Bis-alkylamine quindolone derivatives as new antimalarial leads

Article abstract of DOI:10.1016/j.bmcl.2010.08.043

Quindolone derivatives, designed to target the malaria parasite digestive vacuole and heme detoxification pathway, have been synthesized by reaction with 2-chloro-N,N-diethylethanamine. This reaction gave N,O-, N,N- and O-alkylated products containing one

Full text of DOI:10.1016/j.bmcl.2010.08.043

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5634–5637
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Bis-alkylamine quindolone derivatives as new antimalarial leads
João Lavrado a, Kaamil Gani a, Pedro A. Nobre a, Sofia A. Santos a, Paula Figueiredo b, Dinora Lopes b,
Virgílio do Rosário b, Jiri Gut c, Philip J. Rosenthal c, Rui Moreira a, Alexandra Paulo a,
⇑
a iMed.UL, Faculdade de Farmácia, Universidade de Lisboa, Av Prof. Gama Pinto 1649-003 Lisboa, Portugal
b UEI Malaria, Centro da Malária e Doenças Tropicais, IHMT, Universidade Nova de Lisboa, Rua da Junqueira, 100, P-1349-008 Lisboa, Portugal
c Department of Medicine, San Francisco General Hospital, University of California, San Francisco, Box 0811, San Francisco, CA 94143, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Quindolone derivatives, designed to target the malaria parasite digestive vacuole and heme detoxification
pathway, have been synthesized by reaction with 2-chloro-N,N-diethylethanamine. This reaction gave
N,O-, N,N- and O-alkylated products containing one or two basic side-chains. The compounds were eval-
uated for antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum W2 strain and
for cytotoxicity in HepG2 A16 hepatic cells. By incorporating alkylamine side chains and chlorine atoms
in the quindolone nucleus we transformed the inactive tetracyclic parent quindolones into moderate or
highly active and selective antimalarial compounds. The most active and selective compound, 5c, showed
an IC50 = 51 nM for P. falciparum and a selectivity ratio of 98.
Received 13 July 2010
Revised 6 August 2010
Accepted 7 August 2010
Available online 12 August 2010
Keywords:
Indoloquinoline
Antiplasmodial
Malaria
Ó 2010 Elsevier Ltd. All rights reserved.
Digestive vacuole
Hemozoin
Malaria is one of the most widespread infectious diseases of our
time due to the rapid emergence and spread of multidrug-resistant
strains of Plasmodium falciparum, the mostly lethal of the malaria
parasite species. Although attempts to develop a vaccine for malar-
ia are ongoing, drugs remain a key component of control strategies.
With continued spread of drug resistance, there is an urgent need
for novel antimalarial drugs.1,2
parasite mitochondria.12 However, after introducing
a basic
tertiary amine group, as in the case of 1 (Fig. 1), the acridone accu-
mulates inside the digestive vacuole and kills the parasite by inhi-
bition of hemozoin formation.13
Our research group recognized the antimalarial potential of
indolo[3,2-b]quinolines and acridones. By merging the two scaf-
folds we obtained quindolone 2 (indolo[3,2-b]quinolin-11-one,
Fig. 1). Quindolone and its N5-methyl derivative 3 (Fig. 1) were
first isolated from the African medicinal plant Cryptolepis sangui-
nolenta.10 Although 3 was inactive against chloroquine-resistant
P. falciparum strain K1,14 its tetracyclic aromatic structure and
hydrogen-bond acceptor carbonyl group are believed to be struc-
tural features required for strong binding to heme or hemozoin
During their erythrocytic stage malaria parasites feed on host
hemoglobin, releasing toxic free heme, which is biocrystallized
into hemozoin or malaria pigment, which is harmless to the para-
site.3,4Heme detoxification remains one of the most attractive drug
development targets, mainly due to the immutable nature of heme
molecules.5,6 Several chemotypes of antiplasmodial drugs, such as
4-aminoquinolines (e.g., chloroquine, CQ), xanthones, acridines
and indoloquinolines are reported to act by enhancing free heme
toxicity through inhibition of hemozoin formation.5,7–10 Addition-
ally, we showed that incorporation of a basic side chain at C-11
of the 5-methyl-10H-indolo[3,2-b]quinoline scaffold increased
antiplasmodial activity about 10-fold, possibly by increasing drug
accumulation inside the acidic digestive vacuole with a pH-depen-
dent trapping mechanism.11 Acridones have also been pursued as
leads for new antimalarials. Haloalkoxyacridones were shown to
be very potent and selective antiplasmodial compounds, although
the mechanism of action of these acridone derivatives has been
proposed to be mainly inhibition of the respiratory chain of
⇑
Corresponding author.
E-mail address: mapaulo@ff.ul.pt (A. Paulo).
Figure 1. Structure of bis-alkylamine-acridone (1), quindolone (2), N5-methyl-
quindolone (3) and acridone derivative (KF-A2).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.043

J. Lavrado et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5634–5637
5635
crystals,6 providing the drug can reach its target inside the parasite
Table 1
Alkylation yields of the quindolone derivatives (4–6)
digestive vacuole. Taking advantage of the three nucleophilic
groups of quindolone, N-5, N-10 and O-11, we designed bis-alkyl-
amine quindolone derivatives 4 and 5 (Scheme 1), aiming to target
the parasite’s digestive vacuole and the heme detoxification path-
way. Here we describe the synthesis, in vitro antiplasmodial activ-
ity, cytotoxicity and heme binding affinity constants of these
quindolone derivatives.
Reagent
R1, R2
Alkylation yield (%)
N,N (4)
N,O (5)
O (6)
2a
2b
2c
H, H
Cl, H
Cl, Cl
23
21
19
55
33
34
17
7
5
The synthetic route of quindolones 2 is depicted in Scheme 1
and follows the method previously described.11 The bis-alkylated
quindolone derivatives 4 and 5 and the monoalkylated derivative
6 were obtained after reaction of 2 with four equivalents of 2-
chloro-N,N-diethylethanamine in the presence of a base and NaI,
through a nucleophilic substitution reaction. The structures of
quindolone derivatives 4, 5 and 6 were established on the basis
of bidimensional 1H and 13C heterocorrelation experiments (HMQC
and HMBC), elemental analysis and melting point determination.15
The positions of N-alkylamine side-chains for derivatives 4 and 5
were assigned on the basis of 1H–13C correlations between CH2
protons of the side chain and quaternary carbons of the quindolone
aromatic structure, observed on HMBC spectra and confirmed by
Nuclear Overhauser Effect (NOE) experiments. For instance, the
1H and 13C NMR spectra of derivative 5a (2-(11-(2-(diethyl-
amino)ethoxy)-10H-indolo[3,2-b]quinolin-10-yl)-N,N-diethyle-
thanamine) displayed signals corresponding to the introduction of
two alkylamine side-chains in the quindolone nucleus. Also, the
13C NMR showed signals corroborating the introduction of two
side-chains. The disappearance of the carbonyl carbon signal of
the starting quindolone resonating at 167.77 ppm, replaced by a
phenoxy carbon signal at 144.58 ppm (C11) and a typical ether car-
bon signal (74.51 ppm), correlating in the HMQC with one
deshielded triplet 1H signal of the side chain, indicated the pres-
ence of an alkoxy chain at C-11. Further experiments revealed
the attendance of NOE between the 1H triplet signals of the CH2
of the side-chains with the aromatic protons H-1 and H-9, corrob-
orating alkylation in positions O-11 and N-10, respectively.
Several studies have considered the influence of base and sol-
vent on the regioselectivity of O- versus N-alkylation in pyridones
and quinolones.16,17 Therefore, the alkylation reaction was at-
tempted with different bases, namely, K2CO3, NaH, triethylamine
and N,N-diisoproprylethylamine in dry acetone, tetrahrydrofuran
and dimethoxyethane. The best yields were achieved using
K2CO3in dry acetone. In these conditions the major products were
the bis-alkylated derivatives, with preference for the N,O-deriva-
tives (5), with yields of 34–55% (Table 1). The formation of O-alkyl-
ated compounds as the main products in the alkylation reaction of
quindolones 2 may be justified by the double-tautomeric reso-
nance between the C-11 carbonyl and both NH-5, as in pyridinon-
es,16,18,19 and NH-10, as already discussed for natural quindolone
3.14,20
It is also noteworthy that chlorine atoms, particularly in posi-
tion 3 of the quindolone nucleus, deactivated O-alkylation, as
yields of 5b–c and 6b–c were significantly lower than those of 5a
and 6a (Table 1).
Quindolones 2 and derivatives 4, 5 and 6 were evaluated for
their antiplasmodial activity against P. falciparum chloroquine-
resistant strain W2.21 From the data in Table 2 it can be concluded
that introduction of an alkylamine side chain is essential for anti-
plasmodial activity, as unsubstituted quindolones 2a–c were inac-
tive (IC50 8 to >10
only weakly (IC50 2.6–1.3
l
M) and monoalkylated derivatives 6a–c were
M) or moderately (IC50 of 0.55 M) ac-
l
l
tive, probably due to increased accumulation in the parasite diges-
tive vacuole and affinity for the hemozoin crystal promoted by the
tertiary amine group of the alkyl side chain.5,6 Interestingly, mono-
alkylated quindolone derivative 6a showed antiplasmodial activity
at the same order of magnitude as that reported for the acridone
KF-A2 (Fig. 1), which showed an IC50 of 7.5 lM for the CQ-resistant
P. falciparum Dd2 strain.22 Chlorine atoms at both sides of the tet-
racyclic aromatic structure also improved antiplasmodial activity,
with the monohalogenated derivative 6b two-times more active
than 6a and the dihalogenated derivative 6c even more active.
Scheme 1. Quindolone 2 and derivatives 4, 5 and 6 synthetic pathway. Reagent and conditions: (i) DMF/1,4-dioxane (1:1), bromoacetyl bromide, room temperature,
overnight; (ii) aniline, DMF, 120 °C, 18–48 h; (iv) 2-chloro-N,N-diethylethanaminium, dry acetone, K2CO3 NaI, reflux, overnight.

5636
J. Lavrado et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5634–5637
Table 2
In vitro antiplasmodial activity (IC50) against P. falciparum chloroquine-resistant strain W2, cytotoxicity to hepatic cell line HepG2 A16, selectivity
ratio (SR) and association constants (Kass in MÀ1) for binding to hematin monomer at 25 °C in HEPES buffer pH 5.5 of quindolones 2 and their
derivatives 4, 5 and 6
Compd
R1
R2
IC50 ( SD) P. falciparumW2
IC50 ( SD) cytotoxicity
SRa
Haematin binding
(nM)
HepG2 A16 (
lM)
log Kass ( SD)
CQ
2a
2b
2c
4a
4b
4c
5a
5b
5c
6a
6b
6c
—
H
Cl
Cl
H
Cl
Cl
H
Cl
Cl
H
—
H
H
Cl
H
H
Cl
H
H
Cl
H
H
Cl
138 ( 15)
>10,000
8424 ( 26)
>10,000
267 ( 30)
202 ( 8)
334 ( 14)
539 ( 87)
186 ( 3)
—
—
<2
—
4.9 ( 0.3)
4.7 ( 0.3)
5.0 ( 0.5)
4.9 ( 0.7)
4.9 ( 0.4)
4.9 ( 0.4)
5.1 ( 0.3)
4.9 ( 0.6)
4.9 ( 0.3)
5.2 ( 0.5)
5.0 ( 0.5)
5.1 ( 0.6)
—
21 ( 2)
—
—
—
4.33 ( 0.02)
7 ( 1)
11 ( 2)
7 ( 1)
8 ( 2)
5.0 ( 0.9)
9 ( 1)
—
16
33
31
12
44
98
3.6
—
51 ( 5)
2638 ( 22)
1332 ( 20)
550 ( 40)
Cl
Cl
—
—
HepG2A16
50
a
W2
Selectivity ratio (IC
=IC ).
50
The introduction of a second alkylamine side chain further in-
creased antiplasmodial activity, with derivatives 4 and 5 showing
IC50 values of 51–539 nM. Analyzing the influence of chlorine sub-
stitution on antiplasmodial activity of bis-alkylated derivatives 4
and 5, it can be clearly seen that halogenation increases antiplas-
modial activity of N,O-derivatives 5, following the same pattern
observed for 6, whereas mono or di-halogenation of N,N-deriva-
tives 4 has no effect on antiplasmodial activity. Improved antiplas-
modial activity with introduction of chlorine atoms in quinolines
and acridines has been noted previously, but the reason for this
observation has not been clear.9,23 As a consequence of the struc-
ture–activity relationships discussed above, the dihalogenated
and bis-alkylated quindolone derivative 5c emerges as the most
active compound of the series, with an IC50 of 51 nM, slightly more
active than chloroquine, and as active as the corresponding N,O-
bis-alkylamineacridone 1 (IC50 = 77 nM for the Dd2 strain).13
Investigation of the proposed mechanism of antiplasmodial
activity of quindolone derivatives was performed by determining
cates selectivity against erythrocytic stages of malaria parasites.
The selectivity ratio, determined as the cytotoxicity IC50/antiplas-
modial IC50 ratio, was higher than 10 for all bis-alkylated quindo-
lone derivatives 4 and 5. The most active compound 5c was also
the most selective: it was 100-fold more toxic to the parasite than
to the human hepatic cells.
In conclusion, the introduction of two alkylamine side chains
generally improves antiplasmodial activity, probably by increasing
accumulation in the acidic digestive vacuole. On the other hand,
substitution by chlorine atoms at positions 3 and 7 of the quindo-
lone skeleton improves antiplasmodial activity for N,O-bis-alkyla-
mine derivatives, but not for N,N-disubstituted derivatives, an
interesting finding that deserves further investigation. Addition-
ally, antiplasmodial activity of bis-alkylamine quindolone deriva-
tives cannot be entirely explained by their affinity to hematin
monomers. Taken together, these results indicate that the quindo-
lone nucleus is a suitable scaffold for the design of active and selec-
tive compounds targeting the malaria parasite heme detoxification
pathway and that bis-alkylaminequindolone derivatives are novel
chemotypes with potential for development as antimalarial agents.
equilibrium binding constants (Kass
) with hematin monomer
(FPIX-OH), by UV–vis titration at pH 5.5.24–27 Log Kass shown in Ta-
ble 2 were determined by fitting the experimental data to a bind-
ing model, with stoichiometry of one quindolone molecule to one
FPIX-OH,28 according to the binding stoichiometry determined
Acknowledgments
with Job’s method of continuous variations.29–31
.
This work was supported by Fundação para a Ciência e Tecnolo-
gia (FCT, Portugal); J. Lavrado acknowledges FCT for the Ph.D. Grant
SFRH/BD/29202/2006.
Log Kass values for quindolones 2 and derivatives 4–6 ranged
from 4.7 to 5.2, comparable to the log Kass values determined in
our assay for chloroquine (4.9) and for the bis-alkylamine acridone
1 (4.85) in a similar assay.13 From this experiment it can also be
concluded that binding affinity of quindolone derivatives 4–6 to
hematin monomer is only due to their aromatic tetracyclic struc-
ture, as their log Kass values were not significantly different from
those of parent quindolones 2. Additionally, chlorine atoms in
References and notes
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Cytotoxicity was determined for the hepatic cell line HepG2
A16.32 IC50 values for toxicity of quindolone 2a and derivatives
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K. Nature 2009, 459, 270.
4–6 ranged from 4.3 to 21 lM (Table 2). Introduction of alkylamine
side chains slightly increased cytotoxicity, but no structure–cyto-
toxicity pattern was observed for quindolone derivatives 4–6.
The low cytotoxicity of alkylamine quindolone derivatives indi-

J. Lavrado et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5634–5637
5637
14. Paulo, A.; Gomes, E. T.; Steele, J.; Warhurst, D. C.; Houghton, P. J. Planta Med.
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21. Human red blood cells infected with 1% Plasmodium falciparum strain W2 ring
stage parasites synchronized with 5% Sorbitol, were incubated with tested
compounds in 96-well plates at 37 °C for 48 hrs in medium RPMI-1640,
supplemented with 25 mM HEPES pH 7.4, 10% heat inactivated human serum
15. Synthesis of 4a, 5a, and 6a: To a solution of 2a (40 mg, 0.17 mmol), K2CO3
(352.4 mg, 2.55 mmol, 15 equiv), NaI (101.9 mg, 0.68 mmol, 4 equiv) in dried
acetone (15 mL) was added 2-chloro-N1,N1-diethylethanaminium chloride
(117.0 mg, 0.68 mmol, 4 equiv) and refluxed overnight. At the end of time,
solvent was removed at reduced pressure and the remain solid suspended in
H2O (30 mL). The aqueous solution was extracted with CH2Cl2 (3 Â 30 mL) and
the combined organic extracts, washed with water, brine, dried with
anhydrous Na2SO4 and reduced to small volume. The crude mixture was
purified by preparative thin layer chromatography (P-TLC) using as eluent
CH2Cl2/MeOH (9:1). The compounds 5,10-bis(2-(diethylamino)ethyl)-5H-
(or 0.5% Albumax 2% human serum), and 100 lM Hypoxanthine under
indolo[3,2-b]quinolin-11(10H)-one
(diethylamino)ethoxy)-10H-indolo[3,2-b]quinolin-10-yl)-N,N-
(4a,
Rf
0.43),
2-(11-(2-
atmosphere of 3% O2, 5% CO2, 91% N2. After 48 h the cell were fixed in 2%
HCHO in PBS, and transferred into PBS with 100 mM NH4Cl, 0.1% Triton X-100,
1nM YOYO-1, and analyzed in a flow cytometer (FACSort, Beckton Dickinson;
EX 488 nm, EM 520 nm). IC50 was calculated using GraphPad PRIZM software.
22. Kelly, J. X.; Smilkstein, M. J.; Cooper, R. A.; Lane, K. D.; Johnson, R. A.; Janowsky,
A.; Dodean, R. A.; Hinrichs, D. J.; Winter, R.; Riscoe, M. Antimicrob. Agents
Chemother. 2007, 51, 4133.
diethylethanamine (5a, Rf 0.56) and 2-((10H-indolo[3,2-b]quinolin-11-yl)oxy)-
N,N-diethylethanamine (6a, Rf 0.75), were isolated, as light yellow solids.
Compounds were precipitated as hydrochlorides with HCl in Et2O. 5,10-Bis(2-
(diethylamino)ethyl)-5H-indolo[3,2-b]quinolin-11(10H)-one (4a) mp 228–
231 °C; 1H NMR (400 MHz, CDCl3) dH (ppm) 8.69 (d, J = 8.3 Hz, 1H), 8.25 (d,
J = 8.4 Hz, 1H), 7.70 (m, 2H), 7.59 (d, J = 8.3 Hz, 1H), 7.54 (dd, J = 8.3, 7.6 Hz, 1H),
7.34 (dd, J = 8.3, 7.5 Hz, 1H), 7.24 (dd, J = 8.4, 7.6 Hz, 1H), 5.00 (t, J = 8.0 Hz, 2H),
4.84 (t, J = 7.8 Hz, 2H), 3.03 (t, J = 8.0 Hz, 2H), 2.92 (t, J = 7.8 Hz, 2H), 2.71 (dq,
J = 15.4, 7.1 Hz, 8H), 1.09 (dt, J = 15.4, 7.1 Hz, 12H). 13C NMR (101 MHz, CDCl3)
dC (ppm) 169.13, 139.70, 139.65, 131.38, 130.62, 127.32, 126.86, 124.89,
122.69, 122.60, 120.95, 119.52, 115.14, 114.11, 110.63, 53.17, 50.87, 47.69,
47.35, 43.10, 11.85. Anal. Calcd for C27H36N4OÁ0.4HCl: C, 72.52; H, 8.20; N,
12.53. Found: C, 72.26; H, 8.33; N, 12.27. 2-(11-(2-(Diethylamino)ethoxy)-
10H-indolo[3,2-b]quinolin-10-yl)-N,N-diethylethanamine (5a) mp 144–
146 °C; 1H NMR (400 MHz, CDCl3) dH (ppm) 8.55 (d, J = 7.7 Hz, 1H), 8.40 (d,
J = 8.2 Hz, 1H), 8.33 (d, J = 8.5 Hz, 1H), 7.70 (dd, J = 8.5, 6.8 Hz, 1H), 7.66 (dd,
J = 8.2, 7.4 Hz, 1H), 7.58 (dd, J = 8.2, 6.80 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.35
(dd, J = 7.7, 7.4 Hz, 1H), 4.73 (t, J = 7.70 Hz, 2H), 4.32 (t, J = 6.3 Hz, 2H), 3.10 (t,
J = 6.3 Hz, 2H), 2.79 (t, J = 7.70 Hz, 2H), 2.70 (q, J = 7.1 Hz, 4H), 2.62 (q,
J = 7.1 Hz, 4H), 1.13 (t, J = 7.1 Hz, 6H), 1.00 (t, J = 7.1 Hz, 6H). 13C NMR
(101 MHz, CDCl3) dC (ppm) 148.59, 145.83, 144.80, 144.58, 129.69, 129.31,
126.65, 124.87, 124.68, 122.46, 122.23, 122.08, 121.27, 119.86, 109.17, 74.51,
52.93, 51.71, 47.69, 47.62, 43.51, 12.06. Anal. Calcd for C27H38N4OÁ4HCl: C,
55.87; H, 7.29; N, 9.65. Found: C, 55.43; H, 7.39; N, 9.24. 2-((10H-Indolo[3,2-
b]quinolin-11-yl)oxy)-N,N-diethylethanamine (6a): mp 193–195 °C; 1H NMR
(400 MHz, CDCl3) dH (ppm) 12.63 (s, NH), 8.56 (d, J = 7.8 Hz, 1H), 8.34 (d,
J = 8.3 Hz, 1H), 8.32 (d, J = 8.6 Hz, 1H), 7.67 (dd, J = 8.6, 7.0 Hz, 1H), 7.60 (dd,
J = 9.1, 7.1 Hz, 1H), 7.54 (dd, J = 8.3, 7.0 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.32 (dd,
J = 7.8, 7.1 Hz, 1H), 4.58 (t, J = 7.5 Hz, 2H), 3.07 (t, J = 7.5 Hz, 2H), 2.90 (q,
J = 7.2 Hz, 4H), 1.26 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz, CDCl3) dC 148.68,
145.54, 144.65, 143.52, 129.38, 128.95, 126.47, 124.23, 123.95, 122.58, 122.20,
121.48, 120.96, 119.37, 111.04, 73.99, 55.09, 48.51, 11.33. Anal. Calcd for
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28. Job’s plot of quindolone (2a), and its derivatives 4a, 5a and 6a complexed with
haematin in HEPES buffer pH 5.5 containing 40 % DMSO were performed. The
sum of the concentrations of the FPIX-OH and ligand was kept constant at
[FPIX-OH] + [Ligand] = 10 lM. The cross points were at FPIX-OH mole fractions
of 0.49 (2a), 0.52 (4a), 0.58 (5a) and 0.52 (6a).
29. Job, P. Ann. Chim. France 1928, 9, 113.
30. Ingham, K. C. Anal. Biochem. 1975, 68, 660.
31. Hill, Z. D.; Maccarthy, P. J. Chem. Educ. 1986, 63, 162.
32. HepG2 A16 hepatic cell line viability was determined based on MTT assay.
Briefly, cells were seeded in a 96-well plate at a density of 1 Â 104 cells/well.
Different concentrations of each compound were incubated for 48 h under
standard culture conditions. Concentrations inhibiting 50% of cell growth (IC50
)
were determined from graphics of degree of cell injury over compound
concentration.
C
21H23N3OÁ2.8HCl: C, 57.91; H, 5.97; N, 9.65. Found: C, 57.56; H, 5.82; N, 9.83.