Anti-Plasmodium activity of tetrazolium salts

Article abstract of DOI:10.1016/j.bmc.2007.11.005

We have previously reported that sulfated cyclodextrins inhibit the invasion of Plasmodium merozoites by interacting with receptors present on the surface of erythrocytes. The observation that tetrazolium salts formed stable complexes with the inhibitory sulfated cyclodextrins suggested that tetrazolium salts might have anti-Plasmodium activity as well. Evaluation of commercially available tetrazolium salts indicated that some were active in the low nanomolar range and showed specificity in their inhibition of Plasmodium. Synthesis of a further 54 structures allowed us to determine that activity results from an aromatic component attached to the tetrazolium carbon atom (R¹) and its size is not critical to the activity of the compound. Nitro modifications of active compounds are poorly tolerated, however, the presence of halogen atoms on aromatic groups attached to the nitrogen atoms of the tetrazolium ring (R² and R³) has little effect on activity. Methoxy groups are tolerated on R² and R³ components; however, they are disruptive on the R¹ component. The overall results suggest that the R¹ component is interacting with a specific hydrophobic environment and the R² and R³ components are less constrained. The activity of these compounds in several human and mouse Plasmodium cultures suggests that the compounds interact with a component of the parasite that is both essential and conserved.

Full text of DOI:10.1016/j.bmc.2007.11.005

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1927–1947
Anti-Plasmodium activity of tetrazolium salts
Xilin Cui,a Jason Z. Vlahakis,a Ian E. Crandallb,* and Walter A. Szareka,*
aDepartment of Chemistry, Queen’s University, Kingston, ON, Canada K7L 3N6
bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada M5S 1A8
Received 25 May 2007; revised 23 October 2007; accepted 1 November 2007
Available online 5 November 2007
Abstract—We have previously reported that sulfated cyclodextrins inhibit the invasion of Plasmodium merozoites by interacting
with receptors present on the surface of erythrocytes. The observation that tetrazolium salts formed stable complexes with the inhib-
itory sulfated cyclodextrins suggested that tetrazolium salts might have anti-Plasmodium activity as well. Evaluation of commercially
available tetrazolium salts indicated that some were active in the low nanomolar range and showed specificity in their inhibition of
Plasmodium. Synthesis of a further 54 structures allowed us to determine that activity results from an aromatic component attached
to the tetrazolium carbon atom (R1) and its size is not critical to the activity of the compound. Nitro modifications of active com-
pounds are poorly tolerated, however, the presence of halogen atoms on aromatic groups attached to the nitrogen atoms of the tet-
razolium ring (R2 and R3) has little effect on activity. Methoxy groups are tolerated on R2 and R3 components; however, they are
disruptive on the R1 component. The overall results suggest that the R1 component is interacting with a specific hydrophobic envi-
ronment and the R2 and R3 components are less constrained. The activity of these compounds in several human and mouse Plas-
modium cultures suggests that the compounds interact with a component of the parasite that is both essential and conserved.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ligand/receptor interactions involved in erythrocyte
invasion.6 The invasion process is rapid, typically 15–
30 s,7 and is divided frequently into three stages: (1)
the initial attachment of the merozoite to the erythro-
cyte followed by reorientation of the merozoite such
that its apical end is proximal to the erythrocyte; (2)
the secretion and anchoring of proteins required to form
a ‘tight junction’ with the erythrocyte membrane; and
(3) physical insertion.8 While the receptors required for
tight-junction formation are reasonably well character-
ized9 the identities of the molecules responsible for the
initial contact events are unclear.
The number of people who are at risk of acquiring ma-
laria is not known precisely, however, some estimates
are as high as 3.2 billion people with up to 500 million
clinical cases per year.1 Further, malaria contributes to
the deaths of 1–3 million people per year,2 most of them
young children. The continued spread of resistance to
antimalarials such as chloroquine3 has produced a des-
perate need for safe and inexpensive antimalarial agents
having novel mechanisms of action. Our previous work4
demonstrated that small sulfonated compounds inhib-
ited the entry of merozoites of Plasmodium falciparum
into human erythrocytes, and were able also to suppress
the replication of Plasmodium berghei in mice. The inter-
actions between a merozoite and an erythrocyte that are
responsible for the invasion process are complex;5 how-
ever, individual Plasmodium species are frequently lim-
ited to a particular host, or even a particular stage of
development of an erythrocyte, by the specificity of the
Treatment of erythrocytes with sulfated cyclodextrins
leads to an increase in surface staining with cationic
dyes,10 an observation that is consistent with the com-
pounds’ interaction with an erythrocyte surface compo-
nent. While the inhibitory activity of these compounds
may result entirely from non-specific effects, such as
repulsive negative surface charges,11 alterations in the
conformations of surface receptors,12 or changes in the
multimerization state of the receptor proteins,13 our pre-
vious work10 suggested that a specific mechanism was
responsible since sulfated cyclodextrins could be dis-
placed effectively from erythrocytes by stilbene disulfo-
nates. Stilbene disulfonates, such as SITS (4-
acetamido-40-isothiocyanato-2,20-stilbenedisulfonic acid
Keywords: Plasmodium falciparum; Tetrazolium salts; Malaria.
*
Corresponding authors. Tel.: +1 416 581 7704; fax: +1 416 978 8765
(I.E.C.); tel.: +1 613 533 2643; fax: +1 613 533 6532 (W.A.S.); e-mail
addresses: ian.crandall@utoronto.ca; walter.szarek@chem.queensu.
ca
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.005

1928
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
disodium salt) and DIDS (4,40-diisothiocyanatostilbene-
2,20-disulfonic acid disodium salt), are highly specific
inhibitors of the erythrocyte anion-exchange protein
AE1, also known as band 3.14 An opportunity to test
the hypothesis that sulfated cyclodextrins or stilbene
disulfonates share a surface topology and charge distri-
bution with a merozoite ligand arose when we observed
that commercially available tetrazolium salts, which are
components of our viability assays, could not be re-
duced in the presence of sulfated cyclodextrins or stil-
bene disulfonates. We reasoned that, if a merozoite
ligand were composed of an anionic glycan, then tetra-
zolium salts might be able to prevent merozoite attach-
ment and invasion. Interestingly, it has been reported15
that mono- and bis-thiazolium salts have potent antima-
larial activity; these compounds target the plasmodial
phospholipid metabolism. In the present work, the
anti-Plasmodium activities of eight commercially avail-
able tetrazolium salts were evaluated and it was ob-
served that several compounds inhibited P. falciparum
cultures in the nanomolar range. To explore further
the potential relationship between structure and activity
an additional fifty-four tetrazolium salts were synthe-
sized. Our findings suggest that tetrazolium compounds
may form the basis for a novel class of antimalarials.
concentrations some of the sulfated compounds also
inhibited the LDH assay employed in the parasite viabil-
ity studies.19 Further investigation indicated that sul-
fated cyclodextrins had no effect on the activity of the
P. falciparum or mammalian lactate dehydrogenase en-
zymes or the diaphorase enzyme used in the assay19
(data not shown), however, in the presence of sulfated
cyclodextrins, nitrotetrazolium blue chloride (NBT)
could not be reduced to the corresponding formazan
dye by reducing agents such as glutathione. Non-sul-
fated cyclodextrins had no effect on the reduction of
NBT, a result suggesting that the negatively charged sul-
fate groups formed a high-affinity complex with the elec-
tron-deficient tetrazolium ring thereby stabilizing it
against reduction to the uncharged formazan. We have
hypothesized10 that sulfated cyclodextrins might share
charge and topographical features with stilbene disulfo-
nates such as SITS (4-acetamido-40-isothiocyanato-2,
20-stilbenedisulfonic acid disodium salt) and DIDS (4,40-
diisothiocyanatostilbene-2,20-disulfonic acid disodium
salt), an aspect which might allow sulfated cyclodextrins
to compete with these compounds for the ‘‘DIDS site’’
in AE1.20 DIDS and SITS also inhibited the reduction
of NBT by glutathione. The observation that a tetrazo-
lium salt interacted with stilbene disulfonates or sulfated
cyclodextrins suggested that if a merozoite ligand shared
common structural features with these agents then tetra-
zolium salts should have anti-P. falciparum properties.
To test this hypothesis, the effects of eight commercially
available tetrazolium salts on the viability of P. falcipa-
rum cultures were evaluated (see Table 1) and the
potency was compared to the toxicity observed in a
mammalian cell line, namely Chinese Hamster Ovary
(CHO) cells. Some tetrazolium salts, such as thiazoyl
blue tetrazolium bromide (MTT), nitrotetrazolium blue
chloride (NBT), and tetranitroblue tetrazolium chloride,
produced oxidative erythrocyte lysis; however, others,
such as tetrazolium red and tetrazolium violet, were
found to inhibit parasite viability in the low micromolar
range (see Table 1 and Fig. 4). Microscopic examination
of the treated wells revealed images that were consistent
with merozoite invasion being inhibited; however, other
toxic effects could be possible. Compounds such as tetra-
zolium red and tetrazolium violet were found to be tol-
erated relatively well by mammalian cells (see Table 1
and Fig. 4), a result that suggested that these com-
pounds might inhibit a process that was specific to the
viability of P. falciparum cultures. As regards the bio-
availability of the tetrazolium salts, it is known that such
compounds are able to cross membranes and indeed this
ability is the basis of the MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide) assay.21
2. Synthesis
The QT series of tetrazolium compounds (see Fig. 1, for
generic structure) were synthesized according to the gen-
eral synthetic route shown in Scheme 1. In the first step,
a substituted alkyl- or aryl- aldehyde was condensed
with a substituted phenylhydrazine in acidic MeOH to
form the hydrazone derivative. Concurrently, a substi-
tuted aryldiazonium salt was freshly prepared from a
substituted aromatic amine by treatment with concen-
trated hydrochloric acid and sodium nitrite.16,17 The for-
mazan was then prepared by an azo coupling reaction of
the hydrazone derivative with the diazonium salt in pyr-
idine. Oxidation of the formazan to the tetrazolium salt
was then accomplished using freshly prepared tert-butyl
hypochlorite18 in chloroform. Using this four-step meth-
odology, a wide variety of functionalized tetrazolium
salts were synthesized using three commercially avail-
able starting materials (alkyl- or aryl- aldehydes, aryl-
hydrazines, and arylamines). The wide diversity of
available starting materials and control over the posi-
tion of substituents on the tetrazolium ring allowed us
to explore systematically structure–activity relationships
of almost sixty compounds (see Table 1) with a view of
finding effective antimalarial drug candidates. The R1
substituent (originating from the aldehyde) is attached
at a unique position on the tetrazolium ring, however,
because of charge delocalization in the tetrazolium ring
the R2 and R3 positions are interchangeable (see Fig. 1).
To determine if tetrazolium salts inhibited a process that
was specific to a particular tight-junction receptor the ef-
fects of tetrazolium violet on other laboratory P. falcipa-
rum isolates and two murine malarias were evaluated.
Tetrazolium violet was found to have similar potency
in the laboratory lines ItG, 3D7, and FCR3 (Fig. 5),
and similar results were obtained with the murine mala-
rias, P. berghei and Plasmodium chabaudi chabaudi. P.
falciparum lines that were grown in erythrocyte cultures,
that had been treated either with neuraminidase or tryp-
3. Biological evaluation
When sulfated cyclodextrins were being evaluated for
their ability to inhibit the entry of P. falciparum merozo-
ites into erythrocytes it was observed10 that at higher

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1929
Figure 1. Generic structure for tetrazolium salts.
Scheme 1. General synthetic strategy for the generation of tetrazolium salts.
sin,22,9 were also found to yield similar IC50 values, an
observation suggesting that the process being targeted
was common to many species and lines of Plasmodium
and was not specific to a particular tight-junction
receptor.
zolium ring would either inhibit or enhance this process.
The envisioned complex would appear to be unusually
stable since the results of the assays, involving varying
amounts of sulfated cyclodextrins, when plotted
(Fig. 2) produce a series of lines having similar slopes in-
stead of a series of lines having different slopes that
intersect at the origin, a result suggesting that sulfated
cyclodextrins are effectively removing a stoichiometric
amount of NBT from the reaction well. The observation
that NBT also interacts with the stilbene disulfonates
DIDS and SITS (Fig. 3) compounds that bind with a
high affinity and specificity to a defined site in the eryth-
rocyte anion transport protein,20 is in agreement with
previous reports25,26 that AE1 participates in merozoite
invasion and suggests that tetrazolium salts interact with
either MSP1 or MSP9. While Kariuki et al.26 favor the
hypothesis that the MSP1/MSP9 complex is involved
in tight-junction formation, our data support the
hypothesis that the DIDS site in AE1 is the receptor
used in the initial recognition/tethering interaction of
the merozoite. The ability of tetrazolium salts and sul-
fated cyclodextrins to reduce the viability of multiple
lines and species of Plasmodium is consistent with the
highly conserved nature of the DIDS site.27 Further-
more, the ability of similar concentrations of tetrazo-
lium violet to inhibit the entry of multiple P.
falciparum lines into human erythrocytes and two spe-
cies of murine Plasmodium into murine erythrocytes
(Fig. 5) is consistent with the targeting of a conserved li-
gand structure.
Our analysis of the results obtained with the commer-
cially available tetrazolium salts suggested that further
variation of the R1, R2, and/or R3 moieties might pro-
duce agents with even more desirable anti-Plasmodium
properties. Thus, fifty-four compounds (QT1–QT54)
were synthesized and evaluated in both P. falciparum
and CHO cell cultures (Table 1). A wide range of activ-
ities and specificities for P. falciparum cultures was
observed. Microscopic examination of the inhibited as-
say wells confirmed that the active agents produced
large numbers of extracellular merozoites, a feature
consistent with the hypothesis that inhibition of merozo-
ite entry is the primary mechanism of action of these
compounds (data not shown).
Complex anionic glycans have been shown previously to
inhibit P. falciparum cultures,4,23 a result which was
attributed to their binding to the surface of an erythro-
cyte and creating a surface charge that repelled merozo-
ites.11 The ability of sulfated cyclodextrins to inhibit
P. falciparum cultures might be explained by a similar
mechanism, however, the observation that NBT could
not be reduced to the corresponding formazan in the
presence of these inhibitors permitted a testing of the
hypothesis that sulfated cyclodextrins mimicked a fea-
ture present in a potential merozoite ligand. The interac-
tion of NBT with anions presumably results from the
electron-deficient ring of a tetrazolium salt being at-
tracted by the electron-rich sulfate groups of sulfated
cyclodextrins,24 and the R groups surrounding the tetra-
Variations in the structure of tetrazolium salts can have
several important effects in culture assays. For example,
reduction of the tetrazolium ring to the corresponding
formazan is dependent in part on the presence of elec-
tron-withdrawing groups in the surrounding rings, a fea-

1930
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
Table 1. Activities of tetrazolium salts in CHO and P. falciparum culturesa
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
N
N
N
N
Tetrazolium red
0.3 0.2
71 15
236.7
Cl
N
N
N
N
Tetrazolium violet
0.15 0.05
15
3
100.0
Cl
O2N
N
N
N
N
Iodonitrotetrazolium
chloride
81 16
49 12
0.6
Cl
I
I
N
N
b
c
Thiazoyl blue
tetrazolium
bromide (MTT)
—
N
S
N
Br
H3C
N
H3C
N
N
N
N
Neotetrazolium
chloride
0.35 0.08
11
3
31.4
N
N
N
N
Cl
Cl
N
N
N
N
Tetrazolium
blue chloride
3.8 0.3
>100
—
N
N
N
N
Cl
Cl
H3CO
OCH3

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1931
Table 1 (continued)
Compound
Structure
IC50 (lM) IC50 (lM) IC50 CHO/IC50
P. falciparum CHO cells P. falciparum
NO2
O2N
b
c
Nitrotetrazolium
blue chloride
—
N
N
N
N
N
N
N
N
Cl
Cl
H3CO
OCH3
O2N
NO2
b
Tetranitroblue
tetrazolium
chloride
1.3 0.4
—
N
N
N
N
N
N
N
N
Cl
Cl
OCH3
H3CO
NO2
O2N
N
N
N
N
OCH3
QT1
3.7 0.6
10.0 0.9
4.5 0.6
6
3
1.6
0.7
2.7
0.5
Cl
O2N
O2N
O2N
N
N
N
Br
QT2
QT3
QT4
7
1
N
Cl
N
N
N
12
1
N
Cl
N
N
N
N
O
9
3
4.4 0.8
Cl
O2N
(continued on next page)

1932
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
Table 1 (continued)
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
OCH3
O
N
N
N
O
CH3
QT5
QT6
QT7
QT8
QT9
7.3 0.9
19
4
2.6
40.8
18.1
6.9
N
Cl
OCH3
O2N
N
N
N
N
0.103 0.008
0.26 0.03
0.35 0.03
0.23 0.02
4.2 0.9
4.7 0.4
2.4 0.5
O
Cl
N
N
N
N
O
Cl
OCH3
N
N
N
N
Cl
N
N
7
1
30.4
N
N
Cl
QT10
N
N
2.9 0.2
9
1
3.1
N
N
Cl
N
N
N
QT11
0.43 0.04
19
4
44.2
N
Cl
H3CO

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1933
Table 1 (continued)
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
N
N
N
N
QT12
0.93 0.06
39
6
41.9
Cl
F
N
N
N
N
O
QT13
0.47 0.01
16
2
34.0
Cl
F
N
N
QT14
QT15
QT16
0.81 0.04
4.1 0.6
5.1
N
N
Cl
F
H3CO
N
N
N
N
1.7 0.2
19
2
11.2
Cl
F
N
N
N
N
0.83 0.09
32
3
38.6
Cl
F
N
N
N
QT17
0.39 0.09
44
4
112.8
N
Cl
F
N
N
O
QT18
0.14 0.02
8
1
57.1
N
N
Cl
F
(continued on next page)

1934
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
Table 1 (continued)
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
N
N
N
N
QT19
0.36 0.08
8
1
22.2
Cl
F
N
N
N
N
O
QT20
2.2 0.3
2.7 0.4
1.2
Cl
OCH3
F
N
N
N
N
QT21
QT22
QT23
2.2 0.2
36
3
16.4
10.2
18.8
F
Cl
N
N
N
N
0.42 0.03
4.3 0.5
O
F
Cl
N
N
N
N
0.26 0.03
4.9 0.5
F
Cl
N
N
N
N
QT24
3.2 0.5
6.5 0.7
2.0
O
F
Cl
OCH3
N
N
N
QT25
0.63 0.01
39
3
61.9
N
Cl
Cl

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1935
Table 1 (continued)
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
N
N
N
N
O
QT26
1.14 0.05
4.6 0.8
4.0
Cl
Cl
N
N
N
N
QT27
0.41 0.09
16
32
12
5
8
2
39.0
Cl
Cl
N
N
O
QT28
QT29
QT30
3.8 0.2
8.4
N
N
Cl
OCH3
Cl
H3CO
N
N
N
N
0.40 0.01
30.0
Cl
Cl
N
N
N
N
0.84 0.05
6.3 0.6
7.5
Cl
Cl
N
N
QT31
0.08 0.01
9
4
112.5
O
Cl
N
N
Cl
N
N
N
N
QT32
0.115 0.008
10
2
87.0
Cl
Cl
(continued on next page)

1936
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
Table 1 (continued)
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
N
N
N
N
QT33
0.69 0.07
8
1
11.6
O
Cl
Cl
OCH3
H3CO
N
N
QT34
0.80 0.08
6.5 0.9
8.1
N
N
Cl
H3CO
N
N
N
N
QT35
QT36
QT37
0.32 0.03
9
2
28.1
O
Cl
N
N
9.1 0.5
16.2 0.9
1.8
N
N
Cl
O2N
N
N
N
N
14
2
31
2
2.2
NO2
Cl
H3CO
N
N
QT38
0.29 0.01
16
4
55.2
O
Cl
N
N
Cl
O
N
N
QT39
0.200 0.009
37
4
185.0
N
N
Cl

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1937
Table 1 (continued)
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
O
QT40
0.23 0.01
0.96 0.09
4.2
N
N
N
N
O
Cl
O
N
QT41
QT42
QT43
QT44
0.41 0.02
0.261 0.008
0.10 0.02
0.33 0.03
38
4
92.7
N
N
N
Cl
Cl
O
N
1.7 0.3
6.5
N
N
N
Cl
O
N
14
1
140.0
21.2
93.5
N
N
Cl
N
Cl
N
N
N
7
1
O
Cl
N
Cl
N
N
N
QT45
0.46 0.02
43
5
N
Cl
Cl
N
N
Cl
Cl
N
QT46
0.5 0.1
36
5
72.0
N
Cl
(continued on next page)

1938
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
Table 1 (continued)
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
N
N
Cl
Cl
N
N
QT47
1.3 0.1
21
4
16.2
O
Cl
N
N
QT48
0.6 0.1
6.4 0.5
10.7
Br
N
N
Cl
N
N
N
N
QT49
0.4 0.1
8.1 0.6
20.3
O
Br
Cl
N
N
N
N
QT50
3.4 0.4
21
3
6.2
(CH2)5CH3
Br
N
N
QT51
6.2 0.5
25
1
4.0
N
N
Cl
N
N
N
N
QT52
4
1
0.40 0.03
0.1
O(CH2)10CH3
Cl
N
N
QT53
2.5 0.5
5.0 0.7
2.0
N
N
Cl
O(CH2)10CH3

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1939
Table 1 (continued)
Compound
Structure
IC50 (lM)
P. falciparum
IC50 (lM)
CHO cells
IC50 CHO/IC50
P. falciparum
CH3
CH3
N
N
N
N
QT54
102 19
377 27
3.7
Br
a Each IC50 value represents the mean of four determinations with standard error indicated. The ‘ratio of activities’ given in the fifth column
represents the IC50 value determined for CHO cells divided by the IC50 value determined for P. falciparum cultures. Values greater than unity
indicate that the compound has greater potency in P. falciparum cultures. The strain line used was ItG.
b Oxidizes erythrocytes.
c Not determined.
Figure 2. Effect of b-cyclodextrins on nitrotetrazolium blue chloride (NBT). The ability of b-cyclodextrins to inhibit the reduction of NBT was
determined. Varying concentrations of NBT (indicated on x-axis) and b-cyclodextrin were incubated together prior to the addition of glutathione.
After 10 min the absorbance at 650 nm of the individual wells was determined. The left panel indicates the results obtained with a b-cyclodextrin
having an average of 1 sulfate group per glucose residue; the right panel represents b-cyclodextrin having no sulfate groups. Symbols indicate final
b-cyclodextrin concentrations: e 0 lM; Ç 24 lM; h 48 lM; j 71 lM; , 95 lM; . 119 lM; s 142 lM; and d 167 lM.
ture which determines how well these compounds func-
tion as oxidants. The presence of nitro groups, as in
iodonitrotetrazolium chloride, nitrotetrazolium blue
chloride, tetranitroblue tetrazolium chloride, and QT1–
QT5, appears to make these compounds generally toxic
and therefore they showed little specificity for Plasmo-
dium. Conversely, the absence of nitro groups, as in
tetrazolium red, tetrazolium violet, neotetrazolium chlo-
ride, tetrazolium blue chloride, and the majority of the
QT compounds, appears to allow a selectivity of activity
against Plasmodium. The best example of this general
trend can be seen by comparing the potency of tetrazo-
lium red to that of QT36 and QT37. This finding is con-
sistent with our hypothesis that it is the tetrazolium
form of the compounds that are active because they
complex with components of the parasite that are essen-
tial for its viability. Formazans lack the electron defi-
ciency that we consider to be essential for activity and
therefore reduction of the tetrazolium rings may render
the compounds inactive.
fluoro, chloro, or bromo groups at ortho, meta, or para
positions, all display similar potency and reasonable
selectivity when compared to their parent compound,
tetrazolium red. Multivalency does not seem to be rele-
vant for activity as evidenced by a comparison of the po-
tency of monovalent tetrazolium red and divalent
neotetrazolium chloride.
The effect of changing the R1 components of the tetrazo-
lium compounds was examined by different approaches.
Tetrazolium red had excellent anti-Plasmodium activity,
however, increasing the size of this group, as in QT6,
QT8, and QT9, does not greatly affect potency. How-
ever, when aliphatic or alicyclic groups are added at
the R1 position, as in QT50–54, both potency and selec-
tivity are compromised. The incorporation of halogen
atoms (F or Cl) to compounds having larger R1 groups,
for example, a biphenyl group, is also permissible since
modifying QT9 to produce QT14, QT19, QT23,
QT27, and QT32 gives compounds that are still both ac-
tive and selective. Similarly, incorporation of halogen
into QT6 affords compounds QT13, QT18, QT22,
QT26, QT31, QT44, QT47, and QT49 which have desir-
It is noteworthy that compounds QT12, QT17, QT21,
QT25, QT30, QT45, QT46, and QT48 which contain

1940
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
Figure 3. Effect of DIDS and SITS on nitrotetrazolium blue chloride
(NBT). NBT was added to microwells containing a serial dilution
series of either DIDS (d) or SITS (s) and allowed to incubate for
10 min. To determine the amount of unbound NBT present, glutathi-
one in Tris buffer was added to every well and after 10 min the
absorbance at 650 nm was determined. Data points are the mean of
four determinations with the standard error indicated by error bars.
The IC50 values of individual compounds were determined using a
non-linear regression analysis of the dose–response curve and IC50
values of 31 2 lM and 2.7 0.1 lM were determined for DIDS and
SITS, respectively.
Figure 5. Activities of tetrazolium violet in human and rodent malaria
cultures. Parasite viability was assayed using the SYBR-Green
method.33 Trypsin and neuraminidase treatments of human erythro-
cytes were as described by Lobo et al.9 and the presence of a specific
invasion phenotype was confirmed by attempting to culture parasites
in erythrocytes that had been subjected to the opposite treatment. Bars
represent the mean of four determinations with the standard error
indicated by error bars. The IC50 values of individual compounds were
determined using a non-linear regression analysis of the dose–response
curve. The first three bars represent the results seen in three different
parasite lines; the next three represent results observed for the
laboratory line that had not been selected (3D7); that had been
selected to grow in neuraminidase treated erythrocytes (3D7 N+); or
the line in trypsin treated erythrocytes (3D7 T+). The IC50 values for
P. chabaudi chabaudi (Pcc), P. berghei (Pb), and P. falciparum ItG (Pf)
are indicated.
able potency and selectivity. The addition of methoxy
groups to the R2 and R3groups is also well tolerated
as regards useful effects, for example, QT27 versus
QT29, tetrazolium red versus QT34, and QT9 versus
QT11; however, the addition of a methoxy group to
an R1 group can be disruptive, for example, QT18 ver-
sus QT20, QT22 versus QT24, and QT31 versus QT33.
4. Conclusions
In summary, it would appear from the present series of
compounds that most of the compounds that are active
possess an aromatic group at the R1 position, and the size
of the group is not critical. Nitro modifications of active
compounds are poorly tolerated, however, the presence
of halogen atoms on aromatic R2 and R3 groups has little
effect on potency. Methoxy groups are tolerated on R2
and R3 components, however, they are disruptive when
on the R1 component. These observations suggest that
the R1 component is interacting with a specific hydro-
phobic environment and the R2 and R3 components are
less constrained. The activity of these compounds in sev-
eral human and mouse Plasmodium cultures suggests that
the compounds interact with a component of the parasite
that is both essential and conserved.
5. Experimental
5.1. General
Figure 4. Effect of tetrazolium red and tetrazolium violet on
P. falciparum cultures and CHO cells. CHO cells (circles) and
P. falciparum cultures (inverted triangles) were grown in the presence
of serial dilutions of tetrazolium red (white) or tetrazolium violet
(black) and the viability of the cultures was then determined. Data
points are the mean of four determinations with the standard error
indicated by error bars. The IC50 values of individual compounds were
determined using a non-linear regression analysis of the dose–response
curve and IC50 values of 71 15 lM and 338 150 nM were obtained
for tetrazolium red with CHO and P. falciparum cultures, respectively,
and 15 3 lM and 148 45 nM were obtained for tetrazolium violet
with CHO and P. falciparum cultures, respectively.
Flash column chromatography was performed on Silicy-
˚
cle silica gel (230–400 mesh, 60A). Analytical thin-layer
chromatography was performed on glass-backed pre-
coated silica gel 60 F254 plates (Silicycle), and the com-
pounds were visualized either by UV illumination
(254 nm), or by heating after spraying with phosphomo-
lybdic acid in ethanol. Melting points were taken on a
1
Mel-Temp II apparatus and are uncorrected. H and

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1941
13C NMR spectra were recorded on Bruker Avance 300,
400, and 500 spectrometers in CDCl3, (CD3)2CO,
CD3OD, or (CD3)2SO. The chemical shifts are reported
in d (ppm) relative to tetramethylsilane.28 High-resolu-
tion ES mass spectra were recorded on a Fisons VG
Quattro triple quadrupole mass spectrometer; peaks re-
ported as m/z. Elemental analyses were performed by
MHW Laboratories (Phoenix, AZ).
was cooled to 0 ꢁC using an ice–water bath. Freshly pre-
pared tert-butyl hypochlorite18 (0.15 mmol) was then
added dropwise, the mixture stirred at 0 ꢁC for 1 h, and
then concentrated. Ether was then added and the result-
ing precipitate of the tetrazolium chloride product was
removed by filtration and washed thoroughly with ether.
As previously noted30 the tetrazolium salts are sensitive
to light. The freshly prepared samples change color
within a few hours; NMR analysis indicated the forma-
tion of the corresponding formazans in addition to other
oxidation products. Thus, the tetrazolium salts were iso-
lated and characterized with minimum exposure to light,
and then stored in the dark at ꢀ20 ꢁC until further use.
5.2. Materials
All chemical reagents were obtained from Sigma–Aldrich
and were used without further purification. Tetrazolium
red, tetrazolium violet, iodonitrotetrazolium chloride,
thiazoyl blue tetrazolium bromide, neotetrazolium chlo-
ride, tetrazolium blue chloride, nitrotetrazolium blue
chloride (NBT), and tetranitroblue tetrazolium chloride
were purchased from Sigma–Aldrich. tert-Butyl hypo-
chlorite was freshly prepared as previously described.29
5.6. Characterization of the synthesized tetrazolium salts
5.6.1. 5-(4-Methoxyphenyl)-3-(4-nitrophenyl)-2-phenyl-
3H-tetrazol-2-ium chloride (QT-1). Prepared using 4-
nitrophenylhydrazine, 4-methoxybenzaldehyde, and
aniline as starting materials to give the product; 1H
NMR (300 MHz, DMSO-d6): d 3.93 (s, 3H), 7.32 (d,
J = 8.8 Hz, 2H), 7.74 (t, J = 7.9 Hz, 2H), 7.82–7.95 (m,
3H), 8.15 (d, J = 9.1 Hz, 2H), 8.30 (d, J = 9.0 Hz, 2H),
8.55 (d, J = 9.1 Hz, 2H); HRMS (ES) [MꢀH]ꢀ calcd
for C20H15N5O3Cl: 408.0857. Found: 408.0848; HRMS
(ES) [MꢀCl]+ calcd for C20H16N5O3: 374.1247. Found:
374.1250.
5.3. General procedure for the formation of hydrazones by
way of the condensation of aldehydes and hydrazines, as
outlined in Scheme 1
The hydrazine derivative (10 mmol, usually stabilized by
the addition of 10% by weight H2O) in methanol
(60 mL) was heated at reflux temperature until all of
the solids were dissolved. A solution of the aldehyde
(10 mmol) in methanol (25 mL) was then added in one
portion, followed by a few drops of concentrated HCl.
The mixture was heated at reflux temperature with stir-
ring for 1 h and then concentrated. The crude product
was extracted into ethyl acetate, the organic extract
washed three times with brine, dried over CaCl2, and
then concentrated. The hydrazone product was purified
by recrystallization from either methanol or ethyl ace-
tate and used in the next step.
5.6.2. 5-(4-Bromophenyl)-3-(4-nitrophenyl)-2-phenyl-3H-
tetrazol-2-ium chloride (QT-2). Prepared using 4-nitro-
phenylhydrazine, 4-bromobenzaldehyde, and aniline as
starting materials to give the product; 1H NMR
(300 MHz, DMSO-d6): d 7.16–8.00 (m, 5H), 8.02 (d,
J = 8.5 Hz, 2H), 8.24 (d, J = 5.9 Hz, 2H), 8.30 (d,
J = 8.5 Hz, 2H), 8.56 (d, J = 8.9 Hz, 2H); HRMS (ES)
[MꢀH]ꢀ calcd for C19H12N5O2ClBr: 455.9857. Found:
455.9848;
HRMS
(ES)
[MꢀCl]+
calcd
for
5.4. General procedure for the formation of formazans by
way of the azo coupling of freshly prepared diazonium
salts with hydrazones, as outlined in Scheme 1
C19H13N5O2Br: 422.0247. Found: 422.0243.
5.6.3. 3-(4-Nitrophenyl)-2,5-diphenyl-3H-tetrazol-2-ium
chloride (QT-3). Prepared using 4-nitrophenylhydrazine,
benzaldehyde, and aniline as starting materials to give
To a solution of the aromatic amine (11 mmol) in water
(25 mL) was added concentrated HCl (7.5 mL). The mix-
ture was cooled to 0 ꢁC using an ice–water bath. After
20 min, a solution of sodium nitrite (0.7 g, 10 mmol) in
water (3 mL) was added, and the mixture stirred at 0 ꢁC
for 0.5 h. The freshly prepared diazonium salt solution
was then added dropwise to a stirring solution of the
hydrazone (10 mmol, prepared in Section 5.3) in pyridine
(25 mL) at 0 ꢁC. The mixture was kept at 0 ꢁC for an addi-
tional 2 h, then extracted with ethyl acetate (150 mL). The
organic extract was washed with brine (3 · 30 mL), dried
over CaCl2, and concentrated. Flash column chromatog-
raphy on silica gel (1:3 v/v EtOAc–hexanes) of the residue
gave the crude formazan which was used in the next step.
1
the product; H NMR (300 MHz, DMSO-d6): d 7.42–
7.48 (m, 2H), 7.72–8.02 (m, 6H), 8.22 (d, J = 8.9 Hz,
2H), 8.36 (d, J = 6.8 Hz, 2H), 8.56 (d, J = 9.0 Hz, 2H);
HRMS (ES) [MꢀH]ꢀ calcd for C19H13N5O2Cl:
378.0752. Found: 378.0735; HRMS (ES) [MꢀCl]+ calcd
for C19H14N5O2: 344.1142. Found: 344.1135.
5.6.4. 5-(4-Benzyloxyphenyl)-3-(4-nitrophenyl)-2-phenyl-
3H-tetrazol-2-ium chloride (QT-4). Prepared using 4-
nitrophenylhydrazine, 4-benzyloxybenzaldehyde, and
aniline as starting materials to give the product; 1H
NMR (300 MHz, DMSO-d6): d 5.29 (s, 2H), 7.36–7.53
(m, 8H), 7.74–7.97 (m, 4H), 8.16 (d, J = 9.0 Hz, 2H),
8.33 (d, J = 8.9 Hz, 2H), 8.56 (d, J = 9.1 Hz, 2H);
HRMS (ES) [MꢀH]ꢀ calcd for C26H19N5O3Cl:
484.1190. Found: 484.1170; HRMS (ES) [MꢀCl]+ calcd
for C26H20N5O3: 450.1560. Found: 450.1558.
5.5. General procedure for the formation of tetrazolium
salts by way of the oxidation of formazans using tert-butyl
hypochlorite, as outlined in Scheme 1
The crude formazan (0.1 mmol, prepared in Section 5.4)
was dissolved in chloroform (30 mL) and the solution
5.6.5. 5-(4-Acetoxy-3,5-dimethoxyphenyl)-3-(4-nitrophe-
nyl)-2-phenyl-3H-tetrazol-2-ium chloride (QT-5). Pre-

1942
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
pared using 4-nitrophenylhydrazine, 4-acetoxy-3,5-
dimethoxybenzaldehyde, and aniline as starting materi-
als to give the product; H NMR (300 MHz, DMSO-
d6): d 2.30 (s, 3H), 3.90 (s, 6H), 7.40–8.00 (m, 7H),
8.20 (d, J = 8.9 Hz, 2H), 8.56 (d, J = 9.0 Hz, 2H);
HRMS (ES) [MꢀH]+ calcd for C23H19N5O6Cl:
496.1018. Found: 496.1027; HRMS (ES) [MꢀCOCH3]+
calcd for C21H17N5O5Cl: 454.0912, found: 454.0931.
8.09 (d, J = 8.2 Hz, 2H), 8.40 (d, J = 6.8 Hz, 2H);
HRMS (ES) [MꢀH]+ calcd for C26H20N4OCl:
439.1320. Found: 439.1334; HRMS (ES) [MꢀCl]+ calcd
for C26H21N4O: 405.1709. Found: 405.1703.
1
5.6.12. 3-(4-Fluorophenyl)-2,5-diphenyl-3H-tetrazol-2-
ium chloride (QT-12). Prepared using phenylhydrazine,
benzaldehyde, and 4-fluoroaniline as starting materials
to give the product; H NMR (300 MHz, DMSO-d6):
1
5.6.6. 5-(4-Benzyloxyphenyl)-2,3-diphenyl-3H-tetrazol-2-ium
chloride (QT-6). Prepared using phenylhydrazine, 4-benzyl-
oxybenzaldehyde, and aniline as starting materials to give
d 7.53–8.07 (m, 12H), 8.33 (d, J = 5.9 Hz, 2H); HRMS
(ES) [MꢀH]+ calcd for C19H13N4FCl: 351.0807. Found:
351.0813; HRMS (ES) [MꢀCl]+ calcd for C19H14N4F:
317.1197. Found: 317.1189.
1
the product; H NMR (300 MHz, DMSO-d6): d 5.28 (s,
2H), 7.38–7.53 (m, 9H), 7.69–7.94 (m, 8H), 8.29 (d,
J = 8.7 Hz, 2H); HRMS (ES) [MꢀH]+ calcd for
C26H20N4OCl: 439.1320. Found: 439.1336; HRMS (ES)
[MꢀCl]+ calcd for C26H21N4O: 405.1709. Found: 405.1713.
5.6.13. 5-(4-Benzyloxyphenyl)-3-(4-fluorophenyl)-2-phe-
nyl-3H-tetrazol-2-ium chloride (QT-13). Prepared using
phenylhydrazine, 4-benzyloxybenzaldehyde, and 4-fluo-
1
roaniline as starting materials to give the product; H
5.6.7. 5-(4-Benzyloxy-3-methoxyphenyl)-2,3-diphenyl-
3H-tetrazol-2-ium chloride (QT-7). Prepared using
phenylhydrazine, 4-benzyloxy-3-methoxybenzaldehyde,
and aniline as starting materials to give the product;
1H NMR (300 MHz, DMSO-d6): d 3.91 (s, 3H), 5.30
(s, 2H), 7.39–7.58 (m, 8H), 7.70–7.94 (m, 10H); HRMS
(ES) [MꢀH]+ calcd for C27H22N4O2Cl: 469.1425.
Found: 469.1426.
NMR (300 MHz, DMSO-d6): d 5.29 (s, 2H), 7.38–7.53
(m, 8H), 7.58–7.66 (m, 2H), 7.70–7.75 (m, 1H), 7.80–
7.98 (m, 5H), 8.29 (d, J = 8.4 Hz, 2H); HRMS (ES)
[MꢀH]+ calcd for C26H19N4OFCl: 457.1225. Found:
457.1223; HRMS (ES) [MꢀCl]+ calcd for C26H20N4OF:
423.1615. Found: 423.1631.
5.6.14. 5-Biphenyl-4-yl-3-(4-fluorophenyl)-2-phenyl-3H-tet-
razol-2-ium chloride (QT-14). Prepared using phenylhydr-
azine, biphenyl-4-carboxaldehyde, and 4-fluoroaniline as
starting materials to give the product as a beige solid;
1H NMR (300 MHz, DMSO-d6): d 7.46–7.68 (m, 5H),
7.72–7.78 (m, 2H), 7.82–7.98 (m, 5H), 8.00–8.06 (m,
2H), 8.11 (d, J = 7.4 Hz, 2H), 8.43 (d, J = 8.2 Hz, 2H);
HRMS (ES) [MꢀH]+ calcd for C25H17N4FCl: 427.1120.
Found: 427.1134; HRMS (ES) [MꢀCl]+ calcd for
C25H18N4F: 393.1510. Found: 393.1520.
5.6.8. 5-Naphthalen-1-yl-2,3-diphenyl-3H-tetrazol-2-ium
chloride (QT-8). Prepared using phenylhydrazine, 1-
naphthaldehyde, and aniline as starting materials to give
1
the product; H NMR (300 MHz, DMSO-d6): d 7.50–
8.00 (m, 11H), 8.20 (d, J = 8.9 Hz, 2H), 8.41–8.46 (m,
2H), 8.75–8.84 (m, 2H); HRMS (ES) [MꢀH]+ calcd
for C23H16N4Cl: 383.1058. Found: 383.1061; HRMS
(ES) [MꢀCl]+ calcd for C23H17N4: 349.1447. Found:
349.1455.
5.6.15. 5-Biphenyl-4-yl-3-(4-fluorophenyl)-2-(4-methoxy-
phenyl)-3H-tetrazol-2-ium chloride (QT-15). Prepared
using 4-methoxyphenylhydrazine, biphenyl-4-carboxal-
dehyde, and 4-fluoroaniline as starting materials to give
5.6.9. 5-Biphenyl-4-yl-2,3-diphenyl-3H-tetrazol-2-ium
chloride (QT-9). Prepared using phenylhydrazine,
biphenyl-4-carboxaldehyde, and aniline as starting
materials to give the product as a beige solid; 1H
NMR (300 MHz, DMSO-d6): d 7.46–7.59 (m, 4H),
7.71–8.00 (m, 11H), 8.10 (d, J = 8.1 Hz, 2H), 8.41–8.45
(m, 2H); HRMS (ES) [MꢀH]+ calcd for C25H18N4Cl:
409.1214. Found: 409.1222; HRMS (ES) [MꢀCl]+ calcd
for C25H19N4: 375.1604. Found: 375.1606.
1
the product; H NMR (300 MHz, DMSO-d6): d 3.87 (s,
3H), 7.26 (d, J = 9.1 Hz, 2H), 7.49–7.67 (m, 5H), 7.81 (d,
J = 9.1 Hz, 2H), 7.85 (d, J = 6.3 Hz, 2H), 7.98–8.02 (m,
2H), 8.10 (d, J = 8.4 Hz, 2H), 8.41 (d, J = 8.4 Hz, 2H);
HRMS (ES) [MꢀCl]+ calcd for C26H20N4OF:
423.1615. Found: 423.1617.
5.6.10. 2-Biphenyl-4-yl-5-naphthalen-1-yl-3-phenyl-3H-
tetrazol-2-ium chloride (QT-10). Prepared using phen-
ylhydrazine, 1-naphthaldehyde, and 4-aminobiphenyl
as starting materials to give the product as a white solid;
1H NMR (300 MHz, DMSO-d6): d 7.40–7.60 (m, 4H),
7.74–8.15 (m, 14H), 8.20–8.85 (m, 3H); HRMS (ES)
[MꢀH]+ calcd for C29H20N4Cl: 459.1371. Found:
459.1376; HRMS (ES) [MꢀCl]+ calcd for C29H21N4:
425.1760. Found: 425.1761.
5.6.16. 3-(4-Fluorophenyl)-5-naphthalen-1-yl-2-phenyl-3H-
tetrazol-2-ium chloride (QT-16). Prepared using phen-
ylhydrazine, 1-naphthaldehyde, and 4-fluoroaniline as
starting materials to give the product; 1H NMR
(300 MHz, DMSO-d6): d 7.64–7.70 (m, 2H), 7.77–7.90
(m, 6H), 7.97–8.12 (m, 4H), 8.22 (d, J = 8.0 Hz, 1H),
8.26–8.52 (m, 2H), 8.80 (d, J = 7.9 Hz, 1H); HRMS
(ES) [MꢀH]+ calcd for C23H15N4FCl: 401.0963. Found:
401.0972; HRMS (ES) [MꢀCl]+ calcd for C23H16N4F:
367.1353. Found: 367.1346.
5.6.11. 5-Biphenyl-4-yl-3-(4-methoxyphenyl)-2-phenyl-3H-
tetrazol-2-ium chloride (QT-11). Prepared using 4-
methoxyphenylhydrazine, biphenyl-4-carboxaldehyde,
and aniline as starting materials to give the product;
1H NMR (300 MHz, DMSO-d6): d 3.87 (s, 3H), 7.20–
7.29 (m, 2H), 7.48–7.59 (m, 4H), 7.74–8.00 (m, 8H),
5.6.17. 3-(2-Fluorophenyl)-2,5-diphenyl-3H-tetrazol-2-ium
chloride (QT-17). Prepared using phenylhydrazine,
benzaldehyde, and 2-fluoroaniline as starting materials
to give the product; mp 110–144 ꢁC (dec); 1H NMR
(300 MHz, DMSO-d6): d 7.58–7.63 (m, 1H), 7.70–8.00

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1943
(m, 10H), 8.05–8.14 (m, 1H), 8.34–8.35 (m, 2H); HRMS
(ES) [MꢀH]+ calcd for C19H13N4FCl: 351.0807. Found:
351.0814; HRMS (ES) [MꢀCl]+ calcd for C19H14N4F:
317.1197. Found: 317.1190.
427.1120. Found: 427.1111; HRMS (ES) [MꢀCl]+ calcd
for C25H18N4F: 393.1510. Found: 393.1523.
5.6.24.
5-(4-Benzyloxy-3-methoxyphenyl)-3-(3-fluoroph-
enyl)-2-phenyl-3H-tetrazol-2-ium chloride (QT-24). Pre-
pared using phenylhydrazine, 4-benzyloxy-3-metho-
xybenzaldehyde, and 3-fluoroaniline as starting materials
5.6.18. 5-(4-Benzyloxyphenyl)-3-(2-fluorophenyl)-2-phenyl-
3H-tetrazol-2-ium chloride (QT-18). Prepared using phen-
ylhydrazine, 4-benzyloxybenzaldehyde, and 2-fluoroaniline
as starting materials to give the product; 1H NMR
(300 MHz, DMSO-d6): d 5.30 (s, 2H), 7.36–7.48 (m, 6H),
7.52 (d, J = 7.6 Hz, 2H), 7.56–7.62 (m, 1 H), 7.69–7.77
(m, 2H), 7.82–8.00 (m, 4H), 8.06–8.12 (m, 1H), 8.26–8.30
(m, 2H); HRMS (ES) [MꢀH]+ calcd for C26H19N4OFCl:
457.1225. Found: 457.1225; HRMS (ES) [MꢀCl]+ calcd
for C26H20N4OF: 423.1615. Found: 423.1606.
1
to give the product; H NMR (300 MHz, DMSO-d6): d
3.92 (s, 3H), 5.31 (s, 2H), 7.42–7.58 (m, 7H), 7.69–8.07
(m, 10H); HRMS (ES) [MꢀH]+ calcd for C27H21N4O2FCl:
487.1331. Found: 487.1309.
5.6.25. 3-(4-Chlorophenyl)-2,5-diphenyl-3H-tetrazol-2-
ium chloride (QT-25). Prepared using phenylhydrazine,
benzaldehyde, and 4-chloroaniline as starting materials
to give the product as a beige solid; 1H NMR
(300 MHz, DMSO-d6): d 7.75–7.94 (m, 12H), 8.33–8.37
(m, 2H); HRMS (ES) [MꢀH]+ calcd for C19H13N4Cl2:
367.0511. Found: 367.0521; HRMS (ES) [MꢀCl]+ calcd
for C19H14N4Cl: 333.0901. Found: 333.0889.
5.6.19. 5-Biphenyl-4-yl-3-(2-fluorophenyl)-2-phenyl-3H-
tetrazol-2-ium chloride (QT-19). Prepared using phen-
ylhydrazine, biphenyl-4-carboxaldehyde, and 2-fluoro-
aniline as starting materials to give the product; 1H
NMR (300 MHz, DMSO-d6): d 7.43–7.64 (m, 6H),
7.73–8.05 (m, 9H), 8.10 (d, J = 8.2 Hz, 2H), 8.35–8.43
(m, 1H); HRMS (ES) [MꢀH]+ calcd for C25H17N4FCl:
427.1120. Found: 427.1110; HRMS (ES) [MꢀCl]+ calcd
for C25H18N4F: 393.1510. Found: 393.1518.
5.6.26. 5-(4-Benzyloxyphenyl)-3-(4-chlorophenyl)-2-phe-
nyl-3H-tetrazol-2-ium chloride (QT-26). Prepared using
phenylhydrazine, 4-benzyloxybenzaldehyde, and 4-chlo-
1
roaniline as starting materials to give the product; H
NMR (300 MHz, DMSO-d6): d 5.29 (s, 2H), 7.38–7.53
(m, 8H), 7.74–7.76 (m, 2H), 7.81–7.91 (m, 6H), 8.30
(d, J = 8.8 Hz, 2H); HRMS (ES) [MꢀH]+ calcd for
C26H19N4OCl2: 473.0930. Found: 473.0920; HRMS
(ES) [MꢀCl]+ calcd for C26H20N4OCl: 439.1320.
Found: 439.1341.
5.6.20. 5-(4-Benzyloxy-3-methoxyphenyl)-3-(2-fluoroph-
enyl)-2-phenyl-3H-tetrazol-2-ium chloride (QT-20).
Prepared using phenylhydrazine, 4-benzyloxy-3-
methoxybenzaldehyde, and 2-fluoroaniline as starting
materials to give the product; 1H NMR (300 MHz,
DMSO-d6): d 3.92 (s, 3H), 5.31 (s, 2H), 7.39–7.52 (m,
7H), 7.54–7.62 (m, 2H), 7.68–7.80 (m, 3H), 7.86–8.05
(m, 5H); HRMS (ES) [MꢀH]+ calcd for C27H21
N4O2FCl: 487.1331. Found: 487.1333.
5.6.27. 5-Biphenyl-4-yl-3-(4-chlorophenyl)-2-phenyl-3H-
tetrazol-2-ium chloride (QT-27). Prepared using phen-
ylhydrazine, biphenyl-4-carboxaldehyde, and 4-chloro-
aniline as starting materials to give the product as a
1
5.6.21. 3-(3-Fluorophenyl)-2,5-diphenyl-3H-tetrazol-2-ium
chloride (QT-21). Prepared using phenylhydrazine,
benzaldehyde, and 3-fluoroaniline as starting materials
biege solid; H NMR (300 MHz, DMSO-d6): d 7.48–
7.51 (m, 1H), 7.57 (t, J = 7.0 Hz, 2H), 7.76–7.78 (m,
1H), 7.82–8.00 (m, 10H), 8.10 (d, J = 8.3 Hz, 2H),
8.40–8.44 (m, 2H); HRMS (ES) [MꢀH]+ calcd for
C25H17N4Cl2: 443.0824. Found: 443.0834; HRMS (ES)
[MꢀCl]+ calcd for C25H18N4Cl: 409.1214. Found:
409.1213.
1
to give the product; H NMR (300 MHz, DMSO-d6):
d 7.73–7.89 (m, 9H), 7.99–8.07 (m, 3H), 8.31–8.36 (m,
2H); HRMS (ES) [MꢀH]+ calcd for C19H13N4FCl:
351.0807. Found: 351.0812; HRMS (ES) [MꢀCl]+ calcd
for C19H14N4F: 317.1197. Found: 317.1189.
5.6.28. 5-(4-Benzyloxy-3-methoxyphenyl)-3-(4-chloro-
phenyl)-2-phenyl-3H-tetrazol-2-ium chloride (QT-28).
Prepared using phenylhydrazine, 4-benzyloxy-3-me-
thoxybenzaldehyde, and 4-chloroaniline as starting
materials to give the product; 1H NMR (300 MHz,
DMSO-d6): d 3.91 (s, 3H), 5.30 (s, 2H), 7.32–7.57 (m,
7H), 7.69–8.00 (m, 10H); HRMS (ES) [MꢀH]+ calcd
for C27H21N4O2Cl2: 503.1036. Found: 503.1054.
5.6.22. 5-(4-Benzyloxyphenyl)-3-(3-fluorophenyl)-2-phe-
nyl-3H-tetrazol-2-ium chloride (QT-22). Prepared using
phenylhydrazine, 4-benzyloxybenzaldehyde, and 3-fluo-
1
roaniline as starting materials to give the product; H
NMR (300 MHz, DMSO-d6): d 5.28 (s, 2H), 7.37–7.65
(m, 8H), 7.72–7.94 (m, 5H), 8.00–8.19 (m, 3H), 8.27–
8.37 (m, 2H); HRMS (ES) [MꢀH]+ calcd for
C26H19N4OFCl: 457.1225. Found: 457.1225; HRMS
(ES) [MꢀCl]+ calcd for C26H20N4OF: 423.1615. Found:
423.1611.
5.6.29. 5-Biphenyl-4-yl-3-(4-chlorophenyl)-2-(4-methoxy-
phenyl)-3H-tetrazol-2-ium chloride (QT-29). Prepared
using 4-methoxyphenylhydrazine, biphenyl-4-carboxal-
dehyde, and 4-chloroaniline as starting materials to give
5.6.23. 5-Biphenyl-4-yl-3-(3-fluorophenyl)-2-phenyl-3H-
tetrazol-2-ium chloride (QT-23). Prepared using phen-
ylhydrazine, biphenyl-4-carboxaldehyde, and 3-fluoro-
aniline as starting materials to give the product as a
1
the product; H NMR (300 MHz, DMSO-d6): d 3.87 (s,
3H), 7.26 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 9.3 Hz, 2H),
7.56 (t, J = 7.8 Hz, 2H), 7.84–7.88 (m, 4H), 7.97 (d,
J = 8.9 Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H), 8.41 (d,
J = 8.4 Hz, 2H), 8.67 (d, J = 9.4 Hz, 1H); 13C NMR
(75 MHz, DMSO-d6): d 56.1, 115.5, 117.2, 121.7,
1
beige solid; H NMR (300 MHz, DMSO-d6): d 7.44–
7.81 (m, 11H), 7.93–8.01 (m, 2H), 8.34–8.55 (m, 5H);
HRMS (ES) [MꢀH]+ calcd for C25H17N4FCl:

1944
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
125.1, 127.0, 127.9, 128.2, 128.4, 129.2, 130.6, 131.8,
136.2, 138.3, 138.9, 144.8, 163.0, 163.7; HRMS (ES)
[MꢀH]+ calcd for C26H19N4OCl2: 473.0930. Found:
473.0943; HRMS (ES) [MꢀCl]+ calcd for C26H20
N4OCl: 439.1320. Found: 439.1337.
phenylhydrazine, 4-benzyloxybenzaldehyde, and 4-methox-
yaniline as starting materials to give the product; 1H NMR
(300 MHz, DMSO-d6): d 3.86 (s, 3H), 5.28 (s, 2H), 7.23–7.25
(m, 2H), 7.37–7.64 (m, 8H), 7.72–7.91 (m, 6H), 8.26–8.38 (m,
2H); HRMS (ES) [MꢀH]+ calcd for C27H22N4O2Cl:
469.1425. Found: 469.1418; HRMS (ES) [MꢀCl]+ calcd
for C27H23N4O2: 435.1815. Found: 435.1817.
5.6.30. 3-(3-Chlorophenyl)-2,5-diphenyl-3H-tetrazol-2-
ium chloride (QT-30). Prepared using phenylhydrazine,
benzaldehyde, and 3-chloroaniline as starting materials
5.6.36. 3-(4-Nitrophenyl)-2,5-diphenyl-3H-tetrazol-2-ium
chloride (QT-36). Prepared using phenylhydrazine,
benzaldehyde, and 4-nitroaniline as starting materials
1
to give the product; H NMR (300 MHz, DMSO-d6):
d 7.73–8.12 (m, 12H), 8.34–8.36 (m, 2H); HRMS (ES)
[MꢀH]+ calcd for C19H13N4Cl2: 367.0511. Found:
367.0497; HRMS (ES) [MꢀCl]+ calcd for C19H14N4Cl:
333.0901. Found: 333.0893.
1
to give the product; H NMR (300 MHz, DMSO-d6):
d 7.75–8.01 (m, 7H), 8.10–8.30 (m, 2H), 8.35–8.41 (m,
2H), 8.50–8.61 (m, 2H), 8.69–8.82 (m, 1H); HRMS
(ES) [MꢀH]+ calcd for C19H13N5O2Cl: 378.0752.
Found: 378.0753; HRMS (ES) [MꢀCl]+ calcd for
C19H14N5O2: 344.1142. Found: 344.1155.
5.6.31. 5-(4-Benzyloxyphenyl)-3-(3-chlorophenyl)-2-phe-
nyl-3H-tetrazol-2-ium chloride (QT-31). Prepared using
phenylhydrazine, 4-benzyloxybenzaldehyde, and 3-chlo-
roaniline as starting materials to give the product; mp
169–171 ꢁC; 1H NMR (300 MHz, DMSO-d6): d 5.28
(s, 2H), 7.38–7.45 (m, 5H), 7.51 (d, J = 7.1 Hz, 2H),
7.72–8.08 (m, 9H), 8.32 (d, J = 8.9 Hz, 2H); 13C NMR
(100 MHz, CD3OD): d 70.0, 115.1, 116.0, 124.5, 125.8,
125.9, 127.3, 127.9, 128.3, 129.3, 130.4, 130.7, 131.7,
133.2, 134.0, 134.2, 135.8, 136.4, 152.4, 163.2; HRMS
(ES) [MꢀH]+ calcd for C26H19N4OCl2: 473.0930.
Found: 473.0948; HRMS (ES) [MꢀCl]+ calcd for
C26H20N4OCl: 439.1320. Found: 439.1313.
5.6.37. 5-(4-Nitrophenyl)-2,3-diphenyl-3H-tetrazol-2-ium
chloride (QT-37). Prepared using phenylhydrazine, 4-
nitrobenzaldehyde, and aniline as starting materials to
give the product; 1H NMR (300 MHz, DMSO-d6): d
7.74–7.77 (m, 3H), 7.82–7.97 (m, 7H), 8.58–8.63 (m,
4H); HRMS (ES) [MꢀH]+ calcd for C19H13N5O2Cl:
378.0752. Found: 378.0765; HRMS (ES) [MꢀCl]+ calcd
for C19H14N5O2: 344.1142. Found: 344.1140.
5.6.38. 5-(4-Benzyloxyphenyl)-3-(3-chlorophenyl)-2-(4-
methoxyphenyl)-3H-tetrazol-2-ium chloride (QT-38).
Prepared using 4-methoxyphenylhydrazine, 4-benzyl-
oxybenzaldehyde, and 3-chloroaniline as starting mate-
5.6.32. 5-Biphenyl-4-yl-3-(3-chlorophenyl)-2-phenyl-3H-
tetrazol-2-ium chloride (QT-32). Prepared using phen-
ylhydrazine, biphenyl-4-carboxaldehyde, and 3-chloro-
aniline as starting materials to give the product; 1H
NMR (300 MHz, DMSO-d6): d 7.46–7.59 (m, 4H),
7.76–8.19 (m, 12H), 8.34–8.44 (m, 2H); HRMS (ES)
[MꢀH]+ calcd for C25H17N4Cl2: 443.0824. Found:
443.0832; HRMS (ES) [MꢀCl]+ calcd for C25H18N4Cl:
409.1214. Found: 409.1211.
1
rials to give the product; H NMR (300 MHz, DMSO-
d6): d 3.86 (s, 3H), 5.28 (s, 2H), 7.20–7.29 (m, 2H),
7.38–7.55 (m, 7H), 7.74–8.07 (m, 5H), 8.25–8.30 (m,
2H), 8.60–8.65 (m, 1H); HRMS (ES) [MꢀH]+ calcd
for C27H21N4O2Cl2: 503.1036. Found: 503.1049; HRMS
(ES) [MꢀCl]+ calcd for C27H22N4O2Cl: 469.1425.
Found: 469.1438.
5.6.33. 5-(4-Benzyloxy-3-methoxyphenyl)-3-(3-chloroph-
enyl)-2-phenyl-3H-tetrazol-2-ium
5.6.39. 2-(4-Benzyloxyphenyl)-3,5-diphenyl-3H-tetrazol-
2-ium chloride (QT-39). Prepared using phenylhydr-
azine, benzaldehyde, and 4-benzyloxyaniline as starting
materials to give the product as a beige solid; mp 90–
chloride
(QT-33).
Prepared using phenylhydrazine, 4-benzyloxy-3-
methoxybenzaldehyde, and 3-chloroaniline as starting
materials to give the product; 1H NMR (300 MHz,
DMSO-d6): d 3.40 (s, 3H), 5.31 (s, 2H), 7.38–7.52 (m,
8H), 7.58 (s, 1H), 7.70–7.78 (m, 3H), 7.86–7.96 (m,
4H), 8.00–8.07 (m, 1H); HRMS (ES) [MꢀH]+ calcd
for C27H21N4O2Cl2: 503.1036. Found: 503.1027; HRMS
(ES) [MꢀCl]+ calcd for C27H22N4O2Cl: 469.1425.
Found: 469.1417.
1
120 ꢁC (dec); H NMR (300 MHz, DMSO-d6): d 5.22
(s, 2H), 7.29–7.48 (m, 7H), 7.71–7.98 (m, 10H), 8.32–
8.34 (m, 2H); HRMS (ES) [MꢀH]+ calcd for
C26H20N4OCl: 439.1320. Found: 439.1334; HRMS
(ES) [MꢀCl]+ calcd for C26H21N4O: 405.1709. Found:
405.1706.
5.6.40. 2,5-Bis-(4-benzyloxyphenyl)-3-phenyl-3H-tetra-
zol-2-ium chloride (QT-40). Prepared using phenylhydr-
5.6.34. 2-(4-Methoxyphenyl)-3,5-diphenyl-3H-tetrazol-2-
ium chloride (QT-34). Prepared using phenylhydrazine,
benzaldehyde, and 4-methoxyaniline as starting materi-
azine,
4-benzyloxybenzaldehyde,
and
4-benzy-
loxyaniline as starting materials to give the product;
1H NMR (300 MHz, DMSO-d6): d 5.20 (s, 2H), 5.28
(s, 2H), 7.28–7.53 (m, 15H), 7.73–7.92 (m, 6H), 8.26–
8.29 (m, 2H); HRMS (ES) [MꢀH]+ calcd for
C33H26N4O2Cl: 545.1738. Found: 545.1727; HRMS
(ES) [MꢀCl]+ calcd for C33H27N4O2: 511.2128. Found:
511.2145.
1
als to give the product; H NMR (300 MHz, DMSO-
d6): d 3.86 (s, 3H), 7.21–7.26 (m, 2H), 7.77–7.96 (m,
10H), 8.32–8.34 (m, 2H); HRMS (ES) [MꢀH]+ calcd
for C20H16N4OCl: 363.1007. Found: 363.1023; HRMS
(ES) [MꢀCl]+ calcd for C20H17N4O: 329.1396. Found:
329.1409.
5.6.35. 5-(4-Benzyloxyphenyl)-2-(4-methoxyphenyl)-3-phe-
nyl-3H-tetrazol-2-ium chloride (QT-35). Prepared using
5.6.41. 2-(4-Benzyloxyphenyl)-5-(3-chlorophenyl)-3-phe-
nyl-3H-tetrazol-2-ium chloride (QT-41). Prepared using

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1945
phenylhydrazine, 3-chlorobenzaldehyde, and 4-benzy-
loxyaniline as starting materials to give the product;
1H NMR (300 MHz, DMSO-d6): d 5.22 (s, 2H), 7.29–
7.48 (m, 7H), 7.71–8.02 (m, 9H), 8.28–8.35 (m, 2H);
HRMS (ES) [MꢀH]+ calcd for C26H19N4OCl2:
473.0930. Found: 473.0929; HRMS (ES) [MꢀCl]+ calcd
for C26H20N4OCl: 439.1320. Found: 439.1329.
chlorophenylhydrazine, 4-benzyloxybenzaldehyde, and
3-chloroaniline as starting materials to give the product
as a beige solid; 1H NMR (300 MHz, DMSO-d6): d 5.29
(s, 2H), 7.42–7.62 (m, 9H), 7.75–7.80 (m, 1H), 7.88–8.09
(m, 4H), 8.12 (s, 1H), 8.28–8.31 (m, 2H); HRMS (ES)
[MꢀH]+ calcd for C26H18N4OCl3: 507.0546. Found:
507.0553;
HRMS
(ES)
[MꢀCl]+
calcd
for
C26H19N4OCl2: 473.0935. Found: 473.0936.
5.6.42. 2-(4-Benzyloxyphenyl)-5-biphenyl-4-yl-3-phenyl-
3H-tetrazol-2-ium chloride (QT-42). Prepared using
phenylhydrazine, biphenyl-4-carboxaldehyde, and 4-
benzyloxyaniline as starting materials to give the prod-
5.6.48. 3-(3-Bromophenyl)-2,5-diphenyl-3H-tetrazol-2-ium
chloride (QT-48). Prepared using phenylhydrazine,
benzaldehyde, and 3-bromoaniline as starting materials
1
1
uct as a beige solid; H NMR (300 MHz, DMSO-d6):
to give the product; H NMR (300 MHz, DMSO-d6):
d 5.22 (s, 2 H), 7.31–7.59 (m, 10H), 7.75–7.96 (m, 9H),
8.10 (d, J = 8.4 Hz, 2H), 8.39–8.43 (m, 2H); HRMS
(ES) [MꢀH]+ calcd for C32H24N4OCl: 515.1633. Found:
515.1609; HRMS (ES) [MꢀCl]+ calcd for C32H25N4O:
481.2022. Found: 481.2016.
d 7.67–8.08 (m, 11H), 8.20–8.25 (m, 1H), 8.38–8.46 (m,
2H); HRMS (ES) [MꢀH]+ calcd for C19H13N4ClBr:
411.0006. Found: 411.0020; HRMS (ES) [MꢀCl]+ calcd
for C19H14N4Br: 377.0396. Found: 377.0392.
5.6.49. 5-(4-Benzyloxyphenyl)-3-(3-bromophenyl)-2-phe-
nyl-3H-tetrazol-2-ium chloride (QT-49). Prepared using
phenylhydrazine, 4-benzyloxybenzaldehyde, and 3-bro-
5.6.43. 2-(4-Benzyloxyphenyl)-3-(3-chlorophenyl)-5-phe-
nyl-3H-tetrazol-2-ium chloride (QT-43). Prepared using
3-chlorophenylhydrazine, benzaldehyde, and 4-benzy-
loxyaniline as starting materials to give the product;
1
moaniline as starting materials to give the product; H
NMR (300 MHz, DMSO-d6): d 5.29 (s, 2H), 7.36–7.53
(m, 8H), 7.66–8.14 (m, 8H), 8.29–8.33 (m, 2H); HRMS
(ES) [MꢀH]+ calcd for C26H19N4OClBr: 517.0430.
Found: 517.0458; HRMS (ES) [MꢀCl]+ calcd for
C26H20N4OBr: 483.0820. Found: 483.0810.
1
mp 95–125 ꢁC (dec); H NMR (300 MHz, DMSO-d6):
d 5.44 (s, 2H), 7.36–7.60 (m, 10H), 7.79–7.95 (m, 5H),
8.18–8.33 (m, 2H), 8.69–8.71 (m, 1H); HRMS (ES)
[MꢀH]+ calcd for C26H19N4OCl2: 473.0930. Found:
473.0937;
HRMS
(ES)
[MꢀCl]+
calcd
for
C26H20N4OCl: 439.1320. Found: 439.1341.
5.6.50. 5-Hexyl-2,3-diphenyl-3H-tetrazol-2-ium bromide
(QT-50). Prepared using phenylhydrazine, heptalde-
hyde, and aniline as starting materials to give the prod-
5.6.44. 5-(4-Benzyloxyphenyl)-3-(3-chlorophenyl)-2-phe-
nyl-3H-tetrazol-2-ium chloride (QT-44). Prepared using
1
uct as a white solid; H NMR (300 MHz, CD3OD): d
3-chlorophenylhydrazine,
4-benzyloxybenzaldehyde,
0.97 (t, J = 6.9 Hz, 3H), 1.44–1.46 (m, 4H), 1.61–1.66
(m, 2H), 2.06 (m, 2H), 3.33 (t, J = 7.6 Hz, 2H), 7.65–
7.71 (m, 4H), 7.78–7.83 (m, 6H); 13C NMR (400 MHz,
CD3OD): d 13.0, 22.1, 25.4, 26.6, 28.3, 31.2, 125.9,
130.2, 133.3, 133.8, 168.5; HRMS (ES) [MꢀBr]+ calcd
for C19H23N4: 307.1917. Found: 307.1923.
and aniline as starting materials to give the product;
1H NMR (300 MHz, DMSO-d6): d 5.29 (s, 2H), 7.35–
7.53 (m, 8H), 7.72–8.08 (m, 8H), 8.27–8.36 (m, 2H);
HRMS (ES) [MꢀH]+ calcd for C26H19N4OCl2:
473.0930. Found: 473.0934; HRMS (ES) [MꢀCl]+ calcd
for C26H20N4OCl: 439.1320. Found: 439.1325.
5.6.51.
5-Cyclohexyl-2,3-diphenyl-3H-tetrazol-2-ium
5.6.45. 5-(3-Chlorophenyl)-2,3-diphenyl-3H-tetrazol-2-
ium chloride (QT-45). Prepared using phenylhydrazine,
3-chlorobenzaldehyde, and aniline as starting materials
to give the product; mp 104–128 ꢁC (dec); 1H NMR
(300 MHz, DMSO-d6): d 7.70–7.77 (m, 3H), 7.79–7.87
(m, 4H), 7.90–8.06 (m, 5H), 8.31–8.34 (m, 1H), 8.36–
8.37 (m, 1H); HRMS (ES) [MꢀH]+ calcd for
C19H13N4Cl2: 367.0511. Found: 367.0497; HRMS (ES)
[MꢀCl]+ calcd for C19H14N4Cl: 333.0901. Found:
333.0915.
chloride (QT-51). Prepared using phenylhydrazine,
cyclohexanecarboxaldehyde, and aniline as starting
materials to give the product; 1H NMR (300 MHz,
DMSO-d6): d 1.46 (t, J = 2.1 Hz, 1H), 1.54–1.66 (m,
2H), 1.84–1.92 (m, 3H), 1.96–2.00 (m, 2H), 2.36–2.40
(m, 2H), 3.35–3.50 (m, 1H), 7.65–7.86 (m, 10H); HRMS
(ES) [MꢀH]+ calcd for C19H20N4Cl: 339.1376. Found:
339.1361; HRMS (ES) [MꢀCl]+ calcd for C19H21N4:
305.1766. Found: 305.1755.
5.6.52. 2,3-Diphenyl-5-(4-undecyloxyphenyl)-3H-tetra-
zol-2-ium chloride (QT-52). Prepared using phenylhydr-
azine, 4-undecyloxybenzaldehyde, and aniline as
5.6.46. 2,3-Bis-(3-chlorophenyl)-5-phenyl-3H-tetrazol-2-
ium chloride (QT-46). Prepared using 3-chloro-
phenylhydrazine, benzaldehyde, and 3-chloroaniline as
starting materials to give the product; 1H NMR
(300 MHz, DMSO-d6): d 7.76–7.85 (m, 6H), 7.92–8.08
(m, 4H), 8.17 (s, 1H), 8.34–8.45 (m, 2H); HRMS (ES)
[MꢀH]+ calcd for C19H12N4Cl3: 401.0127. Found:
401.0143; HRMS (ES) [MꢀCl]+ calcd for C19H13N4Cl2:
367.0511. Found: 367.0522.
1
starting materials to give the product; H NMR (300
MHz, DMSO-d6): d 0.86 (t, J = 6.3 Hz, 3H), 1.26–1.46
(m, 16H), 1.75–1.80 (m, 2H), 4.11–4.24 (m, 2H), 7.28
(d, J = 8.7 Hz, 2H), 7.66–7.93 (m, 10H), 8.26 (d, J = 8.7
Hz, 2H); HRMS (ES) [MꢀH]+ calcd for C30H36N4OCl:
503.2577. Found: 503.2582; HRMS (ES) [MꢀCl]+ calcd
for C30H37N4O: 469.2967. Found: 469.2965.
5.6.47. 5-(4-Benzyloxyphenyl)-2,3-bis-(3-chlorophenyl)-
3H-tetrazol-2-ium chloride (QT-47). Prepared using 3-
5.6.53. 2,3-Diphenyl-5-(3-undecyloxyphenyl)-3H-tetra-
zol-2-ium chloride (QT-53). Prepared using phenylhydr-

1946
X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
azine, 3-undecyloxybenzaldehyde, and aniline as start-
ing materials to give the product; H NMR (300 MHz,
plate and then 50 lL of parasite culture (2% hematocrit,
2% parasitemia) was 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 contents of the wells were then
re-suspended using a multi-channel pipettor and a 15-
lL sample was removed from each well and was added
to 100 lL of pLDH enzyme assay mixture.19 After 1 h
the absorbance of the wells at 650 nm was determined
using a ThermoMax microplate reader (Molecular De-
vices, Sunnyvale, CA). The IC50 values of individual
compounds were determined using a non-linear regres-
sion analysis of the data33 using the computer program
SigmaPlot (Jandel Scientific). The IC50 values represent
means standard error calculated from four indepen-
dent determinations. To verify if the poor viability of
the cultures was related to inhibited merozoite invasion,
samples were taken from treated wells and the presence
of extracellular merozoites was confirmed by
microscopy.
1
DMSO-d6): d 0.75–0.90 (m, 3H), 1.10–1.50 (m, 16H),
1.60–1.80 (m, 2H), 4.00–4.25 (m, 2H), 7.30–7.40 (m,
1H), 7.60–7.90 (m, 6H), 7.95–8.25 (m, 6H), 8.75–8.90
(m, 1H); HRMS (ES) [MꢀH]+ calcd for C30H36N4OCl:
503.2577. Found: 503.2579; HRMS (ES) [MꢀCl]+ calcd
for C30H37N4O: 469.2967. Found: 469.2965.
5.6.54. 5-Isopropyl-2,3-diphenyl-3H-tetrazol-2-ium bro-
mide (QT-54). Prepared using phenylhydrazine, isobu-
tyraldehyde, and aniline as starting materials to give
1
the product; H NMR (300 MHz, CD3OD): d 1.65 (d,
J = 7.0 Hz, 6H), 3.66–3.70 (m, 1H), 7.66–7.83 (m,
10H); 13C NMR (75 MHz, CD3OD): d 19.5, 26.8,
125.8, 130.2, 133.4, 133.8, 172.7; HRMS (ES) [MꢀBr]+
calcd for C16H17N4: 265.1447. Found: 265.1456.
5.7. Assays for the inhibition of the reduction of tetrazo-
lium salts
Assays comparing the activity of the compounds in hu-
man and rodent malaria cultures were performed in a
similar fashion, however, parasite viability was assayed
using the SYBR-Green method.33 Rodent malaria sam-
ples were obtained by cardiac puncture of infected mice
and the erythrocytes were then cultured for two days in
compound diluted in RPMI-1640. Trypsin and neur-
aminidase treatment of human erythrocytes was per-
formed as described by Lobo et al.,9 and the presence
of a specific invasion phenotype was confirmed by
attempting to culture parasites in erythrocytes that had
been subjected to the opposite treatment.
Assays were performed in a 96-well plate. Nitrotetrazo-
lium blue chloride (NBT) was added to a final concen-
tration as indicated on the x-axis (see Fig. 2) and
glutathione was added to a final concentration of
7 mM. b-Cyclodextrin, either unsulfated or having an
average of one sulfate group per glucose residue,10 was
added to a final concentration ranging from 0 to
167 lM. All solutions were prepared in 50 mM Tris buf-
fer, pH 9.0.19 Assays were performed by adding 50 lL of
the NBT solution to a microwell followed by 50 lL of
the b-cyclodextrin solution, allowing the plate to incu-
bate for 5 min, and then adding 50 lL of the glutathione
solution. Ten minutes after the addition of the glutathi-
one solution the plates were read at 650 nm in a Thermo-
Max microplate reader (Molecular Devices, Sunnyvale,
CA.). To determine the effect of SITS (4-acetamido-40-
isothiocyanato-2,20-stilbenedisulfonic acid disodium
salt, Molecular Probes, Inc.) and DIDS (4,40-diisothiocy-
anatostilbene-2,20-disulfonic acid disodium salt, Sigma)
on the reduction of NBT serial dilutions of the com-
pounds were prepared in 50 lL of Tris buffer and then
50 lL of 30 lM NBT was added to every well, followed
by 50 lL of 21 mM glutathione. After 10 min, the absor-
bance at 650 nm was determined as above.
5.9. Mammalian strains and culture
CHO cells (ATCC, Manassas, VA) were grown in
RPMI-1640 supplemented with 10% fetal calf serum
(Sigma, St. Louis, MO), 25 mM Hepes, and gentimicin
(RPMI-10). Cells were seeded in 96-well plates and
grown to 50% confluency in 100 lL of RPMI-10 per well
prior to the addition of either DMSO alone, or a 10 mg/
mL solution of a tetrazolium compound in DMSO.
Compound gradients were prepared by adding 90 lL
of RPMI-10 mixed with 10 lL of compound solution
to the first well in the series, mixing, transferring
100 lL to next well, and 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-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma,
St. Louis, MO) in RPMI-10, incubating the plates for
a further hour, and then removing the media and adding
100 lL of DMSO and reading the absorbance at
650 nm.21 The IC50 values of individual compounds
were determined using a non-linear regression analysis
of the dose-response curve using the computer program
SigmaPlot (Jandel Scientific).
5.8. Determination of anti-Plasmodium activity
P. falciparum cultures were grown in O+ blood obtained
by venipuncture of volunteers. Cultures of the labora-
tory line ItG were maintained by the method of Trager
and Jensen31 using RPMI-1640 supplemented with 10%
human serum (a kind gift obtained under ethical consent
from the Chemo Day Care Department of the Princess
Margaret Hospital, Toronto, Canada) and 50 lM hypo-
xanthine (RPMI-A). The effects of the test compounds
on the viability of P. falciparum cultures were deter-
mined using a Lactate Dehydrogenase (LDH) enzyme
assay specific for the enzyme found in Plasmodium falci-
parum (pLDH).19,32 Briefly, compounds to be tested
were dissolved in DMSO to afford a solution having a
concentration of 10 mg/mL. Twofold serial dilutions
were then produced in 50 lL of RPMI-A in a 96-well
Acknowledgments
The authors gratefully acknowledge the generous sup-
port of the Bill and Melinda Gates Foundation, and

X. Cui et al. / Bioorg. Med. Chem. 16 (2008) 1927–1947
1947
´
15. Hamze, 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; Wengelnik, K.; Vidal,
V.; Ancelin, M. L.; Cathiard, A-M.; Morgat, J. L.;
Kocken, C. H.; Calas, M.; Herrera, S.; Thomas, A. W.;
Vial, H. J. Science 2002, 295, 1311.
the award of a CIHR Proof of Principle Grant PPP-
62029 to IC.
References and notes
16. Katritzky, A. R.; Belyakov, S. A.; Cheng, D.; Durst, H. D.
Synthesis 1995, 577.
17. Ashley, J. N.; Davis, B. M.; Nineham, A. W.; Slack, R. J.
Chem. Soc. 1953, 3881.
1. Breman, J. G.; Egan, A.; Keusch, G. T. Am. J. Trop. Med.
Hyg. 2001, 64, iv–vii, See also http://rbm.who.int/
wmr2005/ for the World Health Organization World
Malaria Report 2005.
18. Benson, F. R.; Otten, E. A.; Schachat, R. E. J. Am. Chem.
Soc. 1954, 76, 1695.
19. Prudhomme, J. G.; Sherman, I. W. J. Immunol. Methods
1999, 229, 169.
20. Pimplikar, S. W.; Reithmeier, R. A. J. Biol. Chem. 1986,
261, 9770.
21. Campling, B. G.; Pym, J.; Galbraith, P. R.; Cole, S. P.
Leukocyte Res. 1988, 12, 823.
22. Perkins, M. E.; Holt, E. H. Mol. Biochem. Parasitol. 1988,
27, 23.
23. Clark, D. L.; Su, S.; Davidson, E. A. Glycoconj. J. 1997,
14, 473.
2. Wkly. Epidemiol. Rec. 1997, 72, 277.
3. White, N. J.; Nosten, F.; Looareesuwan, S.; Watkins, W.
M.; Marsh, K.; Snow, R. W.; Kokwaro, G.; Ouma, J.;
Hien, T. T.; Molyneux, M. E.; Taylor, T. E.; Newbold C.
I.; Ruebush, T. K., 2nd; Danis, M.; Greenwood, B. M.;
Anderson, R. M.; Olliaro, P. Lancet 1999, 353, 1965.
4. Kisilevsky, R.; Crandall, I.; Szarek, W. A.; Bhat, S.; Tan,
C.; Boudreau, L.; Kain, K. C. Antimicrob. Agents
Chemother. 2002, 46, 2619.
5. Pasvol, G. Trends Parasitol. 2003, 19, 430.
6. Pasvol, G.; Wainscoat, J. S.; Weatherall, D. J. Nature
1982, 297, 64.
24. Higuti, T.; Arakaki, R.; Kotera, Y.; Takigawa, M.; Tani,
I.; Shibuya, M. Biochim. Biophys. Acta 1983, 725, 1.
25. Goel, V. K.; Li, X.; Chen, H.; Liu, S. C.; Chishti, A. H.;
Oh, S. S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5164.
26. Kariuki, M. M.; Li, X.; Yamodo, I.; Chishti, A. H.; Oh, S.
S. Biochem. Biophys. Res. Commun. 2005, 338, 1690.
27. Casey, J. R.; Reithmeier, R. A. Biochemistry 1993, 32,
1172.
7. Barnwell, J. W.; Galinski, M. R. In Malaria: Parasite
Biology, Pathogenesis, and Protection; Sherman, I. W.,
Ed.; ASM Press: Washington, DC, 1998; pp 93–123.
8. Oh, S. S.; Chishti, A. H. Curr. Top. Microbiol. Immunol.
2005, 295, 203.
9. Lobo, C. A.; de Frazao, K.; Rodriguez, M.; Reid, M.;
Zalis, M.; Lustigman, S. Infect. Immun. 2004, 72, 5886.
10. Crandall, I. E.; Szarek, W. A.; Vlahakis, J. Z.; Xu, Y.;
Vohra, R.; Sui, J.; Kisilevsky, R. Biochem. Pharmacol.
2007, 73, 632.
28. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem.
1997, 62, 7512.
29. Mintz, M. J.; Walling, C. Org. Synth. 1969, 49, 9.
30. Nineham, A. W. Chem. Rev. 1955, 55, 355.
31. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
32. Makler, M. T.; Ries, J. M.; Williams, J. A.; Bancroft, J. E.;
Piper, R. C.; Gibbins, B. L.; Hinrichs, D. J. Am. J. Trop.
Med. Hyg. 1993, 48, 739.
11. Xiao, L.; Yang, C.; Patterson, P. S.; Udhayakumar, V.;
Lal, A. A. Infect. Immun. 1996, 64, 1373.
12. Popov, M.; Li, J.; Reithmeier, R. A. Biochem. J. 1999, 339,
269.
13. Salhany, J. M.; Schopfer, L. M. Biochemistry 1996, 35, 251.
14. Salhany, J. M.; Schopfer, L. M.; Kay, M. M.; Gamble, D.
N.; Lawrence, C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
11844.
33. Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.;
Riscoe, M. Antimicrob. Agents Chemother. 2004, 48, 1803.