Aromatic amino analogues of artemisinin: Synthesis and in vivo antimalarial activity

Article abstract of DOI:10.1007/s00044-009-9209-5

Nine orally active novel artemisinin derivatives were prepared from artemisinin by four-step synthesis, and the compounds were evaluated in the rodent model using multidrug resistant Plasmodium yoelii nigeriensis. All of the compounds exhibited antimalarial activities with the ED₅₀ ranging from 5.41 mg/kg-12.4 mg/kg. Among them, artemisinin derivative bearing N-(4-hydroxy-3- ((4-phenylpiperazin-1-yl)methyl)phenyl) moiety (5f) was found to be the most active compound and was found to be three times more potent than artemisinin (ED₅₀ 16.4 mg/kg). Birkhaeuser Boston 2009.

Full text of DOI:10.1007/s00044-009-9209-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:524–532
DOI 10.1007/s00044-009-9209-5
ORIGINAL RESEARCH
Aromatic amino analogues of artemisinin: synthesis
and in vivo antimalarial activity
Dharmarajan Sriram Æ Ruth Vandana Devakaram Æ
Murugesan Dinakaran Æ Perumal Yogeeswari
Received: 4 November 2008 / Accepted: 15 April 2009 / Published online: 13 May 2009
¨
Ó Birkhauser Boston 2009
Abstract Nine orally active novel artemisinin derivatives were prepared from
artemisinin by four-step synthesis, and the compounds were evaluated in the rodent
model using multidrug resistant Plasmodium yoelii nigeriensis. All of the
compounds exhibited antimalarial activities with the ED50 ranging from 5.41
mg/kg–12.4 mg/kg. Among them, artemisinin derivative bearing N-(4-hydroxy-3-
((4-phenylpiperazin-1-yl)methyl)phenyl) moiety (5f) was found to be the most
active compound and was found to be three times more potent than artemisinin
(ED50 16.4 mg/kg).
Keywords Antimalarial activity Á Artemisinin Á Plasmodium yoelii nigeriensis
Introduction
The occurrence and spread of drug-resistant strains of Plasmodium falciparum
has led to resurgence of malaria, which claims 1–3 million lives annually and to
which 40% of the world’s population remains at risk (www.who.int). P. falci-
parum malaria has been primarily treated with chloroquine and pyrimethamine-
sulphadoxine. However, the emergence of strains resistant to these drugs along
with the reappearance of malaria in well-controlled areas has led to increased
efforts toward the development of new antimalarials. Artemisinin (1) is unique
sesquiterpene peroxide occurring as a constituent of Artemisia annua L. Because
D. Sriram (&) Á R. V. Devakaram Á M. Dinakaran Á P. Yogeeswari
Medicinal Chemistry & Anti-infectives Research Laboratory, Pharmacy Group, Birla Institute
of Technology & Science, Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad 500 078,
Andhra Pradesh, India
e-mail: drdsriram@yahoo.com

Med Chem Res (2010) 19:524–532
525
of the effectiveness of Artemisinin in the treatment of drug-resistant Plasmodium
falciparum and its rapid clearance of cerebral malaria, development of clinically
useful semisynthetic drugs for severe and complicated malaria was prompt (Lin
et al., 1990; Sriram et al., 2004). However, recent reports of fatal neurotoxicity
in animals with artemisinin derivatives, such as artemether, arteethr, and artesu-
nate, have spawned a renewed effort to develop nontoxic analogues of artemis-
inin. The neurotoxicity was the result of its metabolite dihydroartemisinin
(Brewer et al., 1994). In our effort to develop more potent, nonneurotoxic agents
for the oral treatment of drug-resistant malaria, we have synthesized aromatic
amino analogues of artemisinin by molecular integration approach. By using this
approach, we have introduced the 2-(diethylaminoethyl)phenol portion of amo-
diaquine into the artemisinin skeleton and evaluated for their in vivo antimalarial
activity (Fig. 1). This approach is envisaged to target the resistant strain of
malaria by dual action more effectively than the single moiety approach
(Scheme 1).
Fig. 1 Molecular integration approach

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Med Chem Res (2010) 19:524–532
O
O
O
O
O
O
O
O
O
c
a
b
O
O
O
3
2
1
OH
O
O
O
O
O
O
d
O
O
R
Br
Br
O
4
NH
OH
Br
5a-i
a. NaBH4, CH3OH; b. BF3.Et2O; c. Br2, CCl4; d.var. aromatic amines, Et2O , C2H5OH
Scheme 1 Synthetic protocol of artemisinin derivatives
Materials and methods
Synthesis
Melting points were taken on an electrothermal melting point apparatus (Buchi
BM530) in open capillary tubes and are uncorrected 1H-NMR spectra were scanned
on a JEOL Fx 400 MHz NMR spectrometer using DMSO-d6 as solvent. Chemical
shifts are expressed in d (ppm) relative to tertamethylsilane. Elemental analyses (C,
H, and N) were performed on Perkin Elmer model 240C analyzer and the data were
within 0.4% of the theoretical values.
Synthesis of acetamido-a-alkylamino-o-cresols
A mixture of equivalent amounts of acetaminophenol and formaldehyde were taken
along with the appropriate secondary amines [diethyl amine – 2.0 equiv, piperidine
– 2.0 equiv, piperazine – 4.0 equiv, n-methylpiperazine – 2.0 equiv, n-ethylpip-
erazine – 4.0 equiv, 1- benzylpiperazine – 2.2 equiv, 1-(2-pyridyl) piperazine – 4.0
equiv, 1-(3-trifluoromethyl) phenylpiperazine – 2.0 equiv] suspended in 10 ml of
isopropyl alcohol and heated in a steam bath for 3 hours. Upon cooling, a crystalline
mass formed in some cases, which was washed with acetone or alcohol and then
allowed to dry. In certain cases, when a crystalline product could not be readily
obtained by the above-mentioned procedure, the volatile materials were removed
under reduced pressure and the residue taken into ether and treated with excess

Med Chem Res (2010) 19:524–532
527
alcoholic hydrogen chloride. The monohydrochloride of the desired cresol was
obtained as an oil.
Synthesis of 4-amino-a-alkylamino-o-cresols
0.5 g of the cresols obtained in the above step were taken in 20% HCl and heated at
refluxing temperature for approximately 1 hour. The solution was evaporated to
dryness under reduced pressure. Ten milliliters of benzene was added to wash the
residue and the desired salt was precipitated by the addition of a small quantity of
ether.
Synthesis of dihydroartemisinin
Artemisinin (0.1 g, 0.002 M) was taken in 30 ml of carbinol and cooled in an ice
bath to 0–5°C. To the solution was added sodium borohydride (3.6 equ, 0.05 g) in
small portions during a period of 30 minutes. The solution was left to be stirred for
approximately 30 minutes at 0–5°C and then it was neutralized with 5% acetic acid/
methanol and evaporated to dryness under reduced pressure. The white residue
obtained was extracted with ethyl acetate. The ethyl acetate extract was evaporated
to dryness to give a yield of approximately 78%. The compound obtained had a
melting point of 153°C (reported 155°C) (Lin et al., 1992).
Synthesis of 9,10-dehydroartemisinin
Dihydroartemisinin obtained from the above step was dissolved in 30 ml of diethyl
ether and cooled to 0–5°C. Boron trifluoride (5.0 ml in Et2O) was added drop wise
to the solution with stirring. The solution was allowed to stand overnight at room
temperature, after which it was first washed with sodium bicarbonate, then with
saturated aqueous brine solution, and dried over anhydrous sodium sulphate. The
solvent was evaporated to dryness under reduced pressure to give the required
product with the yield of 65%.
Bromination of 9,10-dehydroartemisinin
The semisolid obtained in the above step was dissolved in 5 ml of anhydrous carbon
tetrachloride, to which was added drop wise with stirring a 5% solution of bromine
in anhydrous carbon tetrachloride at 0°C until the mixture turned pale orange in
color. After the mixture was stirred for approximately 30 minutes, the solvent was
removed under reduced pressure at \ 40°C and was used in the succeeding step
without further purification.
General procedure for the condensation with amines
The amine (0.005 M) was dissolved in a mixture of 20 ml of diethyl ether:ethanol
(50:50). The amine solution was added drop wise to the above synthesized di-bromo
compound at 0°C in an ice bath. After the addition was completed, the reaction

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Med Chem Res (2010) 19:524–532
mixture was allowed to stand overnight at room temperature. It was then evaporated
to dryness under reduced pressure to get the desired final crude product.
1
5a. Yield: 71%; M.P: 162°C; H NMR (DMSO-d6) d ppm: 0.87–0.89 (d, 3H),
1.15–1.17 (t, 2 x 3H), 1.25 (s, 3H), 1.27–1.66 (m, 2 x 2H), 1.81–1.82 (d, 3H),
1.83–1.99 (m, 3 x 1H, 2 x 2H), 2.69–2.71 (q, 2 x 2H), 3.72 (s, 2H), 5.23–5.25 (m,
1H), 5.41 (s, 1H), 5.57 (s, 2 x 1H), 6.28–6.29 (m, 1H), 6.55–6.58 (m, 1H), 6.84–
6.86 (m, 1H); Anal (C26H39BrN2O5): calculated C, 57.88; H, 7.29; N, 5.19; found C,
57.79; H, 7.32; N, 5.14.
5b. Yield: 55%; M.P: 48°C; 1H NMR (DMSO-d6) d ppm: 0.87–0.89 (d, 3H), 1.25
(s, 3H), 1.26–1.68 (m, 5 x 2H), 1.81–1.82 (d, 3H), 1.83–1.99 (m, 3 x 1H, 2 x 2H),
2.33–2.41 (m, 2 x 2H), 3.79 (s, 2H), 5.23–5.25 (m, 1H), 5.41 (s, 1H), 5.58 (s,
2 x 1H), 6.24–6.26 (m, 1H), 6.55–6.58 (m, 1H), 6.85–6.88 (m, 1H); Anal
(C27H39BrN2O5): calculated C, 58.80; H, 7.13; N, 5.08; found C, 58.88; H, 7.07;
N, 5.13.
1
5c. Yield: 75%; M.P: 148°C; H NMR (DMSO-d6) d ppm: 0.88–0.89 (d, 3H),
1.25 (s, 3H), 1.26–1.66 (m, 2 x 2H), 1.81–1.82 (d, 3H), 1.83–1.84 (m, 1H), 1.85–
1.99 (m, 2 x 1H, 2 x 2H), 2.25–2.85 (m, 4 x 2H), 3.83 (s, 2H), 5.23–5.25 (m, 1H),
5.41 (s, 1H), 5.44 (s, 3 x 1H), 6.15–6.16 (m, 1H), 6.55–6.58 (m, 1H), 6.80–6.81 (m,
1H); Anal (C26H38BrN3O5): calculated C, 56.52; H, 6.93; N, 7.61; found C, 56.47;
H, 6.92; N, 7.68.
1
5d. Yield: 75%; M.P: 148°C; H NMR (DMSO-d6) d ppm: 0.88–0.89 (d, 3H),
1.25 (s, 3H), 1.26–1.66 (m, 2 x 2H), 1.81–1.82 (d, 3H), 1.83–1.84 (m, 1H), 1.85–
1.99 (m, 2 x 1H, 2 x 2H), 2.3 (s, 3H), 2.45–2.57 (m, 4 x 2H), 3.83 (s, 2H), 5.23–
5.25 (m, 1H), 5.41 (s, 1H), 5.58 (s, 2 x 1H), 6.15–6.16 (m, 1H), 6.55–6.58 (m, 1H),
6.80–6.81 (m, 1H); Anal (C26H40BrN3O5): calculated C, 57.24; H, 7.12; N, 7.42;
found C, 57.20; H, 7.18; N, 7.44.
1
5e. Yield: 61%; M.P: 149°C; H NMR (DMSO-d6) d ppm: 0.88–0.89 (d, 3H),
1.0–1.02 (t, 3H), 1.25 (s, 3H), 1.26–1.66 (m, 2 x 2H), 1.81–1.82 (d, 3H), 1.83–1.84
(m, 1H), 1.85–1.99 (m, 2 x 1H, 2 x 2H), 2.26–2.34 (m, 2 x 2H), 2.47 (q, 2H), 2.77–
2.91 (m, 2 x 2H), 3.83 (s, 2H), 5.23–5.25 (m, 1H), 5.41 (s, 1H), 5.57 (s, 2 x 1H),
6.15–6.16 (m, 1H), 6.55–6.58 (m, 1H), 6.80–6.81 (m, 1H); Anal (C28H42BrN3O5):
calculated C, 57.93; H, 7.29; N, 7.24; found: C, 57.90; H, 7.24; N, 7.28.
1
5f. Yield: 64%; M.P: 112°C; H NMR (DMSO-d6) d ppm: 0.88–0.89 (d, 3H),
1.25 (s, 3H), 1.26–1.66 (m, 2 x 2H), 1.81–1.82 (d, 3H), 1.83–1.84 (m, 1H), 1.85–
1.99 (m, 2 x 1H, 2 x 2H), 2.62–2.70 (m, 2 x 2H), 3.17–3.26 (m, 2 x 2H), 3.83 (s,
2H), 5.23–5.25 (m, 1H), 5.41 (s, 1H), 5.57 (s, 2 x 1H), 6.15–6.16 (m, 1H), 6.55–
6.58 (m, 1H), 6.76–6.78 (m, 1H), 6.80–6.82 (m, 3H), 7.20–7.22 (m, 2H); Anal
(C32H42BrN3O5): calculated C, 61.14; H, 6.73; N, 6.68; found C, 61.12; H, 6.70; N,
6.69.
1
5g. Yield: 60%; M.P: 132 °C; H NMR (DMSO-d6) d ppm: 0.88–0.89 (d, 3H),
1.25 (s, 3H), 1.26–1.66 (m, 2 x 2H), 1.81–1.82 (d, 3H), 1.83–1.84 (m, 1H), 1.85–
1.99 (m, 2 x 1H, 2 x 2H), 2.62–2.70 (m, 2 x 2H), 3.17–3.26 (m, 2 x 2H), 3.83 (s,
2H), 5.23–5.25 (m, 1H), 5.41 (s, 1H), 5.57 (s, 2 x 1H), 6.15–6.16 (m, 1H), 6.55–
6.58 (m, 1H), 6.68–6.70 (m, 1H), 6.80–6.82 (m, 1H), 6.85–6,86 (m, 1H), 7.08–7.10
(m, 1H), 7.35–7.36 (m, 1H); Anal (C33H41BrF3N3O5): calculated C, 56.90; H, 5.93;
N, 6.03; found C, 56.93; H, 5.97; N, 5.97.

Med Chem Res (2010) 19:524–532
529
1
5h. Yield: 49%; M.P: 143°C; H NMR (DMSO-d6) d ppm: 0.88–0.89 (d, 3H),
1.25 (s, 3H), 1.26–1.66 (m, 2 x 2H), 1.81–1.82 (d, 3H), 1.83–1.84 (m, 1H), 1.85–
1.99 (m, 2 x 1H, 2 x 2H), 2.62–2.70 (m, 2 x 2H), 3.32–3.40 (m, 2 x 2H), 3.83 (s,
2H), 5.23–5.25 (m, 1H), 5.42 (s, 1H), 5.58 (s, 2 x 1H), 6.15–6.16 (m, 1H), 6.46–
6.48 (m, 1H), 6.51–6.53 (m, 1H), 6.55–6.58 (m, 1H), 6.80–6.82 (m, 1H), 7.55–7.56
(m, 1H), 8.08–8.10 (m, 1H); Anal (C31H41BrN4O5): calculated C, 59.14; H, 6.56; N,
8.90; found C, 59.15; H, 6.54; N, 8.93.
5i. Yield: 63%; M.P: 46°C; 1H NMR (DMSO-d6) d ppm: 0.88–0.89 (d, 3H), 1.25
(s, 3H), 1.26–1.66 (m, 2 x 2H), 1.81–1.82 (d, 3H), 1.83–1.84 (m, 1H), 1.86–2.0 (m,
2 x 1H, 2 x 2H), 2.41–2.43 (m, 2 x 2H), 2.47–2.49 (m, 2 x 2H), 3.52 (s, 2H), 3.83
(s, 2H), 5.23–5.25 (m, 1H), 5.42 (s, 1H), 5.58 (s, 2 x 1H), 6.15–6.16 (m, 1H), 6.55–
6.58 (m, 1H), 6.80–6.82 (m, 1H), 7.08–7.11 (m, 2H), 7.19–7.20 (m, 2H), 7.26–7.28
(m, 1H); Anal (C33H44BrN3O5): calculated C, 61.68; H, 6.90; N, 6.54; found C,
61.70; H, 6.87; N, 6.58.
In vivo antimalarial activity
In vivo antimalarial activity against murine strain, P. yoelii nigeriensis, was
determined by the 4-day suppressive test. Each group of animals, which consisted of
five female Swiss albino mice, was infected i.p. on day 0 with 2 x 107 parasitized
red blood cells. Thereafter, the synthesized artemisinin derivatives and standard
artemisin were dissolved in dimethyl sulfoxide (DMSO). For 4 consecutive days,
100 ll of the drug solutions were administrated to the mice once daily by p.o. All
experiments included control groups: a sentinel group (the same batch of mice as
treated mice but that were not infected and not treated), a drug-free group (mice that
were infected with Plasmodium but that received no treatment), and an excipient
group (mice that were infected with Plasmodium and that received no drug
treatment but that did receive treatment with the excipient used for the drugs tested).
Parasitemia levels were determined on the day after the last treatment (day 4). The
50% effective dose (ED50) and ED90 values were the doses that led to 50% and 90%
parasite growth inhibition, respectively, compared with the level in the nontreated
control animals. For each dose, at least five animals were treated, and for each route
of administration, ten control animals (vehicle solution) were tested.
Results and discussion
Chemistry
Artemisinin (1) on reduction with sodium borohydride in methanol at 0–5°C
followed by acidification with 5% acetic acid in methanol yielded 78% of
dihydroartemisinin (2). Compound 2 on treatment with boron trifluoride etherate at
room temperature undergoes dehydration to yield 65% of 9,10-dehydroartemisinin
(3). Treatment of 3 with 5% bromine in carbon tetrachloride at 0°C yielded dibromo
derivative 4, which was used in the succeeding step without further purification.
Compound 4 on condensation with two equivalents of corresponding aromatic

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Med Chem Res (2010) 19:524–532
amines in ether:ethanol mixture yielded the target compounds 5a-i. Various
aromatic amines were synthesized from 4-acetamido phenol, first on reaction of
4-acetamido phenol with equimolar concentration of secondary amines and
formaldehyde in isopropyl alcohol medium yielded corresponding Mannich bases,
which on hydrolysis with 20% hydrochloric acid yielded required aromatic amines.
The purity of the compounds was checked by TLC and elemental analyses; and the
1
compounds of this study were identified by spectral data. In the H NMR spectra
the signals of the respective protons of the prepared derivatives were verified on the
basis of their chemical shifts, multiplicities, and coupling constants. The spectra of
all the compounds showed a D2O exchangeable broad singlet at d 5.77 and
5.58 ppm corresponding to NH and OH protons, singlet at 3.72–3.83 ppm
corresponding to Mannich methylene protons, methyl protons at 0.88–0.9, 1.2–
1.25, and 1.81–1.83 ppm. The elemental analysis results were within 0.4% of the
theoretical values.
In vivo antimalarial activity
In vivo antimalarial activity against murine strain, P. yoelii nigeriensis, was
determined by the 4-day suppressive test (Peters and Robinson, 1998). Contrary to
other murine Plasmodium strains, P. yoelii nigeriensis studied on the drug resistance
phenomena (Peters and Robinson, 2000) and this species of the parasite is naturally
resistant to chloroquine. The 50% effective dose (ED50) and ED90 values were the
doses that led to 50% and 90% parasite growth inhibition, respectively, compared
with the level in the nontreated control animals and reported in Table 1. Rapid
glance of the results revealed that all the synthesized compounds showed good
activity with the ED50 ranging from 5.41–12.40 mg/kg and ED50 ranging from
8.72–24.60 mg/kg body weight. Among the nine compounds artemisinin derivative,
which bearing N-(4-hydroxy-3-((4-phenylpiperazin-1-yl)methyl)phenyl) moiety
(5f) was found to be the most active compound and was found to be three times
more potent than artemisinin (ED50 16.4 mg/kg). These enhanced activities were
supported with log P values; all these aromatic amino derivatives have log P value
ranging from 4.4–7.81 and are more lipophilic than artemisinin, which have log P
values 3.38, indicating that increased lipophilicity is accompanied by increase in
oral bioavailability. Highly hydrophobic/lipophilic compounds are known to show
good oral bioavailability, and Frenkel et al. (2005) have proposed a model to
explain this phenomenon. According to this model, hydrophobic compounds form
aggregates of appropriate size in the aqueous environment of the gastrointestinal
tract where they are taken up by microvilli cells and then drained into lymphatic
circulation and emptied into systemic compartments. The advantages of these
aromatic amino analogues are 1) show better activity than artemisinin, 2)
compounds would not give the neurotoxic metabolite dihydroartemisinin because
the keto function has been replaced by an amino, and 3) can be converted into easily
water-soluble substances by adding mineral acids, thereby overcoming the
drawback of artemisinin insolubility in water.

Med Chem Res (2010) 19:524–532
531
Table 1 Physical constants and in vivo antimalarial activity
O
O
O
O
R
Br
NH
OH
No.
R
Yield (%)
Melting
point (°C)
Log Pa
ED50
(mg/kg)
ED90
(mg/kg)
5a
5b
–N(C2H5)2
71
55
162
48
5.53
5.58
12.34
11.26
24.19
23.12
N
5c
5d
5e
75
65
61
148
51
4.23
4.61
4.94
12.40
9.90
9.83
24.60
17.35
16.65
N
N
NH
N
CH3
149
N
N
C2H5
5f
64
60
112
132
6.68
7.6
5.41
6.01
8.72
9.28
N
N
CF3
5g
N
N
5h
49
143
6.06
7.90
12.15
N
N
N

532
Med Chem Res (2010) 19:524–532
Table 1 continued
No.
R
Yield (%) Melting
point (°C)
Log Pa ED50
(mg/kg) (mg/kg)
ED90
5i
63
46
6.34
7.12
12.07
N
N
CH2
Artemisinin
–
–
–
3.38
16.4
27.12
a
Calculated using Chemdraw ultra 11.0
Conclusions
We reported orally active, more potent, and water-soluble aromatic amino
analogues of artemisinin. The high order of antimalarial activity combined with
ease of preparation of these compounds qualifies these compounds as candidates for
further drug development studies.
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