Synthesis and immunosuppressive activity of new artemisinin derivatives. Part 2: 2-[12(β or α)-Dihydroartemisinoxymethyl(or 1′-ethyl)]phenoxyl propionic acids and esters

Article abstract of DOI:10.1016/j.bmc.2006.07.038

Another series of novel dihydroartemisinin derivatives were synthesized and assessed for their cytotoxicity of lymphocyte, inhibitory activity on mitogen-induced spleen lymphocyte proliferation in vitro. Some of the compounds exhibited inhibitory effects on ConA-induced T cell and LPS-induced B cell proliferation comparable to or more potent than parent artemisinin.

Full text of DOI:10.1016/j.bmc.2006.07.038

Bioorganic & Medicinal Chemistry 14 (2006) 8043–8049
Synthesis and immunosuppressive activity of new artemisinin
derivatives. Part 2: 2-[12(b or a)-Dihydroartemisinoxymethyl-
(or 10-ethyl)]phenoxyl propionic acids and esters
Zhong-Shun Yang,a Jun-Xia Wang,b Yu Zhou,b Jian-Ping Zuob,* and Ying Lia,*
aDepartment of Synthetic Chemistry, Shanghai Institute of Materia Medica,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China
bFirst Department of Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China
Received 28 June 2006; revised 20 July 2006; accepted 21 July 2006
Abstract—Another series of novel dihydroartemisinin derivatives were synthesized and assessed for their cytotoxicity of lymphocyte,
inhibitory activity on mitogen-induced spleen lymphocyte proliferation in vitro. Some of the compounds exhibited inhibitory effects
on ConA-induced T cell and LPS-induced B cell proliferation comparable to or more potent than parent artemisinin.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
O O
O
O O
O
The immunosuppressive action of artemisinin and its
derivatives has been studied in China for many years.
Many experimental results in vitro and in vivo suggested
that the new type of antimalarial drugs, such as artemis-
inin (qinghaosu) 1, dihydroartemisinin 2, artemether 3,
and artesunic acid 4 (Fig. 1), possessed definite immuno-
suppressive activity.1–7
O
O
OH
2
O
1
O O
O
O O
O
In search for new potential immunosuppressive agents
with much higher efficacy and lower toxicity, we synthe-
sized a class of novel artemisinin derivatives 5–8 (Fig. 2)
and found that introduction of phen(ox)yl aliphatic acid
and ester into artemisinin nucleus did enhance their
immunosuppressive activity.8
O
O
OMe
OCOCH2CH2COOH
3
4
Figure 1. Artemisinin and its first-generation semi-synthetic
derivatives.
Herein, we will report our continuous work, that is,
synthesis and immunosuppressive activity of another
series of new artemisinin derivatives 9. Compared
with compound 8, the only difference in the chemical
structure of 9 is the link between artemisinin nucleus
and phenoxyl aliphatic acid or ester, that is OCH2
or OCH(Me) group instead of O atom (Fig. 3).
These compounds were tested in MTT assay for
their cytotoxicity, T cell and B cell functional assays
for evaluating their immunosuppressive activity
in vitro.
Keywords: Artemisinin derivatives; Synthesis; Immunosuppressive
activity; Inhibitory effect.
*
Corresponding authors. Tel./fax: +86 21 50806701 (J.-P.Z); Tel.: +86
21 50805832; fax: +86 21 50807088 (Y.L.); e-mail addresses: jpzuo@
mail.shcnc.ac.cn; yli@mail.shcnc.ac.cn
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.038

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Z.-S. Yang et al. / Bioorg. Med. Chem. 14 (2006) 8043–8049
mers and can be separated by column chromatography.
These benzyl ethers then were hydrolyzed with 0.5%
KOH/EtOH solution to give the corresponding free
acids (9a, 9c, 9f, 9h, 9k, and 9m). These free acids finally
were converted into their corresponding aryl esters
(9e, 9j, and 9o) by coupling with o-methoxyphenol in
moderate to good yields.
O O
O
O O
O
O
O
O
O
(CH2)nCOOR
O(CH2)nCOOR
5
6
Synthetic intermediates 10–12 were prepared according
to literature procedure starting with hydroxybenzalde-
hyde or hydroxyacetophenone.10
O O
O
O O
O
Because compounds 9 with one or two chiral centers in
the side chain are diastereoisomeric mixture, the proton
assignment in their 1H NMR spectra became more diffi-
cult. But in the cases of free acids or para substituent
benzyl derivatives, their H NMR data may be easily
confirmed.
O
O
H
H
O
COOR
Me
O
O
COOR
1
8
7
Me
Figure 2. Artemisinin derivatives 5–8.
2.2. Biological activity
The new dihydroartemisinin derivatives were tested in vi-
tro for their cytotoxicity on murine spleen cells, and
inhibitory activity on concanavalin A (ConA)-induced
T cell proliferation or lipopolysaccharide (LPS)-induced
B cell proliferation with artemisinin, artemether, and
artesunate as the controls. The pharmacological results
of these compounds are summarized in Table 1. The
cytotoxicity of each compound was expressed as the
concentration of compound that reduced cell viability
to 50% (CC50). The immunosuppressive activity of each
compound was expressed as the concentration of com-
pound 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.
1
R' = H, Me;
R = H, Et,
10
3
O O
7
O 5
11
12
O
MeO
16
H
O
O
COOR
R'
9
Me
Figure 3. New class of artemisinin derivatives 9.
2. Results and discussion
2.1. Chemistry
The results showed that, among them, 9a, 9e, 9h, and 9j
had 5- to 9-fold higher bioactivity than artemisinin in
the ConA-induced T cell proliferation. In the inhibition
of LPS-induced B cell proliferation, 9e, 9f, 9g, 9j, 9k,
9m, 9n, and 9o exhibited 30- to 88-fold higher bioactivity
than artemisinin. It was unfortunate that the cytotoxic-
ity of compounds 9 examined in MTT assay relatively
increased compared with that of artemisinin. It seemed
The new dihydroartemisinin benzyl ethers (9b, 9d, 9g, 9j,
9l, and 9n) were prepared by treatment of dihydroarte-
misinin with appropriately substituted phenols 10–12
in the presence of boron trifluoride etherate (BF3ÆEt2O)
at room temperature (Schemes 1 and 2). Similar to our
previous work,9 these benzyl ether derivatives (except
for 9n) were composed of almost equal 12b and 12a epi-
Me
COOEt
NaOEt/EtOH, ref.
KBH4/MeOH
0-5 ˚C-r.t.
H2
HO C
H2
HO C
Br
OHC
OH
O
COOEt
OH
13, m
14, p
10, m
11, p
Me
HO
MeO
DCC, DMAP,
ether, r.t.
O O
O O
O
O
0.5%KOH/EtOH
40-50 ˚C
.
BF3 Et2O/CH2Cl2
0-5 ˚C-r.t.
O
9e, 9j
O
2
H2
O C
H2
O C
O
COOH
O
COOEt
9b, 9d, 9g, 9i
9a, 9c, 9f, 9h
Me
Me
Scheme 1. Synthetic routes to 9a–j.

Z.-S. Yang et al. / Bioorg. Med. Chem. 14 (2006) 8043–8049
8045
Me
O
NaBH4/MeOH
0-5 ˚C-r.t.
Br
COOEt
HO
O
COOEt
HO
OH
Me
OH
NaOEt/EtOH, ref.
Me
Me
Me
15
12
HO
MeO
DCC, DMAP,
ether, r.t.
O O
O
O O
O
0.5%KOH/EtOH
40-50 ˚C
.
BF3 Et2O/CH2Cl2
9o
2
O
O
0-5 ˚C-r.t.
O
O
COOEt
O
O
COOH
Me
9l, 9n
Me
Me
Me
9k, 9m
Scheme 2. Synthetic routes to 9k–o.
Table 1. Inhibitory effects of artemisinin and its derivatives 9 on spleen lymphocyte proliferation induced by T cell or B cell mitogen (ConA or LPS)
in vitro
Compound
CC50 (M)
IC50 (M), [SI]a
T lymphocyte
B lymphocyte
9a
9b
1.2 · 10ꢀ5
7.7 · 10ꢀ6
1.1 · 10ꢀ5
8.2 · 10ꢀ6
3.2 · 10ꢀ6
2.7 · 10ꢀ6
8.8 · 10ꢀ6
1.3 · 10ꢀ5
2.4 · 10ꢀ5
2.7 · 10ꢀ6
9.5 · 10ꢀ6
6.6 · 10ꢀ7
1.1 · 10ꢀ5
1.3 · 10ꢀ5
3.2 · 10ꢀ6
2.8 · 10ꢀ5
8.6 · 10ꢀ5
3.3 · 10ꢀ5
6.8 · 10ꢀ7 [17.4]
9.1 · 10ꢀ7 [8.5]
2.4 · 10ꢀ6 [4.4]
4.2 · 10ꢀ6 [2.0]
4.6 · 10ꢀ7 [6.9]
1.4 · 10ꢀ6 [1.9]
1.8 · 10ꢀ6 [4.8]
7.0 · 10ꢀ7 [19.0]
2.4 · 10ꢀ6 [9.8]
8.4 · 10ꢀ7 [3.3]
9.8 · 10ꢀ7 [9.7]
3.6 · 10ꢀ6 [<1]
4.4 · 10ꢀ6 [2.4]
1.4 · 10ꢀ5 [<1]
1.4 · 10ꢀ6 [2.3]
4.4 · 10ꢀ6 [6]
8.0 · 10ꢀ7 [14.8]
5.0 · 10ꢀ7 [15.4]
4.5 · 10ꢀ7 [23.6]
6.7 · 10ꢀ7 [12.2]
2.8 · 10ꢀ7 [11.5]
1.6 · 10ꢀ7 [16.9]
3.0 · 10ꢀ7 [29.8]
7.6 · 10ꢀ7 [17.5]
4.0 · 10ꢀ7 [59.0]
2.2 · 10ꢀ7 [12.3]
1.8 · 10ꢀ7 [52.9]
5.0 · 10ꢀ7 [1.3]
2.8 · 10ꢀ7 [38.2]
1.0 · 10ꢀ7 [128.4]
1.7 · 10ꢀ7 [18.6]
9.0 · 10ꢀ6 [3]
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
Artemisinin
Artemether
Artesunate
3.8 · 10ꢀ6 [23]
4.6 · 10ꢀ6 [7]
1.8 · 10ꢀ6 [48]
9.9 · 10ꢀ7 [33]
a Selectivity index [SI] is determined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50) to the concentration
of the compound needed to inhibit the proliferation to 50% (IC50) of the control value.
that the inhibitory effect of most artemisinin derivatives
9 on ConA-induced T cell proliferation was comparable
to or slightly higher than that of parent artemisinin.
While their inhibitory activity (except for 9l) on LPS-
induced B cell proliferation was much more potent than
that of artemisinin.
In the current study, it was found that when the linking
chain between artemisinin nucleus and phenoxyl ali-
phatic acid or ester, that is, oxygen atom in compound
8 was replaced with OCH2 or OCH(Me) group, their
immunosuppressive activity greatly reduced and was
only comparable to or slightly more potent than that
of parent artemisinin.
3. Conclusion
4. Experimental
4.1. General
In summary, as part of continuous research plan to
search for potential immunosuppressive agents with
higher efficacy and lower toxicity, another new series
of new dihydroartemisinin derivatives 9 were synthe-
sized and assessed for their cytotoxicity of lymphocyte,
inhibitory activity on ConA-induced T cell proliferation
and LPS-induced B cell proliferation in comparison with
artemisinin.
All commercially available reagents were used without
further purification unless otherwise stated. The solvents
used were all of AR grade and were distilled under po-
sitive pressure of dry nitrogen atmosphere where neces-
sary. BF3ÆEt2O was redistilled by the standard methods.

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Z.-S. Yang et al. / Bioorg. Med. Chem. 14 (2006) 8043–8049
The progress of the reactions was monitored by analyt-
ical thin-layer chromatography (TLC) performed on
homemade HSGF254 precoated silica gel plates. Visual-
ization was performed by UV or development using
vanillin solution in sulfuric acid and ethanol (v/v = 4/1).
(1H, q, J = 6.80 Hz, Ar-O–CH-Me); 4.47 (1H, d,
J = 12.49 Hz, 16-Hb); 4.21 (2H, dq, J = 7.15, 1.37 Hz,
–O–CH2-Me); 2.66 (1H, m, 11-H); 2.37 (1H, m, 3-Hb);
1.61 (3H, d, J = 6.73 Hz, Ar-O–CH-Me); 1.45 (3H, s,
4-Me); 1.25 (3H, dt, J = 7.14, 0.96 Hz, –O–CH2-Me);
0.96 (3H, d, J = 6.73 Hz, 10-Me); 0.94 (3H, d,
J = 7.42 Hz, 11-Me).
All melting points were taken in open capillary tubes on
a Buchi-510 melting points apparatus and are uncorrect-
ed. The IR spectra through the range from 4000 to 600
were run on a Perkin-Elmer 599B spectrophotometer
with KBr pellets or as thin films and are reported in
IR (Film, cmꢀ1): 2939, 2874, 1755; 1736 (C@O), 1587;
1489 (C@C), 1448, 1375, 1263, 1194, 1176, 1101, 1022,
878, 785.
1
reciprocal centimeters (cmꢀ1). H NMR spectra were
determined in CDCl3 solution on a Brucker AM 400
spectrophotometer at ambient temperature. Elemental
analyses were performed on a CE 1106 elemental ana-
lyzer and all the results had deviation within 0.4% of
the theoretical values. Yields were of purified com-
pounds and were not optimized.
Anal. Calcd for C27H38O8: C, 66.10; H, 7.81. Found: C,
66.31; H, 7.67.
4.3.2. Ethyl 2-[3-(12-a-artemisinoxymethyl)]phenoxyl
propionate (9d). Colorless oil. Yield: 20%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.24 (1H, t, J = 7.88 Hz,
Ar-H); 6.96 (1H, d, J = 7.55 Hz, Ar-H); 6.88 (1H, br s,
Ar-H); 6.78 (1H, dt, J = 8.23, 1.26 Hz, Ar-H); 5.33
(1H, s, 5-H); 4.93 (1H, d,d, J = 12.59, 1.93 Hz, 16-Ha);
4.74 (1H, dq, J = 6.80,1.50 Hz, Ar-O–CH-Me); 4.59
(1H, d, J = 12.58 Hz, 16-Hb); 4.49 (1H, d, J = 9.41 Hz,
12-H); 4.21 (2H, m, –O–CH2-Me); 2.49 (1H, m, 11-H);
2.38 (1H, m, 3-Hb); 1.61 (3H, d, J = 6.71 Hz, Ar-O–
CH-Me); 1.46 (3H, s, 4-Me); 1.25 (3H, t, J = 7.05, Hz,
–O–CH2-Me); 0.95 (3H, d, J = 5.87 Hz, 10-Me); 0.94
(3H, d, J = 6.04 Hz, 11-Me).
4.2. Preparation of synthetic intermediates 10–12
Ethyl 2-[m or p-hydroxymethyl (or 10-ethyl) phenoxy]
propionate 10–12 were prepared on the basis of the
modified literature procedure,10 which is described be-
low. To a solution of sodium ethoxide, prepared by dis-
solving sodium (2.50 g, 110 mmol) in absolute EtOH
(120 mL), were added consecutively hydroxybenzyl
alcohol 13–15 (100 mmol) prepared by potassium
borohydride reduction of hydroxybenzaldehyde or
hydroxyacetophenone and (dl)-ethyl 2-bromopropio-
nate (15.6 mL, 120 mmol). The mixture solution was re-
fluxed for 5 h, and the solvent was then removed by
distillation. EtOAc (100 mL) was added and the organic
extract was washed with water and brine, dried over
anhydrous MgSO4, and concentrated. The resultant
residue was purified by silica gel chromatography
(petroleum ether/EtOAc, 20:1) to give 10–12 as colorless
oil (yield: 50%).
IR (Film, cmꢀ1): 2937, 2874, 1755; 1736 (C@O), 1610;
1587 (C@C), 1454, 1375, 1263, 1032, 879, 850, 785.
Anal. Calcd for C27H38O8: C, 66.10; H, 7.81. Found: C,
66.15; H, 7.91.
4.3.3. Ethyl 2-[4-(12-b-artemisinoxymethyl)]phenoxyl
propionate (9g). Colorless oil. Yield: 35%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.21 (2H, d, J = 8.79 Hz,
Ar-H); 6.84 (2H, d, J = 8.70 Hz, Ar-H); 5.45 (1H, s,
5-H); 4.87 (1H, d, J = 3.52 Hz, 12-H); 4.81 (1H, d,
J = 11.92 Hz, 16-Ha); 4.73 (1H, q, J = 6.74 Hz, Ar-
O–CH-Me); 4.44 (1H, d, J = 12.02 Hz, 16-Hb); 4.21
(2H, dq, J = 7.13, 1.27 Hz, –O–CH2-Me); 2.64 (1H,
m, 11-H); 2.37 (1H, m, 3-Hb); 1.61 (3H, d,
J = 6.74 Hz, Ar-O–CH-Me); 1.45 (3H, s, 4-Me); 1.25
(3H, dt, J = 7.18, 1.56 Hz, –O–CH2-Me); 0.96 (3H,
d, J = 6.15 Hz, 10-Me); 0.91 (3H, d, J = 7.32 Hz,
11-Me).
4.3. Synthesis of benzyl ether artemisinin derivatives
(9b, 9d, 9g, 9j, 9l, and 9n)
To a solution of dihydroartemisinin (2, 5.7 g, 20.0 mmol)
and ethyl 2-[m or p-hydroxymethyl (or 10-ethyl) phenoxy]
propionate (10–12, 24.0 mmol) in CH2Cl2 (80 mL) was
added BF3ÆEt2O (0.5 mL) 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 chromatog-
raphy with ethyl acetate/petroleum ether (1:25, v/v) as
the eluent to give new ether derivatives (9b, 9d, 9g, 9j,
9l, and 9n), whose yields ranged from 25% to 35%.
IR (Film, cmꢀ1): 2939, 2874, 1751; 1732 (C@O), 1612;
1510 (C@C); 1448, 1375, 1232, 1194, 1136, 1101, 1032,
878, 825.
Anal. Calcd for C27H38O8: C, 66.10; H, 7.81. Found: C,
66.03; H, 7.70.
4.3.1. Ethyl 2-[3-(12-b-artemisinoxymethyl)]phenoxyl
propionate (9b). Colorless oil. Yield: 30%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.22 (1H, t, J = 7.90 Hz,
Ar-H); 6.92 (1H, dd, J = 7.55, 0.82 Hz, Ar-H); 6.84
(1H, br s, Ar-H); 6.77 (1H, dd, J = 8.24, 1.65 Hz,
Ar-H); 5.44 (1H, s, 5-H); 4.89 (1H, d, J = 3.43 Hz,
12-H); 4.85 (1H, dd, J = 12.57, 5.70 Hz, 16-Ha); 4.73
4.3.4. Ethyl 2-[4-(12-a-artemisinoxymethyl)]phenoxyl
propionate (9i). Colorless oil. Yield: 25%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.26 (1H, d, J = 8.51 Hz,
Ar-H); 7.25 (1H, d, J = 8.65 Hz, Ar-H); 6.85 (1H, d,
J = 8.65 Hz, Ar-H); 6.83 (1H, d, J = 8.51 Hz, Ar-H);
5.32 (1H, s, 5-H); 4.88 (1H, d, J = 12.09 Hz, 16-Ha);
4.73 (1H, q, J = 6.73 Hz, Ar-O–CH-Me); 4.56 (1H, d,

Z.-S. Yang et al. / Bioorg. Med. Chem. 14 (2006) 8043–8049
8047
J = 12.09 Hz, 16-Hb); 4.47 (1H, d, J = 9.20 Hz, 12-H);
4.21 (2H, dq, J = 7.14, 1.16 Hz, –O–CH2-Me); 2.47
(1H, m, 11-H); 2.38 (1H, m, 3-Hb); 1.61 (3H, d,
J = 7.56 Hz, Ar-O–CH-Me); 1.46 (3H, s, 4-Me); 1.26
(3H, dt, J = 7.14, 2.06 Hz, –O–CH2-Me); 0.94 (3H, d,
J = 5.91 Hz, 10-Me); 0.86 (3H, dd, J = 7.14, 1.65 Hz,
11-Me).
40–50 ꢁC overnight. After neutralization with acetic
acid, the solution was evaporated in vacuum. The resi-
due was dissolved in EtOAc, washed with brine, dried
over anhydrous MgSO4 and evaporated to dryness un-
der reduced pressure. The resultant solid was purified
by recrystallization or column chromatography (silica
gel, using ethyl acetate as the eluent) to give the corre-
sponding free acid (9a, 9c, 9f, 9h, 9k, and 9m).
IR (Film, cmꢀ1): 2937, 2874, 1751; 1736 (C@O), 1612;
1510 (C@C); 1448, 1375, 1240, 1200, 1134, 1030, 879,
825.
4.4.1. 2-[3-(12-b-Artemisinoxymethyl)]phenoxyl propionic
acid (9a). White amorphous solid. Yield: 95%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.26 (1H, t, J = 7.90 Hz, Ar-
H); 6.95 (1H, d, J = 7.27 Hz, Ar-H); 6.89 (1H, br s, Ar-
H); 6.83 (1H, d, J = 8.33 Hz, Ar-H); 5.44 (1H, s, 5-H);
4.90 (1H, d, J = 2.99 Hz, 12- H); 4.85 (1H, d,d,
J = 12.39, 3.85 Hz, 16-Ha); 4.79 (1H, q, J = 6.95 Hz,
Ar-O–CH-Me); 4.50 (1H, d, J = 12.61 Hz, 16-Hb);
2.68 (1H, m, 11- H); 2.38 (1H, m, 3-Hb); 1.66 (3H, d,
J = 6.84 Hz, Ar-O–CH-Me); 1.46 (3H, s, 4-Me); 0.94
(6H, d, J = 6.41 Hz, 11-Me, 10-Me).
Anal. Calcd for C27H38O8: C, 66.10; H, 7.81. Found: C,
66.18; H, 7.49.
4.3.5. Ethyl 2-[4-(12-b-artemisinoxy)-10-ethyl]phenoxyl
propionate (9l). Colorless oil. Yield: 30%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.20 (2H, d, J = 9.13 Hz,
Ar-H); 6.80 (2H, d, J = 9.20 Hz, Ar-H); 5.50 (1H, s,
5-H); 4.88 (1H, q, J = 6.45 Hz, Ar-O–CH-Me); 4.72
(1H, dq, J = 6.73, 2.20 Hz, Q-O–CH-Me); 4.61 (1H, d,
J = 3.57 Hz, 12-H); 4.20 (2H, m, –O–CH2-Me); 2.50
(1H, m, 11-H); 2.36 (1H, m, 3-Hb); 1.60 (3H, d,
J = 6.18 Hz, Ar-O–CH-Me); 1.44 (3H, s, 4-Me); 1.40
(3H, d, J = 6.45 Hz, Q-O–CH-Me) 1.24 (3H, m, –O–
CH2-Me); 0.97 (3H, d, J = 6.32 Hz, 10-Me); 0.77 (3H,
dd, J = 7.28, 2.00 Hz, 11-Me).
IR (Film, cmꢀ1): 3435 (–OH), 2941, 2874, 1736 (C@O),
1587; 1490 (C@C); 1450, 1377, 1263, 1101, 1030,
1011,876, 826.
Anal. Calcd for C25H34O8: C, 64.92; H, 7.41. Found: C,
64.64; H, 7.61.
IR (Film, cmꢀ1): 2924, 2874, 1755; 1736 (C@O), 1610;
1510 (C@C), 1448, 1375, 1240, 1194, 1134, 1099, 1032,
1011, 984, 876, 829.
4.4.2. 2-[3-(12-a-Artemisinoxymethyl)]phenoxyl propionic
acid (9c). White amorphous solid. Yield: 95%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.24 (1H, t, J = 7.91,
3.50 Hz, Ar-H); 6.97 (1H, dd, J = 7.48, 4.27 Hz,
Ar-H); 6.93 (1H, bs, Ar-H); 6.82 (1H, dt, J = 7.91,
2.59 Hz, Ar-H); 5.34 (1H, s, 5-H); 4.92 (1H, dd,
J = 12.61, 7.91 Hz, 16-Ha); 4.79 (1H, q, J = 6.84 Hz,
Ar-O–CH-Me); 4.60 (1H, d, J = 12.61, 8.33 Hz,
16-Hb); 4.50, 4.45 (1H, d,d, J = 9.40, 9.20 Hz, 12-H);
2.49 (1H, m, H-11); 2.39 (1H, m, 3-Hb); 1.65 (3H, d,
J = 6.84 Hz, Ar-O–CH-Me); 1.46, 1.45 (3H, s, s,
4 -Me); 0.95, 0.94 (3H, d, d, J = 5.99, 5.77 Hz, 10-Me);
0.91, 0.89 (3H, d, d, J = 7.05, 7.05 Hz, 11-Me).
Anal. Calcd for C28H40O8: C, 66.65; H, 7.99. Found: C,
66.60; H, 7.88.
4.3.6. Ethyl 2-[4-(12-artemisinoxy)-10-ethyl]phenoxyl pro-
pionate (9n). A mixture of 12-a and 12-b isomers
(a/b = 1/2). Colorless oil. Yield: 25%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.22 (1H, d, J = 7.69 Hz,
Ar-H); 7.20 (1H, d, J = 8.25 Hz, Ar-H); 6.82 (2H, d,
J = 8.51 Hz, Ar-H); 5.19, 5.17, (1H, s, s, 5-H); 5.03,
4.21 (1H, d,d, J = 3.44, 9.47 Hz, 12-H); 4.96, 4.88 (1H,
q, q, J = 6.46, 6.32 Hz, Ar-O–CH-Me); 4.72 (1H, m,
Q-O–CH-Me); 4.20 (2H, m, –O–CH2-Me); 2.66, 2.42
(1H, m, m, 11-H); 2.36 (1H, m, 3-Hb); 1.61 (3H, d,
J = 6.46 Hz, Ar-O–CH-Me); 1.46, 1.35 (3H, s, s, 4-
Me); 1.41, (3H, m, Q-O–CH-Me) 1.24 (3H, m,
–O–CH2-Me); 0.95, 0.91 (3H, d, d, J = 7.42, 10.30 Hz,
10-Me); 0.89, 0.78 (3H, d, d, J = 10.44, 7.35 Hz, 11-Me).
IR (Film, cmꢀ1): 3427 (–OH), 2928, 2874, 1736 (C@O),
1587; 1490 (C@C); 1454, 1377, 1263, 1155, 1134, 1032,
878, 825.
Anal. Calcd for C25H34O8: C, 64.92; H, 7.41. Found: C,
64.76; H, 7.66.
4.4.3. 2-[4-(12-b-Artemisinoxymethyl)]phenoxyl propionic
acid (9f). White amorphous solid. Yield: 95%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.22 (2H, d, J = 8.56 Hz, Ar-
H); 6.86 (2H, d, J = 8.56 Hz, Ar-H); 5.45 (1H, s, 5-H);
4.88 (1H, d, J = 3.36 Hz, 12-H); 4.80(1H, d,
J = 11.24 Hz, 16-Ha); 4.78 (1H, q, J = 7.05 Hz, Ar-O–
CH-Me); 4.43 (1H, d, J = 11.92 Hz, 16-Hb); 2.64 (1H,
m, 11-H); 2.37 (1H, m, 3-Hb); 1.65 (3H, d, J = 6.88 Hz,
Ar-O–CH-Me); 1.45 (3H, s, 4-Me); 0.94 (3H, d,
J = 6.21 Hz, 10-Me); 0.93 (3H, d, J = 7.39 Hz, 11-Me).
IR (Film, cmꢀ1): 2926, 2873, 1755; 1736 (C@O), 1610;
1512 (C@C), 1448, 1375, 1281, 1240, 1195, 1134, 1099,
1016, 984, 876, 829.
Anal. Calcd for C28H40O8: C, 66.65; H, 7.99. Found: C,
66.77; H, 8.11.
4.4. Synthesis of free acid artemisinin derivatives (9a, 9c,
9f, 9h, 9k, and 9m)
The benzyl ether derivative (9b, 9d, 9g, 9j, 9l, 9n,
4.0 mmol) was dissolved in 0.5% KOH/EtOH solution
(60 mL), and the mixture solution was stirred at about
IR (KBr, cmꢀ1): 3425 (–OH); 3100 (Ar-H), 2941, 2873,
1736 (C@O); 1612; 1512 (C@C), 1450, 1377, 1240,
1134, 1099, 1009, 876, 825.

8048
Z.-S. Yang et al. / Bioorg. Med. Chem. 14 (2006) 8043–8049
Anal. Calcd for C25H34O8: C, 64.92; H, 7.41. Found: C,
64.72; H, 7.67.
4.5. Synthesis of aryl ester artemisinin derivatives (9e, 9j,
and 9o)
4.4.4. 2-[4-(12-a-Artemisinoxymethyl)]phenoxyl propionic
acid (9h). White amorphous solid. Yield: 95%. 1H NMR
(400 MHz, CDCl3, d ppm): 7.28 (2H, d, J = 8.73 Hz,
Ar-H); 6.86 (2H, d, J = 8.56 Hz, Ar-H); 5.33
(1H, s, 5-H); 4.88 (1H, d, J = 11.92 Hz, 16-Ha); 4.78
(1H, q, J = 6.83 Hz, Ar-O–CH-Me); 4.56 (1H, d,
J = 12.09 Hz, 16-Hb); 4.48 (1H, d, J = 9.23 Hz, 12-H);
2.48 (1H, m, 11-H); 2.38 (1H, m, 3-Hb); 1.65 (3H, d,
J = 6.88 Hz, Ar-O–CH-Me); 1.46 (3H, s, 4-Me); 0.95
(3H, d, J = 6.04 Hz, 10-Me); 0.88 (3H, d, d, J = 7.21,
1.51 Hz, 11-Me).
To a solution of above free acid (9a, 9c, 9f, 9h, 9k, 9m,
5.0 mmol) and o-methoxyphenol (0.6 mL, 5.5 mmol) in
dry ether (50 mL), DCC (1.13 g, 5.5 mmol) and DMAP
(cat. 122 mg, 0.5 mmol) were added, and the solution
was stirred for 2–5 h at room temperature. After the
condensation reaction 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 was identified as dicyclohexylurea
(DCU). The acetone-soluble fraction was evaporated
to dryness, and the residue was purified by column chro-
matography (silica gel, using ethyl acetate/petroleum
ether 1:25 v/v as the eluent) to give the target aryl esters
(9e, 9j, and 9o).
IR (KBr, cmꢀ1): 3425; 3250 (–OH), 2937, 2874, 1736
(C@O); 1614; 1512 (C@C), 1454, 1377, 1240-1132,
1032, 876, 825.
4.5.1. 2-Methoxyphenyl 2-[3-(12-b-artemisinoxymeth-
yl)]phenoxyl propionate (9e). White amorphous solid.
Yield: 40%. H NMR (400 MHz, CDCl3, d ppm): 7.27
Anal. Calcd for C25H34O8: C, 64.92; H, 7.41. Found: C,
64.56; H, 7.75.
1
(1H, t, J = 7.84 Hz, Ar-H); 7.20 (1H, dt, J = 7.42,
1.79 Hz, Ar-H); 6.99–6.85 (6H, m, Ar-H); 5.46 (1H, s,
5-H); 5.00 (1H, dq, J = 6.78, 1.65 Hz, Ar-O–CH-Me);
4.92 (1H, d, J = 3.43 Hz, 12-H); 4.89 (1H, dd,
J = 12.24, 2.20 Hz, 16-Ha); 4.51 (1H, d, J = 11.41 Hz,
16-Hb); 3.77 (3H, s, Ar-O-Me); 2.67 (1H, m, 11-H);
2.37 (1H, m, 3-Hb); 1.81 (3H, d, J = 6.74 Hz, Ar-O–
CH-Me); 1.46 (3H, s, 4-Me); 0.96 (3H, d, J = 7.42 Hz,
10-Me); 0.91 (3H, d, J = 6.05 Hz, 11-Me).
4.4.5. 2-[4-(12-b-Artemisinoxy)-10-ethyl]phenoxyl propi-
onic acid (9k). White amorphous solid. Yield: 95%. H
1
NMR (400 MHz, CDCl3, d ppm): 7.21 (2H, dd,
J = 8.62, 1.65 Hz, Ar-H); 6.83 (2H, d, J = 8.62 Hz,
Ar-H); 5.51 (1H, s, 5-H); 4.88 (1H, p, J = 6.32 Hz,
Ar-O–CH-Me); 4.77 (1H, dq, J = 6.78, 2.01 Hz, Q-O–
CH-Me); 4.62, 4.61 (1H,d,d, J = 2.75 Hz, 12-H); 2.52
(1H, m, 11-H); 2.37 (1H, m, 3-Hb); 1.66 (3H, d,
J = 6.59 Hz, Ar-O–CH-Me); 1.44 (3H, s, 4-Me); 1.40
(3H, d, J = 8.07 Hz, Q-O–CH-Me); 0.98 (1H,
J = 6.23, 10-Me); 0.79,0.78 (1H, dd, J = 7.33, 2.57 Hz,
11-Me).
IR (KBr, cmꢀ1): 2941, 2874, 1782 (C@O), 1608; 1502
(C@C), 1456, 1375, 1259-1028, 1109, 1028, 876, 825,
750.
IR (Film, cmꢀ1): 3430 (–OH), 2926, 2860, 1736 (C@O),
1610; 1512 (C@C), 1450, 1377, 1240, 1099, 1032, 984,
876, 827.
Anal. Calcd for C32H40O9: C, 67.59; H, 7.09. Found: C,
67.54; H, 7.17.
4.5.2. 2-Methoxyphenyl 2-[4-(12-b-artemisinoxymeth-
yl)]phenoxyl propionate (9j). White amorphous solid.
Yield: 45%. H NMR (400 MHz, CDCl3, d ppm): 7.26
Anal. Calcd for C26H36O8: C, 65.53; H, 7.61. Found: C,
65.42; H, 7.75.
1
(2H, d, J = 8.41 Hz, Ar-H); 7.20 (1H, td, J = 7.73 Hz,
J = 1.76 Hz, Ar-H); 7.02–6.96 (4H, m, Ar-H); 6.95–
6.84 (1H, m, Ar-H); 5.46 (1H, s, 5-H); 4.99 (1H, q,
J = 6.78 Hz, Ar-O–CH-Me); 4.90 (1H, d, J = 3.13 Hz,
12-H); 4.84 (1H, d, J = 11.74 Hz, 16-Ha); 4.46 (1H, d,
J = 11.93 Hz, 16-Hb); 3.77 (3H, s, Ar-O-Me); 2.66
(1H, m, 11-H); 2.38 (1H, m, 3-Hb); 1.82 (3H, d,
J = 6.85 Hz, Ar-O–CH-Me); 1.47 (3H, s, 4-Me); 0.95
(3H, d, J = 6.85 Hz, 10-Me); 0.93 (3H, d, J = 7.63 Hz,
11-Me).
4.4.6. 2-[4-(2-Artemisinoxy)-10-ethyl]phenoxyl propionic
acid (9m). A mixture of 12-a and 12-b isomers (a/
b = 1/1). White amorphous solid. Yield: 95%. 1H
NMR (400 MHz, CDCl3, d ppm): 7.24 (2H, dd,
J = 8.07, 2.02 Hz, Ar-H); 6.86 (2H, dd, J = 8.70,
2.10 Hz, Ar-H); 5.19 (1H, s, 5-H); 5.05, 4.21 (1H,
d,d, J = 3.49, 9.35 Hz, 12-H); 4.98, 4.88 (1H, q, q,
J = 6.41, 6.41 Hz, Ar-O–CH-Me); 4.79 (1H, m, Q-O–
CH-Me); 2.68, 2.42 (1H, m, m, 11-H); 2.34 (1H, m,
3-Hb); 1.66 (3H, d, d, J = 6.96, 1.10 Hz, Ar-O–CH-
Me); 1.47, 1.42 (3H, s, s, 4-Me); 1.45, 1.37 (3H, m,
Q-O–CH-Me); 0.97, 0.91 (3H, d, d, J = 7.33,
5.68 Hz, 10-Me); 0.90, 0.80 (3H, d, d, J = 7.15,
7.33 Hz, 11-Me).
IR (KBr, cmꢀ1): 2939, 2874, 1782 (C@O), 1610; 1502
(C@C), 1456, 1375, 1248-1159, 1109, 1026, 876, 825,
750.
Anal. Calcd for C32H40O9: C, 67.59; H, 7.09. Found: C,
67.55; H, 7.21.
IR (KBr, cmꢀ1): 3429 (–OH), 2928, 2873, 1736 (C@O),
1610; 1512 (C@C), 1452, 1377, 1240, 1134, 1099, 1011,
984, 876, 827.
4.5.3. 2-Methoxyphenyl 2-[4-(12-b-Artemisinoxy)-10-eth-
yl]phenoxyl propionate (9o). White amorphous solid.
Yield: 40%. 1H NMR (400 MHz, CDCl3, d ppm):
7.28–7.16 (3H, m, Ar-H); 7.00–6.84 (5H, m, Ar-H);
Anal. Calcd for C26H36O8: C, 65.53; H, 7.61. Found: C,
65.47; H, 7.61.

Z.-S. Yang et al. / Bioorg. Med. Chem. 14 (2006) 8043–8049
8049
5.52 (1H, s, 5-H); 4.98 (1H, q, J = 6.78 Hz, Ar-O–CH-
Me); 4.92 (1H, q, J = 6.55 Hz, Q-O–CH-Me); 4.65
(1H, d, J = 3.29 Hz, 12-H); 3.89, 3.77 (3H, s, s, Ar-O-
Me); 2.53 (1H, m, 11-H); 2.38 (1H, m, 3-Hb); 1.81
(3H, d, J = 6.73 Hz, Ar-O–CH-Me); 1.46 (3H, s, 15-
Me); 1.43 (3H, d, J = 6.59 Hz, Q-O–CH-Me); 0.98
(3H, d, J = 6.32 Hz, 10-Me); 0.80 (3H, d, J = 7.41 Hz,
11-Me).
pH 7.2) was added to each well for 6–7 h and the absor-
bance values at 570 nm were collected by microplate
reader (Bio-Rad, Model 550). The percentage of cell
death was determined by using the following calculating
formula:
Compounds ðOD570ÞꢀBackground ðOD570
Þ
Cytotoxicityð%Þ ¼
Control ðOD570ÞꢀBackground ðOD570
ꢁ100
Þ
IR (Film, cmꢀ1): 2924, 2874, 1782 (C@O), 1610; 1502
(C@C); 1456, 1375, 1259, 1097, 1032, 990, 876, 827.
4.6.5. T cell and B cell function assay. Fresh spleen cells
were obtained from BALB/C mice (male, 7–9 weeks
old). 5 · 105 of spleen cells were cultured at the same
conditions to that mentioned above. The cultures were
unstimulated or stimulated with 5 lg/mL of concanava-
lin A (ConA) or 10 lg/mL of lipopolysaccharide (LPS)
to induce T cell or B cell proliferative responses for
48 h, respectively. The compounds were added to cul-
tures with indicated concentrations to test their bioactiv-
ities. Proliferation was assessed in terms of uptake of
[3H]thymidine during 8 h pulsing with 20 kBq of
[3H]thymidine for each well, and then cells will be har-
vested onto glass fiber filters by a Basic 96 harvester.
The incorporated radioactivity was counted by a liquid
scintillation counter (1540 MicroBeta Trilux, Perkin-
Elmer Life Sciences).
Anal. Calcd for C33H42O9: C, 68.02; H, 7.27. Found: C,
68.00; H, 7.13.
4.6. Biological assay
4.6.1. Materials. Stock solution of compounds was
prepared with 100% dimethylsulfoxide (DMSO, Sigma)
and diluted with RPMI 1640 medium containing 10%
fetal bovine serum (FBS). RPMI 1640 medium powder
was purchased from Gibco-BRL, Life Technologies.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-
lium bromide], concanavalin A (ConA), and lipopoly-
saccharide (LPS) were purchased from Sigma.
[3H]Thymidine (1 mci/mL) was purchased from Shang-
hai Institute of Atomic Energy (SIAE). The related in
vitro experimental procedure was performed according
to our previous work.11
Acknowledgment
4.6.2. 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 light/12-h dark photoperiod, 22 1 ꢁC,
55% 5% relative humidity). All husbandry and exper-
imental contacts made with the mice were maintained at
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 Labo-
ratory Animals and were approved by the Bioethics
Committee of the Shanghai Institute of Materia Medica.
Financial support for this project was provided by the
Knowledge Innovation Program of Chinese Academy
of Sciences (No. KSCX2-SW-202).
References and notes
1. Qian, R. Sh.; Li, Zh. L.; Yu, J. L.; Ma, D. J. Zhong Yi Za
Zhi 1981, 22, 63.
2. Zhuang, G. K.; Zou, M. X.; Xu, X.; Zhu, Y. Zhong Hua
Yi Xue Za Zhi 1982, 62, 365.
3. Huang, G. J.; Zhao, Y. Zhong Yi Za Zhi 1983, 3, 171.
4. Shen, M.; Ge, H. L.; He, Y. X.; Song, Q. L.; Zhang, H. Z.
Sci. Sin. [B]. 1984, 27, 398.
5. Sun, X. Z.; Xie, Sh. Sh.; Long, Zh. Zh.; Zhang, Zh. R.;
Tu, Y. Y. Zhong Yi Za Zhi 1991, 11, 37.
6. Lin, P. Y.; Pan, J. Q.; Feng, Z. M.; Zhang, D.; Xiao, L. Y.
Comparison of immunopharmacologic action of artemis-
inin and its derivatives. In Progress in Immuno-pharma-
cology; Zhou, J. H., Li, X. Y., Rong, K. T., Eds.; Chinese
Science and Technique Press: Beijing, 1993; p 325.
7. Yu, Q. B.; Gao, Y. X. Zhongguo Pi Fu Xing Bing Xue Za
Zhi 1997, 30, 51.
8. Yang, Z. S.; Zhou, W. L.; Sui, Y.; Wang, J. X.; Wu, J. M.;
Zhou, Y.; Zhang, Y.; He, P. L.; Han, J. Y.; Tang, W.;
Zuo, J. P.; Li, Y. J. Med. Chem. 2005, 48, 4608.
9. Li, Y.; Yu, P. L.; Chen, Y. X.; Li, L. Q.; Gai, Y. Z.; Wang,
D. S.; Zhang, Y. P. Acta Pharm. Sin. 1981, 16, 429.
10. Hazeldine, S. T.; Polin, L.; Kushner, J.; Paluch, J.; White,
K.; Edelstein, M.; Palomino, E.; Corbett, T. H.; Horwitz,
J. P. J. Med. Chem. 2001, 44, 1758.
4.6.3. 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 3 times with PBS containing 2% FBS and were
resuspended in RPMI 1640 medium at the indicated
concentration.
4.6.4. Cytotoxicity assay. Fresh spleen cells were ob-
tained from BALB/C mice (male, 7–9 weeks old).
5 · 105 of spleen cells were cultured in 96-well flat plates
with 200 lL RPMI 1640 media containing 10% FBS,
100 U/mL penicillin, and 100 lg/mL streptomycin in a
humidified, 37 ꢁC, 5% CO2-containing incubator for
48 h, in the presence or absence of various concentra-
tions of compounds. Eighteen microliters of MTT
(5 mg/mL) was added to each well at the final 5 h cul-
ture. Then 90 lL of lysis buffer (10% SDS, 50% DMF,
11. Feng, Y. H.; Zhou, W. L.; Wu, Q. L.; Li, X. Y.; Zhao, W.
M.; Zuo, J. P. Acta Pharmacol. Sin. 2002, 23, 893.