N1-{4-[(10S)-Dihydroartemisinin-10-oxyl]}phenylmethylene-N 2-(2-methylquinoline-4-yl)hydrazine derivatives as antiplasmodial falcipain-2 inhibitors

Article abstract of DOI:10.1007/s00044-011-9854-3

A series of N¹-{4-[(10S)-dihydroartemisinin- 10-oxyl]}phenylmethylene-N²-(2-methylquinoline-4-yl) hydrazine derivatives 9a-9n possessing 4-quinolylhydrazone and artemisinin cores were herein synthesized and evaluated for their activities against cysteine protease falcipain- 2 of Plasmodium falciparum. The structures were clearly confirmed by elemental analysis, ¹H NMR, and mass spectra. The pharmacological results indicated that all compounds showed excellent activity against recombinant falcipain-2 (IC₅₀ = 0.15-2.28 μM). The best one of this series was compound 9d (IC₅₀ = 0.15 μM). The molecular docking results showed that the compound 9d made close contact with the key active site of cysteine protease falcipain-2.

Full text of DOI:10.1007/s00044-011-9854-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3073–3079
DOI 10.1007/s00044-011-9854-3
ORIGINAL RESEARCH
N1-{4-[(10S)-Dihydroartemisinin-10-oxyl]}phenylmethylene-N2-
(2-methylquinoline-4-yl)hydrazine derivatives as antiplasmodial
falcipain-2 inhibitors
•
Wei Luo Wei-Qiang Lu Kun-Qiang Cui Yang Liu
•
•
•
•
Jian Wang Chun Guo
Received: 11 March 2011 / Accepted: 28 October 2011 / Published online: 9 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of N1-{4-[(10S)-dihydroartemisinin-
10-oxyl]}phenylmethylene-N2-(2-methylquinoline-4-yl)
hydrazine derivatives 9a–9n possessing 4-quinolylhydraz-
one and artemisinin cores were herein synthesized and
evaluated for their activities against cysteine protease fal-
cipain-2 of Plasmodium falciparum. The structures were
clearly confirmed by elemental analysis, 1H NMR, and
mass spectra. The pharmacological results indicated that all
compounds showed excellent activity against recombinant
falcipain-2 (IC50 = 0.15–2.28 lM). The best one of this
series was compound 9d (IC50 = 0.15 lM). The molecular
docking results showed that the compound 9d made close
contact with the key active site of cysteine protease falci-
pain-2.
parasite. Due to the spread of multi-resistant P. falciparum
strains, combined with the lack of effective vaccines
(Kumar et al., 2010; Fattorusso et al., 2008; Li et al.,
2009), there is an urgent need for the rapid development of
new mechanism and effective antimalarial agents.
Cysteine protease falcipain-2 of P. falciparum, stored in
the acidic food vacuole, plays an essential role for the
parasite survival as a major peptide hydrolase within the
hemoglobin degradation pathway (Dasaradhi et al., 2005).
Because of the differences of parasitic cysteine proteases
and its human orthologs in nature, falcipain-2 was emerged
as potential new target for antimalarial chemotherapy (Li
et al., 2009; Verissimo et al., 2008).
Artemisinin has been used successfully against chloro-
quine-resistant malarial parasites. However, its poor solu-
bility, coupled with short plasma half life and relatively
high recrudescence rate, restricts its employment (O’Neill
and Posner, 2004; Galal et al., 2009). Chloroquine has been
the standard antimalarial drug for several decades. Unfor-
tunately, its efficacy was severely compromised by the
spread of resistance. It is important to note that modifica-
tions on the side chain of chloroquine are prevailing,
leading to analogs significantly more potent than chloro-
quine against chloroquine-resistant strains (Kumar et al.,
2010; Fattorusso et al., 2008; Li et al., 2009; Ryckebusch
et al., 2005). Considering that chloroquine analogs incor-
porating a Schiff base hydrazone moiety showed better
antimalarial activity and inhibition against cysteine prote-
ase falcipain-2 (Fattorusso et al., 2008; Gemma et al.,
2009; Chipeleme et al., 2007), the scaffold of 4-quin-
olylhydrazone was attached to artemisinin by combination
principles to get the new hybrid structures which would be
effective against falcipain-2 (Fig. 1). The details of syn-
thesis of target compounds and evaluation of their activities
against falcipain-2 were presented herein.
Keywords Antimalarial ꢀ Falcipain-2 inhibitors ꢀ
Artemisinin derivatives
Introduction
Malaria, the most common parasitic diseases in tropical
and subtropical regions, is one of the major public health
´
problems in the world (Kouznetsov and Gomez-Barrio,
2009). The majority of malaria death is caused by Plas-
modium falciparum, the most virulent human malaria
W. Luo ꢀ Y. Liu ꢀ J. Wang ꢀ C. Guo (&)
Key Laboratory of Structure-Based Drug Design & Discovery
(Shenyang Pharmaceutical University), Ministry of Education,
Shenyang 110016, Liaoning, China
e-mail: chunguo63@yahoo.com.cn
W.-Q. Lu ꢀ K.-Q. Cui
School of Pharmacy, East China University of Science
and Technology, Shanghai 200237, China
123

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Med Chem Res (2012) 21:3073–3079
CH3
Molecular docking
H
O
CH3
CH3
CH3
O
O
H3C
N
HN
N
O
With the aim of better understanding the binding mode of
the target compounds to the biological target, the molecular
modeling study was performed. The AutoDock program
can predict the protein–ligand interactions and free binding
energies. The docked energy for the most potent compound
9d was -11.71 kcal/mol. As shown in Fig. 2, the com-
pound 9d interacted within the receptor pocket surrounded
by residues Cys42, Gly82, Gly83, Gln171, Leu172,
His174, and Asp234. Compound 9d may form hydrophobic
contacts with the residues within the pocket.
H
O
CH3
Cl
H
N
N
CH3
H
O
O
O
H3C
O
H
R1
R2
O
CH3
N
N
N
H
CH3
R1=Cl, F, Br, Me, OMe, Et. R2=Me, Cl.
Pharmacology and discussion
Fig. 1 Structures of the target compounds
Compounds 9a–9n were evaluated for inhibitory activity
against recombinant falcipain-2. An equivalent volume of
DMSO was used as negative control, and the irreversible
standard inhibitor, namely E-64 (Shenai et al., 2000), was
used as positive control. The IC50 values were determined
if the inhibition rates of compounds at 10 lM were larger
than 20%, and the inhibitory rates less than 20% deter-
mined as ineffective. The excellent results were summa-
rized in Table 1.
Results and discussion
Chemistry
The synthesis of the target compounds was carried out
according to Schemes 1 and 2. Commercially available
artemisinin 1 by means of sodium borohydride in methanol
was reduced to dihydroartemisinin 2 which treated with
trifluoroacetic anhydride, and 4-hydroxybenzaldehyde was
converted to compound 3. Compounds 5a–5n could be
prepared by the reaction of corresponding aniline with
ethyl acetoacetate, and cyclization of 5a–5n in refluxing
diphenyl ether afforded the desired 4-hydroxyquinoline
compounds 6a–6n which were converted to 4-chloroquin-
olines 7a–7n using POCl3. Nucleophilic substitution reac-
tion of 7a–7n with refluxing hydrazine monohydrate
afforded molecules 8a–8n, followed by reaction of 8a–8n
with 3 at room temperature to provide desired compounds
9a–9n. All products 9a–9n were 10b-isomers as indicated
by the coupling constant between 9-H and 10-H in 1H
NMR (Lee et al., 2000; Wu et al., 2001).
All target compounds were found to be significant inhib-
itors of falcipain-2 with IC50 values ranging from 0.15 to
2.28 lM. Compounds 9c, 9e, and 9 m displayed a significant
activity, whereas compound 9b turned out to be 2–11 times
less active against the falcipain-2 in the same assay. Hence, it
can be reasonably speculated that the introduction of other
substituents in 9b had an important effect on the pharma-
cological activity. Moreover, with the exception of 9b, all
tested compounds were more potent than compound 9i, and it
suggested that the introduction of substituents on quinoline
ring, especially chloro group on the 60-position, played a
significant role in determining the activity. In addition, it was
noteworthy that compound 9d was the best inhibitor of this
series (IC50 = 0.15 lM). This was surprising given the fact
that the molecule, chloro group on the 60-position, was more
suitable for the key active site of falcipain-2. This may be due
to the reason that compound 9d could form better hydro-
phobic contacts with the residues within the pocket, sup-
ported by the molecular docking results.
Inconclusion,anewclassofnovelmoleculesincorporating
4-quinolylhydrazone and artemisinincores weredesignedand
synthesized, and all compounds showed remarkable activity
versusfalcipain-2.Webelievedthatallinhibitorsdescribedhere
mayrepresentapointforfindingnewmechanismandeffective
antimalarial lead blockingthe cleavage activity of falcipain-2.
Furthermore, further research was underway, focusing on the
modificationofthe20-positioninordertoperfectthestructure–
activityrelationship, as well as the definitemechanism against
falcipain-2.
CH3
CH3
CH3
H
H
H
O
O
O
O
O
O
H3C
H3C
H3C
O
O
O
a
b
H
H
H
O
O
O
CH3
CH3
CH3
O
OH
O
CHO
1
2
3
Scheme 1 Synthesis of the key intermediate 3. Reagents and
conditions: (a) NaBH4, MeOH, 0–5°C, 5 h. (b) 4-Hydroxybenzalde-
hyde, trifluoroacetic anhydride, Et3N, CH2Cl2, 0–5°C, 9 h
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Med Chem Res (2012) 21:3073–3079
3075
O
Scheme 2 Synthesis of the
target compounds 9a–9n.
Reagents and conditions:
(a) MeOH, 40°C, 7 h.
(b) Diphenyl ether, reflux,
30 min. (c) Phosphorus
oxychloride, reflux, 3 h.
(d) Hydrazine hydrate, reflux,
2–3 h. (e) Compound 3, MeOH,
25°C, 7 h
OH
N
c
a
OEt
b
R1
R1
R
NH2
Cl
N
H
5a-n
CH3
CH3
CH3
R2
R2
R2
6a-n
4a-n
H
NHNH2
O
O
H3C
e
d
O
R1
R
H
R1
6'
R2
7'
N
CH3
N
CH3
O
R2
R2
8'
CH3
O
7a-n
8a-n
1'
5'
N
N
N
H
CH
3
2'
4'
3'
R1=Cl, F, Br, Me, OMe, Et. R2=Me, Cl.
9a-n
Table 1 The target compounds 9a–9n and the IC50 values against the
cysteine protease falcipain-2 of Plasmodium falciparum
Compound R1
R2
Inhibition rates (%) IC50 (lM)
Falcipain-
2
9a
9b
9c
9d
9e
9f
80-bromo
H
H
94.14
84.30
0.84
2.28
0.21
0.15
0.39
0.52
0.28
0.67
0.89
0.29
80-methyl
80-methyl
60-chloro
80-methyl
80-ethyl
80-fluoro
60-fluoro
H
70-chloro 82.58
88.16
50-chloro 79.42
H
H
H
H
H
H
85.06
84.65
88.63
94.06
93.69
9g
9h
9ii
9j
60-
methoxyl
9k
9l
60-bromo
70-chloro
80-methyl
H
89.77
88.42
79.41
0.26
0.17
0.84
H
70-
Fig. 2 The interaction mode between compound 9d and falcipain-2
9m
methyl
9n
60-methyl
H
87.19
92
0.78
0.020
0
E-64a
DMSOb
a
Experimental
0
The irreversible standard inhibitor, used as positive control (Shenai
Chemistry
et al., 2000)
b
Negative control
All melting points were determined (uncorrected) on a
melting point apparatus (Bu¨chi Labortechnik, Flawil,
Switzerland). 1H NMR spectra were obtained using
300 MHz spectrometer (Bruker Bioscience, Billerica, MA,
USA) with tetramethylsilane as an internal standard. Mass
spectra (MS) were taken in ESI mode on LC–MS (Agilent
1100, Palo Alto, CA, USA). Elemental analysis was
determined on a Carlo-Erba 1106 elemental analysis
instrument. Reaction progress was monitored by TLC on
silica gel precoated F254 Merck plates. Preparative flash
column chromatography was performed on the Merck 200
mesh silica gel. Unless otherwise noted, all solvents and
reagents were commercially available and used without
further purification.
Preparation of dihydroartemisinin (2)
Dihydroartemisinin 2 was prepared according to a reported
procedure (Brossi et al., 1988).
(10S)-10-(4-formylphenyl)dihydroartemisinin (3)
(10S)-10-(4-formylphenyl)dihydroartemisinin 3 was pre-
pared according to a reported procedure (Liang and Li,
1996). White solid, m.p.:123–124°C. MS m/z: 389.1
(M ? H)?.
123

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Med Chem Res (2012) 21:3073–3079
General procedure for synthesis of compounds (8a–8n)
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(7-chloro-2,8-
dimethylquinoline-4-yl)hydrazine (9c)
Compounds 8a–8n were prepared by using the general
synthetic method outlined in Scheme 2, and following
´
previously described procedures (Kouznetsov and Gomez-
1
Light yellow solid (0.22 g, 18.61%). m.p.:131–132°C. H
NMR (CDCl3) d: 0.98 (d, J = 6.6 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.2 Hz, 3H, 6-CH3), 1.26–1.37 (m, 3H), 1.45 (s, 3H,
3-CH3), 1.49–2.05 (m, 7H), 2.41–2.43 (m, 1H), 2.70 (s, 3H,
quinoline–CH3), 2.82–2.84 (m, 4H, quinoline–CH3, 9-H),
5.50 (s, 1H, 12-H), 5.56 (d, J = 3 Hz, 1H, 10-H), 7.16 (d,
J = 8.4 Hz, 2H, Ar–H), 7.30–7.37 (m, 2H, quinoline–H),
7.59 (1H, quinoline–H), 7.68 (d, J = 8.4 Hz, 2H, Ar–H),
7.99 (s, 1H, CH=N). MS m/z: 592.3 (M ? H)?. Anal.
calcd. For C33H38ClN3O5: C, 66.94; H, 6.47; N, 7.10.
Found: C, 66.97; H, 6.51; N, 7.13.
Barrio, 2009; Gemma et al., 2009).
General procedure for synthesis of compounds (9a–9n)
The corresponding 4-quinolylhydrazine (2.00 mmol) was
added to a solution of 3 (2.00 mmol) in 50 ml methanol
with a drop of acetic acid. The mixture was stirred at room
temperature for 7 h, and the solvent was removed under
reduced pressure. The residue was purified by flash chro-
matography on silica gel using dichloromethane/methanol
as eluent.
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(6-chloro-2-methylquinoline-4-
yl)hydrazine (9d)
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(8-bromo-2-methylquinoline-4-
yl)hydrazine (9a)
1
Light yellow solid (0.25 g, 21.66%). m.p.:137–138°C. H
1
Light yellow solid (0.16 g, 12.88%). m.p.:106–107°C. H
NMR (CDCl3) d: 0.98 (d, J = 5.7 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.5 Hz, 3H, 6-CH3), 1.25–1.35 (m, 3H), 1.45 (s, 3H,
3-CH3), 1.51–2.07 (m, 7H), 2.41–2.43 (m, 1H), 2.67 (s, 3H,
quinoline–CH3), 2.82–2.86 (m, 1H, 9-H), 5.52 (s, 1H,
12-H), 5.56 (d, J = 3.3 Hz, 1H, 10-H), 7.14 (d,
J = 8.7 Hz, 2H, Ar–H), 7.31 (s, 1H, quinoline–H),
7.50–7.56 (m, 1H, quinoline–H), 7.65 (d, J = 8.7 Hz, 2H,
Ar–H), 7.84–7.87 (m, 2H, quinoline–H), 8.06 (s, 1H,
CH=N). MS m/z: 578.1 (M ? H)?. Anal. calcd. For
C32H36ClN3O5: C, 66.48; H, 6.28; N, 7.27. Found: C,
66.43; H, 6.31; N, 7.23.
NMR (CDCl3) d: 0.97 (d, J = 5.7 Hz, 3H, 9-CH3), 1.03 (d,
J = 7.2 Hz, 3H, 6-CH3), 1.26 (s, 3H, 3-CH3), 1.27–1.40
(m, 3H), 1.60-2.06 (m, 7H), 2.34–2.43 (m, 1H), 2.64 (s, 3H,
quinoline–CH3), 2.82–2.85 (m, 1H, 9-H), 5.50(s, 1H,
12-H), 5.55 (d, J = 3 Hz, 1H, 10-H), 7.13 (d, J = 8.7 Hz,
2H, Ar–H), 7.12–7.23 (m, 2H, quinoline–H), 7.64 (d,
J = 8.7 Hz, 2H, Ar–H), 7.85 (d, J = 7.5 Hz, 1H, quino-
line–H), 7.99 (d, J = 8.1 Hz, 1H, quinoline–H) 8.15 (s,
1H, CH=N). MS m/z: 622.4 (M ? H)?. Anal. calcd. For
C32H36BrN3O5: C, 61.74; H, 5.83; N, 6.75. Found: C,
61.71; H, 5.80; N, 6.73.
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(2,8-dimethylquinoline-4-
yl)hydrazine (9b)
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(5-chloro-2,8-
dimethylquinoline-4-yl)hydrazine (9e)
1
1
Light yellow solid (0.23 g, 20.63%). m.p.:125–126°C. H
Light yellow solid (0.58 g, 49.05%). m.p.:132–133°C. H
NMR (CDCl3) d: 0.98 (d, J = 6 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.5 Hz, 3H, 6-CH3), 1.25–1.35 (m, 3H), 1.46 (s, 3H,
3-CH3), 1.59–2.07 (m, 7H), 2.41–2.43 (m, 1H), 2.70 (s, 3H,
quinoline–CH3), 2.73 (s, 3H, quinoline–CH3) 2.84–2.86
(m, 1H, 9-H), 5.50 (s, 1H, 12-H), 5.58 (d, J = 3 Hz, 1H,
10-H), 7.17 (d, J = 8.7 Hz, 2H, Ar–H), 7.23 (s, 1H,
quinoline–H), 7.29–7.31 (m, 1H, quinoline–H), 7.46 (d,
J = 8.7 Hz, 1H, quinoline–H), 7.71 (d, J = 8.7 Hz, 2H,
Ar–H), 7.77 (1H, quinoline–H), 8.16 (s, 1H, CH=N). MS
m/z: 558.1 (M ? H)?. Anal. calcd. For C33H39N3O5: C,
71.07; H, 7.05; N, 7.53. Found: C, 71.01; H, 7.09; N, 7.55.
NMR (CDCl3) d: 0.97 (d, J = 5.7 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.2 Hz, 3H, 6-CH3), 1.21–1.34 (m, 3H), 1.45 (s, 3H,
3-CH3), 1.58–2.06 (m, 7H), 2.38–2.41 (m, 1H), 2.67 (s, 3H,
quinoline–CH3), 2.69 (s, 3H, quinoline–CH3), 2.82–2.86
(m, 1H, 9-H), 5.50 (s, 1H, 12-H), 5.56 (d, J = 3 Hz, 1H,
10-H), 7.17 (d, J = 9 Hz, 2H, Ar–H), 7.52 (s, 1H, quino-
line–H), 7.23 (d, J = 7.5 Hz, 1H, quinoline–H), 7.32 (d,
J = 7.5 Hz, 1H, quinoline–H), 7.68 (d, J = 8.7 Hz, 2H,
Ar–H), 7.91 (s, 1H, CH=N). MS m/z: 592.5 (M ? H)?.
Anal. calcd. For C33H38ClN3O5: C, 66.94; H, 6.47; N, 7.10.
Found: C, 66.97; H, 6.47; N, 7.13.
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Med Chem Res (2012) 21:3073–3079
3077
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(8-ethyl-2-methylquinoline-4-
yl)hydrazine (9f)
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(2-methylquinoline-4-
yl)hydrazine (9i)
1
1
Light yellow solid (0.35 g, 30.63%). m.p.:115–116°C. H
NMR (CDCl3) d: 0.97 (d, J = 6 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.2 Hz, 3H, 6-CH3), 1.27–1.36 (m, 3H), 1.36 (t, 3H,
CH2CH3), 1.45 (s, 3H, 3-CH3), 1.50–2.05 (m, 7H),
2.38–2.41 (m, 1H), 2.68 (s, 3H, quinoline–CH3),
2.82–2.86 (m, 1H, 9-H), 3.25 (q, 2H, CH2CH3), 5.50 (s,
1H, 12-H), 5.56 (d, J = 3.6 Hz, 1H, 10-H), 7.16 (d,
J = 8.4 Hz, 2H, Ar–H), 7.26–7.34 (m, 2H, quinoline–H),
7.47–7.49 (m, 1H, quinoline–H), 7.68–7.69 (m, 1H,
quinoline–H),7.68 (d, J = 8.4 Hz, 2H, Ar–H), 8.01 (s,
1H, CH=N). MS m/z: 572.3 (M ? H)?. Anal. calcd. For
C34H41N3O5: C, 71.43; H, 7.23; N, 7.35. Found: C, 71.39;
H, 7.17; N, 7.40.
Light yellow solid (0.25 g, 23.02%). m.p.:126–127°C. H
NMR (CDCl3) d: 0.98 (d, J = 6 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.5 Hz, 3H, 6-CH3), 1.26–1.35 (m, 3H), 1.46 (s, 3H,
3-CH3), 1.50–2.07 (m, 7H), 2.38–2.41 (m, 1H), 2.69 (s, 3H,
quinoline–CH3), 2.80–2.86 (m, 1H, 9-H), 5.50 (s, 1H,
12-H), 5.57 (d, J = 3 Hz, 1H, 10-H), 7.16 (d, J = 8.7 Hz,
2H, Ar–H), 7.30 (s, 1H, quinoline–H), 7.38–7.40 (m, 1H,
quinoline–H), 7.59–7.61 m(m, 1H, quinoline–H), 7.69 (d,
J = 8.7 Hz, 2H, Ar–H), 7.91–7.94 (m, 2H, quinoline–H),
8.13 (s, 1H, CH=N). MS m/z: 544.2 (M ? H)?. Anal.
calcd. For C32H37N3O5: C, 70.70; H, 6.86; N, 7.73. Found:
C, 70.66; H, 6.88; N, 7.77.
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(2-methyl-6-methoxyquinoline-
4-yl)hydrazine (9j)
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(8-fluoro-2-methylquinoline-4-
yl)hydrazine (9g)
1
Brown solid (0.30 g, 28.09%). m.p.:129–130°C. H NMR
1
Brown solid (0.22 g, 19.60%). m.p.:126–127°C. H NMR
(CDCl3) d: 0.98 (d, J = 6 Hz, 3H, 9-CH3), 1.06 (d,
J = 7.5 Hz, 3H, 6-CH3), 1.26–1.36 (m, 3H), 1.59–2.08 (m,
7H), 2.22 (s, 3H, 3-CH3), 2.40–2.43 (m, 1H), 2.52 (s, 3H,
quinoline–CH3), 2.86–2.89 (m, 1H, 9-H), 3.87 (s, 3H,
OCH3), 5.51 (s, 1H, 12-H), 5.60 (d, J = 3 Hz, 1H, 10-H),
6.79 (s, 1H, quinoline–H), 7.06 (dd, J = 9.3 Hz, J = 9 Hz,
1H, quinoline–H), 7.21 (d, J = 8.7 Hz, 2H, Ar–H), 7.51 (d,
J = 8 Hz, 1H, quinoline–H), 7.58 (1H, quinoline–H), 7.75
(d, J = 8.7 Hz, 2H, Ar–H), 8.66 (s, 1H, CH=N). MS m/z:
574.2 (M ? H)?. Anal. calcd. For C33H39N3O6: C, 69.09;
H, 6.85; N, 7.32. Found: C, 69.11; H, 6.81; N, 7.37.
(CDCl3) d: 0.97 (d, J = 5.7 Hz, 3H, 9-CH3), 1.03 (d,
J = 7.2 Hz, 3H, 6-CH3), 1.26–1.34 (m, 3H), 1.45 (s, 3H,
3-CH3), 1.60–2.06 (m, 7H), 2.38–2.41 (m, 1H), 2.70 (s, 3H,
quinoline–CH3), 2.70–2.76 (m, 1H, 9-H), 5.50 (s, 1H,
12-H), 5.54 (d, J = 3 Hz, 1H, 10-H), 7.13 (d, J = 8.7 Hz,
2H, Ar–H), 7.27–7.36 (m, 3H, quinoline–H), 7.63 (d,
J = 8.7 Hz, 2H, Ar–H), 7.62–7.69 (m, 1H, quinoline–H),
8.02 (s, 1H, CH=N). MS m/z: 562.5 (M ? H)?. Anal.
calcd. For C32H36FN3O5: C, 68.43; H, 6.46; N, 7.48.
Found: C, 68.41; H, 6.45; N, 7.51.
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(6-fluoro-2-methylquinoline-4-
yl)hydrazine (9h)
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(6-bromo-2-methylquinoline-4-
yl)hydrazine (9k)
1
1
Light yellow solid (0.33 g, 29.39%). m.p.:136–137°C. H
Light yellow solid (0.34 g, 27.37%). m.p.:131–132°C. H
NMR (CDCl3) d: 0.98 (d, J = 5.7 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.5 Hz, 3H, 6-CH3), 1.26–1.35 (m, 3H), 1.45 (s, 3H,
3-CH3), 1.51–2.07 (m, 7H), 2.39–2.41 (m, 1H), 2.68 (s, 3H,
quinoline–CH3), 2.80–2.86 (m, 1H, 9-H), 5.51 (s, 1H,
12-H), 5.55 (d, J = 3 Hz, 1H, 10-H), 7.15 (d, J = 8.7 Hz,
2H, Ar–H), 7.30–7.34 (m, 2H, quinoline–H), 7.55–7.57 (m,
1H, quinoline–H), 7.65 (d, J = 8.7 Hz, 2H, Ar–H),
7.90–7.93 (m, 1H, quinoline–H), 8.08 (s, 1H, CH=N).
MS m/z: 562.2 (M ? H)?. Anal. calcd. For C32H36FN3O5:
C, 68.43; H, 6.46; N, 7.48. Found: C, 68.39; H, 6.47; N,
7.52.
NMR (CDCl3) d: 0.98 (d, J = 6 Hz, 3H, 9-CH3), 1.06 (d,
J = 7.5 Hz, 3H, 6-CH3), 1.26–1.36 (m, 3H), 1.46 (s, 3H,
3-CH3), 1.51–2.07 (m, 7H), 2.40–2.43 (m, 1H), 2.67 (s, 3H,
quinoline–CH3), 2.82–2.85 (m, 1H, 9-H), 5.52 (s, 1H,
12-H), 5.56 (d, J = 3.3 Hz, 1H, 10-H), 7.15 (d,
J = 8.7 Hz, 2H, Ar–H), 7.34 (s, 1H, quinoline–H), 7.65 (d,
J = 8.7 Hz, 2H, Ar–H), 7.64–7.67 (m, 1H, quinoline–H),
7.79 (d, J = 9 Hz, 1H, quinoline–H), 8.00–8.02 (m, 2H,
CH=N, quinoline–H). MS m/z: 622.1 (M ? H)?. Anal.
calcd. For C32H36BrN3O5: C, 61.74; H, 5.83; N, 6.75.
Found: C, 61.71; H, 5.87; N, 6.73.
123

3078
Med Chem Res (2012) 21:3073–3079
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(7-chloro-2-methylquinoline-4-
yl)hydrazine (9l)
described procedures (Shenai et al., 2000). IC50 values
against recombinant falcipain-2 were determined as
described previously procedures (Li et al., 2009). Fluo-
rescence was monitored for 30 min at room temperature
with an automated microtiter plate spectrofluorimeter
(Molecular Devices, Flex station). IC50 values were
determined from plots of percent activity over compound
concentration using the GraphPad Prism software.
1
Light yellow solid (0.18 g, 15.60%). m.p.:141–142°C. H
NMR (CDCl3) d: 0.98 (d, J = 6 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.2 Hz, 3H, 6-CH3), 1.26–1.35 (m, 3H), 1.45 (s, 3H,
3-CH3), 1.54–2.07 (m, 7H), 2.40–2.43 (m, 1H), 2.66 (s, 3H,
quinoline–CH3), 2.82–2.85 (m, 1H, 9-H), 5.51 (s, 1H, 12-H),
5.56 (d, J = 3 Hz, 1H, 10-H), 7.15 (d, J = 8.7 Hz, 2H, Ar–
H), 7.31–7.35 (m, 2H, quinoline–H), 7.66 (d, J = 8.7 Hz,
2H, Ar–H), 7.78 (d, 1H, J = 8.7 Hz, quinoline–H), 7.91 (d,
J = 1.8 Hz, 1H, quinoline–H), 8.02 (s, 1H, CH=N). MS m/z:
578.1 (M ? H)?. Anal. calcd. For C32H36ClN3O5: C, 66.48;
H, 6.28; N, 7.27. Found: C, 66.43; H, 6.29; N, 7.23.
Molecular docking
The AutoDock 3.05 was used to perform docking calcu-
lations (Morris et al., 1998). The compound 9d was built
with the Sybyl software package (SYBYL 6.91 Manual.
Tripos Inc., St. Louis, MO, USA. 2004) and minimized
using the Tripos force field and assigned charges using the
Gasteiger–Hu¨ckel method. The crystal structure of falci-
pain-2 (PDB ocde:3BPF) was selected as receptor (Kerr
et al., 2009). Polar hydrogens were added and partial
charges were loaded for protein using the Kollman united
atom charges. Atomic solvation parameters and fragmental
volumes for the proteins were assigned using the addsol
utility in the program package. A grid map of dimensions
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(2,7,8-trimethylquinoline-4-
yl)hydrazine (9m)
1
Light yellow solid (0.20 g, 17.51%). m.p.:144–145°C. H
NMR (CDCl3) d: 0.99 (d, J = 5.7 Hz, 3H, 9-CH3), 1.06 (d,
J = 7.2 Hz, 3H, 6-CH3), 1.25 (s, 3H, 3-CH3), 1.25–1.36 (m,
3H), 1.51–2.08 (m, 7H), 2.20–2.24 (m, 1H), 2.36 (s, 3H,
quinoline–CH3), 2.50 (s, 3H, quinoline–CH3), 2.72 (s, 3H,
quinoline–CH3), 2.86–2.89 (m, 1H, 9-H), 5.52 (s, 1H, 12-H),
5.58 (d, J = 3 Hz, 1H, 10-H), 6.61 (s, 1H, quinoline–H),
7.22 (d, J = 8.4 Hz, 2H, Ar–H), 7.28 (d, J = 8.4 Hz, 1H,
quinoline–H), 7.79 (d, J = 8.4 Hz, 2H, Ar–H), 8.23 (d, 1H,
J = 8.4 Hz, quinoline–H), 8.90 (s, 1H, CH=N). MS m/z:
572.2 (M ? H)?. Anal. calcd. For C32H36ClN3O5: C, 71.43;
H, 7.23; N, 7.35. Found: C, 71.47; H, 7.17; N, 7.31.
˚
22.5 A 9 22.5 A 9 22.5 A, with
˚
˚
a grid spacing of
˚
0.375 A was generated for falcipain-2. Affinity grid fields
were generated using the auxiliary program AutoGrid3.0.
The Lamarckian genetic algorithm (LGA) was used to find
the appropriate binding positions, orientations, and confor-
mations of the ligand. Default parameters were used, except
for the number of generations which was set at 100. The
resulting data were taken from docking experiments in which
the lowest total docking energy was obtained. Ten LGA runs,
each with 300 individuals in the population, were performed.
N1-{4-[(10S)-dihydroartemisinin-10-
oxyl]}phenylmethylene-N2-(2,6-dimethylquinoline-4-
yl)hydrazine (9n)
˚
Results differing by less than 2 A in a positional root mean
square deviation (rmsd) were clustered together. In each
group, the lowest binding energy configuration with the
highest % frequency was selected as the group representative.
1
Yellow solid (0.44 g, 39.46%). m.p.:134–135°C. H NMR
Acknowledgments The authors are grateful to financial support by
the National Science and Technology major projects (Number:
2009ZX09301-012).
(CDCl3) d: 0.98 (d, J = 6 Hz, 3H, 9-CH3), 1.04 (d,
J = 7.2 Hz, 3H, 6-CH3), 1.21–1.35 (m, 3H), 1.45 (s, 3H,
3-CH3), 1.54–2.07 (m, 7H), 2.36–2.40 (m, 1H), 2.45 (s, 3H,
quinoline–CH3), 2.66 (s, 3H, quinoline–CH3), 2.80–2.85 (m,
1H, 9-H), 5.51 (s, 1H, 12-H), 5.56 (d, J = 3.3 Hz, 1H, 10-H),
7.14 (d, J = 8.7 Hz, 2H, Ar–H), 7.26 (s, 1H, quinoline–H),
7.39–7.42 (m, 1H, quinoline–H), 7.65 (d, J = 8.7 Hz, 2H,
Ar–H), 7.77–7.83 (m, 2H, quinoline–H), 8.20 (s, 1H, CH=N).
MS m/z: 558.2 (M ? H)?. Anal. calcd. For C33H39N3O5: C,
71.07; H, 7.05; N, 7.53. Found: C, 71.03; H, 7.06; N, 7.49.
Conflict of interest The authors declare that they have no conflict
of interest.
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