Synthesis and antifungal evaluation of head-to-head and head-to-tail bisamidine compounds

Article abstract of DOI:10.1016/j.bmc.2015.07.006

Herein, we describe the antifungal evaluation of 43 bisamidine compounds, of which 26 are new, having the scaffold [Am]-[HetAr]-[linker]-[HetAr]-[Am], in which [Am] is a cyclic or acyclic amidine group, [linker] is a benzene, pyridine, pyrimidine, pyrazine ring, or an aliphatic chain of two to four carbon, and [HetAr] is a 5,6-bicyclic heterocycle such as indole, benzimidazole, imidazopyridine, benzofuran, or benzothiophene. In the head-to-head series the two [HetAr] units are oriented such that the 5-membered rings are connected through the linker, and in the head-to-tail series, one of the [HetAr] systems is connected through the 6-membered ring; additionally, in some of the head-to-tail compounds, the [linker] is omitted. Many of these compounds exhibited significant antifungal activity against Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans (MIC ≤ 4 μg/ml). The most potent compounds, for example, P10, P19 and P34, are comparable in antifungal activities to amphotericin B (MIC 0.125 μg/ml). They exhibited rapid fungicidal activity (>3 log₁₀ decrease in cfu/ml in 4 h) at concentrations equivalent to 4× the MIC in time kill experiments. The bisamidines strongly inhibited DNA, RNA and cell wall biosynthesis in C. albicans in macromolecular synthesis assays. However, the half-maximal inhibitory concentration for DNA synthesis was approximately 30-fold lower than those for RNA and cell wall biosynthesis. Fluorescence microscopy of intact cells of C. albicans treated with a bisamidine exhibited enhanced fluorescence in the presence of DNA, demonstrating that the bisamidine was localized to the nucleus. The results of this study show that bisamidines are potent antifungal agents with rapid fungicidal activity, which is likely to be the result of their DNA-binding activity. Although it was difficult to obtain a broad-spectrum antifungal compound with low cytotoxicity, some of the compounds (e.g., P9, P14 and P43) exhibited favorable CC₅₀ values against HeLa cells and maintained considerable antifungal activity.

Full text of DOI:10.1016/j.bmc.2015.07.006

Accepted Manuscript
Synthesis and antifungal evaluation of head-to-head and head-to-tail bisamidine
compounds
Son T. Nguyen, Steven M. Kwasny, Xiaoyuan Ding, John D. Williams, Norton
P. Peet, Terry L. Bowlin, Timothy J. Opperman
PII:
S0968-0896(15)00573-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.006
BMC 12435
Reference:
Bioorganic & Medicinal Chemistry
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25 June 2015
4 July 2015
Please cite this article as: Nguyen, S.T., Kwasny, S.M., Ding, X., Williams, J.D., Peet, N.P., Bowlin, T.L., Opperman,
T.J., Synthesis and antifungal evaluation of head-to-head and head-to-tail bisamidine compounds, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.006
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Title: Synthesis and antifungal evaluation of head-to-head and head-to-tail bisamidine
compounds
Authors: Son T. Nguyen, Steven M. Kwasny, Xiaoyuan Ding, John D. Williams, Norton P. Peet, Terry L.
Bowlin, and Timothy J. Opperman*
Affiliations: Microbiotix, Inc., One Innovation Dr., Worcester, MA 01605
*Corresponding author.
Timothy J. Opperman
Microbiotix, Inc.
One Innovation Dr
Worcester, MA 01605
tel: 508-757-2800
email: topperman@microbiotix.com
keywords: bisamidines, antifungal, fungicidal, Candida, Cryptococcus, DNA binding

ABSTRACT
Herein, we describe the antifungal evaluation of 43 bisamidine compounds, of which 26 are new, having
the scaffold [Am]-[HetAr]-[linker]-[HetAr]-[Am], in which [Am] is a cyclic or acyclic amidine group,
[linker] is a benzene, pyridine, pyrimidine, pyrazine ring, or an aliphatic chain of two to four carbon, and
[HetAr] is a 5,6-bicyclic heterocycle such as indole, benzimidazole, imidazopyridine, benzofuran, or
benzothiophene. In the head-to-head series the two [HetAr] units are oriented such that the 5-
membered rings are connected through the linker, and in the head-to-tail series, one of the [HetAr]
systems is connected through the 6-membered ring; additionally, in some of the head-to-tail
compounds, the [linker] is omitted. Many of these compounds exhibited significant antifungal activity
against Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, and Cryptococcus
neoformans (MIC ≤4 µg/ml). The most potent compounds, e.g. P10, P19 and P34, are comparable in
antifungal activities to amphotericin B (MIC 0.125 µg/ml). They exhibited rapid fungicidal activity (>3
log10 decrease in cfu/ml in 4 h) at concentrations equivalent to 4× the MIC in time kill experiments. The
bisamidines strongly inhibited DNA, RNA and cell wall biosynthesis in C. albicans in macromolecular
synthesis assays. However, the half-maximal inhibitory concentration for DNA synthesis was
approximately 30-fold lower than those for RNA and cell wall biosynthesis. Fluorescence microscopy of
intact cells of C. albicans treated with a bisamidine exhibited enhanced fluorescence in the presence of
DNA, demonstrating that the bisamidine was localized to the nucleus. The results of this study show
that bisamidines are potent antifungal agents with rapid fungicidal activity that is likely to be the result
of their DNA-binding activity. Although it was difficult to obtain a broad-spectrum antifungal compound
with low cytotoxicity, some of the compounds (e.g., P9, P14 and P43) exhibited favorable CC50 values
against HeLa cells and maintained considerable antifungal activity.

1. Introduction
Invasive fungal infections are a major clinical problem.1 The mortality rates of infections caused by the
three major fungal pathogens, Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans
are 20%–40%2, 50%–90%2, and 20-70%3, respectively. The majority of these infections affect patients
with compromised immunity. Consequently, the prevalence of these infections has increased as the
numbers of people in the population with compromised immunity has increased due to HIV/AIDs, or
recipients of solid organ or stem cell transplants.4 Presently, there are only three classes of antifungal
agents that are used to treat invasive fungal infections, including the polyenes (e.g. amphotericin B),
azoles (e.g. voriconazole), and echinocandins (e.g. caspofungin). Many of these agents have serious side
effects, especially amphotericin B and ketoconazole. Because invasive fungal infections affect immune-
compromised patients, antifungal compounds that are fungicidal are preferred. However, the
echinocandins are fungistatic, and the fungicidal activity of the newer azoles, such as voriconazole, is
species-specific. In addition, the emergence of drug resistance has resulted in further limitations on the
use of these drugs. It is important, therefore, to explore other classes of compounds for antifungal
activity.
Previously, we have described a series of bisamidine-containing compounds that are potent
antimicrobial agents with a novel mechanism of action. The bisamidines are broad-spectrum
antibacterial compounds, active against Gram-positive (MRSA, MSSA, VRE, MRSE, Group A Strep, B.
anthracis, and C. difficile) and Gram-negative (P. aeruginosa, E. coli, Enterobacter spp., Acinetobacter
spp., Klebsiella spp., and Burckholderia spp.) pathogens5. The bisamidines are rapidly bactericidal
against exponentially growing Gram-positive and Gram-negative bacteria5b and they kill bacteria with a
mechanism of action that is unique among antibacterial compounds. We have shown that these
compounds bind with high affinity to the minor groove of A-T rich DNA.6 Once bound to DNA, the
bisamidines inhibit DNA replication and RNA biosynthesis7, which results in rapid cell death. Because
the bisamidines act through a unique mechanism of action, we investigated the antifungal activity of a
diverse set of bisamidines. Herein, we report the design, synthesis, and evaluation of a panel of 55
bisamidine compounds for antifungal activity against a diverse panel of pathogenic fungi. In addition,
we carried out preliminary mechanism of action studies against Candida albicans. Our results
demonstrate that the bisamidines are potent antifungal compounds that are rapidly fungicidal. As in
bacteria, this class of compounds kills fungi by binding to DNA and inhibiting DNA replication and RNA
synthesis. This non-specific activity of the bisamidines is likely the source of the cytotoxicity of this class
of compounds and is an impediment to their further development for systemic applications.

2. Chemistry
In this study, we synthesized the bisamidine compounds in two scaffolds, head-to-head and head-
to-tail, as shown in Figure 1. All compounds share the generic structure [Am]-[HetAr]-[linker]-[HetAr]-
[Am], in which [Am] is a cyclic or acyclic amidine group, [linker] is a benzene, pyridine, pyrimidine,
pyrazine ring, or an aliphatic chain of two to four carbon, and [HetAr] is a 5,6-bicyclic heterocycle such as
indole, benzimidazole, imidazopyridine, benzofuran, or benzothiophene. When the two [HetAr] units
are oriented such that the link between [HetAr] units is attached through the 5-membered rings, they
are “head-to-head”, and when one of the [HetAr] units is connected through the 6-membered ring, they
are designated as “head-to-tail” compounds. In some of the “head-to-tail” series, the [linker] unit has
been omitted, and the two [HetAr] units are attached directly.
Figure 1. Generic structures of “head-to-head” and “head-to-tail” bisamidine compounds
The syntheses of the symmetrical bisindole compounds P1, P8, P10, P12-P15, P17, P18, P26, P29-
P31; and the unsymmetrical “head-to-head” bisindole analogs P21-P25 have been described
elsewhere.5d, 8 The synthesis of new bisindole compounds with various amidine groups is shown in
Scheme 1. Treatment of the nitrile 18 with 1,2-diaminopropane in the presence of P2S5 provided P9.
Following the Pinner synthesis9, compound 1 was converted to the imidate 210 in the presence of dry HCl
in absolute ethanol. The imidate thus formed was treated with various amines to provide the
compounds P2-P7, P11 and P16.

Scheme 1. Synthesis of new bisindole compounds with various amidine groups. Reagents and conditions
for (a): P1-P7: R1H, EtOH; for P11: CH3NH(CH2)3NH2, EtOH; for P16: NH2CH2C(CH2OH)2CH2NH2, EtOH.
The syntheses of new unsymmetrical bisindole compounds P19, P20 and P27 and a symmetrical
bisbenzofuran compound P28 are shown in Scheme 2. The triaryl cores of these compounds were
assembled by Suzuki-Miyaura coupling of the boronic acid/esters 3, 5, 6 and 8 with appropriate aryl
bromides. The nitrile functionalities were then converted into the corresponding tetrahydropyrimidines
by treatment with P2S5 and 1,3-diaminopropane.
Scheme 2. Synthesis of unsymmetrical bisindole and symmetrical bisbenzofuran compounds. Reagents
and conditions: (a) Pd(Ph3P)4, Na2CO3, DME, H2O, 80 oC; (b) P2S5, 1,3-diaminopropane, 130 oC.
Using a similar strategy, the “head-to-tail” triaryl and diaryl bisamidine compounds were prepared.
The Suzuki-Miyaura coupling partners and the final bisamidine products of this compound series are
shown in Table 1. Details of the synthesis are given in the experimental section.

Table 1. Suzuki-Miyaura coupling partners and the final products of the head-to-tail series
Suzuki-Miyaura coupling partners
Final bisamidine products

3. Results and Discussion
Each compound was evaluated for antifungal activity to obtain Minimal Inhibitory Concentration (MIC)
values against a panel of fungal strains, and the cytotoxicity (CC50) of each analog against the HeLa
mammalian cell line was determined. The results of these assays are shown in Tables 3-5. The strains
used in this study, which represent the major pathogens responsible for invasive infections, and their
susceptibility profile for several antifungal are shown in Table 2.
Table 2. A description of the fungal strains used in this study and their susceptibility to various
antifungal compounds.
MIC (µg/ml)†
Strain
Organism
Source
AMB
0.13
PAD
25
5FC
FLC
KTC
WTBF-50
Candida albicans
Clinical Isolate, Baylor
College of Medicine
1.25
≥32
20.2
WTBF-51
WTBF-86
WTBF-88
WTBF-106
Candida krusei
Clinical Isolate, Baylor
College of Medicine
1
19.8
3.13
12.5
≥100
≥20
0.31
1.25
≥20
≥32
≥32
1
4
Candida glabrata
ATCC 90030
ATCC*
0.25
0.31
32
≥32
0.06
32
Candida parapsilosis ATCC*
ATCC 90018
Cryptococcus
neoformans Cn18
(Serotype C)
Clinical Isolate, UMass
Medical School
32
†: The values shown for MICs are the geometric mean of the results of three independent assays.
*: American Type Culture Collection (Manassas, VA)
Abbreviations: AMB, Amphotericin B; PAD, Pentamidine; 5FC, Fluorocytosine; FLC, Fluconazole; KTC,
Ketoconazole.
All the compounds shown in Table 3 have the same core structure. Thus, the difference in activities
that we observe is caused by the various amidine functionalities. For the acyclic amidines, we noticed
that monoalkylated derivatives (P2-P7) had an improved spectrum of activity and potency when
compared to the non-alkylated amidine P1. The most active compound in this group was P2, which has
the most lipophilic substituent, iso-propyl. The preference for lipophilic amidine substituents also seems
to apply to the cyclic amidine compounds (P8-P16). For example, P9 was generally more active than P8.
It is interesting to note that P9 could be considered to be a cyclized version of P2, and these two
compounds are very similar in their activity against the Candida strains, but P9 is 6-fold more potent
than P2 against Cryptococcus neoformans and 16-fold less cytotoxic against HeLa cells. Also noteworthy

is the 6-membered cyclic amidine P10, which is the constitutional isomer of P9. It is significantly more
potent than P9 against both Candida spp. and C. neoformans, demonstrating the importance of the
amidine ring size. Among the derivatives of P10, analogs that carry a lipophilic substituent on the C(5) of
the tetrahydropyrimidine ring, e.g. methyl (P12) and fluoro (P13), are more active than the others with
more hydrophilic substituents (e.g., P14). Replacing the NH group of the tetrahydropyrimidine ring with
an N-methyl group reduced the potency by 6 to 30 fold (see P11 vs. P10). Bis-substitution at C(5) of the
tetrahydropyrimidine also reduced the potency of the compounds (see P15 and P16 vs. P12).
Compound P14 is unique in that it carries a hydrophilic (OH) substituent at C(5), yet it is very potent
against 4 out of 5 fungal strains that we tested, with especially potent activity against C. neoformans,
and it also showed low cytotoxicity against HeLa cells.
Table 3. The antifungal activity and cytotoxicity of the bis-indoles with different amidine groups
Structure
MIC (µg/ml)†
CC50
C. alb C. kru C. gla C. par C. neo (µg/ml)
#
P1
16
2
4
2
3.13
1.56
7
P2
0.5
0.4
0.2
0.25
3.5
1
1
1.59
1
0.63
0.13
0.5
2
0.5
0.25
0.79
2
1.97
1.97
1.1
5.5
14
P3
P4
0.25
0.79
2.52
2.52
P5
P6
P7
3.5
2
2.48
2.48
24.9
29.8
4
4
2

P8
10.08
0.79
0.25
1.59
0.5
4
0.25
0.25
0.06
1.26
0.13
0.13
0.16
0.63
0.4
2
32.5
66.5
4
P9
0.79
0.13
4
0.25
0.08
1.97
0.25
0.25
0.06
P10
P11
P12
P13
P14
0.1
1
4.7
0.5
0.25
0.25
0.31
5.8
0.5
0.25
12.7
13.9
>80
0.5
P15
P16
4
4
8
2
1
8
2
3.13
3.13
8
1.26
32.4
†, MICs are the geometric mean of three independent assays, MIC values < 1 µg/ml are in blue
Fungal strains: C. alb, Candida albicans (WTBF-50); C. kru, Candida krusei (WTBF-51); C. gla, Candida glabrata
(WTBF-86); C. par, Candida parapsilosis (WTBF-88); C. neo, Cryptococcus neoformans Cn18 (WTBF-106).
In Table 4 are shown MIC and CC50 values for compounds having the “head-to-head” scaffold with
various core structures and the same tetrahydropyrimidine end group. We noticed that chlorination
and bromination at the 3-position of the indole rings led to an increased lipophilicity, but not higher
potency against Candida spp. and C. neoformans (see P17 and P18 vs. P10). These analogs, however,
exhibit higher cytotoxicity against HeLa cells. Somewhat surprisingly, when one of the indole rings is
linked at the 3-position (i.e., P19), the compound remains equally active (as compared to P10) against
Candida spp. and is only less potent than P10 against C neoformans. Yet, when one of the
tetrahydropyrimidine groups is linked to C(5) of the indole, the resulting compound (P20) is much less

active than P10 against both Candida spp. and C. neoformans. Because the topological change is greater
in the case of P19, we expected that its antifungal activity would be more different from P10 than that
of P20.
The effects of exchanging one indole ring with a different 5,6-bicyclic heterocycle are reflected in
compounds P21-P24. Benzofuran- and benzimidazole-containing compounds (P21, P24) are comparable
in their antifungal activities and they are more active than the imidazopyridine-containing compounds
(P22, P23). They are all less potent and less cytotoxic (except P24) than P10. Replacing the phenyl
linker with an N-containing heterocycle reduces lipophilicity and cytotoxicity of the compounds against
HeLa cells (see P25 vs. P24, and P26, P27 vs. P10). Against fungi, the pyridine-linked compound P25 is
equally potent as the phenyl-linked P24, while the pyrazine- and pyrimidine-linked compounds P26 and
P27 are less active than the corresponding phenyl-linked P10, especially against C. neoformans.
Interestingly, replacing both indole rings of P26 with the corresponding benzofuran rings led to a much
less active compound (P28) against Candida spp.; its activity against C. neoformans remained the same.
Notably, the alkyl-linked bisindoles P29-P31 are significantly less active than the aromatic-linked
compounds, and the antifungal activity decreased as the length of the alkyl chain increased. Thus,
scaffold rigidity seems to play a significant role in maintaining the high antifungal activity of these
compounds.
Table 4. Antifungal activity and cytotoxicity of the head-to-head bisindole analogs with varied core
structures.
Structure
MIC (µg/ml)†
CC50
C. alb C. kru C. gla C. par C. neo (µg/ml)
#
P17
0.4
0.25
0.5
0.25
0.5
0.25
0.5
0.78
0.78
0.98
0.65
0.5
P18
P19
0.63
0.25
0.25
0.06
0.16
2.4

P20
P21
P22
P23
7.87
0.4
4
19.8
1
-
-
3.12
0.5
1
13.6
14.1
20.9
19.7
0.13
4
0.16
0.5
6.35
4
0.63
0.25
0.25
3.13
P24
P25
P26
0.5
0.5
1
0.16
0.06
0.25
0.13
0.2
0.25
0.25
3.13
1.24
4.7
0.5
0.5
0.25
15.6
42.7
25.5
0.5
0.06
0.31
≤0.03
0.13
P27
P28
20.16
≥32
20.16
≥32
32
16
1
2
≥32
32
2
3.13
1.59
16
>80
14.7
80.3
24.6
P29
P30
P31
22.63
3.17
12.7
1
8
1
†, MICs are the geometric mean of three independent assays, MIC values < 1 µg/ml are in blue.
Fungal strains: C. alb, Candida albicans (WTBF-50); C. kru, Candida krusei (WTBF-51); C. gla, Candida glabrata (WTBF-86); C.
par, Candida parapsilosis (WTBF-88); C. neo, Cryptococcus neoformans Cn18 (WTBF-106).

In Table 5 are shown MIC and CC50 values for the “head-to-tail” bisamidine analogs with varied core
structures and the same tetrahydropyrimidine end groups. Within this series there are two groups, one
with a phenyl linker, and one without. The general observation is that for ring A, an indole is preferred
(see P34, P36 and P37), and for ring B, benzofuran is preferred (see P32, P33 and P34; P39 and P41).
Thus, the most potent compound of the triaryl group was P34, and of the diaryl group was P41. We
noticed that that switching the position of ring A and ring B could lead to significant changes in MIC
values against Candida spp. For example, P33 was >10-fold more potent than P35, and P41 was
significantly more potent than P39. However, we did not observe the same effect on P37 and P38.
Activity against C. neoformans also did not seem to be affected by this type of change. We observed
only small changes in MIC values when the benzofuran is linked at the C(5) or C(6) (see P41 and P42).
Overall, these “head-to-tail” compounds showed activity and cytotoxicity that was similar to the “head-
to-head compounds”.
Table 5. Antifungal activity and cytotoxicity of the head-to-tail bisindole analogs with varied core
structures
Structure
MIC (µg/ml)†
CC50
C. gla C. par C. neo (µg/ml)
#
C. alb
C. kru
P32
0.5
0.5
0.13
0.13
0.06
32
0.5
0.31
0.13
8
2
0.5
0.13
1
34.6
13.4
1.5
P33
0.79
0.25
16
1
0.5
16
8
P34
P35
P36
P37
20.7
32.5
25.5
8
1.59
0.31
2.52
2
3.94
2
2
8

4
8
1
0.79
1
20.7
P38
P39
P40
4
25.4
16
2.52
5.04
2
2
1
4
40.5
27.5
3.17
0.31
0.4
1
4
4
0.13
1
0.5
0.5
1
0.5
1
17.1
50.5
91.9
P41
P42
P43
1.26
0.16
6.25
†, MICs are the geometric mean of three independent assays, MIC values < 1 µg/ml are in blue.
Fungal strains: C. alb, Candida albicans (WTBF-50); C. kru, Candida krusei (WTBF-51); C. gla, Candida glabrata
(WTBF-86); C. par, Candida parapsilosis (WTBF-88); C. neo, Cryptococcus neoformans Cn18 (WTBF-106).
To determine whether the most active antifungal bis-amidines are fungicidal, we performed time kill
assays using P10, P34, and P19 against C. albicans. The data, shown in Fig. 2, indicate that all three
compounds exhibit dose-dependent fungicidal activity that is comparable to, or superior to, that of the
positive control amphotericin B (AMB) at 4× the MIC. In addition, all three compounds decreased the
numbers of viable cells at a concentration of 1× the MIC. Compound P10 exhibited the most potent
fungicidal activity against C. albicans (Fig. 2A). At a concentration equivalent to 1× the MIC, P10 reduced
the number of viable cells by 3 log10 within 4 h, an activity comparable to that of the positive control.
Compound P10, at concentrations equivalent to 2× the MIC and 4× the MIC, reduced viable cells to
levels below the limit of detection within 8 h and 4 h, respectively. P19 exhibited intermediate
fungicidal activity, reducing viable cells by 1 log10 and 4 log10 within 24 h at 1× the MIC and 2× the MIC,
respectively (Fig. 2C). A concentration of 4× the MIC, P10 reduced viable cells to levels below the limit
of detection within 8 h. The fungicidal activity of P34 at 2× the MIC and 4× the MIC was comparable to
that of AMB (Fig. 2B) at 4× the MIC. Interestingly, the relative fungicidal activity of these compounds is
not correlated with their MICs, as all three compounds exhibit the same MIC against C. albicans (0.25
µg/ml). Because the bisamidines are extremely stable under diverse conditions, including incubation in
liver microsomes (data not shown), it is unlikely that the differences in the fungicidal activity of P10,

P34, and P19 are due to differences in stability. However, it is possible that the fungicidal activity of
P10, P34, and P19 is correlated with their affinity for DNA. Evidence from our laboratory indicates that
the bisamidines readily penetrate bacterial cells, where they bind to the minor groove of DNA with high
affinity6-7 and inhibit the synthesis of DNA and RNA. The inhibitory activity of these compounds is
correlated with their affinity for DNA. Because known inhibitors of bacterial DNA replication
(fluoroquinolones) and RNA synthesis (rifampicin) are bactericidal, it is likely that the potent bactericidal
activity of the bisamidines is the result of the inhibition of DNA synthesis. Therefore, it is likely that the
fungicidal activity of the bisamidines results from the same mechanism of action.
Figure 2. Time kill assays showing the fungicidal activity of the following bisamidines and a positive
control compound amphotericin B (AMB) against C. albicans: A) P10, B) P34, and C) P19. Compound
concentrations are expressed relative to the MIC for C. albicans.
To determine whether the bisamidines inhibit DNA and RNA synthesis in fungi, we performed
macromolecular synthesis assays using C. albicans. In this assay, C. albicans was incubated in the
presence of various concentrations of P10 or P19 in media containing a radiolabeled precursor of each
3
of the following macromolecular synthetic pathways: H-adenine (RNA and DNA), 3H-uridine (RNA only),
3H-leucine (protein), 3H-glucosamine (cell wall), and 3H-acetate (lipid). The results of these assays are
shown in Figures 3A and 3B. We used a curve-fitting algorithm to calculate the half-maximal inhibitory
concentrations (IC50) of P10 and P19 against each MMS pathway (Table 6). The results of these assays
indicate that P10 and P19 inhibit all MMS pathways except lipid biosynthesis. However, both
compounds inhibited the incorporation 3H-adenine into DNA and RNA with the greatest potency, with
IC50 values that were ~30-fold lower than those for the incorporation of uridine into RNA (see Table 6).
Because C. albicans does not have a mechanism to scavenge the DNA-specific precursor thymine from

the medium11, we used 3H-adenine for this experiment, which is incorporated into both DNA and RNA.
By comparing the inhibition of adenine vs. uridine incorporation, however, we can estimate the effect of
each compound on DNA synthesis. Based on our results, it is evident that P10 and P19 are potent
inhibitors of DNA synthesis in C. albicans. The specificity of the bisamidines for DNA synthesis is similar
to that of pentamidine (PAD) (see Fig. 3C and Table 3),an aromatic diamidine with potent antifungal
activity 12 that binds to the minor groove of DNA13. The similarities between the activity of P10, P19, and
PAD demonstrate a strong correlation between the DNA binding activity and inhibition of DNA synthesis.
In contrast, Amphotericin B (AMB), a potent antifungal agent that binds to a component of fungal cell
membranes (ergosterol) and causes leakage of monovalent ions (K+, Na+, H+, and Cl−)14, inhibited all
MMS pathways with equal potency (see Fig. 3D and Table 3), which is consistent with its non-specific
mechanism of action. While DNA synthesis is the pathway that is most sensitive to P10, P19, and PAD,
these compounds also inhibit RNA, protein, and cell wall synthesis at higher concentrations. The effect
of these compounds on other pathways is likely to be the result of either the indirect effects of these
compounds on cellular metabolism and energy production, or a direct inhibition of secondary targets.
Figure 3. Macromolecular synthesis (MMS) assays using radiolabeled precursors for DNA and RNA
(adenine), RNA only (uridine), protein (leucine), cell wall (glucosamine) and lipid (acetate) in the
presence of various concentrations of the following compounds (cmpds): A) P10, B) P19, C)
pentamidine (PAD), and D) amphotericin B (AMB).
Adenine
Uridine
Leucine
Glucosamine
Acetate
2
1.5
1
A
B
0.5
0
2
C
D
1.5
1
0.5
0
0.001
0.01
0.1
1
10
100 .001
0.01
0.1
1
10
100
[CMPD](µg/ml)
[CMPD](µg/ml)

Table 6. The half maximal inhibitory concentrations (IC50) of P10 and P19 against the macromolecular
synthetic pathways in C. albicans. The IC50 values were calculated from the curves shown in Figure 2.
IC50 (µg/ml)† ± stdev for :
Precursor
P10
P19
PAD
0.19 ± 0.046
12.2 ± 5.39
>25
AMB
3H-Adenine
3H-Uridine
0.01 ± 0.001
0.32 ± 0.12
0.59 ± 0.1
0.59 ± 0.07
≥2
0.02 ± 0.002
0.56 ± 0.12
0.86 ± 0.44
0.87 ± 0.31
≥2
0.038 ± 0.03
0.014 ± 0.005
0.052 ± 0.012
0.043 ± 0.019
0.036 ± 0.001
3H-Leucine
3H-Glucosamine
>25
3H-Acetate
>25
†Average of three trials
Abbreviations: PAD, pentamidine; AMB, amphotericin B.
To determine whether bisamidines bind to DNA in living cells of C. albicans, we used fluorescence
microscopy to visualize cells that had been treated with P8 (MBX 1060) and the DNA-intercalating dye
Syto9. Because the structure of P8 is similar to that of DAPI (4',6-diamidino-2-phenylindole), a well-
established dye that fluoresces strongly when bound to the minor groove of A-T rich double stranded
DNA (λex = 358 nm; λem = 461 nm), it fluoresces strongly in the presence of double stranded DNA. As
shown in Fig. 4B, cells treated with P8 exhibit localized fluorescence when viewed with a DAPI-specific
filter. The P8 fluorescence is co-localized with that of Syto9 (see Fig. 4C and D), indicating that P8 is
binding to nuclear DNA. Taken together, our data strongly suggests that bisamidines bind to the
chromosomal DNA of fungi, and inhibits the essential cellular process of DNA replication and RNA
synthesis. In addition, it is likely that this non-specific mechanism of action is responsible for the
cytotoxicity of this class of antimicrobial agents.

Figure 4. Micrographs of C. albicans treated with P8 and Syto9. A) Light micrograph, B) P8
fluorescence (DAPI filter), C) Syto9 fluorescence (GFP filter), D) the P8 and Syto9 fluoresence
images have been merged. Magnification = 400×.
4. Conclusions. Herein, we report the synthesis of 26 new bisamidine compounds that are based on
different “head-to-head” and “head-to-tail” scaffolds. An evaluation of the antifungal activity and
cytotoxicity of 26 new bisamidine compounds and 17 previously reported bisamidine compounds
identified several compounds (e.g P10, P19 and P34) with very potent antifungal activity. The
bisamidines are potent inhibitors of DNA synthesis in C. albicans, which is likely to be the result of the
DNA binding activity of this class of compounds. Because of the mechanism of action of the
bisamidines, we were unable to obtain a broad-spectrum antifungal compound with low cytotoxicity.
However, a few of the bisamidine compounds (e.g., P9, P14 and P43) exhibited favorable CC50 values
against HeLa cells and maintained considerable antifungal activity. It is possible that these compounds
may prove to be useful for treating non-systemic fungal infections.
5. Experimental
5.1 Fungal strains, media, and reagents. The fungal strains used in this study are listed in Table 1.
RPMI 1640 with L-glutamine was purchased from Gibco (Grand Island, NY) and Sabouraud Dextrose
Broth (SDB) was purchased from Difco (Franklin Lakes, NJ) in a dehydrated form that was prepared
according to the manufacturer’s instructions. The following radiolabeled precursors of macromolecular
synthetic pathways were purchased from Perkin Elmer (Waltham, MA): [3H]-adenine (DNA and RNA
synthesis), [3H]-uridine (RNA synthesis), [3H]-leucine (protein synthesis), and [3H]-glucosamine (cell wall).
[3H]-acetate (lipid synthesis) was purchased from Vitrax (Placentia, CA). The following reagents were
purchased from Sigma Aldrich (St. Louis, MO): amphotericin B (AMB), pentamidine (PAD), flucytosine

(5FC), fluconazole (FLC), ketoconazole (KTC). Syto9 was purchased from Molecular Probes (Grand Island,
NY).
5.2 Antifungal activity assay (MIC). The antifungal activity of each compound against Candida species
were measured as the minimal inhibitory concentration (MIC) using the microbroth dilution method as
described in the CLSI protocol15. In the MIC assays using Cryptococcus neoformans, Sabouraud Dextrose
Broth (SDB) was used instead of RPMI medium.
5.3. Cytotoxicity (CC50). The cytotoxicity of compounds against a mammalian cell line (HeLa, ATCC CCL-
2) was determined as described16.
5.4. Time kill assays. C. albicans was grown in RPMI medium at 37 °C until the culture reached an optical
density at 600 nm (OD600) of approximately 1. The culture was diluted 1:10 in RPMI medium containing
various concentrations of test compounds or a control antifungal agent (amphotericin B) and incubated
at 37 °C. At various times, samples were removed for serial dilution and plating on SBD agar to measure
colony forming units per ml (cfu/ml).
5.5. Macromolecular synthesis (MMS) assays. C. albicans was grown in RPMI medium at 37 °C until the
culture reached an optical density at 600 nm (OD600) of approximately 1. Fifty microliters of the culture
was transferred to each well of a 96-well assay plates containing a series of 2-fold serial dilutions of test
compounds and one of the following radiolabeled macromolecular precursors (10 µCi/ml final
concentration) in 50 µl RPMI: 3H-adenine (DNA and RNA), 3H-uridine (RNA), 3H-leucine (protein), 3H-
glucosamine (cell wall), 3H-acetate (lipid). The assay plates were incubated at 37 °C for 90 min, which is
equivalent to one doubling time, and 100 µl 10% TCA was added to each well, and then the radiolabeled
precipitate was collected by filtration and quantified using a scintillation counter. Each compound
concentration was tested in triplicate. The fraction of incorporated label for treated samples as
compared to the untreated control was calculated and plotted as a function of compound
concentration, and a four parameter curve fitting algorithm was used to determine the compound
concentration that resulted in half-maximal inhibition (IC50) for each pathway.
5.6. Fluorescence microscopy. An exponential culture of C. albicans was treated with 4x the MIC of MBX
1066 or 33 µM Syto 9 for 30 min and was analyzed using the DAPI and GFP channels, respectively, of a
Zeiss fluorescence microscope.
5.7. Compound synthesis. The syntheses and physical properties of the compounds described above are
provided in the Supplemental Material.

5.8. Literature preparations. For the synthesis of compounds P1, P8, P10, P12-P15, P17, P18, P26, P29-
P31 see Ref. 8. For the synthesis of compounds P21-P25, and the building blocks 4, 8, 10; 2-(4-
bromophenyl)indole-6-carbonitrile; 2-(4-bromophenyl) imidazo[1,2-a]pyridine-7-carbonitrile; 2-(4-
bromophenyl) imidazo[1,2-a]pyridine-6-carbonitrile see Ref. 5d. The imidate 2 was prepared according
to Ref. 10.
5.9. Commercial building blocks. Compounds 3, 5 and 6 were purchased from Combi-Blocks, Inc. (San
Diego, CA); 6-Bromoindole-2-carbonitrile was purchased from Matrix Scientific (Columbia, SC);
compound 19 was purchased from Ark Pharm, Inc. (Libertyville, IL); compounds 11, 12, 16 and 20 were
supplied by Creagen Biosciences (Woburn, MA).
6. Acknowledgments. The authors would like to thank Dr. Rabih Darouiche for providing fungal strains
from the Baylor College of Medicine clinical laboratory. We thank Dr. Hwa-Ok Kim and the scientists at
Creagen Biosciences (Woburn, MA) for providing the custom-made compounds 11, 12, 16 and 20. This
work was supported by the National Institutes of Health (grants 5 U01 AI 082052-03 and 1R43
AI083032). The content of this article is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.

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Graphical abstract