Synthesis, cytotoxicity, and antiplasmodial and antitrypanosomal activity of new neocryptolepine derivatives

Article abstract of DOI:10.1021/jm011102i

On the basis of the original lead neocryptolepine or 5-methyl-5H-indolo[2,3-b]quinoline, an alkaloid from Cryptolepis sanguinolenta, derivatives were prepared using a biradical cyclization methodology. Starting from easily accessible educts, this approach allowed the synthesis of hitherto unknown compounds with a varied substitution pattern. As a result of steric hindrance, preferential formation of the 3-substituted isomers over the 1-substituted isomers was observed when cyclizing N-(3-substituted-phenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl] carbodiimides. All compounds were evaluated for their activity against chloroquine-sensitive as well as chloroquine-resistant Plasmodium falciparum strains, for their activity against Trypanosoma brucei and T. cruzi, and for their cytotoxicity on human MRC-5 cells. Mechanisms of action were investigated by testing heme complexation using ESI-MS, inhibition of β-hematin formation, DNA interactions (DNA-methyl green assay and linear dichroism), and inhibition of human topoisomerase II. Neocryptolepine derivatives with a higher antiplasmodial activity and a lower cytotoxicity than the original lead have been obtained. This selective antiplasmodial activity was associated with inhibition of β-hematin formation. 2-Bromoneocryptolepine was the most selective compound with an IC₅₀ value against chloroquine-resistant P. falciparum of 4.0 μM in the absence of cytotoxicity (IC₅₀ > 32 μM). Although cryptolepine, a known lead for antimalarials also originally isolated from Cryptolepis sanguinolenta, was more active (IC₅₀ = 2.0 μM), 2-bromoneocryptolepine showed a low affinity for DNA and no inhibition of human topoisomerase II, in contrast to cryptolepine. Although some neocryptolepine derivatives showed a higher antiplasmodial activity than 2-bromocryptolepine, these compounds also showed a higher affinity for DNA and/or a more pronounced cytotoxicity. Therefore, 2-bromoneocryptolepine is considered as the most promising lead from the present work for new antimalarial agents. In addition, 2-bromo-, 2-nitro-, and 2-methoxy-9-cyanoneocryptolepine exhibited antitrypanosomal activity in the micromolar range in the absence of obvious cytotoxicity.

Full text of DOI:10.1021/jm011102i

J . Med. Chem. 2002, 45, 3497-3508
3497
Syn th esis, Cytotoxicity, a n d An tip la sm od ia l a n d An titr yp a n osom a l Activity of
New Neocr yp tolep in e Der iva tives
Tim H. M. J onckers,†,‡ Sabine van Miert,§,‡ Kanyanga Cimanga,§ Christian Bailly,| Pierre Colson,
Marie-Claire De Pauw-Gillet,# Hilde van den Heuvel,§ Magda Claeys,§ Filip Lemie`re,† Eddy L. Esmans,†
J ef Rozenski,∇ Ludo Quirijnen,] Louis Maes,] Roger Dommisse,† Guy L. F. Lemie`re,† Arnold Vlietinck,§ and
Luc Pieters*,§
Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium, Department of
Chemistry, University of Antwerp, B-2020 Antwerp, Belgium, INSERM U-524, Institut de Recherches sur le Cancer de Lille,
F-59045 Lille, France, Institute of Chemistry, University of Lie`ge, Sart-Tilman (B6), B-4000 Lie`ge, Belgium, CRCE,
Histology-Cytology, Institute of Anatomy (L3), University of Lie`ge, B-4020 Lie`ge, Belgium, Rega Institute for Medical
Research, B-3000 Leuven, Belgium, and Tibotec-Virco NV, B-2800 Mechelen, Belgium
Received November 13, 2001
On the basis of the original lead neocryptolepine or 5-methyl-5H-indolo[2,3-b]quinoline, an
alkaloid from Cryptolepis sanguinolenta, derivatives were prepared using a biradical cyclization
methodology. Starting from easily accessible educts, this approach allowed the synthesis of
hitherto unknown compounds with a varied substitution pattern. As a result of steric hindrance,
preferential formation of the 3-substituted isomers over the 1-substituted isomers was observed
when cyclizing N-(3-substituted-phenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]carbodiimides.
All compounds were evaluated for their activity against chloroquine-sensitive as well as
chloroquine-resistant Plasmodium falciparum strains, for their activity against Trypanosoma
brucei and T. cruzi, and for their cytotoxicity on human MRC-5 cells. Mechanisms of action
were investigated by testing heme complexation using ESI-MS, inhibition of â-hematin
formation, DNA interactions (DNA-methyl green assay and linear dichroism), and inhibition
of human topoisomerase II. Neocryptolepine derivatives with a higher antiplasmodial activity
and a lower cytotoxicity than the original lead have been obtained. This selective antiplasmodial
activity was associated with inhibition of â-hematin formation. 2-Bromoneocryptolepine was
the most selective compound with an IC50 value against chloroquine-resistant P. falciparum
of 4.0 µM in the absence of cytotoxicity (IC50 > 32 µM). Although cryptolepine, a known lead
for antimalarials also originally isolated from Cryptolepis sanguinolenta, was more active (IC50
) 2.0 µM), 2-bromoneocryptolepine showed a low affinity for DNA and no inhibition of human
topoisomerase II, in contrast to cryptolepine. Although some neocryptolepine derivatives showed
a higher antiplasmodial activity than 2-bromocryptolepine, these compounds also showed a
higher affinity for DNA and/or a more pronounced cytotoxicity. Therefore, 2-bromoneocryp-
tolepine is considered as the most promising lead from the present work for new antimalarial
agents. In addition, 2-bromo-, 2-nitro-, and 2-methoxy-9-cyanoneocryptolepine exhibited
antitrypanosomal activity in the micromolar range in the absence of obvious cytotoxicity.
In tr od u ction
increasing, especially in tropical and subtropical areas;
in addition, morbidity is rising in the industrialized
world. Therefore, the development of new chemothera-
peutic treatments for this disease is urgently needed.1,2
Malaria is one of the most serious parasitic diseases
confronting both developing and industrialized nations,
causing more than 2.5 million deaths annually and a
staggering amount of chronic ill health. The main
species of human malaria parasites are Plasmodium
falciparum, P. vivax, P. ovale, and P. malariae. Malaria
caused by P. falciparum is the most severe form. A
major problem at the moment in the prevention and
treatment of malaria is the growing resistance of the
malarial parasite P. falciparum to currently available
drugs such as chloroquine. The incidence of malaria is
The first antimalarial compound to be discovered,
which also served as the lead compound for synthetic
antimalarials of the chloroquine/mefloquine type, was
the alkaloid quinine. A lot of other leads for potential
new antimalarial agents have been characterized since
then, and many of them have been isolated from
medicinal plants.3 One of these is the plant alkaloid
cryptolepine (Figure 1) (5-methyl-5H-indolo[3,2-b]quino-
line), the major alkaloid of the African plant Cryptolepis
sanguinolenta. Infusions of Cryptolepis root have a long-
standing reputation in the treatment of malaria in
Central and West Africa (Ghana, Congo). Indeed, cryp-
tolepine showed potent in vitro antiplasmodial activity
and no cross-resistance with chloroquine, and it was also
active in vivo (in mice infected with P. berghei).4,5 In
addition to its antiplasmodial activity cryptolepine also
* To whom correspondence should be addressed. Phone and fax: (32)
3 820 27 09. E-mail pieters@uia.ua.ac.be.
† Department of Chemistry, University of Antwerp.
‡ These authors contributed equally to this work.
§ Department of Pharmaceutical Sciences, University of Antwerp.
| INSERM U-524, Lille.
Institute of Chemistry, University of Lie`ge.
#
Institute of Anatomy, University of Lie`ge.
∇ Rega Institute for Medical Research.
] Tibotec-Virco NV.
10.1021/jm011102i CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/29/2002

3498 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
J onckers et al.
Sch em e 1. General Synthetic Sequence
F igu r e 1. Two constituents of Cryptolepis sanguinolenta.
has antibacterial properties6 and is highly cytotoxic. Its
cytotoxicity is due to interaction with DNA and inhibi-
tion of topoisomerase II.7 Also, the minor alkaloid
neocryptolepine (1a ) (Figure 1) (5-methyl-5H-indolo[2,3-
b]quinoline), from the same plant, showed in vitro
antiplasmodial activity against chloroquine-resistant
strains of P. falciparum.5 However, a direct comparison
of the cytotoxicity of cryptolepine and neocryptole-
pine demonstrated that the latter was much less cyto-
toxic.8 Synthetic δ-carbolines, benzo-δ-carbolines, and
cryptolepine derivatives were evaluated for their cyto-
toxic, antiplasmodial, and antitrypanosomal activities.
Most benzo-δ-carbolines and cryptolepine derivatives
remained relatively cytotoxic; 1-methyl-δ-carboline was
selected as the most promising antimalarial compound.9
Synthetic derivatives of cryptolepine have also been
evaluated for their antihyperglycaemic properties.10
Cryptolepine also displays a series of other pharmaco-
logical effects, such as antimuscarinic, noradrenergic
receptor antagonistic, antihypertensive, vasodilative,
antithrombotic, antipyretic, and antiinflammatory prop-
erties,10 some of which may contribute to the relatively
high systemic toxicity observed during preliminary in
vivo experiments in mice.11
Therefore, and because of its reduced cytotoxicity
compared to cryptolepine, neocryptolepine was selected
in the present work as a lead for the development of
new antimalarial agents. The antiplasmodial properties
of a series of synthetic neocryptolepine derivatives are
discussed, as well as their activity in a series of
functional assays relating to possible mechanisms of
action, to establish structure-activity relationships. In
addition, the antitrypanosomal activity against T. cruzi
and T. brucei was also evaluated.
methyl-2-(2-trimethylsilylethynyl)phenyl]-N′-phenylcar-
bodiimide yielding 9-methyl-11-trimethylsilyl-6H-indolo-
[2,3-b]quinoline, which is an obvious precursor of
9-methylneocryptolepine. Similar to this is Shi’s ther-
molysis of N-[2-(1-alkynyl)phenyl]-N′-phenylcarbodi-
imides in γ-terpinene or p-xylene giving 6H-indolo[2,3-
b]quinolines.17 It is worth noting that in 1992 Saito and
co-workers18 published the Lewis acid induced intramo-
lecular Diels-Alder reaction of conjugated carbodi-
imides leading to indolo[2,3-b] quinolines if the reaction
conditions are carefully chosen. From our point of view,
the methods of Schmittel and Shi seemed to be the most
convenient, since they allow an easy alteration of the
substitution pattern of the A-ring of 1a by starting from
appropriately substituted and commercially available
phenyl isothiocyanates and phenyl isocyanates. Varia-
tion of the D-ring can arise by starting from the
appropriately substituted o-iodoanilines. Moreover, the
possibility of introducing different substituents at posi-
tion 11 of the C-ring arising from the presence of a
suitably substituted alkynyl group19 in the carbodiimide
made this method the most attractive one (Scheme 1).
The reaction sequence starts with a Sonogashira
reaction on o-iodoanilines 2 and 2′ giving the 2-tri-
methylsilylethynylanilines 3 and 3′, respectively, in good
yields. Compound 2′ was synthesized starting from
4-aminobenzonitrile using a combination of ICl and
CaCO3 in MeOH as an iodine source.20 First, we tried
to obtain all the carbodiimides 7a -o via the corre-
sponding thioureas, but unfortunately compound 3′ did
not react with the phenyl isothiocyanates 4a -j. In these
Ch em istr y
During the past 8 years, several research groups
developed synthetic pathways toward 1a . In 1994,
Peczynska-Czoch et al.12 reported the coupling between
substituted 2-chloroquinolines and benzotriazole. The
molecules thus obtained were subjected to a Graebe-
Ullmann reaction with polyphosphoric acid (PPA) to
give the corresponding 6H-indolo[2,3-b]quinolines in
moderate yield. The subsequent transformation of these
molecules into the corresponding neocryptolepine de-
rivatives is straightforward. Molina et al.13,14 reported
the synthesis of 1a via an iminophosphorane methodol-
ogy, while Tima´ri et al.15 synthesized neocryptolepine
via a Pd-catalyzed cross-coupling strategy. Although the
above-mentioned procedures are straightforward for the
synthesis of 1a , using these methods for the synthesis
of derivatives of neocryptolepine with a diverse substi-
tution pattern is not that obvious. A much more promis-
ing procedure has been developed by Schmittel et al.16
who reported a biradical cyclization reaction on N-[4-

Neocryptolepine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3499
Ta ble 1. Biradical Cyclization of Carbodiimides 7a -o to
11-Trimethylsilyl-6H-indolo[2,3-b]quinolines 8a -o
compd
type
compd
type
R′
R
yield,a
%
R′
R
yield,a
%
a
b
c
d
e
f
H
H
H
H
H
H
H
H
H
OCH3
Br
Cl
F
60
92
89
85
82
74
76
62
i
j
H
H
SCH3
CN
H
89
81
70
71
91
74
72
k
l
m
n
o
CN
CN Cl
CN OCH3
CN CF3
CN
F igu r e 2. Cyclization products resistant to methylation.
Sch em e 2. Synthesis of Some Desilylated Compoundsa
I
g
h
CH3
NO2
F
a
Yields given refer to analytically pure compounds.
cases, we synthesized the carbodiimides 7k -o via an
aza-Wittig reaction of the iminophoshorane 5′ with
phenyl isocyanates14 6k -o. Another problem was en-
countered in synthesizing the thioureas. When the
conditions described in the Schmittel paper were used,
the formation of compounds 5a -j was rather trouble-
some. These intermediates could only be obtained in
40-50% yield after 5 days of reaction time and a
problematic chromatographic purification. We therefore
tried to develop a more practical method to overcome
this problem. Replacing the solvent acetone by ethanol
had a dramatic effect on the reaction rate as well as on
the yield. After a solution of 2-(2-trimethylsilylethynyl)-
aniline 3 and the appropriate isothiocyanate in ethanol
was stirred in the presence of a catalytic amount of
(dimethylamino)pyridine (DMAP) for 1-2 h at 40 °C, a
white precipitate is formed, which after filtration and
drying gives the corresponding thioureas 5a -j in good
yields (60-70%). Transformation of the thioureas into
the carbodiimides 7a -j was achieved using the proce-
dure reported by Fell and Coppola.21 Most of these
carbodiimides are quite susceptible to degradation.
Therefore, after their purification, these products were
immediately used in the cyclization reaction. Such
unstable behavior of carbodiimides has been reported
before.22 After biradical cyclization in the presence of
1,4-cyclohexadiene, substituted 11-trimethylsilyl-6H-
indolo[2,3-b]quinolines 8a -o were obtained in good
yields (Table 1). These compounds were subsequently
transformed into the new neocryptolepine derivatives
1a -o by methylation of the 5-nitrogen atom followed
by desilylation.17 Initially ethanol was used instead of
dimethylformamide (DMF) as the solvent for the me-
thylation; the use of the latter, however, greatly en-
hanced the yield. The desilylation can be performed
prior to the methylation, but this in general reduces the
overall yield. The outcome of the biradical cyclization
does not seem to be influenced by the nature of the R
and R′ substituents. This is in contrast to the effect of
the substituent at the alkyne terminus, which deter-
mines the reaction product of the biradical cyclization.16
Besides the products listed in Table 1, we also prepared
7-trimethylsilylnaphtho[1,2-b]-R-carboline 8p and 2,4-
dichloro-11-trimethylsilyl-6H-indolo[2,3-b]quinoline 8q
(via route A), but these compounds could not be methy-
lated even after prolonged reaction times (Figure 2). A
low electron density in the A-ring caused by an electron-
withdrawing substituent may deactivate the 5-nitrogen
atom toward nucleophilic attack. However, this pos-
sibility can be ruled out by the successful synthesis of
2-cyanoneocryptolepine (1j) and 2-nitroneocryptolepine
(1h ). It therefore seems that the presence of a substitu-
a
Compound b: R1 ) OCH3, R2 ) H. Compound d : R1 ) Cl, R2
) H. Compound e: R1 ) F, R2 ) H. Compound q: R1 ) Cl, R2
Cl.
)
Sch em e 3. Neocryptolepine Derivatives 1r -1y
ent at the 4-position prohibits the introduction of a
methyl group at the 5-nitrogen atom because of steric
hindrance.
We were also interested in some non-methylated
derivatives of 1a , and therefore, we prepared 2-methoxy-
6H-indolo[2,3-b]quinoline (10b), 2-chloro-6H-indolo[2,3-
b]quinoline (10d), 2-fluor-6H-indolo[2,3-b]quinoline (10e),
and 2,4-dichloro-6H-indolo[2,3-b]quinoline (10q) by de-
silylation of the corresponding silylated compounds (8b,
8d , 8e, 8q) (Scheme 2).
Besides 2- and 9-substituted neocryptolepine deriva-
tives, we wanted to prepare other isomers of the
products synthesized so far for structure-activity re-
lationship (SAR) studies. As a starting point, a further
modification of the A-ring was investigated. However,
some important remarks have to be made in this
context. First of all, from a chemical point of view, the
iminophosphorane route (route B, Scheme 1) proved to
be more practical than the thiourea pathway (route A,
Scheme 1). Therefore, we decided to use this approach
for the synthesis of the extra set of neocryptolepine
derivatives using the iminophosphorane (5′′).17 Second,
if one uses the biradical cyclization reaction to construct
the tetracyclic core starting from N-(3-substituted-
phenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]carbodi-
imides (7r -u ), one can expect a mixture of the 1- and
3-substituted isomers because two different ring-closure
reactions can occur (Scheme 3, Table 2).
Both isomers were formed with the 3-isomer predomi-
nating in each case. In the case of the chloro-substituted
carbodiimide (7t), only a very low amount of the
1-isomer (8t) was formed (less than 1%). The preferen-
tial formation of the 3-isomers is undoubtedly the result
of the absence of steric hindrance between the substitu-

3500 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Ta ble 2. Synthesis of Isomeric Neocryptolepine Precursors
J onckers et al.
substituent, as in compound 1k (compared to neocryp-
tolepine 1a ), compound 1l (compared to 2-chloroneo-
cryptolepine 1d ), and compound 1m (compared to
2-methoxyneocryptolepine 1b), leads to a loss of cyto-
toxicity but also to a reduction of the antiplasmodial
activity.
compd
type
reaction
carbodiimide 7 product 8 substitution yield,a
%
r
7r
7s
7t
8r
1-OCH3
3-OCH3
1-Br
3-Br
1-Cl
3-Cl
1-CF3
3-CF3
8
41
13
55
<1b
52
4
8v
8v
8w
8t
8x
8u
8y
s
t
(b) DNA In ter a ction s a n d In h ibition of â-Hem a -
tin F or m a tion . All compounds were also evaluated in
functional assays related to possible mechanisms of
action. The first mechanism of action to be proposed for
cryptolepine as an antimalarial agent was intercalation
into DNA, like 9-aminoacridine,4 but conflicting results
have been published.23 For chloroquine, an aminoquino-
line, the hypothesis that interaction with DNA is
responsible for the antiplasmodial effect has been
abandoned. Recent hypotheses propose that quinoline
antimalarials, such as quinine and chloroquine, active
against the intraerythrocytic stages of the Plasmodium
parasite act by interfering with the digestion of hemo-
globin in the acid food vacuole of the Plasmodium
parasite. While the globin part is used as a source of
amino acids, the heme part is toxic to the parasite and
is therefore converted to an insoluble crystalline sub-
stance known as hemozoin or malaria pigment. The
latter process is thought to be inhibited by the quinoline
antimalarials.1 It has been shown that the correspond-
ing in vitro process, the spontaneous polymerization of
hematin to â-hematin in cell-free systems, is also
inhibited by quinoline antimalarials such as chloroquine
and quinine.24 For chloroquine, it has also been claimed
that depolymerization of malarial hemozoin, and/or
inhibition of the glutathione-dependent degradation of
heme in the cytosol of the parasite, may be responsible
in part for its activity.25,26 Cryptolepine, similarly to
chloroquine, was able to inhibit the formation of â-he-
matin in vitro, and it was proposed that this mechanism
is likely to be responsible, at least in part, for its
antiplasmodial activity.11,27
u
7u
39
a
b
Yields given refer to analytically pure compounds. Product
not characterized.
ent and the bulky trimethylsilyl group when the ring-
closure reaction takes place.
Resu lts a n d Discu ssion
(a ) In Vitr o An tip la sm od ia l Activity a n d Cyto-
toxicity. All synthesized compounds and cryptolepine
from natural origin5 were evaluated as their hydrochlo-
ride salts for in vitro antiplasmodial activity against a
chloroquine-sensitive and a chloroquine-resistant P.
falciparum strain and for cytotoxicity on a human cell
line (MRC-5 cells) (Table 3). All compounds were
marginally more active against the chloroquine-resis-
tant strain than against the chloroquine-sensitive one,
so the discussion will be focused on activity against the
first one. It is confirmed that neocryptolepine (1a ), the
original lead in this work, is less cytotoxic than cryp-
tolepine; however, the antiplasmodial activity is also
reduced. Many of the 2-substituted neocryptolepine
derivatives are more antiplasmodially active than neo-
cryptolepine itself but are also more cytotoxic. The most
notable compound in this regard is 2-methylneocryp-
tolepine (1g), with a cytotoxicity and antiplasmodial
activity comparable to those of cryptolepine. This is in
agreement with earlier findings for a series of methyl-
substituted indolo[2,3-b]quinoline derivatives.12 Al-
though 2-methylneocryptolepine was not included in
this study, it was found that the cytotoxicity (against
KB cells) was strongly influenced by the position and
the number of methyl substituents, the most cytotoxic
one being 2,5,9,11-tetramethyl-5H-indolo[2,3-b]quino-
line. The 2-halo-substituted neocryptolepines, on the
other hand (2-bromo-, 2-chloro-, 2-fluoro-, and 2-iodo-
neocryptolepine) (1c, 1d , 1e, and 1f, respectively), are
more active against P. falciparum than neocryptolepine
(1a ) and less cytotoxic, the most active and selective
compound being 2-bromoneocryptolepine (1c) with an
IC50 against cloroquine-resistant P. falciparum of 4.0
µM while the IC50 on the MRC-5 cells is >32 µM.
Removal of the N-methyl group, as in compounds 10d
(compared to 2-chloroneocryptolepine 1d ) and 10e (com-
pared to 2-fluoroneocryptolepine 1e), leads to a complete
loss of biological activity in the concentration range
tested. When comparing 1-, 2-, and 3-substituted neo-
cryptolepine isomers, e.g., in the Br-substituted series,
1-bromo substitution (1s) leads to a loss of antiplasmo-
dial activity; 3-bromoneocryptolepine (1w ) has about the
same antiplasmodial activity against the chloroquine-
resistant strain, but it is more cytotoxic. In the methoxy-
substituted series, 2-methoxyneocryptolepine (1b) and
3-methoxyneocryptolepine (1v) are especially more an-
tiplasmodially active than neocryptolepine (1a ) but also
more cytotoxic. Interestingly, introduction of a 9-cyano
Taking this into account, all compounds synthesized
were evaluated not only for their inhibiting effect on
the polymerization of hematin to â-hematin in cell-free
systems but also in the DNA-methyl green assay, a
colorimetric microassay for the detection of agents that
interact with DNA.28 Compounds interacting with DNA
are able to displace methyl green from a methyl green-
DNA complex, leading to a loss of color and a decrease
in absorbance. IC50 values obtained in this assay (i.e.,
the concentration of test compound leading to a 50%
decrease of absorbance, corresponding to 50% displace-
ment of the dye from the DNA complex), together with
the effect on the formation of polymerized â-hematin,
are displayed in Table 3. Cryptolepine, neocryptolepine
(1a ), and many of the substituted neocryptolepine
derivatives are able to inhibit the formation of â-hema-
tin in cell-free systems. For some compounds, most
notably those lacking the N-methyl group (compounds
10b, 10d , 10e, and 10q), this is not accompanied by an
antiplasmodial activity, so obviously the ability to
inhibit â-hematin formation is not enough to show in
vitro antiplasmodial activity. This may be related to the
presence of the compound at the site of action, i.e., the
heme detoxification process in the plasmodial food
vacuole. Only four compounds show affinity for DNA
in the concentration range tested: cryptolepine, being

Neocryptolepine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3501
Ta ble 3. In Vitro Cytotoxic, Antiplasmodial, and Antitrypanosomal Activity, DNA Interaction (DNA Methyl Green Assay), and
Inhibition of â-Hematin Formation of Synthetic Neocryptolepine Derivatives
Plasmodium falciparum Plasmodium falciparum
cytotoxicity
(MRC-5 cells)
IC50 (µM)
(chloroquine sensitive)
(Ghana strain)
IC50 (µM)
(chloroquine resistant) Trypanosoma Trypanosoma
DNA
interaction
IC50 (µM)
inhibition of
â-hematin
formationa
(W2 strain)
IC50 (µM)
brucei
cruzi
compd
IC50 (µM)
IC50 (µM)
1a
1b
10b
1c
1d
10d
1e
10e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
10q
1r
1s
1u
1v
11.0 ( 1.4
4.0 ( 0.1
>32
27.3 ( 5.7
4.3 ( 0.6
>32
6.0 ( 6.1
21.0 ( 8.9
>32
14.0 ( 1.7
4.7 ( 0.6
>32
4.0 ( 0.1
5.0 ( 0.1
>32
4.7 ( 0.6
>32
6.3 ( 0.6
2.3 ( 0.6
>32
4.0 ( 0.1
0.3 ( 0.1
>32
1.0 ( 0.1
1.3 ( 0.6
>32
4.0 ( 0.1
1.7 ( 0.6
b
>32
12.0 ( 1.7
>32
6.0 ( 1.0
>32
18.3 ( 0.6
b
92.8 ( 9.7
+
-
+
+
+
+
+
-
+
b
b
+
b
+
b
-
b
77.9 ( 4.4
>1000
>400
>32
16.5 ( 0.7
>32
15.0 ( 0.1
>32
16.0 ( 0.1
0.95 ( 0.07
>32
>500
>1000
>500
>1000
>400
b
19.3 ( 3.8
>32
14.3 ( 0.6
>32
17.7 ( 5.1
2.7 ( 2.1
29.0 ( 1.7
4.0 ( 1.0
17.0 ( 1.0
>32
1.0 ( 0.1
0.1 ( 0.1
0.7 ( 0.1
0.8 ( 0.1
4.0 ( 1.0
>32
>32
1.0 ( 0.1
>32
b
>1000
5.0 ( 0.1
16.0 ( 0.1
>32
3.7 ( 0.6
15.3 ( 0.6
>32
4.0 ( 0.1
16.3 ( 0.6
b
>600
>600
>1000
b
>32
>32
>32
b
>32
28.3 ( 3.5
14.0 ( 2.6
16.3 ( 1.2
>32
17.0 ( 6.2
6.7 ( 1.1
14.7 ( 4.9
>32
1.0 ( 0.1
>500
>32
>32
>200
11.0 ( 5.7
>32
4.0 ( 0.1
>32
>32
>600
+
+
-
-
b
b
>1000
>1000
>600
5.0 ( 0.1
16.0 ( 0.1
16.0 ( 0.1
3.5 ( 0.7
17.7 ( 0.6
>32
>32
3.3 ( 0.6
12.3 ( 4.2
>32
>32
1.7 ( 0.6
1.3 ( 0.6
16.7 ( 0.6
>32
4.0 ( 0.1
15.7 ( 0.6
16.3 ( 0.6
1.0 ( 0.1
>400
0.4 ( 0.1
150.3 ( 4.0
(37% inhibition)
>600
-
1w
1x
1y
18.5 ( 0.7
15.5 ( 0.7
18.0 ( 0.1
1.5 ( 0.7
30.0 ( 3.5
21.7 ( 4.0
16.3 ( 0.6
2.3 ( 0.6
4.7 ( 0.6
4.7 ( 0.6
15.7 ( 1.5
2.0 ( 0.1
27.3 ( 4.5
9.3 ( 1.5
16.7 ( 1.5
3.0 ( 0.1
>32
17.0 ( 2.0
>32
b
+
+
+
+
>700
>500
65.7 ( 3.0
cryptolepine
a
b
+, inhibition of â-hematin formation; -, no inhibition of â-hematin formation. Not tested.
the most potent one (IC50 65.7 ( 3.0 µM), followed by
2-methoxyneocryptolepine (1b) (77.9 ( 4.4 µM) and
neocryptolepine (1a ) (92.8 ( 9.7 µM); for 3-methoxy-
neocryptolepine (1v) 37% displacement was observed at
a concentration of 150 µM. Cryptolepine and neocryp-
tolepine (1a ), but not 2-methoxy- (1b) or 3-methoxy-
neocryptolepine (1v), are capable of inhibiting the
formation of â-hematin. Nevertheless, the last two
compounds also show a higher antiplasmodial activity
than neocryptolepine (1a ) itself.
cryptolepine (1a ), 2-methoxy- (1b), 2-bromo- (1c), and
2-chloroneocryptolepine (1d ) (see below).
Earlier work on synthetic indoloquinolines has al-
ready led to compounds with a higher antiplasmo-
dial activity than cryptolepine, such as 11-methylcryp-
tolepine9 and 2,7-dibromocryptolepine.11 However, 11-
methylcryptolepine was also more cytotoxic than cryp-
tolepine. Although the antiplasmodial activity of 2,7-
dibromocryptolepine was confirmed in in vivo experi-
ments in mice infected with Plasmodium berghei with-
out apparent toxicity to the host, this compound also
showed slightly higher in vitro cytotoxicity than cryp-
tolepine, which may limit its clinical usefulness. δ-Car-
bolines such as 1-methyl-δ-carboline showed a selective
antiplasmodial activity and no cytotoxicity on L6 cells.9
This compound was found to be specifically localized in
a parasite structure that could correspond to the
parasite nucleus. Inhibition of â-hematin formation was
not evaluated. Although in the present work we have
not been able to design neocryptolepine derivatives with
a higher antiplasmodial activity than cryptolepine, it
has been possible to improve the antiplasmodial activity
of neocryptolepine while at the same time reducing its
cytotoxicity, i.e., to obtain more selective antiplasmodial
compounds.
From these data, it is obvious that there are at least
two different targets for the antiplasmodial activity of
cryptolepine, neocryptolepine, and the neocryptolepine
derivatives synthesized, and some typical compounds
can be selected. Neocryptolepine derivatives, such as
2-bromoneocryptolepine (1c), are more antiplasmodially
active than neocryptolepine (1a ) itself, are able to inhibit
the formation of â-hematin, and show a reduced or no
cytotoxicity and no DNA interactions in the concentra-
tion range tested. Their antiplasmodial activity is likely
to be due to a specific mechanism of action (inhibition
of the heme detoxification process in the parasite). On
the other hand, a compound such as 2-methoxyneo-
cryptolepine (1b) is also more antiplasmodially active
than neocryptolepine (1a ) but is not able to inhibit
formation of â-hematin. However, it shows affinity for
DNA, and hence, it is also cytotoxic. The antiplasmodial
activity is not due to a selective mechanism of action.
Other compounds, and most notably cryptolepine itself,
show inhibition of â-hematin formation as well as DNA
interactions and most probably have at least two targets
in the Plasmodium parasite: the heme detoxification
process and DNA-containing structures. To support this
hypothesis, more detailed experiments were performed
for some selected compounds, i.e., cryptolepine, neo-
(c) DNA Bin d in g. Electr ic Lin ea r Dich r oism
(ELD) Mea su r em en ts. It is now well established that
cryptolepine and related indoloquinolines (in particular
ellipticines) interact with DNA as intercalators.7,29
Neocryptolepine (1a ) also intercalates into DNA but
exhibits a reduced affinity for DNA compared to cryp-
tolepine.8 Electric linear dichroism (ELD) experiments
provide firm evidence that the newly designed neocryp-
tolepine analogues also behave as typical DNA inter-
calating agents. Figure 3a shows the ELD spectra for

3502 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
J onckers et al.
II may not represent the essential target for the cyto-
toxicity of neocryptolepine.29 However, it is important
to bear in mind that these experiments were performed
with commercially available human topoisomerase II
and it remains possible that these compounds selectively
interfere with the activity of Plasmodium topoisomerase
II. For some compounds known as inhibitors of topo-
isomerase II, antiplasmodial activity has been reported,
e.g., the fluoroquinolone antibiotics, which are gyrase
(bacterial, prokaryotic topoisomerase II) inhibitors.30 For
P. falciparum, two distinct topoisomerase II activities
have been demonstrated: a nuclear eukaryotic type II
and a bacterial (prokaryotic) type II topoisomerase
activity associated with plastid DNA replication.31 This
plastid is an atypical organelle essential for parasite
survival and represents an effective target for parasitic
drug design.32 Therefore, it would be quite useful to
compare the effects of the test drugs on mammalian
versus plasmodial topoisomerase II. This hypothesis will
be explored in our laboratories.
F igu r e 3. (a) ELD spectra for (O) 2-methoxy-, (0) 2-bromo-,
(9) 2-chloro-, and (2) neocryptolepine bound to calf thymus
DNA. (b) Dependence of the reduced dichroism ∆A/A on the
electric field strength for the different drug-DNA complexes
at 350 nm and for DNA alone at 260 nm (b). ELD measure-
ments were performed at a P/D ratio of 20 (200 µM DNA, 10
µM drug) in 1 mM sodium cacodylate buffer, pH 7.0.
(e) Ch a r a cter iza tion of Hem e: Ga s-P h a se Ion
Dr u g Com p lexes by Electr osp r a y Ion iza tion (ESI)
Ma ss Sp ectr om etr y. Recently, Wright et al.33 have
described a mass spectrometric method to detect com-
plexes formed between a test compound and heme,
which was adapted in our laboratory and applied to
cryptolepine, neocryptolepine (1a ), and its 2-methoxy-
(1b), 2-bromo- (1c), and 2-chloro- (1d ) derivatives.
Figure 4a illustrates the ESI mass spectrum obtained
for a mixture containing 40 µM heme and 100 µM
cryptolepine in MeOH/H2O (1:1; v/v; pH 7). Under these
conditions, stable gas-phase cryptolepine complexes
were detected at m/z 848, m/z 1463, and m/z 1481,
corresponding to the complex of the drug with the heme
monomer [FP:C]+, the heme dimer [(2FP - H):C]+, and
the dehydrated dimer of hematin [(FP-O-FP):C]+
respectively. For neocryptolepine and its derivatives,
only the complex with the heme monomer [FP:C]+ and
the dimer [(2PF - H):C]+ could be detected (data not
shown). The relative strength of binding between the
drugs and the monomeric and dimeric heme forms was
assessed using low-energy collision-induced dissociation.
Figure 4b illustrates the collision-induced dissociation
(CID) spectra obtained for the complexes between the
heme monomer and cryptolepine, neocryptolepine (1a ),
and 2-bromoneocryptolepine (1c). Ions corresponding to
the drug complexes with the heme monomer [FP:C]+
were selected and collided with helium. In each case,
an FP+ peak (m/z 616) was produced because of loss of
the drug that, relative to the precursor [FP:C]+ peak,
was about 12-fold weaker with cryptolepine than with
neocryptolepine, indicating a higher binding capacity of
cryptolepine. On the basis of the CID spectral data, the
order of stability for [FP:C]+ complexes of neocryp-
tolepine and its derivatives was 2-methoxy > 2-Cl >
2-Br > neocryptolepine (the ion intensity ratios FP+/
[FP:C]+ were 0.81, 0.88, 0.98, and 1.22, respectively;
relative standard deviation (RSD) ) 0.02). The order of
stability for the complexes with the heme dimer [(2FP
- H):C]+ of neocryptolepine and its derivatives inferred
from the CID spectral data was 2-methoxy > 2-Br >
neocryptolepine > 2-Cl (the ion intensity ratios [2FP -
H]+/[(2FP - H):C]+ were 0.12, 0.24, 0.25, and 0.27,
respectively; RSD ) 0.02). These data demonstrate that
2-methoxy- (1b ), 2-bromo- (1c), and 2-chloroneocryp-
tolepine (1d ) bound to calf thymus DNA. Their mode of
binding to DNA was analyzed on the basis of the highest
ELD values obtained when the drug molecules are fully
bound to DNA, i.e., for P/D ratios of 20. In each case,
the reduced dichroism is negative in sign in the 300-
400 nm region corresponding to the absorption band of
the indoloquinoline unit. The reduced dichroism mea-
sured for the drug-DNA complexes at 320-360 nm is
almost identical to that obtained with DNA alone at 260
nm (Figure 3b). This implies that the drug is oriented
parallel to the DNA base pairs, as expected for an
intercalation binding mode. ELD defines the orientation
of drug bound to DNA, whereas the DNA-methyl green
assay defines the relative affinity for DNA. 2-Chloro-
(1d ) and especially 2-bromoneocryptolepine (1c) have
a low affinity for DNA, which is in agreement with their
reduced cytotoxicity, whereas 2-methoxyneocryptolepine
(1b) has a higher affinity for DNA.
(d ) In h ibition of Top oisom er a se II. In contrast to
cryptolepine, which is a DNA intercalator and a potent
poison for topoisomerase II, neocryptolepine poorly
inhibits the DNA relaxing enzyme. We have shown
recently that cryptolepine is about 5 times more potent
than neocryptolepine at inhibiting the unwinding of
closed circular duplex DNA by human topoisomerase II.8
Both DNA relaxation and cleavage assays with plasmid
DNA and 32P-labeled DNA restriction fragments, re-
spectively, were performed to evaluate the effects of the
2-substituted neocryptolepine analogues on topoisomerase
II activity (human p170 isoform), but none of them
showed an effect superior to that of neocryptolepine. In
the presence of 2-methoxy (1b), 2-bromo- (1c), or 2-chlo-
roneocryptolepine (1d ) (20 µM each), topoisomerase II
mediated DNA cleavage was found to be slightly stimu-
lated, but in all cases, the extent of DNA cleavage never
exceeded 15% of the DNA products (data not shown).
As discussed previously, we consider that topoisomerase

Neocryptolepine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3503
F igu r e 4. (a) ESI mass spectrum of a mixture of 40 µM heme and 100 µM cryptolepine in MeOH/H2O (1:1). The ion at m/z 1253
corresponds to the complex consisting of the heme dimer and methanol [(2FP-H):MeOH]+. (b) CID spectra obtained for the complex
consisting of the heme monomer with cryptolepine, neocryptolepine (1a ), and 2-bromoneocryptolepine (1c).
neocryptolepine and its derivatives show a stronger
binding to the dimeric heme form than to the monomeric
one. Apparently all selected compounds were able to
form complexes with the monomeric and dimeric forms
of heme, but 2-methoxyneocryptolepine (1b) was not
able to inhibit the polymerization of hematin to â-he-
matin in cell-free systems as described above. Whereas
complex formation as detected by MS may be a prereq-
uisite for inhibition of â-hematin formation, it does not
necessarily indicate that the test compound is actually
capable of inhibiting this polymerization reaction.
(f) An titr yp a n osom a l Activity. Because of the
antitrypanosomal activity of cryptolepine,9 all com-
pounds synthesized were also tested against T. brucei
and T. cruzi (Table 3). Cryptolepine showed an IC50
against T. brucei of 3.0 µM, but this is obviously not
due to a selective mechanism of action, in view of its
antiplasmodial and cytotoxic activity. The same is true
for a series of other compounds, most notably 2-meth-
ylneocryptolepine (1g), with an IC50 value against T.
brucei of 0.1 µM and also a significant cytotoxicity (IC50
on MRC-5 cells of 0.95 µM). More specific antitrypano-
somal activity against at least one of the two Trypano-
soma species tested, combined with absence of obvious
cytotoxicity in the concentration range tested, is ob-
served for 2-bromo- (1c), 2-nitro- (1h ), and 2-methoxy-
9-cyanoneocryptolepine (1m ). The last compound showed
an IC50 of 1.0 µM against T. brucei as well as T. cruzi,
whereas the IC50 on the MRC-5 cells was >32 µM.
Therefore, this neocryptolepine derivative could be
considered as a new lead structure with antitrypano-
somal potential. Additional exploration in this area is
warranted.
Con clu sion
In conclusion, some new neocryptolepine (indolo[2,3-
b]quinoline) derivatives were synthesized with a more
selective antiplasmodial activity than the original lead.
This activity is most probably due to inhibition of the
heme detoxification process (formation of hemozoin), as
demonstrated by the investigation of heme complex-
ation and the polymerization of hematin to â-hematin
in cell-free systems. 2-Bromoneocryptolepine (1c) was
the most selective compound with an IC50 value against

3504 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
J onckers et al.
compounds in the following form: column, mobile phase,
retention time. The flow rate in system A was 0.65 mL/min,
and the flow rate was 1.0 mL/min in system B.
chloroquine-resistant P. falciparum of 4.0 µM, in the
absence of obvious cytotoxicity (IC50 > 32 µM). Although
this was higher than the IC50 value of cryptolepine (2.0
µM), 2-bromoneocryptolepine showed a low affinity for
DNA, and no inhibition of human topoisomerase II.
Although some neocryptolepine derivatives with an in
vitro antiplasmodial activity higher than that of 2-bro-
moneocryptolepine were obtained, these compounds also
showed a higher affinity for DNA and/or a more
pronounced cytotoxicity on a human cell line, as ob-
served for cryptolepine as well. From the present work,
2-bromoneocryptolepine is therefore considered as the
most promising lead for potentially new antimalarial
agents. In addition, 2-bromo-, 2-nitro-, and 2-methoxy-
9-cyanoneocryptolepine derivatives exhibited some an-
titrypanosomal activity and could be considered as new
lead structures for antitrypanosomal agents.
4-Am in o-3-iod oben zen eca r bon itr ile (2′). 4-Aminoben-
zonitrile (1.0 g, 8.47 mmol) is dissolved in 20 mL of dry
methanol while stirring. The flask is cooled to 0 °C, after which
CaCO3 (2.5 g, 25 mmol) is added. Then a solution of ICl in
HCl (4 mL, 5 M ICl in concentrated HCl) is added dropwise
in 10 min while maintaining the temperature at 0-5 °C. After
complete addition, the cooling bath is removed and the mixture
is stirred for 30 min at room temperature and further for 2
days at 35 °C. After completion of the reaction, 60 mL of a 0.5
M Na2S2O3 solution is added followed by 100 mL of water. The
mixture is extracted with CH2Cl2 (2 × 80 mL), and after
removal of the solvent the crude product was purified by
column chromatography with CH2Cl2 as the eluent.
2-(2-Tr im eth ylsilyleth yn yl)a n ilin e (3). Pd(PPh3)2Cl2 (280
mg, 0.40 mmol) and CuI (64 mg, 0.33 mmol) are added to a
stirred solution of 2-iodoaniline (8.76 g, 0.04 mol) in Et3N (100
mL). To this suspension, trimethylsilylacetylene (6 mL, 0.43
mol) is added, and the resulting mixture is stirred for 18 h at
room temperature. Then 100 mL of water is added and the
mixture is extracted with CH2Cl2 (3 × 50 mL). The solvent is
removed in vacuo, and the product is purified by column
chromatography using CH2Cl2 as the eluent.
4-Am in o-3-(2-t r im e t h ylsilyle t h yn yl)b e n ze n e ca r b o-
n itr ile (3′). To a stirred solution of 4-amino-3-iodobenzen-
ecarbonitrile (0.60 g, 2.5 mmol) (2′) in THF (10 mL), Et3N (5
mL), Pd(PPh3)2Cl2 (17.2 mg, 0.024 mmol), and CuI (4.2 mg,
0.022 mmol) are added. To this solution trimethylsilylacetylene
(0.36 mL, 2.6 mmol) is added, and the resulting mixture is
stirred for 24 h at room temperature. Then 20 mL of water is
added and the mixture is extracted with CH2Cl2 (3 × 30 mL).
The solvent is removed in vacuo, and the product is purified
by column chromatography using heptane/ether (1:3) as the
eluent.
3-(2-Tr im eth ylsilyleth yn yl)-4-tr ip h en ylp h osp h or a n yli-
d en ea m in oben zen eca r bon itr ile (5′). A flask is charged
with 4-amino-3-(2-trimethylsilylethynyl)benzenecarbonitrile
(3′) (1.30 g, 6.07 mmol), triphenylphosphine (3.28 g, 12.5
mmol), Et3N (12 mL), CCl4 (7 mL), and CH3CN (15 mL). The
resulting mixture is stirred at room temperature for 24 h.
Then, 20 mL of MeOH is added and stirring is continued for
30 min. After the addition of 100 mL of water, the mixture is
extracted with CH2Cl2 (3 × 50 mL), after which the solvent is
removed. The product is then purified by column chromatog-
raphy using EtOAc/MeOH (98:2) as the eluent.
Gen er a l P r oced u r e for th e P r ep a r a tion of Th iou r ea s
5a -j, 5p , a n d 5q. 2-(2-Trimethylsilylethynyl)aniline (3) (1.2
g, 6.35 mmol) and 6.5 mmol of the appropriate 4-substituted
phenyl isothiocyanate (4a -j) are dissolved in EtOH (25 mL).
A few crystals of DMAP (N,N-(dimethylamino)pyridine) are
added, and the resulting solution is stirred at 40 °C in an oil
bath. After 1-2 h, a white precipitate is formed. The solid is
filtered off and washed with 20 mL of cold EtOH and
subsequently dried in vacuo to yield the corresponding thio-
urea. The filtrate is stirred further at 40 °C, giving another
amount of precipitate after a few hours. The following products
were prepared in this manner:
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. NMR spectra were
recorded on Varian Unity and Bruker DRX-400 spectrometers,
operating at 400 MHz for 1H and 100 MHz for 13C and with
CDCl3 as the solvent unless otherwise stated. In every case
tetramethylsilane was used as the internal standard. Chemical
shifts are given in ppm and J values in Hz. Multiplicity is
indicated using the following abbreviations: d for a doublet, t
for a triplet, m for a multiplet, etc. In those cases where
standard 1H and 13C measurements were insufficient, ad-
ditional 2D measurements (i.e., COSY, HETCOR or HSQC,
LR-HETCOR or HMBC) were performed for complete struc-
ture elucidation. IR spectra were recorded on a Bruker Vector
22 spectrometer. Low-resolution mass spectra were recorded
on a triple quadrupole mass spectrometer (Quattro II, Micro-
mass, Manchester, U.K.) using electrospray ionization (ESI).
Samples were dissolved in CH3CN and infused at 5 µL/min
into the mass spectrometer. Fragmentation was induced by
low-energy collisional activation using an Ar gas pressure of
approximately 10-3 mbar and a collision energy between 25
and 35 eV depending on the compound. Exact mass measure-
ments were performed on a quadrupole/orthogonal-acceleration
time-of-flight (Q/oaTOF) tandem mass spectrometer (qTof 2,
Micromass, Manchester, U.K.) equipped with a standard
electrospray ionization (ESI) interface. Samples were diluted
in an appropriate solvent (MeOH/0.1% formic acid 90/10), and
1 µL aliquots were injected at a flow rate of 5 µL/min using
the CapLC system (Waters, Brussels, Belgium). The mass
scale was corrected by an internal lock-mass provided by the
protonated molecule of 2′-deoxyadenosine (m/z 252.1096),
which was added into the LC stream at a flow rate of 2.5 µL/
min (concentrated 10-5 M using the same solvent composition)
using a low dead volume tee union. Combined spectra of the
compound were processed using the Masslynx software suite
(Micromass, Manchester, U.K.). Melting points were deter-
mined on a Bu¨chi B-545 apparatus and are uncorrected. All
reagents were purchased from commercial sources (Acros,
Aldrich, Lancaster) and were used as such. THF was distilled
from sodium benzophenone. Column chromatography was
performed on Kieselgel 60 (Merck), 0.040-0.063 mm. Thin-
layer chromatography was performed on precoated ALUGRAM
SIL G/UV254 Kieselgel 60 0.25 mm TLC plates. For the sake
of brevity, only the data for complete characterization of the
final products (i.e., the new neocryptolepine derivatives) are
given. Characterization data for the intermediate compounds
are supplied in the Supporting Information. Analytical HPLC
using two independent systems was performed to check the
purity of the products: (column A) Merck, Lichrospher Si 600
(4 mm × 250 mm); (column B) Merck, Lichrocart-Lichrospher
100 RP 18 (4 mm × 250 mm). Also, two different solvent
systems were used: (system A) 1-propanol-hexane 5%/water
95:5 or 1-propanol-hexane 5%/water 90:10; (system B) gradi-
ent from 80% to 100% acetonitrile (ACN) in 20 min. Experi-
mental details of the HPLC analysis are reported for all test
N-phenyl-N′-[2-(2-trimethylsilylethynyl)phenyl]thiourea (5a)
N-(4-methoxyphenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
thiourea (5b)
N-(4-bromophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
thiourea (5c)
N-(4-chlorophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
thiourea (5d )
N-(4-Fluorophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
thiourea (5e)
N-(4-iodophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]thio-
urea (5f)
N-(4-methylphenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
thiourea (5g)
N-(4-isothiocyanatophenyl)-N′-[2-(2-trimethylsilylethynyl)-
phenyl]thiourea (5h )

Neocryptolepine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3505
N-(4-methylthiophenyl)-N′-[2-(2-trimethylsilylethynyl)phe-
nyl]thiourea (5i)
N-(3-trifluoromethylphenyl)-N′-[2-(2-trimethylsilylethynyl)-
phenyl]carbodiimide (7u )
N-(4-cyanophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
thiourea (5j)
N-(1-naphthyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]thio-
urea (5p )
N-(2,4-dichlorophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
thiourea (5q)
Gen er a l P r oced u r e for th e Con ver sion of Ca r bod iim -
id es 7a -u in to th e Su bstitu ted 11-Tr im eth ylsilyl-6H-
in d olo[2,3-b]qu in olin es (8a -y). This reaction has to be
performed using freshly prepared carbodiimides. The carbo-
diimide (approximately 2.0 mmol) is dissolved in 30 mL of
mesitylene. Helium gas is bubbled through the solution for
10 min. After the solution was degassed, 1,4-cyclohexadiene
(3 mL, 31.7 mmol) is added and the solution is refluxed for 18
h under a nitrogen atmosphere. The solution is cooled to room
temperature, and the solvent is removed under high vacuum.
Purification of the product is performed by column chroma-
tography using CH2Cl2/EtOAc (7:3) as the solvent. The follow-
ing products were prepared in this manner:
Gen er a l P r oced u r e for th e Syn th esis of Ca r bod iim -
id es 7a -j, 7p , a n d 7q fr om Th iou r ea s 5a -j, 5p , a n d 5q. A
two-necked flask equipped with a drying tube is charged with
the appropriate thiourea (2.13 mmol). Dry CH2Cl2 (25 mL) was
added, followed by Et3N (0.59 mL, 4.25 mmol) and a catalytic
amount of DMAP. The solution is stirred, and CH3SO2Cl (0.5
mL, 6.40 mmol) is added dropwise. It is important that the
rate of addition is kept low because of the very exothermic
nature of the reaction. After complete addition, the solution
is stirred for 15 min at room temperature, after which 50 mL
of water is added. The mixture is extracted with CH2Cl2 (3 ×
30 mL), and the solvent is removed on a rotavapor. Purification
of the product using column chromatography (CH2Cl2 as
eluent) yields the carbodiimide as a viscous oil. The product
is used immediately in the next step to prevent degradation.
The following products were prepared in this manner:
N-phenyl-N′-[2-(2-trimethylsilylethynyl)phenyl]carbodi-
imide (7a )
N-(4-methoxyphenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7b)
N-(4-bromophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7c)
N-(4-chlorophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7d )
N-(4-fluorophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7e)
N-(4-iodophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]carbo-
diimide (7f)
N-(4-methylphenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7g)
N-(4-isothiocyanatophenyl)-N′-[2-(2-trimethylsilylethynyl)-
phenyl]carbodiimide (7h )
N-(4-methylthiophenyl)-N′-[2-(2-trimethylsilylethynyl)phe-
nyl]carbodiimide (7i)
N-(4-cyanophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7j)
N-(1-naphthyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]carbo-
diimide (7p )
N-(2,4-dichlorophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7q)
Gen er a l P r oced u r e for th e Syn th esis of Ca r bod iim -
id es 7k -o fr om Im in op h osp h or a n e 5′ a n d of 7r -u fr om
Im in op h osp h or a n e 5′′. Iminophosphorane 5′ or 5′′ (2.1
mmol) is dissolved in 20 mL of dry toluene. To this solution
the appropriate substituted phenyl isocyanate (2.2 mmol) is
added, and the resulting solution is stirred at 70 °C for 1 h.
The solvent is removed in vacuo, and the product is purified
by column chromatography using CH2Cl2 as the eluent. The
product is used immediately in the next step. The following
products were prepared in this manner:
N-[4-cyano-2-(2-trimethylsilylethynyl)phenyl]-N′-phenylcar-
bodiimide (7k )
N-(4-chlorophenyl)-N′-[4-cyano-2-(2-trimethylsilylethynyl)-
phenyl]carbodiimide (7l)
N-[4-cyano-2-(2-trimethylsilylethynyl)phenyl]-N′-(4-methoxy-
phenyl)carbodiimide (7m )
N-[4-cyano-2-(2-trimethylsilylethynyl)phenyl]-N′-(4-trifluo-
romethylphenyl)carbodiimide (7n )
N-[4-cyano-2-(2-trimethylsilylethynyl)phenyl]-N′-(4-fluorophe-
nyl)carbodiimide (7o)
N-(3-methoxyphenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7r )
11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8a )
2-methoxy-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8b)
2-bromo-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8c)
2-chloro-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8d )
2-fluoro-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8e)
2-iodo-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8f)
2-methyl-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8g)
2-isothiocyanato-11-trimethylsilyl-6H-indolo[2,3-b]quino-
line (8h )
2-methylthio-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8i)
2-cyano-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8j)
9-cyano-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8k )
2-chloro-9-cyano-11-trimethylsilyl-6H-indolo[2,3-b]quino-
line (8l)
9-cyano-2-methoxy-11-trimethylsilyl-6H-indolo[2,3-b]quino-
line (8m )
9-cyano-2-trifluoromethyl-11-trimethylsilyl-6H-indolo[2,3-b]-
quinoline (8n )
9-cyano-2-fluoro-11-trimethylsilyl-6H-indolo[2,3-b]quino-
line (8o)
7-trimethylsilylnaphtho[1,2-b]-R-carboline (8p )
2,4-dichloro-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8q)
1-methoxy-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8r )
1-bromo-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8s)
1-trifluoromethyl-11-trimethylsilyl-6H-indolo[2,3-b]quino-
line (8u )
3-methoxy-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8v)
3-bromo-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8w )
3-chloro-11-trimethylsilyl-6H-indolo[2,3-b]quinoline (8x)
3-trifluoromethyl-11-trimethylsilyl-6H-indolo[2,3-b]quino-
line (8y)
Gen er a l P r oced u r e for th e Con ver sion of th e Su bsti-
tu ted 11-Tr im eth ylsilyl-6H-in d olo[2,3-b]qu in olin es 8b,
8d , 8e, 8q in to th e Desilyla ted 6H-In d olo[2,3-b]qu in olin es
10b, 10d , 10e, 10q. The appropriate 11-trimethylsilyl-6H-
indolo[2,3-b]quinoline (0.35 mmol) is dissolved in ethanol (30
mL), after which NaOH (10 mL, 6 N) is added. The mixture is
stirred for 3 h at 70 °C and cooled to room temperature. Water
(50 mL) is added, and the mixture is extracted with CH2Cl2
(3 × 50 mL). Purification of the product is performed by column
chromatography using CH2Cl2/EtOAc (7:3) as the eluent. The
following products were prepared in this manner:
2-methoxy-6H-indolo[2,3-b]quinoline (10b)
2-chloro-6H-indolo[2,3-b]quinoline (10d )
2-fluoro-6H-indolo[2,3-b]quinoline (10e)
2,4-dichloro-6H-indolo[2,3-b]quinoline (10q)
Gen er a l P r oced u r e for th e Con ver sion of th e Su bsti-
tu ted 11-Tr im eth ylsilyl-6H-in d olo[2,3-b]qu in olin es 8a -y
in to th e Su bstitu ted 11-Tr im eth ylsilyln eocr yp tolep in es
9a -y. The appropriate 11-trimethylsilyl-6H-indolo[2,3-b]-
quinoline (0.625 mmol) is dissolved in DMF (30 mL). To this
solution, CH3I (3 mL, 48 mmol) is added, and the solution is
refluxed overnight. The mixture is then cooled to room
temperature, after which it is poured into 50 mL of a 1 M Na2-
CO3 solution followed by extraction with CH2Cl2 (3 × 50 mL).
After removal of the solvent, the product is purified by column
chromatography using CH2Cl2/EtOAc (7:3) as the solvent. In
two cases, namely, the synthesis of 9-cyano-2-trifluoromethyl-
11-trimethylsilylneocryptolepine (9n ) and 9-cyano-2-fluoro-11-
N-(3-bromophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7s)
N-(3-chlorophenyl)-N′-[2-(2-trimethylsilylethynyl)phenyl]-
carbodiimide (7t)

3506 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
J onckers et al.
trimethylsilylneocryptolepine (9o), formation of the compounds
was accompanied by the presence of a large amount of
desilylated product. Unfortunately, these products could not
be separated from each other. Therefore, the mixture of the
two was used in the desilylation reaction (see below). As a
consequence, no spectral data were recorded for these two
compounds. After the reaction in which substituted 11-
trimethylsilyl-6H-indolo[2,3-b]quinolines were formed, the
1-substituted compound is separated from the 3-substituted
one and other impurities by using a silica column and CH2-
Cl2/EtOAc (7:3) as the eluent. The following products were
prepared in this manner:
In Vitr o Activity a ga in st P . fa lcipa r u m . For the deter-
mination of the antiplasmodial activity, the parasite lactate
dehydrogenase assay was used, with slight modifications, as
previously described by Makler et al.34 Briefly, the assay is
based on the observation that the lactate dehydrogenase (LDH)
enzyme of Plasmodium falciparum has the ability to rapidly
use 3-acetylpyridine NAD (APAD) as a coenzyme in the
reaction leading to the formation of pyruvate from lactate. Test
compounds were added in 2-fold serial dilutions to Plasmo-
dium falciparum (chloroquine-sensitive Ghana strain or chlo-
roquine-resistant W2 strain) cultures (1% parasitaemia, 2%
HCT) in 96-well plates. After 48 h at 37 °C and a gas mixture
of 93% N2, 4% CO2, and 3% O2, the parasite cultures were
frozen at -20 °C to lyse the erythrocytes and to store the plates
until further processing. After thawing, 20 µL of the lysed
culture was added to 100 µL of Malstat reagent (Flow Inc.),
and the formation of APADH was determined. Adding 40 µg
of nitroblue tetrazolium (NBT) and 2 µg of phenazine etho-
sulfate (PES) to the Malstat reagent promoted the spectro-
photometric assessment of LDH activity. As APADH is formed,
the NBT is reduced and forms a blue formazan product that
can be detected visually and measured spectrophotometrically
at 650 nm. Determination of the IC50 values was performed
in triplicate (mean ( SD). Cryptolepine9,11 (Table 3) and
chloroquine were used as positive controls. IC50 values of
chloroquine were 0.01 µM (Ghana) and 0.09 µM (W2).
In Vitr o Acitivity a ga in st T. br u cei Tr yp om a stigotes.
IC50 values against Trypanosoma brucei were determined in
triplicate (mean ( SD) as described before.35 Briefly, blood-
stream forms of T. brucei were cultivated in HMI-9 medium.
In a 96-well microplate, 10 000 hemoflagellates were incubated
at different concentrations of the test compound for 4 days.
Parasite multiplication was measured colorimetrically (490
nm) following addition of MTT, which converts to an water-
soluble formazan product. Suramin was used as positive
control (IC50 ) 0.3 ( 0.1 µM).
In Vit r o Acit ivit y a ga in st In t r a cellu la r T. cr u zi
Am a stigotes. IC50 values against Trypanosoma cruzi were
determined in triplicate (mean ( SD) as described before.35
Briefly, primary mouse peritoneal macrophages were seeded
in 96-well microplates at 30 000 cells per well. After 24 h,
about 10 000 trypomastigotes of T. cruzi were added per well
together with 2-fold dilutions of the drug. The cultures were
incubated at 37 °C in5% CO2/95% air for 4 days. Following
fixation in MeOH and Giemsa staining, the drug activity was
semiquantitatively scored as a percent reduction of the total
parasite load (free trypomastigotes and intracellular amastig-
otes) compared with untreated control cultures. Scoring was
performed microscopically. Nifurtimox was used as a positive
control (IC50 ) 0.4 ( 0.1 µM).
11-trimethylsilylneocryptolepine (9a )
2-methoxy-11-trimethylsilylneocryptolepine (9b)
2-bromo-11-trimethylsilylneocryptolepine (9c)
2-chloro-11-trimethylsilylneocryptolepine (9d )
2-fluoro-11-trimethylsilylneocryptolepine (9e)
2-iodo-11-trimethylsilylneocryptolepine (9f)
2-methyl-11-trimethylsilylneocryptolepine (9g)
2-nitro-11-trimethylsilylneocryptolepine (9h )
2-methylthio-11-trimethylsilylneocryptolepine (9i)
2-cyano-11-trimethylsilylneocryptolepine (9j)
9-cyano-11-trimethylsilylneocryptolepine (9k )
2-chloro-9-cyano-11-trimethylsilylneocryptolepine (9l)
9-cyano-2-methoxy-11-trimethylsilylneocryptolepine (9m )
9-cya n o-2-t r iflu or om et h yl-11-t r im et h ylsilyln eocr yp-
tolepine (9n )
9-cyano-2-fluoro-11-trimethylsilylneocryptolepine (9o)
1-methoxy-11-trimethylsilylneocryptolepine (9r )
1-bromo-11-trimethylsilylneocryptolepine (9s)
1-trifluoromethyl-11-trimethylsilylneocryptolepine (9u )
3-methoxy-11-trimethylsilylneocryptolepine (9v)
3-bromo-11-trimethylsilylneocryptolepine (9w )
3-chloro-11-trimethylsilylneocryptolepine (9x)
3-trifluoromethyl-11-trimethylsilylneocryptolepine (9y)
Gen er a l P r oced u r e for th e Con ver sion of th e Su bsti-
tu ted 11-Tr im eth ylsilyln eocr yp tolep in es 9a -y in to th e
Neocr yp tolep in e Der iva tives 1a -y. The appropriate 11-
trimethylsilylneocryptolepine (0.35 mmol) is dissolved in 30
mL of ethanol, after which NaOH (10 mL, 6 N) is added. The
mixture is stirred for 3 h at 70 °C and cooled to room
temperature. Water (50 mL) is added, and the mixture is
extracted with CH2Cl2 (3 × 50 mL). Purification of the product
is performed by column chromatography using acetone as the
eluent. 3-Methoxyneocryptolepine (1v) is purified by column
chromatography using toluene/diethylamine (9.5:0.5) as the
eluent. The following products were prepared in this manner:
neocryptolepine (1a )
2-methoxyneocryptolepine (1b)
2-bromoneocryptolepine (1c)
Cytotoxicty on MRC-5 Cells. A human diploid embryonic
lung cell line (MRC-5) was used to assess the cytotoxicity of
the test compounds as described before.35 Briefly, MRC-5 cells
were seeded at 5000 cells per well in 96-well microtiter plates.
After 24 h, the cells were washed and 2-fold dilutions of the
drug in 200 µL of standard culture medium (RPMI + 5% FCS)
were added. The final DMSO concentration of the culture
remained below 0.5%. The cultures were incubated with
different concentrations of test compounds at 37 °C in 5% CO2/
95% air for 7 days. Untreated cultures were included as
controls. Cytotoxicity was determined using the colorimetric
MTT assay and scored as a percent reduction of absorption at
540 nm of treated cultures versus untreated control cultures.
Determination of the IC50 values was performed in triplicate
(mean ( SD). Vinblastine was used as a positive control (IC50
< 10 nM).
In h ibition of â-Hem a tin F or m a tion . An in vitro method
was used to measure the inhibition of â-hematin formation,
based on the original method described by Egan et al.24 and
modified by Wright et al.11 Heme refers to Fe(II)protoporphyrin
IX, hemin is Fe(II)protoporphyrin IX chloride, and hematin is
Fe(II)protoporphyrin IX hydroxide. Briefly, 0.1 M NaOH was
added to Fe(II)hemin (Fluka; HPLC purity, >98%) (typically
7.5 mg), which forms hematin. Polymerization to â-hematin,
2-chloroneocryptolepine (1d )
2-fluoroneocryptolepine (1e)
2-iodoneocryptolepine (1f)
2-methylneocryptolepine (1g)
2-nitroneocryptolepine (1h )
2-methylthioneocryptolepine (1i)
2-cyanoneocryptolepine (1j)
9-cyanoneocryptolepine (1k )
2-chloro-9-cyanoneocryptolepine (1l)
9-cyano-2-methoxyneocryptolepine (1m )
9-cyano-2-trifluoromethylneocryptolepine (1n )
9-cyano-2-fluoroneocryptolepine (1o)
1-methoxyneocryptolepine (1r )
1-bromoneocryptolepine (1s)
1-trifluoromethylneocryptolepine (1u )
3-methoxyneocryptolepine (1v)
3-bromoneocryptolepine (1w )
3-chloroneocryptolepine (1x)
3-trifluoromethylneocryptolepine (1y)
Con ver sion to Hyd r och lor id e Sa lts. All compounds were
biologically evaluated as their hydrochloride salts. Compounds
were dissolved in 0.1 N HCl in MeOH, and the solutions were
evaporated to dryness after 30 min.

Neocryptolepine Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3507
which has been characterized as [Fe(III)protoporphyrin IX]2,36
proceeds after addition of a 9.9 M acetate buffer, pH 5. Test
compounds (3 equiv with respect to hemin) were added to the
hematin solutions prior to acidification. After incubation for
40 min at 60 °C, cooling and filtration, the precipitate was
washed with water and dried, and FT-IR (Perkin-Elmer FT-
IR 1760) was used to check the presence of â-hematin, which
shows sharp bands at 1660 and 1207 cm-1. The spectra are
examined on the basis of the presence or absence of the two
typical peaks. When they are absent the compound has
inhibited the formation of â-hematin. A sample containing
hemin but without the test compound, which was not incu-
bated, was used as a negative control (no formation of
â-hematin). A sample containing hemin without the test
compound, which was incubated for 40 min at 60 °C, was used
as a positive control (formation of â-hematin).
Ch a r a cter iza tion of Hem e: Ga s-P h a se Ion Dr u g Com -
p lexes by Electr osp r a y Ion iza tion (ESI) Ma ss Sp ectr om -
etr y. Abbreviations are the following: C, cationic form of the
drug; FP, heme ) ferriprotoporphyrin (depending on the
conditions, FP may be present in an unliganded form [FP]+,
in a dimeric form [2FP - H]+, as a dehydrated dimer of
hematin [FP-O-FP]+, or as the CH3OH complex of the latter
dimeric form [FP-O-FP + MeOH]+). Mixtures of the drugs
(hydrochloride salts) with FP were prepared in MeOH/H2O (1:
1; v/v; pH 7). Positive-ion mass spectra were recorded on an
Autospec-oa TOF mass spectrometer (Micromass, Manchester,
U.K.) employing electrospray ionization. Low-energy collision-
induced dissociation (CID) spectra were acquired at a collision
energy (Elab) of 400 eV using helium as the collision gas.
Helium was introduced into the collision cell until the signal
of the very weak FP+:CH3OH complex (m/z 648) reached 60%
of its original value. Under these conditions, the CID spectrum
of the FP+:MeOH complex revealed the FP+ ion (m/z 616) as
the base peak, and the residual FP+:MeOH ion had a relative
abundance (RA) of 3%. To allow comparisons between binding
strengths of the different [FP:C]+ complexes, the CID experi-
ments were performed on the same day using the same
collision gas pressure.
DNA-Meth yl Gr een Assa y. The DNA-methyl green
assay is a simple microtiter assay for the detection of com-
pounds that bind DNA. Agents that displace methyl green
from a DNA-methyl green complex (deoxyribonucleic acid
methyl green; Sigma) are detected spectrophotometrically
(Labsystems Multiscan MCC/340) by a decrease in absorbance
at 620 nm.28 DNA-methyl green was suspended in 100 mL of
0.05 M Tris-HCl buffer, pH 7.5, containing 7.5 mM MgSO4 and
was stirred at 37 °C for 24 h. The dissolved samples were
dispensed into wells of a 96-well microtiter plate. Solvent was
removed under vacuum, and 200 µL of the DNA-methyl green
solution was added. The initial absorbance was compared with
the final absorbance (after 24 h) in order to calculate the IC50
value (50% displacement of methyl green from DNA). Deter-
mination of the IC50 values was performed in triplicate (mean
( SD). The decrease in absorbance observed represents the
initial rapid displacement of methyl green from DNA by the
drug, followed by the slower reaction with water that yields
the colorless carbinol.
in relaxation buffer (50 mM Tris, pH 7.8, 50 mM KCl, 10 mM
MgCl2, 1 mM dithiothreitol, 1 mM EDTA, and 0.5 mM ATP)
in the presence of varying concentrations of the test compound.
Reactions were terminated by adding SDS to 0.25% and
proteinase K to 250 µg/mL. DNA samples were then added to
the electrophoresis dye mixture (3 µL) and electrophoresed in
a 1% agarose gel containing ethidium bromide (1 µg/mL) at
room temperature for 2 h at 120 V. Gels were washed and
photographed under UV light.
Ack n ow led gm en t . We thank J . Verreydt, J .
Schrooten, N. Verhaert, V. Van Heurck, W. Van Lierde,
and W. Van Dongen for their technical assistance. T.
J onckers thanks the “Vlaams Instituut voor de Bevor-
dering van het Wetenschappelijk Technologisch Onder-
zoek in de Industrie (IWT)” for a scholarship. He is also
indebted to the foundation “Rosa Blanckaert” for a
research grant. Financial support from the Fund for
Scientific Research (FWO-Vlaanderen) (Project Nos.
G.0119.96, G.0082.98, G.0334.00, and 1.5.139.00) and
from the Special Research Fund of the University of
Antwerp (Concerted Research Project No. 99/3/34, fel-
lowship for S. van Miert) is gratefully acknowledged.
Dr. Colin Wright (University of Bradford, U.K.) is kindly
acknowledged for assistance with the assay on inhibi-
tion of â-hematin formation.
Ap p en d ix
Ta ble 4. Degree of Purity for All Compounds
% purity, % purity,
% purity, % purity,
compd system Aa system Bb compd system Aa system Bb
1a
100.0
97.1
98.3
100.0
100.0
100.0
100.0
100.0
100.0
98.0
100.0
99.2
95.3
95.6
100.0
95.7
100.0
95.9
95.5
96.1
95.1
95.5
95.0
1k
1l
100.0
99.0
100.0
96.2
95.7
96.4
96.8
94.5
95.0
93.2
97.1
97.1
95.2
99.5
96.8
95.8
95.2
1b
10b
1c
1d
10d
1e
10e
1f
1g
1h
1i
1m
1n
1o
10q
1r
97.6
100.0
100.0
100.0
98.4
92.8
99.3
1s
1u
1v
1w
1x
1y
95.4
99.4
100.0
100.0
99.5
1j
a
System A: column, MERCK, Lichrospher Si 600 (4 mm × 250
mm); solvent system, 1-propanol-hexane 5%/water 95:5 or 1-pro-
b
panol-hexane 5%/water 90:10. System B: column, MERCK,
Lichrocart-Lichrospher 100 RP 18 (4 mm × 250 mm); solvent
system, gradient from 80% to 100% acetonitrile in 20 min.
Su p p or tin g In for m a tion Ava ila ble: Characterization of
intermediate compounds and target compounds. This mater-
ial is available free of charge via the Internet at http://
pubs.acs.org.
Electr ic Lin ea r Dich r oism (ELD) Mea su r em en ts. ELD
measurements were performed with a computerized optical
measurement system using the procedures previously out-
lined.37 All experiments were conducted with a 10 mm path
length Kerr cell having a 1.5 mm electrode separation. The
samples were oriented under an electric field strength varying
from 1 to 13 kV/cm. The concentration of the test compound
was 10 µM in the presence of DNA at 200 µM unless stated
otherwise. This electrooptical method has proved to be most
useful for determining the orientation of the drugs bound to
DNA. It has the additional advantage that it senses only the
orientation of the polymer-bound ligand; free ligand is isotropic
and does not contribute to the signal.
Refer en ces
(1) O’Neill, P. M.; Bray, P. G.; Hawley, S. R.; Ward, S. A.; Park, B.
K. 4-AminoquinolinessPast, Present, and Future: A Chemical
Perspective. Pharmacol. Ther. 1998, 77, 29-58.
(2) Rang, H. P.; Dale, M. M.; Ritter, J . M. Pharmacology, 4th ed.;
Churchill Livingstone: Edinburgh, U.K., 1999; Chapter 46
(Antiprotozoal Drugs).
(3) Newman, D. J .; Cragg, G. M.; Snader, K. M. The Influence of
Natural Products upon Drug Discovery. Nat. Prod. Rep. 2000,
17, 215-234.
(4) Kirby, G. C.; Paine, A.; Warhurst, D. C.; Noamese, B. K.;
Phillipson, J . D. In Vitro and in Vivo Antimalarial Activity of
Cryptolepine, a Plant-Derived Indoloquinoline. Phytother. Res.
1995, 9, 359-363.
(5) Cimanga, K.; De Bruyne, T.; Pieters, L.; Vlietinck, A.; Turger,
C. A. In Vitro and in Vivo Antiplasmodial Activity of Cryp-
tolepine and Related Alkaloids from Cryptolepis sanguinolenta.
J . Nat. Prod. 1997, 60, 688-691.
Top oisom er a se II Med ia ted DNA Clea va ge Assa y.
Supercoiled pKMp27 DNA (0.5 µg) was incubated with 4 units
of human topoisomerase II (TopoGen Inc.) at 37 °C for 30 min

3508 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
J onckers et al.
(6) Cimanga, K.; De Bruyne, T.; Lasure, A.; Van Poel, B.; Pieters,
L.; Claeys, M.; Vanden Berghe, D.; Kambu, K.; Tona, L.;
Vlietinck, A. In Vitro Biological Activities of Alkaloids from
Cryptolepis sanguinolenta. Planta Med. 1996, 62, 22-27.
(7) Bonjean, K.; De Pauw-Gillet, M.-C.; Defresne, M.-P.; Colson, P.;
Houssier, C.; Dassonneville, L.; Bailly, C.; Greimers, R.; Wright,
C.; Quetin-Leclercq, J .; Tits, M.; Angenot, L. The DNA Interca-
lating Alkaloid Cryptolepine Interferes with Topoisomerase II
and Inhibits Primarily DNA Synthesis in B16 Melanoma Cells.
Biochemistry 1998, 37, 5136-5146.
(8) Bailly, C.; Laine, W.; Baldeyrou, B.; De Pauw-Gillet, M.-C.;
Colson, P.; Houssier, C.; Cimanga, K.; Van Miert, S.; Vlietinck,
A. J .; Pieters, L. DNA Intercalation, Topoisomerase II Inhibition
and Cytotoxic Activity of the Plant Alkaloid Neocryptolepine.
Anti-Cancer Drug Des. 2000, 15, 191-201.
(9) Arzel, E.; Rocca, P.; Grellier, P.; Labae¨ıd, M.; Frappier, F.;
Gue´ritte, F.; Gaspard, C.; Marsais, F.; Godard, A.; Que´guiner,
G. New Synthesis of Benzo-δ-carbolines, Cryptolepines, and
Their Salts: In Vitro Cytotoxic, Antiplasmodial, and Antitry-
panosomal Activities of δ-Carbolines, Benzo-δ-carbolines, and
Cryptolepines. J . Med. Chem. 2001, 44, 949-960.
(10) Bierer, D. E.; Fort, D. M.; Mendez, C. D.; Luo, J .; Imbach, P. A.;
Dubenko, L. G.; J olad, S. D.; Gerber, R. E.; Litvak, J .; Lu, Q.;
Zhang, P.; Reed, M. R.; Waldeck, N.; Bruening, R. C.; Noamesi,
B. K.; Hector, R. F.; Carlson, T. J .; King, S. R. Ethnobotanical-
Directed Discovery of the Antihyperglycemic Properties of Cryp-
tolepine: Its Isolation from Cryptolepis sanguinolenta, Synthesis,
and in Vitro and in Vivo Activities. J . Med. Chem. 1998, 41, 894-
901.
(11) Wright, C. W.; Addae-Kyereme, J .; Breen, A. G.; Brown, J . E.;
Cox, M. F.; Croft, S. L.; Go¨kc¸ek, Y.; Kendrick, H.; Phillips, R.
M.; Pollet, P. L. Synthesis and Evaluation of Cryptolepine
Analogues for Their Potential as New Antimalarial Agents. J .
Med. Chem. 2001, 44, 3187-3194.
(12) Peczynska-Czoch, W.; Pognan, F.; Kaczmarek, L.; Boratynski,
J . Synthesis and Structure-Activity Relationship of Methyl-
Substituted Indolo[2,3-b]quinolines: Novel Cytotoxic, DNA To-
poisomerase II Inhibitors. J . Med. Chem. 1994, 37, 3503.
Kaczmarek, L.; Balicki, R.; Nantka-Namirski, P. Synthesis and
Antineoplastic Properties of some Benzo-iso-R-carbolines. Arch.
Pharm. 1988, 321, 463-467.
(13) Alajar´ın, M.; Molina, P.; Vidal, A. Formal Total Synthesis of the
Alkaloid Cryptotackieine (Neocryptolepine). J . Nat. Prod. 1997,
60, 747-748.
(14) Molina, P.; Fresneda, P. M.; Delgado, S. Iminophosphorane-
Mediated Synthesis of the Alkaloid Cryptotackieine. Synthesis
1999, 326-329.
(15) Tima´ri, G.; Soo´s, T.; Hajo´s G. A Convenient Synthesis of Two
New Indoloquinoline Alkaloids. Synlett 1997, 1067-1068.
(16) Schmittel, M.; Steffen, J . P.; Engels, B.; Lennartz, C. Two Novel
Thermal Biradical Cyclizations in Theory and Experiment: New
Synthetic Routes to 6H-Indolo[2,3-b]quinolines and 2-Amino-
quinolines from Enyne-Carbodiimides. Angew. Chem., Int. Ed.
1998, 37, 2371-2373.
(17) Shi, C.; Zhang, Q.; Wang K. K. Biradicals from Thermolysis of
N-[2-(1-Alkynyl)phenyl]-N′-phenylcarbodiimides and Their Sub-
sequent Transformations to 6H-Indolo[2,3-b]quinolines. J . Org.
Chem. 1999, 64, 925-932.
(18) Saito, T.; Ohmori, H.; Ohkubo, T.; Motoki, S. Periselective, Lewis
Acid-Induced Intramolecular Diels-Alder Reaction of Conju-
gated Carbodiimides: Efficient Synthesis of Nitrogen Hetero-
cycles, Indolo[2,3-b]quinolines and Pyrido[2,3-b]indole. J . Chem.
Soc., Chem. Commun. 1993, 1802-1803.
(19) A trimethylsilyl (TMS) group can be transformed into other
substituents. (a) J acob, L. A.; Chen, B. L.; Stec, D. Further
Studies on the Iodination of Aryltrimethylsilanes. Synthesis
1993, 611-614. (b) Bennetau, B.; Dunogues, J . Unusual Elec-
trophilic Substitution in the Aromatic Series via Organosilicon
Intermediates. Synlett 1993, 171-176.
(20) Chen, C.; Lieberman, D. R.; Larsen, R. D.; Reamer, R. A.;
Verhoeven, T. R.; Reider, P. J . Synthesis of the 5-HT1D
Receptor
Agonist MK-0462 via a Pd-Catalyzed Coupling Reaction. Tet-
rahedron Lett. 1994, 35, 6981-6984.
(21) Fell, J . B.; Coppola, G. M. A Mild and Efficient Preparation of
Carbodiimides. Synth. Commun. 1995, 25 (1), 43-47.
(22) Palomo, C.; Mestres, R. Convenient and Improved Synthesis of
Unstable Carbodiimides. Synthesis 1981, 373.
(23) Wright, C. W.; Phillipson, J . D.; Awe, S. O.; Kirby, G. C.;
Warhurst, D. C.; Leclercq, J .; Angenot, L. The Antimalarial
Action of Cryptolepine Is Unlikely To Be Due to Intercalation
with Parasite DNA. Presented at The U.K. Association of
Pharmaceutical Scientists: Natural Products as Drugs and
Medicines, November 18, 1994; Abstract p 23.
(24) Egan, T. J .; Ross, D. C.; Adams, P. A. Quinoline Anti-Malarial
Drugs Inhibit Spontaneous Formation of â-Haematin (Malaria
Pigment). FEBS Lett. 1994, 352, 54-57.
(25) Pandey, A. V.; Tekwani, B. L. Depolymerization of Malarial
Hemozoin: A Novel Reaction Initiated by Blood Schizonticidal
Antimalarials. FEBS Lett. 1997, 402, 236-240.
(26) Ginsburg, H.; Famin, O.; Zhang, J .; Krugliak, M. Inhibition of
Glutathione-Dependent Degradation of Heme by Chloroquine
and Amodiaquine as a Possible Basis of Their Antimalarial Mode
of Action. Biochem. Pharmacol. 1998, 56, 1305-1313.
(27) Wright, C. W.; Addae-Kyereme, J .; Brown, J . E.; Tranter, D. A.
Evaluation of Cryptolepine and Some Analogues as Leads to
Selective Antimalarial Agents. J . Pharm. Pharmacol. 1997, 49
(Suppl. 4), 36.
(28) Burres, N. L.; Frigo, A.; Rasmussen, R. R.; McAlpine, J . B. A
Colorimetric Microassay for the Detection of Agents That
Interact with DNA. J . Nat. Prod. 1992, 55, 1582-1587.
(29) Dassonneville, L.; Lansiaux, A.; Wattelet, A.; Wattez, N.;
Mahieu, C.; Van Miert, S.; Pieters, L.; Bailly, C. Cytotoxicity
and Cell Cycle Effects of the Plant Alkaloids Cryptolepine and
Neocryptolepine: Relation to Drug-Induced Apoptosis. Eur. J .
Pharmacol. 2000, 409, 9-18.
(30) Nenortas, E. C.; Bodley, A. L.; Shapiro, T. A. DNA Topo-
isomerases: A New Twist for Antiparasitic Chemotherapy?
Biochim. Biophys. Acta 1998, 1400, 349-354.
(31) Weissig, V.; Vetro-Widenhouse, T. S.; Rowe, T. C. Topoisomerase
II Inhibitors Induce Cleavage of Nuclear and 35-kb Plastid DNAs
in the Malarial Parasite Plasmodium falciparum. DNA Cell Biol.
1997, 16, 1483-1492.
(32) Fichera, M. E.; Roos, D. S. A Plastid Organelle as a Drug Target
in Apicomplexan Parasites. Nature 1997, 390, 407-409.
(33) Wright, A.; Wang, H.; Gurrath, M.; Konig, G.; Kocak, G.;
Neumann, G.; Loria, P.; Foley, M.; Tilley, L. Inhibition of Heme
Detoxification Processes Underlies the Antimalarial Activity of
Terpene Isonitrile Compounds from Marine Sponges. J . Med.
Chem. 2001, 44, 873-885.
(34) Makler, M. T.; Hinrichs, D. J . Measurement of the Lactate
Dehydrogenase Activity of Plasmodium falciparum as an As-
sessment of Parasitemia. Am. J . Trop. Med. Hyg. 1993, 48, 205-
210.
(35) Girault, S.; Grellier, P.; Berecibar, A.; Maes, L.; Mouray, E.;
Lemie`re, P.; Debreu, M.-A.; Davioud-Charvet, E.; Sergheraert,
C. Antimalarial, Antitrypanosomal, and Antileishmanial Activi-
ties and Cytotoxicity of Bis(9-amino-6-chloro-2-methoxyacri-
dines): Influence of the Linker. J . Med. Chem. 2000, 43, 2646-
2654.
(36) Pagola, S.; Stephens, P. W.; Bohle, D. S.; Kosar, A. D.; Madsen,
S. K. The Structure of Malaria Pigment â-Haematin. Nature
2000, 404, 307-310.
(37) Colson, P.; Bailly, C.; Houssier, C. Electric Linear Dichroism as
a New Tool To Study Sequence Preference in Drug Binding to
DNA. Biophys. Chem. 1996, 58, 125-140.
J M011102I