The activity of diguanidino and 'reversed' diamidino 2,5-diarylfurans versus Trypanosoma cruzi and Leishmania donovani

Article abstract of DOI:10.1016/S0960-894X(03)00319-6

The in vitro activity of 20 dicationic molecules containing either diguanidino or reversed amidine cationic groups were evaluated versus Trypanosoma cruzi and Leishmania donovani. The most active compounds were in the reversed amidine series and six exhibited IC₅₀ values of less than 1 μmol versus T. cruzi and five gave similar values versus L. donovani.

Full text of DOI:10.1016/S0960-894X(03)00319-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2065–2069
The Activity of Diguanidino and ‘Reversed’ Diamidino
2,5-Diarylfurans versus Trypanosoma cruzi and Leishmania donovani
Chad E. Stephens,a Reto Brun,b Manar M. Salem,c Karl A. Werbovetz,c Farial Tanious,a
W. David Wilsona and David W. Boykina,*
aDepartment of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA
bAntiparasite Chemotherapy, Swiss Tropical Institute, Basel, Switzerland
cDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Received 22 November 2002; accepted 27 January 2003
Abstract—The in vitro activity of 20 dicationic molecules containing either diguanidino or reversed amidine cationic groups were
evaluated versus Trypanosoma cruzi and Leishmania donovani. The most active compounds were in the reversed amidine series and
six exhibited IC50 values of less than 1 mmol versus T. cruzi and five gave similar values versus L. donovani.
# 2003 Elsevier Science Ltd. All rights reserved.
Protozoal parasitic diseases have plagued mankind for
centuries and continue to cause significant public health
problems, particularly in rural developing countries.
Two such diseases are Chagas’ disease and leishma-
niasis. Chagas’ disease (American trypanosomiasis) is
caused by Trypanosoma cruzi and is widespread in
Middle and South America. Cases are reported from the
southern United States to southern Argentina.
Approximately 90 million people are at risk and it is
estimated that 16–18 million are currently infected.1 It is
also estimated that approximately 500,000 new cases
develop per year and that about 50,000 deaths annually
are associated with Chagas’ disease. Current recom-
mended treatment for Chagas’ disease is either nifurti-
mox or benznidazole, although the former is not readily
available.1ꢀ3 Both of these drugs give variable results
when used against the acute form of the disease. No
effective treatment exists for the chronic stage of the
disease.
Approximately 2 million new cases are estimated to
develop annually.5 For decades, Pentostam and Glu-
cantime, pentavalent, antimonial compounds, have been
used to treat leishmaniasis. Both are administered
according to Sb (V) content and are thought to be
equivalent in efficacy and toxicity. These drugs are given
by injection, exhibit considerable toxicity and resistance
to them is developing.6 Amphotericin B has also been
used to treat the visceral form of leishmaniasis.4 How-
ever, it too has toxic side effects, is given by injection
and is expensive. Miltefosine, an orally effective drug, is
showing considerable promise in the treatment of both
visceral and cutaneous leishmaniasis.7,8 The diamidine
pentamidine(4,40 -(pentamethylenedioxy)dibenzamidine)
is also used for the treatment of leishmaniasis,9,10 how-
ever it must be given by injection due to its lack of oral
bioavailability, and exhibits a number of toxic side
effects. From this brief survey of the current status of
chemotherapy of these diseases, there is clearly a need
for development of new effective therapies for both
Chagas’ disease and leishmaniasis.
As many as 15 species of the Leishmania parasite give
rise to human leishmanial disease.4 The three main
clinical variations of leishmaniasis are the cutaneous,
mucocutaneous, and visceral forms. It is currently esti-
mated that leishmaniasis affects people in 88 countries,
with 350 million at risk of contracting the disease.
Broad spectrum antimicrobial activity of aromatic
dicationic compounds, for which pentamidine may be
considered the prototype molecule, has been reported
against a variety of organisms.11ꢀ16 In the pentamidine
type of molecules, the cationic centers are amidine
groups. These type of compounds bind to the minor
groove of DNA at AT sites.17ꢀ19 This binding has been
hypothesized to be important in the mode of anti-
*Corresponding author. Tel.: +1-404-651-3798; fax: +1-404-651-
1416; e-mail: dboykin@gsu.edu
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00319-6

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C. E. Stephens et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2065–2069
microbial action of these compounds possibly leading to
inhibition of DNA dependent enzymes.20ꢀ22 Other
mechanisms of action have also been proposed for pen-
tamidine, including disruption of polyamine metabo-
lism23 and disruption of mitochondrial membrane
potential.24 Diamidine compounds with improved effi-
cacy and reduced toxicity compared to pentamidine
have been developed.15,25 Prodrug strategies for this
class of compounds have provided orally effective
molecules thus obviating an injection dosage regime,
one of the major disadvantages of the pentamidine class
of compounds.26ꢀ28 A prodrug of furamidine [2,5-bis(4-
amidinophenyl)furan] is currently undergoing Phase II
clinical trials against Human African trypanosomiasis
and Pneumocystis carinii pneumonia.
In addition to pentamidine, many of these diamidine
molecules exhibit promising activity against Leishmania.
The antileishmanial activity of several such dicationic
molecules was reported some time ago29ꢀ31 and more
recently several new dicationic molecules have shown
improved in vitro activity against L. donovani compared
to pentamidine.32 A report on the in vitro activity of
mono-amidines against T. cruzi has appeared.33 Eval-
uations of dicationic compounds against T. cruzi appear
to be limited to pentamidine and related analogues,
molecules which have shown only very limited in vivo
activity.34,35
Scheme 1. (a) Pd(PPh3)4, 1,4-dioxane; (b) H2, Pd/C, EtOAc, EtOH or
SnCl2 dihydrate, EtOH, DMSO; (c) S-methyl-di-Bocthiourea, HgCl2,
TEA, DMF; (d) HCl, CH2Cl2, EtOH; (e) S-(2-naphthylmethyl)-2-
pyridylthiomidate, MeCN, EtOH.
reversed amidines 4 in a straightforward manner. The
yields for the reversed amidines were also lower
(ꢁ30%) when a substituent was ortho to the amidino
unit.
In our efforts to identify promising antileishmanial
agents from a large group of dicationic compounds, we
have employed an axenic assay using L. donovani
amastigote-like parasites to quickly screen for intrinsic
antileishmanial activity.32,37 This initial screening assay
indicated that the guanidines possessed good antil-
eishmanial activity, while the reversed amidines dis-
played excellent antileishmanial activity. These
compounds thus merited testing in the Leishmania-
infected macrophage assay, which is more labor-inten-
sive but also a better predictor of in vivo antileishmanial
potential. The structure–activity relationships that have
emerged differ somewhat, which is not surprising con-
sidering the differences between the two assays. For
example, the axenic amastigotes are cultured alone in
medium at pH 5.5, while amastigotes in macrophages
reside within a membrane-bound acidic vacuole.38 Thus,
a prospective antileishmanial agent would need to cross
one membrane in the axenic amastigote assay and three
membranes in the infected macrophage model. These
assays generally agree, in that they show the guanidines
are moderately potent (Table 1) and the reversed ami-
dines are highly potent (Table 2) against L. donovani in
vitro. Note that seven of the eight reversed amidines
reported here have IC50 values in the axenic amastigote
assay that are below 1 mM, while only two of the 58
compounds reported in our previous work with diverse
classes of diamidines30 had IC50 values below 1 mM in
this assay. The fact that the reversed amidines were
identified as potent antileishmanials by the initial axenic
amastigote assay highlights the utility of this screening
approach.
As part of a study to determine the influence of the
structure of the cationic center on the antimicrobial
properties of these minor groove binding molecules, we
are studying replacement of the amidine unit by guani-
dine and reversed amidine units.36 Although anti-
microbial activity has been reported for various classes
of guanidino compounds,12 this type of cationic com-
pounds has not been extensively studied as anti-
protozoan agents. Compounds containing the other
modification of the cationic center being investigated
are referred to as ‘reversed’ amidines. In the reversed
amidines, the imino group is attached to an ‘anilino’
nitrogen in contrast to the original amidino compounds
in which the imino group is directly attached to an aryl
ring. We have recently shown that a group of 2,5-dia-
rylfurans containing modified cationic centers has sig-
nificant in vitro antifungal activity.36 We now report the
in vitro evaluation of 20 dicationic molecules with
modified cationic groups against T. cruzi and L. dono-
vani.
The syntheses of the diguanidino compounds and the
‘reversed’ amidines were achieved starting with 2,5-
bis(tri-n-butylstannyl)furan using the approach which
we previously described36 and which is outlined in
Scheme 1. The 2,5-bis(4-aminophenyl)furan analogues
2 were obtained in good yields and served as the key
common intermediates for both sets of target com-
pounds 3 and 4. The two-step conversion of 2 into the
diguanidino compounds 3 was achieved in good yields.
The yields of the diguanidino compounds bearing sub-
stituents ortho to the guanidino groups were somewhat
lower (ꢁ50%) than the other analogues. The reaction
of 2 with a S-(2-naphthylmethyl)thioimidate gave the
To obtain a qualitative evaluation of the DNA binding
affinity of these drug candidates, melting temperatures
were measured for the compounds in Tables 1 and 2
bound to poly(dA-dT) and the Dickerson–Drew dodec-

C. E. Stephens et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2065–2069
2067
Table 1. In vitro anti- L. donovani and T. cruzi activities, DNA binding affinities and cell toxicities of diguanidinium dications 3a–3k
b (AT)
c (oligo)
Compd
L.d. (amastigote)
IC50, mMa
L.d. (macrophage)
IC50, mMa
T.c.
IC50, mMa
Cell toxicity
IC50, mMa
ÁTm
ÁTm
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
10.9
3.3
18.7
9.65
6.2
>24
>6
5.2
>6
>6.8
>5.6
>19
>29.0
>40
>19
>6.3
9.3
86.0
3.2
1.3
143.0
14.6
20.0
16.0
14.2
11.3
33.4
39.6
74.0
68.0
35.7
4.2
21.6
10.8
17.8
15.2
12.0
26.1
5.9
6.9
2.8
1.5
4.7
0
1.3
6.2
6.8
2.6
2.6
4.8
35.0
27.0
31.2
2.7
29.0
152
114
44.0
7.1
14.8
4.2
3.8
14.5
11.2
22.6
>50
2.6
15.4
16.5
12.2
12.7
12.8
11.7
3k
Pentad
Furame
Miltf
Benzng
2.7
ND23.3
1.0
6.4
25.0
1.27
aSee refs 32, 37, 39, 40 IC50 values are the mean of two independent assays.
bIncrease in thermal melting of poly(dA.dT)2 see ref 25.
cIncrease in thermal melting of d(CGCGAATTCGCG)2 see ref 25.
dPentamidine.
eFuramidine.
fMiltefosine.
gBenznidazole.
Table 2. In vitro anti- L. donovani and T. cruzi activities, DNA binding affinities and cell toxicities of reversed amidine dications 4a–4h
b (AT)
c (oligo)
Compd
L.d. (amastigote)
IC50, mMa
L.d. (macrophage)
IC50, mMa
T.c.
IC50, mMa
Cell toxicity
IC50, mMa
ÁTm
ÁTm
4a
4b
4c
4d
4e
4f
4g
0.55
0.38
0.10
0.66
1.14
0.29
0.35
0.51
>100
2.6
1.45
0.6
0.48
0.13
3.6
0.16
0.15
ND0.5
>8
9.3
ND23.3
1.0
9.6
0.21
0.57
0.49
0.91
1.0
0.15
128
13.7
7.1
17.1
11.3
12.7
8.0
60.6
7.9
7.3
0.6
3.8
4.2
19.6
7.5
8.9
7.8
0
4.2
6.6
0
22.6
19.0
5.2
13.8
15.2
1.0
4h
1.5
Phend
Pentae
Furamf
Miltg
Benznh
28.0
12.8
11.7
15.0
4.8
2.7
6.4
25.0
1.27
aSee refs 32, 37, 39, 40 IC50 values are the mean of two independent assays.
bIncrease in thermal melting of poly(dA.dT)2 see ref 25.
cIncrease in thermal melting of d(CGCGAATTCGCG)2 see ref 25.
d4a with the pyridyl groups replaced by phenyl groups.
ePentamidine.
fFuramidine.
gMiltefosine.
hBenznidazole.
amer d(CGCGAATTCGCG)2. The difference in Tm
values between the drug–DNA complexes and free
DNA in solution provides a useful tool to assess the
interaction strength of the molecules with DNA.
modest lowering of the ÁTm value except for the cases
of 3e with a moderate electron withdrawing chloro
group, 3f with a strong electron withdrawing tri-
fluoromethyl group and 3g which contains multiple
substitution. Generally, a similar pattern of substitutent
effects is seen from the results when the Dickerson–
Drew dodecamer is employed. In general, the ÁTm
values for the pyridyl reversed amidines (Table 2) with
substituents ortho to the cationic center are lower than
the other analogues. This reduction in affinity seems
likely to be due to a combination of steric and electronic
effects. The good antiprotozoan activity (vide infra)
of the compounds which exhibit low DNA affinities
The effect of substitutents on the DNA affinities of these
types of molecules has been commented on previously,34
however in this report we add results for molecules in
which a substitutent is placed ortho to the cationic cen-
ter. Generally, examination of the data obtained with
poly (dA–dT) for the diguanidiums in Table 1 shows
that substituents located either ortho or meta to the
guanidinium groups (cf. 3b–3d or 3 h, 3i) cause a similar

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C. E. Stephens et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2065–2069
suggests a mode of action for which DNA binding does
not play a major role. In general, the DNA affinities for
the substituted compounds in both Tables are reduced
compared to the parent molecules (3a and 4a). How-
ever, the DNA affinities for most of these molecules are
similar to that of pentamidine.
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The antimicrobial activities of the diguanidino com-
pounds are presented in Table 1. The activities of the
diguanidiniums are only modest. Only 3b and 3c show
IC50 values which are comparable to those of pentami-
dine and furamidine. Three of the diguanidino analo-
gues (3b, 3c, 3g) show activity against T. cruzi which is
comparable to that of pentamidine and superior to that
of furamidine.
The series of 2-pyridyl reversed amidines exhibits con-
siderable antimicrobial activity against both protozoan
parasites (Table 2). Again, there is a reasonably good
correlation between the antileishmanial activities
observed for these compounds from both in vitro mod-
els. Both models indicate IC50 values of less than 1 mmol
for five molecules (4b, 4c, 4d, 4f, 4g). The most active
compound (4d) is found to be at least 170 times more
active than pentamidine in the macrophage model. It is
noteworthy that the replacement of the pyridyl rings
with phenyl rings results in significant loss of activity
although the DNA binding affinity is improved. Good
in vitro activity versus T. cruzi is also noted for the
2-pyridyl reversed amidines, with six of these analogues
showing IC50 values of less than 1 mmol. The best com-
pound 4g is 40-fold more active than pentamidine and
exhibits a selectivity index of near 50.
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These studies identify two new classes of dicationic
compounds with activity against Leishmania and
T. cruzi. The reversed amidines are particularly inter-
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antiparasitic activity in vitro. For example, pentami-
dine has little measurable activity against intracellular
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Acknowledgements
This work was supported by awards from the Bill and
Melinda Gates Foundation and NIH (RO1GM61587).
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