Synthesis and biological evaluation of 1,4-naphthoquinones and quinoline-5,8-diones as antimalarial and schistosomicidal agents

Article abstract of DOI:10.1039/c2ob25812a

Improving the solubility of polysubstituted 1,4-naphthoquinone derivatives was achieved by introducing nitrogen in two different positions of the naphthoquinone core, at C-5 and at C-8 of menadione through a two-step, straightforward synthesis based on the regioselective hetero-Diels-Alder reaction. The antimalarial and the antischistosomal activities of these polysubstituted aza-1,4-naphthoquinone derivatives were evaluated and led to the selection of distinct compounds for antimalarial versus antischistosomal action. The Ag^II-assisted oxidative radical decarboxylation of the phenyl acetic acids using AgNO₃ and ammonium peroxodisulfate was modified to generate the 3-picolinyl-menadione with improved pharmacokinetic parameters, high antimalarial effects and capacity to inhibit the formation of β-hematin. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25812a

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PAPER
Synthesis and biological evaluation of 1,4-naphthoquinones and
quinoline-5,8-diones as antimalarial and schistosomicidal agents†
Don Antoine Lanfranchi,a Elena Cesar-Rodo,a Benoît Bertrand,a Hsin-Hung Huang,b Latasha Day,b
Laure Johann,a Mourad Elhabiri,a Katja Becker,c David L. Williamsb and Elisabeth Davioud-Charvet*a
Received 27th April 2012, Accepted 14th June 2012
DOI: 10.1039/c2ob25812a
Improving the solubility of polysubstituted 1,4-naphthoquinone derivatives was achieved by introducing
nitrogen in two different positions of the naphthoquinone core, at C-5 and at C-8 of menadione through a
two-step, straightforward synthesis based on the regioselective hetero-Diels–Alder reaction. The
antimalarial and the antischistosomal activities of these polysubstituted aza-1,4-naphthoquinone
derivatives were evaluated and led to the selection of distinct compounds for antimalarial versus
antischistosomal action. The AgII-assisted oxidative radical decarboxylation of the phenyl acetic acids
using AgNO3 and ammonium peroxodisulfate was modified to generate the 3-picolinyl-menadione with
improved pharmacokinetic parameters, high antimalarial effects and capacity to inhibit the formation of
β-hematin.
Schistosomiasis is a neglected tropical disease caused by
trematodes of the genus Schistosoma. There are five major
Introduction
Malaria is a mosquito-borne infectious disease caused by proto-
zoan parasites in the genus Plasmodium, which is transmitted
via the bites of infected mosquitoes. It is broadly distributed in
the Sub-Saharan Africa, and tropical and sub-tropical countries
worldwide. Each year, there are approximately 300 million cases
of malaria, killing an estimated one million people per year,
mainly infants under five years of age in Sub-Saharan Africa.
Four major species of the Plasmodium parasite can infect
humans; the most serious forms of the disease – cerebral
malaria, severe anemia and death – are largely caused by P. falci-
parum. In humans, the parasites multiply first in the liver, and
then infect red blood cells where they develop the symptomatic
intraerythrocytic stage of the disease. If not treated, malaria can
quickly become life-threatening by increasing the inflammatory
environment and disrupting the oxygen supply to vital organs,
such as the brain. In many parts of the world, the parasites have
developed resistance to a number of malaria medicines.
species of Schistosoma infecting humans, S. mansoni, S. japonicum,
S. haematobium, S. intercalatum and S. mekongi.
According to the World Health Organization, 200 million
people are infected, 600 million people are at risk of infection
and more than 200 000 deaths per year are caused by schisto-
somiasis. The disease is often co-endemic with malaria, found in
most of Africa, Brazil, China, and the Philippines. The inter-
mediate host of the parasite, different species of fresh-water
snails, permits human infection by skin contact with infective
cercariae.1
Our previous work on targeting redox equilibria of malaria
parasites propagating in red blood cells in vitro and in vivo has
led to the selection of a lead series of potent antimalarial
2-methyl-3-benzyl-1,4-naphthoquinones (benzylNQ, Fig. 1).2
aEuropean School of Chemistry, Polymers and Materials (ECPM)
University of Strasbourg, UMR CNRS 7509, 25 Rue Becquerel, F-67087
Strasbourg, France. E-mail: elisabeth.davioud@unistra.fr;
Fax: +33 (0)3 68 85 27 42; Tel: +33 3 68 85 26 20
bDepartment of Immunology/Microbiology, Rush University Medical
Center, 1735 West Harrison Street, Chicago, IL 60612, USA
cInterdisciplinary Research Center, Nutritional Biochemistry, Justus
Liebig University Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen,
Germany
†Electronic supplementary information (ESI) available: Experimental
protocols for enzymic assays and pharmacokinetics; 1H and 13C NMR
spectra of compounds 1a–d, 2a–d, 3a–d, 4a–d, and 11. See DOI:
10.1039/c2ob25812a
Fig. 1 Menadione and its aza-analogs (1–4), and the recently
described antimalarial redox-active lead 5 and metabolite 6 derivatives.
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The antimalarial mode of action was proposed to involve a
cascade of redox reactions in the parasite. The postulated bioacti-
vation of the antimalarial 3-benzyl-menadione is thought to
generate redox-active metabolites which, (i) in their oxidized
form, are reduced by NADPH in glutathione reductase-catalyzed
reactions within the cytosol of infected red blood cells; (ii) in
their reduced forms, can convert methemoglobin(FeIII), the
major nutrient of the parasite, into the very slowly digestible
hemoglobin(FeII).3 Consequently, the antimalarial lead
benzylNQ are suggested to perturb nutrient acquisition and the
major redox homeostasis of the targeted infected red blood cells,
resulting in development arrest and death of the malaria parasite
at the trophozoite stage.2a
Of the crucial physico-chemical properties of the lead anti-
malarial benzylNQ that needed to be significantly improved, the
solubility in aqueous solutions was the first to be targeted. One
possible way to increase aqueous solubility might be to replace
the naphthalene unit of the 1,4-naphthoquinone (NQ) backbone
with a quinoline. Indeed, in addition to its nitrogen-solubilizing
property, the quinoline moiety is frequently found in antimalarial
drugs,4 e.g., chloroquine, in the 4-aminoquinoline series,
quinine, in the 4-methylenealcohol quinoline series, primaquine,
in the 8-aminoquinoline series, etc. Furthermore, the quinoline-
5,8-dione core is often found in antibiotics produced by various
microorganisms. These natural products are reported to exhibit a
broad spectrum of biological activities including antibacterial,
antifungal, antiviral, and antitumor properties.5,6 Examples
include streptonigrin, produced by an actinomycete (Strepto-
myces flocculus)5a lavendamycin (Streptomyces lavendulae),5h,6
and quinolobactin7 (Pseudomonas fluorescens) (Fig. 2).
Scheme 1 General chemical structures of 1,4-naphthoquinones and
related aza-naphthoquinones (aza-NQ) and bioreduction mechanism
through NADPH-dependent disulfide reductase catalysis coupled to
methemoglobin reduction.
chelator (log KLFe = 13.69; log βL2Fe = 26.3; log KL3Fe = 36.9
for 8-hydroxyquinoline)9b was also proposed to exert an anti-
malarial effect.10,11 In the present work, we considered the syn-
thesis of the benzylated aza-menadiones (or the aza-benzylNQ)
analogs, resulting from the merging of both entities – benzylNQ
and aza-menadione – into one molecule in order to increase both
the rate of the toxic redox-cycling in the parasites and the solubi-
lity of the final molecules (Scheme 1).
Results and discussion
Previous studies have highlighted the aza-analogs of mena-
dione (aza-menadiones, Scheme 1) as substrates of both human
glutathione reductase (hGR) and Plasmodium falciparum gluta-
thione reductase (PfGR).8 Electrochemical studies showed that
introduction of one nitrogen atom at C-5 or at C-8 of menadione
increased the oxidant character of the molecule.5d,8 Quinone
reduction catalyzed by GR might generate one or two electrons-
reduced aza-NQH2 species characterized by an oxine (8-hydro-
xyquinoline) backbone (Fig. 2). The oxine unit is known to be a
strong and selective iron (and copper) chelator.9 This property is
the feature of siderophores and contributes to the aza-NQ
cycling between the cytosol, where quinoline-5,8-dione
reduction occurs, and extracellular medium, as was illustrated
with streptonigrin.5e Furthermore, besides its intrinsic redox
cycling properties, the strong affinity for iron(III) of the oxine
Chemistry
The synthesis of aza-analogs of 3-benzylmenadiones was
designed by using the very reliable Jacobsen–Torssell reaction12
(better known as the Kochi–Anderson procedure13) between
commercial phenylacetic acids and “aza-menadiones” 1–4
(quinoline-5,8-diones shown in Fig. 1). None of these quinoline-
5,8-diones are commercially available; 3 and 4 are not even
described compounds. Despite their relatively simple structure,
these “aza-menadiones” are not readily synthesized in bulk.
The quinoline-5,8-dione core is usually obtained by the oxi-
dation of 5- or 8-hydroxy/aminoquinoline derivatives as well as
their 5,8-diamino, 5,8-dihydroxy, or 5,8-hydroxyaminoquinoline
counterparts. Many oxidizing agents have been employed for
that purpose including chromium,14 hypervalent iodine deriva-
tives,15 Fremy’s salt,16 molecular oxygen and salcomine,17 and
photochemical oxidation18 to cite a few. However, in our case,
most of the appropriate 5- or 8-amino/hydroxyl or 5,8-diamino/
dihydroxy/hydroxyamino (di)methylquinolines precursors are
not commercially available or are very expensive, and their prep-
aration uses a tedious route leading to poor yields.
Quinoline-5,8-diones can be synthesized in high yield (>90%)
by oxidative demethylation of 5,8-dimethoxyquinolines,19 which
can be prepared from 2,5-dimethoxyaniline derivatives with the
help of a modified Skraup reaction, although in poor yield
(20%).20 However, once again, the desired aniline derivatives are
not commercially available.
Except for oxidative methods, the hetero-Diels–Alder reaction
between an 1-aza-buta-1,3-diene and a 1,4-(naphtho)quinone is
Fig. 2 Antimalarial and antibiotic drugs possessing either a quinoline,
a redox-active quinoline-5,8-dione, or an oxine core.
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an attractive method to build the quinoline-5,8-dione core.21
Ghosez and coworkers22 have popularized this synthetic route by
adding a dimethylamino group on the N−1 position (acrolein
N,N-dimethylhydrazone derivatives), which consequently
increases the electrophilic character of the aza-diene. Acrolein
N,N-dimethylhydrazone derivatives and quinone compounds are
commercially available or are easily obtained in high yield in
one or two steps.23 Generally, the yields of hetero-Diels–Alder
ranged from good to excellent. However, when applied to our
substrates, the hetero-Diels–Alder reaction between bromo
2-methyl-1,4-quinones and acrolein N,N-dimethylhydrazone pro-
duced aza-menadiones 1 and 2 in very poor yield (only 9% and
7%, respectively).8 In fact, it is well known that the initial N,N-
dimethylhydrazine cycloadduct spontaneously releases dimethyl-
amine that can react with the double bond of the quinonic
moiety (i.e. hetero-Michael addition, Scheme 2, path i), giving
rise to the formation of undesirable by-products and contributing
to lower the yield of the hetero-Diels–Alder reaction.23 Further-
more, dimethylamine, which can be considered as a strong base
(pKa = 10.7724), might react with an acidic proton to generate,
for instance, a (vinyl)enolate (Scheme 2, path ii). The later might
add to ketones or enones also leading to other by-products.
Menadione, which possesses a vinylogous methyl ketone and an
electrophilic site on its quinonic moiety, polymerizes in the pres-
ence of bases via quinone methide formation.25 Moreover,
amino-based compounds add on the menadione double bond or
its methyl group (Scheme 2, path i & ii).26 In this respect, the
aza-menadione core behaves similarly.
Scheme 3 Total synthesis of the aza-menadiones (1–4) and of the
related 6- or 7-methyl-benzylquinoline-5,8-diones (1a–d to 4a–d) in a
two-step sequence. Reagents and conditions: (a) Br2, AcOH, rt, 2h; (b)
CrO3, AcOH, rt, 1 h; (b′) CAN, MeCN, rt, 1 h; (c) 1,1-dimethylhydra-
zine, DCM, AcOH cat., 0 °C, 30 min; (d) Ac2O, MeCN, rt, 2 h; (e) sub-
stituted phenylacetic acid, 0.35 equiv AgNO3, 1.3 equiv (NH4)2S2O8,
MeCN–H2O (3 : 1), 85 °C, 3 h.
With the aim to improve the yield of aza-anthraquinone syn-
thesis, Chigr et al.,25 albeit without success, added acetic anhy-
dride as a dimethylamine scavenger during hetero-Diels–Alder
between juglone (or its O-methyl ether) and crotonaldehyde
N,N-dimethylhydrazone. In our hands, introduction of an excess
of acetic anhydride (15–20 equiv) successfully led to improved
yield of the aza-menadione 1 (43% vs. 7%). As a matter of fact,
we observed the characteristic signals of dimethylacetamide in
Scheme 4 Jacobsen–Torssell reaction versus Minisci reaction in the
preparation of the picolinyl-NQ 11. Reagents and conditions: (a) 1.2
equiv AgNO3, 1 h; (b) menadione, 0.35 equiv AgNO3, 1.3 equiv
(NH4)2S2O8, MeCN–H2O (3 : 1), 85 °C, 3 h.
1
the H NMR spectra of the crude mixture (Scheme 2, path b). It
should be noted that both trifluoroacetic anhydride and acetyl
chloride failed to substitute for acetic anhydride, leading to
intractable mixture. With larger amounts of “aza-menadiones”
1–4 in hand, we thus performed the Jacobsen–Torssell reaction
as depicted in Scheme 3. The silver(II)-mediated radical decar-
boxylation reaction between the four aza-menediones and com-
mercial phenylacetic acids afforded sixteen targeted products
(Scheme 3) in reasonably good yield (33–58% for 2a–d and 4a–d;
31–57% for 1a–d and 3a–d).
We were also interested in introducing a nitrogen atom into
the other aromatic subunit of 3-benzyl-menadiones. The first
attempt to synthesize the picolinyl-NQ 11 (Scheme 4) from com-
mercial menadione and the hydrochloride salt of 4-pyridylacetic
acid under the conditions of the radical decarboxylation of
Kochi–Anderson failed. As chloride anions induced precipitation
of silver cation (AgCl formation) and therefore poisoned the
silver catalyst, we prepared the nitrate salt of 4-pyridylacetic acid
through silver counter-ion exchange (Scheme 4). The desired
picolinyl-NQ 11 was obtained but with very poor yield (10%).
All attempts to improve the yield failed. This was certainly due
to the fact that γ-picoline radical preferred to react with itself (or
the starting pyridine) rather than menadione. Indeed, there is a
competition between Jacobsen–Torssell reaction (radical addition
to quinone) and Minisci reaction (radical addition to pyridine).27
Antiparasitic activities and toxicity against human cells
All the aza-NQ (included the picolinyl-NQ 11) were tested for
antimalarial effects using the 3H-hypoxanthine incorporation-
based assay. Inhibition of the growth of P. falciparum by the
Scheme 2 Improved synthesis of the 6-methyl-quinoline-5,8-dione 1
through hetero-Diels–Alder reaction by addition of an excess of acetic
anhydride. Reagents and conditions: (a) MeCN, rt; (b) Ac2O excess.
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compounds was evaluated by determining the inhibitor concen-
tration required for killing 50% of the parasites (IC50 values). In
a screening assay, all compounds were tested against the CQ-
resistant P. falciparum strain Dd2 (Table 1).
In order to vary the substitution pattern of the benzyl moiety,
the electron-withdrawing and lipophilic p-CF3 group was
replaced by dimethoxy groups on the benzene in 3,5 (1c and 2c)
or in 2,5 (1d and 2d) positions (Fig. 3). Also, a methyl group
was introduced at the meta-position to the nitrogen of the quino-
line-5,8-dione core through the hetero-Diels–Alder reaction in
order to observe the effect on drug metabolism. Interestingly, the
3-methyl-substituted 5-aza 3a–3d provided slight potency
enhancements against Plasmodium (Table 1) compared to the
non-methylated 5-aza-analogs 1a–1d, confirming methyl substi-
tution at the meta-position to the nitrogen is favorable. Again, as
observed with the p-CF3 series, the 3-methyl-substituted 5-aza-
In order to gather more information about the mechanism of
action of our lead compounds, we investigated the antiparasitic
activity of the aza-menadione analogs against another blood-
feeding parasite. It is well documented that both Plasmodium
parasites and Schistosoma worms synthesize heme crystals
similar to hemozoin (called pigment) in vivo.28–30 Heme biocrys-
tallization occurs in the gut of the worm29a and in the intracellu-
lar food vacuole of Plasmodium.30 Both the malarial and the
schistosome pigments have structures similar to synthetic
β-hematin, a physicochemical feature that has been intensively
reviewed.30 Indeed, interference with hemozoin formation rep-
resents an important mechanism of the schistosomicidal action
of antimalarial quinoline methanols including quinine and
quinidine.29b
The structure–activity relationships of the aza-analogs were
also evaluated for S. mansoni worm killing, assayed by visual
examination of cultured worms treated with compound at 50 μM
over a 48 h time course. The experiment was done under two
conditions: one subset with the drug alone and another subset
with the drug in the presence of human red blood cells in order
to stimulate heme-catalyzed drug metabolism due to hemoglobin
digestion. While the lead antimalarial 3-benzylNQ bearing a
p-CF3 group (5, Fig. 1) was very active against the malaria
parasite (Table 1) with observable inhibition of the hemozoin
formation in intact parasites in culture,2a the same compound
displayed no schistosomicidal activity (Table 1). The 5-aza- and
the 8-aza-analogs of 5, 1b and 2b showed reduced activity
against Plasmodium and low activity against ex vivo worms
(Table 1).
Fig. 3 Structures of the polysubstituted 6- or 7-methyl-benzylquino-
line-5,8-diones (1a–d to 4a–d) studied for antiparasitic action.
Table 1 Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase, in vitro antimalarial and antischistosomal activities, and cytotoxicities
against human cells by aza-analogs, the 6- or 7-methyl-benzylquinoline-5,8-diones (1a–d to 4a–d), and the picoliny-NQ 11
Pf 3D7 IC50
(nM)
Pf Dd2 IC50
(nM)
IC50 (μM) or % TGR
h MRC-5 toxicity
IC50(μM)
Ex vivo Sm worm killingb
(% dead)
Compounds
inhibitiona
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
11
5
1700–2400
727
2459.0
1715.0
2172.2
1206.3
9131.2
>3200
>3100
>3100
691.3
364.7
623.5
437.5
>2800
>2900
>3000
>3000
109.4
69.2
12
6.41
4.45
2.58
5.25
5.90
6.93
2.15
1.78
30.05
2.95
4.00
1.87
2.28
9.19
0.67
0.95
3.12
>322a
51.542a
0% (+rbc)
50% ( rbc)
50% (−rbc), < 50% (+rbc)
<40% ( rbc)
∼43% inhibition
3.9
1510.8
1349.7
9516.6
>3200
>3100
>3100
340.25
460.25
442.5
∼70% inhibition
11.2
∼48% inhibition
∼40% inhibition
∼46% inhibition
6.02
∼58% inhibition
20.4
∼48% inhibition
0.332
∼71% inhibition
∼53% inhibition
∼53% inhibition
∼56% inhibition
∼69% inhibition
∼33% inhibition
30% ( rbc)
36% (−rbc), 41% (+rbc)
68% (−rbc), 39% (+rbc)
38% (−rbc), 28% (+rbc)
<40% ( rbc)
93% (+rbc)
65% (−rbc), 43% (+rbc)
71% (−rbc), 38% (+rbc)
0% (+rbc)
100% (+rbc)
0% (+rbc)
391.5
>2800
>2900
>3000
>3000
119.8
100% (+rbc)
0% ( rbc)
67.5
0%c
Chloroquine 7.9
147.7
0%c
a Data were measured in the DTNB reduction assay in 0.1 M phosphate buffer, 10 mM EDTA at 25° C, in the presence of 100 μM NADPH, 10 nM
SmTGR and 50 μM compound. b Data represent the results from three separate experiments carried out at 50 μM drug concentration in the presence or
in the absence of red blood cells (rbc). Data were collected by visual examination of worm movement and shape. c +10 μM human hemoglobin after
24 h.
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3c, 3d and 8-aza-analogs 4b, 4d can decrease S. mansoni worm
survival significantly compared to the non-methylated 5-aza-
1a–1d, and 8-aza-analogs 2a–2d. Likewise, the p-CF3 group
was also replaced by p-Br. No improvement in antiplasmodial or
antischistosomal activity was noted.
The picolinyl-NQ 11 was found to be slightly less active than
the lead 5 against both P. falciparum strains in culture, but both
were ineffective as S. mansoni worm killing agents no matter
whether red blood cells were present or not. The results detailed
in Table 1 showed clear evidence that we can dissociate the anti-
malarial activity from the worm killing capacity by introduction
of the structural diversity at the benzyl chain or/and at the quino-
line ring of the quinoline-5,8-dione core.
of our aza-menadione derivatives was exemplified in the follow-
ing GR assays, by investigating metHb reduction, in the presence
of the picolinyl-NQ 11, and the NADPH-GR system.
Finally, the toxicity of all aza-1,4-naphthoquinone derivatives
was evaluated against the human lung fibroblast cell line
MRC-5. Compared to the starting lead compound 5 which had
low toxicity against two human cell lines, the 5-aza- and the
8-aza-analogs exhibited low IC50 values, suggesting modification
of the partition coefficient of final aza-molecules may have
increased their cell penetration capabilities. Another explanation
could also come from the modified redox potential values8
impacting the specificity of the subversive substrates with
respect to the diaphorase activities of a larger panel of flavo-
enzymes from the host, e.g. human TrxR or LipDH. The least toxic
compound, 3a, was found to be the 3-methyl-5-aza-analog of
the potent lead antimalarial benzylNQ with a p-Br-substitution at
the benzyl chain. The structure–activity relationships and the
rather low molecular weight of the lead benzylNQ 5 (∼300 Da)
suggest that there is a ‘room’ for varying the chemical modifi-
cation of aromatic rings to modulate antiparasitic action vs.
specificity to the targets vs. redox potentials vs. cytotoxicity
against human cells.
In conclusion, all 5-aza- and the 8-aza-menadione analogs
had reduced activity against Plasmodium and minimal ability to
kill ex vivo worms at 50 μM. However, from the structure–
activity relationships, significant enhancements or decreases in
the antiparasitic activities could be drawn.
Enzymatic assays
In a previous work, we investigated the redox-cycling capacity
and the inhibitory effects of a series of six 5-aza- and 8-aza-
menadiones on three disulfide reductases, i.e. the human gluta-
thione reductase and both human and Plasmodium falciparum
thioredoxin reductases (TrxR).8 In the present study, all com-
pounds from the 5-aza- and 8-aza-menadione series were evalu-
ated for their potential to inhibit S. mansoni thioredoxin
glutathione reductase (SmTGR), a hybrid enzyme that carries out
both thioredoxin and glutathione reduction in the worm,31 in the
disulfide reduction assay using the artificial substrate DTNB. A
primary screening SmTGR assay was carried with each com-
pound at 50 μM. Compounds inhibiting the enzyme by ≥80%
were further analyzed. The results of this study are presented in
Table 1. Among all the 5-aza-menadione and 8-aza-menadione
derivatives tested, only six compounds inhibited SmTGR by
≥80%; the remaining inhibited the enzyme by 40 to 70%. The
compounds with the most potent SmTGR inhibitory activities are
11, 1a, 1c, 3a, 3c, 4a, and 2a. Most of these compounds dis-
played moderate SmTGR activities with IC50 values from 3.9 to
20.4 μM. However, 4a displayed a very potent SmTGR inhibi-
tory activity in the nanomolar range with an IC50 value of 332
nM. There was however, no correlation between SmTGR inhi-
bition and worm killing activity. This is possibly due to the fact
that SmTGR interaction with the quinones was investigated
through inhibition of disulfide reduction in this study. It is well
documented that many flavin-binding disulfide reductases show
diaphorase activities catalyzing the reduction by NAD(P)H of
various dyes (e.g. methylene blue) and quinones which act as
hydrogen acceptors. Menadione reduction was not always cor-
related to inhibition of disulfide reduction, as illustrated with
P. falciparum glutathione reductase2a,32a vs. Trypanosoma cruzi
dihydrolipoamide dehydrogenase (LipDH).33a The latter flavo-
enzyme, previously called menadione reductase, did not exhibit
high and significant decreased disulfide reductase activity in the
presence of menadione and analogs.33b The flow of electrons
from the nicotinamide ring of NADPH proceeds via the flavin
ring of GR-bound FAD to menadione.32b The substrate capacity
Physico-(bio)chemical assays
In this preliminary physico-(bio)chemical study, we evaluated
the strength of the interactions between the picolinyl-NQ 11 and
iron(III)-containing targets (hematin, methemoglobin) in solution
under quasi-physiological conditions. Plasmodium (acidic food
vacuole) and Schistosoma (gut of the worm29) parasites digest
methemoglobin (metHb), resulting from endogenous oxidation
of hemoglobin (Hb), as a source of essential nutrients. Both
parasites neutralize these toxic heme species via major detoxifi-
cation processes, mainly in the food vacuole at pH 5.2 where
autoxidation of hemoglobin(FeII) takes place: (i) biocrystalliza-
tion into an insoluble and “inert” crystal called hemozoin29,30
(malaria pigment or Schistosoma pigment), (ii) rapid peroxida-
tive decomposition by reacting with H2O2,34 and in the cytosol
(iii) activities of an efficient thiol redox network based on the
regeneration of thiols catalyzed by NADPH-dependent disulfide
reductases (GR of Plasmodium-infected human erythrocytes and
Schistosoma TGR).2,31,35,36 Several series of antimalarial drugs
have been reported to exert their effects through the enhancement
of free heme toxicity via the inhibition of hemozoin formation
(e.g. 4-aminoquinolines,4,30,37 quinoline methanols,29c,38 endo-
peroxides39 or xanthones40).
Inhibition of hemozoin formation represents the most impor-
tant rational drug development strategy for malaria over the last
few years.4,30 In this context, the ability of a heme binding agent
to strongly interact with heme in solution is usually an important
pre-requisite for further development. Fig. 4A shows the spectro-
photometric titration of hematin binding by picolinyl-NQ 11 by
monitoring changes in absorption of the hematin π–π
dimer.30d,41 Concomitant bathochromic shifts and narrowing of
the Soret band were observed indicating the formation of com-
plexes of picolinyl-NQ 11 with hematin (Fig. 4B). An apparent
dissociation constant (KD = 1.66 μM) and the stoichiometry of
the species formed at equilibrium ([(FeIIIPPIX)2(L)] with log β21
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Fig. 5 Inhibition of β-hematin crystallization by the picolinyl-NQ 11 at
pH 4.5 (12.9 M acetate buffer) measured by UV-visible absorption spec-
trophotometry using pyridine (5% by volume) as reporting reagent.
(solvent: water; pH 7.5 HEPES buffer 200 mM then 20 mM, T =
25 °C). The cross (×) and the square (■) labels correspond to two inde-
pendent replicate, while the solid line corresponds to the averaged
values.
Fig. 4 (A) Absorption spectrophotometric titration of hematin (in a
π–π dimeric state) by the picolinyl-NQ 11 (noted L). Solvent: 0.2 M
HEPES buffer pH 7.5; l = 1 cm; [FeIIIPPIX]tot = 2.93 × 10−5 M. In
panel A, spectra were recorded at varying [L]tot/[FeIIIPPIX]tot ratio, (1)
from [L]tot/[FeIIIPPIX]tot
= 0 = 2.44.
to (2) [L]tot/[FeIIIPPIX]tot
(B) Absorption electronic spectra of the complexes formed between
FeIIIPPIX and the picolinyl-NQ 11 ligand.
= 12.6 0.2 and [FeIIIPPIX)(L)2] with log β12 = 10.6 0.2)
have been determined.
After evaluation of the heme binding capacities of picolinyl-
NQ 11, we undertook a biochemical investigation42 of the
activity of this compound to inhibit hematin crystallization to
β-hematin (i.e. synthetic equivalent of hemozoin). This assay,
performed at pH ∼ 5 (i.e., pH at which hemozoin biomineraliza-
tion efficiently occurs), is based on a classical hematin quantifi-
cation method.43 An IC50 value of 4.3 (Drug (equiv)/Hematin
(equiv)) with ∼80% of inhibition of β-hematin formation by the
picolinyl-NQ 11 was obtained (Fig. 5). Taken together, these pre-
liminary data clearly illustrate that the heme binding properties
observed for the picolinyl-NQ 11 may account for its capacity to
efficiently prevent β-hematin formation in vitro and to a signifi-
cant extent of hemozoin formation in vivo.
As exemplified with our antimalarial lead 3-benzylmenadione
derivatives and with methylene blue by Schirmer’s group,41a,44
the redox-based mechanism of action against malarial parasites
is a recent molecular therapeutic strategy to cure parasitic dis-
eases.2,41 Efficient and fast reduction of methemoglobin(FeIII) to
oxyhemoglobin(FeII) through continuous use of a NADPH flux
under catalysis of disulfide reductases (GR or TGR) in the pres-
ence of redox-active mediators (naphthoquinones,2 aza-naphtho-
quinones8) is expected to significantly slow down metHb(FeIII)
digestion. We therefore investigated the ability of the picolinyl-
NQ 11 to inhibit β-hematin formation, not solely on the basis of
hematin binding (Fig. 5), but also on the basis of electron trans-
fer reactions. We evaluated if, in its reduced states, the picolinyl-
Fig. 6 (A) UV-visible absorption spectra recorded as a function of
time showing the fast metHb(FeIII) Reduction in the coupled assay with
the hGR/NADPH system in the presence of the picolinyl-NQ 11. (B)
Electronic spectra of the reactants and the products of the metHb(FeIII)
reduction. Solvent: water (hGR acetate buffer pH 6.9 + 47 mM K2PO4 +
200 mM KCl); T = 37.0 °C; 40 μM NADPH + 133 nM hGR + 8 μM
metHb + 40 μM picolinyl-NQ 11. In panels A and B, spectra were
recorded as a function of time (1) at t = 0; (2) at t ∼ 18 min.
NQ 11 can trigger metHb(FeIII) reduction to oxyHb(FeII)
(Fig. 6).
The picolinyl-NQ 11 is efficiently reduced by NADPH with
mediation of GR and, in its reduced forms (semi-naphthoquin-
one or dihydronaphthoquinone), catalytically and specifically,
transfers electrons to methemoglobin(FeIII) (kobs = (1.95
red
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0.02) × 10−3 s−1). Both events, oxidizing the reductants (like
NADPH) and reducing the oxidants (like Fe(III)-containing
targets), in a continuous redox cycle are expected to lead to the
death of the parasites. It appeared from our physico-(bio)chemi-
cal investigations that the capacity of the reduced picolinyl-NQ
11 to rapidly and efficiently reduce metHb has also to be taken
into account to rationalize their potential in inhibiting (directly or
indirectly) hemozoin formation.
enveloping the worm is an effective barrier to most hydrophilic
molecules.
Discussion
Malaria and schistosomiasis-associated pathologies are respect-
ively caused by the continuous expansion of Plasmodium para-
sites inside host erythrocytes, and to egg-laying by adult worms
living in the host bloodstream. Both parasites, the protozoan
Plasmodium and the helminth Schistosoma, feed on host hemo-
globin. This process along with the immune response of the host
toward infection causes an intense flux of reactive oxygen
species. To maintain a reducing intracellular milieu in oxygen-
rich environments, malaria and schistosome parasites have
evolved a complex antioxidative network based on two central
electron donors, glutathione and thioredoxin, that are regenerated
by NADPH-dependent disulfide reductases. Plasmodium berghei
GR and thioredoxin reductase are dispensable flavoenzymes for
proliferation of the pathogenic blood stages, but GR-null malaria
parasites, with normal blood stage growth, arrest during develop-
ment in the mosquito.46 As clearly demonstrated, GR is vital for
extracellular parasite development inside the insect vector,
whereas thioredoxin reductase is dispensable during the entire
parasite life cycle. By comparison, Schistosoma worms have a
unique multifunctional NADPH-dependent enzyme, TGR,
which is essential to maintain a proper redox homeostasis during
its survival in the host; the requirement of TGR was assessed by
inhibitor studies and RNA interference experiments.31b These
findings corroborate our drug design projects based on the
development of subversive substrates of GR2,26,32c,41a and
TGR31b combined with the inhibition of hemozoin formation –
two drug targets selected for many years as promising antimalar-
ial and antischistosomal intervention strategies.
Pharmacokinetics
At the lead discovery stage, it is critical that the compounds
identified are not only optimized for their efficacy against the
selected parasites but also to have a characteristic “drug profile”.
This is essential for successful downstream development; the cri-
teria include limited and tolerable side effects, high oral bioavail-
ability, and an appropriate pharmacokinetic pattern.45 When
synthesizing a library of naphthoquinone derivatives we were
further interested in determining if quinoline-5,8-dione core scaf-
fold has suitable properties for oral bioavailability by simply
analyzing the structural physicochemical properties (e.g. number
of hydrogen bonding partners, MW, log D) according to the
Lipinski et al.’s rule, which referred to simple descriptors corre-
lated to oral drug absorption.45 Thus, determination of the solu-
bility (in μM), lipophilicity index (CHI, Chromatographic
Hydrophobicity Index), and log D were determined in order to
predict suitability of the compounds for oral drug administration.
In addition, potential increases in resistance to drug metabolism
were introduced through the hetero-Diels–alder reactions by pre-
paring a series of metabolically-resistant aza-analogues through
the introduction of a methyl group with respect to the nitrogen in
the phenyl rings of the 3-benzylNQ core. The results of these
profiles are shown in Table 2.
In the malaria P. berghei blood stage parasite, even if GR and
TrxR are able to largely compensate for each other, the simul-
taneous interference of both human erythrocytic and Plasmo-
dium GR by redox-active agents is expected to lower the
NADPH flux in infected red blood cells. Two successful
examples of “multi-target-directed redox-cyclers” are illustrated
with the potent antiplasmodial agents, methylene blue and the
3-benzyl menadiones, acting as human and plasmodial GR
redox-cycling substrates in infected red blood cells. Simul-
taneously interfering with redox metabolism by distinct disulfide
reductases and hemoglobin catabolism/heme detoxification rep-
resents a promising approach to antimalarial drug development.
As demonstrated with fluorine-based suicide substrates of
GR2a,32b the bioactivation of the antimalarial benzylNQ
appeared to be essential to observe the antimalarial effects of the
lead series. The objective was not exclusively to inhibit GSSG
reduction in infected red blood cells because, from biochemical47
and molecular biology studies,46 GSSG reduction can be main-
tained by other redox partners of the thiol network, e.g. reduced
thioredoxin. Of greater importance is the NADPH flux, which
can be diverted to a futile cycle in the presence of redox-active
agents by exploiting the GR reductive catalysis for drug bioacti-
vation in infected red blood cells and/or drug combination in
concert with metHb catabolism.2,41 As reported earlier, the high
frequency of the sickle-cell hemoglobinopathy and of glucose-6-
The lead 3-benzylmenadione 5, with the p-CF3 group, has
very low solubility in water. Introduction of the nitrogen at the
phenyl rings of the 3-benzyl-NQ core slightly increased the solu-
bility of the final quinoline-5,8-diones. The most soluble com-
pound was the menadione substituted at C-3 by a 4-picolinyl
chain (see derivative 11). Beyond solubility profiling, the aza-
analogues displayed significantly lower lipophilicities and log D
values, a positive feature for improving the antimalarial drug
candidates. However, with this and other series of compounds,
we observed that antischistosomal molecules required a minimal
lipophilicity, probably because the lipid-rich tegument
Table 2 Solubility and selected ADME propertiesa of the lead
benzylNQ and representative aza-analogs
Compound
CHIb
log D7.4
Solubility (μM)
5
115
96
96
96
90
76
4.68
3.68
3.68
3.71
3.37
2.68
0 (nd)c
4b
3b
3a
1a
11
0.29 0.01
0.08 0.01
0.03 0.00
1.09 0.02
0.29
a Protocols for measurements are given in ESI. b CHI (chromatographic
hydrophobicity index).c nd: not detected.
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phosphate dehydrogenase (G6PD) enzyme deficiency in malaria
endemic regions is believed to be due to a natural genetic protec-
tion against fatal malaria.48 Indeed, numerous hemoglobino-
pathies, G6PD and GR deficiencies have been shown to result in
decreased half-life of red blood cells, due to their enhanced
phagocytosis and rapid elimination from the bloodstream by the
immune system.49 At the biochemical level, these genetic vari-
ations result in protection from severe malaria due to a decreased
flux of NADPH and increased oxidative stress in the erythro-
cytes, making the intracellular milieu hostile for the survival of
Plasmodium.
as 4b which specifically kills 100% of S. mansoni worms, and
11 which, by contrast, is devoid of antischistosomal activity but
possess good antimalarial activity (Table 1). Future studies will
be aimed at metabolically blocking key sites of unproductive
metabolism and modulating lipophilicity and solubility to
improve the pharmacokinetics of the lead compounds while still
maintaining similar oxidant character and specific antiparasitic
potency.
Experimental
Our studies with redox-active agents displaying potent anti-
malarial action should be continued by investigation of the mechan-
ism of interaction of these compounds in the full parasitic cycle,
in particular with Plasmodium GR in mosquito stages. These
studies on optimization of lead antimalarial benzylNQ demon-
strated the tremendous work which remains to be done, likely
because the excellent antimalarial action of the lead benzylNQ
might result from subtle combination of pharmacokinetics para-
meters and interference with different targets in Plasmodium
infecting the human and insect hosts: GR catalysis, metHb
reduction, drug transport, hematin complex formation, etc. For
schistosomiasis, the “multi-target-directed redox-cycler”
approach is directly focused on one sole protein, TGR, which
represents the “bottleneck” of the redox thiol network in Schisto-
soma mansoni worms.31b Although both parasites feed on hemo-
globin, the biology of multicellular Schistosoma worms is
significantly distinct from that of unicellular Plasmodium: the
presence of highly lipophilic tegument enveloping the worm vs.
a complex vesicular system that extends from the parasitophor-
ous vacuole into Plasmodium-infected red blood cell cytoplasm
and the plasma membrane; presence of a gut vs. a food vacuole
for hemoglobin digestion, etc… The present work is a first step
in the development of redox-active agents against blood-feeding
malaria and schistosome parasites.
Chemistry
Melting points were determined on a Büchi melting point appar-
atus and were not corrected, the solvent from which compounds
were crystallized is indicated in brackets. 1H (300 MHz) and 13
C
(75 MHz) NMR spectra were recorded on a Bruker DRX-300
spectrometer, chemical shifts were expressed in ppm relative to
TMS; multiplicity is indicated as s (singlet), d (doublet),
t (triplet), q (quadruplet) and m (multiplet). Elemental analyses
were carried out at the Mikroanalytisches Laboratorium der Che-
mischen Instituteder Universität Heidelberg. EI MS were
recorded at facilities of the Institut für Organische Chemie der
Universität Heidelberg. Analytical TLC was carried out on pre-
coated Sil G-25 UV254 plates from Macherey Nagel. Flash
chromatography was performed using silica gel G60
(230–400 mesh) from Macherey Nagel. Starting materials
6-methylquinoline-5,8-dione 1 and 7-methylquinoline-5,8-dione 2,
were synthesized according to reported procedures,8 but with
introduction of an excess of acetic anhydride (15–20 equiv) prior
to addition of the starting quinone dropwise to the azadiene.
3,6-Dimethylquinoline-5,8-dione 3. 1,1-Dimethyl-2-(2-methyl-
allylidene)hydrazine (2.17 g, 19.37 mmol, 1.3 equiv) was added
in 245 mL of MeCN followed by acetic anhydride (19 mL). The
reaction mixture was stirred at room temperature. 2-bromo-6-
methylcyclohexa-2,5-diene-1,4-dione (3.00 g, 14.9 mmol,
1 equiv) in 85 mL of MeCN was then added dropwise (slowly
during 80 min). The reaction mixture was stirred at room temp-
erature during 120 min. The crude was then concentrated and
purified by column chromatography using diethyl ether and
toluene (70 : 30) to give a brown solid. Yield: 21%; mp: 165 °C
dec. (from Et2O). δH (300 MHz, CDCl3, Me4Si) 8.86–8.87 (d,
Conclusion
Taken together, the available data suggest that improving the
pharmacokinetic properties of our lead compounds need to
address different parameters to facilitate future drug develop-
ment: (i) regioselective introduction of structural elements (nitro-
gen, methyl groups) in the aromatic ring of menadione was
achieved by developing a two step-synthesis of 1,4-naphthoqui-
nones by hetero-Diels–Alder reactions; (ii) increasing the solubi-
lity/decreasing the lipophilicity of our compounds was
accomplished by introduction of nitrogen to the phenyl ring of
the lead benzylNQ, which however increased the cytotoxicity
against the human cells; (iii) introduction of CF3 and methyl
groups and a nitrogen atom resulted in differential antimalarial
vs. antischistosomal activities of compounds and introduction of
a methyl group in meta- to the nitrogen increased the antiparasi-
tic action; (iv) the presence of the pyridine, at the picolinyl- vs.
benzyl-chain increased the binding to hematin, and β-hematin
formation inhibition. Increasing the structural diversity within
the series of benzylNQs might allow for the development of
compounds exclusively targeting either malaria or schistosomes
parasites. This goal was partially achieved with compounds such
4J = 1.7 Hz, 1H), 8.2 (d, J = 2 Hz, 1H), 6.91 (d, J = 1.5 Hz,
1H), 2.54 (s, 3H), 2.28–2.29 (d, 4J = 1.5 Hz, 3H) ppm. δC
(75 MHz, CDCl3, Me4Si) 185.33 (CvO), 183.35 (CvO),
155.14 (CH), 147.64 (Cq), 145.63 (Cq), 138.51 (Cq), 136.09
(CH), 134.32 (CH), 128.74 (Cq), 18.88 (CH3), 16.28 (CH3)
ppm. EIMS (70 eV) m/z: 187.0 ([M]+, 100). Elemental analysis
C11H9NO2: calc: C, 70.58; H, 4.85; N, 7.48. Found: C, 70.40;
H, 5.93; N, 7.37.
4
4
3,7-Dimethylquinoline-5,8-dione 4. The title compound was
prepared in the same manner as 3 but from 2-bromo-5-methyl-
cyclohexa-2,5-diene-1,4-dione. Yield: 23%; mp: 150–151 °C
(from Et2O). δH (300 MHz, CDCl3, Me4Si) 8.85 (s, 1H), 8.22 (s,
4
1H), 6.97–6.98 (d, J = 1.5 Hz, 1H), 2.53 (s, 3H), 2.22–2.23 (d,
4J = 1.5 Hz, 3H).δC (75 MHz, CDCl3, Me4Si) 184.56 (CvO),
183.68 (CvO), 155.09 (CH), 149.04 (Cq), 145.63 (Cq), 138.65
(Cq), 134.86 (CH), 133.94 (CH), 128.67 (Cq), 18.86 (CH3),
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3
3
16.69 (CH3). EIMS (70 eV) m/z: 187.0 ([M]+, 100). Elemental
analysis C11H9NO2: calc: C, 70.58; H, 4.85; N, 7.48. Found: C,
70.35; H, 5.03; N, 7.28.
CDCl3, Me4Si) 9.02 (d, J = 4.5 Hz, 1H), 8.43 (d, J = 7.8 Hz,
1H), 7.66 (dd, 3J = 7.8 Hz, 3J = 4.5 Hz, 1H),: 6.72 (m, 3H), 4.01
(s, 2H), 3.78 (s, 3H), 3.71 (s, 3H), 2.26 (s, 3H) ppm. δC
(75 MHz, CDCl3, Me4Si) 183.9 (CvO), 183.6 (CvO), 154.1
(CH), 153.5 (Cq), 151.5 (Cq), 147.5 (Cq), 145.9 (Cq), 145.1 (Cq),
134.7 (CH), 129.0 (Cq), 127.4 (CH), 127.0 (Cq), 116.4 (CH),
111.2 (CH), 111.1 (CH), 55.9 (CH3), 55.6 (CH3), 27.0 (CH2),
13.3 (CH3) ppm. EIMS (70 eV) m/z: 324.1 ([M + H]+, 24),
323.1 ([M]+, 100); 308.1([M–CH3]+, 54). Elemental analysis
C19H17NO4: calc: C, 70.58; H 5.30; N, 4.33. Found: C, 70.51;
H, 5.38; N, 4.22.
General procedure for synthesis of the related 6- or 7-methyl-
benzylquinoline-5,8-dione derivatives (1a–d to 4a–d). One of the
four aza-menadiones, (1–4) (1 equiv, 0.05 mmol mL−1) and a
substituted phenyl acetic acid derivative (2 equiv) were added to
a stirred solution of MeCN–H2O (3 : 1) and heated at 85 °C
(70 °C in the flask). AgNO3 (0.25 equiv) was added first and
then (NH4)2S2O8 (1.3 equiv, 0.36 mmol mL−1) in MeCN–H2O
(3 : 1) was added dropwise. The reaction mixture was addition-
ally heated for 2–3 h at 85 °C. MeCN was evaporated and the
mixture was extracted with dichloromethane. The crude mixture
was purified by flash chromatography on silica gel using a
mixture diethyl ether and toluene (70 : 30). When necessary, the
final 6- or 7-methyl-benzylquinoline-5,8-dione (1a–d to 4a–d)
was further purified by trituration in diethyl ether.
6-(4-Bromobenzyl)-7-methylquinoline-5,8-dione
2a. Yield:
33%; mp: 105–106 °C (from Et2O). δH (300 MHz, CDCl3,
Me4Si) 9.02 (s, 1H), 8.43 (d, J = 7.5 Hz, 1H), 7.64–7.66 (d, J =
6.1 Hz, 1H), 7.26 ((AB)2 system, J = 8.3 Hz, Δν = 72.4 Hz, 4H),
4.05 (s, 2H), 2.27 (s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si)
184.7 (CvO), 183.0 (CvO), 154.6 (CH), 147.4 (Cq), 145.9
(Cq), 144.1 (Cq), 136.5 (Cq), 134.5 (CH), 131.8 (2 × CH), 130.6
(2 × CH), 128.9 (Cq), 127.6 (CH), 120.5 (Cq), 32.0 (CH2), 13.3
(CH3) ppm. EIMS (70 eV) m/z: 341.0 ([M]+, 17), 325.9 ([M–
CH3]+, 57). Elemental analysis C17H12BrNO2: calc: C, 59.67; H,
3.53; N, 4.09; Br, 23.35. Found: C, 59.57; H, 3.65; N, 4.02; Br,
23.13.
7-(4-Bromobenzyl)-6-methylquinoline-5,8-dione
1a. Yield:
46%; mp: 132–133 °C (from Et2O). δH (300 MHz, CDCl3,
Me4Si) 9.01 (dd, J = 4.6 Hz, 1.7 Hz, 1H), 8.41 (dd, J = 7.9 Hz,
1.7 Hz, 1H), 7.65 (dd, J = 7.9 Hz, 4.6 Hz, 1H), 7.24 ((AB)2
system, J = 8.4 Hz, Δν = 114.9 Hz, 4H), 3.99 (s, 2H), 2.32 (s,
3H) ppm. δC (75 MHz, CDCl3, Me4Si) 184.0 (CvO), 183.5
(CvO), 154.5 (CH), 147.5 (Cq), 145.5 (Cq), 144.3 (Cq), 136.5
(Cq), 134.6 (CH), 131.8 (2 × CH), 130.2 (2 × CH), 128.7 (Cq),
127.5 (CH), 120.5 (Cq), 31.9 (CH2), 13.5 (CH3) ppm. EIMS (70
eV) m/z: 342.9 (22) [M + H]+, 327.9 (92), 325.9 (100), 247.1
(47). Elemental analysis C17H12BrNO2: calc: C 59.67, H 3.53, N
4.09, Br 23.35. Found C 59.56, H 3.68, N 4.18, Br 23.50.
6-(4-(Trifluoromethyl)benzyl)-7-methylquinoline-5,8-dione 2b.
Yield: 51%; mp: 107–109 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 8.95 (dd, J = 4.7 Hz, 1.7 Hz, 1H), 8.36 (dd, J =
7.9 Hz, 1.7 Hz, 1H), 7.59 (dd, J = 7.9 Hz, 4.7 Hz, 1H), 7.38
((AB)2 system, J = 8.5 Hz, Δν = 41.5 Hz, 4H), 4.08 (s, 2H), 2.21
(s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si) 184.0 (CvO), 182.9
(CvO), 154.6 (CH), 147.4(Cq), 145.5(Cq), 144.3(Cq), 134.5
(CH), 129.1 (2 × CH), 127.6, (CH), 128.7 (Cq), 127.6 (CH),
125.6 (q, J = 3.8 Hz, 2 × CH); 122.2 (Cq), 32.3 (CH2), 13.7
(CH3) ppm. EIMS (70 eV) m/z : 331.0 ([M]+, 41), 316.1 ([M–
CH3]+, 100). Elemental analysis C18H12F3NO2: calc: C 65.26, H
3.65, N 4.23. Found C 65.28, H 3.71, N 4.23.
7-(4-(Trifluoromethyl)benzyl)-6-methylquinoline-5,8-dione
1b. Yield: 51%; mp: 124–126 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 9.03 (dd, J = 4.7 Hz, 1.8 Hz, 1H), 8.43 (dd, J =
7.9 Hz, 1.8 Hz, 1H), 7.67 (dd, J = 7.9 Hz, 4.7 Hz, 1H), 7.44
((AB)2 system, J = 8.0 Hz, Δν = 56.4 Hz, 4H), 4.11 (s, 2H), 2.34
(s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si) 184.0 (CvO), 183.5
(CvO), 154.6 (CH), 147.5 (Cq), 145.9 (Cq), 144.0 (Cq), 134.7
(CH), 129.0 (q, J = 33.7 Hz, Cq), 128.9, (CH), 128.7 (Cq), 127.6
(CH), 125.7 (q, J = 3.7 Hz, 2 × CH); 122.2 (Cq), 32.3 (CH2),
13.7 (CH3) ppm. EIMS (70 eV) m/z: 331.0 ([M]+, 23), 316.0
([M–CH3]+, 100). Elemental analysis C18H12F3O2·0.3H2O: calc:
C, H 3.53, N 4.09, Br 23.35. Found C 59.56, H 3.68, N 4.18.
6-(3,5-Dimethoxybenzyl)-7-methylquinoline-5,8-dione 2c. Yield:
55%; mp: 110–111 °C (from Et2O). δH (300 MHz, CDCl3,
Me4Si) 9.02 (dd, J = 4.7 Hz, J = 1.7Hz, 1H), 8.44 (dd, J = 7.9
Hz, J = 1.7 Hz, 1H), 7.66 (dd, J = 7.8 Hz, J = 4.7 Hz, 1H), 6.41
(d, J = 2.2Hz, 2H), 6.29 (t, J = 2.2Hz, 1H), 4.04 (s, 2H), 3.75 (s,
6H), 2.33 (s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si) 184.8
(CvO), 183.0 (CvO), 160.9 (Cq), 154.4 (CH), 147.5 (Cq),
146.0 (Cq), 144.1 (Cq), 139.7 (Cq), 134.4 (CH), 128.9 (Cq),
127.5 (CH), 107.4 (2 × CH), 98.3 (CH), 55.3 (2 × OCH3), 32.6
(CH2), 13.3 (CH3) ppm. EIMS (70 eV) m/z: 323.2 ([M]+, 42),
308.1 ([M − CH3]+, 100), 166.1 (77), 293.1 (58), 173.0 (53),
166.1 (77). Elemental analysis C19H17NO4: calc: C, 70.58; H,
5.30; N, 4.33. Found: C, 70.30; H, 5.41; N, 4.26.
7-(3,5-Dimethoxybenzyl)-6-methylquinoline-5,8-dione 1c.
Yield: 31%; mp: 152–153 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 9.02 (dd, J = 4.7 Hz, J = 1.6 Hz, 1H), 8.43 (d,
4
3J = 7.7 Hz, J = 1.6 Hz, 1H), 7.66 (dd, J = 7.7 Hz, J = 4.7 Hz,
1H), 6.37–6.30 (m, 3H), 4.00 (s, 2H), 3.75 (s, 6H), δ = 2.33
(s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si) 184.0 (CvO),
183.6 (CvO), 160.9 (Cq), 154.3 (CH), 147.6 (Cq), 145.7 (Cq),
144.6 (Cq), 139.7 (Cq), 134.7 (CH), 128.9 (Cq), 127.5 (CH),
106.8 (2 × CH), 98.1 (CH), 55.3 (2 × OCH3), 32.5 (CH2),
13.6 (CH3) ppm. EIMS (70 eV) m/z: 323.1 ([M]+, 69), 308.1
([M–CH3]+, 88), 77.1 (100). Elemental analysis C19H17NO4:
calc: C, 70.58; H, 5.30; N, 4.33. Found: C, 70.56; H, 5.37;
N, 4.21.
6-(2,5-Dimethoxybenzyl)-7-methylquinoline-5,8-dione 2d. Yield:
45%; mp: 135–137 °C (from Et2O). δH (300 MHz, CDCl3,
Me4Si) 9.02 (dd, J = 4.7 Hz, J = 1.7Hz, 1H), 8.44 (dd, J = 7.9
Hz, J = 1.7 Hz, 1H), 7.66 (dd, J = 7.8 Hz, J = 4.7 Hz, 1H),
6.80–6.70 (m, 3H), 4.08 (s, 2H), 3.80 (s, 3H), 3.73 (s, 3H), 2.80
(s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si) 184.9 (CvO), 183.0
(CvO), 154.3 (CH), 153.6 (Cq), 151.5 (Cq), 147.8 (Cq), 146.6
(Cq), 144.4 (Cq), 134.4 (CH), 129.0 (Cq), 127.3 (CH), 127.1
7-(2,5-Dimethoxybenzyl)-6-methylquinoline-5,8-dione 1d.
Yield: 48%; mp: 122–123 °C (from Et2O). δH (300 MHz,
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(Cq), 116.5(CH), 111.6 (CH), 111.3 (CH), 56.0 (CH3), 55.7
(CH3), 27.1 (CH2), 13.1 (CH3) ppm. EIMS (70 eV) m/z: 323.2
([M]+, 53), 308.1([M–CH3]+, 100), 293.1 (43). Elemental analy-
sis C19H17NO4·0.45H2O: calc: C, 68.85; H 5.44; N, 4.23.
Found: C, 68.25; H, 5.30; N, 4.28.
(CvO), 182.9 (CvO), 155.1 (CH), 145.7 (Cq), 145.3 (Cq),
143.8 (Cq), 138.5 (Cq), 136.6 (Cq), 134.2 (CH), 131.7 (2 × CH),
130.5 (2 × CH), 128.5 (Cq), 120.4 (Cq), 31.9 (CH2), 18.9 (CH3),
13.2 (CH3) ppm. EIMS (70 eV) m/z: 357.0 ([M]+, 35), 342.0
([M–CH3]+, 46, 340.0, (100). Elemental analysis C18H14BrNO2:
calc: C, 60.69; H, 3.96; N, 3.93; Br, 22.43. Found: C, 60.92; H,
4.02; N, 3.89; Br, 22.28.
7-(4-Bromobenzyl)-3,6-dimethylquinoline-5,8-dione 3a. Yield:
57%; mp: 166–169 °C (from Et2O). δH (300 MHz, CDCl3,
Me4Si) 8.82 (s, 1H), 8.20 (s, 1H), 7.25 ((AB)2 system, J = 8.4
Hz, Δν = 87.4 Hz, 4H), 3.99 (s, 2H), 2.52 (s, 3H), 2.31 (s, 3H)
ppm. δC (75 MHz, CDCl3, Me4Si) 184.4 (CvO), 183.5 (CvO),
155.1 (CH), 145.4 (Cq), 144.2 (Cq), 138.5 (Cq), 138.0 (Cq),
136.6 (Cq), 134.3 (CH), 131.8 (2 × CH), 130.2 (2 × CH), 128.3
(Cq), 120.5 (Cq), 31.9 (CH2), 18.9 (CH3), 13.5 (CH3) ppm.
EIMS (70 eV) m/z: 357.0 ([M]+, 41), 342.0 ([M–CH3]+, 100).
Elemental analysis C18H14BrNO2: calc: C, 60.69; H, 3.96; N,
3.93; Br, 22.43. Found: C, 60.41; H, 4.15; N, 4.13; Br, 22.43.
6-(4-(Trifluoromethyl)benzyl)-3,7-dimethylquinoline-5,8-dione
4b. Yield: 52%; mp: 121–123 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 8.83 (s, 1H), 8.21 (s, 1H), 7.45 ((AB)2 system,
J = 7.8 Hz, Δν = 41.9 Hz), 4.14 (s, 2H), 2.52 (s, 3H), 2.27 (s,
3H) ppm. δC (75 MHz, CDCl3, Me4Si) 184.9 (CvO), 182.9
(CvO), 155.2 (CH), 145.3 (Cq), 145.2 (Cq), 141.7 (Cq), 143.2
(CH), 138.6 (Cq), 129.1 (2 × CH), 128.9 (q, JC–F = 25.6 Hz,
Cq), 128.5 (Cq), 125.6 (q, JC–F = 3.7 Hz, 2 × CH), 124.0 (q, JC–F
= 272.6 Hz, Cq), 32.3 (CH2), 18.9 (CH3), 13.3 (CH3) ppm.
EIMS (70 eV) m/z: 345.0 ([M]+, 41), 330.0 ([M–CH3]+, 100).
Elemental analysis C19H14F3NO2: calc: C, 66.09; H, 4.09; F,
16.51; N, 4.06. Found: C, 66.15; H, 4.12; N, 4.08.
7-(4-(Trifluoromethyl)benzyl)-3,6-dimethylquinoline-5,8-dione
3b. Yield: 45%; mp: 164–165 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 8.84 (d, J = 2.3 Hz, 1H), 8.21 (d, J = 2.3 Hz,
1H), 7.44 ((AB)2 system, J = 8.1 Hz, Δν = 28.2 Hz, 4H), 4.10 (s,
2H), δ = 2.52 (s, 3H), δ = 2.33 (s, 3H) ppm. δC (75 MHz,
CDCl3, Me4Si) 184.4 (CvO), 183.5 (CvO), 155.2 (CH), 145.7
(Cq), 145.5 (Cq), 143.8 (Cq), 141.8 (Cq), 138.5 (Cq), 134.3 (CH),
128.8 (2 × CH), 128.4 (Cq), 125.6 (q, JC–F = 3.8 Hz, 2 × CH),
122.4 (q, JC–F = 272 Hz, CF3), 32.3 (CH2), 18.9 (CH3), 13.6
(CH3) ppm. EIMS (70 eV) m/z: 345.1 ([M]+, 33), 330.0
([M–CH3]+, 100). Elemental analysis C19H14F3NO2: calc:
C, 66.09; H, 4.09; N, 4.06. Found: C, 65.79; H, 4.29; N, 3.86.
6-(3,5-Dimethoxybenzyl)-3,7-dimethylquinoline-5,8-dione 4c.
Yield: 37%; mp: 106–108 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 8.83 (d, J = 2.1 Hz, 1H), 8.20 (d, J = 2.1 Hz,
1H), 6.41–6.29 (m, 3H), 4.03 (s, 2H), 3.74 (s, 6H), 2.52 (s, 3H),
2.27 (s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si) 185.1 (CvO),
182.9 (CvO), 160.9 (Cq), 155.0 (CH), 145.9 (Cq), 145.3 (Cq),
143.9 (Cq), 139.8 (Cq), 138.4 (Cq), 134.2 (CH), 128.5 (CH),
107.0 (Cq), 98.3 (Cq), 32.5 (CH2), 18.8 (CH3), 13.2 (CH3) ppm.
EIMS (70 eV) m/z: 337.0 ([M]+, 41), 322.0 ([M–CH3]+, 100).
Elemental analysis C20H19NO4: calc: C, 71.20; H, 5.68; N, 4.15.
Found: C, 70.85; H, 5.70.
7-(3,5-Dimethoxybenzyl)-3,6-dimethylquinoline-5,8-dione
3c. Yield: 49%; mp: 140–141 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 8.82 (d, J = 1.7 Hz, 1H), 8.20 (d, J = 1.7 Hz,
1H), 6.36–6.30 (m, 3H), 3.98 (s, 2H), 3.75 (s, 6H), 2.51 (s, 3H),
2.32 (s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si) 184.4 (CvO),
183.6 (CvO), 161.0 (2Cq), 155.0 (CH), 145.5 (Cq), 144.4 (Cq),
139.8 (Cq), 140.1 (Cq), 138.4 (Cq), 134.4 (CH), 128.4 (Cq),
106.8 (2CH), 98.1 (CH), 55.3 (2CH3), 32.5 (CH2), 18.8 (CH3),
13.5 (CH3) ppm. EIMS (70 eV) m/z: 337.1 ([M]+, 80), 322.1
([M–CH3]+, 100). Elemental analysis C20H19NO4: calc: C,
71.20; H, 5.68; N, 4.15. Found: C, 70.88; H, 5.66; N, 4.14.
6-(2,5-Dimethoxybenzyl)-3,7-dimethylquinoline-5,8-dione 4d.
Yield: 58%; mp: 133–135 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 8.81 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 2.1 Hz,
1H), 6.77–6.66 (m, 3H), 4.05 (s, 2H), 3.78 (s, 3H), 3.70 (s, 3H),
2.51 (s, 3H), 2.18 (s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si)
184.3 (CvO), 183.7 (CvO), 154.8 (CH), 153.5 (Cq), 151.5
(Cq), 145.8 (Cq), 145.5 (Cq), 144.9 (Cq), 138.3 (Cq), 134.4 (CH),
128.6 (Cq), 127.2 (Cq), 116.3 (CH), 111.2 (CH), 111.1 (CH),
55.9 (CH3), 55.6 (CH3), 26.9 (CH2), 18.9 (CH3), 13.2 (CH3)
ppm. EIMS (70 eV) m/z: 337.13 ([M]+, 65), 322.1 ([M–CH3]+,
100). Elemental analysis C20H19NO4: calc: C, 71.20; H, 5.68; N,
4.15. Found: C, 71.35; H, 5.75; N, 4.20.
7-(2,5-Dimethoxybenzyl)-3,6-dimethylquinoline-5,8-dione 3d.
Yield: 41%; mp: 129–130 °C (from Et2O). δH (300 MHz,
CDCl3, Me4Si) 8.83 (s, 1H), 8.20 (s, 1H), 6.78–6.62 (m, 3H),
3.79 (s, 3H), 3.71 (s, 3H), 2.51 (s, 3H), 2.24 (s, 3H) ppm. δC
(75 MHz, CDCl3, Me4Si) 184.3 (CvO), 183.7 (CvO), 154.8
(CH), 153.5 (Cq), 151.5 (Cq), 145.8 (Cq), 145.5 (Cq), 144.9 (Cq),
138.3 (Cq), 134.4 (CH), 128.6 (Cq), 127.2 (Cq), 116.3 (CH),
111.2 (CH), 111.1 (CH), 55.9 (CH3), 55.6 (CH3), 26.9 (CH2),
18.9 (CH3), 13.2 (CH3) ppm. EIMS (70 eV) m/z: 337.1 ([M]+,
100); 322.1 ([M–CH3]+, 63). Elemental analysis C20H19NO4:
calc: C, 71.20; H, 5.68; N, 4.1. Found: C, 70.61; H, 5.70; N,
4.16.
2-Methyl-3-(pyridin-4-ylmethyl)naphthalene-1,4-dione 11.
4-Pyridylacetic acid hydrochloride (2.00 g, 11.52 mmol) was
dissolved in 20 mL water and stirred at rt for 5 min. Then
AgNO3 (2.35 g, 13.82 mmol, 1.2 equiv) was added under stir-
ring and a white precipitate appeared in the solution. The precipi-
tate turns black if left under light (AgCl). The precipitate was
filtered and the filtrate containing the pyridinium salt was used
directly in Jacobsen–Torssell reaction with menadione as reac-
tion partner according to the reported general procedure.2a The
residue was dissolved in CH2Cl2 and extracted with K2CO3 satu-
rated solution. The organic phase was evaporated to produce a
brown oil (200 mg). The brown oil was purified through flash
chromatography with the toluene–Et2O system (1 : 1) after a
prior treatment of the column with Et3N. The obtained powder
6-(4-Bromobenzyl)-3,7-dimethylquinoline-5,8-dione 4a. Yield:
50%; mp: 146–148 °C (from Et2O). δH (300 MHz, CDCl3,
Me4Si) 8.83 (d, J = 2.0 Hz, 1H), 8.20 (d, J = 2.0 Hz, 1H), 7.26
((AB)2 system, J = 8.0 Hz, Δν = 72.4 Hz, 4H), 4.04 (s, 2H), 2.52
(s, 3H), 2.26 (s, 3H) ppm. δC (75 MHz, CDCl3, Me4Si) 185.0
Org. Biomol. Chem.
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was recrystallized in 5 mL cyclohexane–EtOAc (10 : 1) to afford
the wished compound 11 as orange crystals. Yield: 10%; mp:
120–122 °C (from cyclohexane–EtOAc). δH (300 MHz, CDCl3,
Me4Si) 8.47–8.49 (m, 2H), 8.07–8.11 (m, 2H), 7.69–7.74 (m,
2H), 7.14–7.15 (m, 2H), 4.02 (s, 2H, CH2), 2.23 (s, 3H, CH3).
δC (75 MHz, CDCl3, Me4Si) 184.9 (CvO), 184.3 (CvO),
150.0 (CH, 2C), 147.2 (Cq), 145.2 (Cq), 143.6 (Cq), 133.8 (CH),
133.7 (CH), 132.0 (Cq), 131.8 (Cq), 126.6 (CH), 126.4 (CH),
123.8 (CH, 2C), 31.9 (CH2), 13.4 (CH3). EIMS (70 eV) m/z:
263.1 ([M]+, 33), 248.1 ([M–CH3]+, 100). Elemental analysis
C17H13NO2: calc: C, 77.55; H, 4.98; N, 5.32. Found: C, 77.48;
H, 4.98; N, 5.30.
10% heat-inactivated fetal calf serum (Cellgro, Mediatech).
Control worms were treated with equal volumes of DMSO
alone. Worms were subsequently observed for motility and mor-
tality. Mortality was scored as a complete lack of movement.
Collection of human red blood cells (RBCs) was as follows.
Whole human blood was collected in a 10 mL heparinized tube
(BD vacutainer, 367874). The samples were centrifuged at 400 ×
g for 10 min without a brake. The plasma (the upper layer) and
white blood cells (interface) were removed. A volume of RPMI
medium equal to that of the whole blood was added to the RBC
pellet and mixed thoroughly. The samples were centrifuged at
400 g for 10 min without a brake. The wash was repeated three
more times. After the final wash the upper layer was removed
and the same volume of RPMI was added to the red blood cells
and then mixed thoroughly. The washed RBCs were stored at
4 °C.
Cyclic voltammetry
Cyclic voltammetry measurements were carried out at 20 °C
using platinum working and auxiliary electrodes (CTV 101T and
platinum wire, respectively) and a potentiostat (VoltaLab Radio-
meter Analytical with PGZ 301). The standard calomel reference
electrodes were separated from the bulk of the solution by a KCl
saturated solution with a glass frit. The compounds (1 mM) were
analyzed in CH2Cl2 solvent using tetrabutylammonium tetra-
fluoroborate salt (0.1 M) as supporting electrolyte. Prior to the
measurements the mixtures were deoxygenated by means of a
stream of dry nitrogen which was maintained above the solutions
during measurements. The sweep rate was 0.2 V s−1. Ipc and Ipa
values are respectively the current intensities (μA) for cathodic
and anodic peaks. The standard redox potentials (E°) were deter-
mined from cyclic voltammograms by averaging the cathodic
(Epc) and the anodic (Epa) peak potentials. The separation of
these peak potentials (ΔEp) can be used to estimate the kinetic
reversibility of the respective redox reaction. Menadione 2 and
methylene blue (MB) were used as reference compounds. The
results are given with reference to the Standard Calomel Elec-
trode (SCE) potential.
Physico-biochemical studies
Materials and methods. Distilled water was purified by
passing it through a mixed bed of ion-exchanger (Bioblock
Scientific R3-83002, M3-83006) and activated carbon (Bioblock
Scientific ORC-83005). All stock solutions were prepared using
an AG 245 Mettler Toledo analytical balance. The [FeIIIPPIX]tot
and [metHb(FeIII)]tot were calculated from the molecular weight
of the corresponding monomers. HEPES (2-[4-(2-hydroxyethyl)
piperazin-1-yl]ethanesulfonic acid, Gerbu Biotechnik) buffer
(0.2 m) was prepared in water and the pH (7.4) was adjusted
with NaOH slurry. pH reading was done on a PHM240 Meter-
Lab® millivoltmeter fitted with a combined glass electrode
(Metrohm 6.0234.500, Long Life, 0.1 m NaCl). Calibration was
done with commercial Merck® buffers (1.68, 4.00, 6.86, 7.41,
and 9.18). Stock solutions of the substrates in DMSO for the
different assays were freshly prepared in Eppendorf tubes just
before the experiments. Hematin (FeIIIPPIX(OH)) solution was
prepared from equine hemin Type III (FeIIIPPIXCl, Sigma-
Aldrich) and 0.1 m NaOH solution vigorously stirred at room
temperature (rt) for 1 h. Methemoglobin was prepared by oxi-
dation of human hemoglobin (Sigma-Aldrich) in solution by
oxygen at rt (the molecular weight of the tetrameric hemoglobin
is 64.458 kDa). NADPH (Biomol, Hamburg, Germany) solution
was prepared in water and kept at 0 °C during the experiments.
Final concentration in the optical cell was calculated from the
absorbance at 340 nm (ε340 = 6.22 mM−1 cm−1).
Parasitology
In vitro assays with Plasmodium falciparum strains in culture.
The chloroquine-sensitive 3D7 and the multi-resistant Dd2
strains of Plasmodium falciparum were cultivated using standard
protocols previously reported.2,32c
Determination of IC50 values against P. falciparum strains.
Drug susceptibility of P. falciparum was studied using a
modified method50a of the protocol described previously for the
3H-hypoxanthine incorporation-based assay.50b All assays
included CQ diphosphate (Sigma, UK) as standard and control
wells with untreated infected and uninfected erythrocytes. IC50
values were derived by sigmoidal regression analysis (Microsoft
xlfit™). All data from in vitro tests were carried out in triplicate
and were given with the 95% confidence limits.
Spectrophotometric titration of hematin π–π dimer by the
substrates. An aliquot of 10 μL of the FeIIIPPIX(OH) stock
solution (∼1.68 mm) was dissolved in 500 μl 0.2 M sodium
HEPES buffer at pH 7.5 in a 1 cm Hellma optical cell. The
absorption spectrophotometric titrations were carried out by
adding microvolumes of a stock solution of the substrate (∼5 ×
10−4 M in DMSO). Aliquots of 5–20 μL of the stock substrate
solution were successively added to the reaction mixture and,
after each addition, a UV-visible absorption spectrum (300 nm <
λ < 800 nm) was recorded with a Uvikon 941 spectrophotometer.
Special care was taken to ensure that equilibrium was attained.
The association constants KA and the stoichiometries of the
species at equilibrium were determined by processing the spec-
trophotometric data with the Specfit program.52 Specfit uses
Assays with ex vivo Schistosoma worms. Adult S. mansoni
worms were obtained from mice 49 days post infection by per-
fusion with RPMI 1640 media as described.51 Compounds were
dissolved in dimethylsulfoxide (DMSO) and added to freshly
perfused worms in RPMI 1640 containing 25 mM HEPES, pH
7, 150 units mL−1 penicillin, 125 μg mL−1 streptomycin, and
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factor analysis to reduce the absorbance matrix and to extract the
eigenvalues prior to the multi-wavelength fit of the reduced data
set according to the Marquardt algorithm. The uncertainties on
the log K values are given as 3σ with σ = standard deviation.
Origin 5.0 was used to process the analytical results. For the
sake of simplicity, charges are omitted in all the chemical
equilibria.
Acknowledgements
This work was funded by US NIH/National Institute of Allergy
and Infectious Disease (NIAID) (grant R01AI065622). L. J. and
D. A. L. are grateful to NIH (L. J., 1st year; D. A. L., 3 years)
and DFG via the SFB 544 (B14 project) for their salaries. K.B.
wishes to thank the DFG for supporting the project (BE1540-15-1).
The Centre National de la Recherche Scientifique (CNRS), the
University of Strasbourg (UMR 7509 CNRS-UdS), and the
International Center for Frontier Research in Chemistry (ic-FRC)
in Strasbourg also partly supported this work. The LC-MS ana-
lyses were performed at the TechMedILL Platform of the Unité
Mixte de Service CNRS-Université de Strasbourg (UMS 3286);
the authors are grateful to Patrick Gizzi for the data and the fruit-
ful discussions. E.D.C is grateful to Prof Louis Maes, Antwerp
University for the primary cytotoxicity assays with the
human MRC-5 cell line. Schistosome materials for this
work were supplied in part through NIH/NIAID contract
HHSN272201000005I to Dr Fred Lewis at the Biomedical
Research Institute, Rockville, MD, USA.
β-Hematin inhibition assay. The β-hematin inhibition assay
used in this work was inspired from that developed by the group
of T. J. Egan42 with some significant improvements in the order
of the addition of the reactants, which were imposed by the low
solubility of some of our compounds.41bb Briefly, drug solutions
(89.1 mM, 53.5 mM, 26.7 mM, 17.8 mM, 13.4 mM, 8.9 mM,
4.5 mM, 0 mM) were prepared by dissolving the drug in DMSO.
Hematin stock solution (1.68 mM) was prepared by dissolving
equine hemin (1.05 mg) in 0.1 M NaOH (960 μL). The solution
was incubated at room temperature for 1 h. In a series of 2 mL
Eppendorf tubes, 2.0 μL of drug solution (or solvent for the
blank reference) were dispensed. Then 20.2 μL of hematin stock
solution were added. The Eppendorf tubes were placed in an
incubator at 60 °C and then, 2 μL of 1 M HCl and 11.7 μL of
12.9 M sodium acetate solution (pH 4.5) pre-incubated at 60 °C
were added. Final hematin concentration was 1 mM. Final drug
concentrations were 4.96 mM, 2.98 mM, 1.45 mM, 0.99 mM,
0.75 mM, 0.5 mM, 0.25 mM and 0 mM and final pH of the solu-
tion was 4.5. The reaction mixtures were incubated at 60 °C for
1 h. After incubation, the reaction mixtures were quenched at
room temperature by adding 900 μL of 0.2 M sodium HEPES
buffer + 5% (v/v) pyridine (pH 8.2) in order to adjust final pH
of the mixture to a value between 7.2 and 7.5. Then 1100 μL of
0.02 M sodium HEPES buffer + 5% pyridine (v/v) (pH 7.5) was
added. The Eppendorf tubes were shaken and the precipitate of
β-hematin was scrapped from the walls of the Eppendorf tubes to
ensure complete dissolution of hematin. The β-hematin was
allowed to settle at room temperature for 1 h. The supernatant
was carefully transferred to a Hellma optical cell (l = 1 cm)
without disturbing the precipitate and the absorption spectra
were measured between 300 and 750 nm.
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Methemoglobin reduction coupled assay with hGR/NADPH.
Stock solutions of 2 mM of the drugs in DMSO were freshly
prepared. Solutions of 397 μM methemoglobin in hGR buffer
and ca. 4 mM NADPH in water were prepared and kept at 0 °C
during the experiment. Final NADPH concentration in the
optical cell was calculated from the absorbance at 340 nm (ε340
= 6.22 mm−1 cm−1). In a Hellma optical cell (l = 1 cm), 945 μL
of the hGR buffer, 20 μL of the methemoglobin solution
(397 μM), 20 μL of the drug solution (2 mM) and 10 μL of the
NADPH solution (ca. 4 mM) were introduced and pre-incubated
at 37.5 °C (Lauda E200/O11 thermostat and a Huber Minichiller
cooler) for 5 min. Then 5 μL of the hGR stock II solution was
added. The reaction was monitored by UV-vis. absorption
spectrophotometry on a Cary 300 spectrophotometer with the
scanning kinetics method. A spectrum was measured every 30 s
between 250 and 500 nm over 1 to 2 h. The kinetic data were
processed with the Specfit program as reported previously.43b
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