Synthesis and investigation of novel benzimidazole derivatives as antifungal agents

Article abstract of DOI:10.1016/j.bmc.2016.06.010

The rise and emergence of resistance to antifungal drugs by diverse pathogenic fungal strains have resulted in an increase in demand for new antifungal agents. Various heterocyclic scaffolds with different mechanisms of action against fungi have been investigated in the past. Herein, we report the synthesis and antifungal activities of 18 alkylated mono-, bis-, and trisbenzimidazole derivatives, their toxicities against mammalian cells, as well as their ability to induce reactive oxygen species (ROS) in yeast cells. Many of our bisbenzimidazole compounds exhibited moderate to excellent antifungal activities against all tested fungal strains, with MIC values ranging from 15.6 to 0.975?μg/mL. The fungal activity profiles of our bisbenzimidazoles were found to be dependent on alkyl chain length. Our most potent compounds were found to display equal or superior antifungal activity when compared to the currently used agents amphotericin B, fluconazole, itraconazole, posaconazole, and voriconazole against many of the strains tested.

Full text of DOI:10.1016/j.bmc.2016.06.010

Accepted Manuscript
Synthesis and investigation of novel benzimidazole derivatives as antifungal
agents
Nishad Thamban Chandrika, Sanjib K. Shrestha, Huy X. Ngo, Sylvie Garneau-
Tsodikova
PII:
S0968-0896(16)30433-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.010
BMC 13066
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 April 2016
27 May 2016
3 June 2016
Please cite this article as: Chandrika, N.T., Shrestha, S.K., Ngo, H.X., Garneau-Tsodikova, S., Synthesis and
investigation of novel benzimidazole derivatives as antifungal agents, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.06.010
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Graphical Abstract
Synthesis and investigation of novel
benzimidazole derivatives as antifungal
agents
Nishad Thamban Chandrika, Sanjib K. Shrestha, Huy X. Ngo, and
Sylvie Garneau-Tsodikova
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536-0596, USA.
N
N
N
N
H
N
N
H
R2
OR1
Antifungal (Candida and Aspergillus)
R1: Et > H > Me
R2: C1 > C2 > C3 > C5 > C12

Bioorganic & Medicinal Chemistry
Synthesis and investigation of novel benzimidazole derivatives as antifungal agents
Nishad Thamban Chandrika,a,† Sanjib K. Shrestha,a,† Huy X. Ngo,a and Sylvie Garneau-Tsodikovaa,*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536-0596, USA.
†These two authors contributed equally to this work.
* Corresponding author: E-mail address: sylviegtsodikova@uky.edu (S. Garneau-Tsodikova)
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The rise and emergence of resistance to antifungal drugs by diverse pathogenic fungal strains
have resulted in an increase in demand for new antifungal agents. Various heterocyclic scaffolds
with different mechanism of action against fungi have been investigated in the past. Herein, we
report the synthesis and antifungal activities of 18 alkylated mono-, bis-, and trisbenzimidazole
derivatives, their toxicities against mammalian cells, as well as their ability to induce reactive
oxygen species (ROS) in yeast cells. Many of our bisbenzimidazole compounds exhibited
moderate to excellent antifungal activities against all tested fungal strains, with MIC values
ranging from 15.6-0.975 g/mL The fungal activity profiles of our bisbenzimidazoles were
found to be dependent on alkyl chain length. Our most potent compounds were found to display
equal or superior antifungal activity when compared to the currently used agents amphotericin
B, fluconazole, itraconazole, posaconazole, and voriconazole against many of the strains tested.
Available online
Keywords:
Aspergillus
Candida
Cytotoxicity
Hoechst 33258
Reactive oxygen species (ROS)
2009 Elsevier Ltd. All rights reserved.
1. Introduction
mechanisms, including (i) inhibition of ergosterol biosynthesis by
targeting the CYP450 14-demethylase (azoles), (ii) inhibition of
At present, there is an increased demand for novel antifungal
drugs for both medical and agricultural purposes because of the
rise in diverse pathogenic fungi and the emergence of resistant
fungal strains.1 Fungal infections can vary in degree of
pathogenicity and severity. For instance, some fungal infections
are merely superficial and simple to treat, but others are systemic
infections and are associated with high mortality rate. These
therefore represent a major health concern worldwide, especially
in immunocompromised patients.2 Aspergillus and Candida
species are responsible for the majority of the documented
infections, which may be endogenous (Candida infections) or
environmentally acquired (Aspergillus and Cryptococcus
infections).3 Candidiasis and aspergillosis account for 80 to 90%
of systemic fungal infections.4 There are seven Candida spp. (C.
tropicalis, C. glabrata, C. parapsilosis, C. stelltoidea, C. krusei,
and C. kyfer) of medical importance, with the most important
being C. albicans.5 The Aspergilli sp. are known to increasingly
cause invasive diseases.6 In addition to A. niger, other species
such as A. flavus, A. terreus, and A. nidulans are increasingly
reported.7, 8 In addition to the known fungal infections, there are
always new fungal pathogens that appear which can cause life-
threatening infections in immunocompromised hosts.9
glucan synthase, which prevents the formation of -glucan
present in fungal cell wall, (iii) inhibition of chitin synthase
causing cell lysis, (iv) ergosterol disruption by formation of
polyene ergosterol complex, (v) inhibition of squalene epoxidase
which converts lanosterol to ergosterol, (vi) interfering with
pyrimidine metabolism, thus inhibiting nucleic acid synthesis,
(vii) inhibiting fungal translation preventing protein synthesis,
and (viii) inhibition of -tubulin protein, which forms
microtubule.10 The low efficacy, emerging resistance to standard
therapy, significant side effects, and the toxicity associated with
the existing antifungal drugs has resulted in a renewed interest in
searching for newer antifungal scaffolds, which can be used
either alone or as a part of combination therapy. In recent years,
our group has been actively involved in developing novel small
molecules as well as aminoglycoside-based antifungal agents to
treat both topical and systemic fungal infections.11-13 Based on a
recent report, a high-throughput screening of more than 100,000
individual compounds with heterocyclic cores, in the presence
fungal pathogens, led to the identification of a promising
benzimidazole scaffold.14
Benzimidazoles (mono-, bis-, and trisbenzimidazoles) are
chemically and biologically relevant molecules, and have been
studied extensively for their antimicrobial, anticancer, and DNA
sequence recognition properties. The DNA-binding affinity of
these benzimidazoles was explored by synthesizing various
mono-, bis-, and trisbenzimidazoles, which were shown to bind
AT-rich DNA minor groove.15-17 The DNA binding depended on
alkyl chain spacers,18 orientation in case of bidentate ligands,19 as
The currently widely used clinical antifungals are azoles (e.g.,
fluconazole (FLC), itraconazole (ITC), posaconazole (POS), and
voriconazole (VOR)), polyenes (e.g., amphotericin B (AmB) and
nystatin), allylamines (e.g., amorolfine, butenafine, and
naftifine), and echinocandins (e.g., caspofungin and micafungin).
These antifungal scaffolds are known to function by different

well as RNA duplex recognition by benzimidazole-
aminoglycoside conjugates.20 Mono- and bisbenzimidazole
analogues were tested for their efficacy as small molecule
inhibitors for RNAs.21-23 Various trisubstituted benzimidazoles
displayed good efficacy against Mycobacterium tuberculosis
(Mtb) FtsZ, Francisella tularensis, methicillin-resistant
tetrahydrofuran (THF), the hydroxyl group of 26 was protected
by using tert-butyldimethylsilyl chloride (TBDMSCl) and the
resulting analogue 33 was reduced with LiAlH4 to the
corresponding aldehydes 34a,b with partial deprotection of the
TBDMS group (Fig. 2A). The other end of the molecules was
functionalized with various alkyl chains (ethyl, propyl, pentyl,
and dodecyl), compounds 42-45 by substitution reaction of 4-
Boc-piperazine (41). The acid mediated removal of the Boc-
protecting group resulted in the corresponding secondary amines,
which were used for SNAr with 5-chloro-2-nitro aniline to form
compounds 51-55 in good yields (38-75%) (Fig. 2B). The mono-
benzimidazole analogue 1 was obtained in 71% yield by
oxidative coupling of 1,2-benzene diamine (51) with aldehyde
22. The bisbenzimidazoles 2-17 were synthesized with oxidative
mismatched coupling of aldehydes 34b-40 with the 1,2-diamino
derivatives 51-55 (Fig. 2C). In order to introduce a third
benzimidazole ring, aldehyde 38 was coupled with 3,4-diamino-
N-methoxy-N-methylbenzamide to yield 56, which was then
reduced at lower temperature to form aldehyde 57 in good yield
(76%). The 4-(4-methylpiperazin-1-yl)-benzene-1,2-diamine was
then coupled with aldehyde 57 in presence of an oxidant to result
in the formation the trisbenzimidazole analogue 18 (Fig. 3). All
the intermediates and final target molecules were characterized
using 1H and 13C NMR (Fig. S1-S111) as well as mass
spectrometry, and were found to be at least 95% pure. The
presence of an alkyne functionality on analogues 1, 14, and 18
gives an opportunity for future structural modifications using
click chemistry.
Staphylococcus
aureus
(MRSA),
and
Acinetobacter
baumannii.24-28 They also showed good activity against a range of
cancer cell lines, such as non-small cell lung cancer, melanoma,
leukemia, and breast cancer cells.29, 30 Various bisbenzimidazole
analogues were found to be efficient EcTopo IA inhibitors, with
potential antibacterial activity with low minimum inhibitory
concentration (MIC) and minimum bactericidal concentration
(MBC) values against various Escherichia coli strains.31 Hoechst
33258-based bisbenzimidazoles containing terminal alkyne
displayed selective inhibition of E. coli topoisomerase I over
human topoisomerase I and II, and effectively inhibited bacterial
growth.32 Hoechst 33258 was shown to bind fungal nucleic acid
and can potentially be used against pneumocystis and possibly
other fungi.33 These studies piqued our curiosity, and we decided
to further synthesize and explore novel benzimidazole derivatives
in a structure-activity-relationship study for their antifungal
properties.
Herein, we report on the synthesis of 18 benzimidazole
derivatives and their antifungal activities against a variety of
Candida albicans and Aspergillus strains (C. albicans ATCC
10231 (A), C. albicans ATCC 64124 (B), C. albicans ATCC
MYA-2876(S) (C), C. albicans ATCC 90819(R) (D), C. albicans
ATCC MYA-2310(S) (E), C. albicans ATCC MYA-1237(R)
(F), C. albicans ATCC MYA-1003(R) (G), C. glabrata. ATCC
2001 (H), C. krusei ATCC 6258 (I), C. parapsilosis ATCC
22019 (J), A. flavus ATCC MYA-3631 (K), A. nidulans ATCC
38163 (L), and A. terreus ATCC MYA-3633 (M)). We also
present in vitro cytotoxicity studies of these compounds as well
as their potential to induce reactive oxygen species (ROS) in
fungi.
N
O
N
N
H
N
1
N
N
N
H
N
N
N
H
R2
OR1
2. Results and discussion
2: R1 = H, R2 = CH3
3: R1 = H, R2 = C3H7
10: R1 = C2H5, R2 = C2H5
11: R1 = C2H5, R2 = C3H7
2.1. Chemistry
4: R1 = H, R2 = C12H25 12: R1 = C2H5, R2 = C5H11
5: R1 = CH3, R2 = CH3 13: R1 = C12H25, R2 = CH3
6: R1 = CH3, R2 = C2H5 14: R1 = CH2C CH, R2 = CH3
7: R1 = CH3, R2 = C3H7 15: R1 = CH2C CH, R2 = C3H7
8: R1 = CH3, R2 = C5H11 16: R1 = (CH2)2OH, R2 = CH3
9: R1 = C2H5, R2 = CH3 17: R1 = (CH2CH2O)2H, R2 = CH3
In order to study the antifungal activity of benzimidazoles, we
synthesized 18 analogues to explore the correlation of antifungal
activity with the number of imidazole rings as well as the identity
of alkyl substituents. We used a divergent strategy to introduce
N
structurally different linkers on either side of the benzimidazole
N
19
moieties.16,
A total of 18 molecules, 1-18, were logically
N
H
N
N
H
N
N
designed and synthesized, where compounds 1, 2-17, and 18
have one, two, and three benzimidazole rings, respectively, with
various substitution patterns (Fig. 1). Analogues 1, 14, and 18
contain an alkyne functionality on the phenyl ring, whereas all of
them have a methyl substitution on the piperazine ring. All the
remaining analogues were designed with various alkyl
substituents on either end of the molecules. The linkers were
introduced on the 4-hydroxybenzaldehyde using a Mitsunobu
reaction (propargyl alcohol (22), diethylene glycol (24)) and
nucleophilic substitution reaction (ethyl iodide (20), 1-
bromododecane (21), and 2-bromoethanol (23)) (Fig. 2). The 4-
substituted benzaldehydes 19-24 were coupled with 3,4-diamine-
N-methoxy-N-methylbenzamide in the presence of an oxidant
(sodium metabisulfite) to yield the corresponding mono
benzimidazole derivatives 26-32 in good yields (54-75%). Low
temperature reaction of analogues 26-32 with lithium aluminum
hydride (LiAlH4) resulted in the reduction of the Weinreb amide
functionality to the corresponding aldehydes 34-40 in 54-86%
yield. Because of the low solubility of compound 26 in
H
O
N
18
Fig. 1. Chemical structures of the 18 benzimidazole derivatives tested in this
study.
2.2. In vitro antifungal assay
In order to investigate their potential as antifungal agents, we
first determined the MIC values for benzimidazole analogues 1-
18 along with those for five known antifungal drugs (AmB, FLC,
ITC, POS, VOR) against 13 fungal strains (yeast and filamentous
fungi, Table 1). We rapidly found that the mono- (1) and
trisbenzimidazole (18) analogues displayed no activity (≥31.2
g/mL) against the 13 fungal strains tested, and therefore decided
to focus our efforts on derivatizing the bisbenzimidazole scaffold.
We also observed that all of our bisbenzimidazole derivatives
displayed no activity (≥31.2 g/mL) against A. flavus ATCC
MYA-3631 (strain K). As we and others previously discovered
that the addition of long alkyl chains to aminoglycosides such as
kanamycin
A
(KANA),34
kanamycin
B

A
OHC
1. R1-X, K2CO3
DMF, 55 °C, 12 h OR
OHC
1. R1-OH, Ph3P, CH2Cl2,
DIAD, THF,
OH
OR1
0 °C to rt, 12 h
19: R1 = CH3 ($)
20: R1 = C2H5 (85%)
1. 25, Pd/C, EtOH
rt, 6 h
N
OR1
21: R1 = C12H25 (72%)
22: R1 = CH2C CH (54%)
23: R1 = (CH2)2OH (70%)
24: R1 = (CH2CH2O)2H (58%)
N
N
H
O
2. Na2S2O5, EtOH
H2O, 90 °C, 12 h
O
26: R1 = H (74%)
27: R1 = CH3 (66%)
28: R1 = C2H5 (75%)
29: R1 = C12H25 (70%)
30: R1 = CH2C CH (54%)
31: R1 = (CH2)2OH (75%)
1. TBDMSCl
DMF, Imd
rt, 16 h
32: R1 = (CH2CH2O)2H (73%)
33: R1 = TBDMS (79%)
1. LAH, THF/Et2O
-78 °C to -20 °C
6 h
N
OR1
N
OHC
H
34a: R1 = TBDMS (37%), 34b: R1 = H (54%)
35: R1 = CH3 (84%)
36: R1 = C2H5 (83%)
1. SOCl2, 80 °C, 4 h
NO2
NO2
NO2
HO
N
37: R1 = C12H25 (84%)
2. CH3ONH2•HCl,
py, 0 °C to rt, 12 h
80%
NO2
O
38: R1 = CH2C CH (76%)
39: R1 = (CH2)2OH (77%)
40: R1 = (CH2CH2O)2H (86%)
O
O
25
B
O
O
1. (Boc)2O, MeOH
0 °C to rt, 36 h
1. R2-X, DIEA
0 °C to rt, 24 h
N
O
N
O
NH
66%
HN
N
HN
R2
41
42: R2 = C2H5 (92%)
43: R2 = C3H7 (69%)
44: R2 = C5H11 (87%)
45: R2 = C12H25 (87%)
1. CF3CO2H, rt
5 min
NO2
NH2
NO2
N
NH
Cl
NH2
N
N
R2
R2
1. K2CO3, DMF
110 °C, 36 h
51: R2 = CH3 (78%)
52: R2 = C2H5 (38%)
53: R2 = C3H7 (56%)
54: R2 = C5H11 (72%)
55: R2 = C12H25 (75%)
46: R2 = CH3 ($)
47: R2 = C2H5 (94%)
48: R2 = C3H7 (90%)
49: R2 = C5H11 (96%)
50: R2 = C12H25 (96%)
C
51 + 22
51 + 34b
53 + 34b
55 + 34b
51 + 35
52 + 35
53 + 35
54 + 35
51 + 36
52 + 36
53 + 36
54 + 36
51 + 37
51 + 38
53 + 38
51 + 39
51 + 40
1 (71%)
2 (60%)
3 (48%)
4 (64%)
5 (56%)
6 (46%)
7 (36%)
8 (55%)
9 (62%)
10 (45%)
11 (40%)
12 (46%)
13 (71%)
14 (67%)
15 (42%)
16 (66%)
17 (45%)
1. Pd/C, EtOH
rt, 6 h
2. Na2S2O5, EtOH
H2O, 90 °C, 12 h
Fig. 2. Synthetic scheme for the preparation of A. benzimidazoles 34-40, B. 4-(4-substituted piperazin-1-yl) 2-nitroaniline intermediates 51-55, and C. mono- (1)
and bisbenzimidazole (2-17) derivatives.
35
13
(KANB),12,
and tobramycin (TOB)11,
results in killing of
broad-spectrum activity against both yeast and filamentous fungi
(MIC values ranging from 0.975 to 15.6 g/mL). In order to
evaluate the role of the alkyl chain, we modified both the
piperazine and phenyl ends of compound 2 with different
substitutions. At this point, it was unclear whether modifications
at one end of the molecule would be more favorable than
modifications at the other end. Therefore, we commenced by
conducting modifications on the piperazine ring. We introduced
linear alkyl chains of 3 and 12 carbons in length (from now on
referred to as C3 and C12) on the piperazine end to form
fungal cells through membrane perturbation, we decided to
functionalize the bisbenzimidazole core with various alkyl chains
hoping for an additive action against fungal growth. The
divergent synthetic strategy allowed us to introduce various alkyl
chains on either side of the molecule efficiently.
We began our study with compound 2, which has a methyl
group on the piperazine ring and a hydroxyl group at the para
position of the phenyl ring. This compound was found to exhibit

compounds 3 and 4, respectively. Analogue 4 exhibited no
activity against all 13 yeast/fungal strains tested, whereas
derivative 3 displayed good activity (7.8 g/mL) against C.
glabrata ATCC 2001 (strain H), C. parapsilosis ATCC 22019
(strain J), and A. nidulans ATCC 38163 (strain L), and low
activity (15.6 g/mL) against A. terreus ATCC MYA-3633
(strain M). This initial analysis provided us valuable information
regarding the effect of substitution pattern, as addition of the C12
chain on the piperazine end (analogue 3) completely obliterated
the activity of the scaffold. To further confirm the relationship of
the regiospecificity of substitutions and their corresponding
antifungal activities, we tested compound 13, which contains a
C12 substituent on the phenyl end. We observed that addition of
this long alkyl chain at this position also resulted in complete loss
of antifungal activity. Collectively, these findings suggested that,
unlike in the case of aminoglycosides, addition of long linear
alkyl chains such as the C12 is detrimental for antifungal potency.
To get a full depiction of activity profile, we decided to also
introduce an ethoxy group on the phenyl ring of these molecules
while keeping the C1 (9), C2 (10), C3 (11), and C5 (12) side chains
on the piperazine ring. As with the methoxy series (compounds
5-8), we found that increasing the length of the alkyl chain on the
piperazine ring resulted in a decrease in antifungal activity, with
compounds 11 (C3) and 12 (C5) being inactive and compound 9
being more active than 10. Interestingly, when comparing the
MIC values for compounds 2, 5, and 9 as well as those of
compound 3, 7, and 10, we discovered that the presence of an
ethoxy group on the phenyl ring conferred equal or better
antifungal activity than that of an hydroxyl group, which itself
conferred better antifungal activity than the methoxy group, as
stated above.
As compounds comprised of a methyl on the piperazine ring
displayed the best antifungal activities, we next explored keeping
this moiety constant while adding a proparyl group on the
hydroxyl moiety of compounds 2 to afford derivative 14.
Surprisingly, compound 14 displayed very similar antifungal
activities to those of its counterparts with ethoxy (compound 9)
and hydroxyl (compound 2) moieties. Analogue 14 showed
excellent activity (1.95 g/mL) against C. parapsilosis ATCC
22019 (strain J) and good activity (7.8 g/mL) against four
additional Candida strains tested (C, E, H, and I). Interestingly,
replacing C1 with C3 on the piperazine ring (compound 15)
significantly reduced its activity.
1. 25, Pd/C, EtOH
rt, 6 h
N
N
38
N
N
O
2. Na2S2O5, EtOH
H2O, 90 °C, 12 h
58%
H
N
O
H
O
56
1. LAH, THF/Et2O
-78 °C to -20 °C
6 h, 76%
1. 51, Pd/C, EtOH
rt, 6 h
N
N
H
18
As compounds 2 and 9 were great antifungal agents, we
thought that combining the structural features of these two R1
substituents, as in compound 16, could potentially be beneficial.
However, the desired effect was not observed with compound 16,
which only showed good activity (7.8 g/mL) against C. krusei
ATCC 6258 (strain I) and low activity against strains J, L, and
M. Finally, to test the influence of the hydrophilicity of the
substituent, we synthesized compound 17 with a diethylene
glycol on the phenyl. Adding hydrophilicity did not improve the
antifungal activity, as 17 was only active against C. krusei ATCC
6258 (strain I) (7.8 g/mL).
N
H
2. Na2S2O5, EtOH
H2O, 90 °C, 12 h
54%
N
H
O
O
57
Fig. 3. Synthetic scheme for the preparation of the trisbenzimidazole
derivative 18.
Based on our finding that addition of a short alkyl (methyl
(C1) (2) and n-propyl (C3) (3)) substituent to the piperazine ring
while having a hydroxyl moiety attached to the phenyl ring led to
good activity against various fungal strains, we pondered if
replacing the hydroxyl moiety of compounds 2 and 3 by a
methoxy group (compounds 5 and 7) would have any effect on
the antifungal activity of these compounds. We also wondered if
increasing the length of the alkyl chain on the piperazine ring
would correlate with a decrease in antifungal activity. To explore
this latter idea, we additionally generated compounds 6 and 8
containing an ethyl (C2) and a pentyl (C5) chain, respectively. In
general, we found that increasing the length of the chain from C1
to C5 on the piperazine ring while keeping the R1 group constant
on the phenyl ring (compounds 5-8) resulted in a gradual
decrease in antifungal activity against almost all strains tested.
Compound 5 with a C1 side chain displayed good activity (3.9-
7.8 g/mL) against four of the tested strains (C. albicans ATCC
MYA-2310(S) (strain E), C. glabrata ATCC 2001 (strain H), and
C. parapsilosis ATCC 22019 (strain J)). As we increased the
chain length we saw a significant drop in activity. Indeed,
compound 6 with a C2 side chain displayed good activity (7.8
g/mL) against two of the tested strains (C. glabrata ATCC 2001
(strain H) and C. parapsilosis ATCC 22019 (strain J)), whereas
analogue 7 with a C3 side chain showed good activity (7.8
g/mL) against only one of the tested strain (C. krusei ATCC
6258 (strain I)), and analogue 8 with a C5 side chain only
displayed low activity (15.6 g/mL) against one of the tested
strain (C. parapsilosis ATCC 22019 (strain J)). When comparing
the antifungal activity of compounds 2 and 5 as well as that of
compounds 3 and 7, we discovered that the presence of a
hydroxyl moiety on the phenyl ring was more beneficial than that
of a methoxy group.
Overall, all the fungal strains used in this study were more
sensitive to analogues 2, 9, and 14 than FLC. Our compounds
were even more potent against some strains when compared to
the clinically potent antifungal agents AmB, ITC, POS, and
VOR. Analogues 2, 9, and 14 displayed better activity against C.
albicans ATCC 64124 (strain B), C. albicans ATCC 90819(R)
(strain D), and C. albicans ATCC MYA-2310(S) (strain E) when
compared to POS and ITC. Both compounds 2 and 9 showed
better activity against C. albicans ATCC MYA-1237(R) (strain
F) than POS and ITC. Finally, compound 2 displayed equal or
better activity than AmB against C. albicans ATCC MYA-
1237(R) (strain F), C. glabrata ATCC 2001 (strain H), C.
parapsilosis ATCC 22019 (strain J), and A. nidulans ATCC
38163 (strain L).
2.3. Antifungal time-kill studies
Data from time-kill studies can provide critical information
regarding the rate and extend of fungicidal activity and other
pharmacodynamics characteristics that are crucial for translation
to in vivo models.36 In order to understand the rate of the
antifungal activity, our best compounds 2 and 9, as well as AmB
(positive control) were selected for time-kill assays against the
azole-resistant fungal strain C. albicans ATCC 64124 (strain B)
(Fig. 4). At 1 MIC, we observed fungistatic properties for both
compounds 2 and 9, while our positive control AmB remained
fungicidal and completely killed fungal cells after 9 h of drug
treatment. Both compounds 2 and 9 displayed similar activities

by reducing the fungal population by 1 log CFU and both
compounds remained fungistatic up to 24 h. To evaluate a
possible dose-response effect, we increased the doses of
analogues 2 and 9 by 2 fold (2 MIC) and treated the fungal
cells. Interestingly, at 2 MIC, our compounds 2 and 9 displayed
fungicidal activity and reduced the cell population by 5 log
CFUs, which were equivalent to one of the most potent FDA-
approved antifungal agents, AmB (also at 2 MIC).
capable of inducing ROS, we investigated the effect of two of our
best compounds, 2 and 9, against C. albicans ATCC 10231
(strain A) in a dose depend manner at 1 and 2 MIC using a
2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye.39
Generally, ROS is generated in mitochondria of eukaryotic cells
as a byproduct during cellular metabolism. Cells neutralize the
excess ROS production by activating their antioxidant functions.
However, overproduction of ROS overwhelms the antioxidant
capacity of cells, which in turn causes cell damage. When we
treated yeast cells with our analogues 2 and 9 at their 1 and 2
MIC values, we observed a significant amount of ROS
production in cells (Fig. 6). Similar results were observed for our
control drug AmB (at 1 and 2 its MIC value). However, no
ROS accumulation was observed for our untreated yeast cells. It
is not clear whether yeast cells treated with our compounds
undergo growth inhibition and cell death solely via ROS
production. Nevertheless, it is one of the contributing factors for
the cell death and it will be interesting in future studies to
1e+11
Control (no drug)
1e+10
1e+9
1e+8
1e+7
1e+6
1e+5
1e+4
1e+3
1e+2
1e+1
1e+0
AmB (1x MIC)
2 (1x MIC)
9 (1x MIC)
AmB (2x MIC)
2 (2x MIC)
9 (2x MIC)
perform
in-depth
mechanistic
analysis
for
these
bisbenzimidazoles and potential additional drug conjugates
comprising these molecules. In order to confirm our hypothesis
that ROS production is a contributing factor for cell death, we
also performed this type of assays with one of our inactive
compounds, molecule 4. We observed no ROS accumulation
with compound 4, confirming the role of ROS production in cell
death.
0
5
10
15
20
25
Time (h)
Fig. 4. Representative time-kill studies of AmB as well as compounds 2 and 9
against C. albicans ATCC 64124 (strain B). At 1 MIC, fungal cells were
either treated with the positive control AmB (white circle) or compounds 2
(black inverted triangle) and 9 (white triangle). At 2 MIC, fungal cells were
treated with the positive control AmB (black square) or compounds 2 (white
square) and 9 (black diamond). The untreated cells or negative control are
presented as black circle.
3. Conclusions
In sum, we have synthesized 18 benzimidazoles analogues
(compounds 1-18) with alkyl chains of various lengths (C1, C2,
C3, C5, and C12) on both ends of the molecules via a convenient
convergent synthetic strategy. Interestingly, we did not observe
any antifungal activities with our mono- and trisbenzimidazoles.
However, we observed chain length dependent antifungal
activities of our alkylated bisbenzimidazole analogues, with C1
displaying superior activity than (>) C2 > C3 > C5 > C12, a trend
opposite to that previously observed with alkylated
aminoglycoside (KANA, KANB, and TOB) derivatives.
Compounds 2, 9, and 14 displayed broad-spectrum antifungal
activities against both yeasts and filamentous fungi. In most
cases, analogues 2, 9, and 14 displayed enhanced or comparable
antifungal activity against fungal strains when compared to the
commercial antifungal drugs FLC, ITC, POS, VOR, and AmB.
We also observed the accumulation of ROS in yeast cells when
treated with our compounds. It is very likely that these
compounds may inhibit fungi by inducing ROS production in
cells. It appears that compounds 2, 9, and 14 show promise as
lead compounds for developing antifungal agents and their in
vivo studies would be encouraging. From these new results, we
can infer that the core structure plays a major role in the
antifungal activity of the molecules, and that adding long alkyl
substituents is not always beneficial for the production of
antifungal agents. The facts that compound 14 displays
significant antifungal activity and that it contains in its structure a
chemical handle (alkyne group) are highly encouraging and set
the stage for the future conjugation of this molecule to other drug
scaffolds. Such studies are currently underway in our laboratory.
2.4. In vitro cytotoxicity assay
A key parameter for developing antifungal drugs is to
selectively target fungal cells without killing mammalian cells.
From our in vitro antifungal studies, we identified that
compounds 2, 5, 9, and 14 were highly potent against pathogenic
fungal strains. However, valuable antifungal compounds should
have a large therapeutic index with high selectivity for fungal
cells compared to mammalian cells since both are eukaryotic
cells with similar biological properties. Thus, we opted to
evaluate these four compounds against two mammalian cell lines,
A549 and BEAS-2B, with the popular FDA-approved antifungal
agent AmB as a positive control (Fig. 5). We observed that our
compounds 5 and 9 displayed some toxic effect against A549 and
BEAS 2B, with IC50 values ranging from 1.95-0.98 g/mL, and
7.8-1.95 g/mL, respectively. Similarly, compound 14 also
exhibited toxicity against BEAS 2B (IC50 = 0.98 g/mL), but
showed only minimal toxicity against A549 (IC50 = 15.6 g/mL),
Intriguingly, we found that compound 2 showed less toxicity
against both cell lines with the IC50 values of ≥31.2 g/mL,
which was better than our reference drug, AmB. Additionally, we
evaluated the cytotoxic effect of these compounds on BEAS 2B
cells at a 6 h time point and compared the results with those
obtained at a 24 h time point (Fig. 5B). As expected, more BEAS
2B cells survived when treated for 6 h instead of 24 h. The only
structural difference between these analogues is the substitution
of the hydroxyl group by a methoxy, ethoxy, or propargyl group.
Considering its low fungal MIC values, analogue 2 could
potentially have a high therapeutic index and would be an
attractive antifungal candidate for further safety evaluation in
vivo.
4. Experimental section / Supplementary material
The Supporting Information includes experimental procedures
1
for all assays performed as well as H and 13C NMR spectra
2.5. ROS assays
(Figs. S1-S111) for all molecules synthesized, which were all at
least 95% pure.
It has previously been reported that various fungicidal drugs
(AmB, miconazole, and ciclopirox) induce ROS.37, 38 However,
ROS induction by antifungal agents remains relatively
unexplored. In order to determine if bisbenzimidazoles are
Acknowledgments

Bright field
Fluorescent
Merge
This work was supported by a grant from the National
Institute of Health NIH AI90048 (to S.G.-T.) and by startup
funds from the University of Kentucky (to S.G.-T.).
Control
(no drug)
2
(1x MIC)
2
(2x MIC)
9
(1x MIC)
9
(2x MIC)
AmB
(1x MIC)
AmB
(2x MIC)
4
(8 g/mL)
4
(16 g/mL)
Fig. 6. Effect of benzimidazole derivatives on intracellular ROS production
by C. albicans ATCC 10231 (strain A). Yeast cells were treated with no drug
(negative control) or compounds 2 or 9, or AmB (positive control), at their 1
and 2 respective MIC values, or with compound 4 at 8 and 16 g/mL for 1 h
at 35 °C. These treated cells were further stained with DCFH-DA (20 g/mL)
for 30 min in the dark and were analyzed using a Zeiss Axovert 200M
fluorescence microscope.
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Table 1: MIC valuesa,b (in g/mL) determined for compounds 1-18 and for five control antifungal agents (AmB, FLC, ITC, POS, and VOR) against various
yeast strains and filamentous fungi.
Yeast strains
F
Filamentous fungi
Cpd #
1
2
3
4
5
6
7
8
A
B
>31.2
7.8
31.2
>31.2
31.2
>31.2
>31.2
>31.2
7.8
C
D
>31.2
7.8
31.2
>31.2
15.6
>31.2
>31.2
>31.2
7.8
E
G
H
I
J
K
>31.2
31.2
>31.2
>31.2
31.2
>31.2
>31.2
>31.2
31.2
L
M
>31.2
7.8-15.6
31.2
>31.2
15.6
>31.2
>31.2
>31.2
7.8
>31.2
7.8-15.6
31.2
>31.2
15.6
>31.2
>31.2
>31.2
7.8
>31.2
3.9-7.8
31.2
>31.2
3.9-7.8
31.2
>31.2
7.8-15.6
31.2
>31.2
>31.2
>31.2
>31.2
>31.2
15.6
>31.2
0.975
7.8
>31.2
3.9
>31.2
7.8
>31.2
0.975
7.8
>31.2
3.9
>31.2
1.95
>31.2
7.8
31.2
>31.2
31.2
31.2
7.8
7.8
>31.2
15.6
>31.2
>31.2
>31.2
3.9
15.6
>31.2
15.6
>31.2
>31.2
>31.2
7.8
>31.2
7.8
>31.2
15.6
>31.2
>31.2
>31.2
7.8
>31.2
>31.2
>31.2
7.8
7.8
7.8
>31.2
>31.2
7.8
31.2
15.6
3.9
>31.2
7.8
9
10
11
12
13
14
15
16
17
18
AmB
FLC
ITC
POS
VOR
15.6
15.6
15.6
15.6
15.6
15.6
>31.2
>31.2
>31.2
>31.2
15.6
>31.2
31.2
>31.2
>31.2
3.9
62.5a
31.2a
15.6a
1.95
31.2
>31.2
>31.2
>31.2
7.8
>31.2
31.2
>31.2
31.2
1.95
>31.2
7.8
15.6
15.6
>31.2
>31.2
7.8
15.6
7.8
7.8
>31.2
3.9
>31.2
0.48
0.06
0.12
3.9
>31.2
>31.2
>31.2
>31.2
>31.2
>31.2
31.2
>31.2
>31.2
15.6
15.6
15.6
>31.2
>31.2
>31.2
15.6
>31.2
15.6
>31.2
31.2
3.9
>31.2
>31.2
>31.2
7.8-31.2
>31.2
>31.2
>31.2
>31.2
3.9
>31.2
>31.2
>31.2
31.2
>31.2
>31.2
>31.2
>31.2
3.9
>125a
>62.5a
>62.5a
3.9
>31.2
>31.2
>31.2
7.8
>31.2
31.2
>31.2
>31.2
>31.2
15.6
>31.2
>31.2
>31.2
>31.2
0.975
>125a
31.2a
31.2a
1.95
>31.2
>31.2
>31.2
7.8
>31.2
31.2
>31.2
>31.2
1.95
>125a
31.2a
31.2a
0.975
>31.2
>31.2
>31.2
>31.2
>31.2
>31.2
>31.2
>31.2
3.9
62.5a
31.2a
15.6a
7.8
>31.2
>31.2
>31.2
1.95
>31.2
15.6
31.2
>31.2
1.95
1.95
0.12
<0.03
<0.03
>31.2
>31.2
>31.2
15.6
>31.2
15.6
>31.2
31.2
15.6
62.5a
0.195a
0.195a
0.03
>31.2
>31.2
1.95
62.5a
0.5a
0.5a
15.6a
7.8a
62.5
0.48
0.24
0.24
62.5
0.975
0.48
0.12
7.8a
0.12
0.06
0.24
1.95
Yeast strains: A = C. albicans ATCC 10231, B = C. albicans ATCC 64124, C = C. albicans ATCC MYA-2876(S), D = C. albicans ATCC 90819(R), E = C.
albicans ATCC MYA-2310(S), F = C. albicans ATCC MYA-1237(R), G = C. albicans ATCC MYA-1003(R), H = C. glabrata ATCC 2001, I = C. krusei
ATCC 6258, J = C. parapsilosis ATCC 22019. NOTE: Here, the (S) and (R) indicate that ATCC reports these strains to be susceptible (S) and resistant (R) to
ITC and FLC.
Filamentous fungi: K = Aspergillus flavus ATCC MYA-3631, L = Aspergillus nidulans ATCC 38163, M = A. terreus ATCC MYA-3633.
Known antifungal agents: AmB = amphotericin B, FLC = fluconazole, ITC = itraconazole, POS = posaconazole, and VOR = voriconazole.
a These values were previously reported in ref #13.
b For yeast strains: MIC-0 values are reported for benzimidazole analogues 1-18 and AmB, whereas MIC-2 values are reported for azoles. For filamentous fungi,
MIC-0 values are reported for all compounds.