Synthesis, in vitro antiplasmodial activity and cytotoxicity of a series of artemisinin-triazine hybrids and hybrid-dimers

Article abstract of DOI:10.1016/j.ejmech.2014.01.040

A series of artemisinin-triazine hybrids and hybrid-dimers were synthesized and their in vitro antimalarial activity against the chloroquine sensitive (CQS), the gametocytocidal (NF54) and the choroquine resistant (CQR) Dd2 strains of Plasmodium falciparu

Full text of DOI:10.1016/j.ejmech.2014.01.040

European Journal of Medicinal Chemistry 76 (2014) 470e481
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro antiplasmodial activity and cytotoxicity of a series of
artemisininetriazine hybrids and hybrid-dimers
,
Theunis T. Cloete a, Carmen de Kock b, Peter J. Smith b, David D. N’Da a
*
a Center of Excellence for Pharmaceutical Sciences, North-West University, Potchefstroom 2520, South Africa
b Department of Pharmacology, University of Cape Town, Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of artemisininetriazine hybrids and hybrid-dimers were synthesized and their in vitro anti-
malarial activity against the chloroquine sensitive (CQS), the gametocytocidal (NF54) and the choroquine
resistant (CQR) Dd2 strains of Plasmodium falciparum determined, while their toxicity against CHO cells
were also established. These compounds were prepared by linking artemisinin and triazine pharmaco-
phores through nucleophilic substitution, using conventional and microwave assisted methods. These
hybrids and hybrid-dimers were all found to be active against all three Plasmodium strains, with the p-
anisidino-substituted triazine hybrid-dimer 22 being the most active of all. It showed good anti-
gametocytocidal activity against the NF54 strain, with a 50% inhibitory concentration value in the
nanomolar range, while having a potency comparable to that of artesunate against the Dd2 strain. This
hybrid-dimer further demonstrated selective toxicity towards the parasitic cells. This dimer hence
showed the necessary potential as candidate for further investigation in the search for malaria trans-
mission blocking drugs so desperately needed.
Received 11 September 2013
Received in revised form
15 January 2014
Accepted 18 January 2014
Available online 18 February 2014
Keywords:
Malaria
Artemisinin
Triazine
Hybrid
Hybrid-dimer
Microwave
Cytotoxicity
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
uncomplicated P. falciparum malaria in most of the areas afflicted by
the disease [3].
Malaria has been a major parasitic disease of man since antiq-
uity and continues to pose an enormous health risk today. Despite
our increased knowledge of this disease, currently around 6.6
billion people are at risk of contracting malaria, with an estimated
655,000 people dying from this disease, annually [1]. The five
species of the Plasmodium (P) parasite that can infect humans are
P. vivax, P. ovale, P. malariae, P. knowlesi and P. falciparum, with the
latter accounting for 91% of all reported cases [1].
Currently used antimalarial drugs fall into one of seven classes,
viz. the 4-aminoquinolines, the arylaminoalcohols, the 8-
aminoquinolines, the antifolates, the hydroxynaphthoquinones,
certain antibiotics (e.g. doxycyclin and clindamycin) and the arte-
misinin class of compounds, comprising artemisinin (ART), dihy-
droartemisinin (DHA), artemether (ArM) and artesunate (AS) [2].
Unfortunately, due to the parasites’ remarkable abilities to acquire
resistance against most of these drugs, only the artemisinin class of
compounds remains a viable first line treatment option for
Although the mechanism of action of the artemisinins is still
under investigation, these compounds have proven to be invaluable
in the fight against malaria, mainly because of their extremely fast
and potent antimalarial action, the lack of cross resistance with
other antimalarial drugs and their actions against the gametocyte
forms of the parasite [4]. Despite these advantages, known disad-
vantages include their poor water (ART, DHA and ArM) and oil
solubilities (AS), biological short half-lives and high parasite
recrudescence after treatment [5].
In an effort to enhance the efficiency of antimalarial drugs and
to counter the spread of resistance, the artemisinin class of com-
pounds is now combined with other antimalarials of a distinct
different class to form artemisinin based combination therapies
(ACTs). These ACTs entail combining a fast acting, highly effective
artemisinin derivative, e.g. AS, with a longer acting partner drug, e.g.
mefloquine, to form a combination that is highly effective in
destroying the parasite and delaying the onset of resistance [6]. The
World Health Organization (WHO) now recommends using an ACT
as the first line of treatment when uncomplicated P. falciparum
malaria is diagnosed [7]. Unfortunately, even with these pre-
cautions in place, resistance against AS has already been observed
in patients from Pailin in Western Cambodia [8]. With the
* Corresponding author. Tel.: þ27 18 299 2256; fax: þ27 18 299 4243.
E-mail address: david.nda@nwu.ac.za (D.D. N’Da).
http://dx.doi.org/10.1016/j.ejmech.2014.01.040
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
471
artemisinin class of compounds being the foundation on which
treatment regimens are currently built, the loss of this important
class of drugs would have dire consequences.
15
6
5
4
5a
7
8
2
O
O
3
14
Another approach to counter the development of resistant
parasite strains is the synthesis of hybrid drug molecules. A hybrid
drug molecule is defined as a chemical entity with more than one
structural domain, each having its own mechanism of action. Aside
from the obvious advantages of these types of molecules, such as
the dual mechanism of action, other advantages may include more
predictable pharmacokinetic properties and the ability of one en-
tity to impart favourable qualities onto the other, e.g. increased
solubility [9].
12a
1
O
8a
13
12
9
O
10
16
11
b
O
Br
a
1
Fig. 2. Structure of 2-bromo-(10b-dihydroartemisinoxy)ethane 1.
In this study, hybrid molecules, consisting of artemisinin and
triazine moieties were proposed as possible structures to address
the shortcomings of the artemisinin drugs. The 1,3,5-triazine
moiety is a common structure found in antifolate drugs (Fig. 1).
Substituted, this moiety has been found to have relatively good
activity against malaria parasites and enzymes, both on its own and
attached to other pharmacophores [10e14].
compound had previously been determined in our research group
by X-ray analysis [21]. This intermediate and the final hybrids and
hybrid-dimers were all in the 10b form, as confirmed by the small
coupling constant, J ¼ 3.4 Hz, between the H-10 and H-9 atoms
[22].
The trichloro-substituted triazine, cyanuric chloride (CyCl) 2,
was then reacted with the relevant amine to produce the mono-
substituted compounds 3 and 4, with yields of 68% and 86%,
respectively. Subsequently, either 2, 3 or 4 was reacted with
different amines, generating the di-substituted compounds, which
upon reaction with ethylenediamine (EDA) resulted in the primary
amine functionalised tri-substituted triazine intermediates 5e13,
with yields ranging between 49 and 90% (Scheme 1). The infrared
(IR) spectra of these intermediates commonly showed absorption
bands in the 3360e3250 cmꢀ1 region, evident of the presence of
both NH2 and NH groups (Supplementary information 1).
Finally, each intermediate was reacted with compound 1
through nucleophilic substitution, using microwave radiation to
render a free base hybrid (14, 15, 16, 18, 20, 23, and 25) and/or a
hybrid-dimer (17, 19, 21, 22, 24, and 26) (Scheme 2), which for
stability reasons were all converted into oxalate salts with yields in
the 3e28% range. The IR spectra of the hybrids and hybrid-dimers
displayed a shoulder in the 3460e3200 cmꢀ1 region, assignable
to vibrations of the secondary amine (NH) groups. A sharp band,
corresponding with the vibrations of an OeO bond, was also pre-
sent in the 2950e2830 cmꢀ1 range, confirming the presence of the
The folate biosynthetic pathway in the parasite consists of a
process in which six enzymes are involved in the conversion of
guanosine triphosphate (GTP) into tetrahydrofolic acid (THF),
which is of vital importance in the synthesis of purines, thymidine
and some amino acids [15]. The folate antagonists, also known as
antifolates, can be divided into two classes, viz. type 1 and type 2
antifolates. The type 1 antifolates include the sulfonamides and
sulfones that mimic p-aminobenzoic acid (PABA), competing for
the active site on dihydropteroate synthase (DHPS), consequently
inhibiting the formation of dihydropteroate. The type 2 antifolates
comprise pyrimethamine (PM), the quinazolines, biguanides and
triazine metabolites. These antifolates inhibit dihydrofolate
reductase (DHFR), preventing the nicotinamide adenine dinucleo-
tide phosphate (NADPH) dependent reduction of dihydrofolate
(DHF) into THF [16]. Not long after the introduction of these anti-
malarials onto the market, worldwide resistance against this class
spread rapidly, making them obsolete in most areas afflicted by
malaria [17]. The combination of both types 1 and 2 antifolates, e.g.
sulfadoxine and pyrimethamine (SP), had proven a synergistic
antimalarial effect, but resistance against these combinations also
developed quickly, preventing them from being used on a signifi-
cant scale against malaria [18].
artemisinin moiety in these structures.
A sharp band at
w1720 cmꢀ1, corresponding with the vibrations of the carboxylate
CeO, also confirmed the presence of an oxalate group
(Supplementary information 2).
The compounds described in this article contain the artemisinin
and 1,3,5-triazine pharmacophores and were designed for activity
against both the sensitive and resistant strains of P. falciparum.
Reported herein are their synthesis, in vitro antimalarial activity
against various strains of P. falciparum and their cytotoxicity.
2.2. Biological activity
The antimalarial activities and cytotoxicities of the synthesized
compounds are summarised in Table 1. All hybrids and hybrid-
dimers were found to be less potent than both DHA and AS
against the NF54 strain, although they were up to six thousand-fold
more active than PM. Compared to chloroquine (CQ), only dimer 22
was at least twice as potent, while the remaining compounds were
either less, or equally potent. No synthesized compound showed
better activity than the equimolar combination of DHA and PM.
With regards to the Dd2 strain, the results were quite different.
All of the hybrids and hybrid-dimers were more potent than both
CQ and PM. More significantly, dimers 22, 24 and 26 displayed
potencies comparable to those of DHA, AS and the DHA:PM mixture
(M). Furthermore, where both the hybrids and counterpart hybrid-
dimers were isolated and screened, the dimer proved to be more
active than its corresponding hybrid, irrespective of the strain being
tested against.
2. Results
2.1. Chemistry
Condensation of dihydroartemisinin with 2-bromoethanol
produced the DHAeethyl bromide derivative 1 in good yield, us-
ing a reported method (Fig. 2) [19,20]. The stereochemistry of this
R1
Cl
NH2
N
4
3
5
N
R1, R2, R3 = Amines
N
N
2
R2
R3
N
1
6
N
H2N
Cycloguanil
2, 4, 6-Trisubstituted-1, 3, 5-trazine moiety
The resistance index (RI) value of most hybrids and hybrid-
dimers was found to be below 2, an indication of a moderate loss
Fig. 1. Structures of cycloguanil and the 2,4,6-tri-substituted-1,3,5-triazine moiety.

472
T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
1'
NH2
HN
Cl
N
2'
3'
N
N
N
N
4' or 5'
R2
(c)
4'
R1
(b)
N
Cl
R1
R2
5-13
N
N
Cl
N
Cl
8'
10'
7'
(a)
2
9'
6'
Cl
HN
HN
O
3:
N
N
R =
Cl
N
R
4:
9'
6'
7' 8'
3-4
Interm
R1 / R2
5' 6'
Interm
R1 / R2
6' 7'
9'
8'
5'
5
10
11
12
13
7'
N
HN
O
6' 7'
6'
5'
5'
8'
6
7
8
HN
N
O
7' 8'
11' 12'
10'
HN
7' 8'
10'
12'
14'
11'
6'
13'
9'
6'
O
HN
HN
O
HN
9'
N
13'
/
/
7' 8'
6'
7' 8'
10'
N
10'
11'
O
11'
12'
O
6'
6'
9'
O
HN
9'
N
/
/
7' 8'
10'
9'
HN
O
/
14'
13'
9
12'
11'
HN
N
Scheme 1. Synthesis of intermediates 5e13. Reagents and conditions: (a) appropriate amine, acetone, Na2CO3, 6 h, 0 ꢁC; (b) appropriate amine, acetone, Na2CO3, 1e3 h, 0 ꢁC; (c) 3e
24 h, r.t.; (d) EDA, 3e24 h, 50e70 ꢁC.
of activity against the CQR strain Dd2, compared to the CQS. This
loss was, however, more significant for CQ, M and DHA with RI
values of 22,12.4 and 9.7, respectively. The synthesized compounds,
together with the equimolar DHA:PM combination proved to be
selectively more toxic to the parasitic cells in the presence of
mammalian cells (SI > 50), with hybrid 19 (SI ¼ 25) and hybrid-
dimer 20 (SI ¼ 34) being the least selective, while PM (SI ¼ 8)
was found to be noticeably toxic to the mammalian cells, but
sparing the parasitic ones.
power settings were 50 ꢁC and 60 W. The reaction was carried out
by increasing the electrical power, until either the temperature or
the hold time was reached. During each cycle, cooled air was
pumped into the reactor, keeping the temperature as low as
possible. The reaction vessel was then cooled to ꢀ8 ꢁC in prep-
aration for the next burst. These bursts were repeated 8 to 13
times, until thin layer chromatography (TLC) indicated comple-
tion of the reaction.
The peroxide bridge of artemisinin is reported to be prone to
facile hydrolysis under basic or acidic conditions and can also
be expected to be heat sensitive [23]. However, in this study,
the spectroscopic data revealed no deviations from the ex-
pected values, confirming that this bridge had been preserved
during microwave radiation, hence supporting previous findings
[20].
3. Discussion
3.1. Chemistry
The artemisinin-triazine hybrids and hybrid-dimers were
synthesized using microwave radiation. The microwave reaction
was carried out in a 250 mL open vessel round bottom flask,
connected to a Liebig cooler, with a ramp and hold time of 1 and
4 min, respectively. The maximum allowed temperature and
In intermediates 5e13, the secondary amine (NH) of the EDA
linker was less nucleophilic as a result of the resonance of the
already di-substituted triazine ring, making it unavailable for sub-
stitution reactions. Subsequently, only the terminal primary amine

T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
473
6
5
2
O
4
5a
O
7
3
O
14
1
12a
12
10
O
8
O
O
13
8a
9
O
S:
O
11
16
1'
b
(a)
O
O
NH
2'
N
R1
4' or 5'
Br
N
R3
O
R3 =
a
1
5 - 13
N
N
CH2
O
4'
R2
H
14-26
R1/R2 of
R1/R2 of
Hybrid/Dimer.
R3 Hybrid/Dimer.
R3
Intermediate
Intermediate
14
15
16
17
18
19
20
5
6
7
7
8
8
9
H
H
H
S
H
S
H
21
22
23
24
25
26
9
S
S
H
S
H
S
10
11
11
12
13
Scheme 2. Synthesis of artemisininetriazine hybrids and hybrid-dimers 14e26. Reagents and conditions: (a) DMF, K2CO3, microwave (60 W, 50 ꢁC, 4 min); (b) 0 ꢁC.
was involved in the substitution reactions, leading to the formation
of the hybrids and/or hybrid-dimers. For stability and solubility
reasons, the free base target compounds were converted into ox-
alate salts, with the unfortunate disadvantage of considerably
reducing the overall yields.
Compound structures were confirmed using IR, high resolution
mass spectrometry (HRMS), and both 1D (1H and 13C nuclear
magnetic resonance) and 2D (correlation spectroscopy (COSY) and
heteronuclear single quantum coherence (HSQC)) techniques, due
to their complex structures.
Table 1
IC50-values of compounds tested in vitro for antiplasmodial activity against NF54 and Dd2 strains of Plasmodium falciparum and their cytotoxicity against CHO cells. Cells were
incubated with compounds at various concentrations for 48 h; antimalarial activity and cytotoxicity were determined using parasite lactate dehydrogenase (pLDH) and MTT-
assays, respectively.
Log Pc
pKa
Activity, IC50 [nM (ng/ml) ꢂ SD]a
Resistance index
RId
Cytotoxicity, IC50
CHO
[m
M (
m
g/ml) ꢂ SD]b
Selectivity index
SIe
c
Comp
NF54
Dd2
14
15
6.7
3.6
3.8
5.8
2.2
4.3
4.9
8.6
20.6 (12.7) ꢂ 3.4
19.3 (12.0) ꢂ 7.9
28.0 (18.3) ꢂ11.5
11.6 (11.2) ꢂ 2.7
38.5 (25.3) ꢂ 1.1
25.8 (24.9) ꢂ 2.4
39.1 (27.9) ꢂ 5.1
47.5 (29.3) ꢂ 1.5
85.0 (52.7) ꢂ 22.9
46.3 (30.3) ꢂ 0.6
13.6 (13.1) ꢂ 2.1
57.4 (37.8) ꢂ 2.1
35.9 (34.7) ꢂ 0.8
74.7 (53.3) ꢂ 2.9
2.3
4.4
1.7
1.2
1.5
1.4
1.9
3.1 (1.9) ꢂ 0.2
3.1 (1.9) ꢂ 0.1
3.0 (2.0) ꢂ 0.2
17.0 (16.4) ꢂ 9.5
3.1 (2.0) ꢂ 0.1
0.9 (0.6) ꢂ 0.0
0.9 (1.0) ꢂ 0.2
150
160
105
1468
81
8.6
8.6
6.4
8.6
6.9
8.6
10.2
10.2
6.9
8.5
6.9
8.5
6.9
6.8
16
17m
18
19m
20
35
23
21m
22m
23
7.0
6.8
4.8
6.9
4.8
4.3
2.9
2.5
31.8 (32.5) ꢂ 5.7
5.5 (5.5) ꢂ 0.4
33.1 (33.9) ꢂ 2.7
10.6 (10.6) ꢂ 1.2
59.1 (37.4) ꢂ 4.3
10.3 (9.7) ꢂ 1.9
28.8 (19.1) ꢂ 1.7
10.25 (9.6) ꢂ 1.7
60,700 (15.1) ꢂ 2900
8.8 (2.5) ꢂ 1.5
1.0
1.9
2.2
0.8
1.5
1.3
1.8
9.7
12.4
22
> (100)
> (100)
16.0 (10.1) ꢂ 3.2
0.7 (0.6) ꢂ 0.3
> (100)
>3077
>18,182
588
27.2 (17.2) ꢂ 3.3
12.9 (12.2) ꢂ 1.9
19.8 (13.1) ꢂ 1.5
7.9 (7.4) ꢂ 0.5
24m
25
54
>5051
>13,514
8
161,539
44,225
e
26m
PMf
DHAg
Mh
> (100)
33,000 (8200) ꢂ 3200
0.9 (0.26) ꢂ 0.2
0.71 ꢂ 0.1 ng/mL
13.8 (4.4) ꢂ 4.7
271.0 (67.4) ꢂ 23.7
147.7 (42.0) ꢂ 50.0
8.9 ꢂ 2.2 ng/mL
300.0 (95.8) ꢂ 2.8
31.4 ꢂ 1.6
mg/mL
CQi
4.69
7.5
10.4
ndl
ASj
<5.2
8.1 (3.1) ꢂ 0.5
ndl
e
EMk
0.1 (0.1) ꢂ 0.02
a
Minimum concentration of compound inducing 50% parasitic cells inhibition, data represents the mean of three independent experiments ꢂ S.D. (standard deviation).
b
c
Minimum concentration of compound selectively inducing 50% of mammalian CHO (Chinese Hamster Ovarian) cells.
Calculated values using ACD Chem/Sketch [31].
Resistance index (RI) ¼ IC50 Dd2/IC50 NF54.
Selectivity index (SI) ¼ IC50 CHO/IC50 NF54.
Pyrimethamine (PM).
d
e
f
g
h
i
Dihydroartemisinin (DHA).
Equimolar DHA:PM mixture (M).
Chloroquine (CQ) was tested as diphosphate salt.
Artesunate (AS).
Emetine (EM) was used as the reference drug in the cytotoxicity study.
Not determined (nd).
j
k
l
m
Hybrid-dimer.

474
T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
The 1H NMR signals of the EDA linker protons were at
3.60 for H-10 and H-20, respectively. The H-10 signal is usually in
close proximity to the H-b signal at 3.25, due to integrating the
four protons together. The COSY was paramount to the correct
allocation of these two signals, as H-b couples with H-a and H-a
whilst H-10 couples with H-20. The 13C NMR signals of C-10 and C-b
showed at 47. The HSQC spectra assisted in confirming the pres-
ence of these two signals. The piperidine moiety in compounds 14,
16 and 17 had distinctive 1H NMR signals at
3.72 and 1.71, arising
from the alicyclic protons, while the alicyclic carbon atoms dis-
played signals at 44.29 and 26.20. The morpholine moiety in
hybrids 15 and 18 and dimers 19 and 26 had 1H NMR signals at
3.71e3.41, correlating with the 13C NMR signals at
66.04 and
d
3.10 and
others [10e14,27,28]. In vivo, most artemisinin derivatives are
metabolized into DHA within a few minutes [28e30]. Arthemether
and arteether, both ether derivatives of artemisinin, stay intact long
enough to exercise their own antimalarial effects, before being
metabolized into DHA, with all three drugs showing comparable
potencies [28]. The synthesized hybrids and hybrid-dimers, also
ethers, likewise exert their own antimalarial activities prior to
metabolism into DHA.
d
a
b,
d
d
The majority of the hybrids and hybrid-dimers either had sec-
ondary and/or tertiary amines, with calculated pKa values, using
ACD Chem/Sketch [31], varying in the 6e10 range. The pKa values of
each synthesized compound, as reported in Table 1, represented
that of the nitrogen linking EDA to the artemisinin pharmacophore
via the C-b carbon. In hybrids 14, 15, 16, 18, 20, 23 and 25, this ni-
trogen was basic and therefore had a pKa value of w8. Such
property was lost in dimers 17, 19, 21, 22, 24 and 26, due to the
attachment of the second artemisinin moiety, which resulted in a
pKa value of w6. Hybrid 20 and its dimer 21 further had the 2-
(isopropylamino)ethylamino substituent on the triazine ring. The
tertiary nitrogen of this group was very basic with a pKa value
around 10, which was comparable to the calculated pKa value of
10.4 (side chain tertiary amine) of CQ. Compound 20 was the least
active against the NF54 strain and the second to least active against
the Dd2 strain. One of the suggested mechanisms of action of CQ is
ion trapping, which relies on the protonation of weakly basic amino
groups. Indeed, in the normal in vivo environment at pH 7.4, amines
tend to be neutral and unprotonated, making it easy for them to
cross lipophilic membranes. Upon reaching the acidic food vacuole
of the malaria parasite, where the pH value is in the 4.5e5.5 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 activity [32,33]. This observation may thus imply
that, unlike with CQ, ion trapping may not play a significant role in
the activities of these amino groups containing compounds. The
high activities of dimers 22 and 26, despite having secondary amine
groups (NH linked to C-b carbon), with low pKa values in the 6e7
range, further supported such assumption.
d
d
d
43.18 in the HSQC spectrum. The p-anisidine in hybrids and hybrid-
dimers 16e22 and 25 showed the characteristic methoxy signal at
d
3.72 and the two aromatic doublets at
NMR spectrum. The methoxy carbon signal was visible at
the 13C NMR spectrum, whilst the aromatic carbon atoms resonated
at 153.61, 129.89, 123.16 and 113.70. The aniline moiety of com-
pounds 23e26 displayed signals in the aromatic region at 7.48,
120.05, 121.59,
d
7.45 and 6.75 in the 1H
d
55.45 in
d
d
7.20 and 7.01 in the 1H NMR spectrum and at
d
128.53 and 140.11 in 13C NMR. The four methyl groups of the 2-
(diisopropylamino)ethylamine moiety in compounds 20 and 21
gave a characteristic 1H NMR signal at
d
1.06 ppm, corresponding
20.24, while the signals resulting from
the two methine groups gave an 1H NMR signal at 2.68 and a 13
NMR signal at 44.19.
with the 13C NMR signal at
d
d
C
d
Conversion of the secondary amine NH of the linker (EDA in the
hybrids) to a tertiary amine in geminal dimers 17, 19, 21, 22, 24 and
13
26 resulted in the C NMR signals of both C-b and C-10 carbon
atoms to shift from roughly
pertaining to C-20 remained at
associated with H-10 and H-b had shifted from
d
47 ppme53 ppm, whilst the signal
37 ppm. Similarly, the signals
3.10 ppme
d
d
3.50 ppm, whilst H-20 remained at the same position more or less,
confirming that indeed the second coupling of compound 1 had
occurred on the nitrogen amine group between C-b and C-10 car-
bons to form the dimers.
3.2. Biological activity
Another plausible explanation for the low activity of hybrid 20
in relation to the high pKa value (10.2) of its tertiary amine could be
that this compound (unlike dimers 22 and 26) was protonated at
physiological pH 7.4. As it had therefore crossed the biomembranes’
phospholipid layers with difficulty, it reached the parasites’ diges-
tive system in lesser concentrations.
All synthesized compounds were screened against the P. fal-
ciparum NF54 and Dd2 strains. The NF54 strain from African origin
is susceptible to all known antimalarial drugs and is often the strain
of choice for testing antigametocytocidal activity, presumably
because of the highest gametocytemia level obtained with this
strain in cell cultures [24]. The Dd2 strain, collected from Indo-
china/Laos, is chloroquine and mefloquine resistant and delivers a
poor level of gametocytemia in cultures [24]. Plasmodium game-
tocytes, responsible for the malaria parasite transmission from
humans to mosquitoes, represent an important target for new
antimalarial drugs in an attempt to achieve malaria eradication.
Gametocytes undergo complex development that is characterized
by five morphologically distinct stages (IeV). The earliest devel-
opmental stages (I and II) of gametocytes are susceptible to most
antimalarial drugs [25]. DHA, the active metabolite of artemisinins
is highly active against stages IeIII gametocytes, but has incomplete
activity against stages IV and V gametocytes [25,26].
It is known that in order for the uptake of a drug molecule to
occur through a membrane, that drug must possess limiting
aqueous solubility and be neither too lipophilic, nor too hydro-
philic. Lipophilic drugs show poor aqueous solubility and tend to be
taken up in fatty globules in the intestine. Once they reach the
blood stream, they may be absorbed into tissue where their slow
release exacerbates toxicity, such as neurotoxicity. Contrary, hy-
drophilic drugs may be excreted directly by the kidneys, or pene-
trate a cell membrane and become entrapped in intracellular
aqueous media. An ideal drug must hence possess balanced lipo-
philic/hydrophilic properties in order to both permeate biological
membranes and be taken up in the systemic circulation. The lipid/
water partition coefficient (Log P) offers a good measure of this
balance, with values between 1 and 5 being targeted, and 1e3
being ideal for better drug absorption through biological mem-
branes [34,35]. Hybrid 18, with calculated Log P value of 2.2, fitting
nicely into the ideal range, happened to be one of the least active
compounds during this study. On the contrary, hybrid-dimer 22,
with a Log P value (6.9) beyond that of both the targeted and ideal
ranges, was found to be the most active against strain NF54 and the
third most active against Dd2. These observations corroborated
Cycloguanil, which contains a triazine pharmacophore, was
unavailable for screening for comparison to the synthesized com-
pounds. A pyrimidine derivative, viz. pyrimethamine, which shares
the same biological mechanism of action (type 2 antifolates), was
hence used as reference instead and screened alongside the syn-
thesized compounds.
Artemisinin and triazine pharmacophores are known for their
activity against malaria, either on their own, or in combination with

T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
475
earlier findings that properties, such as hydrogen bonding, molec-
ular size and shape, polarity, flexibility and the charge/ionization of
a compound/drug molecule as a whole (rather than a single prop-
erty), affect its absorption through membranes [36].
Compounds 17, 19, 21 and 24 are the hybrid-dimers of mono-
meric hybrids 16, 18, 20 and 23, respectively. Each dimer displayed
slightly higher activity than its monomer counterpart against both
the NF54 and Dd2 strains. However, this increase, which may
presumably be as a result of more artemisinin reaching the site of
action, appeared moderately more pronounced against the Dd2
than the NF54 strain.
activities were purely dependent on the artemisinin pharmaco-
phore, or whether their folate biosynthetic pathways would also be
implicated, or both. A dihydrofolate reductase (DHFR) assay would
be required to ascertain their antifolate properties. These com-
pounds also showed good potential as possible candidates for un-
dergoing pharmacokinetic studies in order to establish whether
they possess half-lives longer than those of the current, clinically
available artemisinin derivatives that are known to have short
biological half-lives. Furthermore, a clear identification of the
gametocyte stage of action may be useful in designing new com-
pounds, using the hybrid strategy.
Furthermore, the RI values of most hybrids and hybrid-dimers
were in the 1e4 range, which may have been indicative of the
CQR strain being on the verge of developing resistance against
them. However, this strain was found to be more resistant towards
both DHA (RI ¼ 9) and CQ (RI ¼ 22). As the main pharmacological
shortcoming of most currently used antimalarial drugs is known as
being their decreased activity against the resistant strains of
P. falciparum, the favourable outcomes in this regard, as demon-
strated by the compounds investigated during this study, possibly
made them good candidates for further development and investi-
gation as possible future antimalarials.
Most of the synthesized compounds, especially hybrid-dimers
22 and 26, demonstrated a high level of selectivity towards the
parasitic cells. They were also found to be the most anti-
gametocytocidal of all hybrids and hybrid-dimers and had activities
comparable to those of dihydroartemisinin, artesunate and the
fixed DHA-PM (1:1) combination against the resistant CQ strain.
These two dimers hence showed good potential as transmission
blocking compounds, suitable for further screening/investigation.
None of the hybrid and hybrid-dimers investigated during this
study, however, displayed activities higher than AS or ArM against
the NF54 strain, possibly suggesting that the triazine moiety had
antagonized the antigametocytes action of artemisinin. A better
partner drug would therefore be required in order to achieve
antigametocytocidal synergism, using the artemisinin-based
hybrid strategy. The choice of such a partner drug was further
rendered necessary, due to the most active synthesized compounds
in this study not demonstrating significant advantages over the
equimolar combination of dihydroartemisininepyrimethamine.
5. Materials and methods
5.1. Materials
Dihydroartemisinin (mixture of 10
a and 10b epimers) was
purchased from Changzhou Kaixuan Chemical Co (Chunjiang,
China). Boron trifluoride diethyl etherate (BF3$Et2O), 2-
bromoethanol, N,N-dimethylformamide (DMF), morpholine,
piperidine, aniline, cyanuric chloride (CyCl), tetrahydrofuran (THF),
ethylenediamine (EDA), 2-(diisopropylamino)ethylamine and
ammonia (NH4OH) were purchased from SigmaeAldrich (Johan-
nesburg, South Africa). p-Anisidine was purchased from Merck
Schuchardt (Hohenbrunn, Germany). Oxalic acid was purchased
from BDH Laboratory Reagents (Yorkshire, England). Sodium car-
bonate and potassium carbonate were purchased from Unilab
(Krugersdorp, South Africa). All chemicals, reagents and solvents,
viz. ethyl acetate (EtOAc), petroleum ether (PE), dichloromethane
(DCM) and methanol (MeOH) were of analytical grade and were
used without further purification.
5.2. General chemical and analytical procedures
The 1H and 13C NMR spectra were recorded on a Bruker
AvanceÔ III 600 spectrometer at frequencies of 600.17 and
150.913 MHz, respectively, in deuterated dimethyl sulfoxide
(DMSO-d6), or deuterated chloroform (CDCl3-d). Chemical shifts
were reported in parts per million
d (ppm) with the residual pro-
tons 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), triplet
(t), triplet of doublets (td), quartet (q), multiplet (m).
4. Conclusion
During this study, a series of hybrids and hybrid-dimers were
successfully synthesized by linking artemisinin and triazine phar-
macophores through nucleophilic substitution, involving dihy-
droartemisinin and cyanuric chloride. Their chemical structures
were confirmed through routine IR, NMR and HRMS techniques.
The synthesized compounds were all active against the CQS and
CQR Dd2 strains of P. falciparum, with good selectivity towards
these parasitic cells in the presence of mammalian cells. Although
all synthesized compounds were active against both strains, the
dimers demonstrated higher activity than their monomeric coun-
terparts. Dimers 22 and 26 were distinctively the most active and
most selective, possessing potencies comparable to those of arte-
sunate and dihydroartemisinin against the NF54 strain, while being
slightly less active than both against the CQS strain. Hybrid-dimer
24 was the second most active against the Dd2 strain, but had
lower selectivity index. The antimalarial activities of both com-
pounds in conjunction with their low cytotoxicities may qualify
them as potential drug candidates to be further investigated in
search for novel, antigametocytocidal compounds for blocking
malaria transmission from mosquitoes to humans. It would be
interesting to establish whether their excellent in vitro nanomolar
activities would also be apparent in vivo, and whether their
Mass spectra (MS) were recorded in positive mode by one of two
mass spectrometers. The first was a Thermo Electron LXQÔ ion trap
mass spectrometer, equipped with an APCI source set at 300 ꢁC,
with Xcalibur 2.2 data acquisition and analysis software, utilizing
direct infusion with a Harvard syringe pump at a flow rate of 10
min. A full scan from 100 to 1200 amu was generated in 1 s, at a
capillary voltage of 7 V, while the corona discharge was set at 10 A.
mL/
m
The second instrument used was a Bruker MicroTOF Q II mass
spectrometer, equipped with an APCI or an ESI source set at 300 ꢁC
or 180 ꢁC, respectively, using Bruker Compass DataAnalysis 4.0
software. A full scan from 50 to 1500 m/z was generated at a
capillary voltage of 4500 V, an end plate offset voltage of ꢀ500 V
and a collision cell RF voltage of 100 Vpp
.
Melting points (mp) were determined in duplicate on a BÜCHI
melting point B-545 instrument and reported in degrees Celsius
(ꢁC) (uncorrected). Thin layer chromatography was performed us-
ing silica gel plates (60F254). Column chromatography was carried
out on silica gel (230e240 mesh, G60 Merck) and silica gel 60 (70e
230 mesh ASTM, Fluka).
Microwave radiation was carried out, using a CEM DiscoverÔ,
focused, closed vessel microwave synthesis system. The equipment
consisted of a continuously focused microwave power delivery

476
T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
system with operator selectable power output from 0 to 300 W, a
maximum current of 6.3 A and a frequency of 50/60 Hz. The tem-
perature of the contents of the vessel was monitored using an IR
sensor located underneath the reaction vessel. The contents of the
vessel were stirred by means of a rotating magnetic plate located
below the floor of the microwave cavity and a teflon coated mag-
netic stir bar in the vessel.
in vacuo. The resulting solid residue was washed with ice water
(3 ꢃ 50 mL), filtered and then purified through column chroma-
tography, eluting with various ratios of solvents. In some instances
the product was then further purified through recrystallization
from DCM.
5.4.1.1. 4,6-Dichloro-N-(4-methoxyphenyl)-1,3,5-triazin-2-amine, 3.
The reaction of 2 (43.4 mmol, 8 g), Na2CO3 (86.8 mmol, 9.2 g, 2 eq.)
and p-anisidine (43.4 mmol, 5.3 g, 1 eq.) in acetone (80 mL), fol-
lowed by purification through silica gel column chromatography,
eluting with EtOAc/PE (1:4, v/v) and recrystallization afforded 3 as a
brown powder (8.0 g, 68%); mp: 170.9 ꢁC; 1H NMR (600 MHz,
5.3. Biological study
5.3.1. Determination of antimalarial activity
The synthesized compounds were screened against the NF54
and Dd2 strains of P. falciparum. Continuous in vitro cultures of
asexual erythrocyte stages of P. falciparum were maintained, using a
modified method of Trager and Jensen [37]. Quantitative assess-
ment of antiplasmodial activity in vitro was determined via the
parasite lactate dehydrogenase (pLDH) assay, using a modified
method, as described by Makler et al. [38]. The test samples were all
tested in triplicate on one occasion.
The test samples were each prepared into 20 mg/mL stock so-
lutions in 100% DMSO. These stock solutions were stored at ꢀ20 ꢁC.
Further dilutions were prepared in complete medium on the day of
analysis. Samples were tested as a suspension if not completely
dissolved. Chloroquine (CQ) and artesunate were used as reference
drugs. A full dose response was performed to determine the con-
centration that would inhibit 50% of parasite growth (IC50 value).
DMSO-d6):
d
7.55e7.28 (m, 2H, H-70), 7.02e6.86 (m, 2H, H-80), 3.74
(s, 3H, H-100); 13C NMR (151 MHz, DMSO-d6)
d
169.62 (C-30), 168.59
(C-40), 163.56 (C-50), 156.68 (C-90), 129.51 (C-60), 123.31 (C-70),
114.08 (C-80), 55.31 (C-100); HRMS-APCI m/z: 271.0148 [M þ H]þ
[C10H9Cl2N4O: 271.0175 calcd.].
5.4.1.2. 4,6-Dichloro-N-phenyl-1,3,5-triazin-2-amine, 4. The reac-
tion of 2 (21.7 mmol, 4 g), Na2CO3 (43.4 mmol, 12.4 g, 2 eq.) and
aniline (21.7 mmol, 1.97 mL, 1 eq.) in acetone (50 mL), followed by
purification through silica gel column chromatography, eluting
with DCM afforded 4 as a white powder (4.5 g, 86%); mp: 199.5 ꢁC;
1H NMR (600 MHz, DMSO):
d
7.56 (dd, J ¼ 25.2, 8.1 Hz, 2H, H-70),
7.37 (dt, J ¼ 15.6, 7.9 Hz, 2H, H-80), 7.16 (dt, J ¼ 12.2, 7.4 Hz,1H, H-90);
13C NMR (151 MHz, DMSO-d6):
d
169.59 (30), 168.79 (C-40), 163.84
Test samples were tested at a starting concentration of 10
mg/mL
(C-50), 136.79 (C-60), 129.04 (C-80), 124.86 (C-90), 121.77 (C-70);
and then serially diluted two-fold in complete medium to give a
series of ten concentrations, with the lowest concentration being
HRMS-APCI m/z: 241.0028 [M þ H]þ [C9H7Cl2N4: 241.0070 calcd.].
0.02
mg/mL. The same dilution technique was used for all samples.
5.4.2. Primary amino-functionalized triazines 5e13
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 parasite viability (data
not shown).
The synthesis occurred in two steps. First, to either 2 or 3 or 4,
dissolved in acetone at 0 ꢁC, was added Na2CO3 and then the
relevant amine in four portions. The reaction mixture was allowed
to stir for 1e3 h at 0 ꢁC and then for 3e24 h at room temperature,
whereafter the solvent was evaporated in vacuo. The resulting
residue was dissolved in either EtOAc or DCM and washed several
times with water, then purified through column chromatography,
eluting with various ratios of solvents. The resulting product was
then used without further purification.
In the second step, the above synthesised di-substituted triazine
intermediate was dissolved in EDA and heated with continuous
stirring at 50e70 ꢁC for 3e24 h. Afterwards, the reaction was
quenched with ice cold water and the crude product extracted with
EtOAc (3 ꢃ 100 mL), then purified through column chromatog-
raphy, eluting with various ratios of solvents. The different steps are
illustrated in Scheme 1.
5.3.2. Cytotoxicity assay
Test samples were screened for in vitro cytotoxicity against a
mammalian cell line, Chinese Hamster Ovarian (CHO), using the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)
assay. The MTT assay is used as a colorimetric test for cellular
growth and survival and compares well with other available assays
[39,40]. The tetrazolium salt of MTT was used to measure all growth
and chemosensitivity of the mammalian cells. The samples were all
tested in triplicate on one occasion.
The same stock solutions being prepared for antiplasmodial
testing were used for cytotoxicity testing. Test compounds were
stored at ꢀ20 ꢁC until use. Dilutions were prepared on the day of
analysis. Emetine was used as reference drug in all tests. The initial
5.4.2.1. N-(2-Aminoethyl)-4,6-bis(piperidin-1-yl)-1,3,5-triazin-2-
amine, 5. The reaction of 2 (21.7 mmol, 4 g), Na2CO3 (43.4 mmol,
4.6 g, 2 eq.) and piperidine (43.4 mmol, 4.3 mL, 2 eq.) in acetone
(60 mL), followed by purification through chromatography, eluting
with DCM afforded 3.11 g of the intermediate as a white powder.
The reaction of the intermediate (7.1 mmol, 2 g) with EDA
(224.1 mmol, 15 mL, 32 eq.), followed by chromatography with
solvent mixtures MeOH:DCM:NH4OH (1:13.5:0.5, v/v/v) and
MeOH:NH4OH (19:1, v/v) afforded 5 as a white powder (1.8 g, 83%);
concentration of emetine was 100
diluted in complete medium with ten-fold dilutions to give six
concentrations, the lowest being 0.001 g/mL. The same dilution
mg/mL, which was serially
m
technique was applied to all test samples. The highest concentra-
tion of solvent to which the cells were exposed to had no
measurable effect on the cell viability (data not shown). The 50%
inhibitory concentration (IC50) values were obtained from full dose
response curves, using a non-linear dose response curve, inte-
grating outcomes by using GraphPad Prism v.4 software.
mp: 123.2 ꢁC; 1H NMR (600 MHz, CDCl3): 3.62 (s, 8H, H-50), 3.43e
d
3.34 (m, 2H, H-20), 2.83 (dd, J ¼ 16.6, 10.9 Hz, 2H, H-10), 1.58e1.42
5.4. Synthetic procedures
(m, 14H, H-70, H-60); 13C NMR (151 MHz, CDCl3): 166.58 (C-30),
d
164.89 (C-40), 44.00 (C-50), 42.99 (C-20), 41.95 (C-10),þ25.77 (C-60),
24.83 (C-70); HRMS-APCI m/z: 306.2390 [M þ H] [C15H28N7:
306.2430 calcd.].
5.4.1. Monoamine substituted triazines 3e4
Cyanuric chloride 2 (CyCl) was dissolved in acetone at 0 ꢁC, after
which Na2CO3 was added first and then the relevant amines in four
portions (Scheme 1). The reaction mixture was allowed to stir for
6 h at 0 ꢁC until completion, after which the solvent was evaporated
5.4.2.2. N-(2-Aminoethyl)-4,6-bis(morpholin-4-yl)-1,3,5-triazin-2-
amine, 6. The reaction of 2 (21.7 mmol, 4 g), Na2CO3 (43.4 mmol,

T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
477
4.5 g, 2 eq.) and morpholine (43.4 mmol, 3.8 mL, 2 eq.) in acetone
(50 mL) and extraction with DCM, followed by purification through
chromatography successively with EtOAc:DCM (1:1, v/v) and DCM,
afforded 4.2 g of the intermediate as a white powder.
68.3 ꢁC; IR (ATR)/cmꢀ1: 3391, 3271, 2930, 2859; 1H NMR (600 MHz,
CDCl3):
d
7.45 (s, 2H, H-70), 6.77 (d, J ¼ 8.5 Hz, 2H, H-80), 3.81e3.65
(m, 3H, H-100), 3.40 (d, J ¼ 50.5 Hz, 4H, H-20, H-110), 3.12e2.85 (m,
4H, H-120, H-130), 2.62 (s, 2H, 10), 1.01 (d, J ¼ 6.3 Hz, 12H, 140); 13C
The reaction of the intermediate (7.0 mmol, 2 g) with EDA
(373.5 mmol, 25 mL, 53 eq.), followed by extraction with EtOAc and
then purification through chromatography, using MeOH:DCM:N-
H4OH (1:4:0.5, then 1:9:0.5, v/v/v) afforded 7 as a white powder
(1 g, 49%); mp: 133.2 ꢁC; IR (ATR)/cmꢀ1: 3369, 3263, 3004, 2933,
NMR (151 MHz, CDCl3): d
165.85 (C-30, C-40), 164.07 (C-50), 155.14
(C-90), 132.53 (C-60), 121.61 (C-70), 114.01 (C-80), 55.23 (C-100), 49.24
(C-130), 44.51 (C-10), 41.18 (C-110, C-120), 40.12 (C-20), 20.12 (C-140);
HRMS-APCI m/z: 403.2908 [M þ H]þ [C20H35N8O: 403.2956 calcd.].
2850; 1H NMR (600 MHz, CDCl3):
d
3.85e3.55 (m, 17H, H-50, H-60),
5.4.2.6. 2-N-(2-Aminoethyl)-4-N,6-N-bis(4-methoxyphenyl)-1,3,5-
triazine-2,4,6-triamine, 10. The reaction of 2 (27.1 mmol, 5 g),
Na2CO3 (54.2 mmol, 15.5 g, 2 eq.) and p-anisidine (54.2 mmol, 6.6 g,
2 eq.) in acetone (80 mL), followed by extraction with EtOAc, then
purification through chromatography, eluting with EtOAc:PE (1:4,
v/v) afforded 4.2 g of the intermediate as a brown powder.
3.39 (q, J ¼ 6.0 Hz, 2H, H-20), 2.83 (t, J ¼ 5.9 Hz, 2H, H-10); 13C NMR
(151 MHz, CDCl3):
d
166.56 (C-30), 165.23 (C-40), 66.59 (C-60), 43.5þ0
(C-20, C-50), 41.79 (C-10); HRMS-APCI m/z: 310.1972 [M þ H]
[C13H24N7O2: 310.2013 calcd.].
5.4.2.3. 2N-(2-Aminoethyl)-4N-(4-methoxyphenyl)-6-(piperidin-1-
yl)-1,3,5-triazine-2,4-diamine, 7. The reaction of 3 (14.8 mmol, 4 g),
Na2CO3 (29.6 mmol, 3.1 g, 2 eq.) and piperidine (14.8 mmol, 1.5 mL,
1 eq.) in acetone (40 mL), followed by extraction with EtOAc, then
purification through chromatography, eluting with EtOAc:PE (1:4,
v/v) afforded 5.6 g of the intermediate as an orange oil.
The reaction of the intermediate (11.8 mmol, 4.2 g) with EDA
(373.5 mmol, 25 mL, 32 eq.) and extraction with EtOAc, followed by
purification through chromatography with MeOH:DCM:NH4OH
(1:4:0.2, v/v/v) afforded 3.2 g (71%) of 10 as a white powder; mp:
150.1 ꢁC; IR (ATR)/cmꢀ1: 3267, 2964,1602,1571; 1H NMR (600 MHz,
DMSO-d6):
d
7.64 (s, 4H, H-60), 6.82 (d, J ¼ 7.8 Hz, 5H, H-70), 3.70 (s,
6H, H-90), 3.29e3.26 (m, 2H, H-20), 2.68 (t, J ¼ 6.2 Hz, 2H, H-10); 13C
The reaction of the intermediate (17.5 mmol, 5.6 g) with EDA
(373.5 mmol, 25 mL, 21 eq.), followed by extraction with EtOAc and
NMR (151 MHz, DMSO-d6): d
165.89 (C-30), 163.88 (C-40), 154.29 (C-
purification
through
chromatography,
eluting
with
80), 133.70 (C-50), 121.41 (C-60), 113.42 (C-70), 55.05 (C-90), 43.86 (C-
20), 41.45 (C-10); HRMS-APCI m/z: 382.1959 [M þ H]þ [C19H23N7O2:
382.1923 calcd.].
MeOH:DCM:NH4OH (1:14:0.5, v/v/v) resulted in compound 6 as a
white powder (3.5 g, 58%); mp: 132.8 ꢁC; IR (ATR)/cmꢀ1: 3345,
3279, 2966, 2896, 2858; 1H NMR (600 MHz, CDCl3)
d 7.42 (d,
J ¼ 7.5 Hz, 2H, H-100), 6.81 (d, J ¼ 8.9 Hz, 2H, H-110), 3.90e3.54 (m,
10H, H-60, H-130), 3.42 (dd, J ¼ 11.6, 5.8 Hz, 2H, H-20), 2.85 (t,
J ¼ 5.8 Hz, 2H, H-10), 1.67e1.48 (m, 9H,H-70, H-80); 13C NMR
5.4.2.7. 2-N-(2-Aminoethyl)-4-N,6-N-diphenyl-1,3,5-triazine-2,4,6-
triamine, 11. The reaction of 2 (27.1 mmol, 5 g), Na2CO3 (54.2 mmol,
15.5 g, 2 eq.) and aniline (54.2 mmol, 4.9 mL, 2 eq.) in acetone
(80 mL) and extraction with EtOAc, followed by purification
through chromatography with DCM afforded 5.9 g of the inter-
mediate as a white powder.
The reaction of the intermediate (13.4 mmol, 4 g) with EDA
(373.5 mmol, 25 mL, 28 eq.) and extraction with EtOAc, followed by
purification through chromatography with MeOH:DCM:NH4OH
(1:4:0.2, v/v/v) afforded 11 as a white powder (2.9 g, 67%); mp:
77.7 ꢁC; IR (ATR)/cmꢀ1: 3366, 3280, 3168, 2935, 2835; 1H NMR
(151 MHz, CDCl3) d
139.00 (C-30), 164.92 (C-40), 164.27 (C-50), 155.13
(C-120), 132.52 (C-90), 121.80 (C-100), 113.58 (C-110), 55.47 (C-130),
44.15 (C-60), 43.35 (C-20), 41.83 (C-10), 25.78 (C-70), 24.82 (C-80);
HRMS-APCI m/z: 344.2175 [M þ H]þ [C17H26N7O: 344.2221 calcd.].
5.4.2.4. 2-N-(2-Aminoethyl)-4-N-(4-methoxyphenyl)-6-(morpholin-
4-yl)-1,3,5-triazine-2,4-diamine, 8. The reaction of 3 (14.8 mmol,
4 g), Na2CO3 (29.5 mmol, 8.5 g, 2 eq.) and morpholine (14.8 mmol,
1.3 mL, 1 eq.) in acetone (40 mL) and extraction with EtOAc, fol-
lowed by purification through chromatography with EtOAc:PE (1:1,
v/v) afforded 4.7 g of the intermediate as an orange powder.
The reaction of the intermediate (6.2 mmol, 2 g) with EDA
(373.5 mmol, 25 mL, 60 eq.) and extraction with EtOAc, followed by
purification through chromatography with MeOH:DCM:NH4OH
(1:9:0.5, v/v/v) afforded 8 as a white powder (1.8 g, 85%); mp:
154.6 ꢁC; IR (ATR)/cmꢀ1: 3397, 3265, 2927, 2864, 1574; 1H NMR
(600 MHz, DMSO-d6):
d
7.79 (s, 4H, H-60), 7.24 (s, 5H, H-70), 6.93 (t,
J ¼ 7.2 Hz, 2H, H-80), 3.30 (dd, J ¼ 11.6, 5.6 Hz, 3H, H-20), 2.71 (t,
J ¼ 6.0 Hz, 2H, H-10); 13C NMR (151 MHz, DMSO-d6):
d
165.81 (C-40),
164.00 (C-30), 140.38 (C-50), 128.28 (C-70), 121.40 (C-80), 119.77 (Cþ-
60), 43.85 (C-20), 41.25 (C-10); HRMS-APCI m/z: 322.1754 [M þ H]
[C17H20N7: 322.1780 calcd.].
(600 MHz, DMSO-d6):
d
7.59 (s, 2H, H-70), 6.81 (t, J ¼ 9.6 Hz, 2H, H-
5.4.2.8. 2-N-(2-Aminoethyl)-4-N-(4-methoxyphenyl)-6-N-phenyl-
1,3,5-triazine-2,4,6-triamine, 12. The reaction of 4 (8.3 mmol, 2 g),
Na2CO3 (16.6 mmol, 4.7 g, 2 eq.) and p-anisidine (8.3 mmol, 1.0 g,
1 eq.) in acetone (40 mL), followed by extraction with EtOAc, then
purification through chromatography with EtOAc:PE (1:4, v/v)
afforded 2.3 g of the intermediate as off-white powder.
The reaction of the intermediate (6.1 mmol, 2 g) with EDA
(448.3 mmol, 30 mL, 7 eq.) and extraction with EtOAc, followed by
purification through chromatography with MeOH:DCM:NH4OH
(1:4:0.5, v/v/v) afforded 12 as a purple powder (1.8 g, 84%); mp:
134.5 ꢁC; IR (ATR)/cmꢀ1: 3271, 2961, 2930, 2870, 2834; 1H NMR
80), 3.79e3.55 (m, 12H, H-100, H-110, H-120), 3.49e3.19 (m, 2H, H-20),
2.75e2.56 (m, 2H, H-10); 13C NMR (151 MHz, DMSO-d6):
d 166.79
(C-30), 165.01 (C-40),163.67 (C-50), 153.79 (C-90),133.66 (C-60), 121.17
(C-70), 113.52 (C-80), 66.18 (C-120), 55.02 (C-100), 43.þ27 (C-20, C-110),
41.16 (C-10); HRMS-APCI m/z: 346.1965 [M þ H] [C16H24N7O2:
346.2013 calcd.].
5.4.2.5. 2-N-(2-Aminoethyl)-4-N-{2-[bis(propan-2-yl)amino]ethyl}-
6-N-(4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine,
9.
Compound 3 (7.3 mmol, 2 g), Na2CO3 (14.6 mmol, 4.1 g, 2 eq.) and 2-
(diisopropylamino)ethylamine (7.3 mmol, 1.2 mL, 1 eq.) in acetone
(40 mL) afforded 2.5 g of the intermediate as a brown powder after
extraction with EtOAc, followed by purification through chroma-
tography, using MeOH:DCM (7:3, v/v) as eluent.
The reaction of the intermediate (5.2 mmol, 2 g) with EDA
(448.3 mmol, 30 mL, 86 eq.) and extraction with EtOAc, followed by
purification through chromatography with MeOH:DCM:NH4OH
(1:4:0.5, v/v/v) resulted in 9 as a white powder (2.1 g, 99%); mp:
(600 MHz, DMSO-d6)
d
7.69 (d, J ¼ 65.3 Hz, 4H, H-70, H-110), 7.20 (s,
2H, H-80), 6.85 (d, J ¼ 50.0 Hz, 3H, H-90, H-120), 3.70 (d, J ¼ 15.8 Hz,
3H, H-140), 3.47 (d, J ¼ 91.5 Hz, H-20, H2O), 3.06 (s, 2H, H-10); 13C
NMR (151 MHz, DMSO-d6):
d
165.88 (C-30), 163.90 (C-40, C-50),
154.54 (C-130), 140.22 (C-60), 133.07 (C-100), 128.30 (C-80), 121.92 (C-
90), 121.20 (C-110), 119.83 (C-70), 113.41 (C-120), 55.05 (C-140), 40.32
(C-20), 38.98 (C-10); HRMS-APCI m/z: 352.1864 [M
þ
H]þ
[C18H22N7O: 352.1908 calcd.].

478
T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
5.4.2.9. 2-N-(2-Aminoethyl)-6-(morpholin-4-yl)-4-N-phenyl-1,3,5-
triazine-2,4-diamine, 13. The reaction of 4 (16.6 mmol, 4 g), Na2CO3
(33.2 mmol, 9.4 g, 2 eq.) and morpholine (16.6 mmol, 1.5 mL, 1 eq.)
in acetone (80 mL), followed by extraction with EtOAc, then puri-
fication through chromatography, eluting with EtOAc:PE (1:4, v/v)
afforded 2.2 g of the intermediate as a white powder.
2.16 (td, J ¼ 14.1, 3.7 Hz, 1H, H-4
1.71 (m, 1H, 5 ), 1.68e1.53 (m, 2H, H-8
2.7 Hz, 1H, H-7 ), 1.40e1.19 (m, 6H, H-5
1.02 (m, 1H, H-5a), 0.82 (ddd, J ¼ 32.2, 20.8, 6.4 Hz, 7H, H-7
H-16); 13C NMR (151 MHz, DMSO-d6): 165.72 (C-Oxalate, C-30),
a
), 2.08e1.91 (m, 1H, H-4
, H-8
), 1.48 (dd, J ¼ 12.8,
, H-6, H-8a, H-14), 1.17e
, H-15,
b), 1.84e
a
a
b
b
b
a
d
164.54 (C-40), 103.28 (C-3), 100.91 (C-10), 87.11 (C-12), 80.38 (C-
12a), 66.04 (C-60), 64.23 (C-a), 51.94 (C-5a), 47.36 (C-10), 46.39 (C-b),
43.72 (C-8a), 43.18 (C-50), 37.26 (C-20), 36.42 (C-6), 36.10 (C-4), 34.10
(C-7), 30.06 (C-9), 25.47 (C-14), 23.98 (C-5), 23.64 (C-8), 20.09 (C-
15), 12.55 (C-16); HRMS-APCI m/z: 620.3758 [M þ H]þ [C30H50N7O7:
620.3794 calcd.].
The reaction of the intermediate (6.9 mmol, 2 g) with EDA
(373.5 mmol, 25 mL, 54 eq.), followed by extraction with EtOAc,
then purification through chromatography, eluting with
MeOH:DCM:NH4OH (1:9:0.5, v/v/v) afforded compound 13 as a
white powder (1.8 g, 85%); mp: 95.6 ꢁC; IR (ATR)/cmꢀ1: 3270, 2953,
2923, 2854; 1H NMR (600 MHz, CDCl3): 7.52 (s, 2H, H-90), 7.26 (dd,
d
J ¼ 13.5, 5.9 Hz, 2H, H-100), 6.98 (t, J ¼ 7.3 Hz, 1H, H-110), 3.73 (d,
5.4.3.3. 2-N-(4-Methoxyphenyl)-6-(piperidin-1-yl)-4-N-{2-[(2-(10
dihydroartemisinoxy) ethyl)amino]ethyl}-1,3,5-triazine-2,4-diamine
(oxalate) 16. The reaction of (5.8 mmol, 2.3 g), K2CO3
b-
J ¼ 43.1 Hz, 8H, H-60, H-70), 3.44 (q, J ¼ 5.9 Hz, 2H, H-20), 2.87 (t,
J ¼ 5.8 Hz, 2H, H-10); 13C NMR (151 MHz, CDCl3):
d
166.28 (C-30),
1
165.40 (C-50), 164.26 (C-40), 139.45 (C-80), 128.51 (C-100), 122.29 (C-
110), 120.00 (C-90), 66.58 (C-70), 43.62(C-60þ), 43.11 (C-20), 41.34 (C-
10); HRMS-APCI m/z: 316.1863 [M þ H] [C15H21N7O: 316.1818
calcd.].
(11.6 mmol, 1.6 g, 2 eq.) and 7 (5.8 mmol, 2 g, 1 eq.) in DMF (50 mL),
followed by purification through chromatography with MeOH:E-
tOAc:NH4OH (1:14:0.5 and 19:1:0.5, v/v/v), after conversion
resulted in 16 salt as a white powder (0.5 g, 12%); mp: 151.5 ꢁC;
C
34H51N7O6; IR (ATR)/cmꢀ1: 3266, 2935, 2860, 1719; 1H NMR
5.4.3. Artemisininetriazine hybrids and hybrid-dimers 14e26
DHA derivative 1 and triazine intermediates 5e13, together with
K2CO3 were dissolved in DMF. The resulting mixture was heated in
a microwave reactor in bursts of 60 W and 50 ꢁC for 4 min at a time,
followed by cooling to 0 ꢁC (Scheme 1). This sequence was repeated
until the reaction was complete. The solvent was then evaporated
in vacuo, the resulting residue dissolved in EtOAc and then washed
with H2O. The organic phase was dried over MgSO4 and concen-
trated in vacuo, resulting in an oil. The purification through silica gel
column chromatography, eluting with various ratios of MeOH,
DCM, EtOAc, PE and NH4OH, afforded the target DHA monomeric-
and/or dimeric (in some cases) hybrid free base compounds, which
were later converted into oxalate salts.
(600 MHz, CDCl3):
d
7.45 (d, J ¼ 8.2 Hz, 2H, H-70), 6.75 (d, J ¼ 8.5 Hz,
2H, H-80), 5.40e5.26 (m, 1H, H-12), 4.75 (d, J ¼ 3.4 Hz, 1H, H-10),
4.18e4.00 (m, 1H, H-aa), 3.92e3.54 (m, 10H, H-ab
, H-20, H-130, H-
110), 3.45e3.11 (m, 4H, H-10, H-b), 2.66e2.46 (m, 1H, H-9), 2.30
(ddd, J ¼ 18.4, 17.2, 8.9 Hz, 1H, 4
a
), 2.07e1.88 (m, 1H, H-4
b
a
), 1.86e
1.74 (m, 1H, H-5
H-8
(m, 7H, H-7
a
), 1.60 (d, J ¼ 47.4 Hz, 9H, H-130, H-120, H-8
, H-7b,
b
), 1.47e1.06 (m, 8H, H-6, H-5b, H-8a, H-14, H-5a), 0.96e0.63
a
, H-15, H-16). 13C NMR (151 MHz, CDCl3):
d 164.91 (C-
50), 161.68 (C-30), 156.67 (C-40), 155.98 (C-Oxalate), 153.61 (C-90),
129.89 (C-60),123.16 (C-70),113.70 (C-80),103.93 (C-3),102.10 (C-10),
87.98 (C-12), 81.03 (C-12a), 63.89 (C-a), 55.45 (C-100), 52.44 (C-5a),
47.58 (C-10, C-b), 45.19 (C-110), 44.18 (C-8a), 37.76 (C-6), 37.15 (C-20),
36.35 (C-4), 34.12 (C-7), 30.53 (C-9), 26.17 (C-14), 25.65 (C-120),
24.29(C-5, C-130), 24.14 (C-8), 20.25 (C-15), 12.89 (C-16); HRMS-
APCI m/z: 654.3970 [M þ H]þ [C34H52N7O6: 654.4001 calcd.].
5.4.3.1. (2-{[Bis(piperidin-1-yl)-1,3,5-triazin-2-yl]amino}ethyl)-2-
(10b-dihydroartemisinoxy)ethylamine (oxalate) 14. The reaction of 1
(2.9 mmol, 1.1 g), K2CO3 (5.8 mmol, 0.8 g, 2 eq.) and 5 (2.9 mmol,
0.9 g, 1 eq.) in DMF (25 mL), followed by purification through
chromatography successively with MeOH:DCM (1:4, v/v) and
MeOH, after conversion afforded 14 salt as a white powder (0.4 g,
20%); C32H53N7O5; mp: 152.2 ꢁC; IR (ATR)/cmꢀ1: 3407, 2933, 2551,
5.4.3.4. 2-N-{2-[Bis(2-{10b-dihydroartemisinoxy}ethyl)amino]
ethyl}-4-N-(4-methoxyphenyl)-6-(piperidin-1-yl)-1,3,5-triazine-2,4-
diamine (oxalate) 17. The preceding reaction also yielded a dimeric
compound, which after conversion afforded 17 salt as a white
powder (0.3 g, 5%); mp: 139.4 ꢁC; C51H77N7O11; IR (ATR)/cmꢀ1
:
1721; 1H NMR (600 MHz, CDCl3):
J ¼ 3.2 Hz,1H, H-10), 4.11e3.98 (m, 1H, H-a
, H-20, H-50), 3.35e3.17 (m, 4H, H-10, H-b), 2.61e2.48 (m, 1H, H-9),
2.30 (dt, J ¼ 14.2, 3.7 Hz, 1H, H-4 ), 2.01e1.90 (m, 1H, H-4 ), 1.86e
1.75 (m, 1H, H-5 ), 1.71e1.48 (m, 15H. H-8 , H-8 ),
, H-60, H-70, 7
1.48e1.22 (m, 7H, H-8a, H-5
, H-14, H-6), 1.17 (dt, J ¼ 11.4, 6.8 Hz,
1H, H-5a), 0.98e0.71 (m, 7H, H-7
, H-15, H-16); 13C NMR (151 MHz,
CDCl3):
165.86 (C-oxalate, C-30, C-40), 103.94 (C-3), 102.14 (C-10),
d
5.32 (s, 1H, H-12), 4.73 (d,
), 3.81e3.56 (m,11H, H-
3262, 2939, 2874, 1717; 1H NMR (600 MHz, CDCl3):
d 7.45 (t,
a
J ¼ 18.7 Hz, 2H, H-70), 6.79 (t, J ¼ 22.0 Hz, 2H, H-80), 5.48e5.26 (m,
2H, H-12), 4.78 (d, J ¼ 3.1 Hz, 2H, H-10), 4.26 (dd, J ¼ 29.4, 22.5 Hz,
ab
a
b
2H, H-aa), 4.04e3.41 (m, 17H, H-ab
, H-20, H-110, H-100, H-10, H-b),
a
a
b
b
2.70e2.56 (m, 2H, H-9), 2.31 (td, J ¼ 14.2, 3.7 Hz, 2H, H-4
1.94 (m, 2H, H-4 ), 1.90e1.76 (m, 2H, H-5 ), 1.76e1.52 (m, 12H, H-
, H-8 , H-7 , H-8a, H-
, H-120, H-130), 1.48e1.11 (m, 14H, H-6, H-5
14, H-5a), 0.98e0.70 (m, 13H, H-7
, H-15, H-16); 13C NMR
(151 MHz, CDCl3):
163.22 (C-Oxalate), 161.00 (C-50), 156.51 (C-30),
a), 2.06e
b
b
a
a
8a
b
b
b
d
a
87.84 (C-12), 81.19 (C-12a), 63.73 (C-a), 52.29 (C-5a), 47.65 (C-10, C-
b), 44.31 (C-8a), 44.29 (C-50), 37.78 (C-20), 37.13 (C-6), 36.21 (C-4),
34.42 (C-7), 30.44 (C-9), 26.20 (C-60, C-14), 25.49 (C-70), 24.41 (C-5),
24.19 (C-8), 20.11 (C-15), 12.97 (C-16); HRMS-APCI m/z: 616.4182
[M þ H]þ [C32H54N7O5: 616.4208 calcd.].
d
155.63 (C-40), 153.37 (C-90), 129.65 (C-60), 122.98 (C-70), 113.78 (C-
80), 104.33 (C-3), 102.17 (C-10), 87.89 (C-12), 80.69 (C-12a), 62.71 (C-
a), 55.26 (C-100) 53.01 (C-10, C-b), 52.37 (C-5a), 45.55 (C-110), 43.99
(C-8a), 37.44 (C-6), 36.18 (C-4), 35.91 (C-20), 34.26 (C-7), 30.47 (C-9),
26.01 (C-14), 25.50 (C-120), 24.38 (C-5, C-8), 23.91 (C-130), 20.29 (C-
5.4.3.2. (2-{[Bis(morpholin-4-yl)-1,3,5-triazin-2-yl]amino}ethyl)-2-
15), 12.77 (C-16); HRMS-APCI m/z: 964.5755 [M
[C51H77N7O11: 964.5691 calcd.].
þ
H]þ
(10
b-dihydroartemisinoxy)ethylamine (oxalate) 15. The reaction of 1
(1.6 mmol, 0.6 g), K2CO3 (3.2 mmol, 0.5 g, 2 eq.) and 6 (1.6 mmol,
0.5 g, 1 eq.) in DMF (25 mL), followed by purification through
chromatography with MeOH:DCM (1:9, v/v), afforded after con-
version,15 as a white oxalate salt powder (0.3 g, 28%); mp: 162.1 ꢁC;
5.4.3.5. 2-N-(4-Methoxyphenyl)-6-(morpholin-4-yl)-4-N-{2-[(2-
{10 dihydroartemisinoxy}ethyl)amino]ethyl}-1,3,5-triazine-2,4-
b
diamine (oxalate) 18. The reaction of 1 (2.9 mmol, 1.1 g), K2CO3
(5.8 mmol, 0.8 g, 2 eq.) and 8 (2.9 mmol, 1 g, 1 eq.) in DMF (50 mL),
followed by chromatography successively with MeOH:E-
tOAc:PE:NH4OH (1:10:4:0.5, v/v/v/v) and MeOH:EtOAc:NH4OH
(1:19:0.5, v/v/v) after conversion afforded 18 salt as a white powder
C
30H49N7O7; IR (ATR)/cmꢀ1: 3400, 2920, 2848; 1H NMR (600 MHz,
DMSO-d6):
10), 3.85 (dt, J ¼ 10.6, 5.2 Hz, 1H, H-a
50, H-60, H-20), 3.15e2.85 (m, 4H, H-10, H-b), 2.42e2.30 (m, 1H, H-9),
d
5.31 (d, J ¼ 37.4 Hz,1H, H-12), 4.69 (d, J ¼ 3.3 Hz,1H, H-
a
), 3.71e3.41 (m, 21H, H-a , H-
b

T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
479
(0.3 g, 13%); mp: 140.9 ꢁC; C33H49N7O7; IR (ATR)/cmꢀ1: 3268, 2919,
5.4.3.8. 2-N-{2-[Bis(2-{10
b
-dihydroartemisinoxy}ethyl)amino]
2861, 1719; 1H NMR (600 MHz, CDCl3):
d
7.42 (d, J ¼ 7.7 Hz, 2H, H-
ethyl}-4-N-{2-[bis(propan-2-yl)amino]ethyl}-6-N-(4-
methoxyphenyl)-1,3,5-triazine-2,4,6-triamin (oxalate) 21. The pre-
ceding reaction also gave a hybrid-dimer, which after conversion
resulted in 21 salt as a white powder (0.4 g, 7%); mp: 141.9 ꢁC;
70), 6.81 (dd, J ¼ 47.4, 8.4 Hz, 2H. H-80), 5.37 (d, J ¼ 22.7 Hz, 1H, H-
12), 4.76 (d, J ¼ 3.2 Hz, 1H, H-10), 4.09 (s, 1H, H-a
a), 3.96e3.52 (m,
14H, H-a
b
, H-20, H-120, H-100, H-110), 3.42 (dd, J ¼ 37.3, 30.1 Hz, 4H,
H-10, H-b), 2.57 (s, 1H, H-9), 2.29 (d, J ¼ 12.5 Hz, 1H, H-4
a
), 1.96 (d,
, H-
, H-8a, H-14,
, H-15, H-16); 13C NMR
163.43 (Oxalate), 161.85 (C-40), 156.92 (C-30),
C
54H86N8O11; IR (ATR)/cmꢀ1: 3254, 2947, 2875, 1719; 1H NMR
J ¼ 26.3 Hz, 2H, H-4
b
), 1.81 (s, 1H, H-5
a
), 1.72e1.49 (m, 3H, H-8
a
(600 MHz, DMSO-d6):
d
7.61 (d, J ¼ 19.3 Hz, 2H, H-70), 6.76 (d,
8
b
, H-7 ), 1.28 (ddd, J ¼ 32.8, 24.1, 6.1 Hz, 8H, H-6, H-5
H-5a), 0.86 (dd, J ¼ 29.8, 5.7 Hz, 7H, H-7
(151 MHz, CDCl3):
b
b
J ¼ 8.9 Hz, 2H, H-80), 5.33e5.25 (m, 2H, H-12), 4.65 (s, 2H, H-10),
a
3.78e3.71 (m, 2H, H-a
a
), 3.68 (s, 3H, H-100), 2.97 (dd, J ¼ 12.7,
d
6.3 Hz, 2H, H-130), 2.75e2.64 (m, 6H, H-b, H-10), 2.37 (s, 2H, H-9),
156.00 (C-50), 153.79 (C-90), 129.18 (C-60), 123.60 (C-70), 113.59 (C-
80), 104.15 (C-3),102.36 (C-10), 87.63 (C-12), 80.47 (C-12a), 66.33 (C-
120), 63.86 (C-a), 55.25 (C-100), 52.62 (C-5a), 47.77 (C-10), 47.05 (C-
b), 44.24 (C-8a), 44.06 (C-110), 37.89 (C-20), 36.95 (C-6), 36.39 (C-4),
34.13 (C-7), 30.41 (C-9), 25.96 (C-14), 24.41 (C-5), 24.01 (C-8), 19.93
2.18e2.09 (m, 2H, H-4
a
), 1.93 (t, J ¼ 20.8 Hz, 2H, H-4
, H-8 ), 1.63e1.44 (m, 4H, H-8
, H-14), 1.13e1.06 (m, 2H, H-5a),
b
), 1.73 (dd,
J ¼ 25.1, 12.7 Hz, 4H, H-5
a
a
b
, H-7 ),
b
1.36e1.22 (m, 12H, H-6, H-8a, H-5
b
0.96 (s, 12H, H-140), 0.82 (d, J ¼ 7.1 Hz, 14H, H-7
a C
, H-15, H-16); 13
NMR (151 MHz, DMSO-d6): d
153.95 (C-50), 133.97 (C-60), 121.06 (C-
(C-15), 12.81 (C-16); HRMS-APCI m/z: 656.3752 [M
[C33H50N7O7: 656.3797 calcd.].
þ
H]þ
70), 113.39 (C-80), 103.26 (C-3), 101.20 (C-10), 86.91 (C-12), 80.50 C-
12a), 66.57 (C-a), 55.10 (C-100), 53.99 (C-b), 53.63 (C-10), 52.06 (C-
5a), 48.54 (C-120), 48.10 (C-130), 43.80 (C-8a), 40.13(C-20), 39.78 (C-
110), 36.78 (C-6), 36.18 (C-4), 34.11 (C-7), 30.47 (C-9), 25.50 (C-14),
24.26 (C-5), 23.95 (C-8), 20.57 (C-15), 20.09 (C-140), 12.82 (C-16);
HRMS-APCI m/z: 1023.6457 [M þ H]þ [C54H86N8O11: 1023.6426
calcd.].
5.4.3.6. 2-N-{2-[Bis(2-{10b-dihydroartemisinoxy}ethyl)amino]
ethyl}-4-N-(4-methoxyphenyl)-6-(morpholin-4-yl)-1,3,5-triazine-
2,4-diamine (oxalate) 19. The preceding reaction also yielded a
dimeric compound, which after conversion afforded 19 salt as a
white powder (0.1 g, 4%); mp: 145.3 ꢁC; C50H75N7O12; IR (ATR)/
cmꢀ1: 3248, 2940, 2920, 2874, 1719; 1H NMR (600 MHz, CDCl3):
5.4.3.9. 2-N-{2-[Bis(2-{10b-dihydroartemisinoxy}ethyl)amino]
d
7.41 (t, J ¼ 13.1 Hz, 2H, H-70), 6.81 (dd, J ¼ 37.7, 7.1 Hz, 2H, H-80),
ethyl}-4-N,6-N-bis(4-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine
(oxalate) 22. The reaction of 1 (2.6 mmol, 1 g) and 10 (2.6 mmol,
1.0 g, 1 eq.) with K2CO3 (5.24 mmol, 0.7 g, 2 eq.) in DMF (25 mL),
followed by chromatography successively with EtOAc and
MeOH:DCM (1:14, v/v) after conversion afforded hybrid-dimer 22
salt as a white powder (0.1 g, 3%); mp: 147.4 ꢁC; C53H75N7O12; IR
(ATR)/cmꢀ1: 3269, 2944, 2875,1718; 1H NMR (600 MHz, DMSO-d6):
5.43e5.30 (m, 2H, H-12), 4.84e4.69 (d, J ¼ 3.5, 2H, H-10), 4.32e4.16
(m, 2H, H-aa), 4.04e3.31 (m, 22H, H-ab
, H-b, H-10, H-20, H-100, H-
110, H-120), 2.70e2.54 (m, 2H, H-9), 2.31 (td, J ¼ 14.2, 3.4 Hz, 2H, H-
4
a
), 2.00 (t, J ¼ 23.8 Hz, 2H, H-4
b), 1.90e1.78 (m, 2H, H-5a), 1.61 (dt,
J ¼ 26.8, 12.1 Hz, 6H, H-8
6, H-8a, H-5 , H-14), 0.98e0.74 (m, 15H, H-7
NMR (151 MHz, CDCl3):
162.88 (C-Oxalate), 161.65 (C-40), 156.88
a
, H-8
b
, H-7b
), 1.48e1.13 (m, 16H, H-5a, H-
b
a
, H-15, H-16); 13C
d
d
7.62 (s, 4H, H-60), 6.83 (s, 5H, H-70), 5.34 (s, 2H, H-12), 4.70 (s, 2H,
(C-30), 155.75 (C-50), 153.21 (C-90), 129.20 (C-60), 123.29 (C-70),
113.93 (C-80), 104.39 (C-3), 102.33 (C-10), 87.82 (C-12), 80.69 (C-
12a), 66.35 (C-120), 62.54 (C-a), 55.40 (C-100), 53.17 (C-10), 52.87 (C-
b), 52.35 (C-5a), 44.59 (C-110), 43.98 (C-8a), 37.29 (C-6), 36.17 (C-4),
35.83 (C-20), 34.40 (C-7), 30.46 (C-9), 25.85 (C-14), 24.56 (C-5),
24.38 (C-8), 20.30 (C-15), 12.77 (C-16); HRMS-APCI m/z: 966.5553
[M þ H]þ [C50H75N7O12: 966.5483 calcd.].
H-10), 3.88 (d, J ¼ 11.9 Hz, 3H, H-a
a), 3.79e3.40 (m, 11H, HeH-ab,
H-90, H-20), 3.16 (d, J ¼ 15.0 Hz, 6H, H-10, H-b), 2.39 (s, 2H, H-9), 2.16
(t, J ¼ 12.7 Hz, 2H, H-4
a), 2.05e1.89 (m, 2H, H-4b), 1.85e1.53 (m, 6H,
H-8
H-6, H-5
(dd, J ¼ 11.0, 6.6 Hz, 15H, H-7
DMSO-d6):
165.59 (C-30), 163.77 (C-40), 162.69 (C-oxalate), 154.40
a
, H-5
a
, H-8
, H-8a, H-14), 1.11 (dd, J ¼ 17.4, 10.8 Hz, 2H, H-5a), 0.83
, H-15, H-16); 13C NMR (151 MHz,
b
), 1.47 (d, J ¼ 10.8 Hz, 2H, H-7
b), 1.40e1.16 (m, 12H,
b
a
d
(C-80), 133.31 (C-50), 121.78 (C-60), 113.47 (C-70), 103.44 (C-3), 101.02
(C-10), 86.90 (C-12), 80.46 (C-12a), 55.16 (C-90), 53.15 (C-10, C-b),
52.08 (C-5a), 43.57 (C-8a), 36.63 (C-6, C-20), 36.02 (C-4), 33.70 (C-7),
30.44 (C-9), 25.48 (C-14), 24.12 (C-5), 23.69 (C-8), 20.12 (C-15),
5.4.3.7. 2-N-{2-[Bis(propan-2-yl)amino]ethyl}-4-N-(4-
methoxyphenyl)-6-N-{2-[(2-{10b-dihydroartemisinoxy}ethyl)amino]
ethyl}-1,3,5-triazine-2,4,6-triamine (oxalate) 20. The reaction of 1
(5.0 mmol, 1.9 g), K2CO3 (9.9 mmol, 1.4 g, 2 eq.) and 9 (5.0 mmol,
2.0 g, 1 eq.) in DMF (50 mL), followed by chromatography succes-
sively with MeOH:DCM:NH4OH (1:9:0.5, v/v/v) and MeOH:E-
tOAc:NH4OH (1:14:0.5, v/v/v) after conversion afforded 20 salt as a
white powder (0.5 g, 11%); mp: 152.0 ꢁC; C37H60N8O6; IR (ATR)/
12.55 (C-16); HRMS-APCI m/z: 1002.5512 [M þ H]þ [C53H75N7O12
:
1002.5484 calcd.].
5.4.3.10. 2-N,4-N-Diphenyl-6-N-{2-[(2-{10b-dihydroartemisinoxy}
ethyl)amino]ethyl} 1,3,5-triazine-2,4,6-triamine (oxalate) 23.
The reaction of 1 (2.3 mmol, 0.9 g), K2CO3 (4.6 mmol, 0.6 g, 2 eq.)
and 11 (2.3 mmol, 0.7 g, 1 eq.) in DMF (25 mL), followed by chro-
matography with MeOH:EtOAc:NH4OH (1:29:0.5 and 1:9:0.5, v/v/
v) after conversion afforded 23 salt as a white powder (0.2 g, 14%);
mp: 136.8 ꢁC; C34H45N7O5; IR (ATR)/cmꢀ1: 3270, 2922, 2874, 1710;
cmꢀ1: 3255, 2937, 2877, 1718; 1H NMR (600 MHz, CDCl3):
d 7.45 (s,
2H, H-70), 6.81 (d, J ¼ 8.6 Hz, 2H, H-80), 5.36 (s, 1H, H-12), 4.78 (d,
J ¼ 3.1 Hz, 1H, H-10), 3.95e3.86 (m, 1H, H-a
a
), 3.76 (s, 3H, H-100),
3.48 (dt, J ¼ 48.9, 21.9 Hz, 5H, H-a
b
, H-20, H-110), 3.09 (s, 2H, H-130),
2.80 (dd, J ¼ 11.9, 6.6 Hz, 4H, H-b, H-10), 2.68 (s, 2H, H-120), 2.61e
2.55 (m, 1H, H-9), 2.32 (td, J ¼ 14.1, 3.9 Hz, 1H, H-4
a
), 2.02e1.96 (m,
), 1.67 (dd,
), 1.60e1.54 (m, 1H, H-7 ), 1.47e
, H-14), 1.30e1.19 (m, 3H, H-5a, H-6), 1.06 (s,
14H, H-140), 0.90e0.79 (m, 7H, H-7 , H-15, H-16); 13C NMR
(151 MHz, CDCl3):
155.33 (C-90), 132.41 (C-60), 121.81 (C-70), 113.89
1H NMR (600 MHz, DMSO-d6):
d
7.76 (s, 4H, H-60), 7.25 (dd, J ¼ 13.4,
1H, H-4
J ¼ 25.8, 10.8 Hz, 2H, H-8
1.36 (m, 5H, H-8a, H-5
b
), 1.84 (ddd, J ¼ 13.6, 6.4, 3.3 Hz, 1H, H-5
a
6.9 Hz, 6H, H-70,NH), 6.95 (d, J ¼ 6.4 Hz, 2H, H-80), 5.35 (s, 1H, H-12),
a
, H-8
b
b
4.69 (d, J ¼ 3.2 Hz, 1H, H-10), 3.96e3.87 (m, 1H, H-a
a), 3.71e3.49
b
(m, 3H, H-a
H-9), 2.14 (td, J ¼ 14.2, 3.8 Hz, 1H, H-4
1.79e1.68 (m, 2H, H-5
), 1.60 (dt, J ¼ 26.0, 13.0 Hz, 2H, H-8
1.45 (d, J ¼ 10.2 Hz, 1H, 7 ), 1.37e1.17 (m, 6H, H-6, H-8a, H-5
14), 1.09 (dt, J ¼ 19.1, 6.8 Hz, 3H, H-5a), 0.91e0.59 (m, 8H, H-7
b
, H-20), 3.25e3.13 (m, 4H, H-10, H-b), 2.40e2.30 (m, 1H.
a
a
), 1.99e1.90 (m, 1H, H-4
, H-8
b
b
),
),
d
a
a
(C-80), 104.15 (C-3), 102.09 (C-10), 87.76 (C-12), 81.01 (C-12a), 67.56
(C-a), 55.47 (C-100), 52.41 (C-5a), 48.75 (C-b), 48.54 (C-10), 44.32 (C-
8a), 44.19 (C-120), 40.30 (C-20, C-110), 37.41(C-6), 36.26 (C-4), 34.43
(C-7), 30.72 (C-9), 26.01 (C-14), 24.61 (C-5), 24.48 (C-8), 20.24 (Cþ-
15, C-140), 12.87 (C-16); HRMS-APCI m/z: 713.469 [M þ H]
[C37H61N8O6: 713.444 calcd.].
b
b
, H-
, H-
d
165.73 (C-30), 163.89 (C-
a
15, H-16); 13C NMR (151 MHz, DMSO-d6):
40), 163.06 (C-oxalate), 140.11 (C-50), 128.39 (C-70), 121.83 (C-80),
120.05 (C-60), 103.27 (C-3), 101.03 (C-10), 86.96 (C-12), 80.54 (C-
12a), 63.35 (C-a), 51.94 (C-5a), 46.01 (C-10, C-b), 43.69 (C-8a), 36.78

480
T.T. Cloete et al. / European Journal of Medicinal Chemistry 76 (2014) 470e481
(C-20), 36.47 (C-6), 35.94 (C-4), 34.10 (C-7), 30.22 (C-9), 25.61 (C-
14), 24.29 (C-5), 23.80 (C-8), 20.11 (C-15), 12.55 (C-16); HRMS-APCI
m/z: 632.3540 [M þ H]þ [C34H46N7O5: 632.3582 calcd.].
26.6, 12.1 Hz, 6H, H-8
H-8a, H-14), 1.25e1.11 (m, 3H, H-5a), 0.99e0.70 (m, 14H, H-7
15, H-16); 13C NMR (151 MHz, CDCl3)
164.50 (C-oxalate), 164.07
a
, H-7
b
, H-8
b
), 1.49e1.26 (m, 12H, H-6, H-5
b,
a, H-
d
(C-30, C-5),161.86 (C-40),136.83 (C-80),128.53 (C-100),124.55 (C-110),
121.59 (C-90), 104.14 (C-3), 102.36 (C-10), 87.86 (C-12), 80.91 (C-
12a), 66.38 (C-70), 63.02 (C-a), 53.45(C-10), 52.30 (C-5a, C-b), 44.52
(C-60), 43.86 (C-8a), 37.16 (C-6), 36.21 (C-20,C-4), 34.22 (C-7), 30.42
(C-9), 25.98 (C-14), 24.41 (C-5, C-8), 20.41 (C-15), 13.00 (C-16);
HRMS-APCI m/z: 936.5439 [M þ H]þ [C49H73N7O11: 936.5378
calcd.].
5.4.3.11. 2-N-{2-[Bis(2-{10b-dihydroartemisinoxy}ethyl)amino]
ethyl}-4-N,6-N-diphenyl-1,3,5-triazine-2,4,6-triamine (oxalate) 24.
The preceding reaction also gave hybrid-dimer 24, which after
conversion resulted in an off-white powder salt (0.1 g, 2.5%); mp:
109.3 ꢁC; C51H71N7O10; IR (ATR)/cmꢀ1: 3290, 2941, 2876; 1H NMR
(600 MHz, CDCl3):
d
7.56 (d, J ¼ 24.3 Hz, 4H, H-60), 7.29 (t, J ¼ 7.9 Hz,
4H, H-70), 7.05 (q, J ¼ 7.1 Hz, 2H, H-80), 5.37 (d, J ¼ 16.7 Hz, 2H, H-12),
4.76 (d, J ¼ 5.6 Hz, 2H, H-10), 3.98 (d, J ¼ 4.7 Hz, 2H, H-a
a), 3.54 (dd,
Conflict of interest
J ¼ 57.1, 21.8 Hz, 4H, H-a
b
, H-20), 2.99 (d, J ¼ 124.6 Hz, 6H, H-b, H-10),
2.64e2.56 (m, 2H, H-9), 2.31 (dt, J ¼ 15.0, 10.8 Hz, 2H, H-4
a), 2.05e
The authors declare that they have no conflict of interest to
disclose.
1.96 (m, 4H, H-4
b
, H-5
), 1.56 (dd, J ¼ 13.0, 2.8 Hz, 2H, H-7
, H-14), 1.30e1.17 (m, 4H, H-6, H-5a), 0.93e0.82 (m, 14H, H-7
167.66 (C-30, C-40), 128.69
a
), 1.85e1.81 (m, 2H, H-8
a), 1.71e1.67 (m, 2H,
H-8
b
b
), 1.44e1.35 (m, 10H, H-8a,
H-5b
a
,
Disclaimer
H-15, H-16); 13C NMR (151 MHz, CDCl3):
d
(C-70), 123.44 (C-80), 120.59 (C-60), 104.34 (C-3), 102.17 (C-10), 87.93
(C-12), 80.34 (C-12a), 68.28 (C-a), 54.01 (C-10), 53.83 (C-b), 52.39
(C-5a), 43.97 (C-8a), 37.32 (C-20), 36.64 (C-4), 36.36 (C-6), 34.52 (C-
7), 30.60 (C-9), 25.85 (C-14), 24.88 (C-5), 24.37 (C-8), 20.32 (C-15),
Any opinion, findings and conclusions, or recommendations
expressed in this material are those of the authors and therefore
the NRF does not accept any liability in regard thereto.
12.94 (C-16); HRMS-APCI m/z: 942.5322 [M þ H]þ [C51H71N7O10
:
Acknowledgements
942.5262 calcd.].
This work was based upon research supported by the National
Research Foundation (NRF) of South Africa and the North-West
University, Potchefstroom Campus. We would also like to thank the
German Academic Exchange Service for the study bursary granted
to Mr TT Cloete, Mr A Joubert for NMR acquisition, and Dr J Jordaan
for MS analysis.
5.4.3.12. 2-N-(4-Methoxyphenyl)-4-N-phenyl-6-N-{2-[(2-{10b-dihy-
droartemisinoxy}ethyl)amino]ethyl}-1,3,5-triazine-2,4,6-triamine ox-
alate 25. The reaction of 1 (5.7 mmol, 2.2 g), K2CO3 (11.4 mmol,
1.6 g, 2 eq.) and 12 (5.7 mmol, 2 g, 1 eq.) in DMF (50 mL), followed
by chromatography with MeOH:DCM (1:4, v/v) and
MeOH:DCM:NH4OH (1:9:0.5, v/v/v), after conversion afforded 25
salt as a white powder (0.6 g, 14%); mp: 156.0 ꢁC; C35H47N7O6; IR
(ATR)/cmꢀ1: 3259, 2924, 2873,1710; 1H NMR (600 MHz, DMSO-d6):
Appendix A. Supplementary data
d
7.67 (d, J ¼ 85.8 Hz, 5H, H-70, H-110), 7.23 (d, J ¼ 6.5 Hz, 2H, H-80),
6.98e6.78 (m, 3H, H-90, H-120), 5.34 (s, 1H, H-12), 4.68 (d, J ¼ 3.4 Hz,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.040.
1H, H-10), 3.90 (d, J ¼ 4.9 Hz, 1H, H-a
a
), 3.71 (s, 3H, H-140), 3.57 (m,
H-20, H-a
b
, H2O), 3.25e3.18 (m, 4H, H-10, H-b), 2.36 (dd, J ¼ 7.2,
3.9 Hz, 1H, H-9), 2.14 (td, J ¼ 14.2, 3.7 Hz, 1H, H-4
a), 1.95 (d,
References
J ¼ 13.7 Hz, 1H, H-4
b
), 1.82e1.68 (m, 1H, H-5
), 1.44 (d, J ¼ 10.5 Hz, 1H, H-7 ), 1.36e1.17 (m, 7H, H-6, H-
, H-8a, H-14),1.12e1.03 (m,1H, H-5a), 0.83e0.67 (m, 8H, H-7 , H-
165.89 (C-30), 164.54
a), 1.63e1.52 (m, 2H, H-
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8
5
a
b
, H-8
b
b
a
15, H-16); 13C NMR (151 MHz, DMSO-d6):
d
(Oxalic acid), 163.92 (C-40, C-50), 154.57 (C-130), 128.38 (C-80), 121.95
(C-110), 121.67 (C-90), 119.88 (C-70), 113.64 (C-120), 103.14 (C-3),
101.14 (C-10), 87.27 (C-12), 80.32 (C-12a), 63.36 (C-a), 55.09 (C-140),
51.95 (C-5a), 46.14 (C-10, C-b), 43.74 (C-8a), 36.98 (C-20), 36.35 (C-6),
35.85 (C-4), 34.08 (C-7), 30.28 (C-9), 25.64 (C-14), 24.27 (C-5), 23.84
(C-8), 20.04 (C-15), 12.61 (C-16); HRMS-APCI m/z: 662.3656
[M þ H]þ [C35H48N7O6: 662.3688 calcd.].
5.4.3.13. 2-N-{2-[Bis(2-{10b-dihydroartemisinoxy}ethyl)amino]
ethyl}-6-(morpholin-4-yl)-4-N-phenyl-1,3,5-triazine-2,4-diamine
(oxalate) 26. The reaction of 1 (3.2 mmol, 1.2 g) and 13 (3.2 mmol,
1.0 g, 1 eq.) with K2CO3 (6.3 mmol, 0.9 g, 2 eq.) in DMF (40 mL),
followed by chromatography successively with MeOH:E-
tOAc:NH4OH (1:14:0.5, v/v/v) and EtOAc:PE:NH4OH (39:1:0.5, v/v/
v) afforded hybrid-dimer 26, which after conversion resulted in a
white salt powder (0.3 g,11%); mp: 143.7 ꢁC; C49H73N7O11; IR (ATR)/
cmꢀ1: 3273, 2940, 2914, 2875, 1730; 1H NMR (600 MHz, CDCl3)
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d
7.48 (d, J ¼ 7.9 Hz, 2H, H-90), 7.20e7.08 (m, 2H, H-100), 7.01 (t,
J ¼ 7.4 Hz, 1H, H-110), 5.36 (d, J ¼ 40.0 Hz, 2H, H-12), 4.76 (d,
J ¼ 3.4 Hz, 2H, H-10), 4.23e4.10 (m, 2H, H-a
a), 3.99e3.62 (m, 13H,
H-a
b
, H-60, H-20,H-70), 3.47 (td, J ¼ 24.4, 20.7, 17.9 Hz, 6H, H-10, H-b),
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J ¼ 16.0 Hz, 2H, H-4
b
), 1.90e1.77 (m, 2H, H-5
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