Novel Antimalarial Peroxides: Identification of a

Trioxolane Drug Development Candidate

Jonathan L. Vennerstrom1, Sarah Arbe-Barnes2, Reto Brun3, Susan A. Charman4, Francis C. K. Chiu4, Jacques Chollet3, Yuxiang Dong1, Arnulf Dorn5, Daniel Hunziker5, Hugues Matile5, Kylie McIntosh4, Maniyan Padmanilayam1, Josefina Santo Tomas3, Christian Scheurer3, Bernard Scorneaux3, Yuanqing Tang1, Heinrich Urwyler6, Sergio Wittlin3 & William N. Charman4

1College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE, USA

2Fulcrum Pharma Developments Ltd, Hemel Hempstead, Hertfordshire HP1 1JY, UK

3Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland

4Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia

5F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland

6Basilea Pharmaceutica Ltd., Grenzacherstrasse 487, CH-4058 Basel, Switzerland

Revised Nature Manuscript 2004-03-17397

Supplementary Information (SI)

Supplementary Methods

Reaction of Trioxolane 4 with Ferrous Acetate/TEMPO

To a solution of 4 (277 mg, 1 mmol) and TEMPO (320 mg, 2 mmol) in CH2Cl2 (10 ml) and CH3CN (10 ml) was added iron (II) acetate (261 mg, 1.5 mmol). The resulting mixture was stirred under N2 and at 35 °C for 24 h before being quenched with water (50 ml) and acetic acid (2 ml). After separation of the organic layer, the aqueous layer was extracted with CH2Cl2 (3 x 15 ml). The combined extracts were washed with brine (2 x 50 ml), dried over MgSO4, and filtered. After evaporation, the residue was purified by flash chromatography (silica gel, 50% CH3OH in CH2Cl2) followed by crystallization from 50% aqueous ethanol to afford the aminoxy acid (162 mg, 50%) as a white solid. mp 183–185 °C; 1H NMR (500 MHz, CDCl3)  1.01–1.67 (m, 24H), 2.05–2.29 (m, 6H), 2.41–2.52 (m, 1H), 3.89–4.04 (m, 1H); 13C NMR (125.7 MHz, CDCl3)  17.21, 20.55 (br), 26.01, 28.57, 29.24, 33.65 (br), 35.79, 39.64, 39.80, 60.06, 60.09, 75.48, 180.34 (br). Anal. Calcd for C19H33NO3: C, 70.55; H, 10.28; N, 4.33. Found: C, 70.47; H, 10.30; N, 4.41.

Supplementary Tables

Supplementary Table 1. In vivo activity in P. berghei infected mice following a, a single oral dose or b, three consecutive daily oral doses

a / 1 x 3 mg/kg po / 1 x 10 mg/kg po / 1 x 30 mg/kg po
Compound / Activity (%) / Survival (days) / Activity (%) / Survival (days) / Activity (%) / Survival (days) / Cure*
(%)
control / 0 / 5.2 / 0 / 5.2 / 0 / 5.2 / 0
5 / 50 / 9.0 / 95 / 9.7 / n.t. / --- / ---
6 / 99 / 10.0 / 99.96 / 11.4 / 99.99 / 27.6 / 69
7 / 98 / 9.0 / 99.94 / 10.6 / 99.95 / 10.7 / 0
AS / 33 / 6.6 / 67 / 7.4 / 92 / 9.0 / 0
AM / 56 / 8.0 / 99.36 / 9.7 / 99.73 / 9.0 / 0
CQ / 85 / 7.9 / 99.89 / 8.9 / 99.90 / 9.6 / 0
MF / 18 / 7.0 / 99.78 / 18.3 / 99.63 / 21.8 / 0
b / 3 x 1 mg/kg po / 3 x 3 mg/kg po / 3 x 10 mg/kg po
Compound / Activity (%) / Survival (days) / Activity (%) / Survival (days) / Activity (%) / Survival (days) / Cure*
(%)
control / 0 / 5.2 / 0 / 5.2 / 0 / 5.2 / 0
6 / 97 / 8.9 / 99.99 / 12.8 / >99.99 / 30.0 / 100
7 / 86 / 9.0 / 99.99 / 11.4 / >99.99 / 26.2 / 67
AS / 39 / 7.0 / 70 / 9.2 / 97 / 11.0 / 0
AM / 46 / 7.4 / 79 / 8.5 / >99.99 / 22.3 / 0
CQ / 49 / 8.1 / 99.70 / 9.4 / 99.99 / 18.2 / 0
MF / 6 / 6.6 / 99 / 15.8 / 99.92 / 24.3 / 0

* no detectable parasites at 30 days post infection

# n.t. = not tested

Supplementary Table 2. Prophylactic activity following a single 100 mg/kg oral dose

Treatment* time prior to infection
Compound / -72 h / -48 h / -24 h / 0 h
Activity (%) / Activity (%) / Activity (%) / Activity (%)
6 / 96 / 99.99 / >99.99 / >99.99
7 / --- / --- / 25 / >99.99
AS / --- / --- / --- / 86
AM / --- / --- / --- / >99.99
CQ / --- / 57 / 99.92 / >99.99
MF / 99.97 / 99.92 / >99.99 / >99.99

* all groups of n=5 mice, including an untreated control group, were infected at the same time and parasitemia was determined for each animal on day 3 post-infection and compared with the level of parasitemia in control animals.

Supplementary Table 3. In vitro cross-resistance with various strains of P. falciparum a, IC50 b, IC90

a / IC50 (ng/mL)*
Isolate / Origin / Resistance / 6 / 7 / AS / CQ
K1 / Thailand / CQ, PYR / 0.39 ± 0.06 / 1.0 ± 0.1 / 1.3 ± 0.2 / 62 ± 4
Fc27 / Papua NG / 0.74 ± 0.29 / 1.6 ± 0.5 / 4.2 ± 0.6 / 9.4 ± 2.8
FVO / Vietnam / CQ / 0.64 ± 0.11 / 0.90 ± 0.00 / 2.1 ± 0.5 / 63 ± 0
NF54 / Airport / 0.42 ± 0.06 / 0.91 ± 0.12 / 1.6 ± 0.1 / 5.1 ± 0.8
HB3 / Honduras / PYR / 0.54 ± 0.01 / 1.4 ± 0.1 / 5.0 ± 1.2 / 10 ± 2
FCB1 / Columbia / CQ / 0.63 ± 0.21 / 1.5 ± 0.4 / 1.8 ± 0.5 / 25 ± 3
ITG2-F6 / Brazil / CQ / 0.35 ± 0.07 / 0.83 ± 0.13 / 1.1 ± 0.2 / 32 ± 2
MAD20 / Papua NG / 0.17 ± 0.05 / 0.84 ± 0.13 / 2.6 ± 0.1 / 4.9 ± 0.1
Ro73 / Kenya / PYR / 0.13 ± 0.00 / 0.43 ± 0.02 / 0.74 ± 0.12 / 4.7 ± 0.3
W2 / Indochina / CQ, PYR / 0.18 ± 0.00 / 0.43 ± 0.02 / 0.83 ± 0.03 / 77 ± 5
minimum / 0.13 / 0.43 / 0.74 / 4.7
maximum / 0.74 / 1.6 / 5.0 / 77
b / IC90 (ng/mL)*
Isolate / Origin / Resistance / 6 / 7 / AS / CQ
K1 / Thailand / CQ, PYR / 1.0 ± 0.2 / 2.3 ± 0.2 / 1.9 ± 0.3 / 131 ± 17
Fc27 / Papua NG / 1.5 ± 0.6 / 2.7 ± 0.9 / 6.9 ± 1.1 / 14 ± 4
FVO / Vietnam / CQ / 0.62 ± 0.14 / 1.5 ± 0.0 / 2.0 ± 0.3 / 94 ± 0
NF54 / Airport / 0.86 ± 0.10 / 1.7 ± 0.2 / 2.4 ± 0.2 / 7.7 ± 1.2
HB3 / Honduras / PYR / 0.84 ± 0.07 / 2.7 ± 0.0 / 16 ± 5 / 16 ± 4
FCB1 / Columbia / CQ / 1.2 ± 0.5 / 2.8 ± 0.7 / 2.8 ± 0.8 / 43 ± 6
ITG2-F6 / Brazil / CQ / 0.63 ± 0.02 / 1.7 ± 0.3 / 1.7 ± 0.4 / 55 ± 2
MAD20 / Papua NG / 0.48 ± 0.16 / 2.5 ± 0.2 / 6.7 ± 1.1 / 6.8 ± 0.4
Ro73 / Kenya / PYR / 0.44 ± 0.09 / 0.77 ± 0.04 / 1.2 ± 0.2 / 7.7 ± 1.0
W2 / Indochina / CQ, PYR / 0.41 ± 0.05 / 0.76 ± 0.01 / 1.5 ± 0.2 / 125 ± 14
minimum / 0.41 / 0.76 / 1.2 / 6.8
maximum / 1.5 / 2.8 / 16 / 131

* mean ± SEM, n ≥ 2

Supplementary Figure

Supplementary Figure 1. Plasma concentration versus time profiles following oral administration to rats. AM () was dosed at 50 mg/kg, DHA () concentrations were measured following a dose of 50 mg/kg AS, 6 () was dosed at 18.8 mg/kg, and 7 (▲) was dosed at 17.4 mg/kg. Data points represent the average for n=2-3 rats.

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