2H), 3.86 (s, 3H),
3.58e3.49 (m, 2H), 2.07e2.01 (m, 2H); 13C NMR (150 MHz, DMSO-
(150 MHz, DMSO-d6) d: 167.7, 152.4, 149.3, 136.8, 133.2, 127.3, 124.9,
124.5 (2C), 124.2, 120.8, 116.9, 116.7, 112.2, 98.6, 53.7, 45.3, 42.1, 35.6,
25.1. ESI-HRMS: m/z calcd. for C22H23N4O2 [M ꢁ H]e 375.1826.
Found 375.1828.
d6) d: 180.3,167.4, 158.0,155.3,149.2,138.9,137.4, 131.3, 128.7, 125.9,
124.2(2C), 123.8(2C), 123.2, 121.4, 118.7, 115.8(2C), 115.5, 103.1, 51.7,
45.4, 41.3, 32.6, 30.4. ESI-HRMS: m/z calcd. for C28H28N5O2S
[M þ H]þ 498.1964. Found 498.1965.
5.4. General procedure for the preparation of 2,9-disubstituted 11-
(3-(3-phenylureido)propylamino)-5H-indolo[2,3-b]quinolines
(12aej)
5.4.5. Dimethyl 5-methyl-11-(3-(3-phenylureido)propylamino)-
5H-indolo[2,3-b]quinoline-2,9-dicarboxylate (12e)
2,9-Disubstituted 11-(3-aminopropylamino)-5-methyl-5H-indolo
[2,3-b]quinolines (7e9, 50 mg) was completely dissolved in dry THF
(1 mL), and then a solution of isocyanate (1.1 equiv.) and dry THF (1 mL)
was added drop by drop under stirring at room temperature for 1 h. TLC
monitoring was used to ensure the completion of reaction. After
completion, the reaction mixture was evaporated to dryness under
reduced pressure. The crude product was purified by flash chroma-
tography using AcOEt-2 N ammonia in MeOH (20:1 v/v) as an eluent to
yield pure products as pale yellow solids.
Pale yellow solids, mp: 228e229 ꢀC; 1H NMR (400 MHz, DMSO-
d6)
d
: 9.22 (d, J ¼ 1.7 Hz,1H), 8.58 (d, J ¼ 1.4 Hz,1H), 8.38 (s,1H), 8.29
(dd, J ¼ 9.0, 1.8 Hz, 1H), 8.00 (d, J ¼ 5.9 Hz, 1H), 7.97e7.90 (m, 2H),
7.56 (d, J ¼ 8.4 Hz, 1H), 7.32 (dt, J ¼ 8.8, 1.6 Hz, 2H), 7.20e7.15 (m,
2H), 6.89e6.83 (m, 1H), 6.16 (t, J ¼ 5.7 Hz, 1H), 4.19 (s, 3H), 3.96e
3.89 (m, 5H), 3.86 (s, 3H), 3.23e3.15 (m, 2H), 2.02e1.93 (m, 2H); 13
C
NMR (150 MHz, DMSO-d6) d: 167.3, 165.9, 158.3, 155.5, 149.1, 140.4,
140.1, 130.8, 128.6, 126.3, 125.9, 124.0, 123.7, 122.2, 121.0, 119.1, 117.7,
116.5, 115.8, 115.4, 103.1, 52.3, 51.7, 45.2, 36.4, 32.8, 31.6. ESI-HRMS:
m/z calcd. for C30H30N5O5 [M þ H]þ 540.2247. Found 540.2247.
5.4.1. Methyl 5-methyl-11-(3-(3-phenylureido)propylamino)-5H-
indolo[2,3-b]quinoline-2-carboxylate (12a)
Pale yellow solids, mp: 210e211 ꢀC; 1H NMR (400 MHz,
5.4.6. Dimethyl 5-methyl-11-(3-(3-phenylthioureido)propylamino)-
5H-indolo[2,3-b]quinoline-2,9-dicarboxylate (12f)
DMSO-d6)
d
: 9.16 (d, J ¼ 1.8 Hz, 1H), 8.40 (s, 1H), 8.26 (dd, J ¼ 9.0,
1.6 Hz, 1H), 8.00 (d, J ¼ 7.7 Hz, 1H), 7.91 (d, J ¼ 9.0 Hz, 1H), 7.53 (d,
J ¼ 7.4 Hz, 1H), 7.48 (t, J ¼ 6.0 Hz, 1H), 7.34 (dd, J ¼ 8.6, 1.1 Hz,
2H), 7.32e7.26 (m, 1H), 7.22e7.16 (m, 2H), 7.14e7.06 (m, 1H),
6.92e6.82 (m, 1H), 6.17 (t, J ¼ 5.8 Hz, 1H), 4.17 (s, 3H), 3.93 (s,
3H), 3.88 (dd, J ¼ 13.2, 6.5 Hz, 2H), 3.15 (q, J ¼ 6.5 Hz, 2H), 1.94e
Pale yellow solids, mp: 118e119 ꢀC; 1H NMR (600 MHz, DMSO-
d6) d: 9.44 (s, 1H), 9.21 (s, 1H), 8.56 (s, 1H), 8.31e8.23 (m, 1H), 7.94
(dd, J ¼ 13.9, 8.2 Hz, 3H), 7.69 (s, 1H), 7.56 (dd, J ¼ 8.3, 1.8 Hz, 1H),
7.26e7.19 (m, 4H), 7.06 (dt, J ¼ 8.7, 4.4 Hz, 1H), 4.19 (d, J ¼ 2.9 Hz,
3H), 3.94 (d, J ¼ 0.7 Hz, 5H), 3.86 (s, 3H), 3.55 (s, 2H), 2.09e2.04 (m,
1.86 (m, 2H); 13C NMR (150 MHz, DMSO-d6)
d
: 166.0, 156.3, 155.5,
2H); 13C NMR (150 MHz, DMSO-d6)
d: 180.3, 171.5, 167.3, 165.8,
152.1, 148.2, 140.4, 140.2, 130.4, 128.6, 126.4, 124.8, 124.2, 122.2,
121.4, 121.0, 118.6, 117.7, 117.0, 115.4(2C), 104.9, 52.2, 45.5, 36.5,
32.5, 31.6. ESI-HRMS: m/z calcd. for C28H26N5O3 [M ꢁ H]e
480.2036. Found 480.2024.
149.2, 140.0, 138.9, 130.9, 128.6, 126.3, 125.9, 124.2, 123.8, 123.1,
122.3, 119.4, 117.6, 116.2, 115.9, 115.5, 102.7, 52.3, 51.7, 45.3, 41.2,
33.0, 30.3. ESI-HRMS: m/z calcd. for C30H30N5O4S [M þ H]þ
556.2019. Found 556.2019.
5.4.2. Methyl 5-methyl-11-(3-(3-phenylthioureido)propylamino)-
5H-indolo[2,3-b]quinoline-2-carboxylate (12b)
5.4.7. Methyl 2-chloro-5-methyl-11-(3-(3-phenylureido)
propylamino)-5H-indolo[2,3-b]quinoline-9-carboxylate (12g)
Pale yellow solids, mp: 171e172 ꢀC; 1H NMR (600 MHz, DMSO-
Pale yellow solids, mp: 174e176 ꢀC; 1H NMR (400 MHz, DMSO-
d6)
d
: 9.44 (s,1H), 9.16 (d, J ¼ 1.7 Hz,1H), 8.26 (d, J ¼ 9.0 Hz,1H), 7.94
d6)
d
: 8.74 (d, J ¼ 1.9 Hz, 1H), 8.52 (s, 1H), 8.41 (s, 1H), 7.95e7.88 (m,
(dd, J ¼ 16.7, 8.4 Hz, 2H), 7.72 (s, 1H), 7.53 (d, J ¼ 7.4 Hz, 1H), 7.48 (s,
1H), 7.35e7.28 (m, 1H), 7.23 (d, J ¼ 4.8, 4H), 7.16e7.10 (m, 1H), 7.07
(dt, J ¼ 4.7, 3.5 Hz, 1H), 4.18 (s, 3H), 3.93 (s, 3H), 3.93e3.86 (m, 2H),
3.58e3.46 (m, 2H), 2.04e1.97 (m, 2H); 13C NMR (150 MHz, DMSO-
2H), 7.84 (dd, J ¼ 9.1, 2.1 Hz, 1H), 7.62 (s, 1H), 7.53 (d, J ¼ 8.4 Hz, 1H),
7.32 (d, J ¼ 7.8 Hz, 2H), 7.18 (t, J ¼ 7.9 Hz, 2H), 6.87 (t, J ¼ 7.3 Hz, 1H),
6.18 (t, J ¼ 5.6 Hz, 1H), 4.15 (s, 3H), 3.90e3.86 (m, 2H), 3.85 (s, 3H),
3.20 (dd, J ¼ 12.5, 6.4 Hz, 2H), 1.99e1.94 (m, 2H); 13C NMR
d6)
d
: 180.3, 171.6, 166.0, 156.2, 151.8, 148.2, 140.1, 138.9, 130.4, 128.7,
(150 MHz, DMSO-d6) d: 171.6, 167.3, 158.0, 155.5, 148.1, 140.4, 136.0,
126.3, 124.9, 124.2(2C), 123.2, 122.3, 121.6, 118.8, 116.9, 115.4, 104.5,
52.3, 45.5, 41.4, 32.7, 30.3. ESI-HRMS: m/z calcd. for C28H28N5O2S
[M þ H]þ 498.1964. Found 498.1965.
130.9, 128.6, 126.1(2C), 123.9, 123.7, 123.1, 121.1, 118.9, 117.7(2C),
117.0, 116.1, 103.7, 51.7, 45.2, 36.4, 32.8, 31.5. ESI-HRMS: m/z calcd.
for C28H27ClN5O3 [M þ H]þ 516.1802. Found 516.1805.

510
W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
5.4.8. Methyl 2-bromo-5-methyl-11-(3-(3-phenylureido)
propylamino)-5H-indolo[2,3-b]quinoline-9-carboxylate (12h)
Pale yellow solids, mp: 182e183 ꢀC; 1H NMR (600 MHz, DMSO-
filter then washed with distilled water. The dried filters were
inserted into a plastic foil with 10 mL of scintillation fluid, and
counted in a BetaplateÔ liquid scintillation counter (Wallac, Zürich,
Switzerland). IC50 values were calculated from sigmoidal inhibition
curves by linear regression (Huber 1993) [46] using Microsoft Excel.
d6)
d
: 8.84 (d, J ¼ 2.1 Hz,1H), 8.51 (d, J ¼ 1.4 Hz,1H), 8.42 (s,1H), 7.92
(ddd, J ¼ 8.2, 5.9, 1.8 Hz, 2H), 7.81 (d, J ¼ 9.2 Hz, 1H), 7.63 (t,
J ¼ 5.9 Hz, 1H), 7.53 (d, J ¼ 8.4 Hz, 1H), 7.33 (dd, J ¼ 8.5, 0.9 Hz, 2H),
7.19 (dd, J ¼ 10.7, 5.1 Hz, 2H), 6.89e6.84 (m, 1H), 6.19 (t, J ¼ 5.8 Hz,
1H), 4.13 (s, 3H), 3.89e3.83 (m, 5H), 3.20 (q, J ¼ 6.5 Hz, 2H), 1.99e
6.2. In vitro cytotoxicity against L6 cells
1.94 (m, 2H); 13C NMR (150 MHz, DMSO-d6)
d
: 171.5, 167.3, 158.1,
Assays were performed in 96-well microtiter plates, each well
155.5, 148.0, 140.4, 136.3, 133.5, 128.6, 126.0(2C), 123.9(2C), 121.1,
118.8, 117.8(2C), 117.5, 116.2, 113.8, 103.6, 51.7, 45.2, 36.4, 32.7, 31.5.
ESI-HRMS: m/z calcd. for C28H27BrN5O3 [M þ H]þ 560.1297. Found
560.1295.
containing 100 mL of RPMI 1640 medium supplemented with 1% L-
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) [47,48]. Serial drug dilutions of eleven
3-fold dilution steps covering a range from 100 to 0.002
mg/mL
5.4.9. Methyl 9-bromo-5-methyl-11-(3-(3-phenylureido)
propylamino)-5H-indolo[2,3-b]quinoline-2-carboxylate (12i)
Pale yellow solids, mp: 140e143 ꢀC; 1H NMR (600 MHz, DMSO-
were prepared. After 70 h of incubation the plates were inspected
under an inverted microscope to assure growth of the controls and
sterile conditions. 10 mL of Alamar Blue was then added to each well
d6)
d
: 9.14 (d, J ¼ 1.7 Hz,1H), 8.38 (s,1H), 8.26 (dd, J ¼ 9.0,1.7 Hz,1H),
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) [46] from
the sigmoidal dose inhibition curves using SoftmaxPro software
(Molecular Devices Cooperation, Sunnyvale, CA, USA).
8.12 (d, J ¼ 1.9 Hz, 1H), 7.90 (d, J ¼ 9.0 Hz, 1H), 7.70 (t, J ¼ 6.0 Hz, 1H),
7.47 (d, J ¼ 8.4 Hz, 1H), 7.41 (dd, J ¼ 8.4, 1.9 Hz, 1H), 7.34 (dd, J ¼ 8.6,
1.0 Hz, 2H), 7.21e7.15 (m, 2H), 6.88e6.84 (m, 1H), 6.18 (t, J ¼ 5.8 Hz,
1H), 4.14 (s, 3H), 3.92 (s, 3H), 3.86 (dd, J ¼ 13.0, 6.6 Hz, 2H), 3.16 (dd,
J ¼ 5.7, 2.1 Hz, 2H), 1.94e1.90 (m, 2H); 13C NMR (150 MHz, DMSO-
d6) d: 165.9, 156.5, 155.5, 150.6, 149.2, 140.4(2C), 130.7, 128.6, 127.0,
126.8, 126.1, 123.9, 121.6, 121.0, 118.6, 117.7, 115.6, 114.9, 110.5, 103.4,
52.2, 45.5, 36.4, 32.6, 31.6. ESI-HRMS: m/z calcd. for C28H27BrN5O3
[M þ H]þ 560.1297. Found 560.1298.
6.3. Detergent mediated assay for
b-haematin inhibition
The -haematin formation inhibition assay method described by
b
5.4.10. 1-(3-(9-Bromo-2-chloro-5-methyl-5H-indolo[2,3-b]quinolin-
11-ylamino)propyl)-3-phenylurea (12j)
Carter et al. [32,36] 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-
Pale yellow solids, mp: 145e148 ꢀC; 1H NMR (600 MHz, DMSO-
d6)
d
: 8.67 (d, J ¼ 2.2 Hz,1H), 8.38 (s,1H), 8.01 (d, J ¼ 1.9 Hz,1H), 7.87
(d, J ¼ 9.2 Hz, 1H), 7.82 (dd, J ¼ 9.1, 2.2 Hz, 1H), 7.45 (d, J ¼ 8.4 Hz,
1H), 7.43e7.39 (m, 2H), 7.35e7.30 (m, 2H), 7.20e7.15 (m, 2H), 6.86
(tt, J ¼ 7.4, 1.1 Hz, 1H), 6.17 (t, J ¼ 5.8 Hz, 1H), 4.12 (s, 3H), 3.83 (q,
J ¼ 6.5 Hz, 2H), 3.14 (q, J ¼ 6.5 Hz, 2H), 1.92e1.87 (m, 2H); 13C NMR
centrations ranging from 0 to 1000
total DMSO volume of 10 L in each well after which deionised H2O
(70 L) and NP-40 (20 L; 30.55 M) were added. A 25 mM hae-
matin stock solution was prepared by sonicating haemin in DMSO
for one minute and then suspending 178 L of this in a 1 M acetate
buffer (pH 4.8). The homogenous suspension (100 L) was then
added to the wells to give final buffer and haematin concentrations
of 0.5 M and 100 M respectively. The plate was covered and
mM (final concentration) with a
m
m
m
m
(150 MHz, DMSO-d6) d: 156.6, 155.5, 151.1, 148.1, 140.4, 136.3, 130.7,
128.6, 127.2, 125.9, 125.4, 124.2, 123.3, 121.0, 118.3, 117.7, 117.3, 116.6,
m
110.0, 103.9, 45.5, 36.4, 32.5, 31.5. ESI-HRMS: m/z calcd. for
m
C
26H24BrClN5O [M þ H]þ 536.0853. Found 536.0855.
m
6. Biological testing assay
incubated at 37 ꢀC for 5e6 h in a water bath. Analysis was carried
out using the pyridine-ferrichrome method developed by Ncokazi
and Egan [37]. A solution of 50% (v/v) pyridine, 30% (v/v) H2O, 20%
(v/v) acetone and 0.2 M HEPES buffer (pH 7.4) was prepared and
6.1. Activity against P. falciparum
In vitro activity against erythrocytic stages of P. falciparum was
determined using a 3H-hypoxanthine incorporation assay [42,43],
using the chloroquine and pyrimethamine resistant P. falciparum
K1 strain that originate from Thailand (Thaitong et al., 1983) [44]
and strain susceptible to known antimalarial drugs (P. falciparum
NF54) (Ponnudurai et al., 1981) [45], 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 medium
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 Aþ at 2.5% haematocrit (0.3% para-
sitaemia). Serial drug dilutions of eleven 3-fold dilution steps
32
m
L added to each well to give a final pyridine concentration of 5%
L) was then added to assist with haematin
(v/v). Acetone (60
m
dispersion. The UVevis absorbance of the plate wells was read on a
SpectaMax plate reader. Sigmoidal doseeresponse curves were
fitted to the absorbance data using GraphPad Prism v3.02 to obtain
a 50% inhibitory concentration (IC50) for each compound. Predic-
tion of physical properties and multiple correlation analysis were
carried out using the ChemSW Molecular Modeling Pro Plus v.6.36
software.
Acknowledgements
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 Uni-
versity, and Prof. X.-Q.Yu, Sichuan University, for the HRMS ana-
lyses. Part of this study was supported by Adaptable and Seamless
Technology Transfer Program of JST, and the generous gift of various
indoles from Air Water, Inc, is appreciated. We thank JASSO for
scholarship supports to WJL. TJE acknowledges the National
covering a range from 100 to 0.002 mg/mL were prepared. The 96-
well plates were incubated in a humidified atmosphere at 37 ꢀC; 4%
CO2, 3% O2, 93% N2. After 48 h 50
was added to each well of the plate. The plates were incubated for a
further 24 h under the same conditions. The plates were then
m
L of 3H-hypoxanthine (¼0.5
mCi)
harvested with
a BetaplateÔ cell harvester (Wallac, Zurich,
Switzerland), and the red blood cells transferred onto a glass fibre

W.-J. Lu et al. / European Journal of Medicinal Chemistry 64 (2013) 498e511
511
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J. Wietrzyk, T. Inokuchi, Bioorg. Med. Chem. 20 (2012) 4820e4829.
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