Synthesis and antimicrobial activities of structurally novel S,S′-bis(heterosubstituted) disulfides

Article abstract of DOI:10.1016/j.bmcl.2012.04.056

The central focus of this study is on the antibacterial and antifungal properties of synthetically produced S,S′-bis(heterosubstituted) disulfides as a means to control the growth of various infection-causing pathogens. Staphylococcus aureus, Francisella tularensis and Candida albicans were each found to be highly susceptible to several of these compounds by agar or broth dilution and Kirby-Bauer diffusion assays. These structurally simple, low molecular weight disulfides have shown promising bioactivities and may serve as leads to the development of effective new antibacterials for pathogenic bacteria such as methicillin-resistant S. aureus and F. tularensis.

Full text of DOI:10.1016/j.bmcl.2012.04.056

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3623–3631
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antimicrobial activities of structurally novel
S,S0-bis(heterosubstituted) disulfides
Praveen Ramaraju a, Danielle Gergeres a, Edward Turos a, , Sonja Dickey b, Daniel V. Lim b,
⇑
John Thomas c, Burt Anderson c
a Department of Chemistry, 4202 East Fowler Avenue, CHE 205, University of South Florida, Tampa, FL 33620, USA
b Department of Cell Biology, Microbiology, and Molecular Biology, 4202 East Fowler Avenue, ISA 2015, University of South Florida, Tampa, FL 33620, USA
c Department of Molecular Medicine, 12901 Bruce B. Downs Blvd., MDC 7, University of South Florida, Tampa, FL 33612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 February 2012
Revised 6 April 2012
Accepted 10 April 2012
Available online 19 April 2012
The central focus of this study is on the antibacterial and antifungal properties of synthetically produced
S,S0-bis(heterosubstituted) disulfides as a means to control the growth of various infection-causing patho-
gens. Staphylococcus aureus, Francisella tularensis and Candida albicans were each found to be highly sus-
ceptible to several of these compounds by agar or broth dilution and Kirby-Bauer diffusion assays. These
structurally simple, low molecular weight disulfides have shown promising bioactivities and may serve
as leads to the development of effective new antibacterials for pathogenic bacteria such as methicillin-
resistant S. aureus and F. tularensis.
Keywords:
Disulfides
MRSA
Ó 2012 Elsevier Ltd. All rights reserved.
Francisella tularensis
Candida albicans
Bacteriostatic
The concern over multidrug-resistant bacteria and the need for
more effective antibacterials has truly reached a critical level, given
the high incidence of life-threatening, life-altering drug-resistant
infections. Staphylococcus aureus (Staph), a Gram-positive faculta-
tive anaerobe, resides in the nostrils and on the skin of human
beings, and starts to cause infection once it enters the blood
stream. Antibiotic-resistant variants of S. aureus such as methicil-
lin-resistant S. aureus (MRSA), the single most important bacterial
pathogen causing deadly infections, are of the most immediate
concern. MRSA causes infections of skin and soft tissue,1 as well
as pulmonary,2 osteoarticular,3 hemorrhagic adrenal gland (Water-
house-Friderichsen syndrome4) and ophthalmic infections.5
According to the United States Centers for Disease Control and Pre-
vention (CDC), MRSA accounted for more than 94,000 life-threat-
ening infections and nearly 19,000 deaths in the United States
alone in 2005.6 The CDC also emphasizes that the prevalence of
multi-drug-resistant (MDR) bacteria including MRSAs is rising at
an alarming rate. MRSA mutants that thrive in the community, re-
ferred to as community-acquired MRSA (CA-MRSA), are more
lethal and complex in nature than those originating in hospitals,
which are referred to as hospital-acquired MRSA (HA-MRSA).
A second bacterial pathogen, Francisella tularensis live vaccine
strain (F. tularensis LVS), is a Gram-negative intracellular bacterium
that is the causative agent of tularemia known as rabbit fever.
Symptoms of tularemia include fever, lethargy, anorexia, signs of
septicemia, and in extreme cases can cause death. As few as 10–
50 cfu (colony-forming units) are sufficient to cause an infection.7
Due to its high pathogenicity and ease of spread by aerosol,
F. tularensis is considered for usage in biological warfare, and as
such, is classified as a Class A agent by the U.S. government.8
F. tularensis is susceptible to carbapenems, ceftriazone, ceftazi-
dime, rifampin and certain macrolides, but there remains a lack
of clinical data to recommend any of these compounds for clinical
use.9 Moreover, there is broad resistance to erythromycin among
strains of F. tularensis subspecies holarctica.10 Ciprofloxacin is the
only drug that is considered ideal for the treatment of tularemia,
although with concerns over its continued effectiveness.
Our laboratory has been investigating new types of synthetic
antibacterial agents for use against pathogenic microbes. Previ-
ously, we have reported on N-thiolated b-lactams,11–17 N-thiolated
2-oxazolidinones18 and most recently, aryl–alkyl disulfides19 that
are all effective in vitro growth inhibitors of MRSA and Bacillus bac-
teria (Fig. 1). The mode of action and structure–activity profiles of
these three chemical classes differ dramatically from those of the
traditional b-lactam, oxazolidinone, or disulfide-containing antibi-
otics. Investigations in our laboratory have illustrated that these
highly lipophilic compounds can carry a wide range of substitu-
ents, and are reactive towards thiophilic agents that under certain
conditions (such as in the bacterial cytoplasm) can block metabolic
⇑
Corresponding author. Tel.: +1 813 974 7312; fax: +1 813 974 1733.
E-mail addresses: eturos@usf.edu, eturos@shell.cas.usf.edu (E. Turos).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.056

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P. Ramaraju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3623–3631
Figure 1. Thiophilic antibacterials previously developed in our laboratories.
Table 3
MIC’s of S,S0-bis(alkylamino) disulfides 3a–e against S. aureus
Figure 2. Synthesis of S,S0-bis(heterosubstituted) disulfides 1–6.
Compound
R
MSSA
g/mL)
MRSA
(lg/mL)
(
l
3a
Propyl
8
4
3b
3c
3d
3e
Isopropyl
Butyl
sec-Butyl
Phenyl
32
8
16
4
32
32
16
4
Table 1
MIC’s of S,S0-bis(alkoxy) disulfides 1a–e against S. aureus
Penicillin G (control)
0.25
64
Compound
R
MSSA
g/mL)
MRSA
(lg/mL)
Table 4
MIC’s of S,S0-bis(dialkylamino) disulfides 4a–e against S. aureus
(
l
1a
1b
1c
1d
Propyl
Isopropyl
Butyl
sec-Butyl
Phenyl
32
1
0.25
16
2
32
0.5
8
8
2
1e
Penicillin G (control)
0.25
64
Compound
R
MSS
g/mL)
MRSA
(lg/mL)
Table 2
MIC’s of S,S0-bis(organothio) disulfides 2a–e against S. aureus
(
l
4a
4b
Methyl
Ethyl
0.25
2
0.5
2
4c
4d
4e
Isopropyl
Allyl
Isobutyl
2
1
16
0.25
1
0.5
16
64
Penicillin G (control)
Compound
R
MSSA
g/mL)
MRSA
(lg/mL)
(
l
2a
2b
2c
2d
Propyl
Isopropyl
Butyl
sec-Butyl
Phenyl
32
32
64
32
8
32
32
32
64
8
The antimicrobial activity of these S,S0-bis(heterosubstituted)
disulfide compounds was evaluated against bacterial targets of
interest to us, including methicillin-susceptible S. aureus (MSSA,
ATCC 25923), methicillin-resistant S. aureus (MRSA, ATCC 43300,
b-lactamase-producing), a live vaccine strain (LVS) of F. tularensis
subspecies holarctica, nine subspecies of Bartonella, and Escherichia
coli K12. The testing included the determination of the minimum
inhibitory concentration (MIC), Kirby-Bauer diffusion assay and
growth viability assay. Minimum fungicidal concentration (MFC)
and viability assays were also performed against the fungus,
Candida albicans. To assess antibacterial activity, we conducting
susceptibility screening by determining the MIC values for each
analogue by agar dilution using a 24-well plate, according to the
National Committee for Clinical Laboratory Standards (NCCLS).21
After preparing the agar, the bacteria were allowed to grow for
24 h in the presence of varying concentrations of antibiotic within
the agar. All the antimicrobial assays were performed in triplicate,
using either penicillin G or ciprofloxacin as positive controls and
DMSO as a negative control. Among the bacteria initially examined,
antimicrobial activity was not observed for any of the compounds
against Bartonella ssp. or E. coli K12. However, the disulfides
2e
Penicillin G (control)
0.25
64
pathways used by bacteria for membrane formation and possibly
other vital cellular processes.
This work describes experiments on a fourth series of sulfur-
containing antibacterials, S,S0-bis(heterosubstituted) disulfides. In
this study, we investigated the synthesis and antimicrobial proper-
ties of these intriguing low molecular weight compounds, which at
least structurally are related to naturally-occurring trisulfide and
polysulfide antibiotics.20 These investigations were initiated by
the synthesis of a small library of S,S0-bis(heterosubstituted) disul-
fides to assess the electronic effects of different heteroatoms on
microbiological activity. These derivatives were prepared by treat-
ing an alcohol, amine or thiol with sulfur monochloride at ꢀ20 °C
in the presence of triethylamine as a Lewis base ( Fig. 2). The
desired disulfide products were obtained in 70–90% yields after
column chromatography, and characterized by 1H NMR spectros-
copy, prior to antimicrobial testing.

P. Ramaraju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3623–3631
3625
Figure 3. Chiral S,S0-bis(heterosubstituted) disulfides 5a–g and 6a,b.
1 lg/mL. For this series, antibacterial activity improved with the
Table 5
MIC’s of chiral disulfides 5a–g and 6a,b against S. aureus
increase in the lipophilicity of the alkyl substituent, with exception
for the -butoxy disulfide 1d. There is a 32-fold increase in the MIC
for 1c against the MRSA compared to the MSSA, and we do not
have an explanation or prediction as to the significance of this
finding.
Compound
R
MSSA (
mL)
lg/
MRSA (
mL)
lg/
5a
5b
5c
5d
5e
5f
(S)-sec-butyl
(R)-sec-butyl
( )-1-Phenylpropyl
( )-2-Phenylpropyl
(1S, 2R, 5S)-(+)-menthyl
16
16
16
16
8
32
32
32
16
8
We next examined the anti-staphylococcal activity of the
corresponding tetrasulfide analogs 2. The activity among the S,S0-
bis(organothio) disulfides (Table 2) decreases with increase in
the alkyl chain branching. Surprisingly, these compounds were
not as active as our other disulfides, even with the presence of four
contiguous sulfur atoms, indicating that the number of sulfur–
sulfur linkages is not directly related to the antibacterial activity.
The phenoxy-substituted disulfide (2e) was found to provide the
greatest potency among the five analogues examined.
8
16
L-(ꢀ)-Menthyl
5g
6a
( )-Menthyl
(R)-Benzyl-(1-
phenylethyl)
(S)-Benzyl-(1-
phenylethyl)
8
16
16
64
6b
16
64
64
Penicillin G
(control)
0.25
For the S,S0-bis(alkylamino) disulfides 3a–e, the most active
compound of the five was the S,S0-bis(phenylamino) disulfide 3e,
with an MIC of 4 lg/mL against both MSSA and MRSA (Table 3).
This series provided the weakest potency of all the different disul-
fides we tested, indicating the polarity due to the N–H may in part
lower in vitro efficacy.
Table 6
Bacterial viability assay
S. aureus strain
Concentration of disulfide 1b
g/mL)
Bacterial counts
(cfu/mL)
In the case of the S,S0-bis(alkylamino) disulfides 3 and 4, those
derived from primary amines have weaker activity against
S. aureus than those of secondary amines (Table 4). For instance,
compound 4a, synthesized from the dimethylamine, showed the
most potent activity among the secondary amine disulfides, and
is one of the two most active (with 1b) against MRSA of all the
disulfides we examined. Increase in the chain length and branching
resulted in the loss of activity in this series.
(
l
Before
After
MSSA
0 (Control)
2.11 ꢁ 105
7.60 ꢁ 108
3.86 ꢁ 104
2.04 ꢁ 103
4.02 ꢁ 108
>1.2 ꢁ 106
2.88 ꢁ 103
(ATCC 25923)
2
1.00 ꢁ 105
2.22 ꢁ 105
3.09 ꢁ 105
1.00 ꢁ 105
2.96 ꢁ 105
4
MRSA
0 (Control)
(ATCC 43300)
2
4
Apart from varying the alkyl chains on the heteroatoms, we also
wanted to observe the effects of chirality on the bioactivity. To
accomplish this, various chiral alcohols and amines were used as
substrates for the reaction with sulfur monochloride ( Fig. 3).
Starting materials for compounds 5c, 5d and 5g were racemic,
diastereomeric mixtures, while the rest were prepared enantiome-
rically and diastereomerically pure. It was observed that the
activities of the chiral disulfide analogs 5a–g and 6a, b (Table 5)
were not much different from their racemic counterparts.
exhibited strong inhibitory effects against both S. aureus and F.
tularensis. A breakdown of this data is summarized below.
The MIC values (average from three assays) of the S,S0-bis(alk-
oxy) disulfides 1a–e against MSSA and MRSA are shown in Table
1. The most active of the five analogs tested was identified as the
isopropoxy derivative (1b), whose in vitro MICs are at or below

3626
P. Ramaraju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3623–3631
observed in vitro MIC values (0.5 and 1
these test microbes. The number of MSSA and MRSA cells both de-
creased by ten-fold in the tubes with 2 g/mL disulfide, and 100-
fold with 4 g/mL.
While there was a significant decrease in the number of cells,
not all were killed, indicating a bacteriostatic effect at these
concentrations.
lg/mL, respectively), for
S
O
S
S
l
S
O
l
O2N
S-isopropyl-S'-(4-nitrophenyl) disulfide
S,S'-bis (isopropoxy) disulfide (1b)
In addition to evaluating the biological activity by Kirby-Bauer
diffusion assays, the effect of various additives on the bioactivity
of the S,S0-bis(alkoxy) disulfides was also investigated. For this,
we chose our most active anti-MRSA agent, S,S0-bis(isopropoxy)
disulfide (1b), as a representative. For comparison, an N-thiolated
b-lactam, an alkyl-aryl disulfide, and penicillin G potassium salt
(structures shown in Figure 4) were also tested simultaneously
against MRSA in Mueller-Hinton agar. The experiment involved
placing one of each of the four test compounds in wells cut equidis-
tant from the center of the well, and placing into a center well an
additive that could potentially alter the size or shape of the growth
inhibition zones of the four test compounds by diffusing outward
O
SCH3
H
N
S
N
O
O
N
O
O
-
+
Cl
N-methylthio β-lactam
O K
O
penicillin G potassium salt
Figure 4. Antibacterial compounds examined by Kirby-Bauer well diffusion assay.
from the center of the plate. For testing, we used 10
pyl-S0-(4-nitrophenyl) disulfide,19 20
g of the N-methylthio b-lac-
tam,22 50 g of S,S0-bis(isopropoxy) disulfide (1b) and 10
g of
lg of S-isopro-
l
l
l
penicillin G potassium salt, and 1 mg of the test additive in the
center well (dissolved in DMSO) These particular quantities of
the four test compounds were chosen after optimization to obtain
comparable zones of growth inhibition of each agent, for easier
comparison.
The first assay we did was a simple control using DMSO, to en-
sure that the vehicle did not affect bioactivity of the compounds as
it diffuses outward from the center well (Fig. 5).
In assays in which we placed in the center diffusion well an
additive containing a free thiol group, such as cysteine, coenzyme
A (CoA), and glutathione (GSH) (shown in Figure 6), the in vitro
antibacterial activity of the three thiophilic antibacterial com-
pounds was indeed attenuated (Fig. 7). This was visibly evident
from the indented regions of growth inhibition after 24 h of incu-
bation, which was absent in the case of penicillin G (which is not
sensitive to thiophiles). For each of these, we also noted that there
were no growth inhibition zones appearing around any of the cen-
ter wells containing the additive.
Figure 5. Kirby-Bauer assay showing the lack of a growth inhibition effect by DMSO
(control) on the four test compounds against MRSA.
Other additives that were tested as potential inactivators of
disulfide 1b in these agar diffusion assay experiments included
non-sulfhydryl additives glycine, lysine, valine, S,S0-diisopropyl
disulfide (iPrSSiPr), and triphenylphosphine (PPh3). As expected,
none of these additives had any effect on the bioactivity of the four
test compounds (Figs. 8 and 9). In the case of PPh3, a small inhib-
itory zone was observed around its diffusion well indicative of
weak antimicrobial activity. It is important to note, however, that
none of the growth inhibition zones of the four comparative test
compounds were affected by PPh3.
A bacterial viability assay was carried out to further assess the
antibacterial activity of S,S0-bis(isopropoxy) disulfide (1b), the
most active anti-MRSA compound in our collection. This assay
was conducted by counting the viable bacterial cells before and
after incubation, with an initial count in all tubes of 105 cfu/ml
(Table 6). S. aureus (ATCC 25923) and MRSA (ATCC 43300) used
as test microbes showed a 1000-fold increase in the number of
bacteria in the absence of the disulfide. Disulfide 1b was tested
in concentrations of 2 and 4 lg/mL, slightly above the visibly
Figure 6. Structures of cysteine, coenzyme A and glutathione.

P. Ramaraju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3623–3631
3627
Figure 7. Kirby-Bauer assay showing the effect of CoA, GSH and cysteine on antimicrobial activity of the four test compounds against MRSA.
Figure 8. Kirby-Bauer assay showing the lack of an inhibitory effect of isopropyl disulfide (left) or triphenylphosphine (right) on antimicrobial activity of the four test
compounds against MRSA.
In order to explore the possible mode of action of these structur-
ally interesting disulfide compounds, an experiment was conducted
to check if they induce alterations in the formation of the bacterial
cell wall. Scanning electron microscopy (SEM) was used to observe
MRSA cells both before and after treatment with disulfide 1b during
Kirby-Bauer agar diffusion experiments. Examination of the regions
of the agar bordering the edges of the growth inhibition zones re-
vealed colonies of surviving cells. However, in examining these

3628
P. Ramaraju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3623–3631
Figure 9. Kirby-Bauer assay showing the lack of an inhibitory effect by non-sulfhydryl-containing amino acids valine, lysine and glycine on bioactivity of the four test
compounds against MRSA.
Figure 10. Scanning electron microscopy images of MRSA cells before and after treatment with disulfide 1b. These images (and others not shown here) indicate no
discernible differences in bacterial cell morphology.
cells, we found no obvious changes in their external morphology
(Fig. 10). The cell wall looked intact, indicating that the compound
did not disrupt or interact with bacterial transpeptidases or any cell
wall-related proteins. If the compounds were to interact with these
components, the spherical structure of the cells would have been
disrupted as in the case of exposure to penicillin G.
To further assess antimicrobial activity, all of the S,S0-heterosub-
stituted disulfides were also evaluated against F. tularensis and C.
albicans. The minimum inhibitory concentrations (MIC) of
S,S0-bis(heterosubstituted) disulfides 1–6 were first evaluated by
broth dilution using a live vaccine strain (LVS) of F. tularensis
(Table 7).23

P. Ramaraju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3623–3631
3629
Table 7
MIC’s of S,S0-bis(heterosubstituted) disulfides 1–6 against F. tularensis
Compound
R
Francisella tularensis MIC (lg/mL)
S,S0-Bis(alkoxy) disulfides
1a
1b
1c
Propyl
Isopropyl
Butyl
16
4
4
1d
1e
sec-Butyl
Phenyl
16
1
S,S0-Bis(alkylthio) disulfides
2a
2b
2c
Propyl
Isopropyl
Butyl
1
8
4
2d
2e
sec-Butyl
Phenyl
16
2
S,S0-Bis(alkylamino) disulfides
3a
3b
3c
Propyl
Isopropyl
Butyl
16
2
4
3d
3e
sec-Butyl
Phenyl
8
0.5
S,S0-Bis(dialkylamino) disulfides
4a
4b
4c
4d
Methyl
Ethyl
Isopropyl
Allyl
8
16
32
16
1
4e
Isobutyl
Chiral disulfides
5a
5b
5c
5d
(S)-sec-butyl
(R)-sec-butyl
( )-1-Phenylpropyl
( )-2-Phenylpropyl
16
16
16
8
5e
5f
(
(
D
)-Menthyl
4
4
L)-Menthyl
5g
( )-Menthyl
4
6a
6b
(R)-Benzyl-(1-phenylethyl)
(S)-Benzyl-(1-phenylethyl)
16
16
0.125
Ciprofloxacin (control)
using 1, 0.5, 0.25 and 0.125 lg/mL concentrations of 1b, according
to the prescribed NCCLS protocol for fungi.21 Trypan blue was used
for staining of non-viable cells. Two controls were used as well, a
viable control using only C. albicans in YNB and a dead control
using C. albicans in phosphate buffered saline that was heat-killed
(boiled for 5 min). The MIC of disulfide 1b towards C. albicans was
found to be 0.5 lg/mL. The cells in both the non-treated yeast
nutrient broth (YNB) control, as well as those treated with disulfide
1b, did not incorporate the trypan blue, while the cells in the heat-
killed control sample readily absorbed the dye. This indicates that
the antifungal activity of disulfide 1b towards C. albicans is due to
cytostatic control, and the disulfide is not cytocidal at these con-
centrations. Consequently, the microbiological effects of these
compounds closely mirror those observed previously for N-thiolat-
ed b-lactams, N-thiolated oxazolidinones, and alkyl aryl disulfides.
As a means to ascertain whether the disulfides inflict deleteri-
ous effects on mammalian cells, disulfide 1b was tested for
in vitro cytotoxicity against primary bovine primary aortic endo-
thelial cells and EBM-2 (endothelial basal medium) cells in 96-well
culture dishes. After 48 h, (3-4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) was added to each treated well,
and the cells were further incubated. The formazan product gener-
ated in each well was dissolved in DMSO, and plates were read in a
plate reader at 540 nm to determine cell viability. From these as-
says, disulfide (1b) did not show any cytotoxic activity against
either of the endothelial cells up to a disulfide concentration of
Figure 11. Compounds 1b and 3e, our most active analogs.
These data reveal no obvious structure–activity relationship trends
among these 29 analogues, but some of the compounds do display
good anti-Francisella activities, with MIC’s as low as 0.5 lg/mL. Of
these, disulfides 1b and 3e were the most active compounds in this
selection.
Given that, previously, N-thiolated b-lactams were found to
have antifungal properties,24 we decided to examine the disulfides
against C. albicans, one of the most common human commensal
organisms that lives in the mouth and gastrointestinal tract in
about 80% of healthy individuals.25 Entry of these fungal cells in
the blood stream causes candidiasis, infections which vary from
superficial to systemic, and are often life threatening. Invasive can-
didiasis is very prevalent in intensive care units and is a major
cause of mortality in 33–47% of immuno-compromised individuals
in hospitals.26 Reduced susceptibility of Candida species towards
the commonly used antifungal agents has raised concerns over a
need for effective antifungal agents.
20 lg/mL, a finding again consistent with the cytostatic nature of
these disulfides.
In summary, S,S0-bis(heterosubstituted) disulfides were synthe-
sized in good yields from sulfur monochloride, and most showed
promising growth inhibition activities against S. aureus and sistant
Fungal susceptibility testing of S,S0-bis(isopropoxy) disulfide
(1b) against C. albicans was done in Yeast Nitrogen Broth (YNB)

3630
P. Ramaraju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3623–3631
S. aureus.28 S,S0-Bis(isopropoxy) disulfide (1b) and S,S0-bis(dimeth-
ylamino) disulfide (4a) were found to be the most potent
compounds of those investigated. The data from the Kirby-Bauer
assay concluded that the activity diminished in presence of addi-
tives that contain a free sulfhydryl group. It is likely that this might
be due to the cleavage of the S-heteroatom or disulfide bonds, and
the formation of a biologically inactive mixed disulfide. As of yet,
we have not been able to confirm this in solution, or even to obtain
isolable mixed disulfide adducts by reaction with the various addi-
tives in the absence of the bacterial cells. From the bacterial viabil-
ity assay these disulfides were found to be bacteriostatic in nature.
This is also the case for all our previously reported thiophilic anti-
bacterials, N-thiolated b-lactams, N-thiolated 2-oxazolidinones,
and alkyl-aryl disulfides. It is our contention, at this point, that
all four exert similar if not identical microbiological effects on
the bacteria they each target, altering membrane formation and
proliferation. Further examination of these cellular effects and
pathways for antibiotic-resistance through spontaneous mutation
is now the focus of our efforts. Some of these compounds have also
shown decent activities against F. tularensis, the lowest MIC was
observed for S,S0-bis(phenylamino) disulfide (3e) (Fig. 11).
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8. Dennis, D. T.; Inglesby, T. V.; Henderson, D. A.; Bartlett, J. G.; Ascher, M. S.;
Eitzen, E.; Fine, A. D.; Friedlander, A. M.; Hauer, J.; Layton, M.; Lillibridge, S. R.;
McDade, J. E.; Osterholm, M. T.; O’Toole, T.; Parker, G.; Perl, T. M.; Russell, P. K.;
Tonat, K. J. Am. Med. Assoc. 2001, 285, 2763.
9. Harris, S. Ann. N. Y. Acad. Sci. 1992, 666, 21.
10. Tarnvik, A.; Berglund, L.; Eur. Res. J. 2003, 21, 361.
11. Turos, E.; Long, T. E.; Konaklieva, M. I.; Coates, C.; Shim, J. Y.; Dickey, S.; Lim, D.
V.; Cannons, A. Bioorg. Med. Chem. Lett. 2002, 12, 2229.
12. Long, T. E.; Turos, E.; Konaklieva, M. I.; Blum, A. L.; Amry, A.; Baker, E. A.;
Suwandi, L. S.; McCain, M. D.; Rahman, M. F.; Dickey, S.; Lim, D. V. Bioorg. Med.
Chem. 2003, 11, 1859.
13. Revell, K. D.; Heldreth, B.; Long, T. E.; Jang, S.; Turos, E. Bioorg. Med. Chem. 2007,
15, 2453.
14. Coates, C.; Long, T. E.; Turos, E.; Dickey, S.; Lim, D. V. Bioorg. Med. Chem. 2003,
11, 193.
15. Turos, E.; Coates, C.; Shim, J. Y.; Wang, Y.; Leslie, J. M.; Long, T. E.; Reddy, G. S.;
Ortiz, A.; Culbreath, M.; Dickey, S.; Lim, D. V.; Alonso, E.; Gonzalez, J. Bioorg.
Med. Chem. 2005, 13, 6289.
The SEM images shown in Figure 10 of MRSA treated with S,S0-
bis(isopropoxy) disulfide (1b) showed similarity to those of N-thi-
olated b-lactams. Neither affects the integrity of the bacterial cell
wall, nor alters cell morphology. However, their bioactivity can
be attenuated by the presence of cellular thiols such as cysteine,
glutathione and coenzyme A, a reflection of their sensitivity toward
sulfur-sulfur bond cleavage by thiophilic compounds. It is curious
that these neutralization effects are seen in the presence of bacte-
ria in the form of reduced in vitro bioactivity, but not in the form of
reactions when the thiols and disulfide are mixed in solution in the
absence of bacteria. In the latter instance, no chemical reaction is
observed, even at different pH values (6.5–9 pH). This suggests that
perhaps there may be a need for a cellular component or enzymatic
catalysis that enables the thiophile to react with the disulfide.
It can also be observed that the structural similarity in having a
cleavable S-heteroatom bond in all the three mentioned antibacte-
rials (a sulfur-nitrogen bond in N-thiolated b-lactams, a sulfur-
sulfur bond in aryl–alkyl disulfides and three cleavable
S-heteroatom bonds in S,S0-heterosubstituted disulfides) gives an
understanding of the possible active component and consequently
the similarity in the target moiety. It was established that N-thio-
lated b-lactams target coenzyme A and, subsequently the FabH en-
zyme regulating the bacterial type II fatty acid biosynthetic
pathway.9 It can now be hypothesized there might be a parallel
in the mode of action between the N-thiolated b-lactams and the
S,S0-bis(heterosubstituted) disulfides. Due to the advent of bacte-
rial resistance mechanisms there is always a need for effective
antimicrobial agents with novel modes of action. The present work
shows S,S0-heterosubstituted disulfides as antimicrobial agents
against various bacteria including S. aureus, MRSA, F. tularensis
and the fungus C. albicans. These structurally-simple disulfides
may serve as new leads to the development of effective antibacte-
rials for drug-resistant microbial infections.
16. Heldreth, B.; Long, T. E.; Jang, S.; Reddy, G. S.; Turos, E.; Dickey, S.; Lim, D. V.
Bioorg. Med. Chem. 2006, 14, 3775.
17. Turos, E.; Long, T. E.; Heldreth, B.; Leslie, J. M.; Reddy, G. S.; Wang, Y.; Coates, C.;
Konaklieva, M.; Dickey, S.; Lim, D. V.; Alonso, E.; Gonzalez, J. Bioorg. Med. Chem.
Lett. 2006, 16, 2084.
18. Mishra, R. K.; Revell, K. D.; Coates, C. M.; Turos, E.; Dickey, S.; Lim, D. V. Bioorg.
Med. Chem. Lett. 2006, 16, 2081.
19. Turos, E.; Revell, K. D.; Ramaraju, P.; Gergeres, D.; Greenhalgh, K.; Young, A.;
Dickey, S.; Lim, D. V.; Sathyanarayan, N.; Alhamadsheh, M. M.; Reynolds, K.
Bioorg. Med. Chem. 2008, 16, 6501.
20. Heldreth, B.; Turos, E. Curr. Med. Chem. Anti-Infect. Agents 2005, 4, 295.
21. NCCLS (National Committee for Clinical Laboratory Standards) methods for
dilution of antimicrobial susceptibility tests for bacteria that grow aerobically.
NCCLS document M7-A5, 1997, Vol. 17, No. 2.
22. Turos, E.; Shim, J.-Y.; Wang, Y.; Greenhalgh, K.; Reddy, G. S. K.; Dickey, S.; Lim,
D. V. Bioorg. Med. Chem. Lett. 2007, 17, 53.
23. Ikaheimo, I.; Syrjala, H.; Karhukorpi, J.; Schildt, R.; Koskela, M. J. Antimicrob.
Chemother. 2000, 46, 287.
24. O’Driscoll, M.; Greenhalgh, K.; Young, A.; Turos, E.; Dickey, S.; Lim, D. V. Bioorg.
Med. Chem. 2008, 16, 7832.
25. Gudlaugsson, O.; Gillespie, S.; Lee, K.; Vande Berg, J.; Hu, J.; Messer, S.;
Herwaldt, L.; Pfaller, M.; Diekema, D. Clin. Infect. Dis. 2003, 37, 1172.
26. Grover, N. Indian J. Pharmacol. 2010, 42, 9.
27. Fieser, L. F.; Fieser, M. In Reagents for Organic Synthesis; John Wiley and Sons:
New York, 1967; Vol. 1,
28. Experimental procedures and data on the disulfides 1–6: All the chemicals used
for the synthesis of the disulfide compounds were purchased from Aldrich
Chemical Company and used without further purification. Sulfur monochloride
was purified by freshly distilling from sulfur and charcoal (100:4:1 S2Cl2/sulfur/
charcoal).27 Thin layer chromatography was performed using Silica Gel 60 F254
purchased from EMD Chemicals. A UVG-11 Minera light lamp was used to
visualize the TLC plates. The NMR spectra were recorded in deuterated
chloroform on
a 250 MHz instrument. For further purification of the
compounds, high performance liquid chromatography (HPLC) was done on a
Shimadzu Prominence system using a reverse-phase Shimadzu column (C18,
0.46 ꢁ 5 cm). Samples were eluted using a gradient from 100% of a 10 mM PBS
solution (pH 7.4) to 100% of acetonitrile in 22 min at a flow rate of 1 mL/min. The
detection was performed using a Shimadzu SPD-20A UV-vis detector at 254 nm.
General procedure for the synthesis of S,S0-bis(hetero-substituted) disulfides 1–6: To
a stirred solution of equimolar concentrations (1 mmol) of the selected alcohol,
amine, or thiol and triethylamine in dry dichloromethane at ꢀ20 °C was added
dropwise a solution of sulfur monochloride (0.5 mmol) in dichloromethane. The
reaction was brought to room temperature after the addition was completed and
stirring was continued for about 1 h. The reaction was worked up by the addition
of about 100 mL of ice-cold water. This was stirred for a few minutes, transferred
to a separatory funnel, and the aqueous phase was removed by draining the
organic layer. The organic layer was further washed twice with 50 mL of ice-cold
water to remove the triethylamine hydrochloride, and washed with 50 mL of
brine to dry the organic layer. The dichloromethane layer was further dried by
the addition of anhydrous magnesium sulfate, filtered and concentrated in
vacuo. The product was purified by silica gel chromatography and characterized
Acknowledgment
We thankfully acknowledge funding from the DOD/DARPA
through grant HR0011-08-1-0087 (to B.A. and E.T.).
by 1H NMR
chromatography.
% yields shown are those of isolated pure product after
References and notes
1a: 85% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 3.41 (t, 2H, J = 6.8 Hz),
1.82–1.96 (m, 2H), 1.04 (t, 3H, J = 7.3 Hz).
1. Moran, G. J.; Krishnadasan, A.; Gorwitz, R. J.; Fosheim, G. E.; McDougal, L. K.;
Carey, R. B.; Talan, D. A.; Grp, E. I. N. S. N. Eng. J. Med. 2006, 355, 666.
2. Gillet, Y.; Issartel, B.; Vanhems, P.; Fournet, J. C.; Lina, G.; Bes, M.; Vandenesch,
F.; Piemont, Y.; Brousse, N.; Floret, D.; Etienne, J. Lancet 2002, 359, 753.
1b: 87% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 3.95–4.15 (m, 1H), 1.24–
1.32 (m, 6H).

P. Ramaraju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3623–3631
3631
1c: 83% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 3.43 (t, 2H, J = 6.8 Hz), 1.8–
1.91 (m, 2H), 1.4–1.55 (m, 2H), 0.94 (t, 3H, J = 7.4 Hz).
the appropriate optical density by either diluting with more diluents or adding
more bacteria. Once the appropriate optical density was obtained, a 1:1000
dilution of the culture was prepared in a sterile media storage bottle.
Using a 1 mg/mL stock solution of the test compound in DMSO, the following
volumes were added in each the 12 wells: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25,
1d: 81% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 3.67–3.93 (m, 1H), 1.38–
1.68 (m, 2H), 1.18 (d, 3H, J = 7.5 Hz), 0.86 (t, 3H, J = 6.3 Hz).
1e: 76% as a dark green oil 1H NMR (250 MHz, CDCl3): d 7.27–7.40 (m, 5H).
2a: 86% as a yellow oil; 1H NMR (250 MHz, CDCl3): d 2.94 (t, 2H, J = 7.1 Hz), 1.73–
1.76 (m, 2H), 1.03 (t, 3H, J = 7.3 Hz).
0.125
then added into each of the 12 wells containing the above volume of test drug:
744, 872, 936, 968, 984, 992, 996, 998, 999, 999.50, 999.75, 999.88 L,
lL, respectively. The following volume of molten Mueller-Hinton agar was
2b: 85% as a yellow oil; 1H NMR (250 MHz, CDCl3): d 3.19–3.37 (m, 1H), 1.40 (d,
6H, J = 6.8 Hz).
l
respectively. Each well was mixed using a pipette and allowed the drug-
containing agar to solidify at room temperature before inoculation. This gave the
following order of drug concentrations in the wells: 256, 128, 64, 32, 16, 8, 4, 2, 1,
2c: 90% as a yellow oil; 1H NMR (250 MHz, CDCl3): d 2.68–3.01 (m, 2H), 1.71–1.80
(m, 2H), 1.41–1.51 (m, 2H), 0.96 (t, 3H, J = 7.7 Hz).
2d: 83% as a yellow oil; 1H NMR (250 MHz, CDCl3): d 2.95–3.12 (m, 1H), 1.58–
1.83 (m, 2H), 1.39 (d, 3H, J = 6.2 Hz), 1.01 (t, 3H, J = 7.4 Hz).
2e: 85% as pale yellow crystals; mp 31–33 °C; 1H NMR (250 MHz, CDCl3): d 7.51–
7.55 (m, 2H), 7.25–7.37 (m, 3H).
0.5, 0.25 and 0.125
Optimally, within 15 min after adjusting the turbidity of the inoculum
suspension and preparing 1000-fold dilution, of the appropriate
lg/mL.
a
1 lL
inoculum was added to each well of the 24-well plate, containing solidified
agar/test compound. The plates were then placed into the incubator at 35–37 °C.
After 16–18 h of incubation, each plate was examined for bacterial growth. The
disulfide concentration showing no visible bacterial growth as detected with the
unaided eye is considered the MIC value.
Kirby-Bauer diffusion assay: From a freezer stock in tryptic soy broth (Difco
Laboratories, Detroit, MI) and 20% glycerol, a culture of each microorganism was
transferred with a sterile Dacron swab to TrypticaseÒ Soy Agar (TSA) plates
(Becton Dickinson Laboratories, Cockeysville, MD), streaked for isolation, and
incubated at 37 °C for 24 h. A 108 standardized cell count suspension was then
made in sterile phosphate buffered saline (pH 7.2) and swabbed across fresh TSA
plates. Sterile saline (5 mL) was inoculated with a swab of bacteria with a sterile
cotton plug. The concentration was then adjusted to 0.5 McFarland standard.
This bacterial solution was then streaked across a TSA plate to give an even lawn
of bacteria. Sterile pipet tips were used to drill 6 mm wells into the agar plate,
3a: 65% as a reddish oil; 1H NMR (250 MHz, CDCl3): d 3.14 (t, 2H, J = 7.9 Hz), 1.55–
1.65 (m, 2H), 1.49 (bs, 1H), 0.87 (t, 3H, J = 7.3 Hz).
3b: 65% as a reddish oil; 1H NMR (250 MHz, CDCl3): d 3.79–3.95 (m, 1H), 1.29 (d,
6H, J = 6.5 Hz).
3c: 60% as a reddish oil; 1H NMR (250 MHz, CDCl3): d 3.63–3.71 (m, 2H), 1.80–
1.89 (m, 2H), 1.34–1.48 (m, 2H), 0.96 (t, 3H, J = 7.3 Hz).
3d: 63% as a reddish oil; 1H NMR (250 MHz, CDCl3): d 2.95–3.12 (m, 1H), 1.58–
1.83 (m, 2H), 1.36–1.41 (m, 3H), 0.97–1.05 (m, 3H).
3e: Isolated 65% as a reddish oil; 1H NMR (250 MHz, CDCl3): d 7.14–7.27 (m, 2H),
6.68–6.81 (m, 3H), 3.65 (bs, 1H).
4a: 73% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 2.52 (s, 3H).
4b: 75% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 3.44 (q, 2H, J = 7.4 Hz),
1.68 (t, 3H, J = 7.3 Hz).
4c: 75% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 4.05–4.15 (m, 1H), 1.30 (t,
6H, J = 7 Hz).
then 20 lL of a 1 mg/mL stock solution of the disulfide in DMSO was added into
the well. Plates were incubated overnight at 37 °C.
Anti-Francisella testing: All 29 disulfides were tested against a live vaccine strain
(LVS) of Francisella tularensis subspecies holarctica by the broth dilution
4d: 70% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 5.73–5.90 (m, 4H), 5.07–
5.16 (m, 8H), 3.36 (d, 8H, J = 6.3 Hz).
4e: Isolated 71% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 2.64–2.70 (bs,
1H), 2.44 (d, 1H, J = 7.1 Hz), 1.93–2.13 (m, 1H), 0.91 (d, 6H, J = 6.6 Hz).
technique as described below and in Ref. 23. This was carried out in
biosafety level 3 facility at USF College of Medicine. In a 50 mL conical tube,
320 g of the test disulfide was added to 10 mL of Mueller-Hinton (MH) broth at
room temperature to give a concentration of 32 g/mL. Five millilitre of this
broth was added and mixed into another conical tube containing 5 mL of MH
broth to afford a final disulfide concentration of 16 g/mL. These steps were
repeated to obtain various concentrations. A 5 mL aliquot was removed from the
final tube having a disulfide concentration of 0.25 g/mL. Two full cotton swabs
worth of LVS growth culture was suspended into 10 mL of MH broth at room
temperature. From this stock, 25 L was added into each of the dilution tubes
prepared above. These tubes were incubated at 36 °C in an incubator with 0%
CO2. Three control tubes were also prepared, containing the following: (1) 25
LVS in 5 mL of MH broth in a 50 mL conical tube, (2) 25 L LVS in 5 mL of MH
a
5a: 78%)as a colorless oil; ½a D25
ꢂ
ꢀ10.1° (c 0.29, CH2Cl2); 1H NMR (250 MHz,
CDCl3): d 3.85–3.91 (m, 1H), 1.53–1.69 (m, 2H), 1.26–1.31 (m, 3H), 0.91–0.97 (m,
l
3H).
l
5b: 76% as a colorless oil; ½a D25 +9.2° (c 0.25, CH2Cl2); 1H NMR (250 MHz, CDCl3): d
ꢂ
3.86–3.91 (m, 1H), 1.55–1.67 (m, 2H), 1.26–1.31 (m, 3H), 0.91–0.97 (m, 3H).
5c: 70% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 7.28–7.39 (m, 5H), 4.59–
4.65 (m, 1H), 1.77–1.87 (m, 2H), 0.95 (t, 3H, J = 7.4 Hz).
l
l
5d: 73% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 7.03–7.18 (m, 5H), 3.52 (d,
2H, J = 6.8 Hz), 2.69–2.83 (m, 1H), 1.08 (d, 3H, J = 6.7 Hz).
l
Synthesis of S,S0-bis(heterosubstituted) disulfides made from menthol: To an ice-
cooled suspension of sodium hydride (oil removed by washing with dry
lL
hexanes) (1.5 mmol) in anhydrous tetrahydrofuran (THF),
a
solution of
l
menthol (1 mmol) in THF was added dropwise and was stirred until the
evolution of hydrogen stopped. This was followed by the addition of a solution of
sulfur monochloride (0.5 mmol) in THF dropwise under argon atmosphere. The
reaction was stirred until all the starting material was consumed, monitoring by
TLC. The reaction mixture was then concentrated in vacuo and purified by silica
gel chromatography.
broth in a 50 mL conical tube with dilutions of DMSO added that match those of
the above drug dilutions, and (3) 5 mL of MH broth in a 50 mL conical tube. After
48 h of incubation, all of the tubes were examined visually to define the
concentration of disulfide that inhibits LVS growth.
Fungal cell viability assay: S,S0-Bis(isoproxy) disulfide (1b) was tested for fungal
viability by a Trypan Blue staining protocol. 50
and 50 L of a 0.4% trypan blue solution in water (w/v) were mixed in a 2 mL
plastic microcentrifuge tube. After 5 min, 20 L of the mix was loaded into a
lL of a Candida albicans culture
5e: 60% as a colorless oil; ½a D25
ꢂ
+32.1° (c 0.35, CH2Cl2); 1H NMR (250 MHz, CDCl3):
l
d 3.38–3.46 (m, 1H), 2.15–2.21 (m, 1H), 1.95–2 (m, 1H), 1.59–1.70 (m, 2H), 1.42–
1.45 (m, 1H), 1.25–1.27 (m, 1H), 0.98–1.17 (m, 3H), 0.91–0.95 (m, 6H), 0.81–0.84
(m, 3H).
l
disposable cell-counting chamber (Cellometer (Registered), Nexcelom
Bioscience, Lawrence, MA) and viewed microscopically (Olympus BX60,
Olympus, Center Valley, PA) at 40X magnification.
Cytotoxicity testing: Primary bovine primary aortic endothelial cells and EBM-2
(endothelial basal medium) cell culture medium were purchased from Lonza
Walkersville, Inc. Cells were maintained in culture medium supplemented with
5f: 62% as a colorless oil; ½a D25
ꢂ
ꢀ28.7° (c 0.15, CH2Cl2); 1H NMR (250 MHz, CDCl3):
d 3.37–3.47 (m, 1H), 2.15–2.21 (m, 1H), 1.93–2 (m, 1H), 1.59–1.70 (m, 2H), 1.26–
1.39 (m, 3H), 1.01–1.13 (m, 2H), 0.94–0.95 (m, 3H), 0.91–0.93 (m, 3H), 0.81–0.84
(m, 3H).
5g: 65% as a colorless oil; 1H NMR (250 MHz, CDCl3): d 3.37–3.47 (m, 1H), 2.15–
2.21 (m, 1H), 1.93–2 (m, 1H), 1.59–1.70 (m, 2H), 1.26–1.39 (m, 3H), 1.01–1.13
(m, 2H), 0.94 (d, 3H, J = 4.3 Hz), 0.92 (d, 3H, J = 3.8 Hz), 0.83 (d, 3H, J = 6.8 Hz).
Compounds 6a and 6b were synthesized accordingly from the available
enantiomerically-pure benzylamines.
10% FBS (fetal bovine serum) and 100 IU penicillin/100 lg/mL streptomycin
sulfate in a humidified incubator containing 5% CO2 at 37 °C. For cytotoxicity
testing, cells were trypsinized, counted, centrifuged at 120 ꢁ g, and resuspended
in fresh culture medium. Cells were plated in a volume of 100
well) in 96-well flat-bottom culture dishes. After 16–18 h of incubation, 100 lL
l
L (2.5 ꢁ 104 cells/
6a: 56% as a colorless oil; ½a D25
ꢂ
+23° (c 0.59, CH2Cl2); 1H NMR (250 MHz, CDCl3): d
of medium or medium containing test samples was added to each well. Tests
were performed in triplicate. After 48 h, 20 L of MTT (3-(4,5-dimethylthiazol-
7.13–7.29 (m, 10H), 3.86–3.88 (m, 3H), 1.42–1.46 (m, 3H).
l
6b: 58% as a colorless oil; ½a D25
ꢂ
ꢀ25° (c 0.45, CH2Cl2); 1H NMR (250 MHz, CDCl3): d
2-yl)-2,5-diphenyltetrazolium bromide) (1.25–4 mg/mL in DPBS) was added to
each well, and the cells were further incubated for 2–3.5 h. The medium was
aspirated, and the formazan product generated in each well was dissolved in
7.17–7.27 (m, 10H), 3.82–4.3 (m, 3H), 1.44 (d, 3H, J = 6.6 Hz).
Antimicrobial testing to determine minimum inhibitory concentration: All the
testing was performed according to established NCCLS guidelines.21 10 mL of
Mueller-Hinton broth was added to a sterile test tube. Using a sterile cotton
swab, the broth-containing test tube was inoculated with 3–5 colonies from the
appropriate overnight culture. The test tube was placed in an incubator for
approximately 2 h at 35–37 °C, or until the culture reached an optical density of
0.08–0.10 at 625 nm wavelength. This is equal to 0.5 McFarland standard
[1.5 ꢁ 108 CFU (colony forming units)/mL]. The culture was adjusted to obtain
100 lL DMSO. Plates were read in a BioTek Synergy 2 SLFA plate reader set at
540 nm (background subtract at 660 nm). Cell viability was calculated as
percent of control (sample absorbance/control medium absorbance ꢁ100).
Based on the measured% absorbances relative to an untreated control, S,S0-
bis(isopropoxy) disulfide (1b) did not show any cytotoxic activity against either
of the endothelial cell types up to a final concentration of 20 lg/mL.