Antimalarial, antiproliferative, and antitumor activities of artemisinin-derived, chemically robust, trioxane dimers

Article abstract of DOI:10.1021/jm990363d

Nine C-10 non-acetal derivatives of the natural trioxane artemisinin (1) were prepared as dimers using some novel chemistry. As designed, each dimer was stable chemically. C-10 Olefinic dimers 7 and C-10 saturated dimers 8-13 all showed good to excellent antimalarial and antiproliferative activities in vitro. Dimers 8, 10, and 12 were especially potent and selective at inhibiting growth of some human cancer cell lines in the NCI in vitro 60-cell line assay.

Full text of DOI:10.1021/jm990363d

© Copyright 1999 by the American Chemical Society
Volume 42, Number 21
October 21, 1999
Expedited Articles
An tim a la r ia l, An tip r olifer a tive, a n d An titu m or Activities of
Ar tem isin in -Der ived , Ch em ica lly Robu st, Tr ioxa n e Dim er s
Gary H. Posner,*,† Poonsakdi Ploypradith,† Michael H. Parker,†,1a Hardwin O’Dowd,† Soon-Hyung Woo,†,1b
J ohn Northrop,†,1c Mikhail Krasavin,† Patrick Dolan,‡ Thomas W. Kensler,‡ Suji Xie,§ and Theresa A. Shapiro§
Department of Chemistry, School of Arts and Sciences, The J ohns Hopkins University, Baltimore, Maryland 21218, and
Division of Toxicological Sciences, School of Hygiene and Public Health, and Department of Medicine, School of Medicine,
The J ohns Hopkins University, Baltimore, Maryland 21205
Received J uly 15, 1999
Nine C-10 non-acetal derivatives of the natural trioxane artemisinin (1) were prepared as dimers
using some novel chemistry. As designed, each dimer was stable chemically. C-10 Olefinic
dimers 7 and C-10 saturated dimers 8-13 all showed good to excellent antimalarial and
antiproliferative activities in vitro. Dimers 8, 10, and 12 were especially potent and selective
at inhibiting growth of some human cancer cell lines in the NCI in vitro 60-cell line assay.
In tr od u ction
Some dimeric chemical structures have especially
high biological activities. Examples include bismustard
cross-linked lexitropsins,2 dimeric steroid-pyrazine ma-
rine alkaloids,3 and DNA-cross-linking dimeric benzo-
diazepines4,5 and bis(enediynes).6 Some 1,2,4-trioxane
dimers have high antimalarial, antiproliferative, and
antitumor activities in vitro, but often they are hydro-
lytically unstable.7-10 Most efforts to prepare chemically
more robust semisynthetic derivatives of the natural
antimalarial 1,2,4-trioxane artemisinin (1) have in-
volved replacing the C-10 acetal functionality in ester
and ether derivatives 2-5 by less hydrolytically prone
functional groups.11-18 Recent success along these lines
has led to a series of orally active, hydrolytically stable
antimalarial trioxanes in the artemisinin family.19
Herein, we report synthesis and preliminary biological
evaluation of nine C-10 non-acetal artemisinin-derived
trioxane dimers, some of which are not only potent
antimalarials but also potent antiproliferative and
antitumor agents.
Syn th esis a n d An tim a la r ia l Activities
C-10 Olefinic non-acetal dimers 7a -c were prepared
in high overall yield and in a novel way via a bis-Wittig
coupling reaction with the recently reported artemisi-
nin-derived R,â-unsaturated aldehyde 6 (Scheme 1).20
The coupling reaction was optimized to nearly quantita-
tive yield by generating the bis-phosphonium ylide
reagent via in situ deprotonation of the corresponding
bis-phosphonium bromide salt in the presence of alde-
hyde 6 using in situ generated lithium ethoxide as
base.21 The three geometric isomers 7a -c were sepa-
rated from one another chromatographically and were
assigned trans or cis stereochemistry by 1H NMR
† Department of Chemistry.
‡ Division of Toxicological Sciences.
§ Department of Medicine.
10.1021/jm990363d CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/30/1999

4276 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Posner et al.
Sch em e 1
Sch em e 2
spectroscopy (see Experimental Section). The corre-
sponding p-xylylene dimers were prepared from alde-
hyde 6 in a similar fashion, but they were found to be
unstable, decomposing during several hours in CDCl3.
Using our standard assay,22 the antimalarial poten-
cies of m-xylylene dimers 7 in vitro against chloroquine-
sensitive Plasmodium falciparum (NF54) parasites were
found to be as follows: 7a , IC50 ) 77 nM; 7b, IC50 ) 35
nM; 7c, IC50 ) 18 nM; in comparison, the IC50 value
for artemisinin is 9.7 nM. C-10 Saturated non-acetal
dimers 8-13 were prepared from either artemether (2)
via novel titanium-promoted condensations (eq 1) or
from the recently described artemisinin-derived C-10
fluoride 14 via Friedel-Crafts or aluminum acetylide
condensations (Scheme 2).18 Although benzoylmethylene
dimers 8 and 9 and acetylenic dimers 12 and 13 are
stereochemically â-linked to C-10 of the artemisinin
skeleton, aryl dimers 10 and 11 are R-linked; the basis
for this difference in stereochemistry of attachment is

Activity of Artemisinin-Derived Trioxane Dimers
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4277
Ta ble 1. C-10 Deoxoartemisinin Dimers 8-13
F igu r e 1. Dose-response effects of analogues on keratinocyte
proliferation (96 h).
a
Antimalarial activity was determined against the chloroquine-
sensitive NF54 strain of P. falciparum as reported previously.22
The standard deviation for each set of quadruplicates was an
average of 11% (e 57%) of the mean. R2 values for the fitted curves
were g 0.992. Artemisinin activity is mean ( standard deviation
of concurrent control (n ) 6).
not fully understood. Unlike the bis-acetylene dimers
12 and 13, benzoylmethylene dimers 8 and 9, aryl dimer
10, and furan dimer 11 are considerably more potent
antimalarial agents (IC50 ) 1.3-3.2 nM) than natural
artemisinin (IC50 ) 9.7 nM) (Table 1).
F igu r e 2. Dose-response effects of analogues on keratinocyte
proliferation (96 h).
cell line sensitivity profile for all compounds in the NCI
database and found that most of our dimers are similar
in profile to platinum compounds; such compounds are
DNA intrastrand cross-linking agents that inhibit cell
replication.26 Whether this molecular mechanism is the
biological basis of action of our dimers, however, re-
mains to be established by appropriate additional
experiments in the future.
An tip r olifer a tive a n d An titu m or Activities
Antiproliferative activities, measured in vitro using
murine keratinocytes as described previously,23 are
shown in Figure 1 for C-10 olefinic m-xylylene dimers
7 and in Figure 2 for some of the C-10 saturated dimers
8-13. It is noteworthy that most of these trioxane
dimers are considerably more effective at 1 µM concen-
tration than calcitriol (1R,25-dihydroxyvitamin D3), the
hormonally active form of vitamin D3 that is used
clinically as a topical drug to treat psoriasis,24 a skin
disorder characterized by uncontrolled proliferation of
cells. However, these trioxane dimers are less effective
than calcitriol at nanomolar concentrations.
Growth inhibitory activities at nanomolar to micro-
molar concentrations, measured in vitro as described
previously using a diverse panel of 60 human cancer
cell lines in the National Cancer Institute (NCI’s)
Developmental and Therapeutic Program,25 indicate
that all of our dimers are particularly inhibitory to
leukemia cells and some of these dimers are notably
active in a few other (e.g. colon 205) cancer cell lines.
Furthermore, the NCI COMPARE program25 evaluated
the cell sensitivity profile of our dimers versus the 60-
Preliminary in vivo antitumor evaluation of dimer 8
was performed using the NCI mouse hollow fiber assay.
This mouse model involves implanting intraperitoneally
(ip) and also subcutaneously (sc) polyvinylidene hollow
fibers containing various human cancer cell lines and
then administering the dimer via the ip route. The effect
of a dimer on diminishing the viable cancer cell mass
compared to that of controls was examined. Trioxane
dimer 8 substantially diminished cancer cell mass and
was as effective at the remote sc implant site as at the
ip implant site, suggesting that dimer 8 is potent and
stable in this in vivo assay.
In conclusion, the C-10 non-acetal trioxane dimers
reported here represent a new series of chemically
robust, biologically potent compounds having potential
for diverse therapeutic uses. Further study of these new

4278 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Posner et al.
6.96 (d, J ) 15.6 Hz, 1 H), 6.85 (d, J ) 15.6 Hz, 1 H), 6.50 (d,
J ) 12.0 Hz, 1 H), 6.05 (d, J ) 12.0 Hz, 1 H), 5.70 (s, 1 H),
5.65 (s, 1 H), 2.46-2.35 (m, 2 H), 2.08-1.80 (m, 8 H), 1.86 (s,
3 H), 1.74-1.38 (m, 6 H), 1.44 (s, 3 H), 1.41 (s, 3 H), 1.40 (s, 3
H), 1.30-1.02 (m, 6 H), 0.99 (d, J ) 5.6 Hz, 3 H), 0.97 (d, J )
6.0 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 142.0, 141.3, 137.5,
137.2, 132.3, 128.2, 128.0, 127.8, 127.2, 125.4, 122.8, 119.3,
108.2, 105.5, 104.4, 104.3, 90.0, 89.9, 78.704, 78.695, 51.1, 51.0,
46.9, 45.9, 37.6, 36.3, 36.2, 34.2, 34.1, 29.4, 29.2, 25.8, 24.5,
20.5, 20.2, 16.5, 16.1; HRMS (CI, NH3) m/z calcd for C40H50O8
chemical entities will reveal their mechanism(s) of
action and their broad efficacy/safety profile.
Exp er im en ta l Section
Gen er a l. Unless otherwise noted: Reactions were run in
flame-dried round-bottomed flasks under an atmosphere of
ultrahigh purity (UHP) argon. Diethyl ether (ether) and
tetrahydrofuran (THF) were distilled from sodium benzophe-
none ketyl prior to use. Dichloromethane (CH2Cl2) and tri-
ethylamine (TEA) were distilled from calcium hydride prior
to use. All other compounds were purchased from Aldrich
Chemical Co. Column chromatography was performed using
short path silica gel (particle size < 230 mesh), flash silica gel
(particle size 400-230 mesh), or Florisil gel (200 mesh).
Analytical thin-layer chromatography (TLC) was performed
with silica gel 60 F254 plates (250 µm thickness; Merck). Yields
are not optimized. Nuclear magnetic resonance (NMR) spectra
were obtained using a Varian XL-400 spectrometer, operating
(M+) 658.3506, found 658.3508; 7c [R]23 ) +228 (c ) 0.49,
D
EtOAc); 1H NMR (400 MHz, CDCl3) δ 7.50 (br s, 1 H), 7.43 (d,
J ) 7.6 Hz, 1 H), 7.39 (d, J ) 7.6 Hz, 1 H), 7.18 (t, J ) 8.0 Hz,
1 H), 6.48 (d, J ) 12.0 Hz, 2 H), 5.99 (d, J ) 12.0 Hz, 2 H),
5.66 (s, 2 H), 2.46-2.36 (m, 2 H), 2.04 (m, 2 H), 1.92 (m, 4 H),
1.70-1.38 (m, 10 H), 1.41 (s, 6 H), 1.36 (s, 6 H), 1.28-1.02 (m,
4 H), 0.98 (d, J ) 5.6 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ
141.4, 136.8, 132.7, 129.8, 128.0, 127.6, 122.5, 105.0, 104.4,
89.9, 78.7, 51.2, 45.9, 37.6, 36.4, 34.1, 29.2, 25.9, 24.5, 20.3,
16.5; HRMS (CI, NH3) m/z calcd for C40H50O8 (M+) 658.3506,
found 658.3515.
1
at 400 MHz for H and 100 MHz for 13C, or a Bruker, operating
at 300 MHz for 1H and 75 MHz for 13C. Chemical shifts are
reported in parts per million (ppm, δ) downfield from tetra-
methylsilane. Splitting patterns are described as singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), and broad
(br). Infrared (IR) spectra were obtained using a Perkin-Elmer
1600 FT-IR spectrometer. Resonances are reported in wave-
numbers (cm-1). Low-resolution (LRMS) and high-resolution
(HRMS) mass spectra were obtained on a VG Instruments 70-S
spectrometer run at 70 eV for electronic ionization (EI) and
run with ammonia (NH3) as a carrier for chemical ionization
(CI). High-performance liquid chromatography (HPLC) was
performed using a Rainin HPLX gradient system equipped
with two 25 mL/min preparative pump heads using Rainin
Dynamax 10-mm × 250-mm (semipreparative) columns packed
with 60 Å silica gel (8-mm pore size) as bare silica. Melting
points were measured using a Mel-Temp metal-block ap-
paratus and are uncorrected. Combustion analyses were
conducted by Atlantic Microlab (Norcross, GA).
1,4-Dia cetylben zen e Bis-TMS En ol Eth er . 1,4-Diacetyl-
benzene (1.6 g, 10 mmol) was treated according to the general
procedure 1. The crude was purified by Ku¨gelrohr distillation
to give the desired product (1.8 g, 5.9 mmol, 59%). The mixture
was used for the next step without further purification: 1H
NMR (400 MHz, CDCl3) δ 7.56 (s, 4 H), 4.95 (d, J ) 2.0 Hz, 2
H), 4.45 (d, J ) 2.0 Hz, 2 H), 0.29 (s, 18 H); 13C NMR (100
MHz, CDCl3) δ 155.3, 137.2, 124.9, 91.3, 0.11; IR (neat) 2960,
1687, 1608, 1314, 1253, 1114, 1011 cm-1
.
10-p-Acetylp h en yla r tem isin in Dim er 8. With the use of
a glovebag under argon, neat TiCl4 (0.12 mL, 1.1 mmol) was
added to CH2Cl2 (1 mL) at room temperature. To this pale
yellow mixture at -78 °C was added a -78 °C solution of
â-artemether (2) (0.30 g, 1.0 mmol) in CH2Cl2 (1.5 mL) via
cannula. The resulting mixture was stirred at -78 °C for 5
min. To this mixture at -78 °C was slowly added a -78 °C
solution of 1,4-diacetylbenzene bis-TMS enol ether (0.21 g, 0.55
mmol) in CH2Cl2 (2.5 mL). The reaction mixture was stirred
at -78 °C for 1 h. The mixture was quenched with H2O (5
mL) and diluted with CHCl3 (5 mL). Two phases were
separated and the aqueous phase was extracted with CHCl3
(5 mL × 2). The combined organic layers were washed with
brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The crude mixture was purified by column
chromatography (Florisil, 1%f30% EtOAc/hexanes) to give the
product (0.09 g, 0.13 mmol, 26%). Further purification by
Gen er a l P r oced u r e 1: Syn th esis of TMS En ol Eth er
of Ar yl Meth yl Keton es. To a solution of aryl methyl ketone
(1.0 equiv) in ether (2 mL/mmol of ketone) at 0 °C was added
Et3N (1.1 equiv) via syringe. To this mixture at 0 °C was slowly
added trimethylsilyl trifluoromethanesulfonate (TMSOTf; 1.1
equiv) via gastight syringe. The resulting mixture was stirred
at 0 °C for 15 min, warmed to room temperature, and stirred
for 2 h. Two phases were separated and the ethereal layer was
concentrated under reduced pressure.
m -Ben zen e Dim er s 7. m-Xylylene bis(triphenylphospho-
nium bromide) (0.13 g, 0.17 mmol) and aldehyde 620 (0.10 g,
0.34 mmol) in EtOH (1.0 mL) at 0 °C were treated with lithium
bis(trimethylsilyl)amide (1.0 M in THF, 0.68 mL, 4.0 equiv).
The reaction was stirred for 10 min at 0 °C, then for 10 min
at room temperature. The reaction was concentrated, then
diluted with ether, washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The crude
product was chromatographed on a flash silica gel column with
10% EtOAc/hexanes as eluent to give the desired product 7
as a mixture of all three possible Z/ E isomers as an oil (0.11
HPLC (silica, 25% EtOAc/hexanes, 3.0 mL/min, 254 nm, tR
)
18.3 min) provided the desired p-diacetyl dimer 8 as a white
1
solid: mp ) 87-88 °C; H NMR (400 MHz, CDCl3) δ 8.02 (s,
4 H), 5.33 (s, 2 H), 5.09 (ddd unresolved, 2 H), 3.20 (dABq, J d
) 6.6 Hz, J AB ) 16.0 Hz, ∆νAB ) 84.3 Hz, 4 H), 2.79 (m, 2 H),
2.31 (ddd, J ) 14.4, 13.2, 4.0 Hz, 2 H), 2.00 (ddd, J ) 14.6,
4.8, 3.2 Hz, 2 H), 1.93 (m, 2 H), 1.83 (ddd, J ) 13.6, 8.0, 4.0
Hz, 2 H), 1.71 (m, 4 H), 1.47-1.23 (m, 10 H), 1.31 (s, 6 H),
0.98 (d, J ) 6.0 Hz, 6 H), 0.90 (d, J ) 7.6 Hz, 6 H); 13C NMR
(100 MHz, CDCl3) δ 197.6, 140.0, 128.4, 102.9, 89.6, 80.8, 70.0,
52.0, 44.0, 40.5, 37.5, 36.5, 34.4, 29.9, 25.8, 24.8, 20.1, 13.0;
IR (CHCl3) 2956, 2930, 2877, 1686, 1453, 1378, 1359, 1265,
1
g, 0.17 mmol, 99%), ZE:EE:ZZ ) 6:3:2 (as determined by H
NMR). The three isomers were separated by HPLC (silica
semipreparative column, 10% EtOAc/hexanes at 3 mL/min):
ZZ (tR ) 11 min), EE/ EZ (mixture; tR ) 13 min). Silica
semipreparative column, 80% CH2Cl2/hexanes at 3 mL/min,
1092, 1053, 1011 cm-1; HRMS (CI, NH3) m/z calcd for C40H58
NO10 (M + NH4+) 712.4061, found 712.4054.
-
1,3-Dia cetylben zen e Bis-TMS En ol Eth er . 1,3-Diacetyl-
benzene (1.6 g, 10 mmol) was treated according to the general
procedure 1. The crude was purified by Ku¨gelrohr distillation
to give the desired product (1.8 g, 5.9 mmol, 59%). The mixture
was used for the next step without further purification: 1H
NMR (400 MHz, CDCl3) δ 7.83 (m, 1 H), 7.52 (d, J ) 2.0 Hz,
1 H), 7.50 (d, J ) 2.0 Hz, 1 H), 7.27 (apparent t, J ) 7.8 Hz,
1 H), 4.93 (d, J ) 1.6 Hz, 2 H), 4.44 (d, J ) 1.6 Hz, 2 H), 0.27
(s, 18 H); 13C NMR (100 MHz, CDCl3) δ 155.5, 137.3, 127.8,
125.1, 122.1, 91.2, 0.13; IR (neat) 2962, 1689, 1607, 1312, 1253,
gave separation of the EE/ EZ: mixture; EE tR ) 9.0 min, EZ
1
tR ) 19.0 min; 7a [R]23 ) 0 (c ) 0.48, EtOAc); H NMR (400
D
MHz, CDCl3) δ 7.53 (br s, 1 H), 7.22 (m, 3 H), 6.99 (d, J )
15.6 Hz, 2 H), 6.87 (d, J ) 15.6 Hz, 2 H), 5.72 (s, 2 H), 2.46-
2.36 (m, 2 H), 2.08-1.80 (m, 6 H), 1.87 (s, 6 H), 1.72-1.40 (m,
8 H), 1.46 (s, 6 H), 1.32-1.02 (m, 6 H), 0.99 (d, J ) 5.6 Hz, 6
H); 13C NMR (100 MHz, CDCl3) δ 142.0, 137.9, 128.5, 127.6,
125.4, 125.1, 119.6, 108.5, 104.4, 90.1, 78.7, 51.0, 47.0, 37.6,
36.2, 34.2, 29.4, 25.8, 24.5, 20.2, 16.1; HRMS (CI, NH3) m/z
1114, 1011 cm-1
.
calcd for C40H50O8 (M+) 658.3506, found 658.3515; 7b [R]23
)
D
1
+77 (c ) 0.49, EtOAc); H NMR (400 MHz, CDCl3) δ 7.49 (br
s, 1 H), 7.44 (m, 1 H), 7.29 (m, 1 H), 7.22 (t, J ) 7.6 Hz, 1 H),
10-m -Acetylp h en yla r tem isin in Dim er 9. To a solution
of â-artemether (2) (0.12 g, 0.4 mmol) in CH2Cl2 (1.2 mL) at

Activity of Artemisinin-Derived Trioxane Dimers
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4279
-78 °C was added TiCl4 (1.0 M in CH2Cl2, 0.44 mL, 0.44 mmol)
via gastight syringe. The resulting mixture was stirred at -78
°C for 5 min. To the mixture was added a -78 °C solution of
1,3-diacetylbenzene bis-TMS enol ether (0.070 g, 0.22 mmol)
in CH2Cl2 (1.2 mL) via cannula at -78 °C. The reaction
mixture was stirred at -78 °C for 1 h. The mixture was
quenched with H2O and diluted with CHCl3. Two phases were
separated and the aqueous phase was extracted twice with
CHCl3. The combined organic layers were washed with brine,
dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude mixture was purified by column chroma-
tography (Florisil, 1%f30% EtOAc/hexanes) to give the prod-
uct (0.045 g, 0.065 mmol, 33%). Further purification by HPLC
(silica, 25% EtOAc/hexanes, 3.0 mL/min, 254 nm, tR ) 19.7
min) provided the desired m-diacetyl dimer 9 as a white
solid: mp ) 78-79 °C; 1H NMR (400 MHz, CDCl3) δ 8.51 (m,
1 H), 8.14 (m, 2 H), 7.57 (m, 1 H), 5.34 (s, 2 H), 5.08 (ddd
unresolved, 2 H), 3.22 (dABq, J d ) 6.4 Hz, J AB ) 16.4 Hz, ∆νAB
) 93.4 Hz, 4 H), 2.81 (m, 2 H), 2.31 (ddd, J ) 14.4, 13.2, 4.0
Hz, 2 H), 1.99 (ddd, J ) 14.6, 4.8, 3.2 Hz, 2 H), 1.92 (m, 2 H),
1.84 (ddd, J ) 13.6, 8.0, 4.0 Hz, 2 H), 1.71 (m, 4 H), 1.47-1.22
(m, 10 H), 1.30 (s, 6 H), 0.97 (d, J ) 6.0 Hz, 6 H), 0.91 (d, J )
8.0 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 197.3, 137.2, 132.4,
128.9, 103.0, 89.4, 80.8, 70.2, 52.0, 44.1, 40.0, 37.5, 36.5, 34.4,
29.9, 25.8, 24.8, 20.1, 13.1; IR (CHCl3) 3007, 2956, 2929, 2877,
1688, 1598, 1452, 1433, 1378, 1359, 1180, 1124, 1092, 1053,
1012 cm-1; LRMS (CI, NH3, rel intensity) 712 (M + NH4+, 1),
620 (28), 446 (37), 400 (97), 383 (100), 369 (28), 365 (95), 284
(30), 270 (28), 222 (14), 183 (17); HRMS (CI, NH3) m/z calcd
for C40H58NO10 (M + NH4+) 712.4061, found 712.4069.
Dim eth oxyp h en yl Dim er 10. 12R-(2′,4′-Dimethoxyphen-
yl)-10-deoxoartemisinin (0.065 g, 0.16 mmol) and 10â-fluoro-
10-deoxoartemisinin (14; 0.023 g, 0.08 mmol) were dissolved
in dry dichloromethane (0.8 mL) and the solution was cooled
to -78 °C. Boron trifluoride diethyl etherate (14 mL, 0.11
mmol) was added and the reaction was stirred at -78 °C for
1 h. The solution was warmed to -40 °C over 1 h. After being
stirred at this temperature for an additional 4 h, the reaction
was quenched with saturated aqueous sodium bicarbonate (1
mL). The organic phase was separated, and the aqueous phase
was extracted with dichloromethane (2 × 10 mL). The organic
portion was combined, washed with water, dried with mag-
nesium sulfate, and concentrated under reduced pressure.
Purification by column chromatography on silica gel gave the
product (0.04 g, 0.059 mmol, 74%). Further purification by
4 H), 1.40 (s, 6 H), 1.60-1.00 (m, 12 H), 0.96 (d, J ) 6.0 Hz,
6 H), 0.66 (d, J ) 7.2 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ
152.9, 108.6, 104.1, 92.1, 80.4, 71.2, 52.0, 45.9, 37.3, 36.3, 34.1,
32.0, 26.0, 24.7, 21.3, 20.3, 13.8; IR (CHCl3) 2926, 2873, 1452,
1377, 1197, 1127, 1042, 926, 879, 754 cm-1; HRMS (CI, NH3)
m/z calcd for C34H52NO9 (M + NH4+) 618.3642, found 618.3650.
1,4-Dieth yn ylben zen e. To a solution of lithium diisopro-
pylamide (LDA; Aldrich; 1.5 M, 8.7 mL, 13 mmol) in THF (55
mL) at -78 °C was added a solution of 1,4-diacetylbenzene
(1.0 g, 6.2 mmol) in THF (10 mL) via syringe. The resulting
mixture was stirred at -78 °C for 1 h. To this mixture was
added diethyl chlorophosphate (2.0 mL, 14 mmol) via syringe.
The resulting dark brown mixture was stirred at -78 °C for 5
min, slowly warmed to room temperature, and stirred for 15
min. This orange mixture was cooled to -78 °C and added into
a -78 °C solution of LDA (Aldrich; 1.5 M, 19 mL, 28 mmol) in
THF (55 mL) via cannula. The resulting dark blue-green
mixture was stirred at -78 °C for 40 min, slowly warmed to
room temperature, and stirred for 3.5 h. At this time, TLC
analysis indicated complete consumption of starting material
diketone. The reaction was quenched with a HCl solution (1
M, 3 mL) and diluted with ether (20 mL). Two layers were
separated and the aqueous phase was extracted with ether
(30 mL × 2). The combined organic layers were washed with
a HCl solution (1 M, 10 mL) and a saturated NaHCO3 solution
(10 mL), dried over MgSO4, filtered, and concentrated under
reduced pressure. The crude product was purified by column
chromatography (flash, 1% EtOAc/hexanes) to furnish the
desired product (0.60 g, 4.76 mmol, 77%) as a white solid: mp
1
) 90.0-91.5 °C (lit. mp 96.5 °C); H NMR (400 MHz, CDCl3)
δ 7.44 (s, 4 H), 3.17 (s, 2 H); 13C NMR (100 MHz, CDCl3) δ
132.0, 122.5, 83.0, 79.1. These spectroscopic data matched
those reported previously.27-29
10-p-Dieth yn ylp h en yla r tem isin in Dim er 12. To a solu-
tion of 1,4-diethynylbenzene (0.12 g, 0.97 mmol) in ether (1
mL) at 0 °C was added n-butyllithium (1.6 M, 1.3 mL, 2.0
mmol). The resulting mixture was stirred at 0 °C for 45 min.
To this mixture at 0 °C was slowly added dimethylaluminum
chloride (1.0 M, 2.0 mL, 2.0 mmol) via syringe and the
resulting suspension was stirred at 0 °C for 2 h. To the reaction
mixture at -78 °C was added boron trifluoride diethyl etherate
(0.26 mL, 2.0 mmol) and a solution of â-fluoroartemisinin (14)
(0.58 g, 2.0 mmol) in CH2Cl2 (20 mL). The resulting reaction
mixture was stirred at -78 °C for 20 min, slowly warmed to
-50 °C, and stirred for 3 h. The reaction was quenched with
H2O (5 mL) and diluted with CHCl3 (10 mL). Two layers were
separated and the aqueous phase was extracted with CHCl3
(20 mL × 2). The combined organic layers were washed with
brine (20 mL), dried over MgSO4, filtered, and concentrated
under reduced pressure. The crude product was purified by
column chromatography (Florisil, 10%f20% EtOAc/hexanes)
to furnish the product (0.18 g, 0.27 mmol, 27%). Further
purification by HPLC (silica, 15% EtOAc/hexanes, 3.0 mL/min,
254 nm, tR ) 14.1 min) provided the desired p-diethynyl dimer
12 as a white solid: mp ) 164.5-166.0 °C; 1H NMR (400 MHz,
CDCl3) δ 7.34 (s, 4 H), 5.62 (s, 2 H), 4.96 (d, J ) 5.6 Hz, 2 H),
2.85 (m, 2 H), 2.38 (ddd, J ) 14.4, 13.6, 4.0 Hz, 2 H), 2.19
(apparent dq, J d ) 3.3 Hz, J q ) 13.6 Hz, 2 H), 2.06 (m, 2 H),
1.92-1.78 (m, 6 H), 1.70-1.51 (m, 6 H), 1.45 (s, 6 H), 1.42-
1.25 (m, 4 H), 1.04 (d, J ) 7.6 Hz, 6 H), 0.96 (d, J ) 6.4 Hz, 6
H); 13C NMR (100 MHz, CDCl3) δ 131.4, 122.7, 104.3, 89.6,
88.9, 88.3, 80.9, 67.8, 52.7, 45.4, 37.4, 36.3, 34.6, 30.3, 26.1,
24.6, 23.0, 20.3, 13.9; IR (CHCl3) 2999, 2956, 2928, 2875, 2854,
1508, 1452, 1379, 1369, 1042 cm-1; FAB-MS 659 (M + H+),
658 (M+), 307, 209, 182, 169, 165, 154, 136, 120, 107, 89, 77,
65, 43; HRMS (CI, NH3) m/z calcd for C40H51O8 (M + H+)
659.3584, found 659.3594.
HPLC (silica, 30% EtOAc/hexanes, 3.0 mL/min, 254 nm, tR
)
8.8 min) provided the desired dimer 10 as a white solid: mp
) 168.0-169.2 °C; [R]D25 ) +148.3 (c ) 0.89, CHCl3); 1H NMR
(300 MHz, CDCl3) δ 7.91 (br s, 1 H), 6.31 (s, 1 H), 5.39 (s, 2
H), 4.91 (d, J ) 9.9 Hz, 2 H), 3.76 (s, 6 H), 2.56 (m, 2 H), 2.33
(m, 2 H), 1.99 (m, 2 H), 1.89-0.88 (m, 18 H), 1.43 (s, 6 H),
0.95 (d, J ) 6.2 Hz, 6 H), 0.58 (d, J ) 7.0 Hz, 6 H); 13C NMR
(75 MHz, CDCl3) δ 157.1, 128.8, 122.8, 104.4, 94.2, 92.4, 80.6,
70.0, 56.1, 52.5, 46.8, 37.8, 36.9, 34.8, 26.4, 25.3, 22.0, 20.8,
13.9; IR (KBr) 2934, 2868, 1608, 1509, 1457, 1377, 1294, 1201,
1068, 883, 847 cm-1. Anal. Calcd for C18H54O10: C, 68.03; H,
8.11. Found: C, 67.88; H, 8.11.
F u r a n Dim er 11. 10R-(2′-Furyl)-10-deoxoartemisinin (0.022
g, 0.066 mmol) and 10â-fluoro-10-deoxoartemisinin (14; 0.019
g, 0.066 mmol) were dissolved in dry dichloromethane (1 mL)
and the solution was cooled to -78 °C. Boron trifluoride diethyl
etherate (0.011 g, 10 mL, 0.079 mmol) was added and the
reaction was warmed to -50 °C and kept at -50 °C for 4 h.
Saturated aqueous sodium bicarbonate (1 mL) was added. The
solution was extracted with dichloromethane (3 × 2 mL). The
combined organic solution was dried with magnesium sulfate,
concentrated under vacuum, and chromatographed on Florisil
to give the product (0.022 g, 0.037 mmol, 56%). Further
purification by HPLC (silica, 1% i-PrOH/CH2Cl2, 3.0 mL/min,
236 nm, Rt ) 5.0 min) provided the furan dimer 11 as a white
10-m -Dieth yn ylp h en yla r tem isin in Dim er 13. To a solu-
tion of 1,3-diethynylbenzene (TCI; 0.078 g, 0.62 mmol) in ether
(0.6 mL) at 0 °C was added n-butyllithium (1.6 M, 0.75 mL,
1.2 mmol). The resulting mixture was stirred at 0 °C for 45
min. To this mixture at 0 °C was slowly added dimethylalu-
minum chloride (1.0 M, 1.2 mL, 1.2 mmol) via syringe and the
resulting suspension was stirred at 0 °C for 2 h. To the reaction
25
1
foam: [R]D ) +56.8 (c ) 1.20, CHCl3); H NMR (400 MHz,
CDCl3) δ 6.33 (s, 2 H), 5.35 (s, 2 H), 4.46 (d, J ) 10.8 Hz, 2 H),
2.80-2.70 (m, 2 H), 2.37 (ddd, J ) 14.4, 13.6, 4.0 Hz, 2 H),
2.01 (ddd, J ) 14.4, 4.8, 2.8 Hz, 2 H), 1.88 (m, 2 H), 1.73 (m,

4280 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Posner et al.
(9) Venugopalan, B.; Bapat, C. P.; Karnik, P. J .; Catterjee, D. K.;
Iyer, N.; Lepcha, D. Antimalarial Activity of Novel Ring-
Contracted Artemisinin Derivatives. J . Med. Chem. 1995, 38,
1922-1927.
(10) Posner, G. H.; Ploypradith, P.; Hapangama, W.; Wang, D.;
Cumming, J . N.; Dolan, P.; Kensler, T. W.; Klinedinst, D.;
Shapiro, T. A.; Zheng, Q. Y.; Murray, C. K.; Pilkington, L. G.;
J ayasinghe, L. R.; Bray, J . F.; Daughenbaugh, R. Trioxane
Dimers Have Potent Antimalarial, Antiproliferative, and Anti-
tumor Activities In Vitro. Bioorg. Med. Chem. 1997, 5, 1257-
1265.
(11) Aboubdellah, A.; Be´gue´, J .-P.; Bonnet-Delpon, D.; Gontier, J .-
C.; Nga, T. T.; Thac, T. D. Synthesis and in vivo Antimalarial
Activity of 12R-Trifluoromethyl-hydroartemisinin. Bioorg. Med.
Chem. Lett. 1996, 6, 2717-2720.
(12) Pu, Y. M.; Ziffer, H. Synthesis and Antimalarial Activities of
12â-Allyldeoxoartemisinin. J . Med. Chem. 1995, 38, 613-616.
(13) Vroman, J . A.; Khan, I. A.; Avery, M. A. Copper(I)-Catalyzed
Conjugate Addition of Grignard Reagents to Acrylic Acids:
Homologation of Artemisinic Acid and Subsequent Conversion
to 9-Substituted Artemisinin Analogues. Tetrahedron Lett. 1997,
38, 6173-6176.
(14) 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.
(15) J ung, M.; Lee, S. Synthesis and Cytotoxicity of Novel Artemisinin
Analogues. Bioorg. Med. Chem. Lett. 1997, 7, 1091-1094.
(16) J ung, M.; Lee, S. Stability of Acetal and Nonacetal Type
Analogues of Artemisinin in Simulated Stomach Acid. Bioorg.
Med. Chem. Lett. 1998, 8, 1003-1006.
(17) Haynes, R. K.; Vonwiller, S. C. Efficient Preparation of Novel
Qinghaosu (Artemisinin) Derivatives. Synlett 1992, 481-483.
(18) Woo, S. H.; Parker, M. H.; Ploypradith, P.; Northrop, J .; Posner,
G. H. Direct Conversion of Anomeric OH f F f R in the
Artemisinin Family of Antimalarial Trioxanes. Tetrahedron Lett.
1998, 39, 1533-1536.
(19) Posner, G. H.; Parker, M. H.; Northrop, J .; Elias, J . S.; Ploypra-
dith, P.; Xie, S.; Shapiro, T. A. Orally Active, Hydrolytically
Stable, Semi-synthetic, Antimalarial Trioxanes in the Artemisi-
nin Family. J . Med. Chem. 1999, 42, 300-304.
(20) O’Dowd, H.; Ploypradith, P.; Xie, S.; Shapiro, T. A.; Posner, G.
H. Antimalarial Artemisinin Analogues. Synthesis via Chemose-
lective C-C Bond Formation and Preliminary Biological Evalu-
ation. Tetrahedron 1999, 55, 3625-3636.
mixture at -78 °C was added boron trifluoride diethyl etherate
(0.17 mL, 1.3 mmol) and a solution of â-fluoroartemisinin (14)
(0.38 g, 1.3 mmol) in CH2Cl2 (12 mL). The resulting reaction
mixture was stirred at -78 °C for 20 min, slowly warmed to
-50 °C, and stirred for 3 h. The reaction was quenched with
H2O (5 mL) and diluted with CHCl3 (10 mL). Two layers were
separated and the aqueous phase was extracted with CHCl3
(20 mL × 2). The combined organic layers were washed with
brine (20 mL), dried over MgSO4, filtered, and concentrated
under reduced pressure. The crude product was purified by
column chromatography (Florisil, 10%f20% EtOAc/hexanes)
to furnish the product (0.08 g, 0.12 mmol, 19%). Further
purification by HPLC (silica, 15% EtOAc/hexanes, 3.0 mL/min,
254 nm, tR ) 14.2 min) provided the desired m-diethynyl dimer
13 as a white foam: 1H NMR (300 MHz, CDCl3) δ 7.42-7.24
(m, 4 H), 5.62 (s, 2 H), 4.96 (d, J ) 5.6 Hz, 2 H), 2.85 (m, 2 H),
2.38 (ddd, J ) 14.4, 13.6, 4.0 Hz, 2 H), 2.19 (apparent dq, J d
) 3.3 Hz, J q ) 13.6 Hz, 2 H), 2.05 (m, 2 H), 1.92-1.78 (m, 6
H), 1.70-1.51 (m, 6 H), 1.45 (s, 6 H), 1.42-1.25 (m, 4 H), 1.04
(d, J ) 7.3 Hz, 6 H), 0.96 (d, J ) 6.2 Hz, 6 H); 13C NMR (75
MHz, CDCl3) δ 134.2, 131.4, 128.5, 123.0, 104.3, 89.6, 87.8,
87.7, 80.9, 67.8, 52.7, 45.4, 37.4, 36.3, 34.6, 30.3, 26.1, 24.6,
23.0, 20.3, 13.9; IR (CHCl3) 2925, 2869, 1458, 1372, 1189, 1137,
1050 cm-1
.
Ack n ow led gm en t. We thank the NIH (Grants AI-
34885, CA-44530, and RR-00052) and the Burroughs
Wellcome Fund for financial support, Dr. Anthony B.
Mauger (NCI) for the COMPARE data, the NCI’s
Developmental and Therapeutics program for evalua-
tion of these dimers, and Dr. Melinda Hollingshead
(NIH, Frederick, MD) for the hollow fiber assays.
Su p p or tin g In for m a tion Ava ila ble: Figures 3 and 4 as
bar graphs indicating total growth inhibition (TGI) for dimers
7, 8, 10, and 12 in the NCI 60-human cell line in vitro assay.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(21) Campbell, T. W.; McDonald, R. N. Synthesis of Hydrocarbon
Derivatives by the Wittig Synthesis. I. Distyrylbenzenes. J . Org.
Chem. 1959, 24, 1246-1251.
Refer en ces
(22) For experimental details of the testing protocol, including
references to development of the assay, see: Posner, G. H.;
Gonza´lez, L.; Cumming, J . N.; Klinedinst, D.; Shapiro, T. A.
Synthesis and Antimalarial Activity of Heteroatom-Containing
Bicyclic Endoperoxides. Tetrahedron 1997, 53, 37-50.
(23) Posner, G. H.; Nelson, T. D.; Guyton, K. Z.; Kensler, T. W. New
Vitamin D3 Derivatives Having Unexpected Antiproliferative
Activity: 1-Hydroxymethyl-25-hydroxyvitamin D3 Homologues.
J . Med. Chem. 1992, 35, 3280-3287.
(24) Holick, M. F., Ed. Vitamin D; Humana Press: Totowa, NJ , 1999.
(25) Boyd, M. R.; Paull, K. D. Some Practical Considerations and
Applications of the National Cancer Institute In Vitro Anticancer
Drug Discovery Screen. Drug Dev. Res. 1995, 34, 91-109.
(26) Zipp, A. P.; Zipp, S. G. Pt(NH3)2Cl2 and Cancer. J . Chem. Educ.
1977, 54, 739-741.
(1) (a) Current address: J ohnson & J ohnson, Spring House, PA.
(b) Current address: MethylGene, Inc., Montreal, Quebec. (c)
Recipient of a 1998 Pfizer Undergraduate Summer Fellowship.
(2) Chen, Y.-H.; Liu, J .-X.; Lown, J . W. Design, Synthesis and
Evaluation of Novel Bismustard Cross-linked Lexitropsins.
Bioorg. Med. Chem. Lett. 1995, 5, 2223-2228.
(3) Dro¨gemu¨ller, M.; Flessner, T.; J autelat, R.; Scholz, U.; Winter-
feldt, E. Synthesis of Cephalostatin Analogues by Symmetrical
and Non-Symmetrical Routes. Eur. J . Org. Chem. 1998, 2811-
2831.
(4) Reddy, B. S. P.; Damayanthi, Y.; Lown, J . W. Design and
Efficient Synthesis of Novel DNA Interstrand Cross-Linking
Agents: C2-Linked Pyrrolo[2,1-c][1,4]benzodiazepine Dimers.
Synlett 1999, 7, 1112-1114.
(5) Gregson, S. J .; Howard, P. W.; J enkins, T. C.; Kelland, L. R.;
Thurston, D. E. Synthesis of a Novel C2/C2′-exo Unsaturated
Pyrrolobenzodiazepine Cross-linking Agent With Remarkable
DNA Binding Affinity and Cytotoxicity. J . Chem. Soc., Chem.
Commun. 1999, 797-798.
(6) Borman, S. Higher Yield Synthetic Route to Cyclic Enediynes
Developed. Chem. Eng. News 1995, 28-30.
(27) Negishi, E.; King, A. O.; Klima, W. L. Conversion of Methyl
Ketones into Terminal Acetylenes and (E)-Trisubstituted Olefins
of Terpenoid Origin J . Org. Chem. 1980, 45, 2526-2528.
(28) Hay, A. S. Preparation of m- and p-Diethynylbenzenes. J . Org.
Chem. 1960, 25, 637.
(7) Beekman, A. C.; Barenstsen, A. R. W.; Woerdenbag, H. J .; Van
Uden, W.; Pras, N. Stereochemistry-Dependent Cytotoxicity of
Some Artemisinin Derivatives. J . Nat. Prod. 1997, 60, 325-330.
(8) Galal, A. M.; Ahmad, M. S.; El-Feraly, F. S. Preparation and
Characterization of a New Artemisinin-Derived Dimer. J . Nat.
Prod. 1996, 59, 917-920.
(29) Royles, B. J . L.; Smith, D. M. J . The ‘Inverse Electron-demand’
Diels-Alder Reaction in Polymer Synthesis. Part 1. A Conve-
nient Synthetic Route to Diethynyl Aromatic Compounds. J .
Chem. Soc., Perkin Trans 1 1994, 355-358.
J M990363D