Synthesis and evaluation of cryptolepine analogues for their potential as new antimalarial agents

Article abstract of DOI:10.1021/jm010929+

The indoloquinoline alkaloid cryptolepine 1 has potent in vitro antiplasmodial activity, but it is also a DNA intercalator with cytotoxic properties. We have shown that the antiplasmodial mechanism of 1 is likely to be due, at least in part, to a chloroqu

Full text of DOI:10.1021/jm010929+

J . Med. Chem. 2001, 44, 3187-3194
3187
Syn th esis a n d Eva lu a tion of Cr yp tolep in e An a logu es for Th eir P oten tia l a s New
An tim a la r ia l Agen ts
Colin W. Wright,*,† J onathan Addae-Kyereme,†,‡ Anthony G. Breen,§ J ohn E. Brown,† Marlene F. Cox,†,|
Simon L. Croft, Yaman Go¨kc¸ek,† Howard Kendrick, Roger M. Phillips,§ and Pamela L. Pollet†
The School of Pharmacy and Cancer Research Unit, University of Bradford, West Yorkshire, BD7 1DP, U.K., and the
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street,
London WC1E 7HT, U.K.
Received May 16, 2001
The indoloquinoline alkaloid cryptolepine 1 has potent in vitro antiplasmodial activity, but it
is also a DNA intercalator with cytotoxic properties. We have shown that the antiplasmodial
mechanism of 1 is likely to be due, at least in part, to a chloroquine-like action that does not
depend on intercalation into DNA. A number of substituted analogues of 1 have been prepared
that have potent activities against both chloroquine-sensitive and chloroquine-resistant strains
of Plasmodium falciparum and also have in common with chloroquine the inhibition of
â-hematin formation in a cell-free system. Several compounds also displayed activity against
Plasmodium berghei in mice, the most potent being 2,7-dibromocryptolepine 8, which suppressed
parasitemia by 89% as compared to untreated infected controls at a dose of 12.5 mg kg-1 day-1
ip. No correlation was observed between in vitro cytotoxicity and the effect of compounds on
the melting point of DNA (∆Tm value) or toxicity in the mouse-malaria model.
In tr od u ction
identical to hemozoin5) from hemin in a cell-free sys-
tem.6 Peaks at 1660 and 1210 cm-1 in the FTIR
spectrum of the product confirm the presence of â-he-
matin (see below). To assess cryptolepine analogues for
their potential as leads to selective antimalarial agents,
we have determined their antiplasmodial activities
against chloroquine-resistant and chloroquine-sensitive
strains of Plasmodium falciparum. Those compounds
displaying potent in vitro antiplasmodial activities have
been assessed for in vivo antimalarial activity against
Plasmodium berghei in mice. Selected compounds have
been tested for their abilities to inhibit â-hematin
formation. Thermodenaturation techniques have been
used to examine the effects of compounds on the melting
point of calf thymus DNA as an indication of their
potential to interact with DNA, and preliminary cyto-
toxicity tests have been carried out.
Cryptolepine (5-methyl,10H-indolo[3,2-b]quinoline; 1)
is an indoloquinoline alkaloid found in the west African
climbing shrub Cryptolepis sanguinolenta (family Periplo-
caceae). A decoction of the roots of this species is used
in traditional medicine for the treatment of malaria as
well as for a number of other diseases.1 Previous work
has shown that cryptolepine has potent in vitro anti-
plasmodial activity,2 but this alkaloid also has cytotoxic
properties that are likely due to its abilities to inter-
calate into DNA and inhibit topoisomerase II as well
as DNA synthesis.3
In this paper, we report the synthesis of 15 crypto-
lepine derivatives (of which 11 are novel) to determine
whether it is possible to prepare cryptolepine analogues
that have reduced abilities to interact with DNA (and
hence may be less cytotoxic) but that retain potent
antiplasmodial activities. This hypothesis is supported
by evidence (presented below) which suggests that the
antiplasmodial activity of cryptolepine is due, at least
in part, to a chloroquine-like action that does not depend
on intercalation into DNA. Chloroquine and related
antimalarials appear to act primarily by inhibiting the
formation of hemozoin (malaria pigment), which is
formed in malaria parasites from hemin, the toxic
residue remaining following the digestion of hemoglobin
by the parasite.4 Drugs that have a chloroquine-like
mode of action may be detected by testing their ability
to prevent the formation of â-hematin (shown to be
Ch em istr y
Cryptolepine (1) was isolated from C. sanguinolenta
as described previously2 or prepared by methylation of
quindoline, which was synthesized using methodology
based on that of Holt and Petrow (1947)7 (Scheme 1).
Isatin (2a) was condensed with O,N-acetylindoxyl (3a )
in the presence of KOH under oxygen-free conditions
to give quindoline-11-carboxylic acid (4a ). The latter was
decarboxylated by heating in diphenyl ether to yield
quindoline (5a ), which was then methylated using
iodomethane in tetramethylenesulfone8 (this method
was found to be much more efficient than methylation
with dimethylsulfate or methyl triflate). The 2-bromo
* Corresponding author tel: +44 (0)1274 234739; fax: +44 (0)1274
235600; e-mail: c.w.wright@bradford.ac.uk.
† The School of Pharmacy, University of Bradford.
‡ Present address: Faculty of Pharmacy, University of Science and
Technology, Kumasi, Ghana.
§ Cancer Research Unit, University of Bradford.
| Present address: Department of Chemistry, University of Guyana,
P.O. Box 101110, Georgetown, Guyana.
London School of Hygiene and Tropical Medicine.
10.1021/jm010929+ CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/18/2001

3188 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Wright et al.
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (i) HNO3:HOAc (1:1), room temper-
ature, overnight; (ii) HNO3:HOAc (1:1), reflux; (iii) Sn/HCl; (iv)
Ac2O, reflux.
Sch em e 3a
a
Reagents and conditions: (i) KOH, N2, 10 days; (ii) Ph2O, 250
°C; (iii) CH3I, tetramethylene sulfone, 50 °C; (iv) SOCl2, reflux;
(v) HN4OH.
analogue 6 was prepared similarly using 5-bromoisatin
(2b) and O,N-acetylindoxyl (3a ) as starting materials
while the reaction of isatin (2a ) with 5-bromo-O,N-
acetylindoxyl (3b) provided a route to 7-bromocrypto-
lepine (7). Similarly, 5-bromoisatin (2b) and 5-bromo-
O,N-acetylindoxyl (3b) were used as starting materials
for the synthesis of 2,7-dibromocryptolepine (8). Quin-
doline-11-amide (9) was synthesized by reacting 4a with
thionyl chloride to give the acid chloride, followed by
ammoniolysis as previously described7 (Scheme 1).
Nitration was used as a convenient method of prepar-
ing derivatives directly from 1 (Scheme 2). At room
temperature, nitration of 1 with a mixture of nitric and
glacial acetic acids (1:1) yielded 7-nitrocryptolepine (10)
as the major product together with a smaller amount
of the 9-nitro- isomer 11, which were then separated
by column chromatography over silica gel eluted with
chloroform/methanol. Refluxing the above reaction mix-
ture afforded 7,9-dinitrocryptolepine (12) as the sole
product. Two meta-coupled doublets in the 1H NMR
spectrum of 12 (δ 9.13 and 9.64, J ) 2.2 Hz) together
with the downfield shift of the singlet due to H-11 (δ
9.55) initially suggested that 12 was the 1,3-dinitro
analogue of 1, but X-ray crystallographic analysis of 12
(data not shown)9 proved unequivocally that 12 is the
7,9-dinitro analogue of 1. Reduction of 7-nitrocrypto-
lepine (10) (Sn/HCl) gave the reactive and light-sensi-
tive 7-amino analogue that was isolated and immedi-
ately acetylated to give 7-N-acetylcryptolepine 13
(Scheme 2).
a
Reagents and conditions: (i) bromoacetyl bromide, room
temperature; (ii) reflux, 30 h; (iii) polyphosphoric acid, 130 °C, 2
h; (iv) POCl3, 120 °C, 2 h; (v) CH3I, tetramethylene sulfone, 50
°C, overnight.
The 11-chloro derivative 20 was prepared as previ-
ously described by Bierer et al. (1998),10 and the same
route (Scheme 3) was used to synthesize the novel
dihalogenated derivatives 21-24. Reaction of an anthra-
nilic acid derivative 14 with bromoacetylbromide gave
15, which was treated with an aniline derivative 16. The
resulting anthranilic acid derivative 17 was then cy-
clized using polyphosphoric acid to yield quindolone 18;
treatment with phosphorus oxychloride gave 11-chloro-
quindoline derivative 19, which was then methylated
with iodomethane in tetramethylenesulfone to give the
corresponding cryptolepine analogues, 20-24.
Resu lts a n d Discu ssion
The activities of compounds against P. falciparum in
vitro and against P. berghei in vivo and their effects on

Cryptolepine Analogues as Antimalarial Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3189
Ta ble 1. In Vitro Antiplasmodial and In Vivo Antimalarial Activities of Cryptolepine Derivatives and Chloroquine Diphosphate
against Malaria Parasites and Effects on Formation of â-Hematin
activity vs
activity against
P. berghei (ANKA) in mice
P. falciparum in vitrob
chloroquine-
resistant
strain K1
chloroquine-
sensitive
strain HB3
inhibition of
â-hematin
formation
mean
parasitemia
(% ( SD)
suppression
of parasitemia
(%)
dose schedule
compda
1 (as sulfate)
4a
5a
(mg kg-1 for 4 d)
0.44 ( 0.22(9)
>100
0.27 ( 0.06(3)
NT
NT
yes
no
no
yes
yes
yes
no
yes
NDf
yes
ND
ND
ND
ND
NT
ND
yes
20c
NT
NT
25
NTd
NT
NT
e
NT
NT
5.9
>100
6
0.26 ( 0.094(4)
0.26 ( 0.21(4)
0.049 ( 0.017
>100
0.65 ( 0.28(6)
6.92 ( 1.89(4)
0.65 ( 0.21(6)
0.52 ( 0.21(5)
0.24 ( 0.1(3)
4.75 ( 0.37(3)
7.18 ( 3.5(4)
7.62 ( 2.7(4)
27.0 ( 5.4(3)
0.18 ( 0.025(7)
0.45 ( 0.17(3)
0.19 ( 0.09(3)
0.026 ( 0.005(3)
NT
0.14 ( 0.05(3)
4.14 ( 2.29(3)
0.45 ( 0.22(3)
0.47 ( 0.16(3)
1.67 ( 1.27(3)
NT
21.6 ( 1.9
11.9 ( 2.0
1.4 ( 0.4
NT
6.0 ( 1.2
11.9 ( 2.0
20.3 ( 2.0
13.3 ( 2.1
NT
NT
NT
NT
NT
7 (as hydroiodide)
8
9
10
11
12
13
20
21 (as hydroiodide)
22
23
24
20
41.5
89.1
NT
61.4
23.5
-30.2
14.7
g
NT
NT
NT
NT
93.8
12.5
NT
20
20
20
20
20
NT
NT
NT
NT
10
NT
NT
NT
chloroquine diphosphate
0.023 ( 0.0015(3)
1.3 ( 0.5
a
c
Tested as hydrochloride salt unless stated otherwise. b IC50 µM ( SD(n). n, number of separate determinations. Tested as hydrochloride.
NT, not tested. e Toxic after second dose. f ND, could not be determined as compound has peaks in the IR spectrum close to 1660 cm-1
Toxic after first dose.
.
d
g
Ta ble 2. Cytotoxicity and Effect on DNA Melting Point of Cryptolepine Derivatives
IC50 (µM ( SD) (n ) 3)a
cytotoxicity against
A549 cells
cytotoxicity against
DLD-1 cells
cytotoxicity against
MAC15a cells
effect on DNA
melting point,
∆Tm (°C)
drug
exposure
time 1 h
drug
exposure
time 96 h
drug
exposure
time 1 h
drug
exposure
time 96 h
drug
exposure
time 1 h
drug
exposure
time 96 h
compd
1 (as sulfate)
5a
6
7 (as hydroiodide)
8
10
11
12
13
20
22
24
9
0
4
4
3
5
4
4
6
0
9
9
61.4 ( 18.2
NTb
>100
NT
0.55 ( 0.051
93 ( 29
NT
>100
NT
1.44 ( 0.0015
67.2 ( 26.3
NT
>100
66.2 ( 8.02
NT
9.65 ( 1.37
NT
NT
NT
2.07 ( 0.21
NT
NT
NT
NT
NT
5.04 ( 1.05
NT
2.29 ( 0.67
NT
NT
NT
NT
NT
20.41 ( 1.1
NT
5.79 ( 0.91
NT
6.04 ( 0.49
NT
NT
NT
15.47 ( 4.2
NT
NT
NT
NT
NT
35.6 ( 2.2
90.2 ( 7.8
>100
>100
NT
48.9 ( 7.6
>100
>100
>100
NT
35.4 ( 4.8
>100
28.5 ( 9.3
>100
30.5 ( 6.2
23.1 ( 2.7
14.4 ( 2.8
NT
NT
NT
NT
NT
NT
NT
NT
a
b
n, number of separate determinations. NT, not tested.
â-hematin formation are shown in Table 1; ∆Tm values
(increase in melting point of DNA) and cytotoxic activi-
ties are reported in Table 2.
greater than that of 1. However, in contrast, the
2-bromo,11-chloro derivative (21) was found to be about
20-fold less active than the 2-bromo (6) and 11-chloro
(20) derivatives. Similarly, the other dihalogenated
analogues that were 11-chloro-substituted (22-24) have
little antiplasmodial activity.
In Vitr o An tip la sm od ia l Activity. Cryptolepine (1)
was found to have potent in vitro activity (IC50 ) 0.44
µM) against P. falciparum (multidrug resistant strain
K1) similar to that published in previous reports.2,11
However, quindoline (5a ), quindoline-11-carboxylic acid
(4a ), and its amide 9 were inactive against malaria
parasites (strain K1), indicating that the 5-methyl group
in 1 is a prerequisite for antiplasmodial activity. The
7-nitro (10), 7,9-dinitro (12), and 7-N-acetyl (13) deriva-
tives were of similar potency to 1 against P. falciparum
while 9-nitrocryptolepine (11) was 10-fold less active.
Monohalogenated derivatives 6, 7, and 20 were slightly
more potent than 1 against P. falciparum (strain K1).
Compound 8 (2,7-dibromocryptolepine) was prepared
because both the 2-bromo (6) and the 7-bromo (7)
analogues showed improved antiplasmodial activities as
compared with 1. This strategy was successful as the
antiplasmodial activity of 8 was found to be 9-fold
Compounds active against P. falciparum (strain K1)
were also tested against chloroquine-sensitive strain
HB3 to determine whether they exhibit cross-resistance
with chloroquine. As shown in Table 1, chloroquine was
8-fold less active against chloroquine-resistant strain K1
than against strain HB3. The antiplasmodial activities
of seven of the nine compounds tested were similar
against both parasite strains (less than a 2-fold varia-
tion). One compound, 7-nitrocryptolepine (10), was
4-fold less active against strain HB3 while the 11-choro
analogue 20 was 7-fold more active against chloroquine-
resistant strain K1. These results suggest that crypto-
lepine and its derivatives with the possible exception
of 10 do not show cross-resistance with chloroquine,

3190 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Wright et al.
possible that its effects on DNA synthesis and inhibition
of topoisomerase II may also contribute. This is sup-
ported by a recent study using fluorescence microscopy,
which suggests that 1 accumulates into parasite struc-
tures that may correspond to the parasite nucleus.12 In
contrast, 4, 5, and 9, which are inactive against malaria
parasites, failed to inhibit â-hematin formation (Table
1). Several of the derivatives possessing potent anti-
plasmodial activities (6-8, 10, and 12) like 1 inhibited
â-hematin formation, indicating that this property may
be important for their action against malaria parasites.
It was not possible to determine the effects of 11, 13,
and 20 on â-hematin formation because their IR spectra
have peaks close to 1660 cm-1, which may mask the
peak of â-hematin that occurs at this wavelength.
Cytotoxicity a n d Effect on ∆Tm Va lu e. As 1 is
known to be toxic to cancer cells,3 initial tests for
cytotoxicity were carried out by exposing cancer cells
(A549, DLD-1, and MAC15A) to compounds for 1 h
followed by a 96-h incubation period. The results sug-
gested that three compounds 6, 11, and 13 may be less
toxic than 1 against all three cell lines (Table 2).
However, as the IC50 values for 1 are close to 100 µM,
the highest concentration tested, further experiments
using a drug-cell contact time of 96 h were carried out
to determine the extent by which 6 and 13 are less toxic
than 1; the latter were selected as they possess good
antiplasmodial activities. Against A549 and DLD-1 cells,
6 was found to be about 4- and 2-fold less toxic,
respectively, than 1, but it was about 2-fold more toxic
than 1 against MAC15A cells. The best selectivity was
seen with 13 against DLD-1 cells (about 14-fold less
toxic) and against A549 cells (about 9-fold less toxic),
but little difference was observed with MAC15A cells
(about 1.6-fold less toxic). However, there appears to be
little correlation between the in vitro cytoxicity of these
compounds and toxicity in the mouse-malaria model.
While 10 and 20 were about twice as toxic to MAC15A
cells than 1, in the antimalarial test only 20 was toxic
to mice. Compound 8 was slightly more cytotoxic to
cancer cells than 1, but no apparent toxicity was seen
in mice in striking contrast to 1, which was substan-
tially toxic to mice after two doses, although it must be
F igu r e 1. Inhibition of â-hematin formation by 1. (A) FTIR
spectrum of control reaction product showing peaks at 1663
and 1210 cm-1 characteristic of â-hematin. (B) As above in the
presence of 1, demonstrating inhibition of â-hematin forma-
tion.
although, as discussed below, they share the ability to
inhibit â-hematin formation.
In Vivo An t im a la r ia l Act ivit y. Cryptolepine (1)
was toxic to P. berghei-infected mice by ip injection at
a dose of 12.5 mg kg-1 day-1 (Table 1). Toxicity appears
to be related to the route of administration as no deaths
were reported in previous studies in which subcutane-
ous (at 113 mg-1 kg-1 day)11 or oral (50 mg kg-1 day-1 2
)
routes were used. In the former study, no effect on
parasitemia was observed, but oral administration of 1
at 50 mg kg-1 day-1 for 4 days suppressed parasitemia
by 80.5%2.
In this study, 7, 8, 10, and 11 suppressed parasitemia
by more than 20%. Although 11 displayed only weak in
vitro antiplasmodial activity it was found to have
greater in vivo activity (23% suppression of parasitemia)
than compounds 6, 12, and 13, which all have potent
in vitro antiplasmodial activities. Interestingly, mice
treated with 12 were found to have a mean parasitemia
30% above that of untreated control animals, while in
common with (1), the 11-chloro analogue 20 was toxic
to mice. Compounds 7 and 10 possess moderate in vivo
antimalarial activities (42 and 61%, respectively, at 20
mg kg-1 day-1), but the most potent compound, 2,7-
dibromocryptolepine (8), was found to suppress para-
sitemia by 89% at a daily dose of 12.5 mg kg-1. The in
vivo activity of 8 is comparable to that of chloroquine,
and further studies to determine the oral effectiveness
this compound and its ability to cure malaria in mice
are currently being carried out.
noted that the daily dose of 1 given to mice (20 mg kg-1
was greater than that of 8 (12.5 mg kg-1).
)
The effects of compounds on the melting point of calf
thymus DNA (∆Tm values) were measured in an at-
tempt to obtain an indication of their propensities to
interact with DNA. The value of 9 °C found for 1 is
consistent with its known DNA-intercalating ability3.
With the exceptions of 22 and 24, the ∆Tm values of the
other compounds tested were all 6 °C or less (Table 2),
which may indicate a reduced tendency of the latter to
interact with DNA as compared to 1. However, no
correlation was observed between ∆Tm value and cyto-
toxicity or toxicity in the mouse-malaria model; this is
well illustrated with reference to 1 and 20, which were
both toxic to mice but were found to have ∆Tm values
of 9 and 0 °C, respectively.
Con clu sion
Effect on â-Hem a tin F or m a tion . Cryptolepine (1)
prevented the formation of â-hematin (Figure 1), which
suggests that its antiplasmodial effect depends (at least
in part) on a quinine-like mode of action, although it is
A number of analogues of 1 have been synthesized
that have improved antiplasmodial activities as com-
pared to the parent against both chloroquine-sensitive

Cryptolepine Analogues as Antimalarial Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3191
and tetramethylenesulfone as described below for the prepara-
tion of 7.) 1H NMR of free base (CD3OD, δ): 5.11 (3H, s, NCH3),
7.62 (1H, t, J ) 8.0 Hz, 7 or 8-H), 7.94 (1H, d, J ) 8.8 Hz,
6-H), 8.05 (1H, t, J ) 8 Hz, 7 or 8-H), 8.24 (1H, dd, J ) 9.5,
2.2 Hz, 3-H), 8.62 (1H, d, J ) 9.5 Hz, 4-H), 8.72 (1H, d, J )
2.2 Hz, 1-H), 8.79 (1H, d, J ) 8.4 Hz, 9-H), 9.26 (1H, s, 11-H).
and chloroquine-resistant strains of P. falciparum. With
the possible exception of 10, there was no evidence of
cross-resistance with chloroquine even though these
compounds (like the parent) appear to have a chloro-
quine-like mode of action against malaria parasites.
Several compounds were found to have some activity
against P. berghei in mice, the most potent being 2,7-
dibromocryptolepine (8), which suppressed parasitemia
by 89% at a dose of 12.5 mg kg-1 day-1 with no apparent
toxicity to the mice. There appears to be no correlation
between the in vitro cytotoxicity and the effect of
compounds on the melting point of DNA or toxicity in
the mouse-malaria model.
This study has shown that some derivatives of 1,
particularly 8, are promising leads in the search for new
antimalarial agents. In addition, analogues of 1, which
have enhanced cytotoxicity (such as 24, which was 4-5-
fold more active than 1 against MAC15A cells), may be
worthy of investigation as antitumor agents. This is
supported by recent studies on the cytotoxic properties
of compounds related to 1.13-15
MS (EI, m/z, relative intensity, %) 312 (97), 310 (100) [M+
+
H], 297 (7), 295 (7), 230 (18), 154 (18), 156 (18), 115 (24). Anal.
(C16H11N2Br‚HCl‚H2O) H, N; C: calcd, 52.5; found 52.0.
7-Br om ocr yp tolep in e Hyd r oiod id e (7). 7-Bromoquindo-
line (5b) (0.06 g, 0.18 mmol), tetramethylenesulfone (2 mL),
and iodomethane (0.06 g, 23.4 mmol) were stirred overnight
at 50 °C in a sealed container. After the solution was cooled,
ether (8 mL) and a few drops of methanol were added, and
the resulting precipitate was washed twice with ethyl acetate
(2 × 5 mL) and crystallized from chloroform:methanol (3:1) to
give 7 as orange-brown needles; overall yield 38%. 1H NMR of
free base (CD3OD, δ): 4.70 (3H, s, NCH3), 7.54 (1H, dd, J )
9.2, 1.8 Hz, 8-H), 7.64 (1H, t, J ) 7.3 Hz, 2 or 3-H), 7.71 (1H,
d, J ) 9.2 Hz, 9-H), 7.86 (1H, t, J ) 7.9 Hz, 2 or 3-H), 8.11
(1H, d, J ) 8.8 Hz, 1 or 4-H), 8.20 (1H, d, J ) 8.1 Hz, 1 or
4-H), 8.30 (1H, d. J ) 1.8 Hz, 6-H), 8.80 (1H, s, 11-H). MS (EI,
m/z, relative intensity, %) 312 (100), 310 (100) [M+ + H], 296
(26), 294 (24), 216 (18), 154 (15), 115 (13), 89 (28). Anal.
(C16H11N2Br‚HI) C, H, N.
2,7-Dibr om ocr yp tolep in e Hyd r och lor id e (8). The title
compound was prepared from 5-bromo-O,N-acetylindoxyl (3b)-
(6.84 g, 23 mmol) and 5-bromoisatin (2b) (5.73 g, 25.3 mmol)
as starting materials using the same methodology as for 6
except that the reaction mixture for the first step was refluxed
for 4 h instead of stirring at room temperature. Recrystalli-
zation from chloroform:acetone (3:1) gave 8 as an orange-
Exp er im en ta l Section
Ch em istr y. Chemicals were purchased from Sigma-Aldrich
Chemical Co. Ltd., Poole, U.K. 1H NMR spectra were acquired
on a J EOL GX270 FT NMR spectrometer at 270 MHz. Mass
spectra were run on an AEI MS902 spectrometer equipped
with an MSS data acquisition system, version 10 (Mass
Spectrometer Services, Manchester, U.K.). C, H, and
N
1
yellow solid; overall yield 23%. H NMR of free base (CDCl3,
analyses were carried out by the Chemical and Materials
Analysis Unit, University of Newcastle, U.K., on a Carlo Erba
1106 elemental analyzer. FTIR spectra were recorded on a
Mattson Galaxy 6020 FTIR spectrometer.
δ): 4.72 (3H, s, NCH3), 7.53 (1H, dd, J ) 9.2, 1.8 Hz, 3-H or
8-H), 7.66 (1H, d, J ) 9.2 Hz, 4-H or 9-H), 7.91 (1H, dd, J )
9.2, 2.2 Hz, 3-H or 8-H), 8.0 (1H, d, J ) 9.2 Hz, 4-H or 9-H),
8.19 (1H, d, J ) 1.8 Hz, 1-H or 6-H), 8.27 (1H, d, J ) 2.2 Hz,
1-H or 6-H), 8.74 (1H, s, 11-H). MS (EI, m/z, relative intensity,
%) 392 (46), 390 (92), 388 (38) [M+], 378 (42), 376 (100), 374
(42), 297 (14), 295 (14), 216 (16), 215 (18). Anal. (C16H10N2-
Br2‚1.5HCl) C, H, N.
7-Nitr o- (10) a n d 9-Nitr ocr yp tolep in e Hyd r och lor id e
(11). Cryptolepine (1) as sulfate (1 g, 3 mmol) was dissolved
in 40 mL of nitric acid (69%):glacial acetic acid (1:1) and stirred
at room temperature for 24-48 h. The reaction mixture was
cooled on ice, basified with strong NaOH solution, and filtered,
and the precipitate was washed with ice cold water. The dried
product was column-chromatographed over silica gel under
positive pressure eluted with chloroform containing increasing
amounts of methanol (1-20%) to yield fractions containing 10
(less polar) and 11 (more polar). The total yield of nitrated
products was 56%. These were converted to their hydrochloride
salts with concentrated HCl and crystallized from choroform:
methanol (3:1).
Gen er a l Meth od for P r ep a r a tion of 2-Br om oqu in d o-
lin e (5b) a n d 7-Br om oqu in d olin e (5c). O,N-Acetylindoxyl
(3a ) (5 g, 23.0 mmol) for 5b or 5-bromo-O,N-acetylindoxyl (3b)-
(6.84 g, 23.0 mmol) for 5c and water (50 mL) were stirred
under nitrogen at room temperature. A solution of 5-bromo-
isatin (2b) (5.73 g, 25.3 mmol) for 5b or isatin (2a ) (4.41 g,
25.3 mmol) for 5c and KOH (26 g 0.46 M) in water (50 mL)
was slowly added. Stirring was continued for 10 days at room
temperature and then additional water (20 mL) was added,
and the mixture was heated to 70 °C while air was bubbled
through it for 20 min. The mixture was filtered through Celite,
which was then washed with hot water (60 °C), and an equal
volume of ethanol was added to the filtrate followed by
acidification to pH 1 with concentrated HCl. The product
(bromoquindoline-11-carboxylic acid) 4b or 4c was collected,
washed with 1:1 ethanol:water, and dried. Decarboxylation
was carried out by refluxing the derivative (7.3 g, 21.3 mmol)
and diphenyl ether (50 mL) for 6 h with stirring. After the
mixture was cooled, petroleum ether (55 mL) was added, and
the precipitate was collected and dried, dissolved in methanol
(300 mL), and filtered. Concentration of the filtrate gave the
brominated quindoline derivatives 5b and 5c that were used
without further purification.
2-Br om ocr yp tolep in e Hyd r och lor id e (6). 2-Bromoquin-
doline (5b) (0.116 g, 0.34 mmol), dimethylsulfate (0.265 g, 3.4
mmol), and chloroform (20 mL) were stirred under reflux for
10 days; the course of the reaction was monitored using TLC
over silica gel G with chloroform:methanol:concentrated am-
monia (9:1:0.1). Following evaporation of the solvent, NaOH
solution (10%, 20 mL) was added to the residue, and the
mixture was extracted with chloroform, washed with saturated
NaCl solution, and dried over anhydrous Na2SO4. The dried
product was column-chromatographed over silica gel eluted
with chloroform:methanol:ammonia (9:1:0.1), converted to the
hydrochloride salt with concentrated HCl, and crystallized
from chloroform:methanol (3:1) to give 6 as a yellow-brown
solid; overall yield 13%. (Note: This yield may be increased
significantly if methylation is carried out using iodomethane
10. Yellow solid, yield 45%. 1H NMR of free base (CDCl3,
δ): 4.99 (3H, s, NCH3), 7.78 (1H, t, J ) 7.2 Hz, 7-H), 7.86 (1H,
d, J ) 9.5 Hz, 9-H), 8.01 (1H, t, J ) 7.3 Hz, 8-H), 8.28 (1H, d,
J ) 9.5 Hz, 1-H), 8.33 (1H, d, J ) 8.4 Hz, 6-H), 8.44 (1H, d, J
) 9.5 Hz, 2-H), 9.04 (1H, s, 11-H), 9.38 (1H, s, 4-H). MS (EI,
m/z, relative intensity. %) 277 (16) [M+], 263 (100), 247 (32),
232 (36), 217 (59), 190 (19), 111 (20). Anal. (C16H11N3‚HCl) C,
H, N.
1
11. Yellow solid, yield 11%. HMR of base (270 MHz, CD3-
OD, δ): 5.1 (3H, s, NCH3), 7.54 (1H, t, J ) 8.8 Hz, 7-H), 7.93
(1H, t, J ) 7.7 Hz, 8-H), 8.19 (1H, t, J ) 7.5 Hz, 3-H), 8.48
(1H, d, J ) 8.4 Hz, 6-H), 8.67 (1H, d, J ) 9.2 Hz, 9-H), 8.75
(1H, d, J ) 8.1 Hz, 4-H), 9.11 (1H, d, J ) 8.1 Hz, 2-H), 9.21
(1H, s, 11-H). MS (EI, m/z, relative intensity, %) 277 (9) [M+],
263 (26), 232 (100), 217 (44), 190 (18), 111 (8). Anal. (C16H11N3‚
HCl) C, H, N.
7,9-Din itr ocr yp tolep in e Hyd r och lor id e (12). Crypto-
lepine (1) as sulfate (0.5 g, 1.5 mmol) was refluxed for 30 min
in 30 mL of nitric acid (69%):glacial acetic acid (1:1). The
product was isolated and purified as described above for 10

3192 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Wright et al.
and 11. Recrystallization from chloroform:methanol (3:1) gave
12 as orange-red crystals; yield 69%. 1H NMR of free base
(DMSO-d6, δ): 4.71 (3H, s, NCH3), 7.99 (1H, t, J ) 8.1 Hz,
7-H), 8.69 (1H, t, J ) 7.4 Hz, 8-H), 8.72 (1H, d, J ) 7.3 Hz,
9-H), 8.43 (1H, d, J ) 7.3 Hz, 6-H), 9.13 (1H, d, J ) 2.2 Hz,
4-H), 9.55 (1H, s, 11-H), 9.64 (1H, d, J ) 2.2 Hz, 2-H). MS (EI,
m/z, relative intensity, %) 322 (38) [M+], 308 (100), 292 (14),
262 (35), 230 (22), 216 (56), 188 (10). Anal. (C16H10N4O4‚HCl)
C, H, N.
mL), washed with saturated NaCl solution and water, and
then dried (Na2SO4). Evaporation of the solvent gave the crude
product 19, which was chromatographed over silica gel twice;
the eluent was ethyl acetate:hexane (1:6) followed by chloro-
form:methanol (9.5:0.5).
Step 5. Finally, methylation of 19 was carried out as
described above as in the preparation of 7. Compounds 22-
24 were converted to their hydrochloride salts by the addition
of dilute NH4OH (10%) and extraction with chloroform followed
by the addition of concentrated HCl. The hydrochloride salts
were recrystallized from chloroform:methanol (3:1).
2-Br om o-11-ch lor ocr yp tolep in e Hyd r oiod id e (21). Pre-
pared as above using 5-bromoanthranilic acid (20.1 g, 96.6
mmol) and aniline (26.1 g, 0.28 M) as starting materials. Crude
yields were 70%, 38%, 51%, 70%, and 74% for steps 1-5,
respectively; overall yield of 21 obtained as a yellow-brown
solid, 7%. 1H NMR of free base (DMSO-d6, δ): 4.96 (3H, s,
NCH3), 7.55 (1H, t, J ) 8.0 Hz, 7-H), 7.90 (1H, d, J ) 8.4 Hz,
6-H), 7.99 (1H, t, J ) 7.0 Hz, 8-H), 8.31 (1H, dd, J ) 9.5, 2.2
Hz, 3-H), 8.62 (1H, d, J ) 2.2 Hz, 1-H), 8.76 (1H, d, J ) 9.52,
4-H), 8.79 (1H, d, J ) 8.43, 8-H). MS (EI, m/z, relative
intensity, %) 346 (100), 344 (76) [M+], 332 (78), 330 (60), 250
(10), 215 (36), 173 (12). Anal. (C16H10N2BrCl‚HI) C, H, N.
7-N-Acetylcr yp tolep in e Hyd r och lor id e (13). Compound
10 (0.443 g, 1.6 mmol) was dissolved in 30 mL of methanol:
concentrated HCl (95:5) in the presence of granulated tin (0.2
g, 1.7 mmol) in a nitrogen atmosphere protected from light
and was stirred for 3 h. The reaction mixture was basified with
saturated NaOH solution, filtered, and extracted with chloro-
form. The concentrated chloroform extract was column-chro-
matographed over alumina gradient eluted with chloroform
followed by chloroform containing increasing amounts of
methanol (1-10%) to yield a blue fraction containing 7-amino-
cryptolepine, which is photosensitive. Acetic anhydride (3 mL)
was added to the fraction, which was then refluxed for 15 min,
cooled, basified with saturated NaOH, and extracted with
chloroform:methanol (3:1). Concentrated HCl was added to the
extract, which was then dried to give 13. Crystallization from
chloroform:methanol (3:1) gave 13 as red-brown needles; yield
51%. 1H NMR of hydrochloride (DMSO-d6, δ): 2.15 (3H, s,
CONCH3), 5.01 (3H, s, NCH3), 7.84 (1H, d, J ) 9.2 Hz, 1-H),
7.95 (1H, t, J ) 8.0 Hz, 7-H), 8.04 (1H, d, J ) 8.8 Hz, 6-H),
8.18 (1H, t, J ) 7.9 Hz, 8-H), 8.58 (1H, d, J ) 8.4 Hz, 9-H),
8.73 (1H, d, J ) 9.2 Hz, 2-H), 9.15 (1H, s, 4 or 11-H), 9.30
(1H, s, 4 or 11-H), 10.5 (1H, s, 3-NH), 13.0 (1H, s, 10-NH). MS
(EI, m/z, relative intensity, %) 289 (6) [M+], 246 (8), 232 (3),
159 (2), 149 (4), 60 (32), 44 (100). Anal. (C18H15N3O.HCl‚CHCl3)
C, H, N.
8-Br om o-11-ch lor ocr yp tolep in e Hyd r och lor id e (22).
Prepared from anthranilic acid (13.2 g, 96.6 mmol) and
3-bromoaniline (48.4 g 0.28 M). Crude yields, 74%, 75%, 30%,
66%, and 50% for steps 1-5, respectively; overall yield of 22
1
obtained as a yellow solid, 6%. H NMR of free base (DMSO-
d6, δ): 4.60 (3H, s, NCH3), 6.94 (1H, dd, J ) 8.8, 1.8 Hz, 1 or
4-H), 7.70 (1H, t, J ) 7.3 Hz, 2 or 3-H), 7.73 (1H, d, J ) 1.1
Hz, 9-H), 7.78 (1H, d, J ) 9.2 Hz, 1, 4 or 6-H), 7.91 (1H, t, J
) 7.3 Hz, 2 or 3-H), 8.10 (1H, d, J ) 9.2 Hz, 1, 4 or 6-H), 8.45,
(1H, dd, J ) 9.0, 1.1 Hz, 7-H). MS (EI, m/z, relative intensity,
%) 346 (2), 344 (2) [M+], 328 (98), 326 (100) [M - CH3], 313
(35), 311 (37), 285 (8), 283 (8), 204 (7), 164 (11), 163 (11), 150
(12), 149 (12). Anal. (C16H10N2BrCl‚HCl) C, H, N.
7,11-Dich lor ocr yp tolep in e Hyd r och lor id e (23). Pre-
pared from anthranilic acid (13.2 g, 96.6 mmol) and 4-chloro-
aniline (36 g, 0.28 M). Crude yields, 74%, 43%, 48%, 88%, and
47% for steps 1-5, respectively; overall yield of 23 obtained
as a yellow solid, 6%. 1H NMR of free base (DMSO-d6, δ): 5.08
(3H, s, NCH3), 7.87 (1H, d, J ) 8.9 Hz, 9-H), 7.99 (1H, dd, J )
8.9, 1.8 Hz, 8-H), 8.09 (1H, t, J ) 7.7 Hz, 2 or 3H), 8.29 (1H,
t, J ) 8.1 Hz, 2 or 3-H), 8.75 (1H, d, J ) 9.5 Hz, 1 or 4-H),
8.82 (1H d, J ) 10.1 Hz, 1 or 4-H), 8.82 (1H, d, J ) 1.7 Hz,
6-H). MS (EI, m/z) 302, 300, [M+], 285, 287, 250, 215, 149, 125.
Anal. (C16H10N2Cl2‚HCl‚H2O) N; C: calcd, 54.2; found, 52.9;
H: calcd, 3.66; found, 3.21.
Gen er a l Meth od for P r ep a r a tion of 11-Ch lor o-Su bsti-
tu ted Der iva tives 20-24. Step 1. Anthranilic acid or a
substituted anthranilic acid derivative, 14 (96.6 mmol), di-
methylformamide (35 mL), and dioxane (35 mL) were placed
in
a sealed flask, which was cooled to 0 °C, and then
bromoacetylbromide (19.5 g, 96.6 mmol) was slowly added so
that the temperature did not rise above 1 °C. At the end of
the addition, the temperature was maintained at 0 °C for a
further 10 min, and then the mixture was stirred overnight
at room temperature. The contents of the flask were poured
into water (300 mL), and the resulting precipitate 15 was
filtered, washed with neutral water (3 × 15 mL), and then
dried.
Step 2. Aniline or a substituted aniline derivative, 16 (0.28
M), and the crude acid 15 (90 mmol) were stirred and heated
under reflux at 120 °C for 30 h. After the cooling process, the
reaction mixture was poured onto ice and water (800 mL), and
sufficient KOH solution (5%) was added to dissolve the
precipitate. The pH was checked, and if necessary, more KOH
solution was added to raise the pH to 11. The mixture was
extracted with dichloromethane, and the aqueous phase was
then acidified to pH 3 with hydrobromic acid solution (5%).
The product 17 was collected in the form of a precipitate or as
an oil that solidified on standing overnight at room temper-
ature.
Step 3. The crude product from above 17 (15.8 mmol) and
polyphosphoric acid (150 g) were stirred at 130 °C for 2 h, and
then the mixture was poured into ice/water (1000 mL) and
neutralized with saturated KOH solution. The mixture was
then extracted with ethyl acetate (3 × 250 mL), washed with
saturated NaCl solution and water, and dried (Na2SO4).
Following evaporation of the solvent, the dried product 18 was
column-chromatographed over silica gel eluted with ethyl
acetate:methanol (5:1).
Step 4. The latter product 18 (23.8 mmol) and phosphorus
oxychloride (60 mL) were stirred under reflux at 120 °C for 2
h. After the reaction mixture was cooled, it was poured onto
ice/water (150 mL) and then neutralized with saturated KOH
solution, taking care to prevent the temperature rising above
40 °C. The product was extracted into ethyl acetate (3 × 200
8,11-Dich lor ocr yp tolep in e Hyd r och lor id e (24). Pre-
pared from anthranilic acid (13.2 g, 96.6 mmol) and 3-chlo-
roaniline (36 g, 0.28 M). Crude yields, 74%, 69%, 32%, 41%,
and 48% for steps 1-5, respectively; overall yield of 24, 3%
1
obtained as a yellow solid. H NMR of free base (CD3OD, δ):
5.08 (3H, s, NCH3 ), 7.87 (1H, d, J ) 9.0 Hz, 6-H), 7.99 (1H,
dd, J ) 8.9, 1.8 Hz, 7-H), 8.09 (1H, t, J ) 7.7 Hz, 2 or 3-H),
8.29 (1H, t, J ) 7.4 Hz, 2 or 3-H), 8.75 (1H, d, J ) 9.5 Hz, 1 or
4-H), 8.79 (1H, d, J ) 10.1 Hz, 1 or 4-H), 8.82 (1H, d, J ) 1.7,
9-H). MS (EI, m/z, relative intensity, %) 302 (64), 300 (100)
[M+], 286 (15), 149 (46). Anal. (C16H10N2Cl2‚HCl‚H2O) C, H,
N.
An tip la sm od ia l Assa y. P. falciparum strain K1 was kindly
supplied by Professor D. C. Warhurst (London School of
Hygiene and Tropical Medicine) and P. falciparum strain HB3
was generously provided by Dr L. C. Ranford-Cartwright
(Division of Infection and Immunity, University of Glasgow).
Malaria parasites were maintained in human A+ erythrocytes
suspended in RPMI 1640 medium supplemented with A+
serum and D-glucose according to the methods of Trager and
J ensen (1976)16 and Fairlamb et al. (1985).17 Cultures contain-
ing predominantly early ring stages were used for testing.
Compounds were dissolved or micronized in DMSO and further
diluted with RPMI 1640 medium (the final DMSO concentra-
tion did not exceed 0.5% which did not affect parasite growth).
Twofold serial dilutions were made in 96-well microtiter plates

Cryptolepine Analogues as Antimalarial Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3193
in duplicate, and infected erythrocytes were added to give a
final volume of 100 µL with 2.5% hematocrit and 1% para-
sitemia. Chloroquine diphosphate was used as a positive
control, and uninfected and infected erythrocytes without
compounds were included in each test. Plates were placed into
a modular incubator gassed with 93% nitrogen, 3% oxygen,
and 4% carbon dioxide and incubated at 37 °C for 48 h.
Parasite growth was assessed by measuring lactate dehydro-
genase activity as described by Makler et al. (1993).18 The
reagent used contained the following in each milliliter: acetyl-
pyridine adenine dinucleotide (APAD), 0.74 mg; lithium
lactate, 19.2 mg; diaphorase, 0.1 mg; Triton X-100, 2 µL;
nitroblue tetrazolium, 1 mg; and phenazine ethosulfate, 0.5
mg. Fifty microliters of this reagent was added to each well
and mixed, and plates were incubated for 15 min at 37 °C.
Optical densities were read at 550 nm using a Dynatech
Laboratories MRX microplate reader, and percent inhibition
of growth was calculated by comparison with control values.
IC50 values were determined using linear regression analysis
(Microsoft Excel). A minimum of three separate determinations
was carried out for each compound.
In Vivo An tim a la r ia l Test. This was carried out using
Peters’ 4-day suppressive test19 against P. berghei infection
in mice. Female BALB/C mice, weight 18-20 g, were inocu-
lated with P. berghei (ANKA); each mouse received 1 × 107
infected erythrocytes by iv injection. Drugs were administered
to mice by ip injection in 0.2 mL of inoculum daily for four
consecutive days. Control and test groups all contained 5 mice.
On day 5 of the test a blood smear was taken, and the animals
were killed. The percent suppression of parasitemia was
calculated for each dose level by comparing the parasitemias
present in infected controls with those of test animals. Chloro-
quine diphosphate was used as a positive control.
tion of Animal Cell Cultures (ECACC) and MAC15A (murine
adenocarcinoma of the colon) available in our laboratory. All
cells were routinely maintained as monolayer cultures in RPMI
1640 culture medium supplemented with foetal calf serum
(10%), sodium pyruvate (1 mM), L-glutamine (2 mM), and
penicillin/streptomycin (50 IU mL-1/50 µg mL-1 and buffered
with HEPES (25 mM). Chemosensitivity was assessed using
the MTT assay.21 Briefly, 2 × 103 cells were inoculated into
each well of a 96-well plate and incubated overnight at 37 °C
in an humidified atmosphere containing 5% CO2. All drugs
were dissolved in DMSO and diluted in culture medium to give
a broad range of drug concentrations; the maximum DMSO
concentration in any well was 0.1%. Medium was removed
from each well and replaced with drug solutions (8 wells per
drug concentration). For experiments with a 1-h drug expo-
sure, medium was removed after 1 h, and the cells were
washed twice with Hanks balanced salt solution. RPMI 1640
medium was added (200 µL/well) and cells were incubated at
37 °C for a further 4 days before cell survival was determined
using the MTT assay. Culture medium was replaced with fresh
medium (180 µL) prior to the addition of 20 µL of MTT solution
(0.5 mg mL-1). Following 24-h incubation at 37 °C, medium
plus MTT was removed from each well, and the formazan
crystals were dissolved in DMSO (150 µL/well). Absorbances
of the resulting solutions were read at 550 nm, and cell
survival was calculated as the absorbance of treated cells
divided by the absorbance of the control (RPMI medium plus
0.1% DMSO) wells. Results were expressed in terms of IC50
values (i.e., concentration of drug required to kill 50% of cells),
and all experiments were performed in triplicate.
Ack n ow led gm en t. This work was supported by
WHO TDR Grant 980375. J .A.-K. was supported by the
Ghanaian government. A.G.B. and R.M.P. were funded
by the Cancer Research Campaign Grant SP252301.
M.F.C. is grateful to the Association of Commonwealth
Universities for a scholarship. H.K. and S.L.C. received
financial support from the UNDP/World Bank/WHO
Programme for Research on Tropical Diseases (TDR).
In h ibition of â-Hem a tin F or m a tion . The methodology
used was adapted from that of Egan et al. (1994).6 Hemin (7.5
mg) was dissolved in 1.25 mL of 0.1 M NaOH, and then 0.125
mL of 1 M HCl was added. The 3 mol equiv of the compound
under test (with respect to hemin) was then added followed
by 1.15 mL of 9.78 M acetate buffer at pH 5.0, preincubated
at 60 °C. The pH was adjusted to 5.0 by the addition of glacial
acetic acid, and the mixture was then stirred for 30 min at 60
°C. After being cooled on ice, the precipitate was filtered using
a 5-µm membrane filter and washed with water. After being
dried under vaccuum for 24 h, the FTIR spectrum of the
product was recorded. A control in which no compound was
added was also carried out. The formation of â-hematin was
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