Antileishmanial activities of several classes of aromatic dications

Article abstract of DOI:10.1128/AAC.46.3.797-807.2002

Aromatic dicationic molecules possess impressive activity against a broad spectrum of microbial pathogens, including Pneumocystis carinii, Cryptosporidium parvum, and Candida albicans. In this work, 58 aromatic cations were examined for inhibitory activity against axenic amastigote-like Leishmania donovani parasites. In general, the most potent of the compounds were substituted diphenyl furan and thiophene dications. 2,5-Bis-(4-amidinophenyl)thiophene was the most active compound. This agent displayed a 50% inhibitory concentration (IC₅₀) of 0.42 ± 0.08 μM against L. donovani and an in vitro antileishmanial potency 6.2-fold greater than that of the clinical antileishmanial dication pentamidine and was 155-fold more toxic to the parasites than to a mouse macrophage cell line. 2,4-Bis-(4-amidinopheny)furan was twice as active as pentamidine (IC₅₀, 1.30 ± 0.21 μM), while 2,5-bis-(4-amidinopheny)furan and pentamidine were essentially equipotent in our in vitro antileishmanial assay. Carbazoles, dibenzofurans, dibenzothiophenes, and benzimidazoles containing amidine or substituted amidine groups were generally less active than the diphenyl furans and thiophenes. In all cases, aromatic dications possessing strong antileishmanial activity were severalfold more toxic to the parasites than to a cultured mouse macrophage cell line. These structure-activity relationships demonstrate the potent antileishmanial activity of several aromatic dications and provide valuable information for the future design and synthesis of more potent antiparasitic agents.

Full text of DOI:10.1128/AAC.46.3.797-807.2002

Antileishmanial Activities of Several
Classes of Aromatic Dications
James J. Brendle, Abram Outlaw, Arvind Kumar, David W.
Boykin, Donald A. Patrick, Richard R. Tidwell and Karl A.
Werbovetz
Antimicrob. Agents Chemother. 2002, 46(3):797. DOI:
10.1128/AAC.46.3.797-807.2002.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2002, p. 797–807
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.3.797–807.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 3
Antileishmanial Activities of Several Classes of Aromatic Dications
James J. Brendle,1 Abram Outlaw,1 Arvind Kumar,2 David W. Boykin,2 Donald A. Patrick,3
Richard R. Tidwell,3 and Karl A. Werbovetz4*
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland 209101;
Department of Chemistry, Georgia State University, Atlanta, Georgia 303032; Pathology Department,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275993; and
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,
The Ohio State University, Columbus, Ohio 432104
Received 19 July 2001/Returned for modification 8 October 2001/Accepted 20 November 2001
Aromatic dicationic molecules possess impressive activity against a broad spectrum of microbial pathogens,
including Pneumocystis carinii, Cryptosporidium parvum, and Candida albicans. In this work, 58 aromatic cations
were examined for inhibitory activity against axenic amastigote-like Leishmania donovani parasites. In general,
the most potent of the compounds were substituted diphenyl furan and thiophene dications. 2,5-Bis-(4-
amidinophenyl)thiophene was the most active compound. This agent displayed a 50% inhibitory concentration
(IC50) of 0.42 ؎ 0.08 ␮M against L. donovani and an in vitro antileishmanial potency 6.2-fold greater than that
of the clinical antileishmanial dication pentamidine and was 155-fold more toxic to the parasites than to a
mouse macrophage cell line. 2,4-Bis-(4-amidinopheny)furan was twice as active as pentamidine (IC50, 1.30 ؎
0.21 ␮M), while 2,5-bis-(4-amidinopheny)furan and pentamidine were essentially equipotent in our in vitro
antileishmanial assay. Carbazoles, dibenzofurans, dibenzothiophenes, and benzimidazoles containing amidine
or substituted amidine groups were generally less active than the diphenyl furans and thiophenes. In all cases,
aromatic dications possessing strong antileishmanial activity were severalfold more toxic to the parasites than
to a cultured mouse macrophage cell line. These structure-activity relationships demonstrate the potent
antileishmanial activity of several aromatic dications and provide valuable information for the future design
and synthesis of more potent antiparasitic agents.
Protozoal parasitic diseases continue to pose serious public
health problems in developing parts of the world. One such
disease is leishmaniasis, a spectrum of disease in humans that
is caused by several species of the Leishmania parasite. These
unicellular organisms are related to trypanosomes, the patho-
genic parasites responsible for sleeping sickness in Africa and
Chagas’ disease in South America. Three main clinical variants
of leishmaniasis are known: cutaneous, mucocutaneous, and
visceral, with symptomatic visceral disease often ending in
death if treatment is not provided. In recent years, the coex-
istence of human immunodeficiency virus and Leishmania spe-
cies causing visceral disease has resulted in several hundred
cases of dually infected individuals (4). By current estimates,
leishmaniasis affects people in 88 countries, with 350 million at
risk of contracting the disease and approximately 2 million new
cases each year (www.who.int/emc/diseases/leish/leisdis1.html).
The devastating impact of this disease is exemplified by the
epidemic of visceral leishmaniasis that occurred in the 1990s in
the Sudan (27).
given by injection over a 20- to 28-day course, and common
side effects include nausea, hepatotoxicity, and cardiotoxicity
(4). Reports of unresponsiveness to antimony treatment have
become frequent (24), and a strong correlation between clin-
ical resistance to sodium stibogluconate and decreased in vitro
susceptibility to this drug has been reported (20). Amphoter-
icin B is also used as a treatment for visceral leishmaniasis. Past
implementation of this drug was limited by toxic side effects,
including fever, bone pain, and decreased renal function. New
clinical formulations of amphotericin B in lipid complexes have
proven to be less toxic than amphotericin B but are more
costly, a major problem in treating visceral leishmaniasis in
developing countries. Also, amphotericin B and amphotericin
B-lipid complexes do not appear to be suitable for treatment of
nonvisceral disease (4). Oral miltefosine has shown promise in
the treatment of visceral leishmaniasis (18, 31), but no reports
of efficacy against cutaneous disease have appeared and the
drug has not yet been approved for routine use. Pentamidine is
also used frequently for the treatment of leishmaniasis (16, 30).
The drawbacks of the clinical use of pentamidine are the route
of administration (injection) and the toxicity of the compound.
Administration by injection increases the expense of the treat-
ment and makes the use of the drug less practical in developing
nations, where cost is a major factor. The clinical side effects of
pentamidine include renal and hepatic toxicity, pancreatitis,
hypotension, dysglycemia, and cardiac abnormalities (4, 15).
There is a clear and urgent need for the development of im-
proved treatments for leishmaniasis that are safe, inexpensive,
and orally available.
Pentavalent antimonial compounds have been the first-line
drugs for leishmaniasis since the 1940s, and two forms of Sb(V)
are commonly used. Sodium stibogluconate (Pentostam) and
meglumine antimoniate (Glucantime) are prescribed accord-
ing to Sb(V) content and are generally considered to be equiv-
alent in terms of efficacy and toxicity. These drugs must be
* Corresponding author. Mailing address: Division of Medicinal
Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State
University, 500 West 12th Ave., Columbus, OH 43210. Phone: (614)
292-5499. Fax: (614) 292-2435. E-mail: werbovetz.1@osu.edu.
Dicationic compounds based on pentamidine have shown
797

798
BRENDLE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
solid with a melting point of 280°C dec.; 1H NMR (DMSO-d6/D2O): 7.88 (d, 4H,
J ϭ 8.4 Hz), 7.83 (d, 4 H, J ϭ 8.4 Hz), 4.19 (m, 2H), 2.26 (s, 6H), 2.08–2.04 (m,
4H), 1.74–1.55 (m, 12H). 13C NMR (DMSO-d6): 161.9, 146.3, 134.5, 128.9, 127.0,
124.8, 122.1, 54.2, 31.2, 23.5, 9.4. FABMS: m/e 468 (Mϩϩ1). Analysis calculated
for C30H36N4O · 2HCl · 0.5H2O (550.58): C, 65.44; H, 7.13; N, 10.17. Found: C,
65.02; N, 7.15; N, 9.90.
2,5-Bis{4-(2-tetrahydropyrimidyl)phenyl}-3,4-dimethyl furan dihydrochlo-
ride (compound 16). A suspension of the corresponding imidate ester dihydro-
chloride (11) (0.898 g, 0.002 mol) with 1,3-diaminopropane (0.296 g, 0.004 mol)
in 30 ml of dry ethanol was heated at reflux for 12 h. The solvent was distilled
under reduced pressure. The solid was suspended in ϳ50 ml of water, and the pH
was adjusted to 10 with 2 M NaOH (aqueous). The solid was filtered, washed
with water, and dried to yield a pale yellow crystalline free base (72%) with a
melting point of 159 to 160°C. The solid was dissolved in 15 ml of hot ethanol,
saturated with HCl gas at 0°C, and stirred at 50°C for 2 h. After the addition of
30 ml of dry ether, the precipitated salt was filtered, washed with ether, and dried
under vacuum at 70°C for 24 h to yield 0.77 g (77%) of a yellow crystalline solid
with a melting point of 275 to 278°C dec. 1H NMR (DMSO-d6/D2O) 7.91 (d, 4H,
J ϭ 8.8 Hz), 7.84 (d, 4H, J ϭ 8.8 Hz), 3.05 (t, 8H, J ϭ 6 Hz), 2.26 (s, 6H), 1.99
(q, 4H, J ϭ 6 Hz). 13C NMR (DMSO-d6/D2O) 158.8, 146.6, 134.9, 128.3, 126.6,
125.5, 122.8, 38.9, 17.8, 8.8. FABMS: m/e 413 (Mϩϩ1). Analysis calculated for
impressive antimicrobial activity against a variety of organisms
(2, 13, 25). Dicationic molecules with improved efficacy and
reduced toxicity compared to pentamidine have been reported
in animal models of pneumocystosis and cryptosporidiosis (5,
6, 26). Many of these compounds also possess excellent activity
against Leishmania parasites. The antileishmanial activity of
several such dicationic molecules was reported in 1990 (3).
Since that time, many new aromatic cations have been synthe-
sized and improved assay systems have been developed for the
in vitro testing of candidate drugs against amastigote-like par-
asites (1, 9, 28, 34). We report here the in vitro evaluation of
58 aromatic cations against axenic amastigote-like Leishmania
donovani. Some of the dicationic agents proved to be more
potent than pentamidine in this assay, indicating that the syn-
thesis of new aromatic dications could lead to the development
of more effective antileishmanial agents.
C
26H28N4O · 2HCl · H2O (503.47): C, 62.07; H, 6.40; N, 11.12. Found: C, 62.09;
MATERIALS AND METHODS
N, 6.52; N, 10.84.
2,5-Bis{4-(N-isopropylamidino)phenyl}-thiophene dihydrochloride (com-
pound 24). 2,5-Bis(4-cyanophenyl)thiophene was prepared by Stille coupling of
4-bromobenzonitrile and 2,5-bis(tributylstannyl)thiophene (19). The bis-nitrile
was converted into the corresponding bis imidate ester as previously described
(12). Isopropylamine (0.295 g, 0.005 mol) was added to the bis imidate ester
dihydrochloride (0.9 g, 0.002 mol) in 30 ml of ethanol and stirred at room
temperature for 24 h. The solvent was distilled in a vacuum, the solid was
suspended in 50 ml of water and basified to pH 9 with 2 M NaOH (aqueous), and
the precipitated free base was filtered, washed with water, and dried in a vacuum
at 500°C. The free base was converted into its crystalline yellow dihydrochloride
with saturated ethanolic HCl with a melting point of 358 to 600°C dec. 1H NMR
(DMSO-d6/D2O): 7.84 (d, 4H, J ϭ 8.2 Hz), 7.72 (d, 4H, J ϭ 8.2 Hz), 7.66 (d, 2H),
3.98 (septet, 2H, J ϭ 6.4 Hz), 1.27 (d, 12H, J ϭ 6.4 Hz). 13C NMR (DMSO-d6/
D2O): 162.1, 143.1, 138.2, 129.5, 128.4, 127.7, 126.1, 45.8, 21.4. FABMS: m/e 404
(M ϩ 1). Analysis calculated for C24H28N4S · 2HCl (427.51): C, 60.37; H, 6.53;
N, 11.73. Found: C, 60.62; N, 6.26; N, 11.48.
2(4-Cyanophenyl)thiophene (Fig. 1, c). To a mixture of 2-tributylstannylthio-
phene (Fig. 1, a) (17.0 g, 0.05 mol) and 4-bromobenzonitrile (Fig. 1b) (9.1 g, 0.05
mol) in 45 ml of dry dioxane (under nitrogen) was added Pd(PPh3)4 (1.15 g, 2
mol%). The mixture was heated at reflux for 8 h (monitored by thin-layer
chromatography [TLC]), the solvent was distilled under a vacuum, and the oily
residue was diluted with 100 ml of dichloromethane and 100 ml of 10% KF
(aqueous). The mixture was stirred for 1 h, filtered through Celite, and washed
with water. The organic layer was dried over Na2SO4, filtered, and concentrated
under a vacuum, and the resulting oily residue was chromatographed over silica
gel (elution with hexane [3:7 hexane-benzene ratio]) to give 7.7 g (83%) of a
white solid with a melting point of 85 to 86°C. 1H NMR (DMSO-d6): 7.81 (brs,
4H), 7.67 (brm, 2H), 7.18 (brm, 1H). 13C NMR (DMSO-d6): 141.2, 138.0, 132.9,
128.8, 127.9, 126.0, 125.8, 118.6, 109.5. MS: m/e 185 (Mϩ). Analysis calculated
for C11H7NS (185.25): C, 71.32; H, 3.81; N, 7.56. Found: C, 71.38; N, 3.87; N,
7.59.
Cationic compounds. All of the cationic agents tested in this work were
synthesized in the laboratories of two of the authors (D.W.B. and R.R.T.) as
hydrochloride salts. The synthesis and characterization of 49 of the 58 com-
pounds presented here have been described previously; for the original refer-
ences for these compounds, see Tables 1 to 4. Synthetic details and character-
ization of compounds 13 to 16, 23 to 25, 36, and 37, along with the necessary
synthetic intermediates, are given below.
2,5-Bis{4-(N-isopropylamidino)phenyl}-3,4-dimethyl furan dihydrochloride
(compound 13). A suspension of the bis imidate ester hydrochloride (11) (0.9 g,
0.002 mol) with isopropylamine (0.296 g, 0.005 mol) in 30 ml of dry ethanol was
stirred at room temperature for 24 h. The solvent was removed under reduced
pressure, and the solid was dissolved in ϳ50 ml of water and basified to pH 10
with 2 M NaOH (aqueous). The solid was filtered, washed with water, dried,
dissolved in warm ethanol, saturated with HCl gas at 0 to 5°C, and stirred at
room temperature for 2 h. After the addition of 30 ml of dry ether, the precip-
itated salt was filtered, washed with ether, and dried under vacuum at 60°C for
24 h to yield 0.69 g (69%) of a yellow crystalline solid with a melting point of 243
to 247°C dec. 1H nuclear magnetic resonance (NMR) (dimethyl sulfoxide
[DMSO]-d6): 9.64 (brd, 2H), 9.54 (br, 2H), 9.26 (br, 2H), 7.90 (brs, 8H), 4.20
(brm, 2H), 2.28 (s, 6H), 1.31 (d, 12H, J ϭ 6.4 Hz). 13C NMR (DMSO-d6/D2O):
161.1, 146.3, 134.5, 128.8, 127.2, 124.9, 122.1, 45.0, 21.0, 9.4. Fast atom bombard-
ment mass spectrometry (FABMS): m/e 417 (Mϩϩ1). Analysis calculated for
C26H32N4O · 2HCl · 0.65H2O (501.23): C, 62.30; H, 7.09; N, 11.17. Found: C,
62.63; N, 7.07; N, 10.72.
2,5-Bis{4-(N-cyclopropylamidino)phenyl}-3,4-dimethyl furan dihydrochloride
(compound 14). A suspension of the bis imidate ester dihydrochloride (11) (0.673
g, 0.0015 mol) with cyclopropylamine (0.214 g, 0.00375 mol) in 20 ml of ethanol
was stirred at room temperature for 24 h. The solvent was removed under
reduced pressure, the solid was suspended in ϳ50 ml of water, and the pH was
adjusted to 10 with 2 M NaOH (aqueous). The solid was filtered, washed with
water, dried, dissolved in hot ethanol, saturated with HCl gas at 0°C, and stirred
at room temperature for 2 h. After the addition of 30 ml of dry ether, the
precipitated salt was filtered, washed with ether, and dried under vacuum at 60°C
for 24 h, 0.54 g (72%) to give a yellow crystalline solid with a melting point of 245
to 248°C dec. 1H NMR (DMSO-d6): 10.17 (br, 2H), 9.8 (br, 2H), 9.22 (br, 2H),
7.96 (d, 4H, J ϭ 8 Hz), 7.89 (d, 4H, J ϭ 8 Hz), 2.88 (brs, 2H), 2.28 (s, 6H),
0.97–0.87 (m, 8H). 13C NMR (DMSO-d6): 164.0, 146.3, 134.7, 128.8, 126.1, 124.8,
122.3, 24.6, 9.4, 6.4. FABMS: m/e 413 (Mϩϩ1). Analysis calculated for
2-Bromo-5-(4-cyanophenyl)thiophene (Fig. 1, d). Bromine (1.76 g, 0.011 mol)
in 15 ml of dichloroethane was added in 15 min to a well-stirred solution of
2-(4-cyanophenyl) thiophene (Fig. 1, c) (1.85 g, 0.01 mol) in 50 ml of dichloro-
ethane at 5 to 10°C. The mixture was stirred at room temperature for 2.5 h and
then diluted with 50 ml of water and 50 ml of dichloroethane. The organic layer
was washed with 5% sodium thiosulfate–5% sodium bicarbonate–water, dried
over sodium sulfate, and filtered. The solvent was removed under reduced
pressure, and the residual solid was triturated with ether-hexane (1:4) to yield
2.14 g (81%) of an off-white crystalline solid with a melting point of 98 to 99°C.
1H NMR (DMSO-d6): 7.83 (d, 2H, J ϭ 8.4 Hz), 7.78 (d, 2H, J ϭ 8.4 Hz),7.53 (d,
1H, J ϭ 4 Hz), 7.30 (d, 1H, J ϭ 4 Hz). 13C NMR (DMSO-d6): 142.7, 136.8, 132.8,
131.9, 126.6, 118.3, 112.9, 109.9. MS: m/e 264 (Mϩ). Analysis calculated for
C
26H28N4O · 2HCl · H2O (503.48): C, 62.25; H, 6.40; N, 11.12. Found: C, 62.11;
N, 6.45; N, 11.03.
2,5-Bis{4-(N-cyclopentylamidino)-phenyl}-3,4-dimethyl furan dihydrochlo-
ride (compound 15). Cyclopentylamine (0.425 g, 0.005 mol) was added to a
suspension of the bis imidate ester dihydrochloride (11) (0.9 g, 0.002 mol) in 35
ml of dry ethanol, and this mixture was stirred at room temperature for 12 h. The
solvent was distilled under reduced pressure, and the solid was suspended in 50
ml of water and basified to pH 10 with 2 M NaOH (aqueous). The precipitated
solid was filtered, washed with water, dried, dissolved in ethanol, saturated with
HCl gas at 0 to 5°C, and stirred at room temperature for 2 h. After the addition
of 30 ml of dry ether, the precipitated salt was filtered, washed with ether, and
dried under vacuum at 50°C for 12 h to yield 0.77 g (70%) of a yellow crystalline
C
11H6BrNS (264.14): C, 50.01; H, 2.29; N, 5.30. Found: C, 49.92; N, 2.31; N, 5.16.
2-(3-Cyanophenyl)-5-(4-cyanophenyl)thiophene (Fig. 1, e). Potassium carbon-
ate (3.03 g, 0.022 mol) in 15 ml of water was added to a well-stirred solution of
2-(4-cyanophenyl)-5-bromo-thiophene (Fig. 1, d) (2.64 g, 0.0 1 mol) and 3-cya-
nophenylboronic acid (1.62 g, 0.011 mol) in 30 ml of n-propanol, followed by
addition of tetrakis(triphenylphosphine)palladium (0.23 g, 2 mol%). The reac-
tion mixture was heated at reflux for 12 h (under nitrogen), and then the solvent

VOL. 46, 2002
ANTILEISHMANIAL AROMATIC DICATIONS
799
FIG. 1. Synthesis of thiophene dications 23 and 25.
was removed under reduced pressure. The residue was diluted with 50 ml of
water, filtered, and dried. The resulting solid was dissolved in 100 ml of chloro-
form and filtered through Celite, the solvent was removed, and the solid was
crystallized from ether-benzene to yield 2.1 g (73%) of a bright yellow crystalline
solid with a melting point of 169 to 170°C. 1H NMR (DMSO-d6): 8.13 (dd, 1H,
J ϭ 1.6 Hz), 7.96 (ddd, 1H, J ϭ 0.8 Hz, J ϭ 1.6 Hz, J ϭ 8 Hz), 7.83 (brs, 4H) 7.74
(md, 1H, J ϭ 8Hz), 7.71 (d, 1H, J ϭ 4 Hz), 7.62 (d, 1H, J ϭ 4 Hz), 7.62 (dd, 1H,
J ϭ 8 Hz). 13C NMR (DMSO-d6/D2O): 142.0, 141.5, 137.2, 134.1, 132.7, 131.1,
129.6, 128.3, 127.2,126.7, 125.5, 118.2, 118.0,112.2, 109.8. MS: m/e 286 (Mϩ).
Analysis calculated for C18H10N2S (286.36): C, 75.5; H, 3.52; N, 9.78. Found: C,
75.57; N, 3.62; N, 9.88.
2-{3-(Amidino)phenyl}-5-{4-(amidino)phenyl}-thiophene dihydrochloride (23).
The above-described bis-nitrile (Fig. 1, e) (2.86 g, 0.01 mol) in 70 ml of ethanol
was saturated with dry HCl gas at 0 to 5°C and then stirred at room temperature
for 7 days (monitored by infrared spectroscopy and TLC). Ether was added to
the mixture, and the yellow imidate ester dihydrochloride (Fig. 1, f) was filtered
and washed with ether. The solid was dried at 40°C for 5 h to yield 4.3 g (95%).
The hygroscopic solid was used in the next step without further purification. A
suspension of imidate ester dihydrochloride (0.9 g, 0.002 mol) in 35 ml of ethanol
was saturated with ammonia gas at 0 to 5°C and stirred at room temperature for
2 days, and the solvent was removed under reduced pressure. The solid was
suspended in water, the pH was adjusted to 9, and the pale solid was filtered,
washed with water, and dried. The solid was stirred in saturated ethanolic HCl
(20 ml) at 50°C for 1 h, diluted with ether, filtered, and dried in a vacuum oven
at 75°C for 24 h to yield 0.61 g (78%) of a pale yellow solid with a melting point
of 320 to 321°C dec. 1H NMR (DMSO-d6/D2O): 8.12 (dd, 1H, J ϭ 1.6, J ϭ 8 Hz),
8.01 (brd, 1H, J ϭ 8 Hz), 7.91 (d, 4H, J ϭ 8.8 Hz), 7.88 (d, 4H, J ϭ 8.8 Hz), 7.76
(d, 1H, J ϭ 4 Hz), 7.75 (brd, 1H, J ϭ 8 Hz), 7.72 (d, 1H, J ϭ 4 Hz), 7.69 (dd, 1H,
J ϭ 8 Hz). 13C NMR (DMSO-d6/D2O): 165.4, 165.0, 143.0, 141.9, 138.5, 134.2,
130.6, 130.3, 129.3, 128.9, 127.5, 127.0, 126.6, 125.6, 125.0. FABMS: m/e 321 (M
ϩ 1). Analysis calculated for C18H16N4S · 2HCl (396.35): C, 54.96; H, 4.61, N,
14.24. Found: C, 54.69; H, 4.74; N, 14.02.
2-{3-(2-Imidazolino)phenyl}-5-{4(2-imidazolino)phenyl}thiophene dihydro-
chloride (compound 25). A suspension of the imidate ester dihydrochloride (Fig.
1, f) (0.9 g, 0.002 mol) described above with ethylenediamine (0.24 g, 0.004 mol)
in 30 ml of dry ethanol was heated at reflux for 12 h. The solvent was removed
under reduced pressure, the solid was suspended in ϳ50 ml of water, and the pH
was adjusted to 10 with 2 M NaOH (aqueous). The solid was filtered, washed
with water, and dried (free base: white solid with a melting point of 250 to
253°C). This solid was dissolved in hot ethanol, saturated with HCl gas at 0°C,
and stirred at 50°C for 2 h. After addition of 40 ml of dry ether, the precipitated
salt was filtered, washed with ether, and dried under a vacuum at 70°C for 24 h
to yield 0.76 g (83.7%) of a pale yellow solid with a melting point of 360 to 362°C
dec. 1H NMR (DMSO-d6/D2O): 8.27 (dd, 1H, J ϭ 1.6, J ϭ 8 Hz), 8.02 (md, 1H,
J ϭ 8 Hz), 7.96 (d, 4H, J ϭ 8.4 Hz), 7.89 (d, 4 H, J ϭ 8.4 Hz), 7.84 (md, 1H, J
ϭ 8 Hz), 7.75 (d, 1H, J ϭ 4 Hz), 7.70 (d, 1H, J ϭ 4 Hz), 7.67 (dd, 1H, J ϭ 8 Hz),
4.01 (s, 4H), 3.98 (s, 4H). 13C NMR (DMSO-d6/D2O): 165.1, 164.9, 143.2, 142.0,
139.1, 134.5, 131.3, 130.7, 129.8, 127.9, 127.2, 125.9, 125.4, 123.3, 121.1. FABMS:
m/e 321 (M ϩ 1). Analysis calculated for C22H20N4S · 2HCl · 0.5H2O (454.43):
C, 58.14; H, 5.10, N, 12.33. Found: C, 58.00; H, 5.16; N, 12.21.
9-Carboethoxymethyl-3,6-dicyanocarbazole. Sodium hydride (60% dispersion
in mineral oil, 0.82 g, 0.021 mol) was washed twice in hexane, dried under
reduced pressure, and suspended in dry dimethylformamide (50 ml) under N2.
3,6-Dicyanocarbazole (25) (3.30 g, 0.015 mol) was added, and the mixture was
stirred for 20 min. Ethyl bromoacetate (3.6 ml, 0.0325 mol) was added, and the
mixture was stirred overnight. The reaction mixture was poured into water (800
ml). The crude product was filtered off, dried, and then purified by suspension in
hot acetone (300 ml) to give a white powder (3.19 g, 69.3%) with a melting point
of 275 to 277°C. 1H NMR (DMSO-d6): 8.66 (s, 2H), 7.95 (dd, 2H, J ϭ 8.6 and 1.5
Hz), 7.88 (d, 2H, J ϭ 8.6 Hz), 5.56 (s, 2H), 4.16 (q, 2H, J ϭ 7.1 Hz), 1.21 (t, 3H,
J ϭ 7.1 Hz). Analysis calculated for C18H13N3O2 · 0.2H2O: C, 70.44; H, 4.40; N,
13.69. Found: C, 70.31; H, 4.35; N, 13.64.
9-Carboethoxymethyl-3,6-diisopropylamidinocarbazole dihydrochloride (com-
pound 36). 9-Carboethoxymethyl-3,6-dicyanocarbazole (3.04 g, 0.01 mol) and
anhydrous ethanol (10 ml, 0.17 mol) were added to 1,4-dioxane (125 ml) satu-
rated with hydrogen chloride at Ϫ5°C. The stirred reaction mixture was sealed
and allowed to warm to room temperature. After 10 days, the reaction mixture
was purged with nitrogen and diluted with dry ether. The crude diimidate
intermediate (4.40 g, 93.8%) was filtered off under N2, suspended in anhydrous
ethanol (50 ml), and treated with isopropyl amine (19 ml, 0.22 mol, freshly
distilled from KOH). The reaction mixture was sealed and stirred overnight. The
precipitated product was filtered off, washed with ether, and dried with ether to
give a white powder (2.14 g, 43.2%) with a melting point of 250 to 255°C. 1H
NMR (DMSO-d6): 9.68 (d, 2H, J ϭ 8.0 Hz), 9.50 (s, 2H), 9.21 (s, 2H), 8.76 (s,
2H), 7.91 (dd, 4H, J ϭ 8.9 Hz), 5.61 (s, 2H), 4.20 (m, 2H), 4.15 (q, 2H, J ϭ 7.1
Hz), 1.34 (d, 12H, J ϭ 6.3 Hz), 1.21. (t, 3H, J ϭ 7.1 Hz). FABMS: m/z 422 (MHϩ
for free base). Analysis calculated for C24 H31N5O2 · 0.2HCl · 2H2O: C, 56.25; H,
6.88; N, 13.67; Cl, 13.84. Found: C, 56.33; H, 6.90; N, 13.72; Cl, 13.73.
4؅,5-Dibromo-2-nitrobiphenyl (Fig. 2, i). Copper powder (14.30 g, 225 mg
atoms) was added over 10 min to a mechanically stirred molten mixture of
2,4-dibromonitrobenzene (Fig. 2, g) (21) (21.35 g, 0.076 mol) and 1-bromo-4-
iodobenzene (Fig. 2, h) (26.68 g, 0.094 mol). The mixture was maintained at 150
to 175°C (bath temperature) for 4 h. The reaction mixture was extracted with
ethyl acetate. The extracts were adsorbed onto silica gel (100 g) and chromato-
graphed on a column of the same (1 kg, 8.5 by 40 cm), eluting with 5% ethyl
acetate in hexane. Fractions containing product pure by TLC were evaporated to
a yellow solid (16.34 g). Recrystallization from ethanol gave yellow crystals (12.03
g, 44.3%) with a melting point of 105°C. 1H NMR (CDCl3): 7.79 (d, 1H, J ϭ 8.7
Hz), 7.65 (dd, 1H, J ϭ 8.7 and 2.1 Hz), 7.57 (d, 2H, J ϭ 8.5 Hz), 7.57 (d, 1H, J
ϭ 2.1 Hz), 7.17 (d, 2H, J ϭ 8.5 Hz). Analysis calculated for C12H7Br2NO2: C,
40.37; H, 1.98; N, 3.92. Found: C, 40.45; H, 1.99; N, 3.86.
2,6-Dibromocarbazole (Fig. 2, j). A solution of 4Ј,5-dibromo-2-nitrobiphenyl
(Fig. 2, i) (17.85 g, 0.050 mol) in triethylphosphite (20 ml, 0.117 mol) was stirred
at 165°C (bath temperature) under N2 for 20 h. The residue was chromato-
graphed on a column of silica gel (600 g, 7 by 40 cm) eluting with 5% ethyl
acetate in hexane. Fractions containing product pure by TLC were evaporated to

800
BRENDLE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. Synthesis of carbazole diamidine compound 37.
give a white powder (7.88 g, 48.5%) with a melting point of 173 to 174°C. 1H
NMR (CDCl3): 8.15 (d, 1H, J ϭ 1.8 Hz), 8.08 (brs, 1 H), 7.86 (d, 1H, J ϭ 8.3 Hz),
7.58 (d, 1H, J ϭ 1.6 Hz), 7.51 (dd, 1H, J ϭ 8.5 and 1.8 Hz), 7.36 (dd, 1H, J ϭ 8.3
and 1.6 Hz), 7.31 (d, 1H, J ϭ 8.5 Hz). EIMS: m/e 325 (Mϩ). Analysis calculated
for C12H7Br2N: C, 44.35; H, 2.17; N, 4.31; Br, 49.17. Found: C, 44.44; H, 2.12; N,
4.25; Br, 49.06.
2,6-Dicyanocarbazole (Fig. 2, k). Copper(I) cyanide (7.54 g, 0.0842 mol) was
added to a solution of 2,6-dibromocarbazole (Fig. 2, j) (7.71 g, 0.0237 mol) in
N,N-dimethylacetamide (100 ml). The mixture was stirred at reflux under N2 for
18 h. The resulting precipitate was filtered off and stirred for 1.5 h in water
containing ethylenediamine (25 ml). The solid was filtered off and stirred for 1 h
in water containing NaCN (20 g). The dried crude solid was extracted into CHCl3
in a Soxhlet apparatus. The residue remaining in the thimble (2.4 g) was dis-
solved in DMSO and filtered through Norit-A. The filtrate was diluted with water
to give a precipitate. The solid was filtered off and treated with an aqueous
solution containing FeCl3 (10 g) and concentrated HCl (10 ml). The solid was
filtered off and dried to give an off-white powder (1.58 g, 30.6%) with a melting
point of Ͼ300°C. 1H NMR (DMSO-d6): 12.88 (s, 1H) 8.86 (s, 1H), 8.43 (d, 1H,
J ϭ 8.1 Hz), 8.08 (s, 1H), 7.85 (dd, 1H, J ϭ 8.5 and 1.4 Hz), 7.71 (d, 1H, J ϭ 8.5
Hz), 7.65 (dd, 1H, J ϭ 8.1 and 1.2 Hz). Analysis calculated for C14H7N3 · 0.2H2O:
C, 76.15; H, 3.38; N, 19.03. Found: C, 76.05; H, 3.45; N, 18.98.
tomycin (50 ␮g/ml). Murine monocyte-like J774.G8 macrophages were main-
tained by serial passage in Dulbecco’s modified Eagle medium (DMEM) sup-
plemented with 10% heat-inactivated fetal calf serum, 2.0 mM L-glutamine,
penicillin at 50 U/ml, and streptomycin at 50 ␮g/ml.
Drug susceptibility assays. The susceptibility of the growth of L. donovani
amastigote-like forms to the compounds was measured in a 3-day assay using the
CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit (Promega)
as previously described (34). A SpectraMax Plus microplate reader (Molecular
Devices, Sunnyvale, Calif.) was used to quantitate reduction of the tetrazolium
dye, which is proportional to cell density, by measuring the absorbance in each
well at 490 nm. IC50s (the concentrations of the compounds that inhibited
parasite growth by 50% compared to that of an untreated control) were deter-
mined with the software program SoftMax Pro (Molecular Devices) using the
dose-response equation y ϭ {(a Ϫ d)/[1 ϩ (x/c)b]} ϩ d, where x is the drug
concentration, y is the absorbance at 490 nm, a is the upper asymptote, b is the
slope, c is the IC50, and d is the lower asymptote.
The toxicity of the compounds to J774.G8 macrophages was also measured
with the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit
(Promega). A 1-week-old, confluent culture of J774.G8 macrophages was washed
three times with Hanks balanced salt solution without calcium and magnesium
(Sigma). The cells were then incubated at 37°C in a humidified 5% CO2 incu-
bator for 15 min with a trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA ⅐ 4Na)
solution (GIBCO BRL). Cells were subsequently detached with a cell scraper
and suspended by vigorous pipetting. Macrophages were then resuspended at a
concentration of 5 ϫ 105/ml in DMEM without phenol red indicator supple-
mented with 10% heat-inactivated fetal calf serum containing penicillin (50
U/ml) and streptomycin (50 ␮g/ml). Subsequently, 200 ␮l of this cell solution was
added to individual wells of 96-well flat-bottom plates (Costar) and attachment
of the macrophages to the bottom of the plate was allowed to occur over a period
of 48 h at 37°C in a humidified 5% CO2 incubator. The medium was then
removed, and serial dilutions of drugs in the same DMEM were added to each
well. After 72 h of incubation with the test compounds, again at 37°C in a
humidified 5% CO2 incubator, cell viability was determined with the CellTiter 96
Assay Kit by adding 40 ␮l of assay solution to each well. One to 2 h of incubation
was performed at 37°C to allow color development, and then the absorbance of
each well at 490 nm was measured in a SpectraMax Pro microplate reader
(Molecular Devices). The four-parameter curve given earlier was used to deter-
mine the IC50s of the compounds tested.
2,6-Diamidinocarbazole dihydrochloride (compound 37). 2,6-Dicyanocarba-
zole (Fig. 2, k) (1.10 g, 0.005 mol) was reacted with anhydrous ethanol (5.0 ml,
0.089 mol) in 1,4-dioxane (75 ml) saturated with hydrogen chloride as described
above. After 14 days, the crude diimidate intermediate (2.12 g, 125%) was
collected by filtration, suspended in anhydrous ethanol (15 ml), and treated with
a solution of ammonia (4.0 g, 0.0023 mol) in ethanol (50 ml). The mixture was
sealed and stirred for 62 h. The precipitated product was filtered off and washed
with ether to give a tan powder (1.23 g, 75%) with a melting point of Ͼ300°C. 1
H
NMR (DMSO-d6): 9.39 (brs, 7H), 8.91 (s, 1H), 8.39 (d, 1H, J ϭ 8.1 Hz), 8.08 (s,
1H), 7.96 (d, 1H, J ϭ 8.5 Hz), 7.76 (d, 1H, J ϭ 8.5 Hz), 7.70 (d, 1H, J ϭ 8.1 Hz.
FABMS: m/e 252 (MHϩ of free base). Analysis calculated for C14H13N5 · 2 HCl
· 0.3 H2O: C, 51.01; H, 4.77; N, 21.25; Cl, 21.51. Found: C, 51.25; H, 4.79; N,
21.08; Cl, 21.42.
Other chemicals. Ethyl bromoacetate and 1-bromo-4-iodobenzene were pur-
chased from Aldrich Chemical Co., Milwaukee, Wis. Other chemicals were obtained
from the Sigma Chemical Company unless otherwise indicated. Tissue culture media
and supplements were obtained from either the Sigma Chemical Company or
GIBCO BRL. Stock solutions were prepared in DMSO and stored at 4°C.
Cell lines. L. donovani (World Health Organization designation, MHOM/SD/
62/1S-CL2D) promastigotes and axenic amastigote-like parasites were main-
tained in modified RPMI medium as previously described (34). L. mexicana
(World Health Organization designation, MNYC/BCZ/62/M379) promastigotes
were maintained at 26°C in Schneider’s medium (GIBCO BRL) supplemented
with 20% fetal bovine serum (Sigma) containing penicillin (50 U/ml) and strep-
In the Leishmania-infected macrophage assay, initial steps were identical to
those used in the macrophage toxicity assay. After washing and trypsinization
of the macrophage cell line as described above, J774.G8 cells were mixed with
a late-log-phase culture of L. mexicana promastigotes to yield a solution
containing 5 ϫ 105 macrophages/ml and 25 ϫ 105 promastigotes/ml in the
supplemented DMEM medium described previously. The macrophage-para-
site mixture was then pipetted into 96-well flat-bottom plates at 200 ␮l/well.

VOL. 46, 2002
ANTILEISHMANIAL AROMATIC DICATIONS
801
Infection and attachment of the macrophages were allowed to occur over a
period of 48 h at 33°C in a humidified 5% CO2 incubator. Wells were washed
three times with Hanks balanced salt solution to remove extracellular para-
sites, and then serial dilutions of drugs in supplemented DMEM were added
to each well. The plate was returned to the CO2 incubator for an additional
72 h of incubation at 33°C. Macrophages in each well were subsequently
detached by pipetting and scraping with the tip of a microliter pipettor. A
75-␮l volume of the cell suspension was transferred to a Cytospin funnel, and
the cells were centrifuged onto microscope slides at 800 rpm for 5 min with
a Cytospin centrifuge (Shandon). The slides were allowed to air dry and were
then fixed in methanol for 5 s. After evaporation of the methanol, the slides
were stained with 5% Giemsa stain (Fisher) in phosphate buffer (3.1 mM
dibasic potassium phosphate, 8.3 mM monobasic sodium phosphate) for 45
min. After thorough washing in flowing tap water, the slides were allowed to
air dry before being viewed by oil immersion microscopy to determine the
percentage of infected cells.
Other substitutions to the amidine group in this series essen-
tially inactivate compounds 2, 5, and 6 against Leishmania.
When the 2,5-disubstituted furan possesses a methyl group at
position 3, cyclopropyl or isobutyl substitution of the amidine
group, as in compounds 8 and 9, confers greater antileishma-
nial activity than isopropyl substitution, as in compound 7.
Compound 9 was also 6.1-fold more active against Leishmania
than against the macrophage cell line. Addition of a carboxy-
ethyl group at position 4 of the furan ring deactivates com-
pounds 10 and 11 compared to compounds 7 and 8. In com-
pounds 12 to 16, methyl groups are present at both the 3 and
4 positions of the furan ring. Amidine, isopropyl amidine,
cyclopropyl amidine, and cyclopentyl amidine compounds 12
to 15 have similar activities, while ditetrahydropyrimidine com-
pound 16 is less active. It is interesting that while compound 1
is 6.5-fold more active than its dimethyl furan analog com-
pound 12, cyclopentyl diamidines 4 and 15 are equally active,
while the isopropyl diamidine-substituted dimethyl furan com-
pound 13 is 2.8-fold more active than corresponding furan
compound 3.
Five 2,4-diphenyl furans (compounds 17 to 21) were also
tested. These compounds all possessed activity against L. do-
novani, with 2,4-bis-(4-amidinopheny)furan 17 being about
twice as active as pentamidine and corresponding 2,5-diphenyl
furan compound 1. Compound 17 was also 19-fold more active
against the parasites than against the macrophage cell line.
Isopropyl and cyclopentyl diamidines 18 and 21 were also 5.0-
fold and 3.4-fold more active than their 2,5-disubstituted coun-
terparts (compounds 3 and 4, respectively). Four 2,5-diphenyl
thiophenes (compounds 22 to 25) displayed outstanding in
vitro antileishmanial activity. 2,4-Bis-(4-amidinophenyl)furan
22 exhibited the best activity of any of the compounds studied,
possessing 6.2-fold greater antiparasitic activity than pentami-
dine. This compound was 155-fold more potent against Leish-
mania than against the macrophage cell line. Isomeric diami-
dine compound 23 was 3.2-fold more potent than pentamidine
but was less active and more toxic than compound 22. Isopro-
pyl diamidine compound 24 was less active than diamidine
compounds 22 and 23 but possessed superior antiparasitic ac-
tivity than corresponding 2,5- and 2,4-diphenyl furan isopropyl
diamidine compounds 3 and 13.
RESULTS
Chemistry. The multistep synthesis of novel unsymmetrical
thiophene dications 23 and 25 is given in Fig. 1. Stille coupling
between 2-tributylstannylthiophene (a) and 4-bromobenzoni-
trile (b) gave 2-(4-cyanophenyl)thiophene (c), which was bro-
minated to provide 2-bromo-5-(4-cyanophenyl)thiophene (d).
This thiophene derivative was coupled with 3-cyanophenylbo-
ronic acid to give 2-(3-cyanophenyl)-5-(4-cyanophenyl)-thio-
phene (e). The unsymmetrical dicyanophenyl thiophene was
converted to the corresponding diimidate ester (f) by treat-
ment with ethanolic HCl, and compound f was used to synthe-
size both compounds 23 and 25. The former was obtained by
treatment of the diimidate ester with ethanolic ammonia, while
the latter was provided by reaction of the diimidate ester with
ethylenediamine in ethanol. Figure 2 outlines the multistep
synthesis of unsymmetrical carbazole diamidine (compound
37). A crossed Ullmann reaction of 2,4-dibromonitrobenzene
(g) (21) and 1-bromo-4-iodobenzene (h) gave 4Ј,5-dibromo-2-
nitrobiphenyl (i). Ring closure giving 2,6-dibromocarbazole (j)
was effected with triethyl phosphite. Cyanodebromination with
copper(I) cyanide gave 2,6-dicyanocarbazole (k). Subjection of
the latter to standard Pinner reaction conditions (25) gave the
asymmetric diamidine (compound 37), isolated as the dihydro-
chloride salt.
We examined different classes of cationic molecules for ac-
tivity against L. donovani axenic amastigotes and compared
their activity to that of pentamidine, which possesses an IC50 of
2.59 Ϯ 0.54 ␮M against these parasites (Table 1). For com-
pounds possessing IC50s against the parasites that are near or
less than 10 ␮M, the toxicity of these agents against a J774.G8
macrophage cell line was also determined.
Diphenyl furans and thiophenes. The in vitro antileishma-
nial activities of a wide range of diphenyl furans and thio-
phenes are reported in Table 1. Compounds 1 to 16 are 2,5-
diphenyl furan dications. When the furan ring possesses
hydrogen atoms at positions 3 and 4, as in compounds 1 to 6,
2,5-bis-(4-amidinophenyl)furan (compound 1) is the most ac-
tive of this series, with antileishmanial activity that is indistin-
guishable from that of pentamidine. This compound is 9.7-fold
less toxic to the macrophage cell line than to L. donovani.
Substituting a cyclopentyl group for hydrogen at each of the
amidine groups, as in compound 4, decreases its antileishma-
nial activity 5.7-fold, and a further decrease in activity is ob-
served with the isopropyl substitution found in compound 3.
Carbazoles. Twelve carbazoles were also tested for in vitro
antileishmanial activity (Table 2). These carbazoles were dis-
ubstituted at either the 3 and 6 positions (compounds 26 to 28),
the 2 and 7 positions (compounds 29 to 36), or the 2 and 6
positions (compound 37). Compounds 26 to 28 and compound
37 were inactive against L. donovani. In the 2,7-substituted
carbazole series, however, diamidine 29 was only 3.1-fold less
active than pentamidine and was 8.6-fold more potent against
the parasites than against the macrophage cell line. The iso-
propyl diamidine in this series, compound 35, was nearly as
active as diamidine 29 and was less toxic than compound 29 to
the macrophage cell line. Diimidazoline compound 30 was the
only other compound in this series that possessed measurable
antileishmanial activity. Compound 31, differing from com-
pound 30 only by methylation of the carbazole nitrogen, did
not display antiparasitic activity.
Dibenzofurans and dibenzothiophenes. Closely analogous
to the carbazole dications are dibenzofuran and dibenzothio-
phene compounds 38 to 51 (Table 3). In general, the dibenzo-

802
BRENDLE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Structures and in vitro activities of diphenyl furans and thiophenes
a
IC50 (␮M) vs:
Compound
(reference)
X
Y1
Y2
Y3
L. donovani
J774.G8 cell line
Pentamidine
2.59 Ϯ 0.54
Ͼ100
1 (11)
2 (11)
2.76 Ϯ 0.60
Ͼ100
26.84 Ϯ 2.44
NTb
3 (8)
4 (8)
60.57 Ϯ 17.01
15.75 Ϯ 5.05
NT
NT
5 (7)
Ͼ100
NT
6 (7)
7 (6)
Ͼ100
NT
NT
26.58 Ϯ 7.9
8 (6)
9 (6)
14.21 Ϯ 0.41
10.71 Ϯ 0.48
NT
65.12 Ϯ 13.25
10 (6)
11 (6)
53.67 Ϯ 9.79
68.70 Ϯ 9.39
NT
NT
12 (11)
13
17.96 Ϯ 2.11
21.27 Ϯ 3.2
NT
NT
14
14.20 Ϯ 1.63
NT
Continued on following page

VOL. 46, 2002
ANTILEISHMANIAL AROMATIC DICATIONS
803
TABLE 1—Continued
a
IC50 (␮M) vs:
Compound
(reference)
X
Y1
Y2
Y3
L. donovani
J774.G8 cell line
NT
15
16
13.17 Ϯ 0.75
28.38 Ϯ 5.85
NT
17 (14)
18 (14)
1.30 Ϯ 0.21
24.71 Ϯ 1.71
12.07 Ϯ 0.74
NT
19 (14)
20 (14)
10.39 Ϯ 0.41
7.12 Ϯ 1.90
77.92 Ϯ 24.3
16.28 Ϯ 5.14
21 (14)
4.62 Ϯ 1.18
Ͼ100
22 (12)
23
0.42 Ϯ 0.08
0.82 Ϯ 0.03
65.3 Ϯ 4.6
9.63 Ϯ 0.43
24
25
7.74 Ϯ 0.25
21.26 Ϯ 2.58
12.14 Ϯ 1.81
NT
a Mean Ϯ standard deviation of at least two independent measurements.
b NT, not tested.
furans were ineffective against Leishmania in vitro. Only 3,7-
substituted diamidine compound 42 and corresponding
isopropyl diamidine compound 44 possessed weak antiparasitic
activity. Corresponding dibenzothiophene compounds 48 and
51 were more potent, however. Diamidine compound 48 was
only 4.1-fold less potent than pentamidine, and isopropyl dia-
midine compound 51 possessed 2.5-fold less antileishmanial
activity than pentamidine. Isopropyl diamidine compound 51
was also relatively nontoxic to the macrophage cell line.
Benzimidazoles. Seven benzimidazole-containing cations
have also been examined for activity against L. donovani axenic
amastigotes (Table 4). Cyclopentyl diamidine compound 55
displayed moderate activity against the parasites. When phenyl
substituents were added as spacers between the central 2,5-
disubstituted furan ring and the cyclopentyl amidine-contain-
ing benzimidazole ring systems (compound 58), antileishma-
nial activity decreased 3.9-fold. The only other benzimidazole
compound that possessed measurable antiparasitic activity in
this series was cyclopropyl diamidine-containing compound 57.
Assays with Leishmania-infected macrophages. Agents
with activity against axenic amastigotes comparable or superior
to that of pentamidine (compounds 1, 17, and 21 to 23) were
also tested for activity in J774.G8 cells infected with L. mexi-
cana. In this assay, the clinical antileishmanial agent ampho-
tericin B reduced parasite burdens in infected cells with an
IC50 of 46 Ϯ 6 nM (mean Ϯ standard deviation of four inde-
pendent experiments), while pentamidine was inactive. We
observed no reduction in parasitemia in L. mexicana-infected
macrophages at concentrations of compounds 1, 17, and 21 to
23 that were nontoxic to the J774.G8 cell line.

804
BRENDLE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Structures and in vitro activities of carbazoles
a
IC50 (␮M) vs:
Compound
(reference)
Positions
Y1, Y2
Y1
Y2
R
L. donovani
J774.G8 cell line
NTb
26 (25)
27 (25)
28 (25)
3, 6
H
Ͼ100
Ͼ100
Ͼ100
3, 6
3, 6
H
H
NT
NT
29 (25)
2, 7
H
7.92 Ϯ 2.92
67.88 Ϯ 6.37
30 (25)
31 (25)
32 (25)
33 (25)
2, 7
2, 7
2, 7
2, 7
H
26.45 Ϯ 3.99
Ͼ100
NT
NT
NT
NT
CH3
H
Ͼ100
CH3
Ͼ100
34 (25)
35 (5)
2, 7
2, 7
H
H
Ͼ100
NT
10.19 Ϯ 2.45
Ͼ100
36
37
3, 6
2, 6
Ͼ100
Ͼ100
NT
NT
H
a Mean Ϯ standard deviation of at least two independent measurements.
b NT, not tested.
DISCUSSION
diphenyl furan and thiophene dications described in Table 1
possess the best antileishmanial activity. Three major classes of
diphenyl furans and thiophenes were tested: 2,5-diphenyl
furans, 2,4-diphenyl furans, and 2,5-diphenyl thiophenes. In
each class, the diamidines were the most potent, with 2,5-bis-
Data presented in Tables 1 to 4 indicate that several of the
dications tested possess in vitro antileishmanial activity com-
parable or superior to that of the clinical antileishmanial agent
pentamidine. Of the compounds examined in this work, the

VOL. 46, 2002
ANTILEISHMANIAL AROMATIC DICATIONS
805
(4-amidinophenyl)thiophene (compound 22) possessing the
best activity of all of the agents tested. Since the 2,4-diphenyl
furans were more active than their 2,5-diphenyl furan counter-
parts, it will be of interest to prepare 2,4-diphenyl thiophene-
containing dications in the future.
TABLE 3. Structures and in vitro activities of dibenzofurans and
dibenzothiophenes
Addition of an alkyl group to one of the nitrogen atoms of
the amidine group decreased the antileishmanial potency of
the diphenyl furans and thiophenes. In the 2,5-diphenyl furan
series, substitution of an alkyl group on the amidino nitrogen
generally leads to an increase in DNA binding affinity, and in
particular, dialkylamidine compounds 3 and 4 bind more avidly
to the duplex oligomer d(CGCGAATTCGCG)2 than does 2,5-
bis-(4-amidinopheny)furan compound 1 (8). However, differ-
ent aromatic dication analogs of pentamidine can also have
distinct preferences for specific DNA sequences (32) and it is
conceivable that particular DNA sequences could be targets
for the dications in Leishmania. Although there appears to be
a correlation between DNA binding affinity and the antipneu-
mocystis activity of such compounds (8), DNA binding affinity
may not be the only factor contributing to the antileishmanial
activity of these compounds. Multiple mechanisms of action
have been proposed for pentamidine in kinetoplastid parasites,
including inhibition of serine proteases (22), inhibition of to-
poisomerase II (29), and disruption of polyamine metabolism
(10). With regard to polyamine metabolism, cationic poly-
amine analogs themselves have displayed activity against kin-
etoplastid parasites (35). It is therefore possible that the com-
pounds presented in this work interact at several target sites
within the parasite and that addition of an alkyl group to the
amidine function in the diphenyl furans and thiophenes leads
to unfavorable interactions with an important, as yet uniden-
tified, parasite receptor.
a
IC50 (␮M) vs:
Compound
(reference)
Positions
Y1, Y2
Y
X
J774.G8
cell line
L. donovani
38 (33)
39 (33)
2, 8
2, 8
O
Ͼ100
NTb
O
Ͼ100
NT
40 (33)
41 (33)
2, 8
2, 8
O
O
Ͼ100
Ͼ100
NT
NT
42 (33)
3, 7
O
49.15 Ϯ 5.6
NT
43 (33)
44 (33)
3, 7
3, 7
O
O
Ͼ100
NT
NT
83.96 Ϯ 22.3
The carbazoles, dibenzofurans, dibenzothiophenes, and ben-
zimidazoles presented in Tables 2 to 4 were less active against
L. donovani than were the diphenyl furans and thiophenes.
Since amidine and alkylamidine substitutions also occurred in
the molecules presented in Tables 2 to 4, the geometry of the
substituents imposed by the carbazole, dibenzofuran, dibenzo-
thiophene, and benzimidazole linkers must be less favorable
for antileishmanial activity compared to the diphenyl furans
and thiophenes. In the carbazole series, the 2,7-substituted
diamidines and diisopropyl amidines are active while the
corresponding 3,6-substituted compounds are not. In the diben-
zofuran and dibenzothiophene systems, which are, by conven-
tion, numbered differently than the carbazoles, the 3,7-sub-
stituted compounds are active while the 2,8-substituted
compounds are not. Thus, the positioning of the amidine and
isopropyl amidine groups is more favorable for antileishmanial
activity in the 2,7-substituted geometry (carbazole numbering
system). It is also interesting that in the molecules present in
Tables 2 to 4, the amidines are not always the most active. In
the 2,7-disubstituted carbazole series, diamidine 29 and diiso-
propyl amidine 35 are equally potent within experimental er-
ror. The same is true of 3,7-disubstituted dibenzothiophene
compounds 48 and 51, and in the case of the benzimidazoles,
the diamidines are less active than alkyl-substituted amidines.
The distinct structure-activity relationships among the differ-
ent classes of agents are consistent with the hypothesis that the
aromatic dications have multiple action mechanisms and also
45 (26)
46 (26)
2, 8
2, 8
S
Ͼ100
Ͼ100
NT
NT
SO2
47 (26)
48 (26)
49 (26)
50 (26)
2, 8
3, 7
2, 8
3, 7
S
S
S
S
Ͼ100
10.60 Ϯ 3.9
Ͼ100
NT
NT
NT
NT
Ͼ100
51 (26)
3, 7
S
6.46 Ϯ 1.04 Ͼ100
a Mean Ϯ standard deviation of at least two independent measurements.
b NT, not tested.

806
BRENDLE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 4. Structures and in vitro activities of benzimidazoles
Compound (reference)
52 (13)
X
Y
IC50 (␮M)a
Ͼ100
53 (13)
54 (13)
55 (13)
Ͼ100
Ͼ100
18.24 Ϯ 0.31
56 (17)
57 (17)
Ͼ100
74.70 Ϯ 16.14
58 (17)
71.64 Ϯ 1.89
a Versus L. donovani.
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