New synthesis of benzo-δ-carbolines, cryptolepines, and their salts: In vitro cytotoxic, antiplasmodial, and antitrypanosomal activities of δ-carbolines, benzo-δ-carbolines, and cryptolepines

Article abstract of DOI:10.1021/jm0010419

The paper describes, in its first part, a new synthesis of benzo-δ-carbolines, cryptolepines, and their salts. The strategy is based on the association between halogen-dance and hetero-ring cross-coupling. It is fully convergent and regioselective with in

Full text of DOI:10.1021/jm0010419

J . Med. Chem. 2001, 44, 949-960
949
New Syn th esis of Ben zo-δ-ca r bolin es, Cr yp tolep in es, a n d Th eir Sa lts: In Vitr o
Cytotoxic, An tip la sm od ia l, a n d An titr yp a n osom a l Activities of δ-Ca r bolin es,
Ben zo-δ-ca r bolin es, a n d Cr yp tolep in es
Erwan Arzel,§ Patrick Rocca,*,§ Philippe Grellier,|,† Mehdi Labae¨ıd,| Franc¸ois Frappier, Franc¸oise Gue´ritte,#,‡
Christiane Gaspard,# Francis Marsais,§ Alain Godard,§ and Guy Que´guiner§
Institut National des Sciences Applique´es, UMR 6014, BP 08, 76131 Mont-Saint-Aignan Cedex, France,
Laboratoire de Biologie Parasitaire et Chimiothe´rapie, Muse´um National d’Histoire Naturelle, IFR 63, 61 rue de Buffon,
75231 Paris Cedex 05, France, Laboratoire de Chimie, Muse´um National d’Histoire Naturelle, ESA 8041 CNRS,
63 rue de Buffon, 75231 Paris Cedex 05, France, and Institut de Chimie des Substances Naturelles, CNRS,
Avenue de la Terrasse, Baˆtemont 27, 91198 Gif-sur-Yvette Cedex, France
Received J uly 24, 2000
The paper describes, in its first part, a new synthesis of benzo-δ-carbolines, cryptolepines, and
their salts. The strategy is based on the association between halogen-dance and hetero-ring
cross-coupling. It is fully convergent and regioselective with interesting overall yields from
27% to 70%. A halogen-dance mechanism in quinoline series is also proposed. The formal
synthesis of potential antimalarial compounds and the first total synthesis of 11-isopropyl-
cryptolepine are also described. In the second part, cytotoxic activity against mammalian cells
and activities against Plasmodium falciparum and Trypanosoma cruzi of benzo-δ-carbolines
and δ-carbolines were evaluated in vitro to study the structure-activity relationships. For
benzo-δ-carbolines, methylation at N-5 increases the cytotoxic and antiparasitic activities. A
further alkylation on C-11 generally increases the cytotoxic activity but not the antiparasitic
activity, cryptolepine and 11-methylcryptolepine being the most active on both parasites. Taking
advantage of the fluorescence of the indoloquinoline chromophore, cryptolepine was localized
by fluorescence microscopy in parasite DNA-containing structures suggesting that these
compounds act through interaction with parasite DNA as proposed for cryptolepine on
melanoma cells. For δ-carbolines, methylation at N-1 is essential for the antimalarial activity.
1-Methyl-δ-carboline specifically accumulates in the intracellular parasite. It has weak cytotoxic
activity and can be considered as a potential antimalarial compound.
Ch a r t 1
In tr od u ction
δ-Carbolines and benzo-δ-carbolines1 are very rare in
nature, and the best representatives of this family are
quindoline2 (1a ) and cryptolepine3,4 (2a ) (Chart 1), two
indoloquinoline alkaloids isolated in 1977 and 1929,
respectively, from a West African plant: Cryptolepis
sanguinolenta (Periplocaceae). Benzo-δ-carbolines are
also found in three other plants: Sida acuta (Malvaceae)
from Sri Lanka,5 Microphilis guyanensis (Sapotaccae),
and Genipa americana (Rubiaceae) from Suriname.6
Some minor indoloquinoline alkaloids are functionalized
at the C-11 position. This is the case for cryptomisrine7
(3), cryptolepinone8-12 (4), 11-isopropylcryptolepine13
(2d ), and biscryptolepine14 (5).
Considerable interest in this family has been shown
by several teams throughout the world due to their
various and important biological properties such as:
antimuscarinic, antibacterial, antiviral, antiplasmodial,
and antihyperglycemic activities.15-19 Recently, two
* To whom correspondence should be addressed. Tel: 332 35 52 24
84. Fax: 332 35 52 29 62. E-mail: Patrick.Rocca@insa-rouen.fr.
§ Institut National des Sciences Applique´es.
| Laboratoire de Biologie Parasitaire et Chimiothe´rapie, Muse´um
National d’Histoire Naturelle.
reports mentioned the cytotoxicity of cryptolepine and
analogues toward B16 melanoma cells20 and M109
Madison lung carcinoma.6 Bonjean et al. also showed
that cryptolepine interferes with topoisomerase II and
Laboratoire de Chimie, Muse´um National d’Histoire Naturelle.
#
Institut de Chimie des Substances Naturelles.
† E-mail: grellier@cimrs1.mnhn.fr.
‡ E-mail: Francoise.Gueritte@icsn.cnrs-gif.fr.
10.1021/jm0010419 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/08/2001

950 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6
Ta ble 1. Structures of the Tested δ-Carbolines
Arzel et al.
Sch em e 1
R1
R2
R3
product
H
H
H
H
H
H
H
H
H
Ph
H
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
Me
Et
Ph
2-C6H4NH2
2-pyridyl
2-quinolyl
2-thienyl
H
H
H
H
H
Ph
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
Sch em e 2a
2-C6H4NH2
2-pyridyl
2-quinolyl
2-thienyl
Ph
Ph
H
primarily inhibits DNA synthesis.20 Moreover, some
quindoline derivatives have been described as potent
antitumor-active compounds.21
Recently, we described22,23 a new synthesis of R-sub-
stituted, â-substituted, and R,â-disubstituted δ-carbo-
lines (Table 1). This synthesis was based on metalation
and cross-coupling reactions, starting with simple ben-
zene and pyridine blocks. In a similar process, com-
pound 6p (Table 1) was obtained using a methylated
boronic acid (see Experimental Section). Methylated
carbolines 7-8 were synthesized from δ-carboline fol-
lowing the procedure described further under “Synthesis
of Cryptolepines and Benzo-δ-carbolinium Salts”.
In 1998, we reported a new and very short route24 to
quindoline (1a ) (the benzo-δ-carboline) which involved
the first halogen-dance reaction in the iodoquinoline
series. One year later, using this reaction, we described
the first total synthesis25 of cryptomisrine (3), a bis-
benzo-δ-carboline linked by a keto group. We wish to
report here on an extension of this fruitful method, to
the total synthesis of new C-11-substituted benzo-δ-
carbolines as well as their methylated analogues, called
here “cryptolepines”, and their salt forms: triflates and
chlorides. Cytotoxic activity toward mammalian cells
and activities against the parasites Plasmodium falci-
parum, agent of malaria, and Trypanosoma cruzi, agent
of Chagas disease, of such molecules as well as R-sub-
stituted, â-substituted, and R,â-disubstituted δ-carbo-
lines were evaluated in order to study structure-
activity relationships (SARs) (the skeleton of δ-carbolines
is very close to that of benzo-δ-carboline). In addition,
by taking advantage of the fluorescence of these deriva-
tives, we used fluorescence microscopy for a subcellular
localization of active compounds in parasites.
a
(a) 1 equiv LDA, THF, -78 °C, 2 h (26 f 27) or 4 h; (b) I2,
THF, -78 °C, 2 h; (c) H2O; (d) electrophile, THF, -78 °C, 2 h; (e)
1 equiv (LDA + TMSCl), THF, -78 °C, 2 h; (f) D2O, THF.
Ch em istr y
A retrosynthetic analysis (Scheme 1) suggests that
cryptolepines could be prepared by methylation of the
corresponding benzo-δ-carbolines A. These latter com-
pounds could be obtained by cyclization of the conve-
niently functionalized phenylquinolines B, our key
intermediates in this strategy. These phenylquinolines
could be obtained via a cross-coupling reaction26,27
between the required benzene and quinoline building
blocks. Finally, the trisubstituted quinolines C could be
prepared by an orthometalation-isomerization reaction
on the 3-fluoro-4-iodoquinoline.
3-Fluoroquinoline (9) was metalated26 with LDA at
low temperature to give the 3-fluoro-4-iodoquinoline (10)
in very good yield after reaction of iodine (Scheme 2).
Treatment of 10 by LDA at -75 °C and reaction of the
resulting lithio species with various electrophiles led to
the corresponding 4-substituted 3-fluoro-2-iodoquino-
lines 11-18 in good to high yields (Scheme 2, Table 2).
It should be noted that the methyl group in compound
15 is the only alkyl chain which can be incorporated in
such a position, probably due to the lack of reactivity of
the lithio species toward alkyl iodides. Identification of
compounds 11-18 was inferred from the 1H and 13C

Benzo-δ-carbolines, Cryptolepines, and Their Salts
Ta ble 2. Halogen-Dance
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6 951
A cross-coupling reaction between phenylboronic acid
and 31 led to the corresponding triaryl 34 in quantita-
tive yield (Scheme 4). Cyclization41,42 of the previously
prepared phenylquinolines (23, 25, 31-34) to the cor-
responding benzo-δ-carbolines (1a -f) was best achieved
by treatment with boiling pyridinium chloride at 215
°C followed by a basic workup (Scheme 5, Table 5).
Compound 1a is the natural alkaloid quindoline, and
the chlorinated compound 1f is an intermediate in the
synthesis of potential antimalarial compounds.43 It
should be pointed out that 11-methylquindoline (1b)
(Scheme 5, Table 5) had already been obtained by
chemical modifications of quindoline-11-carboxylic acid.6
Thus, our strategy allowed the formal synthesis of such
compounds. Cooper et al.10 also described this chloro
compound (1f) as a precursor of quindoline itself (1a ).
Syn th esis of Cr yp tolep in es a n d Ben zo-δ-ca r bo-
lin iu m Sa lts. Various cryptolepines were synthesized
from the previously prepared benzo-δ-carbolines 1a -e
using the procedure described by Bierer et al.44 In a first
step, benzo-δ-carbolines 1a -e were treated with methyl
triflate in dry toluene at room temperature to give the
corresponding triflate salts 35a -e. Treatment of these
salts by an aqueous solution of sodium carbonate in the
presence of chloroform and basic alumina gave the
corresponding cryptolepines 2a-e in good yields (Scheme
6, Table 6). Further treatment with HCl yielded the
corresponding hydrochloride compounds 36a -e which
were used for biological tests (see next part).
electrophile
E
product
yield (%)
H2O
D2O
I2
C2Cl6
MeI
MeCHO
PhCHO
HCO2Et
H
D
I
11
12
13
14
15
16
17
18
95
95
98
74
65a
79
75
95
Cl
Me
MeCH(OH)
PhCH(OH)
CHO
a
NMR integration.
NMR spectra: a strong shielding of the carbon bearing
the iodo atom could be observed.27-29
The behavior of iodoquinoline 10 toward LDA is very
similar to that observed in the pyridine series.30 Indeed,
when 3-fluoro-4-iodoquinoline was treated with an
equimolar mixture of LDA and TMSCl, the 2-silylated
compound 19 was observed (Scheme 2), showing that
the first step of this reaction is the fluoro-directed
metalation of the more acidic 2-position under kinetic
control. Without addition of an electrophile such as
TMSCl, a fast isomerization of the 2-lithio derivative
to the more stable 4-lithioquinoline occurred. This
reaction seems to be very fast, since we were unable to
trap the 3-fluoro-4-iodo-2-lithio derivatives by addition
of electrophiles after the lithiation step. However, when
3-fluoro-4-iodoquinoline (10) was treated with LDA at
-78 °C followed by quenching with deuterium oxide
(D2O), three products were obtained, as well as a small
amount of the starting material (Scheme 2).
In all experiments, a small amount of the diiodoquin-
oline 13 was observed (about 0.5%) as well as the
deiodinated compound 20 (about 2%). Moreover, the
diiodo compound 13 seems to play a role as a catalyst
in this halogen-dance reaction. Indeed, when metalation
of 3-fluoro-4-iodoquinoline (10) was carried out with an
additional 10% of diiodoquinoline 13, the rate of the
reaction was increased by 31%. In consequence of these
observations and according to the literature,31-39 we can
propose the following mechanism (Scheme 3): In the
first step, the 2-lithio derivative 10a is generated by
LDA (path 1) and can react with the starting material
(path 2) to form the 3-fluoro-4-lithioquinoline (20a ) and
the diiodo derivative 13. Then two pathways are made
possible, with reaction of the diiodo compound 13 with
either the 2-lithio species 10a (path a) or the 4-lithio-
quinoline 20a (path b), leading to the same expected
4-lithioquinoline 12a .
Syn th esis of Ben zo-δ-ca r bolin es. Palladium-cata-
lyzed cross-coupling reaction between boronic acids 21-
22 and iodoquinolines 11, 13-14 using a Suzuki pro-
cedure26 produced the biaryls 23-28 in good to high
yields (Scheme 4, Table 3). To synthesize other precur-
sors (29-32) of substituted benzo-δ-carbolines, com-
pound 23 was readily functionalized by metalation with
n-BuLi at -78 °C followed by quenching with various
electrophiles (Scheme 4, Table 4).
It can be noted that the chlorinated compound 25 is
easier to obtain according to this route than the previous
one, for yield and purification reasons. Metalation of
compound 32 with KDA40 followed by methylation gave
the isopropyl derivatives 33 with an excellent yield
(Scheme 4). This compound is a precursor of the natural
alkaloid 11-isopropylcryptolepine (2d ).
It should be noted that this general strategy of
cryptolepines allowed the preparation of an analogue
of ellipticine45 (a natural alkaloid with antitumor activi-
ties34) and the first total synthesis of 11-isopropylcryp-
tolepine13 (2d ), isolated in 1999 from the root of C.
sanguinolenta.
Resu lts a n d Discu ssion
In Vitr o Cytotoxicity of Ben zo-δ-ca r bolin es on
KB Cells. To study the influence of the structural
modifications at positions 5 and 11 toward cytotoxicity,
we performed an in vitro cell survival assay on cancer
KB cell lines. The results are shown in Table 7.
As noted by Kingston et al.,6 methylation at the N-5
position led to a large increase of cytotoxicity. Quindo-
line (1a ) is 30 times less cytotoxic on KB cells than
cryptolepine (2a ). In the benzo-δ-carbolines series (com-
pounds 1b-e), alkylation at the C-11 position has no
effect on the activity for most of the compounds.
Compounds 1b-d possess similar cytotoxicity to the
parent compound 1a , but the presence of a phenyl group
(compound 1e) led to a decrease in cytotoxicity. It should
be noted that this lack of cytotoxicity is not due to a
lack of solubility. On the other hand, alkylation at the
C-11 position in the cryptolepine series (compounds 2b-
d ) led to a slight increase in cytotoxicity, except for 11-
isopropylcryptolepine (2d ) which is about 4 times less
active than cryptolepine (2a ). The cytotoxicity of 11-
methylcryptolepine (2b) (IC50 ) 0.53 µM) over cryptol-
epine 2a itself made this compound a suitable candidate
for further pharmacological studies.
In Vitr o An titr yp a n osom a l a n d An tip la sm od ia l
Activities of Ben zo-δ-ca r bolin es. In vitro and in vivo
antiplasmodial activity of cryptolepine and related
benzo-δ-carbolines has been previously reported.6,15-19

952 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6
Arzel et al.
Sch em e 3
However, their activity against T. cruzi, especially
against the intracellular multiplication of the amastig-
ote stage, has only been investigated a little. We
therefore studied the influence of the structural modi-
fications of cryptolepine at N-5 and C-11 positions on
the activity against the T. cruzi Tulahuen strain and
the chloroquine-resistant K1 strain of P. falciparum.
Results are reported in Table 8. Methylation at position
5 is essential for the activity since quindoline (1a ) was
about 100 times less active against both parasites than
cryptolepine 35a . Cryptomisrine (3) showed higher
activity than quindoline, but its index of selectivity (IS)
stayed in a similar range. The IS for T. cruzi was defined
as the ratio between the IC50 value on the L6 cells and
the IC50 value on the intracellular multiplication of the
amastigote forms. The IS for P. falciparum was defined
as the ratio between the IC50 value on the L6 cells and
the IC50 value against P. falciparum. Compounds that
demonstrate a higher selectivity (greater IS number)
should offer the potential of more safer therapy. In the
quindoline series, alkylation at the C-11 position (com-
pounds 1b,c) had no significant effect on the activity
against both parasites, except that 11-ethylquindoline
(1c) showed activity about 10 times higher on the T.
cruzi trypomastigote forms than quindoline. In the
cryptolepine series, alkylation at the C-11 position
(compounds 35b -d ) reduced the activity against P.
falciparum, except for 11-methylcryptolepine 35b which
was about 5 times more active than cryptolepine 35a .
However, as observed for cancer KB cells, C-11 methy-
lation also increased cytotoxicity toward the L6 cells and
the IS value was similar to that of cryptolepine (IS of
22-24). Benzo-δ-carbolines were generally more active
against P. falciparum when used under salt form. As
for P. falciparum, the C-11 alkylation also reduced the
activity against the intracellular multiplication of the
T. cruzi amastigote forms, except for 11-methylcryptol-
epine 35b which is slightly more active than cryptol-
epine 35a . Benzo-δ-carbolines seem to be about one-half
as active against amastigotes when used under their
salt forms. With an IS of about 65, cryptolepine 35a is
the most promising antitrypanosomal compound, which
will require further investigations.
In Vitr o An titr yp a n osom a l a n d An tip la sm od ia l
Activities of δ-Ca r bolin es. Table 9 summaries the
activities of δ-carboline derivatives against T. cruzi, P.
falciparum, and the murin L6 cells. Compound 6a was
found to be the most efficient of the δ-carbolines to
inhibit the dividing forms of T. cruzi (epimastigotes and
amastigotes) with IC50s of around 20 µM, which are
about 10 times lower than the IC50 measured on the L6
cells (around 230 µM). Compound 6a was however not
very effective against the trypomastigote, the infective
but nondividing form of T. cruzi (no effect for concentra-
tions of up to 100 µM). Modifications to increase the
hydrophilic character (hydroxylation at the 8 position,
compound 7a ) or the hydrophobic character (modifica-
tions at the 3 and/or 4 position, e.g. compounds 6d ,e,j)
of δ-carboline 6a , as well as methylation at the 1
position (compounds 7-8), did not result in an increase
of the IS compared to compound 6a . On P. falciparum,
compound 6a showed weak activity with an IS of 2.2.
Increase of hydrophilic or hydrophobic character of
derivatives generally decreased the IS values, except for
compound 6k (IS ) 9.8). In contrast, methylation at the
1 position (compounds 7-8) considerably increased the
activity against P. falciparum with IC50 values in the
low micromolar range (1.5-2.2 µM). As these com-

Benzo-δ-carbolines, Cryptolepines, and Their Salts
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6 953
Ta ble 5. Benzo-δ-carbolines
Sch em e 4a
starting material
R7
benzo-δ-carboline
yield (%)
23
31
32
33
34
25
H
Me
Et
iPr
Ph
Cl
1a
1b
1c
1d
1e
1f
83
75
74
80
85
55
Sch em e 6a
a
(a) MeOTf, toluene, 20 °C, 24 h; (b) 5% Na2CO3; (c) CHCl3,
basic alumina; (d) 1 M HCl/Et2O.
a
(a) cat. Pd(PPh3)4, 2 M K2CO3, EtOH, toluene, reflux under
In tr a cellu la r Loca liza tion of Cr yp tolep in e a n d
1-Meth yl-δ-ca r bolin e. Taking advantage of the fluo-
rescence of the indoloquinoline chromophore, fluores-
cence microscopy was used to localize cryptolepine 35a
inside the human fetal fibroblasts infected by T. cruzi
amastigotes and the red blood cells (RBC) infected by
P. falciparum. In T. cruzi-infected cells, cryptolepine
accumulated mainly in the intracellular amastigotes
(Figure 1). Cytoplasm of fibroblasts was not labeled, and
a weak fluorescence was associated with the host cell
nucleus. In amastigotes, cryptolepine strongly accumu-
lated into barlike structures whose staining corresponds
to a typical staining of kinetoplast DNA. Round struc-
tures were also stained that could indicate accumulation
into the parasite nuclei. For P. falciparum, cryptolepine
also accumulated into specific parasite structures that
could correspond to the parasite nuclei (Figure 2a,b).
No labeling of the cytoplasm of infected RBC or nonin-
fected RBC was observed. Previous studies have shown
the specific accumulation of cryptolepine into the nucleus
of the melanoma cells20 and the DNA-intercalating
properties of cryptolepine.46 These data associated with
the typical staining of the kinetoplast DNA strongly
suggest that cryptolepine acts through interactions with
parasite DNA. As proposed by Dassonneville et al.,46
stimulation of topoisomerase-mediated DNA cleavage
by the DNA-intercalating properties of cryptolepine may
then be its mechanism of action.
argon, 48 h; (b) 2.8 equiv n-BuLi, THF, -78 °C, 3 h; (c) electrophile,
THF, -78 °C, 2 h; (d) H2O; (e) 3.5 equiv KDA, THF, -50 °C, 45
min; (f) CH3I, THF, -78 °C, 1 h.
Ta ble 3. Cross-Coupling
R5 (boronic)
R4 (iodo)
R6 (product)
product
yield (%)
H (21)
OMe (22)
H
OMe
H
H (11)
H (11)
Cl (14)
Cl (14)
I (12)
H
H
Cl
Cl
23
24
25
26
27
28
94
83
58
58
79
54
R5 ) H
R5 ) OMe
OMe
I (12)
Ta ble 4. Metalation of 40
electrophile
E
product
yield (%)
D2O
I2
C2Cl6
MeI
EtI
D
I
Cl
Me
Et
29
30
25
31
32
95
94
80
85
78
Sch em e 5a
a
(a) Pyridinium chloride, 215 °C, 30 min; (b) NH3/H2O.
Fluorescence of 1-methyl-δ-carboline (7b), one of the
most active δ-carboline derivatives tested against P.
falciparum, also allowed to visualize its accumulation
inside the P. falciparum-infected RBC (Figure 2c,d).
Fluorescence was specifically localized in a parasite
pounds have weak cytotoxic activities against the L6
cells (IC50s > 200 µM), they can be considered as
potential antimalarial compounds (IS > 100) and re-
quired further pharmacological studies.

954 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6
Ta ble 6. Benzo-δ-carbolines, Cryptolepines, and Their Salts
Arzel et al.
R7
benzo-δ-carboline
triflate salt
cryptolepine
yield (%)a
chloride salt
H
Me
Et
iPr
Ph
1a
1b
1c
1d
1e
35a
35b
35c
35d
35e
2a
2b
2c
2d
2e
78
59
82
69
51
36a
36b
36c
36d
36e
a
Global yields (2 steps) for pure compounds.
Ta ble 7. KB Cytotoxicity of Benzo-δ-carbolines and
subcellular localization of cryptolepine in parasite DNA-
containing structures (nucleus, kinetoplast) strongly
suggests that compounds of the cryptolepine series act
on the parasite through interaction with DNA, by a
mechanism that could be similar to the one proposed
by Dassonneville et al.47 for cryptolepine on melanoma
cells: stimulation of topoisomerase-mediated DNA cleav-
age by the DNA-intercalating properties. Study of the
SAR of δ-carboline derivatives pointed to the importance
of methylation at N-1 for their antimalarial activity.
1-Methyl-δ-carboline specifically accumulates in intra-
cellular parasites and has weak cytotoxic activity. It can
be considered as a potentiel antimalarial compound that
warrants further investigation. This synthetic strategy
is currently being extended to the preparation of new
alkaloids in these structural families.
Cryptolepinesa
compd
IC50 (µM)
compd
IC50 (µM)
1a
1b
1c
1d
1e
2a
2b
2c
45 ( 10
40 ( 5
40 ( 10
57 ( 3
2d
2e
6.5 ( 0.5
0.75 ( 0.05
0.57 ( 0.35
6.4 ( 1.6
35b
35d
36a
36b
36c
36d
>100
1.75 ( 0.75
0.42 ( 0.08
1.5 ( 0.5
1.5 ( 0.5
0.53 ( 0.07
0.7 ( 0.1
9.25 ( 0.75
a
IC50 values are the means ( standard deviation of 3 indepen-
dent experiments.
structure that could correspond to the parasite nucleus.
However, whether 1-methyl-δ-carboline (7b) possesses
DNA-interacting properties still must be demonstrated.
No labeling of the cytoplasm of infected and noninfected
RBC was observed. Surprisingly, 1-methyl-δ-carboline
accumulation seems to be dependent on the parasite
development. Accumulation was restricted to the young
stage (ring and trophozoite stage); no labeling was
observed for the dividing stage (schizont stage) suggest-
ing either stage-specific targets or stage-specific perme-
ability.
Exp er im en ta l Section
1. Ch em istr y. Gen er a l Da ta . The 1H, 19F and 13C NMR
were obtained from a Brucker Advance 300 (300 MHz fre-
1
quency for H) spectrometer; J values are given in Hz. The IR
spectra were run on a Perkin-Elmer Paragon 500 FT-IR
spectrometer, main absorption frequencies (NH, CH, CdO, Cd
C, CdN) are given in cm-1. Mass spectra were obtained from
a J EOL D700 instrument. Elemental analyses were performed
on a CE instrument apparatus EA 1110 CHNS-O. Tetrahy-
drofuran was distilled from sodium/benzophenone. The water
content of the solvent was estimated to be lower than 25 ppm
by the Karl Fischer method.47 Commercial diisopropylamine
was distilled from calcium hydride under a dry nitrogen
atmosphere. Commercial 2.5 M solution of n-butyllithium in
hexane used and all reactions involving organometallic com-
pounds were carried out under a dry nitrogen atmosphere.
3-Fluoroquinoline (9) was prepared by the Roe and Hawkins27
method starting from 3-aminoquinoline. 3-Fluoro-4-iodoquino-
line (10), cryptomisrine (3) and compounds 6a -o were de-
scribed in previous papers22-23,25 Boronic acids 21-22 were also
described in a previous paper.48
Gen er a l P r oced u r e A: Meta la tion -Isom er iza tion of
3-F lu or o-4-iod oqu in olin e (10). n-Butyllithium in hexane
(10.0 mmol, 4.0 mL, 2.5 M) was added to diisopropylamine
(10.0 mmol, 1.4 mL) in THF (40.0 mL) at -78 °C. After 20
min, a solution of 3-fluoro-4-iodoquinoline (10; 10.0 mmol, 2.73
g) in 8.0 mL of THF was slowly added to the previous solution
(LDA). The resulting solution was stirred for 2 h at -78 °C
and the electrophile (10.0 mmol) in 10.0 mL of THF was slowly
added. Stirring was continued for 2 h at -78 °C before
hydrolysis at 0 °C by 10.0 mL of THF/H2O (9/1). Extraction
with ethyl acetate, drying with MgSO4, filtration and solvent
removal gave a crude product which was purified by flash
chromatography on silica gel.
Con clu sion
The link between metalation and cross-coupling pro-
vides a new convenient and general way to synthesize
benzo-δ-carbolines, as well as cryptolepine and its
substituted analogues. The overall strategy relies on
such key steps such as metalation, halogen-dance, and
hetero-ring cross-coupling. It is fully convergent and
regioselective. Substituted benzo-δ-carbolines 1a -f were
obtained in 4-6 steps from 3-fluoroquinoline (9) with
interesting 37-70% overall yields and cryptolepines
2a -e in 6-8 steps from the same fluorinated compound
in 27-58% overall yields. We also described a formal
synthesis of potential antimalarial compounds and the
first total synthesis of 11-isopropylcryptolepine (2d ),
isolated last year. Biological evaluation of benzo-δ-
carbolines showed that methylation at the N-5 position
(cryptolepine series) led to a large increase of their
cytotoxic activity toward mammalian cells, as well as
their antiplasmodial and antitrypanosomal activities.
Alkylation at the C-11 position in the cryptolepine series
generally increased the cytotoxic activity, 11-methyl-
cryptolepine being the most active among the com-
pounds tested. In contrast, except for 11-methylcryp-
tolepine, alkylation at C-11 did not increase the
antiplasmodial and antitrypanosomal activities. The
3-F lu or o-2-iod oqu in olin e (11). General procedure A, us-
ing water as the electrophile, gave 2.60 g (95%) of 11: mp 87
°C; 1H NMR (CDCl3) δ 8.10 (dd, 1H, H8, J ) 1.0, 8.0), 7.81
(dd, 1H, H5, J ) 1.5, 8.0), 7.75-7.58 (m, 3H, H4, H6 and H7);

Benzo-δ-carbolines, Cryptolepines, and Their Salts
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6 955
Ta ble 8. In Vitro Cytotoxicity and Antitrypanosomial and Antiplasmodial Activities of Benzo-δ-carbolines and Cryptolepinesa
L6 cells
T. cruzi
P. falciparum
compd
IC50 (µM)
epimastigotes IC50 (µM) amastigotes IC50 (µM) trypomastigotes (µM)b ISc K1 strain IC50 (µM)
ISd
1a
1b
1c
1f
2b
2c
2d
2e
48.6 ( 19.7 (3)
18.9 ( 9.1 (3)
21.5 ( 10.6 (3)
43.2 ( 11.5 (5)
3.7 ( 0.6 (3)
5.9 ( 3.8 (4)
23.8 ( 2.8 (3)
5.5 ( 3.1 (4)
15.2 ( 5.9 (3)
8.1 ( 2.3 (3)
1.4 ( 0.9 (3)
6.8 ( 3.2 (3)
15.1 ( 3.8 (3)
6.8 ( 0.4 (3)
8.7 ( 2.3 (3)
3.9 ( 0.4 (3)
2.8 ( 0.4 (3)
6.3 ( 1.2 (3)
ND
3.2 ( 0.6 (3)
ND
0.23 ( 0.03 (3)
>200
1.3 ( 0.2 (3)
0.30 ( 0.05 (3)
0.34 ( 0.17 (3)
0.73 ( 0.07 (3)
0.54 ( 0.32 (3)
14.2 ( 2.3 (4)
4.7 ( 1.7 (3)
5.7 ( 2.0 (5)
6.7 ( 2.4 (3)
ND
0.18 ( 0.13 (3)
ND
0.34 ( 0.13 (5)
5.7 ( 2.6 (3)
0.125 ( 0.008 (4)
0.074 ( 0.009 (4)
0.39 ( 0.09 (5)
3.3 ( 1.1 (6)
0.88 ( 0.17 (5)
50
25
6.25
50
ND
6.25
ND
1.5
100
50
12.5
50
12.5
12.5
3.4
4.0
3.8
6.4
ND
36.2 ( 3.2 (3)
74.6 ( 3.0 (3)
30.9 ( 7.7 (3)
74.6 ( 12.7 (4)
0.101 ( 0.03 (3)
1.1 ( 0.4 (6)
1.3
0.25
0.7
1.7
41.9
5.5
10.6
5.5
33
ND
16.6
2.7
64.8
18.9
17.4
4.6
2.2 ( 0.9 (6)
1.1 ( 0.3 (8)
6.4 ( 2.8 (3)
3
2.4
35a
35b
35c
35d
35e
0.33 ( 0.05 (5)
0.062 ( 0.023 (4)
0.48 ( 0.09 (6)
1.3 ( 0.4 (8)
24.5
22.5
14.1
12.6
6.2
7.7
1.1 ( 0.2 (8)
a
IC50 values are the means ( standard deviation of several independent experiments whose numbers are indicated in parentheses.
b
ND: not determined. Concentration of drug tested with no detectable effects by microscopy on the trypomastigote motility (mean of 2
independent experiments). c Index of selectivity (IS) defined by the ratio IC50 on L6 cells/IC50 on the intracellular amastigote proliferation.
d
Index of selectivity (IS) defined by the ratio IC50 on L6 cells/IC50 on P. falciparum.
Ta ble 9. In Vitro Cytotoxicity and Antitrypanosomial and Antiplasmodial Activities of δ-Carbolinesa
L6 cells
T. cruzi
P. falciparum
compd
IC50 (µM)
epimastigotes IC50 (µM) amastigotes IC50 (µM) trypomastigotes (µM)b ISc K1 strain IC50 (µM)
ISd
2.2
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
7a
7b
8
229 ( 97 (3)
82.4 ( 38.5 (3)
44.9 ( 5.6 (3)
14.3 ( 3.7 (3)
16.2 ( 8.5 (3)
75.1 ( 47.3 (3)
41.6 ( 7.5 (3)
1.8 ( 0.5 (3)
37.2 ( 7.8 (3)
14.3 ( 5.1 (3)
204 (2)
23.2 ( 4.8 (3)
6.6 ( 0.8 (3)
21.4 ( 4.3 (3)
11.1 ( 1.6 (3)
34.4 ( 9.1 (3)
58.4 ( 10.6 (3)
26.4 ( 5.4 (3)
11.2 ( 4.4 (3)
ND
23.5 ( 7.7 (4)
10.4 ( 4.4 (4)
12.2 ( 5.1 (3)
11.9 ( 7.4 (3)
18.9 ( 11.1 (3)
18.8 ( 8.9 (5)
6.4 ( 3.1 (3)
1.8 ( 0.2 (3)
8.6 ( 6.1 (5)
11.2 ( 3.1 (3)
20.8 ( 6.5 (4)
3.3 ( 1.4 (3)
8.4 ( 4.4 (3)
108.4 ( 56.8 (3)
1.9 ( 0.9 (5)
122 ( 54 (4)
199 ( 63 (3)
192 ( 91 (3)
258 ( 137 (3)
100
100
50
25
50
9.7
7.9
3.7
1.2
0.9
4
104 ( 17 (3)
45.6 ( 7.7 (3)
66.8 ( 17.8 (3)
38.5 ( 6.9 (3)
23.9 ( 8.1 (3)
33.1 ( 3.3 (3)
16.3 ( 3.7 (3)
14.8 ( 2.4 (3)
129 ( 16 (4)
29.3 ( 1.9 (3)
22.1 ( 4.1 (3)
14.2 ( 1.7 (3)
16.8 ( 2.4 (3)
259 ( 22 (3)
23.6 ( 1.9 (3)
56.5 ( 9.8 (3)
1.5 ( 0.6 (4)
1.6 ( 0.7 (4)
2.2 ( 0.6 (4)
1.8
0.7
0.4
0.7
2.3
2.6
0.12
0.3
0.5
9.2
1.6
3.2
25
100
200
200
50
6.5
1
4.3
1.3
9.8
6.9
6.4
>1.5
11.2
1.9
>1
>1
>1
10.4 ( 0.6 (3)
49.8 ( 5.3 (3)
18.6 ( 7.8 (3)
6.8 ( 1.6 (3)
>312 (3)
50
22.7 ( 9.5 (3)
54.0 ( 16.8 (4)
>156 (3)
100
200
100
>0.6
0.9
4.2
>100
>138
>100
21.3 ( 12.1 (3)
235 ( 37 (3)
>150 (5)
2.3 ( 0.8 (3)
129 ( 40 (3)
>300 (3)
6.25
200
100
200
200
>230 (5)
>220 (5)
428 ( 38 (3)
>550 (3)
a
IC50 values are the means ( standard deviation of several independent experiments whose numbers are indicated between brackets.
b
ND: not determined. Concentration of drug tested with no detectable effects by microscopy on the trypomastigote motility (mean of 2
independent experiments). c Index of selectivity (IS) defined by the ratio IC50 on L6 cells/IC50 on the intracellular amastigote proliferation.
d
Index of selectivity (IS) defined by the ratio IC50 on L6 cells/IC50 on P. falciparum.
(s, 1C, CHquino), 127.87 (s, 1C, Cquino), 127.10 (d, 1C, C4, J
) 5.0), 110.50 (d, 1C, C2, J ) 31.3); 19F NMR (CDCl3) δ
-105.14 (d, 1F, J ) 8.0); IR (KBr) 1594, 1491, 1396, 1330,
1204, 1170, 1023, 912, 774, 755, 711, 609. Anal. (C9H5FIN) C,
H, N.
4-Deu ter o-3-flu or o-2-iod oqu in olin e (12). General pro-
cedure A, using deuterium oxide as the electrophile, gave 95%
(NMR integration) of 12. This product was not isolated: 1H
NMR (CDCl3) δ 8.10 (dd, 1H, H8, J ) 1.0, 8.0), 7.81 (dd, 1H,
H5, J ) 1.5, 8.0), 7.75-7.58 (m, 2H, H6 and H7); 19F NMR
(CDCl3) δ -105.38 (s, 1F).
2,4-Diiod o-3-flu or oqu in olin e (13). General procedure A,
using iodide as the electrophile, gave 3.91 g (98%) of 13: mp
162 °C; 1H NMR (CDCl3) δ 8.07-7.96 (m, 2H, H5 and H8),
F igu r e 1. Intracellular localization of cryptolepine into T.
7.75-7.63 (m, 2H, H6 and H7); 13C NMR (CDCl3) δ 154.71 (d,
cruzi-infected human fetal fibroblasts. Cells infected by amastig-
1C, C3, J ) 254.0), 146.40 (d, 1C, C1a, J ) 2.7), 130.94 (d, 1C,
Cquino, J ) 5.3), 130.20 (s, 1C, CHquino), 129.85 (d, 1C,
CHquino, J ) 3.0), 129.43 (s, 1C, CHquino), 129.19 (d, 1C,
CHquino, J ) 1.2), 107.83 (d, 1C, C2, J ) 35.2), 93.70 (d, 1C,
C4, J ) 25.8); 19F NMR (CDCl3) δ -79.91 (s, 1F); IR (KBr)
1557, 1371, 1360, 1324, 1307, 1290, 1217, 1146, 756, 734, 641.
Anal. (C9H4FI2N) C, H, N.
ote stages were incubated for 1 h at 37 °C with 5 µM
cryptolepine 35a and were immediately observed after washes
by epifluorescence using UV filters. Nucleus (N) of fibroblast
is weakly labeled. Strong staining is associated with a parasite
barlike structure that corresponds to kinetoplast (k). Round
parasite structure is also stained that could correspond to the
parasite nucleus (n). Bar scale: 50 µm.
4-Ch lor o-3-flu or o-2-iod oqu in olin e (14). General proce-
dure A, using hexachloroethane as the electrophile, gave 2.28
g (74%) of 14: mp 91 °C; 1H NMR (CDCl3) δ 8.10-7.97 (m,
2H, H5 and H8), 7.93-7.59 (m, 2H, H6 and H7); 13C NMR
13C NMR (CDCl3) δ 154.5 (d, 1C, C3, J ) 258.3), 146.69 (s,
1C, C1a), 129.31 (d, 1C, CHquino, J ) 2.7), 128.79 (d, 1C,
CHquino, J ) 0.9), 128.11 (d, 1C, CHquino, J ) 0.8), 127.95

956 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6
Arzel et al.
3-F lu or o-2-iod o-4-qu in olin eca r boxa ld eh yd e (18). Gen-
eral procedure A, using ethyl formate as the electrophile, gave
2.85 g (95%) of 18: mp 106 °C; 1H NMR (CDCl3) δ 10.79 (s,
1H, CHO), 8.98 (comp, 1H, H5 or H8), 8.14 (comp, 1H, H8 or
H5), 7.77 (m, 2H, H6 and H7); 13C NMR (CDCl3) δ 188.15 (d,
1C, CHO, J ) 9.4), 157.20 (d, 1C, C3, J ) 271.4), 147.56 (d,
1C, C8a, J ) 3.9), 130.83 (s, 1Carom), 129.95 (d, 1Carom, J )
2.2), 129.32 (s, 1Carom), 125.14 (d, 1Carom, J ) 5.5), 123.14
(s, 1Carom), 120.28 (d, 1Carom, J ) 5.5), 110.70 (d, 1C, C2, J
) 32.5); 19F NMR (CDCl3) δ -111.95 (s, 1F); IR (KBr) 2925,
1693, 1542, 1494, 1396, 1315, 1181, 1071, 765, 706; MS
(electron impact) m/z 301 (M+, 100%). Anal. (C10H5FINO) C,
H, N.
3-F lu or o-4-iod o-2-tr im eth ylsilylqu in olin e (19). General
procedure A, using trimethylsilyl chloride as the electrophile
(simultaneously introduced with the starting material), gave
95% (1H NMR integration) of 19. This product was not isolated:
1H NMR (CDCl3) δ 8.12-8.03 (m, 2H, H5 and H8), 7.69-7.58
(m, 2H, H6 and H7), 0.61 (s, 9H, SiMe3).
General procedure A, using deuterium oxide as electrophile,
gave 94% (NMR) of 12, 2% (NMR) of 20 and 0.5% (NMR) of
13. These products were not isolated.
Gen er a l P r oced u r e D: Cycliza tion . Anhydrous pyri-
dinium chloride (ø g) at its boiling point (215 °C) was added
to the corresponding phenylquinoline (ø/5 mmol) and the
mixture was refluxed for 30 min. The resulting hot solution
was poured onto a mixture of ice (ø g) and concentrated
ammonia (ø mL). Extraction of the aqueous layer with ethyl
acetate, drying with magnesium sulfate, solvent removal and
flash chromatography on silica (light petroleum/ethyl ac-
etate: 7/3) gave a pure product.
F igu r e 2. Intracellular localization of cryptolepine 35a and
1-methyl-δ-carboline 7b into P. falciparum-infected erythro-
cytes. Cells were incubated for 1 h at 37 °C with 5 µM
cryptolepine (a, b) or 100 µM 1-methyl-δ-carboline (c, d) and
immediately observed after washes using an epifluorescence
microscope fitted with UV filters. (a, b) Schizont-infected
erythrocyte (arrow) labeled with cryptolepine and correspond-
ing phase contrast image, respectively. (c, d) Ring-infected
erythrocyte (arrow) labeled with 1-methyl-δ-carboline and
corresponding phase contrast image, respectively. Bar scale:
5 µm.
11-Meth yl-10H-in d olo[3,2-b]qu in olin e (1b). General pro-
cedure D (ø ) 5.0), using 33, gave 175 mg (75%) of 1b as a
1
white powder: mp >260 °C; H NMR (DMSO-d6) δ 11.47 (s,
1H, NH), 8.38 (d, 1H, H6, J ) 7.9), 8.23 (m, 2H, Harom), 7.71-
7.59 (m, 4H, Harom), 7.31 (t, 1H, Harom, J ) 7.5), 2.92 (s,
3H, Me); 13C NMR (DMSO-d6) δ 144.82 (s, 1C, Carom), 143.94
(s, 1C, Carom), 143.61 (s, 1C, Carom), 131.87 (s, 1C, Carom),
129.53 (s, 1C, CHarom), 129.33 (s, 1C, CHarom), 125.98 (s,
1C, Carom), 125.76 (s, 1C, CHarom), 124.60 (s, 1C, CHarom),
123.35 (s, 1C, CHarom), 121.47 (s, 1C, Carom), 121.40 (s, 1C,
CHarom), 121.25 (s, 1C, Carom), 119.25 (s, 1C, CHarom),
111.46 (s, 1C, CHarom), 12.43 (s, 1C, Me); IR (KBr) 3161, 3072,
1633, 1617, 1560, 1494, 1460, 1395, 1373, 1341, 1224, 1142,
744, 719; MS (electron impact) m/z 232 (M+, 100%), 204 (8%).
Anal. (C16H12N2‚0.25H2O) C, H, N.
(CDCl3) δ 150.42 (d, 1C, C3, J ) 257.9), 148.86 (s, 1C, C1a),
146.31 (d, 1C, C4, J ) 13.2), 130.00 (d, 1C, CHquino, J ) 2.5),
129.21 (s, 1C, CHquino), 128.81 (s, 1C, CHquino), 126.35 (s,
1C, Carom), 123.73 (d, 1C, CHarom, J ) 5.6), 109.12 (d, 1C,
C2, J ) 21.3); 19F NMR (CDCl3) δ -104.00 (s, 1F); IR (KBr)
1578, 1486, 1383, 1331, 1302, 1145, 942, 833, 764, 668. Anal.
(C9H4ClFIN) C, H, N.
3-F lu or o-2-iod o-4-m eth ylqu in olin e (15). General proce-
dure A, using iodomethane as the electrophile, gave 65% (NMR
integration) of 15. This product was not isolated.
3-F lu or o-4-(1-h yd r oxyeth yl)-2-iod oqu in olin e (16). Gen-
eral procedure A, using acetaldehyde as the electrophile, gave
2.51 g (79%) of 16: mp 109 °C; 1H NMR (CDCl3) δ 8.45 (comp,
1H, H8 or H5), 7.91 (comp, 1H, H5 or H8), 7.62-7.49 (m, 2H,
H6 and H7), 5.65 (q, 1H, CH, J ) 6.7), 4.17 (s, 1H, OH), 1.64
(s, 3H, Me, J ) 6.7); 13C NMR (CDCl3) δ 150.64 (d, 1C, C3, J
) 255.0), 147.04 (d, 1C, C1a, J ) 3.0), 132.83 (d, 1C, C4, J )
11.0), 129.25 (d, 1C, Cquino, J ) 1.1), 128.94 (d, 1C, CHquino,
J ) 2.6), 127.60 (s, 1C, CHquino), 125.79 (s, 1C, C4a, J ) 1.5),
125.08 (d, 1C, CHquino, J ) 5.6), 110.63 (d, 1C, C2, J ) 34.3),
11-Eth yl-10H-in d olo[3,2-b]qu in olin e (1c). General pro-
cedure D (ø ) 5.0), using 32, gave 182 mg (74%) of 1c as a
1
pale yellow solid: mp 238 °C; H NMR (DMSO-d6) δ 11.42 (s,
1H, NH), 8.33 (d, 1H, H6, J ) 8,0), 8.20-8.18 (m, 2H, Harom),
7.66-7.54 (m, 4H, Harom), 7.26 (ddd, 1H, H7, J ) 1.4, 6.2,
8.0), 3.39 (q, 2H, CH2 (Et), J ) 7.3), 1.34 (t, 3H, Me (Et), J )
7.3); 13C NMR (DMSO-d6) δ 144.97 (s, 1C, Carom), 143.94 (1C,
Carom), 143.85 (s, 1C, Carom), 130.93 (s, 1C, Carom), 129.58
(s, 1C, CHarom), 129.50 (s, 1C, CHarom), 127.31 (s, 1C,
Carom), 125.77 (s, 1C, CHarom), 124.84 (s, 1C, Carom), 124.77
(s, 1C, CHarom), 123.00 (s, 1C, CHarom), 121.42 (s, 1C,
Carom), 121.37 (s, 1C, CHarom), 119.27 (s, 1C, CHarom),
111.52 (s, 1C, CHarom), 19.65 (s, 1C, CH2 (Et)), 14.35 (s, 1C,
Me (Et)); IR (KBr) 3064, 2967, 2869, 1615, 1565, 1492, 1450,
1397, 1340, 1243, 1220, 1141, 748; MS (electron impact) m/z
246 (M+, 100%), 231 (91%). Anal. (C17H14N2) C, H, N.
64.00 (d, 1C, CH(OH), J ) 3.7), 23.12 (d, 1C, Me, J ) 1.2); 19
F
NMR (CDCl3) δ -108.57 (s, 1F); IR (KBr) 3439, 3333, 2984,
2928, 1579, 1550, 1392, 1365, 1326, 1165, 1037, 762, 684, 636,
579. Anal. (C11H9FINO) C, H, N.
3-Flu or o-4-(1-h ydr oxyben zyl)-2-iodoqu in olin e (17). Gen-
eral procedure A, using benzaldehyde as the electrophile, gave
2.85 g (75%) of 17: mp 138 °C; 1H NMR (CDCl3) δ 8.24 (comp,
1H, Hquino), 8.07 (comp, 1H, Hquino), 7.63 (comp, 1H,
Hquino), 7.54-7.30 (m, 6H, Harom), 6.75 (s, 1C, CH(OH)), 3.51
(s, 1H, OH); 13C NMR (CDCl3) δ 151.5 (d, 1C, C3, J ) 255.4),
147.41 (d, 1C, C1a, J ) 3.3), 140.74 (s, 1C, Carom), 131.10 (d,
1C, C4, J ) 11.1), 129.31 (s, 1C, CHarom), 129.30 (d, 1C,
Carom, J ) 2.2), 128.62 (s, 2C, CHph), 127.80 (d, 1C, CHarom,
J ) 3.9), 125.82 (d, 1C, CHarom, J ) 5.0), 125.80 (s, 1C,
CHarom), 125.54 (s, 1C, CHph), 125.52 (s, 2C, CHph), 110.42
(d, 1C, C2, J ) 34.3), 68.42 (d, 1C, CH(OH), J ) 3.3); 19F NMR
(CDCl3) δ -108.95 (s, 1F); IR (KBr) 3307, 1579, 1497, 1450,
1324, 1160, 1046, 768, 705, 658. Anal. (C16H11FINO) C, H, N.
11-Isop r op yl-10H-in d olo[3,2-b]qu in olin e (1d ). General
procedure D (ø ) 2.5), using 33, gave 129 mg (85%) of 33 as
1
an orange solid: mp 216 °C; H NMR (DMSO-d6) δ 11.04 (s,
1H, NH), 8.39 (d, 1H, H4, J ) 8,4), 8.31 (d, 1H, H6, J ) 7.7),
8.18 (d, 1H, H1, J ) 8,4), 7.65-7.53 (m, 4H, Harom), 7.25 (td,
1H, Harom, J ) 2.2, 7.7), 4.16 (ht, 1H, CH (iPr), J ) 7.1), 1.61
(d, 6H, Me (iPr), J ) 7.1); 13C NMR (DMSO-d6) δ 145.35 (s,
1C, Carom), 144.21 (s, 2C, Carom), 143.76 (s, 2C, Carom),
132.21 (s, 1C, Carom), 130.17 (s, 1C, CHarom), 129.38 (s, 1C,
CHarom), 126.34 (s, 1C, CHarom), 125.15 (s, 1C, CHarom),
124.70 (s, 1C, Carom), 123.80 (s, 1C, CHarom), 121.65 (s, 1C,
CHarom), 120.79 (s, 1C, Carom), 119.77 (s, 1C, CHarom),

Benzo-δ-carbolines, Cryptolepines, and Their Salts
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6 957
112.07 (s, 1C, CHarom), 27.17 (s, 1C, CH (iPr)), 27.05 (s, 2C,
Me (iPr)); IR (KBr) 3154, 3068, 2966, 1624, 1613, 1562, 1492,
1367, 1337, 1217, 769, 745; MS (chemical ionization) m/z 261
(MH+, 100%). Anal. (C18H16N2) C, H, N.
(s, 1C, Carom), 132.13 (s, 1C, CHarom), 126.84 (s, 1C,
CHarom), 126.09 (s, 1C, CHarom), 125.88 (s, 1C, CHarom),
125.18 (s, 1C, Carom), 121.34 (s, 1C, CHarom), 121.03 (q, 1C,
CF3, J ) 321,7), 118.12 (s, 1C, CHarom), 114,03 (s, 1C, Carom),
112.95 (s, 1C, CHarom), 39.85 (s, 1C, NMe), 14.26 (s, 1C, Me
(C11)); 19F NMR (DMSO-d6) δ -73.31 (s, 3F, CF3); IR (KBr)
3206, 1638, 1616, 1587, 1371, 1302, 1283, 1241, 1160, 1027,
755, 638. Anal. (C18H15F3N2O3S) C, H, N, S.
11-Eth yl-5N-m eth ylben zo-δ-ca r bolin iu m Tr ifla te (35c).
General procedure E, using 1c, gave 201 mg (98%) of 35c as
a yellow solid: mp 234 °C; 1H NMR (DMSO-d6) δ 12.86 (s, 1H,
NH), 8.79 (d, 1H, Harom, J ) 8.4), 8.76 (d, 1H, Harom, J )
7.2), 8.70 (d, 1H, H1, J ) 8.7), 8.18 (t, 1H, Harom, J ) 7.0),
8.00-7.91 (m, 2H, Harom), 7.85 (d, 1H, Harom, J ) 8.0), 7.51
(t, 1H, Harom, J ) 8.0), 4.99 (s, 3H, NMe), 3.68 (q, 2H, CH2
(Et), J ) 7.7), 1.42 (t, 3H, Me (Et), J ) 7.7); 13C NMR (DMSO-
d6) δ 145.35 (s, 1C, Carom), 140.82 (s, 1C, Carom), 137.00 (s,
1C, Carom), 135.61 (s, 1C, Carom), 133.71 (s, 1C, CHarom),
132.10 (s, 1C, Carom), 127.07 (s, 1C, Carom), 126.26 (s, 1C,
CHarom), 125.56 (s, 1C, CHarom), 125.56 (s, 1C, Carom),
124.29 (s, 1C, CHarom), 121.52 (q, 1C, CF3, J ) 321.5), 121.38
(s, 1C, CHarom), 118.51 (s, 1C, CHarom), 114.32 (s, 1C,
CHarom), 113.15 (s, 1C, CHarom), 60.12 (s, 1C, NMe), 21.01
(s, 1C, CH2 (Et)), 14.50 (s, 1C, Me (Et)); 19F NMR (DMSO-d6)
δ -73.11 (s, 3F, CF3); IR (KBr) 3161, 3111, 2975, 1634, 1506,
1294, 1237, 1216, 1143, 1029, 754, 630. Anal. (C19H17F3N2O3S)
C, H, N, S.
11-Isop r op yl-5N-m eth ylben zo-δ-ca r bolin iu m Tr ifla te
(35d ). General procedure E, using 1d , gave 98 mg (92%) of
35d as a yellow solid: mp 224 °C; 1H NMR (DMSO-d6) δ 12.42
(s, 1H, NH), 8.85 (d, 1H, H4, J ) 8.3), 8.76 (d, 1H, Harom, J
) 8.2), 8.75 (d, 1H, Harom, J ) 8.8), 8.16 (t, 1H, Harom, J )
7.5), 7.97-7.88 (m, 3H, Harom), 7.51 (t, 1H, Harom, J ) 7.3),
4.99 (s, 3H, NMe), 4.12 (ht, 1H, CH (iPr), J ) 7.2), 1.71 (d,
6H, Me (iPr), J ) 7.2); 13C NMR (DMSO-d6) δ 145.41 (s, 1C,
Carom), 144.18 (s, 1C, Carom), 137.83 (s, 1C, Carom), 135.80
(s, 1C, Carom), 133.86 (s, 1C, CHarom), 132.05 (s, 1C,
CHarom), 131.41 (s, 1C, CHarom), 126.97 (s, 1C, CHarom),
126.27 (s, 1C, CHarom), 126.02 (s, 1C, CHarom), 124.24 (s,
1C, Carom), 121.69 (q, 1C, CF3, J ) 321.5), 121.54 (s, 1C,
CHarom), 118.69 (s, 1C, CHarom), 114.00 (s, 1C, Carom),
113.36 (s, 1C, CHarom), 40.38 (s, 1C, NMe), 28.41 (s, 1C, CH
(iPr)), 20.75 (s, 2C, Me (iPr)); 19F NMR (DMSO-d6) δ -73.33
(s, 3F, CF3); IR (KBr) 3503, 3063, 2979, 2884, 1630, 1508, 1283,
1254, 1226, 1160, 1032, 752, 637. Anal. (C20H19F3N2O3S) C,
H, N, S.
5N-Meth yl-11-ph en ylben zo-δ-car bolin iu m Tr iflate (35e).
General procedure E, using 1e, gave 156 mg (68%) of 35e as
a yellow solid: mp 210 °C; 1H NMR (DMSO-d6) δ 12.31 (s, 1H,
NH), 8.86-8.83 (m, 2H, Harom), 8.19-7.53 (m, 11H, Harom),
5.09 (s, 3H, NMe); 13C NMR (DMSO-d6) δ 146.26 (s, 1C,
Carom), 138.03 (s, 1C, Carom), 136.74 (s, 1C, Carom), 135.84
(s, 1C, Carom), 133.97 (s, 1C, CHarom), 132.19 (s, 1C,
CHarom), 132.09 (s, 1C, Carom), 131.59 (s, 1C, Carom), 130.29
(s, 1C, CHarom), 130.11 (s, 2C, CHph), 129.57 (s, 2C, CHph),
127.44 (s, 1C, CHarom), 127.00 (s, 1C, CHarom), 126.34 (s,
1C, CHarom), 124.96 (s, 1C, Carom), 122.35 (q, 1C, CF3, J )
324.5), 121.64 (s, 1C, Carom), 118.33 (s, 1C, CHarom), 114.17
(s, 1C, Carom), 113.50 (s, 1C, CHarom), 40.41 (s, 1C, NMe);
19F NMR (DMSO-d6) δ -73.32 (s, 3F, CF3); IR (KBr) 3226,
3065, 1625, 1585, 1506, 1294, 1239, 1160, 1028, 761, 636. Anal.
(C23H17F3N2O3S) H, N, S; C: calcd, 60.26; found, 58.22.
11-P h en yl-10H-in d olo[3,2-b]qu in olin e (1e). General pro-
cedure D (ø ) 5.0), using 34, gave 235 mg (80%) of 34 as a
1
pale yellow solid: mp >260 °C; H NMR (DMSO-d6) δ 10.98
(s, 1H, NH), 8.36 (d, 1H, H6, J ) 7.7), 8.25 (dd, 1H, H1, J )
0.8, 8.4), 7.76 (dd, 1H, H4, J ) 0.7, 8.4), 7.72-7.62 (m, 6H,
Harom and H2), 7.59-7.48 (m, 2H, H8 and H3), 7.28 (ddd,
1H, H7, J ) 1.5/6.5/7.5); IR (KBr) 3050, 2958, 2872, 2756, 2670,
1613, 1566, 1489, 1472, 1400, 1382, 1338, 1220, 1141, 745, 697;
MS (electron impact) m/z 294 (M+, 100%), 147 (16%). Anal.
(C21H14N2) C, H, N.
11-Ch lor o-10H-in d olo[3,2-b]qu in olin e (1f). General pro-
cedure D (ø ) 2.5), using 25, gave 147 mg (55%) of 1f as a
1
pale green solid: mp 221 °C; H NMR (DMSO-d6) δ 11.83 (s,
1H, NH), 8.35 (d, 1H, H6, J ) 7.8), 8.31-8.25 (m, 2H, Harom),
7.80-7.60 (m, 4H, Harom), 7.34 (ddd, 1H, H7, J ) 1.8, 6.4,
7.1); 13C NMR (DMSO-d6) δ 146.40 (s, 1C, Carom), 144.49 (s,
1C, Carom), 144.17 (s, 1C, Carom), 130.73 (s, 1C, Carom),
130.45 (s, 1C, CHarom), 129.63 (s, 1C, Carom), 127.19 (s, 1C,
Carom), 126.72 (s, 1C, Carom), 123.96 (s, 1C, CHarom), 122.47
(s, 1C, CHarom), 122.06 (s, 1C, CHarom), 121.53 (s, 1C,
Carom), 120.58 (s, 1C, CHarom), 118.30 (s, 1C, CHarom),
112.38 (s, 1C, CHarom); IR (KBr) 3161, 3057, 1614, 1551, 1489,
1459, 1392, 1372, 1338, 1225, 1144, 1108, 1047, 885, 845, 820,
745, 713; MS (electron impact) m/z 252 (M+, 100%), 216 (15%),
190 (10%), 126 (15%). Anal. (C15H9ClN2) C, H, N.
Gen er a l P r oced u r e E: Tr ifla te Sa lts. To deoxygenated
and freshly distilled toluene (3 mL) were added 0.5 mmol of
the corresponding carboline and 0.95 mmol of methyl triflate.
The resulting solution was stirred for 24 h at room tempera-
ture, filtered and the salt washed with diethyl ether and dried.
1N-Meth yl-δ-ca r bolin iu m Tr ifla te (7a ). General proce-
dure E, using the δ-carboline, gave 84 mg (97%) of 7a as a
1
white solid: mp 230 °C; H NMR (DMSO-d6) δ 12.77 (s, 1H,
NH), 8.83 (d, 1H, H2, J ) 5.8), 8.64 (d, 1H, H4, J ) 8.4), 8.45
(d, 1H, H6, J ) 8.1), 7.97 (dd, 1H, H3, J ) 8.4, 5.8), 7.78-7.76
(m, 2H, H8 and H9), 7.45 (td, 1H, H7, J ) 1.9, 8.1), 4.78 (s,
3H, NMe); 13C NMR (DMSO-d6) δ 141.74 (s, 1C, Carom),
137.79 (s, 1C, Carom), 135.73 (s, 1C, CHarom), 131.04 (s, 1C,
Carom), 131.24 (s, 1C, CHarom), 126.49 (s, 1C, CHarom),
123.99 (s, 1C, CHarom), 121.64 (s, 1C, CHarom), 121.35 (s,
1C, CHarom), 120.74 (q, 1C, CF3, J ) 322.3), 114.33 (s, 1C,
Carom), 113.17 (s, 1C, CHarom), 46.12 (s, 1C, NMe); 19F NMR
(DMSO-d6) δ -73.29 (s, 3F, CF3); IR (KBr) 3185, 3102, 1638,
1462, 1254, 1224, 1162, 725, 63. Anal. (C13H11F3N2O3S) C, H,
N, S.
5N-Meth ylben zo-δ-ca r bolin iu m Tr ifla te (35a ). General
procedure E, using 1a , gave 162 mg (97%) of 35a as an orange
1
solid: mp >260 °C; H NMR (DMSO-d6) δ 10.01 (s, 1H, NH),
9.30 (s, 1H, H11), 8.84-8.75 (m, 2H, Harom), 8.58 (d, 1H, H6
or H9, J ) 7.6), 8.17 (t, 1H, Harom, J ) 8.0), 7.98-7.84 (m,
3H, Harom), 7.52 (t, 1H, Harom, J ) 7.5), 5.04 (s, 3H, NMe);
13C NMR (DMSO-d6) δ 142.21 (s, 1C, Carom), 139.48 (s, 1C,
Carom), 138.84 (s, 1C, Carom), 132.75 (s, 1C, Carom), 129.99
(s, 1C, CHarom), 127.16 (s, 1C, CHarom), 126.79 (s, 1C,
Carom), 125.36 (s, 1C, CHarom), 124.30 (s, 1C, CHarom),
123.34 (s, 1C, CHarom), 123.04 (s, 1C, CHarom), 121.31 (s,
1C, CHarom), 121.23 (q, 1C, CF3, J ) 322.3), 119.87 (s, 1C,
CHarom), 119.47 (s, 1C, Carom), 112.79 (s, 1C, CHarom), 51.05
(s, 1C, NMe); 19F NMR (DMSO-d6) δ -73.31 (s, 3F, CF3); IR
(KBr) 3175, 3119, 3036, 1508, 1273, 1223, 1155, 1031, 753,
745, 633. Anal. (C17H13F3N2O3S) C, H, N, S.
Gen er a l P r oced u r e F : Syn th esis of Cr yp tolep in es. To
an aqueous solution of sodium carbonate (5 mL, 5%) was added
0.25 mmol of the corresponding triflate. The resulting solution
was stirred for 15 min before adding 10 mL of distilled
chloroform. Extraction of the aqueous layer with chloroform,
drying with magnesium sulfate and solvent removal gave a
crude product which was purified by flash chromatography on
alumina (chloroform then chloroform/methanol: 9/1).
5N,11-Dim eth ylben zo-δ-car bolin iu m Tr iflate (35b). Gen-
eral procedure E, using 1b, gave 182 mg (92%) of 35b as a
yellow solid: mp >260 °C; 1H NMR (DMSO-d6) δ 12.84 (s, 1H,
NH), 8.79-8.74 (m, 2H, Harom), 8.65 (d, 1H, Harom, J ) 8.4),
8.20 (t, 1H, Harom, J ) 7.3), 8.00-7.91 (m, 2H, Harom), 7.84
(d, 1H, Harom, J ) 8.0), 7.51 (t, 1H, Harom, J ) 7.7), 5.00 (s,
3H, NMe), 3.23 (s, 3H, Me (C11)); 13C NMR (DMSO-d6) δ
145.17 (s, 1C, Carom), 136.39 (s, 1C, Carom), 135.76 (s, 1C,
Carom), 135.14 (s, 1C, Carom), 133.61 (s, 1C, CHarom), 132.67
1N-Meth yl-δ-ca r bolin e (8). General procedure F, using 7a ,
gave 30 mg (65%) of 8 as an orange solid: mp 202-206 °C
1
dec; H NMR (CDCl3) 8.37 (d, 1H, H2, J ) 8.3), 8.07 (d, 1H,
H6 ou H9, J ) 8.3), 7.91 (d, 1H, H9 or H6, J ) 8.7), 7.70 (d,

958 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6
Arzel et al.
1H, H4, J ) 5.6), 7.55 (td, 1H, H7 or H8, J ) 1.0, 7.9), 7.34
(dd, 1H, H3, J ) 5.6, 8.3), 7.14 (td, 1H, H8 or H7, J ) 1.0,
8.3), 4,58 (s, 3H, NMe); 13C NMR (CDCl3 - 100 MHz - Cr-
(acac)) δ 155.21 (s, 1C, Carom), 146.27 (s, 1C, CHaom), 129.06
(s, 1C, Carom), 128.94 (s, 1C, CHarom), 128.40 (s, 1C, Carom),
121.89 (s, 1C, CHarom), 119.52 (s, 1C, CHarom), 118.34 (s,
1C, CHarom), 116.30 (s, 1C, CHarom), 114.87 (s, 1C, CHarom),
45.49 (s, 1C, NMe); IR (KBr) 3340, 3080, 1624, 1589, 1475,
1434, 1382, 1333, 1315, 1272, 1258, 784, 748, 720; MS (electron
impact) m/z 182 (M+, 100%), 167 (10%). Anal. (C12H10N2) C,
H, N.
microdilution technique of Desjardins et al.52 Stock solutions
of compounds were prepared in DMSO. Drug solutions were
serially diluted with culture medium and added to asynchro-
nous parasite cultures (1% parasitemia and 1% final hemat-
ocrit) in 96-well plates for 24 h, at 37 °C, prior to the addition
of 0.5 µCi of [3H]hypoxanthine (1-5 Ci/mmol; Amersham, Les
Ulis, France) per well, for 24 h. The growth inhibition for each
drug concentration was determined by comparison of the
radioactivity incorporated in the treated culture with that in
the control culture (without drug) maintained on the same
plate. The concentration causing 50% inhibition of parasite
growth (IC50) was obtained from the drug concentration-
response curve, and the results were expressed as the mean
( standard deviation determined from several independent
experiments. The DMSO concentration never exceeded 0.2%
and did not inhibit the parasite growth.
Cyt ot oxicit y Test on Mu r in e Myob la st -Der ived L6
Cells. L6 cells were maintained in DMEM culture medium
supplemented with 25 mM HEPES, pH 7.5, 10% fetal calf
serum, 100 µg/mL penicillin and streptomycin, at 37 °C, under
an atmosphere of 5% CO2. For the cytotoxicity test, L6 cells
were collected and seeded in 96-well microplates at 5 × 103
cells/well. After 24 h, the cells were washed and 2-fold dilutions
of the drug were added in 200 µL culture medium. Untreated
cultures were included as controls. The final DMSO concentra-
tion in the culture remained below 0.5%. Cells were main-
tained for 5 days in culture and cell proliferation was deter-
mined using the XTT-based colorimetric assay (cell proliferation
kit II, Boehringer Mannheim S.A., Meylan, France). Growth
inhibition was determined from the percentage of reduction
of absorption at 540 nm of treated cultures versus untreated
control cultures. IC50 value was calculated from dose-response
curves from several independent experiments.
In Vitr o Activity Aga in st T. cr u zi. Experiments were
performed with the â-galactosidase-expressing parasite Tu-
lahuen LacZ clone 4 kindly provided by W. Van Voorhis.53
Epimastigote forms were grown in liver infusion tryptose
medium containing 10% fetal calf serum, at 28 °C.54 In vitro
activity against epimastigote forms was evaluated in a 96-well
plate (1 × 104 parasite/well) over 5 days in the presence of
2-fold dilutions of the drug. Trypomastigote forms were
obtained from infected culture of murine muscle L6 cells.55
Effects of 2-fold dilutions of the drug (from 200 to 1.5 µM) on
trypomastigotes were scored microscopically by evaluation of
their motility after 48 h of incubation. For the in vitro activity
against the intracellular amasitogte form, L6 cells were seeded
in a 96-well plate at 5 × 103 cells/well. After 24 h, 105
trypomastigotes were added/well for 6 h. Cells were washed
twice to remove extracellular trypomastigotes and incubated
with 2-fold dilutions of the drug, at 37 °C, under a 5% CO2
atmosphere for 5 days. In all these experiments, controls were
constituted by untreated parasite cultures. Growth inhibition
was quantified by measuring â-galactosidase activity using
chlorophenol red â-D-galactopyranoside as the substrate ac-
cording to Buckner et al.54 IC50 value was calculated from
dose-response curves from several independent experiments.
Cellu la r Loca liza tion by F lu or escen ce Micr oscop y.
Human fetal fibroblasts were maintained in 8-well Lab-Tek
culture slides (Nunc, Inc., Naperville, IL) in DMEM supple-
mented with 10% fetal calf serum. Five days after culture
infection by T. cruzi trypomastigotes, cells were washed twice
with culture medium and incubated for 1 h with the drug in
culture medium. After three washes, slides were immediately
observed. P. falciparum-infected red blood cells were incubated
for 1 h with the drug in culture medium, washed twice with
culture medium and immediately observed. Cellular localiza-
tion was realized using a Nikon Eclipse 600 inverted epifluo-
rescence microscope fitted with a UV filter.
5N,11-Dim eth ylin d olo[3,2-b]qu in olin e or 11-Meth yl-
cr yp tolep in e (2b). General procedure F, using 35b, gave 48
1
mg (64%) of 2b as a dark purple solid: mp 232 °C; H NMR
(CDCl3) δ 8.40 (dd, 1H, Harom, J ) 1.1, 8.4), 8.20 (d, 1H,
Harom, J ) 8.4), 8.10 (d, 1H, Harom, J ) 8.7), 7.91 (d, 1H,
Harom, J ) 8.4), 7.81 (ddd, 1H, Harom, J ) 1.5, 7.0, 8.4), 7.63
(td, 1H, Harom, J ) 1.1, 8.4), 7.53 (ddd, 1H, Harom, J ) 1.0,
6.6, 7.5), 4.76 (s, 3H, NMe), 3.27 (s, 3H, Me); IR (KBr) 3045,
1619, 1488, 1459, 1368, 1341, 1300, 1268, 1239, 1128, 741; MS
(chemical ionization) m/z 247 (MH+, 100%), 233 (55%). Anal.
(C17H14N2‚2.8H2O) C, H, N.
11-Eth yl-5N-m eth ylin d olo[3,2-b]qu in olin e or 11-Eth -
ylcr yp tolep in e (2c). General procedure F, using 35c, gave
48 mg (64%) of 2c as a purple solid: mp 200 °C; 1H NMR
(CDCl3) δ 8.33 (dd, 1H, H1 or H4, J ) 1.1, 8.4), 8.04 (dd, 1H,
H4 or H1, J ) 1.1, 8.4), 8.00 (d, 1H, H9 or H6, J ) 8.8), 7.85
(d, 1H, H6 or H9, J ) 8.7), 7.73 (ddd, 1H, Harom, J ) 1.4, 6.6,
7.8), 7.56 (ddd, 1H, Harom, J ) 1.1, 6.6, 7.7), 7.42 (ddd, 1H,
Harom, J ) 1.1, 6.6, 7.5), 6.89 (ddd, 1H, Harom, J ) 1.1, 6.6,
7.5), 4.61 (s, 3H, Me), 4.61 (q, 2H, CH2 (Et), J ) 7.7), 1.45 (t,
3H, Me (Et), J ) 7.7); 13C NMR (CDCl3 - 100 MHz - Cr(acac))
δ 159.92 (s, 1C, Carom), 143.93 (s, 1C, Carom), 142.90 (s, 1C,
CHarom), 137.65 (s, 1C, Carom), 133.22 (s, 1C, Carom), 130.38
(s, 1C, C arom), 128.47 (s, 1C, CHarom), 125.88 (s, 1C,
CHarom), 123.66 (s, 1C, CHarom), 123.36 (s, 1C, CHarom),
122.77 (s, 1C, Carom), 120,37 (s, 1C, CHarom), 117.11 (s, 1C,
CHarom), 115.57 (s, 1C, CHarom), 114.36 (s, 1C, CHarom),
38.16 (s, 1C, NMe), 21.41 (s, 1C, CH2 (Et)), 15.24 (s, 1C, Me
(Et)); IR (KBr) 3240, 2965, 1621, 1585, 1488, 1461, 1367, 1345,
1242, 759, 739; MS (FAB+) 261 (MH+, 85%). Anal. (C18H16N2‚
1.5H2O) C, H, N.
Gen er a l P r oced u r e G: Ch lor id e Sa lts of Cr yp tolep i-
n es. The corresponding cryptolepine (0.25 mmol) was treated
with a 1 M HCl solution in diethyl ether (5 mL). The resulting
yellow salt was filtered and dried at 50 °C. The yield is
quantitative (mp >260 °C).
2. Biologica l Eva lu a tion . Cytotoxicity Assa y. KB cells,
a mouth epidermoid carcinoma,49 were originally obtained from
the American type culture collection. The KB cells were serially
cultured in MEM (minimal essential medium with Earle’s salt
solution) containing 10% fetal calf serum, 2 mM L-glutamine,
60 µg/mL penicillin G and streptomycin sulfate and 40 µg/mL
gentamycin. For the assay, KB cells were grown as monolayers
in NUNC 24-well plastic plates (25 000 cells/well in 1 mL
medium). Serial dilutions of the stock solutions of the com-
pounds under test were made in DMSO (compounds 1a -e,
2a -e) or in water (salts 36a -d ) and added to the cultures
under a volume of 10 µL/well, immediately after plating the
cells. All cultures were incubated at 37 °C in a 95% air-5%
CO2 humidified incubator. After 3 days of incubation, cell
viability was determined by a further 8-16 h of incubation
following addition to each well of 100 µL of a 0.02% solution
in a medium of the vital dye neutral red, followed by washing
the cell monolayers with phosphate-buffred saline, lysis of the
cells with a 1% solution of sodium lauryl-dodecyl sulfate and
photometric quantification of the extracted dye at 540 nm,
using a Uniskan-II microplate reader, as originally described
by Borenfreund and Puerner.50
Ack n ow led gm en t. We are very grateful to Dr.
Georges Werner for his help and valuable discussions
concerning the biological part. This work was partly
supported by the VIHPAL program (French Minister
of Research). We also thank the useful comments of the
In Vitr o Activity Aga in st P . fa lcipa r u m . The chloro-
quine-resistant strain K1/Thailand of P. falciparum was
maintained in continuous culture on human erythrocytes as
described by Trager and J ensen.51 In vitro antimalarial activity
was determined using a modification of the semiautomated

Benzo-δ-carbolines, Cryptolepines, and Their Salts
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6 959
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.
referee and Wendy Theaker (University of East Anglia,
Norwich, U.K.) for assistance with the English lan-
guage.
(19) Grellier, P.; Ramiaramanana, L.; Millerioux, V.; Deharo, E.;
Schre´vel, J .; Frappier, F.; Trigalo, F.; Bodo, B.; Pousset, J .-L.
Antimalarial activity of Cryptolepine and Isocryptolepine, Al-
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