Structure-in vitro activity relationships of pentamidine analogues and dication-substituted bis-benzimidazoles as new antifungal agents

Article abstract of DOI:10.1128/aac.42.10.2495

Twenty analogues of pentamidine, 7 primary metabolites of pentamidine, and 30 dicationic substituted bisbenzimidazoles were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. A majority of the com

Full text of DOI:10.1128/aac.42.10.2495

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1998, p. 2495–2502
0066-4804/98/$04.00ϩ0
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 10
Structure-In Vitro Activity Relationships of Pentamidine Analogues
and Dication-Substituted Bis-Benzimidazoles
as New Antifungal Agents
MAURIZIO DEL POETA,1,2 WILEY A. SCHELL,1 CHRISTINE C. DYKSTRA,3 SUSAN JONES,4
RICHARD R. TIDWELL,4 AGNIESZKA CZARNY,5 MIROSLAV BAJIC,5 MARINA BAJIC,5
ARVIND KUMAR,5 DAVID BOYKIN,5 AND JOHN R. PERFECT1*
Department of Medicine, Division of Infectious Diseases and International Health, Duke University Medical Center,
Durham, North Carolina 277101; Department of Pathobiology, Auburn University, Auburn, Alabama 368493;
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 275994; Department of Chemistry, Georgia State University,
Atlanta, Georgia 303035; and Institute of Infectious Diseases and Public Health,
University of Ancona, Ospedale Umberto I°, 60121 Ancona, Italy2
Received 13 February 1998/Returned for modification 1 April 1998/Accepted 10 July 1998
Twenty analogues of pentamidine, 7 primary metabolites of pentamidine, and 30 dicationic substituted bis-
benzimidazoles were screened for their inhibitory and fungicidal activities against Candida albicans and Crypto-
coccus neoformans. A majority of the compounds had MICs at which 80% of the strains were inhibited (MIC80s)
comparable to those of amphotericin B and fluconazole. Unlike fluconazole, many of these compounds were
found to have potent fungicidal activity. The most potent compound against C. albicans had an MIC80 of <0.09
␮g/ml, and the most potent compound against C. neoformans had an MIC80 of 0.19 ␮g/ml. Selected compounds
were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albi-
cans, and fluconazole-resistant strains of C. albicans and C. neoformans. It is clear from the data presented here
that further studies on the structure-activity relationships, mechanisms of action and toxicities, and in vivo
efficacies of these compounds are warranted to determine their clinical potential.
In the last few years the incidence of fungal infections in the
immunocompromised host has increased greatly. The emer-
gence of these pathogens has been followed by both primary
drug resistance and the secondary development of azole-resis-
tant isolates of Candida albicans and Cryptococcus neoformans
(11, 18, 32, 40). In some severely immunocompromised patients
such as those with AIDS, the concept of lifelong suppressive
therapy has been advocated. It is clear that new antifungal
agents with potent and broad-spectrum fungicidal activity are
needed for the effective management of these infections.
The search for new antifungal agents led to the examination
of a series of dicationic aromatic compounds related to pent-
amidine (Table 1). This class of compounds has been shown to
have excellent activity against a number of pathogenic organ-
isms, including Giardia lamblia (4, 5), Toxoplasma gondii (24),
Pneumocystis carinii (10, 34, 36, 38), Plasmodium falciparum
(6), Leishmania mexicana amazonensis (6), and Trypanosoma
brucei (21). In addition, pentamidine has previously been re-
ported to have an inhibition effect on the growth of C. albicans
(33) and C. neoformans (3).
The current work describes the antifungal activities of ana-
logues of pentamidine (Table 1), metabolites of pentamidine
(Table 2), and a series of compounds derived from the highly
potent anti-P. carinii bis-benzimidazoles (Table 3) (38). All of
the compounds were screened for activity against C. albicans
and C. neoformans. Selected compounds that showed high lev-
els of activity against C. albicans and C. neoformans were tested
for their activities against additional strains of these fungi as
well as other important pathologic yeasts and clinically impor-
tant molds (Table 4). The screening was carried out according
to a broth macrodilution reference method for in vitro anti-
fungal susceptibility testing of yeast by the National Committee
for Clinical Laboratory Standards (29). Examination of the
results from these studies reveal several compounds with po-
tent and broad-spectrum antifungal activities.
MATERIALS AND METHODS
Compounds. All compounds were synthesized in the laboratories of two of the
authors (D.B. or R.R.T.). The synthesis and physical properties of the following
compounds have been described previously: compounds 1 to 4, 7 to 14, and 16 to
20 (37); compounds 5 and 6 (18a); compound 15 (20); compounds 21 to 27 (8);
compounds 28, 30, 32, and 47 (17, 35); and compounds 29, 31, 33, 34, and 48 to
50 (17). The remaining compounds, compounds 35 to 46 and 51 to 57, were newly
synthesized, and brief descriptions of the synthesis as well as the physical prop-
erties of these compounds are given as follows.
Melting points (mp’s) were recorded with a Thomas Hoover (Uni-Melt) cap-
illary melting point apparatus and are uncorrected. The proton (1H) and carbon
(
13C) nuclear magnetic resonance (NMR) spectra of the compounds were re-
corded with a Varian GX400 spectrometer, and chemical shifts (␦) are given in
parts per million relative to tetramethylsilane, and coupling constants (J) are
reported in hertz. Mass spectra (MS) were recorded on a VG Instruments 70-SE
spectrometer (Georgia Institute of Technology, Atlanta). Infrared spectra were
recorded with a Michelson 100 (Bomen, Inc.) instrument. Elemental analyses
were performed by Atlantic Microlab Inc. (Norcross, Ga.) and are within Ϯ0.4 of
the theoretical values. All chemicals and solvents were purchased from Aldrich
Chemical Co., St. Louis, Mo., or Fisher Scientific, Pittsburgh, Pa.
4-(N-Cyclopentylamidino)-1,2-phenylene diamine hydrochloride. 4-(N-Cyclo-
pentylamidino)-1,2-phylene diamine hydrochloride is previously unreported and
was used as an intermediate in the synthesis of compounds 38, 42, 45, 54, and 56.
Distilled cyclopentylamine (1.83 g, 0.021 mol) was added to a stirred suspension
of the imidate ester hydrochloride (4.91 g, 0.02 mol) formed from 4-cyano-2-
nitroaniline under Pinner-type conditions in 30 ml of dry ethanol, and the
mixture was stirred for 12 h at room temperature and for 1 h at 50°C. The solvent
was removed under reduced pressure, and the residual thick oily mass was
triturated with dry ether and dried under vacuum to yield 4.7 g (95%); mp, 168
to 179°C decompose (dec). 1H NMR (dimethyl sulfoxide [DMSO]-d6): 9.29 (br,
3H), 8.45 (d, 2H, J ϭ 2.4 Hz), 803 (s, 2H), 7.77 (dd, 1H, J ϭ 2.4 and 8.8 Hz), 4.20
* Corresponding author. Mailing address: Department of Medicine,
Division of Infectious Diseases and International Health, Duke Uni-
versity Medical Center, P.O. Box 3353, Durham, NC 27710. Phone:
(919) 684-2660. Fax: (919) 684-8902. E-mail: perfe001@mc.duke.edu.
2495

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DEL POETA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
(quintet, 1H, J ϭ 6.0 Hz), 2.04 to 2.0 (m, 2H), 1.73 to 1.65 (m, 4H), 1.57 to 1.49
(m, 2H). 13C NMR (DMSO-d6): 160.4, 148.5, 134.1, 129.4, 127.3, 118.9, 114.6,
54.1, 31.2, 23.5. The 4-(N-cyclopentylamidino)-2-nitroaniline (5.0 g, 0.02 mol;
mp, 238 to 240°C dec) was used directly without further characterization (5.0 g,
0.02 mol), and 1.0 g of 10% Pd/C in 130 ml of dry methanol was subjected to
hydrogenation at 50 lb/in2 for approximately 1 h. The catalyst was filtered over
Celite and washed with hot methanol, the solvent of the filtrate was removed
under reduced pressure, the residue was triturated with dry ether, and the solid
was filtered and dried under vacuum at 45°C for 24 h. The yield of light brown
hygroscopic solid was 3.91 g (72%); mp, 170 to 178°C. 1H NMR (DMSO-d6):
8.97 (br s, 1H), 8.82 (br s, 1H), 8.64 (br s, 1H), 6.89 (s, 1H), 6.88 (d, 1H, J ϭ 8.4
Hz), 6.59 (d, 1H, J ϭ 8.4 Hz), 5.40 (br, 2H), 5.0 (br, 2H) 4.17 (m, 2H), 2.10 to 1.98
(m, 2H), 1.82 to 1.76 (m, 4H). 13C NMR (DMSO-d6): 162.4, 140.8, 134.0, 118.4,
115.6, 113.0, 112.5, 53.7, 31.3, 23.5. MS (fast atom bombardment [FAB]) 219
(Mϩ ϩ 1). Analysis calculated for C12H18N4 ⅐ HCl ⅐ H2O: C, 52.84; H, 7.76; N,
20.54. Found: C, 53.10; H, 7.77; N, 20.72.
2,5-Bis[2-(5-amidino)benzimidazoyl]furan hydrochloride (compound 35). A
solution of furan-2,5-dicaboxaldehyde (28) (0.8 g 2 mmol), 4-amidino-1,2-phe-
nylene diamino hydrochloride hydrate (0.8 g, 4 mmol), and 1,4-benzoquinone
(0.432 g, 4 mmol) in ethanol (40 ml) was heated at reflux for 4 h (under nitrogen)
(1). The reaction mixture was cooled to room temperature and the dark solid was
collected by filtration, washed with cold ethanol and anhydrous ether, and dried
to yield 0.55 g (71%) of the free base. This solid was dissolved slowly in hot
ethanol (300 ml) and filtered. The filtrate volume was reduced to 70 ml and was
acidified with HCl-saturated ethanol. After standing overnight in the refrigera-
tor, the green solid was collected by filtration, washed with anhydrous ether, and
dried under vacuum to yield 0.4 g (52%) yield of a solid; mp, Ͼ300°C. 1H NMR
(DMSO-d6): 9.30 (s, 4H); 8.19 (2s, 2H), 7.81, (d, 2H, J ϭ 8.8 Hz), 7.72 (d, 2H,
J ϭ 8.4 Hz), 7.60 (s, 2H). 13C NMR (DMSO-d6/D2O): 166.8, 146.4, 146.1, 142.2,
139.7, 123.4, 122.7, 117.1, 116.1, 115.4, MS (FAB) m/z 385 (Mϩ ϩ 1); HRMS:
calculated mass (free base), 385.1525 (Mϩ ϩ 1); observed mass, 385.1535. Anal-
ysis calculated for C20H16N8O ⅐ 2HCl ⅐ 1.5 H2O: C, 49.59; H, 4.37; N, 23.14.
Found: C, 49.40; H, 4.31; N, 22.96.
2,5-Bis[2-(5-N-isopropylamidino)benzimidazoyl]pyrrole hydrochloride (com-
pound 41). A protocol similar to that described above was used for the conden-
sation of pyrrole-2,5-dicarboxaldehyde and 4-(N-isopropylamidino)-1,2-phenyl-
ene diamine (17, 35) to yield 79% of a yellow-green solid; mp, 287 to 289°C dec.
1H NMR (DMSO-d6/D2O): 8.06 (s, 2H), 7.81 (d, 2H, J ϭ 8.4 Hz), 7.65 (d, 2H,
J ϭ 8.4 Hz), 7.41 (s, 2H), 4.06 (septet, 2H, J ϭ 6.4 Hz), 1.30 (d, 12H, J ϭ 6.4 Hz).
13C NMR (DMSO-d6/D2O): 162.3, 145.8, 138.6, 135.7, 124.7, 124.2, 123.9, 115.7,
115.5, 114.9, 45.7, 21.4. MS (FAB) m/z 468 (Mϩ ϩ 1). Analysis calculated for
C
26H29N9 ⅐ 4HCl: C, 50.90; H, 5.42; N, 20.55. Found: C, 51.54; H, 5.57; N, 20.30.
2,5-Bis[2-(5-N-cyclopentylamidino)benzimidazoyl]pyrrole hydrochloride (com-
pound 42). A protocol similar to that described above was used for the con-
densation of 4-(N-cyclopentylamidino)-1,2-phenylene diamine with pyrrole-2,5-
dicarboxaldehyde (27) to give a 71% yield of a blue-green solid; mp, 290 to
294°C. 1H NMR (DMSO-d6/D2O): 8.0 (s, 2H), 7.77 (d, 2H, J ϭ 8.4 Hz), 7.60 (d,
2H, J ϭ 8.4 Hz), 7.32 (s, 2H), 4.09 (br m, 2H), 2.11 to 1.97 (m, 4H), 1.77 to 1.62
(m, 8H), 1.61 to 1.50 (m, 4H). 13C NMR (DMSO-d6/D2O): 163.1, 145.7, 138.6,
135.6, 124.6, 124.3, 123.8, 115.8, 115.5, 115.1, 55.0, 31.7, 23.9. MS (FAB) m/z 520
(Mϩ ϩ 1). Analysis calculated for C30H33N9 ⅐ 4HCl ⅐ 0.5 H2O: C, 53.42; H, 5.63;
N, 18.68. Found: C, 53.90; H, 5.75; N, 18.16.
1-Methyl-2,5-bis[2-(5-amidino)benzimidazoyl]pyrrole hydrochloride (compound
43). A protocol similar to that described above was used for the condensation of
4-amidino-1,2-phenylene diamine hydrochloride hydrate (17, 35) with 1-methyl-
pyrrole-2,5-dicarboxaldehyde (12) to give a 70% yield of product; mp, Ͼ300°C.
1H NMR (DMSO-d6): 9.38 (br s, 1H), 9.11 (br s, 1H), 8.19 (s, 2H), 7.80 (d, J ϭ
8.4 Hz), 7.73 (dd, 2H, J ϭ 8 and 1.2 Hz), 7.33 (s, 2H), 4.72 (s, 3H). MS (free base)
m/z 398 (Mϩ ϩ 1). Analysis calculated for C21H19N9 ⅐ 3HCl ⅐ H2O: C, 48.06; H,
4.61; N, 24.02. Found: C, 48.16; H, 4.58; N, 23.93.
2,5-Bis{2-[5-(2-imidazolino)]benzimidazoyl}-1-methylpyrrole hydrochloride
(compound 44). A protocol similar to that described above was used for the
condensation of 2-(3,4-diaminophenyl)imidazoline (17, 35) with 1-methylpyr-
role-2,5-dicarboxaldehyde. A yield of 83% of a solid (mp, Ͼ300°C) was obtained.
1H NMR (DMSO-d6): 10.60 (s, 1H), 8.36 (s, 2H), 7.84 (dd, 4H, J ϭ 8.4 and 8 Hz),
7.30 (s, 2H), 4.72 (s, 3H), 4.04 (s, 8H). MS (free base) m/e 450 (Mϩ ϩ 1). Analysis
calculated for C25H23N9 ⅐ 3HCl ⅐ 3H2O: C, 48.98; H, 5.26; N, 20.57. Found: C,
49.20; H, 4.79; N, 20.51.
2,5-Bis[2-(5-N-cyclopentylamidino)benzimidazoyl]-1-methylpyrrole hydrochlo-
ride (compound 45). A protocol similar to that described above was used for the
condensation of 4-(N-cyclopentylamidino)-1,2-phenylene diamine with 1-methyl-
2,5-pyrrole dicarboxaldehyde (12) to give an 85% yield of a blue solid; mp, 324
to 326°C dec. 1H NMR (DMSO-d6/D2O): 8.0 (s, 2H), 7.73 (d, 2H, J ϭ 8.4 Hz),
7.55 (d, 2H, J ϭ 8.4 Hz), 7.13 (s, 2H), 4.57 (s, 3H), 4.14 (quintet, 2H, J ϭ 5.2 Hz),
2.12 to 2.02 (m, 4H), 1.80 to 1.58 (m, 12H). MS (FAB) m/z 534 (Mϩ ϩ 1).
Analysis calculated for C31H35N9 ⅐ 3HCl ⅐ H2O: C: 56.32; H, 6.10; N, 19.07.
Found: C, 56.90; H, 5.97; N, 18.83.
2,5-Bis{2-[5-(2-imidazolino)]benzimidazoyl}furan hydrochloride (compound
36). A protocol similar to that described above was used for the condensation of
2,5-furandicarboxaldehyde (28) and 2-(3,4-diaminophenyl)imidazoline (17, 35)
to give a 38% yield of a green powder; mp, Ͻ300°C. 1H NMR (DMSO-d6): 10.53
(s, 4H), 8.38 (s, 2H), 7.87 (d, 2H, J ϭ 8.5 Hz), 7.83 (d, 2H, J ϭ 8.2 Hz), 7.62 (s,
2H), 4.04 (s, 8H). 13C NMR (DMSO-d6/D2O): 166.3, 146.2, 146.1, 142.3, 139.8,
123.7, 117.6, 116.9, 116.1, 115.5, 45.0. MS (FAB) m/z 437 (Mϩ ϩ 1); HRMS:
calculated mass (free base), 437.1838 (Mϩ ϩ 1); observed mass, 437.1832. Anal-
ysis calculated for C24H20N8O ⅐ 2HCl ⅐ 5H2O: C, 48.08; H, 5.38; N, 18.69. Found:
C, 48.22; H, 5.25; N, 18.51.
2,5-Bis[2-(5-N-isopropylamidino)benzimidazoyl]furan hydrochloride (com-
pound 37). A protocol similar to that described above (compound 35) was used
for the condensation of 2,5-furandicarboxaldehyde (28) and 4-(N-isopropylami-
dino)-1,2-phenylene diamine (17, 35) to give a 54% yield of a yellow-green
powder; mp, Ͼ300°C. 1H NMR (DMSO-d6): 9.60 (s, 1H), 9.58 (s, 1H), 9.45 (s,
2H), 9.04 (s, 2H), 8.06 (s, 2H), 7.82 (d, 2H, J ϭ 8.4 Hz), 7.69 (s, 2H), 7.62 (d, 2H,
J ϭ 8.2 Hz), 4.09 (m, 2H, J ϭ 7.0 Hz), 1.32 (d, 12H, J ϭ 6.3 Hz). 13C NMR
(DMSO-d6/D2O): 162.8, 145.9, 145.1, 140.9, 138.5, 124.5, 124.0, 116.9, 115.9,
115.8, 45.9, 21.7. MS (FAB) m/z 469 (Mϩ ϩ 1); HRMS: calculated mass (free
base), 469.2464 (Mϩ ϩ 1); observed mass, 469.2475. Analysis calculated for
C26H28N8O ⅐ 3HCl ⅐ 2.5H2O: C, 50.12; H, 5.83; N, 17.99. Found: C, 50.45; H,
5.76; N, 17.64.
2,5-Bis[2-(5-N-cyclopentylamidino)benzimidazoyl]furan hydrochloride (com-
pound 38). A protocol similar to that described above was used for the conden-
sation of 2,5-furandicarboxaldehyde (28) and 4-(N-cyclopentylamidino)-1,2-phe-
nylene diamine to give a 77% yield of a yellow-green powder, mp, 287 to 289°C
dec. 1H NMR (DMSO-d6/D2O): 8.07 (s, 2H), 7.82 (d, 2H, J ϭ 8.4 Hz), 7.66 (s,
2H), 7.63 (d, 2H, J ϭ 8.4 Hz), 4.22 to 4.14 (m, 2H), 2.14 to 2.04 (m, 4H), 1.82 to
1.67 (m, 8H), 1.64 to 1.56 (m, 4H). 13C NMR (DMSO-d6): 163.0, 145.4, 144.5,
140.4, 137.7, 123.9, 116.4, 115.5, 115.2, 54.6, 31.5, 23.7. MS (FAB) m/z 521 (Mϩ
ϩ 1). Analysis calculated for C30H32N8O ⅐ 4HCl: C, 54.06; H, 5.44; N, 16.81.
Found: C, 53.80; H, 5.51; N, 16.68.
2,5-Bis[2-(5-N-isopropylamidino)benzimidazoyl]thiophene hydrochloride (com-
pound 46). A protocol similar to that described above was used for the condensation
of 2,5-thiophenedicarboxaldehyde and 4-(N-isopropylamidino)-1,2-phenylene dia-
mine (17, 35) to give a 75% yield of a green-yellow solid; mp, 290 to 292°C dec. 1
H
NMR (DMSO-d6/D2O): 8.18 (s, 2H), 8.05 (s, 2H), 7.77 (d, 2H, J ϭ 8.4 Hz); 7.60 (d,
2H, J ϭ 8.4 Hz), 4.11 (quintet, 2H, J ϭ 6.4 Hz), 1.31 (d, 12H, J ϭ 6.4 Hz). MS (FAB)
m/z 485 (Mϩ ϩ 1). Analysis calculated for C26H28N8S ⅐ 3HCl ⅐ H2O: C, 51.02; H,
5.43; N, 18.31; Cl, 17.38. Found: C, 51.56; H, 5.54; N, 18.09; Cl, 17.37.
2,6-Bis{2-[5-(2-imidazolino)]benzimidazoyl}pyridine hydrochloride (compound
51). A protocol similar to that described above was used for the condensation of
2,6-pyridine carboxyaldehyde and 2-(3,4-diaminophenyl)imidazoline (17, 35) to give
an 85% yield of a solid; mp, Ͼ300°C. 1H NMR (DMSO-d6): 10.71 (s, 1H), 8.51 to
8.49 (m, 4H), 8.30 (m, 1H), 7.96 (m, 4H), 4.05 (s, 8H). MS (free base) m/e 448 (Mϩ
ϩ 1). Analysis calculated for C25H21N9 ⅐ 3HCl ⅐ 3H2O: C, 49.15; H, 4.94; N, 20.63.
Found: C, 49.14; H, 4.68; N, 20.51.
2,6-Bis[2-(5-amidino)benzimidazoyl]pyridine hydrochloride (compound 52).
A protocol similar to that described above was used to condense 2,6-pyridine
dicarboxaldehyde with 4-amidino-1,2-phenylene diamine hydrochloride hydrate
(17, 35) to give an 89% yield of a solid; mp, Ͼ300°C. 1H NMR (DMSO-d6): 9.45
(br s, 1H), 9.12 (br s, 1H), 8.51 (d, 2H, J ϭ 8 Hz), 8.34 to 8.28 (m, 3H), 7.94 (d,
2H, J ϭ 8.4 Hz), 7.79 (dd, 2H, J ϭ 8.4 and 1.6 Hz), MS (free base) m/z 396 (Mϩ
ϩ 1). Analysis calculated for C21H17N9 ⅐ 3HCl ⅐ 3H2O: C, 45.13; H, 4.69; N,
22.56. Found: C, 45.16; H, 4.58; N, 22.45.
4,4؅-Bis[2-(5-N-isopropylamidino)benzimidazoyl]-1,2-diphenylethane hydro-
chloride (compound 53). 1,2-Bis-(4-cyanophenyl)ethane was prepared in one
step from 2,3-bis-(4-bromophenyl)propanoic acid (13) by the action of CuCN in
dimethylformamide (15) in a 50% yield; mp, 195 to 197°C. 1H NMR (DMSO-d6):
7.68 (d, 4H, J ϭ 8 Hz), 7.40 (d, 4H, J ϭ 8 Hz), 3.01 (s, 4H). 13NMR (DMSO-d6):
146.7, 131.9, 129.3, 118.6, 108.6, 35.8. MS m/e 232 (Mϩ ϩ 1). 1,2-Bis-(4-cyano-
phenyl)ethane was used without further characterization and on treatment with
diisofutylaluminumhydride (DIBAL) gave a white crystalline solid (CHCl3-
ether) of 76% of 1,2-bis-(4-formylphenyl)ethane; mp, 121 to 122°C. 1H NMR
(DMSO-d6): 9.96 (s, 2H), 7.80 (d, 4H, J ϭ 8 Hz), 7.44 (d, 4H, J ϭ 8 Hz), 3.05 (s,
4H). 13C NMR (DMSO-d6/D2O): 192.0, 184.0, 134.2, 129.1, 128.8, 36.0. Analysis
calculated for C16H14O2 ⅐ 0.1 H2O: C, 80.04; H, 5.96. Found: C, 80.09; H, 5.98.
A protocol similar to that described above was used for the condensation of
1,2-bis-(4-formylphenyl)ethane and 4-(N-isopropylamidino)-1,2-phenylene dia-
mine (17, 35) and gave 75% yield of a purple solid; mp, Ͼ320°C. 1H NMR
2,5-Bis[2-(5-amidino)benzimidazoyl]pyrrole hydrochloride (compound 39). A
protocol similar to that described above was used for the condensation of 4-
amidino-1,2-phenylene diamino hydrochloride hydrate (17, 35) with pyrrole-2,5-
dicarboxaldehyde (27) to yield 0.83 g (76%) of a solid; mp, Ͼ300°C. 1H NMR
(DMSO-d6): 9.48 (br s, 1H), 9.18 (br s, 1H), 8.25 (s, 2H), 7.87 (d, 2H, J ϭ 8.4 Hz),
7.80 (dd, 2H, J ϭ 8.8 and 0.8 Hz), 7.54 (s, 2H). MS (free base) m/e 384 (Mϩ
ϩ
1). Analysis calculated for C20H17N9 ⅐ 3HCl ⅐ 3H2O: C, 43.93; H, 4.73; N, 23.05.
Found: C, 43.61; H, 4.62; N, 22.79.
2,5-Bis{2-[5-(2-imidazolino)]benzimidazoyl}pyrrole hydrochloride (com-
pound 40). A protocol similar to that described above was used for the conden-
sation of pyrrole-2,5-dicarboxaldehyde and 2-(3,4-diaminophenyl)imidazoline
(17, 35) to give an 86% yield of a solid; mp, Ͼ300°C. 1H NMR (DMSO-d6): 10.71
(s, 1H), 8.44 (s, 2H), 7.92 (dd, 2H, J ϭ 8.4 and 1.6 Hz), 7.86 (d, 2H, J ϭ 8.8 Hz),
7.39 (s, 2H), 4.04 (s, 8H). MS (free base) m/e 436 (Mϩ ϩ 1). Analysis calculated
for C24H21N9 ⅐ 3HCl ⅐ 4H2O: C, 46.72; H, 5.23; N, 20.43. Found: C, 46.49; H,
5.11; N, 20.28.

VOL. 42, 1998
NEW ANTIFUNGAL AGENTS
2497
(DMSO-d6/D2O): 8.09 (d, 4H, J ϭ 8 Hz), 8.06 (s, 2H), 7.83 (d, 2H, J ϭ 8.4 Hz),
7.65 (d, 2H, J ϭ 8.4 Hz), 7.48 (d, 4H, J ϭ 8 Hz), 4.03 (br m, 2H), 3.07 (br, 4H),
1.29 (d, 12H, J ϭ 6 Hz). 13NMR (DMSO-d6/D2O): 162.4, 153.0, 146.6, 138.3,
135.4, 129.8, 127.9, 124.8, 124.1, 123.5, 115.5, 115.0, 45.6, 36.2, 21.2. MS (FAB)
m/z 583 (Mϩ ϩ 1) Analysis calculated for C36H38N8 ⅐ 4HCl ⅐ 0.5 H2O: C, 58.61;
H, 5.87; N, 15.19. Found: C, 58.31; H, 5.79; N, 15.02.
4,4؅-Bis[2-(5-N-cyclopentylamidino)benzimidazoyl]-2,5-diphenylfuran hydro-
chloride (compound 54). 2,5-Bis(4-formylphenyl)furan was prepared by reduc-
tion of 2,5-bis-(4-cyanophenyl)furan (2) (1.12 g 0.004 mol) with 1 M DIBAL in
CH2Cl2 (1.83 g, 0.012 mol) to yield 0.77 g (70%) of a pale yellow solid; mp, 173
to 174 4°C (CHCl3-ether). 1H NMR (DMSO-d6/D2O) 10.0 (s, 2H), 8.05 (d, 4H,
J ϭ 7.5 Hz), 7.97 (d, 4H, J ϭ 7.5 Hz), 7.37 (s, 2H). 13C NMR (DMSO-d6/D2O)
192.0, 125.7, 135.0, 134.6, 130.1, 123.9, 111.6. MS m/e 276. Analysis calculated for
1H), 8.05 (s, 1H), 7.95 (d, 1H, J ϭ 8.8 Hz), 7.90 (s, 1H), 7.84 (d, 1H, J ϭ 8.8 Hz),
7.79 (d, 1H, J ϭ 8.4 Hz), 7.64 (d, 1H, J ϭ 8.4 Hz), 7.57 (d, 1H, J ϭ 8.8 Hz), 4.14
(br 2H), 2.13 to 2.06 (m, 4H), 1.81 to 1.56 (m, 12H). 13C NMR (DMSO-d6/D2O):
163.4, 163.1, 156.9, 153.2, 147.6, 145.2, 141.2, 138.8, 136.2, 128.9, 126.5, 124.5,
124.1 123.9, 123.5 122.5, 122.4, 116.9, 116.1, 115.8, 115.7, 115.1, 113.2, 108.8,
54.9, 54.8, 31.7, 23.9. MS (FAB) m/z 571 (Mϩ ϩ 1). Analysis calculated for
34H34N8O ⅐ 4HCl: C, 56.99; H, 5.34; N, 15.64. Found: C, 56.89; H, 5.34; N,
15.53.
2,7-Bis[2-(5-N-isopropylamidino)benzimidazoyl]fluorene hydrochloride (com-
C
pound 57). A mixture of 2,7-dibromofluorene (6.48 g, 0.02 mol) and cuprous
cyanide (5.37 g, 0.06 mol) in 35 ml of quinoline was heated under reflux for 2 h
(followed by thin-layer chromatography with silica gel [Kodak] and a benzene
mobile phase). The mixture was cooled and extracted twice with 150 ml of
chloroform; and the organic layer was stirred with 200 ml of 2 M HCl for 2 h,
washed with water, dried over anhydrous Na2SO4, filtered, dried and concen-
trated, redissolved in a minimum amount of chloroform, and chromatographed
over neutral alumina. The column was first eluted with hexane-ether (2:1) to
remove residual quinoline and finally with ether-CHCl3 (1:1) to afford a fluffy
pale solid (2.72 g; 63%); mp 282 to 284°C of 2,7-dicyanofluorene. 1H NMR
(DMSO-d6/D2O): 8.16 (d, 2H, J ϭ 8.4 Hz), 8.04 (s, 2H), 7.83 (d, 2H, J ϭ 8.4 Hz),
4.07 (s, 2H). 13C NMR (DMSO-d6/D2O): 144.2, 143.3, 130.8, 128.5, 121.7, 118.5,
110.1, 36.0. MS m/z 216 (Mϩ). Analysis calculated for C15H8N2: C, 83.21; H,
3.72; N, 12.95. Found: C, 83.21; H, 3.78; N, 12.99. To a stirred solution of
2,7-dicyanofluorene (2.16 g, 0.01 mol) in 150 ml of dry CH2Cl2 was added
DIBAL (1 M in cyclohexane [4.26 g, 0.03 mol]) under nitrogen at room temper-
ature. The suspension was heated at 40°C for 1 h and cooled, 100 ml of 1 M
H2SO4 was added dropwise, the mixture was stirred for 1 h, and the precipitated
yellow solid was filtered and recrystallized from CHCl3-ether to yield 2,7-di-
formylfluorene as pale crystalline solid (1.6 g; 72%); mp, 218 to 220°C. 1H NMR
(DMSO-d6/D2O): 10.08 (s, 2H), 8.16 (d, 2H, J ϭ 8.0 Hz), 8.11 (s, 2H), 7.95 (d,
2H, J ϭ 8.0 Hz), 4.10 (s, 2H). 13C NMR (DMSO-d6/D2O): 191.9, 145.0, 144.7,
135.6, 128.5, 125.4, 121.1, 36.0. MS m/e 222 (Mϩ). Analysis calculated for
C
18H12O3: C, 91.49; H, 5.12. Found: C, 91.22; H, 5.38.
A protocol similar to that described above was used for the condensation of
4-(N-cyclopentylamidino)-1,2-phenylene diamine with 2,5-bis(4-formylphenyl)
furan to give a 77% yield of a yellow solid; mp 295 to 297°C dec. 1H NMR
(DMSO-d6/D2O): 8.30 (d, 4H, J ϭ 8.4 Hz), 8.05 (d, 4H, J ϭ 8.4), 8.01 (s, 1H),
7.77 (d, 2H, J ϭ 8.4 Hz), 7.56 (d, 2H, J ϭ 8.4 Hz), 7.27 (s, 2H), 4.15 (br, 2H), 2.13
to 2.03 (m, 4H), 1.81 to 1.55 (m, 12H). 13C NMR (DMSO-d6/D2O): 163.7, 154.0,
153.1, 141.2, 138.6, 132.8, 127.9, 126.1, 124.6, 123.3, 123.1, 115.9, 115.7, 111.0,
55.7, 32.3, 24.4. MS (FAB) m/z 673 (Mϩ ϩ 1). Analysis calculated for
C
42H40N8O ⅐ 4HCl: C, 61.61; H, 5.42; N, 13.69. Found: C, 62.28; H, 5.74; N,
13.62.
2,5-Bis[2-(5-amidino)benzimidazoyl]benzo[b]furan hydrochloride (compound
55). A protocol similar to that described above was used for the condensation of
benzo[b]furan-2,5-dicarboxaldehyde and 4-amidino-1,2-phenylene diamine hy-
drochloride hydrate (17, 35) to give a 70% yield of a blue-gray solid; mp, 338 to
340°C dec. 1H NMR (DMSO-d6/D2O): 8.6 (s, 1H), 8.27 (d, 1H, J ϭ 8 Hz), 8.19
(d, 2H, J ϭ 9.6 Hz), 7.89 (d, 1H, J ϭ 8.8 Hz), 7.87 (s, 1H), 7.83 (d, 1H, J ϭ 8.4
Hz), 7.78 (d, 1H, J ϭ 8.4 Hz), 7.73 (d, 1H, J ϭ 8.8 Hz), 7.68 (d, 1H, J ϭ 8 Hz).
13C NMR (DMSO-d6/D2O): 166.4, 165.9, 156.6, 153.5, 147.4, 145.4, 141.6, 139.9,
139.1, 136.9, 128.6, 126.2, 123.3, 122.9, 122.6, 122.2, 122.1, 116.9, 115.8, 115.5,
115.0, 112.8, 108.6. MS (FAB) m/z 435 (Mϩ ϩ 1). Analysis calculated for
C
15H10O2 ⅐ 0.1 H2O: C, 80.41; H, 4.49. Found: C, 80.32; H, 4.63. A protocol
similar to that described above was used for the condensation of 2,7-diformylflu-
orene and 4-(N-isopropylamidino)-1,2-phenylene diamine to give a 72% yield of
a green solid; mp, 310 to 313°C dec. 1H NMR (DMSO-d6/D2O): 8.53 (s, 2H),
8.34 (d, 2H, J ϭ 8.4 Hz), 8.22 (d, 2H, J ϭ 8.4 Hz), 8.11 (s, 2H), 7.85 (d, 2H, J ϭ
8.4 Hz), 7.65 (d, 2H, J ϭ 8.4 Hz), 4.23 (s, 2H), 4.09 (quintet, 2H, J ϭ 6.4 Hz), 1.33
(d, 12H, J ϭ 6.4 Hz). 13C NMR (DMSO-d6/D2O) 162.2, 153.1, 144.9, 143.5,
138.7, 136.3, 126.9, 125.7, 124.3, 123.6, 121.7, 115.6, 114.7, 45.3, 36.7, 21.1. MS
m/z (FAB) 567 (Mϩ ϩ 1). Analysis calculated for C35H34N2 ⅐ 4HCl: C, 58.99; H,
5.37; N, 15.73. Found: C, 59.14; H, 5.59; N, 15.43.
C
24H18N8O ⅐ 3HCl ⅐ H2O: C, 51.30; H, 4.12; N, 19.94. Found: C, 51.72; H, 4.14;
N, 15.64.
2,5-Bis[2-(5-N-cyclopentylamidino)benzimidazoyl]benzo[b]furan hydrochloride
(compound 56). 2-Acetyl-5-bromobenzo[b]furan was prepared as reported in the
literature (14) to give an 86% yield; mp, 110 to 111°C (literature mp, 108 to
111°C). 1H NMR (DMSO-d6):8.02 (d, 1H, J ϭ 1.6 Hz), 7.79 (s, 1H), 7.68 (d, 1H,
J ϭ 9.2 Hz), 7.65 (dd, 1H, J ϭ 9.2 and 1.6 Hz), 2.58 (s, 3H). 13C NMR
(DMSO-d6): 181.6, 153.5, 152.9, 130.9, 128.8, 125.7, 115.9, 114.1, 112.9, 26.2. MS
m/z 239 (Mϩ). The acetyl compound was converted into 5-bromobenzo[b]furan-
2-carboxylic acid in a 77% yield as reported previously (14); mp, 258 to 262°C
(literature mp, 258 to 262°C). 1H NMR (DMSO-d6/D2O): 7.95 (d, 1H, J ϭ 1.6
Test organisms. The fungi tested in the study included two reference strains,
C. neoformans var. neoformans H99 and C. albicans A39, and the following clin-
ical isolates: two strains of fluconazole-resistant C. neoformans var. neoformans
(strains 135.95 and 114.96); two strains of C. neoformans var. gattii (strains 119.95
and 114.95); two strains of fluconazole-resistant C. albicans (strains 102.96 and
103.96); and one strain each of Torulopsis glabrata (strain 142.91), Candida
parapsilosis (strain 111.96), Candida krusei (strain 132.91), Candida tropicalis
(strain 110.96), Candida lusitaniae (strain 111.92), Fusarium solani (strain 152.89),
Aspergillus fumigatus (strain 168.95), and Aspergillus flavus (strain 112.96).
Medium. Antifungal susceptibility testing was performed with RPMI 1640
medium (Sigma Chemical Co., St. Louis, Mo.) with glutamine but without so-
dium bicarbonate and buffered at pH 7.0 with 0.165 M morpholinepropanesul-
fonic acid.
In vitro susceptibility testing. Experiments for determination of MICs were
performed by the broth macrodilution method according to the recommenda-
tions of the National Committee for Clinical Laboratory Standards (29). The
only difference compared to the standardized method was the choice of drug
dilutions, which ranged from 100 to 0.09 ␮g/ml. Briefly, this method specifies the
use of an inoculum grown at 35°C and adjusted to a concentration of 0.5 ϫ 103
to 2.5 ϫ 103 CFU/ml, incubation of the culture at 35°C, and reading at 48 h for
all yeasts except for C. neoformans, for which the results are interpreted at 72 h.
The MIC was defined as the culture with the lowest drug concentration in which
a visual turbidity less than or equal to 80% inhibition compared to that produced
by the growth control tube was observed.
Hz), 7.64 (d, 1H, J ϭ 8.8 Hz), 7.58 (dd, 1H, J ϭ 8.8 and 1.6 Hz), 7.57 (s, 1H). 13
C
NMR (DMSO-d6/D2O): 159.5, 153.6, 147.3, 129.9, 128.9, 125.2, 115.8, 113.9,
112.5. MS m/z 241 (Mϩ). 5-Bromobenzo[b]furan-2-carboxylic acid, which was
used without further characterization, was converted by standard procedures via
the acid chloride into 5-bromo-2-carboxamidobenzo[b]furan in an 81% yield;
mp, 208 to 210°C. 1H NMR (DMSO-d6): 8.2 (br s, 1H), 7.99 (s, 1H), 7.77 (br s,
1H), 7.61 (d, 1H, J ϭ 8.8 Hz), 7.57 (dd, 1H, J ϭ 8.8 and 2 Hz), 7.51 (s, 1H). 13
C
NMR (DMSO-d6/D2O): 159.4, 153.0, 150.5, 129.4, 128.3, 125.2, 115.9, 113.9,
109.0. MS m/e 240 (Mϩ). The amide, which was used without further character-
ization, was dehydrated with POCl3 to form 5-bromo-2-cyanobenzo[b]furan in an
88% yield; mp, 147 to 148°C. 1H NMR (DMSO-d6): 8.0 (s, 1H), 7.97 (s, 1H), 7.67
(br s, 2H). 13C NMR (DMSO-d6): 153.7, 131.1 127.2, 127.1, 125.2, 118.8, 116.7,
113.7, 111.1. MS m/e 222 (Mϩ). 5-Bromo-2-cyanobenzo[b]furan, which was used
without further characterization, was converted via a standard procedure (30)
into 2,5-dicyanobenzo[b]furan in a 79% yield; mp, 166 to 167°C. 1H NMR
(DMSO-d6): 8.39 (s, 1H), 8.14 (s, 1H), 7.97 (d, 1H, J ϭ 8.8 Hz) 7.92 (d, 1H, J ϭ
8.8 Hz). 13C NMR (DMSO-d6): 156.3, 131.5 128.5, 128.2, 125.8, 119.3, 117.9,
113.4, 110.8, 107.7. MS m/e 168 (Mϩ). Analysis calculated for C10H4N2O: C,
71.42; H, 2.39; N, 16.16. Found: C, 71.78; H, 2.46; N, 16.51. 2,5-Diformylbenzo
[b]furan was prepared by reduction of the bis-nitrile with DIBAL, which was
added dropwise to a stirred solution of the bis-nitrile (1.68 g, 0.01 mol) in 150 ml
of dry methylene chloride under nitrogen. The mixture was stirred for 15 min and
allowed to reflux for 40 min, and the mixture was cooled and 100 ml of 1 M
H2SO4 was added dropwise while maintaining the solution temperature below
25°C. The methylene chloride layer was separated, the aqueous layer was ex-
tracted with 100 ml of methylene chloride, and the combined organic phases
were washed with 20% NaHCO3 and dried over Na2SO4. The solvent was
removed under reduced pressure, the residue was triturated with ether-hexane
(1:1), and the white solid was filtered and dried in a vacuum to yield 1.2 g (69%);
mp, 141 to 142°C. 1H NMR (DMSO-d6): 10.1 (s, 1H), 9.92 (s, 1H), 8.48 (d, 1H,
J ϭ 0.8 Hz), 8.10 (s, 1H), 8.08 (dd, 1H, J ϭ 0.8 and 8.88 Hz), 7.90 (d, 1H, J ϭ 8.8
Hz). 13C NMR (DMSO-d6): 191.8, 180.6, 158.1, 153.6, 132.9, 128.9, 127.5, 126.9,
118.8, 113.1. Analysis calculated for C10H6N2 ⅐ 0.2 H2O: C, 67.75; H, 3.67. Found:
C, 67.83; H, 3.59. A protocol similar to that described above was used for the
condensation of 4-(N-cyclopentylamidino)-1,2-phenylene diamine with benzo[b]
furan-2,5-dicarboxaldehyde to yield a gray solid in a 73% yield; mp, 290 to 292°C
dec. 1H NMR (DMSO-d6/D2O): 8.62 (s, 1H), 8.27 (d, 1H, J ϭ 8.8 Hz), 8.08 (s,
The methods used in experiments for determination of the minimum fungi-
cidal concentration (MFC) were adapted from a method by McGinnis (26).
Briefly, 10-␮l aliquots from tubes with growth inhibition were plated onto Sab-
ouraud agar plates. The lowest drug concentration that yielded three or fewer
yeasts colonies was recorded as the MFC.
Molds were tested by the same method (29) but with the following modifica-
tions. Isolates were grown on Sabouraud dextrose agar at 30°C and were sub-
cultured twice to ensure viability. After adequate sporulation occurred (after 4 to
12 days), conidia were harvested by flooding the colonies with a sterile solution
of 0.85% NaCl and 0.05% Tween 80 in sterile distilled water. Inocula were
prepared with a hemocytometer for counting and were then diluted with RPMI
1640 medium to obtain a final inoculum size of approximately 0.5 ϫ 103 to 2.5 ϫ
103 CFU/ml. The inoculum size was verified by plating an aliquot of the inocu-
lum. The cultures were incubated at 30°C for 48 to 72 h or until growth in the
control tube was visible.
In each experiment, the quality control included the testing of C. albicans

2498
DEL POETA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Structures and in vitro activities of pentamidine analogues
C. albicans
C. neoformans
Compound
No.
Ya
Position Y
X
R
b
MIC80
MFC
MIC80
MFC
(␮g/ml)
(␮g/ml)
(␮g/ml)
(␮g/ml)
Amphotericin B
1.0
0.25
0.78
3.12
25
NTc
NAd
1.56
100
1.0
2.0
NT
NA
6.25
NT
Fluconazole
Pentamidine
5
2
2
3
3
3
3
3
3
3
3
4
4
4
4
4
4
5
5
6
6
Am
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
2
1
1
2
1
O
O
O
O
O
O
O
O
N
N
N
O
O
O
O
O
O
O
N
O
N
H
H
NH2
H
OCH3
H
OCH3
H
H
NH2
NO2
H
3.12
Ͼ100
Ͼ100
6.25
12.5
Ͼ100
Ͼ100
NT
1.56
25
100
3.12
50
12.5
6.25
Ͼ100
25
1
Am
2
Am
NT
NT
3
Am
3.12
12.5
Ͼ100
Ͼ100
Ͼ100
0.19
12.5
100
3.12
50
NT
NT
Ͼ100
0.39
Ͼ100
100
50
Ͼ100
NT
6.25
12.5
NT
NT
NT
6.25
50
100
6.25
Ͼ100
50
12.5
Ͼ100
25
4
Am
5
AmOH
AmOH
Am
6
7
8
Am
9
Am
10
11
12
13
14
15
16
17
18
19
20
Am
Am
25
Am
OCH3
NH2
H
Ͼ100
6.25
0.78
Ͼ100
12.5
3.12
Յ0.09
1.56
0.78
Am
Im
1.56
Ͼ100
25
ImCH3
Am
H
H
Am
Cl
6.25
0.39
12.5
3.12
1.56
0.78
6.25
0.78
3.12
1.56
12.5
3.12
Am
H
Am
H
Am
NH2
a
b MIC80, MIC at which 80% of isolates are inhibited.
c NT, not tested.
d NA, not active.
A39 and C. neoformans var. neoformans H99 with fluconazole and ampho-
reduction in activity against both organisms. Likewise, substi-
tution of amino groups meta to the amidino groups gave a
reduction in activity for the two- and three-carbon-chain ana-
logs (compare compound 1 to compound 2 and compound 3 to
compound 9). Substitution of chloro groups meta to the ami-
dine moieties of pentamidine (compound 17) resulted in a
reduction in activity against C. albicans, while it afforded a
modest increase in activity against C. neoformans. Moving the
amidino groups from the para to the meta positions with regard
to the ether link produced a marked reduction in activity
against C. albicans for the three-carbon analogues (compare
compounds 3 and 7). However, the same alteration in the four
carbon analogues (compare compounds 11 and 16) resulted in
modestly increased activity against C. albicans and a decrease
in potency against C. neoformans. Perhaps the most striking
structure-activity effects occurred when the oxygens of pen-
tamidine were replaced by nitrogens (compound 18). The
activities of the amino isosteres showed a marked improve-
ment over that of pentamidine against C. albicans and some
improvement over that of pentamidine against C. neofor-
mans. A similar result was noted with the three-carbon-
chain analogue (compare compounds 3 and 8) against C. al-
bicans. A final observation for the pentamidine analogues
was that the conversion of the cationic moieties from an
amidino to an imidazolino group (compare compounds 11
and 14) produced an increase in antifungal activity against
C. albicans.
tericin B.
RESULTS
The MICs of fluconazole and amphotericin B for C. albicans
A39 and C. neoformans var. neoformans H99 were determined
and served as positive controls for comparison with the exper-
imental compounds. The MICs of fluconazole for C. albicans
A39 and C. neoformans var. neoformans H99 were 0.25 and 2
␮g/ml, respectively. The MIC of amphotericin B was 1 ␮g/ml
for both isolates.
Pentamidine analogs. The in vitro activities of pentamidine
and its analogues are summarized in Table 1. Because the
compounds appear to have fungicidal activity, both the MICs
and the MFCs are reported in Table 1.
It is noteworthy that pentamidine exhibits inhibitory activity
that is comparable to those of both amphotericin B and flu-
conazole against both C. albicans and C. neoformans. In addi-
tion, the MFCs of pentamidine for both organisms were de-
termined and they were found to be in the low concentration
range. The variation of the alkyl chain length of pentamidine
(compounds 1 and 3) resulted in a decrease in activity against
C. albicans. Likewise, the two-carbon analogue (compound 1)
had significantly reduced activity against C. neoformans com-
pared to that of the parent molecule. Substitution of methoxy
groups meta to the amidine moieties (compare compound 3 to
compound 4 and compound 11 to compound 12) produced a

VOL. 42, 1998
NEW ANTIFUNGAL AGENTS
TABLE 2. Structures and in vitro activities of pentamidine metabolites
2499
C. albicans
C. neoformans
Compound
Structure
Ya
R
Position R
b
MIC80
MFC
MIC80
MFC
(␮g/ml)
(␮g/ml)
(␮g/ml)
(␮g/ml)
Amphotericin B
1.0
0.25
NTc
NAd
1.56
12.5
12.5
NT
1.0
2.0
NT
NA
6.25
12.5
12.5
NT
Fluconazole
Pentamidine
I
Am
H
0.78
3.12
21
22
23
24
25
26
27
I
Am
OOH
OOH
OH
2
3
6.25
12.5
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
6.25
12.5
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
I
I
II
II
II
II
Am
AmOH
OOH
NT
NT
OO(CH2)4COOH
NT
NT
Ͼ100
NT
OO(CH2)4CH2OH
NT
OOO(CH2)5OOOC6H5OAmOH
NT
a See footnote a of Table 1.
b See footnote b of Table 1.
c See footnote c of Table 1.
d See footnote d of Table 1.
Pentamidine metabolites. Because pentamidine is rapidly
metabolized in the body to at least seven primary metabolites,
it was important to determine the antifungal effects of the
known metabolites (Table 2). When compared to pentamidine,
the chain-hydroxylated metabolites (compounds 21 and 22)
have approximately 10-fold reductions in their inhibitory ac-
tivities against C. albicans and a 2- to 4-fold reductions in their
inhibitory activities against C. neoformans. However, hydroxy-
lation of either one (compound 27) or both (compound 23) of
the amidine groups results in no significant activity against
either organism when the compounds are used at 100 ␮g/ml.
Likewise, all metabolites resulting from the cleavage of an
ether bond of pentamidine (compounds 24 to 26) have little or
no activity at the highest dose tested.
Bis-benzimidazoles. A number of dication-substituted bis-
benzimidazole derivatives have been shown to have excellent
activities against P. carinii pneumonia in the rat model of
disease (20). For this reason we tested a large series of these
compounds against C. albicans and C. neoformans (Table 3).
Structural variations of the compound were accomplished by
altering the bridge connecting the 2-benzimidazolyl moieties
(X in Table 3) and changes in the structure of the cationic
groups (Y in Table 3). Overall, several very potent compounds
were found in this series. In general the bis-benzimidazole
bridged by an alkane chain showed poor antifungal activity
(compounds 28 to 34). Only the benzimidazole joined by eth-
ene bridge (compound 32) exhibited activity within a log range
of the control drugs. Connecting 5-amidino-2-benzimadazoyl
groups with a 2,5-furanyl link (compound 35) produced a com-
pound with potent inhibitory activity against both isolates. In
addition, the MFCs of the compound for C. albicans and
C. neoformans were 0.78 and 3.12 ␮g/ml, respectively. Alter-
ation of the amidino cationic moieties (compounds 36 to 38)
resulted in reduced activity. Isosteric replacement of the furan
oxygen (compound 35) with nitrogen produced the corre-
sponding pyrrole isostere (compound 39). Comparison of the
isosteres (compounds 35 and 39) showed that they had very
similar activities against both yeasts. In the case of the pyrrole-
linked compounds, no difference was seen when the amidine
was replaced by a 2-imidazoline moiety (compound 40). How-
ever, the other alterations of the cation groups (compounds 41
and 42) caused a decrease in activity. Methylation of the pyr-
role nitrogen (compare compounds 39 and 43) had little effect
on the activity against either organism. Interestingly, the cyclo-
pentyl substitution of the amidino group (compound 45) on the
N-methyl pyrrole-bridged compound produced one of the most
potent inhibitors of C. neoformans (MIC, 0.19 ␮g/ml).
Only one thiophene-linked compound was included in this
study (compound 46). However, this compound was highly ef-
fective against C. albicans (MIC, 0.78 ␮g/ml) and gave the high-
est activity of any of the compounds against C. neoformans
(MIC, 0.19 ␮g/ml) and the fungicidal concentrations for these
yeasts were the same as the MICs.
Bridging the benzimidazole rings with a 1,4-phenyl group
produced compounds (compounds 47 to 49) with various
antifungal activities. The most remarkable finding within
this group was the highly selective activity of the amidino-
substituted compound (compound 47) toward the two organ-
isms. Compound 47 was highly effective against C. neoformans,
while at 100 ␮g/ml it was ineffective against C. albicans. The
addition of methyl groups to the phenyl bridge to give a p-
xylene link (compound 50) resulted in decreased antifungal
activity. Bridging of the benzimidazoles with a 2,6-pyridine
linker gave two compounds (compounds 51 and 52) with in-
teresting activities. At 100 ␮g/ml, the amidine-substituted an-
alogue (compound 51) showed no activity against either organ-
ism, while at 1.56 ␮g/ml the imidazoline-substituted compound
(compound 52) exhibited good activity against both organisms.
This is in contrast to the furan- or pyrrole-bridged compounds,
for which little difference in the activities of the imidazoline
and amidine derivatives was noted.
The last five compounds in Table 3 (compounds 53 to 57)
feature multiring bridges between the benzimidazole rings. All
members of this group show good potency against both organ-
isms. The most noteworthy of these compounds has a bridge
consisting of a 2,5-benzo[b]furanyl group and amidino cationic
moieties (compound 55). At concentrations under 1 ␮g/ml the
compound exhibits excellent inhibitory and fungicidal activities
against both yeasts.
Broad-spectrum activities of selected compounds. In order
to examine the spectrum of activity of these compounds, we se-
lected, on the basis of compound activity and availability, com-

2500
DEL POETA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Structures and in vitro activities of benzimidazole compounds
Cryptococcus
neoformans
C. albicans
Compound
X
Ya
b
MIC80
MFC
(␮g/ml)
MIC80
MFC
(␮g/ml)
(␮g/ml)
(␮g/ml)
28
29
30
31
32
33
34
Am
Im
Am
Im
Am
Am
Im
Ͼ100
Ͼ100
12.5
NT
Ͼ100
50
Ͼ100
12.5
Ͼ100
Ͼ100
Ͼ100
100
6.25
25
1.56
Ͼ100
Ͼ100
NT
Ͼ100
25
100
50
6.25
Ͼ100
Ͼ100
3.12
NT
Ͼ100
35
Am
0.78
0.78
0.78
3.12
36
37
38
Im
1.56
Ͼ100
12.5
1.56
NT
Ͼ100
1.56
6.25
1.56
3.12
50
Ͼ12.5
IsopropAm
CyclopentAm
39
Am
0.78
0.78
0.78
0.78
40
41
42
43
44
45
Im
IsopropAm
CyclopentAm
Am
0.78
50
6.25
0.78
1.56
3.12
0.78
NT
0.78
6.25
1.56
0.78
0.78
0.19
0.78
25
6.25
0.78
1.56
Ͼ1.56
50
6.25
3.12
Ͼ25
Im
CyclopentAm
46
IsopropAm
0.78
0.78
0.19
0.19
47
48
Am
IM
Ͼ100
NTc
6.25
0.39
0.78
0.39
1.56
0.78
49
THP
1.56
3.12
0.78
1.56
50
Am
25
100
25
25
51
52
Am
Im
Ͼ100
Ͼ100
Ͼ100
Ͼ100
1.56
1.56
1.56
1.56
53
54
IsopropAm
1.56
1.56
6.25
1.56
0.39
1.56
0.78
6.25
CyclopentAm
55
56
Am
CyclopentAm
0.39
0.78
0.78
0.78
0.19
0.78
0.19
0.78
57
IsopropAm
0.78
1.56
0.78
0.78
a
b See footnote b of Table 1.
c See footnote c of Table 1.

VOL. 42, 1998
NEW ANTIFUNGAL AGENTS
2501
TABLE 4. Extended antifungal spectrum of selected compounds
While pentamidine appears to be a potent in vitro antifungal
agent, the development of pentamidine as a therapeutic drug
may be limited due in part to its known toxic side effects in
humans but may be even more likely to be limited due to
metabolism of the parent drug. For example, it has been dem-
onstrated that pentamidine is readily metabolized to at least
seven primary metabolites (7). It can be seen from the data in
Table 2 that all of the primary metabolites of pentamidine have
reduced antifungal activity and that the metabolites resulting
from the breaking of the ether bond or hydroxylation of an
amidino nitrogen are devoid of activity. In addition to the loss
of activity, recent work in our laboratory indicates that certain
metabolites have increased toxicity (unpublished data). Unfor-
tunately, metabolic breakup of the parent molecule may be a
problem encountered with many of the direct pentamidine
analogues, especially those that contain an ether bond in the
bridge between the cationic moieties. An advantage to the
bis-benzimidazole compounds would be the lack of a cleavage
product resulting from primary metabolism.
Compound 39
Compound 57
MIC80 MFC
Strain
a
MIC80
MFC
(␮g/ml) (␮g/ml) (␮g/ml)
(␮g/ml)
Candida albicans A39
0.78
0.39
0.78
NTc
NT
NT
NT
NT
0.78
NT
Ͼ100
0.19
0.39
0.78
0.78
0.78
NT
NT
NT
NT
NT
0.78
NT
NT
0.78
1.56
1.56
0.78
0.78
0.78
0.78
0.78
0.78
0.78
NT
1.56
12.5
12.5
0.78
3.12
0.78
1.56
0.78
0.78
0.78
NT
Candida albicans 102.96b
Candida albicans 103.96b
Candida krusei 132.91b
Candida lusitaniae 111.92
Candida parapsilosis 111.96
Candida tropicalis 110.96
Torulopsis glabrata 142.91
Cryptococcus neoformans H99
Cryptococcus neoformans 114.96b
Aspergillus flavus 112.96
Aspergillus fumigatus 168.95
Fusarium solani 152.89
3.12
0.39
NT
NT
NT
NT
a See footnote b of Table 1.
b Fluconazole-resistant isolates.
c See footnote c of Table 1.
A key to the further development of these antifungal com-
pounds will be establishment of their mechanisms of action. It
has been proposed that the activity of this class of compounds
against G. lamblia is a result of their binding in the minor
grooves of DNA followed by inhibition of topoisomerase II (4).
While this mechanism appears to be likely for G. lamblia, no
direct correlation was found between the anti-topoisomerase II
inhibition of these compounds and their activity against Crypto-
sporidium parvum (9) or P. carinii (16). It has been hypothe-
sized that the primary cellular target of pentamidine in the
yeast Saccharomyces cerevisiae is the mitochondrion (25), and
more recently, it has been suggested that these compounds
have an effect on another fungus, P. carinii, due to their DNA
binding followed by inhibition of an endo- or exonuclease (19).
A major source of the problem in determining the mechanisms
of action against P. carinii and C. parvum is the lack of effective
in vitro culture systems. Such a problem would not exist for
most classical fungal organisms since they are readily grown in
vitro. Studies to determine the mechanisms of action of these
compounds against selected fungi should have a high proba-
bility of success.
In conclusion, the data presented in this report indicate the
potential of dicationic molecules as antifungal agents. A num-
ber of new molecules have been identified from this initial
study of structure-function relationships, and these new mole-
cules require future synthesis and antifungal testing. An exam-
ple of this need to focus our attention is the development of a
complete series of compounds with a thiophene as the linking
group (compound 46). It is clear that further studies on the
structure-activity relationships, mechanisms of action and tox-
icities, and in vivo efficacies of these compounds are warranted
to determine their clinical potential.
pounds 39 and 57 for testing against other pathogenic fungi
(Table 4). The results of the tests against filamentous fungi
showed that compound 39 is highly effective against Aspergillus
fumigatus, while it shows poor activity against Aspergillus flavus.
Compound 39 also showed excellent in vitro activity against
Fusarium solani, a mold with increasing clinical relevance and
against which few treatment options exist. Moreover, these re-
sults indicate that compounds 39 and 57 have excellent breadths
of antifungal activity against Candida species other than C. albi-
cans and also against fluconazole-resistant strains of C. albi-
cans and C. neoformans.
DISCUSSION
Examination of the in vitro antifungal data for the series of
57 compounds described here reveals a number of structures
that have activities equal to or greater than those of the control
drugs (fluconazole and amphotericin B). The MICs were de-
termined by the standardized methods of the National Com-
mittee for Clinical Laboratory Standards. On the other hand,
the method of determination of MFCs was adapted to the
method of determination of MICs and, although it is not stan-
dardized, showed the apparent fungicidal activities of some
compounds, even though fluconazole does not show fungicidal
activity by this assay. The potential fungicidal properties of
these compounds will need to be assessed with animal models.
Furthermore, selected compounds also appear to have a wide
spectrum of activity, including fungicidal activity against
fluconazole-resistant strains of C. albicans and C. neoformans.
The only data detailing the toxicity of this class of compounds
in humans are based on the findings with pentamidine (22, 34,
39). While pentamidine has been associated with a number of
adverse side effects, it has been proven to be effective in the
treatment of the fungal infection pneumocystosis. It is encour-
aging that a large number of the pentamidine derivatives have
been tested with animal models, and the findings indicate that
the majority of the compounds are considerably less toxic than
the parent compound (20, 23, 31, 34, 38). Furthermore, it has
been shown in the studies cited above that host cell toxicity and
animal toxicity are not linked to the same structural refine-
ments that impart enhanced antimicrobial activity. Given the
in vitro antifungal activity and recent toxicology findings, it is
likely that dication-substituted compounds with an acceptable
antifungal activity to toxicity ratio could be developed.
ACKNOWLEDGMENTS
This work was supported by an NIH Program Project (NAIAD
AI-33363) and awards from Pharm-Eco Pharmaceutical, Inc., Lexing-
ton, Mass., and Immtech International, Inc., Evanston, Ill.
We appreciate the clerical expertise and assistance of Vicki Wing-
ate.
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