Download Synthesis and Antimicrobial Activities of S,S

University of South Florida
Scholar Commons
Graduate Theses and Dissertations
Graduate School
2011
Synthesis and Antimicrobial Activities of S,S'Heterosubstituted Disulfides
Praveen Ramaraju
University of South Florida, [email protected]
Follow this and additional works at: http://scholarcommons.usf.edu/etd
Part of the American Studies Commons, Microbiology Commons, and the Organic Chemistry
Commons
Scholar Commons Citation
Ramaraju, Praveen, "Synthesis and Antimicrobial Activities of S,S'-Heterosubstituted Disulfides" (2011). Graduate Theses and
Dissertations.
http://scholarcommons.usf.edu/etd/3299
This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in
Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact
[email protected].
Synthesis and Antimicrobial Activities of S,S’-heterosubstituted Disulfides
by
Praveen Ramaraju
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Department of Chemistry
College of Arts and Sciences
University of South Florida
Major Professor: Edward Turos, Ph.D.
Kirpal Bisht, Ph.D.
Jianfeng Cai, Ph.D.
Abdul Malik, Ph.D.
Date of Approval
July 14th, 2011
Keywords: Disulfides, Francisella tularensis, Candida albicans, Bacteriostatic, MRSA
Copyright© 2011, Praveen Ramaraju
TABLE OF CONTENTS
LIST OF TABLES
iv
LIST OF FIGURES
v
LIST OF ABBREVIATIONS
x
ABSTRACT
xii
CHAPTER ONE: ORGANOSULFUR ANTIMICROBIALS
1.1
1.2
1.2
Introduction
Biological Targets of Organosulfur Drugs
1.2.1 Glutathione-based Systems
1.2.2 Coenzyme A-based Systems
1.2.3 Thioredoxin-based Systems
1.2.4 Mycothiol-based Systems
1.2.5 Bacillithiol-based Systems
1.2.6 Other Cellular Targets
Classes of Biologically-Active Organosulfur Compounds
1.3.1 Thiol Reagents
1.3.1.1 Antibacterial thiols
1.3.1.2 Antifungal thiols
1.3.1.3 Antiviral thiols
1.3.1.4 Antiparasitic thiols
1.3.2 Sulfide Reagents
1.3.2.1 Antifungal sulfides
1.3.2.2 Antiparasitic sulfides
1.3.3 Disulfide Reagents
1.3.3.1 Antibacterial disulfides
1.3.3.2 Antifungal disulfides
1.3.3.3 Antiviral disulfides
1.3.4 Trisulfide Reagents
1.3.4.1 Antibacterial trisulfides
1.3.4.2 Antifungal trisulfides
1.3.5 Polysulfide Reagents
1.3.5.1 Antibacterial polysulfides
1.3.5.2 Antifungal polysulfides
1.3.5.3 Antiparasitic polysulfides
i
1
1
2
3
4
7
7
8
8
10
10
11
13
14
16
17
18
18
18
23
24
29
29
32
33
33
33
34
1.3.6
1.4
Other organosulfur Reagents
1.3.6.1 N-Thiolated Antibacterials
Conclusions
35
35
37
CHAPTER TWO: SYNTHESIS AND ANTIMICROBIAL ACTIVITIES
OF S,S’-HETEROSUBSTITUTED DISULFIDES
2.1 Introduction
2.2 Synthesis
2.3 Elucidation of 1H NMR spectrum of di sec-butoxy disulfide
2.4 Reactivity of S,S’-heterosubstituted disulfides
2.5 Antimicrobial activity
2.5.1. Determination of minimum inhibitory concentration
of disulfides against bacteria
2.5.2 Bacterial Viability
2.5.3 Assay Anti-staphylococcal assay by Kirby-Bauer testing on
agar plates
2.6 Studies of mode of action of S,S’-heterosubstituted disulfides
2.7 Activity of S,S’-Heterosubstituted disulfides against other microbes
2.8 Anti-Francisella Activity
2.9 Anti-Bartonella Activity
2.10 Anti-fungal Activity
2.10.1 Trypan Blue Staining
2.11 Efforts towards prodrug/ dual action drug synthesis
2.12 Final Conclusions and future directions
38
40
42
44
47
47
53
54
59
62
63
65
67
68
70
73
CHAPTER THREE: MATERIALS AND METHODS
3.1 Antimicrobial Testing by Minimum Inhibitory Concentration
3.1.1 Inoculum Preparation
3.1.2 Preparation of Drug-Containing Mueller Hinton Agar
in 24-well Plates
3.1.3 Inoculation of Drug-Containing Mueller Hinton Agar
3.1.4 Reading the Plates and Interpreting the Results
3.2 Kirby-Bauer Diffusion Assay
3.2.1 Culture preparation
3.2.2 Testing procedure
3.3 Anti-Francisella testing:
3.3.1 Culture preparation
3.3.2 Addition of LVS
3.3.3 Controls
3.4 In vitro Anti-Bartonella activity by disk diffusion method
3.4.1 Preparation of antibiotic containing paper disks
3.4.2 Inoculation of agar plates for disk diffusion testing
ii
76
76
77
77
78
78
78
78
79
79
79
79
82
82
83
3.5 Fungal viability assay
3.6 Cytotoxicity testing
3.7 Synthetic Procedures
3.7.1 General procedure for the synthesis of
S,S’-heterosubstituted disulfides
3.7.2 Synthesis of S,S’-heterosubstituted disulfides of menthol
3.7.3 Synthesis of 7-(4-tert-Butoxycarbonyl-piperazin-1-yl)-1cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid
CHAPTER FOUR:
SPECTRAL DATA
83
84
85
86
89
90
92
REFERENCES
110
iii
LIST OF TABLES
Table (1.1) Isothiazole anti-poliovirus activities
27
Table (1.2) Enediyne antibacterial activities
32
Table (1.3) Enediyne antifungal activities
33
Table (1.4) Anti-malarial activities of Lissoclinotoxin A and a few clinical
standards
34
Table (2.1) Reaction of diethoxy disulfide with monosubstituted hydrazine
47
Table (2.2) MIC’s of S,S’-dialkoxy substituted disulfides
48
Table (2.3) MIC’s of S,S’-dithio substituted disulfides
49
Table (2.4) MIC’s of S,S’-diamino substituted disulfides
50
Table (2.5) MIC’s of S,S’-diamino disubstituted disulfides
51
Table (2.6) MIC’s of chiral disulfides
52
Table (2.7) Bacterial viability assay
54
Table (2.8) MIC’s of S,S’-heterosubstituted disulfides against
Francisella tulerensis
64
Table (2.9) Zone of inhibition data of S,S’-heterosubstituted disulfides
against Bartonella species
66
Table (3.1) Antimicrobial activities of S,S’-heterosubstituted disulfides
80
Table (3.2) Cytotoxicity data of diisopropoxy disulfide (1b)
85
iv
LIST OF FIGURES
Figure (1.1) Biosynthetic pathway of glutathione
3
Figure (1.2) Coenzyme A redox system
4
Figure (1.3) Regular thiol and disulfide interactions with thioredoxins
5
Figure (1.4) Thiol and disulfide bearing drug interactions with thioredoxins
6
Figure (1.5) Mycothiol
7
Figure (1.6) Bacillithiol
7
Figure (1.7) Synthesis of adenosine triphosphate
8
Figure (1.8) Bicyclic β-lactams and sulfa drugs
9
Figure (1.9) Thiosugars and thionucleosides
10
Figure (1.10) Sulfur mustard
10
Figure (1.11) S. aureus-active thiodiazoles
11
Figure (1.12) Substituted 1,3,4-thiadiazolium-5-thiolates
11
Figure (1.13) Mercaptotriazole antifungals
12
Figure (1.14) Antifungal 1,3,4-oxadiazoles
12
Figure (1.15) S-Acetylcysteamine derivatives
13
Figure (1.16) Thiocyanatopyrimidine nucleoside
14
Figure (1.17) Antiprotazoal agent T-Cadinthiol
14
Figure (1.18a) Trypanocidal agents
15
Figure (1.18b) Equilibrium between melarsamine and melarsen oxide in water
16
v
Figure (1.19) Diallyl sulfide, isolated from garlic extract
17
Figure (1.20) Antifungal naphthoquinone sulfides
17
Figure (1.21) Methylthiobenzimidazole fasciolicide
18
Figure (1.22) Formation of mixed disulfides of ajoene
19
Figure (1.23) Gliotoxin, an epipolythiodioxopiperazine (ETP) antibacterial
20
Figure (1.24) Psammaplin A
20
Figure (1.25) Bisaprasin
21
Figure (1.26) Psammaplin D
21
Figure (1.27) Citorellamine
22
Figure (1.28) Aryl-alkyl disulfides synthesized in Dr.Turos’ lab
22
Figure (1.29) Core Structure of the thiarubine group of antifungals
23
Figure (1.30) Dithiole-thione antifungals
24
Figure (1.31) Antiarenavirus disulfides
25
Figure (1.32) Anti-HIV macrocyclic disulfide
26
Figure (1.33) 5,5’-Diphenyl-3,3’-diisothiazole disulfide and thiol
antipoliovirus agents
26
Figure (1.34) Dideoxynucleotides thiryl radical-based reverse transcriptase
inhibitors
27
Figure (1.35) Postulated mechanism of reduction of natural ribonucleoside 5’diphosphates
28
Figure (1.36) Mercaptouridine thiryl radical-based reverse transcriptase
inhibitors
28
Figure (1.37) Nucleosidic disulfide anti-HIV agent
vi
29
Figure (1.38) Anti-infectives from garlic
29
Figure (1.39) 4-Dioxo-1,2,4,6-tetrathiepane
30
Figure (1.40) Enediyne Trisulfides: Calichaemycin (41a),
Esperamicin A1 (41b) and Namenamicin (41c)
31
Figure (1.41) Bioactivation of enediynes
32
Figure (1.42) Lissoclinotoxin A
33
Figure (1.43) Lissoclinotoxin D
34
Figure (1.44) N-Sulfenylated-β-Lactam antibacterial
35
Figure (1.45) Formation of CoA- β-lactam mixed disulfide
35
Figure (1.46) BIT
36
Figure (1.47) Lansoprazole and in vivo Sulfenamide
36
Figure (2.1) Various antibacterials synthesized in the Turos lab
39
Figure (2.2) Various aryl-alkyl disulfides synthesized in Turos lab
40
Figure (2.3) Synthesis of S,S’-heterosubstituted disulfides
41
Figure (2.4) Synthesized S, S’-heterosubstituted disulfides
42
Figure (2.5) 1H NMR spectra of sec-butanol and di sec-butoxy disulfide
43
Figure (2.6) Reactivity of dialkoxy disulfides with thiols and secondary amines
44
Figure (2.7) Reactivity of alkoxy trisulfides (2) with thiols and secondary amines 45
Figure (2.8) Reactivity of alkoxy amino disulfides (3) with thiols and
secondary amines
45
Figure (2.9) Reactivity of dialkoxy disulfides (1) with 1,1 and 1,3- disubstituted
thioureas
Figure (2.10) Reactivity of Dialkoxy disulfide (1) with monosubstituted
vii
46
hydrazine
46
Figure (2.11) Synthesized Chiral S, S’- dihetero substituted disulfides
52
Figure (2.12) Compounds tested by Kirby-Bauer diffusion assay
55
Figure (2.13) Kirby-Bauer Assay showing no growth inhibition by DMSO
56
Figure (2.14) Kirby-Bauer Assay showing the ability of CoA, GSH and cysteine
to inhibit antimicrobial effects of the test compounds on MRSA
Figure (2.15) Structures of cysteine, coenzyme A and glutathione
57
57
Figure (2.16) Kirby-Bauer Assay showing no cancellation effect of
diisopropyl disulfide by isopropyl disulfide or triphenylphosphine
58
Figure (2.17) Kirby-Bauer Assay showing no inhibition effect from the amino
acids valine, lysine and glycine
59
Figure (2.18) Kirby-Bauer assay with compound 1b and glutathione in different
molar ratios in the same well.
60
Figure (2.19) NMR tube experiment of diisopropoxy disulfide and
propane-2-thiol in CDCl3.
61
Figure (2.20) Scanning electron microscopy pictures of MRSA cells before
and after treatment with isopropoxy disulfide
62
Figure (2.21) Trypan Blue
68
Figure (2.22) Trypan Blue staining showing live fungal cells
69
Figure (2.23) Trypan Blue staining of heat killed control showing dead
fungal cells
69
Figure (2.24) Attempted coupling reactions of diisopropoxy disulfide
71
Figure (2.25) Attempted synthesis of ciprofloxacin and penicillin disulfides
72
viii
Figure (2.26) Synthesis of N-Boc ciprofloxacin (7)
72
Figure (2.27) Most active disulfide analogs 1b and 3e
73
ix
LIST OF ABBREVIATIONS
ATP = adenosine triphosphate
ATPase = adenosine triphosphate synthase
Bn = benzyl
o
C = degrees Celsius
CoA = coenzyme A
CoADR = coenzyme A disulfide reductase
Cys = cysteine
δ = delta or chemical shift (NMR)
DBE = disulfide bridge forming enzyme
DID = 5,5’-diphenyl-3,3’-diisothiazole disulfide
dNTP = deoxyribonucleoside triphosphate
DMSO: Dimethylsulfoxide
EC = effective concentration
Et N = triethylamine
3
ETP = epipoly(thiodioxopiperazine)
GDR: glutathione disulfide reductase
GSH: glutathione
1
H NMR= proton nuclear magnetic resonance
HIV = human immunodeficiency virus
HIV-RT = human immunodeficiency virus reverse transcription
Hz = hertz
J = coupling constant (NMR)
JUNV = Junin (agent of Argentine hemorrhagic fever)
MEA = 2-mercaptoethylamine (cysteamine)
MIC = minimum inhibitory concentration
MSH: Mycothiol
µg = micrograms
µM = micromolar
mM = millimolar
MRSA = methicillin-resistant Staphylococcus aureus
MRSE = methicillin-resistant Staphylococcus epidermidis
NAC = N-acetyl-L-cysteine
NCCLS: National Committee for Clinical Laboratory Standards
ng = nanogram
PenG: Penicillin G
Ph = phenyl
ppm = parts per million
x
S = elemental sulfur
8
TCBZ = triclabendazole
TLC = thin layer chromatography
TSA: Trypticase® Soy Agar
trx = thioredoxin
xi
Synthesis and Antimicrobial Activities of S,S’-Heterosubstituted Disulfides
Praveen Ramaraju
ABSTRACT
Antibiotic resistance is a particularly critical health concern and has increased
dramatically over the past two decades. For over a decade the Turos laboratory has
been designing small molecules to target pathogenic microbes such as Staphylococcus
aureus and the resistant variants like methicillin-resistant Staphylococcus aureus
(MRSA). Previously, N-thiolated β-lactams, N-thiolated 2-oxazolidinones and
aromatic disulfides that were synthesized in Dr. Turos’ lab have shown strong activity
against these bacteria. The present work describes the synthesis and antimicrobial
activities of a related structural class called S,S’-heterosubstituted disulfides. For
ages, sulfur (elemental) has been used as an antibacterial for controlling infestation
and bacterial diseases. This is the starting point of this thesis. Chapter 1 discusses the
various sulfur-containing antibiotic compounds and the importance of sulfur
compounds to exhibit inhibitory activity against disease-causing pathogens. Also in
this introductory chapter, the different sulfur functionalities and their respective
modes of action were presented. The synthesis and the antimicrobial activities of the
title compounds are described in chapter 2. S,S’-heterosubstituted disulfides were
found to possess inhibitory activities against Staphylococcus aureus, methicillinxii
resistant Staphylococcus aureus (MRSA), Francisella tulerensis and the fungi
Candida albicans. From the bacterial viability assay and the trypan staining assay,
these compounds were found to be bacteriostatic and fungistatic, and these
structurally-simple disulfides may serve as new leads to the development of effective
antibacterials for drug-resistant staph infections.
X
R
S
S
R
X
X = O, N, S
xiii
CHAPTER ONE
ORGANOSULFUR ANTIINFECTIVES
1.1 Introduction
The use of organosulfur compounds to control infectious diseases has its roots dating
back to early times, when ancient Egyptians recognized the potent medicinal effects of
naturally occurring organosulfur substances from leeks. In 1824 powered sulfur was
shown to treat peach mildew1 and over the centuries, it has successfully been applied as a
fungicide to protect a wide variety of plants against fungal infestation1. Although the
mode of antifungal action of elemental sulfur is not fully defined, there is no doubt of its
dependency on the reaction of sulfur with a biological target.
1.2 Biological Targets of Organosulfur Drugs
Due to the exposure of living organisms to various infection-causing pathogens, over
time they developed defenses against harmful biological oxidants and disinfectants. The
thiol-disulfide equilibrium in cells plays a vital role, creating a natural defense system
that helps rid the cell of potentially damaging chemicals and metabolites that cause
oxidative degradation. Nature protects various bacteria from oxidative stress by
maintaining high thiol:disulfide ratios in the cytosol, typically 19:1 or higher. Disruption
1
of this redox system can alter many vital cellular activities such as regulation of protein
activity, regeneration of enzymatic cofactors and reductases like ribonucleotide
reductase, and a host of other biological processes where an antioxidant is required in the
cell2.
1.2.1 Glutathione-based Systems
For many years, glutathione (1) (γ-L-glutamyl-L-cysteinylglycine), a tripeptide, serves as
the active component of the most common thiol/disulfide redox system in many cells.
Glutathione is produced biologically via the glutaredoxin enzymatic pathway (Fig 1.1).
Biosynthesis of GSH occurs in all cell types via two reactions catalyzed by γ-glutamyl
cysteine synthetase (γ-GCS) and GSH synthetase (GS). γ-GCS catalyzes the formation of
a peptide bond between the γ-carboxyl group of glutamate and the α-amino group of
cysteine. Glutathione synthetase forms the peptide bond between the α-carboxyl of
cysteine in γ-glutamyl cysteine and the α-amino group of glycine.3 The equilibrium
between free thiol and disulfide, at stasis, is typically maintained at a cytoplasmic thiol
concentration of around 90%. Cells with high intracellular glutathione levels are
generally less susceptible to damage by organosulfur drugs. Serving a primary role as an
antioxidant, glutathione is extremely effective in scavenging reactive free radicals,
electrophiles and other destructive oxidants in the cytoplasm. Glutathione can react
directly with various drugs to deactivate them before they are able to cause any damage,
while also serving to reactivate enzymes that have been inhibited as mixed disulfides
formed between a drug and enzyme. In response to this protective mechanism afforded
by glutathione, nature has cleverly designed prodrug molecules such as the endiyne
trisulfides and anthracyclines, which can interact directly with glutathione as a way to be
2
biochemically activated within the target cell. Inhibition of glutathione’s antioxidant
abilities can be induced by formation of mixed glutathione drug disulfides4. Reduction of
glutathione disulfide (GSSG) and mixed disulfides to GSH is catalyzed by glutathione
reductase, which seems to play a crucial role in evolutionary adaptation of organisms to
atmospheric oxygen. Glutathione reductase is widespread among bacteria, fungi, plants,
protozoa, and animals. All glutathione reductases isolated from different sources are
highly homologous, showing high evolutionary conservativeness of this protein.3
O
NH2
O
OH
H2N
HO
OH
NH
γ - glutamylcysteine synthase
OH
H2N
HO
SH
L-cysteine
O
O
O
SH
L-glutamate
O
L-γ - glutamylcysteine
O
Glutathione synthase
H2N
OH
L-glycine
O
NH2
O
NH
HO
O
O
SH
NH
HO
glutathione (1)
Figure (1.1) Biosynthetic pathway of Glutathione
1.2.2 Coenzyme A-based Systems
For many years, glutathione was assumed to be present in all cells. However, it is not
entirely ubiquitous. Some bacteria are totally devoid of glutathione, but in its place have
3
some other thiol/disulfide based redox system. S. aureus does not utilize glutathione at
all, but rather produces millimolar levels of the nucleosidic thiol, coenzyme A (2)
(CoA)5. At stasis, the bacterium maintains a ratio of thiol:disulfide of about 95:5, via
coenzyme A disulfide reductase (CoADR) (Fig 1.2). This redox system is very selective
in its ability to reduce CoA disulfide. Mixed disulfides formed between CoA and
glutathione, or other thiophilic agents, are typically unable to be reduced by CoADR5,6.
The fate of these mixed disulfides is not clearly known. Therefore, anti-infective
compounds that can form CoADR resistant mixed disulfides with CoA can offer an
effective mode of inhibition interfering with the redox buffer, providing an opportunity to
enhance the susceptibility of the bacterial cell to oxidative damage. Indeed, glutathione is
an inhibitor of the redox cycle of CoA, as it forms mixed CoA-glutathione disulfides that
cannot be reductively cleaved, and is detrimental to the growth of this bacterium.
N
N
HO
NH2
N
HO
P
O
P
Antioxidant Activity
O
OH
O
N
O
HO
N
N
O
HO
O
HO
CoADR
O
HO
H
N
HN
2
N
O
OH
O
O
O O
P
O P
HO
OH
NH2
N
O
P
O O
O P
OH
O
HO
H
N
HN
SH
S
O
O
2
Figure (1.2) Coenzyme A redox system
1.2.3 Thioredoxin-based Systems
Another very important group of native cellular thiols / disulfides are the thioredoxins
(trx’s) and a related subfamily, the disulfide bridge forming enzymes (DBE’s), which
4
span through the bacterial membrane connecting the cytoplasm with the periplasm (Fig
1.3). The characteristic Cys-X-X-Cys motif in the structures of the trx’s is highly
conserved in many bacteria. These dithiols undergo reversible oxidation and can quickly
react with non-native thiols or disulfides. Before a drug even passes through the
membrane of a bacterium however, a subfamily of thioredoxins, the DBE’s, could
intervene. Formation of mixed disulfides between the trx’s and an organothio compound
can potentially inhibit enzymatic reduction by disulfide reductases and thus can perturb
proper cellular function.
Reduction/ Oxidation of
Periplasmic Substrates
Periplasm
HS
SH
DBE
S
Normal Electron Flow
Cytoplasm
S
HS
thioredoxin
HS
Figure (1.3) Regular thiol and disulfide interactions with Thioredoxins
DBE’s, for the most part, inhabit the cytoplasmic membrane with exposed (Cys-X-XCys) functionalities on both the cytoplasmic and periplasmic surfaces. DBE’s are
involved in electron transport across the membrane and serve as a signaling mechanism,
5
communicating the oxidation state of the cytoplasmic trx’s with the oxidation state on the
periplasmic side. Disulfide formation involving trx motifs on either side of the membrane
can cause disruption of the DBE’s electron transport abilities, affecting a host of cellular
processes such as respiration and cytochrome syntheses. Although there is evidence that
DBE repair enzymes exist7,8, their effectiveness and versatility against non-native thiols
or sulfides has not been reported.
Inhibition of Redox
Communication
X
X
ug
Dr
S
SH
HS
HS
S
Periplasm
DBE
DBE
Bacterial Membrane
Cytoplasm
S
SH
S
Dr
ug
X
S
S
Dr
S
S
Blocked Electron
Flow
HS
ug
trx
Figure (1.4) Thiol and disulfide bearing drug interactions with Thioredoxins
So, these extraordinarily reactive, native groups represent significant potential as drug
targets.
6
1.2.4 Mycothiol-based Systems
The redox buffer in case of Actinomycetes is that of mycothiol (3), which acts as the
primary antioxidant9. First discovered in a species of Streptomyces, and then identified in
Mycobacterium bovis, mycothiol has since been found to be prevalent only amongst
actinomycetes and is produced in high levels by mycobacteria. Mycothiol undergoes
metal-catalyzed autoxidation at a slower rate than GSH10 and is maintained in the
reduced state by mycothiol disulfide reductase.11
SH
O
O
OH
N
NH H O
OH
O
HO
HO
HO
OH
OH
OH
3
Figure (1.5) Mycothiol
1.2.5 Bacillithiol-based Systems
Bacillithiol (4) is the α-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic
acid and most probably functions as an antioxidant. Bacillithiol, like the structurally
similar mycothiol, may serve as a substitute for glutathione. Bacillithiol is used as a
buffer
system
in
Bacillus
radiodurans.12
species,
SH
Staphylococcus
O
OH
H2 N
O
OH
O
N O
H
O
OH
4
Figure (1.6) Bacillithiol
7
OH
OH
aureus
and
Deinococcus
1.2.6 Other Cellular Targets of Organosulfur Antiinfectives
Another potential intracellular thiol target is adenosine triphosphate synthase (ATPase).
ATPase is responsible for regeneration of the energy source of cells, adenosine
triphosphate (ATP) (5), from adenosine diphosphate and inorganic phosphate (Fig 1.7).
ATPase, which sits on the membrane of mitochondria, contains a reactive, free thiol that
is vital to activity. Inhibition of ATPase, therefore, can be achieved by formation of a
mixed disulfide.
NH2
NH2
N
N N
O
O
O
O
HO P O P O P O
OH OH OH HO
OH
N
N
N
ATP synthase
O
O
O
ON N
O
P
O
HO P O P
OH OH OH HO
Inorganic phosphate
OH
Adenosine diphosphate
Adenosine triphospahte (5)
Figure (1.7) Synthesis of Adenosine Triphosphate (5)
1.3 Classes of Biologically-Active Organosulfur Compounds
Organic compounds that contain sulfur exhibit an extraordinary range of chemical
structures and reactivities. Many of these have biological activity. In the simplest case,
the presence of one or more sulfur atoms in a biologically active molecule may not
actually give the compound its biological effects, but rather may be a non-participant in a
side chain residue or an innocuous constituency of the molecular framework. Important
examples of this are the bicyclic β-lactams, including penicillins (6), cephalosporins (7),
and penems (8), and the sulfa drugs (9). Most of the β-lactam drugs act by inhibiting the
transpeptidation reaction responsible for the synthesis of peptidoglycan, a key component
of the bacterial cell wall that provides rigidity.13, 14 Sulfa drugs are known to inhibit the
8
synthesis of folic acid in bacteria by competing with para-aminobenzoic acid. The antiinfective activity of these drugs does not appear to be directly related to a sulfur-centered
event nor its presence in the molecule.
COOH
COOH
O
O
N
N
S
ROCN
X
S
ROCN
H
H
7
6
COOH
O
N
O R'
S NH
O
R
HN
R
S
OH
9
8
Figure (1.8) Bicyclic β-Lactams and Sulfa Drugs.
In case of thiosugars, the sulfur atom exerts a more definitive, yet subtle, effect on its
biological activity. These sulfur analogs of the natural sugars and nucleosides act as
inhibitors of glycosidases and reverse transcriptases, respectively15-18. It has been
documented that these properties are due to the conformational and stereoelectronic
changes brought about by the replacement of oxygen with sulfur in the heterocyclic ring.
However, even in these molecules, the sulfur atom does not play a central role in the
reaction of the molecule with a biological entity.
9
HO
S
HO
OH
H3 C
OH
HO
S
OH
S
HO
OH
HO
OH
OH
OH
OH
D-5-Thiomannose
L-5-Thiofucose
D-5-Thioglucose
OH
Figure (1.9) Thiosugars and Thionucleosides.
In many other cases, however, the bioactivity of an organosulfur compound may be
directly attributable to the reactivity of the sulfur center, which is certainly the case for
the sulfur mustards (Fig 9). Here, the sulfur atom is responsible for activating the
compound toward nucleophilic attack by displacing a chlorine atom and forming an
episulfonium ion (10). Thus formed, this cationic electrophile is prone to attack by a
biological nucleophile, and can lead to crosslinking of duplex DNA.
Nu
S
Cl
S
Cl
Cl
S
Cl
Nu
10
Figure (1.10) Sulfur Mustard
1.3.1
Thiol Reagents
1.3.1.1 Antibacterial Thiols
Thiol-bearing enzymes often lose some or all of their activity in the presence of nonnative thiols like allyl mercaptan due to the formation of mixed disulfide. Thus, thiolbearing therapeutics are often effective at inhibiting enzymatic pathways regulated by
these proteins. Although there are a large number of small, natural product thiols known
10
to possess antibiotic activities, there are only a few synthetically-derived thiols that have
been shown to be antibacterial. A trio of thiodiazole aromatics (11) is reported to have
minimum inhibitory concentrations (MICs) against Staphylococcus aureus in the 31-62
µg/ml range, but impose no effect on E. coli19 (Fig 1.11).
N N
N
N
X
11
X - o-Me, p-Br, p-Cl
S
SH
Figure (1.11) S. aureus-Active Thiodiazoles.
A similar effect was observed in case of 1,3,4-thiadiazolium-5-thiolates20 (Fig 1.12),
whose MIC’s against Staphylococcus aureus range from 1-8 µg/ml, and which also
exhibit moderate activities against E.coli. The mechanism of action of these compounds
is yet to be studied.
O
O
O
N
N
H2N
N
R
N
H2N
R
S
HN
S
S
N
S
(a) R = CH3, (b) R = C6H5,
(c) R = 2-Cl–5-NO2–C6H3,
(d) R = 3-NO2–C6H4
N
Figure (1.12) Substituted 1,3,4-thiadiazolium-5-thiolates
1.3.1.2 Antifungal Thiols
Due to the proliferation of thiol-bearing enzymes in a large majority of life forms, it is to
be expected that anti-infective thiol compounds could be found in Nature that can
successfully inhibit the growth of fungi by formation of mixed disulfides. Most of the
previously discussed antibacterial thiols have been observed to possess some antifungal
11
activity. Closely related derivatives of the antibacterial mercaptotriazoles (12) inhibit
Candida albicans and Saccharomyces cerevisiae, with minimum fungicidal concentration
(MFC) values in the range of 12.5 to 61 µg/ml21. Both the thiol and amine groups are
believed to be required for antifungal activity, since the thiodiazole aromatics (which lack
the amino moiety) and their precursors have no antifungal properties.
N N
SH
R - 3-Br, 4-CH3, 4-OCH3
N
NH2
R
12
Figure (1.13) Mercaptotriazole antifungals
A new class of 1,3,4-oxadiazole compounds (13) have reportedly shown growth
inhibition zones of 20-25 mm at 100 µg, equivalent to the control antifungal compound
griseofulvin.
HS
N
N
O
R'
N
R
13
R = p-Cl-C6H4, p-F-C6H4, p-CH3-C6H4
R' = F, Cl, NO2
Figure (1.14) Antifungal 1,3,4-oxadiazoles
12
1.3.1.3 Antiviral Thiols
Intracellular redox activity controls replication and virulence of viruses22. The cellular
thiol, glutathione, itself has purported in vitro and in vivo anti-influenza activity. As
levels of glutathione are depleted in mucosal cells lining the oral, nasal and upper
airways, susceptibility to viral infection is enhanced. Decreased intracellular glutathione
levels are also implicated in Human Immunodeficiency Virus (HIV) infections, and
methods to increase glutathione production, in human monocyte derived macrophages
and lymphocytes, have been proposed as a means to stop these infections23-25. N-AcetylL-cysteine (NAC) and 2-mercaptoethylamine (MEA) have been shown to strongly
increase glutathione levels in various cell lines26. Several new N-(N-acetyl–L-cysteinyl)S-acetylcysteamine derivatives (Fig 1.15) have also been reported to actively release
NAC and MEA intracellularly, which in turn improve glutathione levels.
H
N
O
N
H
O H
H
N
SH
O
N
H
O H
S
R
O
HS
HS
H
N
O
S
N
H
O H
S
R
O
R'
O
Figure (1.15) S-Acetylcysteamine derivatives
These compounds display an EC90 (effective concentration for 90% inhibition of virus
yields) ranging from 80 to 380 µM against HIV in human monocyte-derived
macrophages (MDM). S-Acylated derivatives are believed to have increased activity by
13
1) having a protected thiol and 2) increasing lipophilicity relative to the free thiol.
Another way reported to protect a free thiol is by use of a thiocyanate, which is likely
reduced in vivo to the thiol through an equilibrium exchange with a native thiol such as
glutathione27. Examples of this include a group of thiocyanatopyrimidine nucleosides
(14), which are reported to display reasonable activity (EC75 = 100 µM) against vaccinia
virus replication in HeLa cells.
O
H
O
O
SCN
N
R'SH
H
N
R
O
SH
N
N
R
14
Figure (1.16) Thiocyanatopyrimidine nucleoside (R = ribofuranosyl nucleoside).
1.3.1.4 Antiparasitic Thiols
A novel sesquiterpene named T-cadinthiol (15) shows significant antiparasitic
properties28, 29. This terpenoid metabolite, with four fixed stereocenters, displays activity
towards cultured Plasmodium falciparum, a species of malaria, with an IC50 of 3.6
µg/ml. The mode of action of this agent is still unexplored, however, and a number of
derivatives of 15, including the corresponding alcohol analog, were tested and shown to
have absolutely no biological activity. The thiol group appears to be essential for
antiparasitic effects.
H
H
15
Figure (1.17) Antiprotazoal agent T-Cadinthiol.
14
SH
African trypanosomiasis caused by Trypanosoma brucei gambiense and T. brucei
rhodesiense was treated with melaminophenylarsine melarsoprol (MelB), introduced in
194730. MelB is insoluble in water and is administered by intravenous injection as a
propylene glycol solution. This arsenical agent is frequently associated with a number of
serious side effects and the solvent is an irritant that often causes thrombophlebitis.
Water-soluble arsenical agents such as melarsonyl potassium (MelW) showed little
improvement over MelB. Later a new, water-soluble trivalent arsenical agent,
melarsamine hydrochloride (MelCy) (trade name: Cymelarsan), was shown to be very
effective against T. brucei brucei, T. evansi, and T. equiperdum
31, 32
. A successful
attempt to increase the activity of this drug, by derivatizing the thiol groups to get the
level of affinity between arsenic and sulfur atoms optimal for biological activity, has been
reported33. The most active thiol system is the propane-1,3-dithiol (2-melarsenyl).
CH2OH
COOK
S
As
NH2
N
H2N
N
N
NH2
S
N
H2N
N
H
N
N
H
N
MelB
MelW
NH2
N
H2N
SCH2CH2NH2
As
SCH2CH2NH2
N
N
S
As
COOK
N
H
MelCy
NH2
N
H2N
Figure (1.18a) Trypanocidal agents
15
S
As
N
N
N
H
2-Mel
S
S
In fact, 2-melarsenyl is twice as potent as Cymelasan (MelCy) against Trypanosoma
brucei brucei strains (0.025 versus 0.05 µM concentration to terminate all growth in 1
hour). It is believed that in aqueous solution Cymelasan is in equilibrium with the
hydrolyzed oxide form (melarsen oxide), which has lost thiol groups and thus the
activity33.
NH2
N
H2 N
SCH2CH2NH2
As
SCH2CH2NH2
N
N
N
H
MelCy
NH2
N
H2N
SCH2CH2NH2
As
OH
N
N
N
H
NH2
N
H2N
O
As
N
N
N
H
Melarsen oxide
Figure (1.18b) Equilibrium between Cymelarsan and melarsen oxide in water
1.3.2
Sulfide Reagents
Penicillin (6), cephalosporins (7) and penems (8), as discussed earlier in this chapter, are
some of the most potent sulfide anti-infectives. Diallyl sulfide (Fig 1.19), isolated from
16
garlic, is another natural antimicrobial compound which has shown inhibitory activity
against MRSA in mice.34
S
Figure (1.19) Diallyl sulfide, isolated from garlic extract
1.3.2.1 Antifungal Sulfides
Six newly reported sulfide-bearing 1,4-naphthoquinones (16-21) have been found to
display good to potent activities against Candida albicans, Cryptococcus neoformans,
Sporothrix schenckii, Trichophyton mentagraphytes, Aspergillus fumigatus and
Microsporum cannis, with MFCs ranging from less than 0.78 to 50 µg/ml, a marked
improvement over their respective oxygen counterparts35. As of yet, the mechanism of
their activity is not understood, but may in fact be related to the oxidative potential of the
naphthoquinone ring system and not to the presence of the sulfur moieties.
O
O
O
O
S
S
O
16
Cl
OH O
17
18
N
N
N
N
N
S
S
S
19
OH O
O
O
OH O
S(CH2)2CO2H
O
O
O
21
20
Figure (1.20) Antifungal Naphthoquinone Sulfides
17
N
1.3.2.2 Antiparasitic Sulfides
Fasciolosis (Fasciola hepatica) is a serious parasitic disease in humans and livestock.
Few new anti-fasciolitic compounds have been marketed since triclabendazole (TCBZ), a
benzimidazole used routinely in veterinary medicine since 1983 and for human use, in
some regions, since 1989, was patented in 1978. A new bioactive derivative of TCBZ, 5chloro-2-methylthio-6-(1-naphthyloxy)-1H-benzimidazole
(22)
has
been
recently
discovered36. While this analog has an effective dose of 15 mg/kg, the marketed TCBZ
displays a 5 to 10 mg/kg effective dose against Fasciola hepatica. The mechanism of
action of this analog has not been elucidated, and the role of the sulfide moiety is not
known.
H
N
O
CH3
S
N
Cl
22
Figure (1.21) Methylthiobenzimidazole fasciolicide (22)
1.3.3
Disulfide Reagents
1.3.3.1 Antibacterial Disulfides
As therapeutic agents, disulfides usually serve as inactive structural components of
biomolecules or as biological oxidants. In the latter role, disulfides are particularly prone
to react with thiols to give biologically-inert mixed disulfide adducts. Many of the leek
extracts, like Ajoene (23), create the same type of mixed disulfide products with native
thiols that are seen with the corresponding thiol drugs37.
18
When various concentrations of cysteine were added to ajoene-containing media, the
ajoene concentrations were decreased. Similar effects were also observed with
dithiothreitol instead of cysteine. The sulfhydryl groups in cysteine and dithiothreitol are
highly reactive to disulfides and tend to form mixed disulfides, which are biologically
inactive. This likely explains the decrease in concentration of ajoene and inhibitory
activity towards various pathogens. 38
O
S
S
23
OH
OH
SH
SH
O
OH
SH
NH2
cysteine
dithiothreitol
OH
OH
S
S
S
OH
S
SH
O
NH2
S-allylmercaptocysteine
S-allyldithiothreitol
Figure (1.22) Formation of mixed disulfides of Ajoene.
Epipolythiodioxopiperazines (ETP’s) are a class of fungal metabolites of Candida,
Thermoascus and Penicillium, to name a few, that possess characteristic bridged disulfide
piperazinedione six-membered rings. These antibiotics only inhibit Gram-negative
bacteria, which are likely related to their outer membrane permeability, and are prone to
nucleophilic attack on their electrophilic disulfide bridge. Gliotoxin (24) and related
ETP’s are reported to act as oxidants by at least two different pathways: 1) generation of
19
superoxide and hydrogen peroxide via glutathione redox cycling, and 2) sulfenylation of
native thiols of certain proteins to make catalytically defunct mixed disulfides39, 40. Which
sulfur center in gliotoxin is the site of enzymatic attack is still unclear but it is likely that
both may be involved.
Enzyme
Enzyme
S
S O
SH
O
N
H
OH
O
S
SN
H
OH
OCH3
24
N
N
O
SH
OCH3
Figure (1.23) Gliotoxin, an Epipolythiodioxopiperazine (ETP) antibacterial reacts with
thiols.
Isolated from both a species of Psammaplysilla and Thorectopsamma xana, a trio of
closely related disulfides was discovered with antibacterial activity41. Psammaplin A (25)
and the dimer, Bisaprasin (26), along with Psammaplin D (27) all inhibit growth of
Staphylococcus aureus and Bacillus subtilis. Psammaplin D also shows activity against
the Gram-negative bacterium Trichophyton mentagraphytes42.
HO
HO
N
H
N
Br
O
S
S
O
25
Figure (1.24) Psammaplin A
20
N
H
Br
N
OH
OH
HO
HO
N
O
H
N
Br
S
S
O
O
Br
HO
HO
N
S
N
H
Br
N
H
N
N
H
N
S
OH
OH
OH
OH
Br
O
26
Figure (1.25) Bisaprasin
HO
HO
N
O
H
N
Br
S
O
S
N
H
OCH3
27
Figure (1.26) Psammaplin D
Isolated from a tunicate, Polycitorella mariae, a novel disulfide termed Citorellamine
(28), has been reported to have significant antimicrobial activity43. Originally assigned
the structure of a sulfide, the disulfide Citorellamine demonstrates potent antibacterial
activity against Staphylococcus aureus, Bacillus subtilus and Escherichia coli as well as
cytotoxicity in some cancer cell lines44.
Disulfides with interesting antibacterial activities also occur in proteins specifically
synthesized by life forms for defense. These antibiotics are found most commonly in
marine sources, and usually contain two neighboring cysteine residues. One such
antibacterial protein, from a marine decapod, displayed potent inhibition of Planococcus
citreus, Planococcus kocurii, Aerococcus viridans, and Micrococcus luteus and an
21
extraordinarily strong resistance to heat damage due to the presence of disulfide bonds45.
It was found to be cationic, yet hydrophobic, with a molecular mass of about 11.5kDa.
S S
NH
Br
N
H
HN
28
N
H
Br
Figure (1.27) Citorellamine
A series of simple aryl–alkyl disulfides have also been looked at previously in the Turos
lab. These aryl–alkyl disulfides46 demonstrated strong in vitro antimicrobial properties
against S. aureus, MRSA and B. anthracis. The nitrophenyl alkyl disulfides exhibited the
most in vitro potency, and bioactivity seems to require that the aryl substituent (X) be
electron-withdrawing.
SH
RSSO2Me
X
S SR
X
MeOH
X - F, CH3, OCH2CH3, CH2OH, NHAc, NH2, NO2
R - Me, Et, nPr, iPr, nBu, s-Bu
Figure (1.28) Aryl-alkyl Disulfides synthesized in Dr.Turos’ lab.
In vitro microbiological properties of these compounds, determined from agar diffusion
and agar MIC measurements, coincides with the FabH inhibition capabilities, which
points to the likelihood that this key bacterial enzyme is associated with the biological
properties of the disulfide compounds.
22
1.3.3.2 Antifungal Disulfides
Although the mechanism of action of the organodisulfide, Ajoene (23) is still under
active investigation, it is likely that antifungal activity is the result of induced cell wall
damage, since morphological changes in fungal cells treated with these compounds have
been observed via scanning and transmission electron microscopy47. Disulfides are
commonly found as toxins produced by fungi48, however, two groups of disulfides having
antifungal activity have been reported49-51. The first group of compounds contains some
of the most potent antifungal agents ever known, the thiarubines (29), with MIC’s in the
ng/ml range 49. Isolated as natural products from the Compositae (Asteraceae) family of
plants, the thiarubines display activities against Cryptococcus neoformans, Aspergillus
fumigatus, Candida albicans and other species of Candida.
R
R'
S S
29
Figure (1.29) Core Structure of the Thiarubine Group of Antifungals.
The tunicate-generated disulfide, Citorellamine (28), not only possesses strong
antibacterial activity, but is also very active against Saccharomyces cerevisiae and mildly
active towards Pseudomonas aeruginosa
43, 44
. The other group of disulfides, the 1,2-
dithiole-3-thiones (30), is very interesting because it consists of compounds that could
possibly behave as a disulfide, sulfide, or thiol in its activity50. The compounds are
fungicidal against Candida albicans, Candida tropicalis, Cryptococcus neoformans,
Sacharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,
23
Microsporum cannis, Microsporis gypseum, Epidermophyton floccosum, Trichophyton
rubrum and Trichophyton mentagrophytes. The main activity is due to the disulfidethione functionality. Although the pendant sulfide (SR) is not strictly required for
activity51, its presence, as compared to alkyl or aryl groups, increases activity greatly.
After lengthening the sulfur side chain beyond ethyl, an inverse relationship between
chain length and antifungal activity begins to develop. However, against all of the fungi
tested the thiobenzyl analog maintained the greatest potency, with MFC’s ranging from
0.7 to 6.25 µg/ml.
R
S
S
S
S
30
R = ethyl, propyl, butyl, hexyl, decyl, dodecyl, phenyl, benzyl, cyclopentyl
Figure (1.30) Dithiole-Thione antifungals.
1.3.3.3 Antiviral Disulfides
As discussed in relation to antibacterials, organodisulfides can act as in vivo oxidants of
thiols. It is likely that disulfides, and their related thiosulfonates, behave in the same
manner in terms of their activity in viral systems. A group of aromatic disulfides and a
thiosulfonate have been reported with potent antiviral activities against the arena viruses
Junin (JUNV), the causative agent of Argentine hemorrhagic fever, and Tacaribe
(TCRV)52. The disulfides and thiosulfonate (31-34) displayed 50% effective
24
concentration (EC50) values, the concentration at which 50% of the viral yield is
eliminated, ranging from 3.6 to 100 µM towards these microbes. This is at least ten times
lower than the concentrations needed to induce cytotoxic effects.
S S
Cl2PhHCOCNHN
NHNCOCHPhCl2
31
S S
HN(H2N)HCHN
NHCH(NH2)NH
32
O O
S
S
S S
N
S
S
N
HO
OH
33
34
Figure (1.31) Antiarenavirus disulfides.
A large focus of preclinical and clinical development of anti-HIV drugs is in protease
inhibition. However, other processes are certainly important to viral infectivity and
replication. Metabolic pathways of infected cells, such as precursor protein processing,
have been shown to be inhibited by a macrocyclic disulfide (35), 7-methyl-6,7,8,9tetrahydrodibenzo [c,k] [1,2,6,9]-dithiadiazacyclododecine-5,10-dione53. This compound
displays an EC50 of 0.05 µg/ml against HIV-infected macrophages, compared to the
current standard AZT (3’-azido-3’-deoxythymidine) that has an EC50 of 0.004 µg/ml.
25
With a different mode of action from AZT however, the disulfide acts synergistically
with AZT when tested in vitro, and could potentially be used in combination.
O
NH
S
S
NH
O
35
Figure (1.32) Anti-HIV macrocyclic disulfide.
Another disulfide with promising antiviral properties is 5,5’-diphenyl-3,3’-diisothiazole
disulfide (36a) (DID)54. DID induces potent inhibition of plaque-infected cells derived
from invasion of poliovirus type 1, with an IC50 of 0.35 µM. Cytoxicity to healthy human
cells was also examined, and no adverse effects were observed with uninfected cell
cultures at 50 µM concentration of DID. This agent is believed to inhibit an enzyme
associated with RNA synthesis. 50% cytotoxic concentrations (CC50), the concentrations
where normal human cell proliferation is inhibited by 50%, were more than 200 times
higher that the IC50’s, illustrating the exquisite selectivity this compound has for the viral
infected cells55. The reduced form, thiol 36b, has almost the same activity and selectivity
as 36a (Table 1.1).
S N
S
N
S
S N
S
SH
36a
36b
Figure (1.33) 5,5’-Diphenyl-3,3’-diisothiazole disulfide and thiol antipoliovirus agents.
26
Table 1.1 Isothiazole anti-poliovirus activities.
Compound
IC50 (µM)
CC50 (µM)
Selectivity
CC50/ IC50
36a
0.35
89.28
255
36b
0.42
90.75
216
HIV reverse transcription (HIV-RT) and deoxyribonucleoside triphosphate (dNTP)
synthesis are paramount to viral replication, and thus are prime inhibition targets for antiHIV therapy56. A number of sulfur-bearing nucleotide HIV-RT inhibitors, which have
similar effects to AZT, include 3’-mercapto-2’,3’-dideoxynucleotides (37) and 2’- deoxy2’-mercaptouridine-5’-diphosphate (38)57, 58. These nucleosides serve to transfer a radical
to a thiol of the transcriptase as shown in Fig 1.33.
O
O
O
P
O
O
O
P O
O
O P O
O
X
HS
37
X = Adenine, cytosine, guanine, thymine
Figure (1.34) Dideoxynucleotides thiyl radical-based reverse transcriptase inhibitors.
27
S
SHa
PPO
Ha
Base
O
Hb
OHOH
CO2
SH
PPO
H2O
Base
O
PPO
SH
Hb
O O
H
C
O
OH
C
O
H
SH S
SHa
PPO
Ha
HO
Base
O
S
O
SH
PPO
PPO
Base
O
O
Hb
O
S
S
SHa
Hb
H
Base
O
Hb
O
S
O
C
O
SHa
HO
C
O
Hb
H
OH
S
Base
C
O
S
O
H
S
S
Figure (1.35) Postulated mechanism of reduction of natural ribonucleoside 5’-
diphosphates.
A new pyrimidine nucleoside disulfide (39) has been synthesized and shown to inhibit
both HIV-RT and dNTP. The disulfide also has an EC50 of 10 µM and an IC50 of 25 µM
against proliferation of human T-lymphocyte cells. Very interestingly, the corresponding
thiol derivative had no activity at all.
O
O O
P
P
O
OO O
O
NH
O
N
O
HO
Y
38
Y = SH, SSC3H7
Figure (1.36) Mercaptouridine Thiyl Radical-based Reverse Transcriptase Inhibitors.
28
O
NH
HO
O
N
O
H3CSS
39
Figure (1.37) Nucleosidic disulfide anti-HIV agent.
1.3.4 Trisulfide Reagents
1.3.4.1 Antibacterial Trisulfides
Trisulfides found in garlic, such as diallyl trisulfide and allyl methyl trisulfide (Fig 1.38),
can act as antibacterial agents in the same way as disulfides, but with greater efficacy.
S
S
S
S
S
S
Allyl methyl trisulfide
Diallyl trisulfide
Figure (1.38) Anti-infectives from garlic
The antibacterial activity of some cyclic polysulfides like tetrathiepanes can be attributed
to a trisulfide bridge, as much as previous examples owe their activity to a disulfide
bridge48,
59
. 4-Dioxo-1,2,4,6-tetrathiepane (40), an extract from the red alga Chondria
californica, has potent antibacterial activity against Vibrio anguillarium, the causative
agent of a tropical fish disease.
29
O O
S
S
S
S
40
Figure (1.39) 4-Dioxo-1,2,4,6-tetrathiepane.
Also part of the trisulfide family of antibiotics are the enediyne trisulfide
antitumor antibiotics, calichaemycin (41a), a natural product of Micromonospora
echinospora, Namenamicin (41b), and esperamicins, natural products of Actinmadura
verrucosospora. Calichaemycin is at least a thousand times more potent than penicillin G
against S. aureus. Although these antibiotics are not currently used to treat infection
because of their elevated toxicity, in anticancer studies these compounds have been
shown to undergo an intricate cascade of intramolecular reactions initiated by glutathione
attack, resulting in an intensely reactive radical species.
Calicheamycin 41a
O
SSSCH3
O
O
O
R2 =
R1
OH
H3COHOCON
R1 = H
O
R3 = Et
I
OH
O
H
O
S
R2
OH
HN
O
HO
O
R3HN
OCH3
O
OH
O
Esperamicin 41b
OCH3
O
OMe
R1 =
R2 = Me
MeO
O
OH
O
NHCOC(CH2)OMe
30
R3 = iPr
SSSCH3
O
OH
H3COOCHN
HO
H
HO H3CS
S
O
H3C
O
O
HO
O
NH
O
OCH3
41c
Figure (1.40) Enediyne Trisulfides: Calichaemycin (41a), Esperamicin A1 (41b) and
Namenamicin (41c).
In the first step of this process, reaction of the trisulfide group with glutathione generates
a free thiolate anion which in turn undergoes a Michael addition across the α,β-saturated
ketone. This subtle change in orbital hybridization of the carbons allows the enediyne to
undergo Bergman cycloaromatization, producing the phenylene diradical60. These
diradicals are believed to cleave DNA by sequentially stripping off hydrogen atoms along
the minor grove of the double helix. Even though their mechanism of antibacterial action
has not been proven to be the same as that of the anticancer mechanism, it is likely that
the trisulfide moiety is involved as an electrophilic reactant with cellular thiols (Table
1.2).
31
O O
O O
NH
HO
S
O
OCH3
Sugar
NH
HO
HS
O
O O
OCH3
NH
HO
S
Sugar
O
O O
OCH3
NH
HO
S
Sugar
O
OCH3
Sugar
S S
DNA damaging diradical
GSH
Figure (1.41) Bioactivation of Enediynes
Table 1.2 Enediyne Antibacterial Activities
Bacteria
Bacillus subtilis
Staphylococcus aureus
Enterococcus faecium
Escherichia coli
Klebsiella pneumoniae
MIC(µg/ml)
Calichaemycin
0.00005
0.000001
0.00012
0.12
0.25
Namenamicin
0.03
0.001
0.03
0.12
0.06
Penicillin G
0.25
0.015
128
32
128
1.3.4.2 Antifungal Trisulfides
Although not currently used in clinical settings, enediyne antitumor antibiotics also
display potent antifungal activities. Candida albicans, Ustilago maydis, Saccharomyces
cerivisiae, and Neurospora crassa are all inhibited by Calicheamicin and Namenamicin
with MIC’s below 1 µg/ml61. Their mechanism of action is presumably similar to that of
their antibacterial and anticancer properties as DNA cleaving agents triggered by Sthiolation of glutathione or a related cellular thiophile.
32
Table 1.3 Enediyne Antifungal Activities
MIC (µg/ml)
Calichaemycin
0.03
0.001
0.008
0.06
Fungus
C. albicans
U. maydis
S. cerevisiae
N. crassa
Namenamicin
0.25
0.004
0.06
0.25
1.3.5 Polysulfide Reagents
1.3.5.1 Antibacterial Polysulfides
Within the Didemnidae or tunicate family, Lissoclinumi species are rich sources of
organosulfur antibiotics. One such isolate, Lissoclinotoxin A (42), demonstrated potent
growth inhibition of a number of bacteria, including S. aureus, Streptococcus faecalis,
Cirrobacter species, Klebsiella species, E. coli, Enterobacter species, Serratia species,
Salmonella species, Pseudomonas aeruginosa, Acinetobacter, and Proteus species62.
HO
OCH3
S S
S
S S
NH2
42
Figure (1.42) Lissoclinotoxin A.
1.3.5.2 Antifungal Polysulfides
Lissoclinotoxins A (42) and D (43) (disulfide analog), pyridoacridine alkaloids from
ascidians, both display potent antifungal activities against Candida albicans and
33
Trichosporon mentagrophytes, with cell-mediated immunity levels (CMI) of 40 and 20
µg/ml, respectively.
NH2
HO
OCH3
S S
S S
OH
OCH3
NH2
43
Figure (1.43) Lissoclinotoxin D.
1.3.5.3 Antiparasitic Polysulfides
Another anti-malarial agent, Lissoclinotoxin A (42) has also demonstrated potent activity
towards a resistant strain of the parasite Plasmodium falciparum62. Lissoclinotoxin A is
intermediate in activity compared to the usual antimalarials quinine, mefloquine,
halofantrine and chloroquine, with an IC50 of 296 nM (Table 1.4). Although not yet
defined conclusively, its mechanism is likely similar to that described for di- and
trisulfide antibacterials.
Table 1.4 Anti-malarial activities of Lissoclinotoxin A and a few clinical standards.
IC50 (nM)
Anti-Malaria Agent
Lissoclinotoxin A (42)
Quinine
Mefloquine
Halofantrine
Chloroquine
296
350
40
2
580
34
1.3.6 Other Organosulfur agents
1.3.6.1 N-Thiolated Antibacterials
N-Sulfenylated monocyclic β-lactams (44) are another class of sulfur-containing
antibacterial compounds recently discovered to have an unusual mode of action, where
the N-S functionality may interact with cellular thiols in the same fashion as a disulfide63.
Regardless of the presence of a β-lactam ring, the mode of action of these N-thiolated
compounds64 is totally different to that of the penicillins and other beta-lactam drugs,
which act as cell wall biosynthesis inhibitors.
O
N
R1 O
44
S R
3
R2
Figure (1.44) N-Sulfenylated-β-Lactam Antibacterials
It is believed that an intracellular thiol in susceptible bacteria, coenzyme A, attacks the
sulfur atom to form a mixed disulfide which in turn causes inhibition of bacterial growth
by blocking lipid biosynthesis. These thiolated lactams show a narrow range of
antibacterial properties, including Staphylococcus strains such as MRSA and S.
epidermidis, with MIC’s as low as 0.125 µg/ml.
35
N
N
NH2
N
HO
N
P
O
R2
O
HO
R3 S
O
HO
O R
1
N
O
OH
N
O
HO
N
P
O
O
O
O O
P
P
HO O
OH
NH2
N
OH
O
O O
P
O P
HO
OH
O
H
N
HN
O
NH
O
HO
N
O
R1 O
R2
O
HO
H
N
HN
SH
S
S
R3
O
O
Figure (1.45) Formation of CoA – β-lactam mixed disulfide.
Another sulfenamide, 1,2-benzoisothiazolin-3-one (BIT) (45), has shown weak activity
against Staphylococcus aureus, with an MIC around 100 µg/ml [57].
O
NH
S
45
Figure (1.46) BIT.
BIT has been shown to inhibit the action of a number of intracellular thiols such as
glutathione and ATPase. The mode of action has been linked to an inhibitory effect on
cellular respiration upon metabolic uptake. Lansoprazole (46), a drug designed as a
gastric acid pump inhibitor, has been shown to rearrange in the acidic environment of the
stomach to a sulfenamide (47), which is an inhibitor of Helicobacter pylori 65. H. pylori is
considered to be a main culprit in the cause of gastric ulcers and therefore lansoprazole
serves double duty as an antibiotic and an acid production reducer. Even though an
intermediate sulfenic acid derivative of lansoprazole also displays anti-pylori activity, the
sulfinamide affords fast action with an MIC value of 10 µg/ml.
36
F
F
F
F
F
F
H
N
N
N S
N
N S
N
O
47
46
Figure (1.47) Lansoprazole and in vivo Sulfenamide.
1.4
Conclusions
This chapter discusses the various classes of organosulfur compounds which have shown
efficient antimicrobial activities against bacteria, viruses, fungi and various parasites.
Most of these compounds are either made by Nature or inspired from them. The mode of
action of the majority of these organosulfur compounds has not been defined and requires
further investigation. One of the rich sources of natural organosulfur compounds is
Allium sativum (garlic), which has potent antibacterial and antifungal activity. A major
inevitable problem with bacteria is the development of antibiotic resistance. The scope of
these organosulfurs as clinical anti-infectives could possibly be improved if they could be
functionalized innovatively to circumvent the resistance mechanisms or act in multimodal ways.
37
CHAPTER TWO
SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF S, S’HETEROSUBSTITUTED DISULFIDES
2.1 Introduction
The concern over multidrug-resistant bacteria and the need for more effective
antibacterials has reached a critical level, given that successful treatment of common
bacterial infections can no longer be taken for granted. Staphylococcus aureus (Staph), a
gram-positive facultative anaerobe, resides in the nostrils and on the skin of human
beings. S. aureus starts to cause infection once it enters the blood stream. Antibioticresistant variants of S. aureus, mainly methicillin-resistant Staphylococcus aureus
(MRSA), is the single most important pathogen causing infections. The mutants from the
community, referred to as community-acquired MRSA (CA-MRSA), are known to be
more lethal and complex in nature than the variants originating in hospitals, which are
referred to as hospital-acquired MRSA (HA-MRSA). MRSA is known to cause infections
of skin and soft tissue66, as well as pulmonary,67 osteoarticular68, hemorrhagic adrenal
gland (Waterhouse-Friderichsen syndrome69) and opthalmic infections70. According to
the United States Centers for Disease Control and Prevention (CDC), MRSA accounted
for more than 94,000 life-threatening infections and nearly 19,000 deaths in the United
38
States alone in 200571. CDC also emphasizes that the prevalence of multi-drug resistant
bacteria is rising at an alarming rate.
The Turos laboratory has been studying the effects of a variety of synthetic
antibacterial agents against common microbes. Previously we have investigated Nthiolated β-lactams64, 72-77, N-thiolated 2-oxazolidinones78 and recently, a novel family of
aryl-alkyl disulfides,46 are which all effective growth inhibitors of MRSA. The mode of
action and structure–activity profiles of all these substances differ dramatically from
those of other analogs of β-lactams, oxazolidinones, and disulfides. Investigations in our
laboratory have shown that these compounds can each carry a wide range of substituents,
and are reactive towards thiophilic agents under certain conditions (such as in bacterial
cytoplasm).
O
N
R1O
O
SMe
R2
N-Thiolated β-lactams
S
N SMe
O
R1
R2
N-Thiolated oxazolidinones
SR2
R1
Aryl-alkyl disulfides
Figure (2.1) Various antibacterials synthesized in the Turos lab.
The aryl–alkyl disulfides were looked at previously in the Turos lab by Dr. Kevin Revell,
and found to have strong in vitro antimicrobial properties against S. aureus, MRSA and
B. anthracis. The nitrophenyl alkyl disulfides exhibited the most in vitro potency.
39
SH
RSSO2Me
X
S SR
X
MeOH
X - F, CH3, OCH2CH3, CH2OH, NHAc, NH2, NO2
R - Me, Et, nPr, iPr, nBu, s-Bu
Figure (2.2) Various aryl-alkyl disulfides synthesized in the Turos lab.
The fact that the in vitro microbiological properties of these disulfides, determined from
agar diffusion and agar MIC measurements, coincides with the FabH inhibition
capabilities points to the likelihood that this key bacterial enzyme is associated with the
biological properties of the compounds. More detailed studies into this are now being
done in collaboration with Dr. Lindsey Shaw’s laboratory in the USF Biology
department.
This chapter describes experiments on a series of new sulfur-containing
antibacterials, S,S’-heterosubstituted disulfides. In this study, we investigated the
synthesis and antibacterial and antifungal properties of these structurally intriguing
compounds.
2.2 Synthesis
These investigations were initiated by the synthesis of a group of S,S’-heterosubstituted
disulfides shown in figure 2.2. These compounds were chosen as a means to assess the
electronic effects of different heteroatoms on bioactivity. These derivatives were
prepared by treating an alcohol, amine or thiol with sulfur monochloride at -20 °C in the
40
presence of triethylamine as a Lewis base (Fig 2.3). The desired disulfide products were
obtained in 70-90% yields after column chromatography, and characterized by 1H NMR
spectroscopy.
Cl
S
Cl
XR
RXH
S
S
CH2Cl2
Base
-20 oC - rt
( X = O, N, S )
Figure (2.3) Synthesis of S,S’-heterosubstituted disulfides.
41
RX
S
S
S
2
O
S
2b
S
2
H
3a
N S
4a
2
S
2a
N
S
2
N
H
N S
4b
2
S
N
2
H
3c
2
S
S
2
2d
S
N S
1e
S
2c
3b
2
1d
S
4c
2
2e
S
N
2
3d
2
2
O
2
O
1c
S
2
S
S
S
2
O
1b
S
H
2
O
1a
S
S 2
N
4d
H
S
N
2
3e
S 2
N
4e
Figure (2.4) Synthesized S,S’-heterosubstituted disulfides.
2.3 Elucidation of 1H NMR spectrum of di sec-butoxy disulfide (1d):
The structure of the disulfide products 1-4 were determined by 1H NMR spectroscopy.
The 1H NMR spectrum of di-sec-butoxy disulfide (1d) is illustrated below as a
representative example (Fig 2.5). Apart from the obvious lack of the alcohol O-H peak in
1
H NMR spectrum (from the starting alcohol), the chemical shifts of the disulfide protons
42
are slightly downfield as compared to those of sec-butanol. This can be attributed to the
presence of the oxygen-sulfur bond which deshields the neighbouring protons more than
in the case of the alcohol, due to the electronegativity of the O-S bond.
Figure (2.5) 1HNMR spectra of sec-butanol and di sec-butoxy disulfide (1d).
The protons of carbon-4 of butanol (marked in red) have a chemical shift of 0.70-0.76
ppm and appear as a clearly-defined triplet whereas in the disulfide they appear as an
43
unresolved multiplet at 0.83-0.91 ppm. The C-1 methyl protons appear as a doublet at
1.17-1.23 ppm in case of the disulfide and at 0.97-0.99 ppm for the alcohol. The
methylene protons show up at 1.46-1.61 ppm as a multiplet for the disulfide and at 1.221.31 ppm in case of the alcohol. And finally methine (–CH) proton appears at 3.77-3.86
ppm for the disulfide and at 3.49-3.56 ppm in the alcohol. Similar trend was observed in
rest of the disulfides.
2.4. Reactivity of S,S’-heterosubstituted disulfides
The reactivity of S,S’-heterosubstituted disulfides has been previously explored
by Motoki79 etal. Dialkoxy disulfides reportedly react with mercaptans or secondary
amines (Fig 2.6) at reflux conditions in carbon tetrachloride to give alkoxy alkyl
trisulfides (2) or alkoxy amino disulfides (3) with elimination of alcohol. The yields are
typically below 50% for reactions with thiols, and 22-74% for amines.
R'SH 50 °C
ROSSSR'
2
CCl4 3hrs
R"
HN
R"
ROSSOR
1
22% - 50%
CCl4 4hrs
Reflux
R
O
S
S
R"
N
R"
3
22% - 74%
Figure (2.6) Reactivity of dialkoxy disulfides with thiols and secondary amines.
These alkoxy alkyl trisulfides (2) further react with mercaptans or secondary amines at
refluxing conditions in carbon tetrachloride to give unsymmetrical dialkyl tetrasulfides
(4) and alkylamino trisulfides (5) in varying yields.
44
R'
S
S
S
R"
N
R"
5
R"
HN
R"
R"SH
R"SSSSR'
ROSSSR'
CCl4 7hrs
Reflux
CCl4 2hrs
Reflux
2
4
60-63%
60-73%
Figure (2.7) Reactivity of alkoxy trisulfides (2) with thiols and secondary amines.
The alkoxy diamino disulfides (3) also react with thiols and amines in the similar fashion
(Fig 2.8) to yield alkylamino trisulfides (5) and unsymmetrical diamino disulfide (6),
respectively.
R'
S
S
5
S
R"
N
R"
R'SH
CCl4 7hrs
Reflux
R
O
S
S
3
67%
R"
N
R"
HN
R'
R'
CCl4 2hrs
Reflux
R'
R'
N
S
S
R"
N
R"
6
37%
Figure (2.8) Reactivity of alkoxy amino disulfides (3) with thiols and secondary amines.
45
Motoki found that when diethoxy disulfide was reacted with 1,1-disubstituted
thioureas, thiadiazoles (7) were obtained, whereas 1,3-disubstituted thioureas yielded
carbodiimides (9) in moderate yields.80
S
S
R1
R2N C NR1
9
N
H
N
H
R1
R2
CH2Cl2 2 - 23 hrs
N
R1
R1
ROSSOR
CCl4 6 - 11 hrs
1
N S
NH2
N
R1
N
R1
N
R1
R1
7
N
R1
8
CN
Reflux
Reflux
0 - 5%
48 - 75%
19 - 66%
Figure (2.9) Reactivity of dialkoxy disulfides (1) with 1,1 and 1,3- disubstituted
thioureas.
Motoki etal. also studied the reactions of diethoxy disulfide with various arylhydrazines
(Fig 2.10). Diethoxy disulfide on refluxing with arylhydrazines in benzene loses ethanol
and yields aryl ethoxy tetrasulfides (10), aryl benzenes and diaryl sulfides.81
ArNHNH2
ROSSOR
Ar
S
S
PhH 10 - 30 hrs
1
S
S
OEt
ArC6H5
Ar
S
Ar
10
Reflux
Figure (2.10) Reactivity of dialkoxy disulfide (1) with monosubstituted hydrazine.
All these compounds were purified either by recrystallization or column chromatography
and characterized by elemental analysis and 1H NMR spectroscopy. A detailed look at the
various reaction conditions can be seen in table 2.1.
46
Table (2.1) Reaction of diethoxy disulfide with monosubstituted hydrazine.
Hydrazine
(ArNHNH2)
Benzene
refluxing time
(hrs)
Products- Isolated yields (%)
Ar-C6H5
Ar-S4-OEt
Ar-Sn-Ar
Ar
4-NO2C6H4
10
21
29
13 (n=2)
2-Cl-C6H4
30
27
10
48 (n=4)
4-BrC6H4
20
0
5
39 (n=4)
C6H5
12
8
0
37 (n=4)
4-CH3C6H5
15
20
0
31 (n=4)
2-C10H7
15
5
0
47 (n=4)
2.5. Antimicrobial activity
The antimicrobial activity of these S,S’-heterosubstituted disulfide compounds 15 (Fig 2.2) was evaluated against a selection of available common bacteria by
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.
2.5.1. Determination of the minimum inhibitory concentrations of disulfides against
bacteria
According to the National Committee for Clinical Laboratory Standards
(NCCLS), the MIC is the lowest concentration of antimicrobial agent that completely
inhibits visible growth of the organism as detected by the unaided eye. The minimum
47
inhibitory concentrations of the S,S`-heterosubstituted disulfides were evaluated against
Staphylococcus aureus (S.A - ATCC 25923) and a methicillin-resistant strain of
Staphylococcus aureus (MRSA – ATCC 43300) by Danielle Gergeres in Dr. Turos’ lab
by agar dilution using a 24-well plate. This was done by allowing the bacteria to grow for
24 hours in presence of varying concentrations of antibiotic. All the antimicrobial assays
were performed in triplicate. Penicillin G was used as a positive control and DMSO as
negative control in all these assays. The averaged MIC values of the S,S`-dialkoxy
disulfides are shown in table 2.2. For this series, antibacterial activity improved with the
increase in the chain length, with exception for the sec-butoxy derivative. The most
active of the five analogs is the isopropoxy compound (1b).
Table (2.2) MIC’s of S,S’-dialkoxy substituted disulfides.
OR
S
S
RO
Compound
R
S.A
MRSA
(µg/mL)
1a
propyl
32
32
1b
isopropyl
1
0.5
1c
butyl
0.25
8
1d
s-butyl
16
8
1e
phenyl
2
2
The activity among the thiol substituted disulfides (Table 2.3) decreases with increase in
the alkyl chain branching. Surprisingly these compounds were not as active even with the
48
presence of four sulfur atoms, indicating that the number of sulfur atoms is not directly
related to the antibacterial activity. The phenyl substituent (2e) was found to provide the
greatest potency among the five R substituents examined. In general, the activity
decreased with the increase in the hydrophobic nature of the chain. In case of the S,S’diamino disulfides (3a-e), those made from primary amines (Table 2.4) have weaker
activity than those of secondary amines (Table 2.5).
Table (2.3) MIC’s of S,S’-dithio substituted disulfides.
SR
S
S
RS
Compound
R
S.A
MRSA
(µg/mL)
2a
propyl
32
32
2b
isopropyl
32
32
2c
butyl
64
32
2d
s-butyl
32
64
2e
phenyl
8
8
49
Table (2.4) MIC’s of S,S’-diamino substituted disulfides.
R
N H
S
S
R N
H
Compound
R
S.A
MRSA
(µg/mL)
3a
propyl
8
4
3b
isopropyl
32
32
3c
butyl
8
32
3d
s-butyl
16
16
3e
phenyl
4
4
For the S,S’-diamino substituted disulfides also, the most active compound was the
phenyl substituted one, followed by the propyl. Compound 4a, synthesized from the
dimethylamine, showed potent activity among the secondary amine disulfides. Increase in
the chain length and branching only resulted in the loss of activity in this case. In
comparision the S,S’-diamino disubstituted disulfides had a smaller range of MIC values
than the other analogues.
50
Table (2.5) MIC’s of S,S’-diamino disubstituted disulfides.
R
N R
S
S
R N
R
Compound
R
S.A
MRSA
(µg/mL)
4a
methyl
0.25
0.5
4b
ethyl
2
2
4c
isopropyl
2
1
4d
allyl
1
0.5
4e
isobutyl
16
16
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. It
was observed that the activities of the chiral disulfide analogs 5 & 6 (Table 2.6) were not
much different from their racemic counterparts. This was observed in case of sec-butyl
and menthyl disulfide analogs 5a-g. Starting materials for compounds 5c, 5d and 5g were
racemic mixture of diastereomers, while the rest were optically pure.
51
Chiral analogs:
O
O
O
S
S 2
O
S
2
S
2
2
5a
5b
O
5c
S
O
2
5e
5d
S
O
2
5f
S
S
2
5g
S 2
2
N
N
6a
6b
Figure (2.11) Synthesized chiral S, S’- diheterosubstituted disulfides.
Table (2.6) MIC’s of Chiral disulfides 5&6.
Chiral Substrates Compound
R
S.A
MRSA
(µg/mL)
Alcohols
5a
(S) - s-butyl
16
32
5b
(R) - s-butyl
16
32
5c
1-phenylpropyl
16
32
5d
2-phenylpropyl
16
16
5e
(1S, 2R, 5S)-(+)-menthyl
8
8
52
5f
L(-)-menthyl
8
16
5g
(+)-menthyl
8
16
6a
(R)-benzyl-(1-phenylethyl)
16
64
6b
(S)-benzyl-(1-phenylethyl)
16
64
Amines
2.5.2 Bacterial Viability Assay
A bacterial viability assay was carried out by Sonja Dickey in Dr. Daniel Lim’s lab in the
Department of Biology, University of South Florida, to further assess the antibacterial
activity of the most active compound, diisopropoxy disulfide (1b). This assay was
conducted by counting the viable bacterial cells before and after incubation. The initial
plate count in all tubes was 105 cfu/ml. Staphylococcus aureus 25923 and MRSA 43300
used as test microbes showed a 3-log increase in the number of bacteria in the absence of
the disulfide. Diisopropoxy disulfide (1b) was tested against S. aureus 25923 and MRSA
43300 in concentrations of 2 and 4 µg/ml. In the tubes with 2 µg/ml disulfide, S. aureus
25923 was decreased in number of cells by one log, but still not all were killed. In the
MRSA 43300 2 µg/ml tube, it appears that the cell number increased by one log. In fact
there were too many cells (>1200 cfu/ml) to count on those plates; and since plate counts
are considered accurate only when there are 30 – 300 cells on the plate, this cannot be
considered an accurate count. In the tubes with 4 µg/ml of disulfide, the number of cells
decreased by 2 logs in both S. aureus 25923 and MRSA 43300. While there was a
significant decrease in the number of cells, not all were killed. Even with the growth in
53
the 2 µg/ml MRSA tube, it is still less by 2 logs than the control. Also, the minimum
inhibitory concentration of diisopropoxy disulfide (1b) is 2 µg/ml since there was no
visible growth of bacteria in the broth until the concentration of disulfide was lowered to
1 µg/ml. These factors point towards a bacteriostatic nature of the compound.
Table (2.7) Bacterial viability assay.
Drug Concentration
(µg/mL)
S.aureus 25923
MRSA 43300
Before
Incubation
(cfu/mL)
After Incubation
(cfu/mL)
2.11 x 105
7.6 x 108
4
2.22 x 105
2.04 x 103
2
1 x 105
3.86 x 104
Control (no drug)
3.09 x 105
4.02 x 108
4
2.96 x 105
2.88 x 103
2
1 x 105
>1.2 x 106
Control (no drug)
2.5.3 Anti-staphylococcal assay by Kirby-Bauer testing on agar plates
In addition to evaluating the biological activity by Kirby-Bauer diffusion assay,
the effect of various additives on the bioactivity of the dialkoxy disulfides was also
investigated. The intent was to select one of the most bioactive S,S’-heterosubstituted
disulfide compound, diisopropoxy disulfide, as a representative for all the testing. Two
more groups of antibacterials, N-thiolated β-lactams and alkyl-aryl disulfides (Figure
2.12) previously synthesized in Turos’ lab, were also tested simultaneously. All the
54
experiments were performed against MRSA (ATCC 43300) in Mueller-Hinton agar in
duplicates. 10µg of 1-isopropyldisulfanyl-4-nitrobenzene, 20µg of acrylic acid 2-(2chlorophenyl)-1-methylsulfanyl-4-oxo-azetidin-3-yl ester, 50µg of diisopropoxydisulfide
(1b) and 10µg of penicillin G were used for the assay. All the assays were performed by
agar diffusion with 1mg of additive in the centre well. These amounts were considered
after optimization to obtain comparable zones of growth inhibition for easier comparison.
S
O S
S
S O
O2N
Diisopropoxy disulfide
O
H
N
S
N
O
1-isopropyldisulfanyl-4-nitrobenzene
O
Cl
S
N
O
O
O
Acrylic acid 2-(2-chlorophenyl)-1-methyl
sulfanyl-4-oxo-azetidin-3-yl ester
Penicillin G potassium salt
Figure (2.12) Compounds tested by Kirby-Bauer diffusion assay.
55
OK
O
S
SMe
N
O
S
Cl
O
O 2N
DMSO
H
N
O
S
N
O
O
S
OK
O
O
S
Figure (2.13) Kirby-Bauer Assay showing no growth inhibition by DMSO.
The anti-MRSA activity of diisopropoxy disulfide was neutralized by the presence of a
free thiol diffusing away from the centre well. This cancelling effect was also observed in
case of the aryl disulfide and beta-lactam. However penicillin G salt had clear zones with
no inhibition observed. This inhibition was further confirmed by the absence of zones
around the wells containing both disulfide and glutathione (GSH) or coenzyme A (CoA).
Since all the compounds were tested as a solution in DMSO, another assay was
performed to make sure the solvent did not show any inhibitory activity by itself. As
expected, DMSO did not induce, nor diminish, activity of the compounds as it diffuses
outward from the center well (Figure 2.13).
In assays with compounds containing a free thiol group such as coenzyme A (CoA)
glutathione (GSH) and cysteine (Cys) the antibacterial activity of the compounds was
completely cancelled (Figure 2.14). This was evident from the indented regions of growth
inhibition after 24 hours of incubation.
56
O
S
O
S
SMe
N
S
Cl
Coenzyme A
Cysteine
H
N
O
O
S
H
N
N
O OK
S
O
S
O
O
O
N
O
S
S
S
S
O
O OK
O
S
SMe
N Cl
O
O2N
O
O2N
O
O
S
SMe
N
O
Cl
O
O2N
Glutathione
H
N
O
O
S
N
O
O OK
S
S
O
Figure (2.14) Kirby-Bauer assay showing the ability of CoA, GSH and cysteine to inhibit
antimicrobial effects of the test compounds on MRSA.
N
NH2
N
O
OH
O
OH
H2N
SH
N
OH O
P
O OH
N
H
N
HO
O
O
O O
P
P
O
OH
OH
NH2 O
O
O
SH
N H
O
HO
O
HO
H
N
HN
Cysteine
SH
O
Coenzyme A
Figure (2.15) Structures of cysteine, coenzyme A and glutathione.
57
Glutathione
Other additives that were tested as controls included glycine, lysine, valine, isopropyl
disulfide and triphenylphosphine. There was no inhibition observed in any of these cases.
This can be attributed to the absence of free thiol (Figures 2.16 & 2.17). In case of
triphenylphosphine a small zone was observed around its well indicating antimicrobial
activity. The activity may be due to its ability to reduce disulfides responsible for the
redox equilibrium in the bacteria.
O
S
O
S
O
SMe
N
Cl
S
O
O2N
SMe
N
O
Cl
O
S
O2N
iPrDS
H
N
O
O
PPh3
H
N
S
O
N
O OK
S
O
O
O
S
S
N
O OK
S
O
O
S
Figure (2.16) Kirby-Bauer assay showing no cancellation effect of diisopropyl disulfide
by isopropyl disulfide or triphenylphosphine.
58
O
O
S
SMe
N
O
S
S
Cl
O
O2N
S
SMe
N
O
Cl
O
O2N
Lysine
Valine
H
N
H
N
O
O
S
O
S
N
O
N
S
O
O OK
O OK
O
S
O
S
S
O
S
SMe
N
O
S
O
Cl
O
O2N
Glycine
H
N
O
O
S
S
N
O
O
S
O OK
Figure (2.17) Kirby-Bauer Assay showing no inhibition effect from the amino acids
valine, lysine and glycine.
2.6 Studies of the mode of action of S,S’-heterosubstituted disulfides
It can be inferred from the Kirby-Bauer experiments that the interaction between the free
thiol group of the additives and the S,S’-heterosubstituted disulfides is responsible for the
inhibition of the biological activity.
In order to determine the exact concentrations of the coenzyme A and glutathione at
which they can completely inhibit the activity of S,S’-heterosubstituted disulfides,
59
another Kirby-Bauer assay was performed. In this assay both the disulfide and the
additive (coenzyme A or glutathione) were added in the same well, using varying
amounts of the additive, while maintaining a constant initial amount of disulfide.
Thus, disulfide 1b was added along with 0.25, 0.5, 0.75 and 1 molar equivalent of
glutathione into different wells cut into the agar in a Petri plate with MRSA (Fig 2.18)
and incubated for 24 hours at 37 °C. A parallel assay was also done using coenzyme A in
place of glutathione. There were no zones observed around any of the wells, indicating
that the concentration of glutathione and coenzyme A required to inhibit the activity of
compound 1b was less than 0.25 molar equivalents.
1 : 0.25
1 : 0.5
1:1
1 : 0.75
Figure (2.18) Kirby-Bauer assay with compound 1b and glutathione in different molar
ratios in the same well.
To explore more on the type of conjugates formed, compound 1b was reacted directly
with coenzyme A in phosphate buffer solution (pH = ~ 7) at 37 °C. HPLC traces did not
60
show any evidence of formation of a new adduct even after 24 hours. A similar reaction
was also monitored in an NMR tube. Compound 1b was co-mixed with equimolar
amount of propane-2-thiol at room temperature and heated to 50 °C for 24 hours. This
experiment also did not show any change in the starting materials. This finding indicates
something interesting, in that although the mixture was heated to 50 °C, there was no
apparent reaction between the disulfide and the thiol; however, there must be a reaction
occurring between the disulfide and thiol additive in the well (or agar) of the Petri plates
at 37 °C, since the disulfide completely loses its bioactivity. This suggests the possibility
of bacterial enzymes influencing the formation of the biologically inactive conjugates,
perhaps via a thiol transfer process.
Figure (2.19) 1HNMR tube experiment of diisopropoxy disulfide and propane-2-thiol in
CDCl3.
61
In order to explore the possible mode of action, an experiment was conducted to check if
the disulfides were interacting with the formation of the bacterial cell wall.
wall The MRSA
cells were observed
bserved under scanning electron microscope (SEM) before and after the
treatment with diisopropoxy disulfide. It was observed that there was no change in their
external morphology. The cell wall looked intact
intact, indicating that the compound did not
disrupt or interact with bacterial transpeptidases or any cell wall
wall-related
related proteins. If the
compounds were to interact with these components,, the spherical structure of the cells
would most likely have been disrupted as what happens in the case of penicillin G.
Figure (2.20) Scanning electron microscopy pictures of MRSA cells before and after
treatment with diisopropoxy
isopropoxy disulfide.
2.7 Activity of S,S’-heterosubstituted
eterosubstituted disulfides against other microbes
icrobes
To assess the range of antimicrobial activity, S,S’
S,S’-heterosubstituted
heterosubstituted disulfides were also
evaluated against various other bacteria ((Francisella
Francisella tulerensis, Bartonella sp.,
Escherichia coli)) and fungus ((Candida albicans). The testing of Francisella and
Bartonella was carried out by John Thomas in the biosafety level (BSL) 3 labs of Dr.
Burt Anderson at the College of Medicine, University of South Florida.
62
2.8 Anti-Francisella activity
Francisella tularensis is a pathogenic species of gram-negative bacteria and the causative
agent of tularemia, also known as rabbit fever or “cat scratch fever”. Symptoms of
tularemia include fever, lethargy, anorexia and signs of septicemia, and in extreme cases
can cause death. As low as 10-50 cfu82 (colony-forming units) are sufficient to cause an
infection. Due to its high pathogenicity and ease of spread by aerosol they were and still
are considered for usage in biological warfare83,84. Hence F.tularensis is classified as a
Class A agent by the U.S. government. Class A agents have a moderate to high likelihood
for large-scale dissemination or a heightened general awareness that could cause mass
fear and civil disruption. F. tularensis is susceptible to carbapenems, ceftriazone,
ceftazidime, rifampin and certain macrolides, but a lack of clinical data to recommend
any of these compounds for clinical use85. Moreover, there is broad resistance to
erythromycin among strains of F. tularensis subspecies holarctica86. Ciprofloxacin is the
only drug that is considered ideal for the treatment of tularemia and it may not be long
before antibiotic resistance mechanisms initiate. In addition, very little is known about
the molecular basis of this pathogen82. The availability of the genome-sequence will help
better understand this organism. So there is a need for new antibacterial agents and
protocols for treating these infections.
The minimum inhibitory concentrations (MIC) of the 29 S,S`-heterosubstituted disulfides
1-6 were evaluated against a live vaccine strain (LVS) of Francisella tulerensis (F.T) by
broth dilution (Table 2.8).
63
Table (2.8) MIC’s of S,S’-heterosubstituted disulfides against Francisella tularensis.
Substrate
Compound
R
Francisella tularensis
MIC (µg/mL)
S,S`- dialkoxy substituted disulfides
1a
propyl
16
1b
isopropyl
4
1c
butyl
4
1d
s-butyl
16
1e
phenyl
1
2a
propyl
1
2b
isopropyl
8
2c
butyl
4
2d
s-butyl
16
2e
phenyl
2
3a
propyl
16
3b
isopropyl
2
3c
butyl
4
3d
s-butyl
8
3e
phenyl
0.5
4a
methyl
8
4b
ethyl
16
S,S`- dithio substituted disulfides
S,S`- diamino substituted disulfides
S,S`- diamino disubstituted disulfides
64
4c
isopropyl
32
4d
allyl
16
4e
isobutyl
1
5a
(S) - s-butyl
16
5b
(R) - s-butyl
16
5c
1-phenylpropyl
16
5d
2-phenylpropyl
8
5e
(D)-menthyl
4
5f
(L)-menthyl
4
5g
(+)-menthyl
4
6a
(R)-benzyl-(1-phenylethyl)
16
6b
(S)-benzyl-(1-phenylethyl)
16
Chiral S,S’-heterosubstituted disulfides
Chiral amines
Control
Ciprofloxacin
0.125
There was no obvious structure-activity relationship among these 29 analogues, but some
of the compounds showed good activities with MIC’s as low as 0.5 µg/ml. Bisdiaminophenyl disulfide (3e) was observed to be the most active compound.
2.9 Anti-Bartonella activity:
Similarly, a selection of disulfides were tested against various species of Bartonella,
namely B. henselae (houston-1), B. quintana (U-mass), B. henselae Marseille and B.
65
henselae (SA-1) by disk diffusion assay. Rifampicin was used as a positive control along
with DMSO, which was used to solubulize the compounds.
Table (2.9) Zone of inhibition data of S,S’-heterosubstituted disulfides against Bartonella
sp.
Compound
B.henselae
B. Quintana
B. henselae
B. henselae
Houston-1
U-mass
Marseille
SA-1
Zone of inhibition (mm)
S,S`- dialkoxy substituted disulfides
1a
0
0
0
0
1b
12
25
12
11
1c
0
0
0
0
1d
0
0
0
0
1e
0
0
0
0
S,S`- dithio substituted disulfides
2a
0
12
10
0
2b
0
0
10
0
2d
0
0
8
0
2e
17
0
17
8
Rifampin
62
53
22
68
DMSO
0
0
0
0
Surprisingly, most of the disulfides were completely inactive (Table 2.9). Only
compound 1b, diisopropoxy disulfide, which was the most active compound against
66
Staphylococcus aureus, showed a small zone of inhibition. S,S’-heterosubstituted
disulfides were also tested against Escherichia coli and were found to be inactive. MIC’s
of all the disulfides were greater than 128 µg/ml.
2.10 Anti-fungal activity
Given that, previously, N-thiolated β-lactams were found to have antifungal properties87,
we decided to examine the disulfides against Candida albicans, one of the most common
human commensal organisms that lives in the mouth and gastrointestinal tract. It is
present in about 80%88 of healthy individuals. Entry of these fungal cells in the blood
stream causes candidiasis, infections which vary from superficial to systemic, and are
often life threatening. Invasive candidiasis89 is very prevalent in intensive care units and
is a major cause of mortality in 33-47% of immunocompromised individuals in hospitals.
Reduced susceptibility of Candida species towards the commonly used antifungal agents
has raised concerns over a need for effective antifungal agents.
Fungal susceptibility testing of diisopropoxy disulfide (1b) against C. albicans was done,
by Sonja Dickey in Dr. Daniel Lim’s lab in the USF Biology department, according to
NCCLS document M27-A2 in Yeast Nitrogen Broth (YNB) using 1 µg/ml, 0.5 µg/ml,
0.25 µg/ml, and 0.125 µg/ml concentrations of diisopropoxy disulfide (1b). 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 (PBS) that was heat killed (boiled for 5 minutes). The MIC of
diisopropoxy disulfide (1b) towards C. albicans was found to be 0.5 µg/ml.
67
2.10.1 Trypan Blue Staining:
To further assess the antifungal activity of the most active disulfide compound,
diisopropoxy disulfide (1b), a cell staining assay was carried out by Sonja Dickey in Dr.
Daniel Lim’s lab in the Department of Biology, University of South Florida,. This
staining procedure is based on the observation that viable fungal cells do not take up the
trypan blue dye while the non-viable cells do, and indicate cell viability in the presence of
the drug.
HO3S
N
SO3H
SO3H
N
N
HO
N
H2 N
HO3S
OH
NH2
Figure (2.21) Trypan Blue
Trypan staining assay images (Figures 2.22 & 2.23) of C.albicans treated with
diisopropoxy disulfide show that only the cells in the heat-killed control absorb the dye.
None of the cells from the susceptibility test or the viable control from the yeast nutrient
broth (YNB) stained blue, indicating that diisopropoxy disulfide is fungistatic and not
fungicidal.
68
Figure (2.22) Trypan Blue staining showing live fungal cells.
Figure (2.23) Trypan Blue staining of heat killed control showing dead fungal cells.
69
2.11 Efforts towards prodrug/dual action drug synthesis
The advent of multi-drug resistant microbes has made it very difficult to treat the diseases
caused by them. A way to circumvent this would be by means of dual-action drug
approach. In this approach two drugs with different modes of action are connected via a
cleavable covalent linkage and introduced into the microbial cell. Upon entering the cell
the linkage is broken enzymatically and releases both the drugs. These drugs target
different biological pathways and so have a better chance to inhibit the microbe than in
case of a drug with a single target.
In our efforts to make disulfide-based dual-action antibiotics, we unsuccessfully tried to
couple disulfide 1b with 4-(2-chlorophenyl)-3-methoxyazetidin-2-one, ciprofloxacin and
penicillin G (Fig 2.24). All these reactions were tried varying the solvents
(dichloromethane, benzene), bases (triethylamine, diisopropylamine and pyridine) and
temperatures (0 – 50 °C). Ciprofloxacin and penicillin G were used from commercially
available
sources.
4-(2-chlorophenyl)-3-methoxyazetidin-2-one
according to the previously published protocol in Dr. Turos’ lab.
70
was
synthesized
O
S S
O
O
O
NH
X
Cl
MeO
N
S O
Cl
MeO
4-(2-Chlorophenyl)-3-methoxyazetidin-2-one
O
O
O
F
HO
N
O
O
S S
X
N
F
HO
O
N
N
N
NH
S
O
ciprofloxacin
O
H
N
O
O
S
N
S S
O
S
X
N
O
O
O
OH
S
N
O
O
OH
penicillin G
Figure (2.24) Attempted coupling reactions of diisopropoxy disulfide.
Apart from these, the synthesis of disulfide linked ciprofloxacin and penicillin dimers
prepared from sulfur monochloride were also attempted (Fig 2.25). These results also
were not encouraging. The reactions did not yield any new products at ambient
temperature and decomposed at elevated temperatures (50 °C).
71
O
O
O
F
HO
N
S2Cl2 Et3N
O
F
HO
X
N
NH
N
N
N
S
2
CH2Cl2
ciprofloxacin
S2Cl2 Et3N
H
N
O
X
S
N
O
O
S
N
O
S
N
O
CH2Cl2
OH
2
OH
O
penicillin G
Figure (2.25) Attempted synthesis of ciprofloxacin and penicillin disulfides.
The only reaction that worked well was the synthesis of a t-butyl carbamate analog of
ciprofloxacin. This ciprofloxacin prodrug exhibited potent activity, having an MIC <
0.125 µg/ml against Staphylococcus aureus and MRSA, on par with ciprofloxacin.
O
O
O
O
F
HO
N
O
N
NH
ciprofloxacin
O
O
O
F
HO
O
NaOH, THF
N
N
N
O
N-Boc ciprofloxacin (7)
67%
Figure (2.26) Synthesis of N-Boc ciprofloxacin.
2.12 Conclusions and future directions
72
O
S,S’-Heterosubstituted disulfides were synthesized in good yields from sulfur
monochloride, and characterized by 1H NMR. Most of these compounds showed
promising growth inhibition activities against Staphylococcus aureus and methicillinresistant Staphylococcus aureus. Diisopropoxy disulfide (1b) and bisdimethyldiamino
disulfide (4a) were found to be the most potent among the set of compounds. The data
from the Kirby-Bauer assay concluded that the activity diminished in presence of
additives 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
to obtain isolable mixed disulfide adducts. From the bacterial viability assay these
disulfides were found to be bacteriostatic in nature. These compounds have also shown
decent activities against Francisella tulerensis, the lowest MIC was observed for
diphenyl diamino disulfide (3e).
H
N
O
S
S
O
1b - Best Staph inhibitor
(MIC 0.5 µg/ml)
S
S
N
H
3e - Best Francisella inhibitor
(MIC 1 µg/ml)
Figure (2.27) Most active disulfide analogs 1b and 3e
From the various antimicrobial tests and assays performed, S,S’-heterosubstituted
disulfides were observed to be bacteriostatic inhibiting gram-positive bacteria such as
Staphylococcus aureus and its multi-drug resistant variant.
73
The SEM images (Fig 2.20) of MRSA treated with bis-isopropoxy disulfide (1b) also
showed similarity to those of N-thiolated β-lactams. Neither affects the integrity of the
bacterial cell wall, nor alters cell morphology.
These compounds were tested alongside N-thiolated β-lactams and aryl-alkyl disulfides
synthesized previously in Dr. Turos’ lab and have shown comparable zones of inhibition
against MRSA. In addition, it has been also shown that their bioactivity can be cancelled
out in the presence of thiophilic additives, such as cysteine, glutathione and coenzyme A
(Figures 2.14, 2.16 & 2.17).
It can also be observed that the structural similarity in having a cleavable S-heteroatom
bond in all the three mentioned antibacterials (a sulfur-nitrogen bond in N-thiolated βlactams, sulfur-sulfur bond in aryl-alkyl disulfides and three cleavable S-heteroatom
bonds in S,S’-heterosubstituted disulfides) gives an understanding of the possible active
component and consequently the similarity in the target moiety.
It was established that N-thiolated β-lactams target coenzyme A and, subsequently the
FabH64 enzyme regulating bacterial fatty acid pathway. It can now be hypothesized there
might be a parallel in the mode of action between the N-thiolated β-lactams and the S,S’heterosubstituted disulfides.
The next logical step would be to confirm the hypothesized mode of action of these
compounds. With the aid of tools like proteomics, one can get an understanding as to
which bacterial pathway, if any, is being inhibited by the drug. Proteomics is the branch
of genetics that deals with the full set of proteins encoded by the genome. A basic
experiment would include incubating the bacteria with a drug and analyzing the bacterial
74
contents for any over or under expressed proteins with the help of a mass spectrometer.
This would specify the protein and in turn the pathway that is being affected by the drug.
Apart from being antibacterials themselves, the presence of cleavable S-heteroatom
bonds in their structure gives S,S’-heterosubstituted disulfides a potential to be modified
as prodrugs or dual-action drugs. The prodrug nature facilitates the drug to be masked
from the various bacterial metabolic enzymes and gives the drug extra time to show its
inhibitory activity. This helps in lowering the dose of the drug that needs to be
administered. The advantage with dual-action drugs is the presence of two antibacterial
agents with different modes of action. Once these drugs enter the bacterial cell, they are
prone to enzymatic cleavage and can simultaneously target different bacterial metabolic
pathways. This can possibly be a means of overcoming bacterial resistance mechanisms.
Due to the advent of so many different bacterial resistance mechanisms there is always a
need for effective antimicrobial agents with novel modes of action. The present work
shows S,S’-heterosubstituted disulfides as antimicrobial agents against various bacteria
including Staphylococcus aureus, MRSA, Francisella tulerensis and the fungus Candida
albicans. These structurally-simple disulfides may serve as new leads to the development
of effective antibacterials for drug-resistant microbial infections.
75
CHAPTER THREE
MATERIALS AND METHODS
3.1 Antimicrobial Testing to Determine Minimum Inhibitory Concentration
All the testing was performed according to NCCLS guidelines. [NCCLS (National
Committee for Clinical Laboratory Standards) Methods for Dilution of Antimicrobial
Susceptibility Tests for Bacteria that Grow Aerobically. NCCLS Document M7-A5, Vol.
17, No. 2, 1997.]
3.1.1 Inoculum Preparation
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 hours at 35 to 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 [1.5X10^8 CFU (colony
forming units) /ml]. The culture as needed was adjusted to obtain the appropriate optical
density. If the bacterial suspension is too turbid, it can be diluted with more diluent. If the
suspension is not turbid enough, more bacteria can be added. Once the appropriate optical
76
density was obtained, a 1:1000 dilution of the above culture in a sterile media storage
bottle was prepared.
3.1.2 Preparation of Drug-Containing Mueller Hinton Agar in 24-well Plates
Using a 1 mg/mL stock solution of the test drug in DMSO, the following volumes were
added in each the 12 wells: 256 µl, 128 µl, 64 µl, 32 µl, 16 µl, 8 µl, 4 µl, 2 µl, 1 µl, 0.5
µl, 0.25 µl, 0.125 µl, respectively. The following volume of molten Mueller Hinton Agar
was then added into each of the 12 wells containing the above volume of test drug: 744
µl, 872 µl, 936 µl, 968 µl, 984 µl, 992 µl, 996 µl, 998 µl, 999 µl, 999.50 µl, 999.75 µl,
999.875 µl, respectively. Each well was mixed using a pipette and allowed the drugcontaining agar to solidify at room temperature before inoculation. This gives the
following order of drug concentrations in the wells: 256 µg/ml, 128 µg/ml, 64 µg/ml, 32
µg/ml, 16 µg/ml, 8 µg/ml, 4 µg/ml, 2 µg/ml, 1 µg/ml, 0.5 µg/ml, 0.25 µg/ml and 0.125
µg/ml.
3.1.3 Inoculation of Drug-Containing Mueller Hinton Agar
Optimally, within 15 minutes after adjusting the turbidity of the inoculum suspension and
preparing a 1000-fold dilution, 1 µl of the appropriate inoculum was added to each well
of the 24-well plate, containing solidified agar/test drug. The plates were then placed into
the incubator at 35 to 37 °C.
77
3.1.4 Reading the Plates and Interpreting the Results
After 16 to 18 hours of incubation, each plate was examined for bacterial growth. The
drug concentration in the well showing no visible bacterial growth as detected with the
unaided eye is considered the MIC value. These values are recorded in table 3.1.
3.2 Kirby-Bauer Diffusion Assay
3.2.1 Culture preparation
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
Trypticase® 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.
3.2.2 Testing procedure
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 as explained earlier. This
bacterial solution was then streaked across a TSA plate to a give an even lawn of
bacteria. Sterile pipet tips were used to drill 6 mm wells into the agar plate, then 20 µL of
1 mg/mL drug in DMSO was added to the well. Plates were incubated overnight at 37 °C.
78
3.3 Anti-Francisella testing:
All 29 disulfides were tested against a live vaccine strain (LVS) of Francisella tularensis
by the broth dilution technique as described below. This was carried out in a biosafety
level 3 facility by John Thomas in Dr. Burt Anderson’s laboratory at USF College of
Medicine.
3.3.1 Culture preparation
In a 50 ml conical tube, 320 µg of the test disulfide was added to 10 ml of MuellerHinton (MH) broth at room temperature to give a concentration of 32 µg/ml. 5 mL 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 µg/ml. These steps were repeated to obtain
various concentrations. A 5 mL aliquot was removed from the final tube having a
disulfide concentration of 0.25µg/mL.
3.3.2 Addition of LVS
Two full cotton swabs worth of LVS growth culture was suspended into 10 mL of MH
broth at room temperature. From this stock, 25 µl was added into each of the dilution
tubes prepared above. These tubes were incubated at 36 °C in an incubator with 0% CO2.
3.3.3 Controls
Three control tubes were also prepared, containing the following:
1. 25 µl LVS in 5 mL of MH broth in a 50 mL conical tube.
2. 25 µl LVS in 5 mL of MH broth in a 50 mL conical tube with dilutions of DMSO
added that match those of the above drug dilutions.
79
3. 5 mL of MH broth in a 50 mL conical tube.
After 48 hours of incubation, all of the tubes were examined visually to define the
concentration of disulfide that inhibits LVS growth. These values are recorded in table
3.1.
Table 3.1 Antimicrobial activities of S,S’-heterosubstituted disulfides 1-6.
Minimum Inhibitory Concentration
(µg/mL)
Disulfide
Compound
number
alkyl
F.T
S.A
MRSA
R
(RO-S)2
1a
propyl
16
32
32
1b
isopropyl
4
1
0.5
1c
butyl
4
0.25
8
1d
s-butyl
16
16
8
1e
phenyl
1
2
2
2a
propyl
1
32
32
2b
isopropyl
8
32
32
2c
butyl
4
64
32
2d
s-butyl
16
32
64
2e
phenyl
2
8
8
(RS-S)2
80
(RONH-S)2
3a
propyl
16
8
4
3b
isopropyl
2
32
32
3c
butyl
4
8
32
3d
s-butyl
8
16
16
3e
phenyl
0.5
4
4
4a
methyl
8
0.25
0.5
4b
ethyl
16
2
2
4c
isopropyl
32
2
1
4d
allyl
16
1
0.5
4e
isobutyl
1
16
16
5a
(S) - s-butyl
16
16
32
5b
(R) - s-butyl
16
16
32
5c
1-phenylpropyl
16
16
32
5d
2-phenylpropyl
8
16
16
5e
(D)-menthyl
4
8
8
5f
(L)-menthyl
4
8
16
5g
(+)-menthyl
4
8
16
6a
(R)-benzyl-(1-phenylethyl)
16
16
64
6b
(S)-benzyl-(1-phenylethyl)
16
16
64
(R2N-S)2
(R*O-S)2
(R*R*N-S)2
81
Control
Ciprofloxacin
0.125
Penicillin-G
0.125 16
F.T: Francisella tularensis (Live Vaccine Strain)
S.A: Staphylococcus aureus (ATCC-25923)
MRSA: Methicillin-resistant Staphylococcus aureus (ATCC-43300)
3.4 In vitro Anti-Bartonella activity by disk diffusion method
B.henselae (hoston-1), B.quintana (U-mass), B.henselae Marseille and B.henselae (SA-1)
were inoculated onto chocolate agar plates to create a confluent lawn of growth. A 6 mm
diameter paper disk was then placed in the center of the plate before incubation. Plates
were incubated at 37 °C for one week and the diameter of growth inhibition was then
measured and recorded.
3.4.1 Preparation of antibiotic-containing paper disks
The preparation of disks was done in an identical fashion each time, as it is important to
standardize the procedure as much as possible. All antibiotics were predissolved in
DMSO to a final concentration of 1 mg/ml, and added to the paper disks as a 1 mg/ml
solution using a volume of 20 µl. This volume readily adsorbed into the paper disks
quickly, and gave a total standard amount of 20 µg of antibiotic per disk. To load the
antibiotics, 6 mm paper disks were placed on a sheet of aluminum foil in the biological
safety cabinet (BSC). Disks can be easily handled without damage using fine-point
forceps. To each disk was added 20 µl of antibiotic solution at a concentration of 1 mg/ml
82
dissolved in DMSO. A negative control consisting of 20 µl DMSO/disk and a positive
control consisting of 20 µl of rifampicin at 0.1mg/ml in DMSO were also prepared. Each
disk contained 20 µg of the antibiotic being tested while the positive control disk
contained 2.0 µg of rifampicin. The disks were allowed to dry in the BSC for at least 20
minutes, then placed by forceps in a sealable bag with desiccant and stored sealed in the
refrigerator.
3.4.2 Inoculation of agar plates for disk diffusion testing
Bacteria from 4-5 day old plates was harvested and resuspended in 1.0 ml sterile HIB.
Turbidity was adjusted to about McFarland 2.0 by inspection. The suspension was spread
over the surface of a labeled chocolate agar plate using a swab and allowed to dry into the
agar in the BSC for 10-15 minutes. Forceps were used to place the disk in the center of
the plate. The disk was tapped gently to make sure it adhered to the plate. The plates were
then inverted and kept at 35 °C in a CO2 incubator for one week. The zones of inhibition
were then measured and recorded to the nearest mm for each plate.
3.5 Fungal viability assay
Diisopropoxy disulfide was tested for fungal viability by a Trypan Blue staining protocol.
50 µl of a Candida albicans culture and 50 µl of a 0.4% Trypan Blue solution in water
(w/v) were mixed in a 2 ml plastic microcentrifuge tube. After 5 minutes, 20 µl of the
mix was loaded into a disposable cell-counting chamber (Cellometer (Registered),
Nexcelom Bioscience, Lawrence, MA) and viewed microscopically (Olympus BX60,
Olympus, Center Valley, PA) at 40X magnification. Pictures were taken using a Spot
Flex Camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
83
3.6 Cytotoxicity testing
Primary bovine primary aortic endothelial cells and EBM-2 (endothelial basal medium)
cell culture medium were from Lonza Walkersville, Inc. Cells were maintained in culture
medium supplemented with 10% FBS (fetal bovine serum) and 100 IU penicillin/100
µg/ml streptomycin sulfate in a humidified incubator containing 5% CO2 at 37 oC. For
cytotoxicity testing, cells were trypsinized, counted, centrifuged at 120 x g, and
resuspended in fresh culture medium. Cells were plated in a volume of 100 µl (2.5 x 104
cells/well) in 96-well flat-bottom culture dishes. After 16-18 h of incubation, 100 µl of
medium or medium containing test samples was added to each well. Tests were
performed in triplicate. After 48 h, 20 µl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium 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 100 µl 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 x 100).
84
Table 3.2 Cytotoxicity data of diisopropoxy disulfide (1b)
Sample
Concentration Mean
Std.
%
control Appearance
(µg/ml)
Deviation
Absorbance
after 48hr
1b
0.3125
0.248
0.006
90.6
ok
1b
1.25
0.271
0.017
98.9
ok
1b
5
0.299
0.021
109.4
ok
1b
20
0.252
0.010
92.0
ok
Diisopropoxy disulfide was checked for cytotoxicity by MTT assay as explained above.
It was observed that this compound did not show any cytotoxic activity against bovine
aortic endothelial cells upto the concentration of 20 µg/ml.
3.7 Synthetic procedures
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 distilling from sulfur and charcoal (100:4:1 S2Cl2/sulfur/charcoal)
(Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis; John Wiley and Sons: New
York, 1967; Vol. 1). 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 Bruker 250
MHz instrument. 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)
85
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–visible detector at 254 nm.
3.7.1 General procedure for the synthesis of S,S’-heterosubstituted disulfide 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 hr. 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 obtained was purified by silica gel chromatography and
characterized by 1H NMR. Due to the absence of any ionizable functionalities in the S,S’heterosubstituted disulfides, mass spectroscopy was not helpful because no parent ion
could be identified for any of the compounds.
Synthesis of S,S’-dipropoxy disulfide (1a)
To a stirred solution of n-propanol (5 mL, 1mmol) and triethylamine (9.36 mL, 1 mmol)
in 20 mL of anhydrous dichloromethane at -20 °C was added dropwise a solution of
sulfur monochloride (2.68 mL, 0.5mmol) in 10 mL of dichloromethane. The reaction was
brought to room temperature after the addition was completed and stirring was continued
86
for about 1 hr. The reaction was worked up as described in the general procedure. All the
yields mentioned below are after column chromatography.
1
1a: Isolated 77.3 mg (85%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 3.38-3.43 (t,
2H, J= 6.8 Hz), 1.82-1.96 (m, 2H), 1.01-1.07 (t, 3H, J= 7.3 Hz).
1
1b: Isolated 79.2 mg (87%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 3.95-4.15
(m, 1H), 1.24-1.32 (m, 6H).
1
1c: Isolated 87.2 mg (83%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 3.4-3.46 (t,
2H, J= 6.8 Hz), 1.8-1.91 (m, 2H), 1.4-1.55 (m, 2H), 0.91-0.97 (t, 3H, J= 7.4 Hz).
1
1d: Isolated 85 mg (81%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 3.67-3.93 (m,
1H), 1.38-1.68 (m, 2H), 1.08-1.28 (d, 3H, J = 7.5 Hz), 0.75-0.96 (t, 3H, J = 6.3 Hz).
1
1e: Isolated 95 mg (76%) as a dark green oil H NMR (250 MHz, CDCl3): δ 7.27-7.40
(m, 5H).
1
2a: Isolated 92.1 mg (86%) as a yellow oil; H NMR (250 MHz, CDCl3): δ 2.91-2.97 (t,
2H, J= 7.1 Hz), 1.73-1.76 (m, 2H), 1-1.06 (t, 3H, J= 7.3 Hz).
1
2b: Isolated 90.2 mg (85%) as a yellow oil; H NMR (250 MHz, CDCl3): δ 3.19-3.37 (m,
1H), 1.38-1.41 (d, 6H, J= 6.8 Hz).
1
2c: Isolated 108.9 mg (90%) as a yellow oil; H NMR (250 MHz, CDCl3): δ 2.68-3.01
(m, 2H), 1.71-1.80 (m, 2H), 1.41-1.51 (m, 2H), 0.92-0.99 (t, 3H, J= 7.7 Hz).
1
2d: Isolated 100.44 mg (83%) as a yellow oil; H NMR (250 MHz, CDCl3): δ 2.95-3.12
(m, 1H), 1.58-1.83 (m, 2H), 1.36-1.41 (d, 3H, J= 6.2 Hz), 0.97-1.05 (t, 3H, J= 7.4 Hz).
87
1
2e: Isolated 90.2 mg (85%) as pale yellow crystals; mp 31-33 °C; H NMR (250 MHz,
CDCl3): δ 7.51-7.55 (m, 2H), 7.25-7.37 (m, 3H).
1
3a: Isolated 58.5 mg (65%) as a reddish oil; H NMR (250 MHz, CDCl3): δ 3.11-3.17 (t,
2H, J= 7.9 Hz), 1.55-1.65 (m, 2H), 1.46-1.52 (bs, 1H), 0.84-0.89 (t, 3H, J= 7.3 Hz).
1
3b: Isolated 58.5 mg (65%) as a reddish oil; H NMR (250 MHz, CDCl3): δ 3.79-3.95 (m,
1H), 1.28-1.3 (d, 6H, J= 6.5 Hz).
1
3c: Isolated 62.43 mg (60%) as a reddish oil; H NMR (250 MHz, CDCl3): δ 3.63-3.71
(m, 2H), 1.8-1.89 (m, 2H), 1.34-1.48 (m, 2H), 0.93-0.99 (t, 3H, J= 7.3 Hz).
1
3d: Isolated 65.5 mg (63%) as a reddish oil; H NMR (250 MHz, CDCl3): δ 2.95-3.12 (m,
1H), 1.58-1.83 (m, 2H), 1.36-1.41 (m, 3H), 0.97-1.05 (m, 3H).
1
3e: Isolated 80.6 mg (65%) as a reddish oil; H NMR (250 MHz, CDCl3): δ 7.14-7.27 (m,
2H), 6.68-6.81 (m, 3H), 3.65 (bs, 1H).
1
4a: Isolated 55.5 mg (73%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 2.52 (s,
3H).
1
4b: Isolated 78.1 mg (75%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 3.40-3.48
(q, 2H, J= 7.4 Hz), 1.65-1.71 (t, 3H, J= 7.3 Hz).
1
4c: Isolated 99.1 mg (75%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 4.05-4.15
(m, 1H), 1.27-1.33 (t, 6H, J= 7 Hz).
1
4d: Isolated 89.6 mg (70%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 5.73-5.9
(m, 4H), 5.07-5.16 (m, 8H), 3.34-3.37 (d, 8H, J= 6.3 Hz).
88
1
4e: Isolated 113.6 mg (71%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 2.64-2.7
(bs, 1H), 2.42-2.45 (d, 1H, J= 7.1 Hz), 1.93-2.13 (m, 1H), 0.89-0.92 (d, 6H, J= 6.6 Hz).
1
5a: Isolated 81.9 mg (78%) as a colorless oil; [α]25D –10.1° (c 0.29, CH2Cl2); H NMR
(250 MHz, CDCl3): δ 3.85-3.91 (m, 1H), 1.53-1.69 (m, 2H), 1.26-1.31 (m, 3H), 0.91-0.97
(m, 3H).
5b: Isolated 79.8 mg (76%) as a colorless oil; [α]25D +9.2° (c 0.25, CH2Cl2);
1
H NMR
(250 MHz, CDCl3): δ 3.86-3.91 (m, 1H), 1.55-1.67 (m, 2H), 1.26-1.31 (m, 3H), 0.91-0.97
(m, 3H).
1
5c: Isolated 58.45 mg (70%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 7.28-7.39
(m, 5H), 4.59-4.65 (m, 1H), 1.77-1.87 (m, 2H), 0.92-0.98 (t, 3H, J= 7.4 Hz).
1
5d: Isolated 60.95 mg (73%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 7.03-7.18
(m, 5H), 3.50-3.53 (d, 2H, J= 6.8 Hz), 2.69-2.83 (m, 1H), 1.07-1.1 (d, 3H, J= 6.7 Hz).
3.7.2 Synthesis of S,S’-heterosubstituted disulfides of menthol:
To an ice-cooled suspension of sodium hydride (oil removed from hexanes) (1.5 mmol)
in anhydrous tetrahydrofuran (THF), a solution of 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.
89
1
5e: Isolated 56.1 mg (60%) as a colorless oil; [α]25D +32.1° (c 0.35, CH2Cl2); H NMR
(250 MHz, CDCl3): δ 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).
1
5f: Isolated 58 mg (62%) as a colorless oil; [α]25D –28.7° (c 0.15, CH2Cl2); H NMR (250
MHz, CDCl3): δ 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), 0.810.84 (m, 3H).
1
5g: Isolated 60.7 mg (65%) as a colorless oil; H NMR (250 MHz, CDCl3): δ 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), 1.011.13 (m, 2H), 0.94 (d, 3H, J= 4.3 Hz), 0.92 (d, 3H, J= 3.8 Hz), 0.81-0.84 (d, 3H, J= 6.8
Hz).
The compounds 6a and 6b were synthesized according to the general procedure
explained in section 3.6.1.
1
6a: Isolated 67.7 mg (56%) as a colorless oil; [α]25D +23° (c 0.59, CH2Cl2); H NMR (250
MHz, CDCl3): δ 7.13-7.29 (m, 10H), 3.86-3.88 (m, 3H), 1.42-1.46 (m, 3H).
1
6b: Isolated 70.2 mg (58%) as a colorless oil; [α]25D –25° (c 0.45, CH2Cl2); H NMR (250
MHz, CDCl3): δ 7.17-7.27 (m, 10H), 3.82-4.3 (m, 3H), 1.44 (d, 3H, J= 6.6 Hz).
3.7.3 Synthesis of 7-(4-tert-Butoxycarbonyl-piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-
oxo-1,4-dihydro-quinoline-3-carboxylic acid: To a stirred solution of ciprofloxacin (390
90
mg, 1.0 mmol) and 1M sodium hydroxide solution (3 ml, 2.5 mmol) in 20mL of
tetrahydrfuran was added di-tert-butyl dicarbonate (0.20 ml, 1.1 mmol). The reaction was
stirred until all the starting material was consumed by monitoring with TLC. The reaction
mixture was then concentrated in vacuo and purified by silica gel chromatography to give
1
339.4 mg (67%) of the title compound as a white solid. H NMR (250 MHz, CDCl3):
14.95 (bs, 1H), 8.79 (s, 1H), 8.06 (d, 1H, J= 13.0 Hz), 7.36-7.39 (d, 1H, J= 7.3 Hz),
3.66-3.70 (m, 4H), 3.50-3.60 (m, 1H), 3.25-3.35 (m, 4H), 1.51 (s, 9H), 1.40-1.43 (m,
2H), 1.20-1.23 (m, 2H).
91
CHAPTER FOUR
SPECTRA
1
1.04
Spectrum 4.1: H NMR (250 MHz, CDCl3) of compound 1a:
ADS.006.esp
1.0
0.8
3.40
0.9
H3 C
O
S
0.7
S
3.43
3.38
0.5
1.01
1.07
CH3
1.91
1.88
0.4
1.96
0.1
1.82
0.2
1.94
1.85
0.3
7.27
Normalized Intensity
O
0.6
0
2.00
10
9
8
7
6
5
4
Chemical Shift (ppm)
92
2.06
3
2
2.98
1
0
1
0.94
Spectrum 4.2: H NMR (250 MHz, CDCl3) of compound 1c:
ADS.008.esp
1.0
0.9
0.8
O
S
S
0.7
O
0.97
CH3
0.6
0.91
0.5
1.91
1.43
7.27
0.2
1.40
0.3
1.80
1.88
1.85
1.82
0.4
1.49
1.46
3.46
3.40
Normalized Intensity
3.43
H3C
0.1
0
2.00
10
9
8
7
6
5
4
Chemical Shift (ppm)
3
2.03 2.36
3.20
2
1
0
1
7.27
Spectrum 4.3: H NMR (250 MHz, CDCl3) of compound 1d:
PR_sBuDS.esp
1.0
H3C
CH3
0.9
O
O
0.96
H3C
CH3
0.6
1.31
0.5
0.93
1.26
0.4
0.3
1.67
1.64
1.61
1.58
1.55
1.53
0.2
3.91
3.90
3.89
3.88
3.87
3.86
3.85
Normalized Intensity
0.7
S
1.28
S
0.8
0.1
0
0.98
9
8
7
6
5
4
Chemical Shift (ppm)
93
2.14 3.083.00
3
2
1
0
1
1.30
Spectrum 4.4: H NMR (250 MHz, CDCl3) of compound 1b:
Desktop.003.esp
1.0
CH3
1.27
0.9
O
S
0.8
S
CH3
O
0.6
CH3
H3C
1.32
1.26
Normalized Intensity
0.7
0.5
0.4
4.12
4.10
4.07
4.05
7.27
0.2
0.1
1.24
0.3
0
1.02
9
8
7
6
5
Chemical Shift (ppm)
5.86
4
3
2
1
0
1
7.36
Spectrum 4.5: H NMR (250 MHz, CDCl3) of compound 1e:
ADS.010.esp
1.0
0.9
S
S
7.33
O
0.8
O
7.37
7.30
0.6
7.29
7.27
0.5
0.4
0.3
7.40
Normalized Intensity
0.7
0.2
0.1
0
5.00
10
9
8
7
6
5
Chemical Shift (ppm)
94
4
3
2
1
0
1
1.38
Spectrum 4.6: H NMR (250 MHz, CDCl3) of compound 2b:
1.41
spec.009.esp
1.0
0.9
H3C
S
0.8
H3C
S
S
Normalized Intensity
0.7
CH3
S
CH3
0.6
0.5
0.4
0.3
3.37
3.37
3.34
3.31
3.29
3.24
3.22
3.19
7.27
0.2
0.1
0
1.08
10
9
8
7
6
5
4
Chemical Shift (ppm)
6.00
3
2
1
0
1
1.0
0.95
Spectrum 4.7: H NMR (250 MHz, CDCl3) of compound 2c:
spec.011.esp
0.9
H3C
S
S
0.6
CH3
0.4
0.3
3.01
0.2
1.80
1.77 1.77
1.74
1.47
1.51
1.45
1.41
3.00
2.97
2.97
2.89
2.86
0.5
7.27
Normalized Intensity
S
0.92
S
0.7
0.99
0.93
0.8
0.1
0
2.00
10
9
8
7
6
5
4
Chemical Shift (ppm)
95
3
2.322.31 3.40
2
1
0
1
7.29
Spectrum 4.8: H NMR (250 MHz, CDCl3) of compound 2e:
spec.011.esp
1.0
0.9
S
0.8
7.34
S
7.52
0.7
7.55
0.6
0.4
7.28
0.5
7.25
7.55
Normalized Intensity
S
S
0.3
0.2
0.1
0
1.82 1.92 0.90
10
9
8
7
6
5
4
Chemical Shift (ppm)
3
2
1
0
1
1.39
Spectrum 4.9: H NMR (250 MHz, CDCl3) of compound 2d:
spec.013.esp
1.0
H3C
1.00
0.9
CH3
S
S
S
S
1.02
0.7
H3C
CH3
1.41
0.6
1.05
0.99
0.97
0.5
0.4
3.12
3.09
3.06
3.00
2.97
2.95
1.80
1.79
1.66
1.64
1.63
0.3
0.2
7.27
Normalized Intensity
1.36
0.8
0.1
0
0.98
10
9
8
7
6
5
4
Chemical Shift (ppm)
96
3
2.02 2.83 2.92
2
1
0
1
1.04
Spectrum 4.10: H NMR (250 MHz, CDCl3) of compound 2a:
spec.004.esp
1.0
0.9
0.8
S
S
S
CH3
0.5
1.86
1.75
0.4
1.83
2.94
0.6
2.97
2.91
Normalized Intensity
0.7
1.80
1.06
S
1.00
7.27
H3C
0.3
0.2
0.1
0
4.00
16
14
12
10
8
6
4
Chemical Shift (ppm)
4.52 6.68
2
0
-2
-4
1
0
1
1.30
Spectrum 4.11: H NMR (250 MHz, CDCl3) of compound 3b:
AmDS.003.esp
1.28
1.0
CH3
0.9
H3C
0.8
NH S
S
NH
CH3
H3 C
0.6
0.5
0.4
3.95
3.92 3.90
3.87
3.82 3.84
3.79
0.3
0.2
7.27
Normalized Intensity
0.7
0.1
0
0.99
10
9
8
7
6
5
4
Chemical Shift (ppm)
97
5.90
3
2
1
0.96
Spectrum 4.12: H NMR (250 MHz, CDCl3) of compound 3d:
AmDS.008.esp
1.0
0.9
H3C
0.8
NH
S
S
NH
0.99
0.93
CH3
0.6
0.5
0.4
7.27
Normalized Intensity
0.7
3.71 3.68
3.66
3.63
1.89 1.86 1.83
1.80
1.77
1.48
1.43
1.34 1.39
0.3
0.2
0.1
0
1.72
10
9
8
7
1.81 2.11 3.00
6
5
4
Chemical Shift (ppm)
3
2
1
0
1
6.72
Spectrum 4.13: H NMR (250 MHz, CDCl3) of compound 3e:
1.0
6.72
6.69
7.18
AmDS.011.esp
7.14
0.9
NH
NH
6.78
7.21
0.6
6.81
0.5
3.65
0.3
6.81
0.4
7.27
Normalized Intensity
0.7
S
S
6.68
0.8
0.2
0.1
0
2.00 3.11
10
9
8
7
0.89
6
5
Chemical Shift (ppm)
98
4
3
2
1
1
0.86
Spectrum 4.14: H NMR (250 MHz, CDCl3) of compound 3a:
AmDS.013.esp
1.0
0.9
H3C
0.8
NH
S
0.7
S
0.89
0.84
CH3
0.6
7.19
1.49
0.5
0.4
0.3
1.65 1.62
3.17
3.14
3.11
Normalized Intensity
NH
0.2
0.1
0
2.01
10
9
8
7
6
5
4
Chemical Shift (ppm)
2.07 1.10 3.22
3
2
1
0
-2
-4
1
0.92
0.89
Spectrum 4.15: H NMR (250 MHz, CDCl3) of compound 4e:
AmDS.007.esp
1.0
CH3
H3C
0.9
H3C
CH3
0.8
H3C
N
N
S
H3C
0.6
H3C
CH3
0.5
0.4
2.70
2.67
0.1
2.10
1.95
1.93
0.2
2.45
2.42
0.3
7.27
Normalized Intensity
0.7
S
0
1.01 1.00 1.39 6.01
16
14
12
10
8
6
4
Chemical Shift (ppm)
99
2
0
1
2.52
Spectrum 4.16: H NMR (250 MHz, CDCl3) of compound 4a:
AmDS.014.esp
1.0
0.9
CH3
H3C
0.8
N
S
Normalized Intensity
0.7
S
N
0.6
CH3
H3C
0.5
0.4
0.3
0.2
0.1
0
2.99
16
14
12
10
8
6
Chemical Shift (ppm)
4
2
0
-2
-4
1
1.30
Spectrum 4.17: H NMR (250 MHz, CDCl3) of compound 4c:
spec.120.esp
1.0
CH3
H3C
H3C
H3C
CH3
S
N
CH3
CH3
H3C
0.6
0.5
0.4
0.3
4.15 4.13
4.10
4.05 4.08
Normalized Intensity
0.7
S
1.33
1.27
N
0.8
7.27
0.9
0.2
0.1
0
1.02
9
8
7
6
5
Chemical Shift (ppm)
100
4
6.42
3
2
1
1
1.68
Spectrum 4.18: H NMR (250 MHz, CDCl3) of compound 4b:
AmDS.009.esp
1.0
0.9
H3C
CH3
S
N
H3C
0.6
N
S
H3C
3.45
3.42
Normalized Intensity
0.7
1.71
1.65
0.8
0.5
0.4
3.48
3.40
0.3
7.27
0.2
0.1
0
2.00
10
9
8
7
6
5
4
Chemical Shift (ppm)
2.97
3
2
1
0
1
Spectrum 4.19: H NMR (250 MHz, CDCl3) of compound 4d:
1.0 Desktop.002.esp
H
0.9
H
H
H
N
S
0.7
H
N
0.6
H
H
H
3.38
3.34
H
5.07
5.16
5.15
0.1
5.16
0.2
5.74
5.87
0.3
5.80
5.76
0.4
5.08
5.08
0.5
5.90
Normalized Intensity
H
H
S
5.09
H
3.37
3.35
0.8
0
4.00
9
8
7
6
8.00
5
Chemical Shift (ppm)
101
7.27
4
3
2
1
1
7.37
Spectrum 4.20: H NMR (250 MHz, CDCl3) of compound 5c:
spec.130.esp
1.0
0.9
S
O
0.8
S
0.95
CH3
CH3
O
0.6
0.98
0.92
7.29
0.5
0.4
7.39
7.28
0.2
0.1
1.87
1.86 1.84
1.81
1.80
1.78
1.77
7.39
0.3
4.65
4.64
4.63
4.62
4.61
4.60
4.59
Normalized Intensity
0.7
0
10.48
9
8
2.00
7
6
4.57
5
4
Chemical Shift (ppm)
3
6.46
2
1
0
1
0.94
0.93
0.91
0.91
0.84
Spectrum 4.21: H NMR (250 MHz, CDCl3) of compound 5g:
spec.140.esp
1.0
0.95
CH3
0.9
O
H3C
S
CH3
CH3
S
O
0.7
0.6
1.39
CH3
0.3
3.47
3.45
3.45
3.43
3.41
3.39
3.37
2.18
2.17
2.17
2.00
1.95
1.70 1.65
1.59
0.2
0.1
0.78
0.4
1.01 0.98
1.26
1.26
0.5
7.26
Normalized Intensity
H3C
0.80 0.83
0.8
0
2.00
10
9
8
7
6
5
Chemical Shift (ppm)
102
4
2.00 2.08 4.17 6.98 4.37 12.17 6.02
3
2
1
0
1
1.10
1.07
Spectrum 4.22: H NMR (250 MHz, CDCl3) of compound 5d:
spec.150.esp
1.0
0.9
0.8
H3C
S
O
O
S
CH3
7.06
0.6
3.53
3.50
0.5
0.1
7.15
7.18
7.17
0.2
2.83
2.80
2.78
2.75
2.72
2.69
0.3
7.04
7.03
7.03
7.03
0.4
7.14 7.11
Normalized Intensity
0.7
0
5.00
10
9
8
7
2.07
6
5
4
Chemical Shift (ppm)
1.11
3
3.31
2
1
0
0
-2
-4
1
7.26
7.19
Spectrum 4.23: H NMR (250 MHz, CDCl3) of compound 6b:
6a.esp
7.17
1.0
0.9
1.45
1.42
3.87
3.86
S
0.7
S
0.6
1.46
N
7.14
7.13
0.5
0.4
0.3
7.29
Normalized Intensity
7.26
N
0.8
0.2
0.1
0
10.19
16
14
12
10
8
2.48
6
4
Chemical Shift (ppm)
103
2.99
2
1
7.26
Spectrum 4.24: H NMR (250 MHz, CDCl3) of compound 6a:
3_29_0ld.esp
7.20
7.17
1.0
0.9
3.87
3.86
Normalized Intensity
7.27
S
0.7
1.45
1.42
N
0.8
S
N
0.6
0.5
0.4
3.90
3.82
0.3
0.2
0.1
0
10.00
16
14
12
10
8
3.22
3.38
6
4
Chemical Shift (ppm)
2
0
-2
1
Spectrum 4.25: H NMR (250 MHz, CDCl3) of compound 5a:
1.0 PR_(+)_sBuDS.esp
0.9
H3C
CH3
0.8
S
S
1.28
O
0.7
O
0.96
0.6
H3C
1.31
0.5
0.94
0.93
1.26
0.4
1.69
1.67
1.64
1.61
1.58
1.53 1.55
0.2
0.1
0.97
0.92
0.91
0.3
3.91
3.90
3.89
3.88
3.86
3.85
Normalized Intensity
H3C
0
0.98
9
8
7
6
5
4
Chemical Shift (ppm)
104
2.22 3.23 3.07
3
2
1
-4
1
1.0
0.95
0.94
0.93
0.84
0.81
Spectrum 4.26: H NMR (250 MHz, CDCl3) of compound 5e:
ADS.015.esp
0.9
CH3
0.8
0.7
S
CH3
O
S
H3C
0.6
H3C
CH3
CH3
0.5
1.27
1.25
0.4
3.46
3.44
3.42
3.40
3.38
2.21
2.20
2.19
2.17
1.98
0.2
0.1
1.70 1.65
1.64
1.42
0.3
1.12 0.98
Normalized Intensity
O
0
1.99 2.03 1.82 3.61 1.97 1.79 5.02 10.20 5.70
10
9
8
7
6
5
4
Chemical Shift (ppm)
3
2
1
0
1
7.27
Spectrum 4.27: H NMR (250 MHz, CDCl3) of compound 5b:
PR_(-)_sBuDS.esp
1.0
H3C
0.9
CH3
S
0.8
0.6
0.96
H3C
0.7
0.5
0.4
0.94
0.93
1.31
1.28
1.26
H3C
1.67
1.64
1.61
1.58
1.55
0.2
0.1
0.97
0.92
0.91
0.3
3.91
3.89
3.88
3.86
Normalized Intensity
S
1.28
O
O
0
0.99
10
9
8
7
6
5
4
Chemical Shift (ppm)
105
2.253.303.36
3
2
1
0
1
7.27
Spectrum 4.28: H NMR (250 MHz, CDCl3) of compound 5f:
5a.esp
1.0
0.9
CH3
0.8
S
O
0.95
0.94
0.93
0.84
0.81
Normalized Intensity
CH3
O
S
0.7
H3C
H3C
0.6
CH3
CH3
0.5
0.4
0.2
0.1
0.97
1.16 1.00
3.47
3.46
3.44
3.42
3.40
3.38
2.24
2.23
2.21
2.20
2.17
2.15 1.65
1.64
1.64 1.28
0.3
0
2.08 2.03 1.97 4.39 2.04 1.97 5.85 12.08 5.96
9
8
7
6
5
4
Chemical Shift (ppm)
3
2
1
0
1
1.51
Spectrum 4.29: H NMR (250 MHz, CDCl3) of compound 7:
Prodrug.005.esp
1.0
0.9
O
0.8
O
F
OH
CH3
H3C
H3C
O
N
N
N
0.6
O
0.5
7.27
0.4
0.1
1.58
1.42
1.40
1.23
1.22
8.79
8.09
8.03
7.36
0.2
3.70
3.68
3.68
3.66
3.32
3.30
3.28
0.3
14.95
Normalized Intensity
0.7
0
1.19
16
1.11 1.28 1.15
14
12
10
8
3.65 1.54 3.70 8.442.21 2.06
6
4
Chemical Shift (ppm)
106
2
0
-2
-4
Spectrum 4.30: HPLC trace of (1b) and CoA after 30 min in phosphate buffer solution:
CoA + Diisopropoxy disulfide (1b) in PBS after 30min at 254nm
DMSO
CoA
1b
Minutes
107
Spectrum 4.31: HPLC trace of 1b and CoA after 24 hrs in phosphate buffer solution:
CoA + Diisopropoxy disulfide (1b) in PBS after 24 hrs at 254nm
DMSO
CoA
1b
Minutes
108
Spectrum 4.32: HPLC trace of CoA in phosphate buffer solution:
CoA in PBS after 24 hrs at 254nm
DMSO
CoA
Minutes
109
REFERENCES
1.
Williams, J.; Cooper, R. Plant Pathol., 2004, 53, 263.
2.
Cohen, G.; Borovok, I.; Uziel, O.; Schreiber, R.; Aharonowitz, Y., Bacterial thioldisulfide redox metabolism: A new target for rational drug design. Period Biol
2001, 103, (2), 153-5.
3.
Smirnova, G. V.; Oktyabrsky, O. N., Glutathione in bacteria. Biochem (Mosc)
2005, 70, (11), 1199-211.
4.
Szajewski, R.; Whitesides, G., Rate constants and equilibrium constants for thioldisulfide interchange reactions involving oxidized glutathione. J Am Chem Soc
1980, 102, (6), 2011-26.
5.
delCardayre, S. B.; Stock, K. P.; Newton, G. L.; Fahey, R. C.; Davies, J. E.,
Coenzyme A disulfide reductase, the primary low molecular weight disulfide
reductase from Staphylococcus aureus. Purification and characterization of the
native enzyme. J Biol Chem 1998, 273, (10), 5744-51.
6.
Nishimura, J. S.; Mitchell, T.; Hill, K. A.; Collier, G. E., Coenzyme A
thiosulfonate (coenzyme A disulfide-S,S-dioxide), an affinity analog of coenzyme
A. J Biol Chem 1982, 257, (24), 14896-902.
7.
Ritz, D.; Beckwith, J., Roles of thiol-redox pathways in bacteria. Annu Rev
Microbiol 2001, 55, 21-48.
8.
Chung, J.; Chen, T.; Missiakas, D., Transfer of electrons across the cytoplasmic
membrane by DsbD, a membrane protein involved in thiol-disulphide exchange
and protein folding in the bacterial periplasm. Mol Microbiol 2000, 35, (5), 1099109.
9.
Newton, G. L.; Arnold, K.; Price, M. S.; Sherrill, C.; Delcardayre, S. B.;
Aharonowitz, Y.; Cohen, G.; Davies, J.; Fahey, R. C.; Davis, C., Distribution of
thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J
Bacteriol 1996, 178, (7), 1990-5.
10.
Newton, G. L.; Bewley, C. A.; Dwyer, T. J.; Horn, R.; Aharonowitz, Y.; Cohen,
G.; Davies, J.; Faulkner, D. J.; Fahey, R. C., The structure of U17 isolated from
110
Streptomyces-Clavuligerus and its properties as an antioxidant thiol. Eur J
Biochem 1995, 230, (2), 821-5.
11.
Patel, M. P.; Blanchard, J. S., Expression, purification, and characterization of
Mycobacterium tuberculosis mycothione reductase. Biochem 1999, 38, (36),
11827-33.
12.
Newton, G. L.; Rawat, M.; La Clair, J. J.; Jothivasan, V. K.; Budiarto, T.;
Hamilton, C. J.; Claiborne, A.; Helmann, J. D.; Fahey, R. C., Bacillithiol is an
antioxidant thiol produced in Bacilli. Nat Chem Biol 2009, 5, (9), 625-7.
13.
Ghuysen, J. M., Serine β-lactamases and penicillin-binding proteins. Annu Rev
Microbiol 1991, 45, 37-67.
14.
Spratt, B. G., Biochemical and genetic approaches to the mechanism of action of
Penicillin. Philos T Roy Soc B 1980, 289, (1036), 273-83.
15.
Feather, M. S.; Whistler, R. L., Derivatives of 5-Deoxy-5-Mercapto-D-Glucose.
Tetrahedron Lett 1962, (15), 667-8.
16.
Kajimoto, T.; Liu, K. K. C.; Pederson, R. L.; Zhong, Z. Y.; Ichikawa, Y.; Porco,
J. A.; Wong, C. H., Enzyme-catalyzed aldol condensation for asymmetricsynthesis of azasugars - synthesis, evaluation, and modeling of glycosidase
inhibitors. J Am Chem Soc 1991, 113, (16), 6187-96.
17.
Whistler, R. L.; Vanes, T.; Rowell, R. M., Sulfoxide and sulfone derivatives of Dxylothiopyranose. J Org Chem 1965, 30, (8), 2719-21.
18.
Whistler, R. L.; Lake, W. C., Inhibition of cellular transport processes by 5-thioD-glucopyranose. Biochem J 1972, 130, (4), 919-25.
19.
Ghannoum, M. A.; Eweiss, N. F.; Bahajaj, A. A.; Qureshi, M. A., Antimicrobial
activity of some thiol-containing heterocycles. Microbios 1983, 37, (149-150),
151-9.
20.
Asundaria, S. T.; Patel, N. S.; Patel, K. C., Synthesis, characterization, and
antimicrobial studies of novel 1,3,4-thiadiazolium-5-thiolates. Med Chem Res
2011. No pp given yet.
21.
Baranski, K.; Bardos, T. J.; Bloch, A.; Kalman, T. I., 5-Mercaptodeoxyuridine its enzymatic synthesis and mode of action in microbiological systems. Biochem
Pharmacol 1969, 18, (2), 347.
22.
Cai, J.; Chen, Y.; Seth, S.; Furukawa, S.; Compans, R. W.; Jones, D. P., Inhibition
of influenza infection by glutathione. Free Radic Biol Med 2003, 34, (7), 928-36.
111
23.
Mihm, S.; Ennen, J.; Pessara, U.; Kurth, R.; Droge, W., Inhibition of HIV-1
replication and Nf-kappa-β activity by cysteine and cysteine derivatives. AIDS
1991, 5, (5), 497-503.
24.
Oiry, J.; Mialocq, P.; Puy, J. Y.; Fretier, P.; Dereuddre-Bosquet, N.; Dormont, D.;
Imbach, J. L.; Clayette, P., Synthesis and biological evaluation in human
monocyte-derived macrophages of N-(N-acetyl-L-cysteinyl)-S-acetylcysteamine
analogues with potent antioxidant and anti-HIV activities. J Med Chem 2004, 47,
(7), 1789-95.
25.
Kalebic, T.; Kinter, A.; Poli, G.; Anderson, M. E.; Meister, A.; Fauci, A. S.,
Suppression of Human-Immunodeficiency-Virus Expression in Chronically
Infected Monocytic Cells by Glutathione, Glutathione Ester, and NAcetylcysteine. Proc Natl Acad Sci USA 1991, 88, (3), 986-90.
26.
Oiry, J.; Mialocq, P.; Puy, J. Y.; Fretier, P.; Clayette, P.; Dormont, D.; Imbach, J.
L., NAC/MEA conjugate: a new potent antioxidant which increases the GSH
level in various cell lines. Bioorg Med Chem Lett 2001, 11, (9), 1189-91.
27.
Nagamach.T; Fourrey, J. L.; Torrence, P. F.; Waters, J. A.; Witkop, B., Synthesis,
Chemistry, and Biological-Activity of 5-Thiocyanatopyrimidine Nucleosides as
Potential Masked Thiols. J Med Chem 1974, 17, (4), 403-6.
28.
Wright, G. M. K. A. D., New and Unusual Sesquiterpenes: Kelsoene, Prespatane,
Epi-γ-gurjunene, and T-Cadinthiol, from the Tropical Marine Sponge Cymbastela
hooperi. J. Org. Chem. 1997, 62, 3837-40.
29.
Harris, J. C.; Plummer, S.; Turner, M. P.; Lloyd, D., The microaerophilic
flagellate Giardia intestinalis: Allium sativum (garlic) is an effective antigiardial.
Microbiol 2000, 146 Pt 12, 3119-27.
30.
Friedheim, E. A., Mel B in the treatment of human trypanosomiasis. Am J Trop
Med Hyg 1949, 29, (2), 173-80.
31.
Lun, Z. R.; Min, Z. P.; Huang, D.; Liang, J. X.; Yang, X. F.; Huang, Y. T.,
Cymelarsan in the treatment of buffaloes naturally infected with Trypanosoma
evansi in south China. Acta Trop 1991, 49, (3), 233-6.
32.
Zweygarth, E.; Kaminsky, R., Evaluation of an Arsenical Compound (Rm-110,
Mel Cy, Cymelarsan) against susceptible and drug-resistant Trypanosoma-BruceiBrucei and Tb-Evansi. Trop Med Parasitol 1990, 41, (2), 208-12.
33.
Loiseau, P. M.; Lubert, P.; Wolf, J. G., Synthesis and in vitro anthelmintic
properties of some new dithiaarsanes. Arzneimittelforschung 1999, 49, (11), 94450.
112
34.
Tsao, S. M.; Hsu, C. C.; Yin, M. C., Garlic extract and two diallyl sulphides
inhibit methicillin-resistant Staphylococcus aureus infection in BALB/cA mice. J
Antimicrob Chemoth 2003, 52, (6), 974-80.
35.
Tandon, V. K.; Chhor, R. B.; Singh, R. V.; Rai, S.; Yadav, D. B., Design,
synthesis and evaluation of novel 1,4-naphthoquinone derivatives as antifungal
and anticancer agents. Bioorg Med Chem Lett 2004, 14, (5), 1079-83.
36.
Hernandez-Campos, A.; Ibarra-Velarde, F.; Vera-Montenegro, Y.; RiveraFernandez, N.; Castillo, R., Synthesis and fasciolicidal activity of 5-chloro-2methylthio-6-(1-naphthyloxy)-1H-benzimidazole. Chem Pharm Bull (Tokyo)
2002, 50, (5), 649-52.
37.
Naganawa, R.; Iwata, N.; Ishikawa, K.; Fukuda, H.; Fujino, T.; Suzuki, A.,
Inhibition of microbial growth by ajoene, a sulfur-containing compound derived
from garlic. Appl Environ Microb 1996, 62, (11), 4238-42.
38.
San-Blas, G.; San-Blas, F.; Gil, F.; Marino, L.; Apitz-Castro, R., Inhibition of
growth of the dimorphic fungus Paracoccidioides brasiliensis by ajoene.
Antimicrob Agents Chemother 1989, 33, (9), 1641-4.
39.
Waring, P.; Beaver, J., Gliotoxin and related epipolythiodioxopiperazines. Gen
Pharmacol 1996, 27, (8), 1311-6.
40.
Fukuyama, T.; Nakatsuka, S.; Kishi, Y., Total synthesis of gliotoxin,
dehydrogliotoxin and hyalodendrin. Tetrahedron 1981, 37, (11), 2045-78.
41.
Rodriguez, A. D.; Akee, R. K.; Scheuer, P. J., 2 Bromotyrosine cysteine derived
metabolites from a sponge. Tetrahedron Lett 1987, 28, (42), 4989-92.
42.
Pham, N. B.; Butler, M. S.; Quinn, R. J., Isolation of psammaplin A 11'-sulfate
and bisaprasin 11'-sulfate from the marine sponge Aplysinella rhax. J Nat Prod
2000, 63, (3), 393-5.
43.
Moriarty, R. M.; Roll, D. M.; Ku, Y. Y.; Nelson, C.; Ireland, C. M., A Revised
Structure for the Marine Bromoindole Derivative Citorellamine. Tetrahedron Lett
1987, 28, (7), 749-52.
44.
Roll, D. M.; Ireland, C. M., Citorellamine, a New Bromoindole Derivative from
Polycitorella-Mariae. Tetrahedron Lett 1985, 26, (36), 4303-6.
45.
Relf, J. M.; Chisholm, J. R.; Kemp, G. D.; Smith, V. J., Purification and
characterization of a cysteine-rich 11.5-kDa antibacterial protein from the
granular haemocytes of the shore crab, Carcinus maenas. Eur J Biochem 1999,
264, (2), 350-7.
113
46.
Turos, E.; Revell, K. D.; Ramaraju, P.; Gergeres, D. A.; Greenhalgh, K.; Young,
A.; Sathyanarayan, N.; Dickey, S.; Lim, D.; Alhamadsheh, M. M.; Reynolds, K.,
Unsymmetric aryl-alkyl disulfide growth inhibitors of methicillin-resistant
Staphylococcus aureus and Bacillus anthracis. Bioorg Med Chem 2008, 16, (13),
6501-8.
47.
Yoshida, S.; Kasuga, S.; Hayashi, N.; Ushiroguchi, T.; Matsuura, H.; Nakagawa,
S., Antifungal activity of ajoene derived from garlic. Appl Environ Microbiol
1987, 53, (3), 615-7.
48.
Gmelin, R.; Susilo, R.; Fenwick, G. R., Cyclic Polysulfides from Parkia-Speciosa.
Phytochemistry 1981, 20, (11), 2521-3.
49.
Giannini, F. A.; Aimar, M. L.; Sortino, M.; Gomez, R.; Sturniollo, A.; Juarez, A.;
Zacchino, S.; de Rossi, R. H.; Enriz, R. D., In vitro-in vivo antifungal evaluation
and structure-activity relationships of 3H-1,2-dithiole-3-thione derivatives.
Farmaco 2004, 59, (4), 245-54.
50.
Ablordeppey, S. Y.; Fan, P.; Ablordeppey, J. H.; Mardenborough, L., Systemic
antifungal agents against AIDS-related opportunistic infections: current status and
emerging drugs in development. Curr Med Chem 1999, 6, (12), 1151-95.
51.
Aimar, M. L.; Kreiker, J.; de Rossi, R. H., One-pot synthesis of 3H-1,2-dithiole-3thione derivatives from dithiolmalonic esters. Tetrahedron Lett 2002, 43, (11),
1947-9.
52.
Garcia, C. C.; Candurra, N. A.; Damonte, E. B., Mode of inactivation of
arenaviruses by disulfide-based compounds. Antiviral Res 2002, 55, (3), 437-46.
53.
Mahmood, N.; Jhaumeer-Lauloo, S.; Sampson, J.; Houghton, P. J., Anti-HIV
activity and mechanism of action of macrocyclic diamide SRR-SB3. J Pharm
Pharmacol 1998, 50, (12), 1339-42.
54.
Garozzo, A.; Pinizzotto, M. R.; Guerrera, F.; Tempera, G.; Castro, A.; Geremia,
E., Antipoliovirus activity of isothiazole derivatives: mode of action of 5,5'diphenyl-3,3'-diisothiazole disulfide (DID). Arch Virol 1994, 135, (1-2), 1-11.
55.
Pinizzotto, M. R.; Garozzo, A.; Guerrera, F.; Castro, A.; La Rosa, M. G.; Furneri,
P. M.; Geremia, E., In vitro antiviral activity of four isothiazole derivatives
against poliovirus type 1. Antiviral Res 1992, 19, (1), 29-41.
56.
Roy, B.; Chambert, S.; Lepoivre, M.; Aubertin, A. M.; Balzarini, J.; Decout, J. L.,
Deoxyribonucleoside 2'- or 3'-mixed disulfides: prodrugs to target ribonucleotide
reductase and/or to inhibit HIV reverse transcription. J Med Chem 2003, 46, (13),
2565-8.
114
57.
Yuzhakov, A. A.; Chidgeavadze, Z. G.; Beabealashvilli, R., 3'-Mercapto-2',3'dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by
HIV reverse transcriptase. FEBS Lett 1992, 306, (2-3), 185-8.
58.
Coves, J.; Le Hir de Fallois, L.; Le Pape, L.; Decout, J. L.; Fontecave, M.,
Inactivation of Escherichia coli ribonucleotide reductase by 2'-deoxy-2'mercaptouridine 5'-diphosphate. Electron paramagnetic resonance evidence for a
transient protein perthiyl radical. Biochemistry 1996, 35, (26), 8595-602.
59.
Wratten, S. J.; Faulkner, D. J., Cyclic polysulfides from the red alga Chondria
californica. J Org Chem 1976, 41, (14), 2465-7.
60.
Lee, M. D.; Dunne, T. S.; Chang, C. C.; Ellestad, G. A.; Siegel, M. M.; Morton,
G. O.; Mcgahren, W. J.; Borders, D. B., Calichemicins, a novel family of
antitumor antibiotics. 2. Chemistry and structure of Calichemicin-γ-1. J Am Chem
Soc 1987, 109, (11), 3466-8.
61.
Long, B. H.; Golik, J.; Forenza, S.; Ward, B.; Rehfuss, R.; Dabrowiak, J. C.;
Catino, J. J.; Musial, S. T.; Brookshire, K. W.; Doyle, T. W., Esperamicins, a
class of potent antitumor antibiotics: mechanism of action. Proc Natl Acad Sci U
S A 1989, 86, (1), 2-6.
62.
Litaudon, M.; Trigalo, F.; Martin, M. T.; Frappier, F.; Guyot, M.,
Lissoclinotoxins - Antibiotic polysulfur derivatives from the tunicate
Lissoclinum-Perforatum - revised structure of Lissoclinotoxin-A. Tetrahedron
1994, 50, (18), 5323-34.
63.
Long, T. E.; Turos, E., N-Thiolated β-lactams. Curr. Med. Chem. - Anti-Infective
Agents 2002, 1, 251-68.
64.
Revell, K. D.; Heldreth, B.; Long, T. E.; Jang, S.; Turos, E., N-thiolated betalactams: Studies on the mode of action and identification of a primary cellular
target in Staphylococcus aureus. Bioorg Med Chem 2007, 15, (6), 2453-67.
65.
Sachs, G.; Scott, D.; Weeks, D.; Melchers, K., Gastric habitation by Helicobacter
pylori: insights into acid adaptation. Trends Pharmacol Sci 2000, 21, (11), 413-6.
66.
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., Methicillin-resistant S. aureus
infections among patients in the emergency department. New Engl J Med 2006,
355, (7), 666-74.
67.
Gillet, Y.; Issartel, B.; Vanhems, P.; Fournet, J. C.; Lina, G.; Bes, M.;
Vandenesch, F.; Piemont, Y.; Brousse, N.; Floret, D.; Etienne, J., Association
between Staphylococcus aureus strains carrying gene for Panton-Valentine
115
leukocidin and highly lethal necrotising pneumonia in young immunocompetent
patients. Lancet 2002, 359, (9308), 753-9.
68.
69.
Seybold, U.; Talati, N. J.; Kizilbash, Q.; Shah, M.; Blumberg, H. M.; FrancoParedes, C., Hematogenous osteomyelitis mimicking osteosarcoma due to
community associated methicillin-resistant Staphylococcus aureus. Infection
2007, 35, (3), 190-3.
Adem, P. V.; Montgomery, C. P.; Husain, A. N.; Koogler, T. K.; Arangelovich,
V.; Humilier, M.; Boyle-Vavra, S.; Daum, R. S., Brief report: Staphylococcus
aureus sepsis and the waterhouse-friderichsen syndrome in children. New Engl J
Med 2005, 353, (12), 1245-51.
70.
Rutar, T.; Chambers, H. F.; Crawford, J. B.; Perdreau-Remington, F.; Zwick, O.
M.; Karr, M.; Diehn, J. J.; Cockerham, K. P., Ophthalmic manifestations of
infections caused by the USA300 clone of community-associated methicillinresistant Staphylococcus aureus. Ophthalmology 2006, 113, (8), 1455-62.
71.
Klevens, R. M.; Morrison, M. A.; Nadle, J.; Petit, S.; Gershman, K.; Ray, S.;
Harrison, L. H.; Lynfield, R.; Dumyati, G.; Townes, J. M.; Craig, A. S.; Zell, E.
R.; Fosheim, G. E.; McDougal, L. K.; Carey, R. B.; Fridkin, S. K., Invasive
methicillin-resistant Staphylococcus aureus infections in the United States. JAMA
2007, 298, (15), 1763-71.
72.
Turos, E.; Long, T. E.; Konaklieva, M. I.; Coates, C.; Shim, J. Y.; Dickey, S.;
Lim, D. V.; Cannons, A., N-thiolated β-lactams: novel antibacterial agents for
methicillin-resistant Staphylococcus aureus. Bioorg Med Chem Lett 2002, 12,
(16), 2229-31.
73.
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., Effect of
aryl ring fluorination on the antibacterial properties of C4 aryl-substituted Nmethylthio β-lactams. Bioorg Med Chem 2003, 11, (8), 1859-63.
74.
Coates, C.; Long, T. E.; Turos, E.; Dickey, S.; Lim, D. V., N-Thiolated β-lactam
antibacterials: defining the role of unsaturation in the C4 side chain. Bioorg Med
Chem 2003, 11, (2), 193-6.
75.
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., NMethylthio β-lactam antibacterials: effects of the C3/C4 ring substituents on antiMRSA activity. Bioorg Med Chem 2005, 13, (23), 6289-308.
76.
Heldreth, B.; Long, T. E.; Jang, S.; Reddy, G. S.; Turos, E.; Dickey, S.; Lim, D.
V., N-Thiolated β-lactam antibacterials: effects of the N-organothio substituent on
anti-MRSA activity. Bioorg Med Chem 2006, 14, (11), 3775-84.
116
77.
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., Nthiolated β-lactams: a new family of anti-Bacillus agents. Bioorg Med Chem Lett
2006, 16, (8), 2084-90.
78.
Mishra, R. K.; Revell, K. D.; Coates, C. M.; Turos, E.; Dickey, S.; Lim, D. V., Nthiolated 2-oxazolidinones: a new family of antibacterial agents for methicillinresistant Staphylococcus aureus and Bacillus anthracis. Bioorg Med Chem Lett
2006, 16, (8), 2081-3.
79.
Kagami, H.; Motoki, S., Nucleophilic-substitution on dialkoxy disulfides reactions with mercaptans or amines. J Org Chem 1977, 42, (25), 4139-41.
80.
Kagami, H.; Hanzawa, T.; Suzuki, N.; Yamaguchi, S.; Saito, M.; Motoki, S.,
Nucleophilic-substitution on dialkoxy disulfides. 3. Reaction with thioureas. B
Chem Soc Jpn 1980, 53, (12), 3658-60.
81.
Kagami, H.; Motoki, S., Nucleophilic-substitution on dialkoxy disulfides. 2.
Reactions with hydrazine derivatives. B Chem Soc Jpn 1979, 52, (11), 3463-4.
82.
Oyston, P. C. F.; Sjöstedt, A.; Titball, R. W., Tularaemia: bioterrorism defence
renews interest in Francisella tularensis. Nat Rev Microbiol 2004, 2, (12), 967-78.
83.
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., Tularemia as a biological weapon: medical and public health
management. JAMA 2001, 285, (21), 2763-73.
84.
Harris, S., Japanese biological warfare research on humans: a case study of
microbiology and ethics. Ann N Y Acad Sci 1992, 666, 21-52.
85.
Tarnvik, A.; Berglund, L., Tularaemia. Eur Resp J 2003, 21, (2), 361-73.
86.
Ikaheimo, I.; Syrjala, H.; Karhukorpi, J.; Schildt, R.; Koskela, M., In vitro
antibiotic susceptibility of Francisella tularensis isolated from humans and
animals. J Antimicrob Chemother 2000, 46, (2), 287-90.
87.
O’Driscoll, M.; Greenhalgh, K.; Young, A.; Turos, E.; Dickey, S.; Lim, D. V.,
Studies on the antifungal properties of N-thiolated β-lactams. Bioorg Med Chem
2008, 16, (16), 7832-7.
88.
Gudlaugsson, O.; Gillespie, S.; Lee, K.; Vande Berg, J.; Hu, J.; Messer, S.;
Herwaldt, L.; Pfaller, M.; Diekema, D., Attributable mortality of nosocomial
candidemia, revisited. Clin Infect Dis 2003, 37, (9), 1172-7.
117
89.
Grover, N., Echinocandins: A ray of hope in antifungal drug therapy. Indian J
Pharmacol 2010, 42, (1), 9.
118