Synthesis and antimalarial activities of a diverse set of triazole-containing furamidine analogues

Article abstract of DOI:10.1002/cmdc.201100265

Four different series of triazole diamidines have been prepared by the Pinner method from the corresponding triazole dinitriles. Copper-catalyzed "click chemistry" was used for the synthesis of 1,4- and 4,5-substituted triazoles, aryl magnesium acetylide

Full text of DOI:10.1002/cmdc.201100265

MED
DOI: 10.1002/cmdc.201100265
Synthesis and Antimalarial Activities of a Diverse Set of
Triazole-Containing Furamidine Analogues
Olivier Berger,[a] Archana Kaniti,[b] Christophe Tran van Ba,[c] Henri Vial,[c] Stephen A. Ward,[b]
Giancarlo A. Biagini,[b] Patrick G. Bray,[b] and Paul M. O’Neill*[a]
Four different series of triazole diamidines have been prepared
by the Pinner method from the corresponding triazole dini-
triles. Copper-catalyzed “click chemistry” was used for the syn-
thesis of 1,4- and 4,5-substituted triazoles, aryl magnesium ace-
tylide reagents for the 1,5-substituted triazoles, with a thermal
dipolar addition reaction employed for the 2,4-substituted tria-
zoles. In vitro antimalarial activity against two different PfCRT-
modified parasite lines (Science 2002, 298, 210–213) of Plasmo-
dium falciparum and inhibition of hemozoin formation were
determined for each compound. Several diamidines with
potent nanomolar antimalarial activities were identified, and
selected molecules were resynthesized as their diamidoxime
triazole prodrugs. One of these prodrugs, OB216, proved to be
highly potent in vivo with an ED50 value of 5 mgkgꢀ1 (po) and
an observed 100% cure rate (CD100) of just 10 mgkgꢀ1 by oral
(po) administration in mice infected with P. vinckei.
Introduction
Malaria is a major public health issue with about 50% of the
global population at risk.[1] The disease is caused by protozoal
parasites of the genus Plasmodium with Plasmodium falcipa-
rum being responsible for about 80% of all malaria cases and
about 90% of deaths. In spite of global economic develop-
ment, malaria is still present in 106 countries, with approxi-
mately 250 million new infections and one million deaths per
year,[1,2] causing much devastation and a staggering amount of
chronic ill health that not only impedes the economic develop-
ment of these countries, already stricken by poverty, but also
impacts on fertility, population growth, worker productivity,
and premature mortality.[2,3] In the absence of a vaccine and re-
sistance of P. falciparum to the majority of antimalarial drugs in
current clinical use, new antimalarial agents are urgently
needed to aid the prevention and control of malaria.[4] Cross-
resistance is a problem among antimalarial agents due to a
lack of chemical diversity. Therefore, the most promising strat-
egy is the discovery of new chemical entities against novel bio-
logical targets.[5]
Figure 1. Structures of furamidine and pafuramidine and the general struc-
tures of the different triazoles (series 1 and 2) explored in this study.
been tested for the treatment of P. vivax and uncomplicated
P. falciparum infections with a degree of success.[13]
In a program directed at the synthesis of novel analogues of
pentamidine, Das and Boykin inserted a furan unit as a linker
between the two phenylamidine groups to produce 2,5-bis-(4-
amidinophenyl)furan (furamidine; Figure 1), a molecule with
potent antitrypanosomal activity.[6,7] Subsequently, the prodrug
2,5-bis(4-methoxyamidinophenyl)furan (pafuramidine) was de-
veloped by the same groups for the treatment of human Afri-
can trypanosomiasis (HAT).[8,9] Pafuramidine reached phase III
clinical trials against HAT and P. jiroveci,[10,11] although issues
with hepatic and renal toxicity of this molecule in humans are
likely to preclude its further development. Furamidine has
potent antimalarial activity in vitro against chloroquine (CQ)-re-
sistant P. falciparum (IC50 =15.5 nm)[12] with low toxicity towards
mammalian cells. The amidoxy prodrug pafuramidine has also
The use of a triazole functional group as a linker is becom-
ing increasingly popular in medicinal chemistry, and many di-
verse examples of incorporation of this heterocycle into a vari-
[a] Dr. O. Berger, Prof. P. M. O’Neill
Department of Chemistry, University of Liverpool
Crown street, Liverpool, L69 3BX (UK)
E-mail: P.M.oneill01@liverpool.ac.uk
[b] Dr. A. Kaniti, Prof. S. A. Ward, G. A. Biagini, Dr. P. G. Bray
Liverpool School of Tropical Medicine
Pembroke Place, Liverpool, L3 5QA (UK)
[c] C. T. v. Ba, Prof. H. Vial
Dynamique Moleculaire des Interactions Membranaires
UMR 5235 CNRS- Universitꢀ de Montpellier II
34095 Montpellier (France)
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Triazole-Containing Furamidines as Antimalarials
ety of drugs have appeared in the literature.[14–25] Given the
structural diversity possible within the triazole ring system, we
aimed to produce an array of functionalized triazole-containing
diamidines for structure–activity relationship (SAR) analysis. Six-
teen different triazole derivatives with different substitution on
the triazole and aromatic rings (para or meta) were prepared
(Figure 1): seven 1,4-substituted derivatives, two 1,5-substitut-
ed derivatives, two 2,4-substituted derivatives, and six 4,5-sub-
stituted derivatives. Our focus was to study the influence of
the substitution pattern between the two amidine functions
on antimalarial activity, as well as the position of the amidine
functions on the aromatic ring. Based on the initial SAR data,
we also planned to prepare diamidoxime prodrugs of the most
active compounds identified in our in vitro screening, and to
evaluate their in vivo performance versus P. vinckei by both
oral (po) and intraperitoneal (ip) administration.
Scheme 1. Reagents and conditions: a) NaN3, DMF, RT, 4 h; b) CuSO4·5H2O,
Na2CO3, ascorbic acid, l-proline, H2O, DMSO, 658C, 3 h; c) gaseous HCl,
EtOH, RT, 7 d; d) NH3 2m in MeOH, EtOH, RT, 24 h.
Diamidine drugs are impermeable to normal human erythro-
cytes, but are taken up rapidly into P. falciparum-infected eryth-
rocytes through the new permeability pathway (NPP) induced
by the parasite in the host cell membrane.[25] Once inside the
infected cell, it is proposed that this class of drug binds strong-
ly to hematin and kills parasites by inhibition of crystallization/
formation of hemozoin in a similar manner to CQ.[26] Based on
this potential mechanism of action, we planned to investigate
the relationship between antimalarial activity in vitro and the
ability to inhibit hemozoin formation for the triazole series de-
scribed herein.
trile (8) and ethynyltrimethylsilane in the presence of palladi-
um(0) and copper(I) iodide[39] followed by desilylation using
tetra-n-butylammonium fluoride (TBAF) to furnish terminal
alkyne 10 in 53% yield over two steps.[40] Then, 1,4-disubstitut-
ed triazoles 11 a–d were generated using copper(I)-catalyzed
1,3-dipolar cycloaddition in yields ranging from 93 to
97%.[32–34] Diamidines 13a–d were prepared by initial cyana-
tion of the bromine using copper cyanide in DMF[41] followed
by a modified Pinner reaction.[35–37] The two 1,4-disubstituted
prodrugs 14a–b were produced by reacting dinitrile deriva-
tives 12a–b and hydroxylamine hydrochloride under basic
conditions[42] and then alkylating the generated amidoximes
using dimethylsulfate.[41,43]
Results and Discussion
Chemistry
To construct the 1,4-substituted five-membered triazole ring,
several methods have been reported with the most suitable
being the Huisgen 1,3-dipolar cycloaddition of organic azides
to terminal alkynes.[27] This method employs elevated tempera-
tures and generally produces mixtures of 1,4- and 1,5-disubsti-
tuted 1,2,3-triazoles.[28,29] Recently, the use of copper(I) salts
was shown to exclusively produce 1,4-disubstituted deriva-
tives.[30] Therefore, we decided to use copper(I)-catalyzed
azide-alkyne cycloaddition because of the high regioselectivity,
mild reaction conditions, and excellent yields.
Few methods were available for the preparation of the 1,5-
substituted triazole unit from azides and alkynes. These includ-
ed the Huisgen 1,3-dipolar cycloaddition,[27] ruthenium-cata-
lyzed azide-alkyne cycloaddition,[44] and a method using aryl
magnesium acetylides, generated in situ using ethyl magnesi-
um bromide.[45,46] This latter method, developed by Zhang
et al., was preferred because the 1,5-disubstituted derivatives
were obtained exclusively in good yields, and no catalyst prep-
aration is required.
To synthesize the nonaromatic 1,4-substituted derivative 5
(Scheme 1), azide 2 was generated from the corresponding
benzylbromide using sodium azide.[31] The triazole ring was
then generated using copper(I)-catalyzed 1,3-dipolar cycloaddi-
tion of 4-(azidomethyl)benzonitrile (2) to 4-ethynylbenzonitrile
(3) in a mixture of DMSO and water in 98% yield.[32–34] Finally,
The nonaromatic and aromatic 1,5-disubstituted triazole de-
rivatives 18a and 18b were obtained in yields ranging from 89
to 96% using ethyl magnesium bromide in THF.[45,46] Com-
pounds 19a and 19b were allowed to react with copper cya-
nide in DMF to afford dinitrile derivatives 19a and 19b in
moderate yields.[39] Diamidines 20a and 20b were prepared
using a modified Pinner method (Scheme 3).[35–37]
diamidine
5
was prepared using
a
modified Pinner
method.[35–37]
To synthesize the 4,5-disubstituted and the 1,4,5- and 2,4,5-
trisubstituted triazole units (Scheme 4), 4,4’-dicyanostilbene
(22) was prepared as an intermediate for both series of com-
pounds using Sonogashira reaction conditions between the 4-
bromobenzonitrile (21) and the 4-ethynylbenzonitrile (3) in the
presence of palladium(II), copper(I) iodide and triethylamine.[47]
The 1,4,5-trisubstituted triazole derivatives 25a and 25b
were prepared using the copper(II)-catalyzed 1,3-dipolar cyclo-
To synthesize the aromatic 1,4-substituted derivatives 13a–d
and 14a–b (Scheme 2), 1-azido-4-bromobenzene (7a) and 1-
azido-3-bromobenzene (7b) were prepared by diazotization/
azidation of the 4- and 3-bromoaniline (6a and 6b, respective-
ly) using first sodium nitrite in dilute hydrochloric acid at 0–
58C and then sodium azide.[38] The synthesis of alkyne 10 was
performed by Sonogashira coupling between 3-bromobenzoni-
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P. M. O’Neill et al.
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addition of azidobenzene 23a
or azidomethylbenzene 23b to
4,4’-dicyanostilbene (22) at
658C, but the reaction did not
work at this temperature proba-
bly due to steric hindrance;
therefore, the temperature was
increased to 1208C.[32–34] The
Pinner reaction was employed
to obtain compounds 25a and
25b in yields of 22% and 39%,
respectively, over two steps
(Scheme 4).[35–37]
For the synthesis of 4,5-disub-
stituted derivative 27, 4,4’-di-
cyanostilbene (22) was allowed
to react with sodium azide at
reflux in DMF to give the tria-
zole unit in 65% yield
(Scheme 4).[48] This reaction was
attempted using the copper(I)-
catalyzed 1,3-dipolar cycloaddi-
tion,[32–34] but failed possibly be-
cause the sodium azide was de-
graded by the catalyst (no reac-
tion with 1.0 equiv of NaN3, and
only 25% product 26 formation
with 2.0 equiv). Compound 27
was obtained from derivative
26 in 60% yield using the
Pinner reaction.[35–37]
The 2,4,5-trisubstituted tria-
zole derivatives 30a and 30b
were prepared in two steps
(Scheme 4). Compound 28 was
prepared in 90% yield by alkyla-
tion of triazole derivative 26
Scheme 2. A) Synthesis of key nitriles 12a–d; B) Preparation of diamidines 13a–d; C) Preparation of prodrugs 14a
and 14b. Reagents and conditions: a) NaNO2, concd HCl, H2O, 08C, 30 min; b) NaN3, H2O, RT, 1 h; c) ethynyltrime-
thylsilane, Pd(PPh3)4, CuI, Et3N, 808C, 20 h; d) TBAF, ꢀ208C, 30 min; e) CuSO4·5H2O, Na2CO3, ascorbic acid, l-pro-
line, H2O, DMSO, 658C, 3 h; f) CuCN, DMF, 1708C, 24 h; g) gaseous HCl, EtOH, RT, 7 d; h) NH3 2m in MeOH, EtOH,
RT, 24 h; i) NH2OH·HCl, K2CO3, EtOH, 908C, 20 h; j) dimethylsulfate, NaOH 2m, 1,4-dioxane, RT, 20 h.
with iodobenzene using an iron/copper co-catalyzed Ullmann
condensation developed by Taillefer et al.[49] Compound 29
was obtained in 76% yield, again by alkylation of triazole de-
rivative 26, but with benzylchloride in the presence of potassi-
um carbonate and tetrabutylammonium iodide (TBAI).[48]
During this reaction, 19% of 1,4,5-benzyltriazole 24b was also
recovered. The two diamidines 30a and 30b were obtained
using the Pinner methodology in 71% and 66% yields, respec-
tively.[35–37]
For the 2,4-disubstituted series (Scheme 5), 4-phenylethynyl-
benzonitrile (32) was formed in 98% yield using the Sonoga-
shira methodology between 4-bromobenzonitrile (21) and
phenylacetylene (31) in the presence of palladium(II) and cop-
per(I) iodide in dry triethylamine.[47] The triazole unit was then
prepared by reaction between nitrile 32 and sodium azide in
DMF.[48] Compound 33 was unstable and so used immediately
for the next step. Compound 34 was obtained in 32% yield by
alkylation of triazole 33 with 4-bromobenzonitrile using the
iron/copper co-catalysed Ullmann condensation developed by
Taillefer et al.[49] The low yield can be explained by the instabili-
Scheme 3. Reagents and conditions: a) EtMgBr, THF, 508C, 1 h; b) CuCN, DMF,
1708C, 24 h; c) gaseous HCl, EtOH, RT, 7 d; d) NH3 2m in MeOH, EtOH, RT,
24 h.
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Triazole-Containing Furamidines as Antimalarials
Scheme 4. Reagents and conditions: a) PPh3, Pd(PPh3)2Cl2, CuI, Et3N, 1008C, 2 h; b) CuSO4·5H2O, Na2CO3, ascorbic acid, l-proline, H2O, DMSO, 1208C, 15 h;
c) NaN3, DMF, 1708C, 5 h; d) Cs2CO3, CuO, Fe(acac)3, DMF, 908C, 30 h; e) K2CO3, TBAI, acetone, 708C, 20 h; f) gaseous HCl, EtOH, RT, 7 d; g) NH3 2m in MeOH,
EtOH, RT, 24 h.
Scheme 5. Reagents and conditions: a) PPh3, Pd(PPh3)2Cl2, CuI, Et3N, 1008C, 2 h; b) NaN3, DMF, 1708C, 5 h; c) Cs2CO3, CuO, Fe(acac)3, DMF, 908C, 30 h; d) K2CO3,
TBAI, acetone, 708C, 20 h; e) gaseous HCl, EtOH, RT, 7 d; f) NH3 2m in MeOH, EtOH, RT, 24 h.
ty of triazole 33 and also by the duration of the alkylation.
Compound 35 was obtained in 74% yield also by alkylation of
triazole 33 with 4-bromomethyl benzonitrile in the presence of
potassium carbonate and TBAI.[48] The two diamidines 36a and
36b were obtained using the Pinner methodology in 63% and
71% yields, respectively.[35–37]
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In vitro antimalarial activities
between hematin dimerization inhibition and inherent anti-
plasmodial activity versus both CQ-sensitive and CQ-resistant
strains of the parasite.
In vitro antimalarial activities were evaluated for each deriva-
tive using two different strains of P. falciparum with varying CQ
sensitivity: C2GCO3 is a CQ-sensitive strain, and C3Dd2 is a CQ-re-
sistant strain (Table 1).[50] These strains were developed in the
Against the CQ-sensitive C2GC03 strain, the 1,4-diaryl-substi-
tuted triazoles 13a, 13b, and 13c exhibit good antiplasmodial
activity, with IC50 values of 87, 276, and 440 nm, respectively,
while a weak antiplasmodial activity was observed for
compounds 5 and 13d (880 nm and >866 nm, respec-
tively). As expected, the best three triazole-containing
Cytotoxicity IC50 [mm][g]
antimalarial agents expressed better activity versus the
Table 1. Antimalarial activities and heme crystallization of triazole derivatives.
Compd
Series
no.
IC50 ꢁSD [nm][
CQ-resistant strain by between three- and five-fold,
with 13a expressing excellent activity (IC50 =14.2 nm).
Indeed, this pattern of antimalarial potency by diami-
dines versus CQ-sensitive and CQ-resistant parasites has
been noted previously in the literature.[51] By using the
C2GC03 and C3Dd2 clones, we demonstrate higher poten-
cy for diamidines against the CQ-resistant strain, and
furthermore, our results point to a key role in the mem-
brane transporter protein PfCRT mediating sensitivity to
dicationic drugs.[50]
C2GC03[b]
C3Dd2[c]
[mm]
Hemozoin
CQ[a]
NA
11.0ꢁ5.3
95.63ꢁ27.4 117[e]
18
34
45
9.0
Pentamidine NA
5
13a (OB076)
81.1ꢁ29.5
880.5ꢁ64.0
87.2ꢁ7.8
25.0ꢁ8.32
1090ꢁ84
14.2ꢁ5.4
(2.0)[d][52]
46.6[e]
–
8.10[e]
27.5[f]
86.0[e]
29.2[f]
8.6[e]
1
1
13b (OB125)
13c
1
1
1
276.5ꢁ24.0
440.0ꢁ54.3
866.0ꢁ27.5
79.5ꢁ14.0
61
18
6.3
(4.0)[d][52]
145ꢁ24.1
(3.0)[d][52]
13d
300ꢁ26.1
219.0[e]
Notably in previous work, the IC50 values[3] for com-
pounds 13a–c were in the single-digit nanomolar
region[53]—the reason for the discrepancy may be due
to the fact that, in our assays, the total incubation time
was 48 h, whereas 72 h was used previously. This obser-
(21.0)[d][52]
14a (OB216)
14b (OB135)
20a
20b
25a
25b
27
30a
30b
1
1
2
2
2
2
1
2
2
2
2
>1000
>1000
>1000
>1000
55.2[f]
57.1[f]
ND
ND
ND
ND
ND
ND
ND
>100
>100
9.0
199150ꢁ728
28000ꢁ574
4400ꢁ89
75920ꢁ191
7615ꢁ167
1896ꢁ64
41
26
10
129500ꢁ373
571200ꢁ345
808ꢁ56
30490ꢁ676
165010ꢁ403
731.3ꢁ42
738.2ꢁ21.1
331ꢁ88
vation suggests
a time-dependent mechanism of
706
ND
5.0
action, which may be due to stage-specific target pre-
sentation. We are currently performing experiments to
further investigate this observation.
1856ꢁ95
36a
36b
750ꢁ22
ND
ND
51
115
1040ꢁ43
2200ꢁ38
The results show that for compounds 13a–d, IC50
values are influenced by several factors, with the posi-
tion of the amidine function on the phenyl ring signifi-
cantly influencing antimalarial activity. Substitution at
the para position in both aromatic rings provided the
most potent activity, with meta/para combinations less
effective. Remarkably, insertion of a methylene spacer
between the heterocyclic ring system and the aromatic
ring causes a large decrease in activity (5 vs 13a). The
2,4-substituted analogues 36a and 36b exhibit weak
antiplasmodial activities with IC50 values of 750 and
>1 mm for 20a and 20b, respectively, against CQ-sensitive par-
asites.
[a] Chloroquine (CQ). [b] P. falciparum (CQ-sensitive). [c] P. falciparum (CQ-resistant).
C2GC03 and C3Dd2 were produced by allelic modification of the Pfcrt locus in the
GC03 line and express the GC03 wild-type CQ-sensitive sensitive and the Dd2
mutant CQ-resistant pfcrt alleles, respectively; n=3 for IC50 data. [d] K1 strain.
[e] Against L6 rat myoblast cells; values are the mean of duplicate determinations;
values taken from Ref. [52]. [f] Against human HL60 cell lines; average of two de-
terminations. [g] Hemozoin formation inhibition. ND=not determined. NA=not
applicable.
Fidock laboratory to probe the role of PfCRT in CQ resistance.
The C2GC03 line is a recombinant clone generated from the CQ-
sensitive GC03 line transfected with its own wild-type allele,
and it has been shown to display the full CQ-sensitive pheno-
type. C3Dd2 was produced by allelic modification of the pfcrt
locus in the GC03 line and expresses the Dd2 mutant CQ-re-
sistant pfcrt alleles. Inhibition of hemozoin formation was de-
termined by the ability of the different agents to inhibit the
formation of hemozoin according to the method of Stead
et al.[51] For each compound, the IC50 value was determined
and expressed in micromolar (Table 1).
As observed for 13a–d, compound 36a exhibits greater po-
tency against CQ-resistant parasites with a twofold increase in
activity. Compounds 20a, 20b, and 36b exhibit no antiplasmo-
dial activity (IC50 >1000 nm). Unfortunately, the 4,5-substituted
analogues also exhibit poor antiplasmodial activities. Com-
pound 30a exhibited an IC50 value of 808 nm, and only weak
antiplasmodial activity was observed for derivatives 27, 30b,
25a, and 25b. Analogues 30a and 30b also exhibit poor anti-
plasmodial activity against the CQ-resistant strain with IC50
values of 731 and 738 nm, respectively. Compounds 25a, 25b,
and 27 also exhibit poor antiplasmodial activity (>1000 nm).
Thus, a general trend, observed regardless of the actual substi-
tution pattern, was that an a-atom linkage through the central
triazole heterocycle, either on nitrogen or carbon, resulted in
reduced in vitro activity.
The aim of this work was to build on the previous studies of
Tidwell et al.[53] and compare additional analogues within the
triazole diamidine family with the best hits identified in the
previous study. In addition, we were also keen to: 1) explore
the potential of the most promising analogues in their prodrug
amidoxime form; and 2) to investigate potential correlations
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Triazole-Containing Furamidines as Antimalarials
For the more potent analogues, cytotoxicity data against L6
rat myoblast and HL60 cell lines are given (Table 1). For 13a,
13b, and prodrugs 14a, and 14b low cytotoxicity was ob-
served with good overall in vitro therapeutic indices (>500).
Previous work has shown that pentamidine accumulation
and activity can be blocked by inhibitors of hemoglobin diges-
tion, suggesting that pentamidine activity relies on the release
of ferriprotoporphyrin IX (FPIX) during hemoglobin proteoly-
sis.[25] This phenomena is not restricted to pentamidine, and it
was clearly shown by Stead et al.[25] that antimalarial activity
and accumulation of propamidine, stilbamidine, and berenil
were also antagonized by protease enzyme inhibitors, reinforc-
ing the proposal that FPIX liberated from hemoglobin is a key
target for this class of drug. From Table 1, it is clear that all of
the triazole analogues are quite active at inhibiting hemozoin
formation in this assay, and several are more effective than
pentamidine. However, there is no correlation between the
measured IC50 value for hemozoin inhibition and the measured
antimalarial activity. This is particularly striking for 20a and
25b, both of which are more effective than pentamidine in
this assay yet are effectively inactive as antimalarial agents up
to 1000 nm. Possible explanations for this observation could
be that these molecules have poor penetration through the
NPP pathway, or triazole diamidines have additional parasite
targets apart from FPIX.[54–58]
In vivo antimalarial activities
Based on the in vitro activity profiles, the 1,4-bisaryl analogues
emerged as the most promising subset, and amidoxime pro-
drugs 14a and 14b, prepared as described in Scheme 2, were
selected for evaluation in vivo against the P. vinckei petteri (279
BY) strain that infects mice.[59] Both drugs 13a and 13b
showed potent antimalarial activity with complete clearance of
blood parasitemia in infected mice and complete cure without
recrudescence after ip administration. The doses able to
reduce parasitemia by 50% (ED50 values) were 0.41 mgkgꢀ1
and 0.94 mgkgꢀ1 for 13a (OB076) and 13b (OB125), respec-
tively (see Figure 2a). These activities are of the same order of
magnitude as that of CQ, which has an ED50 value (ip) of
1.1 mgkgꢀ1. In addition, compounds 13a and 13b achieved a
total cure of P. vinckei-infected mice (without recrudescence) at
doses of just 0.8 mgkgꢀ1 and 2 mgkgꢀ1 by ip route. Notably,
we observed a large difference between the ED50 values (ip) of
Figure 2. a) In vivo antimalarial properties of 13a (OB076; left) and 13b (OB125; right) at different doses against P. vinckei-infected mice. Treatment consisted
of one daily ip injection (&) or po (&) for four consecutive days. Parasitemia were monitored at on day 5. Results shown are the mean of at least three mice
per dosage ꢁSEM. ED50 (ip) values are 0.41 mgkgꢀ1 and 0.94 mgkgꢀ1 for 13a and 13b, respectively, and ED50 (po) values are 18 mgkgꢀ1 and <60 mgkgꢀ1, re-
spectively. b) In vivo antimalarial properties of the bioprecursors 14a (OB216; left) and 14b (OB135; right) at different doses against P. vinckei-infected mice.
Treatment consisted in one daily ip (&) or po (&) injection for four consecutive days. Parasitemia were monitored at on day 5. Results shown are the mean
of at least three mice per dosage ꢁSEM. ED50 (ip) values are 5.8 mgkgꢀ1 and 18 mgkgꢀ1 for 14a and 14b, respectively, and ED50 (po) values are 5.1 mgkgꢀ1
and 23 mgkgꢀ1, respectively.
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P. M. O’Neill et al.
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prodrugs compared to the ED50 values (ip) of the parent drugs,
that is, 13a versus 14a, and 13b versus 14b. Using ip adminis-
tration, the prodrugs are 14- to 19-fold less active than the
parent drugs (see Figure 2b).
After po administration of 60 mgkgꢀ1, drug 13b is less
active than drug 13a with a decrease in parasitemia of 76%
and 100%, respectively. Further experiments showed that drug
13a, the most potent drug in vitro and in vivo, has an ED50
value by oral (po) route of 18 mgkgꢀ1 and causes a complete
cure at 30 mgkgꢀ1.
Conclusions
Sixteen different triazole derivatives with different substitutions
across both the triazole ring system and on the aromatic ring
(amidine functional group para or meta in A/C rings) were pre-
pared to probe further SAR in the triazole diamidine family of
potential antimalarial agents. Two compounds in this series,
13a (OB076) and 13b (OB125), have good in vitro activity
against human P. falciparum parasites. Moreover, data generat-
ed in the C2GC03 and C3Dd2 clones demonstrate a role for PfCRT
in mediating sensitivity to diamidine-based drugs, and this is
the subject of additional research in our laboratories. We have
established for the first time the potent in vivo activity of
these compounds against the rodent P. vinckei parasite with ip
administration. Interestingly, after a four-day treatment regime,
these two compounds were able to completely cure mice of
malaria infection. Prodrugs 14a (OB216) and 14b (OB135) are
only weakly active in vitro indicating that the prodrug/drug
conversion does not occur in vitro, however, both of these pro-
drugs are capable of achieving a complete cure at po doses
lower than 30 mgkgꢀ1, with 14a representing a lead com-
pound for further studies.
Prodrug 14b exhibited good activity when administered
orally, with an ED50 value of 23 mgkgꢀ1, similar to the ED50
value observed with ip administration of 18 mgkgꢀ1. The ip/po
ratio, which is an indicator of the oral bioavailability, is 78%, in-
dicating a likely high absorption of the prodrug when adminis-
tered by the oral route. Prodrug 14a showed even better ac-
tivity, with similar ED50 values of 5.8 mgkgꢀ1 and 5.1 mgkgꢀ1
for ip and po, respectively. The ip/po ratio of this prodrug is
about 100%, which might indicate similar absorption of the
prodrug when administered by either the ip and oral route.
The in vivo results clearly demonstrate that of the 1,4-biaryl-
substituted analogues, those with amidine groups in the para
position of the A-ring and meta position of C-ring (i.e., 13b)
are less active than their para-position counterparts (i.e., 13a).
The same result was observed for the prodrugs, indicating that
the position of the proamidine moiety is also important for in-
herent potency. The difference in activity between the meta-
substituted C-ring functionalization and para-substituted C-
ring is also found in vitro for drugs 13a and 13b against
C2GC03 and C3Dd2 strains, and this trend was also seen in previ-
ous work by Tidwell et al.[53] Interestingly, against a CQ-sensi-
tive Nigerian strain, similar IC50 values were observed for 13a
and 13b, demonstrating that strain-dependent sensitivity to
this class of compound is apparent.
Experimental Section
Chemistry
General: All reactions requiring anhydrous conditions were con-
ducted using oven-dried glassware with magnetic stirring under
N2, unless otherwise noted. Needles for the transfer of reagents
were dried at 1208C and allowed to cool in desiccators over P2O5
before use. THF was distilled from benzophenone and CH2Cl2 from
CaH2. Other solvents and reagents were used as obtained from
supplier, unless otherwise noted. Reactions were monitored by TLC
using precoated silica gel 60 plates (Merck), or precoated silica
gel 60 RP-18 F254s plates (Merck). Reaction components were visual-
ized initially under UV (254 nm), and then by treatment with acidic
p-anisaldehyde stain or ninhydrin, followed by gentle heating. Or-
ganic extracts were dried using MgSO4, unless stated otherwise.
Column chromatography was carried out on silica gel pore size
60 ꢁ (40–63 mm) or on Polygoprep 100–50 C18 (Macherey–Nagel).
Melting points (mp) were measured on a Gallenkamp capillary
melting point apparatus and are uncorrected.
The curative dose (CD100) values for prodrugs 14a and 14b
are 10 mgkgꢀ1 and 30 mgkgꢀ1, respectively, in this experiment.
Notably, when administered subcutaneously at 30 mgkgꢀ1
daily for four days, therelated triazole 37, with an in vitro activ-
1H NMR and 13C NMR spectra were recorded at 305 K with a Brꢂker
AV400 spectrometer. Samples were prepared in CDCl3 with the re-
sidual CHCl3 peak used as an internal reference (fixed at 7.26 ppm),
or in [D6]DMSO with the residual DMSO peak used as an internal
1
reference (fixed at 2.50 ppm). The H NMR spectra are reported as
follows: chemical shift (d) in parts per million (ppm) (multiplicity,
relative integral, coupling constant(s) (J) in Hertz (Hz), assignment),
where multiplicity is defined as: m=multiplet, s=singlet, d=dou-
blet, t=triplet, q=quadruplet or a combination thereof). 13C NMR
spectra were conducted using a J-modulated sequence. Samples
were prepared in CDCl3, with the residual solvent central peak of
the triplet used as an internal reference (77.00 ppm), or in
[D6]DMSO with the residual solvent central peak of the septuplet
used as an internal reference (39.50 ppm). Mass spectra (MS) were
recorded on a VG analytical 7070E machine and Fisons TRIO spec-
trometers using electrospray (ESI) and chemical ionization (CI). An-
alytical HPLC chromatographs were recorded on a GIBSON appara-
ity of 0.6 nm against K1 P. falciparum, did not reduce parasite-
mia in a mouse model of P. berghei.[53] We previously demon-
strated that membrane-impermeable diamidines gain access
into the infected red blood cell through an induced pathway
termed the NPP. This pathway is absent in P. berghei-infected
blood cells, and as such, diamidines are inactive in this in vivo
model, underlining that alternative murine species of Plasmo-
dia should be considered for the appropriate assessment of for
cationic drugs as potential antimalarial agents.
2100
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ChemMedChem 2011, 6, 2094 – 2108

Triazole-Containing Furamidines as Antimalarials
tus using a Genesis C18 column (4.6 mmꢃ300 mm, 4.0 mm) and
UV photoiode array detection at 230 and 254 nm. Mobile phases
consisted of mixtures of MeCN (10–85%) and water, with both sol-
vents containing formic acid (80 mm), ammonium formate
(20 mm), and Et3N (15 mm). Flow rates were maintained at
1.5 mLminꢀ1. For every derivative, MeCN was increased linearly
from 10 to 85% over 10 min before re-equilibration.
HRMS (CI+): m/z [Mꢀ2NH2]+ calcd for C17H13N5: 286.1327, found:
286.1321; HPLC: tR =3.89 min (100% area).
1-Azido-4-bromobenzene (7a): A mixture of 4-bromoaniline 6a
(3 g, 17.44 mmol), water (50 mL), and concd HCl (50 mL) was
stirred at 0–58C for 30 min. The amine hydrochloride was then
deazotized by the dropwise addition of aq NaNO2 (1.44 g,
20.93 mmol, in 21 mL H2O). The solution was stirred at 0–58C and
treated with aq NaN3 (2.27 g, 34.88 mmol, in 21 mL H2O). After stir-
ring for 1 h, the reaction mixture was extracted with CH2Cl2 (3ꢃ
150 mL). The combined organic layers were washed with water
and brine, dried over MgSO4, filtered and and concentrated in
vacuo to afford the azide as a yellow oil (3.41 g, 98%): 1H NMR
(400 MHz, CDCl3): d=7.39 (d, 2H, J=8.7 Hz, ArH), 6.83 ppm (d, 2H,
J=8.7 Hz, ArH); 13C NMR (100 MHz, CDCl3): d=139.6, 133.2 (2C),
121.1 (2C), 118.2 ppm; MS (EI+): m/z (%): 199 [M+H]+ (15), 197
(15), 171 [M+HꢀN2]+ (56), 169 (51); HRMS (ESI+): m/z [M+H]+
calcd for C6H5BrN3: 198.9589, found: 198.9594.
4-Azidomethylbenzonitrile (2): A mixture of DMF (60 mL) and
benzonitrile 1 (5.88 g, 30.0 mmol) was treated slowly with NaN3
(3.9 g, 60.0 mmol). After 4 h of stirring at RT, the content of the
flask was poured into water (100 mL). The mixture was extracted
with Et2O (3ꢃ100 mL). The combined organic layers were washed
with water (5ꢃ300 mL) and brine (1ꢃ300 mL), dried over MgSO4,
filtered and concentrated in vacuo to afford 2 as a pale-yellow oil
1
(4.63 g, 98%): H NMR (400 MHz, CDCl3): d=7.66 (d, 2H, J=8.3 Hz,
ArH), 7.45 (d, 2H, J=8.3 Hz, ArH), 4.46 ppm (s, 2H, CH2); 13C NMR
(100 MHz, CDCl3): d=141.2, 132.7 (2C), 129.2 (2C), 118.9, 112.5,
54.4 ppm; MS (ESI+): m/z (%): 159 [M+H]+ (100); HRMS (ESI+):
m/z [M+H]+ calcd for C8H7N4: 159.0592, found: 159.0595.
1-Azido-3-bromobenzene (7b): Product 7b was prepared from
6b (3 g, 17.44 mmol) using the same protocol as described for 7a.
Azide 7b was obtained as a yellow oil (3.34 g, 97%): 1H NMR
(400 MHz, CDCl3): d=7.25 (dd, 1H, J=2.1, 8.1 Hz, ArH), 7.19 (t, 1H,
J=8.1 Hz, ArH), 7.16 (t, 1H, J=2.1 Hz, ArH), 6.94 ppm (dd, 1H, J=
2.1, 8.1 Hz, ArH); 13C NMR (100 MHz, CDCl3): d=143.2, 132.5, 129.6,
124.9, 123.8, 119.3 ppm; MS (ESI+): m/z (%): 199 [M+H]+ (100);
HRMS (ESI+): m/z [M+H]+ calcd for C6H5BrN3: 198.9589, found:
198.9594.
1-(4-Cyanobenzyl)-4-(4-cyanophenyl)-[1,2,3]triazole (4): A solu-
tion of benzonitrile 2 (500 mg, 3.16 mmol) and benzonitrile 3
(402 mg, 3.16 mmol), CuSO4·5H2O (1.5 mg, 0.0057 mmol), ascorbic
acid (2.0 mg, 0.0114 mmol), l-proline (3.0 mg, 0.0227 mmol), and
Na2CO3 (2.5 mg, 0.0227 mmol) in DMSO/water (2 mL, 9:1) was
stirred at 658C overnight. After completion, the mixture was
cooled before adding saturated aq NH4Cl (40 mL), the precipitate
obtained was filtered and washed with distilled H2O (100 mL) and
dried in vacuo. The crude product was purified by column chroma-
tography on silica gel (CHCl3) to give 4 as a white powder
3-Trimethylsilanylethynylbenzonitrile (9): A mixture of 8 (18.0 g,
99.00 mmol), Pd(PPh3)4 (1.39 g, 1.98 mmol), and CuI (754 mg,
3.96 mmol) was slowly flushed with N2 followed by the addition of
dry Et3N (250 mL). The stirring mixture was treated with ethynyltri-
methylsilane (20.55 mL, 148.00 mmol), and the solution turned
dark brown. The mixture was heated to 808C under N2 overnight.
The mixture was cooled, concentrated in vacuo, and Et2O (250 mL)
was added to the residue. The mixture was then filtered and con-
centrated, and the brown crude product was purified by chroma-
tography on silica gel (hexane/EtOAc, 98:2) to afford 9 as a white
1
(890 mg, 99%): mp: 156–1578C; H NMR (400 MHz, CDCl3): d=7.93
(d, 2H, J=8.6 Hz, ArH), 7.84 (s, 1H, CH triazole), 7.71 (d, 2H, J=
8.6 Hz, ArH), 7.70 (d, 2H, J=8.6 Hz, ArH), 7.41 (d, 2H, J=8.6 Hz,
ArH), 5.68 ppm (s, 2H, CH2); 13C NMR (100 MHz, CDCl3): d=147.2,
139.8, 134.9, 133.4 (2C), 133.2 (2C), 128.9 (2C), 126.5 (2C), 121.2,
119.0, 118.4, 113.5, 112.3, 54.1 ppm; MS (CI+): m/z (%): 286 [M+
H]+ (100); HRMS (CI+): m/z [M+H]+ calcd for C17H12N5: 286.1014,
found: 286.1009.
1
solid (17.0 g, 86%): H NMR (400 MHz, CDCl3): d=7.63 (t, 1H, J=
1.4 Hz, ArH), 7.55 (ddd, 1H, J=1.3, 1.4, 7.9 Hz, ArH), 7.47 (ddd, 1H,
J=1.3, 1.4, 7.9 Hz, ArH), 7.31 (t, 1H, J=7.9 Hz, ArH), 0.15 ppm (s,
9H, CH3); 13C NMR (100 MHz, CDCl3): d=136.4, 135.6, 131.7,
128.8,123.0, 116.5, 112.1, 103.2, 99.8, 0.0 ppm (3C); MS (CI+): m/z
(%): 199 [M+H]+ (100); HRMS (CI+): m/z [M]+ calcd for C12H13NSi:
199.0817, found: 199.0821.
1-(4-Amidinobenzyl)-4-(4-amidinophenyl)-[1,2,3]triazole dihydro-
chloride (5): Triazole 4 (1 g, 3.5 mmol) in dry EtOH (40 mL) was
added to a flask, and the mixture was cooled in an ice-water bath.
To saturate the reaction medium with HCl, gaseous HCl was bub-
bled through the solution for 20 min before the flask was sealed.
The mixture was stirred at RT for one week. The reaction mixture
was diluted with dry Et2O (100 mL), and the precipitate was filtered
off under a stream of N2, rinsed with anhyd Et2O and dried in
vacuo to give an unstable yellow powder. The imidate intermedi-
ate was suspended in a mixture of dry NH3 (2m in EtOH, 25 mL)
and dry EtOH (25 mL), and stirred under N2 at RT for 24 h. After
evaporating the solvent, the crude material was solubilized in aq
NaOH (1m, 20 mL) and MeCN (20 mL) to generate the free base.
After stirring for 1 h, the formed precipitate was washed with
MeCN, water and Et2O. A mixture of MeOH and concd HCl (50:50)
was used to give the desired HCl salt as a white solid (790 mg,
3-Ethynylbenzonitrile (10): Benzonitrile 9 (16.5 g, 82.80 mmol) was
dissolved in dry THF (200 mL). This solution was then cooled to
ꢀ208C using an acetone/dry ice bath. TBAI (91.06 mL, 1m in THF,
91.06 mmol) was added, and the mixture was stirred at ꢀ208C for
30 min. Water (250 mL) was added, and the mixture was extracted
with Et2O (3ꢃ250 mL). The combined organic layers were washed
with brine, dried over MgSO4, filtered and concentrated in vacuo.
The crude product was purified by chromatography on silica gel
(hexane/EtOAc, 95:5) to give 10 as a white solid (6.5 g, 62%): mp:
42–448C; 1H NMR (400 MHz, CDCl3): d=7.76 (t, 1H, J=1.4 Hz,
ArH), 7.70 (ddd, 1H, J=1.4, 1.4, 7.9 Hz, ArH), 7.63 (ddd, 1H, J=1.4,
1.4, 7.9 Hz, ArH), 7.50–7.55 (m, 3H, ArH), 7.45 (t, 1H, J=7.9 Hz,
ArH), 3.20 ppm (s, 1H, CH); 13C NMR (100 MHz, CDCl3): d=136.6,
135.9, 132.5, 129.7, 124.1, 118.3, 113.3, 81.6, 80.2 ppm; MS (EI+):
m/z (%): 127 [M]+ (100); HRMS (CI+): m/z [M]+ calcd for 127.0422,
found: 127.0423.
1
55%): mp: >3608C; H NMR (400 MHz, [D6]DMSO): d=9.53 (s, 4H,
NH2 or NH·HCl), 9.36 (s, 4H, NH2 or NH·HCl), 9.00 (s, 1H, CH tria-
zole), 8.09 (d, 2H, J=8.0 Hz, ArH), 7.97 (d, 2H, J=8.0 Hz, ArH), 7.89
(d, 2H, J=8.0 Hz, ArH), 7.58 (d, 2H, J=8.0 Hz, ArH), 5.84 ppm (s,
2H, CH2); 13C NMR (100 MHz, [D6]DMSO): d=165.7, 165.5, 145.7,
141.9, 136.0, 129.4 (2C), 129.1 (2C), 128.7 (2C), 128.2, 127.3, 125.6
(2C), 123.9, 52.9 ppm; MS (CI+): m/z (%): 286 [Mꢀ2NH2]+ (100);
ChemMedChem 2011, 6, 2094 – 2108
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2101

P. M. O’Neill et al.
MED
and dried in vacuo. The crude product was purified by column
chromatography on silica gel (CH2Cl2) to give 12a as a pale-yellow
solid (810 mg, 75%): mp: 250–2528C; 1H NMR (400 MHz,
[D6]DMSO): d=9.64 (s, 1H, CH triazole), 8.17 (m, 4H, ArH), 8.12 (d,
2H, J=8.2 Hz, ArH), 7.99 ppm (d, 2H, J=8.2 Hz, ArH); 13C NMR
(100 MHz, [D6]DMSO): d=146.4, 139.7, 134.8 (2C), 134.7, 133.5
(2C), 126.3 (2C), 121.9, 120.9 (2C), 119.1, 118.4, 111.7, 111.1 ppm; MS
(CI+): m/z (%): 272 [M+H]+ (100); HRMS (CI+): m/z [M+H]+ calcd
for C16H10N5: 272.0936, found: 272.0932.
1-(4-Bromophenyl)-4-(4-cyanophenyl)-[1,2,3]triazole (11a): The
same protocol used for compound 4 was followed to generate
product 11 a, using bromobenzene 7a (2.34 g, 11.80 mmol) and
benzonitrile 3 (1.5 g, 11.80 mmol). Compound 11 a was obtained as
a
pale-yellow solid (3.6 g, 94%): mp: 190–1928C; 1H NMR
(400 MHz, CDCl3): d=8.28 (s, 1H, CH triazole), 8.02 (d, 2H, J=
8.6 Hz, ArH), 7.76 (d, 2H, J=8.6 Hz, ArH), 7.70 ppm (m, 4H, ArH);
13C NMR (100 MHz, CDCl3): d=147.2, 136.1, 134.8, 133.5 (2C), 133.3
(2C), 126.7 (2C), 123.3, 122.4 (2C), 119.0, 119.0, 112.4 ppm; MS
(CI+): m/z (%): 327 (37), 325 [M]+ (38), 247 [M+HꢀBr]+ (100);
HRMS (CI+): m/z [M]+ calcd for C15H10N4Br: 325.0089, found:
325.0081.
1-(3-Cyanophenyl)-4-(4-cyanophenyl)-[1,2,3]triazole (12b): The
same protocol described for compound 12a was used to generate
12b from triazole 11 b (2.32 g, 7.13 mmol). The crude product was
purified by column chromatography on silica gel (9:1 CH2Cl2/
EtOAc) to give 12b as a pale-yellow solid (1.47 g, 76%): mp: 214–
1-(3-Bromophenyl)-4-(4-cyanophenyl)-[1,2,3]triazole (11b): The
same protocol as described for compound 4 was used to generate
11 b from bromobenzene 7b (1.56 g, 7.87 mmol) and benzonitrile
3 (1 g, 7.87 mmol). The crude product was purified by column
chromatography on silica gel (CH2Cl2) to give 11 b as a pale-yellow
powder (2.47 g, 97%): mp: 176–1778C; 1H NMR (400 MHz,
[D6]DMSO): d=9.56 (s, 1H, CH triazole), 8.19 (t, 1H, J=2.0 Hz, ArH),
8.11 (d, 2H, J=8.6 Hz, ArH), 8.01 (ddd, 1H, J=2.0, 2.1, 8.1 Hz, ArH),
7.98 (d, 2H, J=8.6 Hz, ArH), 7.74 (ddd, 1H, J=2.0, 2.1, 8.1 Hz, ArH),
7.61 ppm (t, 1H, J=8.1 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO):
d=145.7, 137.4, 134.4, 133.1 (2C), 131.9, 131.6, 125.8 (2C), 122.5,
122.4, 121.5, 119.0, 118.7, 110.5 ppm; MS (ESI+): m/z (%): 327 [M+
H]+ (100), 325 [M+H]+ (92); HRMS (ESI+): m/z [M]+ calcd for
C15H10N4Br: 325.0089, found: 325.0074.
1
2158C; H NMR (400 MHz, [D6]DMSO): d=9.60 (s, 1H, CH triazole),
8.46 (s, 1H, ArH), 8.33 (d, 1H, J=8.2 Hz, ArH), 8.10 (d, 2H, J=
8.2 Hz, ArH), 8.02 (d, 1H, J=8.2 Hz, ArH), 8.00 (d, 2H, J=8.2 Hz,
ArH), 7.87 ppm (t, 1H, J=8.2 Hz, ArH); 13C NMR (100 MHz,
[D6]DMSO): d=146.3, 137.2, 134.8, 133.6 (2C), 132.9, 131.8, 126.2
(2C), 125.1, 123.9, 122.0, 119.1, 118.1, 113.2, 111.1 ppm; MS (ESI+):
m/z (%): 272 [M+H]+ (100); HRMS (ESI+): m/z [M+H]+ calcd for
C16H10N5: 272.0936, found: 272.0937.
1-(4-Cyanophenyl)-4-(3-cyanophenyl)-[1,2,3]triazole (12c): The
same protocol as described for compound 12a was used to gener-
ate 12c from triazole 11 c (5.5 g, 16.91 mmol). The crude product
was purified by column chromatography on silica gel (9:1 CH2Cl2/
EtOAc) to give 12c as a white solid (3.64 g, 79%): mp: 202–2058C;
1H NMR (400 MHz, [D6]DMSO): d=9.60 (t, 1H, J=1.8 Hz, CH tria-
zole), 8.33 (t, 1H, J=1.6 Hz, ArH), 8.27 (d, 1H, J=7.8 Hz, ArH), 8.16
(m, 4H, ArH), 7.88 (d, 1H, J=7.8 Hz, ArH), 7.74 ppm (t, 1H, J=
7.8 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO): d=146.2, 139.7, 134.8
(2C), 132.3, 131.5, 130.9, 130.1, 129.1, 121.4, 120.8 (2C), 118.8, 118.4,
112.6, 111.7 ppm; MS (CI+): m/z (%): 272 [M+H]+ (100); HRMS
(CI+): m/z [M+H]+ calcd for C16H10N5: 272.0936, found: 272.0930.
1-(4-Bromophenyl)-4-(3-cyanophenyl)-[1,2,3]triazole (11c): The
same protocol as described for compound 4 was used to generate
11 c from bromobenzene 7a (3.9 g, 19.70 mmol) and benzonitrile
10 (2.5 g, 19.70 mmol). The crude product was purified by column
chromatography on silica gel (CH2Cl2) to give 11 c as a white solid
1
(5.95 g, 93%): mp: 164–1678C; H NMR (400 MHz, CDCl3): d=8.26
(s, 1H, CH triazole), 8.18 (t, 1H, J=1.4 Hz, ArH), 8.17 (ddd, 1H, J=
1.4, 1.5, 7.9 Hz, ArH), 7.70 (m, 4H, ArH), 7.66 (ddd, 1H, J=1.4, 1.5,
7.9 Hz, ArH), 7.59 ppm (t, 1H, J=7.9 Hz, ArH); 13C NMR (100 MHz,
CDCl3): d=147.0, 136.1, 133.5 (2C), 132.3, 131.8, 130.4, 130.3, 129.7,
123.3, 122.4 (2C), 118.8, 118.5, 113.7 ppm; MS (ESI+): m/z (%): 327
(100), 325 [M+H]+ (97), 247 [M+HꢀBr]+ (90); HRMS (ESI+): m/z
[M]+ calcd for C15H10N4Br: 325.0089, found: 325.0080.
1,4-Bis-(3-cyanophenyl)-[1,2,3]triazole (12d): The same protocol
as described for compound 12a was used to generate 12d from
triazole 11 d (5.5 g, 16.91 mmol). The crude product was purified
by column chromatography on silica gel (9:1 CH2Cl2/EtOAc) to give
12d as a white solid (3.45 g, 75%): mp: 198–2008C; 1H NMR
(400 MHz, [D6]DMSO): d=9.54 (t, 1H, J=2.0 Hz, CH triazole), 8.42
(t, 1H, J=1.5 Hz, ArH), 8.30 (dd, 1H, J=1.2, 7.7 Hz, ArH), 8.29 (t,
1H, J=1.2 Hz, ArH), 8.24 (dd, 1H, J=1.5, 8.0 Hz, ArH), 8.00 (dd, 1H,
J=1.2, 7.7 Hz, ArH), 7.86 (dd, 1H, J=1.5, 8.0 Hz, ArH), 7.85 (dt, 1H,
J=1.5, 7.9 Hz, ArH), 7.73 ppm (dt, 1H, J=1.2, 7.9 Hz, ArH); 13C NMR
(100 MHz, [D6]DMSO): d=146.0, 137.2, 132.8, 132.2, 131.8, 131.5,
130.9, 130.0, 129.0, 124.9, 123.7, 121.4, 118.8, 118.1, 113.2,
112.6 ppm; MS (ESI+): m/z (%): 272 [M+H]+ (100); HRMS (ESI+):
m/z [M+H]+ calcd for C16H10N5: 272.0936, found: 272.0930.
1-(3-Bromophenyl)-4-(3-cyanophenyl)-[1,2,3]triazole (11d): The
same protocol as described for compound 4 was used to generate
11 d from bromobenzene 7b (3.9 g, 19.70 mmol) and benzonitrile
10 (2.5 g, 19.70 mmol). The crude was purified by column chroma-
tography on silica gel (CH2Cl2) to give the product as a white solid
1
(6.13 g, 96%): mp: 162–1658C; H NMR (400 MHz, CDCl3): d=8.29
(s, 1H, CH triazole), 8.17–8.19 (m, 1H, ArH), 8.17 (ddd, 1H, J=1.4,
1.6, 7.8 Hz, ArH), 8.00 (t, 1H, J=2.0 Hz, ArH), 7.76 (ddd, 1H, J=1.9,
2.0, 8.2 Hz, ArH), 7.66 (ddd, 1H, J=1.4, 1.6, 7.8 Hz, ArH), 7.62 (ddd,
1H, J=1.9, 2.0, 8.2 Hz, ArH), 7.60 (dt, 1H, J=1.6, 7.8 Hz, ArH),
7.45 ppm (t, 1H, J=8.2 Hz, ArH); 13C NMR (100 MHz, CDCl3): d=
146.9, 138.1, 132.6, 132.3, 131.8, 131.7, 130.4, 130.3, 129.7, 124.0,
123.9, 119.4, 118.8, 118.6, 113.7 ppm; MS (ESI+): m/z (%): 327 (100),
325 [M+H]+ (98); HRMS (ESI+): m/z [M]+ calcd for C15H10N4Br:
325.0089, found: 325.0079.
1,4-Bis-(4-benzamidine)-1H-[1,2,3]triazole
dihydrochloride
(13a):[53] The same protocol as described for the synthesis of com-
pound 5 was used to generate amidine 13a from triazole 12a
(1 g, 3.69 mmol). A mixture of MeOH and and concd HCl (1:1) was
used to give the desired HCl salt as a white solid (1.23 g, 84%):
1
mp: 357–3598C; H NMR (400 MHz, [D6]DMSO): d=9.84 (s, 1H, CH
triazole), 9.59 (s, 4H, NH·HCl or NH2), 9.45 (s, 4H, NH·HCl or NH2),
8.25 (d, 2H, J=8.2 Hz, ArH), 8.18 (d, 2H, J=8.9 Hz, ArH), 8.15 (d,
2H, J=8.9 Hz, ArH), 8.03 ppm (d, 2H, J=8.2 Hz, ArH); 13C NMR
(100 MHz, [D6]DMSO): d=165.5, 165.0, 146.6, 140.2, 135.3, 130.7
(2C), 129.5 (2C), 128.2, 127.7, 125.8 (2C), 122.0, 120.2 ppm (2C); MS
(ESI+): m/z (%): 306 [M+H]+ (100), 153.5 [M+H]2+ (55); HRMS
1,4-Bis-(4-cyanophenyl)-[1,2,3]triazole (12a): A suspension of tria-
zole 11 a (1.3 g, 4.0 mmol) and CuCN (716 mg, 8.0 mmol) in dry
DMF (35 mL) was heated to reflux under N2 for 24 h. The cooled
reaction was poured into a mixture of water (50 mL) and saturated
aq NH4Cl (30 mL). The suspension was filtered, washed with water,
2102
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2094 – 2108

Triazole-Containing Furamidines as Antimalarials
(ESI+): m/z [M+H]+ calcd for C16H16N7: 306.1467, found: 306.1475;
HPLC: tR =5.03 min (98.1% area).
and the mixture was stirred at RT overnight. Then, the solvent was
evaporated in vacuo, and the crude product was washed with
water and Et2O. The residue was purified by column chromatogra-
phy on silica gel (hexane/EtOAc, 6:4!5:5) to afford the free ami-
doxime as a white solid. This solid was solubilized in MeOH/concd
HCl (1:1) to generate the dihydrochloride salt as a white solid
1-(3-Amidinophenyl)-4-(4-amidinophenyl)-[1,2,3]triazole
dihydrochloride (13b).[53] The same protocol as described for the
synthesis of compound 5 was used to generate amidine 13b from
triazole 12b (900 mg, 3.32 mmol). Purification by column chroma-
tography on reversed phase silica gel (water) gave the product as
a white solid (1.01 g, 73%): mp: 330–3328C; 1H NMR (400 MHz,
[D6]DMSO): d=9.89 (s, 1H, CH triazole), 9.76 (s, 2H, NH·HCl or NH2),
9.52 (s, 4H, NH·HCl or NH2), 9.32 (s, 2H, NH·HCl or NH2), 8.62 (s, 1H,
ArH), 8.36 (d, 1H, J=8.0 Hz, ArH), 8.18 (d, 2H, J=8.4 Hz, ArH), 8.03
(d, 2H, J=8.4 Hz, ArH), 8.01 (d, 1H, J=8.0 Hz, ArH), 7.91 ppm (t,
1H, J=8.0 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO): d=165.5,
165.0, 146.5, 136.9, 135.4, 131.2, 130.0, 129.5 (2C), 128.8, 127.8,
125.8 (2C), 125.2, 122.1, 120.4 ppm; MS (ESI+): m/z (%): 306 [M+
H]+ (100); HRMS (ESI+): m/z [M+H]+ calcd for C16H16N7: 306.1467,
found: 306.1455; HPLC: tR =4.66 min (100% area).
1
(1.34 g, 35% over two steps): mp: 210–2138C; H NMR (400 MHz,
[D6]DMSO): d=9.73 (s, 1H, CH triazole), 8.15 (m, 4H, ArH), 8.04 (d,
2H, J=8.4 Hz, ArH), 7.95 (d, 2H, J=8.4 Hz, ArH), 3.89 (s, 3H, CH3),
3.87 ppm (s, 3H, CH3); 13C NMR (100 MHz, [D6]DMSO): d=157.6
(2C), 146.7, 139.0, 138.9, 134.5, 129.5 (2C), 129.1 (2C), 125.8 (2C),
121.5 (2C), 120.1 (2C), 63.6, 62.9 ppm; MS (ESI+): m/z (%): 366 [M+
H]+ (100); HRMS (ESI+): m/z [M+H]+ calcd for C18H20N7O2:
366.1678, found: 366.1678; HPLC: tR =8.48 min (100% area).
1-(3-N-Methoxyamidinophenyl)-4-(4-N-methoxyamidinophenyl)-
[1,2,3]triazole dihydrochloride (14b): The same protocol as de-
scribed for compound 14a was used to generate product 14b
from triazole 12b (1.70 g, 6.27 mmol). This solid was solubilised in
MeOH/concd HCl (1:1) to generate the dihydrochloride salt as a
1-(4-Amidinophenyl)-4-(3-amidinophenyl)-[1,2,3]triazole
dihydrochloride (13c):[53] The same protocol as described for the
synthesis of compound 5 was used to generate imidate 13c from
triazole 12c (1 g, 3.69 mmol). Purification by column chromatogra-
phy on reversed phase silica gel (water) gave the product as a
white solid (1.08 g, 65%): mp: 266–2688C; 1H NMR (400 MHz,
[D6]DMSO): d=9.83 (s, 1H, CH triazole), 9.66 (s, 4H, NH2 and
NH·HCl), 9.42 (s, 4H, NH2 and NH·HCl), 8.56 (s, 1H, ArH), 8.30 (d,
1H, J=7.8 Hz, ArH), 8.25 (d, 2H, J=8.7 Hz, ArH), 8.16 (d, 2H, J=
8.7 Hz, ArH), 7.89 (d, 1H, J=7.8 Hz, ArH), 7.78 ppm (t, 1H, J=
7.8 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO): d=165.9, 165.0, 146.8,
140.3, 131.0, 130.7 (2C), 130.6, 130.3, 129.3, 128.3 (2C), 125.4, 121.4,
120.2 ppm (2C); MS (ESI+): m/z (%): 306 [M+H]+ (100), 153.5 [M+
H]2+ (20); HRMS (ESI+): m/z [M+H]+ calcd for C16H16N7: 306.1467,
found: 306.1470; HPLC: tR =4.74 min (100% area).
1
white solid (1.28 g, 40% over two steps): mp: 180–1838C; H NMR
(400 MHz, [D6]DMSO): d=9.82 (s, 1H, CH triazole), 8.50 (s, 1H,
ArH), 8.24 (d, 1H, J=7.8 Hz, ArH), 8.16 (d, 2H, J=8.0 Hz, ArH), 7.98
(d, 2H, J=8.0 Hz, ArH), 7.95 (d, 1H, J=7.8 Hz, ArH), 7.82 (t, 1H, J=
8.0 Hz, ArH), 3.91 (s, 3H, CH3), 3.90 ppm (s, 3H, CH3); 13C NMR
(100 MHz, [D6]DMSO): d=156.4, 153.6, 144.5, 134.8, 132.9, 128.9,
127.7, 127.4, 126.1 (2C), 123.8, 123.5 (2C), 121.7, 119.9, 117.6, 62.0,
61.2 ppm; MS (ESI+): m/z (%): 366 [M+H]+ (100); HRMS (ESI+):
m/z [M+H]+ calcd for C18H20N7O2: 366.1678, found: 366.1674;
HPLC: tR =8.99 min (98.8% area).
1-Azidomethyl-4-bromobenzene (16): The same protocol as de-
scribed for compound 2 was used to generate product 16 from 15
(2 g, 8.0 mmol). The combined organic extracts after the workup
were concentrated in vacuo to afford the product as a pale-yellow
oil (1.64 g, 97%): 1H NMR (400 MHz, CDCl3): d=7.49 (d, 2H, J=
8.4 Hz, ArH), 7.17 (d, 2H, J=8.4 Hz, ArH), 4.28 ppm (s, 2H, CH2);
13C NMR (100 MHz, CDCl3): d=134.8, 132.4 (2C), 130.3 (2C), 122.8,
54.5 ppm; MS (ESI+): m/z (%): 213 [M+H]+ (100); HRMS (ESI+):
m/z [M+H]+ calcd for C7H7BrN3: 212.9745, found: 212.9738.
1,4-Bis-(3-amidinophenyl)-[1,2,3]triazole
dihydrochloride
(13d):[53] The same protocol as described for the synthesis of com-
pound 5 was used to generate imidate 13d from triazole 12d (1 g,
3.69 mmol). Purification by column chromatography on reversed
phase silica gel (water) gave the product as a white solid (1.15 g,
1
72%): mp: >3608C; H NMR (400 MHz, [D6]DMSO): d=9.76 (t, 1H,
1,5-Bis-(4-bromophenyl)-1H-[1,2,3]triazole (18a): A solution of 17
(0.9 g, 4.97 mmol) in dry THF (3 mL) was treated with EtMgBr (1m
in THF, 4.97 mL, 4.97 mmol) at RT under N2. The reaction mixture
was heated to 508C for 15 min. After cooling to RT, a solution of
7a (1.0 g, 4.97 mmol) in dry THF (3 mL) was added. The resulting
solution was stirred at RT for 30 min, and then heated to 508C for
1 h before quenching with saturated aq NH4Cl (20 mL). The layers
were separated, and the aqueous layer was extracted CH2Cl2 (3ꢃ
30 mL). The combined organic layers were washed with water and
brine, dried over MgSO4, filtered and concentrated in vacuo. Purifi-
cation by column chromatography on silica gel (24:1 CH2Cl2/
EtOAc) gave the product as a yellow solid (1.81 g, 96%): mp: 142–
J=1.2 Hz, CH triazole), 9.51 (s, 8H, NH2 and NH·HCl), 8.54 (t, 1H,
J=1.2 Hz, ArH), 8.47 (t, 1H, J=1.2 Hz, ArH), 8.34 (d, 1H, J=7.9 Hz,
ArH), 8.27 (dd, 1H, J=1.2, 7.8 Hz, ArH), 7.99 (d, 1H, J=7.9 Hz, ArH),
7.91 (t, 1H, J=7.9 Hz, ArH), 7.85 (dd, 1H, J=1.2, 7.8 Hz, ArH),
7.78 ppm (t, 1H, J=7.8 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO):
d=165.9, 165.0, 146.7, 136.9, 131.3, 131.1, 130.6, 130.3, 130.2,
129.5, 128.8, 128.3, 125.2, 125.1, 121.4, 120.3 ppm; MS (ESI+): m/z
(%): 306 [M+H]+ (100), 153.5 [M+H]2+ (14); HRMS (ESI+): m/z
[M+H]+ calcd for C16H16N7: 306.1467, found: 306.1475; HPLC: tR =
4.72 min (99.7% area).
1,4-Bis-(4-N-methoxyamidinophenyl)-[1,2,3]triazole
dihydro-
1
1448C; H NMR (400 MHz, CDCl3): d=7.86 (s, 1H, CH triazole), 7.59
chloride (14a): NH2OH·HCl (1.57 g, 22.56 mmol) was suspended in
abs EtOH (50 mL), K2CO3 (2.08 g, 15.04 mmol) was added, and the
solution was stirred at RT for 1 h. Triazole 12a (2.04 g, 7.52 mmol)
was added to the solution, and the reaction mixture was stirred at
reflux (908C) for 24 h. After cooling, the mixture was filtered, and
the filtrate was concentrated in vacuo. The solid obtained was
washed with water and Et2O. The crude product was then dis-
solved in 1,4-dioxane (80 mL), and the solution was cooled to 08C.
A solution of 2n NaOH (150 mL) was added slowly, followed by
dropwise addition of Me2SO4 (5.7 g, 45.12 mmol) in 1,4-dioxane
(40 mL). After addition of the reagents, the ice bath was removed,
(d, 2H, J=8.5 Hz, ArH), 7.52 (d, 2H, J=8.4 Hz, ArH), 7.24 (d, 2H, J=
8.5 Hz, ArH), 7.10 ppm (d, 2H, J=8.4 Hz, ArH); 13C NMR (100 MHz,
CDCl3): d=137.1, 135.7, 134.1, 133.2 (2C), 132.8 (2C), 130.5 (2C),
126.9 (2C), 125.8, 124.4, 123.9 ppm; MS (ESI+): m/z (%): 400 (45),
402 [M+Na]+ (100), 404 (40), 434 [M+Na+MeOH]+ (26); HRMS
(ESI+): m/z [M+Na]+ calcd for C14H9Br2NaN3: 401.9040, found:
401.9029.
1-(4-Bromobenzyl)-5-(4-bromophenyl)-[1,2,3]triazole (18b): The
same protocol as described for compound 18a was used to gener-
ChemMedChem 2011, 6, 2094 – 2108
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2103

P. M. O’Neill et al.
MED
ate product 18b from 17 (0.9 g, 4.97 mmol) and 4-bromobenzene
16 (1.05 g, 4.97 mmol). Purification by column chromatography on
silica gel (CH2Cl2/hexane, 9:1, then 100% CH2Cl2) gave the product
[D6]DMSO): d=165.5, 165.2, 141.8, 136.8, 134.6, 131.6, 129.3 (2C),
129.0 (2C), 129.0 (2C), 128.9, 127.9, 127.6 (2C), 51.5 ppm; MS
(ESI+): m/z (%): 382 [M+Cu]+ (100), 320 [M+H]+ (14), 160.5 [M+
2H]2+ (24); HRMS (ESI+): m/z [M+H+Cu]+ calcd for C17H17N7Cu:
382.0841, found: 382.0826; HPLC: tR =3.12 min (99.3% area).
1
as a yellow solid (1.73 g, 89%): H NMR (400 MHz, CDCl3): d=7.73
(s, 1H, CH triazole), 7.57 (d, 2H, J=8.2 Hz, ArH), 7.42 (d, 2H, J=
8.4 Hz, ArH), 7.10 (d, 2H, J=8.4 Hz, ArH), 6.95 (d, 2H, J=8.2 Hz,
ArH), 5.48 ppm (s, 2H, CH2); 13C NMR (100 MHz, CDCl3): d=137.5,
134.6, 133.9, 132.8 (2C), 132.5 (2C), 130.8 (2C), 129.2 (2C), 126.0,
124.6, 122.9, 51.7 ppm; MS (ESI+): m/z (%): 416 [M+Na]+ (100);
HRMS (ESI+): m/z [M+Na]+ calcd for C15H11Br2NaN3: 414.9197,
found: 414.9209.
4,4’-Dicyanostilbene (22): A flask was charged with compounds 3
(1 g, 7.87 mmol) and 21 (1.43 g, 7.87 mmol), PPh3 (41 mg,
0.16 mmol), Pd(PPh3)2Cl2 (110 mg, 0.16 mmol), and dry Et3N
(30 mL). The mixture was stirred for 10 min with Ar bubbling
through the solution, and then CuI (60 mg, 0.31 mmol) was added.
The mixture was then heated to reflux with stirring under Ar for
2 h. Following the removal of Et3N in vacuo, CHCl3 was added, and
the solution was filtered, washed with 15% aq K2CO3, water, and
brine, dried over MgSO4, filtered, and the CHCl3 was removed. The
crude product was purified by column chromatography on silica
gel (hexane/CH2Cl2, 6:4!5:5!4:6!3:7) to give the product as a
white solid (1.63 g, 91%): mp: 249–2518C; 1H NMR (400 MHz,
[D6]DMSO): d=7.68 (d, 4H, J=8.3 Hz, ArH), 7.64 ppm (d, 4H, J=
8.3 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO): d=132.7 (4C), 132.6
(4C), 127.5 (2C), 118.7 (2C), 112.8 (2C), 92.0 ppm (2C); MS (EI+): m/z
(%): 228 [M]+ (100); HRMS (EI+): m/z [M]+ calcd for C16H8N2:
228.0687, found: 228.0695.
1,5-Bis-(4-cyanophenyl)-1H-[1,2,3]triazole (19a): The same proto-
col as described for compound 12a was used to generate product
19a from triazole 18a (1.65 g, 4.35 mmol). For this reaction,
4.0 equiv of CuCN (1.56 g, 17.4 mmol) were used. Purification by
column chromatography on silica gel (8:2 CH2Cl2/EtOAc) gave the
product as a pale-yellow solid (560 mg, 45%): mp: 176–1788C;
1H NMR (400 MHz, CDCl3): d=7.96 (s, 1H, CH triazole), 7.79 (d, 2H,
J=8.6 Hz, ArH), 7.72 (d, 2H, J=8.3 Hz, ArH), 7.51 (d, 2H, J=8.6 Hz,
ArH), 7.37 ppm (d, 2H, J=8.3 Hz, ArH),); 13C NMR (100 MHz, CDCl3):
d=139.7, 136.5, 135.1, 134.1 (2C), 133.4 (2C), 131.0, 129.6 (2C),
125.8 (2C), 118.1, 117.7, 114.2, 114.1 ppm; MS (ESI+): m/z (%): 272
[M+H]+ (100); HRMS (ESI+): m/z [M+H]+ calcd for C16H10N5:
272.0936, found: 272.0928.
Azidobenzene (23a): The same protocol as described for com-
pound 7a was used to generate product 23a from aniline (5 g,
53.70 mmol). The product was obtained after concentrating the or-
ganic extracts in vacuo as an orange oil (5.85 g, 91.5%): 1H NMR
(400 MHz, CDCl3): d=7.33 (t, 2H, J=8.2 Hz, ArH), 7.12 (t, 1H, J=
8.2 Hz, ArH), 7.01 ppm (d, 2H, J=8.2 Hz, ArH); 13C NMR (100 MHz,
CDCl3): d=140.5, 130.2 (2C), 125.3, 119.5 ppm (2C).
1-(4-Cyanobenzyl)-5-(4-cyanophenyl)-[1,2,3]triazole (19b): The
same protocol as described for compound 12a was used to gener-
ate product 19b from triazole 18b (1.64 g, 4.17 mmol). For this re-
action, 4.0 equiv of CuCN were used. Purification by column chro-
matography on silica gel (CH2Cl2/EtOAc, 10:0!9:1!8:2) gave the
product as a pale-yellow solid (700 mg, 62%): 1H NMR (400 MHz,
[D6]DMSO): d=8.14 (s, 1H, CH triazole), 7.96 (d, 2H, J=8.0 Hz,
ArH), 7.78 (d, 2H, J=8.0 Hz, ArH), 7.69 (d, 2H, J=8.0 Hz, ArH), 7.18
(d, 2H, J=8.0 Hz, ArH), 5.88 ppm (s, 2H, CH2); 13C NMR (100 MHz,
[D6]DMSO): d=141.5, 136.8, 134.6, 133.3 (2C), 133.1 (2C), 131.2,
129.7 (2C), 128.3 (2C), 118.8, 118.7, 112.4, 111.1, 51.4 ppm; MS
(ESI+): m/z (%): 286 [M+H]+ (100); HRMS (ESI+): m/z [M+H]+
calcd for C17H12N5: 286.1014, found: 286.1021.
Azidomethylbenzene (23b): The same protocol as described for
compound 2 was used to generate product 23b from benzylbro-
mide (2 g, 11.7 mmol). The product was obtained as a pale-yellow
oil after concentrating the organic extracts in vacuo. (1.48 g, 95%):
1H NMR (400 MHz, CDCl3): d=7.29–7.41 (m, 5H, ArH), 4.32 ppm (s,
2H, CH2); 13C NMR (100 MHz, CDCl3): d=135.8, 129.3 (2C), 128.7,
128.7 (2C), 55.2 ppm.
1,5-Bis-(4-amidinophenyl)-1H-[1,2,3]triazole
dihydrochloride
1-Phenyl-4,5-bis-(4-cyanophenyl)-[1,2,3]triazole (24a): A solution
of 23a (522 mg, 4.38 mmol), 22 (1 g, 4.38 mmol), CuSO4·5H2O
(55 mg, 0.22 mmol), ascorbic acid (77 mg, 0.44 mmol), l-proline
(101 mg, 0.88 mmol) and Na2CO3 (93 mg, 0.88 mmol) in DMSO/
water (9:1, 8 mL) was stirred at 1208C overnight (15 h). The mix-
ture was cooled, and a saturated aq NH4Cl was added (100 mL).
The suspension was filtered, washed with water, and dried in
vacuo. The crude product was purified by column chromatography
on silica gel (CH2Cl2/hexane, 9:1, then 100% CH2Cl2) to give the
product as a brown pale solid (1.18 g, 77%): 1H NMR (400 MHz,
CDCl3): d=7.70 (d, 2H, J=8.4 Hz, ArH), 7.67 (d, 2H, J=8.8 Hz, ArH),
7.64 (d, 2H, J=8.8 Hz, ArH), 7.43–7.49 (m, 3H, ArH), 7.33 (d, 2H, J=
8.4 Hz, ArH), 7.27 ppm (dt, 2H, J=1.8, 6.7 Hz, ArH); 13C NMR
(100 MHz, CDCl3): d=144.0, 136.0, 135.0, 133.5 (2C), 133.3, 133.0
(2C), 132.2, 131.2 (2C), 130.3, 130.1 (2C), 128.2 (2C), 125.6 (2C),
118.9, 118.1, 114.4, 112.4 ppm; MS (ESI+): m/z (%): 370 [M+Na]+
(100); HRMS (ESI+): m/z [M+Na]+ calcd for C22H13N5Na: 370.1069,
found: 370.1062.
(20a): The same protocol as described for the synthesis of com-
pound 5 was used to generate amidine 20a from triazole 19a
(650 mg, 2.40 mmol). Purification by column chromatography on
reversed phase silica gel (water) gave the product as a white solid
1
(520 mg, 55%): mp: 278–2818C; H NMR (400 MHz, [D6]DMSO): d=
9.60 (s, 4H, NH·HCl and NH2), 9.43 (s, 4H, NH·HCl and NH2), 8.33 (t,
1H, J=0.8 Hz, CH triazole), 8.04 (d, 2H, J=8.3 Hz, ArH), 7.93 (d, 2H,
J=8.1 Hz, ArH), 7.72 (d, 2H, J=8.1 Hz, ArH), 7.56 ppm (d, 1H, J=
8.3 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO): d=165.1, 165.1, 140.2,
137.1, 134.8, 131.5, 130.2 (2C), 129.5, 129.4 (2C), 129.1 (2C), 128.9,
126.5 ppm (2C); MS (ESI+): m/z (%): 306 [M+H]+ (100); HRMS
(ESI+): m/z [M+H]+ calcd for C16H16N7: 306.1467, found: 306.1456;
HPLC: tR =3.07 min (100% area).
1-(4-Amidinobenzyl)-5-(4-amidinophenyl)-[1,2,3]triazole dihydro-
chloride (20b): The same protocol as described for the synthesis
of compound 5 was used to generate imidate 20b from triazole
19b (450 mg, 1.66 mmol). Purification by column chromatography
on reversed phase silica gel (water) gave the product as a white
solid (370 mg, 46%): mp: 252–2558C; 1H NMR (400 MHz,
[D6]DMSO): d=9.46 (s, 8H, NH2 and NH·HCl), 8.18 (s, 1H, CH tria-
zole), 7.98 (d, 2H, J=8.1 Hz, ArH), 7.79 (d, 4H, J=8.1 Hz, ArH), 7.21
(d, 2H, J=8.1 Hz, ArH), 5.93 ppm (s, 2H, CH2); 13C NMR (100 MHz,
1-Benzyl-4,5-bis-(4-cyanophenyl)-[1,2,3]triazole (24b): The same
protocol as described for compound 24a was used to generate
product 24b from 23b (583 mg, 4.38 mmol) and 22 (1 g,
4.38 mmol). Purification by column chromatography on silica gel
(CH2Cl2) gave the product as a pale-yellow solid (950 mg, 60%):
2104
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2094 – 2108

Triazole-Containing Furamidines as Antimalarials
mp: 178–1808C; 1H NMR (400 MHz, CDCl3): d=7.74 (d, 2H, J=
8.2 Hz, ArH), 7.60 (d, 2H, J=8.5 Hz, ArH), 7.56 (d, 2H, J=8.5 Hz,
ArH), 7.23–7.32 (m, 5H, ArH), 6.98 (d, 2H, J=8.2 Hz, ArH), 5.45 ppm
(s, 2H, CH2); 13C NMR (100 MHz, CDCl3): d=143.9, 135.1, 134.8,
133.5 (2C), 132.9 (2C), 132.4, 131.2 (2C), 129.4 (2C), 129.1 (2C), 127.7
(2C), 127.5 (2C), 118.9, 118.1, 114.7, 112.1, 53.1 ppm; MS (ESI+): m/z
(%): 362 [M+H]+ (100); HRMS (ESI+): m/z [M+H]+ calcd for
C23H16N5: 362.1406, found: 362.1409.
[M+2H]2+ (26); HRMS (ESI+): m/z [M+H]+ calcd for C16H16N7:
306.1467, found: 306.1452; HPLC: tR =3.45 min (100% area).
2-Phenyl-4,5-bis-(4-cyanophenyl)-[1,2,3]triazole (28): A flask was
charged with Fe(acac)3 (453 mg, 1.28 mmol), CuO (61 mg,
0.43 mmol), triazole 26 (1.16 g, 4.28 mmol), iodobenzene (1.31 g,
6.41 mmol), and Cs2CO3 (2.79 g, 8.55 mmol). Then, dry DMF (12 mL)
was added, and the flask was closed and heated to 908C for 30 h.
After cooling the mixture to RT, dilution with CH2Cl2 and subse-
quent filtration followed. The filtrate was washed with water (5ꢃ
20 mL), and the organic layer was dried over MgSO4, filtered and
concentrated in vacuo. Purification by column chromatography on
silica gel (CH2Cl2/hexane, 5:5!6:4!7:3!8:2) gave the product as
1-Phenyl-4,5-bis-(4-amidinophenyl)-[1,2,3]triazole dihydrochlor-
ide (25a): The same protocol as described for the synthesis of
compound 5 was used to generate amidine 25a from triazole 24a
(1 g, 2.86 mmol). Purification by column chromatography on re-
versed phase silica gel (water) gave the product as a white solid
1
a white powder (1.33 g, 90%): mp: 169–1708C; H NMR (400 MHz,
1
(716 mg, 53%): mp: 245–2508C; H NMR (400 MHz, [D6]DMSO): d=
CDCl3): d=8.17 (dd, 2H, J=1.2, 8.7 Hz, ArH), 7.74 (m, 8H, ArH),
7.54 (t, 2H, J=7.3 Hz, ArH), 7.43 ppm (tt, 1H, J=1.2, 7.3 Hz, ArH);
13C NMR (100 MHz, CDCl3): d=144.9 (2C), 139.6, 135.1 (2C), 133.1
(4C), 129.9 (2C), 129.3 (4C), 128.8 (2C), 119.4 (2C), 118.8, 113.2 ppm
(2C); MS (CI+): m/z (%): 370 [M+Na]+ (100); HRMS (CI+): m/z
[M+Na]+ calcd for C22H13N5Na: 370.1069, found: 370.1065.
9.47 (s, 8H, NH2 and NH·HCl), 7.95 (d, 2H, J=8.4 Hz, ArH), 7.89 (d,
2H, J=8.4 Hz, ArH), 7.69 (d, 4H, J=8.4 Hz, ArH), 7.46–7.53 ppm (m,
5H, ArH); 13C NMR (100 MHz, [D6]DMSO): d=165.5, 165.2, 143.2,
135.9, 135.7, 134.7, 132.5, 131.4 (2C), 130.4, 129.9 (2C), 129.5, 129.2
(2C), 129.2 (2C), 127.8, 127.2 (2C), 126.4 ppm (2C); MS (ESI+): m/z
(%): 382 [M+H]+ (16), 191.5 [M+H]2+ (100); HRMS (ESI+): m/z
[M+H]+ calcd for C22H20N7: 382.1780, found: 382.1769; HPLC: tR =
6.57 min (100% area).
2-Benzyl-4,5-(cyanophenyl)-[1,2,3]triazole (29): A solution of tria-
zole 26 (750 mg, 2.76 mmol) in dry acetone (75 mL) was treated
with K2CO3 (1.91 g, 13.8 mmol), BnCl (1.75 g, 13.8 mmol), and TBAI
(102 mg, 10% mol). The reaction mixture was heated to reflux
under N2 overnight. Thereafter, the reaction mixture was concen-
trated in vacuo. Purification by column chromatography on silica
gel (CH2Cl2/hexane, 7:3!8:2!9:1,then 100% CH2Cl2) gave the
1-Benzyl-4,5-bis-(4-amidinophenyl)-[1,2,3]triazole dihydrochlor-
ide (25b): The same protocol as described for the synthesis of
compound 5 was used to generate amidine 25b from triazole 24b
(860 mg, 2.38 mmol). Purification by column chromatography on
reversed phase silica gel (water) gave the product as a white solid
1
product as a white solid (760 mg, 76%): H NMR (400 MHz, CDCl3):
1
(430 mg, 36%): mp: 218–2208C; H NMR (400 MHz, [D6]DMSO): d=
d=7.67 (d, 4H, J=8.3 Hz, ArH), 7.63 (d, 4H, J=8.3 Hz, ArH), 7.46
(dd, 2H, J=1.3, 7.3 Hz, ArH), 7.40 (t, 1H, J=7.3 Hz, ArH), 7.38 (t,
2H, J=7.3 Hz, ArH), 5.66 ppm (s, 2H, CH2); 13C NMR (100 MHz,
CDCl3): d=143.9 (2C), 135.4 (2C), 134.8, 133.3 (4C), 129.4 (2C),
129.2 (5C), 128.8 (2C), 118.8 (2C), 112.9 (2C), 59.7 ppm; MS (ESI+):
m/z (%): 384 [M+Na]+ (100); HRMS (ESI+): m/z [M+Na]+ calcd for
C23H15N5Na: 384.1225, found: 384.1222.
9.60 (s, 2H, NH2 or NH·HCl), 9.42 (s, 2H, NH2 or NH·HCl), 9.36 (s, 2H,
NH2 or NH·HCl), 9.18 (s, 2H, NH2 or NH·HCl), 8.01 (d, 2H, J=8.1 Hz,
ArH), 7.81 (d, 2H, J=8.1 Hz, ArH), 7.67 (d, 2H, J=8.1 Hz, ArH), 7.63
(d, 2H, J=8.1 Hz, ArH), 7.29 (m, 3H, ArH), 7.01 (m, 2H, ArH),
5.53 ppm (s, 2H, CH2); 13C NMR (100 MHz, [D6]DMSO): d=165.4,
165.2, 143.0, 135.9, 135.5, 134.3, 132.4, 131.1 (2C), 129.7, 129.4 (2C),
129.1 (2C), 129.0 (2C), 128.5, 127.7 (2C), 127.6, 127.0 (2C),
52.0 ppm; MS (ESI+): m/z (%): 396 [M+H]+ (30), 198.6 [M+H]2+
(100); HRMS (ESI+): m/z [M+H]+ calcd for C23H22N7: 396.1937,
found: 396.1938; HPLC: tR =6.82 min (98.4% area).
2-Phenyl-4,5-bis-(4-amidinophenyl)-[1,2,3]triazole dihydrochlor-
ide (30a): The same protocol as described for the synthesis of
compound 5 was used to generate amidine 30a from triazole 28
(1 g, 2.88 mmol). Purification by column chromatography on re-
versed phase silica gel (water) gave the product as a white solid
(1.2 g, 74%): mp: 298–3008C; 1H NMR (400 MHz, [D6]DMSO): d=
9.63 (s, 4H, NH2 and NH·HCl), 9.41 (s, 4H, NH2 and NH·HCl), 8.17 (d,
2H, J=7.8 Hz, ArH), 8.01 (d, 4H, J=8.4 Hz, ArH), 7.83 (d, 4H, J=
8.4 Hz, ArH), 7.65 (t, 2H, J=7.8 Hz, ArH), 7.53 ppm (t, 1H, J=
7.8 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO): d=164.9 (2C), 144.7
(2C), 138.6, 134.7 (2C), 129.8 (2C), 128.7 (4C), 128.5 (5C), 128.3 (2C),
118.7 ppm (2C); MS (ESI+): m/z (%): 382 [M+H]+ (30), 191.5 [M+
H]2+ (65); HRMS (ESI+): m/z [M+H]+ calcd for C22H20N7: 382.1780,
found: 382.1785; HPLC: tR =7.72 min (99.7% area).
4,5-Bis-(4-cyanophenyl)-[1,2,3]triazole
(26):
NaN3
(1.14 g,
17.52 mmol) was added slowly to a mixture of DMF (40 mL) and
22 (2 g, 8.76 mmol). After stirring at reflux for 6 h, the solvent was
evaporated. The crude product was purified by column chroma-
tography on silica gel (CH2Cl2/EtOAc, 9:1!8:2) to afford the prod-
1
uct as a yellow solid (1.55 g, 65%): H NMR (400 MHz, [D6]DMSO):
d=7.89 (s, 1H, NH), 7.84 (d, 4H, J=8.2 Hz, ArH), 7.63 ppm (d, 4H,
J=8.2 Hz, ArH); 13C NMR (100 MHz, [D6]DMSO): d=163.2 (2C),
134.9 (2C), 133.2 (4C), 129.1 (4C), 118.9 (2C), 111.5 ppm (2C); MS
(ESI+): m/z (%): 272 [M+H]+ (100); HRMS (ESI+): m/z [M+H]+
calcd for C16H10N5: 272.0936, found: 272.0939.
2-Benzyl-4,5-bis-(4-amidinophenyl)-[1,2,3]triazole dihydrochlor-
ide (30b): The same protocol as described for the synthesis of
compound 5 was used to generate amidine 30b from triazole 29
(840 mg, 2.32 mmol). Purification by column chromatography on
reversed phase silica gel (water) gave the product as a pale-yellow
solid (830 mg, 66%): mp: 230–2348C; 1H NMR (400 MHz,
[D6]DMSO): d=9.44 (s, 8H, NH2 and NH·HCl), 7.94 (d, 4H, J=8.4 Hz,
ArH), 7.70 (d, 4H, J=8.4 Hz, ArH), 7.34–7.48 (m, 5H, ArH), 5.80 ppm
(s, 2H, CH2); 13C NMR (100 MHz, [D6]DMSO): d=165.4 (2C), 143.6
(2C), 135.6 (2C), 135.6, 129.2 (2C), 129.1 (4C), 128.7 (6C), 128.6 (2C),
128.4, 79.6 ppm; MS (ESI+): m/z (%): 396 [M+H]+ (28), 198.5 [M+
4,5-Bis-(4-amidinophenyl)-[1,2,3]triazole dihydrochloride (27):
The same protocol as described for the synthesis of compound 5
was used to generate amidine 27 from triazole 26 (900 mg,
3.32 mmol). Purification by column chromatography on reversed
phase silica gel (water) gave the product as a white solid (790 mg,
60%): mp: 310–3128C; 1H NMR (400 MHz, [D6]DMSO): d=9.54 (s,
4H, NH2 or NH·HCl), 9.31 (s, 4H, NH2 or NH·HCl), 7.94 (d, 4H, J=
8.3 Hz, ArH), 7.74 ppm (d, 4H, J=8.3 Hz, ArH); 13C NMR (100 MHz,
[D6]DMSO): d=165.4 (2C), 144.5 (2C), 129.1 (4C), 128.7 (2C), 128.6
(4C), 128.2 ppm (2C); MS (ESI+): m/z (%): 306 [M+H]+ (100), 153.5
ChemMedChem 2011, 6, 2094 – 2108
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2105

P. M. O’Neill et al.
MED
1
2H]2+ (100); HRMS (ESI+): m/z [M+H]+ calcd for C23H22N7:
(390 mg, 63%): mp: 248–2528C; H NMR (400 MHz, [D6]DMSO): d=
9.40 (s, 8H, NH·HCl and NH2), 8.34 (d, 2H, J=8.2 Hz, ArH), 8.10 (d,
2H, J=8.2 Hz, ArH), 7.94 (d, 2H, J=7.9 Hz, ArH), 7.82 (d, 2H, J=
7.9 Hz, ArH), 7.59–7.63 (m, 2H, ArH), 7.50–7.55 ppm (m, 3H, ArH);
13C NMR (100 MHz, [D6]DMSO): d=165.4, 165.0, 147.7, 145.6, 142.4,
135.0, 130.6 (2C), 130.0, 129.6, 129.4 (2C), 129.1 (2C), 128.9, 128.8
(3C), 128.8 (2C), 127.6, 118.9 ppm; MS (ESI+): m/z (%): 382 [M+H]+
(18), 191.5 [M+2H]2+ (100); HRMS (ESI+): [M+H]+ calcd for
C22H20N7: 382.1780, found: 382.1785; HPLC: tR =7.45 min (99.3%
area).
396.1937, found: 396.1926; HPLC: tR =5.90 min (98.9% area).
4-Phenylethynylbenzonitrile (32): The same protocol as described
for compound 22 was used to generate 32 from 31 (2 g,
19.58 mmol) and 21 (3.56 g, 19.58 mmol). Purification by column
chromatography on silica gel (hexane/CH2Cl2, 5:5!4:6) gave the
product as a white solid (3.91 g, 98%): mp: 95–978C; 1H NMR
(400 MHz, CDCl3): d=7.64 (d, 2H, J=8.4 Hz, ArH), 7.60 (d, 2H, J=
8.4 Hz, ArH), 7.55 (d, 1H, J=7.5 Hz, ArH), 7.54 (d, 1H, J=7.5 Hz,
ArH), 7.37–7.41 ppm (m, 3H, ArH); 13C NMR (100 MHz, CDCl3): d=
132.5 (2C), 132.5 (2C), 132.2 (2C), 129.6, 128.9 (2C), 128.7, 122.6,
119.0, 111.9, 94.2, 88.1 ppm; MS (ESI+): m/z (%): 226 [M+Na]+
(100); HRMS (ESI+): m/z [M+Na]+ calcd for C15H9NNa: 226.0633,
found: 226.0622.
2-(4-Amidinobenzyl)-4-(4-amidinophenyl)-5-phenyl-[1,2,3]tria-
zole dihydrochloride (36b): The same protocol as described for
the synthesis of compound 5 was used to generate amidine 36b
from triazole 35 (1 g, 2.77 mmol). Purification by column chroma-
tography on reversed phase silica gel (water) gave the product as
a white solid (920 mg, 71%): mp: 215–2208C; 1H NMR (400 MHz,
[D6]DMSO): d=9.47 (s, 4H, NH·HCl and NH2), 9.37 (s, 4H, NH·HCl
and NH2), 7.90 (d, 2H, J=8.7 Hz, ArH), 7.88 (d, 2H, J=8.2 Hz, ArH),
7.68 (d, 2H, J=8.7 Hz, ArH), 7.63 (d, 2H, J=8.2 Hz, ArH), 7.43–7.51
(m, 5H, ArH), 5.92 ppm (s, 2H, CH2); 13C NMR (100 MHz, [D6]DMSO):
d=165.8, 165.5, 145.4, 143.1, 141.5, 135.8, 130.4, 129.3, 129.3 (2C),
129.0 (4C), 128.9 (2C), 128.5 (2C), 128.4 (2C), 128.3, 128.1,
57.9 ppm; MS (ESI+): m/z (%): 396 [M+H]+ (30), 198.5 [M+2H]2+
(100); HRMS (ESI+): [M+H]+ and [M+H]2+ calcd for C23H22N7:
396.1937, found: 396.1939; HPLC: tR =7.04 min (99.2% area).
4-(4-Cyanophenyl)-5-phenyl-[1,2,3]triazole (33): A solution of 32
(1.9 g, 9.35 mmol) in DMF (30 mL) was treated slowly with NaN3
(1.22 g, 18.70 mmol). After 6 h at reflux with stirring, water
(100 mL) was added to the reaction flask. The mixture was extract-
ed with CH2Cl2 (3ꢃ100 mL). The combined organic layers were
washed with water (300 mL) and brine, dried over MgSO4, filtered
and concentrated in vacuo to afford the product as an orange
solid. This product was unstable, and so used in the subsequent re-
action without further purification.
2,4-Bis-(4-cyanophenyl)-5-phenyl-[1,2,3]triazole (34): The same
protocol as described for compound 28 was used to generate
product 34 from benzonitrile 33 (1.10 g, 4.47 mmol) and 21
(1.63 g, 8.93 mmol). Purification by column chromatography on
silica gel (CH2Cl2/hexane, 5:5!6:4!7:3!8:2) gave the product as
a white powder (490 mg, 32% over two steps): mp: 126–1288C;
1H NMR (400 MHz, CDCl3): d=8.32 (d, 2H, J=8.8 Hz, ArH), 7.83 (d,
2H, J=8.8 Hz, ArH), 7.78 (d, 2H, J=8.5 Hz, ArH), 7.70 (d, 2H, J=
8.5 Hz, ArH), 7.59 (d, 1H, J=7.2 Hz, ArH), 7.58 (d, 1H, J=7.8 Hz,
ArH), 7.46–7.50 ppm (m, 3H, ArH); 13C NMR (100 MHz, CDCl3): d=
148.3, 145.7, 142.4, 135.0, 134.0 (2C), 132.9 (2C), 130.1, 129.8, 129.4
(2C), 129.2 (2C), 129.0 (2C), 119.5 (2C), 118.9, 118.6, 113.1,
111.6 ppm; MS (ESI+): m/z (%): 370 [M+Na]+ (100); HRMS (ESI+):
m/z [M+Na]+ calcd for C22H13N5Na: 370.1069, found: 370.1055.
Biology
In vitro growth inhibition assay of P. falciparum (GC03 and Dd2):
P. falciparum cultures consisted of a 2% suspension of O+ erythro-
cytes in RPMI-1640 medium (R8758, glutamine, and NaHCO3) sup-
plemented with 10% pooled human AB+ serum, 25 mm 4-(2-hy-
droxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.4, and
20 mm gentamicin sulfate. Cultures were grown under a gaseous
headspace of 4% O2, 3% CO2 in N2 at 378C. Parasite growth was
synchronized by treatment with sorbitol. Drug sensitivity experi-
ments were performed as described by Smilkstein et al.[59] IC50
values were calculated by using the four-parameter logistic
method (Grafit program; Erithacus Software, UK).
2-(4-Cyanobenzyl)-4-(4-cyanophenyl)-5-phenyl-[1,2,3]triazole
(35): A solution of triazole 33 (1.1 g, 4.47 mmol) in dry acetone
(120 mL) was treated with K2CO3 (3.09 g, 22.33 mmol), 4-(bromo-
methyl)benzonitrile (4.38 g, 22.33 mmol), and TBAI (165 mg, 10%
mol). The reaction mixture was heated to reflux under N2 over-
night. Thereafter, the reaction mixture was concentrated in vacuo.
Purification by column chromatography on silica gel (CH2Cl2/
hexane, 5:5!6:4!7:3!8:2) gave the product as a white solid
(1.2 g, 74% over two steps): mp: 49–518C; 1H NMR (400 MHz,
CDCl3): d=7.69 (d, 2H, J=8.4 Hz, ArH), 7.68 (d, 2H, J=8.6 Hz, ArH),
7.63 (d, 2H, J=8.6 Hz, ArH), 7.52 (d, 2H, J=8.4 Hz, ArH), 7.47–7.50
(m, 2H, ArH), 7.40–7.44 (m, 3H, ArH), 5.71 ppm (s, 2H, CH2);
13C NMR (100 MHz, CDCl3): d=146.5, 143.8, 140.2, 135.6, 133.2 (2C),
132.8 (2C), 130.4, 129.6, 129.3 (2C), 129.2 (2C), 129.0 (2C), 128.8
(2C), 119.0, 118.8, 113.0, 112.5, 59.7 ppm; MS (ESI+): m/z (%): 384
[M+Na]+ (100); HRMS (ESI+): m/z [M+Na]+ calcd for C23H15N5Na:
384.1225, found: 384.1241.
In vitro inhibition of hemozoin formation: For each product, the IC50
value was determined and expressed in micromolar. To measure
this, NaOAc (0.5m, pH 5.2), ghost membranes, a 3 mm solution of
iron(III) FPIX (dissolved in 0.1m NaOH), a 0.1m solution of HCl, and
the different drug dilutions (1 mm, 500 mm, 100 mm, 50 mm, 10 mm,
1 mm, 500 nm, and 100 nm) were introduced in eppendorf tubes.
The eppendorf tubes were then incubated at 378C for 72 h. After
incubation, the different solutions were centrifuged at 14000 rpm
and the supernatant was discarded. The pellet was washed with a
2% solution of sodium dodecyl sulfate (SDS) and also a 1m solu-
tion of Na2CO3 (pH 9). The pellet was then dissolved in a 1m solu-
tion of NaOH, and the absorbance of this solution was measured
at 400 nm. A graph was plotted for each product, and the IC50
value was determined by comparison with a control (without
drug).
In vivo growth inhibition assay of P. vinckei:[60,61] Groups of three
female Swiss mice OF1 (22–26 g, Charles River Laboratories France,
Domaine de Oncins–BP 0109–69592 L’Arbresle Cedex) were acclim-
atized one week before experiments. Each animal was weighted
the first day of the experiment in order to adjust dosage to mice
weight and identified individually by a ring on their ear. Female
Swiss mice were infected at day 0 by intravenous (iv) injection in
2,4-Bis-(4-amidinophenyl)-5-phenyl-[1,2,3]triazole dihydrochlor-
ide (36a): The same protocol as described for the synthesis of
compound 5 was used to generate amidine 36a from triazole 34
(450 mg, 1.30 mmol). Purification by column chromatography on
reversed phase silica gel (water) gave the product as a white solid
2106
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2094 – 2108

Triazole-Containing Furamidines as Antimalarials
the caudal vein of 107 infected erythrocytes in 200 mL 0.9% NaCl.
These injections lead to an initial parasitemia at day 1 between
0.5% and 1.5%. Mice were treated once a day for four days
(days 1–4). The compounds were dissolved in DMSO and adminis-
tered in 100 mL ip and/or po. On day 5, parasitemia levels were
monitored in Giemsa-stained blood smears and by flow cytometry
on blood samples (Yoyo-1 iodide (491/509), Invitrogen). In the ab-
sence of treatment, the P. vinckei malaria infection was lethal to the
mice, and death followed on day 6–7 with parasitemia higher than
85%. The ED50 value was determined on day 5 only, when treat-
ment induced a microscopically verified clearance of blood parasi-
temia. In addition, survival of mice that had cleared their parasite-
mia was monitored until at least one month after the end of treat-
ment. Absence of parasitemia and lethality within the 30 days
shows a complete cure without recrudescence. All protocols used
for in vivo experiments were approved by the local ethics commit-
tee at CNRS-Universitꢄ de Montpellier II, 34095 Montpellier
(France)
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