Synthesis and antimalarial activity of metal complexes of cross-bridged tetraazamacrocyclic ligands

Article abstract of DOI:10.1016/j.bmc.2014.05.003

Using transition metals such as manganese(II), iron(II), cobalt(II), nickel(II), copper(II), and zinc(II), several new metal complexes of cross-bridged tetraazamacrocyclic chelators namely, cyclen- and cyclam-analogs with benzyl groups, were synthesized and screened for in vitro antimalarial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum. The metal-free chelators tested showed little or no antimalarial activity. All the metal complexes of the dibenzyl cross-bridged cyclam ligand exhibited potent antimalarial activity. The Mn ²⁺ complex of this ligand was the most potent with IC₅₀s of 0.127 and 0.157 μM against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) P. falciparum strains, respectively. In general, the dibenzyl hydrophobic ligands showed better anti-malarial activity compared to the activity of monobenzyl ligands, potentially because of their higher lipophilicity and thus better cell penetration ability. The higher antimalarial activity displayed by the manganese complex for the cyclam ligand in comparison to that of the cyclen, correlates with the larger pocket of cyclam compared to that of cyclen which produces a more stable complex with the Mn²⁺. Few of the Cu²⁺ and Fe²⁺ complexes also showed improvement in activity but Ni²⁺, Co²⁺ and Zn²⁺ complexes did not show any improvement in activity upon the metal-free ligands for anti-malarial development.

Full text of DOI:10.1016/j.bmc.2014.05.003

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antimalarial activity of metal complexes
of cross-bridged tetraazamacrocyclic ligands
b
a
a
Timothy J. Hubin a, , Prince N.-A. Amoyaw , Kimberly D. Roewe , Natalie C. Simpson ,
⇑
Randall D. Maples a, TaRynn N. Carder Freeman a, Amy N. Cain a, Justin G. Le a, Stephen J. Archibald c,
Shabana I. Khan d, Babu L. Tekwani d, M. O. Faruk Khan b,
⇑
a Department of Chemistry, Southwestern Oklahoma State University, 100 Campus Drive, Weatherford, OK 73096, United States
b College of Pharmacy, Southwestern Oklahoma State University, 100 Campus Drive, Weatherford, OK 73096, United States
c Department of Chemistry, University of Hull, Cottingham Road, Kingston Upon Hull HU6 7RX, United Kingdom
d National Center for Natural Products Research, University of Mississippi, University, MS 38677, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Using transition metals such as manganese(II), iron(II), cobalt(II), nickel(II), copper(II), and zinc(II), several
new metal complexes of cross-bridged tetraazamacrocyclic chelators namely, cyclen- and cyclam-analogs
with benzyl groups, were synthesized and screened for in vitro antimalarial activity against chloroquine-
resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum. The metal-free chelators
tested showed little or no antimalarial activity. All the metal complexes of the dibenzyl cross-bridged
cyclam ligand exhibited potent antimalarial activity. The Mn2+ complex of this ligand was the most potent
Received 6 March 2014
Revised 25 April 2014
Accepted 2 May 2014
Available online xxxx
Keywords:
with IC50s of 0.127 and 0.157 lM against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) P.
Antimalarial agents
Metal complexes
Tetraazamacrocycles
Cross-bridged tetraazamacrocycles
Synthesis
falciparum strains, respectively. In general, the dibenzyl hydrophobic ligands showed better anti-malarial
activity compared to the activity of monobenzyl ligands, potentially because of their higher lipophilicity
and thus better cell penetration ability. The higher antimalarial activity displayed by the manganese com-
plex for the cyclam ligand in comparison to that of the cyclen, correlates with the larger pocket of cyclam
compared to that of cyclen which produces a more stable complex with the Mn2+. Few of the Cu2+ and Fe2+
complexes also showed improvement in activity but Ni2+, Co2+ and Zn2+ complexes did not show any
improvement in activity upon the metal-free ligands for anti-malarial development.
Published by Elsevier Ltd.
Biological activity
1. Introduction
antimalarial drugs has been of high priority for the control of
malaria. Over the past two decades, a number of antimalarial
Malaria is a major health problem, especially in the developing
countries1 of South Saharan Africa, Southeast Asia and in South
America. Today, almost all the existing antimalarial drugs, particu-
larly the 4-aminoquinoline based drugs, such as chloroquine (CQ)
that have historically been used to treat the disease effectively,
have been rendered less effective due to chemo-resistance prob-
lems.2 Artemisinin and its derivatives offer promising curative
alternatives but due to their thermal instability3,4 and high cost
of therapy5 in the economically disadvantaged regions of the world
where the disease is prevalent, access to them is difficult, and their
therapeutic use has been severely limited.
agents, particularly the 4-aminoquinoline-based drugs, have been
developed and tested against chloroquine-resistant parasites.6,7
Although there has been substantial improvement in the conven-
tional organic synthetic strategies used for the development of
antimalarial agents, researchers have sought out ways to develop
more innovative approaches in order to develop more efficacious
drugs to cure the disease. One of the most promising new
approaches involves the use of transition metal ion complexes to
produce novel antimalarial drugs.8
Metal-based chemotherapies have existed for centuries, but in
recent years there has been an increasing interest in the applica-
tion of transition metal complexes or organometallic complexes
in medicine and in other areas of biological sciences.9–12 Metal
complexes have been used as drugs in a variety of diseases, as
exemplified by the continued success of the platinum complex,
cis-PtCl2(NH3)2 (cisplatin), as an anticancer drug.13–15 This impor-
tant breakthrough has indeed stimulated a renewed interest in
Due to the prevalence of Plasmodium falciparum strains resistant
to chloroquine and other antimalarial drugs, the search for new
⇑
Corresponding authors. Tel.: +1 580 774 3026; fax: +1 580 774 3115 (T.J.H.);
tel.: +1 580 774 3064; fax: +1 580 774 7020 (M.O.F.K.).
E-mail address: faruk.khan@swosu.edu (M.O. Faruk Khan).
http://dx.doi.org/10.1016/j.bmc.2014.05.003
0968-0896/Published by Elsevier Ltd.
Please cite this article in press as: Hubin, T. J.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.003

2
T. J. Hubin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
metal complex based chemotherapy. Today, other metal-contain-
ing drugs have been developed in a variety of therapeutic areas
including malaria. To ensure that effective metal containing anti-
malarial agents are produced, the present study exploited the
metal-drug synergism approach.16–19
complexes have been extensively utilized in applications of a vari-
ety of diagnostic and magnetic resonance imaging (MRI) contrast
agents,34 their metal based derivatives are still relatively unex-
plored in antimalarial drug development. A recent report, in which
the 7-chloro-4-(1,4,7,10-tetraaza-cyclododec-1-yl)-quinoline-Zn2+
complex was found to demonstrate a high antimalarial activity
has motivated us to pursue the present investigation.35 We
hypothesized that the particularly kinetically inert transition metal
complexes of cross-bridged tetraazamacrocycles36–49 (Fig. 1, L1–
L3) would be stable, potentially active antimalarials against
drug-sensitive and -resistant strains of malarial parasites.35 These
complexes have several properties that suggested this activity to
us: (1) tight-binding metal complexation that will prevent loss of
the metal ion even in vivo; (2) aromatic functional groups repro-
ducing the aromaticity of chloroquine; and (3) multiple amine
functional groups, as also present in chloroquine.
The ligands L1–L3 in Figure 1 represent different combinations
of aromatic and tetraazamacrocycle units for screening to deter-
mine which combination of groups maximizes antimalarial effi-
cacy. Ligands L1 and L2 both contain two aromatic groups and
only one tetraazamacrocycle. Ligand L3 contains only one aromatic
group and two tetraazamacrocycles. L3 was also chosen because it
has proven to be biologically active against other disease states.
Bis-tetraazamacrocycles are potent CXCR4 antagonists, as exempli-
fied by the FDA approved drug Plerixafor. Cross-bridged analogs,
particularly in their transition metal complexes, have demon-
strated CXCR4 binding efficiency even superior to Plerixafor.50–54
Development of this route for biological activity has encouraged
us to explore other potential disease states which may benefit from
these cross-bridged bis-tetraazamacrocycle complexes. Finally, we
also screened six different metal ions in their complexes with each
ligand. This approach was taken to allow us to draw a conclusion
about which metal ion is most appropriate for inclusion in metal
complex antimalarials.
Thus far, several reports have shown that incorporation of tran-
sition metal ions into organic pharmacophores offer new opportu-
nities to design unique metal-containing compounds which
compliment the molecular diversity created by purely organic scaf-
folds.20,21 These reports show that the incorporation of transition
metal ions into rationally designed ligands can result in enhance-
ment of the biological activity.22 There are also several reports of
enhancement of the efficacy of existing drugs, for example, chloro-
quine, when transition metal ions were coordinated to the parent
drug structures.23 A thorough literature review revealed that sev-
eral transition metal complexes exhibit high antimalarial activity
against chloroquine-sensitive and -resistant strains of P. falciparum
and, consequently, have become antimalarial drug candidates. It is
also well documented that many metal complexes of chloroquine
or other 4-aminoquinoline based antimalarials have activities
superior to that of chloroquine, which is one of the most successful
drugs currently being used for antimalarial chemotherapy. The
consistent enhancement of these drugs when coordinated to metal
ions reinforces the fact that metal complexes are important
resources for the generation of structural or chemical diversity in
the area of antimalarial drug development.20,23
In a recent report, a gold-chloroquine antimalarial agent was
obtained by coordinating chloroquine (CQ) to a [Au(PPh3)]+ frag-
ments to give a new compound, [Au(PPh3)(CQ)]PF6, which is more
activethanCQaloneinvitroagainstculturesofchloroquineresistant
strains of P. falciparum and also against Plasmodium berghei both
in vitro and in vivo.23 Also, it has been reported that the coordination
complexes of gold(I) and thiosemicarbazone exhibited enhanced
antiplasmodial activity against two P. falciparum strains (D10 and
W2) in vitro when compared to the activity of the ligand alone.24
Many other enhancements of antimalarial activity by metal
complexation of a variety of ligands compared to the antimalarial
activity of the free ligands themselves have been extensively doc-
umented. For example, a reaction of the chloroquine (CQ) free base
with [Rh(COD)Cl]2 (COD = 1,5-cyclooctadiene) yielded Rh(COD)
(CQ)Cl, which has comparable in vitro antimalarial activity to that
of chloroquine diphosphate and reduced parasitemia 1.33 times
more than chloroquine in vivo, without any sign of acute toxicity
observed up to 30 days.25,26
2. Results and discussion
2.1. Chemistry
The ligands L1, L2 and L3 have all been published previously.55
Transition metal complexes of these ligands were generally formed
from anhydrous metal salts in nonprotic solvents in an inert atmo-
sphere glove box. Mn2+, Fe2+, and Co2+ divalent complexes are often
air sensitive and must be protected from oxygen. Additionally, it is
known that protection of cross-bridged tetraazamacrocycle ligands
from sources of water, or even protic solvents, is helpful in com-
plexation reactions as they are strongly basic and protonation
can defeat complexation.
It has been documented that because of the avidity of Plasmo-
dium parasites for free iron, one way of using iron in antimalarial
drug design is by adding iron to an existing drug such as chloro-
quine to effectively remove the chloroquine resistance.27–29 Sev-
eral studies have shown that organometallic complex based drug
molecules are very promising in overcoming various types of drug
resistance and allow improved specificity and drug targeting, thus
minimizing side effects associated with chemotherapy.30 For
example, ferrocene serves as a reliable organometallic scaffold
for the construction of an ordered structure via hydrogen bond-
ing.31,32 It is worth noting that as a consequence of this drug devel-
opment effort, the novel antimalarial drug, ferroquine (FQ), in
which ferrocene group is incorporated into chloroquine structure,
is being developed at Sanofi-Avantis.33
2.1.1. Manganese complex [Mn2(L3)Cl4]
This complexation reaction is representative of all of the L3
complexes. Complexation of the ligand was carried out using the
anhydrous metal chloride salt in anhydrous DMF in an inert atmo-
sphere glove box (Scheme 1). The anhydrous MnCl2 is not very sol-
uble at room temperature in DMF, so the reaction was heated to
ꢀ50 °C and it proceeded smoothly at this temperature with stirring
overnight. This complex is air sensitive, as indicated by formation
of a brown solution when the reaction solution was exposed to air.
In order to circumvent drug resistance caused by the P. falcipa-
rum parasites, we have employed the metal coordination approach
to prepare series of metal complexes using cross-bridged and side-
bridged tetraazamacrocyclic ligands L1, L2, and L3 (Fig. 1). The
effect of metal coordination on the ligands was investigated for
antimalarial activity.
2.1.2. Copper complex [Cu(L1)(OAc)]PF6
This complexation is representative of all of the dibenzyl mac-
rocycle complexes of L1 and L2. Complexation of the ligand was
carried out using the anhydrous metal acetate salt in anhydrous
DMF in an inert atmosphere glove box and proceeded smoothly
at room temperature with overnight stirring (Scheme 2). Once
the complexation had occurred, the reaction solution was removed
Even though the synthetic tetraazamacrocyclic compounds,
particularly, cyclen and cyclam, and their analogs or their metal
Please cite this article in press as: Hubin, T. J.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.003

T. J. Hubin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
N
Cl
CH3
O
O
O
H
NH HN
NH HN
H3C
N
NH
H
NH HN
NH HN
H3C
O
O
H
Cyclen
H3C
H3C
CH3
Cyclam
Artemisinin
Chloroquine
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H3C
L3
CH3
L2
L1
Figure 1. Structures of ligands used for the preparation of complexes and standard drugs (chloroquine and artemisinin) used as controls.
N
Mn
N
N
Mn
N
N
N
N
N
N
N
N
N
N
Cl
Cl
Cl
Cl
N
N
N
CH3
H3C
CH3
H3C
[Mn(L3)Cl4]
(60%)
L3
Scheme 1. Synthetic procedure of metal complex [Mn(L3)Cl4]. Reagents and conditions: MnCl2, anhydrous DMF, 50 °C, 18 h, 60%.
from the glove box and concentrated to dryness, which yielded a
viscous oil product. In order to produce a more easily handled solid
complex, as well as to purify the product, an anion metathesis
reaction with NH4PF6 in dry methanol was carried out. With only
two cis coordination sites available, only one acetate anion was
able to coordinate to the metal ion, leaving an uncoordinated ace-
tate anion that could be replaced with PF6À. Hexafluorophosphate
anions precipitated the complex cation from methanol as a micro-
crystalline powder.
RPMI-1640 medium supplemented with 10% human serum. Inhibi-
tion of the uptake of [3H] hypoxanthine served as a measure of par-
asite viability56,57 and the potencies of all the test compounds
against chloroquine resistant strains of P. falciparum were also con-
firmed by [3H] hypoxanthine incorporation. Artemisinin and chlo-
roquine were used as drug controls and DMSO was included as a
vehicle control. The selectivity index (SI) and cytotoxicity to mam-
malian cells were also determined.
As shown in Table 1, while the Mn2+ and Co2+ complexes of the
dibenzyl cross-bridged cyclam ligand L1 demonstrated a higher
antimalarial activity against the chloroquine resistant (W2) strain
of the parasite than the D6 strain, the Fe2+, Ni2+, Cu2+, and Zn2+
complexes of the same ligand all showed lower antimalarial activ-
ity towards W2 than towards D6. The manganese complex is
clearly the most active complex against both strains of the malarial
parasites, with nearly an order of magnitude greater SI than some
of the other metals. In fact, this manganese complex was the most
active of all of the compounds tested.
2.2. Biological evaluation
2.2.1. In vitro antimalarial activity
Both the free ligands and their metal complexes were tested for
in vitro antimalarial activity against the D6 (chloroquine sensitive)
and W2 (chloroquine resistance) strains of P. falciparum. The test
performed was based on the determination of plasmodial lactate
dehydrogenase (LDH) activity. Varying concentrations of the test
samples of either the complexes or the free ligands were added
to a suspension of red blood cells infected with P. falciparum (D6
or W2) strains containing 2% parasitemia and 2% hematocrit in
For the metal complexes of the dibenzyl cross-bridged cyclen
ligand L2, the Cu2+ complex showed a higher antimalarial activity
against the chloroquine resistant (W2) strain of the parasite, while
+
N
1
2
N
N
N
N
O
N
-
PF6
Cu
N
N
O
L1
[Cu(L1)(OAc)]PF6
(83%)
Scheme 2. Synthetic procedure for [Cu(L1)(OAc)]PF6. Reagents and conditions: (1) Cu(OAc)2, anhydrous DMF, rt, 18 h. (2) NH4PF6, MeOH.
Please cite this article in press as: Hubin, T. J.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.003

4
T. J. Hubin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 1
which is the most potent, is 4.24 times less potent than chloro-
quine and 6.54 times less potent than artimisinin against the chlo-
roquine-sensitive (D6) strains of P. falciparum. However, it also
shows signs of toxicity as indicated by the Vero IC50 value. It is also
worth noting that this complex is superior to chloroquine and it is
5.35 times more potent than chloroquine against the chloroquine-
resistant (W2) strain of P. falciparum although it is 32 times less
potent than artimisinin.
These results suggest that the higher antimalarial activity dis-
played by the manganese complex for the cyclam ligand compared
to that of the cyclen may be due to the larger pocket of the cyclam
which makes it form more stable complexes with manganese.40
The cavity size difference also results in adjustments in the redox
properties of the two complexes,45 which may also be important,
depending on the mechanism of action. We should be able to tune
multiple properties of these complexes by adjusting the cavity size
and changing the benzyl substituents to other groups. Interest-
ingly, the manganese complex of L1 exhibits superior antimalarial
activity against the chloroquine-resistant strains of P. falciparum in
comparison to chloroquine.
In vitro antimalarial activities of ligands and complexes against D6 and W2 strains of
Plasmodium falciparum
Compound
D6 IC50
M)
D6 SI W2 IC50
M)
W2 SI Vero IC50 (ng/
mL)
(
l
(l
L1
2.679
0.157
0.324
NA
0.825
0.423
0.342
1.350
0.265
1.384
NA
1.110
0.147
2.057
1.278
0.957
NA
>4.4
4.358
>2.7
NC
Mn(L1)Cl2
Fe(L1)Cl2
38.45 0.127
>27.6 0.426
47.793 3222.8
>21
NC
NC
NC
NC
[Co(L1)(OAc)]PF6
[Ni(L1)(OAc)]PF6
[Cu(L1)(OAc)]PF6
[Zn(L1)(OAc)]PF6
L2
—
>8.6
5.162
1.326
>1.4
>5.4
>8.7
>16.7 0.809
>20.6 0.593
>11.9 NC
>6.3 NC
>17.3 NC
>9.3
2.008
Mn(L2)Cl2
Fe(L2)Cl2
>35.6 0.545
>6.8
—
2.139
NA
>4.4
—
>4.8
NC
NC
NC
[Co(L2)(OAc)]PF6
[Ni(L2)(OAc)]PF6
[Cu(L2)(OAc)]PF6
[Zn(L2)(OAc)]PF6
L3
[Mn2(L3)Cl4]
Fe(L3)2Cl4
[Co2(L3)(OAc)2](PF6)2 NA
[Ni2(L3)(OAc)2](PF6)2 NA
[Cu2(L3)(OAc)2](PF6)2 NA
[Zn2(L3)(OAc)2](PF6)2 NA
>6.7
1.538
>50.3 0.312
>23.6 NC
>3.6
>6.4
>6
—
3.814
1.414
0.959
NA
>1.9
>5.8
>5.9
—
—
—
NC
NC
NC
NC
NC
NC
NC
NC
—
NA
—
NA
The fact that all the free ligands tested showed fairly low anti-
malarial activity, suggests that the metal complexation may have
played a critical role in making the complexes exhibit antimalarial
activity, rather than the roles played by the free ligand or the metal
alone.22 Additionally, the generally low cytotoxicity of these
ligands against mammalian cells, also suggests that the ligands
are attractive for further development of new metal complex based
antimalarial drugs.22
—
NA
—
—
—
NA
Artemisinin (ART)
Chloroquine (CQ)
0.024
0.037
>>35.5 0.004
20.4 0.679
>211.4 NC
>1.1 NC
NC, no cytotoxicity up to about 10 lM; NA, no activity; SI, selectivity index (IC50 for
Vero cells/IC50 for P. falciparum).
the Mn2+ complex demonstrated a lower, but comparable antima-
larial activity against the parasites. The Fe2+, Co2+, Ni2+, and Zn2+
complexes were generally less effective against the chloroquine
3. Conclusions
resistant parasite, and less active overall than either Mn2+ or Cu2+
.
The screening of the antimalarial activity metal complexes of
the tetraazamacrocyclic compounds generated by reactions of
cyclam or cyclen cross-bridged and side-bridged ligands with the
All of the metal complexes showed low to moderate antimalarial
activity against the chloroquine sensitive (D6) strain of the parasite.
Bis-macrocyclic ligand L3 demonstrated an activity towards
both malaria strains similar to that of the monocyclic dibenzyl
ligands L1 and L2. Interestingly, the ligand made from the combina-
tion of two tetraazamacrocycles and one aromatic group (L3) exhib-
its nearly the same activity as the combination of two aromatic
groups and one tetraazamacrocycle, at least for the free ligands
themselves. However, a striking difference is seen when comparing
the transition metal complexes of L3. Only the manganese complex
of L3 was active against either malarial strain. Why the linking of
two such macrocycle complexes together greatly reduces the activ-
ity is not readily apparent, although the higher positive charge car-
ried by these bimetallic complexes may prevent them from readily
crossing the parasite’s membranes. It is also worth emphasizing,
once again, the superior activity of the manganese complex, com-
pared to the other metal ions. This appears to be a general trend
of these tetraazamacrocyclic complexes, and is one that we hope
to test further in future studies on other manganese analogs.
With regard to the cyclam L1 analogs, the activity ratio of metal
complex:free ligand, with the ligand value normalized to 1.00 as cal-
culatedby dividingtheIC50 (free ligand)/IC50 (metal complex)for the
antimalarialactivityagainst chloroquine sensitivity (D6) strains of P.
falciparum was in the range of 3.25–17.1. For the antimalarial activ-
ity against chloroquine-resistant (W2) strains of P. falciparum, the
ratio was in the range of 3.29–34.3. The increasing order of potency
of antimalarial activity (W2 and D6) for these complexes is Co2+ < -
Ni2+ < Cu2+ < Zn2+ < Fe2+ 6 Mn2+. In contrast, for the cyclen L2 ana-
logs, the same ratio for the D6 strains of the P. falciparum, was in
the range of 0.656–9.18 and for the W2 strains of the P. falciparum,
the ratio was in the range of 0.526–6.44; with the copper complex
exhibiting the most potency followed by the manganese complex.
In comparison of these complexes to the positive controls, chlo-
roquine and artemisinin, the cyclam based manganese complex
respective transition metal ions of Mn2+, Fe2+, Co2+, Ni2+, Cu2+
,
and Zn2+, was carried out. The results revealed that coordination
of a transition metal ion to a biologically active molecule may
result in an improvement of the activity of the resulting molecule
depending on the identity of the metal ion chosen. As shown in
Table 1, this trend is illustrated by the coordination of Mn2+, Fe2+
,
Co2+, Ni2+, Cu2+, and Zn2+ ions to the dibenzyl cross-bridged cyclam
ligand L1, which produced complexes with increased potency,
whereas only the Co2+ complex of the same ligand L1 did not result
in an enhancement of activity compared to the free ligand. This
data demonstrates that although the biological activity of a mole-
cule can be enhanced through metal ion coordination, the
improvement occurs only when a suitable metal–ligand pairing
is made. Interestingly, only Cu2+ and Mn2+ enhanced the activity
of L2, emphasizing that a suitable metal–ligand pairing is required.
The same metals (Fe2+, Ni2+ and Zn2+) which enhance the activity of
L1 do not enhance potency when incorporated into a very similar
analog, L2. Apparently, there is a fine balance between the proper-
ties of the organic ligand and the metal ion that must be optimized
to produce enhancement in biological activity. Finally, the metal
complexes generated using ligand L3, are mostly inactive. The
use of two tetraazamacrocycle pharmacophores and only one aro-
matic group does not appear to lead to improved antimalarial
activity for the resulting metal complexes, even though the ligand
itself has activity similar to L1 and L2, and L3 complexes are known
to have other biological activity.50–54 Perhaps the additional posi-
tive charge and polarity accompanying the second metal ion are
too much to overcome in order to get the drug molecule into the
parasite. It should be noted, however, that the Mn2+ complex of
L3 does in fact increase its activity. In all three ligand cases, Mn2+
improves the activity. This property of Mn2+ will be explored fur-
ther with other tetraazamacrocyclic ligands.
Please cite this article in press as: Hubin, T. J.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.003

T. J. Hubin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Based on the findings of this investigation, a significant insight
into the design of new transition metal based antimalarial drugs
was obtained. These results will support the further development
of this promising new class of compounds with potential for the
development of new drug(s) for malaria chemotherapy that may
contribute to improving future therapeutic potentials. Further-
more, although certainly not an exhaustive survey, certain metal
ions which were found to be suitable for the design of the new
metal complexes of tetraazamacrocyclic derivatives for malaria
chemotherapy have been identified. Depending on the ligand used,
manganese(II), copper(II), and iron(II) have shown to be the most
suitable metals that can form the basis for rational antimalarial
drug design.
solution. Overnight stirring at this temperature resulted in the for-
mation of a white precipitate. The reaction was stopped after stir-
ring for ꢀ18 h and cooled to room temperature. Inside the glove
box, the white solid product was collected by filtration on a fine
glass frit and washed with DMF followed by ether, then dried
under vacuum to give the product (0.256 g, 60%). FAB+ mass spec-
tral analysis yielded peaks at m/z = 797, consistent with Mn2LCl+3,
and m/z = 379, consistent with Mn2L3Cl22+. Elemental analysis (%)
Calcd [Mn2C34H62N8Cl4] (834.599 g/mol):
13.43; Found C 48.54, H 7.59, N 13.24.
C 48.93, H 7.49, N
4.3.2. Synthesis of acetato(4,11-dibenzyl-1,4,8,11-tetraazabicy
clo[6.6.2]hexadecane)copper(II)hexafluorophosphate
[Cu(L1)(OAc)]PF6
4. Experimental section
4.1. Chemistry
Ligand L1, (0.406 g, 1.00 mmol) and anhydrous copper(II) ace-
tate salt (0.182 g, 1.00 mmol) were added to 25 ml of dry DMF in
an inert atmosphere glove box. The reaction was stirred at room
temperature for 18 h. The crude blue-green [Cu(L1)(OAc)][(OAc)]
solution was removed from the glove box, filtered to remove any
trace solids, and evaporated to dryness, giving a blue-green oil.
The crude product was dissolved in 10 ml of dry methanol, to
which was added dropwise, a 5 ml dry methanol solution of 5
equiv (0.815 g, 5.00 mmol) of NH4PF6. A blue-green powder of
the [Cu(L1)(OAc)]PF6 salt precipitated immediately, and was stored
overnight in a freezer at À5 °C to complete the precipitation. The
solid was collected on a fine glass frit, washed with cold methanol
and ether, and dried under vacuum to give the final product
(0.573 g, 83%). Elemental analysis (%) Calcd [CuC26H38N4(C2H3O2)]-
PF6ÁH2O (692.185 g/mol): C 48.59, H 6.26, N 8.09; Found C 48.65, H
6.18, N 8.21. MS (ES) m/z 528.3 and 530.3 [CuL(OAc)]+.
4.1.1. General
All the materials were reagent grade, and used as supplied. The
reactions were performed in anhydrous solvents under inert atmo-
sphere unless otherwise indicated. Anhydrous solvents (acetoni-
trile and DMF) as well as all other reagents were used as
received from a commercial source. Elemental analysis was carried
out by Quantitative Technologies, Inc. in Whitehouse, NJ. Electro-
spray MS was obtained using a Shimadzu LCMS-2020 in 1:1 meth-
anol/water mixture. 1H and 13C NMR spectra were recorded at
300 MHz and 75 MHz, respectively on Bruker 300 spectrometer
with TMS as internal standard.
4.2. Synthesis of ligands
4.4. Biological evaluation
Ligand L1, 4,11-dibenzyl-1,4,8,11-tetraazabicyclo[6.6.2]hexa-
decane and L2, 4,10-dibenzyl-1,4,7,10-tetraazabicyclo[5.5.2]
tetradecane were prepared as outlined in reference.55 Ligand L3, 4-
methyl-11-[4-(4-methyl-1,4,8,11-tetraaza-bicyclo[6.6.2]hexadec-
11-ylmethyl)-benzyl]-1,4,8,11-tetraaza-bicyclo[6.6.2]hexadecane
was prepared according to the literature procedure.58
4.4.1. In vitro antimalarial activity
The in vitro antimalarial activity was measured by a colorimet-
ric assay that determines the parasitic lactate dehydrogenase
(pLDH) activity. The assay was performed in a 96-well microplate
using two P. falciparum clones, [Sierra Leone D6 (chloroquine-sen-
sitive) and Indochina W2 (chloroquine-resistant)]. For the assay, a
suspension of red blood cells infected with P. falciparum (D6 or W2)
4.3. General synthetic procedure for complexes M(L)Cl2 and
[M(L)(OAc)]PF6
strains (200
1640 medium supplemented with 10% human serum and 60
mL amikacin) was added to the wells of a 96-well plate containing
10 L of test samples at various concentrations. The plate was
l
L, with 2% parasitemia and 2% hematocrit in RPMI-
lg/
Reactions of MnCl2 or FeCl2 with ligands L1 and L2 in a 1:1 ratio
under mild conditions in a glove box gave the complexes Mn(L)Cl2
and Fe(L)Cl2, respectively.45 The complexes were isolated in good
yields as air stable white solid Mn(L)Cl2 and/or pale brown
Fe(L)Cl2. Complexes [M(L)(OAc)]PF6, where M = Cu, Zn, Ni, and
Co, were also synthesized in two steps by first reacting the appro-
priate metal acetates with the ligands L1, L2, and L3 in CH3CN or
DMF at room temperature for 24 h, followed by solvent removal
to give an oily product which was subsequently dissolved in meth-
anol and reacted with 5 equiv of NH4PF6 to give the desired com-
plexes.58 All these new complexes were characterized by
electrospray, MS, and elemental analyses. Complexes Mn(L1)Cl2,
Fe(L1)Cl2, Mn(L2)Cl2 and Fe(L1)Cl2 were synthesized45,58 and com-
plex [Ni(L1)OAc]PF6 was synthesized according to the literature
procedure.59
l
flushed with a gas mixture of 90% N2, 5% O2, and 5% CO2 in a mod-
ular incubation chamber (Billups-Rothenberg, 4464 M) and incu-
bated at 37 °C for 72 h. Plasmodial LDH activity was determined
by using Malstat reagent (Flow Inc., Portland, OR) as described ear-
lier.56,57 The IC50 values were computed from the dose–response
curves generated by plotting percent growth against test concen-
trations. DMSO (0.25%), artemisinin, and chloroquine were
included in each assay as vehicle and drug controls, respectively.
The selectivity indices (SI) were determined by measuring the
cytotoxicity of samples toward mammalian cells (Vero; monkey
kidney fibroblast).
4.4.2. Cytotoxicity assay
The in vitro cytotoxic activity was determined against noncan-
cerous Vero cell lines (monkey kidney fibroblast), obtained from
the American Type Culture Collection (ATCC, Rockville, MD). The
assay was performed in 96-well tissue culture treated microplates.
Cells (25000 cells/well) were seeded in the wells of the plate and
incubated for 24 h. Samples were added and plates were again
incubated for 48 h. The number of viable cells was determined
using Neutral Red according to a modification of the procedure of
Borenfreund et al.60,61
4.3.1. Synthesis of tetrachloro(4-methyl-11-[4-(4-methyl-
1,4,8,11-tetraaza-bicyclo[6.6.2]hexadec-11-ylmethyl)-benzyl]-
1,4,8,11-tetraaza-bicyclo[6.6.2]hexadecane)manganese(II)
[Mn2(L3)Cl4]
The ligand, L3 (0.300 g, 0.511 mmol) and anhydrous manga-
nese(II) chloride salt (0.129 g, 1.02 mmol) were added to 20 ml of
dry DMF in an inert atmosphere glove box. Upon stirring and
heating (ꢀ50 °C) both reactants dissolved to give a colorless
Please cite this article in press as: Hubin, T. J.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.003

6
T. J. Hubin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
30. Biot, C.; Castro, W.; Botte, C. Y.; Navarro, M. Dalton Trans. 2012, 41, 6335.
Acknowledgments
31. Allardyce, C. S.; Butler, P. A.; Dyson, P. J.; Fontecilla-Camps, J. C.; Krautler, B.;
Hirao, T.; Le Maux, P.; Moriuchi, T.; Severin, K.; Simonneaux, G.; Volbeda, A. In
This project as well as the publication was supported by the
National Institute of General Medical Sciences of the National Insti-
tutes of Health through Grant Number 8P20GM103447 (T.J.H. and
M.O.F.K.). T.J.H. acknowledges the Research Corporation (CC6505),
the Oklahoma Center for the Advancement of Science and Technol-
ogy (HR13-157), and the Henry Dreyfus Teacher-Scholar Awards
Program for support of this work.
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