Tideglusib and Its Analogues As Inhibitors of Staphylococcus aureus SrtA

Article abstract of DOI:10.1021/acs.jmedchem.0c00803

Sortase A (SrtA) anchors surface proteins to the cell wall envelope, and it has attracted increasing interesting as a potential antivirulence target. Several small-molecule inhibitors for SrtA have been developed, but target validation remains largely und

Full text of DOI:10.1021/acs.jmedchem.0c00803

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Article
Tideglusib and its analogs as inhibitors of Staphylococcus aureus SrtA
Teng Yang, Tao Zhang, Xiangna Guan, Ze Dong, Le-Fu Lan, Song Yang, and Cai-Guang Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00803 • Publication Date (Web): 08 Jul 2020
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Tideglusib and its analogs as inhibitors of Staphylococcus aureus SrtA
Teng Yang†,‡,§, Tao Zhang , Xiang-Na Guan , Ze Dong , Lefu Lan‡,¶,, Song Yang†,*,
‡,§
‡,¶
‡
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Cai-Guang Yang‡
,¶,*
†
State Key Laboratory Breeding Base of Green Pesticide and Agricultural
Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Center for R&D of Fine Chemicals, Guizhou University,
Guizhou 550025, China
‡
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China
¶
University of the Chinese Academy of Sciences, Beijing 100049, China

School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced
Study, UCAS, Hangzhou 310024, China
§
These authors contributed equally to this work
*Correspondence: jhzx.msm@gmail.com (S.Y.) or yangcg@simm.ac.cn (C.-G.Y.)
ABSTRACT
Sortase A (SrtA) anchors surface proteins to the cell wall envelope, and it has
attracted increasing interesting as a potential antivirulence target. Several
small-molecule inhibitors for SrtA have been developed, but target validation
remains largely underexplored. Herein, we report a new class of SrtA inhibitors that
supports antivirulence therapy through small-molecule targeting of SrtA. Tideglusib
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(TD), a drug candidate for myotonic dystrophy, was outstanding in high-throughput
screening. A concise synthetic route quickly provided TD analogs, and the
structure-activity relationships for SrtA inhibition have been established from those
analogs. Several compounds largely retained the in vitro potency and exhibited a
better solubility than TD. Additionally, TD attenuated virulence-related phenotypes
in vitro and protected mice against lethal S. aureus USA300 bacteremia. Our study
indicates that TD and its analogs could be new candidates as SrtA inhibitors with
potential in the development of new antivirulence agents.
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Key words: S. aureus, tideglusib, antivirulence, sortase A, inhibitor.
INTRODUCTION
Staphylococcus aureus is a major gram-positive pathogenic bacteria that causes
1
serious suppurative infection of tissues as well as pneumonia and sepsis. Antibiotics,
either bactericidal or bacteriostatic, are widely used clinically for antiinfective
2
therapy . However, the inappropriate use and abuse of antibiotics have led to the
emergence of drug-resistant bacteria, such as methicillin-resistant S. aureus (MRSA),
which is the most common cause of the nosocomial infections and difficult to treat
3
-6
due to resistance against many different drugs. In order to alleviate antimicrobial
resistance, the continued research for new antibiotics is necessary. In addition,
antibacterial drug discovery should highlight the needs for new antiinfective agents,
particularly those with new modes of action, such as antivirulence compounds.7-11
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The sortase A (SrtA) transpeptidase is a biologically proven target ideal for the
development of antivirulence drugs.12, 13 Genome sequencing revealed that all S.
aureus isolates encode 17-21 surface proteins with LPXTG sorting signals, which
fulfill diverse functions in the development of pathogenesis.14, 15 Without these
surface proteins anchored on the cell wall, the bacteria display low virulence. SrtA
specifically recognizes and incorporates these surface proteins on the cell wall
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envelope using active-site cysteine biochemistry. The srtA mutant lost its function
in assembling surface proteins into the envelope and was unable to trigger
bacteremia or sepsis in mice,12, 16 but it is not essential for bacterial viability.
Additionally, SrtA is a membrane enzyme that is much more easily accessible.
Recent research efforts have been directed at developing antivirulence therapies
against S. aureus, focusing on small-molecule inhibitors for sortase which interferes
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7
with assembly of surface proteins. These inhibitors can be grouped into three
classes based on the resources of the compounds: natural products,18-23 synthetic
small molecules,24-29 and substrate-derived peptides.30-33 On the basis of their mode
of action, SrtA inhibitors can be classified into two groups, covalent and noncovalent
inhibitors.34, 35 Covalent inhibitors react with the active-site cysteine to form a
covalent bond,36, 37 whereas noncovalent inhibitors bind reversibly to SrtA. While
these studies identified both competitive and non-competitive inhibitors, many SrtA
inhibitors exhibited moderate potency, undetermined cytotoxicity, and unknown in
vivo antiinfective activity. Even worse, some SrtA inhibitors diminished or blocked
staphylococcal growth, which indicated that they are not selective inhibitors of the
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sortase enzyme. Therefore, it has not been fully established whether the SrtA
protein could be chemically attenuated for antivirulence therapy against S. aureus.
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Herein, we report the screening and structure-activity relationship (SAR) study of
tideglusib and its analogs as the inhibitors of S. aureus SrtA.
RESULTS AND DISCUSSION
High-throughput screening (HTS) for SrtA inhibitors. Previously, we developed a
reversible SrtA inhibitor (sodium
3
-(3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)benzenesulfonate, 6e)
that may be a useful antiinfective therapeutic to prevent S. aureus infection without
the side effects of antibiotics (Figure S1). 38 In addition to demonstrating
antiinfective efficacy in vivo, to our knowledge, this is the first inhibitor that has been
shown to inhibit the sortase-mediated cleavage of surface-protein sorting signals in
vivo. To search for new inhibitors of S. aureus SrtA, we performed HTS on a
compound library consisting of 2,400 clinical drugs and candidates using
fluorescence resonance energy transfer (FRET) to measures the transpeptidation
activity of recombinant SrtAΔN24 on a fluorogenic peptide substrate
1
5
(
Abz-LPATG-Dnp). In addition to the FRET-based assay, the inhibitory activities of
the hit compounds were further validated by an orthogonal PAGE-based method we
previously established, in which the surface protein IsdA64-323 was assayed as an
SrtAΔN24 substrate to rule out interference from false-positive effects of fluorogenic
compounds.38
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Interestingly, tideglusib (TD) was outstanding in the initial FRET-based HTS (Figure 1),
and it efficiently inhibited SrtA-mediated transpeptidation on the protein substrate
in the PAGE-based assay. TD has been granted fast-track designation for the
treatment of congenital myotonic dystrophy and orphan drug designation for
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myotonic dystrophy type 1. In addition, the thiadiazolidinone scaffold in TD has
been demonstrated as an efficient pharmacophore in drug discovery for Alzheimer’s
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disease, although in-depth SAR investigations remain largely underexplored. These
data highlight the excellent drug-like properties of TD, which make this promising
chemical space for the development of SrtA inhibitors.
Figure 1. Screening hit TD provides a chemical scaffold for new inhibitors of SrtA.
Synthesis of TD analogs. To explore the SARs of the new set of SrtA inhibitors, we
performed a concise synthesis of TD and its analogs by incorporating different
4
1
substituents (R and R ) into the core structure of TD (Figure 1 and Scheme 1).
1
2
Commercially available isothiocyanates 1 were mixed with isocyanates 2 in the
presence of sulfuryl chloride at 0 °C; then, the reaction was warmed to rt. Resulting
intermediate S-chloroisothiocarbamoyl chlorides 3 were further transformed in situ
to provide TD and analogs TD1-TD31 in good yields. Generally, commercially
unavailable isocyanate 2’ was prepared from 3,5-dimethylisoxazol-4-amine 4 by
reaction with triphosgene, and 2’ was then reacted with different isothiocyanates 1
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to afford analogs TD32-TD40. The concise synthetic route made it possible to quickly
evaluate the effects of R and R on the SrtA inhibitory activities.
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Scheme 1. General synthesis of TD and its analogs.
Conditions: (a) SO Cl , THF, 0 °C-rt, overnight; (b) air, 30 min; (c) triphosgene, THF, 50
2
2
°
C, 1 hr.
SAR analysis. The inhibitory activities of the designed analogs were evaluated by
both FRET-based and PAGE-based assays on SrtAΔN24 transpeptidation of peptide and
protein substrates, and the results are given as FRET IC and PAGE IC , respectively
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(
Table 1). Although a small IC values were usually observed in the FRET-based assay,
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interfere with the IC value in the PAGE-based assay. We also monitored the
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minimal inhibitory concentration (MIC) to rule out the bactericidal or bacteriostatic
compounds because the ideal inhibitors for SrtA should have minimal impacts on
bacterial viability. Indeed, TD and several analogs minimally inhibited the growth of
the S. aureus Newman strain at concentrations up to 100 μg/mL (Table 1), which
might indicate that TD analogs would be unlikely to function in the same manner as
conventional antibiotics.
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Table 1. Effects of substituted benzyl substituents at R1
FRET IC50
μM)a
PAGE IC50
Comp.
TD
MIC (μg/mL)c cLogPd
(
(μM)b
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0
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0
0.6 ± 0.2
1.3 ± 0.2
5.9 ± 0.1
>100.0
>100.0
5.2
4.6
TD1
32.2 ± 0.7
TD2
1.6 ± 0.3
9.2 ± 0.3
>100.0
4.6
TD3
TD4
TD5
TD6
2.6 ± 0.2
4.1 ± 0.2
0.7 ± 0.3
0.9 ± 0.1
1.2 ± 0.2
4.7 ± 0.8
5.9 ± 1.1
23.1 ± 0.3
14.4 ± 0.1
12.6 ± 0.7
100.0
100.0
100.0
50.0
4.6
5.4
5.9
6.5
5.1
TD7
100.0
a
Measured by monitoring the cleavage of a fluorogenic peptide substrate
b
(
Abz-LPATG-Dnp) by SrtA∆N24 in a FRET-based assay. Measured by monitoring the
c
transpeptidation of IsdA64-323 by SrtA∆N24 in a PAGE-based assay. Against growth of
d
the S. aureus Newman strain. Predicted in ChemBioDraw Professional 14.
First, we kept the naphthalene ring constant to explore the effect of the functionality
on the benzyl group in R for SrtA inhibition. As shown in Table 1, the introduction of
1
hydroxyl substituents (TD1-TD3) appeared to lower compound cLogP in comparison
to that for TD, while slightly attenuated the SrtA inhibitory activity measured in the
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FRET-based assay. However, the position of the hydroxy moiety on the phenyl ring
dramatically alters the SrtA inhibitory activity in the PAGE-based assay. Compounds
TD3 and TD4 bearing hydroxy or fluoro substituents at the para-position of the
phenyl ring largely retain the activity of TD, as observed in the PAGE-based assay.
Introducing chloro or methyl substituents to the phenyl ring gave analogs TD5-TD7,
which exhibited weaker inhibition of SrtAΔN24, as detected in the PAGE-based assay.
Taken together, the substituents on the benzyl group were further altered to
prepare more analogs, which displayed inhibitory activities comparable to that of the
parent compound, TD.
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Next, we explored whether the incorporation of more flexible alkyl chains as the R1
group could affect the inhibitory activity of the SrtAΔN24 inhibitors. As shown in Table
2
, the one or two carbon-long alkyl substituents in compounds TD8 or TD9 are
favorable and result in the lower cLogP values. Although all the tested compounds,
including TD10 and TD11, bearing larger or bulky alkyl groups retained the inhibitory
activity for the SrtAΔN24 enzyme, as observed in the FRET-based assay, the moderate
activity detected in the PAGE-based assay implies that flexible substituents in the R1
positions are not favorable.
Table 2. SAR of TD derivatives bearing flexible alkyl substituents at R1
FRET IC50
PAGE IC50
Comp.
MIC (μg/mL)c cLogPd
(μM)a
(μM)b
TD8
0.8 ± 0.2
8.0 ± 0.3
100.0
3.4
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TD9
0.7 ± 0.3
0.9 ± 0.2
8.2 ± 0.1
>100.0
100.0
4.0
5.0
TD10
12.6 ± 0.8
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TD11
0.6 ± 1.1
27.5 ± 1.0
>100.0
5.5
a,b,c,d
Same definitions used as in Table 1.
Given that the bulky naphthalene might generally make the TD derivatives too rigid
for SrtA binding, we fixed R as a benzyl group and prepared a new set of derivatives
2
by varying the substituents at the R position (Table 3). A short alkyl group at
1
position R , typically less than four carbons, improves lipid-water partition
1
coefficient; for example, consider compounds TD12, TD14 and TD15. Those
derivatives of TD bearing a benzyl group at the R position and an aliphatic
2
substituent in R showed dramatically decreased activity, as detected in both assays,
1
such as compound TD12-TD14. Similarly, compounds TD17-TD20, with two benzyl
substituents, showed significantly decreased inhibitory activities as SrtAΔN24
inhibitors. Moreover, the introduction of sterically bulky groups at R , such as
1
isopropyl (TD15) and fluorophenyl (TD16), abolished the SrtAΔN24 inhibitory activity.
Additionally, all TD analogs bearing a benzyl group at the R position displayed
2
increased activity against the growth of the S. aureus Newman strain in vitro with
moderate MIC values, which indicates that these analogs do not truly function as
SrtA inhibitors. Consistently, the thiadiazolidinedione derivatives were previously
identified to function as antibiotics through selectively inhibiting bacterial
dihydroorotate dehydrogenase (DHODH), which is a critical enzyme in de novo
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pyrimidine biosynthesis in prokaryotic and eukaryotic cells. However, it remains
underexplored whether the bactericidal activities of our TD analogs could also likely
be attributed to the selective inhibition of bacterial DHODH. Taken together, we
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observed that all the tested derivatives bearing a benzyl groups at R exhibited
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decreased inhibitory activity toward SrtA and inhibited bacterial growth
unanticipatedly, which emphasizes the contribution of the rigid aromatic moiety at
the R position.
2
Table 3. SAR of TD derivatives with benzyl groups at R2
FRET IC50
μM)a
PAGE IC50
Comp.
MIC (μg/mL)c cLogPd
(
(μM)b
TD12
TD13
TD14
TD15
3.5 ± 1.5
8.4 ± 0.3
2.0 ± 0.5
>100
23.6 ± 0.2
21.2 ± 0.8
16.7 ± 0.5
>50.0
12.5
6.2
2.3
3.9
2.9
3.1
25.0
6.2
TD16
>100
>50.0
12.5
4.2
TD17
TD18
TD19
7.0 ± 0.2
5.1 ± 4.0
3.4 ± 0.8
2.9 ± 1.3
12.9 ± 0.3
35.7 ± 2.6
28.0 ± 1.1
19.7 ± 0.1
12.5
12.5
12.5
12.5
4.1
4.6
4.8
5.4
TD20
a,b,c,d
Same definitions used as in Table 1.
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We then explored the incorporation of more flexible R groups (Table 4). The
2
aliphatic groups in R are unfavorable for inhibition since none of the synthesized
2
compounds (TD21-TD28) showed enzymatic inhibition of SrtAΔN24 in either assays.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
These analogs with an aliphatic R moiety displayed even better bactericidal activity
2
than did the analogs bearing a benzyl group at the R position (Table 3), which is
2
consistent with the previous observation that aliphatic and/or benzyl substituted
4
3
thiadiazolidinones could function as antibiotics. These data, alongside the SAR
observations listed in Table 3, indicate that rigid R substituents are likely important
2
for SrtA inhibition by TD analogs.
Table 4. SAR of TD derivatives bearing flexible alkyl substituents at both positions
FRET IC50
μM)a
PAGE IC50
Comp.
TD21
TD22
TD23
TD24
TD25
MIC (μg/mL)c cLogPd
(
(μM)b
8.7 ± 0.9
>50.0
>50.0
>50.0
>50.0
>50.0
6.2
6.2
6.2
3.1
6.2
3.5
3.3
3.1
3.7
4.0
16.7 ± 2.4
16.3 ± 0.9
43.9 ± 1.4
10.2 ± 0.6
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TD26
TD27
10.1 ± 0.5
>100
42.7 ± 3.2
>50.0
12.5
12.5
25.0
4.4
4.6
3.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TD28
>100
>50.0
a,b,c,d
Same definitions used as in Table 1.
According to the SAR analysis above, a benzyl substituent at R is favorable for
1
SrtAΔN24 inhibition activity, while analogs with alkyl groups in R exhibited decreased
2
inhibitory activity. Next, we sought to investigate the effects of different aromatic R2
groups on SrtAΔN24 inhibition when R was fixed as a benzyl group (Table 5). Both
1
TD29 and TD32 remained highly active compared to TD, and minimally impaired the
viability of the S. aureus Newman strain. It should be highlighted that TD32 might
exhibit improved solubility after introducing heterocycle moiety compared to that of
TD. Indeed, the solubility of TD32 was estimated to be 144 g/mL in
phosphate-buffered saline (PBS) in the presence of 1% DMSO, while the solubility of
TD is about 11 g/mL. The derivatives bearing benzocycloalkanes, compounds TD30
and TD31, were much less active than TD. These data further support the necessity
of having an aromatic substituent at the R position for SrtA inhibition.
2
Table 5. Effect of R when R is a benzyl group
2
1
FRET IC50
PAGE IC50
Comp.
MIC (μg/mL)c
cLogPd
(μM)a
(μM)b
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TD29
3.4 ± 0.2
4.7 ± 0.2
>100.0
4.0
TD30
TD31
4.4 ± 0.4
45.6 ± 2.1
31.9 ± 1.6
4.6 ± 0.1
>100.0
50.0
5.0
5.6
3.0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6.0 ± 0.2
0.8 ± 0.1
TD32
>100.0
a,b,c,d
Same definitions applied as in Table 1.
Last, we fixed R as the motif derived from TD32, and then explored the effects of
2
different substituents at R on the inhibition of SrtAΔN24. As shown in Table 6, in
1
general, the alkyl group in R influenced the SrtA
inhibition. Compounds with
1
ΔN24
short aliphatic chains, such as compounds TD33 and TD34, largely retained the
activity against SrtAΔN24 transpeptidation in both assays, while compound TD35, with
a sterically bulky group, made the TD analog inactive for SrtAΔN24 inhibition. For
derivatives bearing substituted benzyl groups at the R position, the presence of
1
methyl, methoxy, or chloro substituents on the phenyl ring of TD32, such as in
compounds TD36-TD40, resulted in activities comparable to that of compound TD32.
In addition, the MIC data show that these analogs are inactive against S. aureus in
vitro, which indicates that the TD32 analogs might not function as traditional
antibiotics for treating infection. Taken together, several analogs bearing a
3
,5-dimethylisoxazole substituent at R position exhibited good inhibitory activity on
2
SrtAΔN24
.
Table 6. SAR of R with the R motif derived from TD32
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
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5
5
5
5
5
6
FRET IC50
PAGE IC50
MIC (μg/mL)c
cLogPd
Comp.
μM)a
(μM)b
(
TD33
TD34
1.5 ± 0.3
1.1 ± 0.1
6.3 ± 0.3
4.1 ± 1.0
>100.0
>100.0
1.8
2.8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TD35
TD36
TD37
TD38
TD39
12.6 ± 0.1
1.1 ± 0.1
1.2 ± 0.1
0.9 ± 0.1
0.6 ± 0.4
1.0 ± 0.4
>50.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
2.1
3.5
2.9
4.3
3.7
3.7
4.7 ± 0.2
5.0 ± 0.2
3.4 ± 0.1
3.7 ± 0.5
4.1 ± 0.5
TD40
a,b,c,d
Same definitions applied as in Table 1.
TD and analogs are reversible inhibitors of SrtA and are not PAINS-containing. TD is
4
4
an irreversible inhibitor of glycogen synthase kinase-3β (GSK-3β), and a derivative
of TD covalently binds to regulators of G protein signaling protein 4 by forming a
4
5
disulfide bond. Additionally, a TD analog was found to effectively inactivate
bacterial DHODH enzymes regardless of the presence of cysteine residues in their
46
primary structures. However, TD compounds do not inhibit cysteine protease
papain.41, 45 The covalent modification of cysteine residues is thus unlikely to be the
general mechanism of action for TD compounds, but they might depend on the
environment in the binding site of the target. We investigated whether TD and its
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analog function as covalent inhibitors for SrtA transpeptidation. Using ESI-LC/MS
analysis, SrtAΔN24 was determined at an m/z of 22967.5 Da (Figure 2A), while a 120.5
Da m/z shift was observed after SrtAΔN24 was incubated with the generic covalent
alkylator, N,N,N-trimethyl-2-[(methylsulfonyl)sulfanyl]ethanaminium bromide
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(MTSET) as a positive control at rt for 12 hr (Figures 2B and S1), and this shift in mass
perfectly matched to the fragment of MTSET covalently added to SrtAΔN24. No m/z
shift was observed when SrtAΔN24 was exposed to either the TD or TD32 inhibitor
under the same conditions as used for the control groups (Figures 2C and 2D), which
revealed that TD compounds could not covalently modify the SrtAΔN24 protein in vitro.
However, whether they covalently modify SrtA in cells remains unclear. Next, we
measured the recovery of sortase activity in a reversible inhibition assay that has
3
8
been published. As shown in Figure 2E, the covalent alkylator MTSET abolished the
transpeptidation activity of SrtAΔN24, while this activity was mostly recovered in the
groups of treating with TD inhibitors and following dilution of SrtAΔN24/inhibitor at 10
fold IC as compared to mock treated SrtAΔN24, which indicate that TD and TD32 are
5
0
reversible inhibitors. More generally, a time-dependent increase in the apparent
inhibitory potency suggests irreversible binding. The inhibition effects of TD and
TD32 on SrtAΔN24 minimally changed over time (Figure S2A). Taken together, the TD
inhibitors inhibit the transpeptidation in a reversible manner.
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1
2
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2
2
2
2
2
3
3
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3
3
4
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0
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0
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0
1
2
3
4
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0
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2
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8
9
0
Figure 2. Characterization on the mode of actions of the TD inhibitors. MS analysis of
SrtAΔN24 protein treated with DMSO (A), the covalent alkylator MTSET (B), the
inhibitor TD (C), and the inhibitor TD32 (D). (E) Determination of the reversibility of
the SrtAΔN24 inhibition. Two independent experiments were performed. (F) NMR
spectra of TD32 interacting with the SrtAΔN59 protein. The CPMG-NMR spectra were
recorded for TD32 without SrtAΔN59 (red) and with SrtAΔN59 at 5.0 μM (blue), 10.0 μM
(
magenta), 20.0 μM (green), and 40.0 μM (cyan). Two independent experiments
were performed. Docking of TD (G) and TD32 (H) to the solution structure of SrtAΔN59
PDB code ID: 2KID) performed in AutoDock 4.2. Protein is shown as a cartoon in
(
cyan, the surface is presented in gray, the side chains are in yellow, and the
inhibitors are in magenta.
We further investigated the interactions between SrtA and the TD inhibitors. Since
4
7
the solution structure of SrtAΔN59 bound with peptide has been determined, we
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performed Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance (NMR)
titrations by using the SrtAΔN59 truncation. Dose-dependent attenuation of the
signals was observed in CPMG NMR titrations (Figures 2F), which suggested direct
interactions between SrtAΔN59 and the inhibitor TD32. When SrtAΔN24 was incubated
in 10-fold excesses of TD and TD32, we observed no obvious changes in the circular
dichroism (CD) spectra (Figure S2B), which means TD and its derivatives cause no
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
8
gross structural perturbations in SrtA folding. Additionally, we performed a docking
study to reveal the potential binding modes between the TD inhibitors and SrtAΔN59
(Figures 2G and 2H). Both inhibitors could bind in the substrate binding pocket of
SrtA. The naphthyl ring of TD forms a - stacking with His120. A hydrogen bond
forms between the sulfur atom in TD and the side chain of Arg197 in SrtAΔN59, while
the 3,5-dimethylisoxazole ring in TD32 forms a hydrogen bond with the side chain of
Arg197. These observed differences in hydrogen-binding modes might explain why
the heterocycle of thiadiazolidinedione posed differently in binding to TD and TD32.
In the docked pose of the inhibitors binding to SrtAΔN59, the methylene of the benzyl
group is close to the Ile182 residue in SrtA, which is a constrained position for
introducing more or bulkier substituents. This is consistent with the experimental
observations that analogs with bulky groups, for example, isopropyl (TD15, TD28 and
TD35), fluorophenyl (TD16) and cyclohexyl (TD25 and TD27), at R showed no
1
inhibitory activities. There is space in the benzyl group binding pocket for the binding
of an extra substituent, which also explains why the introduction of methyl (TD36),
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
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4
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methoxy (TD37), or chloro (TD38-TD40) moieties on the phenyl ring minimally
affected the inhibitory activity on SrtA transpeptidation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Furthermore, we investigated whether TD compounds are PAINS-containing
inhibitors of SrtA.49, 50 TD and TD32 are not intrinsically fluorescent (Figure S2C). If
their inhibitory activities are attenuated by small amounts of nonionic detergent, the
inhibitors are likely aggregators. The presence of 0.01% v/v freshly prepared Triton
X-100 minimally affected the inhibitory activities of TD and TD32 in a typical
FRET-based assay (Figure S2D). These data suggest that the TD compounds are not
PAINS-containing inhibitors of SrtA.
Determination of the enzymatic activity of SrtA inhibitors. After filtration of the
PAINS properties, we chose TD and TD32 for further investigations as SrtA inhibitors,
while the covalent inhibitor MTSET was assayed as a positive control, and compound
(E)-3-phenyl-5-(2-(phenylsulfonyl)vinyl)-2-(propylamino)-4a,8a-dihydronaphthalene-
36
1
,4-dione (63) was assayed as a negative control (Figure S1). Both TD and TD32
efficiently inhibited the cleavage of the fluorogenic peptide substrate
Abz-LPATG-Dnp) by SrtA∆N24 in a concentration-dependent manner, and their IC50
(
values were calculated to be 0.6 μM and 0.8 μM, respectively (Figure 3A). TD32 also
inhibited the transpeptidation of IsdA64-323 protein by SrtA∆N24 in a dose-dependent
manner, and its activity was comparable to that of TD (Figure 3B). Deletion of the
srtA gene minimally attenuated the in vitro growth of the S. aureus Newman strain;12
consistent with this result, neither TD nor TD32 inhibited the growth of the S. aureus
Newman strain at concentrations up to 100 μM (Figure 3C), while TD at 200 μM
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attenuated bacterial growth, which means at least it has bacteriostatic properties
and therefore is not a true antivirulence inhibitor at this high concentration.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Inhibition of SrtA transpeptidation. (A) Determination of the IC values of
5
0
the inhibition of the SrtAΔN24 enzyme by a FRET-based assay. (B) Effect of the
inhibitors on the SrtA∆N24-mediated transpeptidation of IsdA64-323 protein in
PAGE-based assay. The migratory positions of the IsdA64-323 substrate precursor (M)
and mature transpeptidation product (P) are indicated. MTSET was assayed as a
positive control, while 63 was assayed as a negative control. (C) Effects of the
inhibitors on the growth of the S. aureus Newman strain. The srtA mutant S. aureus
Newman strain was assayed as a positive control. Three independent experiment
were performed for these assays.
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Effect of the TD inhibitors on virulence-related phenotypes in S. aureus. The
inhibition of SrtA activity in staphylococci by compounds TD and TD32 was then
evaluated. SrtA anchors several surface proteins in the staphylococcal envelope,
including SpA.12, 38, 51-53 Since SpA can specifically bind FITC-labeled human IgG, the
abundance of IgG-bound SpA in the bacterial cell wall envelope can be roughly
estimated from the fluorescence. In the control assay, the srtA deletion mutant S.
aureus strain almost completely lost its ability to anchor SpA to the cell wall (Figure
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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0
1
2
3
4
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0
1
2
3
4
5
6
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9
0
4
A). The covalent inhibitor MTSET reduced the abundance of anchored SpA, while
the negative control (63) had minimal effect on the abundance of anchored SpA
when the S. aureus Newman strain was exposed to the test compounds at a
concentration of 100 μM. As expected, both TD and TD32 reduced the amount of
anchored SpA in a concentration-dependent manner (Figure 4A). The reduction in
cell wall-anchored SpA induced by the SrtA inhibitors also could be clearly visualized
in the Western blot assay (Figure 4B). Biofilm formation is a critical pathogenic
5
4
process of S. aureus. MRSA biofilms increase its resistance to antimicrobial action
5
from external agents. In this context, the development of strategies to prevent,
remove, or disperse biofilms is critical for the treatment of MRSA infections. It has
been demonstrated that srtA gene deletion or inhibition of the SrtA protein can
2
0
reduce the ability of in S. aureus to form biofilms. To investigate whether TD and
TD32 inhibit biofilm formation, we performed a gentian violet staining assay to
calculate biofilm biomass. Noncovalent inhibitor 6e was assayed as a positive control
3
8
(
Figure S1). Compared to the wild-type control groups, a significant decrease in
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biofilm biomass was observed when S. aureus was exposed to TD or TD32, and the
decreases were dose dependent (Figure 4C). Taken together, the TD-derived SrtA
inhibitors inhibit virulence-related phenotypes in S. aureus.
1
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
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Figure 4. Effect of SrtA inhibitors on virulence-related phenotypes in the S. aureus
Newman strain. (A) Effects of SrtA inhibitors on cell wall-anchored SpA estimated
based on the fluorescence of FITC-labeled human IgG. MTSET and the srtA mutant
S. aureus Newman strain were assayed as positive controls, while 63 was used as a
negative control. Statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001) was
determined using the unpaired, two-tailed Student’s t-test (n = 3). (B) Effects of SrtA
inhibitors on cell wall-anchored SpA detected by Western blot. The srtA mutant S.
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aureus Newman strain was assayed as a positive control. The Cytoplasmic protein
ClpP of S. aureus was used as a loading control. The non-specific band of Sbi was
indicated. (C) Effects of SrtA inhibitors on biofilm formation in the S. aureus Newman
strain. The top panel shows a photograph of a biofilm stained with crystal violet, and
the bottom panel shows the quantification of biofilm determined by measuring the
absorbance at a wavelength of 600 nm. Compound 6e and the srtA mutant S.
aureus Newman strain were assayed as positive controls, while 63 was assayed as a
negative control. Statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001) was
determined using the unpaired, two-tailed Student’s t-test (n = 4). Three
independent experiments were performed for all assays.
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Antiinfective therapy with TD. Although TD32 displayed activity comparable to that
of TD and better solubility than TD, the biosafety of TD has already been established
since it is a drug candidate undergoing phase II clinical trials for congenital myotonic
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dystrophy treatment. In addition, the S. aureus USA300 strain is a
community-associated MASR strain and resistant to many drugs. Therefore, we
evaluated the in vivo antiinfective efficacy of TD against the S. aureus USA300 strain.
As shown in Figure 5A, TD reduced the virulence factor of the S. aureus USA300
strain, biofilm formation for example. Also, the inhibitor TD at 150 μg/mL slightly
attenuated the in vitro growth of the tested strain (Figure 5B), which suggested that
it is unlikely that TD inactivates S. aureus by a mechanism similar to those of
conventional antibiotics. A previous pharmacokinetic study of TD revealed that the
postintraperitoneal administration maximum concentrations in plasma were low
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since the Cmax was only 137 ng/mL when a single dose of 10 mg/kg TD was
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administered to mice. Therefore, a high dose of TD is necessary to achieve a
sufficient high concentration for SrtA inhibition in mouse. The antiinfective
properties of TD were assessed in a mouse model of systemic infection via i.v.
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inoculation of 1  10 colony-forming units (CFU) of S. aureus USA300, which load is
a lethal load for healthy mice. As shown in Figure 5C, placebo-treated mice died
rapidly on the third day to the sixth day. However, the mice that had received i.p.
treatment of compound TD at a dose of 40 mg/kg/day displayed a prolonged time to
death and 40% survival at the endpoint of monitoring, which showed that the
placebo and TD had different in vivo effects (*, P < 0.05). This in vivo assessment
indicated that the chemical scaffold of TD is promising for the development of SrtA
inhibitors, which might be useful as antiinfective agents in preventing S. aureus
bloodstream infections in community-associated settings.
Figure 5. Antiinfective therapy with TD. (A) Effects of TD on biofilm formation in the
S. aureus USA300 strain. Compound 6e was assayed as positive controls, while 63
was assayed as a negative control. Statistical significance (*, P < 0.05; ***, P < 0.001)
was determined using the unpaired, two-tailed Student’s t-test (n = 4). Three
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independent experiments were performed. (B) Effects of TD on the growth of the S.
aureus USA300 strain. Three independent experiments were performed. (C)
Antiinfective effects of TD on BALB/c mice (n = 10) infected by the S. aureus USA300
strain. The survival rate was recorded over a 10-day period. Statistical significance
was examined with the log-rank test (placebo vs. TD, P < 0.05).
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CONCLUSION
Currently, all antibiotics in therapeutic application or in development pipelines are
bactericidal or bacteriostatic, which has put pressure on bacteria to evolve
resistance. To address the challenge posed by the rapid emergence of new
resistance, antibacterial drug discovery should emphasize the need to find new
antiinfective agents, particularly those with novel modes of action. Targeting
bacterial virulence is one of the most promising strategies, and it represents a
complementary or alternative strategy to conventional antibiotics. Sortases and cell
wall anchored surface proteins contribute to the virulence of many different
Gram-positive bacteria. Thus, antivirulence strategy with sortase inhibitors may be
useful to broadly prevent bacterial infections with antibiotic-resistant bacterial
pathogens. However, the identification of sortase inhibitors as antiinfective
therapeutics has not been fully demonstrated.
In this study, we employed FRET-based HTS on a library consisting of over 2,400
clinical drugs and candidates. TD was identified as a hit compound for the in vitro
inhibition of SrtAΔN24 in the low micromolar range. Subsequently, an SAR exploration
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of SrtA inhibitors was quickly established via the concise synthesis of TD analogs.
Some analogs are effective inactivators of the growth of S. aureus, while others
displayed weaker inhibitory activities on the SrtAΔN24 enzyme. Several analogs
bearing the 3,5-dimethylisoxazole moiety retained inhibitory activities comparable
to that of TD and exhibited solubility superior to that of TD. The anchoring of SpA
was reduced when S. aureus was exposed to TD and TD32, which also led to the
attenuation of biofilm formation. Importantly, TD is effective in protecting mice
against lethal infection by the S. aureus USA300 strain. Whether the potential
off-target effect and/or the slight bacteriostatic property of TD would influence the
observed outcome in mouse infection warrants further study. In summary, TD and
its analogs represent a new chemical scaffold for SrtA inhibitors, making them
promising lead compounds in the development of new antivirulence drugs.
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EXPERIMENTAL SECTION
General methods. All chemicals were analytically pure and were used as received.
Reactions were monitored by thin-layer chromatography (TLC) using silica gel-coated
TLC plates, and detection was performed by UV absorbance at a wavelength of 254
1
nm. H NMR spectra were recorded on a Bruker-400 (400 MHz) spectrometer using
DMSO-d or CDCl as the solvent. The data are reported as chemical shifts or δ values
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(
ppm). C NMR spectra were obtained on a Bruker-500 (125 MHz) spectrometer
using DMSO-d or CDCl as the solvent. Agilent 1200 (HPLC) and 6110 (MSD)
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instruments were utilized for generating HPLC traces, obtaining mass spectrometry
data, and evaluating purity. The system was equipped with a PDA UV detector and
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an Xbridge C18 column (3.5 μm, 4.6  50 mm), and the separations were achieved at
rt. The HPLC gradient program utilized 5% to 95% MeCN in H O with 0.01%
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trifluoroacetic acid over 5 min with a 1.5 mL/min flow rate. The purity of the final
compound was assessed at a wavelength of 254 nm by HPLC analysis.
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Reversed-phase preparatory purifications were performed on a Lisui EZ Plus 100 D.
This system utilized a PDA detector and a Kromat flash C18 column (20-40 μm, 120 g).
Purification methods used a 30-min gradient from 5% to 70% MeCN in H O. The
2
purity of each target compound was >95% by HPLC.
General synthesis of compounds TD and TD1-TD32. Sulfuryl chloride (238 mg, 2.0
mmol, 1.0 eq) was added into a solution of isothiocyanate (1, 1.0 eq) and isocyanate
(2, 1.0 eq) in anhydrous THF at 0 °C and the mixture was warmed to rt and then
stirred for 4-10 hr. The precipitate was separated by filtration and washed with cold
THF (2  10 mL) and hexane (2  10 mL). Alternatively, the reaction mixture was
concentrated under vacuum and purified by reversed-phase C18 (RP-C18) column
chromatography to give the target compound.
4
-benzyl-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD). Obtained in 80%
1
yield; white powder; H NMR (400 MHz, CDCl ) δ 8.01 – 7.93 (m, 2H), 7.80 (m, J = 7.8,
3
1
3
1
.9 Hz, 1H), 7.65 – 7.51 (m, 6H), 7.46 – 7.36 (m, 3H), 5.02 (s, 2H); C NMR (125 MHz,
DMSO-d ) δ 166.5, 152.5, 136.1, 134.4, 131.4, 130.6, 130.4, 129.2, 129.0, 128.3,
6
+
1
1
28.1, 128.1, 127.4, 126.2, 122.7, 46.1; LC-MS [M + H] : 335.1; HPLC purity at 254 nm,
00.0%.
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4-(2-hydroxybenzyl)-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD1).
1
Obtained in 75% yield; white powder; H NMR (400 MHz, DMSO-d ) δ 9.78 (s, 1H),
6
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.10 (t, J = 7.2 Hz, 2H), 7.93 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 7.3 Hz, 1H), 7.77 – 7.55 (m,
1
1
1
1
1
1
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2
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0
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3H), 7.13 (d, J = 7.3 Hz, 2H), 6.85 (dd, J = 15.4, 7.6 Hz, 2H), 4.86 (s, 2H); C NMR (125
MHz, CDCl ) δ 166.7, 155.7, 153.7, 134.6, 132.5, 131.0, 130.9, 130.2, 130.2, 128.8,
3
+
127.9, 127.4, 127.2, 125.4, 122.0, 121.1, 120.8, 118.5, 42.2; LC-MS [M + H] : 351.1;
+
HRMS [M + H] (ESI - TOF) calcd for C H N O S 351.0803, found 351.1332; HPLC
1
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purity at 254 nm, 100.0%.
4
-(3-hydroxybenzyl)-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD2).
1
Obtained in 70% yield; white powder; H NMR (400 MHz, DMSO-d ) δ 9.54 (s, 1H),
6
8
3
.10 (t, J = 8.2 Hz, 2H), 7.98 – 7.84 (m, 1H), 7.79 (d, J = 7.1 Hz, 1H), 7.74 – 7.51 (m,
H), 7.19 (t, J = 8.0 Hz, 1H), 6.82 (d, J = 6.5 Hz, 2H), 6.73 (d, J = 8.8 Hz, 1H), 4.81 (s,
1
3
2H); C NMR (125 MHz, CDCl ) δ 165.7, 155.5, 152.0, 136.3, 134.1, 130.5, 130.0,
3
1
4
3
29.7, 129.6, 129.3, 128.3, 127.3, 127.0, 126.6, 125.0, 121.8, 121.1, 115.5, 115.1,
+
+
5.7; LC-MS [M + H] : 351.1; HRMS [M + H] (ESI - TOF) calcd for C H N O S
1
9 14 2 3
51.0803, found 351.0801; HPLC purity at 254 nm, 100.0%.
4-(4-hydroxybenzyl)-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD3).
1
Obtained in 78% yield; white powder; H NMR (400 MHz, DMSO-d ) δ 9.51 (s, 1H),
6
8
7
.09 (dd, J = 10.0, 5.9 Hz, 2H), 7.85 (dd, J = 6.1, 3.5 Hz, 1H), 7.77 (d, J = 7.0 Hz, 1H),
1
3
.69 – 7.57 (m, 3H), 7.23 (d, J = 8.5 Hz, 2H), 6.78 (d, J = 8.5 Hz, 2H), 4.77 (s, 2H); C
NMR (125 MHz, CDCl ) δ 165.8, 155.3, 152.1, 134.1, 130.6, 130.0, 128.3, 127.2, 127.2,
3
+
+
126.9, 125.0, 121.8, 115.1, 45.4; LC-MS [M + H] : 351.1; HRMS [M + H] (ESI - TOF)
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calcd for C H N O S 351.0803, found 351.0792; HPLC purity at 254 nm, 96.5%.
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4
-(4-fluorobenzyl)-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD4).
1
0
1
2
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4
5
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9
0
1
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Obtained in 26% yield; off-white powder; H NMR (400 MHz, CDCl ) δ 7.97 (d, J = 7.5
3
Hz, 2H), 7.78 (s, 1H), 7.71 – 7.48 (m, 6H), 7.09 (d, J = 7.9 Hz, 2H), 4.98 (d, J = 7.5 Hz,
1
3
2
1
H). C NMR (125 MHz, CDCl ) δ 166.2, 163.8, 161.8, 152.4, 134.6, 131.1, 130.5,
3
+
28.8, 127.7, 127.4, 127.0, 125.4, 122.2, 115.8, 115.7, 45.7; LC-MS [M + H] : 353.1;
+
HRMS [M + H] (ESI - TOF) calcd for C H FN O S 353.0760, found 353.0753; HPLC
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purity at 254 nm, 97.8%.
4
-(4-chlorobenzyl)-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD5).
1
Obtained in 67% yield; white powder; H NMR (400 MHz, CDCl ) δ 8.38 (d, J = 8.1 Hz,
3
1
H), 7.80 (d, J = 8.2 Hz, 1H), 7.75 – 7.59 (m, 3H), 7.58 – 7.48 (m, 3H), 7.38 (d, J = 8.3
1
3
Hz, 2H), 4.96 (s, 2H); C NMR (125 MHz, CDCl ) δ 165.9, 152.3, 134.6, 133.5, 131.8,
3
1
31.4, 130.7, 130.0, 129.1, 128.5, 128.2, 127.4, 125.8, 125.6, 122.7, 45.7; LC-MS [M +
+
+
H] : 369.1; HRMS [M + H] (ESI - TOF) calcd for C H ClN O S 369.0465, found
1
9
13
2 2
3
69.0461; HPLC purity at 254 nm, 99.6%.
4-(3,4-dichlorobenzyl)-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD6).
1
Obtained in 75% yield; white powder; H NMR (400 MHz, DMSO-d ) δ 8.30 (d, J = 8.3
6
Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.85 (s, 2H), 7.83 – 7.75 (m, 2H), 7.73 – 7.62 (m, 3H),
1
3
7
1
.43 (d, J = 8.2 Hz, 1H), 4.90 (s, 2H); C NMR (125 MHz, DMSO-d ) δ 166.7, 152.4,
6
37.2, 132.9, 131.8, 131.6, 131.4, 131.2, 131.1, 131.0, 130.5, 129.1, 129.1, 128.9,
+
+
128.7, 126.8, 125.0, 124.0, 45.1; LC-MS [M + H] : 403.0; HRMS [M + H] (ESI - TOF)
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calcd for C H Cl N O S 403.0069, found 403.0075; HPLC purity at 254 nm, 99.6%.
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9
12
2 2 2
4
-(4-methylbenzyl)-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD7).
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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5
5
5
5
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0
1
2
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0
1
2
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4
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0
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0
Obtained in 70% yield; white powder; H NMR (400 MHz, CDCl ) δ 8.41 – 8.34 (m,
3
1
7
H), 7.85 – 7.81 (m, 1H), 7.72 – 7.66 (m, 2H), 7.64 (d, J = 7.9 Hz, 1H), 7.59 (s, 1H),
.53 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 7.8 Hz, 2H), 4.97 (d, J = 3.3
1
3
Hz, 2H), 2.39 (s, 3H); C NMR (125 MHz, CDCl ) δ 165.9, 152.5, 138.4, 134.3, 132.2,
3
1
31.8, 131.5, 130.2, 129.5, 129.2, 128.4, 128.1, 127.4, 125.8, 125.5, 122.8, 46.3, 21.2;
+
+
LC-MS [M + H] : 349.1; HRMS [M + H] (ESI - TOF) calcd for C H N O S 349.1011,
2
0 16 2 2
found 349.1011; HPLC purity at 254 nm, 95.7%.
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-methyl-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD8). Obtained in 56%
1
yield; white powder; H NMR (400 MHz, CDCl ) δ 7.97 (t, J = 7.6 Hz, 2H), 7.87 (d, J =
3
1
3
8.1 Hz, 1H), 7.68 – 7.50 (m, 4H), 3.40 (s, 3H); C NMR (125 MHz, DMSO-d ) δ 166.0,
6
1
52.2, 134.2, 130.6, 130.1, 130.1, 128.3, 127.3, 127.0, 126.6, 125.1, 121.9, 28.5;
+
+
LC-MS [M + H] : 259.1; HRMS [M + H] (ESI - TOF) calcd for C H N O S 259.0541,
1
3 10 2 2
found 259.0532; HPLC purity at 254 nm, 99.0%.
4-ethyl-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione (TD9). Obtained in 47%
1
yield; white powder; H NMR (400 MHz, DMSO-d ) δ 8.09 (dd, J = 10.5, 5.2 Hz, 2H),
6
7
7
1
.95 – 7.88 (m, 1H), 7.78 (d, J = 7.3 Hz, 1H), 7.64 (dq, J = 15.1, 7.5 Hz, 3H), 3.76 (q, J =
1
3
.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ 166.3, 152.4,
6
34.5, 131.6, 130.6, 130.5, 129.0, 128.4, 128.3, 128.1, 127.4, 126.3, 122.9, 38.2, 13.4;
+
+
LC-MS [M + H] : 273.1; HRMS [M + H] (ESI - TOF) calcd for C H N O S 273.0698,
1
4 12 2 2
2
9
ACS Paragon Plus Environment