Bis(N-amidinohydrazones) and N-(amidino)-N′-aryl-bishydrazones: New classes of antibacterial/antifungal agents

Article abstract of DOI:10.1016/j.bmc.2016.10.009

The emergence of multidrug-resistant bacterial and fungal strains poses a threat to human health that requires the design and synthesis of new classes of antimicrobial agents. We evaluated bis(N-amidinohydrazones) and N-(amidino)-N′-aryl-bishydrazones for

Full text of DOI:10.1016/j.bmc.2016.10.009

Accepted Manuscript
Bis(N-amidinohydrazones) and N-(amidino)-N ’-aryl-bishydrazones: New
classes of antibacterial/antifungal agents
Sanjib K. Shrestha, Liliia M. Kril, Keith D. Green, Stefan Kwiatkowski, Vitaliy
M. Sviripa, Justin R. Nickell, Linda P. Dwoskin, David S. Watt, Sylvie Garneau-
Tsodikova
PII:
S0968-0896(16)30924-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.009
BMC 13336
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 September 2016
6 October 2016
7 October 2016
Please cite this article as: Shrestha, S.K., Kril, L.M., Green, K.D., Kwiatkowski, S., Sviripa, V.M., Nickell, J.R.,
Dwoskin, L.P., Watt, D.S., Garneau-Tsodikova, S., Bis(N-amidinohydrazones) and N-(amidino)-N ’-aryl-
bishydrazones: New classes of antibacterial/antifungal agents, Bioorganic & Medicinal Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bmc.2016.10.009
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Graphical Abstract
Bis(N-amidinohydrazones) and N-(amidino)-
N'-aryl-bishydrazones: New classes of
antibacterial/antifungal agents
Sanjib K. Shrestha, Liliia M. Kril, Keith D. Green, Stefan Kwiatkowski, Vitaliy M. Sviripa, Justin R. Nickell,
Linda P. Dwoskin, David S. Watt, and Sylvie Garneau-Tsodikova
Department of Pharmaceutical Sciences and Center for Pharmaceutical Research and Innovation (College of Pharmacy), Department of Molecular and
Cellular Biochemistry and Lucille Parker Markey Cancer Center (College of Medicine), University of Kentucky, 789 South Limestone Street, Lexington,
KY, 40536-0596, USA.

Bioorganic & Medicinal Chemistry
Bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones: New classes of
antibacterial/antifungal agents
Sanjib K. Shrestha,a Liliia M. Kril,a,b Keith D. Green,a Stefan Kwiatkowski,b,d Vitaliy M. Sviripa,b,c Justin
R. Nickell,a Linda P. Dwoskin,a David S. Watt,b,c,d,* and Sylvie Garneau-Tsodikovaa,*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536-0596, USA.
bDepartment of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, 40536-0509, USA. cCenter for
Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, Lexington, KY, 40536-0596, USA. dLucille Parker Markey Cancer
Center, University of Kentucky, Lexington, KY, 40536-0093, USA.
* Corresponding author: E-mail address: dwatt@uky.edu (D.S. Watt) or sylviegtsodikova@uky.edu (S. Garneau-Tsodikova)
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The emergence of multidrug-resistant bacterial and fungal strains poses a threat to human health
that requires the design and synthesis of new classes of antimicrobial agents. We evaluated
bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones for their antibacterial and
antifungal activities against panels of Gram-positive/Gram-negative bacteria as well as fungi.
We investigated their potential to develop resistance against both bacteria and fungi by a multi-
step, resistance-selection method, explored their potential to induce the production of reactive
oxygen species, and assessed their toxicity. In summary, we found that these compounds
exhibited broad-spectrum antibacterial and antifungal activities against most of the tested strains
with minimum inhibitory concentration (MIC) values ranging from <0.5->500 M against
bacteria and 1.0->31.3 g/mL against fungi; and in most cases, they exhibited either superior or
similar antimicrobial activity compared to those of the standard drugs used in the clinic. We also
observed minimal emergence of drug resistance to these newly synthesized compounds by
bacteria and fungi even after 15 passages, and we found weak to moderate inhibition of the
human Ether-à-go-go-related gene (hERG) channel with acceptable IC50 values ranging from
1.12-3.29 M. Overall, these studies show that bis(N-amidinohydrazones) and N-(amidino)-N'-
aryl-bishydrazones are potentially promising scaffolds for the discovery of novel antibacterial
and antifungal agents.
Available online
Keywords:
Bishydrazones
Candida albicans
Cytotoxicity
hERG channel
Resistance
Staphylococcus aureus
2009 Elsevier Ltd. All rights reserved.
1. Introduction
The incidence of invasive fungal infections is also on the rise
due to an increasing population of critically ill patients as a result
The emergence of multidrug-resistant bacteria and fungi as
human pathogens warrants a continued focus on the development
of new pharmacophores for the treatment of these devastating
and often fatal infections. The rise of multidrug-resistant bacteria,
such as methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant Enterococci (VRE), adversely affects the
efficacy of many known, standard-of-care, antibacterial agents.1
Evidence of the impact of these multidrug-resistant strains
appears in a 2011 report from the Centers for Disease Control
and Prevention (CDC) that estimates that the national incidence
of invasive MRSA infections was 80,461 cases and 650 deaths.
This mortality rate is among the highest recorded for bacterial
infections.2 Likewise, listerosis, which is a common foodborne
illness caused by Listeria monocytogenes, represents a serious
illness afflicting elderly people, newborns, and those with
impaired immune systems. Estimates are that 19% of deaths
associated with the consumption of contaminated foods in the
United States are due to L. monocytogenes.3
of the human immunodeficiency virus (HIV), systemic diseases
such as cancer, and the increasing occurrence of organ
transplantation.4 The National Healthcare Safety Network
(NHSN) at the CDC reported that Candida spp. ranked fifth
amongst hospital-acquired pathogens.5 Candida spp. are reported
as the fourth most common causative pathogens of nosocomial
and of fatal bloodstream infections.6 Eukaryotic C. albicans
shares a close evolutionary relationship, as well as many cellular
mechanisms, with their human hosts, and represents challenges
for treatment of systemic fungal infections. There is a clear need
for new antimicrobials that selectively inhibit these
microorganisms without causing host toxicity.
Pentamidine represents an archetypical, biscationic antibiotic
with
a symmetrical structure containing two amidinium
functional groups separated by a flexible 1,5-diphenoxypentane
spacer.7 Developed initially as an antiprotozoal agent,
pentamidine currently finds applications in both the treatment of
protozoan diseases, such as Trypanosoma brucei gambiense

Scheme 1. Synthetic scheme for the preparation of A. the bis(N-amidinohydrazones) and B. N-(amidino)-N'-aryl-bishydrazones.
(West African trypanosomiasis), as well as systemic fungal
infections caused by Pneumocystis jirovecii, often seen in
patients with HIV. Related compounds include symmetrical
bisamidines, (e.g., furimidazoline),8 developed principally as
topoisomerase inhibitors for cancer treatment. In addition to
these biscationic compounds, other hydrazone-containing and
guanidine-containing molecules possess a range of promising
biological activities, including antitubercular,9,10 anti-HIV,11,12
yeast cells, determined their in vitro cytotoxicity and their
affinity for the human Ether-à-go-go-related gene (hERG)
potassium channel. The combination of testing new antimicrobial
agents and early screening for resistance and toxicity represents
an avenue that may produce pharmacophores of potential utility
in the treatment of diseases.
2. Results and discussion
anticonvulsant,13
anticancer,14
anti-inflammatory,15,16
2.1. Design and chemical synthesis of biscationic compounds
with two chemically identical termini
antimalarial,17,18 antibacterial,19,20 and antifungal activities.21,22
Recently, bis(N-amidinohydrazones) were reported to inhibit the
calcium-dependent serine endoprotease, furin, which activates
immature proteins to their functional, mature forms.23
The reported syntheses of biscationic compounds containing
two identical termini bearing guanidinium, amidinium, or
pyridinium groups attached to aryl or alkyl spacers typically
involved the modification of spacers with termini bearing
primary halides, carbonyl, or nitrile groups.24-26 Alkylation of
,-dihaloalkanes, for example, with 2-aminopyridine led to
bis(2-aminopyridinium) salts.24 Modification of the nitrile group
in 5-(4-cyanophenyl)furan-2-carboxylic acid and completion of
the spacer by linking the carboxylate groups led to bisamidinium
agents.27 In the same fashion, we modified the carbonyl groups in
either bisaldehyde 1 or bisketone 2, which in some cases were in
regiochemically distinct positions in the spacer (e.g., 1B, 2B,
2H), with N-aminoguanidine hydrochloride to obtain the
biscationic products 3 and 4, respectively (Scheme 1A).
Inspired by the above successes, we were intrigued by the
possibility that biscationic pharmacophores in either the bis(N-
amidinohydrazones) or the N-(amidino)-N'-aryl-bishydrazone
family with either a flexible or a rigid spacer would offer
advantages in terms of antibacterial and antifungal activities not
found in the flexible bisamidines, as well as in the hydrazone-
and guanidine-containing molecules currently in the literature. To
investigate the potential of such compounds, we synthesized nine
symmetrical bis(N-amidinohydrazones) (3A-B and 4A,C-H) and
eight asymmetrical N-(amidino)-N'-aryl-bishydrazones (7Aa-Ag
and 8Aa) (Scheme 1). Further, we evaluated the antibacterial and
antifungal activities of these novel compounds against panels of
bacterial strains (four Gram-positive, six Gram-negative, and one
mycobacterial) and seven Candida albicans strains. Because the
development of resistance represents a crucial problem in
antimicrobial drug development, we also established the potential
for resistance development by bacteria and fungi against these
compounds. Additionally, we measured the production of
reactive oxygen species (ROS) of some of these compounds in
2.2. Design and chemical synthesis of biscationic compounds
with chemically non-identical termini
Synthetic approaches to bicationic agents with two
chemically non-identical termini often involved the construction
of the spacer as the ultimate step. For example, the coupling of an
amidino-substituted naphthol with
a
guanidine-substituted

benzoic acid secured biscationic esters with an amidinium group
at one terminus and a guanidinium group at the other.28 Our
approach differed from this strategy in that we utilized a
stepwise, chemoselective modification of bisaldehydes 1 or
bisketones 2 in order to arrive at biscationic systems with
different cationic groups at each terminus. Condensations of one
equivalent of N-aminoguanidine hydrochloride with 1A and 2A
led to the efficient production of monosubstituted N-
amidinohydrazones 5A and 6A, respectively. The hydrochloride
salts of these monocatioinic products were readily crystallized
free from starting materials. The subsequent treatment of 5A and
6A with N-arylhydrazines provided the desired, biscationic
agents 7Aa-Ag and 8Aa, respectively, which we describe as N-
(amidino)-N'-aryl-bishydrazones (Scheme 1B). Cavallini
described the monocationic N-amidinohydrazones as antibacterial
agents some years ago, but the range of organisms and MIC50
values were, in general, unimpressive.29
ATCC 19115 (strain B, Lmo) (MIC <0.5-7.8 M), methicillin-
resistant Staphylococcus aureus (strain C, MRSA) (MIC = 1.0-
7.8 M), and vancomycin-resistant enterococcus (strain D, VRE)
(MIC = 1.0-7.8 M). All of the novel compounds displayed poor
activity against Bacillus subtilis 168 (strain A, Bsu), with the
exception of compound 4F, which displayed excellent activity
(MIC = 7.8 M) against this strain. Likewise, compound 7Ae
exhibited excellent antibacterial activity against L.
monocytogenes ATCC 19115 (strain B, Lmo; MIC = 7.8 M) and
VRE (strain D; MIC = 2.0-3.9 M), but only moderate
antibacterial activity against B. subtilis 168 (strain A, Bsu; MIC =
31.3 M) and MRSA (strain C; MIC = 15.6 M), respectively. In
general, compounds 4A, 4E (except against strain D, VRE), 4G,
4H, and 8Aa (except against strain D) showed poor antibacterial
activity (MIC = 62.5-≥500 M) against all four Gram-positive
bacterial strains tested. Overall, compound 4F was the most
active against Gram-positive bacteria.
With these first generations of bis(N-amidinohydrazones) 3
and 4 and N-(amidino)-N'-aryl-bishydrazones 7 and 8 in hand, we
aimed to answer the following eight apriori questions in terms of
structure-activity-relationship (SAR). We included the numbers
of the compounds used to answer these questions into
parentheses following the questions. The eight questions are as
follows: (i) are linkers comprised of only one aromatic ring
sufficient to confer antimicrobial activity? (compounds 4G and
4H); (ii) is the presence of methyl groups on the carbons alpha to
the linker beneficial for antimicrobial activity? (compounds 3A
versus 4A, and 7Aa versus 8Aa); (iii) how does the substitution
pattern of the biphenyl linker affect antimicrobial activity?
(compounds 3A and 3B); (iv) how does the length of the flexible
linear alkyl spacer between the two phenyl rings affect
antimicrobial activity? (compounds 4A, 4C, and 4D); (v) is the
introduction of rigidity in the linker beneficial for antimicrobial
activity? (compound 4C versus 4F); (vi) is the presence of an
oxygen atom in the linker beneficial for antimicrobial activity?
(compound 4C versus 4E); (vii) how does the identity of the
substituent, when located at the same position on the aryl ring of
the side chain, affect antimicrobial activity? (compounds 7Aa
versus 7Ab, 7Ac versus 7Ad versus 7Ae, and 7Af versus 7Ag);
and finally, (viii) how does the substitution pattern of the aryl
ring (ortho versus para-monosubstituted versus ortho,para-
disubstituted) affect antimicrobial activity? (compounds 7Aa
versus 7Ad versus 7Ag).
On the other hand, the majority of the novel compounds
remained inactive against most of the Gram-negative bacterial
strains that we tested with MIC values ranging from 62.5->500
M. As one of several exceptions, compound 4A exhibited
excellent activity against P. aeruginosa ATCC 27853 (strain I,
Pae; MIC = 2.0 M) and moderate activity against Klebsiella
pneumoniae ATCC 27736 (strain H, Kpn; MIC = 15.6 M).
Similarly, compound 4F also displayed excellent antibacterial
activities against Pae strain I (MIC = 1.0-2.0 M) and S. enterica
ATCC 14028 (strain J, Sen; MIC = 3.9 M), as well as moderate
to low activity against Kpn strain H (MIC = 31.3-62.5 M).
Additionally, 7Ae showed only moderate antibacterial activities
against Kpn strain H (MIC = 15.6-31.3 M), strain I (MIC =
31.3-62.5 M), and Sen strain J (MIC = 15.6 M). Compounds
3B, 4C-D, 4F, 7Aa-Ad, and 7Af-Ag exhibited excellent
antibacterial activity (MIC = 1.0-3.9 M) against M. smegmatis
MC2-155 (strain K, Msm), whereas compounds 4H and 7Ae
showed only moderate activity (MIC = 15.6-31.3 M) and
compounds 3A, 4A, 4E, 4G, and 8Aa showed low activity (MIC
= 125->500 M) against this mycobacterial strain. It is
noteworthy that when compared to clinically relevant
antibacterial drugs, such as AMK (MIC = 3.9-125 M), AMP
(MIC ≥250 M), and OFX (MIC ≤0.5-31.3 M), the novel
compounds showed either superior or comparable antibacterial
activity against all bacterial strains tested.
2.4. Antifungal activity
2.3. Antibacterial activity
Having established the antibacterial activity profile of the
novel bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-
bishydrazones, we next determined their antifungal activity
against a panel of seven Candida albicans strains using a
concentration range of 0.5-31.3 g/mL (Table 2). We used the
common antifungal agent amphotericin B (AmB) as a positive
control testing in the same concentration range of 0.5-31.3
g/mL. For antifungal activity, we use the following descriptors:
excellent (MIC = 0.5-3.9 g/mL), moderate (MIC = 7.8-15.6
g/mL), and poor (MIC = 31.3 g/mL). We found that
compounds 3B, 4F, 7Aa, 7Ab, and 7Af exhibited excellent
antifungal activities against all fungal strains tested (MIC = 1.0-
3.9 g/mL), with 4F being overall the most active. The only
exception to the aforementioned statement was 7Af, which
displayed only moderate activity against C. albicans ATCC
90819 (MIC = 7.8 g/mL). Similarly, compound 4A also
displayed excellent antifungal activities (MIC = 2.0-3.9 g/mL)
against most strains, except against C. albicans ATCC 1003
(MIC = 7.8 g/mL) and C. albicans ATCC 1237 (MIC = 31.3
g/mL). Compounds 7Ad, 7Ae, 7Ag, and 8Aa showed only
To determine if our novel bis(N-amidinohydrazones) and N-
(amidino)-N'-aryl-bishydrazones displayed antibacterial activity,
we first evaluated compounds 3A-8Aa against a panel of Gram-
positive (4 strains), Gram-negative (6 strains), and one
mycobacterial strain in a concentration range of 0.5-500 M
(Table 1). We used the aminoglycoside amikacin (AMK), the -
lactam ampicillin (AMP), and the fluoroquinolone ofloxacin
(OFX) as positive controls. In general, the novel compounds
displayed excellent (MIC values ≤0.5-7.8 M), intermediate (also
referred to as moderate) (15.6-31.3 M), or low (also referred to
as poor) (62.5-≥500 M) antibacterial activity against the
bacterial strains tested. It is important to note that these MIC
value ranges are used from here on to qualitatively described all
compounds tested, including the positive controls.
Overall, when analyzing the MIC data obtained against the
Gram-positive bacterial strains (strains A-D), we note that most
of the novel bis(N-amidinohydrazones) (3A,B, 4C,D, 4F) and N-
(amidino)-N'-aryl-bishydrazones (7Aa-Ad, and 7Af-Ag) showed
excellent antibacterial activity against Listeria monocytogenes

Table 1: MIC values ing/mL and (M)a for compounds 3A,B, 4A,C-H, 7Aa-Ag, and 8Aa against various bacterial strains.
Gram-positive
Gram-negative
Cpd
AMK
A (Bsu)
3.0-12.2
(3.9-15.6)
>92.8
(>250)
≤0.2
(≤0.5)
>198
(>500)
>198
(>500)
>212
(>500)
>219
(>500)
>226
(>500)
54.9
(125)
3.4
(7.8)
86.8
(250)
86.8
(250)
>215
(>500)
>235
(>500)
>205
(>500)
>214
(>500)
13.1
(31.3)
>214
(>500)
>231
(>500)
26.6-53.3 26.6-53.3
(62.5-125) (62.5-125)
B (Lmo) C (MRSA) D (VRE) E (Aba)
F (Ecl) G (Eco)
H (Kpn)
24.5
(31.3)
>92.8
(>250)
I (Pae)
48.9
(62.5)
>92.8
(>250)
1.4
J (Sen)
3.0-6.1
(3.9-7.8)
92.8
K (Msm)
12.2
(15.6)
>92.8
(>250)
5.6
(15.6)
>198
(>500)
0.4
3.0
(3.9)
92.8
(250)
0.4
24.5
(31.3)
92.8
(250)
0.4
97.7
(125)
92.8
(250)
2.8
24.5
(31.3)
>92.8
(>250)
≤0.2
48.9
(62.5)
>92.8
(>250)
≤0.2
24.5
(31.3)
>92.8
(>250)
1.4-2.8
AMP
OFX
3A
(250)
5.6-11.3
5.6-11.3
(15.6-31.3)
>198
(>500)
>198
(>500)
>212
(>500)
>219
(>500)
>226
(1.0)
0.4
(1.0)
1.5
(7.8)
0.8
(≤0.5)
>198
(≤0.5)
>198
(>500)
>198
(>500)
52.9
(3.9-7.8) (15.6-31.3)
(3.9)
>198
(>500)
>198
(>500)
26.5
>198
(>500)
>198
(>500)
6.6
(15.6)
>219
(>500)
>226
(>500)
27.5
(62.5)
13.6-27.2
(31.3-62.5) (1.0-2.0)
173
(500)
>173
(>500)
>215
(>500)
>235
>198
(>500)
>198
(>500)
0.8
(1.0)
0.4
(3.9)
1.5
(2.0)
1.5
(>500)
>198
3B
(1.0)
>212
(>500)
1.7
(3.9)
>212
(>500)
1.7
(3.9)
>212
(>500)
0.9
(>500)
>212
(>500)
>219
(1.0)
>212
(>500)
0.9
(2.0)
0.5
4A
(125)
>219
(>500)
>226
(>500)
110
(62.5)
>219
(>500)
>226
(>500)
54.9
(2.0)
>219
(>500)
>226
(>500)
27.5
4C
(3.9)
0.5
(1.0)
27.5
(3.9)
0.5-0.9
(1.0-2.0)
54.9
(2.0)
0.5
(1.0)
1.7
(>500)
>226
(>500)
27.5
4D
(>500)
54.9
(125)
1.7
(1.0)
54.9-110
(125-250)
0.4
4E
(62.5)
<0.2
(<0.5)
86.8
(250)
86.8
(250)
0.4-0.9
(1.0-2.0)
0.9
(125)
0.4-0.9
(1.0-2.0)
>173
(>500)
>173
(>500)
0.9
(3.9)
0.4
(62.5)
218
(500)
173
(500)
>173
(>500)
>215
(>500)
>235
(250)
(125)
(62.5)
0.4-0.9
218
218
4F
(1.0)
43.4
(125)
43.4
(125)
0.9
(500)
>173
(>500)
>173
(>500)
>215
(>500)
>235
(>500)
>205
(>500)
>214
(>500)
209
(500)
>173
(>500)
>173
(>500)
>215
(>500)
>235
(>500)
>205
(>500)
(3.9)
(1.0)
86.8
(250)
173
(500)
>215
(>500)
>235
(>500)
>205
(>500)
86.8-173
(250-500)
21.7-86.8
(62.5-250)
>215
(>500)
>235
(>500)
>205
(>500)
>214
(>500)
6.5
(15.6)
>214
(>500)
>231
(>500)
26.3-53.3
(62.5-125)
>173
(>500)
5.4
(15.6)
1.7
(3.9)
1.8
(3.9)
1.6
4G
4H
7Aa
7Ab
7Ac
7Ad
7Ae
7Af
7Ag
8Aa
(2.0)
0.9
(2.0)
0.9
(2.0)
0.8
(2.0)
3.2
(2.0)
1.6
(>500)
>205
(>500)
>205
(>500)
>214
(>500)
(2.0)
1.7
(7.8)
3.3
(3.9)
1.7
(>500)
>214
(3.9)
1.7
(3.9)
>214
(>500)
26.1-52.2
>214
(>500)
13.1-26.1
(3.9)
3.3
(7.8)
6.5
(3.9)
0.8-1.6
(>500)
6.5-13.1
6.5-13.1
6.5-13.1
(15.6-31.3)
0.9-1.7
(2.0-3.9)
1.8
(3.9)
213
(500)
(7.8)
0.9-1.7
(2.0-3.9)
3.6
(15.6)
1.7
(3.9)
3.6
(2.0-3.9) (15.6-31.3) (500) (62.5-125) (15.6-31.3) (31.3-62.5)
1.7
(3.9)
3.6
>214
(>500)
>231
>214
(>500)
>231
(>500)
213
>214
(>500)
>231
(>500)
213
>214
(>500)
>231
(>500)
26.6-53.3
(62.5-125)
>214
(>500)
>231
(>500)
213
(7.8)
(7.8)
(7.8)
(>500)
53.3
1.7
26.6
(125)
(3.9)
(62.5)
(500)
(500)
(500)
a These antibacterial MIC values were determined originally in M using a range of 0.5 to 500 M. These values are presented into parentheses.
The values in g/mL are presented for comparison with the antifungal MIC values, which were determined originally in g/mL.
Gram-positive: A (Bsu) = B. subtilis 168, B (Lmo) = L. monocytogenes ATCC 19115, C = MRSA, D = VRE.
Gram-negative: E (Aba) = A. baumannii ATCC 19606, F (Ecl) = E. cloacae ATCC 13047, G (Eco) = E. coli MC1061, H (Kpn) = K. pneumoniae
ATCC 27736, I (Pae) = P. aeruginosa ATCC 27853, J (Sen) = S. enterica ATCC 14028.
Mycobacterial: K (Msm) = M. smegmatis MC2-155. Note: All strains without an ATCC number are either clinical isolates or obtained from a
different source than ATCC (source provided in supporting information).
Control antibiotics: AMK = amikacin, AMP = ampicillin, OFX = ofloxacin.
moderate fungal growth inhibition (MIC = 7.8-15.6 g/mL)
against the majority of the fungal strains, with the exception of C.
albicans ATCC 64124 (MIC = 31.3 g/mL for 7Ae, 7Ag, and
8Aa), C. albicans ATCC 90819 (MIC = 31.3 g/mL for 7Ag), C.
albicans ATCC 1003 (MIC = 3.9 g/mL for 7Ad) and C.
albicans ATCC 2310 (MIC = 3.9 g/mL for 7Ad). Compounds
7Ac also exhibited excellent growth inhibition against most of
the fungal strains tested, except against C. albicans ATCC 10231
(moderate activity, MIC = 7.8 g/mL) and C. albicans ATCC
64124 (poor activity, MIC = 31.3 g/mL). Just as observed
against all bacterial strains, compounds 4G and 4H displayed no
activity (MIC ≥31.3 g/mL) against all fungal strains tested. It is
important to emphasize that most of our bis(N-
amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones
displayed either superior or comparable antifungal activities
against the majority of the fungal strains tested, when compared
to the clinically-relevant antifungal agent, AmB (MIC = 2.0-3.9
g/mL).
Using the antibacterial and antifungal results presented above
(Tables 1 and 2), we addressed the eight apriori questions posed:
(i) Are linkers comprised of only one aromatic ring sufficient
to confer antimicrobial activity? We concluded that bis(N-
amidinohydrazones) with linkers comprised of a single phenyl
ring, regardless of its substitution pattern (para or meta, as in
compounds 4G and 4H, respectively), displayed insufficient
activity as antimicrobial agents to warrant further study of their
antibacterial or antifungal activity.
(ii) Is the presence of methyl groups on the carbons alpha to
the linker beneficial for antimicrobial activity? Comparing the
MIC values of compounds 3A and 4A as well as 7Aa and 8Aa,
which only differ by the absence or presence of a methyl group
on the carbons alpha to the phenyl rings, led us to conclude that
the methyl group was detrimental to the antibacterial activity of
these compounds. However, the presence of the methyl group
appeared beneficial in terms of antifungal activity. Compound
4A was overall a much more active antifungal agent than
compound 3A.
2.5. SAR analysis

Table 2: MIC values in g/mLa and (M) for compounds 3A,B, 4A,C-H, 7Aa-Ag, and 8Aa against various C. albicans strains.
C. albicans ATCC strain #
1003a
1237b
2310c
2876c
10231b
64124b
90819b
Cpd
AmB
3A
3B
4A
4C
4D
4E
4F
3.9 (4.2)
7.8 (19.7)
3.9 (9.9)
3.9 (4.2)
15.6 (39.5)
3.9 (9.9)
3.9 (4.2)
>31.3 (>79.2)
3.9 (9.9)
3.9 (4.2)
7.8 (19.7)
3.9 (9.9)
2.0 (4.7)
3.9 (8.9)
3.9 (8.6)
3.9 (8.9)
1.0 (2.3)
>31.3 (>90.1)
7.8 (22.5)
2.0 (4.6)
3.9 (8.3)
3.9 (9.5)
7.8 (18.3)
15.6 (37.3)
3.9 (9.1)
15.6 (33.8)
15.6 (34.3)
3.9 (4.2)
15.6 (39.5)
3.9 (9.9)
2.0-3.9 (2.1-4.2) 2.0 (2.1)
>31.3 (>79.2)
3.9 (9.9)
>31.3 (>79.2)
3.9 (9.9)
3.9 (9.2)
7.8 (17.8)
7.8 (17.3)
7.8 (17.8)
2.0 (4.6)
>31.3 (>90.1)
>31.3 (>90.1)
2.0 (4.6)
7.8 (18.4)
7.8 (17.8)
15.6 (34.6)
7.8 (17.8)
1.0 (2.3)
>31.3 (>90.1)
>31.3 (>90.1)
2.0 (4.6)
2.0 (4.1)
2.0 (4.7)
3.9 (9.1)
7.8 (18.7)
2.0 (4.5)
>31.3 (>73.9)
7.8 (17.8)
15.6 (34.6)
7.8 (17.8)
2.0 (4.6)
>31.3 (>90.1)
31.3 (90.1)
3.9 (9.2)
3.9 (9.2)
2.0-3.9 (4.7-9.2)
7.8 (17.8)
7.8 (17.3)
7.8 (17.8)
1.0-2.0 (2.3-4.6)
>31.3 (>90.1)
>31.3 (>90.1)
2.0 (4.6)
2.0 (4.7)
7.8 (17.8)
31.3 (69.3)
7.8 (17.8)
1.0 (2.3)
>31.3 (>90.1)
31.3 (90.1)
2.0 (4.6)
3.9 (8.3)
3.9 (9.5)
3.9 (9.1)
15.6 (37.3)
3.9 (9.1)
7.8 (17.8)
>31.3 (>69.3)
7.8 (17.8)
1.0 (2.3)
31.3 (90.1)
>31.3 (>90.1)
3.9 (9.2)
4G
4H
7Aa
7Ab
7Ac
7Ad
7Ae
7Af
7Ag
8Aa
3.9 (8.3)
2.0 (4.1)
3.9 (8.3)
3.9 (8.3)
3.9 (9.5)
7.8 (19.0)
15.6 (36.5)
15.6 (37.3)
3.9 (9.1)
15.6 (33.8)
15.6 (34.3)
7.8 (19.0)
7.8 (18.3)
15.6 (37.3)
1.0 (2.3)
7.8 (16.8)
15.6 (34.3)
31.3 (76.2)
15.6 (36.5)
31.3 (74.9)
3.9 (9.1)
31.3 (67.8)
31.3 (68.7)
7.8 (18.3)
15.6 (37.3)
7.8 (18.2)
31.3 (67.8)
15.6 (34.3)
7.8 (16.8)
15.6 (34.3)
7.8 (16.8)
15.6 (34.3)
a These antifungal MIC values were determined originally in g/mL using a range of 0.5 to 31.3 g/mL. The values in M into parentheses are
presented for comparison with the antibacterial MIC values, which were determined originally in M.
b Indicates strains that are resistant to FLC, ITC, and VOR according to ATCC.
c Indicates strains that are susceptible to FLC, ITC, and VOR according to ATCC.
Control antifungal agent: AmB = amphotericin B.
(iii) How does the substitution pattern of the biphenyl linker
affect antimicrobial activity? When evaluating compounds 3A
and 3B, the antibacterial MIC values were similar in all cases,
except against strain K where the MIC value for compound 3B
(MIC = 1 M) was 500 times lower than that for compound 3A
(MIC >500 M). From these data, we deduced that the
substitution pattern of the biphenyl linker had minimal effect on
antibacterial activity. However, we found the substitution pattern
of the biphenyl linker to have a substantial effect on antifungal
activity. For example, compound 3B was a much more active
antifungal agent than compound 3A.
antibacterial (CN > halogen) and antifungal (halogen > CN)
activity. We also observed that the o,p-difluorinated and o,p-
dichlorinated aryl moieties of compounds 7Af and 7Ag had
similar overall antibacterial activity, but different antifungal
activity, in which the o,p-difluorinated group possessed improved
antifungal activity.
(viii) How does the substitution pattern of the aryl ring (ortho
versus para-monosubstituted versus ortho,para-disubstituted)
affect antimicrobial activity? Finally, comparison of the activity
resulting from different chlorination patterns in compounds 7Aa
(ortho), 7Ad (para), and 7Ag (o,p-diCl), revealed minimal
effects on antibacterial activity, but substantial effects on
antifungal activity. The o-Cl N-(amidino)-N'-aryl-bishydrazone
was superior to the p-Cl analogue, which in turn was much better
than o,p-diCl analogue. In summary, the similarity and
differences in the trends observed in this promising, preliminary
SAR will be used to lay the foundation for the future discovery of
compounds capable of selectively targeting either bacteria or
fungi.
(iv) How does the length of the flexible linear alkyl spacer
between the two phenyl rings affect antimicrobial activity? When
comparing the MIC values of compounds 4A, 4C, and 4D, again
we observed opposing trends in antibacterial and antifungal
activities. Longer flexible alkyl linkers between the two phenyl
rings correlated with a decrease in antibacterial MIC values, but
correlated with an increase in antifungal MIC values.
(v) Is the introduction of rigidity in the linker beneficial for
antimicrobial activity? By contrasting the MIC value profile of
compound 4C containing a flexible linker to that of compound
4F containing a rigid linker, we concluded that introducing
rigidity in the linker was overall beneficial for antimicrobial
activity.
2.6. Development of bacterial and fungal resistance studies
The emergence of antibiotic resistance by microbes is an
inevitable process; however, the frequency at which resistance
develops varies from one antibiotic to another. It is critical to
assess the ability of new antimicrobials to evade, as long as
possible, the development of bacterial and fungal resistance early
in the drug discovery process. To establish if the bis(N-
amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones
evade resistance in bacteria, we first performed a multi-step
resistance selection experiment with compounds 4D, 4F, and
7Aa as well as with AMK as a reference drug against four
bacterial strains (Figure 1). L. monocytogenes ATCC 19115
(strain B, Lmo), MRSA (strain C), VRE (strain D), and P.
aeruginosa ATCC 27853 (strain I, Pae) were exposed to sub-
inhibitory concentrations of compounds 4D, 4F, 7Aa, and AMK,
and were sub-cultured for 15 serial passages to determine if an
increase in MIC values occurred for each compound against the
strains tested. L. monocytogenes ATCC 19115 (strain B, Lmo),
MRSA (strain C), VRE (strain D), and P. aeruginosa ATCC
(vi) Is the presence of an oxygen atom in the linker beneficial
for antimicrobial activity? A comparison of the MIC value
profiles of compounds 4C and 4E indicated that replacing the
methylene bridging the two phenyl groups of the linker by an
oxygen atom had no effect on antifungal activity, but was
detrimental to antibacterial activity.
(vii) How does the identity of the substituent, when located at
the same position on the aryl ring of the side chain, affect
antimicrobial activity? By comparing compounds 7Aa-7Ag, we
observed that the nature of the substituent in the ortho position of
the mono-substituted aryl group of compounds 7Aa and 7Ab did
not affect antibacterial or antifungal activity, whereas the nature
of the substituent in the para position of the mono-substituted
aryl group of compounds 7Ac-7Ae had opposing effects on

27853 (strain I, Pae) did not develop resistance to compound 4F,
as demonstrated by the one-fold increase in MIC values after 15
serial passages. Likewise, strains B (Lmo) and D (VRE) did not
develop resistance to compound 7Aa, but strains C (MRSA) and
I (Pae) developed resistance to this compound after twelve serial
passages. In contrast, we observed the rapid development of
resistance to compound 4D in strains C (MRSA) and D (VRE)
with a 16-fold increase in MIC values after fifteen serial
passages. This development of resistance against compound 4D
was observed also in strains B (Lmo) and I (Pae), where
increases in relative MIC values by 8- and 4-folds, respectively,
were observed after 15 passages. Interestingly, in strains D
(VRE) and I (Pae), resistance to the control antibiotic, AMK,
was observed also after 3 and 5 passages, respectively; whereas
in strains C (MRSA) and B (Lmo), resistance developed after 12
and 15 passages, respectively. Overall, these results indicate that
there is a low probability of emergence of resistance to these
promising novel biscationic compounds 4D, 4F, and 7Aa.
dichlorodihydrofluorescein diacetate (DCFH-DA) dye.33 We
found that treatment of C. albicans ATCC 10231 (strain A) with
compounds 4F and 7Aa at their 1 and 2 MIC values increased
intracellular ROS production in this fungal strain (Figure 2). As
expected, the H2O2 positive control (at 1 mM) also induced ROS
production in yeast cell, whereas no ROS induction was observed
with untreated yeast cells (negative control). Although it remains
to be determined if ROS production is critical for growth
inhibition and death of yeast cells, these results suggest one
direction for future mechanistic exploration, which is outside of
the scope of the current proof-of-principle exploration for the
first generations of bis(N-amidinohydrazones) and N-(amidino)-
N'-aryl-bishydrazones as antimicrobials.
Bright field
Fluorescent
Merge
Control
(no drug)
H2O2
(1 mM)
positive
control
4F
(1x MIC)
4F
(2x MIC)
7Aa
(1x MIC)
7Aa
(2x MIC)
Figure 1. Changes in MIC values of L. monocytogenes ATCC 19115 (strain
B), MRSA (strain C), VRE (strain D), and P. aeruginosa ATCC 27853
(strain I) treated with AMK (green), 7Aa (purple), 4F (blue), 4D (orange), as
well as C. albicans ATCC 10231 treated with AmB (yellow), 7Aa (purple),
and 4F (blue) over 15 cycles. Numbers above the bars represent the passage
when either bacterial or fungal cells developed resistance.
Figure 2. Effect of compounds 4F and 7Aa on intracellular ROS production
by C. albicans ATCC 10231. Yeast cells were treated in the absence of drug
(negative control), 1 mM of H2O2 (positive control), or 4F and 7Aa, at 1 and
2 respective MIC values for 1 h at 35 °C. After staining with DCFH-DA (20
g/mL), the samples were analyzed using a Zeiss Axovert 200M fluorescence
microscope.
We next investigated the development of drug resistance in C.
albicans ATCC 10231 to our best antifungal compounds 7Aa
and 4F, and we found that C. albicans ATCC 10231 did not to
develop drug resistance to either 7Aa or 4F, despite repeated
treatments with sub-MIC drug concentrations. Only a slight 2-
fold shift in MIC values was observed after 13 passages. These
results also suggest the low probability of onset of drug
resistance by fungi to these novel compounds.
Table 3: The cytotoxicity (IC50, g/mL) of selected bis(N-
amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones against A549
and BEAS-2B cell lines. Values are presented as mean ± SDEV.
Mammalian cell line
Cpd
AmB
3A
3B
4A
4C
4D
4E
4F
A549
BEAS-2B
17.7 ± 6.4
3.0 ± 0.7
5.7 ± 1.8
1.7 ± 0.5
2.9 ± 0.9
1.8 ± 0.6
2.9 ± 0.9
1.8 ± 0.6
33.0 ± 4.6
2.3 ± 1.0
3.4 ± 1.3
6.0 ± 1.2
6.0 ± 1.1
1.9 ± 0.8
5.8 ± 1.2
18.2 ± 4.5
6.6 ± 1.7
13.9 ± 4.1
6.3 ± 2.2
4.3 ± 1.3
2.9 ± 0.9
3.9 ± 0.9
2.5 ± 1.4
31.2 ± 7.2
2.8 ± 0.9
6.7 ± 2.3
4.4 ± 1.4
4.9 ± 1.1
4.2 ± 1.4
4.8 ± 1.3
2.7. Measurement of ROS induction in fungal cells
Various antifungal drugs, such as AmB and miconazole, as
well as novel antifungal agents, were shown to mediate their
inhibitory effect by inducing intracellular ROS production.30-32
Universally, eukaryotic cells produce basal amount of ROS in
mitochondria as a byproduct of cellular metabolism. In response,
the cellular enzymatic antioxidants, including superoxide
dismutase and glutathione peroxidase, scavenge ROS in cells.
However, overproduction of deleterious ROS perturbs the
delicate, intracellular equilibrium between ROS production and
scavenging, and the result is cellular damage.
4H
7Aa
7Ab
7Ac
7Ad
7Ae
7Af
In order to investigate the ability of compounds 4F and 7Aa
to alter ROS production in C. albicans ATCC 10231 (strain A),
we performed
a fluorescent-based assay using a 2',7'-
2.8. Cytotoxicity

Another important aspect to consider during the development
of antimicrobial agents is their potential toxicity towards
Figure 3. Mammalian cell cytotoxicity of selected bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones, as well as AmB (as a control) against A.
A549 cell line and B. BEAS-2B cell line.
Figure 4. IC50 curves for hERG interaction by representative bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones along with a table (bottom right)
summarizing these IC50 values.
mammalian cells. Having established the potent antimicrobial
activities of the novel bis(N-amidinohydrazones) and N-
(amidino)-N'-aryl-bishydrazones against bacteria and fungi, we
determined the toxicity profile of these compounds against two
mammalian cell lines (A549 and BEAS-2B; Figure 3) and
measure the IC50 values for these compounds (Table 3). The
majority of the novel compounds showed concentration-
dependent toxicity with IC50 values of 1.7-6.7 g/mL.
Compounds 3B, 7Ac, 7Ad, and 7Af, with excellent antibacterial
MIC values of <0.4-3.3 g/mL, displayed IC50 values of 4.4-13.9
g/mL against both mammalian cell lines. It is important to note
that compounds 7Ac and 7Af also displayed excellent antifungal

MIC values, indicating that these compounds are important leads
for future evaluation.
Acknowledgments
This work was supported by startup funds from the College of
Pharmacy (to S.G.-T.) and by the Office of the Dean of the
College of Medicine (to D.S.W.) at the University of Kentucky.
It was also supported by NIH grant AI090048 (to S.G.-T.); and
NIH grants U01 DA013519, UL1TR000117 and T32 DA016176
(to L.P.D. and J.R.N.). D.S.W. was also supported in part through
collaborations involving NIH grants P20 RR020171 (to L.
Hersh), CA172379 (to C. Liu), and CA187273 (to V.
Rangnekar). The manuscript’s contents are solely the
responsibility of the authors and do not necessarily represent the
official views of the NIH, NIAID, or NIGMS.
2.9. hERG inhibition assay
The hERG encodes a voltage gated potassium channel that plays
an essential role in regulating hearth rhythm.34 Inhibition of the
hERG channel disrupts the heart rhythm and may lead to cardiac
death. Recently, drugs (e.g., terfendadine and cisapride) were
withdrawn from the market due to their interaction with the
hERG channel.35 Currently, the U.S. Food and Drug
Administration (FDA) and the European Medicines Agency
(EMA) require evaluation at hERG for drug candidates.
We performed a [3H]-dofetilide competition binding assay
using HEK-293 cell membranes stably expressing the hERG
channel to evaluate the activity of our most potent bis(N-
amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones at
hERG (Figure 4). Compounds exhibiting IC50 values of >10 M
are considered to have low affinity for the hERG channel.
Compounds displaying IC50 values in the range of 1-10 M are
considered to have moderate affinity, whereas compounds
exhibiting IC50 values of <1 M are considered to have high
affinity for the hERG channel.36 The majority of the novel
compounds displayed IC50 values within the acceptable range of
1-10 M (Figure 4). Although compound 7Ab displayed
excellent antifungal activities (2-3.9 g/mL), it exhibited in vitro
mammalian cell toxicity and was found to potently interact with
the hERG channel (IC50 = 0.44 ± 0.10 M), suggesting that the o-
bromophenyl substituent is an unfavorable structural feature.
However, the p-fluorophenyl and the 2,4-difluorophenyl groups
in compounds 7Ac and 7Af displayed only moderate interaction
with hERG (IC50 = 3.29 ± 0.63 M and 2.24 ± 0.70 M,
respectively). Strikingly, both of these compounds displayed
excellent antifungal activities and also exerted low mammalian
cell toxicity. Similarly, compounds 4F and 7Aa displayed
moderate affinity for hERG (IC50 = 1.12 ± 0.32 M and 1.14 ±
0.27 M, respectively); however, they displayed some toxicity
against mammalian cells. Therefore, we concluded that
compounds 7Ac and 7Af are the most promising N-(amidino)-N'-
aryl-bishydrazone compounds.
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