Synthesis and immunosuppressive activity of new artemisinin derivatives. 1. [12(β or α)-dihydroartemisininoxy]phen(ox)yl aliphatic acids and esters

Article abstract of DOI:10.1021/jm048979c

A series of novel dihydroartemisinin derivatives were synthesized and evaluated on their immunosuppressive activity in the search for potential immunosuppressive agents with high efficacy and low toxicity. These compounds were assayed in their cytotoxicity of lymphocyte, inhibition activity on concanavalin A (ConA) induced T cell proliferation and lipopolysaccharide (LPS) induced B cell proliferation. Among them, 11b, 13b, 14d, 15b, 16, and 17 remarkably exhibited lower cytotoxicity and higher inhibition activity on the mitogen-induced T cell and B cell proliferation in comparison with artemisinin, artesunate, and artemether in vitro. More significantly, compound 11b displayed reduced cytotoxicity by over 100-fold compared with cyclosporin A (CsA) and comparable inhibition activity (SI = 848) on ConA-induced T cell proliferation to CsA (SI = 963) and more than 4000 times the inhibitory effect (SI = 28473) on LPS-induced B cell proliferation compared with CsA (SI = 7) in vitro. The in vivo experimental results showed that compound 16 could inhibit 2,4-dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH) reaction and sheep red blood cell (SRBC) induced antibody production, respectively. The structure and activity relationships (SAR) of these compounds were also discussed.

Full text of DOI:10.1021/jm048979c

4608
J. Med. Chem. 2005, 48, 4608-4617
Synthesis and Immunosuppressive Activity of New Artemisinin Derivatives. 1.
[12(â or r)-Dihydroartemisininoxy]phen(ox)yl Aliphatic Acids and Esters
Zhong-Shun Yang,§ Wen-Liang Zhou,‡ Yi Sui,§ Jun-Xia Wang,‡ Jin-Ming Wu,§ Yu Zhou,‡ Yu Zhang,§
Pei-Lan He,‡ Ji-Ye Han,§ Wei Tang,‡ Ying Li,*,§ and Jian-Ping Zuo*,‡
Department of Synthetic Chemistry and First Department of Pharmacology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences,
Chinese Academy of Sciences, Shanghai 201203, China
Received December 17, 2004
A series of novel dihydroartemisinin derivatives were synthesized and evaluated on their
immunosuppressive activity in the search for potential immunosuppressive agents with high
efficacy and low toxicity. These compounds were assayed in their cytotoxicity of lymphocyte,
inhibition activity on concanavalin A (ConA) induced T cell proliferation and lipopolysaccharide
(LPS) induced B cell proliferation. Among them, 11b, 13b, 14d, 15b, 16, and 17 remarkably
exhibited lower cytotoxicity and higher inhibition activity on the mitogen-induced T cell and B
cell proliferation in comparison with artemisinin, artesunate, and artemether in vitro. More
significantly, compound 11b displayed reduced cytotoxicity by over 100-fold compared with
cyclosporin A (CsA) and comparable inhibition activity (SI ) 848) on ConA-induced T cell
proliferation to CsA (SI ) 963) and more than 4000 times the inhibitory effect (SI ) 28473) on
LPS-induced B cell proliferation compared with CsA (SI ) 7) in vitro. The in vivo experimental
results showed that compound 16 could inhibit 2,4-dinitrofluorobenzene (DNFB)-induced
delayed-type hypersensitivity (DTH) reaction and sheep red blood cell (SRBC) induced antibody
production, respectively. The structure and activity relationships (SAR) of these compounds
were also discussed.
Introduction
Since Murry succeeded in carrying out the first kidney
transplant operation in 1954,1 organ transplantation
has grown tremendously during the last part of the 20th
century.2 The knowledge that the T-lymphocyte played
an integral role in transplant rejection brought cyclo-
sporine A (CsA) and tacrolimus (FK506) to the fore as
therapeutic immunosuppressants.3 However, the cur-
rently clinically used immunosuppressive drugs such as
glycocorticoids (e.g., cortisol), cyclosporin A (CsA), tac-
rolimus (FK506), and sirolimus (rapamycin), etc., de-
spite their undeniable clinical advantages, were proved
Figure 1. Artemisnin and its semisynthetic derivatives.
to possess rather serious side effects including renal
toxicity, liver toxicity, infection, malignancy, cosmetic
effects, and other bad responses.4-8
been studied in China. The experimental results sug-
gested that qinghaosu 1, dihydroartemisinin 2, arte-
mether 3, arteether 4, and artesunic acid 5 (Figure 1)
had definite immunosuppressive activity.11-18 Qinghao
extracts and qinghaosu were tried for treatment of the
systemic lupus erythematosus (SLE) patients.19 Besides,
high immunosuppressive action of sodium artesunate
had been demonstrated on SLE mice model and patients
with lupus erythematosus.20 Recently, both in vivo
(delayed-type hypersensitivity (DTH) response) and in
vitro (spectrofluorometry) studies indicated the activity
of artemisinin as an immunosuppressive agent was
more than that of cyclosporin A (CsA).21
In the search for new potential immunosuppressive
agents with high efficacy and low toxicity, we turned
our attention to the traditional Chinese medicine arte-
misinin (qinghaosu, 1). It is well-known that artemisinin
opened a new era in malaria chemotherapy.9 In fact,
artemisinin and its derivatives have a variety of bio-
activities besides their outstanding antimalarial activ-
ity. In general, antimalarial drugs possess immuno-
suppressive action and have been used for the treat-
ment of dermatosis.10 Since the 1980s, the immuno-
suppressive action of artemisinin and its derivatives has
Currently, nonsteroidal antiinflammatory drugs
(NSAIDs) and some immunosuppressive agents remain
the first-line therapy for most people with arthritis. In
addition, lessons from allograft tolerance could be
extended to the treatment of autoimmune diseases22
such as rheumatoid arthritis (RA)23-29 and SLE.30
* To whom correspondence should be addressed. For Y.L.: (address)
555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai, 201203,
China; (phone) +86-21-50805832; (fax) +86-21-50807088; (e-mail)
yli@mail.shcnc.ac.cn. For J.-P.Z.: (phone) +86-21-50806701; (fax) +86-
21-50806701; (e-mail) jpzuo@mail.shcnc.ac.cn.
§ Department of Synthetic Chemistry.
‡ First Department of Pharmacology.
10.1021/jm048979c CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/14/2005

Artemisinin Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4609
Chemistry
Dihydroartemisinin 2 was prepared by sodium boro-
hydride reduction of artemisinin 1 as previously re-
ported.32 Dihydroartemisinin acetate, 25, and 26
were prepared according to the literature proce-
dure.33 To probe their structure-activity relationships
(SAR), we synthesized eight classes of novel structure-
related dihydroartemisinin derivatives 10-17 (Figure
3).
Figure 2. Several important nonsteroidal anti-inflammatory
drugs (NSAIDs).
Considering the certain relevance of NSAIDs with the
immunosuppressive activity, we are interested in com-
bining artemisinin and the NSAIDs to develop a new
class of immunosuppressive agents. 2-Arylpropanoic
acids are a type of widely used nonsteroidal antiinflam-
matory drugs with excellent antipyretic, antiinflamma-
tory and analgesic effects. Among them, ibuprofen 7,
naproxen 8, and guaiacol ester of ibuprofen 9 were the
typical representatives31 with a general formula, 6
(Figure 2). We observed that each of them consists of
both a hydrophilic part (carboxylic group) and a lipo-
philic part (aromatic ring); thus, we supposed that
amphiphilicity may be one of the most important
features of these compounds.
In view of the aforementioned observations, we at-
tempted to link the artemisinin nucleus with various
arylaliphatic acids and esters, which are similar to
compound 6 in structure, to improve their bioactivity
and bioavailability. Thus, as part of an ongoing research
project in the search for highly potent, low toxicity,
specific-selective, and tolerance-inducing immuno-
suppressive agents, a series of [12(â or R)-dihydro-
artemisininoxy]phen(ox)yl aliphatic acids and esters
10-17 were synthesized and tested in cytotoxicity T cell
and B cell functional assays for evaluating their immuno-
suppressive activity in vitro. Furthermore, some of them
have been tested in vivo for their immunosuppressive
effects. Compound 16, the representative of these new
artemisinin derivatives, was investigated in the animal
models of 2,4-dinitrofluorobenzene (DNFB) induced
delayed-type hypersensitivity (DTH) reaction and sheep
red blood cell (SRBC) induced antibody production.
The new aryl ethers (10b, 11b, 12b, 12d, 13b, 14b,
14d, 15b) of dihydroartemisinin were prepared by
treatment of dihydroartemisinin acetate 18 with ap-
propriately substituted phenols 19-24 in the presence
of trifluoacetic acid (TFA) or boron trifluoride etherate
(BF3‚Et2O) at room temperature (Schemes 1-4). Similar
to previous work,33,34 these aryl ether derivatives are
almost all 12-â epimers under the catalysis of TFA, with
the exception of compound 12b wherein the 12-R epimer
is predominant. In our former work, the reaction of 18
with resorcinol afforded the 12-C glycoside compound
12h.35 However, a similar reaction of 18 with 21, the
monoether of resorcinol, gave not the 12-C glycoside
isomer 12g but only the 12-O glycoside isomer 12d
(Scheme 2). This result may be caused by the steric
hindrance effect of substrate 21 with a bulky substitu-
ent. These aryl ethers, after purification by column
chromatography, were hydrolyzed with 0.5% KOH/
EtOH solution followed by acidification and then re-
crystallization or column chromatography of the crude
products to furnish the corresponding free acids (10a,
11a, 12a, 12c, 13a, 14a, 14c, 15a), whose composition
(R/â) was the same as that of precursory esters. The free
acids then were converted into the corresponding target
esters (11c, 12e, 12f, 13c, 15c) in moderate to good
yields by condensation with the appropriate alcohol (for
11c) or phenol. It is worth noting that an additional
chiral center present in compounds 12a-f, 13a-c, and
15a-c was due to the employment of material (dl)-ethyl
Figure 3. Eight classes of novel dihydroartemisinin derivatives.

4610 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Scheme 1. Synthetic Routes to 10a,b and 11a-c
Yang et al.
Scheme 2. Synthetic Routes to 12a-h, 13a-c
Scheme 3. Synthetic Routes to 14a-d
2-bromopropionate or nonstereoselective preparation of
intermediate 24.
It was interesting that the racemization of the 11-
methyl group in the artemisinin nucleus from compound
11a arose during the course of hydrolysis, which was
embodied in its proton NMR spectra.
[12-â-Dihydroartemisininoxy]phenoxylsuccinic acid
16, structurally close to artesunate, was synthesized by
treatment of 25 and succinic anhydride in the presence
of pyridine and DMAP at room temperature. Compound
17 was prepared by condensation between 26 and
H-Gly-OEt‚HCl in the presence of HOBt at room tem-
perature (Scheme 5).
The starting substituted phenols 19, 20, and 23 were
prepared by methyl esterification of commercially avail-
able acids catalyzed with concentrated sulfuric acid.
Synthetic intermediates 21, 22, and 24 were prepared
according to the literature procedure.36,37

Artemisinin Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4611
Scheme 4. Synthetic Routes to 15a-c
Scheme 5. Synthetic Routes to 16 and 17
Results and Discussion
less potent than cyclosporin A (SI ) 963). However, the
cytotoxicity of compounds 10a, 11a, 11b, 13a, 13b, 14a,
and 17 was about 10- to 100-fold less than that ofarte-
mether and cyclosporin A. The SI values of inhibition
on ConA-induced T cell proliferation showed that com-
pounds 11b, 13b, 14d, 15b, 16, and 17 were much more
effective than artemether, among which compound 11b
(SI ) 848) is the most active one and comparable to
cyclosporin A (SI ) 963). The SI values of inhibition on
LPS-induced B cell proliferation indicated that the
derivatives of 11b, 13a, 13b, 13c, 14c, 14d, 16, and 17,
especially 11b (SI ) 28473), had much higher inhibition
activity than artemether (SI ) 49), even cyclosporin A
(SI ) 7). Compound 16, as a representative of these
derivatives, has been under systematic investigations
both in vitro and in vivo experiments. The immuno-
suppressive activity of compound 16 was tested in
BALB/c mice by DNFB-induced DTH response in com-
parison to CsA (Figure 4). The DTH reaction is a cell-
mediated pathologic response involved with T cell
activation and the production of many cytokines.38,39
Compound 16 could effectively reduce the ear swelling
in DTH mice at a dose of 20 mg/kg. Compound 16 was
also assessed on its immunosuppressive activity by
The new dihydroartemisinin derivatives were tested
in vitro for their cytotoxicity on murine spleen cells,
inhibition activity on concanavalin A (ConA) induced T
cell proliferation, and lipopolysaccharide (LPS) induced
B cell proliferation, with cyclosporin A (CsA), artemisi-
nin, artesunate, and artemether as the controls. The
pharmacological results of these compounds are sum-
marized in Table 1. The cytotoxicity of each compound
was expressed as the concentration of compound that
reduced cell viability to 50% (CC50). The immunosup-
pressive activity of each compound was expressed as the
concentration of compound that inhibited ConA-induced
T cell proliferation and LPS-induced B cell proliferation
to 50% (IC50) of the control value. The selective index
(SI) value was used to evaluate the bioactivity of
compounds. In the experiments for testing bioactivity
of each compound, results were expressed as the mean
( standard error. Student’s t-test was used to determine
variances between groups where appropriate, although
these data are not shown in Table 1. On the basis of
the inhibition of ConA-induced T lymphocyte prolifera-
tion, artemether (SI ) 23) was more potent than
artemisinin (SI ) 6) and artesunate (SI ) 7) but far

4612 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Yang et al.
Table 1. Inhibitory Effects of Cyclosporin A, Artemisinin, and
Its Derivatives on Spleen Lymphocyte Proliferation Induced by
Mitogens in Vitro
IC50 (M) [SIb]
inhibition of
ConA-induced
T lymphocyte
proliferation
inhibition of
LPS-induced
B lymphocyte
proliferation
cytotoxicity
CC50 (M)
compda
10a
11a
11b
11c
12a
12b
12c
12d
12e
12f
13a
13b
13c
14a
14b
14c
14d
15a
15b
15c
16
17
CsA
artemisinin
artemether
artesunate
3.25 × 10-4 5.25 × 10-6 [62]
3.54 × 10-4 5.34 × 10-6 [66]
2.07 × 10-4 2.44 × 10-7 [848]
2.08 × 10-6 1.96 × 10-6 [1]
3.51 × 10-5 8.60 × 10-6 [4]
1.36 × 10-6 1.73 × 10-6 [1]
5.54 × 10-6 7.64 × 10-6 [1]
2.99 × 10-6 3.59 × 10-6 [1]
2.99 × 10-6 4.12 × 10-6 [1]
8.23 × 10-6 5.73 × 10-6 [1]
1.70 × 10-5 [19]
5.98 × 10-6 [59]
7.27 × 10-9 [28473]
4.96 × 10-8 [42]
7.40 × 10-7 [47]
3.50 × 10-7 [4]
1.05 × 10-6 [5]
5.60 × 10-7 [5]
5.70 × 10-7 [5]
8.20 × 10-7 [10]
Figure 5. Suppressive effects of compound 16 on anti-SRBC
antibody production in vivo. BALB/c mice were given with
vehicle, compound 16 (20 mg/kg), or DEX (10 mg/kg) for 4 days.
Quantitative hemolysis of SRBC was determined as previously
described. Values represent the mean ( SEM ((///) p < 0.001))
compared with vehicle. n ) 12 mice/group. Three individual
experiments were performed with similar results.
>1.00 × 10-4 7.27 × 10-6 [>14] 8.60 × 10-7 [>116]
>1.00 × 10-4 2.60 × 10-7 [>385] 8.50 × 10-7 [>118]
3.91 × 10-5 2.33 × 10-6 [17]
>1.00 × 10-4 1.18 × 10-5 [>9]
8.00 × 10-6 7.13 × 10-7 [11]
1.04 × 10-5 4.00 × 10-6 [3]
4.17 × 10-5 1.38 × 10-7 [302]
5.96 × 10-5 3.60 × 10-6 [17]
6.53 × 10-6 1.37 × 10-8 [477]
5.33 × 10-6 4.80 × 10-7 [11]
5.27 × 10-5 3.27 × 10-7 [161]
1.30 × 10-4 2.53 × 10-6 [51]
1.29 × 10-6 1.34 × 10-9 [963]
2.83 × 10-5 4.43 × 10-6 [6]
8.60 × 10-5 3.82 × 10-6 [23]
3.29 × 10-5 4.58 × 10-6 [7]
2.70 × 10-7 [145]
1.02 × 10-5 [>10]
1.79 × 10-7 [45]
1.07 × 10-7 [97]
1.78 × 10-8 [2343]
8.60 × 10-6 [7]
Structure-Activity Relationships (SAR)
From the preliminary in vitro immunosuppressive
evaluation in Table 1, we could found the following. (i)
All the methyl or ethyl esters (except 14b) in the new
dihydroartemisinin derivatives showed a much more
potent inhibitory effect on T cell proliferation than their
corresponding acids, with their SI ratios of 11b to 11a
(848/66), 13b to 13a (385/14), 14d to 14c (302/3), and
15b to 15a (477/17) ranging from 13 to 101. (ii) All the
free acids (except 16) and their other esters exhibited
activity similar to that of artemether, even if their
structures differ from each other. (iii) As a group,
dihydroartemisinin derivatives 10a, 12a-f, and 14a,b
are almost equipotent but less potent than artemether
and CsA. Compounds 13a-c are more potent than their
homologous region isomers 12a-f. (iv) In comparison
with 14b, compounds 11b, 14d, and 15b structurally
has one more O atom between the benzene ring and
methoxycarbonylmethyl group, one more methano
(-CH2-) group in the aliphatic side chain, and another
methyl group at the vicinal position of carbonyl group.
However, the last three displayed similar immuno-
suppressive activity to each other but much more
efficacy than 14b. (v) Compound 13b was no more active
than 15b, although holding another O atom between
the benzene ring and the 2-substituted propionate
moiety. (vi) Compound 16 represented a more power-
ful inhibitory effect on T cell proliferation than its
structure-related prototype artesunate. Apparently, free
carboxylic acids, the hydrophilic part of some dihydro-
artemisinin derivatives, could not increase the immuno-
suppressive activity in vitro as we anticipated. Esteri-
fication of the free acids instead with proper small
aliphatic groups such as methyl and ethyl groups did
improve greatly their immunosuppressive activity.
7.80 × 10-7 [8]
9.47 × 10-8 [56]
2.65 × 10-7 [199]
5.37 × 10-8 [2421]
1.84 × 10-7 [7]
8.96 × 10-6 [3]
1.78 × 10-6 [48]
9.89 × 10-7 [33]
a The compounds tested for immunosuppressive activity were
consistent with the description in the Experimental Section;
b Selectivity index [SI] is determined as the ratio of the concentra-
tion of the compound that reduced cell viability to 50% (CC50) to
the concentration of the compound needed to inhibit the prolifera-
tion to 50% (IC50) of the control value.
Figure 4. Inhibitory effects of compound 16 on DNFB-induced
ear swelling in BALB/c mice. BALB/c mice were initially
sensitized with DNFB on days 0 and 1 and then challenged
with DNFB on day 9. Vehicle, compound 16 (20 mg/kg), and
CsA (20 mg/kg) were administered on days 1-3 before and
0.5 h after challenge. Ear swelling was calculated as the
difference between the weights of left (DNFB treated) and
right (untreated) ear punches 30 h after challenge. Values
represent the mean ( SEM ((/) P < 0.05, (//) P < 0.01)
compared with vehicle. n ) 12 mice/group. Three individual
experiments were performed with similar results.
Conclusions
quantitative hemolysis of SRBC (QHS) assay, which is
based on the principle that B lymphocytes are activated
in vivo by SRBC and could produce anti-SRBC specific
antibody ex vivo. Administration of compound 16 at a
dose of 20 mg/kg for 4 days effectively reduced the
hemolysis of SRBC more than dexamethasone (DEX)
(Figure 5).
In summary, a series of novel dihydroartemisinin
derivatives, potential immunosuppressants, have been
synthesized and assessed for their cytotoxicity of lym-
phocyte, inhibition activity on ConA-induced T cell
proliferation, and LPS-induced B cell proliferation in
comparison with artemisinin, artesunate, artemether,
and cyclosporin A (CsA) in vitro. Among them, 11b, 13b,

Artemisinin Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4613
then allowed to stand overnight, and the solvent was removed
under vacuum. To the residue was added 50% sodium hydrox-
ide solution (10 mL) and water (10 mL). The aqueous mixture
was then heated under reflux for 5 h and was then cooled and
made acidic to pH 2.0 with hydrochloride. To it was added
ethyl acetate (50 mL), and the two-phase mixture was stirred
for 5 min. It was then filtered through a filter aid pad, and
the pad was rinsed with EtOAc (20 mL). The organic layer of
the filtrate was separated, dried over anhydrous MgSO4, and
evaporated under vacuum to dryness. The residue was taken
up in ethyl acetate (30 mL) and petroleum ether (30 mL) and
stirred in an ice bath. The product crystallized within a few
minutes, and the suspension was stirred for 1 h before being
filtered. The solids were dried under vacuum at 40 °C to obtain
the desired product 24 (2.6 g, 80%). Its melting point was 128-
130 °C.
General Method for Preparation of New Dihydro-
artemisinin Derivatives. 1. Condensation of Dihydro-
artemisinin Acetate (18) with Substituted Phenols (19-
24) To Prepare New Aryl Ether Derivatives of Dihydro-
artemisnin (10b, 11b, 12b, 12d, 13b, 14b, 14d, 15b). To a
solution of dihydroartemisinin acetate (18, 3.26 g, 10 mmol)
and appropriate ethyl (or methyl) esters of the corresponding
substituted phenol (21-25, 12 mmol) in CH2Cl2 (60 mL) was
added 1.2 equiv of trifluoroacetic acid (TFA, 0.9 mL, 12 mmol)
or several drops BF3‚Et2O (for 21) at 0 °C. The mixture was
stirred at room temperature until the condensation reaction
was complete (monitored by TLC) and then washed by
saturated aqueous NaHCO3 solution, H2O, and brine, dried
over anhydrous MgSO4, and evaporated to dryness under
reduced pressure. The resultant crude products were purified
using silica gel chromatography with ethyl acetate-petroleum
ether (1:20, v/v) as the eluent to give new ether derivatives
(10b, 11b, 12b, 12d, 13b, 14b, 14d, 15b), whose yields ranged
from 30% to 50%. The Lewis acid TFA was employed for the
syntheses of all the new aryl ethers, with the exception of
compound 12d under the catalysis of BF3‚Et2O.
14d, 15b, 16, and 17 obviously exhibited their lower
cytotoxicity and higher inhibition activity on the mito-
gen-induced T cell and B cell proliferation compared
with the controls. It is worth noting that compound 11b
displayed the most outstanding immunosuppressive
effects in vitro, and it will be tested in additional in vitro
studies and in vivo animal model experiments. From
the preliminary in vivo tests of compound 16, the results
outlined the great potential of these compounds for
further exploitation as immunosuppressants.
In view of the above observations and analyses, we
presumed that one important factor contributing to high
immunosuppressive potency is the presence of a lipo-
philic artemisinin nucleus and an appropriate aliphatic
ester side chain. Further modification of analogue and
optimization of their synthetic strategy are now in
progress. The qualitative structure-activity relation-
ship was expected to be useful in guiding the design of
new immunosuppressive agents with high efficacy and
low toxicity.
Experimental Section
Chemistry. All commercially available reagents were used
without further purification unless otherwise stated. The
solvents used were all AR grade and were distilled under
positive pressure of dry nitrogen atmosphere where necessary.
BF3‚Et2O was redistilled by the standard methods. The
progress of the reactions was monitored by analytical thin-
layer chromatography (TLC) performed on homemade HSGF254
precoated silica gel plates. Visualization was performed by UV
or development using vanillin solution in sulfuric acid and
ethanol (4/1 v/v).
All melting points were taken in open capillary tubes on a
Buchi-510 melting point apparatus and are uncorrected. The
IR spectra through the range from 4000 to 600 cm-1 were run
on a Perkin-Elmer 599B spectrophotometer with KBr pellets
or as thin films and are reported in reciprocal centimeters
Methyl [3-(12-â-Artemisininoxy)]phenoxylpropionate
(10b). White crystals. Mp: 115-117 °C (from ethyl acetate-
1
petroleum ether). Yield: 36%. H NMR (400 MHz, CDCl3, δ
ppm): 6.83-7.26 (4H, m, Ar-H), 5.49 (1H, s, H-5), 5.48 (1H,
d, J ) 3.66 Hz, H-12), 4.33 (2H, t, J ) 7.69 Hz, Ar-OCH2-),
3.65 (3H, s, -OMe), 2.78 (1H, m, H-11), 2.62 (2H, d, J ) 7.13
Hz, -CH2CO-), 2.36 (1H, m, H-3â), 1.44 (3H, s, Me-15), 0.98
(3H, d, J ) 7.33 Hz, Me-13), 0.97 (3H, d, J ) 4.76 Hz, Me-14).
IR (KBr, cm-1): 1740 (CdO, ester), 1591 (CdC, benzene ring).
Anal. (C25H34O7) C, H.
1
(cm-1). H NMR spectra were determined in CDCl3 solution
on a Brucker AM-400 spectrophotometer at ambient temper-
ature. The chemical shifts are expressed as δ values in parts
per million (ppm) downfield from TMS as the internal stand-
ard, and the coupling constants (J) are given in hertz (Hz).
The peak patterns are indicated by the following abbrevia-
tions: bs ) broad singlet, d ) doublet, m ) multiple, p )
penta, q ) quarter, s ) singlet, and t ) triplet. Elemental
analyses were performed on a CE 1106 elemental analyzer,
and all the results had deviation within (0.4% of the theoreti-
cal values. Yields were of purified compounds and were not
optimized.
Methyl [4-(12-â-Artemisininoxy)]phenoxylacetate (11b).
White catkin/flossy crystals. Mp: 97-99 °C (from ethyl
1
acetate-petroleum ether). Yield: 30%. H NMR (400 MHz,
CDCl3, δ ppm): 7.02 (2H, d, J ) 9.15 Hz, Ar-H), 6.82 (2H, d,
J ) 9.16 Hz, Ar-H), 5.49 (1H, s, H-5), 5.36 (1H, d, J ) 3.29
Hz, H-12), 4.57 (2H, s, Ar-O-CH2-), 3.78 (3H, s, -OMe), 2.75
(1H, m, H-11), 2.36 (1H, m, H-3â), 1.42 (3H, s, Me-15), 0.99
(3H, d, J ) 7.32 Hz, Me-13), 0.94 (3H, d, J ) 6.22 Hz, Me-14).
IR (KBr, cm-1): 1770 (CdO, ester), 1508 (CdC, benzene ring).
Anal. (C24H32O8) C, H.
Preparation of Synthetic Intermediates 21, 22, 24.
Ethyl 2-(m or p-hydroxyphenoxy)propionate 21 and 22 were
prepared on the basis of the modified literature procedure,36
which is detailed below.
Ethyl 2-[3-(12-Artemisininoxy)phenoxylpropionate
To a solution of sodium ethoxide, prepared by dissolving
sodium (2.30 g, 100 mmol) in absolute EtOH (50 mL), there
was added consecutively resorcinol or hydroquinone (5.50 g,
50 mmol). Ethyl 2-bromopropionate (3.8 mL, 5.7 g, 33 mmol)
was added over 0.5 h at reflux. The mixture was refluxed
overnight, and the solvent was then removed by distillation.
After drying, EtOAc (50 mL) was added, and the organic
extract was washed with water and brine, dried over anhy-
drous MgSO4, and concentrated before purifying by chroma-
tography (20:1, petroleum ether/EtOAc) to give 21 (5.2 g, 50%)
or 22 (5.2 g, 50%) as a colorless liquid.
2-(4-Hydroxyphenyl)propanoic acid 24 was synthesized from
p-methoxyacetophenone according to the modified U.S. patent
method,37 and the detailed procedure modified is as follows.
DMF (50 mL) and sodium cyanide (2.7 g, 20.4 mmol) were
added to a solution of 1-(4-hydroxyphenyl)ethanol in DMF (10
mL). The mixture was stirred for 6 h at 130 °C, which was
(12b). 12-R + 12-â mixture, R/â ) 2/1. Colorless oil. Yield:
1
45%. H NMR (400 MHz, CDCl3, δ ppm): 7.16 (1H, m, Ar-
H), 6.78 (1H, m, Ar-H), 6.73 (1H, m, Ar-H), 6.49 (1H, m, Ar-
H), 5.54, 5.47 (1H, d × 2, J ) 2.20, 10.71 Hz, H-12), 5.46 (1H,
s × 2, H-5), 4.72 (1H, m, Ar-O-CH-Me), 3.93 (2H, m, -O-
CH2-Me), 2.78 (1H, m, H-11), 2.37 (1H, m, H-3â), 1.60 (3H,
m, Ar-O-CH-Me), 1.43 (3H, s × 2, Me-15), 1.26 (3H, m,
-OCH2Me), 1.00 (3H, d × 2, J ) 7.41, 7.42 Hz, Me-13), 0.96
(3H, d × 2, J ) 7.96, 8.98 Hz, Me-14). IR (film, cm-1): 1755,
1732 (CdO, ester), 1593, 1489 (CdC, benzene ring). Anal.
(C26H36O8) C, H.
Ethyl 2-[3-(12-r-Artemisininoxy)]phenoxylpropionate
(12d). Colorless oil. Yield: 40%. 1H NMR (400 MHz, CDCl3, δ
ppm): 7.15 (1H, td, J ) 8.16, 1.65 Hz, Ar-H), 6.76 (1H, m,
Ar-H), 6.65 (1H, t + d, J ) 5.05, 5.50 Hz, Ar-H), 6.49 (1H,
dt, J ) 8.25, 2.66 Hz, Ar-H), 5.47 (2H, s + d, J ) 8.25 Hz,

4614 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Yang et al.
H-5, H-12), 4.73 (1H, q × 2, J ) 6.78, 6.78 Hz, Ar-O-CH-
Me), 4.22 (2H, m, -O-CH2-), 2.78 (1H, m, H-11), 2.37 (1H,
m, H-3â), 1.60 (3H, d, J ) 6.78 Hz, Ar-O-CH-Me), 1.44 (3H,
s, Me-15), 1.26 (3H, m, -O-CH2-Me), 1.01 (3H, d × 2, J )
7.33, 7.31 Hz, Me-13), 0.97 (3H, d, J ) 6.05 Hz, Me-14). IR
(film, cm-1): 1755, 1734 (CdO, ester), 1593, 1489 (CdC,
benzene ring). Anal. (C26H36O8) C, H.
Ethyl 2-[4-(12-â-Artemisininoxy)]phenoxylpropionate
(13b). Colorless oil. Yield: 45%. 1H NMR (400 MHz, CDCl3, δ
ppm): 6.92 (2H, d, J ) 9.14 Hz, Ar-H), 6.72 (2H, dd, J ) 9.07,
2.27 Hz, Ar-H), 5.42 (1H, s, H-5), 5.27 (1H, d, J ) 3.43 Hz,
H-12), 4.56 (1H, q, J ) 6.82 Hz, Ar-O-CH-Me), 4.12 (2H, q
× 2, J ) 7.14, 7.00 Hz, -O-CH2-Me), 2.68 (1H, m, H-11),
2.29 (1H, m, H-3â), 1.50 (3H, d, J ) 6.73 Hz, Ar-O-CH-
Me), 1.35 (3H, s, Me-15), 1.18 (3H, m, -O-CH2-Me), 0.92
(3H, d, J ) 7.42 Hz, Me-13), 0.87 (3H, d, J ) 7.00 Hz, Me-14).
IR (film, cm-1): 1755, 1738 (CdO, ester), 1593, 1506 (CdC,
benzene ring). Anal. (C26H36O8) C, H.
ppm): 7.04 (2H, d, J ) 9.22 Hz, Ar-H), 6.84 (2H, d, J ) 9.20
Hz, Ar-H), 5.53, 5.49 (1H, s × 2, H-5), 5.68, 5.38 (1H, d × 2,
J ) 6.29, 3.35 Hz, H-12), 4.60 (2H, s, -OCH2-), 3.85 (1H, bs,
-OH), 2.76 (1H, m, H-11), 2.36 (1H, m, H-3â), 1.42 (3H, s,
Me-15), 1.00 (3H, d, J ) 7.17 Hz, Me-13), 0.94 (3H, d, J )
5.93 Hz, Me-14). IR (KBr, cm-1): 3429 (OH), 1724 (CdO,
carboxylic acid), 1662, 1506 (CdC, benzene ring). Anal.
(C23H30O8) C, H.
2-[3-(12-Artemisininoxy)]phenoxylpropionic Acid (12a).
12-R + 12-â mixture, R/â ) 2/1. White amorphous solid.
Yield: 95%. 1H NMR (400 MHz, CDCl3, δ ppm): 7.14 (1H, m,
Ar-H), 6.78-6.70 (1H, m, Ar-H), 6.53-6.42 (2H, m, Ar-H),
5.54 (1H, s, H-5), 5.48 (1H, d × 2, J ) 4.36, 10.70 Hz, H-12),
4.78 (1H, m, Ar-O-CH-Me), 2.80 (1H, m, H-11), 2.40 (1H,
m, H-3â), 1.65 (3H, d × 2, J ) 6.38, 7.38 Hz, Ar-O-CH-
Me), 1.43 (3H, s × 2, Me-15), 0.99 (3H, d × 2, J ) 7.39, 6.05
Hz, Me-13), 0.96 (3H, d × 2, J ) 6.05, 7.05 Hz, Me-14). IR
(KBr, cm-1): 3429 (OH), 1732 (CdO, carboxylic acid), 1603,
1491 (CdC, benzene ring). Anal. (C24H32O8) C, H.
2-[3-(12-r-Artemisininoxy)]phenoxylpropionicAcid(12c).
White amorphous solid. Yield: 95%. 1H NMR (400 MHz,
CDCl3, δ ppm): 7.19 (1H, t, J ) 8.25 Hz, Ar-H), 6.80 (1H, m,
Ar-H), 6.71 (1H, q, J ) 2.20 Hz, Ar-H), 6.54 (1H, d, J ) 8.11
Hz, Ar-H), 5.48 (2H, s + d, J ) 9.49 Hz, H-5, H-12), 4.81 (1H,
q × 2, J ) 6.97, 6.73 Hz, Ar-O-CH-Me), 2.79 (1H, m, H-11),
2.38 (1H, m, H-3â), 1.65 (3H, d, J ) 6.87 Hz, Ar-O-CH-
Me), 1.44 (3H, s, Me-15), 1.01 (3H, d, J ) 7.25 Hz, Me-13),
0.97 (3H, d, J ) 7.14 Hz, Me-14). IR (KBr, cm-1): 3417 (OH),
1732 (CdO, carboxylic acid), 1603, 1593, 1491(CdC, benzene
ring). Anal. (C24H32O8) C, H.
2-[4-(12-â-Artemisininoxy)]phenoxylpropionic Acid
(13a). White amorphous solid. Yield: 95%. 1H NMR (400 MHz,
CDCl3, δ ppm): 7.05 (2H, m, Ar-H), 6.85 (2H, d, J ) 9.07 Hz,
Ar-H), 5.50 (1H, s, H-5), 5.39 (1H, d, J ) 3.69 Hz, H-12), 4.72
(1H, q, J ) 6.72 Hz, Ar-O-CH-Me), 2.78 (1H, m, H-11), 2.36
(1H, m, H-3â), 1.62 (3H, d × 2, J ) 7.05, 6.71 Hz, Ar-O-
CH-Me), 1.43 (3H, s, Me-15), 1.01 (3H, d, J ) 7.38 Hz, Me-
13), 0.96 (3H, d, J ) 6.84 Hz, Me-14). IR (KBr, cm-1): 3435
(OH), 1732 (CdO, carboxylic acid), 1506 (CdC, benzene ring).
Anal. (C24H32O8) C, H.
Methyl [4-(12-â-Artemisininoxy)]phenylacetate (14b).
Colorless crystals. Mp: 108-112 °C (from ethyl acetate-
1
petroleum ether). Yield: 30%. H NMR (400 MHz, CDCl3, δ
ppm): 7.19 (2H, d, J ) 8.43 Hz, Ar-H), 7.06 (2H, d, J ) 8.61
Hz, Ar-H), 5.48 (1H, d, J ) 4.09 Hz, H-12), 5.43 (1H, s, H-5),
3.64 (3H, s, -OMe), 3.52 (2H, s, Ar-CH2-), 2.79 (1H, m, H-11),
2.37 (1H, ddd, J ) 4.03, 3.85, 3.85 Hz, H-3â), 1.43 (3H, s, Me-
15), 1.00 (3H, d, J ) 7.33 Hz, Me-13), 0.96 (3H, d, J ) 6.05
Hz, Me-14). IR (KBr, cm-1): 1734 (CdO, ester), 1512 (CdC,
benzene ring). Anal. (C24H32O7) C, H.
Methyl 3-[4-(12-â-Artemisininoxy)]phenylpropionate
(14d). White needle crystals. Mp: 82-84 °C (from ethyl
1
acetate-petroleum ether). Yield: 35%. H NMR (400 MHz,
CDCl3, δ ppm): 7.11 (2H, d, J ) 8.52 Hz, Ar-H), 7.03 (2H, d,
J ) 8.67 Hz, Ar-H), 5.49 (1H, s, H-5), 5.46 (1H, d, J ) 2.88
Hz, H-12), 3.67 (3H, s, -OMe), 2.89 (2H, t, J ) 7.84 Hz, Ar-
CH2-), 2.77 (1H, m, H-11), 2.59 (2H, t, J ) 7.77 Hz, -CH2-
CO-), 2.36 (1H, m, H-3â), 1.43 (3H, s, Me-15), 1.01 (3H, d, J
) 7.29 Hz, Me-13), 0.96 (3H, d, J ) 5.78 Hz, Me-14). IR (KBr,
cm-1): 1736 (CdO, ester), 1608, 1512 (CdC, benzene ring).
Anal. (C25H34O7) C, H.
Methyl 2-[4-(12-â-Artemisininoxy)]phenylpropionate
(15b). Colorless oil. Yield: 40%. 1H NMR (400 MHz, CDCl3, δ
ppm): 7.20 (2H, dd, J ) 8.69, 1.56 Hz, Ar-H), 7.06 (2H, dd, J
) 6.73, 2.06 Hz, Ar-H), 5.49 (1H, d, J ) 3.30 Hz, H-12), 5.47
(1H, s, H-5), 3.67 (1H, q, J ) 6.50 Hz, Ar-CH-Me), 3.64 (3H,
s, -OMe), 2.78 (1H, m, H-11), 2.36 (1H, m, H-3â), 1.46 (3H, d,
J ) 7.14 Hz, Ar-CH-Me), 1.43 (3H, s, Me-15), 1.00 (3H, d, J
) 7.42 Hz, Me-13), 0.95 (3H, d, J ) 9.62 Hz, Me-14). IR (film,
cm-1): 1738 (CdO, ester), 1610, 1510 (CdC, benzene ring).
Anal. (C25 H34O7) C, H.
2. Hydrolysis of Ethers To Prepare Corresponding
Free Acids 10a, 11a, 12a, 12c, 13a, 14a, 14c, 15a. The ether
(10b-15b, 12d-14d; 10 mmol) was dissolved in 0.5% KOH/
EtOH solution (150 mL), and the mixture was stirred at about
40-50 °C overnight. After neutralization with acetic acid, the
solution was evaporated in vaccum. The residue was dissolved
in EtOAc, washed with brine, dried over anhydrous MgSO4,
and evaporated to dryness under reduced pressure. The
resultant solid was purified by recrystallization or column
chromatography (silica gel, using ethyl acetate as the eluent)
to give the corresponding free acids (10a, 11a, 12a, 12c, 13a,
14a, 14c, 15a).
[4-(12-â-Artemisininoxy)]phenylacetic Acid (14a). White
crystals. Mp:154-156 °C (from ethyl acetate-petroleum ether).
Yield: 90%. 1 H NMR (400 MHz, CDCl3, δ ppm): 7.20 (2H, d,
J ) 8.79 Hz, Ar-H), 7.08 (2H, d, J ) 8.66 Hz, Ar-H), 5.49
(1H, d, J ) 3.57 Hz, H-12), 5.47 (1H, s, H-5), 3.59 (2H, s, Ar-
CH2-), 2.79 (1H, m, H-11), 2.38 (1H, m, H-3â), 1.43 (3H, s,
Me-15), 1.00 (3H, d, J ) 7.42 Hz, Me-13), 0.96 (3H, d, J )
6.04 Hz, Me-14). IR (KBr, cm-1): 3267 (OH), 1730 (CdO,
carboxylic acid), 1610, 1512 (CdC, benzene ring). Anal.
(C23H30O7) C, H.
3-[4-(12-â-Artemisininoxy)]phenylpropionic Acid (14c).
White amorphous solid. Yield: 90%. 1H NMR (400 MHz,
CDCl3, δ ppm): 7.07 (2H, d × 2, J ) 8.42, 8.42 Hz, Ar-H),
7.01, 6.75 (2H, d ×2, J ) 8.79, 8.43 Hz, Ar-H), 5.49 (1H, s,
H-5), 5.45 (1H, d, J ) 3.30 Hz, H-12), 2.87 (2H, td, J ) 7.51,
3.66 Hz, Ar-CH2-), 2.77 (1H, m, H-11), 2.62 (2H, t, J ) 7.69
Hz, -CH2-CO-), 2.36 (1H, m, H-3â), 1.42 (3H, s, Me-15), 0.99
(3H, d, J ) 7.32 Hz, Me-13), 0.94 (3H, d, J ) 5.86 Hz, Me-14).
IR (KBr, cm-1): 3400 (chel OH), 1709 (CdO, carboxylic acid),
1612, 1512 (CdC, benzene ring). Anal. (C24H32O7) C, H.
2-[4-(12-â-Artemisininoxy)]phenylpropionic Acid (15a).
White particle crystals. Mp: 152-154 °C (from ethyl acetate-
[3-(12-â-Artemisininoxy)]phenoxylacetic Acid (10a).
1
1
White amorphous solid. Yield: 90%. H NMR (400 MHz,
petroleum ether). Yield: 95%. H NMR (300 MHz, CDCl3, δ
CDCl3, δ ppm): 7.18 (1H, m, Ar-H), 6.77 (1H, d, J ) 8.05 Hz,
Ar-H), 6.70 (1H, s, Ar-H), 6.54 (1H, d, J ) 8.34 Hz, Ar-H),
5.48 (1H, d, J ) 3.35 Hz, H-12), 5.46 (1H, s, H-5), 4.64 (2H, s,
-OCH2-), 3.98 (1H, bs, -OH), 2.76 (1H, m, H-11), 2.36 (1H,
m, H-3â), 1.42 (3H, s, Me-15), 0.99 (3H, d, J ) 7.26 Hz, Me-
13), 0.94 (3H, d, J ) 5.98 Hz, Me-14). IR (KBr, cm-1): 3435
(OH), 1740 (CdO, carboxylic acid), 1595, 1491 (CdC, benzene
ring) Anal. (C23H30O8) C, H.
ppm): 7.24 (2H, m, Ar-H), 7.08 (2H, d, J ) 8.52 Hz, Ar-H),
5.49 (1H, d, J ) 3.30 Hz, H-12), 5.47 (1H, s, H-5), 3.68 (1H, q,
J ) 7.23 Hz, Ar-CH-Me), 2.79 (1H, m, H-11), 2.37 (1H, m,
H-3â), 1.48 (3H, d, J ) 7.14 Hz, Ar-CH-Me), 1.44 (3H, s,
Me-15), 1.00 (3H, d, J ) 7.42 Hz, Me-13), 0.96 (3H, d, J )
6.04 Hz, Me-14). IR (KBr, cm-1): 3400 (chel OH), 1725, 1705
(CdO, carboxylic acid), 1608, 1510 (CdC, benzene ring). Anal.
(C24H32O7) C, H.
[4-(12-â-Artemisininoxy)]phenoxylacetic Acid (11a).
3. Condensation of Acid with N,N-Dimethylethanol or
o-Methoxyphenol To Prepare Corresponding Esters 11c,
12e, 12f, 13c, 15c. To a solution of free acid (11a, 12a, 12c,
White needle crystals. Mp: 132-132.5 °C (from ethyl acetate-
1
petroleum ether). Yield: 90%. H NMR (400 MHz, CDCl3, δ

Artemisinin Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4615
13a, 15a; 2.0 mmol) and o-methoxyphenol (0.24 mL, 2.2 mmol)
or N,N-dimethylethanol (2.2 mmol, for 11a) in dry ether (50
mL), DCC (453.2 g, 2.2 mmol) and DMAP (cat. 24.4 mg, 0.1
mmol) were added, and the solution was stirred for 2-5 h at
room temperature. After the condensation was complete, the
solvent was evaporated and the solid residue was macerated
with cold acetone. The acetone-insoluble portion melted at
225-227 °C and identified as dicyclohexylurea (DCU). The
acetone-soluble fraction was evaporated to dryness, and the
residue was purified by column chromatography (silica gel,
using ethyl acetate-petroleum ether 1:20 v/v as the eleunt)
to give the target ester (11c, 12e, 12f, 13c, 15c).
Dimethylaminoethyl [4-(12-â-Artemisininoxy)]phen-
oxylacetate (11c). White amorphous solid. Yield: 45%. 1H
NMR (400 MHz, CDCl3, δ ppm): 7.04 (2H, d, J ) 9.14 Hz,
Ar-H), 6.83 (2H, d, J ) 9.16 Hz, Ar-H), 5.51 (1H, s, H-5),
5.37 (1H, d, J ) 3.30 Hz, H-12), 4.68 (2H, s, Ar-OCH2-), 3.93,
3.66 (2H, m, -COO-CH2-), 2.77 (1H, m, H-11), 2.38 (1H, m,
H-3â), 1.69 (2H, m, -CH2N-), 1.62 (6H, s, -NMe2), 1.43 (3H,
s, Me-15), 1.01 (3H, d, J ) 7.34 Hz, Me-13), 0.96 (3H, d, J )
6.24 Hz, Me-14). IR (KBr, cm-1): 1700, 1660 (CdO, ester),
1545, 1510 (CdC, benzene ring). Anal. (C27H39O8N) C, H, N.
2-Methoxyphenyl 2-[3-(12-â-Artemisininoxy)]phenoxyl-
propionate (12e). White amorphous solid. Yield: 40%. 1H
NMR (400 MHz, CDCl3, δ ppm): 7.20 (2H, td, J ) 8.24, 2.74
Hz, Ar-H), 7.00 (1H, d, J ) 7.83 Hz, Ar-H), 6.93 (2H, q, J )
7.96 Hz, Ar-H), 6.86-6.78 (1H, m + brd, J ) 8.52 Hz, Ar-
H), 6.76 (1H, dt, J ) 6.96, 2.27 Hz, Ar-H), 6.65 (1H, dt, J )
8.24, 2.74 Hz, Ar-H), 5.49 (1H, s, H-5), 5.51 (1H, d, J ) 3.43
Hz, H-12), 4.99 (1H, q, J ) 6.73 Hz, Ar-O-CH-Me), 3.78 (3H,
s, Ar-OMe), 2.79 (1H, m, H-11), 2.37 (1H, m, H-3â), 1.79 (3H,
d, J ) 6.73 Hz, Ar-O-CH-Me), 1.43 (3H, s, Me-15), 1.01 (3H,
d, J ) 7.42 Hz, Me-13), 0.92 (3H, d, J ) 6.05 Hz, Me-14). IR
(KBr, cm-1): 1782 (CdO, ester), 1606, 1502 (CdC, benzene
ring). Anal. (C31H38O9) C, H.
2-Methoxyphenyl 2-[3-(12-r-Artemisininoxy)]phenoxyl-
propionate (12f). White amorphous solid. Yield: 30%. 1H
NMR (400 MHz, CDCl3, δ ppm): 7.18 (2H, m, Ar-H), 6.94
(3H, m, Ar-H), 6.77 (2H, m, Ar-H), 6.65 (1H, m, Ar-H), 5.48
(1H, s, H-5), 5.06 (1H, d, J ) 9.34 Hz, H-12), 4.99 (1H, q, J )
6.86 Hz, Ar-OCH-Me), 3.77 (3H, s, Ar-OMe), 2.71 (1H, m,
H-11), 1.80 (3H, d × 2, J ) 6.87, 6.73 Hz, Ar-OCH-Me), 1.43
(3H, s, Me-15), 0.99 (3H, d, J ) 7.00 Hz, Me-13), 0.97 (3H, d,
J ) 7.14 Hz, Me-14). IR (KBr, cm-1): 1782 (CdO, ester), 1606,
1502 (CdC, benzene ring). Anal. (C31H38O9) C, H.
2-Methoxyphenyl 2-[4-(12-â-Artemisininoxy)]phenoxyl-
propionate (13c). White particle crystal (from ethyl acetate-
petroleum ether). Mp: 142-144 °C. Yield: 45%. 1H NMR (400
MHz, CDCl3, δ ppm): 7.20 (1H, td, J ) 7.65, 1.97 Hz, Ar-H),
7.14 (2H, d, J ) 10.18 Hz, Ar-H), 6.98 (1H, m, Ar-H), 6.94
(4H, m, Ar-H), 5.52 (1H, s, H-5), 5.39 (1H, d, J ) 3.44 Hz,
H-12), 4.92 (1H, q × 2, J ) 6.73, 6.86 Hz, Ar-O-CH-Me),
3.79 (3H, s, Ar-OMe), 2.78 (1H, m, H-11), 2.38 (1H, m, H-3â),
1.78 (3H, d, J ) 6.87 Hz, Ar-O-CH-Me), 1.03 (3H, d, J )
7.28 Hz, Me-13), 0.97 (3H, d, J ) 6.18 Hz, Me-14). IR (KBr,
cm-1): 1774 (CdO, ester), 1610, 1504 (CdC, benzene ring).
Anal. (C31H38O9) C, H.
2-Methoxylphenol 2-[4-(12-â-Artemisininoxy)]phenyl-
propionate (15c). White amorphous solid. Yield: 75%. 1H
NMR (400 MHz, CDCl3, δ ppm): 7.34 (2H, d, J ) 8.40 Hz,
Ar-H), 7.16 (1H, m, Ar-H), 7.11 (2H, dd, J ) 8.80, 0.80 Hz,
Ar-H), 6.96-6.87 (3H, m, Ar-H), 5.52 (1H, d, J ) 2.40 Hz,
H-12), 4.49 (1H, s, H-5), 3.96 (1H, q, J ) 7.20 Hz, Ar-CH-
Me), 3.74 (3H, s, Ar-OMe), 2.80 (1H, m, H-11), 2.38 (1H, m,
H-3â), 1.59 (3H, d, J ) 7.20 Hz, Ar-CH-Me), 1.44 (3H, s,
Me-15), 1.02 (3H, d, J ) 7.68 Hz, Me-13), 0.96 (3H, d, J )
6.00 Hz, Me-14). IR (KBr, cm-1): 1759 (CdO, ester), 1608,
1600, 1582 (CdC, benzene ring). Anal. (C31H38O8) C, H.
then washed with 10% HCl, H2O, and brine and dried over
anhydrous MgSO4. After evaporation of the solvent under
reduced pressure, the crude products were purified with a
silica gel chromatography using ethyl acetate-petroleum ether
(1:5, v/v) as the eluent to give the target acid 16 (3.1 g, 63%)
as a white amorphous solid. 1H NMR (400 MHz, CDCl3, δ
ppm): 7.25 (1H, s, Ar-H), 7.00 (1H, dd, J ) 8.51, 2.23 Hz,
Ar-H), 6.84 (1H, t, J ) 2.23 Hz, Ar-H), 6.72 (1H, dd, J )
7.80, 2.01 Hz, Ar-H), 5.46 (1H, d, J ) 3.55 Hz, H-12), 5.45
(1H, s, H-5), 2.98 (1H, bs, -OH), 2.85 (2H, t, J ) 6.87 Hz,
-CH2COOH), 2.77 (2H, t, J ) 6.67, 7.12 Hz, Ar-OCOCH2-),
2.36 (1H, m, H-3â), 1.42 (3H, s, Me-15), 0.98 (3H, d, J ) 7.50
Hz, Me-13), 0.94 (3H, d, J ) 6.10 Hz, Me-14). IR (KBr, cm-1):
3300 (chel OH), 1786, 1765 (CdO, ester), 1740, 1713 (CdO,
carboxylic acid), 1608, 1600, 1487 (CdC, benzene ring). Anal.
(C25H32O9‚3/4H2O) C, H.
Synthesis of Ethyl [4-(12-â-Artemisininoxy)]benzoyl-
aminoacetate (17). A solution of 26 (4.0 g, 10 mmol), H-Gly-
OEt‚HCl (2.8 g, 20 mmol), triethylamine (TEA, 2.8 mL, 20
mmol), and 1-hydroxybenzotriazole (HOBt, 2.0 g, 15 mmol) in
dry dichloromethane (DCM, 80 mL) was stirred at room
temperature for 5 h. After completion, the reaction mixture
was washed with water and brine and and dried over anhy-
drous MgSO4. Concentration and purification with silica gel
chromatography using ethyl acetate-petroleum ether (1:20,
v/v) as the eluent afforded the target compound 17 (3.9 g, 80%)
as a white amorphous solid. 1H NMR (400 MHz, CDCl3, δ
ppm): 7.78 (2H, d, J ) 8.79 Hz, Ar-H), 7.15 (2H, d, J ) 8.80
Hz, Ar-H), 6.59 (1H, t, J ) 4.88 Hz, -NH-), 5.55 (1H, d, J )
3.29 Hz, H-12), 5.45 (1H, s, H-5), 4.26 (2H, q, J ) 7.15 Hz,
-OCH2-), 4.22 (2H, d, J ) 5.08 Hz, -NCH2-), 2.83 (1H, m,
H-11), 2.37 (1H, m, H-3â), 1.44 (3H, s, Me-15), 1.31 (3H, t, J
) 7.08 Hz, -OCH2Me), 1.03 (3H, d, J ) 7.42 Hz, Me-13), 0.96
(3H, d, J ) 6.18 Hz, Me-14). IR (KBr, cm-1): 3371 (NH), 1755
(CdO, ester), 1645 (CdO, acylamide), 1608, 1504 (CdC,
benzene ring). Anal. (C26H35O8N) C, H, N.
Biological Assay. Materials. Stock solutions of compounds
were prepared with 100% dimethylsulfoxide (DMSO, Sigma)
and diluted with RPMI 1640 medium containing 10% fetal
bovine serum (FBS). RPMI 1640 medium powder was pur-
chased from Gibco BRL, Life Technologies. MTT [3-(4,5-
dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide],
Concanavalin A (ConA), and lipopolysaccharide (LPS) were
purchased from Sigma. [3H]Thymidine (1 mCi/mL) was pur-
chased from Shanghai Institute of Atomic Energy (SIAE). The
related in vitro experimental procedure was performed ac-
cording to our previous work.40
Animals. BALB/c mice, used at 6-8 weeks of age were
purchased from Shanghai Experimental Animal Center and
were housed in a controlled environment (12 h of light/12 h of
dark photoperiod, 22 ( 1 °C, 55% ( 5% relative humidity).
All husbandry and experimental contacts made with the mice
were conducted under specific pathogen-free conditions. All
mice were allowed to acclimatize in our facility for 1 week
before any experiment started. All experiments were carried
out according to the NIH Guide for Care and Use of Laboratory
Animals and were approved by the Bioethics Committee of the
Shanghai Institute of Materia Medica.
Preparation of Spleen Cell from Mice. BALB/C mice
were sacrificed, and spleens were removed aseptically. A single
cell suspension was prepared after cell debris, and clumps were
removed. Erythrocytes were depleted with ammonium chloride
buffer solution. Lymphocytes were washed three times with
PBS containing 2% FBS and were resuspended in RPMI 1640
medium at the indicated concentration.
Cytotoxicity Assay. Fresh spleen cells were obtained from
BALB/C mice (male, 7-9 weeks old). Spleen cells (5 × 105 cells)
were cultured in 96-well flat plates with 200 µL of RPMI 1640
media containing 10% FBS, 100 U/mL penicillin, and 100 µg/
mL streptomycin in a humidified, 37 °C, 5% CO2-containing
incubator for 48 h in the presence or absence of various
concentrations of compounds. An amount of 18 µL of MTT (5
mg/mL) was added to each well at the final 5 h of culture. Then
90 µL of lysis buffer (10% SDS, 50% DMF, pH 7.2) was added
Synthesis of [3-(12-â-Artemisininoxy)]phenoxylsuc-
cinic Acid (16). To a solution of 25 (3.6 g, 10 mmol) in dry
CH2Cl2 (60 mL) was added succinic anhydride (SAA, 1.2 g, 12
mmol), pyridine (1.0 mL, 12 mmol), and catalytic DMAP (1.2
mg, 1 mmol) at room temperature. The mixture was stirred
for 2 h at the same conditions until the reaction was complete,

4616 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Yang et al.
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to each well for 6-7 h and the absorbance values at 570 nm
were collected by microplate reader (Bio-Rad, model 550). The
percentage of cell death was determined using the following
formula:
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cytotoxicity (%) )
compounds (OD570) - background (OD570
)
× 100
control (OD570) - background (OD570
)
T Cell and B Cell Function Assay. Fresh spleen cells
were obtained from BALB/C mice (male, 7-9 weeks old). The
5 × 105 spleen cells were cultured at the same conditions as
those mentioned above. The cultures were unstimulated or
stimulated with 5 µg/mL of concanavalin A (ConA) or 10 µg/
mL of lipopolysaccharide (LPS) to induce T cells or B cells
proliferative responses, respectively. The compounds were
added to cultures with indicated concentrations to test their
bioactivities. Proliferation was assessed in terms of uptake of
[3H]thymidine during 8 h of pulsing with 20 kBq of [3H]thy-
midine for each well, and then cells will be harvested onto glass
fiber filters by a Basic 96 harvester. The incorporated radio-
activity was counted by a liquid scintillation counter (1540
MicroBeta Trilux, PerkinElmer Life Sciences).
DNFB-Induced Delayed-Type Hypersensitivity (DTH)
Response. The DTH assay was carried out as previously
described with some modifications.40 Briefly, mice were ini-
tially sensitized with 0.5% DNFB (2,4-dinitrofluorobenzene,
Pharmigen) dissolved in acetone-olive oil (4:1) on each hind
foot on days 0 and 1. On day 9, mice were challenged with
0.4% DNFB on both sides of their left ear. Vehicle, compound
16 (20 mg/kg), and cyclosporine A (CsA, 20 mg/kg) were
administered to each group intraperitoneally (ip) on days 1-3,
before and 0.5 h after challenge. At least 12 BALB/c mice were
prepared for each group. Ear swelling was expressed as the
difference between the weight of the left and right ear patches
obtained from 8 mm punches 30 h after challenge. The punches
were obtained in a blinded manner.
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Acknowledgment. This study was supported by the
Knowledge Innovation Program of Chinese Academy of
Sciences (Grant No. KSCX2-SW-202). We also thank
Prof. Y. Q. Long for her advice and encouragement.
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Supporting Information Available: Elemental analysis
data for the target compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
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