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Supporting information

Synthesis and antiplasmodial activity of new indolone N-oxide derivatives

Françoise Nepveu1,2 *

, Sothea Kim1,2

, Jeremie Boyer1,2

, Olivier Chatriant3, Hany Ibrahim

1,2,

Karine Reybier1,2

, Marie-Carmen Monje1,2

, Severine Chevalley1,2

, Pierre Perio1,2

, Barbora H.

Lajoie1,2,

, Jalloul Bouajila1,2

, Eric Deharo1,2

, Michel Sauvain1,2

, Rachida Tahar4, Leonardo

Basco4, Antonella Pantaleo

5, Francesco Turini

5, Paolo Arese

5, Alexis Valentin

1,2, Eloise

Thompson, Livia Vivas6, Serge Petit

3, Jean-Pierre Nallet

3.

1 Université de Toulouse, UPS; UMR 152 (Laboratoire de pharmacochimie des substances naturelles et

pharmacophores redox), F-31062 Toulouse cedex 9, France 2

IRD, UMR 152, F-31062 Toulouse cedex 9, France 3 IDEALP-PHARMA, Bâtiment CEI, 66 Bd Niels Bohr, BP 2132, 69603 Villeurbanne cedex, France

4 Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), BP 288,

Yaoundé, Cameroun 5 University of Turin Medical School, Via Santena 6 bis, 10126 Torino, Italy

6 London School of Hygiene and Tropical Medicine (LSHTM), Keppel Street, London C1E 7HT United

Kingdom.

Table of contents

1. Hematin binding assays

2. Metabolism of some selected compounds by liver microsomes

3. Lactate dehydrogenase (LDH) assay

4. Elemental analysis

5. Selected 1H- and

13C-NMR spectra

6. Selected IR spectra

7. References cited in Supporting Information

* Corresponding author:

Françoise Nepveu Tel: +33-5-62-25-68-69; Fax: +33-5-62-25-98-02; e-mail: nepveu@cict.fr

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1. Hematin binding assays

Degradation of hemoglobin by malaria parasites releases ferriprotoporphyrin IX (FP)

that is detoxified by crystallization into hemozoin (HZ). Antimalarial drugs such as

chloroquine and chloroquine-like derivatives bind to FP and impair this process. To approach

the mechanisms of action of the new series, we examine the capacity of indolone-N-oxides to

interfere with hematin dimerization, to bind to iron or to form heme-adducts. First, the drug

concentration required to inhibit β-hematin formation by 50% (IC50) was determined for

selected analogues from the new series of indolone-N-oxide using the UV-visible

spectrophotometric assay of Deharo et al1. The results are given in Table 1 for five selected

compounds and are the results of two different experiments performed in triplicate.

Table 1. Comparison of IC50 required to inhibit P.f parasite growth (FCB1) to IC50

required to inhibit β-hematin formation for seven selected compound compared to

chloroquine.

Compound IC50/P.f. FcB1 (nM) IC50/FBIT (nM)

1 66 ± 28 238

49 3 940 ± 200 2 840

52 899 ± 88 inactif

51 3 560 ± 400 39

15 156 ± 57 inactif

CQ 151 ± 6 28

β-hematin formation inhibition UV-visible assay:

The analysis were done using 96-well plates non sterile with a flat bottom (Greiner

Bio-one, Poitiers, France). The well plates were charged by the addition of the following

solutions (N.B. the ordre of addition is important): i) 50 μL hemin chloride (0.5 mg/mL)

freshly prepared into DMSO, ii) 100 μL sodium acetate buffer (0.5 M, pH = 4.4), iii) 50 μL

tested compound/ DMSO (50 μL DMSO for the blank). The final pH must be adjusted

between 5 and 5.2. After incubation at 37 °C for 18-24 h, the well plates were centrifuged at

3000 rpm for 8 minutes. The supernatant was rejected and the residue was re-suspended using

200 μL de DMSO, to eliminate the non-reacted hematine. The well plate was centrifuged

again and the supernatant was rejected. The residue, ß-hematine, was dissolved into 200 μL

de NaOH 0.1 M and the absorbance was measured at 405 nm using microplate reader

(Metertech S960, Avantech, Illkirch, France). The results were expressed as a percentage

inhibition of hemin biocrystallization calculated by the following equation:

%inhibiton = 100 x (Ablank – Atest)/Ablank.

The experiments were done in triplicate (n = 3).

2. Metabolism of some selected compounds by liver microsomes

Liver microsomes pooled from male mouse (CD-1) 20 mg protein/mL and NADPH

were obtained from Biopredic International (Rennes, France). Microsomes (20 mg protein/mL

in 250 mM sucrose, stored at - 80 °C) were quickly thawed at 37 °C using a water bath and

kept on ice until use. NADPH (10 mM in phosphate buffer) was prepared in the previous

prepared buffer solution.

Due to the poor aqueous solubility, the cosolvent method2 was used as follows. DMSO

stock solutions of test compounds were prepared at 0.5 mM concentration. Diluted solutions

of test compounds were prepared by adding 50 µL of each DMSO stock solution to 200 µL of

acetonitrile to make 0.1 mM solutions in 20% DMSO/ 80% acetonitrile. Mouse liver

microsomal solution was prepared by adding 1.582 mL of concentrated mouse liver

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microsomes (20 mg/mL protein concentration) to 48.291 mL of pre-warmed (to 37 °C) 0.1 M

potassium phosphate buffer (pH 7.4) containing 127 µL of 0.5 M EDTA to make a 0.6329

mg/mL (protein) microsomal solution. 11.2 µL of each test compound diluted solution was

each added directly to 885 µL of mouse liver microsomal solution (allowing direct binding of

drugs to microsomal proteins and lipids to minimize precipitation and non-specific binding to

the plastic ware).

This solution was mixed and 180 µL was transferred to Time 0 and Time 15 min

plates (each in duplicate wells). For the Time 15 min plate, the cofactor NADPH (45 µL, 10

mM) was added to each well to initiate the reaction; the plate was incubated at 37 °C for 15

min, followed by quenching of the reaction by adding 450 µL of cold acetonitrile to each

well. For the Time 0 plate, 450 µL of cold acetonitrile was added to each well, followed by

addition of NADPH (45 µL) and no incubation. All of the plates were centrifuged at 3000

rpm for 15 min and the supernatants were transferred to other well plates for analysis by LC–

MS. The final aqueous volume of acetonitrile was 0.8% and that of DMSO was 0.2%.

Verapamil hydrochloride was used as a reference standard (T1/2 = 6 min).

3. Lactate dehydrogenase (LDH) assay

Evaluation of parasitemia by lactate dehydrogenase assay: RPMI 1640 medium

(BioWhittaker, Cambrex, Belgium) containing L-glutamine (BioWhittaker), 25 mM HEPES

(BioWhittaker), and 10% human serum (EFS, Toulouse, France), was used to cultivate P.

falciparum. Human RBCs (group O±) for parasite culture were obtained from EFS in

transfusion vials. They were washed with RPMI medium to discard plasma and leukocytes.

Leucocyte-free erythrocytes were stored at 50% hematocrit (1 volume of RPMI + 1 volume of

packed RBC) for a maximum period of 21 days. P. f. asexual blood stage parasites were

propagated by incubation at 37 °C in P.f. culture media at 3-5% hematocrit in controlled

atmosphere (5% CO2, 100% relative humidity)3. Parasitized RBCs were maintained in 25 cm

2

culture flasks (TPP, Switzerland). Reference drugs, chloroquine (CQ) and artemether (ART)

were obtained from Sigma and Cambrex, respectively. CQ was dissolved in culture medium

and ART in ethanol (stock solutions: 10 mg/mL) and stored at - 20 °C prior to testing.

Solution containing the co-enzymz acetyl pyridine adenine dinucleotide APAD (MALSTAT)

was prepared by dissolving Triton in distilled water (0.13 % w/v), warmed to 50 °C to ensure

complete dissolution, followed by addition of L-lactate (20% w/v), Tris (5.5% w/v) and

APAD (3.7% w/v), the pH of the MALSTAT final solution was adjusted to 9.2 using NaOH

(1M). Solution containing nitro blue tetrazolium (NBT) was freshly prepared by dissolving

NBT (1.6% w/v) and phenazine ethosulphate (8% w/v) into distilled water. For drug assays,

serial drug dilutions were made in P.f. culture media and added to 96-well (TPP) culture

plates. All drugs were tested in triplicate. Plasmodium-infected RBCs (PRBC) were

distributed at 1-2% parasitaemia (1.5% hematocrit) in 96-well microtiter plates with different

drug concentrations and incubated for 48 h at 37 °C and 5% of CO2. At the end of incubation

(48 h), microtiter plates were frozen and thawed. After thawing, the MALSTAT reagent was

added and the reaction was stopped after 30 min (in the dark) by addition of 25% acetic acid

into each well, the absorbance were measured at 650 nm. Growth-inhibition percentages were

plotted as a semi logarithmic function of drug concentration. The IC50 values were determined

by linear regression analysis on the linear segments of the curves. Assays were done in

triplicate. Controls were carried out to assess the background (non-treated RBC, negative

control) and parasite growth (parasitized RBC, positive control).

% inhibition = [(Mean of PRBC- mean treated RBC)/mean PRBC] X 100

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4. Elemental analysis

Compound C H N

Cal. Found Cal. Found Cal. Found

1 59.72 59.16 2.67 2.13 4.64 4.48

3 71.14 71.05 4.38 4.32 5.53 5.55

5 75.24 75.18 4.32 4.11 4.62 4.64

7 70.19 70.13 3.65 3.51 3.90 3.83

26 68.91 68.65 5.44 5.12 9.45 9.31

49 70.92 70.33 6.45 6.24 6.89 6.80

50 69.83 69.71 5.86 5.78 7.40 7.25

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5. Selected 1H and

13C NMR

1H NMR spectrum of 1

13

C NMR spectrum of 1

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HSQC spectrum of 1

HMBC spectrum of 1

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6. Selected IR Spectra

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7. References cited in the supporting information

(1) Deharo, E.; Garcia, R. N.; Oporto, P.; Gimenez, A.; Sauvain, M.; Jullian, V.; Ginsburg, H.

A non-radiolabelled ferriprotoporphyrin IX biomineralisation inhibition test for the high

throughput screening of antimalarial compounds. Exp. Parasitol., 2002, 100, 252-256.

(2) Di, L.; et al. Experimental design on single-time-point high-throughput microsomal

stability assay. J. Pharmaceut. Sci. 2004, 93, 1537–1544.

(3) Benoit, F.; Valentin, A; Pélissier, Y.; Diafouka, F.; Marion, C.; Kone-Bamba, D.; Koné,

M.; Mallié, M.; Yapo, A.; Bastide, J.M. In vitro antimalarial activity of vegetal extracts used

in west african traditional medicine. Am. J. Trop. Med. Hyg. 1996, 54, 67-71.