Synthesis and biological activities of novel artemisinin derivatives as cysteine protease falcipain-2 inhibitors

Article abstract of DOI:10.1007/s12272-012-0902-4

A series of novel artemisinin derivatives were synthesized from artemisinin and different anilines. All compounds were obtained as β-isomers. The target compounds were evaluated for inhibition activity against Plasmodium falciparum falcipain-2 in vitro, and most of them exhibited potent inhibition in the low micromolar range and proved to be new types of falcipain-2 inhibitors.

Full text of DOI:10.1007/s12272-012-0902-4

Arch Pharm Res Vol 35, No 9, 1525-1531, 2012
DOI 10.1007/s12272-012-0902-4
Synthesis and Biological Activities of Novel Artemisinin Derivatives
as Cysteine Protease Falcipain-2 Inhibitors
Yang Liu1, Wei-Qiang Lu2, Kun-Qiang Cui2, Wei Luo1, Jian Wang1, and Chun Guo1
1Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engi-
2
neering, Shenyang Pharmaceutical University, Shenyang 110016, China and School of Pharmacy, East China University
of Science and Technology, Shanghai 200237, China
(Received February 18, 2011/Revised June 26, 2011/Accepted July 21, 2011)
A series of novel artemisinin derivatives were synthesized from artemisinin and different
anilines. All compounds were obtained as β-isomers. The target compounds were evaluated for
inhibition activity against Plasmodium falciparum falcipain-2 in vitro, and most of them
exhibited potent inhibition in the low micromolar range and proved to be new types of falci-
pain-2 inhibitors.
Key words: Falcipain-2 inhibitor, Artemisinin derivatives, (thio)Semicarbazone derivatives,
Synthesis, Inhibition activity, Antimalarial
INTRODUCTION
Malaria is one of the most prevalent parasitic dis-
eases and especially Plasmodium falciparum malaria
has developed resistance to most available antimalar-
ial agents (Begue and Bonnet, 2005; WHO, 2010 ). The
new endoperoxide sesquiterpene lactone artemisinin
(1), isolated from Artemisia annua L., and its deriv-
atives 2a-d (Fig. 1) as new generation antimalarial
drugs have been used successfully in the treatment of
multidrug-resistant Plasmodium falciparum strains
(Klayman, 1985; Muraleedharan and Mitchell, 2009).
However, as artemisinin derivatives have been widely
used and drug resistance has emerged, development
Fig. 1. Structures of artemisinin and its derivatives
of antimalarial drugs against new targets is an urgent is a meaningful work.
priority. Among promising new targets, Plasmodium
Thiosemicarbazone derivatives have a broad spectrum
falciparum cysteine protease falcipain-2, which is re- of chemotherapeutic properties including antimalarial
sponsible for host hemoglobin hydrolysis to provide activities due to potent falcipain-2 inhibition (Klayman
amino acids for parasite protein biosynthesis, has been et al., 1979; Chipeleme et al., 2007). Iron is essential
perceived as one of the most attractive targets (Rosenthal for the biological activity of a number of plasmodial
et al., 2002; Ettari et al., 2010). Therefore, the discovery proteins and the thiosemicarbazone moiety is a class
of falcipain-2 inhibitors as potential antimalarial drugs of iron-chelators which have shown activities at pre-
venting growth of the malarial parasite (Walcourt et
Correspondence to: Chun Guo, Key Laboratory of Structure-
Based Drug Design & Discovery of Ministry of Education,
School of Pharmaceutical Engineering, Shenyang Pharmaceuti-
cal University, Shenyang 110016, China
Tel: 86-24-2398-6425, Fax: 86-24-2398-6425
E-mail: chunguo63@yahoo.com.cn
al., 2004). Furthermore, semicarbazones, which can be
regarded as urea derivatives, have recently gained
considerable importance in the design of enzyme inhibi-
tors. The urea linkage has remarkable resistance to
proteolytic degradation by enzymes in the gastrointest-
1525

1526
Y. Liu et al.
inal tract, which opens the opportunity for oral deliv-
ery. In addition, the hydrogen bond forming capacity
of the urea unit assist in rendering the urea compounds
more water soluble (Chipeleme et al., 2007).
General procedure for the synthesis of 4-[(10S)
dihydroartemisinin-10-oxy]benzaldehyde 4-(sub-
stituted phenyl)(thio)semicarbazones (10a-p)
To a stirred solution of artemisinin (1, 2 g, 7.08
Based on the above observations, a series of novel mmol) in CH3OH, NaBH4 (0.40 g, 10.62 mmol) was
10-substituted artemisinin derivatives (10a-p) were added at 0-5oC over a period of 20 min. After being
designed and synthesized by incorporating the two stirred for an additional 3 h under the same condition,
biologically active scaffolds, namely artemisinin and the mixture was neutralized with glacial acetic acid
(thio)semicarbazone. All compounds were evaluated while the temperature was maintained at 0-5oC, con-
against Plasmodium falciparum falcipain-2 in vitro
.
centrated by evaporating most of the CH3OH, diluted
with cold water (100 mL) and stirred for 15 min at
room temperature. The precipitate was collected, washed
MATERIALS AND METHODS
Instruments and reagents
with water (100 mL
× 3) and dried for further use. To
a CH2Cl2 solution (50 mL) of dihydroartemisinin (2a
,
Melting points (m.p.) were taken on glass slides 11.36 g, 40 mmol) and triethylamine (11.08 mL, 80
with X-4 digital display microscopic melting point ap- mmol), TFAA (11.12 mL, 80 mmol) in CH2Cl2 (30 mL)
paratus and are uncorrected. Infrared spectra were was added dropwise at −
5-0oC. After 1 day, 4-hydroxy-
recorded on a Bruker IFS-55 spectrometer. 1H-NMR benzaldehyde (9.76 g, 80 mmol) was added and stirred
and 13C-NMR spectra were recorded using a Bruker for 12 h at the same temperature. The reaction solu-
ARX-300 spectrometer using CDCl3 as solvent and tion was quenched with saturated NaHCO3 solution
TMS as internal standard. Electrospray ionization mass and washed by saturated NaHCO3 solution (50 mL
spectra (ESIMS) were recorded on a Waters Quattro 5) and water (50 mL 5), respectively. The organic
micro API. All reagents were commercially available layer was dried (Na2SO4) and concentrated in vacuo
and were used after purification as needed. Chroma- The residue was purified on a silica gel column with
tography was performed on silica gel (200-300 mesh). petroleum ether-ethyl acetate (8:1) to afford . Equimolar
All reactions were monitored by TLC (silica gel plates quantities of (2 mmol) and 5a-h (2 mmol) or 7a-h (2
×
×
.
9
9
with fluorescence F254 were used).
mmol) was dissolved in EtOH (20 mL) catalyzed by
glacial acetic acid (3 drops) and stirred for 2 h at room
temperature. The precipitate was filtered and purified
on a silica gel column with petroleum ether-ethyl acetate
General procedures for the synthesis of 4-(sub-
stituted phenyl)thiosemicarbazides (5a-h)
To an EtOH solution (50 mL) of aniline (0.1 mol), (3:1) to afford 10a-h or with CH2Cl2-CH3OH (80:1) to
concentrated ammonia (20 mL) was slowly added. The afford 10i-p
.
mixture was cooled below 20oC and CS2 (8 mL) was
added dropwise for 15 min. After 1 h, sodium monochlo- 4-[(10S)dihydroartemisinin-10-oxy]benzaldehyde
roacetate (14 g, 0.12 mol) was added and vigorously 4-phenylthiosemicarbazone (10a)
stirred for 30 min followed by addition of hydrazine Yield 29.74%; pale yellow solid; m.p. 124-126oC; ESI-
hydrate (80%, 12.5 mL). After 1 h, the mixture was MS m/z: 536 (M-H)−; IR (KBr) cm−1: 3317, 2921, 2870,
cooled overnight and the crude reaction mixture was 1602, 1543, 1508, 1445, 1238, 1193, 1096, 1068, 1034,
filtered and recrystallized from EtOH.
978, 956, 937, 876, 832, 745, 693; 1H-NMR (300 MHz,
CDCl3): 9.22 (1H, s), 9.17 (1H, s), 7.79 (1H, s), 7.62
(2H, d, = 9.0 Hz), 7.17 (2H, d, = 8.7 Hz), 5.57 (1H,
d, = 3.3 Hz), 5.46 (1H, s), 2.82-2.87 (1H, m), 2.34-2.45
= 7.2 Hz), 0.97 (3H,
= 6.0 Hz); 13C-NMR (75 MHz, CDCl3):
175.8,
δ
J
J
General procedure for the synthesis of 4-(sub-
stituted phenyl)semicarbazides (7a-h)
To a mixture of aniline (0.05 mol), pyridine (5 mL) (1H, m), 1.44 (3H, s), 1.03 (3H, d,
and CH2Cl2 (30 mL), phenyl chloroformate (6.3 mL, d,
J
J
J
δ
0.05 mol) was added dropwise on ice water and then 159.9, 143.3, 138.2, 129.1, 126.5, 124.9, 117.3, 104.6,
reacted at room temperature for 4 h. The mixture was 100.6, 88.6, 81.3, 52.8, 44.6, 37.7, 36.6, 34.9, 31.2, 26.4,
then evaporated under reduced pressure and the 24.9, 24.7, 20.6, 13.2.
residue was salted out by pouring into a saturated
NaCl solution. The precipitate was filtered and dried 4-[(10S)dihydroartemisinin-10-oxy]benzaldehyde
followed by refluxing in hydrazine hydrate (80%, 10 4-(2-methylphenyl)thiosemicarbazone (10b)
mL) for 12 h. After cooling, the crude reaction mixture Yield 59.82%; pale yellow solid; m.p. 153-155oC; ESI-
was filtered and recrystallized from EtOH.
MS m/z: 552 (M+H)+; IR (KBr) cm−1: 3313, 2924,
2864, 1603, 1587, 1545, 1500, 1457, 1231, 1211, 1172,

Artemisinin Derivatives as Falcipain-2 Inhibitors
1527
1097, 1069, 1035, 978, 958, 939, 876, 832, 743; 1H-NMR 137.4, 134.5, 134.1, 131.7, 129.3, 127.6, 127.5, 127.3,
(300 MHz, CDCl3): 9.50 (1H, s), 8.99 (1H, s), 7.85 (1H, 117.2, 104.6, 100.6, 88.6, 81.2, 52.8, 44.6, 37.7, 36.6,
s), 7.63 (2H, d, = 8.7 Hz), 7.18 (2H, d, = 8.7 Hz), 34.9, 31.2, 26.4, 24.9, 24.7, 21.4, 20.6, 18.3, 13.2.
5.58 (1H, d, = 3.3 Hz), 5.48 (1H, s), 2.83-2.86 (1H,
m), 2.39 (3H, s), 1.46 (3H, s), 1.05 (3H, d, = 7.2 Hz), 4-[(10
δ
J
J
J
J
S)dihydroartemisinin-10-oxy]benzaldehyde
4-(3,4-dimethylphenyl)thiosemicarbazone (10f)
0.99 (3H, d,
J
= 6.0 Hz); 13C-NMR (75 MHz, CDCl3):
δ
176.6, 159.8, 143.3, 136.7, 134.3, 131.0, 129.3, 127.5, Yield 32.70%; pale yellow solid; m.p. 117-118oC; ESI-
127.2, 126.8, 117.2, 104.6, 100.6, 88.6, 81.2, 52.8, 44.6, MS m/z: 566 (M+H)+; IR (KBr) cm−1: 3320, 2922, 2871,
37.7, 36.6, 34.9, 31.2, 36.4, 24.9, 24.7, 20.6, 18.3, 13.2.
1604, 1540, 1508, 1450, 1376, 1282, 1236, 1195, 1163,
1121, 1096, 1067, 1034, 978, 956, 936, 875, 724, 533;
4-[(10S
)dihydroartemisinin-10-oxy]benzaldehyde 1H-NMR (300 MHz, CDCl3):
δ
9.81 (1H, s), 9.05 (1H,
= 8.7 Hz), 7.15 (2H, d,
J = 3.3 Hz), 5.46 (1H, s), 2.81-
4-(4-methylphenyl)thiosemicarbazone (10c)
s), 7.86 (1H, s), 7.61 (2H, d,
J
J
Yield 36.25%; pale yellow solid; m.p. 124-126oC; ESI- = 8.7 Hz), 5.56 (1H, d,
MS m/z: 552 (M+H)+; IR (KBr) cm−1: 3324, 2923, 2.86 (1H, m), 2.34-2.44 (2H, m), 2.29 (3H, s), 2.27 (3H,
2850, 1605, 1510, 1449, 1236, 1192, 1173, 1096, 1068, s), 1.44 (3H, s), 1.03 (3H, d,
1034, 978, 956, 937, 876, 832, 736; 1H-NMR (300 = 6.0 Hz); 13C-NMR (75 MHz, CDCl3):
MHz, CDCl3): 9.27 (1H, s), 9.08 (1H, s), 7.79 (1H, s), 143.1, 137.4, 135.8, 135.2, 130.2, 129.3, 127.2, 126.5,
7.62 (2H, d, = 8.4 Hz), 7.16 (2H, d, = 8.7 Hz), 5.57 122.9, 117.2, 104.6, 100.6, 88.6, 81.2, 52.8, 44.6, 37.7,
(1H, d, = 3.3 Hz), 5.48 (1H, s), 2.82-2.87 (1H, m), 2.37 36.6, 34.9, 31.2, 27.2, 26.3, 24.9, 24.7, 20.6, 19.7, 13.2.
(3H, s), 1.45 (3H, s), 1.03 (3H, d, = 7.2 Hz), 0.97 (3H,
d, 176.1, 4-[(10
= 5.7 Hz); 13C-NMR (75 MHz, CDCl3):
J
= 7.5 Hz), 0.97 (3H, d,
J
δ
176.1, 159.8,
δ
J
J
J
J
J
δ
S)dihydroartemisinin-10-oxy]benzaldehyde
159.8, 143.3, 136.4, 135.6, 129.7, 129.3, 127.2, 125.3, 4-(4-methoxylphenyl)thiosemicarbazone (10g)
117.2, 104.6, 100.6, 88.6, 81.3, 52.8, 44.6, 37.7, 36.6, Yield 35.23%; pale yellow solid; m.p. 119-120oC; ESI-
34.9, 31.2, 26.4, 24.9, 24.7, 21.4, 20.6, 19.1, 13.2.
MS m/z: 566 (M-H)−; IR (KBr) cm−1: 3320, 2924, 2850,
1604, 1511, 1378, 1299, 1241, 1193, 1174, 1121, 1096,
4-[(10
4-(2,3-dimethylphenyl)thiosemicarbazone (10d)
Yield 46.84%; pale yellow solid; m.p. 120-121oC; ESI- = 7.5 Hz), 1.44 (3H, s), 2.34-2.44 (1H, m), 2.81-2.86
MS m/z: 566 (M+H)+; IR (KBr) cm−1: 3331, 3212, 2921, (1H, m), 3.83 (3H, s), 5.46 (1H, s), 5.56 (1H, d,
= 3.3
2872, 1603, 1531, 1506, 1375, 1237, 1208, 1170, 1096, Hz), 7.15 (2H, d, = 8.7 Hz), 7.61 (2H, d, = 8.7 Hz),
1067, 1034, 978, 957, 937, 876, 830, 727, 534; 1H-NMR 7.87 (1H, s), 9.01 (1H, s), 9.89 (1H, s); 13C-NMR (75
S
)dihydroartemisinin-10-oxy]benzaldehyde 1068, 1035, 978, 956, 937, 876, 831, 529; 1H-NMR (300
MHz, CDCl3):
δ 0.97 (3H, d, J = 5.7 Hz), 1.03 (3H, d, J
J
J
J
(300 MHz, CDCl3):
(1H, s), 7.60 (2H, d,
δ
9.88 (1H, s), 8.91 (1H, s), 7.87 MHz, CDCl3):
= 8.7 Hz), 7.15 (2H, d, = 8.7 127.2, 117.2, 114.3, 104.6, 100.6, 88.6, 81.2, 55.8, 52.8,
= 3.3 Hz), 5.46 (1H, s), 2.81-2.86 44.6, 37.7, 36.6, 34.9, 31.2, 26.4, 24.9, 24.7, 20.6, 13.2.
(1H, m), 2.34 (3H, s), 2.24 (3H, s), 1.44 (3H, s), 1.02
(3H, d, = 7.5 Hz), 0.97 (3H, d,
= 6.0 Hz); 13C-NMR 4-[(10
(75 MHz, CDCl3):
δ 176.5, 159.8, 158.3, 143.2, 131.1, 129.3,
J
J
Hz), 5.56 (1H, d,
J
J
J
S)dihydroartemisinin-10-oxy]benzaldehyde
δ
177.1, 159.7, 143.3, 138.1, 136.6, 4-(4-chlorophenyl)thiosemicarbazone (10h)
133.9, 129.3, 127.3, 126.0, 117.2, 104.6, 100.6, 88.6, Yield 35.83%; pale yellow solid; m.p. 126-127oC; ESI-
81.2, 52.8, 44.6, 37.7, 36.6, 34.9, 31.2, 26.4, 24.9, 24.7, MS m/z: 570 (M-H)−; IR (KBr) cm−1: 3317, 2923, 2871,
20.8, 20.6, 14.6, 13.2.
1604, 1540, 1508, 1399, 1296, 1238, 1192, 1173, 1121,
1095, 1068, 1034, 978, 956, 937, 876, 830, 770; 1H-NMR
4-[(10
4-(2,4-dimethylphenyl)thiosemicarbazone (10e)
Yield 28.28%; pale yellow solid; m.p. 125-126oC; ESI- (1H, d,
MS m/z: 566 (M+H)+; IR (KBr) cm−1: 3326, 2922, 2.44 (1H, m), 1.44 (3H, s), 1.03 (3H, d,
2871, 1604, 1537, 1508, 1449, 1376, 1297, 1238, 1194, 0.97 (3H, d,
= 5.7 Hz); 13C-NMR (75 MHz, CDCl3):
1173, 1121, 1096, 1068, 1035, 978, 957, 937, 976, 831, 175.8, 160.0, 143.5, 136.7, 131.8, 129.4, 129.2, 126.8,
S
)dihydroartemisinin-10-oxy]benzaldehyde (300 MHz, CDCl3):
s), 7.62 (2H, d, = 8.7 Hz), 7.16 (2H, d,
= 3.3 Hz), 5.46 (1H, s), 2.82-2.87 (1H, m), 2.34-
= 7.2 Hz),
δ
9.67 (1H, s), 9.13 (1H, s), 7.85 (1H,
J
J
= 8.7 Hz), 5.67
J
J
J
δ
1
755, 533; H-NMR (300 MHz, CDCl3):
8.87 (1H, s), 7.87 (1H, s), 7.60 (2H, d,
(2H, d, = 8.7 Hz), 5.56 (1H, d, = 3.3 Hz), 5.46 (1H,
s), 2.81-2.86 (1H, m), 2.35 (3H, s), 2.32 (3H, s), 1.44 4-[(10
(3H, s), 1.03 (3H, d, = 7.2 Hz), 0.97 (3H, d, = 5.7 4-phenylsemicarbazone (10i)
Hz); 13C-NMR (75 MHz, CDCl3): 176.9, 159.7, 143.3, Yield 68.06%; white solid; m.p. 161-162oC; ESI-MS m/
δ
9.89 (1H, s), 126.1, 117.3, 104.6, 100.6, 88.6, 81.2, 52.8, 44.6, 37.7,
= 9.0 Hz), 7.15 36.6, 34.9, 31.2, 26.4, 24.9, 24.7, 20.6, 13.2.
J
J
J
S)dihydroartemisinin-10-oxy]benzaldehyde
J
J
δ

1528
Y. Liu et al.
z
: 520 (M-H)−; IR (KBr) cm−1: 3370, 3229, 2928, 2868, 154.8, 141.7, 137.5, 136.1, 128.6, 128.2, 126.4, 126.2,
1694, 1668, 1604, 1538, 1151, 1447, 1231, 1126, 1097, 120.9, 117.2, 104.6, 100.7, 88.6, 81.3, 52.8, 44.7, 37.7,
1068, 1035, 979, 958, 939, 877, 831, 752, 532; 1H-NMR 36.7, 34.9, 31.3, 26.4, 24.9, 24.7, 21.0, 20.6, 13.9, 13.2.
(300 MHz, CDCl3):
(1H, s), 7.60 (2H, d,
δ
J
8.71 (1H, s), 8.13 (1H, s), 7.74
= 8.7 Hz), 7.16 (2H, d, = 8.7 4-[(10
= 3.3 Hz), 5.48 (1H, s), 2.82-2.87 4-(2,4-dimethylphenyl)semicarbazone (10m)
(1H, m), 2.34-2.45 (1H, m), 1.45 (3H, s), 1.04 (3H, d,
Yield 80.05%; white solid; m.p. 172-173oC; ESI-MS m/
= 7.5 Hz), 0.97 (3H, d,
= 5.7 Hz); 13C-NMR (75 MHz, : 550 (M+H)+; IR (KBr) cm−1: 3381, 3217, 2949, 2866,
CDCl3): 159.2, 154.3, 142.1, 138.3, 129.3, 128.7, 127.9, 1695, 1605, 1535, 1450, 1233, 1138, 1097, 1067, 1035,
123.7, 120.0, 117.2, 104.6, 100.6, 88.6, 81.3, 52.8, 44.7, 978, 956, 938, 876, 831, 486; H-NMR (300 MHz,
37.7, 36.6, 34.9, 31.2, 26.4, 24.9, 24.7, 20.6, 13.2. CDCl3): 8.66 (1H, s), 8.03 (1H, s), 7.73 (1H, s), 7.57
(2H, d, = 8.7 Hz), 7.15 (2H, d, = 8.7 Hz), 5.55 (1H,
d, = 3.3 Hz), 5.48 (1H, s), 2.33 (3H, s), 2.81-2.86 (1H,
m), 2.31 (3H, s), 1.45 (3H, s), 1.03 (3H, d, = 7.2 Hz),
= 5.7 Hz); 13C-NMR (75 MHz, CDCl3):
J
S)dihydroartemisinin-10-oxy]benzaldehyde
Hz), 5.56 (1H, d,
J
J
J
z
δ
1
δ
J
J
4-[(10S)dihydroartemisinin-10-oxy]benzaldehyde
J
4-(2-methylphenyl)semicarbazone (10j)
J
Yield 84.28%; white solid; m.p. 166-167oC; ESI-MS 0.97 (3H, d,
J
δ
m/z: 584 (M+Na)+; IR (KBr) cm−1: 3376, 3229, 2927, 159.1, 154.6, 141.6, 133.8, 131.4, 128.5, 127.6, 122.1,
2866, 1696, 1669, 1605, 1589, 1539, 1511, 1460, 1231, 117.2, 104.6, 100.7, 88.6, 81.3, 52.8, 44.7, 37.7, 36.7,
1097, 1036, 978, 958, 940, 877, 831, 755; 1H-NMR (300 34.9, 31.2, 26.4, 24.7, 21.1, 18.0, 13.2.
MHz, CDCl3):
7.58 (2H, d,
(1H, d, = 3.3 Hz), 5.48 (1H, s), 2.81-2.86 (1H, m), 2.38 4-(3,4-dimethylphenyl)semicarbazone (10n)
(3H, s), 1.45 (3H, s), 1.03 (3H, d,
= 7.5 Hz), 0.97 (3H, Yield 75.50%; white solid; m.p. 139-141oC; ESI-MS m/
d, 159.2,
= 5.7 Hz); 13C-NMR (75 MHz, CDCl3): : 572 (M+Na)+; IR (KBr) cm−1: 3384, 3216, 2922, 2865,
δ
9.01 (1H, s), 8.17 (1H, s), 7.77 (1H, s),
J
= 9.0 Hz), 7.16 (2H, d, = 8.7 Hz), 5.55 4-[(10
J
S)dihydroartemisinin-10-oxy]benzaldehyde
J
J
J
δ
z
154.4, 141.7, 136.5, 130.7, 128.6, 128.1, 128.0, 127.2, 1694, 1606, 1591, 1535, 1542, 1235, 1123, 1097, 1068,
124.0, 121.4, 117.3, 104.6, 100.7, 88.6, 81.3, 52.8, 44.7, 1035, 978, 957, 938, 876, 832, 497; 1H-NMR (300 MHz,
37.7, 36.7, 34.9, 31.2, 36.4, 24.9, 24.7, 20.6, 18.0, 13.2.
CDCl3):
(2H, d,
)dihydroartemisinin-10-oxy]benzaldehyde d, = 3.3 Hz), 5.48 (1H, s), 2.81-2.86 (1H, m), 2.34-
4-(4-methylphenyl)semicarbazone (10k) 2.44 (1H, m), 2.28 (3H, s), 2.24 (3H, s), 1.45 (3H, s),
Yield 84.01%; white solid; m.p. 166-168oC; ESI-MS m/ 1.04 (3H, d, = 6.0 Hz); 13C-
= 7.5 Hz), 0.97 (3H, d,
: 534 (M-H)−; IR (KBr) cm−1: 3409, 3115, 2942, 2863, NMR (75 MHz, CDCl3):
159.2, 154.3, 141.8, 137.4,
δ
9.09 (1H, s), 8.02 (1H, s), 7.76 (1H, s), 7.59
J
= 8.7 Hz), 7.16 (2H, d, = 8.7 Hz), 5.56 (1H,
J
4-[(10S
J
J
J
z
δ
1687, 1603, 1521, 1447, 1321, 1231, 1136, 1096, 1067, 135.9, 132.0, 130.3, 128.7, 128.0, 121.6, 117.7, 117.2,
1035, 977, 956, 936, 875, 812, 751, 486; 1H-NMR (300 104.6, 100.7, 88.6, 81.3, 52.8, 44.7, 37.7, 36.7, 34.9,
MHz, CDCl3):
7.59 (2H, d,
(1H, d, = 3.0 Hz), 5.48 (1H, s), 2.82-2.85 (1H, m), 2.36- 4-[(10
2.42 (1H, m), 2.34 (3H, s), 1.45 (3H, s), 1.04 (3H, d, 4-(4-methoxylphenyl)semicarbazone (10o)
7.2 Hz), 0.97 (3H, d,
= 6.6 Hz); 13C-NMR (75 MHz, Yield 84.30%; white solid; m.p. 167-168oC; ESI-MS m/
CDCl3): 159.2, 154.3, 141.8, 135.7, 133.3, 129.8, 128.7,
: 552 (M+H)+; IR (KBr) cm−1: 3369, 3203, 3118, 2941,
128.0, 120.2, 117.2, 104.6, 100.7, 88.6, 81.3, 52.8, 44.7, 2879, 1685, 1604, 1516, 1281, 1229, 1172, 1096, 1036,
δ
9.24 (1H, s), 8.07 (1H, s), 7.77 (1H, s), 31.2, 26.4, 24.9, 24.7, 20.6, 20.2, 19.4, 13.2.
J
= 9.0 Hz), 7.15 (2H, d, = 8.4 Hz), 5.55
J
J
S)dihydroartemisinin-10-oxy]benzaldehyde
J
=
J
δ
z
1
37.7, 36.6, 34.9, 31.3, 26.4, 24.9, 24.7, 21.1, 20.6, 13.2.
977, 956, 937, 876, 829, 532; H-NMR (300 MHz,
CDCl3): 8.78 (1H, s), 7.98 (1H, s), 7.73 (1H, s), 7.59
)dihydroartemisinin-10-oxy]benzaldehyde (2H, d, = 8.7 Hz), 7.15 (2H, d, = 8.7 Hz), 5.56 (1H,
4-(2,3-dimethylphenyl)semicarbazone (10l) d, = 3.3 Hz), 5.48 (1H, s), 3.81 (3H, s), 2.81-2.86 (1H,
Yield 86.41%; white solid; m.p. 166-168oC; ESI-MS m/ m), 2.34-2.45 (1H, m), 1.45 (3H, s), 1.04 (3H, d,
= 7.5
= 6.0 Hz); 13C-NMR (75 MHz,
159.2, 156.3, 154.6, 141.8, 131.3, 128.7, 128.0,
δ
4-[(10S
J
J
J
J
z
: 572 (M+Na)+; IR (KBr) cm−1: 3387, 3221, 2948, 2924, Hz), 0.97 (3H, d,
J
2865, 1698, 1665, 1605, 1541, 1510, 1474, 1232, 1098, CDCl3):
δ
1
1035, 979, 958, 939, 877, 832; H-NMR (300 MHz, 122.2, 117.1, 114.5, 104.6, 100.6, 88.6, 81.3, 55.8, 52.8,
CDCl3):
(2H, d,
= 3.3 Hz), 5.48 (1H, s), 2.81-2.86 (1H, m), 2.34 (3H, s), 4-[(10
2.27 (3H, s), 1.45 (3H, s), 1.03 (3H, d, = 7.2 Hz), 0.97 4-(4-chlorophenyl)semicarbazone (10p)
(3H, d,
= 6.0 Hz); 13C-NMR (75 MHz, CDCl3): 159.1, Yield 77.33%; white solid; m.p. 167-168oC; ESI-MS m/
δ
8.85 (1H, s), 8.09 (1H, s), 7.75 (1H, s), 7.57 44.7, 37.7, 36.6, 34.9, 31.2, 26.4, 24.9, 24.7, 20.6, 13.2.
J
= 8.7 Hz), 7.15 (2H, d, = 8.7 Hz), 5.55 (1H, d,
J
J
S)dihydroartemisinin-10-oxy]benzaldehyde
J
J
δ

Artemisinin Derivatives as Falcipain-2 Inhibitors
1529
z
: 554 (M-H)−; IR (KBr) cm−1: 3371, 3233, 2954, 2922, nm) was monitored for 30 min at room temperature
1697, 1605, 1590, 1528, 1405, 1280, 1231, 1124, 1096, with an automated microplate spectrofluorimeter
1036, 978, 955, 939, 877, 829, 446; 1H-NMR (300 MHz, (Biotek, Synergy 2). The inhibition rate (%) was evalu-
CDCl3):
(2H, d,
δ
8.72 (1H, s), 8.12 (1H, s), 7.74 (1H, s), 7.59 ated at 10
µ
M of compound and was calculated using
J
= 8.7 Hz), 7.16 (2H, d, = 8.7 Hz), 5.56 (1H, the equation: [1
J
−
(
F460 F460 control)] × 100%. All values
/
d,
2.45 (1H, m), 1.45 (3H, s), 1.04 (3H, d,
0.97 (3H, d,
= 5.7 Hz); 13C-NMR (75 MHz, CDCl3):
J
= 3.3 Hz), 5.48 (1H, s), 2.81-2.85 (1H, m), 2.34- are the means of three independent determinations
= 7.2 Hz), and the deviations were less than 10% of the mean
value. IC50 values were determined from plots of
J
J
δ
159.4, 154.1, 142.4, 136.9, 129.3, 128.8, 127.7, 121.2, percent activity over compound concentration using
117.2, 104.6, 100.6, 88.6, 81.3, 52.8, 44.6, 37.7, 36.6, GraphPad Prism software. IC50s were determined
34.9, 31.2, 26.4, 24.9, 24.7, 20.6, 13.2.
from three separate experiments and each compound
concentration was tested in triplicate. Attempts to
determine IC50 values were made if the inhibition rate
Falcipain-2 inhibition assay
Recombinant falcipain-2 (30 nM) was incubated for at 10
30 min at room temperature in 100 mM NaOAc, pH concentrations of the tested compounds were 50
5.5, 10 mM dithiothreitol (DTT), with different concen-
µM was greater than 20% and the maximum
µM.
trations of 10a-p. Compound solutions were prepared
from stock in DMSO (maximum concentration of
DMSO in the assay was 1%). After incubation, the
substrate benzyloxycarbonyl-Leu-Arg-7-amino-4-methyl-
RESULTS AND DISCUSSION
Chemistry
The target compounds 10a-p were synthesized through
coumarin (Z-Leu-Arg-AMC) with the same buffer was a general condensation procedure of 4-substituted
added to a final concentration of 25 M. The increase benzaldehyde with thiosemicarbazides 5a-h or semi-
in fluorescence (excitation, 355 nm and emission, 460 carbazides 7a-h, respectively, which were obtained
µ
9
Scheme 1. The synthetic route for compounds 10a-p. Reagents and conditions: (a) CS2, NH3
ClCH2COONa, rt, 30 min; (c) NH2NH2 H2O, rt, 1 h; (d) phenyl chloroformate, pyridine, CH2Cl2, ice-water bath, rt, 4 h; (e)
NH2NH2
H2O, reflux, 12 h; (f) NaBH4, CH3OH, 0-5oC, 3 h; (g) TFAA, Et3N, CH2Cl2, 5-0oC, 1 d; (h) 4-hydroxybenzaldehyde,
5-0oC, 12 h; (i) 5a-h or 7a-h, CH3COOH, EtOH, rt, 2 h.
·H2O, EtOH, rt, 1 h; (b)
·
·
−
−

1530
Y. Liu et al.
from different anilines (Scheme 1).
Hz) was observed in
α
-arteether (Brossi et al., 1988)
Compound
9 was the key intermediate during the and the similar rule was found in artemisinin aromatic
synthetic process which was accomplished by an in- ethers by the other reports (O’Neil et al., 2001; Yang
direct method rather than the generally direct etheri- et al., 2005). For compound
fication catalyzed by BF3
Artemisinin was reduced by NaBH4 based on an
9
, this coupling constant
·
Et2O (Liang and Li, 1996). was 3.3 Hz indicating that it was a
β-isomer.
1
effective reaction to yield dihydroartemisinin 2a (Brossi
Inhibition activity in vitro
et al., 1988) which was activated by trifluoroacetic
The in vitro inhibition assay against Plasmodium
anhydride (TFAA) in the presence of Et3N to form tri- falciparum falcipain-2 was carried out according to a
fluoroacetate . Without separation, compound previously reported method by taking E-64 [ -(L-
reacted with 4-hydroxy benzaldehyde to give com- 3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-amido(4-
pound which was purified on a silica gel column. guanido)butane, an irreversible inhibitor of falcipain-
8
8
N
9
The anilines reacted with carbon disulphide in the 2, as positive control and DMSO as the vehicle control
presence of concentrated ammonia yielded dithio- (Shenai et al., 2000; Li et al., 2009). Activity of the
caramates
sodium monochloroacetate, followed by condensation
with hydrazine hydrate gave thiosemicarbazides 5a-h 2 with inhibition rates greater than 20% at 10
(Dimri and Parmar, 1978). Semicarbazides 7a-h were IC50 values ranged from 0.46 to 4.93 M. Semicar-
also obtained from anilines through acylation with bazones were more effective than thiosemicarbazones
phenyl chloroformate to give the carbamates , which with the same substitution on the aromatic ring (e.g.
were condensed with hydrazine hydrate to yield 7a-h 10f and 10n). Furthermore, with similar scaffolds, the
3
. These compounds on treatment with tested compounds is presented in Table I.
All compounds were found to be inhibitors of falcipain-
M and
µ
µ
6
(Cerecetto et al., 2000).
introduction of methyl, methoxyl and chloro group at
All these compounds were obtained as
β
-isomers due C-4 position on the aromatic ring improved activity
1
to the configuration of compound
9
confirmed by H- (e.g. 10h
,
10m, and 10o). These data will provide a
NMR. The configuration at C-10 was assigned based starting point for drug design in future research.
on the coupling constant between H-9 and H-10. A
small coupling constant (
J
= 3.6 Hz) was observed in
= 9.2
Molecular docking studies
β-arteether while a large coupling constant (
J
Molecular docking studies were performed to inves-
tigate the binding affinities and interaction modes for
the inhibitors using AutoDock 3.05 (Morris et al.,
1998). As illustrated in Fig. 2, residues Cys42, Gly82,
Table I. Inhibition activity of compounds 10a-p against
Plasmodium falciparum falcipain-2 in vitro
b
Com-
pound
Inhibitiona IC50
X
R1
R2
(%)
(µM)
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
E-64
DMSO
S
S
S
S
S
S
S
S
O
O
O
O
O
O
O
O
H
H
H
2-CH3
2-CH3
3-CH3
H
H
H
H
H
H
67.59
84.28
71.75
62.27
78.43
78.20
63.87
89.89
87.30
66.28
94.38
83.02
87.62
81.86
92.99
74.34
100.00
0000.0
1.78
4.00
2.70
4.93
0.97
1.35
1.71
0.46
1.35
1.66
1.16
1.33
0.680
0.501
0.46
1.07
0.0197
2-CH3
4-CH3
3-CH3
4-CH3
4-CH3
4-OCH3
4-Cl
H
2-CH3
4-CH3
3-CH3
4-CH3
4-CH3
4-OCH3
4-Cl
2-CH3
2-CH3
3-CH3
H
H
aInhibition was evaluated at 10
µ
M; bIC50 values were
Fig. 2. Proposed binding mode of compound 10h in the
active site of falcipain-2
determined if inhibition was larger than 20%.

Artemisinin Derivatives as Falcipain-2 Inhibitors
1531
Klayman, D. L., Bartosevich, J. F., Griffin, T. S., Mason, C.
J., and Scovill, J. P., 2-Acetylpyridine thiosemicarbazones.
1. a new class of potential antimalarial agents. J. Med.
Chem., 22, 855-862 (1979).
Gly83, Gln171, Leu172, His174, and Asp234 formed a
pocket for ligand binding, and the most potent com-
pound 10h formed hydrogen bonds with Gly83, Leu172,
and Asp234.
Klayman, D. L., Qinghaosu (artemisinin): an antimalarial
drug from China. Science, 228, 1049-1055 (1985).
Li, H., Huang, J., Chen, L., Liu, X., Chen, T., Zhu, J., Lu, W.,
Shen, X., Li, J., Hilgenfeld, R., and Jiang, H., Identification
of novel falcipain-2 inhibitors as potential antimalarial
agents through structure-based virtual screening. J. Med.
Chem., 52, 4936-4940 (2009).
Liang, J. and Li, Y., Synthesis of the aryl ether derivatives of
artemisinin. Chin. J. Med. Chem., 6, 22-25 (1996).
Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart,
W. E., Belew, R. K., and Olson, A. J., Automated docking
using a Lamarckian genetic algorithm and an empirical
binding free energy function. J. Comput. Chem., 19, 1639-
1662 (1998).
In conclusion, a total of 16 artemisinin derivatives
(
10a-p) were synthesized from the natural product
artemisinin ( ) through a simplified strategy with con-
tinuous operation. All target compounds were obtain-
1
1
ed as
key intermediate
β
-isomers confirmed by H-NMR through the
9
. Biological evaluation and mole-
cular docking studies indicated that this new class of
artemisinin derivatives were identified as potential
falcipain-2 inhibitors and serve as a foundation for
further investigation of antimalarial drugs.
ACKNOWLEDGEMENTS
This work was supported by the National Science &
Technology Major Project of China (No. 2009ZX09301-
012).
Muraleedharan, K. M. and Mitchell, A. A., Progress in the
development of peroxide-based anti-parasitic agents. Drug
Discov. Today, 14, 793-803 (2009).
O’Neil, P. M., Miller, A., Bishop, L. P. D., Hindley, S., Maggs,
J. L., Ward, S. A., Roberts, S. M., Scheinmann, F., Stachulski,
A. K., Posner, G. H., and Park, B. K., Synthesis, antimalarial
activity, biomimetic iron(II) chemistry, and in vivo meta-
bolism of novel, potent C-10-phenoxy derivatives of di-
hydroartemisinin. J. Med. Chem., 44, 58-68 (2001).
Rosenthal, P. J., Sijwali, P. S., Singh, A., and Shenai, B. R.,
Cysteine proteases of malaria parasites: Targets for chemo-
therapy. Curr. Pharm. Des., 8, 1659-1672 (2002).
Shenai, B. R., Sijwali, P. S., Singh, A., and Rosenthal, P. J.,
Characterization of native and recombinant falcipain-2, a
principal trophozoite cysteine protease and essential hemo-
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