Discovery and development of substituted thiadiazoles as inhibitors of Staphylococcus aureus Sortase A

Article abstract of DOI:10.1016/j.bmc.2019.115043

High-throughput screening of small-molecule libraries has led to the identification of thiadiazoles as a new class of inhibitors against Staphylococcus aureus sortase A (SrtA). N-(5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide (IC₅₀

Full text of DOI:10.1016/j.bmc.2019.115043

Journal Pre-Proof
Discovery and Development of Substituted Thiadiazoles as Inhibitors of Staph-
ylococcus aureus Sortase A
Patrick M. Wehrli, Ivana Uzelac, Thomas Olsson, Tomas Jacso, Daniel Tietze,
Johan Gottfries
PII:
S0968-0896(19)30394-3
https://doi.org/10.1016/j.bmc.2019.115043
BMC 115043
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
5 April 2019
8 July 2019
6 August 2019
Please cite this article as: Wehrli, P.M., Uzelac, I., Olsson, T., Jacso, T., Tietze, D., Gottfries, J., Discovery and
Development of Substituted Thiadiazoles as Inhibitors of Staphylococcus aureus Sortase A, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115043
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Discovery and Development of Substituted Thiadiazoles
as Inhibitors of Staphylococcus aureus Sortase A
Patrick M. Wehrli a, b, †, Ivana Uzelac a, †, Thomas Olsson a, *, Tomas Jacso c, d, Daniel Tietzea,
and Johan Gottfries a, b
aDepartment of Chemistry and Molecular Biology, Medicinal Chemistry, University of
Gothenburg, Gothenburg, Sweden
bCentre for Antibiotic Resistance Research (CARe) at University of Gothenburg, Gothenburg,
Sweden
cStructure & Biophysics, Discovery Sciences, AstraZeneca R&D, Sweden
dEarly Discovery, Department of Biology, Nuevolution AB, Denmark
KEYWORDS: Staphylococcus aureus, Sortase A, SrtA inhibitors, Anti-virulence drugs,
Antimicrobial resistance.
ABSTRACT
High-throughput screening of small-molecule libraries has led to the identification of thiadiazoles
as a new class of inhibitors against Staphylococcus aureus sortase A (SrtA). N-(5-((4-
nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide (IC50 = 3.8 µM) was identified as a potent
inhibitor of SrtA after synthetic modification of hit compounds. Additional ligands developed in
this study displayed affinities in the low micromolar range without affecting bacterial growth in
vitro. The study also suggest a new mode of action through covalent binding to the active site
cysteine.
*Corresponding Author.
e-mail adress: thomas.olsson@chem.gu.se
†The authors contributed equally to this work.
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1. Introduction
Staphylococcus aureus is an opportunistic pathogen that can cause a range of life-threatening
diseases with a high incidence of drug resistance. The rise of bacterial infections caused by
antibiotic-resistant strains, including methicillin-resistant S. aureus (MRSA), has become a serious
pathogenic threat to public health.1-2 MRSA is widespread and among the most commonly
identified antibiotic-resistant pathogens in Europe, the Americas, North Africa, the Middle East,
and East Asia.2 In addition, MRSA is often resistant to several widely used antibiotics,
complicating the burden to clinical treatment and resulting in increased risk of morbidity and
mortality.3-4 Multi-drug-resistant pathogens are a challenge for existing therapeutic options, and
their increasing occurrence mandates the discovery and development of novel treatment
strategies.5
Bacterial virulence enables bacteria to infect and damage a host, inhibition of virulence is
therefore an alternative approach to combat bacterial infections. Most virulence traits promote
infection and persistence but are not vital to bacterial survival or growth.6-8 Consequently, anti-
virulence therapies would have to rely solely on the host immune defence to clear the infection.
Disarming rather than killing the pathogen would reduce the selection pressure for drug-resistant
mutations and lead to fewer of the undesirable effects on the host microbiota that are associated
with current antimicrobials.6-7, 9 Virulence-associated proteins that are displayed on the surface of
S. aureus are essential for infection.10 The most prevalent group of surface proteins are microbial
surface component recognizing adhesive matrix molecules (MSCRAMMs) which contain a
sorting signal comprising LPXTG (Leu-Pro-X-Thr-Gly; where X is any amino acid).10 These
surface proteins mediate adherence to host tissue and are involved in the acquisition of nutrients,
biofilm formation, and immune evasion.10-11
The sortase A (SrtA) enzyme plays a critical role in the attachment of surface proteins to the
bacterial cell wall.12-13 In S. aureus, surface proteins are expressed with a C-terminal sorting signal,
including an LPXTG motif. SrtA catalyses the transpeptidation reaction between the LPXTG motif
of the protein and lipid II, resulting in an LPXT-lipid-II precursor which is incorporated into the
cell wall during cell wall biosynthesis.14 S. aureus mutants, lacking SrtA, are defective in
establishing an infection because they fail to display surface proteins.13, 15 These in vivo studies
clearly validate the importance of SrtA and the virulence factors in the establishment of infections.
In light of the conservation and widespread use of SrtA by various pathogens inhibition of SrtA
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by small molecules presents a powerful anti-virulence strategy.16 In 2015, recent advances in the
development of SrtA inhibitors were comprehensively reviewed by Cascioferro et al.17 A number
of compounds with SrtA inhibitory activity have been identified as a result of screening efforts
using natural product or small molecule libraries, and via computational strategies. These have
shown IC50 values of compounds with the greatest potency to range between 0.2 and 167 µM.17
The proof of concept of small molecule SrtA inhibitors as anti-infective drugs has been
demonstrated in vivo.9, 18 However, many of the currently reported SrtA inhibitors have limited
potency, undesirable structural moieties or poor selectivity, and require further optimisation in
order to be therapeutically useful.
In this study, we performed high-throughput screening of ~ 28500 compounds, originated from
ChemBridge (San Diego, CA) and Biovitrum AB (Sweden) and selected based on chemical
diversity, to identify structurally new SrtA inhibitors. The most promising hits were synthetically
optimised for increased inhibitory potency.
2. Result and Discussion
Two small-molecule libraries were screened for potential SrtA inhibition using a FRET-based
functional assay.19 Primary hits were evaluated using concentration-response curves, 2D
heteronuclear multiple-quantum coherence (HMQC) NMR experiments, 1D carr-purcell-
meiboom-gill (CPMG) sequence NMR experiments, LCMS (purity and ID), and were also
selected based on favourable structural properties, such as reversibility and novelty. Synthetic
modification of the most promising hit was performed as hit-to-lead optimisation where
derivatives were characterised by enzymatic inhibition, NMR binding studies, binding
reversibility, and their minimum inhibitory concentration (MIC) for bacterial growth.
2.1 HTS results and compound selection
High-throughput screening of two small-molecule libraries with a total of ~ 28500 compounds
yielded 110 primary hits with an apparent inhibitory activity of more than 60% at 10 µM.
Concentration-response analysis identified 60 compounds with a concentration-dependent assay
readout. The previously known S. aureus SrtA inhibitor Morin (IC50 = 37.39 ± 3.14 µM) was used
as a positive control.20 The hits were structurally evaluated to identify pan-assay interference
compounds (PAINS), which are able to interfere in a range of assays and with various target
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proteins.21-22 Identified compounds with possible assay interference included aryl imides (n = 21),
benzisothiazolinones (n = 12), rhodanines (n = 10), aryl sulfonamides (n = 10), and alkenyl
barbiturates (n = 5, Figure 1). With the exception of aryl imides all of the above-mentioned
compound classes have been described as PAINS.21 However, PAINS classification alone does
not necessarily exclude a compound from further investigation. Both, rhodanines and substituted
benzisothiazolinones have previously been explored as SrtA inhibitors.23-24 Hit structures were
excluded from further investigations when PAINS classification was accompanied by an
unfavourable structure-activity relationship (SAR) or when the structures had previously been
explored as SrtA inhibitors.
Figure 1. Classes of Assay Interference Compounds found in Primary Hits.
Hit compounds 1–4 (Figure 2) were confirmed as SrtA binders by ligand detected protein
binding studies (1D CPMG NMR experiments). Compounds 1 and 3 might exhibit a common
pharmacophore. Hit compound 2 has previously been reported as a potent SrtA inhibitor (IC50 =
1.4 µM) after synthetic optimisation by Suree et al. and inhibits SrtA by covalent binding.23
Caffeine derivative hit structure 4 was validated as a non-reversible binder and later found to have
three analogues, which were recovered via auto-fluorescence model (See Supporting Information).
Compound 1 was a confirmed binder in both ligand- and protein-detected NMR experiments. In
addition, it displayed reversible binding properties, confirmed by NMR displacement experiments,
and contained multiple structural features for possible optimisation of its affinity to SrtA.
Therefore, 1 was selected as the starting point for structural hit-to-lead optimisation. Previously,
structurally related compounds have been shown to be cytotoxic;25 therefore, an objective of the
hit-to-lead optimisation was to remove the key structural features responsible for this action.
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Figure 2. Top Hit Structures
2.2 Chemistry
The synthetic routes for compounds 5a–h, 6a–g, 7a–c, 8a–b, 9a–d, and 10a–b are presented in
Scheme 1. Structurally simplified first-generation analogues of 1 (Figure 2) are phenyl sulphides
(7a-c). Phenyl sulphide 7a was prepared from 5-amino-1,3,4-thiadiazole-2-thiol and commercially
available 4-nitrobenzenediazonium tetrafluoroborate in 77% yield. For the preparation of 7b-c, the
corresponding diazonium salts were prepared in situ. However, due to low conversion and
purification problems, the final products (7b-c) were obtained in low yields (1–7%). Isolation was
achieved only after Phenolverkochung26 of unreacted diazonium salt subsequent to the coupling.
Benzyl- and phenethyl sulphides 5a-g (Table 1) were synthesised to explore how elongation of the
sulphide substituent and the electronic nature of the benzyl substituent effect the activity. 5-
Amino-1,3,4-thiadiazole-2-thiol was reacted with the appropriate bromide under basic conditions,
yielding the desired products (5a-g) in moderate to high yields (62–97%). The amine was changed
to an amide function (Tables 2 and 3) by acetylation of 5a-b which was achieved upon treatment
with acetic anhydride in acetic acid at 65 °C for 10 min to afford amides 6a and 6b (37–41%).27
Similarly, treatment of 5a-b with the appropriate acid chloride obtained amides 6c-g in moderate
yields (44–81%).28 Nicotinoyl- and isonicotinoyl chloride, used in the preparation of 6d-g, were
prepared in situ from their corresponding carboxylic acids by refluxing in SOCl2 for 4 h.29
Sandmeyer-type bromination of amines 5a-b using tert-butyl nitrite and copper(II) bromide in
acetonitrile at −10 °C gave bromothiadiazole 8a-b in 38–44% yield.30 To establish whether this
part of the pocket could be expanded and whether it may require an amine, compounds 9a-d and
10a-b were prepared. Buchwald–Hartwig amination of bromothiadiazole 8a-b with the
appropriate amine using Pd2(dba)3/BINAP as the catalyst system gave piperazines and
morpholines 9a-d in moderate yields (29–69%).31-32 Aryl amines 10a-b, were obtained via the p-
toluenesulfonic acid catalysed reaction of bromothiadiazole 8a with the appropriate
aminopyridine.33 Due to the instability of the free-base form of 10a, the compound was isolated as
tosylate in 92% yield.
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Scheme 1. General Methods for the Synthesis of Compounds 5a–h, 6a–g, 7a–c, 8a–b, 9a–d, and
a
10a–ba. Reagents and conditions: (a) appropriate benzyl- or phenethyl bromide, NaOH, H2O,
22 °C, 15 h, 6297%; (b) 6ab: Ac2O, AcOH, 65 °C, 10 min, 37%; 6cg: acid chloride (in some
cases prepared from the corresponding carboxylic acid, SOCl2, reflux, 4 h), pyridine, 0→22 °C,
15 h, 4481%; (c) 7a: 4-nitrobenzenediazonium tetrafluoroborate, CH3CN, 21 °C, 5 min, 77%;
7bc: diazonium salt (prepared from appropriate aniline, HClaq, NaNO2, H2O, 0 °C, 20 min), H2O,
t
0→22 °C, 1.5 h; 17%; (d) CuBr2, CH3CN, BuONO, -10→21 °C, 15 h, 3844%; (e) amine,
Pd2(dba)3, BINAP, NaOtBu, 1,4-dioxane, 100 °C, 19 h, 2964%, (f) appropriate aniline, p-TsOH,
i-PrOH, 80 °C, 315 d, 392%.
2.3 Structure-Activity-Relationship (SAR) Studies
A series of modification of 1 (Table 1) included the removal of the oxadiazole moiety, which
has been shown to cause cytotoxicity,25 elongation of the sulphide substituent, and addition of
benzyl derivatives having different electronic effects. A previously reported FRET-based assay19
with an internally quenched fluorescent substrate analogue was used to study the activity of SrtA
and 1D CPMG and 2D HMQC NMR experiments were used to detect ligand binding. Removal of
the oxadiazole moiety resulted in significantly decreased activity (7a) while elongation of the
sulphide substituent by introducing of benzyl-derivatives (5a–h) restored the binding capability
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and increased inhibitory activity (5a–c). The most potent compound identified in the series was
4-nitrobenzyl derivative 5b (IC50 = 26 µM). Removal of the nitro group significantly lowered its
potency (5d).
Table 1. Evaluation results of compounds 1, 5a–h, and 7a–c in FRET assay, and 1D CPMG
NMR experiments.
FRET
FRET
NMR
CPMG
cmpd
1
R1
Inhib (%)a,b,c IC50 (µM)d,e,f
101 ± 0.7b,c
6.2 ± 0.2
active
5a
5b
5c
5d
5e
5f
11 ± 4.3
101 ± 1.6
1 ± 3.8
521 ± 52
26 ± 0.7
1745 ± 129
n.d.
active
active
active
0 ± 3.8
non-active
non-active
non-active
5 ± 3.9
n.d.
2 ± 3.6
n.d.
5g
5h
7a
7b
13 ± 3.3
13 ± 2.8
35 ± 2.3c
25 ± 3.7
n.d.
n.d.
n.a.
n.d.
non-active
sol. Issue
non-active
non-active
aInhibition ± standard deviation (SD) (n = 3) at 200 µM with 15 min incubation time. bInhibition
± SD (n = 2) at 167 µM with 15 min incubation time. cFluorescence quenching properties. dIC50 ±
SD (n = 3). en.a. = not applicable due to FRET assay interference. fn.d. = not determined. sol. Issue
= solubility issue.
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The binding activity appeared to be nitro-group specific rather than depending on the electronic
nature of the substituent (5e, 5f). Ortho or para substituted nitrothiophenol ethers further show
fluorescence quenching properties (1, 7a, 7c). No FRET assay interference was observed for any
other compounds in table 1.
Table 2. Evaluation results of compounds 5b, 6b, 6f–g, 8b and 9c–d, in FRET assay, and 1D
CPMG NMR experiments.
FRET
FRET IC50
(µM)b
NMR
CPMG
cmpd
5b
R2
Inhib (%)a
101 ± 1.6
94 ± 0.8
99 ± 0.5
26 ± 0.7
127 ± 19
42 ± 2
active
6b
6f
active
non-active
6g
100 ± 0.4
3.8 ± 0.3
non-active
9c
104 ± 0.7
80 ± 1.3
9 ± 0.6
71 ± 2
active
active
9d
aInhibition ± standard deviation (SD) (n = 3) at 200 µM with 15 min preincubation time. bIC50 ±
SD (n = 3). sol. Issue = solubility issue.
The compound series in Table 2 are derivatives of 5b and included modifications of the amino
function and inhibit SrtA in the same range as 5b (~ 100 % at 200 µM). Considering the structural
diversity probed, the exocyclic amino group of 5b is likely not required for binding. Furthermore,
the size of the substituent in 6f may indicate unfilled volume in the binding pocket or protrusion
of the ligand from the protein. Compound 6g emerged as the most potent inhibitor (IC50 = 3.8 µM).
Ligand detected 1D CPMG NMR experiments confirmed binding and showed binding for 5b, 6b,
9c and 9d. Compound 8b exhibited solubility issues and 6g could be a false negative result due to
strong binding or slow exchange. Compound 6g was retested using the protein detected NMR
experiment, HMQC, which confirmed 6g as a binder (SI Figure 4).
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Table 3. Evaluation results of compounds 5a–b, 6a–d, 8, 9a–b, and 10a–b in FRET assay and 1D
CPMG NMR experiments.
FRET
NMR
CPMG
cmpd
5a
R2
Inhib (%)a
11 ± 4.3
18 ± 3.8
8 ± 2.9
active
sol. Issue
active
6a
6d
6e
13 ± 3.6
sol. Issue
6c
8a
9a
9b
6 ± 2.7
19 ± 2.2
29 ± 3.7
27 ± 2.7
sol. Issue
sol. Issue
active
non-active
10b
37 ± 2.3
sol. Issue
aInhibition ± standard deviation (SD) (n = 3) at 200 µM with 15 min preincubation time. sol. Issue
= solubility issue.
The compound series in Table 3 are derivatives of 5a and include similar modifications to those
described in Table 2. However, the modifications had no significant effects on the activity. A
longer pre-incubation time (extended from 15 min to 6.3 h), granting slow-binding ligands more
time to bind to the protein, slightly increased the potency of 9b (27% to 38% inhibition at 200 µM).
An apparent increase in the potency of other compounds, such as 8a and 10b, is likely attributable
to protein-ligand precipitation. Figure 3 summarises the SAR of the compound series.
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Figure 3. Structure activity relationship for the thiadiazoles in this study.
2.4 Mode of action
When adding DTT (500 µM) to the buffer most activity of the protein was regained when testing
1, 5b, and 6g, which could indicate that SrtA inhibition is caused by inactivation through oxidation
or disulfide bond formation to the active site cysteine (SI Figure 2). According to Melvin et al.,34
SrtA has an unusually high reduction potential which is why a reducing agent is rarely used in the
buffer when testing for SrtA inhibition. We found that DTT itself reacts with 1 which explains the
decrease in inhibitory activity; however, we observed no reaction between DTT and 5b or 6g.
Morin was used as a reference which retained its inhibitory activity (SI Figure 3).
The oxidation states of the active site cysteine was further determined using LCMS. Four
samples were analysed, protein +/- compounds 5b (200 µM) and 6g (200 µM), and reference +/-
dmso. All four samples showed similar results where mono-, di-, and trioxidation was observed
throughout all samples. Additional oxidation was not observed with addition of compounds which
makes the hypothesis of oxidation unlikely. However, other modifications were found, amino-
thiadiazolyl thio (+130.96119 Da, +C2HN3S2) was observed in the sample containing 5b,
nicotinamido-thiadiazolyl thio (+235.98265 Da, +C8H4N4OS2) in 6g, and nitrobenzyl thio
(+167.00410, +C7H5NO2S) was found in both 5b and 6g. This suggest that the mode of action
goes through a cysteine thiol reaction resulting in a disulfide bond which can be reduced with a
reducing agent.
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Figure 4. A) Docking results of 6g for Induced fit (carbon atoms colored green-yellow) and non-
covalent docking (carbon atoms colored orange, general coloring scheme: nitrogen – blue, oxygen
– red, Sulphur – green, protein backbone is colored in grey) B) Covalent docking of the
nicotinamide-thiadiazole thiol fragment of 6g and C) Superposed with the covalently bound
substrate (LPAX, carbon atoms colored cyan) of SrtA (PDB ID: 2KID).
2.5 Molecular modeling and docking
Experimental observations were followed up by molecular docking studies in which 5b and 6g
were docked in the active site of SrtA. Dockings were performed using both standard and induced
fit. Induced fit docking (IFD) was used because of the large flexibility of the active site and because
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structures such as 1T2W (crystal structure) and 2KID (NMR model) have significantly different
binding sites when it comes to size, positioning of amino acid residues, and loops β6-β7 and β7-
β8, even though their active sites are conserved. Suliman et al. argue that 1T2W represents an
initial substrate-SrtA binding mode followed by a large conformational change which leads to the
catalytic event where 2KID represents the catalytically competent enzyme.35 For both docking
approaches respective orientations were identified among the best scoring docking clusters,
suggesting a pre-orientation of the inhibitor in the binding pocket, which might facilitate
disulphide bond formation (Figure 4A). Both docking approaches with the full ligand 6g showed
that the ligand mainly occupies the binding pocket of the substrate. However, the orientation of
the ligand differed by 180° between IFD and non-covalent VINA docking. The covalent docking
of the thiadiazol fragment (Figure 4B) results in a conformation which aligns well with the bound
substrate of 2KID (Figure 4C). In both non-covalent and covalent docking Arg197 and His120
show hydrophobic interactions to the p-nitro phenyl and the pyridine rings. IFD shows pi-pi
interactions between His120 and the p-nitro phenyl and H-bonds between Arg197 and the amide
and Thr164 to the pyridine nitrogen.
2.6 Inhibition of bacterial growth
It has been demonstrated that defects in SrtA activity due to deletion or mutation of the srtA
gene does not affect in vitro growth of S. aureus,12-13 Bacillus anthracis,36 and lactobacilli.37
However, deletion of srtA reduced pathogenicity and mortality in vivo.38 This may suggest that
selective inhibition of SrtA, via small molecule inhibitors, may have a therapeutic effect in vivo
but would not impair bacterial growth in rich medium. To test the susceptibility of bacterial growth
we determined the minimum inhibitory concentration (MIC) of 5a–c, 6a, 6d, 9a, and 9b for two
strains of S. aureus, and E. coli. The MIC for all compounds tested was higher than the upper test
limit of 256 mg/L, indicating that the compounds are not intrinsically toxic to bacteria.
3. Conclusion
In this study we have identified promising small-molecule SrtA inhibitors which inhibit the
enzyme with IC50 values in the low micromolar range and are amongst the most potent SrtA
inhibitors published so far. An initial assessment of their selectivity and toxicity showed that they
do not affect bacterial growth which is of importance is the development of anti-virulence agents
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because it will not exert strong selective pressure towards development of drug resistance. Even
though SrtA is known for having a high reduction potential and therefore not prone to oxidation
we found that when using a reducing agent in the FRET assay the enzymatic activity was regained.
LCMS studies further showed that all samples with and without active compound have both
reduced and oxidized protein forms present (mono-, di-, and trioxidation). Interestingly, we found
modifications corresponding to fragments of 5b and 6g covalently bound through a disulfide bond
to the active site cysteine, which might result from a preoriented ligand towards the thiol of Cys184
as suggested from our docking studies.
4. Experimental section
4.1 High-throughput screening
A total of 28671 small-molecule compounds were screened at the Laboratories for Chemical
Biology Umeå (LCBU), Umeå University. The compound collections screened originated from
ChemBridge (San Diego, CA) and Biovitrum AB (Sweden) and were selected based on chemical
diversity. For the high-throughput screening, a fluorescence resonance energy transfer (FRET)
assay was used. The assay utilizes an internally quenched fluorescent substrate analog. The
hydrolysis of the substrate by SrtA is monitored as increase in fluorescence signal. In brief, SrtA
enzyme (recombinant SrtA enzyme, 17 678 Da) in FRET buffer (50 mM HEPES, 150 mM NaCl,
5 mM CaCl2, 0.005% Tween20, pH = 7.5) was pre-incubated for 30 min at 23 °C together with
library compounds (final assay concentration: 10 µM) before after addition of the substrate
solution (75 µM Abz-Leu-Pro-Glu-Thr-Gly-Lys(Dnp)-NH2, 15 mM H-Gly3-OH in FRET buffer)
and subsequent incubation for 60 min at 23 °C. The enzymatic activity was stopped by addition of
EDTA solution (0.5 M, pH = 8.0). The plates were read out (excitation 320 nm, emission 420 nm)
during pre-incubation (counter screen) and after addition of EDTA solution. Assay data from the
primary screen were evaluated in MScreen (Center for Chemical Biology, University of
Michigan). Screening was performed using white 384 well-plates whereby 64 wells of each plate
were dedicated to negative controls. The DMSO final assay concentration was 1.3%. More detailed
information about the FRET assay and materials and methods used for screening may be found in
supporting information.
4.2 Confirmation screen
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Primary hits from the HTS were further evaluated with concentration-response curves (CRC)
under conditions as used in the primary screen. Thereby, compounds concentrations were varied
in fivefold serial dilutions from 0.16 to 500 µM.
To test if the activity of the compounds is affected by addition of reducing agent, DTT (0.5 mM)
or TCEP (0.5 µM) was added to the buffer used in the primary screen and concentration response
curves were measured for varying concentrations (0.05166 µM) of 1, 5b, and 6g. Morin was used
as reference.20
4.3 NMR orthogonal screen
1D CPMG relaxation dispersion NMR experiments39 were performed for ligand-detected protein
binding and to assess binding reversibility. The CPMG pulse sequence consisted of 96 repetitive
constant 1H 180 pulse blocks (τ-180-τ, τ = 0.5 ms), 256 transient scans, and a relaxation delay of
2.5 s. Residual water suppression was achieved through excitation sculpting.40 Spectra of
compound solutions (100 µM) in buffer (50 mM 2H-Tris, pH 7.6, 150 mM NaCl, 2 mM CaCl2, in
95% H2O/ 5% D2O) were recorded and compared to spectra after addition of SrtA (final
concentration 10 µM). Ligand-binding was identified through decrease in compound signal, due
to T2 relaxation line-broadening, after addition of SrtA. Reversibility of binding was assessed
through competitive binding experiments by subsequent addition of a substrate analog (Abz-Leu-
Pro-Glu-Thr-Gly-Lys(Dnp)-NH2, final concentration of 100 µM). Successive recovery of
compound signal indicates reversible binding. All CPMG NMR experiments were measured at
293 K on a Bruker 800 MHz spectrometer equipped with a triple resonance cryogenic probe and
analysed in Topspin 3.0 (Bruker Biospin, Karlsruhe, Germany) or MestReNova 6.1.1 software
(Mestrelab Research, Escondido, CA).
Protein detected 2D SOFAST-HMQC41 NMR experiments were used in addition to CPMG
experiments to confirm binding by successive titration of the compounds (3300 µM) to 20 µM
of uniformly 15N labeled SrtA and 1% DMSO-d6. 1H-15N chemical shift changes were monitored
by SOFAST-HMQC experiments.
4.4 Sample Preparation for Liquid Chromatography-Mass Spectrometry Analysis
Aliquots of 25 µl (1/2 of the total amount) of the reaction mixture or control in the buffer
containing 50 mM HEPES, 150 mM NaCl and 5mM CaCl2 5mM were diluted with 70 µl of 50
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mM triethylammonium bicarbonate (TEAB) solution and incubated with mixing with 250 ng of
trypsin (Pierce Trypsin Protease, MS Grade, Thermo Fisher Scientific) at 37 °C for 2h; then, 100
ng more of trypsin was added to each vial and the reaction mixtures were incubated overnight. The
digested samples were acidified using 10% formic acid and purified using the Pierce C18 Spin
columns (Thermo Fisher Scientific) according to the manufacturer’s protocol. The purified
samples were dried on Speedvac and reconstituted in 3% acetonitrile, 0.2% formic acid for
analysis.
4.5 Liquid Chromatography-Mass Spectrometry
Each sample analyzed on an Orbitrap Fusion Tribrid mass spectrometer interfaced with Easy-
nLC1200 liquid chromatography system (both Thermo Fisher Scientific). Peptides were trapped
on an Acclaim Pepmap 100 C18 trap column (100 μm x 2 cm, particle size 5 μm, Thermo Fischer
Scientific) and separated on an in-house packed analytical column (75 μm x 30 cm, particle size 3
μm, Reprosil-Pur C18, Dr. Maisch) using a linear gradient from 5% to 32% B over 47 min followed
by an increase to 100% B in 3 min, and 100% B for 10 min at a flow of 300 nL/min. Solvent A
was 0.2% formic acid in water and solvent B was 80% acetonitrile, 0.2% formic acid. MS1 scans
were performed at 120 000 resolution, m/z range 350-1500, the most abundant doubly or multiply
charged precursors from the MS1 scans were selected with a “top speed” cycle of 3 s, isolated with
a 1.2 Da window, fragmented via the higher-energy collision induced dissociation (HCD) at the
energy setting of 28 with a maximum injection time of 200 ms, and the MS2 spectra were detected
in the Orbitrap at 30 000 resolution. Dynamic exclusion was enabled with 10 ppm tolerance and
30 s duration.
4.6 Mass Spectrometry Data Analysis
Database search and further processing of the LC-MS files was performed using Proteome
Discoverer version 2.2 (Thermo Fisher Scientific). The fasta sequence database contained the
sortase A sequence and the common proteomic contaminants (74 sequences in total). The database
search was performed using the Mascot search engine v. 2.5.1 (Matrix Science, London, UK) with
precursor mass tolerance of 10 ppm and fragment ion tolerance of 0.02 Da. Trypsin/P was used as
a cleavage rule with 2 allowed missed cleavage; the following dynamic modifications on Cysteine
residue were used: monooxidation (+15.99491 Da, +O), dioxidation (+31.98983 Da, +2O),
trioxidation (+47.98474 Da, +3O), amino-thiadiazolyl thio (+130.96119 Da, +C2HN3S2),
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nicotinamido-thiadiazolyl thio (+235.98265 Da, +C8H4N4OS2), nitrobenzyl thio (+167.00410,
+C7H5NO2S). FixedPSM node was used to validate the identification results. Minora feature
detection node in Proteome Discoverer 2.2 was used to calculate the peptide signal abundances.
4.7 Molecular modeling.
All molecular modeling and in silico analysis steps were performed employing the Induced Fit
Docking protocol Schrödinger Suite 2019-242 or YASARA molecular modeling program.43-44
Molecular graphics were created with YASARA (www.yasara.org) and POVRay
(www.povray.org). The already known structures of SrtA (PDB ID: 2KID and 1T2W) were used
for the modeling.
4.8 Energy minimization and simulation protocol
The structure of the sortase (pdb ID: 2KID) and the individual in-silico built inhibitors were
energy minimized prior to the docking experiments.
To remove bumps and correct the covalent geometry, the structure of the protein was energy-
minimized with the AMBER14 force field,45-47 whereas the Yasara2 force field48 was used for the
ligands. A 8 Å force cutoff and the Particle Mesh Ewald algorithm49 was applied to treat long-
range electrostatic interactions. After removal of conformational stress by a short steepest descent
minimization, the procedure continued by simulated annealing (timestep 2 fs, atom velocities
scaled down by 0.9 every 10th step) until convergence was reached and the energy improved by
less than 0.05 kJ/mol per atom during 200 steps.
9.9 Docking experiments
The structure of SrtA was derived from PDB (ID: 2KID and 1T2W). Docking of the non-
covalently bound ligand was performed using Vina50 (default parameters) and the default Induced
Fit Docking protocol in Schrödinger Suite. The covalent docking was performed using
Autodock.51 The setup was done with the YASARA molecular modeling program. Results were
chosen from 25 (VINA) or 128 (Autodock) independent docking runs in which the thioether
moiety of the inhibitor was the closest to Cys184 of the catalytic triad. To guide the docking runs
the docking cell was placed around the putative binding site around Cys184. Ligands and the side
chains of the receptor residues were kept flexible during the docking runs.
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The structure 1T2W contains a mutation of the active site cysteine to alanine which was restored
prior to processing. The default setup in Schrödinger Protein Preparation Wizard was used for
processing of the protein and the bound substrate (LPETG) was used to define the docking site in
all Induced Fit Docking. All docked compounds in Schrödinger were scored using Glide XP
docking score.
4.10 Minimum inhibitory concentration (MIC)
The method used to determine the MIC was adopted from ISO 20776-1 2006 with changes.52
The MIC was determined via broth microdilution assay and defined as the lowest concentration of
compound (in mg/L) that prevents the appearance of visible bacterial growth under the defined in
vitro conditions stated below. Bacterial strains used in this study included S. aureus (CCUG
15915), S. aureus (CCUG 17621), and E. coli (CCUG 17620). Cultures of bacteria, were diluted
with phosphate buffered saline (PBS) to achieve a turbidity equivalent to 0.5 McFarland standard
(108 CFU/mL) using a DensiCHECK™ Plus instrument (BioMérieux Inc). Subsequent dilution
(1:10) gave the inoculum, which was used to inoculate MHB supplemented with test compound in
a two-fold serial dilution (50 µL + 50 µL, final assay concentration 0.125–256 mg/L, triplicate,
final inoculation concentration 5 × 105 CFU/mL). Control experiments included blank (not
inoculated), vehicle, ciprofloxacin as positive control, and viability tests (CFU count of
innoculum). After incubation (1824 h) the MIC was read out visually.
4.11 General procedure: Chemistry
All starting materials and reagents were obtained from commercial suppliers and used without
prior purification unless otherwise stated. All reactions were monitored by thin-layer
chromatography (TLC) on silica-plated aluminum sheets (Silica gel 60 F254, E. Merck) under UV
light (254 nm) or by visualization with staining solutions, or by LCMS analysis on an API SCIEX
150 EX Perkin Elmer ESI-MS (30 eV) connected to a Perkin Elmer gradient pump system and C8
column (Gemini) using acetonitrile and MilliQ-water with 1% formic acid as mobile phase with a
gradient of 5 to 95% acetonitrile over 4 min. Purification by flash chromatography was performed
on an automatic Biotage SP4 Flash+® instrument using prefabricated columns (surface area
1
500 m2/g, porosity 60 Å, particle size 40-63 µm). H and 13C NMR spectra for compound
identification were measured at 400 and 101 MHz, respectively, with a Varian 400-MR
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spectrometer at ambient temperature. Chemical shifts are reported in ppm with the solvent residual
peak as internal standard [acetone-d6 δH 2.05, δC 29.84; DMSO-d6 δH 2.5, δC 39.52; CD3CN δH
1.94].
4.11.1 General Procedure A for the Synthesis of Benzyl- and Phenethyl sulfides (5a–h).
5-Amino-1,3,4-thiadiazole-2-thiol (1.00 mmol) was dissolved in NaOHaq (2 mL, 0.5 M) at
22 °C. The appropriate benzyl bromide (1.00 mmol) was added as either a solid, suspension or
solution in acetonitrile or methanol at 22 °C and stirred for 15 h. The reaction mixture was diluted
with water (10 mL) and extracted with EtOAc (30 mL), the organic layer was washed with water
(3 × 10 mL), brine (10 mL), dried (MgSO4), and the solvent was evaporated under reduced
pressure. The crude material was purified by flash chromatography or recrystallization.
4.11.1.1 5-((3-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-amine (5a).
The title compound was synthesized according to the general procedure A from 5-amino-1,3,4-
thiadiazole-2-thiol (2.50 mmol, 333 mg), 3-nitrobenzyl bromide (2.50 mmol, 540 mg) to afford 5a
(618 mg, 92%) as yellow solid. 1H NMR (DMSO-d6) δ 8.23 (dd, J = 2.2, 1.7 Hz, 1H), 8.13 (ddd,
J = 8.3, 2.4, 1.1 Hz, 1H), 7.81 (ddd, J = 7.7, 1.8, 1.1 Hz, 1H), 7.63 (dd, J = 8.2, 7.6 Hz, 1H), 7.31
13
(s, 2H), 4.45 (s, 2H); C NMR (DMSO-d6) δ 170.5, 148.9, 148.1, 140.5, 136.1, 130.4, 124.0,
122.7, 37.7; HRMS (ESI, m/z): calculated for C9H8N4O2S2 [M+H]+ 269.0167; found: 269.0165.
4.11.1.2 5-((4-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-amine (5b)
The title compound was synthesized according to the general procedure A from 5-amino-1,3,4-
thiadiazole-2-thiol (1.00 mmol, 133 mg) and 4-nitrobenzyl bromide (1.00 mmol, 216 mg) to
obtain 5b (216 mg, 85%) as pale yellow crystals. 1H NMR (DMSO-d6) δ 8.22–8.16 (m, 2H), 7.64–
7.59 (m, 2H), 7.31 (s, 2H), 4.43 (s, 2H); 13C NMR (DMSO-d6) δ 170.1, 148.4, 146.6, 145.7, 130.2,
123.6, 37.5; HRMS (ESI, m/z): calculated for C9H8N4O2S2 [M+H]+ 269.0167; found: 269.0160.
4.11.1.3 2-(((5-Amino-1,3,4-thiadiazol-2-yl)thio)methyl)benzonitrile (5c).
The title compound was synthesized according to the general procedure A from 5-amino-1,3,4-
thiadiazole-2-thiol (1.00 mmol, 133 mg) and 2-(bromomethyl)benzonitrile (1.00 mmol, 97%,
202 mg). Purification by flash column chromatography (EtOAc/pentane, 4:6→10:0) afforded 5c
(153 mg, 62%) as white crystals. 1H NMR (DMSO-d6) δ 7.84 (ddd, J = 7.7, 1.4, 0.5 Hz, 1H), 7.66
(td, J = 7.7, 1.4 Hz, 1H), 7.54 (ddd, J = 7.9, 1.3, 0.6 Hz, 1H), 7.48 (td, J = 7.6, 1.3 Hz, 1H), 7.36
13
(s, 2H), 4.43 (s, 2H); C NMR (DMSO-d6) δ 170.6, 147.6, 140.8, 133.3, 133.2, 130.5, 128.4,
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117.2, 111.8, 37.1; HRMS (ESI, m/z): calculated for C10H8N4S2 [M+H]+ 249.0269; found:
249.0258.
4.11.1.4 5-(Benzylthio)-1,3,4-thiadiazol-2-amine (5d).
The title compound was synthesized according to the general procedure A from 5-amino-1,3,4-
thiadiazole-2-thiol (1.00 mmol, 133 mg) and benzyl bromide (1.00 mmol, 98%, 174 mg) to afford
1
5d (214 mg, 96%) as off-white crystals. H NMR (CD3CN) δ 7.37–7.25 (m, 5H), 5.69 (s, 2H),
4.28 (s, 2H); 13C NMR (DMSO-d6) δ 169.8, 149.4, 137.1, 129.0, 128.5, 127.4, 38.4; HRMS (ESI,
m/z): calculated for C9H9N3S2 [M+H]+ 224.0316; found: 224.0298.
4.11.1.5 5-((4-Fluorobenzyl)thio)-1,3,4-thiadiazol-2-amine (5e).
The title compound was synthesized according to the general procedure A from 5-amino-1,3,4-
thiadiazole-2-thiol (1.00 mmol, 133 mg) and 4-fluorobenzyl bromide (1.00 mmol, 97%, 195 mg)
to afford 5e (234 mg, 97%) as white crystals. 1H NMR (DMSO-d6) δ 7.42–7.35 (m, 2H), 7.28 (s,
13
2H), 7.18–7.10 (m, 2H), 4.29 (s, 2H); C NMR (DMSO-d6) δ 169.9, 161.4 (d, J = 243.7 Hz),
149.1, 133.5 (d, J = 3.1 Hz), 131.0 (d, J = 8.3 Hz), 115.2 (d, J = 21.4 Hz), 37.6; 19F NMR
(376 MHz, DMSO-d6) δ −114.97 (tt, J = 9.1, 5.5 Hz); HRMS (ESI, m/z): calculated for C9H8FN3S2
[M+H]+ 242.0222; found: 242.0221.
4.11.1.6 4-(((5-Amino-1,3,4-thiadiazol-2-yl)thio)methyl)benzonitrile (5f).
The title compound was synthesized according to the general procedure A from 5-amino-1,3,4-
thiadiazole-2-thiol (1.00 mmol, 133 mg) and 4-(bromomethyl)benzonitrile (1.00 mmol, 196 mg)
1
to afford 5f (188 mg, 76%) as white crystals. H NMR (DMSO-d6) δ 7.82–7.76 (m, 2H), 7.57–
7.50 (m, 2H), 7.31 (s, 2H), 4.37 (s, 2H); 13C NMR (DMSO-d6) δ 170.1, 148.6, 143.4, 132.3, 130.0,
118.7, 110.1, 37.8; HRMS (ESI, m/z): calculated for C10H8N4S2 [M+H]+ 249.0269; found:
249.0270.
4.11.1.7 5-((3-Nitrophenethyl)thio)-1,3,4-thiadiazol-2-amine (5g).
The title compound was synthesized according to the general procedure A from 5-amino-1,3,4-
thiadiazole-2-thiol (0.50 mmol, 67 mg) and 3-nitrophenethyl bromide (0.50 mmol, 115 mg).
Purification by recrystallization (acetone) afforded 5g (106 mg, 75%) as white crystals. 1H NMR
(DMSO-d6) δ 8.15 (t, J = 2.0 Hz, 1H), 8.09 (ddd, J = 8.2, 2.5, 1.0 Hz, 1H), 7.74 (dt, J = 7.6, 1.3 Hz,
1H), 7.61 (t, J = 7.9 Hz, 1H), 7.30 (s, 2H), 3.39 (t, J = 7.3 Hz, 2H), 3.11 (t, J = 7.3 Hz, 2H); 13C
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NMR (DMSO-d6) δ 170.0, 150.0, 148.2, 142.2, 136.2, 130.2, 123.9, 121.9, 35.4, 34.8; HRMS
(ESI, m/z): calculated for C10H10N4O2S2 [M+H]+ 283.0323; found: 283.0303.
4.11.1.8 5-((Naphthalen-2-ylmethyl)thio)-1,3,4-thiadiazol-2-amine (5h).
The title compound was synthesized according to the general procedure A from 5-amino-1,3,4-
thiadiazole-2-thiol (1.00 mmol, 133 mg) and 2-(Bromoethyl)naphthalene (1.00 mmol, 96%,
1
230 mg) to afford 5h (188 mg, 69%) as a pale yellow solid. H NMR (Acetone-d6) δ 7.92–7.83
(m, 4H), 7.57 (dd, J = 8.4, 1.8 Hz, 1H), 7.54–7.46 (m, 2H), 6.54 (s, 2H), 4.51 (s, 2H); 13C NMR
(DMSO-d6) δ 169.9, 149.2, 134.7, 132.7, 132.2, 128.2, 127.58, 127.55, 127.5, 127.0, 126.3, 126.1,
38.8; HRMS (ESI, m/z): calculated for C13H11N3S2 [M+H]+ 274.0473; found: 274.0476.
4.11.2 N-(5-((3-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)acetamide (6a).
A suspension of 5-((3-nitrobenzyl)thio)-1,3,4-thiadiazol-2-amine 5a (0.12 mmol, 32 mg) in
acetic acid (0.2 mL) and acetic anhydride (0.12 mL) was heated to 65 °C and stirred for 10 min.
After cooling, cold water (0.5 mL) was added, the precipitate was filtered off under suction and
washed with cold (2 × 10 mL, 0 °C) water. Purification by recrystallization (acetone) afforded 6a
(18 mg, 37%) as a pale yellow solid. 1H NMR (DMSO-d6) δ 12.58 (s, 1H), 8.30 (td, J = 1.9, 1.0 Hz,
1H), 8.13 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 7.86 (ddd, J = 7.7, 1.7, 1.1 Hz, 1H), 7.66–7.60 (m, 1H),
4.63 (s, 2H), 2.16 (s, 3H); 13C NMR (DMSO-d6) δ 168.7, 159.2, 157.0, 147.7, 139.7, 135.7, 130.0,
123.6, 122.4, 36.3, 22.3; HRMS (ESI, m/z): calculated for C11H10N4O3S2 [M+H]+ 311.0273;
found: 311.0231.
4.11.3 N-(5-((4-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)acetamide (6b).
A suspension of 5-((3-nitrobenzyl)thio)-1,3,4-thiadiazol-2-amine 5b (0.12 mmol, 32 mg) in
acetic acid (0.2 mL) and acetic anhydride (0.12 mL) was heated to 65 °C and stirred for 15 h. After
cooling, cold water (1 mL) was added, the precipitate was filtered off under suction and washed
with cold (3 × 2 mL, 0 °C) water. Purification by recrystallization (acetone) afforded 6b (15 mg,
1
41%) as a white solid. H NMR (DMSO-d6) δ 12.59 (s, 1H), 8.22–8.16 (m, 2H), 7.70–7.64 (m,
2H), 4.61 (s, 2H), 2.16 (s, 3H); 13C NMR (DMSO-d6) δ 168.8, 159.3, 156.9, 146.7, 145.3, 130.2,
123.6; HRMS (ESI, m/z): calculated for C11H10N4O3S2 [M+H]+ 311,0267; found: 311,0266.
4.11.4 N-(5-((3-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)furan-2-carboxamide (6c).
To a suspension of 5a (0.50 mmol, 134 mg) in THF (1 mL), a solution of 2-furoyl chloride
(0.70 mmol, 1.4 equiv, 95%, 96 mg) in THF (1 mL) was added dropwise under vigorous stirring
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at 22 °C. After 15 h, water (5 mL) was added and the solvent was reduced in vacuo. The resulting
solid residue was washed with Et2O (10 mL) and water (10 mL). Purification by recrystallization
(EtOH) afforded 6c (80 mg, 44%) as an off-white solid. 1H NMR (DMSO-d6) δ 13.10 (s, 1H), 8.33
(ddd, J = 2.3, 1.7, 0.5 Hz, 1H), 8.14 (ddd, J = 8.3, 2.4, 1.1 Hz, 1H), 8.04 (dd, J = 1.8, 0.8 Hz, 1H),
7.89 (ddd, J = 7.7, 1.7, 1.0 Hz, 1H), 7.69 (d, J = 3.6 Hz, 1H), 7.64 (ddd, J = 8.2, 7.7, 0.4 Hz, 1H),
6.76 (dd, J = 3.6, 1.7 Hz, 1H), 4.66 (s, 2H); 13C NMR (DMSO-d6) δ 157.6, 147.8, 147.7, 145.0,
139.7, 135.7, 130.0, 123.7, 122.4, 117.6, 112.5, 36.3; HRMS (ESI, m/z): calculated for
C14H10N4O4S2 [M+H]+ 363.0222; found: 363.0221. Carbons with signals δC 145.0 and δC 157.58
were identified by HMBC while carbonyl-C and thiadiazole-C-NH were not identified.
4.11.5 General procedure B for the Synthesis of Thiadiazol-2-yl Amides (6d–g).
A mixture of the appropriate benzoic acid (1.10 mmol) and thionyl chloride (1.1 mL) was
refluxed for 4 h. Excess thionyl chloride was removed under reduced pressure. After addition of
pyridine (1.1 mL), the reaction mixture was cooled to 0 °C, the appropriate amine (1.00 mmol)
was added and stirred for 15 h allowing to reach 22 °C. Water (5 mL) was added at 0 °C, the
precipitation was filtered and washed with water (3 × 10 mL) to yield thiadiazol-2-yl amides (6d–
g).
4.11.5.1 N-(5-((3-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)isonicotinamide (6d).
The title compound was synthesized according to the general procedure B from isonicotinic acid
(1.10 mmol, 135 mg), thionyl chloride (1.1 mL) and 5a (0.50 mmol, 0.5 equiv, 134 mg) to afford
6d (142 mg, 76%) as a pale yellow solid. 1H NMR (DMSO-d6) δ 8.88–8.83 (m, 2H), 8.34 (t, J =
2.0 Hz, 1H), 8.14 (ddd, J = 8.3, 2.4, 1.0 Hz, 1H), 8.05–8.00 (m, 2H), 7.90 (ddd, J = 7.7, 1.8, 1.0 Hz,
13
1H), 7.65 (t, J = 7.9 Hz, 1H), 4.69 (s, 2H); C NMR (DMSO-d6) δ 164.0, 160.3, 158.3, 148.7,
147.7, 140.7, 139.6, 135.7, 130.0, 123.7, 122.9, 122.5, 36.2; HRMS (ESI, m/z): calculated for
C15H11N5O3S2 [M+H]+ 374.0382; found: 374.0363.
4.11.5.2 N-(5-((3-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide (6e).
The title compound was synthesized according to the general procedure B from nicotinic acid
(1.10 mmol, 135mg), thionyl chloride (1.1 mL) and 5a (0.50 mmol, 134 mg) to afford 6e (152 mg,
81%) as an off-white solid. 1H NMR (DMSO-d6) δ 13.35 (s, 1H), 9.19 (dd, J = 2.4, 0.9 Hz, 1H),
8.81 (dd, J = 4.8, 1.7 Hz, 1H), 8.41 (ddd, J = 8.0, 2.4, 1.7 Hz, 1H), 8.34 (dd, J = 2.2, 1.7 Hz, 1H),
8.14 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 7.90 (ddd, J = 7.7, 1.7, 1.1 Hz, 1H), 7.64 (dd, J = 8.2, 7.7 Hz,
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1H), 7.59 (ddd, J = 8.0, 4.8, 0.9 Hz, 1H), 4.68 (s, 2H); 13C NMR (DMSO-d6) δ 164.2, 160.0, 158.0,
153.3, 149.3, 147.7, 139.7, 136.1, 135.7, 130.0, 127.4, 123.7, 123.6, 122.5, 36.2; HRMS (ESI,
m/z): calculated for C15H11N5O3S2 [M+H]+ 374.0382; found: 374.0377.
4.11.5.3 N-(5-((4-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)isonicotinamide (6f).
The title compound was synthesized according to the general procedure B from isonicotinic acid
(0.88 mmol, 2.2 equiv, 108 mg), thionyl chloride (0.9 mL) and 5b (0.4 mmol, 107 mg) to afford
1
6f (87 mg, 58%) as white solid. H NMR (DMSO-d6) δ 13.48 (s, 1H), 8.81 (d, J = 5.4 Hz, 2H),
8.24–8.17 (m, 2H), 7.99–7.93 (m, 2H), 7.74–7.67 (m, 2H), 4.67 (s, 2H); 13C NMR (DMSO-d6) δ
164.9, 158.3, 150.5, 146.8, 145.2, 139.2, 130.2, 123.6, 121.9, 36.9; HRMS (ESI, m/z): calculated
for C15H11N5O3S2 [M+H]+ 374,0376; found: 374,0370. With signals δC 164.9, δC 158.3 and δC
139.2 were identified by HMBC while C-NO2 and thiadiazole-C-NH were not identified.
4.11.5.4 N-(5-((4-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide (6g).
The title compound was synthesized according to the general procedure B from nicotinic acid
(0.88 mmol, 2.2 equiv, 108 mg), thionyl chloride (0.9 mL) and 5b (0.4 mmol, 107 mg) to afford
6g (149 mg, 15%) as an white solid. 1H NMR (DMSO-d6) δ 9.18 (s, 1H), 8.73 (m, 1H), 8.37 (d,
1H), 8.18 (d, 2H), 7.67 (d, 2H), 7.53 (t, 1H), 4.61 (s, 2H); HRMS (ESI, m/z): calculated for
C15H11N5O3S2 [M+H]+ 374,0376; found: 374,0362.
4.11.6 5-((4-Nitrophenyl)thio)-1,3,4-thiadiazol-2-amine (7a).
A suspension of 5-Amino-1,3,4-thiadiazole-2-thiol (0.20 mmol) in methanol (1 mL) was added
to a solution of commercially available 4-nitrobenzenediazonium tetrafluoroborate (0.20 mmol) in
CH3CN (1 mL) at 21ꢀ°C. After stirring for 5 min, the reaction mixture was diluted with water
(10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layer was washed with
NaOHaq (1 M, 5 mL), water (3 × 5 mL), brine (5 mL), dried (MgSO4), filtered and the solvent was
removed under reduced pressure to yield the title compound 7a (39 mg, 77%) as a bright brown
1
solid. H NMR (DMSO-d6) δ 8.24–8.17 (m, 2H), 7.79 (s, 2H), 7.54–7.46 (m, 2H); 13C NMR
(DMSO-d6) δ 173.4, 146.5, 144.4, 143.5, 128.1, 125.0; HRMS (ESI, m/z): calculated for
C8H6N4O2S2 [M+H]+ 255.0010; found: 255.0006.
4.11.7 General Procedure C for the Synthesis of Phenyl Sulfides 7b–c.
The appropriate aniline (3.00 mmol) was dissolved in dilute HClaq (2.25 mL, 4 M) and cooled
to 0 °C before a solution of NaNO2 (3.3 mmol, 1.1 equiv) in water (1.5 ꢀmL) was added dropwise
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under vigorous stirring. The reaction mixture was stirred at 0 °C for 20 min. Crystals of urea were
added until KI/starch paper indicated no excess nitrous acid. A suspension of 5-Amino-1,3,4-
thiadiazole-2-thiol (3.0 mmol) in water (1.5 mL) was added under vigorous stirring. After stirring
for 1.5 h at 22 °C, water (20 mL) was added to the reaction mixture and heated to boiling point for
2 min. Next, the precipitation was filtered off, the filtrate was basified (NaOHaq, 1 M) and
extracted with EtOAc (2 × 10 mL). The combined organic phase was washed with NaOHaq, (1 M)
until phenolic impurities were removed, then washed with water (10 mL), brine (10 mL), dried
(MgSO4), filtered and the excess solvent was removed under reduced pressure. Purification by
column chromatography afforded the phenyl sulfides 7b–c.
4.11.7.1 5-(Phenylthio)-1,3,4-thiadiazol-2-amine (7b).
The title compound was synthesized according to the general procedure C from aniline
(3.00 mmol, 280 mg) and 5-Amino-1,3,4-thiadiazole-2-thiol (3.00 mmol, 400 mg). Purification by
flash chromatography (EtOAc/hexane, 6:4) to afforded 7b (7.9 mg, 1%) as a pale brown solid. 1H
NMR (Acetone-d6) δ 7.50–7.34 (m, 5H), 6.76 (s, 2H); HRMS (ESI, m/z): calculated for C8H7N3S2
[M+H]+ 210.0160; found: 210.0159.
4.11.8 2-Bromo-5-((3-nitrobenzyl)thio)-1,3,4-thiadiazole (8a).
A suspension of 5a (1.70 mmol, 456 mg) and copper(II) bromide (1.87 mmol, 1.1 equiv,
418 mg) in acetonitrile (10 mL) was cooled to −10 °C. After dropwise addition of tert-butyl nitrite
(2.21 mmol, 1.3 equiv, 228 mg) the reaction mixture was stirred for 1 h at −10 °C before stirring
for a further 15 h at 21 °C. Next, NH4Claq (25%, 10 mL) was added to the reaction mixture and
stirred for 1 h at 21 °C, before diluting with water (10 mL) and extracting with Et2O (3 × 10 mL).
Combined organic layer was washed with water (10 mL) and brine (5 mL), dried (MgSO4) and the
solvent was evaporated under reduced pressure. Purification by recrystallization (ethanol) yielded
the title compound 8a (248 mg, 44%) as a pale brown solid. 1H NMR (DMSO-d6) δ 8.35 (ddd, J
= 2.3, 1.7, 0.5 Hz, 1H), 8.15 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 7.92 (ddd, J = 7.7, 1.7, 1.0 Hz, 1H),
7.65 (t, J = 8.0 Hz, 1H), 4.73 (s, 2H); 13C NMR (DMSO-d6) δ 168.7, 148.2, 139.6, 136.2, 130.5,
124.2, 123.0, 36.5; HRMS (ESI, m/z): calculated for C9H6BrN3O2S2 [M+H]+ 331.9163; found:
331.9189. The thiadiazole-C-Br was not identified.
4.11.10 4-(5-((3-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)morpho-line (9a).
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An oven-dried tube was charged with 8a (0.33 mmol, 110 mg), morpholine (1.1 equiv,
0.36 mmol, 32 mg), Pd2(dba)3 (2.76 mg, 1.8 mol% Pd), BINAP (5.64 mg, 2.74 mol%), NaOtBu
(11.6 mg, 0.12 mmol), and 1,4-dioxane (3 mL), degassed with N2, sealed, and heated to 100 °C
for 2 d. Next, the reaction mixture was diluted with water (10 mL) and extracted with Et2O (3 ×
10 mL). Combined organic layer was washed with water (5 mL) and brine (5 mL), dried (MgSO4),
filtered, and the solvent was removed under reduced pressure to afford a brown oil. Purification
by recrystallization (EtOAc/Pentane) afforded the title compound 9a (33 mg, 29%) as a pale brown
solid. 1H NMR (DMSO-d6) δ 8.24 (t, J = 2.0 Hz, 1H), 8.13 (ddd, J = 8.2, 2.4, 1.1 Hz, 1H), 7.82
(ddd, J = 7.7, 1.8, 1.1 Hz, 1H), 7.63 (t, J = 7.9 Hz, 1H), 4.51 (s, 2H), 3.71–3.65 (m, 4H), 3.40–
3.34 (m, 4H); 13C NMR (DMSO-d6) δ 173.2, 150.5, 147.7, 139.8, 135.7, 130.0, 123.6, 122.4, 65.2,
49.4, 37.2; HRMS (ESI, m/z): calculated for C13H14N4O3S2 [M+H]+ 339.0586; found: 339.0574.
4.11.11 2-((3-Nitrobenzyl)thio)-5-(piperazin-1-yl)-1,3,4-thiadiazole (9b).
An oven-dried tube was charged with 8a (0.80 mmol, 266 mg), piperazine (5 equiv, 4.0 mmol,
345 mg), Pd2(dba)3 (11.0 mg, 3.0 mol% Pd), BINAP (22.6 mg, 4.5 mol%), NaOtBu (46.4 mg,
0.12 mmol), and 1,4-dioxane (5 mL), degassed with N2, sealed, and heated to 100 °C for 19 h.
Next, the reaction mixture was reduced in vacuo, diluted with water (10 mL) and extracted with
CH2Cl2 (3 × 15 mL). Combined organic layer was washed with brine (5 mL), dried (MgSO4),
filtered, and the solvent was removed under reduced pressure to afford an orange oil. Purification
by flash column chromatography (1. CH2Cl2/MeOH, 9:1; 2. 1% TEA in CH2Cl2) afforded the title
compound 9b (185 mg, 69%) as a viscous orange residue. 1H NMR (DMSO-d6) δ 8.23 (ddd, J =
2.3, 1.7, 0.5 Hz, 1H), 8.13 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 7.82 (ddd, J = 7.7, 1.8, 1.1 Hz, 1H), 7.63
(dd, J = 8.2, 7.7 Hz, 1H), 4.49 (s, 2H), 3.39–3.33 (m, 4H), 2.89–2.80 (m, 4H); 13C NMR (DMSO-
d6) δ 173.0, 150.1, 147.7, 139.9, 135.7, 130.0, 123.6, 122.4, 49.6, 44.0, 37.3; HRMS (ESI, m/z):
calculated for C13H154N5O2S2 [M+H]+ 338.0745; found: 338.0731.
4.11.12 4-(5-((4-Nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)morpho-line (9c).
An oven-dried tube was charged with 5b (0.33 mmol, 110 mg), morpholine (1.1 equiv,
0.36 mmol, 32 mg), Pd2(dba)3 (2.76 mg, 1.8 mol% Pd), BINAP (5.64 mg, 2.74 mol%), NaOtBu
(11.6 mg, 0.12 mmol), and 1,4-dioxane (3 mL), degassed with N2, sealed, and heated to 100 °C
for 4 d. Next, the reaction mixture was diluted with water (10 mL) and extracted with Et2O (3 ×
10 mL). Combined organic layer was washed with water (5 mL) and brine (5 mL), dried (MgSO4),
filtered, and the solvent was removed under reduced pressure to afford a brown oil. Purification
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by recrystallization (EtOAc/Pentane) afforded the title compound 9c (55 mg, 49%) as a pale brown
solid. 1H NMR (DMSO-d6) δ 8.23–8.16 (m, 2H), 7.67–7.60 (m, 2H), 4.49 (s, 2H), 3.72–3.64 (m,
4H), 3.41–3.34 (m, 4H); 13C NMR (DMSO-d6) δ 173.2, 150.5, 146.7, 145.5, 130.2, 123.6, 65.2,
49.3, 37.4; HRMS (ESI, m/z): calculated for C13H14N4O3S2 [M+H]+ 339.058; found: 339.0593.
4.11.13 2-((4-Nitrobenzyl)thio)-5-(piperazin-1-yl)-1,3,4-thiadiazole (9d).
An oven-dried tube was charged with 5b (0.25 mmol, 83 mg), piperazine (5 equiv, 1.25 mmol,
108 mg), Pd2(dba)3 (11.0 mg, 3.0 mol% Pd), BINAP (22.6 mg, 4.5 mol%), NaOtBu (46.4 mg,
0.12 mmol), and 1,4-dioxane (5 mL), degassed with N2, sealed, and heated to 100 °C for 4 d. Next,
the reaction mixture was diluted with water (30 mL) and extracted with Et2O (12 × 5 mL).
Combined organic layer was washed with brine (5 mL), dried (MgSO4), filtered, and the solvent
was removed under reduced pressure to afford an yellow oil. Purification by flash column
chromatography (1. CH2Cl2/MeOH, 9:1; 2. 1% TEA in CH2Cl2) afforded the title compound 9d
(53 mg, 63%) as a bright yellow solid. 1H NMR (DMSO-d6) δ 8.23–8.14 (m, 2H), 7.69–7.57 (m,
13
2H), 4.47 (s, 2H), 3.33–3.26 (m, 4H), 2.81–2.70 (m, 4H); C NMR (DMSO-d6) δ 173.3, 149.6,
146.7, 145.5, 130.2, 123.6, 50.6, 44.7, 37.5; HRMS (ESI, m/z): calculated for C13H15N5O2S2
[M+H]+ 338.0740; found: 338.0728.
4.11.14 5-((3-Nitrobenzyl)thio)-N-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine,
4-methylbenzenesulfonate salt (10a).
Bromide 8a (0.70 mmol, 233 mg), 4-aminopyridine (1.05 mmol, 1.5 equiv, 98%, 101 mg), and
p-toluenesulfonic acid monohydrate (0.35 mmol, 0.5 equiv, 67 mg) were suspended in i-PrOH
(5 mL) and stirred at 80 °C for 3 d. The yellow precipitate was filtered off and washed with i-
PrOH. The yellow solid was triturated with Et2O to afford the title compound 10a (223 mg, 92%)
with a cation-anion ratio of 2:1. 1H NMR (DMSO-d6) δ 9.09 (s, 2H), 8.64–8.57 (m, 2H), 8.39 (t, J
= 2.1 Hz, 1H), 8.15 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 7.96 (ddd, J = 7.7, 1.8, 1.1 Hz, 1H), 7.66 (t, J
= 8.0 Hz, 1H), 7.50–7.45 (m, 1H), 7.14–7.08 (m, 1H), 7.02–6.93 (m, 2H), 4.77 (s, 2H), 2.28 (s,
1H); 13C NMR (DMSO-d6) δ 165.2, 163.5, 160.6, 147.7, 145.7, 141.0, 139.22, 137.6, 135.9, 130.1,
128.0, 125.5, 123.8, 122.6, 109.7, 36.0, 20.8; HRMS (ESI, m/z): calculated for C14H11N5O2S2
[M+H]+ 346.0432; found: 346.0421.
4.11.15 5-((3-Nitrobenzyl)thio)-N-(pyridin-3-yl)-1,3,4-thiadiazol-2-amine (10b).
3-aminopyridine was purified by sublimation under reduced pressure prior to use. Bromide 8a
(0.80 mmol, 266 mg), 3-aminopyridine (1.05 mmol, 1.5 equiv, 113 mg), and p-toluenesulfonic
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acid monohydrate (1.60 mmol, 2.0 equiv, 304 mg) were suspended in i-PrOH (6 mL) and stirred
at 80 °C for 9 d. The reaction mixture was decanted to obtain the crude material as brown residue.
1
Purification by flash column chromatography afforded 10b (7.4 mg, 3%) as tan solid. H NMR
(DMSO-d6) δ 10.59 (s, 1H), 8.68 (s, 1H), 8.33–8.30 (m, 1H), 8.24–8.17 (m, 1H), 8.16–8.13 (m,
1H), 8.10 (ddd, J = 8.4, 2.7, 1.3 Hz, 1H), 7.90–7.86 (m, 1H), 7.65 (t, J = 8.2, 7.7 Hz, 1H), 7.37 (s,
1H), 4.59 (s, 2H); 13C NMR (DMSO-d6) δ 165.2, 153.0, 148.2, 143.3, 140.3, 139.6, 136.3, 130.5,
124.5, 124.1, 122.9, 37.1; HRMS (ESI, m/z): calculated for C14H11N5O2S2 [M+H]+ 346.0432;
found: 346.0432.Carbon with signal δC 143.3 was identified by HMBC, two carbon signals could
not be identified.
Acknowledgements
We wish to acknowledge the Laboratories for Chemical Biology Umeå (LCBU), Sweden, and
the Swedish NMR center, Gothenburg. Further we would like to thank Erika Lindberg, Department
of Infectious Disease/Clinical Bacteriology, University of Gothenburg, Sweden, for the help with
the MIC assessments. LC-MS analysis was performed at the Proteomics Core Facility of
Sahlgrenska Academy, University of Gothenburg by Egor Vorontsov. The Proteomics Core
Facility is grateful to the Inga-Britt and Arne Lundbergs Forskningsstiftlese for the donation of the
Orbitrap Fusion Tribrid MS instrument.
Abbreviations used
Abz, 2-aminobenzoyl; BINAP, 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl; CCUG, Culture
Collection University of Gothenburg; CFU, colony forming units; CPMG, Carr-Purcell-Meiboom-
Gill; DMSO, dimethyl sulfoxide; DBA, direct binding assay; dba, dibenzylideneacetone; 2,4-
dinitrophenyl; EDTA, ethylenediamine-N,N,N',N'-tetraacetic acid; ESI, electrospray ionization;
FRET, fluorescence resonance energy transfer; HEPES, 4-(2-Hydroxyethyl)piperazine-1-
ethanesulfonic acid; HMBC, heteronuclear multiple bond correlation; HMQC, multiple-quantum
coherence; HTS, high-throughput screening; IC50, the half maximal inhibitory concentration;
LCBU, Laboratories for Chemical Biology Umeå; LCMS, liquid chromatography mass
spectrometry; HRMS, high mass resolution mass spectrometry; MHB, Mueller-Hinton broth;
MIC, minimum inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus;
NMR, nuclear magnetic resonance; PAINS, pan-assay interference compounds; SAR, structure-
27

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activity relationship; SrtA, Sortase A enzyme; srtA, gene coding for SrtA; TCEP, Tris(2-
carboxyethyl)phosphin; THF, tetrahydrofuran; TLC, thin layer chromatography; Tris,
tris(hydroxymethyl)amino-methane; p-TSA, p-toluenesulfonic acid.
Competing interests
Declarations of interest: none
Appendix A. Supplementary data
Supplementary data to this article can be found online at:
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