Synthesis and Antimalarial Activity of Dihydroperoxides and Tetraoxanes Conjugated with Bis(benzyl)acetone Derivatives

Article abstract of DOI:10.1111/j.1747-0285.2012.01345.x

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 (1.0μm≤IC₅₀≤5.0μm) against blood forms of the human malaria parasite Plasmodium falciparum.

Full text of DOI:10.1111/j.1747-0285.2012.01345.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01345.x
Chem Biol Drug Des 2012; 79: 790–797
Research Article
Synthesis and Antimalarial Activity of
Dihydroperoxides and Tetraoxanes Conjugated
with Bis(benzyl)acetone Derivatives
Lucas Lopardi Franco1, Mauro Vieira de
ment of antimalarials, which are structurally less complex than arte-
misinin such as the tetraoxanes. The latter have two endoperoxide
Almeida1, Luiz Francisco Rocha e Silva3,5
,
Pedro Paulo Ribeiro Vieira2, Adrian Martin
Pohlit3 and Marcelo Siqueira Valle4,*
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 tetraox-
anes is via dihydroperoxides that are known key intermediates in
the synthesis of many classes of peroxides, many of which have
antimalarial activity (Figure 1).
1Departamento de Quꢀmica, Universidade Federal de Juiz de Fora,
Campus Universitꢁrio, s ⁄ n, Martelos – Juiz de Fora – MG 36036-
900, Brazil
2GerÞncia da Malꢁria, Fundażo de Medicina Tropical do Amazonas,
Avenida Pedro Teixeira, 25, Dom Pedro – Manaus – AM 69040-000,
Brazil
Curcumin is an a,b-unsaturated ketone, which is obtained from the
roots of Curcuma longa (Figure 2) (7). It has proven antimalarial
activity in vitro (IC50 = 5 lM) 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-Try-
panosoma properties (11).
3Coordenażo de Tecnologia e Inovażo, Instituto Nacional de
Pesquisa da Amazꢂnia, Av. Andrꢃ Araffljo, 2936, Aleixo – Manaus –
AM 69060-001, Brazil
4Departamento de CiÞncias Naturais, Universidade Federal de S¼o
Jo¼o del Rei, Campus Dom Bosco, PraÅa Dom Helvꢃcio, 74 –
Fꢁbricas – S¼o Jo¼o del Rei – MG 36301-160, Brazil
5Programa de Pós-Graduażo em Biotecnologia - Universidade
Federal do Amazonas. Av General Rodrigo Otꢁvio, 60200, Coroado,
Manaus - AM 69077-000, Brazil
*Corresponding author: Marcelo Siqueira Valle,
marcelovalle@ufsj.edu.br
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).
Dihydroperoxides and tetraoxanes derived from
symmetrically substituted bis(arylmethyl)acetones
were synthesized in modest to good yields using
several methods. Three of these compounds exhi-
bit an important in vitro antimalarial activity
(1.0 lM £ IC50 £ 5.0 lM) against blood forms of the
human malaria parasite Plasmodium falciparum.
Methods and Materials
Key words: antiplasmodial, antiprotozoal, malaria, Plasmodium fal-
ciparum
Melting points were measured in a Microquꢀmica MQAPF melting
point apparatus and are uncorrected. Infrared spectra were recorded
Received 12 June 2011, revised 11 January 2012 and accepted for pub-
lication 12 January 2012
Me
H
O
Me
Me
O
O
O
O
Many compounds having a peroxide functional group exhibit anti-
malarial 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 develop-
O
O
HOO
OOH
H
Me
Me
O
O
A Dihydroperoxide
Artemisinin
WR148999
Figure 1: Structures of O–O bond containing antimalarials and
1,1-dihydroperoxycyclohexane.
790

New Dihydroperoxides and Tetraoxanes as Antimalarials
O
OH
R1
O
R10
MeO
HO
OMe
OH
R2
R3
R9
R8
R5
R6
R4
R7
Curcumin
(1E,4E)-1,5-Bis(phenyl)pentadien-3-ones
Figure 2: Antimalarial natural product curcumin and patented synthetic analogs.
HOO
R
OOH
R
HOO
OOH
R
Dihydroperoxides
R
O
Molecular
Hibridization
New Peroxide Conjugates
R
R
Symmetric Bisbenzylacetones
R
O
O
R
O
O
R
R
O
O
R
O
O
R
R
R
Tetraoxanes
Scheme 1: Reactions between dihydroperoxides and tetraoxanes and bisbenzylacetones.
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 (d) were
reported in parts per million (ppm) with reference to tetramethylsi-
lane (TMS) as internal standard. The following abbreviations were
removed by filtration, and the resulting solution was evaporated
under reduced pressure. The residue was purified by chromatogra-
phy 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
1
used for the H 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 sup-
ported 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 lm, 70–230 mesh ASTM; Merck, Waltham, MA, USA).
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 reac-
tion 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.
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.
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 stir-
red at room temperature for 24 h and was then diluted with CH2Cl2
(10 mL) and H2O (20 mL). The organic layer was washed with satu-
rated aqueous NaCl, dried with Na2SO4, and filtered. The solvents
were evaporated to dryness under reduced pressure, and the result-
ing 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.
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
Chem Biol Drug Des 2012; 79: 790–797
791

Franco et al.
HCl in CH2Cl2 ⁄ MeCN procedure
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.
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 chroma-
tography on silica gel (hexane ⁄ EtOAc 9:1) to afford dihydroperoxides
11 or 13.
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 (m, ⁄ cm):
3054, 3026, 1651, 1627, 1592, 1496, 1443, 1343, 1195, 983, 762,
1
H2O2 in ether procedure
696; UV (k, nm): 326.0; H NMR (300 MHz, CDCl3, ppm): d 7.10 (d,
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 fil-
tered. The solvents were evaporated to dryness under reduced pres-
sure, and the resulting residue was purified by column
chromatography on silica gel (EtOAc ⁄ hexane, 2:1) to afford dihydr-
operoxides 11, 13, 14, and 15.
J = 13.0 Hz, 2H, H2, H4), 7.40–7.63 (m, 10H, H arom), 7.75 (d,
J = 13.0 Hz, 2H, H1, H5); 13C NMR (75 MHz, CDCl3, ppm): d 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 (m, ⁄ cm): 2961, 2841,
1653, 1630, 1597, 1572, 1421, 1251, 1178, 1031, 981, 830; UV (k,
nm): 361.0; 1H NMR (300 MHz, CDCl3, ppm): d 3.82 (s, 6H,
2 · OCH3), 6.90 (d, J = 8.7 Hz, 4H, H arom), 6.94 (d, J = 15.9 Hz,
2H, H2, H4), 7.54 (d, J = 8.7 Hz, 4H, H arom), 7.68 (d, J = 15.9 Hz,
2H, (H1, H5); 13C NMR (75 MHz, CDCl3, ppm): d 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): Yel-
low crystals, mp 130.1–131.4 ꢀC; Yield: 76% IR (m, ⁄ cm): 3015,
3030, 2818, 2842, 1626; H NMR (300 MHz, CDCl3, ppm): d 3.91 (s,
18H, 6 · OCH3); 6.94 (s, 4H, H arom); 7.00 (d, J = 12.0 Hz, 2H, H2,
H4), 7.67 (d, J = 12.0 Hz, 2H, H1, H5); 13C NMR (75 MHz, CDCl3,
ppm): d 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
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 fur-
nishing the tetraoxane 17 (0.20 g; 0.8 mmol, 8% yield).
1,5-Bis(phenyl)pentan-3-one (7): Yellow oil; Yield: 95%; IR (m, ⁄ cm):
1
3109, 3000, 2960, 2855, 1713; H NMR (300 MHz, CDCl3, ppm): d
2.67 (t, J = 7.5 Hz, 4H, H1, H1¢, H5, H5¢); 2.88 (t, J = 7.5 Hz, 4H,
H2, H2¢, H4, H4¢), 7.14 (d, J = 7.0 Hz, 4H, H arom); 7.27 (m,
J = 7.0 Hz, 6H, H arom); 13C NMR (75 MHz, CDCl3, ppm): d 29.7
(C1, C5), 44.4 (C2, C4), 126.4, 129.2 (CH arom), 141.0 (C arom),
208.9 (C=O).
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 stir-
red at room temperature for 24 h and was then diluted with CH2Cl2
(10 mL) and water (20 mL). The organic layer was washed with sat-
urated aqueous NaCl, dried with Na2SO4, and filtered. The solvents
were evaporated to dryness under reduced pressure, and the result-
ing 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
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 (m, ⁄ cm): 3109,
1
3033, 2830, 2905, 1705. H NMR (300 MHz, CDCl3, ppm): d 2.65 (t,
J = 7.5 Hz, 4H, H1, H1¢, H5, H5¢), 2.81 (t, J = 7.5 Hz, 4H, H2, H2¢,
H4, H4¢), 3.76 (s, 6H, 2 · OCH3), 6.79 (d, J = 8.5 Hz, 4H, H arom),
7.05 (d, J = 8.5 Hz, 4H, H arom); 13C NMR (75 MHz, CDCl3, ppm): d
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).
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
1,5-Bis(3,4,5-trimethoxyphenyl)pentan-3-one (9): White solid, mp
53.2–54.5 ꢀC; Yield: 51%; IR (m, ⁄ cm): 3010, 3025, 2920, 1700; H
NMR (300 MHz, CDCl3, ppm): d 2.74 (t, J = 7.0 Hz, 4H, H2, H2¢,
1
792
Chem Biol Drug Des 2012; 79: 790–797

New Dihydroperoxides and Tetraoxanes as Antimalarials
13C NMR (75 MHz, CDCl3, ppm):
26.6 (PhCH2CH2), 36.2
H4, H4¢), 2.81 (t, J = 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): d
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).
d
(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 (m, ⁄ cm): 3224 (O-O-H), 3117,
3008, 2902, 2814, 2775; H NMR (300 MHz, CDCl3, ppm): d 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): d 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).
4-tert-Butyl-1,1-dihydroperoxycyclohexane (11): Yield: 89%1; H NMR
(300 MHz, CDCl3, ppm): d 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): d 23.5 (C2), 27.7 (C6), 29.9 (C2), 41.5
(C5), 47.6 (C4), 110.5 (C1).
1
1,1¢-Peroxybis(4-tert-butyl(hydroperoxy)cyclohexane) (12): White
solid, mp 80.9–81.3 ꢀC; Yield: 41%; IR (m, ⁄ cm): 3411, 3428 (O-O-H),
2891, 2910; 1H NMR (300 MHz, CDCl3, ppm): d 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): d 23.2 (C3), 23.5 (C2), 27.8 (9 · CH3), 30.1
(C(CH3)3), 47.6 (C4), 111.3 (C1).
3,3-Bis(3-phenyloxiran-2-yl)-1,2,4,5-tetraoxaspiro[5.5]undecane (19):
White solid, 87.5–89.1ꢀC; Yield: 18%; IR (m, ⁄ cm): 3012, 3184 (O-O-
H), 2992, 3002, 2765, 2916; H NMR (300 MHz, CDCl3, ppm): d 1.26
1
(m, 2H, CH2CH2CH2CH2CH2), 1.31 (m, 6H, CH2CH2CH2CH2CH2), 1.39
(m, 4H, CH2CH2CH2CH2CH2), 4.13 (m, 1H,
), 4.37 (m, 1H,
),
7.26 (d, J = 7.0 Hz, 2H, H arom), 7.72 (d, J = 7.0 Hz, 4H, H arom),
8.06 (d, J = 7.0 Hz, 4H, H arom); 13C NMR (75 MHz, CDCl3, ppm):
3,3-Dihydroperoxy-1,5-(bisphenyl)pentane (13): White solid, mp 85.4–
86.7 ꢀC; Yield: 84%; IR (m, ⁄ cm): 3278, 3415 (O-O-H), 3024, 3091,
2888, 2940; H NMR (300 MHz, CDCl3, ppm): d 2.07 (t, J = 8.3 Hz,
d
14.5 (CH2CH2CH2CH2CH2), 29.9 (CH2CH2CH2CH2CH2), 41.4
(CH2CH2CH2CH2CH2), 60.7 ( ), 61.1 ( ), 118.5 (C3, C6), 128.2
(CH arom), 130.4 (CH arom), 167.2 (C arom).
1
4H, H1, H1¢, H5, H5¢), 2.73 (t, J = 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):
d 30.1 (C2, C4), 31.3 (C1, C5), 113.9 (C3), 126.4, 128.7 (CH arom),
141.3 (C arom).
Antimalarial activity
In vitro culture of human malaria parasite
Plasmodium falciparum
3,3-Dihydroperoxy-1,5-bis(4¢-methoxyphenyl)pentane (14): White
solid, mp 170.2–173.1 ꢀC; Yield: 50%; IR (m, ⁄ cm): 3435, 3250 (O-O-
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,
Eusꢁbio, CE, Brazil), 25 mM HEPES (Sigma-Aldrich), 40 lg ⁄ mL gen-
tamycin, 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).
H), 2885, 2720; 1H NMR (300 MHz, CDCl3, ppm):
d 1.25
(t, J = 6.5 Hz, 4H, H1, H1¢, H5, H5¢), 2.05 (t, J = 6.5 Hz, 4H, H2,
H2¢, H4, H4¢), 3.72 (s, 6H, 2 · OCH3), 6.81 (d, J = 8.5 Hz, 4H, H
arom), 7.08 (d, J = 8.5 Hz, 4H, H arom), 9.78 (large s, 2H, OOH); 13C
NMR (75 MHz, CDCl3, ppm): d 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): Yel-
low oil; Yield: 31%; IR (m, ⁄ cm): 3125, 3319 (O-O-H), 2884, 2920,
1
2762, 2809. H NMR (300 MHz, CDCl3, ppm): d 2.06 (t, J = 7.0 Hz,
4H, H2, H2¢, H4, H4¢), 2.72 (t, J = 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): d 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).
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) concen-
trations of 100–6.4 · 10)3 lg ⁄ 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 concentra-
tion of DMSO. The final volume in each well was 200 lL. Refer-
ence 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,
[3,3¢-Peroxybis-(3-hydroperoxypentane-5,3,1-triyl)]tetrabenzene (16):
White solid, mp 140.9–141.3 ꢀC; Yield: 23%; IR (m, ⁄ cm): 3370,
3458 (O-O-H), 2916, 3021, 2991, 2844; 1H NMR (300 MHz, CDCl3,
ppm): d 2.11 (d, J = 8.0 Hz, 8H, 2 · (H1, H1¢, H5, H5¢)), 2.78 (d,
J = 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): d 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 (m, ⁄ cm): 3075, 3244 (O-O-H), 2990,
1
3966, 2830, 2974; H NMR (300 MHz, CDCl3, ppm): d 2.04 (m, 8H,
4 · PhCH2CH2), 2.72 (m, 8H, 4 · PhCH2CH2), 7.27 (m, 2OH, H arom);
Chem Biol Drug Des 2012; 79: 790–797
793

Franco et al.
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. Par-
asitemia was expressed as a percentage of the number of parasit-
ized RBCs found in the total number of RBCs counted:
Results
Preparation of dibenzalacetones and their
hydrogenated 1,2-bisphenylmethyl acetone
derivatives
a,b-Unsaturated ketones 4, 5, and 6 are structurally analogous to
curcumin and were obtained from benzaldehyde (1), 4-methoxybenz-
aldehyde (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).
No. of parasitized RBCs ꢀ 100
Parasitemia ð%Þ ¼
No. of RBCs total
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
lg ⁄ mL were plotted against parasite viability (ratio of the average
parasitemias of test wells to the average parasitemias of the con-
trol wells) for each concentration using the sigmoidal fitting analy-
sis function to generate a smooth curve. The IC50 value is obtained
from the graph and corresponds to a viability of 0.5.
Ketones 4–9, cyclohexanone, and tert-butylcyclohexanone were
used as substrates in the synthesis of dihydroperoxides and tetraox-
anes. 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.
O
O
3
1
5
acetone,
NaOH
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
R1
R
R1
R
R1
R
H
2
4
MeOH/H2O,
0 °C ꢀ rt
R1
R = R1 = H
R = OMe, R1 = H
R1
R1
4: R = R1 = H (95%)
5:
1:
2:
R = OMe, R1 = H (91%)
3: R = R1 = OMe
6: R = R1 = OMe (76%)
Table 1: Yields of dihydroperoxides 11 and 12 by treating 1
with 30% H2O2 under different conditions
Pd/C,H2
EtOH
O
Yield (%)
R1
R
R1
R
Entry
Method 30% H2O2
11
12
R1
R1
1
2
3
4
5
I2, MeCN
MTO, TFE
MTO, CH2Cl2
HCl, CH2Cl2 ⁄ MeCN
Et2O, PMA
61
30
18
30
89
–
18
41
–
7: R = R1 = H (95%)
R = OMe, R1 = H (78%)
R = R1 = OMe (51%)
8:
9:
–
Scheme 2: Preparation of ketones 4–9.
t-Bu
O
HOO
OOH
Conditions
Table 1
O
O
OOH
+
HOO
t-Bu
11
t-Bu
10
12
t-Bu
Scheme 3: Synthesis of dihydroperoxides 11 and 12 from 4-tert-butylcyclohexanone.
794
Chem Biol Drug Des 2012; 79: 790–797

New Dihydroperoxides and Tetraoxanes as Antimalarials
HOO
OOH
R1
R
R1
R
7-9
O
O
OOH
R1
13:
R1
HOO
R = R1 = H
14: R = OMe, R1 = H
15: R = R1 = OMe
16
Scheme 4: Synthesis of dihydroperoxides 13–15 and dimer 16 from ketones 7–9.
Table 2: Yields of dihydroperoxides 13–16 using H2O2 under
7
4
18 (28%)
different conditions
O
Entry Method 30% H2O2 Substrate Product % Yield Product % Yield
H2O2 30%, MTO
TFE
1
2
3
4
5
6
7
I2, MeCN
MTO, TFE
MTO, CH2Cl2
HCl, CH2Cl2 ⁄ MeCN
7
7
7
7
7
8
9
13
30
15
15
15
84
50
31
–
16
11
23
O
O
O
O
–
–
–
–
13
14
15
O
O
Et2O, PMA
19 (18%)
Scheme 6: Synthesis of tetraoxanes 18 and 19.
with ketones 7–9, similar results were observed furnishing dihydr-
operoxides 13–15 and dimer 16 (Scheme 4, Table 2).
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 deriva-
tives were isolated when unsaturated ketones 4–6 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 tetra-
oxanes 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).
Alternatively, the synthesis of non-symmetric tetraoxanes can be
achieved directly from the corresponding ketones via a one-pot
procedure described by Daniꢂle-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 unex-
pected tetraoxane diepoxide product 19 was isolated in 18%
yield.
H2O2 30%,
H2SO4,
EtOH, H2O
7
8%
O
O
O
O
7
The biphenylidene acetone (4), three bi-benzylmethyl ketones 7–9,
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 concentra-
tion (IC50). Only compounds containing O–O bonds were found
22%
17
18
H2O2 30%,
HBF4
13
TFE
O
O
O
O
O
38%
Scheme 5: Synthesis of tetraoxanes 17 and 18.
Chem Biol Drug Des 2012; 79: 790–797
795

Franco et al.
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
R1
O
O
R
R1
O
O
O
HOO OOH
HOO O O OOH
R
R
R
R R
R
R
R
R
A
B
C
D
Classes
Compound
Class
R
R1
P. falciparum, IC50 (lM)
Result
4
A
A
A
A
B
B
B
C
C
D
D
—
—
E-PhCH=CH-
PhCH2CH2-
p-CH3OPhCH2CH2-
3,4,5-(CH3O)3PhCH2CH2-
PhCH2CH2-
(CH3)3CCH(CH2CH2-)2
p-CH3OPhCH2CH2-
(CH3)3CCH(CH2CH2-)2
PhCH2CH2-
PhCH2CH2-
Ph(CH2)2-
—
—
—
—
—
—
—
—
—
32
66
42
I
I
I
I
7
8
9
1.1 · 102
1.4
13
11
14
12
16
17
18
CQ
QN
A
I
—
I
A
A
—
VA
VA
64
Hemolysis
31
3.3
1.6
PhCH2CH2-
-(CH2)5-
—
Hemolysis
0.25
—
—
—
0.012
VA, very active (IC50 < 1.0 lM), A, active (1.0 £ IC50 £ 5 lM); I, inactive (IC50 > 10 lM); CQ, chloroquine; QN, quinine (controls).
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 lM) were active (Table 3).
that ferric ion (toxic to parasites) metabolism is also affected in
the digestive vacuole by the natural 1,2,4-trioxane (peroxide) arte-
misinin and its derivatives (28). The relatively flat or planar sub-
structures and peroxy moieties in the synthesized compounds may
thus be acting in the digestive vacuole by stabilizing heme ⁄ inhibit-
ing 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).
While specific mechanistic studies were not performed to estab-
lish 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 7–9. 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 interac-
tion 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 com-
pounds 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 sub-
stance, which is toxic to the parasite, is polymerized in the diges-
tive 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
Conclusion
Synthesis of dihydroperoxides derived from hydrogenated dibenz-
alacetones was achieved using four different methods. The most
efficient method was the treatment of ketone with hydrogen perox-
ide 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 pro-
vided compounds 17 and 18 in 22% and 38% yield, respectively.
Dihydroperoxide 13, dihydroperoxy peroxide 16, and 1,2,4,5-tetra-
oxane 17 exhibit significant antimalarial activity against chloro-
quine-resistant human malaria parasite P. falciparum. These
compounds will be further evaluated for in vivo antimalarial and
against other parasite species.
Acknowledgements
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 Quꢀmica (RQ-MG) supported by FAPEMIG (Project: REDE-
796
Chem Biol Drug Des 2012; 79: 790–797

New Dihydroperoxides and Tetraoxanes as Antimalarials
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.
16. Lambros C., Vanderberg J.P. (1979) Syncronization of Plasmodium
falciparum erythrocytic stages in culture. J Parasitol;65:418–420.
17. Rieckmann K.H., Vampbell G.H., Sax L.J. (1978) Drug sensitivity
of P. falciparum an ``in vitro'' microtechnique. Lancet;7:22–23.
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Pinto M.C.F.R., Pinto A.V., Zalis M.G., Carvalho L.H., Krettli A.U.
(2004) Antimalarial activity of phenazines from lapachol, b-
lapachone and its derivatives against Plasmodium falciparum in
vitro and Plasmodium berghei in vivo. Bioorg Med Chem
Lett;14:1145–1149.
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