1,2,3-Triazole-gold(I)-triethylposphine derivatives active against resistant Gram-positive pathogens

Article abstract of DOI:10.1016/j.bmcl.2021.127879

Innovative organogold(I) antibacterial compounds were synthesized by click chemistry with triethylphosphine-gold(I) azides and an alkyne derivative. The resulting organo-gold(I) compounds exhibit high levels of antibacterial activity against Gram-positive pathogens, with particularly low MICs against Clostridium difficile.

Full text of DOI:10.1016/j.bmcl.2021.127879

Bioorg. Med. Chem. Lett. 40 (2021) 127879
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,2,3-Triazole-gold(I)-triethylposphine derivatives active against resistant
Gram-positive pathogens☆
Mathieu Michaute, Alexandre Steffene, Jean-Marie Contrerase, Christophe Moricee,
,
b
,
c
d
a,b,c,*
Isabelle J. Schalka , Patrick Plesiat , Gaetan L.A. Mislin
´
¨
a
´
CNRS, UMR7242 Biotechnologie et Signalisation Cellulaire, 300 Boulevard Sebastien Brant, 67400 Illkirch-Graffenstaden, France
b
c
´
Universite de Strasbourg, Institut de Recherche de l’Ecole de Biotechnologie de Strasbourg (IREBS), 67400 Illkirch-Graffenstaden, France
´
Interdisciplinary Thematic Institute for Innovative Vectorization (ITI InnoVec), Universite de Strasbourg, 67000 Strasbourg, France
d
´
´
´
´
Laboratoire de Bacteriologie, UMR 6249 CNRS Chrono-Environnement, UFR Sante, Universite Bourgogne Franche-Comte, 25000 Besançon, France
e Prestwick Chemical, PC SAS, 220 Boulevard Gonthier d’Andernach, 67400 Illkirch-Graffenstaden, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
Innovative organogold(I) antibacterial compounds were synthesized by click chemistry with triethylphosphine-
gold(I) azides and an alkyne derivative. The resulting organo-gold(I) compounds exhibit high levels of anti-
bacterial activity against Gram-positive pathogens, with particularly low MICs against Clostridium difficile.
Antibiotic
Gold(I) complex
Azide-alkyne Huisgen cycloaddition
Clostridium difficile
Staphylococcus sp.
Enterococcus sp.
Resistance to antibiotics will be a major threat for mankind in the
forthcoming decades. The development of bacterial resistance to anti-
biotics is a natural Darwinian process which has been greatly amplified
by the massive use of antibiotics against human infections, and the
misuse of antibiotics to promote animal growth in farming.1 The raise of
increasingly antibiotic-resistant strains is also the result of a lack of
innovation in anti-infective discovery over the four last decades. Novel
antibacterial strategies are therefore urgently needed to regain control
on resistant pathogens.2
patients.4,5 Hybrid drugs made of two conjugated antibacterial mole-
cules were shown to improve greatly the biological and/or pharmaco-
logical properties of the combined molecules.6 Even so hybrids between
antibacterial metal complexes and approved antibiotics have been only
scarcely reported in the literature. Based on a β-lactam substructure
ferrocene and ruthenocene hybrids were described and shown to have
promising antibiotic activities.7,8 We recently added a new string to the
bow by reporting a new family of metallo-antibiotics namely chryso-
lactams 2 to 5, based on the penam scaffold of ampicillin (Amp) 1 and
containing a gold(I) ion (Fig. 1).9
Drugs used in the current antibiotic arsenal are exclusively consti-
tuted by elements from the first to the third periods of the Mendeleev
table (carbone, hydrogen, sulfur, oxygen, nitrogen, phosphorus). With
only the notable exception of boron3 and bismuth4 no metals were
included in approved drugs dedicated to antibacterial strategies. Com-
pounds containing other elements from the periodic table have been
developed mainly against cancer, and applications of organometallic
compounds in the treatment of bacterial infections have been put on
standby so far for toxicity issues. However, the diffusion of ever more
resistant bacterial strains and therefore the use of fully organic last
resort antibiotics with significant side effects make now organometallic
species competitive in the benefit vs risk balance for hard-to-treat
In the present article, we describe the synthesis and biological
properties of a second generation of chryso-lactams and a set of gold(I)-
containing fragments with inhibitory activities on Gram-positive path-
ogenic bacteria, including Clostridium difficile.
Amongst the five chryso-lactams described previously9 compounds 2
and 3 where shown to be those having promising in vitro antibacterial
activities. Triethylphosphane derivative 3 was much more easily syn-
thesized compared to the trimethylphosphane 2 analog, for only a
moderate difference in antimicrobial effects. Of note gold(I)-
triethylphosphine complexes are already approved for a use in human
health,10 and known to inhibit bacterial growth.11–13 We therefore built
☆
This article is dedicated to the late Pr. Ermanno Candolfi in memory of its outstanding contributions in microbiology
´
* Corresponding author at: CNRS, UMR7242 Biotechnologie et Signalisation Cellulaire, 300 Boulevard Sebastien Brant, 67400 Illkirch-Graffenstaden, France.
E-mail address: mislin@unistra.fr (G.L.A. Mislin).
https://doi.org/10.1016/j.bmcl.2021.127879
Received 18 November 2020; Received in revised form 6 January 2021; Accepted 10 February 2021
Available online 24 February 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

M. Michaut et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127879
compound 25 was synthesized starting from D-phenylglycine methyl
ester hydrochloride 21. The reaction of compound 21 with propargyl-
chloroformate led to the alkyne 22. The later compound was then
treated with benzyl azide in the presence of Cp*RuCl(cod),16,17 and the
resulting N-benzyl-triazole 23 was converted into the carboxylate 24 by
hydrolysis with lithium hydroxide and finally converted in the expected
compound 25 by catalytic hydrogenation under 6 bars (Scheme 2).
The antibacterial properties of the chryso-lactams 9 and 12 and
fragments 13, 14, 19, 20 and 25 were then assessed with characterized
microorganisms, and compared with those of ampicillin (Amp) 1 and the
chryso-lactam 3 reported previously.9 Minimal inhibitory concentra-
tions (MICs) were determined for Escherichia coli, a Gram-negative
bacterium, and a set of Gram-positive pathogenic bacteria, namely
Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, E. faecium and
Clostridium difficile (Table 1). This latter microorganism is a common
cause of diarrhea in healthcare facilities and, for the most aggressive
strains of hard-to-treat pseudomembranous colitis.18
Fig. 1. Structure of ampicillin 1 and chryso-lactams 2 to 5.
the next generation of organogold(I) compounds using solely the trie-
thylphosphine as co-ligand for the gold(I) center. Phosphane gold(I) was
introduced on the organic moiety by a Huisgen 1,3-dipolar cycloaddi-
tion, between (triethylphosphine)Au(I) azide 69,14 and the terminal
alkyne of an organic scaffold. In a second step, a migration of the
phosphane gold(I) moiety occurs, resulting in the formation of a 1,2,3-
triazole ring with, in the final conjugate, the gold(I) ion connected in
position 5 of the triazole through a
σ
-gold-carbon bond.14,15
E. coli was resistant to chryso-lactams as well as to all fragments, in
the range of concentrations tested (from 0.06 to 8 µg.mLꢀ 1) (Table 1).
Such a resistance of Gram-negative bacteria to organometallics is well
described,4,5,19 and is attributed to the permeability barrier exerted by
the bacterial outer membrane toward relatively hydrophobic mole-
cules.13,19 Interestingly, chryso-lactams 3, 9 and 12 and fragments 13,
14, 19 and 20 proved to be much more effective that compound 25, a
fully organic molecule, on the whole set of strains tested, with C. difficile
exhibiting a particularly high susceptibility to organometallics. These
results confirmed the crucial role played by gold(I) ion in the antibac-
terial activity of such compounds.12,13 Chryso-lactam 3 was previously
found to be very active against both S. aureus and S. epidermidis, and to a
lower extent against E. faecalis and E. faecium.9 Chryso-lactams 9 and 12,
as well as the fragments 13, 14, 19 and 20 exhibited anti-staphylococcal
and anti-enterococcal activities very similar to that of compound 3, that
In this second generation of organogold(I) compounds we aimed
more specifically to investigate the influence of the size of the penam
moiety involved in the hybrid conjugate on the antibacterial properties.
For this purpose, 4-ethynyl-benzoic acid 7 and 3-ethynyl-benzoic acid
10 were treated with (triethylphosphine)Au(I) azide 6. The carboxylate
functions of resulting organogold(I) compounds 8 and 11 were further
activated under the form of pentafluorophenyl-esters. These activated
esters were not purified and reacted with ampicillin 1 trihydrate to
generate penam derivatives 9 and 12 (Scheme 1).
In the frame of a SAR study, we aimed also to assess the contribution
of the β-lactam ring to the antibacterial activity of organogold(I) com-
pounds, by synthesizing gold(I) compounds including organic fragments
of the penam structure. Compounds 13 and 14 were obtained by the
reaction of D-phenylglycine with the activated ester of gold(I) com-
pounds 8 and 11 respectively. Smaller fragments 19 and 20 bearing only
the phenylglycine moiety were also synthesized. For this purpose, D-
phenylglycine 15 and L-phenylglycine 16 were treated with propargyl-
chloroformate under basic conditions. The resulting alkynes 17 and
18 were further combined with the phosphine-gold(I) azide 6 to
generate fragments 19 and 20. We previously reported that antimicro-
bial properties of chryso-lactams are mostly related to the presence of
the gold(I) ion.9 To confirm this observation, the fully organic
Scheme 2. Synthesis of chryso-lactam fragments 13, 14, 19, 20 and fully
organic fragment 25. i. C6F5OH, EDCI.HCl, CH2Cl2, 20 ◦C. ii. D-phenylglycine,
NEt3, THF, 20 ◦C. iii. Propargyl-chloroformate, NaOH, THF/H2O, 0◦ to 20 ◦C. iv.
6, THF, 20 ◦C. v. benzyl azide, Cp*RuCl(cod), toluene, 80 ◦C, sealed tube. vi.
LiOH⋅H2O, THF/H2O, 20 ◦C. vii. H2 (6 bars), Pd/C, MeOH, 50 ◦C.
Scheme 1. Synthesis of ampicillin-Au(I) conjugates 9 and 12. i. 6, THF, 20 ◦C.
ii. C6F5OH, EDCI.HCl, CH2Cl2, 20 ◦C. iii. 1, NEt3, THF/H2O, 0 ◦C to 20 ◦C.
2

M. Michaut et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127879
Table 1
Minimal inhibitory concentrations (MICs) in µg.mLꢀ 1 of ampicillin (Amp) 1, chrysolactams 3, 9, and 12, gold(I) conjugates 13, 14, 19 and 20, and of gold-free
fragment 25 for a panel of wild-type and resistant bacterial strains.a.
Bacteria tested
Amp (1)
3
9
12
13
14
19
20
25
S. aureus
ATCC 25923b
ATCC 700699c,d
ATCC 29213b
ST20131365c,d
S. epidermidisATCC 14990b
ATCC 35984c
ST20140436c,d
ST20150446c,d
E. faecalis
0.5
>8
0.5
4
0.125
0.25
0.125
0.25
0.125
0.25
≤0.06
0.125
0.125
0.125
≤0.06
0.125
0.125
≤0.06
0.125
0.125
0.125
0.125
≤0.06
≤0.06
≤0.06
0.125
0.125
0.25
0.125
0.25
>8
>8
>8
>8
>8
>8
>8
>8
0.125
0.25
≤0.06
0.25
0.125
0.5
0.125
0.125
≤0.06
≤0.06
≤0.06
0.125
0.25
0.25
1
≤0.06
≤0.06
≤0.06
0.125
≤0.06
0.125
0.125
0.125
≤0.06
≤0.06
≤0.06
≤0.06
≤0.06
≤0.06
≤0.06
≤0.06
>8
4
8
JH2-2b
0.5
0.5
0.5
0.5
1
0.5
0.5
1
0.25
0.125
0.25
0.25
0.25
0.5
0.25
0.25
0.5
0.5
>8
>8
>8
UCN4d
0.25
0.5
0.25
0.5
V583d
1
1
E. faecium
ATCC 19434 Tb
BM 4147c,d
0.5
8
1
0.5
0.5
1
0.25
0.5
0.25
0.5
0.25
0.5
0.25
0.25
0.5
>8
>8
>8
>8
>8
2
0.5
AUS0004c,d
>8
2
1
0.5
0.5
>8
0.25
>8
0.25
>8
0.5
E. coliATCC 25922b
>8
0.125
>8
>8
>8
C. difficileATCC70057b
0.5
≤0.06
≤0.06
≤0.06
≤0.06
≤0.06
0.125
a
The MIC values displayed are the highest values obtained in two independent experiments.
b
c
wild-type reference strain.
β-lactam-resistant strain;
glycopeptide-resistant strain.
d
were not impaired by expression of resistance mechanisms to β-lactams
Declaration of Competing Interest
and/or glycopeptides in the strains. Of note, as exemplified by com-
pounds 19 and 20, the chirality of the phenyl-glycine core, common to
all our gold(I) derivatives, appeared to have no notable impact on the
antimicrobial properties of the series. The same applies to the penam
scaffold which appears as dispensable. Since antibacterial activities of
the chryso-lactams and fragments synthetized relies on gold(I) ion,
emergence of cross-resistance with currently used antibiotic families
under treatment with organometallics seems very unlikely.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
G.L.A.M. and I.J.S. thank the Centre National de la Recherche Scien-
tifique (CNRS) for global financial support. G.L.A.M. and I.J.S. thank also
TTO Conectus for investment in the valorization and patenting process.
M.M. thanks Nathalie Wagner for purifying compounds by semi-
preparative HPLC and the entire Prestwick Quality Control team for
compound analysis. P.P thanks Mr Loïs Andrey for his technical assis-
tance with drug susceptibility testing. Reference strains of Staphylo-
coccus sp. and Enterococcus sp. were kindly provided by the French
Heavy and noble metals derivatives are known to be more toxic for
eukaryotic cells than their fully organic homologs. We thus compared
the toxicity of the fragments 13 and 19 with that of chryso-lactams 3 and
9 on human healthy hepatocytes. As previously reported, ampicillin
remained non-cytotoxic over the entire range of concentrations tested.9
Penam derivatives 3 and 9 exhibited EC50s of 23.14 ± 0.18 µM (18.12
± 0.14 µg.mLꢀ 1) and 17.57 ± 1.29 µM (14.91 ± 1.08 µg.mLꢀ 1
)
respectively, while the EC50 of fragment 13 was 11.71 ± 0.56 µM (7.61
± 0.36 µg.mLꢀ 1). EC50 of the smallest fragment 19 could not be
determined due to solubility issues in the concentration range used for
the other compounds. However, with 8% viable cells at 7 µM and 0% at
12 µM 19 proved to have a much higher cytotoxicity compared to 3, 9 or
13 (See ESI). As already mentioned for chryso-lactams and other gold(I)
derivatives with potential anti-infective applications, the metal center is
essential for the expected activity.9,11,12,19 Our results prove that the
smaller is the organic part of gold(I) compound, the higher is the
eukaryotic cytotoxicity with, in parallel, only modest changes in anti-
bacterial activity. The organic counterpart is therefore of great impor-
tance to finely tune up the biological properties of organo-gold(I)
antibacterial compounds.
´ ´
national reference laboratories of Lyon (Frederic Laurent) and Rennes
(Vincent Cattoir), respectively.
Appendix A. Supplementary data
Protocols and analytical data concerning the synthesis of compounds
9, 12, 13, 14, 19, 20 and 25. Experimental conditions for Minimum
Inhibitory Concentrations assessments and for the in vitro cytotoxicity
assays of compounds 3, 9, 13 and 19 on hepatocytes can be found online
DOI
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127879.
In conclusion, this study describes the synthesis and the biological
activities of unprecedented gold(I) derivatives including a new genera-
tion of chryso-lactams. All these organometallics exhibit high antibac-
terial activities in vitro against major Gram-positive pathogens,
including multidrug resistant strains. While ineffective against Gram-
negative bacteria like E. coli, they were able to inhibit C. difficile at
very low concentrations (≤0.125 µg/mL), confirming that chryso-
antibiotics are a promising and untapped source of therapeutic inno-
vation against this drug recalcitrant Gram-positive pathogen.
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