Synthesis and biological evaluation of new bis-indolone-N-oxides

Article abstract of DOI:10.1016/j.bioorg.2013.03.001

A series of bis-indolone-N-oxides, 1a-f, was prepared from bis(ethynyl)benzenes and o-halonitroaryls and studied for their in vitro antiplasmodial activities against Plasmodium falciparum and representative strains of bacteria and candida as well as for their cytotoxicity against a human tumor cell line (MCF7). They did not cause any haemolysis (300 μg mL^-1). Of the synthesized bis-indolones, compound 1a had the most potent antiplasmodial activity (IC₅₀ = 0.763 μmol L^-1 on the FcB1 strain) with a selectivity index (CC₅₀ MCF7/IC ₅₀ FcB1) of 35.6. No potency against the tested microbial strains was observed.

Full text of DOI:10.1016/j.bioorg.2013.03.001

Bioorganic Chemistry 48 (2013) 16–21
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of new bis-indolone-N-oxides
Ennaji Najahi a,b, , Alexis Valentin a,b, Nathan Téné c, Michel Treilhou c, Françoise Nepveu a,b
⇑
a Université de Toulouse III, UPS, UMR 152 PHARMA-DEV, 118 Route de Narbonne, F-31062 Toulouse cedex 9, France
b IRD, UMR 152, F-31062 Toulouse cedex 9, France
c Université de Toulouse, CUFR JF Champollion, EA 4357, Equipe VacBio, Place de Verdun, F-81012 Albi, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 November 2012
Available online 6 April 2013
A series of bis-indolone-N-oxides, 1a–f, was prepared from bis(ethynyl)benzenes and o-halonitroaryls
and studied for their in vitro antiplasmodial activities against Plasmodium falciparum and representative
strains of bacteria and candida as well as for their cytotoxicity against a human tumor cell line (MCF7).
They did not cause any haemolysis (300
the most potent antiplasmodial activity (IC50 = 0.763
(CC50 MCF7/IC50 FcB1) of 35.6. No potency against the tested microbial strains was observed.
Ó 2013 Elsevier Inc. All rights reserved.
l
g mLꢀ1). Of the synthesized bis-indolones, compound 1a had
l
mol Lꢀ1 on the FcB1 strain) with a selectivity index
Keywords:
Bis-indolone-N-oxides
Plasmodium falciparum
Bacteria
Cytotoxicity
1. Introduction
rum-infected erythrocytes, through a mechanism triggered by a
redox signal that activates a tyrosine kinase leading to a hyper-
phosphorylation of the band 3 protein. The infected erythrocyte
becomes not only less hospitable for parasite maturation, but
also less mechanically stable and capable of harboring the devel-
oping parasite [7]. We also demonstrated the rapid uptake and
biotransformation of INODs into an active metabolite in human
erythrocytes [8]. This biotransformation is a bioreduction of
the indolone-N-oxide structure, which is thiol and enzyme
dependent. These reduction reactions are highly probable in
Malaria remains a major cause of morbidity and mortality
throughout the world despite many efforts in recent years to
control it. The number of malaria cases has fallen over the past
decade and it is estimated that nearly 750,000 lives have been
saved in Africa alone (Global Plan for ARTs resistance contain-
ment, WHO, Report, 2011), but this progress is very fragile. Nat-
ural and hemisynthetic artemisinins (ARTs) are used in
artemisinin-based combination therapies (ACTs), which are one
of the reference treatments for Plasmodium falciparum malaria.
However, resistance to ARTs is becoming a major challenge in
malaria control [1]. Resistance to artemisinins (ARTs resistance)
has spread to new locations in Asia, out of the Greater Mekong
sub-region (Thailand, Myanmar, China) [2]. No other antimalarial
drugs with the same level of efficacy and tolerability of ARTs are
available and they are essential also for the treatment of severe
malaria to decrease its mortality [3]. No other drugs are ready
for deployment, and drug development efforts are not expected
to yield new antimalarials until the end of this decade [4]. Thus,
there is an urgent need for new molecules to prepare and devel-
op drugs for the near future.
erythrocytes where molecular reducers (glutathione, L-cysteine)
and reductases are concentrated. The nature of the reductases
responsible for this biotransformation is still not identified nor
is the contribution of the chemical versus enzymatic bioreduc-
tion that is the starting point of the bioactivity of the indo-
lone-N-oxides. In order to better understand the role played by
the reducible scaffold of these molecules we decided to design
and prepare new molecules with two indolone-N-oxide cores
to examine if their increased H and electron pumping properties
would increase the antiplasmodial activity. We also tested the
impact of these redox properties on bacteria and fungi (Pseudo-
monas aeruginosa, Cronobacter sakazakii, Bacillus subtilis, Staphylo-
coccus aureus, Geotrichum candidum, and Candida albicans). The
effect on the biological properties of the meta or para position
of the second indolone-N-oxide core in respect to the 2-phenyl
indolone-N-oxide was examined and compared to the activities
of representative mono-indolone-N-oxide. Six new compounds
were synthesized using the Sonogashira coupling reaction of
bis(ethynyl)benzene with different o-halo-nitroaryls followed by
Recently, we have developed a series of indolone-N-oxides
(INODs) with potent antimalarial activity [5] affording a new
antimalarial molecular scaffold [5,6]. We have shown that INODs
cause a membrane destabilization and vesiculation of P. falcipa-
⇑
Corresponding author at: Université de Toulouse III, UPS, UMR 152 PHARMA-
DEV, 118 Route de Narbonne, F-31062 Toulouse cedex 9, France. Fax: +33
562259802.
a
cycloisomerization of bis(2-(2-nitrophenyl)ethynyl)benzene
systems by an electrophilic palladium(II) complex.
E-mail address: ennaji.najahi@univ-tlse3.fr (E. Najahi).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.03.001

E. Najahi et al. / Bioorganic Chemistry 48 (2013) 16–21
17
Table 1
Structure, antiplasmodial and cytotoxic activities of bis-indolone-N-oxide derivatives.
c
d
Compounds
Structures
MW (g molꢀ1)/logPcalc
IC50
(
l
mol Lꢀ1) FcB1
CC50
19.5
(
l
mol Lꢀ1
)
MCF7
Selectivity index MCF7/FcB1
22
Ia
O
223.23/2.06
0.889
N
O
1a
368.34/2.19
0.763
>27.2
>35.6
O
N
O
O
N
O
1b
IIa
1c
O
O
368.34/2.22
253.26/2.02
428.39/2.11
2.720
0.184
3.738
>27.2
13.4
>10
>10
73
N
O
N
O
O
O
N
O
>3
O
O
N
O
O
O
O
N
O
O
O
1d
428.39 /2.17
267.24/1.51
6.074
1.272
>10
>1.6
O
N
O
N
O
IIIa
O
b
b
O
O
N
O
1e
456.36/1.47
3.508
>21.9
>6
O
O
O
N
O
O
O
O
N
O
O
O
1f
456.36/1.48
4.324
>21.9
>5
O
O
O
N
N
O
O
O
Chloroquine
Sodium artesunate
319/5.28
406/2.29
0.202
19.4
9.8
96
1633
0.006d
a
b
c
Ref. [5].
Not determined.
LogPcalcd; calculated with VCCLAB (http://www.virtuallaboratory.org/lab/alogps/start.html).
The drug concentration needed to cause a 50% decrease in cell viability. The IC50 SD were always lower than 10% and were discarded for maximum lisibility.
d
under reflux (Scheme 1). Compounds 6 were obtained by electro-
2. Results
philic nitration of o-bromoaryl aldehydes using a fuming nitric
acid/acetic acid mixture [5]. Di-iodobenzenes 5 were coupled with
two equivalents of trimethylsilylacetylene to provide 4[10,11] in
high yield. Then, the alkynes were deprotected to afford the diyne
3[10,12] in quantitative yield (Scheme 1).
The structures of bis-isatogens were determined from spectro-
scopic data. The IR spectra showed bands in the range of 1690–
1710 cmꢀ1 which confirmed the presence of C@O functions. In
the 13C NMR spectra the most significant information was the dis-
appearance of signals at d = 86 and 95 ppm corresponding to the
2.1. Chemistry
A series of six analogues of bis-indolone-N-oxide 1a–f (Table 1)
was prepared. The synthesis of the bis-isatogens 1 was divided into
two sub-steps: the first involved Sonogashira coupling of 2-halo-
nitroaryls 6 with diynes 3, followed by the nitro-alkyne cycloiso-
merization of the bis(2-(2-nitrophenyl)ethynyl)benzenes 2 in the
presence of catalytic amounts of Pd(CH3CN)2Cl2[9] in acetonitrile

18
E. Najahi et al. / Bioorganic Chemistry 48 (2013) 16–21
Scheme 1. Synthesis of bis-indolone-N-oxides derivatives. Reagents and conditions: (i) (CH3)3SiAC„H, Pd(PPh3)2Cl2, CuI, Et3N, N2, rt; (ii) CH3OH, CH2Cl2, K2CO3; (iii) AcOH,
HNO3 fuming; (iv) Pd(PPh3)2Cl2, CuI, NEt3, N2, rt; and (v) Pd(CH3CN)2Cl2, CH3CN, N2, reflux 86 °C.
Table 2
Antimicrobial activity of bis-indolone-N-oxide derivatives.
Compounds
Pseudomonas aeruginosa
Cronobacter sakazakii
MIC
Bacillus subtilis
MIC
Staphylococcus aureus
MIC
Geotrichum candidum
MIC
Candida albicans
MIC
MICa
1a
1b
1c
1d
1e
1f
>814.46
430.28
>700.3
>700.3
437.22
437.22
56.52
>814.46
341.78
>700.3
>700.3
245.86
245.86
17.86
430.31
341.8
>814.46
85.79
84.16
95.71
91.04
70.12
67.93
–
534.83
304.07
271.49
b
b
>700.3
>700.3
275.66
275.86
71.15
b
b
250.9
246.73
2.25
304.58
309.62
–
–
Tetracycline
Ampicillin
Amphotericinc
DMSO
71.89
14.34
14.48
0.56
–
–
–
–
–
6.77
0.32
b
b
b
b
b
b
a
b
c
MICs: minimum inhibitory concentrations (
Inactive.
Ref. [14].
l
mol Lꢀ1).
ethynylene group present in the intermediate reagent 2a–f and the
appearance of signals at d = 186–188 ppm indicating the presence
of the carbonyl functions in the compounds 1a–f. The color of
the bis-isatogens was dark orange to dark red.
This was confirmed by the SYTOX Green assay which shows no
membrane destabilization against micro-organisms. Cytotoxicity
was assayed on the mammalian MCF7 cell line and was higher
The logP values (Table 1) calculated with the VCCLAB software
are in the range 1.47–2.22 and show a slight increase in the lipo-
philicity for compounds 1a–d, and a slight decrease for compounds
1e–f, compared to those of the parent mono-indolone-N-oxides.
Traditionally, the values of the n-octanol/water partition coeffi-
cient, logPo/w, are measured using the ‘‘shake-flask’’ method. How-
ever, the reducibility character of the N-oxide derivatives in
solution makes the use of this time-consuming classical method
limited. The prediction of logP by the RP-HPLC analysis can replace
the shake-flask method and is appropriate for the N-oxide deriva-
tives as recently demonstrated by Moreno et al. [13]. The logP val-
ues of the new synthesized derivatives 1a–f (1.47–2.22) are in the
same range as those determined by RP-HPLC for 3-methylquinox-
aline-2-carboxylic acid benzylamide 1,4-di-N-oxide derivatives
(1.76–2.72) [13].
than 10 l
mol Lꢀ1 (Table 1).
By comparing the new bis-analogues with the mono-analogues
(2-phenyl-3H-indol-3-one-N-oxide, I, 5-methoxy-1-oxy-2-phenyl-
indol-3-one, II, and 5-oxy-6-phenyl-[1,3]dioxolo-[4,5-f]indol-7-
one, III) previously reported [5], it appears that mono parent com-
pounds present better antiplasmodial activity (IC50
0.184–1.27) than the bis-derivatives 1a–f, (IC50
mol Lꢀ1: 0.76–
6.07) (Table 1). It should be noted that the activity of the bis-ana-
logue 1a (IC50 = 0.76
mol Lꢀ1) was moderate but comparable to
that of the mono-analogue, I (IC50 = 0. 89
mol Lꢀ1), 1a and I bear-
/l :
mol Lꢀ1
/l
l
l
ing not substituted cycles. When substituents are introduced, the
meta substitution gave always better results than the para position
but a strong decrease of the activity is observed. Replacement of R1
(H) by a methoxyl group leads to a significant decrease or loss of
activity (CI50
the replacement of the H (R1 = R2) by a dioxymethylene group
(OACH2AO) (CI50
mol Lꢀ1: III = 1.272, 1e = 3.50, 1f = 4.32). Selec-
/l
mol Lꢀ1: II = 0.184, 1c = 3.74, 1d = 6.07) as well as
2.2. Pharmacology
/l
tivity index defined as the ratio of the CC50 value obtained with
2.2.1. In vitro haemolytic, cytotoxic and antiplasmodial activities
The haemolytic assay determined on human erythrocytes was
the first step used to determine a potential cytotoxicity. It is also
a classical test to evaluate the membrane perturbation power of
compounds. No haemolysis was found, even with high concentra-
MCF7 cells to the IC50 value with CQ-resistant P.f. strain FcB1 were
in the
lmolar range, one log higher than the antiplasmodial IC50
value varying from 10 to 27.2 l
mol Lꢀ1 (Table 1).
2.2.2. Antimicrobial activities
tions (300
l
g mLꢀ1). It shows that the antiplasmodial activities ob-
Compounds 1a–f were evaluated for their in vitro antibacterial
activity against representative Gram-positive and Gram-negative
bacteria and against two fungi using standard techniques. Mini-
served (see later) were not correlated with a potential haemolytic
activity and are presumed to be the result of an intracellular action.

E. Najahi et al. / Bioorganic Chemistry 48 (2013) 16–21
19
mum inhibitory concentrations (MICs) were calculated to express
the antimicrobial activity and were defined as the concentration
of the compound required to give complete inhibition of bacterial
growth (Table 2). The data show that the compounds have no con-
siderable potency against all the tested micro-organisms. The best
activities observed were against B. subtilis.
LCQ Deca XP Max) using electrospray as an ionization source. High-
resolution mass spectra (HRMS) were recorded on a Bruker Maxis
spectrometer (Service Commun, ICT, Toulouse, France). Melting
points were determined with an Electrothermal 9300 capillary
melting point apparatus and are uncorrected. The purity of all
compounds was determined by LC–PDA–MS methods and was
found to be in the range 96–99%.
3. Discussion
5.2. General procedure for the Sonogashira coupling
New bis-analogues had the same antiplasmodial activities as
mono-analogues when no substituents were present at R1, R2 and
at R3 (meta or para positions of the phenyl group). Introducing sub-
stituents led to a strong decrease in the antiplasmodial activities.
For example, when the methoxy group was introduced at the R1
position in the mono-indolone series, the IC50 value decreased by
Pd(PPh3)2Cl2 (0.2 mmol) was added to a solution of bis(ethy-
nyl)benzene, 3 (1 mmol) and 2-halonitroaryl, 6 (2.2 mmol) in
freshly distilled triethylamine (20 mL) and DMF (10 mL), and the
mixture was de-aerated with argon for 30 min. CuI (0.2 mmol)
was added, and the mixture was de-aerated with argon for
10 min and stirred at room temperature for 6 h. The reaction mix-
ture was partitioned between ethyl acetate and water. The organic
layer was separated, washed with brine, dried (NaSO4), and con-
centrated, and the residue obtained was purified by column chro-
matography (cyclohexane in dichloromethane) to afford an
intermediate, 2.
about 0.7
introduction of this substituent in the bis-analogue series increased
the IC50 value by about 3.35 mol units from compound 1a to com-
lmol units from compound I to compound II, while the
l
pound 1c. The pharmacomodulation on the mono-indolone-N-
oxide series gave hits with IC50 values in the nanomolar range,
by introducing various substituents at R1, R2 and/or R3 positions
[5]. The results obtained in this current study show that the same
substituents had the opposite effect, increasing the IC50 values. The
activity of the bis-indolone-N-oxides seems limited by steric ef-
5.2.1. 1,3-Bis(2-(2-nitrophenyl)ethynyl)benzene 2a
Yellow solid, yield: 53%, mp: 134–137 °C. 1H NMR (300 MHz,
DMSO-d6) d: 7.36–7.40 (m, 3H), 7.45–7.52 (m, 2H), 7.58–7.63 (m,
5H), 8.07 (d, J = 1.2, 7.9 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) d:
86.1 (2C„C), 95.1 (2C„C), 117 (2C), 122.8 (2C), 125.4 (2CH),
128.4 (CH), 129.7 (2CH), 130.9 (2CH), 133 (2CH), 134.3 (2CH),
135.5 (CH), 149.7 (2C). IR (KBr, cmꢀ1): 3019, 2412, 1605, 1579,
1523, 1465, 1343.
fects. The IC50 value variation (0.7–6.0 l
mol Lꢀ1) is more depen-
dent on substituent and steric effects than on logP values (1.74–
2.22). We previously reported that the indolone-N-oxides were
bio-reducible in red blood cells, through thiol and enzyme depen-
dent reactions [8]. However, the twofold increase in the reducible
character of the molecule obtained in this study did not improve
the antiplasmodial activity. This result strongly suggests that an
enzymatic reduction of these molecules is necessary to induce a re-
dox signal that activates the syk tyrosine kinase leading to a hyper-
phosphorylation of band 3 of the erythrocyte [7]. Moreover, a
decrease in the antiplasmodial activity was observed with the
more bulky derivatives (1a–b; 1e–f) supporting the idea that indo-
lone-N-oxides might be the substrates of some reductases present
in the erythrocyte, and that the steric hindrance generated in bis-
analogues may impair the enzymatic bioreduction.
5.2.2. 1,4-Bis(2-(2-nitrophenyl)ethynyl)benzene 2b
Yellow solid, yield: 68%, mp: 151–153 °C. 1H NMR (300 MHz,
DMSO-d6) d: 7.35–7.41 (m, 2H), 7.43–7.52 (m, 2H), 7.64–7.75 (m,
6H), 8.06 (d, J = 1.2, 7.9 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) d:
86.1 (2C„C), 95.1 (2C„C), 117.2 (2C), 122.8 (2C), 125.3 (2CH),
131.1 (2CH), 131.1 (2CH), 129.6 (2CH), 133 (2CH), 134.3 (2CH),
149.7 (2C). IR (KBr, cmꢀ1): 3019, 2412, 1607, 1579, 1524, 1465,
1343.
Concerning the other antimicrobial activities, the introduction
of a second indolone-N-oxide moiety did not increase the antibac-
terial and antifungal activities [15].
5.2.3. 1,3-Bis(2-(5-methoxy-2-nitrophenyl)ethynyl)benzene 2c
Yellow solid, yield: 47%, mp: 131–133 °C. 1H NMR (300 MHz,
DMSO-d6) d: 3.91 (s, 6H, 2 CH3), 7.14–7.43 (m, 4H), 7.58–7.67
(m, 4H), 8.15 (m, 2H); 13C NMR (75 MHz, DMSO-d6) d: 56.3
(2CH3), 87 (2C„C), 97.1 (2C„C), 116.1 (2CH), 117.9 (2C), 118.5
(2CH), 123.6 (2C), 124.9 (2CH), 128.1 (CH), 131 (2CH), 134.7
(CH), 145.5 (2C), 162.1 (2C). IR (KBr, cmꢀ1): 3054, 2936, 1653,
1610, 1578, 1520, 1477, 1447, 1385, 1357, 1260.
4. Conclusion
Six novel bis-indolone-N-oxide compounds have been prepared
in comparison with mono-indolone-N-oxides analogues. The in-
creased number of reducible functions does not improve the anti-
microbial activities of the indolone-N-oxide derivatives.
5.2.4. 1,4-Bis(2-(5-methoxy-2-nitrophenyl)ethynyl)benzene 2d
Yellow solid, yield: 54%, mp: 156–158 °C. 1H NMR (300 MHz,
DMSO-d6) d: 3.90 (s, 6H, 2 CH3), 7.14–7.42 (m, 6H), 7.62–7.68
(m, 2H), 8.17 (m, 2H). 13C NMR (75 MHz, DMSO-d6) d: 56.3
(2CH3), 86.94 (2C„C), 97.1 (2C„C), 116.2 (2CH), 118 (2C), 118.5
(2CH), 123.7 (2C), 125.1 (2CH), 131.2 (2CH), 131.2 (2CH), 145.7
(2C), 161.4 (2C). IR (KBr, cmꢀ1): 3054, 2950, 1653, 1611, 1576,
1520, 1476, 1447, 1385, 1356, 1260.
5. Experimental
5.1. General
Commercially reagent grade chemicals were used as received
without additional purification. All reactions were followed by
TLC (E. Merck Kieselgel 60 F-254), with detection by UV light at
254 nm. Column chromatography was performed on silica gel
(60–200 mesh E. Merck). IR spectra were recorded on a Perkin–El-
mer PARAGON 1000 FT-IR spectrometer. 1H and 13C NMR spectra
were recorded on an AC Bruker spectrometer at 300 MHz (1H)
and 75 MHz (13C) using (CD3)2SO as solvent with (CD3)2SO (dH
2.5) or (CD3)2SO (dC 39.5). Chemical shifts (d) are reported in parts
per million (ppm) relative to tetramethylsilane (0 ppm). The mass
spectra were recorded on an ion trap mass spectrometer (Finnigan
5.2.5. 1,3-Bis(2-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethynyl)benzene 2e
Pale brown solid, yield: 58%, mp: 134–136 °C. 1H NMR
(300 MHz, DMSO-d6) d: 6.14 (s, 4H, 2CH2), 7.04 (s, 2H), 7.35–7.59
(m, 6H). 13C NMR (75 MHz, DMSO-d6) d: 86.7 (2C„C), 96.4
(2C„C), 97.3 (2CH2), 105.4 (2CH), 112.4 (2CH), 114.7 (2C), 122.5
(2C), 128.5 (CH), 129.7 (2CH), 132.1 (CH), 144.4 (2C), 147.8 (2C),
151.6 (2C). IR (KBr, cmꢀ1): 3077, 3020, 2400, 1615, 1557, 1522,
1493, 1443, 1342, 1297, 1210.

20
E. Najahi et al. / Bioorganic Chemistry 48 (2013) 16–21
5.2.6. 1,4-Bis(2-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethynyl)benzene 2f
Pale brown solid, yield: 61%, mp: 162–164 °C. 1H NMR
(300 MHz, DMSO-d6) d: 6.15 (s, 4H, 2 CH2), 7.04 (s, 1H), 7.34–
7.60 (m, 6H); 13C NMR (75 MHz, DMSO-d6) d: 86.7 (2C„C), 96.3
(2C„C), 97.3 (2CH2), 105.6 (2CH), 112.4 (2CH), 114.7 (2C), 122.5
(2C), 130.9 (2 CH), 131 (2CH), 144.6 (2C), 147.9 (2C), 151.6 (2C).
IR (KBr, cmꢀ1): 3077, 3019, 2400, 1614, 1557, 1524, 1493, 1443,
1342, 1297, 1211.
127.9, 128.1, 128.4, 130.6, 131.2, 143.9, 144.6, 149.7, 151.7,
152.4, 186.8 (C-3), 187.1 (C-30). IR (KBr, cmꢀ1): 3100, 3092, 2922,
1702, 1632, 1586, 1506, 1490, 1445, 1392, 1350, 1277, 1168,
1029, 924, 775. MS-(+)ESI, m/z: 457 [M+H]+. HR-MS [M+H]+ calcd
for C24H13N2O8 457.0672 found: 457.0695.
5.3.6. Bisisatogen 1f
Red solid, yield: 54%, mp: 268–261 °C. 1H NMR (300 MHz,
DMSO-d6) d: 6.13 (s, 2H), 6.14 (s, 2H), 7.02–7.15 (m, 4H), 7.53–
786 (m, 4H). 13C NMR (75 MHz, DMSO-d6) d: 97.2 (CH2), 97.4
(CH2), 104.5, 105.1, 105.5, 105.7, 116.3, 117.1, 126.4, 126.7,
128.6, 130.3, 130.7, 144.8, 145.1, 147.5, 147.9, 149.6, 150.1, 186
(C-3), 186.3 (C-30). IR (KBr, cmꢀ1): 3100, 3090, 2917, 1702, 1632,
1587, 1505, 1489, 1445, 1390, 1350, 1273, 1168, 1029, 919, 775.
MS-(+)ESI, m/z: 457 [M+H]+. HR-MS [M+H]+ calcd for C24H13N2O8
457.0672 found: 457.0691.
5.3. General procedure for the cycloisomerization
Pd(CH3CN)2Cl2 (0.025 mmol, 6.5 mg 5 mol-%) was added to a
solution of diethynylene benzene 2 (0.5 mmol) in CH3CN (15 mL),
and the mixture was refluxed for 3 h in an argon atmosphere.
The precipitate formed was isolated by filtration and purified by
column chromatography (ethyl acetate in petroleum ether) to give
compound 1.
5.4. Methodology for in vitro biological evaluation
5.3.1. Bisisatogen 1a
Dark orange solid, yield: 53%, mp: 251–253 °C. 1H NMR
(300 MHz, DMSO-d6) d: 7.55–7.77 (m, 8H), 8.48–8.81 (m, 4H). 13C
NMR (75 MHz, DMSO-d6) d: 114.3, 114.4, 122,4, 122.6, 123.9,
125.1, 127.5, 128.3, 128.5, 129.3, 130.7, 130.9, 131.1, 131.3,
134,4, 134.5, 147.4, 147.7, 186.8 (C-3), 186.9 (C-30). IR (KBr,
cmꢀ1): 3054, 2986, 1702, 1602, 1570, 1527, 1511, 1498, 1422,
1375, 1273, 1178, 1046, 1029, 736, 705. MS-(+)ESI, m/z: 369
[M+H]+. HR-MS [M+H]+ calcd for C22H13N2O4 369.0875 found:
369.0888.
5.4.1. Haemolytic activity and SYTOX Green uptake assays
The haemolytic activity of the different compounds was deter-
mined against human erythrocytes from healthy donors. Details
of both assays are given in Supplementary data.
5.4.2. In vitro antiplasmodial and cytotoxicity assays
The in vitro antiplasmodial assay was carried out against the
chloroquine-resistant strain (FcB1) of P. falciparum and the
in vitro cytotoxicity was determined on human breast cancer cells
(MCF7) [16,17]. All these assays were performed as previously re-
ported [5] and details are given in the Supplementary data.
5.3.2. Bisisatogen 1b
Dark orange solid, yield: 71%, mp: 252–254 °C. 1H NMR
(300 MHz, DMSO-d6) d: 7.45–7.70 (m, 8H), 8.53–8.61 (m, 4H). 13C
NMR (75 MHz, DMSO-d6) d: 114.3, 114.9, 121.7, 122.1, 122.3,
124.8, 125.2, 127.7, 127.9, 128.4 (2C), 130.3, 131.1, 131.3, 134.4,
134.5, 147.9, 148, 186.4 (C-3), 186.7 (C-30). IR (KBr, cmꢀ1): 3054,
2986, 1706, 1601, 1569, 1527, 1512, 1496, 1422, 1372, 1261,
1180, 1045, 1029, 741, 705. MS-(+)ESI, m/z: 369 [M+H]+. HR-MS
[M+H]+ calcd for C22H13N2O4 369.0875 found: 369.0884.
5.4.3. Antimiocrobial assays
All microorganisms were grown in broth medium with contin-
uous shaking (200 rpm). B. subtilis (CIP 5262), S. aureus (CIP 53156)
were used as Gram-positive strains. The Gram-negative strains
used were P. aeruginosa (CIP 82118), and C. sakazakii (CIP
103183). Fungal strains used were C. albicans (CIP 4872) and G.
candidum (ATCC 204307). The minimal inhibitory concentrations
(MICs) were obtained as previously reported [15] and details are
given in the Supplementary data.
5.3.3. Bisisatogen 1c
Dark orange solid, yield: 47%, mp: 255–257 °C. 1H NMR
(300 MHz, DMSO-d6) d: 3.86 (s, 3H), 3.89 (s, 3H), 7.11–7.59 (m,
6H), 8.01–8.56 (m, 4H). 13C NMR (75 MHz, DMSO-d6) d: 56.6
(CH3), 56.8 (CH3), 107.9, 108.1, 114.7, 115.2, 118.3, 118.9, 124.3,
125.1, 126.8, 128.8, 129.6, 130.8, 131.1, 140.8, 141.1, 149.8,
161.8, 161.9, 186.2 (C-3), 186.9 (C-30). IR (KBr, cmꢀ1): 3032,
2997, 1704, 1597, 1523, 1481, 1447, 1386, 1283, 1182, 1020,
875, 758, 685. MS-(+)ESI, m/z: 429 [M+H]+. HR-MS [M+H]+ calcd
for C24H17N2O6 429.1087 found: 429.1098.
Acknowledgment
This work was supported by the French Research National
Agency (ANR-10-BLAN-0726, Mechanisms of Action and Targets
of new antimalarial Redox molecules, MATURE).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2013.
03.001.
5.3.4. Bisisatogen 1d
Dark orange solid, yield: 51%, mp: 257–259 °C. 1H NMR
(300 MHz, DMSO-d6) d: 3.88 (s, 3H), 3.90 (s, 3H), 7.16–7.60 (m, 6
H), 8.12–8.58 (m, 4H). 13C NMR (75 MHz, DMSO-d6) d: 56.6
(CH3), 56.7 (CH3), 108.2, 108.4, 113.8, 114.8, 118.7, 119.1, 124.3,
124.5, 129.1, 131.6, 131.9, 141, 141.1, 151.6, 151.7, 159.9, 160.7,
186.6 (C-3), 186.8 (C-30). IR (KBr, cmꢀ1): 3032, 2998, 1704, 1581,
1531, 1483, 1447, 1386, 1281, 1185, 1020, 876, 756. MS-(+)ESI,
m/z: 429 [M+H]+. HR-MS [M+H]+ calcd for C24H17N2O6 429.1087
found: 429.1096.
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