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Keywords:

  • antiplasmodial;
  • antiprotozoal;
  • malaria;
  • Plasmodium falciparum

Abstract

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Conclusion
  6. Acknowledgements
  7. References

Dihydroperoxides and tetraoxanes derived from symmetrically substituted bis(arylmethyl)acetones were synthesized in modest to good yields using several methods. Three of these compounds exhibit an important in vitro antimalarial activity (1.0 μ≤ IC50 ≤ 5.0 μm) against blood forms of the human malaria parasite Plasmodium falciparum.

Many compounds having a peroxide functional group exhibit antimalarial activity as exemplified by the natural product artemisinin and its semi-synthetic derivatives (Figure 1) (1). Artemisinin-based antimalarials are highly potent, and little or no cross-resistance has been observed for this class of compound that contains a unique 1,2,4-trioxane heterocycle as pharmacophore. Although artemisinin produces an initially rapid clearance of parasites from human blood, its clinical utility has been limited by early recrudescence and low oral activity (2–4). These limitations have stimulated the development of antimalarials, which are structurally less complex than artemisinin such as the tetraoxanes. The latter have two endoperoxide bonds and are inexpensive, easy to synthesize, and highly active antimalarial agents (5) as illustrated by di-spiro-1,2,4,5-tetraoxane WR 148999 (Figure 1) (6). An interesting way to synthesize tetraoxanes is via dihydroperoxides that are known key intermediates in the synthesis of many classes of peroxides, many of which have antimalarial activity (Figure 1).

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Figure 1.  Structures of O–O bond containing antimalarials and 1,1-dihydroperoxycyclohexane.

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Curcumin is an α,β-unsaturated ketone, which is obtained from the roots of Curcuma longa (Figure 2) (7). It has proven antimalarial activity in vitro (IC50 = 5 μm) against blood forms of chloroquine-resistant Plasmodium falciparum and is also active in vivo against Plasmodium berghei when administered orally in mice (8). Curcumin also has significant antitumor activity, and bis(arylidene)acetones were introduced as curcumin analogs having improved solubility and other properties (9,10). Furthermore, a series of ring-substituted 1,5-bis-(phenyl)pentadien-3-ones have been prepared as curcumin analogs and been patented for their anti-Plasmodium and anti-Trypanosoma properties (11).

image

Figure 2.  Antimalarial natural product curcumin and patented synthetic analogs.

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In this context, we report herein the synthesis of dihydroperoxides and tetraoxanes having bis-(substituted)phenylethyl substructural units. Based on the antimalarial activity of curcumin and patented 1,5-bis-(phenyl)pentadien-3-ones and of the (endo)peroxide moiety, these compounds were believed to have potential as antimalarial agents (Scheme 1).

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Figure Scheme 1:.  Reactions between dihydroperoxides and tetraoxanes and bisbenzylacetones.

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Methods and Materials

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Conclusion
  6. Acknowledgements
  7. References

Melting points were measured in a Microquímica MQAPF melting point apparatus and are uncorrected. Infrared spectra were recorded with a BOMEM-FTIR MB102 spectrometer. Ultraviolet (UV) spectra were recorded with a UV-1601 PC Shimadzu. 1H and 13C NMR spectra were recorded in deuterochloroform (CDCl3) with a Bruker Avance DRX300 (300 MHz) spectrometer. Chemical shifts (δ) were reported in parts per million (ppm) with reference to tetramethylsilane (TMS) as internal standard. The following abbreviations were used for the 1H multiplicities: singlet (s), doublet (d), triplet (t), and multiplet (m). Coupling constants (J) were reported in Hertz (Hz). Thin-layer chromatography (TLC) was performed on silica gel supported on glass plates (Silica Gel F254; Merck) with a fluorescent indicator and visualized under a UV lamp (254 nm) and/or in I2 vapor and/or with a solution of 1% anisaldehyde with H2SO4 (1% v/v) in EtOH. Column chromatography was performed using silica gel 60G (63–200 μm, 70–230 mesh ASTM; Merck, Waltham, MA, USA).

General procedure for the preparation of dibenzalacetones 4–6

A solution of aldehyde (30 mmol) in EtOH (10 mL) was added to a solution of acetone (25 mmol) and NaOH (3 g, 75 mmol) in EtOH (10 mL) at 0 °C. The reaction mixture was stirred for 15 min and allowed to come to room temperature. The precipitate was filtered, washed with a cold mixture of EtOH/H2O, and recrystallized from a mixture of hexane/EtOAc to yield dibenzalacetone derivatives 4–6.

General procedure for the synthesis of pentan-3-one derivatives 7–9

To a solution of dibenzalacetone 4–6 (10 mmol) in EtOAc (30 mL) was added 10% Pd/C (30 mg, 0.1 mmol). The suspension was hydrogenated for 6 h at 4 psi for 12–24 h. The catalyst was removed by filtration, and the resulting solution was evaporated under reduced pressure. The residue was purified by chromatography on silica gel (hexane/EtOAc 8:2) to give pentan-3-ones 7–9.

Representative synthesis of dihydroperoxides using 30% H2O2 and I2 in MeCN

I2 (0.23 g, 1 mmol) and 30% H2O2 (1.36 g, 40 mmol) were added to a solution of ketone 10 or 7 (10 mmol) in MeCN (30 mL). The reaction mixture was stirred at room temperature for 24 h (72 h when dibenzalacetone derivatives were used as starting materials) and was then diluted with CH2Cl2 (10 mL) and H2O (20 mL). The organic layer was successively washed with saturated aqueous NaCl, dried with Na2SO4, and filtered. The solvents were evaporated to dryness under reduced pressure, and the resulting residue was purified by column chromatography on silica gel (hexane/EtOAc 8:2) to afford dihydroperoxides 11 or 13, respectively.

MTO in TFE procedure

H2O2 30% (1.36 g, 40 mmol) and methyltrioxorhenium (MTO) (0.0003 g, 0.001 mmol) were added to a solution of ketone 10 or 7 (10 mmol) in trifluoroethanol (15 mL). The reaction mixture was stirred at room temperature for 24 h and was then diluted with CH2Cl2 (10 mL) and H2O (20 mL). The organic layer was washed with saturated aqueous NaCl, dried with Na2SO4, and filtered. The solvents were evaporated to dryness under reduced pressure, and the resulting residue was purified by column chromatography on silica gel (hexane/EtOAc 9:1) to afford dihydroperoxide 11 and dimer 12 or dihydroperoxide 13 and dimer 16, respectively.

HCl in CH2Cl2/MeCN procedure

H2O2 30% (3.4 g, 100 mmol) and concentrated HCl (six drops) were added to a solution of ketone 10 or 7 (10 mmol) in a mixture of CH2Cl2 (10 mL) and MeCN (30 mL). The reaction mixture was stirred at room temperature for 24 h and was then diluted with CH2Cl2 (10 mL) and water (20 mL). The organic layer was successively washed with saturated aqueous NaHCO3 (30 mL), water (30 mL), and saturated aqueous NaCl. The organic phase was dried with Na2SO4 and filtered. The solvents were removed under reduced pressure, and the resulting residue was purified by column chromatography on silica gel (hexane/EtOAc 9:1) to afford dihydroperoxides 11 or 13.

H2O2 in ether procedure

Preparation of H2O2 in ether (12).  Thirty percent H2O2 solution (50 mL) was saturated with NaCl, and the mixture was stirred at room temperature for 10 min. After the initial cloudy solution became clear, it was extracted with Et2O (3 × 30 mL). The organic phase was separated and dried with Na2SO4, filtered and used without purification. Ketones 7–10 (10 mmol) were dissolved in the freshly prepared solution of H2O2 in Et2O (30 mL) described above, and phosphomolybdic acid (10 mg) was added. The reaction mixture was stirred at room temperature for 24–72 h and was then diluted with EtOAc (10 mL) and water (20 mL). The organic layer was washed with saturated aqueous NaCl, dried with Na2SO4, and filtered. The solvents were evaporated to dryness under reduced pressure, and the resulting residue was purified by column chromatography on silica gel (EtOAc/hexane, 2:1) to afford dihydroperoxides 11, 13, 14, and 15.

Representative synthesis of tetraoxanes using 30% H2O2 and H2SO4, EtOH, H2O

Ketone 7 (10 mmol) and 30% H2O2 (0.34 g, 10 mmol) were added to a solution of concentrated H2SO4 (20 mL) in EtOH (15 mL) and H2O (15 mL). The reaction mixture was stirred at 0 °C for 14 h. The precipitated material was filtered and recrystallized in MeCN furnishing the tetraoxane 17 (0.20 g; 0.8 mmol, 8% yield).

HBF4 in TFE

Dihydroperoxide 13 (2.88 g, 10 mmol) and 30% H2O2 (0.34 g, 10 mmol) were added to a solution of ketone 7 or cyclohexanone (10 mmol) in trifluoroethanol (15 mL). The reaction mixture was stirred at room temperature for 24 h and was then diluted with CH2Cl2 (10 mL) and water (20 mL). The organic layer was washed with saturated aqueous NaCl, dried with Na2SO4, and filtered. The solvents were evaporated to dryness under reduced pressure, and the resulting residue was purified by column chromatography on silica gel (hexane/EtOAc 9:1) to afford compound 17 or 18 as a white solid in 22 or 38% yield, respectively

MTO/HBF4 in TFE

30% H2O2 (0.07 g, 2 mmol) and methyltrioxorhenium (0.0003 g, 0.001 mmol) were added to a solution of cyclohexanone (1 mmol) in trifluoroethanol (2 mL). The reaction mixture was stirred for 3 h at room temperature; then, ketone 4 or 7 (1 mmol) and HBF4 were added (0.08 g, 1 mmol). After stirring for 24 h at room temperature, the reaction was diluted with CH2Cl2 (1 mL) and H2O (2 mL). The organic layer was washed with saturated aqueous NaCl, dried with Na2SO4, and filtered. The solvents were evaporated to dryness under reduced pressure, and the resulting residue was purified by column chromatography on silica gel (hexane/EtOAc 9:1) to afford compounds 18 or 19 in 28 or 18% yield, respectively.

Data

(1E,4E)-1,5-Bis(phenyl)-penta-1,4-dien-3-one (4): Yellow crystals, mp 109.9–110.6 °C (lit. (13)): 111.0–113.0 °C); Yield: 95%; IR (ν, /cm): 3054, 3026, 1651, 1627, 1592, 1496, 1443, 1343, 1195, 983, 762, 696; UV (λ, nm): 326.0; 1H NMR (300 MHz, CDCl3, ppm): δ 7.10 (d, = 13.0 Hz, 2H, H2, H4), 7.40–7.63 (m, 10H, H arom), 7.75 (d, = 13.0 Hz, 2H, H1, H5); 13C NMR (75 MHz, CDCl3, ppm): δ 125.4 (C2, C4), 128.4 (C arom), 128.9, 130.4, 134.8 (CH arom), 143.3 (C1, C5), 188.6 (C=O).

(1E,4E)-1,5-Bis(4-methoxyphenyl)penta-1,4-dien-3-one (5): Yellow crystals, mp 129.3–129.8 °C; Yield: 91% IR (ν, /cm): 2961, 2841, 1653, 1630, 1597, 1572, 1421, 1251, 1178, 1031, 981, 830; UV (λ, nm): 361.0; 1H NMR (300 MHz, CDCl3, ppm): δ 3.82 (s, 6H, 2 × OCH3), 6.90 (d, = 8.7 Hz, 4H, H arom), 6.94 (d, = 15.9 Hz, 2H, H2, H4), 7.54 (d, = 8.7 Hz, 4H, H arom), 7.68 (d, = 15.9 Hz, 2H, (H1, H5); 13C NMR (75 MHz, CDCl3, ppm): δ 55.3 (2 × OCH3), 114.4 (CH arom), 123.5 (C2, C4), 127.6 (C arom), 130.0 (CH arom), 142.5 (C1, C5), 161.5 (C arom), 188.7 (C=O).

(1E,4E)-1,5-Bis(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (6): Yellow crystals, mp 130.1–131.4 °C; Yield: 76% IR (ν, /cm): 3015, 3030, 2818, 2842, 1626; 1H NMR (300 MHz, CDCl3, ppm): δ 3.91 (s, 18H, 6 × OCH3); 6.94 (s, 4H, H arom); 7.00 (d, = 12.0 Hz, 2H, H2, H4), 7.67 (d, = 12.0 Hz, 2H, H1, H5); 13C NMR (75 MHz, CDCl3, ppm): δ 56.5 (6 × OCH3), 105.4 (4 × CH arom), 125.0 (C2, C4), 130.4 (C arom), 145.6 (3 × C arom), 155.7 (C1, C5), 188.6 (C=O).

1,5-Bis(phenyl)pentan-3-one (7): Yellow oil; Yield: 95%; IR (ν, /cm): 3109, 3000, 2960, 2855, 1713; 1H NMR (300 MHz, CDCl3, ppm): δ 2.67 (t, = 7.5 Hz, 4H, H1, H1′, H5, H5′); 2.88 (t, = 7.5 Hz, 4H, H2, H2′, H4, H4′), 7.14 (d, = 7.0 Hz, 4H, H arom); 7.27 (m, = 7.0 Hz, 6H, H arom); 13C NMR (75 MHz, CDCl3, ppm): δ 29.7 (C1, C5), 44.4 (C2, C4), 126.4, 129.2 (CH arom), 141.0 (C arom), 208.9 (C=O).

1,5-Bis(4-methoxyphenyl)pentan-3-one (8): White solid, mp 53.2–54.5 °C [lit. (14) mp 55.0–55.2 °C]; Yield: 78%; IR (ν, /cm): 3109, 3033, 2830, 2905, 1705. 1H NMR (300 MHz, CDCl3, ppm): δ 2.65 (t, = 7.5 Hz, 4H, H1, H1′, H5, H5′), 2.81 (t, = 7.5 Hz, 4H, H2, H2′, H4, H4′), 3.76 (s, 6H, 2 × OCH3), 6.79 (d, = 8.5 Hz, 4H, H arom), 7.05 (d, = 8.5 Hz, 4H, H arom); 13C NMR (75 MHz, CDCl3, ppm): δ 29.0 (C1, C5), 44.9 (C2, C4), 55.4 (2 × OCH3), 114.0, 129.4 (CH arom), 133.2 (C arom), 158.1 (C arom), 209.6 (C=O).

1,5-Bis(3,4,5-trimethoxyphenyl)pentan-3-one (9): White solid, mp 53.2–54.5 °C; Yield: 51%; IR (ν, /cm): 3010, 3025, 2920, 1700; 1H NMR (300 MHz, CDCl3, ppm): δ 2.74 (t, = 7.0 Hz, 4H, H2, H2′, H4, H4′), 2.81 (t, = 7.0 Hz, 4H, H1, H1′, H5, H5′), 3.81 (s, 18H, 6 × OCH3), 6.38 (s, 4H, H arom). 13C NMR (75 MHz, CDCl3, ppm): δ 30.2 (C1, C5), 44.8 (C2, C4), 56.1 (6 × OCH3), 105.3 (CH arom), 136.9 (C arom), 153.3 (C arom), 209.1 (C=O).

4-tert-Butyl-1,1-dihydroperoxycyclohexane (11): Yield: 89%1; H NMR (300 MHz, CDCl3, ppm): δ 0.87 (s, 14H, 3 × CH3, H3, H4), 1.35 (m, 2H, H2, Ha, Ha’), 2.26 (m, 2H, H2, Hb, Hb’), 8.10 (sl, 2H, H7); 13C NMR (75 MHz, CDCl3, ppm): δ 23.5 (C2), 27.7 (C6), 29.9 (C2), 41.5 (C5), 47.6 (C4), 110.5 (C1).

1,1′-Peroxybis(4-tert-butyl(hydroperoxy)cyclohexane) (12): White solid, mp 80.9–81.3 °C; Yield: 41%; IR (ν, /cm): 3411, 3428 (O-O-H), 2891, 2910; 1H NMR (300 MHz, CDCl3, ppm): δ 1.05 (s, 18H, 6 × CH3); 1.26 (m, 2H, 2 × H4), 1.49 (m, 8H, 2 × H2, H2′, H6, H6′), 2.32 (m, 8H, 2 × H3, H3′, H5, H5′), 9.62 (s, 2H, 2 × OOH); 13C NMR (75 MHz, CDCl3, ppm): δ 23.2 (C3), 23.5 (C2), 27.8 (9 × CH3), 30.1 (C(CH3)3), 47.6 (C4), 111.3 (C1).

3,3-Dihydroperoxy-1,5-(bisphenyl)pentane (13): White solid, mp 85.4–86.7 °C; Yield: 84%; IR (ν, /cm): 3278, 3415 (O-O-H), 3024, 3091, 2888, 2940; 1H NMR (300 MHz, CDCl3, ppm): δ 2.07 (t, = 8.3 Hz, 4H, H1, H1′, H5, H5′), 2.73 (t, = 8.0 Hz, 4H, H2, H2′, H4, H4′), 7.22 (m, 10H, H arom), 9.28 (s, 2H, OOH); 13C NMR (75 MHz, CDCl3, ppm): δ 30.1 (C2, C4), 31.3 (C1, C5), 113.9 (C3), 126.4, 128.7 (CH arom), 141.3 (C arom).

3,3-Dihydroperoxy-1,5-bis(4′-methoxyphenyl)pentane (14): White solid, mp 170.2–173.1 °C; Yield: 50%; IR (ν, /cm): 3435, 3250 (O-O-H), 2885, 2720; 1H NMR (300 MHz, CDCl3, ppm): δ 1.25 (t, = 6.5 Hz, 4H, H1, H1′, H5, H5′), 2.05 (t, = 6.5 Hz, 4H, H2, H2′, H4, H4′), 3.72 (s, 6H, 2 × OCH3), 6.81 (d, = 8.5 Hz, 4H, H arom), 7.08 (d, = 8.5 Hz, 4H, H arom), 9.78 (large s, 2H, OOH); 13C NMR (75 MHz, CDCl3, ppm): δ 29.1 (C2, C4), 31.6 (C1, C5), 113.4 (C3), 114.1, 129.4 (CH arom), 133.6 (C arom), 158.0 (2 × OCH3).

3,3-Dihydroperoxy-1,5-bis(3′,4′,5′-trimethoxyphenyl)pentane (15): Yellow oil; Yield: 31%; IR (ν, /cm): 3125, 3319 (O-O-H), 2884, 2920, 2762, 2809. 1H NMR (300 MHz, CDCl3, ppm): δ 2.06 (t, = 7.0 Hz, 4H, H2, H2′, H4, H4′), 2.72 (t, = 7.0 Hz, 4H, H1, H1′, H5, H5′), 3.84 (s, 18H, 6 × OCH3), 6.43 (s, 4H, H arom), 9.50 (s, 2H, OOH); 13C NMR (75 MHz, CDCl3, ppm): δ 30.6 (C2, C4), 31.5 (C1, C5), 56.3, 61.2 (6 × OCH3), 105.6 (CH arom), 112.4 (CH arom), 133.6 (C arom), 153.3 (OCH3).

[3,3′-Peroxybis-(3-hydroperoxypentane-5,3,1-triyl)]tetrabenzene (16): White solid, mp 140.9–141.3 °C; Yield: 23%; IR (ν, /cm): 3370, 3458 (O-O-H), 2916, 3021, 2991, 2844; 1H NMR (300 MHz, CDCl3, ppm): δ 2.11 (d, = 8.0 Hz, 8H, 2 × (H1, H1′, H5, H5′)), 2.78 (d, = 8.0 Hz, 8H, 2 × (H2, H2′, H4, H4′)), 7.18 (m, 4H, H arom), 7.30 (m, 16H, H arom), 9.70 (s, 2H, 2 × OOH); 13C NMR (75 MHz, CDCl3, ppm): δ 30.3 (2 × C1, C5), 31.8 (2 × C2, C4), 114.3 (C3), 126.6 (4 × CH arom), 128.7 (16 × CH arom), 141.2 (4 × C arom).

3,3,6,6-Tetraphenethyl-1,2,4,5-tetraoxane (17): White solid, mp 149.7–150.9 °C; Yield: 22%; IR (ν, /cm): 3075, 3244 (O-O-H), 2990, 3966, 2830, 2974; 1H NMR (300 MHz, CDCl3, ppm): δ 2.04 (m, 8H, 4 × PhCH2CH2), 2.72 (m, 8H, 4 × PhCH2CH2), 7.27 (m, 2OH, H arom); 13C NMR (75 MHz, CDCl3, ppm): δ 26.6 (PhCH2CH2), 36.2 (PhCH2CH2), 110.2 (C3, C6), 141.9 (C arom), 125.4, 127.6, 129.7 (CH arom).

3,3-Diphenethyl-1,2,4,5-tetraoxaspiro[5.5]undecane (18): White solid, mp 114.5–115.3 °C; Yield: 38%; IR (ν, /cm): 3224 (O-O-H), 3117, 3008, 2902, 2814, 2775; 1H NMR (300 MHz, CDCl3, ppm): δ 1.47 (s, 10H, cyclohexyl), 2.28 (m, 8H, 2 × (CH2)2Ph), 7.20–7.30 (m, 10H, H arom); 13C NMR (75 MHz, CDCl3, ppm): δ 21.6 (CH2CH2CH2CH2CH2), 25.6 (CH2CH2Ph), 32.1 (CH2CH2CH2CH2CH2, CH2CH2Ph), 110.1 (C3), 126.2 (CH arom), 128.2 (C arom), 128.6 (C arom).

3,3-Bis(3-phenyloxiran-2-yl)-1,2,4,5-tetraoxaspiro[5.5]undecane (19): White solid, 87.5–89.1°C; Yield: 18%; IR (ν, /cm): 3012, 3184 (O-O-H), 2992, 3002, 2765, 2916; 1H NMR (300 MHz, CDCl3, ppm): δ 1.26 (m, 2H, CH2CH2CH2CH2CH2), 1.31 (m, 6H, CH2CH2CH2CH2CH2), 1.39 (m, 4H, CH2CH2CH2CH2CH2), 4.13 (m, 1H, inline image), 4.37 (m, 1H, inline image), 7.26 (d, = 7.0 Hz, 2H, H arom), 7.72 (d, = 7.0 Hz, 4H, H arom), 8.06 (d, = 7.0 Hz, 4H, H arom); 13C NMR (75 MHz, CDCl3, ppm): δ 14.5 (CH2CH2CH2CH2CH2), 29.9 (CH2CH2CH2CH2CH2), 41.4 (CH2CH2CH2CH2CH2), 60.7 (inline image), 61.1 (inline image), 118.5 (C3, C6), 128.2 (CH arom), 130.4 (CH arom), 167.2 (C arom).

Antimalarial activity

In vitro culture of human malaria parasite Plasmodium falciparum

Chloroquine-resistant P. falciparum K1 strain was obtained from the Malaria Research Reference Reagent Resource Center (MR4/Manassas, VA, USA) and maintained in continuous culture using the Trager and Jensen (15) method at 5% hematocrit using type A+ human erythrocytes in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 2 mm l-glutamine (Gibco, Eusébio, CE, Brazil), 25 mm HEPES (Sigma-Aldrich), 40 μg/mL gentamycin, 10% A+ human plasma, and 25 mm NaHCO3. Cultures were maintained under a mixture of 5% O2, 5% CO2, and 90% N2 and incubated at 37 °C. When cultures attained parasitemias of 4–5%, they were synchronized with 5% sorbitol (16).

Test for in vitro inhibition of blood forms of Plasmodium falciparum

This assay was performed according to the method of Rieckmann et al. (17) with modifications described by Andrade-Neto et al. (18,19). Briefly, 5 mg/mL stock solutions of hydroperoxides 11–14 and 16 were prepared in DMSO. Stock solutions were serially diluted in culture medium (RPMI 1640) by a factor of 1:5 to obtain seven dilutions of each sample having final (sample well) concentrations of 100–6.4 × 10−3 μg/mL. Each diluted sample was tested in duplicate. Diluted samples were transferred to 96-well microtest plates containing parasitized red blood cell (RBC) suspension with 3% hematocrit and initial parasitemia of 1% of synchronized young trophozoites (ring form). Control wells contained 1% final concentration of DMSO. The final volume in each well was 200 μL. Reference antimalarial compounds (chloroquine and quinine) were tested in the concentrations recommended by WHO (20). The microplate was incubated for 48 h at 37 °C under a mixture of gases (5% O2, 5% CO2, and 90% N2). After the incubation period, thin smears of the contents of each well were evaluated by optical microscopy (100× magnification). In this procedure, the number of parasites present in a total of approximately 2000 RBCs was determined. Parasitemia was expressed as a percentage of the number of parasitized RBCs found in the total number of RBCs counted:

  • image

The half-maximal inhibitory (IC50) responses as compared to the drug-free controls were estimated by interpolation using Microcal Origin software. The test concentration values in units of log μg/mL were plotted against parasite viability (ratio of the average parasitemias of test wells to the average parasitemias of the control wells) for each concentration using the sigmoidal fitting analysis function to generate a smooth curve. The IC50 value is obtained from the graph and corresponds to a viability of 0.5.

Results

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Conclusion
  6. Acknowledgements
  7. References

Preparation of dibenzalacetones and their hydrogenated 1,2-bisphenylmethyl acetone derivatives

α,β-Unsaturated ketones 4, 5, and 6 are structurally analogous to curcumin and were obtained from benzaldehyde (1), 4-methoxybenzaldehyde (2), and 3,4,5-trimethoxybenzaldehyde (3), respectively, in 76–95% yields (Scheme 2). Ketones 7, 8, and 9 were prepared from dibenzylacetones 4, 5, and 6, respectively, by hydrogenation in 51–93% yields (Scheme 2).

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Figure Scheme 2:.  Preparation of ketones 4–9.

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Ketones 49, cyclohexanone, and tert-butylcyclohexanone were used as substrates in the synthesis of dihydroperoxides and tetraoxanes. For this purpose, four different methods of direct preparation were tested: (i) 30% H2O2 with I2 in MeCN;(21) (ii) 30% H2O2 with methyltrioxorhenium (MTO) in trifluoroethanol (TFE) or CH2Cl2; (22) (iii) 30% H2O2 with HCl in CH2Cl2 and MeCN; (23) (iv) 30% H2O2 with phosphomolybdic acid (PMA) in Et2O (24).

Synthesis of dihydroperoxides

Initial attempts to obtain dihydroperoxides were carried out starting with inexpensive and readily available tert-butylcyclohexanone applying the methods described above (Scheme 3). The results are summarized in Table 1.

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Figure Scheme 3:.  Synthesis of dihydroperoxides 11 and 12 from 4-tert-butylcyclohexanone.

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Table 1.   Yields of dihydroperoxides 11 and 12 by treating 1 with 30% H2O2 under different conditions
EntryMethod 30% H2O2Yield (%)
1112
1I2, MeCN61
2MTO, TFE3018
3MTO, CH2Cl21841
4HCl, CH2Cl2/MeCN30
5Et2O, PMA89

Preparation of dihydroperoxide was best performed with 30% H2O2 in Et2O catalyzed by PMA (Entry 5) providing compound 11 in 89% yield. Using the MTO method, dihydroperoxide 11 was obtained along with dimeric dihydroperoxide 12, which contains a peroxide function (Entries 2 and 3). Using these same conditions and starting with ketones 79, similar results were observed furnishing dihydroperoxides 13–15 and dimer 16 (Scheme 4, Table 2).

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Figure Scheme 4:.  Synthesis of dihydroperoxides 13–15 and dimer 16 from ketones 7–9.

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Table 2.   Yields of dihydroperoxides 13–16 using H2O2 under different conditions
EntryMethod 30% H2O2SubstrateProduct% YieldProduct% Yield
1I2, MeCN71330
2MTO, TFE7151611
3MTO, CH2Cl271523
4HCl, CH2Cl2/MeCN715
5 71384
6Et2O, PMA81450
7 91531

Thirty per cent H2O2/PMA in Et2O was superior to other methods and provided 13 in good yield (84%) (Entry 5). Lower yields were obtained for the synthesis of analogs 14 and 15 (Entries 6 and 7). Other methods were less satisfactory providing yields of 15–30% (Entries 1–4). Dimer 16 was obtained as a side product during the synthesis of 13 using the MTO method. No dihydroperoxide derivatives were isolated when unsaturated ketones 46 were used as substrates in the methods described. Instead, oxidation or double bond cleavage products were observed.

Synthesis of tetraoxanes

Three different methods were employed in the synthesis of tetraoxanes 17–19. Symmetric tetraoxane 17 was prepared in 8% yield using 30% H2O2, H2SO4, EtOH, and H2O (Scheme 5). A better yield of 17 (22%) was obtained by reacting dihydroperoxide 13 with ketone 7 in 30% H2O2 and HBF4 in TFE. The reaction of 13 and cyclohexanone under the same conditions leads to non-symmetric tetraoxane 18 in 38% yield (Scheme 5).

image

Figure Scheme 5:.  Synthesis of tetraoxanes 17 and 18.

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Alternatively, the synthesis of non-symmetric tetraoxanes can be achieved directly from the corresponding ketones via a one-pot procedure described by Danièle-Bonnet and coworker (25). Treatment of a mixture of cyclohexanone and ketone 7 with 30% H2O2 and MTO/HBF4 in TFE (Scheme 6) gave tetraoxane 18 in poor yield (28%). Using the same reaction conditions and a mixture of cyclohexanone and unsaturated ketone 4, an unexpected tetraoxane diepoxide product 19 was isolated in 18% yield.

image

Figure Scheme 6:.  Synthesis of tetraoxanes 18 and 19.

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The biphenylidene acetone (4), three bi-benzylmethyl ketones 79, three dihydroperoxides 11, 13, 14, two peroxydihydroperoxides 12 and 16, and two 1,2,4,5-tetroxanes 17 and 18 were initially screened at two concentrations for in vitro inhibition of the growth of blood stages of the human malaria parasite P. falciparum and hemolytic action. Dihydroperoxide 14 and 18 caused hemolysis of the human blood containing culture medium and were not further tested. Next, the nine remaining compounds were further tested at different concentrations to establish quantitative concentration–activity relationships and calculate the median inhibitory concentration (IC50). Only compounds containing O–O bonds were found to have significant antimalarial activities. Among the seven O-O-containing compounds which were tested, compounds 13, 16, and 17 (IC50 = 1.4–3.3 μm) were active (Table 3).

Table 3.   Inhibition of the in vitro growth of the K1 strain of Plasmodium falciparum by selected dihydroperoxide (B), dihydroperoxyperoxide (C), and 1,2,4,5-tetraoxane (D) compounds Thumbnail image of

While specific mechanistic studies were not performed to establish the mode of antimalarial action of the compounds synthesized in this study, the preliminary antiplasmodial data presented in Table 3 allow for speculations as to the possible modes of action of the synthesized compounds. First, low antiplasmodial activity is associated with the starting bisbenzyl acetone (7), and its aryl ring substituted analogs 8 and 9 and no general cytotoxicity or membrane destabilization (hemolysis) was observed for bisbenzyl acetones 79. On the other hand, high antiplasmodial activity was observed for gem-dihydroperoxide 13, dihydroperoxy peroxide 16, and 1,2,4,5-tetraoxane 17, and hemolytic activity was observed for gem-dihydroperoxide 15 and tetraoxane 18. These preliminary data may be an indication of a strong general interaction of this class of compound with the surface membranes of RBCs and perhaps those of free parasites (merozoites). Another possibility is that the highly active hydroperoxy and peroxide compounds are able to penetrate the erythrocyte and P. falciparum membranes and end up in the digestive vacuole of P. falciparum cells. This vacuole is the specific organelle wherein the infected RBC’s hemoglobin (a protein) is digested by the parasite. An important by-product of hemoglobin digestion is heme. This substance, which is toxic to the parasite, is polymerized in the digestive vacuole to a stable hemozoin complex (26). There is evidence that common antimalarials such as chloroquine and artemisinin and highly antiplasmodial compounds having planar substructures such as ellipticine derivatives, xanthones act within the parasite digestive vacuole and kill the parasites by stabilizing soluble heme and preventing hemozoin formation (27). Furthermore, it is believed that ferric ion (toxic to parasites) metabolism is also affected in the digestive vacuole by the natural 1,2,4-trioxane (peroxide) artemisinin and its derivatives (28). The relatively flat or planar substructures and peroxy moieties in the synthesized compounds may thus be acting in the digestive vacuole by stabilizing heme/inhibiting hemozoin formation and also by affecting the metabolism of Fe3+. Only through specific studies on mechanism and structure–activity relationships will the mechanism(s) of antiplasmodial action be forthcoming (29).

Conclusion

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Conclusion
  6. Acknowledgements
  7. References

Synthesis of dihydroperoxides derived from hydrogenated dibenzalacetones was achieved using four different methods. The most efficient method was the treatment of ketone with hydrogen peroxide in ether catalyzed by phosphomolybdic acid, which provided the desired dihydroperoxides in 31–84% yields. Among the methods explored for the synthesis of tetraoxanes, the most efficient provided compounds 17 and 18 in 22% and 38% yield, respectively. Dihydroperoxide 13, dihydroperoxy peroxide 16, and 1,2,4,5-tetraoxane 17 exhibit significant antimalarial activity against chloroquine-resistant human malaria parasite P. falciparum. These compounds will be further evaluated for in vivo antimalarial and against other parasite species.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Conclusion
  6. Acknowledgements
  7. References

This work was supported by FAPEMIG, CAPES and CNPq. This work is part of a collaboration research project of members of the Rede Mineira de Química (RQ-MG) supported by FAPEMIG (Project: REDE-113/10). This work was also supported by the CNPq Brazilian Malaria Network (partial or complete grants from: FAPESP, CNPq 555.669/2009-2 & 555.665/2009-7, PRONEX/FAPEAM 9/2009) and grants from DTI/INPA/CNPq and FAPEAM/PPP.

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  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Conclusion
  6. Acknowledgements
  7. References
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