Allicin-inspired thiolated fluoroquinolones as antibacterials against ESKAPE pathogens

Article abstract of DOI:10.1016/j.bmcl.2016.10.002

Thiolated fluoroquinolones were synthesized from ciprofloxacin and evaluated for antimicrobial activity against a panel of pathogenic bacteria. Gram-positive species including methicillin-resistant Staphylococcus aureus (MRSA) exhibited the highest level of increased sensitivity toward ciprofloxacin bound with a N-propylthio substituent. Evidence was found that the antibiotics form disulfides with low molecular weight thiols in bacteria and potentiate generation of cytosolic reactive oxygen species (ROS). In final analysis, the enhanced anti-MRSA activity of thiolated fluoroquinolones was attributed to increased cell permeability and reaction with cytosolic thiols that yields an inactive disulfide metabolite and the parent drug ciprofloxacin as an inhibitor of DNA synthesis.

Full text of DOI:10.1016/j.bmcl.2016.10.002

Accepted Manuscript
Allicin-inspired thiolated fluoroquinolones as antibacterials against ESKAPE
pathogens
Jordan G. Sheppard, Timothy E. Long
PII:
S0960-894X(16)31030-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.002
BMCL 24305
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
29 July 2016
30 September 2016
1 October 2016
Please cite this article as: Sheppard, J.G., Long, T.E., Allicin-inspired thiolated fluoroquinolones as antibacterials
against ESKAPE pathogens, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.10.002
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1
Bioorganic & Medicinal Chemistry Letters
Allicin-inspired thiolated fluoroquinolones as antibacterials against ESKAPE
pathogens
Jordan G. Sheppard and Timothy E. Longa,b
a
a
Department of Pharmaceutical Science and Research, School of Pharmacy, Marshall University, Huntington, WV 25755
Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Thiolated fluoroquinolones were synthesized from ciprofloxacin and evaluated for
antimicrobial activity against a panel of pathogenic bacteria. Gram-positive species including
methicillin-resistant Staphylococcus aureus (MRSA) exhibited the highest level of increased
sensitivity toward ciprofloxacin bound with a N-propylthio substituent. Evidence was found
that the antibiotics form disulfides with low molecular weight thiols in bacteria and potentiate
generation of cytosolic reactive oxygen species (ROS). In final analysis, the enhanced anti-
MRSA activity of thiolated fluoroquinolones was attributed to increased cellular permeability
and reaction with cytosolic thiols that yields an inactive disulfide metabolite and the parent
drug ciprofloxacin as an inhibitor of DNA synthesis.
Available online
Keywords:
fluoroquinolone
allicin
antibiotic
MRSA
2
009 Elsevier Ltd. All rights reserved.
glutathione
Multidrug resistant (MDR) bacteria pose a major ongoing
threat to public health [1]. The majority of MDR infections are
healthcare-acquired and involve evasive, toxin-producing strains.
Among the most dangerous are species in the Center for Disease
Control (CDC)-designated list of “ESKAPE” bacteria
(Enterococcus spp., Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, and Enterobacter spp.). Each of the ESKAPE
species, which includes vancomycin-resistant Enterococcus
Figure 1. Chemical structures of allicin and ciprofloxacin.
Synthetic allicin was tested in combination with 10 µM CIP by
the conventional disc diffusion (Kirby-Bauer) assay on Mueller
Hinton agar (Table 1). Of the ESKAPE species evaluated, S.
aureus (MRSA) was the only bacterium with increased
susceptibility to the combination, which was attributed to an
additive effect, rather than synergism between the antibiotics.
Concomitantly, the study corroborated the reported activity
spectrum of allicin [7] and prompted an investigation on whether
a labile, electrophilic N-alkylthio residue could enhance the
antibacterial properties of CIP. In this report, the biological
activity of thiolated fluoroquinolones against ESKAPE pathogens
is examined.
(
VRE) and methicillin-resistant S. aureus (MRSA), have the
ability to cause severe disease and exhibit rapid rates of
resistance development [2]. Despite having limited coverage for
MDR variants of ESKAPE bacteria, fluoroquinolones remain one
of the most effective antibiotic classes for curing infections.
Fluoroquinolones are broad spectrum, bactericidal antibiotics
that bind DNA topioisomerases and inhibit nucleic acid
synthesis. Resistant bacteria (e.g., ESKAPE) often possess
signaling pathways as a protective mechanism against genotoxic
fluoroquinolone exposure [3]. Activation of the state of system
(SOS) distress response when DNA synthesis is impaired by
Table 1: Antibacterial activity of allicin and ciprofloxacin combinations.
these antibiotics [4] induces expression of error-prone DNA
polymerases [4], efflux pumps [4,5] and biofilm-forming
mediators [6]. Interestingly, allicin (1, Figure 1), a natural
electrophilic (δ ) thiosulfinate from Allium sativum (garlic) [7], is
reported to inhibit biofilm formation and quorum-sensing in P.
aeruginosa [8]. Moreover, allicin exhibits anti-MRSA activity
compound | species
allicin (1)
test amount (µM) | zone of inhibition (mm)
50
0
0
25
10
10
25
22
20
28
31
50
10
17
29
24
25
27
31
+
ciprofloxacin (CIP)
E. faecium (VRE)
S. aureus (MRSA)
K. pneumoniae (MDR)
A. baumannii (MDR)
P. aeruginosa
10
0
18
28
13
24
0
20
22
0
[9] and synergism with vancomycin (VAN) against VRE [10].
These prior findings lead us to synthesize allicin from diallyl
disulfide [11] to assess for synergistic activity with the
fluoroquinolone ciprofloxacin (CIP, Figure 1) on a panel of
ESKAPE bacteria.
29
31
E. cloacae
15
—
——

Corresponding author. Tel.: +1-304-696-7393; fax: +1-304-696-7309; e-mail: longt@marshall.edu

2
N-Thiolated fluoroquinolones 3 were synthesized from CIP
with use of the phthalimide-based sulfenylating agents 2 (Scheme
). The reagents were easily prepared from phthalimide and their
corresponding sulfenyl chlorides, which was generated in situ
from sulfuryl chloride and its respective thiol or disulfide [12].
Trituration in hexanes-EtOAc afforded thiophthalimide 2 in high
yield. The sulfenylating agents and CIP (free-base) were then
reacted with NaHCO in MeCN:DMF at 80 °C [13]. Purification
3
by silica gel flash chromatography or precipitation in hexanes-
acetone gave N-alkylthio fluoroquinolones 3a-h.
vancomycin-intermediate resistant S. aureus (VISA) and VRE to
both CIP and N-alkylthio fluoroquinolones was negligible due to
undiscerned mechanisms of resistance. Analogs bound with
shorter straight alkylthio chains (e.g., 3c R = n-Pr) were generally
more active than those having longer chains (e.g., 3h R = n-
hexyl). Branched alkylthio derivatives 3d, 3f, and 3g
demonstrated similar antibacterial efficacies to the n-propyl
analog 3c of equal chain length. It was concluded from these data
that alkylthio chains of three carbons confer the greatest increase
in activity. Moreover, the structure-activity relationship (SAR)
against Gram-positive bacteria was in good agreement with the
SAR of N-thiolated β-lactams previously reported by the Turos
laboratory [14,15].
1
The N-alkylthio fluoroquinolones 3a-h also exhibited
antibacterial activity against the Gram-negative ESKAPE
members. Overall, the n-propyl derivative 3c proved again to be
the most active growth inhibitor. Other analogs with slightly
improved activity over CIP (Table 2) were fluoroquinolones 3b
(R = Et) for P. aeruginosa and 3g (R = 3-methyl-2-butyl) for K.
pneumoniae. Curiously, CIP-resistant A. baumannii had weak
susceptibility to N-methylthio fluoroquinolone 3a. Potentially
related cysteine-modifying disulfide inhibitors of thioredoxin-A
(TrxA) have also demonstrated antibacterial activity against A.
baumannii including MDR variants [16]. Based on these results,
it was concluded that attachment of short chain alkylthio
substituents did not significantly alter fluoroquinolone potency
against Gram-negative ESKAPE bacteria.
Scheme 1. Synthesis of N-alkylthio fluoroquinolones 3a-h.
Table 2: Antibacterial activity of N-alkylthio fluoroquinolones 3a-h.
a
b
Other thiolated fluoroquinolones including arylthio analogs 3i-l
(Table 3) were synthesized and evaluated for antibacterial
activity. The compounds were similarly prepared from CIP and
their respective N-(arylthio)phthalimide reagent (2, Scheme 1).
Most of the arylthio derivatives were weaker antibacterials
compared to CIP (Table 2) with the exception of the p-
nitrophenylthio substituted 3k, which exhibited increased
antistaphylococcal activity. Likewise, the analog was more active
than its unsubstituted- (3i), p-chloro- (3j) and o-nitro- (3l) phenyl
counterparts.
species
zone of inhibition (mm)
3b 3c 3d 3e 3f 3g 3h CIP
c
3
a
E. faecium (VRE)
E. faecalis
0
0
0
0
0
8
0
8
0
21 25 28 23 23 22 25 15 22
24 24 29 21 22 24 28 18 23
23 22 24 23 20 23 23 18 20
S. aureus (MSSA)
S. aureus (MRSA)
S. aureus (VISA)
11 11 11 10 11 11
9
9
9
K. pneumoniae (MDR) 22 23 25 23 21 20 26 16 22
A. baumannii
A. baumannii (MDR)
P. aeruginosa
E. cloacae
23 24 27 23 22 22 26 15 23
10
0
0
0
0
0
0
0
0
29 32 32 31 28 27 31 23 29
23 25 29 25 25 23 29 19 31
36 37 38 36 37 35 32 30 34
33 32 37 34 34 32 29 26 31
Table 3: Antibacterial activity of N-arylthio fluoroquinolones 3i-l.
a
b
species
zone of inhibition (mm)
B. anthracis
3i
Ph
10
20
21
22
12
20
23
0
3j
p-ClPh
8
3k
3l
o-NO
0
S. epidermidis
R =
p-NO
8
2
Ph
2
Ph
a
E. faecium 70021 [vancomycin-resistant (VRE)]; E. faecalis 29212
vancomycin-susceptible); S. aureus 29213 [methicillin-susceptible (MSSA)],
E. faecium (VRE)
E. faecalis
(
20
25
29
24
11
21
23
0
20
18
19
0
BAA-1747 [methicillin-resistant (MRSA)], 700699 [Mu50, vancomycin-
intermediate resistant (VISA)]; K. pneumoniae K6 700603 [ESBL-producer (β-
lactam-resistant)]; A. baumannii 19606, BAA-1605 [MDR (ciprofloxacin-
resistant)]; P. aeruginosa 15442; E. cloacae 23355; S. epidermidis 14990; B.
anthracis Sterne 34F2
S. aureus (MSSA)
S. aureus (MRSA)
S. aureus (VISA)
21
17
12
b
mean diameter of visible growth inhibition for test compounds at 10 µM
CIP: ciprofloxacin
K. pneumoniae (MDR)
A. baumannii
20
9
c
20
19
0
A. baumannii (MDR)
P. aeruginosa
0
The thiolated fluoroquinolones 3a-h were evaluated for
antibacterial activity by the disc diffusion assay (Table 2).
Compounds were tested in DMSO at 10 µM against a diverse
panel of characterized ESKAPE strains. In addition, the
susceptibility of Staphylococcus epidermidis and Bacillus
27
26
26
27
23
9
E. cloacae
22
19
B. anthracis
36
23
34
29
32
36
27
23
S. epidermidis
+
-
a
anthracis Sterne 34F2 (pXO1 , pXO2 ) was assessed with CIP as
a comparator. As expected, CIP-susceptible organisms exhibited
similar sensitivities to the N-alkylthio derivatives. Increases in
antibacterial activity (i.e., larger zone diameters) were observed
for most compounds against the Gram-positive species
Enterococcus faecalis, B. anthracis, S. epidermidis, methicillin-
susceptible S. aureus (MSSA), and MRSA. The susceptibility of
refer to Table 2 for strain information
mean diameter of visible growth inhibition for test compounds at 10 µM
b
Following preliminary evaluation of the fluoroquinolones 3a-l
by disc diffusion analysis, the N-propylthio analog 3c was
selected for further activity comparison by the broth
microdilution assay (Table 4). Against the three S. aureus

3
variants, compound 3c exhibited
a
minimum inhibitory
ROS detoxification and metabolism, inactivation of CoASH
through disulfide formation is an additional mechanism by which
thiolated fluoroquinolones could potentially decrease cell
viability.
concentrations (MIC) range of 0.1 – 50 µM/mL, which correlated
with the zone data results (Table 2). In comparison with standard
drugs, CIP displayed equipotent efficacy while the activity of
VAN was retained against MRSA, but not against the VISA
strain Mu50. The minimum bactericidal concentrations (MBCs)
were also delineated for the antibiotics and as expected,
fluoroquinolone 3c exhibited a bactericidal profile based on the
MIC and MBC data. The MBC:MIC ratios for the three bacteria
were less than or equal to 4:1, a frequently metric used to
designate bactericidal agents [17]. Interestingly, compound 3c
appeared to have enhanced bactericidal effects in comparison to
CIP against MSSA and MRSA.
Table 4: Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) data comparison of N-propylthio fluoroquinolone 3c
with standard drugs.
a
b
b
c
species | strain
S. aureus (MSSA)
compd
3c
CIP
VAN
3c
CIP
VAN
3c
CIP
VAN
MIC
MBC
0.20
0.78
0.10
1.56
3.13
0.10
50
ratio
0.10
0.10
0.10
1.56
1.56
0.10
50
50
0.78
2:1
4:1
1:1
1:1
2:1
1:1
1:1
1:1
1:1
2
9213
S. aureus (MRSA)
BAA-1747
S. aureus (VISA)
7
00699
50
0.78
Figure 2. MS spectrum reveals incubation of N-propylthio fluoroquinolone
3c with glutathione (GSH) results in formation of ciprofloxacin (CIP) and S-
(propylthio)glutathione (GS-S-Pr).
a
b
c
CIP: ciprofloxacin; VAN: vancomycin
values reported in M/mL
MBC:MIC
To further probe if the antimicrobial effects can be attributed to
antagonism of the thiol-redox buffer system, the cytosolic ROS
level of fluoroquinolone-treated cells was measured in a
fluorometric assay [21]. A 5-fold dilution of an overnight culture
of S. aureus was incubated for 0.5 h with either 25 µM of the
fluoroquinolones 3c or CIP. The cells were then washed with
PBS and labeled with 2’,7’-dicholorodihydrofluorescin-diacetate
The apparent increase in bactericidal activity against S. aureus
was partially attributed to greater cell membrane permeability of
N-propylthio fluoroquinolone 3c (clogP 2.22) compared to CIP
clogP 0.65). Moreover, it is thought that antagonistic effects due
(
to chemical modification of low molecular weight thiols in the
cytoplasm was partly influential on activity. A key cellular thiol
believed to cleave the electrophilic N-alkylthio group (-N-S-R) of
thiolated fluoroquinolones 3 is coenzyme A (CoASH). It is
hypothesized that the resulting S-(alkylthio)-coenzyme A product
i.e., CoAS-S-R) could function as an inhibitor of biosynthetic
pathways that require acyl coenzyme A (CoASAc) metabolites
18]. β-Ketoacyl-acyl carrier protein synthase III (FabH), a
bacterial enzyme involved in fatty acid biosynthesis, is a
potential pharmacological target of CoAS-S-R disulfides [19]. In
addition, many Gram-positive bacteria including Staphylococcus
and Bacillus spp. are deficient in endogenous glutathione (γ-
glutamyl-cysteinyl-glycine, GSH) and alternatively use CoASH
and/or bacillithiol (BSH) in their thiol-redox buffer system to
detoxify reactive oxygen species (ROS) [20]. Decreased
availability of these intracellular thiols to attenuate cytosolic
ROS levels could evoke an oxidative stress response (e.g., SOS)
and inhibit cell growth.
(DCFH-DA). Upon uptake, intracellular DCFH-DA is
deacetylated by esterases and oxidized by nascent ROS to the
fluorophore 2’7’-dichlorofluorescin, which can be detected by
fluorescence (λex 485 nm, λem 535 nm). After 20 h, a 2 to 3 fold
increase in cytosolic ROS was detected for both fluoroquinolones
compared to untreated cells (Figure 3). Despite observing
increased levels of oxidative stress, these results were not
conclusive as many antibiotics are known to incite ROS
generation [22]. Prior studies have shown that fluoroquinolones
are inducers of oxidative stress [23] and a SOS response in S.
aureus [24]. With these results, additional evidence was sought
to determine if induction of oxidative stress plays a role in the
mechanism of action.
(
[
0
6
210
*
0
6
1
10
To test whether thiolated fluoroquinolones could react with the
antioxidant and metabolic thiols in bacterial cells, a 1.2 µM
0 5
510
2
aqueous solution of GSH was incubated in dH O with 1 µM of
N-propylthio fluoroquinolone 3c in DMSO at 37 °C and the
0
control
3c
CIP
mixture was analyzed by electrospray ionization mass
+
spectrometry in positive ion mode (ESI MS). After 2 h, a
Figure 3. Comparison of effects of 3c and CIP on endogenous ROS
generation in Staphylococcus aureus shown as mean ± SEM (n=3) [25].
reaction between thiolated fluoroquinolone 3c and GSH was
observed with formation of S-(propylthio)glutathione (GS-S-Pr)
and CIP (Figure 2). With this result, it is believed that thiolated
fluoroquinolones have the ability to react with endogenous low
molecular weight thiols in bacteria. In GSH-deficient species
To investigate the mechanism of growth inhibition in S. aureus
further, thiolated fluoroquinolone 3c and CIP were tested in a
double-diffusion assay with the antioxidant vitamin E (α-
(e.g., S. aureus, B. anthracis) [20] reliant on CoASH for both

4
tocopherol). In the experiment, 0.5 M vitamin E acetate was
dispensed into a well bored in the center of a freshly streaked
agar plate. Cellulose discs were then placed 1 cm from the well
and impregnated with the antibiotics. Following overnight
incubation, the effect of the antioxidant on antimicrobial activity
was qualitatively assessed by examining the size and appearance
of the zone of inhibition. On inspection, a slight increase in
growth density was observed along the central interface of the
zones (Figure 4a). This result lent support for the potential role
of ROS generation in the inhibitory activity of fluoroquinolones.
Figure 5. Proposed mechanisms of action of thiolated fluoroquinolones.
Acknowledgements
This research was supported by the Marshall University School
of Pharmacy FRS Grant Program. The authors also thank
Marshall University Department of Chemistry for use of the
NMR facility and Dr. Mohammad F. Hossain for mass
spectroscopy analyses.
References and notes
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Figure 4. Double-diffusion assay with (a) vitamin E (α-tocopherol) acetate
and (b) glutathione (GSH) against Staphylococcus aureus.
The double-diffusion assay was also employed to probe
whether GSH could attenuate the antistaphylococcal activity of
thiolated fluoroquinolone 3c. No inhibitory effects on stationary
cell growth was evident for fluoroquinolone 3c (Figure 4b) or
CIP (not shown); however, marked antagonism was detected for
allicin (1). The inhibition of allicin by GSH was attributed to
cleavage of the thiosulfinate bond in allicin resulting in formation
of inactive S-(allylthio)glutathione [26], a reaction similarly
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2. Preparation of N-(alkylthio)- and N-(arylthio)phthalimide 2; general
procedure: Sulfuryl chloride (3.37 g, 25 mmol) in 5 mL was added
dropwise to an ice-chilled solution of thiol (25 mmol) or disulfide (12.5
1
1
1
1
In final analysis, this investigation established that attachment
of a N-alkylthio residue could improve the pharmacological
properties of CIP against MRSA and other Gram-positive
organisms. The enhanced bactericidal activity observed against
MRSA was attributed to increased cellular uptake and a reaction
of the thiolated fluoroquinolones with low molecular weight
thiols (e.g., CoASH) in the cytoplasm that yields the parent drug
CIP and an inactive metabolite (e.g., CoAS-S-R). Evidence was
found that the compounds could react with antioxidant thiols
such as GSH to form disulfides and potentiate ROS generation in
bacteria. Based on these findings, it is hypothesized that
increased intracellular drug concentration and the inhibition of
metabolic pathways requiring CoASAc confer the “additive”
antimicrobial effect of thiolated fluoroquinolones with inhibition
of DNA topioisomerase in MRSA (Figure 5). Current research
efforts are focused now on deciphering the pharmacology and
activity spectrum of other thiolated antibiotics derived from
FDA-approved antibacterials by the same design strategy.
mmol) and cat. Et
.5 h at 0 °C, the mixture was warmed to rt and added by cannula to an
ice-chilled suspension of phthalimide (3.68 g, 25 mmol) and Et N (4.6
mL, 33 mmol) in 25 mL of dry CH Cl . The reaction mixture was
stirred for 1.5 h while warming to rt. The solution was then filtered into
a separatory funnel, washed with water and brine, dried over MgSO
3 2 2
N (50 µL) in 25 mL of dry CH Cl . After stirring for
0
3
2
2
4
,
filtered, and concentrated under reduced pressure. The final product was
obtained as a white solid by trituration in 0-25% EtOAc in hexanes and
used without further purification.
2
7
-Propylsulfanyl-isoindole-1,3-dione (2c): Yield 63%; white solid, m.p.
1
4–76 °C; TLC (SiO
2 f
) R 0.61 (3:1 hexanes:EtOAc); H NMR (400
MHz, CDCl
3
) δ 7.88−7.86 (m, 2H), 7.75−7.73 (m, 2H) 2.80 (t, 2H, J =
1
3
7
.3 Hz), 1.57 (sxt, 2H, J = 7.3), 0.98 (t, 3H, J = 7.3 Hz); C NMR (101
MHz, CDCl ) δ 168.6, 134.7, 132.1, 123.9, 40.7, 21.8, 13.1; ESI–MS:
3
+
m/z 222.0 [M+H] .
1
3. Synthesis of N-thiolated fluoroquinolones 3; general procedure:
Ciprofloxacin (76 mg, 0.23 mmol), phthalimide 2 (0.23 mmol), and
NaHCO
(1:1) and stirred for 14-22 h at 80 °C. The mixture was then cooled to rt,
diluted with CH Cl , washed with 5% citric acid and water, dried over
MgSO , filtered, and concentrated in vacou. Trituration or flash silica
3
(39 mg, 0.46 mmol) were combined in 1 mL of MeCN:DMF
2
2
4
gel chromatography with 50-100% acetone in hexanes afforded the
product (3) as a powdery solid.
1
-Cyclopropyl-6-fluoro-4-oxo-7-(4-propylsulfanyl-piperazin-1-yl)-1,4-
dihydro-quinoline-3-carboxylic acid (3c): Yield 68%; white solid, m.p.
1
1
2 f
71–173 °C; TLC (SiO ) R 0.65 (100% EtOAc); H NMR (400 MHz,

5
DMSO-d
6
) δ 14.86 (bs, 1H), 8.39 (s, 1H), 7.59 (m, 1H), 7.29 (d, 1H, J =
.4 Hz), 3.56 (m, 1H), 3.10−3.07 (m, 4H), 2.90−2.88 (m, 4H), 2.50 (t,
H, J = 7.2 Hz), 1.32 (sxt, 2H, J = 7.2 Hz), 1.10−1.05 (m, 2H),
7
2
0
1
3
.95−0.93 (m, 2H), 0.73 (t, 3H, J = 7.2 Hz); C NMR (101 MHz,
) δ 169.2. 165.9, 147.9, 139.1, 134.3, 132.6, 122.9, 106.6 (d,
app JC-F = 17.9 Hz), 55.6, 50.2, 35.8, 33.4. 21.7, 13.4, 7.6; ESI–MS: m/z
DMSO-d
6
+
4
06.1 [M+H] .
1
1
4. Bhattacharya, B.; Turos, E. Tetrahedron 2012, 68, 10665.
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Lim, D. V. Bioorg Med Chem. 2006, 14, 3775.
1
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2
015009699 A1, 2015.
1
1
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Allicin-inspired Thiolated Fluoroquinolones as
Antibacterials Against ESKAPE Pathogens
Jordan G. Sheppard, Timothy E. Long
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