1,2,4-Triazolidine-3-thiones as narrow spectrum antibiotics against multidrug-resistant acinetobacter baumannii

Article abstract of DOI:10.1021/acsmedchemlett.6b00296

With only two new classes of antibiotics developed in the last 40 years, novel antibiotics are desperately needed to combat the growing problem of multidrug-resistant and extensively drug resistant bacteria, particularly Gram-negative bacteria. Described in this letter is the synthesis and antibiotic activity of 1,2,4-triazolidine-3-thiones as narrow spectrum antibiotics. Optimization of the 1,2,4-triazolidine-3-thione scaffold identified a small molecule with potent antibiotic activity against multiple strains of multidrug-resistant and extensively drug-resistant Acinetobacter baumannii. This small molecule also shows single dose, in vivo activity in a Galleria mellonella infection model with A. baumannii and represents a promising start in the development of a class of drugs that can target this bacterial pathogen.

Full text of DOI:10.1021/acsmedchemlett.6b00296

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Letter
pubs.acs.org/acsmedchemlett
1,2,4-Triazolidine-3-thiones as Narrow Spectrum Antibiotics against
Multidrug-Resistant Acinetobacter baumannii
William M. Huggins,† Bradley M. Minrovic,† Brendan W. Corey,‡ Anna C. Jacobs,‡ Roberta J. Melander,†
Roger D. Sommer,† Daniel V. Zurawski,‡ and Christian Melander*,†
†Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, North Carolina 27695, United States
‡Wound Infections Department, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, United States
S
* Supporting Information
ABSTRACT: With only two new classes of antibiotics developed
in the last 40 years, novel antibiotics are desperately needed to
combat the growing problem of multidrug-resistant and
extensively drug resistant bacteria, particularly Gram-negative
bacteria. Described in this letter is the synthesis and antibiotic
activity of 1,2,4-triazolidine-3-thiones as narrow spectrum anti-
biotics. Optimization of the 1,2,4-triazolidine-3-thione scaffold
identified a small molecule with potent antibiotic activity against multiple strains of multidrug-resistant and extensively drug-
resistant Acinetobacter baumannii. This small molecule also shows single dose, in vivo activity in a Galleria mellonella infection
model with A. baumannii and represents a promising start in the development of a class of drugs that can target this bacterial
pathogen.
KEYWORDS: antibiotic resistance, antibiotics, Acinetobacter baumannii
ntibiotic resistance has become one of the forefront issues
in global health because of the emergence of multidrug-
spectrum antibiotics, namely, the possibility of slower resistance
generation and a reduction in detrimental effects on the gut
microbiome including a decreased risk of antibiotic-associated
colitis.6 As more rapid and sensitive diagnostic tests are
developed, the use of narrow-spectrum antibiotics is becoming
more feasible, and the EMA and the FDA have recently
included antibacterial compounds active against a narrow
spectrum of MDR pathogens in a list of treatments that qualify
for a shorter route to registration.7 Of the Gram-negative
ESKAPE pathogens, A. baumannii has recently come under the
microscope because of its high mortality rates in the ICU
(>50%), prevalence in wound infections in U.S. servicemen that
have been injured in the conflicts in Iraq and Afghanistan,8 as
well as its ability to survive in hospital environments and the
isolation of PR strains.9
A
resistant (MDR) bacteria. According to the Centers for Disease
Control and Prevention (CDC), an estimated two million
people each year acquire MDR bacterial infections, of which
23,000 are fatal.1 Even more alarming is the dearth of antibiotic
options emerging to treat these infections. Only two antibiotics
belonging to novel structural classes have been brought into the
clinic in the past 40 years, daptomycin and linezolid.2 Of great
concern is that both of these antibiotics are only effective
against Gram-positive bacteria, which leaves treatment options
for MDR Gram-negative bacteria as an urgent unmet medical
need. Prominent Gram-negative pathogens include Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,
and Enterobacter species, which along with the Gram-positive
species Enterococcus faecium and Staphylococcus aureus make up
the bacterial species that are often referred to as “ESKAPE”
pathogens.3 Currently, the most effective option to treat MDR
Gram-negative bacteria remains the polymyxin colistin, which
has significant side effects including nephrotoxicity.4 These side
effects, coupled with colistin’s effectiveness, have made it a last
resort antibiotic against MDR Gram-negative bacteria. Worry-
ingly, colistin-resistant strains of bacteria are being isolated with
greater frequency, and recently, a colistin-resistance gene was
found on a plasmid that could easily move between bacterial
species.5 Therefore, the threat of superbugs, bacteria becoming
extensively drug resistant (XDR) and pandrug-resistant (PR), is
a real possibility. As a result, new antibiotics with activity
against Gram-negative pathogens are sorely needed.
In 2009 compound 1 (Scheme 1) was reported to have
antifungal activity against Candida albicans.10 Recently, genomic
Scheme 1. Synthesis of 1,2,4-Triazolidine-3-thiones12,13
Received: July 29, 2016
The development of narrow spectrum antibiotics has some
advantages over the traditional approach of developing broad-
Accepted: October 31, 2016
© XXXX American Chemical Society
A
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ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
sequencing has revealed that A. baumannii harbors genes that
share homology to a long chain fatty acid elongation pathway in
fungi that was the supposed target of compound 1. Testing of
1,2,4-triazolidine-3-thiones, for antibiotic activity against A.
baumannii identified compound 1 as a promising antibacterial
lead.11 Using this compound as a structural template, we have
synthesized analogues of the 1,2,4-triazolidine-3-thione core
structure with variations introduced at the N-1, N-2, and C-5
positions as well as alkylations of the sulfur atom at C-3 in an
attempt to identify new, more biologically active compounds.
Herein, we report the synthesis of analogues of 1,2,4-
triazolidine-3-thiones as well as their antibiotic activity against
MDR-A. baumannii. Moreover, we report a compound that has
minimum inhibitory concentrations (MICs) of up to 4-fold
lower against multiple strains of MDR-A. baumannii compared
to compound 1 and, unlike parent compound 1, shows activity
in a Galleria mellonella model of infection.
The 1,2,4-triazolidine-3-thione scaffold is readily accessible
through a three-component reaction between ketones or
aldehydes, hydrazines, and potassium thiocyanate in hydro-
chloric acid for 16 h in the dark (Scheme 1). The resulting
triazolidines typically precipitate during the reaction, allowing
for simple filtration followed by recrystallization from methanol
to deliver purified material.12,13 Moreover, this synthetic
procedure easily allows for modifications to be made at the
N-2 and C-5 positions on the ring, thus allowing rapid
assessment of the structure−activity relationship of 1,2,4-
triazolidine-3-thiones.
Initially, we evaluated the effect that substitution at carbon 5
had upon activity by varying the identity of the aliphatic
substituents. A series of 14 compounds that were mono- or di-
substituted with alkyl or aryl groups were synthesized and
screened for antibiotic activity against AB5075 under standard
CLSI broth microdilution conditions14 (Table 1). A. baumannii
strain AB5075 is an XDR primary clinical isolate and was
chosen to evaluate the 1,2,4-triazolidine-3-thiones because of its
virulence and XDR properties.15
Compound 1 exhibited an MIC of 8 μg/mL against AB5075,
and both the dimethyl (compound 2a) and diethyl (compound
2e) derivatives exhibited identical activity to the parent
compound. All other modifications at the C-5 position yielded
either no change, or an increase in MIC compared to the parent
compound (Table 1).
As we did not observe an increase in antibiotic activity
through modification of the 5-position we next shifted our
focus to the substitution at the N-2 position of the ring. The
diethyl substituted derivatives were chosen instead of the
parent methyl ethyl substituted derivatives to avoid racemic
mixtures. Benzyl, cyclohexyl, and 2-pyridinyl substitutions of
the N-2 position completely abolished biological activity
(supporting information), which led to the conclusion that
the phenyl ring at the N-2 position was required for activity
(compound 3, Scheme 1).
Given the significant impact that changing the 2-phenyl
substituent to a 2-benzyl had upon activity, we then
investigated whether substitution of the phenyl ring itself had
any effect on the MIC (Table 3). The 4-chloro derivative 4a
exhibited increased activity compared to the parent compound,
with an MIC of 2 μg/mL. Encouraged by this result, additional
analogues were synthesized with various halogen substitutions
in different positions on the phenyl ring. Unfortunately, only a
decrease in activity was observed compared to the 4-chloro
compound. Other substitutions on the phenyl ring were also
made, but only halogen substitutions were tolerated (Table 2).
Table 2. MIC Values for Various Phenyl Ring Substitutions
against AB5075, Where R1 = R2 = Ethyl
a
compd
R3
MIC
4a
4b
4c
4d
4e
4f
(4-chlorophenyl)
(4-bromophenyl)
(4-iodophenyl)
2
4
8
(4-fluorophenyl)
(2-fluorophenyl)
(3-fluorophenyl)
(2-chlorophenyl)
(3-chlorophenyl)
(3-chloro-4-fluoro)
(3,5-difluorophenyl)
(3,4-dichlorophenyl)
(4-isopropylphenyl)
(4-nitrophenyl)
4
32
8
4g
4h
4i
128
8
32
4j
>128
>128
>128
>128
>128
>128
4k
4l
4m
4n
4o
(4-cyanophenyl)
(4-trifluoromethylphenyl)
a
Table 1. MIC Values for C-5 Modifications against AB5075,
All concentrations are in μg/mL.
Where R3 = Phenyl
a
compd
R1
R2
ethyl
MIC
With a more active derivative 4a identified, we next tested
1
methyl
methyl
methyl
methyl
methyl
ethyl
8
the lead compound and parent compound against multiple
strains of MDR-A. baumannii (representative examples shown
in Table 3). The dimethyl analogue of compound 4a,
compound 516−18 (Scheme 1), was also prepared to investigate
whether the lipophilicity of the compounds could be tuned
without affecting the activity of the compounds (note: our
2a
2b
2c
2d
2e
2f
methyl
isopropyl
phenyl
H
8
8
>128
>128
8
ethyl
ethyl
isopropyl
H
32
>128
16
32
32
64
64
8
2g
2h
2i
ethyl
Table 3. MIC Results for Compounds 1, 4a, and 5 against
a
n-propyl
n-propyl
n-propyl
n-propyl
phenyl
cyclopentyl
cyclohexyl
methyl
ethyl
Multiple MDR Strains of A. baumannii
compd AB-4878 AB-3785 AB-3806 AB-4957 BAA-1605 AB-3638
2j
n-propyl
H
2k
2l
1
8
4
2
4
4
1
4
4
1
8
4
2
8
4
2
4
4
1
H
4a
5
2m
2n
8
a
The resistance profiles of these strains have been previously
published.19 All concentrations are in μg/mL.
a
All concentrations are in μg/mL.
B
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ACS Medicinal Chemistry Letters
Letter
spectral data for compound 5 did not match previously
reported data; however, the crystal structure of our synthetic
sample was determined to unambiguously establish identity).
Compound 4a has a predicted log D value of 3.85, while
compound 5 has a predicted log D value of 2.58 at pH 7.
All three compounds exhibited activity against multiple
strains of A. baumannii, while compound 5 was more active
against several strains of A. baumannii than either compound 4a
or the parent compound 1 (Table 3). Additional MIC results
against more strains of A. baumannii can be found in the
Supporting Information.
After the identification of compound 5 as the most active
analogue against several A. baumannii strains, we next opted to
investigate further structure−activity relationships of com-
pound 5, beginning with the effect of alkylating the sulfur atom
at the C-3 position. Briefly, compound 5 was reacted with the
corresponding alkyl halide in methanol at room temperature
overnight to yield the thioethers 6a−b (Scheme 2). The methyl
than128 μg/mL, demonstrating the need for the free N−H at
the N-1 position on the 1,2,4-triazolidine-3-thione ring for
biological activity.
The final structural modification that was investigated was to
move the 4-chlorophenyl substituent from the N-2 position to
the N-4 position of compound 5 (Scheme 2C). The reverse
1,2,4-triazolidine-3-thione scaffold is accessed through the
reaction of N-(4-chlorophenyl) hydrazinecarbothioamide with
acetone in hydrochloric acid for 16 h in the dark (Scheme 2C).
As with compounds 1−4o, the resulting triazolidine precipi-
tated during the reaction, allowing for simple filtration followed
by recrystallization from methanol to deliver purified materi-
al.12,13 The observed MIC of compound 10 was greater than
128 μg/mL, which is a greater than 64-fold increase in MIC
compared to compound 5, indicating that the original
substitution pattern of the 1,2,4-triazolidine-3-thiones is the
more active motif.
We next investigated whether compounds 5 was acting in a
bactericidal or bacteriostatic manner by constructing a time kill
curve. An antibiotic is defined as bactericidal when it kills
≥99.9% of bacteria at a concentration no greater than four
times the MIC.21 As seen in the time-kill curve below (Figure
1), compound 5 is bactericidal, effecting a greater than six log
reduction in colony forming units per milliliter (CFU/mL) at
double the MIC and greater.
Scheme 2. Synthetic Routes To Allow for the Alkylation of
the Thiol Moiety (A), N-1 Position (B), and Move of 4-
a
Chloro Substituent (C) of Compound 5
Figure 1. Time-kill curve for compound 5 against AB5075.
In C. albicans and A. baumannii, compound 1 distorts the
ratio of unsaturated to saturated fatty acids.10,11 It was also
established that fatty acid supplementation of media abrogated
the bactericidal effects of compound 1 against A. baumannii.11
In order to test whether compound 5 exhibited similar
behavior, an MIC assay under fatty acid supplementation was
performed. As with compound 1, when the media was
supplemented with 0.02% linoleic acid, the activity of
compound 5 against AB5075 was completely abolished with
an observed MIC of >128 μg/mL.
In order to probe the antibiotic spectrum of compound 5,
MICs against Escherichia coli, P. aeruginosa, K. pneumoniae, and
methicillin resistant S. aureus (MRSA) were recorded. An MIC
of ≥128 μg/mL was observed for all bacterial species other
than A. baumannii, establishing that as with the original lead 1,
compound 5 is a narrow spectrum antibiotic. The synergistic
potential of compound 5 with other antibiotics was also
investigated utilizing checkerboard assays.22 Compound 5 did
not show synergy with Meropenem or colistin against AB5075,
or with colistin against the colistin-resistant AB3941.
a
Reagents and conditions: (a) Alloc-Cl, DMAP, Et3N, THF rt, 16 h;
(b) RX, NaH, DMF 0° C to rt, 4 h; (c) NaBH4, Pd(PPh3)4, EtOH 0°
C to rt, 1 h; (d) 12 N HCl (pH 2.5-3), rt, 4 h.
and benzyl analogues were prepared, and the observed MIC for
both compounds was greater than 128 μg/mL, showing the
necessity of the unsubstituted sulfur atom for biological activity.
To further probe the SAR of the 1,2,4-triazolidine-3-thione
core, effect of alkylation of the N-1 position of compound 5 was
also investigated (Scheme 2).
In order to alkylate the N-1 position, the thiol was protected
with an alloc protecting group. The N-1 position was then
alkylated using sodium hydride and the corresponding alkyl
halide. The alloc group was removed using sodium borohydride
and tetrakis(triphenylphosphine)palladium(0) in ethanol at 0
°C, and the solution was then acidified with 12 N HCl at room
temperature.20 The MICs of compounds 9a−b were all greater
Given the potential hydrolytic instability of 5, we performed
a time-dependent stability study. Compound 5 was dosed in
C
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ACS Medicinal Chemistry Letters
Letter
cation adjusted Mueller−Hinton Broth (CAMHB) and
incubated at 37 °C. The mixture was then analyzed by taking
liquid chromatography. We determined that compound 5 has a
half-life of about 2.5 h in these conditions and is almost fully
degraded after 16 h (Supporting Information). After observing
the decomposition of the compound, compound 5 was dosed
in CAMHB at 4 μg/mL and incubated for 1, 2, 4, and 8 h. The
activity of each of the incubated solutions was then tested
against A. baumannii 5075, and compound 5 was found to have
no activity at its MIC after incubation in MHB for any of the
time points. This indicates that compound 5, and not any
degradation products, is most likely the active chemical species.
The toxicity of compound 5 to eukaryotic cell membranes
was also investigated by measuring its hemolytic activity against
mechanically defibrinated sheep blood as previously reported.23
Compound 5 did not lyse the red blood cells at any
concentration tested (up to 512 μg/mL, Supporting
Information).
Finally, we compared the activity of compound 1 and 5 in a
G. mellonella infection model with AB5075.15 Compound 1 was
inactive against AB5075 in this model (data not shown,
Supporting Information). However, compound 5 exhibited
modest in vivo activity, with 50 mg/kg of compound 5 saving
22% of worms after 6 days, and 100 mg/kg saving 32% of
worms after 6 days. In comparison, 100% of untreated, infected
worms were dead after 6 days (Figure 2). The comparison is
against multiple strains of A. baumannii compared with
compound 1, as well as returning a lower MIC than compound
4a in most cases. Compound 5 was found to be bactericidal,
and an in vivo study showed positive survival results when
compound 5 was used against AB5075 in a G. mellonella model
of infection. While these results were modest, it is important to
note that just a single dose was provided, and most antibiotics
are dosed multiple times a day. Furthermore, AB5075 is a more
virulent strain than most other A. baumannii isolates,14 which
provides a higher bar when evaluating activity. Future studies
are now underway to develop structural modifications and
conduct biological evaluations against A. baumannii using
compound 5 as our current lead in an attempt to further
augment in vitro activity and improve in vivo results.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00296.
Compound characterization for all novel compounds, 1H
and 13C NMR spectra, MIC and full in vivo testing data
(PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: ccmeland@ncsu.edu. Phone: (919) 513-2960.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
The authors from N.C. State would like to thank the university
for support. The authors from Wound Infection Department at
WRAIR would like to acknowledge the constant support of the
Military Infectious Diseases Research Program.
Notes
The material has been reviewed by the Walter Reed Army
Institute of Research. There is no objection to its presentation
and/or publication. The opinions or assertions contained
herein are the private views of the authors, and are not to be
construed as official, or as reflecting true views of the
Department of the Army or the Department of Defense.
The authors declare no competing financial interest.
Figure 2. Percent survival of G. mellonella after AB5075 infection and
compound 5 treatment.
more stark at 3 days, where almost 90% of the worms are dead
in the untreated group, but in contrast, 42.5% and 45% survival
are seen with the 50 mg/kg and 100 mg/kg treatments,
respectively (Figure 2).
ACKNOWLEDGMENTS
■
Dr. Zurawski would like to thank the Multidrug-resistant
Organism Repository and Surveillance Network (MRSN) for
providing the isolates used in this study.
In conclusion, following the identification of the 1,2,4-
triazolidine-3-thiones as possessing antibiotic activity against A.
baumannii we synthesized a diverse array of analogues in
attempts to identify a compound with increased activity. After
making modifications at the C-5 and N-2 positions on the 1,2,4-
triazolidine-3-thione core structure to no effect, substitutions
were made to the phenyl ring at position 2 to yield compound
4a, which displayed a 4-fold improvement in MIC against A.
baumannii compared to compound 1. Compound 5 was also
synthesized and tested against multiple strains of A. baumannii
along with compound 1 and compound 4a in order to ascertain
whether the lipophilicity could be tuned without affecting the
MIC. Compound 5 displayed an up to 4-fold improvement
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