Synthesis and Structure-Activity Relationship Study of Antimicrobial Auranofin against ESKAPE Pathogens

Article abstract of DOI:10.1021/acs.jmedchem.9b00550

Auranofin, an FDA-approved arthritis drug, has recently been repurposed as a potential antimicrobial agent; it performed well against many Gram-positive bacteria, including multidrug resistant strains. It is, however, inactive toward Gram-negative bacteria, for which we are in dire need of new therapies. In this work, 40 auranofin analogues were synthesized by varying the structures of the thiol and phosphine ligands, and their activities were tested against ESKAPE pathogens. The study identified compounds that exhibited bacterial inhibition (MIC) and killing (MBC) activities up to 65 folds higher than that of auranofin, making them effective against Gram-negative pathogens. Both thiol and the phosphine structures influence the activities of the analogues. The trimethylphosphine and triethylphosphine ligands gave the highest activities against Gram-negative and Gram-positive bacteria, respectively. Our SAR study revealed that the thiol ligand is also very important, the structure of which can modulate the activities of the Au^I complexes for both Gram-negative and Gram-positive bacteria. Moreover, these analogues had mammalian cell toxicities either similar to or lower than that of auranofin.

Full text of DOI:10.1021/acs.jmedchem.9b00550

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Article
Synthesis and Structure-Activity Relationship Study of
Antimicrobial Auranofin Against ESKAPE Pathogens
Bin Wu, Xiaojian Yang, and Mingdi Yan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00550 • Publication Date (Web): 06 Aug 2019
Downloaded from pubs.acs.org on August 7, 2019
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Synthesis and Structure-Activity Relationship Study
of Antimicrobial Auranofin Against ESKAPE
Pathogens
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Bin Wu, Xiaojian Yang, Mingdi Yan*
Department of chemistry, The University of Massachusetts Lowell, MA 01854
ABSTRACT: Auranofin, an FDA-approved arthritis drug, has recently been repurposed
as a potential antimicrobial agent where it performed well against many Gram-positive
bacteria, including multidrug resistant strains. It is however inactive towards Gram-
negative bacteria, which are in dire need for new therapies. In this work, 40 auranofin
and analogs were synthesized by varying the structures of the thiol and phosphine
ligands, and their activities were tested against ESKAPE pathogens. The study
identified compounds that exhibited up to over 2 logs higher bacterial inhibition (MIC)
and killing (MBC) activities than auranofin, making them effective against Gram-
negative pathogens. Both thiol and the phosphine structures influence the activities of
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the analogs. The trimethylphosphine and triethylphosphine ligand gave the highest
activities against Gram-negative and Gram-positive bacteria, respectively. Our SAR
study revealed that the thiol ligand is also very important, the structure of which can
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modulate the activities of the Au complexes for both Gram-negative and Gram-positive
bacteria. Moreover, these analogs had either similar or lower mammalian cell toxicity
compared to auranofin.
INTRODUCTION
The increasing prevalence of resistance to the majority of existing antibiotics has
1
generated a pressing global healthcare crisis. To undermine the actions of antibiotics,
bacteria have developed powerful resistance mechanisms including mutational
2
alteration of the target proteins, loss of membrane porins, enzymes that degrade
antibiotics,3-5 and over-expression of efflux pumps that drive antibiotics out of the
bacterium.6-8 Certain highly resistant bacteria have acquired multiple mechanisms
against all available antibiotics.9-11 The situation is especially dire for Gram-negative
bacteria. The recent addition of antibiotics in the clinical pipeline has been limited to
treating Gram-positive infections, and there is no new class of clinically-approved
antibiotics for Gram-negative bacteria since the discovery of quinolones in 1968.12
Untreatable antimicrobial resistance (AMR) is rapidly emerging in, for example,
Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii
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that are resistant to all commonly used antibiotics, including fluoroquinolones, -
lactams, macrolides, aminoglycosides, tetracyclines, contributing to the majority of
deaths caused by hospital-acquired infections. To combat AMR, new strategies are
being developed, like the use of efflux inhibitors,13 bacteriophages,14 and
nanoparticles15-17 as antibacterial agents. However, these endeavors are often associated
with lengthy and costly development processes, for which reason other avenues are
needed. One such route is drug repurposing, which is the redirecting of known anti-
inflammatory, anticancer and other drugs into antimicrobial use, providing viable
candidates and potentially low cost ways in the fight against AMR.18-20
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Auranofin is a gold compound consisting of a tetraacetylated thioglucose and a
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triethylphosphine coordinated to Au (1, Fig. 1). The medicinal use of gold compounds
th
can be traced back to the 19 century in treating tuberculosis, syphilis, and
inflammatory rheumatoid diseases.21-22 Early studies showed that coordination of
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phosphine to Au gave non-ionic complexes that had high lipid solubility. In the
search for better anti-arthritic drugs, Sutton et al. at Smith Kline and French
Laboratories
discovered
2,3,4,6-tetra-O-acetyl-1-thio--D-glucopyranosato-S-
(triethylphosphine) gold, i.e., auranofin, which gave excellent therapeutic efficacy and
24
high serum gold concentration. Auranofin was subsequently approved by the FDA in
1985 as an oral anti-inflammatory aid in the treatment of rheumatoid arthritis.25-26 Its
safety has been well-documented, with no reported concerns on carcinogenicity, serious
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side effects or other long-term safety issues.27 Auranofin has shown broad activity
towards a number of thiol-dependent reductases that are essential for maintaining
intracellular levels of free thiols and redox homeostasis. Inhibition of these enzymes by
auranofin decreases cellular reducing capacity and weakens their defense against
oxidative stress. Its broad activity has sparked increasing interests to repurpose this
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drug as potential therapy for other diseases. For example, the anticancer activity has
been reported for different types of tumors, including melanoma and leukemia,29-30 and
auranofin compares well with the currently marketed drug cisplatin.31-32 Auranofin has
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also shown strong activity against parasites that cause malaria and severe diarrhea,
as well as invasive fungi that are particularly difficult to target.35-36 Auranofin is
currently undergoing a phase II clinical trial for recurrent epithelial ovarian cancer.37
The results from an NIH-sponsored phase I trial to characterize the pharmacokinetics
and safety of auranofin in healthy volunteers against the parasites Entamoeba histolytica
and Giardia intestinalis supported the safety of auranofin as a broad-spectrum
antiparasitic drug.25, 38
The precise molecular mechanism of auranofin's mode of action is still unclear. It is
generally believed that upon administration of auranofin, the tetraacetylated
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thioglucose ligand is rapidly displaced by the blood thiols. The Au or [(PEt )Au]
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species emanating from auranofin then binds to the redox-active selenocysteine or
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cysteine residues in thioredoxin reductase (TrxR) or other reductases. X-ray structure
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analyses are however inconclusive. In some cases, no gold was found in the active sites
of, for example, E. histolytica TrxR41 or Schistosoma mansoni thioredoxin-glutathione
reductase.40 In the case of Leishmania infantum trypanothione reductase, the
tetraacetylated thioglucose was also found at the trypanothione binding site in addition
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to Au , hinting at a dual mechanism of inhibition.
Auranofin has also been assessed for its antimicrobial activity against different
bacteria, where it showed no activity towards Gram-negative bacteria but it performed
well compared to current antibiotics against many Gram-positive bacteria, including
multidrug resistant strains and Mycobacterium tuberculosis.18, 43-47 The source of this
activity was postulated as the inhibition of the bacterial TrxRs, a process that led to
lower intracellular thiol concentration, disruption of the bacterial thiol-redox
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homeostasis, and weakening of the bacterial defense against oxidative stress. Other
mechanisms have also been proposed, including disruption of the selenium metabolism
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by forming a stable Au-Se adduct in the case of Clostridium difficile, and inhibition of
cell wall, DNA, protein and toxin syntheses in the case of methicillin-resistant
Staphylococcus aureus (MRSA).48 The thiophilic nature of auranofin, which may react
with other cysteine-containing enzymes, and the suggested multiple modes of action,
argue well for the potential of auranofin against drug-resistant bacteria and in
preventing the emergence of resistance. In fact, studies have shown the inability to
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48
generate auranofin-resistant mutants of M. tuberculosis or S. aureus.
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The ineffectiveness of auranofin towards Gram-negative bacteria was suggested to be
the result of their glutathione system – glutathione/glutathione reductase – that could
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compensate for the loss of reducing capability in Trx-TrxR system by auranofin. An
alternative hypothesis was also proposed, suggesting that the outer membrane of
Gram-negative bacteria was an effective barrier to auranofin.48 This argument was
supported by results showing that auranofin could be made susceptible to Gram-
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negative strains by co-administration with a membrane permeable agent like
polymyxin B.48-49
There are limited SAR (structure-activity relationship) studies on auranofin.24, 49-50
These and other investigations generally conclude that the thioglucose ligand is not
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essential, and the pharmacophore is likely either Au or the [(PEt )Au] in complex with
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the target. In this study, we synthesized 40 auranofin and analogs by varying the
structures of the thiol and phosphine ligands, and tested their antimicrobial activities
against Enterococcus faecium, Staphylococcus aureus, Klebiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa and Enterobacter cloacae, the six ESKAPE pathogens
that have shown growing multidrug resistant virulence and causing two-thirds of all
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clinical infections, as well as a control strain of Gram-negative E. coli. A preliminary
in vitro toxicity study was also carried out on A549 cells. Our results demonstrate that
(1) Auranofin can be made active towards Gram-negative pathogens by manipulating
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the ligands coordinated to Au . While auranofin itself has no activity against all Gram-
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negative pathogens, some analogs become active even for P. aeruginosa; (2) The thiol
ligand is important for the activity; (3) The analogs showed either similar or lower
mammalian cell toxicity compared to auranofin.
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RESULTS
Design and synthesis of auranofin analogs. Auranofin is a monomeric linear complex
consisting of Au(I) core coordinated with a peracetylated thioglucose and a
triethylphosphine ligand (1, Fig. 1). Gold is a late transition metal with the most
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common oxidation states of +1 (aurous Au ) and +3 (auric Au ). Approved gold drugs,
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including sodium aurothiomalate and auranofin, are Au compounds probably due to
the high toxicity of AuIII complexes (e.g., HAuCl4).27 Au is a soft acid. Au salts
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52
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coordinated with a relatively hard base like chloride are unstable, and generally
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27
disproportionate into metallic gold and Au in the presence of water. As such, the
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most stable Au complexes and nanoclusters have soft ligands like thiols and
phosphines. The roles of phosphine and the thiol ligands were investigated by Sutton et
al., where the in vivo antiarthritic activities were studied on 13 auranofin and analogs
prepared by combining different alkylphosphine ligands (Me, Et, i-Pr, n-Bu) with either
24
Cl or a thio glucose ligand. It was concluded that the phosphine ligand played a major
role in the activity of the gold complex, whereas the non-phosphine ligand had less
effect. Triethylphosphine PEt , the ligand in auranofin, gave the best therapeutic
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efficacy and the highest serum gold concentration. The authors also showed that the
non-phosphine ligand could modulate the cytotoxicity where the acetylated thio
glucose ligand rendered auranofin less toxic than triethylphosphine gold chloride (the
rat acute oral LD of 265 vs. 79.0 mg/kg, respectively), which was further confirmed by
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other studies.53
Here, we conducted a SAR study by varying the structures of the thiol and the
phosphine ligands on auranofin. Three groups of compounds were initially synthesized:
Group 1 are analogs with different thio sugar structures (2–14); Group 2 are analogs
with aromatic or aliphatic thiols (15–23); and Group 3 are analogs with varying
phosphine structures (24–36). Subsequent antibacterial activity studies of these
compounds led to the identification of structural features which were then applied to
the design and syntheses of analogs 37–40.
In Group 1 (Fig. 1), the peracetylated thioglucose (Glc(Ac) ) ligand in auranofin was
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replaced by peracetylated galactose (Gal(Ac) , 2), N-acetylglucosamine (GlcNAc(Ac) , 3,
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, 8), maltose (Mal(Ac) , 5), lactose (Lac(Ac) , 6) or trehalose (Tre(Ac) , 7). De-acetylated
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analogs of Glc (9), Gal (10), Man (11), GlcNAc (12), Lac (13) and Mal (14) were also
included to test the impact of lipophilicity on the activities of the analogs.
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OAc
O
AcO
AcO
OAc
O
OAc
O
OAc
O
AcO
AcO
AcO
AcO
AcO
AcO
S
S
S
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Au
S
Au
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PEt3
Au
NHAc
PEt3
NH
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PEt3
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O
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O
AcO
AcO
AcO
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O
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O
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NH
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OH
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HO
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S
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HO
S
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HO
Au
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OH
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OH
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OH
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OH
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OH
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OH
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HO
HO
OH
O
S
Au
S
Au
NHAc
PEt3
HO
O
OH
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S
Au
HO
14
OH
PEt3
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Figure 1. Auranofin (1), and Group 1 analogs with protected and unprotected thio
sugar ligands (2–14).
Compounds 1–6 were synthesized from -glycosyl thiol 1c–6c and Et PAuCl
3
according to reported methods (Scheme 1).54-57 Briefly, α-glycosyl halides 1a–6a were
treated with KSAc to give peracetylated thioglycosides 1b–6b through a
straightforward S 2 reaction. S-Deacetylation of the anomeric thioacetate 1b–6b by
N
transthioesterification using dithiothreitol (DTT) afforded the glycosyl thiols 1c–6c,
which was then treated with Et PAuCl under mild basic conditions for 1-1.5 h to give
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the desired auranofin 1 and its analogs 2–6 in good to excellent yields.
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O
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S
Au
X
PEt3
1
a-6a
1b-6b
1c-6c
1-6
X=Cl or Br
Scheme 1. Synthesis of auranofin 1 and analogs 2–6: a) KSAc, acetone, RT, 3–4 h (82–
0%); b) DTT, NaHCO , DMF, RT, 1–1.5 h (90–95%); c) Et PAuCl, K CO , DCM/H O, 0
0
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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
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
3
3
2
3
2
oC–RT, 1–1.5 h (61–94%).
The synthesis of Tre(Ac) analog 7 follows the procedure shown in Scheme 2.
7
Reaction of D-trehalose 7a with benzaldehyde dimethyl acetal catalyzed by TsOH gave
a mixture of trehalose, trehalose benzylidene and trehalose dibenzylidene, which was
purified after acetylation to give peracetylated trehalose benzylidene 7b in 40% overall
yield. The N-bromosuccinimide (NBS)-mediated cleavage of benzylidene acetal 7b gave
6-bromo trehalose derivative 7c in 89% yield. The bromide 7c was then converted to
thioacetate 7d through the iodide intermediate in an overall 89% yield. Deprotection of
the benzoyl group by hydrolysis followed by acetylation afforded 7e (72%, 2 steps),
which was then S-deacetylated via DTT-mediated transthioesterification to afford the
thiol derivative 7f in 89% yield. Direct coupling of 7f with Et PAuCl gave only the
3
dinuclear gold complex 7. Even after reducing the amount of Et PAuCl, no
3
mononuclear gold (I) complex was observed. Stronger base like DBN gave similar
results, whereas NaOMe or NaOH removed the acetyl groups instead. The formation of
the dinuclear gold structure was supported by NMR and high-resolution mass
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1
spectrometry (HRMS). The integrations of peaks at 1.80 ppm and 1.17 ppm in the H
NMR (Fig. S34), assigned to CH and CH in PEt , respectively, corresponded to two
2
3
3
3
1
PEt ligands in the complex. The P NMR gave a single peak at 34.66 ppm (Fig. S36),
3
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
whereas for all other triethylphosphine mononuclear gold complexes synthesized in
this work, the chemical shifts of P in PEt appeared at 36–39 ppm instead. The structure
3
was further supported by HRMS, where the highest intensity peak at 1281.2744
corresponded to the molecular ion of 7. The thiolate in 7f, formed from a less acidic
thiol at C-6, is more nucleophilic than those thiolates formed from a more acidic
anomeric thiol at C-1 in compounds 1–6, allowing the C-6 thiolate to react further with
Et PAuCl to form the dinuclear gold complex 7. There are similar literature reports on
3
the formation of dinuclear gold complexes, for example, from the reaction of a
mononuclear gold (I) complex bearing a cysteine-containing dipeptide ligand with
5
8
[
Au(OTf)(PPh )].
3
Br
SAc
O
OH
O
Ph
O
O
O
HO
HO
O
BzO
AcO
BzO
AcO
a
b
c
AcO
HO
AcO
AcO
AcO
O
O
O
O
OH
OH
OAc
OAc
OAc
OAc
OAc
OAc
O
O
O
O
AcO
7d
HO
AcO
AcO
AcO
HO
AcO
AcO
7a
7b
7c
PEt3
Au
SAc
O
SH
Au PEt3
S
d
AcO
AcO
e
AcO
AcO
O
f
O
AcO
AcO
Cl
AcO
AcO
O
O
AcO
O
OAc
OAc
OAc
OAc
O
O
OAc
OAc
AcO
AcO
O
AcO
AcO
AcO
AcO
7e
7f
7
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8
9
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
Scheme 2. Synthesis of analog 7: a) i: PhCH(OMe) , TsOH, DMF; ii Ac O, Et N, DMF
2
2
3
(
40% over 2 steps); b) NBS, CaCO , CCl (89%); c) i: KI, DMF, 60 °C; ii: KSAc, DMF, RT,
3
4
8 h (87%); d) i: NaOMe, MeOH, RT; ii: Ac O, pyridine, 0 °C–RT (72%); e) DTT, NaHCO ,
2
3
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
DMF, RT, 24 h (93%); f) Et PAuCl, DBU, DCM, RT (62%).
3
Compound 8 was synthesized from the D-glucosamine hydrochloride 8a (Scheme 3).
Reaction with p-anisaldehyde gave the Schiff base glucosamine imine 8b, which was
then peracetylated to afford 8c. Subsequent hydrolysis in HCl/acetone gave
peracetylated glucosamine hydrochloride 8d, which reacted with chloroacetic
anhydride to afford 8e. A final substitution by 1,4-benzenedithiol followed by coupling
with Et PAuCl gave the gold complex 8 in 96% yield.
3
OAc
O
OH
O
OH
O
a
b
HO
HO
AcO
AcO
HO
HO
OAc
OH
NH HCl
OH
8b
N
N
2
8a
8c
MeO
MeO
c
OAc
O
OAc
O
OAc
O
e
d
AcO
AcO
AcO
AcO
AcO
AcO
OAc
OAc
OAc
NH
O
NH
O
NH ·HCl
2
8e
8d
S
Cl
OAc
O
f
AcO
AcO
OAc
8f
NH
HS
O
Et P
3
S
Au
8
S
Scheme 3. Synthesis of analog 8: a) p-anisaldehyde, NaOH, H O, RT, 1 h (80%); b) Ac O,
2
2
pyridine, RT, overnight (81%); c) 5 M HCl, acetone, reflux, 30 min (86%); d) chloroacetic
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anhydride, pyridine, DCM, 0 °C–RT (89%); e) 1,4-benzenedithiol, TEA, DCM, 16 h (91%);
f) Et PAuCl, DBU, DCM, RT, 1.5 h (96%).
3
The analogs containing an unprotected thio sugar ligand 9–14 were synthesized by
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
treating the corresponding peracylated thio sugar analogs with NaOMe/MeOH
followed by Et PAuCl in a one-pot reaction (Scheme 4).
3
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8
9
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
a
b
O
O
O
HO
S
AcO
SAc
HO
SNa
Au
PR3
9-14
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
Scheme 4. Synthesis of analogs 9–14: a) NaOMe, MeOH, RT, 2-4 h; b) Et PAuCl, MeOH,
3
1
-2 h, 57%–83% over 2 steps.
Analogs 15–23 in Group 2 are compounds with the thio sugar replaced by an aromatic
or aliphatic thiol ligand (Fig. 2). Both electron-donating and electron-withdrawing
groups are included to test the electronic effect of the thiol ligand on the antimicrobial
activity. For the synthesis of gold (I) complexes with an aromatic thiol ligand (15, 16,
1
8–20), the corresponding thiol was treated with Et PAuCl in DCM in the presence of
3
DBU, and the product was obtained after a straightforward purification by column
chromatography (condition a, Scheme 5). Analogs having an aliphatic thiol ligand 21–
2
3 were prepared by reacting the corresponding thiol with Et PAuCl in the presence of
3
one equivalent of NaOMe in methanol (condition b, Scheme 5). Analog 17 was also
synthesized following this method and was purified by recrystallization
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NH2
S
S
S
Au
Au
PEt3
Au
Au
PEt3
PEt3
PEt3
H N
2
15
16
17
S
S
S
Au
Au
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
PEt3
PEt3
MeO
HO
O N
2
F3C
1
8
1
19
22
20
O
S
S
F3C
S
Au
O
Au
Au
PEt3
PEt3
PEt3
2
23
Figure 2. Group 2 analogs having aromatic and aliphatic thiol ligands.
a or b
S
RSH
R
Au
PEt3
Scheme 5. Synthesis of Group 2 analogs 15–23 having an aromatic and aliphatic thiol
ligand. a) For compounds 15, 16, 18–20: Et PAuCl, DBU, DCM, RT, 1-2 h (62%–91%). b)
3
For compounds 17, 21–23: Et PAuCl, NaOMe, MeOH, RT, 1-2 h (59%–92%).
3
Analogs 24–36 in Group 3 are compounds having different phosphine structures (Fig.
3
). Aliphatic phosphines with varying chain length (methyl, ethyl, n-butyl) and
aromatic phosphines with either electron-donating (OMe) or electron-withdrawing (F,
CF ) groups were prepared as both gold chloride and gold thio sugar complexes. AuCl
3
was generated in situ by reducing HAuCl with 2,2'-thiodiethanol, which was treated
4
immediately with a cold solution of 1.05 equiv. of the corresponding triphosphine in
EtOH/DCM to give triphosphine gold chlorides 24–30 (reaction a, Scheme 6).24
Auranofin analogs 31–36 were prepared straightforwardly by treating the
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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
corresponding triphosphine gold chlorides (24, 26–30) with peracetylated thioglucose 1c
in high yields (reaction b, Scheme 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
P
Au Cl
P
Au Cl
P
Au Cl
P
Au Cl MeO
P
Au Cl
F
P
Au Cl
F3C
P
Au Cl
3
3
3
3
24
25
26
27
28
29
30
OAc
O
OAc
O
OAc
O
AcO
AcO
AcO
AcO
AcO
AcO
S
S
S
Au
Au
Au
OAc
P
OAc
P
OAc
P
31
32
1
OAc
O
OAc
OAc
O
OAc
O
O
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
S
S
S
S
Au
Au
Au
P
Au
OAc
P
OAc
P
OMe
OAc
F
OAc
P
CF3
3
3
3
3
33
34
35
36
Figure 3. Group 3 analogs having different phosphine ligand structures.
HAuCl •3H O
4
2
a
OAc
O
AuCl
b
AcO
AcO
S
PR3
R P Au Cl
3
Au
OAc
PR3
2
2
4, R=Me
5, R=Et
31, R=Me
32, R=n-Bu
33, R=Ph
26, R=n-Bu
2
2
2
7, R=Ph
8, R=p-OMe-Ph
9, R=p-F-Ph
34, R=p-OMe-Ph
35, R=p-F-Ph
36, R=p-CF -Ph
3
30, R=p-CF -Ph
3
Scheme 6. Synthesis of auranofin analogs having different phosphine ligand structures:
a) 2,2'-thiodiethanol, H O/EtOH/DCM (72%–94%); b) 1c, DBU, toluene, RT, 2 h (83%–
2
90%).
In vitro antimicrobial activities. The antimicrobial activities of auranofin and analogs
were tested against ESKAPE pathogens, which include 4 Gram-negative strains (A.
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baumannii, P. aeruginosa, E. cloacae, K. pneumonia) and 2 Gram-positive strains (S. aureus,
E. faecium). A quality control strain, E. coli ATCC 25922, was included in all assays and
validated with ciprofloxacin and teicoplanin in every trial. The results are presented in
Tables 1–3 (in µM) and Tables S1–S3 (in µg/mL) as MIC (minimal inhibitory
concentration), the lowest concentration that inhibits the growth of the bacterium, and
MBC (minimal bactericidal concentration), the lowest concentration that kills the
bacterium. Auranofin 1 showed potent activities against both Gram-positive strains, S.
aureus and E. faecium, with the MIC (MBC) of 0.03 (0.06) and 0.12 (0.25) µg/mL,
respectively (Table S1). It showed weak activity for A. baumannii and E. coli, with
MIC/MBC at 32 and 16 µg/mL, respectively. Auranofin lacks significant activity against
the P. aeruginosa, E. cloacae, and K. pneumonia. These results are in agreement with
previous reports that consistently showed Gram-positive activities of auranofin.18, 43-44,
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
7-48, 59-60
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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
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
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a
Table 1. MIC/MBC (µM) of Group 1 analogs having varying thio sugar structures.
P.
K.
A. baumannii
E. cloacae
S. aureus
E. faecium
E. coli
aeruginosa
pneumoniae
LogP
NCTC 13420
NCTC 13405
JE2 (USA300)
ATCC 700221
ATCC 25922
NCTC 13437
377 (377)
377 (377)
189 (189)
>328
ATCC 700603
377 (377)
>377
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
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
1
2
47 (47)
47 (94)
189 (189)
377 (377)
47 (94)
0.04 (0.09)
0.04 (0.09)
0.2/0.09 (0.4)
0.2/0.4 (0.4)
0.09 (0.4)
24 (24)
24 (24)
12 (12)
21 (21)
17 (17)
33 (33)
24 (24)
152/323 (152)
31 (31)
16 (16)
16 (16)
7/4 (7)
0.56
0.38
3
24 (47)
189 (189)
>328
0.04/0.09 (0.09)
0.01 (0.01)
0.26
4f
5f
6g
7c
8f
9
82 (164)
33 (33)
>328
0.08 (0.2)
1.01
132 (132)
132 (132)
>194
66 (66)
>265
0.008 (0.06)
0.03/0.06 (0.1)
0.1 (0.3)
0.45
66 (132)
12 (12)
265 (265)
24 (24)
>265
0.03 (0.1)
1.04
>194
0.003/0.006 (0.006)
0.04/0.009 (0.1)
0.06/0.1 (0.5)
0.02 (0.1)
0.05 (0.09)
0.04/0.07 (0.3)
0.1 (2)
-0.28
-0.36
-0.81
-0.73
-0.59
-0.89
-1.81
-1.69
76/19 (19)
63/16 (251)
31 (31)
>303
152 (303)
125 (125)
63 (63)
>303
502 (502)
251 (251)
502 (502)
464 (464)
381 (381)
381 (381)
502 (502)
251 (251)
502 (502)
464 (464)
381 (381)
>381
10
11
12
0.1/0.2 (0.5)
0.1/0.06 (0.5)
0.05/0.1 (0.5)
0.09 (0.4)
31 (63)
125 (251)
116 (232)
190 (190)
190 (190)
0.02/0.06 (0.1)
0.02 (0.05)
15/29 (116)
24/48 (190)
24 (24)
1
1
3
4
0.02/0.05 (0.09)
0.09/0.04 (0.09)
24 (24)
12 (12)
0.09/0.2 (0.4)
aAssays were repeated twice. Only one value is presented unless both data are shown.
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1
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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
b
c
d
e
f
g
Lowest precipitation concentration at 4, 8, 16, 32 64, 128 µg/mL.
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
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9
The results of Group 1 compounds, i.e., analogs with varying thio sugar structure (Fig.
), are shown in Table 1. Changing Glc(Ac) in auranofin (1) to Gal(Ac) (2) did not
1
4
4
cause much difference in activity against any of the strains. The GlcNAc analog 3
showed 2-4 times increase in activity against all Gram-negative strains and E. faecium.
Substituting the N-acetyl group of GlcNAc with a N-trichloroacetyl group (4)
completely diminished the enhancement in the activity of all Gram-negative strains, so
was the GlcNAc analog having an aromatic linker (8). However, compound 4 showed
excellent activity against S. aureus, lowering the MIC/MBC of auranofin by up to 4 times.
The disaccharide maltose analog 5 also enhanced the activity against S. aureus, E.
faecium, P. aeruginosa and E. cloacae by 2-4 times. This was however not observed for the
other disaccharide lactose analog 6, which showed no improvement for Gram-positives
and was worse for all Gram-negative strains. The trehalose analog 7 increased the
activity against E. cloacae by 8 folds and was 15 times better than auranofin against S.
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
+
58
aureus with MIC/MBC of 6 nM, perhaps owing to the presence of two AuPEt in 7.
3
The de-acetylated auranofin analog 9 had similar activity as auranofin 1 for all strains.
Among other de-acetylation analogs, compound 10 showed 3 times enhancement in
activity against E. cloacae, and compound 12 was 2-3 times better than auranofin against
S. aureus and E. coli .
Table 2 lists MIC/MBC of Group 2 analogs that contain either an aromatic or an
aliphatic thiol ligand (15–23, Fig. 2). Among compounds bearing an aromatic thiol
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9
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
ligand (15–20), those having an electron-donating group on the phenyl ring (16–18) are
generally more active than auranofin. Compounds 17 having an p-aminophenyl group
showed the highest activity enhancement of up to 10 times over auranofin against all
strains. The electron-withdrawing groups (NO and CF ), on the other hand, made the
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
2
3
analogs 19 and 20 completely inactive towards all Gram-negative strains tested.
Compounds 19 is however very active against S. aureus, lowering the MIC/MBC of
auranofin by up to 10 times. Replacing the thio sugar in auranofin with an aliphatic
thiol ligand generally increased the activity (21–23). For example, compound 21 had 19
times lower MIC than auranofin against E. cloacae as well as K. pneumoniae, and showed
activity against P. aeruginosa with a MIC/MBC of 41 M, which is 9 times better than
auranofin. For E. faecium, the activity of most analogs were similar to auranofin with the
exception of compounds 16 and 17 which showed more than 7 times lower MIC than
auranofin. With the exception of compound 17, all other analogs showed higher
activities than auranofin against S. aureus.
Compounds in Group 3 are analogs having a trialkylphosphine of varying chain
length (24–26, 31–32) or a triarylphosphine with different electronic property (27–30, 33–
3
6, Fig. 3), and the MIC/MBC are shown in Table 3. For the Gram-negative strains, the
activity generally decreased with increasing the alkyl chain length (24–>25–>26, and 31–
>1–>32). Replacing triethylphosphine with trimethylphosphine resulted in a drastic
enhancement in activity. For example, the trimethylphosphine analog 31 improved the
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activity of auranofin against all Gram-negatives tested. The MIC of 31 for E. cloacae and
K. pneumoniae was 60 and 30 folds lower, making them active against these two strains,
whereas auranofin is inactive for both. On the other hand, the tri-n-butylphosphine
ligand completely diminished the activity of the resulting analog 32 against all Gram-
negatives. Aromatic phosphine ligands are also detrimental. Analogs with a
triarylphosphine ligand having either electron-withdrawing or electron-donating group
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
(33–36) showed almost no activity against all Gram-negative strains.
For the two Gram-positive strains, replacing triethylphosphine in auranofin with
trimethylphosphine (31) had almost no impact on the activity. The longer tri-n-
butylphosphine ligand decreased the activity by more than an order of magnitude (32
vs. 1, 26 vs. 25), and so were all triarylphosphine ligands (27–30, 33–36).
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2
3
4
5
6
7
8
9
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
a
Table 2. MIC/MBC (µM) of Group 2 analogs having an aromatic or aliphatic thiol ligand.
K.
E. faecium
E. coli
A. baumannii P. aeruginosa
E. cloacae
S. aureus
Log
P
pneumoniae
ATCC
700221
ATCC
25922
NCTC 13420
NCTC 13437
NCTC 13405
JE2 (USA300)
ATCC 700603
377 (377)
>603
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
1
47 (47)
19/75 (>603)
18/36 (18)
18(18)
377 (377)
302 (302)
291 (291)
146 (146)
282 (282)
189 (189)
75 (603)
0.04 (0.09)
0.02 (0.6)
0.2/0.09 (0.4)
0.6 (1)
24 (24)
151 (151)
36 (36)
9 (9)
0.56
2.20
0.61
1.36
2.01
1
5f
6g
7d
1
1
18/73 (36/73)
36 (36)
146 (146)
36 (36)
0.009 (0.6)
0.07/0.02 (0.07)
0.02 (0.3)
0.02 (0.1)
0.02 (0.1)
0.02/0.1 (0.3)
1
8f
35 (35)
141 (141)
141 (141)
35 (35)
0
.0004/0.02
0.02)
1
9e
>546
>546
>546
>546
0.06/0.1 (0.5)
>546
>3.28
(
2
2
0e
1e
520 (>520)
10 (82)
>520
>520
>520
20 (20)
76 (76)
149 (149)
0.02/0.1 (0.2)
0.03 (0.03)
0.02 (0.2)
0.2/ 0.3 (0.6)
0.1/0.3 (0.6)
0.6 (1)
>520
>3.18
1.19
1.75
2.08
41 (41)
5/10 (82)
38 (152)
37 (149)
10 (10)
76 (76)
74 (74)
2
2d
19 (76)
305 (305)
149 (149)
0.005/0.02 (0.02)
0.009 (0.1)
23g
37/74 (149)
aAssays were repeated twice. Only one value is presented unless both data are shown.
b
c
d
e
f
g
Lowest precipitation concentration at 4, 8, 16, 32 64, 128 µg/mL
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Page 25 of 77
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
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
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
a
Table 3. MIC/MBC (µM) of Group 3 analogs having a trialkyl- or triaryl-phosphine ligand.
A. baumannii P. aeruginosa
E. cloacae
K. pneumoniae
ATCC 700603
S. aureus
E. faecium
E. coli
Log P
NCTC 13420 NCTC 13437 NCTC 13405
JE2 (USA300) ATCC 700221 ATCC 25922
2
2
4
5
7/13 (13)
11/23 (23)
74/147 (147)
52 (52)
92 (183)
74 (74)
>518
3 (3)
23 (23)
>589
7/13 (13)
46 (46)
147 (147)
>518
0.1/0.2 (1.6)
0.4 (3)
7 (7)
23 (23)
>589
>518
>438
>467
>366
6 (6)
0.16
1.74
0.001/0.02 (0.2) 0.09/0.2 (0.7)
2
6e
2 (4)
1/2 (4)
2 (3)
5 (5)
4 (4)
>3.99
>3.94
>4.09
>4.04
>3.94
0.35
2
7d 16/129 (518)
>518
2
8c
>438
29/58 (233)
>366
>438
>438
>438
3 (3)
2
9d
>467
>467
>467
1/ (4)
2/4 (4)
e
3
3
0
>366
>366
>366
3/1 (11)
0.1 (0.2)
0.04 (0.09)
3 (3)
>366
1g
6/13 (101)
47 (47)
101 (101)
377 (377)
>336
3 (3)
13 (13)
377 (377)
>336
0.2/0.4 (0.8)
0.2/0.09 (0.4)
3/5 (3)
1
189 (189)
>336
24 (24)
>336
>311
0.56
3
2e
3c
84 (>336)
>311
>4.32
>3.87
3
>311
>311
>311
1/2 (2)
5 (5)
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6
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8
9
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
3
3
3
4c
5c
>281
>292
>249
>281
>292
>249
>281
>292
>249
>281
>292
>249
1 (2)
1 (2)
4 (8)
2/4 (4)
2/5 (5)
31 (62)
>281
>292
>249
>3.03
>3.04
>3.55
e
6
aAssays were repeated twice. Only one value is presented unless both data are shown.
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
b
c
d
e
f
g
Lowest precipitation concentration at 4, 8, 16, 32 64, 128 µg/mL.
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These results demonstrated that the Gram-negative activity of auranofin can be
effectively modulated by altering the thiol and phosphine ligands. The following
conclusions can be drawn from these data with regard to Gram-negative activities. (1)
De-protected thio sugars do not decrease the activity (e.g., 9–14, Table 1). It was
reported that following oral administration, auranofin metabolized to yield
deacetylated glucose.61 (2) Among the non-sugar thiol ligands, mercaptoethanol
showed the best activity enhancement against Gram-negatives (21, Table 2). (3)
Trimethylphosphine is a substantially better ligand than triethylphosphine against
Gram-negatives (24, 31, Table 3). Based on these observations, analogs 37–40 were
designed. Compounds 37–39, which have a trimethylphosphine ligand and a de-
protected Glc, Gal or GlcNAc thio sugar, respectively, were prepared following reaction
b in Scheme 4 except Me PAuCl was used instead of Et PAuCl. All 3 compounds
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
3
3
showed substantial enhancement in activity compared to auranofin against almost all
Gram-negative strains (Table 4). Except for P. aeruginosa, the MIC was lowered by 3-12
folds for A. baumannii, K. pneumoniae and E. coli, and by up to 44 folds for E. cloacae. The
highest activity was observed for compound 38, the de-acetylated Gal and
trimethylphosphine analog, reaching MIC/MBC of 4.5 and 34 µM against E. cloacae and
K. pneumoniae, which are otherwise ineffective by auranofin.
HO
OH
O
OH
O
OH
O
HO
HO
HO
HO
S
S
S
S
HO
Au
Au
HO
Au
Au
OH
37
PMe3
OH
38
PMe3
NHAc
PMe3
PMe3
39
40
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8
9
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
Figure. 4. Auranofin analogs having a trimethylphosphine and a de-protected thio
sugar (37–39) or mercaptoethanol ligands (40).
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
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a
Table 4. MIC/MBC (µM) of analogs having trimethylphosphine ligand.
A.
baumannii
P. aeruginosa
E. cloacae
K. pneumoniae
S. aureus
E. faecium
E. coli
Log P
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
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
NCTC
NCTC 13437
NCTC 13405
ATCC 700603
JE2 (USA300)
ATCC 700221
ATCC 25922
1
3420
1
47 (47)
377 (377)
101 (101)
>547
189 (189)
3 (3)
377 (377)
13 (13)
34 (34)
34 (34)
31 (126)
11 (23)
0.04 (0.09)
0.09 (0.2)
0.3/0.5 (1)
0.3 (0.3)
0.2/0.09 (0.4)
0.2/0.4(0.8)
0.3/0.5 (0.5)
0.3/0.5 (0.5)
0.2 /0.5 (0.5)
0.3 (0.3)
24 (24)
6 (6)
0.56
31g 6/13 (101)
0.35
37
38
39
17/4 (17)
17/9 (17)
16/8 (16)
4/9 (17)
4 (4)
9 (9)
-1.63
-1.88
-2.03
-0.15
>547
9 (9)
>503
8/16 (16)
3 (3)
0.5 (0.5)
8/31 (31)
1/6 (11)
40f 6/3 (23)
23/91 (91)
0.3 (0.7)
aAssays were repeated twice. Only one value is presented unless both data are shown.
b
c
d
e
f
g
Lowest precipitation concentration at 4, 8, 16, 32 64, 128 µg/mL.
ACS Paragon Plus Environment
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