Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin–resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmcl.2018.09.038

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64 μg/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.

Full text of DOI:10.1016/j.bmcl.2018.09.038

Accepted Manuscript
Structure-Activity Relationships of Pyrazole-4-carbodithioates as Antibacteri-
als against Methicillin−Resistant Staphylococcus aureus
Hiwa Majed, Tatiana Johnston, Celine Kelso, Enrico Monachino, Slobodan
Jergic, Nicholas E. Dixon, Eleftherios Mylonakis, Michael J. Kelso
PII:
S0960-894X(18)30780-7
https://doi.org/10.1016/j.bmcl.2018.09.038
BMCL 26059
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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23 September 2018
28 September 2018
Please cite this article as: Majed, H., Johnston, T., Kelso, C., Monachino, E., Jergic, S., Dixon, N.E., Mylonakis,
E., Kelso, M.J., Structure-Activity Relationships of Pyrazole-4-carbodithioates as Antibacterials against Methicillin
−Resistant Staphylococcus aureus, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/
j.bmcl.2018.09.038
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Structure-Activity Relationships of Pyrazole-4-carbodithioates as Antibacterials against
Methicillin−Resistant Staphylococcus aureus
Hiwa Majeda, Tatiana Johnstonb, Celine Kelsoa, Enrico Monachino,a Slobodan Jergic,a
Nicholas E. Dixon,a Eleftherios Mylonakisb, Michael J. Kelsoa*
aSchool of Chemistry, University of Wollongong, and Illawarra Health and Medical Research
Institute, Wollongong, New South Wales 2522, Australia; bDepartment of Infectious Disease,
Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI, 02903,
USA.
*To whom correspondence should be addressed. Email: mkelso@uow.edu.au, Tel.: +61 (0)2
4221 5085, Fax: +61 (0)2 4221 4287
Keywords: antibacterial, Staphylococcus aureus, MRSA, pyrazole-4-carbodithioate, MgrA
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-
acquired infections and is responsible for significant morbidity and mortality in residential
care facilities. New agents against MRSA are needed to combat rising resistance to current
antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate
(HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel
mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA
structure-activity relationships evaluated and selectivity versus human HKC-8 cells
determined. Minimum inhibitory concentrations (MIC) ranged from 0.5−64 g/mL and up to
16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for
activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The
study supports further work aimed at elucidating the molecular targets of this interesting new
class of anti-MRSA agents.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most common cause of hospital-
acquired infections1,2 and a frequent source of skin and soft tissue infections in the North and
Latin Americas, Europe and Asia.3,4 In the USA, MRSA accounts for almost 60% of clinical
S. aureus strains isolated from intensive care units5 and it is widespread in residential care
facilities.6,7 Worryingly, pathogenic strains are also becoming more prevalent in the community
(i.e. community-acquired MRSA).8 Vancomycin has been used extensively for several decades
to treat complicated S. aureus and other Gram-positive infections. However, concerns have
been growing over the increasing minimum inhibitory concentration (MIC) of the drug against
MRSA isolates,9 and its use has other well-known shortcomings (e.g. nephrotoxicity, complex
pharmacokinetics, requirement for slow intravenous infusion).10
It is evident that vancomycin’s effectiveness will continue to wane in the coming
years, thus compelling the discovery of effective new antibiotics against this major human
pathogen.
We recently reported the discovery of 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-
carbodithioate (HMPC) 1 (Scheme 1) as a new bacteriostatic agent against MRSA.11 The
compound showed the same minimum inhibitory concentration (MIC 4 g/mL) against
MRSA MW2 (lab strain) and six recent clinical isolates and was able to rescue
Caenorhabditis elegans from an MRSA infection. Whole-genome sequencing of mutants
resistant to 1 highlighted a role for the global defense regulator MgrA12 in its mechanism and
the compound displayed a S. aureus promoter-lux array luminescence profile distinct from all
major classes of antibiotics. HMPC 1 appears to exert anti-staphylococcal effects through a
novel, uncharacterized mechanism that involves an MgrA-mediated defense response. In the
current report, we explored the structural requirements for anti-MRSA activity and eukaryotic
cell selectivity in the pyrazole-4-carbodithioate class.

Initial efforts sought to understand the role of the 4-carbodithioate by replacing the
group with amide 2, thioamide 3 and ester 5 isosteres (Scheme 1). Analogs 2 and 3 were
formed by quenching the enolate of pyrazolin-3-one 7 (generated using K2CO3) with n-propyl
isocyanate and n-propyl isothiocyanate, respectively. Ester 5 was obtained by first generating
ketene dithioacetal 4 from 7 using K2CO3, CS2 and excess CH3I via the reported method.13
Subsequent reaction of 4 with sodium in n-PrOH at 80 °C delivered 5. Compounds 2, 3, 5
and intermediate 4 all showed no activity against MRSA MW2 (MIC > 64 g/mL),
establishing the critical importance of the 4-carbodithioate function.
The role of the neighboring 3-OH substituent was investigated next but attempting to prepare
the O-Me ether of 1 by treatment with K2CO3 and excess CH3I instead delivered ketene
dithioacetal 6 (44% yield). Compound 6 was also found to be inactive against MRSA (MIC >
128 g/mL).
S
O
O
S
NH
OH
O
N
N
N
O
OH
N
N
N
e
a
b
c
2
4
5
N
O
N
S
S
S
NH
OH
S
S
d
N
N
N
O
OH
N
N
N
7
f
3
1
6
HMPC
Scheme 1. Reagents and conditions: a. K2CO3, n-propyl isocyanate, DMF/benzene, rt, 56%;
b. K2CO3, n-propyl isothiocyanate, DMF/benzene, 50 °C, 80% c. K2CO3, CS2, CH3I,
DMF/benzene, 0 °C, 66%; d. n-BuLi, CS2, n-bromopropane, THF, 0-25 °C, 52%,14 e. Na(s),
n-PrOH, 80 °C, 91%;15 f. K2CO3, CH3I, DMF 0-25 °C, 90%.

Replacement of the carbodithioate n-propyl chain with Me, Et, allyl, n-Bu, Bn and 3-
pyridyl groups was explored next. Each of these was obtained by forming the enolate of 7
with n-butyllithium and successively quenching with CS2 and the appropriate alkyl halide
(Figure 1(a)). Shortening the chain to one carbon 8a led to a 4-fold drop in potency, while Et
derivative 8b produced a 2-fold loss. Introduction of an alkene 8c maintained or slightly
reduced potency and extension by 1 carbon 8d increased potency 2-fold. Addition of steric
bulk and hydrophobicity with a benzyl group 8e resulted in a 2-fold increase in activity, but a
significant drop in potency occurred when a nitrogen atom was introduced into the benzylic
substituent 8f (MIC 16-32 mg/mL, Figure 1(b)).
In our previous report,11 we showed that HMPC 1 does not cause hemolysis of human
red blood cells at concentrations up to 64 g/mL but is cytotoxic towards eukaryotic HKC-8
and HepG2 cells at concentrations around its MRSA MIC. Here, cytotoxicity of analogs 8a-e
was tested in HKC-8 cells and selectivity indices were calculated (SI = HKC-8 IC50/MRSA-
MW2 MIC, Figure 1(b)). No MRSA selectivity was observed for 8a, 8b or 8d and modest
selectivity (2-4 fold) was seen with allyl derivative 8c. Benzylic derivative 8e delivered the
highest selectivity (8-16 fold) in this series.
A variety of halo, electron donating and electron withdrawing substituents were added
to the 4-position of the pyrazole N-phenyl group. Commercially available 4-substituted
phenylhydrazine.HCl salts 9a-g were condensed with ethyl acetoacetate to form pyrazol-3-
one intermediates 10a-g in 25-87% yield. Treating ketones 10a-g with n-butyllithium and
quenching the enolates with CS2 followed by n-bromopropane gave targets 11a-g in 32-75%
yield (Figure 1(a)).
Addition of a Me group 11a gave no change in activity (relative to 1) while
introducing an electron donating methoxy group 11b produced a 2-fold loss. Halogen
substituents 11c-e gave slight increases in potency (2-4 fold), as did electron withdrawing

cyano and nitro groups, with the p-NO2-substituted analog 11g showing the highest activity
(MIC 0.5-1 g/mL). Corresponding increases in eukaryotic cell cytotoxicity were observed
though, with 11g showing no MRSA selectivity (Figure 1(b)).
R1
(b)
S
(a)
IC50
R3 g/mL g/mL SI
MIC
S
R1
R2
N
N
a
O
OH
1
8a
8b
n-Pr
Me
Et
4
16
8
4
16
8
1
1
1
N
N
8c
8d
8e
8f
allyl
n-Bu
Bn
4-8
2-4
2-4
16-32
4
16-32 2-4
4
1-2
S
7
HMPC 1, 8a-f
32
8-16
S
NH2.HCl
+
HN
3-pyridyl
11a
11b
11c
11d
11e
11f
11g
14a
14b
14c
14d
14e
14f
14g
14h
14i
O
O
Me
OMe
F
Cl
Br
N
O
N
N
OH
b
N
c
8
OEt
2-4
2-4
1-2
2
0.5-1 0.5-1
2
R2
9a-g
R2
R2
CN
NO2
10a-g
11a-g
1
R3
R3
H
32
8
8
16
4-8
4
Me 1-2
OMe 2
S
NH2.HCl
O
O
S
HN
b
c
F
Cl
Br
1
4
4
+
OEt
0.5-1
0.5
4
4-8
N
N
N
O
OH
N
R3
12a-i
CF3 32-64
CN
NO2
2
1
0.5
0.5
13a-i
14a-i
Figure 1. (a) Synthesis of pyrazole-4-carbodithioate analogs. Reagents and conditions: a. n-
BuLi, CS2, alkyl halide, THF, 0-25 °C, 59-84%; b. glacial acetic acid, EtOH, 45-50 °C, 19-
90%; c. n-BuLi, CS2, n-bromopropane, THF, 0-25 °C. (b) Antibacterial activities (MIC
g/mL) against MRSA-MW2, cytotoxicity towards HKC-8 cells (IC50 g/mL) and selectivity
indices (SI).
Replacement of the 3-methyl group of 1 with a phenyl ring was explored next.
Condensation of phenylhydrazine.HCl with ethyl benzoylacetate 12a produced pyrazol-3-one
13a, which upon base treatment and successive quenching with CS2 and n-bromopropane
afforded 3-phenyl derivative 14a in 90% yield. Compound 14a showed a 2-fold increase in
MRSA potency and a 16-fold increase in selectivity. The promising selectivity obtained upon
addition of the phenyl ring at the 3-position led to exploration of para-substituted analogs
carrying halo, electron donating and electron withdrawing substituents. Condensation of p-

substituted ethylbenzoylacetates 12b-i with phenylhydrazine.HCl and appending n-propyl
dithioate groups to the resulting ketones 13b-i delivered target analogs 14b-i. Addition of the
Me group 14b led to a slight increase in potency (MIC 1-2 g/mL) relative to 1 but reduced
selectivity. The methoxy group 14c did not change activity against MRSA but selectivity was
reduced. Larger increases in antibacterial potency were achieved with halo groups 14d-f
(MIC 0.5-1 g/mL) but no improvements in selectivity were seen. A large drop in potency
was observed with the CF3 group 14g (MIC 32-64 mg/mL), while other strongly electron
withdrawing cyano 14h and nitro 14i substituents maintained activity but reduced selectivity.
We previously showed that treatment of a S. aureus promoter-lux array with HMPC 1
produces a unique luminescence profile (suggesting a unique mechanism of action), but some
similarities to DNA-damaging agents and/or DNA replication inhibitors were noted.11 This
led to speculation that the anti-MRSA and apparent general cytotoxicity of 1 might arise from
DNA binding. However, UV/vis experiments measuring the binding of 1 to calf thymus DNA
and zone of growth inhibition disk measurements performed with 1 in the presence/absence
of calf thymus or S. aureus genomic DNA appeared to rule this out. Nevertheless, the similar
levels of HKC-8 toxicity observed in the current study with the majority of analogs of 1,
combined with the absolute requirement of a carbodithioate function for anti-MRSA activity,
led us to examine more closely whether 1 (and hence the class) may exert effects through
non-specific nucleic acid and/or protein reactivity.
The DNA-reactivity of 1 was probed by electrospray ionization mass spectrometry
(ESI-MS) using a panel of single stranded, double stranded and G-quadruplex DNA
oligonucleotides. When incubated with up to 10-fold excesses of HMPC 1 under a variety of
conditions, no evidence for any DNA:1 adducts was observed (Supporting Information
Figure S1). Similarly, no adducts were observed by ESI-MS when 1 was incubated with RNA
oligonucleotides (data not shown).

The effects of HMPC 1 on DNA replication in vitro were explored next. In this assay,
all of the enzymes, ancillary proteins, nucleotide precursors, DNA template and other
molecular components required for duplication of circular bacterial DNA are present and able
to effect replication in a cell-free environment.16 Covalent reactivity with any of the reaction
components would be expected to read out as inhibition of replication. However, only slight
inhibition of replication was observed with 1 at concentrations > 160 M, well above its MIC
against MRSA (Supporting Information Figure S2). In vitro RNA transcription assays
similarly showed no inhibition by 1 at relevant concentrations (data not shown). The absence
of effects for 1 in these assays rules out non-specific nucleic acid or protein reactivity as a
contributor to the anti-MRSA mechanism of the pyrazole-4-carbodithioate class.
In summary, pyrazole-4-carbodithioates are a new class of anti-MRSA agents that
require the 4-carbodithioate function for activity. Non-specific covalent reactivity appears not
to be part of the mechanism but intrinsic reactivity of the 4-carbodithioate may still play a
role. We showed previously that MgrA-mediated defense responses are triggered by 1.11
MgrA is an oxidation-sensing mechanism used by MRSA to counter challenges of reactive
oxygen and nitrogen species. Upon detecting these species, a unique cysteine residue (Cys12)
located at the dimer interface of the protein is oxidized to cysteine sulfenic acid, causing
dissociation of MgrA from DNA and initiation of signalling pathways that turn on antibiotic
resistance.17 We speculate that intracellular redox reactivity of the 4-carbodithioate function
(aided by the neighboring OH group) triggers oxidative stress that leads to MgrA activation.
Alternatively, metal chelation by the 4-carbodithioate and neighboring OH group may be
involved.18 While limited selectivity (maximum 16-fold) for MRSA over eukaryotic HKC-8
cells was achieved with the analogs explored here, further increases seem possible with a
larger analog set. Studies to fully elucidate the anti-MRSA mechanism and identify the
discrete intracellular targets using such selective analogs would undoubtedly prove insightful.

Author Contributions
H.M completed all synthetic chemistry. T.J completed all MRSA MIC and HKC-8
cytotoxicity measurements. Mass spectrometry studies were performed by C.K and
replication assays by E.M and S.J. N.D, E.M and M.K directed the project. All authors
contributed to writing the manuscript.
Notes
The authors declare no competing financial interests.
Acknowledgements
We thank the University of Wollongong (Wollongong, Australia) and Brown University (RI,
USA) for supporting this work. The study was partly funded by a P01 grant (AI083214,
National Institutes of Health) to Eleftherios Mylonakis and a Discovery Project grant
(DP150100956, Australian Research Council) to Nicholas Dixon.
Supplementary Data
Supplementary data associated with this article can be found, in the online version, at
XXXXX
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Highlights
 Twenty nine pyrazole-4-carbodithioates synthesized and tested for anti-MRSA
activity
 MIC range 0.5−64 g/mL with up to 16-fold selectivity over human HKC-8 cells
 Carbodithioate essential for activity

R1
S
R3
S
N
OH
N
Anti-MRSA pyrazoles
29 analogs (MIC 0.5-64 ug/mL)
4-carbodithioate essential
R2