The anti-malarial activity of bivalent imidazolium salts

Article abstract of DOI:10.1016/j.bmc.2011.06.002

A series of compounds containing bivalent imidazolium rings and one triazolium analog were synthesized and evaluated for their ability to inhibit the replication of Plasmodium falciparum cultures. The activity and selectivity of the compounds for P. falciparum cultures were found to depend on the presence of electron-deficient rings that were spaced an appropriate distance apart. The activity of the compounds was not critically dependent on the nature of the linker between the electron-deficient rings, an observation that suggests that the rings were responsible for the primary interaction with the molecular target of the compounds in the parasite. The bivalent imidazolium and triazolium compounds disrupted the process whereby merozoites gain entry into erythrocytes, however, they did not appear to prevent merozoites from forming. The compounds were also found to be active in a murine Plasmodium berghei infection, a result consistent with the compounds specifically interacting with a parasite component that is required for replication and is conserved between two Plasmodium species.

Full text of DOI:10.1016/j.bmc.2011.06.002

Bioorganic & Medicinal Chemistry 19 (2011) 6525–6542
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The anti-malarial activity of bivalent imidazolium salts
Jason Z. Vlahakis a, Simona Mitu a, Gheorghe Roman a, E. Patricia Rodriguez c, Ian E. Crandall b,
,
⇑
Walter A. Szarek a,
⇑
a Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6
b Department of Pharmaceutical Sciences, University of Toronto, and The Sandra Rotman Laboratories, McLaughlin-Rotman Centre, Toronto, Ontario, Canada M5S 1A8
c Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of compounds containing bivalent imidazolium rings and one triazolium analog were synthe-
sized and evaluated for their ability to inhibit the replication of Plasmodium falciparum cultures. The
activity and selectivity of the compounds for P. falciparum cultures were found to depend on the presence
of electron-deficient rings that were spaced an appropriate distance apart. The activity of the compounds
was not critically dependent on the nature of the linker between the electron-deficient rings, an obser-
vation that suggests that the rings were responsible for the primary interaction with the molecular target
of the compounds in the parasite. The bivalent imidazolium and triazolium compounds disrupted the
process whereby merozoites gain entry into erythrocytes, however, they did not appear to prevent mer-
ozoites from forming. The compounds were also found to be active in a murine Plasmodium berghei infec-
tion, a result consistent with the compounds specifically interacting with a parasite component that is
required for replication and is conserved between two Plasmodium species.
Received 15 February 2011
Revised 26 May 2011
Accepted 2 June 2011
Available online 16 June 2011
Keywords:
Plasmodium falciparum
Imidazolium salts
Triazolium salts
Malaria
Selective inhibitors
Antimalarial drugs
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
share the same mechanism of action. Triazolium and imidazolium
structures are preferable in biological systems because they cannot
The development and spread of drug resistance is a major con-
cern in the current efforts to treat malaria. Therefore, there is a
continuing need to develop new antimalarial compounds,1 partic-
ularly those having a novel mechanism of action2 based on novel
chemical structures. Recent estimates suggest that as many as
3.2 billion people live in areas with a risk of malaria transmission,
with the heaviest burden occurring in the low-resource regions of
sub-Saharan Africa.3 Antimalarial agents capable of widespread
use in the developing world need to be inexpensive, effective,
and safe even when used repeatedly by children and pregnant wo-
men. Unfortunately, many of the agents currently in use do not
meet these criteria. We have previously observed that tetrazolium,
triazolium, and imidazolium salts are preferentially toxic to Plas-
modium falciparum cultures4,5 because they appear to interfere
with a step involved in the invasion of merozoites into erythro-
cytes. The structural motif that appears to make active agents both
potent and selective towards Plasmodium cultures is comprised of
an electron-deficient ring surrounded by hydrophobic side-groups.
This motif was originally examined using tetrazolium-based struc-
tures,4 however, compounds based on triazolium and imidazolium
rings were found to be equally selective and potent5 and appear to
be easily reduced to a neutral structure, as is the case with
tetrazolium structures which yield a neutral formazan, and hence
triazolium- and imidazolium-based compounds are amenable to
evaluation of their antimalarial potential in a malaria animal
model.
When triazolium- or imidazolium-containing compounds are
added to P. falciparum cultures they inhibit the Plasmodium
merozoite invasion process.5 The initial events that allow a mero-
zoite to recognize and tether itself to an erythrocyte are thought to
be mediated by proteins present on the surface of the merozoite,
particularly MSP-1. Merozoite Surface Proteins (MSPs) consist of
a family of proteins with at least 10 members.6 MSPs-1, -2, -4,
-5, -8, and -10 are anchored to the merozoite membrane by a
glycosylphosphatidylinositol (GPI)-lipid anchor, and the anchor
also functions as a toxin that leads to host signalling through TLR
molecules.7 MSP-1 is a prime candidate for mediating the initial
adhesion event8 as MSP-1 is the most abundant protein on the
surface of the merozoite.9 MSP-1 is synthesized as
a large
(180–250 kDa) protein which then undergoes proteolytic process-
ing until a 19 kDa fragment, referred to as MSP-119, is left on the
GPI anchor. The role of MSP-119 in merozoite invasion remains
controversial, however, transfection studies have established that
MSP-1 is required for merozoite survival10 and therefore MSP-119
is currently a candidate for merozoite invasion-blocking vac-
cines.11 The exact mechanism by which tetrazolium, triazolium,
⇑
Corresponding authors. Tel.: +1 613 533 2643 (W.A.S.); +1 416 978 6627 (I.E.C.).
E-mail addresses: ian.crandall@utoronto.ca (I.E. Crandall), walter.szarek@chem.
queensu.ca (W.A. Szarek).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.002

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
and imidazolium salts impair merozoite invasion is unknown;
however, their capacity to inhibit P. falciparum cultures was origi-
nally examined because the electron-deficient rings in tetrazolium
salts have been observed to form tight complexes with sulfated or
phosphorylated glycans,4 some of which can also be potent inhib-
itors of the invasion process. Our current hypothesis is that tetra-
zolium, triazolium, and imidazolium compounds function by
interfering with a phosphorylated moiety in a hydrophobic envi-
ronment and that this target is either the parasite ligand itself, or
is an essential component of the merozoite invasion process.5 This
description appears to fit the GPI anchor of the MSP proteins and
therefore it is possible that tetrazolium, triazolium, and imidazoli-
um salts inhibit merozoite invasion by interfering with the synthe-
sis or function of the GPI anchor on MSP molecules. If GPI anchors,
or other potential targets, are present in merozoites at a suffi-
ciently high density, then bivalent compounds, that is, compounds
containing two electron-deficient rings, should be able to interact
with two target motifs simultaneously if the active centers, that
is, the electron-deficient rings, are spaced sufficiently far apart. In
the present work we have explored the effect of combining two
such centers into a single molecule to produce bivalent com-
pounds. Bivalency should have the beneficial effect of permitting
the compound to interact with two target domains at once, leading
to a higher avidity-interaction with the Plasmodium cultures. In
this article we report the synthesis and evaluation of bivalent ana-
logs of a triazolium salt and of imidazolium salts having alkyl spac-
ers of varying lengths as well as varying types of spacers. The
activity of the compounds was found to depend strongly on the
length of the spacer, but less so on the nature of the spacer.
by treatment with H3PO4–P2O5–KI. 1,15-Diiodopentadecane and
1,16-diiodohexadecane were prepared from pentadecanolide and
hexadecanolide, respectively, by reduction of the cyclic ester using
LiAlH4 in THF,13 followed by treatment of the resulting diol with
H3PO4–P2O5–KI. The bis-1,2,4-triazole compound 38 was prepared
by treatment of 1,12-dibromododecane with 1,2,4-triazole–NaOH–
DMSO.
Compounds 27 and 29, having phenyl-group linkers, were pre-
pared from 1,4-dibromobenzene and 4,40-dibromobiphenyl,
respectively, by treatment with imidazole–K2CO3–CuSO4.14 Simil-
arily, the benzimidazolium analog 35 was prepared from 4,40-
dibromobiphenyl by treatment with benzimidazole–K2CO3–CuSO4.
Compound 31 was prepared by the treatment of 4,40-bis(chloro-
methyl)-1,10-biphenyl with imidazole–NaOH–DMSO. Compound
36 was prepared by the dialkylation of hydroquinone using
1,3-dibromopropane, followed by treatment of the resulting
dibromide with imidazole–NaOH–DMSO.
Alkylation of 27, 29, 31, 36, and 38 using iodomethane in 1-pro-
panol gave the salts 28, 30, 32, 37, and 39, respectively; the
reaction of 11 with iodoethane or iodopropane afforded the corre-
sponding salts 25 and 26. Treatment of 29 with benzyl bromide in
1-propanol gave 33, whereas treatment of 29 with 4-nitrobenzyl
bromide in 1-propanol gave 34.
2.2. Biological evaluation
2.2.1. Structure–activity relationships
Previous work4,5 suggested that select tetrazolium, triazolium,
and imidazolium compounds had anti-Plasmodium activity in cul-
tures because they contained electron-deficient rings substituted
by hydrophobic side groups. To determine if combining two elec-
tron-deficient centers in one molecule produced molecules that
would selectively interact with Plasmodium cultures with a high
avidity, a series of compounds that consisted of either imidazole
or imidazolium rings linked by alkyl chains having 4, 6, 8, 10–16,
18, and 20 carbon atoms were synthesized (Table 1). The activity
of compounds 1–24 in P. falciparum and mammalian (CHO) cell
cultures was then determined (Table 1). Increasing the length of
the alkyl chain in the bivalent (neutral) imidazole compounds led
to a moderate decrease in the IC50 values observed in P. falciparum
and CHO cultures (Fig. 1, top-left panel). Further, similar IC50 val-
ues were observed in both culture systems indicating that these
compounds were not preferentially toxic to malaria parasites. In
contrast, bivalent imidazolium compounds with spacers consisting
2. Results
2.1. Chemical synthesis
The synthesis of the bis-imidazolium salts is shown in Scheme
1, and involved the conversion of an
a,x-dihaloalkane into a bis-
imidazole which, on treatment with an alkyl halide, afforded the
corresponding bis-imidazolium salt. The non-commercially avail-
able dihaloalkanes were synthesized as follows. 1,13-Diiodotride-
cane and 1,18-diiodooctadecane were prepared by the method of
Wang et al.12 in two steps, namely, the reduction of the
corresponding dicarboxylic acid using LiAlH4 in THF, followed by
treatment of the resulting diol with H3PO4–P2O5–KI. Similarly,
1,14-diiodotetradecane was prepared from 1,14-tetradecanediol
Scheme 1. Synthesis of selected bis-imidazoles and bis-imidazolium salts.

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
6527
Table 1
Activities of compounds in P. falciparum and CHO cell cultures.
IC50 CHO
cells/ IC50
IC50 (µM)
IC50 (µM)
Compound
1 · 2HCl
Structure
P. falciparum
CHO cells
P. falciparum
2HCl
N
N
N
N
1066 314
67 22
1900
122
0
1.8
1.8
N
N
N
N
2I
2
3
N
N
2HCl
3 · 2HCl
90
6
899 80
146 14
10
56
N
N
N
N
2I
4
2.6 0.8
N
N
N
N
N
N
2HCl
5 · 2HCl
6
6
2
140 28
452 49
23
96
N
N
N
N
4.7 0.9
9.2 0.9
2I
N
2HCl
N
7 · 2HCl
15 11
1.6
N
N
N
2 I
N
8
9
0.082 0.009
1.9 0.1
57
20
5
5
695
11
N
N
N
N
N
N
N
0.0061 0.0006
N
2I
N
10
42
3
6885
N
N
N
N
N
2HCl
11 · 2HCl
12
4.1 0.8
1.4 0.2
0.3
N
N
N
N
2I
0.006 0.002
26
5
4333
(continued on next page)

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
Table 1 (continued)
IC50 CHO
cells/ IC50
IC50 (µM)
IC50 (µM)
Compound
Structure
P. falciparum
CHO cells
P. falciparum
N
N
2HCl
N
13 · 2HCl
14
0.56 0.06
3.0 0.3
5.4
N
N
N
2I
0.007 0.003
51 11
7286
N
N
2HCl
N
N
15 · 2HCl
16
1.6 0.3
4.4 0.7
2.8
3438
3.0
N
N
2I
0.0032 0.0002
N
N
11
2
N
N
N
N
N
2HCl
17 · 2HCl
18
0.93 0.07
2.8 0.4
1.2 0.2
N
N
N
N
0.002 0.001
600
2I
N
N
N
19
20
0.65 0.03
1.30 0.09
2.7 0.1
2.0
N
N
N
0.0005 0.0002
5400
N
2I
N
N
N
N
21
22
0.4 0.1
1.2 0.1
1.4 0.3
3.0
N
N
N
2I
N
0.0009 0.0002
1556
N
N
N
N
23
2.5 0.4
8.0 0.6
3.2
N
N

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
6529
Table 1 (continued)
IC50 CHO
cells/ IC50
IC50 (µM)
IC50 (µM)
Compound
Structure
P. falciparum
CHO cells
P. falciparum
N
N
24
1.4 0.2
667
0.0021 0.0005
2I
N
N
25
26
34
7
2833
N
N
N
0.0120 0.0005
0.0008 0.0001
N
2I
78 14
97500
N
N
N
N
2I
N
N
N
N
27
28
29
30
93
4
1106 127
317 64
12
28
N
N
2I
N
N
11.2 0.5
3.1 0.1
N
N
N
N
27
9
7
9
N
N
0.009 0.001
2
1000
N
N
2I
31
32
N
N
1.7 0.1
65 15
38
N
N
0.18 0.04
125 13
694
2I
N
N
N
N
N
N
33
34
N
N
0.022 0.003
0.023 0.003
12.1 0.6
550
609
2Br
N
N
N
N
14
2
2Br
O2
N
NO2
(continued on next page)

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
Table 1 (continued)
IC50 CHO
cells/ IC50
IC50 (µM)
IC50 (µM)
Compound
Structure
P. falciparum
CHO cells
P. falciparum
N
N
N
N
2I
35
0.07 0.01
4.9 0.9
98 24
1400
10
N
2HCl
2I
N
N
O
O
N
36 · 2HCl
37
50
4
N
N
N
O
O
N
0.045 0.004
113 12
2511
0.6
N
N
2HCl
N
N
38 · 2HCl
19
7
11.7 0.5
N
N
N
N
N
N
2I
39
0.008 0.002
52
9
6500
N
N
For each indicated structure the IC50 value was determined in both P. falciparum and CHO cell cultures. Values represent the mean of four independent determinations with
the standard error of the mean indicated. Parallel experiments with chloroquine were included as a positive control.
Figure 1. Effect of the alkyl spacer and charge on the activity of compounds in P. falciparum and CHO cell cultures. Compounds were synthesized either as bivalent
imidazolium salts, or as neutral bivalent imidazole structures (see compounds 1–24 in Table 1). The compounds were then evaluated for activity in P. falciparum and CHO cell
cultures as described in Section 4. When neutral imidazole compounds (top-left panel) were assayed, a moderate increase in activity was observed as the alkyl chain length
increased, however, little, or no, selective toxicity was observed for P. falciparum cultures. When the experiment was repeated using similar bivalent imidazolium salts (top-
right panel) it was observed that increasing the length of the alkyl chain between the imidazolium moieties resulted in a moderate increase in toxicity in CHO cultures
throughout the range, however, a significant increase in activity is observed in P. falciparum cultures when an alkyl chain of more that 8 carbon atoms was present. When the
data were plotted to allow a direct comparison of the effect of the imidazole and imidazolium compounds in P. falciparum cultures alone (bottom-left panel) it was observed
that the presence of electron-deficient imidazolium rings was only a significant determinant of activity at alkyl spacer lengths greater than eight carbon atoms. Conversely,
bivalent compounds containing either imidazole or imidazolium rings were found to have similar activities in CHO cell cultures (bottom-right panel).

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
6531
of 4, 6, and 8 carbon atoms (compounds 2, 4, and 6) had IC50 values
in the micromolar range; the potency of the compounds in P. falci-
parum cultures increased dramatically as the length of the spacer
was increased to 10 atoms and beyond. The activity of the bivalent
imidazolium compounds in CHO cell cultures showed a general
trend towards greater toxicity as the alkyl-spacer length increased,
however, the effect was far less dramatic compared to that seen in
P. falciparum cultures (Fig. 1, top-right panel). For example, the IC50
substituents on a imidazolium compound having a 12-carbon
spacer did result in increased potency and selectivity as was seen
in the series 12/25 (methyl/ethyl) and 26 (propyl).
In order to determine if the nature of the spacer significantly af-
fected the activity of a compound, bivalent imidazole- and imi-
dazolium-containing compounds were synthesized having either
a phenyl (27, 28), a biphenyl (29, 30), or a methylbiphenylmethyl
(31, 32) spacer. Consistent with our hypothesis of the necessity
for the presence of a charged center, the uncharged compounds
27, 29, and 31 had poor activities in both culture systems. Com-
pound 27 having a relatively short phenyl spacer did not display
a high degree of potency or selectivity when assayed in CHO and
P. falciparum cultures, however, the biphenyl imidazolium com-
pound 30 was potent and selective (Fig. 2), as was 32. In the case
of 36ꢁ2HCl and 37 which have a 1,4-dialkoxyphenyl spacer, the re-
sults obtained were consistent with the dependence of activity on
charge, however, the presence of this particular spacer did not re-
sult in a loss of activity or selectivity. In summary, the activities of
the compounds are more dependent on the length of the spacer
than on its type.
To determine if the substituents on the heterocyclic rings signif-
icantly influenced the potency and selectivity of the compounds, a
limited series of compounds, namely, 30 (methyl), 33 (benzyl), and
34 (4-nitrobenzyl), was examined. Relatively minor differences
were seen in the IC50 values of these compounds in both CHO
and P. falciparum cultures.
Interestingly, compound 35, which contains benzimidazolium
rings, was also both potent and selective.
In the present work an analog (39) containing two 1,2,4-triazo-
lium rings was synthesized and evaluated (Fig. 3). Interestingly, it
of compound 2 with a 4-carbon spacer was 67 22 lM in P. falci-
parum cultures, whereas the IC50 of compound 24 with a 20-carbon
spacer compound was 2.1 0.5 nM; the corresponding values ob-
served in CHO cell cultures for 2 and 24 were 122
3 lM and
1.4 0.2 M, respectively. These results indicate that increasing
l
the alkyl-chain length resulted in an 87-fold increase in potency
in CHO cell cultures, and an ꢀ32,000-fold increase in potency in
P. falciparum cultures. The capacity of imidazolium compounds to
be selectively toxic to P. falciparum cultures is also apparent when
the IC50 values for imidazole and imidazolium compounds are plot-
ted for P. falciparum cultures alone (Fig. 1, bottom-left panel) or
CHO cell cultures alone (Fig. 1, bottom-right panel); these results
are consistent with our hypothesis that imidazolium rings are
interacting with a specific component of the parasite and that
there is a minimum distance that is required between the imidazo-
lium rings for them to interact with two target-domains. The imi-
dazolium series has also been assayed using RAW cells (murine
macrophage cells) and results similar to those seen for the CHO
cells were observed (data not shown), an observation suggesting
that the interaction is not a CHO-cell specific phenomenon. The ef-
fect of adding alkyl groups to the periphery of the imidazolium
rings was also determined; thus, increasing the length of the alkyl
Figure 2. Effect of spacer chemistry and charge on the activity of the compound in P. falciparum and CHO cell cultures. Bivalent imidazole or imidazolium compounds with
various spacers were synthesized. Compounds were then evaluated for activity in P. falciparum and CHO cell cultures as described in Section 4. Compounds with a phenyl
spacer (27 and 28) were observed to have both low potency and selectivity for P. falciparum cultures. Increasing the spacer size to a biphenyl structure conferred activity and
selectivity when the imidazolium rings were present (30, 35, 33, and 34). Increasing the spacer length by two carbon atoms did not result in a significant change in activity
(31 and 32). Adding phenyl groups (35 and 33) or 4-nitrophenyl groups (34) to the exterior of the imidazolium rings also did not significantly change the compounds’
activities and the presence of a 1,4-dialkoxy moiety (36ꢁ2HCl and 37) was equally well tolerated.

6532
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
Figure 3. The effect of compound 39 on P. falciparum cultures and CHO cells.
Compound 39 was serially diluted across the plate to produce twofold dilutions. A
constant number of CHO cells or P. falciparum parasites were added to each well and
the plate was allowed to incubate for 72 h before viability (vertical axis) was
determined for each concentration of compound 39 (horizontal axis) using either a
pLDH or MTT assay (see Methods). Each point represents the average value with
standard error indicated. IC50 values were calculated using Sigma Plot and represent
the mean of four determinations.
Figure 4. Determination of parasitemia of treated and untreated cultures. A Percoll
gradient was used to synchronize the parasite cultures at the mature stage of
development one day prior to the start of the parasite development assay. The
cultures were then adjusted to a parasitemia of 2.4% and were treated for various
periods of time with compounds (see x-axis) before the parasitemia present was
determined by examining Giemsa-stained samples of the cultures. Untreated
was found that 39 was a highly potent and selective compound
with an IC50 value in P. falciparum cultures of 8 2 nM and with
an IC50 value in CHO cells of 52
9 lM leading to a selectivity value
cultures showed
a marked increase in parasitemia from Day 1 to Day 2,
of 6500. The observation that the uncharged analog (38ꢁ2HCl) of 39
was neither potent nor selective is consistent with our hypothesis
of the presence of a charged species, however, the noteworthy val-
ues obtained with 39 warrant further exploration of 1,2,4-triazoli-
um analogs, a study currently underway.
corresponding to the time of reinvasion, but no increase in parasitemia was
observed between Day 0 to Day 1, or Day 2 to Day 3, corresponding to the time of
intracellular maturation. Chloroquine-treated cultures produced results consistent
with an intracellular action preventing maturation and a drop in parasitemia was
observed between Days 1 and 2 with sulfated b-cyclodextrin (sodium salt)-treated
cultures. When compound 39 was present at a concentration corresponding to five
times its IC50 value the apparent parasitemia increased between Days 1 and 2,
however, increasing the concentration to ten times the IC50 prevented an increase
in parasitemia over time.
2.2.2. Effect of compounds on P. falciparum cultures
Previous work has indicated that negatively charged sulfated
cyclodextrins interact with a receptor on the surface of an erythro-
cyte15 while positively charged compounds that interact with
these sulfated cyclodextrins might interact with a parasite compo-
nent.5 Examination of treated cultures suggests that bivalent imi-
dazolium and triazolium compounds are active, at least in part,
because they too inhibit merozoite invasion and their presence
during the merozoite invasion event renders a culture unviable
(Table 1). However, to exclude the possibility that these com-
pounds are affecting intracellular parasite maturation, we deter-
mined the ability of parasites to go through their normal
developmental stages in the presence of the bis-triazolium com-
pound 39 by tracking the parasitemia of treated cultures over a
period corresponding to two replication cycles, or 0–3 days
(Fig. 4). Untreated synchronized culture parasites should be ring
forms on Day 0, mature forms on Day 1, will be released to rein-
vade erythrocytes and become new ring forms on Day 2, and will
develop into mature forms again on Day 3. As expected untreated
parasite cultures displayed an increase in parasitemia only be-
tween Days 1 and 2, a period that coincides with the production
and release of merozoites. Chloroquine prevents parasite matura-
tion16 and a drop in parasitemia was observed between Days 1
and 2. Cultures treated with sulfated b-cyclodextrin (sodium salt)
showed a reduced parasitemia on Day 2, a result consistent with
released merozoites not being able to associate with erythro-
cytes.15 When compound 39 was present at a concentration corre-
sponding to five times its IC50 value, the apparent parasitemia
increased between Days 1 and 2, a result indicating that merozoite
production was taking place; however, increasing the concentra-
tion to ten times the IC50 value prevented an increase in
parasitemia over time, an observation suggesting that at this
concentration the compound either prevented parasite association,
as was seen with the sulfated cyclodextrin, or was toxic to intracel-
lular parasites, as was seen with chloroquine.
We further examined the effect of compound 39 on parasite
growth and development by analyzing treated cultures by flow
cytometry (Fig. 5). Untreated cultures were observed to increase
in parasitemia from 5% to 29% during this period, with a significant
percentage of the DNA present not associated with the erythro-
cytes. The dissociation of DNA and erythrocytes suggests the pres-
ence of free merozoites. As intimated in Figure 4, treatment with
chloroquine prevented the increase of parasitemia, however, treat-
ment with putative invasion inhibitors resulted in a number of par-
asite particles (total bar heights in the graph in Fig. 5) that was
equivalent to the control; this suggests that the production of
DNA-containing particles, presumably merozoites, was normal
but that the particles were less likely to be associated with eryth-
rocytes. Treatment of the cultures with active compounds (Fig. 5)
resulted in an increased number of parasite forms with a decreased
size (Fig. 5, grey-bar section), however, treatment with sulfated b-
cyclodextrin (sodium salt) produced the clearest indication that
merozoite entry was not occurring. Further, when cultures treated
with imidazolium or triazolium compounds were examined micro-
scopically, it was observed that forms of the parasite appeared to
adhere to the external surface of the erythrocytes (see picture in
Fig. 5), a result suggesting that merozoites were still able to tether
to erythrocytes, but that the entry process was inhibited. The

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
6533
Figure 5. Flow cytometry analysis of treated cultures. Parasite cultures were synchronized as ring-stage cultures by sorbitol lysis. The cultures were then adjusted to a
parasitemia of 5% and a hematocrit of 0.5% and were treated with various compounds for 48 h before 0.2 L per mL of stock SYBR-Green solution was added and then the
l
samples were subjected to flow cytometry. Each graph represents ꢀ200,000 events and the x-axis represents forward scatter (size) while the y-axis indicates relative
fluorescence. Diagrams represent data obtained from a culture stained with SYBR-Green that contained no parasites (top-left), an untreated parasite culture (top-middle), a
parasite culture treated with 1 lM chloroquine (top-right), a culture treated with 20 lM compound 40 (bottom-left), a culture treated with 20 nM of compound 39 (bottom-
middle), or a culture treated with 0.2 mg/mL sulfated b-cyclodextrin (sodium salt) (bottom-right). The data contained in the flow cytometry plots was quantitated by
comparing the number of events observed in the lower-right quadrant (non-parasitized erythrocytes) compared to the upper-right quadrant (parasitized erythrocytes) and
upper-left quadrant (small-sized material containing DNA) to the total number of erythrocytes present (upper- and lower-right quadrants). The sulfated b-cyclodextrin
clearly prevented the association of parasite material with the erythrocytes in culture, however, the effect observed with compounds 12, 40, and 39 was less pronounced.
Microscopic examination of the individual cultures suggested that in cultures treated with compounds 12, 40, and 39, a significant number of parasite forms adhered to, but
were not inside, the erythrocytes in the cultures (top-left photo, untreated; top-right photo, treated with compound 40; bottom-left, treated with compound 39; bottom-right
photo, treated with the sulfated b-cyclodextrin).
maturation process ends in the production of 20–40 merozoites,
followed by erythrocyte rupture and immediate reinvasion of the
merozoites into new erythrocytes. It is interesting to note that
the untreated culture contained a significant population of smaller
DNA-containing elements which are presumably merozoites which
have not completed the invasion process. This result is not

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J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
unexpected since the reinvasion efficiency of P. falciparum cultures
is frequently well below the maximum possible, for example, a
5–10-fold increase is seen per cycle compared to the 20–40-fold
increase in parasitemia that would be expected if the process were
100% successful (see Ref. 6). The presence of chloroquine prevented
the appearance of an increased number of parasite forms contain-
ing DNA (bar graph, Fig. 5), and SYBR-Green I-staining populations
(top-right panel, Fig. 5) are consistent with chloroquine treatment,
since it would be expected to produce a diverse population of
parasite forms and debris. In contrast, the presence of the other
agents did not significantly affect the production of DNA-contain-
ing particles in the treated cultures, since the total height of the
corresponding bars in Figure 5 are approximately equal to that of
the control culture. The presence of moderate numbers of apparent
ring-stage parasites detected by flow cytometry in cultures treated
with 39 (as well as 12 or 40) was surprising, since a result similar
to that seen for the sulfated b-cyclodextrin (sodium salt)-treated
sample was expected. However, examination of compound-treated
cultures indicated that the parasite material appeared to be associ-
ated with the exterior of the erythrocytes; this suggests that mer-
ozoite production and release continue to occur, but that it is
merozoite entry that is being aborted. The relative size and fluores-
cence of ring-stage infected erythrocytes and erythrocytes having
adhered merozoites should be similar. The capacity of sulfated
cyclodextrins to greatly reduce the number of parasites associated
with red blood cells may reflect the fact that these molecules alter
the surface charge of an erythrocyte.15
Other bis-cationic compounds have been studied for anti-Plas-
modium activity, including bis-(quaternary ammonium) salts,17–20
bis-pyridinium salts,21 and bis-thiazolium salts.22,23 In addition
to inhibiting the formation of hemzoin inside the parasite food vac-
uole, many of these reported bis-cationic agents are considered
mimics of choline, and target the membrane biogenesis of
parasites in the erythrocytic stage by blocking the biosynthesis of
phosphatidylcholine, the main component for the cytoplasmic
membrane of the parasite. Although the possibility of reduced
phosphatidylcholine production occurring inside the infected
erythrocyte can not be eliminated in the case of our compounds,
microscopic examination experiments suggest that the reported
bis-imidazolium and bis-triazolium compounds appear to inhibit
the process of merozoite invasion into the erythrocyte, and that
the immobilization of the merozoite to the exterior surface of the
erythrocyte is the main cause of parasite death. Thus, our com-
pounds may act primarily by way of a mechanism of action similar
to that seen using our tetrazolium salt compounds.4 It is also pos-
sible that alteration of the merozoite surface proteins, GPI anchors,
or other such merozoite features (while being constructed inside
an infected erythrocyte), as a result of treatment with the bis-
cationic compounds could lead to the inability of newly formed
defected merozoites to reinvade other erythrocytes. These mecha-
nistic aspects are currently under investigation in our laboratories.
It is recognized that the compounds in Table 1 may have surfac-
tant properties (see, e.g., Refs. 24 and 25) and that the selective
toxicity of the charged compounds in P. falciparum cultures may
be attributable to surfactant effects. Surfactants can have the
desirable property of interacting with, and crossing, biological
membranes; however, this interaction depends on the relative con-
tributions of the hydrophobic and hydrophilic components of the
molecule. It should be noted that the environment found within
P. falciparum-containing erythrocytes is known to differ from that
of mammalian cells.26,27 Three pieces of evidence suggest that
the surfactant properties of the compounds do not explain the
results found in Table 1. Firstly, erythrocytes parasitized with
Plasmodium preferentially interact with surfactants having a higher
hydrophilic content,26 whereas the current compounds that are
most active are more hydrophobic. Secondly, there appears to be
a fair degree of latitude in the structure of the linker in active com-
pounds, for example, in the series comprised of 12, 30, 32, and 37,
and, therefore, there does not appear to be a direct relationship
between hydrophobicity and activity, as seen also in the series
comprised of 30, 33, and 34. In the series comprised of 12, 25,
and 26, in which the only structural difference is the size of the
alkyl substituents on the rings, there is a systematic decrease in
toxicity in the CHO cell cultures. Thirdly, the IC50 values of the
most active imidazolium compounds are sub-nanomolar, a result
which is not consistent with a surfactant-like action.28,29
2.2.3. In vivo activity
The observation that some of the compounds were selectively
active in P. falciparum cultures compared to mammalian cell
cultures suggested that these compounds might suppress
a
Plasmodium infection in a mouse model. Preliminary work using
Plasmodium berghei-parasitized blood suggested that randomly se-
lected compounds had similar, but not identical, activities in both
P. falciparum cultures and P. berghei ex vivo assays (Table 2).
P. berghei would be expected to differ from P. falciparum in at least
two aspects: (1) P. berghei has a replicative cycle of 24 h versus the
48 h cycle seen in P. falciparum; and (2) P. berghei may vary from
P. falciparum in its use of specific initial contact and tight-junction
receptors. The similar activities of the compounds observed for
both species (Table 2) suggests that a process common to both
species is being inhibited by the compounds.
To determine if select compounds could positively influence
the outcome of a murine malaria infection, mice were infected
with 106 P. berghei parasites on Day 0 and their parasitemia
was followed until parasites were observed in the peripheral cir-
culation of the mice (Day 5, Fig. 6). The mice were then infused
once daily for three days with: (1) a bivalent imidazolium salt
having a 13-carbon spacer (compound 14), (2) a bivalent triazo-
lium salt compound having a 12-carbon spacer (compound 39),
(3) the equivalent amount of solvent (DMSO) in RPMI 1640, or
(4) chloroquine, which functioned as a positive control. It was
observed that the mice that were infused with 14 showed a less
rapid development of parasitemia, however, mice infused with
39 showed both a delayed development of parasitemia and a
lower final parasitemia, when compared to the RPMI 1640 con-
trol group (Fig. 6). Compound 39 was then subjected to a 4-day
test30 at two different doses, namely, 3.75 and 15 mg/kg, and it
was observed that the parasitemia on the day following the final
infusion was reduced by ꢀ50% in the mice receiving the lower
dose and by ꢀ75% in the mice receiving the higher dose. Sul-
fated cyclodextrins, which bind to the corresponding receptor
domain in the erythrocyte protein AE1, were also found to be
effective in a mouse model, a result which is consistent with
the hypothesis that P. falciparum and P. berghei share similar tar-
gets for both receptor inhibitors, for example, sulfated carbohy-
drates or the putative ligand inhibitors presented here. The
results obtained suggest that complete suppression of a Plasmo-
dium infection in vivo can be attained using compounds similar
to those reported here.
3. Conclusions
Compounds containing two imidazolium rings (but not imidaz-
ole rings) show potent and selective activity in P. falciparum cul-
tures and in
a murine malaria model. A minimal spacing
between the rings, for example, of more than eight carbon atoms,
is necessary for both selectivity and potency, however, various
spacers can be used to equal effect. Further, it is intimated that
1,2,4-triazolium rings can be substituted for imidazolium rings
and the activity of the pharmacophore, both in vitro, and in vivo,
is retained.

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
6535
Table 2
Activities of compounds in P. falciparum and P. berghei cell cultures.
IC50 (µM)
IC50 (µM)
Compound
Structure
P. falciparum
P. berghei
N
N
2I
N
10
25
0.0061 0.0006
0.0120 0.0005
0.05 0.02
0.12 0.03
N
N
N
N
N
2I
N
N
N
O
O
N
2I
37
40
0.045 0.004
2.6 0.1
0.046 0.009
4.2 0.1
Cl
Cl
H
Br
N
N
O
O
For each indicated structure the IC50 value was determined in both P. falciparum and P. berghei ex vivo cultures. Values represent the mean of four independent determi-
nations with the standard error of the mean indicated. Compound 40 has been previously reported.5
CD3OD, or DMSO-d6. The chemical shifts are reported in d (ppm)
relative to tetramethylsilane.31 The compounds synthesized were
deemed >95% pure by 1H NMR analysis. High-resolution ESI mass
spectra were recorded on an Applied Biosystems/MDS Sciex QSTAR
XL mass spectrometer with an Agilent HP1100 Cap-LC system.
Samples were run in 50% aqueous MeOH at a flow rate of 6 lL/
min. High-resolution EI mass spectra were recorded on a Waters/
Micromass GC-TOF instrument.
4. Experimental
4.1. General
Flash column chromatography was performed on Silicycle silica
gel (230–400 mesh, 60 Å). Analytical thin-layer chromatography
was performed on glass-backed pre-coated Silica Gel 60 F254
plates (Silicycle), and the compounds were visualized either by
UV illumination (254 nm), or by heating after spraying with phos-
phomolybdic acid in ethanol. Melting points were measured on a
Mel-Temp II apparatus and are uncorrected. 1H and 13C NMR spec-
tra were recorded on a Bruker Avance 400 spectrometer in CDCl3,
4.2. Materials
1,3-Dibromopropane, 1,4-dibromobutane, 1,6-dibromohexane,
1,8-dibromooctane, 1,10-dibromodecane, 4,40-dibromobiphenyl,
1,4-dibromobenzene,
4,40-bis(chloromethyl)-1,10-biphenyl,
1,11-undecanedicarboxylic acid, and hydroquinone were obtained
from Sigma–Aldrich. 1,11-Dibromoundecane and 1,14-tetrade-
canediol were obtained from Acros Organics. 1,12-Dibromodode-
cane and 16-hexadecanolide were obtained from Alfa Aesar.
1,20-Dibromoeicosane was obtained from Karl Industries Inc.
Octadecanedioic acid was obtained from TCI America. Sulfated
b-cyclodextrin (sodium salt) was obtained from Sigma–Aldrich
and contained 7–11 mol of sulfate residues per mol of b-cyclodex-
trin. All of the other chemicals were obtained from Sigma–Aldrich.
4.3. Procedures for the formation of
a,x-diiodoalkanes
4.3.1. 1,13-Diiodotridecane
This compound was prepared in two steps (94% overall yield)
from 1,11-undecanedicarboxylic acid following the procedure for
the formation of 1,18-diiodooctadecane given below and was ob-
tained as a beige solid; mp 33–34 °C; 1H NMR (400 MHz, CDCl3):
d 1.18–1.45 (m, 18H), 1.77–1.88 (m, 4H), 3.19 (t, J = 7.0 Hz, 4H);
13C NMR (100 MHz, CDCl3): d 7.5, 28.7, 29.5, 29.6 (2C), 30.6, 33.7;
HRMS (EI) [M]+. Calcd for C13H26I2: 436.0124. Found: 436.0121.
Figure 6. In vivo evaluation of compounds 14 and 39. Groups of 5 female Balb/c
mice were infected with 106 P. berghei parasites and their parasitemia was followed
until parasites were observed in the peripheral circulation of the mice (Day 5). The
mice were then infused once daily for three days (denoted by arrows) with: 1% (v/v)
DMSO in RPMI-1640 (d); 60 lg per day of compound 14 (.); 75 lg per day of
compound 39 (open triangle); or 15 mg/kg per day of chloroquine (s, which
functioned as a positive control). Parasitemia was determined by microscopic
examination of Giemsa-stained peripheral blood films and the results are repre-
sented as mean with standard error shown using bars.
4.3.2. 1,14-Diiodotetradecane
Under an atmosphere of N2, a 97% H3PO4 solution (8.7 equiv) was
prepared by adding P2O5 (426 mg, 3.00 mmol, 1.4 equiv) to 85%

6536
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
H3PO4 (0.86 mL, 1.22 g H3PO4, 12.41 mmol, 5.8 equiv). To this solu-
tion was added KI (2.06 g, 12.41 mmol, 5.8 equiv) and then 1,14-
tetradecanediol (493 mg, 2.14 mmol, 1 equiv). The reaction mixture
was stirred at ꢀ115 °C for 4 h, cooled to rt, diluted with Et2O
(50 mL), and water (50 mL) was added. The organic layer was sepa-
rated, and the aqueous phase extracted with an additional 50 mL of
Et2O. The combined organic extracts were washed with brine
(30 mL), washed with a concentrated aqueous solution of Na2S2O3
(10 mL), dried (Na2SO4), and then concentrated. High-vacuum drying
gave 1,14-diiodotetradecane (842 mg, 1.87 mmol, 87%) as a white so-
lid; mp 46–47 °C; 1H NMR (400 MHz, CDCl3): d 1.23–1.33 (m, 16H),
1.34–1.42 (m, 4H), 1.77–1.87 (m, 4H), 3.19 (t, J = 7.0 Hz, 4H); 13C
NMR (100 MHz, CDCl3): d 7.5, 28.7, 29.5, 29.6, 29.7, 30.7, 33.7;
HRMS (EI) [M]+. Calcd for C14H28I2: 450.0281. Found: 450.0276.
mixture was filtered through Celite and the filter cake washed with
THF (100 mL) and Et2O (100 mL). The organic filtrate was washed
twice with brine, dried (Na2SO4), and then concentrated. High-vac-
uum drying gave 1,18-octadecanediol (1.40 g, 4.89 mmol, 77%) as a
white solid; mp 93–94 °C; 1H NMR (400 MHz, CD3OD): d 1.27–1.38
(m, 28H), 1.48–1.57 (m, 4H), 3.54 (t, J = 6.6 Hz, 4H); 13C NMR
(100 MHz, CD3OD): d 26.9, 30.6, 30.7 (5C), 33.7, 63.0; HRMS (ESI)
[M+H]+. Calcd for C18H39O2: 287.2950. Found: 287.2947.
Under an atmosphere of N2, a 97% H3PO4 solution (8.7 equiv) was
prepared by adding P2O5 (894 mg, 6.30 mmol, 1.4 equiv) to 85%
H3PO4 (1.78 mL, 2.55 g H3PO4, 26.02 mmol, 5.8 equiv). To this solution
was added KI (4.33 g, 26.10 mmol, 5.8 equiv) and then 1,18-octade-
canediol (1.29 g, 4.50 mmol, 1 equiv). The reaction mixture was stirred
at ꢀ115 °C for 5 h, cooled to rt, diluted with Et2O (20 mL), and water
(20 mL) was added. The organic layer was separated, and the aqueous
phase further extracted with Et2O (3 ꢂ 100 mL). The combined organic
extracts were washed with brine (20 mL), washed with a concentrated
aqueous solution of Na2S2O3 (10 mL), dried (Na2SO4), and then concen-
trated. High-vacuum drying gave 1,18-diiodooctadecane (2.27 g,
4.48 mmol, 99%) as a beige solid; mp 59–60 °C; 1H NMR (400 MHz,
CDCl3): d 1.20–1.44 (m, 28H), 1.77–1.86 (m, 4H), 3.19 (t, J = 7.0 Hz,
4H); 13C NMR (100 MHz, CDCl3): d 7.5, 28.7, 29.6, 29.70, 29.76,
29.80 (2C), 30.7, 33.7; HRMS (EI) [M]+. Calcd for C18H36I2:
506.0907. Found: 506.0921.
4.3.3. 1,15-Diiodopentadecane
Under an atmosphere of N2, a suspension of LiAlH4 (228 mg,
6.01 mmol, 11.6 equiv) in dry THF (6 mL) was stirred at rt for
5 min. The mixture was cooled to 0 °C, and a solution of pentade-
canolide (500 mg, 2.08 mmol, 1 equiv) in THF (6 mL) was added.
The reaction mixture was brought to rt and then stirred at reflux
temperature for 2 h. The mixture was cooled to 0 °C and then an
aqueous solution of NaOH (2 M, 2 mL) was added. A white precip-
itate was formed and the mixture was stirred at reflux temperature
for 0.5 h. The mixture was extracted with Et2O (2 ꢂ 50 mL). The
combined organic extracts were washed with brine (2 ꢂ 20 mL),
dried (Na2SO4), and concentrated. High-vacuum drying gave
1,15-pentadecanediol (482 mg, 1.97 mmol, 95%) as a white solid;
mp 87–88 °C; 1H NMR (400 MHz, CD3OD): d 1.27–1.39 (m, 22H),
1.48–1.58 (m, 4H), 3.54 (t, J = 6.6 Hz, 4H); 13C NMR (100 MHz,
CD3OD): d 27.0, 30.6, 30.8 (4C), 33.7, 63.0; HRMS (ESI) [M+H]+.
Calcd for C15H32O2: 245.2481. Found: 245.2475.
Under an atmosphere of N2, a 97% H3PO4 solution (8.7 equiv) was
prepared by adding P2O5 (300 mg, 2.10 mmol, 1.4 equiv) to 85%
H3PO4 (0.6 mL, 0.85 g H3PO4, 8.67 mmol, 5.8 equiv). To this solution
was added KI (1.44 g, 8.70 mmol) and then 1,15-pentadecanediol
(367 mg, 1.50 mmol, 1 equiv). The reaction mixture was stirred at
ꢀ115 °C for 4 h, cooled to rt, diluted with Et2O (20 mL), and water
(20 mL) was added. The organic layer was separated, and the aque-
ous phase extracted with an additional 50 mL of Et2O. The combined
organic extracts were washed with brine (20 mL), washed with a
concentrated aqueous solution of Na2S2O3 (10 mL), dried (Na2SO4),
and then concentrated. High-vacuum drying gave 1,15-diiodopen-
tadecane (683 mg, 1.47 mmol, 70%) as a white solid; mp 46–47 °C;
1H NMR (400 MHz, CDCl3): d 1.19–1.45 (m, 22H), 1.77–1.87 (m,
4H), 3.19 (t, J = 7.2 Hz, 4H); 13C NMR (100 MHz, CDCl3): d 7.5, 28.7,
29.5, 29.6, 29.7 (2C), 30.7, 33.7; HRMS (EI) [M]+. Calcd for C15H30I2:
464.0437. Found: 464.0436.
4.4. Representative procedure for the formation of bis-
imidazole compounds, as outlined in Scheme 1
4.4.1. 1,4-Bis-(1H-imidazol-1-yl)butane dihydrochloride
(1ꢁ2HCl)
Under an atmosphere of N2, a mixture of imidazole (1.70 g,
25 mmol, 2 equiv) and sodium hydroxide (1.00 g, 25 mmol, 2 equiv)
in dimethyl sulfoxide (5 mL) was stirred at 60 °C for 1.5 h. To this
mixture was added carefully (very exothermic) 1,4-dibromobutane
(2.70 g, 12.5 mmol, 1 equiv) and the mixture stirred at 60 °C for
2.5 h. The temperature was increased to ꢀ100 °C and a stream of
N2 gas was blown over the mixture to remove dimethyl sulfoxide;
the mixture solidified after 2 h. The mushy solid was dried under
high vacuum leaving a beige solid which was extracted into benzene
(3 ꢂ 75 mL) with excessive stirring and without the addition of
water. The combined organic extracts were dried (MgSO4), filtered,
and the filtrate concentrated to a clear oil. {Alternatively, in cases
where the alkane was longer than dodecane, the free base was iso-
lated simply by cooling the DMSO-containing reaction mixture to
0 °C, diluting with water, collecting thesolid precipitate by filtration,
and washing the solid free base with water.} High-vacuum drying
gave the crude free base as a white solid (1.22 g, 6.41 mmol, 51%).
To a solution of the free base in warm 2-propanol (2 mL) was added
a solution of 37% aqueous HCl (1.46 g, 14.8 mmol, 2.3 equiv) in 2-
propanol (2 mL). The mixture was concentrated and dried under
high vacuum. The residue was recrystallized from a mixture of
methanol/ethanol/2-propanol. High-vacuum drying gave 1ꢁ2HCl
(881 mg, 3.35 mmol, 27%) as a white solid; mp >260 °C; Rf = 0.9
(MeOH); 1H NMR (400 MHz, CD3OD): d 1.94–2.02 (m, 4H), 4.33–
4.41 (m, 4H), 7.60 (s, 2H), 7.73 (s, 2H), 9.07 (s, 2H); 13C NMR
(100 MHz, CD3OD): d 28.0, 49.8, 121.2, 123.3, 136.5; HRMS (ESI)
[M-H-2Cl]+. Calcd for C10H15N4: 191.1297. Found: 191.1288.
4.3.4. 1,16-Diiodohexadecane
This compound was prepared in two steps (84% overall yield)
from 16-hexadecanolide following the procedure for the formation
of 1,15-diiodopentadecane given above and was obtained as a white
solid; mp 49–50 °C; 1H NMR (300 MHz, CDCl3): d 1.18–1.45 (m,
24H), 1.75–1.89 (m, 4H), 3.19 (t, J = 7.1 Hz, 4H); 13C NMR (75 MHz,
CDCl3): d 7.5, 28.7, 29.6, 29.69, 29.74, 29.77, 30.7, 33.7; HRMS (EI)
[M]+. Calcd for C16H32I2: 478.0594. Found: 478.0590.
4.3.5. 1,18-Diiodooctadecane
4.5. Characterization of bis-imidazole compounds synthesized
following the representative procedure (as given for compound 1)
Under an atmosphere of N2, octadecanedioic acid (2.00 g,
6.36 mmol, 1 equiv) was dissolved in freshly distilled THF (100 mL)
and the solution was cooled to 0 °C; LiAlH4 (228 mg, 6.01 mmol,
11.6 equiv) was added in small portions. The mixture was stirred at
rt for 5 days then at reflux temperature for 5 h. The mixture was
cooled to 0 °C and quenched with wet THF, then water, and finally
an aqueous solution of NaOH (2 M, 2 mL) was added (pH ꢀ10). The
4.5.1. 1,6-Bis-(1H-imidazol-1-yl)hexane dihydrochloride
(3ꢁ2HCl)
This compound was prepared in one step (24% yield) from 1,6-
dibromohexane and was obtained as a white solid (recrystallized

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
6537
from
a
mixture of methanol/ethanol/2-propanol); mp
lized); mp 142–143 °C; 1H NMR (400 MHz, CD3OD): d 1.20–1.44
(m, 20H), 1.85–1.98 (m, 4H), 4.20–4.35 (m, 4H), 7.58 (s, 2H), 7.68
(s, 2H), 8.99 (s, 2H); 13C NMR (100 MHz, CD3OD): d 27.4, 30.1,
30.6, 30.7, 30.8, 31.3, 50.7, 121.2, 123.4, 136.4; HRMS (ESI)
[Mꢃ2Cl]2+. Calcd for C20H36N4: 166.1464. Found: 166.1463.
220–221 °C; Rf = 0 (EtOAc); 1H NMR (400 MHz, CD3OD):
d
1.38–1.49 (m, 4H), 1.88–2.00 (m, 4H), 4.30 (t, J = 7.2 Hz, 4H), 7.59
(s, 2H), 7.71 (s, 2H), 9.04 (s, 2H); 13C NMR (100 MHz, CD3OD): d
26.6, 30.9, 50.4, 121.1, 123.4, 136.3; HRMS (ESI) [M-H-2Cl]+. Calcd
for C12H19N4: 219.1610. Found: 219.1599.
4.5.8. 1,15-Bis-(1H-imidazol-1-yl)pentadecane dihydrochloride
(17ꢁ2HCl)
4.5.2. 1,8-Bis-(1H-imidazol-1-yl)octane dihydrochloride
(5ꢁ2HCl)
This compound was prepared in one step (100% yield) from
1,15-diiodopentadecane and was obtained as a yellow solid (not
This compound was prepared in one step (77% yield) from 1,8-
dibromooctane and was obtained as a white solid (recrystallized
from EtOH/2-propanol): mp 176–177 °C; Rf = 0 (EtOAc); 1H NMR
(400 MHz, CD3OD): d 1.30–1.46 (m, 8H), 1.84–1.98 (m, 4H), 4.28
(t, J = 7.4 Hz, 4H), 7.59 (app t, J = 1.6 Hz, 2H), 7.69 (app t,
J = 1.8 Hz, 2H), 9.02 (s, 2H); 13C NMR (100 MHz, CD3OD): d 27.1,
29.8, 31.1, 50.6, 121.1, 123.3, 136.3; HRMS (ESI) [M-H-2Cl]+. Calcd
for C14H23N4: 247.1923. Found: 247.1918.
recrystallized); mp 98–99 °C; 1H NMR (400 MHz, CD3OD):
d
1.24–1.43 (m, 22H), 1.85–1.98 (m, 4H), 4.20–4.35 (m, 4H), 7.57
(s, 2H), 7.67 (s, 2H), 8.98 (s, 2H); 13C NMR (100 MHz, CD3OD): d
18.3, 27.4, 30.2, 30.6, 30.7, 30.8, 31.3, 50.9, 121.2, 123.5, 136.6;
HRMS (ESI) [M-2Cl]2+. Calcd for C21H38N4: 173.1542. Found:
173.1544.
4.5.9. 1,16-Bis-(1H-imidazol-1-yl)hexadecane (19)
This compound was prepared in one step (98% yield) from 1,16-
diiodohexadecane and was obtained as a yellow solid (not recrys-
tallized); mp 46–48 °C; 1H NMR (400 MHz, CDCl3): d 1.20–1.44 (m,
24H), 1.68–1.82 (m, 4H), 3.90 (t, J = 6.8 Hz, 4H), 6.88 (s, 2H), 7.03 (s,
2H), 7.44 (s, 2H); 13C NMR (100 MHz, CDCl3): d 26.6, 29.2, 29.5,
29.6, 29.7 (2C), 31.2, 47.1, 118.9, 129.4, 137.2; HRMS (ESI)
[M+H]+. Calcd for C22H39N4: 359.3174. Found: 359.3196.
4.5.3. 1,10-Bis-(1H-imidazol-1-yl)decane dihydrochloride
(7ꢁ2HCl)
This compound was prepared in one step (84% yield) from 1,10-
dibromodecane and was obtained as a white solid (recrystallized
from EtOH/2-propanol); mp 143–144 °C; Rf = 0 (EtOAc); 1H NMR
(400 MHz, CD3OD): d 1.28–1.42 (m, 12H), 1.85–1.96 (m, 4H), 4.27
(t, J = 7.4 Hz, 4H), 7.59 (app t, J = 1.6 Hz, 2H), 7.69 (app t,
J = 1.6 Hz, 2H), 9.02 (s, 2H); 13C NMR (100 MHz, CD3OD): d 27.3,
30.0, 30.4, 31.2, 50.6, 121.1, 123.3, 136.3; HRMS (ESI) [M-H-2Cl]+.
Calcd for C16H27N4: 275.2236. Found: 275.2229.
4.5.10. 1,18-Bis-(1H-imidazol-1-yl)octadecane (21)
This compound was prepared in one step (93% yield) from 1,18-
diiodooctadecane and was obtained as a beige solid (not recrystal-
lized); mp 56–58 °C; 1H NMR (400 MHz, CDCl3): d 1.08–1.40 (m,
28H), 1.68–1.80 (m, 4H), 3.91 (t, J = 7.0 Hz, 4H), 6.89 (s, 2H), 7.04
(s, 2H), 7.44 (s, 2H); 13C NMR (100 MHz, CDCl3): d 26.7, 29.2,
29.5, 29.6, 29.7 (2C), 29.8, 31.2, 47.2, 118.9, 129.5, 137.2; HRMS
(EI) [M+H]+. Calcd for C24H43N4: 387.3487. Found: 387.3499.
4.5.4. 1,11-Bis-(1H-imidazol-1-yl)undecane (9)
This compound was prepared in one step (96% yield) from 1,11-
dibromoundecane and was obtained as a golden oil (the solid free
base isolated turned into an oil upon high-vacuum drying); 1H
NMR (400 MHz, CDCl3): d 1.15–1.34 (m, 14H), 1.68–1.80 (m, 4H),
3.90 (t, J = 7.2 Hz, 4H), 6.88 (s, 2H), 7.03 (s, 2H), 7.43 (s, 2H); 13C
NMR (100 MHz, CD3OD): d 26.6, 29.1, 29.4 (2C), 31.1, 47.1, 118.9,
129.4, 137.1; HRMS (ESI) [M]+. Calcd for C17H28N4: 288.2314.
Found: 288.2321.
4.5.11. 1,20-Bis-(1H-imidazol-1-yl)eicosane (23)
This compound was prepared in one step (68% yield) from 1,20-
dibromoeicosane and was obtained as an off-white solid (isolated
as the free base, not recrystallized); mp 58–59 °C; 1H NMR
(400 MHz, CDCl3): d 1.18–1.36 (m, 32H), 1.70–1.80 (m, 4H), 3.91
(t, J = 7.0 Hz, 4H), 6.89 (s, 2H), 7.04 (s, 2H), 7.45 (s, 2H); 13C NMR
(100 MHz, CDCl3): d 26.7, 29.2, 29.55, 29.64, 29.7, 29.77, 29.81
(2C), 31.2, 47.2, 118.9, 129.5, 137.2; HRMS (EI) [M]+. Calcd for
4.5.5. 1,12-Bis-(1H-imidazol-1-yl)dodecane dihydrochloride
(11ꢁ2HCl)
This compound was prepared in one step (89% yield) from 1,12-
dibromododecane and was obtained as a white solid (recrystallized
from 2-propanol/Et2O); mp 164–165 °C; Rf = 0 (EtOAc); 1H NMR
(400 MHz, CD3OD): d 1.27–1.42 (m, 18H), 1.86–1.96 (m, 4H), 4.27
(t, J = 7.4 Hz, 4H), 7.58 (app t, J = 1.6 Hz, 2H), 7.69 (app t,
J = 1.6 Hz, 2H), 9.01 (s, 2H); 13C NMR (100 MHz, CD3OD): d 27.3,
30.1, 30.5, 30.6, 31.2, 50.6, 121.1, 123.3, 136.3; HRMS (ESI) [M-H-
2Cl]+. Calcd for C18H31N4: 303.2549. Found: 303.2535.
C26H46N4: 414.3722. Found: 414.3748.
4.6. Representative procedure for the formation of bis-
imidazolium compounds, as outlined in Scheme 1
4.6.1. 1,4-Bis-(3-methyl-1H-imidazolium-1-yl)butane diiodide
(2)
The dihydrochloride salt 1ꢁ2HCl (400 mg, 1.52 mmol) was dis-
solved in water (2 mL) and the solution basified with an excess of
Na2CO3 (400 mg, 3.77 mmol). The mixture was extracted with
EtOAc (3ꢂ), and the combined organic extracts were dried (Na2SO4),
concentrated, and dried under high vacuum leaving the free base 1
(167 mg, 0.88 mmol, 58%). The free base 1 (167 mg, 0.88 mmol,
1 equiv) was dissolved in 1-propanol (2 mL) at rt and to this solution
was added iodomethane (0.22 mL, 3.52 mmol, 4 equiv). The mixture
was heated at reflux temperature for 24 h, then cooled to 0 °C. The
resulting oil was washed with Et2O (3 ꢂ 10 mL, decanting off the
Et2O layer each time), then concentrated at 60 °C and dried under
high vacuum leaving a beige solid (395 mg, 0.83 mmol, 94%). The so-
lid was recrystallized from boiling 2-propanol (4 mL), and washed
with Et2O. High-vacuum drying gave 2 (383 mg, 0.81 mmol, 92%)
as a white solid; mp 103–104 °C; 1H NMR (400 MHz, DMSO-d6): d
1.70–1.86 (m, 4H), 3.85 (s, 6H), 4.15–4.26 (m, 4H), 7.71 (s, 2H),
4.5.6. 1,13-Bis-(1H-imidazol-1-yl)tridecane dihydrochloride
(13ꢁ2HCl)
This compound was prepared in one step (88% yield) from 1,13-
diiodotridecane and was obtained as an orange oil; 1H NMR
(400 MHz, CD3OD): d 1.25–1.42 (m, 18H), 1.85–1.95 (m, 4H), 4.27
(t, J = 7.4 Hz, 4H), 7.59 (s, 2H), 7.69 (s, 2H), 9.02 (s, 2H); 13C NMR
(100 MHz, CD3OD): d 27.3, 30.1, 30.5, 30.6, 30.7, 31.2, 50.6, 121.1,
123.3, 136.3; HRMS (ESI) [M-H-2Cl]+. Calcd for C19H33N4:
317.2699. Found: 317.2696.
4.5.7. 1,14-Bis-(1H-imidazol-1-yl)tetradecane dihydrochloride
(15ꢁ2HCl)
This compound was prepared in one step (99% yield) from 1,14-
diiodotetradecane and was obtained as a white solid (not recrystal-

6538
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
7.76 (s, 2H), 9.11 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d 26.1, 36.0,
48.1, 122.3, 123.7, 136.6; HRMS (ESI) [M-2I]2+. Calcd for C12H20N4:
110.0838. Found: 110.0840.
NMR (100 MHz, DMSO-d6): d 25.5, 28.4, 28.9, 29.0 (2C), 29.4,
35.8, 48.8, 122.2, 123.6, 136.4; HRMS (ESI) [M-2I]2+. Calcd for
C21H38N4: 173.1542. Found: 173.1544.
4.7. Characterization of the bis-imidazolium compounds
synthesized following the representative procedure shown for
2, unless otherwise stated
4.7.7. 1,14-Bis-(3-methyl-1H-imidazolium-1-yl)tetradecane
diiodide (16)
This compound was prepared in one step (95% yield) from free
base 15 and was obtained as a amber oil; 1H NMR (400 MHz,
DMSO-d6): d 1.15–1.35 (m, 20H), 1.70–1.85 (m, 4H), 3.85 (s, 6H),
4.10–4.20 (m, 4H), 7.71 (s, 2H), 7.77 (s, 2H), 9.12 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 25.5, 28.4, 28.9, 29.0, 29.1, 29.4,
35.8, 48.8, 122.3, 123.6, 136.5; HRMS (ESI) [M-2I]2+. Calcd for
4.7.1. 1,6-Bis-(3-methyl-1H-imidazolium-1-yl)hexane diiodide
(4)
This compound was prepared in one step (95% yield) from free
base 3 and was obtained as a white solid (recrystallized from 2-pro-
panol/EtOH); mp 156–157 °C; 1H NMR (400 MHz, DMSO-d6): d
1.20–1.32 (m, 4H), 1.71–1.83 (m, 4H), 3.85 (s, 6H), 4.16 (t, J = 7.0 Hz,
4H), 7.70 (s, 2H), 7.77 (s, 2H), 9.12 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): d 24.9, 29.2, 35.9, 48.7, 122.3, 123.6, 136.5; HRMS (ESI)
[Mꢃ2I]2+. Calcd for C14H24N4: 124.0994. Found: 124.0998.
C22H40N4: 180.1621. Found: 180.1622.
4.7.8. 1,15-Bis-(3-methyl-1H-imidazolium-1-yl)pentadecane
diiodide (18)
This compound was prepared in one step (80% yield) from free
base 17 (using instead 2-propanol as reaction solvent) and was ob-
tained as an amber oil; 1H NMR (400 MHz, DMSO-d6): d 1.15–1.35
(m, 22H), 1.70–1.85 (m, 4H), 3.85 (s, 6H), 4.15 (t, J = 7.2 Hz, 4H),
7.70 (s, 2H), 7.77 (s, 2H), 9.11 (s, 2H); 13C NMR (100 MHz, DMSO-
d6): d 25.4, 28.3, 28.8, 28.9, 29.0, 29.3, 35.7, 48.7, 122.2, 123.5,
136.4; HRMS (ESI) [M-2I-H]2+. Calcd for C23H41N4: 373.3325.
Found: 373.3326.
4.7.2. 1,8-Bis-(3-methyl-1H-imidazolium-1-yl)octane diiodide
(6)
This compound was prepared in one step (88% yield) from free
base 5 and was obtained as a white–orange solid (recrystallized
from 2-propanol/Et2O); mp 115–116 °C; 1H NMR (400 MHz,
DMSO-d6): d 1.16–1.32 (m, 8H), 1.71–1.82 (m, 4H), 3.85 (s, 6H),
4.15 (t, J = 7.2 Hz, 4H), 7.70 (s, 2H), 7.76 (s, 2H), 9.11 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 25.4, 28.2, 29.4, 35.9, 48.8, 122.3,
123.6, 136.4; HRMS (ESI) [M-I]+. Calcd for C16H28N4I: 403.1358.
Found: 403.1368.
4.7.9. 1,16-Bis-(3-methyl-1H-imidazolium-1-yl)hexadecane
diiodide (20)
This compound was prepared in one step (90% yield) from free
base 19 and was obtained as a brown oil; 1H NMR (400 MHz,
DMSO-d6): d 1.10–1.40 (m, 24H), 1.70–1.85 (m, 4H), 3.85 (s, 6H),
4.14 (t, J = 6.8 Hz, 4H), 7.70 (s, 2H), 7.76 (s, 2H), 9.11 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 25.5, 28.4, 28.8, 29.0, 29.05, 29.09,
29.4, 35.8, 48.8, 122.2, 123.6, 136.4; HRMS (ESI) [M-2I]2+. Calcd
for C24H44N4: 194.1777. Found: 194.1783.
4.7.3. 1,10-Bis-(3-methyl-1H-imidazolium-1-yl)decane diiodide
(8)
This compound was prepared in one step (76% yield) from free
base 7 and was obtained as a brown oil; 1H NMR (400 MHz, DMSO-
d6): d 1.16–1.32 (m, 12H), 1.71–1.81 (m, 4H), 3.85 (s, 6H), 4.14 (t,
J = 7.2 Hz, 4H), 7.69 (s, 2H), 7.76 (s, 2H), 9.10 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 25.5, 28.4, 28.7, 29.4, 35.8, 48.8, 122.3,
123.6, 136.4; HRMS (ESI) [MꢃI]+. Calcd for C18H32N4I: 431.1671.
Found: 431.1658.
4.7.10. 1,18-Bis-(3-methyl-1H-imidazolium-1-yl)octadecane
diiodide (22)
This compound was prepared in one step (90% yield) from free
base 21 and was obtained as a brown oil; 1H NMR (400 MHz,
DMSO-d6): d 1.10–1.40 (m, 28H), 1.70–1.85 (m, 4H), 3.85 (s, 6H),
4.15 (t, J = 7.0 Hz, 4H), 7.70 (s, 2H), 7.77 (s, 2H), 9.11 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 25.5, 28.4, 28.8, 28.96, 29.03, 29.07
4.7.4. 1,11-Bis-(3-methyl-1H-imidazolium-1-yl)undecane
diiodide (10)
This compound was prepared in one step (96% yield) from free
base 9 and was obtained as a brown oil; 1H NMR (400 MHz, DMSO-
d6): d 1.12–1.34 (m, 14H), 1.68–1.84 (m, 4H), 3.85 (s, 6H), 4.15 (t,
J = 7.2 Hz, 4H), 7.71 (s, 2H), 7.78 (s, 2H), 9.13 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 25.5, 28.4, 28.8, 28.9, 29.4, 35.8, 48.7,
122.2, 123.6, 136.4; HRMS (ESI) [Mꢃ2I]2+. Calcd for C19H34N4:
159.1386. Found: 159.1380.
(2C), 29.3, 35.8, 48.8, 122.2, 123.6, 136.4; HRMS (ESI) [M-2I]2+
Calcd for C26H48N4: 208.1934. Found: 208.1946.
.
4.7.11. 1,20-Bis-(3-methyl-1H-imidazolium-1-yl)eicosane
diiodide (24)
This compound was prepared in one step (70% yield) from free
base 23 and was obtained as a yellow solid (recrystallized from 2-
propanol/Et2O); mp 78–80 °C; 1H NMR (400 MHz, DMSO-d6): d
1.18–1.30 (m, 32H), 1.72–1.82 (m, 4H), 3.85 (s, 6H), 4.15 (t,
J = 7.2 Hz, 4H), 7.70 (s, 2H), 7.77 (s, 2H), 9.11 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 25.5, 28.4, 28.8, 28.9, 29.1 (4C), 29.3,
35.8, 48.7, 122.2, 123.6, 136.4; HRMS (ESI) [M-2I]2+. Calcd for
4.7.5. 1,12-Bis-(3-methyl-1H-imidazolium-1-yl)dodecane
diiodide (12)
This compound was prepared in one step (97% yield) from free
base 11 and was obtained as a brown oil; 1H NMR (400 MHz,
DMSO-d6): d 1.15–1.32 (m, 16H), 1.70–1.82 (m, 4H), 3.85 (s, 6H),
4.14 (t, J = 7.2 Hz, 4H), 7.69 (s, 2H), 7.76 (s, 2H), 9.11 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 25.5, 28.4, 28.8, 28.9, 29.4, 35.8,
48.8, 122.3, 123.6, 136.4; HRMS (ESI) [M-I]+. Calcd for C20H36N4I:
459.1984. Found: 459.1977.
C28H52N4: 222.2090. Found: 222.2089.
4.8. Other synthetic procedures
4.8.1. 1,12-Bis-(3-ethyl-1H-imidazolium-1-yl)dodecane diiodide
(25)
4.7.6. 1,13-Bis-(3-methyl-1H-imidazolium-1-yl)tridecane
diiodide (14)
This compound was prepared in one step (96% yield) from free
base 13 and was obtained as a brown oil; 1H NMR (400 MHz,
DMSO-d6): d 1.16–1.32 (m, 18H), 1.71–1.82 (m, 4H), 3.85 (s, 6H),
4.15 (t, J = 7.2 Hz, 4H), 7.71 (s, 2H), 7.77 (s, 2H), 9.12 (s, 2H); 13C
The free base 11 (205 mg, 0.68 mmol, 1 equiv) was dissolved in
1-propanol (2 mL) at rt and to this solution was added iodoethane
(0.49 mL, 955 mg, 6.12 mmol, 9 equiv). The mixture was stirred at
reflux temperature (ꢀ110 °C) for 24 h, another portion of iodoeth-
ane (0.25 mL) was added, and the mixture stirred at ꢀ110 °C for

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
6539
3 h. The mixture was cooled to 0 °C, and Et2O was added. The
resulting insoluble oil was washed with Et2O (3 ꢂ 10 mL, Et2O
layer removed using a pipette each time), then dried under high
vacuum leaving an amber oil (323 mg, 0.53 mmol, 78%). The oil
(Rf = 0, EtOAc) was purified using preparatory-scale thin-layer
chromatography: the plate was loaded using MeOH, dried, and
then eluted twice using 95:5 (v/v) EtOAc–MeOH, and then twice
using 90:10 (v/v) EtOAc–MeOH; the bottom of the plate was ex-
cised and extracted using MeOH, the extract was filtered, and the
filtrate concentrated. High-vacuum drying gave 25 (189 mg,
0.31 mmol, 46%) as an amber oil; 1H NMR (400 MHz, DMSO-d6):
d 1.12–1.35 (m, 16H), 1.42 (t, J = 7.2 Hz, 6H), 1.73–1.87 (m, 4H),
4.10–4.28 (m, 8H), 7.79 (s, 2H), 7.81 (s, 2H), 9.21 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 15.0, 25.5, 28.4, 28.8, 28.9, 29.3,
44.2, 48.8, 122.1, 122.4, 135.6; HRMS (ESI) [Mꢃ2I]2+. Calcd for
4.8.5. 4,40-Bis-(1H-imidazol-1-yl)biphenyl (29)
Under an atmosphere of N2, a mixture of 4,40-dibromobiphenyl
(5.00 g, 16.03 mmol, 1 equiv), imidazole (4.58 g, 67.33 mmol,
4.2 equiv), K2CO3 (6.98 g, 50.50 mmol, 3.2 equiv), and CuSO4
(51.08 mg, 0.32 mmol, 0.02 equiv) was stirred at ꢀ180 °C for
24 h. The mixture was cooled to rt, water (200 mL) was added,
and the solid collected by filtration. The solid was dissolved in
warm EtOH (200 mL), the solution filtered, and the filtrate concen-
trated and dried under high vacuum. The resulting off-white solid
was purified using flash column chromatography on silica gel (sil-
ica plug was made using MeOH, washed with EtOAc, and eluted
with MeOH). High-vacuum drying gave 29 (0.87 g, 3.04 mmol,
19%) as an off-white solid; mp 288–289 °C; 1H NMR (400 MHz,
DMSO-d6): d 7.14 (s, 2H), 7.78 (d, J = 8.4 Hz, 4H), 7.83 (s, 2H),
7.88 (d, J = 8.8 Hz, 4H), 8.35 (s, 2H); 13C NMR (100 MHz, DMSO-
d6): d 117.9, 120.7, 128.0, 130.0, 135.5, 136.3, 137.3; HRMS (EI)
[M]+. Calcd for C18H14N4: 286.1218. Found: 286.1219.
C22H40N4: 180.1627. Found: 180.1623.
4.8.2. 1,12-Bis-(3-propyl-1H-imidazolium-1-yl)dodecane
diiodide (26)
4.8.6. 4,40-Bis-(3-methyl-1H-imidazolium-1-yl)biphenyl
diiodide (30)
The free base 11 (265 mg, 0.88 mmol, 1 equiv) was dissolved
in 1-propanol (7 mL) at rt and to this solution was added 1-iodo-
propane (854 mg, 5.26 mmol, 6 equiv). The mixture was stirred
at reflux temperature for 42 h and then concentrated. To the res-
idue were added MeOH (5 mL) and Et2O (20 mL) and the mixture
concentrated, and then dried under high vacuum leaving an im-
pure solid (525 mg). The solid (Rf ꢀ0, 9:1 EtOAc–MeOH) was
purified using flash column chromatography (colored impurities
eluted first using 9:1 (v/v) EtOAc–MeOH, and then the product
using 1:1 (v/v) EtOAc–MeOH). High-vacuum drying gave 26
(40 mg, 0.06 mmol, 7%) as a beige solid; mp 121–123 °C; 1H
NMR (400 MHz, DMSO-d6): d 0.84 (t, J = 7.4 Hz, 6H), 1.16–1.32
(m, 16H), 1.74–1.86 (m, 8H), 4.10–4.18 (m, 8H), 7.79 (s, 2H),
7.80 (s, 2H), 9.18 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d
10.4, 22.7, 25.5, 28.3, 28.8, 28.9, 29.3, 48.9, 50.3, 122.4 (2C),
135.9; HRMS (ESI) [M-2I]2+. Calcd for C24H44N4: 194.1777. Found:
194.1774.
Under an atmosphere of N2, a mixture of 4,40-bis-(1H-imidazol-
1-yl)biphenyl (29) (200 mg, 0.70 mmol,
1 equiv), 1-propanol
(3 mL), and iodomethane (0.44 mL, 994 mg, 7.00 mmol, 10 equiv)
was stirred at ꢀ85 °C for 6 h. The solution was stirred at rt for
24 h, then diluted with Et2O. The solid was collected by filtration
and washed with Et2O. High-vacuum drying gave 30 (365 mg,
0.64 mmol, 91%) as
a
yellow solid; mp >300 °C; 1H NMR
(400 MHz, DMSO-d6): d 3.98 (s, 6H), 7.93 (d, J = 8.4 Hz, 4H), 8.00
(s, 2H), 8.11 (d, J = 8.0 Hz, 4H), 8.39 (s, 2H), 9.87 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 36.3, 120.9, 122.3, 124.5, 128.5, 134.5,
136.0, 139.4; HRMS (ESI) [M-2I]2+. Calcd for C20H20N4: 158.0838.
Found: 158.0857.
4.8.7. 4,40-Bis-(1H-imidazol-1-yl-methyl)biphenyl (31)
Under an atmosphere of N2, a mixture of imidazole (569 mg,
8.36 mmol, 2.1 equiv) and sodium hydroxide (334 mg, 8.36 mmol,
2.1 equiv) in dimethyl sulfoxide (3 mL) was stirred at 70–80 °C for
1 h. The mixture was cooled to rt and a solution of 4,40-bis(chloro-
methyl)-1,10-biphenyl (1.00 g, 3.98 mmol, 1 equiv) in dimethyl
sulfoxide (5 mL) was slowly added. The mixture was stirred at
70–80 °C for 24 h, then cooled to rt and diluted with water
(300 mL). The resulting white precipitate was collected by filtra-
tion, washed with water (100 mL), dissolved in MeOH (10 mL),
and the solution was concentrated. High-vacuum drying gave 31
(981 mg, 3.12 mmol, 78%) as an off-white solid; mp 161–162 °C;
1H NMR (400 MHz, CDCl3): d 5.17 (s, 4H); 6.94 (s, 2H), 7.12 (s,
2H), 7.23 (d, J = 8.0 Hz, 4H), 7.55 (d, J = 7.6 Hz, 4H), 7.60 (s, 2H);
13C NMR (100 MHz, CDCl3): d 50.6, 119.4, 127.7, 127.9, 130.1,
135.7, 137.6, 140.5; HRMS (EI) [M]+. Calcd for C20H18N4:
314.1531. Found: 314.1519.
4.8.3. 1,4-Bis-(1H-imidazol-1-yl)benzene (27)
Under an atmosphere of N2, a mixture of 1,4-dibromobenzene
(2.50 g, 10.60 mmol, 1 equiv), imidazole (3.03 g, 44.52 mmol,
4.2 equiv), K2CO3 (4.69 g, 33.93 mmol, 3.2 equiv), and CuSO4
(34 mg, 0.21 mmol, 0.02 equiv) was stirred at ꢀ180 °C for 24 h.
The mixture was cooled to rt, and diluted with water (100 mL).
The resulting solid was collected by filtration and washed with
water (100 mL). The brown solid obtained was dissolved in hot
EtOH (200 mL), the solution filtered, and the filtrate concentrated.
High-vacuum drying gave 27 (1.87 g, 8.90 mmol, 84%) as a white
solid; mp 208–209 °C; 1H NMR (400 MHz, CDCl3): d 7.23 (s, 2H),
7.30 (s, 2H), 7.52 (s, 4H), 7.87 (s, 2H); 13C NMR (100 MHz, CDCl3):
d 118.3, 123.0, 131.1, 135.7, 136.6; HRMS (EI) [M]+. Calcd for
C12H10N4: 210.0905. Found: 210.0900.
4.8.8. 4,40-Bis-(3-methyl-1H-imidazolium-1-yl-methyl)biphenyl
diiodide (32)
4.8.4. 1,4-Bis-(3-methyl-1H-imidazolium-1-yl)benzene diiodide
(28)
Under an atmosphere of N2, a mixture of 4,40-bis-(1H-imidazol-
1-yl-methyl)biphenyl (31, 200 mg, 0.64 mmol, 1 equiv), 1-propa-
nol (3 mL), and iodomethane (0.40 mL, 908 mg, 6.40 mmol,
10 equiv) was stirred at ꢀ85 °C for 24 h, then stirred at rt for
24 h. The resulting yellow precipitate was collected by filtration
and washed with Et2O (50 mL). High-vacuum drying gave 32
(346 mg, 0.58 mmol, 91%) as yellow solid; mp 215–216 °C; 1H
NMR (400 MHz, DMSO-d6): d 3.87 (s, 6H), 5.48 (s, 4H), 7.53 (d,
J = 8.0 Hz, 4H), 7.73 (d, J = 6.8 Hz, 4H), 7.74 (s, 2H), 7.84 (s, 2H),
9.26 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d 35.9, 51.5, 122.3,
124.0, 127.2, 129.0, 134.3, 136.7, 139.7; HRMS (ESI) [M-2I]2+. Calcd
for C22H24N4: 172.0994. Found: 172.0992.
Under an atmosphere of N2, a mixture of 1,4-bis-(1H-imidazol-1-
yl)benzene (27) (200 mg, 0.95 mmol, 1 equiv), 1-propanol (3 mL),
and iodomethane (0.57 mL, 1.30 g, 9.50 mmol, 10 equiv) was stirred
at rt for 1 h, then stirred at ꢀ85 °C for 24 h. The mixture was cooled
to rt and diluted with Et2O (30 mL). The resulting white precipitate
was collected by filtration and washed with Et2O (20 mL). High-vac-
uum drying gave 28 (335 mg, 0.68 mmol, 72%) as a white solid; mp
324–325 °C; 1H NMR (400 MHz, DMSO-d6): d 3.98 (s, 6H), 8.01 (s,
2H), 8.11 (s, 4H), 8.40 (s, 2H), 9.91 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): d 36.4, 120.9, 123.4, 124.6, 135.2, 136.4; HRMS (ESI)
[M–2I]2+. Calcd for C14H16N4: 120.0681. Found: 120.0680.

6540
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
4.8.9. 4,40-Bis-(3-benzyl-1H-imidazolium-1-yl)biphenyl
dibromide (33)
d 33.6, 114.0, 125.8, 128.9, 130.9, 131.9, 133.1, 140.3, 143.2; HRMS
(ESI) [Mꢃ2I]2+. Calcd for C28H24N4: 208.0994. Found: 208.0992.
Under an atmosphere of N2, 4,40-bis-(1H-imidazol-1-yl)biphe-
nyl (29, 200 mg, 0.70 mmol, 1 equiv) was suspended in 1-propanol
(3 mL) and to this mixture was added benzyl bromide (0.33 mL,
479 mg, 2.80 mmol, 4 equiv). The mixture was stirred at ꢀ85 °C
for 24 h, and then slowly cooled to 0 °C, to give a white solid pre-
cipitate. The solid was collected by filtration and washed with cold
1-propanol and then with Et2O. The white solid obtained was
recrystallized from hot MeOH. High-vacuum drying gave 33
(176 mg, 0.28 mmol, 40%) as a white solid; mp 308–309 °C; 1H
NMR (400 MHz, DMSO-d6): d 5.56 (s, 4H), 7.41–7.50 (m, 6H),
7.98 (d, J = 8.4 Hz, 4H), 7.98 (d, J = 8.4 Hz, 4H), 8.11 (d, J = 8.8 Hz,
6H), 8.45 (s, 2H), 10.21 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d
52.4, 121.6, 122.4, 123.3, 128.4, 128.6, 128.9, 129.0, 134.5, 134.5,
4.8.12. 1,4-Bis[3-(1H-imidazol-1-yl)propoxy]benzene
dihydrochloride (36ꢁ2HCl)
Under an atmosphere of N2, a mixture of hydroquinone (2.75 g,
25 mmol, 1 equiv), 1,3-dibromopropane (20.2 g, 100 mmol,
2 equiv), and anhydrous K2CO3 (10.35 g, 75 mmol, 6 equiv) in ace-
tone (75 mL) was stirred at reflux temperature for 24 h. The solid
was removed by filtration and discarded, and the filtrate was con-
centrated. The semi-solid residue was diluted with hexanes
(125 mL), and the resulting solid was removed by filtration and
discarded. The filtrate was set aside overnight, and the crystals that
formed were collected by filtration (2.05 g). The filtrate was
concentrated to a yellow oil that was diluted with 2-propanol
135.6, 139.4; HRMS (ESI) [Mꢃ2Br]2+
.
Calcd for C32H28N4:
(10 mL) and refrigerated overnight to afford a second crop
234.1151. Found: 234.1148.
(1.28 g) of crystals. The combined crystalline material was recrys-
tallized from ethanol (10 mL). High-vacuum drying gave 1,4-bis
(3-bromopropyloxy)benzene (2.88 g, 65%) as a yellow solid; mp
72–73 °C; 1H NMR (400 MHz, CDCl3): d 2.25–2.34 (m, 4H), 3.60
(t, J = 6.6 Hz, 4H), 4.06 (t, J = 5.8 Hz, 4H), 6.84 (s, 4H); 13C NMR
(100 MHz, CDCl3): d 30.2, 32.6, 66.2, 115.7, 153.2; HRMS (EI)
[M]+. Calcd for C12H16Br2O2: 349.9512. Found 349.9521.
4.8.10. 4,40-Bis-[3-(4-nitrobenzyl)-1H-imidazolium-1-
yl]biphenyl dibromide (34)
Under an atmosphere of N2, 4,40-bis-(1H-imidazol-1-yl)biphe-
nyl (29) (200 mg, 0.70 mmol, 1 equiv) was suspended in 1-propa-
nol (6 mL) and to this mixture was added 4-nitrobenzyl bromide
(907 mg, 4.20 mmol, 6 equiv). The mixture was stirred at ꢀ110 °C
for 72 h, and then slowly cooled to 0 °C, to give a solid precipitate.
The solid was collected by filtration and washed with cold 1-pro-
panol and then with Et2O. The yellow solid obtained was recrystal-
lized from hot MeOH (20 mL). High-vacuum drying gave 34
(127 mg, 0.18 mmol, 26%) as a yellow solid; mp 302–303 °C; 1H
NMR (400 MHz, DMSO-d6): d 5.74 (s, 4H), 7.83 (d, J = 8.4 Hz, 4H),
7.98 (d, J = 8.4 Hz, 4H), 8.12 (d, J = 7.2 Hz, 6H), 8.31 (d, J = 8.4 Hz,
4H), 8.49 (s, 2H), 10.20 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d
51.5, 121.7, 122.4, 123.4, 124.0, 128.4, 129.8, 134.5, 136.2, 139.5,
Under an atmosphere of N2, a mixture of imidazole (1224 mg,
18 mmol, 6 equiv) and NaOH (720 mg, 18 mmol, 6 equiv) in DMSO
(8 mL) was stirred at 70–80 °C for 1 h, then 1,4-bis(3-bromopro-
pyloxy)benzene (1056 mg, 3 mmol, 1 equiv) was added, and the
mixture stirred at 70–80 °C overnight. The mixture was partitioned
between water (120 mL) and ethyl acetate (30 mL), the organic
phase separated, and the aqueous phase was further extracted
with ethyl acetate (2 ꢂ 20 mL). The combined organic extracts
were washed with water (50 mL) and then with brine (20 mL),
dried over anhydrous Na2SO4, and concentrated to a brown solid
(784 mg, 80%). A portion of this solid free base (228 mg, 0.70 mmol,
1 equiv) was dissolved in methanol (3 mL) and to this solution was
added 37% aqueous HCl (300 mg, 3.05 mmol, 2.2 equiv). The mix-
ture was concentrated and the residue recrystallized from 2-propa-
nol. High-vacuum drying gave 36ꢁ2HCl (146 mg, 0.37 mmol, 53%
from free base, 42% overall) as a hygroscopic tan solid; mp 203–
204 °C; 1H NMR (400 MHz, DMSO-d6): d 2.20–2.31 (m, 4H), 3.92
(t, J = 6.0 Hz, 4H), 4.38 (t, J = 6.8 Hz, 4H), 6.81 (s, 4H), 7.69 (t,
J = 1.0 Hz, 2H), 7.84 (t, J = 1.0 Hz, 2H), 9.26 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 29.1, 46.1, 65.1, 115.3, 119.7, 122.1,
141.6, 147.7; HRMS (ESI) [M-2Br]2+
279.1002. Found: 279.1002.
. Calcd for C32H26N6O4:
4.8.11. 4,40-Bis-(3-methyl-1H-benzimidazolium-1-yl)biphenyl
diiodide (35)
Under an atmosphere of N2, a mixture of 4,40-dibromobiphenyl
(1.00 g, 3.20 mmol, 1 equiv), benzimidazole (1.89 g, 16.00 mmol,
5 equiv), K2CO3 (1.37 g, 9.92 mmol, 3.1 equiv), and CuSO4 (16 mg,
0.10 mmol, 0.03 equiv) was stirred at ꢀ180 °C for 24 h. The mix-
ture was cooled to rt, water (200 mL) was added, and the solid col-
lected by filtration. The solid was dissolved in hot MeOH (200 mL),
the solution filtered, and the filtrate concentrated and dried under
high vacuum. The resulting grey solid was washed with hot water
(3 ꢂ 50 mL) and then recrystallized from hot EtOAc (30 mL). High-
vacuum drying gave 4,40-bis-(1H-benzimidazol-1-yl)biphenyl
(91 mg, 0.24 mmol, 8%) as a brown solid; mp 258–259 °C; 1H
135.3, 152.4; HRMS (ESI) [Mꢃ2Cl]2+
. Calcd for C18H24N4O2:
164.0944. Found 164.0953.
4.8.13. 1,4-Bis[3-(3-methyl-1H-imidazolium-1-yl)propoxy]-
benzene diiodide (37)
Under an atmosphere of N2 atmosphere, the free base 36
(458 mg, 1.40 mmol, 1 equiv) was dissolved in 1-propanol (4 mL)
at rt and to this solution was added iodomethane (1.99 g,
14.00 mmol, 10 equiv). The mixture was stirred at reflux tempera-
ture overnight, and then cooled to rt. The dark-brown solution was
diluted with diethyl ether (25 mL), and the solution was stirred for
2 h resulting in an insoluble oil. The supernatant was removed using
a pipette, and the oil washed with diethyl ether (2 ꢂ 25 mL). The
brown oil was dissolved in hot ethanol (10 mL), diluted with ethyl
acetate (10 mL), and stirred vigorously overnight. The solid that
formed was collected by filtration. High-vacuum drying gave 37
NMR (400 MHz, DMSO-d6):
d 7.30–7.42 (m, 4H), 7.72 (d,
J = 7.6 Hz, 2H), 7.81 (d, J = 7.6 Hz, 2H), 7.85 (d, J = 8.4 Hz, 4H),
8.04 (d, J = 8.4 Hz, 4H), 8.66 (s, 2H); 13C NMR (100 MHz, DMSO-
d6): d 110.8, 120.1, 122.6, 123.6, 124.2, 128.4, 135.6, 138.3; HRMS
(ESI) [M+H]+. Calcd for C26H19N4: 387.1609. Found: 387.1607.
Under an atmosphere of N2, a mixture of 4,40-bis-(1H-ben-
zimidazol-1-yl)biphenyl (59 mg, 0.15 mmol, 1 equiv), 1-propanol
(2 mL), and iodomethane (0.09 mL, 213 mg, 1.50 mmol, 10 equiv)
was stirred at ꢀ85 °C for 24 h and then stirred at rt for 24 h. Et2O
(50 mL) was added, and the resulting solid precipitate was col-
lected by filtration, washed with Et2O, and dried under high vac-
uum to obtain a brown solid. The solid was recrystallized from
hot MeOH (7 mL) with a few drops of Et2O. High-vacuum drying
gave 35 (20 mg, 0.03 mmol, 20%) as orange solid; mp >300 °C; 1H
NMR (400 MHz, DMSO-d6): d 4.21 (s, 6H), 7.70–8.05 (m, 10H),
8.15–8.25 (m, 6H), 10.19 (s, 2H); 13C NMR (100 MHz, DMSO-d6):
(510 mg, 60%) as
a
brown solid; mp 127–128 °C; 1H NMR
(400 MHz, DMSO-d6): d 2.14–2.29 (m, 4H), 3.85 (s, 6H), 3.95 (t,
J = 6.0 Hz, 4H), 4.34 (t, J = 6.8 Hz, 4H), 6.84 (s, 4H), 7.72 (t,
J = 1.6 Hz, 2H), 7.81 (t, J = 1.6 Hz, 2H), 9.17 (s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 29.1, 35.8, 46.4, 64.9, 115.4, 122.4, 123.5,
136.7, 152.4; HRMS (ESI) [Mꢃ2I]2+
. Calcd for C20H28N4O2:
178.1100. Found 178.1100.

J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
6541
4.8.14. 1,12-Bis(1H-1,2,4-triazol-1-yl)dodecane dihydrochloride
(38ꢁ2HCl)
The IC50 values of individual compounds were determined using
a non-linear regression analysis of the data35 using the computer
program SigmaPlot (Jandel Scientific). The IC50 values represent
the mean SEM, n = 4. Parallel experiments using chloroquine
were included as a positive control. The determination of the activ-
ities of compounds in P. berghei cultures presented in Table 2 was
performed using the SYBR-Green method35 as previously de-
scribed.4 P. berghei was obtained by cardiac puncture of an infected
mouse (parasitemia of 5%); the blood was washed 3 times in RPMI
and was then plated at a 2% hematocrit in a gradient of compounds
as above. After 24 h the contents of the well were agitated to effect
merozoite release36 and the plate was incubated for a further 24 h
before SYBR-Green I was added and the relative fluorescence was
determined using a Fluostar Optima plate reader (BMG Labtech,
Offenburg, Germany).
Under an atmosphere of N2,
a mixture of 1,2,4-triazole
(1035 mg, 15.00 mmol, 2.5 equiv) and NaOH (600 mg, 15.00 mmol,
2.5 equiv) in DMSO (4 mL) was stirred at 70–80 °C for 1 h, then
1,12-dibromododecane (1.97 g, 6.00 mmol, 1 equiv) was added,
and the mixture stirred at 70–80 °C overnight. The mixture was di-
luted with water (50 mL), and the solid that formed was collected
by filtration and air-dried to give the crude bis-triazole product
(1.50 g, 4.93 mmol, 82%). The solid was dissolved in ethanol
(10 mL), and then a 37% aqueous solution of HCl (2 g, 20.30 mmol,
2.1 equiv) in ethanol (2 mL) was added. The precipitate that
formed was collected by filtration and recrystallized from ethanol.
High-vacuum drying gave 38ꢁ2HCl (1.45 g, 64%) as a white solid;
mp 192–193 °C; 1H NMR (400 MHz, DMSO-d6): d 1.21 (br s, 16H),
1.72–1.83 (m, 4H), 4.20 (t, J = 7.0 Hz, 4H), 8.24 (s, 2H), 8.91 (s,
2H); 13C NMR (100 MHz, DMSO-d6): d 25.6, 28.3, 28.8, 28.9, 49.6,
4.10. Mammalian strains and culture
142.8, 148.0; HRMS (ESI) [Mꢃ2Cl]2+
. Calcd for C16H30N6:
153.1261. Found 153.1265.
The effect of the compounds on CHO cell cultures was deter-
mined by using the MTT assay37 as described previously.5 CHO
cells (ATCCÒ, Manassas, VA, USA) were grown in RPMI-1640 sup-
plemented with 10% fetal calf serum (Sigma–AldrichÒ Canada,
Ltd, Oakville, ON, Canada), 25 mM HEPES, and gentimicin (referred
to as RPMI-10). Cells were seeded in 96-well plates and grown to
4.8.15. 1,12-Bis(4-methyl-1H-1,2,4-triazolium-1-yl)dodecane
diiodide (39)
A sample of 38ꢁ2HCl (530 mg, 1.40 mmol) was suspended in
water (30 mL) and the mixture basified with an aqueous solution
of NaOH (2 M). The mixture was extracted with ethyl acetate
(30 mL). The organic phase was washed successively with water
(30 mL) and brine (10 mL), dried (Na2SO4), and concentrated.
High-vacuum drying gave the free base 38 (417 mg, 1.37 mmol,
98%) as a white solid. Under an atmosphere of N2, the free base
38 (417 mg, 1.37 mmol, 1 equiv) was dissolved in warm 1-propa-
nol (7 mL) and the solution was treated with iodomethane
(1.95 g, 13.72 mmol, 10 equiv). The mixture was stirred at reflux
temperature overnight, and then it was cooled to rt and diluted
with diethyl ether (20 mL). The solid that formed was collected
by filtration, washed with diethyl ether, and then recrystallized
from methanol. High-vacuum drying gave 39 (492 mg, 61%) as an
off-white solid; mp 203–204 °C; 1H NMR (400 MHz, DMSO-d6): d
1.18–1.34 (m, 16H), 1.77–1.89 (m, 4H), 3.89 (s, 6H), 4.35 (t,
J = 7.0 Hz, 4H), 9.13 (s, 2H), 10.05 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): d 25.4, 28.1, 28.3, 28.8, 28.9, 34.1, 51.4, 142.9, 145.4;
50% confluency in 100
of either DMSO alone, or a 10 mg/mL solution of a test compound
in DMSO. Compound gradients were prepared by adding 90 L of
RPMI-10 mixed with 10 L of compound solution to the first well
in the series, mixing, transferring 100 L to the next well, and
lL of RPMI-10 per well prior to the addition
l
l
l
repeating until the next-to-last well was reached. After 48 h, the
viability of the cells was determined by discarding the media in
the wells and adding 100 lL of 10 mg/mL of 3-[4,5-dimethylthia-
zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma–AldrichÒ
Canada, Ltd, Oakville, ON, Canada) in RPMI-10, incubating the
plates for a further 30 min, and then removing the media and add-
ing 100 lL of DMSO and reading the absorbance at 650 nm (see
Ref. 37). The IC50 values of individual compounds were determined
using a non-linear regression analysis of the sigmoidal dose-re-
sponse curves using the computer program SigmaPlot (Jandel Sci-
entificÒ, San Rafael, CA, USA).
HRMS (ESI) [Mꢃ2I]2+
. Calcd for C18H34N6: 167.1417. Found
167.1419.
4.11. Examination of treated parasite cultures using light
microscopy
4.9. Determination of anti-Plasmodium activity
A Percoll gradient38 was used to synchronize 3D7 parasite cul-
tures at the mature stage of development one day prior to the start
of the parasite development assay. The cultures were then adjusted
to have a hematocrit of 1% and a parasitemia of 2%. Parasites were
then treated with either 40 nM or 83 nM of compound 39 and
sampled every 24 h over a period of 4 days. Controls included
uninfected RBCs, untreated cultures, treatment with sulfated
b-cyclodextrin (sodium salt, 7–11 mol of sulfate residues per mol
of b-cyclodextrin), and treatment with chloroquine.
P. falciparum cultures were grown in O+ blood obtained by veni-
puncture of volunteers. Cultures of the laboratory line ItG were
maintained by the method of Trager and Jensen32 using RPMI-
1640 supplemented with 10% human serum and 50 lM hypoxa-
nthine (RPMI-A). The effects of the test compounds on the viability
of P. falciparum cultures were evaluated using a lactate dehydroge-
nase (LDH) enzyme assay specific to the enzyme found in P. falcipa-
rum (pLDH).33,34 Briefly, compounds to be tested were dissolved in
DMSO to afford a solution having a concentration of 10 mg/mL.
Fifty microliters of RPMI-A were added to every well in a 96-well
4.12. Flow cytometry analysis
plate, then 5
lL of the 10 mg/mL compound solution mixed with
45 L of RPMI-A were added to the first well, and then serially
l
Parasite cultures were synchronized as ring-stage cultures by
sorbitol lysis.38 The cultures were then adjusted to a parasitemia
of 5% and a hematocrit of 0.5% and were treated with various com-
pounds for 48 h before an equal volume of RPMI-1640 containing
diluted across the plate to produce a compound gradient with
two-fold dilutions. Fifty microliters of parasite culture (2% hemat-
ocrit, 2% parasitemia) were added to each well and the plates were
then incubated at 37 °C in 95% N2, 3% CO2, and 2% O2 for 72 h. The
0.2 lL per mL of stock SYBR-Green solution was added, and the
contents of the wells were then re-suspended and 15-
lL samples
mixture was allowed to incubate for 1 h in the dark. The samples
were subjected to flow cytometry using a FACS Calibur Flow
Cytometer in a level II BSC. Data from the sample were collected
and analysed using the FlowJo software program.
were removed and added to 100
lL of pLDH enzyme assay mixture
in the corresponding well of a second 96-well plate.33 After 1 h the
absorbance of the wells at 595 nm was determined using a Ther-
moMax microplate reader (Molecular Devices, Sunnyvale, CA).

6542
J. Z. Vlahakis et al. / Bioorg. Med. Chem. 19 (2011) 6525–6542
11. Hoffman, S. L. Malaria Vaccine Development:
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4.13. In vivo evaluation of compounds
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8980. Supplementary data.
13. Ganske, F.; Meyer, H. H.; Deutz, H.; Bornscheuer, U. Eur. J. Lipid Sci. Technol.
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14. Fan, J.; Hanson, B. E. Chem. Commun. 2005, 18, 2327.
15. Crandall, I. E.; Szarek, W. A.; Vlahakis, J. Z.; Xu, Y.; Vohra, R.; Sui, J.; Kisilevsky,
R. Biochem. Pharmacol. 2007, 73, 632.
16. Foley, M.; Tilley, L. Int. J. Parasitol. 1997, 27, 231.
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Female 6–8 week-old Balb/c mice (Charles River) were infected
with 106 P. berghei parasites on Day 0 and their parasitemia was
followed by producing Giemsa-stained peripheral blood films until
parasites were observed in the peripheral circulation of the mice.
The mice were then infused once daily for three days with either
0.5 mL of RPMI-1640 or RPMI-1640 containing compound. The
health of the animals was monitored closely and the parasitemia
of the mice was determined by examining Giemsa-stained periph-
eral blood films. Experimental groups contained five mice each and
had unrestricted access to food and water during the experiment.
20. Calas, M.; Ancelin, M.-L.; Cordina, G.; Portefaix, P.; Piquet, G.; Vidal-Sailhan, V.;
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Acknowledgments
This work was funded by a CIHR Proof-of-Principle Grant (CIHR
PPP-84167) and a BioDiscovery Grant (BDT-2012-S3a). Expert help
with the flow cytometry was provided by Dr. Constance Finney and
Kathleen Zhong.
22. Ahiboh, H.; Djaman, A. J.; Yapi, F. H.; Edjeme-Aké, A.; Hauhouot-
Attoungbré, M.-L.; Yayo, E. D.; Monnet, D. J. Enzyme Inhib. Med. Chem.
2009, 24, 911.
23. Hamzé, A.; Rubi, E.; Arnal, P.; Boisbrun, M.; Carcel, C.; Salom-Roig, X.;
Maynadier, M.; Wein, S.; Vial, H.; Calas, M. J. Med. Chem. 2005, 48, 3639.
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