Synthesis and antimalarial activity of novel dihydro-artemisinin derivatives

Article abstract of DOI:10.3390/molecules16064527

The Plasmodium falciparum cysteine protease falcipain-2, one of the most promising targets for antimalarial drug design, plays a key role in parasite survival as a major peptide hydrolase within the hemoglobin degradation pathway. In this work, a series of novel dihydroartemisinin derivatives based on (thio)semicarbazone scaffold were designed and synthesized as potential falcipain-2 inhibitors. The in vitro biological assay indicated that most of the target compounds showed excellent inhibition activity against P. falciparum falcipain-2, with IC₅₀ values in the 0.29-10.63 μM range. Molecular docking studies were performed to investigate the binding affinities and interaction modes for the inhibitors. The preliminary SARs were summarized and could serve as a foundation for further investigation in the development of antimalarial drugs.

Full text of DOI:10.3390/molecules16064527

Molecules 2011, 16, 4527-4538; doi:10.3390/molecules16064527
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antimalarial Activity of Novel
Dihydro-Artemisinin Derivatives
Yang Liu 1, Kunqiang Cui 2, Weiqiang Lu 2, Wei Luo 1, Jian Wang 1, Jin Huang 2 and
Chun Guo 1,*
1
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China;
E-Mails: syphuly0325@yahoo.com.cn (Y.L.); l198278w@yahoo.com.cn (W.L.);
wjmed@163.com (J.W.)
2
School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China;
E-Mails: cscs12683@126.com (K.C.); luweiqiangnb@126.com (W.L.);
huangjin@ecust.edu.cn (J.H.)
* Author to whom correspondence should be addressed; E-Mail: chunguo63@yahoo.com.cn;
Tel.: +86-24-23986425; Fax: +86-24-23986425.
Received: 18 April 2011; in revised form: 20 May 2011 / Accepted: 24 May 2011 /
Published: 30 May 2011
Abstract: The Plasmodium falciparum cysteine protease falcipain-2, one of the most
promising targets for antimalarial drug design, plays a key role in parasite survival as a
major peptide hydrolase within the hemoglobin degradation pathway. In this work, a series
of novel dihydroartemisinin derivatives based on (thio)semicarbazone scaffold were
designed and synthesized as potential falcipain-2 inhibitors. The in vitro biological assay
indicated that most of the target compounds showed excellent inhibition activity against
P. falciparum falcipain-2, with IC50 values in the 0.29–10.63 μM range. Molecular docking
studies were performed to investigate the binding affinities and interaction modes for the
inhibitors. The preliminary SARs were summarized and could serve as a foundation for
further investigation in the development of antimalarial drugs.
Keywords: falcipain-2 inhibitors; dihydroartemisinin derivatives; inhibition activity;
molecular docking studies; SARs

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1. Introduction
Malaria is the most common parasitic disease in the World. Plasmodium falciparum is the major
cause of sever malaria and death and has developed resistance to most available antimalarial drugs [1].
Artemisinin (1), isolated from the Artemisia annua L., and its derivatives 2a-d (Figure 1) are effective
antimalarial drugs against multidrug-resistant P. falciparum [2]. The WHO has recommended
artemisinin-based combination therapies (ACTs) as first-line treatment for uncomplicated
P. falciparum malarial since 2001 [3]. However, as P. falciparum resistance to artemisinin has
emerged [4], development of antimalarial drugs against new targets is an urgent priority.
Figure 1. Structures of artemisinin and its derivatives.
Among the promising new targets, P. falciparum cysteine protease falcipain-2, which plays a key
role for the parasite survival as a major peptide hydrolase within the hemoglobin degradation pathway,
is one of the most attractive targets for antimalarial drug design [5]. Falcipain-2 inhibitors can block
parasite protein biosynthesis by preventing host hemoglobin hydrolysis [6]. Therefore, discovery of
new falcipain-2 inhibitors with acceptable properties is a meaningful work.
Iron is essential for the biological activities of some plasmodial proteins and due to the metal
chelating properties, the (thio)semicarbazone moiety has shown activity as a potential facipain-2
inhibitor aimed at preventing the growth of malarial parasites [7-9].
In this work, a series of novel dihydroartemisinin derivatives 10a-l and 12a-f were designed and
synthesized by incorporating the above two biologically active scaffolds, dihydroartemisinin and
(thio)semicarbazone. The in vitro biological assay was carried out against P. falciparum falcipain-2.
The preliminary SARs were summarized and could benefit to the subsequent drug design process.
2. Results and Discussion
2.1. Chemistry
The target compounds 10a-l and 12a-f were synthesized through a general condensation procedure
of 4-substituted benzaldehydes 9 and 11 with thiosemicarbazides 5a-f or semicarbazides 7a-f
respectively (Scheme 1).
Compounds 9 and 11 were the key intermediates of the synthetic process. Compound 9 was
synthesized by an indirect method rather than the generally direct etherification catalyzed by BF3⋅
Et2O [10]. Artemisinin (1) was reduced with NaBH4 based on an effective route to dihydroartemisinin
(2a) [11] which was activated by trifluoroacetic anhydride (TFAA) in the presence of Et3N to
form trifluoroacetate 8. Without separation, compound 8 reacted with 4-hydroxybenzaldehyde to

Molecules 2011, 16
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give compound 9 which was purified on a silica gel column. Compound 11 was synthesized
directly through the esterification of dihydroartemisinin 2a and 4-formylbenzoic acid catalyzed by
dicyclohexylcarbodiimide (DCC) and 4-N,N-dimethylaminopyridine (DMAP) and was also purified on
a silica gel column [12]. Thiosemicarbazides 5a-f were obtained from different anilines by a literature
method [13]. The anilines reacted with carbon disulphide in the presence of concentrated ammonia to
yield dithiocarbamates 3. These compounds on treatment with sodium monochloroacetate followed by
condensation with hydrazine hydrate gave 5a-f. Semicarbazides 7a-f were also obtained from anilines
through acylation with phenyl chloroformate to give carbamates 6, which were condensed with
hydrazine hydrate to yield 7a-f [14].
Scheme 1. The synthetic routes for compounds 10a-l and 12a-f.
Reagents and conditions: (a) CS2, NH3⋅H2O, EtOH, rt, 1 h; (b) 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, 0–5 °C, 3 h; (g) TFAA, Et3N, CH2Cl2, −5–0 °C, 1
d; (h) 4-hydroxybenzaldehyde, −5–0 °C, 12 h; (i) 5a-f or 7a-f, CH3COOH, EtOH, rt, 2 h; (j)
4-formylbenzoic acid, DCC, DMAP, ice-water bath, rt, 1 d; (k) 7a-f, CH3COOH, EtOH, rt, 2 h.
All the target compounds were identified as single isomers due to the configuration of compounds 9
and 11 confirmed by 1H-NMR. The configuration at C-10 was assigned based on the coupling constant
between H-9 and H-10. A small coupling constant (J = 3.6 Hz) was observed in β-arteether, while a
large coupling constant (J = 9.2 Hz) was observed in α-arteether [11] and a similar rule was found in
dihydroartemisinin aromatic ethers in other reports [15,16]. In compounds 9 and 11, these coupling
constants were 3.3 Hz and 9.6 Hz, respectively, which indicated the former was a β-isomer and the
latter was an α-isomer.

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2.2. Biological Assay in Vitro
The in vitro P. falciparum falcipain-2 inhibition assay against was carried out according to a
previously reported method by taking N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl)-amido(4-
guanido)butane (E-64) as a positive control and DMSO as a negative control [17]. Falcipain-2
could hydrolyse the fluorogenic substrate benzyloxycarbonyl-Leu-Arg-7-amino-4-methylcoumarin
(Z-Leu-Arg-AMC) to produce coumarin and its activity could be evaluated by monitoring the
fluorescence of the coumarin. Activity of all the tested compounds is shown in Table 1.
Table 1. Inhibition activity of compounds 10a-l and 12a-f against P. falciparum falcipain-2 in vitro.
Compd.
10a
10b
10c
X
S
S
S
S
R1
H
H
R2
2-F
4-F
Inhibition ratea (%) IC50b (μM)
72.48
59.76
87.42
75.28
74.06
75.62
76.74
84.60
72.70
59.01
87.88
81.82
79.32
69.25
75.89
76.61
71.62
74.67
100.0
2.51
2.17
0.52
0.55
1.35
0.984
2.25
1.02
0.7
2.28
1.03
2.54
0.47
0.65
0.29
2.96
0.57
10.63
0.0197
H
4-OCH2CH3
5-CH3
5-CH3
2-CH3
2-F
10d
10e
10f
10g
10h
10i
10j
10k
10l
12a
12b
12c
12d
12e
2-CH3
3-CH3
3-Cl
H
H
H
2-CH3
3-CH3
3-Cl
H
H
H
2-CH3
3-CH3
3-Cl
S
S
O
O
O
O
O
O
O
O
O
O
O
O
4-F
4-OCH2CH3
5-CH3
5-CH3
2-CH3
2-F
4-F
4-OCH2CH3
5-CH3
5-CH3
2-CH3
12f
E-64
DMSO
0
b
a
Inhibition rates were evaluated at 10 μM concentration level; IC50 values were determined if
above inhibition rates were larger than 20%.
All the tested compounds were found to be inhibitors of falcipain-2 with inhibition rates greater
than 20% at 10 μM concentration level and IC50 values ranging from 0.29 to 10.63 μM. The
preliminary SARs showed that the ester linker in compounds 12a-f was more effective than the ether
linkers in compounds 10a-l with the same substitution on the aromatic ring (e.g., 12a and 10a) and the
latter displayed equivalent efficacy between semicarbazones and thiosemicarbazones (e.g., 10e and
10k). Furthermore, in the same kind of scaffolds, the introduction of fluoro or ethoxyl groups at the
C-4 position as monosubstituents on the aromatic ring could improve the activity (e.g., 12b and 10c)
and among disubstituted compounds, the ones with methyl group disubstitution at the C-3 and C-5
positions on the aromatic ring presented better activities (e.g., 10k and 12e). These implications would
benefit a drug design in future research.

Molecules 2011, 16
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2.3. Molecular Docking Studies
Molecular docking studies were performed to investigate the binding affinities and interaction
modes for the inhibitors using AutoDock 3.05 [18]. As illustrated in Figure 2, residues Trp206, Cys42,
His174, Gly82, Gly83, Leu172, Asp234 and Gln171 form a pocket for ligand binding, and the most
potent compound 12c forms hydrogen bonds with Cys42 and Gly83.
Figure 2. Proposed binding mode of compound 12c in the active site of falcipain-2.
3. Experimental
3.1. Instruments and Reagents
Melting points were taken on glass slides with X-4 digital display microscopic melting point
1
apparatus and were presented uncorrected. H-NMR and 13C-NMR spectra were recorded using a
Bruker ARX-300 spectrometer with CDCl3 as solvent and TMS as internal standard. Electrospray
ionization mass spectra (ESIMS) were recorded on a Waters Quattro micro API. All reagents were
commercially available and were used with purification as needed. Chromatography was carried on
silica gel (200–300 mesh). All reactions were monitored by TLC (silica gel plates with fluorescence
F254 were used).
3.2. General Procedure for the Synthesis of N4-(Substitued Phenyl)thiosemicarbazides 5a-f
To an EtOH solution (50 mL) of aniline (0.1 mol), concentrated ammonia (20 mL) was slowly
added. The mixture was cooled below 20 °C and CS2 (8 mL) was added dropwise during a period of
15 min. After 1 h, sodium monochloroacetate (14 g, 0.12 mol) was added and vigorously stirred for
30 min followed by adding hydrazine hydrate (80%, 12.5 mL). After 1 h, the mixture was cooled
overnight and the crude reaction mixture was filtered and recrystallized from EtOH.
3.3. General Procedure for the Synthesis of N4-(Substitued Phenyl)semicarbazides 7a-f
To a mixture of aniline (0.05 mol), pyridine (5 mL) and CH2Cl2 (30 mL), phenyl chloroformate
(6.3 mL, 0.05 mol) was added dropwise under ice-water bath and reacted at room temperature for 4 h.
The mixture was then evaporated under reduced pressure and the residue was poured into saturated

Molecules 2011, 16
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NaCl solution for salting out. The precipitate was filtered and dried followed by refluxing in hydrazine
hydrate (80%, 10 mL) for 12 h. After cooling, the crude reaction mixture was filtered and recrystallized
from EtOH.
3.4. General Procedure for the Synthesis of Dihydroartemisinin (2a)
To a stirred solution of artemisinin (1, 2 g, 7.08 mmol) in CH3OH, NaBH4 (0.40 g, 10.62 mmol)
was added at 0–5 °C over a period of 20 min. After being stirred for an additional 3 h under the same
condition, the mixture was neutralized with glacial acetic acid while the temperature was maintained at
0–5 °C, concentrated by evaporating most CH3OH, diluted with cold water (100 mL) and stirred for
15 min at room temperature. The precipitate was collected, washed with water (100 mL × 3) and dried.
Yield 97.15%, m.p. 143–145 °C.
3.5. General Procedure for the Synthesis of 4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde
N4-(substitued phenyl)(thio)semicarbazones 10a-l
To a CH2Cl2 solution (50 mL) of dihydroartemisinin (2a, 11.36 g, 40 mmol) and triethylamine
(11.08 mL, 80 mmol), TFAA (11.12 mL, 80 mmol) in CH2Cl2 (30 mL) was added dropwise at −5–0 °C.
After 1 d, 4-hydroxylbenzaldehyde (9.76 g, 80 mmol) was added and stirred for 12 h at the same
temperature. The reaction solution was quenched with saturated NaHCO3 solution and washed by
saturated NaHCO3 solution (50 mL × 5) and water (50 mL × 5), respectively. The organic layer was
dried (Na2SO4) and concentrated in vacuo. The residue was purified on silica gel column with
petroleum ether/ethyl acetate (8:1) to afford 9. Yield 40.29%, m.p. 88–90 °C. MS (ESI) m/z: 411
1
(M+Na); H-NMR (300 MHz, CDCl3) δ: 0.97 (3H, d, J = 6.0 Hz, 16-CH3), 1.03 (3H, d, J = 7.2 Hz,
15-CH3), 1.45 (3H, s, 14-CH3), 2.34–2.44 (1H, m, 4-H), 2.83–2.88 (1H, m, 9-H), 5.44 (1H, s, 12-H),
5.62 (1H, d, J = 3.3 Hz, 10-H), 7.23 (2H, d, J = 8.7 Hz, AA’-2H), 7.84 (2H, d, J = 8.7 Hz, BB’-2H),
9.90 (1H, s, C-H); 13C-NMR (75 MHz, CDCl3) δ: 191.17, 162.65, 132.20, 131.03, 116.95, 104.63,
100.40, 88.65, 81.11, 52.74, 44.52, 37.69, 36.57, 34.85, 31.10, 26.29, 24.88, 24.67, 20.56, 13.13.
Equimolar quantities of 9 (2 mmol) and 5a-f (2 mmol) or 7a-f (2 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 silica gel column with petroleum ether/ethyl acetate (3:1) to
afford 10a-f or with CH2Cl2/CH3OH (80:1) to afford 10g-l.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(2-fluorophenyl)thiosemicarbazone (10a). Yield
1
19.90%, m.p. 108–109 °C. MS (ESI) m/z: 556 (M+H); H-NMR (300 MHz, CDCl3) δ: 0.97 (3H, d,
J = 6.0 Hz, 16-CH3), 1.03 (3H, d, J = 7.2 Hz, 15-CH3), 1.45 (3H, s, 14-CH3), 2.34–2.44 (1H, m, 4-H),
2.82–2.87 (1H, m, 9-H), 5.47 (1H, s, 12-H), 5.57 (1H, d, J = 3.0 Hz, 10-H), 7.16 (2H, d, J = 8.7 Hz,
AA’-2H), 7.63 (2H, d, J = 9.0 Hz, BB’-2H), 7.84 (1H, s, C-H), 9.37 (1H, s, E-H), 9.53 (1H, s, F-H);
13C-NMR (75 MHz, CDCl3) δ: 176.32, 159.78, 143.30, 136.74, 134.31, 130.96, 129.30, 127.53,
127.22, 126.77, 117.25, 104.63, 100.58, 88.63, 81.25, 52.79, 44.61, 37.72, 36.63, 34.90, 31.21, 26.36,
24.92, 24.73, 20.62, 13.22.

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4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(4-fluorophenyl)thiosemicarbazone (10b). Yield
1
42.29%, m.p. 117–118 °C. MS (ESI) m/z: 556 (M+H); H-NMR (300 MHz, CDCl3) δ: 0.97 (3H, d,
J = 6.0 Hz, 16-CH3), 1.03 (3H, d, J = 7.2 Hz, 15-CH3), 1.44 (3H, s, 14-CH3), 2.34–2.44 (1H, m, 4-H),
2.82-2.87 (1H, m, 9-H), 5.46 (1H, s, 12-H), 5.57 (1H, d, J = 3.0 Hz, 10-H), 7.16 (2H, d, J = 8.7 Hz,
AA’-2H), 7.62 (2H, d, J = 9.0 Hz, BB’-2H), 7.85 (1H, s, C-H), 9.07 (1H, s, E-H), 9.68 (1H, s, F-H);
13C-NMR (75 MHz, CDCl3) δ: 175.82, 159.98, 143.54, 136.74, 131.76, 129.45, 129.17, 126.81,
126.11, 117.28, 104.64, 100.57, 88.64, 81.23, 52.78, 44.60, 37.73, 36.62, 34.90, 31.20, 26.35, 24.92,
24.74, 20.60, 13.20.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(4-ethoxyphenyl)thiosemicarbazone (10c). Yield
1
31.80%, m.p. 114–116 °C. MS (ESI) m/z: 580 (M-H); H-NMR (300 MHz, CDCl3) δ: 0.96 (3H, d,
J = 6.0 Hz, 16-CH3), 1.02 (3H, d, J = 7.2 Hz, 15-CH3), 1.44 (3H, s, 14-CH3), 2.33–2.44 (1H, m, 4-H),
2.81–2.86 (1H, m, 9-H), 4.05 (2H, q, J = 6.9 Hz, 4’-OCH2CH3), 5.46 (1H, s, 12-H), 5.55 (1H, d,
J = 3.3 Hz, 10-H), 7.14 (2H, d, J = 8.7 Hz, AA’-2H), 7.60 (2H, d, J = 8.7 Hz, BB’-2H), 7.91 (1H, s,
13
C-H), 9.02 (1H, s, E-H), 10.25 (1H, s, F-H); C-NMR (75 MHz, CDCl3) δ: 176.53, 159.79, 158.31,
143.24, 131.07, 129.34, 127.22, 117.22, 114.34, 104.62, 100.57, 88.62, 81.25, 55.77, 52.79, 44.62,
37.72, 36.63, 34.90, 31.21, 26.35, 24.92, 24.73, 20.60, 19.33, 13.20.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(2,5-dimethylphenyl)thiosemicarbazone (10d).
Yield 38.00%, m.p. 120–122 °C. MS (ESI) m/z: 566 (M+H); 1H-NMR (300 MHz, CDCl3) δ: 0.96 (3H,
d, J = 5.7 Hz, 16-CH3), 1.02 (3H, d, J = 7.2 Hz, 15-CH3), 1.44 (3H, s, 14-CH3), 2.32 (3H, s, 2’-CH3),
2.37 (3H, s, 5’-CH3), 2.81–2.86 (1H, m, 9-H), 5.46 (1H, s, 12-H), 5.55 (1H, d, J = 3.3 Hz, 10-H), 7.14
(2H, d, J = 8.7 Hz, AA’-2H), 7.60 (2H, d, J = 8.7 Hz, BB’-2H), 7.90 (1H, s, C-H), 8.93 (1H, s, E-H),
13
10.13 (1H, s, F-H); C-NMR (75 MHz, CDCl3) δ: 176.91, 159.74, 143.29, 137.45, 134.48, 134.14,
131.69, 129.28, 127.64, 127.47, 127.29, 117.23, 104.62, 100.59, 88.62, 81.24, 52.79, 44.63, 37.72,
36.64, 34.92, 31.21, 26.36, 24.92, 24.74, 21.42, 20.61, 18.28, 13.21.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(3,5-dimethylphenyl)thiosemicarbazone (10e).
1
Yield 16.19%, m.p. 125–127 °C. MS (ESI) m/z: 566 (M+H); H-NMR (CDCl3) δ: 0.97 (3H, d,
J = 5.7 Hz, 16-CH3), 1.03 (3H, d, J = 7.2 Hz, 15-CH3), 1.44 (3H, s, 14-CH3), 2.35 (6H, s, 3’-CH3,
5’-CH3), 2.81–2.86 (1H, m, 9-H), 5.46 (1H, s, 12-H), 5.56 (1H, d, J = 3.3 Hz, 10-H), 7.16 (2H, d,
J = 8.7 Hz, AA’-2H), 7.61 (2H, d, J = 8.7 Hz, BB’-2H), 7.80 (1H, s, C-H), 9.08 (1H, s, E-H), 9.38
13
(1H, s, F-H); C-NMR (75 MHz, CDCl3) δ: 176.56, 159.77, 143.10, 137.43, 135.80, 135.15, 130.18,
129.32, 127.19, 126.46, 122.87, 117.23, 104.62, 100.58, 88.61, 81.24, 52.80, 44.62, 37.72, 36.64,
34.91, 31.21, 27.21, 26.35, 24.92, 24.73, 20.60, 19.68, 13.20.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(3-chloro-2-methylphenyl)thiosemicarbazone
1
(10f). Yield 26.44%, m.p. 150–152 °C. MS (ESI) m/z: 584 (M-H); H-NMR (CDCl3) δ: 0.97 (3H, d,
J = 6.0 Hz, 16-CH3), 1.03 (3H, d, J = 7.2 Hz, 15-CH3), 1.44 (3H, s, 14-CH3), 2.39 (3H, s, 2’-CH3),
2.81–2.86 (1H, m, 9-H), 5.46 (1H, s, 12-H), 5.56 (1H, d, J = 3.3 Hz, 10-H), 7.15 (2H, d, J = 8.7 Hz,
AA’-2H), 7.61 (2H, d, J = 8.7 Hz, BB’-2H), 7.89 (1H, s, C-H), 8.95 (1H, s, E-H), 10.00 (1H, s, F-H);
13C-NMR (75 MHz, CDCl3) δ: 177.06, 159.74, 143.29, 138.08, 136.65, 133.88, 129.28, 127.32,

Molecules 2011, 16
4534
126.04, 117.22, 104.62, 100.58, 88.62, 81.25, 52.79, 44.62, 37.72, 36.64, 34.91, 31.21, 26.36, 24.93,
24.74, 20.84, 20.62, 14.33, 13.22.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(2-fluorophenyl)semicarbazone (10g). Yield
1
72.28%, m.p. 175–177 °C. MS (ESI) m/z: 538 (M-H); H-NMR (CDCl3) δ: 0.97 (3H, d, J = 6.0 Hz,
16-CH3), 1.04 (3H, d, J = 7.5 Hz, 15-CH3), 1.45 (3H, s, 14-CH3), 2.34–2.45 (1H, m, 4-H), 2.81–2.86
(1H, m, 9-H), 5.48 (1H, s, 12-H), 5.56 (1H, d, J = 3.3 Hz, 10-H), 7.16 (2H, d, J = 8.7 Hz, AA’-2H),
13
7.61 (2H, d, J = 8.7 Hz, BB’-2H), 7.77 (1H, s, C-H), 8.49 (1H, s, E-H), 8.98 (1H, s, F-H); C-NMR
(75 MHz, CDCl3) δ: 159.21, 154.37, 141.72, 136.54, 130.66, 128.57, 128.05, 128.00, 127.17, 124.02,
121.44, 117.25, 104.60, 100.69, 88.63, 81.26, 52.82, 44.66, 37.73, 36.65, 34.93, 31.25, 26.39, 24.94,
24.75, 20.63, 13.25.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(4-fluorophenyl)semicarbazone (10h). Yield
1
44.63%, m.p. 124–126 °C. MS (ESI) m/z: 538 (M-H); H-NMR (CDCl3) δ: 0.97 (3H, d, J = 6.0 Hz,
16-CH3), 1.04 (3H, d, J = 7.5 Hz, 15-CH3), 1.45 (3H, s, 14-CH3), 2.34-2.45 (1H, m, 4-H), 2.82–2.87
(1H, m, 9-H), 5.48 (1H, s, 12-H), 5.56 (1H, d, J = 3.0 Hz, 10-H), 7.16 (2H, d, J = 8.7 Hz, AA’-2H),
13
7.59 (2H, d, J = 8.7 Hz, BB’-2H), 7.74 (1H, s, C-H), 8.08 (1H, s, E-H), 8.76 (1H, s, F-H); C-NMR
(75 MHz, CDCl3) δ: 159.27, 154.12, 142.40, 136.95, 129.28, 128.78, 127.72, 121.17, 117.20, 104.61,
100.63, 88.62, 81.26, 52.81, 44.64, 37.73, 36.64, 34.92, 31.24, 26.37, 24.94, 24.74, 20.61, 13.23.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(4-ethoxyphenyl)semicarbazone (10i). Yield
1
52.15%, m.p. 158–159 °C. MS (ESI) m/z: 564 (M-H); H-NMR (CDCl3) δ: 0.97 (3H, d, J = 5.7 Hz,
16-CH3), 1.04 (3H, d, J = 7.2 Hz, 15-CH3), 1.42 (3H, t, J = 7.2 Hz, 4’-OCH2CH3), 1.45 (3H, s,
14-CH3), 2.34–2.44 (1H, m, 4-H), 2.81–2.86 (1H, m, 9-H), 4.03 (2H, q, J = 6.9 Hz, 4’-OCH2CH3),
5.48 (1H, s, 12-H), 5.55 (1H, d, J = 3.3 Hz, 10-H), 7.15 (2H, d, J = 8.7 Hz, AA’-2H), 7.58 (2H, d,
13
J = 8.7 Hz, BB’-2H), 7.73 (1H, s, C-H), 7.97 (1H, s, E-H), 8.81 (1H, s, F-H); C-NMR (75 MHz,
CDCl3) δ: 159.18, 156.34, 154.57, 141.83, 131.32, 128.66, 128.03, 122.24, 117.15, 114.53, 104.58,
100.64, 88.60, 81.27, 55.82, 52.81, 44.66, 37.72, 36.65, 34.93, 31.25, 26.37, 24.93, 24.74, 20.61, 19.50,
13.23.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(2,5-dimethylphenyl)semicarbazone (10j). Yield
1
80.05%, m.p. 172–173 °C. MS (ESI) m/z: 548 (M-H); H-NMR (CDCl3) δ: 0.97 (3H, d, J = 5.7 Hz,
16-CH3), 1.03 (3H, d, J = 7.2 Hz, 15-CH3), 1.45 (3H, s, 14-CH3), 2.33 (3H, s, 2’-CH3), 2.36 (3H, s,
5’-CH3), 2.81–2.86 (1H, m, 9-H), 5.48 (1H, s, 12-H), 5.55 (1H, d, J = 3.3 Hz, 10-H), 7.16 (2H, d,
J = 8.7 Hz, AA’-2H), 7.57 (2H, d, J = 8.7 Hz, BB’-2H), 7.76 (1H, s, C-H), 8.12 (1H, s, E-H), 8.95
13
(1H, s, F-H); C-NMR (75 MHz, CDCl3) δ: 159.15, 154.62, 141.61, 133.83, 131.36, 128.57, 127.63,
122.14, 117.22, 104.59, 100.70, 88.62, 81.26, 52.83, 44.67, 37.72, 36.65, 34.95, 31.25, 26.38, 24.74,
21.12, 20.62, 18.34, 13.24.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(3,5-dimethylphenyl)semicarbazone (10k). Yield
84.60%, m.p. 169–171 °C. MS (ESI) m/z: 548 (M-H); 1H-NMR (CDCl3) δ: 0.97 (3H, d,
J = 6.0 Hz, 16-CH3), 1.04 (3H, d, J = 7.2 Hz, 15-CH3), 1.45 (3H, s, 14-CH3), 2.33 (6H, s, 3’-CH3,
5’-CH3), 2.81–2.86 (1H, m, 9-H), 5.48 (1H, s, 12-H), 5.56 (1H, d, J = 3.3 Hz, 10-H), 7.16 (2H, d,

Molecules 2011, 16
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J = 8.7 Hz, AA’-2H), 7.59 (2H, d, J = 8.7 Hz, BB’-2H), 7.76 (1H, s, C-H), 8.05 (1H, s, E-H), 9.01
13
(1H, s, F-H); C-NMR (75 MHz, CDCl3) δ: 159.19, 154.33, 141.79, 137.44, 135.94, 132.01, 130.30,
128.67, 128.04, 121.58, 117.71, 117.17, 104.59, 100.67, 88.60, 81.27, 52.83, 44.67, 37.73, 36.66,
34.94, 31.26, 26.37, 24.94, 24.75, 20.61, 20.24, 19.03, 13.23.
4-[(10S)-Dihydroartemisinin-10-oxy]benzaldehyde N4-(3-chloro-2-methylphenyl)semicarbazone (10l).
1
Yield 92.97%, m.p. 177–179 °C. MS (ESI) m/z: 568 (M-H); H-NMR (CDCl3) δ: 0.97 (3H, d,
J = 5.7 Hz, 16-CH3), 1.03 (3H, d, J = 7.5 Hz, 15-CH3), 1.45 (3H, s, 14-CH3), 2.81–2.86 (1H, m, 9-H),
5.48 (1H, s, 12-H), 5.56 (1H, d, J = 3.3 Hz, 10-H), 7.57 (2H, d, J = 8.7 Hz, BB’-2H), 7.76 (1H, s,
13
C-H), 8.20 (1H, s, E-H), 8.98 (1H, s, F-H); C-NMR (75 MHz, CDCl3) δ: 159.15, 154.79, 141.71,
137.49, 136.14, 128.56, 128.24, 126.43, 126.24, 120.86, 117.21, 104.59, 100.69, 88.62, 81.26, 52.83,
44.67, 37.73, 36.66, 34.94, 31.25, 26.38, 24.94, 24.74, 21.03, 20.62, 13.73, 13.24.
3.6. General Procedure for the Synthesis of 4-[(10S)dihydroartemisinin-10-oxycarbonyl]benzaldehyde
N4-(substitued phenyl)semicarbazones 12a-f
To a CH2Cl2 solution (100 mL) of dihydroartemisinin (2a, 11.36 g, 40 mmol) and 4-formylbenzoic
acid (7.21 g, 48 mmol), DCC (9.90 g, 48 mmol) and DMAP (1.47 g, 12 mmol) were added at 0–5 °C
for 1 h. After 1 d reaction at room temperature, the mixture was filtered and filtrate was concentrated
in vacuo. The residue was purified on silica gel column with petroleum ether/ethyl acetate (6:1) to
afford 11. Yield 40.46 %, m.p. 81–82 °C. MS (ESI) m/z: 439 (M+Na); 1H-NMR (CDCl3) δ: 0.94 (3H,
d, J = 7.2 Hz, 16-CH3), 0.99 (3H, d, J = 5.7 Hz, 15-CH3), 1.43 (3H, s, 14-CH3), 2.35–2.45 (1H, m,
4-H), 2.74–2.81 1H, m, 9-H), 5.54 (1H, s, 12-H), 6.03 (1H, d, J = 9.6 Hz, 10-H), 7.97 (2H, d, J = 8.1
13
Hz, AA’-2H), 8.28 (2H, d, J = 8.1 Hz, BB’-2H), 10.12 (1H, s, C-H); C-NMR (75 MHz, CDCl3) δ:
190.89, 168.31, 162.30, 133.17, 117.52, 104.61, 100.73, 88.56, 81.13, 52.69, 44.62, 37.70, 36.63,
34.89, 31.10, 26.34, 24.72, 23.67, 20.60, 13.11. Equimolar quantities of 11 (2 mmol) and 7a-f
(2 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 silica gel column with
CH2Cl2/CH3OH (80:1) to afford 12a-f.
4-[(10S)-Dihydroartemisinin-10-oxycarbonyl]benzaldehyde N4-(2-fluorophenyl)semicarbazone (12a).
1
Yield 17.62%, m.p. 145–147 °C. MS (ESI) m/z: 590 (M+Na); H-NMR (CDCl3) δ: 0.94 (3H, d,
J = 7.2 Hz, 16-CH3), 0.99 (3H, d, J = 5.7 Hz, 15-CH3), 1.44 (3H, s, 14-CH3), 2.35–2.46 (1H, m, 4-H),
2.74–2.81 (1H, m, 9-H), 5.55 (1H, s, 12-H), 6.02 (1H, d, J = 9.9 Hz, 10-H), 7.72 (2H, d, J = 8.4 Hz,
AA’-2H), 7.89 (1H, s, C-H), 8.14 (2H, d, J = 8.4 Hz, BB’-2H), 8.20 (1H, s, E-H), 9.46 (1H, s, F-H);
13C-NMR (75 MHz, CDCl3) δ: 166.32, 158.65, 154.63, 142.01, 136.62, 130.66, 128.63, 128.14,
128.03, 127.22, 124.03, 121.35, 117.33, 104.62, 100.70, 88.45, 81.27, 52.80, 44.51, 37.67, 36.73,
34.89, 31.20, 36.37, 24.90, 24.71, 20.59, 13.12.
4-[(10S)-Dihydroartemisinin-10-oxycarbonyl]benzaldehyde N4-(4-fluorophenyl)semicarbazone (12b).
1
Yield 81.92%, m.p. 139–141 °C. MS (ESI) m/z: 590 (M+Na); H-NMR (CDCl3) δ: 0.93 (3H, d,
J = 7.2 Hz, 16-CH3), 0.99 (3H, d, J = 5.7 Hz, 15-CH3), 1.43 (3H, s, 14-CH3), 2.35–2.45 (1H, m, 4-H),
2.73–2.81 (1H, m, 9-H), 5.55 (1H, s, 12-H), 6.02 (1H, d, J = 9.9 Hz, 10-H), 7.71 (2H, d, J = 8.4 Hz,

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AA’-2H), 7.91 (1H, s, C-H), 8.12 (2H, d, J = 8.1 Hz, BB’-2H), 8.13 (1H, s, E-H), 9.99 (1H, s, F-H);
13C-NMR (75 MHz, CDCl3) δ: 166.02, 159.32, 154.34, 142.36, 136.89, 129.33, 128.78, 128.91,
121.20, 117.15, 104.61, 100.60, 88.63, 81.32, 52.77, 44.56, 37.73, 36.55, 34.90, 31.22, 26.41, 24.91,
24.70, 20.62, 13.16.
4-[(10S)-Dihydroartemisinin-10-oxycarbonyl]benzaldehyde N4-(4-ethoxyphenyl)semicarbazone (12c).
1
Yield 42.11%, m.p. 152–153 °C. MS (ESI) m/z: 616 (M+Na); H-NMR (CDCl3) δ: 0.93 (3H, d,
J = 7.2 Hz, 16-CH3), 0.98 (3H, d, J = 5.1 Hz, 15-CH3), 1.43 (3H, s, 14-CH3), 2.35–2.45 (1H, m, 4-H),
2.73–2.80 (1H, m, 9-H), 4.04 (2H, q, J = 6.9 Hz, 4’-OCH2CH3), 5.54 (1H, s, 12-H), 6.01 (1H, d,
J = 9.6 Hz, 10-H), 7.70 (2H, d, J = 8.1 Hz, AA’-2H), 7.90 (1H, s, C-H), 8.03 (1H, s, E-H), 8.12 (2H, d,
J = 8.1 Hz, BB’-2H), 10.11 (1H, s, F-H); 13C-NMR (75 MHz, CDCl3) δ: 166.87, 159.52, 154.62,
142.78, 131.31, 128.70, 128.03, 122.17, 117.22, 114.51, 104.59, 100.60, 88.56, 81.32, 55.83, 52.79,
44.65, 37.71, 36.63, 34.90, 31.21, 26.36, 24.90, 24.72, 20.60, 19.45, 13.21.
4-[(10S)-Dihydroartemisinin-10-oxycarbonyl]benzaldehyde
N4-(2,5-dimethylphenyl)semicarbazone
(12d). Yield 85.69%, m.p. 155–156 °C. MS (ESI) m/z: 600 (M+Na); 1H-NMR (CDCl3) δ: 0.94 (3H, d,
J = 7.2 Hz, 16-CH3), 0.99 (3H, d, J = 5.7 Hz, 15-CH3), 1.43 (3H, s, 14-CH3), 2.73–2.80 (1H, m, 9-H),
5.54 (1H, s, 12-H), 6.02 (1H, d, J = 9.6 Hz, 10-H), 7.67 (2H, d, J = 8.4 Hz, AA’-2H), 7.89 (1H, s,
13
C-H), 8.11 (1H, s, E-H), 8.13 (2H, d, J = 8.4 Hz, BB’-2H), 9.68 (1H, s, F-H); C-NMR (75 MHz,
CDCl3) δ: 167.14, 159.02, 154.61, 141.70, 134.23, 131.41, 128.48, 127.55, 121.11, 117.20, 104.62,
100.60, 88.56, 81.31, 52.78, 44.72, 37.66, 36.69, 34.90, 31.24, 26.42, 24.67, 21.10, 18.33, 13.21.
4-[(10S)-Dihydroartemisinin-10-oxycarbonyl]benzaldehyde
N4-(3,5-dimethylphenyl)semicarbazone
(12e). Yield 63.18%, m.p. 154–155 °C. MS (ESI) m/z: 600 (M+Na); 1H-NMR (CDCl3) δ: 0.93 (3H, d,
J = 7.2 Hz, 16-CH3), 0.98 (3H, d, J = 5.7 Hz, 15-CH3), 1.43 (3H, s, 14-CH3), 2.73–2.80 (1H, m, 9-H),
5.54 (1H, s, 12-H), 6.01 (1H, d, J = 9.9 Hz, 10-H), 7.68 (2H, d, J = 8.4 Hz, AA’-2H), 7.93 (1H, s,
13
C-H), 8.08 (1H, s, E-H), 8.11 (2H, d, J = 8.4 Hz, BB’-2H), 10.06 (1H, s, F-H); C-NMR (75 MHz,
CDCl3) δ: 167.71, 159.22, 154.31, 141.77, 134.90, 132.01, 130.86, 128.66, 128.02, 121.56, 117.71,
117.20, 104.61, 100.68, 88.61, 81.32, 52.80, 44.73, 37.67, 36.70, 34.86, 31.20, 26.43, 24.91, 24.73,
20.60, 20.16, 19.21, 13.21.
4-[(10S)-Dihydroartemisinin-10-oxycarbonyl]benzaldehyde N4-(3-chloro-2-methylphenyl)semicarbaz-
one (12f). Yield 63.54%, m.p. 173–174 °C. MS (ESI) m/z: 620 (M+Na); 1H-NMR (CDCl3) δ: 0.94 (3H,
d, J = 6.9 Hz, 16-CH3), 0.98 (3H, d, J = 5.7 Hz, 15-CH3), 1.43 (3H, s, 14-CH3), 2.73–2.80 (1H, m,
9-H), 5.54 (1H, s, 12-H), 6.01 (1H, d, J = 9.9 Hz, 10-H), 7.67 (2H, d, J = 8.4 Hz, AA’-2H), 7.92 (1H,
s, C-H), 8.12 (2H, d, J = 8.1 Hz, BB’-2H), 8.21 (1H, s, E-H), 10.19 (1H, s, F-H); 13C-NMR (75 MHz,
CDCl3) δ: 166.32, 159.14, 154.81, 141.68, 137.11, 136.10, 128.61, 128.15, 126.42, 126.21, 120.94,
117.20, 104.56, 100.72, 88.61, 81.26, 52.80, 44.66, 37.70, 36.74, 34.92, 31.31, 26.42, 24.89, 24.70,
21.01, 20.64, 13.71, 13.20.

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3.7. Inhibition Assays
Recombinant falcipain-2 (30 nM) was incubated for 30 min at room temperature in 100 mM
NaOAc, pH 5.5, 10 mM dithiothreitol (DTT), with different concentrations of 10a-l and 12a-f. These
solutions were prepared from stock in DMSO (maximum concentration of DMSO in the assay was
1%). After incubation, the substrate Z-Leu-Arg-AMC with the same buffer was added to a final
concentration of 25 μM. The increase in fluorescence (excitation at 355 nm and emission at
460 nm) was monitored for 30 min at room temperature with an automated microplate
spectrofluorimeter (Biotek, Synergy 2). IC50 values were determined from plots of percent activity
over compound concentration using the GraphPad Prism software if inhibition rates were larger than
20% at 10 μM concentration level.
4. Conclusions
In conclusion, a total of 18 dihydroartemisinin derivatives, 10a-l and 12a-f, were synthesized from
the natural product artemisinin (1) and anilines through simplified procedures. All target compounds
were obtained as single isomers as confirmed by 1H-NMR through the key intermediates 9 and 11. The
biological evaluation and molecular docking studies indicated that this new class of dihydroartemisinin
derivatives could be identified as potential falcipain-2 inhibitors and the preliminary SARs could serve
as a foundation for further investigation of antimalarial drugs.
Acknowledgements
This work was supported by the National Science & Technology Key Specific Project for
Significant Creation of New Drugs (2009ZX09301-012).
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Sample Availability: Samples of the compounds 9, 11, 10a-l and 12a-f are available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).