Synthesis and in vitro biological evaluation of aminoacridines and artemisinin-acridine hybrids

Article abstract of DOI:10.1016/j.ejps.2014.01.014

During this study, 9-aminoacridine and artemisinin-acridine hybrid compounds were synthesized and the in vitro for antimalarial activity against both the chloroquine sensitive but also gametocytocidal strain (NF54), and chloroquine resistant (Dd2) strains of Plasmodium falciparum was determined. In vitro cytotoxicity against CHO cells, apoptosis of HepG2 and SH-SY5Y as well as anticancer activity against HeLa cell lines were assessed. The hybrids were synthesized, using a microwave-assisted radiation method by covalently linking artemisinin and acridine pharmacophores by means of a liable, aminoethyl ether linker. The synthesized compounds were found active against both the Plasmodium strains and displayed superior selective toxicity towards the parasitic cells. Hybrid 7, however, containing ethylenediamine linker, proved the most active of all of the synthesized compounds. It had seven-fold higher antigametocytocidal activity compared to chloroquine and was also found to be seven-fold more potent than chloroquine against the Dd2 strain, with highly selective action towards the parasitic cells. This hybrid also showed favourable anti-cancer activity against the HeLa cells, three- and eight-fold higher than those of chloroquine and melphalan, respectively. This hybrid may therefore stand as drug candidate for further investigation in the search for new and effective drugs against malaria and cervical cancer.

Full text of DOI:10.1016/j.ejps.2014.01.014

European Journal of Pharmaceutical Sciences 56 (2014) 16–27
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Synthesis and in vitro biological evaluation of aminoacridines
and artemisinin–acridine hybrids
a
a
b
c
b,
⇑
Juan P. Joubert , Frans J. Smit , Lissinda du Plessis , Peter J. Smith , David D. N’Da
a
Department of Pharmaceutical Chemistry, North-West University, Potchefstroom 2520, South Africa
Centre of Excellence for Pharmaceutical Sciences (PHARMCEN), North-West University, Potchefstroom 2520, South Africa
Department of Pharmacology, University of Cape Town, Groote Schuur Hospital, Observatory 7925, South Africa
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
During this study, 9-aminoacridine and artemisinin–acridine hybrid compounds were synthesized and
Received 4 December 2013
Received in revised form 13 January 2014
Accepted 29 January 2014
Available online 18 February 2014
the in vitro for antimalarial activity against both the chloroquine sensitive but also gametocytocidal strain
(NF54), and chloroquine resistant (Dd2) strains of Plasmodium falciparum was determined. In vitro cyto-
toxicity against CHO cells, apoptosis of HepG2 and SH-SY5Y as well as anticancer activity against HeLa
cell lines were assessed. The hybrids were synthesized, using a microwave-assisted radiation method
by covalently linking artemisinin and acridine pharmacophores by means of a liable, aminoethyl ether
linker. The synthesized compounds were found active against both the Plasmodium strains and displayed
superior selective toxicity towards the parasitic cells. Hybrid 7, however, containing ethylenediamine lin-
ker, proved the most active of all of the synthesized compounds. It had seven-fold higher antigametocy-
tocidal activity compared to chloroquine and was also found to be seven-fold more potent than
chloroquine against the Dd2 strain, with highly selective action towards the parasitic cells. This hybrid
also showed favourable anti-cancer activity against the HeLa cells, three- and eight-fold higher than those
of chloroquine and melphalan, respectively. This hybrid may therefore stand as drug candidate for further
investigation in the search for new and effective drugs against malaria and cervical cancer.
Ó 2014 Elsevier B.V. All rights reserved.
Keywords:
Malaria
Artemisinin
Acridine
Hybrids
Cytotoxicity
Apoptosis
1
. Introduction
Malaria is a life threatening disease to 3.3 billion people glob-
parasite’s intrinsic ability to attain drug resistance through various
mechanisms (Gregson and Plowe, 2005), has rendered traditionally
used antimalarial drugs, such as chloroquine, (CQ) therapeutically
ineffective, whilst having elevated the artemisinins as the only via-
ble first line treatment option for uncomplicated P. falciparum.
Artemisinin therapeutic proficiency is limited by its low solubil-
ity in both oil and water and by its short plasma half-life, which
had paved the way for the synthesis of semi-synthetic drugs with
superior pharmacological properties (Ploypradith, 2004). Despite
these improved properties of the semi-synthetic derivatives, the
artemisinins, as a class, still possess clear disadvantages, viz. short
half-lives, irregular absorption, liability towards acid hydrolysis,
increased dosage frequency and high recrudescence, all of which
prohibit their use as monotherapy (Meshnick, 2002; Price et al.,
1996). These shortcomings restrict therapeutic efficacy, patient
compliance and increase the possible development of drug
resistance.
ally, of which predominantly children under the age of five, preg-
nant women, geriatrics and immunocompromized individuals
(WHO, 2012) are at risk of contracting it. In 2011, the estimated
malaria related deaths were 660,000 out of 219 million reported
cases. 81% of these cases and 91% of malaria related mortality oc-
curred in the African region (WHO, 2011). As infectious disease,
malaria accounts for the second highest death rate in Africa, fol-
lowing HIV/Aids, whilst malaria comprizes the fifth deadliest dis-
ease, worldwide (Global Malaria action Plan, 2008).
The armamentariums, currently used to treat malaria, are from
seven drug classes, namely 4-aminoquinolines (chloroquine),
arylaminoalcohols, 8-aminoquinolines, artemisinins (Fig. 1),
antifolates, hydroxynaphthoquinones, inhibitors of the respiratory
chain and antibiotics (Rudrapal, 2011; Schlitzer, 2008). The ideal
malaria pharmacotherapy should preferably be a cost effective
and easily obtainable monotherapy. However, the malaria
Efforts to enhance their efficiency and to avoid the development
of parasitic resistance to the artemisinin class, had led to the
incorporation of a partner drug that meets with preordained spec-
ifications to the artemisinins, resulting in artemisinin based combi-
nation therapies (ACTs). ACTs combine fast acting, highly potent
⇑
Corresponding author. Tel.: +27 18 299 2256; fax: +27 18 299 4243.
E-mail address: david.nda@nwu.ac.za (D.D. N’Da).
http://dx.doi.org/10.1016/j.ejps.2014.01.014
928-0987/Ó 2014 Elsevier B.V. All rights reserved.
0

J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
17
malaria during World War II, but was discontinued shortly there-
after, due to reported toxicity (Elueze et al., 1996). Acridines
reportedly own their biological activity to various mechanisms of
action, including DNA intercalators, inhibitors of mammalian
topoisomerases I and II and acetylcholinesterase (Ciesielska et al.,
O
O
O
O
O
H
O
H
H
H
O
O
O
OR
1
997; Valdés, 2011). Recently, various synthetic and semi-syn-
Artemisinin
thetic acridinone analogues have been synthesized. Screen tests
of 3-(5,6,6,6-tetrafluoro-5-trifluoromethyl-hexyloxy)-6-chloroacr-
idinone exhibited potent antimalarial activity, with a 50% inhibi-
tory concentration (IC50) value of 1 nM against the D6 sensitive
strain of P. falciparum, as well as low cytotoxicity towards murine
splenic lymphocytes (Winter et al., 2006). The quinacrine-floxa-
crine hybrid, WR 243251, exhibited nanomolar IC50 values of 11
nM against CQ sensitive D6, and 25 nM against CQ resistant W2
P. falciparum strains (Berman et al., 1994), with limited side effects
(Biagini et al., 2008).
In search for new antimalarial drugs and in light of the above
considerations, hybrids, synthesized through the chemical combi-
nation of acridine and artemisinin pharmacophores with various
linkers were investigated. In this paper, the synthesis, in vitro anti-
malarial activity cytotoxicity and apoptotic/anticancer activity are
reported.
Dihydroartimisinin R = H
Artemether R = OCH3
Artesunate R = CO(CH ) COOH
2
2
Fig. 1. Structures of artemisinins. The artemisinins class comprises artemisinin and
its oil-soluble semi-synthetic derivatives dihydroartemisinin, artemether and
arteether, and water-soluble, artesunate.
artemisinin derivatives with a longer acting partner drug from an-
other class. In that combination, the short acting, highly potent
artemisinin derivative reduces the parasitemia, after which the
slower, eliminated partner compound prevents parasite recrudes-
cence (Price, 2013). The World Health Organization (WHO) recom-
mends ACTs as first line treatment for uncomplicated P. falciparum
malaria (Amuasi et al., 2012; Fink et al., 2013). However, artemis-
inins’ tolerance has already been confirmed through prolonged,
parasite clearance times, despite adequate drug exposure in the
Greater Mekong countries, like Cambodia, Myanmar, Thailand
and Vietnam (Fairhurst et al., 2012). This worrying predicament
can have dire consequences, since the artemisinin class serves as
the foundation for current treatment regimes, thus emphasizing
the urgent search for new and effective therapies to be identified
and developed.
2
. Materials and methods
2.1. Materials
Ethylenediamine, N-methyl-1,3-propylenediamine, piperazine,
2
-methylpiperazine, 1-(2-aminoethyl)piperazine, 6,9-dichloro-2-
A new approach to impede drug resistance development is the
synthesis of hybrid molecules (Burgess et al., 2006). Hybrids are
two or more chemical entities with differing structural domains
and biological activity, covalently linked together (Hulsman et al.,
methoxyacridine, magnesium sulphate (MgSO
ate (NaHCO ), potassium carbonate (K CO ), acetonitrile (ACN),
ammonium hydroxide solution (NH OH), N,N-dimethylformamide
DMF), tetrahydrofuran (THF), deuterated dimethyl sulfoxide
DMSO-d ), deuterated chloroform (CDCl -d), boron trifluoride
–Et O) and 2-bromoethanol were all pur-
4
), sodium bicarbon-
3
2
3
4
(
(
2
007; Meunier, 2008). These distinct chemical entities should have
6
3
independent pharmacophores (Meunier, 2008) and in addition, for
the dual therapeutic action, should have distinct abilities to impart
favourable physicochemical properties of one entity to another
diethyl etherate (BF
3
2
chased from Sigma–Aldrich (South-Africa). Potassium iodide (KI)
was obtained from Merck (South-Africa). All solvents used were
acquired from Associated Chemical Enterprises (ACE, South Africa).
(
Meunier, 2008; Walsh et al., 2007). The linker can be chosen care-
fully to allow the intact hybrid to dissociate into its individual
components following administration (cleavable conjugates),
whilst synergism in activity is aimed at, or to remain fixed when
metabolically resistant linker units are chosen. The latter is the
preferred option for overcoming resistance (Walsh and Bell, 2009).
Various artemisinin based hybrids have demonstrated promis-
ing biological activity and enhanced physicochemical properties,
compared to the individual parent compounds (Capela et al.,
Dihydroartemisinin (mixture of 10-a and 10-b epimers) was pur-
chased from Changzhou Kaixuan Chemical Co (Chunjiang, China).
All chemicals, i.e. methanol (MeOH), dichloromethane (DCM),
ethyl acetate (EtOAc) and reagents were of analytical grade and
were used without further purification.
2.2. General procedures
2
011; Lai et al., 2013; Muregi and Ishih, 2010; Njogu et al., 2013;
The 1H and 13C NMR spectra were recorded on a Bruker Ad-
vance™ III 600 spectrometer at a frequency of 600 MHz and
150.913 MHz, respectively, in DMSO-d or CDCl -d. Chemical shifts
are reported in parts per million d (ppm), with the residual protons
of the solvent as reference. The splitting pattern abbreviations are
as follows: singlet (s), doublet (d), doublet of doublet (dd), doublet
of doublet of doublets (ddd), doublet of triplets (dt), doublet of
quartets (dq), triplet (t), triplet of doublets (td), triplet of triplets
Xie et al., 2011), but none has yet been approved from a regulatory
perspective.
Acridine based compounds are the foundation of antimalarial
drug research (Pérez et al., 2013). Indeed, mepacrine (Fig. 2), the
first blood schizontocide, had been used as first line treatment of
6
3
N
(
tt), quartet of doublets (qd), multiplet (m).
High resolution mass spectrometry (HRMS) was recorded on a
Bruker MicroTOF Q II mass spectrometer, equipped with an APCI
or an ESI source set at 200 °C or 180 °C, respectively, with Bruker
Compass DataAnalysis 4.0 software. A full scan from 50 to
HN
O
1
500 m/z was generated, at a capillary voltage of 4500 V, an end
Cl
N
plate offset voltage of ꢀ500 V, the nebulizer set at 1.6 and
0
.4 bar, respectively and a collision cell RF voltage of 100 Vpp.
Infrared (IR) spectra were recorded on a Bruker Alpha-P FTIR
Mepacrine
instrument. Melting points (mp) were determined on a BÜCHI
melting point B-545 instrument in triplicate and were uncorrected.
Fig. 2. Structure of mepacrine. Mepacrine was the first synthetic antimalarial drug
but abandoned for toxicity reason.

1
8
J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
Thin layer chromatography was performed using silica gel plates
60F254) obtained from Merck. Column chromatography was per-
through column chromatography by successively eluting with var-
ious ratios of MeOH, DCM, EtOAc and NH OH.
(
4
formed using MN silica gel 60, 70–230 mesh ASTM, supplied by
Macerey–Nagel (Germany).
2.3.2.1. N-(2-aminoethyl)-6-chloro-2-methoxyacridin-9-amine, 2. The
All microwave assisted reactions were carried out in a CEM Dis-
covery™ microwave synthesizer focused closed vessel. The ma-
chine consisted of a continuously focused microwave power
delivery system, selectable power output between 0 and 300 W,
a maximum current of 6.3 amps and a frequency of 50/60 Hz.
The temperature of the vessel’s contents was continuously moni-
tored via an IR sensor, located underneath the reaction vessel.
The reaction mixture was stirred with a Teflon coated magnetic stir
bar via a rotating magnetic plate, located below the microwave
cavity floor.
reaction of 6,9-dichloro-2-methoxyacridine and ethylenediamine,
followed by successive purification through chromatography, eluting
4 4
withDCM:MeOH:NH OH(8:1.5:0.5, v/v/v)thenMeOH:NH OH(19:1,
v/v) afforded 2 (1.6 g, 75%) a yellow–brown powder; mp: 164 °C; IR
ꢀ
1
1
(
ATR)
m
max/cm : 3343, 1631, 1557, 750, 580; H NMR (600 MHz,
0
CDCl
3
) d (ppm): 8.06 (d, J = 9.1 Hz, 1H, H-8 ), 8.01–7.95 (m, 1H, H-
), 7.93 (d, J = 9.3 Hz, 1H, H-4 ), 7.37 (dd, J = 9.4, 2.7 Hz, 1H, H-3 ),
.30 (d, J = 2.7 Hz, 1H, H-1 ), 7.24–7.22 (m, 1H, H-7 ), 5.90 (s, 1H, H-
1 ), 3.91 (s, 3H, H-10 ), 3.63 (t, J = 5.5 Hz, 2H, H-12 ), 2.94 (dd,
J = 6.3, 4.8 Hz, 2H, H-13 ); C NMR (151 MHz, CDCl
55.84 (C-2 ), 150.20 (C-9 ), 148.38, 146.74, 134.62 (C-6 ), 131.31
C-4 ), 128.08 (C-5 ), 124.46 (C-8 ), 124.24 (C-7 ), 118.28, 116.16,
9.30 (C-1 ), 55.43 (C-10 ), 51.50 (C-12 ), 41.92 (C-13 ); APCI-HRMS
0
0
0
5
7
1
0
0
0
0
0
0
13
3
) d (ppm):
0
0
0
1
(
9
0
0
0
0
2
2
.3. Synthesis
0
0
0
0
+
3
m/z [M + H] : 302.1051 (calcd. for C16H17ClN O: 302.1054).
.3.1. 2-Bromo-(10b-dihydroartemisinoxy)ethane, 1
2
-Bromo-(10b-dihydroartemisinoxy)ethane 1 (Fig. 3) analogue
of DHA was synthesized in moderate yields (71%) from DHA and
-bromoethanol by using a reported method of Li et al. (2000), rou-
tinely used by our research group.
2
.3.2.2. {3-[(6-Chloro-2-methoxyacridin-9-yl)amino]propyl}(methyl)
2
amine, 3. The reaction of 6,9-dichloro-2-methoxyacridine and
N-methyl-1,3-diaminopropane, followed by successive purification
1
H NMR (600 MHz, CDCl
J = 3.4 Hz, 1H, H-10), 4.13 (dt, J = 10.8, 5.4 Hz, 1H, H-a
J = 10.9, 5.3 Hz, 1H, H-ab), 3.53 (t, 2H, H-b), 2.66 (dt, J = 7.7,
.7 Hz, 1H, H-9), 2.33 (tt, J = 20.2, 10.0 Hz, 1H, H-4 ), 2.04–1.97
m, 1H, H-4b), 1.91–1.80 (m, 2H, H-8 , H-5 ), 1.73 (ddd, J = 14.2,
.5, 3.6 Hz, 1H, H-8b), 1.65–1.55 (m, 1H, H-7b), 1.52–1.37 (m, 5H,
3
) d (ppm): 5.46 (s, 1H, H-12), 4.82 (d,
through chromatography, eluting with DCM:MeOH:NH
8:1.5:0.5, v/v/v) then MeOH:NH OH (19:1, v/v) afforded 3 (1.9 g,
9%) an off-white to yellow powder; mp: 165 °C; IR (ATR)
4
OH
a
), 3.76 (dt,
(
7
4
m
max
/
)
ꢀ1
1
5
a
cm : 3374, 1633, 1563, 675, 560; H NMR (600 MHz, DMSO-d
d (ppm): 8.31 (d, J = 9.3 Hz, 1H, H-8 ), 7.82 (d, J = 2.2 Hz, 1H, H-
5
6
(
a
a
0
7
0
0
0
), 7.78 (d, J = 9.3 Hz, 1H, H-4 ), 7.55 (d, J = 2.7 Hz, 1H, H-1 ), 7.38
H-8a, H-5b, H-14), 1.36–1.28 (m, 1H, H-6), 1.25–1.17 (m, 1H, H-
a), 0.89 (m, 7H, H-7 ) d
, H-15, H-16); 13C NMR (151 MHz, CDCl
ppm): 104.10 (C-3), 102.02 (C-10), 88.12 (C-12), 81.02 (C-12a),
0
0
(
dd, J = 9.2, 2.6 Hz, 1H, H-3 ), 7.25 (dd, J = 9.3, 2.3 Hz, 1H, H-7 ),
5
a
3
0
0
3
.91 (s, 3H, H-10 ), 3.86 (q, J = 5.9, 5.2 Hz, 2H, H-12 ), 3.56–3.09
(
0
(
m, 4H, H
2
O), 2.61 (t, J = 6.1 Hz, 2H, H-14 ), 2.49 (p, J = 1.8 Hz, 4H,
0
0
0
13
6
3
2
3
8.15 (C-a), 52.55 (C-5a), 44.33 (C-8a), 37.37 (C-6), 36.37 (C-4),
H-16 ), 2.26 (s, 2H, H-15 ), 1.81 (p, J = 6.4 Hz, 2H, H-13 ); C NMR
4.63 (C-7), 31.41 (C-b), 30.83 (C-9), 26.12 (C-14), 24.62 (C-5),
0
0
(
6
151 MHz, DMSO-d ) d (ppm): 154.90 (C-2 ), 150.25 (C-9 ),
+
4.34(C-8), 20.31(C-15), 12.91 (C-16); APCI-HRMS m/z [M + H] :
0
0
0
0
1
1
48.31, 133.32 (C-6 ), 130.49 (C-4 ), 126.78 (C-5 ), 123.90 (C-8 ),
92.1400 (Calcd. for C21
H
36NO
6
: 391.1115).
0
0
0
22.07 (C-7 ), 116.53, 113.95, 100.59 (C-1 ), 55.50 (C-10 ), 49.81
0
0
0
0
(
C-12 ), 48.88 (C-13 ), 36.24 (C-16 ), 29.57 (C-14 ); APCI-HRMS m/
+
3
z [M + H] : 330.1354 (Calcd. for C18H21ClN O: 330.1367).
2.3.2. 9-Aminoacridines, 2–6
An adapted literature method of Ishikawa et al. (2001) served to
synthesize the 9-aminoacridines. It is described as follows: 6,9-di-
chloro-2-methoxyacridine (7.1 mmol, 2.0 g, 1 eq.) was dissolved in
anhydrous DMF (40 ml) upon stirring at 60–80 °C for 5–10 min and
2.3.2.3. 6-Chloro-2-methoxy-9-(piperazin-1-yl)acridine, 4. The reac-
tion of 6,9-dichloro-2-methoxyacridine and piperazin, followed
by successive purification through chromatography, eluting with
K
2
CO
3
(7.2 mmol, 1.0 g, 1 eq.) added, followed by the appropriate
DCM:EtOAc (1:1, v/v) then MeOH:NH
4
OH (19:1, v/v) afforded 4
ꢀ1
amine, either di- or triamine (143.8 mmol, 20 eq.) (Scheme 1).
The resulting mixture was stirred for an additional 3–6 h until
completion of the reaction (Scheme 1). Thereafter, the reaction
mixture was filtered and the filtrate was spun to dryness in vacuo.
The residue was dissolved in DCM (50 ml) and the organic phase
(1.7 g, 73%) a yellow powder; mp: 163 °C; IR (ATR)
mmax/cm
:
1
3532, 3293, 1627, 766, 564; H NMR (600 MHz, DMSO-d
6
) d
0
0
(ppm): 8.39 (d, J = 9.2 Hz, 1H, H-8 ), 8.08 (d, J = 2.2 Hz, 1H, H-5 ),
0
0
0
0
7.99 (d, J = 9.3 Hz, 1H, H-4 ), 7.53–7.49 (m, 3H, H-1 , H-3 , H-7 ),
0
0
0
3.96 (s, 2H, H-10 ), 3.49 (t, J = 4.6 Hz, 4H, H-12 , H-14 ), 3.39 (s,
0
0
13
washed with saturated NaHCO
water (50 ml), and dried over MgSO
in vacuo, resulting in a yellow powder/oil, which was purified
3
(3 ꢁ 50 ml), brine (50 ml) and
61H,
2
H O), 3.05 (t, J = 4.6 Hz, 4H, H-13 , H-15 );
C
NMR
0
0
. The solvent was removed
(151 MHz, DMSO-d
6
)
d
(ppm): 156.30 (C-2 ), 152.54 (C-9 ),
4
0
0
0
148.23, 147.41, 133.51 (C-6 ), 131.42 (C-4 ), 127.87 (C-5 ),
0
0
0
1
1
1
26.81 (C-8 ), 125.55 (C-7 ), 125.24 (C-3 ), 124.57, 122.28,
0
0
0
0
0
00.68 (C-1 ), 55.51 (C-10 ), 52.83 (C-12 , C-14 ), 46.64 (C-13 , C-
0
+
1
5
3
5 ); APCI-HRMS m/z [M + H] : 328.1200 (Calcd. for C18H19ClN O:
5
328.1211).
4
5
a
O
O
6
7
8
3
1
1
4
8
a
2.3.2.4. 6-Chloro-2-methoxy-9-(3-methylpiperazin-1-yl)acridine, 5. The
reaction of 6,9-dichloro-2-methoxyacridine and 2-methylpiperazin,
followed by successive purification through chromatography, eluting
O
1
2a
3 12
9
O
10
1
1
1
6
with DCM:MeOH:NH
4 4
OH (8:1.5:0.5, v/v/v) then MeOH:NH OH
O
a
(19:1, v/v) afforded 5 (1.2 g, 45%) a yellow–reddish powder; mp:
b
Br
ꢀ
1
1
165 °C; IR (ATR)
m
max/cm : 3275, 1628, 1417, 169, 652; H NMR
1
0
(
600 MHz, CDCl
3
) d (ppm): 8.36 (d, J = 9.2 Hz, 1H, H-8 ), 8.07 (s, 1H,
0
0
0
0
0
H-5 ), 7.98 (d, J = 9.3 Hz, 1H, H-4 ), 7.57–7.49 (m, 3H, H-1 , H-3 , H-7 ),
Fig. 3. The 2-bromo-(10b-dihydroartemisinoxy)ethane analogue of DHA. Com-
pound 1 was synthesized from DHA and 2-bromoethanol using routinely methods.
0
0
0
0
2
3.95 (s, 3H, H-10 ), 3.36 (s, 46H, H O, H-14 , H-13 , H-15 ), 3.09–3.04

J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
19
15
5
H
4
O
O
6
7
8
3
5a
14
8a
O
12a
1
2
H
1
9
O
10
6
Cl
RH
O
a
b
8
'
9'
1'
4'
R
9
2' O 10'
2
O
10'
7'
8'
1'
4'
9'
7'
2'
3'
O
(
i)
(ii)
10'
6'
6
3'
Cl
N
Cl
N
5'
Cl 6' 5'
N
2
- 6
7
- 11
Compound
2
R
Compound
7
R
1
1'
13'
11'
13'
13'
NH
14'
NH
14'
HN
HN
12'
1
2'
11'
15'
3
4
11'
HN 12'
13'
15'
N
8
9
HN 12'
14'
N
1
6'
14'
16'
1
2' 13'
12' 13'
1
1'
14'
11'
14'
N
N
N
N
1
5' 16'
15' 16'
1
7'
1
7'
1
2'
1
2'
11'
13' 16'
N N
1
1'
13' 16'
N
5
6
10
11
N
1
4' 15'
1
4' 15'
1
5' 16'
15' 16'
1
4'
N19'
14'
12'
N19'
1
2'
N
N
N
H
N
1
3'
13'
1
1'
17' 18'
11'
H
17' 18'
Scheme 1. Multi-step synthesis of artemisinin–acridine hybrids 7–11. Reagents and conditions: (i) appropriate amine, DMF, 3–6 h, 60–80 °C and (ii) 1, 2–7, ACN, KI, K
microwave (150 W, 45 °C).
2 3
CO ,
0
0
(
m, 2H, H-12 ), 1.03 (d, J = 6.1 Hz, 2H, H-17 ); 13C NMR (151 MHz,
2.3.3. Artemisinin–acridine hybrids, 6–11
9-Aminoacridine, 2–6 (1 eq.) and 1 (1.5 eq.) were dissolved in
ACN (40 ml) at room temperature, and NaHCO (1 eq.) and KI (cat-
alytic amount) were added. The reaction mixture was transferred
into a 100 ml round-bottomed flask and placed in a CEM Discov-
ery™ microwave synthesizer explorer carousel (Scheme 1). The
mixture was heated to 45 °C while stirring for 10 min (150 W,
0
0
CDCl
(
1
1
3
) d (ppm): 156.93 (C-2 ), 152.49 (C-9 ), 148.67, 148.01, 133.43
C-6 ), 131.38 (C-4 ), 127.87 (C-5 ), 126.84 (C-8 ), 125.41 (C-7 ),
25.15 (C-3 ), 124.47, 122.17, 100.70 (C-1 ) 59.78 (C-14 ), 55.45 (C-
0 ), 52.48 (C-12 ), 51.54 (C-13 ), 46.76 (C-15 ), 19.12 (C-17 ); APCI-
0
0
0
0
0
3
0
0
0
0
0
0
0
0
+
3
HRMS m/z [M + H] : 342.1364 (Calcd. for C19H20ClN O: 342.1367).
5
0 psi), then left to cool for 10 min at room temperature. This pro-
cedure was repeated, until completion of the reaction. Afterwards,
the mixture was filtered and the filtrate spun to dryness, leaving a
yellow residue, which was dissolved in DCM. The organic phase
2
.3.2.5.
amine, 6. The reaction of 6,9-dichloro-2-methoxyacridine and
-(2-aminoethyl)piperazine, followed by successive purification
through chromatography, eluting with DCM:MeOH:NH OH
OH (19:1, v/v) resulted in 6
1.0 g, 45%) an off-white to yellow powder; mp: 166 °C; IR (ATR)
6-Chloro-2-methoxy-N-[2-(piperazin-1-yl)ethyl]acridin-9-
1
was washed with NaHCO
50 ml) and dried over MgSO
and the residue purified through column chromatography, eluting
with various ratios of MeOH, DCM, EtOAc and NH OH to afford the
3
(3 ꢁ 50 ml), brine (50 ml) and water
4
(
4
. The solvent was removed in vacuo
(
8:1.5:0.5, v/v/v) then MeOH:NH
4
(
m
CDCl
ꢀ
1
1
max/cm : 3407, 3332, 2819, 1631, 557; H NMR (600 MHz,
4
0
target hybrids. The IR, NMR and HRMS data are reported below.
3
) d (ppm): 8.12 (d, J = 9.2 Hz, 1H, H-8 ), 8.07–8.05 (m, 1H,
0
0
H-5 ), 8.01–798 (d, J = 9.4 Hz, 1H, H-4 ), 7.42 (dd, J = 9.4, 2.7 Hz,
1
1
J = 4.9 Hz, 4H, H-16 , H-18 ), 2.67 (t, J = 5.7 Hz, 2H, H-15 ), 2.68–
2
0
0
0
H, H-3 ), 7.27 (tt, J = 4.7, 2.4 Hz, 2H, H-1 , H-7 ), 6.24 (s, 1H, H-
2.3.3.1. {N-(2-aminoethyl)-6-chloro-2-methoxyacridin-9-amine}-2-
(10b-dihydroartemisinoxy)ethane, 7. The reaction of 1 (5.0 mmol,
0
0
0
1 ), 3.98 (s, 3H, H-10 ), 3.80 (t, J = 5.7 Hz, 2H, H-12 ), 3.00 (t,
0
0
0
1.9 g), 2 (3.3 mmol, 1.0 g) and K
by purification on silica gel, eluting with DCM:MeOH;NH
(9:0.5:0.5, v/v/v) afforded 7 (0.2 g, 7%) yellow–brown crystals;
2
CO
3
(5.0 mmol, 0.7 g), followed
0
0
13
.56 (m, 4H, H-13 , H-17 ); C NMR (151 MHz, CDCl
3
) d (ppm):
4
OH
0
0
0
1
55.76 (C-2 ), 150.15 (C-9 ), 148.41, 146.51, 134.70 (C-6 ), 131.27
0
0
0
0
0
ꢀ1
1
(
C-4 ), 127.99 (C-5 ), 124.50 (C-8 ), 124.42 (C-7 ), 124.26 (C-3 ),
mp: 166 °C; IR (ATR)
m
max/cm : 3310, 2920, 1630, 1012, 563; H
0
0
0
1
5
1
3
23.98 117.51, 115.41, 99.10 (C-1 ), 57.88 (C-13 ), 55.46 (C-10 ),
NMR (600 MHz, CDCl
8 ), 7.93 (d, J = 2.1 Hz, 1H, H-5 ), 7.88 (d, J = 9.3 Hz, 1H, H-4 ), 7.32
(d, J = 2.7 Hz, 1H, H-1 ), 7.16 (dd, J = 9.2, 2.4 Hz, 1H, H-3 ), 7.07
(dd, J = 9.1, 2.1 Hz, 1H, H-7 ), 5.37 (s, 1H, H-12), 4.82 (d,
3
) d (ppm): 8.07 (dd, J = 15.0, 9.3 Hz, 1H, H-
0
0
0
0
0
0
0
5.42, 53.87 (C-15 , C-17 ), 46.94 (C-16 ), 46.33 (C-18 ), 45.62 (C-
2 ); APCI-HRMS m/z [M + H] : 371.1619 (Calcd. for C20
0
+
0
0
H24ClN
4
O:
0
71.1633).

2
0
J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
0
0
0
0
0
0
J = 3.5 Hz, 1H, H-10), 4.27–4.11 (m, 2H, H-13 ), 4.05 (dt, J = 10.4,
(C-2 ), 134.91 (C-6 ), 131.15 (C-4 ), 128.01 (C-5 ), 126.17 (C-8 ),
0
0
0
5
.2 Hz, 1H, H-a
a
), 3.94 (s, 4H, H-10 ), 3.89–3.72 (m, 1H), 3.64
ddd, J = 14.3, 10.0, 5.5 Hz, 1H, H-ab), 3.31 (dh, J = 12.2, 6.7 Hz,
H, H-12 ), 3.03 (t, J = 5.4 Hz, 2H, H-b), 2.62 (qt, J = 7.5, 3.9 Hz,
), 2.14 (s, 1H), 2.06–1.91 (m, 2H,
), 1.50–1.15 (m, 15H, H-8b, H-
a, H-14, H-7, H-6, H-5a), 0.93–0.70 (m, 15H, H-16, H-15);
) d (ppm): 158.18, 156.15 (C-2 ), 152.68 (C-
), 139.30, 134.95 (C-6 ), 131.85 (C-4 ), 128.70 (C-5 ), 126.28 (C-
), 125.92 (C-7 ), 125.69 (C-3 ), 124.10, 122.91, 104.13 (C-3),
02.35 (C-10), 101.23 (C-1 ), 98.45, 87.96, 87.88 (C-12), 80.93 (C-
125.64 (C-7 ), 125.48 (C-3 ), 122.84, 104.12 (C-3), 102.07 (C-10),
100.40 (C-1 ), 87.97 (C-12), 81.05 (C-12a), 65.54 (C-a), 60.37 (C-
13 , C-15 ), 58.20 (C-b), 55.72 (C-10 ), 55.72, 54.71, 52.51 (C-5a),
51.93, 51.85, 44.34 (C-8a), 37.61 (C-6), 36.39 (C-4), 34.65 (C-7),
30.80 (C-9), 26.16 (C-14), 24.74 (C-8), 24.45 (C-5), 20.38 (C-15),
0
(
2
1
0
0
0
0
H, H-9), 2.44–2.23 (m, 2H, H-4
a
H-4b), 1.92–1.53 (m, 5H, H-5, H-8
8
a
1
3
+
C
13.10 (C-16); APCI-HRMS m/z [M + H] : 638.3005 (Calcd. for C35-
H45ClN O : 638.2991).
3 6
0
NMR (151 MHz, CDCl
3
0
0
0
0
9
8
1
1
0
0
0
0
2
(
.3.3.4. 6-Chloro-2-methoxy-9-(2-methylpiperazin-1-yl)acridine-2-
10b-dihydroarte misinoxy)ethane, 10. The reaction between 1
4.4 mmol, 1.7 g), 5 (2.9 mmol, 1.0 g) and K CO (4.40 mmol,
.6 g), followed by purification on silica gel, eluting with
DCM:MeOH;NH OH (9:0.5:0.5, v/v/v) afforded 10 (0.5 g, 64%) yel-
4
low crystals; mp: 168 °C; IR (ATR)
024, 568; H NMR (600 MHz, CDCl
H, H-8 ), 8.16–8.15 (m, 1H, H-5 ), 8.07 (d, J = 9.5 Hz, 1H, H-4 ),
.50 (t, J = 3.7 Hz, 1H, H-1 ), 7.42 (dd, J = 9.3, 2.7 Hz, 1H, H-3 ),
.36 (ddd, J = 10.9, 5.2, 2.2 Hz, 1H, H-7 ), 5.54 (d, J = 30.9 Hz, 1H,
H-12), 4.86 (dd, J = 7.2, 3.5 Hz, 2H, H-10), 4.08 (q, J = 17.1,
3.4 Hz, 1H, H-a
H, H-ab), 3.55–3.33 (m, 2H, H-12 , H-14 ), 3.19 (d, J = 36.3 Hz,
H, H-13 ), 2.89 (d, J = 75.2 Hz, 1H, H-15 ), 2.67–2.65 (m, 1H, H-
), 2.37 (tt, J = 14.0, 4.5 Hz, 1H, H-4 ), 2.04 (ddt, J = 14.8, 4.9,
.3 Hz, 1H, H-4b), 1.90–1.73 (m, 3H, H-8), 1.61 (ddq, J = 12.9, 6.7,
.5 Hz, 1H, H-5b), 1.43 (d, J = 1.5 Hz, 3H, H-14), 1.23 (dtt, J = 13.4,
.9, 4.0 Hz, 4H, H-17 , H-6), 0.96–0.90 (m, 7H, H-7b, H-16, H-15);
C NMR (151 MHz, CDCl
), 148.64 (C-9 ), 135.22 (C-6 ), 131.38, 131.25 (C-4 ), 128.28,
28.27 (C-5 ), 128.15, 126.67, 126.12, 125.39, 125.20, 123.39,
20.92, 118.70, 104.13 (C-3), 102.36 (C-10), 100.37, 97.42 (C-1 ),
7.99 (C-12), 87.95, 81.05 (C-12a), 81.03, 70.52, 63.34 (C-a),
5.67 (C-10 ), 52.63 (C-12 , C-14 ), 52.50 (C-5a), 44.31 (C-8a),
7.60, 37.57 (C-6), 36.40 (C-4), 36.38, 34.60 (C-7), 30.81, (C-9),
6.16 (C-14), 24.77 (C-8), 24.70 (C-5), 24.45, 20.36 (C-16), 13.16
C-15), 13.09 (C-17 ); ESI-HRMS m/z [M + H] : 652.3142 (Calcd.
for C36 47ClN : 652.3148).
0
2a), 67.73 (C-a), 66.83, 55.92(C-10 ), 55.60, 53.40, 52.48, 52.38
0
0
(
C-5a), 48.49 (C-b), 47.77 (C-12 ), 47.06 (C-13 ), 46.08, 44.69,
4.22 (C-8a), 37.41 (C-6), 36.32 (C-4), 34.47 (C-7), 31.90, 30.79
C-9), 29.67, 29.34, 26.15, 26.10 (C-14), 24.60 (C-8), 24.56 (C-5),
2.67, 20.22 (C-15), 14.10, 13.06 (C-16); ESI-HRMS m/z [M + H] :
12.2825 (Calcd. for C33 43ClN : 612.2835).
(
2
3
4
0
(
2
+
ꢀ1
m
max/cm : 3419, 2919, 1629,
) d (ppm): 8.26 (d, J = 9.3 Hz,
6
H
3 6
O
1
1
1
7
7
3
0
0
0
2
.3.3.2. {3-[(6-Chloro-2-methoxyacridin-9-yl)amino]propyl}(methyl)
0
0
amine-2-(10b-dihydroartemisinoxy)ethane, 8. The reaction between
(4.6 mmol, 1.8 g), 3 (3 mmol, 1.0 g) and K CO (5.0 mmol, 0.7 g),
followed by purification on silica gel, eluting with
DCM:MeOH;NH OH (9:0.5:0.5, v/v/v) afforded 8 (1.1 g, 59%) yel-
low crystals; mp: 171 °C; IR (ATR)
0
1
2
3
0
1
1
1
9
2
3
6
a
), 3.97 (d, J = 2.6 Hz, 3H, H-10 ), 3.74–3.59 (m,
4
0
0
ꢀ
1
m
max/cm : 3309, 2920, 1630,
0
0
1
1
1
2
2
4
023, 665; H NMR (600 MHz, CDCl
3
) d (ppm): 8.03 (d, J = 2.1 Hz,
a
0
0
0
H, H-5 ), 8.00 (dd, J = 9.3, 4.6 Hz, 2H, H-8 , H-4 ), 7.33 (dd, J = 9.3,
0
0
.6 Hz, 1H, H-3 ), 7.28 (d, J = 2.6 Hz, 1H, H-1 ), 7.17 (dd, J = 9.2,
0
.1 Hz, 1H, H-7 ), 5.33 (s, 1H, H-12), 4.77 (d, J = 3.7 Hz, 1H, H-10),
.08–4.03 (m, 3H, H-13 , H-aa), 3.92 (s, 3H, H-10 ), 3.61 (ddd,
J = 10.9, 6.4, 4.4 Hz, 1H, H-ab), 2.79 (dddd, J = 10.9, 6.5, 4.4,
.9 Hz, 2H, H-b), 2.71 (dddd, J = 19.1, 12.8, 6.6, 4.2 Hz, 2H), 2.55
0
0
0
1
3
3
) d (ppm): 159.79, 157.18, 156.91 (C-
0
0
0
0
2
1
1
8
5
3
2
1
(
0
0
dp, J = 11.5, 4.3, 3.8 Hz, 1H, H-9), 2.42 (s, 3H, H-16 ), 2.26 (td,
0
J = 14.0, 3.9 Hz, 1H, H-4
a
), 1.90 (dtd, J = 12.9, 7.6, 6.2, 3.8 Hz, 3H,
), 1.58 (qd, J = 13.7,
), 1.49 (dq, J = 14.1, 3.8 Hz, 1H, H-8b), 1.37 (s,
H-4b), 1.69 (ddt, J = 13.6, 6.7, 3.5 Hz, 1H, H-8
a
0
0
0
3
3
1
(
(
1
(
5
1
2
.5 Hz, 1H, H-5
a
0
H, H-14 ), 1.31 (dt, J = 13.5, 4.6 Hz, 1H, H-6), 1.25–1.13 (m, 2H),
.07–1.05 (m, 2H, 5a), 0.98–0.73 (m, 5H, H-7
a
, H-16), 0.63–0.61
) d (ppm): 155.22
0
+
(
1
3
m, 4H, H-7b, H-15); C NMR (151 MHz, CDCl
C-2 ), 151.72 (C-9 ), 125.07 (C-8 ), 123.83 (C-7 ), 123.30 (C-3 ),
15.86, 113.35, 104.06 (C-3), 102.59 (C-10), 101.75 (C-1 ), 87.91
C-12), 80.92 (C-12a), 66.42 (C-a), 58.25 (C-b), 57.34 (C-13 ),
5.66 (C-10 ), 52.25 (C-5a), 51.32 (C-12 ), 44.12 (C-8a), 43.31 (C-
6 ), 37.22 (C-6), 36.29 (C-4), 34.30 (C-7), 30.71 (C-9), 26.62,
6.11 (C-14), 24.47 (C-8), 24.34 (C-5), 20.03 (C-15), 12.92 (C-16);
ESI-HRMS m/z [M + H] : 640.3134 (Calcd. for
3
H
3 6
O
0
0
0
0
0
0
0
0
0
2.3.3.5.
amine-2-(10b-dihydroartemisinoxy)ethane,
between 1 (4.1 mmol, 1.6 g), 6 (2.7 mmol, 1.0 g) and K
(4.1 mmol, 0.6 g), followed by purification on silica gel, eluting
with DCM:MeOH;NH OH (9:0.5:0.5, v/v/v) afforded 11 (1.3 g,
1%) yellow crystals; mp: 168 °C; IR (ATR)
2937, 1630, 1022, 563; H NMR (600 MHz, CDCl
(d, J = 9.2 Hz, 1H, H-8 ), 8.03 (d, J = 2.0 Hz, 1H, H-5 ), 7.98 (d,
J = 9.4 Hz, 1H, H-4 ), 7.32 (d, J = 2.6 Hz, 1H, H-3 ), 7.22 (d,
J = 2.6 Hz, 1H, H-1 ), 7.20 (d, J = 2.2 Hz, 1H, H-7 ), 5.45 (s, 1H, H-
12), 4.80 (d, J = 3.5 Hz, 1H, H-10), 3.95 (s, 5H, H-13 , H-10 ), 3.88
(t, J = 6.4 Hz, 3H, H-a
2.73 (t, J = 5.7 Hz, 2H, H-b, H-9), 2.34 (td, J = 14.0, 3.9 Hz, 1H, H-
), 2.00 (ddd, J = 14.6, 4.9, 3.0 Hz, 1H, H-4b), 1.86–1.72 (m, 3H,
H-8), 1.71 (dq, J = 14.2, 3.9 Hz, 1H, H-5 ), 1.58 (dq, J = 12.9,
6-Chloro-2-methoxy-N-[2-(piperazin-1-yl)ethyl]acridin-9-
11. The reaction
CO
0
2
3
+
35 3 6
C H47ClN O :
6
40.3148).
4
ꢀ
1
7
mmax/cm : 3334,
1
2.3.3.3.
6-Chloro-2-methoxy-9-(piperazin-1-yl)acridine-2-(10b-
3
) d (ppm):): 8.07
0
0
dihydroartemisinoxy)ethane, 9. The reaction between 1 (1.8 mmol,
.7 g), 4 (1.2 mmol, 0.4 g) and K CO (1.8 mmol, 0.3 g), followed
by purification on silica gel, eluting with DCM:MeOH;NH OH
9:0.5:0.5, v/v/v) afforded 9 (1.2 g, 64%) yellow crystals; mp:
max/cm : 3466, 2953, 1629, 1015, 772;
) d (ppm): 8.25 (d, J = 9.2 Hz, 1H, H-8 ),
.17 (d, J = 2.1 Hz, 1H, H-5 ), 8.08 (d, J = 9.3 Hz, 1H, H-4 ), 7.49
d, J = 2.8 Hz, 1H, H-1 ), 7.43 (dd, J = 9.3, 2.7 Hz, 1H, H-3 ), 7.36
0
0
0
2
3
0
0
4
0
0
(
1
ꢀ
1
1
0
64.2 °C; IR (ATR)
m
H
a
H-12 ), 3.55 (dt, J = 10.9, 5.8 Hz, 1H, H-ab),
0
NMR (600 MHz, CDCl
8
(
(
3
0
0
0
4a
0
a
0
dd, J = 9.2, 2.2 Hz, 1H, H-7 ), 5.52 (s, 1H, H-12), 4.86 (d,
3.3 Hz, 1H, H-5b), 1.46–1.37 (m, 6H, H-14), 1.36–1.32 (m, 1H, H-
J = 3.4 Hz, 1H, H-10), 4.09–4.08 (m, 1H, H-a
a
), 3.98 (s, 3H, H-
6), 1.23–1.20 (m, 2H, H-5a), 0.92–0.86 (m, 9H, H-15, H-16, H-7);
0
0
0
13
0
0
1
6
2
0 ), 3.72–3.64 (m, 5H, H-ab, H-12 , H14 ), 2.87 (t, J = 37.4 Hz,
C NMR (151 MHz, CDCl
3
) d (ppm): 155.90 (C-2 ), 150.89 (C-9 ),
0
0
0
0
0
H, H13 , H-15 H-b), 2.65 (qt, J = 7.4, 4.1 Hz, 1H, H-9), 2.43–
.26 (m, 1H, H-4 ), 2.04–2.02 (m, 1H, H-4b), 1.90–1.76 (m, 2H,
), 1.63 (dq, J = 13.2,
.4 Hz, 1H, H-5b), 1.48 (ddt, J = 21.9, 13.4, 4.7 Hz, 2H), 1.42 (s,
124.98 (C-7 ), 124.86 (C-3 ), 124.05 (C-8 ), 104.03 (C-3), 101.97
0
a
(C-10), 99.54 (C-1 ), 87.92 (C-12), 81.09 (C-12a), 65.92 (C-a),
0
0
0
0
H-8), 1.75 (dq, J = 14.3, 3.8 Hz, 1H, H-5
a
57.92 (C-b), 56.90 (C-16 , C-18 ), 55.61 (C-10 ), 53.80 (C-15 , C-
0
0
3
3
1
0
17 ), 52.65, 52.52 (C-5a), 45.40 (C-12 ), 44.38 (C-8a), 37.50 (C-6),
36.39 (C-4), 34.64 (C-7), 30.82 (C-9), 26.17 (C-14), 24.70 (C-8),
H, H-14), 1.37 (dddd, J = 16.1, 12.5, 6.5, 3.8 Hz, 1H, H-6), 1.27–
.22 (m, 2H, H-5a), 0.94 (dd, J = 12.3, 6.8 Hz, 6H, H-15, H-16),
+
24.37 (C-5), 20.32 (C-15), 13.05 (C-16); ESI-HRMS m/z [M + H] :
1
3
.90–0.78 (m, 1H); C NMR (151 MHz, CDCl
3
) d (ppm): 156.93
4 6
681.3403 (Calcd. for C37H50ClN O : 681.3413).

J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
21
Table 1
TGA and DSC data of compounds 7–11 and DHA. DSC served to determine the both the physical state and transition phases while TGA was used to determine the thermal stability.
a,b
a,b
% weight loss-T2 a
b
b
b
Heat loss (mJ)b
Compd.
T1 (°C)
T2 (°C)
Peak (°C)
Peak onset (°C)
Peak endset (°C)
7
8
9
1
1
25
25
25
25
25
25
240
240
240
240
240
240
13.6
10.7
8.9
8.9
9.2
60.5
*
*
58.3
61.9; 96.7
*
54.1
*
*
53.2
58.5; 102.6
*
66.4
*
*
64.0
64.7; 98.4
*
22.6
*
*
12.9
23.1; 3.5
*
0
1
DHA
86.9
*
= No data.
a
TGA data determined by a Shimadzu DTG-60 and data interpreted by Schimadzu software).
DSC data determined by a Shimadzu DSC-60 A and data interpreted by Schimadzu software.
b
2.4. Physicochemical properties
Table 2
Differential scanning calorimetry (DSC) was performed, using a
Predicted physicochemical parameters of compounds 2–11 (Accelrys Discovery
Shimadzu DSC-60, a LABCON low temperature water bath and a
computer system. Data was collected for 2–4 mg samples weighed
into aluminium crimp cells, covered and sealed with a crimping
tool from Shimadzu. The samples were placed onto the auto sam-
pler. This process was repeated with a reference that was placed
manually on the left side of the furnace of the DSC apparatus.
The DSC system parameters were set with a starting temperature
of 25 °C, a maximum temperature of 300 °C, a heating rate of
Studio 3.1 served to calculate the predicted values).
Compound LogPa pK b
a
ADMET
ADMET
Absorption
leveld
ADMET
BBB
Solubility
c
levele
level
2
3
4
5
6
7
8
9
0
1
2.98
3.48
3.58
3.96
3.37
6.40
7.00
7.11
7.49
6.90
9.13
2
2
2
1
2
0
1
0
0
1
0
0
0
0
0
2
3
3
3
2
2
1
1
1
1
4
4
4
4
4
9.90
9.37
9.37
9.40
8.45
9.03
9.37
9.37
7.23
1
0 °C/min and the nitrogen gas flow rate at 35 ml/min. The thermal
process was started after the DSC trace was produced and ana-
lyzed, using the Schimadzu software. The results are reported in
Table 1.
1
1
Thermogravimetric analysis (TGA) was performed by means of
a Shimadzu DTG-60 and its computer system. Two empty alumin-
ium crimp cells were placed on the furnace of the TGA and manu-
ally set to tear the microbalance. Data was collected for that
quantity of sample that was placed in an aluminium cell and
placed on the right hand side of the furnace, after which the system
was closed. The system parameters were set with the starting tem-
perature of 25 °C, a maximum temperature of 300 °C, a heating rate
of 10 °C/min and the nitrogen gas flow rate at 35 ml/min. The sam-
ple was weighed automatically before starting the thermal process.
A TGA trace was generated and analyzed, using the Schimadzu
software. The results are reported in Table 1.
a
b
c
Calculated using Accelrys Discovery Studio 3.1.
Calculated using ACD.
w
ADMET aqueous solubility level (LogS ) at 25 °C and pH = 7, values = 0, 1, 2 and
indicate extremely low, very low, low and good solubility, respectively.
ADMET human intestinal absorption, values = 0, 1, 2 and 3 indicate good,
3
d
moderate, low and very low absorption, respectively.
e
Blood brain barrier values = 0, 1, 2, 3, and 4 indicating, very high, high, medium,
low and undefined blood–brain barrier penetration, respectively.
a starting concentration of 100
luted two-fold in complete medium to give ten concentrations
per compound, with the lowest concentration being 0.2 g/ml.
lg/ml, which was then serially di-
l
Accelrys Discovery Studio 3.1 was used to calculate the absorp-
tion, distribution, metabolism, and excretion – toxicity (ADMET)
properties, including logP, aqueous solubility, human intestine
absorption and blood brain barrier (BBB) levels of compounds
The same dilution technique was used for all samples. References
were tested at a starting concentration of 1000 ng/ml. The highest
concentration of solvent to which the parasites were exposed to
had no measurable effect on the parasites’ viability (data not
shown). The IC50 values were obtained using a non-linear, dose re-
sponse, curve fitting analysis via Graph Pad Prism v.4.0 software.
The results are reported in Table 3.
2–11, and the results are summarized in Table 2.
2
2
.5. In vitro biological evaluation
.5.1. Antimalarial activity
2.5.2. Cytotoxicity and anticancer activity
The test samples were screened in triplicate for the in vitro anti-
plasmodial activity determinations against a chloroquine sensitive
CQS) strain of P. falciparum (NF54) and against the chloroquine
resistant (CQR) strain of P. falciparum (Dd2). Continuous in vitro
cultures of asexual erythrocyte stages of P. falciparum were main-
tained, using a modified method of Trager and Jensen (1976).
Quantitative assessment of antiplasmodial activity in vitro was
determined by means of the parasite lactate dehydrogenase assay,
using a modified method as described by Makler et al. (1993).
The test samples were prepared as 20 mg/ml stock solutions in
Test samples were screened for cytotoxicity and anticancer
activity against the Chinese hamster ovarian (CHO) and HeLa cell
lines, respectively, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) assay. The MTT assay is used
as a colorimetric assay for the determination of cellular growth
and survival, and compares well with other available assays (Mos-
mann, 1983; Rubinstein et al., 1990). The tetrazolium salt MTT was
used to measure all growth and chemosensitivity. The test samples
were tested in triplicate. The same stock solutions prepared for
antiplasmodial testing were used for cytotoxicity testing. Test
compounds were stored at ꢀ20 °C, until use. Dilutions were pre-
pared on the day of the experiment. Emetine was used as the ref-
erence drug in all experiments. The initial concentration of
(
1
00% DMSO and were stored at ꢀ20 °C. Further dilutions were pre-
pared in complete medium on the day of the experiment. Samples
were tested as a suspension if not completely dissolved. Dihyd-
roartemisinin (DHA), artesunate (AS) and chloroquine (CQ) were
used as reference drugs. A full dose response analysis was per-
formed to determine the IC50-values. Test samples were tested at
emetine was 100
medium with ten-fold dilutions to give six concentrations per com-
pound, the lowest being 0.001 g/ml. The same dilution technique
lg/ml, which was serially diluted in complete
l

2
2
J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
Table 3
In vitro biological activity of compounds. Cells were incubated with compounds at various concentrations for 48 h; antimalarial activity and cytotoxicity against CHO and HeLa
cells were determined using parasite lactate dehydrogenase (pLDH) and MTT-assays, respectively. Apoptosis against HepG2 and SH-SY5Y was determined using FITC annexin V/
dead cell apoptosis assay.
Compound
Antimal. activity,
IC50 (nM) ±S.D.
Resistance
index
Cytotoxicity
Antic. activity
Apoptosis EC50
(l
M) ±S.D.d
Selectivity
indexe
a
b
c
c
IC50
(l
M) ±S.D.
IC50
(lM) ±S.D.
NF54
Dd2
RI
CHO
HeLa
HepG2
SH-SY5Y
SI
2
3
4
5
6
7
8
9
1
1
DHA
AS
13.9 ± 2.3
9.1 ± 0.9
1491.8 ± 68.8
158.3 ± 7.9
7.3 ± 1.4
2.6 ± 0.0
12.2 ± 1.6
266.8 ± 122.4
17.0 ± 2.1
14.1 ± 7.5
<7
80.7 ± 8.3
116.7 ± 15.8
1672.2 ± 152.2
384.0 ± 45
141.8 ± 4.1
35.3 ± 3.6
38.5 ± 1.6
429.9 ± 10.2
41.3 ± 5.8
50.4 ± 2.9
7 ± 2
5.8
12.8
1.1
1.0 ± 0.1
0.5 ± 0
3.4 ± 0
9.2 ± 1.2
2.2 ± 1.3
12.3 ± 1.1
3.3 ± 1.2
0.4 ± 1.3
5.2 ± 1.4
4.7 ± 1.3
2.7 ± 1.8
3.3 ± ndj
0.8 ± 2.5
0.6 ± 1.3
0.5 ± 1.3
17.7 ± 1.3
55.2 ± 1.1
47.4 ± 1.1
56.8 ± 1.1
15.1 ± 1.2
58.7 ± 1.4
55.1 ± 1.1
54.6 ± 1.1
33.6 ± 1.2
55.6 ± 1.1
58.2 ± 1.2
44.3 ± 1.1
58.0 ± 1.1
58.2 ± 1.2
43.8 ± 1.1
36.0 ± 1.2
55.7 ± 1.1
24.6 ± 1.2
42.4 ± 1.1
50.3 ± 1.2
48.8 ± 1.1
26.9 ± 1.1
58.1 ± 1.1
45.8 ± 1.1
39.0 ± 1.1
57.1 ± 1.1
58.1 ± 1.2
nd
72
55
2
35
2.4
5.6 ± 0
19.4
13.6
3.2
1.6
2.4
3.6
<1
<2.9
14.7
nd
0.3 ± 0.1
1.6 ± 0.2
1.2 ± 0.3
>156.9
6.0 ± 1.1
0.9 ± 0.2
20.1 ± 6.4
1.6 ± 0.5
>312.6
0.1
41
615
98
>588
353
64
>2871
>308
>16,897
nd
0
1
f
g
<5.2
18.5 ± 6.3
nd
15.1 ± 0.5
271.7 ± 104.4
nd
h
CQ
EMi
Melphalan
40 ± 2.3k
a,c
Antimalarial activity/cytotoxicity/apoptotic activity, IC50 value is the minimum concentration causing 50% cells growth inhibition, data represents the mean of three
independent experiments SD (standard deviation).
b
Resistance index (RI) = IC50Dd2/IC50NF54.
d
Effective concentration of compound inducing 50% reduction in parasitic cells count, data represents the mean of three independent experiments SD (standard deviation),
significance was set at p 6 0.05.
e
Selectivity index (SI) = IC50CHO /IC50NF54.
Dihydroartemisinin (DHA).
Artesunate (AS).
Chloroquine (CQ) was tested as diphosphate salt.
Emetine (EM) was used as the reference drug in the cytotoxicity study against CHO cells.
Not determined (nd).
IC50 value as reported by Ibrahim et al. (2013).
f
g
h
i
j
k
was applied to all test samples. The highest concentration of sol-
vent to which the cells were exposed to had no measurable effect
on the cell viability (data not shown). The IC50 values were ob-
tained from full dose response curves, using a non-linear, dose re-
sponse, curve fitting analysis via GraphPad Prism.
15–20 min at room temperature and protected from light. Thereaf-
ter, 400
l
l of 1ꢁ annexin binding buffer was gently mixed into the
cells and kept on ice. After incubation, the stained cells underwent
quantitative assessment through flow cytometry, quantifying fluo-
rescence excitation/emission wavelengths at 530 nm and >575 nm.
The cells were detected in three separate groups, i.e. live cells with
a low level of green fluorescence, apoptotic cells with green fluo-
rescence and dead cells with red and green fluorescence.
2.5.3. Apoptosis
Human hepatocellular cells (HepG2) and human neuroblastoma
The data was processed with FCS Express v.4 software. Gates
were set on the dot plot FSC and SSC during analysis. The geometric
means of fluorescence for all of the parameters were calculated
from the respective histograms, or from two parameter fluores-
cence dot plots. The results are expressed in arbitrary units as
mean fluorescence intensity (MFI). The statistical significance
was determined with one way analysis of variance (ANOVA) with
Dunn’s multiple comparison tests for non-parametric statistics.
The data is presented as mean ± standard deviation (SD), whilst
significance was set at p 6 0.05. The EC50 values were obtained
from full dose response curves, using a non-linear, dose response,
curve fitting analysis via GraphPad Prism™ v.5 software and are
summarized in Table 3.
cells (SH-SY5Y) were used for the apoptosis assay. Dilutions of the
compounds were prepared on the day of the experiment. The ini-
tial concentration of 200
ium with two-fold dilutions to give eight concentrations per
compound, the lowest being 0.01 M. The same dilution technique
lM was serially diluted in complete med-
l
was applied to all test samples. The highest concentration of sol-
vent to which the cells were exposed to had no measurable effect
on the cell viability (data not shown). An FITC annexin V/dead cell
apoptosis kit, equipped with FITC annexin V and PI for flow cytom-
etry were used to simultaneously determine the numbers of live,
dead and apoptotic cells.
Apoptosis was induced for 4 h in the cells through the addition
of 1 ml of the stock solution (1 mM). A negative control was pre-
pared simultaneously, without the inducing agent and the positive
control was staurosporine (500 nM). The cells were harvested and
washed in 100 ml cold phosphate buffered saline (PBS) solution,
while the required annexin binding buffer was prepared. A
3. Results
3.1. Chemistry
1
00
lg/ml PI solution was prepared by diluting 100 ll of the
1
mg/ml PI stock solution in 45
l
L of 1ꢁ annexin binding buffer.
Condensation of dihydroartemisinin with 2-bromoethanol pro-
duced the DHA-ethyl bromide derivative 1 in good yield. The ste-
reochemistry of this compound had previously been determined
by our research group through X-ray analysis (Lombard et al.,
2010). The configuration at C-10 stereocenter is assigned based
on the vicinal coupling constant JH-9:H-10 (Venugopalan et al.,
1995). A large coupling constant (7–10 Hz) is generally found for
After the washed cells had been re-centrifuged and the resulting
supernatant removed, the cells were re-suspended in 1ꢁ annexin
binding buffer. The cell density was determined and adjusted
6
where needed to the required concentration of ꢂ1 ꢁ 10 cells/ml.
To each 100
of the 100
ll of cell suspension, 5 ll of FITC annexin V and 1 ll
l
g/ml PI were added. The cells were incubated for

J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
23
the 10
a
-isomer (Lin et al., 1997), indicating the relative trans-con-
brids 7–11, with logP values in the 6–7.5 range (Table 2). These
values were consistent with the intermediates and hybrids pos-
sessing low and extremely aqueous solubility levels. The amines
also had better predicted human intestinal absorption and BBB
penetration abilities than the hybrids.
figuration. The 10b-isomer, indicating cis-configuration at C-10, on
the other hand, has a smaller coupling constant (3.6–5 Hz) (Lin
et al., 1989).
This intermediate and the final artemisinin–acridine hybrids 7–
9
and 11 were therefore all in the 10b form, as confirmed by the
The synthesized hybrids have a secondary/tertiary amino group
that is the nitrogen N point linked to C-b and connecting both arte-
misinin and acridine pharmacophores. This N is for most com-
small coupling constant, J = 3.4–3.7 Hz between the H-10 and
H-9 atoms. The hybrid 10, on the contrary, is a racemate mixture
of both the 10
doublet of doublet (dd) with JH9–H10 values of 3.5 Hz (10b) and
.2 Hz (10 ).
The 9-aminoacridine intermediates 2–6 were also prepared in
a
and 10b forms, as can be observed from the
pounds, the most basic with a calculated pK
a
value ꢂ9 therefore
will not protonate at the physiological pH of 7.4. It is, however,
likely to be protonated in an acidic environment, such as that
occurring in the malaria parasite digestive vacuole.
7
a
low to high yields (45–79%) through nucleophilic substitution of
the reactive chlorine in position 9 of 6,9-dichloro-2-methoxyacri-
dine, with various amines by using the literature adapted method
by Ishikawa et al. (2001). The IR spectra of these intermediates
3
.3. In vitro antimalarial activity and cytotoxicity
The biological activities are summarized in Table 3.
The synthesized compounds were all active against both the
ꢀ1
commonly showed absorption bands in the 3500–3200 cm re-
gion, evident of the presence of both NH and NH groups in their
NF54 and Dd2 strains. However, they were more active against
NF54, than against the Dd2 strain, with resistance index (RI) values
in the 2–19 range. The 9-aminoacridines 2–6 were less active than
their corresponding hybrids, irrespective of the strain considered.
All compounds were also found less potent than DHA and AS
against both strains, while possessing either comparable or higher
potencies than CQ. 9-Aminoacridine 4 and its corresponding hy-
brid 9 were the least active, while hybrids 7 and 10 were the most
and second most active compounds, respectively.
All compounds were less cytotoxic than emetine against the
CHO cells. The hybrids 7–11 appeared less toxic to CHO cells than
their corresponding 9-aminoacridine intermediates, resulting in
selective antimalarial action against the parasitic cells, with
SI > 90, as compared to SI < 70 for the intermediates 2–6. The anti-
cancer activity picture against the HeLa cells was different. Indeed,
all synthesized compounds were found to be more potent than CQ
and melphalan, while showing lesser potency than both DHA and
AS. In particular, hybrids 7 and 10 were found to be 3- and 8-,
and 5- and 12-fold more potent than CQ and melphalan, respec-
tively. The intermediates 2 and 4 showed less toxicity than their
corresponding hybrids 5 and 9, respectively, whereas the opposite
was observed for compounds 3 and 6, compared to their hybrids, 8
and 11, respectively.
2
structures. Each amino-functionalized acridine intermediate was
reacted with compound 1 through nucleophilic substitution, using
microwave radiation (Scheme 1) to render a free base artemisinin–
acridine hybrid. The hybrids 7–11 were obtained with yields in the
1
0–75% range.
The IR spectra of hybrids 7, 8 and 11 displayed shoulders in the
ꢀ1
3
500–3300 cm region, assignable due to the vibrations of sec-
ondary (N–H), while compounds 9 and 10 showed broad bands
ꢀ1
in the 1210–1150 cm region, as a result of the presence of ter-
tiary amine groups in their structures. A sharp band, corresponding
with the stretching of the C–O bond of the endoperoxide bridge
ꢀ1
was also present in the 2950–2900 cm region of the spectra of
all hybrids, confirming the presence of the artemisinin moiety in
these structures.
3.2. Physicochemical properties
Three different techniques were used to determine the physico-
chemical properties of the prepared compounds. These included
DSC, which was used to determine both the physical state of the
hybrids and their different transition phases. The TGA, on the other
hand, served to determine the thermal stability of the compounds.
Calculated ADMET properties, such as logP, solubility, absorption
and BBB levels were determined to investigate the compounds’
drug-likeness properties.
All synthesized compounds (except 5), CQ, DHA and AS showed
comparable apoptotic activity against both HepG2 and SH-SY5Y
cell lines.
Due to the reported instability of the artemisinin class of anti-
malarials (Boreen et al., 2003; Lin et al., 1985), DSC and TGA were
used to explore as to whether the addition of the 9-aminoacridine
intermediate would have the ability to impart more stability onto
the artemesinin pharmacophore. The hybrid compounds showed
exceptional stability with a 9–14% weight loss in comparison with
the 87% of DHA at 240 °C (Table 1).
Hybrids 7 and 10 displayed small endothermic phase transi-
tions, with onset temperatures of 60.5 °C and 58.3 °C, respectively,
which were consistent with glass transitions. Hybrid 11, on the
other hand, had two onset temperatures, viz. 58.5 °C and
4. Discussion
4.1. Chemistry
The artemisinin–acridine hybrids were synthesized using
microwave radiation. The peroxide bridge of artemisinin is re-
ported to be prone to simplistic hydrolysis under basic or acidic
conditions, and can also be expected to be heat sensitive (Torok
and Ziffer, 1995). However, in this study, the spectroscopic data re-
vealed no deviations from the expected values, confirming that this
bridge was preserved during microwave radiation, which was in
accordance with previous findings (Cloete et al., 2012).
In intermediates 2, 3 and 6, the secondary amine (NH) of the
linkers was rendered less nucleophilic as a result of the resonance
of the acridine ring, making it unavailable for substitution reac-
tions. Subsequently, only the terminal, primary amine (in 2) and
secondary amine (in 3 and 6) were involved in the substitution
reactions, leading to the formation of hybrids 7, 8 and 11. The
intermediate 4 (also 5) has a tertiary amine, which is a group
non-susceptible to nucleophilic substitution. Subsequently, only
the secondary amine of the piperazine linker was involved in the
reaction with 1 to form hybrid 9 (and 10).
1
02.6 °C, an indication that the equilibrium in phase transition
was inadequate at lower temperature and that another cooperative
molecular motion was required for internal re-adjustments (Souda,
2
012). The rubberlike characteristics of compound 11 were more
pronounced after the second phase transition.
Overall, the DSC thermograms suggested that all of the com-
pounds 7–11 possessed amorphous structures.
Preliminary ADMET calculations were performed in order to
determine the drug-likeness properties of the synthesized
compounds.
The 9-aminoacridines 2–6, with predicted logP values between
3
and 4 were more hydrophilic, compared to their respective hy-

2
4
J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
Substitution on the methyl-piperazine linker transpired on
thermodynamic conditions, occurring in the generally tropical, ma-
laria endemic countries. The TGA data of 7–11 revealed weight
losses between 9% and 14% at an extreme temperature of 240 °C,
in contrast to the total degradation of DHA at the same tempera-
ture. DSC data revealed that compounds 7, 10 and 11 underwent
endothermic phase transitions, characteristic of a glass transition
and possessed amorphous structures. Overall, hybrids 7–11 had
minimum weight losses, with predictable proven stability in ex-
treme conditions to withstand those harsh conditions prevailing
in the malaria endemic countries.
Antimalarial drug research and development are furthermore
affected by the pharmacokinetic and toxic profiles of potential
compounds (Na-Bangchang and Karbwang, 2009). The physico-
chemical properties of antimalarial drugs are important in the
sense that they can either help improve dose regimens and clinical
efficiency, or encumber them. The importance of favourable solu-
bility and absorption levels are paramount in increasing the plas-
ma bioavailability, ensuring positive therapeutical responses
(Ades, 2011) and are imperative in achieving the recommended
>95% cure rate (Nosten and White, 2007).
The pharmacokinetic limitations of artemisinin had led to the
development of synthetic and semi-synthetic derivatives to over-
come its poor water solubility, absorption and bioavailability
(Crespo-Ortiz and Wei, 2012). Since most antimalarial drugs are
administrated orally, water solubility is necessary to ensure
absorption and therapeutically viable plasma drug concentrations
(Kasim et al., 2004). A high degree of water solubility is associated
with hydrophilic compounds, allowing optimum absorption,
whereas lipophilic compounds have low water solubility, but a
high degree of cell permeation. Novel antimalarial compounds
should hence possess balanced hydrophilic/lipophilic properties
to permeate biological cell membranes and barriers to ensure pre-
dicable systemic drug concentrations (Avdeef, 2001).
The n-octanol/water partition coefficient (LogP) and aqueous
solubility level provide predictable solubility characteristics in
aqueous and organic solvents, with values between 1–5 being tar-
geted and 1–3 being ideal for maximum absorption (Caron et al.,
1999; Lipinski et al., 2001). The aqueous solubility level had been
derived from a predictive model, generated from a comprehensive
dataset by Cheng and Merz (2003) at 25 °C and pH 7 mimicking hu-
man blood, while the calculated human intestinal absorption mod-
el had been developed by incorporating various descriptors (Egan
et al., 2000). Combined exploration of the logP, aqueous solubility
level, absorption level and BBB penetration resulted in intermedi-
ates 2–6 exhibiting logP values in the targeted range, i.e. low sol-
ubility and good absorption, but high to medium blood brain
penetration. Differences in the chain length or addition of second-
ary and tertiary amines had no definitive effect on the calculated
drug-likeness properties of solubility and absorption, but increased
BBB penetration of these 9-aminoacridine.
N-11 as a result of steric hindrance of the methyl group on C-13
and impeded the accessibility of N-16 for nucleophilic substitution
to yield 5.
Even though C-13 was a racemate, no indication of carbon split-
ting was observed on the 13C NMR. The NMR data ensuing artemis-
inin–acridine hybrid 10, however, displayed the presence of
stereoisomers.
Furthermore, free base artemisinin-quinoline hybrids, previ-
ously synthesized by Lombard et al. (2011), using the same linkers,
were found unstable, with very short shelf-lives after purification
and isolation, causing them to thus convert into oxalate salts. No
such instability was experienced among the artemisinin–acridine
hybrids during this study, presumably due to the bulkiness and
rigidity of the acridine ring. These compounds were therefore sub-
jected to further investigations, namely physicochemical proper-
ties determinations and their in vitro biological evaluations as
free bases.
Characteristic signals, indicative of the 9-aminoacridine portion
of the artemisinin–acridine hybrids, represented the tricycle
aromatic scaffold of the 9-aminoacridines with six-aryl protons,
with integration signals ranging between d 8.26–7.07 ppm and
0
0
0
0
0
0
3
.91–3.98 ppm for protons H-1 , H-3 , H-4 , H-5 , H-7 , H-8 .
The protons for 7–11 on positions a, 4, 5, 7 and 8 were non-
equivalent, splitting into an AB pattern for H-a, H-4, H-5, H-7,
H-8 as follows: d 4.13–3.88 (a ), 3.77–3.55 (ab), 2.44–2.23 (4 ),
), 1.62–1.13 (5b), 1.53–0.78 (7
) and 1.53–1.00 (8b) ppm. The characteristic pro-
a
a
2
7
.07–1.91 (4b), 1.75–1.59 (5
a
a,
b), 1.94–1.53 (8
a
tons of H-b gave signals at d 3.03–2.73 ppm. The protons on posi-
tions H-a, H-b and H-5 underwent chemical shifts upfield, due to
the substitution of bromine with an electron donating amine.
The protons on H-b of compound 7 were adjacent to a secondary
amine and showed a chemical shift of 3.03 ppm (3.53 ppm for 1),
whereas 8 and 9, adjacent to tertiary amines, reported chemical
shifts of 2.79 ppm and 2.88 ppm, respectively.
For all hybrid compounds the proton on H-9 gave a characteris-
tic doublet of quartets of doublets signal at d 2.73–2.55 ppm, while
the protons of H-12 resulted in a singlet and H-10 in a doublet, giv-
ing signals at d 5.52–5.33 ppm and 4.86–4.77 ppm, respectively.
The most characteristic 13C NMR signals were C-3, C-10, C-12,
C-12a, C-a, C-b and C-9, at d 104.13–104.03, 102.59–101.97,
8
3
7.99–87.88, 81.09–80.92, 67.73–63.34, 58.25–48.49 and 30.82–
0.71 ppm, respectively. The hybrid carbons 3, 10, 12, 12a and 9
underwent no major chemical shifts with regards to 1. The resul-
tant chemical environment of C-a and C-b in the artemisinin–acri-
dine hybrid led to C-a shifting upfield but C-b shifting downfield.
Furthermore, with the exception of compound 10, in all other
hybrid compounds only one isomer (10b) was observed. A mixture
of isomers for 10 (10a and 10b) was confirmed by NMR data. This
substantiated the racemic nature of the 3-methylpiperazin linker.
In all of these structures, the theoretical number of alkyl groups
in the linker was in accordance with that of DEPT135. COSY, HSQC
and DEPT135, which further indisputably, indicated the presence
of artemisinin and acridine moieties. These observations were val-
idated by HRMS data.
The corresponding artemisinin–acridine hybrids 7–11, how-
ever, displayed high log P values, ranging across the limits of both
the targeted and ideal ranges, i.e. extreme to very low solubility
levels, very low-to-low absorption levels and undefined BBB pene-
tration levels. All of the hybrids therefore possessed unfavourable
drug-likeness properties. The undesirable logP values and solubil-
ity and absorption levels at physiological conditions could be
attributed to blocking of the polar primary/secondary amine
groups in the intermediates, causing a reduction in the hydrogen
bonding formation, and the addition of the lipophilic artemisinin
moiety.
The artemisinin–acridine hybrids had no predicted physico-
chemical/pharmacokinetic benefits over their precursor, 9-amino-
acridines and therefore were expected to be biologically less active.
However, since logP values, solubility and absorption levels, and
other factors as a whole (rather than a single property) affect the
4
.2. Physicochemical/pharmacokinetic properties
The stability of drugs in general plays a fundamental role in
ensuring their safety and potency, long after the manufacturing
date. The stability of antimalarial drugs is impeded by economic
and climatic factors in malaria endemic countries, where high heat
and humidity conditions prevail (Brown, 2010) and storage
facilities generally are inadequate. In this context, the hybrids
during this study were subjected to TGA and DSC in order to
establish whether they would be able to withstand the demanding

J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
25
extent of a compound’s ability to cross biological membranes and
absorption into the systemic circulation (Bigucci et al., 2008), both
intermediate and hybrids may have contracting biological activity
profiles.
protein (PfCRT) is involved in this efflux (Sanchez et al., 2003;
Howard et al., 2001; Wellems, 2002).
The majority of the synthesized intermediates and hybrids were
found active against the NF54 strain, with significant losses in activ-
ity against Dd2, which resulted in high RI values in the 2–19 range.
This is in accordance with the above previous finding. The hybrids
showing different level of activity against the Dd2 strain suggest a
difference in efflux level as a result of the difference in absorption
through the cellular membrane. These values above 1 underlined
the inability of the compounds to overcome resistance against
Dd2. No such activity loss was experienced with DHA though. The
combined analysis of the activity data of compounds 2–11 and
DHA hence suggested that the presence of the acridine moiety
had antagonized the activity of artemisinin, and that the hybrid
strategy might not be the best option when wishing to achieve syn-
ergistic activity from these antimalarial pharmacophores.
Furthermore, compound 7, the most active hybrid, showed a
six-fold higher activity loss against the CQR strain, compared to
10 (RI: 13.6 vs. 2.4), the second most active compound. However,
hybrid 7 displayed six times more activity than 10 against the
NF54 strain. Gametocytes are responsible for malaria transmission
from human to mosquitoes. Thus, a potentially effective and effi-
cient antimalarial drug candidate should have the ability to com-
pletely eliminate gametocytes from the parasite’s life cycle.
Therefore, despite the higher RI value, the hybrid 7 appears a more
promising antimalarial drug candidate than 10.
The previously used acridine based antimalarial drug, viz. mep-
acrine, had been abandoned due to reported toxicity. Hepatotoxic-
ity of acridine and neurotoxicity of DHA have also been reported
(Grace et al., 1997). An attempt to ascertain whether the synthe-
sized artemisinin–acridine hybrids could convey the same proper-
ties as the parent drugs, led in this study to screen all compounds
2–11 for their cytotoxicity and apoptotic activity against various
mammalian cell line types of both animal and human origins.
These included CHO (Chinese hamster ovarian, animal), HeLa (cer-
vical cancer, human), HepG2 (hepatocellular carcinoma, human)
and SH-SY5Y (neuroblastoma, human) cell lines. The latter cells
are often used as in vitro models of neuronal function and differen-
tiation and are therefore a viable cell line to investigate neurotoxic
properties of compounds.
4.3. In vitro biological evaluation
The compounds were screened against the P. falciparum strains
NF54 and Dd2. NF54 is a strain of P. falciparum from African origin
that is susceptible to all known antimalarial drugs, and it is often
the strain of choice to test for antigametocytocidal activity, pre-
sumably because of the highest gametocytemia obtained with this
strain in cell cultures (Roncales et al., 2012). Contrary, the Dd2
strain is collected in Indochina/Laos, it is CQ and mefloquine resis-
tant and delivers poor levels of gametocytemia in cultures (Ron-
cales et al., 2012).
Plasmodium gametocytes, responsible for malaria parasite
transmission from humans to mosquitoes, represent a crucial tar-
get for future new antimalarial drugs, in order to achieve malaria
eradication.
Artemisinin and acridine pharmacophores are known to be ac-
tive against malaria parasites (O’Neill et al., 2010; Valdés, 2011). In
vivo, most artemisinin derivatives are within a few minutes metab-
olized into DHA (Grace et al., 1997; Karbwang et al., 1997; Wood-
row et al., 2005). Arthemether and arteether, both ether derivatives
of artemisinin, stay intact long enough to exercise their own anti-
malarial effect before being metabolized into DHA, with all three
drugs showing comparable potencies (Woodrow et al., 2005). The
synthesized hybrids also being ethers, will likewise exert their
own antimalarial activities prior to metabolism into DHA, which
is unlikely to occur in vitro. The reported IC50 values were therefore
intrinsic to the hybrids, rather than due to any active metabolite.
The trend of ADMET predicted drug-likeable factors did not cor-
roborate that of antimalarial activity, since 9-aminoacridines 2–6,
with more favourable predicted properties, displayed less activity
than their corresponding hybrids. These results are therefore in
agreement with previous reports that properties, such as hydro-
gen-bonding, molecular size and shape, polarity, flexibility and
the charge/ionization of a compound/drug molecule as a whole
(
rather than a single property) affect its absorption through mem-
branes (Bigucci et al., 2008).
Compounds 2–11 had primary, secondary and/or tertiary amine
a
groups with calculated pK (ACD/ChemSketch, 2000) values being
in the 8–10 range. Compound 4 and its corresponding hybrid 9
were the least active compounds against both strains. They possess
a tertiary amine with calculated pK
the most active compound, has a secondary amine with pK
Analysis of the IC50 values revealed all compounds to be more
toxic to the mammalian cell line of animal origin (lower IC50
against CHO), than to those of human origin. The 9-aminoacridines
2–6 were more cytotoxic to CHO and were found to be less selec-
tive in their antimalarial actions against the parasitic cells (lower SI
values), than were hybrids 7–11, DHA, AS and CQ. This suggested
that the presence of the artemisinin pharmacophore had contrib-
uted in reducing the cytotoxicity of the acridine moiety against
the CHO cells in the hybrids.
a
value of 9.37, while hybrid 7,
value
a
of 8.45. Hybrid 7 is thus less basic than 9, and yet more active. One
of the mechanisms suggested to could explain the antimalarial
activity of basic drugs, such as CQ (pK
a
10.2), is ion trapping, which
The majority of hybrids appeared less cytotoxic than DHA, but
displayed more toxicity than their precursor 9-aminoacridines
against the HeLa cells, indicating that covalently linking acridine
and artemisinin pharmacophores had led to a reduction in the cer-
vical anticancer activity of the latter. Antagonism of anticaner
activity, using the hybrid strategy, therefore occurred.
All synthesized compounds (except 5) were found to be as
apoptotic as DHA, AS and CQ against the HepG2 and SH-SY5Y cells.
This indicated that both acridine and artemisinin pharmacophores
had transferred their hepatotoxicity and neurotoxicity characteris-
tics over into the hybrids although these properties were less ex-
pressed than the cytotoxicity and anticancer activity against CHO
and HeLa cells, respectively.
relies on the protonation of weakly basic amino groups. Indeed, in
the normal in vivo environment at pH 7.4, amines tend to be neu-
tral and unprotonated, making it easy for them to cross lipophillic
membranes. Upon reaching the acidic food vacuole of the malaria
parasite, where the pH value is in the 4.5–5.5 range, these amines
become protonated, making them less membrane permeable. The
net effect of this mechanism is the increased accumulation of these
amines in the digestive vacuole, giving rise to an increase in activ-
ity (Egan, 2003; Hindley et al., 2002). This observation may thus
imply that, unlike with CQ, ion trapping may not play a significant
role in the activities of these amino group containing compounds.
The mechanism of resistance of the Dd2 strain is still not com-
pletely elucidated. However, it has been shown that CQ-resistant
including Dd2 parasites expel much more rapidly the CQ from
the red blood cell (RBC) than CQ-sensitive parasites, and many
On account of activity, resistance index, selectivity and cytotox-
icity/apoptosis, hybrid 7 stood as a possible future antimalarial
drug candidate while hybrid 10 may stand for possible exploration
as an anticancer drug.
observations indicated that
a P. falciparum trans-membrane

2
6
J.P. Joubert et al. / European Journal of Pharmaceutical Sciences 56 (2014) 16–27
Avdeef, A., 2001. Physicochemical profiling: solubility, permeability and charge
5
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a
hybrids were synthesized in one- and three step synthetic routes,
respectively. All structures were confirmed by means of NMR,
HRMS and IR techniques. The 9-aminoacridines displayed better
ADMET predicted drug-likeness than their corresponding hybrids.
However, in screens alongside DHA, AS and CQ, although both
compound types were active against the NF54 and Dd2 strains of
P. falciparum, the hybrids possessed higher activity than their pre-
cursor 9-aminoacridines. No resistance against the CQR strain Dd2
was overcome during this study, since all synthesized compounds
had lost their activities against that strain in comparison with
NF54. The majority of hybrids showed more antimalarial potency
than CQ, but less than DHA and AS against both strains. They were
more selective in their antiparasitic actions. The hybrids displayed
better anticancer activity than the aminoacridines and CQ, but less
than both DHA and AS. All synthesized and reference drugs pos-
sessed comparable hepatotoxicity and neurotoxicity.
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R., Bray, P.G., Owen, A., O’Neill, P.M., Ward, S.A., 2008. Acridinediones: selective
and potent inhibitors of the malaria parasite mitochondrial bc1 complex. Mol.
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Hybrid 7, containing ehtylenediamine linker, was the most
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It had 7-fold higher antimalarial potency than of CQ against both
the NF54 and Dd2 strains, with high selective action towards the
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potent than CQ and melphalan, respectively. This compound may
therefore stand as drug candidate for further investigation in
search for new anti-cervical cancer drugs, rather than malaria.
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Any opinions, findings and conclusions or recommendations ex-
pressed in this material are those of the author(s) and therefore the
NRF does not accept any liability in regard thereto.
Acknowledgements
This work was based upon research financially supported by the
National Research Foundation (NRF) and the North-West
University. The authors thank Dr. J.H.L. Jordaan for MS analysis,
Mr. A. Joubert for NMR analysis, Prof. W. Liebenberg for TGA and
DSC and Prof. M. van de Venter and Ms. M.C. Pruissen for cytotox-
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejps.2014.01.014.
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