Synthesis and antimalarial evaluation of novel isocryptolepine derivatives

Article abstract of DOI:10.1016/j.bmc.2011.10.037

A series of mono- and di-substituted analogues of isocryptolepine have been synthesized and evaluated for in vitro antimalarial activity against chloroquine sensitive (3D7) and resistant (W2mef) Plasmodium falciparum and for cytotoxicity (3T3 cells). Di-h

Full text of DOI:10.1016/j.bmc.2011.10.037

Bioorganic & Medicinal Chemistry 19 (2011) 7519–7525
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antimalarial evaluation of novel isocryptolepine derivatives
Louise R. Whittell a,b, Kevin T. Batty a,b, Rina P.M. Wong c, Erin M. Bolitho a,b, Simon A. Fox a,b
,
Timothy M.E. Davis c, Paul E. Murray a,b,
⇑
a School of Pharmacy, Curtin University, Bentley, GPO Box U1987, Perth, Western Australia 6845, Australia
b Curtin Health Innovation Research Institute, Curtin University, Bentley, Western Australia, Australia
c School of Medicine and Pharmacology, University of Western Australia, Crawley, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of mono- and di-substituted analogues of isocryptolepine have been synthesized and evaluated
for in vitro antimalarial activity against chloroquine sensitive (3D7) and resistant (W2mef) Plasmodium
falciparum and for cytotoxicity (3T3 cells). Di-halogenated compounds were the most potent derivatives
and 8-bromo-2-chloroisocryptolepine displayed the highest selectivity index (106; the ratio of
cytotoxicity (IC50 = 9005 nM) to antimalarial activity (IC50 = 85 nM)). Our evaluation of novel isocryptole-
pine compounds has demonstrated that di-halogenated derivatives are promising antimalarial lead
compounds.
Received 2 August 2011
Revised 5 October 2011
Available online 20 October 2011
Keywords:
Isocryptolepine
Cryptolepis sanguinolenta
Antimalarial
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
derivatives have demonstrated improved bioactivity, with 2,7-dib-
romocryptolepine the most promising antimalarial candidate
The development of new antimalarial therapies represents an
important contribution to global health and natural products are
(IC50 = 49 nM; K1 strain P. falciparum).18
Of the other alkaloids isolated from Cryptolepis sanguinolenta,
neocryptolepine (5-methyl-5H-indolo[2,3-b]quinoline) (2) and iso-
cryptolepine (5-methyl-11H-indolo[3,2-c]quinoline) (3a) were the
only ones found to possess significant antimalarial activity, albeit
less potent than 1.19 Several synthetic and biological investigations
of substituted neocryptolepine derivatives have been undertaken
but no compound has proved sufficiently bioactive for therapeutic
use.20–22 Many of its derivatives, however, possess increased anti-
malarial activity and lower cytotoxicity in comparison to the par-
ent alkaloid.
A small number of isocryptolepine derivatives have previously
been synthesized,23,24 and the synthesis of two compounds (2-chlo-
roisocryptolepine and 8-chloroisocryptolepine), coincidental to our
work, was recently reported.25 However, the antimalarial activity of
these compounds has not been described thus far.
a
valuable source of potential lead compounds.1,2 Invariably
semi-synthetic derivatives of natural compounds are developed
to improve biopharmaceutical and/or pharmacological properties,
as demonstrated with artemisinin.3
The West African climbing shrub Cryptolepis sanguinolenta is a
component of several traditional herbal remedies used to treat a vari-
ety of maladies, including malaria.4 Cryptolepine (5-methyl-5H-indo-
lo[3,2-b]quinoline) (1) was the major bioactive alkaloid isolated from
this shrub and has been found to possess antibacterial,5,6 antihyper-
glycemic,7 antimuscarinic,8 antifungal9 and antimalarial activity
(Fig. 1).10,11
Cryptolepine (1) has also been found to intercalate non-specifically
into DNA and inhibit topoisomerase II, resulting in undesirable cyto-
toxicity that could negate its therapeutic potential.12–14 Investigations
of cryptolepine analogues have found that the cytotoxicity and anti-
malarial activity of the parent alkaloid can be improved by the addi-
tion of ring substituents. A range of alkylated, halogenated and
nitrated cryptolepine derivatives have been synthesized and biologi-
cally evaluated in recent years.15–18 Typically, the halogenated
N
N
N
N
N
N
Abbreviations: CQ, chloroquine; CQR, chloroquine resistant; CQS, chloroquine
sensitive; NBS, N-bromosuccinimide; NCS, N-chlorosuccinimide; PPA, polyphos-
phoric acid; NOE, nuclear overhauser effect.
1
2
3a
⇑
Corresponding author. Tel.: +618 9266 2082; fax: +618 9266 2769.
E-mail address: P.Murray@curtin.edu.au (P.E. Murray).
Figure 1. Structures of cryptolepine (1), neocryptolepine (2) and isocryptolepine
(3a).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.037

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L. R. Whittell et al. / Bioorg. Med. Chem. 19 (2011) 7519–7525
Although both 3a and 2 are less active than 1, they have similar
method.20 Substituted 4-chloroquinolines 4b–d were coupled to
benzotriazole, producing 4-(1-benzotraizolyl)quinolines 5b–d in
good yields (70–78%). Subsequent cyclisation gave the chloro
substituted 11H-indolo[3,2-c]quinolines 6b and 6d in moderate
yield (77–78%) but low yield of the bromo substituted 11H-indo-
lo[3,2-c]quinoline 6c (54%). Intermediates 6b–d were treated with
iodomethane to give the isocryptolepine derivatives 3a–d and the
reaction proceeded smoothly when acetonitrile was used as the
reaction solvent. Higher yields were obtained with a substituent
at R1 (88–90%) while N-methylation was less efficient with a sub-
stituent at R2 (61%).
In contrast to the above halogenated compounds, the 9-methyl
analogue 3e was synthesized by a modification of the method
reported by Jonckers et al.31 for 3a (Scheme 2). Initially
5-methyl-2-bromoaniline was coupled to 4-bromoquinoline (7)
via a palladium catalyzed Buchwald-Hartwig reaction to generate
the intermediate 4-(2-bromo-5-methylphenylamino)quinoline (9)
in moderate yield (76%). Intermediate 8 was subjected to cycliza-
tion via a palladium catalyzed intramolecular Heck reaction to
yield 9-methyl-11H-indolo[3,2-c]quinoline (6e) in moderate yield
(69%). Finally treatment of the cyclic intermediate 6e with iodo-
methane in acetonitrile afforded 9-methylisocryptolepine (3e) in
good yield (84%).
selectivity indices19 and it is unclear why 3a has been neglected.
We report the synthesis and antimalarial activity of eight
isocryptolepine derivatives that were assessed for potential as lead
compounds for new antimalarial agents. Derivatives with substitu-
ents in similar positions to derivatives of 1 and 217,18,21 were syn-
thesized, to enable direct comparison. Compounds were evaluated
for their in vitro antiplasmodial activity and cytotoxicity.
2. Results and discussion
2.1. Chemistry
The parent alkaloid, isocryptolepine (3a) was prepared as
described previously by Molina et al.26 (Scheme 1). 4-Chloroquin-
oline (4a) was initially thermally coupled with benzotriazole to
give 4-(1-benzotraizolyl)quinoline (5a). Intermediate 5a was sub-
sequently cyclized, in the presence of polyphosphoric acid (PPA),
to yield 11H-indolo[3,2-c]quinoline (6a). Compound 6a was
N-methylated with iodomethane to yield the hydroiodide salt of
3a, which was converted to its free base and characterization data
were consistent with previous reports.11,26–30
Our synthetic strategy for analogues of 3a was inspired by a re-
cent report of the synthesis of neocryptolepine derivatives from
substituted 2-chloroquinolines, via a modification of the Molina
A further range of halogenated derivatives 9a–e and 10 were
prepared via aromatic electrophilic substitution (Scheme 3).
N
N
HN
N
N
Cl
N
N
R1
R2
R1
R2
R1
R2
R1
R2
ii
iii
i
N
N
4a R1=H R2=H
4b R1=Cl R2=H
4c R1=Br R2=H
4d R1=H R2=Cl
5a R1=H R2=H
5b R1=Cl R2=H
5c R1=Br R2=H
5d R1=H R2=Cl
6a R1=H R2=H
6b R1=Cl R2=H
6c R1=Br R2=H
6d R1=H R2=Cl
3a R1=H R2=H
3b R1=Cl R2=H
3c R1=Br R2=H
3d R1=H R2=Cl
Scheme 1. Synthesis of compounds 3a–d. aReagents and Conditions: (i) Benzotriazole, 110–120 °C, 30 min; (ii) PPA, 130–140 °C, 3–4 h; (iii) CH3I, CH3CN, reflux, 20 h.
CH3
CH3
CH3
HN
HN
N
Br
Br
i
iii
ii
N
N
N
N
8
6e
3e
7
Scheme 2. Synthesis of compound 3e. aReagents and Conditions: (i) 2-bromo-5-methylaniline, Pd2(dba)3 (1 mol %), XANTPHOS (2 mol %), Cs2CO3, dioxane, reflux, 24 h; (ii)
Pd(OAc)2 (2 mol %), BINAP (2 mol %), K2CO3, DMF, 150 °C, 24 h; (iii) CH3I, CH3CN, reflux, 20 h.
R3
R3
N
N
N
Cl
Br
R1
R2
R1
R2
b
a
9a R1=H R2=H R3=H
9b R1=Cl R2=H R3=H
9c R1=Br R2=H R3=H
9d R1=H R2=Cl R3=H
9e R1=H R2=H R3=CH3
N
N
N
3a-3e
10
Scheme 3. Synthesis of compounds 10 and 9a–e. aReagents and Conditions: (a) NBS, 150 °C; (b) NCS, 150 °C.

L. R. Whittell et al. / Bioorg. Med. Chem. 19 (2011) 7519–7525
7521
Bromination of 3a and mono-substituted derivatives 3b–e was
achieved by reaction with N-bromosuccinimide (NBS) via adaption
of literature methods.32 At room temperature, or elevated temper-
atures, the use of one molar equivalent of NBS exclusively pro-
duced 8-substituted derivatives 9a–e in good yield (71–80%).
Attempts to chlorinate 3a with N-chlorosuccinimide (NCS) gave
8-chloroisocryptolepine (10) in low yield (41%), possibly due to
the lower reactivity of this halogenating reagent. The ring position
of electrophilic substitution, in compounds 9a–e and 10, was con-
firmed by selective 1D NOE NMR spectroscopic analysis.
The compound 8-bromo-2-chloroisocryptolepine (9b) was an
exception, being 4-fold less cytotoxic than the parent alkaloid.
The most potent antimalarial derivatives, 9b, 9c and 9d had selec-
tivity indices of 106, 23 and 26 respectively. Therefore compound
9b (8-bromo-2-chloroisocryptolepine) represents the most selec-
tive of this series of compounds.
3. Conclusion
Eight derivatives of isocryptolepine have been evaluated for
in vitro antiplasmodial activity against CQS and CQR strains of
P. falciparum. All were found to be more active than the parent com-
pound, with 8-bromo-2-chloroisocryptolepine (9b) being the most
potent derivative. In vitro cytotoxicity against 3T3 cells has identi-
fied 9b as the only derivative to have reduced cytotoxicity in com-
parison to the parent compound and a selectivity index >100. As
compound 9b displays antimalarial activity in the low nano-molar
range, comparable to lumefantrine33 (IC50 = 56 nM; CQR W2mef),
9b represents a novel promising lead in antimalarial drug develop
ment.
2.2. In vitro antiplasmodial activity
The hydrochloride salts of isocryptolepine derivatives, and the
parent 3a, were evaluated for their in vitro antiplasmodial activity
against the chloroquine sensitive strain (CQS) 3D7 and the chloro-
quine resistant strain (CQR) W2mef of P. falciparum. The results are
summarized in Table 1. The parent alkaloid 3a was found to pos-
sess in vitro antimalarial activity (IC50 = 1177 nM) against CQR
W2mef comparable to previous data (IC50 = 780 nM).19 Similarly
CQ displayed in vitro antimalarial activity (IC50 = 144 nM) against
the CQR strain at comparable levels to published data
(IC50 = 171–246 nM).17–19,33 The mono-substituted isocryptolepine
compounds 3d, 3e, 9a and 10a showed improved activity com-
pared to 3a, against both strains. Di-substituted derivatives 9b–e
were also more active than 3a against both strains, and generally
more active than the mono-substituted counterparts. The di-halo-
genated derivative 9b showed the greatest activity (IC50 = 85 nM,
CQR W2mef) and our order of activity is the same as that reported
for a series of cryptolepine derivatives.17 Onyeibor et al.17 found
that 7-bromo-2-chlorocryptolepine, 7-bromo-3-chlorocryptole-
pine and 2,7-dibromocryptolepine were the most active deriva-
tives and the analogous derivatives in the present study were 9b,
9d and 9c. The di-substituted derivatives with substituents on
opposing sides of the molecule (i.e., 9b) were more active than
those with substituents on the same aromatic ring (i.e., 9e). The
di-bromo compound 9c was the only derivative to have greater
activity against CQR W2mef compared to CQS 3D7.
4. Experimental section
4.1. Chemistry
Starting materials 4b, 4c and 7 were prepared as previously re-
ported.34–36DMF was dried over CaSO4 and distilled under reduced
pressure. Dioxane was dried over sodium before distillation under
nitrogen from sodium/benzophenone. Other solvents, reagents and
reactants were available commercially and used as received from
the supplier. Analytical TLC was performed on silicagel 60 F254
(Macherey–Nagel) and flash chromatography was performed using
Silica gel 60 (0.040–0.063 mm, Fluka). Characterization of novel
compounds was carried out using NMR spectroscopy and mass
spectrometry. NMR spectra were recorded with Varian-Gemini
(200 MHz, 1H; 50 MHz, 13C), Bruker AV400 (400 MHz, 1H;
100 MHz, 13C) or Bruker AV600 (600 MHz, 1H) spectrometers. Cou-
pling constants (J) are expressed in Hertz (Hz) and assignment of
1H and 13C spectra were routinely made with the aid of 2D exper-
iments. Mass spectra (MS) were obtained using a HP5896 or VG
Autospec high-resolution mass spectrometer. High resolution mass
spectrometry (HRMS) was used to determine the accurate mass of
the molecular ion in-lieu of elemental analysis. Melting points
(Mp) were recorded with a Barnstead Electrothermal digital melt-
ing-point apparatus. The purity of derivatives for biological testing
were assessed by analytical HPLC (purity >96%) on an Apollo C18
2.3. In vitro cytotoxicity
The hydrochloride salts of isocryptolepine derivatives, and the
parent 3a, were evaluated for their in vitro cytotoxicity against
3T3 cells (mouse embryonic fibroblasts). The results are summa-
rized in Table 1 and selectivity indices were calculated using the
CQR (W2mef) antiplasmodial activity data. The majority of deriva-
tives (3d, 3e, 9a, 9c–e and 10) had a similar cytotoxicity compared
to the parent 3a, with IC50 values ranging from 1970 to 2640 nM.
column (4.6 ꢀ 150 mm, 5
lm) eluting with 20–80% acetonitrile in
water (0.5% formic acid) over ten min at a flow rate of 1.5 mL/min.
Table 1
In vitro antiplasmodial activity against P. falciparum, in vitro cytotoxicity against 3T3 cells and selectivity indices of substituted isocryptolepines
c
Compounda
Antiplasmodial activityb (nM)
Cytotoxicityb (nM)
Selectivity index
3D7
W2mef
3a, Isocryptolepine
665 221
130 12
448 83
85 33
57 14
127 98
1177 390
316 205
760 268
184 47
85 5.6
112 12
100 16
131 55
218 35
144 11
2188 351
2263 460
2067 331
1970 360
9005 3754
2585 533
2640 663
2500 352
2104 304
71,954 19,532
1.9
7.3
2.7
11
106
23
26
19
9.6
500
3d, 3-Chloroisocryptolepine
3e, 9-Methylisocryptolepine
9a, 8-Bromoisocryptolepine
9b, 8-Bromo-2-chloroisocryptolepine
9c, 2,8-Dibromoisocryptolepine
9d, 8-Bromo-3-chloroisocryptolepine
9e, 8-Bromo-9-methylisocryptolepine
10, 8-Chloroisocryptolepine
CQ
50
4
62 39
117 16
20 27
a
b
c
Tested as hydrochloride salts.
Mean IC50 standard deviation; at least three separate determinations.
Cytotoxic/antiplasmodial ratio.

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L. R. Whittell et al. / Bioorg. Med. Chem. 19 (2011) 7519–7525
4.1.1. General procedure for the synthesis of 4-(1-
benzotriazolyl)quinolines (5a–d)
4.1.7. 11H-indolo[3,2-c]quinoline (6a)
Prepared from 5a (204 mg, 0.83 mmol) and PPA (7.17 g). Cream
Benzotriazole and the appropriate quinoline compound (4a–d)
were heated at 110–120 °C for 30 min. The resulting solid was
cooled to room temperate and collected by filtration (washing with
H2O). The solid obtained was subsequently recrystallized from eth-
anol to give 5a–d.
solid; yield 84% (152 mg). Mp >300 °C (Lit.26 mp 336–337 °C).
4.1.8. 2-Chloro-11H-indolo[3,2-c]quinoline (6b)
Prepared from 5b (608 mg, 2.17 mmol) and PPA (19.48 g).
Cream solid; yield 77% (422 mg). Mp >350 °C. 1H NMR (600 MHz,
DMSO-d6): d 7.36 (1H, td, J = 7.5, 0.6 Hz, H-8), 7.52 (1H, td,
J = 7.8, 0.6 Hz, H-9), 7.73–7.75 (2H, m, H-3 and H-10), 8.14 (1H,
d, J = 9.0 Hz, H-4), 8.33 (1H, dd, J = 7.8, 0.6 Hz, H-7), 8.64 (1H, d,
J = 2.4 Hz, H-1), 9.61 (1H, s, H-6). 13C NMR (50 MHz, DMSO-d6): d
111.9, 114.7, 117.8, 120.1, 120.7, 121.1, 121.5, 125.8, 128.1,
129.8, 131.4, 138.7, 143.6, 145.1. MS (EI): 252 (100), 253 (18),
254 (34). MS (EI): 217 (11), 252 (100), 253 (18), 254 (34). HRMS
(EI): 252.0453 (C15H9N2Cl [M]+ requires 252.0454).
4.1.2. 4-(1-Benzotriazolyl)quinoline (5a)
Prepared from 4a (1.62 g, 9.89 mmol) and benzotriazole (1.30 g,
10.88 mmol). White crystalline solid; yield 77% (1.87 g). Mp
132–133 °C (Lit.37 mp 132–133 °C).
4.1.3. 4-(1-Benzotriazolyl)-6-chloroquinoline (5b)
Prepared from 4b (425 mg, 2.15 mmol) and benzotriazole
(274 mg, 2.30 mmol). White crystalline solid; yield 77% (464 mg).
Mp 186–187 °C. 1H NMR (200 MHz, CDCl3): d 7.45–7.55 (3H, m,
H-40, H-50 and H-60), 7.61 (1H,d, J = 4.8 Hz, H-3), 7.75 (1H, dd,
J = 8.8, 2.2 Hz, H-7), 7.86 (1H, d, J = 2.2 Hz, H-5), 8.20 (1H, d,
J = 8.8 Hz, H-8), 8.22 (1H, dd, J = 8.8, 1.2 Hz, H-70), 9.10 (1H, d,
J = 4.8 Hz, H-2). 13C NMR (50 MHz, CDCl3): d 109.1, 116.7, 119.9,
121.6, 122.9, 124.2, 128.3, 130.9, 132.8, 133.7, 138.9, 145.3,
147.7, 149.7. MS (EI): 99 (44), 126 (15), 127 (18), 134 (31), 162
(49), 164 (19), 190 (29), 216 (17), 217 (81), 252 (100), 253 (19),
254 (35), 280 (37). HRMS (EI): 280.0515 (C15H9N4Cl [M]+ requires
280.0516).
4.1.9. 2-Bromo-11H-indolo[3,2-c]quinoline (6c)
Prepared from 5c (675 mg, 2.08 mmol) and PPA (16.71 g). Wash
with MeOH; cream solid; yield 54% (332 mg). Mp >350 °C. 1H NMR
(400 MHz, DMSO-d6): d .34 (1H, td, J = 7.5, 0.8 Hz, H-8), 7.50 (1H,
ddd, J = = 8.2, 7.0, 0.8 Hz, H-9), 7.75 (1H, dt, J = 7.2, 0.8 Hz, H-10),
7.83 (1H, dd, J = 8.8, 2.0 Hz, H-3), 8.06 (1H, d, J = 9.2 Hz, H-4),
8.32 (1H, dt, J = 7.6, 0.8 Hz, H-7), 8.85 (1H, d, J = 2.0 Hz, H-1), 9.62
(1H, s, H-6). 13C NMR (100 MHz, DMSO-d6): d 112.3, 114.9, 118.3,
118.7, 120.3, 120.7, 121.7, 124.7, 125.9, 130.8, 131.7, 139.0,
139.2, 144.0, 145.4. MS (EI): 190 (19), 216 (22), 217 (40), 296
(100), 297 (22), 298 (98), 299 (17). HRMS (EI): 295.9944
(C15H9N2Br [M]+ requires 295.9949).
4.1.4. 4-(1-Benzotriazolyl)-6-bromoquinoline (5c)
Prepared from 4c (401 mg, 1.65 mmol) and benzotriazole
(212 mg, 1.78 mmol). White crystalline solid; yield 70% (376 mg).
Mp 181–182 °C. 1H NMR(200 MHz, CDCl3): d 7.46–7.57 (3H, m,
H-40. H-50 and H-60), 7.61 (1H,d, J = 4.4 Hz, H-3), 7.91 (1H, dd,
J = 9.2, 2.2 Hz, H-7), 8.05 (1H, d, J = 1.8 Hz, H-5), 8.14 (1H, d,
J = 8.8 Hz, H-8), 8.24 (1H, dd, J = 7.6, 1 Hz, H-70), 9.13 (1H, d,
J = 4.6 Hz, H-2). 13C NMR (50 MHz, CDCl3): d 109.1, 116.6, 119.9,
121.8, 123.4, 124.2, 124.9, 128.3, 130.9, 132.9, 133.5, 138.8,
145.4, 147.9, 149.8. MS (EI): 100 (19), 127 (20), 190 (29), 206
(18), 208 (17), 216 (23), 217 (100), 218 (20), 296 (55), 298 (58),
324 (17), 326 (17). HRMS (EI): 324.0005 (C15H9N4Br [M]+ requires
324.0011).
4.1.10. 3-Chloro-11H-indolo[3,2-c]quinoline (6d)
Prepared from 5d (993 mg, 3.54 mmol) and PPA (31.32 g).
Cream solid; yield 78% (695 mg). Mp >310 °C. 1H NMR (600 MHz,
DMSO-d6): d 7.35 (1H, td, J = 7.5, 0.6 Hz, H-8), 7.51 (1H, ddd,
J = 8.4, 7.2, 0.6 Hz, H-9), 7.73 (1H, dd, J = 8.4, 0.6 Hz, H-10), 7.74
(1H, dd, J = 8.7, 2.1 Hz, H-2), 8.16, (1H, d, J = 2.4 Hz, H-4), 8.33
(1H, dd, J = 7.5, 0.6 Hz, H-7), 8.56 (1H, d, J = 8.4 Hz, H-1), 9.62
(1H, s, H-6), 12.88 (1H, br s, N-H). 13C NMR (50 MHz, DMSO-d6):
d 111.8, 114.5, 115.5, 120.0, 120.6, 121.5, 124.0, 125.7, 125.9,
128.1, 132.2, 138.7, 139.3, 145.7, 145.9. MS (EI): 217 (16), 252
(100), 253 (20), 254 (33). HRMS (EI): 252.0454 (C15H9N2Cl [M]+re-
quires 252.0454).
4.1.5. 4-(1-Benzotriazolyl)-7-chloroquinoline (5d)
4.1.11. 9-Methyl-11H-indolo[3,2-c]quinoline (6e)
Prepared from 4d (392 mg, 1.98 mmol) and benzotriazole
(265 mg, 2.22 mmol). White crystalline solid; yield 78% (432 g).
Mp 190–192 °C. 1H NMR (600 MHz, CDCl3) d: 7.48 (1H, d,
J = 8.4 Hz, H-40), 7.52 (1H, t, J = 7.8 Hz, H-60), 7.56 (1H, dd, J = 9.0,
2.4 Hz, H-6), 7.59 (1H, t, J = 7.8 Hz, H-50), 7.62 (1H, d, J = 4.8 Hz,
H-3), 7.83 (1H, d, J = 9.0 Hz, H-5), 8.24 (1H, d, J = 8.4 Hz, H-70),
8.29 (1H, d, J = 1.8 Hz, H-8), 9.14 (1H, d, J = 4.2 Hz, H-2). 13C NMR
(100 MHz, CDCl3) d: 110.3, 117.0, 120.9, 125.2, 129.1, 129.3,
129.4, 133.8, 137.2, 141.0, 146.4, 150.4, 151.5. MS (EI): 99 (20),
135 (17), 162 (38), 190 (21), 217 (62), 252 (100), 253 (21), 254
(34), 280 (25). HRMS (EI): 280.0520 (C15H9N4Cl [M]+ requires
280.0516).
To a degassed solution of Pd(OAc)2 (4.2 mg, 2 mol %) and BINAP
(12 mg, 2 mol %) in dry DMF (8 mL), K2CO3 (2.51 g, 18.16 mmol)
and 8 (282 mg, 0.90 mmol) was added. The flask was flushed with
nitrogen and the mixture heated at 150 °C for 24 h under nitrogen.
After cooling, the mixture was filtered through celite and washed
with DCM (40 mL). The solvent was removed in vacuo and the res-
idue purified by flash column chromatography eluting with DCM
and EtOAc (1:1 increasing to 1:0). Cream solid; yield 69%
(144 mg). Mp >340 °C (d). 1H NMR (600 MHz, DMSO-d6): 2.52
(3H, s, CH3), 7.16 (1H, dd, J = = 7.8, 1.2 Hz, H-8), 7.51 (1H, d,
J = 0.6 Hz, H-10), 7.66 (1H, ddd, J = 8.1, 6.9, 1.2 Hz, H-2), 7.72 (1H,
ddd, J = 8.1, 6.9, 1.2 Hz, H-3), 8.11 (1H, dd, J = 8.4, 0.6 Hz, H-4),
8.17 (1H, d, J = 7.8 Hz, H-7), 8.53 (1H, dd, J = 8.4, 1.2 Hz, H-1),
9.53 (1H, s, H-6). 13C NMR (100 MHz, DMSO-d6): d 21.7, 111.7,
114.4, 117.2, 119.6, 119.8, 122.0, 122.2, 125.6, 127.8, 129.5,
135.2, 139.3, 139.6, 144.6, 145.3. MS (EI): 231 (63), 232 (100),
233 (19). HRMS (EI): 232.1002 (C16H12N2 [M]+ requires 232.1000).
4.1.6. General procedure for the synthesis of 11H-indolo[3,2-
c]quinolines (6a–d)
To the appropriate 4-(10-benzotriazolo)quinoline (5a-5d),
polyphosphoric acid (PPA) was added and the mixture heated at
130–140 °C until the formation of nitrogen ceased (1–4 h). The
reaction mixture was cooled, quenched with water and the precip-
itate collected by filtration. The residue was re-suspended in water,
made alkaline with 10% NaOH (aq) and the precipitate collected by
filtration. The product was purified by washed the solid with DCM
(unless otherwise stated) to give 6a–d.
4.1.12. General procedure for the synthesis of 3a–e
To a solution of the appropriate 11H-indolo[3,2-c]quinoline
(6a–e) in CH3CN, iodomethane was added and the mixture re-
fluxed for 20 h under nitrogen. The reaction mixture was then
cooled, the solvent removed in vacuo and the residue dissolved

L. R. Whittell et al. / Bioorg. Med. Chem. 19 (2011) 7519–7525
7523
in a 1:1 solution of 30% NH4OH (aq) and DCM. The organic layer
was extracted with DCM, dried (MgSO4) and the residue purified
by flash column chromatography. A gradient solvent mixture of
DCM:EtOH:NH3 was used, starting with 100:0:1 and increasing
to 100:4:1 to give the indicated isocryptolepine 3a–e.
117.9 (C-10), 119.1 (C-7), 120.4 (C-11b), 121.5 (C-8), 122.7
(C-6b), 123.8 (C-1), 125.1 (C-2), 129.2 (C-3), 134.9 (C-9), 135.3
(C-4a), 137.8 (C-6), 151.8 (C-11a), 153.7 (C-10a). MS (EI): 231.1
(19), 245.1 (35), 246.1 (100), 247.1 (19). HRMS (EI): 246.1151
(C17H14N2 [M]+ requires 246.1157). HPLC purity, 98.7%.
4.1.13. Isocryptolepine (3a)
4.1.18. 4-(2-Bromo-5-methylphenylamino)quinoline (8)
Prepared from 6a (128 mg, 0.58 mmol) and iodomethane
(3.5 mL, 56.22 mmol). Yellow crystalline solid; yield 94%
(128 mg). Mp 138–139 °C (Lit.38 mp 134–135 °C). HPLC purity,
99.9%.
To a degassed solution of Pd2(dba)3 (21.7 mg, 1 mol %) and
XANTPHOS (29.8 mg, 2.1 mol %) in dry dioxane (10 mL), Cs2CO3
(1.08 g, 3.31 mmol), 4-bromoquinoline (500 mg, 2.40 mmol) and
2-bromo-5-methylaniline (490 mg, 2.63 mmol) were added. The
flask was flushed with nitrogen and the mixture refluxed for 24 h
under nitrogen. After cooling, the reaction mixture was filtered
through Celite, washing with DCM (120 mL). The solvent was re-
moved in vacuo and the residue purified by flash column chroma-
tography eluting with DCM and EtOAc (50:50 increasing to 20:80).
Off-white solid; yield 76% (572 mg). Mp 155–156 °C. 1H NMR
(600 MHz, CDCl3): d 2.33 (3H, s, CH3), 6.84 (1H, dd, J = 8.4,
2.4 Hz, H-40), 7.02 (1H, d, J = 5.4 Hz, H-3), 7.34 (1H, d, J = 2.4 Hz,
H-60), 7.52 (1H, d, J = 7.8 Hz, H-30), 7.54 (1H, ddd, J = 8.4, 7.2,
1.2 Hz, H-6), 7.71 (1H, ddd, J = 8.4, 7.2, 1.2 Hz, H-7), 8.01 (1H, d,
J = 8.4 Hz, H-8), 8.09 (1H, d, J = 8.4 Hz, H-5), 8.63 (1H, d,
J = 4.8 Hz, H-2). 13C NMR (100 MHz, CDCl3): d 21.3, 102.8, 114.5,
119.8, 120.8, 124.2, 126.3, 127.1, 128.3, 130.5, 133.3, 137.4,
139.0, 147.1, 148.4, 148.9. MS (EI): 218 (31), 231 (15), 232 (23),
233 (100), 234 (18), 312 (27), 314 (28). HRMS (EI): 312.0263
(C16H13N2Br [M]+ requires 312.0262).
4.1.14. 2-Chloroisocryptolepine (3b)
Prepared from 6b (51 mg, 0.20 mmol) and iodomethane
(1.3 mL, 20.9 mmol). Yellow crystalline solid; yield 88% (47 mg).
Mp 248–249 °C. 1H NMR (600 MHz, DMSO-d6): d 4.19 (3H, s,
NCH3), 7.26 (1H, t, J = 7.5 Hz, H-8), 7.45 (1H, t, J = 7.5 Hz, H-9),
7.79–7.81 (2H, m, H-3 and H-10), 8.02 (1H, d, J = 9.0 Hz, H-4),
8.10 (1H, d, J = 7.2 Hz, H-7), 8.66 (1H, d, J = 2.4 Hz, H-1), 9.25 (1H,
s, H-6). 13C NMR (50 MHz, DMSO-d6): d 42.0 (NCH3), 116.4
(C-6a), 118.5 (C-4), 119.5 (C-10), 119.7 (C-7), 120.0 (C-8), 121.9
(C-11b), 122.4 (C-1), 125.4 (C-6b), 125.5 (C-9), 128.8 (C-3), 129.5
(C-2), 133.8 (C-4a), 138.1 (C-6), 151.3 (C-11a), 154.4 (C-10a). MS
(EI): 205 (28), 266 (100), 267 (20), 268 (34). HRMS (EI): 266.0603
(C16H11N2Cl [M]+ requires 266.0611). The spectra data acquired
was in agreement with that previously published.25
4.1.15. 2-Bromoisocryptolepine (3c)
Prepared from 6c (206 mg, 0.69 mmol) and iodomethane
(4.3 mL, 69.07 mmol). Yellow crystalline solid; yield 90%
(194 mg). Mp 262–263 °C. 1H NMR (600 MHz, DMSO-d6): d 4.19
(3H, s, NCH3), 7.26 (1H, t, J = 7.5 Hz, H-8), 7.45 (1H, ddd, J = 7.8,
7.2, 0.6 Hz, H-9), 7.80 (1H, d, J = 7.8 Hz, H-10), 7.92 (1H, dd,
J = 9.6, 2.1 Hz, H-3), 7.96 (1H, d, J = 9.0 Hz, H-4), 8.10 (1H, d,
J = 7.2 Hz, H-7), 8.82 (1H, d, J = 2.4 Hz, H-1), 9.27 (1H, s, H-6). 13C
NMR (50 MHz, DMSO-d6): d 42.0 (NCH3), 116.6 (C-6a), 117.7 (C-
2), 118.6 (C-10), 119.5 (C-4), 119.9 (C-7), 120.0 (C-8), 122.3 (C-
11b), 125.5 (C-6b), 125.6 (C-9 and C-1), 131.4 (C-3), 134.1 (C-4a),
138.1 (C-6), 151.2 (C-11a), 154.5 (C-10a). MS (EI): 189 (16), 231
(44), 310 (100), 311 (19), 312 (88), 313 (18). HRMS (EI):
310.0113 (C16H11N2Br [M]+ requires 310.0106).
4.1.19. General procedure for the synthesis of 9a–d and 10
To a solution of the appropriate isocryptolepine (3a-3e) in dry
DMF, N-bromosuccinimide (NBS) or N-chlorosuccinimide (NCS)
was added and the solution heated at 150 °C overnight (unless
otherwise stated). The reaction was then cooled, quenched with
water and basified with 10% NaOH (aq). The precipitated obtained
was collected and purified by flash column chromatography (elut-
ing with DCM:EtOH:NH3; 100:0:1 increasing to 100:4:1) or recrys-
tallisation (from the solvent indicated) to give the respective
isocryptolepine derivative 9a–d or 10.
4.1.20. 8-Bromoisocryptolepine (9a)
Prepared from 3a (395 mg, 1.70 mmol) and NBS (344 mg,
1.93 mmol), 20 h. Recrystallisation solvent: EtOH; yellow crystal-
line solid; yield 74% (392 mg). Mp 257–258 °C. 1H NMR
(600 MHz, DMSO-d6): d 4.25 (3H, s, NCH3), 7.54 (1H, dd, J = 8.4,
1.8 Hz, H-9), 7.71–7.74 (2H, m, H-2 and H-10), 7.85 (1H, ddd,
J = 8.7, 7.2, 1.5 Hz, H-3), 8.05 (1H, d, J = 9.0 Hz, H-4), 8.30 (1H, d,
J = 2.4 Hz, H-7), 8.76 (1H, dd, J = 8.1, 1.5 Hz, H-1), 9.39 (1H, s, H-
6). 13C NMR (50 MHz, DMSO-d6): d 42.3 (NCH3), 111.7 (C-8),
115.2 (C-6a), 117.5 (C-4), 120.0 (C-10), 121.0 (C-11b), 122.0 (C-
7), 123.8 (C-1), 125.4 (C-2), 127.5 (C-6b), 127.6 (C-9), 129.4 (C-3),
135.4 (C-4a), 139.2 (C-6), 153.0 (C-10a and C-11a). MS (EI): 189
(17), 215 (16), 216 (20), 231 (21), 310 (100), 311 (22), 312 (100),
313 (19). HRMS (EI): 310.0110 (C16H11N2Br [M]+ requires
310.0106). HPLC purity, 99.5%.
4.1.16. 3-Chloroisocryptolepine (3d)
Prepared from 6d (438 mg, 1.73 mmol) and iodomethane
(11 mL, 176.7 mmol). Yellow crystalline solid; yield 61%
(281 mg). Mp 268–270 °C. 1H NMR (600 MHz, DMSO-d6): d 4.21
(3H, s, NCH3), 7.25 (1H, t, J = 7.5 Hz, H-8), 7.44 (1H, ddd, J = 7.8,
7.2, 0.6 Hz, H-9), 7.71 (1H, dd, J = 9.0, 1.8 Hz, H-2), 7.79 (1H, br d,
J = 8.4 Hz, H-10), 8.09–8.10 (2H, m, H-4 and H-7), 8.73 (1H, d,
J = 9.0 Hz, H-1), 9.27 (1H, s, H-6). 13C NMR (100 MHz, DMSO-d6):
d 42.2 (NCH3), 116.8 (C-6a), 117.2 (C-4), 118.6 (C-10), 119.6 (C-
11b), 119.7 (C-7), 120.1 (C-8), 125.4 (C-2), 125.6 (C-6b), 125.7 (C-
1), 125.8 (C-9), 133.9 (C-3), 136.2 (C-4a), 138.4 (C-6), 152.1 (C-
11a), 154.7 (C-10a). MS (FAB): 147 (18), 267 (100), 268 (23), 269
(35). HRMS (FAB): 267.0685 (C16H12N2Cl [M+H]+ requires
267.0689). HPLC purity, 98.9%.
4.1.21. 8-Bromo-2-chloroisocryptolepine (9b)
Prepared from 3b (116 mg, 0.44 mmol) and NBS (86.6 mg,
0.49 mmol), 2 h. Yellow crystalline solid; yield 77% (118 mg). Mp
265–266 °C. 1H NMR (600 MHz, DMSO-d6): d 4.22 (3H, s, NCH3),
7.54 (1H, dd, J = 8.4, 2.1 Hz, H-9), 7.73 (1H, d, J = 8.4 Hz, H-10),
7.85 (1H, dd, J = 9.6, 2.7 Hz, H-3), 8.08 (1H, d, J = 9.6 Hz, H-4),
8.30 (H, d, J = 2.4 Hz, H-7), 8.66 (1H, d, J = 2.4 Hz, H-1), 9.36 (1H,
s, H-6). 13C NMR (50 MHz, DMSO-d6): d 42.4 (NCH3), 112.1 (C-8),
115.5 (C-6a), 120.0 (C-4), 120.2 (C-10), 122.0 (C-11b) 122.1 (C-7),
122.4 (C-1), 127.4 (C-6b), 127.9 (C-9), 129.1 (C-3), 129.9 (C-2),
133.9 (C-4a), 139.3 (C-6), 151.8 (C-11a), 153.0 (C-10a). MS (EI):
4.1.17. 9-Methylisocryptolepine (3e)
Prepared from 6e (222 mg, 0.95 mmol) and iodomethane
(5.9 mL, 94.77 mmol). Yellow crystalline solid; yield 84%
(197 mg). Mp 259–260 °C. 1H NMR (600 MHz, CDCl3): d 2.58 (3H,
s, CH3), 3.97 (3H, s, NCH3), 7.09 (1H, ddd, J = 7.8, 1.2, 0.6 Hz, H-8),
7.52–7.55 (2H, m, H-2 and H-4), 7.61 (1H, ddd, J = 9.0, 7.8, 1.2 Hz,
H-3), 7.73 (1H, d, J = 7.8 Hz, H-7), 7.77 (1H, br s, H-10), 8.12 (1H,
s, H-6), 8.86 (1H, dd, J = 7.2, 2.1 Hz, H-1). 13C NMR (50 MHz,
DMSO-d6): d 21.7 (CH3), 42.1 (NCH3), 116.0 (C-6a), 117.4 (C-4),

7524
L. R. Whittell et al. / Bioorg. Med. Chem. 19 (2011) 7519–7525
188 (17), 215 (21), 310 (22), 312 (22), 344 (72), 346 (100), 347
(19), 348 (25). HRMS (EI): 343.9727 (C16H10N2ClBr [M]+ requires
343.9716). HPLC purity, 98.3%.
4.1.26. Conversion of 3a, 3d, 3e, 9a–9e and 10 to hydrochloride
salts
Compounds were dissolved in a minimal amount of methanol,
and concentrated HCl added dropwise. The volume of solvent
was reduced in vacuo, the resultant precipitate collected by filtra-
tion and dried under high vacuum, before purity analysis by HPLC
and biological evaluation.
4.1.22. 2,8-Dibromoisocryptolepine (9c)
Prepared from 3c (175 mg, 0.56 mmol) and NBS (106 mg,
0.59 mmol), 20 h. Yellow crystalline solid; yield 71% (155 mg).
Mp 324–326 °C. 1H NMR (400 MHz, DMSO-d6): d 4.29 (3H, s,
CH3), 7.55 (1H, dd, J = 8.8, 2.2 Hz, H-9), 7.74 (1H, d, J = 8.8 Hz, H-
10), 8.00 (1H, dd, J = 8.8, 2.2 Hz, H-3), 8.05 (1H, d, J = 9.2 Hz, H-4),
8.33 (H, d, J = 2.0 Hz, H-7), 8.83 (1H, d, J = 2.0 Hz, H-1), 9.44 (1H,
s, H-6). 13C NMR (50 MHz, DMSO-d6): d 42.4 (NCH3), 112.2 (C-8),
115.6 (C-6a), 118.2 (C-2), 120.3 (C-4 and C-10), 122.2 (C-7),
122.4 (C-11b), 125.6 (C-1), 127.4 (C-6b), 128.0 (C-9), 131.9 (C-3),
134.3 (C-4a), 139.6 (C-6), 151.8 (C-11a), 152.9 (C-10a). MS (EI):
215 (18), 309 (21), 311 (20), 388 (52), 390 (100), 391 (19), 392
(50). HRMS (EI): 387.9189 (C16H10N2Br2 [M]+ requires 387.9211).
HPLC purity, 96.5%.
4.2. Biological evaluation
4.2.1. In vitro activity against P. falciparum
The laboratory-adapted Plasmodium falciparum strains 3D7 and
W2mef were cultured in RPMI 1640 HEPES (Sigma–Aldrich) supple-
mented with 92.6 mg/L
L-glutamine (Sigma–Aldrich), 500 lg/L gen-
tamicin, 50 mg/L -hypoxanthine (Sigma–Aldrich) and 10% v/v
L
pooled human plasma.33,39,40 Cultures were incubated at 37 °C in
a low oxygen atmosphere (3–9%). Stock solutions were prepared
in 50% v/v DMSO and distilled water (3a, 3e, 9a, 9e and 10), 80%
v/v DMSO and distilled water (3d, 9b, 9c and 9d), or distilled water
(CQ) and stored in the absence of light at 0 °C. Aliquots were freshly
diluted to a working standard with RPMI and added in triplicate to
96-well plates (final DMSO concentration <0.05% per well and had
no effect on parasite growth). Infected erythrocytes were added (fi-
nal parasitemia 0.5% and 1.5% hematocrit) and parasite growth was
measured using the [3H]-hypoxanthine growth inhibition as-
say.33,41 To the drug-parasite suspensions, [3H]-hypoxanthine (Per-
4.1.23. 8-Bromo-3-chloroisocryptolepine (9d)
Prepared from 3d (202 mg, 0.76 mmol) and NBS (149 mg,
0.84 mmol), 24 h. Recrystallisation solvent: MeOH and H2O; yellow
crystalline solid; yield 71% (187 mg). Mp 247–250 °C. 1H NMR
(600 MHz, DMSO-d6): d 4.23 (3H, s, CH3), 7.54 (1H, dd, J = 8.7,
2.1 Hz, H-9), 7.73 (1H, d, J = 8.4 Hz, H-10), 7.76 (1H, dd, J = 8.4,
1.8 Hz, H-2), 8.16 (1H, d, J = 1.8 Hz, H-4), 8.31 (H, d, J = 1.8 Hz, H-
7), 8.74 (1H, d, J = 8.4 Hz, H-1), 9.40 (1H, s, H-6). 13C NMR (50 MHz,
DMSO-d6): d 42.4 (NCH3), 112.0 (C-8), 115.7 (C-6a), 117.4 (C-4),
119.6 (C-11b), 120.2 (C-10), 122.2 (C-7), 125.6 (C-2), 125.7 (C-1),
127.4 (C-6b), 127.9 (C-9), 134.0 (C-3), 136.1 (C-4a), 139.6 (C-6),
152.4 (C-11a), 153.2 (C-10a). MS (FAB): 344 (18), 345 (80), 346
(39), 347 (100), 348 (25), 349 (26). HRMS (FAB): 344.9798
(C16H11N2ClBr [M+H]+ requires 344.9794). HPLC purity, 97.6%.
kin Elmer) was added (final concentration of 0.5 lCi/well). The
plates were incubated for 48 h and then subjected to a freeze-thaw
process before harvesting onto 96-well glass-fibre filtermats using
a Havaster 96 (Tomtec Incorporated). Filtermats were counted on a
1450 Microbeta Plus liquid scintillation counter (Wallac). Chloro-
quine diphosphate was used as a positive control for antimalarial
activity and drug-free controls (uninfected and infected) were in-
cluded in each test. The assay was performed a minimum of three
separate times for each compound and IC50 values were determined
by non-linear regression analysis of log-dose–response curves
(Graphpad Prism 4.0).
4.1.24. 8-Bromo-9-methylisocryptolepine (9e)
Prepared from 3e (160 mg, 0.65 mmol) and NBS (137 mg,
0.77 mmol), 20 h. Yellow crystalline solid; yield 80% (168 mg).
Mp 266–267 °C. 13C NMR (400 MHz, DMSO-d6): d 2.53 (3H, s,
CH3), 4.23 (3H, s, NCH3), 7.70 (1H, t, J = 7.2 Hz, H-2), 7.75 (1H, s,
H-10), 7.84 (1H, ddd, J = 8.4, 7.2, 1.4 Hz, H-3), 8.03 (1H, d,
J = 8.8 Hz, H-4), 8.32 (1H, s, H-7), 8.74 (1H, dd, J = 8.0, 1.2 Hz, H-
1), 9.32 (1H, s, H-6). 13C NMR (50 MHz, DMSO-d6): d 23.2 (CH3),
42.1 (NCH3), 114.9 (C-8), 115.3 (C-6a), 117.4 (C-4), 120.1 (C-10),
121.0 (C-11b), 122.5 (C-7), 123.8 (C-1), 125.1 (C-2), 125.4 (C-6b),
129.2 (C-3), 133.1 (C-9), 135.4 (C-4a), 138.3 (C-6), 153.3 (C-11a),
154.1 (C-10a). MS (EI): 98 (54), 229 (21), 230 (21), 245 (54), 246
(16), 324 (100), 326 (94), 327 (17). HRMS (EI): 324.0255
(C17H13N2Br [M]+ requires 324.0262). HPLC purity, 97.5%.
4.2.2. In vitro cytotoxicity
3T3 cells (mouse embryonic fibroblasts) were cultured and
maintained in RPMI 1640 (Invitrogen) supplemented with 2 mM
L-alanyl-L-glutamine (GlutaMAX; Invitrogen), 100 U/mL penicillin
(Invitrogen), 100
lg/mL streptomycin (Invitrogen) and 10% fetal
calf serum. 24 h prior to testing, cells were added to 96-well plates
pre-coated with 1% gelatine at 7500 cells per well (final well vol-
ume 100 lL) and incubated at 37 °C in a 5% CO2 humidified atmo-
sphere. Stock solutions were prepared as for the antimalarial
evaluation. Aliquots were freshly diluted with RPMI to a working
standard, serial dilutions conducted and 100 lL of each solution
4.1.25. 8-Chloroisocryptolepine (10)
added in quadruplicate to 96-well plates. The potential DMSO ef-
fect was countered by including a vehicle control arm to the exper-
iments. The plates were incubated for 48 h and chemosensitivity
was assessed using the MTT assay.42,43 The media was removed
Prepared from 3a (222 mg, 0.96 mmol) and NCS (146 mg,
1.09 mmol), 24 h. Recrystallisation solvent: EtOH; yellow crystal-
line solid; yield 41% (99 mg). Mp 257–259 °C. 1H NMR (600 MHz,
DMSO-d6): d 4.25 (3H, s, CH3), 7.40 (1H, dd, J = 9.0, 2.1 Hz, H-9),
7.72 (1H, t, J = 7.5 Hz, H-2), 7.77 (1H, d, J = 9.0 Hz, H-10), 7.85
(1H, ddd, J = 8.5, 7.0, 1.2 Hz, H-3), 8.05 (1H, d, J = 8.4 Hz, H-4),
8.16 (1H, d, J = 2.4 Hz, H-7), 8.75 (1H, dd, J = 7.8, 1.2 Hz, H-1),
9.38 (1H, s, H-6). 13C NMR (50 MHz, DMSO-d6): d 42.6 (NCH3),
114.8 (C-6a), 117.8 (C-4), 118.7 (C-10), 119.2 (C-7), 120.2
(C-11b), 123.8 (C-1), 124.3 (C-8), 125.4 (C-2), 125.9 (C-9), 126.2
(C-6b), 129.9 (C-3), 135.4 (C-4a), 140.0 (C-6), 150.5 (C-11a),
151.6 (C-10a). MS (EI): 266 (100), 267 (20), 268 (33). HRMS (EI):
266.0602 (C16H11N2Cl [M]+ requires 266.0611). HPLC purity,
97.6%. The spectra data acquired was in agreement with that
previously published.25
from each well and 100
added. Plates were incubated for 60 min at 37 °C, the supernatant
was removed and 100 L DMSO added to each well. The absor-
lL of 1 mg/mL MTT (Sigma) in RPMI was
l
bance of each plate was measured using an automated plate reader
(Bio-Rad Laboratories) at a wavelength of 595 nm. Isocryptolepine
hydrochloride was used as a positive control for cytotoxicity and a
drug-free control was included in each test. The assay was per-
formed a minimum of three separate times for each compound
and IC50 values were determined by non-linear regression analysis
of log-dose–response curves, after correction for the DMSO effects
(Graphpad Prism 4.0).

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7525
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Acknowledgments
We thank Dr Lindsay Byrne and Dr Tony Reeder, The University
of Western Australia, and Mr. Mike Boddy, Curtin University, for
their technical assistance. We thank Curtin University for an inter-
nal research grant (KTB and PEM) to support this work. TMED is a
recipient of a National Health and Medical Research Council Prac-
titioner Fellowship and LRW is a recipient of an Australian Post-
graduate Award.
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