Antimalarial simplified 3-aryltrioxanes: Synthesis and preclinical efficacy/toxicity testing in rodents

Article abstract of DOI:10.1021/jm0102396

A streamlined five-step chemical synthesis of rationally designed, simplified 3-aryltrioxane 8a is described. A noteworthy feature of this synthetic scheme is use of air rather than expensive molecular oxygen as the source of the pharmacologically critica

Full text of DOI:10.1021/jm0102396

3054
J . Med. Chem. 2001, 44, 3054-3058
An tim a la r ia l Sim p lified 3-Ar yltr ioxa n es: Syn th esis a n d P r eclin ica l Effica cy/
Toxicity Testin g in Rod en ts
Gary H. Posner,*,‡ Heung Bae J eon,‡ Michael H. Parker,‡ Mikhail Krasavin,‡ Ik-Hyeon Paik,‡ and
Theresa A. Shapiro†
Department of Chemistry, School of Arts and Sciences, The J ohns Hopkins University, Baltimore, Maryland 21218;
Division of Clinical Pharmacology, Department of Medicine, School of Medicine, The J ohns Hopkins University,
Baltimore, Maryland 21205
Received May 30, 2001
A streamlined five-step chemical synthesis of rationally designed, simplified 3-aryltrioxane
8a is described. A noteworthy feature of this synthetic scheme is use of air rather than expensive
molecular oxygen as the source of the pharmacologically critical peroxide unit in trioxane 8a .
This simplified acetal trioxane carboxylic acid 8a is thermally stable, and it is hydrolytically
stable in water even at 40 °C and pH 7.4 for at least 7 days. Preclinical evaluation of this
water-soluble synthetic trioxane 8a in rodents shows it to have at least as good a therapeutic
index (efficacy/toxicity) as that of water-soluble semisynthetic trioxane artelinic acid (5).
In tr od u ction
On the basis of our detailed understanding at the
molecular level of the chemical cascade leading from
antimalarial trioxane to various cytotoxic chemical
intermediates,13,14 we have rationally designed and
easily synthesized a series of structurally simple 3-aryl-
trioxanes 6. Water-insoluble 3-p-fluorophenyl trioxane
7a , described by us previously,15 has now undergone
preclinical toxicity evaluation in rodents. It is safer than
the currently used antimalarial drug arteether (3).
Solubility in water is important for convenient intra-
venous (iv) administration of any antimalarial trioxane.
Water-soluble 3-p-carboxyphenyltrioxanes 8, described
here for the first time, are prepared in only five steps
from cyclohexanone, including practical use of air
(rather than pure O2)16 for IR lamp-induced photochemi-
cal introduction of the peroxide unit. Because pure 12R-
methoxy diastereoisomer 8a is more easily obtained
chromatographically pure than 12â-methoxy diastereo-
isomer 8b, we have selected the new chemical entity
8a for detailed study.
Over two billion people worldwide are currently at
risk of contracting malaria.1 Unfortunately, no vaccine
is now available for effective protection against malaria,
and chemotherapy of this infectious disease is becoming
more and more difficult due to the widespread resis-
tance of the Plasmodium falciparum malaria parasite
to standard quinoline-based antimalarial drugs such as
chloroquine and mefloquine.2 A promising nonalkaloid
class of new, potent, and fast-acting antimalarial tri-
oxanes, based on ancient Chinese folk herbal medicine,
is now being developed for clinical use; natural trioxane
artemisinin (1) and especially its semisynthetic deriva-
tives artemether (2), arteether (3), and artesunic acid
(4) are currently used drugs for treating individuals who
are infected with malaria.3-10 Semisynthetic water-
soluble trioxane artelinic acid (5), designed by the U.S.
Walter Reed Army Institute of Research to be more
stable than artesunic acid (4), is currently a leading
candidate for antimalarial drug development.11,12
Ch em istr y
Five-step preparation of synthetic trioxanes 8 is
outlined in Scheme 1. Highlights of this streamlined
synthetic route include the following: (1) use of a
styrene carbon-carbon double bond as a masked form
of a water-solubilizing carboxyl group; (2) use of readily
available and inexpensive air instead of expensive
cylinders of purified molecular oxygen for IR lamp-
induced formation of styryl trioxane 10; (3) optimization
of the photooxygenation step after trying diverse silyl
triflate and tertiary amines; and (4) final oxidative
cleavage of the vinyl group in styryl trioxane 10 to form
benzoic acid trioxanes 8 without disturbing the trioxane
peroxide linkage that is the crucial antimalarial phar-
macophore. Noteworthy also is formation of styryl
ketone 9 using the corresponding styryllithium orga-
nometallic reagent formed in situ via tert-butyllithium
reaction with commercial p-bromostyrene, without an-
ionic polymerization of the styrene system.17
* Author to whom correspondence should be addressed.
‡ Department of Chemistry.
† Division of Clinical Pharmacology.
10.1021/jm0102396 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/21/2001

Antimalarial Simplified 3-Aryltrioxanes
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3055
Ta ble 1. Antimalarial Efficacy in Mice Against P. berghei
ED50, mg/kga (µmol/kg)
ED90, mg/kga (µmol/kg)
compd
R
sc
iv
sc
iv
7a (12R-OMe)
8a (12R-OMe)
8b (12â-OMe)
arteether (3)
artelinic acid (5)
chloroquine
F
4.7 (15.3)
22.5 (67.4)
10.5 (31.4)
0.9 (2.9)
6.6 (15.8)
1.0 (2.0)
4.0 (13.0)
19.5 (58.4)
16.5 (49.4)
0.46 (1.5)
4.3 (10.3)
5.0 (10.1)
8.3 (27.0)
112 (335)
19.5 (58.4)
1.4 (4.5)
8.0 (26.0)
55.0 (165)
48.0 (144)
HOOC
HOOC
14.0 (33.6)
15.5 (37.2)
a
Four different doses (1, 3, 10, and 30 mg/kg) were administered each day for four days to five mice per dose regimen to establish the
ED values indicated here using a previously described protocol.18
Sch em e 1
hematocrit) and increase in reticulocyte count (Figure
1B and data not shown); and (iii) increase in liver
function tests (alanine aminotransferase, aspartate
aminotransferase, and total bilirubin; Figure 1C and
data not shown). At necropsy, there were neuronal
abnormalities in brain tissue. These changes indicate
toxicity to circulating red cells, liver cells, and brain
cells. In contrast, synthetic trioxane 7a at the same dose
caused a less marked reduction in red cell mass and
increase in reticulocytes, with no changes in weight
gain, liver function tests, or brain histopathology (Figure
1A-C and data not shown).
Water-soluble 3-carboxyphenyltrioxane 8a was evalu-
ated also in the same manner, in comparison with
water-soluble artelinic acid (5). In mice, synthetic tri-
oxane 8a is about 4-fold less efficacious against P.
berghei malaria parasites than semisynthetic artelinic
acid (5) via sc administration and about 5-fold less
efficacious via iv administration (Table 1).18 Synthetic
trioxane 8a is 3-5 times more soluble in water at pH
7.4 than is artelinic acid (5). Relative to buffer controls,
artelinic acid (5) at 26 µmol/kg/day (the limit of solubil-
ity) caused a time-dependent (i) reduction in red blood
cell mass (red cell count, hemoglobin, and hematocrit)
and increase in reticulocyte count (Figure 2B and data
not shown) and (ii) increase in alanine aminotransferase
(Figure 2C), indicative of red cell and liver toxicity.
However, water-soluble 8a , at a dose 6 times greater
than that of artelinic acid, caused no detectable toxicity.
Thus, the therapeutic index for water-soluble synthetic
trioxane 8a is at least as good as that of artelinic acid
(5).
To be a promising antimalarial drug candidate, any
new trioxane must have a long shelf life. The thermal
stability of synthetic acetal trioxane carboxylic acid 8a
is very good. For example, heating this crystalline
trioxane in air at 60 °C for 24 h caused less than 5%
decomposition, as judged by 1H NMR spectroscopy. Also,
heating a pH 7.4 phosphate-buffered 0.1 N NaCl solu-
tion of trioxane 8a in air at 40 °C for 7 days also caused
less than 5% decomposition, as determined by 1 H NMR
spectroscopy.
Biology
Water-insoluble 3-fluorophenyltrioxane 7a was evalu-
ated for preclinical efficacy and toxicity in rodents. In
mice, efficacy against P. berghei malaria parasites was
studied using both subcutaneous (sc) and intravenous
(iv) modes of administration.18 Results are summarized
in Table 1. Relative to water-insoluble semisynthetic
arteether (3), synthetic fluorophenyl trioxane 7a is
about 5-fold less efficacious via sc administration and
about 8-fold less efficacious via iv administration. In
rats, a 14-day iv toxicity study, however, showed
synthetic trioxane 7a to be significantly safer than
arteether (3). Relative to vehicle controls, arteether (3)
at 160 µmol/kg/day caused a time-dependent (i) reduc-
tion in expected weight gain (Figure 1A); (ii) reduction
in red blood cell mass (red cell count, hemoglobin, and
In conclusion, synthetic water-soluble trioxane 8a is
a promising antimalarial drug candidate. More direct
side-by-side preclinical testing of water-soluble trioxanes
5 and 8a in larger animals is now appropriate to
establish the generality of the physiological observations
reported here and to allow a reliable ranking of the

3056 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Posner et al.
F igu r e 1. Toxicity study of water-insoluble trioxanes. Com-
pounds were formulated in an emulsion and administered
intravenously to rats once daily for 14 days, at 160 µmol/kg/
day arteether (open squares); 160 µmol/kg/day 7a (open
triangles); or comparable volume of vehicle (open circles). Body
weight (A), hemoglobin (B), and alanine aminotransferase (C)
were measured on the indicated days of dosing. Values are
means from five animals. Double asterisk, value different from
vehicle control at p < 0.01.
F igu r e 2. Toxicity study of water-soluble trioxanes. Com-
pounds were dissolved in phosphate-buffered saline and
administered intravenously to rats once daily for 14 days, at
26 µmol/kg/day artelinic acid (closed square); 160 µmol/kg/day
8a (closed triangle); or comparable volume of buffer (closed
circles). Body weight (A), hemoglobin (B), and alanine amino-
transferase (C) were measured on the indicated days of dosing.
Values are means from five animals. Single asterisk, value
different from buffer control at p < 0.05; double asterisk, p <
0.01.
therapeutic potential of these two promising water-
soluble antimalarial drug candidates.
from Aldrich Chem. Co.) and dry THF (180 mL) and then
cooled to -78 °C. To this solution was added n-BuLi (1.6 M in
hexane, 43.4 mL, 69.4 mmol) over 10 min by syringe. The
resultant red ylide solution was then warmed to room tem-
perature, stirred for 3 h, and cooled to -78 °C. To this ylide
solution was added 2-cyanoethylcyclohexanone19 (7.0 g, 46.3
mmol) in dry THF (50 mL) via cannula over 10 min. After
Exp er im en ta l Section
P r ep a r a tion of 2-(2-Cya n oeth yl)-1-(m eth oxym eth yl-
en e)cycloh exa n e. An oven-dried 500 mL one-necked round-
bottomed flask was charged with (methoxymethyl)triphen-
ylphosphonium chloride (23.8 g, 69.4 mmol, used as received

Antimalarial Simplified 3-Aryltrioxanes
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3057
being stirred for 1 h at -78 °C, the reaction mixture was
warmed to room temperature slowly, stirred for 10 h at room
temperature, and then cooled to 0 °C, quenched with water
(50 mL), and diluted with ether (50 mL). The organic layer
was separated from aqueous layer and further extracted with
ether (70 mL × 2), combined, and washed with saturated NaCl
solution (60 mL). The combined organic layer was dried over
MgSO4, filtered, and concentrated. The residue was subjected
to column chromatography with EtOAc-hexane (1:12) as
eluent to give the product (6.7 g, 81%) as yellow oil, having
the same spectroscopic properties as those of an authentic
sample.19, 20
δ 7.52 (dt, J d ) 8.4 Hz, J t ) 2.0 Hz, 2H), 7.40 (dt, J d ) 8.4 Hz,
J t ) 2.0 Hz, 2H), 6.71 (dd, J ) 17.6, 10.8 Hz, 1H), 5.76 (dd, J
) 17.6, 0.8 Hz, 1H), 5.26 (dd, J ) 10.8, 0.8 Hz, 1H), 5.14 (d, J
) 1.2 Hz, 1H), 3.65 (s, 3H), 2.78 (ddd, J ) 14.4, 13.2, 3.6 Hz,
1H), 2.30 (ddd, J ) 14.4, 4.4, 3.2 Hz, 1H), 1.88-1.99 (m, 2H),
1.60-1.82 (m, 6H), 1.17-1.34 (m, 3H). 13C NMR (100 MHz,
CDCl3) δ 140.2, 137.9, 136.3, 126.0, 125.5, 114.5, 105.1, 105.0,
83.8, 57.2, 47.4, 39.1, 35.6, 30.8, 26.8, 25.0, 23.8. Due to the
tendency of styryl systems to polymerize, styryl trioxane 10
was used immediately in the next step.
P r ep a r a tion of p-Styr yl Tr ioxa n e 10 Mor e Con ve-
n ien tly a t -78 °C. A 100 mL three-necked round-bottom flask
equipped with dispersion bubbler gas inlet, gas outlet, and a
septum was charged with p-styryl ketone 9 (E/Z ) ∼1/1, 101
mg, 0.36 mmol) in dry CH2Cl2 (35 mL), and methylene blue
(1.3 mg) was added. To the solution maintained at -78 °C by
a dry ice in acetone bath was bubbled dry air (flow rate ) ∼240
mL/min) with a 250W IR lamp (General Electric) at 1 in.
distance from the reaction flask. TLC analysis after 20 min
showed no starting material, at which time the IR lamp was
removed. The air bubbler and outlet also were removed, and
the reaction was placed under Ar atmosphere. A precooled
(-78 °C) solution of Me3SiOTf (TMSOTf, 0.09 mL, 0.47 mmol)
in CH2Cl2 (5 mL) was added slowly via cannula during 2 min.
The reaction was stirred for 1 h at -78 °C, then quenched with
1-ethylpiperidine (0.15 mL, 1.1 mmol) by syringe. The reaction
mixture was allowed to warm to room temperature and then
concentrated. The residue was subjected to column chroma-
tography with EtOAc-hexane (1:15) as eluent to give 10b (13
mg, 11%) as colorless oil and then 10a (38 mg, 33%) subse-
quently as a sticky white solid. Due to the tendency of styryl
systems to polymerize, styryl trioxane 10 was used im-
mediately in the next step.
P r ep a r a tion of p-Styr yl Keton e 9. To a solution of
p-styryl bromide (1.1 mL, 8.0 mmol) in ether (30 mL) at -78
°C was added t-BuLi (5.4 mL, 1.7 M in pentane, 9.2 mmol)
via syringe over 1 min. The resulting dark-red solution was
stirred at -78 °C for 15 min. A precooled (-78 °C) solution of
the above nitrile (1.1 g, 6.1 mmol) in ether (25 mL) was then
added dropwise via cannula during 3 min. The resulting
mixture was stirred at -78 °C for 15 min, then the cooling
bath was removed, and the reaction mixture was allowed to
reach room temperature. At this point TLC analysis indicated
full consumption of the starting material. The reaction was
quenched with 10 mL of saturated aqueous NaHCO3, poured
into a separatory funnel containing 200 mL of ether and 50
mL of water. The organic layer was further washed with 50
mL of saturated aqueous NaHCO3, dried over MgSO4, and
concentrated. The residue was subjected to column chroma-
tography with EtOAc-hexane (1:15) as eluent to give the
1
ketone 9 (1.4 g, 81%) as pale yellow oil. H NMR (400 MHz,
CDCl3) δ 7.90 (m, 2H), 7.45 (m, 2H), 6.73 (dd, J ) 17.6, 5.4
Hz, 1H), 5.85 (dm, J ) 8.8 Hz, 1H), 5.78 (br s) and 5.72 (s) -
1H total, 5.37 (dt, J d ) 9.6 Hz, J t ) 0.8 Hz) and 5.36 (dt, J d
)
10.8 Hz, J t ) 0.8 Hz) - 1H total, 3.48 (m) and 3.39 (m) - 3H
total, 2.88 (m, 2H), 2.27 (dt, J d ) 14.0 Hz, J t ) 4.8 Hz, 1H),
2.00 (m, 3H), 1.43-1.83 (m, 6H), 1.16-1.42 (m, 1H). 13C NMR
(100 MHz, CDCl3) δ 200.2, 200.0, 141.7, 141.5, 140.4, 139.6,
136.4, 136.2, 135.9, 135.8, 128.3, 126.2, 126.1, 119.7, 119.0,
116.4, 116.2, 59.2, 59.0, 38.5. 36.8, 36.7, 33.5, 32.6, 31.6, 28.2,
27.2, 26.4, 26.2, 25.8, 23.0, 22.6, 21.6. IR (neat) 2925, 2852,
1682, 1604, 1448, 1404, 1233, 1123, 989, 915, 841 cm-1. Due
to the tendency of styryl systems to polymerize, styryl ketone
9 was used immediately in the next step.
P r ep a r a tion of p-Ca r boxyp h en yl Tr ioxa n e 8a . A solu-
tion of p-styryl trioxane 10a (215 mg, 0.68 mmol) and KMnO4
(430 mg) in acetone (40 mL) was stirred for 1 h (no starting
material by TLC) at room temperature, generating a precipi-
tate. The precipitate was filtered and washed with more
acetone, then it was dissolved in MeOH (50 mL). The filtrate
was rotary evaporated, and the residue was dissolved in water
(7 mL). The aqueous solution was acidified with aqueous 0.5
N HCl solution to pH 2, generating a white solid, which was
filtered and recrystallized from MeOH/H2O at room temper-
ature to give 122 mg (56%) of 8a . Mp 164.5-165.5 °C (white
solid). 1H NMR (400 MHz, CD3OD) δ 7.92 (d, J ) 8.4 Hz, 2H),
7.51 (d, J ) 8.4 Hz, 2H), 5.26 (s, 1H), 3.56 (s, 3H), 2.80 (ddd,
J ) 14.4, 12.8, 3.6 Hz, 1H), 2.30 (m, 1H), 2.18 (ddd, J ) 14.4,
4.4, 2.4 Hz, 1H), 1.86 (m, 1H), 1.58-1.76 (m, 5H), 1.24-1.37
(m, 3H), 1.11 (m, 1H). 13C NMR (100 MHz, CD3OD) δ 174.9,
144.4, 138.8, 130.5, 126.0, 105.4, 97.6, 85.1, 56.3, 47.0, 39.1,
34.7, 33.6, 28.4, 26.6, 24.1. IR (neat) 3382, 2927, 1595, 1556,
1409, 1100, 1043, 1012, 784 cm-1. Anal. calcd for C18H22O6: C
64.66, H 6.63, found: C 64.61, H 6.57.
P r ep a r a tion of p-Styr yl Tr ioxa n e 10 a t -90 °C. A 100
mL three-necked round-bottom flask equipped with dispersion
bubbler gas inlet, gas outlet, and a septum was charged with
p-styryl ketone 9 (E/Z ) ∼1/1, 80 mg, 0.28 mmol) in dry CH2-
Cl2 (30 mL), and methylene blue (1.1 mg) was added. To the
solution maintained at -90 °C by liquid N2 in MeOH was
bubbled dry air (flow rate ) ∼240 mL/min) with a 250W IR
lamp (General Electric) at 1 in. distance from the reaction
flask. During the reaction, the temperature was maintained
between -80 °C and -84 °C. TLC analysis after 15 min
showed no starting material, at which time the IR lamp was
removed. The air bubbler and outlet also were removed, and
the reaction was placed under Ar atmosphere. A precooled
(-78 °C) solution of Me3SiOTf (TMSOTf, 0.08 mL, 0.42 mmol)
in CH2Cl2 (5 mL) was added slowly via cannula during 2 min.
The reaction was stirred for 1 h at -90 °C, then quenched with
1-ethylpiperidine (0.12 mL, 0.84 mmol) by syringe. The reac-
tion mixture was allowed to warm to room temperature and
then concentrated. The residue was subjected to column
chromatography with EtOAc-hexane (1:15) as eluent to give
10b (13 mg, 15%) as colorless oil and then 10a (32 mg, 36%)
subsequently as a sticky white solid. Compound 10a : 1H NMR
(400 MHz, CDCl3) δ 7.51 (dt, J d ) 8.4 Hz, J t ) 2.0 Hz, 2H),
7.39 (dt, J d ) 8.4 Hz, J t ) 2.0 Hz, 2H), 6.70 (dd, J ) 17.6, 10.8
Hz, 1H), 5.76 (dd, J ) 17.6, 0.8 Hz, 1H), 5.26 (dd, J ) 10.8,
0.8 Hz, 1H), 5.18 (s, 1H), 3.61 (s, 3H), 2.83 (ddd, J ) 14.8,
13.6, 4.0 Hz, 1H), 2.42 (m, 1H), 2.27 (ddd, J ) 14.8, 4.8, 3.2
Hz, 1H), 1.89 (m, 1H), 1.70-1.83 (m, 4H), 1.63 (m, 1H), 1.15-
1.32 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 139.9, 137.9, 136.3,
126.0, 125.6, 114.5, 103.9, 96.1, 83.6, 55.9, 45.4, 37.5, 33.4, 32.5,
27.1, 25.2, 23.1. Compound 10b: 1H NMR (400 MHz, CDCl3)
P r ep a r a tion of p-Ca r boxyp h en yl Tr ioxa n e 8b. A solu-
tion of p-styryl trioxane 10b (20 mg, 0.063 mmol) and KMnO4
(40 mg) in acetone (4 mL) was stirred for 0.5 h (no starting
material by TLC) at room temperature, generating a precipi-
tate. The precipitate was filtered and washed with more
acetone, then it was dissolved in MeOH (4 mL). The filtrate
was rotary evaporated, and the residue was dissolved in water
(2 mL). The aqueous solution was acidified with aqueous 0.5
N HCl solution to pH 2, which was concentrated. The residue
was recrystallized from MeOH/EtOAc at room temperature to
1
give 9 mg (45%) of 8b. Mp 151-153 °C (white solid). H NMR
(400 MHz, CD3OD) δ 7.96 (d, J ) 8.0 Hz, 2H), 7.52 (d, J ) 8.0
Hz, 2H), 5.13 (s, 1H), 3.65 (s, 3H), 2.78 (dt, J d ) 3.6 Hz, J t
)
14.0 Hz, 1H), 2.24 (dt, J d ) 14.4 Hz, J t ) 3.8 Hz, 1H), 1.85-
2.00 (m, 2H), 1.55-1.79 (m, 7H), 1.14-1.28 (m, 2H). 13C NMR
(100 MHz, CD3OD) δ 175.8, 139.7, 132.1, 130.5, 126.0, 106.6,
106.4, 84.9, 57.6, 40.4, 36.8, 32.1, 29.9, 28.1, 26.4, 25.1. IR
(neat) 3401, 2931, 2858, 1598, 1559, 1413, 1276, 1206, 1137,
1104, 1014, 786 cm-1. Anal. calcd for C18H22O6: C 64.66, H
6.63, found: C 64.71, H 6.55.

3058 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Posner et al.
(7) Haynes, R. K.; Vonwiller, S. C. From Qinghao, Marvelous Herb
of Antiquity, to the Antimalarial Trioxane Qinghaosu-and Some
Remarkable New Chemistry. Acc. Chem. Res. 1997, 30, 73-79.
(8) Meshnick, S. R.; Taylor, T. E.; Kamchonwongpaisan, S. Arte-
misinin and the Antimalarial Endoperoxides: From Herbal
Remedy to Targeted Chemotherapy. Microbiol. Rev. 1996, 60,
301-315.
(9) Thuma, P. P.; Bhat, G. J .; Mabeza, G. F.; Osborne, C.; Biemba,
G.; Shakankale, G. M.; Peeters, P. A. M.; Oosterhuis, B.; Lugt,
C. B.; Gorduek, V. R. A Randomized Controlled Trial of Artemotil
(â-Arteether) in Zambian Children with Cerebral Malaria. Am.
J . Trop. Med. Hyg. 2000, 62, 524-529.
(10) Van Vugt, M.; Angus, B. J .; Price, R. N.; Mann, C.; Simpson, J .
A.; Poletto, C.; Htoo, S. E.; Looareesuwan, S.; White, N. J .;
Nosten, F. A Case-Control Auditory Evaluation of Patients
Treated with Aretmisinin Derivatives for Multidrug-Resistant
Plasmodium falciparum Malaria. Am. J . Trop. Med. Hyg. 2000,
62, 65-69.
(11) Lin, A. J .; Klayman, D. L.; Milhous, W. K. Antimalarial Activity
of New Water-Soluble Dihydroartemisinin Derivatives. J . Med.
Chem. 1987, 30, 2147-2150.
(12) Li, Q.-G.; Peggins, J . O.; Lin, A. J .; Masonic, K. J .; Troutman,
K. M.; Brewer, T. G. Pharmacology and Toxicology of Artelinic
Acid: Preclinical Investigations on Pharmacokinetics, Metabo-
lism, Protein and Red Blood Cell Binding, and Acute and
Anorectic Toxicities. Trans. Royal Soc. Trop. Med. Hyg. 1998,
92, 332-340.
(13) Posner, G. H.; Cumming, J . N.; Krasavin, M. Carbon-Centered
Radicals and Rational Design of New Antimalarial Peroxide
Drugs. In Biomedical Chemistry: Applying Chemical Principles
to the Understanding and Treatment of Disease; Torrence, P. F.,
Eds.; Wiley-Interscience: 2000; 289-309.
Toxicity Testin g. Compound 7a and artelinic acid were
dissolved in phosphate-buffered saline (Life Technologies);
compound 8a and arteether were formulated in a vegetable
oil/aqueous emulsion. Groups of five male Sprague-Dawley
rats (about 165 g at initiation of study) were dosed intrave-
nously by tail vein once daily for 14 consecutive days with 26
µmol/kg/day artelinic acid (limit of solubility); 160 µmol/kg/
day arteether, or 40, 80, or 160 µmol/kg/day 7a or 8a . Doses
were administered at 10 mL/kg (7a , 8a , arteether, controls)
or 20 mL/kg (artelinic acid). Each dose was administered to 5
rats. Animals were evaluated at least once daily during dosing
for mortality or signs of morbidity. Body weights were obtained
before dosing, on days 1, 4, 8, and 12 of dosing, and on day 15
after the start of dosing. Blood was obtained from the retro-
orbital sinus on day 8 and at scheduled necropsy on day 15,
and was analyzed for red cell count, hematocrit, hemoglobin,
mean corpuscular volume, mean corpuscular hemoglobin,
mean corpuscular hemoglobin concentration, white cell count
and differential, platelet count, reticulocyte count, alanine
aminotransferase, alkaline phosphatase, aspartate aminotrans-
ferase, urea nitrogen, creatine phosphokinase, creatinine,
glucose, total protein, albumin, total bilirubin, calcium, chlo-
ride, cholesterol, globulin, phosphorus, potassium, and sodium.
On day 15, animals were sacrificed by sodium pentothal
overdose and perfused transcardially with 10% formalin.
Brain, kidneys, liver, and eyes were harvested, weighed, and
fixed for histopathologic examination.
(14) Cumming, J . N.; Ploypradith, P.; Posner, G. H. Antimalarial
Activity of Artemisinin (Qinghaosu) and Related Trioxanes:
Mechanism of Action. Adv. Pharmacol. 1997, 37, 253-297.
(15) Posner, G. H.; Cumming, J . N.; Woo, S.-H.; Ploypradith, P.; Xie,
S.; Shapiro, T. A. Orally Active Antimalarial 3-Substituted
Trioxanes: New Synthetic Methodology and Biological Evalu-
ation. J . Med. Chem. 1998, 41, 940-951.
(16) Trabanco, A. A.; Montalban, A. G.; Rumbles, G.; Barrett, A. G.
M.; Hoffman, B. M. A seco-Porhyrazine: Superb Sensitizer for
Singlet Oxygen Generation and Endoperoxide Synthesis. Synlett
2000, 7, 1010-1012.
(17) Seymour, R. B. Polymer Chemistry; Marcel Dekker: New York,
1981.
(18) Peters, W.; Robinson, B. L.; Rossiter, J . C.; J efford, C. W. The
Chemotherapy of Rodent Malaria. XLVIII. The Activities of
Some Synthetic 1,2,4-Trioxanes Against Chloroquine-Sensitive
and Chloroquine-Resistant Parasites. Part 1: Studies Leading
to the Development of Novel cis-Fused Cyclopenteno Derivatives.
Ann. Trop. Med. Parasitol. 1993, 87, 1-7.
(19) Posner, G. H.; Oh. C. H.; Gerena, L.; Milhous, W. K. Synthesis
and Antimalarial Activities of Structurally Simplified 1,2,4-
Trioxanes Related to Artemisinin. Heteroat. Chem. 1995, 6, 105-
116.
(20) J efford, C. W.; Velarde, J . A.; Bernardinelli, G.; Bray, D. H.;
Warhurst, D. C.; Milhous, W. K. 198. Synthesis, Structure, and
Antimalarial Activity of Tricyclic 1,2,4-Trioxanes Related to
Artemisinin. Helv. Chim. Acta 1993, 76, 2775-2788.
Ack n ow led gm en t. We thank the NIH (A1-34885)
and the Burroughs Wellcome Fund for generous finan-
cial support, Professor Wallace Peters and Mr. Brian
Robinson for the efficacy testing, the NIH NIAID
contract services for toxicity testing, and Dr. Paul
O’Neill and Mr. J effrey Elias for pioneering our photo-
oxygenations at J ohns Hopkins using air rather than
molecular oxygen.
Refer en ces
(1) Rogers, D. J .; Randolph, S. E. The Global Spread of Malaria in
a Future, Warmer World. Science 2000, 289, 1763-1766.
(2) Foley, M.; Tilley, L. Quinoline Antimalarials: Mechanisms of
Action and Resistance. Int. J . Parasitol. 1997, 27, 231-240.
(3) Klayman, D. L. Qinghaosu (Artemisinin): An Antimalarial Drug
from China. Science 1985, 228, 1049-1055.
(4) Avery, M. A.; Alvim-Gaston, M.; Woolfrey, J . R. Synthesis and
Structure Activity Relationships of Peroxidic Antimalarials
Based on Artemisinin. Adv. Med. Chem. 1999, 4, 125-217.
(5) Posner, G. H. Antimalarial Peroxides in the Qinghaosu (Arte-
misinin) and Yingzhaosu Families. Exp. Opin. Ther. Patents
1998, 8, 1487-1494.
(6) Ziffer, H.; Highet, R. J .; Klayman, D. L. Artemisinin: An
Endoperoxide Antimalarial from Artemisia annua L. Prog.
Chem. Org. Nat. Prod. 1997, 72, 121-214.
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