Synthesis and antiprotozoal activity of original porphyrin precursors and derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.06.002

Importance of heme in African trypanosomes, Leishmania sp. and Plasmodium sp. metabolisms justifies considering the potential of porphyrins and their precursors and derivatives as potential antiparasitic agents by interfering with heme metabolism. Consequ

Full text of DOI:10.1016/j.ejmech.2013.06.002

European Journal of Medicinal Chemistry 67 (2013) 158e165
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antiprotozoal activity of original porphyrin precursors
and derivatives
Zahra Abada a, Sandrine Cojean b, Sébastien Pomel b, Laurent Ferrié a, Bernardin Akagah c,
,
Anh Tuan Lormier c, Philippe M. Loiseau b, Bruno Figadère a
*
a UMR CNRS 8076, LERMIT, Université Paris-Sud, Laboratoire de Pharmacognosie, UFR de Pharmacie, Châtenay-Malabry F-92296, France
b UMR CNRS 8076, LERMIT, Université Paris-Sud, Groupe Chimiothérapie Antiparasitaire, UFR de Pharmacie, Châtenay-Malabry F-92296, France
c Alpha-Chimica, 5 rue Jean-Baptiste Clément, F-92296 Châtenay-Malabry, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Importance of heme in African trypanosomes, Leishmania sp. and Plasmodium sp. metabolisms justifies
considering the potential of porphyrins and their precursors and derivatives as potential antiparasitic
agents by interfering with heme metabolism. Consequently, twenty-four porphyrin precursors and de-
rivatives were evaluated against Leishmania donovani, Trypanosoma brucei and Plasmodium sp. The best
active compound against Trypanosoma brucei brucei was a new porphyrin derivative; compound 4i, with
Received 25 February 2013
Received in revised form
28 May 2013
Accepted 2 June 2013
Available online 12 June 2013
a MEC value of 6.25 mM justifying further in vivo evaluation. Whereas these compounds were not active
against intramacrophage amastigotes of L. donovani, another new porphyrin derivative, compound 4f
was active in vitro against Plasmodium falciparum at 20 nM and a slight delay of mice survival was
Keywords:
Leishmanicid
Anti-malaric
Trypanocid
observed on the Plasmodium berghei/Swiss mice model at 50
m
mol/kg/day ꢀ 4. Pharmacomodulations
should be further developed relying on a better knowledge on the porphyrin behaviour into the parasites
comparatively to host cells.
Metal complexes
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Leishmania amastigote model [6]. These data suggest that de-
rivatives of porphyrins could be efficient antileishmanial drugs by
Malaria, leishmaniasis and human African trypanosomiasis are
parasitic diseases responsible for high mortality and disability in
tropical and sub-tropical areas [1]. The main limitations of classical
drugs rely on their toxicity, and problems of drug resistance justi-
fying the search for new compounds [2]. In order to get more
specific drugs, a rational approach in the research of new antipar-
asitic compounds is to take advantage of different biochemical
pathways (e.g.) between the parasite and its mammalian host.
Trypanosomatid protozoa such as Leishmania and Trypanosoma
parasites require an exogenous source of heme for growth and
transformation because of their inability to synthesize heme since
they lack several key enzymes in the heme biosynthetic pathway
[3]. Heme is a critical prosthetic group for proteins involved in
metabolism and electron transport in these parasites. Leishmanias
transform and grow only in medium containing either heme,
usually supplied as hemin or protoporphyrin IX [4]. On one hand,
compounds complexing heme, such as xanthones are toxic for
Leishmania [5]. On the other hand, heme plays an important role in
the cell hosteparasite relationships on the in vitro intramacrophage
interfering with parasite heme metabolism.. Thus, dimethyl and
diethyl carbaporphyrin ketals inhibit the growth of Leishmania
tarentolae promastigotes in vitro [7].
Plasmodium parasites use host hemoglobin for their nutrition
into erythrocytes, as a source of amino-acids, releasing the heme
scaffold. Heme is highly toxic for Plasmodium parasites and heme
detoxification is a critical step in the life cycle of the parasite,
achieved by crystallization into physiologically insoluble hemozoin
[8]. Porphyrins are interesting compounds to be tested against
Plasmodium parasites because of their structural analogy with
those of heme. Thus, any inhibition of heme detoxification is a
rational strategy to fight the parasite.
Thus, all these data justify considering porphyrin derivatives as
potential antiparasitic agents against Trypanosoma, Leishmania and
Plasmodium parasites.
Consequently, this paper reports on the synthesis of
dipyrrinato-metallates (3aed), obtained from dipyrromethenes
(2aee), the latter being prepared from the known corresponding
dipyrromethanes (1aee). Porphyrins (4aek) were also prepared
through a known procedure and the synthesized products were
evaluated for their antileishmanial, trypanocidal and antimalarial
properties.
* Corresponding author.
E-mail address: bruno.figadere@u-psud.fr (B. Figadère).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.002

Z. Abada et al. / European Journal of Medicinal Chemistry 67 (2013) 158e165
159
2. Chemistry
Dipyrromethanes 1aee were known and prepared through a
published procedure from pyrrole and the desired aromatic alde-
hydes (Scheme 1) in the presence of trifluoroacetic acid [9,10] in
excellent yields (62e89%).
Dipyrromethenes 2aee were prepared, through a published
method [11] from corresponding dipyrromethanes 1aee by DDQ
mediated oxidation (Scheme 2) in good yields (53e95%).
Dipyrrinato-metallates 3aed were prepared for the first time
by mixing the corresponding dipyrromethenes 2c,d and the iron (II)
[12] or manganese (II) [13] salts (Scheme 3) in the presence of NEt3
in a 1:1 mixture of MeOH : chloroform under reflux (39e98% yield)
[14].
Porphyrins 4aee, g and k were prepared as already reported by
us [15]. Unreported porphyrin 4i was prepared from dibromopor-
phyrin 4d by a palladium-mediated cross coupling with pyrrolidi-
none in 11% yield as a secondary product besides major porphyrin
4j [16]. Unknown porphyrin 4f was prepared in 52% yield by
saponification of porphyrin 4g (Scheme 4).
Scheme 2.
formazan product by the mitochondria of viable cells. After in-
cubation of the cells with the MTT reagent, a detergent solution
(Triton X100, HCl) was added to lyse the cells and dissolve the
coloured crystals. The absorbance at 570 nm, directly proportional
to the number of viable cells, was measured using an ELISA plate
reader. The results are expressed as the concentrations inhibiting
parasite growth by 50% (IC50) ꢂ SD after a 3-day incubation
period.
3. Biology
3.1. Antileishmanial evaluation
Promastigote forms of Leishmania donovani (MHOM/ET/67/HU3)
were grown in M-199 medium supplemented with 40 mM HEPES,
3.1.2. In vitro antileishmanial evaluation on intramacrophage
amastigotes
100
m
M adenosine, 0.5 mg/L haemin, 10% heat-inactivated foetal
bovine serum (FBS) and 50
m
g/mL gentamycin at 26 ꢁC in a dark
Concerning the amastigote in vitro model, the peritoneal mac-
rophages of Swiss mice (female, 18e20 g, Janvier, France) were
infected after a 24 h incubation period with promastigote forms of
L. donovani in a stationary phase at a ratio of 10 parasites per
macrophage, to obtain 87% of infected macrophages and 10 ꢂ 3
amastigotes per macrophage. At 18 h after the promastigotes had
entered macrophages, the free promastigotes were eliminated and
intramacrophagic amastigotes were treated at various concentra-
tions of the compounds.
environment under an atmosphere of 5% CO2. All the experiments
were performed with parasites in their logarithmic phase of
growth. Differentiation of promastigotes into axenic amastigotes
was achieved by dilution of 1 ꢀ106 promastigotes in 5 mL of axenic
amastigote media (15 mM KCl; 8 mM glucose; 5 mM glutamine, 1 X
M-199, 2.5% BBL_trypticase_peptone, 4 mM haemin, and 20% FBS).
The pH was adjusted to pH 5.5. Axenic amastigotes were grown at
37 ꢁC in 5% CO2.
Pentamidine and amphotericin B were used as reference com-
pounds. Each experiment was performed in triplicate. The culture
3.1.1. In vitro antileishmanial evaluation on promastigote forms
Promastigote forms from logarithmic phase culture were sus-
pended to yield 106 cells/mL. Miltefosine was used as anti-
leishmanial reference compound. Compounds to be evaluated and
miltefosine were distributed in the plates by making a serial
dilution. The final concentrations used were between 100 mM and
50 nM. Triplicates were used for each concentration. After a 3-day
incubation period at 27 ꢁC in the dark and under a 5% CO2 atmo-
sphere, the viability of the promastigotes was assessed using the
tetrazolium-dye (MTT) colorimetric method, which measures the
reduction of a tetrazolium component (MTT) into an insoluble
Scheme 1.
Scheme 3.

160
Z. Abada et al. / European Journal of Medicinal Chemistry 67 (2013) 158e165
Pd2dba3
O
Xantphos
O
N
H
N
NH
N
N
NH
N
N
NH
N
Cs2CO3
N
Br
N
N
Br
Br
+
HN
HN
dioxane 100°C
HN
O
O
4j (89%)
4i (11%)
4d
OMe
OMe
MeO
OMe
MeO
OMe
O
O
NaOH
N
NH
N
N
NH
N
N
H
N
H
CH2Cl2
MeOH
HN
HN
O
O
MeO
HO
4f
52%
4g
OMe
MeO
OMe
MeO
OMe
OMe
Scheme 4.
medium was renewed 48 h later and a new culture medium con-
taining the drug was added. The experiment was stopped at day 5,
and the percentage of infected macrophages was evaluated
microscopically after Giemsa staining. The 50% inhibitory concen-
trations (IC50) were determined by linear regression analysis, and
3.3.2. In vivo antimalarial evaluation Plasmodium berghei
NK-173, a strain free of contamination with Eperythrozoon coc-
coides and sensitive to chloroquine, was used for in vivo antima-
larial evaluation. This strain is known to induce high mortality in
mice, providing a good model to estimate survival and antimalarial
efficacy in reducing parasitemia, and is sensitive to all currently
used antimalarial drugs.
expressed in
mM ꢂ SD.
Animal experiments were carried out according to the Principles
of Laboratory Animal Care and legislation in force in France. Female
outbred eperythrozoon-free Swiss albino mice weighing 18e20 g
were obtained from Janvier, France. They were kept in a normal
diurnal cycle and had free access to food and water throughout the
experiments.
3.2. In vitro trypanocidal evaluation
The drug incubation infectivity test (DIIT) was used for com-
pound evaluation following a protocol previously described [17].
3.3. Antimalarial evaluation
The four-day test was performed as described by Peters et al.
[20]. An infective inoculum was prepared from a previously
infected donor mouse with rising parasitemia (20%). On day
0 the mice were infected i.v. with 106 P. berghei-parasitized RBC
in 0.2 ml of phosphate-buffered saline. They were randomly
divided into groups of 10 and treated once daily, by the i.p. route
with the compound for four consecutive days (days 0e3). The
treatment was stopped when some toxicity was observed.
3.3.1. In vitro antimalarial evaluation
Plasmodium falciparum 3D7 strain was maintained in Oþ human
erythrocytes in albumin RPMI supplemented medium under
continuous culture using the candle-jar method [18]. The parasites
were synchronized to the ring stage by repeated sorbitol treatment
[19]. A 5%(v/v) erythrocytes suspension with 0.5% parasitemia
(number of parasites per 100 red blood cell) was incubated with the
compounds to be tested, previously dissolved in DMSO. Parasites
were also incubated with culture medium (negative control) or
Control groups received 100 mmoles chloroquine/kg. Thin blood
smears were made from tail blood from untreated controls
and from treated animals on days 4, 7, 8, 9, 10 and 14 after
infection. Levels of parasitemia were measured in Giemsa-
stained smears, and RBC numbers were determined on the
same days. If mice were free from Plasmodium after at least 200
fields (magnification, ꢀ1000) were checked, they were consid-
ered cured but were kept under observation for 60 days in case
of relapse.
with 4 mM chloroquine (positive control) in 96-well culture plates.
After 44 h incubation at 37 ꢁC, the plates were subjected to 3 freeze-
thaw cycles to achieve complete hemolysis. The parasite growth
was determined by the ELISA-Malaria antigentest (DiaMed, France),
for the detection of P. falciparum lactate dehydrogenase (pLDH), as
well as by microscopic examination under oil immersion of
Giemsa-stained thin blood smears. Results were expressed as the
percentage of reduction of parasite growth over the control
receiving only the culture medium. Blood smears were prepared
after 24 and 44 h by Giemsa staining. Parasitemia were determined
by microscopic counting of at least 5000 erythrocytes under oil
immersion.
3.4. Cytotoxicity
The toxicity of the compounds was evaluated on mouse
peritoneal macrophages, which were then used for the

Z. Abada et al. / European Journal of Medicinal Chemistry 67 (2013) 158e165
161
intramacrophagic amastigotes in vitro model. Peritoneal macro-
4.4. Antimalarial activity
phages were harvested from female Swiss mice (Janvier, France)
3 days after an intraperitoneal injection of 1.5 ml of sodium
thioglycolate (Biomérieux, France) and were dispensed into
eight-well chamber slides (LabTek Ltd.) at a concentration of
ml/well) in RPMI 1640 medium supplemented
with 10% heat-inactivated fetal calf serum, 25 mM Hepes, and
Only six porphyrin derivatives were evaluated in vitro against
P. falciparum and new compound 4f was the most active with an
IC50 value of 20 nM (Table 1). This compound was poorly active
against leishmania and African trypanosomes. Compound 4d was
also active but with an IC50 value of 200 nM and this activity was
selective towards Plasmodium. To the best of our knowledge, this is
the first report of antimalarial activity of a porphyrin derivative. The
activity of compound 4f justified an in vivo evaluation on the
5 ꢀ 104/well (400_
2 mM L-glutamine (Life Technologies, Cergy-Pontoise, France).
Four hours after the macrophages were plated, they were washed
to eliminate fibroblasts. The culture medium was renewed 48 h
later and a new culture medium containing the drug was added.
The experiment was stopped at day 5, and the viability of
macrophages was checked using the tetrazolium-dye (MTT)
P. berghei/mice model. Compound 4f was toxic at 130
a slight delay of the animal death (Table 2). A dose regimen at
50
mol/kg/day ꢀ 4 allowed a survival time of about 2 days
mmol/kg with
m
colorimetric method. The cytotoxic concentrations 50% (CC50
were determined by linear regression analysis, expressed in
SD.
)
comparatively to those of controls treated only with the excipient.
Pyrroglutamic acid itself, a part of compound 4f, was able to delay
the animal death on about one day. Despite the dose regimen
optimized to get the maximum antimalarial activity, discrimination
between antimalarial activity and toxicity in animals could not be
obtained. Thus, it was not possible to observe a dose for which an
antimalarial activity was significant at Day 14 without toxicity.
m
M
4. Results and discussion
4.1. Antileishmanial activity
5. Conclusion
Twenty-four compounds were first evaluated against the pro-
mastigote forms of L. donovani, as a primary screening and seven
Among the four chemical series synthesized, three of
them, 5-aryldipyrromethenes, dipyrrinato-metallates and 5,15-
diarylporphyrins, can be considered as interesting leads for
further pharmacomodulations because of their activity against
three major parasites, leishmania, African trypanosomes and
Plasmodium. Whereas these compounds were not active against
intramacrophage amastigotes of L. donovani, one of them, new
compound 4i is worth of in vivo evaluation on the T. brucei brucei/
Swiss mice model. More interestingly, new compound 4f delayed
the mice survival of P. berghei infected mice. The pharmacomo-
dulations of these series will be optimized to get more active and
less toxic analogues on the basis of a better knowledge of the
porphyrin transport and action in Plasmodium, Leishmania and
trypanosomes parasites.
of them exhibited an IC50 value of less than 10
most active compound was compound 2c with an IC50 value at
3.6 M, which belongs to the dipyrrin series. Eventhough tetra-
pyrroles and dipyrroles were reported to show cytotoxicity against
tumour cells [21], it is the first time that antileishmanial proper-
ties are disclosed for such compounds. Among this series,
5-aryldipyrromethanes 1aed were inactive or poorly active
whereas 5-aryldipyrromethenes 2aee were homogeneously active
mM (Table 1). The
m
in the same concentration range (3.61e5.55
dipyrrinato-metallates, compound 3b with manganese exhibited a
similar activity (IC50 ¼ 4.52 M) whereas analogs were poorly
active or inactive. To the best of our knowledge, this is the first
report of antileishmanial activity for such complex. 5,15-
diarylporphyrins 4aek were poorly active except new compound
4i with an IC50 value of 6.37 M. These results are quite disap-
mM). Among
m
a
m
6. Experimental
pointing in view of previous activities observed with tetraaryl
porphyrins [7,22e24]. All compounds with IC50 values less than
6.1. General
10
mM were evaluated on L. donovani intramacrophage amasti-
gotes and were found inactive at 20
cells at this concentration.
mM, without toxicity for the
1H NMR spectra were recorded on 300 or 400 MHz in CDCl3 and
data are reported as follows: chemical shift in parts per million
from tetramethylsilane as an internal standard, multiplicity
(br ¼ broad signal, s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
quint ¼ quintuplet, m ¼ multiplet or overlap of non-equivalent
resonances), integration. 13C NMR spectra were recorded at
75 MHz in CDCl3 (unless otherwise specified) and data were re-
ported as follows: chemical shift in parts per million from tetra-
methylsilane with the solvent as an internal indicator (CDCl3
4.2. Trypanocidal activity
The best active compound against Trypanosoma brucei brucei
was new porphyrin derivative 4i, with a MEC value of 6.25
(Table 1). This result has to be compared with other metallated
porphyrins showing some activities against trypanosomes [25]. All
5-aryldipyrromethenes 2aee were active in a range from 12.5 to
mM
d
77.0 ppm), multiplicity with respect to proton (deduced from
50
mM and one dipyrrinato-metallate, compound 3b, was active at
JMOD experiments, br ¼ broad signal, s ¼ quaternary C, d ¼ CH,
t ¼ CH2, q ¼ CH3). Low Resolution Mass Spectra were recorded on
an LC/MS equipment with ElectroSpray Ionisation method.
Elemental analyses were recorded by our internal service. High-
resolution mass spectra (HRMS) were performed by the service
de masse (Gif-sur-Yvette IMAGIF e ICSN (Université Paris-Sud) on
the spectrometer (LC) ESI/TOF (LCT, Waters) and MALDI-TOF
(Voyager DE-STR, Applied Biosystems). UVevisible spectra were
recorded on a Xenon source light wave diode spectrophotometer.
DMF was distilled from CaH2. Dichloromethane, THF, Et2O, Toluene
and were dried by filtration through activated molecular sieves.
MeOH was dried by filtration through alumina. Other reagents
were obtained from commercial suppliers and used as received.
12.5
m
M. However, these levels of activity did not justify an in vivo
evaluation, except for compound 4i.
4.3. Relationship between antileishmanial and trypanocidal
activities
Except compound 3b, which was active against L. donovani and
not active against T. brucei brucei, these chemical series exhibited a
strong positive correlation between antileishmanial and trypano-
cidal activities. This homogeneity to the drug susceptibility could
suggest that a target common to leishmania and African trypano-
somes could be affected.

162
Z. Abada et al. / European Journal of Medicinal Chemistry 67 (2013) 158e165
Table 1
In vitro antileishmanial, antitrypanosomal, antimalarial and cytotoxic effects of porphyrins and their precursors and derivatives.
R
R1
R2
Compound
L. donovani
T. brucei brucei
P. falciparum
Cytotoxicity
promastigote
MEC (
m
M)
IC50 ꢂ SD (
m
M)
CC50 ꢂ SD (
mM)
IC50 ꢂ SD (
mM)
DIPYRRINS
H
2-Br
2,3,4,5,6-F5
3,4,5-(OMe)3
H
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
1a
1b
1c
1d
2a
2b
2c
2d
ꢃ100
>100
>100
>100
100
25
50
12.5
25
12.5
12.5
>100
>100
e
e
e
e
e
e
e
e
e
e
e
e
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
44.74 ꢂ 2.80
44.1 ꢂ 3.67
ꢃ100
5.23 ꢂ 0.41
5.38 ꢂ 0.47
3.61 ꢂ 0.43
4.71 ꢂ 0.23
5.55 ꢂ 0.28
56.79 ꢂ 3.84
4.52 ꢂ 0.33
ꢃ 100
2-Br
2,3,4,5,6-F5
3,4,5-(OMe)3
2,6-Cl2
3,4,5-(OMe)3
3,4,5-(OMe)3
2,3,4,5,6-F5
2,3,4,5,6-F5
2e
3a (Fe)
3b (Mn)
3c (Fe)
3d (Mn)
e
e
e
58.77 ꢂ 6.15b
100
Porphyrins
H
H
3-Br
H
H
H
H
H
Br
H
4a
4b
4c
4d
4e
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
>100
>100
>100
>100
>100
>20
>20
>20
0.2
>100
>100
>100
>100
>100
CHO
CHO
Br
3,4,5-(OMe)3
H
>20
3,4,5-(OMe)3
H
H
4f
57.23 ꢂ 3.13
25
0.02
e
>100
>100
>100
>100
>100
>100
3,4,5-(OMe)3
4g
4h
4i
ꢃ100
e
3,4,5-(OMe)3
ꢃ100
100
e
H
H
H
Br
6.37 ꢂ 0.42
ꢃ100
6.25
e
4j
>100
e
4k
53.01 ꢂ 4.40
50
e
REF
Pentamidine
Chloroquine
Miltefosine
e
e
e
3.10 ꢂ 0.21
e
5
e
e
26.3 ꢂ 2.1
e
15e25
4.26 ꢂ 0.40
e
15.8 ꢂ 0.9
Analytical thin layer chromatography (TLC) was performed on silica
gel plates visualized either with a UV lamp (254 nm), or by using
solutions of p-anisaldehyde/sulfuric acid/acetic acid in EtOH or
KMnO4/K2CO3 in water followed by heating. Flash chromatogra-
phies were performed on silica gel (230e400 mesh). All the re-
actions were carried out under N2 atmosphere unless specified.
Only spectroscopic data of new compounds are described. It
is worth noting that NMR analyses of dipyrromethene complexes
3aee were not satisfying because of the presence of Fe (II) or
Mn(II), thus HRMS analyses were crucial for their structural
determinations.
6.2. General protocol for the synthesis of dipyrromethenes 2aee
To 5-aryldipyrromethane (1 g, 3.20 mol) in a 9:1/CH2Cl2:acetone
mixture was added at 0 ꢁC a solution of DDQ (799 mg, 3.52 mol, 1.1

Z. Abada et al. / European Journal of Medicinal Chemistry 67 (2013) 158e165
163
Table 2
In vivo antimalarial activity of compound 4f on the P. berghei/Swiss mice model.
R1
Comp.
R
R
R
R
N
HN
1a
H
1b 2-Br
5
15
1c 2,3,4,5,6-F
1d 3,4,5-OMe
5
NH
N
NH HN
5-aryldipyrromethanes 1a-d
R2
5,15-diarylporphyrins 4a-k
R
R
N
N
N
N
Comp.
R
R1
R2
5
M
15
4a
4b
H
H
H
H
H
CHO
CHO
Br
4c 3-Br
4d
H
H
Br
H
4e 3,4,5-OMe
4f 3,4,5-OMe
H
5-aryldipyrrinato metallate 3a-d
H
O
COOH
N
Comp.
M
R
3a Fe
3,4,5-OMe
4g 3,4,5-OMeO
4h 3,4,5-OMe
H
Comp.
R
H
3b Mn 3,4,5-OMe
COOMe
N
N
O
3c Fe
2,3,4,5,6-F
R
COOMe
2a
3d Mn 2,3,4,5,6-F
2b 2-Br
2c 2,3,4,5,6-F
2d 3,4,5-OMe
2e 2,6-Cl
5
O
N
O
4i
H
H
H
Br
NH
N
5-aryldipyrromethenes 2a-e
N
4j
Ph
Ph
OH
O
N
4k
Compound
Dose
Route of
administration
Reduction
parasitemia
at Day 4 (%)
Quantal survival by daysa
Toxicity: number
of dead mice
[day of death
post-infection]
% Surviving
mice at
Day 30
(! moles/kg/
day ꢀ n)
mg/kg/
day ꢀ n
Day 7
Day 8
Day 9
Day 10
Day 14
130 ꢀ 2
100 ꢀ 1
50 ꢀ 1
100 ꢀ 2
76.9 ꢀ 1
38.5 ꢀ 1
38.5 ꢀ 4
100 ꢀ 4
i.p.
i.p.
i.p.
i.p.
i.p.
60
40
0
5/10
5/10
6/10
5/10
10/10
10/10
5/10
5/10
0/10
7/10
5/10
4/10
0/10
0/10
3/10
0/10
0/10
0/10
0/10
0/10
0/10
3[Day 2]; 2[Day 4]
No toxicity
No toxicity
No toxicity
No toxicity
0
0
0
0
0
10/10
10/10
10/10
10/10
50 ꢀ 4
Pyrroglutamic
acid
774 ꢀ 4
/
Chloroquine
Excipient
100 ꢀ 4
51.5 ꢀ 4
i.p.
i.p.
100
0
10/10
10/10
10/10
6/10
10/10
0/10
10/10
0/10
10/10
0/10
No toxicity
No toxicity
100
0
/
/
a
The numerators represent the number of surviving mice each day relative to the total number of mice in each group.
equiv.) in acetone (8 mL). Additional acetone (5 mL) was added and
the reaction mixture was stirred at room temperature for 45 min.
After concentration, the residue was stirred 10 min in cold CHCl3/
DCM (1:1), filtered, and washed with cold CHCl3. The filtered was
concentrated and the crude material was then purified over neutral
alumina DCM/AcOEt þ1%NEt3 to yield the corresponding
dipyrromethene.
6.2.2. 5-(2-Bromophenyl)dipyrromethene (2b)
Reaction from 5-(2-bromophenyl)dipyrromethane 1b (50.0 mg,
0.166 mmol) and DDQ (41.5 mg, 0.183 mmol, 1.1 equiv) following
the general protocol afforded dipyrromethene 2b (47.3 mg, 95%). 1H
(CDCl3, 300 MHz) d 6.38 (m, 4H, CH), 7.32 (m, 1H), 7.40 (m, 2H), 7.64
(s, 2H), 7.68 (d, J ¼ 7.8 Hz,1H),11.8 (brs,1H, NH). 13C (CDCl3, 75 MHz)
d
144.1 (2CH), 140.5 (C), 139.3 (C), 137.9 (C), 132.6 (CH), 131.6 (CH),
129.9 (CH), 127.9 (2CH), 126.5 (CH), 123.6 (CeBr), 117.9 (2CH).
6.2.1. 5-Phenyldipyrromethene (2a)
LRMS-ESI: m/z 299 [M þ H]þ
(
79Br). HRMS-ESI: m/z calculated for
[M þ H] þ 299.0184, obtained 299.0185. Rf 0.49 (CH2Cl2).
Reaction from 5-phenyldipyrromethane 1a (50 mg,
0.225 mmol) and DDQ (56.2 mg, 0.47 mmol, 1.1 equiv.) following
the general protocol afforded dipyrromethene 2a (26 mg, 53%). 1H
6.2.3. 5-Pentafluorophenyldipyrromethene (2c)
(CDCl3, 300 MHz)
d
6.40 (m, 2H, CH), 6.60 (m, 2H, CH), 7.47 (m, 5H,
Reaction from 5-(pentafluorophenyl)dipyrromethane 1c
(983 mg, 3.02 mmol) and DDQ (760 mg, 3.32 mmol, 1.1 equiv.)
following the general protocol afforded dipyrromethene 2c
Ph-H), 7.65 (s, 2H, CH), 11.5 (brs, 1H, NH). 13C (CDCl3, 75 MHz)
d
143.6 (CH), 142.0 (CH), 140.9 (Cq), 137.3 (Cq), 130.8 (CH), 128.8
(CH), 127.6 (CH), 117.6 (CH). LRMS-ESI: m/z 221 [M þ H]þ. HRMS-
ESI: m/z calculated for [M þ H]þ C15H13 N2 221.1079, obtained
221.1070.
(638.9 mg, 68%). 1H (CDCl3, 400 MHz)
d
6.42 (dd, J ¼ 4.2, 1.2 Hz, 2H),
6.48 (d, J ¼ 4.2 Hz, 2H), 7.66 (s, 2H), 12.3 (brs, 1H, NH). 13C (CDCl3,
100 MHz)
d 145.4 (2CH), 143.5 (Cq), 140.4 (Cq), 139.5 (dm,

164
Z. Abada et al. / European Journal of Medicinal Chemistry 67 (2013) 158e165
J ¼ 190 Hz, CeF), 138.6 (m, Cq), 136.2 (m, Cq), 126.9 (2CH), 123.9
(Cq), 118.9 (2CH). 19F (CDCl3, 188 MHz)
14.8 Hz, 2F), ꢄ152.83 (tt, J ¼ 20.9, 1.7 Hz, 2F), ꢄ138.76 (m, 1H).
[M þ H]þ. HRMS-ESI: m/z calculated for [M þ H]þ 311.0608,
obtained 311.0608. Rf 0.63 (CH2Cl2).
general protocol afforded bis-(5-pentafluoro-phenyl)dipyrrinato)
iron (II) complex 3c (302.1 mg, 72%). 19F (CDCl3, 188 MHz)
d
ꢄ161.41 (dd, J ¼ 20.9,
d
ꢄ135.77
(brd, J ¼ 11.3 Hz, 4F), ꢄ151.91 (t, J ¼ 20.7 Hz, 2F), ꢄ160.23 (brt,
J ¼ 18.8 Hz, 4F). LRMS-APCI: m/z 674.2 [M]þ. HRMS-APCI: m/z
calculated for [M
674.0267. Rf 0.87 (Cyclohexane/AcOEt:7/3).
þ
H]þ. C30H13F10FeN4 674.0267, obtained
6.2.4. 5-(3,4,5-Trimethoxyphenyl)dipyrromethene (2d)
Reaction from 5-(3,4,5-methoxyphenyl)dipyrromethane 1d (1 g,
3.20 mmol) and DDQ (799 mg, 3.52 mmol) following the general
protocol afforded dipyrromethene 2d (690.8 mg, 69%). 1H (CDCl3,
6.3.4. Bis-(5-pentafluorophenyl)dipyrrinato)manganese (II)
complex (3d)
Reaction from 5-(3,4,5-methoxyphenyl)dipyrromethane 2c
(200 mg, 0.65 mmol) and Mn(OAc)2 (10 equiv.) bis-(5-penta-
fluorophenyl)dipyrrinato)manganese (II) complex 3d (84 mg, 39%).
300 MHz)
J ¼ 4.1 Hz, 2H), 6.71 (d, J ¼ 4.1 Hz, 2H), 6.75 (s, 2H), 7.65 (s, 2H), 12.6
(brs, 1H, NH). 13C (CDCl3, 75 MHz)
152.3 (Cq), 143.6 (CH), 141.7
d 3.86 (s, 6H, 2OCH3), 3.94 (s, 3H, OCH3), 6.41 (d,
d
19F (CDCl3, 188 MHz)
d
ꢄ142.32 (brs, 4F), ꢄ153.45 (t, J ¼ 20.7 Hz,
(Cq), 140.7 (Cq), 138.5 (Cq), 132.6 (Cq), 128.6 (CH), 117.5 (CH), 108.4
(CH), 60.9 (4OCH3), 56.2 (3OCH3). LRMS-APCI: m/z 310 [M þ H]þ.
HRMS-ESI: m/z calculated for [M þ H]þ C18H18N2O3 311.1396, ob-
tained 311.1396. Rf 0.62 (CH2Cl2).
2F), ꢄ161.60 (brs, 4F). LRMS-APCI: m/z 673.0 [M þ H]þ. HRMS-APCI:
m/z calculated for [M]þ. (55Mn) C30H12F10MnN4 673.0283, obtained
673.0277. Rf 0.7 (CH2Cl2).
6.4. Synthesis of porphyrins (4i) and (4j)
6.2.5. 5-(2,6-Dichlorophenyl)dipyrromethene (2e)
Reaction from 5-(2,6-dichlorophenyl)dipyrromethane 1e
(500 mg, 1.71 mmol) and DDQ (428 mg, 1.89 mmol, 1.1 equiv.)
following the general protocol afforded dipyrromethene 2e
To a round bottom flask, containing a magnetic bar, solid
reagents were weighed : 5-bromo-10,15-diphenylporphyrin 4d
(31 mg, 0.05 mmol, 1 equiv.), Pd2dba3 (1.6 mg, 0.0018 mmol, 10 mol
%), Xantphos (4.1 mg, 0.0071 mmol, 40 mol%) and Cs2CO3 (23.2 mg,
0.071 mmol, 2 eq.) and the flask was purged with nitrogen. A so-
(73.4 mg, 15%). 1H (CDCl3, 300 MHz)
d
6.37 (m, 4H, CH), 7.34 (dd,
J ¼ 9.0, 6.9 Hz, 1H), 7.40 (d, J ¼ 7.0 Hz, 1H), 7.45 (d, J ¼ 1.49 Hz, 1H),
7.64 (s, 2H), 11.8 (brs, 1H, NH). 13C (CDCl3, 75 MHz)
144.4 (CH),
d
lution of pyrrolidinone (30.8 ml, 0.40 mmol, 8 equiv.) in Dioxane was
139.9 (CH), 135.6 (C), 134.9 (CeCl), 134.8 (CeCl), 130.1 (C), 127.9
(CH), 126.7 (CH), 118.3 (CH). LRMS-ESI: m/z 290 [M þ H]þ. HRMS-
ESI: m/z calculated for [M þ H]þ 289.0299, obtained 289.0290. Rf
0.67 (CH2Cl2).
added and the flask was purged again with nitrogen. The reaction
mixture was stirred at 100 ꢁC for 24 h. The flask tube was cooled
down to room temperature and then concentrated. The crude re-
action mixture was then purified on a silica gel column (solid de-
posit) and purified affording the title porphyrin 4j (28 mg, 89%)
along mono coupled porphyrin 4i (4 mg, 11%).
6.3. General protocol for the synthesis of dipyrromethenes
complexes 3aee
6.4.1. 5,15-Di-(N-(20-Pyrrolidinone-yl)-10,20-diphenylporphyrin
To a solution of dipyrromethene 2aee in 1:1/CHCl3:MeOH was
added Et3N (1 equiv) followed by the metal salt, e.g. FeCl2$2H2O or
Mn(OAc)2 (10 equiv). The reaction mixture was then heated to
reflux and controlled by TLC until complete conversion. The reac-
tion mixture was then diluted with CH2Cl2, washed with brine and
deionized water, dried over sodium sulfate, filtered, and concen-
trated under vacuum. The residue was purified over neutral
alumina (CH2Cl2: Et3N, 99:1) to furnish complexes (39e98%)
(4j) [16]
1H (CDCl3, 300 MHz)
d
ꢄ2.87 (s, 2H), 2.83 (m, 4H), 3.24 (m, 4H),
4.73 (td, 4H, J ¼ 6.7 Hz, J ¼ 14.3 Hz), 7.79 (m, 6H, Ar), 8.17 (m, 4H,
Ar), 8.90 (d, 4H, J ¼ 4.9 Hz), 9.13 (d, 4H, J ¼ 4.7 Hz). 13C (CDCl3,
75 MHz)
d 178.3 (NeC]O), 141.2 (Cq), 134.6 (CH), 127.8 (CH), 41.9
(CH2), 32.0 (CH2), 20.2 (CH2). UVevisible (nm,
3 , c 0.06 mM,
CH2Cl2): 421 (36,860), 513 (7805), 547 (2623), 590 (2670), 646
(1696). LRMS-APCI: m/z 629 [M þ H]þ. HRMS-MALDI: m/z calcu-
lated for [M þ H]þ C40H33N6O2 629.2659, obtained 629.2629. IR
(cmꢄ1): 2037, 2023, 1397, 1277, 750. Rf 0.27 (AcOEt).
6.3.1. Bis-(5-(3,4,5-methoxyphenyl)dipyrrinato)iron (II) complex
(3a)
Reaction from 5-(3,4,5-methoxyphenyl)dipyrromethene 2d
(345 mg, 1.11 mmol) and FeCl2$H2O (10 equiv.) following the gen-
eral protocol afforded bis-(5-(3,4,5-methoxy-phenyl)dipyrrinato)
6.4.2. 5-Bromo-15-(N-(20-pyrrolidinone-yl)-10,20-
diphenylporphyrin (4i)
1H (CDCl3, 400 MHz)
d
ꢄ2.78 (s, 2H, 2NH), 2.82 (quint, J ¼ 7.5 Hz,
2H), 3.23 (t, J ¼ 7.9 Hz, 2H), 4.71 (t, J ¼ 7.1 Hz, 2H), 7.78 (m, 6H, Ar),
8.17 (m, 4H, Ar), 8.86 (m, 4H, -H), 9.65
-H), 9.10 (d, J ¼ 4.5 Hz, 2H,
(d, J ¼ 4.5 Hz, 178.1 (NeC]O), 141.3
-H). 13C (CDCl3, 100 MHz)
iron (II) complex 3a (255.8 mg, 68%). 1H (CDCl3, 300 MHz)
d 10.46 (s,
4H), 4.90 (s, 12H), 4.66 (s, 6H). LRMS-APCI: m/z 674.4 [M]þ. HRMS-
APCI: m/z calculated for [M þ H]þ. C36H35FeN4O6 674.1828, ob-
tained 674.1815. Rf 0.26 (Cyclohexane/AcOEt: 7/3).
b
b
b
d
(Cq), 134.7 (CH), 134.6 (CH), 134.5 (CH), 128.1 (CH), 126.9 (CH), 126.8
(CH),121.1 (Cq),114.6 (Cq), 58.4 (CH2), 32.0 (CH2), 20.2 (CH2). LRMS-
APCI: m/z 624 [M þ H]þ. HRMS-MALDI: m/z calculated for [M þ H]þ
C36H27N5OBr 624.1393, obtained 624.1395. UVevisible (nm,
6.3.2. Bis-(5-(3,4,5-methoxyphenyl)dipyrrinato)manganese (II)
complex (3b)
Reaction from 5-(3,4,5-methoxyphenyl)dipyrromethene 2d
(345 mg,1.11 mmol) and Mn(OAc)2 (10 equiv.) following the general
protocol afforded bis-(5-(3,4,5-methoxy-phenyl)dipyrrinato)man-
3
0.1 mM, CHCl3): 421 (19,923), 517 (2070), 551 (1017), 593 (892),
652 (732). Rf 0.56 (AcOEt).
ganese (II) complex 3b (395.1 mg, 98%). 1H (CDCl3, 300 MHz)
d
7.46
6.5. 5,15-Di-(3,4,5-trimethoxyphenyl)-10-(5-oxopyrrolidine-2(S)-
carboxylate)) porphyrin (4f)
(brs, 4H), 3.91 (s, 6H), 3.81 (s, 12H). LRMS-APCI: m/z 674.2 [M þ H]þ.
HRMS-APCI: m/z calculated for [M þ H]þ. C36H35MnN4O6 674.1932,
obtained 674.1921. Rf 0.17 (Cyclohexane/AcOEt: 7/3).
Methyl ester 4g (133 mg, 0.17 mmol, 1 equiv.) was dissolved in a
CH2Cl2/MeOH (1:1) mixture and NaOH 3M (1 ml) was added. The
mixture was stirred at room temperature for 4 h then the reaction
was neutralized with a solution of 10% HCl until pH 4e5 was
reached. The solution was extracted with dichloromethane several
6.3.3. Bis-(5-pentafluorophenyl)dipyrrinato)iron (II) complex (3c)
Reaction from 5-(3,4,5-methoxyphenyl)dipyrromethane 2c
(388.9 mg, 1.25 mmol) and FeCl2$H2O (10 equiv.) following the

Z. Abada et al. / European Journal of Medicinal Chemistry 67 (2013) 158e165
165
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times and the different organic phases were combined, washed
with distilled water and the solvent was removed on rotavapor
affording carboxylic acid porphyrin 4f after column chromatog-
raphy purification on silica gel (68.7 mg, 52%). 1H (CDCl3, 300 MHz)
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d
ꢄ2.55 (bs, 2H, NH), 2.41e2.65 (m, 2H, CH2), 2.66e2.96 (m,
2H,CH2), 3.79 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.92 (s, 3H, OMe), 3.94
(s, 3H, OMe), 4.01 (s, 3H, OMe), 4.17 (s, 3H, OMe), 4.92 (m, 1H, CHe
N), 7.32 (s, 2H, Ar), 7.38 (s, 2H, Ar), 8.48 (s, 1H), 8.90 (s, 2H), 9.02 (dd,
J ¼ 4.3 Hz, J ¼ 13.5 Hz, 2H), 9.23 (s, 1H), 9.81 (s,1H), 10.06 (s, 1H),
10.25 (s, 1H), 12.43 (s, 1H, C(O)OH). 13C (CDCl3, 75 MHz)
d 177.9 (Ne
C]O), 151.5 (Cq), 137.8 (Cq), 136.5 (Cq), 134.0 (CH), 132.9 (CH), 132.3
(CH), 131.3 (CH), 130.4 (CH), 128.4 (CH), 125.8 (CH), 125.8 (CH), 120.1
(Cq), 112.7 (CH), 106.4 (CH), 61.2 (OCH3), 61.1 (OCH3), 56.3(OCH3),
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53.4 (CH2). UVevisible (nm, , c 0.05 mM, MeOH): 419 (44,013), 508
3
(9449), 542 (2848), 583 (3406), 641 (1520). LRMS-APCIꢄ: m/z 768
[M ꢄ H]ꢄ. HRMS-MALDI: m/z calculated for [M þ H]þ C43H40N5O9
770.2821, obtained 770.2850. IR (cmꢄ1): 3300, 2366, 1581, 1408,
1126. Rf 0.17 (CH2Cl2/MeOH : 8/2).
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Acknowledgements
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Our laboratory BioCIS-UMR 8076 is a member of the laboratory
of Excellence LERMIT supported by a grant from ANR (ANR-10-
LABX-33). We thank J.C. Jullian for the NMR experiments and K.
Leblanc for HPLC analyses. We thank the referees for pointing out
some important features and helping the quality of manuscript
improvement.
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Appendix A. Supplementary material
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.06.002.
References
[1] http://www.who.int/tdr/diseases/en/.