Orally active, antimalarial, anticancer, artemisinin-derived trioxane dimers with high stability and efficacy

Article abstract of DOI:10.1021/jm020461q

In only two steps and in 70% overall yield, naturally occurring trioxane artemisinin (1) was converted on a gram scale into C-10-carba trioxane dimer 3. This new, very stable dimer was then transformed easily in one additional step into four different dimers 4-7. Alcohol and diol dimers 4 and 5 and ketone dimer 7 are 10 times more antimalarially potent in vitro than artemisinin (1), and alcohol and diol dimers 4 and 5 are strongly growth inhibitory but not cytotoxic toward several human cancer cell lines. Water-soluble carboxylic acid derivatives 8a and 9 were easily prepared in one additional step from dimers 4 and 5. Carboxylic acid dimers 8a and 9 are thermally stable even at 60 °C for 24 h, are more orally efficacious as antimalarials in rodents than either artelinic acid or sodium artesunate, and are strongly inhibitory but not cytotoxic toward several human cancer cell lines.

Full text of DOI:10.1021/jm020461q

1060
J . Med. Chem. 2003, 46, 1060-1065
Or a lly Active, An tim a la r ia l, An tica n cer , Ar tem isin in -Der ived Tr ioxa n e Dim er s
w ith High Sta bility a n d Effica cy
Gary H. Posner,*,†,§ Ik-Hyeon Paik,†,§ Surojit Sur,†,§ Andrew J . McRiner,†,§ Kristina Borstnik,†,§ Suji Xie,‡,§ and
Theresa A. Shapiro‡,§
Department of Chemistry, School of Arts and Sciences, The J ohns Hopkins University, 3400 North Charles Street,
Baltimore, Maryland 21218-2685, Division of Clinical Pharmacology, Department of Medicine, School of Medicine,
The J ohns Hopkins University, Baltimore, Maryland 21205, and The J ohns Hopkins Malaria Research Institute,
Bloomberg School of Public Health, Baltimore, Maryland 21205
Received October 17, 2002
In only two steps and in 70% overall yield, naturally occurring trioxane artemisinin (1) was
converted on a gram scale into C-10-carba trioxane dimer 3. This new, very stable dimer was
then transformed easily in one additional step into four different dimers 4-7. Alcohol and diol
dimers 4 and 5 and ketone dimer 7 are 10 times more antimalarially potent in vitro than
artemisinin (1), and alcohol and diol dimers 4 and 5 are strongly growth inhibitory but not
cytotoxic toward several human cancer cell lines. Water-soluble carboxylic acid derivatives 8a
and 9 were easily prepared in one additional step from dimers 4 and 5. Carboxylic acid dimers
8a and 9 are thermally stable even at 60 °C for 24 h, are more orally efficacious as antimalarials
in rodents than either artelinic acid or sodium artesunate, and are strongly inhibitory but not
cytotoxic toward several human cancer cell lines.
Sch em e 1
In tr od u ction
Several 1,2,4-trioxane dimers have high in vitro anti-
malarial, antiproliferative, and antitumor activities,1-5
and one has high in vivo anticancer activity.6 When such
dimers are semisynthesized from the natural Chinese
antimalarial trioxane artemisinin (1), C-10 acetal de-
rivatives are often unstable (i.e., easily hydrolyzed) in
water.4,7 Therefore, making hydrolytically stable C-10
non-acetal carba derivatives has become a high priority
internationally.7-16 Important also for development of
practical new antimalarial drugs is keeping their syn-
thesis short and their cost low.
Resu lts
Pursuing our interest in trioxane dimers,4-6 we now
describe high-yield conversion of easily prepared C-10
acetate 2 in one step directly into C-10 non-acetal
trioxane dimer 3 (Scheme 1). Inspiration for this bis-
allylation of C-10 acetate 2 was based on the pioneering
trioxane monoallylations of Ziffer9 and O’Neill17,18 using
allylsilane and based also on allylic bis-silane chemis-
try.19 The requisite allylic bis-silane was easily prepared
in one step from the corresponding commercial allylic
dichloride (Scheme 1); in the presence of tin tetrachlo-
ride, the allylic bis-silane converted acetate 2 on a gram
unsaturated three-carbon atom linker between the two
trioxane units, is stable in air and light at room
temperature for at least 6 months, and its preparation
on a much larger industrial scale should be feasible.
In contrast to most simple peroxides that are easily
cleaved by reducing agents and by reactive organome-
tallics,22 the peroxide linkage in artemisinin-like triox-
anes is relatively inert.23 Therefore, we have been able
to perform several different chemical transformations
chemospecifically involving only the linker isobutylene
carbon-carbon double bond in dimer 3 (Scheme 2).
Borane reduction and in situ oxidation produced bis-
trioxane primary alcohol 4. Dihydroxylation using cata-
lytic osmium tetroxide gave bis-trioxane vicinal diol 5.
Dimethyldioxirane formed bis-trioxane epoxide 6. Oxi-
1
scale into dimer 3, characterized by H NMR spectros-
copy as done before in structurally related trioxanes.20,21
This double-substitution reaction undoubtedly pro-
ceeded sequentially via initial monoallylation, producing
an intermediate C-10 trioxane allylic silane that then
reacted with another molecule of trioxane acetate 2 to
form the product dimer 3. This new dimer 3, with an
* To whom correspondence should be addressed. Phone: 410-516-
4670. Fax: 410-516-8420. E-mail: ghp@jhu.edu.
† School of Arts and Sciences.
§ Bloomberg School of Public Health.
‡ School of Medicine.
10.1021/jm020461q CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/15/2003

Trioxane Dimers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1061
Sch em e 2
Ta ble 1. Antimalarial Activities in Vitroa
dative cleavage using catalytic osmium tetroxide and
oxone led to bis-trioxane ketone 7. Bis-trioxanes 4-7
are stable even when heated neat in air for 24 h at 60
°C; 1H NMR spectrometry showed less than 5% decom-
position under these accelerated aging conditions.
To illustrate further the chemical stability of the
peroxide unit in these trioxane dimers and especially
to generate some water-soluble dimers that can be easily
administered in vivo, we have converted each of the
dimers 4-7 into a carboxylic acid (Scheme 2). Primary
alcohol 4 opened succinic anhydride to form bis-artesu-
nate 8a in high yield. Esterification of primary alcohol
4 gave isonicotinate ester 8b . Diol 5 opened succinic
anhydride to produce the tertiary alcohol primary
succinate ester 9. Epoxide 6 reacted chemospecifically
with a substituted benzenethiol in the presence of
chromatographic alumina24 to give â-hydroxysulfide 10a
in high yield; sulfide to sulfone oxidation gave dimer
benzoate ester 10b that was saponified into benzoic acid
10c. Finally, ketone 7 underwent chemospecific addition
of styryllithium to afford styryl tertiary alcohol 11a ;
oxidative cleavage of the styrene double bond produced
benzoic acid 11b. Each of these new carboxylic acid
dimers is at least as soluble in water buffered at pH
7.4 as is the antimalarial drug candidate artelinic acid,25
and tertiary alcohol primary succinate ester 9 is close
trioxane
IC50 (nM)
trioxane
8b
IC50 (nM)
3
24
1.7
3.0
2.4
2.1
4
5
6
0.87
0.59
2.8
9
10c
11b
7
8a
0.91
2.0
artemisinin
9.0 ( 1.9
a
The standard deviation for each set of quadruplicates was an
average of 9% (e22%) of the mean. R2 values for the fitted curves
were g0.987. Artemisinin activity is the mean ( standard devia-
tion of the concurrent control (n ) 10).
to 30 times more water-soluble than artelinic acid.
Trioxane dimer carboxylic acids 8a , 9, and 10c are
stable in air even at 60 °C for 24 h; 1H NMR spectrom-
etry indicated <5% decomposition under these condi-
tions.
By use of our standard assay,26 the antimalarial
potencies of these dimers in vitro against chloroquine-
sensitive Plasmodium falciparum (NF 54) parasites
were determined to be as shown in Table 1. In sharp
contrast to the potencies of the natural trioxane arte-
misinin (IC50 ) 9.0 nM) and of the initial olefinic dimer
3 (IC50 ) 24 nM), alcohol and diol dimers 4 and 5 and
ketone dimer 7 all have substantially enhanced poten-
cies, with IC50 values below 1 nM. Also, water-soluble

1062 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Posner et al.
Ta ble 2. Antimalarial Efficacies in Vivoa
(1)] dimer carboxylic acids 8a and 9, therefore, deserve
further preclinical evaluation as potential drug candi-
dates for chemotherapy of malaria and cancer.29-32
% parasite
trioxane
administration
route
suppression
ED50 ED90 at 10 mg/kg
b
b
carboxylic acid
Exp er im en ta l Section
8a
iv
po
iv
2.2
9.0
11.0
10.0
6.3
15.0
25.0
39.0
81.0
79.2
54.7
52.8
Syn th esis of r-Dih yd r oa r tem isin in Aceta te (2).33 A 250
mL round-bottomed flask was charged with artemisinin (1)
(1.49 g, 5.29 mmol, 1.0 equiv) and anhydrous dichloromethane
(45.0 mL), and this solution was cooled to -78 °C. DIBAL, 1.0
M, in toluene (6.5 mL, 6.5 mmol, 1.2 equiv) was added
dropwise at -78 °C. Upon the complete consumption of
starting material (about an hour, checked by TLC), pyridine
(1.50 mL, 18.5 mmol, 3.5 equiv) and 4-(N,N-dimethylamino)-
pyridine (780 mg, 6.36 mmol, 1.2 equiv) were added. Then
acetic anhydride (2.00 mL, 21.2 mmol, 4.0 equiv) was added
at -78 °C. The reaction mixture was stirred vigorously at -78
°C for 3 h and slowly warmed to room temperature and stirred
overnight. The reaction was quenched with 20 mL of saturated
NH4Cl. Then the organic layer was separated and washed with
brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The crude mixture was purified by flash
column chromatography (14% EtOAc/hexanes) to afford the
desired product 2 (1.60 g, 4.90 mmol, 92%) as a white solid:
artelinic acid
sodium artesunate
po
9
iv
po
iv
po
iv
po
2.4
4.8
2.7
7.5
5.6
5.5
11.0
34.0
10.0
35.0
43.0
70.0
83.4
55.4
83.4
46.1
42.4
50.9
10c
artelinic acid
sodium artesunate
Reference 27. mg kg-1 day-1 × 4 days.
a
b
carboxylic acid dimers 8a , 9, 10c, and 11b all are several
times more potent than artemisinin (1).
The in vivo antimalarial efficacies of water-soluble
dimer carboxylic acids 8a , 9, and 10c (11b not tested),
as measured in mice according to a published protocol,27
are shown in Table 2 as two separate groups of experi-
ments. In all cases, these water-soluble dimeric triox-
anes are more efficacious than the drug candidate
artelinic acid administered intravenously (iv) and more
efficacious than the clinically used drug sodium arte-
sunate administered orally (po). At a dose of 10 mg/kg
of mouse body weight, each of the dimeric trioxanes 8a ,
9, and 10c suppressed P. berghei NY malaria parasite
growth considerably better (>80%) than did artelinic
acid or sodium artesunate. Neither overt toxicity nor
behavioral modification was observed in the mice due
to drug administration in any of these experiments.
Preliminary growth inhibitory activities at nanomolar
to micromolar concentrations, measured in vitro as
described previously using a diverse panel of human
cancer cell lines in the National Cancer Institute’s
(NCI’s) Developmental and Therapeutic Program,28
indicate that diol dimer 5 is selectively and strongly
growth inhibitory but not lethal (very low LC50 values)
to the renal cancer cell line ACHN. The epoxide dimer
6 and the ketone dimer 7 are less inhibitory. Without
being cytotoxic (very low LC50 values), water-soluble
dimer 9 is especially selective and potent at inhibiting
growth of only colon cancer KM 12 cells, central nervous
system SF-295 cells, and ovarian cancer OVCAR-3 cells.
Water-soluble dimers 8a and 10c are also not cytotoxic
but are somewhat less selective than dimer 9 in cancer
cell growth inhibition.
In conclusion, new C-10 non-acetal trioxane dimer 3,
easily prepared on a gram scale and thermally stable,
can be used to make a diverse series of three-carbon-
atom-linked oxygenated dimers 4-7 without destroying
the critical pharmacophore peroxide bond. Each of the
new trioxane dimers 4, 5, and 7 is 10 times more
antimalarially potent in vitro than the natural trioxane
artemisinin (1), and alcohol and diol dimers 4 and 5 are
strongly growth inhibitory but not cytotoxic toward
several human cancer cell lines. Moreover, water-soluble
trioxane dimers 8a , 9, and 10c are orally active new
antimalarials that are more efficacious than artelinic
acid, are more efficacious than sodium artesunate in
mice, and also are selective and potent inhibitors of
cancer cell growth without being cytotoxic. These semi-
synthetic new chemical entities 4 and 5 and especially
easily synthesized [four steps from natural artemisinin
1
mp ) 129-132 °C (ref 34, 128-129 °C); H NMR (400 MHz,
CDCl3) δ 5.78 (d, J ) 9.6 Hz, 1H), 5.44 (s, 1H), 2.60-2.51 (m,
1H), 2.41-2.33 (m, 1H), 2.12 (s, 3H), 1.43 (s, 3H), 0.96 (d, J )
6.0 Hz, 3H), 0.84 (d, J ) 7.2 Hz, 3H).
Syn th esis of Tr ioxa n e Isobu tylen e Dim er 3. A solution
of dihydroartemisinin acetate 2 (2.18 g, 6.68 mmol) and the
allylic bis-silane linker (1.14 g, 5.70 mmol) in dry dichloro-
methane (120 mL) was cooled to -78 °C. Tin(IV) chloride
(6.7 mL, 6.7 mmol, 1.0 M solution in dichloromethane) was
further diluted with dry dichloromethane (7 mL) and was
added to the reaction mixture by cannula in a dropwise
manner. The reaction mixture was stirred at -78 °C for 1 h,
at which time TLC confirmed the complete consumption of
starting material. Water (10 mL) was added, and the reaction
mixture was allowed to warm to room temperature. Additional
water (50 mL) was added, and the organics were extracted
with dichloromethane (3 × 50 mL), dried (MgSO4), and
concentrated in vacuo to give a solid. Gradient column chro-
matography on silica, eluting with 5-10% ethyl acetate in
hexanes isolated the trioxane isobutylene dimer 3 as a white
1
solid (1.52 g, 2.58 mmol, 76%): mp ) 132-133 °C; H NMR
(400 MHz, CDCl3) δ 5.41 (s, 2H), 4.87 (s, 2H), 4.30 (ddd J )
10.4, 5.6, 4.0 Hz, 2H), 2.75-2.67 (m, 2H), 2.61-2.54 (m, 2H),
2.37-2.23 (m, 4H), 2.04-1.98 (m, 2H), 1.91-1.84 (m, 2H),
1.81-1.75 (m, 2H), 1.67-1.56 (m, 4H), 1.52-1.32 (m, 12H,
including singlet at 1.39), 1.23-1.20 (m, 2H), 0.94 (d, J ) 6.0
Hz, 6H), 0.90 (d, J ) 7.6 Hz, 6H), 0.98-0.86 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 147.4, 113.1, 103.1, 88.6, 81.2, 75.5, 52.4,
44.5, 37.1, 36.7, 35.2, 34.5, 30.6, 26.2, 24.7, 24.6, 20.2, 13.3;
IR(CHCl3) 3060, 2938, 2875, 1641, 1451, 1375, 1121, 1092,
1054, 1007, 878, 732 cm-1; HRMS (ES) m/z calcd for C34H52O8-
Na (M + Na) 611.3560, found 611.3555. Anal. (C34H52O8) C, H.
Syn th esis of Bis-tr ioxa n e P r im a r y Alcoh ol 4. A solution
of trioxane isobutylene dimer 3 (0.89 g, 1.5 mmol) in dry
tetrahydrofuran (25 mL) was cooled to 0 °C. Borane/dimethyl
sulfide complex (2.0 M solution in diethyl ether, 0.90 mL, 1.80
mmol) was carefully added, and the reaction mixture was
warmed to room temperature and stirred for 3 h. At this time
TLC analysis confirmed that no starting material remained.
A suspension of NaBO3‚4H2O (1.17 g, 7.60 mmol) in water (12
mL) was slowly added, and the resulting suspension was
stirred for 17 h. Water (10 mL) and dichloromethane (50 mL)
were added, and organics were extracted with dichloromethane
(3 × 20 mL), dried (Na2SO4), and concentrated in vacuo to give
a white solid. Gradient column chromatography on silica,
eluting with 20%, 30%, and finally 40% ethyl acetate/
petroleum ether isolated bis-trioxane primary alcohol 4 as a
white solid (0.85 g, 1.40 mmol, 93%): mp ) 81-82 °C; 1H NMR
(CDCl3, 400 MHz) δ 5.35 (s, 1H), 5.34 (s, 1H), 4.47-4.40 (m,
1H), 4.33 (qt, 1H, J ) 6.0), 3.83-3.75 (m, 1H), 3.67-3.60 (m,
1H), 3.17 (dd, 1H, J ) 7.6, J ) 6.1), 2.64 (qt, 1H, J ) 6.9),

Trioxane Dimers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1063
2.59 (qt, 1H, J ) 6.9), 2.32 (t, br, 2H, J ) 14.1), 2.06-1.97 (m,
3H), 1.95-1.87 (m, 2H), 1.86-1.74 (m, 2H), 1.70-1.55 (m, 8H),
1.50-1.30 (m, 14H, including singlet at 1.40), 0.99-0.90 (m,
2H), 0.95 (d, 6H, J ) 5.8), 0.87 (apparent t, 6H, J ) 6.9); 13C
NMR (CDCl3, 100 MHz) δ 103.12, 102.97, 89.47, 89.22, 81.11
(2), 73.86, 71.27, 65.12, 52.20, 52.07, 44.18, 44.01, 37.68, 37.46,
37.43, 36.52, 36.51, 34.40, 34.37, 31.26, 30.74 (2), 30.65, 25.95,
25.89, 24.83 (2), 24.73, 24.70, 20.15, 20.10, 12.89, 12.61; IR
(CHCl3) 3490 cm-1; HRMS (EI, m/z) calcd for C34H54O9Na (M
+ Na) 629.3660, found 629.3697
mixture was then diluted with water and extracted with
dichloromethane. Flash column chromatography (25% EtOAc/
hexanes) yielded the desired ketone 7 (21 mg, 0.035 mmol,
70%) as a white solid: mp ) 120-121 °C; 1H NMR (400 MHz,
CDCl3) δ 5.34 (s, 2H), 4.75 (m, 2H), 2.90 (m, 2H), 2.75 (m, 2H),
2.59 (dd, 2H, J ) 15.6 Hz, J ) 4 Hz), 2.33 (dt, 2H, J ) 15.6
Hz, J ) 4 Hz), 2.04-1.96 (m, 2H), 1.94 (m, 2H), 1.79 (m, 2H),
1.68-1.57 (m, 6H), 1.48-1.22 (m, 14H, including singlet at
1.35), 0.99 (d, 6H, J ) 6.4 Hz), 0.88 (d, 6H, J ) 7.2 Hz); 13C
NMR (100 MHz, CDCl3) δ 207.8, 103.1, 88.9, 80.8, 71.2, 52.1,
44.1, 43.9, 37.3, 36.4, 34.3, 29.9, 25.9, 24.6, 24.4, 20.0, 13.0;
IR(CHCl3) 1722 cm-1; HRMS (ES) m/z calcd for C33H50O9Na
(M + Na) 613.3347, found 613.3303.
Syn th esis of Bis-tr ioxa n e Su ccin a te Mon oester 8a . To
a solution of the bis-trioxane primary alcohol 4 (50 mg, 0.082
mmol) and succinic anhydride (24 mg, 0.24 mmol) in dichlo-
romethane (10 mL) was added 4-(N,N-dimethylamino)pyridine
(10 mg, 0.082 mmol) at 0 °C. The reaction mixture was allowed
to reach room temperature and stirred for 12 h, after which
TLC showed complete consumption of the starting materials.
The mixture was then washed with brine and extracted with
EtOAc (3 × 30 mL), and the crude mixture was purified by
flash column chromatography (50% EtOAc/hexanes) to furnish
Syn th esis of Bis-tr ioxa n e Vicin a l Diol 5. A 10 mL round-
bottomed flask was charged with isobutylene dimer 3 (21.0
mg, 0.036 mmol, 1.0 equiv) and 4-methylmorpholine N-oxide
(5.0 mg, 0.043 mmol, 1.2 equiv). The mixture was dissolved
in acetone (2.0 mL). To this solution was added osmium
tetroxide (0.016 mL, 25 mg/2 mL of aqueous solution, 0.02
equiv). The reaction mixture was stirred vigorously at room
temperature for 24 h. The reaction mixture was quenched with
saturated aqueous NaHSO3 solution (2.0 mL) and stirred for
an additional 30 min. Then the color turned to pale-orange.
The reaction mixture was poured into a mixture of 20 mL of
diethyl ether and 20 mL of saturated aqueous NH4Cl solution.
The aqueous layer was extracted with ethyl acetate (30 mL ×
2). Then organic layer was combined and washed with brine,
dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude mixture was purified by flash column
chromatography (50% EtOAc/hexanes) to afford the desired
product 5 as a white solid (20.3 mg, 0.033 mmol, 92%): mp )
the desired product 8a as a white solid (40 mg, 0.068 mmol,
1
84%): mp ) 77-80 °C; [R]23.9 46.7 (CHCl3, c 0.10); H NMR
D
(400 MHz, CDCl3) δ 5.39 (s, 1H), 5.31 (s, 1H), 4.36 (m, 2H),
4.22 (m, 3H), 2.74-2.54 (m, 6H), 2.32 (m, 2H), 2.17 (m, 1H),
2.01 (m, 2H), 1.90 (m, 2H), 1.84-1.60 (m, 11H), 1.48-1.20 (m,
16H, including two singlets at 1.40 and 1.39), 1.00-0.80 (m,
12H); 13C NMR (100 MHz, CDCl3) δ 175.4, 172.0, 102.9, 103.6,
89.4, 88.5, 81.1, 74.1, 71.1, 67.1, 52.4, 52.1, 44.6, 44.1, 37.4,
37.3, 36.6, 36.5, 34.4, 34.3, 30.5, 30.4, 30.0, 29.7, 29.4, 29.0,
26.0, 25.9, 24.7, 20.3, 20.1, 13.3, 12.7; HRMS (ES) m/z calcd
for C38H58O12Na (M + Na) 729.3820, found 729.3795. Anal.
(C38H60O13) C, H.
1
159-160 °C; H NMR (400 MHz, CDCl3) δ 5.36 (s, 1H), 5.35
(s, 1H), 4.74-4.70 (m, 1H), 4.56-4.51 (m, 1H), 4.09 (s, 1H),
3.71-3.62 (m, 2H), 3.12 (t, J ) 7.2 Hz, 1H), 2.64-2.52 (m, 2H),
2.36-2.26 (m, 2H), 2.04-1.99 (m, 2H), 1.96-1.63 (m, 12H),
1.46-1.20 (m, 14H, including two singlets at 1.40 and 1.39),
0.96 (d, J ) 6.0 Hz, 3H), 0.95 (d, J ) 6.0 Hz, 3H), 0.89 (d, J )
7.6 Hz, 3H), 0.88 (d, J ) 7.6 Hz, 3H), 0.98-0.86 (m, 2H); 13C
NMR (100 MHz, CDCl3) δ 103.0, 102.9, 89.7, 89.5, 81.1, 81.0,
74.7, 70.6, 70.5, 68.4, 52.0, 43.8, 43.7, 37.8, 37.5, 36.5, 34.9,
34.3, 31.0, 30.9, 25.91, 25.86, 24.9, 24.82, 24.75, 20.1, 12.5, 12.4;
IR(CHCl3) 3499.4, 2951.2, 2875.8, 1452.9, 1377.5, 1207.4,
1108.3, 1053.5, 1009.0, 911.5, 878.2, 843.8, 731.6 cm-1; HRMS
(ES) m/z calcd for C34H54O10Na (M + Na) 645.3609, found
645.3559.
Syn th esis of Bis-tr ioxa n e Ison icotin a te Ester Dim er
8b. To a stirring suspension of dimer alcohol 4 (30.4 mg, 0.050
mmol) and isonicotinic acid (20.1 mg, 0.163 mmol) in dry
dichloromethane (1 mL) was added 4-(N,N-dimethylamino)-
pyridine (23.5 mg, 0.192 mmol) and 1-(3-(dimethylamino)-
propyl)-3-ethylcarbodiimide (EDC) hydrochloride (39.2 mg,
0.204 mmol). A further 1.5 mL of dry dichloromethane was
added to wash down the flask walls, and the reaction mixture
was stirred at room temperature for 3 h, at which time TLC
analysis showed full consumption of starting material. Water
(5 mL), saturated NaHCO3 solution (5 mL), and dichloro-
methane (5 mL) were added, and the organics were extracted
with dichloromethane (3 × 20 mL), dried (Na2SO4), and
concentrated in vacuo to give a white solid. Gradient column
chromatography on silica (crude was dry-loaded), eluting first
with 25% ethyl acetate in petrol and then 30% ethyl acetate
in petrol, isolated the isonicotinate dimer 8b as a white solid
Syn th esis of Bis-tr ioxa n e Ep oxid e 6. To a 25 mL round-
bottomed flask was added isobutylene dimer 3 (34.1 mg, 0.058
mmol, 1.0 equiv) and anhydrous dichloromethane (10.0 mL).
This solution was cooled to -78 °C. Dimethyldioxirane (3.8
mL, 0.29 mmol, 5.0 equiv, 0.08 M solution in acetone) was
added rapidly. The mixture was then stirred for 30 min at -78
°C and was slowly warmed to room temperature while
monitoring was done with TLC. Solvents were removed under
reduced pressure, which afforded a yellow oil. The crude
mixture was purified by flash column chromatography (20%
EtOAc/hexanes) to afford the desired product 6 as a white solid
(28.1 mg, 0.047 mmol, 80%): mp ) 147-148 °C; 1H NMR (400
MHz, CDCl3) δ 5.48 (s, H), 5.43 (s, H), 4.45 (dd J ) 10.0, 6.0
Hz, 1H), 4.22 (dd J ) 10.0, 6.0 Hz, 1H), 2.82-2.72 (m, 2H),
2.68-2.62 (m, 3H), 2.43-2.33 (m, 3H), 2.02-1.97 (m, 2H),
1.88-1.82 (m, 2H), 1.75-1.70 (m, 2H), 1.62-1.58 (m, 2H),
1.54-1.26 (m, 17H, including singlet at 1.39), 0.98-0.88 (m,
3H), 0.93 (d, J ) 6.4 Hz, 6H), 0.92 (d, J ) 6.4 Hz, 6H), 0.80 (d,
J ) 7.6 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 103.43, 103.40,
87.94, 87.93, 81.10, 81.07, 75.44, 74.37, 60.43, 54.46, 52.54,
52.50, 44.58, 36.91, 36.70, 34.40, 33.50, 33.01, 30.40, 30.36,
26.18, 24.45, 24.35, 24.32, 20.22, 20.19, 13.69, 13.51; IR(CHCl3)
2939, 2875, 1452, 1376, 1280, 1208, 1188, 1123, 1091, 1057,
1006, 941, 878, 754 cm-1; HRMS (ES) m/z calcd for C34H52O9-
Na (M + Na) 627.3509, found 627.3478.
(32.6 mg, 0.046 mmol, 91%); mp ) 74-78 °C; [R]24.5 77.8
D
(CHCl3, c 0.06); 1H NMR (CDCl3, 400 MHz) δ 8.78 (s, br, 2H),
7.86 (s, br, 2H), 5.33 (s, 1H), 5.29 (s, 1H), 4.55 (s, 1H), 4.54 (s,
1H), 4.51-4.44 (m, 1H), 4.37-4.30 (m, 1H), 2.70 (st, 1H, J )
7.0), 2.59 (st, 1H, J ) 7.0), 2.45-2.36 (m, br, 1H), 2.31 (td,
2H, J ) 14.0, J ) 3.7), 2.05-1.96 (m, 2H), 1.95-1.73 (m, 6H),
1.69-1.50 (m, 6H), 1.41-1.15 (m, 14H, including two singlets
at 1.40 and 1.39), 0.98-0.92 (m, 2H), 0.96 (d, 3H, J ) 5.9),
0.94 (d, 3H, J ) 5.9), 0.88 (d, 3H, J ) 7.4), 0.87 (d, 3H, J )
7.4); 13C NMR (CDCl3, 100 MHz) δ 164.99, 150.49 (2), 137.93,
122.91 (2), 103.16, 102.87, 89.52, 88.83, 81.12, 81.11, 73.23,
70.83, 68.10, 52.30, 52.07, 44.32, 44.06, 37.50, 37.47, 36.64,
36.54, 34.42, 34.39, 33.95, 30.61, 30.58, 30.49, 30.15, 26.02,
26.01, 24.97, 24.89, 24.76, 24.67, 20.17, 20.10, 13.08, 12.72;
IR (CHCl3) 2942, 1866, 1726, 1452, 1407, 1372, 1321, 1276,
1210, 1123, 1106, 1057, 1046, 1006, 931, 876, 756, 707 cm-1
;
Syn th esis of Bis-tr ioxa n e Keton e 7. Trioxane isobutylene
dimer 3 (30 mg, 0.051 mmol) was dissolved in anhydrous DMF
(0.250 mL). OsO4 (0.1 mol %, 2.5% in t-BuOH) was added to
it. The reaction mixture was stirred for 5 min, and oxone (63
mg, 0.2 mmol) was added to it in one portion. After the reaction
mixture was stirred for 2 h, it was quenched with saturated
aqueous Na2SO3 solution and stirred for an additional 1 h. The
HRMS (EI, m/z) calcd for C40H57NO10Na (M + Na) 734.3875,
found 734.3855. Anal. (C40H57NO10) C, H.
Syn th esis of Ter tia r y Alcoh ol P r im a r y Su ccin a te
Ester 9. A 25 mL round-bottomed flask was charged with
vicinal diol 5 (52.9 mg, 0.085 mmol, 1.0 equiv) and anhydrous
dichloromethane (3.0 mL). To this solution, succinic anhydride

1064 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Posner et al.
(25.5 mg, 0.255 mmol, 3.0 equiv) and 4-N,N-(dimethylamino)-
pyridine (10.4 mg, 0.085 mmol, 1.0 equiv) were added, and the
reaction mixture was stirred for 24 h at room temperature
while being monitored by TLC. Upon complete consumption
of starting material, the reaction mixture was poured into a
mixture of 30 mL of dichloromethane and 30 mL of saturated
aqueous NH4Cl solution. Then the organic layer was separated
and washed with brine, dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude mixture was
purified by flash column chromatography (2-propanol/dichlo-
romethane/ethyl acetate, 1:3:18) to afford the desired product
9 as a white solid (52.0 mg, 0.071 mmol, 85%): mp ) 95-97
of diethyl ether was added and the resulting mixture was
stirred for an additional 10 min. Then, the reaction mixture
was dried over MgSO4 and filtered through a Celite pad. The
solid was washed with ethyl acetate (30 mL) and then
concentrated under reduced pressure. The crude mixture was
purified by flash column chromatography (30% EtOAc/hex-
anes) to afford the desired product 10b as a white solid (32.0
mg, 0.040 mmol, 76%): mp ) 115-117 °C; 1H NMR (400 MHz,
CDCl3) δ 8.21 (ABq, J AB ) 8.4 Hz, ∆νAB ) 73.6 Hz, 4H), 5.70
(s, 1H), 5.36 (s, 1H), 5.19 (dd, J ) 10.8, 6.0 Hz, 1H), 4.88 (s,
1H), 4.58 (dd, J ) 9.6, 6.4 Hz, 1H), 3.95 (s, 3H), 3.81 (ABq,
J AB ) 13.6 Hz, ∆νAB ) 278.4 Hz, 2H), 2.90-2.82 (m, 1H), 2.70-
2.56 (m, 2H), 2.43-2.30 (m, 2H), 2.20-1.20 (m, 22H, including
two singlets at 1.54 and 1.24), 0.96 (d, J ) 6.0 Hz, 6H), 0.93
(d, J ) 8.0 Hz, 3H), 0.88 (d, J ) 7.6 Hz, 3H), 0.98-0.86 (m,
2H); 13C NMR (100 MHz, CDCl3) δ 165.8, 145.3, 134.3, 129.9,
128.5, 103.6, 102.8, 90.1, 88.5, 81.2, 73.6, 71.6, 71.1, 60.1, 52.6,
52.5, 51.8, 45.0, 43.3, 39.5, 37.5, 37.3, 36.5, 34.5, 34.3, 30.5,
30.4, 26.4, 26.0, 25.0, 24.8, 24.6, 24.3, 20.3, 20.0, 13.7, 11.9;
IR (CHCl3) 3458, 2951, 2875, 1731, 1435, 1377, 1320, 1280,
1153, 1105, 1051, 1015, 911, 880, 733 cm-1; HRMS (ES) m/z
calcd for C42H60NaO13S (M + Na) 827.3646, found 827.3661.
°C; [R]23.5 56.6 (CHCl3, c 0.16); 1H NMR (400 MHz, CDCl3) δ
D
5.38 (s, 1H), 5.34 (s, 1H), 4.61-4.52 (m, 2H), 4.29 (ABq, J AB
)
18.8 Hz, ∆νAB ) 11.6 Hz, 2H), 2.72-2.66 (m, 4H), 2.58-2.50
(m, 2H), 2.36-2.24 (m, 2H), 2.04-1.58 (m, 13H), 1.46-1.18
(m, 17H, including two singlets at 1.40 and 1.39), 0.95 (d, J )
6.0 Hz, 6H), 0.88 (d, J ) 7.6 Hz, 3H), 0.87 (d, J ) 7.6 Hz, 3H),
0.98-0.86 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 176.4, 171.7,
103.2, 102.9, 89.6, 89.0, 81.1, 81.0, 73.3, 70.6, 70.3, 68.6, 52.1,
51.9, 44.1, 43.7, 37.5, 36.6, 36.5, 36.3, 35.1, 34.4, 34.3, 30.8,
30.7, 29.2, 29.1, 25.9, 25.8, 24.8, 24.7, 20.2, 20.1, 12.8, 12.5;
IR(CHCl3) 3502, 2950, 2872, 1737, 1713, 1453, 1378, 1208,
1168, 1106, 1054, 1009, 942, 878, 844, 735 cm-1; HRMS (ES)
m/z calcd for C38H58NaO13 (M + Na) 745.3769, found 745.3726.
Anal. (C38H58O13) C, H.
Syn th esis of â-Hyd r oxysu lfon e Ben zoic Acid 10c. A 10
mL round-bottomed flask was charged with â-hydroxysulfone
ester 10b (19.7 mg, 0.024 mmol, 1.0 equiv) and then dissolved
in 2.5% KOH/MeOH (1.0 mL). This solution was stirred
vigorously at room temperature while being monitored with
TLC. Upon the complete consumption of starting material
(about 3 h), the solution was evaporated to dryness under
reduced pressure and then was dissolved in 10 mL of water.
This solution was acidified with acetic acid until pH 4.0 was
attained and was extracted with EtOAc (30 mL × 2). Then
the organic layer was combined and dried over MgSO4, filtered,
and concentrated under reduced pressure. The crude mixture
was purified by flash column chromatography (2-propanol/
dichloromethane/ethyl acetate, 1:3:18) to afford the desired
product 10c as a white solid (14.7 mg, 0.019 mmol, 79%): mp
Wa ter Solu bility Test. To a 1 dram vial, 0.5 mL of pH 7.4
phosphate buffer solution and 2 mg of trioxane dimer were
added, and the mixture was vigorously stirred for 1 h. In some
cases, compound was added again until there was remaining
compound that was not dissolved. Then the solution was
transferred via pipet into a small sintered glass filter. The
filtrate was evaporated under reduced pressure, and the
weight was measured. The buffer solution (0.5 mL for blank
run) was dried, and the weight was measured. The weight of
phosphate blank was subtracted from the observed weight for
the trioxane dimer. Succinate ester 9 had a solubility of 16.6
mg/mL, compared to 0.6 mg/mL for artelinic acid.
) 143-145 °C; [R]23.9 105.2 (CHCl3, c 0.07); 1H NMR (400
D
MHz, CDCl3) δ 8.21 (ABq, J AB ) 8.4 Hz, ∆νAB ) 76.4 Hz, 4H),
5.71 (s, 1H), 5.38 (s, 1H), 5.19 (dd, J ) 10.8, 6.4 Hz, 1H), 4.63
Syn th esis of â-Hyd r oxysu lfid e Ester 10a . A 50 mL
round-bottomed flask was charged with bis-trioxane epoxide
6 (80.0 mg, 0.132 mmol, 1.0 equiv), methyl 4-mercaptobenzoate
(44.4 mg, 0.264 mmol, 2.0 equiv), and neutral aluminum oxide
(1.0 g, type W 200 super I, Woelm Pharma, Germany), and
then anhydrous diethyl ether (10.0 mL) was added to make a
slurry, which was stirred for 3 h at room temperature while
being monitored by TLC. Upon complete consumption of
starting material, the reaction mixture was filtered through
a Celite pad and the solid was washed with ethyl acetate (30
mL × 2) and then concentrated under reduced pressure. The
crude mixture was purified by flash column chromatography
(25% EtOAc/hexanes) to afford the desired product 10a as a
(dd, J ) 9.2, 6.0 Hz, 1H), 3.82 (ABq, J AB ) 13.6 Hz, ∆νAB
)
266.4 Hz, 2H), 2.85-2.82 (m, 1H), 2.70-2.56 (m, 2H), 2.43-
2.30 (m, 2H), 2.20-1.20 (m, 22H, including two singlets at 1.56
and 1.25), 0.96 (d, J ) 6.0 Hz, 6H), 0.93 (d, J ) 8.0 Hz, 3H),
0.88 (d, J ) 7.6 Hz, 3H), 0.98-0.86 (m, 2H); 13C NMR (100
MHz, CDCl3) δ 168.0, 145.7, 133.5, 130.4, 128.5, 103.8, 102.9,
90.1, 88.7, 81.3, 73.7, 71.8, 71.2, 60.2, 52.6, 51.9, 44.9, 43.3,
39.4, 37.5, 37.4, 37.3, 36.5, 34.5, 34.3, 30.6, 30.5, 26.1, 25.9,
25.0, 24.8, 24.5, 24.4, 20.2, 20.0, 13.6, 11.9; IR (CHCl3) 3459,
2939, 2875, 1723, 1432, 1403, 1378, 1319, 1300, 1228, 1154,
1099, 1051, 1015, 911, 877, 732, 616 cm-1; HRMS (ES) m/z
calcd for C41H58NaO13S (M + Na) 813.3490, found 813.3500.
Anal. (C41H58O13) C, H.
1
sticky solid (81.2 mg, 0.105 mmol, 80%): H NMR (400 MHz,
CDCl3) δ 7.67 (ABq, J AB ) 8.4 Hz, ∆νAB ) 154.4 Hz, 4H), 5.36
(s, 1H), 5.35 (s, 1H), 4.69 (dd, J ) 10.4, 6.0 Hz, 1H), 4.69 (dd,
J ) 10.0, 6.0 Hz, 1H), 3.88 (s, 3H), 3.52 (ABq, J AB ) 12.4 Hz,
∆νAB ) 44.4 Hz, 2H), 2.65-2.55 (m, 1H), 2.52-2.42 (m, 1H),
2.34-2.24 (m, 2H), 2.08-1.58 (m, 12H), 1.46-1.18 (m, 10H,
including two singlets at 1.36 and 1.31), 0.94 (d, J ) 6.0 Hz,
6H), 0.85 (d, J ) 6.8 Hz, 3H), 0.84 (d, J ) 7.2 Hz, 3H), 0.98-
0.86 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 166.9, 145.0, 129.6,
127.7, 126.4, 102.9, 102.8, 89.7, 89.3, 81.1, 81.0, 74.9, 70.8, 70.5,
52.1, 51.9, 43.9, 43.6, 42.4, 38.3, 37.4, 36.5, 36.4, 36.1, 34.4,
34.3, 30.8, 30.7, 26.0, 25.9, 24.8, 24.7, 20.1, 20.0, 12.7, 12.4;
IR (CHCl3) 3498, 2951, 2876, 1721, 1594, 1430, 1377, 1276,
Syn th esis of Bis-tr ioxa n e Styr yl Ter tia r y Alcoh ol 11a .
A solution of styryllithium was prepared by adding t-BuLi (1.2
mL, 1.7 M in hexanes) to a solution of 4-bromostyrene (0.13
mL, 1 mmol) in diethyl ether (5.0 mL). The deep-red solution
was stirred for 30 min to ensure complete formation of
styryllithium. To a solution of ketone 7 (30 mg, 0.051 mmol)
in THF was added a solution of styryllithium (0.35 mL, ∼0.07
mmol) at -78 °C, and the reaction mixture was stirred for 30
min at the same temperature. The reaction was then worked
up by adding a saturated solution of ammonium chloride and
extracting with dichloromethane. Flash column chromatogra-
phy (10-15% EtOAc/petroleum ether) yielded the desired
tertiary alcohol 11a (26 mg, 0.037 mmol, 74%) as a viscous
oil: 1H NMR (400 MHz, CDCl3) δ 7.45 (d, 2H), 7.35 (d, 2H),
6.68 (dd, 1H), 5.70 (d, 1H), 5.35 (s, 2H), 5.2 (d, 2H), 4.46 (m,
1H), 4.19 (m, 1H), 2.78-2.42 (m, 4H), 2.32 (m, 2H), 2.17 (m,
1H), 2.01 (m, 2H), 1.90 (m, 4H), 1.84-1.60 (m, 11H), 1.48-
1.20 (m, 16H, including singlet at 1.38), 1.00-0.80 (m, 12H).
1182, 1110, 1054, 1008, 942, 880, 845, 762, 736 cm-1
.
Syn th esis of â-Hyd r oxysu lfon e Ester 10b.35 A 50 mL
round-bottomed flask was charged with â-hydroxysulfide ester
10a (40.0 mg, 0.052 mmol, 1.0 equiv), which was then dissolved
in tetrachloromethane (1.5 mL). Then acetonitrile (1.5 mL) and
H2O (2.3 mL) were added. To the reaction mixture ruthenium-
(III) chloride hydrate (catalytic amount) and periodic acid (23.7
mg, 0.104 mmol, 2.0 equiv) was added. (Upon addition of
ruthenium chloride, the solution was turned black.) After the
mixture was stirred for 30 min at room temperature, 30 mL
Syn th esis of Bis-tr ioxa n e Ter tia r y Alcoh ol Ben zoic
Acid 11b. Styryl tertiary alcohol 11a (20 mg, 0.028 mmol) was
dissolved in acetone (3 mL), and KMnO4 was added to it as a

Trioxane Dimers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1065
(15) Posner, G. H.; Parker, M. H.; Northrop, J .; Elias, J . S.; Ploy-
pradith, P.; Xie, S.; Shapiro, T. A. Orally Active, Hydrolytically
Stable, Semisynthetic, Antimalarial Trioxanes in the Artemisi-
nin Family. J . Med. Chem. 1999, 42, 300-304.
(16) O’Dowd, H.; Ploypradith, P.; Xie, S.; Shapiro, T. A.; Posner, G.
H. Antimalarial Artemisinin Analogs. Synthesis via Chemo-
selective C-C Bond Formation and Preliminary Biological
Evaluation. Tetrahedron 1999, 55, 3625-3636.
(17) O’Neill, P. M.; Searle, N. L.; Kan, K.-W.; Storr, R. C.; Maggs, J .
L.; Ward, S. A.; Raynes, K.; Park, B. K. Novel, Potent, Semi-
synthetic Antimalarial Carba Analogues of the First-Generation
1,2,4-Trioxane Artemether. J . Med. Chem. 1999, 42, 5487-5493.
(18) O’Neill, P. M.; Pugh, M.; Stachulski, A. V.; Ward, S. A.; Davies,
J .; Park, B. K. Optimisation of the Allylsilane Approach to C-10
Deoxo Carba Analogues of Dihydroartemisinin: Synthesis and
in Vitro Antimalarial Activity of New, Metabolically Stable C-10
Analogues. J . Chem. Soc., Perkin Trans. 1 2001, 2682-2689.
(19) Rychnovsky, S. D.; Fryszman, O.; Khire, U. R. Convergent
Synthesis of a Polyol Chain with 4-Acetoxy-1,3-dioxanes Using
a 1,1-Bis((trimethylsilyl)methyl)ethene Linchpin. Tetrahedron
Lett. 1999, 40, 41-44.
solid. The reaction mixture was stirred at room temperature
for 6 h. The reaction mixture was then treated with 2-propanol
to quench excess KMnO4. The solvent was evaporated, and
the product was washed with water and extracted with ethyl
acetate. Flash column chromatography (90% EtOAc/petroleum
ether) yielded the desired product 11b (19 mg, 0.014 mmol,
50%) as a white solid: mp ) 97-101 °C; [R]24.1 17.6 (CHCl3,
D
c 0.06); 1H NMR (400 MHz, CD3OD) δ 7.98 (d, J ) 8.4 Hz,
2H), 7.61 (d, J ) 8.4 Hz, 2H), 5.47 (s, 1H), 5.29 (s, 1H), 4.36
(m, 1H), 4.08 (m, 1H), 2.59-2.48 (m, 2H), 2.40-2.20 (m, 5H),
2.02-1.76 (m, 8H), 1.58-1.10 (m, 18H, including two singlets
13
at 1.27 and 1.20), 1.00-0.70 (m, 14H); C NMR (100 MHz,
CD3OD) δ 167.4, 132.5 130.6, 130.0, 127.7, 104.7, 104.6, 90.5,
89.9, 82.3, 82.1, 78.4, 74.1, 73.6, 54.1, 53.8, 46.4, 45.9, 43.4,
39.8, 38.6, 38.5, 37.8, 37.7, 35.9, 35.8, 32.1, 31.9, 26.4, 26.1,
26.0, 25.9, 25.8, 25.7, 20.8, 20.7, 13.9, 13.6; HRMS (ES) m/z
calcd for C40H56NaO11 (M + Na) 735.3715, found 735.3717.
Ack n ow led gm en t. We thank the NIH (Grants AI
34885 and RR-00052) and the Burroughs Wellcome
Fund for generous financial support.
(20) Ma, J .; Katz, E.; Ziffer, H. A New Synthetic Route to 10â-
Alkyldeoxoartemisinins. Tetrahedron Lett. 1999, 40, 8543-8545.
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selective C-C Bond Formation and Preliminary Biological
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