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FULL PAPERS Parasite fighters: A series of tetraoxane–vinyl sulfone hybrids generate toxic radicals once activated inside malaria parasites, releasing highly active falcipain-2 (FP-2) inhibitors in situ. These hybrids are active within the low nanomolar range against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains and may present alternatives to current antimalarial chemotherapeutics.
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R. Oliveira, A. S. Newton, R. C. Guedes, D. Miranda, R. K. Amewu, A. Srivastava, J. Gut, P. J. Rosenthal, P. M. O’Neill,* S. A. Ward, F. Lopes, R. Moreira* && – && An Endoperoxide-Based Hybrid Approach to Deliver Falcipain Inhibitors Inside Malaria Parasites
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DOI: 10.1002/cmdc.201300202
An Endoperoxide-Based Hybrid Approach to Deliver Falcipain Inhibitors Inside Malaria Parasites Rudi Oliveira,[a] Ana S. Newton,[a] Rita C. Guedes,[a] Daniela Miranda,[a] Richard K. Amewu,[b] Abhishek Srivastava,[c] Jiri Gut,[d] Philip J. Rosenthal,[d] Paul M. O’Neill,*[b] Stephen A. Ward,[c] Francisca Lopes,[a] and Rui Moreira*[a] The emergence of artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia has reinforced the urgent need to discover novel chemotherapeutic strategies to treat and control malaria. To address this problem, we prepared a set of dual-acting tetraoxane-based hybrid molecules designed to deliver a falcipain-2 (FP-2) inhibitor upon activation by iron(II) in the parasite digestive vacuole. These hybrids are active in the low nanomolar range against chloroquine-sensitive and chloroquine-resistant P. falciparum strains. We also demonstrate that in the presence of FeBr2 or within infected red blood cells,
these molecules fragment to release falcipain inhibitors with nanomolar protease inhibitory activity. Molecular docking studies were performed to better understand the molecular interactions established between the tetraoxane-based hybrids and the cysteine protease binding pocket residues. Our results further indicate that the intrinsic activity of the tetraoxane partner compound can be masked, suggesting that a tetraoxanebased delivery system offers the potential to attenuate the offtarget effects of known drugs.
Introduction Malaria remains one of the major infectious diseases in man, with three billion people at risk and about 660 000 deaths worldwide in 2010.[1] Artemisinin (ART, 1, Figure 1)-based combination therapies (ACTs) are the mainstay for first-line treatment of uncomplicated Plasmodium falciparum malaria, replacing older drugs such as chloroquine (CQ) that became ineffective due to the spread of drug resistance.[2] Nevertheless, the recently reported emergence of P. falciparum with decreased ART sensitivity at the Cambodia–Thailand border has reinforced the urgent need to discover novel chemotherapeutic strategies to treat and control malaria.[3] Endoperoxide-based hybrid compounds represent an attractive alternative to ACTs.[4] ART and other endoperoxides contain
[a] R. Oliveira, A. S. Newton, Dr. R. C. Guedes, D. Miranda, Dr. F. Lopes, Prof. R. Moreira iMed.UL and Department of Pharmaceutical and Medicinal Chemistry Faculty of Pharmacy, University of Lisbon Av. Prof. Gama Pinto, Lisbon, 1649-003 (Portugal) E-mail:
[email protected] [b] Dr. R. K. Amewu, Prof. P. M. O’Neill Department of Chemistry University of Liverpool, Liverpool, L69 3BX (UK) E-mail:
[email protected] [c] Dr. A. Srivastava, Prof. S. A. Ward Liverpool School of Tropical Medicine Pembroke Place, Liverpool, L3 5QA (UK) [d] Dr. J. Gut, Prof. P. J. Rosenthal Department of Medicine University of California, San Francisco, CA 94143 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201300202.
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an endoperoxide core that is reductively activated by iron(II)– heme—a by-product of host hemoglobin degradation—to form carbon-centered radicals capable of reacting with heme and proteins.[5] Intracellular localization studies suggest that endoperoxides preferentially accumulate in the P. falciparum digestive vacuole (DV),[6] the organelle in which host hemoglobin is digested during the intraerythrocytic life cycle of malaria parasites.[7] Falcipains-2 and -3 are cysteine proteases that localize in the DV and play a key role in the hydrolysis of host hemoglobin into amino acids that are essential to parasite growth.[8] Falcipain inhibitors such as Michael-acceptor dipeptidyl vinyl sulfones (e.g. 2, Figure 1) were reported to inhibit the development of cultured erythrocytic parasites by blocking the hydrolysis of host hemoglobin and to cure mice infected with lethal malaria infections.[9] We have previously reported that hybrid compounds combining a semisynthetic ART derivative with a dipeptidyl vinyl sulfone moiety (2) displayed potent antiplasmodial activity with IC50 values in the low nanomolar range against CQ-resistant and CQ-sensitive P. falciparum strains.[10] However, these compounds were only moderately active against falcipain-2 (FP-2), with IC50 values in the low micromolar range, nearly two orders of magnitude higher than those of vinyl sulfones such as 3.[11] Based on the prodrug concept, we also synthesized hybrid molecules containing the 1,2,4-trioxolane core as a protease inhibitor carbonyl-masking group (e.g. 4, Figure 1).[12] These molecules were designed to target the parasite DV through activation by iron(II) to release a carbonyl protease inhibitor, with simultaneous formation of potentially cytotoxic carbon-radical species. Using a similar approach, a 1,2,4-trioxolane-based hybrid was designed to release a partner cysteine ChemMedChem 0000, 00, 1 – 10
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erate carbon-centered radicals that lead to the formation of drug derived-heme adducts, with concomitant formation of a ketone.[17] Based on this information, we reasoned that tetraoxane-based hybrid molecules (5, Scheme 1 A) containing dipeptidyl vinyl sulfone partners could deliver the FP-2 inhibitor once activated in the parasite DV. Herein, we report the synthesis and structure–activity profiling of tetraoxanes 5 with regard to their antiplasmodial and FP-2 inhibitory activities. As previous structure–activity relationship (SAR) studies for vinyl sulfone inhibitors of FP-2 highlighted a preference for the Leu residue at P2,[11, 18] we retained this structural feature in most of compounds 5 and investigated the influence of the P1 and P1’ constituents of vinyl sulfones, as well as the role of the linker between the dipeptide and tetraoxane moieties, on enzyme inhibitory potency. For comparison purposes, we also prepared a 1,2,4-trioxolane counterpart (6; Scheme 1 A). Finally, a mechanistic Figure 1. Structure of artemisinin (1), endoperoxide–vinyl sulfone hybrid 2, vinyl sulfone study was performed to assess the ability of tetraox3, and 1,2,4-trioxolane-based hybrid 4. ane hybrids 5 to deliver dipeptidyl vinyl sulfones 7 to erythrocytic parasites in the presence of iron(II) broprotease inhibitor upon fragmentation of the carbonyl intermide (Scheme 1 B for compounds 5 a–h and figure S1, Supportmediate formed following activation by iron(II).[13] ing Information, for 5 i). 1,2,4,5-Tetraoxanes are a class of cyclic peroxides endowed with potent in vitro and in vivo antimalarial activity.[14] The O’Neill group has designed and prepared simple, achiral, and Results and Discussion highly potent dispirotetraoxanes that incorporate the adamanSynthesis tyl group, which is known to increase stability of the endoperoxide motif,[15] some of which have improved pharmacokinetic Based on the SAR for vinyl sulfones as inhibitors of FP-2,[11, 18] [16] profiles relative to those of ART derivatives. As observed we initially focused our attention on target hybrids 5 a–f and with ART and other endoperoxides, tetraoxanes are rapidly and 5 i, which contain an amide linker between the tetraoxane and efficiently activated by iron(II) in the presence of heme to gendipeptidyl vinyl sulfone moieties. Compounds 5 a–f and 5 i
Scheme 1. A) 1,2,4,5-Tetraoxane–vinyl sulfone (5 a–i) and 1,2,4-trioxolane–vinyl sulfone hybrids (6) synthesized in this study. B) FeII activation of hybrids 5 a–h into carbon-centered radical species and vinyl sulfone FP-2 inhibitors (7).
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Scheme 2. Reagents and conditions: a) HCO2H, H2O2 50 %, CH3CN, RT, 45 min; b) adamantan-2-one for 9 a,b or cyclohexanone for 9 c, Re2O7, CH2Cl2, RT, 3 h; c) NaOH, MeOH/H2O (2:1), 80 8C, 2 h; d) Et3N, TBTU, HOBt, 10 a, 10 b, or 10 c, dry DMF, RT, overnight.
pared by reductive amination of tetraoxane ketone 14, which was, in turn, synthesized using a procedure previously reported by O’Neill and co-workers.[21] The trioxolane-based hybrid 6 was synthesized as described in Scheme 4. Griesbaum co-ozonolysis of adamantan-2-one methyl oxime 15 and cyclohexanone 8 a afforded trioxolane ester 16.[22] Hydrolysis of 16, followed by coupling of the resulting acid 17 to Leu-hPhe vinyl sulfone 11 a in the presence of TBTU, HOBt, and triethylamine gave the desired hybrid 6 in moderate yield. To decrease the peptidic nature of
were prepared via tetraoxane intermediates 10 a–c as described in Scheme 2. Briefly, synthesis of the tetraoxane core was achieved in a one-pot reaction. Appropriately 4-substituted cyclohexanones 8 were treated with hydrogen peroxide and formic acid to give the corresponding gem-dihydroperoxide. Adamantan-2-one or cyclohexanone and Re2O7 were then Scheme 4. Reagents and conditions: a) O3, pentane/CH2Cl2 (4:1), 8 a, 30 min; added to complete conversion into 1,2,4,5-tetraoxane esters b) NaOH, MeOH/H2O (2:1), 80 8C, 2 h; c) 17, Et3N, TBTU, HOBt, dry DMF, RT, 9 a–c in reasonable to good yields. Compounds 9 were subseovernight. quently hydrolyzed to the corresponding acids 10 a–c using a modified procedure reported by Ghorai and Dussault.[19] Ficompounds 5, we removed one amino acid from the vinyl sulnally, coupling of dipeptidyl vinyl sulfones 11 a–e, prepared by fone partner. In this context, we designed compounds 18, Wadsworth–Emmons chemistry,[10, 11, 20] with tetraoxane acids which contain hPhe (Scheme 5). Hybrids 18 were synthesized 10 a–c in the presence of TBTU, HOBt, and triethylamine affordfollowing a procedure similar to that described for hybrids 5. ed the corresponding hybrids 5 a–f and 5 i in moderate to good yields. The synthesis of hybrids 5 g and 5 h, containing amine linkers between the two pharmacophores, was carried out as shown in Scheme 3. Tetraoxane acid 10 a was converted into the corresponding Weinreb amide 12 which, upon reduction with lithium aluminum hydride, gave aldehyde 13. Reductive amination of intermediate 13 with Leu-hPhe vinyl sulfone 11 a and sodium triacetoxyborohydride in acetic acid af- Scheme 5. Reagents and conditions: a) Et3N, TBTU, HOBt, dry DMF, RT, overnight; b) AcOH, NaBH(OAc)3, MeOH, RT, overnight. forded compound 5 g. Similarly, hybrid 5 h was preFalcipain-2 inhibition
Scheme 3. Reagents and conditions: a) Et3N, TBTU, N,O-dimethylhydroxylamine, CH2Cl2, RT, overnight; b) LiAlH4, dry THF, 0 8C, 1 h; c) 11 a, AcOH, NaBH(OAc)3, MeOH, RT, overnight.
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Tetraoxane hybrids 5 a–i, 18 a and 18 b, and the trioxolane derivative 6 were screened against recombinant FP-2. The dipeptidyl vinyl sulfone derivatives 5 a– i and 6 displayed weak to moderate inhibitory activity against FP-2, with IC50 values ranging from 1 mm to 10 mm, in line with the activity data reported for ChemMedChem 0000, 00, 1 – 10
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their ART-based vinyl sulfone counterparts (Table 1).[10] These results contrast sharply with those for the dipeptidyl vinyl sulfone 7 (R1 = CH2CH2Ph, R2 = CH2CH(CH3)2, R3 = Ph), which inhibited FP-2 in the low nanomolar range (Table 1). This strongly suggests that appending bulky groups to the N-terminus (P3) of the dipeptide core is detrimental to FP-2 inhibitory activity. Replacement of an adamantyl group for a more flexible cyclohexyl group (5 a vs. 5 i) increased inhibitory potency versus FP2 by roughly twofold, while replacement of the tetraoxane core with a smaller trioxolane (5 a vs. 6) exerted the opposite effect, indicating that structural modification in the distal peroxide moiety allows fine tuning of inhibitory activity but does not significantly improve potency relative to vinyl dipeptidyl sulfones. Inspection of the FP-2 activity data presented in Table 1 also reveals that the inhibitory activity of hybrids 5 a–i is strongly dependent on the nature of the amino acids at P1 and P2. For example, replacing hPhe with Phe at P1 (5 a vs. 5 b) and Leu with Phe at P2 (5 a vs. 5 c) led to an approximate fivefold decrease in potency. Interestingly, hybrids 18 a and 18 b, lacking the P2 residue, were at least tenfold less potent than their LeuhPhe counterparts 5 a and 5 g, respectively, consistent with the absolute requirement of a P2 moiety for substrate recognition by the enzyme.[23] To decrease lipophilicity, we prepared compound 5 d with a methyl group at P1’, which was equipotent to its phenyl counterpart 5 a, indicating that decreasing the size of the P1’ substituent did not affect FP-2 inhibitory activity. To further decrease the lipophilicity and molecular weight to more drug-like values, we synthesized compound 5 e with a Leu at P1, which was also equipotent to 5 a against FP-2. Overall, these results are consistent with the preference of FP-2 for the Leu-hPhe sequence reported for dipeptidyl vinyl sulfones.[24] The nature of the linker between the vinyl sulfone and tetraoxane moieties also significantly affected the FP-2
enzyme inhibition of the hybrid compounds 5, as shown by the approximate fivefold decrease in potency observed when an amide linker was replaced with an amine (e.g. 5 h and 5 g vs. 5 a). This result, which suggests that amine linkers are detrimental to FP-2 inhibitory activity, is further supported by the observation of a similar trend in potency for compounds 18. To explain the inhibitory activity of hybrids 5, molecular docking calculations were performed to better understand the molecular interactions established between the tetraoxanebased hybrids and the cysteine protease binding pocket residues. GOLD software was used to perform covalent docking, and top ranking solutions, selected according to their GoldScore fitness function, were visually analyzed for hydrophobic interactions and hydrogen bonds between the inhibitor and the enzyme surface. Inspection of the best poses for hybrids 5 a, 5 f, 5 g, and 5 i inside the FP-2 binding pocket revealed that all compounds orient with the vinyl sulfone moiety in the large hydrophobic S1’ binding pocket, while the dipeptidyl moiety fits into the S1, S2, and S3 binding sites, as shown in Figure 2 for hybrids 5 a and 5 i (see Supporting Information for a closer view of the binding site). The top ranking binding poses for the most potent inhibitor, 5 i, show the amide group of Gly 83 oriented toward the nitrogen atom of the amide linker, with the Leu residue in P2 partially occupying the S2 subsite and the tetraoxane moiety blocking the access to the S2 subsite. A similar pose is found for hybrid 5 a, with the Leu in P2 occupying also the S2 subsite. However, in this case, the hPhe residue interacts poorly with S1, which could explain the decreased inhibitory activity shown by this compound. In contrast to 5 a and 5 i, the top ranking conformations for weak inhibitors 5 f and 5 g present a more exposed S2 subsite (see Supporting Information for a closer view of the binding site).
Table 1. Falcipain-2 (FP-2) inhibition and antiplasmodial activity of hybrids 5 a–i, 6, and 18 a,b, and of vinyl sulfone 7, against chloroquine-sensitive (3D7) and chloroquine-resistant (W2) strains of P. falciparum.[a]
Compd
X
R1
R2
R3
FP-2
5a 5b 5c 5d 5e 5f 5g 5h 5i 6 7 18 a 18 b ART CQ 3
CONH CONH CONH CONH CONH CH2CONH NH CH2NH CONH CONH CONH CONH NH – – –
CH2CH2Ph CH2Ph CH2CH2Ph CH2CH2Ph CH2CH(CH3)2 CH2CH2Ph CH2CH2Ph CH2CH2Ph CH2CH2Ph CH2CH2Ph CH2CH2Ph CH2CH2Ph CH2CH2Ph – – –
CH2CH(CH3)2 CH2CH(CH3)2 CH2Ph CH2CH(CH3)2 CH2CH(CH3)2 CH2CH(CH3)2 CH2CH(CH3)2 CH2CH(CH3)2 CH2CH(CH3)2 CH2CH(CH3)2 CH2CH(CH3)2 – – – – –
Ph Ph Ph Me Me Ph Ph Ph Ph Ph Ph Ph Ph – – –
1802 374 8093 1092 9784 942 1530 388 1698 221 8739 705 10 453 1085 6762 28.3 964.8 686 2929 139 69.1 14.7 18 460 156 > 50 000 ND ND 29.0 1.2
IC50 [nm][b] 3D7 28.4 13.9 67.2 2.68 60.1 4.24 17.6 4.59 18.5 1.68 41.1 1.56 38.8 0.78 45.5 4.48 175 15.1 77.1 10.6 287.6 55.2 69.5 14.3 22.4 2.14 8.37 1.15 17.7 7.98 97.4 11.4
W2 18.5 0.09 38.2 2.43 33.4 2.31 10.7 2.47 18.5 0.09 16.5 3.46 23.7 2.19 37.1 1.01 20.2 0.62 53.8 3.40 26.2 5.40 47.4 1.09 8.80 3.14 6.98 0.38 187 60 7.32 2.36
[a] Methods were as previously described.[25] [b] Values represent the mean SD for duplicate assays; ND: not determined.
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www.chemmedchem.org hemoglobin hydrolysis caused by inhibition of falcipains.[11, 26] Hemoglobin degradation was blocked by low (4.6 nm) concentrations of both 5 i and parent 3,[18] despite the large difference in their potencies against FP-2. Thus, the hybrid strategy allowed delivery of the protease inhibitor to the DV, contributing to the observed antimalarial effect against P. falciparum.
Activation of tetraoxanes with FeII and in human erythrocytes
Figure 2. Superimposition of docked conformations of tetraoxane-based hybrids 5 a (yellow) and 5 i (blue) in the active site of FP-2.
Antiplasmodial activity and inhibition of hemoglobin degradation Hybrids 5, 6, and 18 were screened against CQ-sensitive (3D7) and CQ-resistant (W2) strains of P. falciparum. Dipeptide vinyl sulfone hybrids displayed potent antiplasmodial activity, with IC50 values in the low nanomolar range against both strains of P. falciparum (Table 1). Hybrids 5 a and 5 i, as well as dipeptidyl vinyl sulfone 3, had similar activity against cultured parasites. In contrast, dipeptidyl vinyl sulfone 3 was much more potent against FP-2 than 5 a or 5 i (IC50 value of 29 nm vs. 1802 and 965 nm, respectively). These results may suggest that activation and inhibitor release contributed to the potent parasite growth inhibition observed with the hybrid molecules. To examine the potential intraparasitic release of FP-2 inhibitor 7, we assessed the morphological effects of hybrid 5 i on P. falciparum infected erythrocytes. When cultured parasites were incubated with 5 i for 24 h beginning at the ring stage, the parasites developed swollen DVs with the staining characteristics of erythrocytic cytoplasm (Figure 3). It has previously been shown that this specific abnormality is indicative of a block in
Figure 3. P. falciparum digestive vacuole swelling caused by hybrid 5 i. A) Untreated infected erythrocytes with normal white-staining digestive vacuoles. B) Infected erythrocyte treated with compound 5 i (4.6 nm); the digestive vacuole is swollen and stains the same color as the erythrocyte cytosol.
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A mechanistic study of the activation of tetraoxane-based vinyl sulfone hybrids 5 a and 5 i with the parent ketone vinyl sulfone 7 was performed in vitro using LC–MS/MS. In a preliminary experiment, hybrids 5 a and 5 i were reacted with iron(II) bromide in acetonitrile/dichloromethane (1:1) (Figure 4 A). The reaction proceeded to completion, with 7 formed quantitatively from both hybrid compounds (Figure 4 B). Definitive proof of inhibitor release within the malaria parasite was obtained by incubating 5 a with erythrocytes infected with late ring/early trophozoite stage P. falciparum (3D7 strain), using a previously described protocol.[12] LC–MS/MS analysis of the lysates obtained from erythrocytes at three incubation time points revealed that the parent vinyl sulfone 7 was formed in near quantitative yields after 4 h of incubation (Figure 4 C), indicating that activation occurs rapidly inside the parasites. This contrasts with the slower turnover of 5 a observed in uninfected erythrocytes. Detection of 7 after 48 h of incubation suggests that the vinyl sulfone inhibitor has the potential to remain functional for many hours after the tetraoxane begins to kill parasites via formation of carbon-centered radicals, a feature that may be convenient in combination with fast-acting peroxide antimalarials.[27]
Conclusions We successfully designed and synthesized a new class of tetraoxane–vinyl sulfone-based hybrid compounds endowed with excellent antimalarial activity against both CQ-sensitive and CQ-resistant strains of P. falciparum. Although these hybrid molecules displayed weak to moderate FP-2 inhibitory activity, their activation in the presence of iron(II) bromide and in infected erythrocytes led to the rapid and efficient release of the parent vinyl sulfone inside malaria parasites. Importantly, we have shown that, in spite of decreased inhibitory activity against FP-2, the hybrid compounds inhibited hemoglobin digestion at low nanomolar concentration. Detection of intact parent vinyl sulfone in infected erythrocytes 48 h post-treatment with the hybrid compounds strongly suggests that the inhibitor remained within infected erythrocytes long after the fast-acting endoperoxide moiety had exerted its effects. The results presented for 5 i indicate that the intrinsic activity of the tetraoxane partner compound can be masked, suggesting that a tetraoxane-based delivery system offers the potential to attenuate the off-target effects of known drugs. ChemMedChem 0000, 00, 1 – 10
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Figure 4. In vitro activation of hybrids 5 a and 5 i. A) Hybrid activation scheme. B) Chromatograms showing ketone 7 after reaction with iron(II) bromide, with activation of 5 a (top) and 5 i (bottom). C) Chromatograms from lysed infected erythrocytes showing ketone 7 at three time points, as indicated, after treatment with hybrid 5 a.
Experimental Section Chemistry All chemicals and solvents were of analytical reagent grade and were purchased from Alfa Aesar or Sigma–Aldrich. Tetrahydrofuran was dried before use. Thin layer chromatography was performed using Merck silica gel 60F254 aluminum plates with visualization by UV light, iodine, potassium permanganate dip, and/or p-anisaldehyde dip. Flash column chromatography was performed using Merck silica gel 60 (230–400 mesh ASTM), eluting with various solvent mixtures and using an air aquarium pump to apply pressure. NMR spectra were collected using a Bruker 400 Ultra-Shield (400 MHz) in CDCl3 ; chemical shifts, d, are expressed in ppm, and coupling constants, J, are expressed in Hz. Infrared spectra were collected on a Shimadzu IRAffinity-1 IR spectrophotometer. Mass analyses were determined by LCLEM (Faculty of Pharmacy from 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
the University of Lisbon, Portugal) using a Micromass Quattro Micro API spectrometer equipped with a Waters 2695 HPLC module and a Waters 2996 photodiode array detector. All compounds tested in biological assays were determined by LCLEM to be > 95 % pure by elemental analysis (for C, H, and N), determined using a FLASH 2000 analyzer. Melting points were determined using a Kofler Bock Monoscop M and are uncorrected. See Supporting Information for experimental information and data on all intermediates. General procedure for the synthesis of tetraoxane–vinyl sulfones 5 a–f, 5 i, 6, and 18 a: HOBt (0.13 mmol) and TBTU (0.14 mmol) were added to a solution of carboxylic acid 10 a–c or 17 (0.12 mmol) and Et3N (0.13 mmol) in dry DMF (1.5 mL) at 0 8C under nitrogen atmosphere. The mixture was stirred for 30 min at 0 8C, then a stirred solution of the appropriate amine 11 or 19 (0.13 mmol) and Et3N (0.14 mmol) in dry DMF (1.5 mL) was added. ChemMedChem 0000, 00, 1 – 10
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CHEMMEDCHEM FULL PAPERS The mixture was allowed to warm to room temperature and was stirred overnight under nitrogen atmosphere. The reaction was diluted with EtOAc (20 mL) and washed with 1 n HCl (20 mL), saturated NaHCO3 (20 mL), and brine (20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography gave the pure compound. Compound 5 a: White solid (42 mg, 49 % yield): mp: 108–110 8C; H NMR (400 MHz, CDCl3), d = 7.87 (d, J = 8.0 Hz, 2 H), 7.64 (t, J = 7.2 Hz, 1 H), 7.55 (t, J = 8.0 Hz, 2 H), 7.27 (t, J = 7.2 Hz, 2 H), 7.19 (t, J = 7.0 Hz, 1 H), 7.08 (d, J = 7.6 Hz, 2 H), 7.00 (d, J = 8.4 Hz, 1 H), 6.90 (dd, J = 15.0 Hz and 5.2 Hz, 1 H), 6.44 (d, J = 15.0 Hz, 1 H), 6.10 (d, J = 8.0 Hz, 1 H), 4.64 (m, 1 H), 4.43 (m, 1 H), 2.58 (m, 2 H), 2.22 (m, 1 H), 1.45–2.05 (m, 27 H), 0.89 (d, J = 6.0 Hz, 3 H), 0.85 ppm (d, J = 6.0 Hz, 3 H); 13C NMR (101 MHz, CDCl3), d = 175.2, 171.7, 145.4, 140.3, 140.1, 133.6, 130.9, 129.4, 128.6, 128.4, 127.7, 126.3, 110.6, 106.9, 51.7, 49.1, 43.7, 36.9, 35.5, 33.1, 31.8, 27.0, 24.8, 22.8, 22.2 ppm; IR (NaCl): n˜ = 1640 (C=O), 1539 (C=C), 1321 (S=O asymmetric), 1308 (S=O asymmetric), 1149 (S=O symmetric), 844 cm 1 (O O); HRMS-ESI: m/z [M + H] + calcd for C40H53N2O8S: 721.3517, found: 721.3526; Anal. calcd for C40H53N2O8S·0.15 H2O: C 66.39, H 7.30, N 3.87, found: C 66.00, H 7.42, N 3.74. 1
Compound 5 b: White solid (63 mg, 72 % yield): mp: 115–120 8C; H NMR (400 MHz, CDCl3): d = 7.81 (d, J = 7.6 Hz, 2 H), 7.63 (t, J = 7.6 Hz, 1 H), 7.54 (t, J = 7.6 Hz, 2 H), 7.26–7.17 (m, 3 H), 7.11 (d, J = 6.4 Hz, 2 H), 7.00 (d, J = 8.4 Hz, 1 H), 6.94 (dd, J = 15.0 Hz, 5.2 Hz, 2 H), 6.35 (dd, J = 15.0, 1.2 Hz, 1 H), 6.00 (br s, 1 H), 4.96 (m, 1 H), 4.39 (m, 1 H), 2.90 (d, J = 7.2 Hz, 2 H), 2.14 (m, 1 H), 2.09–1.39 (m, 25 H), 0.87 (d, J = 6.2 Hz, 3 H), 0.83 ppm (d, J = 6.2 Hz, 3 H); 13C NMR (101 MHz, CDCl3): d = 174.9, 171.6, 144.8, 140.0, 135.7, 133.5, 131.0, 129.3, 129.3, 128.7, 127.6, 127.1, 110.6, 106.9, 51.6, 50.4, 43.6, 40.3, 40.0, 36.9, 33.1, 27.0, 24.8, 22.8, 22.1 ppm; IR (NaCl): n˜ = 1643 (C= O), 1544 (C=C), 1320 (S=O asymmetric), 1308 (S=O asymmetric), 1148 (S=O symmetric), 847 cm 1 (O O); HRMS-ESI: m/z [M + Na] + calcd for C39H50N2NaO8S: 729.3180, found: 729.3159; Anal. calcd for C39H50N2O8S: C 66.26, H 7.14, N 3.96, found: C 66.48, H 6.89, N 3.51. 1
Compound 5 c: White solid (55 mg, 83 % yield): mp: 185–186 8C; H NMR (400 MHz, CDCl3): d = 7.85 (d, J = 7.6 Hz, 2 H), 7.66 (t, J = 7.2 Hz, 1 H), 7.57 (t, J = 7.6 Hz, 2 H), 7.30–7.15 (m, 6 H), 7.12 (d, J = 6.8 Hz, 2 H), 7.06 (d, J = 7.2 Hz, 2 H), 6.78 (dd, J = 15.2, 4.8 Hz, 1 H), 6.52 (d, J = 8.4 Hz, 1 H), 6.27 (d, J = 7.6 Hz, 1 H), 6.02 (d, J = 15.2 Hz, 1 H), 4.70–4.55 (m, 2 H), 3.00 (d, J = 7.2 Hz, 2 H), 2.62–2.47 (m, 2 H), 2.19 (m, 1 H), 2.07–1.39 ppm (m, 24 H); 13C NMR (101 MHz, CDCl3): d = 174.8, 170.6, 145.2, 140.2, 136.1, 133.5, 130.6, 129.3, 129.1, 128.8, 128.6, 128.3, 127.7, 127.3, 126.3, 110.6, 106.9, 54.6, 49.1, 43.6, 38.2, 36.9, 35.5, 33.1, 31.7, 27.0 ppm; IR (NaCl): n˜ = 1640 (C=O), 1539 (C=C), 1311 (S=O asymmetric), 1134 (S=O symmetric), 834 cm 1 (O O); HRMS-ESI: m/z [M + Na] + calcd for C43H50N2NaO8S: 777.3180, found: 777.3147; Anal. calcd for C43H50N2O8S·1.5 H2O: C 66.04, H 6.85, N 3.58, found: C 66.03, H 7.23, N 3.86.
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Compound 5 d: White solid (60 mg, 52 % yield): mp: 118–121 8C; H NMR (400 MHz, CDCl3): d = 7.27 (d, J = 7.5 Hz, 3 H), 7.20 (dd, J = 12.3, 7.5 Hz, 2 H), 7.11 (d, J = 7.5 Hz, 2 H), 6.84 (dd, J = 15.0, 5.1 Hz, 1 H), 6.50 (d, J = 15.0 Hz, 1 H), 6.22 (d, J = 5.9 Hz, 1 H), 4.62 (m, 1 H), 4.51 (m, 1 H), 3.14 (br s, 2 H), 2.94 (s, 3 H), 2.61 (m, 2 H), 2.25 (m, 1 H), 2.06–1.41 (m, 31 H), 0.96 (d, J = 5.2 Hz, 3 H), 0.92 ppm (d, J = 5.2 Hz, 3 H); 13C NMR (101 MHz, CDCl3): d = 175.3, 172.0, 147.0, 140.3, 129.8, 128.7, 128.4, 126.4, 110.6, 106.9, 51.7, 49.2, 43.8, 42.8, 40.4, 36.9, 35.4, 33.1, 31.8, 29.7, 27.0, 24.9, 23.0, 22.1 ppm; IR (NaCl): n˜ = 1640 (C=O), 1539 (C=C), 1310 (S=O asymmetric), 1134 (S=O symmetric), 834 cm 1 (O O); HRMS-ESI: m/z [M + Na] + calcd for
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www.chemmedchem.org C35H50N2NaO8S: 681.3180, found: 681.3158; Anal. calcd for C35H50N2O8S: C 63.80, H 7.66, N 4.25, found: C 63.33, H 7.54, N 4.20. Compound 5 e: White solid (69 mg, 80 % yield): mp: 133–135 8C; H NMR (400 MHz, CDCl3): d = 7.09 (d, J = 7.1 Hz, 1 H), 6.84 (dd, J = 15.1, 5.4 Hz, 1 H), 6.51 (d, J = 15.1 Hz, 1 H), 6.23 (br s, 1 H), 4.69 (m, 1 H), 4.50 (m, 1 H), 2.95 (s, 3 H), 2.27 (m, 1 H), 1.63 (m, 28 H), 1.00– 0.82 ppm (m, 12 H); 13C NMR (101 MHz, CDCl3): d = 175.2, 171.9, 147.6, 129.3, 110.6, 106.9, 51.7, 47.9, 43.8, 42.8, 42.7, 40.5, 36.9, 33.1, 27.0, 24.9, 24.7, 22.9, 22.8, 22.2, 21.9 ppm; IR (NaCl): n˜ = 1641 (C=O), 1539 (C=C), 1312 (S=O asymmetric), 1137 (S=O symmetric), 832 cm 1 (O O); HRMS-ESI: m/z [M + Na] + calcd for C31H50N2NaO8S: 633.3186, found: 633.3178; Anal. calcd for C31H50N2O8S·0.15 H2O: C 60.69, H 8.28, N 4.57, found: C 60.34, H 8.18, N 4.55. 1
Compound 5 f: White solid (96 mg, 52 % yield): mp: 109–111 8C; H NMR (400 MHz, CDCl3): d = 7.81–7.75 (dd, J = 8.0, 0,8 Hz, 2 H), 7.51 (m, 1 H), 7.47 (t, J = 8 Hz, 2 H), 7.22–7.16 (m, 2 H), 7.11 (t, J = 7.2 Hz, 1 H), 7.01 (d, J = 7.2 Hz, 2 H), 6.82 (dd, J = 15.1, 5.3 Hz, 1 H), 6.69 (br s, 1 H), 6.35 (dd, J = 15.1, 1.4 Hz, 1 H), 5.90 (br s, 1 H), 4.58 (m, 1 H), 4.33 (m, 1 H), 2.60–2.45 (m, 2 H), 2.06–1.32 (m, 30 H), 0.82 (dd, J = 6.0 Hz, 3 H), 0.78 ppm (d, J = 6.0 Hz, 3 H); 13C NMR (101 MHz, CDCl3): d = 181.8, 171.6, 145.3, 140.3, 140.0, 133.6, 130.9, 129.4, 128.7, 128.4, 127.7, 126.4, 110.5, 107.5, 51.8, 49.2, 43.0, 40.3, 36.9, 35.7, 34.0, 33.1, 31.8, 27.0, 24.8, 22.8, 22.1 ppm; IR (NaCl): n˜ = 1639 (C=O), 1546 (C=C), 1320 (S=O asymmetric), 1308 (S=O asymmetric), 1149 (S=O symmetric), 847 cm 1 (O O); HRMS-ESI: m/z [M + H] + calcd for C41H55N2O8S: 735.3674, found: 735.3671; Anal. calcd for C41H54N2O8S·0.5 H2O: C 66.19, H 7.47, N 3.77, found: C 65.84, H 7.27, N 3.78.
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Compound 5 i: White solid (42 mg, 56 % yield): mp: 84–87 8C; H NMR (400 MHz, CDCl3): d = 7.87 (d, J = 7.6 Hz, 2 H), 7.64 (t, J = 7.3 Hz, 1 H), 7.55 (t, J = 7.6 Hz, 2 H), 7.30–7.23 (m, 2 H), 7.19 (t, J = 7.2 Hz, 1 H), 7.08 (d, J = 7.2 Hz, 2 H), 7.00 (d, J = 8.3 Hz, 1 H), 6.90 (dd, J = 15.1, 5.3 Hz, 1 H), 6.44 (d, J = 15.1 Hz, 1 H), 6.12 (d, J = 8.0 Hz, 1 H), 4.63 (m, 1 H), 4.44 (m, 1 H), 2.66–2.51 (m, 2 H), 2.40– 2.16 (m, 3 H), 1.98–1.36 (m, 23 H), 0.89 (d, J = 6.1 Hz, 3 H), 0.85 ppm (d, J = 6.1 Hz, 3 H); 13C NMR (101 MHz, CDCl3): d = 175.1, 171.7, 145.4, 140.3, 140.1, 133.6, 130.9, 129.4, 128.6, 128.4, 127.7, 126.3, 108.5, 107.0, 82.5, 51.7, 49.1, 43.7, 40.4, 35.5, 31.8, 29.7, 25.3, 24.8, 22.8, 22.2 ppm; IR (NaCl): n˜ = 1652 (C=O), 1640 (C=O), 1539 (C=C), 1321 (S=O asymmetric), 1308 (S=O asymmetric), 1149 (S=O symmetric), 849 cm 1 (O O); HRMS-ESI: m/z [M + H] + calcd for C36H49N2O8S: 669.3204, found: 669.3201; Anal. calcd for C36H48N2O8S·0.75 H2O: C 63.36, H 7.33, N 4.11, found: C 63.36, H 7.05, N 4.59. 1
Compound 6: White solid (77 mg, 51 % yield): mp: 107–110 8C; H NMR (400 MHz, CDCl3): d = 7.85 (d, J = 7.5 Hz, 2 H), 7.62 (t, J = 7.5 Hz, 1 H), 7.53 (t, J = 7.5 Hz, 2 H), 7.25 (t, J = 7.1 Hz, 2 H), 7.18 (t, J = 7.1 Hz, 1 H), 7.07 (d, J = 7.1 Hz, 2 H), 6.88 (m, 3 H), 6.41 (d, J = 15.0 Hz, 1 H), 5.97 (d, J = 7.9 Hz, 1 H), 4.61 (m, 1 H), 4.39 (m, 1 H), 2.64–2.49 (m, 2 H), 2.11 (m, 1 H), 2.04–1.40 (m, 27 H), 0.88 (d, J = 6.1 Hz, 3 H), 0.83 ppm (d, J = 6.1 Hz, 3 H); 13C NMR (101 MHz, CDCl3): d = 175.39, 171.66, 145.35, 140.31, 140.11, 133.51, 130.91, 129.34, 128.61, 128.37, 127.64, 126.31, 111.59, 107.58, 51.69, 49.09, 43.36, 40.28, 36.75, 36.35, 35.55, 34.80, 34.77, 33.45, 33.40, 31.80, 29.69, 27.03, 26.88, 26.85, 26.45, 24.83, 22.74, 22.18 ppm; IR (NaCl): n˜ = 1640 (C=O), 1539 (C=C), 1321 (S=O asymmetric), 1308 (S=O asymmetric), 1147 (S=O symmetric), 844 cm 1 (O O); HRMS-ESI: m/z [M + H] + calcd for C40H53N2O7S: 705.3568, found 705.3566; Anal. calcd for C40H52N2O7S: C 68.15, H 7.45, N 3.97, found: C 67.84, H 7.64, N 3.87. 1
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CHEMMEDCHEM FULL PAPERS Compound 18 a: White solid (77 mg, 67 % yield): mp: 93–95 8C; 1 H NMR (400 MHz, CDCl3): d = 7.85 (d, J = 7.7 Hz, 2 H), 7.63 (t, J = 7.4 Hz, 1 H), 7.54 (t, J = 7.6 Hz, 2 H), 7.32–7.23 (t, J = 7.4 Hz, 2 H), 7.20 (t, J = 7.2 Hz, 1 H), 7.11 (d, J = 7.4 Hz, 2 H), 6.90 (dd, J = 15.0, 4.9 Hz, 1 H), 6.36 (d, J = 15.0 Hz, 1 H), 5.63 (d, J = 8.5 Hz, 1 H), 4.75 (m, 1 H), 2.62 (m, 2 H), 2.12 (m, 1 H), 2.05–1.37 ppm (m, 24 H), 13C NMR (101 MHz, CDCl3): d = 174.0, 145.9, 140.4, 139.9, 133.7, 130.9, 130.7, 129.5, 128.8, 128.4, 127.7, 126.5, 110.6, 106.9, 48.9, 43.9, 36.9, 35.5, 33.1, 32.0, 31.5, 30.2, 29.7, 27.0 ppm; IR (NaCl): n˜ = 1647 (C=O), 1534 (C=C), 1319 (S=O asymmetric), 1308 (S=O asymmetric), 1147 (S=O symmetric), 831 cm 1 (O O); HRMS-ESI: m/z [M + Na] + calcd for C34H41NNaO7S: 630.2496, found: 630.2470; Anal. calcd for C34H41N1O7S: C 67.19, H 6.81, N 2.31, found: C 67.06, H 7.16, N 2.20. General procedure for the synthesis of tetraoxane–vinyl sulfones 5 g,h and 18 b: A solution of compound 13 or 14 (0.16 mmol) and the appropriate amine 11 a or 19 (0.17 mmol) was stirred in dry MeOH (1.5 mL) for 30 min at room temperature under nitrogen atmosphere. Acetic acid (0.032 mL) was added, followed by sodium triacetoxyborohydride (0.24 mmol), and the mixture was stirred overnight at room temperature under nitrogen. The reaction mixture was then adjusted to pH 8–9 with a 1 n solution of NaOH. Water (20 mL) and diethyl ether (20 mL) were added, and the layers were separated. The organic phase was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography gave the pure compound. Compound 5 g: White solid (67 mg, 61 % yield): mp: 82–85 8C; H NMR (400 MHz, CDCl3): d = 7.85 (d, J = 7.6 Hz, 2 H), 7.62 (t, J = 7.6 Hz, 1 H), 7.55 (t, J = 7.5 Hz, 2 H), 7.38 (d, J = 9.1 Hz, 1 H), 7.27 (t, J = 7.0 Hz, 2 H), 7.19 (t, J = 7.0 Hz, 1 H), 7.12 (d, J = 7.0 Hz, 2 H), 6.90 (dd, J = 15.0, 5.2 Hz, 1 H), 6.35 (d, J = 15.0 Hz, 1 H), 4.68 (m, 1 H), 3.23–3.10 (m, 2 H), 3.02 (br s, 1 H), 2.71–2.55 (m, 2 H), 2.38 (m, 1 H), 2.07–1.28 (m, 27 H), 0.97–0.86 ppm (m, 6 H); 13C NMR (101 MHz, CDCl3): d = 175.56, 145.92, 140.82, 140.49, 134.01, 131.41, 129.84, 129.08, 128.74, 128.06, 126.74, 111.04, 107.57, 77.74, 77.42, 77.10, 60.80, 48.93, 43.99, 37.32, 36.21, 33.53, 32.35, 27.44, 25.51, 23.79, 22.08 ppm; IR (NaCl): n˜ = 1659 (C=O), 1653 (C=O), 1532 (C=C), 1319 (S=O asymmetric), 1308 (S=O asymmetric), 1148 (S=O symmetric), 840 cm 1 (O O); HRMS-ESI: m/z [M + H] + calcd for C39H53N2O7S: 693.3568, found: 693.3546; Anal. calcd for C39H52N2O7S·1.5 H2O: C 65.06, H 7.72, N 3.89, found: C 65.42, H 7.97, N 3.49.
www.chemmedchem.org J = 7.1 Hz, 1 H), 7.11 (d, J = 7.2 Hz, 2 H), 6.85 (dd, J = 15.0, 7.1 Hz, 1 H), 6.47 (d, J = 15.0 Hz, 1 H), 3.34 (s, 1 H), 2.84 (s, 1 H), 2.63 (t, J = 7.6 Hz, 2 H), 2.52 (m, 1 H), 2.09–1.53 ppm (m, 24 H); 13C NMR (101 MHz, CDCl3): d = 140.8, 140.5, 133.5, 129.4, 128.5, 128.3, 127.6, 126.2, 110.5, 107.4, 67.8, 55.3, 52.5, 36.9, 36.5, 33.1, 31.9, 29.7, 27.0 ppm; IR (NaCl): n˜ = 1447 (C=C), 1317 (S=O asymmetric), 1307 (S=O asymmetric), 1146 (S=O symmetric), 824 cm 1 (O O); HRMSESI: m/z [M + H] + calcd for C33H42NO6S: 580.2727, found: 580.2721; Anal. calcd for C33H41N1O6S·0.15 H2O: C 68.04, H 7.16, N 2.41, found: C 67.75, H 7.36, N 2.15. Iron(II) bromide fragmentation of 5 a or 5 i: FeBr2 (0.134 mmol) was added to a solution of 5 a or 5 i (0.067 mmol) in CH3CN/CH2Cl2 (1:1, 1.5 mL). The reaction was stirred overnight at 35 8C under nitrogen atmosphere and then concentrated. The crude product was redissolved in EtOAc, washed with water and brine, dried over MgSO4, and concentrated. The crude product was then reconstituted with water/MeOH (400 mL:3600 mL for 5 a and 400 mL:4600 mL for 5 i). Samples were sonicated for 30 min and analyzed by LC– MS/MS spectroscopy to detect compound 7.
Computational studies Computational tools: All calculations were performed on the iMedUL scientific cluster. GOLD (version 4.0.1)[28] and Molecular Operating Environment (MOE) software (version 2011.10)[29] were used for docking calculations, to build and optimize small molecules, and for enzyme structure refinement.
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Compound 5 h: White solid (26 mg, 23 % yield): mp: 60–61 8C; H NMR (400 MHz, CDCl3): d = 7.86 (d, J = 7.4 Hz, 2 H), 7.62 (t, J = 7.4 Hz, 1 H), 7.55 (t, J = 7.4 Hz, 2 H), 7.37 (d, J = 8.4 Hz, 1 H), 7.27 (t, J = 7.3 Hz, 2 H), 7.20 (t, J = 7.3 Hz, 1 H), 7.13 (d, J = 7.3 Hz, 2 H), 6.91 (dd, J = 15.0, 5.2 Hz, 1 H), 6.36 (d, J = 15.0 Hz, 1 H), 4.69 (m, 1 H), 3.18 (br s, 2 H), 3.03 (m, 1 H), 2.72–2.55 (m, 2 H), 2.47 (m, 1 H), 2.30 (m, 1 H), 2.09–1.39 (m, 27 H), 1.39–1.10 (m, 5 H), 0.94 (d, J = 6.2 Hz, 3 H), 0.90 ppm (d, J = 6.2 Hz, 3 H); 13C NMR (101 MHz, CDCl3): d = 174.46, 145.58, 140.41, 140.06, 133.58, 130.94, 129.41, 128.65, 128.33, 127.68, 126.34, 110.52, 107.76, 77.34, 77.02, 76.70, 61.64, 54.74, 48.48, 42.91, 37.37, 36.94, 35.95, 33.15, 31.94, 29.69, 27.06, 25.27, 23.20, 21.81 ppm; IR (NaCl): n˜ = 1662 (C=O), 1657 (C=O), 1517 (C=C), 1319 (S=O asymmetric), 1308 (S=O asymmetric), 1148 (S=O symmetric), 840 cm 1 (O O); HRMS-ESI: m/z [M + H] + calcd for C40H55N2O7S: 707.3724, found: 707.3719; Anal. calcd for C40H54N2O7S·0.4 H2O: C 67.27, H 7.75, N 3.92, found: C 66.99, H 7.76, N 3.90.
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Compound 18 b: White solid (82 mg, 70 % yield): mp: 45–48 8C; H NMR (400 MHz, CDCl3): d = 7.90 (d, J = 7.5 Hz, 2 H), 7.65 (t, J = 7.2 Hz, 1 H), 7.57 (t, J = 7.5 Hz, 2 H), 7.28 (t, J = 7.2 Hz, 2 H), 7.20 (t,
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Protein preparation: In the present study, the crystal structure of FP-2 complexed with epoxysuccinate E64 at a resolution of 2.9 (PDB code: 3BPF) was employed in the docking calculations.[23a] The crystallographic complex has four chains (A, B, C, and D); however, for this study, we decided to work only with the A chain. The E64 inhibitor and all crystallographic water molecules were removed from the coordinate set using MOE software.[29] Hydrogen atoms were added to this reduced crystal structure, and the protein was protonated to pH 5 to mimic the acidic environment of the P. falciparum food vacuole (pH between 4 and 6), where the enzyme is located. The enzyme was then submitted to restrained molecular mechanics refinement using the AMBER99 force field implemented in MOE software.[29] The final structure of the FP-2 enzyme was used for the docking calculations. Docking studies: To establish our docking procedure, a preliminary structure validation study was carried out by redocking E64 to the obtained FP-2 enzyme structure. For docking calculations, a ligandbinding domain with a radius of 15 centered on the sulfur atom of the catalytic Cys 42 residue was included in the binding site definition. The GoldScore fitness function was applied to rank the compounds, and the following GOLD parameters were employed: 1000 runs, population size of 100, 100 000 genetic algorithm operations, and five islands at the normal time speed up setting were conducted for each compound.[30] The 20 top-ranked compounds were visually inspected with PyMOL.[28]
Biological assays Inhibitory activity of FP-2 was determined using an assay previously described for ART-based vinyl sulfone compounds in ref. [10]. The assays used to determine antiplasmodial activity and inhibition of hemoglobin degradation for various compounds followed previously described assay protocols in refs. [11] and [18], respectively. ChemMedChem 0000, 00, 1 – 10
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Analysis of compound activation by FeBr2 and in erythrocytes was performed essentially as described in ref. [12]. Cell incubation assay with compound 5 a: Plasmodium falciparum (3D7 strain)-infected human erythrocytes (2 % hematocrit, 5 % parasitemia, trophozoite stages) in serum-free (albumax [0.5 w/v] and hypoxanthine [40 mm]) supplemented complete media were incubated for 48 h with hybrid 5 a (1 mm, final concentration) at 37 8C. Incubations were performed in separate flasks for three different time points (0, 4, and 48 h), with a total incubation volume of 10 mL for each time point. After incubation, the parasite culture (35 mL) was separated by centrifugation (2000 g, 5 min), and the resulting infected erythrocyte pellet was lysed by the addition of 1 mL CH3CN/MeOH (50:50). The lysed material was separated by centrifugation (3000 g, 5 min). The lysis solution was then cleaned by centrifugation (13 000 g, 20 min) and analyzed by LC–MS/MS to detect compound 7.
Acknowledgements This work was supported by the Fundażo para a CiÞncia e Tecnologia (grants PEst-OE/SAU/UI4013/2011 and REDE/1501/REM/ 2005; fellowships SFRH/BD/63200/2009 and SFRH/BD/30418/2006 to R.O. and A.S.N., respectively) and Fundażo Luso-Americana (award to R.M.). Keywords: antimalarial agents peroxides · vinyl sulfones
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